A fragment based click chemistry approach towards hybrid G-quadruplex ligands: design, synthesis and biophysical evaluation

A library of hybrid oxazoleetriazole based compounds containing contiguously linked aromatic units were synthesised as G-quadruplex binding ligands. The design of these ligands was based upon combining features of our first generation of G-quadruplex bis-triazole ligands and the natural product telomestatin. The syntheses and biophysical studies of these ligands are described. 2011 Elsevier Ltd. Open access under CC BY license.


Introduction
G-Quadruplex structures are organised polymorphic tertiary assemblies formed by oligonucleotides and nucleic acids containing G-tracts. 1,9 A core of hydrophobic G-quartets and a periphery of negatively charged loops characterise these structures, which exhibit higher stability over other possible conformations in the presence of monovalent cations, such as Na þ or K þ . Putative Gquadruplex sequences have been identified in the genome, 2 with over-representation in guanine rich sequences, such as telomeres, 3 in the promoter regions of a number of proto-oncogenes, 4 such as c-myc 5a and c-kit, 5b in 5 0 untranslated regions 6a and in introns. 6b Unsurprisingly, several of these structures have become attractive targets for cancer chemotherapy.
For example, inducing the single-stranded telomeric DNA overhang to fold into G-quadruplexes has been shown to inhibit telomerase activity 7a and cancer cell growth. 7b This is significant, since in >80% of cancers telomerase is up-regulated and contributes to the malignant phenotype by maintaining cancer cell immortalization. 7c In addition to perturbing telomere elongation, the induced G-quadruplex structures result in telomere uncapping, which leads to rapid growth arrest or apoptosis. 8 This creates a window of opportunity for the design and synthesis of G-quadruplex binding ligands with a view to employ them as therapeutic agents. 9,10 In recent years numerous G-quadruplex binding ligands have been identified, the vast majority possessing a large aromatic/heteroaromatic unit, usually locked into a planar disposition. 9,10 These aromatic moieties p-stack onto the terminal tetraplex of the quadruplex providing extra stabilisation, and can even induce folding into the higher order structures. 9,11 Side chains, which have a positive charge or that can become protonated at physiological pH, are another common feature, and provide points for electrostatic interactions with the negatively charged phosphate residues in the grooves. 10 However, ligands that selectively target specific Gquadruplex structures are limited, presenting a significant challenge for further development. 10b We have previously reported the synthesis and biological evaluation of a family of triazole based quadruplex binding 'click' ligands (e.g., 1, Scheme 1), which showed strong G-quadruplex stabilising effects, telomerase inhibition and anti-cancer activity. 12 The design of these compounds was inspired by the non-fused complex polycyclic aromatic structure of telomestatin (2), a very potent telomerase inhibitor isolated from Streptomyces annulatus. 13 We proposed that the 1,4-triazole moiety would serve as a suitable mimic of the oxazole functionality of telomestatin (2), and could be readily installed using the powerful CuAAC click reaction, thus facilitating the synthesis of libraries of potential ligands. Indeed, the 'click' ligands (e.g., 1) displayed impressive selectivity for G-quadruplex structures over duplex DNA, comparative to that of telomestatin (2). 12 Although 2 is an incredibly potent and selective quadruplex binder, it is also a complex synthetic target requiring lengthy syntheses, 14 whereas our ligands were readily accessible in significant quantities.
We have since developed a second generation of tristriazole ligands (3), which were designed in order to examine whether Gquadruplexes could be stabilised by end-on hydrogen atomep interactions. 15 However, whilst the loss of effective pep binding interactions by the inclusion of such tetrahedral centres was not balanced by potential hydrogenep bonding interactions, the quadruplex/duplex selectivity of the ligands once again remained high, suggesting that the rotational freedom of non-fused polyaromatic systems is important with regard to selectivity.
Taking these two factors into consideration, we have designed a third generation of ligands, again based on a non-fused polyaromatic system but excluding excessive conformational flexibility.
Since p-stacking interactions had proven to be particularly important, we opted to increase the number of heteroaromatic units in the ligand cf. 1, in an attempt to maximise binding between electronrich ligand heterocycles and guanine residues. The general design of these hybrid ligands was to incorporate the oxazole motifs of telomestatin (2), and the 1,4-triazole and side chain functionalities present in our first generation (e.g., 1) (Scheme 1). Keeping in mind the proposed use of click chemistry, we opted to target a series of triazole-centred ligands (e.g., 4) flanked by phenyloxazoles (Scheme 1). Synthetically, the ligands would be accessed through a CuAAC reaction 16 of the corresponding alkynes 5 and azides 6.

Results and discussion
The synthesis of our library began using the procedure of Scanlan et al., with a Sonogashira reaction of 4-bromobenzamide 7 with ethynyltrimethylsilane to give 8 in 88% yield (cf. 53%, Scanlan, Scheme 2). 17 Typical Bl€ umleineLewy oxazole synthesis conditions (8, ethyl bromopyruvate, EtOH, reflux) provided oxazole 9 in low yield (<30%). Hence, we opted for the two step procedure described by Panek,18 which provided the oxazole 9 in good overall yield. The TMS-protected alkyne was then unmasked and the ester hydrolysed in one step to afford the acid 10, which was converted to the acid chloride 11 by treatment with (COCl) 2 /DMF. Compound 11 was subsequently reacted with various N,N-dialkyldiamines to afford the alkyne coupling partners 12e17 for the ensuing click reaction.
The complementary azide-coupling partners were accessed via an analogous route. Hence, 4-aminobenzamide 18 was converted to the corresponding azide, which was then subjected to the same oxazole forming conditions as before to give 19 (Scheme 2). Hydrolysis of 19 was followed by conversion to the acid chloride 20, to which a series of N,N-dialkyldiamines were added providing the amides 21e26. Finally, the azide 5 and alkyne 6 coupling partners were reacted together, under our previously developed microwave conditions, 19 to deliver the new library of hybrid triazoleebisoxazole ligands 27e35 in good yield (69e87%, Scheme 2).
The ability of these compounds to stabilise G-quadruplex and double helix DNA was investigated using a high-throughput FRET (fluorescence resonance energy transfer) assay. 20 Table 1  showed moderate to good stabilisation of the G-quadruplex structure with excellent selectivity over duplex DNA. Ligand 30 was by far the best binder (DT m ¼15 C) but it did not discriminate between duplex and quadruplex DNA quite as effectively as 31, 34 and 35, which is essential for development as potential therapeutics. The ligands with side chains containing three methylene units (n/ n 0 ¼2) were uniformly stronger quadruplex binders than the analogous ligands with side chains containing two methylene units (n/ n 0 ¼1) i.e., 27 cf. 30, 28 cf. 31, 29 cf. 32, 33 cf. 34. This may be due to the fact that the longer arms allow more effective positioning of the protonated amines for interaction with the phosphate backbone, or simply due to providing more interactions with the guanine quartet. It appears that the smaller ammonium ions interact more effectively than larger analogues, regardless of the length of the side chain, i.e., 27 cf. 29 and 30 cf. 32, suggesting that there is a pocket with some steric constraints where the protonated amines and phosphate backbone interact. At a concentration of 1 mM the ligands were quite poor at binding G-quadruplex forming DNA, the unsymmetrical ligand 35 being the only ligand to show any real affinity for h-Tel. Although these ligands show comparable quadruplex binding ability to some previously reported ligands, 21 they were rather disappointing bearing in mind our first generation of ligands, i.e., 1 (Scheme 1), which exhibited DT m ¼18 C at a concentration of 1 mM for the h-Tel G-quadruplex forming sequence.
However, one critical feature was prominent from these studies; the selectivity of the ligands for quadruplex DNA over duplex DNA remained consistently high, even at higher concentrations of the Scheme 1. Rational design and retrosynthesis of new hybrid G-quadruplex 'click' ligands.
ligands. Although non-specific binding of the ligands to quadruplex DNA cannot be ruled out, it is noteworthy that this series of nonfused polyaromatic ligands have displayed excellent selectivity for quadruplex DNA over duplex DNA. In this context perhaps ligand 35 is most interesting, which displayed complete selectivity for quadruplex DNA over duplex DNA, even at a concentration of 4 mM, and showed a small amount of stabilisation of h-Tel at a concentration of 1 mM.

Conclusion
In summary, we have prepared a small library of hybrid Gquadruplex binding ligands inspired by the natural product telomestatin and our first generation 'click' ligands. These non-fused polycyclic ligands comprised both triazole and oxazole functionality with pendant side chains and demonstrated modest, yet selective, affinity for the intramolecular h-Tel G-quadruplex.
We undertook SAR studies, which further confirmed the advantageous nature of designing ligands with non-fused polyaromatic motifs with regard to selectivity of the ligand for quadruplex DNA over duplex DNA. We believe that these results provide important information that will aid in the design of new Gquadruplex binding ligands that could potentially lead to novel cancer therapeutics.
4.1.4. Ethyl 2-(4-azidophenyl)oxazole-4-carboxylate (19). 4-Aminobenzamide 18 (10.0 g, 73.4 mmol) was suspended in THF (600 mL) and cooled to 0 C then concd HCl (35 mL) was added dropwise followed by t-BuONO (22.0 mL, 186 mmol). The reaction was stirred for 2 h then NaN 3 (14.2 g, 218 mmol) was added in one portion. H 2 O (150 mL) was then added very carefully and after ½ h the reaction was warmed to room temperature. After 2 ½ h a saturated solution of NaHCO 3 (80 mL) was added cautiously and the reaction was neutralised by the careful addition of solid NaHCO 3 . The THF was removed in vacuo and the product was extracted with EtOAc (5Â200 mL), which was dried (MgSO 4 ), filtered and concentrated. The pale yellow solid was used directly in the next step without further purification.
The crude azide was suspended in anhydrous THF (300 mL), under an Ar atmosphere and NaHCO 3 (21.8 g, 260 mmol) then ethyl bromopyruvate (9.40 mL, 74.7 mmol) were added. The reaction was heated to reflux overnight and a second charge of ethyl bromopyruvate (9.40 mL, 74.7 mmol) was added in the morning. After a further 6 h reflux the reaction was cooled and filtered through Celite, washing with EtOAc. The filtrate was concentrated then redissolved in anhydrous THF (250 mL) under N 2 atmosphere. The mixture was cooled to 0 C and trifluoroacetic anhydride (31.2 mL, 223 mmol) was added slowly. The reaction was allowed to warm to room temperature and stirred overnight before the slow addition of a saturated solution of NaHCO 3 (150 mL) at 0 C. The reaction was neutralised by the careful addition of solid NaHCO 3 and the THF was evaporated in vacuo. The crude oxazole was extracted with EtOAc (3Â250 mL), which was washed with brine (150 mL), dried (MgSO 4 ), filtered and concentrated. The oxazole was recrystallised from EtOAc/petrol to give 19 as light orange needles (9.90 g, 38.4 mmol). The mother liquors were purified by flash chromatography (9:1, petrol/EtOAc) to give the title compound 19 as a pale yellow solid (2.30 g, 8.91 mmol, total 47.3 mmol, 64% over three steps). R f 0.16 (9:1, petrol ethers/EtOAc); n max /cm À1 3012, 2130, 2097, 1734, 1610, 1598; (20). The azide 19 (9.87 g, 38.3 mmol) was dissolved in THF/H 2 O (4.3:1, 160 mL) and cooled to 0 C. LiOH (1.39 g, 57.9 mmol) in H 2 O (30 mL) was added slowly then the reaction was stirred ½ h before being warmed to room temperature. After a further 1 ½ h H 2 O was added until all the solids had dissolved then the pH was adjusted to w2 with 10% HCl. The mixture was extracted with EtOAc repeatedly (150 mL portions) until all the solids had dissolved. The organics were combined, dried (MgSO 4 ), filtered and concentrated. The crude material was used directly in the next step.

2-(4-Azidophenyl)oxazole-4-carbonyl chloride
The crude acid was suspended in anhydrous CH 2 Cl 2 /THF (7:1, 175 mL) under an Ar atmosphere with anhydrous DMF (0.2 mL) and cooled to 0 C. (COCl) 2 (6.50 mL, 77.3 mmol) was added slowly, and after 1 h the reaction was allowed to come to room temperature and was stirred overnight. The solvents were removed in vacuo and the crude brown solid was used directly in the next step without further purification.