Design and synthesis of C3-pyrazole/chalcone-linked beta-carboline hybrids: Antitopoisomerase I, DNA-interactive, and apoptosis-inducing anticancer agents

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DOI: 10.1002/cmdc.201300406

Design and Synthesis of C3-Pyrazole/Chalcone-Linked

Beta-Carboline Hybrids: Antitopoisomerase I, DNA-

Interactive, and Apoptosis-Inducing Anticancer Agents

Ahmed Kamal,*^{[a, b]}Vunnam Srinivasulu,^[a]V. Lakshma Nayak,^[a]Manda Sathish,^[a]

Nagula Shankaraiah,^[b]Chandrakant Bagul,^{[a, b]}N. V. Subba Reddy,^[a]Nandini Rangaraj,^[c]and

Narayana Nagesh*^[c]

A series of b-carboline hybrids bearing a substituted phenyl

and a chalcone/(N-acetyl)-pyrazole moiety at the C1 and C3

positions, respectively, was designed, synthesized, and evaluat-

ed for anticancer activity. These new hybrid molecules showed

significant cytotoxic activity, with IC₅₀values ranging from

<2.0 mm to 80 mm, and the structure–activity relationships

(SAR) associated with substitutions at positions 1 and 3 of

these hybrids was clearly addressed. Further, induction of

apoptosis was confirmed by Annexin V-FITC, Hoechst staining,

and DNA fragmentation analysis. In addition, DNA photocleav-

age studies proved that two of the hybrids, (E)-1-(furan-2-yl)-3-

(1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indol-3-yl)prop-2-

en-1-one (7d) and 1-(3-(furan-2-yl)-5-(1-(4-(trifluoromethyl)-

phenyl)-9H-pyrido[3,4-b]indol-3-yl)-4,5-dihydro-1H-pyrazol-1-

yl)ethanone (8d) could effectively cleave pBR322 plasmid DNA

upon irradiation with UV light. Active hybrid 8d inhibited DNA

topoisomerase I activity efficiently and preserved DNA in the

supercoiled form. To further corroborate the biological activi-

ties, as well as to understand the nature of the interaction of

these hybrids with DNA, spectroscopic studies were also per-

formed. Unlike simple b-carboline alkaloids, the binding mode

of these new hybrid molecules with DNA was not similar, and

both biophysical as well as molecular docking studies speculat-

ed a combilexin-type of interaction with DNA. Further, an in

silico study of these b-carboline hybrids revealed their drug-

like properties.

Introduction

In recent years, discovery of new hybrid molecules that can

recognize DNA and display anticancer activity has emerged as

an important research area.^[1,2]A majority of the anticancer

drugs that are in the clinic act by interacting with DNA directly

or preventing the proper relaxation of DNA through the inhibi-

tion of DNA-bound proteins/enzymes.^[3]Indeed, the anticancer

activity of these drugs is due to their significant interactions

with the double helix of DNA or associated enzymes by form-

ing hydrogen bonds and/or stable complexes leading to con-

formational changes, which affect the usual mechanisms of

DNA replication, transcription, and repair.^[4]In general, most of

the anticancer drugs interact non-covalently with DNA via

1) groove binding through a combination of hydrophobic,

electrostatic, and hydrogen bonding interactions, and 2) inter-

calation, in which a planar aromatic moiety inserts between

the DNA base pairs. Moreover, studies of the interactions be-

tween these types of molecules and DNA, especially the effects

of structural features of the molecules on DNA binding mode

and strength, could offer some insights into the design of new

DNA-targeting antitumor drugs.^[5]In addition, the combilexin

class of molecules, with mixed DNA groove binding and inter-

calating properties, are capable of enhancing DNA binding af-

finity due to their double mode of DNA interaction, as seen

with NetAmsa (Figure 1E). In this context, designing a hybrid

molecule with such dual properties could be considered attrac-

tive, in light of their stronger interaction compared to mole-

cules with a single mode of action.^[6,7]Additional impetus for

the development of such new DNA-targeting chemotherapeu-

tic agents comes from the recent approval of Belotecan (Fig-

ure 1D).^[8]

[a] Dr. A. Kamal, V. Srinivasulu, V. L. Nayak, M. Sathish, C. Bagul, N. V. S. Reddy

Medicinal Chemistry & Pharmacology

CSIR-Indian Institute of Chemical Technology

Hyderabad 500 007 (India)

E-mail: ahmedkamal@iict.res.in

[b] Dr. A. Kamal, Dr. N. Shankaraiah, C. Bagul

Department of Medicinal Chemistry

As a majority of the anticancer drugs that are presently in

clinical use are derived from natural product scaffolds, it is logi-

cal to design improved DNA-binding agents based on natural

products. A number of b-carboline alkaloids offer excellent op-

portunities for rational structural modifications towards this

end (Figure 1).^[9]The b-carbolines, which contain planar tricyclic

systems, are a class of synthetic and naturally occurring com-

pounds that possess a large spectrum of important pharmaco-

National Institute of Pharmaceutical Education & Research (NIPER)

Hyderabad 500 037 (India)

[c] Dr. N. Rangaraj, Dr. N. Nagesh

CSIR-Centre for Cellular & Molecular Biology

Hyderabad-500 007 (India)

E-mail: nagesh@ccmb.res.in

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cmdc.201300406.

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In the present investigation,

an effort has been made to find

new b-carboline congeners that

possess potential antitumor ac-

tivity, as well as to study the in-

fluence of various groups at the

C1 and C3 positions, apart from

understanding their mode of

DNA interaction. In this context,

we designed and synthesized

new chalcone-/(N-acetyl)-pyra-

zole-linked b-carboline hybrids.

These new hybrid molecules

comprising two pharmacophoric

groups (i.e., a b-carboline ring

and (N-acetyl)-pyrazole or chal-

cone moieties) exhibit promising

cytotoxicity and significant DNA

binding affinity, apart from in-

ducing apoptosis. It was specu-

lated that these new hybrids

behave similarly to the combilex-

in class of compounds.

Figure 1. Chemical structures of b-carboline derivatives (A, B, C, and F); a recently approved DNA targeting drug

(D); a typical example of combilexin (E); and designed chalcone/(N-acetyl)-pyrazole-linked b-carboline hybrids 7a–

m and 8a–m.

Recently topoisomerases I and

II have become

a potential

logical properties, including potent antitumor activity.^[10]Litera-

ture reports show that b-carbolines can induce DNA damage

or intercalate with DNA, in addition to acting as DNA topoiso-

merase I (topo I) inhibitors.^[11–13]Structure–activity relationship

(SAR) analysis revealed that the introduction of appropriate

groups at the C1 and C3 positions of the b-carboline nucleus

accentuated antitumor activity as well as DNA binding abili-

ty.^[13]Previous studies demonstrated that b-carboline deriva-

tives containing a substituted phenyl group at the C1 position

and certain biologically important heterocyclic scaffolds, such

as 1,3,4-oxadiazole, 1,2,4-triazole, 4-benzylidene-4H-oxazol-5-

one, or a substituted carbohydrazide moiety at the C3 position

demonstrated significant antitumor properties.^[14–16]In addition,

some of the specially designed b-carboline derivatives bearing

a flexible amino side chain, and their dimers, were also found

to possess promising antitumor activity with significant DNA

interacting ability.^[17,18]

target for cancer chemotherapy. Topo I catalyzes the cleavage

and passage of single-stranded DNA through the single strand

break, whereas topo II catalyzes the cleavage of double-strand-

ed DNA and its passage through a double strand break.^[25]

Compounds that inhibit topoisomerase block the religation of

DNA and generate single and double strand breaks in DNA,

subsequently leading to apoptosis. Until recently, very few

topo I inhibitors were known, but after the synthesis of camp-

tothecin derivatives, several topo I inhibitors such as harmine,

harmine derivatives, and norharman were identified.^[26]As

a result, the search for new molecules that inhibit DNA topoi-

somerases has gained importance; thus, the role of newly syn-

thesized b-carboline hybrids to act as topo I inhibitors was

studied.

Photodynamic therapy (PDT) has gained increased attention

recently, due to effective action of PDT molecules on cancer in

an affected area upon irradiation with light.^[27]Therefore, the

development of photochemical DNA cleaving agents, in the

presence of a specific wavelength of UV radiation without any

additives such as metals and reducing agents, has attained sig-

nificant importance in the field of medicine.^[28]In this context,

the DNA photocleavage potential of these new hybrid mole-

cules was also examined using pBR322 plasmid DNA.

Hybrid generation from b-carboline derivatives requires in-

corporation of carefully selected appendages that are expected

to enhance the DNA binding and anticancer potential of the

individual components. a,b-Unsaturated ketones (chalcones)

constitute one such class of compounds that may be hybrid-

ized with b-carbolines to achieve this goal. Chalcones are

useful synthetic intermediates in the preparation of pharmaco-

logically active heterocycles, such as pyrazoles, which possess

potent antitumor activity.^[19,20]Additionally, chalcones and pyra-

zoles are known for their DNA damaging ability, without an

obvious mode of binding.^[21–23]In addition, cytotoxicity studies

indicate that the toxicity of individual components of present

hybrids (i.e., b-carboline and chalcone/pyrazole derivatives)

was considerably higher in tumor cells compared to the

normal cells.^[18,24]

Here we report the effect of structural variations on the b-

carboline scaffold and the role played by different pharmaco-

phores on cytotoxicity, as well as the apoptosis-inducing ability

of these b-carboline hybrids on representative human cancer

cell lines. The main focus of this study is the effect of these hy-

brids on DNA, which is the most vital and genetically impor-

tant macromolecule in the cell, by examining topo I inhibition,

DNA binding, conformation, and cleavage studies. The possible

ways in which these hybrids might interact with DNA was also

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elucidated by molecular docking studies. Further investigations

to determine the detailed mechanism of their biological action

are actively underway in our laboratory.

thesized by a barium hydroxide-mediated Claisen–Schmidt

condensation reaction between 6a–d and a variety of aromat-

ic, as well as heteroaromatic, ketones. Finally, these chalcones

(7a–m), upon reaction with hydrazine monohydrate in the

presence of acetic acid produced the required corresponding

C1-(phenyl)-C3-(N-acetyl)-pyrazole-substituted b-carboline hy-

brids (8a–m), which are detailed in the Supporting Information

(Table S1).

Results and Discussion

Chemistry

Synthetic strategies followed for the preparation of 1,3-disub-

stituted chalcone- and (N-acetyl)-pyrazole-linked b-carboline

hybrids are depicted in Scheme 1. Initially, a Pictet–Spengler

condensation of l-tryptophan methyl ester (2) with various

substituted benzaldehydes in the presence of para-toluenesul-

fonic acid (PTSA) afforded the corresponding 1,2,3,4-tetrahydro

b-carboline derivatives (3a–d). Aromatization of 3a–d was ach-

ieved using sulfur in xylene, resulting in the formation of

methyl b-carboline-3-carboxylate derivatives (4a–d). The ester

group at the C3 position of 4a–d was then reduced to its cor-

responding alcohol by lithium aluminum hydride in dry THF to

provide 5a–d, which were further oxidized by Dess–Martin pe-

riodinane in CH₂Cl₂to afford 1-phenyl-3-carboxaldehyde b-car-

boline derivatives (6a–d). Next, the required C1-phenyl-substi-

tuted C3-chalcone-linked b-carboline hybrids (7a–m) were syn-

Biology

Cytotoxic activity

Preliminary screening of the synthesized hybrids was per-

formed to evaluate cytotoxic activity against a panel of select-

ed human cancer cell lines, including A-549 (lung), DU-145

(prostate), MCF-7 (breast), HeLa (cervical), ACHN (renal) and

normal human embryonic kidney (HEK-293) cell lines, by em-

ploying the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoli-

um bromide (MTT) assay. The results of this cytotoxicity data,

expressed as IC₅₀values, is summarized in Table 1 in compari-

son with positive controls such as doxorubicin and harmine.

Gratifyingly, all of the hybrids were selectively cytotoxic to

cancer cells with a weak effect

on normal cells. They exhibited

good to moderate cytotoxicity

against cancer cells, with IC₅₀

values ranging from <2.0 mm to

80 mm. Among a series of 26 hy-

brids, 13 of the hybrids (7c–d,

7 f–h, 7j, 7l, 8c–d, 8g–h, 8j,

and 8l) exhibited significant cy-

totoxicity against all cell lines ex-

amined (<10.0 mm). These hy-

brids showed higher cytotoxic

activity in the MCF-7 breast

cancer cell line compared to

other cell lines used in the pres-

ent study. The most active hy-

brids in the series were 7d, 7h,

8d, and 8h with IC₅₀values

ranging from 1.86 mm to 5.29 mm

in the cell lines tested. It was ob-

served that the cytotoxicity of

these 1,3-disubstituted b-carbo-

line hybrids is dependent on the

nature of the substituent present

on the C1-phenyl ring (R¹) and

the heterocyclic ring attached to

the chalcone/pyrazole moiety of

the b-carboline scaffold (R²).

Among all of the hybrids synthe-

sized, 7d and 8d, which possess

Scheme 1. Synthetic pathway for the preparation of new chalcone/(N-acetyl)pyrazole-linked C1-phenyl-substituted

b-carboline hybrids. Reagents and conditions: a) SOCl₂, MeOH, 0^oC!RT, 4 h, 90%; b) PTSA, toluene, reflux, 12 h;

c) sulfur, xylene, reflux, overnight, then 4^oC, 3 h, 70–80%; d) 1. LiAlH₄, THF, 0^oC!RT, 4 h; 2. DMP, CH₂Cl₂, 0^oC!RT,

3 h, 80–85%; e) Ba(OH)₂, MeOH, RT 64–85%; f) NH₂NH₂·H₂O, acetic acid, reflux, 51–85%. Reaction times for steps e

and f can be found in the Supporting Information (Table S1).

a trifluoromethyl substituent at

the C1-phenyl and furan-con-

taining chalcone/pyrazole moiet-

ies at C3 displayed potent cyto-

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Table 1. Cytotoxicity of b-carboline hybrids 7a–m and 8a–m against a panel of human cancer and normal cell lines.

Compd

IC₅₀[mm]^[a]

A549

DU-145

MCF-7

HeLa

ACHN

HEK-293

7a

7b

7c

7d

7e

7 f

7g

7h

7i

60.22ꢀ1.37

18.62ꢀ0.30

2.08ꢀ0.06

2.57ꢀ0.08

10.23ꢀ0.33

2.95ꢀ0.04

3.31ꢀ0.11

3.80ꢀ0.06

14.79ꢀ0.04

6.76ꢀ0.12

15.22ꢀ0.03

9.54ꢀ0.31

11.22ꢀ0.37

89.12ꢀ2.36

15.84ꢀ1.79

2.81ꢀ0.41

3.31ꢀ0.37

13.83ꢀ0.40

7.94ꢀ0.66

8.91ꢀ0.30

4.16ꢀ1.07

14.68ꢀ0.19

5.59ꢀ0.18

22.38ꢀ1.08

7.07ꢀ0.12

23.44ꢀ3.49

48.97ꢀ4.13

12.02ꢀ0.08

2.39ꢀ0.94

1.86ꢀ0.18

7.58ꢀ0.04

2.88ꢀ0.10

2.39ꢀ0.12

2.29ꢀ0.45

14.61ꢀ0.39

3.88ꢀ1.32

10.47ꢀ0.35

5.62ꢀ0.09

8.70ꢀ0.04

77.62ꢀ1.14

13.18ꢀ1.48

7.24ꢀ0.35

2.75ꢀ0.13

9.54ꢀ0.16

6.76ꢀ1.24

6.91ꢀ0.11

4.81ꢀ0.80

23.0ꢀ0.37

7.56ꢀ0.07

23.44ꢀ0.38

6.76ꢀ0.44

27.54ꢀ2.96

61.65ꢀ0.78

17.78ꢀ1.44

5.37ꢀ0.52

2.13ꢀ2.23

16.59ꢀ2.201

9.97ꢀ0.98

9.54ꢀ0.49

4.04ꢀ0.27

16.21ꢀ0.53

7.29ꢀ0.57

19.95ꢀ3.34

6.91ꢀ0.34

17.78ꢀ0.88

74.30ꢀ3.82

47.05ꢀ0.49

43.96ꢀ2.66

69.99ꢀ1.14

26.69ꢀ0.04

42.24ꢀ1.65

60.32ꢀ3.92

96.61ꢀ1.58

47.85ꢀ3.20

55.39ꢀ0.12

27.69ꢀ3.64

40.97ꢀ0.34

76.78ꢀ3.75

7j

7k

7l

7m

8a

8b

8c

8d

8e

8 f

8g

8h

8i

40.8ꢀ0.51

15.37ꢀ0.01

2.28ꢀ0.16

2.39ꢀ0.07

12.3ꢀ0.58

13.18ꢀ0.21

2.31ꢀ1.32

3.20ꢀ1.05

52.8ꢀ3.16

7.76ꢀ0.25

9.77ꢀ0.33

2.51ꢀ0.13

10.96ꢀ0.18

50.11ꢀ3.34

23.44ꢀ1.16

4.81ꢀ3.49

3.63ꢀ0.69

13.96ꢀ1.28

24.54ꢀ0.40

7.81ꢀ4.69

3.66ꢀ0.43

69.18ꢀ4.31

7.59ꢀ0.03

9.94ꢀ0.10

4.90ꢀ0.87

19.76ꢀ0.55

37.15ꢀ4.34

13.75ꢀ1.15

5.39ꢀ0.06

1.94ꢀ0.41

7.58ꢀ1.35

10.00ꢀ0.16

2.29ꢀ0.43

2.19ꢀ0.32

57.77ꢀ0.61

4.78ꢀ0.39

5.37ꢀ0.08

2.13ꢀ0.45

7.76ꢀ0.90

64.05ꢀ4.18

16.05ꢀ1.23

6.24ꢀ0.24

2.63ꢀ0.30

19.03ꢀ1.39

21.42ꢀ0.23

4.51ꢀ1.44

3.81ꢀ0.11

66.06ꢀ1.02

8.7ꢀ0.28

42.1ꢀ1.78

14.12ꢀ0.69

6.47ꢀ1.47

2.75ꢀ0.62

5.77ꢀ2.15

15.84ꢀ0.25

6.40ꢀ0.69

5.29ꢀ2.27

52.2ꢀ0.61

8.09ꢀ0.09

15.12ꢀ0.43

6.60ꢀ0.54

8.08ꢀ0.13

37.46ꢀ0.43

66.40ꢀ3.58

63.86ꢀ3.12

95.36ꢀ0.13

78.57ꢀ0.34

73.71ꢀ0.60

45.46ꢀ4.24

60.99ꢀ2.98

55.39ꢀ0.12

75.00ꢀ2.49

67.06ꢀ0.79

45.72ꢀ1.49

28.92ꢀ2.05

8j

8k

8l

10.0ꢀ0.33

3.38ꢀ1.12

10.23ꢀ0.04

8m

Harmine

Doxorubicin

7.76ꢀ0.22

1.86ꢀ0.38

12.02ꢀ0.59

2.13ꢀ0.92

10.78ꢀ0.52

2.18ꢀ0.21

16.21ꢀ1.06

1.73ꢀ0.28

23.96ꢀ0.80

1.81ꢀ0.81

60.70ꢀ1.49

NT

[a] Lung cancer (A549), prostate cancer (DU-145), breast cancer (MCF-7), cervical cancer (HeLa), renal cancer (ACHN), or normal human embryonic kidney

(HEK-293) cells. Half-maximal inhibitory concentration (IC₅₀) values are the mean ꢀSD of three individual experiments performed in duplicate. NT: not

tested. Harmine and doxorubicin were included as reference agents.

Cell cycle analysis

toxic activity compared to other hybrids that had different sub-

stituents such as 3-chloro, 3,4-diflouro, and 3,4,5-trimethoxy at

R¹, as well as pyrrole, thiophene, and anilino at R². Further, hy-

brids with a trimethoxy group at R¹and a furan ring at R²(7h

and 8h) were also considerably active against almost all cell

lines tested; however, hybrids with a 3-chloro substituent at R¹

were less active within this series.

These results imply that the furan ring at R²represents the

most optimal structure for this class of hybrids, which exhibit

remarkable cytotoxicity. From SAR studies, some interesting

trends were observed in these hybrids. It appears that a trifluor-

omethyl substitution at R¹and a furan ring at R²are crucial for

cytotoxicity. Some hybrids with 3,4-difluoro substitution at C1

also showed good cytotoxic activity. Acylation of the amine

group of the 4-amino phenyl attached to the (N-acetyl)-pyra-

zole moiety has no effect on the cytotoxicity. Based on results

obtained from the MTT assay, the most cytotoxic hybrids (7d

and 8d) were used for further mechanistic studies, such as

topo I inhibition and apoptosis induction. In addition, these hy-

brids were also investigated for DNA binding and cleavage, as

well as molecular docking studies.

MTT assay results indicated that, among the hybrids tested, 7d

and 8d exhibited the lowest IC₅₀values (1–2 mm) in the MCF-7

breast cancer cell line. In order to understand the unique po-

tential of these hybrids at their respective IC₅₀values and to

shed more light on various factors responsible for the cytotox-

ic effects of 7d and 8d, we examined their influence on cell

cycle progression in MCF-7 cells; the results are displayed in

Figure 2. After 48 h of treatment, hybrids 7d and 8d caused

a small increase in the percentage of cells in the sub-G₁phase

and a reduction in the percentage of cells in the S-phase com-

pared to the control, which indicates the onset of apoptosis.^[29]

However, a higher accumulation of cells in the sub-G₁phase

was observed in case of hybrid 8d in comparison with 7d.

Apoptosis induction studies

Annexin V-FITC

As the observed drift of cells into the sub-G₁phase was mar-

ginal in cell cycle analysis, the effect of hybrids 7d and 8d on

apoptosis was further studied using an Annexin V-FITC/PI (AV/

PI) dual staining assay. It was also useful to examine the occur-

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hybrids in generating free radicals in the system. In

general, photocleavage reactions proceed via the

generation of molecular oxygen or hydroxyl radical

species. However, these reactions involving triplet

oxygen state (³O₂) could proceed by two major

mechanistic pathways. In the first, the excited singlet

state of the molecule may undergo intersystem

crossing to generate an excited triplet state which, in

turn, activates molecular oxygen from its triplet

(ground) state to a more reactive singlet state

oxygen (¹O₂).^[33]In an alternate pathway, the excited

state molecule could reduce molecular oxygen to

generate a highly reactive hydroxyl radical. The abili-

ty of hybrids 7d and 8d to cleave DNA upon irradia-

tion with UV light of 365 nm wavelength, followed

by agarose gel electrophoresis, depicts that these hy-

brids generate free radicals and function as potent

Figure 2. Effect of b-carboline hybrids 7d and 8d on cell cycle progression of MCF-7

cells: Data are expressed as the percentage cell count in each phase of the cell cycle in-

duced by each compound. (* denotes p<0.05 vs. control).

rence of phosphatidylserine externalization, as well as to un-

derstand whether it was due to physiological apoptosis

or nonspecific necrosis.^[30]Phosphatidylserine is

a marker of cell death, moving from the inner cell

membrane to the outer cell membrane during the

cascade of apoptosis.^[31]Annexin V is a cellular pro-

tein of the annexin group which selectively binds to

phosphatidylserine. In this study, MCF-7 cells were

treated with 7d and 8d for 48 h at 2 mm concentra-

tion, and we found that these hybrids displayed sig-

nificant apoptosis, as evidenced in Figure 3a. The re-

sults indicated that 7d and 8d showed 58.29% and

62.64% apoptosis at a concentration of 2 mm, where-

as 0.03% of apoptosis was observed in the control. It

was also clear that hybrid 8d was a better inducer of

apoptosis than 7d.

nuclease agents. When pBR322 plasmid DNA, in the presence

of ligands, was subjected to electrophoresis, relatively fast mi-

Hoechst staining

Apoptosis is one of the major pathways that lead to

cell death, whereas chromatin condensation and

fragmented nuclei are known as the classic character-

istics of apoptosis.^[32]Flow cytometry experiments

demonstrated that 7d and 8d were effective in in-

ducing apoptosis in MCF-7 cells. However, for visuali-

zation of the apoptosis-inducing effect of these two

hybrids, Hoechst (H33258) staining was performed in

MCF-7 cells. H33258 is a membrane-permeable fluo-

rescent blue dye that stains cell nuclei. Manual field

quantification of apoptotic cells based on cellular

condensation, presence of apoptotic bodies, and nu-

clear fragmentation under microscope in the pres-

ence of tested hybrids (7d and 8d) revealed a signifi-

cant increase in the percentage of apoptotic cells in

treated cells compared to control (Figure 3b).

DNA photocleavage studies

DNA photocleavage reactions were carried out in the

Figure 3. a) Annexin V-FITC/PI (AV/PI) dual staining assay. b) Hoechst (H33258) staining

presence of 7d and 8d to find the efficiency of these assay.

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gration was observed for the supercoiled form (Form I). If scis-

sion occurs on one strand, the supercoiled form relaxes to gen-

erate a relaxed circular form (Form II). Gel electrophoresis sepa-

ration of pBR322 DNA after incubation with two different con-

centrations (100 mm and 200 mm) of 7d and 8d upon irradia-

tion for 1 h is shown in Figure 4a. Upon further analysis of the

gel electrophoresis results, control (irradiated DNA solution

alone) as well as DNA with hybrids (200 mm) incubated in the

absence of light did not show any considerable cleavage. How-

ever, DNA samples irradiated along with hybrids exhibited re-

markable photocleavage, as evidenced by a decrease in the in-

tensity of Form I. Moreover, at a higher concentration of hy-

brids (200 mm), photocleavage was more significant compared

to that at 100 mm concentration. On the other hand, 8d was

considerably active, even at the lower concentration (100 mm),

indicating its potential to generate more free radicals that ef-

fectively cleave DNA. The photocleavage activity of these hy-

brids may be due to the presence of -NH moieties and polyar-

omatic rings, which could be involved in n–p* and p–p* transi-

tions upon intercalation with DNA.

Form II was enhanced by ~75%, representing the higher pho-

tocleavage capacity of 8d (Figure 4b).

DNA–topo I inhibition studies

Topo I has been identified as the potential target of several an-

ticancer drugs used clinically today. As topo I is involved in

replication and proliferation processes, overproduction of

topo I is observed in cancer cells compared to normal cells. In

general, topo I controls changes in the DNA structure by cut-

ting single-stranded DNA and rejoining the phosphate back-

bone during the normal cell cycle. The mechanism of topo I in-

hibition is known to occur in two ways. The inhibitors may

bind topo I directly, or they may bind to DNA and alter its

structure, so that it cannot be recognized by topo I. In the

present study, the potential of the synthesized b-carboline hy-

brids to inhibit topo I was studied. Though the actual mecha-

nism of topo I inhibition by these hybrids is not known, they

have potential to inhibit topo I through direct DNA interaction

or may bind to the DNA–topoisomerase cleavage complex and

interfere with its relegation step, which was further investigat-

ed via molecular docking studies.

In addition, with a goal of understanding the effect of DNA

photocleavage as a function of irradiation time, 8d (100 mm)

was irradiated at different time intervals (15 min, 30 min,

60 min, 90 min, and 120 min). It was observed that the intensi-

ty of the band corresponding to Form I diminished gradually,

whereas that of Form II increased upon increasing the irradia-

tion time. After 2 h, the intensity of the band corresponding to

Cao and coworkers recently reported that camptothecin in-

hibits topo I at a concentration of 100 mm, whereas harmine

derivatives inhibit topo I at 150 mm.^[34]Interestingly, the present

hybrids (7d and 8d) also showed significant topo I inhibition

at concentrations of 100 mm as well as 200 mm (Figure 4c). This

indicates that the incorporation

of phenyl and chalcone or (N-

acetyl)-pyrazole groups at posi-

tions C1 and C3 of the b-carbo-

line nucleus enhance the poten-

tial of these hybrids to inhibit

topo I. The observed low IC₅₀

values for hybrids 7d and 8d

may be the result of effective

topo I inhibition, as well as

better DNA interaction. In addi-

tion, we tested the DNA cleav-

age ability of these hybrids in

the absence of DNA–topo I and

did not observe considerable

cleavage (Figure S4, Supporting

Information).

DNA fragmentation study

From the topo I inhibition assay,

it was evident that these hybrids

could cleave DNA, either by in-

teraction with DNA or through

forming a ternary DNA–topo I

complex. To further study the

Figure 4. a) Photo-activated cleavage of pBR322 in the presence of hybrids 7d and 8d at different concentrations.

b) Effect of hybrid 8d as a function of irradiation time; Form II: relaxed circular DNA; Form I: supercoiled DNA;

Control: DNA solution irradiated in the absence of compounds; Dark: DNA+hybrid in the absence of light.

c) Effect of hybrids 7d and 8d on topo I inhibition; Control: DNA solution alone. d) DNA Fragmentation assay in

MCF-7 cells; Marker: 100 bp; Control: untreated; Doxorubicin (Doxo).

effect of these hybrids, as well as

to examine permeability across

the cellular membrane, a DNA

fragmentation assay was per-

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formed using MCF-7 cells. It is another hallmark of apoptosis

to determine the cleavage of DNA by endonucleases into mul-

tiple 180 bp fragments (and multiples thereof), which form

a ladder pattern when subjected to gel electrophoresis.^[35]In

brief, 2 mm concentrations of 7d and 8d were incubated with

MCF-7 cells for 48 h. After lysing the cells, DNA was eluted and

subjected to electrophoresis by loading onto a 0.8% agarose

gel. Both 7d and 8d were effective in cleaving DNA; however,

8d exhibited better performance in comparison with 7d (Fig-

ure 4d). It was also noticeable that DNA fragmentation in-

creased with an increase in concentration of the hybrids, as

well as duration of treatment (data was not shown). In general,

molecules that cleave DNA effectively at low concentrations

have higher potential to induce apoptosis in cells. Though the

actual mechanism of cellular uptake of these hybrid molecules

is unknown, Bringmann reported that b-carboline derivatives

could easily enter human serotonergic cells.^[36]The results indi-

cate that these hybrids could cross the cell membrane barrier,

then remain in the cell and effectively cleave DNA.

The CD spectrum of calf thymus (CT) DNA exhibits a positive

band at 275 nm and a negative band at 245 nm, due to p–p

base stacking and right-hand helicity. The addition of hybrid

7d to CT DNA solution at a lower concentration (hybrid/DNA,

1:1) exhibited slight hyperchromicity of the positive CD band,

which is an indication of DNA stabilization. Interestingly, upon

increasing the hybrid (7d) concentration (ligand/DNA, 2:1) the

positive CD band exhibited significant hypochromicity, indicat-

ing unwinding and conformational change in the DNA upon

further interaction of 7d with CT DNA. A similar interaction

with DNA was observed in our previous studies with pyrrolo

benzodiazepine (PBD) ligands.^[38]On the other hand, the ob-

served interaction of 8d with DNA was slightly different from

that of 7d. Upon addition of 8d to DNA solution, the positive

CD band did not show hyperchromicity; however, 7d exhibit-

ed continuous hypochromicity at both concentrations, demon-

strating its potential to unwind DNA (Figure 5a).

Fluorescence spectroscopy

Fluorescence titration is yet another valuable technique for un-

derstanding the binding mode of small molecules to DNA at

comparatively lower concentrations.^[39]As these hybrid mole-

cules exhibit fluorescence properties, their interaction with CT

DNA could provide some insight regarding the nature of their

interaction with DNA. The emission spectrum was shown in

Figure 5b for hybrids 7d and 8d in the absence and presence

of varying amounts of CT DNA. Upon addition of CT DNA to

each of the hybrids, the intensity of fluorescence for the emis-

sion peak exhibited remarkable hypochromicity, along with

a slight red shift. The extent of fluorescence hypochromicity at

DNA binding studies

To further corroborate the biological activities of these active

hybrids (7d and 8d) and to understand their nature of interac-

tion with DNA, spectroscopic studies were performed.

Circular dichroism spectroscopy

Circular dichroism (CD) is a potential technique to study nucle-

ic acid conformational change due to interaction of DNA with

small molecules or changes in environmental conditions.^[37]

Figure 5. CD spectra (left), fluorescence spectra (middle) and UV–visible spectra (right) of hybrids a) 7d and b) 8d in the absence or presence of increasing

amount of hybrids (for CD spectra) or CT DNA (for fluorescence and UV–visible spectra).

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485 nm was a little higher for 8d compared to 7d. However,

higher fluorescence quenching in the case of 8d was ob-

served, probably due to the surface binding to DNA.^[40]Indeed,

simple b-carboline alkaloids such as harmine and harmane are

well known DNA intercalators, having a fully planar nature,

whereas the mode of binding of these b-carboline hybrids is

slightly different (combilexin-type binding), which could be re-

lated to their structure. However, the combilexin character is

greater for 8d compared to 7d, presumably due to having less

structural planarity (Figure S2, Supporting Information).

these values are in good agreement with Lipinski’s rules. More-

over, the calculated percentage of absorption of all hybrids

ranged between 69.29% and 93.21%, demonstrating that

these hybrids may have the required cell membrane permea-

bility.

Molecular docking studies

b-Carboline derivatives are known to bind topo I, resulting in

significant anticancer activity.^[32]The present C1- and C3-substi-

tuted b-carboline hybrids show efficient topo I inhibition and

in vitro cytotoxic activity against various human cancer cell

lines. Other biological studies also demonstrated that these hy-

brids are potential anticancer agents. To gain further insight

into the binding mode of these hybrids to topo I, molecular

docking studies were performed. A crystal structure of a DNA–

topo 1 complex was obtained from the Protein Data Bank

(PDB ID: 1SC7).^[44]The structure was prepared using the Protein

Preparation Wizard by Schrodinger. All hybrid molecules were

optimized using the PM3 method in Gaussian 09.^[45]The geom-

etry of the optimized hybrids shows that the plane of C1

phenyl substitution was not in the plane of the b-carboline

moiety, with an angle greater than 358 in all molecules. For the

chalcone series, C3 substitution was in the plane of the b-car-

boline ring system, but for the pyrazole series, it was not in

the plane of the b-carboline (Figure S2, Supporting Informa-

tion).

UV-visible spectroscopy

In order to further investigate the mode of binding of these

hybrid molecules, the UV absorption spectra of active hybrids

7d and 8d in the presence and absence of increasing concen-

trations of CT DNA was examined (Figure 5c). In all cases, bind-

ing of the hybrids to CT DNA resulted in considerable spectral

changes, characterized by marked hypochromicity of the

ligand soret band. The hypochromicity of the ligand soret

band at 298 nm is characteristic of intercalation, mostly due to

the interaction between the electronic states of the hybrids

and those of the DNA bases. The extent of hypochromicity ex-

hibited by the ligand soret band is an indication of strength of

intercalation.^[41]UV spectrophotometric titration curves exhibit-

ed isosbestic points at 295 nm and 290 nm, indicating that the

hybrids interact well with CT DNA and the interaction takes

place in single step.

Docking studies were performed using Gold docking soft-

ware. Docking poses for all hybrids showed that all intercalat-

ed DNA at the topo I binding site. However, these new hybrids

bearing chalcone and pyrazole motifs showed a different bind-

ing mode (Figure 6). For 7d, the C3 substituent and the b-car-

boline ring were stacked within the DNA, whereas the C1 sub-

stituent extended out of the minor groove of the DNA. The ni-

trogen atom in the last b-carboline ring showed a hydrogen

bonding interaction with the guanidine group of Arg364 as

shown by co-crystallization with the ligand. The C3 substituent

was oriented towards chain A (cleaved strand), the furan ring

was stacked with Cys112 with the enone region forming inter-

actions with Ala113, and the b-carboline ring stacked with

TGP11 and Thr10. For 8d, The C1 substituent and the b-carbo-

line ring were stacked within the DNA, and the C3 substituent

extended out of the major groove. The oxygen atom of the

furan ring exhibited hydrogen bonding interactions with the

amine group of Asn352, whereas stacking interactions were

shown by the phenyl ring with TGP11 and b-carboline with

Cys112 and Ala113. As the C3 substituent of 7d was in the

plane of the b-carboline, it could fit in the intercalation cavity,

while the off-planar phenyl substituent extended out of the

minor groove. However for 8d, the C3 substituent was not in

the plane of b-carboline and could not fit into the intercalating

cavity. The portion of the hybrid that projected out of the

plane is marked and shown in Figure S2 (Supporting Informa-

tion). This may be a reason why the chalcones and pyrazoles

exhibited different type of binding with DNA. It is clear from

the present experimental and docking results that the inhibito-

ry potential and cytotoxicity of b-carboline hybrids varies by

In silico studies

An in silico computational study for these b-carboline hybrids

(7a–m and 8a–m) was undertaken to determine the Lipinski

parameters for topological polar surface area (TPSA) and per-

centage of absorption (%ABS).^[42]All calculations were per-

formed using the Molinspiration Online Property Calculation

toolkit.^[43](http://www.molinspiration.com). The percentage of

absorption was determined using the equation: %ABS=109–

0.345ꢁTPSA, and data is presented in the Supporting Informa-

tion (Table S2). The in vivo absorption of these new b-carboline

hybrids was assessed by means of theoretical calculations fol-

lowing Lipinski’s Rules of Five, which implies that the absorp-

tion of an orally administered drug is likely to be better if it

agrees with the following rules: ꢁ5 hydrogen bond donors

(OH and NH groups), ꢁ10 hydrogen bond acceptors (N and O

atoms), molecular weight <500 Da, and a calculated logP<

5.0.^[42]Compound that violate more than one of these aspects

could face bioavailability problems.

The in silico results for these b-carboline hybrids are shown

in Table S2. Most of these new hybrid molecules violated only

one of the Lipinski’s rules, except 7m, 8e, 8 f, 8h, and 8m. In-

terestingly, three of the hybrids (7i, 8k, and 8l) in the series

satisfied all of Lipinski’s rules. Moreover, hybrids that violated

Lipinski’s rules either have a molecular weight larger than

500 Da or showed octanol–water partition coefficients (logP)>

5.0. All of these hybrids have a number of hydrogen bond ac-

ceptors between 3 and 9 and 1–3 hydrogen bond donors;

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Figure 6. Full view of topo I–ligand complex (b) and docking poses (in zoom) for 7d (a) and 8d (c) in topo I. Hydrogen bonds are shown as black dotted

lines.

the inclusion and placement of different groups. For better un-

derstanding the contribution of the trifluoromethyl group and

the factors responsible for the better activity of 7d and 8d, we

also analyzed the docking poses of the structurally similar hy-

brids (7a and 7c). Details are presented in the Supporting In-

formation (Figure S3).

which exhibit promising cytotoxicity as well as significant DNA

binding affinity, apart from inducing apoptosis. Apoptosis was

further confirmed by Annexin V-FITC and Hoechst staining, as

well as DNA fragmentation analysis. In addition, the effect of

these hybrids on topo I and their role as topo I inhibitors were

also studied. Docking and binding studies of these hybrids

demonstrated that topo I inhibition was mostly due to the for-

mation of a ternary complex with DNA–topo I, as well as

a two-fold mode of interaction with DNA. Moreover, DNA pho-

tocleavage studies confirmed that these hybrids could effec-

tively cleave the pBR322 plasmid DNA in the presence of UV

light. Furthermore, a DNA fragmentation assay demonstrated

that these hybrids can cross the cell membrane barrier and

cause DNA damage under normal cellular conditions. The

mode of binding of these new hybrids with DNA is different

from that of simple b-carboline alkaloids, which was supported

by both biophysical as well as molecular docking studies, sug-

gesting a combilexin-type of interaction with DNA. Overall,

two very potent hybrids were found, with 8d found to be su-

perior to 7d.

In addition, topo I inhibition may also occur due to the for-

mation of a hydrogen bond between the b-carboline ring and

the DNA–topo 1 complex. The presence of nitrogen atoms in

the b-carboline ring may also be associated with increased

topo I inhibition, as a similar observation was reported by

Beerman.^[46]

Biophysical studies speculated that these hybrids could

behave like combilexin-type molecules. In this regard, docking

studies were performed on two different DNA crystal struc-

tures. Docking studies performed with CT DNA (PDB ID:

1NAB)^[47]suggested intercalation as the binding mode, in

which the b-carboline ring was stacked within the DNA base

pairs, and the nitrogen atom showed hydrogen binding inter-

actions with the carbonyl of cytosine (Figure S1A and B, Sup-

porting Information). The second DNA structure on which

docking studies were performed was a complex with netropsin

(PDB ID: 195D), which showed minor groove-binding interac-

tions.^[48](Figure S1C and D, Supporting Information). The bind-

ing pose of hybrids 7d and 8d were shown, in which the C1

substituent and the b-carboline ring were facing towards the

minor groove and the C3 substituent was extending outwards.

These studies may be useful for understanding the role of

pharmacophores and their relative positions on a naturally oc-

curring b-carboline ring system that exhibits favorable biologi-

cal activities, as well as understanding the role of various side

chains attached to b-carboline in demonstrating higher cyto-

toxicity, apoptosis, inhibition of topo I, and photocleavage of

DNA.

Conclusions

Experimental Section

New b-carboline hybrids with significant antitumor activity, by

modulation of different motifs at the C1 and C3 positions,

were developed in addition to evaluating their mode of inter-

action with DNA. We designed and synthesized some b-carbo-

line hybrids by placing a substituted phenyl at the C1 position

and a chalcone/(N-acetyl)-pyrazole moiety at the C3 position.

These hybrids comprise two pharmacophoric groups (i.e., b-

carboline ring and (N-acetyl)-pyrazole or chalcone moieties),

Chemistry

General: Chemical reagents were purchased from Sigma–Aldrich

and were used without further purification. Anhydrous CH₂Cl₂, THF,

CH₃CN, toluene, and ether used in reactions were prepared by dis-

tillation under nitrogen over Na/benzophenone, CaH₂, Na/P₂O₅,

and CaH₂/molecular sieves, respectively. Solvents for extraction and

column chromatography were distilled prior to use. TLC analyses

were performed with silica gel plates (0.25 mm, E. Merck, 60 F₂₅₄

)

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using iodine, KMnO₄, and a UV lamp for visualization. H and ¹³C

NMR experiments were performed at 300 and 75 MHz, respectively,

on a Bruker Avance. Melting points were determined on an Electro-

thermal melting point apparatus and are uncorrected. Chemical

shifts are reported in parts per million (ppm) downstream from the

internal tetramethylsilane standard. Spin multiplicities are de-

scribed as s (singlet), bs (broad singlet), d (doublet), dd (double

doublet), t (triplet), q (quartet), or m (multiplet). Coupling con-

stants are reported in Hertz (Hz). FT-IR spectra for all compounds

were recorded using thin film as well as a KBr disk. Mass spectra

were recorded by electrospray ionization mass spectrometry (ESI-

MS).

7.52 Hz, 1H), 8.21 (d, J=7.54 Hz 1H), 8.75 (s, 1H), 9.38 ppm (bs,

1H); MS (ESI): m/z 393 [M+H]⁺.

1

Methyl-1-(3,4-difluorophenyl)-9H-pyrido[3,4-b]indole-3-carboxyl-

ate (4d): Off-white solid (5.51 g; 71%): mp: 236–2398C; ¹H NMR

(300 MHz, [D₆]DMSO): d=3.94 (s, 3H), 7.32–7.37 (m, 1H), 7.42–7.49

(m, 1H), 7.60–7.78 (m, 4H), 8.46 (d, J=7.93 Hz, 1H), 8.97 (s, 1H),

12.05 ppm (bs, 1H); MS (ESI): m/z 339 [M+H]⁺.

General procedure for the preparation of compounds 5a–d:

LiAlH₄(22 mmol) was added to a fine suspension of compounds

4a–d (20 mmol) in dry THF (60 mL) at 08C and was stirred for 4 h

at RT. The reaction mixture was then quenched with saturated aq

NH₄Cl and diluted with EtOAc (150 mL). The organic layer was

dried over anhydrous Na₂SO₄and concentrated under vacuum. The

obtained crude was used directly for the next step without any fur-

ther purification.

(S)-Methyl 2-amino-3-(1H-indol-3-yl)propanoate (2):^[16]SOCl₂

(8.02 mL, 0.11 mol) was added dropwise to a stirred solution of

commercially available l-tryptophan (20.4 g, 0.1 mol) in MeOH

(50 mL) at 08C and was stirred for 4 h at RT. Next, excess solvent

was removed under vacuum, and the crude product was washed

with toluene (2ꢁ10 mL) to obtain a clean solid. The resulting solid

was then dissolved in CH₂Cl₂(500 mL), basified with saturated aq

NaHCO₃(200 mL), and extracted into CH₂Cl₂(2ꢁ500 mL). The or-

ganic layer was dried over anhydrous Na₂SO₄and concentrated

under vacuum to obtain the pure product as a white solid

General procedure for the preparation of compounds 6a–d:

Dess–Martine periodinane (11.0 mmol) was added to a solution of

compound 5a–d (10.0 mmol) in CH₂Cl₂at 08C and was stirred at

RT for 3 h. The reaction mixture was then quenched with a 1:1 mix-

ture of aq sodium thiosulfate and NaHCO₃(40 mL) and diluted

with CH₂Cl₂(50 mL). The organic layer was dried over anhydrous

Na₂SO₄and concentrated under vacuum to obtain the crude prod-

uct. The resulting crude product was purified by column chroma-

tography to afford products 6a–d in high purity.

1

(19.62 g; 90%): mp: 103–1048C; H NMR (300 MHz, CDCl₃): d=2.00

(s, 2H), 3.00 (dd, J=4.12, 13.8 Hz, 1H), 3.25 (dd, J=4.45, 13.8 Hz,

1H), 3.73 (s, 3H), 3.84 (t, J=4.90, 5.42 Hz, 1H), 6.90 (s, 1H), 7.08 (t,

J=6.90, 1H), 7.14 (t, J=7.14 Hz, 1H), 7.25 (d, J=7.90 Hz, 1H), 7.58

(d, J=7.90 Hz, 1H), 8.81 ppm (bs, 1H); MS (ESI): m/z 219 [M+H]⁺.

1-(3-Chlorophenyl)-9H-pyrido[3,4-b]indole-3-carbaldehyde (6a):

1

Pale yellow solid (2.58 g; 84%): mp: 240–2438C; H NMR (300 MHz,

[D₆]DMSO): d=7.34–7.39 (m, 1H), 7.61–7.74 (m, 4H), 8.01–8.05 (m,

2H), 8.46 (d, J=7.93 Hz, 1H), 8.84 (s, 1H), 10.16 (s, 1H), 12.14 ppm

(s, 1H); MS (ESI): m/z: 307 [M+H]⁺.

General procedure for the preparation of compounds 3a–d: A

catalytic amount of PTSA was added to a mixture of the l-trypto-

phan ester (2, 0.023 mol) and substituted benzaldehyde

(0.023 mol) in toluene (70 mL), and the mixture was stirred at

110 8C for 12 h. After completion of the reaction, the toluene was

removed under vacuum, and the obtained crude was dissolved in

EtOAc (150 mL) and washed with water. Next, the organic layer

was dried over anhydrous Na₂SO₄and concentrated under

vacuum. The resulting diastereomeric mixture was purified by

column chromatography to obtain products 3a–d with maximum

purity.

1-(4-(Trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indole-3-carbalde-

hyde (6b): Pale yellow solid (2.77 g; 81%): mp: 280–2838C;

¹H NMR 300 MHz, [D₆]DMSO): d=7.34–7.39 (m, 1H), 7.61–7.71 (m,

2H), 8.01 (d, J=8.12 Hz, 2H), 8.28 (t, J=8.12 Hz, 2H), 8.44 (d, J=

7.93 Hz, 1H), 8.86 (s, 1H), 10.16 (s, 1H), 12.15 ppm (s, 1H); MS (ESI):

m/z: 341 [M+H]⁺.

1-(3,4,5-Trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbalde-

hyde (6c): Pale yellow solid (2.91 g; 80%): mp: 213–2148C;

¹H NMR (300 MHz, [D₆]DMSO): d=3.84 (s, 6H), 3.91 (s, 3H), 7.06 (s,

2H), 7.35–7.41 (m, 1H), 7.62 (d, J=7.54 Hz, 1H), 7.60–7.64 (m, 1H),

8.23 (d, J=7.54 Hz, 1H), 8.67 (s, 1H), 9.52 (bs, 1H), 10.23 ppm (s,

1H); MS (ESI): m/z 363 [M+H]⁺.

General procedure for the preparation of compounds 4a–d: A

suspension of compounds 3a–d (0.025 mol) and sulfur (0.075 mol)

in xylene (100 mL) was heated at 1208C for 8 h. The mixture was

then cooled to RT and allowed to stand at 48C for 3 h to obtain

precipitate. The precipitate was filtered, washed with hexane, and

dried. The obtained solid was recrystallized from EtOAc to afford

products 4a–d in high purity.

1-(3,4-Difluorophenyl)-9H-pyrido[3,4-b]indole-3-carbaldehyde

(6d): Pale yellow solid (2.60 g; 84%): mp: 225–2288C; ¹H NMR

(300 MHz, [D₆]DMSO): d=7.34–7.39 (m, 1H), 7.44–7.49 (m, 1H),

7.62–7.80 (m, 2H), 7.92–8.03 (m, 2H), 8.48 (d, J=7.93 Hz, 1H), 8.88

(s, 1H), 10.16 ppm (s, 1H); MS (ESI): m/z: 309 [M+H]⁺.

Methyl-1-(3-chlorophenyl)-9H-pyrido[3,4-b]indole-3-carboxylate

(4a):^[49]Pale yellow solid (6.2 g; 80%): mp: 209–2118C; ¹H NMR

(300 MHz, [D₆]DMSO): d=3.94 (s, 3H), 7.27–7.32 (m, 1H), 7.47- 7.71

(m, 4H), 7.98 (d, J=10.38 Hz, 2H), 8.31 (s, 1H), 8.87 (s, 1H),

11.93 ppm (bs, 1H); MS (ESI): m/z 337 [M+H]⁺.

General procedure for the preparation of chalcone derivatives

7a–m: The corresponding ketone (0.5 mmol) was added to a stirred

solution of aldehydes 6a–d (0.5 mmol) in MeOH, followed by addi-

tion of Ba(OH)₂(0.75 mmol), and stirring was continued at RT for

the desired time (see Table S1, Supporting Information). The sol-

vent was then removed under vacuum, and the residue was dis-

solved in EtOAc (100 mL) and washed with water. The organic

layer was dried over anhydrous Na₂SO₄and concentrated under

vacuum to obtain the crude product, which was purified by

column chromatography to offered products 7a–m in high purity.

Methyl-1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indole-3-

carboxylate (4b):^[50]Pale yellow solid (6.14 g; 72%): mp: 252–

2548C; ¹H NMR (300 MHz, [D₆]DMSO): d=3.94 (s, 3H), 7.30–7.37

(m, 1H), 7.56–7.61 (m, 1H), 7.68 (d, J=8.12 Hz, 1H), 7.92 (d, J=

8.12 Hz, 2H), 8.23 (d, J=7.74 Hz, 2H), 8.36 (d, J=7.74 Hz, 1H), 8.93

(s, 1H), 11.99 ppm (bs, 1H); MS (ESI): m/z 371 [M+H]⁺.

Methyl-1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-car-

boxylate (4c):^[50]Yellow solid (6.85 g; 76%): mp: 229–2308C;

¹H NMR (300 MHz, CDCl₃): d=3.77 (s, 6H), 3.84 (s, 3H), 4.03 (s, 3H),

6.94 (s, 2H), 7.32–7.38 (m, 1H), 7.56–7.61 (m, 1H), 7.60 (d, J=

(E)-3-(1-(3-Chlorophenyl)-9H-pyrido[3,4-b]indol-3-yl)-1-(1H-

pyrrol-2-yl)prop-2-en-1-one (7a): Yellow solid (139 mg; 70%): mp:

1

190–1948C; H NMR (300 MHz, [D₆]DMSO+CDCl₃): d=6.32 (s, 1H),

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7.11 (s, 1H), 7.19 (s, 1H), 7.31 (t, J=7.47 Hz, 1H), 7.48–7.61 (m, 4H),

7.66 (d, J=8.08 Hz, 1H), 7.96- 8.01 (m, 1H), 8.03–8.06 (m, 1H), 8.12

(s, 1H), 8.16–8.22 (m, 2H), 11.20 (bs, 1H), 11.25 ppm (s, 1H);

¹³C NMR (75 MHz, [D₆]DMSO+CDCl₃): d=110.2, 112.7, 115.9, 116.9,

120.2, 121.0, 121.6, 123.0, 126.3, 127.2, 128.2, 128.6, 128.7, 130.1,

130.7, 133.1, 133.3, 133.6, 139.9, 140.6, 141.5, 141.6, 142.7,

177.9 ppm; MS (ESI): m/z: 398 [M+H]⁺; HRMS (ESI): m/z calcd for

C₂₄H₁₇ON₃Cl: 398.10547; found: 398.10446 [M+H]⁺.

(ESI): m/z: 458 [M+H]⁺; HRMS (ESI): m/z calcd for C₂₇H₁₉ON₃F₃:

458.14747; found: 458.14596 [M+H]⁺.

(E)-1-(Thiophen-2-yl)-3-(1-(3,4,5-trimethoxyphenyl)-9H-pyrido-

[3,4-b]indol-3-yl)prop-2-en-1-one (7g): Yellow solid (173 mg; 74%

yield): mp: 217–2208C; ¹H NMR (300 MHz, [D₆]DMSO): d=3.79 (s,

3H), 3.96 (s, 6H), 7.26–7.39 (m, 4H), 7.56–7.61 (m, 1H), 7.66 (d, J=

8.12 Hz, 1H), 7.92–8.16 (m, 3H), 8.22 (s, 1H), 8.27 (d, J=8.12 Hz,

1H), 8.71 (s, 1H), 11.62 ppm (s, 1H); ¹³C NMR (75 MHz, [D₆]DMSO):

d=55.9, 60.0, 105.9, 112.8, 116.3, 120.2, 121.1, 121.5, 128.9, 129.4,

133.0, 133.2, 133.5, 135.2, 138.1, 141.3, 141.9, 142.8, 144.1, 145.4,

153.1, 181.6 ppm; FT-IR: n˜ =638, 738, 786, 971, 1006, 1236, 1313,

1405, 1462, 1500, 1583, 1733, 2360, 2851, 2923, 3430 cm^ꢂ1; MS

(ESI): m/z: 471 [M+H]⁺; HRMS (ESI): m/z calcd for C₂₇H₂₃N₂O₄S:

471.13623; found: 471.13587 [M+H]⁺.

(E)-3-(1-(3-Chlorophenyl)-9H-pyrido[3,4-b]indol-3-yl)-1-(furan-2-

yl)prop-2-en-1-one (7b): Yellow solid (159 mg; 80%): mp: 188–

1908C; ¹H NMR (300 MHz, CDCl₃): d=6.74 (s, 1H), 7.41–7.47 (m,

1H), 7.49 (s, 1H), 7.51–7.55 (m, 1H), 7.63 (d, J=7.93 Hz, 1H), 7.65–

7.71 (m, 2H), 7.78 (d, J=8.08 Hz, 1H), 7.82 (s, 1H), 8.12–8.16 (s,

1H), 8.21–8.26 (m, 2H), 8.29–8.31 (m, 2H), 10.95 ppm (s, 1H);

¹³C NMR (75 MHz, [D₆]DMSO+CDCl₃): d=111.9, 112.2, 116.5, 117.2,

119.8, 120.8, 121.3, 126.6, 127.9, 128.2, 129.6, 129.7, 133.5, 134.0,

139.7, 141.1, 141.9, 143.5, 146.1, 153.1, 177.7 ppm; MS (ESI): m/z:

399 [M+H]⁺; HRMS (ESI): m/z calcd for C₂₄H₁₆O₂N₂Cl: 399.08948;

found: 399.08905 [M+H]⁺.

(E)-1-(Furan-2-yl)-3-(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-

b]indol-3-yl)prop-2-en-1-one (7h): Yellow solid (186 mg; 82%):

mp: 215–2178C; ¹H NMR (300 MHz, [D₆]DMSO): d=3.79 (s, 3H),

3.95 (s, 6H), 6.79–6.81 (m, 1H), 7.28–7.35 (m, 3H), 7.59 (t, J=

7.55 Hz, 1H), 7.66–7.70 (m, 2H), 7.99 (s, 2H), 8.08 (s, 1H), 8.30 (d,

J=7.36 Hz, 1H), 8.66 (s, 1H), 11.83 ppm (s, 1H); ¹³C NMR (75 MHz,

[D₆]DMSO): d=55.9, 60.0, 105.9, 112.8, 116.5, 118.7, 120.2, 121.1,

121.5, 128.4, 129.4, 133.2, 133.5, 138.1, 141.3, 141.8, 142.9, 143.8,

148.1, 153.1, 177.0 ppm; FT-IR: n˜ =590, 630, 705, 722, 750, 802,

847, 862, 881, 975, 1010, 1047, 1086, 1128, 1182, 1244, 1324, 1362,

1392, 1424, 1463, 1568, 1645, 2359, 2925, 3293 cm^ꢂ1; MS (ESI): m/z:

455 [M+H]⁺; HRMS (ESI): m/z calcd for C₂₇H₂₃N₂O₅: 455.15770;

found: 455.15884 [M+H]⁺.

(E)-1-(1H-Pyrrol-2-yl)-3-(1-(4-(trifluoromethyl)phenyl)-9H-pyrido-

[3,4-b]indol-3-yl)prop-2-en-1-one (7c): Yellow solid (140 mg;

65%): mp: 222–2248C; ¹H NMR (300 MHz, [D₆]DMSO): d=6.30 (s,

1H), 7.19 (s, 1H), 7.29–7.36 (m, 2H), 7.56–7.69 (m, 2H), 7.93–8.01

(m, 4H), 8.31 (d, J=7.97 Hz, 3H), 8.73 (s, 1H), 11.89 (s, 1H),

12.01 ppm (s, 1H); ¹³C NMR (75 MHz, [D₆]DMSO): d=110.2, 112.7,

116.2, 116.9, 120.2, 121.0, 121.7, 122.5, 123.2, 125.6, 126.3, 128.7,

129.1, 129.4, 130.2, 133.1, 133.5, 140.6, 141.5, 141.7, 142.8,

177.9 ppm; MS (ESI): m/z: 432 [M+H]⁺; HRMS (ESI): m/z calcd for

C₂₅H₁₇F₃N₃O: 432.13076; found: 432.13049 [M+H]⁺.

(E)-1-(4-Aminophenyl)-3-(1-(3,4,5-trimethoxyphenyl)-9H-pyrido-

[3,4-b]indol-3-yl)prop-2-en-1-one (7i): Yellow solid (153 mg; 64%

yield): mp: 229–2318C; ¹H NMR (300 MHz, [D₆]DMSO): d=3.95 (s,

3H), 3.96 (s, 6H), 4.14 (bs, 2H), 6.68 (d, J=8.43 Hz, 2H), 7.18 (s,

2H), 7.35 (t, J=7.92 Hz, 1H), 7.55 (t, J=7.78 Hz, 1H), 7.56 (d, J=

8.16 Hz, 1H), 7.96 (d, J=15.10 Hz, 1H), 8.01 (d, J=8.42 Hz, 2H,),

8.11 (d, J=15.10 Hz, 1H), 8.18 (d, J=7.92 Hz, 1H,), 8.10 (s, 1H),

8.11 ppm (s, 1H); ¹³C NMR (75 MHz, [D₆]DMSO): d=55.8, 59.9,

112.8, 112.6, 115.2, 119.9, 121.0, 121.1, 121.5, 122. 1, 125.5, 128.2,

129.5, 130.8, 133.2, 133.4, 138.0, 141.3, 142.5, 142.4, 142.7, 153.0,

153.7, 186.0 ppm; FT-IR: n˜ =3401, 3258, 3001, 2927, 1647, 1614,

1582, 1427, 1312, 1231, 1127, 1037, 820, 753 cm^ꢂ1; MS (ESI): m/z:

480 [M+H]⁺; HRMS (ESI): m/z calcd. for C₂₉H₂₆N₃0₄: 480.1923

found: 480.1925 [M+H]⁺.

(E)-1-(Furan-2-yl)-3-(1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-

b]indol-3-yl)prop-2-en-1-one (7d): Yellow solid (183 mg; 85%):

mp: 240–2438C; ¹H NMR (300 MHz, [D₆]DMSO+CDCl₃): d=6.68–

6.70 (m, 1H), 7.28 (t, J=7.33 Hz, 1H), 7.51–7.55 (m, 1H), 7.62 (d, J=

8.06 Hz, 1H), 7.89 (d, J=8.06 Hz, 3H), 7.97 (s, 2H), 8.12 (s, 1H),

8.22–8.30 (m, 3H), 8.54 (s, 1H), 11.84 ppm (s, 1H); ¹³C NMR

(75 MHz, [D₆]DMSO): d=112.7, 112.8, 117.3, 118.7, 120.3, 120.9,

121.7, 125.6, 128.7, 129.4, 130.0, 133.7, 140.9, 141.5, 141.6, 142.1,

143.5, 148.07, 153.1, 176.9 ppm; MS (ESI): m/z: 433 [M+H]⁺; HRMS

(ESI): m/z calcd for C₂₅H₁₆O₂N₂F₃: 433.11584; found: 433.11466

[M+H]⁺.

(E)-1-(Thiophen-2-yl)-3-(1-(4-(trifluoromethyl)phenyl)-9H-pyrido-

[3,4-b]indol-3-yl)prop-2-en-1-one (7e): Yellow solid (179 mg;

(E)-3-(1-(3,4-Difluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)-1-(thio-

phen-2-yl)prop-2-en-1-one (7j): Yellow solid (156 mg; 75% yield):

mp: 238–2428C; ¹H NMR (300 MHz, [D₆]DMSO): d=7.23–7.45 (m,

3H), 7.50–7.88 (m, 5H), 7.92–8.17 (m, 2H), 8.24 (d, J=18.31 Hz,

2H), 8.64–8.87 (m, 1H), 10.18 ppm (s, 1H); ¹³C NMR (75 MHz,

[D₆]DMSO): d=111.4, 111.7, 112.6, 114.2, 117.0, 119.9, 120.2, 120.6,

121.4, 121.9, 127.9, 128.4, 128.6, 128.9, 130.2, 132.8, 133.3, 134.9,

139.4, 141.1, 141.4, 143.6, 145.3, 181.5 ppm; FT-IR: n˜ =534, 621,

680, 718, 792, 837, 859, 968, 1046, 1082, 1113, 1233, 1245, 1352,

1402, 1496, 1567, 1591, 1624, 1638, 1681, 2356, 2852, 2922, 3084,

3298 cm^ꢂ1; MS (ESI): m/z: 417 [M+H]⁺; HRMS (ESI): m/z calcd for

C₂₄H₁₅F₂N₂OS: 417.08454; found: 417.08539 [M+H]⁺.

1

80%): mp: 250–2548C; H NMR (300 MHz, [D₆]DMSO): d=7.18–7.27

(m, 1H), 7.33 (t, J=7.3 Hz, 1H), 7.48–7.70 (m, 3H), 7.75 (d, J=

4.7 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.96–8.11 (m, 3H), 8.20 (d, J=

7.9 Hz, 1H), 8.24–8.36 (m, 3H), 11.42 ppm (s, 1H); ¹³C NMR

(75 MHz, [D₆]DMSO+CDCl₃): d=111.6, 113.3, 113.9, 118.5, 120.6,

121.7, 122.7, 125.0, 125.6, 125.9, 128.7, 130.6, 131.1, 133.3, 133.4,

133.7, 135.7, 136.8, 137.6, 138. 4, 144.0, 145.2, 181.0 ppm; MS (ESI):

m/z: 449 [M+H]⁺; HRMS (ESI): m/z calcd for C₂₅H₁₆ON₂F₃S:

449.09299; found: 449.09164 [M+H]⁺.

(E)-1-(4-Aminophenyl)-3-(1-(4-(trifluoromethyl)phenyl)-9H-

pyrido[3,4-b]indol-3-yl)prop-2-en-1-one

(7 f):

Yellow

solid

(148 mg; 65% yield): mp: 221–2238C; ¹H NMR (300 MHz,

[D₆]DMSO+CDCl₃): d=4.97 (s, 2H), 6.67–6.82 (m, 1H), 7.26–7.41 (m,

1H), 7.48–7.74 (m, 3H), 7.85–8.10 (m, 5H), 8.11–8.41 (m, 5H), 11.31

(s, 1H) ppm; ¹³C NMR (75 MHz, [D₆]DMSO+CDCl₃): d=112.1, 113.0,

116.0, 119.7, 120.8, 120.9, 122.1, 125.0, 126.7, 128.0, 128.8, 129.9,

130.5, 133.4, 140.7, 141.2, 141.6, 142.1, 142.7, 152.2, 187.2 ppm; FT-

IR: n˜ =543, 609, 689, 741, 831, 971, 1014, 1064, 1110, 1236, 1323,

1390, 1444, 1493, 1599, 2359, 2852, 2921, 3353, 3427 cm^ꢂ1; MS

(E)-1-(4-Aminophenyl)-3-(1-(3,4-difluorophenyl)-9H-pyrido[3,4-

b]indol-3-yl)prop-2-en-1-one (7k): Yellow solid (140 mg; 66%

yield): mp: 195–1988C; ¹H NMR (300 MHz, [D₆]DMSO): d=6.04 (s,

2H), 6.66 (d, J=8.30 Hz, 2H), 7.31 (t, J=7.36 Hz, 1H), 7.55–7.74 (m,

4H), 7.90 (d, J=7.93 Hz, 2H), 7.98 (s, 1H), 8.04–8.29 (m, 3H), 8.57

(s, 1H), 11.77 ppm (s, 1H); ¹³C NMR (75 MHz, [D₆]DMSO): d=112.6,

112.7, 115.9, 117.6, 117.7, 117.9, 120.1, 121.0, 121.6, 122.4, 125.4,

125.6, 128.6, 130.1, 130.5, 130.9, 131.5, 133.1, 139.9, 141.4, 142.2,

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142.8, 153.8, 186.0 ppm; FT-IR: n˜ =574, 607, 736, 825, 978, 1022,

1116, 1176, 1240, 1290, 1306, 1357, 1377, 1405, 1436, 1464, 1543,

1634, 1687, 1718, 2854, 2924, 3056, 3339, 3402 cm^ꢂ1; MS (ESI): m/z:

426 [M+H]⁺; HRMS (ESI): m/z calcd for C₂₆H₁₈F₂N₃O: 426.14125;

found: 426.14005 [M+H]⁺.

1-(3-(1H-Pyrrol-2-yl)-5-(1-(4-(trifluoromethyl)phenyl)-9H-pyrido-

[3,4-b]indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (8c): Off-

white solid (87 mg; 72% yield): mp: 326–3308C; H NMR (300 MHz,

1

[D₆]DMSO+[D₆]acetone): d=2.33 (s, 3H), 3.48 (dd, J=4.53,

17.53 Hz, 1H), 3.83 (dd, J=11.51, 17.37 Hz, 1H), 5.80 (dd, J=4.34,

11.14 Hz, 1H), 6.14 (s, 1H), 6.54 (s, 1H), 6.96 (s, 1H), 7.26 (t, J=

7.36 Hz, 1H), 7.52–7.57 (m, 1H), 7.64 (d, J=8.30 Hz, 1H), 7.90 (d,

J=7.93 Hz, 2H), 8.08 (s, 1H), 8.22–8.33 (m, 3H), 11.44 (s, 1H),

11.59 ppm (s, 1H); ¹³C NMR (75 MHz, CDCl₃): d=21.5, 40.9, 60.3,

108.9, 111.8, 112.3, 119.5, 120.8, 121.9, 122.0, 123.9, 125.4, 126.1,

128.3, 128.4, 129.0, 130.4, 142.1, 145.2, 148.3, 149.0, 167.1 ppm; MS

(ESI): m/z: 488 [M+H]⁺; HRMS (ESI): m/z calcd for C₂₇H₂₁F₃N₅O:

488.16927; found: 488.16765 [M+H]⁺.

(E)-3-(1-(3,4-Difluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)-1-(furan-

2-yl)prop-2-en-1-one (7l): Yellow solid (154 mg; 77% yield): mp:

224–2268C; ¹H NMR (300 MHz, [D₆]DMSO): d=6.93–6.98 (m, 1H),

7.60–7.68 (m,1H), 7.75–7.77 (m, 2H), 7.86–7.92 (m, 2H), 7.99 (d, J=

8.12 Hz, 1H), 8.05–8.07 (m, 1H), 8.25–8.32 (m, 1H), 8.35–8.45 (m,

2H), 8.45–8.53 (m, 2H), 11.52 ppm (s, 1H); ¹³C NMR (75 MHz,

[D₆]DMSO): d=111.7, 112.1, 112.3, 116.4, 117.16, 117.2, 117.4, 119.0,

119.8, 120.8, 121.0, 121.3, 124.9, 128.1, 129.8, 133.2, 140.0, 141.2,

141.7, 143.3, 144.0, 146.5, 153.1, 177.2 ppm; MS (ESI): m/z: 401

[M+H]⁺.

1-(3-(Furan-2-yl)-5-(1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-

b]indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (8d): Off-

white solid (104 mg; 85%): mp: 217–2008C; ¹H NMR (300 MHz,

CDCl₃): d=2.50 (s, 3H), 3.48 (dd, J=4.99, 17.99 Hz, 1H), 3.73 (dd,

J=11.99, 16.99 Hz, 1H), 5.88 (dd, J=3.99, 10.99 Hz, 1H), 6.50 (s,

1H), 6.75 (d, J=2.99 Hz, 1H), 7.23 (d, J=6.99 Hz, 1H), 7.49–7.54 (m,

2H), 7.55 (s, 1H), 7.72 (d, J=6.99 Hz, 1H), 7.76 (s, 1H), 7.79 (s, 1H),

7.91 (d, J=6.99 Hz, 2H), 7.94 (d, J=7.99 Hz, 1H), 8.77 ppm (s, 1H);

¹³C NMR (75 MHz, CDCl₃): d=21.8, 40.6, 60.5, 112.1, 112.4, 113.7,

119.7, 120.7, 121.8, 125.5, 128.5, 128.8, 129.1, 130.3, 132.4, 139.7,

141.6, 141.9, 148.5, 167.7 ppm; FT-IR: n˜ =436, 592, 619, 682, 746,

857, 884, 899, 1164, 1240, 1358, 1394, 1451, 1466, 1492, 1562,

1624, 1642, 2924, 3229 cm^ꢂ1; MS (ESI): m/z: 489 [M+H]⁺; HRMS

(ESI): m/z calcd for C₂₇H₂₀F₃N₄O₂: 489.15329; found: 489.15158

[M+H]⁺.

(E)-3-(1-(3,4-Difluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)-1-(3,4,5-

trimethoxyphenyl)prop-2-en-1-one (7m): Yellow solid (187 mg;

75%): mp: 213–2168C; ¹H NMR (300 MHz, CDCl₃+[D₆]DMSO): d=

3.78 (s, 3H), 3.92 (s, 6H), 7.18–7.26 (m, 1H), 7.32–7.38 (m, 1H), 7.42

(s, 2H), 7.59–7.64 (m, 1H), 7.68–7.72 (m, 2H), 7.99 (s, 1H), 8.16 (s,

1H), 8.28 (d, J=7.93 Hz, 1H), 8.31 (d, J=7.93 Hz, 1H), 8.78 (s, 1H),

11.92 ppm (s, 1H); ¹³C NMR (75 MHz, [D₆]DMSO): d=55.9, 60.0,

105.5, 105.8, 112.5, 116.2, 117.3, 117.5, 120.0, 120.9, 121.4, 121.9,

125.1, 128.3, 130.0, 133.2, 135.1, 139.8, 141.4, 141.7, 142.1, 144.3,

152.7, 188.1 ppm; MS (ESI): m/z: 501 [M+H]⁺; HRMS (ESI): m/z

calcd for C₂₉H₂₃F₂N₂O₄: 501.16204; found: 501.16026 [M+H]⁺.

General procedure for the preparation of chalcone derivatives

8a–m: NH₂-NH₂·H₂O (0.5 mmol) was added to a stirred solution of

chalcones 7a–m (0.25 mmol) in acetic acid at 08C, and the mixture

was stirred at reflux for the desired time (see Table S1). The reac-

tion mixture was then cooled, water was added, and the mixture

was allowed to stand for 2 h at 08C to obtain precipitate. The re-

sulting crude product was purified by column chromatography to

offered products (8a–m) in high purity.

1-(3-(Thiophen-2-yl)-5-(1-(4-(trifluoromethyl)phenyl)-9H-pyrido-

[3,4-b]indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone

(8e):

White solid (100 mg; 80%): mp: 230–2358C; ¹H NMR (300 MHz,

CDCl₃): d=2.49 (s, 3H), 3.53 (dd, J=4.53, 17.37 Hz, 1H), 3.80 (dd,

J=11.33, 17.37 Hz, 1H), 5.90 (dd, J=4.53, 11.33 Hz, 1H), 7.02–7.06

(m, 1H), 7.21–7.28 (m, 2H), 7.42 (d, J=4.53 Hz, 1H), 7.48–7.56 (m,

2H), 7.72–7.76 (m, 3H), 7.94 (dd, J=8.30, 18.12 Hz, 3H), 8.76 ppm

(s, 1H); ¹³C NMR (75 MHz, CDCl₃): d=21.7, 41.5, 61.1, 112.3, 119.6,

120.7, 121.8, 123.2, 125.3, 125.5, 127.9, 128.5, 128.8, 129.1, 129.3,

130.2, 132.4, 134.3, 139.7, 141.6, 141.8, 148.5, 150.4, 167.4 ppm; FT-

IR: n˜ =437, 583, 683, 704, 841, 857, 899, 963, 1016, 1160, 1251,

1284, 1416, 1498, 1525, 1561, 1642, 2359, 2924, 3209,

3434 cm^ꢂ1;MS (ESI): m/z: 504 [M+H]⁺; HRMS (ESI): m/z calcd for

C₂₇H₂₀F₃N₄OS: 505.13044; found: 505.13025 [M+H]⁺.

1-(5-(1-(3-Chlorophenyl)-9H-pyrido[3,4-b]indol-3-yl)-3-(1H-pyrrol-

2-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (8a): Off-white solid

1

(90 mg; 80%): mp: 231–2338C; H NMR (300 MHz, [D₆]DMSO): d=

2.33 (s, 3H), 3.49–3.51 (m, 1H), 3.78 (dd, J=11.33, 17.37 Hz, 1H),

5.69–5.80 (m, 1H), 6.14 (s, 1H), 6.54 (s, 1H), 6.96 (s, 1H), 7.23–7.28

(m, 1H), 7.50–7.67 (m, 5H), 7.96 (m, 2H), 8.26 (d, J=7.74 Hz, 1H),

11.50 (ds, 1H), 11.59 ppm (bs, 1H); ¹³C NMR (75 MHz, [D₆]DMSO):

d=21.7, 40.1, 60.1, 109.2, 111.4, 112.2, 112.4, 118.0, 119.6, 120.7,

121.7, 122.3, 123.7, 126.9, 128.0, 128.4, 129.2, 130.2, 130.5, 132.1,

133.5, 140.1, 141.6, 148.5, 148.7, 167.1 ppm; MS (ESI): m/z: 454

[M+H]⁺.

N-(4-(1-Acetyl-5-(1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-

b]indol-3-yl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)acetamide (8 f):

White solid (76 mg; 55%): mp: 218–2208C; ¹H NMR (300 MHz,

[D₆]DMSO): d=2.06 (s, 3H), 2.34 (s, 3H), 3.43–3.56 (m, 1H), 3.79–

3.95 (m, 1H), 5.79 (dd, J=5.09, 11.70 Hz, 1H), 7.26 (t, J=7.74,

8.12 Hz, 1H), 7.53–7.63 (m, 2H), 7.60 (d, J=8.68 Hz, 2H), 7.77 (d,

J=8.68, 2H), 7.91 (d, J=7.93 Hz, 2H), 8.09 (s, 1H), 8.18 (d, J=

7.93 Hz, 2H), 8.30 (d, J=7.74 Hz, 1H), 10.16 (s, 1H), 11.62 ppm (s,

1H); ¹³C NMR (75 MHz, CDCl₃): d=21.8, 24.0, 40.8, 60.9, 112.3,

113.3, 118.7, 119.6, 120.7, 121.9, 123.2, 125.5, 125.8, 127.3, 128.5,

129.1, 130.2, 132.4, 139.7, 141.0, 141.6, 141.9, 148.8, 154.1, 167.5,

168.5 ppm; FT-IR: n˜ =503, 620, 690, 743, 899, 956, 1015, 1126,

1168, 1243, 1267, 1365, 1409, 1452, 1530, 1636, 2921, 3298,

3304 cm^ꢂ1; MS (ESI): m/z: 556 [M+H]⁺; HRMS (ESI): m/z calcd for

C₃₁H₂₅O₂N₅F₃: 556.19549; found: 556.19377 [M+H]⁺.

1-(5-(1-(3-Chlorophenyl)-9H-pyrido[3,4-b]indol-3-yl)-3-(furan-2-

yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (8b): Off-white solid

1

(96 mg; 85%): mp: 245–2488C; H NMR (300 MHz, CDCl₃): d=2.50

(s, 3H), 3.52 (dd, J=5.14, 17.99 Hz, 1H), 3.74 (dd, J=11.57,

17.99 Hz, 1H), 5.89 (dd, J=3.85, 11.57 Hz, 1H), 6.49 (s, 1H), 6.74 (d,

J=3.85 Hz, 1H), 7.22 (d, J=7.71 Hz, 1H), 7.38–7.44 (m, 2H), 7.46–

7.52 (m, 2H), 7.54 (s, 1H), 7.73 (d, J=7.71 Hz, 1H), 7.79 (s, 1H), 7.84

(s, 1H), 7.99 (d, J=7.71 Hz, 1H), 8.66 ppm (s, 1H); ¹³C NMR

(75 MHz, CDCl₃): d=21.7, 40.5, 60.4, 111.7, 112.0, 112.3, 113.6, 119.6,

120.6, 121.7, 126.8, 127.9, 128.3, 130.2, 130.4, 132.1, 133.4, 139.5,

140.0, 141.5, 145.2, 146.0, 146.4, 148.2, 167.5 ppm; FT-IR: n˜ =476,

621, 669, 747, 763, 866, 896, 972, 1006, 1046, 1144, 1243, 1317,

1392, 1493, 1564, 1593, 1642, 2360, 3232 cm^ꢂ1; MS (ESI): m/z: 455

[M+H]⁺; HRMS (ESI): m/z calcd for C₂₆H₂₀ClN₄O₂: 455.12693; found:

455.12714 [M+H]⁺.

1-(3-(Thiophen-2-yl)-5-(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-

b]indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (8g): White

solid (79 mg; 68% yield): mp: 160–1628C; ¹H NMR (300 MHz,

CDCl₃): d=2.44 (s, 3H), 3.60–3.68 (m, 1H), 3.74–3.80 (m, 1H), 3.85

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(s, 6H), 3.90 (s, 3H), 5.95 (dd, J=4.53, 11.33 Hz, 1H), 7.04–7.07 (m,

1H), 7.13 (s, 2H), 7.26–7.29 (m, 2H), 7.40 (d, J=5.28 Hz, 1H), 7.45–

7.53 (m, 2H), 7.88 (s, 1H), 8.06 (d, J=7.55 Hz, 1H), 8.59 ppm (s,

1H); ¹³C NMR (75 MHz, CDCl₃): d=21.9, 41.6, 56.2, 60.8, 61.3, 105.6,

110.9, 111.6, 120.1, 121.9, 127.5, 128.3, 128.4, 128.6, 130.6, 132.6,

134.0, 135.3, 138.6, 140.9, 142.0, 148.3, 150.8, 153.6, 169.0 ppm; FT-

IR: n˜ =582, 628, 724, 747, 789, 840, 899, 938, 963, 1003, 1033, 1125,

1182, 1232, 1268, 1324, 1360, 1394, 1452, 1503, 2830, 2993, 3093,

3272 cm^ꢂ1; MS (ESI): m/z: 527 [M+H]⁺; HRMS (ESI): m/z calcd for

C₂₉H₂₇N₄O₄S: 527.17235; found: 527.17303 [M+H]⁺.

830, 938, 981, 1009, 1107, 1179, 1199, 1238, 1318, 1364, 1496, 1510,

1623, 1634, 2925, 3228 cm^ꢂ1; MS (ESI): m/z: 482 [M+H]⁺; HRMS

(ESI): m/z calcd for C₂₈H₂₂F₂N₅O: 482.17869; found: 482.17695

[M+H]⁺.

1-(5-(1-(3,4-Difluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)-3-(furan-

2-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (8l): White solid

(72 mg; 64%): mp: 210–2128C; ¹H NMR (300 MHz, [D₆]acetone):

d=2.30 (s, 3H), 3.53–3.63 (m, 1H), 3.77–3.90 (m, 1H), 5.86 (dd, J=

4.91, 11.70 Hz, 1H), 6.58–6.64 (m, 1H), 6.93 (d, J=3.39 Hz, 1H),

7.25–7.32 (m, 1H), 7.42–7.64 (m, 4H), 7.74 (s, 1H), 7.85–7.91 (m,

1H), 8.09 (s, 1H), 8.24 (d, J=7.74 Hz, 1H), 10.80 ppm (s, 1H);

¹³C NMR (75 MHz, [D₆]acetone): d=23.1, 42.3, 62.7, 113.8, 114.1,

114.4, 119.0, 119.2, 119.5, 121.7, 123.5, 126.8, 130.3, 132.7, 134.3,

141.2, 143.6, 146.7, 147.9, 149.4, 150.7, 169.7 ppm; MS (ESI): m/z:

457 [M+H]⁺; HRMS (ESI): m/z calcd for C₂₆H₁₉O₂N₄F₂: 457.14706;

found: 457.14620 [M+H]⁺.

1-(3-(Furan-2-yl)-5-(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-

b]indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (8h): White

1

solid (95 mg; 75%): mp: 195–1978C; H NMR (300 MHz, CDCl₃): d=

2.47 (s, 3H), 3.56 (dd, J=4.34, 17.37 Hz, 1H), 3.72 (dd, J=11.33,

17.37 Hz, 1H), 3.87 (s, 6H), 3.90 (s, 3H), 5.95 (dd, J=3.96 Hz,

11.14 Hz, 1H), 6.49–6.51 (m, 1H), 6.75 (d, J=3.39 Hz, 1H), 7.13 (s,

2H), 7.26–7.29 (m, 1H), 7.45–7.52 (m, 2H), 7.55 (s, 1H), 7.87 (s, 1H),

8.09 (d, J=7.93 Hz, 1H), 8.54 ppm (s, 1H); ¹³C NMR (75 MHz,

CDCl₃): d=22.1, 40.8, 56.3, 60.3, 60.9, 105.6, 110.8, 111.6, 111.8,

112.6, 120.1, 121.9, 128.4, 130.6, 132.7, 134.0, 138.6, 140.9, 142.1,

144.6, 146.8, 147.2, 148.4, 153.6, 169.2 ppm; FT-IR: n˜ =592, 630,

696, 747, 789, 846, 884, 899, 915, 958, 1006, 1035, 1125, 1183,

1234, 1325, 1360, 1390, 1494, 1583, 1638, 2359, 2832, 2935,

3292 cm^ꢂ1; MS (ESI): m/z: 511 [M+H]⁺; HRMS (ESI): m/z calcd for

C₂₉H₂₇N₄O₅: 511.19519; found: 511.19537 [M+H]⁺.

1-(5-(1-(3,4-Difluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)-3-(3,4,5-

trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (8m):

White solid (99 mg; 71%): mp: 165–1688C; ¹H NMR (300 MHz,

[D₆]DMSO): d=2.36 (s, 3H), 3.51–3.60 (m, 1H), 3.69 (s, 3H), 3.82 (s,

6H), 3.86–3.96 (m, 1H), 5.80 (dd, J=4.76, 11.73 Hz, 1H), 7.09 (s,

2H), 7.22–7.29 (m, 1H), 7.50–7.71 (m, 3H), 7.85 (s, 1H), 7.93–8.03

(m, 1H), 8.05 (s, 1H), 8.30 (d, J=7.70 Hz, 1H), 11.60 ppm (s, 1H);

¹³C NMR (75 MHz, CDCl₃): d=2.8, 41.0, 55.9, 60.1, 61.0, 104.0, 111.5,

112.3, 117.2, 117.7, 119.6, 120.7, 121.8, 125.2, 126.8, 128.4, 130.3,

132.0, 135.5, 138.9, 139.3, 141.6, 148.6, 150.7, 153.0, 154.6,

167.6 ppm; FT-IR: n˜ =609, 747, 1004, 1127, 1240, 1320, 1369, 1415,

1461, 1523, 1578, 1643, 2933, 3430 cm^ꢂ1; MS (ESI): m/z: 557

[M+H]⁺; HRMS (ESI): m/z calcd for C₃₁H₂₇F₂N₄O₄: 557.19949; found:

557.19861 [M+H]⁺.

1-(3-(4-Aminophenyl)-5-(1-(3,4,5-trimethoxyphenyl)-9H-pyrido-

[3,4-b]indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone

(8i):

White solid (70 mg; 52%): mp: 165–1678C; ¹H NMR (300 MHz,

CDCl₃): d=2.47 (s, 3H), 3.51–3.63 (m,1H), 3.65–3.76 (m, 1H), 3.85

(s, 6H), 3.90 (s, 3H), 3.97 (s, 2H), 5.89–5.99(m, 1H), 6.65 (d, J=

8.30 Hz, 2H), 7.12 (s, 2H), 7.25 (s, 1H), 7.45–7.53 (m, 2H), 7.61 (d,

J=7.55 Hz, 2H), 7.85 (s, 1H), 8.08 (d, J=8.30 Hz, 1H), 8.58 ppm (s,

1H); ¹³C NMR (75 MHz, CDCl₃): d=21.7, 40.5, 55.5, 60.0, 60.2, 105.6,

111.2, 112.3, 113.3, 113.8, 118.4, 119.4, 120.9, 121.6, 128.0, 129.6,

132.0, 133.5, 137.8, 141.3, 141.4, 148.3, 150.8, 152.9, 155.4,

166.7 ppm; FT-IR: n˜ =628, 724, 747, 834, 959, 1002, 1029, 1125,

1234, 1326, 1362, 1419, 1452, 1503, 1585, 1606, 1624, 2850, 2926,

3362, 3424 cm^ꢂ1; MS (ESI): m/z: 536 [M+H]⁺; HRMS (ESI): m/z calcd

for C₃₁H₃₀N₅O₄: 536.22923; found: 536.22809 [M+H]⁺.

Biological methods

MTT assay: The anticancer activity of the hybrids was determined

using an MTT assay.^[51]Cells (1ꢁ10⁴cells per well) were seeded in

100 mL Dulbecco’s modified Eagle’s medium (DMEM), supplement-

ed with 10% fetal bovine serum (FBS), in each well of 96-well mi-

croculture plates and were incubated for 24 h at 378C in a CO₂in-

cubator. Compounds, diluted to the desired concentrations in cul-

ture medium, were added to the wells with respective vehicle con-

trol. After 48 h of incubation, 10 mL MTT (5 mgmL^ꢂ1) was added to

each well, and the plates were further incubated for 4 h. The su-

pernatant from each well was then carefully removed, the forma-

zan crystals were dissolved in 100 mL of DMSO, and absorbance

was recorded at a wavelength of 540 nm using an ELx800 micro-

plate reader (BioTek, USA).

1-(5-(1-(3,4-Difluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)-3-(thio-

phen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (8j): White solid

(100 mg;

85%):

mp:

248–2508C;

¹H NMR

(300 MHz,

[D₆]DMSO+CDCl₃): d=2.29 (s, 3H), 3.49–3.57 (m, 1H), 3.89 (dd, J=

11.51, 17.56 Hz, 1H), 5.80 (dd, J=4.53, 11.51 Hz, 1H), 7.12–7.15 (m,

1H), 7.23–7.28 (m, 1H), 7.46 (d, J=3.58 Hz, 1H), 7.52–7.57 (m, 1H),

7.59–7.65 (m, 2H), 7.70 (d, J=5.09 Hz, 1H), 7.83 (s, 1H), 7.92–7.99

(m, 1H), 8.05 (s, 1H), 8.28 (s, 1H), 11.59 ppm (s, 1H); ¹³C NMR

(75 MHz, CDCl₃): d=21.6, 41.3, 60.9, 111.5, 112.2, 117.0, 117.2, 119.4,

120.6, 121.4, 124.1, 124.8, 127.6, 128.1, 128.8, 129.4, 130.2, 134.4,

135.3, 138.8, 141.5, 147.9, 150.3, 167.4 ppm; FT-IR: n˜ =580, 605,

641, 710, 743, 771, 831, 868, 901, 939, 1008, 1047, 1106, 1199,

1245, 1270, 1322, 1381, 1409, 1441, 1515, 1562, 1637, 1699, 3040,

3254 cm^ꢂ1; MS (ESI): m/z: 473 [M+H]⁺; HRMS (ESI): m/z calcd for

C₂₆H₁₉ON₄F₂S: 473.12421; found: 473.12327 [M+H]⁺.

Cell cycle analysis: Flow cytometry analysis was performed to evalu-

ate the distribution of the cells in each of the cell cycle phases.

MCF-7 cells were incubated with hybrids 7d and 8d at concentra-

tions of 1 mm and 2 mm for 48 h. Untreated and treated cells were

harvested, washed with phosphate-buffered saline (PBS), fixed in

cold EtOH (70%), and stained with propidium iodide (PI) (Sigma–

Aldrich). Cell cycle analysis was performed using a flow cytometer

(Becton Dickinson FACS Caliber).^[48]

1-(3-(4-Aminophenyl)-5-(1-(3,4-difluorophenyl)-9H-pyrido[3,4-

b]indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (8k): White

solid (61 mg; 51%): mp: 190–1928C; H NMR (300 MHz, [D₆]DMSO):

d=2.31 (s, 3H), 3.37–3.54 (m, 1H), 3.73–3.86 (m, 1H), 5.58–5.83 (m,

2H), 6.60 (d, J=8.68 Hz, 1H), 7.25 (t, J=7.36 Hz, 1H), 7.48–7.69 (m,

6H), 7.74 (d, J=8.68 Hz, 1H), 7.83 (s, 1H), 7.93–8.05 (m, 2H), 8.28

(d, J=7.93 Hz, 1H), 11.57 ppm (s, 1H); FT-IR: n˜ =536, 606, 746, 769,

Annexin V-FITC assay: MCF-7 cells (1ꢁ10⁶cells per well) were

seeded in six-well plates and allowed to grow overnight. Next, cells

were treated with hybrids 7d and 8d at concentrations of 1 mm

and 2 mm for 48 h with vehicle alone (0.001% DMSO) as a control.

After 48 h of treatment, cells from the supernatant and adherent

monolayer cells were harvested by trypsinization, washed with

PBS, and centrifuged at 3000 rpm. The cells were then stained with

1

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Annexin V-FITC and propidium iodide using the Annexin V–PI

apoptosis detection kit (Sigma–Aldrich, India). Flow cytometry was

performed using a FACScan (Becton Dickinson) equipped with

a single 488 nm argon laser as previously reported.^[52]Annexin V-

FITC was analyzed using excitation and emission settings of

488 nm and 535 nm (FL-1 channel) and 488 nm and 610 nm (FL-2

channel).

50 mL of TE buffer and DNA laddering was determined using 2%

agarose gel electrophoresis in TE.

UV-visible spectroscopy titrations: UV-visible spectroscopic titrations

were performed using an ABI Lambda 40 UV-Vis spectrophotome-

ter (Foster City, CA, USA) at 258C using a 1 cm path length quartz

cuvette. Stock solutions of 10 mm test molecules and 10 mm CT

DNA were prepared in 100 mm KBPES buffer (pH 7.0). A 1 mm

stock solution of b-carboline hybrids was prepared in DMSO and

diluted to the required concentrations in suitable buffer solutions.

The quartz cells were thoroughly cleaned with distilled water, fol-

lowed by nitric acid (~0.1 n) after each experiment. UV-visible ab-

sorption titrations were performed by adding CT DNA stock solu-

tion (200–350 nm) to the quartz cuvette containing approximately

a 1 mm compound solution prepared in the same buffer. Prepara-

tion of CT DNA and test samples were done on the same day that

the experiment was performed. Titrations were carried out until

the molecule soret band remained at a fixed wavelength upon suc-

cessive additions of CT DNA.

Morphological analysis by Hoechst staining: Cells were seeded at

a density of 10000 cells over 18 mm cover slips and incubated for

24 h. Then, cells were treated with hybrids 7d and 8d at 1 mm for

48 h with vehicle alone (0.001% DMSO) as a control. After 48 h of

treatment, Hoechst 33258 (Sigma–Aldrich) was added to the

medium at a concentration of 0.5 mgmL^ꢂ1containing 4% parafor-

maldehyde. After 30 min, cells from each dish were captured from

randomly selected fields under a fluorescence microscope (Leica

TCS SP5, Heidelberg, Germany) to qualitatively determine the pro-

portion of viable and apoptotic cells, based on their relative fluo-

rescence and nuclear fragmentation.

Fluorescence spectroscopy titrations: Fluorescence emission spectra

were measured at 258C using a Hitachi F4500 spectrofluorimeter

(Maryland, USA) using a 1 cm path length quartz cuvette. Quartz

cuvettes were thoroughly washed with distilled water and dilute

nitric acid (~0.1 n) to minimize non-specific binding of the mole-

cules to the surface of the cuvette. Throughout the fluorescence

experiment, the concentration of the test molecules was kept con-

stant (10 mm) and titrated with increasing concentrations of CT

DNA (multiples of 5 mm). Fluorescence spectra were recorded after

each addition of CT DNA to the fluorescent cuvette. After each ex-

periment, the quartz cuvettes were thoroughly washed with dis-

tilled water and dilute nitric acid (~0.1 n) to remove traces of mol-

ecules binding to the walls of quartz cuvette. Molecules 7d and

8d were excited at 242.4 nm and 520.2 nm, respectively, and emis-

sion spectra for each titration were recorded from 470 nm to

510 nm for 7d and 700 nm to 900 nm for 8d. Each spectrum was

recorded three times, and the average of the three scans was

taken.

DNA photocleavage assay: This assay was carried out according to

the protocol reported by Toshima and coworkers.^[53]pBR322 plas-

mid DNA (0.45 mg) in Tris-HCl buffer (50 mm, pH 7.5) was treated

with different concentrations of 7d and 8d, with the total volume

maintained at 20 mL. DNA samples with test molecules in TPP

tissue culture test plates were irradiated with UV light (8 W,

365 nm, 4 cm distance). After irradiation, the samples were collect-

ed and mixed with 2 mL of loading dye (50% sucrose and 0.25%

bromophenol blue). Then, samples were analyzed by gel electro-

phoresis on

a 0.8% agarose horizontal slab gel containing

0.5 mgmL^ꢂ1ethidium bromide in Tris-EDTA buffer (40 mm Tris,

20 mm acetic acid, and 1 mm EDTA, pH 8.0) at 10 Vcm^ꢂ1. Gels were

photographed under UV light with a Bio-Rad Gel Doc XR digital

camera and analyzed with Gel-Pro software (version 3.1).

DNA–topo I inhibition assay: pBR322 plasmid DNA was purchased

from Sigma–Aldrich (St. Louis, MO, USA), and 0.5 mg of DNA was in-

cubated with 1 U topo I enzyme (Invitrogen) in 1X NEB buffer

(50 mm potassium acetate, 20 mm Tris acetate, 10 mm magnesium

acetate, and 1 mm dithiothreitol (DTT)). Next, hybrids 7d and 8d

were added to the topo I–DNA complex and incubated at 378C for

30 min, allowing the formation of ternary enzyme–DNA–ligand

complexes. Then, the enzyme was inactivated by increasing the

temperature to 658C for 20 min. After incubation, the samples

were resolved using 0.8% agarose gel electrophoresis, which ena-

bles the visualization of cleavage products. Camptothecin (100 mm)

and harmine (100 mm) were used as positive controls.

Circular dichroism (CD) spectroscopy studies: CD experiments were

carried out using a JASCO 815 CD spectropolarimeter (Jasco,

Tokyo, Japan). CD spectra were recorded from 220–340 nm to de-

termine the conformational changes in the CT DNA after addition

of test molecules to the DNA solution. For each CD experiment, 5ꢁ

10^ꢂ6m of CT DNA was initially used. Further, for the characteriza-

tion of molecule–DNA interactions, CD spectra were recorded in

1:0, 1:1, and 1:2 molar ratios of CT DNA and test molecules, respec-

tively. CD titrations were performed in 100 mm KBPES buffer

(pH 7.0) at 258C. Each spectrum was recorded three times, and the

average of the three scans was taken.

DNA fragmentation assay: This experiment was performed per the

protocol published previously by us, in which MCF-7 cells were

seeded (1ꢁ10⁶cells per well) in six-well plates and incubated for

24 h.^[54]The cells were treated with hybrids 7d and 8d at a concen-

tration of 2 mm for 48. Next, cells were collected and centrifuged at

2500 rpm for 5 min at 48C. The pellet was collected and washed

with PBS. Lysis buffer (100 mL) was added, the mixture was centri-

fuged at 3000 rpm for 5 min at 48C, and the supernatant was col-

lected. Next, 10% sodium dodecyl sulfate (SDS, 10 mL) and

50 mgmL^ꢂ1RNase A (10 mL) were added, and the mixture was in-

cubated for 2 h at 568C. Proteinase K (10 mL, 25 mgmL^ꢂ1) was

added, and the mixture was further incubated at 378C for 2 h. Am-

monium acetate (10 m, 65 mL) and cold EtOH (500 mL) were added,

and the reaction was mixed thoroughly. The samples were incubat-

ed at 808C for 1 h and centrifuged at 12000 rpm for 20 min at

48C. The pellet was washed with 80% EtOH and air dried for

10 min at room temperature. Finally, the pellet was dissolved in

Molecular docking studies

DNA coordinates for a DNA–topo 1 complex were obtained from

the Protein Data Bank. Unless otherwise noted, all ligand molecules

were optimized by the PM3 method using the Gaussian 09 pack-

age. Necessary corrections to the structure were performed using

the Protein Preparation Wizard in Schrodinger. Docking studies

were performed using the Glide docking tool in Schrodinger.

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[28] B. Armitage, Chem. Rev. 1998 , 98 , 117 1 –1 200.

[29] M. A. Hotz, J. Gong, F. Traganos, Z. Darzynkiewicz, Cytometry 1994 , 15 ,

23 7 –2 44.

Acknowledgements

The authors thank the Council of Scientific and Industrial Re-

search (CSIR), New Delhi (India) for financial support under the

12th Five Year Plan project “Affordable Cancer Therapeutics

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Keywords: anticancer activity

photocleavage · topoisomerase I inhibition · beta-carboline

hybrids

·

DNA binding

·

DNA

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Published online on && &&, 0000

ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 0000, 00, 1 – 16

&15

&

ÞÞ

These are not the final page numbers!

FULL PAPERS

A. Kamal,* V. Srinivasulu, V. L. Nayak,

M. Sathish, N. Shankaraiah, C. Bagul,

N. V. S. Reddy, N. Rangaraj, N. Nagesh*

Hybridization Station: A series of new

b-carboline hybrids were designed, syn-

thesized, and evaluated for their anti-

cancer activity. Induction of apoptosis

was confirmed by Annexin V-FITC,

Hoechst staining, and DNA fragmenta-

tion analysis. The interaction of these

hybrids with DNA, as well as with topo-

isomerase I, was also studied using

docking experiments.

&& – &&

Design and Synthesis of C3-Pyrazole/

Chalcone-Linked Beta-Carboline

Hybrids: Antitopoisomerase I, DNA-

Interactive, and Apoptosis-Inducing

Anticancer Agents

ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 0000, 00, 1 – 16

&16

&

ÞÞ

These are not the final page numbers!

Article Doi

Design and synthesis of C3-pyrazole/chalcone-linked beta-carboline hybrids: Antitopoisomerase I, DNA-interactive, and apoptosis-inducing anticancer agents

DOI: 10.1002/cmdc.201300406

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Authors:

Article abstract of DOI:10.1002/cmdc.201300406

Full text of DOI:10.1002/cmdc.201300406

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