Synthesis of novel chiral TBBt derivatives with hydroxyl moiety. Studies on inhibition of human protein kinase CK2α and cytotoxicity properties

Article abstract of DOI:10.1016/j.ejmech.2014.07.019

The efficient method for the synthesis of novel 4,5,6,7-tetrabromo-1H- benzotriazole (TBBt) derivatives bearing a single stereogenic center has been developed. New compounds with a variety of substituents at the meta- and para-position of the phenyl ring are reported. All of the presented compounds were obtained using classical synthetic methods, such as bromination of benzotriazole, and its subsequent alkylation by monotosylated arylpropane-1,3-diols, which in turn have been synthesized through reduction of the corresponding prochiral β-keto esters, and the selective monotosylation of the primary hydroxyl group. The influence of the new and previously reported N-hydroxyalkyl TBBt derivatives on the activity of human protein kinase CK2α catalytic subunit was examined. The most active were derivatives with N-hydroxyalkyl substituents (IC₅₀ in 0.80-7.35 μM range). A binding mode of (R)-1-(4,5,6,7-tetrabromo-2H-benzotriazol-2-yl)butan-3-ol 7b to hCK2α has been proposed based on in silico docking studies. Additionally, MTT-based cytotoxicity tests demonstrated high activities of novel 1-aryl-3-TBBt-propan-1-ol and 3-TBBt-propan-1,2-diol derivatives against human peripheral blood T lymphoblast (CCRF-CEM), and moderate anti-tumor activities against human breast adenocarcinoma (MCF7) cell lines.

Full text of DOI:10.1016/j.ejmech.2014.07.019

Accepted Manuscript
Synthesis of novel chiral TBBt derivatives with hydroxyl moiety. Studies on inhibition
of human protein kinase CK2α and cytotoxicity properties
Paweł Borowiecki, Adam M. Wawro, Patrycja Wińska, Monika Wielechowska, Maria
Bretner
PII:
S0223-5234(14)00625-4
10.1016/j.ejmech.2014.07.019
EJMECH 7139
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 February 2014
Revised Date: 26 June 2014
Accepted Date: 6 July 2014
Please cite this article as: P. Borowiecki, A.M. Wawro, P. Wińska, M. Wielechowska, M. Bretner,
Synthesis of novel chiral TBBt derivatives with hydroxyl moiety. Studies on inhibition of human protein
kinase CK2α and cytotoxicity properties, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.07.019.
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European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com
Synthesis of novel chiral TBBt derivatives with hydroxyl moiety. Studies on
inhibition of human protein kinase CK2α and cytotoxicity properties.
Paweł Borowiecki ∗ , Adam M. Wawro, Patrycja Wińska, Monika Wielechowska, and Maria Bretner
Warsaw University of Technology, Faculty of Chemistry, Noakowskiego St. 3, 00-664 Warsaw, Poland.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The efficient method for the synthesis of novel 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt)
derivatives bearing a single stereogenic center has been developed. New compounds with a
variety of substituents at the meta- and para-position of the phenyl ring are reported. All of the
presented compounds were obtained using classical synthetic methods, such as bromination of
benzotriazole, and its subsequent alkylation by monotosylated arylpropane-1,3-diols, which in
turn have been synthesized through reduction of the corresponding prochiral β-keto esters, and
the selective monotosylation of the primary hydroxyl group. The influence of the new and
previously reported N-hydroxyalkyl TBBt derivatives on the activity of human protein kinase
CK2α catalytic subunit was examined. The most active were derivatives with N-hydroxyalkyl
substituents (IC50 in 0.80–7.35 µM range). A binding mode of (R)-1-(4,5,6,7-tetrabromo-2H-
benzotriazol-2-yl)butan-3-ol 7b to hCK2α has been proposed based on in silico docking studies.
Additionally, MTT-based cytotoxicity tests demonstrated high activities of novel 1-aryl-3-TBBt-
propan-1-ol and 3-TBBt-propan-1,2-diol derivatives against human peripheral blood T
lymphoblast (CCRF-CEM), and moderate anti-tumor activities against human breast
adenocarcinoma (MCF7) cell lines.
Available online
Keywords:
4
,5,6,7-Tetrabromo-1H-benzotriazole
Protein kinase CK2
Inhibitors
Molecular docking
Cytotoxicity evaluations
Anti-tumor therapies
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
In the last decade a great number of inhibition studies have led
to the development of various classes of ATP-site directed
inhibitors of CK2 [15-18]. Among them, condensed polyphenolic
compounds [19,20], polyhalogenated tetrabromobenzimidazole /
triazole derivatives [21,22], indolo- [23] and pyrrolo- [24]
quinazolines were suggested as the most promising drug
candidates. It has been reported that among the above mentioned
inhibitors, the most efficient and selective are 4,5,6,7-tetrabromo-
Human protein kinase CK2 (formerly known as casein kinase
II) catalyzes the reversible phosphorylation of proteins, which is
the most common post-translational modification in signal
transduction. CK2 plays a key role in the regulation of numerous
signaling pathways and diverse cellular functions such as: cell
cycle progression, development, signal transduction, apoptosis,
metabolism, differentiation (proliferation), cell morphology and
migration as well as gene expression and secretion of cellular
proteins [1-6]. Besides all the above mentioned fundamental
processes of normal eukaryotic cell life, in which CK2 protein
kinase participates, it has been experimentally confirmed that
when CK2 becomes over-expressed or up-regulated as a result of
i.e. mutations [7-9], it can display an uncontrolled activity
leading to tumor growth promotion. As CK2 abnormal level was
functionally associated with different cancer types [10-14],
including prostate, lung, kidney, mammary gland, head and neck,
it became obvious that this enzyme constitutes an attractive
druggable target for cancer therapy. Therefore, the development
of highly specific, cell-permeable, and ATP-competitive CK2
inhibitors appears as a promising strategy for treatment of several
tumors.
1
2
H-benzotriazole TBBt (K = 0.4 ꢀM) [25,26] and its derivative
-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole DMAT
i
(
K = 0.04 ꢀM) [27]. However, the cytotoxicity tests showed
i
TBBt as almost inactive anti-cancer compound [28]. Attempts to
modify the TBBt scaffold in order to increase its selectivity
and/or inhibitory activity toward CK2 were not successful up to
now. In turn, the recent report [29] showed highly potent
inhibitor ARC-1502 based on 4,5,6,7-tetrabromo-1H-
benzimidazole (TBBi) conjugated with oligo-aspartate-
containing peptide, which possess sub-nanomolar affinity (K =
i
0
.5 nM) towards both CK2α, and the holoenzyme. However, to
our best knowledge the inhibition of tumor cell lines growth was
not reported for this compound.
—
——
∗
Corresponding author. Tel.: +48 22 660 53 42; fax: +48 22 628 27 41; e-mail: pawel_borowiecki@onet.eu or pborowiecki@ch.pw.edu.pl

2
European Journal of Medicinal Chemistry
The aim of this study was to obtain novel TBBt derivatives,
The reason of this was a multiple side-products formation,
ACCEPTED MANUSCRIPT
efficient inhibitors of CK2 with better cellular permeability than
the parent TBBt-structure. Since the binding of TBBt at CK2
active site is based on hydrophobic interactions between the
enzyme ATP-binding pocket and bromine atoms as well as
hydrogen bond between benzotriazole ring and conserved water
molecule deep inside the pocket [28], therefore to improve the
activity of TBBt derivatives it seemed reasonable to introduce
polar functional group on appropriately long rotatable
hydrocarbon chain. Bearing in mind that this modification may
allow for the interaction of the inhibitor molecule with polar
what also significantly hampered the product isolation procedure.
To develop a more efficient method we replaced LiAlH by
4
NaBH as a less powerful reducing agent. Therefore, the reaction
4
condition was changed to milder according to De Castro et. al.
[39] and Kim et. al. [40] suggestions, respectively. In our hands,
neither of these methods proved efficient, with respect to very
low reaction rates. Looking for more favorable conditions we
examined methanolic solution of sodium borohydride (NaBH4-
MeOH system) as the source of in situ generated NaHB(OCH3)3
complex, which is capable to reduce carbonyl group in both:
ketones and esters [41]. Sodium borohydride was used in
considerable 8-fold molar excess at reflux temperature. Thereby,
with the optimal conditions in hand, 2a-g were isolated in high
78-96% yields (see the Experimental Section).
175
residues located around hydrophobic pocket (e.g. Asp
and
6
8
Lys ) [30,31], we have followed our previous research concept
as its rational continuation [32-34]. Using the reported TBBt
inhibitor core structure, as the lead compound, this study was set
up to verify the above mentioned hypothesis, and thus evaluate
whether incorporation of hydroxyl moiety with different
substituents into a side chain of the newly designed and
synthesized compounds, could increase the affinity for the
enzyme and hence lower inhibitory concentration. Those
modifications of parent TBBt-structure was also motivated by
desire of increasing solubility and cellular permeability of
potential inhibitors.
In the next step, 1-aryl-propano-1,3-diols 2a-g were
submitted to the selective tosylation of their primary hydroxyl
groups using 1 equiv of p-toluenesulfonyl chloride (TosCl) in the
presence of 1.1 equiv of freshly distilled triethylamine and
catalytic amount of 4-(dimethylamino)pyridine (DMAP)
dissolved in dry CH Cl according to the method reported in the
2
2
literature [42-44]. The adopted procedure allowed the reaction to
proceed smoothly, affording the expected products in moderate to
high yields (33-82%) (see the Experimental Section).
2
. Results and discussion
The required 4,5,6,7-tetrabromo-1H-benzotriazole 5 was
primarily prepared by exhaustive bromination of benzotriazole 4
with Br in concentrated HNO , according to the original [45]
Herein, we disclose our study on designing and synthesis of
new benzo-fused nitrogen heterocycles corresponding to TBBt,
using simple synthetic methodologies. The planned four-step
reaction sequence (Scheme 1) consists of the synthesis of
appropriate 1,3-diols 2a-g from corresponding β-keto esters 1a-g,
which after selective tosylation yield monotosylated derivatives
a-g further used as alkylation agents for 4,5,6,7-tetrabromo-1H-
benzotriazole 5, synthesized from commercially available
benzotriazole 4.
2
3
and generic [46] literature procedures. However, the first
attempted bromination reaction failed giving mixture of di-, tri-
and tetra-bromobenzotriazoles even after elongation of the
reaction time up to 5 days. The negative result of this process
was likely due to larger scale used by us in comparison with the
parent generic procedure [46]. Therefore, in order to solve the
up-scaling problems, a modified synthetic strategy was proposed.
This includes additional UV irradiation of the reacting mixture. It
was performed to verify if the synthesis of tetrabromo benzo-
fused nitrogen heterocycles of this type was possible not only by
3
2
.1. Synthesis of 1-aryl-3-TBBt-propan-1-ol derivatives 6a-g
The simultaneous reduction of carbonyl and carboxyl group
of β-keto esters 1a-g is well-known, and several protocols of
such convenient reaction were reported [35-38]. Surprisingly,
means of an in situ generated electrophilic bromonium cation
+
(
Br ) formed in the presence of strong oxidizing agent (HNO )
2
3
using very common reducing agent (LiAlH ) in several variants
4
in course of the reaction, but if it was partially dependent on free-
radical halogenation mechanism (Br·) as well. The employed
change in the reaction condition resulted in desired sole product
of >99% purity and in 59% yield accomplished after 48 h.
of the reaction resulted in the obtaining of desired 1,3-diols 2a-g
with very poor yields (ca. 25%).
Scheme 1. Synthesis of 1-aryl-3-TBBt-propan-1-ol derivatives. Reagents and conditions: (i) NaBH
DMAP (10 mg), TosCl (1 equiv), CH Cl anh., 12 h at -15 °C, then 1 h at rt; (iii) Br (5.99 equiv), 69% HNO
equiv), K CO (3 equiv), acetone/acetonitrile (anh. mixture, 1:1, v/v), 48 h at reflux.
4
(8 equiv), MeOH, 12 h at reflux; (ii) dry Et
3
N (1.1 equiv),
2
2
2
3
and 100% HNO , hν, 48 h at 60 °C; (iv) TBBt 5 (1
3
2
3

3
This was confirmed by nuclear magnetic resonance
ACCEPTED MANUSCRIPT
spectroscopy and high-resolution mass spectrometry analyses. It
is worth to note that this methodology in contrast to the original
one [45], ensures good reproducibility of the reaction yield.
Bearing in mind that regioselective N-alkylation of TBBt 5 is
often problematic, we decided to investigate how the most crucial
parameters affect the overall outcome of this reaction. These
included both: TBBt-deprotonation approach and the selection of
the appropriate reaction medium. Adopting the general procedure
described by us previously [33], several bases were investigated
(
NaH, KOH, K CO ). It turned out that the sequential
2 3
deprotonation in the presence of 3-fold molar excess of
anhydrous K CO proved to be sufficient toward strong acidic
2
3
proton of TBBt, thus allowing the anion formation, and
subsequent reaction with monotosylated derivative 3a as the
model compound. In comparison to other tested bases, anhydrous
K CO proved to be the best, also concerning problems of the
2
3
product isolation. From among several solvents applied
acetonitrile, acetone, DMF), the mixture composed of acetone
(
Scheme 2. Synthesis of 3-TBBt-propane-1,2-diol. Reagents and conditions:
and acetonitrile (1:1, v/v) turned out to be the medium of choice,
since it was the only one in which the reaction ran with a
reasonable rate. Under the optimized reaction conditions, at least
the alkylation at N-1 atom has been significantly suppressed, thus
improving the yield of desired N-2 substituted compound up to
(i) dry Et
3
N (1.1 equiv), DMAP (10 mg), TosCl (1 equiv), CH
Cl
2 2
anh., 12 h
at -15 °C, then 1 h at rt; (ii) TBBt 5 (1 equiv), K
2
CO
3
(3 equiv),
acetone/acetonitrile (anh. mixture, 1:1, v/v), 48 h at reflux; (iii) dry Et
3
N (1.5
equiv), DMAP (0.1 equiv), TosCl (1.2 equiv), CH
HCl aq., 6 h at reflux.
2 2
Cl anh., 5 h at 0-5 °C; (iv)
4
2%. The other very important observation made, that should be
briefly noted herein, refers to the manner of reaction progress
monitoring. In the course of the experiments, it turned out that
visualization of the TLC plates with the standard UV light (λ =
2
.3. Inhibitory activity against human CK2α in vitro
The in vitro inhibitory activities against human protein kinase
2
54 nm) did not allow to follow the alkylation reaction rate, since
the spots representing monotosylated substrates 3a-g and the
CK2 catalytic subunit (hCK2α) of the obtained 1-aryl-3-TBBt-
propan-1-ol derivatives 6a-g, and previously synthesized
compounds 7a-c of a similar structure [49] as well as novel 3-
TBBt-propane-1,2-diol 13 are presented and compared with
TBBt 5 in Table 1. Inhibition of hCK2α by above mentioned
TBBt derivatives was determined using P81 filter isotopic assay
[50] (see the Experimental Section and the Supporting
Information for details). Herein studied TBBt derivatives 6a-g,
7a-c, 13 contain a stereogenic center, which has not been
reported for this class of inhibitors heretofore. We decided to
determine biochemical activity of all the compounds 6a-7c and
corresponding major products 6a-g had the same R values in
f
various eluent systems applied. Initially, we had problems to
recognize this phenomenon as it is not obvious to foresee that
two such different structures in terms of molecular weight and
polarity could behave identically on the silica matrix. Trying to
overcome the above drawback, we found that the expected
product could be distinguished from the corresponding
monotosyl-derivative in UV light of λ = 312 nm. The isolated
yields of the obtained products 6a-g were in the range of 33-42%
(see the Experimental Section).
1
3 as racemic mixtures at first, since activity of racemate should
2
.2. Synthesis of 3-TBBt-propane-1,2-diol 13
not drastically differs from the activity of a pure enantiomer.
With the aim to explore if an addition of one more hydroxyl
group into side chain of 3-TBBt-propan-1-ol structure could
influence kinase CK2 inhibitory activity, we also designed novel
-TBBt-propane-1,2-diol 13. To obtain desired compound 13, we
followed at first the synthetic approach shown in Scheme 2
Method A), which included selective tosylation of glycerol 8
However, since the aliphatic derivatives 7a-c separation had
been performed before [49] and isolated enantiopure (>99% ee)
compounds were available, we determined the activity of those
compounds as well. Basing on these results, we tried to
investigate structure-activity relationship of TBBt derivatives in
terms of their stereochemistry. The structure optimization also
assumed the introduction of electron-withdrawing and electron-
donating groups to the para- and meta-position of the phenyl ring
attached directly to the chiral carbon, which contains hydroxyl
group. This allows us to evaluate not only the effect of the phenyl
ring itself, but also the influence of delocalized electron density
by the type of substitution.
3
(
and its subsequent reaction with TBBt 5. Since this strategy
suffered from tedious purification procedure, we have planned an
alternative synthetic route (Scheme 2, Method B). Our main
objective was to eliminate the difficulties in separation of TBBt 5
and 3-TBBt-propane-1,2-diol 13 present in the crude reaction
mixture. Therefore, we decided to start from ready-to-use
commercially available 1,2-O-isopropylidene glycerol (IPG) 10,
also called solketal, which was smoothly converted to tosyl
derivative 11 with very high yield (81%) according to the
published procedure [47]. The tosylated solketal 11 was
subsequently reacted with TBBt 5 yielding TBBt-ketal 12.
Finally, acidic hydrolysis of ketal 12 using 0.5 N HCl according
to Oh et al. [48], allowed to afford 3-TBBt-propane-1,2-diol 13
in fairly good 30% overall yield after two-steps. In accordance
with our predictions the isolation and purification of compound
Biochemical assays revealed that aliphatic 7a-c, 13 and
aromatic 6a-g derivatives of 3-TBBt-propan-1-ol show very
different affinity towards hCK2α subunit. The aliphatic chiral
derivatives 7a-c, 13 showed slightly lower inhibitory activity
than the lead structure 5, IC50 values within the range 0.8-7.2 ꢀM.
On the other hand, derivatives with aromatic, bulky substituent
6
3
a-g have shown hardly any inhibitory activity (Table 1). In turn,
-TBBt-propane-1,2-diol 13 was almost equipotent with (S)-7b
and 7c (IC50 in 2.17–2.50 µM range), what suggest that in spite of
our expectations this compound turned out to be moderate
inhibitor.
1
3 obtained through alternative pathway (Method B) went more
easily when compared to Method A.

4
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
Table 1. hCK2α inhibitory activity of 4,5,6,7-tetrabromo-1H-benzotriazole derivatives with hydroxyl-aryl (6a-g) and -alkyl (7a-
c, 13) substituents.
a
CK2α activity (%)
IC (µM)
d
5
0
Compound
R1
-
R2
-
logP
CK2α
2
0 µM
200 µM
5
0.32
-
-
3
5
6
6
5
6
6
5
1
.74
b
6
a
CH
2
2
2
2
2
2
2
CH
2
2
2
2
2
2
2
C
6
H
5
N.D.
93.00
31.09
.52
.22
.13
.58
.22
.13
.41
.25
-
b
6
b
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
p-Br-C
p-Cl-C
p-F-C
m-Br-C
m-Cl-C
m-OMe-C
CH
6
H
4
N.D.
97.42
33.45
b
6
c
6
H
4
N.D.
95.60
45.38
b
6
d
6
H
4
N.D.
91.42
35.35
b
6
e
6
H
4
N.D.
96.61
43.36
b
6
f
6
H
4
N.D.
74.90
23.77
b
6
7
g
a
6
H
4
N.D.
95.12
87.51
CH
5.40
5.60
7.35
4.16
2.50
0.80
2.50
3.60
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
3
c
c
(
S)-7a
CH2
CH2
CH3
CH3
(
R)-7a
-
7
b
CH
2
CH
2
2
2
CH
3
1
1
.35
-
c
c
(
S)-7b
CH CH
CH3
CH3
2
(
R)-7b
CH CH
-
2
7
c
CH
2
CH
2
CH
3
3
3
.73
-
c
c
(
S)-7c
CH2
CH2
CH CH
2
b
(
R)-7c
CH CH
N.D.
-
2
1
3
CH
2
CH
2
OH
2.17
0.39
a
hCK2α kinase assays were performed in the absence or presence of a 20 or 200 µM solution of tested compound. Results are expressed as a percentage of the
control activity without inhibitor.
Not determined.
b
c
d
Obtained in absolutely enantiopure form (>99% ee) (see Ref. [49]).
Logarithm of the partition coefficient of a given inhibitor between n-octanol and water according to ChemBioDraw Ultra 13.0 software indications.
Fig. 1. The binding mode of compound (R)-7b in complex with the human CK2α active site (PDB code: 1J91). The inhibitor molecule shown as ball-and-stick
model with carbon atoms in grey, hydrogen in white, oxygen in red, nitrogen in blue and bromine in brown from three different perspectives. Enzyme model is
presented as a wire model. View a corresponds to that presented in Fig. 2. High-resolution graphics were generated with UCSF Chimera.

5
ACCEPTED MANUSCRIPT
Fig. 2. The binding mode of compound (R)-7b in complex with the human CK2α active site (PDB code: 1J91). The enzyme residues are shown with the
molecular surface colored a) according to the residue atom type (see Fig. 1), b) according to the residue hydrophobicity. Hydrophilic residues are depicted in
blue, while hydrophobic are orange-red. Hydrophobic ATP-binding pocket is surrounded by hydrophilic residues. Molecular surfaces were generated with UCSF
Chimera.
The lowest IC50 value (0.80 µM) was obtained for enantiopure
R)-7b compound, which was twice as active as the racemic
range 100-300 ꢀM. This could be explained by an unfavorable
interaction between the phenyl ring and the amino acid residues
surrounding the ATP-binding site, which suggest that bulky
substituent of hydrophobic nature is less willingly present in a
distance of about 5 Å from the pocket. An additional conclusion
is that those compounds with para-substituted phenyl ring 6b-d
were relatively more active comparing to meta-derivatives 6e-g.
(
mixture 7b, and almost 4-fold more potent than the opposite (S)-
enantiomer (S)-7b. The difference in affinities of (S)- and (R)-
enantiomers of compound 7 may be explained with assistance of
molecular docking. We have docked all of the synthesized
compounds with Autodock 4.2 package and received ligand-
receptor structures, which are useful in the explanation of this
phenomenon. For the most potent inhibitor (R)-7b binding model
for its complex with hCK2α is presented in Fig. 1 and 2. While in
2
.4. In vitro anticancer screening
As CK2 inhibitory activity does not always correlate with
1
16
both cases polar interactions of the hydroxyl group with Val
cytotoxicity properties of the studied compounds, the influence
on the cell viability of newly 6a-g, 13 and previously synthesized
compounds 7a-b was evaluated in MCF7 (human breast
adenocarcinoma), and CCRF-CEM (acute lymphoblastic
leukemia) cell lines (Table 2 and 3). Such an evaluation was
motivated by the fact that the apoptosis of tumor cells can be
induce not necessarily by down-regulation of a particular CK2
enzyme, but also with disparate molecular mechanism of action,
which discovery is always of considerable interest.
118
and Asn were observed, in (S)-molecule, in order to avoid
clash of the terminal methyl group, hydroxyl group is located
116
118
farther from Val
backbone and carbonyl group of Asn
residue, thus may form weaker hydrogen bonds.
All the aromatic-substituted derivatives 6a-g have shown
considerably lower inhibitory activity, with an IC50 value in the
Table 2. Effects of TBBt derivatives (6a-g, 7a-b, 13) on viability of MCF7 adherent cells (human breast adenocarcinoma) after
4 h and 48 h treatment (by MTT assay).
2
a
Cell viability % ± SD
Compound
After 24 h incubation
After 48 h incubation
1
2.5µM
25 µM
108 ± 14
99 ± 3
59 ± 1
96 ± 8
101 ± 9
52 ± 9
96 ± 3
71 ± 4
96 ± 6
50 µM
120 ± 2
28 ± 8
10 ± 0
19 ± 2
16 ± 1
14 ± 2
28 ± 4
22 ± 7
60 ± 2
58 ± 5
90 ± 6
105 ± 6
102 ± 4
98 ± 2
100 µM
109 ± 3
13 ± 0
0 ± 0
0 ± 0
0 ± 0
12.5µM
N.D.
25 µM
N.D.
50 µM
N.D.
23 ± 8
15 ± 4
9 ± 5
100 µM
N.D.
b
b
b
b
5
111 ± 16
115 ± 2
82 ± 1
90 ± 5
116 ± 7
81 ± 7
6
6
a
b
113 ± 3
107 ± 1
100 ± 4
109 ± 6
102 ± 8
107 ± 1
97 ± 6
99 ± 4
101 ± 3
79 ± 1
105 ± 4
105 ± 5
92 ± 2
97 ± 6
106 ± 2
101 ± 7
96 ± 2
83 ± 3
64 ± 5
73 ± 1
89 ± 6
77 ± 6
111 ± 3
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0.5±1
6 ± 0
6 ± 2
8 ± 2
0 ± 0
6 ± 1
0 ± 1
8 ± 3
6
c
6
d
7 ± 1
6
e
1 ± 0
0 ± 0
11 ± 2
12 ± 2
20 ± 2
16 ± 2
10 ± 2
76 ± 2
87 ± 0
64 ± 1
109 ± 1
6
f
97 ± 7
6
7
g
a
121 ± 5
118 ± 5
119 ± 5
99 ± 5
112 ± 6
109 ± 5
145 ± 4
16 ± 2
15 ± 2
25 ± 4
57 ± 1
31 ± 2
18 ± 2
53 ± 5
(
S)-7a
67 ± 1
7
b
102 ± 6
102 ± 4
105 ± 3
156 ± 5
(
S)-7b
100 ± 1
86 ± 3
116 ± 5
(
R)-7b
1
3
a
Standard deviation; the results are expressed in percentage of cell viability relative to control (cells without inhibitor in 0.5% DMSO) and are divided into two
groups for each tumor cell line depending on the treatment time (24 h and 48 h). Experiments were performed in triplicate and the % of viable cells are mean
values of three independent experiments.
Not determined.
b

6
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
Table 3. Effects of TBBt derivatives (6a-g, 7a-b, 13) on viability of CCRF-CEM suspension cells (human acute lymphoblastic
leukemia) after 24 h and 48 h treatment (by MTT assay).
a
Cell viability % ± SD
Compound
After 24 h incubation
After 48 h incubation
1
2.5µM
25 µM
105 ± 24
123 ± 4
109 ± 1
106 ± 3
104 ± 4
123 ± 8
118 ± 2
106 ± 8
102 ± 7
104 ± 12
113 ± 8
112 ± 6
81 ± 7
50 µM
118 ± 27
66 ± 2
58 ± 0
56 ± 2
46 ± 6
54 ± 6
71 ± 8
58 ± 4
108 ± 4
72 ± 7
89 ± 7
86 ± 4
69 ± 4
47 ± 6
100 µM
61 ± 18
21 ± 2
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
48 ± 4
9 ± 6
9 ± 3
12.5µM
N.D.
25 µM
N.D.
50 µM
N.D.
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
2 ± 1
53 ± 5
58 ± 5
45 ± 4
42 ± 3
23 ± 4
27 ± 1
100 µM
N.D.
b
b
b
b
5
115 ± 12
122 ± 2
101 ± 6
105 ± 2
110 ± 4
102 ± 5
110 ± 4
110 ± 4
125 ± 4
104 ± 7
106 ± 5
126 ± 8
88 ± 8
6
6
a
b
89 ± 2
89 ± 4
93 ± 4
101 ± 1
118 ± 8
70 ± 9
96 ± 6
83 ± 8
105 ± 2
94 ± 4
92 ± 9
94 ± 7
60 ± 4
84 ± 6
69 ± 4
69 ± 4
88 ± 8
54 ± 3
68 ± 8
50 ± 2
78 ± 9
92 ± 3
86 ± 4
89 ± 8
71 ± 9
56 ± 5
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0 ± 0
2 ± 0
0 ± 0
0 ± 0
0 ± 0
10 ± 3
24 ± 3
1 ± 0
6
c
6
d
6
e
6
f
6
7
g
a
(
S)-7a
7
b
3 ± 1
(
S)-7b
36 ± 2
36 ± 2
8 ± 1
(
R)-7b
1
3
78 ± 3
89 ± 3
a and b
see Table 2.
We found that most of the tested compounds showed
cytotoxicity toward both MCF7 and CCRF-CEM cells at
micromolar ranges. Among them, compounds 6b-f exerted a
strong effect on MCF7 cells viability (Table 2). Only 10-20% of
the cells survived in the presence of 6b-e at 50 ꢀM concentration
after 24 h treatment. In turn, treatment of MCF7 for 48 h resulted
in the most cytotoxic effect for para-fluoro substituted derivative
The analysis of the obtained data revealed that the nature of
the aryl substituent has little influence on cytotoxic activity of
particular compound. The compounds with bromine 6b, 6e and
fluorine 6d substituent located at the appropriate meta- or para-
position of the phenyl ring showed slightly increased cytotoxicity
comparing to unsubstituted aryl derivative 6a, and the meta-
methoxy substituted 6g, which decreased viability of both cell
lines only to 13-48% range at 100 ꢀM after 24 h treatment.
Undoubtedly, study of structural optimization of TBBt
derivatives should be continued.
6
d. The cytotoxicity data obtained for CCRF-CEM (Table 3)
revealed the cytotoxic activity of all compounds bearing a phenyl
ring with the exception of 6a and 6g, when CCRF-CEM cells
were treated with 100 ꢀM solution of the compounds. The most
active compounds 6c-e exhibited significant effect on CCRF-
CEM cells viability (46-56%) at a concentration of 50 ꢀM after
3
. Conclusion
Although 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt, 5) is
2
4 h of incubation. After 48 h of treatment, tumor cell viability
considered as a promising precursor for the synthesis of CK2
inhibitors, it has no obvious effect on studied cancer cells (Jurkat,
HL-60) treated in typical culture conditions (medium
supplemented with 10% serum) [28]. Therefore, it was of interest
to modify its structure in order to obtain new compounds active
against hCK2α and/or inhibiting tumor cells growth. This was
achieved by straightforward, four-step synthesis leading through
simple organic reaction methodology and with the use of cheap
chemical materials. The products were obtained via the following
reached 50% of the control in the presence of 25 ꢀM
concentration of 6g derivative.
The logarithm of a partition coefficient (logP) is an important
parameter, which has some influence on pharmacological
properties of the biologically active compounds. We have
employed the ChemBioDraw Ultra 13.0 software to define the
values of logP for each of TBBt derivatives 6a-g, 7a-b, 13 and
the results are summarized in Table 1. The lipophilicity of
phenyl-containing compounds 6a-g (logP 5.41-6.22) were higher
compared with TBBt 5 (logP 3.74), and its alkyl derivatives 7a-b
and 13 (logP 0.39-1.73), what may result in enhanced ability of
those compounds to penetrate into the cell and may explain
higher antitumor activity. Importantly, new derivatives exhibited
significantly higher cytotoxicity activities than almost inactive
parent TBBt 5. We found, that the inhibitory activities towards
hCK2α were inconsistent with MTT cytotoxicity assay results,
since compounds containing phenyl ring 6a-g in spite of being
weak inhibitors of hCK2α, turned out to display anti-tumor
activity at an acceptable level, and vice versa, TBBt alkyl-
containing derivatives 7a-b, 13 with promising hCK2α inhibitory
activity showed a moderate cytotoxic properties. In general,
compounds with alkyl substituents 7a-b, 13 were substantially
less active than those with additional aryl moiety 6a-g in both
cell lines what may suggest that aromatic substituent in this
position is important for the TBBt derivatives to exhibit
cytotoxicity.
reaction sequence: (i) NaBH -reduction of the appropriate β-keto
4
esters 1a-g, (ii) selective monotosylation of a primary hydroxyl
group of 2a-g to obtain 3a-g, (iii) bromination of benzotriazole
to obtain 4,5,6,7-tetrabromo-1H-benzotriazole 5, (iv) subsequent
alkylation of 5 with the appropriate derivative 3a-g. Human
CK2α inhibitory activity of the newly 6a-g, 13 and previously
synthesized compounds 7a-c was estimated at micromolar
concentrations. After the in vitro screening we found that
aliphatic TBBt derivatives 7a-c and 13 suppressed the activity of
hCK2α more efficiently than the new compounds with phenyl
ring 6a-g.
Experimental data obtained for (R)-7b revealed that this
compound is the most active TBBt derivative studied in this
work, with similar affinity for the hCK2α as TBBt 5. This ability
is connected with an optimal orientation of the hydroxyl group of
that inhibitor in the ATP binding pocket, which is consistent with
the predictions made by the computational docking method and
suggests that such algorithms can serve as an aid in the designing
of TBBt-based inhibitors. Despite the low hCK2α inhibitory

7
activity of compounds 6a-g, 13 and moderate activity of
previously received alkyl derivatives 7a-b, cytotoxic effect of all
compounds was tested on two human tumor cell lines and led to
the discovery of their antitumor activity. In vitro biological
evaluation showed that most of the compounds exhibit moderate
cytotoxicity against human breast cancer cells (MCF7), in
particular compounds 6b-f posses antitumor activity against the
leukemic cells (CCRF-CEM). This higher cytotoxicity than for
alkyl derivatives 7a-b, 13 is likely due to their cell penetration
ability and may be related to some other mechanism of action
rather than the result of CK2α kinase inhibition.
consumed (approx. 12 h, TLC). The cooled mixture was
ACCEPTED MANUSCRIPT
concentrated under reduced pressure and partitioned between
distilled water (35 mL) and EtOAc (40 mL). The layers were
separated, and the aqueous phase was back-extracted with EtOAc
(3 x 40 mL). The combined extracts were dried (Na SO ),
2
4
concentrated under reduced pressure, and purified by
chromatography on silica gel using mixture of PhCH /AcOEt
3
(1:1, v/v) as an eluent to give the corresponding 1,3-diol 2a-g.
4
.1.1.1. 1-Phenylpropane-1,3-diol (2a):
Colorless oil; 82% yield; R [PhCH /AcOEt (1:1, v/v)] 0.29;
H-NMR (CDCl , 400 MHz) δ: 1.84-2.02 (m, 2H, CH ), 3.26 (br.
s., 2H, OH), 3.74-3.84 (m, 2H, CH OH), 4.90 (dd, J=8.8, 3.8 Hz,
H, CH), 7.24-7.38 (m, 5H, Ph); C-NMR (CDCl , 100 MHz) δ:
f
3
1
3
2
A new strategy for the herein presented synthesis allows the
preparation of novel TBBt derivatives 6a-g and 13 with
significantly increased solubility in common organic solvents,
what can be considered as advantage as compared with low
solubility of TBBt 5.
2
13
1
4
3
0.3 (CH ), 61.1 (CH OH), 73.9 (CH), 125.6 (2C, o-Ph), 127.5
2
2
(p-Ph), 128.4 (2C, m-Ph), 144.2 (Ph).
4
.1.1.2. 1-(4-Bromophenyl)propane-1,3-diol (2b):
4
. Experimental
Colorless oil; 96% yield; R [PhCH /AcOEt (1:1, v/v)] 0.24;
f
3
4
.1. General details
1
H NMR (CDCl , 400 MHz) δ: 1.81-2.00 (m, 2H, CH ), 2.94 (br.
3
2
s., 2H, OH), 3.77-3.88 (m, 2H, CH OH), 4.90 (dd, J=8.4, 3.8 Hz,
All commercially available reagents (Aldrich, Fluka and
2
1
3
1
H, CH), 7.18-7.25 (m, 2H, o-Ph), 7.43-7.51 (m, 2H, m-Ph);
C
POCH) for chemistry purposes were used without further
purification. Solvents (methylene chloride, acetonitrile, acetone)
were dried by simply allowing them to stand over activated
NMR (CDCl , 100 MHz) δ: 40.3 (CH ), 61.3 (CH OH), 73.6
3
2
2
(CH), 121.2 (p-Ph), 127.4 (2C, o-Ph), 131.5 (2C, m-Ph), 143.2
(Ph).
(
predried at 300 °C for 24 h) 3 Å molecular sieves [20%
mass/volume (m/v) loading of the desiccant] at least for 48 h
before use [51]. Dimethyl Sulphoxide (DMSO), Molecular
Biology grade used as a solvent for all stocks of the chemical
agents was obtained from Roth, [gamma-P32] ATP (3000
Ci/mmol) was obtained from Hartmann Analytic GmbH; P81
4
.1.1.3. 1-(4-Chlorophenyl)propane-1,3-diol (2c):
Yellowish oil; 81% yield; R [PhCH /AcOEt (1:1, v/v)] 0.21;
f
3
1
H-NMR (CDCl , 400 MHz) δ: 1.82-2.02 (m, 2H, CH ), 2.77 (br.
3
2
s., 2H, OH), 3.78-3.90 (m, 2H, CH OH), 4.94 (dd, J=8.6, 3.6 Hz,
1H, CH), 7.27-7.35 (m, 4H, Ph); C-NMR (CDCl , 100 MHz) δ:
40.4 (CH ), 61.4 (CH OH), 73.6 (CH), 126.9 (2C, o-Ph), 128.5
2
13
(2.3 cm) circles were from Whatman. hCK2 substrate peptide
RRRDDDSDDD) was purchased from Biaffin GmbH & Co KG.
3
(
2
2
All other reagents used in this study were of analytical grade.
Melting points were obtained with MPA100 Optimelt SRS
apparatus. Thin-layer chromatography was carried on TLC
aluminium plates with silica gel Kieselgel 60 F254 (Merck) (0.2
mm thickness film) using UV light as a visualizing agent.
Preparative separations were carried out by: (i) column
chromatography using Silica gel with grain size 40-63 ꢀm or 15-
(2C, m-Ph), 133.0 (p-Ph), 142.6 (Ph).
4
.1.1.4. 1-(4-Fluorophenyl)propane-1,3-diol (2d):
Colorless oil; 78% yield; R [PhCH /AcOEt (1:1, v/v)] 0.16;
f
3
1
H-NMR (CDCl , 400 MHz) δ: 1.81-2.01 (m, 2H, CH ), 3.20 (s,
3
2
2
1
H, OH), 3.74-3.86 (m, 2H, CH OH), 4.90 (dd, J=8.8, 3.6 Hz,
2
13
H, CH), 6.97-7.07 (m, 2H, m-Ph), 7.27-7.34 (m, 2H, p-Ph); C-
4
0 ꢀm, respectively or by (ii) PLC Silica gel 60 F254 (20 × 20 cm
NMR (CDCl , 100 MHz) δ: 40.4 (CH ), 61.2 (CH OH), 73.5
3
2
2
1 13
with 2 mm thickness layer) glass plates from Merck. H and
C
(CH), 115.2 (d, J=21.0 Hz, 2C, m-Ph), 127.2 (d, J=7.6 Hz, 2C, o-
NMR spectra were measured with a Varian Mercury 400BB
Ph), 140.0 (d, J=2.9 Hz, 1C, Ph), 162.1 (d, J=245.3 Hz, 1C, p-
Ph).
1
13
spectrometer operating at 400 MHz for H and 100 MHz for
C
nuclei, chemical shifts (δ) are given in parts per million (ppm) on
the delta scale. The solvent peak was used as reference value;
signal multiplicity assignment: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; the type of signals: br., broad; coupling
constant (J) are given in hertz (Hz). All of the fid documents of
4
.1.1.5. 1-(3-Bromophenyl)propane-1,3-diol (2e):
Yellowish oil; 85% yield; R [PhCH /AcOEt (1:1, v/v)] 0.24;
f
3
1
H-NMR (CDCl , 400 MHz) δ: 1.86-2.01 (m, 2H, CH ), 2.65 (br.
3
2
1
13
s., 2H, OH), 3.79-3.90 (m, 2H, CH
1
2
OH), 4.92 (dd, J=8.1, 4.1 Hz,
H and C NMR spectra were analyzed by ACD/NMR Processor
H, CH), 7.17-7.30 (m, 2H, m-Ph and one of o-Ph), 7.33-7.43
Academic Edition 12.0 (freeware software). Mass spectrometry
was recorded on Micro-mass ESI Q-TOF spectrometer at IBB
PAN Warsaw. IR spectra of neat samples were recorded on a
Perkin Elmer System 2000 FTIR Spectrometer equipped with a
Pike Technologies GladiATR attenuated total reflectance (ATR)
accessory with a monolithic diamond crystal stage and a pressure
clamp; FTIR spectra were recorded in transmittance mode in the
13
(m, 1H, p-Ph), 7.50-7.54 (m, 1H, o-Ph-situated closer to Br); C-
NMR (CDCl , 100 MHz) δ: 40.3 (CH ), 61.4 (CH OH), 73.6
3
2
2
(CH), 122.5 (m-Ph-Br), 124.2 (o-Ph), 128.7 (o-Ph-situated closer
to Br), 130.0 (m-Ph), 130.4 (p-Ph), 146.5 (Ph).
4.1.1.6. 1-(3-Chlorophenyl)propane-1,3-diol (2f):
-
1
Yellowish oil; 86% yield; R [PhCH /AcOEt (1:1, v/v)] 0.20;
3
00-4000 cm range, in ambient air at room temperature, with 2
f
3
1
-
1
H-NMR (CDCl , 400 MHz) δ: 1.85-2.01 (m, 2H, CH ), 2.99 (br.
cm resolution and accumulation of 32 scans.
3
2
s., 2H, OH), 3.76-3.90 (m, 2H, CH OH), 4.92 (dd, J=8.1, 4.3 Hz,
1
much further from Cl); C-NMR (CDCl , 100 MHz) δ: 40.3
2
4
.1. 1. General procedure for the synthesis 1,3-diols (2a-g) from
H, CH), 7.18-7.30 (m, 3H, Ph), 7.33-7.38 (m, 1H, o-Ph-situated
13
β-keto esters (1a-g):
3
(
CH ), 61.3 (CH OH), 73.5 (CH), 123.7 (o-Ph), 125.8 (o-Ph-
2 2
NaBH (8 equiv) was added portion-wise to the solution of an
4
situated closer to Cl), 127.5 (p-Ph), 129.7 (m-Ph), 134.3 (m-Ph-
Cl), 146.2 (Ph).
appropriate β-keto ester 1a-g (1.5 g) in MeOH (15 mL) at room
temperature. After 20 min the heterogeneous white reaction
mixture was heated to reflux until all starting material was

8
European Journal of Medicinal Chemistry
4
.1.1.7. 1-(3-Methoxyphenyl)propane-1,3-diol (2g):
21.6 (CH ), 38.0 (CH ), 67.3 (CH OSO ), 69.4 (CH), 127.0 (2C,
3 2 2 2
ACCEPTED MANUSC
Yellowish oil; 95% yield; R [PhCH /AcOEt (1:1, v/v)] 0.22;
RIPT
o-Ph), 127.9 (2C, m-Ph), 128.6 (2C, o-PhTos), 129.9 (2C, m-
PhTos), 132.7 (p-PhTos), 133.4 (p-Ph), 141.9 (Ph), 144.9
f
3
1
H-NMR (CDCl , 400 MHz) δ: 1.84-2.06 (m, 2H, CH ), 2.92 (br.
3
2
(PhTos).
s., 2H, OH), 3.80 (s, 3H, OCH ), 3.81-3.87 (m, 2H, CH OH),
3
2
4
.1.2.4.
3-(4-Fluorophenyl)-3-hydroxypropyl
4-
4
6
.91 (dd, J=8.6, 3.8 Hz, 1H, CH), 6.78-6.83 (m, 1H, Ph), 6.90-
.95 (m, 2H, Ph), 7.22-7.29 (m, 1H, Ph); C-NMR (CDCl , 100
13
methylbenzenesulfonate (3d):
3
MHz) δ: 40.4 (CH ), 55.2 (OCH ), 61.3 (CH OH), 74.1 (CH),
2
3
2
Colorless oil; 66% yield; R [AcOEt/Cyclohexene (1:1, v/v)]
f
1
1
11.1 (o-Ph-situated closer to OCH ), 112.9 (p-Ph), 117.8 (o-Ph),
29.4 (m-Ph), 145.9 (Ph), 159.6 (m-Ph-OCH3).
1
3
0
2
.71; H-NMR (CDCl , 400 MHz) δ: 1.94-2.06 (m, 2H, CH ),
3
2
.22 (br. s., 1H, OH), 2.45 (s, 3H, CH ), 3.98-4.06 (m, 1H,
3
4
.1.2. General procedure for the synthesis 3a-g by the
CH OSO ), 4.22-4.31 (m, 1H, CH OSO ), 4.79 (dd, J=7.9, 5.7
2 2 2 2
monotosylation of the 1,3-diols (2a-g):
Hz, 1H, CH), 6.95-7.03 (m, 2H, m-Ph), 7.20-7.27 (m, 2H, o-
PhTos), 7.33-7.36 (m, 2H, m-PhTos), 7.74-7.80 (m, 2H, o-Ph);
To a flame-dried flask, containing the 1,3-diol 2a-g (1 g)
13
C-NMR (CDCl , 100 MHz) δ: 21.6 (CH ), 38.1 (CH ), 67.4
3
3
2
dissolved in dry CH Cl (15 mL), freshly distilled triethylamine
2
2
(
CH OSO ), 69.5 (CH), 115.3 (d, J=21.2 Hz, 2C, m-Ph), 127.3
2 2
(1.1 equiv) and 4-(dimethylamino)pyridine (10 mg) were added
(d, J=7.6 Hz, 2C, o-Ph), 127.8 (2C, o-PhTos), 129.8 (2C, m-
at -15 °C under an nitrogen atmosphere. After 15 min, p-
toluenesulfonyl chloride (1 equiv) suspended in anhydrous
CH Cl (6 mL) was added dropwise via syringe with stirring over
PhTos), 132.7 (p-PhTos), 139.2 (d, J=3.8 Hz, 1C, Ph), 144.9
PhTos), 162.2 (d, J=246.3 Hz, 1C, p-Ph).
(
2
2
4
.1.2.5. 3-(3-Bromophenyl)-3-hydroxypropyl
4-
a period of 1 h under fast stream of nitrogen. Upon completion of
the addition, the reaction mixture was maintained at -15 °C for 12
h and then allowed to warm to room temperature and stirred for a
further 1 h. Next, the content of the flask was cooled to 0 °C,
portion of CH Cl (30 mL) was added, and the solution was
methylbenzenesulfonate (3e):
Yellowish oil; 82% yield; R [AcOEt/Cyclohexene (4:1, v/v)]
f
1
0
2
1
4
7
.67; H-NMR (CDCl , 400 MHz) δ: 1.97-2.01 (m, 2H, CH ),
3
2
2
2
.06 (br. s., 1H, OH), 2.46 (s, 3H, CH ), 4.06 (dt, J=10.2, 5.2 Hz,
3
quenched successively by the careful addition of cold 1% HCl (2
x 15 mL), washed with saturated sodium bicarbonate (2 x 15
mL), brine (2 x 15 mL), dried over anhydrous MgSO , and
concentrated in vacuo to yield the crude product. The purification
by column chromatography on silica gel with mixture of ethyl
acetate/cyclohexane (4:1 or 1:1, v/v – depending on the substance
purified) as an eluent afforded the desired monotosylated alcohol
H, CH OSO ), 4.30 (ddd, J=10.1, 8.1, 5.3 Hz, 1H, CH OSO ),
2
2
2
2
.79 (dd, J=8.4, 5.0 Hz, 1H, CH), 7.14-7.22 (m, 2H, Ph), 7.31-
4
13
.47 (m, 5H, Ph), 7.76-7.83 (m, 2H, Ph); C-NMR (CDCl , 100
3
MHz) δ: 21.7 (CH ), 38.1 (CH ), 67.2 (CH OSO ), 69.4 (CH),
3
2
2
2
1
22.7 (m-Ph-Br), 124.2 (o-Ph), 127.9 (2C, o-PhTos), 128.7 (o-
Ph-situated closer to Br), 129.9 (2C, m-PhTos), 130.1 (m-Ph),
1
30.8 (p-Ph), 132.7 (p-PhTos), 144.9 (Ph), 145.8 (PhTos).
.1.2.6. 3-(3-Chlorophenyl)-3-hydroxypropyl
3
a-g.
4
4-
4
.1.2.1. 3-Hydroxy-3-phenylpropyl 4-methylbenzenesulfonate
methylbenzenesulfonate (3f):
(3a):
Yellowish oil; 40% yield; R [AcOEt/Cyclohexene (4:1, v/v)]
f
Yellowish oil; 41% yield; R [AcOEt/Cyclohexene (4:1, v/v)]
.67; H-NMR (CDCl , 400 MHz) δ: 1.97-2.06 (m, 2H, CH ),
3 2
1
f
1
0.71; H-NMR (CDCl
3
, 400 MHz) δ: 1.98 (dtd, J=8.0, 5.2, 5.2,
0
2
3
4
.1 Hz, 2H, CH ), 2.27 (br. s., 1H, OH), 2.45 (s, 3H, CH ), 3.99-
2
3
.10 (br. s., 1H, OH), 2.45 (s, 3H, CH ), 4.00-4.10 (m, 1H,
3
.08 (m, 1H, CH OSO ), 4.29 (ddd, J=10.1, 7.8, 5.4 Hz, 1H,
2 2
CH OSO ), 4.22-4.33 (m, 1H, CH OSO ), 4.76-4.83 (m, 1H,
2
2
2
2
13
CH OSO ), 4.78 (dd, J=8.0, 5.3 Hz, 1H, CH), 7.11-7.17 (m, 1H,
2 2
CH), 7.24-7.38 (m, 7H, Ph), 7.76-7.82 (m, 2H, Ph); C-NMR
Ph), 7.20-7.28 (m, 3H, Ph), 7.33-7.37 (m, 2H, Ph), 7.75-7.81 (m,
(
7
1
CDCl , 100 MHz) δ: 21.7 (CH ), 38.0 (CH ), 67.6 (CH OSO ),
13
3
3
2
2
2
2
6
H, Ph); C-NMR (CDCl , 100 MHz) δ: 21.7 (CH ), 38.0 (CH ),
3
3
2
0.15 (CH), 125.5 (2C, o-Ph), 127.8 (p-Ph), 127.8 (2C, o-PhTos),
28.5 (2C, m-PhTos), 129.8 (2C, m-Ph), 132.8 (p-PhTos), 143.4
Ph), 144.7 (PhTos).
7.3 (CH OSO ), 69.4 (CH), 123.8 (o-Ph), 125.8 (o-Ph-situated
2
2
closer to Cl), 127.8 (3C, p-Ph and o-PhTos), 129.8 (m-Ph), 129.9
2C, m-PhTos), 132.7 (p-PhTos), 134.4 (m-Ph-Cl), 144.9 (Ph),
145.6 (PhTos).
(
(
4
.1.2.2. 3-(4-Bromophenyl)-3-hydroxypropyl
4-
methylbenzenesulfonate (3b):
4
.1.2.7.
3-(3-Methoxyphenyl)-3-hydroxypropyl
4-
methylbenzenesulfonate (3g):
Colorless oil; 61% yield; R [AcOEt/Cyclohexene (1:1, v/v)]
f
1
0
2
1
.76; H-NMR (CDCl , 400 MHz) δ: 1.98 (td, J=6.55, 5.42 Hz,
3
Yellowish oil; 39% yield; R [AcOEt/Cyclohexene (4:1, v/v)]
f
H, CH ), 2.23 (br. s., 1H, OH), 2.46 (s, 3H, CH ), 3.96-4.07 (m,
1
2
3
0
.64; H-NMR (CDCl , 400 MHz) δ: 1.96-2.04 (m, 2H, CH ),
3
2
H, CH OSO ), 4.27 (dt, J=10.0, 6.6 Hz, 1H, CH OSO ), 4.79 (t,
2
2
2
2
2
.05 (br. s., 1H, OH), 2.45 (s, 3H, CH ), 3.79 (s, 3H, OCH ),
3
3
J=6.6 Hz, 1H, CH), 7.11-7.17 (m, 2H, Ph), 7.32-7.38 (m, 2H,
13
4.01-4.11 (m, 1H, CH OSO ), 4.27 (dt, J=9.9, 6.7 Hz, 1H,
2 2
Ph), 7.39-7.45 (m, 2H, Ph), 7.74-7.80 (m, 2H, Ph); C-NMR
CH OSO ), 4.78 (t, J=6.7 Hz, 1H, CH), 6.76-6.88 (m, 3H, Ph),
2
2
(
6
1
CDCl , 100 MHz) δ: 21.7 (CH ), 38.0 (CH ), 67.3 (CH OSO ),
3 3 2 2 2
7
.17-7.27 (m, 1H, Ph), 7.29-7.39 (m, 2 H, Ph), 7.79-7.81 (m, 2H,
9.5 (CH), 121.5 (p-Ph), 127.3 (2C, o-Ph), 127.9 (2C, o-PhTos),
29.9 (2C, m-PhTos), 131.6 (2C, m-Ph), 132.7 (p-PhTos), 142.4
Ph), 144.9 (PhTos).
13
Ph); C-NMR (CDCl , 100 MHz) δ: 21.6 (CH ), 38.0 (CH ),
3
3
2
5
5.2 (OCH ), 67.6 (CH OSO ), 70.1 (CH), 111.0 (o-Ph-situated
3 2 2
(
closer to OCH ), 113.3 (p-Ph), 117.8 (o-Ph), 127.9 (2C, m-
3
4
.1.2.3. 3-(4-Chlorophenyl)-3-hydroxypropyl
4-
PhTos), 129.6 (m-Ph), 129.9 (2C, o-PhTos), 132.8 (p-PhTos),
144.8 (Ph), 145.2 (PhTos), 159.7 (OCH3).
methylbenzenesulfonate (3c):
4
.1.3. Synthesis of 4,5,6,7-tetrabromo-1H-benzotriazole (5):
Yellowish oil; 33% yield; R [AcOEt/Cyclohexene (4:1, v/v)]
f
1
0
2
.73; H-NMR (CDCl , 400 MHz) δ: 1.94-2.03 (m, 2H, CH ),
3
2
1
H-Benzotriazole 4 (6 g, 50.4 mmol) was dissolved in a
.33 (br. s., 1H, OH), 2.45 (s, 3H, CH ), 3.98-4.05 (m, 1H,
3
mixture of 69% HNO (150 mL) and fuming 100% HNO (10
mL). Next, the solution was heated to a temperature of 80 °C and
Br (48.3 g, 302 mmol, 15 mL) was added dropwise within 1 h.
3
3
CH OSO ), 4.22-4.30 (m, 1H, CH OSO ), 4.79 (t, J=6.7 Hz, 1H,
2
2
2
2
CH), 7.17-7.22 (m, 2H, Ph), 7.23-7.29 (m, 2H, Ph), 7.31-7.38 (m,
13
2
2
H, Ph), 7.73-7.79 (m, 2H, Ph); C-NMR (CDCl , 100 MHz) δ:
3

9
The reaction mixture was stirred vigorously using mechanical
stirrer at 60 °C for 48 h and irradiated by exposure to UV light.
4.1.4.2.
1-(4-Bromophenyl)-3-(4,5,6,7-tetrabromo-2H-
ACCEPTED MANUSCRIPT
benzotriazol-2-yl)propan-1-ol (6b):
Subsequently, content of the flask was cooled to room
temperature, the excess of unreacted bromine was removed in the
gentle flow of nitrogen and trapped into a 20 % solution of
sodium pyrosulfite (150 mL). The suspension was poured into
mixture of ice-cold H O (300 mL) and saturated Na S O (20
White solid; 33% yield; mp 125-126 °C; R [CHCl /acetone
f
3
1
(
95:5, v/v)] 0.55; H-NMR (CDCl , 400 MHz) δ: 1.97 (br. s., 1H,
3
OH), 2.44-2.61 (m, 2H, CH ), 4.78 (dd, J=7.8, 5.1 Hz, 1H, CH),
2
4
2
(
(
(
.83-5.02 (m, 2H, CH N), 7.14-7.25 (m, 2H, o-Ph), 7.38-7.47 (m,
2
2
2
2
5
13
H, m-Ph); C-NMR (CDCl , 100 MHz) δ: 38.4 (CH ), 54.2
3
2
mL). The resulting yellowish precipitate was filtered off, and
CH N), 70.8 (CH), 113.7 (TBBt-internal), 121.7 (p-Ph), 126.5
2
washed with H O (100 mL) and EtOH (100 mL), respectively.
2
2C, TBBt-external), 127.4 (2C, o-Ph), 131.6 (2C, m-Ph), 141.9
The obtained crude product was refluxed several times in the
mixture of MeOH (50 mL) and i-PrOH (25 mL), and hot
saturated solution was filtered off yielding white crystals 5 (13 g,
+
+
calcd
-
Ph), 143.1 (TBBt=N); HRMS (ESI , m/z): [M+H]
=
=
+
-
6
6
49.6758, [M+H]
= 649.7219; (ESI , m/z): [M+H]
= 647.7287; FTIR νmax(neat): 3417, 1588,
found
calcd
-
47.6601, [M+H]
found
2
9.9 mmol, 59%). The purity of the compound 5 was confirmed
1 13
1533, 1486, 1426, 1309, 1295, 1274, 1176, 1159, 1073, 1011,
79, 926, 876, 823, 772, 750, 718, 609, 544, 509.
by high-resolution mass spectrometry (HRMS). The H-, C-
NMR and m.p. were consistent with the literature data [46].
9
White solid; mp 264-266 °C; yield: 40%; R [CHCl /MeOH
f
3
4.1.4.3. 1-(4-Chlorophenyl)-3-(4,5,6,7-tetrabromo-2H-
benzotriazol-2-yl)propan-1-ol (6c):
+
+
(
[
99:1, v/v)] 0.16; HRMS (ESI , m/z): [M+H]
= 435.6941,
calcd
+
M+H]
= 435.6629.
found
White solid; 41% yield; mp 112-114 °C; R [CHCl /Et O (6:4,
f
3
2
4
.1.4. General procedure for the synthesis of 6a-g:
1
v/v)] 0.51; H-NMR (CDCl , 400 MHz) δ: 2.42-2.58 (m, 2H,
3
CH ), 2.71 (br. s., 1H, OH), 4.79 (dd, J=8.1, 5.0 Hz, 1H, CH),
2
To the solution of TBBt 5 (1 equiv) in the mixture of absolute
1
3
4
.91 (dt, J=19.1, 6.9 Hz, 2H, CH N), 7.23-7.27 (m, 4H, Ph); C-
2
acetone (15 mL) and dry acetonitrile (10 mL), anhydrous K CO3
2
NMR (CDCl , 100 MHz) δ: 38.5 (CH ), 54.1 (CH N), 70.6 (CH),
3
2
2
(
3 equiv) was added. The content of the flask was flushed with
nitrogen for several times, and stirred for 3 h at reflux. Next, the
reaction mixture was cooled to room temperature and the
1
13.6 (2C, TBBt-internal), 126.4 (2C, TBBt-external), 127.0 (2C,
m-Ph), 128.6 (2C, o-Ph), 133.5 (p-Ph), 141.4 (Ph), 142.9
+
+
(
[
[
Tbbt=N); HRMS (ESI , m/z): [M+H]
=
603.7283,
calcd
solution
of
appropriate
3-hydroxy-3-arylpropyl-4-
+
-
-
M+H]
M+H]
= 603.7826; (ESI , m/z): [M+H]
= 601.7653; FTIR νmax(neat): 3387, 1739, 1701,
= 601.7127,
found
calcd
methylbenzenesulfonate 3a-g (400 mg) in acetonitrile (5 mL)
was added dropwise. After stirring at reflux for 48 h, the crude
reaction mixture was cooled to room temperature, and both the
white precipitate as well as the red colored solution were
transferred into separator and washed with saturated solution of
potassium carbonate (100 mL). Water phase was extracted with
-
found
1
9
490, 1426, 1366, 1270, 1230, 1218, 1174, 1089, 1074, 1012,
78, 832, 770, 750, 607, 530.
4.1.4.4.
1-(4-Fluorophenyl)-3-(4,5,6,7-tetrabromo-2H-
benzotriazol-2-yl)propan-1-ol (6d):
CHCl (2 x 150 mL) and AcOEt (3 x 100 mL), and the collected
3
White solid; 33% yield; mp 73-75 °C; R [CHCl /acetone
organic layers were partially condensed on rotator evaporator,
and dried (Na SO ). The purification procedure was performed
f
3
1
(
95:5, v/v)] 0.44; H-NMR (CDCl , 400 MHz) δ: 2.10 (br. s., 1H,
3
2
4
OH), 2.47-2.57 (m, 2H, CH ), 4.80 (dd, J=7.4, 5.7 Hz, 1H, CH),
on preparative layer plates (PLC) coated with unmodified silica
gel matrices, using mixture of CHCl /Et O (7:3 or 6:4, v/v) or
2
4
(
(
.84-5.02 (m, 2H, CH N), 6.98-7.06 (m, 2H, m-Ph), 7.29-7.37
2
13
3
2
m, 2H, o-Ph); C-NMR (CDCl , 100 MHz) δ: 38.7 (CH ), 54.3
CHCl /acetone (95:5, v/v), depending on substance purified. The
3
2
3
CH N), 70.7 (CH), 113.7 (2C, TBBt-internal), 115.5 (d, J=21.9
appropriate fraction was removed, placed in the round-bottomed
2
Hz, 2C, m-Ph), 126.4 (2C, TBBt-external), 127.3 (d, J=7.6 Hz,
flask, suspended in the mixture of CHCl /acetone (150 mL, 2:1,
3
2
(
5
5
1
1
C, o-Ph), 138.8 (d, J=2.9 Hz, 1C, Ph), 143.1 (TBBt=N), 162.3
v/v), and vigorously stirred for 1 h at room temperature. Next, the
+
+
d, J=246.4 Hz, 1C, p-Ph); HRMS (ESI , m/z): [M+H]
=
=
silica gel was filtered, and washed with CHCl /acetone (2 x 50
calcd
3
+
-
-
87.7579, [M+H]
= 587.8105; (ESI , m/z): [M+H]
mL, 1:1, v/v). After partial solvent removal in vacuo, solution of
the filtrate (approx. 1.5 mL) was kept in the refrigerator until
white precipitate occurred. Next, the liquid phase was decanted
off by Pasteur pipette tipped with cotton wool, and the remaining
solid was washed with ice-cold acetone (1 mL), and then ethanol
found
calcd
-
85.7422, [M+H]
= 585.7770; FTIR νmax(neat): 3401, 1604,
found
531, 1507, 1426, 1395, 1344, 1298, 1271, 1217, 1174, 1157,
100, 1073, 1013, 977, 929, 883, 835, 771, 746, 608, 567, 544.
4
.1.4.5.
1-(3-Bromophenyl)-3-(4,5,6,7-tetrabromo-2H-
(1 mL) respectively to obtain desired product 6a-g as a white
benzotriazol-2-yl)propan-1-ol (6e):
solid, which was further high-vacuum-dried in the desiccator
over phosphorus pentoxide (P O ) overnight.
White solid; 36% yield; mp 88-91 °C; R
[CHCl /acetone
3
2
5
f
1
(
95:5, v/v)] 0.55; H-NMR (CDCl , 400 MHz) δ: 2.22 (br. s., 1H,
3
4
.1.4.1.
1-Phenyl-3-(4,5,6,7-tetrabromo-2H-benzotriazol-2-
OH), 2.44-2.63 (m, 2H, CH ), 4.79 (dd, J=8.2, 4.6 Hz, 1H, CH),
2
yl)propan-1-ol (6a):
4
1
(
.84-5.02 (m, 2H, CH N), 7.16-7.22 (m, 1H, Ph), 7.23-7.29 (m,
2
13
H, Ph), 7.35-7.40 (m, 1H, Ph), 7.48-7.51 (m, 1H, Ph); C-NMR
White solid; 40% yield; mp 135-136 °C; R [CHCl /Et O (7:3,
f
3
2
1
CDCl , 100 MHz) δ: 38.5 (CH ), 54.1 (CH N), 70.6 (CH), 113.6
v/v)] 0.67; H-NMR (CDCl , 400 MHz) δ: 2.31 (br. s., 1H, OH),
3
2
2
3
(
2C, TBBt-internal), 122.7 (m-Ph-Br), 124.2 (2C, TBBt-
2
5
1
.48-2.62 (m, 2H, CH ), 4.82 (dd, J=7.5, 5.6 Hz, 1H, CH), 4.85-
2
13
external), 126.4 (o-Ph), 128.7 (m-Ph), 130.2 (o-Ph-situated closer
.02 (m, 2H, CH N), 7.23-7.40 (m, 5H, Ph); C-NMR (CDCl3,
2
+
to Br), 130.9 (p-Ph), 143.0 (TBBt=N), 145.3 (Ph); HRMS (ESI ,
00 MHz) δ: 38.6 (CH ), 54.4 (CH N), 71.4 (CH), 113.7 (2C,
2
2
+
+
-
m/z): [M+H]
= 647.6778, [M+H]
= 645.6622, [M+H]
= 647.7335; (ESI ,
TBBt-internal), 125.6 (2C, TBBt-external), 126.3 (2C, o-Ph),
28.0 (2C, m-Ph), 128.6 (p-Ph), 143.0 (Ph), 143.1 (TBBt=N);
calcd
found
-
-
m/z): [M+H]
= not determined;
1
calcd
found
+
+
+
FTIR νmax(neat): 3397, 1740, 1591, 1573, 1534, 1478, 1426,
342, 1305, 1275, 1241, 1146, 1172, 1100, 1070, 1035, 1014,
004, 977, 878, 800, 788, 753, 693, 667, 608, 585, 511, 443.
HRMS (ESI , m/z): [M+H]
= 569.7673, [M+H]
=
=
calcd
found
-
-
-
1
1
5
5
1
69.8090; (ESI , m/z): [M+H]
67.7919; FTIR νmax(neat): 3549, 3417, 1533, 1490, 1430, 1272,
178, 1162, 1086, 1057, 975, 765, 748, 698, 606, 516.
= 567.7516, [M+H]
calcd found

1
0
European Journal of Medicinal Chemistry
4
.1.4.6.
1-(3-Chlorophenyl)-3-(4,5,6,7-tetrabromo-2H-
equimolar amounts of both reagents: 11 (400 mg, 1.4 mmol) and
ACCEPTED MANUSCRIPT
benzotriazol-2-yl)propan-1-ol (6f):
5 (610 mg, 1.4 mmol) in the presence of 3-fold molar excess of
K CO (582 mg, 4.21 mmol) were stirred in refluxing mixture of
absolute acetone (15 mL) and dry acetonitrile (10 mL) for 48 h.
2
3
White solid; 42% yield; mp 114-115 °C; R [CHCl /acetone
f
3
1
(95:5, v/v)]; H-NMR (CDCl , 400 MHz) δ: 1.68 (br. s., 1H,
3
Crude product 12 was partially purified by simple filtration on
short silica pad by eluting with mixture of CHCl /acetone (7:3,
OH), 2.44-2.64 (m, 2H, CH ), 4.80 (dd, J=8.3, 4.5 Hz, 1H, CH),
2
3
4
.83-5.05 (m, 2H, CH N), 7.16-7.29 (m, 3H, Ph), 7.34 (t, J=1.8
2
13
v/v) just to remove unreacted 5. As the high resolution mass
spectrometry analysis confirmed presence of desired product 12,
and absence of compound 5, it was allowed to use in the next
Hz, 1H, Ph); C-NMR (CDCl , 100 MHz) δ: 38.5 (CH ), 54.2
3
2
(CH N), 70.7 (CH), 113.7 (2C,TBBt-internal), 123.7 (2C, TBBt-
2
external), 125.8 (o-Ph), 126.4 (o-Ph-situated closer to Cl), 128.0
+
+
calcd
step without further purification. HRMS (ESI , m/z): [M+H]
(
(
[
m-Ph), 129.9 (p-Ph), 134.5 (m-Ph-Cl), 143.1 (Ph), 145.1
TBBt=N); HRMS (ESI , m/z): [M+H]
M+H]
+
=
549.7622, [M+H]
= 549.7753.
+
+
calcd
found
= 603.7283,
+
= 603.7560; FTIR νmax(neat): 3429, 1596, 1573,
4.1.7. Synthesis of 3-(4,5,6,7-tetrabromo-2H-benzotriazol-2-
yl)propane-1,2-diol (13):
found
1
1
7
535, 1476, 1445, 1428, 1370, 1335, 1277, 1201, 1173, 1146,
099, 1072, 1051, 1041, 1010, 977, 881, 868, 798, 791, 777, 767,
18, 700, 688, 609, 500, 455.
To the compound 12 (165 mg, 0.3 mmol) dissolved in MeOH
(3 mL), HCl (0.5 N, 0.3 mL) was added dropwise, and the
4
.1.4.7.
1-(3-Methoxyphenyl)-3-(4,5,6,7-tetrabromo-2H-
resulting mixture was heated to reflux. After 4 h acetone and
methanol were slowly distilled off. Additional portion of MeOH
benzotriazol-2-yl)propan-1-ol (6g):
(
1 mL) and HCl (0.5 N, 0.2 mL) was added, and the mixture was
kept at room temperature until ketal hydrolysis was completed
approx. 2 h, TLC). The mixture was diluted with careful
White solid; 33% yield; mp 79-80 °C; R [CHCl /acetone
f
3
1
(
95:5, v/v)] 0.42; H-NMR (CDCl , 400 MHz) δ: 2.00 (br. s., 1H,
3
(
OH), 2.50-2.61 (m, 2H, CH ), 3.80 (s, 3H, OCH ), 4.79 (dd,
2
3
addition of saturated NaHCO until effervescence ceased, and the
3
J=7.4, 5.5 Hz, 1H, CH), 4.93 (td, J=13.7, 6.4 Hz, 2H, CH N),
2
water phase was extracted with EtOAc (3 x 10 mL). The organic
extracts were combined, washed with brine (5 mL), dried over
anhydrous Na SO , filtered, concentrated under reduced pressure,
and purified by preparative chromatography (PLC) using mixture
of CHCl /acetone (9:1, v/v) as an eluent, affording target
compound 13 as hygroscopic white solid (95 mg, 0.19 mmol,
6
1
6
.77-6.80 (m, 1H, Ph), 6.89-6.94 (m, 2H, Ph), 7.21-7.26 (m, 1H,
13
Ph); C-NMR (CDCl , 100 MHz) δ: 38.5 (CH ), 54.4 (CH N),
3
2
2
2
4
5
5.2 (OCH ), 71.3 (CH), 111.1 (p-Ph), 113.3 (o-Ph-situated
3
closer to OCH ), 113.7 (o-Ph), 117.8 (TBBt-internal), 126.3
3
3
(
1
TBBt-external), 129.7 (m-Ph), 143.1 (TBBt=N), 144.7 (Ph),
+
+
59.8 (m-Ph-OCH ); HRMS (ESI , m/z): [M+H]
= 599.7779,
= 597.7622,
calcd
3
calcd
2%). White semi-solid; 30% yield (after two steps: from 11 to
+
-
-
[
[
2
1
9
M+H]
= 599.8348; (ESI , m/z): [M+H]
= not determined; FTIR νmax(neat): 3529, 3447, 2932,
1
found
3); R [CHCl /acetone (9:1, v/v)] 0.78; H-NMR (MeOD, 400
-
f
3
M+H]
found
MHz) δ: 3.59-3.74 (m, 2H, CH OH), 4.16-4.42 (m, 1H, CHOH)
4
2
831, 1735, 1604, 1579, 1533, 1482, 1429, 1377, 1306, 1284,
273, 1256, 1235, 1176, 1166, 1146, 1095, 1062, 1041, 995, 977,
33, 913, 886, 834, 785, 770, 739, 700, 605, 566, 525, 475.
13
.76 (s, 1H, CH N), 4.93-5.21 (m, 1H, CH N); C-NMR
2
2
(
(
(
DMSO-d , 100 MHz) δ: 53.2 (CH N), 63.5 (CH OH), 71.0
CHOH), 115.7 (TBBt-internal), 125.4 (TBBt-external), 142.6
6
2
2
+
+
calcd
TBBt=N); HRMS (ESI , m/z): [M+H]
= 509.7309,
4
.1.5. Synthesis of (2,2-Dimethyl-1,3-dioxolan-4-yl)methyl 4-
methylbenzenesulfonate (11):
+
[M+H] found = 509.7447.
4
4
.1.8. Biological Assays
Tosyl chloride (2.07 g, 10.89 mmol) was added in small
portions (during 1 h) to a solution of 2,2-dimethyl-1,3-dioxolane-
.1.8.1. Cloning, expression and purification of human CK2α:
4
-methanol 10 (1.20 g, 9.08 mmol), triethylamine (1.38 g, 13.62
mmol, 1.90 mL), and DMAP (112 mg, 0.9 mmol) in dry CH Cl
The coding region of human CK2α was amplified by
polymerase chain reaction using the following primers: 5’-
CGCGGATCCGTCGGGACCCGTGCCAA (upstream primer)
2
2
(
20 mL), with magnetic stirring at 0-5 °C. After 5 h, Et O (15
2
mL) was added, and the solution was washed with 10% water
solution of HCl (2 x 15 mL), saturated aqueous NaHCO (20
and
5’-CCCAAGCTTCTGCTGAGCGCCAGCGGCA
3
mL), dried over anhydrous MgSO , and concentrated in vacuo.
(downstream primer) and I.M.A.G.E. clone as a template. The
product was cloned into the vector pETDuet-1 (MCS1) using the
restriction sites BamHI and HindIII and the bacterial strain
DH5α. The sequence of the obtained clone was confirmed.
Expression of the resulting N-terminal histidine-tagged hCK2α
was done in the bacterial strain BL21(DE3)pLysS growing in
superbroth medium after induction with 0.5 mM IPTG for 20 h at
20 °C. The cell pellet was resuspended in extraction buffer
[composed of: 20 mM NaH PO (pH 8.0), 500 mM NaCl, 10 mM
imidazole, O-complete inhibitor cocktail (Roche), lysozyme (1
mg/mL)] and sonicated. The supernatant of the pellet from 200
mL of bacterial culture was loaded onto Ni-NTA agarose
(Qiagene) column (2 mL bed volume). hCK2α was eluted with
300 mM imidazole. Fractions containing His-tagged hCK2α were
dialyzed against 20 mM Tris-HCl (pH 8.5), 500 mM NaCl, 1 mM
DTT, 0-20% glycerol and stored at -20 °C. The protein
concentration in final solution was 12.68 mg/mL (determined by
Bradford method and bovine serum albumin as the standard)
4
The crude product was purified by column chromatography over
silica gel using mixture of hexane/ethyl acetate (5:1, v/v) as an
eluent, to give the product 11 as white solid (2.1 g, 7.33 mmol,
8
1%) with >99% purity according to GC. White solid; 81%
yield; mp 50-52 °C [Lit. [52]: mp 49-50 °C after recrystallization
from petroleum ether/diethyl ether (10:1, v/v)]; R [hexane/ethyl
f
1
acetate (5:1, v/v)] 0.36; H-NMR (CDCl , 400 MHz) δ: 1.31 (d,
3
J=11.5 Hz, 6H, 2xCH ), 2.43 (s, 3H, p-CH ), 3.74 (dd, J=8.8, 5.2
3
3
2
4
Hz, 1H, partially CH ), 3.92-4.06 (m, 3H, CH and partially CH2
2
and partially CH OSO ), 4.21-4.31 (m, 1H, partially CH OSO ),
2
2
2
2
13
7
.34 (d, J=8.1 Hz, 2H, Ph), 7.78 (d, J=8.1 Hz, 2H, Ph); C-NMR
(
(
CDCl , 100 MHz) δ: 21.6 (p-CH ), 25.0 (CH ), 26.5 (CH ), 66.0
3
3
3
3
CHCH O), 69.4 (CH OSO ), 72.8 (CH), 109.9 (C), 127.9 (2C,
2
2
2
m-Tos), 129.8 (2C, o-Tos), 132.5 (p-Tos), 145.0 (PhOSO );
2
+
+
calcd
+
HRMS (ESI , m/z): [M+H]
= 287.0953, [M+H]
=
found
2
87.0884; GC [100-260 (10 °C/min)]: t = 14.82 min.
R
4
.1.6. Synthesis of 4,5,6,7-tetrabromo-2-[(2,2-dimethyl-1,3-
[
53].
dioxolan-4-yl)methyl]-2H-benzotriazole (12):
This compound was prepared by using the procedure as
described in synthesis detail for compounds 6a-g. Briefly,

1
1
4
.1.8.2. Computational docking methods
glycine, 0.1 M NaCl, pH 10.5) was added (25 ꢀL per well).
ACCEPTED MANUSCRIPT
The absorbance were measured at 570 nm using Synergy H4
BioTek microplate reader. All measurements were carried out in
triplicate and the results are expressed in percentage of cell
viability relative to control (cells without inhibitor in 0.5%
DMSO). At such conditions, IC50 for standard anti-cancer
inhibitor - doxorubicin was 1.4 µM for both MCF7 and CCRF-
CEM lines.
Computer molecular dynamics simulations (docking studies)
were
carried
out
using
AutoDock
4.2
program
(http://autodock.scripps.edu/) [54]. Ligand molecules were
prepared
with
ChemAxon
MarvinSketch
5.12.3
(
http://www.chemaxon.com/marvin/) and saved as .PDB files.
The crystallographic structure of CK2α (PDB code: 1J91) was
obtained from RCSB Protein Data Bank (PDB,
http://www.rcsb.org/pdb/). Non-protein molecules were removed,
polar hydrogen were added and Gasteiger charges were
calculated with AutoDock Tools 1.5.6 package (ADT,
http://mgltools.scripps.edu/) [55]. Docking was performed in 70
x 70 x 70 units grid box centered on the enzyme hydrophobic
pocket with the grid spacing of 0.325 Å. For each ligand
molecule 100 independent runs were performed, using
Acknowledgments
We gratefully acknowledge the financial support from the
project „Biotransformations for Pharmaceutical and Cosmetics
Industry” No. POIG.01.03.01-00-158/09, funded by the
European Union within the European Regional Development
Fund. The study was co-financed by NCN grant No
6
Lamarckian genetic algorithm, with at most 10 energy
2
011/01/B/ST5/00849, and by Warsaw University of
evaluations and maximum number of generations of 27 000.
Remaining parameters were set as default. The docking results
were clustered into groups with RMSd lower than 2.0 Å. The
docked ligand conformations were analyzed using ADT and
Chimera 1.8 package (http://www.cgl.ucsf.edu/chimera/) [56].
Technology, Faculty of Chemistry. In addition, we are deeply
indebted to Professor Jan Plenkiewicz for his insightful guidance
and continuous support throughout this work. Adam Wawro
thanks Dr. Takahiro Muraoka and Prof. Kazushi Kinbara from
Tohoku University for their kind support during manuscript
preparation. We would also like to extend our thanks to
undergraduate students: Magdalena Kurowska, Martyna
Poprzeczko and Karolina Wasielak for their skillful technical
assistance during chemical and biological studies.
4
.1.8.3. Assays of CK2α subunit activity and inhibition studies:
The activity of hCK2α was tested using P81 filter isotopic
assay [50]. The reaction mixture contained 20 mM Tris-HCl, pH
.5, 20 mM MgCl , 20 µM DTT, 50 µM CK2 substrate peptide
7
2
Appendix A. Supplementary data
(RRRDDDSDDD), 10 µM ATP (200-300 CPM/pmol) and 200
ng hCK2α. The reaction was initiated with enzyme in a total
volume of 50 µl, incubated at 30 C, and performed for 20 min.
°
Supplementary data related to this article can be found at (…)
1
0 µl of a reaction mixture was spotted onto P81 paper circle.
References
The filter papers were washed 3 x with 0.6 % phosphoric acid
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were determined at 4% DMSO with minimum 7 concentrations
of each tested inhibitor at the range of 0.064-1000 µM and
calculated by fitting the data to sigmoidal dose-response (variable
slope) Y = Bottom + (Top-Bottom) / (1+10^((LogIC50-
X)*HillSlope) equation in GraphPad Prism Software (La Jolla,
CA, USA).
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019-6023.

ACCEPTED MANUSCRIPT
ꢀ
ꢀ
ꢀ
ꢀ
A series of 4,5,6,7-tetrabromo-1H-benzotriazole derivatives were synthesized.
The most potent inhibitor of CK2α was the (R)-enantiomer of 7b (IC 0.80 ꢀM).
The obtained results were rationalized by means of in silico docking experiments.
5
0
Cytotoxicity was assessed in parallel on the human CCRF-CEM and MCF7 cell lines.

ACCEPTED MANUSCRIPT
Supporting Information
for
Synthesis of novel chiral TBBt derivatives with hydroxyl
moiety. Studies on inhibition of human protein kinase
CK2α and cytotoxicity properties.
Paweł Borowiecki,* Adam Wawro, Patrycja Wińska,
Monika Wielechowska, and Maria Bretner
Address: Warsaw University of Technology, Faculty of Chemistry, Noakowskiego St. 3, 00-
6
64 Warsaw, Poland;
Email: Paweł Borowiecki* - pawel_borowiecki@onet.eu
Corresponding author
*
Table of contents
General details ……………………………………………………………..S2-S3
Compound characterization data and representative synthetic procedures S4-S13
Biological Assays ……………………………………………………….S13-S14
Copies of NMR, HRMS and IR spectra …………………………….…..S15-S55
References ……………………………………………………………………S56
S1

General details
ACCEPTED MANUSCRIPT
All commercially available reagents (Aldrich, Fluka and POCH) for chemistry purposes were
used without further purification. Solvents (methylene chloride, acetonitrile, acetone) were
dried by simply allowing them to stand over activated 3Å molecular sieves [20%
mass/volume (m/v) loading of the desiccant] at least for 48 h before use [1].
Dimethyl Sulphoxide (DMSO), Molecular Biology grade used as a solvent for all stocks of
the chemical agents was obtained from Roth, [gamma-P32] ATP (3000 Ci/mmol) was
obtained from Hartmann Analytic GmbH; P81 (2.3 cm) circles were from Whatman. CK2
substrate peptide (RRRDDDSDDD) was purchased from Biaffin GmbH & Co KG. All other
reagents used in this study were of analytical grade. Melting points were obtained with an
MPA100 Optimelt SRS apparatus. Thin-layer chromatography was carried on TLC
aluminium plates with silica gel Kieselgel 60 F254 (Merck) (0.2 mm thickness film) using UV
light as a visualizing agent. Preparative separations were carried out by: (i) column
chromatography using silica gel with grain size 40-63 μm or 15-40 μm, respectively or by (ii)
PLC PSC-Fertigplatten Kieselgel 60 F254 (20 × 20 cm with 2 mm thickness layer) glass plates
1
13
from Merck. H and C NMR spectra were measured with a Varian Mercury 400BB
1
13
spectrometer operating at 400 MHz for H and 100 MHz for C nuclei, chemical shifts (δ) are
given in parts per million (ppm) on the delta scale. The solvent peak was used as reference
value; signal multiplicity assignment: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
the type of signals: br., broad; coupling constant (J) are given in hertz (Hz); All samples were
performed in fully deuterated chloroform (CDCl
3
) or methanol (CD OD), respectively. All of
3
1
13
the H and C NMR spectra were created by ACD/NMR Processor Academic Edition 12.0.
Mass spectrometry was recorded on Micro-mass ESI Q-TOF spectrometer at IBB PAN. IR
spectra of neat samples were recorded on a Perkin Elmer System 2000 FTIR Spectrometer
equipped with a Pike Technologies GladiATR attenuated total reflectance (ATR) accessory
with a monolithic diamond crystal stage and a pressure clamp; FTIR spectra were recorded in
-1
transmittance mode in the 300-4000 cm range, in ambient air at room temperature, with 2
-1
cm resolution and accumulation of 32 scans. Computer molecular dynamics simulations
(
[
(
docking studies) were carried out using AutoDock 4.2 program (http://autodock.scripps.edu/)
2]. Ligand molecules were prepared with ChemAxon MarvinSketch 5.12.3
http://www.chemaxon.com/marvin/) and saved as .PDB files. The crystallographic structures
of CK2α (codes: 1daw, 1j91) were obtained from RCSB Protein Data Bank (PDB,
http://www.rcsb.org/pdb/). Non-protein molecules were removed, polar hydrogen were added
and Gasteiger charges were calculated with AutoDock Tools 1.5.6 package (ADT,
S2

http://mgltools.scripps.edu/) [ 3A] .C DC oEc Pk iTn gE Dw aMs Ap eNr fUo rSm Ce dR I iPn T70 x 70 x 70 units grid box
centered on the enzyme hydrophobic pocket with the grid spacing of 0.325 Å. For each ligand
molecule 100 independent runs were performed, using Lamarckian genetic algorithm, with at
6
most 10 energy evaluations and maximum number of generations of 27 000. Remaining
parameters were set as default. The docking results were clustered into groups with RMSd
lower than 2.0 Å. The docked ligand conformations were analyzed using ADT and Chimera
1
.8 package (http://www.cgl.ucsf.edu/chimera/) [4].
S3

Compound characterization Ad aCt aC aEn Pd Tr Ee pD re Ms e An t Na t Ui v Se Cs yRn It Ph eT tic procedures:
General procedure of preparing 1,3-diols (2a-g) from β-keto ester (1a-g):
NaBH
4
(8 equiv) was added portion-wise to a solution of β-keto ester 1a-g (1.5 g) in MeOH
(
15 mL) at room temperature. After 20 min the heterogeneous white reaction mixture was
heated to reflux until all starting material was consumed (approx. 12 h, TLC). The cooled
mixture was concentrated under reduced pressure and partitioned between distilled water (35
mL) and EtOAc (40 mL). The layers were separated, and the aqueous phase was back-
extracted with EtOAc (3 x 40 mL). The combined extracts were dried (Na
under reduced pressure, and purified by chromatography on silica gel using mixture of
PhCH /AcOEt (1:1, v/v) as an eluent to give the corresponding 1,3-diol 2a-g.
2
SO ), concentrated
4
3
1
-Phenylpropane-1,3-diol (2a):
Colorless oil; yield: 82%; R [PhCH
), 3.26 (br. s., 2H, OH), 3.74-3.84 (m, 2H, CH
1
f
3
/AcOEt (1:1, v/v)] 0.29; H-NMR (CDCl
3
, 400 MHz) δ:
1
3
.84-2.02 (m, 2H, CH
2
2
OH), 4.90 (dd, J=8.81,
1
3
.84 Hz, 1H, CH), 7.24-7.38 (m, 5H, Ph); C-NMR (CDCl
3
, 100 MHz) δ: 40.32 (CH ), 61.12
2
(
CH OH), 73.94 (CH), 125.60 (2C, o-Ph), 127.46 (p-Ph), 128.41 (2C, m-Ph), 144.21 (Ph).
2
1
-(4-Bromophenyl)propane-1,3-diol (2b):
[PhCH
1
Colorless oil; yield: 96%; R
f
3
/AcOEt (1:1, v/v)] 0.24; H NMR (CDCl
3
, 400 MHz) δ:
1
.81-2.00 (m, 2H, CH
.84 Hz, 1H, CH), 7.18-7.25 (m, 2H, o-Ph), 7.43-7.51 (m, 2H, m-Ph); C NMR (CDCl
), 61.25 (CH OH), 73.56 (CH), 121.20 (p-Ph), 127.35 (2C, o-Ph), 131.51
2C, m-Ph), 143.24 (Ph).
2
), 2.94 (br. s., 2H, OH), 3.77-3.88 (m, 2H, CH
2
OH), 4.90 (dd, J=8.36,
1
3
3
3
, 100
MHz) δ: 40.25 (CH
2
2
(
1
-(4-Chlorophenyl)propane-1,3-diol (2c):
[PhCH /AcOEt (1:1, v/v)] 0.21; H-NMR (CDCl
), 2.77 (br. s., 2H, OH), 3.78-3.90 (m, 2H, CH OH), 4.94 (dd, J=8.58,
1
Yellowish oil; yield: 81%; R
f
3
3
, 400 MHz) δ:
1
3
.82-2.02 (m, 2H, CH
2
2
1
3
.61 Hz, 1H, CH), 7.27-7.35 (m, 4H, Ph); C-NMR (CDCl
3
, 100 MHz) δ: 40.41 (CH ), 61.39
2
(
CH OH), 73.64 (CH), 126.92 (2C, o-Ph), 128.50 (2C, m-Ph), 133.04 (p-Ph), 142.64 (Ph).
2
1
-(4-Fluorophenyl)propane-1,3-diol (2d):
[PhCH
1
Colorless oil; yield: 78%; R
f
3
/AcOEt (1:1, v/v)] 0.16; H-NMR (CDCl
3
, 400 MHz) δ:
1
.81-2.01 (m, 2H, CH
2
), 3.20 (s, 2H, OH), 3.74-3.86 (m, 2H, CH
2
OH), 4.90 (dd, J=8.81, 3.57
S4

1
3
Hz, 1H, CH), 6.97-7.07 (m, 2A HC , Cm E- PP hT ) E, D7 .2 M7 - A7 . 3N 4U( Sm C, R2 HI P, Tp -Ph); C-NMR (CDCl
MHz) δ: 40.38 (CH ), 61.20 (CH OH), 73.47 (CH), 115,22 (d, J=20.95 Hz, 2C, m-Ph), 127.23
d, J=7.60 Hz, 2C, o-Ph), 139.97 (d, J=2.85 Hz, 1C, Ph), 162.06 (d, J=245.33 Hz, 1C, p-Ph).
, 100
3
2
2
(
1
-(3-Bromophenyl)propane-1,3-diol (2e):
Yellowish oil; yield: 85%; R [PhCH /AcOEt (1:1, v/v)] 0.24; H-NMR (CDCl
), 2.65 (br. s., 2H, OH), 3.79-3.90 (m, 2H, CH OH), 4.92 (dd, J=8.13,
.07 Hz, 1H, CH), 7.17-7.30 (m, 2H, m-Ph and one of o-Ph), 7.33-7.43 (m, 1H, p-Ph), 7.50-
1
f
3
3
, 400 MHz) δ:
1
4
7
.86-2.01 (m, 2H, CH
2
2
1
3
.54 (m, 1H, o-Ph-situated closer to Br); C-NMR (CDCl
3
, 100 MHz) δ: 40.34 (CH ), 61.37
2
(
CH OH), 73.61 (CH), 122.54 (m-Ph-Br), 124.15 (o-Ph), 128.69 (o-Ph-situated closer to Br),
2
1
29.97 (m-Ph), 130.44 (p-Ph), 146.51 (Ph).
1
-(3-Chlorophenyl)propane-1,3-diol (2f):
1
Yellowish oil; yield: 86%; R
f
[PhCH
3
/AcOEt (1:1, v/v)] 0.20; H-NMR (CDCl
3
, 400 MHz) δ:
OH), 4.92 (dd, J=8.13,
.29 Hz, 1H, CH), 7.18-7.30 (m, 3H, Ph), 7.33-7.38 (m, 1H, o-Ph-situated much further from
1
4
.85-2.01 (m, 2H, CH
2
), 2.99 (br. s., 2H, OH), 3.76-3.90 (m, 2H, CH
2
1
3
Cl); C-NMR (CDCl
3
, 100 MHz) δ: 40.29 (CH
2
), 61.26 (CH OH), 73.54 (CH), 123.67 (o-
2
Ph), 125.75 (o-Ph-situated closer to Cl), 127.48 (p-Ph), 129.66 (m-Ph), 134.25 (m-Ph-Cl),
1
46.22 (Ph).
1
-(3-Methoxyphenyl)propane-1,3-diol (2g):
1
Yellowish oil; yield: 95%; R
.84-2.06 (m, 2H, CH ), 2.92 (br. s., 2H, OH), 3.80 (s, 3H, OCH
CH OH), 4.91 (dd, J=8.58, 3.84 Hz, 1H, CH), 6.78-6.83 (m, 1H, Ph), 6.90-6.95 (m, 2H, Ph),
.22-7.29 (m, 1H, Ph); C-NMR (CDCl
CH OH), 74.07 (CH), 111.05 (o-Ph-situated closer to OCH
29.39 (m-Ph), 145.88 (Ph), 159.55 (m-Ph-OCH ).
f
[PhCH
3
/AcOEt (1:1, v/v)] 0.22; H-NMR (CDCl
3
, 400 MHz) δ:
1
2
3
), 3.81-3.87 (m, 2H,
2
1
3
7
3
, 100 MHz) δ: 40.38 (CH
2
), 55.22 (OCH ), 61.33
3
(
2
3
), 112.85 (p-Ph), 117.82 (o-Ph),
1
3
General procedure of preparing (3a-g) by the monotosylation of the 1,3-diols (2a-g):
To a flame-dried flask, containing the 1,3-diol 2a-g (1 g) dissolved in dry CH
2
Cl (15 mL),
2
freshly distilled triethylamine (1.1 equiv) and 4-(dimethylamino)pyridine (10 mg) were added
at -15 °C under an nitrogen atmosphere. After 15 min, p-toluenesulfonyl chloride (1 equiv)
suspended in anhydrous CH
2
Cl (6 mL) was added dropwise via syringe with stirring over a
2
period of 1 h under fast stream of nitrogen. Upon completion of the addition, the reaction
S5

mixture was maintained at -15 A° CC fCo Er 1P 2T hE aD n dM t hAe nN aUl l So wC eRd I tPo Twarm to room temperature and
stirred for a further 1 h. Next, the content of the flask was cooled to 0 °C, portion of CH Cl
30 mL) was added, and the solution was quenched successively by the careful addition of
cold 1% HCl (2 x 15 mL), washed with saturated sodium bicarbonate (2 x 15 mL), brine (2 x
5 mL), dried over anhydrous MgSO and concentrated in vacuo to yield the crude product.
2
2
(
1
4
The purification by column chromatography on silica gel with mixture of ethyl
acetate/cyclohexane (4:1 or 1:1, v/v – depending on the substance purified) as an eluent
afforded the desired monotosylated alcohol 3a-g.
3
-Hydroxy-3-phenylpropyl 4-methylbenzenesulfonate (3a):
Yellowish oil; yield: 41%; R [AcOEt/Cyclohexene (4:1, v/v)] 0.67; H-NMR (CDCl
MHz) δ: 1.97-2.06 (m, 2H, CH ), 2.10 (br. s., 1H, OH), 2.45 (s, 3H, CH ), 4.00-4.10 (m, 1H,
CH OSO ), 4.22-4.33 (m, 1H, CH OSO ), 4.76-4.83 (m, 1H, CH), 7.24-7.38 (m, 7H, Ph),
.76-7.82 (m, 2H, Ph); C-NMR (CDCl , 100 MHz) δ: 21.66 (CH ), 38.03 (CH ), 67.57
CH OSO ), 70.15 (CH), 125.54 (2C, o-Ph), 127.75 (p-Ph), 127.82 (2C, o-PhTos), 128.49
2C, m-PhTos), 129.79 (2C, m-Ph), 132.79 (p-PhTos), 143.36 (Ph), 144.74 (PhTos).
1
f
3
, 400
2
3
2
2
2
2
1
3
7
3
3
2
(
(
2
2
3
-(4-Bromophenyl)-3-hydroxypropyl 4-methylbenzenesulfonate (3b):
1
Colorless oil; yield: 61%; R
f
[AcOEt/Cyclohexene (1:1, v/v)] 0.76; H-NMR (CDCl
3
, 400
), 3.96-
), 4.79 (t, J=6.66 Hz, 1H,
CH), 7.11-7.17 (m, 2H, Ph), 7.32-7.38 (m, 2H, Ph), 7.39-7.45 (m, 2H, Ph), 7.74-7.80 (m, 2H,
MHz) δ: 1.98 (td, J=6.55, 5.42 Hz, 2H, CH
2
), 2.23 (br. s., 1H, OH), 2.46 (s, 3H, CH
3
4
.07 (m, 1H, CH OSO ), 4.27 (dt, J=9.99, 6.63 Hz, 1H, CH
2
2
2
OSO
2
1
3
Ph); C-NMR (CDCl
3
, 100 MHz) δ: 21.65 (CH
CH), 121.52 (p-Ph), 127.34 (2C, o-Ph), 127.85 (2C, o-PhTos), 129.87 (2C, m-PhTos), 131.61
2C, m-Ph), 132.68 (p-PhTos), 142.43 (Ph), 144.93 (PhTos).
3
), 37.98 (CH
2
), 67.27 (CH
2
OSO ), 69.49
2
(
(
3
-(4-Chlorophenyl)-3-hydroxypropyl 4-methylbenzenesulfonate (3c):
1
Yellowish oil; yield: 33%; R
f
[AcOEt/Cyclohexene (4:1, v/v)] 0.73; H-NMR (CDCl
3
, 400
), 3.98-4.05 (m, 1H,
), 4.79 (t, J=6.66 Hz, 1H, CH), 7.17-7.22 (m, 2H,
MHz) δ: 1.94-2.03 (m, 2H, CH
CH OSO ), 4.22-4.30 (m, 1H, CH
Ph), 7.23-7.29 (m, 2H, Ph), 7.31-7.38 (m, 2H, Ph), 7.73-7.79 (m, 2H, Ph); C-NMR (CDCl
2
), 2.33 (br. s., 1H, OH), 2.45 (s, 3H, CH
3
2
2
2
OSO
2
1
3
3
,
1
1
00 MHz) δ: 21.64 (CH
3
), 38.00 (CH
2
), 67.33 (CH
2
OSO
2
), 69.43 (CH), 127.00 (2C, o-Ph),
27.85 (2C, m-Ph), 128.63 (2C, o-PhTos), 129.87 (2C, m-PhTos), 132.65 (p-PhTos), 133.36
(
p-Ph), 141.94 (Ph), 144.93 (PhTos).
S6

3
-(4-Fluorophenyl)-3-hydrox Ay pCr Co pEy Pl T4 - Em De t hM y Al b Ne n Uz eSn Ce sRu Il Pf o Tn ate (3d):
1
Colorless oil; yield: 66%; R
MHz) δ: 1.94-2.06 (m, 2H, CH
CH OSO ), 4.22-4.31 (m, 1H, CH
H, m-Ph), 7.20-7.27 (m, 2H, o-PhTos), 7.33-7.36 (m, 2H, m-PhTos), 7.74-7.80 (m, 2H, o-
f
[AcOEt/Cyclohexene (1:1, v/v)] 0.71; H-NMR (CDCl
3
, 400
), 3.98-4.06 (m, 1H,
), 4.79 (dd, J=7.86, 5.71 Hz, 1H, CH), 6.95-7.03 (m,
2
), 2.22 (br. s., 1H, OH), 2.45 (s, 3H, CH
3
2
2
2
OSO
2
2
1
3
Ph); C-NMR (CDCl
CH), 115.34 (d, J=21.23 Hz, 2C, m-Ph), 127.28 (d, J=7.60 Hz, 2C, o-Ph), 127.84 (2C, o-
PhTos), 129.84 (2C, m-PhTos), 132.74 (p-PhTos), 139.19 (d, J=3.80 Hz, 1C, Ph), 144.89
PhTos), 162.17 (d, J=246.26 Hz, 1C, p-Ph).
3
, 100 MHz) δ: 21.60 (CH
3
), 38.08 (CH
2
), 67.41 (CH
2
OSO ), 69.49
2
(
(
3
-(3-Bromophenyl)-3-hydroxypropyl 4-methylbenzenesulfonate (3e):
1
Yellowish oil; yield: 82%; R
f
[AcOEt/Cyclohexene (4:1, v/v)] 0.67; H-NMR (CDCl
3
, 400
), 4.06 (dt, J=10.22,
OSO ), 4.79 (dd, J=8.36,
.97 Hz, 1H, CH), 7.14-7.22 (m, 2H, Ph), 7.31-7.47 (m, 5H, Ph), 7.76-7.83 (m, 2H, Ph); C-
, 100 MHz) δ: 21.68 (CH ), 38.06 (CH ), 67.24 (CH OSO ), 69.43 (CH), 122.68
m-Ph-Br), 124.24 (o-Ph), 127.87 (2C, o-PhTos), 128.69 (o-Ph-situated closer to Br), 129.90
MHz) δ: 1.97-2.01 (m, 2H, CH
2
), 2.06 (br. s., 1H, OH), 2.46 (s, 3H, CH
3
5
4
.17 Hz, 1H, CH
2
OSO
2
), 4.30 (ddd, J=10.11, 8.07, 5.31 Hz, 1H, CH
2
2
1
3
NMR (CDCl
3
3
2
2
2
(
(
(
2C, m-PhTos), 130.14 (m-Ph), 130.82 (p-Ph), 132.73 (p-PhTos), 144.94 (Ph), 145.83
PhTos).
3
-(3-Chlorophenyl)-3-hydroxypropyl 4-methylbenzenesulfonate (3f):
1
Yellowish oil; yield: 40%; R
f
[AcOEt/Cyclohexene (4:1, v/v)] 0.71; H-NMR (CDCl
3
, 400
MHz) δ: 1.98 (dtd, J=7.99, 5.21, 5.21, 3.05 Hz, 2H, CH
2
), 2.27 (br. s., 1H, OH), 2.45 (s, 3H,
CH
3
), 3.99-4.08 (m, 1H, CH
2
OSO
2
), 4.29 (ddd, J=10.05, 7.79, 5.42 Hz, 1H, CH OSO ), 4.78
2
2
(
dd, J=8.02, 5.31 Hz, 1H, CH), 7.11-7.17 (m, 1H, Ph), 7.20-7.28 (m, 3H, Ph), 7.33-7.37 (m,
1
3
2
6
H, Ph), 7.75-7.81 (m, 2H, Ph); C-NMR (CDCl
3
, 100 MHz) δ: 21.65 (CH
3
), 38.00 (CH ),
2
7.27 (CH
2
OSO ), 69.44 (CH), 123.75 (o-Ph), 125.75 (o-Ph-situated closer to Cl), 127.84
2
(
3C, p-Ph and o-PhTos), 129.82 (m-Ph), 129.90 (2C, m-PhTos), 132.66 (p-PhTos), 134.39 (m-
Ph-Cl), 144.94 (Ph), 145.58 (PhTos).
3
-(3-Methoxyphenyl)-3-hydroxypropyl 4-methylbenzenesulfonate (3g):
1
Yellowish oil; yield: 39%; R
MHz) δ: 1.96-2.04 (m, 2H, CH
OCH ), 4.01-4.11 (m, 1H, CH OSO
f
[AcOEt/Cyclohexene (4:1, v/v)] 0.64; H-NMR (CDCl
3
, 400
), 3.79 (s, 3H,
OSO ), 4.78 (t,
2
), 2.05 (br. s., 1H, OH), 2.45 (s, 3H, CH
3
3
2
2
), 4.27 (dt, J=9.94, 6.66 Hz, 1H, CH
2
2
J=6.66 Hz, 1H, CH), 6.76-6.88 (m, 3H, Ph), 7.17-7.27 (m, 1H, Ph), 7.29-7.39 (m, 2 H, Ph),
S7

1
3
7
.79-7.81 (m, 2H, Ph); C-N AM CR C (EC PD TC El
OCH ), 67.55 (CH OSO ), 70.06 (CH), 110.99 (o-Ph-situated closer to OCH
Ph), 117.81 (o-Ph), 127.87 (2C, m-PhTos), 129.61 (m-Ph), 129.85 (2C, o-PhTos), 132.78 (p-
PhTos), 144.82 (Ph), 145.15 (PhTos), 159.73 (OCH ).
3
D, 1M0 0A MN HU zS) Cδ R: I2 P1 T. 64 (CH
3
), 37.97 (CH
2
), 55.20
(
3
2
2
3
), 113.30 (p-
3
Preparation of 4,5,6,7-tetrabromo-1H-benzotriazole (5):
H-Benzotriazole 4 (6 g, 50.4 mmol) was dissolved in a mixture of 69% HNO
fuming 100% HNO (10 mL). Next, the solution was heated to a temperature of 80 °C and Br
48.3 g, 302 mmol, 15 mL) was added drop by drop within 1 h. The reaction mixture was
1
3
(150 mL) and
3
2
(
stirred vigorously by using mechanical stirrer at 60 °C for 48 h and irradiated by exposure to
UV light. Subsequently, content of the flask was cooled to room temperature, the excess of
unreacted bromine was removed in the gentle flow of nitrogen and trapped into a 20 %
solution of sodium pyrosulfite (150 mL). After this, the suspension was poured into mixture
of ice-cold distilled H
2
O (300 mL) and saturated Na
2
S
2
5
O (20 mL). The resulting yellowish
precipitate was filtered off, and washed with H
2
O (100 mL) and EtOH (100 mL), respectively.
The obtained crude product was refluxed several times in the mixture of MeOH (50 mL) and
i-PrOH (25 mL), and hot saturated solution was filtered off yielding white crystals 5 (13 g,
2
9.9 mmol, 59%). The purity of the desired compound 5 was confirmed by high-resolution
1
13
mass spectrometry (HRMS). The rest of the analyses made (vide H-, C-NMR, m.p.) were
consistent with the literature data [5]. White solid; m.p.: 264-266 °C; yield: 40%; R
f
+
+
+
[
CHCl
3
/MeOH (99:1, v/v)] 0.16; HRMS (ESI , m/z): [M+H] calcd = 435.6941, [M+H] found
=
4
35.6629.
General procedure for the introduction of the Tbbt moiety [synthesis of (6a-g)]:
To a solution of TBBt 5 (1 equiv) in the mixture of absolute acetone (15 mL) and dry
acetonitrile (10 mL) anhydrous K CO (3 equiv) was added. The content of the flask was
2
3
flushed with nitrogen for several times, and stirred for 3 h at reflux. Next, the reaction mixture
was cooled to room temperature and the solution of appropriate 3-hydroxy-3-arylpropyl-4-
methylbenzenesulfonate 3a-g (400 mg) in acetonitrile (5 mL) was added drop by drop by
using syringe over 1 h. After stirring the mixture at reflux for 48 h, the crude reaction was
cooled to room temperature, and both the white precipitate as well as the red colored solution
were transferred into separator and washed with saturated solution of potassium carbonate
(
100 mL). Water phase was extracted with CHCl (2 x 150 mL) and AcOEt (3 x 100 mL), and
3
the collected organic layers were partially condensed on rotator evaporator, and dried over dry
S8

Na
with unmodified silica matrices, using mixture of CHCl
CHCl /acetone (95:5, v/v), depending on substance purified. The appropriate fraction was
removed from the glass plate with SiO , placed in the round-bottomed flask, suspended in the
mixture of CHCl /acetone (150 mL, 2:1, v/v), and stirred for 1 h at room temperature. Next,
the silica gel was filtered on a sintered glass funnel, and washed with CHCl /acetone (2 x 50
2
SO
4
. The purification proc Ae d Cu rCe Ew Pa Ts Ep eDr f oMr mA e Nd Uo nS Cp rRe pI aP r Ta tive layer plates (PLC) coated
3
/Et O (7:3 or 6:4, v/v) or
2
3
2
3
3
mL, 1:1, v/v). After partial solvent removal in vacuo, solution of the filtrate (approx. 1.5 mL)
was kept in the refrigerator until white precipitate occurred. Next, the liquid phase was
decanted off by Pasteur pipette tipped with cotton wool, and the remaining solid was washed
with portions of ice-cold acetone (1 mL), and then ethanol (1 mL) respectively to obtained
desired product 6a-g as a white solid, which was further high-vacuum-dried in the desiccator
over phosphorus pentoxide (P
2
5
O ) overnight.
1
-Phenyl-3-(4,5,6,7-tetrabromo-2H-benzotriazol-2-yl)propan-1-ol (6a):
White solid; m.p.: 135-136 °C; yield: 40%; R [CHCl /Et O (7:3, v/v)] 0.67; H-NMR
CDCl , 400 MHz) δ: 2.31 (br. s., 1H, OH), 2.48-2.62 (m, 2H, CH ), 4.82 (dd, J=7.45, 5.65
Hz, 1H, CH), 4.85-5.02 (m, 2H, CH N), 7.23-7.40 (m, 5H, Ph); C-NMR (CDCl , 100 MHz)
δ: 38.61 (CH ), 54.42 (CH N), 71.40 (CH), 113.69 (2C, TBBt-internal), 125.61 (2C, TBBt-
external), 126.32 (2C, o-Ph), 127.98 (2C, m-Ph), 128.62 (p-Ph), 143.01 (Ph), 143.08
1
f
3
2
(
3
2
1
3
2
3
2
2
+
+
+
-
(
[
TBBt=N); HRMS (ESI , m/z): [M+H] calcd = 569.7673, [M+H] found = 569.8090; (ESI , m/z):
-
-
M+H] calcd = 567.7516, [M+H] found = 567.7919; FTIR νmax(neat): 3549.27, 3416.85, 1533.15,
1
6
489.52, 1429.48, 1271.50, 1177.95, 1162.39, 1086.05, 1056.91, 974.64, 765.01, 747.77,
97.57, 606.17, 515.80.
1
-(4-Bromophenyl)-3-(4,5,6,7-tetrabromo-2H-benzotriazol-2-yl)propan-1-ol (6b):
1
White solid; m.p.: 125-126 °C; yield: 33%; R
CDCl , 400 MHz) δ: 1.97 (br. s., 1H, OH), 2.44-2.61 (m, 2H, CH
Hz, 1H, CH), 4.83-5.02 (m, 2H, CH N), 7.14-7.25 (m, 2H, o-Ph), 7.38-7.47 (m, 2H, m-Ph);
, 100 MHz) δ: 38.40 (CH ), 54.15 (CH N), 70.79 (CH), 113.68 (TBBt-
internal), 121.70 (p-Ph), 126.52 (2C, TBBt-external), 127.35 (2C, o-Ph), 131.60 (2C, m-Ph),
f
[CHCl
3
/acetone (95:5, v/v)] 0.55; H-NMR
(
3
2
), 4.78 (dd, J=7.79, 5.08
2
1
3
C-NMR (CDCl
3
2
2
+
+
+
1
6
3
1
5
41.87 (Ph), 143.08 (TBBt=N); HRMS (ESI , m/z): [M+H] calcd = 649.6758, [M+H] found =
-
-
-
49.7219; (ESI , m/z): [M+H] calcd = 647.6601, [M+H] found = 647.7287; FTIR νmax(neat):
417.18, 1588.37, 1533.29, 1485.73, 1425.84, 1308.92, 1295.42, 1274.19, 1175.75, 1159.37,
073.38, 1011.10, 979.00, 926.17, 876.54, 822.85, 771.60, 750.05, 718.53, 608.83, 543.95,
08.55.
S9

1
-(4-Chlorophenyl)-3-(4,5,6,7 A- t Ce t Cr aEb Pr oT mE oD- 2 MH -Ab eN n Uz oSt Cr i aR z IoPl -T2 -yl)propan-1-ol (6c):
1
White solid; m.p.: 112-114 °C; yield: 41%; R
CDCl , 400 MHz) δ: 2.42-2.58 (m, 2H, CH
Hz, 1H, CH), 4.91 (dt, J=19.14, 6.92 Hz, 2H, CH
CDCl , 100 MHz) δ: 38.46 (CH ), 54.14 (CH N), 70.57 (CH), 113.57 (2C, TBBt-internal),
26.42 (2C, TBBt-external), 126.96 (2C, m-Ph), 128.60 (2C, o-Ph), 133.51 (p-Ph), 141.39
f
[CHCl
3
/Et
2
O (6:4, v/v)] 0.51; H-NMR
(
3
2
), 2.71 (br. s., 1H, OH), 4.79 (dd, J=8.13, 4.97
1
3
2
N), 7.23-7.27 (m, 4H, Ph); C-NMR
(
3
2
2
1
+
+
+
(
(
Ph), 142.94 (Tbbt=N); HRMS (ESI , m/z): [M+H] calcd = 603.7283, [M+H] found = 603.7826;
-
-
-
ESI , m/z): [M+H] calcd = 601.7127, [M+H] found = 601.7653; FTIR νmax(neat): 3387.41,
1
1
739.34, 1700.79, 1489.54, 1425.63, 1365.48, 1270.44, 1229.73, 1217.47, 1173.57, 1089.40,
074.43, 1012.15, 977.71, 832.05, 770.11, 749.58, 607.20, 529.61.
1
-(4-Fluorophenyl)-3-(4,5,6,7-tetrabromo-2H-benzotriazol-2-yl)propan-1-ol (6d):
1
White solid; m.p.: 73-75 °C; yield: 33%; R
CDCl , 400 MHz) δ: 2.10 (br. s., 1H, OH), 2.47-2.57 (m, 2H, CH
Hz, 1H, CH), 4.84-5.02 (m, 2H, CH N), 6.98-7.06 (m, 2H, m-Ph), 7.29-7.37 (m, 2H, o-Ph);
, 100 MHz) δ: 38.70 (CH ), 54.33 (CH N), 70.72 (CH), 113.67 (2C, TBBt-
f
[CHCl
3
/acetone (95:5, v/v)] 0.44; H-NMR
(
3
2
), 4.80 (dd, J=7.38, 5.71
2
1
3
C-NMR (CDCl
3
2
2
internal), 115.47 (d, J=21.86 Hz, 2C, m-Ph), 126.43 (2C, TBBt-external), 127.33 (d, J=7.60
Hz, 2C, o-Ph), 138.82 (d, J=2.85 Hz, 1C, Ph), 143.09 (TBBt=N), 162.29 (d, J=246.43 Hz, 1C,
+
+
+
-
p-Ph); HRMS (ESI , m/z): [M+H] calcd = 587.7579, [M+H] found = 587.8105; (ESI , m/z):
-
-
[
M+H] calcd = 585.7422, [M+H] found = 585.7770; FTIR νmax(neat): 3400.54, 1603.65, 1530.65,
1
1
507.41, 1425.71, 1394.99, 1344.03, 1297.48, 1270.62, 1216.71, 1174.22, 1156.57, 1100.42,
073.21, 1013.16, 976.66, 928.84, 882.96, 835.06, 770.74, 745.98, 607.49, 566.47, 544.29.
1
-(3-Bromophenyl)-3-(4,5,6,7-tetrabromo-2H-benzotriazol-2-yl)propan-1-ol (6e):
1
White solid; m.p.: 88-91 °C; yield: 36%; R
CDCl , 400 MHz) δ: 2.22 (br. s., 1H, OH), 2.44-2.63 (m, 2H, CH
Hz, 1H, CH), 4.84-5.02 (m, 2H, CH N), 7.16-7.22 (m, 1H, Ph), 7.23-7.29 (m, 1H, Ph), 7.35-
.40 (m, 1H, Ph), 7.48-7.51 (m, 1H, Ph); C-NMR (CDCl
CH N), 70.56 (CH), 113.64 (2C, TBBt-internal), 122.68 (m-Ph-Br), 124.20 (2C, TBBt-
external), 126.44 (o-Ph), 128.70 (m-Ph), 130.15 (o-Ph-situated closer to Br), 130.90 (p-Ph),
f
[CHCl
3
/acetone (95:5, v/v)] 0.55; H-NMR
(
3
2
), 4.79 (dd, J=8.24, 4.63
2
1
3
7
3
, 100 MHz) δ: 38.48 (CH ), 54.12
2
(
2
+
+
+
1
6
43.01 (TBBt=N), 145.33 (Ph); HRMS (ESI , m/z): [M+H] calcd = 647.6778, [M+H] found =
-
-
-
47.7335; (ESI , m/z): [M+H] calcd = 645.6622, [M+H] found = not determined; FTIR
max(neat): 3396.57, 1740.41, 1591.39, 1573.26, 1533.54, 1477.79, 1426.25, 1341.93,
ν
1
9
304.67, 1274.68, 1240.74, 1145.62, 1172.03, 1100.35, 1069.74, 1035.20, 1014.10, 1004.29,
77.15, 877.76, 800.31, 788.28, 753.38, 692.91, 666.99, 608.00, 585.16, 511.23, 442.87.
S10

1
-(3-Chlorophenyl)-3-(4,5,6,7 A- t Ce t Cr aEb Pr oT mE oD- 2 MH -Ab eN n Uz oSt Cr i aR z IoPl -T2 -yl)propan-1-ol (6f):
1
White solid; m.p.: 114-115 °C; yield: 42%; R
f
[CHCl
3
/acetone (95:5, v/v)]; H-NMR (CDCl
3
,
4
00 MHz) δ: 1.68 (br. s., 1H, OH), 2.44-2.64 (m, 2H, CH
2
), 4.80 (dd, J=8.33, 4.52 Hz, 1H,
1
3
CH), 4.83-5.05 (m, 2H, CH
2
N), 7.16-7.29 (m, 3H, Ph), 7.34 (t, J=1.79 Hz, 1H, Ph); C-NMR
), 54.15 (CH N), 70.68 (CH), 113.67 (2C,TBBt-internal),
(
CDCl
3
, 100 MHz) δ: 38.49 (CH
2
2
1
23.73 (2C, TBBt-external), 125.81 (o-Ph), 126.44 (o-Ph-situated closer to Cl), 127.99 (m-
+
Ph), 129.87 (p-Ph), 134.53 (m-Ph-Cl), 143.06 (Ph), 145.09 (TBBt=N); HRMS (ESI , m/z):
+
+
[
M+H] calcd = 603.7283, [M+H] found = 603.7560; FTIR νmax(neat): 3429.45, 1596.23,
1
1
7
573.06, 1534.71, 1476.43, 1444.67, 1428.30, 1370.38, 1334.89, 1276.90, 1201.11, 1172.60,
146.23, 1099.14, 1072.06, 1051.12, 1040.93, 1009.52, 976.79, 880.73, 867.62, 798.02,
91.15, 776.73, 766.58, 718.30, 700.06, 687.80, 608.63, 500.13, 455.35.
1
-(3-Methoxyphenyl)-3-(4,5,6,7-tetrabromo-2H-benzotriazol-2-yl)propan-1-ol (6g):
1
White solid; m.p.: 79-80 °C; yield: 33%; R
f
[CHCl
, 400 MHz) δ: 2.00 (br. s., 1H, OH), 2.50-2.61 (m, 2H, CH
dd, J=7.38, 5.47 Hz, 1H, CH), 4.93 (td, J=13.69, 6.43 Hz, 2H, CH
3
/acetone (95:5, v/v)] 0.42; H-NMR
(
CDCl
3
2
), 3.80 (s, 3H, OCH
N), 6.77-6.80 (m, 1H, Ph),
, 100 MHz) δ: 38.51 (CH ),
), 71.34 (CH), 111.14 (p-Ph), 113.30 (o-Ph-situated closer to
), 113.70 (o-Ph), 117.81 (TBBt-internal), 126.31 (TBBt-external), 129.69 (m-Ph),
3
), 4.79
(
2
1
3
6
5
.89-6.94 (m, 2H, Ph), 7.21-7.26 (m, 1H, Ph); C-NMR (CDCl
3
2
4.37 (CH N), 55.24 (OCH
2
3
OCH
3
+
+
1
5
43.08 (TBBt=N), 144.66 (Ph), 159.77 (m-Ph-OCH ); HRMS (ESI , m/z): [M+H] calcd =
3
+
-
-
-
99.7779, [M+H] found = 599.8348; (ESI , m/z): [M+H] calcd = 597.7622, [M+H] found = not
determined; FTIR νmax(neat): 3528.67, 3446.53, 2932.07, 2830.51, 1735.04, 1604.03,
1
1
8
579.07, 1533.16, 1482.10, 1429.36, 1376.82, 1305.88, 1284.04, 1272.96, 1256.28, 1235.27,
176.17, 1166.31, 1145.49, 1095.20, 1062.25, 1040.59, 995.10, 977.14, 933.18, 913.19,
86.37, 833.89, 785.01, 770.28, 739.31, 700.25, 604.77, 566.11, 525.32, 474.47.
Preparation of (2,2-Dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate (11):
Procedure was adopted from literature [6]. Tosyl chloride (2.07 g, 10.89 mmol) was added in
small portions (during 1 h) to a solution of 2,2-dimethyl-1,3-dioxolane-4-methanol 10 (1.20 g,
9
.08 mmol), triethylamine (1.38 g, 13.62 mmol, 1.90 mL), and DMAP (112 mg, 0.9 mmol) in
dry dichloromethane (20 mL), with magnetic stirring at 0-5 °C. After 5 h, Et O (15 mL) was
added, and the solution was washed with 10% water solution of HCl (2 x 15 mL), saturated
aqueous NaHCO (20 mL), dried over anhydrous MgSO , and concentrated in vacuo. The
crude product was chromatographed over silica gel using mixture of hexane/ethyl acetate
2
3
4
(
5:1, v/v) as an eluent, obtaining the product 11 as an white solid (2.1 g, 7.33 mmol, 81%)
S11

with >99% purity according to AG CC C. WE Ph Ti t eE sDo l Mi d; Am N. pU. : S5 C0 -R5 2I P° TC [Lit [7]: m.p.: 49-50 °C after
recrystallization from petroleum ether/diethyl ether (10:1, v/v)]; yield: 81%; R
f
[hexane/ethyl
),
), 3.92-4.06 (m, 3H, CH and
), 4.21-4.31 (m, 1H, partially CH OSO ), 7.34 (d, J=8.13
, 100 MHz) δ: 21.55 (p-CH ),
OSO ), 72.78 (CH), 109.91 (C),
27.85 (2C, m-Tos), 129.80 (2C, o-Tos), 132.47 (p-Tos), 144.95 (PhOSO
1
acetate (5:1, v/v)] 0.36; H-NMR (CDCl
3
, 400 MHz) δ: 1.31 (d, J=11.52 Hz, 6H, 2xCH
3
2
.43 (s, 3H, p-CH
partially CH and partially CH
Hz, 2H, Ph), 7.78 (d, J=8.13 Hz, 2H, Ph); C-NMR (CDCl
3
), 3.74 (dd, J=8.81, 5.19 Hz, 1H, partially CH
2
2
2
OSO
2
2
2
1
3
3
3
2
1
5.03 (CH
3
), 26.52 (CH
3
), 66.03 (CHCH
2
O), 69.40 (CH
2
2
+
2
); HRMS (ESI ,
+
+
m/z): [M+H] calcd = 287.0953, [M+H] found = 287.0884; GC [100-260 (10 °C/min)]: t = 14.82
R
min.
Preparation
of
4,5,6,7-tetrabromo-2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]-2H-
benzotriazole (12):
This compound was prepared by using the procedure as described in synthesis detail for
compounds 6a-g. Briefly, equimolar amounts of both reagents: 11 (400 mg, 1.4 mmol), and 5
(
610 mg, 1.4 mmol) in the presence of 3-fold molar excess of K
were reacted]. Crude product 12 was partially purified by simple filtration on short silica pad
by eluting with mixture of CHCl /acetone (7:3, v/v) just to remove unreacted 5. As the high
2
CO
3
(582 mg, 4.21 mmol)
3
resolution mass spectrometry analysis confirmed presence of desired product 12, and absence
of compound 5 it was allowed to use in the next step without further purification. HRMS
+
+
+
(
ESI , m/z): [M+H] calcd = 549.7622, [M+H] found = 549.7753.
Preparation of 3-(4,5,6,7-tetrabromo-2H-benzotriazol-2-yl)propane-1,2-diol (13):
Procedure was adopted from literature [8]. Compound 12 (165 mg, 0.3 mmol) was dissolved
in MeOH (3 mL). Then, 0.5 N HCl (0.3 mL) was added dropwise, and the resulting mixture
was heated to reflux. After 4 h acetone and methanol were slowly distilled off. Additional
portion of MeOH (1 mL) and 0.5 N HCl (0.2 mL) was added, and the mixture was kept at
room temperature until ketal hydrolysis was completed (approx. 2 h, TLC). The mixture was
diluted with careful addition of saturated NaHCO
phase was extracted with EtOAc (3 x 10 mL). The organic extracts were combined, washed
with brine (5 mL), dried over anhydrous Na SO , filtered, concentrated under reduced
pressure, and purified by preparative chromatography (PLC) using mixture of CHCl /acetone
9:1, v/v) as an eluent, affording target compound 13 as hygroscopic white solid (95 mg, 0.19
3
until effervescence ceased, and the water
2
4
3
(
mmol, 62%). White semi-solid; yield: 30% (after two steps: from 11 to 13); R
f
1
[
CHCl
3
/acetone (9:1, v/v)] 0.78; H-NMR (MeOD, 400 MHz) δ: 3.59-3.74 (m, 2H, CH
2
OH),
S12

1
3
4
d
1
.16-4.42 (m, 1H, CHOH) 4.7 6A (Cs ,C1 EH P, TC EH D
2
N M) , 4A. 9N3 -U5 .S2 C1 R( mI P, 1T H, CH
2
N); C-NMR (DMSO-
6
, 100 MHz) δ: 53.23 (CH N), 63.53 (CH
2
2
OH), 71.02 (CHOH), 115.72 (TBBt-internal),
+
+
25.40 (TBBt-external), 142.60 (TBBt=N); HRMS (ESI , m/z): [M+H] calcd = 509.7309,
+
[
M+H] found = 509.7447.
Biological Assays
Cloning, expression and purification of hCK2α:
The coding region of human CK2α was amplified by polymerase chain reaction using the
following primers: 5’-CGCGGATCCGTCGGGACCCGTGCCAA (upstream primer) and 5’-
CCCAAGCTTCTGCTGAGCGCCAGCGGCA (downstream primer) and I.M.A.G.E. clone
as a template. The product was cloned into the vector pETDuet-1 (MCS1) using the restriction
sites BamHI and HindIII and the bacterial strain DH5α. The sequence of the obtained clone
was confirmed. Expression of the resulting N-terminal histidine-tagged hCK2α was done in
the bacterial strain BL21(DE3)pLysS growing in superbroth medium after induction with 0.5
°
mM IPTG for 20 h at 20 C. The cell pellet was resuspended in extraction buffer [composed
of: 20 mM NaH
2
PO (pH 8.0), 500 mM NaCl, 10 mM imidazole, O-complete inhibitor
4
cocktail (Roche), lysozyme (1 mg/mL)] and sonicated. The supernatant of the pellet from 200
mL of bacterial culture was loaded onto Ni-NTA agarose (Qiagene) column (2 mL bed
volume). hCK2α was eluted with 300 mM imidazole. Fractions containing His-tagged hCK2α
were dialyzed against 20 mM Tris-HCl (pH 8.5), 500 mM NaCl, 1 mM DTT, 0-20% glycerol
°
and stored at -20 C. The protein concentration in final solution was 12.68 mg/mL
(
determined by Bradford method and bovine serum albumin as a standard) [9].
Assays of CK2 alpha subunit activity and inhibition studies:
The activity of hCK2α was tested using P81 filter isotopic assay [10]. The reaction mixture
contained 20 mM Tris-HCl, pH 7.5, 20 mM MgCl , 50 µM DTT, 20 µM hCK2 substrate
2
peptide (RRRDDDSDDD), 10 µM ATP (200-300 CPM/pmol) and 200 ng hCK2α. The
°
reaction was initiated with enzyme in a total volume of 50 µl, incubated at 30 C, and
performed for 20 min. 10 µl of a reaction mixture was spotted onto P81 paper circle. The
filter papers were washed 3 x with 0.6 % phosphoric acid and once with 95% ethanol before
counting in a scintillation counter (Canberra-Packard). IC50 values for studied compounds
were determined at 4% DMSO with minimum 7 concentrations of each tested inhibitor at the
range of 0.064-1000 µM and calculated by fitting the data to sigmoidal dose-response
S13

(
variable slope) Y = Bottom +A (CT Co pE - PB To tEt oDm )M / A( 1N+ U1 0 S^ C( ( LR oI gP ITC 50-X)*HillSlope) equation in
GraphPad Prism.
Cell culture and treatment:
MCF7 adherent cells (human breast cancer cell line) were cultured in DMEM advanced
medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 2 mM
L-glutamine, antibiotics (100 U/ml penicillin, 100 µg/mL streptomycin) and 10 µg/mL of
human recombinant insulin. CCRF-CEM suspension cells (human peripheral blood T
lymphoblast cell line) were cultured in RPMI medium (Sigma-Aldrich) supplemented with
1
0% fetal bovine serum and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin). Cells
2
were grown in 25 cm cell culture flasks (Sarstedt), in a humidified atmosphere of CO
2
/air
(
5/95%) at 37 °C.
MTT-based cytotoxicity assay:
Before the treatment MCF7 cells were trypsinized in 0.25% trypsin-EDTA solution (Sigma-
4
Aldrich) and seeded into 96-well microplates at a density of 1.5-3 x 10 cells/well. Cells were
treated with specific compounds dissolved in DMSO or DMSO (0.5%) at the corresponding
concentrations 18 h after plating (at 70% of confluency). CCRF-CEM were seeded at 2-3 x
4
1
0 cells/well and treated with compounds. MTT stock solution (Sigma-Aldrich) was added to
each well to a final concentration of 0.5 mg/mL. After 4 h of incubation at 37 °C water-
insoluble dark blue formazan crystals were dissolved in DMSO (200 µL) (37 °C/10 min
incubation), and Sorensen’s glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5) was added
(
25 μL per well). Optical densities were measured at 570 nm using Synergy H4 BioTek
microplate reader. All measurements were carried out in triplicate and the results are
expressed in percentage of cell viability relative to control (cells without inhibitor in 0.5%
DMSO). At such conditions, IC50 for standard anti-cancer inhibitor - doxorubicin was 1.4 µM
for both MCF7 and CCRF-CEM lines.
S14