Symmetric and dissymmetric carbohydrate-phenyl ditriazole derivatives as DNA G-quadruplex ligands: Synthesis, biophysical studies and antiproliferative activity

https://doi.org/10.1016/j.bioorg.2020.103786Get rights and content

Highlights

  • Synthesis of carbohydrate-phenyl ditriazole derivatives as DNA G-quadruplex ligands.

  • Binding studies to G-quadruplexes using FRET, CD, MS and NMR spectroscopy.

  • Different binding modes to the HIV321 g-quadruplex sequence were observed for meta- and para-phenyl ditriazole ligands.

  • Antiproliferative activity was measured for the new G-quadruplex ligands.

Abstract

Here we present a novel G4-binding family of compounds based on a central core of phenyl ditriazole (PDTZ) modified with carbohydrates and phenyl pyrrolidinyl side-chains. Their synthesis was achieved using controlled click chemistry conditions to obtain both, symmetric and dissymmetric carb-PDTZ derivatives without any intermediate protecting steps through an optimized methodology. Binding of the new carb-PDTZ to a variety of G-quadruplex motifs was examined using different biophysical techniques. The symmetric carb-PDTZ derivatives were not able to stabilize G4, but the dissymmetric ones (containing one sugar and one phenyl pyrrolidinyl side-chain) did. Interestingly, the dissymmetric carb-PDTZ derivatives showed much higher G4 vs duplex DNA selectivity than the control compound PDTZ 1, which contains two phenyl pyrrodilinyl side-chains and no carbohydrates. Their potential antitumoral activity was also investigated by in vitro cytotoxicity measurements on different cancerous cell lines. All carb-PDTZ derivatives showed higher IC50 values than the control PDTZ 1, probably due to the lack of compound stability of some derivatives and to lower cellular uptake.

Introduction

G-quadruplexes (G4) are promising targets in cancer [1], [2]. In fact, many G4 ligands have been reported as potential antitumor drugs in the past years [3], [4]. G4 are secondary structures that are formed by guanine-rich DNA or RNA sequences. Four guanines are associated through Hoogsteen hydrogen bonding to constitute the basic G-quartet unit. These G-quartet motifs stack on top of each other with a metal cation hosted in the central cavity to form G4 structures. Various topologies are possible, and classified as parallel, antiparallel, or hybrid [5] depending on strands orientation. Preference for a given fold depends mainly on the DNA/RNA sequence, the syn or anti conformation of the glycosidic bond, the metal cation and the loops.

Some genome searches have found around 300,000 or more potential G4 sequences inside the human genome, mostly located in introns, promoters and repetitive motifs including telomeres [6], [7], [8]. Additionally, they have also been located in non-human genomes including parasites [9], viruses [10], [11], [12], [13], [14], and bacteria [15]. The biological role of the quadruplexes has not been completely solved yet, but two main hypotheses exist regarding their function: (a) In telomeric regions, the formation of G4s seems to inhibit telomerase activity [16], which is responsible for maintaining the length of telomeres and is overexpressed in most cancers [17]. (b) At oncogene promoter regulatory regions, G4s may have the potential to down-regulate their expression when formed and stabilized, and repress the formation of the corresponding oncoprotein. In another proposed model for these regions, G4s are formed at the non-coding DNA facilitating an open conformation of coding DNA strand and therefore, enhancing the expression of a specific gene [18], [19], [20].

G4 ligands display a large variety of structures and many of these have notable antiproliferative activity [21], [22]. They tend to share some common structural features [23], such as the presence of (i) a large aromatic core to interact with the terminal G-quartet through π-π aromatic interactions and (ii) cationic side-chains to interact with the phosphates of the DNA backbone. In general, G4 ligands do not possess drug-like features due to their high molecular weight, low hydrophilicity and poor pharmacokinetic properties. In addition, their selectivity towards G4s is often limited when compared to their binding to duplex DNA. Quarfloxin [24], APTO-253 [25], [26], and CX-5461 [27] are the only G4 ligands which have reached clinical trials stage for the treatment of advanced solid tumors or lymphomas, acute leukemia and BRCA1/2 deficient tumours, respectively.

Recently, we proposed the conjugation of carbohydrates to G4 ligands intending to improve their pharmacological features [28], [29], to increase selective binding to G4 structures versus dsDNA and to take advantage of the overexpression of glucose transporters (GLUT) [30] at the surface of cancerous cells. Carbohydrate conjugation to chemotherapeutic agents has been previously used to improve drug selectivity and diminish drug toxicity. Examples include glufosfamide [31], some glucose-platinum conjugates [32], [33] and 2́-paclitaxel-methyl-2-glucopyranose [32]. Several G-quadruplex ligands conjugated with carbohydrate have also been described, including a pyridostatin-glucose derivative [34]. Our group reported how carbohydrate naphthalene diimide conjugates stabilized several G4 structures, with preference towards the human telomeric quadruplex sequence [35]. These conjugates showed antiproliferative activity on different human cancer cell lines, with some selectivity for cancerous cells versus non-cancerous cells linked to cell uptake through GLUT4 transporters. We also observed differences in efficacy depending on the presentation of the sugar motif in the conjugate. Recently, these conjugates have demonstrated their binding to G4 sequences present in parasites such as Trypanosoma brucei. Their antiparasitic activity (as IC50 values) was in the nanomolar range for T.brucei and in the submicromolar range for Leishmania major and Plasmodium falciparum [36], [37].

In this work, we present a new family of carbohydrate G4 ligand conjugates inspired on 1,3-di(1,2,3-triazole-4-yl) benzene derivatives, such as compound 1, described by Neidle’s group (Fig. 1) [38], [39]. Our design consists on the partial or full replacement of the pyrrolidinyl side-chains with mono- or disaccharides. Binding studies of the new carbohydrate phenyl ditriazole derivatives (carb-PDTZ) to G4 structures were carried out using a variety of biophysical methodologies. Cytotoxicity assays were also performed on cancer (HT-29, MCF-7, LN229 and HepG2) and non-cancer cell lines (MRC-5, HEK-293).

Section snippets

Synthesis of carbohydrate-phenyl ditriazole derivatives

We prepared two different types of phenyl ditriazole analogues based on the reported G4 ligand 1. Derivatives of the first family were symmetric with two sugar motifs attached to each side of the phenyl ditriazole central core, whilst derivatives of the second family were asymmetric, with a carbohydrate on one side and the phenyl pyrrolidinyl residue employed by Moorhouse et al. [38], [39] on the other side. The synthesis was achieved using click-chemistry based on the Huisgen 1,3-dipolar

Conclusions

We have designed and prepared a new family of carbohydrate conjugated G-quadruplex ligands based on a phenyl ditriazole central core. CuAAC click chemistry was used for conjugation and it was optimized to synthesize symmetrical and dissymmetrical carb-PDTZ derivatives containing a phenyl pyrrolidinyl side-chain. The compounds were evaluated as G4-binders for several G-quadruplex sequences using FRET-melting assay, CD, mass spectrometry and NMR techniques. FRET-melting assays demonstrated that

Materials and methods

All reagents and solvents were purchased to Sigma Aldrich, Carbosynth, Fluka or Merck, and used without further purification. All reactions were monitored by TLC, on silica gel plates (60 F254 (Merck), and visualized with UV light or with mostain (500 mL of 10% H2SO4, 25 g of (NH4)6Mo7O24·4H2O, 1 g Ce(SO4)2 4H2O), sulphuric acid (10% H2SO4 in ethanol), anisaldehyde (450 mL ethanol, 25 mL anisaldehyde, 25 mL H2SO4 and 1 mL AcOH), or ninhydrin (0.25 g ninhydrin, 100 mL ethanol) as staining

Declaration of Competing Interest

Author(s) declared no conflict of interest

Acknowledgements

This work was supported by the Spanish Ministerio de Economía y Competitividad (CTQ2012-35360 and CTQ2015-64275-P MINECO/FEDER, UE) and the Symbit project (Reg. no. CZ.02.1.01/0.0/0.0/15 003/0000477) financed by the ERDF. M.A-R. thanks Ministerio de Educación, Cultura y Deporte for a FPU predoctoral fellowship and A. De Rache for the supervision of the FRET melting assay, the CD studies and the NMR titration experiments.

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    Actual address: Life Sciences Department, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga, 4715-330 Braga, Portugal.

    2

    Actual address: Laboratorium für Organische Chemie, ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland.

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