Bispyridinium cyclophanes: Novel templates for human choline kinase inhibitors

Article abstract of DOI:10.1021/jm030792i

The synthesis and biological activities of four novel bispyridinium cyclophanes as choline kinase (ChoK) inhibitors are presented. Their synthetic methodology has been optimized according to dilution, temperature, and reaction time and provides pure bispy

Full text of DOI:10.1021/jm030792i

3754
J . Med. Chem. 2003, 46, 3754-3757
Brief Articles
Bisp yr id in iu m Cyclop h a n es: Novel Tem p la tes for Hu m a n Ch olin e Kin a se
In h ibitor s
Ana Conejo-Garc´ıa, J oaqu´ın M. Campos, Rosario M. Sa´nchez-Mart´ın, Miguel AÄ . Gallo, and Antonio Espinosa*
Departamento de Quı´mica Farmace´utica y Orga´nica, Facultad de Farmacia, c/ Campus de Cartuja s/ n,
18071 Granada, Spain
Received J anuary 28, 2003
The synthesis and biological activities of four novel bispyridinium cyclophanes as choline kinase
(ChoK) inhibitors are presented. Their synthetic methodology has been optimized according to
dilution, temperature, and reaction time and provides pure bispyridinium cyclophanes in high
yields very easily. One of these cyclophanes (6, 4,8-diaza-3(1,4),9(4,1)-dipyridina-1(1,4),6(1,3)-
dibenzenacyclodecaphan-31,91-bis(ilium) dibromide) has an IC50(ChoK) of 0.3 µM and is the most
potent human ChoK inhibitor described to date.
Ch a r t 1
In tr od u ction
Ras genes encode proteins that regulate key cellular
signaling pathways.1 Several lines of evidence have
indicated that among other cellular changes the phos-
pholipid metabolism was also altered as a result of
oncogene-induced transformation.2,3 Phospholipid mol-
ecules and their metabolites are presumed to participate
in the processes of oncogene-induced transformation.4
Phosphatidylcholine (PC), the major component of the
plasma membrane, is hydrolyzed by phospholipase D
(PLD) to yield phosphatidic acid (PA) and choline. PA
is then hydrolyzed to generate diacylglycerol (DAG). On
the other hand, choline is phosphorylated by choline
kinase (ChoK) to generate phosphorylcholine (PCho).
Increased levels of ChoK activity and PCho production
in human cancers have been found,5,6 in keeping with
previous reports using nuclear magnetic resonance.7,8
These observations have resulted in the development
of an antitumoral strategy focused on ChoK. Research
on ChoK inhibitors has identified hemicholinium-3 (HC-
3, Chart 1) as a relatively potent and selective blocker.9
This choline homologue with a biphenyl structure has
been used for the design of new antitumoral drugs. Since
HC-3 is a potent respiratory paralyzant, it is not a good
candidate for use in the clinic. The synthesis of several
derivatives was based on structural modifications of
HC-3 that improve the ChoK inhibitory activity and
omit the toxic effect. We have correlated the inhibitory
effect on proliferation of symmetrical bisquaternary
compounds10 with their ability to inhibit the production
of PCho in whole cells. When the 1,2-ethylene-p-(bis-
benzyldimethyldiyl) (1, Chart 1) moiety was used as a
linker between the two 4-substituted pyridinium cat-
ionic heads, the structures were screened for their
activity inhibiting isolated ChoK (under ex vivo condi-
tions). The R4 group, which is a tertiary amine, made a
substantial contribution, and it was suggested11 that its
role was electronic, via delocalization of the positive
charge. The importance of frontier orbital energies
(LUMO) of model compounds has been emphasized and
interpreted.12 Fifty-six biscationic dibromides with dis-
tinct heads [bis(4-substituted)pyridinium, bis(4-amino-
quinolinium), bisquinolinium, and bisisoquinolinium
moieties] and several spacers between the two charged
nitrogen atoms were synthesized. The electron charac-
teristic of the substituent at position 4 of the heterocycle
* To whom correspondence should be addressed. Phone: +34 958
243850. Fax: +34 958 243845. E-mail: aespinos@ugr.es.
10.1021/jm030792i CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/27/2003

Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3755
Sch em e 1a
and the theoretical lipophilic character of the whole
molecule were found to significantly affect the antitumor
activity.13 We have lately studied the role played by a
third positive charge on both ChoK inhibitory and
antiproliferative activities. A benzene ring was used as
a linker in order to carry out such a study because it
allows us to obtain symmetrical bis- and triscationic
compounds (2, Chart 1). Trispyridinium compounds are
more potent than the bispyridinium ones as inhibitors
of human ChoK. Actually, the triscationic compound 2
(Chart 1, R4 ) 4-chloro-N-methylanilino) was the most
potent ChoK inhibitor so far described (IC50 ) 1.4 µM).14
Nevertheless, the trispyridinium compounds are less
active than the bispyridinium ones as antiproliferative
agents because the latter show better lipophilicity to
cross the cytosolic membranes. We have very recently
published a review on ChoK inhibitors.15
a
(i) Phenol; (ii) 1,3- or 1,4-bis(bromomethyl)benzene, refluxing
Up to now, all the structures synthesized by our group
are open compounds and accordingly are highly flexible
molecules. However, rigidification has been a habitual
tactic used to increase the activity of a drug or to reduce
its side effects. Incorporating the skeleton of a flexible
drug into a ring is the usual way of “locking” a
conformation that, moreover, can give information on
the active conformation of the compound. As a first stage
in the design of this new class of ChoK inhibitors, we
have embarked upon the synthesis of the most simple
model of cyclic compounds that have only one benzene
ring as a linker. The purpose is to study how the rigidity
increase affects the ChoK inhibitory and/or antiprolif-
erative activities, and hence, the following bispyri-
dinium cyclophane family (3-6, Chart 1) has been
prepared. These compounds differ from each other by
the substitution pattern shown by the benzene rings.
The first prefix takes into account the upper linker that
connects the amino groups, whereas the second one is
related to the disubstitution pattern of the lower
benzene ring that links the N+ atoms. In the present
study, we report the synthesis and biological activities
of several bispyridinium cyclophanes as human ChoK
inhibitors that are antiproliferative agents.
acetronitrile.
of the dibromide drop by drop to the dipyridine struc-
ture; in this way the obtention of the cyclophanes is
favored and, accordingly, polymer formation is kept to
1
a minimum. Nevertheless, the H NMR spectra of the
structures obtained show the presence of a second
compound very similar to the desired compounds. High-
resolution liquid secondary ion mass spectrometry
(LSIMS) data and elemental analyses demonstrated
that the impurity should have the same molecular
formula as our targets. This led us to think that the
substance obtained was the open monocationic com-
pound (see Supporting Information) as a consequence
of an incomplete cyclization process.
Thus, a 50% mixture was obtained, which made the
separation of both by recrystallization impossible be-
cause of the similarity of their physicochemical proper-
ties and forced us to improve the synthesis method in
order to oblige the monocationic salt to cyclize. The
monoquaternized salt has to be dissolved to undergo the
cyclization step with the participation of the other
pyridine nitrogen atom. Therefore, increment of dilu-
tion, the use of a solvent more polar than acetonitrile
and/or extending the reaction time facilitated the mac-
rocycle formation.
As can be seen in Chart 1 (3-6), compounds derivated
from both ortho benzenes have not been obtained
because of the lack of solubility of the intermediates that
makes the final cyclization reaction impossible (data not
shown).
Similar bisquinolinium cyclophanes17,18 needed to be
purified by tedious reverse-phase preparative HPLC
because conventional purification methods failed to give
analytically pure samples for biological testing, despite
having been obtained under high-dilution conditions
(0.001-0.002 M). In our case, this represents a great
advantage for the accessibility of such an interesting
class of compounds.
Ch em istr y
The cyclophanes were synthesized according to Scheme
1. The dipyridines 7 and 8 (para and meta isomers,
respectively) are novel and were prepared from the
commercially available diamines and 4-bromopyridine
in the presence of phenol, which is known to catalyze
the reaction in the case of 2- and 4-haloquinolines.16 As
a reaction medium, phenol reduces both the reaction
time and temperature of halogen-replacement reactions.
Dipyridines 7 and 8 were characterized as the bis-
hydrobromide compounds after recrystallization from
methanol. The conversion of 7 and 8 to the desired
cyclophanes was carried out under high-dilution condi-
tions in acetonitrile at the reflux temperature of the
mixture.
Biologica l Testin g
Compounds 3-6 were tested in an ex vivo system
using human ChoK as a target. This assay allowed us
to evaluate the affinity of the compounds for ChoK,
without considering the possible passage through mem-
branes. The effects on cell proliferation by the ChoK
inhibitors in ras-transformed cells were next investi-
gated on the HT-29 cell line (in vitro assay). This cell
line was established from a colon adenocarcinoma, one
of the most frequent solid cancers in humans that are
mainly resistant to chemotherapy,19 making these cells
appropriate for the search of new antitumor drugs. IC50
To start with, the initial concentration was 0.04 M
and the reaction was carried out by adding a solution

3756 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Brief Articles
Ta ble 1. ChoK Inhibitory and Antiproliferative Activities of
Cyclophanes 3-6
Exp er im en ta l Section
Ch em istr y. Melting points were taken in open capillaries
on an Electrothermal melting point apparatus and are uncor-
rected. Nuclear magnetic resonance spectra were recorded on
a 400 MHz 1H and 100 MHz 13C NMR Bruker ARX 400 or
300 MHz 1H and 75 MHz 13C NMR Bruker AMX-300 spec-
trometers, and chemical shifts (ppm) are reported relative to
the solvent peak. Signals are designated as follows: s, singlet;
bs, broad singlet; d, doublet; dd, doublet of doublets; t, triplet;
pst, pseudotriplet; m, multiplet. All the final products had
compd
isomera
IC50(ChoK) (µM)
IC50(HT-29) (µM)
6
3
4
5
m,p
p,p
m,m
p,m
0.3
2.1
13.2
24.8
28.8
36.9
>100
58.6
a
The first prefix takes into account the upper linker that
connects the amino groups, whereas the second one is related to
the disubstitution pattern of the lower benzene ring that links the
N+ atoms.
satisfactory (within (0.4%) C, H, and
N analyses. The
compounds gave accurate mass spectra, having the correct
isotope abundance and no extraneous peaks. High-resolution
liquid secondary ion mass spectra (HR LSIMS) were carried
out on a VG AutoSpec Q high-resolution mass spectrometer
(Fisons Instruments). All compounds were dried at 40 °C and
0.1 mmHg for 15 h, but many held on tenaciously to water,
which appears to be a solvate. The final compounds (3-6) have
been named according to IUPAC Recommendations for phane
structures.20,21
values were obtained from a nonlinear least-squares fit
of the Hill equation to the data. The activity in the in
vitro assay reflects the pharmacodynamic properties of
the compounds rather than their affinity for the enzyme.
Resu lts a n d Discu ssion
The biological results of the four bispyridinium cy-
clophanes are shown in Table 1. Compound 6 is ap-
proximately 5-fold more potent than the most effective
inhibitor of human ChoK previously reported (see ref
14 and Chart 1, triscationic compound 2, R4 ) 4-chloro-
N-methylanilino).
The first thing that draws attention is the fact that
four structurally isomeric compounds present biological
activities that are so different. This indicates that the
substitution model of the cyclophane linkers is very
significant for biological activity.
Gen er a l P r oced u r e for th e P r ep a r a tion of 1,4-Bis-
[(p yr id in -4-yl)a m in om eth yl]ben zen e Hyd r obr om id e (7)
a n d 1,3-Bis[(p yr id in -4-yl)a m in om eth yl]ben zen e Hyd r o-
br om id e (8). 4-Bromopyridine and the respective diamine
(molar ratio 1:2) in a high excess of phenol were heated at
180 °C for 7 h. After cooling to room temperature and being
diluted with methanol, the reaction mixture was acidified up
to pH 1-2 with HBr in 33% glacial AcOH. On addition of
diethyl ether, a solid precipitated and was collected by vacuum
filtration and washed thoroughly with the same solvent to
remove phenol. Compounds were purified by recrystallization
from methanol, obtaining the target molecules 7 and 8 as white
solids (yields 65%).
On one hand, regarding the ChoK inhibitory activity,
it can be accepted that the lower benzene ring must be
para because the two most active compounds (6 and 3)
correspond to this pattern. If the IC50(ChoK) values of the
two compounds with the meta pattern for the lower
linker (4 and 5) are compared between them and if the
two compounds with the para pattern for the lower one
(6 and 3) are compared too, it can be inferred that the
inhibitory activity notably increases when the upper
benzene ring is meta.
On the other hand, the influence of the isomerism of
the lower linker on the antiproliferative activity follows
the same trends shown in the ChoK activity. The fact
that the enzymatic inhibition and the antiproliferative
activity do not display an exact relationship may be
explained on the basis that IC50(ChoK) only assesses the
affinity of the ligand by the enzyme whereas in the
IC50(HT-29), other biological factors intervene such as the
passage through the cellular membranes. Although the
anticancer activities of the cyclophanes are modest, the
increase in lipophilicity should favorably affect the
passage through the cytoplasmic membrane.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of t h e Bis-
p yr id in iu m Cyclop h a n es (3-6). Over a suspension of the
dipyridine (7 and 8) in refluxing acetonitrile a solution of the
appropriate dibromide in acetonitrile was added dropwise
(molar ratio 1:1). The minimun dilution for cyclization to take
place was 0.01 M. The solution was heated under reflux for
24-72 h. During the course of the reaction, a white precipitate
formed. The solvent was removed in vacuo, and the precipitate
was washed with ethyl acetate and diethyl ether. The com-
pounds were purified by recrystallization from methanol. In
the Supporting Information, a comparative analysis of dilu-
tions and reaction times is undertaken to obtain each of the
four cyclophanes.
P h a r m a cology. The ex vivo ChoK inhibition22,23 and an-
tiproliferative assays against HT-29 cells9 were followed in
accordance with the protocols previously reported. The results
are recorded in the table.
Ack n ow led gm en t. We thank the Spanish CICYT
(Project SAF98-0112-C02-01) for financial support. The
award of grants from the Ministerio de Educacio´n,
Cultura y Deporte to A.C.G. and from the J unta de
Andaluc´ıa to R.M.S. is gratefully acknowledged. We are
deeply indebted to J . C. Lacal (Instituto de Investiga-
ciones Biome´dicas, Consejo Superior de Investigaciones
Cient´ıficas, Madrid, Spain) and his group for the
biological testing of compounds described in this study.
Con clu sion
The synthesis and biological activities of novel bis-
pyridinium cyclophanes as ChoK inhibitors have been
described. The ChoK inhibition activities of the cyclo-
phanes (3-6) strongly depend on the disubstitution
model of the upper and lower benzene rings. Compound
6 is the most potent human ChoK inhibitory agent
reported to date, showing activity in the low micromolar
range. Clearly the bispyridinium cyclophane derivatives
that we have described here are useful pharmacological
tools for the further investigation of ChoK inhibitors as
antiproliferative agents.
Su p p or tin g In for m a tion Ava ila ble: Detailed methods
for the cyclization process and 1H and 13C NMR assignments
of all new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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