Synthesis of Fluorescent Binaphthyl Amines That Bind c-MYC G

Jul 21, 2016 - (7) It has been proposed that the c-MYC G-quadruplex present in the ..... Elemental analysis (C, H, N) was performed with a PerkinElmer...
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Synthesis of Fluorescent Binaphthyl Amines That Bind c‑MYC G‑Quadruplex DNA and Repress c‑MYC Expression Ajay Chauhan,† Rakesh Paul,‡ Manish Debnath,‡ Irene Bessi,§ Samir Mandal,‡ Harald Schwalbe,§ and Jyotirmayee Dash*,†,‡ †

Department of Chemical Sciences, Indian Institute of Science Education and Research, Mohanpur, West Bengal 741252, India Department of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India § Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, Max-von-Laue Strasse 7, 60438 Frankfurt am Main, Germany ‡

S Supporting Information *

ABSTRACT: Two novel binaphthyl amines have been designed and synthesized using Buchwald amination and oxidative homocoupling as key steps. The binaphthyl amine containing two triazole rings shows higher affinity for c-MYC G-quadruplex, exhibits fluorescence “turn-on” response with cMYC, and stains the nucleus in cells. The triazolyl binaphthyl amine shows cytotoxicity for cancer cells by inducing G2/M phase cell cycle arrest and apoptosis. Moreover, both ligands can downregulate c-MYC expression at transcriptional and translational levels.

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INTRODUCTION G-quadruplexes are four-stranded nucleic acid structures involved in a wide range of human malignancies and emerged as a therapeutic target in oncology.1,2 G-Quadruplexes are present in many regulatory regions in the human genome, including telomeric ends (h-TELO),2 and in oncogene promoters such as c-MYC,3 c-KIT,4 k-RAS,5 etc.6 Thus, stabilization of G-quadruplex structures may play an important regulatory role and therefore can be considered as a promising new class of targets for the design of anticancer drugs.7 It has been proposed that the c-MYC G-quadruplex present in the nuclease hypersensitive element III 1 (NHE III 1), located upstream of the P1 promoter of c-MYC, controls 80−90% of the c-MYC expression.3,8 Therefore, the c-MYC G-quadruplex is critical for transcriptional silencing and it can be targeted by small molecules.8,9 A number of small molecules have been reported that can stabilize the c-MYC quadruplex.10−16 Small molecules such as porphyrins derivatives TMPyP4,11 Se2SAP,12 quindolines,13 quarfloxin,14 berberines,15 and carbazoles16 have shown the potential to inhibit c-MYC expression in cellular studies. The design and synthesis of novel c-MYC inhibitors operating at the transcriptional level would expand our understanding of the molecular basis of interaction of small molecules with G-quadruplexes that may lead to the possibility of using this promising strategy for anticancer therapeutics. Furthermore, G-quadruplexes are highly polymorphic and it is quite challenging to design small molecules that are selective toward a particular G-quadruplex. In this report, we delineate the synthesis of two novel binaphthyl amines functionalized with amide and triazole © XXXX American Chemical Society

moieties (8 and 12) for selective interaction with quadruplex structures over double-stranded (ds) DNA. The interaction of these binaphthyl derivatives with G-quadruplexes were investigated using Förster resonance energy transfer (FRET) melting analysis, UV−vis, fluorescence, NMR, and circular dichroism (CD) spectroscopic experiments. The effect of these ligands on human cancer cells (HeLa) was investigated using MTT assay, quantitative real time PCR (qRT-PCR), Western blotting, and flow cytometric assay.

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RESULTS AND DISCUSSION

Synthesis. As shown in Scheme 1, the binaphthyl ligands 8 and 12 were synthesized starting from 1-naphthol 1. First, 1naphthol 1 was treated with sodium hydride in the presence of commercially available nonafluorobutanesulfonyl fluoride (NfF) to obtain the corresponding nonaflate derivative 2 (Scheme 1).17 The Buchwald amination of the naphthylnonaflate 2 with methyl 4-aminobenzoate 3 using a catalytic system consisting of Pd2dba3 (1.2 mol %) and XPhos (2.8 mol %) in the presence of DBU (2.5 equiv) in toluene at 80 °C afforded the naphthyl derivative 4 in high yield (Scheme 1). The oxidative homocoupling18 of the amine 4 using FeCl3 in the presence of K2CO3, as the base provided 1,1-binaphthyl-4,4diamine derivative 5 in 95% yield. Hydrolysis of diester 5 afforded the corresponding diacid 6 (Scheme 1), which was used as a building block to synthesize the binaphthyl ligands (Scheme 2). Amide coupling of diacid 6 with 3Received: March 15, 2016

A

DOI: 10.1021/acs.jmedchem.6b00328 J. Med. Chem. XXXX, XXX, XXX−XXX

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RAS, c-KIT1, and c-KIT2) and a control ds DNA (Supporting Information (SI), Figure S1). The diamide ligand 8 displayed moderate G-quadruplex stabilization potential, with ΔTm values ranging from 7 to 18 °C (Table 1 and SI, Figure S1). Ligand 12 containing the triazole modifications exhibited an enhanced stabilization potential (ΔTm ∼ 11−23 °C) for quadruplexes at 1.0 μM ligand concentration, whereas the compound 6 with −COOH end groups shows least affinity for all the sequences. Next, we have performed a concentration-dependent FRET melting experiment using the promoter quadruplexes and ds DNA. Ligand 8 shows a maximum ΔTm value of 23 ± 1 °C (i.e., a Tm of 93 °C) for c-MYC at a concentration of 3.0 μM (Figure 1a). Compared to this, 12 attained the maximum ΔTm

Scheme 1. Synthesis of a Binaphthyl Diacid 6

Scheme 2. Synthesis of Binaphthyl Ligands 8 and 12

Figure 1. G-Quadruplex stabilization by ligands in FRET melting assay. Thermal shift profiles for (a) 8 and (b) 12 upon stabilizing to quadruplexes and ds DNA in 60 mM K+-cacodylate buffer, pH 7.4. (c) FRET competitive assay of 8 and 12 (1 μM) for G-quadruplexes (100 nM) in the presence of ds DNA (100 nM, 1 μM, and 10 μM).

(dimethylamino)propylamine 7 using EDC, HOBT, and NMM in DMF afforded the corresponding diamide 8 (Scheme 2). Next, we have incorporated triazole containing side chains by using Cu(I) catalyzed azide−alkyne cycloaddition. The amide coupling of 6 with propargylamine 9 provided the dialkyne 10, which was subsequently treated with 3-azidopropylamine 11 to give the triazole functionalized binaphthyl ligand 12 in 72% yield (Scheme 2). FRET Melting Assay. FRET melting analysis19 was employed to determine stabilization potential (change in melting temperature, ΔTm) and selectivity of binaphthyl ligands 8 and 12 on the dual labeled (5′-FAM and 3′TAMRA) G-rich oncogenic promoter sequences (c-MYC, k-

value (∼23 °C) for c-MYC at 1 μM ligand concentration (Figure 1b). However, 3−5-fold higher concentrations of 8 and 12 were required to attain maximum ΔTm values for k-RAS, cKIT1, and c-KIT2, suggesting its high affinity toward the c-MYC quadruplex. In addition to that, 8 and 12 do not significantly alter the melting temperature of ds DNA at 5.0 μM ligand concentration. A FRET competitive experiment was performed to evaluate the selectivity of ligands for G-quadruplexes over duplex DNA

Table 1. Stabilization Potential of G-Quadruplexes Determined by FRET Melting Analysisa ΔTm at 1 μM (°C) DNA sequences

6

8

12

c-MYC: 5′ FAM-d(TGAG3TG3TAG3TG3TA2)-TAMRA 3′ c-KIT1: 5′ FAM-d(G3AG3CGCTG3AG3AG3)-TAMRA 3′ c-KIT2: 5′ FAM-d(G3CG3CGCGAG3AG4)-TAMRA 3′ k-RAS: 5′ FAM-d(AG3CG2TGTG3A2GAG3A2GAG5AG2)-TAMRA 3′ ds DNA: 5′ FAM-d(CCAGTTCGTAGTAACCC)-3′ 3′ TAMRA-d(GGTCAAGCATCATTGGG)-5′

4.5 2.5 2.8 3.8 0.5

15 9 7 18 1

23 11 15 22 2

The Tm values of the quadruplexes in 60 mM potassium cacodylate buffer, pH 7.4, in the absence of ligands are c-MYC (70 ± 1), k-RAS (46 ± 1), cKIT1 (57 ± 1), c-KIT2 (69 ± 1), and ds DNA (60 ± 1) °C; maximum measurable Tm = 93 °C. a

B

DOI: 10.1021/acs.jmedchem.6b00328 J. Med. Chem. XXXX, XXX, XXX−XXX

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Tris·HCl, pH 7.4). Fluorimetric titration of 8 (1.0 μM) with increasing concentration of the c-MYC shows a 1.2-fold increase in fluorescence intensity (Figure 2e), while 12 exhibits a 3.5fold increase in fluorescence intensity in the presence of c-MYC quadruplex (Figure 2f). The dissociation constant (Kd) values indicate a 5-fold higher affinity of 12 (Kd = 4.2 μM) compared to 8 (Kd = 21.4 μM) for the c-MYC G-quadruplex. The fluorescence titrations further reveal that ligand 12 binds cKIT1, c-KIT2, and k-RAS with higher dissociation constants (Kd) of 10.15, 11.13, and 11.4 μM, respectively (SI, Table S2). Ligand 8 exhibited much higher Kd values for all the quadruplexes, which indicates that it binds with less affinity compared to 12. However, both 8 and 12 barely show any enhancement in fluorescence intensity even after the addition of 20 equiv of ds DNA (SI, Figure S4). These results indicate that ligand 12 shows selectivity for c-MYC, and it can be considered as an effective light-up probe for the c-MYC quadruplex. NMR Titration. The target c-MYC G-quadruplex used for the interaction studies has been previously characterized by the Yang group.21,22 Both the ligands showed comparable effects on the 1D 1H NMR spectrum of c-MYC G-quadruplex, although 12 was found to be less soluble than 8 in the concentration range used for NMR study. Upon addition of 8, at [ligand]: [DNA] < 1, strong line broadening and chemical shift perturbations (CSP) were detected for the imino protons belonging to residues G6, G10, and G15 (Figure 3a, yellow boxes), as well as for the aromatic protons belonging to the 3′flanking nucleotides T20, A21, and A22 (Figure 3b, yellow boxes, Figure 3c). At [ligand]:[DNA] ≥ 1, additional weak CSPs, highlighted with gray boxes in Figure 3a,b, were observed in the imino region (with the exception of G5 and G9, belonging to the inner G-tetrad) and in the aromatic region (H8-G13 and H8-G2, as well as several unassigned signals in the region 7.6−7.9 ppm). The strongest effects of 12 on the DNA proton signals are highlighted with yellow spheres on the NMR structure of the c-MYC G-quadruplex in Figure 3d. The mapping of the CSPs/line broadening suggests that the ligand interacts with the 3′-end G-tetrad, possibly by restructuring the TAA-3′ capping. Similar effects were observed with 8 (SI, Figure S5). Circular dichroism (CD) spectroscopy suggests that the binding of ligands 8 and 12 does not disrupt the parallel topology of the c-MYC quadruplex (SI, Figure S6). Cell Cytotoxicity Assay. The growth inhibitory properties of 8 and 12 toward human cervical cancer cells (HeLa), human alveolar basal epithelial cancer cells (A549) and normal mouse myoblast cells (C2C12) were evaluated using MTT assay (SI, Figure S7).23 Cells treated with 0.1% DMSO served as control. Ligand 8 shows IC50 value of 7.24 ± 2.6 μM and displays lower antiproliferative activity relative to 12, which shows an IC50 value of 5.53 ± 1.7 μM. Compared to this, 8 and 12 exhibited significantly lower cytotoxicity for A549 (SI, Table S3) and normal cells (IC50 ≥ 40 μM). Effect of Ligands on the Endogenous c-MYC Transcription. Ligands 8 and 12 were examined for their effect on c-MYC transcription in HeLa cells (Figure 4). HeLa cells were treated with various concentrations (1.0, 2.5, and 5.0 μM) of 8 and 12 for 24 h, and the expression of c-MYC mRNA was normalized against the constitutively expressed housekeeping gene, glyceraldehyde-3 phosphate dehydrogenase (GAPDH). Ligands 8 and 12 reduced the c-MYC mRNA level by ∼27% and ∼58%, respectively (Figure 4a), at 5.0 μM concentration relative to the control (cell treated with 0.1% DMSO).

(Figure 1c). The melting of 100 nM dual labeled c-MYC, kRAS, c-KIT1, and c-KIT2 quadruplexes were carried out with 1 μM of 8 and 12 in the presence of competitor ds DNA. The results indicate that ligands 8 and 12 retain the high ΔTm values for the quadruplexes, even in the presence of 100 mol equiv excess of ds DNA. UV−vis Binding Titrations. The UV−vis absorption spectra of 8 and 12 exhibit two weak bands at ∼298 and ∼362 nm in 100 mM KCl and 10 mM Tris·HCl at pH 7.4 (SI, Figure S2). Because the ligand peak at 298 nm overlapped with that of the c-MYC, the equilibrium dissociation constant (Kd) were calculated using the band at 362 nm. Upon incremental addition of the preannealed c-MYC, 8 shows up to ∼18% hypochromicity (Figure 2a) and 12 produces hypochromicity

Figure 2. UV−vis titration spectra of 20 μM (a) 8 and (b) 12 upon titration with the c-MYC (0−1.4 equiv) in buffer solution containing KCl (100 mM) and Tris·HCl (10 mM) at pH 7.4. Dissociation constants (Kd) of (c) 8 and (d) 12 were determined from the UV−vis studies using Hill 1 equation. Fluorescence titration spectra of 1 μM (e) 8 and (f) 12 upon titration with the c-MYC (0−20 equiv) in buffer solution containing KCl (100 mM) and Tris·HCl (10 mM) at pH 7.4.

up to ∼37% (Figure 2b). The observed hypochromicity suggests that the planar core of 8 and 12 interact with the terminal G-quartets of c-MYC quadruplex by π−π stacking. The dissociation constants (Kd) of 8 and 12 for the c-MYC were determined to be 22.6 and 4.4 μM, respectively (Figure 2c,d). Similar UV−vis titrations reveals that 12 exhibits higher Kd values for c-KIT1, c-KIT2, and k-RAS quadruplexes (SI, Figure S3, Table S1). Ligand 8 shows relatively less binding affinity for the quadruplexes compared to 12 (SI, Figure S3, Table S1). However, negligible changes in the hypochromicity were observed upon titration of 8 and 12 with ds DNA (SI, Figure S3). These results suggest that ligand 12 exhibits higher binding affinity for c-MYC quadruplex compared to other promoter quadruplexes and ds DNA. Fluorimetric Titration. The fluorescence spectroscopy20 reveals that 8 and 12 exhibit a maxima at 430 nm upon excitation at 350 nm in buffer solution (100 mM KCl, 10 mM C

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Figure 4. Effect of ligands 8 and 12 on the expression of c-MYC protein in HeLa cancer cells: (a) qRT-PCR for c-MYC in HeLa cells after treatment with DMSO (Control) and 1, 2.5, and 5 μM of 8 or 12 for 24 h. (b) Densitometric analyses of immunoblots showing concentration dependent reduction in MYC protein level. (c) Western Blot analysis to determine the expression of c-MYC in HeLa cells treated with DMSO (Control) and 1, 2.5, and 5 μM of 8 or 12 for 24 h. GAPDH was used as a loading control.

GAPDH expression was observed in both treated and control cells. Together, these results indicate that the c-MYC Gquadruplex binding ligands 8 and 12 reduces the c-MYC protein expression in cancer cells. Structure−Activity Relationship. The binaphthyl moiety is a planar structure which can interact with the terminal Gquartet by π−π stacking. The G-quadruplexes differ in their intermediate loop composition. The triazole motifs can interact through π−π stacking with the G-quadruplex loop bases.16,24 The amine containing side chains are cationic at physiological pH and can promote electrostatic interaction with the phosphate containing backbone.25 Hence we envisioned that a ligand with a binaphthyl core and triazole side chains can efficiently bind G-quadruplexes. The biophysical analysis including FRET melting assay, UV−vis, and fluorescence spectroscopy reveals that the triazole containing ligand 12 shows higher affinity toward c-MYC compared to other quadruplexes and duplex DNA. On the other hand the anionic −COOH containing compound 6 shows least affinity. Ligand 8, containing amide side chains but no triazole groups, shows intermediate binding affinity. Further the more potent Gquadruplex binder 12 shows higher potency in downregulating c-MYC expression compared to less potent G-quadruplex binder 8. Effect of Binaphthyl Ligands on Cell Cycle Regulation. To examine whether the inhibitory effect of ligands 8 and 12 in HeLa cell proliferation was associated with cell cycle arrest, we conducted PI-metric cell cycle analysis using flow cytometry (Figure 5a). The analysis of cell cycle histograms reveals a marked increase in Sub G1 population (2.2% to 39.2%) upon treatment with 5.0 μM 8. Upon treatment with 12 (5.0 μM), we observed an increase in the population of both Sub G1 phase (2.2% to 32.2%) and G2/M phase (25.5% to 29.9%). The observed growth arrest in the G2/M phase (DNA damage response) by 12 indicates that it can induce growth inhibition in HeLa cells, while arrest in Sub G1 population indicates that these ligands may induce apoptotic and/or necrotic cell death.

Figure 3. (a) Imino and (b) aromatic region of 1D 1H NMR spectrum of c-MYC quadruplex DNA with 12 at different [ligand]:[DNA] ratios. Partial assignment of DNA signals is reported on the spectrum, according to the numbering shown in (c). Effect of ligands on the DNA signals are highlighted with yellow (strong CSP/line broadening at [ligand]:[DNA] < 1) or gray (CSP at [ligand]:[DNA] ≥ 1) boxes. Experimental conditions: 100 μM DNA, 25 mM Tris·HCl buffer (pH 7.4) containing 100 mM KCl in 10% DMSO-d6/90% H2O, 600 MHz, 298 K. (c) Sequence and numbering of the oligonucleotide used for NMR studies. (d) Mapping of the protons showing the strongest perturbations (strong CSP/line broadening at [ligand]:[DNA] < 1) on the NMR model of c-MYC [PDB code: 1XAV].21 Guanine, adenine, and thymine bases are colored in dark gray, cyan and blue, respectively. Perturbations are highlighted with yellow spheres.

Interestingly, negligible changes in GAPDH mRNA expression were observed. These results suggest that 12 effectively decreases c-MYC expression at transcriptional level with higher efficacy than 8 but they do not alter the expression of housekeeping genes. We then employed Western blot analysis to investigate the effect of the ligands 8 and 12 upon c-MYC expression at the protein level (Figure 4b,c). Densitometric analysis showed that the treatment of 5 μM 8 and 12 reduced the expression of c-MYC protein by ∼36% and ∼68%, respectively, compared to the control. Negligible reduction in D

DOI: 10.1021/acs.jmedchem.6b00328 J. Med. Chem. XXXX, XXX, XXX−XXX

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downregulate the c-MYC expression in transcriptional and translational level. In addition, the binaphthyl amine derivatives inhibit cancer cell growth by inducing Sub G1 phase cell cycle arrest and apoptosis.

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EXPERIMENTAL SECTION

Chemistry. All anhydrous solvents were dried or purified by standard techniques. All starting materials were used as supplied without purification. All reactions were generally carried out in ovendried flasks under inert atmosphere of argon unless otherwise noted. TLC was performed on Kieselgel60 F254 plates, and spots were visualized under UV light. Products were purified by flash chromatography on silica gel (100−200 mesh). 1H NMR spectra were recorded at 500 and 400 MHz instruments at 278 K. 13C NMR spectra were recorded on either 100 or 125 MHz with complete proton decoupling. Chemical shifts (δ) are reported in ppm. Coupling constants are quoted in hertz and are denoted as J. Mass spectra were recorded on a Micromass Q-Tof (ESI) spectrometer by +ve mode electrospray ionization. Elemental analysis (C, H, N) was performed with a PerkinElmer 2400 II series CHNS analyzer. All compounds were analyzed for purity by HPLC using UV absorbance detectors. All compounds showed ≥95% purity. Synthesis of Naphthyl Nonaflate 2. Under argon atmosphere, 1-naphthol 1 (200 mg, 1.4 mmol, 1.0 equiv) in dry THF (10 mL) and NaH (100.8 mg, 4.2 mmol, 3.0 equiv) was added. Then nonafluorobutanesulfonylfluoride (1.25 mL, 7.0 mmol, 5.0 equiv) was added dropwise at rt and stirred for 12 h, quenched with water, extracted with diethyl ether (3 × 10 mL), dried with Na2SO4, filtered, and concentrated. Residue was purified by column chromatography with 0.5% EtOAc/hexane to afford compound 2 (590 mg, 99%) as a colorless oil. 1H NMR (CDCl3, 500 MHz): δ 8.11 (1H, d, J = 8.5 Hz), 7.92 (1H, d, J = 8.1 Hz), 7.89−7.87 (1H, m), 7.69−7.64 (1H, m), 7.62−7.59 (1H,m), 7.51−7.48 (2H, m). 13C NMR (CDCl3, 500 MHz): δ 148.8, 134.9, 128.5, 128.0, 127.8, 127.3, 126.4, 125.1, 120.8, 117.8. HRMS (ESI) calcd for C14H8F9O3S [M + H]+, 427.0050; found, 427.0060. Elemental Analysis Calcd (%) for C14H7F9O3S: C, 39.45; H, 1.66; F, 40.11; O, 11.26; S, 7.52. Found: C, 39.49; H, 1.61; F, 40.25; O, 11.56; S, 7.54. Synthesis of Naphthyl Derivative 4. An oven-dried microwave tube containing a magnetic stirrer bar was charged with Pd2dba3 (11.0 mg, 0.012 mmol, 0.024 equiv) and XPhos (13.0 mg, 0.028 mmol, 0.056 equiv). The aryl nonaflate 2 (213 mg, 0.5 mmol, 1.0 equiv), amine 3 (106.0 mg, 0.7 mmol, 1.3 equiv), DBU (187 μL, 1.3 mmol, 2.5 equiv), and toluene (2 mL) were added at rt under an argon atmosphere and microwave irradiated for 3 h at 80 °C. The mixture was cooled to rt and diluted with water and EtOAc. The organic layer was separated, dried, and concentrated. The crude material was purified by column chromatography with EtOAc−hexane (5/95 to 10/ 90) to obtain naphthylamine 4 (127 mg, 92%) as greenish crystal; mp 131.2 °C. 1H NMR (CDCl3, 400 MHz): δ 7.97 (1H, d, J = 9.7 Hz), 7.90−7.88 (3H, m), 7.74−7.69 (1H, m), 7.24−7.50 (1H, m), 7.50− 7.49 (1H, m), 7.48−7.46 (2H, m), 6.83 (2H, d, J = 8.6 Hz), 6.21 (1H, s), 3.86 (3H, s). 13C NMR (CDCl3, 100 MHz): δ 167.1, 149.9, 136.3, 134.6, 131,4, 129.0, 128.5, 126.3, 126.1, 125.8, 125.3, 122.2, 120.4, 120.2, 114.1, 51.6. HRMS (ESI) calcd for C18H16NO2 [M + H]+, 278.1181; found, 278.1258. Elemental Analysis Calcd (%) for C18H15NO2: C, 77.96; H, 5.45; N, 5.05; O, 11.54. Found: C, 77.70; H, 5.29; N, 5.09; O, 11.60. Synthesis of Diester 5. To K2CO3 (193 mg, 1.4 mmol, 2.0 equiv) and naphthylamine 4 (200 mg, 0.7 mmol, 1.0 equiv) in a flask, 1,2dichloroethane (8 mL) was added and was stirred for 5 min at rt. To the mixture, anhydrous FeCl3 (130 mg, 0.8 mmol, 1.2 equiv) was added and stirred for 2 h at rt. Then triethylamine (1 mL) was added to the mixture and stirred for another 5 min and filtered through silica gel washed with ethyl acetate (20 mL × 3). The combined organic phases were concentrated and purified by column chromatography with EtOAc−hexane (5/95 to 10/90) to obtain compound 5 (367 mg, 95%), as a white solid; mp 195.3 °C. 1H NMR (CDCl3, 400 MHz): δ 8.10 (2H, d, J = 11.0 Hz), 7.95 (4H, d, J = 10.4 Hz), 7.60 (2H, d, J =

Figure 5. (a) Flow cytometric analysis of cell cycle parameters after incubation of HeLa cancer cells with ligands 8 and 12 (5 μM). P1, P2, P3, and P4 represent cell population at Sub G1, G0/G1, S, and G2/M, respectively. (b) Flow cytometric analysis of the mode of cancer cell death after treatment with 8 or 12 (5 μM) in HeLa cancer cells; Q3, Q4, Q2, and Q1 indicate healthy cells, early, late apoptotic, and necrotic cells, respectively. (c) Fluorescence microscopic images of localization of 12 in HeLa cells.

Binaphthyl Ligands Drive Cell toward Apoptosis. The mode of cell death induced by ligands 8 and 12 was investigated by flow cytometry using Annexin V and PI dual staining assay (Figure 5b). HeLa cells were treated with 5.0 μM of 8 and 12 for 24 h. The analyses of dot-plots show a dosedependent increase in the population of apoptotic cells from ∼1.1% to ∼37.9% for 8 and ∼1.1% to ∼55.9% for 12, respectively. However, the populations of necrotic cells were only 0.5% and 0.6% for 8 and 12, respectively, relative to the control (cell treated with 0.1% DMSO). These results indicate that G-quadruplex binding ligands 8 and 12 cause cancer cell death by inducing apoptosis. Binaphthyl Ligands as Fluorescent Probes for Cell Imaging. Fluorescence microscopy was employed to examine the cellular internalization of binaphthyl ligands. HeLa cells were treated with 8 and 12 (5.0 μM) for 2 h, and the fluorescence microscopic images were taken. The merged images shows that both the ligands 8 (SI, Figure S8) and 12 (Figure 5c) localizes inside the nucleus and cytoplasm. These results suggest that both the ligands are cell permeable and may bind to the cellular DNA.

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CONCLUSION We have synthesized two novel derivatives of binaphthyl amine that selectively bind c-MYC G-quadruplex over ds DNA. Binaphthyl amine containing two additional triazole moieties binds the c-MYC G-quadruplex with higher affinity. NMR analysis suggests that both the ligands interact with the 3′-end G-tetrad of c-MYC, possibly by restructuring the TAA-3′ capping. In vitro cellular assays reveal that both ligands E

DOI: 10.1021/acs.jmedchem.6b00328 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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9.6 Hz), 7.51−7.48 (6H, m), 7.38−7.34 (2H, m), 6.97 (4H, d, J = 10.6 Hz), 6.31 (2H, s), 3.88 (6H, s). 13C NMR (CDCl3, 100 MHz): δ 167.2, 150.0, 136.5, 135.5, 134.2, 132.0, 129.0, 128.2, 127.4, 126.6, 126.2, 122.4, 120.8, 119.4, 114.5, 51.8. HRMS (ESI) calcd for C36H28N2NaO4 [M + Na]+, 575.1947; found, 575.2221. Elemental Analysis Calcd (%) for C36H28N2O4: C, 78.24; H, 5.11; N, 5.07; O, 11.58. Found: C, 77.95; H, 5.13; N, 5.05; O, 11.59. Synthesis of Diacid 6. To diester 5 (100.0 mg, 0.2 mmol) in methanol and THF (1:1, 10 mL), NaOH (5 mL, 2N solution in water) was added at 0 °C. After stirring for 12 h at 60 °C, solvents were removed in vacuo and the residue was dissolved in water and acidified with formic acid. The precipitate formed was filtered and purified by column chromatography with MeOH−CH2Cl2 (2/98 to 10/90) to afford 6 (86 mg, 82%) as a light-yellow solid; mp 225−226 °C. 1H NMR (DMSO-d6, 400 MHz): δ 12.33 (2Hbr, s), 8.95 (2H, s), 8.20 (2H, d, J = 8.5 Hz), 7.80 (4H, d, J = 8.7 Hz), 7.60 (2H, d, J = 7.6 Hz), 7.55−7.48 (4H, m), 7.41−7.37 (2H, m), 7.32 (2H, d, J = 8.5 Hz), 7.06 (4H, d, J = 8.7 Hz). 13C NMR (DMSO-d6, 100 MHz): δ 167.3, 150.3, 137.2, 133.6, 131.3, 128.4, 128.2, 127.5 127.4, 126.6, 125.7, 123.4, 120.1, 118.3, 114.2. HRMS (ESI) calcd for C34H24N2NaO4 [M + Na]+, 547.1634, found, 547.1686. Elemental Analysis Calcd (%) for C34H24N2O4: C, 77.85; H, 4.61; N, 5.34; O, 12.20. Found: C, 78.05; H, 4.65; N, 5.38; O, 12.22. Synthesis of 8. To compound 6 (200 mg, 0.4 mmol, 1.0 equiv) in dry DMF (25 mL), EDC·HCl (306 mg, 1.6 mmol, 4.0 equiv), NMM (359 μL, 3.2 mmol, 8.0 equiv), and 1-hydroxy-1H-benzotriazole (HOBT) (122 mg, 0.8 mmol, 2.0 equiv) were added and the mixture was stirred for 15 min. Then amine 7 (135 μL, 1.2 mmol, 3.0 equiv) was added dropwise to the reaction mixture and was strirred for 12 h at rt. The solvent was removed in vacuo and was purified by column chromatography with MeOH−CH2Cl2 (2/98 to 5/95) to afford 8 (210 mg, 76%) as a light-yellow solid. 1H NMR (DMSO-d6, 500 MHz): δ8.70 (2H, s), 8.26−8.21 (4H, m), 7.73 (4H, d, J = 8.7 Hz), 7.56−7.50 (4H, m), 7.55 (2H, d, J = 7.6 Hz), 7.52 (2H, t, J = 7.5 Hz), 7.46 (2H, d, J = 7.6 Hz), 7.38 (2H, t, J = 7.9 Hz), 7.32 (2H, d, J = 8.4 Hz), 3.27−3.23 (4H, m), 2.26 (4H, t, J = 7.1 Hz), 2.14 (12 H, s), 1.65−1.62 (4H, m). 13C NMR (DMSO-d6, 100 MHz): δ 166.0, 148.3, 138.0, 134.0, 133.0, 129.0, 128.6, 128.2, 128.0, 126.5, 125.5, 125.0, 123.3, 117.0, 115.0, 57.0, 45.2, 39.2, 27.3. HRMS (ESI) calcd for C44H48N6NaO2 [M + Na]+, 715.3736; found, 715.3925. Elemental Analysis Calcd (%) for C44H48N6O2: C, 76.27; H, 6.98; N, 12.13; O, 4.62. Found: C, 76.31; H, 7.02; N, 12.18; O, 4.59. Synthesis of Dialkyne 10. Dialkyne 10 (light-yellow solid) was prepared from 6 (200.0 mg, 0.4 mmol, 1.0 equiv) and propargylamine 9 (128 μL, 2.0 mmol, 5.0 equiv) using the same procedure as 8. 1H NMR (MeOD-d4, 500 MHz): δ 8.17 (2H, d, J = 8.5 Hz), 7.72 (4H, d, J = 8.7 Hz), 7.56 (2H, d, J = 7.4 Hz), 7.45 (4H, d, J = 7.3 Hz), 7.38 (2H, d, J = 8.4 Hz), 7.36−7.31 (2H, m), 7.01 (4H, d, J = 8.7 Hz), 4.14 (4H, d, J = 2.4 Hz), 2.57 (2H, s). 13C NMR (MeOD-d4, 100 MHz): δ 170.0, 151.5, 139.0, 136.1, 135.5, 130.4, 130.8, 129.2, 128.0, 127.3, 126.7, 124.4, 124.2, 119.2, 115.5, 81.2, 72.0, 30.0. HRMS (ESI) calcd for C40H30N4O2Na[M + Na]+, 621.2266; found, 621.1959. Elemental Analysis Calcd (%) for C40H30N4O2: C, 80.25; H, 5.05; N, 9.36; O, 5.34. Found: C, 80.63; H, 5.03; N, 9.29; O, 5.29. Synthesis of 12. A mixture of 10 (120 mg, 0.2 mmol, 1.0 equiv), azide 11 (79 mg, 0.6 mmol, 3.0 equiv), Na ascorbate (32 mg, 0.16 mmol, 0.8 equiv), and CuSO4·5H2O (15 mg, 0.06 mmol, 0.3 equiv) in H2O/tBuOH (7:3, 10 mL) was stirred at rt for 10 h. The reaction mixture was concentrated and purified by column chromatography with MeOH−CH2Cl2 (2/98 to 5/95) to afford 12 (123 mg, 72%) as a colorless solid. 1H NMR (MeOD-d4, 500 MHz): δ 8.16 (2H, d, J = 8.5 Hz), 7.92 (2H, s), 7.74 (4H, d, J = 8.7 Hz), 7.56 (2H, d, J = 7.5 Hz), 7.46−7.43 (4H, m), 7.38 (2H, d, J = 8.4 Hz), 7.33−7.29 (2H, m), 7.01 (4H, d, J = 8.8 Hz), 4.62 (4H, s), 4.44 (4H, t, J = 6.8 Hz), 2.48 (4H, sbr), 2.35 (12H, s), 2.14−2.08 (4H, m). 13C NMR (MeOD-d4, 100 MHz): δ 170.1, 151.5, 139.0, 136.2, 135.5, 130.4, 130.1, 129.2, 128.1, 127.3, 126.7, 124.5, 124.3, 124.2, 120.0, 115.4, 57.0, 45.0, 36.1, 31.0, 28.3. HRMS (ESI) calcd for C50H54N12NaO2 [M + Na]+, 877.4390; found, 877.4507. Elemental Analysis Calcd (%) for C50H54N12O2: C,

70.23; H, 6.37; N, 19.66; O, 3.74. Found: C, 70.25; H, 6.33; N, 19.74; O, 3.73.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00328. NMR data, CD spectroscopic data, additional data of FRET, UV−vis titration, fluorescence titration, NMR titration, cytotoxicity data, and cell image (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +91 33 2473 4971, Ext 1405. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Department of Science and Technology (DST), Department of Biotechnology (DBT), CSIR-India, for funding. R.P. and M.D. thank DST for INSPIRE fellowships. The work was supported by LOEWE program: SYNCHEMBIO. H.S. is a member of the DFG-funded cluster of excellence: macromolecular complexes. B.M.R.Z. is supported by the state of Hesse.

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ABBREVIATIONS USED MTT, Methyl thiazolyl tetrazolium; PI, Propidium iodide; r.t, room temperature REFERENCES

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