Symmetrical bis-quinolinium compounds: New human choline kinase inhibitors with antiproliferative activity against the HT-29 cell line

Article abstract of DOI:10.1021/jm049061o

Studies have been aimed at the establishment of structure-activity relationships that define choline kinase inhibitory and antiproliferative activities of 40 bisquinolinium compounds. These derivatives have electron-releasing groups at position 4 of the q

Full text of DOI:10.1021/jm049061o

3354
J. Med. Chem. 2005, 48, 3354-3363
Symmetrical Bis-Quinolinium Compounds: New Human Choline Kinase
Inhibitors with Antiproliferative Activity against the HT-29 Cell Line
Rosario Sa´nchez-Mart´ın,† Joaqu´ın M. Campos,† Ana Conejo-Garc´ıa,† Olga Cruz-Lo´pez,† Mo´nica Ba´n˜ez-Coronel,‡
Agust´ın Rodr´ıguez-Gonza´lez,‡ Miguel A. Gallo,† Juan C. Lacal,‡ and Antonio Espinosa*,†
Departamento de Quı´mica Farmace´utica y Orga´nica, Facultad de Farmacia, Universidad de Granada, c/ Campus de Cartuja
s/n, 18071 Granada, Spain, and Instituto de Investigaciones Biome´dicas, CSIC, c/ Arturo Duperier 4, 28029 Madrid, Spain
Received November 19, 2004
Studies have been aimed at the establishment of structure-activity relationships that define
choline kinase inhibitory and antiproliferative activities of 40 bisquinolinium compounds. These
derivatives have electron-releasing groups at position 4 of the quinolinium ring. It is found
that the enzymatic inhibition is closely related to the size of the linker, the 3,3′-biphenyl moiety
being the most suitable. On the other hand, the antiproliferative activity against the HT-29
cancer cell line is less influenced by the linker type and by substituent R4. The corresponding
QSAR equation was obtained for the whole set of compounds for the antiproliferative activity,
the electronic parameter σR of R4, the molar refractivity of R8, and the lipophilic parameters
clog P and πlinker. The most potent antiproliferative agent so far described is 40 for which an
IC50 ) 0.45 µM was predicted by the QSAR equation, while its experimental value is IC50
0.20 µM.
)
Introduction
screened for their activity inhibiting isolated ChoK
(under ex vivo conditions). The 4-NR2 group made a
substantial contribution and it was suggested8 that the
role of the 4-NR2 group was electronic, via the delocal-
ization of the positive charge. The importance of frontier
orbital energies (LUMO) of model compounds has been
emphasized and interpreted.9 We have very recently
published a review on ChoK inhibitors.10 It has been
reported that increased activity of ChoK was observed
in a variety of human breast carcinomas.11 It has
recently been reported that ChoK dysregulation is a
frequent event found in a variety of human tumors such
as lung, colorectal, and prostate tumors.12 All these
encouraging results are the basis of the development of
a novel strategy in cancer treatment.
Trispyridinium compounds (3, Figure 1) are more
potent than the bispyridinium ones (2, Figure 1) as
inhibitors of human ChoK.13 Nevertheless, the tris-
pyridinium structures are less active than the bispyri-
dinium ones as antiproliferative agents because the
latter show better lipophilicity to cross the cytosolic
membranes. On the other hand, cyclizing open struc-
tures or creating an additional ring system in a given
structure represents one of the useful methods in the
search for biologically active conformations. The result
is a more constrained molecule with an imposed con-
formation. To begin with, we designed and synthesized
the most simple model of macrocyclic compounds that
have only one benzene ring as a linker (4-7).14 These
compounds are dissimilar to each other in the substitu-
tion pattern shown by the upper (first prefix) and lower
(second prefix) benzene rings (Figure 1). We have
recently studied the conformational dynamics of cyclo-
phane 415 and the inhibitory activities against human
ChoK of a set of 25 bispyridinium compounds with
electron-releasing groups at position 4 and found that
the 3,3′-biphenyl linker is the most suitable.16 We have
Choline kinase (ChoK) is the first enzyme in the
Kennedy pathway for the synthesis of phosphatidylcho-
line (PC), and it phosphorylates choline to phosphoryl-
choline (PCho) using adenosine 5′-triphosphate (ATP)
as phosphate donor.1 ras genes are the most extensively
studied oncogenes in human cancer to date. Ras proteins
play a pivotal role in cellular signal transduction and
help to regulate cellular proliferation and terminal
differentiation.2 The family of ras oncogenes has been
implicated in up to 30% of all human tumors and in
some of them up to 90%.3 Overexpression of several
oncogenes induces increased levels of ChoK and the
intracellular levels of PCho.4 Additional evidence gives
support for a role of ChoK in the generation of human
tumors. Studies using nuclear magnetic resonance
(NMR) techniques have demonstrated elevated levels
of PCho in human tumoral tissues with respect to
normal ones, including breast, colon, lung, and prostate
tumors among others.5 It is generally accepted that ras
is the most widely studied oncogene in human carcino-
genesis, and inhibition of ChoK has been demonstrated
to be a novel efficient antitumor strategy in oncogene-
transformed cells.6 These primary observations were
later extrapolated in vivo in the nude mice system.7
Research on ChoK inhibitors has correlated the
inhibitory effect on proliferation by symmetrical bis-
quaternary compounds with the ability to inhibit the
production of PCho in whole cells.6c When the 1,2-
ethylene-p-(bisbenzyldimethyl-diyl) moiety was used as
a linker between the two 4-substituted pyridinium
cationic heads (1, Figure 1),8 the structures were
* Address for correspondence: Departamento de Qu´ımica Farma-
ce´utica y Orga´nica, Facultad de Farmacia, c/ Campus de Cartuja s/n,
18071 Granada, Spain. Tel: +34 958 243850. Fax: +34 958 243845.
E-mail: aespinos@ugr.es.
† Universidad de Granada.
‡ Instituto de Investigaciones Biome´dicas.
10.1021/jm049061o CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/13/2005

Symmetrical Bis-Quinolinium Compounds
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3355
Figure 1.
proposed a model for the inhibition of ChoK by using
cyclophanes 4-7.17
With the aim of obtaining new structure-activity
relationships and studying in depth the structural
parameters that define the ChoK inhibitory and anti-
proliferative activities, the synthesis of a new set of
compounds is proposed based on changing the pyri-
dinium for quinolinium moieties of the biscationic
acyclic compounds.
The aim of this paper specifically focuses on the two
following aspects: (1) the effect to be expected on the
ex vivo human ChoK inhibitory activity by a variation
in the linker that connects the quinolinium cations
having electron-releasing groups at their position 4,
with other different groups at positions 3, 7, and 8 of
the heterocycle (compounds 8-48, Figure 1); (2) the
influence of the factors that control the antiproliferative
activities of such compounds.
Chemistry. Forty end compounds, included in three
series (according to their cationic heads), of which each
is divided in three subseries (according to their linker),
have been synthesized (Tables 1-3). They are biscat-
ionic compounds that consist of a linker and two cationic
heads which are 4-substituted quinolinium rings with
tertiary cyclic and acyclic amino groups. Figure 2 and
Scheme 1 (compound 68) show the quinoline structures
that are to be quaternized.
The synthesis of 7-unsubstituted-4-aminoquinolines
such as 4-aminoquinoline (49),18 4-(dimethylamino)-
quinoline (50),19 and 4-N-(methylanilino)quinoline (53)20
have been reported. Although 4-anilinoquinoline (52)21
Figure 2. Structures of quinolines 49-65. Reagents: (a) R4H,
heating; (b) SnCl2, glacial acetic acid.
was first reported by Backeberg, we decided to prepare
it by heating at reflux a mixture of 4-chloroquinoline
and aniline in glacial acetic acid (88%). When this
procedure was applied for the reaction between 4-chlo-
roquinoline and 4-chloro-N-methylaniline, the new com-
pound 4-(4-chloro-N-methylanilino)quinoline (54) was
obtained (97%).
7-Chloro-4-aminoquinoline (55)22 was prepared ac-
cording to a known procedure. Although 7-chloro-4-

3356 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Sa´nchez-Martı´n et al.
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) Peracetic acid, glacial acetic acid,
reflux, 3 h. (b) HNO3/H2SO4, 100 °C, 3 h. (c) H2, Pd/C, MeOH, 3 h.
(dimethylamino)quinoline (56)23 was first reported by
Gupton et al., we decided to apply the same methodology
used for the synthesis of 50, but changing 4-chloro-
quinoline for 4,7-dichloroquinoline. The target molecule
was obtained with a 40% yield, while in the original
paper23 compound 56 was obtained with a 24% yield.
Although the synthesis of 7-chloro-4-N-(methylanilino)-
quinoline (58)24 was known, a different route from the
one published was used. The procedure used for the
synthesis of 52 was applied for the synthesis of 57
between 4,7-dichloroquinoline and pyrrolidine (50%
yield), 4,7-dichloroquinoline and N-methylaniline (to
yield 58‚HBr with a 75% yield, after adding a solution
of hydrogen bromide in glacial acetic acid), and 4,7-
dichloroquinoline and 4-chloro-N-methylaniline (to pro-
duce 59 with a 59% yield). Before the quaternization
reaction, 58‚HBr was converted to the free base by
treating it with a sodium hydroxyde aqueous solution
and subsequent extraction with diethyl ether.
For the quinolines belonging to the third series,
4-chloro-7-nitro-8-methylquinoline25 was treated with
ammonia gas in phenol to produce 60 (with a 65% yield),
with N-methylaniline in glacial acetic acid at reflux to
give 62 (with a 60% yield), and with 4-chloro-N-
methylaniline in glacial acetic acid at reflux to provide
64 (with a 50% yield). Reduction of nitro compounds 60,
62, and 64 with SnCl2 in glacial acetic acid rendered
61 (75%), 63 (75%), and 65 (70%), respectively.
Finally, the starting material for the synthesis of
4-amino-3-methylquinoline (68) was 3-methylquinoline
(Scheme 1). The sequence of reactions was the follow-
ing: (a) oxidation with peracetic acid in glacial acetic
acid under reflux to yield 66 (53%); (b) nitration at
position 4 of the heterocyclic structure to yield 67 (70%);
and (c) reduction of both the nitro and the N-oxide
groups to produce 68 (82%).
Three different types of linkers have been used. The
3,3′-bis(bromomethyl)biphenyl linker was obtained
through a radical benzylic halogenation of the com-
mercially available 3,3′-dimethylbiphenyl with N-bro-
mosuccinimide (NBS) and benzoyl peroxide as initia-
tor.26 The syntheses of 4,4′-bis(bromomethyl)biphenyl27,28
and 4,4′-bis(bromomethyl)bibenzyl29 were carried out by
reaction between biphenyl (or bibenzyl), formaldehyde
(or its polymers), and hydrogen bromide in the presence
of o-phosphoric acid.
a Reagents and conditions: (a) Butanone, sealed tube, 100 °C.
carried out in a sealed tube and at a temperature of 100
°C. Compounds 8, 15, and 22 have been previously
described by one of us.30
Biological Testing. Compounds 8-48 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 biological membranes. The effects on
cell proliferation by the ChoK inhibitors in ras-trans-
formed cells were next investigated on the HT-29 cell
line (in vitro assay for 6 days). This cell line was
established from a colon adenocarcinoma, one of the
most frequent solid human cancers that is resistant to
chemotherapy,31 making these cells appropriate for the
search of new antitumor drugs. IC50 values were ob-
tained from nonlinear least-squares fit of the Hill
equation to the data. The activity in the in vitro assay
reflected the pharmacodynamic properties of the com-
pounds rather than their affinity for the enzyme.
Results and Discussion
Structure-Activity Relationships (SAR). Biologi-
cal results of compounds 8-48 are shown in Tables 1,
2, and 3, corresponding to series A, B and C, as a
function of group located at position 7 of the quinolinium
ring. Each series has been divided into three subseries
depending on the linker nature.
7-Unsubstituted Bisquinolium Derivatives (ser-
ies A). Biological results of compounds 8-27 (series A)
are shown in Table 1. From the biological results
obtained in the subfamily with the 3,3′-biphenyl linker,
it can be deduced that a good correlation generally exists
between ChoK inhibition and antiproliferative activity.
Compound 11 shows an excellent IC50 against ChoK
inhibition, and it suggests that the introduction of
cycloalkylamino groups favors the inhibitory activity.
Moreover, its antiproliferative activity is equivalent to
the ChoK inhibition. When the substituent R4 is an
anilino group, it is observed that the three derivatives
12, 13, and 14 are potent ChoK inhibitors, the N-
methylanilino group being better than anilino and
4-chloro-N-methylanilino. This might indicate that the
N-methylanilino group is the most appropriate sub-
stituent in this type of biscationic structures. If this
series is analyzed as a group it is observed that the
The synthesis of the acyclic final compounds was
carried out by heating the corresponding bromide and
heterocyclic derivatives (in a molar ratio 1:2) using
butanone as solvent (Scheme 2). The reaction was

Symmetrical Bis-Quinolinium Compounds
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3357
Table 1. IC50 ChoK and HT-29 Values for the Bisquinolinium
Compounds (series A)
Table 2. IC50 ChoK and HT-29 Values for the Bisquinolinium
Compounds (series B)
a 4CNMA ) 4-chloro-N-methylanilino.
interfere with the interaction between the compound
and the enzyme.
a 4CNMA ) 4-chloro-N-methylanilino.
7-Chloro-Substituted Bisquinolium Derivatives
(series B). Table 2 shows the biological results for
compounds 28-42 (series B). The synthesis of series B
of this bis(7-chloroquinolinium) compounds was carried
out to analyze the influence that an electron-withdraw-
ing group has on the ChoK inhibitory activity. If the
human ChoK inhibitory activities of series B is com-
pared with the same values for the 7-unsubstituted
series (series A), it is deduced that such an inhibition
is influenced by the chlorine atom. Thus, the inhibitory
activity always decreases regardless of the linker na-
ture. Nevertheless, the antiproliferative activity is
barely affected by the introduction of the chlorine atom.
The bis(7-chloroquinolinium) compounds with the
3,3′-biphenyl spacer are more active than compounds
with the 4,4′-biphenyl spacer, as happened with the bis-
(7-unsubstituted quinolinium) salts. It must be pointed
out that cycloalkylamino or arylamino substituents are
markedly better enzymatic inhibitors than their amino
or dimethylamino derivatives. Within series B the most
active antiproliferative agent against HT-29 so far
described, 40, is found with an IC50 in the in vitro assay
) 0.20 µM.
linker type has an influence on the ChoK inhibition, the
3,3′-biphenyl being the most suitable one. The IC50
values against ChoK of 4,4’-bibenzyl derivatives are
slightly poorer, and a clear decrease of such an activity
is observed with the 4,4’-biphenyl spacer. This fact
reaffirms previous results related to the influence of the
linker on the ChoK activity.16 Thus, the bibenzyl spacer
allows the compounds to adopt the appropriate confor-
mation on binding to the enzyme, due to the higher
flexibility caused by the ethylene bridge between the
two phenyl rings. On the other hand, the antiprolifera-
tive capacity against the human colon cell line HT-29
is less influenced by both the linker spacer and the R4
substituent, the obtained values being similar for the
three subseries. This fact leads us to think that these
structures could act, apart from on ChoK, at another
level in the cellular signaling triggered by the activation
of ras oncogene. It can be deduced that substituents R4
that give rise to higher ChoK inhibitory and antipro-
liferative activities are cycloalkylamino and phenyl-
amino groups.
Influence of the Introduction of an Amino Group
at Position 7 of the Quinolinium Ring (series C).
The biological results of compounds 43-48 (series C)
are shown in Table 3. In the following phase of this
study, the biscationic structures with an amino group
at position 7 of the quinolinium ring were assayed.
Moreover, in such compounds a methyl group was
introduced at position 8 to simplify the synthesis. The
On the other hand, if each subfamily is analyzed
individually, on comparing the bis(4-aminoquinolinium)
derivatives (8 and 15) with the analogous derivatives
with a methyl group at position 3 (9 and 16, respec-
tively), both the ChoK inhibitory and the antiprolifera-
tive activities decrease (Table 1). This may be due to a
steric hindrance caused by the methyl group that might

3358 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Sa´nchez-Martı´n et al.
Table 3. IC50 ChoK and HT-29 Values for the Bisquinolinium
Compounds (series C)
the more potent is the compound, together with the
descriptors necessary for the establishment of the
corresponding QSAR equation. The octanol-water par-
tition coefficient, used in its logarithmic form (log P), is
the most widely accepted measure of lipophilicity.
Reproducibility and accuracy of experimental log P
determinations are not exact for extremely lipophilic
and/or hydrophilic compounds such as the bisquino-
linium structures 8-48. Fragmental methods make it
possible to create data banks and to perform log P
calculations by computer.
The clog P values of the bis-salts were calculated by
using the Ghose-Crippen modified atomic contribution
system32 (ATOMIC5 option) of the PALLAS 2.0 pro-
33
gram.
π
is the substituent constant for the linker
spacer
calculated by using the Ghose-Crippen modified atomic
contribution system32 (ATOMIC5 option) of the PALLAS
2.0 program.33 One of the most important chemicophysi-
cal properties used in QSAR studies is the molar
refractivity (MR). It has been shown to be related to
lipophilicity, molar volume, and steric bulk.34 Most often
the MR values are scaled by a factor of 0.1 to achieve
reasonable values of the regression coefficients of the
resulting QSAR equations. The significance of MR in
QSAR equations of some ligand-enzyme interactions
has being interpreted with the help of 3D structures.
These investigations showed that substituents modeled
by MR bind in polar areas, while substituents modeled
by π bind in a hydrophobic space.35,36 Correspondingly,
a positive sign of MR in a QSAR equation can be
explained by binding the substituents to a polar surface,
while a negative sign or a nonlinear relationship
indicates a limited area or steric hindrance at this
binding site.34 The Hammett-type σR values for the
amino, dimethylamino, and anilino groups were taken
from the Hansch and Leo tables,37 while we have
previously published38 such values for the pyrrolidino,
perhydroazepino, and N-methylanilino groups. The
same methodology38 was used to estimate the unknown
σR value for the 4-chloro-N-methylanilino group by using
the 13C chemical shifts of the previously reported
compounds 1,1′-(biphenyl-3,3′-diylmethylene)bis[4-(4-
chloro-N-methylanilino)pyridinium] dibromide and 1,1′-
(biphenyl-4,4′-diylmethylene)bis[4-(4-chloro-N-methyl-
anilino)pyridinium] dibromide.16
a 4CNMA ) 4-chloro-N-methylanilino.
ChoK inhibitory activity of these bis(7-amino-8-meth-
ylquinolinium) compounds (series C) was clearly lower
than the corresponding series A and B. 46 and 48 were
the only compounds, whose substituent at position 4
is the 4-chloro-N-methylanilino group, that showed
notable IC50 ex vivo values (although such activities
were not outstanding compared with the values ob-
tained for the previously mentioned bisquinolinium
derivatives). Interestingly, despite showing a marginal
ChoK inhibitory activity, compounds of series C showed
antiproliferative activities against HT-29, which rein-
forced the above-mentioned hypothesis that these types
of structures could act in another point of the trans-
duction process (it must be remembered that the mech-
anism by which PCho transmits the mitogenic signal
to the nucleus is still unknown). The starting hypothesis
supposed that the introduction of an electron-releasing
group at position 7 should increase the ChoK inhibitory
activity in these biscationic type structures. On the
contrary, the enzymatic inhibitory potency was drasti-
cally diminished. It is probable that the methyl group
at position 8, introduced to simplify the synthetic route,
caused a steric hindrance in its interaction with the
enzyme that would explain the decrease of ChoK activ-
ity. Therefore, it was not possible to assess the influence
of the amino group at position 7 of the quinolinium ring
with the results obtained. The future synthesis and
biological tests of their analogous compounds without
the methyl group in such a position will confirm this
hypothesis.
When the volume effects (MR8) (the subscript refers
to the position of the substituent), calculated global
lipophilicity (clog P), the substituent constant of the
linker, and the electronic parameters (σR4) of the R4
substituent were taken into account for the antiprolif-
erative activity, eq 1 was obtained:
QSAR of the Antiproliferative Activity against
the HT-29 Cell Line. We have tried to correlate ChoK
inhibitory activity for the whole set of compounds (series
A, B, and C) with the electronic and lipophilic param-
eters, but all the attempts turned out to be fruitless. In
general it can be deduced (see Tables 1, 2, and 3) that
the activity against the HT-29 cell line is greater than
the corresponding activity against ChoK, with which the
symmetrical bisquaternized salts could act on another
point of the pathway triggered by ras activation. Table
4 (see Supporting Information) shows all the ChoK
inhibitors arranged according to decreasing antiprolif-
2
p(IC50)HT-29 ) - 2.66 - 0.03 (( 0.00) MR8
+
0.10 (( 0.02) clog P + 1.05 (( 0.31) πlinker
-
3.73 (( 0.71) σR (1)
n ) 40, r ) 0.920, s ) 0.223, F4,35
)
47.856, R < 0.001
where n is the number of compounds, r is the correlation
coefficient, and s is the standard deviation between
estimated and actual antiproliferative values. The
Fisher test is highly significant here (R < 0.001). The
number in parentheses accounts for the standard error
of the regression coefficients.
erative activity, where p(IC50)HT-29 ) -log(IC50)HT-29
,
bearing in mind that the higher the value of p(IC50)HT-29

Symmetrical Bis-Quinolinium Compounds
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3359
p(IC50)HT-29 exptl
)
- 0.35 + 1.05 (( 0.08) p(IC50)HT-29 theoret (2)
n ) 40, r ) 0.916, s ) 0.220, F1,38 ) 198.854,
R < 0.001
Conclusions
From the biological assays obtained under ex vivo
conditions on human ChoK from all the final com-
pounds, it can be deduced that an increase of lipophi-
licity generated by the introduction of two quinolinium
rings as cationic heads (in relation to the two pyridinium
rings16) improves the antiproliferative activity. On the
other hand, the ChoK inhibition is influenced by the
introduction of a chlorine atom at position 7 of the
quinolinium rings. Thus, the inhibitory activity against
the enzyme always decreases regardless of the nature
of the linker. Nevertheless, the antiproliferative activity
is scarcely affected by the introduction of a chlorine
atom, which is logical because many other factors have
an influence on the HT-29 activity. The bis(7-amino-
quinolinium) compounds show antiproliferative activi-
ties despite showing a marginal ChoK inhibitory activ-
ity. This strongly supports the hypothesis that these
types of structures might act either in another point of
the signaling pathway triggered after the activation of
the ras oncogene or in another pathway involved in the
cellular proliferation. Finally, the ChoK inhibition is
influenced by the type of arylalkyl spacer of compounds.
Thus, the best spacer is the 3,3′-biphenyl. Moreover, the
positive influence of the introduction of electron-releas-
ing substituents at position 4 in the quinolinium ring
is confirmed, and the introduction of a methyl group at
position 3 of the quinolinium ring gives rise to a
decrease in the ChoK inhibitory activity.
Figure 3. Graph between experimental and predicted anti-
proliferative activities for the test set.
In deriving eq 1, 16 was not included because this
compound is not a ChoK inhibitor (see Table 1). Equa-
tion 1 gives a good cross-validated r2 value (q2) of
CV
0.837. A most significant aspect of this study is that
every data point was included in the formulation of eq
1. Such an outcome is rarely found and merits a special
consideration. We find this to be quite unusual since
one usually finds some outliers in QSAR works which
must be omitted to obtain a high correlation.
From eq 1 the following aspects must be highlighted:
(i) the coefficient of MR8 is a squared negative term,
and hence the presence of the methyl group at position
8 is detrimental in relation to the antiproliferative
activity; accordingly, it is advisable that a hydrogen
atom be in this position because this atom has a smaller
value for MR; (ii) there is no electronic contribution of
the group located at position 7 of the quinolinium ring.
This is most remarkable in the case of the 7-NH2 group,
which is located at the same relative position (although
in the other aromatic ring) as the 4-substituent in
relation to the N+ atom, and its presence should have
facilitated even more the delocalization of the positive
charge of the endocyclic N atom. A plausible explanation
is that the 8-methyl group exerts a steric hindrance
making it impossible for the 7-NH2 group to adopt a
coplanar disposition in relation to the aromatic ring.
This is necessary for the electronic delocalization to take
place. Therefore, 7-substituent contributes only to the
global lipophilicity of the molecules; (iii) finally, lipo-
philicity contributes in two aspects to the antiprolifera-
tive activity: on one hand, a global contribution (clog
P) and on the other, a contribution at a specific site on
the molecules (πlinker). Both descriptors are orthogonal,
and therefore the participation of both is justified in the
QSAR equation. The relative contribution of πlinker in
eq 1 is higher than that of log P, and it can be
hypothesized that favorable hydrophobic interactions
between the enzyme and the linker would modulate the
coupling inhibitor-ChoK. Although, according to eq 1 the
increase in the global lipophilicity and in the lipophi-
licity of the linker would augment the antiproliferative
activity, the solubility was the reason for limiting the
spacers to the 3,3′-, 4,4′-biphenyl, and 4,4′-bibenzyl
moieties.
Experimental Section
(a) Chemistry. For general procedures see ref 14. All
compounds were dried at 40 °C and 0.1 mmHg for 24 h, but
many which appear to be solvates held tenaciously to water.
49,18 50,19 53,20 55,22 4-chloro-7-nitro-8-methylquinoline,25 3,3′-
Bis(bromomethyl)biphenyl,26 4,4′-bis(bromomethyl)biphenyl,27,28
and 4,4′-bis(bromomethyl)bibenzyl29 were synthesized accord-
ing to literature procedures. In the NMR data, the abbreviation
pst means a pseutotriplet.
General Experimental Procedure for the Preparation
of Bisquinolinium Compounds. A solution of the linker [bis-
(bromomethyl) compound]26-29 and the corresponding (4-sub-
stituted)quinoline (in a 1/2 molar ratio) was heated at 100 °C
in a sealed tube for a period of time that went from 15 and
192 h. After filtration and thorough washing with butanone,
ethyl acetate, and diethyl ether, the solid was purified by
recrystallization from EtOH or MeOH after adding diethyl
ether to turbidity.
1,1′-(Biphenyl-3,3′-diylmethylene)bis(4-amino-3-meth-
ylquinolinium) dibromide (9): Yield: 77%. Mp 291-292 °C.
1H NMR (300 MHz, DMSO-d6) δ 9.23 (bs, 2H); 8.93 (s, 2H);
8.60 (d, J ) 8.4, 2H); 8.46 (bs, 2H); 8.04 (d, J ) 8.4, 2H); 7.89
(pst, J ) 7.1, 2H); 7.74 (s, 2H); 7.68 (pst, J ) 7.5, 2H); 7.56 (d,
J ) 7.7, 2H); 7.41 (t, J ) 7.7, 2H); 7.12 (d, J ) 7.7, 2H); 5.88
(s, 4H); 2.31 (s, 6H). HR LSIMS (m/z) calcd for C34H32N4Br2
(M - HBr - Br)+ 495.2549; found 495.2549. Anal. (C34H32N4-
Br2‚H2O) C, H, N.
1,1′-(Biphenyl-3,3′-diylmethylene)bis(4-dimethylami-
noquinolinium) dibromide (10): Yield: 60%. Mp 278-279
°C. 1H NMR (400 MHz, DMSO-d6) δ 8.93 (d, J ) 7.6, 2H); 8.41
(d, J ) 8.6, 2H); 8.10 (d, J ) 8.7, 2H); 7.91 (pst, J ) 8.1, 2H);
7.80 (s, 2H); 7.63 (pst, J ) 8.1, 2H); 7.58 (d, J ) 7.7, 2H); 7.43
When the experimental p(IC50)HT-29 values are cor-
related with the theoretical ones (see Table 4) calculated
by eq 1, eq 2 is obtained corresponding to the straight
line represented in Figure 3:

3360 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Sa´nchez-Martı´n et al.
(t, J ) 7.7, 2H); 7.17 (d, J ) 7.7, 2H); 7.12 (d, J ) 7.6, 2H);
5.93 (s, 4H); 3.49 (s, 12H). HR LSIMS (m/z) calcd for C36H36N4-
Br2 (M - Br)+ 603.2123; found 603.2123. Anal. (C36H36N4Br2‚
H2O) C, H, N.
1,1′-(Biphenyl-3,3′-diylmethylene)bis[4-(perhydroaze-
pino)quinolinium] dibromide (11): Yield: 84%. Mp 293-
294 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.83 (d, J ) 7.7, 2H);
8.34 (d, J ) 8.2, 2H); 8.05 (d, J ) 8.5, 2H); 7.89 (pst, J ) 7.8,
2H); 7.76 (s, 2H); 7.61 (pst, J ) 7.5, 2H); 7.57 (d, J ) 7.7, 2H);
7.43 (t, J ) 7.7, 2H); 7.15 (m, 4H); 5.90 (s, 4H); 4.00 (t, 8H);
1.94 (bs, 8H); 1.58 (bs, 8H). HR LSIMS (m/z) calcd for C44H48N4-
Br2 (M - Br)+ 711.3062; found 711.3062. Anal. (C44H48N4Br2‚
H2O) C, H, N.
°C. 1H NMR (300 MHz, DMSO-d6) δ 9.15 (d, J ) 7.4, 2H); 8.09
(d, J ) 8.9, 2H); 7.80 (pst, J ) 8.3, 2H); 7.66 (d, J ) 8.3, 2H);
7.52-7.38 (m, 20H); 7.32 (t, J ) 7.6, 2H); 6.03 (s, 4H); 3.75 (s,
6H). HR LSIMS (m/z) calcd for C46H40N4Br2 (M - HBr - Br)+
647.3175; found 647.3177. Anal. (C46H40N4Br2‚H2O) C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis[4-(4-chloro-N-
methylanilino)quinolinium] dibromide (21): Yield: 30%.
Mp 255-257 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.19 (d, J )
7.4, 2H); 8.12 (d, J ) 8.9, 2H); 7.83 (pst, J ) 7.5, 2H); 7.66 (d,
J ) 8.2, 2H); 7.55 (d, J ) 8.8, 4H); 7.44 (d, J ) 8.9, 4H); 7.56-
7.39 (m, 12H); 6.05 (s, 4H); 3.73 (s, 6H). HR LSIMS (m/z) calcd
for C46H38N4Cl2Br2 (M - Br)+ 795.1657; found 795.1658. Anal.
(C46H38N4Cl2Br2‚2H2O) C, H, N.
1,1′-(Biphenyl-3,3′-diylmethylene)bis(4-anilinoquino-
linium) dibromide (12): Yield: 65%. Mp 316-318 °C. 1H
NMR (400 MHz, DMSO-d6) δ 11.11 (s, 2H); 8.95 (d, J ) 7.4,
2H); 8.84 (d, J ) 8.5, 2H); 8.20 (d, J ) 8.9, 2H); 8.02 (pst, J )
7.8, 2H); 7.82 (pst, J ) 7.7, 2H); 7.76 (s, 2H); 7.62-7.53 (m,
8H); 7.48-7.41 (m, 6H); 7.21 (d, J ) 7.7, 2H); 6.87 (d, J ) 7.4,
2H); 5.99 (s, 4H). HR LSIMS (m/z) calcd for C44H36N4Br2 (M
- HBr - Br)+ 619.2862; found 619.2861. Anal. (C44H36N4Br2)
C, H, N.
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis[4-
(dimethylamino)quinolinium] dibromide (23): Yield: 93%.
Mp 255-257 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.82 (d, J )
7.6, 2H); 8.40 (d, J ) 8.6, 2H); 7.99 (d, J ) 8.7, 2H); 7.88 (pst,
J ) 8.1, 2H); 7.62 (pst, J ) 7.9, 2H); 7.20 (d, J ) 8.3, 4H); 7.17
(d, J ) 8.3, 4H); 7.10 (d, J ) 7.6, 2H); 5.81 (s, 4H); 3.48 (s,
12H); 2.76 (s, 4H). HR LSIMS (m/z) calcd for C38H40N4Br2 (M
- Br)+ 631.2436; found 631.2437. Anal. (C38H40N4Br2‚H2O) C,
H, N.
1,1′-(Biphenyl-3,3′-diylmethylene)bis[4-(N-methylanili-
no)quinolinium] dibromide (13): Yield: 64%. Mp 274-275
°C. 1H NMR (300 MHz, DMSO-d6) δ 9.25 (d, J ) 7.4, 2H); 8.17
(d, J ) 8.9, 2H); 7.85 (s, 2H); 7.81 (pst, J ) 8.0, 2H); 7.63 (d,
J ) 8.3, 2H); 7.52-7.39 (m, 16H); 7.32 (pst, J ) 7.7, 2H); 7.23
(d, J ) 7.4, 2H); 6.09 (s, 4H); 3.76 (s, 6H). HR LSIMS (m/z)
calcd for C46H40N4Br2 (M - Br)+ 727.2436; found 727.2436.
Anal. (C46H40N4Br2‚3H2O) C, H, N.
1,1′-(Biphenyl-3,3′-diylmethylene)bis[4-(4-chloro-N-
methylanilino)quinolinium] dibromide (14): Yield: 45%.
Mp 217-218 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.24 (d, J )
7.4, 2H); 8.18 (d, J ) 8.9, 2H); 7.84 (s, 2H); 7.63 (d, J ) 7.5,
2H); 7.56-7.43 (m, 18H); 7.23 (d, J ) 7.4, 2H); 6.08 (s, 4H);
3.74(s, 6H). HR LSIMS (m/z) calcd for C46H38N4Cl2Br2 (M -
Br)+ 795.1657; found 795.1656. Anal. (C46H38N4Cl2Br2‚3H2O)
C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis(4-amino-3-meth-
ylquinolinium) dibromide (16): Yield: 64%. Mp 299-301
°C. 1H NMR (300 MHz, DMSO-d6) δ 9.31 (bs, 2H); 8.93 (s, 2H);
8.56 (d, J ) 8.5, 2H); 8.47 (bs, 2H); 8.00 (d, J ) 8.2, 2H); 7.90
(m, 8H); 7.89 (pst, J ) 8.2, 2H); 7.68 (pst, J ) 8.5, 2H); 5.87
(s, 4H); 2.25 (s, 6H). HR LSIMS (m/z) calcd for C34H32N4Br2
(M - HBr - Br)+ 495.2549; found 495.2549. Anal. (C34H32N4-
Br2‚H2O) C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis[4-(dimethylami-
no)quinolinium] dibromide (17): Yield: 61%. Mp 224-225
°C. 1H NMR (400 MHz, DMSO-d6) δ 8.86 (d, J ) 7.6, 2H); 8.41
(d, J ) 8.6, 2H); 8.03 (d, J ) 8.9, 2H); 7.90 (pst, J ) 8.1, 2H);
7.63 (pst, J ) 8.1, 2H); 7.61 (d, J ) 8.3, 4H); 7.34 (d, J ) 8.3,
4H); 7.12 (d, J ) 7.6, 2H); 5.90 (s, 4H); 3.48 (s, 12H). HR
LSIMS (m/z) calcd for C36H36N4Br2 (M - Br)+ 603.2123; found
603.2123. Anal. (C36H36N4Br2‚H2O) C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis[4-(perhydroaze-
pino)quinolinium] dibromide (18): Yield: 82%. Mp 273-
274 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.78 (d, J ) 7.7, 2H);
8.34 (d, J ) 7.9, 2H); 8.00 (d, J ) 8.5, 2H); 7.88 (pst, J ) 7.8,
2H); 7.62 (d, J ) 8.4, 4H); 7.60 (pst, J ) 7.5, 2H); 7.33 (d, J )
8.4, 4H); 7.14 (d, J ) 7.7, 2H); 5.87 (s, 4H); 4.01 (t, 8H); 1.94
(bs, 8H); 1.59 (bs, 8H). HR LSIMS (m/z) calcd for C44H48N4Br2
(M - Br)+ 711.3062; found 711.3060. Anal. (C44H48N4Br2‚
0.5H2O) C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis[4-(anilino)quino-
linium] dibromide (19): Yield: 66%. Mp 326-327 °C. 1H
NMR (400 MHz, DMSO-d6) δ 11.10 (s, 2H); 8.88 (d, J ) 7.4,
2H); 8.83 (d, J ) 8.5, 2H); 8.14 (d, J ) 8.9, 2H); 8.02 (pst, J )
7.8, 2H); 7.82 (pst, J ) 7.7, 2H); 7.62 (d, J ) 8.1, 4H); 7.61 (t,
J ) 7.8, 4H); 7.53 (d, J ) 7.8, 4H); 7.47 (t, J ) 7.3, 4H); 7.35
(d, J ) 8.1, 4H); 6.90 (d, J ) 7.4, 2H); 5.97 (s, 4H). HR LSIMS
(m/z) calcd for C44H36N4Br2 (M - HBr - Br)+ 619.2862; found
619.2862. Anal. (C44H36N4Br2‚H2O) C, H, N.
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis[4-
(perhydroazepino)quinolinium] dibromide (24): Yield:
1
76%. Mp 292-293 °C. H NMR (400 MHz, DMSO-d6) δ 8.76
(d, J ) 7.7, 2H); 8.33 (d, J ) 8.2, 2H); 7.96 (d, J ) 8.5, 2H);
7.87 (pst, J ) 7.8, 2H); 7.60 (pst, J ) 7.5, 2H); 7.20 (d, J )
8.5, 4H); 7.17 (d, J ) 8.5, 4H); 7.13 (d, J ) 7.7, 2H); 5.79 (s,
4H); 3.99 (t, 8H); 2.77 (s, 4H); 1.94 (bs, 8H); 1.58 (bs, 8H). HR
LSIMS (m/z) calcd for C46H52N4Br2 (M - Br)+ 739.3375; found
739.3375. Anal. (C46H52N4Br2‚1H2O) C, H, N.
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis[4-
(anilino)quinolinium] dibromide (25): Yield: 77%. Mp
1
284-286 °C. H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 2H);
8.85 (d, J ) 7.4, 2H); 8.83 (d, J ) 8.5, 2H); 8.01 (d, J ) 8.9,
2H); 8.00 (pst, J ) 7.8, 2H); 7.82 (pst, J ) 7.7, 2H); 7.60 (t, J
) 7.6, 4H); 7.53 (d, J ) 7.6, 4H); 7.46 (t, J ) 7.6, 4H); 7.19 (s,
8H); 6.88 (d, J ) 7.4, 2H); 5.88 (s, 4H); 2.77 (s, 4H). HR LSIMS
(m/z) calcd for C46H40N4Br2 (M - HBr - Br)+ 647.3174; found
647.3176. Anal. (C46H40N4Br2‚1.9H2O) C, H, N.
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis[4-(N-
methylanilino)quinolinium] dibromide (26): Yield: 64%.
Mp 279-280 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.14 (d, J )
7.4, 2H); 8.06 (d, J ) 8.9, 2H); 7.79 (pst, J ) 8.3, 2H); 7.52-
7.39 (m, 14H); 7.31 (t, J ) 7.6, 2H); 7.24 (s, 8H); 5.96 (s, 4H);
3.74 (s, 6H); 2.80 (s, 4H). HR LSIMS (m/z) calcd for C48H44N4-
Br2 (M - Br)+ 755.2749; found 755.2749. Anal. (C48H44N4Br2‚
H2O) C, H, N.
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis[4-(4-
chloro-N-methylanilino)quinolinium] dibromide (27):
1
Yield: 20%. Mp 212-214 °C. H NMR (300 MHz, DMSO-d6)
δ 9.19 (d, J ) 7.4, 2H); 8.10 (d, J ) 8.9, 2H); 7.82 (pst, J ) 7.5,
2H); 7.54 (d, J ) 8.8, 4H); 7.44 (d, J ) 8.9, 4H); 7.52-7.39 (m,
6H); 7.24 (s, 8H); 5.98 (s, 4H); 3.73 (s, 6H); 2.80 (s, 4H). HR
LSIMS (m/z) calcd for C48H42N4Cl2Br2 (M - Br)+ 823.1970;
found 823.1972. Anal. (C48H42N4Cl2Br2‚H2O) C, H, N.
1,1′-(Biphenyl-3,3′-diylmethylene)bis(4-amino-7-chlo-
roquinolinium) dibromide (28): Yield: 91%. Mp 277-278
1
)
°C. H NMR (300 MHz, DMSO-d6 δ 9.38 and 9.31 (bs , 4H);
8.76 (d, J ) 7.3, 2H); 8.54 (d, J ) 9.0, 2H); 8.17 (d, J ) 1.5,
2H); 7.81 (dd, J ) 9.0, 1.5, 2H); 7.68 (s, 2H); 7.59 (d, J ) 7.7,
2H); 7.45 (t, J ) 7.7, 2H); 7.18 (d, J ) 7.7, 2H); 6.93 (d, J )
7.3, 2H); 5.89 (s, 4H). HR LSIMS (m/z) calcd for C32H26N4Br2-
Cl2 (M - HBr - Br)+ 535.1456; found 535.1457. Anal.
(C32H26N4Br2Cl2‚0.2H2O) C, H, N.
1,1′-(Biphenyl-3,3′-diylmethylene)bis[7-chloro-4-(di-
methylamino)quinolinium] dibromide (29): Yield: 45%.
Mp 285-286 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.85 (d, J )
7.7, 2H); 8.42 (d, J ) 9.2, 2H); 8.18 (d, J ) 2.0, 2H); 7.78 (s,
2H); 7.65 (dd, J ) 9.2, 2.0, 2H); 7.59 (d, J ) 7.7, 2H); 7.46 (t,
J ) 7.7, 2H); 7.18 (d, J ) 7.7, 2H); 7.13 (d, J ) 7.7, 2H); 5.93
(s, 4H); 3.49 (s, 12H). HR LSIMS (m/z) calcd for C36H34N4Br2-
Cl2 (M - HBr - Br)+ 591.2082; found 591.2081. Anal.
(C36H34N4Br2Cl2‚0.7H2O) C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis[4-(N-methylanili-
no)quinolinium] dibromide (20): Yield: 58%. Mp 276-278

Symmetrical Bis-Quinolinium Compounds
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3361
1,1′-(Biphenyl-3,3′-diylmethylene)bis[7-chloro-4-(pyr-
rolidino)quinolinium] dibromide (30): Yield: 48%. Mp
299-301 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.83 (d, J ) 7.7,
2H); 8.58 (d, J ) 9.3, 2H); 8.16 (d, J ) 1.8, 2H); 7.78 (s, 2H);
7.65 (dd, J ) 9.3 and 1.8, 2H); 7.60 (d, J ) 7.7, 2H); 7.46 (t, J
) 7.7, 2H); 7.18 (d, J ) 7.7, 4H); 6.95 (d, J ) 7.7, 2H); 5.93 (s,
4H); 4.16-3.77 (bd, 8H); 2.04 (bs, 8H). HR LSIMS (m/z) calcd
for C40H38N4Br2Cl2 (M - Br)+ 723.1657; found 723.1657. Anal.
(C40H38N4Br2Cl2‚H2O) C, H, N.
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis[7-
chloro-4-(dimethylamino)quinolinium] dibromide (39):
1
Yield: 38%. Mp 252-253 °C. H NMR (400 MHz, DMSO-d6)
δ 8.77 (d, J ) 7.7, 2H); 8.41 (d, J ) 9.2, 2H); 8.07 (d, J ) 2.0,
2H); 7.65 (dd, J ) 9.0, J ) 2.0, 2H); 7.20 (d, J ) 8.3, 4H); 7.17
(d, J ) 8.3, 4H); 7.11 (d, J ) 7.7, 2H); 5.81 (s, 4H); 3.48 (s,
12H); 2.79 (s, 4H). HR LSIMS (m/z) calcd for C38H38N4Br2Cl2
(M - HBr - Br)+ 619.2395; found 619.2393. Anal. (C38H38N4-
Br2Cl2‚0.8H2O) C, H, N.
1,1′-(Biphenyl-3,3′-diylmethylene)bis[7-chloro-4-(N-
methylanilino)quinolinium] dibromide (31): Yield: 40%.
Mp 275-277 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.15 (d, J )
7.4, 2H); 8.09 (s, 2H); 7.80 (d, J ) 8.3, 2H); 7.73 (s, 2H); 7.66
(d, J ) 8.3, 2H); 7.52-7.32 (m, 16H); 7.24 (d, J ) 7.7, 2H);
6.03 (s, 4H); 3.75 (s, 6H). HR LSIMS (m/z) calcd for C46H38N4-
Br2Cl2 (M - Br)+ 795.1657; found 795.1657. Anal. (C46H38N4-
Br2Cl2‚3.9H2O) C, H, N.
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis[7-
chloro-4-(pyrrolidino)quinolinium] dibromide (40): Yield:
1
63%. Mp 285-286 °C. H NMR (400 MHz, DMSO-d6) δ 8.70
(d, J ) 7.7, 2H); 8.57 (d, J ) 9.2, 2H); 8.03 (d, J )2.0, 2H);
7.64 (dd, J ) 9.2, J ) 2.0, 2H); 7.20 (d, J ) 8.2, 4H); 7.15 (d,
J ) 8.2, 4H); 6.93 (d, J ) 7.7, 2H); 5.78 (s, 4H); 4.16-3.76 (m,
8H); 2.78 (s, 4H); 2.04 (bs, 8H). HR LSIMS (m/z) calcd for
C42H42N4Br2Cl2 (M - HBr - Br)+ 671.2708; found 671.2709.
Anal. (C42H42N4Br2Cl2‚0.5H2O) C, H, N.
1,1′-(Biphenyl-3,3′-diylmethylene)bis[7-chloro-4-(4-chlo-
ro-N-methylanilino)quinolinium] dibromide (32): Yield:
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis[7-
1
45%. Mp 220-221 °C. H NMR (300 MHz, DMSO-d6) δ 9.19
chloro-4-(N-methylanilino)quinolinium] dibromide (41):
1
(d, J ) 7.5, 2H); 8.29 (d, J ) 1.7, 2H); 7.85 (s, 2H); 7.64 (d, J
) 7.2, 2H); 7.57-7.45 (m, 16H); 7.25 (d, J ) 7.7, 2H); 6.08 (s,
4H); 3.73 (s, 6H). HR LSIMS (m/z) calcd for C46H36N4Cl4Br2
(M - HBr - Br)+ 783.1616; found 783.1616. Anal. (C46H36N4-
Cl4Br2‚1.5H2O) C, H, N.
Yield: 43%. Mp 240-242 °C. H NMR (400 MHz, DMSO-d6)
δ 9.08 (d, J ) 7.4, 2H); 8.14 (s, 2H); 7.51-7.38 (m, 16H); 7.23
(s, 8H); 5.95 (s, 4H); 3.73 (s, 6H); 2.81 (s, 4H). HR LSIMS (m/
z) calcd for C48H42N4Br2Cl2 (M - Br)+ 823.1970; found 823.1968.
Anal. (C48H42N4Br2Cl2‚4H2O) C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis(4-amino-7-chlo-
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis[7-
roquinolinium) dibromide (33): Yield: 70%. Mp 273-274
chloro-4-(4-chloro-N-methylanilino)quinolinium] dibro-
1
1
°C. H NMR (400 MHz, DMSO-d6) δ 9.38 and 9.31 (bs , 4H);
mide (42): Yield: 48%. Mp 256-257 °C. H NMR (400 MHz,
8.73 (d, J ) 7.3, 2H); 8.54 (d, J ) 9.0, 2H); 8.14 (d, J ) 1.8,
2H); 7.80 (dd, J ) 9.0, 1.8, 2H); 7.64 (d, J ) 8.3, 4H); 7.32 (d,
J ) 8.3, 4H); 6.92 (d, J ) 7.3, 2H); 5.87 (s, 4H). HR LSIMS
(m/z) calcd for C32H26N4Br2Cl2 (M - Br)+ 615.0718; found
615.0718. Anal. (C32H26N4Br2Cl2‚0.7H2O) C, H, N.
DMSO-d6) δ 9.11 (d, J ) 7.4, 2H); 8.18 (d, J ) 1.5, 2H); 7.55
(d, J ) 8.8, 4H); 7.46 (d, J ) 8.8, 4H); 7.56-7.44 (m, 6H); 7.24
(s, 8H); 5.97 (s, 4H); 3.72 (s, 6H); 2.82 (s, 4H). HR LSIMS (m/
z) calcd for C48H40N4Cl4Br2 (M - HBr - Br)+ 811.1927; found
811.1926. Anal. (C48H40N4Cl4Br2‚2H2O) C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis[7-chloro-4-(di-
methylamino)quinolinium] dibromide (34): Yield: 45%.
Mp 225-226 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.81 (d, J )
7.7, 2H); 8.42 (d, J ) 9.2, 2H); 8.13 (d, J ) 2.0, 2H); 7.69-7.62
(m, 6H); 7.34 (d, J ) 8.3, 4H); 7.13 (d, J ) 7.7, 2H); 5.90 (s,
4H); 3.49 (s, 12H). HR LSIMS (m/z) calcd for C36H34N4Br2Cl2
(M - HBr - Br)+ 591.2082; found 591.2082. Anal. (C36H34N4-
Br2Cl2‚1.2H2O) C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis[7-chloro-4-(pyr-
rolidino)quinolinium] dibromide (35): Yield: 47%. Mp
288-289 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.76 (d, J ) 7.7,
2H); 8.58 (d, J ) 9.3, 2H); 8.10 (s, 2H); 7.65 (m, 6H); 7.33 (d,
J ) 8.2, 4H); 6.95 (d, J ) 7.7, 2H); 5.89 (s, 4H); 4.16-3.77 (m,
8H); 2.04 (bs, 8H). HR LSIMS (m/z) calcd for C40H38N4Br2Cl2
(M - HBr - Br)+ 643.2395; found 643.2396. Anal. (C40H38N4-
Br2Cl2‚1.5H2O) C, H, N.
1,1′-(Biphenyl-3,3′-diylmethylene)bis[7-amino-8-meth-
yl-4-(N-methylanilino)-quinolinium] dibromide (43):
1
Yield: 25%. Mp 327-329 °C. H NMR (400 MHz, DMSO-d6)
δ 8.43 (d, J ) 7.0, 2H); 7.57 (s, 2H); 7.46-7.32 (m, 12H); 7.29
(d, J ) 7.8, 4H); 7.23 (d, J ) 7.8, 2H); 7.09 (d, J ) 9.7, 2H);
6.98 (d, J ) 7.0, 2H); 6.74 (d, J ) 9.7, 2H); 4.72 (s, 2H); 4.70
(s, 2H); 3.59 (s, 6H); 2.40 (s, 6H). HR LSIMS (m/z) calcd for
C48H46N6Br2 (M - HBr - Br)+ 705.3706; found 705.3706. Anal.
(C48H46N6Br2‚2.1H2O) C, H, N.
1,1′-(Biphenyl-3,3′-diylmethylene)bis[7-amino-4-(4-chlo-
ro-N-methylanilino)-8-methylquinolinium] dibromide
(44): Yield: 55%. Mp 314-315 °C. 1H NMR (300 MHz, DMSO-
d6) δ 8.47 (d, J ) 6.9, 2H); 7.58 (s, 2H); 7.40 (m, 6H); 7.39 (d,
J ) 8.7, 2H); 7.34 (t, J ) 7.7, 2H); 7.27 (d, J ) 7.7, 2H); 7.23
(d, J ) 8.7, 2H); 7.13 (d, J ) 9.7, 2H); 7.03 (d, J ) 6.9, 2H);
6.82 (d, J ) 9.7, 2H); 4.58 (s, 2H); 4.56 (s, 2H); 3.56 (s, 6H);
2.41 (s, 6H). HR LSIMS (m/z) calcd for C48H44N6Cl2Br2 (M -
HBr - Br)+ 773.2926; found 773.2916. Anal. (C48H44N6Cl2Br2‚
H2O) C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis[7-chloro-4-(N-
methylanilino)quinolinium] dibromide (36): Yield: 34%.
Mp 284-286 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.12 (d, J )
7.5, 2H); 8.21 (s, 2H); 7.70 (d, J ) 8.3, 2H); 7.50 (t, J ) 8.3
and 6.9, 2H); 7.46-7.40 (m, 20H); 6.06 (s, 4H); 3.75 (s, 6H).
HR LSIMS (m/z) calcd for C46H38N4Br2Cl2 (M - Br)+ 795.1657;
found 795.1657. Anal. (C46H38N4Br2Cl2‚1.5H2O) C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis[7-chloro-4-(4-chlo-
ro-N-methylanilino)-quinolinium] dibromide (37): Yield:
1,1′-(Biphenyl-4,4′-diylmethylene)bis[7-amino-8-meth-
yl-4-(N-methylanilino)quinolinium] dibromide (45): Yield:
1
45%. Mp 311-313 °C. H NMR (400 MHz, DMSO-d6) δ 8.46
(bs, 2H); 7.47 (d, J ) 8.2, 4H); 7.41-7.29 (m, 14H); 7.21 (d, J
) 8.2, 4H); 7.07 (d, J ) 9.4, 2H); 6.94 (d, J ) 6.9, 2H); 6.64 (d,
J ) 9.4, 2H); 4.68 (s, 2H); 4.66 (s, 2H); 3.55 (s, 6H); 2.43 (s,
6H). HR LSIMS (m/z) calcd for C48H46N6Br2 (M - HBr - Br)+
705.3706; found 705.3706. Anal. (C48H46N6Br2‚2.1H2O) C, H,
N.
1
48%. Mp 276-277 °C. H NMR (400 MHz, DMSO-d6) δ 9.14
(d, J ) 7.4, 2H); 8.23 (d, J ) 1.6, 2H); 7.73 (d, J ) 8.3, 2H);
7.69 (d, J ) 8.4, 4H); 7.56 (d, J ) 8.8, 4H); 7.46 (d, J ) 8.9,
4H); 7.50-7.46 (m, 6H); 7.41 (d, J ) 8.4, 4H); 6.04 (s, 4H);
3.73(s, 6H). HR LSIMS (m/z) calcd for C46H36N4Cl4Br2 (M -
HBr - Br)+ 783.1616; found 783.1614. Anal. (C46H36N4Cl4Br2)
C, H, N.
1,1′-(Biphenyl-4,4′-diylmethylene)bis[7-amino-4-(4-chlo-
ro-N-methylanilino)-8-methylquinolinium] dibromide
(46): Yield: 42%. Mp 315-317 °C. 1H NMR (300 MHz, DMSO-
d6) δ 8.46 (d, J ) 6.9, 2H); 7.51 (d, J ) 8.1, 4H); 7.41 (m, 8H);
7.34 (d, J ) 8.1, 4H); 7.23 (d, J ) 8.7, 4H); 7.12 (d, J ) 9.7,
2H); 7.02 (d, J ) 6.9, 2H); 6.80 (d, J ) 9.7, 2H); 4.56 (s, 2H);
4.54 (s, 2H); 3.56 (s, 6H); 2.41 (s, 6H). HR LSIMS (m/z) calcd
for C48H44N6Cl2Br2 (M - HBr - Br)+ 773.2926; found 773.2926.
Anal. (C48H44N6Cl2Br2‚H2O) C, H, N.
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis(4-
amino-7-chloroquinolinium) dibromide (38): Yield: 47%.
Mp 285-290 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.42 and
9.32 (bs , 4H); 8.72 (d, J ) 7.3, 2H); 8.55 (d, J ) 9.0, 2H); 8.07
(s, 2H); 7.76 (dd, J ) 9.0, J ) 1.1, 2H); 7.17 (d, J ) 8.2, 4H);
7.14 (d, J ) 8.2, 4H); 6.94 (d, J ) 7.3, 2H); 5.79 (s, 4H); 2.77
(s, 4H). HR LSIMS (m/z) calcd for C34H30N4Br2Cl2 (M - HBr
- Br)+ 563.1769; found 563.1769. Anal. (C34H30N4Br2Cl2‚
1.5H2O) C, H, N.
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis[7-
amino-8-methyl-4-(N-methylanilino)quinolinium] dibro-
1
mide (47): Yield: 37%. Mp 288-290 °C. H NMR (400 MHz,
DMSO-d6) δ 8.41 (d, J ) 7.0, 2H); 7.44 (t, J ) 7.8, 4H); 7.41

3362 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Sa´nchez-Martı´n et al.
(8) Campos, J.; Nu´n˜ez, M. C.; Rodr´ıguez, V.; Gallo, M. A.; Espinosa,
A. QSAR of 1,1′-(1,2-ethylenebisbenzyl)bis(4-substitutedpyri-
dinium) dibromides as choline kinase inhibitors: a different
approach for antiproliferative drug design. Bioorg. Med. Chem.
Lett. 2000, 10, 767-770.
(9) Campos, J.; Nu´n˜ez, M. C.; Rodr´ıguez, V.; Entrena, A.; Herna´n-
dez-Alcoceba, R.; Ferna´ndez, F.; Lacal, J. C.; Gallo, M. A.;
Espinosa, A. LUMO energy of model compounds of bispyridinium
compounds as an index for the inhibition of choline kinase. Eur.
J. Med. Chem. 2001, 36, 215-225.
(10) Campos, J.; Nu´n˜ez, M. C.; Conejo-Garc´ıa, A.; Sa´nchez-Mart´ın,
R. M. Herna´ndez-Alcoceba, R.; Rodr´ıguez-Gonza´lez, A.; Lacal,
J. C.; Gallo, M. A.; Espinosa, A. QSAR-derived choline kinase
inhibitors: how rational can antiproliferative drug design be?
Curr. Med. Chem. 2003, 10, 1095-1112.
(11) Ram´ırez de Molina, A.; Gutie´rrez, R.; Ramos, M. A.; Silva, J.
M.; Silva, J.; Sa´nchez, J. J.; Bonilla, F.; Lacal, J. C. Oncogene
2002, 21, 4317-4322.
(m, 8H); 7.39 (t, J ) 7.8, 2H); 7.26 (m, 8H); 7.18- 6.97 (m,
4H); 6.70 (d, J ) 9.4, 2H); 4.62 (s, 2H); 4.59 (s, 2H); 3.61 (s,
6H); 2.79 (s, 4H); 2.27 (s, 6H). HR LSIMS (m/z) calcd for
C50H50N6Br2 (M - HBr - Br)+ 733.4019; found 733.4019. Anal.
(C50H50N6Br2‚3.5H2O) C, H, N.
1,1′-[Ethylenebis(benzene-1,4-diylmethylene)]bis[7-
amino-4-(4-chloro-N-methylanilino)-8-methylquinolini-
1
um] dibromide (48): Yield: 36%. Mp 299-300 °C. H NMR
(300 MHz, DMSO-d6) δ 8.45 (d, J ) 7.0, 2H); 7.41 (d, J ) 8.7,
4H); 7.23 (m, 8H); 7.17 (d, J ) 8.2, 4H); 7.10 (d, J ) 8,2, 4H);
7.09 (d, J ) 9.8, 2H); 7.02 (d, J ) 7.0, 2H); 6.75 (d, J ) 9.7,
2H); 4.48 (s, 2H); 4.45 (s, 2H); 3.56 (s, 6H); 2.78 (s, 4H); 2.38
(s, 6H). HR LSIMS (m/z) calcd for C50H48N6Cl2Br2 (M - HBr
- Br)+ 801.3240; found 801.3238. Anal. (C50H48N6Cl2Br2‚H2O)
C, H, N.
(b) Pharmacology. ChoK Inhibition and Cell Prolif-
eration Assays. The ex vivo human ChoK inhibition and
antiproliferative assays against HT-29 cells were followed in
accordance with the protocols previously reported.39,40 The
results are recorded in Tables 1-3.
(12) Ram´ırez de Molina, A.; Rodr´ıguez-Gonza´lez, A.; Gutie´rrez, R.;
Mart´ınez-Pin˜ero, L.; Sa´nchez, J. J.; Bonilla, F.; Rosell, R.; Lacal,
J. C. Overexpression of choline kinase is a frequent feature in
human tumor-derived cell lines and in lung, prostate, and
colorectal human cancers. Biochem. Biophys. Res. Commun.
2002, 296, 580-583.
Acknowledgment. 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., from Junta de Andaluc´ıa
to R.M.S.-M., from Fondo de Investigacio´n Sanitaria
(Instituto de Salud Carlos III, ref.: CPC/CLC) to
A.R.de M., and from Gobierno Vasco (ref. BFI98.124)
to A.R.-G. is gratefully acknowledged.
(13) Conejo-Garc´ıa, A.; Campos, J.; Sa´nchez, R. M.; Rodr´ıguez-
Gonza´lez, A.; Juan C. Lacal, Gallo, M. A.; Espinosa, A. Choline
kinase inhibitory effect and antiproliferative activity of new
1,1′,1′′-(benzene-1,3,5-triylmethylene)tris{4-[(disubstituted)ami-
no]pyridinium} tribromides. Eur. J. Med. Chem. 2003, 38, 109-
116.
(14) Conejo-Garc´ıa, A.; Campos, J. M.; Sa´nchez-Mart´ın, R. M.; Gallo,
M. A.; Espinosa, A. Bispyridinium cyclophanes: novel templates
for human choline kinase inhibitors. J. Med. Chem. 2003, 46,
3754-3757.
(15) Conejo-Garc´ıa, A.; Campos, J. M.; Entrena, A.; Sa´nchez-Mart´ın,
R. M.; Gallo, M. A.; Espinosa, A. Conformational dynamics of a
bispyridinium cyclophane. J. Org. Chem. 2003, 68, 8697-8699.
(16) Conejo-Garc´ıa, A.; Ba´n˜ez-Coronel, M.; Sa´nchez-Mart´ın, R. M.;
Rodr´ıguez-Gonza´lez, A.; Ramos, A.; Ram´ırez de Molina, A.;
Espinosa, A.; Gallo, M. A.; Campos, J. M.; Lacal, J. C. Influence
of the linker in bispyridinium compounds on the inhibition of
human choline kinase. J. Med. Chem. 2004, 47, 5433-5440.
(17) Conejo-Garc´ıa, A.; Entrena, A.; Campos, J. M.; Sa´nchez-Mart´ın,
R. M.; Gallo, M. A.; Espinosa, A. Towards a model for the
inhibition of choline kinase by a new type of inhibitor. Eur. J.
Med. Chem. 2004, 315-319.
Supporting Information Available: Table 4 and full
experimental procedures and characterization data for quino-
lines 51, 52, 54, 56-59, and 62-68. This material is available
free of charge via Internet at http://pubs.acs.org.
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