Synthesis of β-carboline-based N-heterocyclic carbenes and their antiproliferative and antimetastatic activities against human breast cancer cells

Article abstract of DOI:10.1021/acs.jmedchem.5b00016

A series of novel β-carboline-based N-heterocyclic carbenes was prepared via Mannich reaction between methyl 1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carboxylate, formaldehyde, and primary amines. All compounds were evaluated for their antiproliferati

Full text of DOI:10.1021/acs.jmedchem.5b00016

Article
pubs.acs.org/jmc
Synthesis of β‑Carboline-Based N‑Heterocyclic Carbenes and Their
Antiproliferative and Antimetastatic Activities against Human Breast
Cancer Cells
Shashikant U. Dighe,†,⊥ Sajid Khan,‡,⊥ Isha Soni,‡ Preeti Jain,†,# Samriddhi Shukla,‡ Rajeev Yadav,§
Pratik Sen,*,§ Syed M. Meeran,*,‡,∥ and Sanjay Batra*,†,∥
‡
†Medicinal and Process Chemistry Division, and Endocrinology Division, CSIR-Central Drug Research Institute, Sector 10,
Jankipuram Extension, Sitapur Road, Lucknow 226031, India
§Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208016, India
∥Academy of Scientific and Innovative Research, New Delhi 110025, India
S
* Supporting Information
ABSTRACT: A series of novel β-carboline-based N-hetero-
cyclic carbenes was prepared via Mannich reaction between
methyl 1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-car-
boxylate, formaldehyde, and primary amines. All compounds
were evaluated for their antiproliferative activity using human
breast cancer and lung cancer cell lines. Three compounds, 3c,
3j, and 3h, were discovered to display IC50 less than 10 μM
against human breast cancer MDA-MB-231 cells at 24 h of
treatment. Pharmacologically these compounds lead to G2/M
phase cell cycle arrest and induction of cellular apoptosis by triggering intrinsic apoptotic pathway through depolarization of
mitochondrial membrane potential and activation of caspases. At lower concentrations, these compounds also showed
antimigratory and antiinvasive effects against highly metastatic human breast cancer MDA-MB-231 cells via aberration of MAP-
kinase signaling and by the inhibition of matrix metalloproteinases. However, these analogues lack in vivo effect in mouse model
which may be attributed to their strong affinity to HSA that was investigated spectroscopically with compound 3h.
property.3 It is widely reported that β-carboline derivatives elicit
INTRODUCTION
Naturally occurring β-carboline alkaloids and synthetic
analogues containing β-carboline subunit are endowed with
■
antitumor and anticancer properties via DNA intercalation,4
inhibition of topoisomerase I and II,5 cyclin-dependent kinase
(CDK),6 and IkK kinase complex (IkK).7 In one of the recent
diverse pharmacological properties including antimalarial, anti-
HIV, antibacterial, antitumor, and anticancer activities.1 Among
studies, Lee’s and Su’s groups disclosed the antitumor activity
of bis(hydroxymethyl)indolizino[6,7-b]indoles (II).8 These
these derivatives, the naturally occurring β-carboline alkaloids
such as harmicine, fascaplysin, and callophycin A (Figure 1) are
compounds were described to be the hybrid of β-carboline
which was responsible for the topoisomerase I and II inhibition
and bis(hydroxymethyl)pyrrole which accounted for the DNA-
reported to display antiproliferative effect against many cancer
cell lines.2 In addition, several synthetic analogues (I, II, Figure
cross-linking property. In one of our research endeavors, we
have been exploring the potential of 1-formyl-9H-β-carboline
for preparing chemical libraries of β-carboline analogues which
are ascribed with anticancer properties. The N-heterocyclic
carbenes (NHC) and their metal derivatives are widely
reported to show significant anticancer property by affecting
mitochondrial integrity.9 Therefore, we reasoned that merging
the β-carboline subunit with NHC may lead to discovery of
novel anticancer agents. Accordingly, we synthesized 2-
substituted-2,11-dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indoles
(III) which may be considered as the hybrid of β-carboline and
imidazole-based NHC. Screening of this series of compounds
led to identification of new β-carboline-based antiproliferative
1) structurally related to these alkaloids or containing β-
carboline-core are also reported to display potent anticancer
Figure 1. Natural and synthetic β-carboline derivatives with antitumor
and anticancer properties.
Received: January 6, 2015
© XXXX American Chemical Society
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Scheme 1. Synthesis of β-Carboline-Based Aryl, Alkyl, and Cycloalkyl Substituted N-Heterocyclic Carbenes
and antimetastatic agents against human breast cancer cells.
The details of synthesis and biological activity of these
compounds are disclosed herein.
significant antiproliferative activity with IC50 values less than 10
μM in breast as well as lung cancer cells (Table 1, also see
Figure S-1, Supporting Information). We found that these three
compounds exhibited maximum growth inhibition in the case
of highly aggressive and metastatic MDA-MB-231 breast cancer
cells. The IC50 values of 3c, 3j, and 3h in MDA-MB-231 cells at
24 h were 7.98 0.47 μM, 7.28 0.24 μM, and 4.49 0.76
μM, respectively (see Figure S-1, Supporting Information).
Considering their potent antiproliferative activity against
metastatic breast cancer, we further considered evaluating the
mode of actions of these compounds in MDA-MB-231 cells at
sub-IC50 concentrations. One of the most unique characteristic
of cells undergoing apoptosis is the capacity to bind with the
protein annexin V (AV) at their cell membrane.12 Dual staining
with AV and propidium iodide (PI) facilitates in the
discrimination of live cells (AV−/PI−), early apoptotic cells
(AV+/PI−), late apoptotic cells (AV+/PI+), and necrotic cells
(AV−/PI+). As shown in Figure 2A, MDA-MB-231 cells
treated with compounds 3c, 3j, and 3h showed an
accumulation of single and double-positive cells (early and
late apoptotic cells, respectively) in comparison with the
control, in a concentration-dependent manner. Further, JC-1
assay revealed that 3c, 3j, and 3h lead to a depolarization of the
mitochondrial membrane potential in MDA-MB-231 cells,
which is considered to be the characteristic feature of induction
of intrinsic pathway of apoptosis. Mitochondrial depolarization
is indicated by a decrease in the red/green fluorescence
intensity ratio. The ratio of red/green fluorescence decreased
with the treatment of compounds 3c, 3j, and 3h in MDA-MB-
231 cells (Figure 2B,C). These results suggest that compounds
3c, 3j, and 3h induced apoptosis in MDA-MB-231 cells through
activation of intrinsic apoptotic pathway.
In order to further validate the mechanism of the induction
of cellular apoptosis, we assessed the expressions of regulatory
proteins of apoptotic pathway such as Bax, Bcl-2, Bcl-XL,
cleaved caspase-3, and cleaved caspase-9 in MDA-MB-231 cells.
It was found that treatment with compounds 3c, 3j, and 3h
increased the expression of proapoptotic marker such as
cleaved caspase-3 and decreased the expressions of anti-
apoptotic proteins such as Bcl-2, Bcl-XL, and survivin in
MDA-MB-231 cells at 24 h (Figure 3). The proapoptotic
proteins promote the release of cytochrome c, whereas
antiapoptotic proteins antagonize the actions of proapoptotic
proteins and prevent the loss of mitochondrial membrane
potential.13 In agreement with these observations, we found
that antiapoptotic proteins Bcl-2 and Bcl-XL were down-
regulated after 24 h of treatment with 3c, 3j, and 3h. The
activation of caspases is required for the induction and
RESULTS AND DISCUSSION
■
Chemistry. Recently, Hutt and Aron reported that treating
piconaldehyde with formalin and a primary amine in the
presence of HCl in ethanol results in formation of imidazo[1,5-
a]pyridinium ions.10 In contrast our initial attempts of reacting
methyl 1-formyl-9H-β-carboline-3-carboxylate (2)11 with form-
aldehyde and aniline (a) with HCl in ethanol did not yield the
desired result. Therefore, in a modification of the protocol, we
considered generating the ethanolic HCl in situ by replacing
HCl with AcCl, and to our delight this reaction condition gave
a product in 92% yield. The spectroscopic analysis of this
product revealed it to be the required 2-phenyl-2,11-dihydro-
imidazo[1′,5′:1,2]pyrido[3,4-b]indole 3a (Scheme 1). Since the
medium of the reaction was acidic, we considered performing
the reaction with methyl 1-(dimethoxymethyl)-9H-pyrido[3,4-
b]indole-3-carboxylate (1) directly instead of the aldehyde 2, as
it may reduce a step in the sequence. Gratifyingly the reaction
of 1 with formaldehyde and aniline gave an identical product
without attenuation of the yield. Next we tested the scope of
the methodology by reacting 1 and formaldehyde with several
aromatic, cyclic, and aliphatic primary amines under the
optimized conditions and discovered that the respective
products (3b−v) were isolated in each case as chloride salt in
good yields (Table 1). It was noted that as compared to
arylamines, the aliphatic amines furnished the product in
relatively lower yields (compare entries 18−20 with 1−17,
Table 1). Mechanistically the reaction proceeds via initial imine
I formation from the amine and formaldehyde in the presence
of ethanolic HCl generated from AcCl and ethanol (Scheme 2).
The nucleophilic attack of the pyridine nitrogen onto the imine
I lead to intermediate II followed by intramolecular cyclization
to afford the product 3 as hydrochloride salt.
Subsequently we also investigated the success of the protocol
with the substrate where the indole nitrogen was protected. It
was observed that the reaction between methyl 9-(3,4,5-
trimethoxybenzyl)-1-(dimethoxymethyl)-9H-pyrido[3,4-b]-
indole-3-carboxylate 4, formaldehyde, and 4-chlorobenzylamine
was successful to furnish 5 in 85% yield (Scheme 3).
Biological Activity. With the chemical library of β-
carboline-based N-heterocyclic carbenes in hand, growth
inhibitory effect of all test compounds was evaluated using
three different human breast cancer cells including MDA-MB-
231, MCF-7, and MDA-MB-468 and lung cancer H1299 cells.
Among the set, three compounds 3c, 3j, and 3h displayed
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Table 1. Yields of the Isolated Products and Their in Vitro Antiproliferative Effects on Different Human Cancer Cell Lines
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Table 1. continued
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Table 1. continued
a
b
All reactions were performed with 0.1 g of 1, 1.5 equiv of HCHO, and 1.2 equiv of amine (a−v). Yields of 3a−v, 5 are isolated yields after column
chromatography.
execution of cellular apoptosis.14 As shown in Figure 3,
compounds 3c, 3j, and 3h induced proteolytic cleavage of
caspase-3, which is in a good agreement with the mitochondrial
depolarization. Survivin is the member of inhibitors of
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Scheme 2. Plausible Mechanism for the Formation of N-Heterocyclic Carbene
Scheme 3. Synthesis of Product from N-Protected Starting β-Carboline
apoptosis protein (IAP) family which functions through direct
interactions to inhibit the activity of several caspases including
caspase-3, caspase-7, and caspase-9.15 Treatment with these
compounds at their IC50 concentrations induced the cleavage of
DNA repair enzyme, poly(ADP-ribose) polymerase (PARP) in
MDA-MB-231 cells, which is a characteristic feature of cells
undergoing apoptosis (Figure 3). PARP is cleaved by caspase-3
from its full-length active 116 kDa form to an inactive 89 kDa
form during execution of apoptosis.16 Altogether, our results
suggest that compounds 3c, 3j, and 3h were able to induce
caspase-dependent intrinsic pathway of apoptosis in MDA-MB-
231 cells.
Cell cycle checkpoints are important control mechanisms
that ensure the irreversible execution of cell cycle events. G2/M
checkpoint blocks the entry into mitosis in response to DNA
damaging agents or those which target cytoskeleton assembly.17
Cell cycle regulatory effects of 3c, 3j, and 3h were analyzed
using flow cytometry-based total DNA content analysis after 24
h of treatment in MDA-MB-231 cells. All three compounds led
to significant induction of G2/M phase cell cycle arrest in a
concentration-dependent manner as evidenced from the
increasing population in peak corresponding to G2/M phase
which is accompanied by a proportionate reduction in cells in
other phases of the cell cycle (Figure 4A). G2/M transition is
largely dependent on cyclin B1/CDK1 (CDC2) activity which
is mainly regulated by the positive regulator CDC25C
phosphatase. CDC25C dephosphorylates CDK1 leading to
the activation of cyclin B1/CDK1 complex and execution of
G2/M transition.18 In response to DNA damage or other
changes leading to G2/M arrest, phosphorylation of CDC25C
leads to its degradation and sequestration in the cytoplasm,
which eventually destabilizes cyclin B/CDK complexes.19 In
accordance, treatment with compounds 3c, 3j at concentrations
of 7.5, 10 μM and with compound 3h at concentrations of 5,
7.5 μM caused a drastic decrease in the expression of cyclin B,
CDC25C, and CDK1 in MDA-MB-231 cells (Figure 4B).
These results clearly demonstrate that compounds 3c, 3j, and
3h potentially induce G2/M phase cell cycle arrest in MDA-
MB-231 cells, which might be the reason for their
antiproliferative effect.
Cell invasion and migration are the crucial steps in the
progression of tumor metastasis.20 We performed wound
healing and transwell migration assays on human breast cancer
MDA-MB-231 cells in response to treatment with 3c, 3j, and
3h. Compounds 3c and 3j showed significantly reduced
number of migrated cells at concentrations of 1−7.5 μM,
whereas 3h significantly inhibited migration at 2.5 and 5 μM
concentrations as shown in Figure 5A,B. Similar results were
also obtained in transwell migration assay, where treatment
with all three compounds considerably inhibited cell migration
at concentrations of 2.5 μM or higher (Figure 5C). In addition
to cell migration, we have also observed a considerable decrease
in the number of invaded cells at as low as at 2.5 μM
concentration of 3c, 3j and 1 μM concentration of 3h after 48 h
of treatment (Figure 5D). Thus, these compounds significantly
inhibited cell migration and invasion at concentrations where
no significant antiproliferative and proapoptotic effects were
observed. This indicates that the inhibition of cell migration
and invasion was not due to antiproliferative and/or
proapoptotic effects of these compounds.
Elevated levels of matrix metalloproteinases (MMPs) such as
MMP-2 and MMP-9 have been well correlated with cell
invasion and migration in many cancers including breast
cancer.21,22 We observed considerable decreases in the activities
of MMP-2 and MMP-9 after treatment with compounds 3c, 3j,
and 3h in MDA-MB-231 cells (Figure 6A). MAPK pathway
plays an important role in the activation of ECM-degrading
MMPs leading to enhanced cell invasion and migration.23,24
Therefore, we further assessed the phosphorylations of ERK1/
2, SAPK/JNK, and p38 MAPKs after treatment of MDA-MB-
231 cells with these compounds. The activation of ERK and
JNK is generally associated with cell survival, invasion, and
tumor metastasis, while p38 MAPK activity has been found to
be involved in induction of cellular apoptosis. As shown in
Figure 6B, compounds 3c, 3j, and 3h suppressed the
phosphorylation of ERK1/2 and SAPK/JNK MAPKs but
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Figure 2. Compounds 3c, 3j, and 3h induce apoptosis in MDA-MB-
231 cells. (A) Flow cytometric analysis of MDA-MB-231 cells after 24
h of treatment with 3c, 3j, and 3h at the indicated concentrations. The
lower right quadrant of the FACS histogram is showing the percentage
of early apoptotic cells, and the upper right quadrant is showing the
late apoptotic cells. (B) FACS histograms are showing assessment of
mitochondrial membrane potential (Δψmt) after treatment of MDA-
MB-231 cells with compounds 3c, 3j, and 3h for 24 h. In the lower
right quadrant the percentage of cells that emit only green fluorescence
can be attributed to depolarized Δψmt. (C) Bar graphs depicting
reduction in red to green fluorescence intensity ratio indicative of
reduction in Δψmt, in a dose-dependent manner. Each value has been
normalized with the red/green ratio of control. The results were
obtained from three independent experiments, mean SEM, (∗) p <
0.05, and (∗∗) p < 0.01.
Figure 4. Compounds 3c, 3j, and 3h induce G2/M phase cell cycle
arrest in MDA-MB-231 cells. MDA-MB-231 cells were treated with
compounds 3c, 3j, and 3h at the indicated concentrations for 24 h. (A)
Flow cytometric histograms represent percentage of cells in each phase
of the cell cycle The results were obtained from three independent
experiments, mean SEM, (∗) p < 0.05, and (∗∗) p < 0.01 against
respective control cells at G2/M phases. (B) MDA-MB-231 cells were
treated for 24 h with the indicated concentrations of compounds 3c,
3j, and 3h. The whole cell lysates were analyzed for the detection of
cyclin B, CDK1, and CDC25C using Western blot. β-Actin was used
as an equal loading control. The blots are representative of three
independent experiments.
increased the phosphorylation of p38 MAPK. These results
suggest that in response to treatment of 3c, 3j, and 3h, cell
survival pathways particularly MAPK/ERK and SAPK/JNK
were suppressed, which was consistently associated with the
decreased cell migration and invasion. In contrast, treatment
with 3c, 3j, and 3h led to the up-regulation of cell death
inducing pathway, i.e., p38 MAPK in MDA-MB-231 breast
cancer cells.
In the next stage of the study, 3c, 3j, and 3h were
investigated for their in vivo breast anticancer activity using
4T1-syngeneic mouse mammary cancer model. The com-
pounds were administered via oral gavage at 10 mg/kg body
weight doses. The administration of these compounds had no
significant reduction in body weight, which indicates that these
compounds are not toxic to the animals (Figure S-2,
Supporting Information). Further, tumor volume graph
indicated that compound 3c resulted in about 20% decrease
in the tumor volume compared with control, whereas
Figure 3. Treatment with compounds 3c, 3j, and 3h alters the
expression of pro- and antiapoptotic proteins in MDA-MB-231 cells at
24 h. β-Actin was used as an equal loading control. The blots are
representative of three independent experiments.
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Figure 5. Compounds 3c, 3j, and 3h inhibit migration and invasion capacities of highly metastatic human breast cancer MDA-MB-231 cells. (A)
Effect of 3c, 3j, and 3h on cell motility assessed by wound healing assay. Subconfluent monolayer cells were wounded and then treated with 0, 1, 2.5,
5, 7.5 μM 3c, 3j and 0, 1, 2.5, 5 μM 3h. At 0, 12, and 24 h after wounding, the wound areas were photographed using a phase contrast microscope
under 100× magnification. (B) Wound closure area was expressed as a control in bar graphs. Each bar represents the mean SEM calculated from
three independent experiments, (∗∗) p < 0.01. (C) Effect of 3c, 3j, and 3h on cell migration using highly metastatic MDA-MB-231 cells. The cell
migration was assessed by the transwell migration assay in MDA-MB-231 cells incubated in the absence or presence of indicated concentrations of
3c, 3j, and 3h for 48 h in transwell chambers placed in 24-well tissue culture plates. (D) Effect of 3c, 3j, and 3h on cell invasion in MDA-MB-
231cells. For invasion assay, Matrigel coated the transwell chambers. The migrated cells (C) and invaded cells (D) were stained with crystal violet
and photographed under 100× magnification. The images are representative of three independent experiments.
compounds 3j and 3h had no significant effect on tumor
growth in vivo (Figure S-2, Supporting Information).
3h (Figure S-4, Supporting Information). The pH dependence
experiment inferred that the binding was viable from pH 5.8 to
8. The single Trp residue of HSA, which is present in domain
II, can be selectively excited at 295 nm and shows emission
maxima at 340 nm in native state. However, the emission
spectrum of 3h displayed a peak at 437 nm on excitation with
295 nm, which has no significant overlap with Trp emission of
HSA. Hence, any detectable change in the emission spectra of
Trp inside HSA on addition of 3h should give the binding
information. It was observed that on continuous addition of 3h
from 0 to 50 μM, the fluorescence intensity of Trp inside HSA
(10 μM) decreases continuously without any change in the
emission maximum (Figure S-5, Supporting Information),
thereby suggesting the binding of 3h with HSA. Subsequently
with the objective to assess the effects of temperature on
binding and to measure the thermodynamic parameters for
binding of 3h to HSA, the same experiment was performed at
three different temperatures 283, 293, and 303 K (Figure S-5,
The lack of any in vivo effect for this series of compounds
prompted us to examine their human serum albumin (HSA)
binding, as it is known that high affinity to HSA results in lack
of in vivo effect.25 In this context, we studied the interaction of
3h with HSA spectroscopically. From the crystallographic data
of HSA it is known that it contains a single chain of 585 amino
acid residues consisting of three different homologous domains
(I, II, and III) and each divided into two subdomains (A and
B).26 The absorption spectrum of 3h in 0.05 M phosphate
buffer (pH 7.4) displayed three peaks at 292, 320, and 371 nm
(see Figure S-3, Supporting Information), and the molar
extinction coefficient for 3h was measured as 38500 500 L
mol−1 cm−1 at 292 nm. The absorption spectrum of HSA in
0.05 M phosphate buffer (pH 7.4) shows a peak at 278 nm. A
small red shift in the absorption maximum with a concomitant
increase in absorbance was observed on continuous addition of
H
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Figure 7. Stern−Volmer plot for fluorescence of HSA by 3h. Red line
denotes the best fit for eq 1, and blue line is just an eye guide.
almost constant, which suggested that the quenching of Trp
fluorescence by 3h is static in nature. This inferred that the
quenching of fluorescence was due to the nonfluorescent
complex formation between HSA and 3h. The quenching
constant was calculated at each temperature using average
lifetime of Trp inside HSA in the absence of 3h (Table S-2,
Supporting Information). The high order of kq at each
temperature further asserted that the quenching is static in
nature, as the maximum value of kq for dynamic quenching is
on the order of 1010 L mol−1 s−1. Subsequently with these
quenching data, we estimated the binding constant between
HSA and 3h using modified Stern−Volmer equation as27
Figure 6. Compounds 3c, 3j, and 3h inhibit MMP-2 and MMP-9
activities and alter MAPKs expression in MDA-MB-231 cells. (A)
MDA-MB-231 cells were plated in six-well plates at a density of 1 ×
105 cells/well and then treated with 0, 1, 2.5, 5, 7.5 μM compounds 3c,
3j and 0, 1, 2.5, 5 μM 3h for 24 h. Conditioned medium was collected
and centrifuged. The activities of MMP-2 and MMP-9 were measured
by gelatin zymography. (B) MDA-MB-231 cells were treated for 24 h
with the indicated concentrations of compounds. The cells were
harvested and lysed for the whole cell lysates extraction. Samples were
subjected to 10% SDS−PAGE followed by Western blot analysis for
the detection of p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, and p38
MAPK. β-Actin antibody was used as an equal loading control. The
blots are representative of three independent experiments.
log[(F − F)/F] = log Kb + n log[Q]
(2)
0
where K, n, and [Q] stand for the binding constant, number of
3h molecules that bind to HSA, and concentration of 3h,
respectively.
The change in log[(F0 − F)/F] with log[Q] and the values of
Kb and n were estimated from the intercept and slope of the
best linear fit as shown in Figure 8. At 283 K, the estimated
Supporting Information). At each temperature the fluorescence
intensity of Trp inside HSA was quenched by 3h. However, as
the extent was different at different temperatures, it was
analyzed using the Stern−Volmer equation as follows.27b
F
F
τ0
τ
0
=
= 1 + kqτ0[Q] = 1 + KSV[Q]
(1)
where F0 and F are the fluorescence intensities in absence and
presence of quencher, i.e., 3h, [Q] is the concentration of 3h, kq
is quenching constant, KSV is the Stern−Volmer quenching
constant, and τ0 is the excited state lifetime in the absence of
quencher. The change in F0/F value with concentration of 3h
was determined (Figure S-6, Supporting Information), and by
fitting these data points using eq 1, Stern−Volmer quenching
constants were calculated for all the three temperatures (Table
S-1, Supporting Information). Linearity of the Stern−Volmer
plot for all temperatures suggested that the quenching is either
static or dynamic in nature. Next to evaluate the mechanism of
quenching, fluorescence transients of Trp inside HSA were
recorded at room temperature (25 °C) in the absence and
presence of different concentrations of 3h (see Figure S-7,
Supporting Information). The observed lifetime at each
concentration of 3h was analyzed in terms of the Stern−
Volmer equation. The change in the value of τ0/τ with the
concentration of 3h as shown in Figure 7 was observed to be
Figure 8. Plot of log((F0 − F)/F)against log[2h] at three different
temperatures 283 K (red circle), 293 K (blue circle), 303 K (green
circle). The solid lines show the best fit at each temperature by using
eq 2.
value of the binding constant is 9.77 × 104 L mol−1, which
decreases to 8.91 × 104 and 7.94 × 104 L mol−1 at 293 and 303
K, respectively. The value of n is around 1 for all the
temperatures, suggesting that 3h binds to a single site of HSA.
In order to investigate the binding sites of 3h in HSA,
molecular docking was carried out using AutoDock Tools (4.2)
and the initial coordinates of HSA from PDB entry 1HA2.28 On
the basis of the results of the docking studies, it was inferred
that 3h binds to domain I of HSA.
I
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To gain insight into the interaction behavior, thermodynamic
parameters of binding between 3h and HSA were estimated.
Free energy (ΔG0binding), entropy (ΔS0), and enthalpy (ΔH0)
of binding are the main parameters that play an important role
for binding and can be estimated by
cancer MDA-MB-231 cells by decreased activities of MMPs and
the down-regulation of ERK1/2 and SAPK/JNK signaling.
Interestingly, these compounds act like antiproliferative and
proapoptotic at higher doses, whereas at lower doses these
compounds inhibited metastasis hallmarks (e.g., migration and
invasion) in breast cancer cells. However, these compounds did
not elicit significant bioresponse during the in vivo inves-
tigations in mouse model which may be attributed to their
strong affinity for the HSA protein which is evident from the
results of the present study. Nevertheless the purpose of
designing new β-carboline-based anticancer agents by merging
the two subunits which are individually known to display
anticancer property was achieved. Further work toward
improving the in vivo profile of this core via chemical
modifications is underway and will be reported in the future.
ΔG0
= −2.303RT log Kb
= ΔH0 − TΔS0
binding
(3)
ΔG0
binding
(4)
where R is the universal gas constant. The values of ΔG0
binding
were calculated as −11.74, −12.06, and −12.34 kJ/mol at 283,
293, and 303 K, respectively. The negative values of ΔG0
binding
at all the temperatures indicated the spontaneous nature of
binding of 3h with HSA. The other thermodynamic parameters
were calculated by fitting free energy values at different
temperatures (Figure S-8 and Table S-3, Supporting
Information). It is worth mentioning that the positive value
of ΔS0 and a very small value of ΔH0, either positive or
negative, are responsible for electrostatic interactions in the
binding process.29 Thus, the observed values of ΔH0 and ΔS0
as −3.21 kJ/mol and 30.15 J mol−1 K−1, respectively, inferred
that electrostatic interactions played a major role in the binding
of 3h with HSA. Since domain I contains a lot of positively
charged amino acid residues and 3h is in ionized form, it was
not surprising to discover that in this case ionic interaction
prevailed for binding.
To explore the structural change of HSA by addition of 3h,
circular dichroism (CD) measurements were performed for
HSA in the presence of several concentrations of 3h. As
expected for HSA, CD spectrum displayed strong negative
ellipticity at 208 and 222 nm which is the signature of α-helicity
(Figure S-9, Supporting Information).30 Experimental data
were expressed in terms % α-helix of HSA and are shown in
Figure 9. These observations delineated that the secondary
structure of HSA remains unchanged until 40 μM 3h.
EXPERIMENTAL SECTION
■
General. All experiments were monitored by analytical TLC
performed on precoated silica gel plates. After elution, the plate was
visualized under UV illumination at 254 nm for UV active materials. IR
spectra were recorded using a FTIR spectrophotometer. 1H NMR and
13C NMR spectra were recorded on 400 MHz spectrometer, using
TMS as an internal standard (chemical shifts in δ). Peak multiplicities
of NMR signals were designated as s (singlet), bs (broad singlet), d
(doublet), dd (doublet of doublet), t (triplet), m (multiplet), etc. The
ESI-MS data were recorded on an ion trap mass spectrometer, and the
HRMS spectra were recorded as ESI-HRMS data on a Q-TOF LC-
MS/MS mass spectrometer. The synthesized compounds were more
than 95% pure according to the HPLC chromatograms (Agilent 1200
with PDA detector, RP C-18 column (250 mm × 4.5 mm, 5 μm) using
a gradient of 20−90% acetonitrile and water in 25 min at a flow rate of
1 mL/min). Human serum albumin (HSA, fatty acid free) was
purchased from Sigma-Aldrich, USA. Analytical grade disodium
hydrogen phosphate and sodium dihydrogen phosphate was purchased
from Merck, India, and used for preparing buffer solution. The steady
state absorption and emission spectra were recorded in a commercial
UV−vis spectrophotometer and a spectroflurimeter, respectively.
Fluorescence transients were collected at magic angle polarization
using a commercial TCSPC setup (Fluorolog 3, Jobin-Yvon, USA).
Circular dichroism (CD) measurements were performed on a
commercial spectropolarimeter.
General Procedure for the Synthesis of 3a−v. To a round-
bottom flask containing dry ethanol (10 mL) was added acetyl
chloride (1.5 equiv) at 0 °C under stirring. After 5 min formaldehyde
(1.5 equiv) and appropriate amine from a−v (1.2 equiv) were added
dropwise at the same temperature. The reaction was allowed to
proceed for 20 min after which methyl 1-(dimethoxymethyl)-9H-
pyrido[3,4-b]indole-3-carboxylate (1) (1.0 equiv) was added under
stirring. The reaction mixture was allowed to warm to room
temperature and continued for 12 h. The separated solid in the
reaction mixture was filtered and washed with ice cold ethanol (2 mL)
to obtain the analytically pure products as solids.
5-(Methoxycarbonyl)-2-phenyl-2,11-dihydroimidazo-
[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride (3a). Yield, 92%
(0.136 g from 0.1 g); white solid, mp 217−219 °C; Rf = 0.48
(CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 896, 923, 1141, 1563,
Figure 9. Change in % α-helicity of HSA with 3h concentration.
1
1762, 3414 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm) = 4. 06
CONCLUSIONS
■
(s, 3H), 7.34 (t, J = 7.4 Hz, 1H), 7.49 (t, J = 6.9 Hz, 1H), 7.65−7.78
(m, 4H), 7.98 (d, J = 7.1 Hz, 2H), 8.28 (d, J = 7.5 Hz, 1H), 9.95 (s,
1H), 9.22 (s, 1H), 10.61 (s, 1H), 14.11 (s, 1H). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) = 53.6, 112.1, 112.7, 113.2, 115.8, 121.1, 123.8,
124.1, 128.1, 130.3, 130.8, 131.2, 135.6, 139.3, 162.1. MS (ESI+): m/z
= 342.1. ESI-HR-MS calculated for C21H16N3O2 [M]+: 342.1237.
Found: 342.1239.
2-(4-Chlorophenyl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3b). Yield, 94% (0.152 g from 0.1 g); white solid, mp 204−206
°C; Rf = 0.46 (CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 919, 1123,
We have disclosed an efficient synthesis and antiproliferative
activity against breast cancer cells of a new series of fused β-
carbolines that can be readily prepared via Mannich reaction
between methyl 1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-
3-carboxylate, formaldehyde, and primary amines. Three of the
compounds 3c, 3j, and 3h from the series display significant
antiproliferative and proapoptotic effects in breast cancer cells.
Further, these compounds were found to suppress migration
and invasion capacities of highly metastatic human breast
J
DOI: 10.1021/acs.jmedchem.5b00016
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
1560, 1750, 3424 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm)
(3h). Yield, 85% (0.144 g from 0.1 g); yellow solid, mp 206−208
°C; Rf = 0.42 (CH2Cl2/MeOH, 9:1, v/v). IR (neat) νmax: 937, 1248,
4.06 (s, 3H), 7.36 (t, J = 7.4 Hz, 1H), 7.48−7.73 (m, 6H), 8.30 (d, J =
7.8 Hz, 1H), 8.64 (s, 1H), 8.97 (s, 1H), 10.73 (s, 1H), 13.68 (s, 1H).
13C NMR (100 MHz, DMSO-d6): δ (ppm) = 55.4, 112.1, 112.5, 113.2,
1
1554, 1762, 3455 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm)
4.06 (s, 3H), 7.36 (bs, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.60 (s, 1H), 7.70
(d, J = 7.3 Hz, 1H), 7.99 (s, 1H), 8.30 (d, J = 7.3 Hz, 1H), 9.02 (s,
1H), 9.16 (s, 1H), 10.82 (s, 1H), 14.15 (s, 1H). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) = 53.6, 110.5, 112.6 (d, J = 110.0 Hz), 115.2 (d, J
= 150.0 Hz), 119.3, 121.1 121.1, 121.8, 122.5 (d, J = 10.0 Hz), 123.3,
(d, J = 50.0 Hz), 123.9, 124.2, 125.1, 126.6, 126.8, 127.2, 129.1, 130.5,
133.5, 136.7, 139.4, 145.4, 147.8, 162.1. MS (ESI+): m/z = 394.1. ESI-
HR-MS calculated for C21H14ClFN3O2 [M]+: 394.0753. Found:
394.0758.
116.3, 121.1, 122.4, 123.1, 123.6, 126.7, 129.3, 129.5, 129.8, 131.1,
134.2, 134.3, 139.3, 162.3. MS (ESI+): m/z = 376.1. ESI-HR-MS
calculated for C21H15ClN3O2 [M]+: 376.0847. Found: 376.0850.
5-(Methoxycarbonyl)-2-(4-(trifluoromethyl)phenyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3c). Yield, 98% (0.172 g from 0.1 g); white solid, mp 210−212 °C;
Rf = 0.39 (CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 1223, 1598, 1754,
3410 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm) 4.08 (s, 3H),
1
7.38−7.43 (m, 1H), 7.53−7.57 (m, 1H), 7.77 (d, J = 8.2 Hz, 1H), 8.18
(d, J = 8.6 Hz, 2H), 8.24 (d, J = 8.6 Hz, 2H), 8.37 (d, J = 7.9 Hz, 1H),
9.11 (s, 1H), 9.17 (d, J = 1.8 Hz, 1H), 10.81 (d, J = 1.8 Hz, 1H), 13.91
(s, 1H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 53.6, 112.3,
113.4, 116.0, 121.2, 122.7, 123.2, 123.9, 125.6, 126.9, 127.9, 128.0,
129.1, 129.2, 139.4, 162.1. MS (ESI+): m/z = 410.1. ESI-HR-MS
calculated for C22H15F3N3O2 [M]+: 410.1111. Found: 410.1115.
2 - ( 2 - I o d o p h e n y l ) - 5 - ( m e t h o x y c a r b o n y l ) - 2 , 1 1 -
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3d). Yield, 88% (0.174 g from 0.1 g); gray solid, mp 232−234 °C;
Rf = 0.48 (CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 1152, 1536, 1756,
2-(2-Bromo-4-fluorophenyl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3i). Yield, 92% (0.167 g from 0.1 g); yellow solid, mp 221−223
°C; Rf = 0.43 (CH2Cl2/MeOH, 9:1, v/v). IR (neat) νmax: 932, 1256,
1
1546, 1745, 3425 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm)
4.05 (s, 3H), 7.37 (t, J = 7.5 Hz, 1H), 7.52 (t, J = 7.4 Hz, 1H), 7.64−
7.71 (m, 2H), 8.04−8.08 (m, 2H), 8.31 (d, J = 7.9 Hz, 1H), 9.03 (s,
1H), 9.11 (d, J = 1.1 Hz, 1H), 10.79 (d, J = 1.4 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6): δ (ppm) = 53.6, 112.8 (d, J = 107.0 Hz),
115.6, (d, J = 77.0 Hz)116.6, 116.8, 121.2, 121.4, 122.5, 122.6, 123.1,
123.4, 126.8, 129.1, 131.1, 131.4, 131.5, 131.8, 139.4, 162.1. (ESI+):
m/z = 428.2. ESI-HR-MS calculated for C21H14BrFN3O2 [M]+:
438.0248. Found: 438.0246.
2-(2-Bromo-4-methylphenyl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3j). Yield, 90% (0.166 g from 0.1 g); light yellow solid, mp 218−
220 °C; Rf = 0.38 (CH21Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 868,
1254, 1767, 3431 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm)
2.09 (s, 3H), 4.06 (s, 3H), 7.39 (dd, J1 = 7.2 Hz, J2 = 0.7 Hz, 1H), 7.53
(dd, J1 = 8.2 Hz, J2 = 1.1 Hz, 2H), 7.75 (d, J = 8.2 Hz, 1H), 7.81 (d, J =
8.1 Hz, 1H), 7.88 (s, 1H), 8.37 (d, J = 7.9 Hz, 1H) 9.03 (d, J = 1.7 Hz,
1H), 9.10 (s, 1H), 14.09 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ
(ppm) = 20.9, 53.6, 112.2, 113.3, 115.1, 116.2, 119.2, 121.2, 122.5,
122.6, 123.2, 123.4, 126.9, 129.2, 129.3, 130.1, 130.8, 132.5, 134.4,
139.4, 144.1, 162.1. MS (ESI+): m/z = 434.2. ESI-HR-MS calculated
for C22H17BrN3O2 [M]+: 434.0499. Found: 434.0503.
1
3453 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm) 4.15 (s, 3H),
7.47−7.83 (m, 6H), 8.12−8.17 (m, 2H), 8.97 (d, J = 10.4 Hz, 2H),
10.57 (s, 1H), 13.42 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ
(ppm) = 53.2, 111.7, 112.4, 112.6, 115.4, 120.5, 121.5, 122.1, 122.5,
123.2, 126.4, 127.5, 128.4, 128.6, 130.7, 131.6, 135.8, 138.7, 161.6. MS
(ESI+): m/z = 468.2. ESI-HR-MS calculated for C21H15IN3O2 [M]+:
468.0203. Found: 468.0189.
2-(2-Bromophenyl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3e). Yield, 90% (0.161 g from 0.1 g); white solid; mp 212−214
°C; Rf = 0.42 (CH2Cl2/MeOH, 9:1, v/v). IR (neat) νmax: 931, 1568,
1
1749, 3443 cm−1. H NMR (300 MHz, DMSO-d6): δ (ppm) 4.21 (s,
3H), 7.27 (t, J = 7.4 Hz, 1H), 7.40−7.48 (m, 4H), 7.61 (d, J = 7.9 Hz,
1H), 7.79 (d, J = 7.6 Hz, 1H), 8.17 (d, J = 7.7 Hz, 1H), 8.71 (s, 1H),
8.79 (s, 1H), 10.62 (s, 1H), 13.99 (s, 1H). 13C NMR (75 MHz,
DMSO-d6): δ (ppm) = 53.5, 111.7, 112.9, 113.2, 115.8, 120.8, 121.5,
122.2, 122.8, 123.4, 123.5, 126.5, 129.1, 130.0, 130.9, 131.2, 131.6,
133.6, 134.4, 139.1, 162.1. MS (ESI+): m/z = 420.1. ESI-HR-MS
calculated for C21H15BrN3O2 [M]+: 420.0342. Found: 420.0348.
2-(2-Chloro-4-methylphenyl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3f). Yield, 96% (0.172 g from 0.1 g); white solid, mp 214−216 °C;
Rf = 0.40 (CH2Cl21/MeOH, 9:1, v/v). IR (KBr) νmax: 899, 1265, 1578,
1758, 3423 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm) 3.61 (s,
3H), 6.86 (t, J = 7.6 Hz, 1H), 7.01(t, J = 7.6 Hz, 1H), 7.16 (d, J = 8.2
Hz, 1H), 7.26 (t, J = 8.1 Hz, 1H), 7.48 (dd, J1 = 0.8 Hz, J2 = 0.9 Hz,
1H), 7.54 (dd, J1 = 1.5 Hz, J2 = 1.5 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H),
7.84 (d, J = 1.7 Hz, 1H), 8.38 (d, J = 5.7 Hz, 1H), 8.72 (d, J = 1.6 Hz,
1H), 10.20 (d, J = 1.7 Hz, 1H), 13.62 (s, 1H). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) = 53.5, 111.8, 112.9, 113.1, 115.6, 120.9, 122.2,
122.3, 122.8, 122.9, 123.5, 123.6, 126.7, 127.3, 128.5, 128.9, 132.5,
134.1, 136.7, 139.2, 161.9. MS (ESI+): m/z = 420.2. ESI-HR-MS
calculated for C21H15BrN3O2 [M]+: 420.0342. Found: 420.0346.
5-(Methoxycarbonyl)-2-(3-(trifluoromethyl)phenyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3g). Yield, 95% (0.166 g from 0.1 g); white solid, mp 216−218
°C; Rf = 0.44 (CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 952, 1257,
2-(4-Chloro-2-methylphenyl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3k). Yield, 93% (0.156 g from 0.1 g); yellow solid, mp 212−214
°C; Rf = 0.46 (CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 899, 1216,
1
1764, 3450 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm) 4.05 (s,
3H), 7.28−7.41 (m, 3H), 7.52 (t, J = 7.4 Hz, 1H), 7.63 (dd, J1 = 1.9
Hz, J2 = 2.0 Hz, 1H), 7.69−7.73 (m, 1H), 7.78 (d, J = 8.4 Hz, 1H),
8.33 (d, J = 7.8 Hz, 1H), 9.03 (s, 1H), 9.08 (d, J = 1.5 Hz, 1H), 10.62
(d, J = 1.5 Hz, 1H), 14.21 (s, 1H). 13C NMR (100 MHz, DMSO-d6):
δ (ppm) = 17.5, 53.5, 112.1, 113.2, 114.8, 116.2, 121.1, 122.3, 122.4,
123.1, 123.6, 126.8, 127.1, 127.6, 129.3, 130.4, 131.6, 132.3, 134.1,
134.4, 135.9, 136.9, 139.3, 162.2. MS (ESI+): m/z = 390.1. ESI-HR-
MS calculated for C22H17ClN3O2 [M]+: 390.1004. Found: 390.1009.
-(2-Chloro-4-methylphenyl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3l). Yield, 94% (0.157 g from 0.1 g); yellow solid, mp 202−204
°C; Rf = 0.43 (CH2Cl2/MeOH, 9:1, v/v). IR (neat) νmax: 918, 1221,
1
1758, 3442 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm) 2.49 (s,
3H), 4.05 (s, 3H), 7.36−7.86 (m, 6H), 8.29 (s, 1H), 8.99−9.09 (m,
2H), 10.71 (s, 1H), 14.18 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ
(ppm) = 21.1, 53.6, 112.1, 113.2, 115.1, 116.1, 121.1, 122.5, 123.0,
123.4, 126.8, 129.2, 129.7, 130.8, 131.2, 139.4, 143.9, 162.2. MS (ESI
+): m/z = 390.1. ESI-HR-MS calculated for C22H17ClN3O2 [M]+:
390.1004. Found: 390.1011.
1
1566, 1760, 3445 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm)
4.05 (s, 3H), 7.26 (t, J = 7.3 Hz, 1H), 7.42 (t, J = 7.2 Hz, 1H), 7.60 (d,
J = 7.9 Hz, 1H), 7.99 (t, J = 7.6 Hz, 1H), 8.08 (d, J = 7.4 Hz, 1H), 8.15
(d, J = 7.6 Hz, 1H), 8.28 (d, J = 7.3 Hz, 1H), 8.41 (s, 1H), 8.81 (s,
1H), 8.99 (s, 1H), 10.74 (s, 1H), 13.58 (s, 1H). 13C NMR (75 MHz,
DMSO-d6): δ (ppm) = 53.4, 111.9, 112.6, 112.9, 115.7, 120.7, 121.8,
122.1, 122.3, 122.7, 123.4, 125.6, 126.7, 127.7, 128.5, 128.8, 130.9,
131.4, 131.9, 136.1, 138.9, 161.8. MS (ESI+): m/z = 410.3. ESI-HR-
MS calculated for C22H15F3N3O2 [M]+: 410.1111. Found: 410.1118.
2-(2-Chloro-4-fluorophenyl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
2-(3,4-Dimethoxyphenyl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3m). Yield, 83% (0.142 g from 0.1 g); white solid; mp 198−200
°C; Rf = 0.36 (CH2Cl2/MeOH, 9:1, v/v). IR (neat) νmax: 854, 1242,
1
1560, 1742, 3430 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm)
3.68 (s, 6H), 3.97 (s, 3H), 6.68 (s, 2H), 6.99 (s, 2H), 7.36−7.40 (m,
1H), 7.63−7.67 (m, 1H), 7.83 (d, J = 8.2 Hz, 1H), 8.47 (d, J = 7.8 Hz,
1H), 9.17 (s, 1H), 10.27 (s, 1H), 12.4 (s, 1H). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) = 52.7, 56.1, 56.2, 108.1, 109.9, 113.9, 120.6,
K
DOI: 10.1021/acs.jmedchem.5b00016
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
121.7, 121.8, 122.8, 130.1, 132.0, 135.2, 135.8, 137.1, 142.9, 148.9,
165.7. MS (ESI+): m/z = 402.2. ESI-HR-MS calculated for
C23H20N3O4 [M]+: 402.1448. Found: 402.1448.
10.71 (d, J = 1.2 Hz, 1H) 14.03 (s, 1H). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) = 21.2, 45.8, 53.4, 111.8, 112.7, 113.1, 116.1,
120.8, 121.4, 122.3, 122.9, 126.6, 129.4, 129.5, 131.5, 132.2, 138.2,
138.9, 139.2, 162.2. MS (ESI+): m/z = 365.2. ESI-HR-MS calculated
for C21H25N4O2 [M]+: 365.1972. Found: 365.1976.
2-Benzyl-5-(methoxycarbonyl)-2,11-dihydroimidazo-
[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride (3n). Yield, 89%
(0.137 g from 0.1 g); white solid, mp 212−214 °C; Rf = 0.47
(CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 868, 1168, 1547, 1748,
2-(Adamantan-1-yl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3t). Yield, 68% (0.091 g from 0.1 g); yellow solid; mp 166−168
°C; Rf = 0.48 (CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 1238, 1560,
1
3464 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm) 4.05 (s, 3H),
5.98 (s, 2H), 7.33 (t, J = 7.1 Hz, 1H), 7.46−7.67 (m, 7H), 8.28 (d, J =
7.5 Hz, 1H), 8.69 (s, 1H), 8.93 (s, 1H), 10.72 (s, 1H), 13.85 (s, 1H).
13C NMR (100 MHz, DMSO-d6): δ (ppm) = 53.5, 55.3, 112.1, 112.5,
1
1740, 3432 cm−1. H NMR (300 MHz, DMSO-d6): δ (ppm) 1.28−
1.39 (m, 4H), 4.04 (s, 3H), 4.29−4.38 (m, 1H), 7.29−7.39 (m, 1H),
7.44−7.53 (m, 1H), 7.72 (t, J = 9.8 Hz, 1H), 8.25 (dd, J1 = 7.9 Hz, J2 =
7.6 Hz, 1H), 8.51 (s, 1H), 8.69 (d, J = 3.9 Hz, 1H), 10.71 (s, 1H)
13.26 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ (ppm) = 11.5, 33.5,
53.4, 111.6, 113.1, 115.8, 120.8, 121.3, 121.4, 122.2, 122.9, 123.4,
126.6, 129.6, 132.4, 139.2, 162.2. MS (ESI+): m/z = 306.1. ESI-HR-
MS calculated for C18H16N3O2 [M]+: 306.1237. Found: 306.1241.
2-Cyclohexyl-5-(methoxycarbonyl)-2,11-dihydroimidazo-
[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride (3u). Yield, 80%
(0.121 g from 0.1 g); yellow solid, mp 178−180 °C; Rf = 0.43
(CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 726, 823, 916, 1256, 1560,
113.2, 116.3, 121.1, 122.4, 123.7, 126.7, 129.3, 129.5, 129.8, 131.1,
134.2, 134.3, 139.3, 162.3. MS (ESI+): m/z = 356.1. ESI-HR-MS
calculated for C19H28N2O2 [M]+: 356.1394. Found: 356.1397.
2-(4-Fluorobenzyl)-5-(methoxycarbonyl)-2, 11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3o). Yield, 91% (0.146 g from 0.1 g); white solid; mp 228−230
°C; Rf = 0.45 (CH2Cl2/MeOH, 9:1, v/v). IR (neat) νmax: 756, 1235,
1
1575, 1753, 3396 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm)
4.05 (s, 3H), 5.96 (s, 2H), 7.30−7.36 (m, 3H), 7.46−7.51 (m, 1H),
7.66−7.71 (m, 3H), 8.29 (d, J = 7.9 Hz, 1H), 8.65 (d, J = 1.6 Hz, 1H),
8.95 (s, 1H), 10.71 (d, J = 1.5 Hz, 1H), 13.72 (s, 1H). 13C NMR (100
MHz, DMSO-d6): δ (ppm) = 52.7, 53.5, 111.9, 112.8 (d, J = 64.0 Hz),
116.2, 116.3, 116.5, 121.1, 121.5, 122.3, 123.1, 123.6, 126.6, 129.3,
129.7, 131.6, 131.6, 139.3, 162.3. MS (ESI+): m/z = 374.1. ESI-HR-
MS calculated for C19H28N2O2 [M]+: 374.1299. Found: 374.1301.
5-(Methoxycarbonyl)-2-(4-methylbenzyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3p). Yield, 94% (0.150 g from 0.1 g); white solid; mp 223−225
°C; Rf = 0.47 (CH2Cl2/MeOH, 9:1, v/v). IR (neat) νmax: 868, 956,
1
1755, 3428 cm−1. H NMR (500 MHz, DMSO-d6): δ (ppm) 1.48−
1.83 (m, 10H), 2.43−2.48 (m, 1H), 3.93 (s, 3H), 7.34 (t, J = 7.4 Hz,
1H), 7.62 (t, J = 7.8 Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H), 8.40 (d, J = 7.8
Hz, 1H), 8.70 (s, 1H), 8.70 (s, 1H), 8.96(s, 1H), 10.37 (s, 1H), 13.22
(s, 1H). 13C NMR (125 MHz, DMSO-d6): δ (ppm) = 24.8, 25.1, 25.9,
50.4, 69.3, 114.0, 118.8, 121.1, 121.3, 122.5, 129.3, 129.8, 135.3, 136.7,
137.7, 141.9, 160.9, 166.2. MS (ESI+): m/z = 348.2. ESI-HR-MS
calculated for C21H22N3O2 [M]+: 348.1707. Found: 348.1711.
2-(Adamantan-1-yl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3v). Yield, 64% (0.109 g from 0.1 g); light yellow solid, mp 158−
160 °C; Rf = 0.47 (CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 968,
1259,1590, 1746, 3469 cm−1. 1H NMR (400 MHz, DMSO-d6): δ
(ppm) 1.54−1.65 (m, 10H), 3.96 (s, 3H), 7.37 (bs, 1H), 7.65−7.84
(m, 3H), 8.46 (s, 1H), 9.16 (s, 1H), 10.27 (s, 1H), 12.45 (s, 1H). 13C
NMR (100 MHz, DMSO-d6): δ (ppm) = 28.7, 35.5, 51.4, 52.8, 113.9,
120.6, 121.7, 121.8, 122.8, 130.1, 131.9, 135.2, 135.8, 137.1, 142.9,
165.7. MS (ESI+): m/z = 400.2. ESI-HR-MS calculated for
C25H26N3O2 [M]+: 400.2020. Found: 400.2026.
1
1240, 1752, 3380 cm−1. H NMR (400 MHz, DMSO-d6): δ (ppm)
2.51 (s, 3H), 4.03 (s, 3H), 5.91 (s, 2H), 7.19 (d, J = 7.6 Hz, 1H), 7.27
(d, J = 7.2 Hz, 2H), 7.37−7.44 (m, 2H), 7.49 (d, J = 7.6 Hz, 2H), 7.61
(d, J = 8.0 Hz, 1H), 8.17 (d, J = 7.2 Hz, 1H), 8.79 (s, 1H), 10.59 (s,
1H), 13.85 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) =
21.2, 45.8, 53.4, 111.8, 112.7, 113.1, 116.1, 120.8, 121.4, 122.3, 122.9,
126.6, 129.4, 129.5, 131.5, 132.2, 138.2, 138.9, 139.2, 162.2. MS (ESI
+): m/z = 370.1. ESI-HR-MS calculated for C23H20N3O2 [M]+:
370.1550. Found: 370.1553.
5-(Methoxycarbonyl)-2-phenethyl-2,11-dihydroimidazo-
[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride (3q). Yield, 90%
(0.143 g from 0.1 g); white solid, mp 190−192 °C; Rf = 0.45
(CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 918, 1260, 1598, 1757,
3427 cm−1. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 3.31−3.34 (m,
6H), 4.04 (s, 3H), 4.96 (t, J = 7.4 Hz, 1H), 7.26−7.37 (m, 2H), 7.51
(t, J = 7.3 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 8.28 (d, J = 7.8 Hz, 1H),
8.68 (s, 1H), 8.93 (s, 1H), 10.52 (s, 1H), 13.86 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): δ (ppm) = 31.2, 52.7, 55.4, 112.1, 112.6,
113.2, 116.3, 121.1, 121.6, 122.4, 123.1, 123.7, 126.7, 129.3, 129.5,
129.9, 131.2, 134.2, 134.4, 139.3, 162.3. MS (ESI+): m/z = 370.2. ESI-
HR-MS calculated for C23H20N3O2 [M]+: 370.1550. Found: 370.1555.
2-Allyl-5-(methoxycarbonyl)-2,11-dihydroimidazo[1′,5′:1,2]-
pyrido[3,4-b]indol-4-ium Chloride (3r). Yield, 77% (0.103 g from
0.1 g); yellow solid, mp 194−196 °C; Rf = 0.36 (CH2Cl2/MeOH, 9:1,
v/v). IR (KBr) νmax: 756, 941, 1563, 1760, 3440 cm−1. 1H NMR (300
MHz, DMSO-d6): δ (ppm) 4.03 (s, 3H), 5.37−5.52 (m, 4H), 6.15−
6.27 (m, 1H), 7.32 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.3 Hz, 1H), 7.66
(d, J = 8.2 Hz, 1H), 8.22 (d, J = 7.8 Hz, 1H), 8.72 (s, 1H), 8.83 (s,
1H), 10.47 (s, 1H), 14.11 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ
(ppm) = 52.6, 53.4, 111.6, 113.1, 115.8, 120.8, 121.3, 121.4, 122.2,
122.9, 123.4, 126.6, 129.6, 132.4, 139.2, 162.2 MS (ESI+): m/z =
306.1. ESI-HR-MS calculated for C18H16N3O2 [M]+: 306.1237.
Found: 306.1239.
2-(4-Chlorophenyl)-5-(methoxycarbonyl)-11-(3,4,5-trime-
thoxybenzyl)-2,11-dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]-
indol-4-ium Chloride (5). Yield, 86% (0.200 g from 0.1 g); white
solid; mp 198−200 °C; Rf = 0.62 (CH2Cl2/MeOH, 9:1, v/v). IR
(neat) νmax: 916, 1023, 1256, 1760, cm−1. 1H NMR (400 MHz,
DMSO-d6): δ (ppm) 3.55 (s, 9H), 4.11 (s, 3H), 6.09 (s, 2H), 7.42 (t, J
= 7.5 Hz, 1H), 7.54 (t, J = 7.4 Hz, 1H), 7.85−7.92 (m, 3H), 8.16 (d, J
= 8.8 Hz, 2H), 8.40 (d, J = 7.8 Hz, 1H), 9.09 (s, 1H), 9.60 (d, J = 1.3
Hz, 1H), 10.77 (d, J = 1.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6):
δ (ppm) = 53.7, 56.4, 60.4, 104.9, 112.2, 112.4, 112.7, 116.4, 121.3,
126.1, 127.2, 128.6, 129.5, 130.5, 132.4, 134.2, 135.8, 137.5, 140.5,
153.6, 162.1. MS (ESI+): m/z = 556.1. ESI-HR-MS calculated for
C31H27ClN3O5 [M]+: 556.1634. Found: 556.1631.
Biological Assays. Chemicals and Antibodies. The chemical
compounds were synthesized within CSIR-CDRI. The annexin V-
conjugated Alexa Fluor 488 apoptosis assay kit and JC-1 mitochondrial
membrane potential detection kits were purchased from Invitrogen,
Carlsbad, CA. The primary antibodies against cyclin B1, CDC25C,
and ECL substrate were purchased from Millipore, Billerica, MA.
CDK1 primary antibody was from BD Biosciences. Bax, Bcl-2, Bcl-XL,
cleaved caspase-3, cleaved caspase-9, PARP, phospho-ERK1/2, ERK1/
2, phospho-JNK, JNK, phospho-p38, p38, and β-actin were purchased
from Cell Signaling Technology, Danver, MA. The respective HRP-
conjugated secondary antibodies were purchased from Santa Cruz
Biotechnology, Santa Cruz, CA. All other chemicals and culture media
were purchased from Sigma-Aldrich, St. Louis, MO, unless otherwise
specified.
2-(2-(Diethylamino)ethyl)-5-(methoxycarbonyl)-2,11-
dihydroimidazo[1′,5′:1,2]pyrido[3,4-b]indol-4-ium Chloride
(3s). Yield, 74% (0.116 g from 0.1 g); yellow solid, mp 172−174
°C; Rf = 0.36 (CH2Cl2/MeOH, 9:1, v/v). IR (KBr) νmax: 1456, 1569,
1753, 3413 cm−1. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 1.29 (t, J
= 7.2 Hz, 6H), 3.15−3.27 (m, 6H), 4.05 (s, 3H), 5.24 (t, J = 6.3 Hz,
2H), 7.34 (dd, J1 = 7.2 Hz, J2 = 0.6 Hz, 1H), 7.48−7.52 (m, 1H), 7.70
(d, J = 8.2 Hz, 1H), 8.27 (d, J = 7.9 Hz, 1H), 8.87−8.91 (m, 2H),
Cell Culture and Treatment. Human breast cancer MDA-MB-
231, MCF-7, and MDA-MB-468 cells, human lung cancer H1299 cells,
and mouse mammary cancer 4T1 cells were obtained from the
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Article
American Type Culture Collection (ATCC, Manassas, VA). The cells
were cultured in RPMI-1640 medium supplemented with 10% fetal
bovine serum, 100 μg/mL penicillin−streptomycin solutions and
maintained in 5% CO2 at 37 °C. For all treatments, compounds were
dissolved in DMSO at a stock concentration of 10 mM. The
subconfluent cells were treated with different concentrations of
compounds in complete cell growth medium.
then fixed with ice-cold methanol followed by staining in 0.1% crystal
violet. The membranes were removed gently with the help of scalpel
and put upside down on a glass slide, and then images were captured
using Olympus IX51 microscope equipped with a digital camera. The
numbers of invaded cells were counted at least in 10 random fields,
and results were expressed as an average number of invaded/migrated
cells compared with control.
Cell Proliferation Assay. MTT-based cell proliferation assay was
performed as described previously.21 Briefly, cells were seeded in 96-
well plates at a density of approximately 5000 cells/well and grown for
24 h. The growth medium was then replaced with fresh medium
containing the compounds to be tested at different concentrations.
The cells were incubated for 24, 48, and 72 h in a humidified CO2
incubator. After incubation, 10 μL of MTT solution (5 mg/mL) was
added to each well, and the cells were incubated for another 2 h until
purple-colored formazan crystals formed. The MTT−formazan
crystals were dissolved in 200 μL of DMSO, and then the absorbance
was measured at 595 nm using iMARK spectrophotometer (Biorad,
Hercules, CA). Data were represented as percent cell proliferation
compared with DMSO control.
Cellular Apoptosis Analysis by Flow Cytometry. Approx-
imately 2 × 105 human breast cancer MDA-MB-231 cells were seeded
in six-well plates in triplicate. The cells were treated with different
concentrations of compounds for 24 h. Cellular apoptosis was
determined by annexin V-PI staining using Alexa Fluor 488 apoptosis
detection kit (Invitrogen, Carlsbad, CA) following the manufacturer’s
instructions as described earlier.21 The results were expressed as a
cumulative percentage of cells in early and late apoptotic phases
compared with control.
Mitochondrial Membrane Potential Assay. MDA-MB-231 cells
were seeded in 60 mm dishes. After incubation for 24 h, cells were
treated with different concentrations of compounds for 24 h.
Mitochondrial membrane depolarization was determined by using
JC-1 detection kit (Invitrogen) following the manufacturer’s protocol
as described previously.31 The JC-1 stained cells were then subjected
to flow-cytometric analysis using FACS Calibur instrument.
Cell-Cycle Analysis. Cell cycle analysis was performed as
described previously.31 Briefly, approximately 2 × 105 cells were
seeded in six-well plates in triplicate. Cells were serum-starved for 12 h
and treated with different concentrations of compounds for 24 h. Cells
were then harvested, fixed with ice-cold absolute methanol, and stained
with 4 μg/mL PI and 0.1 mg/mL RNase A in PBS. Samples were
incubated for 30 min in the dark at room temperature and then
subjected to flow-cytometric analysis using FACS Calibur instrument.
Data were analyzed using the ModFit LT 3.2.1 software.
Wound Healing Assay. The migration capacity of highly
metastatic human breast cancer MDA-MB-231 cells was assessed by
in vitro wound healing assay as described previously.21 Briefly, 2 × 105
cells were plated in six-well plates and a wound was created by
scraping the cells with a sterile 200 μL pipet tip in the middle of the
culture well. After removal of cellular debris, the cells were treated with
different concentrations of test compounds. Images of the wound area
were captured at 0, 12, and 24 h of post-treatments. Wound area was
measured by ImageJ software, and data were expressed as percent
wound closure compared with control.
Transwell Invasion and Migration Assays. The capacity of
human breast cancer MDA-MB-231 cells to pass through a
polycarbonate membrane (6.5 mm diameter; 8 μm pore size) was
measured using transwell chambers (Corning Life Sciences) in 24-well
tissue culture plates as described earlier.21 For transwell invasion assay,
growth factor reduced Matrigel was diluted in serum free culture
medium and spread on the top of the membrane, and then the plate
was kept in a CO2 incubator at 37 °C for 2 h. To assess the transwell
migration capacity of cells, uncoated chambers were used. Cells were
counted and seeded (30 000 cells/well for invasion assay and 20 000
cells/well for migration assay) in RPMI-1640 medium with 1% FBS
containing treatments, whereas 10% FBS in medium was applied to
the lower chamber as a chemoattractant. After incubation for 48 h,
noninvaded or nonmigrated cells on upper surface of the membrane
were carefully removed with a cotton swab; the membrane insert was
Determination of the Effect of Compounds on MMP-2 and
MMP-9 Activities by Gelatin Zymography. MDA-MB-231 cells
were treated with test compounds at different concentrations. The
conditioned medium from treated and untreated cells was centrifuged,
and the supernatant was collected. After determination of the protein
concentration in conditioned media, an equal amount of protein was
loaded on 8% SDS−PAGE with 0.1% gelatin added in the separating
gel as described previously.21 Electrophoresis was carried out at
constant voltage of 80 V until the tracking dye reached the periphery.
The gels were washed twice for 40 min in wash buffer (10 mM Tris
(pH 8.0) containing 2.5% (v/v) Triton X-100) to remove residual
SDS and incubated with developing buffer (50 mM Tris-HCl, pH 7.5,
0.2 M NaCl, 10 mM CaCl2, and 1 mM ZnCl2) for 20 h at 37 °C. Gels
were stained using 0.5% Coomassie blue R-250 in 5% (v/v) methanol
and 10% (v/v) acetic acid for 1 h and destained in 10% (v/v)
methanol, 5% (v/v) acetic acid until the bands are visualized against
the dark background. The gels were scanned on Image Quant
LAS4010 detection system.
Western Blot Analysis. MDA-MB-231 cells were treated with
different concentrations of test compounds. After 24 h of treatment,
cell lysates were prepared and protein was extracted using RIPA lysis
buffer (Millipore) as described earlier.21 Protein was quantified, and 40
μg of protein was separated on 10−15% SDS−PAGE and transferred
to PVDF membrane. After blocking with 5% non fat dry milk,
membrane was incubated with the primary antibodies specific for
cyclin B, CDC25C, CDK1 (Millipore, Billerica, MA), Bax, Bcl-2, Bcl-
XL, survivin, cleaved caspase-3, cleaved caspase-9, PARP, phospho-
ERK1/2, ERK1/2, phospho-JNK, JNK, phospho-p38, p38, and β-actin
(Cell Signaling Technology, Danver, MA) at 4 °C overnight. After
primary antibody incubation, membrane was washed thrice in TBS
containing 0.1% Tween-20 for 10 min each and then incubated with
appropriate HRP-conjugated secondary antibody. The immunoreac-
tive protein bands were visualized by enhanced chemiluminescence
(ECL) on Image Quant LAS4010 chemilumenescent detection
system.
Statistical Analysis. The differences between control and
compounds-treated samples were determined by one-way ANOVA
with Dunnet’s post hoc test using GraphPad Prism, version 3.00, for
Windows. GraphPad software data were the mean of two or three
independent experiments performed in triplicate SEM, (∗) p < 0.05,
(∗∗) p < 0.01, and (∗∗∗) p < 0.001.
In Vivo. 4T1-Syngeneic Mammary Tumor Model. All studies
were conducted under approved protocol by the Institutional Animal
Ethics Committee (IAEC) of CSIR-CDRI. The experiment was
performed as described previously.21 Briefly, 4T1 cells (7 × 105) were
injected in the second right mammary fat pad of 8-week-old female
Balb/c mice. After 4 days, approximately 5 × 105 cells were injected as
booster dose. After 1 week of booster dose, compounds were
administered orally at a single dose of 10 mg/kg body weight on
alternate days for 5 weeks (n = 6). Control group animals were
administered with vehicles. The body weight and tumor size were
measured twice a week. The tumor volume was calculated using the
formula (LW2) × 0.5, where L is the tumor length and W is the tumor
width.
ASSOCIATED CONTENT
■
S
* Supporting Information
Figures concerning in vitro and in vivo assays and spectroscopic
1
investigations of HSA binding with 3h and H and 13C NMR
spectra of all compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
M
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Journal of Medicinal Chemistry
Article
AUTHOR INFORMATION
REFERENCES
■
■
(1) (a) Cao, R.; Peng, W.; Wang, Z.; Xu, A. β-Carboline alkaloids:
biochemical and pharmacological functions. Curr. Med. Chem. 2007,
14, 479−500. (b) Huang, H.; Yao, Y.; He, Z.; Yang, T.; Ma, J.; Tian,
X.; Li, Y.; Huang, C.; Chen, X.; Li, W.; Zhang, S.; Zhang, C.; Ju, J.
Antimalarial β-carboline and indolactam alkaloids from marinactino-
spora thermotolerans, a deep sea isolate. J. Nat. Prod. 2011, 74, 2122−
2127. (c) Kuo, P.-C.; Shi, L.-S.; Damu, A. G.; Su, C.-R.; Huang, C.-H.;
Ke, C.-H.; Wu, J.-B.; Lin, A.-J.; Bastow, K. F.; Lee, K.-H.; Wu, T.-S.
Cytotoxic and antimalarial β-carboline alkaloids from the roots of
Eurycoma longifolia. J. Nat. Prod. 2003, 66, 1324−1327. (d) Ang, K. K.
H.; Holmes, M. J.; HIGA, T.; Hamann, M. T.; Kara, U. A. K. In vivo
antimalarial activity of the β-carboline alkaloid manzamine A.
Antimicrob. Agents Chemother. 2000, 44, 1645−1649. (e) Brahmbhatt,
K. G.; Ahmed, N.; Sabde, S.; Mitra, D.; Singh, I. P.; Bhutani, K. K.
Synthesis and evaluation of β-carboline derivatives as inhibitors of
human immunodeficiency virus. Bioorg. Med. Chem. Lett. 2010, 20,
4416−4419. (f) Wang, Y.-H.; Tang, J.-G.; Wang, R.-R.; Yang, L.-M.;
Dong, Z.-J.; Du, L.; Shen, X.; Liu, J.-K.; Zheng, Y.-T. Flazinamide, a
novel β-carboline compound with anti-HIV actions. Biochem. Biophys.
Res. Commun. 2007, 355, 1091−1095. (g) Liu, Y.; Song, H.; Huang, Y.;
Li, J.; Zhao, S.; Song, Y.; Yang, P.; Xiao, Z.; Liu, Y.; Li, Y.; Shang, H.;
Wang, Q. Design, synthesis, and antiviral, fungicidal, and insecticidal
activities of tetrahydro-β-carboline-3-carbohydrazide derivatives. J.
Agric. Food Chem. 2014, 62, 9987−9999. (h) Meurer, L. C.;
Guthikonda, R. N.; Huber, J. L.; DiNinno, F. The synthesis and
antibacterial activity of 2-carbolinyl-carbapenems: potent anti-MRSA/
MRCNS agents. Bioorg. Med. Chem. Lett. 1995, 5, 767−772.
Corresponding Authors
*P.S.: e-mail, psen@iitk.ac.in; phone, +91-512-2596312.
*S.M.M.: e-mail, s.musthapa@cdri.res.in; phone, +91-522-
2772450, Ext-4491.
*S.B.: e-mail, batra_san@yahoo.co.uk; phone, +91-522-
2772450, Ext 4727.
Author Contributions
⊥S.U.D. and S.K. contributed equally.
S.U.D. and S.B. designed the synthesis, whereas S.U.D. and
P.J. synthesized the compounds. S.K. and S.M.M. designed the
in vitro and in vivo experiments, whereas S.K., I.S., and S.S.
performed the biological experiments. R.Y. and P.S. designed
and performed the spectroscopic studies to investigate the HSA
binding. The manuscript was written through contributions of
all authors. All authors have given approval to the final version
of the manuscript.
Notes
The authors declare no competing financial interest.
#P.J. from Department of Chemistry, Malaviya National
Institute of Technology, Jaipur, Rajasthan, India worked as
National Science Academy Summer Research Trainee at
Medicinal and Process Chemistry Division, CDRI between
May 7 and July 2, 2012.
This is CDRI Communication No. 8953.
(2) (a) Simmon, T. L.; Andrianasolo, E.; McPhail, K.; Flatt, P.;
Gerwick, W. H. Marine natural products as anticancer drugs. Mol.
Cancer Ther. 2005, 4, 333−342. (b) Zhang, P.; Sun, X.; Xu, B.; Bijjan,
K.; Wan, S.; Li, G.; Alaoui-Jamali, M.; Jiang, T. Total synthesis and
biological activity of marine alkaloid eudistomins Y1−Y7 and their
analogues. Eur. J. Med. Chem. 2011, 46, 6089−6097. (c) Bharate, S. B.;
Manda, S.; Mupparapu, N.; Battini, N.; Vishwakarma, R. A. Chemistry
and biology of fascaplysin, a potent marine-derived CDK−4 inhibitor.
Mini-Rev. Med. Chem. 2012, 12, 650−664.
ACKNOWLEDGMENTS
■
S.U.D., S.K., and S.S. acknowledge CSIR, New Delhi, India,
whereas R.Y. and I.S. acknowledge UGC, New Delhi, India, and
ICMR, New Delhi, India, for the financial support in the form
of fellowships. P.J. acknowledges the National Science
Academy, Bangalore, India, for financial assistance in the
form of Summer Research Fellowship. S.B., S.M.M., and P.S.
acknowledge SERB-DST for financial grant in the form of
project. Authors acknowledge SAIF Division of CDRI for
providing the spectroscopic data.
́ ́
(3) (a) Frederick, R.; Bruyere, C.; Vancraeynest, C.; Reniers, J.;
Meinguet, C.; Pochet, L.; Backlund, A.; Masereel, B.; Kiss, R.;
Wouters, J. Novel trisubstituted harmine derivatives with original in
vitro anticancer activity. J. Med. Chem. 2012, 55, 6489−6501.
(b) Zheng, C.; Fang, Y.; Tong, W.; Li, G.; Wu, H.; Zhou, W.; Lin,
Q.; Yang, F.; Yang, Z.; Wang, P.; Peng, Y.; Pang, X.; Yi, Z.; Luo, J.; Liu,
M.; Chen, Y. Synthesis and biological evaluation of novel tetrahydro-β-
carboline derivatives as antitumor growth and metastasis agents
through inhibiting the transforming growth factor-β signaling pathway.
J. Med. Chem. 2014, 57, 600−612. (c) Shen, L.; Park, E.-J.;
Kondratyuk, T. P.; Guendisch, D.; Marler, L.; Pezzuto, J. M.;
Wright, A. D.; Sun, D. Design, synthesis, and biological evaluation
of callophycin A and analogues as potential chemopreventive and
anticancer agents. Bioorg. Med. Chem. 2011, 19, 6182−6195. (d) Cao,
R.; Fan, W.; Guo, L.; Ma, Q.; Zhang, G.; Li, J.; Chen, X.; Ren, Z.; Qui,
L. Synthesis and structure−activity relationships of harmine derivatives
as potential antitumor agents. Eur. J. Med. Chem. 2013, 60, 135−143.
(e) Chauhan, S. S.; Singh, A. K.; Meena, S.; Lohani, M.; Singh, A.;
Arya, R. K.; Cheruvu, S. H.; Sarkar, J.; Gayen, J. R.; Datta, D.;
Chauhan, P. M. S. Synthesis of novel β-carboline based chalcones with
high cytotoxic activity against breast cancer cells. Bioorg. Med. Chem.
Lett. 2014, 24, 2820−2824.
ABBREVIATIONS USED
■
MAP, mitogen-activated protein; MMP, matrix metalloprotei-
nase; HIV, human immunodeficiency virus; CDK, cyclin-
dependent kinase; IkK, inhibitor of nuclear factor κ kinase;
SEM, standard error of mean; Bax, Bcl-2-associated X protein;
Bcl-2, B-cell lymphoma 2; Bcl-XL, B-cell lymphoma-extra-large;
HSA, human serum albumin; ECM, extracellular matrix; ERK,
extracellular signal-regulated kinase; MAPK, mitogen-activated
protein kinase; DMSO, dimethyl sulfoxide; SAPK, stress-
activated protein kinase; JNK, c-Jun N-terminal kinase; PDB,
Protein Data Bank; FTIR, Fourier transform infrared spectros-
copy; kDa, kilodalton; HPLC, high-performance liquid
chromatography; HRMS, high-resolution mass spectrometry;
IC50, half-maximal inhibitory concentration; ESI-HRMS,
electrospray ionization high resolution mass spectrometry;
LC−MS/MS, liquid chromatography−tandem mass spectrom-
etry; m/z, mass-to-charge ratio; ESI-MS, electrospray ionization
mass spectrometry; CH3CN, acetonitrile; EtOH, ethanol; HCl,
hydrochloric acid; AcCl, acetyl chloride; CaCl2, calcium
chloride; ZnCl2, zinc chloride; MS, mass spectrometry; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
NMR, nuclear magnetic resonance; IR, infrared spectroscopy;
TLC, thin-layer chromatography
(4) Guan, H.; Chen, H.; Peng, W.; Ma, Y.; Cao, R.; Liu, X.; Xu, A.
Design of β-carboline derivatives as DNA-targeting antitumor agents.
Eur. J. Med. Chem. 2006, 41, 1167−1179.
(5) Funayama, Y.; Nishio, K.; Wakabayashi, K.; Nagao, M.; Shimoi,
K.; Ohira, T.; Hasegawa, S.; Saijo, N. Effects of beta- and gamma-
carboline derivatives of DNA topoisomerase activities. Mutat. Res.
1996, 349, 183−191.
(6) Song, Y.; Kesuma, D.; Wang, J.; Deng, Y.; Duan, J.; Wang, J. H.;
Qi, R. Z. Specific inhibition of cyclin-dependent kinases and cell
N
DOI: 10.1021/acs.jmedchem.5b00016
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
proliferation by harmine. Biochem. Biophys. Res. Commun. 2004, 317,
128−132.
(7) Castro, A. C.; Dang, L. C.; Soucy, F.; Grenier, L.; Mazdiyasni, H.;
Hottelet, M.; Parent, L.; Pien, C.; Palombella, V.; Adams, J. Novel IKK
inhibitors: beta-carbolines. Bioorg. Med. Chem. Lett. 2003, 13, 2419−
2422.
(8) Chaniyara, R.; Tala, S. K.; Chen, C.-W.; Zang, X.; Kakadiya, R.;
Lin, L.-F.; Chen, C.-H.; Chien, S.-I.; Chou, T.-C.; Tsai, T.-H.; Lee, T.-
C.; Shah, A.; Su, T.-L. Novel antitumor indolizino[6,7-b]indoles with
multiple modes of action: DNA cross-linking and topoisomerase I and
II inhibition. J. Med. Chem. 2013, 56, 1544−1563.
integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2).
Oncogene 2010, 29, 3100−3109.
(25) (a) Kragh-Hansen, U. Molecular aspects of ligand binding to
serum albumin. Pharmacol. Rev. 1981, 33, 17−53. (b) Trainor, G. L.
The importance of plasma protein binding in drug discovery. Expert
Opin. Drug Discovery 2007, 2, 51−64. (c) Zhang, F.; Xue, J.; Shao, J.;
Jia, L. Compilation of 222 drugs’ plasma protein binding data and
guidance for study designs. Drug Discovery Today 2012, 17, 475−485.
(d) Yang, F.; Zhang, Y.; Liang, H. Interactive association of drugs
binding to human serum albumin. Int. J. Mol. Sci. 2014, 15, 3580−
3595.
(26) (a) He, X. M.; Carter, D. C. Atomic structure and chemistry of
human serum albumin. Nature 1992, 358, 209−215. (b) Dugiaczyk,
A.; Law, S.; Dennison, O. E. Nucleotide sequence and the encoded
amino acids of human serum albumin mRNA. Proc. Natl. Acad. Sci.
U.S.A. 1982, 79, 71−75. (c) Curry, S.; Mandelkow, H.; Brick, P.;
Franks, N. Crystal structure of human serum albumin complexed with
fatty acid reveals an asymmetric distribution of binding sites. Nat.
Struct. Biol. 1998, 5, 827−835. (d) Sugio, S.; Kashima, A.; Mochizuki,
S.; Noda, M.; Kobayashi, K. Crystal structure of human serum albumin
at 2.5 Å resolution. Protein Eng. 1999, 12, 439−446.
(27) (a) Anand, U.; Jash, C.; Mukherjee, S. Spectroscopic probing of
the microenvironment in a protein-surfactant assembly. J. Phys. Chem.
B 2010, 114, 15839−15845. (b) Lakowicz, J. R. Principles of
Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006.
(28) Petitpas, I.; Bhattacharya, A. A.; Twine, S.; East, M.; Curry, S.
Crystal structure analysis of warfarin binding to human serum albumin
anatomy of drug site I. J. Biol. Chem. 2001, 276, 22804−22809.
(29) (a) Ross, P. D.; Subramanian, S. Thermodynamics of protein
association reactions: forces contributing to stability. Biochemistry
1981, 20, 3096−3102. (b) Rahman, M. H.; Maruyama, T.; Okada, T.;
Yamasaki, K.; Otagiri, M. Study of interaction of carprofen and its
enantiomers with human serum albumin-I. Mechanism of binding
studied by dialysis and spectroscopic methods. Biochem. Pharmacol.
1993, 46, 1721−1731.
(9) (a) Aher, S. B.; Muskawar, P. N.; Thenmozhi, K.; Bhagat, P. R.
Recent developments of metal N-heterocyclic carbenes as anticancer
agents. Eur. J. Med. Chem. 2014, 81, 408−419. (b) Hindi, K. M.;
Panzner, M. J.; Tessier, C. A.; Cannon, C. L.; Youngs, W. J. The
medicinal applications of imidazolium carbene-metal complexes. Chem.
Rev. 2009, 109, 3859−3884. (c) Hickey, J. L.; Ruhayel, R. A.; Barnard,
P. J.; Baker, M. V.; Berners-Price, S. J.; Filipovska, A. Mitochondria-
targeted chemotherapeutics: the rational design of gold(I) N-
heterocyclic carbene complexes that are selectively toxic to cancer
cells and target protein selenols in preference to thiols. J. Am. Chem.
Soc. 2008, 130, 12570−12571.
(10) Hutt, J. T.; Aron, Z. D. Efficient, single-step access to
imidazo[1,5-a]pyridine N-heterocyclic carbene precursors. Org. Lett.
2011, 13, 5256−5259.
(11) Singh, V.; Hutait, S.; Batra, S. Baylis−Hillman reaction of 1-
formyl-β-carboline: one-step synthesis of the canthin-6-one framework
via an unprecedented cascade cyclization. Eur. J. Org. Chem. 2009,
6211−6216.
(12) Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutelingsperger,
C. A novel assay for apoptosis. Flow cytometric detection of
phosphatidylserine expression on early apoptotic cells using
fluorescein labelled annexin V. J. Immunol. Methods 1995, 184, 39−51.
(13) Debatin, K. M. Apoptosis pathways in cancer and cancer
therapy. Cancer Immunol. Immunother. 2004, 53, 153−159.
(14) Thornberry, N. A. Caspases: key mediators of apoptosis. Chem.
Biol. 1998, 5, R97−R103.
(30) Woody, R. W. Theory of circular dichroism of proteins. In
Circular Dichroism and Conformational Analysis of Biomolecules;
Fasman, G. D., Ed.; Plenum Publishing Corp.: New York, 1996; p 25.
(31) Meeran, S. M.; Katiyar, S. K. Grape seed proanthocyanidins
promote apoptosis in human epidermoid carcinoma A431 cells
through alterations in Cdki-Cdk-cyclin cascade, and caspase-3
activation via loss of mitochondrial membrane potential. Exp Dermatol.
2007, 16, 405−415.
(15) Deveraux, Q. L.; Reed, J. C. IAP family proteinssuppressors of
apoptosis. Genes Dev. 1999, 13, 239−252.
(16) Soldani, C.; Scovassi, A. I. Poly(ADP-ribose) polymerase-1
cleavage during apoptosis: an update. Apoptosis 2002, 7, 321−328.
(17) Choi, J. A.; Kim, J. Y.; Lee, J. Y.; Kang, C. M.; Kwon, H. J.; Yoo,
Y. D.; Kim, T. W.; Lee, Y. S.; Lee, S. J. Induction of cell cycle arrest
and apoptosis in human breast cancer cells by quercetin. Int. J. Oncol.
2001, 19, 837−844.
(18) Kastan, M. B.; Bartek, J. Cell-cycle checkpoints and cancer.
Nature 2004, 432, 316−323.
(19) Boutros, R.; Lobjois, V.; Ducommun, B. CDC25 phosphatases
in cancer cells: key players? Good targets? Nat. Rev. Cancer 2007, 7,
495−507.
(20) Gotzmann, J.; Mikula, M.; Eger, A.; Schulte-Hermann, R.;
Foisner, R.; Beug, H.; Mikulits, W. Molecular aspects of epithelial cell
plasticity: implications for local tumor invasion and metastasis. Mutat.
Res. 2004, 566, 9−20.
(21) Khan, S.; Shukla, S.; Sinha, S.; Lakra, A. D.; Bora, H. K.; Meeran,
S. M. Centchroman suppresses breast cancer metastasis by reversing
epithelial-mesenchymal transition via downregulation of HER2/
ERK1/2/MMP-9 signalling. Int. J. Biochem. Cell Biol. 2015, 58, 1−16.
(22) Salo, T.; Makela, M.; Kylmaniemi, M.; Autio-Harmainen, H.;
̈
̈
̈
Larjava, H. Expression of matrix metalloproteinase-2 and -9 during
early human wound healing. Lab. Invest. 1994, 70, 176−182.
(23) Krueger, J. S.; Keshamouni, V. G.; Atanaskova, N.; Reddy, K. B.
Temporal and quantitative regulation of mitogen-activated protein
kinase (MAPK) modulates cell motility and invasion. Oncogene 2001,
20, 4209−4218.
(24) Aziz, M. H.; Hafeez, B. B.; Sand, J. M.; Pierce, D. B.; Aziz, S. W.;
Dreckschmidt, N. E.; Verma, A. K. Protein kinase Cvarepsilon
mediates Stat3Ser727 phosphorylation, Stat3-regulated gene expres-
sion, and cell invasion in various human cancer cell lines through
O
DOI: 10.1021/acs.jmedchem.5b00016
J. Med. Chem. XXXX, XXX, XXX−XXX