Synthesis and biological activity of new bispyridinium salts of 4,4′-bispyridyl-5,5′-perfluoroalkyl-2,2′-bisoxazoles

Article abstract of DOI:10.1016/j.ejmech.2011.09.046

A series of bispyridinium compounds were synthesized by a short sequence of reactions from symmetric diamides. All compounds were tested for their antiproliferative activity against HT-29, a cell line derived from a human colon adenocarcinoma, and their i

Full text of DOI:10.1016/j.ejmech.2011.09.046

European Journal of Medicinal Chemistry 46 (2011) 5662e5667
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of new bispyridinium salts of 4,40-bispyridyl-5,
50-perfluoroalkyl-2,20-bisoxazoles
*
Y. Martín-Cantalejo , B. Sáez, M.I. Monterde, M.T. Murillo, M.F. Braña
Departamento de Ciencias, Escuela Politécnica, Universidad Europea de Madrid, Villaviciosa de Odón 28670, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of bispyridinium compounds were synthesized by a short sequence of reactions from symmetric
diamides. All compounds were tested for their antiproliferative activity against HT-29, a cell line derived
from a human colon adenocarcinoma, and their inhibitory activity against choline kinase (ChoK), a novel
anticancer molecular target already in clinical trials. Most of the compounds analyzed showed good
antiproliferative activities, in the micromolar range, with the identification of promising lead molecules
as a new family of potential inhibitors of ChoK.
Received 13 June 2011
Received in revised form
23 September 2011
Accepted 26 September 2011
Available online 2 October 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Bisoxazol
Bispyridinium
Antiproliferative
Choline kinase
Inhibitory activity
1. Introduction
structures within this family has inspired the development of
methods to synthesize specific substitution patterns, most of them
One of the targets recently identified for antitumor drug
discovery is choline kinase (ChoK) [1e3]. This enzyme plays a vital
role in cell signaling pathways and regulation of cell growth, being
involved in malignant transformation through ras oncogenes. ChoK
is overexpressed in some of the most relevant human tumors such
as breast, colon, lung, prostate, hepatic lymphomas and ovary [4].
Generation of ChoK inhibitors will provide new substances with
potential anticancer activity [5e8].
As a part of our work related to the development of new anti-
tumor agents, we have synthesized several families of 4,40-bispyr-
idyl-2,20-bisoxazoles (2) and their salts (3) [9,10], molecules
structurally related to the potent ChoK inhibitor hemicolinium (HC-
3) and its bispyridinium derivatives [5] (Fig. 1).
Oxazoles and bisoxazoles represent an important class of five-
membered heterocycles, found in a variety of biologically signifi-
cant natural products [11]. Compounds wearing this structure have
been reported to exhibit antiproliferative [12,13], anti-
inflammatory [14], hypoglucemic [15], antibacterial [16,17] and
antifungal activities [18]. Even if the proliferation of interesting
are focused, in the case of bisoxazoles [19], on the construction of
a 2,40 linkage by the stepwise formation of the rings [20] or by cross
coupling of previously synthesized heterocycles [21,22]. Symmet-
rical and especially 2,20-bisoxazoles are scarcely reported [23e25].
Usually their synthesis involve long stepwise procedures, instead of
a simultaneous construction of both rings, and/or low reaction
yields.
We have developed a new convergent synthesis of 4,40-bispyr-
idyl-2,20-bisoxazoles 5,50-methyl or perfluoralkyl disustituted 2
starting from symmetric diamides [9]. Quaternization of 4,40-bis-
pyridyl-5,50-perfluoralkyl-2,20-bisoxazoles with alkyl groups
(methyl, allyl) have provided compounds 3 with antitumor activity
in the micromolar range against HT-29 cells and other cell lines
[10,26], even if unexpectedly, some of the generated structures
showed very little effects on ChoK. We also described [10] oxazole
3b, bearing a hydroxyethyl group, which displayed antiproliferative
properties in tumor cell lines and prevented in vivo tumor growth
in nude mice. Here we report the synthesis, antiproliferative and
ChoK inhibitory activity of new bisoxazoles related to compound
3b. We further explore the influence of the Z linker and per-
fluoroalkyl substituent of 2,20 bisoxazoles in activity, extending the
range of alkylating agents to those providing electron donating
groups, and describing other counteranions besides iodine.
* Corresponding author. Tel.: þ34 91 2115619.
E-mail address: yolanda.martin@uem.es (Y. Martín-Cantalejo).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.046

Y. Martín-Cantalejo et al. / European Journal of Medicinal Chemistry 46 (2011) 5662e5667
5663
R
R
O
O
Z
N
N
N
N
N
N
N
HO
OH
O
O
2
R
R
N
N
O
O
CH3
H3C
CH3
H3C
Z
Hemicolinium
N
R'
R'
X
X
3
3b
Z = p-biphenyl, R = CF3, R' = CH2-CH2OH, X = I
Fig. 1. 4,40-Bispiridyl-2,20-bisoxazoles 5,50-disustituted previously synthesized and hemicolinium.
2. Results and discussion
between the linker and ChoK inhibitory activity is not
straightforward.
2.1. Chemistry
It is more difficult to see a clear relationship between the per-
fluoroalkyl sustituent and the biological activity. In most cases,
a CF3CF2 chain seems to afford better antiproliferative activity than
CF3 or CF3CF2CF2 (3a vs 3c and 3d, 3i vs 3j, 3l vs 3m and 3n), but not
always (3e and 3g vs 3h). Regarding the alkylating agent, both
CH2eCH2OH and CH2eCON(CH3)2 (which have in common the
ability to form hydrogen bonds), provided good antiproliferative
activity, but the best values for ChoK inhibition were obtained with
the CH2eCON(CH3)2 sustituent (3g, 3p, 3h). The introduction of
a chloroethyl sustituent, unable to form hydrogen bonds, lowers
the antiproliferative activity (3l, 3m, 3n). The use of different
counterions (chlorine, iodine, methylsulfonate) has not been
studied thoroughly as this is not a key issue at this point of the
development of the potential drugs.
In summary, the synthesis and characterization of a new series
of bispyridinium salts of 4,40-bispyridyl-5,50-perfluoroalkyl-2,20-
bisoxazol have been described. The tested compounds displayed
antiproliferative activity against human adenocarcinoma HT-29
cells, and inhibitory activity towards ChoK. Compounds bearing
a p-biphenyl linker and an alkylating agent able to form hydrogen
bonds could serve as lead chemical entities for further modification
to render them clinically useful drug agents.
Sixteen final compounds 3 have been synthesized, using the one
pot method described by our lab [9,10] to convert N,N0-bis(4-
pyridylmethylen)diamides into bisoxazoles using perfluoroalkyl
anhydrides as a fluorine source.
In this way (Scheme 1), the treatment of symmetric diamides 1
(easily prepared from the corresponding diacid chloride) with
perfluoroalkyl anhydrides (bearing three, five, or seven fluorine) in
dry toluene in the presence of pyridine provided 5,50-bisper-
fluoroalkyl-2,20-bisoxazoles 2 in low (41%) to excellent (94%)
yields. Three different linkers (p-biphenyl, PheC(CF3)2ePh, p-
phenyl) were used. Quaternization of the pyridine nitrogen with
functionalized alkylating agents afforded the target molecules 3.
The reaction was performed either in a dry solvent (acetone or
alcohols) or without solvent, and a sealed tube was needed in
some cases.
2.2. Biological activity
All the compounds were tested for antitumoral activity against
the human adenocarcinoma HT-29 cells. They were also tested in
an ex vivo system using recombinant human ChoK as target as
described in the experimental section, in order to evaluate the
inhibitory potency of the compounds toward ChoK without the
possible interference due to permeability through membranes.
Table 1 summarizes the activity of the pyridinium salts of bisox-
azoles prepared, indicated as IC50. The table is arranged in
descending order of antiproliferative activity.
Compounds 3ae3l showed potent cytotoxicity against the
human tumor-derived cell line tested, in the range described for
compound 3b. This confirmed our prediction that alkylating groups
wearing electrondonating atoms would increase the potency of
symmetric perfluoroalkyl-2,20-bisoxazoles 3 as antiproliferative
agents. Even if the initial design was focused on the generation of
inhibitors against ChoK, the results show that in most cases anti-
proliferative and inhibition data correlates (3a, 3e, 3f, 3g, 3h, 3i,
3k), but not always (3b, 3c and 3o, 3p).
3. Experimental section
3.1. Chemistry
3.1.1. General
The 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were
recorded in a Bruker AC 300 (300 MHz) spectrometer. Chemical
shifts were reported in ppm (d) relative to the middle of solvent
signals. Signals were designated as follows: s, singlet; bs, broad
singlet; d, doublet; t, triplet; m, multiplet. IR spectra were taken on
a PerkineElmer 1330 spectrometer. ESI mass spectra were obtained
on Esquire-LC_00126 mass spectrometer from Bruker. Melting
points were determined on a GallenKamp capillary melting point
apparatus and were not corrected. For purification of crude reaction
mixtures by flash chromatography, Merck silica gel (230e240
mesh) was used as the stationary phase. Identification of products
was made by analytical TLC (Merck Kieselgel 60F-254) and UV light
It is observed clearly that antiproliferative activity increases
using p-biphenyl as linker. The distance between the cationic heads
must have with this linker the optimum value. Once such a distance
is exceeded (when spacer PheC(CF3)2ePh is used) the anti-
proliferative activity decreases slightly, and when it is shortened
(using a p-phenyl spacer) it drops completely. The relationship
(
l
¼ 254 mm). All the commercially available reagents were ob-
tained from Aldrich and used without further purification. Reagents
and solvents were handled by using standard syringe techniques.
All solvents used were reagent grade.

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Y. Martín-Cantalejo et al. / European Journal of Medicinal Chemistry 46 (2011) 5662e5667
O
O
Cl
Z
Cl
N
H
Z
N
H
N
CH2NH2
+
N
N
O
O
1
1a
Z = p-biphenyl
1b Z = p,p'-Ph-C(CF3)2-Ph
1c Z = p-phenyl
(RCO)2O
Pyridine
R
R
R
R
O
O
O
O
R'-X
Z
N
N
Z
N
N
N
N
R' = CH2-CH2OH
CH2-CH2Cl
N
N
X
R'
R'
2
3
CH2-CON(CH3)2
X
2a
Z = p-biphenyl, R = CF3
2b Z = p-biphenyl, R = CF3CF2
X = Cl, SO3CH3, I
2c Z = p-biphenyl, R = CF3CF2CF2
2d
Z = p,p'-Ph-C(CF3)2-Ph, R = CF3
2e Z = p,p'-Ph-C(CF3)2-Ph, R = CF3CF2
2f Z = p,p'-Ph-C(CF3)2-Ph, R = CF3CF2CF2
2g
Z = p-phenyl, R = CF3
2h Z = p-phenyl, R = CF3CF2
Scheme 1. Synthesis of bispyridinium salts 3.
3.1.2. General procedure for the synthesis of symmetrical bisamides,
1
previously sonicated, pyridine was added (12 mmol). The mixture
was cooled to 0 ꢀC and the corresponding perfluoroalkyl anhydride
(6 mmol) was added dropwise. The mixture was left to attain room
temperature for 48 h. The solvent was evaporated, the obtained solid
was dissolved in CH2Cl2, extracted with water and washed with 5%
Na2CO3. The combined organics extracts were dried with MgSO4. The
solvent was evaporated and purification of crude was accomplished
by flash column chromatography or by recrystallization in adequate
solvent to obtain compounds 2c, 2e, 2f and 2h. Bisoxazoles 2a, 2b, 2d
and 2g were described previously [6].
Bisamides 1a, 1b y 1c, were synthesized by acylation of 4-
aminomethylpyridine (8.2 mmol, 2 eq) with different acid chlo-
rides(4.1 mmol,1 eq) using 5 mL of CH2Cl2 or AcOEt as solvent in Et3N
(13mmol, 3eq)usingdimethylaminepyridineascatalyst(0.82 mmol,
0.2 eq) [6]. The bisamides were obtained in moderate to good yields
(1a: 75%; 1b: 42%; 1c: 85%) after purification of crude by flash
chromatography using as eluent a mixture of CH2Cl2:MeOH (95:5).
3.1.3. General procedure for the synthesis of 4,40-bispyridyl-5,50-
perfluoroalkyl-2,20-bisoxazoles, 2
Toastirredsolutionof therespectivebisamide 1a,1b,1c, (1mmol)
in anhydrous toluene (10 mL), kept under argon atmosphere and
3.1.3.1. 4,4-Bis[5-heptafluoropropyl-4-(4-pyridyl)oxazolyl]byphenyl,
2c. Obtained from 1a (131.3 mg, 0.311 mmol) and hepta-
fluorobutyric anhydride (0.46 mL, 3.732 mmol) as a white solid
Table 1
Antiproliferative and ChoK inhibitory activity of novel pyridinium salts 3.
b
b
Compound
Z
R
R0
X
Yield
IC50
(m
M) antiproliferative
IC50 (mM) ex vivo
3a
3ba
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
p-biphenyl
p-biphenyl
p-biphenyl
p-biphenyl
p-biphenyl
p-biphenyl
p-biphenyl
p-biphenyl
p,p0-PheC(CF3)2ePh
p,p0-PheC(CF3)2ePh
p,p0-PheC(CF3)2ePh
p,p0-PheC(CF3)2ePh
p,p0-PheC(CF3)2ePh
p,p0-PheC(CF3)2ePh
p,p0-PheC(CF3)2ePh
p-phenyl
CF3CF2
CF3
CF3
CH2eCH2OH
CH2eCH2OH
CH2eCH2OH
CH2eCH2OH
CH2eCON(CH3)2
CH2eCH2OH
CH2eCON(CH3)2
CH2eCON(CH3)2
CH2eCH2OH
CH2eCH2OH
CH2eCH2OH
CH2eCH2Cl
CH2eCH2Cl
CH2eCH2Cl
CH2eCON(CH3)2
CH2eCON(CH3)2
CH2eCON(CH3)2
Cl
I
Cl
Cl
Cl
I
Cl
Cl
Cl
Cl
I
SO3CH3
SO3CH3
SO3CH3
Cl
Cl
Cl
70
36
55
31
94
66
50
91
65
70
58
45
42
41
91
89
65
0.84 ꢁ 0.005
1.50 ꢁ 0.03
1.85 ꢁ 0.01
2.26 ꢁ 0.03
2.50 ꢁ 0.02
3.60 ꢁ 0.02
4.00 ꢁ 0.06
5.50 ꢁ 0.01
6.00 ꢁ 0.04
6.35 ꢁ 0.01
7.70 ꢁ 0.01
9.30 ꢁ 0.01
13.75 ꢁ 1.5
15.50 ꢁ 1.5
>20
9.65 ꢁ 0.04
23.80 ꢁ 2.5
>20
CF3CF2CF2
CF3CF2CF2
CF3CF2
CF3
CF3CF2
CF3CF2
CF3CF2CF2
CF3CF2
CF3CF2
CF3
17.30 ꢁ 1.5
4.25 ꢁ 0.06
3.10 ꢁ 0.01
0.30 ꢁ 0.003
2.00 ꢁ 0.03
4.40 ꢁ 0.02
13.50 ꢁ 1.5
4.20 ꢁ 0.01
14.20 ꢁ 2.5
12.50 ꢁ 1.7
11.60 ꢁ 3.5
6.15 ꢁ 0.02
1.35 ꢁ 0.01
15.00 ꢁ 4.5
CF3CF2CF2
CF3
CF3
>50
>50
p-phenyl
CF3CF2
a
Described in reference [10].
IC50 values are means ꢁ SEM of at least three independent measurements.
b

Y. Martín-Cantalejo et al. / European Journal of Medicinal Chemistry 46 (2011) 5662e5667
5665
with a yield of 30% after recrystallization from CHCl3/MeOH/Et2O.
Mp: 192e194 ꢀC. 1H NMR (CDCl3)
: 8.79 (d, J ¼ 5.5 Hz, 4H, Py), 8.26
(d, J ¼ 8.5 Hz, 4H, Ph), 7.82 (d, J ¼ 7.9 Hz, 4H, Ph), 7.76 (d, J ¼ 5.5 Hz,
0.09 mmol) and 2-chloroethanol (1 mL, 15 mmol) after refluxing
under argon atmosphere for four hours. After cooling, acetone was
added until a solid precipitated. It was filtered and washed with
acetone to obtain 35 mg of compound 3c as a beige solid. Yield: 55%.
d
4H, Py); 13C NMR (CDCl3)
d: 162.9, 148.8, 143.2, 142.3, 138.2, 135.3,
136.7, 127.8, 127.7, 124.9, 123.3, 109.3; IR (cmꢂ1) (KBr): 1614, 1589,
1545, 1378, 1232, 1199, 1116, 877.
Mp: >250 ꢀC.1H NMR (DMSO-d6)
d
: 9.17(d, 4H, Pyr, J ¼ 6.27 Hz); 8.47
(d, 4H, Pyr, 6.09 Hz); 8.31(d, 4H, Ph, J ¼ 7.92 Hz); 8.12 (d, 4H, Ph,
J ¼ 8.55 Hz); 5.45 (bs, 2H, OH); 4.76 (bs, 4H, CH2N); 3.92 (bs, 4H,
3.1.3.2. 2,2-Bis[5-pentafluoroethyl-4-(4-pyridyl)oxazolyl]hexa-
fluoropropane, 2e. Obtained from 1b (300 mg, 0.52 mmol) and
pentafluoropropionic anhydride (0.61 mL, 3.14 mmol) as a white
solid with a yield of 50% after flash chromatography with a mixture
CH2OH); 13C NMR (DMSO-d6)
d: 164.2, 147.0, 146.9, 144.9, 138.3,
129.3, 129.0, 127.51, 125.9, 120.2, 66.9, 65.1, 61.6.
3.1.4.3. 4,40-Bis[(5-heptafluoropropyl-4-(hydroxiethylpyridinium-4-
yl)oxazolyl)]biphenyldichloride, 3d. Obtained from bisoxazol 2c
(50 mg, 0.064 mmol) and 2-chloroethanol (1 mL, 15 mmol) after
heating at 110 ꢀC in a sealed tube under argon atmosphere for three
days. After cooling, acetone (2 mL) was added and then diethyl
ether until a yellow solid was precipitated. The solid was filtered
and purified by recristallization from MeOH/Et2O/acetone to obtain
40.1 mg of compound 3d as a pale yellow solid. Yield: 31%. Mp:
CH2Cl2/MeOH 3%. Mp: 193e195 ꢀC. 1H NMR (CDCl3)
d: 8.8 (bs, 4H,
Py), 8.19 (d, 4H, J ¼ 8.7 Hz, Ph), 7.8 (d, 4H, J ¼ 5.0 Hz, Py), 7.6 (d, 4H,
J ¼ 8.2 Hz, Ph); 13C NMR (CDCl3)
d: 161.2, 147.11, 140.8, 137.9, 135.8,
134.5, 134.1, 129.9, 126.3, 125.2, 122.6; IR (cmꢂ1) (KBr): 1619, 1592,
1550, 1505, 1375, 1341, 1170, 901.
3.1.3.3. 2,2-Bis[5-heptafluoropropyl-4-(4-pyridyl)oxazolyl]hexa-
fluoropropane, 2f. Obtained from 1b (678 mg, 1.185 mmol) and
heptafluorobutyric anhydride (1.74 mL, 7.11 mmol) as a foaming
solid with a yield of 37% after flash chromatography with a mixture
>250 ꢀC. 1H NMR (MeOH-d4)
d
: 9.15 (d, J ¼ 6.7 Hz, 4H, Py), 8.51 (d,
J ¼ 6.12 Hz, 4H, Py), 8.34 (d, J ¼ 7.9 Hz, 4H, Ph), 8.04 (d, J ¼ 7.9 Hz,
4H, Ph), 4.80 (t, J ¼ 4.3 Hz, 4H, CH2Py), 4.06 (t, J ¼ 4.3 Hz, 4H,
CH2Cl2/MeOH 3%. Mp: 195e197 ꢀC. 1H NMR (CDCl3)
d: 8.78 (d, 4H,
CH2Py); 13C NMR (MeOH-d4)
d: 165.3, 147.1, 146.8, 145.1, 141.0, 138.1,
J ¼ 5.49 Hz, Py), 8.19 (d, 4H, J ¼ 8.5 Hz, Ph), 7.77 (d, 4H, J ¼ 5.49 Hz,
137.6, 129.3, 128.1, 125.8, 119.8, 110.8, 65.1, 61.6.
Py), 7.59 (d, 4H, J ¼ 7.9 Hz, Ph); 13C NMR (CDCl3)
d: 162.0, 150.0,
142.9, 136.9, 136.6, 134.2, 130.9, 127.1, 126.3, 122.9, 111.3.
3.1.4.4. 4,40-Bis[(5-heptafluoropropyl-4-(4-N,N0-dimethylacetamide
pyridinium-4-yl)oxazolyl)]biphenyl dichloride, 3e. Obtained from
bisoxazol 2c (41 mg, 0.053 mmol) and 2-chloro-N,N0-dimethyla-
cetamide (0.9 mL, 8.75 mmol) after heating at 110 ꢀC in a sealed
tube under argon atmosphere for 48 h. After cooling, acetone was
added until a solid precipitated. It was filtered to obtain 62.6 mg of
compound 3e as a white solid. Yield: 94%. Mp: >250 ꢀC. 1H NMR
3.1.3.4. 1,4-Bis[5-pentafluoropropyl-4-(4-pyridyl)oxazolyl]benzene,
2h. Obtained from 1c (226 mg, 0.65 mmol) and penta-
fluoropropionic anhydride (0.8 mL, 4.1 mmol) as a beige solid with
a yield of 21% after flash chromatography with a mixture CH2Cl2/
MeOH 3%. Mp: 189e191 ꢀC. 1H NMR (CDCl3)
d: 8.77 (d, 4H, Py,
J ¼ 5.5 Hz, Py), 8.3 (s, 4H, J ¼ 8.2 Hz, Ph), 7.69 (d, 4H, J ¼ 5.5 Hz, Py);
(MeOH-d4)
8.34 (d, 4H, J ¼ 8.5 Hz, Ph), 8.04 (d, 4H, J ¼ 8.5 Hz, Ph), 5.85 (s, 4H,
CH2), 3.19 (s, 6H, CH3), 3.05 (s, 6H, CH3); 13C NMR (MeOH-d4)
165.8, 165.4, 148.3, 147.1, 145.0, 141.3, 137.7, 129.3, 129.2, 127.7, 125.8,
119.8, 114.1, 110.8, 62.8, 36.6, 36.3.
d
: 9.03 (d, 4H, J ¼ 6.7 Hz, Py), 8.54 (d, 4H, J ¼ 6.1 Hz, Py),
13C NMR (CDCl3)
d: 161.9, 151.2, 142.8, 136.7, 134.5, 128.3, 127.8,
122.9, 118.0. 109.3; IR (cmꢂ1) (KBr): 1702, 1590, 1565, 1496, 1340,
1332, 1311, 993.
d:
3.1.4. General procedure for the synthesis of pyridinium salts of
4,40-bispyridyl-5,50-perfluoroalkyl-2,20-bisoxazoles, 3
3.1.4.5. 4,40-Bis[(5-pentafluoroethyl-4-(hydroxiethylpyridinium-4-yl)
oxazolyl)]biphenyldiiodide, 3f. Obtained from bisoxazol 2b (45 mg,
0.04 mmol) and 2-hydroxyethyl iodide (0.45 mL, 6.4 mmol) after
refluxing for 72 h. The precipitated solid was filtered and washed
with acetone to obtain 92 mg of compound 3f as a beige solid.
Excess of the alkylating agent (10 mmol) (2-hydroxyethyl iodide,
2-hydroxyethyl chloride, 2-chloroethyl methanosulphonate, 2-
chloro-N,N0-dimethylacetamide) was added to a stirred solution of
bisoxazol (1 mmol) in a dry solvent (50 mL) (acetone or isopropanol)
or without solvent and kept under argon atmosphere. After reflux-
ing until total consumption of the starting material (in some cases in
a sealed tube), the reaction mixture was cooled and then the
resulting solid was filtered, washed with dry solvent and dried.
Crude was purified by recrystallization when it was necessary.
Yield: 90%. Mp: >250 ꢀC. 1H NMR (DMSO-d6)
d: 9.16 (d, 4H,
J ¼ 6.7 Hz, Py), 8.44 (d, 4H, J ¼ 6.1 Hz, Py), 8.26 (d, 4H, J ¼ 8.5 Hz, Ph),
8.10 (d, 4H, J ¼ 8.5 Hz, Ph), 5.33 (t, 2H, J ¼ 5.5 Hz, OH), 4.74 (bs, 4H,
CH2eN), 3.91 (bs, 4H, CH2OH); 13C NMR (DMSO-d6)
d: 162.8, 145.9,
143.8, 142.4, 139.7, 134.7, 127.9, 126.4, 124.1, 124.0, 110.0, 63.3, 59.9;
IR (cmꢂ1) (KBr): 3422, 1639, 1613, 1458, 1403, 1373, 1328, 1212, 1119.
MS: 384 (C36H26F10I2N4O4, [Mþ ꢂ 2Iꢂ/2]).
3.1.4.1. 4,40-Bis[(5-pentafluoroethyl-4-(hydroxiethylpyridinium-4-yl)
oxazolyl)]biphenyldichloride, 3a. Obtained from bisoxazol 2b
(60 mg, 0.09 mmol) and 2-chloroethanol (1 mL, 14.9 mmol) after
refluxing under argon atmosphere for two hours. After cooling,
acetone was added and a solid was precipitated. It was filtered and
washed with acetone to obtain 51 mg of compound 3a as a white
3.1.4.6. 4,40-Bis[(5-trifluoromethyl-4-(N,N0-dimethylacetamide-pyr-
idinium-4-yl)oxazolyl)]biphenyl dichloride, 3g. Obtained from
bisoxazol 2a (50 mg, 0.086 mmol) and 2-chloro-N,N0-dimethyla-
cetamide (2.5 mL, 40 mmol) after heating at 110 ꢀC in a sealed tube
for five days. After cooling, acetone was added until a solid was
obtained. The solid was filtered and purified by recristallization
from MeOH/Et2O to obtain 35 mg of compound 3g as a beige solid.
solid. Yield: 70%. Mp: >250 ꢀC. 1H NMR (DMSO-d6)
d: 9.17 (d, 4H, Py,
J ¼ 6.72 Hz); 8.43 (d, 4H, Py, J ¼ 6.72 Hz); 8.25 (d, 4H, Ph,
J ¼ 8.52 Hz); 8.10 (d, 4H, Ph, J ¼ 8.55 Hz); 5.44 (t, OH, 2H,
J ¼ 5.49 Hz); 4.74 (bs, 4H, CH2N); 3.90 (bs, 4H, CH2OH); 13C NMR
(MeOH-d4): 165.3, 147.1, 146.7, 145.0, 141.0, 138.1, 137.6, 129.6, 128.0,
125.8,119.8,110.8, 65.1, 61.6; IR (cmꢂ1) (KBr): 3392.6,1645.6,1615.2,
Yield: 50%. Mp: >250 ꢀC. 1H NMR (MeOH-d4)
d: 9.02 (d, 4H,
J ¼ 5.5 Hz, Py), 8.56 (d, 4H, J ¼ 5.4 Hz, Py), 8.34 (d, 4H, J ¼ 6.8 Hz, Ph),
8.02 (d, 4H, J ¼ 6.6 Hz, Ph), 5.84 (s, 4H, CH2), 3.2 (s, 6H, CH3), 3.06 (s,
1224.7,
1115.4,
940;
MS
m/e
(electrospray):
383.9
6H, CH3); 13C NMR (MeOH-d4)
d: 165.9, 164.2, 148.3, 146.9, 144.8,
(C36H26F10Cl2N4O4, [Mþ ꢂ (2 ꢃ Clꢂ)]/2).
139.6, 138.2, 129.2, 129.1, 127.2, 125.8, 120.5, 62.7, 36.4, 36.6.
3.1.4.2. 4,40-Bis[(5-trifluoromethyl-4-(hydroxiethylpyridinium-4-yl)
oxazolyl)]biphenyldichloride, 3c. Obtained from bisoxazol 2a (50 mg,
3.1.4.7. 4,40-Bis[(5-pentafluoroethyl-4-(N,N0-dimethylacetamidepyr-
idinium-4-yl)oxazolyl)]biphenyl dichloride, 3h. Obtained from

5666
Y. Martín-Cantalejo et al. / European Journal of Medicinal Chemistry 46 (2011) 5662e5667
bisoxazol 2b (50 mg, 0.074 mmol) and 2-chloro-N,N0-dimethyla-
cetamide (0.9 mL, 8.75 mmol) after heating at 110 ꢀC in a sealed
tube for 48 h. After cooling, acetone was added until a solid was
obtained. The solid was filtered and washed with Et2O to give
62.6 mg of compound 3h as a white solid. Yield: 91%. Mp: >250 ꢀC.
137.7,128.2,128.1,127.3,126.9,110.7,119.5, 63.0, 43.4, 39.5; IR (cmꢂ1
(KBr): 3422.9, 1647.8, 1177.4, 1058.7, 939.9.
)
3.1.4.12. 2,2-Bis[5-pentafluoroethyl-4-(4-chloroethyl-pyridinium-4-
yl)oxazolyl]hexafluoropropane dimethylsulfonide, 3m. Obtained
from bisoxazol 2d (65 mg, 0.09 mmol) and 2-chloroethyl-meth-
anesulfonate (1 mL, 8.76 mmol) after heating at 100 ꢀC under argon
atmosphere for nine hours. After cooling Et2O and acetone were
added. The precipitated solid was filtered and washed with Et2O to
obtain 38.5 mg of compound 3m as a white solid. Yield: 42%. Mp:
1H NMR (MeOH-d4)
d
: 9.02 (d, 4H, J ¼ 7.3 Hz, Py), 8.54 (d, 4H,
J ¼ 6.1 Hz, Py), 8.34 (d, 4H, J ¼ 8.5 Hz, Ph), 8.03 (d, 4H, J ¼ 8.5 Hz, Ph),
5.84 (s, 4H, CH2), 3.19 (s, 6H, CH3), 3.05 (s, 6H, CH3); 13C NMR
(MeOH-d4) d: 165.8, 165.2, 148.3, 147.1, 145.0, 140.9, 137.7, 129.3,
127.6, 125.8, 119.8, 117.9, 110.8, 62.8, 36.6, 36.3. MS: 849.3
(C40H32Cl2F10N6O4, [Mþ]); 764 (C40H32Cl2F10N6O4, [Mþ ꢂ 85þ]).
>250 ꢀC. 1H NMR (MeOH-d4)
d
: 9.20 (d, 4H, Py, J ¼ 6.72 Hz); 8.56 (d,
4H, Py, J ¼ 6.72 Hz); 8.33 (d, 4H, Ph, J ¼ 8.55 Hz); 7.71 (d, 4H, Ph,
3.1.4.8. 2,2-Bis[5-pentafluoroethyl-4-(4-hydroxiethyl-pyridinium-4-
yl)oxazolyl]hexafluoropropane dichloride, 3i. Obtained from bisox-
azol 2e (73 mg, 0.09 mmol) and 2-chloroethanol (1 mL, 15 mmol)
after refluxing under argon atmosphere for five and a half hours.
After cooling, Et2O was added. The precipitated solid was filtered
and washed with Et2O and CHCl3 to obtain 57 mg of compound 3i
J ¼ 8.55 Hz); 5.09 (t, 4H, CH2N, J ¼ 4.89 Hz); 4.24 (t, 4H, CH2Cl,
J ¼ 4.89 Hz); 2.69 (s, 6H, CH3SO3); 13C NMR (MeOH-d4)
d: 162.9,
146.6, 139.5, 138.9, 137.7, 131.8, 128.2, 127.3, 126.9, 119.5, 110.6, 66.4,
63.0, 43.4, 38.9. IR (cmꢂ1) (KBr): 3433.3, 1647.2, 1387.4, 1209.0,
1111.0, 1059.0, 993.1.
as a white solid. Yield: 65%. Mp: >250 ꢀC. 1H NMR (MeOH-d4)
d:
3.1.4.13. 2,2-Bis[5-heptafluoroprophyl-4-(4-chloroethyl-pyridinium-
4-yl)oxazolyl]hexafluoropropane dimethylsulfonide, 3n. Obtained
from bisoxazol 2f (50 mg, 0.054 mmol) and 2-chloroethyl-meth-
anesulfonate (1 mL, 9 mmol) after heating at 100 ꢀC under argon
atmosphere for 40 h. After cooling 4 mL of mixture Et2O/acetone
(20:1) was added. The precipitated solid was filtered and purified
by trituration with Et2O to obtain 27.6 mg of compound 3n as
9.13 (d, 4H, Py, J ¼ 6.72 Hz); 8.50 (d, 4H, Py, J ¼ 6.72 Hz); 8.30 (d, 4H,
Ph, J ¼ 9.15 Hz); 7.71 (d, 4H, Ph, J ¼ 8.55 Hz); 4.79 (t, 4H, CH2N,
J ¼ 4.89 Hz); 4.05 (t, 4H, CH2OH, J ¼ 4.89 Hz); 13C NMR (MeOH-d4)
d
: 164.4, 147.1, 146.5, 141.0, 138.3, 138.0, 137.5, 132.4, 128.7, 128.0,
127.3, 120.0, 110.5, 65.1, 61.6; IR (cmꢂ1) (KBr): 3417, 1647.1, 1339.9,
1212.0, 939.5, 752.4, 722.7.
a white solid. Yield: 41%. Mp: >250 ꢀC. 1H NMR (MeOH-d4)
d: 9.21
3.1.4.9. 2,2-Bis[5-heptafluoroprophyl-4-(4-hydroxyethyl-pyridinium-
4-yl)oxazolyl]hexafluoropropane dichloride, 3j. Obtained from
bisoxazol 2f (50 mg, 0.054 mmol) and 2-chloroethanol (1 mL,
15 mmol) after heating at 100 ꢀC under argon atmosphere for 32 h.
After cooling it was added Et2O and acetone (20:1). The precipi-
tated solid was filtered and washed with Et2O. The resulting solid
was purified by recristallization from MeOH/CHCl3/Et2O to obtain
15 mg of compound 3j as a white solid. Yield: 70%. Mp: >250 ꢀC. 1H
(d, 4H, J ¼ 6.7 Hz, Py), 8.54 (d, 4H, J ¼ 6.7 Hz, Py), 8.31 (d, 4H,
J ¼ 9.1 Hz, Ph), 7.72 (dd, 4H, J ¼ 8.5 Hz, Ph), 5.1 (t, 4H, J ¼ 5.5 Hz,
CH2Py), 4.24 (t, 4H, J ¼ 5.5 Hz, CH2Cl), 2.69 (s, 12H, CH3SO3).
3.1.4.14. 2,2-Bis[5-trifluoromethyl-4-(4-N,N0-dimethylacetamide-
pyridinium-4-yl)oxazolyl]hexafluoro
propanedichloride,
3o. Obtained from bisoxazol 2d (88 mg, 0.1208 mmol) and 2-
chloro-N,N0-dimethylacetamide (0.9 mL, 8.75 mmol) after heating
at 110 ꢀC in a sealed tube for four days. After cooling, 2 mL of Et2O/
CH2Cl2 (20:1) was added until a solid precipitated. The solid was
filtered and purified by recristallization from MeOH/Et2O to give
71.4 mg of compound 3o as a white solid. Yield: 61%. Mp: >250 ꢀC.
NMR (MeOH-d4)
d
: 9.13 (d, 4H, J ¼ 5.5 Hz, Py), 8.49 (d, 4H, J ¼ 5.5 Hz,
Py), 8.31 (d, 4H, J ¼ 7.3 Hz, Ph), 7.72 (d, 4H, J ¼ 7.9 Hz, Ph), 4.79 (t, 4H,
J ¼ 4.3 Hz, CH2Py), 4.05 (t, 4H, J ¼ 4.8 Hz, CH2OH); 13C NMR (MeOH-
d4) d: 168.4,163.4,148.1,146.9,140.0,138.2,132.4,131.6,128.8,128.7,
127.4, 126.8, 120.4, 110.6, 62.0.
1H NMR (MeOH-d4)
d
: 9.01 (d, 4H, J ¼ 5.5 Hz, Py), 8.54 (d, 4H,
J ¼ 6.7 Hz, Py), 8.32 (d, 4H, J ¼ 8.5 Hz, Ph), 7.72 (d, 4H, J ¼ 7.95 Hz,
3.1.4.10. 2,2-Bis[5-pentafluoroethyl-4-(4-hydroxiethyl-pyridinium-
4yl)oxazolyl]hexafluoropropane diiodide, 3k. Obtained from bisox-
azol 2e (69 mg, 0.088 mmol) and 2-chloroethanol (0.13 mL,
1.41 mmol) after heating in isopropanol at 110 ꢀC for five days. After
cooling the precipitated solid was filtered and washed with acetone
to obtain 60 mg of compound 3k as a beige solid. Yield: 58%. Mp:
Ph), 5.83 (s, 4H, CH2), 3.19 (s, 6H, CH3), 3.04 (s, 6H, CH3); 13C NMR
(MeOH-d4) d: 165.8, 163.4, 148.3, 146.8, 140.0, 139.3, 138.3, 138.1,
132.4, 128.7, 127.4, 127.2, 126.6, 120.4, 62.7, 36.5.
3.1.4.15. 1,4-Bis[(trifluoromethyl-4-(4-N,N0-dimethylacetamide-pyr-
idinium-4-yl)oxazolyl)]benzene dichloride, 3p. Obtained from
bisoxazol 2g (45.2 mg, 0.089 mmol) and 2-chloro-N,N0-dimethyla-
cetamide (1 mL, 9.7 mmol) after heating at 100 ꢀC in a sealed tube
for ten days. After cooling, acetone was added until a solid was
obtained. The solid was filtered and washed with Et2O to obtain
59.8 mg of compound 3p as a white solid. Yield: 89%. Mp: >250 ꢀC.
>250 ꢀC. 1H NMR (DMSO-d6)
d
: 9.17 (d, 4H, J ¼ 6.7 Hz, Py), 8.42 (d,
4H, J ¼ 6.7 Hz, Py), 8.28 (d, 4H, J ¼ 8.5 Hz, Ph), 7.70 (d, 4H, J ¼ 8.5 Hz,
Ph), 4.74 (t, 4H, CH2eN, J ¼ 4.89 Hz), 3.91 (t, 4H, CH2OH,
J ¼ 4.89 Hz); 13C NMR (DMSO-d6)
d: 162.3, 146.3, 146.1, 143.8, 139.9,
135.9, 135.2, 131.1, 127.8, 126.6, 125.7, 110.5, 63.4, 60.1; IR (cmꢂ1
)
(KBr): 3446, 2360, 1643, 1560, 1507, 1460, 1420, 1212; MS: 827
1H NMR (MeOH-d4)
J ¼ 5.7 Hz, Py), 8.45 (s, 4H, Ph), 5.82 (s, 4H, CH2), 3.18 (s, 6H, CH3),
3.04 (s, 6H, CH3); 13C NMR (MeOH-d4)
: 165.8, 163.3, 149.6, 147.1,
d: 9.01 (d, 4H, J ¼ 5.3 Hz, Py), 8.54 (d, 4H,
(C39H26F16I2N4O4, [Mþ ꢂ 2 ꢃ CF3]).
d
3.1.4.11. 2,2-Bis[5-pentafluoroethyl-4-(4-chloroethyl-pyridinium-4-
yl)oxazolyl]hexafluoropropane dimethylsulfonide, 3l. Obtained from
bisoxazol 2e (73 mg, 0.09 mmol) and 2-chloroethyl-methanesulfo-
nate (1 mL, 8.76 mmol) after heating at 100 ꢀC under argon atmo-
sphere for four hours. After cooling Et2O and acetone were added.
The precipitated solid was filtered and washed with Et2O to obtain
39 mg of compound 3l as a white solid. Yield: 45%. Mp: >250 ꢀC. 1H
139.8, 130.1, 128.6, 127.2, 120.4, 114.9, 62.8, 36.6, 36.3.
3.1.4.16. 1,4-Bis[(pentafluoropropyl-4-(4-N,N0-dimethylacetamide-
pyridinium-4-yl)oxazolyl)]benzene dichloride, 3q. Obtained from
bisoxazol 2h (52.9 mg, 0.087 mmol) and 2-chloro-N,N0-dimethy-
lacetamide (1 mL, 9.7 mmol) after heating at 100 ꢀC in a sealed tube
for twelve days. After cooling, acetone was added until a solid was
obtained. The solid was filtered and purified by recrystallization
from MeOH/Et2O to obtain 48.2 mg of compound 3q as a white
NMR (MeOH-d4)
J ¼ 7.32 Hz); 8.30 (d, 4H, Ph, J ¼ 8.52 Hz); 7.31 (d, 4H, Ph, J ¼ 8.55 Hz);
5.08 (t, 4H, CH2N, J ¼ 5.49 Hz); 4.24 (t, 4H, CH2Cl, J ¼ 5.49 Hz); 2.69 (s,
d
: 9.21 (d, 4H, Py, J ¼ 6.69 Hz); 8.54 (d, 4H, Py,
solid. Yield: 65%. Mp: >250 ꢀC. 1H NMR (MeOH-d4)
J ¼ 5.3 Hz, Py), 8.55 (d, 4H, J ¼ 5.7 Hz, Py), 8.45 (s, 4H, Ph), 5.82 (s,
d: 9.01 (d, 4H,
6H, CH3SO3); 13C NMR (MeOH-d4)
d: 165.3, 147.3, 145.0, 140.8, 137.8,

Y. Martín-Cantalejo et al. / European Journal of Medicinal Chemistry 46 (2011) 5662e5667
5667
4H, CH2), 3.19 (s, 6H, CH3), 3.05 (s, 6H, CH3); 13C NMR (MeOH-d4)
d:
Acknowledgments
165.8, 164.3, 148.4, 146.9, 141.1,138.3, 129.6, 129.4, 127.7,118.8,110.9,
62.8, 36.6, 36.3.
This work was supported by Translational Cancer Drug Pharma
(TCD Pharma). We thank Dr. Juan Carlos Lacal of Instituto de
Investigaciones Biomédicas (CSIC) for the biological assays. We also
thank Dr. Ma Jesús Villa for fruitful discussions, and Dr. Lourdes
García-Oroz and Ma Angeles Ramos for technical assistance.
3.2. In vitro inhibitory activity and cell proliferation inhibitory
activity
3.2.1. Choline kinase ex vivo activity assays
All the compounds were tested in an ex vivo system in order to
evaluate their inhibitory effect towards choline kinase as previously
reported [5]. The inhibitory effect against the choline kinase activity
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is achieved expressing the human ChoKa isoform in Escherichia coli
bacteria lacking such enzyme activity; therefore the activity that
could be observed is exclusively from the human recombinant
expressed isoform. Cell extracts were incubated for 30 min at 37 ꢀC
in a buffer containing 50
10 mM MgCl2,10 mM ATP, and 200
(55 mCi/nmol, 2 mCi/ml; Amersham Biosciences, UK)] for ChoK
assay. The reaction was stopped in ice by adding 10 l of 500 mM
EDTA. Thirty l of every sample were resolved by thin layer chro-
m
l of reaction buffer [100 mM Tris pH 8,
m
M [methyl-14C] choline chloride
a
m
m
matography plates (LK6D Silica gel 60 A, Whatman Inc. New Jersey)
using as liquid phase 0.9% NaCl:methanol; ammonium hydroxide
50:70:5; V:V:V. Radioactive metabolites (Choline and Phos-
phocholine) were automatically quantifed by an electronic auto
radiographysystem. Theinhibitoryconcentrations at which 50%was
reached (IC50) were calculated using the ratio PCho/(PCho þ Cho)
versus concentration of the inhibitors. IC50 values were obtained
from nonlinear least-squares fit of the Hill equation to the data.
3.2.2. Cell proliferation assays
Human HT-29 (colorectal adenocarcinoma) cells were grown in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with
10% Fetal Bovine Serum (FBS) under standard conditions of
temperature (37 ꢀC), humidity (95%) and CO2 (5%). Cells were
seeded on 24-well plates and incubated for 24 h in growth medium.
Then, cells were incubated in fresh medium containing increasing
concentrations (quadruplicates for each concentration) of the
compounds for 72 h. The colorimetric assay MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used
to asses cell viability. The reaction correlates with absorbance at
595 nm in a VersaMax Microplate Reader (Molecular Devices,
Sunnyvale, CA, USA). The IC50 (50% inhibitory concentration of
a substance) is quantified by plotting the logOD (optical density)
versus log drug concentration.
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