A convenient method for synthesis of tetraoxazole peptide macrocycles

G-quadruplex DNA plays important regulatory roles in the maintenance of telomere length and transcriptions inhibition of such oncogenes as c-myc, and thus has become an attractive target for the development of anti-cancer therapeutic agents. Poly-oxazole macrocycles are a promising new class of G-quadruplex binding ligands. Herein is described the synthesis of a tetraoxazole peptide macrocycle and an epimerization product. The synthetic unit was prepared by cyclization-oxidation of a diserine with one hydroxyl protected. Two-cycle coupling of the unit gave a linear tetraoxazole amide. The targeted molecule and the epimerization product were obtained after macrocyclization of the linear precursor and following removal of the protecting benzyl groups. With structural similarities to the most potent G-quadruplex stabilizer telomestatin, these two molecules might potentially be used to probe the biological significance of G-quadruplex’ in vivo.


Introduction
G-quadruplex stabilization by small molecules is a promising strategy to develop anticancer therapeutic drugs. 1 Many small molecules have been reported to bind to and stabilize Gquadruplex and some of them have proven to be efficacious in cancer cell lines and xenoraft tumor models. 2Telomestatin 1, (Figure 1) a natural product isolated from Streptomyces anulatus 3533-SV4, is the most potent G-quadruplex stabilizer identified 3 and widely used to probe the in vivo significance of G-quadruplex. 4Telomestatin 1 has a unique macrocyclic structure consisting of seven oxazole rings and one thiazoline ring (Figure 1).Molecular modeling studies suggest that the exceptional activity of telomestatin may be attributed to the π-π stacking interaction between the oxazole rings and the end G-tetrads of G-quadruplex. 5The unique structure and significance of biological activity have rendered telomestatin an attractive synthetic target. 6The total synthesis of telomestatin was successfully completed in 2006. 7However, the synthesis involved many steps and thus telomestatin is still not available in great quantity.Alternatively, some poly-oxazole macrocycles have emerged as a promising new class of anticancer agents that target G-quadruplex DNA. 8Here, we report the synthesis of a tetraoxazole peptide macrocycle 2a and an epimerization product 2b.With structural similarities with telomestatin, these two molecules might potentially be used to probe G-quadruplex biological significance in vivo.

Results and Discussion
The biosynthesis of telomestatin has not been elucidated.A reasonable hypothesis is that it is constructed from a cyclooctapeptide containing five serine, two threonine and one cysteine.According to this, we selected Boc-L-Serine(Bzl)-OH 3 and H-L-Serine-OMe•HCl 4 as starting materials for the synthesis of 2a.(Scheme 1).Scheme 1. Synthesis of 2,4-disubstituted oxazole unit 6.
The resulting 2,5-disubstituted oxazole 6 was then divided into two portions, with one portion treated with 4 M HCl in ethyl acetate to remove the Boc group to give ammonium chloride 6a 6a and the other portion hydrolyzed with LiOH to give carboxylic acid 6b.(Scheme 2) Coupling of 6a and 6b using PyBroP 13 -DIEA 14 gave a di-oxazole amide 7 in 69% yield.Then, the same protocol was used again to treat 7.These two-cycle couplings afforded the linear tetraoxazole amide 8.
The precursor for macrocyclization was prepared by successive use of the hydrolysis and acidolysis treatments on 8 to remove both the methyl and Boc group (Scheme 3).
We tried both HATU 15 and DPPA 16 -HOBt as coupling reagents for macrolactamization of 8, but only the latter in the presence of DMAP 17 gave the desired product in a satisfactory yield.(Scheme 3) The reaction was performed by adding a solution of the linear precursor (10 μmol/mL) to a solution of DPPA-HOBt (30 μmol/mL) at room temperature.The crude macrocyclization product was purified by flash chromatography was then loaded on a semipreparative HPLC for further purification.The desired product 9a and an epimerization product 9b (Scheme 3) were obtained in an overall isolated yield of 20% (9a : 9b = 3 : 1).This is easy to understand since racemization is inevitable in the long reaction time for hydrolysis, acidolysis and macrolactamization.The two products were then treated by 20 wt% Pd(OH)2/C in a solution of MeOH : CH2Cl2 = 1 : 1 respectively to remove the protecting benzyl groups.The target molecule 2a and a stereomer 2b, corresponding to 9a and 9b respectively, were obtained in a same yield (71%).

Scheme 2. Synthesis of linear tetraoxazole amide 8.
Poly-oxazole macrocycles have been demonstrated to selectively bind to G-quadruplex DNA and but not to duplex DNA. 8It remains to be seen whether compounds 2a and 2b could be useful in this regard.Very recently, a family of azole-modified cyclic peptides which have a core

Experimental Section
General.NMR spectra were recorded on Varian 300 (300 MHz for 1 H, 75 MHz for 13 C) or a Bruker 400 MHz NMR instrument in the indicated solvent.Chemical shifts are reported in parts per million (ppm) relative to the residual proton solvent signal for internal tetramethylsilane (7.25 ppm for 1 H) for solutions in CDCl3.NMR spectral data are reported as follows: chloroform (7.26 ppm for 1 H) or chloroform-d (77.1 ppm for 13 C) when the internal standard is not indicated.Multiplicities are reported by using the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; J, coupling constants in Hertz.ESI-MS spectra were obtained using a Finnigan LCQ Deca XP Plus mass spectrometer.HRMS (ESI) spectra were obtained using Bruker Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (APEX IV).IR spectra were recorded on AVATAR 330FT-IR THERMO NICOLET spectrophotometer.Only the strongest and/or structurally important IR absorption data is reported, given in cm -1 .Optical rotations were measured with a Perkin-Elmer 341-LC polarimeter.CD spectra were recorded on a Jasco J-810 spectropolarimeter.Spectra were baseline-corrected and the signal contributions of the buffer were subtracted.Semi-preparative reversed-phase HPLC (UV 215 nm) was performed on a Waters 600 system with 2996 Photodiode Array Detector.The column used was Waters Symmetry Prep C18 Column, 7 μm, 78x300 mm.
After being stirred at the same temperature for 5 h, the mixture was poured into saturated aqueous NaHCO3 at 0 °C.The aqueous layer was extracted twice with CH2Cl2.The organic layer was washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4, and filtered.The filtrate was concentrated in vacuo.The residue was used for the next reaction without further purification.