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Synthesis of #-carboline-based N-heterocyclic carbenes and their antiproliferative and anti-metastatic activities against human breast cancer cells Shashikant Uttam Dighe, Sajid Khan, Isha Soni, Preeti Jain, Samriddhi Shukla, Rajeev Yadav, Pratik Sen, Syed Musthapa Meeran, and Sanjay Batra J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Apr 2015 Downloaded from http://pubs.acs.org on April 3, 2015

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Synthesis of β-carboline-based N-heterocyclic carbenes and their anti-proliferative and antimetastatic activities against human breast cancer cells Shashikant U. Dighe,a, ‡ Sajid Khan,b, ‡ Isha Soni,b Preeti Jain,a,# Samriddhi Shukla,b Rajeev Yadav,c Pratik Sen,*,c Syed M. Meeran, *,b,d and Sanjay Batra*,a,d a

Medicinal and Process Chemistry Division and bEndocrinology Division, CSIR-Central Drug

Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow-226031, India; c

Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur–208016, India;

d

Academy of Scientific and Innovative Research, New Delhi, India

KEYWORDS. β-Carboline • N-heterocyclic carbene • anticancer • breast cancer • HSA binding.

ABSTRACT. 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 anti-proliferative 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

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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 anti-migratory and anti-invasive effects against highly metastatic human breast cancer MDA-MB-231 cells via aberration of MAP-kinase signalling 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.

Introduction Naturally occurring β-carboline alkaloids and synthetic analogues containing β-carboline subunit are endowed with diverse pharmacological properties including antimalarial, anti-HIV, antibacterial, antitumor and anticancer activities.1 Amongst these derivatives, the naturally occurring β-carboline alkaloids such as harmicine, fascaplysin and callophycin A (Fig. 1) are reported to display anti-proliferative effect against many cancer cell lines.2 In addition, several synthetic analogues (I-II, Fig. 1) structurally related to these alkaloids or containing β-carbolinecore are also reported to display potent anticancer property.3 It is widely reported that βcarboline derivatives elicit 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 work, Lee’s and Su’s groups disclosed the antitumor activity of bis(hydroxymethyl)indolizino[6,7-b]indoles (II).8 These 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-crosslinking 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

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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 βcarboline and imidazole-based NHC. Screening of this series of compounds led to identification of new β-carboline-based anti-proliferative and -metastatic agents against human breast cancer cells. The details of synthesis and biological activity of these compounds are disclosed herein.

Figure 1. Natural and synthetic β-carboline derivatives with anti-tumor and anti-cancer properties. 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 into 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 formaldehyde 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%

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Scheme1. Synthesis of β-carboline-based aryl. alkyl and cycloalkyl substituted N-heterocyclic carbenes. CO2Me

CO2Me NH2 N H Tryptophan

Ref. 11

N N H MeO 1

+ OMe

CO2Me AcCl, HCHO R-NH2 a-v

EtOH, rt, 12 h

N Cl N H

N 3a-v

AcOH, reflux, 5 h CO2Me

R

PhNH2 (a), AcCl, HCHO EtOH, rt, 12 h

N N H 2

CHO

yield. The spectroscopic analysis of this product revealed it to be the required 2-phenyl-2,11dihydroimidazo[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)-9Hpyrido[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 product (3b-v) were isolated in each case as chloride salt in good yields (Table 1). It was noticed that as compared to aryl amines, the aliphatic amines furnished the product in relatively lower yields (compare entries 18-20 with 117, 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-

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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 2. Plausible mechanism for the formation of N-heterocyclic carbene

Scheme 3. Synthesis of product from N-protected starting β-carboline.

Table 1. Yields of the isolated products and their in vitro anti-proliferative effects on different human cancer cell lines a

entry

b

amine

product 2 (yield %)

1

IC50 (µM) MDA-MB-231

MCF-7

MDA-MB-468

H1299

>30

>30

5.82±0.02

8.87±1.6

9.38 ±0.28

25.7±0.59

10.21±0.05

15.43±0.12

a

3a (92) 2

b

3b(94)

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7.98±0.47

14.16±0.86

11.11±1.71

4.48±1.24

7.80±1.9

5.96±0.44

8.02±0.52

6.46±0.8

20.73±3.2

>30

10.96±0.62

>30

17.86± 1.22

>30

15.12±0.53

26.95±2.4

14.55± 1.4

>30

10.54±0.24

10.92±0.81

4.49±0.76

6.80±0.49

5.94±0.53

5.13±0.34

10.82±1.15

13.26±0.23

9.51±0.59

16.42±1.2

c

3c(98)

4 d

3d(88)

CO2Me

5

N e

N H

Cl Br N

3e(90)

6

f

3f(96)

7

g 3g(95)

8

h

3h(85) 9

i

3i(92)

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10

7.28±0.24

6.9±0.57

5.78±0.62

7.80±1.73

23.38±1.6

>30

20.25±1.4

>30

13.96±1.23

>30

>30

>30

29.91±0.08

>30

11.54±0.66

16.44±1.6

11.78±0.13

6.26±1.6

9.27±1.03

23.73±0.24

12.82± 1.6

>30

>30

>30

11.19±1.0

>30

>30

>30

j

3j(90)

11

k

3k(93) 12

l

3l(94)

13

m

3m(83) 14

n 3n(89)

15

o 3o(91)

16

p 3p(94)

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>30

>30

>30

>30

22.98±1.02

>30

>30

>30

>30

>30

>30

>30

>30

>30

>30

>30

>30

>30

18.18±2.3

>30

>30

>30

>30

>30

>30

>30

>30

>30

q

3q(90)

18 r

3r(77)

19 s

3s(74) 20 t

3t(68) 21 u

3u(80)

22 v

3v(64) 23

b

5(86)

a

All reactions were performed with 0.1 g of 1, 1.5 equiv of HCHO and 1.2 equiv of amine (av). bYields of 3a-v, 5 are isolated yields after column chromatography.

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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 significant anti-proliferative activity with IC50 values less than 10 µM in breast as well as lung cancer cells (Table 1, also see Fig. S-1, SI). 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 Fig. S-1, SI). Considering their potent anti-proliferative activity against metastatic breast cancer, we further considered in evaluating the mode of actions of these compounds in MDAMB-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 Fig. 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 (Fig. 2B & C).

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These results suggest that compounds 3c, 3j and 3h induced apoptosis in MDA-MB-231 cells through activation of intrinsic apoptotic pathway.

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 and the upper right quadrant is showing the late apoptotic cells. B) FACS histograms are showing assessment of mitochondrial membrane potential (∆ψmt) after treatment of MDA-MB-231 cells with compounds 3c, 3j and 3h for 24 h. Lower right quadrant, the percentage of cells that emit only green fluorescence can be attributed to depolarized ∆ψmt. C) Bar graphs depicting reduction in red to green fluorescence intensity ratio indicative of

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reduction in ∆ψmt, 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. 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, 3h and 3j increased the expression of pro-apoptotic 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 after 24 h of treatment (Fig. 3). The pro-apoptotic proteins promote the release of cytochrome C, whereas anti-apoptotic proteins antagonize the actions of pro-apoptotic proteins and prevent the loss of mitochondrial membrane potential.13 In agreement with these observations, we found that anti-apoptotic 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 execution of cellular apoptosis.14 As shown in Fig. 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 apoptosis protein (IAP) family which functions through direct interactions to inhibit the activity of several caspases including caspase3, 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 MDAMB-231 cells, which is a characteristic feature of cells undergoing apoptosis (Fig. 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.

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Figure 3. Treatment with compounds 3c, 3j and 3h alter 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. 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 (Fig. 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

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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 (Fig. 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 anti-proliferative effect.

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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. βactin was used as an equal loading control. The blots are representative of three independent experiments. 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 Fig. 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 (Fig. 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 (Fig. 5D). Thus, these compounds significantly inhibited cell migration and invasion at concentrations where no significant anti-proliferative and pro-apoptotic effects were observed. This indicates that the inhibition of cell migration and invasion was not due to anti-proliferative and/or pro-apoptotic effects of these compounds.

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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 (Fig. 6A). MAPK pathway plays an important role in the activation of ECM-degrading MMPs leading to enhanced cell invasion and

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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 of 3c, 3j and 0, 1, 2.5, 5 µM of 3h. At 0, 12, and 24 h after wounding, the wound areas were photographed using a phase contrast microscope under 100X 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-MB231cells. For invasion assay, matrigel was coated in transwell chambers. The migrated cells (in panel C) and invaded cells (in panel D) were stained with crystal violet and photographed under 100X magnification. The images are representative of three independent experiments. 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 are 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 Fig. 6B, compounds 3c, 3j and 3h suppressed the phosphorylation of ERK1/2 and SAPK/JNK MAPKs, whereas 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

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migration and invasion. In contrast, treatment with 3c, 3j and 3h lead to the upregulation of cell death inducing pathway i.e. p38 MAPK in MDA-MB-231 breast cancer cells.

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 6 well plates at a density of 1×105 cells/well and then treated with 0, 1, 2.5, 5, 7.5 µM of compounds 3c, 3j and 0, 1, 2.5, 5 µM of 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

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MAPK. β-actin antibody was used as an equal loading control. The blots are representative of three independent experiments. 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 compounds 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 (Fig. S-2, SI). Further, tumor volume graph indicated that compound 3c resulted in about 20% decrease in the tumor volume compared with control, whereas compounds 3j and 3h had no significant effect on tumor growth in vivo (Fig. S-2, SI). 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 sub domains (A and B).26 The absorption spectrum of 3h in 0.05 M phosphate buffer (pH 7.4) displayed three peaks at 292 nm, 320 nm and 371 nm (see Fig. S-3, SI) 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 3h (Fig. S-4, SI). 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

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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 µM to 50 µM, the fluorescence intensity of Trp inside HSA (10 µM) decreases continuously without any change in the emission maximum (Fig. S-5, SI), 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 K, 293 K and 303 K (Fig. S-5, SI). 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 Stern-Volmer equation as fellows.27b

 

=

 

= 1 +  = 1 + 

(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 absence of quencher. The change in F0/F value with concentration of 3h was determined (Fig. S-6, SI) and by fitting these data points using equation 1, Stern–Volmer quenching constant were calculated for all the three temperatures (Table S-1, SI). 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 absence and presence of different concentrations of 3h (see Fig. S-7, SI). The observed lifetime at each concentration of 3h was analyzed in terms of Stern–Volmer equation. The change in the value of τ0/τ with the

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concentration of 3h as shown in Fig. 7 was observed to be 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 non-fluorescent 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, SI). 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 in 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

   / =  + 

(2)

where, K, n and Q stands for the binding constant, number of 3h molecules that bind to HSA and concentration of 3h, respectively.

Figure 7. Stern-Volmer plot for fluorescence of HSA by 3h. Red line denotes the best fit for equation 1 and blue line is just eye guide. The change in    / with  and the values of Kb and n were estimated from the intercept and slope of the best leaner fit as shown in Fig. 8. At 283 K, the estimated value of

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the binding constant is 9.77 x 104 L mol-1, which decreases to 8.91 x 104 L mol-1 and 7.94 x 104 L mol-1 at 293 K 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 ID 1HA2.28 Based on the results of the docking studies it was inferred that 3h binds to domain I of HSA.

Figure 8. A plot of log((F0-F)/F)against log[2h] at three different temperatures, 283K (red circle), 293K (blue circle),303K (green circle).The solid lines shows the best fit at each temperature by using equation 2. To gain insight into the interaction behavior, thermodynamic parameters of binding between 3h and HSA were estimated. Free energy (∆G0binding), entropy (∆S0) and enthalpy (∆H0) of binding are the main parameters that play an important role for binding and can be estimated by

∆ = 2.303 &' 

(3)

= ∆(  '∆)

∆

(4)

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where R is universal gas constant. The values of ∆G0binding were calculated as -11.74 kJ/mole, 12.06 kJ/mole and -12.34 kJ/mole at 283 K, 293 K and 303 K respectively. The negative values of ∆G0binding 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 (Fig. S-8 and Table S-3, SI). It is worth mentioning that the positive value of ∆S0 and a very small value of ∆H0, either positive or negative are responsible for electrostatic interactions in the binding process.29 Thus the observed values of ∆H0 and ∆S0 as -3.21 JK/mole and 30.15 kJ/mole, respectively inferred that electrostatic interactions played a major role in the binding of 3h with HSA. Since domain I contain a lots 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 nm and 222 nm which is the signature of α-helicity (Fig. S-9, SI).30 Experimental data was expressed in terms % α-helix of HSA and is shown in Fig. 9. These observations delineated that the secondary structure of HSA remains unchanged till 40 µM of 3h.

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Figure 9. The change in % α-helicity of HSA with 3h concentration. Conclusions In conclusion, we have disclosed an efficient synthesis and anti-proliferative activity against breast cancer cells of a new series of fused β-carbolines which 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 anti-proliferative and pro-apoptotic effects in breast cancer cells. Further, these compounds were found to suppress migration and invasion capacities of highly metastatic human breast cancer MDA-MB-231 cells by decreased activities of MMPs and the downregulation of ERK1/2 and SAPK/JNK signaling. Interestingly, these compounds act like anti-proliferative and pro-apoptotic 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 bio response during the in vivo investigations 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

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merging the two subunits which are individually known to display anti-cancer property was achieved. Further work towards improving the in vivo profile of this core via. chemical modifications is underway and will be reported in future. Experimental Section General. All experiments were monitored by analytical TLC performed on pre-coated silica gel plates. After elution, plate was visualized under UV illumination at 254 nm for UV active materials. IR spectra were recorded using a FTIR spectrophotometer. 1H NMR and

13

C 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 were recorded on Ion Trap Mass spectrometer and the HRMS spectra were recorded as ESI-HRMS 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 (250x 4.5 mm, 5µm) 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 di-Sodium hydrogen phosphate and sodium di-hydrogen 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 eq) at 0 oC under stirring. After 5 min. formaldehyde (1.5

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eq) and appropriate amine from a-v (1.2 eq) were added dropwise at same temperature. The reaction was allowed to proceed for 20 min after which methyl 1-(dimethoxymethyl)-9Hpyrido[3,4-b]indole-3-carboxylate (1) (1.0 eq) 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 oC; Rf = 0.48 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax: 896, 923, 1141, 1563, 1762, 3414 cm-1. 1H NMR (400 MHz, DMSO-d6): δ (ppm): 4. 06 (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[3,4b]indol-4-ium chloride (3b).Yield: 94% (0.152 g from 0.1 g); solid, mp 204-206 oC; Rf = 0.46 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax: 919, 1123, 1560, 1750, 3424 cm-1. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 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);

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C NMR (100 MHz,

DMSO-d6): δ (ppm) = 55.4, 112.1, 112.5, 113.2, 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.

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5-(Methoxycarbonyl)-2-(4-(trifluoromethyl)phenyl)-2,11dihydroimidazo[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 oC; Rf = 0.39 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax: 1223, 1598, 1754, 3410 cm-1. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 4.08 (s, 3H), 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);

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C NMR (100 MHz, C 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-Iodophenyl)-5-(methoxycarbonyl)-2,11-dihydroimidazo[1',5':1,2]pyrido[3,4-b]indol4-ium chloride (3d).Yield: 88% (0.174 g from 0.1 g); grey solid, mp 232-234 oC; Rf = 0.48 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax: 1152, 1536, 1756, 3453 cm-1. 1H 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[3,4b]indol-4-ium chloride (3e).Yield: 90% (0.161 g from 0.1 g); white solid; mp 212-214 oC; Rf = 0.42 (CH2Cl2: MeOH, 9:1, v/v); IR (neat) νmax: 931, 1568, 1749, 3443 cm-1. 1H 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,

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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)-2,11dihydroimidazo[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 oC; Rf = 0.40 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax: 899, 1265, 1578, 1758, 3423 cm-1. 1H 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);

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C 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)-2,11dihydroimidazo[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 oC; Rf = 0.44 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax: 952, 1257, 1566, 1760, 3445 cm-1. 1H 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);

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C 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,

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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)-2,11dihydroimidazo[1',5':1,2]pyrido[3,4-b]indol-4-ium chloride (3h).Yield: 85% (0.144 g from 0.1 g); yellow solid, mp 206-208 oC; Rf = 0.42 (CH2Cl2: MeOH, 9:1, v/v); IR (neat) νmax: 937, 1248, 1554, 1762, 3455 cm-1. 1H NMR (400 MHz, C 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);

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C 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. 2-(2-Bromo-4-fluorophenyl)-5-(methoxycarbonyl)-2,11dihydroimidazo[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 oC; Rf = 0.43 (CH2Cl2: MeOH, 9:1, v/v); IR (neat) νmax: 932, 1256, 1546, 1745, 3425 cm-1. 1H 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, DMSOd6): 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.

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2-(2-Bromo-4-methylphenyl)-5-(methoxycarbonyl)-2,11dihydroimidazo[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 oC; Rf = 0.38 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax: 868, 1254, 1767, 3431 cm-1. 1H 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. 2-(4-Chloro-2-methylphenyl)-5-(methoxycarbonyl)-2,11dihydroimidazo[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 oC; Rf = 0.46 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax: 899, 1216, 1764, 3450 cm-1. 1H 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);

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C 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)-2,11dihydroimidazo[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 oC; Rf = 0.43 (CH2Cl2: MeOH, 9:1, v/v); IR (neat) νmax: 918,

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1221, 1758, 3442 cm-1. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 2.49 (s, 3H), 4.05 (s, 3H), 7.367.86 (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. 2-(3,4-Dimethoxyphenyl)-5-(methoxycarbonyl)-2,11-dihydroimidazo[1',5':1,2]pyrido[3,4b]indol-4-ium chloride (3m).Yield: 83% (0.142 g from 0.1 g); white solid; mp 198-200 oC; Rf = 0.36 (CH2Cl2: MeOH, 9:1, v/v); IR (neat) νmax: 854, 1242, 1560, 1742, 3430 cm-1. 1H NMR (400 MHz, C 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);

13

C NMR (100 MHz, DMSO-d6): δ (ppm) = 52.7, 56.1, 56.2, 108.1, 109.9,

113.9, 120.6, 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. 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 oC; Rf = 0.47 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax: 868, 1168, 1547, 1748, 3464 cm-1. 1H NMR (400 MHz, DMSOd6): 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);

13

C NMR (100 MHz,

DMSO-d6): δ (ppm) = 53.5, 55.3, 112.1, 112.5, 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.

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Journal of Medicinal Chemistry

2-(4-Fluorobenzyl)-5-(methoxycarbonyl)-2,11-dihydroimidazo[1',5':1,2]pyrido[3,4b]indol-4-ium chloride (3o).Yield: 91% (0.146 g from 0.1 g); white solid; mp 228-230 oC; Rf = 0.45 (CH2Cl2: MeOH, 9:1, v/v); IR (neat) νmax: 756, 1235, 1575, 1753, 3396 cm-1. 1H 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[3,4b]indol-4-ium chloride (3p).Yield: 94% (0.150 g from 0.1 g); white solid; mp 223-225 oC; Rf = 0.47 (CH2Cl2: MeOH, 9:1, v/v); IR (neat) νmax: 868, 956, 1240, 1752, 3380 cm-1. 1H 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-4ium chloride (3q).Yield: 90% (0.143 g from 0.1 g); white solid, mp 190-192 oC; 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),

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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 oC; Rf = 0.36 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax:, 756, 941, 1563, 1760, 3440 cm-1. 1H NMR (300 MHz, DMSOd6): 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-(2-(Diethylamino)ethyl)-5-(methoxycarbonyl)-2,11-dihydroimidazo[1',5':1,2]pyrido[3,4b]indol-4-ium chloride (3s).Yield: 74% (0.116 g from 0.1 g); yellow solid, mp 172-174 oC; 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), 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.

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2-(Adamantan-1-yl)-5-(methoxycarbonyl)-2,11-dihydroimidazo[1',5':1,2]pyrido[3,4b]indol-4-ium chloride (3t).Yield: 68% (0.091 g from 0.1 g); yellow solid; mp 166-168 oC; Rf = 0.48 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax:, 1238, 1560, 1740, 3432 cm-1. 1H 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);

13

C 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-4ium chloride (3u).Yield: 80% (0.121 g from 0.1 g); yellow solid, mp 178-180 oC; Rf = 0.43 (CH2Cl2: MeOH, 9:1, v/v); IR (KBr) νmax: 726, 823, 916, 1256, 1560, 1755, 3428 cm-1. 1H 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[3,4b]indol-4-ium chloride (3v).Yield: 64% (0.109 g from 0.1 g); light yellow solid, mp 158-160 o

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-

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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. 2-(4-Chlorophenyl)-5-(methoxycarbonyl)-11-(3,4,5-trimethoxybenzyl)-2,11dihydroimidazo[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 oC; 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); 13

C 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 AlexaFluor 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, phosphop38, p38 and β-actin were purchased from Cell Signalling 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, until and unless specified.

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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 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 37oC. For all treatments, compounds were dissolved in DMSO at a stock concentration of 10 mM. The sub-confluent cells were treated with different concentrations of compounds in complete cell growth medium. 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 purplecolored 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. Approximately 2 x 105 human breast cancer MDA-MB-231 cells were seeded in six well plates in triplicates. 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.

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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 depolarisation 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 x 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 dark at room temperature, and then subjected to flow-cytometric analysis using FACS Calibur instrument. Data was 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 x 105 cells were plated in 6 well plates and a wound was created by scraping the cells with a sterile 200 µL pipette tip in the middle of the culture well. After removing 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-MB231 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

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Journal of Medicinal Chemistry

described earlier.21 For transwell invasion assay, growth factor reduced matrigel was diluted in serum free culture medium and coated 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, non-invaded or non-migrated cells on upper surface of the membrane were carefully removed with a cotton swab; the membrane insert was 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. 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 determining the protein concentration in conditioned media, 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 80V 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 37oC. Gels were stained using 0.5% Coomassie blue R-250 in 5% (v/v) methanol and

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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 B1, CDC25C, Cdk1 (Millipore, Billerica, MA), Bax, Bcl-2, Bcl-XL, surviving, cleaved caspase3, 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 immunoreactive 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 triplicates ± SEM. (*) p

Synthesis of β-Carboline-Based N-Heterocyclic Carbenes and Their Antiproliferative and Antimetastatic Activities against Human Breast Cancer Cells.

A series of novel β-carboline-based N-heterocyclic carbenes was prepared via Mannich reaction between methyl 1-(dimethoxymethyl)-9H-pyrido[3,4-b]indol...
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