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                5-N-Methylated Quindoline Derivatives as Telomeric

                日期:2022-05-18 13:54
                瀏覽次數:5825
                摘要:
                5-N-Methylated Quindoline Derivatives as Telomeric G-QuadruplexStabilizing Ligands: Effects
                of 5-N Positive Charge on Quadruplex Binding Affinity and CellProliferation
                Yu-Jing Lu,?,? Tian-Miao Ou,?,? Jia-Heng Tan,? Jin-Qiang Hou,?Wei-Yan Shao,? Dan Peng,? Ning Sun,? Xiao-Dong Wang,?
                Wei-Bin Wu,? Xian-Zhang Bu,? Zhi-Shu Huang,?,* Dik-Lung Ma,§Kwok-Yin Wong,§ and Lian-Quan Gu?,*
                School of Pharmaceutical Sciences, Sun Yat-sen UniVersity,Guangzhou 510080, People’s Republic of China, Department of AppliedBiology
                and Chemical Technology and the Central Laboratory of the Instituteof Molecular Technology for Drug DiscoVery and Synthesis, andThe
                Hong Kong Polytechnic UniVersity, Hung Hom, Kowloon, Hong Kong,China, People’s Republic of China
                ReceiVed April 30, 2008
                A series of 5-N-methyl quindoline (cryptolepine) derivatives (2a-x)as telomeric quadruplex ligands was
                synthesized and evaluated. The designed ligands possess a positivecharge at the 5-N position of the aromatic
                quindoline scaffold. The quadruplex binding of these compounds wasevaluated by circular dichroism (CD)
                spectroscopy, fluorescence resonance energy transfer (FRET) meltingassay, polymerase chain reaction (PCR)
                stop assay, nuclear magnetic resonance (NMR), and molecularmodeling studies. Introduction of a positive
                charge not only significantly improved the binding ability but alsoinduced the selectivity toward antiparallel
                quadruplex, whereas the nonmethylated derivatives tended tostabilize hybrid-type quadruplexes. NMR and
                molecular modeling studies revealed that the ligands stacked on theexternal G-quartets and the positively
                charged 5-N atom could contribute to the stabilizing ability.Long-term exposure of human cancer cells to
                2r showed a remarkable cessation in population growth and cellularsenescence phenotype and accompanied
                by a shortening of the telomere length.
                1. Introduction
                Telomeres are DNA-protein assemblies that cap the end of
                linear chromosomes. Their function is to protect the termini of
                chromosomes from recombination, end-to-end fusion, and
                degradation. Telomere shortening occurs progressively during
                cell divisions. When telomeres reach a critical short size,cells
                enter a stage of senescence replication, followed by cellcrisis
                and apoptosis. Human telomere is composed of random repeats
                of guanine-rich sequence d[(TTAGGG)n].1-3 The telomerase
                overexpressed in most tumor cells is a RNA-dependent DNA
                polymerase that uses its endogenous RNA template to catalyze
                the elongation of telomere, thus maintaining the telomerelength
                and rendering the tumor cells with an almost infinite capacity
                to divide and to be immortal.4-8 This unique telomeraseactivity
                and telomere capping function are becoming powerful and
                promising targets for cancer chemotherapy.9-15
                In addition to targeting the enzyme itself, an alternative
                approach is to stabilize higher-order quadruplex structures
                formed by G-rich sequence d[(TTAGGG)n], which is used by
                telomerase as a primer during the elongation phase.16 In vitro,
                a number of small molecule ligands have been identified to
                stabilize G-quadruplex structures and inhibit telomeraseactivity.
                These include the natural product telomestatin,17 cationic
                porphyrins,18 substituted acridines,19-21 polycyclicaceidines,22
                and perylenetetracarboxylic diimide derivatives.23,24Furthermore,
                in cellular experiments, several classes of these compounds
                have been shown to have pronounced effects on cancer
                cell lines, suggesting that the postulated action mechanism of
                these compounds is a denial of telomerase access to the
                telomere.25-28
                Quindoline and cryptolepine (Figure 1b,c) belong to the
                indoloquinoline family, a relatively rare group of alkaloids in
                nature.29 Cryptolepine and its hydrochloride salt possessinteresting
                biological properties and have been used as antimalarial
                drugs in Central and Western Africa for centuries.30,31 Further
                mechanistic investigation at the molecular level demonstrated
                that cryptolepine could interact with DNA throughintercalation.
                32 Most recent studies further revealed that some structural
                derivatives of quindoline were capable of interacting with the
                telomeric G-quadruplex structure and showed inhibitory effect
                on telomerase.33-35 Quindoline derivative 1 (SYUIQ-5, Figure
                1d) had been developed by us as the quadruplex stabilizing
                ligand and potent telomerase inhibitor.36,37 Subsequent studies
                on this ligand indicated that the 11-alkylamino group on
                quindoline could provide the in situ protonation ability of the
                5-N atom. Molecular modeling studies on the binding mode of
                these quindolines with quadruplex revealed that the crescent
                aromatic core stacked on two guanine residues of the G-quartet
                and the 5-N electropositive center overlapped with the cation
                channel of the quadruplex, thereby achieving effectivestabilization
                and selective recognition of G-quadruplex. However, the
                * To whom correspondence should be addressed. Phone:8620-39332679
                (Z.-S.H.); 8620-39332678 (L.-Q.G.). Fax: 8620-39332678 (Z.-S.H.and
                L.-Q.G.). E-mail: ceshzs@mail.sysu.edu.cn(Z.-S.H.); cesglq@mail.sysu.edu.cn
                (L.-Q.G.).
                ? These authors contributed equally to this paper.
                ? School of Pharmaceutical Sciences, Sun Yat-sen University,Guangzhou
                510080, China.
                § Department of Applied Biology and Chemical Technology and the
                Central Laboratory of the Institute of Molecular Technology forDrug
                Discovery and Synthesis, The Hong Kong Polytechnic University,Hung
                Hom, Kowloon, Hong Kong, China.
                Figure 1. Structures of the G-quartet (a), quindoline (b),cryptolepine
                (c, salt form), 1 (d), and 11-alkylamines substituted5-N-methyl
                quindoline derivatives (e).
                J. Med. Chem. 2008, 51, 6381–6392 6381
                10.1021/jm800497p CCC: $40.75 ? 2008 American Chemical Society
                Published on Web 09/27/2008
                pKa values of the 5-N atom of 11-aminoquindolines were
                8.2-8.436 and thus could be influenced easily by the solution
                condition. Besides the introduction of the positive charge via
                in situ protonation, an alternative pathway was N-methylation
                at 5-position.38 The advantage of this design was multiple,
                including introduction of a steady positive charge for the
                electrostatic interaction and increasing π-stacking interaction
                due to the reduction of the electron density of the aromaticcore
                of ligand. On this basis, a series of 5-N-methyl quindoline
                (cryptolepine) derivatives were designed for screening the
                ligands with better binding ability and selective recognitionof
                G-quadruplex. We reported here our biophysical, biochemical,
                and cellular evaluation studies on the binding of the5-N-methyl
                quindoline derivatives to telomeric quadruplex DNA and its
                relationship with telomere biological functions. CD datarevealed
                that the 5-N-methyl quindoline derivatives selectively induced
                the formation of the antiparallel G-quadruplex in K+ solution,
                whereas the nonmethylated quindoline derivatives could only
                induce hybrid-type quadruplexes (Supporting Information). All
                the results revealed that the introduction of a positive charge
                by methylation at the 5-N position of 11-aminoquindoline
                significantly improved the binding ability and inhibitoryeffect
                on the telomere biological functions. To investigate howchanges
                in electronic distribution on the skeleton of the ligand could
                modify its ability to stack onto the G-quadruplex, one or more
                fluorine substituents were introduced on some of 5-N-methyl
                quindoline derivatives. In addition, NMR and molecular modeling
                studies were used to investigate the structure-activity
                relationships and to probe the binding modes.
                2. Results and Discussion
                2.1. Chemistry. The key intermediates of 11-chloroquindoline
                3 was prepared following the procedure reported by Bierer
                et al.30 The selective N-5 methylation of 11-chloroquindoline
                with iodomethane was achieved in the presence of sulfolane
                with an excellent yield (88-93%). Sulpholane was reported as
                a solvent for certain N-quarternization reactions that wereused
                for selective N-alkylation of quindoline.39 The substitution
                reaction of compounds 4 with various alkylamines gave the final
                products of 11-amino 5-N-methyl quindoline derivatives 2a-2x
                (Scheme 1). All the substitution reactions were completed in
                high yields (80-92%) within 30 min.
                2.2. Converting the Preformed Hybrid-Type G-Quadruplex
                to the Antiparallel G-Quadruplex by 5-N-Methyl
                Quindoline Derivatives in the Presence of K+. Circular
                dichroism (CD) spectroscopy was employed to determine the
                formation of G-quadruplex in the presence of 5-N-methyl
                quindoline derivatives. It had been reported that the human
                telomeric sequence d[G3(T2AG3)3] (HTG21) formed a typical
                antiparallel quadruplex DNA structure in the presence of Na+,
                with a large positive band at 295 nm and a negative band at
                265 nm in CD spectra. On the other hand, the CD spectra of
                HTG21 in the presence of K+ exhibited a large positive band
                at 290 nm, a small positive band at 270 nm, and a negative
                band at 235 nm, which suggested that HTG21 might exist as a
                hybrid-type of quadruplex DNA containing parallel andantiparallel
                structure.40-43 Upon addition of compound 2b to
                HTG21 in buffer containing K+, the CD spectra changed with
                a disappearance of the small positive band at 270 nm,indicating
                the possible destruction of the parallel structure of the mixed
                G-quadruplex conformations, while the positive band at about
                290 nm increased obviously and the negative band at about 260
                nm appeared, suggesting the formation of an antiparallel
                structure (Figure 2A). CD studies in sodium solution were also
                carried out to examine the effects of compounds on different
                G-quadruplex structures, although they were lessphysiologically
                relevant. However, upon addition of 2b to HTG21 in buffer
                containing Na+, the CD spectra changed only slightly with a
                small increase at about 290 nm (Supporting Information). The
                other derivatives induced similar CD changes under the same
                conditions (Supporting Information). Results from these CD
                experiments suggest that 5-N-methyl quindoline derivatives
                could convert the preformed hybrid-type G-quadruplex structure
                into antiparallel G-quadruplex. Also, the stoichiometry of the
                binding of 5-N-methyl quindoline derivative to G-quadruplex
                was determined from CD spectra. When 2b was titrated against
                the HTG21 in the presence of K+, the band at 290 nm gradually
                increased until a ratio of 2b to HTG21 equal to 2:1 wasreached.
                The changes at 290 nm as a function of 2b/HTG21 (r) were
                plotted in Figure 2B, and the curve was fitted to anexponential
                function that suggested the formation of a 2:1 2b-HTG21
                complex.
                2.3. Thermodynamic Stability of the Telomeric G-Quadruplex
                by 5-N-Methyl Quindoline Derivatives. Thermodynamic
                stability of 5-N-methyl quindoline derivatives to the
                G-quadruplex DNA was determined from the melting temperature
                of the G-quadruplex DNA incubated with 5-N-methyl
                quindoline derivatives using a fluorescence resonance energy
                transfer (FRET) assay.44,45 All melting temperature assays were
                carried out in triplicate, and the quindoline derivative 136,37
                previously reported by us (Figure 1d) was used as a reference
                compound. It was known that 1 caused a rise of the melting
                Scheme 1. Synthesis of 5-N-Methyl Quindoline Derivativea
                a Reaction conditions: (a) CH3I, sulpholane, 55 °C, overnight; (b)substituted alkylamine, 2-ethoxyethanol, 120 °C, 30 min.
                6382 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 Lu etal.
                temperature of the native quadruplex DNA in 60 mM potassium
                from 60 to 69 °C.
                The effects of compounds 2a-2x on the Tm of the Gquadruplex
                are summarized in Table 1. A comparison of the
                FRET assay results for the reference compound 1 and 2b (both
                with the same side chain substituent) revealed thatintroduction
                of the positive charge by methylation at the 5-N position of
                quindoline significantly improved the stability of thequadruplex
                structure. Previous reports on nonmethylated quindolinederivatives36
                indicated that derivatives with a pyrrole ring in the
                aromatic core showed a comparatively higher Tm value than
                those with a furan ring. However, a similar trend was not
                observed in the 5-N-methyl quindoline derivatives. Thissuggested
                that the effects of pyrrole ring or furan ring in the
                aromatic core on the electron density were limited when a
                positive charge of 5-N position presented in the quindoline
                skeleton. Similarly, to study whether changes in the electronic
                distribution of the core could influence the binding ability of
                the ligand with the G-quadruplex, a fluorine substituent was
                introduced in 2q-2x. The electron-withdrawing effect of the
                fluorine could further reduce the electron density of theskeleton,
                which might favor a stronger interaction with the electron-rich
                π-system of the G-quartet. However, the introduction of the
                fluorine did not show an obvious effect either on quadruplex
                binding or biological activity. Nevertheless, the attachment of
                weakly basic groups (e.g., hydroxyl) to the side chain could
                decrease the binding affinity with G-quadruplex DNA, suggesting
                that electrostatic interaction between the ligand side chain
                and the G-quadruplex structure was an important factor for the
                recognition process.
                2.4. Inhibition of Amplification in HTG21 by 5-NMethyl
                Quindoline Derivatives. To further evaluate the ability
                of 5-N-methyl quindoline derivatives to stabilize G-quadruplex
                DNA, polymerase chain reaction (PCR) stop assay was carried
                out. In the presence of 5-N-methyl quindoline derivatives, the
                template sequence HTG21 was induced into a G-quadruplex
                structure that blocked the hybridization with a complementary
                primer sequence. In that case, 5′ to 3′ primer extension by DNA
                Taq polymerase was arrested and the final double-stranded DNA
                PCR product could not be detected.46,47 Concentrations of
                derivatives 2a-x and 1 that inhibited amplification by 50%
                (IC50) are listed in Table 1. A correlation between the PCRstop
                assay results and FRET data could be drawn, and derivatives
                with greater stabilizing power of G-quadruplex structure were
                in general better inhibitors of amplification in HTG21.Compounds
                2b, 2d, 2j, 2l, 2t, and 2x, with dimethylpropane-1,3-
                diamine or diethylpropane-1,3-diamine side chain, were most
                effective in these aspects.
                2.5. Binding Mode between 5-N-Methyl Quindoline
                Derivatives and G-Quadruplex. To further investigate the
                binding affinity and binding modes between the 5-N-methyl
                quindoline derivatives and G-quadruplex structures, nuclear
                magnetic resonance (NMR) studies and molecular modeling
                studies were performed.23 NMR titration experiments clearly
                showed a 2:1 stoichiometry for binding of ligand 2r to the
                intermolecular four-stranded parallel quadruplex [d(T2AG3)]
                derived from human telomeric DNA sequences (Supporting
                Information). The more pronounced change of the G4 and G6
                imino proton inferred that the ligand stacks over 3′- and5′-Gquartet.
                The thermal denaturation behavior of the G-quadruplex
                in the absence and presence of ligand 2r revealed that the
                interaction of ligand 2r increased the Tm by about 25 °C
                (Supporting Information).
                Molecular docking studies were performed on some of
                G-quadruplex-ligand complexes to investigate the best binding
                mode between the G-quadruplex and the 5-N-methylated
                quindoline derivatives. The crystal structure of the propeller
                telomeric G-quadruplex (d[AG3(T2AG3)3], PDB code: 1KF148)
                with potassium was used as the starting point for the modeling
                because it might be the more biologically relevant form.49 This
                G-quadruplex structure could be characterized by two external
                G-quartet planes. The 5′ G-quartet surface was relatively more
                hydrophobic for favoring π-π stacking interactions, whereas
                the 3′ G-quartet surface was more favored for electronic
                interactions (Supporting Information). Our study showed that
                5-N-methylated quindoline derivatives could stack on both
                external G-quartet planes. However, results from clustering
                analysis of molecular docking indicated that in most cases
                derivatives prefer to stack on the 3′ G-quartet plane(Supporting
                Information). The docking results also showed the terminal
                protonated amino group could interact with phosphate diester
                backbone by electrostatic interaction and hydrogen bond. The
                Figure 2. (A) CD titration spectra of HTG21 in the presence ofvarious
                concentrations of 2b in Tris-HCl buffer containing KCl. (B)Curve
                representing the changes in CD titration spectra was fitted toan
                exponential function and a 2:1 stoichiometry was assigned fromthe
                function. All spectra were collected in a strand concentration of 5μM
                in 10 mM Tris-HCl buffer (pH 7.4).
                Table 1. G-Quadruplex FRET and PCR Stop Assay Data by 1 and
                2a-xa
                compd ΔTm/°C
                IC50/
                μM compd ΔTm/°C
                IC50/
                μM compd ΔTm/°C
                IC50/
                μM
                2a 13 10.3 2i 11 11.5 2q 14 13
                2b 17 4.5 2j 14 6.1 2r 14 8
                2c 12 12 2k 12 13 2s 12 18
                2d 19 5.1 2l 15 5.7 2t 18 5
                2e 10 >20 2m 9 >20 2u 11 >20
                2f 12 >20 2n 13 18 2v 15 9
                2g 5 >20 2o 4 >20 2w 11 15
                2h 7 >20 2p 10 >20 2x 15 5.5
                1 9 >20
                a The melting point of native DNA quadruplex was 60 °C. ΔTm,change
                in melting temperature at 1 μM compound concentration and 200 nMDNA
                concentration; IC50, concentration of compound required to achieve50%
                inhibition of PCR.
                5-N-Methylated Quindolines as Telomeric Stabilizing Ligands Journalof Medicinal Chemistry, 2008, Vol. 51, No. 20 6383
                complexes obtained from docking studies were used for
                subsequent molecular dynamics studies.
                The MD runs for the complexes showed that the crescentlike
                quindoline skeleton could interact with at least two guanines,
                thus stabilized the G-quadruplex. The methylation on the 5-N
                atom of quindoline could significantly increase theelectrostatic
                interaction between the positively charged center of thequindoline
                skeleton and the negatively charged carbonyl channel
                of G-quadruplex. The terminal protonated amino group tended
                to interact with phosphodiester backbone by electrostatic
                interaction and hydrogen bonds. Compounds with a hydroxyl
                end could also interact with phosphodiester backbone by forming
                hydrogen bonds but much weaker than those with terminal
                amino group (Figure 3). Elongation of the side chains by one
                methylene caused the protonated amino group in the side chain
                to reach the phosphodiester backbone, accompanied by a
                correspondingly increased binding affinity. Compounds 2b, 2d,
                2j, and 2t, with dimethylpropane-1,3-diamine or diethylpropane-
                1,3-diamine side chain, possessed the most favorableinteraction
                (lower binding free energy). These results approximatelyparallel
                the trend in the FRET assay data and PCR stop assay data. The
                correlation between FRET assay results and theMM-GBSAcalculated
                binding energy (Table 2) had been explored in Figure
                4, and the correlation coefficient was 0.75.
                2.6. Competition Dialysis Assay. Competition dialysis assay
                was used to evaluate the selectivity of 5-N-methyl quindoline
                derivatives toward various G-quadruplex and other DNA
                structures. In this assay, the various nucleic acids weredialyzed
                simultaneously against a free ligand solution. Higher binding
                affinity was reflected by the higher concentration of ligands
                accumulated in the dialysis tube containing the specific form
                of DNA.50 The various forms of DNA used in the assay include
                HTG21 and Pu27 [d(TG4AG3TG4AG3TG4A2G2)], which
                form the intramolecular G-quadruplex structures, HT-7
                [d(T2AG3T)], the intermolecular G-quadruplex structure, HTC21
                [d(C3[TA2C3]3)], the i-motif structure, (dT21)2/dA21, which
                associate to give a triplex structure, dT21/dA21, a duplex
                structure, HTds, a 21-mer duplex structure (from human
                telomere sequence), HTG21mu [d(GAG[T2AGAG]3)], a singlestrand
                structure (a mutant oligomer of human telomere sequence
                containing multiple mutant sites), and dA21 and dT21, which
                were single-strands of purine and pyridine respectively. Thedata
                on the amount of the ligands bound to the 10 structurally
                different nucleic acids, which should be proportional to the
                binding affinity for each conformational form of DNA, are
                shown in Figure 5.
                As shown in Figure 5, 5-N-methyl quindoline derivative 2b
                displayed stronger binding to different types of DNA than the
                quindoline derivative 1, especially for G-quadruplexes.Compound
                2b interacted preferentially with the G-quadruplex DNA
                of Pu27 and HTG21, while little binding to single strands was
                observed. The binding affinities for double strands and triplex
                were lower than those for quadruplexes. Overall, these studies
                indicated that 2b binds preferentially to the quadruplexes and
                had a better selectivity for G-quadruplex DNA than 1.
                2.7. Inhibition of Telomerase Activity in Cell-Free
                System. The contribution of the positive charge by methylation
                on the quindoline for telomerase inhibition was investigated
                using telomerase repeat amplification protocol (TRAP) assay.51
                Although the limitation of TRAP was reported recently,52 the
                data from this experiment could be used to make the comparison
                between different quindoline ligands. In this experiment,solutions
                of quindoline derivatives at certain concentrations were
                added to the telomerase reaction mixture containing extractfrom
                cracked MCF-7 breast carcinoma cell lines and the inhibitory
                concentrations by half (TelIC50) values of these compounds are
                listed in Table 3. It was found that the inhibitory effects on
                telomerase activity of 5-N-methyl quindoline derivatives were
                significantly enhanced when compared to the quindolinederivative
                1. Thus introducing a positive charge by methylation
                on the 5-N of quindoline could improve its inhibitory ability
                Figure 3. View onto the plane of the 3′ surface of humanG-quadruplex
                complex with compound 2b (A) and 2h (B), showing theinteraction
                between compound and G-quadruplex. Picture was created with
                PyMOL.
                Table 2. Estimated Free Energy of Binding in MM-GBSACalculations
                compd ΔG/kcal · mol-1 compd ΔG/kcal · mol-1
                2a -18.2 2j -26.38
                2b -25.35 2l -23.34
                2c -17.57 2r -18.76
                2d -29.81 2t -28.47
                2f -20.89 2v -21.24
                2h -15.61 2x -19.56
                1 -13.21
                Figure 4. Plots of Calculated Binding Energies vs FRET AssayData.
                Figure 5. Results of competition dialysis assay. A 1 μM solutionof
                1 or 2b was dialyzed against 10 different nucleic acid structures(45
                μM) for 24 h. The amount of bound ligand, 1 (white) or 2b(gray),
                was plotted against each DNA structure as a bar.
                6384 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 Lu etal.
                against telomerase. These results were in line with the current
                thinking that a stronger ligand of the G-quadruplex was also a
                good inhibitor of telomerase activity. In this way, 5-N-methyl
                quindoline derivatives might act as a “pseudo-potassium ion”,
                which could induce the formation and stabilization of the
                G-quadruplex and thus exhibited a strong inhibitory effect on
                telomerase activity.
                2.8. Shortening of the Telomere Length by 2r. Inhibition
                of telomerase and interaction with telomere G-overhang in
                cancer cells was predicted to disrupt telomere lengthmaintenance
                and caused telomeres to erode. To investigate whether
                2r could cause telomeres to shorten, the telomere length was
                evaluated using the telomeric restriction fragment (TRF) length
                assay. The results showed that 0.06 μM of 2r triggered telomere
                shortening about 1.1 kb against HL60 cells, and telomere
                shortening was also observed after 0.03 μM of 2r treatment
                (Figure 6A). Similar reduction in telomere length was observed
                in CA46 cells; 0.06 μM of 2r caused telomere shortening of
                about 1.4 kb against CA46 cells (Figure 6B). The results
                indicated that the derivative 2r could inhibit the elongationof
                telomeres in two cell lines of HL60 and CA46, although the
                real action mechanism of this inhibition needed to furtherstudy.
                2.9. Senescence Induced by 5-N-Methyl Quindoline
                Derivative 2r. To examine the effect of ligand 2r on leukemia
                cell HL60 and lymphoma cell CA46, short-term cell viability
                was determined in a two-day cytotoxic assay (MTT assay) first.
                Results showed that 2r had a potent inhibitory effect with an
                IC50 value of 1.21 μM in HL60 and 1.36 μM in CA46.
                To avoid acute cytotoxicity and other nonspecific events that
                could lead to difficulty in result interpretation, subcytotoxic
                concentrations (0.03 and 0.06 μM) of 2r were evaluated on
                HL60 and CA46 cells for long-term exposure. Treatment of
                HL60 cells with 0.06 μM 2r resulted in an arrest in cell growth
                at day 12, and even with 0.03 μM 2r, the population doubling
                time increased substantially compared to the control (Figure7A).
                The similar results were observed in another human lymphoma
                cell line CA46 cells. Morphologic examination of the cells at
                the plateau phase displayed an increased proportion of flat and
                giant cells with phenotypic characteristics of senescence53,54as
                revealed by the senescence-associated -galactosidase (SA--
                Gal) assay method (Figure 8). For dysfunctional telomeres that
                could activate p53 to initiate cellular senescence or apoptosis
                to suppress tumorigenesis, this effect of 2r to inducesenescence
                might result from the shortening effect to telomere length.
                3. Conclusions
                A series of 5-N-methyl quindoline (cryptolepine) derivatives
                (2a-x) as telomeric quadruplex stabilizing ligands havesynthesized
                and evaluated by CD spectroscopy, FRET-melting
                assay, PCR stop assay, NMR study, molecular modeling,
                competition dialysis assay, inhibitory telomerase activitytest,
                Figure 6. Effect of quindoline derivative 2r on telomere length.TRF of cancer cells treated or untreated with 2r was analyzed usingthe Telo
                TAGGG telomere length assay. (A) TRF analysis of HL60 cells treatedor untreated with 2r for 16 days. Lane 1, 0.1% DMSO; lane 2, 0.03μM
                2r; lane 3, 0.06 μM 2r. (B) TRF analysis of CA46 cells treated oruntreated with 2r for 16 days. Lane 1, 0.1% DMSO; lane 2, 0.03 μM2r; lane
                3, 0.06 μM 2r.
                Figure 7. Long-term exposure of HL60 (A) and CA46 cells (B)with
                2r at subcytotoxic concentrations. Cells were exposed toindicated
                concentrations of 2r or 0.1% DMSO, respectively. Every 4 days,the
                cells in control and drug-exposed flasks were counted andflasks
                reseeded with cells. Each experiment was performed three times ateach
                point. This experiment was a representative of threeexperiments.
                Table 3. Telomerase Inhibition by 1 and 5-N-Methyl Quindolinesin
                Cell-Free Assay
                1 2b 2d 2j 2r 2t 2v 2x
                TelIC50 (μM) 0.63 0.22 0.16 0.37 0.31 0.20 0.40 0.27
                5-N-Methylated Quindolines as Telomeric Stabilizing Ligands Journalof Medicinal Chemistry, 2008, Vol. 51, No. 20 6385
                and series cellular studies. All of the results clearly showedthat
                these ligands were capable of inducing the formation of
                antiparallel G-quadruplex in the presence of K+. NMR study
                and molecular modeling revealed the binding mode was
                external-stacking on the G-tetrad, and the positively charged
                5-N position of quindoline core could contribute to the overall
                stabilizing ability. Treatment with the ligand 2r remarkably
                inhibited the telomerase activity in a cell-free system.Longterm
                exposure assays of HL60 leukemia cells and CA46
                lymphomas cells showed ligand 2r could induce a remarkable
                cessation in population growth, cellular senescence phenotype,
                and shortening of telomere length.
                4. Experimental Section
                Synthesis and Characterization. Melting points were recorded
                on a Leica Galen III hot-stage melting point apparatus and were
                uncorrected. 1H NMR spectra were recorded on a 300 MHz
                Mercury-Plus spectrometer using TMS as an internal standard in
                DMSO-d6, CDCl3, or CD3OD. 13C NMR spectra were recorded
                on a Varian Unity 400 NMR instrument at 100 MHz. Mass spectra
                were recorded on a VG ZAB-HS (fast atom bombardment)
                spectrometer. High-resolution mass spectra were obtained with a
                MAT95XP (Thermo) mass spectrometer. Elemental analyses were
                carried out on an Elementar Vario EL CHNS elemental analyzer.
                All compounds were routinely checked by TLC with Merck silica
                gel 60F-254 glass plates. 11-Chloroquindoline 3a-d wassynthesized
                as reported.30 Analytical data for compound11-chloro-10Hindolo[
                3,2-b]quinoline (3a) and 11-chlorobenzofuro[3,2-b]- quinoline
                (3b) have been previously presented.36
                11-Chloro-7-fluoro-10H-indolo[3,2-b]quinoline (11-Chloro-
                7-fluoro-quindoline) (3c). Compound 3c was synthesized by a
                literature procedure;30 mp 207-209 °C. 1H NMR (300 MHz,
                CDCl3): δ 11.86 (s, 1H), 8.26 (m, 2H), 8.10(d, J ) 8.4 Hz, 1H),
                7.75 (m, 2H), 7.61 (m, 1H), 7.53 (t, J ) 9.0 Hz, 1H). FAB-MS
                m/z: 271 [M + 1]+. Anal. (C15H8ClFN2) C, H, N.
                11-Chloro-7,9-difluoro-10H-indolo[3,2-b]quinoline (11-Chloro-
                7,9-difluoro-quindoline) (3d). Compound 3d was synthesized by
                a literature procedure;30 mp 204-206 °C. 1H NMR (DMSO-d6,
                300 Hz): δ 12.22 (s, 1H), 8.21 (t, J ) 7.2 Hz, 2H), 7.91 (dd, J)
                7.8, 2.1 Hz, 1H), 7.73 (m, 2H), 7.57 (td, J ) 9.3, 2.4 Hz, 1H).
                FAB-MS m/z: 289 [M + 1]+. Anal. (C15H7ClF2N2) C, H, N.
                General Method39 for the Preparation of the 11-Iodo-5-Nmethyl-
                quindolinium Iodide Derivatives (4). A suspension of 3
                (3 mmol), iodomethane (8 g), and sulfolane (15 g) was heated in
                a sealed flask overnight at 55 °C. A yellow precipitate wasformed.
                The reaction mixture was allowed to cool to room temperatureand
                then placed in an ice bath, precipitated further with a mixtureof
                ice cold anhydrous diethyl ether (50 mL) and methanol (1 mL),
                filtered, andd washed thoroughly with anhydrous ethyl acetateto
                afford 4 as a yellow solid.
                11-Iodo-5-N-methyl-10H-indolo[3,2-b]quinolinium Iodide
                (4a). Prepared as above using 3a as starting materials. Yield91%;
                mp 263-266 °C. 1H NMR (DMSO-d6, 300 Hz): δ 8.67 (d, J )
                6.0 Hz, 1H), 8.56 (d, J ) 9.0 Hz, 1H), 8.34 (d, J ) 6.0 Hz,1H),
                8.01 (t, J ) 6.0 Hz, 1H), 7.83 (d, J ) 9.0 Hz, 1H), 7.72 (m,2H),
                7.37 (t, J ) 6.0 Hz, 1H), 4.61 (s, 3H). ESI-MS m/z 359 [M -I]+.
                Anal. (C16H12I2N2 · 1.3H2O) C, H, N.
                11-Iodo-5-N-methyl-benzofuro[3,2-b]quinolinium Iodide (4b).
                Prepared as above using 3b as starting materials. Yield 93%; mp
                227-230 °C. 1H NMR (DMSO-d6, 300 Hz): δ 8.85 (d, J ) 6.0
                Hz, 1H), 8.77 (d, J ) 9.0 Hz, 1H), 8.57 (d, J ) 9.0 Hz, 1H),8.31
                (t, J ) 6.0 Hz, 1H), 8.91 (d, J ) 6.0 Hz, 1H), 8.13 (q, J ) 9.0,6.0
                Hz, 2H), 7.79 (t, J ) 6.0 Hz, 1H), 4.91 (s, 3H). ESI-MS m/z 360
                [M - I]+. Anal. (C16H11I2NO· 0.5H2O) C, H, N.
                7-Fluoro-11-iodo-5-N-methyl-10H-indolo[3,2-b]quinolinium
                Iodide (4c). Prepared as above using 3c as starting materials.Yield
                88%; mp 255-257 °C. 1H NMR (DMSO-d6, 300 Hz): δ 12.76 (s,
                1H), 8.78 (d, J ) 9.0 Hz, 1H), 8.69 (d, J ) 9.0 Hz, 1H), 8.55(d,
                J ) 8.1 Hz, 1H), 8.20 (t, J ) 7.5 Hz, 1H), 8.02 (t, J ) 7.8 Hz,
                1H), 7.89 (m, 2H), 4.93 (s, 3H). FAB-MS m/z 377 [M - I]+. Anal.
                (C16H11FI2N2) C, H, N.
                7,9-Difluoro-11-iodo-5-N-methyl-10H-indolo[3,2-b]quinolinium
                Iodide (4d). Prepared as above using 3d as starting materials.
                Yield 90%; mp 235-238 °C. 1H NMR (DMSO-d6, 300 Hz): δ
                8.87 (d, J ) 9.0 Hz, 1H), 8.77 (t, J ) 9.0 Hz, 1H), 8.53 (d, J )9.0
                Hz, 1H), 8.00 (m, 1H), 7.96 (d, J ) 9.0 Hz, 1H), 7.86 (d, J )9.0
                Hz, 1H), 4.94 (s, 3H). FAB-MS m/z 395 [M - I]+. Anal.
                (C16H10F2I2N2 · 1.6H2O) C, H, N.
                General Method for the Preparation of the 11-Alkylamido-
                5-N-methyl-quindolinium Iodide Derivatives (2). A suspension
                of 4 (0.4 mmol), N,N-dialkylalkylamine (10 mmol), and2-ethoxyethanol
                (8 mL) was heated at 120 °C for 30 min. The reaction
                solution was allowed to cool to room temperature, added withicecold
                anhydrous diethyl ether (20 mL), and then placed at 4 °C
                overnight. A yellow precipitate was formed, which was filteredand
                washed thoroughly with anhydrous ethyl acetate to afford 2 as a
                yellow solid. Further purification was carried out byrecrystallization
                from methanol--diethyl ether (1:3).
                N′-(5-N-Methyl-10H-indolo[3,2-b]quinolin-5-ium)-N,N-dimethylethane-
                1,2-diamine iodide (2a). Yield 80%; mp 208-209 °C. 1H
                NMR (CDCl3, 300 Hz): δ 9.26 (d, J ) 9.0 Hz, 1H), 8.29 (d, J )
                9.0 Hz, 1H), 7.90 (t, J ) 6.0 Hz, 2H), 7.67 (m, 3H), 7.33 (t, J)
                9.0 Hz, 1H), 4.60 (s, 3H), 4.43 (m, 2H), 3.24 (d, J ) 6 Hz,2H),
                2.58 (s, 6H). 13C NMR (DMSO-d6, 100 Hz): δ 143.0, 141.5, 136.9,
                135.6, 132.1, 130.5, 124.2, 123.9, 123.7, 120.6, 117.3, 116.8,115.1,
                114.6, 113.5, 57.4, 43.8, 41.8, 38.0. FAB-MS m/z 319 [M - I]+.
                FAB-HRMS m/z: calcd for C20H23N4 [M - I]+ 319.1917, found
                319.1917. Anal. (C20H23IN4 · 2.7H2O) C, H, N.
                N′-(5-N-Methyl-10H-indolo[3,2-b]quinolin-5-ium)-N,N-dimethylpropane-
                1,3-diamine Iodide (2b). Yield 84%; mp 207-209 °C.
                1H NMR (DMSO-d6, 300 Hz): δ 8.73 (d, J ) 8.4 Hz, 1H), 8.54
                (d, J ) 8.4 Hz, 1H), 8.34 (d, J ) 8.7 Hz, 1 H), 8.01 (t, J )
                7.2 Hz, 2H), 7.91 (d, J ) 8.7 Hz, 1H), 7.71 (m, 2H), 7.37 (t, J)
                7.5 Hz, 1H), 4.60 (s, 3H), 4.23 (t, J ) 6.3 Hz, 2H), 3.22 (t, J )6.4
                Hz, 2H), 2.76 (s, 6H), 2.22 (t, J ) 7.2 Hz, 2H). 13C NMR(DMSOd6,
                100 Hz): δ 143.4, 142.6, 137.0, 135.4, 132.2, 130.4, 124.1,
                124.1, 123.8, 120.6, 117.2, 116.2, 115.0, 114.3, 113.5, 54.1,42.7,
                42.2, 42.2, 37.9, 24.6. FAB-MS m/z 333 [M - I]+. FAB-HRMS
                m/z: calcd for C21H25N4 [M - I]+ 333.2079, found 333.2070.Anal.
                (C21H25IN4 · 1.9H2O) C, H, N.
                Figure 8. Expression of SA--Gal in HL60 and CA46 cells aftercontinuous treatment with 2r. HL60 cells were treated with 0.06 μM2r (A) or
                0.1% DMSO (B) continuously for 16 days. CA46 cells were alsotreated with 0.06 μM 2r (C) or 0.1% DMSO (D) continuously for 16days. Then,
                cells were fixed, stained with SA--Gal staining kit, andphotographed (×400). This experiment was repeated twice.
                6386 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 Lu etal.
                N′-(5-N-Methyl-10H-indolo[3,2-b]quinolin-5-ium)-N,N-diethyl-
                ethane-1,2-diamine Iodide (2c). Yield 78%; mp 180-182 °C.
                1H NMR (CDCl3, 300 Hz): δ 9.31 (d, J ) 8.7 Hz, 1H), 8.29(d, J
                ) 8.4 Hz, 1H), 7.88 (t, J ) 7.2 Hz, 2 H), 7.66 (m, 3H), 7.33 (t,J
                ) 7.4 Hz, 1H), 4.60 (s, 3 H), 4.39 (brs, 2H), 3.28 (brs, 2H),2.85
                (m, 4H), 1.13 (t, J ) 6.3 Hz, 6H). 13C NMR (DMSO-d6, 100 Hz):
                δ 143.2, 142.8, 136.7, 135.6, 132.0, 130.3, 124.1, 123.8,123.7,
                122.9, 120.4, 117.1, 115.0, 114.4, 113.3, 54.8, 46.5, 46.5,41.9,
                37.8, 10.7, 10.7. FAB-MS m/z 347 [M - I]+. FAB-HRMS m/z:
                calcd for C22H27N4 [M - I]+ 347.2230, found 347.2287. Anal.
                (C22H27IN4 · 2.1H2O) C, H, N.
                N′-(5-N-Methyl-10H-indolo[3,2-b]quinolin-5-ium)-N,N-diethyl-
                propane-1,3-diamine Iodide (2d). Yield 87%; mp 206-208 °C.
                1H NMR (DMSO-d6, 300 Hz): δ 8.54 (m, 2H), 8.31 (t, J ) 9.0
                Hz, 1H), 7.99 (dd, J ) 9.0, 2.0 Hz, 1 H), 7.67 (m, 3H), 7.35 (t,J
                ) 9.0 Hz, 1H), 4.53 (s, 3H), 4.32 (t, J ) 6.0 Hz, 2H), 2.87 (q,J
                ) 3.0 Hz, 2H), 2.74 (brs, 2H), 2.07 (m, 2H), 0.97 (s, 6H). 13C
                NMR (DMSO-d6, 100 Hz): δ 144.7, 143.9, 136.7, 134.3, 131.8,
                130.2, 124.1, 123.4, 122.6, 120.3, 117.2, 116.4, 115.4, 114.4,113.5,
                47.9, 45.8, 45.8, 43.5, 37.5, 25.5, 9.6, 9.6. FAB-MS m/z 361 [M-
                I]+. FAB-HRMS m/z: calcd for C23H29N4 [M - I]+ 361.2387, found
                361.2415. Anal. (C23H29IN4 · 1.7H2O) C, H, N.
                N′-(5-N-Methyl-10H-indolo[3,2-b]quinolin-5-ium)-(2-morpholin-
                4-yl-ethyl)-amine Iodide (2e). Yield 90%; mp 240-242 °C.
                1H NMR (DMSO-d6, 300 Hz): δ 8.59 (dd, J ) 8.4, 3.3 Hz, 2H),
                8.35 (d, J ) 8.7 Hz, 1H), 8.02 (t, J ) 8.4 Hz, 1H), 7.87 (t, J )7.2
                Hz, 1H), 7.74 (dd, J ) 7.8, 6.9 Hz, 2H), 7.39 (t, J ) 7.8 Hz,1H),
                4.62 (s, 3H), 4.21 (t, J ) 5.7 Hz, 2H), 3.51 (t, J ) 4.5 Hz,4H),
                2.83 (t, J ) 5.7 Hz, 2H), 2.53 (s, 4H). 13C NMR (DMSO-d6, 100
                Hz): δ 143.7, 142.4, 137.0, 135.4, 132.2, 130.5, 124.3, 124.0,123.5,
                120.7, 117.3, 116.7, 115.1, 114.7, 113.4, 65.9, 65.9, 57.3,53.3,
                53.3, 42.9, 37.9. FAB-MS m/z 361 [M - I]+. FAB-HRMS m/z:
                calcd for C22H25N4O [M - I]+ 361.2023, found 361.2047. Anal.
                (C22H25IN4O· 1.1H2O) C, H, N.
                N′-(5-N-Methyl-10H-indolo[3,2-b]quinolin-5-ium)-(3-morpholin-
                4-yl-propyl)-amine Iodide (2f). Yield 90%; mp 220-223 °C.
                1H NMR (CDCl3, 300 Hz): δ 9.33 (m, 1H), 8.67 (d, J ) 9.0 Hz,
                1H), 8.17 (dd, J ) 6.0, 3.0 Hz, 2H), 7.96 (m, 2H), 7.60 (t, J )9.0
                Hz, 1H), 7.50 (t, J ) 9.0 Hz, 1H), 4.55 (s, 3H), 3.82 (t, J )6.0
                Hz, 4H), 2.74 (t, J ) 3.0 Hz, 2H), 2.61 (t, J ) 3.0 Hz, 4H),2.29
                (m, 2H). 13C NMR (DMSO-d6, 100 Hz): δ 143.8, 142.4, 137.1,
                135.5, 132.2, 130.4, 124.2, 123.9, 123.7, 117.3, 116.2, 115.1,114.5,
                113.4, 65.8, 65.8, 55.5, 53.1, 53.1, 43.9, 37.9, 25.6. FAB-MSm/z
                375 [M - I]+. FAB-HRMS m/z: calcd for C23H27N4O [M - I]+
                375.2185, found 375.2234. Anal. (C23H27IN4O· 0.9H2O) C, H, N.
                N′-(5-N-Methyl-10H-indolo[3,2-b]quinolin-5-ium)-2-aminoethanol
                Iodide (2g). Yield 92%; mp 235-237 °C. 1H NMR
                (DMSO-d6, 300 Hz): δ 8.67 (d, J ) 6.0 Hz, 1H), 8.56 (d, J ) 9.0
                Hz, 1H), 8.34 (d, J ) 9.0 Hz, 1H), 8.02 (t, J ) 6.0 Hz, 1H),7.83
                (d, J ) 9.0 Hz, 1H), 7.73 (m, 2H), 7.38 (t, J ) 9.0 Hz, 1H),4.62
                (s, 3H), 4.17 (m, 2H), 3.85 (m, 2H). 13C NMR (DMSO-d6, 100
                Hz): δ 144.1, 142.2, 137.0, 135.3, 132.1, 130.4, 124.1, 123.8,123.7,
                120.6, 117.2, 116.3, 115.1, 114.5, 113.4, 60.1, 47.7, 37.8.FABMS
                m/z 292 [M - I]+. FAB-HRMS m/z: calcd for C18H18N3O
                [M - I]+ 292.1444, found 292.1470. Anal. (C18H18IN3O·H2O) C,
                H, N.
                N′-(5-N-Methyl-10H-indolo[3,2-b]quinolin-5-ium)-3-aminopropanol
                Iodide (2h). Yield 88%; mp 203-207 °C. 1H NMR
                (DMSO-d6, 300 Hz): δ 8.63 (d, J ) 9.4 Hz, 1H), 8.55 (d, J ) 8.4
                Hz, 1H), 8.34 (d, J ) 9.3 Hz, 1H), 8.01 (t, J ) 7.5 Hz, 1H),7.74
                (m, 3H), 7.39 (t, J ) 6.9 Hz, 1H), 4.61 (s, 3H), 4.18 (m, 2H),3.64
                (m, 2H), 2.02 (m, 2H). 13C NMR (DMSO-d6, 100 Hz): δ 143.7,
                142.4, 137.1, 135.2, 132.1, 130.3, 124.1, 123.8, 123.7, 120.5,117.2,
                116.2, 115.0, 114.3, 113.3, 57.8, 42.4, 37.8, 32.3. FAB-MS m/z
                306 [M - I]+. FAB-HRMS m/z: calcd for C19H20N3O [M - I]+
                306.1601, found 306.1590. Anal. (C19H20IN3O· 2.5H2O) C, H, N.
                N′-(5-N-Methyl-benzofuro[3,2-b]quinolin-5-ium)-N,N-dimethyl-
                ethane-1,2-diamine Iodide (2i). Yield 85%; mp 162-163 °C.
                1H NMR (DMSO-d6, 300 Hz): δ 8.62 (d, J ) 9.0 Hz, 2H), 8.32
                (d, J ) 8.7 Hz, 1H), 8.07 (t, J ) 7.5 Hz, 1H), 7.78 (m, 1H),7.65
                (t, J ) 6.9 Hz, 1H), 4.54 (s, 3H), 4.20 (m, 2H), 2.74 (m, 2H),2.21
                (s, 6H). FAB-MS m/z 320 [M - I]+. 13C NMR (DMSO-d6, 100
                Hz): δ 156.6, 142.2, 138.4, 138.0, 133.2, 132.7, 131.4, 125.3,124.9,
                124.5, 123.9, 117.9, 116.8, 116.5, 113.0, 58.8, 45.2, 45.2,43.3,
                37.5 FAB-HRMS m/z: calcd for C20H22N3O [M - I]+ 320.1757,
                found 320.1773. Anal. (C20H22IN3O· 3.2H2O) C, H, N.
                N′-(5-N-Methyl-benzofuro[3,2-b]quinolin-5-ium)-N,N-dimethyl-
                propane-1,2-diamine Iodide (2j). Yield 83%; mp 190-192 °C.
                1H NMR (CDCl3, 300 Hz): δ 8.58 (d, J ) 8.7 Hz, 1H), 8.44 (d, J
                ) 7.8 Hz, 1H), 8.18 (t, J ) 8.7 Hz, 1H), 7.78 (m, 2H), 7.60 (m,
                2H), 4.65 (s, 3H), 4.40 (m, 2H), 2.74 (m, 2H), 2.42 (s, 6H),2.13
                (m, 2H). 13C NMR (DMSO-d6, 100 Hz): δ 156.6, 142.1, 138.2,
                137.9, 133.1, 132.6, 131.4, 125.3, 124.8, 124.5, 123.7, 117.8,116.7,
                116.4, 113.0, 56.6, 44.8, 44.8, 44.5, 37.4, 27.3. FAB-MS m/z334
                [M - I]+. FAB-HRMS m/z: calcd for C21H24N3O [M - I]+
                334.1914, found 334.1939. Anal. (C21H24IN3O· 1.8H2O) C, H, N.
                N′-(5-N-Methyl-benzofuro[3,2-b]quinolin-5-ium)-N,N-diethylethane-
                1,2-diamine Iodide (2k). Yield 84%; mp 179-180 °C. 1H
                NMR (DMSO-d6, 300 Hz): δ 8.60 (d, J ) 8.7 Hz, 1H), 8.35 (d, J
                ) 9.0 Hz, 1H), 8.08 (t, J ) 7.2 Hz, 1H), 7.93 (m, 2H), 7.78 (t,J
                ) 7.8 Hz, 1H), 7.67 (m, 2H), 4.54 (s, 3H), 4.20 (t, J ) 6.6 Hz,
                2H), 2.85 (t, J ) 6.9 Hz, 2H), 2.60 (q, J ) 6.9 Hz, 4H), 0.92 (t,J
                ) 6.9 Hz, 6H). 13C NMR (DMSO-d6, 100 Hz): δ 156.2, 142.1,
                138.1, 137.7, 132.9, 132.5, 131.2, 131.2, 125.1, 124.7, 124.3,123.6,
                117.7, 116.5, 116.2, 112.7, 52.3, 46.6, 46.6, 43.5, 37.3,11.7,11.7.
                FAB-MS m/z 348 [M - I]+. FAB-HRMS m/z: calcd for C22H26N3O
                [M - I]+ 348.2070, found 348.2052. Anal. (C22H26IN3O· 2.2H2O)
                C, H, N.
                N′-(5-N-Methyl-benzofuro[3,2-b]quinolin-5-ium)-N,N-diethylpropane-
                1,2-diamine Iodide (2l). Yield 80%; mp 168-170 °C.
                1H NMR (CDCl3, 300 Hz): δ 8.58 (d, J ) 8.4 Hz, 1H), 8.46 (d, J
                ) 8.1 Hz, 1H), 8.22 (t, J ) 9.0 Hz, 1H), 7.96 (t, J ) 7.2 Hz,2H),
                7.75 (m, 2H), 7.60 (m, 2H), 4.67 (s, 3H), 4.40 (t, J ) 6.3 Hz,2H),
                2.86 (t, J ) 6.9 Hz, 2H), 2.76 (q, J ) 7.2 Hz, 4H), 2.12 (m,2H),
                1.12 (t, J ) 7.2 Hz, 6H). 13C NMR (DMSO-d6, 100 Hz): δ 156.5,
                142.1, 138.2, 137.9, 133.1, 132.6, 131.5, 125.1, 124.8, 124.5,123.8,
                117.8, 116.8, 116.5, 112.9, 49.9, 46.1, 46.1, 44.8, 37.4, 27.0,11.3,
                11.3. FAB-MS m/z 362 [M - I]+. FAB-HRMS m/z: calcd for
                C23H28N3O [M - I]+ 362.2227, found 362.2252. Anal.
                (C23H28IN3O· 1.6H2O) C, H, N.
                N′-(5-N-Methyl-benzofuro[3,2-b]quinolin-5-ium)-(2-morpholin-
                4-yl-ethyl)-amine Iodide (2m). Yield 89%; mp 186-187 °C.
                1H NMR (DMSO-d6, 300 Hz): δ 8.61 (d, J ) 9.0 Hz, 1H), 8.35
                (d, J ) 9.0 Hz, 1H), 8.07 (t, J ) 9.0 Hz, 1H), 7.94 (m, 2H),7.77
                (t, J ) 9.0 Hz, 1H), 7.65 (t, J ) 9.0 Hz, 1H), 4.52 (s, 3H), 4.19(t,
                J ) 6.0 Hz, 2H), 3.65 (m, 4H), 3.65 (t, J ) 6.0 Hz, 4H), 2.77(t,
                J ) 6.0 Hz, 2H). 13C NMR (DMSO-d6, 100 Hz): δ 156.6, 142.2,
                138.5, 137.9, 133.2, 132.8, 131.4, 125.4, 124.9, 124.6, 123.8,118.0,
                116.7, 116.3, 113.0, 66.1, 66.1, 58.0, 53.2, 53.2, 42.5, 37.6.FABMS
                m/z 362 [M - I]+. FAB-HRMS m/z: calcd for C22H24N3O2
                [M - I]+ 362.1863, found 362.1858. Anal. (C22H24IN3O2 · 1.3H2O)
                C, H, N.
                N′-(5-N-Methyl-benzofuro[3,2-b]quinolin-5-ium)-(3-morpholin-
                4-yl-propyl)-amine Iodide (2n). Yield 87%; mp 193-195 °C.
                1H NMR (CDCl3, 300 Hz): δ 9.21 (d, J ) 9.0 Hz,
                1H), 8.40 (d, J ) 9.0 Hz, 1H), 8.03 (d, J ) 9.0 Hz, 1H), 7.89(t,
                J ) 9.0 Hz, 1H), 7.81 (t, J ) 6.0 Hz, 1H), 7.73 (d, J ) 6.0 Hz,
                1H), 7.60 (m, 2H), 4.61 (s, 3H), 4.36 (t, J ) 9.0 Hz, 2H), 3.69(t,
                J ) 3.0 Hz, 2H), 2.69 (t, J ) 6.0 Hz, 2H), 2.55 (t, J ) 3.0 Hz,
                4H), 2.17 (m, 2H). 13C NMR (DMSO-d6, 100 Hz): δ 157.2, 142.8,
                139.1, 138.5, 133.8, 133.4, 131.9, 125.9, 125.5, 125.2, 124.6,118.5,
                117.3, 117.0, 113.6, 66.6, 66.6, 56.3, 53.8, 53.8, 44.7, 38.2,27.3.
                FAB-MS m/z 376 [M - I]+. FAB-HRMS m/z: calcd for
                C23H26N3O2 [M - I]+ 376.2020, found 376.2029. Anal.
                (C23H26IN3O2 · 0.8H2O) C, H, N.
                N′-(5-N-Methyl-benzofuro[3,2-b]quinolin-5-ium)-2-aminoethanol
                Iodide (2o). Yield 92%; mp 145-146 °C. 1H NMR
                (DMSO-d6, 300 Hz): δ 8.67 (d, J ) 9.0 Hz, 1H), 8.59 (d, J ) 9.0
                Hz, 1H), 8.31 (d, J ) 9.0 Hz, 1H), 8.05 (t, J ) 9.0 Hz, 1H),7.91
                (m, 2H), 7.72 (t, J ) 9.0 Hz 1H), 7.63 (t, J ) 9.0 Hz, 1H), 4.51(s,
                3H), 4.19 (t, J ) 6.0 Hz, 2H), 3.82 (t, J ) 6.0 Hz, 2H). 13CNMR
                (DMSO-d6, 100 Hz): δ 156.3, 142.3, 138.2, 137.6, 133.0, 132.5,
                5-N-Methylated Quindolines as Telomeric Stabilizing Ligands Journalof Medicinal Chemistry, 2008, Vol. 51, No. 20 6387
                131.2, 125.0, 124.6, 124.3, 123.9, 117.6, 116.4, 116.2, 112.9,60.1,
                47.7, 37.3. FAB-MS m/z 293 [M - I]+. FAB-HRMS m/z: calcd
                for C18H17N2O2 [M - I]+ 293.1285, found 293.1286. Anal.
                (C18H17IN2O2 · 0.4H2O) C, H, N.
                N′-(5-N-Methyl-benzofuro[3,2-b]quinolin-5-ium)-3-aminopropanol
                Iodide (2p). Yield 91%; mp 170-172 °C. 1H NMR
                (DMSO-d6, 300 Hz): δ 8.60 (t, J ) 7.5 Hz, 2H), 8.34 (d, J ) 8.7
                Hz, 1H), 8.06 (t, J ) 7.5 Hz, 1H), 7.92 (m, 2H), 7.76 (t, J )7.5
                Hz, 1H), 7.64 (t, J ) 7.2 Hz, 1H), 4.51 (s, 3H), 4.16 (t, J )6.9
                Hz, 2H), 3.63 (t, J ) 6.0 Hz, 6H), 1.99 (m, 2H). FAB-MS m/z 307
                [M - I]+. 13C NMR (DMSO-d6, 100 Hz): δ 156.6, 142.1, 138.4,
                137.8, 133.1, 132.6, 131.2, 125.2, 124.8, 124.5, 123.9, 117.8,116.6,
                116.3, 113.1, 58.2, 43.0, 37.5, 33.2. FAB-HRMS m/z: calcd for
                C19H19N2O2 [M - I]+ 307.1441, found 307.1446. Anal.
                (C19H19IN2O2 · 1.9H2O) C, H, N.
                N′-(7-Fluoro-5-N-methyl-10H-indolo[3,2-b]quinolin-5-ium)-
                N,N-dimethyl-ethane-1,2-diamine Iodide (2q). Yield 83%; mp
                245-247 °C. 1H NMR (DMSO-d6, 300 Hz): δ 8.61 (d, J ) 8.4
                Hz, 1H), 8.37 (m, 2H), 8.01 (t, J ) 8.1 Hz, 1 H), 7.84 (q, J )4.8
                Hz, 1H), 7. 67 (m, 2H), 4.57 (s, 3H), 4.19 (t, J ) 5.1 Hz, 2H),
                2.95 (m, 2H), 2.44 (s, 6H). 13C NMR (DMSO-d6, 100 Hz): δ 157.6,
                155.3, 144.2, 139.9, 137.0, 134.7, 132.2, 123.7, 123.5, 118.7,117.2,
                115.3, 114.7, 108.9, 108.6, 58.5, 44.6, 44.6, 43.2, 37.6.FAB-MS
                m/z 337 [M - I]+. FAB-HRMS m/z: calcd for C20H22FN4
                [M - I]+ 337.1823, found 337.1768. Anal. (C20H22FIN4 · 1.2H2O)
                C, H, N.
                N′-(7-Fluoro-5-N-methyl-10H-indolo[3,2-b]quinolin-5-ium)-
                N,N-dimethyl-propane-1,3-diamine Iodide (2r). Yield 84%; mp
                230-232 °C. 1H NMR (CDCl3, 300 Hz): δ 9.74 (brs, 1H), 9.07
                (d, J ) 8.7 Hz, 1H), 7.88 (m, 3H), 7.73 (q, J ) 4.5 Hz, 1H),7.61
                (t, J ) 7.8 Hz, 1H), 7.32 (td, J ) 8.7, 2.1 Hz, 1H), 4.52 (s,3H),
                4.46 (t, J ) 5.1 Hz, 2H), 2.72 (t, J ) 5.7 Hz, 2H), 2.43 (s,6H),
                2.37 (m, 2H). 13C NMR (DMSO-d6, 100 Hz): δ 157.5, 155.1, 144.9,
                140.5, 136.9, 135.2, 132.1, 123.4, 119.1, 117.1, 115.5, 114.7,114.4,
                108.7, 108.5, 54.1, 43.3, 43.3, 42.9, 37.5, 25.8. FAB-MS m/z351
                [M - I]+. FAB-HRMS m/z: calcd for C21H24FN4 [M - I]+
                351.1980, found 351.1953. Anal. (C21H24FIN4 · 3.2H2O) C, H, N.
                N′-(7-Fluoro-5-N-methyl-10H-indolo[3,2-b]quinolin-5-ium)-
                N,N-diethyl-ethane-1,2-diamine Iodide (2s). Yield 80%; mp
                232-234 °C. 1H NMR (CDCl3, 300 Hz): δ 8.58 (d, J ) 7.8 Hz,
                1H), 8.41 (d, J ) 9.0 Hz, 1H), 8.35 (d, J ) 9.0 Hz, 1H), 8.00(t,
                J ) 7.5 Hz, 1H), 7.77 (q, J ) 4.5 Hz, 1H), 7.69 (t, J ) 7.5 Hz,
                1H), 7.62 (td, J ) 9.0, 2.4 Hz, 1H), 4.57 (s, 3 H), 4.14 (t, J )5.4
                Hz, 2H), 3.00 (t, J ) 5.4 Hz, 2H), 2.85 (q, J ) 6.9 Hz, 4H),0.96
                (t, J ) 6.9 Hz, 6H). 13C NMR (DMSO-d6, 100 Hz): δ 158.2, 155.8,
                145.0, 140.6, 137.6, 135.4, 132.8, 124.3, 123.9, 119.8, 117.9,115.8,
                115.3, 109.6, 109.3, 53.4, 47.7, 47.7, 44.7, 38.1, 11.1, 11.1.FABMS
                m/z 365 [M - I]+. FAB-HRMS m/z: calcd for C22H26FN4
                [M - I]+ 365.2136, found 365.2193. Anal. (C22H26FIN4 · 2.4H2O)
                C, H, N.
                N′-(7-Fluoro-5-N-methyl-10H-indolo[3,2-b]quinolin-5-ium)-
                N,N-diethyl-propane-1,3-diamine Iodide (2t). Yield 82%; mp
                216-218 °C. 1H NMR (DMSO-d6, 300 Hz): δ 8.54 (d, J ) 8.9
                Hz, 1H), 8.32 (t, J ) 9.0 Hz, 1H), 7.98 (t, J ) 9.0 Hz, 1 H),7.77
                (q, J ) 6.0 Hz, 1H), 7.57 (td, J ) 9.0, 3.0 Hz, 1H), 4.54 (s,3H),
                4.26 (t, J ) 6.0 Hz, 2H), 2.77 (q, J ) 6.0 Hz, 2H), 2.05 (m,2H),
                0.96 (t, J ) 6.0 Hz, 6H). 13C NMR (DMSO-d6, 100 Hz): δ 158.0,
                155.7, 145.1, 141.3, 137.5, 135.5, 132.6, 124.0, 124.0, 119.2,117.7,
                116.3, 115.1, 109.2, 109.0, 48.8, 46.5, 46.5, 44.4, 38.1, 26.2,10.5,
                10.5. FAB-MS m/z 379 [M - I]+. FAB-HRMS m/z: calcd
                for C23H28FN4 [M - I]+ 379.2293, found 379.2319. Anal.
                (C23H28FIN4 · 1.5H2O) C, H, N.
                N′-(7,9-Difluoro-5-N-methyl-10H-indolo[3,2-b]quinolin-5-ium)-
                N,N-dimethyl-ethane-1,2-diamine Iodide (2u). Yield 80%; mp
                238-240 °C. 1H NMR (DMSO-d6, 300 Hz): δ 8.56 (d, J ) 9.0
                Hz, 1H), 8.29 (d, J ) 9.0 Hz, 1H), 8.14 (d, J ) 9.0 Hz, 1 H),7.94
                (t, J ) 9.0 Hz, 1H), 7.62 (t, J ) 9.0 Hz, 1H), 7.47 (t, J ) 9.0Hz,
                1H), 4.53 (s, 3H), 4.44 (t, J ) 6.0 Hz, 2H), 3.37 (t, J ) 6.0Hz,
                2H), 2.75 (s, 6H). 13C NMR (DMSO-d6, 100 Hz): δ 155.7, 153.2,
                146.1, 137.2, 135.2, 132.1, 130.2, 125.0, 124.0, 123.3, 117.4,116.3,
                114.5, 104.7, 103.4, 59.4, 44.3, 44.3, 42.6, 37.8. FAB-MS m/z355
                [M - I]+. FAB-HRMS m/z: calcd for C20H21F2N4 [M - I]+
                355.1729, found 355.1733. Anal. (C20H21F2IN4 · 1.4H2O) C, H, N.
                N′-(7,9-Difluoro-5-N-methyl-10H-indolo[3,2-b]quinolin-5-ium)-
                N,N-dimethyl-propane-1,3-diamine Iodide (2v). Yield 86%; mp
                250-253 °C. 1H NMR (DMSO-d6, 300 Hz): δ 8.59 (d, J ) 8.4
                Hz 1H), 8.26 (d, J ) 9.0 Hz, 1H), 8.11 (d, J ) 9.0 Hz, 1H),7.92
                (t, J ) 7.5 Hz, 1H), 7.59 (t, J ) 8.4 Hz, 1H), 7.42 (t, J ) 9.0Hz,
                1H), 4.51 (s, 3H), 4.51 (m, 2H), 3.14 (t, J ) 5.7 Hz, 2H), 2.68(s,
                6H), 2.20 (m, 2H). 13C NMR (DMSO-d6, 100 Hz): δ 154.7, 152.5,
                149.7, 146.6, 136.5, 135.1, 131.4, 125.6, 123.6, 122.3, 116.6,115.9,
                113.9, 103.6, 102.3, 62.7, 52.4, 41.5, 41.5, 37.0, 25.1. FAB-MS
                m/z 369 [M - I]+. FAB-HRMS m/z: calcd for C21H23F2N4 [M -
                I]+ 369.2185, found 369.2194. Anal. (C21H23F2IN4 · 2.3H2O) C,
                H, N.
                N′-(7,9-Difluoro-5-N-methyl-10H-indolo[3,2-b]quinolin-5-ium)-
                N,N-diethyl-ethane-1,2-diamine Iodide (2w). Yield 83%; mp
                240-243 °C. 1H NMR (CD3OD, 300 Hz): δ 8.48 (d, J ) 8.4 Hz,
                1H), 8.23 (d, J ) 9.0 Hz, 1H), 8.06 (d, J ) 9.0 Hz, 1H), 7.97(t,
                J ) 7.2 Hz, 1H), 7.65 (t, J ) 7.5 Hz, 1H), 7.35 (t, J ) 9.0 Hz,
                1H), 4.56 (s, 3 H), 4.32 (t, J ) 4.2 Hz, 2H), 3.30 (m, 2H), 2.98(q,
                J ) 7.2 Hz, 4H), 1.10 (t, J ) 7.2 Hz, 6H). 13C NMR (DMSO-d6,
                100 Hz): δ 154.7, 152.4, 145.2, 136.3, 134.5, 131.3, 124.7,123.2,
                122.4, 116.6, 115.9, 115.8, 113.6, 103.9, 102.6, 52.9, 47.8,47.8,
                42.1, 37.0, 8.7, 8.7. FAB-MS m/z 383 [M - I]+. FAB-HRMS m/z:
                calcd for C22H25F2N4 [M - I]+ 383.2042, found 383.2049. Anal.
                (C22H25F2IN4 · 1.1H2O) C, H, N.
                N′-(7,9-Difluoro-5-N-methyl-10H-indolo[3,2-b]quinolin-5-ium)-
                N,N-diethyl-propane-1,3-diamine Iodide (2x). Yield 82%; mp
                253-254 °C. 1H NMR (DMSO-d6, 300 Hz): δ 8.60 (d, J ) 8.7
                Hz, 1H), 8.26 (d, J ) 9.0 Hz, 1H), 8.11 (m, 1 H), 7.93 (t, J )8.1
                Hz, 1H), 7.59 (t, J ) 7.5 Hz, 1H), 7.42 (m, 1 H), 4.57 (m, 2H),
                4.51 (s, 3H), 3.22 (t, J ) 6.6 Hz, 2H), 3.14 (q, J ) 5.4 Hz,4H),
                2.20 (m, 2H), 0.98 (t, J ) 5.4 Hz, 6H). 13C NMR (DMSO-d6, 100
                Hz): δ 157.2, 154.8, 144.3, 140.4, 136.7, 134.7, 131.8, 123.1,119.0,
                118.3, 116.9, 115.4, 114.3, 108.4, 108.1, 48.0, 45.6, 45.6,43.6,
                37.2, 25.4, 9.7, 9.7. FAB-MS m/z 397 [M - I]+. FAB-HRMS m/z:
                calcd for C23H27F2N4 [M - I]+ 397.2198, found 397.2221. Anal.
                (C23H27F2IN4 · 0.9H2O) C, H, N.
                Materials. All oligomers/primers used in this study were
                purchased from Invitrogen (China). Acrylamide/ bisacrylamide
                solution and N,N,N′,N′-tetramethyl-ethylenediamine werepurchased
                from Sigma. Taq DNA polymerase was purchased from Sangon,
                China. Stock solutions of all the derivatives (10 mM) were made
                using DMSO (10%) or double-distilled deionized water. Further
                dilutions to working concentrations were made withdouble-distilled
                deionized water. All tumor cell lines were obtained from the
                American Type Culture Collection (ATCC, Rockville, MD). The
                cell culture was maintained in a RPMI-1640 medium supplemented
                with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/
                mL streptomycin in 25 cm2 culture flasks at 37 °C humidified
                atmosphere with 5% CO2.
                CD Measurements. CD measurements were performed on a
                Chirascan circular dichroism spectrophotometer (AppliedPhotophysics)
                using a quartz cuvettes of 2 mm optical path length and
                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
                d[GGG(TTAGGG)3] at a final concentration of 5 μM wasresuspended
                in Tris-HCl buffer (10 mM, pH 7.4) containing the specific
                cations and the compounds to be tested. The samples were heated
                to 95 °C for 5 min, then gradually cooled to room temperatureand
                incubated at 4 °C overnight. A buffer baseline was collected inthe
                same cuvette and subtracted from the sample spectra. The CD
                spectra were obtained by taking the average of at least threescans
                at 25 °C. Then, CD titration was performed at a fixed HTG21
                concentration (5 μM) with various concentrations (0-5 molequiv)
                of the compounds in Tris-HCl buffer with 100 mM KCl. After each
                addition of compound, the reaction was stirred and allowed to
                equilibrate for at least 15 min (until no elliptic changes were
                observed) and a CD spectrum was collected. Final analysis ofthe
                data was carried out using Origin 7.5 (OriginLab Corp.).
                6388 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 Lu etal.
                FRET Assay. FRET assay was carried out on a real-time PCR
                apparatus (Roche LightCycler 2) following previously published
                procedures.45 The fluorescent labeled oligonucleotide F21T [5′-
                FAM- d(GGG[TTAGGG]3)-TAMRA-3′], donor fluorophore FAM,
                6-carboxyfluorescein, acceptor fluorophore TAMRA, and6-carboxytetramethylrhodamine]
                used as the FRET probes were diluted from
                stock to the correct concentration (400 nM) in Tris-HCl buffer(10
                mM, pH 7.4) containing 60 mM KCl and then annealed by heating
                to 90 °C for 5 min, followed by cooling to room temperature.
                Samples were prepared by aliquoting 10 μL of the annealed F21T
                (at 2× concentration, 400 nM) into LightCycler capillaries,followed
                by 10 μL of the compound solutions (at 2× concentration, 2 μM)
                and further incubated for 1 h. Measurements were made intriplicate
                on a RT-PCR with excitation at 470 nm and detection at 530 nm.
                Fluorescence readings were taken at intervals of 1 °C over therange
                37-99 °C, with a constant temperature being maintained for 30 s
                prior to each reading to ensure a stable value. Final analysis ofthe
                data was carried out using Origin 7.5 (OriginLab Corp.).
                PCR Stop Assay. The PCR stop assay was conducted according
                to a modified protocol of the previous study.47 Theoligonucleotide
                HTG21 d[GGG(TTAGGG)3] and the corresponding complementary
                sequence d(ATCGCT2CTCGTC3TA2C2) were used here. The
                reaction were performed in 1× PCR buffer, containing with 10
                pmol of each oligonucleotide, 0.16 mM dNTP, 2.5 U Taq
                polymerase, and the compounds to be tested. Reaction mixtures
                were incubated in a thermocycler with the following cycling
                conditions: 94 °C for 3 min, followed by 30 cycles of 94 °C for
                30 s, 58 °C for 30 s, and 72 °C for 30 s. Amplified productswere
                resolved on 15% nondenaturing polyacrylamide gels in 1× TBE
                and silver stained. IC50 values were calculated using opticaldensity
                read from LadWorks software.
                NMR Spectroscopy. NMR experiments were performed on a
                Bruker AVANCE AV 400 MHz spectrometer. All of the titration
                experiments were carried out at 25 °C in a 90% H2O/10% D2O
                solution containing 150 mM KCl and 25 mM potassium phosphate
                buffer (pH 7.0). Water suppression was achieved by theWatergate
                method.23,55 The oligonucleotide d(T2AG3) was purified by HPLC,
                and the concentration was 0.5 mM for the NMR measurements.
                For the thermal denature experiments, spectra were taken atintervals
                of 5 °C over the range 25-90 °C, with a constant temperaturebeing
                maintained for 15 min prior to each reading to ensure a stablesignal.
                Molecular Modeling. The crystal structure of the parallel 22-
                mer telomeric G-quadruplex (PDB ID 1KF1)48 was used as an
                initial model to study the interaction between the quindoline
                derivatives and telomeric DNA. The terminal 5′ adenine residue
                was removed to generate a 21-mer structure. Water moleculeswere
                removed from the PDB file, whereas the missing hydrogen atoms
                were added to the system using the Biopolymer moduleimplemented
                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 anonbond
                cutoff of 12 ? and a convergence of 0.01 kcal mol-1/? over10000
                steps using the Powell conjugate-gradient algorithm.
                Docking studies were carried out using the AUTODOCK 4.0
                program.56 Using ADT,57 nonpolar hydrogens of telomericGquadruplex
                was 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 performed a precalculated
                atomic affinity grid maps for each atom type in the ligand plusan
                electrostatics map and a separate desolvation map present inthe
                substrate molecule. The dimensions of the active site box,which
                was placed at the center of the G-quadruplex, were set to 50 ? × 50? × 50 ? with the grid points 0.375 ? apart. Docking
                calculations were carried out using the Lamarckian geneticalgorithm
                (LGA). Initially, we used a population of random individuals
                (population size: 150), a maximum number of 5000000 energy
                evaluations, a maximum number of generations of 27000, and a
                mutation rate of 0.02. One hundred independent docking runswere
                done for each ligand. The resulting positions were clustered
                according to a root-mean-square criterion of 0.5 ?.
                Molecular dynamics simulations were performed with the FF99
                version of the Cornell et al. force field58 using the sandermodule
                of the Amber 10.0 program suite. The nucleic acids studied were
                as treated using the parm99 parameters. Partial atomic chargesfor
                the ligand molecules were calculated using Gasteiger method,while
                force-field parameters were taken from the generalized Amberforce
                field (GAFF)59 using ANTECHAMBER module. A formal positive
                charge was manually assigned to the ammonium group present in
                the side chain and the 5-position of aromatic core. The K+radius
                was kept at 2.025 ?.60 Periodic boundary conditions wereapplied
                with the particle-mesh Ewald (PME) method61 used to treatlongrange
                electrostatic interactions. The quadruplex and ligand complexes
                were solvated in a rectangular box of TIP3P62 water
                molecules with solvent layers 8 ?, and the potassiumcounterions
                were added to neutralize the complexes.
                The hydrogen bonds were constrained using SHAKE.63 For the
                nonbonded interactions, a residue-based cutoff of 10 ? wasused.
                Temperature regulation was achieved by Langevin coupling with
                a collision frequency of 1.0. The solvated structures weresubjected
                to initial minimization to equilibrate the solvent and countercations.
                The G-quadruplex and inner K+ ions were initially fixed withforce
                constants of 100 kcal mol-1. The system was then heated from 0
                to 300 K in a 100 ps simulation and followed by a 100 pssimulation
                to equilibrate the density of the system. Afterward, constantpressure
                MD simulation of 1 ns was then performed in an NPT ensemble at
                1 atm and 300 K. The output and trajectory files were savedevery
                0.1 and 1 ps for the subsequent analysis, respectively. Alltrajectory
                analysis was done with the Ptraj module in the Amber 10.0 suite
                and examined visually using the VMD software package.64
                The MM/GBSA method65 implemented in the AMBER 10.0
                suite was used to calculate the binding free energy between the
                G-quadruplex and the quindoline derivatives. All the waters and
                counterions were stripped off but including the K+ presentwithin
                the negatively charged central channel. The set of 500snapshots
                Table 4. Nucleic Acid Conformation and Samples Used in CompetitionDialysis Experiments
                conformation DNA/oligonucleotide ε/Μ-1 · μ-1 description
                single strand DNA dA21 255400 multiple mutant oligomer of HTG21that may not form G-quadruplex
                dT21 170700
                HTG21mu:d[GAG(TTAGAG)3] 257400
                duplex DNA dA21:dT21 12000 human telomere/complementary
                HTds:d[G3(T2AG3)3]/d[C3(TA2C3)3] 11403
                triplex DNA dA21: (dT21)2 17200
                quadruplex DNA HTG21:d[G3(T2AG3)3] 73000 partial sequence in humantelomere may form intramolecular
                G-quadruplex
                Pu27:d(TG4AG3TG4AG3TG4A2G2) 45309 partial sequence oncogene c-mycthat may form G-quadruplex
                HT-7:d(T2AG3T) 19500 partial sequence of HTG21 that may formintermolecular G-quadruplex
                i-motif HTC21:d[C3(TA2C3)3] 148720 complementary sequence of HTG21that may form an i-motif structure
                5-N-Methylated Quindolines as Telomeric Stabilizing Ligands Journalof Medicinal Chemistry, 2008, Vol. 51, No. 20 6389
                from MD trajectories were collected to calculate the bindingfree
                energies. The formula was used as:
                ΔG)ΔEMM+ΔGsolv+ΔGnp-TΔSMM
                EMM ) Eint + Eelstat + EvdW
                EMM is the internal molecular mechanics energy (comprises
                internal bonding energy terms, nonbonding electrostatic, andvan
                der Waals interations), Gsolv is the solvation free energycalculated
                by solving generalized Born65 equations, Gnp is the nonpolarpart
                of the solvation free energy calculated from solvent-accessible
                surface area (SASA),66 and TΔS is the solute entropy, which is
                usually estimated by normal-mode analysis method.67
                Competition Dialysis Experiment. Competition dialysisexperiments
                were performed as previously described.50 A Tris-HCl buffer
                (10 mM, pH 7.4) containing 100 mM NaCl was used for all
                experiments. For each competition dialysis assay, test ligands(1
                mM concentration, 200 mL volume) were dialyzed against the
                nucleic acid array. A volume of 0.5 mL (at 45 μM monomeric
                unit) of each of the DNA samples was pipetted into a separate0.5
                mL Spectro/Por DispoDialyzer unit with a 1000 molecular weight
                cutoff tubing (Spectrum, Laguna Hills, CA). A panel of 10different
                nucleic acid structures used was listed in Table 4. The entiredialysis
                units were then placed in the beaker containing the dialysate
                solution. At the end of the 24 h equilibration period at room
                temperature, DNA samples were carefully removed to microfuge
                tubes and were taken to a final concentration of 1% (w/v)sodium
                dodecyl sulfate (SDS). The total concentration of ligand (Ct)within
                each dialysis tube was then measured spectrophotometrically at425
                nm with an extinction coefficient of 10780 M-1 ·cm-1 for 2b and
                at 415 nm with an extinction coefficient of 11410 M-1 ·cm-1 for
                1. The free ligand concentration (Cf) was determinedspectrophotometrically
                using an aliquot of the dialysate solution. The amount
                of bound ligand was determined by the difference between thetotal
                ligand concentration and the free ligand concentration (Cb ) Ct-
                Cf). Final analysis of the data was carried out using the Origin7.5
                software (OriginLab Corp.).
                Cell-Free Telomerase Activity Assay. The ability of ligands
                to inhibit telomerase in a cell-free system was assessed with a
                modified TRAP using the extracts from exponentially growing
                MCF-7 breast carcinoma cells as described previously.51Briefly,
                the TRAP assay was carried out in two steps includingtelomerasemediated
                primer-elongation and PCR amplification of the telomerase
                products to enable detection. PCR was performed in a final
                50 μL reaction volume composed of a 45 μL reaction mix
                containing 20 mM Tris-HCl (pH 8.0), 50 μM dNTPs, 1.5 mM
                MgCl2, 63 mM KCl, 1 mM EGTA, 0.005% Tween 20, 20 μg/mL
                BSA, 3.5 pmol of primer TSG4 d(G3AT2G3AT2G3AT2G3T2), 18
                pmol of primer TS d(A2TC2GTCGAGCAGAGT2), 22.5 pmol of
                primer CXext d(GTGC3T2AC3T2AC3T2AC2CTA2), 7.5 pmol of
                primer NT d(ATCGCT2CTCG2C2T4), 0.01 amol of TSNT internal
                control d(AT2C2GTCGAGCAGAGT2A3G2C2GAGA2GCGAT), 2.5
                units of Taq DNA polymerase, and 100 ng of the extracts.
                Compounds or distilled water were added under a volume of 5 μL.
                PCR were performed in an Eppendorf Mastercycler equipped with
                a hot lid and incubated for 30 min at 30 °C, followed by 92 °C
                30 s, 52 °C 30 s, and 72 °C 30 s for 30 cycles. Afteramplification,
                8 μL of loading buffer (containing 5× Tris-Borate-EDTA buffer
                (TBE buffer), 0.2% bromphenol blue, and 0.2% xylene cyanol)were
                added to the reaction. A 15 μL aliquot was loaded onto a 10%
                nondenaturing acrylamide gel (19:1) in 1× TBE buffer and run at
                200 V for 1 h. Gels were fixed and then stained with AgNO3.TelIC50
                values were then calculated from the optical density with the
                LadWorks software.
                Telomere Length Assay. 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 fixed
                on the wet blotting membrane by baking the membrane at 120 °C
                for 20 min. Membrane was hybridized with a DIG-labeled
                hybridization probe for telomeric repeats and incubated withanti-
                DIG-alkaline phosphatase. TRF was performed bychemiluminescence
                detection.
                Short-Term Cell Viability. HL60 and CA46 cells were seeded
                on 96-well plates (1.0 × 103/well) and exposure to various
                concentrations of ligands. After 48 h of treatment at 37 °C ina
                humidified atmosphere of 5% CO2, 10 μL of 5 mg/mL methyl
                thiazolyl tetrazolium (MTT) solution was added to each well and
                further incubated for 4 h. The cells in each well were thentreated
                with dimethyl sulfoxide (DMSO) (200 μL for each well) and the
                optical density (OD) was recorded at 570 nm. All drug doseswere
                parallel tested in triplicate, and the IC50 values were derivedfrom
                the mean OD values of the triplicate tests versus drugconcentration
                curves.
                Long-Term Cell Culture Experiments. Long-term proliferation
                experiments were carried out using the HL60 leukemia cell line
                and CA46 lymphoma cell line. Cells were grown in T80 tissue
                culture flasks at 1.0 × 105 per flask and exposed to asubcytotoxic
                concentration of ligand or an equivalent volume of 0.1% DMSO
                every 4 days. The cells in control and drug-exposed flasks were
                counted and flasks reseeded with 1.0 × 105 cells. The remaining
                cells were collected and used for measurements described below.
                This process was continued for 16 days.
                SA--Gal Assay.21 Cells treated with the ligand were incubated
                for 16 days. After the incubation, the growth medium wasaspirated
                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 thenstained
                using the -Gal stain solution containing 1 mg/mL of 5-bromo-4-
                chloro-3-indolyl--D-galactoside, followed by incubationovernight
                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.
                Acknowledgment. We thank the National Nature Science
                Foundation of China (20472117 and 20772159), the NSFC/RGC
                Joint Research Scheme (30731160006 and N_PolyU 508/06),
                the Science Foundation of Guangzhou (2006Z2-E402), the
                Science Foundation of Zhuhai (grant PC20041131), the NCET,
                the Shenzhen Key Laboratory Fund, and the University Grants
                Committee Areas of Excellence Scheme in Hong Kong (AoE
                P/10-01) for financial support of this study. We also thank Dr.
                Hai-Bin Luo, School of Pharmaceutical Sciences in Sun Yatsen
                University, for his assistance with the molecular modeling
                studies.
                Note Added after ASAP Publication. This paper published
                ASAP on September 27, 2008 without the author affiliation
                information. The correct version was published on October 16,
                2008.
                Supporting Information Available: CD spectra of HTG21 with
                5-N-methyl quindolines 2a-x and nonmethylated quindolines,
                NMR spectra of [d(T2AG3)]4 with ligand 2r, molecular modeling
                studies, MS, and 1H NMR spectra of compounds 3a-d and 4a-d,
                HRMS and 1H and 13C NMR spectra of compounds 2a-x. This
                material is available free of charge via the Internet athttp://
                pubs.acs.org.
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