Distribution of furamidine analogues in tumor cells: Influence of the number of positive charges

Article abstract of DOI:10.1021/jm010539n

Fluorescence microscopy has been used to study the cellular distribution properties of a series of DNA binding cationic compounds related to the potent antiparasitic drug furamidine (DB75). The compounds tested bear a diphenylfuran or a phenylfuranbenzimi

Full text of DOI:10.1021/jm010539n

1994
J . Med. Chem. 2002, 45, 1994-2002
Distr ibu tion of F u r a m id in e An a logu es in Tu m or Cells: In flu en ce of th e Nu m ber
of P ositive Ch a r ges
Ame´lie Lansiaux,† Laurent Dassonneville,† Michae¨l Facompre´,† Arvind Kumar,‡ Chad E. Stephens,‡
Miroslav Bajic,‡ Farial Tanious,‡ W. David Wilson,‡ David W. Boykin,‡ and Christian Bailly*,†
INSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, Place de Verdun,
59045 Lille, France, and Department of Chemistry and Laboratory for Chemical and Biological Sciences, Georgia State
University, Atlanta, Georgia 30303
Received November 27, 2001
Fluorescence microscopy has been used to study the cellular distribution properties of a series
of DNA binding cationic compounds related to the potent antiparasitic drug furamidine (DB75).
The compounds tested bear a diphenylfuran or a phenylfuranbenzimidazole unfused aromatic
core substituted with one or two amidine or imidazoline groups. The synthesis of five new
compounds is reported. The B16 melanoma cell line was used to compare the capacities of
mono-, bis-, and tetracations to enter the cell and nuclei. The high-resolution fluorescence
pictures show that in the furamidine series, the compounds with two or four positive charges
selectively accumulate in the cell nuclei whereas, in most cases, those bearing only one positive
charge show reduced cell uptake capacities. One of the monocationic compounds, DB607,
distributes in the cytoplasm, possibly in mitochondria, with no distinct nuclear accumulation.
In sharp contrast, furamidine and benzimidazole analogues, including the drug DB293 that
forms DNA minor groove dimers, efficiently accumulate in the cell nuclei and the intranuclear
distribution of these DNA minor groove binders is significantly different from that seen with
the DNA intercalating drug propidium iodide. The results suggest that the presence of two
amidine terminal groups plays a role in facilitating nuclear accumulation into cells, probably
as a result of nucleic acid binding. The determination of DNA melting temperature increases
on addition of these compounds supports the importance of DNA binding in nuclear uptake.
In tr od u ction
ases.5-7 To bind to DNA, the drug must first enter the
cell and then reach the nuclear compartment. The
mechanism by which such positively charged, highly
water-soluble drugs manage to penetrate into cells is
unknown at present, as is the influence of compound
structure on cell uptake. In this report, we have ad-
dressed the issue of cell and nuclear uptake by means
of fluorescence microscopy. The strong fluorescence
properties of the diphenylfuran provide unique op-
portunities to investigate their subcellular localization.
We chose a series of furamidine analogues that differ
by (i) the number of positive charges, from one to four
charges, (ii) the structure of the terminal group, either
an amidine, a more planar imidazoline group, or an
amide alkylamine, and (iii) the nature of the central
unfused aromatic system, with a symmetrical diphe-
nylfuran or an asymmetrical phenylfuranbenzimidazole
system.
Furamidine (DB75; Chart 1) is a bis-amidine diphe-
nylfuran derivative endowed with high antimicrobial
activities. The drug is most active against Pneumocystis
carinii, a widely spread pathogen responsible for pneu-
monia that often infects immunodeficient patients,
including those with acquired immune deficiency syn-
drome. In addition, furamidine is active against diverse,
highly infectious parasites such as Giardia lamblia,
Plasmodium falciparum, and Trypanosoma rhode-
sience.1,2 Recently an amidoxime prodrug of furamidine
was selected for clinical evaluation3 and is currently
undergoing phase II clinical trials against human
African trypanosomes.
In addition to their antimicrobial properties, certain
diphenylfuran derivatives exhibit significant cytotoxic
activities. Although furamidine is weakly toxic to hu-
man cells, in contrast its imidazoline analogue, furimi-
dazoline (DB60), has shown significant antiproliferative
activities against various tumor cell lines, including cells
resistant to cisplatinum.4
Resu lts
DB75. The high intrinsic fluorescence properties of
furamidine (and structurally related compounds) greatly
facilitate the fluorescence microscopy assessment of its
cellular uptake and intracellular distribution. Figure 1
shows fluorescence images of B16 melanoma cells
exposed to DB75. The cells (selected for their extended
morphology) were treated with 2 µM DB75 for 18 h at
37 °C, washed, fixed with 2% paraformaldehyde, and
then labeled with the green fluorescent dye 3,3-dihexy-
loxacarbocyanine iodide (20 nM DiOC6) and/or with the
red fluorescent dye propidium iodide (0.2 µg/mL PI).
The main cellular target of furamidine and related
diphenylfuran analogues is considered to be DNA. On
the basis of a number of results, it is assumed that these
drugs operate at the nucleic acids level by interfering
with DNA-interacting enzymes such as topoisomer-
* To whom correspondence should be addressed. E-mail: bailly@
lille.inserm.fr.
† INSERM U-524.
‡ Georgia State University.
10.1021/jm010539n CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/05/2002

Cellular Distribution of Furamidine Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 1995
F igu r e 1. (A) Fluorescence micrographs of B16 melanoma cells stained with 2 µM DB75 or DB607 (blue) with propidium iodide
(PI in red) or with 3,3-dihexyloxacarbocyanine iodide (DiOC6 in green). Images on the right side of the figure show the overlay of
the DB compound with PI (blue + red) or with DiOC6 (blue + green). The cells were incubated with the test drug for 18 h,
washed, fixed with 2% paraformaldehyde, and then labeled with PI (0.2 µg/mL PI) or DiOC6 (20 nM) prior to the microscopy
observation (×63). Panel B shows the cells at a higher magnification (×100) after treatment with 2 µM DB75 or DB607, which
accumulate selectively in the nucleus and in the cytoplasm of the cells, respectively. The cells were also stained with PI, and an
overlay of the blue and red signals are shown on the right side.
Like ethidium bromide, PI binds strongly to nucleic
acids and is routinely used to stain cell nuclei and to
label DNA in cytometry studies. In contrast, the DiOC6
dye is known to bind selectively to mitochondria (pos-
sibly to reticulum also) and is commonly used to
measure variations of the mitochondrial membrane
potential,8 in particular during drug-induced apopto-
sis.9,10 Here, DiOC6 was used as a probe to label the
cytoplasm of B16 cells. The three colors (blue, green,
and red) can be easily differentiated by fluorescence
microscopy.
The illustrations in Figure 1A show clearly that
essentially all of the DB75 molecules are sequestered
in the cell nuclei. The massive accumulation of the blue
fluorescence in the nuclear compartment contrasts with
the extensive green fluorescence detected in the cyto-
plasm. Although DB75 essentially colocalizes in the
nucleus with PI, the two drugs do not distribute identi-
cally within the nucleus of the cells. Significantly
distinct intranuclear patterns can be detected (compare
the red-blue overlay images in Figure 1A). The differ-
ences are very pronounced when the cells are observed
at a higher magnification (Figure 1B). A few relatively
large and bright-red nucleolar-like bodies were observed
with PI, whereas the blue staining with DB75 is more
uniformly distributed throughout the nucleus. However,
close examination of the micrographs show small puncta
that were patchy throughout the nuclei. This different
distribution may be due to different DNA sequence
selectivity. DB75, but not PI, exhibits a high preference
for AT-rich sequences in DNA,11-13 and therefore, its
binding to the GC-rich nature of nucleolar DNA must
be disfavored. Altogether, the images in Figure 1 leave
no room for doubt that the dicationic compound DB75
accumulates selectively in the cell nuclei and that this
process is likely driven by the drug binding to nucleic
acids.
St r u ct u r e Dist r ib u t ion R ela t ion sh ip s. Eleven
DB75 analogues (Chart 1) were selected to compare
their cell distribution profiles. The selected compounds
bear a diphenylfuran or a phenylfuranbenzimidazole
unfused aromatic core substituted with one or two
amidine or imidazoline groups or with two amide
alkylamines. They all fluoresce in blue (λexc
) 360-
max
380 nm; λem
) 450-500 nm), but the relative
max
fluorescence intensity varies with the compound struc-
ture. The monocations are less fluorescent than the
dications, and therefore, the exposition time (ET in
Table 1) had to be adjusted to obtain images of equal
intensity. These compounds were specifically chosen to
provide some insight into what molecular factors of
DB75 play the most significant roles in preventing or
favoring cytoplasmic and/or nuclear distribution of the
drug.
Figure 2A shows images of (left) three diphenylfuran
compounds with one (DB607), two (DB75), and four

1996 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Lansiaux et al.
Ch a r t 1
Ta ble 1. Cytotoxicity and DNA Binding
pounds bear the expected number of positive charges
in cells. The dications DB75 and DB293 show identical
distribution patterns with their bright-blue fluorescence
specifically located in the cell nuclei. The phenyl-to-
benzimidazole substitution (DB75 f DB293) profoundly
affect DNA sequence recognition16 but has no detectable
effect on cell distribution.
b
ETa (s)
∆Tm (°C)
IC50c (µM)
DB60
DB75
2.3
0.5
0.4
0.2
0.2
23.9
24.7
>28
24.0
>28
>28
8.9
1.1 ( 0.06
9.2 ( 0.34
2.5 ( 0.85
43.2 ( 1.8
nd
DB182
DB293
DB302
DB340
DB501
DB606
DB607
DB654
DB771
DB772
0.4
>100
>100
1.7 ( 0.31
13.9
20.0
0.6
10.0
6.2
The compounds with only one amidine group show
reduced uptake capacities. The monocationic diphenyl-
furan derivative DB607 was found essentially in the
cytoplasm of the cells. The overlay of the DiOC6 signal
and DB607 signal results in a light-blue color where the
green and blue signals colocalize. Little or no blue
fluorescence was observed in the nuclei. Another set of
images obtained with this compound is presented in
Figure 1A where the green and red colors are used to
show the position of the cytoplasm (DiOC6) and the
nucleus (PI), respectively. The uptake differences be-
tween DB75 and DB607, which differ only by the nature
of one of the terminal groups (amidine vs methoxy), are
exemplary. DB75 is clearly inside, whereas DB607 is
exclusively outside the nucleus and they produce com-
5.2
5.2
5.7
6.9
3.5 ( 0.15
2.3 ( 0.40
51.4 ( 2.74
8.6 ( 0.05
0.3
8.3
a
Exposure time required to obtain digital images of equal
b
fluorescence intensity. Variation in melting temperature, ∆Tm
complex
) Tm
- Tmpoly(dA)‚(dT). Tm measurements were performed
in MES10 buffer. c Drug concentration that inhibits B16 cell
growth by 50% after 72 h of incubation.
(DB182) positive charges and (right) three phenylfuran-
benzimidazole compounds with one (DB771), two
(DB293), and four (DB340) positive charges. The pKa
values of benzamidine is sufficiently high (pKa ) 11.2
in 50% EtOH)14,15 to consider that the studied com-

Cellular Distribution of Furamidine Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 1997
F igu r e 2. (A) Intracellular distribution of the mono- (DB607, DB771), bis- (DB75, DB293), and tetracationic (DB182, DB340)
diphenylfuran (left) and phenylfuranbenzimidazole (right) derivatives (2 µM each) in B16 melanoma cells visualized by fluorescence
microscopy (×63) after 18 h of incubation. (B) Intracellular distribution of the monocations (DB606, DB772, DB501) and dications
(DB60, DB302, DB654) (2 µM each) in B16 melanoma cells visualized by fluorescence microscopy (×63) after 18 h of incubation.
The green images correspond to the subsequent incubation of the same cells with 20 nM DiOC6, and the images on the right side
correspond to the superimposed blue + green fluorescence.
pletely opposite distribution patterns in Figure 1A.
Figure 1B shows additional views of the cells (at a
higher magnification) labeled with the two compounds,
allowing full visualization of their totally opposite
intracellular spatial distribution. These observations
demonstrate that the substitution of a methoxy group
in DB607 for one of the amidine groups in DB75 has a
major impact on the cell distribution properties of the
compound. Conversion of the amidine to a -OCH3 group
with the loss of one positive charge precludes the uptake
of the compound in the cell nuclei. Because this infor-
mation is essential for future drug design, it is impor-
tant to mention here that identical observations were
made using two other cell lines, P388 murine leukemia
and HT29 human colon carcinoma (data not shown).
The different cellular distribution properties of DB75
vs DB607 are absolutely not a unique feature of the B16
melanoma cell line used throughout this study.
The situation is comparable in the imidazoline series.
The cell distribution profiles obtained with furimidazo-
line (DB60) are indistinguishable from that seen with
furamidine (DB75). The conversion of the amidine into
a five-membered cyclic form is known to enhance DNA
intercalation as opposed to minor groove binding12 but
has no perceptible effect on cell distribution because
both compounds accumulate in the nuclei. But here
again, the replacement of one of the two imidazoline
groups with a methoxy group leads to a loss of nuclear
uptake, and this is in perfect agreement with the above
observations. The result is not as clear with DB606 as
it is with DB607 because an excessively long exposure
time was required to observe the cells under the
microscope with DB606 (compared the exposure times
for DB606 and DB607 in Table 1), but nevertheless, it
can be seen clearly that this monocationic imidazoline
compound is poorly taken up into the nuclei. Very
limited nuclear uptake was also observed with the
monocations in the phenylfuranbenzimidazole series.
DB771 showed almost no cell uptake, and its imidazo-
line counterpart DB772 was considerably less concen-
trated in the nuclei of the cells than the corresponding
dication DB302. Interestingly, the amidine DB771
shows a higher tendency to enter the nucleus than the
corresponding imidazolidine DB772 (both the profiles
and exposure times are different). A similar observation
can be made when comparing the methoxy compounds,
amidine DB607 vs imidazolidine DB606. The difference
might be accounted for by their different capacities to
bind to DNA. Previous DNA binding studies have
revealed that the imidazolidines have a tendency to
intercalate into GC-rich DNA sequences as well as into
a favorable AT-specific minor groove interaction mode.
The amidine derivatives display a much weaker ten-

1998 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Lansiaux et al.
F igu r e 3. Fluorescence of B16 melanoma cells labeled with DB293. The cells were treated with 2 µM DB293 for 15 min to 6 h
(left) or for 18 h with increasing concentrations of DB293 (right). Other details are as for Figure 2.
dency to intercalate and behave as much more specific
minor groove binders in AT sequences.12,17
cases, the blue fluorescence was clearly concentrated in
the nucleus of the cells with no apparent blue fluores-
cence in the cytoplasm. Therefore, the presence of four
positive charges is clearly not an obstacle to the nuclear
binding. The dimethylaminopropyl side chains of DB182
attached on the amidine moieties do not restrict the
capacity of the drug to enter into the nuclei, but it seems
that the transfer into the nucleus is entirely driven by
the amidines. Indeed, the dication DB654, for which the
amidine of DB182 has been replaced with an amide
linkage, is practically not detected in the cells and no
blue signal is detected in the nuclei of cells labeled with
this compound. We conclude, therefore, that the amidine
groups (or equivalent cyclic forms) are the key structural
elements necessary for the accumulation of the drugs
in the nuclear compartment.
DB293. The benzimidazole compound DB293 is clearly
distinct from all other compounds of the present series
in terms of DNA binding. We have recently shown
that this compound forms a stacked dimer in the DNA
minor groove16 and preferentially recognizes ATGA
sequences.19,20 It represents a lead compound for the
design of sequence-specific gene regulatory molecules.21
For this reason, its cell uptake capacities were investi-
gated in more detail. Figure 3 shows micrographs
obtained from a kinetics experiment with this com-
pound. A time course study was carried out with 1 µM
DB293, but little or no differences was observed when
the cells were incubated for 15 min or 6 h prior to
fluorescence microscopy. The nuclear uptake is too fast
to be monitored by this technique. In all cases, DB293
seems to have “privileged” access to the nucleus. In fact,
nuclear staining with DB293 was observed in less than
5 min after the incubation began. Similarly, cell nuclei
become fluorescent within minutes of exposure to DB75
(data not shown). The concentration dependence of the
nuclear accumulation process was also studied. At a low
It is important to mention that upon binding to DNA,
the fluorescence of these diamidines decreases (by about
20-25% for the groove binders, like DB75, and up to
70-90% for the compounds than can intercalate into
DNA, like DB60). Therefore, the extent of nuclear
uptake must be underestimated. The bright-blue fluo-
rescence detected in the nuclei of the cells treated with
compounds like DB75, DB60, and DB293 must reflect
a massive nuclear accumulation.
The dications bearing either two amidines (DB75,
DB293) or two imidazolidines (DB60, DB302) show
highly preferential nuclear uptake, whereas the corre-
sponding monocations accumulates preferentially into
the cytoplasm of the cells (DB607) or they stain the cells
very poorly, probably because they have difficulties
entering the cells (DB771, DB772, DB606). The deletion
of one of the two the amidine or imidazolidine groups
or their substitution for methoxy groups reduces or
abolishes nuclear uptake. Therefore, we may conclude
that the presence of two positive charges favors nuclear
uptake. However, there is an exception to the rule
because the monocation DB501 with an amino group
in place of the amidine of DB75 still shows preferential
nuclear accumulation, but a long exposure time was
required to detect this compound in the cells. The
relatively weak intrinsic fluorescence of the monocations
makes their detection in cells more difficult.
Interestingly, incubation of the cells with the ex-
tended compound DB182 produced a dense and bright
accumulation of blue fluorescence in the nuclear region,
demonstrating that the cells are capable of massive
internalization of this tetracation. In the benzimidazole
series, the tetracation DB340, which binds strongly and
selectively to certain hairpin RNA,18 also retains the
ability to concentrate in the nuclei like DB75. In both

Cellular Distribution of Furamidine Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 1999
concentration (0.05 µM), the drug DB293 is exclusively
detected in the cell nuclei with no distinct cytoplasmic
staining. Raising the concentration 100-fold to 5 µM
increased the fluorescence intensity but did not change
the internalization profile. No blue fluorescence was
observed in the cytoplasm, indicating that the drug is
concentrated exclusively in the nuclei. This was further
confirmed by means of confocal microscopy (data not
shown).
interact with DNA much more weakly than the corre-
sponding dimers. For example, the Tm values measured
with DB293 are considerably higher than those obtained
with DB771 containing one amidine group. But here
again, we cannot deduce any direct relationship between
DNA binding and cytotoxicity or cell distribution. As a
consequence, it will be difficult to use direct imaging of
diphenylfuran derivatives within cells to guide analogue
screening for potentially advantageous antiproliferative
properties. The degree of nuclear retention may be a
secondary factor in determining cellular sensitivity to
diphenylfurans. The nucleus may serve as a reservoir
and perhaps as a slow-release device for furamidine-
type compounds.
Rela tion to Cytotoxicity a n d DNA Bin d in g. A
conventional tetrazolium-based MTS assay was applied
to determine the drug concentration required to inhibit
cell growth by 50% after incubation in the culture
medium for 72 h. As indicated by the IC50 values in
Table 1, the cytotoxicity of the test compounds varies
considerably from one to another and no direct relation-
ship with the distribution profile can be proposed. For
example, the tetracation DB340, which was found to
enter efficiently into the nuclei of the B16 cells, is totally
inactive whereas the other tetracation DB182 proved
to be highly toxic to the cells under identical conditions.
In general but not systematically, the compounds in the
benzimidazole series are less toxic than those in the
equivalent phenyl-containing analogues and the imida-
zoline compounds show higher toxicity compared to the
amidines (e.g., DB60 vs DB75 and DB772 vs DB771).
This general trend can be deduced from additional
studies with a larger number of compounds (unpub-
lished data).
Discu ssion
The natural fluorescence of some drugs enables
distribution of the molecules to be monitored both
throughout the cell population and within intracellular
compartments of intact viable cells. But the benefits of
imaging fluorescent DNA-binding compounds in live
cells have, until now, been underexploited. The cellular
distribution of some fluorescent DNA intercalating
drugs, in particular antitumor agents from the anthra-
cycline and ellipticine families (e.g., daunomycin, adria-
mycin,22,23 mitoxantrone,24 ditercalinium25), has been
investigated. But relatively few studies have been
performed with nonintercalating drugs26 and in par-
ticular with DNA minor groove binders. The dyes DAPI
and Hoechst 33258/33342 have been shown to enter cell
nuclei,27-29 but as far as we know, there are no reports
in the literature of structure cell distribution relation-
ship studies. The present study is unique in comparing
the cellular localization of a homogeneous series of DNA
minor groove binders. These are the first data to show
that this type of cationic ligand readily penetrates into
cells and exhibits selective nuclear uptake capacity. The
high degree of accumulation of DB75 into the cell nuclei
is an essential component in the development of gene-
targeted drugs.
The nuclei of B16 cells retain the dication DB75,
where it would be expected to exert a cytotoxic effect.
In contrast, the monocation does not enter the nuclei,
but despite the lack of nuclear fluorescence in DB607-
treated cells, the compound is among the most cytotoxic
agents in the series. The IC50 value measured with
DB607 is even superior to that of DB75, indicating that
no simple relationship exists among the level of nuclear-
bound drug and intranuclear fluorescence intensity.
Nuclear uptake is not associated with cytotoxicity.
To determine the relative affinities of the compounds
used in the cell penetration studies for their DNA
recognition sites, melting temperature (Tm) values of the
compound-DNA complexes were compared to values for
free DNA (Table 1). All of the compounds caused
increases in the DNA Tm value, but the magnitude of
the changes was quite different. The compounds fall into
two different binding classes based on their Tm increases
with DNA. The dications and tetracations all have Tm
increases that are 24 °C or greater. This magnitude of
Tm increase is consistent with binding constants in the
range of 108 M-1 and higher for this set of com-
pounds.12,13,18 With the monocations, the Tm increases
are much smaller and can be grouped between a 5 and
9 °C increase. This magnitude of increase is consistent
with a binding constant below 105 M-1 for the com-
pound-DNA complex. Clearly the dication and tetra-
cation groups bind to the AT DNA recognition site
approximately 1000 times more strongly than the
monocations of corresponding structure. Interestingly,
the only compound that clearly does not enter the
nucleus, DB607, exhibits the weakest affinity for DNA,
as judged from the melting temperature measurements
performed with poly(dA)‚(dT) (Table 1) and calf thymus
DNA (data not shown). The other monocations also
The molecular mechanism(s) directing DB75 and
DB293 to the nucleus remains unknown at present and
awaits further investigation. These charged compounds
enter cells rapidly, and a pure diffusion process seems
unlikely. Specific transporters, analogous to those iden-
tified for the entry of the related drug pentamidine in
parasites,30 likely promote intracellular accumulation
of these compounds. Normal and malignant cells possess
a high-affinity transport system that controls the entry
of polyamines (putrescine, spermidine, spermine) and
guanidines (e.g., methylglyoxal bis(guanylhydrazone)).31
This membrane transport system may well be exploited
by the amidines to cross the plasma membrane. Inter-
estingly, the potent accumulation of the DNA-binding
dicationic compounds, like DB75 and DB293, in nuclei
contrasts with the limited intracellular capacity of
related polyamide-type minor groove binders. In a
recent study with a fluorescently tagged DNA-binding
polyamide related to distamycin, it was shown that the
conjugate molecule selectively accumulates in mitochon-
dria of adenocarcinoma SKOV-3 cells rather than in the
nucleus.32 No evidence of nuclear uptake was reported,
and the selective mitochondrial uptake was attributed
to the relatively high electronegativity of the mitochon-

2000 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10
Lansiaux et al.
1
(69%), mp 227-228 °C dec. H NMR (DMSO-d6): δ 8.06 (d,-
2H, J ) 8.8 Hz), 7.91(d,2H, J ) 8.8 Hz), 7.60 (dd,2H, J ) 3.2,
6.4 Hz),7.38 (d, 1H, J ) 3.6 Hz), 7.32(d, 1H, J ) 3.6 Hz), 7.23
(dd,2H, J ) 3.2, 6.4 Hz). 13C NMR (DMSO-d6): δ 152.1, 146.0,
142.7, 138.7, 133.2, 132.6, 124.1, 122.3, 118.4, 114.9, 112.5,
111.1, 109.8. MS, m/e: 285 (M+). Anal. Calcd for C18H11N3O:
C, H, N.
drial membranes, which can restrict uptake of cationic
compounds.33 The findings reported here do not support
this view and in contrast reveal that cationic drugs can
be delivered selectively to the cell nucleus. The Tm
studies in Table 1 suggest a correlation between DNA
binding affinity and nuclear uptake for the furan-based
compounds in this report. Apparently there is no direct
relationship between nuclear accumulation and cyto-
toxicity in the cells tested in this study. Rather, the
biological activity may depend on the capacity of these
compounds to recognize specific sequences in DNA and
consequently to affect the profiles of gene expression.
The specific distribution of the diamidines within some
critical portions of the genome is currently investigated.
All results together indicate that amidine or related
functions and strong DNA binding are required for
significant nuclear uptake in this series.
2-(2-Ben zim id a zolyl)-5-[4-(a m id in o)p h en yl]fu r a n Di-
h yd r och lor id e (DB771). The above cyano compound (2.85
g, 0.01 mol) in 60 mL of ethanol was saturared with dry HCl
gas at 0-5 °C. The reaction mixture was stirred at room
temperature for 12 days (monitored by IR and TLC). The
mixture was diluted with ether, and the yellow imidate ester
hydrochloride was filtered, washed with ether, and dried under
vacuum for 6 h, giving 3.73 g (92%) of solid. The solid was
used in next step without further purification. A suspension
of the imidate ester hydrochloride (0.808 g, 0.002 mol) in 35
mL of ethanol was saturated with ammonia gas at 0-5 °C and
was stirred for 24 h at room temperature. The solvent was
reduced to one-third under reduced pressure, diluted with
ether, and filtered. The yellow solid was resuspended in 10
mL of ethanol, treated with 4 mL of saturated ethanolic HCl,
and stirred at 35 °C for 2 h. The solvent was removed under
vacuum, and the residue was triturated with ether, filtered,
washed with ether, and dried under vacuum at 45 °C for 24 h
to yield 0.61 g (81%) of a yellow solid, mp >280 °C dec. 1H
NMR (DMSO-d6/D2): δ 8.15 (d, 2H, J ) 8.7 Hz), 7.93 (d, 2H,
J ) 8.7 Hz), 7.78 (d, 1H, J ) 3.6 Hz), 7.75 (dd, 2H, J ) 3, 6.3
Hz), 7.50 (d, 1H, J ) 3.6 Hz), 7.49 (dd, 1H, J ) 3, 6.3 Hz). 13C
NMR (DMSO-d6): δ 165.0, 155.9, 139.8, 139.7, 133.4, 132.4,
129.3, 127.8, 126.3, 125.2, 119.5, 114.3, 112.0. FAB-MS, m/e:
303 (M+ + 1). Anal. Calcd for C18H14N4O‚2HCl: C, H, N.
2-(2-Ben zim id a zolyl)-5-[4-(2-im id a zolin o)p h en yl]fu -
r a n Dih yd r och lor id e (DB772). A mixture of the imidate
ester hydrochloride (0.808 g, 0.002 mol) from above and
ethylenediamine (0.12 g, 0.002 mol) in 20 mL of dry ethanol
was heated at reflux for 12 h. The solvent volume was reduced
to 8 mL under reduced pressure and diluted with ether. The
resultant solid was filtered and dried. This solid was dissolved
in 35 mL of hot ethanol and was saturated with HCl gas at
room temperature. The mixture was stirred at 50 °C for 2 h
and was concentrated under reduced pressure, and 30 mL of
dry ether was added. The precipitated yellow salt was filtered,
washed with ether, and dried under vacuum at 70 °C for 24 h
to yield 0.69 g (84%) of a yellow solid, mp >300 °C dec. 1H
NMR (DMSO-d6/D2): δ 8.06 (d, 2H, J ) 8.7 Hz), 7.91 (d, 2H,
J ) 8.7 Hz), 7.71 (dd, 2H, J ) 3, 6 Hz), 7.64 (d, 1H, J ) 3.9
Hz), 7.47 (dd, 1H, J ) 3, 6.3 Hz), 7.44 (d, 1H, J ) 3.9 Hz),
3.94 (s, 4H). 13C NMR (DMSO-d6): δ 164.6, 155.7, 140.3, 140.1,
133.9, 132.9, 129.7, 126.7, 125.4, 122.1, 119.2, 114.6, 112.5,
44.8. FAB-MS, m/e: 303 (M+ + 1). Anal. Calcd for C20H16N4O‚
2HCl‚0.5H2O: C, H, N.
1-(4-Br om op h en yl)-4-(4-m eth oxyp h en yl)bu ta n e-1,4-d i-
on e. To a stirred mixture of 1-(4′-bromophenyl)-3-dimethy-
laminopropane-1-one hydrochloride (14.0 g, 0.05 mol), 3-benzyl-
5-(2-hydroxyethyl)-4-methylthiazolium chloride (0.68 g, 0.0025
mol), and triethylamine (15.15 g, 0.15 mol) in 200 mL of
anhydrous dioxane was added 4-methoxybenzaldehyde (6.8 g,
0.05 mol), and the mixture was heated at reflux for 12 h (under
nitrogen). The solvent was removed, and the residue was
stirred with 200 mL of water. The gummy mass was extracted
with 3 × 120 mL of warm chloroform, washed with 2 × 100
mL of 10% NaHCO3, dried over Na2SO4, and filtered. Solvent
was removed, and the residual mass was stirred with 30 mL
of ether/ethanol (1:2) to yield a white crystalline solid, 6.1 g
(35.3%), mp 121-123 °C. 1H NMR (DMSO-d6): δ 8.0 (d, 2H, J
) 8.8 Hz), 7.88 (d, 2H, J ) 8.8 Hz), 7.60 (d, 2H, J ) 8.8 Hz),
6.94 (d, 2H, J ) 8.8 Hz), 3.86 (s, 1H), 3.40-3.38 (m, 4H). 13C
NMR (DMSO-d6): δ 197.8, 196.9, 136.2, 131.9, 130.4, 130.1,
129.7, 128.1, 113.8, 55.5, 32.6, 32.3. MS, m/e: 347 (M+). Anal.
Calcd for C17H15BrO3: C, H, N.
Exp er im en ta l Section
Syn th esis. Compounds DB60 and DB75,1 DB182,34 DB293
and DB302,35 DB340,18 and DB50119 have been previously
described. The experimental protocols for the synthesis of
compounds DB606, DB607, DB654, DB771, and DB772 are
described below.
2,5-Bis[4-(4-ch lor obu tyr am ido)ph en yl]fu r an . To a chilled
solution of 2,5-bis(4-aminophenyl)furan36 (0.50 g, 2.0 mmol)
in dry acetonitrile (20 mL) was added triethylamine (0.42 g,
4.15 mmol) followed dropwise by 4-chlorobutyryl chloride (0.58
g, 4.1 mmol), and the resulting suspension was stirred at room
temperature for 2 h. Water (20 mL) was then added, and the
solid was collected, rinsed with water followed by methanol,
and finally air-dried to give a light-tan solid (0.88 g, 96%), mp
268-269 °C. 1H NMR (DMSO-d6): δ 10.09 (broad s, 2H), 7.64-
7.74 (partially coalescing dd, J ) 9.0, 9.0 Hz, 8H), 6.92 (s, 2H),
3.70 (t, 4H), 2.40-2.50 (t, 4H, partially obscured by DMSO
solvent), 2.00-2.14 (m, 4H). MS (EI), m/z: 386 (M - 2HCl).
2,5-Bis[4-[4-(d im e t h yla m in o)b u t yr a m id o]p h e n yl]-
fu r a n Dih yd r och lor id e (DB654). To a solution of the
4-chlorobutyramide (0.66 g, 1.44 mmol) and NaI (50 mg) in
DMF (15 mL) was added 40% aqueous dimethylamine (7 mL).
After the sealed mixture was stirred at room temperature for
1 week, excess brine solution was added and the precipitate
was collected and dissolved in 1 N HCl. After extraction with
EtOAc, the acidic aqueous layer was made basic (NaOH) and
extracted again with EtOAc. This second extract was washed
with brine, dried (MgSO4), and concentrated to a solid that
consisted of two spots on TLC. Column chromatrography
(silica) eluting with EtOAc/MeOH/triethylamine (5:4:1) and
collection of the more polar/slower-moving product gave, upon
concentration of homogeneous fractions and trituration with
ether, a light-tan solid (0.14 g, 20%), mp 218-220 °C. To
prepare the dihydrochloride, a solution of the free base in
EtOH (40 mL) was treated with anhydrous HCl and then was
concentrated to near dryness to give a suspension. Following
dilution with ether, the tan solid was collected and dried in
vacuo. Yield: 0.16 g (20% overall). 1H NMR (DMSO-d6): δ
10.38 (broad s, 2H), 10.27 (s, 2H), 7.67-7.74 (coalescing dd, J
) 9.0, 9.0 Hz, 8H), 6.92 (s, 2H), 2.75 (s, 6H), 3.05-3.10 (t, 4H),
2.43-2.49 (t, 4H, partially obscured by DMSO solvent), 1.94-
2.02 (m, 4H). 13C NMR (DMSO-d6): δ 170.1, 152.2, 138.5,
125.2, 123.9, 119.4, 107.1, 56.0, 42.0, 33.0,19.7. MS (EI), m/z:
476 (M - 2HCl). Anal. Calcd for C28H36N4O3‚2HCl: C, H, N.
2-(2-Ben zim id a zolyl)-5-[4-cya n op h en yl]fu r a n . A mix-
ture of 5-[4-cyanophenyl]-2-furancarboxaldehyde (1.97, 0.01
mol), 1,2-phenylenediamine (1.06 g, 0.01mol), and 1,4-benzo-
quinone (1.08 g, 0.01mol) in 50 mL of dry ethanol was heated
at reflux (under nitrogen) for 8 h. The reaction mixture was
cooled, diluted with ether, and filtered. The solid was collected
and stirred with a 1:3 mixture of EtOH and ether for 20 min,
and the yellow-brown solid was filtered, washed with ether,
and dried in a vacuum at 70 °C for 12 h, which yielded 1.96 g
2-(4-Br om op h en yl)-5-(4-m eth oxyp h en yl)fu r a n . A solu-
tion of the above diketone (5.55 g, 0.016 mol) in 150 mL of
CHCl3/CH3OH (7:3) was saturated with dry HCl gas at 0-5

Cellular Distribution of Furamidine Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 10 2001
106.7, 55.1, 44.0. FAB-MS, m/e: 319 (M+ + 1). Anal. Calcd for
°C and was stirred at 25 °C for 2 h (monitored by TLC silica
gel ether/CHCl3, 7:3), and the solvent was removed under
reduced pressure. The residue was stirred with water, and the
solid was filtered, washed with water, resuspended in 10%
NaHCO3 (150 mL), filtered, washed with water, dried, and
recrystallized from CHCl3/ether (3:7) to give a white solid, 4.7
C
20H18N2O2‚1HCl: C, H, N.
Cell Cu ltu r es a n d Su r viva l Assa y. B16 melanoma cells
were obtained from Dr. M.-C. De Pauw (University of Lie`ge,
Belgium). The melanocytes were maintained as monolayers
in 150 cm2 culture flasks using culture medium consisting of
DMEM-glutaMAX medium supplemented with 10% fetal
bovine serum, penicillin (100 IU/mL), and streptomycin (100
µg/mL). Cells were grown in a humidified atmosphere at 37
°C under 5% CO2. In the exponential phase, the doubling time
of these melanocytes ranged from 15 to 18 h and the confluence
stage was achieved at a cell density of 3 × 105 cells/cm2. B16
cells were harvested by trypsinization and plated 20 h before
treatment with the test drug. The cytotoxicity of the drugs was
assessed using a cell proliferation assay developed by Promega
(CellTiter 96 AQueous one solution cell proliferation assay).
Briefly, 2 × 104 exponentially growing cells were seeded in
96-well microculture plates with various drug concentrations
in a volume of 100 µL. After 72 h of incubation at 37 °C, a
total of 20 µL of MTS37 was added to each well and the samples
were incubated for a further 3 h at 37 °C. Plates were analyzed
on a Labsystems Multiskan MS reader at 492 nm.
1
g (89%), mp 193-194 °C. H NMR (DMSO-d6): δ 7.65 (d, 2H,
J ) 8.8 Hz), 7.59 (d, 2H, J ) 8.8 Hz), 7.51 (d, 2H, J ) 8.8 Hz),
6.94 (d, 2H, J ) 8.8 Hz), 6.77 (d, 1H, J ) 3.6 Hz), 6.63 (d, 1H,
J ) 3.6 Hz), 3.84 (s, 3H). 13C NMR (DMSO-d6): δ 158.4, 152.8,
150.4, 130.8, 128.8, 124.2, 124.0, 122.5, 119.5, 113.4, 107.1,
104.9, 54.3. MS, m/e: 329 (M+). Anal. Calcd for C17H13BrO2:
C, H, N.
2-(4-Cya n op h en yl)-5-(4-m eth oxyp h en yl)fu r a n . A mix-
ture of the above bromo analogue (4.6 g, 0.014 mol) and CuCN
(1.87 g, 0.021 mol) in 60 mL of N-methyl-2-pyrrolidinone was
heated at reflux (under nitrogen) for 2.5 h. The mixture was
cooled, diluted with 100 mL water, and stirred with 100 mL
of 10% NaCN(aq) for 3 h. The solid was filtered, washed with
water, and dried, and the crude product was dissolved in
chloroform and chromatographed over neutral alumina to yield
1
a pale solid, 2.95 g (77%), mp 148-149 °C. H NMR (DMSO-
F lu or escen ce Micr oscop y. The cells (20 000 cells/cm2)
were incubated at 37 °C with the test compound, usually at 2
µM for 18 h unless otherwise stated. The medium was
removed, and cells were rinsed with ice-cold PBS (10 min) prior
to fixation with a 2% paraformaldehyde solution for 20 min
at 4 °C. After being washed, the cells were incubated with
fluorescent probes DiOC6 (20 nM, 3,3-dihexyloxacarbocyanine
iodide, Molecular Probes, Inc.) for 5 min at 37 °C in the dark,
washed again with PBS, and then incubated with a solution
of propidium iodide (0.2 µg/mL) for 5 min at room temperature.
A drop of antifade solution was added, and the treated portion
of the slide was covered with a glass coverslip. The fluorescence
of the drug was detected and localized by fluorescence micros-
copy using a Zeiss microscope with a ×63 or ×100 oil objective.
Images were captured using the software Quips Smart Cap-
ture (Vysis).
DNA Th er m a l Meltin g. Thermal melting experiments
were conducted with Cary 3 and Cary 4 spectrophotometers
interfaced to microcomputers as previously described.38 MES10
buffer [10 mM 2-(N-morpholino)ethanesulfonic acid, 1 mM
EDTA, pH 6.25 with 0.1 M NaCl] was used in the experiments
with poly(dA)‚(dT). A thermistor fixed to a reference cuvette
was used to monitor the temperature. In Tm experiments, DNA
was added to buffer in 1 cm path length reduced volume quartz
cells, and the concentration was determined by measuring the
absorbance at 260 nm. The experiments were generally
conducted at a concentration of 5 × 10-5 M DNA base pairs
and a ratio of 0.6 compound per base pair of DNA.
d6): δ 7.93 (d,2H, J ) 8.8 Hz), 7.83 (d, 2H, J ) 8.8 Hz), 7.77
(d, 2H, J ) 8.8 Hz), 7.25 (d, 1H, J ) 3.6 Hz), 7.02 (d, 2H, J )
8.8 Hz), 6.95 (d,1H, J ) 3.6 Hz), 3.81 (s, 3H). 13C NMR (DMSO-
d6): δ 159.2, 154.3, 150.0, 133.9, 132.5, 125.2, 123.4, 122.4,
118.6, 111.4, 108.7, 106.6, 55.1. MS, m/e: 275 (M+). Anal. Calcd
for C18H13BrO2: C, H, N.
2-(4-Am id in op h en yl)-5-(4-m et h oxyp h en yl)fu r a n H y-
d r och lor id e (DB607). The above cyano compound (2.75 g,
0.01 mol) in 60 mL of ethanol was saturated with dry HCl gas
at 0-5 °C. The reaction mixture was stirred at room temper-
ature for 8 days (monitored by IR and TLC) and was diluted
with ether. The yellow imidate ester hydrochloride was
filtered, washed with ether, and dried under vacuum for 6 h
to give 3.25 g (91%) of a solid. The solid was used in the next
step without further purification. A suspension of the imidate
ester hydrochloride (1.07 g, 0.003 mol), in 25 mL of ethanol
was saturated with ammonia gas at 0-5 °C and was stirred
for 24 h at room temperature. The solvent was removed under
reduced pressure, diluted with water, and basified with 2 N
NaOH(aq) to pH 10. The solid was filtered, washed with water,
and dried. The solid was dissolved in 15 mL of ethanol, treated
with 2 mL of saturated ethanolic HCl, and stirred at 35 °C for
2 h. The solvent was removed under reduced pressure, and
the residue was triturated with ether, filtered, washed with
ether, and dried under vacuum at 45 °C for 24 h to yield 0.66
g (67%) of a yellow solid, mp >280 °C dec. 1H NMR(DMSO-
d6): δ 9.44 (br, 2H), 9.29 (br, 2H), 9.26 (br, 2H), 7.98 (d, 2H, J
) 8.8 Hz), 7.95 (d, 2H, J ) 8.8 Hz), 7.78 (d, 2H, J ) 8.8 Hz),
7.29 (d, 2H, J ) 8.8 Hz), 7.02 (d, 2H, J ) 8.8 Hz), 6.97 (d, 1H,
J ) 3.6 Hz), 3.80 (s, 3H). 13C NMR (DMSO-d6): δ 164.9, 159.2,
154.2, 150.2, 134.7, 128.6, 125.4, 125.3, 122.4, 114.4, 111.2,
106.7, 55.1. FAB-MS, m/e: 293 (M+ + 1). Anal. Calcd for
C18H16N2O2‚1HCl: C, H, N.
2-[4-(2-Im idazolyl)ph en yl]-4-(m eth oxyph en yl)fu r an Hy-
d r och lor id e (DB606). A mixture of the imidate ester hydro-
chloride (1.07 g, 0.003 mol) from above and ethylenediamine
(0.18 g, 0.003 mol) in 30 mL of dry ethanol was heated at reflux
for 12 h. The solvent was distilled under reduced pressure.
The solid was suspended in 50 mL of water, and the pH was
adjusted to 10 with 2 N NaOH(aq). The solid was filtered,
washed with water, and dried to yield the crystalline free base
(72%), mp 159-160 °C. The solid was dissolved in 15 mL of
hot ethanol and was saturated with HCl gas at 0 °C. The
mixture was stirred at 50 °C for 2 h, and 30 mL of dry ether
was added. The precipitated salt was filtered, washed with
ether, and dried under vacuum at 70 °C for 24 h to yield 0.78
g (73%) of a yellow crystalline solid, mp >300 °C dec. 1H NMR
(DMSO-d6): δ 10.8 (br, 2H), 8.16 (d, 2H, J ) 8.8 Hz), 7.99 (d,
2H, J ) 8.8 Hz), 7.78 (d, 2H, J ) 8.8 Hz), 7.31 (d, 1H, J ) 3.6
Hz), 7.02 (d, 2H, J ) 8.8 Hz), 6.97 (d, 1H, J ) 3.6 Hz), 3.99 (s,
3H), 3.81 (s, 3H). 13C NMR (DMSO-d6): δ 164.0, 159.2, 154.5,
150.0, 135.1, 129.2, 125.3, 123.0, 122.3, 119.7, 114.3, 111.7,
Ack n ow led gm en t. This work was done under the
support of research grants (to C.B.) from the Ligue
Nationale Contre le Cancer (Equipe labellise´e LA
LIGUE) (to W.D.W and D.W.B.), NIH Grant GM61587,
the Georgia Research Alliance, and a Gates Foundation
Grant. W.D.W. acknowledges an INSERM “Poste
Orange” fellowship for research in Lille at INSERM
U-524.The authors thank the Service Commun d’Imagerie
Cellulaire de l’IFR22 for access to the fluorescence
microscope.
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