5-N-Methylated Quindoline Derivatives as Telomeric

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.
References
(1) Blackburn, E. H. Structure and function of telomeres. Nature1991,
350, 569–573.
(2) Blackburn, E. H. Switching and signaling at the telomere. Cell2001,
106, 661–673.
(3) Harley, C. B.; Futcher, A. B.; Greider, C. W. Telomeres shortenduring
aging of human Fibroblasts. Nature 1990, 345, 458–460.
(4) Greider, C. W.; Blackburn, E. H. Identification of a specifictelomere
terminal transferase activity in Tetrahymena extracts. Cell 1985,43,
405–413.
(5) Feng, J.; Funk, W.; Wang, S.-S.; Weinrich, S. L.; Avilion, A.A.; Chiu,
C.-P.; Adam, R. R.; Chang, E. The RNA component of human
telomerase. Science 1995, 269, 1236.
6390 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 Lu etal.
(6) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.;West,
M. D.; Ho, P. L.; Coviello, G. M.; Wright, W. E.; Weinrich, S.L.;
Shay, J. W. Specific association of human telomerase activitywith
immortal cell and cancer. Science 1994, 266, 2011–2015.
(7) Bryan, T. M.; Cech, T. R. Telomerase and maintenance ofchromosome
ends. Curr. Opin. Cell Biol. 1999, 11, 318–322.
(8) Masutomi, K.; Yu, E. Y.; Khurts, S.; Ben-Porath, I.; Currier,J. L.;
Metz, G. B.; Brooks, M. W.; Kaneko, S.; Murakami, S.; DeCaprio,
J. A.; Weinberg, R. A.; Stewart, S. A.; Hahn, W. C. Telomerase
maintains telomere structure in normal human cells. Cell 1999,114,
241–253.
(9) Herbert, B. S.; Pitts, A. E.; Baker, S. I.; Hamilton, S.E.;Wright, W. E.;
Shay, J. W.; Corey, D. R. Inhibition of human telomerase inimmortal
human cells leads to progressive telomere shortening and celldeath.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14276–14281.
(10) Neidle, S.; Parkinson, G. Telomere maintenance as a targetfor
anticancer drug discovery. Nat. ReV. Drug DiscoVery 2002, 1,383–
393.
(11) Herbert, B. S.; Pitts, A. E.; Baker, S. I.; Hamilton, S.E.;Wright, W. E.;
Shay, J. W.; Corey, D. R. Inhibition of human telomerase inimmortal
human cells leads to progressive telomere shortening and celldeath.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14276–14281.
(12) Mergny, J.-L.; Riou, J.-F.; Mailliet, P.; Teulade-Fichou,M.-P.; Gilson,
E. Natural and pharmacological regulation of telomerase. NucleicAcids
Res. 2002, 30, 839–865.
(13) Rezler, E. M.; Rearss, D. J.; Hurley, L. H. Telomeres andtelomerases
as drug targets. Curr. Opin. Pharmacol. 2002, 2, 415–423.
(14) De Cian, A.; Lacroix, L.; Douarre, C.; Temime-Smaali, N.;Trentesaux,
C.; Riou, J.-F.; Mergny, J.-L. Targeting telomeres andtelomerase.
Biochime 2008, 90, 131–155.
(15) Ou, T.-M.; Lu, Y.-J.; Tan, J.-H.; Huang, Z.-S.; Wong, K.-Y.;Gu,
L.-Q. G-quadruplexes: targets in anticancer drug designChemMed-
Chem 2008, DOI:10.1002/cmdc.200700300.
(16) Neidle, S.; Parkinson, G. N. The structure of telomeric DNA.Curr.
Opin. Struct. Biol. 2003, 13, 275–283.
(17) Kim, M.-Y.; Vankayalapati, H.; Shin-ya, K.; Wierzba, K.;Hurley,
L. H. Telomestatin, a Potent Telomerase Inhibitor That InteractsQuite
Specifically with the Human Telomeric IntramolecularG-Quadruplex.
J. Am. Chem. Soc. 2002, 124, 2098–2099.
(18) Wheelhouse, R. T.; Sun, D.; Han, H.; Han, F. X.; Hurley, L.H.
Cationic Porphyrins as Telomerase Inhibitors: the Interactionof
Tetra(N-methyl-4-pyridyl)porphine with Quadruplex DNA. J. Am.
Chem. Soc. 1998, 120, 3261–3262.
(19) Read, M. A.; Wood, A. A.; Harrison, J. R.; Gowan, S. M.;Kelland,
L. R.; Dosanjh, H. S.; Neidle, S. Molecular Modeling Studies on
G-Quadruplex Complexes of Telomerase Inhibitors: Structure-
Activity Relationships. J. Med. Chem. 1999, 42, 4538–4546.
(20) Read, M.; Harrison, R. J.; Romagnoli, B.; Tanious, F. A.;Gowan,
S. H.; Reszka, A. P.; Wilson, W. D.; Kelland, L. R.; Neidle, S.
Structure-based design of selective and potent Gquadruplex-mediated
telomerase inhibitors. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,4844–
4849.
(21) Moore, M. J. B.; Schultes, C. M.; Cuesta, J.; Cuenca, F.;Gunaratnam,
M.; Tanious, F. A.; Wilson, W. D.; Neidle, S. Trisubstitutedacridines
as G-quadruplex telomere targeting agents. Effects of extensionsof
the 3,6- and 9- side chains on quadruplex binding, telomeraseactivity,
and cell proliferation. J. Med. Chem. 2006, 49, 582–599.
(22) Cookson, J. C.; Heald, R. A.; Stevens, M. F. G. Antitumorpolycyclic
acridines. 17. Synthesis and pharmaceutical profiles ofpentacyclic
acridinium salts designed to destabilize telomeric integrity. J.Med.
Chem. 2005, 48, 7198–7207.
(23) Fedoroff, O. Y.; Salazar, M.; Han, H.; Chemeris, V. V.;Kerwin, S. M.;
Hurley, L. H. NMR-based model of a telomerase-inhibitingcompound
bound to G-quadruplex DNA. Biochemistry 1998, 37, 12367–12374.
(24) Kern, J. T.; Wang, T. P.; Kerwin, S. M. The relationshipbetween
ligand aggregation and G-quadruplex DNA selectivity in a seriesof
3,4,9,10-perylenetetracarboxylic acid diimides. Biochemistry 2002,41,
11379–11389.
(25) Gomez, D.; Aouali, N.; Renaud, A.; Douarre, C.; Shin-ya, K.;Tazi,
J.; Martinez, S.; Trentesaux, C.; Morjani, H.; Riou, J.-F.Resistance
to senescence induction and telomere shortening by aG-quadruplex
ligand inhibitor of telomerase. Cancer Res. 2003, 63,6149–6153.
(26) Izbicka, E.; Wheelhouse, R. T.; Raymond, E.; Davidson, K.K.;
Lawrence, R. A.; Sun, D.; Windle, B. E.; Hurley, L. H.; VonHoff,
D. D. Effects of cationic porphyrins as G-quadruplex interactiveagents
in human tumor cells. Cancer Res. 1999, 59, 639–644.
(27) Missailidis, S.; Stanslas, J.; Modi, C.; Ellis, M. J.; Robins,R. A.;
Laughton, C. A.; Stevens, M. F. G. Antitumor polycyclicacridines.
Part 12. Physical and biological properties of8,13-diethyl-6-methylquino[
4,3,2-kl]acridinium iodide: a lead compound in anticancer drug
design. Oncol. Res. 2002, 13, 175–189.
(28) Gowan, S. M.; Heald, R.; Stevens, M. F. G.; Kelland, L. R.Potent
inhibition of telomerase by small-molecule pentacyclicacridines
capable of interacting with G-quadruplexes. Mol. Pharmacol.2001,
60, 981–988.
(29) Holt, S. J.; Petrow, V. Cabarzole, carbolines, and relatedcompounds
[Part 1]. J. Chem. Soc. 1947, 607–611.
(30) Bierer, D. E.; Dubenko, L. G.; Zhang, P. Antihyperglycemicactivities
of cryptolepine analogues: an ethnobotanical lead structurefrom
Cryptolepis sanguinolenta. J. Med. Chem. 1998, 41, 2754–2764.
(31) Yang, S.-W.; Abdel-Kader, M.; Malone, S.; Werkhoven, M. C.;Wisse,
J. H.; Bursuker, I.; Neddermann, K.; Fairchild, C.;Raventos-Suarez,
C.; Menendez, A. T.; Lane, K.; Kingston, D. G. Synthesis and
biological evaluation of analogues of cryptolepine, an alkaloidisolated
from Suriname Rainforest. J. Nat.Prod 1999, 62, 976–983.
(32) Lisgarten, J. N.; Coll, M.; Portugal, J. W. W. C.; Aymami, J.The
antimalaral and cytotoxic drug cryptolepine intercalates into DNAat
cytosine-cytosine sites. Nat. Struct. Biol. 2002, 9, 57–60.
(33) Caprio, V.; Guyen, B.; Opoku-Boahen, Y.; Mann, J.; Gowan, S.M.;
Kelland, L. M.; Read, M. A.; Neidle, S. A novel inhibitor ofhuman
telomerase derived from 10H-indolo-[3,2-b]quinoline. Bioorg.Med.
Chem. Lett. 2000, 10, 2063–2066.
(34) Guyen, B.; Schultes, C. M.; Hazel, P.; Mann, J.; Neidle, S.Synthesis
and evaluation of analogs of 10H-indolo[3,2-b]quinoline asGquadruplex
stabilizing ligands and potential inhibitors of the enzyme
telomerase. Org. Biomol. Chem. 2004, 2, 981–988.
(35) LeSanna, C.; Huddlestona, J.; Mann, J. Synthesis andpreliminary
evaluation of novel analogues of quindolines as potentialstabilisers
of telomeric G-quadruplex DNA. Tetrahedron 2007, 63,12903–12911.
(36) Zhou, J.-L.; Lu, Y.-J.; Ou, T.-M.; Zhou, J.-M.; Huang, Z.-S.;Zhu,
X.-F.; Du, C.-J.; Bu, X.-Z.; Ma, L.; Gu, L.-Q.; Li, Y.-M.; Chan,A.
S.-C. Synthesis and evaluation of quindoline derivatives asGquadruplex
inducing and stabilizing ligands and potential inhibitors
of telomerase. J. Med. Chem. 2005, 48, 7315–7321.
(37) Zhou, J. M.; Zhu, X. F.; Lu, Y. J.; Deng, R.; Huang, Z. S.;Mei, Y. P.;
Wang, Y.; Huang, W. L.; Liu, Z. C.; Gu, L. Q.; Zeng, Y. X.
Senescence and telomere shortening induced by novel potentGquadruplex
interactive agents, quindoline derivatives, in human cancer
cell lines. Oncogene 2006, 25, 503–511.
(38) Monchaud, D.; Teulade-Fichou, M. P. A hitchhiker’s guideto
G-quadruplex ligands. Org. Biomol. Chem. 2008, 6, 627–636.
(39) Fan, P.; Ablordeppey, S. Y. An alternative synthesis of10H-Indolo[3,2-
b]quinoline and its selective N-alkylation. J. Heterocycl. Chem.1997,
34, 1789–1794.
(40) Li, W.; Wu, P.; Ohmichi, T.; Sugimoto, N. Characterizationand
thermodynamic properties of quadruplex/duplex competition. FEBS
Lett. 2002, 526, 77–81.
(41) Balagurumoorthy, P.; Brahmachari, S. K.; Mohanty, D.; Bansal,M.;
Sasisekharan, V. Hairpin and parallel quartet structures fortelomeric
sequences. Nucleic Acids Res. 1992, 20, 4061–4067.
(42) Rezler, E. M.; Seenisamy, J.; Bashyam, S.; Kim, M.-Y.; White,E.;
Wilson, W. D.; Hurley, L. H. Telomestatin and diselenosapphyrin
bind selectively to two different forms of the human telomeric
G-quadruplex structure. J. Am. Chem. Soc. 2005, 127, 9439–9447.
(43) Paramasivan, S.; Rujan, I.; Bolton, P. H. Circular dichroismof
quadruplex DNAs: applications to structure, cation effects andligand
binding. Methods 2007, 43, 324–331.
(44) Mergny, J.-L.; Lacroix, L.; Teulade-Fichou, M.-P.; Hounsou,C.;
Guittat, L.; Hoarau, M.; Arimondo, P. B.; Vigneron, J.-P.; Lehn
J.-M.; Riou, J.-F.; Garestier, T.; Helene, C. Telomerase inhibitorsbased
on quadruplex ligands selected by a fluorescence assay. Proc.Natl.
Acad. Sci. U.S.A. 2001, 98, 3062–3067.
(45) De Cian, A.; Guittat, L.; Kaiser, M.; Sacca, B.; Amrane,S.;
Bourdoncle, A.; Alberti, P.; Teulade-Fichou, M. P.; Lacroix,L.;
Mergny, J. L. Fluorescence-based melting assays for studyingquadruplex
ligands. Methods 2007, 42, 183–95.
(46) Han, H.; Hurley, L. H.; Salazar, M. A DNA polymerase stopassay
for G-quadruplex-interactive compounds. Nucleic Acids Res.1999,
27, 537–542.
(47) Lemarteleur, T.; Gomez, D.; Paterski, R.; Mandine, E.;Mailliet, P.;
Riou, J.-F. Stabilization of the c-myc gene promoter quadruplexby
specific ligands’ inhibitors of telomerase. Biochem. Biophys.Res.
Commun. 2004, 323, 802–808.
(48) Parkinson, G. N.; Lee, M. P. H.; Neidle, S. Crystal structureof parallel
quadruplexes from human telomeric DNA. Nature 2002, 417, 876–
880.
(49) Bates, P.; Mergny, J. L; Yang, D. Quartets in G-major. EMBORep.
2007, 8, 1003–1010.
(50) Ragazzon, P.; Chaires, J. B. Use of competition dialysis inthe
discovery of G-quadruplex selective ligands. Methods 2007, 43,313–
323.
(51) Gomez, D.; Mergny, J.-L.; Riou, J.-F. Detection oftelomerase
inhibitors based on G-quadruplex ligands by a modifiedtelomeric
repeat amplification protocol assay. Cancer Res. 2002, 62,3365–3368.
5-N-Methylated Quindolines as Telomeric Stabilizing Ligands Journalof Medicinal Chemistry, 2008, Vol. 51, No. 20 6391
(52) De Cian, A.; Cristofari, G.; Reichenbach, P.; De Lemos, E.;Monchaud,
D.; Teulade-Fichou, M.-P.; Shin-ya, K.; Lacroix, L.; Lingner,J.;
Mergny, J.-L. Reevaluation of telomerase inhibition byquadruplex
ligands and their mechanisms of action. Proc. Natl. Acad. Sci.USA
2007, 104, 17347–17352.
(53) Hwang, E. S. Replicative senescence and senescence-like stateinduced
in cancer-derived cells. Mech. Aging DeV. 2002, 123, 1681–1694.
(54) Roninson, I. B. Tumor senescence as a determinant of drugresponse
in vivo. Drug Resist. Updates 2002, 5, 204–208.
(55) Mita, H.; Ohyama, T.; Tanaka, Y.; Yamamoto, Y. Formation ofa
complex of5,10,15,20-tetrakis(N-methyl-pyridinium-4-yl)-21H,23Hporphyrin
with G-quadruplex DNA. Biochemistry 2006, 45, 6765–
6772.
(56) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.;Hart, W. E.;
Belew, R. K.; Olson, A. J. Automated docking using a Lamarckian
genetic algorithm and an empirical binding free energyfunction.
J. Comput. Chem. 1998, 19, 1639–1662.
(57) Sanner, M. F. Python: a programming language for softwareintegration
and development. J. Mol. Graphics Modell. 1999, 17, 57–61.
(58) Cornell, W. D.; Cieplak, C. I.; Bayly, I. R.; Gould, I. R.;Merz, K. M.;
Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.;Kollman,
P. A. A second generation force field for the simulation ofproteins,
nucleic acids and organic molecules. J. Am. Chem. Soc. 1995,117,
5179–5197.
(59) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case,D. A.
Development and testing of a general amber force field. J.Comput.
Chem. 2004, 25, 1157–1174.
(60) Hazel, P.; Parkinson, G. N.; Neidle, S. Predictive modellingof topology
and loop variations in dimeric DNA quadruplex structures.Nucleic
Acids Res. 2006, 34, 21172127.
(61) Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald: An Nlog-
(N) method for Ewald sums in large systems. J. Chem. Phys.1993,
98, 10089–10092.
(62) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R.W.;
Klein, M. L. Comparison of simple potential functions forsimulating
liquid water. J. Chem. Phys. 1983, 79, 926–935.
(63) Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. Numericalintegration
of the cartesian equations of motion of a system withconstraints:
molecular dynamics of n-alkanes. J. Comput. Phys. 1977, 23,327–341.
(64) Humphrey, W.; Dalke, A.; Schulten, K. VMD: visualmolecular
dynamics. J. Mol. Graphics 1996, 14, 33–38.
(65) Kollman, P. A.; Massova, I.; Reyes, C.; Kuhn, B.; Huo, S. H.;Chong,
L.; Lee, M.; Lee, T.; Duan, Y.; Wang, W.; Donini, O.; Cieplak,P.;
Srinivasan, J.; Case, D. A.; Cheatham, T. E. Calculatingstructures
and free energies of complex molecules: combining molecular
mechanics and continuum models. Acc. Chem. Res. 2000, 33, 889–
897.
(66) Sitkoff, D.; Sharp, K. A.; Honig, B. Accurate calculation ofhydration
free energies using macroscopic solvent models. J. Phys. Chem.1994,
98, 1978–1988.
(67) Kottalam, J.; Case, D. A. Langevin modes ofmacromoleculessapplications
to Crambin and DNA hexamers. Biopolymers 1990, 29,
1409–1421.
JM800497P