Synthesis and Biological Evaluation of Novel 2-Aryl Benzimidazoles as Chemotherapeutic Agents

Article abstract of DOI:10.1002/jhet.2575

Here, we describe the synthesis and preliminary biological evaluation of novel N-unsubstituted and N-methylated 2-aryl benzimidazole derivatives that contain fluorinated or hydroxylated alkyl substituents in the 4-N-aryl position and different substitution patterns (H vs Br vs I) in the benzimidazole ring. For the selected compounds and for comparison purposes, the congener benzothiazoles were also tested. The cytotoxic effect of 11 benzazole derivatives was evaluated in a panel of human cancer cell lines, such as breast (MCF7), melanoma (A375), cervix (HeLa), and glioblastoma (U87). In general, the compounds exerted a moderate cytotoxic activity against all cells tested. In particular, for the A375 and HeLa cells, the N-unsubstituted benzimidazoles 2 and 3 displayed a better cytotoxic profile than the respective N-methylated benzimidazole congeners (5 and 7). The biodistribution of compound 2, which has shown the highest cytotoxic activity active in the U87 glioblastoma cells (IC₅₀= 45.2 ± 13.0), was evaluated in CD1 mice using its¹⁸F-labeled counterpart ([¹⁸F]2). These studies showed that compound 2 can cross the blood brain barrier with a reasonable brain uptake (1.24 and 2.81%I.A./g at 5 and 60 min p.i., respectively), which is a crucial issue for systemic chemotherapy of glioblastoma. Altogether, the in vitro antitumoral activity of benzimidazole 2 against the U87 cells and the ability of its¹⁸F-congener to cross the blood brain barrier provide a strong rationale to consider the reported fluoroalkylated 2-aryl benzimidazoles as lead candidates for the generation of chemotherapeutic agents, in particular, against highly aggressive brain tumors such as glioblastoma.

Full text of DOI:10.1002/jhet.2575

Month 2015
Synthesis and Biological Evaluation of Novel 2-Aryl Benzimidazoles as
Chemotherapeutic Agents
Goreti Ribeiro Morais [1], Elisa Palma, Fernanda Marques,a Lurdes Gano,a Maria Cristina Oliveira,a
a
a,b
*
Antero Abrunhosa,c Hugo Vicente Miranda,d,e Tiago F. Outeiro,d,e,f,g Isabel Santos, and Antonio Paulo
a
a
*
aCentro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Bobadela, Portugal
bCentro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais 1, 1049-001
Lisboa, Portugal
cICNAS, Instituto de Ciências Nucleares Aplicadas à Saúde, Universidade Coimbra, Coimbra, Portugal
dInstituto de Medicina Molecular, Lisboa, Portugal
eCEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal
fInstituto de Fisiologia, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal
gDepartment of Neurodegeneration and Restorative Research, University Medical Center Göttingen, Göttingen, Germany
*
E-mail: apaulo@ctn.ist.utl.pt; gribeiro@bradford.ac.uk
Additional Supporting Information may be found in the online version of this article.
Received June 28, 2015
DOI 10.1002/jhet.2575
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
Here, we describe the synthesis and preliminary biological evaluation of novel N-unsubstituted and
N-methylated 2-aryl benzimidazole derivatives that contain fluorinated or hydroxylated alkyl substituents
in the 4-N-aryl position and different substitution patterns (H vs Br vs I) in the benzimidazole ring. For
the selected compounds and for comparison purposes, the congener benzothiazoles were also tested. The
cytotoxic effect of 11 benzazole derivatives was evaluated in a panel of human cancer cell lines, such as
breast (MCF7), melanoma (A375), cervix (HeLa), and glioblastoma (U87). In general, the compounds
exerted a moderate cytotoxic activity against all cells tested. In particular, for the A375 and HeLa cells,
the N-unsubstituted benzimidazoles 2 and 3 displayed a better cytotoxic profile than the respective
N-methylated benzimidazole congeners (5 and 7). The biodistribution of compound 2, which has shown
the highest cytotoxic activity active in the U87 glioblastoma cells (IC50 = 45.2 13.0), was evaluated in
CD1 mice using its 18F-labeled counterpart ([18F]-2). These studies showed that compound 2 can cross the
blood brain barrier with a reasonable brain uptake (1.24 and 2.81%I.A./g at 5 and 60 min p.i., respectively),
which is a crucial issue for systemic chemotherapy of glioblastoma. Altogether, the in vitro antitumoral
activity of benzimidazole 2 against the U87 cells and the ability of its 18F-congener to cross the blood brain
barrier provide a strong rationale to consider the reported fluoroalkylated 2-aryl benzimidazoles as lead
candidates for the generation of chemotherapeutic agents, in particular, against highly aggressive brain tumors
such as glioblastoma.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
Since then, benzimidazole derivatives were shown to present
a broad spectrum of biological activity, such as antimicrobial
[3,4], anticancer [5–8], antiviral [9], antihypertensive [10],
antioxidant [11], antidiabetic [12], and anti-inflammatory
[13] properties, to cite a few examples. There is a wide struc-
tural diversity for benzimidazole derivatives displaying anti-
tumoral activity. For example, Hoechst 33258 is a
fluorescent dye used to stain DNA and a known topoisomer-
ase inhibitor, which corresponds to a bis-benzimidazole with a
hydroxy aryl substituent at the 2-position of one of the
Benzimidazole derivatives started to be envisaged as
compounds with biological and medical relevance since
the discovery of 5,6-dimethyl-1-(α-D-ribofuranosyl)
benzimidazole as part of the structure of vitamin B12 in
1950 [2]. Later on, this heterocycle-fused ring system
was recognized as a relevant pharmacophore in drug
design, and extensive research has been reported in this
field profiting from the versatile modification of its scaffold.
© 2015 HeteroCorporation

G. R. Morais, E. Palma, F. Marques, L. Gano, M. C. Oliveira, A. Abrunhosa,
H. V. Miranda, T. F. Outeiro, I. Santos, and A. Paulo
Vol 000
heterorings [14]. More recently, the two 2-arylbenzimidazole
derivatives NU1085 (2-(4-hydroxyphenyl)benzimidazole-4-
carboxamide) and a 2-(4-oxadiazolyphenyl) analog were de-
scribed as chemosensitizers and radiosensitizers, respectively
[15,16]. Both compounds were shown to be potent poly
(ADP-ribose) polymerase (PARP-1) inhibitors with remark-
able chemotherapeutic effect.
of the heterocycle in the antitumor properties of the com-
pounds. The preclinical studies reported herein have also
comprised biodistribution in mice for the 18F-labeled analog
([18F]-2) of compound 2-[N-methyl-N-(2′-fluoroethyl)-4′-
aminophenyl]-1H-benzo[d]imidazole (2), which has emerged
as the most promising candidate for further evaluation as an
anticancer drug.
Following our work on antitumoral compounds [17–20],
we focused our attention on novel fluoroalkylated benz-
imidazoles derivatives (Fig. 1). By including alkyl substitu-
ents in the structure of these compounds, we have
considered the possibility of preparing the corresponding
radiofluorinated congeners to evaluate their in vivo
biological behavior. In fact, the generation of the radioac-
tive counterparts of putative drugs is crucial in all stages
of the drug development process, including the preclinical
phase where promising leads are selected or excluded
[21]. Besides classical biodistribution studies, the radioac-
tive probes may also allow the in vivo imaging of drug
biodistribution if the proper radionuclides are used. For
in vivo imaging, there are two different nuclear imaging mo-
dalities: single photon emitted computed tomography
(SPECT) and positron emission tomography (PET), which
are highly sensitive techniques that allow the quantification
and monitoring of drug distribution and its pharmacokinet-
ics. SPECT measurements are mainly based on the use of
gamma emitters such as radiometals (e.g., 99mTc), while
PET explores positron emitter radionuclides such as 11C
and 18F. Nowadays, 18F is still the most relevant PET radio-
nuclide because of its favorable nuclear decay properties
(T1/2 =109.8min, Eβ+max = 0.69MeV). Moreover, contrary
to the use of radiometals, no deep structural change occurs
when 18F is incorporated into the compounds to be tested,
and thus, no alterations of the pharmacokinetics are expected.
Herein, we describe the synthesis of a series of new
arylbenzimidazoles that contain hydroxyalkyl or fluoroalkyl
substituents in the 4-N-aryl position and different halogen
(Br or I) substituents in the benzimidazole ring (Fig. 1), as
well as the screening of their cytotoxic activity against differ-
ent human tumor cell lines. N-methylated and benzothiazole
congeners of some compounds were also synthesized and
biologically evaluated using the same panel of cell lines,
aiming to study the effect of the imidazolic proton and nature
RESULTS AND DISCUSSION
Chemistry and radiochemistry. We have considered a
number of structurally related 1-H and 1-methyl
2-arylbenzimidazole derivatives (Fig. 1) whose aniline nitro-
gen atom is bi-substituted with a methyl group and with
2-hydroxyethyl or 2-fluoroethyl substituents. This new family
of compounds includes unsubstituted (1, 2, 4, 5) and
halogenated 2-aryl benzimidazoles (3, 6–9), as we sought to
verify the effect of different substitution patterns on their
antitumoral activity. To assess the role of the heterocycle core
in the biological performance of the compounds, the
benzothiazole congeners (10 and 11) of 1 and 2 were also
prepared and evaluated.
The new benzazole derivatives were obtained based on a
multistep synthesis that started with the formation of the
heterocycle rings by the oxidative cyclocondensation of
1,2-diamine benzenes with adequate 4-substituted benzal-
dehydes (Scheme 1). For BOC-protected benzaldehyde
derivatives, the cyclocondensation reactions were carried
out in the presence of sodium metabisulfite in refluxing
DMF, leading to compounds 17–19 that were obtained in
high yield [11]. When an O-tosylated benzaldehyde was
employed, no oxidant was used and the cyclocondensation
was performed at lower temperatures (50°C) to limit
hydrolysis processes. In this way, the respective benzimid-
azoles (15 and 16) were obtained in fair to good yields
(Scheme 1). The N-methylated congeners of 17–19, com-
pounds 20–24, were prepared by their reaction with methyl
iodide under basic conditions (Scheme 1). In the case of
the 5-substituted compounds, the N-methylation led to the
formation of two pairs of regioisomers (21/22 and 23/24)
that were obtained in equal ratio. The structures of
regioisomers 21 and 22 were determined in solution by
Figure 1. General chemical structures of the novel benzazole derivatives.
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Scheme 1. Reaction reagents and conditions: (a) for 1: N-methyl-N-(2′-hydroxyethyl)-4-aminobenzaldehyde, Na2S2O5, DMF, 80°C; for 15 and 16: N-
methyl-N-(2′-O-tosyloxyethyl)-4-aminobenzaldehyde, DMF, 50°C; (b) TBAF, THF, 65°C; (c) N-methyl-N-(2′-O-tert-butylcarbonate)ethyl)-4-
aminobenzaldehyde, Na2S2O5, DMF, 120°C; (d) MeI, acetone, NaOH, water; (e) TFA, CH2Cl2; (f) p-TsCl, Et3N, CH2Cl2; and (g) TBAF, THF, reflux.
NMR NOESY experiments and in solid state by X-ray dif-
fraction studies, as described elsewhere [22]. Assignment
of the structures of the related iodinated regioisomers 21
the fluorination of the N-unsubstituted benzimidazoles,
15 and 16, was achieved using a lower amount of fluoride
ions (1.5 vs 5 eq) and lower reaction temperature in order
to minimize competitive nucleophilic alkylation reactions
involving the imidazole nitrogen. Under these conditions,
fluorinated benzimidazoles 2 and 3 were obtained in fair
to good yield (η = 59–71%) (Scheme 1).
1
and 22 was achieved by comparing their H NMR data
with those of the brominated counterparts 24 and 25. The
final fluorinated benzimidazoles (2, 3, and 5–9) were pre-
pared by nucleophilic substitution of the correspondent
O-tosylated precursors in good to excellent yields (η =
59–85%). If compared with the N-methylated counterparts,
Similarly, the benzothiazole scaffold of compound 10 was
generated by cyclocondensation of o-aminothiophenol with
Scheme 2. Reaction reagents and conditions: (a) N-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde, pyridine, reflux; (b) p-TsCl, Et3N, CH2Cl2, RT; (c)
TBAF, THF, reflux.
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G. R. Morais, E. Palma, F. Marques, L. Gano, M. C. Oliveira, A. Abrunhosa,
H. V. Miranda, T. F. Outeiro, I. Santos, and A. Paulo
Vol 000
the adequate benzaldehyde under basic conditions (Scheme 2).
Thereafter, its sequential tosylation and fluorination afforded
the desired fluoroalkylated 2-arylbenzothiazole derivative
(compound 11).
As previously mentioned, the presence of fluorine atoms
in the structures of this new family of benzazole derivatives
offered the possibility of synthesizing the corresponding
[18F]-labeled congeners. Such congeners may be applied
in biodistribution and metabolism studies to corroborate
the application of the synthesized compounds in the design
of new anticancer drugs. Thus, we selected compound 2
because it showed the most promising biological profile
among the tested compounds, as discussed later. As shown
in Scheme 3, the radiofluorinated counterpart of 2 (com-
pound [18F]2) was prepared by direct aliphatic nucleophilic
substitution of the tosylated group in 15 using activated
radiofluoride ion (18FÀ) in the form of [18F]KF/K2.2.2
complex.
Figure 2. High-performance liquid chromatography profile of compound
2
triethylamine (pH = 10.2) 40/60, 1 mL/min, 254 nm, tR = (UV) 6.88 min,
(γ) 7.17 min. The slight difference in retention time between the radioac-
tive peak and the UV peak is due to difference in the void volume of
the detector system.
(top) and [
18F]2 (bottom). HPLC conditions: CH3CN/0.1%
[18F]2 was synthesized and purified in 70 min total
synthesis time with a 30% radiochemical yield (decay
corrected) from [18F]fluoride, and with radiochemical
purity >99% at the end of synthesis. The chemical identi-
fication of [18F]2 was performed by HPLC comparison
with the non-radioactive congener 2 as shown in Figure 2.
To our knowledge, [18F]2 is a relatively rare example of
radiofluorinated benzimidazole derivatives that has been
obtained using a direct labeling strategy.
Biological studies: cytotoxicity assays and biodistribution. In
vitro preliminary screening of the cytotoxic activity of the 2-
arylbenzimidazole (1–9) and 2-arylbenzothiazole (10 and 11)
derivatives was assessed in a panel of representative human
tumor cells such as MCF7, A375, HeLa and U87 from
breast, melanoma, cervix carcinoma, and glioblastoma,
respectively. Cells were treated with 100 and 200 μM of the
tested compounds for 48h, and the cellular viability
evaluated by MTT, a metabolic assay in which the cellular
reduction of a yellow tetrazolium salt yields a purple
formazan in proportion to the number of viable cells [23]. At
100 μM concentration and for the large majority of
compounds, the A375 and HeLa cells were the less sensitive
and the U87 glioblastoma cells the less resistant ones
(Fig. 3). In general, the benzimidazoles 2, 3, 5, 6, and 7
exhibited a more favorable cytotoxicity profile against all
cancer cells, particularly against glioblastoma in a dose-
dependent manner.
lines, suggesting that there is a negative effect of the
N-methylation on the antiproliferative activity. The pres-
ence of different substituents at the benzimidazole ring also
influences the activity of the compounds. This can be
verified by comparing the antiproliferative activity of the
iodinated benzimidazole 9 with the brominated congener 7
(Fig. 3), which shows that the replacement of a bromo by
an iodo substituent tends to decrease the cytotoxicity of the
compounds. The nature of the different N-methylated
regioisomers (6/7 and 8/9) also affects the biological activity
of the compounds, particularly in the case of the iodinated
benzimidazoles. In fact, the 6-iodinated compound 8 has a
higher antiproliferative activity than the corresponding
5-iodinated regioisomer 9 in all tested cell lines. Finally,
we confirmed that there is an influence of the fluoroalkylated
group on the antiproliferative activity of the compounds, as
the fluorinated benzimidazoles 2 and 3 are more active than
their hydroxyl-alkylated counterpart, 4 and 5, respectively.
By comparing the antiproliferative activity of the fluorinated
derivatives 2 with 11, it was possible to confirm that the
presence of a benzimidazole ring, instead of a benzothiazole,
enhances the cytotoxicity of the compounds. This finding
suggests that the nature of the heterocycle ring has an impor-
tant influence on the biological activity of the reported
benzazoles.
Based on the preliminary screening of the in vitro antipro-
liferative activity of the different compounds, we have
selected 2, 3, 5, and 7 to determine the concentration that
Compounds 2 and 3 were more active than the
N-methylated counterparts 5 and 7 in almost all cancer cell
Scheme 3. Radiosynthesis of [18F]2.
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Figure 3. In vitro anticancer screening of 2-arylbenzimidazoles (1–9) and 2-arylbenzothiazoles (10 and 11) against a panel of human tumor cell lines
(MCF7, A375, HeLa, and U87). Data are presented as the percentage of cellular viability compared with controls (cells with no treatment) obtained after
48 h treatment with the compounds at 100 and 200 μM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
causes a 50% reduction of the cellular viability (IC50 values)
in the same cancer cell lines (MCF7, A375, HeLa, and U87).
The IC50 values found for the selected compounds are shown
in Table 1.
Using the MTT assay and the same experimental conditions
used for the benzimidazole derivatives (i.e., 48h treatment
at 37°C) the IC50 value found for DOX in the U87 cells
was 16.6 2.5 μM (from two experiments performed with
six replicates).
The MTT assay showed that the fluorinated aryl-
benzimidazoles 2, 3, 5, and 7 inhibited the cell growth of
MCF7, HeLa, and U87 cells after 48 h treatment. In
general, the compounds exerted less cytotoxic effect on
the A375 melanoma cells. In particular, compound 2 showed
the highest antiproliferative activity (IC50 = 45.2 13.0 μM)
against the U87 glioblastoma cells, which are representative
of an aggressive malignancy often characterized by resis-
tance to cytotoxic agents [24,25]. Doxorubicin (DOX) a
widely used antitumor compound was used as a positive
control for the U87 cells. Although important toxicity, to-
gether with poor distribution and limited penetration into
solid tumors, has limited its full therapeutic potential, sev-
eral studies have indicated its significant activity against a
variety of human cancers, including glioblastoma [26,27].
The promising cytotoxic profile of compound 2 against
the U87 glioblastoma cells (45.2 13.0 μM) prompted us
to proceed with the radiosynthesis of its radioactive
counterpart [18F]2 in order to assess its biodistribution, par-
ticularly the capability of crossing the BBB to reach the brain.
The biodistribution studies of [18F]2 were performed in
CD1 Charles River mice, at 5 and 60min post-injection
(p.i.) times to assess its ability to cross the BBB. The ob-
tained biodistribution data (Table 2) showed moderate ini-
tial uptake in most of the organs, including the brain. Most
importantly, the 18F-radioactivity uptake in the brain in-
creases over time (1.26 0.47%ID/g and 2.81 0.15%ID/
g at 5 and 60min p.i., respectively), suggesting that the
compound can reach the central nervous system.
Table 1
Cytotoxic activity of selected fluorinated aryl-benzimidazoles measured by the MTT assay. Data shown are the IC50 values obtained after 48 h treatment
with the compounds at serial concentrations. Results are mean ( SD) of two experiments carried out with six replicates.
IC50 (μM)
Compound
MCF7
A375
HeLa
U87
2
3
5
7
71.1 18.0
90.2 15.0
45.5 16.0
68.4 32.0
106 44.0
91.6 31.0
104 64.0
>200
62.7 15.0
17.8 3.00
75.1 19.0
122 37.0
45.2 13.0
62.8 18.0
67.8 25.0
71.9 30.0
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Table 2
benzimidazole that was obtained based on a direct
radiofluorination strategy. Biodistribution studies showed
that [18F]2 has a significant brain uptake, which
increases over time. Altogether, our results represent a
strong starting point for the generation of a novel class of
chemotherapeutic agents. In particular, the satisfactory
in vitro antitumoral activity of 2 against the U87 glioma
cell line, together with its ability to cross the BBB, sug-
gests this compound might be used in the design of drugs
for the glioblastoma treatment.
Biodistribution data of [18F]2 in CD1 Charles River mice (n = 4).
% I.A./g SD
Organs
5 min p.i.
2.54 0.25
60 min p.i.
Blood
Liver
Intestine
Spleen
Lung
Kidneys
Femur
Pancreas
Brain
2.00 0.54
2.83 0.32
2.37 0.09
2.86 0.12
2.39 0.29
2.89 0.13
2.84 0.16
1.90 0.15
2.81 0.15
1.40
2.39 0.80
2.22 1.30
2.52 1.45
2.15 1.1
3.64 0.56
1.50 0.64
1.92 1.18
1.26 0.47
0.49
EXPERIMENTAL
Brain/blood
Chemistry.
Diamine benzenes 12 and 13 were
commercially obtained from Sigma-Aldrich (Taufkirchen,
Germany). 4-Iodo-diaminebenzene (14) was prepared by
reducing 4-iodo-2-nitroaniline (SnCl2, conc HCl, EtOH).
N-Methyl-N-(2-O-tosyloxyethyl)-4-aminobenzaldehyde and
N-methyl-N-(2′-O-tert-butylcarbonate)ethyl)-4-aminobenzaldehyde
were prepared as described [28]. Synthesis and characterization
of compounds 18, 21, and 22 have been described elsewhere
[22]. Melting points were measured in a Stuart SMP3
apparatus. NMR spectra were recorded on a Varian Unity
300 NMR spectrometer at the frequencies of 300 MHz (1H),
75MHz (13C), and 282MHz (19F). Chemical shifts are
Moreover, [18F]2 displayed a rather slow clearance from
the blood compartment (2.54 0.25% and 2.00 0.54% at
5 and 60min p.i., respectively) and main organs. Owing
to the slow clearance from most organs, except the kid-
neys, the excretion pathway cannot be clearly defined at
these early time points. However, the activity in the liver
and intestines suggests a relevant contribution of the
hepatobiliar excretion with a small contribution of the uri-
nary route. Such biodistribution profile can probably be at-
tributed to the high lipophilic character of [18F]2
(LogP = 3.34). Nevertheless the slow blood clearance
points out to a rather large in vivo half-life of the non-
radioactive congener, compound 2, favoring its delivery
to organ and tissues.
1
reported in parts per million. H and 13C chemical shifts (δ)
are reported in ppm relative to residual solvent signals
1
(CDCl3: 7.26ppm for H NMR, 77.0ppm for 13C NMR).
19F chemical shifts were referenced externally to α,α′,α″-
trifluorotoluene (0.05% in C6D6; δ=À63.3). Electrospray
ionization mass spectrometry (ESI-MS) was performed on a
Bruker HCT quadrupole ion trap instrument in positive
ionization mode. High-resolution mass spectrometry (HRMS)
measurements were performed on an Extrel–Finnigan Fourier
transform ion cyclotron resonance instrument by electron
ionization. Elemental analyses of the tested compounds were
recorded on an CE Instruments EA 1110. Analytical
thin-layer chromatography was performed on precoated silica
plates 60 F254 (Merck). Visualization of the plates was carried
out using UV light (254 and 365 nm) and/or in an iodine
chamber. Column chromatography was carried out on silica
gel (Merck).
CONCLUSIONS
In summary, we have introduced a set of new heterocy-
cles, including nine structurally related benzimidazoles and
two benzothiazole derivatives. Their cytotoxicity was
screened against a panel of representative human cancer
cell lines. Considering the cellular viability results, four
compounds (2, 3, 5, and 7) were selected for further
determination of their IC50 values. The results showed that
compound 2 displayed the most promising in vitro properties
to be applied in the design of new anticancer drugs for treat-
ment of glioblastoma. In fact, compound 2, having a
non-substituted benzimidazole core and a 2-fluoroethyl chain
at the aniline nitrogen, presented a reasonable cytotoxic activity
against the U87 glioblastoma cell line (IC50 =45.2 13.0μM)
if compared with DOX (IC50 = 16.6 2.5 μM), which is a
widely used antitumor drug. This compound was selected
for further in vivo evaluation, using its radiofluorinated
congener [18F]2 as a surrogate. [18F]2 was synthesized in
fair radiochemical yield and high radiochemical purity.
[18F]2 represents a rare example of an N-unprotected
2-[N-methyl-N-(2-hydroxyethyl)-4′-aminophenyl]-1H-
benzo[d]imidazole (1).
A mixture of 12 (200mg,
1.85mM), N-methyl-N-(2′-hydroxyethyl)-4-aminobenzaldehyde
(330 mg, 1.85 mM), and Na2S2O5 (351mg, 1.85mM) in
DMF (8 mL) was refluxed for 2 h. Ice water (50 mL) was
added, and the precipitate formed was collected by
filtration, washed with water, and dried under vacuum to
give 1 in quantitative yield. Rf (CH2Cl2:MeOH 95:5)
= 0.14; 1H NMR (CD3OD, 300MHz) δ 3.06 (s, 3H,
3
NCH3), 3.57 (t, 2H, J 5.5 Hz, NCH2CH2OH), 3.77 (t,
2H, 3J 5.5 Hz, NCH2CH2OH), 6.87 (d, 2H, 3J 7.5 Hz,
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H3′and H5′), 7.18–7.21 (m, 2H, H5 and H6), 7.52–7.55
(m, 2H, H4 and H7), 7.93 (d, 2H, J 7.5 Hz, H2′ and
and concentrated. The resulting residue was subjected to
column chromatography on silica gel (n-hexane/EtOAc
3
H6′); 13C NMR (CD3OD, 75 MHz) δ 39.31 (NCH3),
55.32 (CH2), 60.21 (CH2), 112.84 (2C, Carom), 115.16
(2C,Carom), 117.86 (2C,Carom), 123.20 (2C, Carom),
129.00 (2C, Cquart), 152.28 (Cquart), 154.50 (Cquart); ES+
MS C16H17N3O (267.1) m/z 268.0 [M + H]+; HRMS (EI
+) found 267.13646, calcd for C16H17N3O 267.13661
1:1) to afford compounds 2 or 3.
2-[N-methyl-N-(2′-fluoroethyl)-4′-aminophenyl]-1H-benzo
[d]imidazole (2). Yield = 71%; Rf (n-hexane/EtOAc 1:1)
= 0.24; m.p. = 230–235°C; 1H NMR (CDCl3, 300 MHz) δ
3.06 (s, 3H, NCH3), 3.69 (dt, 2H, JH,H 4.8 Hz, JH,F
24.6 Hz, NCH2CH2F), 4.60 (dt, 2H, JH,H 4.8 Hz, JH,F
47.1 Hz, NCH2CH2F), 6.72 (d, 2H, J 8.1 Hz, H3′ and
3
3
3
2
3
[M]+.
H5′), 7.19–7.21 (m, 2H, H5 and H6), 5.57-7.60 (m, 2H,
General procedure for the preparation of benzimidazole
3
H4 and H7), 7.95 (d, 2H, J 8.1 Hz, H2′ and H6′); 13C
derivatives 15 and 16. A mixture of 1,2-diaminebenzene
(12 or 13) (1 mM) and N-methyl-N-(2-tosyloxyethyl)-4-
aminobenzaldehyde (1 mM) in DMF (3 mL) was stirred at
50°C for 2h. Then the solvent was concentrated under
vacuum, and the residue was submitted to column
chromatography on silica gel (CH2Cl2:CH3OH 99:1) to
NMR (CDCl3, 75 MHz) δ 39.04 (NCH3), 52.26 (d, JC,F
20.85 Hz, NCH2CH2F), 81.42 (d, JC,F 169.2 Hz,
NCH2CH2F), 110.90 (Cquart), 111.91 (2C, Carom),
115.10 (Carom), 115.67 (Carom), 117.21 (Carom), 128.10
(2C, Carom), 150.60 (Cquart), 153.05 (Cquart); 19F NMR
(CDCl3, 282 MHz) -222.642 (m); ES+ MS C16H16FN3
(269.1) m/z 270.0 [M + H]+; HRMS (EI+) found
269.13212, calcd for C16H16FN3 269.13228 [M]+;
Anal. calcd. for C16H16FN3.0.4H2O: C 69.50, H 6.12, N
afford compounds 15 or 16.
2-[N-methyl-N-(2′-O-tosyloxyethyl)-4′-aminophenyl]-1H-
benzo[d]imidazole (15). Yield: 63%; Rf (n-hexane/EtOAc
1
1:2) = 0.47; H NMR (CDCl3, 300MHz) δ 2.32 (s, 3H,
CH3), 2.87 (s, 3H, NCH3), 3.60 (t, 2H, 3J 5.7 Hz,
15.20; found C 69.09, H 7.84, N 15.53.
3
6-Bromo-2-[N-methyl-N-(2′-fluoroethyl)-4′-aminophenyl]-
NCH2CH2OTs), 4.14 (t, 2H, J 5.7 Hz, NCH2CH2OTs),
3
1H-benzo[d]imidazole (3).
Yield =59%; Rf (n-hexane/
6.53 (d, 2H, J 8.8 Hz, H3′ and H5′), 7.16–7.21 (m, 4H,
1
EtOAc 1:1) = 0.37; m.p. = 207–210°C; H NMR (CDCl3,
H5, H6 and H3″and H5″), 7.54–7.57 (m, 2H, H4 and
3
3
300 MHz) δ 3.02 (s, 3H, NCH3), 3.63 (dt, 2H, JH,H
H7), 7.64 (d, 2H, J 8.1 Hz, H2″ and H6″), 7.85 (d, 2H,
3
3
3J 8.8Hz, H2′ and H6′); 13C NMR (CDCl3, 75MHz) δ
21.58 (CH3), 38.94 (NCH3), 50.86 (CH2), 66.84 (CH2),
111.75 (2C, Carom), 114.68 (2C, Carom), 117.63 (Cquart),
122.37 (2C, Carom), 127.75 (2C, Carom), 127.85 (2C,
Carom), 129.83 (2C, Carom), 132.39 (2C, Cquart), 145.06
(Cquart), 149.46 (Cquart), 152.29 (Cquart); ES+ MS
4.8 Hz, JH,F 24.6Hz, NCH2CH2F), 4.56 (dt, 2H, JH,H
2
4.8 Hz, JH,F 47.1 Hz, NCH2CH2F), 6.65 (d, 2H, 3J
3
8.4 Hz, H3′ and H5′), 7.26 (d, 1H, J 8.4 Hz, H5), 7.38
(d, 3J 8.4 Hz, H4), 7.65 (s, 1H, H7), 7.90 (d, 2H, 3J
8.4 Hz, H2′ and H6′); 13C NMR (CDCl3, 75MHz) δ
39.04 (NCH3), 52.26 (d, JC,F 20.85 Hz, NCH2CH2F),
81.58 (d, JC,F 169.2Hz, NCH2CH2F), 111.78 (Carom),
111.91 (2C, Carom), 115.59 (Carom), 115.77 (Cquart), 117.31
(Cquart), 125.80 (Carom), 128.25 (2C, Carom), 150.65
(Cquart), 152.94 (Cquart); 19F NMR (CDCl3, 282MHz):
δ =À222.81 (m); ES+ MS C16H15BrFN3 (347.0 calcd
for 79Br) m/z 348.1 [M+H]+; Anal. calcd. for C16H15BrFN3: C
C23H23N3O3S (421.1) m/z 422.0 [M+ H]+.
6-Bromo-2-[N-methyl-N-(2′-O-tosyloxyethyl)-4′-aminophenyl]-
1H-benzo[d]imidazole (16).
EtOAc 1:2) =0.68; H NMR (CDCl3, 300MHz) δ 2.31
(s, 3H, CH3), 2.83 (s, 3H, NCH3), 3.56 (t, 2H, J 5.7 Hz,
Yield: 42%; Rf (n-hexane/
1
3
3
NCH2CH2OTs), 4.11 (t, 2H, J 5.7 Hz, NCH2CH2OTs),
3
6.46 (d, 2H, J 8.7 Hz, H3′ and H5′), 7.17–8. 21 (m, 4H,
55.19, H 4.34, N 12.07; found C 55.20, H 4.91, N 11.68.
3
General procedure for the preparation of benzimidazole
H5, H6 and H3″ and H5″), 7.36 (d, 1H, J 8.4Hz, H4),
3
derivatives 17 and 19.
Compounds 17 and 19 were
7.61 (s, 1H, H7), 7.63 (d, 2H, J 8.4 Hz, H2″ and H6″),
3
7.82 (d, 2H, J 8.7 Hz, H2′ and H6′); 13C NMR (CDCl3,
obtained as previously described [22]. Briefly a mixture
of 1,2-diaminebenzene (12 or 14) (1mM), N-methyl-N-
((2′-O-tert-butylcarbonate)ethyl)-4-aminobenzaldehyde (1mM),
and Na2S2O5 (1 mM) in DMF (3 mL) was refluxed. After
2 h iced water was added and the formed precipitate
filtered. Then, the filtrate was redissolved in MeOH, the
solvent was concentrated, and the residue was submitted
to column chromatography on silica gel (n-hexane/EtOAc
1:1) to afford compounds 17 or 19.
75MHz) δ 21.60 (CH3), 38.85 (NCH3), 50.82 (CH2),
66.88 (CH2), 111.67 (2C, Carom), 115.45 (Carom), 115.69
(Cquart), 117.20 (Cquart), 125.88 (Carom), 127.76 (2C, Carom),
128.25 (2C, Carom), 129.89 (2C, Carom), 132.38 (Cquart),
145.15 (Cquart), 150.07 (Cquart), 152.69 (Cquart); ES+ MS
C23H22BrN3O3S (499.0 calcd for 81Br) m/z 500.2 [M +H]+.
General procedure for the fluorination of N-unsubstituted
benzimidazole derivatives (2 and 3).
A solution of
tosylate precursor (15 or 16) (1mM) and anhydrous
TBAF (1.5 mM) in anhydrous THF (40 mL) was stirred at
65°C for 25 min. Thereafter, the solvent was
concentrated, and the crude product was taken up in
CH2Cl2 (50 mL). The organic phase was extracted with
sat sol NaHCO3 (50 mL), dried over Na2SO4, filtered,
2-[N-methyl-N-(2′-O-tert-butylcarbonatethyl)-4′-aminophenyl]-
1H-benzo[d]imidazole (17).
Yield: quantitative; Rf (n-
1
hexane/EtOAc 1:2) = 0.66; H NMR (CD3OD, 300MHz)
δ 1.43 (s, 9H, C(CH3)3), 3.10 (s, 3H, NCH3), 3.75 (t, 2H,
3J 5.5 Hz, NCH2CH2OBoc), 4.31 (t, 2H, 3J 5.5Hz,
3
NCH2CH2OBoc), 6.93 (d, 2H, J 9.0 Hz, H3′and H5′),
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

G. R. Morais, E. Palma, F. Marques, L. Gano, M. C. Oliveira, A. Abrunhosa,
H. V. Miranda, T. F. Outeiro, I. Santos, and A. Paulo
Vol 000
7.21–7.24 (m, 2H, H5 and H6), 7.57 (b, 2H, H4 and H7),
evaporated, the reaction mixture was diluted with water
(50 mL) and was extracted with CH2Cl2 (2 × 50mL). The
organic phase was dried over Na2SO4, filtered, and the
filtrate was concentrated under vacuum to give a mixture
of regioisomers 23 and 24 (378 mg, 93%), in an
3
7.97 (d, 2H, J 9.0 Hz, H2′and H6′); 13C NMR (CD3OD,
75MHz) δ 27.92 (C(CH3)3), 38.89 (NCH3), 51.75 (CH2),
65.09 (CH2), 82.93 (C(CH3)3), 112.99 (2C, Carom),
118.30 (2C, Carom), 123.24 (2C, Carom), 129.02 (2C,
Carom), 151.99 (Cquart), 154.40 (Cquart), 155.05 (C = O);
ES+ MS C21H25N3O3 (367.2) m/z 368.0 [M +H]+; HRMS
(EI+) found 367.18874, calcd for C21H25N3O3 367.18904
approximate 1:1
M ratio. The regioisomers were
separated by column chromatography on silica gel (n-
hexane/EtOAc 2:1).
[M]+.
6-Iodo-1-methyl-2-[N-methyl-N-(2′-O-tert-butylcarbonatethyl)-
5-Iodo-2-[N-methyl-N-(2′-O-tert-butylcarbonatethyl)-4′-
4′-aminophenyl]-1H-benzo[d]imidazole (23).
EtOAc 1:1)=0.68; H NMR (CDCl3, 300 MHz) δ 1.44 (s,
Rf (n-hexane/
1
aminophenyl]-1H-benzo[d]imidazole (19).
Yield = 77%;
Rf (n-hexane/EtOAc 1:1) = 0.52; 1H NMR (CDCl3,
300MHz) δ 1.44 (s, 9H, C(CH3)3), 3.00 (s, 3H, NCH3),
3
9H, C(CH3)3), 3.05 (s, 3H, NCH3), 3.67 (t, 2H, J 6.3Hz,
NCH2CH2OBoc), 3.81 (s, 3H, NCH3), 4.24 (t, 2H, 3J
3
3
3
3.62 (t, 2H, J 6.0 Hz, NCH2CH2OBoc), 4.20 (t, 2H, J
6.3Hz, NCH2CH2OBoc), 6.80 (d, 2H, J 9.0 Hz, H3′ and
3
3
6.0Hz, NCH2CH2OBoc), 6.67 (d, 2H, J 9.0 Hz, H3′ and
H5′), 7.52 (s, 2H, H4 and H5), 7.63 (d, 2H, J 9.0 Hz, H2′
H5′), 7.31 (d, 1H, 3J 8.4 Hz, H7), 7.45 (dd, 1H, 4J
and H6′), 7.66 (s, 1H, H7); 13C NMR (CDCl3, 75MHz) δ
27.71 (3C, C(CH3)3), 31.96 (NCH3), 38.70 (NCH3), 50.82
(CH2), 63.45 (CH2), 82.42 (C(CH3)3), 85.23 (Cquart),
104.73 (Carom), 111.62 (2C, Carom), 118.45 (Cquart), 120.90
(Carom), 130.63 (2C, Carom), 131.12 (Cquart), 142.27 (Cquart),
149.96 (Cquart), 153.37 (Cquart); ES+ MS C22H26IN3O3
3
3
1.5Hz, J 8.4 Hz, H6), 7.86 (d, 2H, J 9.0 Hz, H2′ and
H6′), 7.87 (d, 1H, 4J 1.5 Hz, H4); 13C NMR (CDCl3,
75MHz) δ 27.68 (3C, (C(CH3)3), 38.73 (NCH3), 50.71
(CH2), 63.45 (CH2), 82.56 (C(CH3)3), 85.89 (Cquart),
111.70 (2C, Carom), 114.88 (Carom), 123.08 (Carom),
128.34 (2C, Carom), 131.57 (Cquart), 150.70 (Cquart),
152.29 (Cquart), 153.30 (C =O); ES+ MS C21H24IN3O3
(507.1) m/z 508.2 [M+ H]+.
5-Iodo-1-methyl-2-[N-methyl-N-(2′-O-tert-butylcarbonatethyl)-
(493.1) m/z 494.2 [M + H]+.
1-Methyl-2-[N-methyl-N-(2′-O-tert-butylcarbonatethyl)-4′-
4′-aminophenyl]-1H-benzo[d]imidazole (24).
EtOAc 1:1)=0.72; H NMR (CDCl3, 300 MHz) δ 1.43 (s,
Rf (n-hexane/
1
3
aminophenyl]-1H-benzo[d]imidazole (20).
To a solution
9H, C(CH3)3), 3.04 (s, 3H, NCH3), 3.66 (t, 2H, J 6.3 Hz,
NCH2CH2OBoc), 3.80 (s, 3H, NCH3), 4.23 (t, 2H, 3J
of NaOH (230 mg, 5.6 mM) in water (0.5 mL) were added
17 (300 mg, 0.8 mM), DMF (1 mL), acetone (4mL), and
methyl iodide (60 μL, 0.97mM), dropwise and using a
cooling bath of ice water. The reaction mixture was
stirred for 2h at RT. Then, the solvents were evaporated,
the reaction mixture was diluted with water (100mL),
and was extracted with CH2Cl2 (100 mL). The organic
phase was dried over Na2SO4, filtered, and the filtrate
was dried under vacuum to afford 20 (256 mg, 83%). Rf
(CH2Cl2/MeOH 100:2) = 0.57; 1H NMR (CDCl3,
300MHz) δ 1.42 (s, 9H, C(CH3)3), 3.04 (s, 3H, NCH3),
3.66 (t, 2H, 3J 6.3 Hz, NCH2CH2OBoc), 3.84 (s, 3H,
3
6.3Hz, NCH2CH2OBoc), 6.79 (d, 2H, J 8.7Hz, H3′ and
3
4
H5′), 7.08 (d, 1H, J 8.5Hz, H7), 7.50 (dd, 1H, J 1.8 Hz
3
3
and J 8.5Hz, H6), 7.61 (d, 2H, J 8.7Hz, H2′ and H6′),
8.08 (d, 1H, 4J 1.8Hz, H4); 13C NMR (CDCl3, 75MHz) δ
27.67 (3C, C(CH3)3), 31.92 (NCH3), 38.68 (NCH3), 50.75
(CH2), 63.41 (CH2), 82.40 (C(CH3)3), 85.26 (Cquart),
111.08 (Carom), 111.56 (2C, Carom), 116.68 (Carom), 128.01
(Cquart), 130.61 (2C, Carom), 136.09 (Carom), 144.54 (Cquart),
149.90 (Cquart), 153.33 (Cquart), 154.98 (C=O); ES+ MS
C22H26IN3O3 (507.1) m/z 508.2 [M+H]+.
General procedure for the hydrolysis of t-Boc group:
3
synthesis of 4 and 25–28. A solution of 20–24 (1mM) in
CH2Cl2 (8 mL) and TFA (2mL) was stirred at RT for 1 h.
Then, the solvents were concentrated under vacuum. The
residue was taken up in CH2Cl2 (100mL), and NEt3 was
added until neutral pH. The organic phase was extracted
with sat sol NaHCO3 (100mL), was dried over Na2SO4,
was filtered, and the filtrate was concentrated. The crude
product was subjected to column chromatography on
silica gel (CH2Cl2/MeOH 94:6) to afford the title
NCH3), 4.23 (t, 2H, J 6.3 Hz, NCH2CH2OBoc), 6.79 (d,
3
2H, J 8.7Hz, H3′ and H5′), 7.23–7.28 (m, 2H, H5 and
3
H6), 7.32–7.35 (m, 1H, H7), 7.64 (d, 2H, J 8.7Hz, H2′
and H6′), 7.75–7.77 (m, 1H, H4); 13C NMR (CDCl3,
75MHz) δ 27.65 (3C, C(CH3)3), 31.74 (NCH3), 38.62
(NCH3), 50.78 (CH2), 63.45 (CH2), 82.31 (C(CH3)3),
109.25 (2C, Carom), 111.53 (2C, Carom), 117.60 (Cquart),
119.20 (Carom), 122.05 (Carom), 130.53 (2C, Carom),
136.60 (Cquart), 142.91 (Cquart), 149.66 (Cquart), 153.33
(Cquart), 154.39 (C =O); ES+ MS C22H27N3O3 (381.2)
compounds.
m/z 382.0 [M + H]+.
Synthesis of the N-methylated regioisomers 23 and 24
[22]. To a solution of NaOH (280 mg, 7 mM) in water
(0.5 mL) were added 19 (390 mg, 0.8 mM), acetone
(4 mL), and methyl iodide (60 μL, 0.95 mM), dropwise
and using a cooling ice water bath. The reaction mixture
was stirred for 2 h 30 min at RT. Then, the solvents were
1-Methyl-2-[N-methyl-N-(2′-hydroxyethyl)-4′-aminophenyl]-
1H-benzo[d]imidazole (4).
Yield = 86%; Rf (CH2Cl2/
MeOH 94:6)= 0.45;1H NMR (CDCl3, 300MHz) δ 3.03
3
(s, 3H, NCH3), 3.54 (t, 2H, J 6.0 Hz, NCH2CH2OH),
3.82 (t, 2H, 3J 6.0 Hz, NCH2CH2OH), 3.83 (s, 3H,
3
NCH3), 6.77 (d, 2H, J 8.7 Hz, H3′ and H5′), 7.26–7.31
(m, 2H, H5 and H6), 7.32–7.36 (m, 1H, H7), 7.59 (d, 2H,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Fluoroalkylated 2-Aryl Benzimidazoles as Lead Candidates for the Generation of
Chemotherapeutic Agents
3J 8.7 Hz, H2′ and H6′), 7.7–7.8 (m, 1H, H4); 13C NMR
(CDCl3, 75MHz) δ 31.77 (NCH3), 38.74 (NCH3), 54.75
(CH2), 59.41 (CH2), 109.33 (Carom), 111.67 (2C, Carom),
116.82 (Carom), 118.94 (Cquart), 122.14 (Carom), 122.20
(Carom), 130.44 (2C, Carom), 136.47 (Cquart), 142.60 (Cquart),
150.49 (Cquart), 154.51 (Cquart); ES+ MS C17H19N3O (281.2)
m/z 282.0 [M + H]+; Anal. calcd. for C17H19N3O C 72.57,
H 6.81, N 14.94; found C 72.02, H 7.66, N 14.99.
(CDCl3, 300MHz) δ 1.90 (bs, 1H), 3.05 (s, 3H, NCH3),
3
3.56 (t, 2H, J 5.7 Hz, NCH2CH2OH), 3.84–3.86 (m, 5H,
3
NCH3 and NCH2CH2OH), 6.83 (d, 2H, J 8.7 Hz, H3′
3
3
and H5′), 7.11 (d, 1H J 8.4 Hz, H7), 7.54 (d, 1H, J
3
8.4 Hz, H6), 7.65 (d, 2H, J 8.7 Hz, H2′ and H6′), 8.13
(s, 1H, H4); 13C NMR (CDCl3, 75MHz) δ 32.17
(NCH3), 38.89 (NCH3), 54.64 (CH2), 60.07 (CH2),
111.29 (Carom), 111.79 (Cquart), 112.00 (2C, Carom),
127.38 (Carom), 130.78 (2C, Carom), 131.27 (Carom); ES+
MS C17H18IN3O (407.0) m/z 408.0 [M + H]+.
6-Bromo-1-methyl-2-[N-methyl-N-(2′-hydroxyethyl)-4′-
aminophenyl]-1H-benzo[d]imidazole (25). Yield=74%;
Rf (n-hexane/EtOAc 1:2)=0.13; 1H NMR (CDCl3,
General procedure for the O-tosylation of N-
methylbenzimidazole derivatives: synthesis of 29–33.
A
3
300MHz) δ 3.04 (s, 3H, NCH3), 3.56 (t, 2H, J 5.7Hz,
solution of 4/25-28 (1 mM), NEt3 (0.5 mL), and p-TsCl
(3 mmol) in CH2Cl2 (12 mL) was stirred at RT for 4 h.
Thereafter, the reaction mixture was diluted with CH2Cl2
(100 mL), and the organic phase was extracted with a
saturated solution of NaHCO3 (100mL). The organic
extract was dried over Na2SO4, filtered, and the filtrate
was concentrated. The residue was subjected to column
chromatography on silica gel (CH2Cl2:MeOH 98:2) to
afford the title compounds.
NCH2CH2OH), 3.81 (s, 3H, NCH3), 3.84 (t, 2H, 3J
3
5.7Hz, NCH2CH2OH), 6.82 (d, 2H, J 9.0 Hz, H3′ and
4
3
H5′), 7.36 (dd, 1H, J 1.8 Hz, J 8.4 Hz, H5), 7.49 (d,
4
3
1H, J 1.8Hz, H7), 7.61 (d, 2H, J 9.0 Hz, H2′ and H6′),
7.62 (d, 1H, J 8.4 Hz, H4); 13C NMR (CDCl3, 75MHz)
3
δ 32.11 (NCH3), 38.84 (NCH3), 54.67 (CH2), 60.08
(CH2), 104.75 (Carom), 111.99 (2C, Carom), 119.99,
125.94 (Carom), 130.62 (2C, Carom), 150.98 (Cquart);
ES+ MS C17H18BrN3O (359.1 calcd for 79Br) m/z 360.2
[M + H]+.
1-Methyl-2-[N-methyl-N-(2′-tosyloxyethyl)-4′-aminophenyl]-
1H-benzo[d]imidazole (29).
MeOH 98:2) = 0.45; H NMR (CDCl3, 300 MHz) δ 2.40
Yield = 80%; Rf CH2Cl2:
1
5-Bromo-1-methyl-2-[N-methyl-N-(2′-hydroxyethyl)-4′-
aminophenyl]-1H-benzo[d]imidazole (26). Yield = 93%;
Rf (n-hexane/EtOAc 1:2) = 0.18; 1H NMR (CDCl3,
3
(s, 3H, CH3), 2.98 (s, 3H, NCH3), 3.70 (t, 2H, J 6.0Hz,
NCH2CH2OTs), 3.85 (s, 3H, NCH3), 4.21 (t, 2H, 3J
3
3
300MHz) δ 3.04 (s, 3H, NCH3), 3.55 (t, 2H, J 5.7 Hz,
6.0 Hz, NCH2CH2OTs), 6.69 (d, 2H, J 8.7 Hz, H3′ and
NCH2CH2OH), 3.82 (s, 3H, NCH3), 3.83 (t, 2H, 3J
H5′), 7.26–7.30 (m, 4H, H5, H6, H3″ and H5″), 7.35–
3
3
5.7Hz, NCH2CH2OH), 6.82 (d, 2H, J 8.7 Hz, H3′ and
7.38 (m, 1H, H7), 7.63 (d, 2H, J 8.7 Hz, H2′ and H6′),
3
4
3
H5′), 7.19 (d, 1H; J 8.7 Hz, H7), 7.35 (dd, 1H, J 1.8 Hz
7.73 (d, 2H, J 8.4 Hz, H2″ and H6″`), 7.78–7.81 (m, 1H,
3
3
H4); 13C NMR (CDCl3, 75 MHz) δ 21.65 (CH3), 31.84
(NCH3), 39.00 (NCH3), 51.03 (CH2), 66.73 (CH2),
109.37 (2C, Carom), 111.59 (2C, Carom), 119.20 (Cquart),
122.31 (2C, Carom), 127.82 (2C, Carom), 129.87 (2C,
Carom), 130.61 (2C, Carom), 132.59 (Cquart), 136.50
(Cquart), 145.04 (Cquart), 149.14 (Cquart), 154.12 (Cquart);
ES+ MS C24H25N3O3S (435.2) m/z 436.0 [M + H]+.
6-Bromo-1-methyl-2-[N-methyl-N-(2′-tosyloxyethyl)-4′-
and J 8.7Hz, H6), 7.61 (d, 2H, J 8.7 Hz, H2′ and H6′),
7.9 (d, 1H, J 1.8 Hz, H4); 13C NMR (CDCl3, 75 MHz) δ
4
32.07 (NCH3), 38.87 (NCH3), 54.69 (CH2), 60.03 (CH2),
110.62 (Carom), 111.95 (2C, Carom), 115.48 (Carom),
121.68 (Carom), 125.36 (Cquart), 130.64 (2C, Carom),
135.35 (Cquart), 150.92 (Cquart), 155.18 (Cquart); ES+ MS
C17H18BrN3O (359.1 calcd for 79Br) m/z 360.2 [M +H]+;
HRMS (EI+) found 359.06264, calcd for C17H18BrN3O
359.06277 [M] + .
aminophenyl]-1H-benzo[d]imidazole (30).
(n-hexane/EtOAc 1:1)=0.26; H NMR (CDCl3, 300MHz)
δ 2.32 (s, 3H, CH3), 2.89 (s, 3H, NCH3), 3.61 (t, 2H, J
Yield=54%; Rf
1
6-Iodo-1-methyl-2-[N-methyl-N-(2′-hydroxyethyl)-4′-
3
aminophenyl]-1H-benzo[d]imidazole
(27).
Yield =
quantitative; Rf (n-hexane/EtOAc 1:2)= 0.14; 1H NMR
(CDCl3, 300 MHz) δ 1.96 (bs, 1H), 3.05 (s, 3H, NCH3),
3.56 (t, 2H, 3J 5.7 Hz, NCH2CH2OH), 3.81 (s, 3H,
5.7Hz, NCH2CH2OTs), 3.74 (s, 3H, NCH3), 4.12 (t, 2H,
3
3J 5.7Hz, NCH2CH2OTs), 6.60 (d, 2H, J 9.0Hz, H3′ and
3
H5′), 7.21 (d, 2H, J 8.7Hz, H3″ and H5″), 7.29 (dd, 1H,
3
4J 1.8Hz, 3J 8.7Hz, H5), 7.43 (d, 1H, 4J 1.8Hz, H7), 7.53
NCH3), 3.84 (t, 2H, J 5.7 Hz, NCH2CH2OH), 6.83 (d,
3
3
3
2H, J 9.0 Hz, H3′ and H5′), 7.54 (s, 2H, H4 and H5),
(d, 2H, J 9.0Hz, H2′ and H6′), 7.54 (d, 1H, J 8.7Hz,
3
H4), 7.64 (d, 2H, J 8.7Hz, H2″ and H6″); 13C NMR
3
7.63 (d, 2H, J 9.0Hz, H2′ and H6′), 7.68 (s, 1H, H7);
13C NMR (CDCl3, 75 MHz) δ 32.08 (NCH3), 38.87
(NCH3), 54.67 (CH2), 60.07 (CH2), 111.97 (2C, Carom),
118.58 (Carom), 120.51 (Cquart), 130.67 (2C, Carom),
131.49 (Carom), 135.54 (Cquart), 147.17 (Cquart), 150.95
(Cquart); ES+ MS C17H18IN3O (407.0) m/z 407.9 [M+ H]+.
5-Iodo-1-methyl-2-[N-methyl-N-(2′-hydroxyethyl)-4′-
(CDCl3, 75MHz) δ 21.60 (CH3), 31.93 (NCH3), 38.92
(NCH3), 50.97 (CH2), 66.69 (CH2), 111.58 (2C, Carom),
112.50 (Carom), 115.22 (Cquart), 117.25 (Carom), 120.38
(Cquart), 125.43 (Carom), 127.77 (2C, Carom), 129.84 (2C,
Carom), 130.50 (2C, Carom), 132.59 (Cquart), 137.70 (Cquart),
141.79 (Cquart), 145.01 (Cquart), 149.28 (Cquart), 154.94
(Cquart); ES+ MS C24H24BrN3O3S (513.1 calcd for 79Br)
m/z 514.2 [M+H]+.
aminophenyl]-1H-benzo[d]imidazole
= quantitative; Rf (n-hexane/EtOAc 1:2) = 0.16; H NMR
(28).
Yield
1
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

G. R. Morais, E. Palma, F. Marques, L. Gano, M. C. Oliveira, A. Abrunhosa,
H. V. Miranda, T. F. Outeiro, I. Santos, and A. Paulo
Vol 000
5-Bromo-1-methyl-2-[N-methyl-N-(2′-tosyloxyethyl)-4′-
aminophenyl]-1H-benzo[d]imidazole (31).
sat sol NaHCO3 (50 mL), dried over Na2SO4, filtered,
and the filtrate was concentrated. The resulting residue
was subjected to column chromatography on silica gel
(n-hexane/EtOAc 1:1).
Yield= 75%; Rf
1
(n-hexane/EtOAc 1:1)=0.36; H NMR (CDCl3, 300MHz)
3
δ 2.39 (s, 3H, CH3), 2.97 (s, 3H, NCH3), 3.69 (t, 2H, J
5.7Hz, NCH2CH2OTs), 3.84 (s, 3H, NCH3), 4.19 (t, 2H,
1-Methyl-2-[N-methyl-N-(2′-fluoroethyl)-4′-aminophenyl]-
3J 5.7Hz, NCH2CH2OTs), 6.68 (d, 2H, 3J 8.7Hz, H3′ and
1H-benzo[d]imidazole (5).
EtOAc 1:1)=0.25; m.p. = 118–120°C; H NMR (CDCl3,
Yield = 72%; Rf (n-hexane/
3
3
1
H5′), 7.21 (d, 1H, J 8.4Hz, H7), 7.27 (d, 2H, J 8.4Hz,
4
3
3
H3″ and H5″), 7.37 (dd, 1H, J 1.5Hz and J 8.4Hz, H6),
300MHz) δ 3.02 (s, 3H, NCH3), 3.65 (dt, 2H, JH,H
7.62 (d, 2H, 3J 8.7Hz, H2′ and H6′), 7.70 (d, 2H, 3J
3
3
5.1Hz, JH,F 24.3 Hz, NCH2CH2F), 4.56 (dt, 2H, JH,H
4
8.4Hz, H2″ and H6″), 7.92 (d, 1H, J 1.5Hz, H4); 13C
2
5.1Hz, JH,F 47.1Hz, NCH2CH2F), 6.73 (d, 2H, 3J
NMR (CDCl3, 75MHz) δ 21.65 (CH3), 32.08 (NCH3),
38.99 (NCH3), 51.02 (CH2), 66.68 (CH2), 110.65 (Cquart),
111.67 (2C, Carom), 115.56 (Carom), 121.76 (Cquart), 125.47
(Carom), 127.82 (2C, Carom), 129.90 (2C, Carom), 130.71
(2C, Carom), 132.61 (Cquart), 145.08 (Cquart); ES+ MS
C24H24BrN3O3S (513.1 calcd for 81Br) m/z 514.2 [M+H]+.
6-Iodo-1-methyl-2-[N-methyl-N-(2′-tosyloxyethyl)-4′-
8.7Hz, H3′ and H5′), 7.18–7.22 (m, 2H, H5 and H6),
7.24–7.29 (m, 1H, H7), 7.59 (d, 2H, J 8.7Hz, H2′ and
3
H6′), 7.70–7.73 (m, 1H, H4) Falta um NCH3; 13C NMR
(CDCl3, 75MHz) δ 31.76 (NCH3), 39.03 (NCH3), 52.37
(d, JC,F 20.8Hz, NCH2CH2F), 81.65 (d, JC,F 169.2Hz,
NCH2CH2F), 109.28 (Carom), 111.70 (2C, Carom), 117.84
(Carom), 119.26 (Cquart), 122.08 (2C, Carom), 130.57 (2C,
C
arom), 136.63 (Cquart), 142.98 (Cquart), 149.66 (Cquart),
aminophenyl]-1H-benzo[d]imidazole (32).
Yield=60%; Rf
154.37 (Cquart); 19F NMR (CDCl3, 282MHz)
δ =À222.681 (m); ES+ MS C17H18FN3 (283.1) m/z 284.0
[M+H]+; HRMS (EI+) found 283.14824, calcd for
C17H18N3F 283.14793 [M]+; Anal. calcd. for
C17H18FN.30.3H2O: C 70.71, H 6.49, N 14.55; found C
71.08, H 6.87, N 14.28.
(n-hexane/EtOAc 1:1) =0.33; 1H NMR (CDCl3,
300MHz) δ 2.38 (s, 3H, CH3), 2.96 (s, 3H, NCH3), 3.69
(t, 2H, 3J 5.8Hz, NCH2CH2OTs), 3.81 (s, 3H, NCH3),
3
3
4.18 (t, 2H, J 5.8Hz, NCH2CH2OTs), 6.67 (d, 2H, J
3
9.0Hz, H3′ and H5′), 7.27 (d, 2H, J 8.1 Hz, H3″ and
H5″), 7.54 (s, 2H, H4 and H5), 7.61 (d, 2H, J 9.0Hz,
H2′ and H6′), 7.69 (s, 1H, H7), 7.70 (d, 2H, J 8.1Hz,
3
3
6-Bromo-1-methyl-2-[N-methyl-N-(2′-fluoroethyl)-4′-
H2″ and H6″); 13C NMR (CDCl3, 75MHz) δ 21.90
(CH3), 32.29 (NCH3), 39.26 (NCH3), 51.27 (CH2), 66.95
(CH2), 85.80 (Cquart), 111.91 (2C, Carom), 118.83 (Carom),
120.97 (Cquart), 128.08 (2C, Carom), 130.15 (2C, Carom),
130.94 (2C, Carom), 131.67 (Carom), 132.87 (Carom), 149.74
(Cquart); ES+ MS C24H24IN3O3 (561.1) m/z 562.2 [M+H]+.
5-Iodo-1-methyl-2-[N-methyl-N-(2′-tosyloxyethyl)-4′-
aminophenyl]-1H-benzo[d]imidazole (6).
Yield=87%; Rf
(n-hexane/EtOAc 1:1)=0.44; m.p. = 146–150°C; 1H
NMR (CDCl3, 300MHz) δ 3.07 (s, 3H, NCH3), 3.71 (dt,
3
3
2H, JH,H 5.1Hz and JH,F 24.6Hz, NCH2CH2F), 3.80 (s,
3
2
3H, NCH3), 4.62 (dt, 2H, JH,H 5.1Hz and JH,F 47.4Hz,
NCH2CH2F), 6.77 (d, 2H, J 8.7Hz, H3′ and H5′), 7.34
(dd, 1H, J 1.2Hz and J 8.4Hz, H5), 7.47 (d, 1H, J
3
4
3
4
3
3
1.2Hz, H7), 7.60 (d, 1H, J 8.4Hz, H4), 7.63 (d, 2H, J
aminophenyl]-1H-benzo[d]imidazole (33).
(n-hexane/EtOAc 1:1)=0.37; H NMR (CDCl3, 300MHz)
δ 2.38 (s, 3H, CH3), 2.96 (s, 3H, NCH3), 3.68 (t, 2H, J
Yield =41%; Rf
8.7Hz, H2′ and H6′); 13C NMR (CDCl3, 75MHz) δ 31.96
1
2
3
(NCH3), 39.05 (NCH3), 52.34 (d, JC,F 20.85Hz,
g
NCH2CH2F), 81.63 (d, JC,F 169.2Hz, NCH2CH2F),
5.7Hz, NCH2CH2OTs), 3.81 (s, 3H, NCH3), 4.18 (t, 2H,
3J 5.7Hz, NCH2CH2OTs), 6.67 (d, 2H, 3J 8.7Hz, H3′ and
111.70 (2C, Carom), 112.49 (Cquart), 115.21 (Carom), 116.93
(Carom), 120.36 (Cquart), 125.44 (Carom), 130.57 (2C,
Carom), 137.69 (Cquart), 141.71 (Cquart), 149.89 (Cquart),
155.05 (Cquart); 19F (CDCl3, 282MHz) δ=À223.18 (m);
ES+ MS C17H17BrFN3 (361.1 calcd for 79Br) m/z 362.1
[M+H]+; Anal. calcd. for C17H17BrFN3: C 56.36, H 4.73,
N 11.60; found C 56.45, H 5.33, N 11.26.
3
3
H5′), 7.10 (d, 1H, J 8.4Hz, H7), 7.26 (d, 2H, J 8.4Hz,
4
3
H3″ and H5″), 7.52 (dd, 1H, J 1.5Hz and J 8.4Hz, H6),
7.59 (d, 2H, 3J 8.7Hz, H2′ and H6′), 7.70 (d, 2H, 3J
8.4Hz, H2″ and H6″), 8.09 (d, 1H, J 1.5Hz, H4); 13C
4
NMR (CDCl3, 75MHz) δ 21.64 (CH3), 31.96 (NCH3),
38.98 (NCH3), 51.00 (CH2), 66.69 (CH2), 85.41 (Cquart),
111.15 (Carom), 111.62 (2C, Carom), 127.80 (2C, Carom),
128.04 (Carom), 129.87 (2C, Carom), 130.64 (2C, Carom),
130.78 (Carom), 132.58 (Cquart), 136.06 (Cquart), 145.05
(Cquart), 149.37 (Cquart), 154.75 (Cquart); ES+ MS
C24H24IN3O3S(561.1) m/z 562.2 [M+H]+.
General procedure for the fluorination of N-methyl-
benzimidazole derivatives: synthesis of 5–9. A solution of
29–33 (1mM) and anhydrous TBAF (5mM) in anhydrous
THF (40mL) was refluxed for 10min. Thereafter, the
solvent was concentrated, and the crude was taken up in
CH2Cl2 (50 mL). The organic phase was extracted with
5-Bromo-1-methyl-2-[N-methyl-N-(2′-fluoroethyl)-4′-
aminophenyl]-1H-benzo[d]imidazole (7).
Yield = 75%; Rf
(n-hexane/EtOAc 1:1) = 0.55; m.p. = 154–156°C; 1H
NMR (CDCl3, 300 MHz) δ 3.08 (s, 3H, NCH3), 3.72 (dt,
3
3
2H, JH,H 5.1 Hz and JH,F 24.6Hz, NCH2CH2F), 3.84 (s,
3
2
3H, NCH3), 4.62 (dt, 2H, JH,H 5.1 Hz and JH,F 47.1Hz,
NCH2CH2F), 6.79 (d, 2H, J 8.7 Hz, H3′ and H5′), 7.20
(d, 1H, J 8.7 Hz, H7), 7.36 (dd, 1H, J 1.8 Hz and J
3
3
4
3
3
8.7 Hz, H6), 7.65 (d, 2H, J 8.7 H2′ and H6′), 7.90 (d,
4
1H, J 1.8 Hz, H4); 13C NMR (CDCl3, 75MHz) δ 32.00
2
(NCH3), 39.06 (NCH3), 52.36 (d, JC,F 20.85 Hz,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Fluoroalkylated 2-Aryl Benzimidazoles as Lead Candidates for the Generation of
Chemotherapeutic Agents
1
1
NCH2CH2F), 81.63 Hz (d, JC,F 169.2Hz, NCH2CH2F),
110.54 (Cquart), 111.74 (2C, Carom), 115.22 (Carom),
121.90 (Carom), 125.15 (Cquart), 130.65 (2C, Carom),
135.52 (Cquart), 149.98 (Cquart), 155.27 (Cquart); 19F NMR
(CDCl3, 282 MHz): δ= À223.00 (m); ES+ MS
C17H17BrFN3 (361.1 calcd for 79Br) m/z 362.2 [M +H]+;
Anal. calcd. for C17H17BrFN3: C 56.37, H 4.73, N 11.60;
found C 56.28, H 5.31, N 11.36.
= 0.24; m.p. = 185–187°C; H NMR (CDCl3, 300 MHz)
3
δ 1.78 (bs, 1H, OH), 3.07 (s, 3H, NCH3), 3.58 (t, 2H, J
5.7 Hz, NCH2CH2OH), 3.86 (t, 2H, 3J 5.7 Hz,
NCH2CH2OH), 6.79 (d, 2H, 3J 8.7 Hz, H3′ and H5′),
3
3
3
7.29 (dd, 1H, J 8.1 Hz, J 8.1 Hz, H5), 7.42 (dd, 1H, J
8.1 Hz, J 8.1 Hz, H6), 7.82 (d, 1H, J 8.1 Hz, H4), 7.94
3
3
3
3
(d, 2H, J 8.7 Hz, H2′ and H6′), 7.98 (d, 1H, J 8.1 Hz,
H7); 13C (CDCl3, 75MHz) δ 39.05 (NCH3), 54.67 (CH2),
60.17 (CH2), 104.72 (Cquart), 111.98 (2C, Carom), 121.37
(Carom), 122.15 (Carom), 124.38 (Carom), 126.14 (Carom),
129.07 (2C, Carom); ES+ MS C16H16N2OS (284.1) m/z
284.9 [M]+; Anal. calcd. for C16H16N2OS: C 67.58, H 5.67,
N 9.85, S 11.28; found C 67.17, H 6.26, N 9.74, S 11.41.
2-[N-methyl-N-(2′-O-tosyloxyethyl)-4′-aminophenyl]-
6-Iodo-1-methyl-2-[N-methyl-N-(2′-fluoroethyl)-4′-
aminophenyl]-1H-benzo[d]imidazole (8).
Yield=65%; Rf
1
(n-hexane/EtOAc 1:1)=0.47; m.p.=137–140°C; H NMR
(CDCl3, 300MHz) δ 3.08 (s, 3H, NCH3), 3.71 (dt, 2H,
3JH,H 5.1Hz and JH,F 24.3Hz, NCH2CH2F), 3.80 (s, 3H,
3
3
2
NCH3), 4.62 (dt, 2H, JH,H 5.1Hz and JH,F 47.1Hz,
3
NCH2CH2F), 6.78 (d, 2H, J 9.0Hz, H3′ and H5′), 7.52
benzothiazole (35).
As described for the synthesis and
purification of 20 – Yield=96%; Rf (n-hexane/EtOAc 1:1)
3
(s, 2H, H4 and H5), 7.64 (d, 2H, J 9.0Hz, H2′ and H6′),
7.67 (s, 1H, H7); 13C NMR (CDCl3, 75MHz) δ 31.95
=0.42; H NMR (CDCl3, 300MHz) δ 2.41 (s, 3H, CH3),
1
2
(NCH3), 39.07 (NCH3), 52.36 (d, JC,F 21.4Hz,
NCH2CH2F), 81.63 (d, JC,F 169.6Hz, NCH2CH2F),
3.02 (s, 3H, NCH3), 3.74 (t, 2H, 3J 5.7Hz,
1
3
NCH2CH2OTs), 4.25 (t, 2H, J 5.7Hz, NCH2CH2OTs),
3
85.27 (Cquart), 111.73 (2C, Carom), 118.46 (Carom), 120.90
(Cquart), 130.63 (2C, Carom), 131.13 (Carom), 138.21
(Carom), 142.32 (Cquart), 142.79 (Cquart), 149.93 (Cquart);
19F NMR (CDCl3, 282MHz): δ =À222.64; ES+ MS
C17H17FIN3 (409.0) m/z 409.9 [M+ H]+; HRMS (EI+)
found 409.04447, calcd for C17H17FIN3 409.04457 [M]
+Anal. calcd. for C17H17FIN.30,15H2O: C 49.35, H 4.26,
N 10.16; found C 49.31, H 4.75, N 9.87.
6.63 (d, 2H, 3J 8.5Hz, H3′ and H5′), 7.28 (d, 2H, J
3
3
7.8Hz, H3″ and H5″), 7.37 (dd, 1H, J 7.5Hz, J 7.5Hz,
3
3
H5), 7.50 (dd, 1H, J 7.5Hz, J 8.1Hz, H6), 7.73 (d, 2H,
3J 7.8Hz, H2″), 7.84 (d, 2H, 3J 8.5Hz, H2′ and H6′), 7.90
(d, 1H, J 7.5Hz, H4), 8.04 (d, 1H, J 8.1Hz, H7); 13C
NMR (CDCl3, 75MHz) δ 21.62 (CH3), 39.20 (NCH3),
50.90 (CH2), 66.60 (CH2), 111.55 (2C, Carom), 121.39
(Carom), 122.22 (Carom), 124.45 (Carom), 126.17 (Carom),
127.77 (2C, Carom), 128.98 (2C, Carom), 129.82 (2C,
3
3
5-Iodo-1-methyl-2-[N-methyl-N-(2′-fluoroethyl)-4′-
arom); ES+ MS C23H22N2O3S2 (438.1) m/z 439.0 [M+ H]+.
2-[N-methyl-N-(2′-fluoroethyl)-4′-aminophenyl]-benzothiazole
(11). As described for the synthesis and purification of
5 – Yield=87%; Rf (n-hexane/EtOAc 4:1)=0.37; m.p. =
138–140°C; H NMR (CDCl3, 300MHz) δ 3.09 (s, 3H,
NCH3), 3.72 (dt, 2H, JH,H 5.1Hz and JH,F 24.3Hz,
NCH2CH2F), 4.62 (dt, 2H, JH,H 5.1Hz and JH,F 47.1Hz,
NCH2CH2F), 6.74 (d, 2H, J 8.7Hz, H3′ and H5′), 7.29
(dd, 1H, J 7.2Hz and J 7.2Hz, H5), 7.42 (dd, 1H, J
aminophenyl]-1H-benzo[d]imidazole (9).
Yield=49%; Rf
C
(n-hexane/EtOAc 1:1)=0–57; m.p. = 147–149°C; 1H
NMR (CDCl3, 300MHz) δ 3.08 (s, 3H, NCH3), 3.71 (dt,
3
3
2H, JH,H 5.2Hz, JH,F 24.6Hz, NCH2CH2F), 3.81 (s, 3H,
3
2
1
NCH3), 4.62 (dt, 2H, JH,H 5.2Hz, JH,F 47.1Hz,
3
3
3
NCH2CH2F), 6.78 (d, 2H, J 8.7Hz, H3′ and H5′), 7.08
3
4
3
3
2
(d, 1H, J 8.4Hz, H7), 7.51 (dd, 1H, J 1.5Hz, J 8.4Hz,
3
4
3
H6), 7.63 (d, 2H, J 8.7Hz, H2′ and H6′), 8.09 (d, 1H, J
1.5Hz, H4); 13C NMR (CDCl3, 75MHz) δ 31.92 (NCH3),
39.06 (NCH3), 52.35 (d, JC,F 20.8Hz, NCH2CH2F), 81.62
3
3
3
3
3
7.2Hz and J 7.8Hz, H6), 7.82 (d, 1H, J 7.2Hz, H4),
2
3
(d, JC,F 169.2Hz, NCH2CH2F), 85.29 (Cquart), 111.09
7.95 (d, 2H, 3J 8.7Hz, H2′ and H6′), 7.97 (d, 1H, J
(Carom), 111.73 (2C, Carom), 128.08 (Carom), 130.67 (2C,
Carom), 136.13 (Cquart), 149.93 (Cquart); 19F NMR (CDCl3,
282MHz): δ=À222.73 (m); ES+ MS C17H17FIN3 (409.0)
m/z 409.9 [M+H]+; HRMS (EI+) found 409.04446, calcd
for C17H17FIN3 409.04457 [M]+; Anal. calcd. for
C17H17FIN3: C 48.89, H 4.19, N 10.27; found C 49.17, H
4.61, N 9.87.
2-[N-methyl-N-(2′-hydroxyethyl)-4′-aminophenyl]-
benzothiazole (10). A solution of o-aminothiophenol (34)
(550 mg, 4.4 mM) and N-methyl-N-(2-tosyloxyethyl)-4-
aminobenzaldehyde (650 mg, 3.6 mM) in pyridine (6 mL)
was refluxed for 32 h. After cooling, the reaction mixture
was acidified by addition of 2 M HCl. The resulting
precipitate was filtered off and dried under vacuum to
afford 10 (805 mg, 79%) - Rf (EtOAc/n-hexane 1:1)
7.8Hz, H7); 13C NMR (CDCl3, 75MHz) δ 39.17 (NCH3),
2
1
52.33 (d, JC,F 20.8 Hz, NCH2CH2F), 81.61 (d, JC,F
169.7Hz, NCH2CH2F), 111.70 (2C, Carom), 121.36
(Cquart), 122.29 (2C, Carom), 124.31 (Carom), 126.04
(Carom), 129.04 (2C, Carom), 134.34 (Cquart), 150.81
(Cquart); 19F NMR (CDCl3, 282MHz): δ=À222.77; ES+
MS C16H15FN2S (286.1) m/z 287.0 [M+H]+; Anal. calcd.
for C16H15FN2S: C 67.11, H 5.28, N 11.20, S 11.20;
found C 66.87, H 5.90, N 9.69, S 11.31.
Radiochemistry.
No-carrier-added aqueous [18F]
fluoride was produced in a CYCLONE 18/9 cyclotron
(IBA) by irradiation of [18O]H2O via the 18O(p,n)18F
nuclear reaction. Resolubilization of the aqueous [18F]
fluoride (0.8–1.0 GBq) was accomplished with
Kryptofix® 2.2.2 and K2CO3 in a conical vial and
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

G. R. Morais, E. Palma, F. Marques, L. Gano, M. C. Oliveira, A. Abrunhosa,
H. V. Miranda, T. F. Outeiro, I. Santos, and A. Paulo
Vol 000
azeotropically removing water with acetonitrile in a stream
of nitrogen [29]. Finally, the dried [18F]KF was
resolubilized in 500 μL of anhydrous acetonitrile and
added to 15 (4.1 mg) in a conical glass vial. The vial was
sealed and heated for 20 min at 100°C in an oil bath.
After cooling, the mixture was subjected to semi-preparative
HPLC (Phenomenex Luna C18, 10× 250mm, 5μm) using
an isocratic eluent of 40/60 acetonintrile/water(0.1% NEt3,
pH=10.2) with a flow rate of 6mL/min originated by a
Knauer K-501 pump. The products were monitored by UV
detector (Knauer K-200) at 254nm and by gamma
detection with a scintillation detector (Bioscan Reform).
The radiolabeled product [18F]2 eluting at 10–11min
was collected, diluted with 30 mL of water, and the whole
solution applied to a C18 cartridge. The cartridge was
washed with 5 mL of water and the radiolabeled product
[18F]2 eluted with 0.4 mL of ethanol and reconstituted with
1.6 mL of E153 electrolyte infusion solution. This solution
was used for the biodistribution studies.
Each experiment was repeated at least two times, and each
concentration was tested in at least six replicates. Results are
expressed as the percentage of cellular viability with respect
to control wells (non-treated cells). The IC50 values were
calculated from plots for cell survival (%) versus com-
pound concentration with the GraphPad Prism software
(version 4.0).
Biodistribution.
Animal studies were carried out in
conformity with the national law and with the EU
Guidelines for Animal Care and Ethics in Animal
Experimentation. The animals were housed in
a
temperature and humidity-controlled room with a 12h
light/dark schedule.
The biodistribution of the [18F]2 was studied in groups
of four female CD-1 mice (randomly bred, Charles River)
weighting approximately 30–35g each. Animals were in-
travenously injected with 100 μL (0.7–1.7 MBq) of each
preparation via the tail vein and maintained on normal diet
ad libitum. At 5 min and 1h after administration, each mice
group was sacrificed by cervical dislocation. The radioac-
tive dosage administered in the animal was measured in a
dose calibrator (Capintec CRC25R). Blood samples were
taken by cardiac puncture at sacrifice. Tissue samples of
the main organs were removed, weighted, and counted in
a well counter (Capintec CRC-55tW). Accumulation of
radioactivity in the tissues was calculated and expressed
as percentage of the injected radioactivity per gram of
organ (% I.A./g) (Table 2).
HPLC analyses of the radiolabeled product [18F]2 were
performed using an Agilent 1200 Series system (Agilent
Technologies, USA) equipped with a multi-wavelength
UV detector and a GABIstar NaI(Tl) radiometric detector
(Raytest Isotopenmessgeraete GmbH, Straubenhardt,
Germany) using an Agilent Zorbax Eclipse XDB C18
column, 5μm, 4.6 × 150mm and the indicated isocratic
40/60 acetonitrile/water (0.1% NEt3, pH= 10.2) with a flow
rate of 1.0mL/min.
Cytotoxicity studies.
Cell culture growth conditions:
The human cancer cell lines MCF7 breast, A375
melanoma, HeLa cervical, and U87 glioblastoma
(American Type Culture Collection, ATCC) were used in
this study. The cells were maintained in DMEM
(Dulbecco’s Modified Eagle Medium) with Glutamax I
(Gibco) supplemented with 10% (v/v) fetal bovine serum
(FBS) and 1% antibiotics (Invitrogen). Cells were
maintained in flasks at 37°C in a 5%CO2 incubator
(Heraus, Germany) in a humidified atmosphere. For the
assays, cells in exponential growth were detached with
trypsin–EDTA, suspended in medium and seeded in
96-well plates at a density of between 1–3 × 104cells/well.
Cytotoxicity assays: For evaluation of cellular viability,
cells were treated with selected concentrations of the compounds
previously dissolved in DMSO (final concentration<1%)
diluted in medium and incubated for 48h at 37°C, 5%
CO2. Analysis of cell survival was carried out by the MTT
[MTT = [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] colorimetric assay at 37°C 48h incubation. After
incubation, the medium was discarded, and a solution of
MTT dissolved in PBS (0.5mg/mL) was added to each well
(200μL) and the plates incubated at 37°C for another 3–4h.
Then, 200μL of DMSO was added to each well to dissolve
the formazan crystals. Absorbance was measured at 570nm
with a plate spectrophotometer (Power Wave Xs, Bio-TeK).
Acknowledgments. The authors thank the Fundação para a Ciência
e a Tecnologia (FCT) for the “Ciência 2008” program (GRM)
and for the grants SFRH/BPD/80758/2011 (EP) and SFRH/BPD/
64702/2009 (HVM). The authors thank Sergio do Carmo (ICNAS)
for his support in the operation of the cyclotron and production
of fluorine-18. Elizabete Correia and Vitor Alves are acknowledged
for technical support in the biodistribution studies and HPLC,
respectively. J. Marçalo is acknowledged for the mass
spectrometry analyses performed on QIT and FTICR instruments
that are part of RNEM - Rede Nacional de Espectrometria de
Massa, supported by the FCT. TFO is supported by the DFG
Center for Nanoscale Microscopy and Molecular Physiology of
the Brain (CNMPB).
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