CK2 inhibition, lipophilicity and anticancer activity of new: N 1versus N 2-substituted tetrabromobenzotriazole regioisomers

Article abstract of DOI:10.1039/d0nj01194k

A new series of antiproliferative casein kinase 2α (CK2α) inhibitors were synthesized incorporating either a hydrophilic group (carboxylic or hydrazide) or a hydrophobic group (ester) at N1 or N2 of 4,5,6,7-tetrabromobenzotriazole (TBBt). New compounds were prepared via N-alkylation of TBBt followed by base-catalysed hydrolysis or hydrazinolysis. All the compounds demonstrated low sub-micromolar inhibition of CK2α and antiproliferative activity against both breast and lung cancer cell lines (MCF-7, and A549, respectively), at low micromolar concentrations with N2-regioisomers exhibiting higher activity than their corresponding N1-isomers. The most active compound incorporates an acetic acid hydrazide moiety at the N2 of the TBBt triazole nucleus with IC50 at 0.131 μM (CK2α), 9.1 μM (MCF-7) and 6.3 μM (A549). It induced apoptosis in the MCF-7 cell line through upregulation of bax (pro-apoptotic gene) four to five times higher than the corresponding ester or acid analogues. Molecular docking suggests that the hydrophilic group at N2 of the TBBt triazole nucleus provides binding with important residues (Asp175, Lys68 and Trp176) in the ATP binding site of the CK2α enzyme. We are the first to experimentally estimate the lipophilicity of TBBt derivatives. Our study demonstrated that TBBt hydrazides are more lipophilic than their corresponding acids in contrast to the contradicting calculated lipophilicity using four well-known software programs. This may explain the higher anticancer activity of the most active hydrazide over its corresponding acid despite their nearly equipotent enzyme inhibition. This journal is

Full text of DOI:10.1039/d0nj01194k

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CK2 inhibition, lipophilicity and anticancer activity of new N1
versus N2-substituted tetrabromobenzotriazole regioisomers
Ahmed El-Kardocy 1, Yaser A. Mostafa2, Noha G. Mohamed1, Mohammad Nabil Abo-Zeid3, Nivin A.
Received 00th January 20xx,
Accepted 00th January 20xx
Hassan4, Helal F. Hetta5,6, Abu-Baker M. Abdel-Aal1,2,
*
DOI: 10.1039/x0xx00000x
Abstract: A new series of antiproliferative Casein Kinase 2α (CK2α) inhibitors were synthesized
incorporating either a hydrophilic group (carboxylic or hydrazide) or a hydrophobic group (ester)at
N1 or N2 of 4,5,6,7-tetrabromobenzotriazole (TBBt). New compounds were prepared via N-
alkylation of TBBt followed by base-catalysed hydrolysis or hydrazinolysis. All compounds
demonstrated low sub-micromolar inhibition of CK2α and antiproliferati ve activity against both
breastand lung cancer cell lines (MCF-7, A549, respectively) atlowmicromolar concentrations with
N2-regioisomers exhibiting higher activity than their corresponding N1-isomers. The most active
compound incorporates acetic acid hydrazide moiety at the N2 of TBBt triazolenucleus with IC50 at
0.131 μM (CK2α), 9.1 μM (MCF-7) and 6.3 μM (A549). Itinduced apoptosis in MCF-7 cell linethrough
upregulation of bax (pro-apoptotic gene) four to five times higher than the corresponding ester or
acid analogues. Molecular docking suggests thehydrophilic group at N2 of theTBBttriazolenucleus
provides binding with importantresidues (Asp175, Lys68 and Trp176) in the ATP binding siteof the
CK2α enzyme. We are the first to experimentally estimate lipophilicity of TBBt derivatives. Our
study demonstrated that TBBt hydrazides are more lipophilic than their corresponding acids in
contrastto thecontradicting calculated lipophilicity using four well-known softwareprograms.This
may explain the higher anticancer activity of themost activehydrazideover its corresponding acid
despite their nearly equipotent enzyme inhibition.
Keywords: Kinase CK2; anticancer; apoptosis; lipophilicity; benzotriazole
these proteins 3. Inhibition of protein kinases is becoming the
most attractive therapeutic strategy with over 20 kinase
targeting drugs have been approved as anticancer agents over
Introduction
Oncogenic transformations are associated with activation and
overexpression of several protein kinases involved in the
the past decade and hundreds are currently in clinical trials for
various diseases4-6
For instance, casein kinase 2 (CK2) is
.
a
regulation of critical cellular processes such as growth,
serine/threonine protein kinase overexpressed in many cancers
including acute myeloid leukaemia, breast, prostate and lung
cancer7, 8. In the last decade, a great number of CK2 inhibitors
2
differentiation, signalling and transduction1,
. Protein kinases
activate other proteins by transferring a phosphate group from
adenosine triphosphate (ATP) to serine, threonine or tyrosine in
have
benzimidazoles, polyhalogenated benzotriazoles, indolo and
pyrroloquinolines with the most successful example,
been
developed
including
polyhalogenated
silmitasertib (CX-4945, Figure 1), currently in clinical trials
a. 1
Student research unit, Faculty of Pharmacy, Assiut University, Assiut 71526,
9-17
Egypt.
(ClinicalTrials.gov identifier: NCT02128282)
.
b. 2
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Assiut University, Assiut 71526, Egypt
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy,
Assiut University, Assiut 71526, Egypt
Cancer Biology Department, South Egypt Cancer Institute, Assiut University,
Assiut, Egypt
Department of Medical Microbiology and Immunology, Faculty of Medicine,
Assiut University, Assiut, Egypt
c.
3
4,5,6,7-tetrabromo-1H-benzotriazole (TBBt, Figure 1) is the
most potent and selective CK2 inhibitor (Ki = 0.4 μM) among
polyhalogenated benzotriazoles and second after DMAT in the
d. 4
e. 5
polyhalogenated benzimidazoles18-21
CK2α is relatively smaller than the active sites of other kinases22
. The catalytic center of
.
f.
6
Department of Internal Medicine, University of Cincinnati College of Medicine,
The presence of four bromine atoms is optimal for binding of
the molecule to a hydrophobic pocket in the ATP-binding site of
the enzyme forming halogen bonds and hydrophobic
interactions with essential hydrophobic amino acid residues23,
Cincinnati, OH, USA
*
g.
Correspondence: abubaker.elsayed@pharm.aun.edu.eg; Tel.: +02-0101-932-
9596
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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24. Despite the potency and selectivity of TBBt as CK2 inhibitor,
it was found to be of low anti-proliferative potency and does
not affect cell viability of cancer cell lines below 50 μM 25. The
human clinical trial candidate CX-4945 (Figure 1) which is
structurally unrelated to polyhalogenated benzotriazole,
View Article Online
DOI: 10.1039/D0NJ01194K
occupies the same hydrophobic pocket in the ATP binding site
25,
of CK2α13,
26. The carboxyl group of CX-4945 is strategically
positioned to provide extra hydrophilic interactions between
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CX-4945 and CK2α (Lys68, Glu81, Asp175 and Trp176) which is
13, 26, 27
not possible in the case of TBBt
.
In the current study, we envisaged introduction of polar
hydrophilic moieties (carboxyl acid or hydrazide) at the TBBt
triazole ring (Figure 1) may provide novel derivatives capable of
forming strong polar interactions with the area outside the
hydrophobic pocket of the CK2 ATP binding site compared to
their hydrophobic ester analogues. Introduction of the polar
groups at N1 or N2 of the triazole nucleus would give information
about the importance of their orientation in the ATP binding site
for CK2 inhibitory activity. Hydrophilic groups will alter
important physicochemical properties of the polyhalogenated
benzotriazole scaffold such as lipophilicity which affect their cell
membrane penetration and anti-proliferative activity. To the
best of our knowledge, there are no reports for experimentally
Figure 1: Design of new anti-proliferative CK2 inhibitors.The
polar ester, hydrazide or acid functional groupsareintroduced
at either N1 or N2 of the TBBt scaffold.
estimated lipophilicity of
derivatives.
polyhalogenated benzotriazole
Scheme 1: Synthesis of N1 and N2-substituted TBBt regioisomers (2-7). Reagents and conditions: a) Br2, conc. HNO3, reflux, 48h; b)
ClCH2COOCH2CH3, K2CO3, acetone, reflux, 6h; c) NH2NH2.H2O, ethanol, reflux, 6h; d) 5% KOH, 50% aqueous etha nol, reflux, 1h.
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In the current study, we report the lipophilicity of TBBt and our
new N1 and N2-substituted TBBt regioisomers as estimated
theoretically and experimentally to find correlations between
lipophilicity and anticancer activity. The aim was to modify the
polyhalogenated benzotriazole scaffold with
a minimal polar
functional group to improve CK2 inhibitory activity and to
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obtain polyhalogenated TBBt derivatives capable of inhibiting
cancer cell proliferation at low concentrations.
2. Results and discussion
2.1. Chemistry
4,5,6,7-tetrabromobenzotriazole
extensive bromination of
(1) was
benzotriazole
synthesized
via
in refluxing
concentrated nitric acid as previously reported28. New target
compounds 2-7 were efficiently synthesized via N-alkylation
followed by hydrazinolysis or base catalyzed hydrolysis, Scheme
1. N-alkylation was achieved by refluxing
1 with ethyl
chloroacetate in acetone, potassium carbonate and tetrabutyl
ammonium bromide as phase transfer catalyst. A mixture of N1
and N2-acetate regioisomers (2 and 3) was obtained in a ratio of
3:2 in a good total yield. Separation of these two regioisomers
Figure 2: C13-NMR spectra demonstrate distinct patternforpurified
regioisomers 2 and 3.
was successfully performed using silica
gel
column
chromatography and gradient elution of hexane: ethyl acetate
(12:1 to 7:1). N-alkylation was confirmed by the appearance of
ester carbonyl group and aliphatic C-H stretching vibrations in
the IR spectrum of 2 (1734.6 and 2925.2 cm-1) and 3 (1743.2,
2967.9 cm-1). Additionally, the N-methylene protons appears as
singlet peak at 5.89 and 5.94 ppm in the H1-NMR spectrum of 2
and 3, respectively. While, the two isomers exhibit quite similar
IR and H1-NMR spectra, their C13-NMR spectra (Figure 2) are
fundamentally distinguishable. For instance, the N1-acetate
isomer (2) has six peaks in the aromatic region (100-150 ppm)
corresponding to the benzotriazole ring carbons. In contrast,
the N2-acetate isomer (3) has only three peaks in the same
region due to the existence of a plane of molecular symmetry.
Having the two esters in hand, we were able to obtain the
corresponding hydrazides and acids. Hydrazinolysis was
achieved by refluxing each ester (2-3) in excess hydrazine
hydrate and ethanol for 6 h. Pure hydrazide derivatives (4-5)
simply crystallized out when the reaction was left to cool
overnight and obtained in a satisfactory yield by simple filtration
and washing with ethanol. Hydrazides were identified by the
appearance of NH and carbonyl vibrations in the IR spectra of 4
(3316 and 1663 cm-1) and 5 (3304 and 1661 cm-1). Moreover,
the ethyl protons of the starting esters disappear in the H1-NMR
spectra of the product hydrazides. Instead, additional peaks
appear at 4.4 ppm and 9.5 ppm corresponding to the NH2 and
NH protons with upfield shifting of the N-methylene protons to
5.51 ppm. The acid derivatives (6-7) were obtained by base-
catalyzed hydrolysis of the corresponding esters (2-3) using 5%
KOH. The products were obtained pure after acidification of the
reaction mixture and were confirmed by the appearance of
broad OH stretching vibration at 3200-2500 cm-1 and acid
carbonyl stretching vibration at 1736 and 1732 cm-1 in the IR
spectrum of 6 and 7, respectively. The H1-NMR spectra of both
compounds show only one singlet peak for the N-methylene at
5.7-5.8 ppm. The purity of all compounds was confirmed using
elemental analysis and were found to exceed 99.6%.
2.2. CK2 enzyme inhibition and molecular modelling study
All tested compounds have the TBBt scaffold and differ only in
the position and nature of substituent at the triazole N1 or N2.
Except for compound 2, other tested compounds showed CK2
inhibitory activity at sub-micromolar concentrations (Figure 3a).
The most potent derivatives (5 and 7) were five times more
potent than the parent TBBt (1) with their CK2 IC50 at 0.131 and
0.098 µM, respectively. Both compounds have their hydrophilic
groups (hydrazide and carboxyl) substituted at N2 of the triazole
nucleus. Their respective regioisomers (4 and 6) were four to
five times less potent which indicates the importance of the
position of hydrophilic group for CK2 enzyme inhibitory activity.
Similarly, ester derivatives (2 and 3) were three to four times
less potent than their corresponding acid derivatives (6 and 7).
Molecular modelling study was done to explain the differences
in CK2 enzymatic inhibitory activity of tested compounds in
comparison to CX-4549. For instance, CX-4549 binds to key
amino acids in the ATP binding site of CK2α including direct
binding to Val116 and Lys68 and indirect binding via two
26
conserved water molecules to Asp175, Glu81 and Trp176
.
Docking of compounds 2-7 showed that these compounds
occupy the same hydrophobic pocket in the CK2 ATP binding
site like their parent compound (1) and CX-4945 (Figure 3b-c,
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Supporting information S2). The most potent CK2 enzyme essential direct interactions with Val116, Lys68 and Asp175
inhibitors of the tested compounds (5 and 7) establish the and indirect interactions with Glu81 and Trp176 through two
View Article Online
DOI: 10.1039/D0NJ01194K
5
6
7
8
Figure 3: a) CK2 IC50 values of tested compounds (1-7) represented as means ±sdofthreeindependentexperimentsstatisticallyanalyzed
using t-test, **; p <0.0, *; p <0.05, ns; p >0.05), b) Superposition of CX-4945 and compound (5) at ATP bindingsiteofhumanCK2enzyme
PDB code 3PE1, c) 2D representation of (2), (5) and (7) docked at ATP binding site of CK2α, d) Dose dependentCK2αinhibitionoftested
compounds (2, 5 and 7) at concentration range 0.0001-5 μM represented as mean ± sd of three independent experiments
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water molecules. The triazole ring assesses
interaction with
hydrophobic binding of hydrazide group in
Val66 (5) or Ile 174 (7). Moreover, the However, there was
variation between them in the N1
and carboxyl group of are super- isomers. As shown in supporting information S1c, hydrazide
5 and carboxyl group in 7.
a
hydrazide group of
5
7
positioned as the carboxyl group of CX-4945 (Figure 3b). Both group in 4 couldn’t establish the direct bond to Asp125 while
compounds have their hydrophilic groups (hydrazide and carboxyl group in 6 doesn’t establish the indirect interaction
carboxyl, respectively) substituted at N2 of the triazole nucleus. with Glu81 and Trp176 via water molecule. This may explain the
The marked decrease in CK2 enzyme inhibition by compounds 4 converging IC50 of 5 and 7 (0.098 μM and 0.13 μM respectively)
and 6 compared to their corresponding isomers (5 and 7) may and the variable IC50 in
be explained by the loss of direct binding of 4 to Asp175 and respectively). The docking results are consistent with the
water mediated binding of to Trp176 and Glu81 due to concentration dependent CK2α inhibition of tested compounds
4 and 6 (0.39 μM and 0.54 μM
6
changing the position of hydrophilic groups (carboxyl and
hydrazide) from N2 to N1 of the triazole nucleus. Similarly,
esterification of the carboxylic acid group in compound 2 and 3
leads to loss of the direct essential inhibitory hydrogen bonding
to Val 116 and water bridged bonding to Glu81 and Trp176. In
the N2 regioisomers there was no difference in the mode of
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and their order of inhibitory activity (2 < 5 < 7) as shown in group of tested compounds are critical factVoiresw Arftioclre Ontlhinee
Figure 3d. Both CK2α inhibition assay and docking studies interaction of tested compounds with theDCOKI:210e.1n0z3y9m/De.0NJ01194K
illustrated clearly that the nature and position of substituting
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Figure 4: Antiproliferative activities of tested compounds: a) cell viabilityofhuman lungcancercellline(A549)treatedwithtested
compounds at 5 μM for 48 h.; (b) cell viabilityof human breast cancer cell line (MCF -7)treatedwithtestedcompoundsat5μMfor
48 h.; c) IC50 (MCF-7) of tested compounds, 1IC50 (A549) of 5 is 6.31μM; d) Fold change in expressionlevelsofBAXmRNAfollowing
treatment of MCF-7 cells with tested compounds at 10 μM for 48 h; e) Fold change in expressionlevelsofBCL2mRNAfollowing
treatment of MCF-7 cells with tested compounds at 10 μM for 48 h; f) Effect of compound 5 concentration on cell viabilityof
cancer cell lines represented bysolid line (A549) and dashed line (MCF-7); g) Effect of compound4concentrationonBAXmRNA
expression; h) Effect of compound 4 concentration on BCL2 mRNA expression.
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2.3. In-vitro Anti-proliferative activity
hydrazide (5) and the acid (7) achieved similar lVeievwelAsrticolefOnCliKn2e
enzyme inhibition which is three times more potent than their
DOI: 10.1039/D0NJ01194K
The anti-proliferative assay was performed against human
breast and lung adenocarcinoma cell lines (MCF-7 and A549;
corresponding ester 3. The hydrazide 5 was significantly more
cytotoxic and apoptotic than both 3 and 7 (Figure 4d-e). The
unparallel between CK2 enzyme inhibitory and cytotoxic
activities is not fully explained. In order to find a possible
explanation of these differences, we decided to estimate the
lipophilicity of the tested compounds both theoretically and
experimentally as lipophilicity may affect cell penetration
properties and hence explain the difference between enzyme
and cell-based activities.
respectively)
using
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) colorimetric assay as
described by T. Mosmann29. Cell viability of cancer cell lines
were evaluated after 48 h treatment with tested compounds,
Figure 4. Tested compounds exhibited comparable pattern of
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anti-proliferative activity in both cell lines at
5
μM
concentration where hydrazides (4 and 5) were more effective
than their corresponding esters (2 and 3). A549 cells were
slightly more sensitive to the tested compounds than MCF-7
2.5. Lipophilicity study
cells which lack caspase
Compound demonstrated
proliferative effect than its corresponding regioisomer 4.
3
and have impaired apoptosis.
Lipophilicity is a physicochemical property that influences
pharmacokinetics, pharmacodynamics and toxicityof drugsasa
result of its impact on their passive diffusion through biological
membranes. According to IUPAC, lipophilicityis the affinityof a
compound for a lipophilic environment. It is generallyassessedby
the distribution behavior of the drug in biphasic systemssuchas
liquid-liquid systems (e.g. 1-octano/water) orsolid-liquidsystems
(e.g. RP-HPLCor RP-TLC)30. A common parameterusedforexpression
of the lipophilicityof a drug molecule, is the partitioncoefficient,P,
that represents partitioning of a drug between an aqueousanda
non-aqueous phase. 1-octanol/water solvent system has been
recognized as the reference system usingtheshake-flasktechnique
for determination of the partition coefficient, PO/W, of drug.
Lipophilicityis usuallyexpressed as logPO/Wwhichisdefinedasthe
logarithm of the ratio of concentration of a neutral, non-ionized
substance, in 1-octanol to that in water31.
Due to practical difficulties of the shake -flask method,reversed-
phase (RP) chromatographyis frequentlyusedfortheestimationof
lipophilicityof drugs, mainlyRP-HPLCand RP-TLC, relyingontheir
retention factors via simulating 1-octanol–water system. RP-TLC
offers manyadvantages over other methods as it is costeffective,
rapid and high throughput technique, consumes smallvolume of
solvents and requires simple equipmentandhenceitiswidelyused
for experimental estimation of lipophilicity32.
5
significantly higher anti-
All compounds were tested on MCF-7 at several concentration
in the range of 2.5-20 μM to determine their IC50 (Figure 4c, f
and supporting information S2). IC50 is the concentration of a
tested compound which decreases the percentage of viable
cells to a half in comparison to negative control cells treated
with only media and positive control cells treated with
doxorubicin as a reference anticancer drug. Compounds 2, 4, 6
and
7 demonstrated moderate anti-proliferative activity with
IC50 for MCF-7 cell line in the range of 32-37.5 μM which is
markedly better than the parent 1. Compound 3 which has the
ester functional group at N2 of the triazole ring was more potent
with IC50
functional group at N2 of the triazole ring exhibited the highest
potency in MCF-7 and A549 cell lines (IC50 9.1, 6.3 μM,
= 23 μM. Compound 5 which has the hydrazide
=
respectively, Figure 4c) and was equipotent to CX-4945, the
most successful CK2 inhibitor in clinical trials. It exhibited a
concentration dependent antiproliferative activity in both
cancer cell lines at concentration range (0-20 μM) as shown in
Figure 4f.
2.4. Quantitative real-time PCR evaluation of apoptotic genes
expression levels.
Computation methods using different computer programscanbe
used to generate calculated Log P valuesforthestudiedcompounds
based on atom contributions, fragment contributions,
atom/fragment contributions or atom-typeelectrotopological-state
indices and neural network modeling for instance, MOE,Molsoft,
ChemDraw and ALOGPS.
Bax and Bcl-2 are two genes playing opposing roles in the
regulation of cellular apoptosis. Their expression leads to two
different proteins: bax (pro-apoptotic) and bcl-2 (anti-
apoptotic) . In this study, the levels of mRNA expression of Bax
and Bcl-2 genes in MCF-7 cells, treated with tested compounds
at 10 μM, was determined using quantitative real-time PCR
(qRT-PCR) technique. Interestingly, all treated cells (Figure 4d-
e) exhibited increase of the Bax mRNA expression levels
(upregulation) and non-significant change of Bcl-2 m-RNA
expression levels. Both hydrazide derivatives (4-5) showed four
to five times higher levels of Bax m-RNA than the corresponding
ester (3) or acid (7). The most potent compound (4)
demonstrated a concentration-dependent up-regulation of Bax
m-RNA (Figure 4g) with non-significant change in Bcl-2 m-RNA
expression levels (Figure 4h) at the tested concentration range
(0-20μM).
2.5.1. Theoretical lipophilicity calculation of N1 and N2-
substituted TBBt derivatives
Lipophilicity of tested compounds were theoretically calculated
using four different software programs (Molsoft, MOE,
ChemDraw and ALOGPS 2.1). Calculated LogP (o/w) values of
tested compounds (1-7, Table 1) were in the range of 1.92–5.04.
The order of lipophilicity of tested compounds were different
amongst different software programs. The highest lipophilic
compound is the ester derivative (3) as calculated by Molsoft,
ChemDraw and ALOGPS 2.1 software programs. MOE software
predicts the parent compound (1) to be the highest lipophilic in
the series. The highest hydrophilic compound as calculated by
all software is the hydrazide derivative (4) with Calcd. LogP
A comparison of results from the CK2 enzyme inhibition Figure
3a) and anti-proliferative activity (Figure 4c), gave different
order of activity for tested compounds. For instance, both the
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(o/w) in the range 1.92-3.20 which is about one digit less than is basic feature of the chromatographic deteVrimewinAartticiolenOnlinoef
the calculated values of the acid derivatives (6 and 7). lipophilicity. C0 represents the concentration
DOI: 10.1o0f39/aDn0NJo0r1g1a9n4iKc
In conclusion, lipophilicity prediction software programs give modifier in the mobile phase for which the solute is equally
contradicting results despite the structural similarity among the distributed between the two phases, in other words, RM = 0, RF
tested compounds and are not reliable to comprehend on the = 0.5. Moreover, C0 represents the hydrophobicity per unit of
difference in antiproliferative activity of tested compounds. specific hydrophobic surface area and It is considered more
Previous studies on polyhalogenated benzotriazoles and reliable in QSAR analysis as it embraces both the specific
5
6
7
8
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benzimidazoles were relying on calculated lipophilicity which hydrophobic surface
area
of
the
solute
and the
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may have complicated interpretation of their anticancer activity chromatographically-measured lipophilicity index (RM0). High
33,
34. However up to our knowledge there was no attempts to values of correlation coefficients were obtained (r > 0.9831,
experimentally measure lipophilicity of any polybrominated supporting information S3-5) in all mobile phases, which proves
benzimidazole or benzotriazole derivatives. We thought the high significance of obtained descriptors for estimation of
experimental estimation of TBBt derivatives lipophilicity would lipophilicity. The type of organic modifier (methanol,
give unprecedent information and may provide clues about acetonitrile and acetone, supporting information S3-5) has
lipophilicity-antiproliferative activity relationship.
small influence on relative lipophilicity (expressed as RM0) or
lipophilic parameter (C0). significant correlation exists
between RM0 and slope (b) with r ≈ -0.9406, -0.9958 and -0.8379
A
Table 1: Calculated log P valuesusing different software programs
Comp. No.
Calcd. LogP (o/w)
in acetonitrile/water, acetone/water and methanol/water;
respectively, which suggests
retention mechanism for this congeneric series of studied
derivatives. Moreover, good correlation was obtained
between RM0 and the lipophilic parameter C0 with r ≈ 0.9250,
0.9820 and 0.8043 in acetonitrile/water, acetone/water and
methanol/water; respectively.
a
similar chromatographic
MOE
Molsoft
ChemDraw
ALOGPS 2.1
1
2
3
4
5
6
7
4.67
4.16
4.31
2.41
2.56
3.55
3.70
3.91
4.05
4.44
1.92
2.31
3.22
3.62
4.26
4.07
5.04
2.71
3.04
3.84
4.15
3.94
4.28
4.38
3.20
3.55
3.41
3.76
a
Within the same solvent system, the difference in RM0 values
midst tested compounds are attributed to the variation in their
substituents, which affects considerably their lipophilicity.
Higher RM0 values were found for the relatively lipophilic
derivatives containing ester group (3 and 2, Table 2) followed
by hydrazide derivatives group (5) which were more lipophilic
than TBBt itself. N2 derivatives were slightly more lipophilic than
their corresponding N1 derivatives. This finding is critical in
understanding the differences between N1 and N2 regioisomers
regarding their antiproliferative activity. The lowest values of
RM0 were obtained for the more hydrophilic derivatives
containing hydrazide and carboxylic acid group (4, 7 and 6,
Table 2). Compound 3 (containing ester group) is the highest
lipophilic compound. It possesses the highest value of RM0, b
and C0 in all organic modifier/water systems. According to C0
values, compound 6 (containing carboxylic acid group) is the
highest hydrophilic compound followed by its structural isomer
(7).
2.5.2. Experimental lipophilicity estimation of N1 and N2-
substituted TBBt derivatives using RP-TLC
Lipophilicity parameters were experimentally measured using
RP-TLC method as described elsewhere 31. Three mobile phases
were used consisting of different proportions of water and
organic modifiers (acetonitrile, acetone and methanol). Briefly,
the RF and RM values were obtained for each organic
modifier/water system ratio (supporting information S3-5) and
a
linear regression analysis was achieved to obtain three
lipophilicity chromatographic descriptors (RM0 , b, C0 Table 2).
RM0 is known as "the relative lipophilicity" as it describes the
solute partitioning between pure water and nonpolar stationary
phase. On the other hand, b (slope of the regression line)
describes the specific hydrophobic surface area of the
compound. In a series of structurally related compounds, b is
linearly correlated with RM0 resulting in a linear relationship that
Table 2: RP-TLC measured lipophilicity parameters RM0, b and C0
RM 0
b
C0
Comp. No.
ACN
acetone
MeOH
ACN
acetone
MeOH
ACN
acetone
MeOH
1
2
3
4
2.02
3.42
3.64
1.39
3.16
4.15
4.51
3.07
3.59
4.01
4.69
2.26
-0.0268
-0.0404
-0.0417
-0.0240
-0.0428
-0.0512
-0.0543
-0.0412
-0.0416
-0.0429
-0.0488
-0.0286
75.36
84.52
87.27
57.78
73.77
81.13
83.06
74.53
86.34
93.64
96.13
79.05
5
6
7
2.56
1.45
1.28
3.69
1.51
2.11
3.88
2.62
3.38
-0.0326
-0.0304
-0.0258
-0.0470
-0.0271
-0.0351
-0.0438
-0.0397
-0.0465
78.49
47.79
49.85
78.50
55.66
60.06
88.55
65.83
72.69
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Experimental results of lipophilicity were completely different Synthesis was performed by brominaDtiOnIg: 10.110H3V-9bie/ewDnA0zrNtoicJtlr0ei1aO1zn9ol4inlKee
in comparison with Calcd. LogP values. In contrast to Calcd. LogP according to published method28.
4.2.2. Synthesis of N1 and N2 -acetate esters (2 and 3)
(o/w), the highest lipophilic compounds as measured by RP-TLC
method are the derivatives containing ester group (3 and 2)
followed by the hydrazide derivative (5) while, the highest
hydrophilic compounds in the series are the derivatives
containing carboxylic group (6 and 7) followed by the hydrazide
derivative (4).
Ethyl chloroacetate (2.82 g, 23.0 mmol, 5 equiv.) was added to
a mixture of 1 (2.00 g, 4.6 mmol, 1.0 equiv) and K2CO3 (1.27 g,
9.20 mmol, 2 equiv) and few crystals of tetrabutylammonium
bromide as phase transfer catalyst in acetone (10 mL). The
mixture was stirred at 60oC for 6 h. The reaction was monitored
by TLC and when completed, the solvent was evaporated to
dryness. The residue was purified by column chromatography
using silica gel and hexane: ethyl acetate (15:1 to 7:1) as eluent.
Two regioisomers (2 and 3) were separated in 80% total yield.
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A comparison of results from the CK2 enzyme inhibition (Figure
3a), anti-proliferative activity (Figure 4c) and experimentally
estimated lipophilicity (Table 2) reveal that the hydrazide (5)
and its acid (7) are nearly equipotent as CK2 inhibitors but quite
different in terms of lipophilicity and antiproliferative activity.
The hydrazide (5) is three times more potent as antiproliferative
than the acid (7) which may be attributed to its increased
lipophilicity as it would have better chance to penetrate cell
membranes. There is also a significant difference between the
two hydrazide regioisomers (4 and 5) in terms of lipophilicity,
antiproliferative activity and enzyme inhibitory. While the ester
3 is more lipophilic than both 5 and 7, it exhibited reduced
cytotoxic response as a result of its reduced enzyme inhibitory
activity.
Ethyl (4,5,6,7-tetrabromo-1H-benzo[d]triazol-1-yl)-acetate (2)
Yield: 1.15 g (48%), white crystals, m.p 140 ºC. IR (KBr, cm-1):
1734.6 (CO), 2925.2 (C2H5). 1H NMR (DMSO-d6, 400MHz,)
δ
ppm: 5.89 (s, 2H, CH2), 4.23 (q, 2H, J = 8.0 Hz, CH2), 1.24 (t, 3H,
J = 4.0 Hz, CH3). 13C NMR (DMSO-d6, 100 MHz) δ ppm: 14.07
(CH3), 51.31 (ester CH2), 62.67 (N-CH2), 105.64 (C5), 117.08 (C6),
124.73 (C4), 129.68 (C7), 132.33 (C8), 145.63 (C9), 166.35 (C=O).
Anal. calcd/found. for C10H7Br4N3O2 (520.723): C, 23.06/23.42;
H, 1.35/1.59; N, 8.07/8.24.
Ethyl (4,5,6,7-tetrabromo-2H-benzo[d]triazol-2-yl )-acetate (3)
Yield: 0.766 g (32%), white crystals, m.p.182-183 ºC. IR (KBr, cm-
1): 1743.2 (CO), 2967.9 (C2H5). 1H NMR (DMSO-d6, 400MHz,) δ
ppm: 5.94 (s, 2H, CH2), 4.24 (q, 2H, J = 8.0 Hz, CH2), 1.24 (t, 3H,
4. Materials and Methods
4.1. General
J = 4.0 Hz,
CH3). 13C NMR (DMSO-d6, 100M Hz) δ ppm: 14.34
All commercially available solvents and reagents were used
without further purification. Melting points were measured on
an electrothermal melting point apparatus [model 9100, UK],
and were reported as uncorrected. Pre-coated TLC silica gel
plates (kieselgel 0.25 mm, 60G F254, Merck, Germany) were
used for reactions monitoring and TLC silica gel G 60 RP–18 F254
aluminum sheets (Merck, Germany) were used for lipophilicity
estimation. TLC spots were detected using ultraviolet lamp at
254 nm wavelength (Spectroline, model CM-10, USA). Infrared
spectra (KBr discs) were recorded on thermo scientific nicolet
IS10 FT-IR spectrometer (thermo Fischer scientific, USA) at the
Faculty of science, Assiut University, Assiut, Egypt. 1H-NMR and
(CH3), 58.20 (CH2), 62.50 (N-CH2), 117.20 (C5 &C6), 126.82 (C4
&C7), 143.37 (C8 &C9), 166.50 (C=O). Anal. calcd/found. for
C10H7Br4N3O2 (520.723): C, 23.06/23.27; H, 1.35/1.57; N,
8.07/7.92.
4.2.3. Hydrazinolysis of 2 and 3
The mixture of 2 or 3 (0.120 g, 2.3 mmol, 1.0 equiv.) and
hydrazine hydrate (99 %, 0.6 g, 18.5 mmol, 8.0 equiv.) in ethanol
(10 mL) was stirred and refluxed for 6 h. The mixture was left to
cool for crystallization and filtered to give pure hydrazide
products (4 or 5 respectively).
13C-NMR spectra
were
scanned on AVANCE-III
High
2-(4,5,6,7-tetrabromo-1H-benzo[d]triazol-1-yl)acetohydrazide
(4)
Performance FT-NMR spectrometer (400 MHz Brucker) at
Faculty of Science, Sohag University, Sohag, Egypt. Mass spectra
were carried out on Direct Probe Controller Inlet Part TO Single
Quadropole mass analyzer in Thermo Scientific GCMS model
ISQ LT using Thermo X-Calibur software at the Regional Center
for Mycology and Biotechnology (RCMB), Faculty of Science, Al-
Yield: 0.074 g (63.5%). White crystals, mp. > 300 ºC. IR (KBr,cm-
1): 3316.3, 2925.1, 1663.3, 1626.6, 1545.1, 1430.2. 1H NMR
(DMSO-d6, 400MHz,) δ ppm: 4.46 (br s, NH2), 5.51 (s, CH2), 9.47
(s, NH). 502.59 (21.03%), 504.32 (14.54%). EI-MS [m/z (%)]:
502.59 (M+, 21.03%), 504.32 (M++2,14.54%). Anal. calcd/found.
for C8H5Br4N5O (506.774): C, 18.96/19.24; H, 0.99/1.23; N,
13.82/14.09.
Azhar University,
Nasr city, Cairo,
Egypt. Elemental
microanalyses were performed on elemental analyzer model
flash 2000 thermo fisher at the Regional Center for Mycology
and Biotechnology (RCMB), Faculty of Science, Al-Azhar
University, Nasr city, Cairo, Egypt.
2-(4,5,6,7-tetrabromo-2H-benzo[d]triazol-2-yl)acetohydrazide
(5)
Yield: 0.084 g (70.5%). White crystals, mp. > 300 ºC.. IR (KBr, cm-
1): 3304.2, 3053.5, 2924.0, 1661.4, 1623.6, 1545.9. 1H NMR
(DMSO-d6, 400MHz,) δ ppm: 4.45 (br s, NH2), 5.51 (s, CH2), 9.59
(s, NH). 13C NMR (DMSO-d6, 100M Hz) δ ppm: 58.48 (CH2),
4.2. Synthesis
4.2.1. 4,5,6,7-tetrabromo-1H-benzotriazole (1)
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114.11 (C5 & C6), 126.38 (C4 & C7), 143.27 (C8 & C9), 163.94 automatically calculated. The obtained databasVeiewwAratiscle Otnhleinne
(C=O). Anal. calcd/found. for C8H5Br4N5O (506.774): C, saved as MDB file to be used in theDOdIo: c10ki.1n0g39c/Dal0cNuJla0t1i1o9n4sK.
18.96/19.23; H, 0.99/1.07; N, 13.82/14.09.
Docking of the energy minimized conformations was done using
MOE- dock wizard. London dG was used as the scoring function
(S).
4.2.4. Base catalyzed hydrolysis of 2 and 3
The suspension of 2 or 3 (0.075 g, mmol) in a solution of 5%
NaOH (5 mL) and 2 mL ethanol was stirred and refluxed for 1 h.
The mixture became clear solution. The solution was brought to
pH 2-3 with conc. HCl. The white precipitate was filtered and
washed with water to give pure products (6 or 7 respectively).
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4.5. MTT-based cytotoxicity assay
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Cell viability was assessed using MTT assay as described
elsewhere29. Briefly, cells were seeded into 96-well tissue
culture plates in DMEM containing 10% FBS to a final volume of
200 μL/well. After 24 h of seeding, cells were treated with
tested compounds, doxorubicin (positive control), or 0.5%
DMSO (negative control). After 48 h incubation, media were
removed and replaced with 200 μL DMEM containing 0.5
2-(4,5,6,7-tetrabromo-1H-benzo[d]triazol-1-yl)acetic acid (6)
Yield 0.024 g (33.8%), white crystals mp 216 ºC. IR (KBr, cm-1):
3200-2500, 3007.4, 296.4, 1736.0, 1630.3. 1H NMR (DMSO-
d6,400MHz,)δ ppm: 5.77(s, 2H,CH2). 13C NMR (DMSO-d6, 100M
Hz) δ ppm: 52.1 (CH2), 97.44, 115.95. Anal. calcd/found. for
C8H3Br4N3O2 (492.774): C, 19.50/19.78; H, 0.61/0.87; N,
mg/mL MTT. After
2 h incubation, the supernatant was
removed, and the precipitated formazan was dissolved in 200
μL DMSO and absorbance measured at 570 nm using Varioskan
flash 4.00.53 microplate reader. All experiments were
performed in six replicates and were calculated by subtracting
blank readings. Tested compounds were screened to find the
most active compound(s) capable of significantly influencing
cell viability of both cell lines at low micromolar concentration
(5 μM). IC50 was calculated as the concentration of tested
compounds required to achieve 50% inhibition of cell viability
.
8.53/8.78.
2-(4,5,6,7-tetrabromo-2H-benzo[d]triazol-2-yl)acetic acid (7)
Yield = 0.025 g (35.2%), white crystals mp 263. IR (KBr, cm-1):
3200-2500, 2991.8, 2946.2, 1732.2, 1553.1. 1H NMR (DMSO-
d6,400MHz,)δ ppm: 5.8 (s, 2H, CH2). 13C NMR (DMSO-d6, 100M
Hz) δ ppm: 58.47 (CH2), 114.16 (C5 & C6), 126.60 (C4 & C7),
143.26 (C8
& C9), 167.86 (C=O). Anal. calcd/found. for
C8H3Br4N3O2 (492.774): C, 19.50/19.78; H, 0.61/0.87; N,
8.53/8.78
4.6 Evaluating the effect of tested compounds on Bax and
Bcl2 genes Using Quantitative Real-Time PCR (qRT-PCR)
4.3. CK2α inhibition assay
4.6.1- RNA extraction and reverse transcription
The CK2α activity was measured using P81 filter isotopic assay
Total RNA was extracted, from MCF-7 cells after overnight
treatment with tested compounds, using the Direct-zol™ RNA
MiniPrep (Zymoresearch, Catalog No. R2053, CA, USA). The
purity and concentration of RNA determined by biotech
nanodrop. complementary DNA (cDNA) was synthesized using
Thermo Scientific Revert Aid Reverse kit (Thermo, Waltham,
as described elsewhere35. Briefly,
a reaction solution was
prepared using 20 μM substrate peptide (RRRDDDSDDD) and 10
μM ATP dissolved in 40mM HEPES buffer (50 μL, pH 7.5) with
130 mM KCl, 10 mM MgCl2, and 50 μM DTT. The enzyme
(hCK2α, Sigma-Aldrich) was added to the reaction mixture and
was kept for 15 min at 30 ºC. 10 μL of the phosphorylation
reaction mixture was spotted onto the P81 filter paper followed
by washing three times with 0.5 % phosphoric acid once with
ethanol before scintillation counting. IC50 values for tested
compounds were determined by measuring CK2α activity at
concentrations ranging 0.001-5.0 μM of each compound. All
tested compounds were dissolved in 0.5% DMSO.
MA, US).
4.6.2- Quantitative real-time PCR (qRT-PCR)
QRT-PCR was performed by 7500 fast real time PCR (Applied
Biosystems, CA, USA) using QuantiTect SYBR Green PCR Kit
(Qiagen, Germany) under the following conditions, hot start
step at 95 °C for 7, initial denaturation for 20 sec at 95 °C,
annealing and extension for 60 sec at 59 °C in 40 cycles.
4.4. Molecular Modelling
Table 3: Primer sequences of the genes used for qRT‐PCR
All molecular modeling studies were carried out on an Intel(R) experiments in this study
Xeon(R) CPU E5-1650 3.20 GHz processor, 16 GB memory with
Windows 10 Professional operating system. Molecular
Operating Environment (MOE 2014.0901, Chemical Computing
Group, Canada) was used as the computational software. Pymol
2.3.2 was used as viewer. The crystal structure of human
Protein Kinase CK2 Catalytic Subunit co-crystallized with CX-
4945 Inhibitor was downloaded from PDB (code: 3pe1). The
Studied compounds were built using the builder interface of the
MOE software and subjected to conformational search.
Genes
Forward primer
Reverse primer
Bax
5′-
5′-
CCCGAGAGGTCTTTTT
CCGAG-3′
CCAGCCCATGATGGTTCT
GAT-3′
5′-
ATCGCTCTGTGGATGACT
GAGTAC-3′
5′-
Bcl2
5′-
ATCGCTCTGTGGATGA
CTGAGTAC-3′
5′-
β‐Actin
Conformers were subjected to energy minimization until
RMSD gradient of 0.01 Kcal/mol and RMS distance of 0.1 Å with
MMFF94X force-field and the partial charges were
a
TGTTGTCCCTGTATGC
CTCT-3′
TAATGTCACGCACGATTT
CC-3′
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The fold change in expression of Bax and Bcl2 genes was
calculated according to the 2−ΔΔCT method using β‐actin as
internal control gene to normalize the relative level of gene
expression. The sequence of primers used in this study are listed
in Table 3.
5. Conclusions
View Article Online
The current study reports synthesiDs,OI: 1p0u.1r0if3i9c/aDt0ioNnJ011a94nKd
characterization of N1 and N2-substituted regioisomers of the
most potent CK2 enzyme inhibitor; TBBt. We have shown that
incorporation of hydrophilic substituents at N2 improved TBBt
CK2 enzyme inhibition and antiproliferative activities. We are
first to report experimental estimation of lipophilicity for TBBt
derivatives and its contradiction with their calculated values.
This would shed light on the complex relationship between
enzyme inhibition, lipophilicity and antiproliferative activity of
TBBt derivatives. Further studies are needed to measure CK2
inhibitory activity of TBBt regioisomers in treated cells and to
determine their CK2 enzyme specificity and their ability to
target other kinases. This may improve our understanding of
TBBt regioisomers structure-activity relationships and would
impact the design of future CK2 based anticancer agents.
4.7. Lipophilicity estimation of tested compounds using RP-
TLC chromatographic method
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Chromatographic analysis was carried out on TLC silica gel G 60
RP–18 F254 aluminum sheets (20
× 20 cm, 0.20 mm layer
thickness) that were obtained from Merck, Darmstadt,
Germany. The mobile phases were prepared by mixing different
proportions of double-distilled water and organic modifier.
Methanol (SD Fine-Chem Limited, India), acetonitrile (Fisher
Scientific U.K. Limited, United Kingdom) and acetone (Merck,
Germany) were used as organic modifier for preparation of the
different mobile phase systems. Standard solution (0.1 mg/mL)
of each of the studied compounds was prepared in Methanol
(MeOH). The content of the organic modifier; methanol,
Supplementary Materials: The following are available online at
acetonitrile or acetone was varied between 50 and 95% (v/v) www.xxx.com/xxx/s1, S1: Molecular modelling and docking
with CK2α, S2: Cell viability of adenocarcinoma cell lines
treated with 0-20 μM of compound 5 for 48 h; Table S3: The
RM0 Values, C (the percentage of ACN), b (Slope) and r
(Correlation Coefficient) of the Equation RM = RM0 + bC for
the studied compounds ; Table S4: The RM0 Values, C (the
percentage of acetone), b (Slope) and r (Correlation
Coefficient) of the Equation RM = RM0 + bC for the studied
compounds; Table S5: The RM0 Values, C (the percentage of
MeOH), b (Slope) and r (Correlation Coefficient) of the
Equation RM = RM0 + bC for the studied compounds. Figures
S6: IR, H-NMR and C13-NMR spectra.
with an increment of 5%. The upper and lower content of
organic modifier depends on the linearity between RM and
organic modifier concentration. The concentration ranges for
the tested organic modifiers were used where the upper value
of the range indicates that the analyte elutes near the solvent
front and the lower value implies that the analyte is very near
to the starting line. Then, the chromatographic chambers were
saturated with the mobile phase for 0.5 h. TLC was performed
on RP TLC plates (20 × 5 cm pieces). The standard solutions (5
μL) were spotted in triplicate on the plates 5 mm apart, 10 mm
from the bottom edges and 5 mm from the side edges. The
plates were air-dried then developed in the chromatographic
chambers. Linear ascending plate development was performed
Author Contributions: Conceptualization and experimental
design, A. M. A.; Methodology and experimental, A. B. A., Y. A.
M, M. N. A., N. A. H, N. G. M. and H. F. H; Data analysis and
interpretation, A. B. A., Y. A. M, M. N. A., N. A. H., H. F. H., N. G.
M; A. M. A; Manuscript writing A. B. A, N. G. M, M. N. A., A. M.
A.; All authors have read and approved the final manuscript.
until
a migration distance of 35 mm from the origin was
reached. After development, the plates were removed and air-
dried. Then spots were localized under UV light at λ = 254 nm.
Finally, the RF values were calculated.
Rm values were derived using Rf of solutes in mobile solvent
systems including water and an organic modifier such as
acetone or methanol followed by construction of a relationship
between Rm and C (percentage of organic modifier). A linear
regression analysis was carried out using Excel 2016 (Microsoft
Office) to obtain lipophilicity chromatographic descriptors (RM0
, b, C0) as calculated from the following equations.
Conflicts of interest
There are no conflicts to declare.
Notes and references
RM0
RM = log (R1 − 1),
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C0 = −
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