Design and synthesis of new benzimidazole-carbazole conjugates for the stabilization of human telomeric DNA, telomerase inhibition, and their selective action on cancer cells

Article abstract of DOI:10.1021/jm500427n

Cell-permeable small molecules that enhance the stability of the G-quadruplex (G4) DNA structures are currently among the most intensively pursued ligands for inhibition of the telomerase activity. Herein we report the design and syntheses of four novel benzimidazole-carbazole conjugates and demonstrate their high binding affinity to G4 DNA. S1 nuclease assay confirmed the ligand mediated G-quadruplex DNA protection. Additional evidence from Telomeric Repeat Amplification Protocol (TRAP-LIG) assay demonstrated efficient telomerase inhibition activity by the ligands. Two of the ligands showed IC ₅₀ values in the sub-micromolar range in the TRAP-LIG assay, which are the best among the benzimidazole derivatives reported so far. The ligands also exhibited cancer cell selective nuclear internalization, nuclear condensation, fragmentation, and eventually antiproliferative activity in long-term cell viability assays. Annexin V-FITC/PI staining assays confirm that the cell death induced by the ligands follows an apoptotic pathway. An insight into the mode of ligand binding was obtained from the molecular dynamics simulations.

Full text of DOI:10.1021/jm500427n

Article
pubs.acs.org/jmc
Design and Synthesis of New Benzimidazole−Carbazole Conjugates
for the Stabilization of Human Telomeric DNA, Telomerase
Inhibition, and Their Selective Action on Cancer Cells
Basudeb Maji,† Krishan Kumar,† Mangesh Kaulage,†,§ K. Muniyappa,§ and Santanu Bhattacharya*,†,‡
†Department of Organic Chemistry, Indian Institute of Science, Bangalore, Karnataka 560 012, India
‡Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, Karnataka 560 012, India
§Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka 560 012, India
S
* Supporting Information
ABSTRACT: Cell-permeable small molecules that enhance
the stability of the G-quadruplex (G4) DNA structures are
currently among the most intensively pursued ligands for
inhibition of the telomerase activity. Herein we report the
design and syntheses of four novel benzimidazole−carbazole
conjugates and demonstrate their high binding affinity to G4
DNA. S1 nuclease assay confirmed the ligand mediated G-
quadruplex DNA protection. Additional evidence from
Telomeric Repeat Amplification Protocol (TRAP-LIG) assay
demonstrated efficient telomerase inhibition activity by the
ligands. Two of the ligands showed IC50 values in the sub-
micromolar range in the TRAP-LIG assay, which are the best
among the benzimidazole derivatives reported so far. The
ligands also exhibited cancer cell selective nuclear internalization, nuclear condensation, fragmentation, and eventually
antiproliferative activity in long-term cell viability assays. Annexin V-FITC/PI staining assays confirm that the cell death induced
by the ligands follows an apoptotic pathway. An insight into the mode of ligand binding was obtained from the molecular
dynamics simulations.
In recent years, a number of G4 DNA stabilizing small
molecules has been synthesized and tested for telomerase
inhibition. Among these, the natural product telomestatin7 and
the synthetic molecules, e.g., BRACO-198 and BMSG-SH-3,9
are among the most promising candidates. Benzimidazole
derivatives belong to another class of molecules which possess
sequence-selective DNA binding ability.10 These include
Hoechst 33258 which is known for its specific affinity toward
AT-rich minor grooves in double-stranded DNA. Neidle and
co-workers demonstrated for the first time the symmetric bis-
benzimidazoles for their potent sequence-specific duplex-DNA
binding activity.11 Li et al. were able to utilize a bis-
(benzimidazole)pyridine derivative for selective G4 stabiliza-
tion.12
We have previously reported 1,3-phenylene-bis-benzimida-
zole derivatives for their higher G4 DNA selectivity and
stabilizing ability (Figure 1A).13a−d On the other hand, cationic
carbazole derivatives are known for their ability to inhibit
topoisomerase activity, and they also function as antimicrobial
agents.14 Recently, carbazole conjugates with a vinylic-
pyridinium moiety were shown to bind and stabilize telomeric
INTRODUCTION
■
Throughout the eukaryotic kingdom, the ends of linear
chromosomes are capped by telomeres. The telomere in
mammalian cells consists of a tandem repeat of guanine rich
hexameric (5′-TTAGGG-3′) arrays of up to 5−15 kb from the
extreme 3′-end.1 In somatic cells, telomeres shorten due to the
“end-replication problem” and consequently lead to various
abnormalities of which the most important one involves an
“end-to-end fusion”.2 A ribonucleoprotein complex called
telomerase is responsible for telomeric length maintenance
through the addition of TTAGGG repeats.3 Initially identified
in ciliates, human telomerase consists of three subunits:
telomerase reverse transcriptase (hTERT), telomerase RNA
(hTR), and Dyskerin. Certain types of cancer cells contain
considerably high levels of telomerase activity, whereas it has
not been detected in most of the normal somatic cells.4 The
terminal 3′-telomeric DNA sequence, being both single-
stranded and G-rich in nature, has a high propensity to fold
into the G4 structures by Hoogsteen H-bonding between the
guanine bases under physiological ionic conditions.5 The
telomeric DNA folded into the G4 DNA is unable to serve
as a substrate for telomerase. Therefore, stabilization of the G4
DNA is considered as a potential target in cancer chemo-
therapy.6
Received: March 18, 2014
© XXXX American Chemical Society
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Figure 1. (A−C) The molecular structures of the ligands used in the study. (D) A superimposed structure of the G-tetrad18 and the ligand (1) in
real scale showing efficient overlapping. The ligand is shown in blue color. The mono-(bis-benzimidazole) derivatives (1−3) possess more similarity
with the G-tetrad dimension than the corresponding di-(bis-benzimidazole) derivative (4).
DNA G-quadruplexes and inhibit telomerase activity.15 A few
carbazole−benzimidazole conjugates are known for the
sequence-specific double-stranded DNA binding.16 However,
there is hardly any example of the G4 DNA selective ligands
that inhibit telomerase activity in this family.17 It occurred to us
that it would be logical to modify carbazole−benzimidazole
conjugates so that they are transformed into the G4 DNA
binders and do not prefer the duplex DNA. Finally, they should
also be capable of targeting the cancer cells selectively. Herein,
we report the syntheses of four new benzimidazole derivatives
with a carbazole coreCMP (1), CHP (2), CBM (3), and
CBhoe (4)and show that they possess a high potential for
the G4 DNA stabilization. The structure−activity relationship
has been investigated with functional variation from mono-(bis-
benimidazole) to di-(bis-benzimidazole) along with different
substitutions at the termini ranging from piperazine to
morpholine (Figure 1). The variation in the number of
benzimidazoles which leads to an alteration in the molecular
dimension, the extent of crescent shape in the molecular
structure, along with the modification in the end-functionality
enabled us to investigate their influence on the G4 DNA
binding and telomerase inhibition efficiency. Finally, the
experimental results were rationalized with appropriate
computational studies.
To optimize their interaction with DNA, we have incorporated
a piperazine/morpholine moiety in the three termini which
should induce hydrogen bonding interactions and get
protonated at pH 7.4 to facilitate the ligand’s interaction with
the phosphate groups of DNA.19
The synthesis started with the N-alkylation of carbazole upon
reaction with 1,4-dibromobutane by the PTC method to
furnish the N-4-bromobutyl-carbazole (9) in ∼72% yield. The
bromo derivative (9) was then reacted with N-methylpiperizine
followed by formylation of the carbazole nucleus to obtain the
dialdehyde (11) in 75% yield. Oxidative coupling of 11 with
each of the individual diamines 6, 7, and 8 in the presence of
Na2S2O5 afforded the final compounds 1, 2, and 3, respectively,
in 60−80% isolated yield (Scheme 1).
In a separate flask, 4-aminobenzonitrile was N-acetylated in
98% yield. This was followed by its nitration (85%) and the
resulting product was then deacetylated to furnish 4-amino-3-
nitrobenzonitrile (14) in 95% yield. The compound 14 was
then reacted with dry HCl in the presence of freshly dried
ethanol to afford the corresponding imino-ether hydrochloride
(15) in 85% yield. The resulting compound 15 was reacted
with freshly prepared diamine 6 to obtain the nitro amino
benzimidazole derivative (16) in 78% yield. Subsequent
reduction of compound 16 afforded the corresponding diamine
(17) which was used for the next reaction without isolation.
The coupling of freshly prepared diamine 17 with the
dialdehyde (11) afforded the desired di-(bis-bezimidazole)
derivative (4) in 50% yield (Scheme 2). All the new
compounds were adequately characterized by IR, NMR, mass,
and elemental analysis as given in the Materials and Methods.
UV−vis Absorption Spectral Titrations. To confirm
whether each ligand interacts with the G4 DNA, UV−vis
titrations of each compound with preformed G4 DNA were
individually performed in 10 mM Tris−HCl (pH 7.4)
RESULTS AND DISCUSSION
■
Synthesis. Herein we have designed and synthesized
benzimidazole derivatives based on carbazole as the central
pharmacophore. We have considered a few aspects in the ligand
design like the dimension, shape, extended planarity, and nature
of protonatable sites to investigate their role in the structure−
activity relationship. The molecular dimension and the crescent
character of the ligands have been tuned by varying the number
of benzimidazole units attached to the central carbazole core.
B
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a
Scheme 1
a
Reagents, conditions, and yields: (a) K2CO3, dry DMF, 110 °C, 24 h, 89%; (b) Pd/C−H2, EtOH, rt, 24 h; (c) 1,4-dibromobutane, TBAI, 50%
NaOH, benzene, rt, 12 h, 72%; (d) N-methyl-piperazine, K2CO3, acetonitrile, rt, 6 h, 89%; (e) ZnCl2, POCl3/DMF, 0 °C → 100 °C, 24 h, 75%; (f)
6, 7, or 8, Na2S2O5, EtOH, 80 °C, 24 h, 60−80%.
containing 0.1 M NaCl or KCl and 0.1 mM EDTA. This was
compared with their binding with a telomeric duplex DNA or
calf-thymus (CT) DNA in 10 mM Tris−HCl (pH 7.4)
containing 40 mM NaCl and 0.1 mM EDTA.
induced collisional quenching in the aqueous medium (Figure
2A and B and Supporting Information, Figures S3 and S4).
When an increasing amount of the preformed G4 DNA was
added to the ligand solution in Tris−HCl buffer of pH 7.4,
sharp increases in the fluorescence intensity were observed,
which was followed by saturation for each ligand.
Ligand solutions of 5 μM were titrated by gradual addition of
aliquots of preformed G4 DNA (50 μM). This resulted in a
strong hypochromism, suggesting the involvement of π-stacking
interactions between the ligands and the G4 DNA. The extent
of hypochromicity observed with the duplex DNA (either
telomeric duplex or CT DNA) during titration was
insignificant, indicating considerably weaker interactions of
the ligands with the duplex DNA (Supporting Information,
Figures S1 and S2). The absorption spectral data (at 320 nm
for each of CMP, CHP, and CBM and 340 nm for CBhoe)
were analyzed using the linear Scatchard equation r/Cf = Ka(n
− r) and plotted to determine the binding constants (Table 1
and Supporting Information, Table S1) following a previously
reported protocol (discussed in the Supporting Informatio-
n).13a−e
Fluorescence Spectroscopy. Each ligand when solubi-
lized in the experimental buffer of pH 7.4 displayed significant
fluorescence emission following excitation at either 300 or 330
nm. Accordingly, we have used fluorescence spectroscopy to
monitor the interaction of each ligand with the preformed G4
DNA in various buffer solutions in the presence of KCl or
NaCl. All the ligands CMP, CHP, CBM, and CBhoe showed
low fluorescence in Tris−HCl buffer possibly due to solvent
The increase in fluorescence intensity indicates that the
ligands come to the proximity of the G4 DNA and achieve a
hydrophobic environment to retain their intrinsic fluorescence
emission manifesting their high affinity toward the G4 DNA.
The extent of increments in the fluorescence emission was
different for the ligands in buffer solutions according to their
binding efficiency (Figure 2A and B and Supporting
Information, Figures S3 and S4). Moreover, the ligands showed
a higher enhancement in the fluorescence intensity in KCl
buffer than the corresponding NaCl buffer possibly due to their
higher affinity toward the K+-ion-stabilized G4 DNA (Figure
2C and Supporting Information, Figures S5 and S6, Table S2).
The fluorescence images showed selective light-up ability
toward the G4 DNA upon ligand binding (Figure 2D and
Supporting Information, Figure S7).
Each ligand solution was also titrated against CT DNA in 10
mM Tris−HCl (pH 7.4), 0.1 mM EDTA, and 40 mM NaCl.
The titrations did not show any significant change in the
fluorescence emission spectra (Figure 2C). This observation
indicates the minimal affinity of the ligands toward the double-
stranded CT DNA.
C
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a
Scheme 2
a
Reagents, conditions, and yields: (a) Ac2O (4 equiv), NaOAc, rt, 20 min, 98%; (b) KNO3 (2.1 equiv), c. H2SO4, <0 °C, 1 h, 85%; (c) 10% H2SO4,
heat, 30 min, 95%; (d) anhyd. HCl(g), dry EtOH, 7 days of stirring, rt, 85%; (e) 4-(4-methylpiperazin-1-yl)benzene-1,2-diamine (Scheme 1;
compound 6) EtOH:AcOH (2:1), reflux, 24 h, 78%; (f) Pd/C−H2, EtOH, rt, 12 h, 100%; (g) 9-(4-(4-methylpiperazin-1-yl)butyl)-9H-carbazole-3,6-
dicarbaldehyde (Scheme 1; compound 11), Na2S2O5, EtOH, 80 °C, 12 h, 50%.
Table 1. Dissociation Constants (Kd) of the Ligands with the Preformed Hum21 G4 DNA and CT DNA
a
a
b
Hum21 (NaCl)
Hum21 (KCl)
Kd (μM)
CT DNA
ligand
Kd (μM)
n
n
Kd (μM)
selectivity toward the G4 DNA over the duplex DNA
CMP
CHP
0.34 0.01
0.05 0.002
0.48 0.02
0.38 0.03
2.5
2.3
2.9
3.2
0.13 0.01
0.03 0.002
0.33 0.04
0.23 0.02
2.7
3.2
2.6
3.4
196 12
576
500
650
234
25
312 14
89
2
CBM
CBhoe
4
a
Dissociation constants (Kd) were calculated from the binding assays performed with the preformed Hum21 G4 DNA in 10 mM Tris−HCl (pH 7.4)
b
having 0.1 M NaCl/KCl and 0.1 mM EDTA. The notation “n” represents the binding stoichiometry of the ligands with the G4 DNA. Binding
assays were performed with CT DNA in 10 mM Tris−HCl (pH 7.4) having 40 mM NaCl and 0.1 mM EDTA.
G4-FID Assays. Fluorescent intercalator displacement
assays have been performed to examine each ligand’s relative
affinity toward the G4 DNA and selectivity over the double-
stranded CT DNA or telomeric duplex DNA. For this purpose,
the standard G4 DNA intercalator thiazole orange (TO) has
been used.20 All the ligands showed efficient TO displacement
ability from the G4 DNA−TO complex. The mono-(bis-
benzimidazole) ligands (CMP, CHP, and CBM) showed a
higher efficiency than the di-(bis-benzimidazole) ligand
(CBhoe), suggesting a higher G-tetrad affinity of the former
ligand (Figure 3). In contrast, all the ligands showed a
considerably lower TO displacement efficiency (Table 2) while
interacting with the duplex DNA (CT DNA and Telo ds
DNA).
Circular Dichroism Spectroscopy. The preformed Hum21
G4 DNA structures were established by recording their CD
spectra both in 0.1 M NaCl and 0.1 M KCl buffer. These
spectral profiles were consistent with those reported earlier.21,22
The G4 DNA in the KCl buffer showed a negative peak at 240
nm and a positive peak at 292 nm along with two shoulders at
250 and 270 nm which matched with the reported intra-
molecular hybrid DNA structure as described earlier.19 The G4
DNA in NaCl buffer showed two positive peaks at 295 and 246
nm along with a negative peak at 266 nm which is similar to
D
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Figure 2. Fluorescence titration spectra of 0.4 μM ligand (A) CHP and (B) CMP in 10 mM Tris−HCl (pH 7.4), 0.1 mM EDTA, and 0.1 M KCl
with 10 μM preformed Hum21 G4 DNA. (C) Relative enhancement in the fluorescence intensities of the ligands (CMP, CHP, CBM, and CBhoe) in
10 mM Tris−HCl (pH 7.4), 0.1 mM EDTA, and 0.1 M NaCl upon interaction with the Hum21 G4 DNA/CT DNA. (D) Fluorescence images of 20
μM ligand CHP and ligand−DNA complexes (with 40 μM Hum21, CT DNA, and Telo ds DNA) under UV light (λex = 365 nm) in 10 mM Tris−
HCl (pH 7.4) and 0.1 mM EDTA having either 0.1 M KCl (for G4 DNA) or 0.04 M NaCl (for CT DNA and Telo ds DNA).
Figure 3. (A) TO displacement assay by the ligand CHP from Hum21 G4 DNA−TO complex. (B) Relative TO displacement plots for the ligands
CMP, CHP, CBM, and CBhoe.
that reported for an intramolecular antiparallel G4 DNA
structure in solution.22
Table 2. DC50 Values (Ligand Concentrations for the
Displacement of 50% of the Bound TO from DNA) of the
Ligands as Determined from Fluorescent Intercalator
The titration of the G4 DNA with both CMP and CHP led
to an increase in the positive peak at 295 nm followed by the
appearance of a new peak in the range of the ligand’s
absorption maxima (300−350 nm) at higher ligand concen-
trations (Figure 4 and Supporting Information, Figures S8 and
S9). CBM and CBhoe also showed sharp enhancements in the
CD intensity, emphasizing their strong association with the G4
DNA structure (Supporting Information, Figure S9C and D).
Though the spectra maintained an iso-dichroic point in the
initial stage of titration, they started to deviate at higher ligand
a
Displacement Assays
ligand
DC50 (μM)
ligand
DC50 (μM)
CMP (G4-K+)
CHP (G4-K+)
CHP (CT DNA)
0.61 0.04
1.12 0.03
7.71 0.08
CBM (G4-K+)
CBhoe (G4-K+)
CHP (Telo ds)
1.01 0.03
1.36 0.05
>8
a
The results are the average of two independent experiments.
E
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Figure 4. (A, B) CD spectral titrations of 4 μM Hum21 G4 DNA with increasing concentration of CHP in 10 mM Tris−HCl (pH 7.4) containing
0.1 mM EDTA and 0.1 M NaCl.
Figure 5. Formation of the G4 DNA in the presence of (A) CMP and (B) CHP in 10 mM Tris−HCl (pH 7.4), 0.1 M KCl, and 0.1 mM EDTA.
concentrations with simultaneous appearance of an induced
circular dichroism (ICD) band (Figure 4). This suggests the
generation of more than one form of DNA−ligand complexes
possibly due to the groove binding at higher ligand
concentrations. For all the ligands, the ICD started appearing
from a [L]:[DNA] ratio of ∼5. Most probably at lower
concentration, the ligands prefer to interact with the G-tetrad of
the G4 DNA structure, whereas, at a higher concentration of
the ligands, these molecules begin to interact with the chiral
groove and the chirality gets transferred to the DNA groove
bound achiral ligands, giving ICD followed by saturation.13a−e
The CD titration of CT DNA with each of the ligands CMP,
CHP, and CBM did not, however, show any significant change.
This indicates their poor binding affinity toward the duplex
DNA (Supporting Information, Figure S8C). In contrast, the
ligand CBhoe showed appreciable enhancement in the ICD
region upon binding with CT DNA (Supporting Information,
Figure S8D). Thus, the CD spectral titration studies with both
the G- quadruplex and the duplex DNA suggest that the ligands
containing one (bis-benzimidazole) moiety have better
selectivity toward the G4 DNA than the ligands consisting of
two (bis-benzimidazole) units.
Formation of the G4 DNA in the Presence of the
Ligand. When the G4 DNA was formed in the presence of
ligand CMP or CHP (at the [L]:[DNA] ratio of 7.5) in NaCl
buffer, the positive peak at 295 nm did not shift, except that
there was a change in the peak intensity. However, the positive
peak at 246 nm showed an ∼3 nm red-shift with intensity
changes in both cases. Additionally, there was a chirality
inversion in the region of the negative peak around 266 nm
along with a large red-shift of about 13 nm. Probably the Na+-
stabilized antiparallel G4 DNA structure is maintained with
some distortion in the parent G4 structure due to the strong
association with the ligands (Supporting Information, Figure
S10A and B).
Surprisingly, in KCl buffer, the phenomenon was totally
different. Both of the ligands showed structural inversion from
the K+-stabilized mixed hybrid G4 DNA structure to a stable,
biologically nonrelevant, telomeric parallel G4 DNA struc-
ture.23 The positive peaks at 292, 271, and 251 nm merged
together to give a positive peak at 263 nm and a negative peak
at 241 nm, indicating the formation of the parallel G4 DNA
(Figure 5 and Supporting Information, Figure S11). Though
telomeric G4 DNA is known to form a hybrid structure under
physiological conditions, it has been shown to adopt a parallel
conformation in a molecularly overcrowded environment.23,24
Interestingly, for CMP at [L]:[DNA] = 5, the presence of the
292 nm peak provides evidence of incomplete structural
inversion, whereas, at [L]:[DNA] = 7.5, the inversion was
complete. We note that CHP showed a lower ligand
concentration ([L]:[DNA] = 5) for the complete structural
inversion. The ligand CBhoe also showed a similar
phenomenon, but the topological transformation was incom-
plete even at [L]:[DNA] = 7.5 (Supporting Information, Figure
S10D). Interestingly, the ligand CBM did not show any kind of
structural inversion (Supporting Information, Figure S10C).
Thus, the studies suggest that the two terminal piperazines
attached next to the benzimidazole moiety (CMP, CHP, and
CBhoe) are essential for the topological transformation.
DNA Melting Studies. We incubated the preformed G4
DNA with each of CMP, CHP, CBM, and CBhoe separately
either in NaCl or KCl buffer for 12 h and determined their
F
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Figure 6. (A) Melting profiles of the preformed G4 DNA alone and the G4 DNA incubated with ligands for 12 h in specified concentrations in KCl
buffer, monitored at a wavelength of 295 nm. (B) Melting profiles of the preformed G4 and the G4 DNA formed in the presence of specified ligand
with indicated concentrations in KCl buffer, monitored at a wavelength of either 295 nm (for CBM) or 263 nm (for CMP, CHP, and CBhoe).
a
Table 3. Summary of the DNA Melting Studies
ligand
NaCl (preformed), ΔTm (°C)
NaCl (with ligand), ΔTm (°C)
KCl (preformed), ΔTm (°C)
KCl (with ligand), ΔTm (°C)
CMP
CHP
7
10
4
12
15
8
13
15
4
16
19
7
CBM
CBhoe
7
10
7
10
a
ΔTm values were obtained from the differences in the melting temperatures of the ligand bound and uncomplexed G4 DNA in 10 mM Tris−HCl
(pH 7.4) containing 0.1 M KCl/NaCl and 0.1 mM EDTA. The results are the average of two independent experiments and are within 0.5 °C of
each other.
thermal denaturation profile by following the changes in CD
spectral profiles as a function of temperature. Also, we checked
the melting data of the G4 DNA formed in the presence of
ligand in both NaCl and KCl buffers. The melting experiments
for the mixed-hybrid G4 DNA in KCl buffer and antiparallel
basket type G4 DNA in NaCl buffer incubated with each of the
ligands separately afforded sigmoidal melting curves at 295 nm,
indicating significant enhancement in the melting temperature
compared with that of the G4 DNA alone (Figure 6 and
Supporting Information, Figure S12A and B). Similarly, the
melting experiments for the G4 DNA formed in the presence of
each ligand in NaCl buffer were performed at 295 nm.
On the other hand, the melting studies in KCl buffer were
followed at 263 nm because of the peak shift due to a structural
alteration in the presence of CMP, CHP, and CBhoe (Figure
5). A [L]:[DNA] ratio of ∼7.5 was used for CMP and CBhoe,
which showed a total structural inversion in KCl buffer, whereas
for CHP the corresponding ratio is ∼5 in KCl buffer and ∼7.5
in NaCl buffer (Figure 5 and Supporting Information, Figure
S11D).
All the CD melting experiments showed significant thermal
stabilization of the G4 DNA formed in the presence of each
ligand, compared to the G4 DNA alone (Table 3). Upon
increasing the ligand concentrations from r = 5 to r = 7.5, the
denaturation temperatures of the G4 DNA also increased
(Supporting Information, Figure S13). The elevated thermal
stabilization may have originated from the additional groove
binding at higher ligand concentrations which is also evident
from the concomitant appearance of the ICD band.25 The
melting curves of the preformed G4 DNA incubated with each
of the individual ligands showed hysteresis in the reverse scan
(Supporting Information, Figure S12C). This was possibly due
to the formation of the G4 structure in a kinetically slower
process. Interestingly, the G4 DNA formed in the presence of
CMP, CHP, and CBhoe did not show any hysteresis in the
reverse melting scan. This phenomenon may be explained
considering the formation of thermodynamically and kinetically
more stable parallel G4 DNA structures (Supporting
Information, Figure S12D).
S1 Nuclease Assay. A single-stranded DNA can adopt
various secondary structures like A-motif, i-motif, G-quad-
ruplex, etc., depending upon its sequence and experimental
conditions.26 Topology specific interaction is a useful technique
in the identification of DNA secondary structures. S1 is an
endonuclease which cleaves preferentially the single-stranded
DNA over the duplex DNA, G4 DNA, and i-motif DNA in the
presence of Zn2+ ion.27
We observed that the Hum21 ODN even in the presence of
50 mM KCl (Figure 7, lane 1) was sufficiently cleaved,
suggesting that the G4 DNA was not stable and remained in
equilibrium with its random single-stranded form. The two
lower major bands represent the extent of highly cleaved ODN
products. However, as we incubated the ODN in 50 mM KCl
along with increasing concentration of each ligand (lanes 2−6),
the lower bands corresponding to highly digested products
were reduced significantly, indicating protection from the S1
nuclease cleavage. The ss-DNA (Figure 7, lane 7) was unable to
resist the S1 nuclease induced cleavage, giving two intensely
populated bands of higher mobility. Thus, this study provides
additional evidence in support of the G4 DNA stabilizing
capability of the ligands.
TRAP-LIG Assay. A modified TRAP assay was performed
using a three-step Telomeric Repeat Amplification Protocol
(TRAP-LIG) procedure.28 This involved (1) primer elongation
by telomerase in the presence and absence of ligand, (2)
removal of the bound and unbound ligand from the elongated
G
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at ∼3 μM (Supporting Information, Figure S14A). To the best
of our knowledge, the present set of compounds CMP and
CHP are the most potent telomerase inhibitors in the bis-
benzimidazole category. The quantification in UVI-Tech gel
documentation station using UVI-Band Map software (version
97.04) enabled determination of the IC50 values for CMP,
CHP, CBM, and CBhoe which were 0.9, 0.6, 1.6, and 1.8 μM,
respectively (Table 4 and Supporting Information, Figure
S14B).
a
Table 4. IC50 Values Calculated from the TRAP-LIG Assay
ligand
IC50 (μM)
ligand
IC50 (μM)
CMP
CHP
0.9 0.02
0.6 0.01
CBM
1.6 0.02
1.8 0.03
CBhoe
a
The results are the average of two independent experiments.
Cancer-Cell-Specific Toxicity. The cytotoxicity studies of
the ligands (CMP, CHP, CBM, and CBhoe) were performed
in telomerase negative human foreskin fibroblast normal cells
(HFF),29 representative cancer cells (adenocarcinomic human
alveolar basal epithelial cellsA549, human cervical cancer
cellsHeLa, and human embryonic kidney transformed
cellsHEK 293T, to demonstrate their cancer cell specificity
in a short-term (72 h) cell viability assay.
Figure 7. S1 nuclease cleavage of the human telomeric G4 DNA in the
presence and absence of CMP and CHP. Lane 1: Hum21 in 50 mM
KCl. Lanes 2, 3, and 4: Hum21 + CMP (2.5, 5, and 7.5 μM) in 50 mM
KCl. Lanes 5 and 6: Hum21 + CHP (2.5 and 5 μM) in 50 mM KCl.
Lane 7: Hum21 in the absence of KCl (single strand). Lane 8: A+G
ladders corresponding to the Hum21 DNA.
At first, the cell viability experiments were performed in
mortal HFF cells where no noticeable reduction in cell
viabilities was observed in the presence of ligands (Figure 9
and Supporting Information, Figures S15 and S16). On the
other hand, we noticed a significant decrease in cell viability in
cancer cells at the same concentrations under identical
conditions (Figure 9 and Supporting Information, Figures
S15 and S16).
Long-Term Cell Viability Assay. It must be noted that the
ligand’s moderate toxicity to the cancer cells (IC50 ∼8 μM) in a
short-term (72 h) viability assay cannot be correlated with their
activity through a telomerase inhibition pathway.30 Therefore,
long-term cell viability assays (15 days), which allow for a
sufficient time tag for telomere shortening to take place, were
performed using a sub-cytotoxic ligand concentration of 2 and
5 μM. Long-term cell proliferation experiments were carried
out using human cervical cancer cells (HeLa) and human
embryonic kidney transformed cells (HEK 293T).
primer, and (3) PCR amplification of the telomerase elongated
product, as given below.
Since telomerase is a potential drug target toward the
anticancer therapy, we examined the efficiency of the
synthesized ligands CMP, CHP, CBM, and CBhoe toward
telomerase inhibition by modified three-step TRAP-LIG
assay.28 Ligands were tested at concentrations ranging from
0.01 to 5 μM for their ability to inhibit the telomerase activity
against the telomerase enzyme extracted from the human lung
carcinoma A549 cell lines in vitro. Both the ligands CMP and
CHP showed total inhibition at ∼1 μM ligand concentration
(Figure 8), whereas CBhoe and CBM showed a total inhibition
All the ligands showed effective antiproliferative activity in
accordance with their activity observed in a short-term cell
viability assay. The ligands CMP and CHP were found to be
more efficient than CBM and CBhoe toward this end (Figure
10 and Supporting Information, Figure S15C and D). Thus,
such ligand’s acute activity in a long-term (15 days) cell viability
assay further confirms the possibility of a telomerase inhibition
pathway. This observation suggests that the telomerase activity,
which is more pronounced in cancer cells, may be targeted
efficiently with these G-quadruplex DNA binding ligands.31
Annexin V-FITC and PI Staining Assay. To further
ascertain whether the nature of ligand induced cell death was
caused due to an apoptotic pathway, an Annexin-V and PI dual
staining assay was performed to detect the presence of
apoptotic cells after the treatment with ligands. Interestingly,
for HeLa cells treated with ligands (10 μM CHP and CMP) for
a period of 12 h, significant evidence of apoptosis was obtained.
Ligand CHP and CMP showed ∼55 and ∼25% apoptotic cell
populations, respectively. In contrast, HeLa cells not treated
with any of the ligands (control) did not show any evidence of
Figure 8. (A) Lane 1: Negative control (absence of enzyme and
ligand). Lane 12: positive control. Lanes 2, 3, 4, 5, and 6: with
increasing concentrations of CMP (0.01, 0.1, 0.2, 1, and 3 μM). Lanes
7, 8, 9, 10, and 11: with increasing concentrations of CHP (0.01, 0.1,
0.2, 1, and 3 μM). (B) Lane 1: positive control. Lanes 2, 3, 4, 5, 6, and
7: with increasing concentrations of CBM (0.5, 1, 2, 3, 4, and 5 μM).
H
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Figure 9. Effect of CMP and CHP on the cell viability after a short-term exposure (72 h) to different (A) cancer (A549, HeLa, and HEK 293T) cells
and human foreskin fibroblast (HFF) normal cells as measured using the MTT assay. Cells were treated with each concentration in triplicates in
individual experiments, and the results shown are based on at least three independent experiments. Representative bright field images of the cell
morphology of human foreskin fibroblast (HFF) normal cells (B) before and (C) after the treatment with 4 μM CHP and HeLa cells (D) before and
(E) after the treatment with 4 μM CHP.
Figure 10. Effect of ligands on the cell viability upon long-term exposure (15 days) to (A) human cervical cancer cells (HeLa) and (B) human
embryonic kidney transformed cells (HEK 293T) as measured using the MTT assay.
Figure 11. Representative dot plots for Annexin-V and PI staining of (A) untreated cells (HeLa) as control and after incubation with ligands (B)
CHP and (C) CMP. The different stages of cells were assigned as alive (LL), early apoptotic (LR), late apoptotic (UR), and necrotic cells (UL). The
depiction of apoptotic cell population is made in the lower right quadrant which originated due to the Annexin V-FITC staining only.
apoptotic cell populations (Figure 11). These observations
suggest that the ligand induced cell death was primarily due to
apoptosis consistent with an earlier report.32
Confocal Microscopy. To provide visual evidence,
confocal microscopy experiments were performed to follow
the cellular internalization of ligands. This also afforded critical
I
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Figure 12. Representative confocal microscopic images depicting untreated cells (A) and cellular internalization of ligands at a concentration of 10
μM CHP (B) and CMP (C) for 12 h in HeLa cells. PI was used as a nuclear counterstain. Panels A, B, and C represent (left to right) bright field,
ligand fluorescence (blue), PI nuclear counterstain (red), and overlay of the previous three images.
evidence toward differences in cell death induced by apoptosis
in various cells. The ligand treated HeLa cells showed a
prominent nuclear localization after 12 h of incubation (Figure
12). Nuclear localization of ligands was confronted using PI
(propidium iodide) as a nuclear counterstain. On the other
hand, normal HFF cells treated with ligands showed only
cytoplasmic distribution (Supporting Information, Figure S17).
To identify the apoptotic cells as a result of the ligand
treatment, PI was used to counterstain the nuclei (Figure 13).
Condensed and fragmented nuclei were observed in ligand
treated HeLa cells, which is a characteristic appearance of the
apoptotic cells (Figure 13B, C, E, and F). On the contrary, we
did not notice any change in the nuclear morphology of normal
HFF cells treated with ligands under the same conditions
(Figure 13H, I, K, and L).
The MD simulation was performed using the Amber 934
software package with a 6 ns production run (Figure 14 and
Supporting Information, Figure S19). The molecule CMP was
found to prefer stacking in the 3′-end. The central
pharmacophore is stacked over the G-quartet DNA plane,
whereas the three piperazine arms approach the grooves
interacting with the anionic phosphodiester backbone (Figure
14). Though a 1:1 ligand−DNA interaction shows stacking as
the preferable interaction over the groove binding, at higher
ligand concentrations, the ligand may also interact with the
grooves, which has been experimentally observed with the
manifestation of the ICD bands (Figure 5).15,25,35 Hence, the
MD simulation studies demonstrate the various types of
possible interaction in favor of the strong G4 DNA binding
efficiency of each ligand.
Computational Studies. Each ligand possesses a planar
aromatic moiety connected via C−C single bonds, and this
makes the molecule flexible. The three pendent flexible
piperazine/morpholine residues also contribute to the molec-
ular features being dynamic in shape. Initially, the molecular
structures were energy optimized using the B3LYP level of
theory with 6-31G* as the basis set in Gaussian 03.33 The
energy optimized structures show that the central pharmaco-
phore which consists of three aromatic residues is quite planar
in nature. The two piperazine/morpholine residues connected
to the benzimidazole moiety remain in the molecular plane.
The pendant butyl-piperazine spacer attached to the carbazole-
N projects out of the central molecular plane.
CONCLUSIONS
■
In this work, we present the design and synthesis of four novel
carbazole based benzimidazole derivatives. We have inves-
tigated their interaction with the Hum21 G4 DNA by various
techniques under different experimental conditions including
physical, biological, and theoretical approaches. We have
unambiguously shown that all the compounds are highly
effective in the stabilization of preformed Hum21 G4 DNA
structure. The mono-(bis-benzimidazole) derivatives (CMP,
CHP, and CBM) showed a higher preference (>500-fold)
toward the G4 DNA over the duplex DNA than the
corresponding di-(bis-benzimidazole) ligand, CBhoe. Similarity
in the molecular dimension of mono-(bis-benzimidazole)
J
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Figure 13. Representative bright field (A−C, HeLa cells; G−I, HFF cells) and fluorescence microscopic images (D−F, HeLa cells; J−L, HFF cells)
of cells using PI as a nuclear counterstain. Parts A, D, G, and J show images of cells without ligand treatment, and parts B, E, H, and K and C, F, I,
and L show images of cells treated with ligand CHP and CMP (10 μM), respectively, for 24 h.
Figure 14. (A) Simulated structures of CMP and Hum21 G4 DNA (PDB 1KF1) DNA showing excellent stacking capability of the central
pharmacophore while its side-arms interact with the grooves. The ligand is shown in stick model and is green in color, and the DNA is shown in red.
(B) The side-view of the simulated complex shows that the ligand conserves its planarity while stacking upon the planar G-tetrad.
derivatives and G-tetrad may account for their higher selectivity
toward the G4 DNA. On the other hand, the extended crescent
shape in the molecular structure of the di-(bis-benzimidazole)
ligand, CBhoe, could be responsible for its inferior structural
K
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z = 267.1457 [M + H]+; Calcd = 267.1457 [M + H]+; mp 163 °C (lit.
163 °C).13a
discrimination ability between the G4 DNA and the duplex
DNA. The observation of excellent fluorescence emission
enhancement upon binding to the G4 DNA may be useful for
the staining of DNA stretches that form G4 structures in vivo.36
The ligands stabilize both the preformed hybrid and antiparallel
G4 structures, rendering increases in their melting temper-
atures. The ligands containing three piperazine residues (CMP,
CHP, and CBhoe) induced a structural alteration in the G4
DNA structure from hybrid to unusual but more stable, parallel
telomeric G4 DNA in the presence of K+ ion, whereas the one
with morpholine termini, CBM, was found to be inefficient in
this regard. Thus, SAR established that the piperazine residues
attached to the benzimidazole moiety were crucial toward the
topological alteration in the G4 DNA structure. The high
melting temperature of the parallel G4 DNA formed in the
presence of each ligand reveals that the equilibrium between G4
structure and the single-stranded form is shifted significantly
toward the higher order structures. The ligands CMP and CHP
were also highly effective toward eliciting telomerase inhibition,
and the IC50 below 1 μM indicates that these are among the
best of the benzimidazole derivatives reported so far. Moreover,
the carbazole based ligands were found to be remarkably
superior to the first generation phenyl based benzimidazole
ligands (Figure 1C, ligand 5),13a−d as evident from both
physical and biological experiments (Supporting Information,
Figure S20). Manifestation of the selective cancer cell nuclear
internalization and nuclear fragmentation, cytotoxicity at the
low ligand concentration, and ability to induce cell death
through an apoptotic pathway reveals their possible utility as an
anticancer drug.
5-Morpholino-2-nitrobenzenamine (8a). A mixture of 5-chloro-2-
nitrobenzenamine (1 g, 1.8 mmol), morpholine (2 mL), and dry
K2CO3 (1.2 g, 9 mmol) was taken along with 5 mL of dry DMF and
heated at 110 °C under a nitrogen atmosphere for 12 h. The reaction
mixture was then dried under a vacuum, and 50 mL of cold water was
added to it to form a yellow colored precipitate which was filtered off
and washed several times with distilled water and then dried, affording
a yellow colored product which was adjudged to be pure by TLC (2%
MeOH/CHCl3 on precoated silica gel) (1.23 g, 95%).
1H NMR (400 MHz, DMSO-d6): δ ppm 7.82 (d, J = 10, 1H), 7.28
(s, 2H), 6.39 (dd, J = 9.8, J = 2.8, 1H), 6.2 (d, J = 2.8, 1H), 3.70 (t, J =
4.8, H), 3.27 (t, J = 4.8, 4H); IR (KBr): 3448, 3335, 3179, 3088, 2973,
2928, 2872, 2846, 1618, 1234, 1123, 892 cm−1; HRMS: m/z =
246.0858 [M + Na]+; Calcd = 246.0855 [M + Na]+; mp 188 °C.
4-(4-Methylpiperazin-1-yl)benzene-1,2-diamine (6). Compound
6a (206 mg) was taken along with 100 mg of Pd/C (10%) in ethanol
and stirred under a H2 atmosphere (1 atm pressure) for 12 h when the
yellow solution turned colorless. The reaction mixture was then passed
through a Celite bed under nitrogen flow and used for the next
reaction without any further purification, as the diamine product was
found to be highly unstable.
2-(4-(3,4-Diaminophenyl)piperazin-1-yl)ethanol (7). Compound
7 has been synthesized from 7a following a similar procedure as
described for compound 6.
4-Morpholinobenzene-1,2-diamine (8). Compound 8 has been
synthesized from 8a following a similar procedure as described for
compound 6.
9-(4-Bromobutyl)-9H-carbazole (9). To a mixture of TBAI (277
mg, 0.75 mmol), carbazole (3 g, 18 mmol), and 11.67 g (54 mmol) of
1,4-dibromobutane in benzene, aqueous 50% NaOH solution (9 mL)
was added at rt. The mixture was then stirred for 6 h at the same
temperature and then poured into water and extracted with DCM.
The organic layer was passed through a bed of dry Na2SO4 and
evaporated to get a crude product which was purified by silica gel
column chromatography with a hexane/ethyl acetate mixture as the
eluent, yielding a white solid (5.4 g, 72%).
MATERIALS AND METHODS
■
Materials. All starting materials were from the best known
commercial sources and used as received. All solvents were from
Merck, and they were distilled and/or dried prior to use whenever
necessary. All tested ligands were found to be at least >95% pure by
elemental analysis.
1H NMR (400 MHz, CDCl3): δ ppm 8.1 (d, J = 7.6, 2H), 7.47 (m,
4H), 7.24 (dd, J = 4.5, J = 7.8, 2H), 4.36 (t, J = 6.8, 2H), 3.38 (t, J =
6.8, 2H), 2.07 (m, 2H), 1.91 (m, 2H); HRMS: m/z = 302.0545 [M +
H]+; Calcd = 302.0544 [M + H]+; mp 101 °C (lit. 100 °C).37
9-(4-(4-Methylpiperazin-1-yl)butyl)-9H-carbazole (10). Com-
pound 9 (900 mg, 2.98 mmol), 596 mg (5.96 mmol) of N-
methylpyperazine, and 823 mg of dry K2CO3 were stirred together in
30 mL of dry acetonitrile for 6 h. Then, the reaction mixture was dried
and partitioned between water and chloroform. The organic layer was
separated and passed through a bed of dry Na2SO4. The filtrate was
collected and evaporated to get an off-white sticky solid product which
was adjudged to be pure by TLC (3% MeOH/CHCl3 on precoated
silica gel) (852 mg, 89%).
Synthesis. 5-(4-Methylpiperazin-1-yl)-2-nitroaniline (6a). 5-
Chloro-2-nitroaniline (2 g, 11.6 mmol) was taken in dry DMF (5
mL), and to that, 4-methylpiperazine (1.4 g, 13.8 mmol) and
anhydrous K2CO3 (2.5 g, 18 mmol) were added. The mixture was then
heated at 110 °C for 12 h under a N2 atmosphere until TLC showed
the disappearance of 5-chloro-2-nitroaniline. The crude compound
was suspended in water, and the product was extracted with ethyl
acetate. The organic layer was washed twice with water, dried over
anhydrous Na2SO4, and concentrated. This afforded a product which
was adjudged as pure by TLC (1% MeOH/CHCl3 on precoated silica
gel) and isolated as a bright yellow solid (2.46 g, 90% yield).
1H NMR (CDCl3, 300 MHz): δ ppm 2.34 (s, 3H). 2.52 (t, J = 4.8,
4H), 3.37 (t, J = 4.8, 4H), 5.94 (s, 1H), 6.14 (bs), 6.30 (d, J = 9.3 Hz,
1H), 8.02 (d, J = 9.3 Hz, 1H); HRMS: m/z = 259.1271 [M + Na]+;
Calcd = 259.1273 [M + Na]+; mp 154 °C (lit. 154 °C).13a
1H NMR (300 MHz, CDCl3): δ ppm 8.1 (d, J = 7.8, 2H), 7.44 (m,
4H), 7.23 (dd, J = 4.5, J = 7.8, 3H), 4.33 (t, J = 6.8, 2H), 2.35 (m,
10H), 2.27 (s, 3H), 1.91 (m, 2H), 1.58 (m, 2); HRMS: m/z =
322.2283 [M + H]+ ; Calcd = 322.2283 [M + H]+.
9-(4-(4-Methylpiperazin-1-yl)butyl)-9H-carbazole-3,6-dicarbalde-
hyde (11). Compound 10 (1 g, 3.12 mmol) and 850 mg (6.24 mmol)
of anhydrous ZnCl2 were taken together in a round-bottom flask (100
mL) along with 6 mL of dry DMF and heated at 100 °C for 15 min.
This was followed by cooling to rt. To this, 2.9 mL (31.2 mmol) of
POCl3 was added dropwise upon cooling over an ice-bath for 15 min
followed by heating at 100 °C for 24 h. Then, the reaction mixture was
evaporated under a vacuum to remove the excess DMF and quenched
carefully by addition of ice-cold water followed by neutralization with
concentrated KOH solution. The mixture was finally extracted using
ethyl acetate, and the organic layer was passed through a bed of dry
Na2SO4. The filtrate was collected and evaporated to get a brown
gummy mass which was purified by silica gel column chromatography
using chloroform/methanol as the eluent. The final product came at
3% methanol/chloroform as a light yellow solid which was adjudged to
2-[4-(3-Amino-4-nitro-phenyl)-piperazin-1-yl]-ethanol (7a). 5-
Chloro-2-nitroaniline (2 g, 11.6 mmol) was taken in dry DMF (5
mL), and 2-piperazin-1-yl-ethanol (3 g, 23.08 mmol) and K2CO3 (4.8
g, 34.6 mmol) were added to the solution. This mixture was then
heated at 110 °C under a N2 atmosphere for 12 h until TLC showed
the disappearance of 5-chloro-2-nitroaniline. The crude compound
was suspended in water, and the product was extracted with ethyl
acetate. The organic layer was washed twice with water, dried over
anhydrous Na2SO4, and concentrated. This afforded a pure product as
confirmed by TLC (1% MeOH/CHCl3 on precoated silica gel) and
was isolated as a bright yellow solid (2.93 g, 95% yield).
1H NMR (300 MHz, CDCl3): δ ppm 2.59−2.65 (m, 6H), 3.4 (t, J =
4.8, 4H), 3.7 (t, J = 4.8, 2H), 5.95 (d, J = 2.7 Hz, 1H), 6.1 (bs, 2H,
NH2), 6.29 (dd, J = 9.3, J = 2.7, 1H), 8.02 (d, J = 9.3, 1H); HRMS: m/
L
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be pure by TLC (5% MeOH/CHCl3 on precoated silica gel) (881 mg,
75%).
3,6-Bis(5-(5-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-
1H-benzo[d]imidazol-2-yl)-9-(4-(4-methylpiperazin-1-yl)butyl)-9H-
carbazole (4, CBhoe). The N-substituted carbazole-3,6-dicarbalde-
hyde 11 (50 mg, 0.133 mmol) was taken with 86 mg (0.266 mmol) of
freshly prepared diamine (17) in 20 mL of ethanol and heated at 80
°C. An aqueous solution of Na2S2O5 (30 mg) was added to the
reaction mixture and refluxed with stirring for 12 h. The reaction
mixture was then cooled, filtered, and dried under reduced pressure.
The crude solid material was dissolved in methanol and repeatedly
precipitated by adding ethyl acetate. The product was then further
purified with preparative thin layer chromatography with a 1:1
methanol/chloroform mixture.
1H NMR (300 MHz, CDCl3): δ ppm 10.14 (s, 2H), 8.67 (s, 2H),
8.09 (d, J = 8.7, 2H), 7.58 (d, J = 8.4, 2H), 4.43 (t, J = 6.9, 2H), 2.42−
2.37 (m, 6H), 2.31 (s, 3H), 2.05−1.96 (m, 6H), 1.6 (m, 2H); IR
(KBr): 3052, 2964, 2936, 2786, 2736, 1683, 1593, 1488, 1385, 1345,
1285, 1202, 1126, 1014, 812 cm−1. HRMS: m/z = 378.2183; Calcd =
378.2182 [M + H]+; mp 218 °C; Anal. (calcd for C23H27N3O2): C,
73.18; H, 7.21; N, 11.13; found: C, 73.82; H, 7.13; N, 11.21.
3-(5-(4-Methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-6-(6-(4-
methylpiperazin-1-yl)-1H-benzo [d]imidazol-2-yl)-9-(4-(4-methylpi-
perazin-1-yl)butyl)-9H-carbazole (1, CMP). Freshly prepared com-
pound 6 (180 mg, 0.87 mmol) was taken with 164 mg (0.44 mmol) of
compound 11 in 30 mL of ethanol. To that, 87 mg (0.45 mmol) of
Na2S2O5 dissolved in 1 mL of water was added and refluxed for 12 h
until the reactants were consumed as indicated from TLC. Then, the
reaction mixture was cooled and filtered. The supernatant was
evaporated and purified by repetitive precipitation from a methanol/
ethyl acetate mixture, yielding a light brown powder (195 mg, 60%).
1H NMR (400 MHz, CD3OD): δ ppm 8.79 (s, 2H), 8.06 (d, J = 8,
2H), 7.5 (d, J = 8.4, 2H), 7.45 (d, J = 8.4, 2H), 7.12 (s, 2H), 7 (d, J =
8.8, 2H), 4.36 (s, 2H), 3.8 (s, 4H), 2.99 (s, 9H), 2.87 (s, 4H), 2.85 (s,
2H), 2.71 (s, 6H), 2.5 (s, 4H), 2.4 (m, 6H), 1.8(s, 2H), 1.48(s, 2H);
13C NMR (CD3OD): δ ppm 152.24, 148.31, 142.52, 137.89, 133.21,
1H NMR (400 MHz, DMSO-d6): δ ppm 9.21 (s, 2H), 8.42 (d, J =
8.8, 2H), 8.37 (s, 2H), 8.04 (d, J = 8.8), 7.88 (d, J = 8.8, 2H), 7.73 (d, J
= 8.4, 2H), 7.46 (d, J = 7.2, 2H), 7.03 (s, 2H), 6.94 (dd, J = 1.6, J = 8.6,
2H), 4.54 (s, 2H), 3.17−3.13 (m, 10H), 2.30−2.25 (m, 18H), 2.12 (s,
6H), 1.87 (s, 2H), 1.69 (s, 2H), 1.51 (s, 2H); 13C NMR (DMSO-d6):
δ ppm 162.39, 154.97, 141.60, 128.05, 125.47, 122.45, 121.41, 119.13,
110.34, 79.09, 56.98, 54.84, 54.71, 52.51, 49.95, 48.57, 45.73, 45.69,
35.78, 30.78, 26.28, 23.48; mp >300 °C; IR (KBr): 3455, 2930, 2817,
2706, 1638, 1622, 1604, 1463, 1381, 1373, 1241, 1143, 1119, 1013,
961, 817, 806 cm−1; HRMS: m/z = 982.5469; Calcd = 982.5469 [M +
H]+; Anal. (calcd for C59H63N15): C, 72.15; H, 6.46; N, 21.39; found:
C, 71.98; H, 6.41; N, 21.48.
Oligonucleotides. HPLC purified oligodeoxyribonucleotide
(ODN) d[G3(T2AG3)3], abbreviated as Hum21, was purchased from
Sigma Genosys, Bangalore. Their purity was confirmed using high
resolution sequencing gel. The molar concentration of each ODN was
determined from absorbance measurements at 260 nm based on its
molar extinction coefficients (ε260) of 215 000 for d[G3(T2AG3)3].
G4 DNA Formation. Hum21 [5′-G3(T2AG3)3-3′] sequence was
incubated in a buffer containing 10 mM Tris−HCl (pH 7.4), 0.1 mM
EDTA, and 0.1 M of indicated salt and heated at 95 °C for 5 min and
cooled slowly to 24 °C over 24 h. The formation of G4 DNA has been
confirmed by circular dichroic spectral signature with that reported in
the literature13c−e as well as by PAGE.
Circular Dichroism Spectroscopy. The CD experiments were
performed in a Jasco J-815 CD spectropolarimeter equipped with a
Peltier temperature controller in 1 cm quartz cuvette at 20 °C. To the
preformed G4 (4 μM) in 10 mM Tris−HCl (pH 7.4), 0.1 M either
NaCl or KCl, and 0.1 mM EDTA, aliquots of ligand solutions
(prepared in the corresponding buffer) were added and incubated for
15 min prior to recording with a scan rate of 50 nm/min.
DNA Melting Experiment. The melting experiments were
performed in a Jasco J-815 CD spectropolarimeter equipped with a
Peltier temperature controller. DNA and DNA−ligand complexes
were placed in a 1 cm quartz cuvette, charged in the temperature range
from 20 to 90 °C, and monitored with an increase in the temperature
range of 0.5 °C/min. All the experiments were repeated twice, and an
average value has been reported with an error of 0.5 °C. The data
were plotted with Origin 8.0 software and the CD signal intensities
were normalized to the range between 1 and 0.13,38 The ligand
solutions of 10 mM have been prepared in Milli-Q water/biological
grade DMSO and diluted in the corresponding buffer related to the
experiment.
Fluorescence Spectroscopy. Fluorescence emission spectra were
recorded on a Carey Eclipse Varian spectrophotometer using quartz
cells with a path length of 1 cm. The temperature of the sample
component was maintained at 20 °C using a Peltier controller. To 0.4
μM of the ligand in specified buffer, 4 μL of 10 μM preformed G4
DNA of the corresponding buffer was added and incubated for 10 min
prior to recording. The ligand solutions were excited at 300 nm (for
CMP, CHP, and CBM) or 330 nm (for CBhoe) with a slit width of
10/10 nm. The data have been processed using Origin 8.0 software.
G4-FID Assays. A solution of 1 μM TO in Tris−HCl (pH 7.4)
buffer containing 0.1 M KCl and 0.1 mM EDTA was taken in a 500 μL
cell. The displacement assays were performed by adding increasing
ligand concentration to the preformed DNA−TO complex (1 μM TO
+ 0.5 μM G4 DNA; 1 μM TO + 0.33 μM CT DNA; 1 μM TO + 0.33
μM Telo ds DNA) followed by measuring the emission spectra upon
125.47, 123.43, 120.09, 119.22, 116.14, 110.62, 101.81, 65.19, 57.50,
55, 54.03, 51.56, 44.32, 44.18, 27.13, 24.02, 15.48; IR (KBr): 3458,
2931, 2702, 1638, 1602, 1459, 1384, 1240, 1140, 1023, 963, 814 cm−1;
HRMS: m/z = 750.4717 [M + H]+; Calcd = 750.4720 [M + H]+; mp
>280 °C; Anal. (calcd for C45H55N11·0.5H2O): C, 71.21; H, 7.44; N,
20.30; found: C, 71.42; H, 7.40; N, 20.27.
3-(5-(4,2-Hydroxyethylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-
6-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)-9-(4-(4-
methylpiperazin-1-yl)butyl)-9H-carbazole (2, CHP). Compound 2
was synthesized following a similar procedure as described for 1. Yield
= 60%.
1H NMR (400 MHz, CD3OD): δ ppm 8.72 (s, 2H), 7.91 (d, J = 8.4,
2H), 7.36 (m, 4H), 6.98 (s, 2H), 6.85 (d, J = 8.4, 2H), 4.2 (s, 2H),
3.69 (t, J = 5.6, 4H), 2.81 (s, 8H), 2.7 (t, J = 5.2, 8H), 2.6 (d, J = 2,
4H), 2.53 (s, 4H), 2.42 (s, 4H), 2.29 (m, 6H), 1.73 (s, 2H), 1.44 (s,
2H); 13C NMR (CD3OD): δ ppm 153.39, 149.07, 142.77, 139.35,
135.24, 125.82, 124.07, 121.46, 120.20, 116.48, 115.82, 110.69, 101.83,
65.04, 60.81, 58.89, 57.99, 54.78, 54.34, 52.35, 50.67, 44.96, 27.41,
24.50, 22.79, 15.44; IR (KBr): 3440, 2943, 2823, 1633, 1603, 1451,
1403, 1239, 1188, 1138, 1020, 966, 814 cm−1; HRMS: m/z = 810.4930
[M + H]+; Calcd = 810.4931 [M + H]+; mp >250 °C; Anal. (calcd for
C47H59N11O2): C, 69.69; H, 7.34; N, 19.02; found: C, 69.42; H, 7.40;
N, 19.08.
9-(4-(4-Methylpiperazin-1-yl)butyl)-3,6-bis(6-morpholino-1H-
benzo[d]imidazol-2-yl)-9H-carbazole (3, CBM). The freshly prepared
diamine 8 (205 mg, 1.1 mmol) was mixed with the dialdehyde 11 (200
mg, 0.53 mmol) in 50 mL of ethanol, and to that, an aqueous solution
of 110 mg of Na2S2O5 was added and the mixture was refluxed for 12
h. The reaction mixture was then cooled and filtered. The filtrate was
evaporated to dryness which afforded a crude yellow mass which was
dissolved in 5 mL of methanol and precipitated with the addition of
ethyl acetate. The precipitation process was repeated twice to get a
yellowish product which was adjudged to be pure by TLC (10%
MeOH/CHCl3 on precoated silica gel) (325 mg, 85%).
1H NMR (400 MHz, DMSO-d6): δ ppm 12.67 (br, 2H), 9.06 (s,
2H), 8.31 (d, J = 8.8, 2H), 7.82 (d, J = 8.8, 2H), 7.48 (d, J = 8, 2H),
7.05 (s, 2H), 6.95 (d, J = 8, 2H), 4.5 (s, 2H), 3.76 (s, 8H), 3.1 (s, 4H),
2.7−2.34 (m, 10H), 1.82 (s, 2H), 1.52 (s, 2H); 13C NMR (DMSO-
d6): δ ppm =154.12, 151.77, 148.20, 147.78, 141.27, 127.31, 124.70,
123.79, 122.52, 122.38, 122.02, 118.54, 113.27, 110.25, 105.49, 98.93,
66.41, 61.44, 59.82, 56.30, 53.09, 50.48, 43.75, 26.27, 23.07, 15.22; IR
(KBr): 2955, 2856, 2822, 2690, 1633, 1604, 1449, 1402, 1378, 1352,
1299, 1240, 1186, 1138, 1115, 980, 965, 900, 812 cm−1; HRMS: m/z =
724.4086; Calcd = 724.4087 [M + H]+; mp >300 °C; Anal. (calcd for
C43H49N9O2): C, 71.34; H, 6.82; N, 17.4; found: C, 71.82; H, 6.76; N,
17.28.
M
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Article
incubation for 5 min. The percentage of TO displacement is calculated
from the fluorescence intensity (at λ = 531 nm, λex = 501 nm) using
the following equation: percentage of displacement = 100 − [(Ft/F0)
× 100], where Ft is the fluorescence intensity at each titration point
and F0 is the fluorescence of TO bound to DNA without any added
ligand as described earlier.20 The DC50 values have been calculated
from the plot of the percentage of fluorescence intercalator
displacement vs added ligand concentration.
S1 Nuclease Assay. S1 nuclease assay was performed as described
in the reported protocol.39 The 5′-end of Hum21 DNA was labeled
with 32P-ATP and purified using gel electrophoresis. The radioactively
labeled DNA was then mixed with cold DNA and annealed in the
presence of KCl to form the G4 DNA structure. The G4 DNA was
incubated with the indicated ligand for 4 h before performing the S1
nuclease assay. A sample of 2 μM (cold + labeled) of 25 μL of DNA
(ss- or G4-) was treated with 20 U of S1 nuclease in 1X S1 nuclease
buffer for 2 min, and the reaction was quenched by the addition of 100
μL of stop buffer (0.3 M sodium acetate, 0.25 μg/mL CT DNA, 0.1
mM EDTA) followed by ethanol precipitation and washing with 70%
ethanol. The reaction products were then vacuum-dried and
resuspended in formamide dye and loaded on a 15% polyacrylamide
urea sequencing gel along with a reference A+G ladder.
final concentration of 1 μg/mL for 5 min. Samples were then analyzed
using an FACS Calibur flow cytometer (Becton-Dickinson). Apoptotic
cells were shown to be stained only with Annexin V-FITC conjugate.
Data obtained from the flow cytometry were analyzed using WinMDI
software by considering a gated cell population for the detection of %
apoptotic cells.
Confocal Microscopy. To visualize the intracellular internalization
of ligands and apoptotic nuclei, confocal microscopic studies were
undertaken. In a typical experiment, cells were cultured on glass
coverslips placed in 12-well cell culture plates. Cells were treated with
ligands (12 h, internalization; 24 h, apoptosis) followed by proper
washing with DPBS buffer three times and fixed in 4%
paraformaldehyde solution for 10 min. Cells were then rinsed again
with DPBS buffer three times and treated with 0.1% Triton-X-100 for
5 min to permeabilize the cell membrane. After a proper wash again
with DPBS buffer, the glass coverslips were taken out and treated with
propidium iodide (PI) for nuclear counterstaining. Finally, after a
repeated wash with DPBS buffer and autoclaved Milli-Q to control
overstaining, the coverslips were mounted on glass slides and viewed
under a confocal fluorescence microscope (Leica confocal microscope,
SP5).
The reference A+G ladder was prepared following a reported
protocol.40 The 5′-end labeled Hum21 DNA (10 μL) was incubated
with 25 μL of 100% formic acid at 25 °C for 5 min and terminated
with 200 μL of hydrazine stop buffer (0.3 M sodium acetate, 250 μg/
mL CT DNA, 0.1 mM EDTA) followed by ethanol precipitation. The
product was vacuum-dried and treated with 10% of 70 μL of piperidine
at 90 °C for 30 min. It was dried under a vacuum centrifuge, excess
piperidine was removed by evaporating the sample twice from water,
and it was resuspended in formamide dye before loading on a 15%
polyacrylamide urea sequencing gel for 90 min against a voltage of
1800 V. Gel was transferred to a Whatman (3 mm) paper, dried, and
exposed to a phosphor imaging screen, and the resulting image was
captured using a Fuji-5000 phosphorimager.
ASSOCIATED CONTENT
* Supporting Information
Synthesis of compounds 11−16, characterization of the key
compounds, additional UV−vis titration, fluorescence, CD
spectroscopic data, telomerase inhibition data, cytotoxicity data,
and computational results. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: sb@orgchem.iisc.ernet.in. Phone: (91)-80-2293 2664.
Fax: (91)-80-2293 0529.
■
Cell Viability Assay. The cells were seeded in 96-well plates (15.0
× 103/well). Cells were grown for 72 h before treatment to obtain
>70% confluency and exposed to either various concentrations of
ligands or an equivalent volume of DMSO (0.1%) in the presence of
10% FBS. After 72 h of incubation at 37 °C in a humidified
atmosphere of 5% CO2, the old medium was replaced with the fresh
medium containing 10% FBS in DMEM and cells were further grown
post treatment. Then, 20 μL of 5 mg/mL methyl thiazolyl tetrazolium
(MTT) reagent was added to 200 μL of the medium present in each
well and cells were further incubated for 4 h. The old medium was
discarded, formazan crystals were dissolved in DMSO, and a reading
(fluorescence emission intensity, FI) was taken at 595 nm in the
ELISA plate reader. All ligand doses were parallel tested in triplicate.
The percentage of cell viability was calculated using the following
formula: % cell viability = [(FI(595) of treated cells − FI(595) of plain
DMSO)/(FI(595) of untreated cells − FI(595) of plain DMSO)] × 100.
Long-Term Cell Viability Assay. In a typical long-term viability
assay protocol, cells were grown in 6-well tissue culture plates at 5.0 ×
104 per well and exposed to a sub-cytotoxic concentration of 2 μM or
an equivalent volume of 0.1% DMSO. The cells in the control and
ligand-exposed wells were counted, and wells were reseeded with a half
population of cells. This process was continued for 15 days. Cell
population versus time (days) graphs were generated.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by J. C. Bose Fellowship, DST, to S.B.
B.M. acknowledges CSIR for a senior research fellowship. The
human foreskin fibroblast (HFF) cells were a kind gift from the
Manipal University, Manipal.
ABBREVIATIONS USED
■
G4 DNA, G-quadruplex DNA; ODNs, oligodeoxynucleotides;
CD, circular dichorism; ICD, induced circular dichorism;
TRAP, telomerase repeat amplification protocol; FI, fluores-
cence intensity; THF, tetrahydrofuran; DMF, dimethylforma-
mide; TBAI, tetrabutyl ammonium iodide; EDTA, ethyl-
enediaminetetraacetic acid; MTT, methyl thiazolyl tetrazolium;
PI, propidium iodide; DMSO, dimethyl sulfoxide; NMR,
nuclear magnetic resonance; IR, infrared
Annexin V-FITC and PI Staining Assay.32 The Annexin V-FITC
Apoptosis Detection kit (Sigma) was used to detect apoptosis induced
by the ligands in HeLa cells after 12 h of incubation. In a typical
experiment, cells were seeded at a density of 0.25 million in 6-well cell
culture plates. After 24 h, cells were incubated with the ligands for 12 h
at a final concentration of 10 μM. At the end, cells were washed
properly with DPBS buffer followed by trypsinization and resuspended
in 1X binding buffer (HEPES buffer containing 0.14 M NaCl and 2.5
mM CaCl2). Thereafter, Annexin V-FITC conjugate was added to
each of the cell suspensions and incubation was carried out for 10 min
at room temperature while protecting from light. Then, each cell
suspension was incubated with propidium iodide (PI) solution at a
■
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