Design, synthesis, theoretical calculations and biological evaluation of new non-symmetrical choline kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.01.050

Inhibition of Choline Kinase (ChoK) has been reported as a therapeutical target in the treatment of some kinds of tumor. In this paper, the design and synthesis of new non-symmetrical monocationic ChoK inhibitors is described, bearing a cationic head and an adenine moiety connected by linkers of different lengths. Docking studies indicate that the cationic head of these compounds could be inserted into the choline binding site of the enzyme, while the adenine moiety could be stabilized into the ATP binding site. Docking studies also support the difference of activity of the synthesized compounds, which depends on both the substituent at position 4 of the cationic head and the linker length, being dimethylamine and 1,4-diphenylbutane respectively, the most appropriate ones. Compounds 14 (IC₅₀ = 10.70 ± 0.40 μM) and 17 (IC₅₀ = 6.21 ± 0.97 μM) are the most potent ChoK inhibitors and suitable for further modification with a view to obtain more potent antitumor compounds.

Full text of DOI:10.1016/j.ejmech.2012.01.050

European Journal of Medicinal Chemistry 50 (2012) 154e162
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, theoretical calculations and biological evaluation of new
non-symmetrical choline kinase inhibitors
Belén Rubio-Ruíz a, Ana Conejo-García a, Pablo Ríos-Marco b, María Paz Carrasco-Jiménez b,
,
Josefa Segovia b, Carmen Marco b, Miguel A. Gallo a, Antonio Espinosa a, Antonio Entrena a
*
a Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Campus de Cartuja, 18071 Granada, Spain
b Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias, Campus de Fuentenueva, 18071 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of Choline Kinase (ChoK) has been reported as a therapeutical target in the treatment of some
kinds of tumor. In this paper, the design and synthesis of new non-symmetrical monocationic ChoK
inhibitors is described, bearing a cationic head and an adenine moiety connected by linkers of different
lengths. Docking studies indicate that the cationic head of these compounds could be inserted into the
choline binding site of the enzyme, while the adenine moiety could be stabilized into the ATP binding
site. Docking studies also support the difference of activity of the synthesized compounds, which
depends on both the substituent at position 4 of the cationic head and the linker length, being dime-
thylamine and 1,4-diphenylbutane respectively, the most appropriate ones. Compounds 14
Received 9 September 2011
Received in revised form
17 January 2012
Accepted 24 January 2012
Available online 3 February 2012
Keywords:
Antiproliferative agents
Choline kinase inhibitors
Pyridinium compounds
Molecular modeling
Docking studies
(IC50 ¼ 10.70 ꢀ 0.40
for further modification with a view to obtain more potent antitumor compounds.
Ó 2012 Elsevier Masson SAS. All rights reserved.
m
M) and 17 (IC50 ¼ 6.21 ꢀ 0.97
mM) are the most potent ChoK inhibitors and suitable
1. Introduction
In mammalian cells, the three known isoforms of ChoK (ChoK
a1,
ChoK 2, and ChoK ) are encoded by two genes: chok and chokb.
a
b
a
Choline (Cho) is phosphorylated by choline kinase (ChoK) to
generate phosphocholine (PCho), which represents the first step in
the biosynthesis of a membrane phospholipid, phosphatidylcholine
[1]. During the tumor formation the phospholipid metabolism is
altered, which consequently leads to an increase in the PCho levels
[2,3] associated with the overexpression of ChoK. Platelet-derived,
epidermal, insulin dependent and vascular endothelial growth
factors enhance the ChoK activity during the tumor production [4].
None of the isoforms are active as monomers and the active
enzyme consists of homo- or heterodimers [1]. Recent studies on
the biological function of ChoK isozymes revealed that ChoK
play a more prominent role in cancer development as compared to
ChoK , as only ChoK was upregulated in breast cancer cell lines [9]
and specific depletion of the ChoK isoform by shRNA selectively
induced apoptosis in several tumor-derived cell lines without
affecting the viability of normal primary cells [10]. The ChoK
a may
b
a
a
a
In addition, several oncogenes such as ras or rhoa increase ChoK
a
isoform has also been proposed as a new prognostic marker for
predicting the clinical outcome in patients with non-small-cell lung
cancer [11]. These observations have resulted in the development of
an antitumoral strategy focused on ChoK inhibition.
The synthesis of several derivatives was based on structural
modifications of Cho uptake inhibitor hemicholinium-3 (HC-3,
Fig. 1). We have previously reported a series of compounds namely
bis-pyridinium (1e3) [12], bis-quinolinium (4) [13], tris-pyridinium
(5) [14], cyclophanes (6) [15,16] and bicyclophanes (7) [17] as ChoK
inhibitors. All these compounds show a symmetrical structure,
bearing two or three cationic heads connected through a linker.
Our research group prepared a homology model of the human
activity resulting in higher intracellular levels of PCho [5,6].
Recently it has been observed that hypoxia can induce ChoK
expression in cancer cells [7]. Thus, an activation of ChoK and the
resulting increase in PCho levels have been proposed as necessary
events for the proliferation of certain cell types [8].
Abbreviations: Cho, choline; ChoK, choline kinase; PCho, phosphocholine; HC-3,
hemicholinium-3; PHC-3, phosphohemicholinium-3; CKA2, Caenorhabditis elegans
choline kinase; SAR, structureeactivity relationship; HepG2, human hepatocellular
carcinoma; FBS, fetal bovine serum; MEM, minimal essential medium; MD,
molecular dynamic; SFXC, surflex-dock geomx; LDH, lactate dehydrogenase.
* Corresponding author. Tel.: þ34 958 243848; fax: þ34 958 243845.
E-mail address: aentrena@ugr.es (A. Entrena).
choline kinase isoform ChoKa [18] based on the 3D structure of
Caenorhabditis elegans choline kinase (CKA2 PDB id: 1NW1) [19]. In
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.050

B. Rubio-Ruíz et al. / European Journal of Medicinal Chemistry 50 (2012) 154e162
155
Fig. 2. Chemical structures of mono-quaternary pyridinium salts 8e20.
1,2-diphenylethane with formaldehyde and hydrogen bromide in
the presence of H3PO4 yields 4,40-bis(bromomethyl)biphenyl [21]
and 4,40-bis(bromomethyl)bibenzyl [22], respectively. Finally, 1,4-
Bis[4-(bromomethyl)phenyl]butane was prepared by reduction of
trans,trans-1,4-diphenyl-1,3-butadiene with Pd/C in glacial acetic
acid, followed by the bisbromomethylation in the previous
mentioned conditions [22].
The synthesis of the target molecules 8e20 has been carried
out as described in Scheme 1. Reaction between the appropriate
bisbromomethylated linker and the adequate pyridine derivative
in butanone, at room temperature during 3 h, yields the inter-
mediates 21e28. 1H NMR of the crude reaction showed the pres-
ence of the bisquaternisation structures as side products which
have to be separated from the monocationic intermediates by
filtration and thorough washing with butanone, ethyl acetate and
diethyl ether or flash column chromatography on silica gel using
CH2Cl2/MeOH (10/0.5) as eluent (see experimental protocols for
details).
The reaction between the intermediates 21e28 and the purine
moiety was carried out in DMF using K2CO3 as a base. The reac-
tion mixture was microwave-irradiated at a temperature of
130 ꢁC for 30 min. The preferential N-9 substitution on adenine
was accompanied by formation of the corresponding N-3
isomers. In general the formation ratio of the N-9:N-3 isomers is
1.5:1, except for 15 in which the ratio has shown to be reversed
(1:1.5). When the total yield of the N-3 isomer is smaller than 10%
it was no possible to isolate these structures (N-3 isomers of 9, 11
and 12).
Fig. 1. Chemical structures of bis- and tris-quaternary ammonium salts 1e7.
that paper, preliminary docking studies of some of our previously
synthesized ChoK inhibitors were also included. It was observed
that the size of symmetrical biscationic inhibitors is appropriated to
bind simultaneously in both the ATP and choline putative binding
sites of the protein model (Fig. S1, Supplementary data).
Simultaneously to the publication of our ChoK
a homology
model, the crystal structure of human ChoK 2 isoform was pub-
a
lished [20]. Three crystal structures corresponding to the apo form
(PDB id: 2CKO) and two complexes with the products of the
enzymatic reaction, ADP (PDB id: 2CKP) and PCho (PDB id: 2CKQ)
were described in this paper. These structures allow the correct
identification of both the ATP and Cho binding pockets (Fig. S2).
The preliminary docking studies performed on the homology
model, and the fact that the nature of the residues that constitute
the ATP binding site is quite different to those residues that form
the Cho binding site, suggested the idea of preparing non-
symmetric monocationic inhibitors that could bind in both
binding sites. Such inhibitors should conserve one cationic head
that could be inserted into the Cho binding site, but the second
cationic head will be substituted by another fragment that could
mimic the ATP adenine moiety.
In this paper, the first family of these new inhibitors is presented
(Fig. 2). In these compounds, adenine itself was used to mimic the
ATP adenine moiety, and 4-dimethylamino- or 4-pyrrolidino-pyr-
idinium salts were used as cationic head. Both fragments are
separated by several linkers that have been chosen to study the
influence of the linker size in the inhibition of ChoK.
2. Results
2.1. Chemistry
Four different types of linkers have been used, benzene,
biphenyl, 1,2-diphenylethane and 1,4-diphenylbutane, that are
bisbromomethylated in the adequate position in order to insert
both the adenine and the cationic head. 1,4-Bis(bromomethyl)
benzene is commercially available. Reaction between biphenyl or
Scheme 1. Synthesis of the N-9 and N-3 mono-quaternary pyridinium salts 8e20.
Reagents and conditions: i) Butanone, rt, 3 h; ii) Adenine, K2CO3, DMF, 130 ꢁC, MW,
30 min.

156
B. Rubio-Ruíz et al. / European Journal of Medicinal Chemistry 50 (2012) 154e162
2.2. Pharmacology
a slightly higher antiproliferative activity than the corresponding
dimethylamine analogs (8,10,12 and 14) probably due to the higher
lipophilicity of these structures as a consequence of the presence of
two additional methylene groups in the 4-pyrrolidine derivatives
that allows a better penetration into HepG2 cells. Finally, in the N-3
series, compounds 19 and 20 show a similar antiproliferative
activity, and no other relationships can be established due to the
lack of several derivatives of this series.
Compounds 8e20 were tested in an in vitro system using human
ChoK as a target. This assay allows the evaluation of the affinity of
the compounds for ChoK without considering the possible passage
through biological membranes. The effects on cell proliferation by
the ChoK inhibitors were also investigated on the human hepatoma
HepG2 cell line. Because of their high degree of morphological and
functional differentiation in vitro, HepG2 cells are a suitable model
for the search of new antitumor drugs. Two initial assays were
The index of cytotoxicity used in this study was the measure-
ment of lactate dehydrogenase (LDH) leakage [23]. Toxicity was
performed for each molecule using a final concentration of 10
mM
only apparent after treatment with 50 mM of 15 (data not shown).
and 50 M, with the object of identifying the more potent
m
For the rest of the compounds the decrease in cell number observed
after 24 h of treatments was not related to any acute cytotoxicity
produced by plasma membrane leakage since we did not detect
cytotoxicity determined by measurements LDH leakage.
compounds. IC50 values for ChoK inhibition and antiproliferative
activity were also measure for selected compounds obtained in the
initial assays by using the ED50plus v1.0 software. The activity in
the antiproliferative assay reflected not only their affinity for the
enzyme but also other properties like the capacity of the compound
to pass trough the cell membrane.
2.3. Molecular modeling and docking studies
Table 1 shows the ChoK inhibition and the antiproliferative
activity for 8e20. The differences in the enzyme inhibition of each
N-9 derivative and the corresponding N-3 regioisomer are not too
much significant and the antiproliferative activity between the N-9
and the corresponding N-3 derivative are similar too. The biological
Recently the 3D structure of both human ChoK
and ChoK (PDB id: 3FEG) isoforms in complex with HC-3, the first
known inhibitor of this enzyme, has been published [24]. In the
crystal structure of ChoK 1 both HC-3 and ADP are present, while
in the ChoK structure HC-3 is present in a phosphorilated form
a1 (PDB id: 3G15)
b
a
b
effect is dependent on the linker length although
a clear
(PHC-3), since it was phosphorilated during the crystallization
process [24].
structureeactivity relationship between the size of this moiety and
the inhibition of ChoK and/or the HepG2 antiproliferative activity is
not observed. The most potent ChoK inhibitor of the N-9 series is
compound 14 that presents as a linker a 1,4-diphenylbutane group
Human choline kinase ChoK
the docking studies by two reasons: i) ChoK
pressed in a large number of human tumors, playing a key role in
the carcinogenic process, while the role of ChoK is still not so clear
a1 (PDB id: 3G15) was chosen for
a1 isoform is overex-
and shows an IC50 ¼ 10.70 ꢀ 0.40
m
M. The antiproliferative IC50 of
M.
b
14 measured on HepG2 cell line gives a value of 49.70 ꢀ 1.78
m
[25]; and ii) In this crystal structure both the HC-3 inhibitor and
ADP are presents. Molecular modeling studies were performed
using Sybyl-X program [26].
IC50 ChoK inhibition value has also been calculated for the
most active compound belonging to the N-3 series (17, IC50
ChoK ¼ 6.21 ꢀ 0.97
proliferative agent of this series is compound 19 that presents an
IC50 value of 38.9 ꢀ 1.02 M.
mM). The most potent structure as anti-
Docking studies indicate that there are different modes for the
binding of the inhibitors inside the enzyme. The length of the linker
in compounds 8 and 9 (and also 16) is too short to allow these
molecules to be allocated in both ATP and Cho binding sites
simultaneously. The obtained poses for these compounds indicate
that they can be inserted into the Cho binding site or into the ATP
binding site.
Fig. 3 shows as an example the poses obtained for compounds 8
and 9. The cationic head of compound 8 is inserted into the
hydrophobic pocket of the Cho binding site and is stabilized by
m
In general the dimethylamine substituent at position 4 of the
pyridinium fragment lead to higher ChoK inhibitory potency. In the
N-9 series, compounds 8, 12 and 14 show higher inhibition
percentage than compounds 9, 13 and 15, respectively, with the
only exception of compound 11 that presents a pyrrolidine group
and is more potent than 10. Unfortunately, in the N-3 series some
compounds could not be isolated and this relationship between the
dimethylamine and pyrrolidine derivatives can not be confirmed,
except for 19 bearing a dimethylamine substituent which is more
potent than compound 20.
Regarding the antiproliferative activity, in the N-9 series can be
observed that the 4-pyrrolidine derivatives (9, 11, 13 and 15) show
Table 1
ChoK inhibition and HepG2 antiproliferative activity for the target compounds
8e20.
Comp.
Inhib.ChoK (%)
10
Antiprolif. (%)
m
M
50
mM
10
m
M
50 mM
8
9
46.7 ꢀ 0.9
0
54.1 ꢀ 2.7
27.0 ꢀ 2.8
51.0 ꢀ 2.5
72.8 ꢀ 2.9
81.0 ꢀ 1.7
46.9 ꢀ 3.7
89.7 ꢀ 3.1
41.9 ꢀ 3.3
50.2 ꢀ 2.9
71.4 ꢀ 2.1
24.8 ꢀ 3.1
68.2 ꢀ 2.4
59.9 ꢀ 3.3
0
0
0
5.1 ꢀ 0.7
15.5 ꢀ 0.5
10.3 ꢀ 0.6
45.0 ꢀ 2.2
10.5 ꢀ 0.7
45.0 ꢀ 4.2
45.5 ꢀ 2.5
90.1 ꢀ 1.6
2.5 ꢀ 0.3
10
11
12
13
14
15
16
17
18
19
20
37.6 ꢀ 0.4
50.2 ꢀ 3.9
38.6 ꢀ 4.2
28.6 ꢀ 2.7
66.7 ꢀ 3.8
36.9 ꢀ 3.8
25.0 ꢀ 3.4
62.3 ꢀ 3.5
0.1 ꢀ 0
10.4 ꢀ 1.0
10.5 ꢀ 1.5
30.1 ꢀ 2.2
10.2 ꢀ 1.8
15.4 ꢀ 2.8
10.3 ꢀ 1.7
10.0 ꢀ 1.9
11.6 ꢀ 1.8
14.3 ꢀ 1.3
13.7 ꢀ 1.1
Fig. 3. The most stable poses obtained for compounds 8 (right, carbon atoms in orange
color) and 9 (left, carbon atoms in yellow color) in the docking studies. The pyridinium
moiety of compound 8 is inserted into the hydrophobic pocket of the Cho binding site
(Tyr333, Tyr354, Tyr440, Trp420 and Trp423, carbon atoms in cyan color), while the
phenyl and the adenine moieties are stabilized by a
p-p interaction with Phe435 and
Phe361 (magenta). Compound 9 is inserted into the ATP binding site (Glu206, Gln207
and Ile209, carbon atoms in blue slate color), being the adenine moiety stabilized by
three H-bonds with these residues and by two additional H-bonds with Arg146
(carbon atoms in magenta color). (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
7.0 ꢀ 0.4
30.0 ꢀ 2.1
56.1 ꢀ 0.7
46.5 ꢀ 2.1
61.3 ꢀ 2.8
22.6 ꢀ 3.6

B. Rubio-Ruíz et al. / European Journal of Medicinal Chemistry 50 (2012) 154e162
157
means of
interaction with Phe435. The adenine moiety points toward
the surface of the protein and is parallel to Phe361, making a
p
-cation interactions, and the phenyl ring is stabilized by
adopts an opposite orientation in relation to the ADP adenine
observed in the crystal structure. Figure S8 and S9 show the two
main poses of both compounds.
a p-p
p-p
interaction with this residue. This pose can be superimposed to the
HC-3 inhibitor inserted into the crystal structure of the enzyme
(Fig. S3). A similar pose has been obtained for compound 9 (Fig. S4),
but the higher volume of the 4-pirrolidine substituent in relation to
the 4-dimethylamine of compound 8 prevents a correct orientation
of the molecule inside the Cho binding site, and the interaction
between the benzene moiety and Phe435 does not exist in
compound 9.
In the most stable pose of compound 9, the molecule is inserted
into the ATP binding pocket, and is stabilized by hydrogen bonds
between the adenine moiety and Ile209, Asn207, Asp206 and
Arg146 (Fig. 3). The adenine moiety can be superimposed with the
ADP adenine that is present in the crystal structure of the enzyme
(Fig. S5). No similar pose has been obtained for compound 8,
probably because the union to the Cho binding site is more favored.
On the other hand, compounds 10e15 bear longer linkers,
whose length is enough to allow these molecules to bind
simultaneously in both ATP and Cho binding sites. Fig. 4a shows
as an example the best poses obtained for compounds 11, 13 and
15. It can be observed that the adenine moiety of the three
compounds is situated on the ATP binding site, performing
hydrogen bonds interactions with Ile209 and Asn207, similarly to
Like compounds 10e15, compounds 17e20 (and the non iso-
lated N-3 isomers of 11 and 12) have a longer linker and can be
accommodated simultaneously in both ATP and Cho binding sites.
Fig. 4b shows as an example the main poses obtained for 17 and for
compound 20. In this case, the orientation of the adenine moiety of
17 adopts an orientation similar to that described for the N-3
isomer of compound 9 Fig. S9), rotated in an opposite direction to
the ADP adenine fragment. This orientation seems to be necessary
since the linker is connected to N-3 atom and the length of the
linker is not enough to allow the insertion of the cationic head into
the Cho binding site when the adenine adopts an orientation
similar to that of the ADP adenine.
Compound 20 shows an adenine orientation similar to the N-9
derivatives above described. In this case, the length of the linker of
compound 20 is higher and allows the cationic head to be inserted
into the Cho binding site. Even more, the linker is less folded than in
compound 15. Figures S10 and S11 show a more detailed view of
the pose of each molecule.
3. Discussion
N-9 derivatives 8e15 were designed as new monocationic
inhibitors of ChoK. Nevertheless, during the synthesis of these
molecules, N-3 derivatives 16e20 were also obtained as subprod-
ucts of the synthetic reactions, and their biological activity as ChoK
inhibitors were also evaluated.
the interactions between the ADP and the ChoKa1 in the crystal
structure of the complex (PDB id: 3G15). The cationic head of
these molecules is inserted into the Cho binding site, with the
4-pyrrolidinopyridinium moiety parallel to Trp423. The proximity
of both fragments stabilizes the inhibitor inside the hydrophobic
Biological assays were performed at two different concentra-
pocket by means of
p
-
p
or
p
-cation interactions. The linker of
tions: 10 and 50
mM in order to determine the more potent ChoK
compounds 11 and 13 can adopt an extended conformation and
their main poses for both molecules are almost parallels each
other, being the cationic head of compound 13 slightly near to
Trp423. The longer linker of compound 15 made this molecule to
adopt a more folded conformation, and the cationic head adopts
a different disposition into the Cho binding site in comparison
with those of compounds 11 and 13. Figures S6 and S7 show
a more detailed view of the pose of each molecule.
Regarding N-3 derivatives, the overall behavior of these mole-
cules is similar to that of N-9 derivatives. Compound 16 and the N-
3 isomer of compound 9 have a short linker and can only occupy
one of the binding sites, the ATP or the Cho binding site.
Compound 16 shows a similar pose to that of compound 8 inside
into the Cho binding site, with the benzene moiety interacting
inhibitors. The IC50 values were measured only for the most potent
compounds of each series (14 and 17).
Among N-9 derivatives, and regarding the ChoK inhibition,
some structure relationships can be established. It can be
observed that the inhibition activity depends on both the nature
of the cationic head and the length of the linker. Compounds
bearing
a 4-dimethylaminopyridinium moiety as a cationic
head are more potent inhibitors when the molecules bear
a benzene, 1,2-diphenylethane or 1,4-diphenylbutane linker, while
4-pyrrolidinopyridinium moiety originates more potent inhibitors
only when a biphenyl fragment is the linker of the inhibitor.
In compounds 8 and 9, with the benzene linker, the higher
activity of the 4-dimethylaminopyridinium derivative (8) can be
easily explained in base to the preferred poses obtained for this
molecule. In the preferred pose for 8, the cationic head is inserted
with Phe345 trough a
p-p interaction. The N-3 isomer of 9 is
inserted into the ATP binding site, and established hydrogen bonds
with Ile209, Asn207 and Glu246. The main difference with the
pose of compound 9 is that the adenine moiety is rotated and
into the Cho binding site, and is stabilized by a
between the benzene moiety of the linker and Phe435 that adopts
parallel orientation each other (Fig. 3). Nevertheless, the
p-p interaction
a
Fig. 4. (a) The most stable poses obtained in the docking studies of compounds 11 (magenta), 13 (yellow) and 15 (blue) inside the ChoKa1. (b) The most stable poses obtained in the
docking studies of compounds 17 (yellow), and 20 (magenta) inside the ChoK 1. All compounds are simultaneously situated inside both ATP and Cho binding sites. Adenine moieties
a
adopt an opposite orientation in each compound to allow the cationic head to be inserted into the Cho binding site. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)

158
B. Rubio-Ruíz et al. / European Journal of Medicinal Chemistry 50 (2012) 154e162
equivalent pose of compound 9 into the Cho binding site is not
stabilized by such type of interaction (Fig. S4) due to the higher
volume of the 4-pyrrolidine substituent that prevents the correct
orientation of the ligand inside the Cho binding site. In fact, in the
most stable pose obtained for compound 9, the inhibitor is inserted
into the ATP binding site, and not in the Cho binding site. This
difference could be the explanation of the higher inhibition activity
of compound 8 in relation to compound 9: compound 8 binds more
efficiently inside the Cho binding site.
In compounds 10 and 11, the biphenyl linker is long enough to
allow the insertion of the adenine moiety into the ATP binding site
and of the cationic head into the Cho binding site. In this pair of
compounds, the 4-pyrrolidinopyridinium cationic head gives place
to a more potent inhibitor. This could be due to the smaller volume
of the dimethylamine substituent. The depth of the insertion of
both cationic head inside the hydrophobic pocket is similar and the
length of the linker controls it. Nevertheless, in compound 10 the
cationic head is smaller and it can be situated nearer to Tyr354 and
slightly far to Trp423 (6.6 Å). In compound 11, the higher volume of
the pyrrolidine moiety brings the cationic head closer to Trp423
Regarding antiproliferative activity (Table 1), the SAR can be
hardly established, since it depends not only on the affinity of these
molecules as ChoK inhibitors, but also on their facility to pass trough
the membrane cells. In general, it can be observed that 4-pyrrolidine
derivatives show a slightly higher activity than the corresponding
dimethylamine analogs. This fact could be due to the higher lip-
ophilicity of these molecules due to the presence of two additional
carbon atoms. On the other hand, it can also be observed that
compounds with longer linker also show higher antiproliferative
activity and this could also be due to their higher lipophilicity.
4. Conclusions
Compounds described on this paper present inhibitory
activity against ChoK. Docking studies indicate that in most cases,
these molecules could bind simultaneously in both ATP and
Cho binding sites of this enzyme, except when the linker is
a benzene moiety. The adenine moiety binds into the ATP binding
site in a similar mode to the ADP adenine fragment present in the
crystal structure of the ChoK/ADP/HC-3 complex. In general,
(4.3 Å), originating a more potent
p-p interactions between the
4-dimethylaminopyridinium moiety acts as
head since it permits a better insertion inside the Cho binding
site. Only when the linker is biphenyl fragment, the 4-
pyrrolidinopyridinium is better as a cationic head. Unfortu-
nately, a direct relationship between the inhibition of ChoK and
the antiproliferative activity has not been found. Nevertheless,
these molecules could serve as a pattern for the design of new and
more potent ChoK inhibitors.
a better cationic
cationic head of compound 11 with Trp423. This could be the
reason for the higher activity of compound 11 in relation to
compound 10 (Fig. 5a).
a
In compounds 12e15, the length of the linker is higher, and
consequently, allows a more flexible orientation of the cationic head
inside the Cho binding site. Fig. 5b shows as an example the
difference in the orientation of the cationic head in compounds 14
and 15. In these molecules the linker is very long and must be folded
to allow the accommodation of the adenine and the cationic head.
Nevertheless, in compound 14, the smaller volume of the dime-
thylamine substituent allows a more deep insertion of the cationic
head inside the hydrophobic pocket of the Cho binding site. A
similar situation occurs in compound 12 and 13. The longer length of
the linker and the smaller volume of the dimethylamine moiety in
compounds 12 and 14 allow a deeper insertion of the cationic head
inside the hydrophobic pocket of the Cho binding site, and this fact
could be an explanation of the higher activity of these molecules in
relation to their corresponding compounds 13 and 15, respectively.
The differences in activity of the N-3 derivatives in relation to
their corresponding N-9 analogs is really smaller. Furthermore,
since some of these molecules could not be isolated, the
structureeactivity relationships (SAR) cannot be completely
established, but docking studies indicates similar preference for the
ATP and/or choline binding sites. Only compounds 19 and 20 can be
compared and their inhibition activity is similar to that of
compounds 14 and 15. This fact is expected since the linker and the
cationic head are similar in these molecules.
5. Experimental protocols
5.1. Chemistry
Melting points were taken in open capillaries on a Stuart
Scientific SMP3 electrothermal melting point apparatus and are
uncorrected. Elemental analyses were performed on the Thermo
Scientific Flash 2000 analyzer only on the final compounds tested
as ChoK inhibitors. The measured values for C, H, and N agreed to
within ꢀ0.40% of the theoretical values. Analytical thin-layer
chromatography (TLC) was performed using Merck Kieselgel 60
F254 aluminum plates and visualized by UV light or iodine. All
evaporation was carried out in vacuo with a Büchi rotary evaporator
and the pressure controlled by a Vacuubrand CVCII apparatus. For
flash chromatography, Merck silicagel 60 with a particle size of
0.040e0.063 mm (230e400 mesh ASTM) was used. Nuclear
magnetic resonance spectra were recorded on a 500 MHz 1H and
125 MHz 13C NMR Varian NMR-System-TM 500, a 400 MHz 1H NMR
Varian NMR-System-TM 400 or 300 MHz 1H and 75 MHz 13C NMR
Fig. 5. (a) Comparison between the most stable poses obtained for compounds 10 (yellow) and 11 (magenta). The cationic head of compound 11 is situated near to Trp423. (b)
Detailed view of the cationic heads of compounds 14 (magenta) and 15 (yellow) inserted into the hydrophobic pocket of the Cho binding site. The cationic head of compound 14 can
be inserted more deeply inside the hydrophobic pocket. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

B. Rubio-Ruíz et al. / European Journal of Medicinal Chemistry 50 (2012) 154e162
159
Varian Inova-TM spectrometers at ambient temperature. Chem-
ical shifts ( ) are quoted in parts per million (ppm) and are
7.50 (d, J ¼ 8.1 Hz, 2H); 7.45 (d, J ¼ 8.1 Hz, 2H); 6.87 (d, J ¼ 7.5 Hz,
2H); 5.40 (s, 2H); 4.62 (s, 2H); 3.56 (t, J ¼ 6.6 Hz, 4H); 2.12 (q,
d
referenced to the residual solvent peak. Spin multiplicities are
given as s (singlet), bs (broad singlet), d (doublet), t (triplet), q
(quintuplet) and m (multiplet). High-resolution Nano-Assisted
Laser Desorption/Ionization (NALDI-TOF) or Electrospray Ioniza-
tion (ESI-TOF) mass spectra were carried out on a Bruker Autoflex
J ¼ 6.6 Hz, 4H). 13C NMR (125 MHz, CD3OD)
d
155.03, 142.99 ꢂ 2,
142.34, 141.13, 139.25, 135.52, 130.83 ꢂ 2, 130.04 ꢂ 2, 128.78 ꢂ 2,
128.24 ꢂ 2, 109.73 ꢂ 2, 61.36, 49.86 ꢂ 2, 33.81, 26.07 ꢂ 2. HRMS (m/
z) calcd for C23H24N2Br (M ꢃ Br)+ 407.1123; found 407.1109.
or
a
Waters LCT Premier Mass Spectrometer, respectively.
5.2.5. 1-{4-[4-(bromomethyl)phenethyl]benzyl}-4-
(dimethylamino)pyridinium bromide (25)
Microwave-assisted synthesis was carried out in an Initiator 2.0
single-mode microwave instrument producing controlled irradi-
ation at 2.450 GHz (Biotage AB, Upsala). Reaction time refers to
hold time at 130 ꢁC, not to total irradiation time. The temperature
was measured with an IR sensor on the outside of the reaction
vessel. All compounds were dried at 40 ꢁC and 0.1 mmHg for
24 h, but many held on tenaciously to water which appear to
be solvates. Adenine, 4-(pyrrolidino)pyridine, 4-(N,N-dimethyla-
mino)pyridine and 1,4-bis(bromomethyl)benzene were purchased
from Aldrich. 4,40-Bis(bromomethyl)biphenyl [21], 4,40-bis(-
bromomethyl)bibenzyl [22] and 1,4-bis[4-bromomethyl)phenyl]
butane [22] were synthesized according to literature procedures.
Yield: 74%. M.p. 242 ꢁC (gel) e 245 ꢁC (foam). 1H NMR (300 MHz,
CD3OD)
2H); 6.99 (d, J ¼ 8 Hz, 2H); 5.32 (s, 2H); 4.52 (s, 2H); 3.24 (s, 6H);
2.91 (m, 4H). 13C NMR (75 MHz, CD3OD)
157.99, 144.24, 143.12,
d
8.21 (d, J ¼ 8 Hz, 2H); 7.29e7.19 (m, 6H); 7.12 (d, J ¼ 8.1 Hz,
d
143.03 ꢂ 2, 137.31, 133.67, 130.62 ꢂ 2, 130.15 ꢂ 2, 129.91 ꢂ 2,
129.37 ꢂ 2, 109.08 ꢂ 2, 61.58, 40.28 ꢂ 2, 38.34, 38.30, 34.13. HRMS
(m/z) calcd for C23H26N2Br (M ꢃ Br)+ 409.1272; found 409.1276.
5.2.6. 1-{4-[4-(bromomethyl)phenethyl]benzyl}-4-
pyrrolidinopyridinium bromide (26)
Yield: 60%. M.p. 243e245 ꢁC 1H NMR (400 MHz, CD3OD)
d 8.16
(d, J ¼ 7.1 Hz, 2H); 7.28e7.19 (m, 6H); 7.11 (d, J ¼ 7.8 Hz, 2H); 6.84 (d,
5.2. General procedure for the synthesis of the monocationic
intermediates
J ¼ 7.1 Hz, 2H); 5.30 (s, 2H); 4.52 (s, 2H); 3.55 (t, J ¼ 6.3 Hz, 4H); 2.91
(m, 4H); 2.12 (q, J ¼ 6.3 Hz, 4H). 13C NMR (75 MHz, CD3OD)
d 155.20,
144.18, 143.01, 142.96 ꢂ 2, 137.25, 133.87, 130.60 ꢂ 2, 130.15 ꢂ 2,
129.91 ꢂ 2, 129.33 ꢂ 2, 109.67 ꢂ 2, 61.57, 49.71 ꢂ 2, 38.34, 38.30,
34.02, 26.12 ꢂ 2. HRMS (m/z) calcd for C25H28N2Br (M ꢃ Br)+
435.1436; found 435.1427.
A butanone solution (40 mL) of the linker (2 equiv.) and the
corresponding 4-substituted pyridine (1 equiv.) was stirred at room
temperature for 3 h 21, 22, 23, 25 and 26 were isolated after
filtration and thorough washing with butanone, ethyl acetate and
diethyl ether. In the case of 24, 27 and 28, the reaction mixture was
evaporated, and the crude products purified by flash column
chromatography on silica gel using CH2Cl2/MeOH (10/0.5) as eluent.
5.2.7. 1-(4-{4-[4-(bromomethyl)phenyl]butyl}benzyl)-4-
(dimethylamino)pyridinium bromide (27)
Yield: 64%. M.p. 193e198 ꢁC 1H NMR (400 MHz, CD3OD)
d 8.20
(d, J ¼ 7.7 Hz, 2H); 7.30e7.21 (m, 6H); 7.12 (d, J ¼ 7.9 Hz, 2H); 6.99 (d,
5.2.1. 1-[4-(bromomethyl)benzyl]-4-(dimethylamino)pyridinium
J ¼ 7.7 Hz, 2H); 5.31 (s, 2H); 4.54 (s, 2H); 3.24 (s, 6H); 2.66e2.60 (m,
bromide (21)
4H); 1.64e1.60 (m, 4H). 13C NMR (75 MHz, CD3OD)
d 157.92, 145.13,
Yield: 76%. M.p. 186e189 ꢁC 1H NMR (300 MHz, CD3OD)
d
8.22
144.06,143.01 ꢂ 2,136.98,133.46,130.41 ꢂ 2,130.14 ꢂ 2,129.75 ꢂ 2,
129.41 ꢂ 2, 109.08 ꢂ 2, 61.56, 40.39 ꢂ 2, 36.29, 36.25, 34.27, 32.07,
31.98. HRMS (m/z) calcd for C25H30N2Br (M ꢃ Br)+ 437.1593; found
437.1596.
(d, J ¼ 7.9 Hz, 2H); 7.48 (d, J ¼ 8.2 Hz, 2H); 7.36 (d, J ¼ 8.2 Hz, 2H);
7.01 (d, J ¼ 7.9 Hz, 2H); 5.37 (s, 2H); 4.57 (s, 2H); 3.25 (s, 6H). 13C
NMR (125 MHz, DMSO-d6)
d
155.88, 141.99 ꢂ 2, 138.54, 135.82,
129.87 ꢂ 2, 128.23 ꢂ 2, 108.01 ꢂ 2, 58.90, 39.77 ꢂ 2, 33.72. HRMS
(m/z) calcd for C15H18N2Br (M ꢃ Br)+ 305.0653; found 305.0644.
5.2.8. 1-(4-{4-[4-(bromomethyl)phenyl]butyl}benzyl)-4-
pyrrolidinopyridinium bromide (28)
5.2.2. 1-[4-(bromomethyl)benzyl]-4-pyrrolidinopyridinium
bromide (22)
Yield: 63%. M.p. 180 ꢁC (gel) e 185 ꢁC (foam). 1H NMR (400 MHz,
CD3OD)
d
8.18 (d, J ¼ 7.5 Hz, 2H); 7.30e7.21 (m, 6H); 7.12 (d, J ¼ 7.9 Hz,
Yield: 77%. M.p. 203 ꢁC (gel) e 241 ꢁC (foam). 1H NMR (400 MHz,
2H); 6.84 (d, J ¼ 7.5, 2H); 5.30 (s, 2H); 4.53 (s, 2H); 3.54 (t, J ¼ 6.8 Hz,
CD3OD)
d
8.18 (d, J ¼ 7.6 Hz, 2H); 7.48 (d, J ¼ 8.1 Hz, 2H); 7.34 (d,
4H); 2.72e2.57 (m, 4H); 2.11 (q, J ¼ 6.8 Hz, 4H); 1.62 (m, 4H).13C NMR
J ¼ 8.1 Hz, 2H); 6.86 (d, J ¼ 7.6 Hz, 2H); 5.35 (s, 2H); 4.57 (s, 2H); 3.55
(125 MHz, CD3OD)
d
155.19,145.19,144.08,142.95 ꢂ 2,137.04,133.52,
(t, J ¼ 6.8 Hz, 4H); 2.12 (q, J ¼ 6.8 Hz, 4H). 13C NMR (75 MHz, CD3OD)
130.44 ꢂ 2, 130.13 ꢂ 2, 129.75 ꢂ 2, 129.33 ꢂ 2, 109.66 ꢂ 2, 61.67,
49.70 ꢂ 2, 36.29, 36.26, 34.19, 32.05, 31.96, 26.11 ꢂ 2. HRMS (m/z)
calcd for C27H32N2Br (M ꢃ Br)+ 463.1749; found 463.1741.
d
155.18, 143.04 ꢂ 2, 140.87, 136.35, 131.11 ꢂ 2, 129.64 ꢂ 2,
109.76 ꢂ 2, 61.32, 49.74 ꢂ 2, 33.079, 26.129 ꢂ 2. HRMS (m/z) calcd
for C17H20N2Br (M ꢃ Br)+ 331. 0810; found 331.0811.
5.3. General procedure for the synthesis of the target compounds
5.2.3. 1-{4-[4-(bromomethyl)phenyl]benzyl}-4-(dimethylamino)
pyridinium bromide (23)
A mixture of adenine (1 equiv.), the corresponding mono-
cationic intermediate (1 equiv.) and K2CO3 (2 equiv.) in DMF (4 mL)
was microwave-irradiated at a set temperature of 130 ꢁC for 30 min.
Solvent was evaporated and the crude product was purified by flash
column chromatography using as eluent CH2Cl2/methanol (10/
0.5/7/3) to afford 8e20. The discrimination between N-9 and N-3
substituted derivatives has been clarified through the HMBC
experiment to three bonds.
Yield: 70%. M.P. 209e210 ꢁC 1H NMR (400 MHz, CD3OD)
d 8.25
(d, J ¼ 7.5 Hz, 2H); 7.70 (d, J ¼ 8 Hz, 2H); 7.61 (d, J ¼ 8 Hz, 2H);
7.52e7.45 (m, 4H); 7.02 (d, J ¼ 7.5 Hz, 2H); 5.41 (s, 2H), 4.62 (s, 2H),
3.26 (s, 6H). 13C NMR (75 MHz, CD3OD)
d
158.00, 143.12 ꢂ 2, 142.61,
141.28, 139.33, 135.39, 130.83 ꢂ 2, 129.99 ꢂ 2, 128.88 ꢂ 2,
128.30 ꢂ 2, 109.16 ꢂ 2, 61.41, 40.39 ꢂ 2, 33.69. HRMS (m/z) calcd for
C21H22N2Br (M ꢃ Br)+ 381.0966; found 381.0964.
5.2.4. 1-{4-[4-(bromomethyl)phenyl]benzyl}-4-
5.3.1. 1-{4-[(6-amino-9H-purin-9-yl)methyl]benzyl}-4-
(dimethylamino)pyridinium bromide (8)
pyrrolidinopyridinium bromide (24)
Yield: 61%. M.p. 106e109 ꢁC 1H NMR (400 MHz, CD3OD)
d
8.22
Yield: 41%. M.p. 271 ꢁC (decomposition). 1H NMR (400 MHz,
(d, J ¼ 7.5 Hz, 2H); 7.70 (d, J ¼ 8.1 Hz, 2H); 7.61 (d, J ¼ 8.1 Hz, 2H);
CD3OD)
d
8.19e8.16 (m, 4H); 7.40 (d, J ¼ 8.3 Hz, 2H); 7.35 (d,

160
B. Rubio-Ruíz et al. / European Journal of Medicinal Chemistry 50 (2012) 154e162
J ¼ 8.3 Hz, 2H); 6.97 (d, J ¼ 7.8 Hz, 2H); 5.45 (s, 2H); 5.34 (s, 2H); 3.23
(s, 6H). 13C NMR (125 MHz, CD3OD)
158.02, 157.37, 153.93, 150.70,
26.12 ꢂ 2. HRMS (m/z) calcd for C30H32N7 (M ꢃ Br)+ 490.2720;
found 490.2721. Anal. C30H32N7Br $0.8H2O (C, H, N).
d
143.09 ꢂ 2, 142.60, 138.86, 136.15, 129.88 ꢂ 2, 129.70 ꢂ 2, 120.01,
109.14 ꢂ 2, 61.28, 47.62, 40.36 ꢂ 2. HRMS (m/z) calcd for C20H22N7
(M ꢃ Br)+ 360.1937; found 360.1948. Anal. C20H22N7Br $1.2H2O
(C, H, N).
5.3.7. 1-[4-(4-{4-[(6-amino-9H-purin-9-yl)methyl]phenyl}butyl)
benzyl]-4-(dimethylamino)pyridinium bromide (14)
Yield: 54%. M.p. >350 ꢁC (decomposition). 1H NMR (500 MHz,
CD3OD)
d
8.20e8.19 (m, 3H); 8.14 (s, 1H); 7.26 (d, J ¼ 8.1 Hz, 2H);
5.3.2. 1-{4-[(6-amino-9H-purin-9-yl)methyl]benzyl}-4-
pyrrolidinopyridinium bromide (9)
7.21 (d, J ¼ 8.1 Hz, 2H); 7.18 (d, J ¼ 8.1 Hz, 2H); 7.12 (d, J ¼ 8.1 Hz,
2H); 6.97 (d, J ¼ 7.7 Hz, 2H); 5.38 (s, 2H); 5.31 (s, 2H); 3.23 (s, 6H);
2.61e2.57 (m, 4H); 1.57 (q, J ¼ 3.3, 7.1, 4H). 13C NMR (125 MHz,
Yield: 56%. M.p. >350 ꢁC (decomposition). 1H NMR (300 MHz,
DMSO-d6)
d
8.38 (d, J ¼ 7.5 Hz, 2H); 8.27 (s, 1H); 8.11 (s, 1H); 7.38 (d,
CD3OD)
d
157.96, 157.32, 153.84, 150.68, 145.16, 143.89, 143.00 ꢂ 2,
J ¼ 8.3 Hz, 2H); 7.35 (d, J ¼ 8.3 Hz, 2H); 7.20 (bs, 2H); 6.86 (d,
142.63, 134.96, 133.40, 130.41 ꢂ 2, 129.99 ꢂ 2, 129.38 ꢂ 2,
128.74 ꢂ 2, 119.96, 109.07 ꢂ 2, 61.59, 47.84, 40.36 ꢂ 2, 36.23, 36.21,
31.93 ꢂ 2. HRMS (m/z) calcd for C30H34N7 (M ꢃ Br)+ 492.2876;
found 492.2877. Anal. C30H34N7Br $1.6H2O (C, H, N).
J ¼ 7.5 Hz, 2H); 5.39 (s, 2H); 5.37 (s, 2H); 3.45 (t, J ¼ 6.4 Hz, 4H); 1.97
(q, J ¼ 6.4 Hz, 4H). 13C NMR (75 MHz, DMSO-d6)
d 155.92, 152.97,
152.57, 149.36, 141.88 ꢂ 2, 140.74, 137.61, 135.33, 128.31 ꢂ 2,
128.16 ꢂ 2, 118.64, 108.54 ꢂ 2, 58.91, 48.28 ꢂ 2, 45.75, 24.59 ꢂ 2.
HRMS (m/z) calcd for C22H24N7 (M ꢃ Br)+ 386.2094; found
386.2093. Anal. C22H24N7Br $0.6H2O (C, H, N).
5.3.8. 1-[4-(4-{4-[(6-amino-9H-purin-9-yl)methyl]phenyl}butyl)
benzyl]-4-pyrrolidinopyridinium bromide (15)
Yield: 26%. M.p. 244 ꢁC (gel) e 259 ꢁC (foam). 1H NMR (500 MHz,
5.3.3. 1-(4-{4-[(6-amino-9H-purin-9-yl)methyl]phenyl}benzyl)-4-
(dimethylamino)pyridinium bromide (10)
CD3OD)
d
8.21 (s, 1H); 8.16 (d, J ¼ 7.6 Hz, 2H); 8.12 (s, 1H); 7.26e7.17
(m, 6H); 7.12 (d, J ¼ 8 Hz, 2H); 6.83 (d, J ¼ 7.6 Hz, 2H); 5.38 (s, 2H);
5.29 (s, 2H); 3.53 (t, J ¼ 6.8 Hz, 4H); 2.61e2.57 (m, 4H); 2.11 (q,
J ¼ 6.8 Hz, 4H); 1.58 (q, J ¼ 7.1 Hz, 4H). 13C NMR (125 MHz, CD3OD)
Yield: 26%. M.p. >350 ꢁC (decomposition). 1H NMR (400 MHz,
DMSO-d6)
d
8.42 (d, J ¼ 7.1 Hz, 2H); 8.28 (s, 1H); 8.15 (s, 1H); 7.67 (d,
J ¼ 7.9 Hz, 2H); 7.62 (d, J ¼ 7.9 Hz, 2H); 7.46 (d, J ¼ 7.9 Hz, 2H); 7.40 (d,
J ¼ 7.9 Hz, 2H); 7.20 (bs, 2H); 7.05 (d, J ¼ 7.1 Hz, 2H); 5.42 (s, 2H); 5.41
d
157.34, 155.14, 153.70, 150.69, 145.15, 143.89, 142.91 ꢂ 2, 142.61,
134.96, 133.50, 130.41 ꢂ 2, 129.99 x2, 129.32 ꢂ 2, 128.73 x2, 119.97,
109.64 x2, 61.64, 49.70 x2, 47.84, 36.23, 36.21, 31.92 x2, 26.12 x2.
HRMS (m/z) calcd for C32H36N7 (M ꢃ Br)+ 518.3032; found 518.3041.
Anal. C32H36N7Br $0.8H2O (C, H, N).
(s, 2H); 3.18 (s, 6H). 13C NMR (125 MHz, DMSO-d6)
d 155.99, 155.91,
152.66, 149.46, 142.03 ꢂ 2, 140.87, 139.98, 138.80, 136.69, 135.03,
128.65 ꢂ 2, 128.21 ꢂ 2, 127.25 ꢂ 2, 127.03 ꢂ 2, 118.68, 107.99 ꢂ 2,
58.93, 48.58 ꢂ 2, 45.84. HRMS (m/z) calcd for C26H26N7 (M ꢃ Br)+
436.2250; found 436.2240. Anal. C26H26N7Br $1.6H2O (C, H, N).
5.3.9. 1-{4-[(6-amino-3H-purin-3-yl)methyl]benzyl}-4-
(dimethylamino)pyridinium bromide(16)
5.3.4. 1-(4-{4-[(6-amino-9H-purin-9-yl)methyl]phenyl}benzyl)-4-
pyrrolidinopyridinium bromide (11)
Yield: 24%. M.p. 271 ꢁC (decomposition). 1H NMR (400 MHz,
CD3OD)
d
8.43 (s, 1H); 8.16 (d, J ¼ 7.8 Hz, 2H); 7.88 (s, 1H); 7.46 (d,
Yield: 43%. M.p. 245e246 ꢁC 1H NMR (500 MHz, DMSO-d6)
J ¼ 8.2 Hz, 2H); 7.35 (d, J ¼ 8.2 Hz, 2H); 6.96 (d, J ¼ 7.8 Hz, 2H); 5.60
d
8.43 (d, J ¼ 7.6 Hz, 2H); 8.30 (s, 1H); 8.14 (s, 1H); 7.66 (d, J ¼ 8.3 Hz,
(s, 2H); 5.34 (s, 2H); 3.23 (s, 6H). 13C NMR (125 MHz, CD3OD)
2H); 7.62 (d, J ¼ 8.3 Hz, 2H); 7.46 (d, J ¼ 8.3 Hz, 2H); 7.40 (d,
J ¼ 8.3 Hz, 2H); 7.23 (bs, 2H); 6.91 (d, J ¼ 7.6 Hz, 2H); 5.43 (s, 2H);
5.41 (s, 2H); 3.48 (t, J ¼ 6.7 Hz, 4H); 1.98 (q, J ¼ 6.7 Hz, 4H). 13C NMR
d
158.02, 156.81, 153.11, 150.73, 145.68, 143.09 ꢂ 2, 137.78, 136.52,
129.85 ꢂ 4, 120.99, 109.12 ꢂ 2, 61.24, 53.62, 40.32 ꢂ 2. HRMS (m/z)
calcd for C20H22N7 (M ꢃ Br)+ 360.1937; found 360.1935. Anal.
C20H22N7Br $0.8H2O (C, H, N).
(125 MHz, DMSO-d6)
d
155.99, 153.03, 152.63, 149.45, 142.00 ꢂ 2,
140.79, 139.96, 138.77, 136.68, 135.07, 128.55 ꢂ 2, 128.17 ꢂ 2,
127.23 ꢂ 2, 127.01 ꢂ 2, 118.60, 108.61 ꢂ 2, 59.06, 48.32 ꢂ 2, 45.82,
24.64 ꢂ 2. HRMS (m/z) calcd for C28H28N7 (M ꢃ Br)+ 462.2406;
found 462.2426. Anal. C28H28N7Br $0.6H2O (C, H, N).
5.3.10. 1-(4-{4-[(6-amino-3H-purin-3-yl)methyl]phenyl}benzyl)-4-
(dimethylamino)pyridinium bromide (17)
Yield: 23%. M.p. >350 ꢁC (decomposition). 1H NMR (500 MHz,
CD3OD)
d
8.46 (s, 1H); 8.24 (d, J ¼ 7.7 Hz, 2H); 7.94 (s, 1H); 7.66 (d,
5.3.5. 1-(4-{4-[(6-amino-9H-purin-9-yl)methyl]phenethyl}benzyl)-
J ¼ 8.2 Hz, 2H); 7.61 (d, J ¼ 8.2 Hz, 2H); 7.48 (d, J ¼ 8.2 Hz, 2H); 7.45
4-(dimethylamino)pyridinium bromide (12)
(d, J ¼ 8.2 Hz, 2H); 7.01 (d, J ¼ 7.7 Hz, 2H); 5.64 (s, 2H); 5.40 (s, 2H);
Yield: 72%. M.p.163e166 ꢁC 1H NMR (300 MHz, CD3OD)
d
8.21 (s,
3.25 (s, 6H). 13C NMR(125 MHz, CD3OD)
d 158.05, 156.74, 153.00,
1H); 8.17 (d, J ¼ 7.9 Hz, 2H); 8.11 (s, 1H); 7.25e7.18 (m, 6H); 7.13 (d,
150.20, 145.78, 143.11 ꢂ 2, 142.57, 141.64, 136.13, 135.39, 129.92 ꢂ 2,
129.52 ꢂ 2, 128.92 ꢂ 2, 128.58 ꢂ 2, 120.85, 109.15 ꢂ 2, 61.44, 53.76,
40.36 ꢂ 2. HRMS (m/z) calcd for C26H26N7 (M ꢃ Br)+ 436.2250;
found 436.2233. Anal. C26H26N7Br $1.3H2O (C, H, N).
J ¼ 8.2 Hz, 2H); 6.98 (d, J ¼ 7.9 Hz, 2H); 5.38 (s, 2H); 5.29 (s, 2H);
3.24 (s, 6H); 2.89 (m, 4H). 13C NMR (75 MHz, CD3OD)
d 157.97,
157.33, 153.84, 150.68, 144.22, 142.99 ꢂ 2, 142.86, 142.60, 135.21,
133.59, 130.60 ꢂ 2, 130.15 ꢂ 2, 129.34 ꢂ 2, 128.76 ꢂ 2, 119.96,
109.05 ꢂ 2, 61.57, 47.84, 40.38 ꢂ 2, 38.29, 38.23. HRMS (m/z)
calcd for C28H30N7 (M ꢃ Br)+ 464.2563; found 464.2574. Anal.
C28H30N7Br $1.2H2O (C, H, N).
5.3.11. 1-(4-{4-[(6-amino-3H-purin-3-yl)methyl]phenethyl}
benzyl)-4-pyrrolidinopyridinium bromide (18)
Yield: 36%. M.p. 203e207 ꢁC 1H NMR (500 MHz, CD3OD)
d
8.33 (s, 1H); 8.14 (d, J ¼ 7.4 Hz, 2H); 7.91 (s, 1H); 7.27 (d,
5.3.6. 1-(4-{4-[(6-amino-9H-purin-9-yl)methyl]phenethyl}benzyl)-
J ¼ 7.9 Hz, 2H); 7.23 (d, J ¼ 7.9 Hz, 2H); 7.18 (d, J ¼ 7.9 Hz, 2H);
7.14 (d, J ¼ 7.9 Hz, 2H); 6.83 (d, J ¼ 7.4 Hz, 2H); 5.53 (s, 2H); 5.28
(s, 2H); 3.53 (t, J ¼ 6.6 Hz, 4H); 2.89 (m, 4H); 2.11 (t, J ¼ 6.6 Hz,
4-pyrrolidinopyridinium bromide (13)
Yield: 41%. M.p. 203e207 ꢁC 1H NMR (400 MHz, CD3OD)
d 8.21
(s, 1H); 8.14 (d, J ¼ 7.7 Hz, 2H); 8.10 (s, 1H); 7.27e7.19 (m, 6H); 7.13
(d, J ¼ 7.9 Hz, 2H); 6.83 (d, J ¼ 7.7 Hz, 2H); 5.37 (s, 2H); 5.28 (s, 2H);
3.54 (t, J ¼ 6.8 Hz, 4H); 2.89 (m, 4H); 2.11 (q, J ¼ 6.8 Hz, 4H). 13C NMR
4H). 13C NMR (125 MHz, CD3OD)
d 156.70, 155.21, 153.18, 150.81,
145.48, 144.17, 143.32, 142.91 ꢂ 2, 134.05, 133.65, 130.61 ꢂ 2,
130.25 ꢂ 2, 129.29 ꢂ 2, 129.00 ꢂ 2, 121.12, 109.63 ꢂ 2, 61.66,
53.86, 49.70 ꢂ 2, 38.20 ꢂ 2, 26.11 ꢂ 2. HRMS (m/z) calcd for
C30H32N7 (M ꢃ Br)+ 490.2720; found 490.2711. Anal. C30H32N7Br $
1.3H2O (C, H, N).
(75 MHz, CD3OD)
d
157.33, 155.13, 153.84, 150.67, 144.16, 142.92 ꢂ 2,
142.87, 142.61, 135.20, 133.72, 130.58 ꢂ 2, 130.15 ꢂ 2, 129.32 ꢂ 2,
128.77 ꢂ 2, 119.95, 109.64 ꢂ 2, 61.58, 49.73 ꢂ 2, 47.83, 38.29, 38.24,

B. Rubio-Ruíz et al. / European Journal of Medicinal Chemistry 50 (2012) 154e162
161
5.3.12. 1-[4-(4-{4-[(6-amino-3H-purin-3-yl)methyl]phenyl}butyl)
benzyl]-4-(dimethylamino)pyridinium bromide (19)
centrifugation at 12000 g for 3 min. Aliquots of the supernatant
were applied to the origin of Silica Gel plates in the presence of
phosphocholine (0.1 mg) and choline (0.1 mg) as carriers. The
chromatography was developed in methanol/0.6% NaCl/28%
NH4OH in water (50:50:5, v/v/v) as solvent. Phosphocholine was
visualized under exposure to iodine vapor and the corresponding
spot was scraped and transferred to scintillation vials for
measurement of radioactivity by a Beckman 6000-TA (Madrid,
Spain) liquid scintillation counter.
Yield: 23%. M.p. >350 ꢁC (decomposition). 1H NMR (500 MHz,
CD3OD)
d
8.39 (s, 1H); 8.19 (d, J ¼ 7.8 Hz, 2H); 7.92 (s, 1H); 7.28 (d,
J ¼ 8.1 Hz, 2H); 7.25 (d, J ¼ 8.1 Hz, 2H); 7.19 (d, J ¼ 8.1 Hz, 2H); 7.14
(d, J ¼ 8.1 Hz, 2H); 6.98 (d, J ¼ 7.8 Hz, 2H); 5.54 (s, 2H); 5.30 (s, 2H);
3.24 (s, 6H); 2.62e2.58 (m, 4H); 1.58 (q, J ¼ 7, 4H). 13C NMR
(125 MHz, CD3OD)
d 158.00, 156.69, 153.02, 150.43, 145.66, 145.20,
144.36, 143.01 ꢂ 2, 133.85, 133.39, 130.43 ꢂ 2, 130.07 ꢂ 2,
129.36 ꢂ 2, 128.94 ꢂ 2, 120.63, 109.07 ꢂ 2, 61.61, 53.86, 40.34 ꢂ 2,
36.23 ꢂ 2, 31.90 ꢂ 2. HRMS (m/z) calcd for C30H34N7 (M ꢃ Br)+
492.2876; found 492.2874. Anal. C30H34N7Br $1.3H2O (C, H, N).
5.4.2. Cell proliferation
HepG2 cells were seeded onto 96-well plates (10 000 cells/well)
and maintained in MEM containing 10% FBS for 24 h. Then, the
culture medium was replaced with fresh medium/10% FBS and the
cells were incubated for 24 h in the absence or presence of different
amounts of ChoK inhibitors before analyses. The antiproliferative
effect of the distinct compounds was assessed by the crystal violet-
staining assay using a cell number-based standard curve. The
medium was removed and the cells were fixed by addition of 100 ml
of a 1.1% glutaraldehyde solution. After shaking (50 rpm for 15 min),
plates were washed eight times with ultrapure water. Plates were
5.3.13. 1-[4-(4-{4-[(6-amino-3H-purin-3-yl)methyl]phenyl}butyl)
benzyl]-4-pyrrolidinopyridinium bromide (20)
Yield: 38%. M.p. 244 ꢁC (gel) e 259 ꢁC (foam). 1H NMR (500 MHz,
CD3OD)
d
8.38 (s, 1H); 8.16 (d, J ¼ 7.6 Hz, 2H); 7.92 (s, 1H); 7.28 (d,
J ¼ 8 Hz, 2H); 7.25 (d, J ¼ 8 Hz, 2H); 7.17 (d, J ¼ 8 Hz, 2H); 7.12 (d,
J ¼ 8 Hz, 2H); 6.82 (d, J ¼ 7.6 Hz, 2H); 5.54 (s, 2H); 5.29 (s, 2H); 3.52 (t,
J ¼ 6.8 Hz, 4H); 2.60e2.56 (m, 4H); 2.10 (q, J ¼ 6.8 Hz, 4H); 1.57 (q,
J ¼ 7, 4H).13C NMR (125 MHz, CD3OD)
d 156.65,155.14,153.01,150.77,
145.61, 145.11, 144.35, 142.92 ꢂ 2, 133.83, 133.49, 130.39 ꢂ 2,
130.06 ꢂ 2, 129.34 ꢂ 2, 128.97 ꢂ 2, 120.92, 109.65 x2, 61.64, 53.86,
49.70 x2, 36.21 x2, 31.86 x2, 26.11 x2. HRMS (m/z) calcd for C32H36N7
(M ꢃ Br)+ 518.3032; found 518.3039. Anal. C32H36N7Br $1.2H2O
(C, H, N).
air-dried and stained by addition of 200 ml of a 0.1% solution of
crystal violet dissolved in water. After shaking (30 rpm for 20 min),
the dye was removed and plates were extensively washed with
deionized water. Samples were air-dried prior to dye solubilisation
in 100 ml of 10% acetic acid. The absorbance of crystal violet in each
well was measured at a wavelength of 590 nm directly in plates
using a microplate reader.
5.4. Biological assays
[Methyl-14C]choline chloride (55 mCi/mmol) was supplied by
Perkin Elmer (Massachusetts, USA). Fetal bovine serum (FBS) was
from The Cell Culture Company (Pasching, Austria). Minimal
essential medium (MEM) was from SigmaeAldrich (Madrid, Spain).
Thin-layer chromatography (TLC) plates of Silica Gel 60 A was
acquired from Whatman (Kent, UK). Microwell plates and culture
dishes were obtained from NuncTM (Langenselbold, Germany). All
other reagents were of analytical grade. The human hepatoma
HepG2 cell line was from The European Collection of Animal Cell
Cultures (Salisbury, UK). Cells were cultured in MEM containing
5.5. Molecular modeling and docking studies
Molecular modeling studies were performed by using Sybyl
program [26]. Crystal structure of human ChoKa1 in complex with
HC-3 was obtained from the Protein Data Bank (PDB id: 3G15). This
structure contains a homodimeric protein, and Chain A was chosen
for docking calculations since in Chain B there are more missing
residues. All water molecules and unknown residues were deleted,
and only ADP and HC-3 were conserved in order to define the
binding sites.
10% heat-inasctivated FBS, supplemented with 2 mM
1% nonessential amino acids, 100 U/ml penicillin and 100
L
-glutamine,
Protein was refined by using the Structure Preparation Tool
module of Sybyl. Missing side chains of those residues situated far
away from the binding sites were added, and protein N-terminal
and C-terminal were fixed with ACE and NME, respectively.
Hydrogens and charges were also added and protonation type of
Glu, Asp, Gln and Asp was analyzed and fixed. Hydrogen orienta-
tions were also checked in order to maintain intramolecular
hydrogen bonds into the protein. Finally, ADP and HC-3 atom types
and connectivity were carefully checked to assure the correction of
these molecules.
Ligands were constructed from standard fragments of the
Libraries of the Sybyl program. As described previously [16], a new
type of atom was necessary to define in order to build the mole-
cules: N.ar4, the quaternary nitrogen of the pyridinium fragments.
Additional parameters were also developed from ab initio calcula-
tions to optimize the geometry of these molecules [16]. Charges
were calculated by means of Gaussian Program [27], and optimi-
zations were tackled using the BFGS method.
The Surflex-Dock [28] module implemented in the Sybyl
program was used for docking studies. Surflex-Dock Protomol was
prepared using both ADP and HC-3 ligands inserted into the 3G15
crystal structure, with a threshold value of 0.5 and a Bloat of 0 A.
Surflex-Dock GeomX (SFXC) protocol was used, the search grid was
expanded in 3 A, 50 additional starting conformation were used for
each molecule and 30 conformations per fragment. Results were
analyzed using Sybyl program and the most stable pose for each
mg/ml
streptomycin. Cells were grown in a humidified atmosphere with
5% CO2 at 37 ꢁC and were subcultured at a 1:10 ratio once a week.
Cells were plated on tissue-culture dishes at a density of 5 x 104
cells/cm2 and maintained in culture at approximately 70% conflu-
ence. Cytosol was obtained as described [23] with minor modifi-
cations. Briefly, HepG2 cells were seeded onto 6-well dishes and
maintained in MEM containing 10% FBS until confluence. Then,
cells were scraped into ice-cold 275 mM Tris (pH 8.5). Cells in
suspension were sonicated for 3 s with a microprobe in an ice bath,
and the homogenate was centrifuged immediately at 12000 g for
5 min at 4 ꢁC. After centrifugation the cell supernatant was
immediately frozen in liquid nitrogen and stored at ꢃ80 ꢁC until
use.
5.4.1. Choline kinase inhibition
ChoK activity was assayed by measurement of the rate of
incorporation of 14C from [methyl-14C]choline into phosphocholine.
Briefly, the final reaction mixture contained 100 mM Tris (pH 8.5),
10 mM MgCl2, and 10 mM ATP, approximately 50 mg of cytosolic
protein and different amounts of ChoK inhibitors. The reaction was
initiated with 1 mM [methyl-14C]choline (4500 dpm/nmol) and it
was incubated at 37 ꢁC for 20 min, being the final volume of 50
ml.
The assay was stopped by immersion of the reaction tubes in
boiling water for 3 min and protein precipitate was removed by

162
B. Rubio-Ruíz et al. / European Journal of Medicinal Chemistry 50 (2012) 154e162
Barón, J.C. . Lacal, Expression of choline kinase alpha to predict outcome in
patients with early-stage non-small-cell lung cancer: a retrospective study,
Lancet Oncol. 8 (2007) 889e897.
molecule was chosen as the preferred one inside the ChoK enzyme.
Figures were built using the PyMOL program [29].
[12] A. Conejo-García, M. Bañez-Coronel, R. Sánchez-Martín, A. Rodríguez-Gon-
zález, A. Ramos, A. Ramírez de Molina, A. Espinosa, M.A. Gallo, J. Campos,
J.C. Lacal, Influence of the linker in bispyridinium compounds on
the inhibition of human choline kinase, J. Med. Chem. 47 (2004)
5433e5440.
[13] R. Sanchez-Martín, J.M. Campos, A. Conejo-García, O. Cruz-López, M. Bañez-
Coronel, A. Rodríguez-González, M.A. Gallo, J.C. Lacal, A. Espinosa, Symmet-
rical bis-quinolinium compounds: new human choline kinase inhibitors with
antiproliferative activity against the ht-29 cell line, J. Med. Chem. 48 (2005)
3354e3363.
[14] A. Conejo-García, J. Campos, R.M. Sanchez, A. Rodríguez-Gonzalez,
J.C. Lacal, M.A. Gallo, A. Espinosa, Choline kinase inhibitory effect and
antiproliferative activity of new 1,1’,1’’-(benzene-1,3,5-triylmethylene)tris
{4-[(disubstituted)amino]pyridinium} tribromides, Eur. J. Med. Chem. 38
(2003) 109e116.
Acknowledgments
We thank the “Consejería de Innovación, Ciencia y Empresa,
Junta de Andalucía” (Excellence Research Project no. P07-CTS-
03210) and the “ Ministerio de Ciencia e Innovación” (Project no.
SAF2009-11955) for the financial support. The award of grants from
the “Ministerio de Educación” to B ReR. and P.R-M is gratefully
acknowledged. We thank Xiomara Gálvez for her technical support.
Appendix. Supplementary material
[15] A. Conejo-García, J.M. Campos, R.M. Sánchez-Martín, M.A. Gallo, A. Espinosa,
Bispyridinium cyclophanes: novel templates for human choline kinase
inhibitors, J. Med. Chem. 46 (2003) 3754e3757.
[16] A. Conejo-García, J.M. Campos, A. Entrena, R.M. Sánchez-Martín, M.A. Gallo,
A. Espinosa, Conformational dynamics of a bispyridinium cyclophane, J. Org.
Chem. 68 (2003) 8697e8699.
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.01.050.
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