Article pubs.acs.org/jmc
Unexpected DNA Affinity and Sequence Selectivity through Core Rigidity in Guanidinium-Based Minor Groove Binders Padraic S. Nagle,† Caitriona McKeever,† Fernando Rodriguez,†,§ Binh Nguyen,‡ W. David Wilson,‡ and Isabel Rozas*,† †
School of Chemistry, Trinity College Dublin, Dublin 2, Ireland Department of Chemistry, Georgia State University, Atlanta, Georgia 30302, United States
‡
S Supporting Information *
ABSTRACT: In this paper we report the design and biophysical evaluation of novel rigid-core symmetric and asymmetric dicationic DNA binders containing 9H-fluorene and 9,10-dihydroanthracene cores as well as the synthesis of one of these fluorene derivatives. First, the affinity toward particular DNA sequences of these compounds and flexible core derivatives was evaluated by means of surface plasmon resonance and thermal denaturation experiments finding that the position of the cations significantly influence the binding strength. Then their affinity and mode of binding were further studied by performing circular dichroism and UV studies and the results obtained were rationalized by means of DFT calculations. We found that the fluorene derivatives prepared have the ability to bind to the minor groove of certain DNA sequences and intercalate to others, whereas the dihydroanthracene compounds bind via intercalation to all the DNA sequences studied here.
■
series of bis-guanidine and N-alkyl bis-guanidine fluorene derivatives that bind into the minor groove in the same way as for previously reported dicationic carbazoles8 that were cytotoxic toward Pneumocystis jirovecii9 (cause of Pneumocystis pneumonia, PCP). These bis-guanidine fluorenes showed encouraging in vitro activity against both Trypanosoma brucei rhodesiense and Plasmodium falciparum.7 In vivo efficacy assays were also performed showing that, for example, the dicationic N-isopropylguanidino-9H-fluorene derivative resulted in 4/4 cures of the treated animals in the STIB900 animal model for stage 1 African trypanosomiasis.7 DNA minor groove binding agents normally adopt a crescent shape that fits into the minor groove, though recently it has been found that linear compounds can also bind to the minor groove.10 Athri et al. have shown by X-ray crystallographic analysis of complexes with AATT sequence that linear compounds such as DB921 (Figure 1) display a higher binding affinity than the equivalent crescent shaped derivatives by forming indirect contacts with the bases through highly structured water molecules.5,11−13 Moreover, Nguyen et al. have reported a compound (CGP 40215A, Figure 1) having little curvature that exhibits excellent antitrypanosomal activity by forming a “seesaw” complex with the minor groove.14 Interestingly, Baily et al. developed compound DB950 (Figure 1),15 which is a guanidinium derivative of the
INTRODUCTION Drugs that target the DNA minor groove, such as pentamidine and furamidine, have demonstrated widespread therapeutic applications as antiprotozoal or anticancer agents due to their ability to recognize AT rich sequences. These compounds and their analogues such as furimidazole (an imidazole analogue of furamidine) displayed exceptional uptake into tumor cells, and it has been postulated that they gain entry into the cell via the human organic cation transporter 1 (hOCT1).1 Moreover, other DNA minor groove binders, such as distamycin derivatives, have been found to act as antibacterial agents;2 specifically, Suckling has reported a series of bis-alkenyl minor groove binders with potent activity against Staphylococcus aureus and Streptococcus faecalis (Gram-positive bacteria) and in a thorough NMR study has proved that very specific hydrogen bonds (HBs) together with hydrophobic interactions are responsible for this activity.3 Additionally, Dervan and coworkers have described how a sequence specific DNA binding polyamide inhibits the expression of a number of androgen receptor regulated genes in prostate cancer cells.4 Previous research by Dardonville, Rozas, and co-workers reported a series of diaromatic bis-(2-aminoimidazolinium) derivatives that strongly bind to the DNA minor groove displaying very good antitrypanosomal and antiplasmodial activity.5 Wilson et al.6 have reported several minor groove binders such as furamidine and pentamidine used to treat a number of diseases including trypanosomiasis, leishmaniasis, Pneumocystis pneumonia, and malaria. Arafa et al.7 prepared a © XXXX American Chemical Society
Received: May 23, 2014
A
dx.doi.org/10.1021/jm5008006 | J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Article
Figure 1. Structure of known DNA minor groove binders.
Figure 2. General structure of the fluorene (left) and dihydroanthracene (right) dicationic (guanidinium/2-aminoimidazolinium) derivatives studied.
Scheme 1a
a
Reagents and conditions: (a) HgCl2, Et3N, Ch2Cl2, rt, 16 h; (b) TFA/CH2Cl2, Amberlyte resin IRA400 basic form (Cl−), rt, 24 h.
less likely to interact with the minor groove via their two cationic functionalities which are oriented in opposite directions. In each family, we have varied the nature of the cation through the replacement of the guanidine group for the 2-aminoimidazoline functionality (Figure 2) to examine whether the extra hydrophobic ethylene bridge increases the binding affinity. In addition, we have studied some flexible derivatives for the sake of comparison. To investigate the binding of all these compounds to DNA, different biophysical techniques have been used such as surface plasmon resonance (SPR) and DNA thermal denaturation experiments to obtain information on their sequence selectivity, and circular dichroism (CD) and ultraviolet (UV) spectroscopies were used to measure their binding strength and mode of binding.
phenanthridinium dye ethidium bromide (a prototypical DNA intercalating agent), and they observed that this compound binds to DNA much more tightly than ethidium bromide and displays distinct DNA-dependent absorption and fluorescence properties. Circular and linear dichroism showed that this rigidcore molecule, instead of intercalating DNA, preferentially binds to its minor groove. Continuing with our interest in DNA minor groove binders with guanidinium-like cations and flexible diaromatic cores, we have now studied the DNA affinity and sequence selectivity of a series of guanidine/2-aminoimidazoline derivatives with different rigid structures (9H-fluorene and 9,10-dihydroantracene) connecting such cations with different orientations (Figure 2). Our aim is to learn how the different orientation of these cations connected to both rigid cores affects the strength and mode of binding as well as the sequence selectivity. Thus, we have studied two rigid-core families, one with a 9Hfluorene system and another with a 9,10-dihydroanthracene (DHA) structure, being the underlining difference between them the curvature that can be achieved upon substitution. Hence, 2,7-disubstituted fluorenes exhibit a curvature that complements the convex shape of the minor groove, whereas 2,6-disubstituted DHA derivatives are linear and, in principle,
■
RESULTS AND DISCUSSION Chemistry. The synthesis of some of the compounds here studied was previously reported by us. Thus, the preparation of the 2,6-bis-guanidinium (1) and 2,6-bis-2-aminoimidazolinium (2) 9,10-dihydroanthracene derivatives (see generic structures in Figure 2) and the 2,7-bis-2-aminoimidazolinium-9H-fluorene derivative (3) was reported by us in 2008.16 Similarly, the B
dx.doi.org/10.1021/jm5008006 | J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Article
Table 1. Equilibrium Binding Constants (×105 M−1) for Rigid-Core Compounds 2 and 3 and Flexible Core Compounds 4−7 Calculated from SPR Experiments with AATT, (AT)4, and (CG)4 DNA Hairpinsa
compd
core
2 -DHA3 -fluorene4 -Ph-O-Ph5 -Ph-S-Ph6 -Ph-CH2CH2-Ph7 -Ph-piperidine-Phproflavine
K [AATT]
K [(AT)4]
K [(GC)4]
2.7 15
