Synthesis of new analogs of benzotriazole, benzimidazole and phthalimide-potential inhibitors of human protein kinase CK2

Article abstract of DOI:10.1016/j.bmc.2008.12.071

New derivatives of 4,5,6,7-tetrabromo-1H-1,2,3-benzotriazole (TBBt), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi), and N-substituted tetrabromophthalimides were synthesized and their effect on the activity of human protein kinase CK2 was examined. The most active were derivatives with N-hydroxypropyl substituents (IC₅₀ in 0.32-0.54 μM range) whereas derivatives of phthalimide were almost ineffective.

Full text of DOI:10.1016/j.bmc.2008.12.071

Bioorganic & Medicinal Chemistry 17 (2009) 1573–1578
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of new analogs of benzotriazole, benzimidazole and
phthalimide—potential inhibitors of human protein kinase CK2
a
Andzelika Najda-Bernatowicz a, Maja Łebska a, Andrzej Orzeszko b,c, Katarzyna Kopanska ,
´
a
a
a,d,
*
_
´
´
Ewa Krzywinska , Grazyna Muszynska , Maria Bretner
a Institute of Biochemistry and Biophysics, Pawin´skiego 5A, 02-106 Warsaw, Poland
b Warsaw University of Life Sciences, Institute of Chemistry, ul. Nowoursynowska 159C, 02-787 Warsaw, Poland
c Military University of Technology, Kaliskiego 2, 00-908 Warsaw, Poland
d Warsaw University of Technology, Faculty of Chemistry, Noakowskiego 3, 00-664 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
New derivatives of 4,5,6,7-tetrabromo-1H-1,2,3-benzotriazole (TBBt), 4,5,6,7-tetrabromo-1H-benzimid-
azole (TBBi), and N-substituted tetrabromophthalimides were synthesized and their effect on the activity
of human protein kinase CK2 was examined. The most active were derivatives with N-hydroxypropyl
Received 19 September 2008
Revised 29 December 2008
Accepted 31 December 2008
Available online 7 January 2009
substituents (IC50 in 0.32–0.54
l
M range) whereas derivatives of phthalimide were almost ineffective.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Benzotriazoles
Benzimidazoles
Inhibitors of CK2
1. Introduction
zation, as well as differences in phosphorylation pattern and in
interaction with protein partners.
Protein phosphorylation, the most prevalent post-translational
process in living cells, is catalyzed by protein kinases. The first pro-
tein kinase activity described in 1954 by Burnett and Kennedy1
was found in rat liver mitochondria using casein as a substrate,
hence the enzymes were later called casein kinase 1 (CK1) and
casein kinase 2 (CK2). Subsequently, more than 500 protein kinases
were discovered and their role in the control of nearly all aspects of
cell life and death has been studied widely. It is now recognized
that protein kinases are often functionally interlinked and highly
regulated, forming a complex communicative network.2 Abnormal
phosphorylation of proteins mediated by kinases may result in dis-
eases, which include cancer, diabetes, rheumatoid arthritis and
hypertension.
CK2 may function as a sensor of cell integrity due to antiapoptotic
function caused by its ability to phosphorylate proteins that would
be designated for caspase mediated degradation during apoptosis.4
Inhibitors of CK2 trigger apoptosis and increase the susceptibility
of cancer cells to chemotherapeutic agents.5,6 The increased expres-
sion of CK2 is observed in response to various growth stimuli, and its
activity is aberrantly elevated in various tumor types, like breast car-
cinoma, adenocarcinoma of the lung,7 prostate carcinoma8 and lym-
phomas. Genetic studies in yeast9 indicate that CK2 is required for
progression through both G1/S and G2/M transitions. In mammalian
cells, forced expression of kinase-inactive CK2a
or CK2a0 subunits
compromises cell proliferation.10 Because inhibition of CK2 has a po-
tential role in the treatment of breast and other cancers, therefore
the major efforts are currently devoted to the development of spe-
cific CK2 inhibitors.
Several classes of CK2 inhibitors, effective in low micromolar
ranges, have been reported. The specific inhibitors of CK2,
4,5,6,7-tetrabromo-1H-1,2,3-benzotriazole11 (TBBt), 4,5,6,7-tetrab-
romo-1H-benzimidazole (TBBi) and 4,5,6,7-tetrabromo-1H-benz-
imidazole-2-N,N-dimethylamine (DMAT)12 are used very widely
to elucidate the role of CK2 phosphorylation of a variety of proteins
with important metabolic functions.13–15 Bearing in mind the role
of CK2 in regulation of cell proliferation and the fact that TBBt, TBBi
and DMAT can induce apoptosis,16,17 we undertook modification of
TBBt and TBBi in order to obtain more potent inhibitors of CK2.
Protein kinase CK2 is an evolutionarily conserved serine/threo-
nine kinase of tetrameric form, composed of two catalytic
a (and/
or a0) subunits and two regulatory b subunits that form native
structures exhibiting the stoichiometry: a
2b2, a02b2 and/or aa0b2.
Another catalytic isoform—CK2a00 and its proapoptotic function
was reported in human cells.3 There is strong evidence supporting
functional specialization of CK2 catalytic subunits; CK2
a and
CK2a0 show cell cycle-dependent differences in subcellular locali-
* Corresponding author. Tel.: +48 22 592 3508/234 7570.
E-mail addresses: mbretner@ch.pw.edu.pl, majka@ibb.waw.pl (M. Bretner).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.071

1574
A. Najda-Bernatowicz et al. / Bioorg. Med. Chem. 17 (2009) 1573–1578
The basis for TBBt and TBBi selectivity is provided by the hydro-
phobic pocket adjacent to the ATP/GTP binding site which is smaller
in CK2, than in the majority of other protein kinases. Structural infor-
Derivatives 12 and 13 were obtained by reaction of 1-methylpiper-
azine with 10 and 11 respectively.
N-Substituted tetrabromophthalimides (19–21) were obtained by
reaction of tetrabromophthalic anhydride with different amines.
The latter compound in reaction with hydrazine gave 5,6,7,8-tet-
rabromo-2,3-dihydrophthalazine-1,4-dione (22) (Scheme 3).
mation regarding the CK2a subunit in complex with various inhibi-
tors18,19 indicates conformational plasticity around the ATP binding
site. SinceTBBtandderivativesofTBBimayhaveaneffectonthecon-
formation of the Gly-rich loop at the active site of CK2, it seemed
interesting to explore the effect of alkyl substituents with different
hydrophobic, steric and electrophilic features attached to TBBt and
TBBi on their inhibitory potency. Another group of compounds that
can fit into the small ATP binding pocket could be derivatives of
phthalimide. Phthalimide rings are present in numerous biologically
active compounds. For example, naphthalimides such as amonafide,
dendric imides with N-polyamine tails were synthesized and stud-
ied as antitumor agents,20 whereas 50-N-phthaloyl-30-azido-20,30-
dideoxythymidine derivatives were evaluated for their antiviral
activity against HIV-1, HIV-2 and Moloney murine sarcoma virus.
Compound with the tetrabromophthaloyl moiety inhibited both
HIV-1 and HIV-2 in the low micromolar range.21 Thalidomide, as
well as simple halogenated phthalimides have shown bidirectional
2.2. Expression of subunits of CK2 and determination of CK2
activity
The pT7-7 vector carrying human CK2a subunit (hCK2a) cDNA
and the pT7-7 vector carrying human CK2b subunit (hCK2b) cDNA
were a kind gift of Dr. Stefania Sarno, University of Padova, Italy.
Expression and purification of rhCK2a and rhCK2b proteins was
done according to published methods.27,28
The reaction mixture (final volume of 50
tion of CK2 activity (apoenzyme CK2 or holoenzyme CK2
contained: casein substrate (75 g) or peptide substrate
(RRRDDDSDDD, Biosyntan, Germany, 20 M), Tris–HCl pH 7.5
(20 mM), MgCl2 (20 mM), M, 100–200 cpm
32P]ATP (10
pmolꢀ1) and appropriate concentrations of inhibitor in 1
L DMSO.
After 20 min of incubation at 30 °C, 40 L of the assay mixture was
l
L) for the determina-
a
a
2b2)
l
l
c[
l
TNF-a
production regulating properties.22
l
We designed new derivatives of TBBt, TBBi and phthalimide and
evaluated their effect on human CK2 alfa and human holoenzyme
activities.
l
spotted onto a square (2 cm ꢁ 2 cm) of Whatman 3 MM (for case-
in) or P81 (for peptide) paper, which was immediately immersed in
cold 5% (w/v) trichloroacetic acid containing 0.3% o-phosphoric
acid (10 mL per square), and washed with H2O five times for
10 min. Then the squares were washed in 96% ethanol and allowed
to dry. The radioactivity was quantified using a LKB Flexi-Vial li-
quid scintillation counter.
2. Material and methods
2.1. Chemistry
The activity was calculated as the percentage of incorporated
32P (measured in scintillation counter) in the presence of various
concentrations of the studied compounds versus control with
appropriate concentration of DMSO. Microcal Origin Version: 6.0
and Excel (Microsoft) were used to calculate the data.
The starting materials—4,5,6,7-tetrabromo-1H-benzimidazole
(TBBi) and 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) were syn-
thesized by bromination of 1H-benzimidazole23 or 1H-benzotria-
zole24 according to published methods. The N-alkyl derivatives
( 4, 5, 10, 11, 18) were synthesized by alkylation of TBBt or TBBi
with the use of alkyl halides in the presence of DBU, NaH or KOH
as bases (Scheme 1).
TM
3. Results and discussion
N-Hydroxyalkyl derivatives (2, 3, 7, 8, 17) were prepared by alkyl-
ation of TBBt or TBBi with the use of NaH, KOH or DBU as bases and
appropriate hydroxyalkyl halide as reported in Refs. 25,26. The ratio
of N1 to N2 substituted anomers of TBBt depends on the reaction con-
ditions and on the length of the alkyl chain. For the synthesis of com-
pounds 14 and 15 a different strategy was used. The hydroxyl group
of 4-bromobutan-1-ol was protected with 3,4-dihydro-2H-pyran
before using it in the alkylation reaction. The yield of 14 was very
low, so that compound was not used for biological tests.
Our search for new inhibitors of protein kinase CK2 was based on
the knowledge of the inhibitory activity of benzimidazole and ben-
zotriazole derivatives. The four atoms of bromine in the benzene
ring seem to be an essential requirement for biological activity. An-
other substituents in the benzene ring, four atoms of chlorine or four
methyl groups, leads to significantly lower inhibitory effect versus
Saccharomyces cerevisiae23, rat and recombinant human CK2. Thus,
we focused on TBBt and TBBi as the leading compounds used for
modification. We compared the effect of N1 versus N2-alkyl deriva-
tives of TBBt on their inhibitory potency. Additionally, we examined
the effect of the length of the alkyl substituent on CK2 activity.
Compound 6 was obtained by reaction of dimethylamine with
derivative 5 (Scheme 2). The phosphorylation of compound 8 with
the use of POCl3 and subsequent purification gave derivative 9.
Br
Br
Br
Br
Br
Br
Br
Br
Br
N
N
N
N
N
N
N
N
a
b
N
Z
N
N
N
+
Z
H
Br
H
Br
Br
2
4
7
N1-EtOH-TBBt Z=C2H4OH
N1-EtBr-TBBt
Z=C2H4Br
N1-PrOH-TBBt Z=C3H6OH
Z=C3H6Cl
14 N1-BuOH-TBBt Z=C4H8OH
N2-EtOH-TBBt Z= C2H4OH
N2-EtBr-TBBt Z=C2H4Br
N2-PrOH-TBBt Z=C3H6OH
Bt
3
5
8
1 TBBt
11 N2-PrCl-TBBt
15 N2-BuOH-TBBt Z=C4H8OH
Z=C3H6Cl
10 N1-PrCl-TBBt
Scheme 1. Synthesis of derivatives of 4,5,6,7-tetrabromo-1H-benzotriazole: (a) Br2, HNO3; (b) 2 and 3: Br(CH2)2OH, DBU, CH3CN, reflux, 12 h; 4 and 5: Br(CH2)2Br, KOH in
CH3CN, 60 °C, 12 h; 7 and 8: Br(CH2)3OH, DBU, CH3CN, RT; 10 and 11: Cl(CH2)3Br, NaH in dry DMF, rt; 14 and 15: 4-chloro-1-(2-tetrahydropyranyloxy)-butane, NaH, DMF, p-
toluenesulfonic acid, rt, 48 h.

A. Najda-Bernatowicz et al. / Bioorg. Med. Chem. 17 (2009) 1573–1578
1575
Br
Br
differed slightly with a change of triazole on the imidazole ring.
The small differences between N1 versus N2 substituents may
prove the plasticity of the binding pocket of CK2.
Br
Br
Br
Br
Br
Br
N
N
N
N
c
N
N
The change oftriazole ringinthe phthalimidemoiety (compounds
19–21) or phthalazine (22) results in loss of inhibitory potency.
The attempt to correlate the inhibitory activity of the synthe-
sized compounds with theirs lipophilicity was undertaken (Table
1). When ClogP of derivatives is compared, it can be seen that for
lower ClogP values the inhibitory potency is higher, albeit no strict
relationship can be observed. The difference between ClogP of
known inhibitor DMAT and compound 6 (4.74 vs 5.14) may par-
tially explain the difference in inhibitory potency of those two
compounds, albeit this is not observed for compounds 2 or 12 with
low ClogP (4.25 and 4.63, respectively) and low inhibitory potency
Z
R
10 N1-PrCl-TBBt
Z=C3H6Cl
12 N1-PrMePip-TBBt
R=C3H6NC4H8NCH3
Br
Br
Br
Br
N
N
N
N
c
N
R
N
Z
Br
Br
(9.25
lV and 11.20 lV vs CK2a).
Br
N2-EtDMeN-TBBt R=C2H4N(CH3)2
N2-PrP-TBBt
R=C3H6OPO(OH)2
Br
5
8
N2-EtCl-TBBt Z=C2H4Cl
N2-PrOH-TBBt Z=C3H6OH
6
9
4. Conclusions
In the present study we report the evaluation of new TBBt, TBBi
and phthalimide derivatives as inhibitors of CK2 protein kinase.
To improvetheinhibitoryactivityversus CK2, TBBtand TBBiwere
subjected to chemical modifications. Most modifications either par-
tially or completely abrogated the ability of the new derivatives to
inhibit CK2. However, selected modifications with the hydroxypro-
pyl substituent resulted in compounds with comparable or better
inhibitory activity than the parent compounds. In particular,
3-(4,5,6,7-tetrabromo-1H-1,2,3-benzotriazol-1-yl)propan-1-ol (7),
3-(4,5,6,7-tetrabromo-2H-1,2,3-benzotriazol-2-yl)propan-1-ol (8),
and 3-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)propan-1-ol (17)
showed a small improvement in CK2 inhibitory activity.
11 N2-PrCl-TBBt Z=C3H6Cl
13 N2-PrMePip-TBBt R=C3H6NC4H8NCH3
Scheme 2. Reagents and conditions (c) 6 (CH3)2NH/EtOH, 70 °C, 24 h; 9 TMP, POCl3,
0 °C to rt; 12 and 13 C4H9N2CH3, K2CO3, CH3CN, 70 °C.
Br
O
Br
NH
NH
Br
Br
O
O
O
Br
Br
Br
Br
O
R
NH2
22
5. Experimental
O
Br
Melting points were determined in open capillary tubes using
Büchi apparatus B504 and are uncorrected. UV absorption spectra
were recorded on a Cary 300 spectrophotometer. Thin-layer chro-
matography (TLC) was performed on 0.2 mm Merck silica gel 60
F254 plates. Preparative separations were carried out by column
chromatography using silica gel (230–400 mesh), or PLC silica gel
60 F254 2 mm glass plates from Merck. 1H and 13C NMR spectra were
recorded on Varian 500, 400 or 200 MHz spectrometer at 298 K.
Chemical shifts (d) are reported in parts per million units (ppm) rel-
ative to tetramethylsilane (Me4Si, d = 0 ppm). Mass spectrometry
was recorded on Micro-mass ESI Q-TOF spectrometer at IBB PAN.
Starting materials: 1H-benzimidazole and 1H-1,2,3-benzotriazole,
were purchased from Aldrich Co; 1H-1,2,3-benzotriazole and 1H-
benzimidazole derivatives were synthesized adapting the methods
developed in our laboratory: 4,5,6,7-tetrabromo-1H-1,2,3-benzotria-
zole (1, TBBt),24 2-(4,5,6,7-tetrabromo-1H-1,2,3-benzotriazol-1-
yl)ethanol (2, N1-EtOH-TBBt) and 2-(4,5,6,7-tetrabromo-2H-1,2,3-
benzotriazol-2-yl)ethanol (3, N2-EtOH-TBBt) as reported in Ref. 25,
4,5,6,7-tetrabromo-1H-benzimidazole (16, TBBi) as published in Ref.
23, 3-(4,5,6,7-tetrabromo-1H-1,2,3-benzotriazol-1-yl)propan-1-ol
(7, N1-PrOH-TBBt), 3-(4,5,6,7-tetrabromo-2H-1,2,3-benzotriazol-2-
yl)propan-1-ol (8, N2-PrOH-TBBt) and 3-(4,5,6,7-tetrabromo-1H-ben-
zimidazol-1-yl)propan-1-ol (17, N-PrOH-TBBi) were synthesized as
reported in patent application P 380112. The ClogP values were ob-
tained using ChemDraw Pro 8.0 software.
Br
Br
N
R
O
Br
19, R = CH2CH2N(CH3)2
20, R = CH2CN
21, R = CH2CF3
Scheme 3. Synthesis of derivatives of tetrabromophthalic anhydride.
The influence of the synthesized compounds on human CK2
a
subunit and on the holoenzyme activity was determined as de-
scribed in Materials and Methods. The results are shown in Table
1. For compounds which exerted a small effect on the activity of
CK2
IC50 was much higher than 10
ence of 5 M concentration of the tested compound was evaluated.
a
, the inhibition of holoenzyme was not determined. When
l
M, the CK2 activity at the pres-
a
l
Some of the new analogs showed IC50 similar to that of the par-
ent compounds (1 and 16) and their inhibitory activity depended
on the length of the alkyl substituents. The 3-(4,5,6,7-tetrab-
romo-1H-benzimidazol-1-yl)propan-1-ol (17) exhibited a higher
potency (IC50 0.54 0.15
lM) than the parent compound TBBi
5.1. 4,5,6,7-Tetrabromo-1-(2-bromoethyl)-1H-1,2,3-
benzotriazole (4, N1-EtBr-TBBt) and 4,5,6,7-tetrabromo-2-(2-
bromoethyl)-2H-1,2,3-benzotriazole (5, N2-EtBr-TBBt)
(IC50 1.30 0.13 M). The 3-(4,5,6,7-tetrabromo-1H-1,2,3-ben-
zotriazol-1-yl)propan-1-ol (7) and 3-(4,5,6,7-tetrabromo-2H-
1,2,3-benzotriazol-2-yl)propan-1-ol (8) also exhibited higher po-
l
tency against the catalytic subunit of CK2
IC50 0.32 0.15 and 0.34 0.12
0.50 0.07 M for 1. Derivatives with shorter (3, 4, 5) or longer
chains (12, 13, 15) showed lower inhibitory activity. The activity
a
that TBBt (1), with
To the solution of TBBt (200 mg, 0.46 mmol) in acetonitrile
(2.5 mL) the solution of KOH (112 mg, 2 mmol) in MeOH (1 mL)
was added, followed by 1,2-dibromoethane (1.6 mL), and the reac-
l
M
lM compared to
l

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A. Najda-Bernatowicz et al. / Bioorg. Med. Chem. 17 (2009) 1573–1578
Table 1
Inhibition of the CK2 protein kinase activity by derivatives of 1H-benzotriazole, 1H-benzimidazole and phthalimide
Compound
CK2
a
activitya (%)
IC50
[lM] CK2
a
IC50[l
M] CK2
a2b2
ClogP
1
2
4
5
6
7
8
9
10
11
12
13
15
16
17
18
19
20
21
22
TBBt
0.50 0,07
9.25 1.09
0.5 0.12
2.61 0.72
4.53
4.25
5.6
N1-EtOH-TBBt
N1-EtBr-TBBt
N2-EtBr-TBBt
N2-EtDiMetN-TBBt
N1-PrOH-TBBt
N2-PrOH-TBBt
N2-PrP-TBBt
N1-PrCl-TBBt
N2-PrCl-TBBt
N1-PrMePip-TBBt
N2-PrMePip-TBBt
N2-BuOH-TBBt
TBBi
60
79
75
5.97
5.14
4.36
4.73
5.27
5.58
5.96
4.63
5.0
5.19
4.13
3.95
5.18
4.3
0.32 0.15
0.34 0.12
2.2 0.22
0.48 0.14
0.82 0.19
2.6 0.17
5.87 2.24
87
69
11.20 4.84
7.3 4.89
1.73 0.15
1.30 0.23
0.54 0.15
2.9 0.20
2.50 0.59
8.6 3.41
1.52 0,30
0.93 0.08
0.71 0.04
1.1 0.14
N-PrOH-TBBi
N-PrCl-TBBi
N-EtdMeN-TBiI
N-MeCN-TBiI
N-tFEt-TBiI
88
80
88
95
4.05
5.28
3.58
TBPht
a
CK2a activity at the presence of 5 lM concentration of the tested compound.
tion mixture was heated at 60 °C for 12 h. The same amount of
KOH and 1,2-dibromoethane were added twice again to complete
the reaction. The resulting precipitate was filtered, washed with
water, dissolved in chloroform and the products were separated
by chromatography with the use of CH3Cl:hexane 1:1. After elution
and evaporation the products 4 (42 mg, 16.8% yield) and 5 (54 mg,
21.7% yield) were obtained.
289 nm (8600), 308 nm (6000); 1H NMR (Me2SO-d6) d: 2.06–2.11
(qui, 2H, 20-CH2); 3.50 (q, 2H, 30-CH2); 4.64 (t, 1H, OH); 4.98 (t,
2H, 10-CH2); 13C NMR (Me2SO-d6) d: 33.72; 47.54; 57.45; 106.70;
115.62; 123.34; 128.33; 131.67; 144.84. MS [M+H]+ m/z calcd for
C9H8Br4N3O+ 493.7360, found 493.7782.
5.6. 3-(4,5,6,7-Tetrabromo-2H-1,2,3-benzotriazol-2-yl)propan-
1-ol (8, N2-PrOH-TBBt)
5.2. (4, N1-EtBr-TBBt)
Mp 139.6–139.9 °C; Rf 0.71 (CHCl3:MeOH 9:1), UV pH 7 kmax
(e):
Mp 198–199.5 °C, Rf 0.39 (CHCl3:hexane 1:1); 0.89
300 (12,800), 307 (13,400); MeOH kmax ): 297 (13,000), 306
(e
1
(CHCl3:MeOH 98:2). UV (MeOH + 4% CH3CN) kmax
(e) 277 (8400),
(13,500); H NMR (Me2SO-d6) d: 2.17–2.22 (qui, 2H, 20-CH2); 3.49
(q, 2H, 30-CH2); 4.72 (t, 1H, OH); 4.86 (t, 2H, 10-CH2); 13C NMR
(Me2SO-d6) d: 32.38; 54.28; 57.37; 113.47; 125.36; 142.41. MS
[M+H]+ m/z calcd for C9H8Br4N3O+ 493.7360, found 493.7855.
288 (8300), 310 (5500), 321 (3800); 1H NMR (Me2SO-d6) d: 4.03
(t, 2H, 20-CH2), 5.35 (t, 2H, 10-CH2); MS [M+H]+ m/z calcd for
C8H5Br5N3+, 541.6359, found, 541.6775.
5.3. (5, N2-EtBr-TBBt)
5.7. 3-(4,5,6,7-Tetrabromo-2H-1,2,3-benzotriazol-2-yl)propyl
dihydrogen phosphate (9, N2-PrP-TBBt)
Mp 163.7–164.7 °C, Rf 0.63 (CHCl3:hexane 1:1); 0.9 (CHCl3:
MeOH 98:2). UV (MeOH + 7% CH3CN) kmax
(e) 284 (11,000), 298
A solution of N2-PrOHTBBt (200 mg, 0.4 mmol) and POCl3,
(0.300 mL) in trimethylphosphate (1.6 mL) was cooled to 0 °C for
30 min., stirred at rt for a few hours, and neutralized with NaHCO3.
The product was separated with the use of DEAE–Sephadex, with a
gradientofTEABbuffer.Aftertheevaporationandlyophylisationcom-
pound 9, (N2-PrP-TBBt) was obtained. Rf 0.54 (i-prop:NH3:H2O 7:1:2);
UV (MeOH) kmax 283, 296, 306; 1H NMR (D2O) d: 2.47 (m, 2H, 20-CH2),
3.98 (dt, 2H, 30-CH2), 4.95 (t, 2H, 10-CH2), 31P NMR (D2O) d: 2.32; MS
[MꢀH]ꢀ m/z calcd for C9H7Br4N3O4Pꢀ 571.6867, found 571.6853.
(14,400), 307 (15,000), 321 (7000) 1H NMR (Me2SO-d6) d: 4.14 (t,
2H, 20-CH2), 5.27 (t, 2H, 10-CH2); MS [M+H]+ m/z calcd for
C8H5Br5N3+, 541.6359, found 541.6575.
5.4. N,N-Dimethyl-N-[2-(4,5,6,7-tetrabromo-2H-1,2,3-
benzotriazol-2-yl)ethyl]amine (6, N2-EtDMeN-TBBt)
The mixture of 4,5,6,7-tetrabromo-2-(2-chloroethyl)-2H-1,2,3-
benzotriazole25 (200 mg, 0.40 mmol) and ethanolic dimethylamine
(12 mL, 20%) was heated in steel autoclave at 70 °C for 20 h. After
cooling the resulting precipitate was crystallized from MeOH/diox-
ane to obtain 6, (126 mg, 60% yield) mp 125–126 °C, Rf 0.035
5.8. 4,5,6,7-Tetrabromo-1-(3-chloropropyl)-1H-1,2,3-
benzotriazole (10, N1-PrCl-TBBt) and 4,5,6,7-tetrabromo-2-(3-
chloropropyl)-2H-1,2,3-benzotriazole (11, N2-PrCl-TBBt)
(CHCl3:hexane 1:1); UV (MeOH + 25% CH3CN) kmax
(e) 284
(10,000), 297 (12,000), 306 (12,600); 1H NMR (Me2SO-d6) d: 2.18
(s, 6H, 2 ꢁ CH3), 2.95 (t, 2H, CH2), 4.91 (t, 2H, CH2); 13C NMR
(Me2SO-d6) d: 45.6, 55.7, 58.5, 114.3, 126.2, 143.2; EI ASC 506.8
To a suspension of 60% sodium hydride (36 mg, 0.9 mmol) in
DMF (4.5 mL) cooled to 0 °C TBBt (200 mg, 0.46 mmol) was added
and the mixture was stirred for 30 min. To this solution 1-bromo-
3-chloropropan (0.500 mL) was added and the reaction was stirred
overnight at reflux. The solution was poured into cold water, the
resulting precipitate was filtered off, dissolved in CHCl3 and prod-
ucts were separated with the use of PLC (CHCl3:hexane 1:1). After
elution and evaporation compounds 10 (37 mg, 15.8% yield) and 11
(142 mg, 60% yield) were obtained.
+
[M+H]+, 528.8 [M+Na]+; MS [M+H]+ m/z calcd for C10H11Br4N4
506.7676, found 506.8237.
5.5. 3-(4,5,6,7-Tetrabromo-1H-1,2,3-benzotriazol-1-yl)propan-
1-ol (7, N1-PrOH-TBBt)
Mp 180–182 °C, Rf 0.65 (CHCl3:MeOH 9:1); UV: pH 7 kmax
(
e
)
Compound 10: mp 170.0–171.2 °C; Rf 0.40 (CHCl3:hexane 1:1),
1
281 (8500), 291 (8900), 311 (6300); MeOH kmax ) 278 (8700),
(e
0.89 (CHCl3:MeOH 98:2); H NMR (Me2SO-d6) d: 2.41 (qui, 2H, 20-

A. Najda-Bernatowicz et al. / Bioorg. Med. Chem. 17 (2009) 1573–1578
1577
CH2), 3.71 (t, 2H, 30-CH2), 5.06 (t, 2H, 10-CH2). 13C chemical shifts
The solvent was removed under reduced pressure, residue poured
into dichloromethane, washed with 2 N NaOH, and water, dried
with Na2SO4, and products were separated with the use of SiO2
plates (ethyl acetate:methanol 20:1). After elution and evaporation
4-(4,5,6,7-tetrabromo-1H-benzotriazol-1-yl)butan-1-ol (14, 3 mg,
1.28% yield) and 4-(4,5,6,7-tetrabromo-2H-benzotriazol-2-yl)bu-
tan-1-ol (15, 125 mg, 53.6%) were obtained.
Compound 14: Rf 0.017 (CHCl3:hexane 1:1); 0.49 (CHCl3: MeOH
98:2), MS [M+H]+ m/z calcd for C10H10Br4N3O+ 507.7516, found
507.7692.
13
from C HSQC and HMBC (Me2SO-d6) d: 33.16 (20-CH2), 41.94
(30-CH2), 47.59 (10-CH2), 132.02 (7a-C); MS [M+H]+ m/z calcd for
+
C9H7Br4ClN3 511.7021, found 5.1182.
Compound 11: mp 160–162 °C, Rf 0.75 (CHCl3:hexane 1:1), 0.93
(CHCl3:MeOH 98:2) UV (MeOH + 14% dioxane) kmax (e) 270 (7600),
277 (8500), 288 (8000), 310 (5200) 1H NMR (Me2SO-d6) d: 2.58
(qui, 2H, 20-CH2), 3.69 (t, 2H, 30-CH2), 4.95 (t, 2H, 10-CH2) MS
+
[M+H]+ m/z calcd for C9H7Br4ClN3 511.7021, found 511.7951.
5.9. 4,5,6,7-Tetrabromo-1-[3-(4-methylpiperazin-1-yl)propyl]-
Compound 15: mp 179–179.9 °C, Rf 0.03 (CHCl3:hexane 1:1);
0.59 (CHCl3 MeOH 98:2) UV (MeOH + 17% CH3CN) 284 (10,700),
297 (13,700), 306 (14,300), 320 (6600). 1H NMR (Me2SO-d6) d:
1.45 (qui, 2H, 30-CH2), 2.07 (qui, 2H, 20-CH2), 3.43 (q, 2H, 40-CH2),
4.49 (t, 1H, OH), 4.82 (t, 2H, 10-CH2). 13C chemical shifts from 13C
HSQC and HMBC: (Me2SO-d6) d: 25.92 (20-CH2), 29.81 (30-CH2),
57.00 (10-CH2), 59.70 (40-CH2). MS [M+H]+ m/z calcd for
C10H10Br4N3O+ 507.7516, found 507.8037.
1H-1,2,3-benzotriazole (12, N1-PrMePip-TBBt)
To the mixture of 4,5,6,7-tetrabromo-1-(3-chloropropyl)-1H-
1,2,3-benzotriazole (40 mg, 0.078 mmol) and K2CO3 (40 mg), in
1.5 mL of acetonitrile, N1-methyl piperazine (0.052 mL,
0.469 mmol) was added and heated at 70 °C for 72 h. After cooling,
the reaction mixture was evaporated and the products were sepa-
rated by PLC with the use of CHCl3:MeOH 9:1. After the elution and
evaporation compound 12 was obtained (10 mg, 22.3% yield). 1H
NMR (Me2SO-d6) d: 1.90–2.20 (br s and br m; 13H, 20-CH2 and
piperazine), 2.34 (t, 2H, 30-CH2), 4.99 (t, 2H, 10-CH2); 1H NMR
(Me2SO-d6, t = 323 K) d: 1.92–2.00 (br s, 4H, piperazine), 2.02 (s,
3H, CH3), 2.08 (m, 2H, 20-CH2), 2.14–2.18 (br s, 4H, piperazine),
2.35 (t, 2H, 30-CH2), 4.99 (t, 2H, 10-CH2); MS [M+H]+ m/z calcd for
5.12. 3-(4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)propan-1-
ol; (17, N-PrOH-TBBi)
Mp 196.4–197.2 °C, Rf 0.41 (CHCl3:MeOH 9:1), UV: pH 7 (9%
dioxane) kmax (e) 269 nm (9700), 274 nm (9650), 301 nm (3800);
1H NMR (Me2SO-d6) d: 1.93–1.98 (qui, 2H, 20-CH2); 3.41 (q, 2H,
30-CH2); 4.57 (t, 2H, 10-CH2); 4.68 (t, 1H, OH); 8.46 (s, 1H, 2-CH);
13C NMR: 34.21; 43.81; 57.23; 106.49; 116.43; 120.26; 122.24;
131.28; 143.60; 148.94. MS [M+H+] m/z calcd for C10H9Br4N2O+
492.7407, found 492.7268.
+
C14H18Br4N5 575.8255, found 575.7589.
5.10. 4,5,6,7-Tetrabromo-2-[3-(4-methylpiperazin-1-yl)propyl]-
2H-1,2,3-benzotriazole (13, N2-PrMePip-TBBt)
To the mixture of 4,5,6,7-tetrabromo-2-(3-chloropropyl)-2H-
benzotriazole (160 mg, 0.312 mmol) and K2CO3 (140 mg) in aceto-
nitrile (4 mL), 1-methylpiperazine (0.200 mL, 1.8 mmol) was added
and heated to reflux for 48 h. After cooling, the reaction mixture
was partitioned between water and chloroform. The organic layer
was concentrated and the products were separated by PLC with the
use of CHCl3:MeOH 98:2. After the elution and evaporation com-
pound 13 was obtained (80 mg, 44.5% yield). Mp 97–99 °C, Rf
0.04 (CHCl3:MeOH 98:2); UV: (MeOH) 284 (9000), 298 (11,400),
306.5 (11,700); 1H NMR (Me2SO-d6) d: 1.90–2.40 (br s, br m, br t;
15H, 20-CH2, 30-CH2 and piperazine), 4.83 (t, 10-CH2); MS [M+H]+
5.13. 4,5,6,7-Tetrabromo-1-(3-chloropropyl)-1H-benzimidazole
(18, N-PrCl-TBBi)
Obtained as for 10, purified by chromatography (CHCl3:MeOH
99:1) 125 mg, (53% yield) mp 141–142 °C, Rf 0.73 (CHCl3:MeOH
98:2); UV (MeOH + 10%CH3CN) kmax (e): 268 (10,400), 273 (10,350),
302 (3750); 1H NMR (Me2SO-d6) d: 2.29 (m, 2H, 20-CH2); 3.69 (t, 2H,
30-CH2), 4.63 (t, 2H, 10-CH2), 8.49 (s, 1H, 2-CH); MS [M+H]+ m/z calcd
for C10H8Br4ClN2+ 510.7069, found 510.7082.
5.14. General procedure for N-substituted
tetrabromophthalimides
+
m/z calcd for C14H18Br4N5 575.8255, found 575.7624.
5.11. 4-(4,5,6,7-Tetrabromo-1H-1,2,3-benzotriazol-1-yl)butan-
1-ol (14, N1-BuOH-TBBt) and 4-(4,5,6,7-tetrabromo-2H-1,2,3-
benzotriazol-2-yl)butan-1-ol (15, N2-BuOH-TBBt)
4,5,6,7-Tetrabromophthalic anhydride (10 mmol) and proper
amine (10 mmol) were dissolved in dry DMF (15 mL) and refluxed
for 2 h. Then the mixtures were cooled and poured into water.
Crude products were filtered and crystallized from 90% ethanol.
4-Chlorobutanol-1 (0.45 mL) in ether (4.6 mL) containing two
drops of concd HCl was stirred and cooled to 0 °C. 3,4-Dihydro-
2H-pyran (0.63 mL) was added and the solution was stirred at
room temperature overnight. The solution was neutralized with
NaHCO3 and extracted with ether. The organic layer was washed
with water and brine, dried (Na2SO4) and evaporated. After evapo-
ration 846 mg of 4-chloro-1-(2-tetrahydropyranyloxy)-butane as
an oil was obtained.
To the mixture of TBBt (200 mg, 0.46 mmol) in DMF (1.5 mL)
with 60% sodium hydride (24 mg, 0.6 mmol), K2CO3 (33 mg) was
added and the mixture was stirred and heated to 100 °C for
30 min. The solution of 4-chloro-1-(2-tetrahydropyranyloxy)-bu-
tane (280 mg) in DMF (1 mL) was added and the reaction mixture
was heated overnight. After cooling, the precipitate was filtered
off; the filtrate was evaporated, the residue was diluted with chlo-
roform, extracted with water and the organic layer was evapo-
rated. p-Toluenesulfonic acid (150 mg) in ethanol (4 mL) was
added to the residue and stirred for 48 h at room temperature.
5.15. 4,5,6,7-Tetrabromo-2-[2-(dimethylamino)ethyl]-1H-
isoindole-1,3(2H)-dione (19, N-EtDMeN-TBiI)
Mp 225 °C, UV (MeOH) kmax (e
) 244 (28,350), 335 (1400).1H
NMR (Me2SO-d6) d: 4.05 (t, 2H, J = 6.9 Hz), 2.99 (t, 2H, J = 6.9 Hz),
2.02 (s, 6H, 2 ꢁ CH3). IR (KBr, cmꢀ1): 1776, 1726 (C@O). Anal. Calcd
for C12H10Br4N2O2: C, 27.00; H, 1.89; N, 5.25. Found: C, 27.04; H,
1.94, N, 5.22.
5.16. (4,5,6,7-Tetrabromo-1,3-dioxo-1,3-dihydro-2H-isoindol-
2-yl)acetonitrile (20, N-MeCN-TBiI)
Mp 305 °C, UV (MeOH) kmax (e
) 246 (31,000), 340 (2500). 1H
NMR (Me2SO-d6) d: 4.58 (s, 2H, CH2). IR (KBr, cmꢀ1): 2228 (–CN),
1778, 1725 (C@O). Anal. Calcd for C10H2Br4N2O2: C, 23.94; H,
0.40; N, 5.58. Found: C, 23.90; H, 0.42; N, 5.55.

1578
A. Najda-Bernatowicz et al. / Bioorg. Med. Chem. 17 (2009) 1573–1578
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1,3(2H)-dione (21, N-tFEt-TBiI)
Mp 213 °C, UV (MeOH), kmax (e
) 246 (33,000), 338 (2300). 1H
8. Ahmed, K. Cell. Mol. Biol. Res. 1994, 40, 1.
NMR (Me2SO-d6) d: 3.96 (q, J = 13 Hz, CH2). IR (KBr, cmꢀ1): 1777,
1726 (C@O). Anal. Calcd for C10H2Br4F3NO2: C, 22.05; H, 0.37; N,
2.57. Found: C, 22.06; H, 0.39; N, 2.60.
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TBPht)
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Tetrabromophthal-anhydride (10 mmol) in dry ethanol (20 mL)
were refluxed over 30 min. with hydrazine monohydrate
(15 mmol). Then 10 mL of 10% HCl was added and heated during
next 30 min. The crude product was filtered and crystallized from
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Biochim. Biophys. Acta 2005, 1754, 271.
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dioxane; mp 299 °C UV (MeOH) kmax (e) 254 (25,500), 330 (2900).
1H NMR (Me2SO-d6) d: 4.22 (s, 2H, 2 ꢁ CH) IR (KBr, cmꢀ1): 1740
(C@O). Anal. Calcd for C8H2Br4N2O2: C, 20.11; H, 0.42; N, 5.86.
Found: C, 20.06; H, 0.39; N, 5.90.
´
21. Balzarini, J.; De Clercq, E.; Kaminska, B.; Orzeszko, A. Antiviral Chem. Chemother.
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Acknowledgments
´
22. Orzeszko, A.; Lasek, W.; Switaj, T.; Stoksik, M.; Kamin´ ska, B. Farmaco 2003, 58,
371.
These studies were supported by the Polish Ministry of Higher
Education Grant No. PBZ-MIN 014/P05/2004 and partially by War-
saw University of Technology.
23. Zien´ , P.; Bretner, M.; Zasta˛piło, K.; Szyszka, R.; Shugar, D. Biochem. Biophys. Res.
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Lipniacki, A.; Piasek, A.; Kulikowski, T.; Borowski, P. Antiviral Chem. Chemother.
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