Asymmetrical diaromatic guanidinium/2-aminoimidazolinium derivatives: Synthesis and DNA affinity

Article abstract of DOI:10.1021/jm901017t

In this paper we report the synthesis of three families of new amidine-based aromatic derivatives as potential DNA minor groove binding agents for the treatment of cancer. The preparation of monoguanidine, mono-2-aminoimidazoline, and asymmetric diphenylg

Full text of DOI:10.1021/jm901017t

J. Med. Chem. 2009, 52, 7113–7121 7113
DOI: 10.1021/jm901017t
Asymmetrical Diaromatic Guanidinium/2-Aminoimidazolinium Derivatives:
Synthesis and DNA Affinity
†
Padraic S. Nagle, Fernando Rodriguez, Amila Kahvedzic, Susan J. Quinn,*,‡ and Isabel Rozas*
ꢀ ꢁ
School of Chemistry, University of Dublin, Trinity College, Dublin 2, Ireland. †Present address: Grupo de Sintesis Quimica de La Rioja,
Departamento de Quimica, Universidad de La Rioja, UA-CSIC, E-26006 Logrono, Spain. ‡Present address: School of Chemistry and
Biochemistry, University College Dublin, Ireland.
~
Received July 10, 2009
In this paper we report the synthesis of three families of new amidine-based aromatic derivatives as
potential DNA minor groove binding agents for the treatment of cancer. The preparation of
monoguanidine, mono-2-aminoimidazoline, and asymmetric diphenylguanidine/2-aminoimidazoline
derivatives (compounds 1a-c to 8a-c) is presented. The affinity of these substrates and of a family of
mono- and bis-isoureas (previously prepared in Rozas’ laboratory) for DNA was evaluated by means of
DNA thermal denaturation measurements. In particular, compounds 2c, 5c, 6c, 7c, and 8c were found to
bind strongly both to natural DNA and to adenine-thymine oligonucleotides, showing a preference for
the adenine-thymine base pair sequences.
Introduction
toxicity has prevented any therapeutic use. However, some
dicationic aromatic diamidines such as furamidine (Figure 1)7
have shown promising antiproliferative activity against dif-
ferent tumor cell lines. The furamidine derivative furimidazo-
line is one particularly good example.8 It is worth noting that
fluorescence microscopy indicates the rapid nuclear accumu-
lation of some furamidines in tumor cells.9 These findings
together open new possibilities for developing novel aromatic
dicationic derivatives with enhanced interaction and selecti-
vity within the DNA minor-groove and possibly with more
selective toxicity.
We have previously prepared a large number of symmetric
diaromatic bis-guanidinium and bis-2-aminoimidazolinium
derivatives, which are structurally related to furamidine, with
potential to treat sleeping sickness (Figure 1).10 These com-
pounds showed good affinity for DNA as indicated by the
increment of the melting temperature (ΔTm) values obtained
in thermal denaturation measurements. The results encour-
aged us to develop new ligands that might more efficiently
bind to DNA.
Hence, we have further developed this set by preparing
three new additional families (Figure 2), comprising a series of
diaromatic monoguanidinium and mono-2-aminoimidazoli-
niumderivatives (family I, compounds1g,h to8g,h), a seriesof
diaromatic mono- and bis-isouroniums (family II, com-
pounds 9i,j to 11i,j), and a series of asymmetric diaromatic
guanidinium/2-aminoimidazolinium dications (family III:,
compounds 1c to 8c). Consideration of the properties of each
of these families has allowed us to explore the requirements
for improved binding to the DNA minor-groove. All of the
compounds described here contain the basic furamidine scaf-
fold which has been modified by systematically changing both
the terminal cationic arms and the bridging furan moiety.
The cationic groups may bind to DNA in two ways, first
through direct H-bonds through the NH groups and second
through water-mediated interactions with the bases. These
The DNA minor-groove is the interaction site for many
replicative and repair enzymes and transcription control
proteins. It also provides an opportunity to design drugs that
are capable of selective binding and hence the potential to
interfere with these processes. Minor-groove binding agents
typically have long and planar structures that allow them to
adopt a crescent shape that fits into the minor-groove, form-
ing close contacts in the deep, narrow space formed between
the two DNA strands. These DNA/minor-groove binder
complexes are typically stabilized by hydrogen bonds
(HBsa), van der Waals contacts, and/or ionic interactions.1
Binding is typically driven by hydrophobic interactions, and if
the molecule is positively charged, this is usually accompanied
by the liberation of a large number of water molecules. The
positively charged natural products distamycin and netropsin
were among the first examples of molecules that specifically
target the minor-groove. Since their discovery, many drugs
that target the minor-groove have been developed. They are
highly selective, and they compromise the fundamental bio-
chemistry of a cell through inhibition of the actions of DNA-
dependent enzymes, for example, by direct inhibition of
transcription.2-4
Sophisticated synthesis has yielded oligoamides capable of
highly specific binding;5 further development has also wit-
nessed the emergence of hairpin polyamides to latch around
the groove.6 While many different minor-groove binders have
been synthesized and tested as anticancer agents, their severe
*To whom correspondence should be addressed. For S.J.Q.: phone,
+353 1 716 2407; fax, +353 1 716 1178; e-mail, susan.quinn@ucd.ie.
For I.R.: phone, þ353 1 896 3731; fax, þ353 1 671 2826; e-mail,
rozasi@tcd.ie.
a Abbreviations: HB, hydrogen bond; ΔTm, increment in DNA
denaturation temperature; AT, adenine-thymine pairs; MES, 2-(N-
morpholino)ethanesulfonic acid; P/D, ratio between base pairs and
ligand (drug).
r
2009 American Chemical Society
Published on Web 10/29/2009
pubs.acs.org/jmc

7114 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Nagle et al.
Table 1. Results Obtained from the Thermal Melting Experiments Showing the Change in Melting Temperature in the Presence of the Monoamidine-
like Cations (1g,h-6g,h, 7g, 8g,h, 9i-11i) to Salmon Sperm DNA (ΔTm(SS), °C)a
a Melting temperature in phosphate buffer (10 mM) is 69 °C.
Figure 1. Structure of furamidine (left) and general structure of the
related compounds previously reported by Rozas’ group10 that have
shown good affinity toward DNA.
Figure 2. Structures of the three families studied.
cations are larger than in the furamidine series, making this
central group smaller should ensure optimization of the
two mechanisms of binding mean that different interactions,
distance between the cationic moieties (guanidinium and 2-
depending on the nature of the cation, can exist. In this work
aminoimidazolinium) for DNA binding.
the amidinium cations have been exchanged with guanidi-
To assess the binding to DNA, we have performed thermal
nium, 2-aminoimidazolinium, and isouronium cations. In
denaturation experiments in natural DNA (salmon sperm,
these cases, we sought to enhance HB contacts in the minor
68% adenine-thymine base pair (AT) content). Further-
groove by increasing the number of HB donors (NH groups in
more, since binding to the minor groove requires the presence
the guanidinium and 2-aminoimidazolinium cations) or in-
of a run of five AT base pairs, binding experiments were also
troducing an additional HB acceptor (an O atom in the
conducted in the presence of double stranded AT homopoly-
isouronium cation). Furthermore, these are more articulated
mers [poly(dA dT)2 and poly(dA) poly(dT)] to assess their
3
3
cations than amidinium. The structure of the supporting
molecular scaffold or linker is very important if we wish to
capitalize on these multiple binding interactions. The opti-
mum linker will allow for a better orientation of the positive
charges within the groove and introduce the curvature or
geometry best suited to fitting. However, it is now known that
curvature is not essential to fit into the minor groove, since
Boykin et al. have shown that linear molecules are also
capable of strongly binding to the minor-groove.11 Work by
Nguyen et al. showed that in the linear molecule CPG-
40215A, the N,N0-diaminoguanidine bridging group served
as a “seesaw” hinge facilitating a variety of interactions with
the bases through motions that position the terminal amidine
cations in a range of binding modes.12
Moreover, to investigate the optimal linking scaffold, we
have exchanged the furan moiety of furamidine for a number
of smaller functional groups (O, S, NH, CO, CH2, CH2CH2,
or NHCONH). We hypothesized that such a substitution
shouldafford a 2-fold advantagebyfacilitating theinteraction
with DNA through HBs (as donors or acceptors), and as our
potential selectivity for the minor groove.
Results and Discussion
Chemistry. On the basis of our previous experience
and taking into account the good results obtained by our
group,10,13,14 the guanidinylation and 2-aminoimidazoliny-
lation of the present target molecules were performed follow-
ing our established methodology. This consists of the
reaction of a deactivated aromatic amine with N,N0-bis-
(tert-butoxycarbonyl)thiourea (Boc protected guanidine
precursor) or N,N0-di(tert-butoxycarbonyl)imidazoline-2-
thione13 (Boc protected 2-aminoimidazoline precursor) as-
sisted by mercury(II) chloride in the presence of an excess of
triethylamine. Deprotection of the Boc-intermediates of the
first stage and further treatment with Amberlyte resin in
water lead to the hydrochloride salts of the target mole-
cules.10,13-15 Preparation of the corresponding monoiso-
uronium (9i, 10i, 11i, Table 1) and bis-isouronium deriva-
tives (9j, 10j, 11j, Table 2) has been reported by our group in a

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7115
Table 2. Results Obtained in the Thermal Melting Experiments Showing the Change in Melting Temperature of DNA in the Presence of the Bis-
isouronium Derivatives (9j-11j) and Asymmetric Diphenyl Dicationic Compounds (1c-8c)
a DNA melting temperature in phosphate buffer (10 mM) is 69 °C. b P/D = 10. Poly(dA dT)2 melting temperature in phosphate buffer (10 mM) is
3
47 °C. Experiments were performed in TCD. c P/D = 3. Poly(dA) poly(dT) melting temperature in MES buffer (10 mM) is 43 °C. Experiments were
performed by W. D. Wilson et al. at Georgia State University, GA.
3
Scheme 1a
previous article16 using a similar synthetic approach to the
one just described starting from the corresponding aromatic
alcohols.
Synthesis of Asymmetric Guanidine/2-Aminoimidazoline
Derivatives (Family III). All our starting materials are sym-
metric, and hence, the two amino moieties have the same
reactivity. As we intend to introduce different groups at both
ends of the molecule, an excess of the diamines was required
to avoid difunctionalization. Thus, a total of 3 equiv of each
of the corresponding diamines was treated with just 1 equiv
of N,N0-bis(tert-butoxycarbonyl)thiourea, 1 equiv of mer-
cury(II) chloride, and an excess of triethylamine to afford the
desired monofunctionalized Boc-protected guanidines
(1a-8a) as main products with yields ranging from 29% to
82% (see Scheme 1). Under these conditions, less than 5% of
the difunctionalized products was observed.
In the second stage of the synthesis, 1 equiv of each of the
previous intermediates was reacted with 1 equiv of N,N0-
di(tert-butoxycarbonyl)imidazoline-2-thione, a slight excess
of mercury(II) chloride, and an excess of triethylamine to
afford the asymmetric Boc-protected derivatives (1b-8b) in
moderate to good yields. Standard deprotection of the Boc
groups with an excess of trifluoroacetic acid in dichloro-
methane followed by treatment with Amberlyte resin in
water led to the hydrochloride salts of the asymmetric
guanidine- and 2-aminoimidazoline containing products
(1c-8c).
a Reagents and conditions: (A) N,N0-bis(tert-butoxycarbonyl)thiour-
ea, HgCl2, TEA, DCM or DMF, room temp; (B) N,N0-di(tert-butoxy-
carbonyl)imidazoline-2-thione, HgCl2, TEA, DCM or DMF, room
temp; (C) (i) TFA, DCM, room temp; (ii) Amberlyte resin, H2O, room
temp.
Synthesis of Aminoguanidinium and Amino-2-aminoimida-
zolinium Derivatives (Family I). Regarding the synthesis ofthe
monoguanidine and mono-2-aminoimidazoline containing
derivatives, in the first step we decided to protect one of the
amino groups of the starting amines with Boc (see Scheme 2)
to increase the solubility of the intermediates and allow a
faster development of the column chromatography.
Following the same strategy as mentioned above, a total of
3 equiv of each of the starting diamines was treated with

7116 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Nagle et al.
Scheme 2a
a Reagents and conditions: (A) Boc2O, TEA, DCM or DMF, room temp; (B) N,N0-di(tert-butoxycarbonyl)imidazoline-2-thione, HgCl2, TEA, DCM
or DMF, room temp; (C) N,N0-bis(tert-butoxycarbonyl)thiourea, HgCl2, TEA, DCM or DMF, room temp; (D) (i) TFA, DCM, room temp;
(ii) Amberlyte resin, H2O, room temp; (E) 4-nitrobenzoisocyanate, DCM, 0 °C; (F) H2 (3 bar), Pd-C, MeOH.
1 equiv of Boc anhydride. The mono-Boc-protected amines
(1d-6d) were obtained as the main products in good yields.
The urea derivative 8d was prepared following a different
route (see Scheme 2). Thus, reaction of 4-nitrobenzoisocya-
nate with mono-Boc-protected 4-aminoaniline yielded the
corresponding Boc protected 4-[3-(4-nitrophenyl)ureido]-
aniline (12), which upon hydrogenation produced derivative
8d. However, the carbonyl group proved to be too deactivat-
ing and compound 7d (and subsequently 7e, 7f and 7h) could
not be prepared. All these compounds are new except for 1d17
and 3d,18 which have been already described.
In the second stage, the mono-Boc-protected amines were
treated with 1 equiv of N,N0-bis(tert-butoxycarbonyl)thiour-
ea or N,N0-di(tert-butoxycarbonyl)imidazoline-2-thione, a
slight excess of mercury(II) chloride, and triethylamine.
The guanidine precursors (1e-6e and 8e) were purified by
column chromatography in silica gel, whereas for the 2-
aminoimidazoline ones (1f-6f and 8f), neutral alumina
was used instead. Finally, deprotection of the Boc groups
afforded the final monoamidinium-like products after three
stages in moderate to good overall yields. It should be
mentioned that the aminoguanidine derivatives 1g-8g could
also be obtained after Boc deprotection of 1a-8a. In fact,
this was the only possible route to prepare compound 7g and
proved to be a shorter and more efficient methodology,
producing higher overall yields.
small molecules to DNA. These assays, for example, in
unspecific salmon sperm DNA, can be used as a general
screening for the suitability of the compounds studied as
DNA binders. Afterward, the preference for specific base
sequences can be explored.
Denaturation Studies of Families I-III in the Presence of
Natural DNA. The interaction of all the molecules with DNA
was first examined by performing thermal denaturation
experiments using a mixed sequence salmon sperm DNA
with a 32% GC content. The melting temperature of salmon
sperm DNA (Tm(SS)) was measured in the presence of the
different families of molecules.
The results found for some of the monoamidinium-like
derivatives (guanidinium, 2-aminoimidazolinium, and iso-
uronium derivatives from families I and II) are presented in
Table 1. The monoamidinium-like compounds tested were
found to have a slight stabilizing interaction with natural
DNA, as indicated by the moderate increase in melting
temperature, with ΔTm(SS) values of around 3 °C being
obtained.
Progressing from the monocationic species to the dicatio-
nic symmetric bis-isouronium derivatives (9j-11j) resulted
in moderately greater changes (Table 2). For these com-
pounds, the O linker (10j) was found to have the greatest
effect on the melting temperature, resulting in an increase of
6 °C in the Tm value.
All the diamines used as starting materials are commer-
cially available from Aldrich or Fluka except for the 1,4-(bis-
4-aminophenyl)piperazine, whose synthesis has been de-
scribed previously.19 It is also worth mentioning that the
unreacted starting diamines could be recovered from the
column eluting with a more polar solvent system.
More pronounced changes were observed when the mea-
surements were repeated for the asymmetric dicationic com-
pounds of family III (1c-8c). These compounds were found
to significantly increase the Tm by a range of 6 and 12 °C,
indicating an enhanced stabilization of the duplex structure
that is attributed to improved binding.
Biophysical Results: Thermal Denaturation Assays. Ther-
mal denaturation experiments are easy to perform and
provide quick and reliable information on the binding of
Comparison of the ΔTm(SS) values obtained for the sym-
metric bis-isouronium (9j-11j) and the asymmetric dicatio-
nic (1c, 3c, 4c) possessing the CH2, O, or S linker revealed

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7117
found to stabilize the homopolymer to the same extent as
natural DNA.
However, in the case of the ethylene linker (2c) the melting
temperature showed a 2-fold increment compared to salmon
sperm DNA. This would imply that while 2c binds to AT rich
domains, which are characterized by a narrow minor groove,
1c is unlikely to bind in the same fashion, as the increased
availability of binding sites in the AT homopolymer is not
accompanied by increased binding affinity. Thus, it may be
concluded that the restricted degree of rotation conferred by
the -CH2- bridge does not favor insertion into the narrower
minor groove present in the AT DNA and the molecule most
probably binds more effectively through an alternative
mode. However, the higher degree of rotation due to the
extra CH2 in 2c allows the molecule to wrap around the
minor groove better than 1c.
Figure 3. Graph showing the ΔTm for 1c (ΔTm = 6 °C, red) and 5c
(ΔTm = 12 °C, green) in salmon sperm DNA (blue).
Next, we considered the influence of the other bridging
groups in the selective binding between the different DNA
systems. By comparison of molecules 1c, 3c, 4c, and 5c
(linkers -CH2-, -O-, -S-, and -NH-, respectively), it
was noted that compound 5c had the highest melting tem-
very little difference in the melting temperature between the
compounds with different cations.
When we compare the melting results of the molecules
with the methylene (1c; see thermal melting experiment
results in Figure 3) and ethylene (2c) bridges in the presence
of natural DNA, we observe that the ethylene linker gave a
slightly better melting result, which is indicative of improved
binding. A possible reason for this is that the extra CH2
increases hydrophobicity, thereby improving the hydropho-
bic interactions. Another possible conclusion for this is that
the more flexible ethylene linker provides another point of
contact with the DNA, allowing for a better binding.
A comparable change was also found in the case of the
bulky piperazine linker (6c), which yielded intermediate
stabilization of DNA. This is interesting given the linear
and bulky nature of the piperazine linker, and thus, the
combination of both features could play a role in its entry
and fitting into the minor groove. The greatest change was
observed for the NH (5c; see thermal melting experiment
results in Figure 3) and NHCONH (8c) species (∼12 °C).
Interestingly, both of these compounds are capable of strong
hydrogen bonding interactions. The strong binding of com-
pound 8c to salmon sperm DNA (Table 2) could possibly be
explained because it has a urea linker that could provide
enough freedom of rotation, allowing a good induced fit
inside the DNA minor groove. However, it is noted that the
changes do not seem to reflect the difference in the length of
the linker in the cases of 5c and 8c.
perature in both salmon sperm DNA and poly(dA dT)2.
3
This was followed by molecules 3c and 4c that have the same
melting temperature in salmon sperm DNA but slightly
different in poly(dA dT)2 DNA (by 1 °C). As noted pre-
3
viously, the smallest change was observed for compound 1c.
These results seem to indicate the importance of hydrogen
bonding with a possible HB donation to the O2 of thymine
being a potential dominant factor. The fact that 3c and 4c
gave better melting results in comparison to 1c illustrates
further the order of binding, since the oxygen and sulfur
linkers could be HB acceptors, but the methylene bridge is a
known weak HB donor.
However, if we consider the relative sensitivity of the
melting temperature (ΔTrel) defined to the DNA type
(ΔTm(AT) - ΔTm(SS); see Table 2), two distinct groupings
emerge. The first group contains the compounds 2c
(-CH2CH2-), 5c (-NH-), 6c (-piperazine-), and 7c
(-CO-) and shows a ΔTrel change of about 6 °C. The
second grouping shows significantly less sensitivity to the
two forms of DNA, 1c (-CH2-), 8c (-NHCONH-), 4c
(-S-), and 3c (-O-), with the ether linker showing the
greatest difference of 3 °C.
When the results for molecules 1c, 5c, and 7c (linkers
-CH2-, -NH-, and -CO-, respectively) are compared,
the primary difference is the HB characteristics of the
corresponding linkers, with the methylene bridge being a
weak HB donor, the -NH- a stronger HB donor, and the
carbonyl group a HB acceptor. The order of binding to DNA
and AT oligonucleotides, from best to worst, was found to be
as follows: strong HB donor (NH) > HB acceptor (CO) >
weak HB donor (CH2). This indicates that, in the synthesis of
future molecules, incorporating more HB donors would be
advantageous. The geometry around the bridging linker
must also be considered, as it is likely to play an important
role. Here, there are two important factors. First, the steric
nature of the geometry (tetrahedral -CH2- vs trigonal
planar -CO- and trigonal pyramidal -NH-) will influence
the approach to and binding in the pocket. Second, the
rotational freedom conferred by the bridging moiety would
be expected to influence the binding. There is no freedom of
rotation in 7c (trigonal planar), whereas there is freedom of
rotation around the linker for molecules 1c (tetrahedral
linker) and 5c (trigonal pyramidal linker). It is interesting
Denaturation Studies of Family III in the Presence of
Poly(dA dT)2 and Poly(dA) Poly(dT). The experiments
3
3
carried out on the asymmetric series in the presence
of salmon sperm DNA revealed that, considering the
linkers, the order of affinity is -NH- ∼ -NHCONH- >
-piperazine-, -CO-, -CH2CH2-, -O-, -S-, -CH2-.
In light of the promising results found for family III, the
experiments were repeated in the presence of poly(dA dT)2
3
using similar experimental conditions. Here, the absence of
the G C base pair reduces the width of the groove, allowing
3
for a tighter fit, with minor groove complexes typically
forming with a run of four A T base pairs. In almost all
3
experiments, a greater change in melting temperature was
found in the presence of the asymmetric dications (2c-8c) in
the AT oligonucleotides. This result was taken as a strong
indication of a preference for the minor groove binding site
afforded in homopolymer DNA and indicates sequence
selectivity for the AT domains of DNA (Table 2). The
exception was found for the CH2 species (1c) which was

7118 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Nagle et al.
to note that compound 6c shows a medium affinity not only
toward natural DNA but also to poly(dA dT)2. This linker is
3
longer than the rest of the groups considered, and the
relatively good results obtained could be justified by the
large size and linear characteristics of the piperazine ring.
The ΔTm of netropsin (a well-known minor groove binder)
was also measured in this homopolymer for the sake of
comparison. Even though this compound shows a 2-fold
binding strength compared to the asymmetric dications, this
can be explained by the large number of HB donor and
acceptor groups present in this molecule.
AT sequence selectivity between different five-base-pair
binding sites has previously been reported for the different
binding modes (1:1 versus 2:1) of the classic minor groove
binder distamycin.20 In the study by Chen et al., a prefer-
ence for the ATATA over AAAAA was observed. In
addition, small molecules can bind in a 2:1 and a 1:1 fashion
in the minor-groove, and this seems to strongly depend on
the sequence. It has been found by Vesnaver et al.21 that the
narrowest groove is that of five base pairs of AT and
netropsin prefers 1:1 binding in such a system. For this
reason, we also considered the interesting results already
published by us in collaboration with W. D. Wilson
(Georgia State University, GA) on the thermal denatura-
tion of the symmetric bis-guanidines and bis-2-aminoimi-
dazolines8 and pursued further collaboration with this
group using those reported conditions with the asymmetric
Figure 4. Correlation found between the ΔTm poly(dA dT)2 vs
3
ΔTm poly(dA) poly(dT).
3
derivatives have been prepared following the synthetic path-
ways previously described by us.10,13,14 Second, regarding
family II, the preparation of the asymmetric guanidinium/
2-aminoimidazolinium dications hasbeenperformedfrom the
corresponding monoguanidinium derivatives and subsequent
introduction of the 2-aminoimidazoline group. Third, regard-
ing family III, the preparation of the mono- and bis-isou-
ronium derivatives has been reported in a previous paper from
our group.16
The ability of these compounds to bind to DNA has been
evaluated by thermal denaturation experiments not only with
mixed sequence DNA (salmon sperm), which comprises all
four bases, but also with poly(dA dT)2 and poly(dA) poly-
3
3
dications and the poly(dA) poly(dT) oligonucleotide
3
(dT) oligonucleotides. Despite showing cationic groups at
both ends of the diaromatic moieties, monoamidinium-like
derivatives (ammonium and guanidinium or 2-aminoimida-
zolinium) and bis-isouroniums (two isouronium cations) have
been shown to poorly/moderately bind to DNA. Neverthe-
less, in general, asymmetric bis-amidinium-based derivatives
displayed a good affinity toward DNA and a preference for
the AT sequences. In particular, compounds 2c, 5c, 6c, 7c, and
8c strongly bind to both unspecific and AT oligonucleotides.
Taking into consideration these promising results, we
believe that these asymmetric compounds deserve more in-
vestigation as DNA targeting agents, and thus, more biophy-
sical studies are ongoing and will be reported soon.
(Table 2). It was felt that these experiments would help to
confirm the selectivity of the asymmetric dications toward
the AT sequences.
The most significant differences in conditions were, on the
one hand, the higher loading of the drug to DNA, with a
ratio between base pairs and ligand (P/D) of 3 rather than
that of 10 used above and, on the other hand, the buffer used,
10 mM 2-(N-morpholino)ethanesulfonic acid (MES), as
opposed to 10 mM phosphate buffer. Because of these
differences, the results obtained cannot be quantitatively
compared to the poly(dA dT)2 results. However, the trend
3
observed is similar and a good correlation was found be-
tween both sets of results, as shown in Figure 4. By compar-
ison of the results previously published with Wilson’s group
on the thermal denaturation of the symmetric bis-guanidines
and bis-2-aminoimidazolines10 and those found now using
the same Wilson’s conditions (Table 2), it is interesting to
observe how the ΔTm values obtained for the asymmetric
compounds are almost an average of those measured for the
bis-guanidines (smaller) and those obtained for the bis-2-
aminoimidazolines (larger). This would imply a similar
binding mode in both AT DNAs and most probably linked
to a 1:1 mode, as would be expected given the bis-cationic
nature of the compounds under study.
Experimental Section
Chemistry. All the commercial chemicals were obtained from
Sigma-Aldrich or Fluka and were used without further purifica-
tion. Deuterated solvents for NMR use were purchased from
Apollo. Dry solvents were prepared using standard procedures,
according to Vogel, with distillation prior to use. Chromato-
graphic columns were run using silica gel 60 (230-400 mesh
ASTM) or aluminum oxide (activated, Neutral Brockman I
STD grade 150 mesh). Solvents for synthesis purposes were used
at GPR grade. Analytical TLC was performed using Merck
Kieselgel 60 F254 silica gel plates or Polygram Alox N/UV254
aluminum oxide plates. Visualization was by UV light (254 nm).
NMR spectra were recorded in a Bruker DPX-400 Avance
spectrometer, operating at 400.13 and 600.1 MHz for 1H
NMR and 100.6 and 150.9 MHz for 13C NMR. Shifts are
referenced to the internal solvent signals. NMR data were
processed using Bruker Win-NMR 5.0 software. Electrospray
mass spectra were recorded on a Mass Lynx NT V 3.4 on a
Waters 600 controller connected to a 996 photodiode array
detector with methanol, water, or ethanol as carrier solvents.
Melting points were determined using an Electrothermal
IA9000 digital melting point apparatus and are uncorrected.
Infrared spectra were recorded on a Mattson Genesis II
FTIR spectrometer equipped with a Gateway 2000 4DX2-66
From these studies, it can be concluded that hydrophobic
interactions, hydrogen bonding, electrostatic interactions,
and molecular conformation are all important factors in
DNA minor groove binding for the asymmetric dications.
More importantly, these show strong binding to DNA
showing AT-sequence specificity.
Conclusions
We have prepared three different families of mono- and bis-
amidinium-based derivatives in order to explore their poten-
tial as DNA minor-groove binders. First, with respect to family I,
the monoguanidinium and mono-2-aminoimidazolinium

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7119
workstation and on a Perkin-Elmer Spectrum One FT-IR
spectrometer equipped with Universal ATR sampling acces-
sory. Elemental analyses (C, H, N) of the target compounds,
which were performed at the Microanalysis Laboratory (School
of Chemistry and Chemical Biology, University College Dublin,
Ireland), are within (0.4% of the calculated values, confirming
g95% purity. Fractional moles of water frequently found in
amidinium-like salts could not be prevented despite 24-48 h of
drying in vacuum.
General Method for the Synthesis of Monofunctionalized Boc-
Protected Guanidines: Method 1 [Scheme 1, Conditions A]. Over
a solution containing an excess (9.0 mmol) of each of the starting
diamines, 3.0 mmol of N,N0-di(tert-butoxycarbonyl)thiourea,
and 1.3 mL (9.3 mmol) of TEA in DCM (5 mL) at 0 °C, a total of
3.3 mmol of HgCl2 was added. The resulting mixture was stirred
at 0 °C for 1 h and for the appropriate duration at room
temperature. Then the mixture was diluted with EtOAc and
filtered through a pad of Celite. The filter cake was rinsed with
EtOAc. The organic phase was washed with water (2 ꢀ 30 mL),
washed with brine (1 ꢀ 30 mL), dried over anhydrous Na2SO4,
and concentrated under vacuum to give a residue that was
purified by silica gel column chromatography, eluting with the
corresponding hexane/EtOAc mixture to yield the desired
compound as a solid. Less than 5% of the disubstituted sub-
strate was observed.
General Procedure for the Synthesis of Boc-Protected Asym-
metric Guanidine/2-Aminoimidazoline, Boc-Protected Amino/2-
Aminoimidazolidine, and Boc-Protected Amino/Guanidine Deri-
vatives: Method 2 [Scheme 1, Conditions B; Scheme 2, Conditions
B and C]. Each of the corresponding amines was treated in DCM
at 0 °C with 1.1 equiv of mercury(II) chloride, 1.0 equiv of N,N0-
di(tert-butoxycarbonyl)imidazolidine-2-thione (for the 2-ami-
noimidazoline precursors) or N,N0-di(tert-butoxycarbonyl)thi-
ourea (for the guanidine precursors) and 3.1 equiv of TEA. The
resulting mixture was stirred at 0 °C for 1 h and for the
appropriate duration at room temperature. Then the reaction
mixture was diluted with EtOAc and filtered through a pad of
Celite to get rid of the mercury sulfide formed. The filter cake
was rinsed with EtOAc. The organic phase was washed with
water (2 ꢀ 30 mL), washed with brine (1 ꢀ 30 mL), dried over
anhydrous Na2SO4, and concentrated under vacuum to give a
residue that was purified by silica gel (guanidine precursors) or
neutral alumina column flash chromatography (2-aminoimida-
zoline precursors), eluting with the appropriate hexane/EtOAc
mixture.
General Procedure for the Synthesis of Mono-Boc-Protected
4,40-Diphenyldiamines: Method 3. Compounds 1d-6d [Scheme 2,
Conditions A]. Over a solution containing an excess (30.0 mmol)
of each of the corresponding diamines in DCM (200 mL),
10.0 mmol of TEA and 10.0 mmol of di-tert-butyl dicarbonate
(Boc2O) were added at 0 °C. The resulting mixture was stirred at
0 °C for 1 and 16 h more at room temperature. Then the mixture
was concentrated under vacuum to give a residue that was
purified by silica gel column chromatography, eluting with the
appropriate hexane/EtOAc mixture. The mono-Boc-protected
compound was obtained as a solid. The excess of unreacted
starting material diamine was recovered from the column. Less
than 10% formation of the di-Boc-protected compound was
observed.
Dihydrochloride Salt of N-{4-[4-(imidazolidin-2-ylideneami-
no)benzyl]phenyl}guanidine (1c): Method 4. White solid (96%);
mp 78-80 °C; 1H NMR (D2O) δ 3.58 (s, 4H, CH2), 3.76 (s, 2H,
PhCH2Ph), 6.98 (d, 2H, J = 8.0 Hz, Ar.), 7.01 (d, 2H, J =
8.0 Hz, Ar.), 7.15 (d, 2H, J = 8.0 Hz, Ar.), 7.16 (d, 2H, J =
8.0 Hz, Ar.); 13C NMR (D2O) δ 39.6 (PhCH2Ph), 42.2 (CH2 Im),
123.3, 125.1, 129.6, 129.7, 131.5, 132.6, 139.7, 140.4 (Ar.), 155.5,
157.6 (CN); HRMS (ESIþ) m/z 309.1728 calcd [M þ H]þ; found
309.1732. Anal. (C17H22Cl2N6 1.5H2O) C, H, N.
3
Dihydrochloride Salt of N-(4-{2-[4-(Imidazolidin-2-ylidenea-
mino)phenyl]ethyl}phenyl)guanidine (2c): Method 4. White solid
(94%); mp, decomposes over 215 °C; 1H NMR (D2O) δ 2.84 (s,
4H, CH2), 3.67 (s, 4H, CH2 Im), 7.03-7.13 (m, 4H, Ar.),
7.15-7.25 (m, 4H, Ar.); 13C NMR (D2O) δ 35.4, 35.5 (2CH2),
42.2 (CH2 Im), 123.3, 125.1, 129.4, 129.5, 131.3, 132.3, 140.3,
140.9 (Ar.), 155.6, 157.8 (CN); HRMS (ESIþ) m/z 323.1984
calcd [M þ H]þ; found 323.1988. Anal. (C18H24Cl2N6 0.8H2O)
3
C, H, N.
Dihydrochloride Salt of N-{4-[4-(Imidazolidin-2-ylideneami-
no)phenoxy]phenyl}guanidine (3c): Method 4. Yellowish solid
(94%); mp 48-50 °C; 1H NMR (D2O) δ 3.70 (s, 4H, CH2 Im),
6.97-7.06 (m, 4H, Ar.), 7.17-7.26 (m, 4H, Ar.); 13C NMR
(D2O) δ 42.3 (CH2), 119.5, 119.6, 125.8, 127.4, 129.0, 130.1,
154.8, 155.4 (Ar.), 155.8, 158.1 (CN); HRMS (ESIþ) m/z
311.1594 calcd [M þ H]þ; found 311.1595. Anal. (C16H20Cl2-
N6O 2.8H2O) C, H, N.
3
Dihydrochloride Salt of N-{4-[4-(Imidazolidin-2-ylideneami-
no)phenylsulfanyl]phenyl}guanidine (4c): Method 4. Yellowish
solid (95%); mp 94-96 °C; 1H NMR (D2O) δ 3.67 (s, 4H, CH2),
7.05-7.12 (m, 4H, Ar.), 7.19-7.27 (m, 4H, Ar.); 13C NMR
(D2O) δ 42.2 (CH2), 123.7, 125.5, 131.5, 132.0, 132.4, 132.9,
133.6, 134.1 (Ar.), 155.3, 157.4 (CN); HRMS (ESIþ) m/z
327.1293 calcd [M þ H]þ; found 327.1295. Anal. (C16H20Cl2-
N6S 1.8H2O) C, H, N.
3
Dihydrochloride Salt of N-{4-[4-(Imidazolidin-2-ylideneami-
no)phenylamino]phenyl}guanidine (5c): Method 4. Green solid
1
(94%); mp 66-68 °C; H NMR (D2O) δ 3.60 (s, 4H, CH2),
6.95-7.09 (m, 8H, Ar.); 13C NMR (D2O) δ 42.2 (CH2), 117.8,
118.0, 125.2, 126.0, 126.9, 127.3, 141.3, 142.0 (Ar.), 155.9, 158.1
(CN); HRMS (ESIþ) m/z 310.1722 calcd [M þ H]þ; found
310.1724. Anal. (C16H21Cl2N7 1.7H2O) C, H, N.
3
Tetrahydrochloride Salt of N-(4-{4-[4-(Imidazolidin-2-ylide-
neamino)phenyl]piperazin-1-yl}phenyl)guanidine (6c): Method
1
4. Brownish solid (93%); mp 168-170 °C; H NMR (D2O) δ
3.62 (s, 4H, CH2 Im), 3.71 (s, 8H, CH2 Pip), 7.23-7.31 (m, 4H,
Ar.), 7.37 (d, 2H, J = 9.0 Hz, Ar.), 7.41 (d, 2H, J = 8.5 Hz, Ar.);
13C NMR (D2O) δ 42.3 (CH2 Im), 50.2, 50.9 (CH2 Pip), 120.0,
120.3, 125.0, 126.6, 131.4, 133.0, 141.9, 143.1 (Ar.), 155.6, 157.8
(CN); HRMS (ESIþ) m/z 379.2359 calcd [M þ H]þ; found
379.2362. Anal. (C20H30Cl4N8 1.0H2O) C, H, N.
3
Dihydrochloride Salt of N-{4-[4-(Imidazolidin-2-ylideneami-
no)benzoyl]phenyl}guanidine (7c): Method 4. White solid
1
(94%); mp 99-101 °C; H NMR (D2O) δ 3.80 (s, 4H, CH2),
7.38 (d, 2H, J = 8.5 Hz, Ar.), 7.42 (d, 2H, J = 8.5 Hz, Ar.),
7.78-7.84 (m, 4H, Ar.); 13C NMR (D2O) δ 42.3 (CH2), 121.8,
123.5, 131.5, 131.6, 133.7, 134.2, 138.9, 139.5 (Ar.), 155.3, 157.5
(CN), 197.5 (CO); HRMS (ESIþ) m/z 323.1620 calcd [M þ H]þ;
found 323.1619. Anal. (C17H20Cl2N6O 2.0H2O) C, H, N.
3
Dihydrochloride Salt of 1-(4-Guanidinophenyl)-3-[4-(imida-
zolidin-2-ylideneamino)phenyl]urea (8c): Method 4. White solid
(94%); mp, decomposes over 200 °C; 1H NMR (D2O) δ 3.57 (s,
4H, CH2), 6.98 (d, 2H, J = 8.5 Hz, Ar.), 7.03 (d, 2H, J = 8.5 Hz,
Ar.), 7.16 (d, 2H, J = 8.5 Hz, Ar.), 7.20 (d, 2H, J = 8.5 Hz, Ar.);
13C NMR (D2O) δ 42.1 (CH2), 120.0, 120.1, 123.4, 125.4, 128.3,
129.3, 136.1, 136.8 (Ar.), 153.4, 155.4, 157.3 (CO, CN); HRMS
(ESIþ) m/z 353.1838 calcd [M þ H]þ; found 353.1832. Anal.
General Procedure for the Synthesis of the Hydrochloride
Salts: Method 4. Each of the corresponding Boc-protected
precursors (0.5 mmol) was treated with 15 mL of a 50% solution
of trifluoroacetic acid in DCM for 3 h. After that time, the
solvent was eliminated under vacuum to generate the trifluoro-
acetate salt. This salt was dissolved in 20 mL of water and
treated for 24 h with IRA400 Amberlyte resin in its Cl- form.
Then the resin was removed by filtration and the aqueous
solution washed with DCM (2 ꢀ 10 mL). Evaporation of the
water afforded the pure hydrochloride salt. Absence of the
trifluoroacetate salt was checked by 19F NMR.
(C17H22Cl2N8O 2.0H2O) C, H, N.
3
DNA Binding Assays. DNA Salmon Sperm and Poly(dA dT)2
Experiments. Thermal melting experiments were conducted with
a Varian Cary 300 Bio spectrophotometer equipped with a 6 ꢀ 6
3

7120 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Nagle et al.
multicell temperature-controlled block. Temperature was mon-
itored with a thermistor inserted into a 1 mL quartz cuvette
containing the same volume of water as in the sample cells.
Absorbance changes at 260 nm were monitored from a range of
20 to 90 °C with a heating rate of 1 °C/min and a data collection
rate of five points per °C. The salmon sperm DNA was
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purchased from Sigma Aldrich (extinction coefficient ε260
=
6600 cm-1 M-1 base). A quartz cell with a 1 cm path length was
filled with a 1 mL solution of DNA polymer or DNA-
compound complex. The DNA polymer (150 μM base) and
the compound solution (15 μM) were prepared in a phosphate
buffer [0.01 M Na2HPO4/NaH2PO4], adjusted to pH 7) so that a
compound to DNA base ratio of 0.1 was obtained. The thermal
melting temperatures of the duplex or duplex-compound com-
plex obtained from the first derivative of the melting curves are
reported.
Poly(dA) Poly(dT) Experiments. Thermal melting experi-
3
ments were conducted with a Cary 300 Bio UV-visible spectro-
photometer (Varian, Inc.) in 1 cm quartz cells. The solutions
were prepared in MES buffer containing 10 mM 2-(N-morpho-
lino)ethanesulfonic acid, 0.1 M NaCl, 1 mM EDTA, pH 6.25.
The DNA polymer poly(dA) poly(dT) was purchased from
3
Amersham Biosciences (NJ) and used with an extinction coeffi-
cient ε260 = 6000 cm-1 M-1 base, room temperature, MES
buffer. The samples were mixed with ratio of one compound per
two base pairs and scanned up and down a temperature range of
25-95 °C with a heating/cooling rate of 0.5 °C/min. Tempera-
ture was recorded with a temperature probe inserted into a
cuvette filled with water. The procedure was repeated twice to
generate four thermal melting curves for each sample. One
strong transition was observed for every curve. The data were
processed with a software package included with the instrument.
Acknowledgment. We truly thank Dr. W. David Wilson
and Dr. Binh Nguyen at Georgia State University, GA, for
providing us with DNA thermal denaturation experiments for
our compounds. We express our gratitude to Prof. John M.
Kelly for his comments and advice. This research was sup-
ported by Science Foundation Ireland (Grant SFI-CHE275).
P.S.N. and A.K. thank SFI for generous funding. F.R. thanks
the Consejeria de Educacion Cultura y Deporte de la Comu-
nidad Autonoma de La Rioja for his grant.
Supporting Information Available: Preparation details and 1H
NMR, 13C NMR, and MS data of all new Boc-protected
derivatives (1a-8a, 1b-8b, 1d-6d, 8d, 1e-6e, 8e, 1f-6f, 8f,
12) and all monoguanidines and mono-2-aminoimidazolines
(1g-8g, 1h-6h, 8h); a table containing the combustion analysis
data of the new target compounds; 1H and 13C NMR spectra for
the final compounds (1c-8c). This material is available free of
charge via the Internet at http://pubs.acs.org.
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