Organofluorine Hydrazone Derivatives as Multifunctional Anti-Alzheimer's Agents with CK2 Inhibitory and Antioxidant Features

Article abstract of DOI:10.1002/cmdc.202100047

A set of novel hydrazone derivatives were synthesized and analyzed for their biological activities. The compounds were tested for their inhibitory effect on the phosphorylating activity of the protein kinase CK2, and their antioxidant activity was also de

Full text of DOI:10.1002/cmdc.202100047

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Organofluorine Hydrazone Derivatives as Multifunctional
Anti-Alzheimer’s Agents with CK2 Inhibitory and
Antioxidant Features
Andrea Baier,*[a] Anne Kokel,[b] William Horton,[b] Ewa Gizińska,[c] Garima Pandey,[b]
Ryszard Szyszka,[c] Béla Török,[b] and Marianna Török*[b]
A set of novel hydrazone derivatives were synthesized and
analyzed for their biological activities. The compounds were
tested for their inhibitory effect on the phosphorylating activity
of the protein kinase CK2, and their antioxidant activity was
also determined in three commonly used assays. The hydra-
zones were evaluated for their radical scavenging against the
DPPH, ABTS and peroxyl radicals. Several compounds have
been identified as good antioxidants as well as potent protein
kinase CK2 inhibitors. Most hydrazones containing a 4-N(CH3)2
residue or perfluorinated phenyl rings showed high activity in
the radical-scavenging assays and possess nanomolar IC50
values in the kinase assays.
Introduction
spectrum. Up to date more than 350 of its protein substrates
have been identified.[11] CK2 is known to phosphorylate tyrosine
besides the serine and threonine residues, thus it is considered
a dual-specificity kinase.[10] Although there is no broad agree-
ment regarding the mechanisms of CK2 regulation, its potential
role in pathogenesis associated with inflammation has been
proposed.[12]
A wide range of compounds have been identified as
inhibitors of CK2,[13,14] including 4,5,6,7-tetrabromo-1H-benzo-
triazole (TBB).[15] This compound is an ATP-competitive inhibitor
that has been commonly applied as a molecular probe to
identify the function of CK2. Silmitasertib (CX-4945) is another
selective, ATP-competitive inhibitor that can modulate the
activity of the α and α’ catalytic subunits of CK2.[16,17] It is the
first CK2 inhibitor that has reached the level of clinical trials in
2010 for the treatment of various forms of cancer.[17]
In addition to its potential as an anticancer drug target, the
role of CK2 has also been evaluated in the hippocampus and
temporal cortex of AD patients in comparative studies. The
effects of CK2 inhibitors on IL-6 and MCP-1 secretion have also
been investigated in stimulated human primary astrocytes and
U373 cells. These reports highlight the recent sentiment
regarding a possible role of CK2 in neuroinflammation and
thus, in the pathogenesis of AD. It has been proposed, that CK2
could serve a potential drug-able target to modulate inflamma-
tory response in AD.[18]
In Alzheimer’s disease therapy, the application of CK2
inhibitors could prove to be advantageous alleviating the effect
of hyperphosphorylated tau and neurofibrillary tangles. In
addition, the reduction of insulin receptor internalization and
improvement of the Aβ disabled fast axonal transport could
improve the condition of AD patients.[19] In our earlier
publications we described the inhibition of various CK2 isoforms
by natural and synthetic compounds such as halogenated
benzimidazoles and flavonoids.[20–22]
Due to the ever-increasing number of patients affected by
Alzheimer’s disease (AD), the development of new therapeutics
for AD is of high importance. AD is a complex disease, and
identifying the related biochemical processes and signaling
pathways significantly contributes to finding new therapeutic
targets.[1,2] The major targets include protein kinases due to
their involvement in many cellular processes and a plethora of
signaling pathways. An additional advantage of kinases is that
they are drugable, thus they are frequently considered
therapeutic targets for AD drug development.[3]
Protein kinase CK2 was found in the AD affected brain as
early as 1990 and has been proposed to play a role in the
pathology of the disease.[4,5] The CK2 holoenzyme possesses
two catalytic α subunits of slightly different sizes (39 and
45 kDa) and two regulatory β subunits (26 kDa), however, all
subunits also exist in their monomeric forms in the cell.[6] In
addition, the catalytic α subunit can be found in several
different isoforms, the three most common ones are the α, α’
and α’’.[7,8] CK2 is constitutively active and uses both adenosine
triphosphate (ATP) and guanosine triphosphate (GTP) as
phosphate donors.[5,9,10] This protein kinase has
a broad
[a] Prof. A. Baier
Department of Animal Physiology and Toxicology
The John Paul II Catholic University of Lublin
ul. Konstantynów 1i, 20-708 Lublin (Poland)
E-mail: baier@kul.pl
[b] Dr. A. Kokel, Dr. W. Horton, Dr. G. Pandey, Prof. B. Török, Prof. M. Török
Department of Chemistry
University of Massachusetts Boston
100 Morrissey Blvd, Boston, MA 02125 (USA)
E-mail: marianna.torok@umb.edu
[c] E. Gizińska, Prof. R. Szyszka
Department of Molecular Biology
The John Paul II Catholic University of Lublin
ul. Konstantynów 1i, 20-708 Lublin (Poland)
Hydrazones, as largely nontoxic entities, have attracted
significant attention in drug development. They are considered
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100047
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as part of hybrid compounds when designing more effective, Results and Discussion
often multitarget therapeutics.[23–26] Hydrazones exhibit a broad
spectrum of biological activities and have been described in the
treatment of tuberculosis,[27] malaria[28] or as antiviral[29,30] and
anticancer agents.[31] Furthermore, hydrazones have also been
identified as strong radical scavengers.[32,33] Their multitarget
features have been combined as potential AD therapeutics;
several hydrazones were found to be excellent inhibitors of
amyloid beta fibril and oligomer formation as well as showed
highly effective radical scavenging properties.[34]
Continuing our efforts in the development of multifunc-
tional anti-AD agents, herein we describe the synthesis and
evaluation of novel organofluorine hydrazones as protein kinase
CK2 inhibitors and free radical scavengers.
For this study, several novel hydrazone derivatives were
designed and prepared (Scheme 1, Figure 1). Our earlier studies
indicated that hydrazones are potent multitarget compounds
to combat AD. They appeared to be excellent inhibitors of Aβ
self-assembly, including both oligomer and fibril formation and
also acted as effective radical scavengers.[34] In this study it was
intended to observe how similar hydrazone derivatives would
affect protein kinase CK2 activity that has been connected to
the development of AD as well. The first generation of
hydrazones (Figure 1A) included in this work were compounds
possessing the hydrazone core unit and a variety of substitu-
ents. The major goal was to investigate the role of different
substituents and identify compounds with CK2 inhibitory
activities that potentially could be improved by further design.
Based on the preliminary data tabulated in Table 1, it was
found that hydrazones that possessed one or more fluorine
atom showed the highest potency. This is in agreement with
our earlier findings with different scaffolds that the presence of
multiple halogens, such as bromine or iodine atoms to
benzimidazoles resulted in a significant increase in their
inhibitory potential.[20] Combining the efficacy of the hydrazone
scaffold with the beneficial effects of fluorine incorporation it
was decided to design and synthesize compounds with
gradually growing number of fluorine atoms (Figure 1B). In
general, two types of fluorine incorporation was considered; the
aromatic F, and the CF3 groups. Both of these substituents
exhibit high stability during a wide range of chemical and
biological conditions,[35] thus by their application one can avoid
the formation of highly toxic metabolites. The identity and
purity of the new compounds were confirmed by gas
chromatography-mass spectrometry (GC-MS) and NMR spectro-
scopy (see the Supporting Information). The structures of the
compounds prepared for the current study are summarized in
Figure 1.
Scheme 1. General synthesis of hydrazones.
Table 1. Inhibition of different CK2 isoforms by the first generation
hydrazones (1–15).
Compound
IC50 [μM]
CK2α
CK2α’
CK2α2β2
CK2α’2β2
1
2
>50
>50
>50
>50
17.8�1.4
>50
>50
>50
3
4
5
6
7
8
>50
>50
>50
>50
9.8�0.8
>50
>50
>50
16.5�1.2
15.4�1.2
>50
12.1�1.1
16.6�1.8
>50
>50
>50
>50
10.8�0.6
8.4�0.6
>50
22.4�2.5
13.8�1.1
>50
>50
9
25.1�2.8
>50
>50
>50
>50
1.2�0.1
10.3�0.9
15.5�1.0
14.7�1.2
>50
15.2�1.6
15.1�1.6
>50
>50
>50
>50
13.6�1.4
10
11
12
13
14
15
>50
7.3�0.5
>50
1.7�0.1
14.4�1.4
14.6�1.7
>50
>50
>50
13.2�0.9
>50
Figure 1. Structure of the A) first and B) second generation hydrazones used
in this study.
IC50 values�standard deviation are expressed as the mean of three
independent experiments.
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In the first step we tested 15 hydrazone derivatives
Table 2. Inhibition of different CK2 isoforms by the second generation
hydrazones (16–31).
(Figure 1A) containing varying substituents. The inhibitory
effect, described as IC50, was determined by using a series of
concentrations of inhibitors and using yeast acidic ribosomal
protein P2B as phosphoacceptor. From these enzyme inhibition
studies the activity of the compounds has been determined.
The obtained results are summarized in Table 1. Analyzing the
data in connection to the structural features of the molecules, it
was observed that compounds with either the 2-OHÀ 4-OCH3 or
4-N(CH3)2 substituent exhibited an improved effect on CK2α’.
Compounds 3, 6, 7, 9, 11 and 13 showed the best inhibitory
effect towards CK2α’ subunit. Those compounds seem to be
selective inhibitors for the CK2α’ subunit with IC50 values
ranging from 1 to 11 μM. Whereas inhibitors 1–6, 10–13 and 15
were largely ineffective on CK2α, decreasing its activity only to
70% at a concentration of 50 μM, compounds 7 and 9 also
possessed moderate inhibitory activity towards CK2α.
Afterwards, we analyzed the potential of these compounds
towards both CK2 holoenzymes. Only one hydrazone (com-
pound 9) possessed activity against all four CK2 isoforms.
Several compounds (4, 5, 10) were effective only towards the
holoenzymes without influencing the enzymatic activities of the
free catalytic subunits. Interestingly, compounds which showed
no or only moderate effect towards monomeric CK2α possessed
activity on its holoenzyme CK2α2β2. Similar tendency was
already reported in our former work with flavonoid
compounds.[22] Compounds 1, 4, 5, 7, 9, 10, 11 and 13
possessed modest inhibitory effect (IC50 =10–20 μM) against
CK2α2β2. Some compounds (3, 6, 7, 11 and 13) showing
inhibitory influence on the monomeric CK2α’ subunit lost this
activity towards the holoenzyme CK2α’2β2. Within all tested
compounds three (2, 8, 12) had no inhibitory effect on any of
the four examined CK2 isoforms.
Based on the information obtained by screening the general
1st generation hydrazones as well as earlier evidence on the
beneficial effect of halogen incorporation to other CK2 inhib-
itors a new set of hydrazones (noted as 2nd generation,
Figure 1 B) were designed and synthesized as described above
(Scheme 1). Sixteen hydrazones, fluorinated to various extents
were included in these assays with the aim of further improving
the inhibitory properties of the compounds. The preparation of
fluorinated test compounds was limited to the addition of
aromatic F and CF3 residues based on the results from the first
series as well as to ensure the high metabolic stability of these
substituents. In addition to the general positive features of
fluorine introduction to drug candidates, a major reason to
replace other halogens was the high stability of these groups.[35]
In earlier studies we tested several brominated, chlorinated and
iodinated compounds, which usually possess higher cytotoxicity
and are less stable.[20] The CK2 inhibition data obtained with the
second generation hydrazones are tabulated in Table 2.
Compound
IC50 [μM]
CK2α
CK2α’
CK2α2β2
CK2α’2β2
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
18.9�1.9
21.2�2.2
16.4�1.4
>50
0.1�0.01
0.2�0.1
32.5�2.4
34.3�2.6
>50
39.1�2.6
37.3�2.5
>50
17.7�1.2
18.5�1.3
18.7�1.3
19.2�1.4
15.1�1.1
25.7�1.9
>50
>50
>50
>50
0.9�0.04
0.2�0.01
0.1�0.01
19.8�1.8
16.8�1.3
1.3�0.07
13.2�1.1
11.4�1.0
14.9�1.3
34.7�2.5
12.2�1.1
17.3�1.3
13.6�0.9
8.2�0.7
>50
14.8�1.1
>50
36.2�2.3
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
38.2�2.7
>50
31.4�2.7
28.7�1.9
>50
28.9�2.1
32.6�2.3
>50
>50
>50
>50
>50
>50
>50
IC50 values�standard deviation are expressed as the mean of three
independent experiments.
tendency observed in these experiments is that the addition of
fluorine atoms improves the inhibitory activity of the com-
pounds. It is worth noting that both compounds (16, 17) that
exhibit strong to excellent activity in the inhibition of all four
forms of the enzyme, contain perfluorophenyl group on either
side of the hydrazone core. Furthermore, only one of the six
high activity compounds (16–21) does not contain the perfluor-
ophenyl unit, emphasizing its importance in ensuring favorable
molecular characteristics for inhibition of CK2. Interestingly,
several compounds are active against the free CK2α’ subunit
and the CK2α2β2 holoenzyme. This is a similar mode as in case
of natural flavonoids we tested previously.[22] The regulatory
subunit affects the inhibitory activity leading to better inhib-
ition of CK2α’ than the holoenzyme CK2α’2β2, but increased
inhibition for CK2α2β2 than the free CK2α subunit.
To evaluate the mechanism of the hydrazones in the
inhibition of the CK2 activity different ATP concentrations (10,
20 and 40 μM) were evaluated in the reaction mixture. All
inhibitors decreasing the catalytic activities acted via a purely
ATP-competitive mode of inhibition. The lowest Ki values were
estimated for compounds 16 and 17 with 107 and 69 nM,
respectively (Figure 2).
As it was shown for benzimidazoles and flavonoids[36,37] the
efficacy of many ATP-binding site competitive inhibitors
depends on hydrophobic amino acids, namely V66, M163, I174
and V67, M164, I175 in CK2α and CK2α’, respectively. Those
amino acids are replaced with less bulky residues in most other
protein kinases which give the pocket in CK2 its specific narrow
characteristics. Table 3 summarizes the results obtained with
the CK2α’ mutants V67A, I117A and I175A. The inhibitory effect
of the hydrazone derivatives on all three tested mutants is
decreased when compared to the wild-type. This proves that
the compounds fit into the ATP-binding pocket and the hinge
region.
The addition of fluorine atoms led to highly selective
inhibitors towards the CK2α’ subunit. As can be seen
extensively fluorinated compounds are highly effective inhib-
itors. Derivatives 16–20 possessed IC50 values in the 100–
900 nM range. All of these compounds have at least one (often
two) pentafluorophenyl ring highlighting its importance. The
As oxidative stress plays a significant role in the develop-
ment of AD, incorporating antioxidant features into multitarget
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Figure 3. Radical scavenging potential of the hydrazone derivatives (1–31)
used in this study, in comparison to known standards such as ascorbic acid
(AA), resveratrol (Res) and Trolox. The activity was measured in ORAC (black),
ABTS (gray) and DPPH (pattern) assays at 10 μM compound concentration.
The values are shown as mean of % radical scavenging�standard deviation,
where the number of independent repeats is 3.
and compounds containing the 2-OH and 4-OCH3 motifs
showed high activity with recorded radical scavenging higher
than that of ascorbic acid and Trolox. In the ORAC assay, the 4-
N(CH3)2 containing compounds were of the highest activity
ranging from 83% to 94% for compounds 11, 13, 17, 19 and
22, which is double of the potency of ascorbic acid and Trolox,
and about the same as that of resveratrol. The next highest
compound activity in the ORAC assays was for the hydrazone
derivatives containing the 2-OH and 4-OCH3 moiety, with the
highest activity ranging from 49 to 87% for compounds 7, 9,
25, 26 and 30. In the DPPH assay, the 2-OH 4-OCH3 and 4-
N(CH3)2 containing compounds dominated, showing the high-
est levels of activity with compounds 7, 9, 22, 28 and 30
ranging from 28 to 54%. In the ABTS assay, the highly
fluorinated compounds (pentafluorophenyl rings) 16, 18, 20,
21, and 24 all showed high activity with radical scavenging
percentages from 34 to 53%. The 2-OH and 4-OCH3 containing
compounds performed well with 30 to 41% radical scavenging
as well as the 4-N(CH3)2 compounds ranging from 28 to 35%
radical scavenging activity in the ABTS assay. Compound 8 with
a bicyclic structure and no NH-group, revealed the importance
of the hydrazone moiety for the radical scavenging activity of
these compounds having a radical scavenging of less than zero
percent in all three radical scavenging assays. Compounds with
two hydrazone moieties did not show much improvement in
radical scavenging activity with many of these compounds
having limited potency. Overall the hydrazones had generally
higher radical scavenging activity than the Trolox and ascorbic
acid control compounds in all three of the radical scavenging
assays. Several compounds such as 7, 9, 15, 19, 22, 26, 28 and
30 all had equal or higher activity than resveratrol in the DPPH
assay. These compounds show promise as future candidates for
the development of free radical scavenging agents.
Figure 2. Inhibition of CK2α’ by compounds A) 16 and B) 17. Lineweaver–
Burk double reciprocal plots are shown, the insets refer to the Km/Vmax vs.
inhibitor concentration replot. Inhibitor concentration=a) 0, b) 500 nM, c)
1000 nM, and d) 5000 nM.
Table 3. IC50 values for the inhibition of CK2α’ WT and mutants by selected
second generation hydrazones.
Compound
IC50 [μM]
CK2α’ wt
CK2α’V67A
CK2α’I117A
CK2α’I175A
16
17
18
19
20
23
0.1�0.01
0.2�0.01
0.9�0.04
0.2�0.01
0.1�0.01
1.3�0.07
18.4�1.6
12.6�1.1
15.3�1.2
11.9�1.1
8.1�0.7
2.5�0.1
3.7�0.2
5.8�0.2
2.8�0.2
2.6�0.1
9.8�0.8
15.1�1.3
11.8�1.1
25.7�2.1
13.4�1.2
5.2�0.4
15.7�1.1
40.8�3.4
IC50 values�standard deviation are expressed as the mean of three
independent experiments.
anti-AD compounds is highly desirable. Thus, in addition to the
evaluation of the CK2 enzyme inhibitory potential, the antiox-
idant potential of the compounds was also determined using
broadly accepted radical scavenging assays. The first and
second sets of hydrazone derivatives were evaluated for their
radical scavenging activity by the 2,2-diphenyl-1-picryl-hydra-
zyl-hydrate (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-
sulfonic acid (ABTS), and Oxygen Radical Absorbance Capacity
(ORAC) assays.[38] The data are illustrated in Figure 3.
Several trends both for compounds with excellent radical
scavenging activity and poor scavenging activity were ob-
served. In all three assays, compounds with the 4-N(CH3)2 group
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The analysis of the combined results of CK2 inhibition data Acknowledgements
and the radical scavenging activities reveals important details
about the structure-activity relationship of the compounds.
Although the hydrazone scaffold showed overall promising
behavior in both CK2 inhibition and radical scavenging, every
compound that showed strong activity against all four isoforms
of the enzyme (9, 16, 17) and moderate (16) to excellent (9, 17)
This research was supported by The John Paul II Catholic
University of Lublin-University of Massachusetts Boston Strategic
Partnership Joint Seed Funding Program.
radical scavenging, had multiple F atoms in their structure, Conflict of Interest
either in the form of a perfluorophenyl ring or a CF3 group. This
is also true for compounds that inhibited at least three isoforms
of the enzyme (7, 18–21) and showed moderate (7, 18, 20, 21)
to excellent (7, 19) radical scavenging. Based on the data it is
reasonable to propose that the incorporation of multiple
fluorine atoms to the scaffold strongly improves the enzyme
inhibitory potential, while it is somewhat neutral regarding the
antioxidant features in vitro. In addition to these effects, these
organofluorine hydrazones possess much better membrane
transport properties in vivo. The radical scavenging potency is,
however, enhanced by the presence of substituents that can
back donate an electron pair to the system, such as À N(CH3)2, or
OH and OCH3, which increase the mobility of the NÀ H hydrogen
of the hydrazone unit contributing to effective radical scaveng-
ing via the HAT mechanism.[23,38] Concerning the activities in all
assays the best combination appears to be when one aromatic
ring possesses the À N(CH3)2 substituents with a perfluorinated
ring as a counterpart (17). Although based on a limited number
of hydrazones, the structure-activity relationship is not abso-
lutely clear, the activity data provide sufficient guidance for
further structural refinement.
The authors declare no conflict of interest.
Keywords: Alzheimer’s disease · antioxidants · hydrazones ·
organofluorine · protein kinase CK2 · radical scavenging
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Conclusions
In conclusion, the findings in this study corroborate our former
results concerning the antioxidant features of hydrazone
derivatives. To our knowledge this is the first report identifying
hydrazones as inhibitors of the protein kinase CK2. Up to now
they were known as inhibitors of other kinases, like RSK2, CDK4
and MARK4.[39–41]
The role of CK2 in the brain is not completely clear and still
remains elusive. It was reported that CK2 subunits (α, α’, β) are
differently distributed in the major brain regions.[42] Whereas
CK2α is found at equal level in the different regions, the
hippocampus and the prefrontal cortex contain significantly
higher amounts of CK2α’ when compared to other analyzed
regions. It seems that CK2α’ participates in cell migration and
adhesion.[43] In AD patients, it was shown that CK2α/α’, and
thereby enzyme activity, is significantly overexpressed in the
hippocampus and temporal cortex when compared to the
control group.[18]
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doi.org/10.1002/cmdc.202100047
ChemMedChem
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Manuscript received: January 19, 2021
Accepted manuscript online: March 12, 2021
Version of record online: ■■■, ■■■■
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These are not the final page numbers!

FULL PAPERS
Prof. A. Baier*, Dr. A. Kokel, Dr. W.
Horton, E. Gizińska, Dr. G. Pandey,
Prof. R. Szyszka, Prof. B. Török,
Prof. M. Török*
1 – 7
Organofluorine Hydrazone Deriva-
tives as Multifunctional Anti-Alz-
heimer’s Agents with CK2 Inhibi-
tory and Antioxidant Features
Seeing off CK2: Organofluorine diaryl-
hydrazones are effective multitarget
compounds that alleviate the activity
of protein kinase CK2 and show
strong antioxidant effects in in vitro
radical-scavenging assays. They
contain a phenyl CÀ F bond and/or the
CF3 group. As both CK2 activity and
oxidative stress appear to contribute
to the development of Alzheimer’s
disease, the compounds are potential
anti-AD agents.