9-Amino acridines undergo reversible amine exchange reactions in water: Implications on their mechanism of action in vivo

Article abstract of DOI:10.1021/ol9019925

9-Amino substituted acridities undergo a reversible amine exchange reaction In water under near-physiological conditions via an unstable hemiaminal intermediate. This thermodynamically controlled reaction may have Implications In understanding the mode of

Full text of DOI:10.1021/ol9019925

ORGANIC
LETTERS
2009
Vol. 11, No. 21
4894-4897
9-Amino Acridines Undergo Reversible
Amine Exchange Reactions in Water:
Implications on Their Mechanism of
Action in Vivo
Alexis Paul and Sylvain Ladame*
ISIS UniVersite´ de Strasbourg, 8 alle´e Gaspard Monge, BP 70028, 67083 Strasbourg, France
s.ladame@isis.u-strasbg.fr
Received August 27, 2009
ABSTRACT
9-Amino substituted acridines undergo a reversible amine exchange reaction in water under near-physiological conditions via an unstable
hemiaminal intermediate. This thermodynamically controlled reaction may have implications in understanding the mode of action of
9-aminoacridines in vivo and in the future design of drugs based on this scaffold.
Known since the 19th century, acridines have been exten-
sively used in medical sciences for their therapeutic proper-
ties. These pharmacophores were first developed for their
antibacterial,1 trypanocidal,2 or antimalarial3 activities. Since
the 70s, acridines have also been identified as highly potent
antitumor agents.4 The anticancer activity of acridines relies
essentially on their capacity to interact with nucleic acids,
either (i) via intercalation between double-stranded DNA
base pairs and inhibition of a DNA topoisomerase II enzyme
(e.g., AMSA5) or (ii) via stabilization of alternative four-
stranded DNA structures called G-quadruplexes (e.g., BRACO-
196). Interestingly, a large majority of acridines showing
anticancer properties have an amino substituent at position
C9, the nature of which proved critical for biological activity
(Figure 1).7,8 Because of their high DNA binding affinity,
acridines have also been used as vectors to direct unspecific
drugs (e.g., cis-platinum, aziridin) to specific DNA sites.9
Once again, those alkylating agents were almost exclusively
introduced on the same C9 position.
(5) (a) Rene, B.; Fosse, P.; Khelifa, T.; Jacquemin-Sablon, A.; Bailly,
C. Mol. Pharmacol. 1996, 49, 343. (b) Denny, W. A. Anti-Cancer Drug
Des. 1989, 4, 241.
(6) Campbell, N. H.; Parkinson, G. N.; Reszka, A. P.; Neidle, S. J. Am.
Chem. Soc. 2008, 130, 6722.
(1) Wainwright, M. J. Antimicrob. Chemother. 2001, 47, 1.
(2) Gamage, S. A.; Figgitt, D. P.; Wojcik, S. J.; Ralph, R. K.; Ransijn,
A.; Mauel, J.; Yardley, V.; Snowdon, D.; Croft, S. L.; Denny, W. A. J. Med.
Chem. 1997, 40, 2634.
(7) Moore, M. J.; Schultes, C. M.; Cuesta, J.; Cuenca, F.; Gunaratnam,
M.; Tanious, F. A.; Wilson, W. D.; Neidle, S. J. Med. Chem. 2006, 49,
582
.
(3) Chauhan, P. M.; Srivastava, S. K. Curr. Med. Chem. 2001, 8, 1535.
(4) For a recent reviews, see: Belmont, P.; Bosson, J.; Godet, T.; Tiano,
M. Anti-Cancer Agents Med. Chem. 2007, 7, 139.
(8) Acridines lacking a 9-amino substituent (e.g., DACA) have also been
reported as potent anticancer drugs. For example, see: Bridewell, D. J.;
Finlay, G. J.; Baguley, B. C. Anti-Cancer Drug Des. 2001, 16, 317
.
10.1021/ol9019925 CCC: $40.75
Published on Web 10/01/2009
 2009 American Chemical Society

As a model system, a series of water-soluble 9-anili-
noacridines was synthesized from commercially available
anthranilic acid and 2-bromobenzoic acid, via a 4-carboxylic
acid acridone intermediate.13 Reaction of the acridone with
thionyl chloride followed by a mild and selective hydrolysis
of the acyl chloride intermediate led to the formation of
9-chloro-4-carboxylic acid acridine. Reaction with an ap-
propriate aromatic amine (p-aminobenzoic acid, aniline,
p-sulfanilic acid, or 2,3-xylidine) finally afforded the desired
9-anilinoacridines of interest.14 All four 9-aminoacridines
proved stable in aqueous potassium phosphate buffer (100
mM, pH 7.4). Their reactivity with respect to other aromatic
amines was then investigated (Scheme 1). In a typical
experiment, a solution of acridine (50 µM) in potassium
phosphate buffer (100 mM, pH 7.4) was reacted with a
mixture of four aromatic amines (15 mM each, 300 equiv),
and the reaction was monitored by LC-MS.
Figure 1. Structures of the two anticancer agents m-AMSA and
BRACO-19.
The anticancer activity of 9-aminoacridines is clearly
established in vitro, and selected molecules have also been
successfully tested in vivo.10 However, although rarely
commented upon, the relative instability of this class of
molecules is also well established.11 While 9-alkylami-
noacridines can readily hydrolyze at alkaline pH to generate
the corresponding acridone, it was also recently shown that
9-aminoacridines can undergo partial amine exchange reac-
tion in organic solutions.12
When either 9-anilino-4-carboxyacridine (A3) or 9-sulfa-
nylo-4-carboxyacridine (A1) were reacted with a mixture of
aniline, p-aminobenzoic acid, p-sulfanilic acid, and 2,3-
xylidine, the slow formation of all four 9-aminoacridines was
observed until equilibrium was reached after 6 days (Figure
2). Although all four possible 9-aminoacridines were ob-
Scheme 1. General Principle for Reversible Synthesis of Four
9-Aminoacridines, A1-A4, from 9-Anilino and 9-Sulfanylo
Acridine
Figure 2. HPLC traces of an equilibrating mixture of 50 µM
9-anilinoacridine A3 (left) or 9-sulfaniloacridine A1 (right) in
potassium phosphate buffer (100 mM, pH 7.4) containing a mixture
of p-aminobenzoic acid, aniline, p-sulfanilic acid, and 2,3-xylidine
(15 mM each). All mixtures were analyzed by LC-MS at t ) 0
(black), 3 days (red), and 6 days (blue).
Herein, we report that water-soluble 9-aminoacridines can
undergo a reversible and thermodynamically controlled
amine exchange reaction under near-physiological aqueous
conditions, which has potential implications for the under-
standing of the possible mode of action of 9-aminoacridines
in vivo and on the future design of acridine-based therapeutic
agents.
served in both reactions, they were not present at equilibrium
in identical proportions: A3 > A2 ≈ A4 > A1 (Figure 2).
However, the same distribution was obtained when starting
from either the 9-anilino (A3) or the 9-sulfanilic acid (A1)
acridine, thus demonstrating that this amine exchange
reaction is reversible and under thermodynamic control.
Because of the possibility for 9-aminoacridines to coexist
in solution with their tautomeric imino form, we propose
that the amine exchange reaction proceeds reversibly via
formation of a 9,9-diaminoacridine hemiaminal intermediate
(Scheme 2). This mechanism is in agreement with that
proposed by Bierbach et al. to explain the formation of
unusual 9-spirocyclic acridines.15 However, formation of this
unstable intermediate can proceed either via a SNAr-like
mechanism or via a transimination reaction depending on
whether the reactive species is the amino or the imino
acridine, respectively.
(9) (a) Sundquist, W. I.; Bancroft, D. P.; Lippard, S. J. J. Am. Chem.
Soc. 1990, 112, 1590. (b) Su, T. L.; Lin, Y. W.; Chou, T. C.; Zhang, X.;
Bacherikov, V. A.; Chen, C. H.; Liu, L. F.; Tsai, T. J. J. Med. Chem. 2006,
49, 3710. (c) Ma, Z.; Rao, L.; Bierbach, U. J. Med. Chem. 2009, 52, 3424.
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M. J.; Double, J. A.; Neidle, S. Cancer Res. 2005, 65, 1489. (b) Oppegard,
L. M.; Ougolkov, A. V.; Luchini, D. N.; Schoon, R. A.; Goodell, J. R.;
Kaur, H.; Billadeau, D. D.; Ferguson, D. M.; Hiasa, H. Eur. J. Pharmacol.
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(11) (a) Su, T. L.; Chou, T. C.; Kim, J. Y.; Huang, J. T.; Ciszewska,
G.; Ren, W. Y.; Otter, G. M.; Sirotnak, F. M.; Watanabe, K. A. J. Med.
Chem. 1995, 38, 3226. (b) Goodell, J. R.; Svensson, B.; Ferguson, D. M.
J. Chem. Inf. Model. 2006, 46, 876.
(12) Sebest´ık, J.; Safar´ık, M.; Stibor, I.; Hlava´cek, J. Biopolymers 2006,
84, 605.
Org. Lett., Vol. 11, No. 21, 2009
4895

ethylene diamine was present at equilibrium. Although
9-anilinoacridine A3 formed faster (Figure 3, t ) 3 days),
the system subsequently re-equilibrated toward the formation
of thermodynamically more stable 9-benzylaminoacridine A6.
As already observed with the mixture of aromatic amines
(Figure 2), the correlation between the relative concentration
of each aminoacridine at equilibrium and the pKa value of
the corresponding protonated amine is very poor. Our results
show that as steric interactions around the C9 position of
acridine increase, the C9-N bond weakens, thus facilitating
the amine exchange reaction (e.g., benzylamine vs aniline
Figure 3). For compounds of comparable steric hindrance,
electronic delocalization between the acridine platform and
the 9-amino substituent becomes the main stabilizing force
of the C9-N bond. The unexpectedly low stability of
acridine A5 (bearing a N,N-dimethyl-ethylene diamine sub-
stituent) can be explained by the formation of a five-
membered intramolecular ring resulting in increased steric
hindrance, as previously proposed by Ferguson et al.11b
(Figure 4).
Scheme 2. Proposed Mechanism for the Reversible Amine
Exchange Reaction on 9-Aminoacridines
To the previous mixture of aromatic amines were added
two primary aliphatic amines: benzylamine and N,N-dime-
thyl-ethylene diamine. At physiological pH, N,N-dimethyl-
ethylene diamine (pKa ) 6.6) is present in solution almost
exclusively as a free amine, whereas benzylamine (pKa )
9.3) is present under its ammonium form. Two solutions
containing either A1 or A3 (50 µM) were reacted with this
new mixture of 6 amines (15 mM each). Again, a position
of equilibrium was obtained after 7 days that was identical
when starting from either 9-anilino (A3) or 9-sulfanylo (A1)
acridine (Figure 3).
Figure 4. Structures of acridines A3, A5, and A6. Destabilizing steric
clashes are highlighted in red.
To demonstrate the general applicability of our discovery
to differently substituted acridine scaffolds, a 9-p-aminoben-
zoic acid analogue of the DNA binding ligand BRACO-19
(A7) was synthesized16 and subjected to an amine exchange
reaction in potassium phosphate buffer under near physi-
ological conditions of pH (7.4) and temperature (37 °C).
When A7 (50 µM) was reacted with an equimolar solution
of aniline, p-aminobenzoic acid, p-sulfanilic acid, and
p-fluoroaniline (15 mM each), the formation of all four
possible 9-aminoacridines was observed (Figure 5) until
equilibrium was reached after 5 days. Interestingly, a similar
distribution of the four possible 9-aminoacridines was
obtained at equilibrium when the acridine-4-carboxylic acid
A2 was subjected to the same conditions (see Supporting
Information). This demonstrated that the substitution pattern
(i.e., carboxylic acids or amides at positions 3, 4, and 6) of
the acridine has very little effect on the reversible amine
exchange at position C9. Chloroquine,17 however, proved
Figure 3. HPLC traces of an equilibrating mixture of 50 µM
9-anilinoacridine A3 (left) or 50 µM 9-sulfanyloacridine A1 (right)
in potassium phosphate buffer pH 7.4 (100 mM) containing a
mixture of p-aminobenzoic acid, aniline, p-sulfanilic acid, xylidine,
benzylamine, and N,N-dimethylethylene diamine (15 mM each).
All mixtures were analyzed by LC-MS at t ) 0 (black), 3 days
(red), and 7 days (blue).
Despite the fact that benzylamine is protonated under the
conditions of the exchange experiment (pH 7.4), the 9-ben-
zylaminacridine A6 was the predominant acridine species at
equilibrium. In contrast to that, only a very small proportion
of acridine A5 obtained from free amine N,N-dimethyl-
(13) Carlson, C. B.; Beal, P. A. Org. Lett. 2000, 2, 1465.
(14) 9-Amino-4-carboxylic acid acridines were chosen as a model system
because of their high solubility in water at near-physiological pH and
because of their structural analogy with drugs from the AHMA and DACA
families.
(15) Ma, Z.; Day, C. S.; Bierbach, U. J. Org. Chem. 2007, 27, 5387.
(16) The synthesis was adapted from Redman, J. E.; Granadino-
Rolda´n, J. M.; Schouten, J. A.; Ladame, S.; Reszka, A. P.; Neidle, S.;
Balasubramanian, S. Org. Biomol. Chem. 2009, 7, 76.
(17) Cooper, R. G.; Magwere, T. Indian J. Med. Res. 2008, 127, 305.
4896
Org. Lett., Vol. 11, No. 21, 2009

ological conditions. Although incubation of A1 with a large
excess of NR-acetyl-lysine and NR-acetyl-arginine at pH 7.4
did not result in any detectable amine exchange reaction19
(see Supporting Information), we cannot rule out that such
reaction may occur in vivo in a specific environment, for
instance, nearby the DNA where pH can locally vary.
In addition, this amine exchange reaction in water repre-
sents a novel example of reversible chemistry appropriate
for dynamic combinatorial chemistry (DCC).20 DCC has
recently proven to be a very powerful tool to rapidly identify
ligands21 or inhibitors22 of biomacromolecules, and the
discovery of novel reversible covalent reactions usable in
DCC becomes very valuable to create dynamic combinatorial
libraries of increased chemical diversity.23 Since 9-ami-
noacridines display a wide range of biological properties,
the design of 9-aminoacridine-based dynamic combinatorial
libraries could find valuable applications in drug discovery.
The selection via a DCC approach of optimized 9-ami-
noacridines as specific nucleic acid binders is currently
underway in our laboratory.
Figure 5. Reaction of 9-aminoacridine A7 (50 µM) with a mixture
of four amines (15 mM each).
unable to undergo an amine exchange reaction when
subjected to the same mixture of aromatic amines, thus
suggesting the reversible amine exchange is not applicable
to 4-amino substituted quinolines.
Acknowledgment. A.P. thanks the French Ministry of
Research and Technology for a doctoral Fellowship. S.L.
thanks the International Center for Frontier Research in
Chemistry for financial support.
To determine the influence of the concentration of amines
on the reversible exchange reaction, A7 (50 µM) was reacted
with various concentrations of the same stoichiometric
mixture of four amines: 15, 5, and 1 mM concentrations of
each amine (i.e., 300, 100, and 20 equiv, respectively). The
amine exchange reaction proved slower with decreased amine
concentration. Nevertheless the same distribution at equi-
librium was obtained when reacting A7 with either 15 or 5
mM amines. When the amine concentration was decreased
further (i.e., 1 mM), all four possible 9-aminoacridines were
detectable in solution after only 1 day, but less than 60%
conversion of A7 was observed even after 7 days (see
Supporting Information).
The possibility for 9-aminoacridines to undergo a revers-
ible amine exchange reaction under physiological conditions
may have implications on the mode of action of 9-ami-
noacridines in vivo. Recent study on the derivative BRACO-
19 revealed that it could decompose in physiological media
mainly via hydrolysis of the amido bonds in positions 3 and
6.18 We demonstrate herein that BRACO-19 may also
undergo an amine exchange reaction in vivo under physi-
Supporting Information Available: General experimental
procedures and spectroscopic data for new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL9019925
(19) No amine exchange reaction was observed with spermidine or other
primary amines that are protonated in solution at near-physiological pH of
7.4.
(20) For recent reviews, see: (a) Ladame, S. Org. Biomol. Chem. 2008,
6, 219. (b) Lehn, J. M. Chem. Soc. ReV. 2007, 36, 151. (c) Corbett, P. T.;
Leclaire, J.; Vial, L.; West, K. R.; Wietor, J. L.; Sanders, J. K.; Otto, S.
Chem. ReV. 2006, 106, 3652.
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Rodriguez, R.; Sanders, J. K.; Balasubramanian, S. Angew. Chem., Int. Ed.
2008, 47, 2677. (b) Ludlow, R. F.; Otto, S. J. Am. Chem. Soc. 2008, 130,
12218. (c) Milanesi, L.; Hunter, C. A.; Sedelnikova, S. E.; Waltho, J. P.
Chem.sEur. J. 2008, 12, 1081.
(22) For recent examples, see: (a) Gareiss, P. C.; Sobczak, K.; Mc-
Naughton, B. R.; Palde, P. B.; Thornton, C. A.; Miller, B. L. J. Am. Chem.
Soc. 2008, 130, 16254. (b) Shi, B.; Stevenson, R.; Campopiano, D. J.;
Greaney, M. F. J. Am. Chem. Soc. 2006, 128, 8459.
(23) For recent examples of novel reversible covalent chemistries suitable
for DCC, see: (a) Saiz, C.; Wipf, P.; Manta, E.; Mahler, G. Org. Lett. 2009,
11, 3170. (b) Giuseppone, N.; Schmitt, J. L.; Lehn, J. M. J. Am. Chem.
Soc. 2006, 128, 16748. (c) Cacciapaglia, R.; Di Stefano, S.; Mandolini, L.
J. Am. Chem. Soc. 2005, 127, 13666. (d) Shi, B.; Greaney, M. F. Chem.
Commun. 2005, 7, 886.
(18) Taetz, S.; Mu¨rdter, T. E.; Zapp, J.; Boettcher, S.; Baldes, C.;
Kleideiter, E.; Piotrowska, K.; Schaefer, U. F.; Klotz, U.; Lehr, C. M. Int.
J. Pharm. 2008, 357, 6.
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