Synthesis of G-quadruplex-targeting flexible macrocyclic molecules via click reactions

Four flexible macrocycles, such as cTz ( 1 ), cTN ( 2 ), cPT ( 3 ), and cPTN ( 4 ), were efficiently synthesized through cyclodimerizing triazo-alkynyl-containing monomers via Cu(I)-catalyzed click reactions with optimized conditions. The starting materials were pyrrole and triazole derivatives containing amine and carboxyl groups, followed by amide coupling to introduce the triazo and alkynyl groups to prepare the cyclization precursors. The electrospray ionization mass spectrometry (ESI-MS) results indicated that cTN ( 2 ) and cPT ( 3 ) showed the ability to bind with c-myb G-quadruplex. Therefore, cTN ( 2 ) and cPT ( 3 ) molecules might be potential leading compounds for anti-cancer drug discovery.


Introduction
DNA G-rich sequences widely exist in human genome and are potential to form G-quadruplex structures. 1,2The G-quadruplex involved in the significant regions such as promoters and telomeres have been proven to play important roles in life process, including gene transcription and expression, cell division and apoptosis. 1,3-102][13] Further studies showed that Myc-associated zinc finger protein can bind to the G-quadruplex and repress c-myb promoter activity. 14Thus, the G-rich sequences in c-myb promoter region can act as a critical element to regulate the expression of the c-myb.Furthermore, the small molecules that selectively bind with c-myb G-quadruplex and stabilize this structure are considered as potential anti-cancer drugs.

Results and Discussion
First, we optimized the Cu(I)-catalyzed click reaction conditions using azidoacetic acid 5 and Boc-protected propynylamine 6 as model substrates, as shown in Table 1.In presence of TEMED (N,N,N',N'-tetramethylethylenediamine) as the ligand (100 mol%), CuCl (20 mol%) showed the higher catalytic activity than CuI and CuBr (entry 1-3).The reaction was promoted more effectively in THF than acetonitrile, and as the addition of water, the product yield increased to 71% when the THF / H2O ratio reached 5 / 1 (entry 3-5).However, the further increasing amount of water suppressed the reaction due to the decreasing solubility of 6 (entry 6, 7).
As shown in Scheme 1, propynylamine, and azidoacetic acid 5 or its derivative 9 were efficiently linked to the C-and N-terminal of 7 by amide bonds to afford cyclization precursors 10 and 11, respectively.In our case, uronium-based coupling reagent HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and phosphonium-based coupling reagent BOP (benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate) were utilized to activate the carboxylic acid of 7, and 5 or 9 in order to facilitate the amide-bond coupling reaction.The triazo-alkynyl-containing precursors were readily cyclized by the formation of two triazoles with high yields at the concentration of 2 mM in the optimized click reaction condition.In addition, our starting material 12 for cPT (3) and cPTN (4) was obtained from pyrrole via five steps according to the procedures reported 25,26 .cPT (3) and cPTN (4) were also synthesized from triazole derivative 7 by the similar method (Scheme 2).
The binding affinity of compound 1-4 towards the c-myb G-quadruplex (Q1) was evaluated by ESI-MS.The mass spectra showed that as the molar ratio of ligand/Q1 was 4:1, the complex ion of Q1 with one cTN ([Q1+cTN] 5-) at m/z 1672.4 appeared in the spectrum with an intensity of nearly 20% (Figure 2a).For cPT, the complex ion ([Q1+cPT] 5-) appeared at m/z 1649.5 with an intensity of 50%, and that of Q1 with two cPT ([Q1+2cPT] 5-) at m/z 1748.0 with an intensity of 10% (Figure 2b).The results indicated cTN (2) and cPT (3) exhibited the ability to bind with Q1 (Figure 2).To evaluate the binding affinity of cTN (2) and cPT (3) to Q1, the parameter IRa 27-30 was defined as the relative abundance ratio of bound ions ( to that of both unbound and bound species ( The IRa values were 0.15 and 0.29 for cTN (2) and cPT (3), respectively.

Experimental Section
General.Low

Azidoacetic acid (5).
To a solution of ethyl bromoacetate (20.0 g, 120 mmol) in DMF (20 mL) was added sodium azide (12.5 g, 192 mmol) with stirring at 0 °C.The mixture was allowed to warm to rt and stirred for 24 h.Saturated sodium carbonate solution was added and the aqueous layer was extracted three times with Et2O.The organic layer was combined and washed by saturated aqueous NaHCO3 and brine, dried over Na2SO4.The solvent was evaporated to obtain colorless liquid.Without further purification, 1 M NaOH (60 mL) and methanol (60 mL) were added and stirred at 40 o C. Methanol was evaporated after 5 h.The pH of the remaining aqueous solution was adjusted about 2 by adding 2 M HCl.The aqueous layer was extracted three times with Et2O.The organic layer was combined and washed with brine, dried over Na2SO4.The solvent was evaporated to give 5 as a colorless liquid (4.9 g，48.5 mmol，81%). 1H NMR (400 MHz, CDCl3): δ 6.97 (s, 1 H), 3.95 (s, 2 H). 13

(R)-2-Azido-3-((tert-butoxycarbonyl)amino)propanoic acid) azidoacetic acid derivative (9).
To a solution of (S)-isoserine (5.00 g, 47.6 mmol) in ethanol (200 mL) was added hydrogen chloride gas with stirring at 0 o C for 5 h.The mixture was allowed to warm to rt and stirred overnight.Ethanol was evaporated and EtOAc was added.Boc2O (15.0 g, 68.8 mmol) and triethylamine (9.44 g, 93.5 mmol) were added and stirred at 0 o C. The mixture was allowed to warm to rt and stirred overnight.The solvent was evaporated and the residue was washed with petroleum ether to obtain white solid intermediate.The intermediate compound was dissolved in CH2Cl2 (100 mL) and triethylamine (8.7 g, 86.3 mmol) was added.The mixture was treated dropwise with a solution of tosyl chloride (9.8 g, 51.3 mmol) in CH2Cl2 (80 mL).The mixture was allowed to warm to rt and stirred overnight.The organic solution was washed by 1% HCl solution, saturated aqueous NaHCO3, and brine, dried over Na2SO4.The solvent was evaporated and the residue was dissolved in DMF (75 mL).Sodium azide (7.0 g, 108 mmol) was added with stirring at rt.The reaction mixture was warmed to 60 o C and stirred overnight at this temperature.
The mixture was then cooled to rt. saturated aqueous NaHCO3 was added and the aqueous layer was extracted three times with EtOAc.The organic layer was washed with brine, dried over Na2SO4.The solvent was evaporated and the residue was dissolved in methanol (60 mL). 1 M NaOH (60 mL) was added with stirring at 40 o C. Methanol was evaporated after 5 h.The aqueous layer was washed twice with Et2O.The pH of the remaining aqueous solution was adjusted about 2 by adding 2 M HCl.The aqueous layer was extracted three times with Et2O.The organic layer was combined and washed with brine, dried over Na2SO4.The solvent was evaporated to give 9 as a light yellow solid (7.1 g，30.9 mmol，65%). 1H NMR (400 MHz, CDCl3): δ 7.80 (d, 3 J (H,H) 7.8 Hz, 1 H), 7.18 (s, 1 H), 4.17 (m, 1 H), 3.60 (m, 1 H), 3.42 (m, 1 H), 1.44 (s, 9 H). 13 C NMR (100 MHz, CDCl3): δ 176.5, 171.9, 80.7, 61.5, 41.7, 28.To a suspension of 8 (0.250 g, 0.853 mmol) in CH2Cl2 (4 mL) was added the same volume of TFA (4 mL) under N2.After being stirred at rt for 1 h, the solvent was evaporated to give a yellow solid and added to the mixture of 5 (0.130 g, 1.29 mmol), BOP (0.860 g, 1.94 mmol), and DIEA (0.5 mL, 3 mmol) in DMF (5 mL) was stirred under N2 at rt overnight.The solution was added to EtOAc, followed by washing by 1% HCl solution, saturated aqueous NaHCO3, and brine, dried over Na2SO4.The solvent was evaporated and the residue was purified by column chromatography on silica gel (CHCl3: CH3OH 15: 1 -10: 1) to give 10 as a light yellow solid (0.188 g, 0.681 mmol, 80%