Benzofuroquinoline Derivatives Had Remarkable Improvement of their

Aug 8, 2012 - ABSTRACT: In order to improve the selectivity of 5-N-methyl quindoline (cryptolepine) derivatives as telomeric quadruplex binding...
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Benzofuroquinoline Derivatives Had Remarkable Improvement of their Selectivity for Telomeric G‑Quadruplex DNA over Duplex DNA upon Introduction of Peptidyl Group Yi Long, Zeng Li, Jia-Heng Tan, Tian-Miao Ou, Ding Li, Lian-Quan Gu, and Zhi-shu Huang* School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China S Supporting Information *

ABSTRACT: In order to improve the selectivity of 5-N-methyl quindoline (cryptolepine) derivatives as telomeric quadruplex binding ligands versus duplex DNA, a series of peptidyl-benzofuroquinoline (PBFQ) conjugates (2a−2n) were designed and synthesized. Their interactions with telomeric quadruplex and duplex DNA were examined by using the fluorescence resonance energy transfer (FRET) melting assay, surface plasmon resonance (SPR), circular dichroism spectroscopy (CD), and molecular modeling studies. Introduction of a peptidyl group at 11-position of the aromatic benzofuroquinoline scaffold not only effectively increased its binding affinity, but also significantly improved its selectivity toward telomeric quadruplex versus duplex DNA. Combined with the data for their inhibitory effects on telomerase activity, their structure−activity relationships (SARs) studies showed that the types of amino acid residues and the length of the peptidyl side chains were important for the improvement of their interactions with the telomeric G-quadruplex. Long-term exposure of human cancer cells to 2c showed a remarkable cessation in population growth and cellular senescence phenotype, and accompanied by a shortening of the telomere length.

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INTRODUCTION Cancer cells mostly maintain their indefinite telomere lengths via the synthesis of further telomeric DNA repeats using the telomerase enzyme, which will trigger cellular immortalization, a key step along the pathway to tumorigenesis.1,2 Human telomeres comprise tandem repeats of the DNA motif (TTAGGG) together with associated telomeric proteins,3,4 and the terminal 150−250 nucleotides at the extreme 3′-ends of telomeres are single-stranded, which could easily adopt Gquadruplex structures from stacked tetrads of hydrogen-bonded guanine bases.5,6 Stabilization of telomeric G-quadruplexes by the binding of small molecules can lead to a range of biological effects,7 including inhibition of the end-capping8 and catalytic functions9−11 of the telomerase enzyme. Thus, the design of drugs targeting at the telomeric G-quadruplex is an appropriate and promising approach for cancer chemotherapy. However, further efforts are still needed to improve the G-quadruplex selectivity over duplex DNA because ligand interaction with duplex DNA results in acute toxic and drastic side effects on normal tissues.12 In the past several years, a number of small molecules have been reported to bind with G-quadruplex DNA and effectively inhibit telomerase activity. Most of these molecules share a common feature of polycyclic aromatic cores that stack by π−π interactions over the terminal planar G-tetrads of a quadruplex,13 such as acridines,14 anthraquinones,15 indoloquinolines,16 and porphyrins.17 In addition, cationic side chains © 2012 American Chemical Society

can interact with the negatively charged phosphate backbones in G-quadruplexes.13 Our group has endeavored to explore potent and selective G-quadruplex ligands for several years. Indoloquinoline or benzofuroquinoline derivatives modified from cryptolepine are some among those ligands reported by us.16,18 These ligands were capable of interacting with the telomeric G-quadruplex structure and showed inhibitory effect on telomerase; however, they also can intercalate into two base pairs of duplex DNA to a certain extent.19 Thus, further efforts were undertaken for the purpose of improving selectivity of these compounds. As is well-known, G-quadruplex DNA binding proteins are potentially useful for investigating the biologic role of Gquadruplex DNA. The specificity of recognition between Gquadruplex DNA binding proteins and G-quadruplex structures depends on the specific peptide sequences of those binding proteins.20 For example, the specificity of G-quadruplex recognition depends on the guanidinium group of the arginine in the Arg-Gly-Gly repeat (RGG) domain in the C-terminal of Ewing’s sarcoma protein (EWS).21 In the meantime, it has also been reported that the combination of selected peptides to DNA binding scaffolds would greatly increase their selectivity to G-quadruplex versus duplex DNA.22 Additionally, a Received: March 13, 2012 Revised: July 21, 2012 Published: August 8, 2012 1821

dx.doi.org/10.1021/bc300123m | Bioconjugate Chem. 2012, 23, 1821−1831

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The Fmoc protected various amino acids (4 equiv ratio excess to the resin) were coupled orderly to the Rink amide AM resin (500 mg) using DIC/HOBT (190 mg/200 mg) in DMF (4 mL) for 3 h, and the deprotection of the Fmoc group was carried out with piperidine/DMF 25% (2 mL) for 30 min. Then, intermediate compound 2 (322 mg) was mixed with deprotected peptidyl side chains using DIC/HOBT (475 mg/ 500 mg) in DMF (4 mL), with shaking for 12 h. After coupling with the compound 2, the resin was treated with the cleavage reagent (95% TFA, 5% dd H2O) for 2 h, washed with dry ether (30 mL), and then evaporated under vacuum. The crude products were purified by using preparative RP-HPLC with CH3CN/H2O (40:60) elution, and lyophilized to give compounds 2a−2n. 11-(Carboxymethylamino)-5-methylbenzofuro[3,2-b]quinolin-5-ium (II). Compound 1 was treated with glycin in 2-ethoxyethanol solution gave the intermediate compound 2 as a white solid with a yield of 41%. mp 217−219 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.75 (t, J = 6.3 Hz, 1H), 8.67 (t, J = 8.4 Hz, 2H), 8.44 (d, J = 8.9 Hz, 1H), 8.17−8.07 (m, 1H), 7.96−7.87 (m, 2H), 7.84 (t, J = 7.5 Hz, 1H), 7.65 (t, J = 7.1 Hz, 1H), 4.80 (d, J = 6.3 Hz, 2H), 4.59 (s, 3H); HRMS (ESI+) m/ z: Calc. for C18H15N2O3+: 307.1077 [M+H]+. Found 307.1087 [M+H]+. (S)-11-(2-(1-Amino-5-guanidino-1-oxopentan-2-ylamino)-2-oxoethylamino)-5-methylbenzofuro[3,2-b]quinolin-5-ium (2a). Peptidyl coupling order is arginine, following the general procedure to give the desired compound 2a as a straw yellow solid with a yield of 32%. mp 202−203 °C. 1 H NMR (400 MHz, DMSO-d6) δ 9.71 (t, J = 6.4 Hz, 1H), 8.71 (d, J = 8.5 Hz, 1H), 8.63 (d, J = 8.2 Hz, 1H), 8.56 (d, J = 8.2 Hz, 1H), 8.43 (d, J = 9.0 Hz, 1H), 8.15−8.07 (m, 1H), 7.87 (ddd, J = 21.5, 15.3, 8.0 Hz, 3H), 7.64 (t, J = 7.4 Hz, 2H), 7.49 (s, 1H), 7.13 (s, 2H), 4.79 (d, J = 6.4 Hz, 2H), 4.58 (s, 3H), 4.25 (dd, J = 14.7, 7.0 Hz, 2H), 3.04 (dd, J = 13.1, 7.4 Hz, 2H), 1.73 (td, J = 10.5, 3.9 Hz, 1H), 1.63−1.28 (m, 4H); HRMS (ESI+) m/z: Calc. for C24H28N7O3+: 462.2248 [M+H]+. Found 462.2232 [M+H]+. 11-(2-(2-(2-Amino-2-oxoethylamino)-2-oxoethylamino)-2-oxoethylamino)-5-methylbenzofuro[3,2-b]quinolin-5-ium (2b). Peptidyl coupling order is glycine− glycine, following the general procedure to give the desired compound 2b as a white solid with a yield of 29%. mp 200−212 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.72 (t, J = 6.5 Hz, 1H), 8.70 (t, J = 8.4 Hz, 2H), 8.64 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 8.9 Hz, 1H), 8.12 (dd, J = 9.8, 6.4 Hz, 2H), 7.93 (t, J = 5.9 Hz, 2H), 7.87−7.77 (m, 1H), 7.65 (ddd, J = 8.2, 5.8, 2.4 Hz, 1H), 7.23 (s, 1H), 7.02 (s, 1H), 4.78 (d, J = 6.4 Hz, 2H), 4.59 (s, 3H), 3.83 (d, J = 5.7 Hz, 2H), 3.60 (d, J = 5.8 Hz, 2H); HRMS (ESI+) m/z: Calc. for C22H22N5O3+: 420.1666 [M+H]+. Found 420.1658 [M+H]+. (S)-11-(2-(2-(1-Amino-5-guanidino-1-oxopentan-2ylamino)-2-oxoethylamino)-2-oxoethylamino)-5methylbenzofuro[3,2-b]quinolin-5-ium (2c). Peptidyl coupling order is arginine−glycine, following the general procedure to give the desired compound 2c as a straw yellow solid with a yield of 28%. mp 195−196 °C. 1H NMR (400 MHz, DMSOd6) δ 9.68 (t, J = 5.7 Hz, 1H), 8.66 (dd, J = 20.8, 8.5 Hz, 3H), 8.43 (d, J = 8.8 Hz, 1H), 8.17−8.09 (m, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.92 (s, 2H), 7.87−7.78 (m, 1H), 7.63 (dd, J = 13.2, 5.6 Hz, 2H), 7.35 (s, 1H), 7.05 (s, 1H), 4.77 (d, J = 5.6 Hz, 2H), 4.59 (s, 3H), 4.25−4.14 (m, 3H), 3.88−3.81 (m, 2H), 3.04 (d, J = 5.5 Hz, 2H), 1.72−1.63 (m, 1H), 1.58−1.27 (m,

significant role of ionic interactions has been confirmed, since N-terminal protonatable amino acids (Lys, Arg) largely incremented G-quadruplex binding.23 Inspired by these findings, introduction of alkaline peptidyl side chains to classic benzofuroquinoline scaffold may be a feasible strategy through mimicking specific peptide sequences to improve its binding selectivity for telomeric G-quadruplex DNA. On this basis, a series of 11-peptidyl benzofuroquinoline (PBFQ) derivatives were designed for developing the ligands with better selective recognition and stabilizing ability of Gquadruplex. Peptidyl side chains mainly consist of alkaline and aromatic amino acid residues, such as Arg, Lys, His, and Phe. The peptidyl sequences are arranged as follows: Arg/Lys/ His are located at the terminal of peptide chains, which can form alkaline positive center interacting with grooves and loops of G-quadruplex, Phe is located at the middle of peptide chains forming a certain steric conformation, and Gly played a joint role connecting with the scaffold. According to this principle of arrangement, a series of various peptidyl sequences were designed for this study, and some of these sequences just for control test purpose. We report here our research on the synthesis of P-BFQ conjugates, their interactions with telomeric G-quadruplex versus duplex DNA using biophysical and biochemical assays, and their inhibitory effects on telomerase activity by using TRAP-LIG assay, with compound 1 (reported earlier)16 as reference compound. The ligand−quadruplex interactions were further explored by using molecular modeling studies, and cellular studies were performed to evaluate the cell senescence and telomere shortening effect induced by these PBFQ derivatives.

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EXPERIMENTAL PROCEDURES Materials. The Fmoc protected amino acids, Rink amide AM resin, and others used for peptide synthesis were purchased from GL Biochem (Shanghai) Ltd. All oligomers/primers used in this study were purchased from Invitrogen and Sangon Biotech (China). All other chemicals or solvents were of analytical grade or better. Stock solutions of all the derivatives (10 mM) were made using DMSO (10%) or double-distilled deionized water. Synthesis and Characterization of P-BFQ Conjugates. 1 H and 13C NMR spectra were recorded using TMS as the internal standard in DMSO-d6 with a Bruker BioSpin GmbH spectrometer at 400 MHz, mass spectra (MS) were recorded on a Shimadzu LCMS-2010A instrument with an ESI or ACPI mass selective detector, and high resolution mass spectra (HRMS) on Shimadzu LCMS-IT-TOF. Melting points (m.p.) were determined using a SRS-OptiMelt automated melting point instrument without correction. Flash column chromatography was performed with silica gel (200−300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. The purification of all tested compounds was processed with preparative HPLC using a Varian Pro Star equipped with an UV−vis detector (BioRad) and a Phenomenex C18 (10.0 × 250 mm, 5 μm) column. The purities of all compounds were confirmed to be higher than 95% by using analytical HPLC with a dual pump Shimadzu LC-20AB system equipped with a Ultimate XB-C18 column (4.6 × 250 mm, 5 μm), and eluted with methanol/ water (40:60 to 65:35) containing 0.1% TFA at a flow rate of 1 mL/min. General Procedure for Preparation of Compounds 2a−2n. Peptidyl side chains were synthesized using solid-phase methodology by manual operation of a peptide synthesizer. 1822

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4H); HRMS (ESI+) m/z: Calc. for C26H31N8O4+: 519.2463 [M +H]+. Found 519.2446 [M+H]+. (S)-11-(2-(2-(1-Amino-1-oxo-3-phenylpropan-2-ylamino)-2-oxoethylamino)-2-oxoethylamino)-5methylbenzofuro[3,2-b]quinolin-5-ium (2d). Peptidyl coupling order is phenylalanine-glycine, following the general procedure to give the desired compound 2d as a straw yellow solid with a yield of 27%. mp 200−202 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.73 (t, J = 6.0 Hz, 1H), 8.71 (d, J = 8.5 Hz, 1H), 8.60 (dd, J = 13.1, 6.7 Hz, 2H), 8.42 (d, J = 8.9 Hz, 1H), 8.18−8.07 (m, 2H), 7.89 (d, J = 3.5 Hz, 2H), 7.82 (t, J = 7.6 Hz, 1H), 7.67−7.60 (m, 1H), 7.43 (s, 1H), 7.26−7.17 (m, 4H), 7.15 (d, J = 5.1 Hz, 1H), 7.07 (s, 1H), 4.74 (d, J = 5.9 Hz, 2H), 4.57 (s, 3H), 4.43 (dd, J = 13.0, 8.5 Hz, 1H), 3.85 (d, J = 21.8 Hz, 2H), 3.68 (d, J = 16.6 Hz, 2H); HRMS (ESI+) m/z: Calc. for C29H28N5O4+: 510.2136 [M+H]+. Found 510.2140 [M +H]+. 11-(2-(2-((2S)-1-Amino-3-(4H-imidazol-4-yl)-1-oxopropan-2-ylamino)-2-oxoethylamino)-2-oxoethylamino)-5methylbenzofuro[3,2-b]quinolin-5-ium (2e). Peptidyl coupling order is histidine-glycine, following the general procedure to give the desired compound 2e as a straw yellow solid with a yield of 29%. mp 188−189 °C. 1H NMR (400 MHz, DMSOd6) δ 9.90 (t, J = 6.1 Hz, 1H), 8.91 (s, 1H), 8.82−8.71 (m, 2H), 8.61 (d, J = 8.3 Hz, 1H), 8.41 (d, J = 9.0 Hz, 1H), 8.35 (d, J = 8.3 Hz, 1H), 8.12−8.05 (m, 1H), 7.91−7.85 (m, 2H), 7.83− 7.78 (m, 1H), 7.62 (ddd, J = 8.1, 5.1, 3.0 Hz, 1H), 7.43 (s, 1H), 7.28 (s, 1H), 7.21 (s, 1H), 4.78 (d, J = 6.0 Hz, 2H), 4.57 (s, 3H), 4.53−4.45 (m, 2H), 3.87−3.77 (m, 2H), 3.13 (dd, J = 15.2, 4.5 Hz, 1H), 2.93 (dd, J = 15.2, 9.2 Hz, 1H); HRMS (ESI +) m/z: Calc. for C26H26N7O4+: 500.2041 [M+H]+. Found 500.2056 [M+H]+. (S)-11-(2-(2-(1,6-Diamino-1-oxohexan-2-ylamino)-2oxoethylamino)-2-oxoethylamino)-5-methylbenzofuro[3,2-b]quinolin-5-ium (2f). Peptidyl coupling order is lysineglycine, following the general procedure to give the desired compound 2f as a straw yellow solid with a yield of 26%. mp 201−202 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.79 (t, J = 6.4 Hz, 1H), 8.75−8.67 (m, 2H), 8.63 (d, J = 8.2 Hz, 1H), 8.43 (d, J = 9.0 Hz, 1H), 8.13−8.09 (m, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.92 (dd, J = 7.0, 4.8 Hz, 2H), 7.84 (s, 1H), 7.64 (ddd, J = 8.2, 5.8, 2.5 Hz, 1H), 7.35 (s, 1H), 7.02 (s, 1H), 4.77 (d, J = 6.4 Hz, 2H), 4.58 (s, 3H), 4.20−4.11 (m, 2H), 3.85 (t, J = 5.5 Hz, 2H), 2.75−2.66 (m, 2H), 1.68−1.60 (m, 1H), 1.59−1.32 (m, 4H), 1.25 (dt, J = 16.3, 8.7 Hz, 2H); HRMS (ESI+) m/z: Calc. for C26H31N6O4+: 491.2401 [M+H]+. Found 491.2420 [M+H]+. 11-(2-((S)-5-amino-6-((S)-1-amino-5-guanidino-1-oxopentan-2-ylamino)-6-oxohexylamino)-2-oxoethylamino)-5-methylbenzofuro[3,2-b]quinolin-5-ium (2g). Peptidyl coupling order is arginine-lysine, following the general procedure to give the desired compound 2g as a yellow solid with a yield of 24%. mp 212−213 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.80 (t, J = 12.5 Hz, 1H), 8.86 (s, 1H), 8.72 (d, J = 8.3 Hz, 1H), 8.61 (d, J = 8.1 Hz, 1H), 8.40 (d, J = 8.9 Hz, 1H), 8.21 (d, J = 6.4 Hz, 1H), 8.12−8.05 (m, 1H), 7.89 (t, J = 7.8 Hz, 2H), 7.79 (d, J = 7.2 Hz, 3H), 7.65−7.60 (m, 1H), 7.22 (s, 1H), 6.95 (s, 1H), 4.81 (d, J = 5.2 Hz, 2H), 4.55 (s, 3H), 4.26 (dd, J = 11.8, 5.6 Hz, 1H), 4.10 (dd, J = 12.8, 6.9 Hz, 1H), 3.09 (d, J = 5.6 Hz, 1H), 2.99 (s, 2H), 2.67 (s, 2H), 1.76−1.58 (m, 4H), 1.51 (dd, J = 14.3, 8.8 Hz, 4H), 1.43−1.19 (m, 5H); HRMS (ESI+) m/z: Calc. for C30H40N9O4+: 590.3198 [M +H]+. Found 590.3209 [M+H]+.

11-(2-((S)-1-((S)-1-Amino-5-guanidino-1-oxopentan-2ylamino)-1-oxo-3-phenylpropan-2-ylamino)-2-oxoethylamino)-5-methylbenzofuro[3,2-b]quinolin-5-ium (2h). Peptidyl coupling order is arginine-phenylalanine, following the general procedure to give the desired compound 2h as a straw yellow solid with a yield of 26%. mp 204−205 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.56 (t, J = 6.4 Hz, 1H), 8.71− 8.59 (m, 3H), 8.43 (d, J = 9.0 Hz, 1H), 8.21 (d, J = 7.9 Hz, 1H), 8.15−8.09 (m, 1H), 7.89 (t, J = 7.8 Hz, 1H), 7.86−7.81 (m, 1H), 7.64 (t, J = 7.7 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.43−7.39 (m, 1H), 7.23 (dd, J = 7.3, 3.9 Hz, 5H), 7.11 (dd, J = 11.3, 8.2 Hz, 5H), 4.71 (dd, J = 17.3, 6.0 Hz, 2H), 4.59 (s, 3H), 4.18 (dd, J = 13.3, 7.3 Hz, 1H), 3.00 (ddd, J = 36.8, 13.2, 4.7 Hz, 4H), 1.61 (dd, J = 13.1, 6.8 Hz, 1H), 1.48−1.29 (m, 4H); HRMS (ESI+) m/z: Calc. for C33H37N8O4+: 609.2932 [M +H]+. Found 609.2932 [M+H]+. 11-(2-((S)-1-((S)-1-Amino-5-guanidino-1-oxopentan-2ylamino)-3-(1H-imidazol-4-yl)-1-oxopropan-2-ylamino)2-oxoethylamino)-5-methylbenzofuro[3,2-b]quinolin-5ium (2i). Peptidyl coupling order is arginine-histidine, following the general procedure to give the desired compound 2i as a straw yellow solid with a yield of 28%. mp 193−194 °C. 1 H NMR (400 MHz, DMSO-d6) δ 9.74 (t, J = 12.4 Hz, 1H), 8.90 (d, J = 8.0 Hz, 1H), 8.74 (s, 1H), 8.69 (d, J = 8.4 Hz, 1H), 8.60 (d, J = 8.2 Hz, 1H), 8.42 (d, J = 9.0 Hz, 1H), 8.31 (d, J = 7.2 Hz, 1H), 8.13−8.08 (m, 1H), 7.92−7.87 (m, 1H), 7.81 (t, J = 7.4 Hz, 2H), 7.73 (d, J = 8.4 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.50 (s, 1H), 7.26 (s, 1H), 7.13 (s, 1H), 4.97−4.63 (m, 4H), 4.57 (s, 3H), 4.13 (dd, J = 13.0, 7.9 Hz, 2H), 3.16 (dd, J = 14.8, 5.3 Hz, 2H), 3.01−2.90 (m, 3H), 1.68−1.60 (m, 1H), 1.58−1.43 (m, 2H), 1.40−1.33 (m, 2H); HRMS (ESI+) m/z: Calc. for C30H35N10O4+: 599.2766 [M+H]+. Found 599.2780 [M+H]+. (S)-11-(1-Amino-6-carbamoyl-1-imino-8,11,14-trioxo2,7,10,13-tetraazapentadecan-15-ylamino)-5methylbenzofuro[3,2-b]quinolin-5-ium (2j). Peptidyl coupling order is arginine-glycine-glycine, following the general procedure to give the desired compound 2j as a straw yellow solid with a yield of 20%. mp 187−188 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.78 (s, 1H), 8.71 (d, J = 8.1 Hz, 1H), 8.65 (d, J = 8.1 Hz, 1H), 8.43 (d, J = 8.9 Hz, 1H), 8.22 (s, 1H), 8.14−8.10 (m, 1H), 8.02 (d, J = 6.1 Hz, 1H), 7.96 (d, J = 8.1 Hz, 3H), 7.86−7.82 (m, 1H), 7.66 (s, 4H), 7.37 (s, 1H), 7.02 (s, 1H), 4.80 (d, J = 3.5 Hz, 2H), 4.60 (s, 3H), 4.21− 4.15 (m, 4H), 3.10 (dd, J = 3.8, 1.6 Hz, 2H), 1.68 (dd, J = 2.6, 1.6 Hz, 1H), 1.52−1.48 (m, 4H); HRMS (ESI+) m/z: Calc. for C28H34N9O5+: 576.2677 [M+H]+. Found 576.2699 [M+H]+. 11-((6S,9S)-1-Amino-9-benzyl-6-carbamoyl-1-imino8,11,14-trioxo-2,7,10,13-tetraazapentadecan-15-ylamino)-5-methylbenzofuro[3,2-b]quinolin-5-ium (2k). Peptidyl coupling order is arginine-phenylalanine-glycine, following the general procedure to give the desired compound 2k as a straw yellow solid with a yield of 22%. mp 215−217 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.73−9.58 (m, 1H), 8.69 (d, J = 8.2 Hz, 1H), 8.64 (d, J = 8.1 Hz, 1H), 8.59 (s, 1H), 8.43 (d, J = 9.0 Hz, 1H), 8.29 (d, J = 4.5 Hz, 1H), 8.20 (d, J = 7.3 Hz, 1H), 8.15−8.09 (m, 1H), 7.90 (d, J = 4.5 Hz, 2H), 7.87−7.79 (m, 2H), 7.69 (s, 3H), 7.57 (s, 1H), 7.34 (d, J = 6.8 Hz, 1H), 7.21 (t, J = 8.4 Hz, 4H), 7.15−7.12 (m, 1H), 7.06 (s, 1H), 4.75 (d, J = 3.8 Hz, 2H), 4.59 (s, 3H), 4.24 (d, J = 5.2 Hz, 1H), 3.01 (d, J = 13.7 Hz, 2H), 2.73 (dd, J = 13.4, 6.9 Hz, 4H), 1.72−1.64 (m, 2H), 1.35−1.26 (m, 3H); HRMS (ESI+) m/z: Calc. for C35H40N9O5+: 666.3147 [M+H]+ . Found 666.3132 [M+H]+. 1823

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and detection at 530 nm were taken at intervals of 1 °C over the range 37−99 °C, with a constant temperature being maintained for 30 s prior to each reading to ensure a stable value. The melting of the G-quadruplex was monitored alone or in the presence of various concentrations of compounds and/or of double-stranded competitor ds26 (5′-CAATCGGATCGAATTCGATCCGATTG-3′). Final analysis of the data was carried out using Origin 7.5 (OriginLab Corp.). Surface Plasmon Resonance. SPR measurements were performed on a ProteOn XPR36 Protein Interaction Array system (Bio-Rad Laboratories, Hercules, CA) using a Neutravidin-coated GLH sensor chip. In a typical experiment, biotinylated HTG21 (5′-d(GGG[TTAGGG]3)-3′) was folded in filtered and degassed running buffer (50 mM Tris-HCl, pH 7.2, 100 mM KCl). The DNA samples were then captured (∼1000 RU) in flow cell 1, leaving the fourth flow cell as a blank. Ligand solutions (at 4, 2, 1, 0.5, 0.25, 0.125 μM) were prepared with running buffer by serial dilutions from stock solution. Five concentrations were injected simultaneously at a flow rate of 100 μL/min for 150 s of association phase, followed with 300 s of dissociation phase at 25 °C. The GLH sensor chip was regenerated with short injection of 50 mM NaOH between consecutive measurements. The final graphs were obtained by subtracting blank sensorgrams from quadruplex sensorgrams. Data were analyzed with ProteOn manager software, using the Equilibrium method for fitting kinetic data. CD Measurements. CD experiments were performed on a Chirascan circular dichroism spectrophotometer (Applied Photophysics). A quartz cuvette with 4 mm path length was used for the spectra recorded over a wavelength range of 230− 450 at 1 nm bandwidth, 1 nm step size, and 0.5 s time per point. The oligomer HTG21 (5′-d(GGG[TTAGGG]3)-3′) was diluted from stock to the required concentration (5 μM) in 10 mM Tris-HCl buffer, pH 7.2, 100 mM KCl, and then annealed by heating at 95 °C for 5 min, gradually cooled to room temperature, and incubated at 4 °C overnight. Then, CD titration was performed at a fixed HTG21 concentration (5 μM) with various concentrations (0−5 mol equiv) of the ligands in buffer containing 100 mM KCl at 25 °C. After each addition of ligand, the reaction was stirred and allowed to equilibrate for at least 10 min (until no elliptic changes were observed), and then a CD spectrum was collected. A buffer baseline was collected in the same cuvette, and subtracted from the sample spectra. Final analysis of the data was carried out using Origin 7.5 (OriginLab Corp.). Molecular Modeling. The crystal structure of the parallel 22-mer telomeric G-quadruplex (PDB ID 1KF1)25 was used as an initial model to study the interaction between the P-BFQ derivatives and telomeric DNA. For comparison with the DNA d(GGG[TTAGGG]3) we used in the FRET and CD experiments, we removed the terminal 5′ adenine residue from the propeller-type structure and five adenines from each end of the hybrid-type structure to generate the 21-mer structure. Water molecules were removed from the PDB file, while the missing hydrogen atoms were added to the system using the Biopolymer module implemented in the SYBYL7.3.5 molecular modeling software from Tripos Inc. (St. Louis, MO). Ligand structures were constructed by adopting the empirical Gasteiger-Huckel (GH) partial atomic charges, and then were optimized (Tripos force field) with a nonbond cutoff of 12 Å and a convergence of 0.01 kcal·mol−1/Å over 10 000 steps using the Powell conjugate-gradient algorithm.

(S)-11-(1-Amino-6-carbamoyl-1-imino-8,11,14,17-tetraoxo-2,7,10,13,16-pentaazaoctadecan-18-ylamino)-5methylbenzofuro[3,2-b]quinolin-5-ium (2l). Peptidyl coupling order is arginine-glycine-glycine-glycine, following the general procedure to give the desired compound 2l as a straw yellow solid with a yield of 19%. mp 182−183 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.82 (t, J = 6.4 Hz, 1H), 8.75 (d, J = 7.6 Hz, 2H), 8.63 (d, J = 8.2 Hz, 1H), 8.42 (d, J = 8.9 Hz, 1H), 8.29 (t, J = 5.6 Hz, 1H), 8.17 (t, J = 5.7 Hz, 1H), 8.13−8.08 (m, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.91 (dd, J = 9.2, 5.5 Hz, 2H), 7.81 (dd, J = 14.0, 6.8 Hz, 2H), 7.64 (ddd, J = 8.2, 5.9, 2.2 Hz, 1H), 7.37 (s, 1H), 7.09 (s, 1H), 4.80 (d, J = 6.3 Hz, 2H), 4.58 (s, 3H), 4.19 (dd, J = 13.2, 8.2 Hz, 3H), 3.85 (d, J = 5.6 Hz, 2H), 3.77−3.66 (m, 4H), 3.08 (dd, J = 12.2, 6.3 Hz, 2H), 1.76− 1.68 (m, 1H), 1.59−1.36 (m, 4H); HRMS (ESI+) m/z: Calc. for C30H37N10O6+: 633.2892 [M+H]+. Found 633.2895 [M +H]+. 11-((6S,9S,12S)-9-((4H-Imidazol-4-yl)methyl)-1-amino12-benzyl-6-carbamoyl-1-imino-8,11,14,17-tetraoxo2,7,10,13,16-pentaazaoctadecan-18-ylamino)-5methylbenzofuro[3,2-b]quinolin-5-ium (2m). Peptidyl coupling order is arginine-histidine-phenylalanine-glycine, following the general procedure to give the desired compound 2m as a straw yellow solid with a yield of 16%. mp 171−173 °C. 1H NMR (400 MHz, DMSO-d6) δ 14.17 (s, 2H), 9.66 (t, J = 5.5 Hz, 1H), 8.93 (s, 1H), 8.69 (d, J = 8.4 Hz, 1H), 8.63 (d, J = 8.2 Hz, 1H), 8.57 (t, J = 5.2 Hz, 1H), 8.42 (d, J = 9.0 Hz, 1H), 8.35 (d, J = 7.7 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.15− 8.07 (m, 1H), 8.00 (d, J = 7.3 Hz, 1H), 7.96−7.74 (m, 3H), 7.71−7.57 (m, 2H), 7.50 (s, 1H), 7.33 (s, 1H), 7.16−7.12 (m, 1H), 4.78 (d, J = 5.4 Hz, 2H), 4.59 (s, 3H), 4.50 (d, J = 10.2 Hz, 1H), 4.18 (dd, J = 12.5, 7.2 Hz, 2H), 3.87−3.78 (m, 5H), 3.08 (dd, J = 13.8, 5.8 Hz, 3H), 2.97 (dd, J = 14.0, 5.0 Hz, 2H), 2.73 (dd, J = 13.6, 9.9 Hz, 1H), 1.77−1.65 (m, 1H), 1.64−1.34 (m, 3H); HRMS (ESI+) m/z: Calc. for C41H47N12O6+: 402.1904 [M+H]2+. Found 402.1902 [M+H]2+. 11-((7S,10S,13S)-17-Amino-7-benzyl-13-carbamoyl10-(3-guanidinopropyl)-2,5,8,11-tetraoxo-3,6,9,12-tetraazaheptadecylamino)-5-methylbenzofuro[3,2-b]quinolin-5-ium (2n). Peptidyl coupling order is lysinearginine-phenylalanine-glycine, following the general procedure to give the desired compound 2n as a straw yellow solid with a yield of 17%. mp 177−179 °C. 1H NMR (400 MHz, DMSOd6) δ 9.64 (s, 1H), 8.69 (d, J = 8.2 Hz, 1H), 8.64 (d, J = 8.2 Hz, 1H), 8.59 (s, 1H), 8.43 (d, J = 9.0 Hz, 1H), 8.30 (d, J = 6.1 Hz, 1H), 8.20 (d, J = 7.5 Hz, 1H), 8.15−8.09 (m, 1H), 7.86 (dt, J = 15.7, 6.0 Hz, 5H), 7.69 (s, 2H), 7.57 (s, 1H), 7.34 (d, J = 6.9 Hz, 1H), 7.20 (t, J = 12.1 Hz, 4H), 7.14 (d, J = 6.9 Hz, 1H), 7.06 (s, 1H), 4.75 (d, J = 5.0 Hz, 2H), 4.59 (s, 3H), 4.21−4.08 (m, 2H), 3.04 (dd, J = 25.8, 15.3 Hz, 4H), 2.75 (dd, J = 19.3, 6.2 Hz, 4H), 1.74−1.64 (m, 2H), 1.60−1.43 (m, 8H), 1.38− 1.24 (m, 4H); HRMS (ESI+) m/z: Calc. for C41H52N11O6+: 397.7085 [M+H]2+. Found 397.7101 [M+H] 2+. FRET Assays. FRET assay was carried out on a real-time PCR apparatus following previously published procedures.24 The labeled oligonucleotides F21T: 5′-FAM-d(GGG[TTAGGG]3)-TAMRA-3′ and F10T: 5′-FAM-dTATAGCTATA-HEG-TATAGCTATA-TAMRA-3′ were used as the FRET probes. Fluorescence melting curves were determined with a Roche LightCycler 2 real-time PCR machine, using a total reaction volume of 20 μL, with 0.2 μM of labeled oligonucleotide in 10 mM Tris-HCl buffer, pH 7.2, containing 60 mM KCl. Fluorescence readings with excitation at 470 nm 1824

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Docking studies were carried out using the AUTODOCK 4.0 program.26 By using ADT,27 nonpolar hydrogens of telomeric G-quadruplex were merged to their corresponding carbons, and partial atomic charges were assigned. The nonpolar hydrogens of the ligands were merged, and rotatable bonds were assigned. The resulting G-quadruplex structure was used as an input for the AUTOGRID program. AUTOGRID gave a precalculated atomic affinity grid maps for each atom type in the ligand, plus an electrostatics map and a separate desolvation map present in the substrate molecule. The dimensions of the active site box, which was placed at the center of the G-quadruplex, were set to 60 Å × 60 Å × 60 Å with the grid points 0.375 Å apart. Docking calculations were carried out using the Lamarckian genetic algorithm (LGA). Initially, we used a population of random individuals (population size: 150), a maximum number of 25 000 000 energy evaluations, a maximum number of generations of 27 000, and a mutation rate of 0.02. One hundred independent docking runs were carried out for each ligand. The resulting positions were clustered according to a root-mean-square criterion of 0.5 Å. TRAP-LIG Assay. The activity of P-BFQ conjugates to inhibit telomerase in a cell-free system was assessed with the TRAP-LIG assay following previously published procedures.28 Protein extracts from exponentially growing A549 leukemia cells were used. Briefly, 0.1 μg of TS forward primer (5′AATCCGTCGAGCAGAGTT-3′) was elongated by telomerase (500 ng protein extract) in TRAP buffer (20 mM Tris-HCl, pH 8.3, 68 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, and 0.05% Tween 20) containing 125 μM dNTPs and 0.05 μg BSA. The mixture was added to tubes containing freshly prepared ligand at various concentrations and to a negative control containing no ligand. The initial elongation step was carried out for 20 min at 30 °C, followed with 95 °C for 5 min, and a final maintenance of the mixture at 20 °C. To purify the elongated product and to remove the bound ligands, the quick nucleotide purification kit (Tiangen) was used according to the manufacturer’s instructions. The purified extended samples were then subjected to PCR amplification. In this step, a second PCR master mix was prepared consisting of 1 μM ACX reverse primer (5′-GCGCGG[CTTACC]3CTAACC-3′), 0.1 μg TS forward primer (5′-AATCCGTCGAGC-AGAGTT-3′), TRAP buffer, 5 μg BSA, 0.5 mM dNTPs, and 2 units of Taq polymerase. A 10 μL aliquot of the master mix was added to the purified telomerase-extended samples, and amplified for 35 cycles at 94 °C for 30 s, at 61 °C for 1 min, and at 72 °C for 1 min. Samples were separated on a 16% PAGE, and visualized with silver-staining. IC50 values were then calculated from the optical density quantitated from the Quantity One software. Long-Term Cell Culture Experiments. Long-term proliferation experiments were carried out using the HL60 Human promyelocytic leukemia cells. Cells were grown in T80 tissue culture flasks at 1.0 × 105 per flask, and exposed to a subcytotoxic concentration of ligand or an equivalent volume of 0.1% DMSO every 4 days. The cells in control and drugexposed flasks were counted and flasks reseeded with 1.0 × 105 cells. The remaining cells were collected and used for the measurements described below. This process was continued for 16 days. SA-β-Gal Assay. Cells treated with the ligand were incubated for 16 days. After the incubation, the growth medium was aspirated and the cells were fixed in 2% formaldehyde/0.2% glutaraldehyde for 15 min at room temperature. The fixing solution was removed, and the cells

were gently washed twice with PBS, and then stained with the β-Gal staining solution containing 1 mg/mL of 5-bromo-4chloro-3-indolyl-β-D-galactoside, followed with incubation overnight at 37 °C. The staining solution was removed, and the cells were washed three times with PBS. The cells were viewed under an optical microscope and photographed. Telomere Length Assay. Cells treated with the ligand were incubated for 16 days. To measure the telomere length, genomic DNA was digested with Hinf1/Rsa1 restriction enzymes. The digested DNA fragments were separated on 0.8% agarose gel, transferred to a nylon membrane, and the transferred DNA was fixed on the wet blotting membrane by baking the membrane at 120 °C for 20 min. The membrane was hybridized with a DIG-labeled hybridization probe for telomeric repeats, and incubated with anti-DIG-alkaline phosphatase. TRF was performed through chemiluminescence detection.

■

RESULTS AND DISCUSSION Chemistry. The synthesis of P-BFQ derivatives followed the general pathway as shown in Scheme 1. The key intermediate I Scheme 1. Synthesis of P-BFQ Derivativesa

Reagents: (i) glycine, 2-ethoxyethanol, 55 °C, 12 h; (ii) synthesized peptides, HOBT, DIC, DMF, RT 12 h.

a

of 11-iodo-5-methylbenzofuro [3,2-b]quinolin-5-ium was prepared following the procedure previously reported by Lu et al.16,29,30 The substitution reaction of I mixed with glycin in 2ethoxyethanol solution gave the intermediate II, which had a carboxyl group that can take condensation reaction with the amino terminal of peptidyl side chain. All synthesized sequences of peptidyl side chain are shown in Table 1. The solid-phase synthesis of these short peptides was carried out manually using standard Fmoc strategy on a Rink Amide AM resin (4-(2′,4′-dimethoxyphenyl-Fmocaminomethyl)phe-noxyacetamido-norleucylaminomethyl resin).31 The coupling procedure was performed with 4 equiv of N-Fmoc-protected amino acid, 4 equiv of DIC, and HOBt in DMF for 3 h. The coupling was monitored using standard Kaiser’s test, and the Fmoc group was removed by incubation with 25% piperidine solution in DMF twice for about 30 min. Then, 3 equiv of II were added into Fmoc deprotected peptidyl side chain with 10 equiv of DIC and HOBt in DMF for 12 h. The final cleavage of various P-BFQ conjugates (2a− 2n) from the resin was carried out using a mixture of trifluoroacetic acid and water (95:5), and conjugates were purified by using preparative RP-HPLC. The compounds used in the biological activity tests were at least 95% pure according to the analytical HPLC. The structures of the synthesized compounds were confirmed by using HR MS/MS and NMR analysis. Fluorescence Melting Assays. All synthesized compounds were evaluated for their thermodynamic stability and selectivity to telomeric G-quadruplex over duplex DNA through FRET melting experiments24,32,33 with F21T and F10T, respectively, and the benzofuroquinoline derivative 1 previously reported by our group was used as a reference compound. 1825

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Table 1. G-Quadruplex Stabilization (ΔTm) Potential Obtained from FRET-Melting, Equilibrium Binding Constants (KD) Measured with SPR, and Telomerase Inhibition (IC50) by P-BFQ Conjugates in Cell-Free Assay FRET (ΔTm/°C)b a

compound

R1

2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 2l 2m 2n 1

R-NH2 GG-NH2 GR-NH2 GF-NH2 GH-NH2 GK-NH2 KR-NH2 FR-NH2 HR-NH2 GGR-NH2 GFR-NH2 GGGR-NH2 GFHR-NH2 GFRK-NH2 --

F21T 12.3 3.9 20.6 5.6 7.7 14.8 24.3 13.1 17.4 10.9 9.7 9.6 12.5 11.2 13.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 0.1 0.2 0.6 0.3 0.2 0.8 0.6 0.6 0.3 0.5 0.3 0.2 0.2 0.4

SPR (KD/μM)

F10T

G4

duplex

duplex/G4

telomerase inhibition (IC50/ μM)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.27 2.29 0.65 1.91 0.32 0.66 0.19 0.42 0.39 0.39 0.89 0.76 0.70 0.62 0.76

19.3 12.6 45.2 25.4 21.8 86.6 9.5 23.4 12.0 5.3 21.7 11.8 14.7 13.3 1.82

70.4 5.5 69.0 13.3 67.9 130.2 50.5 55.0 30.7 13.6 24.2 15.5 20.9 21.2 2.4

12.0 >30 7.4 26.3 >20 13.8 5.5 14.6 10.5 19.9 21.2 22.7 18.8 20.4 13.3

1.4 0.1 1.9 0.1 0.5 1.2 3.3 1.0 2.1 1.3 1.2 1.3 0.6 0.2 10.1

0.2 0.0 0.3 0.1 0.2 0.1 0.3 0.1 0.2 0.0 0.1 0.1 0.2 0.0 0.1

a R1 is the peptidyl sequences of these conjugates’ side chains, starting from the terminal of scaffold. R, G, F, H, and K are abbreviations of Arginine, Glycine, Phenylalanine, Histidine, and Lysine, respectively. bΔTm = Tm(DNA + ligand) − Tm (DNA). ΔTm values of 0.2 μM F21T or F10T incubated with 1.0 μM compound in the presence of 60 mM KCl.

F21T (5′-FAM-d(GGG[TTAGGG]3)-TAMRA-3′) represents the human telomeric DNA sequence, while F10T (5′-FAMdTATAGCTATA-HEG-TATAGCTATA- TAMRA-3′) is a self-complementary duplex DNA hairpin. The results of Tm values from the FRET melting experiments (Table 1 and SI Figure S1) indicated that the peptidylsubstituted benzofuroquinoline derivatives had a wide range of telomeric G-quadruplex DNA stabilizing activity with ΔTm ranging from 3.9 to 24.3 °C, and the activity of some compounds (2c, 2f, 2g, and 2i) are better than that of the reference compound 1. The structure−activity relationships could be obtained from the stabilizing activity of the tested compounds toward telomeric G-quadruplex. In particular, it appeared that the introduction of arginine at a certain position of dipeptidyl side chain, such as compounds 2c, 2g, 2h, and 2i, remarkably increased their G-quadruplex stabilizing efficiency. This effect was weakened when amino acid residues changed from arginine (or lysine) to phenylalanine (2d), histidine (2e), or glycine (2b). This suggests that the positive charge and alkyl chain-length of the peptidyl side chain are important for its interaction with G-quadruplex. We compared ligands with different peptidyl length containing arginine, and the results showed that ligands 2c and 2g (ΔTm values of 20.6 and 24.3 °C, respectively) with dipeptidyl side chain were better than other ligands with shorter or longer chain-length. From these data, we can determine that the distance between the scaffold and the positive charge of arginine must be moderate, which is essential to stabilize G-quadruplex DNA. This result was consistent with our molecular modeling studies shown below. As shown in Table 1, all ligands had a rather weak effect on the thermal stability of the duplex DNA F10T, compared to reference compound 1, suggesting their weak binding activity to the duplex DNA.34,35 To further characterize the selectivity of these ligands for G-quadruplex DNA over duplex DNA, competitive FRET experiment was employed with a nonfluorescent duplex DNA competitor (ds26).35,36 In these experiments, the labeled oligonucleotide F21T was melted in the presence of excess ds26 (15- and 50-fold excess). The result of competitive FRET melting assay was plotted in Figure 1.

Figure 1. Competitive FRET results for P-BFQ ligands (2a−2n) and compound 1 (1 μM), without (blank) and with 15-fold (3 μM; grid) or 50-fold (10 μM, black) excess of duplex DNA competitor (ds26). The concentration of F21T was 0.2 μM.

The results clearly indicated a high level of stabilization of these ligands on telomeric G-quadruplex, which was only a little affected in the presence of 50-fold competitor ds26. Compared with reference compound 1, selectivity of these compounds was highly improved. The results also showed that these compounds had high selectivity for G-quadruplex DNA over duplex DNA, which can be attributed to the introduction of the peptidyl side chain. Accordingly, the combined results of these assays demonstrated that peptidyl−benzofuroquinoline (PBFQ) conjugates are a new class of potent and highly selective telomeric G-quadruplex binding ligands. Surface Plasmon Resonance. In order to investigate the binding affinity of P-BFQ ligands for their quadruplex target and their level of specificity for quadruplex versus duplex DNA, the surface plasmon resonance (SPR) method was employed. The equilibrium constants for the binding of the ligands to DNA were determined using SPR with biotinylated DNA attached to a streptavidin-coated sensor chip.37,38 A range of 1826

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acid residues are very important to their activity. Peptide sequences with arginine and lysine were optimal for their interactions with telomeric G-quadruplex DNA, while the introduction of glycine, phenylalalnine, or some other amino acid residues without positive charge on peptiyl side chain was unfavorable for their binding interactions and selectivity. Overall, SPR experiments indicated that P-BFQ conjugates had good selectivity for telomeric G-quadruplex, which were in agreement with our FRET data. Circular Dichroism. Circular dichroism (CD) spectroscopy was used to determine the structural type of G-quadruplex DNA and the effect of ligand binding on quadruplex structures.39 The interactions of synthesized conjugates with telomeric G-quadruplex DNA were further explored by using this method. The particular signature of the CD spectrum of the G-quadruplex is well-characterized. In the presence of 100 mM K+, the CD spectrum of HTG21 in the absence of any compound showed a major positive band at 290 nm, a shoulder at around 270 nm, a small positive band at 250 nm, and a minor negative band near 234 nm. This indicated a mixture of antiparallel and parallel conformations, possibly including hybrid-types as well. As shown in Figure 3A, upon the addition of compound 2f (5 mol equiv of HTG21) to the above solution, the CD spectrum significantly changed, with a

concentrations of ligands were injected simultaneously to the immobilized various DNA and the blank reference. The binding constants were determined using equilibrium analysis as shown in Table 1. The binding constants of the PBFQ ligands to telomeric G-quadruplex and duplex DNA fell into a wide range (telomeric KD, 0.19−2.29 μM; duplex KD, 5.3−86.6 μM). All tested ligands were found to bind to the human telomeric G-quadruplex more tightly than their binding to duplex DNA. From these data, we can clearly see that introduction of peptidyl side chain onto the scaffold of BFQ induced, for most designed peptide sequences, gives a significant improvement of its specificity for quadruplex over duplex DNA compared with compound 1 [Kd(ds)/Kd(Htelo) = 2.4]. Compound 2b had weak interaction with G-quadruplex based on FRET data, and was also found to have weak binding activity and specificity [Kd(ds)/Kd(Htelo) = 5.5]. Other compounds showed a discrimination of at least 10-fold for the G-quadruplex structure [Kd(ds)/Kd(Htelo) values ranging from 13.3 to 130.2]. For example, compound 2c, which is a representative compound of these series, had excellent binding affinity and selectivity to telomeric G-quadruplex DNA as shown in Figure 2. All these results were approximately consistent with their ΔTm values obtained from FRET melting. Both FRET and SPR experimental results indicated that BFQ scaffold with introduced peptidyl side chain had greatly improved binding capability and selectivity to telomeric Gquadruplex DNA. Functional groups on side chain of amino

Figure 3. (A) CD spectra of HTG21 in 10 mM Tris-HCl buffer, pH 7.2, 100 mM KCl. CD spectra of HTG21 (dashed line), and HTG21 in the presence of 5 equiv of 2d, 2f, 2k, 2l, and 2m, respectively. (B) CD titration spectra of HTG21 (5 μM) at increasing concentrations of 2c (0−4 mol equiv; dashed line indicated HTG21 in the absence of 2c).

Figure 2. (A) SPR sensorgram overlay for binding of compound 2c to telomeric quadruplex HTG21. Concentration range: 0.125, 0.25, 0.5, 1, 2, 4 μM from the bottom curve upward. (B) Binding plots used to determine the Kd value for 2c with telomeric G-quadruplex and duplex DNAs, running buffer, 50 mM Tris-HCl, pH 7.2, 100 mM KCl. 1827

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dramatic increase of maximum band at 290 nm and the emergence of the shoulder at 270 nm, indicating the possible destruction of the parallel structure of the mixed G-quadruplex conformations; meanwhile, the small positive band at 250 nm disappeared, which led to the appearance of a positive band at 240 nm and a major negative band at 260 nm, suggesting the formation of an antiparallel structure. Similar results were also observed upon addition of other compounds (2d, 2k, 2l, 2m) but with less significant changes. In order to further study the binding interactions, CD spectra of HTG21 (5 μM) were also recorded in the presence of increasing concentrations of compound 2c (2.5−20 μM, corresponding to 0.5−4 equiv of compound 2c) (Figure 3B). The band with maximum at 291 nm significantly increased and shifted toward 288 nm, while the shoulder at 270 nm was also gradually enhanced and merged into the band at 288 nm. In the meantime, the small positive band at 250 nm gradually decreased. The changes of the CD spectra were notably concentration dependent, as shown in Figure 3B. All these CD studies illustrate that tested ligands might transform the preformed hybrid-type G-quadruplex structure into antiparallel G-quadruplex, which is consistent with the performances of the Quindoline derivatives we reported previously,16 indicating manifest interactions of ligands with telomeric G-quadruplex DNA. Molecular Modeling Studies. In order to understand the properties of the interactions between P-BFQ conjugates and the telomeric G-quadruplex DNA, molecular docking studies were performed. The crystal structure of the propeller telomeric G-quadruplex (d[AG3(T2AG3)3], PDB code: 1KF1) with potassium ion was used as a template for the modeling studies because it might be a more biologically relevant form.40 Compound 2c was selected for this study as one of the best binding ligands; meanwhile, 2b, 2j, and 2l were also selected to study the effect of different peptide lengths on telomeric Gquadruplex. Our results from clustering analysis of molecular docking showed that compound 2c could stack on both external G-quartet planes, and the peptidyl side chain with protonated amino group was just embedded into the grooves, and interacted with the phosphate diester backbone through hydrogen bonding and electrostatic interaction. Meanwhile, other tested compounds (2b, 2j, 2l) with longer or shorter side chains could not favorably interact with grooves (SI Figure S2). Therefore, the appropriate peptidyl side chain is critical in ligand−quadruplex interactions, which was confirmed by using biophysical and biochemical methods. Furthermore, 2c had the same binding mode as reference compound 1, and more hydrogen bonding interactions with grooves and loops might make 2c have lower binding energy, as shown in Figure 4. In

this experiment, 100 random starting structures were generated, and structures with lowest energy (average intermolecular binding energy, ΔG = −12.53 kcal/mol for 2c and −9.87 kcal/ mol for 1) were obtained after the docking protocols were analyzed. Telomerase Inhibition. To determine the inhibitory ability of P-BFQ conjugates to human telomerase, a modified TRAP assay was adopted. 28 The TRAP-LIG assay provided quantitative measurement of telomerase inhibition by the small molecules, which is a reliable and reproducible approach to evaluate telomerase inhibitory effects without a separate Taq polymerase assay. Considering the compounds might interact with the extension reaction products thus interfering with the PCR step, the ligands were removed after incubation with the enzyme and prior to the amplification step. In the experiments, solutions of compounds were added to the telomerase reaction mixture containing extract from A549 leukemia cells, and their concentrations causing half enzyme inhibition (TelIC50) were obtained as shown in Table 1. The TelIC50 values of these ligands ranged from 5.5 to 30 μM, which indicated that most of the tested compounds had remarkable inhibitory effects on telomerase activity. Thus, introduction of peptidyl side arms on BFQ scaffold could maintain or improve its inhibitory activity against telomerase. Compound 2g with a dipeptide fragment of -Lys-Arg had the most potent inhibitory effect (TelIC50 = 5.5 μM), while compounds (2b, 2d, and 2e) without arginine at their side chain had low inhibitory activity. Compared with reference compound 1 (TelIC50 = 13.3 μM), reported telomerase inhibitor by our group previously,16 some tested compounds with dipeptidyl side chain (contained argininyl/lysyl) had more potent inhibitory effects as shown in Table 1. In addition, compound 2g also had an equivalent effect of inhibition relative to classical telomerase inhibitors such as BRACO-19 and TMPyP4 (TelIC50 = 6.3 μM and 8.9 μM, respectively).28 These results were consistent with those data obtained from previous experiments (FRET, SPR). Interestingly, the observed inhibition of telomerase elongation was well-related to Gquadruplex binding thermodynamics, as shown by the almost linear dependence of TelIC50 for telomerase inhibition on ΔTm/ Tm, where Tm is the melting temperature in the presence of 5 equiv of the compound (Figure 5).41 The R2 value of this linear equation reached 0.90, which indicated that the effect of a series of P-BFQ compounds had a good structure−activity relationship. Cell Senescence Induced by Compound 2c. To examine the effect of representative compound 2c on leukemia cell HL60, short-term cell viability was first determined in a 2day cytotoxic assay (MTT assay). The results showed that 2c had a moderate inhibitory effect, with an IC50 value of 45 μM for HL60 cells. This result prompted us to further investigate the long-term effect of this compound. To evaluate the longterm effect of 2c on HL60, subcytotoxic concentrations (1.25 and 5 μM) of 2c were adopted to avoid acute cytotoxicity and other nonspecific events that could lead to difficulty in result interpretation. Treatment of HL60 cells with 5 μM compound 2c resulted in a significant inhibitory effect after 12 days. In the presence of even 1.25 μM compound 2c, a discernible difference was observed between the control and treated cells (Figure 6A). Morphologic examination of the cells during long-term studies showed an increased proportion of enlarged and flattened cells with phenotypic characteristics of senes-

Figure 4. Side view of compound (a) 1 and (b) 2c making stacking interactions with the 3′ G-quartet. The ligand side chain is lying in the grooves formed by the TTA loops. Pictures were generated by PyMOL. 1828

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Figure 5. Correlation between telomerase elongation inhibition (IC50) and thermal stabilization (ΔTm/Tm) of telomeric G-quadruplex measured for all tested ligands. Figure 7. Effect of compound 2c on telomere length. TRF analysis of HL60 cells treated with or without compound 2c for 16 days. Lane 1, 0.1% DMSO; lane 2, 1.25 μM 2c; lane 3, 2.5 μM 2c; lane 4, 5 μM 2c.

cence.42,43 These flattened cells also stained positively for the senescence-associated β-galactosidase (SA-β-Gal) activity after continuous treatment by compound 2c (Figure 6B,C). Results indicated that compound 2c induced accelerated senescence of HL60 cancer cells. Telomere Shortening by Compound 2c. Treatment of cancer cells with telomeric G-quadruplex ligands had been previously reported to disrupt telomere length maintenance and cause telomeres to erode.44−46 To investigate whether 2c could shorten telomeres, the telomere length was evaluated using the telomeric restriction fragment (TRF) length assay. The results showed that 5 μM compound 2c triggered telomere shortening about 1.4 kb for HL60 cells, and telomere shortening was also observed after 2.5 μM and 1.25 μM of 2c treatment (Figure 7). Dysfunctional telomere could activate p53 to initiate cellular senescence or apoptosis to suppress tumorigenesis16 as reported, so the senescence induced by compound 2c might result from this shortening of telomere length. This result is consistent with the activity expected for effective telomeric G-quadruplex ligand and telomerase inhibitor.47

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CONCLUSIONS The design of compounds targeting at the telomeric Gquadruplex DNA is a rational and promising strategy. Our previous work has confirmed that cryptolepine derivatives are powerful compounds for this target. On the basis of this result, a series of novel peptidyl derivatives were designed, synthesized, and evaluated as effective and selective Gquadruplex ligands. The present study showed that P-BFQ conjugates were a promising class of telomeric G-quadruplex binding ligands with significant selectivity against duplex DNA in vitro. The guanidine group of arginine could greatly contribute to the overall binding ability. Moreover, their cellular effects indicated that some potent compounds had more effective inhibitory activities than typical telomerase inhibitors, which were also consistent with the properties expected for potent telomeric G-quadruplex ligands and telomerase inhibitors. These results proved that introduction of peptidyl side chains is a novel approach for obtaining more efficient and selective telomeric G-quadruplex ligands.

Figure 6. Senescence induced by compound 2c on HL60 cells. (A) Long-term incubation of HL60 with compound 2c at subcytotoxic concentrations. Expression of SA-β-Gal in HL60 cells after treatment with (B) 0.1% DMSO or (C) 5 μM compound 2c continuously for 16 days. 1829

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 8620-39943056; Fax: 8620-39943056; E-mail: ceshzs@ mail.sysu.edu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Natural Science Foundation of China (Grants U0832005, 21172272), the International S&T Cooperation Program of China (2010DFA34630), the Specialized Research Fund for the Doctoral Program of Higher Education (20110171110051), and the Science Foundation of Guangzhou (2009A1-E011-6) for financial support of this study.

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