mTOR in breast cancer cells

Accepted Manuscript Pyranocarbazole derivatives as potent anti-cancer agents triggering tubulin polymerization stabilization induced activation of caspase-dependent apoptosis and downregulation of Akt/mTOR in breast cancer cells Om P.S. Patel, Ashutosh Arun, Pankaj K. Singh, Deepika Saini, Sharanbasappa Shrimant Karade, Manish K. Chourasia, Rituraj Konwar, Prem P. Yadav PII:

S0223-5234(19)30113-8

DOI:

https://doi.org/10.1016/j.ejmech.2019.02.003

Reference:

EJMECH 11095

To appear in:

European Journal of Medicinal Chemistry

Received Date: 1 October 2018 Revised Date:

1 February 2019

Accepted Date: 1 February 2019

Please cite this article as: O.P.S. Patel, A. Arun, P.K. Singh, D. Saini, S.S. Karade, M.K. Chourasia, R. Konwar, P.P. Yadav, Pyranocarbazole derivatives as potent anti-cancer agents triggering tubulin polymerization stabilization induced activation of caspase-dependent apoptosis and downregulation of Akt/mTOR in breast cancer cells, European Journal of Medicinal Chemistry (2019), doi: https:// doi.org/10.1016/j.ejmech.2019.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Pyranocarbazole derivatives as potent anti-cancer agents triggering tubulin polymerization stabilization induced activation of caspasedependent apoptosis and downregulation of Akt/mTOR in breast cancer

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cells Om P. S. Patela,1, Ashutosh Arunb,1, Pankaj K. Singhc, Deepika Sainia,d, Sharanbasappa Shrimant Karadee, Manish K. Chourasiac, Rituraj Konwarb,*, and Prem P. Yadava,d,* a

Division of Medicinal and Process Chemistry,

b

Division of Endocrinology,

c

Division of

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Pharmaceutics, dAcademy of Scientific and Innovative Research eDivision of Molecular Structural Biology, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram Extension, Sitapur

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Road, Lucknow 226031, Uttar Pradesh, India

*Corresponding authors. E-mail addresses: [email protected]; [email protected] (P. P. Yadav)

[email protected]; [email protected] (R. Konwar) 1

These authors contributed equally to this work.

Me

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Graphical abstract:

O

O

N H

R1

R1 = H; Koenimbine (1a) R1 = OCH3; Koenidine (1b)

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Structural modification

Me

In silico tubulin polymerization

Bax Bcl-2

AC C

MMP

Pan caspases Caspase

In vitro mechanistic study

O

O

3bgF O

N

formulation of 3bg

Cl 3bg 3bg MDA-MB-231 = 3.8 µM DU145 = 7.6 µM PC3 = 5.8 µM HepG2 = 11.3 µM

3bgF Cmax = 3.139 µg/mL T1/2 = 7.6996 h AUC = 73.8233 µg.h.ml-1 MRT = 18.1928 h CL = 0.1355 mL/h/kg

Drug release study

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ABSTRACT A series of new pyranocarbazole derivatives were synthesized via semi-synthetic modification of koenimbine (1a) and koenidine (1b) isolated from the leaves of Murraya

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koenigii. Among all, compound 3bg displayed significant anti-cancer activity against MDA-MB-231, DU145 and PC3 cell lines with the IC50 values of 3.8, 7.6 and 5.8 µM, respectively. It was also observed that the halogenated-benzyl substitution at N-9 position, C-3 Methyl and C-7 methoxy group on carbazole motif are favoured for anti-cancer

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activity. The detailed investigation was carried out with compound 3bg and its SEDDS (self-emulsifying drug delivery systems) formulation 3bgF. The in vivo drug release behavior study showed that the formulation enhanced slow release and better

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bioavailability at a tumor site. Compound 3bg and its formulation (3bgF) significantly inhibited cell proliferation and colony formation, induced G2/M arrest, reduced cellular ROS generation and induced caspase-dependent apoptosis in MDA-MB-231 cells. 3bg also induced significant alteration of Bax/Bcl expression ratio suggesting involvement of mitochondrial apoptosis. Additionally, 3bg caused down-regulation of mTOR/Akt survival

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pathway. 3bg do not bind to DNA, but interacts with tubulin as observed with in silico molecular docking studies. This interaction results in stabilization of tubulin polymerization similar to paclitaxel as detected in cell-free assay. Oral administration 3bgF for 30 days at dose rate of 10 and 20 mg/kg body weight significantly reduced tumor growth

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in syngenic rat LA-7 mammary tumor model. These results indicated that the pyranocarbazole natural product based N-substituted analogues can act as potential anticancer lead.

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Keywords: Pyranocarbazoles; Tubulin; Breast cancer; Akt/mTOR Pathway; Apoptosis; SEDDS.

Abbreviations

SEDDS, Self-emulsifying drug delivery systems; IC50, Half-maximal inhibitory concentration; SAR, Structure Activity Relationship; MMP, Mitochondrial membrane potential; DMEM, dulbecco’s modified eagle’s medium; FACS, fluorescence-activated cell sorting; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ROS, reactive oxygen species; SD, Sprague dawley; SEDDS, self-emulsifying drug delivery systems.

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1. Introduction Fundamental alteration in normal properties of cells through a series of molecular genetic changes promoting their excessive proliferation, invasion and migration into other tissues ultimately leads to fatal cancer. Microtubules are vital elements involved in various

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cellular processes such as the maintenance of the cell shape, transportation of vesicles, regulation of motility, and cell division. The rapid proliferation of cancer cells is highly dependent on the dynamic cellular process of tubulin polymerization/depolymerization [1]. The interruption in microtubule polymerization can lead to cytostasis and cell death by

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apoptosis. Thus, the interference with tubulin dynamics is a clinically proven approach for the development of highly efficient cancer chemotherapeutics [2-5].

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Natural products (NPs) and semi-synthetic NPs are exquisite entities as they offer a diverse range of biological activities with immense scope of new drug development [6-12]. In the area of cancer, NPs and NPs derived molecules constitute about 49% of all small molecule drugs approved around 1940-2014[13]. Amongst natural product motifs, carbazole alkaloids and their derivatives reported to exhibit various biological activities [9, 14-16] such as anti-diabetic [17], anti-inflammatory [18], anti-microbial [19], anti-oxidant [20-22], [23], anti-HIV

[24]

and

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anti-psychotic

anti-tumor

[25-30].

In

recent

years,

modifications/substitutions of the carbazole moiety either into nitrogen atom or benzene ring have become one of the emerging approach to generate novel anti-cancer agents. In this aspect, Chan and co-workers reported that HYL-6d (Fig. 1, iii) exhibited potential anti-

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angiogenic activity and promoted apoptosis in MCF-7 cells (Fig. 1) [31]. Molatlhegi et al. reported anti-cancer potential of ECAP (Fig. 1, vi) against lung cancer cells and its

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mechanism of action (Fig. 1) [32]. Chen et al. documented the potential of BC3EE2,9B (Fig. 1, v), a synthetic carbazole, to upregulate autophagy and sensitize drug- resistant glioblastoma cells [33]. Pal and co-workers reported that EWGs bearing N-benzoyl derivative of carbazole (Fig. 1, iv) demonstrated considerable activity against oral cancer (CAL 27) and breast cancer (MDA-MB-231) cells (Fig. 1) [34]. Carbazole alkaloids are reported to exhibit potential anti-cancer activity through targeting of DNA or tubulin and have undergone clinical trials [27]. Recently, we established that the carbazole alkaloids isolated from the leaves of Murraya koenigii have potential anti-diabetic [35] and anticancer activities [36]. In our ongoing pursuit to explore the biological activities of natural products and synthetic molecules [35-39] and supported by previous literatures [9, 14, 40, 41], we incorporated

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structural modification at nitrogen atom and C-3 position of isolated pyranocarbazole alkaloids to obtain a series of synthetic analogues (Fig. 1 and Table 1). After establishing their molecular structure, compounds were evaluated for anti-cancer activity against various cancer cell lines. The most active carbazole derivative 3bg along with its

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formulation 3bgF were explored using in vitro and in vivo models for their pharmacokinetic property and anti-tumor efficacy. The details of synthesis and biological activity of these compounds are disclosed herein.

OH

O

O

N H

N H

(ii) Mahanimbine, active against various cancer cells

O N

O

O

R1

R1

O

R1 = H; Koenimbine (1a) R1 = OCH3; Koenidine (1b)

Present work

R1 = H, OCH3 R2 = alkyl, benzyl groups R3 = CHO, CH3

R1 = OCH3 group is favored

N H

R2 = m-chlorobenzyl group is optimum for activity

AC C

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(viii) O-methylmurrayamine A (active against DLD-1) (our previous work)35,36

(order of activity = Cl>Br>Me>OMe)

3

R = CH3 (favoured for R2 halobenzyl) O O N Br

Br

O

N R2

N H

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(vii) Murryazoline (active against DLD-1)

Cl

R3

O

O

N

N

(iii) HYL-6d, active against MCF-7

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(i) Mahanine, active against various cancer cells

O

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N

HO

N

O H N N

Cl

(iv) Active against CAL 27 and MDA-MB231 cells

(v) BC3EE2,9B, active (vi) ECAP, active against lung against brain cancer cancer

Fig. 1. Representative examples of carbazole alkaloids and nitrogen substituted carbazoles having potential anti-cancer activity.

2. Results and Discussion 2.1. Chemistry

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The carbazole alkaloids koenimbine (1a) and koenidine (1b) were isolated in sufficient amount from the leaves of M. koenigii [35, 36] and have been studied for their cytotoxicity against various cancer cells (MCF-7, MDA-MB-231, DU145, PC3, U937, HepG2, DLD-1 and A549) and normal cells (HaCaT and HE-293). We reported previously that these

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compounds (1a and 1b) exhibited moderate toxicity against HepG2 and DLD-1 cells with excellent safety profile towards normal cells up to 100 µM [36]. Owing to enough quantity and safety profile of these compounds (1a and 1b), we have performed semi-synthetic modifications at N-9 and C-3 positions to synthesize a series of new compounds for the

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evaluation of anti-cancer activity.

Initially, we have synthesized new analogs of pyranocarbazoles by the alkylation of 1a and 1b with benzyl bromide/chloride (2) in the presence of Cs2CO3 in dimethylformamide

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(DMF) at room temperature for 12 h (Scheme 1, A). Various benzylated carbazoles derivatives bearing different substituents like electron-withdrawing (-Cl, -Br, -F, -diF, and –diCl) and electron-donating (-CH3 and –OCH3) groups at various positions (-ortho, -meta and –para) were prepared. Moreover, the propargylated (3af and 3bf) and allylated (3bh) compounds were also prepared by using propargyl bromide (2f) and allyl bromide (2h) under the identical reaction conditions. Another interesting bis-iodo-alkenyl substituted

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carbazole derivative (4bf) was prepared from compound 3bf by using I2 (1.5 equiv) in dichloromethane (DCM) at room temperature for 24 h (Scheme 1 A). Next, the oxidation of compounds 1a and 1b was carried out in presence of 2,3-dichloro5,6-dicyanobenzoquinone (DDQ) as an oxidizing agent in MeOH and H2O (10:1) at room

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temperature to afford C-3 oxidized products 5a and 5b, in 55% and 57% yields, respectively (Scheme 1, B). These formylated products were further alkylated by using

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suitable alkylating agents under standard reaction conditions to obtain the corresponding products (6aa, 6bg and 6bi). After synthesis of these derivatives, the molecular structure was established on the basis of 1H and analysis (Supplementary data).

13

C NMR spectroscopy followed by ESI-HRMS

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2.2. Biology

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Scheme 1. Synthesis of pyranocarbazole derivatives. Reagents and conditions: (i) benzyl/propargyl/allyl bromides/chlorides (2a-2j), 1.1 equiv), Cs2CO3 (1.5 equiv), DMF, RT, 12 h; (ii) I2 (1.5 equiv), DCM, RT, 24 h; (iii) DDQ (1.5 equiv), CH3OH: H2O (10:1), RT, 20 h.

2.2.1. Anti-cancer activity of carbazole derivatives against various cancer cell lines

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Semi-synthetically modified koenimibine (1a) and koenidine (1b) derivatives were evaluated for their anti-cancer activity against different cancer cell lines using the MTT

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assay (Table 1). Human breast cancer (MCF-7 and MDA-MB-231), prostate cancer (PC3), liver cancer (HepG2), colon cancer (DLD-1) and lung cancer (A549) cells were used to assess the effect of the semi-synthetic carbazole alkaloids on cancer cell inhibition using the MTT assay. Human embryonic kidney (HEK-293) cell line was used as non-cancer cell whereas tamoxifen citrate and doxorubicin were used as standard drug control. Among all, nine compounds (3ad, 3bb, 3be, 3bg, 3bf, 3bj, 4bf, 5a, and 5b) displayed promising antiproliferative activity below 10 µM concentrations against different cancer cells (Table 1). The N-benzylated or alkylated carbazole derivatives (3ad, 3bb, 3be, 3bg, 3bf, 3bj, 4bf) demonstrated significant activity against MDA-MB-231, DU145, PC3, HepG2 and DLD-1 cells in the range of 3.8-9.7 µM (see; Table 1). More importantly, compound 3bg bearing 3-chlorobenzyl group at N-9 position exhibited most significant anti-proliferative activity

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against various cancer cell lines such as MDA-MB-231, DU145 and PC3 with the IC50 values of 3.8, 7.6 and 5.8 µM, respectively, without having cytotoxicity towards HEK-293 cells up to 100 µM concentration. Compounds 5a and 5b bearing C-3 formyl group with free NH, exhibited high activity against breast cancer (MDA-MB-231) cells with the IC50

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values of 2.6 and 8.3 µM, respectively as compared to the positive control tamoxifen. However, compound 5a showed significant cytotoxicity against HEK-293 cells (normal cell model) at the similar concentration (2.5 µM) to the IC50 value. Overall, these studies suggested that several new leads against different cancer types were obtained from semi-

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synthetic modification of koenimibine (1a) and koenidine (1b) suggesting that pyranocarbazole motifs generally contain significant anti-cancer potential. In order to limit our studies to our resources, we prioritized the most promising lead from this primary

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screening. We used most potent compound 3bg against multiple cell lines as a selection criterion. 3bg was found to be most active, with the IC50 of values 3.8 µM (MDA-MB231), 7.6 µM (DU145), 5.8 µM (PC3) and 12.05 µM (LA-7) as illustrated in Table 3. Therefore, the compound 3bg was explored further for its efficacy and mechanism of action in vitro and in vivo.

Based on anti-cancer activity results of newly synthesized pyranocarbazole analogues, the

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preliminary SARs were investigated. The benzylated compounds (3aa-3af) derived from 1a i.e. koenimbine demonstrated poor to moderate activities against various cancer cells. Notably, the compounds (3bb, 3be, 3bg, 3bf, 3bj, and 4bf) bearing C-7 methoxy group (derived from 1b) showed significant anti-cancer activity in different cancer cell lines. The

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additional C-7 methoxy group plays a major role to achieve optimum activity. Further, the EWGs bearing benzyl substitution at N-9 position of carbazoles (3bg and 3be) exhibited

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comparatively better activity (Cl>Br) as compared to the EDGs bearing compounds (Me>OMe). More importantly, the corresponding C3-formyl product (6bg) for most potent compound (3bg), showed significantly diminished anti-cancer activity despite the presence of C-7 methoxy and EWGs containing N-benzyl substitution (6bg vs. 3bg). The observed SAR results clearly indicated that the key structural features for optimum anti-cancer activity in pyranocarbazole scaffolds are C-7 methoxy substitution, C-3 methyl and N-9 position substituted by halogenated benzyl moieties.

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Table 1. In vitro anti-cancer activities of compounds against different cancer cells MCF-7

3aa

>100

3ab

MDA-MB-

DU145

PC3

HepG2

DLD-1

A549

HEK-293

46.5±3.69

>100

74.9±2.6

41.8±11.08

52.8±6.08

64.0±19.84

>100

48.2±6.42

46.5±8.92

55.7±2.04

35.5±1.88

34.6±5.88

73.1±5.58

35.7±4.67

>100

3ac

>100

23.2±3.93

>100

13.3±3.34

36.8±4.08

34.1±9.32

88.0±8.29

>100

3ad

51.8±1.44

>100

>100

33.1±6.10

9.1±3.2

54.2±5.38

53.3±12.82

>100

3ae

59.1±9.97

>100

>100

15.2±3.74

16.0±2.78

>100

56.5±7.07

>100

3af

>100

52.4±7.43

>100

>100

>100

50.1±5.80

>100

>100

3ba

71.9±12.46

28.5±4.59

>100

29.2±2.11

47.1±4.73

42.1±5.69

80.1±8.45

>100

231

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Entry

64.9±7.81

28.0±3.11

>100

14.1±1.86

9.6±2.44

73.4±7.25

96.6±8.06

>100

3be

70.7±9.94

36.7±2.95

36.6±3.81

9.3±0.96

8.2±1.85

35.9±4.45

24.4±5.97

>100

3bg

64.3±3.25

3.8±1.06

7.6±1.91

5.8±1.76

11.3±1.78

23.7±4.60

30.9±6.89

>100

3bf

>100

9.7±2.1

>100

76.1±2.77

38.8±4.71

>100

51.5±5.95

>100

3bh

>100

36.5±5.48

>100

>100

42.9±3.91

75.3±5.41

>100

>100

3bi

65.6±1.47

33.4±3.54

>100

3bj

29.4±4.37

19.7±2.70

>100

4bf

44.9±7.12

4.4±1.05

5a

3.9±1.1

5b

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3bb

20.0±2.60

41.8±4.28

56.1±5.13

29.1±11.5

NT

7.4±1.91

33.8±5.40

35.4±8.27

14.8±5.24

NT

47.6±7.27

9.6±1.16

15.5±3.47

8.5±2.46

71.7±6.77

>100

2.6±1.13

>100

56.4±3.40

33.3±1.59

54.7±7.78

>100

2.5±1.07

>100

8.3±1.41

70.0±3.78

27.3±3.00

35.5±4.13

>100

33.6±9.60

>100

32.9±3.20

42.4±7.57

>100

36.6±11.09

NT

70.5±4.37

>100

>100

>100

NT

32.8±2.05

30.7±3.85

>100

72.4±2.03

>100

>100

6bi

>100

>100

>100

77.2±3.82

>100

>100

>100

NT

Tam citrate

6.2±2.05

12.8±2.08

11.2±4.00

11.7±2.24

NT

NT

NT

27.3±2.98

Doxorubicin

NT

NT

NT

NT

8.0±2.31

10.2±3.03

NT

NT

a

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6aa 6bg

Data expressed as the mean IC50 ± SEM; NT means not tested.

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2.2.2. Formulation development with compound 3bg SEDDS of compound 3bg were developed after rigorous optimization of all the parameters

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i.e. amount of PEG-400 and other surfactants. Optimized formulation, 3bgF (formulation of compound 3bg) showed mean particle diameter 453.94±70.85 nm with acceptable PDI (0.4670±0.103). Moreover, the zeta potential (-29.6±1.61) of formulation 3bgF is quite excellent in term of stability of formulation (Table 2). Fig. 2A and 2B showing a histogram of mean particle diameter and zeta potential of 3bgF. Table 2. Showing mean particle diameter, PDI and zeta potential of optimized formulation 3bgF

S. No. 1 2 3 4

Batches F1 F2 F3 Mean

Particle size (nm) 533.80 429.40 398.62 453.94±70.85

PDI 0.541 0.511 0.349 0.4670±0.103

Zeta potential -30.1 -30.9 -27.8 -29.6±1.61

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Fig. 2. Morphological evaluation of developed nanoparticles formulation. Mean particle size (A) and Zeta potential (B) of 3bgF

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2.2.3. Cytotoxicity of compound 3bg and 3bgF towards normal cells

Compound 3bg and 3bgF were also re-evaluated against MDA-MB-231, LA-7 cells for

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comparison of their in vitro activity using MTT assay. The formulation 3bgF showed better activity than compound 3bg against MDA-MB-231 cells. However, its activity against LA-7 cell remained within similar range. In addition, possible non-selective cytotoxicity against normal cells MCF-10A was estimated. Both compound 3bg and formulation 3bgF did not show inhibition of MCF-10A up to 100 µM concentration. This

(Table 3).

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further validated that both compound 3bg and formulation 3bgF have good safety index

Table 3. Anti-cancer activity of compound 3bg and 3bgF in different cell lines

3bg 3bgF

Activity in terms of IC50 (Mean± SEM, in µM) MDA-MB-231 LA-7 MCF-10A 3.8±1.06 12.05±0.14 >100 1.0±2.19 14.3±3.79 >100

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Compound code

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2.2.4. Effect of compound 3bg and formulation 3bgF on cancer cell morphology MDA-MB-231 cells were treated with various concentrations of 3bg and 3bgF for 24 h in 24-well culture plates and random fields under a phase-contrast inverted microscope were acquired. We observed visible rounding, decrease in number and size of cells in treated groups in comparison to the untreated control group (Fig. 3C and D).

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Fig. 3. Colony formation after treatment with 3bg (A) and 3bgF (B) in MDA-MB-231 cells. Representative phase-contrast microphotograph (4X) after 24 h treatment with compound 3bg (C) and 3bgF (D) in MDA-MB-231 cells.

2.2.5. Effect of 3bg and 3bgF on colony formation potential of MDA-MB-231 cells Colony formation assay is commonly used to determine long-term effects of cytotoxic agents on cancer cell growth in vitro [42]. Colony formation assay can be used not only to measure direct immediate impact of compounds on cancer cells but also to estimate antiproliferative potency or long-term recurrence prevention efficacy of compounds. Here, both 3bg and 3bgF treated groups, a significant decrease in a number of colony formation of MDA-MB-231 cells on the 7th day after the removal of initial treatment was observed as

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compared to the untreated control group grown for the same time point (Fig. 3A and B). This suggests that both 3bg and 3bgF inhibit cancer cell proliferation even after removal of therapy. 2.2.6. Effect of 3bg and 3bgF on cell cycle distribution in MDA-MB-231 cells

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For cell cycle analysis, MDA-MB-231 cells were grown in T25 cell culture flask and treated with various concentrations of 3bg and 3bgF for 24 h. Neither 3bg nor 3bgF induced any cell cycle arrest in MDA-MB-231 cells with lower concentrations. However, only at higher concentrations both 3bg and 3bgF significantly induced cell arrest at the

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G2/M phase. Compound 3bg at 8 µM increased G2/M populations to 10.3% as compared to 5.4% that of untreated control. Similarly, 3bgF at 2.5 µM concentration resulted 20%

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increased G2/M populations as compared to that of 9.2% in untreated control MDA-MB-

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231 cells (Fig. 4A and B).

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Fig. 4. Effect of compound 3bg and 3bgF in cell cycle (A & B) and apoptosis (C & D) of MDAMB-231 breast cancer cells. For cell cycle, PI-stained cells were analyzed using flow cytometry. The percentage of cells in different phases of cell cycle were calculated based on their PI-stained DNA content vs cell size. The percentage of cells undergoing apoptosis was determined using Annexin V-FITC & PI double staining assay and flow cytometry (FACS Calibur, BectonDickinson, San Jose, CA, USA). Dot plot of cells in each quadrant of flow cytogram was presented based on their Annexin-V-FITC and PI staining characteristics using the CellQuest software. The upper left quadrant of represented cytogram is positive for only PI (necrotic), upper right for both Annexin-V and PI (late apoptotic), lower right for Annexin-V only (early apoptotic) and lower left negative for both Annexin-V and PI (live). Each quadrant data presented using histogram based on their corresponding percent of total cell count. All values are expressed as mean with their standard errors (Mean ± SEM, n=3) derived from three independent cytometry assay. Statistical analysis of each parameter for the compound treated groups was compared with non-treated groups using oneway ANOVA (non-parametric) with Newman-Keuls post-hoc test. The difference was considered statistically significant if *P<0.05. **P<0.01 and ***P<0.001 vs control.

2.2.7. Effect of 3bg and 3bgF on apoptosis of MDA-MB-231 cells

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Treatment of MDA-MB-231 cells with compound 3bg and its formulation 3bgF for 24 h resulted in a significant decrease in live cells counts with a concomitant significant increase in total apoptotic cells counts. Cell death was enhanced up to 21.4% at the highest concentration of treatment with 3bg as compared to the 16.1% in the untreated control group. Similarly, formulation 3bgF treated MDA-MB-231 cells showed about 22.4%

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apoptotic cells for 2.5 µM concentration which is significantly higher as compared to the untreated control group (Fig. 4C and D).

2.2.8. Effect of 3bg and 3bgF on ROS generation in MDA-MB-231 cells MDA-MB-231 cells were further treated with compound 3bg and 3bgF at various

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concentrations for 24 h and ROS generation was estimated using DCFDA staining and flow cytometry. Both 3bg and 3bgF significantly decreased ROS level. However, 3bgF

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appeared to be more potent in reducing ROS generation than compound 3bg for their respective treatment concentrations (Fig. 5C and D). 2.2.9. Effect of 3bg and 3bgF on mitochondrial membrane potential (MMP) MDA-MB-231 cells were treated with various concentrations of 3bg and 3bgF for 24 h. A significant increase in green fluorescence was observed in the treated group in comparison to the untreated group. JC-1 dye forms aggregates and emits red fluorescence when mitochondrial membrane potential is intact suggestive of healthy live cells. Green fluorescence of JC-1 is indicative of monomeric form that is observed when there is a loss of mitochondrial membrane potential usually due to disruption of mitochondrial membrane integrity. Therefore, the results suggest that 3bg and 3bgF induced mitochondrial

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depolarization. These compounds-induced depolarized mitochondria are probably involved in the process of earlier observed apoptosis and could suggest activation of mitochondria-

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mediated intrinsic apoptosis (Fig. 5A and B).

Fig. 5. Compound 3bg and 3bgF induced mitochondrial depolarisation determined by JC-1 dye (A & B) and ROS generation determined by DCFH dye (C & D) of MDA-MB-231 cells. All values of ROS are expressed as percentage of ROS generation derived from CellQuest software-based analysis of acquired cytometric data. 10 µM CCCP was used as positive control to induce mitochondrial depolarization and added 2 h before harvesting of cells for flow cytometric analysis. Data of mitochondrial depolarisation are expressed as % gated population of monomeric JC-1 with green fluorescence indicative of low ∆Ψm and aggregates JC-1 with red fluorescence indicative of high ∆Ψm derived from cytometry (FACS Calibur, Becton-Dickinson, San Jose, CA, USA). Statistical analysis of each parameter for the compound 3bg and 3bgF treated groups were compared with untreated groups using one-way ANOVA (non-parametric) with Newman-Keuls post-hoc test. The difference was considered statistically significant if *P<0.05, **P<0.01 and ***P<0.001 vs control.

2.2.10. Effect of 3bg and 3bgF on caspase-dependent apoptosis in MDA-MB-231 cells

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MDA-MB-231 cells were treated with 3bg and 3bgF in presence or absence of 50 µM of zVAD-FMK (Sigma, USA). z-VAD-FMK is a peptide pan-caspase inhibitor commonly used to inhibit caspase-dependent apoptosis in experimental models. In presence of pancaspase inhibitor, there is a significant decrease in total apoptosis induced by 3bg and 3bgF

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in comparison to their respective compound alone group. The inhibition of apoptosis by caspase inhibitor is comparable with untreated control group even in presence of the compound. Thus, this data establishes that both 3bg and 3bgF induced apoptosis is

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caspase-dependent (Fig. 6A and B).

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Fig. 6. Compound 3bg (A) and 3bgF (B) induced caspase-dependent apoptosis of MDA-MB-231 cells determined by Annexin V-FITC & PI double staining assay using flow cytometry. Dot plot of cells in each quadrant of flow cytogram was presented with the percent of total cell count based on their Annexin-V-FITC and PI staining characteristics using the CellQuest software. Here, 50 µM zVAD-FMK (pan-caspase inhibitor) was used as a caspase inhibitor. Statistical analysis of each parameter for the compound 3bg and 3bgF treated in presence of z-VAD-FMK was compared with compound 3bg and 3bgF alone treated groups using one-way ANOVA (non-parametric) with Newman-Keuls post-hoc test. The difference was considered statistically significant if *P<0.05, **P<0.01 and ***P<0.001 3bg and 3bgF in presence of z-VAD-FMK vs 3bg and 3bgF alone treated group.

2.2.11. DNA binding capacity of 3bg Several carbazole derived bioactive compounds have been reported to exhibit DNA binding property [43-45]. Therefore, we tested if the lead compound 3bg could bind DNA using a gel shift assay. Compound 3bg in tested concentrations range of 0.25 to 5 mM did not bind to DNA, unlike the doxorubicin which was used as positive control (Fig. 7A). 2.2.12. Effect 3bg on tubulin polymerisation in cell-free system

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Recently, several carbazole analogues have been showed to interact with tubulin and thereby influencing its polymerization [46, 47]. This tubulin binding property of carbazole derivatives is suggested as the major mechanism responsible for its anti-cancer activity. We evaluated the effect of 3bg at 1 and 10 µM concentrations on tubulin polymerization in

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cell-free system (Cytoskelton, USA). Here, 10 µM of paclitaxel was used a positive drug control. We observed a significant effect of 3bg on tubulin polymerization similar to paclitaxel as compared to untreated control. Thus, it appears that 3bg binds to tubulin and stabilizes the microtubules (Fig. 7B).

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2.2.13. Molecular docking of 3bg onto tubulin

Since the compound 3bg stabilized tubulin polymerization similar to the paclitaxel in cell

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free system. Therefore we aimed to study the in silico prediction of its structural interaction with tubulin. Both the first crystal structure of the tubulin heterodimer resolved from swine (Sus scrofa) by Nogales et al in 1998 (PDB:1TUB), [48] as well as the recently solved human neuronal tubulin beta subunit crystal structure (PDB: 5JCO) is used for this study. The human neuronal tubulin beta subunit (PDB: 5JCO) used as a model for docking while swine tubulin heterodimer (PDB:1TUB) was superimposed to compare binding

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pockets. Docking was performed using Autodock Vina software available at vina.scripps.edu [49]. Compound 3bg was drawn using ChemDraw (PerkinElmer, USA) and energy minimized. The docking grid was set into paclitaxel binding site in the βtubulin subunit of the microtubule. The docking energy of 3bg and paclitaxel was -7.5

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Kcal/mol. The Fig. 7C shows binding of paclitaxel in 1TUB [50], paclitaxel is shown as magenta and the binding pocket residues are shown in gray stick. The docked compound is

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shown as green and corresponding residues in gray sticks (Fig. 7D) and pocket with cyan surface and compound in (Fig. 7E). 1TUB was superimposed on 5JCO to show the binding pocket (Fig. 7F). Thus, the docking studies suggested that the compound 3bg could easily fit into the paclitaxel binding pocket of tubulin and provides justification for its tubulin stabilizing property and anti-cancer activity in biological assays. We further checked the tubulin polymerization properties of other active compounds (3be, 3bf, 3bj, 4bf and 5b) due to their pharmacophoric resemblance to 3bg (see Supplementary Data). Among these compounds, 3bj has the highest docking energy (-7.4 Kcal/mol) followed by 3bf (-7.1 Kcal/mol), 3be (-7.0 Kcal/mol), 5b (-6.6 Kcal/mol) and 4bf (-6.3 Kcal/mol). Thus, the docking studies suggested that the compounds could easily fit into the paclitaxel binding pocket of tubulin as shown in case of 3bg with similar docking energy.

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Therefore these compounds might have tubulin stabilizing property and thereby possess

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anti-cancer activity in the biological assays.

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Fig. 7. (A) Effect of 3bg on DNA binding capability. Doxorubicin is used as a positive control for DNA binding. DNA was incubated with different concentration of 3bg and run in 2% agarose gel electrophoresis. (B) Effect of 3bg on tubulin polymerization. Compound 3bg was added into prewarmed 96 well plates along with tubulin buffer and absorbance was recorded at 340 ηm for 60 min at 37 °C using spectrophotometer with kinetic assay capability. Paclitaxel at 10 µM is used as a positive control along with untreated vehicle control. (C, D, E & F) Molecular docking of 3bg onto tubulin. Intermolecular binding prediction of 3bg and tubulin. 5JCO and TUB1A structures were retrieved from the online database (RCSB). The docking study was performed on the human neuronal tubulin structure (5JCO). TUB1A structures were superimposed to show the binding pocket. Docking was done using Autodock Vina software. Figures were prepared with the help of Maestro 10.4 (EPSRC UK National Service for Computational Chemistry Software, London, UK).

2.2.14. Western blot analysis Death receptor-mediated extrinsic apoptosis pathway and mitochondria-mediated intrinsic pathways involve a different subset of cellular signaling proteins [51]. In case of the intrinsic pathway of apoptosis, pro-apoptotic (example, Bax) and anti-apoptotic (example,

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Bcl-2) proteins are activated or inhibited depending upon the upstream signaling and therefore their cellular levels are employed as distinctive markers [52]. Cells undergoing intrinsic mitochondrial apoptosis show down-regulation of Bcl-2 and up-regulation of Bax. To investigate the pathway involved in compound 3bg mediated apoptosis; MDA-MB-231

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cells were treated with 3bg for 24 h. From each sample, an equal amount of protein was resolved according to molecular weight using SDS-PAGE gel, transferred onto PVDF membrane and immunoblotted with specific antibodies. We observed that 3bg at 3.8 µM concentration induced significant down-regulation of Bcl-2 and up-regulation of Bax (Fig.

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8A). This result clearly establishes that 3bg perturbs key proteins involved in the intrinsic apoptosis pathway. Analysis of Bax/Bcl-2 ratio confirmed a dose-dependent increase which is characteristic of mitochondrial apoptosis (Fig. 8A). Caspases, which are the final

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facilitator of apoptosis, comprised of two distinct classes, the initiators and the executioner [53]. Caspase-3 is an executioner caspase which is cleaved and activated during apoptosis [54]. Significant cleavage of caspase-3 with 3bg treatment was observed, thus overall confirming that 3bg induces caspase-dependent intrinsic apoptosis in MDA-MB-231 cells. 2.2.15. Effect of compounds 3bg on Akt/mTOR phosphorylation

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PI3K/Akt/mTOR pathway is a critical determinant of cell survival and apoptosis [55]. This pathway is also one of the most frequently dysregulated signaling cascades in human malignancies and is implicated in a wide variety of different neoplasms [56]. Compound 3bg significantly decreased phospho-Akt (Ser473) and phospho-mTOR (Ser2448)

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expression in MDA-MB-231 cells essential for its survival. Thus, compound 3bg downregulates Akt/mTOR pathway in breast cancer cells by inhibiting activation of Akt

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and mTOR. This inhibition of Akt/mTOR pathway could possibly activate in the intrinsic apoptotic program (Fig. 8B).

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Fig. 8. (A) Effect of compound 3bg on apoptosis pathway proteins in MDA-MB-231 cells. (B) Effect of 3bg on in phospho-Akt (Ser473) and phospho-mTOR (Ser2448) expression in MDA-MB231 cells. 20 µg of protein lysate was electrophoresed in 12% SDS-PAGE and transferred onto PVDF membrane for probing with the primary and secondary antibody. Image was acquired by a gel documentation system (GE Image quanta). Housekeeping β-actin signal was used for normalization of loading differences. Each experiment was repeated three times and quantitation of band intensity was performed by densitometry using Quantity One® software (v.4.6.6). Statistical analysis of each parameter for the compound treated groups was compared with non-treated groups using one-way ANOVA (non-parametric) with Newman-Keuls post-hoc test. The difference was considered statistically significant if *P<0.05, **P<0.01 and ***P<0.001 vs control.

2.2.16. Drug release study with 3bg and 3bgF

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The formulated 3bgF showed sustain release behavior with 40% release of compound 3bg after 12 h, unlike the pure 3bg suspension that showed an immediate release of about 81% of compound 3bg. Sustain release behavior of the developed formulation is thought to be due to the formation of micro-emulsion encapsulating 3bg inside the core thereby retarding the release of the compound (Fig. 9A). Estimation of 3bg in plasma was performed by protein precipitation method with good resolution. The 3bgF plasma concentration vs time profile after oral administration is shown in Fig. 9B. In addition, both 3bg and 3bgF administration through oral route to the animals did not elicit any acute reaction and apparently healthy during the entire course of the experimentation. This could be considered as a preliminary indication of the pre-clinical safety of the formulation. Winnonlin version 5.1 (Certara, USA) was utilized to derive principal pharmacokinetic

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parameters such as peak plasma concentration (Cmax), half-life (T1/2), mean residence time (MRT0-t), area under curve (AUC0-t), AUMC and clearance (CL), summarised in Table 4.

3.139 µg/mL

T1/2

7.6996 h

AUC0-t

73.8233 µg.h.ml-1

AUMC

1343.0524 h.h.µg/ml

MRT0-t

18.1928 h

CL

0.1355 mL/h/kg

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2.2.17. Tissue distribution profile 3bg and 3bgF

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Table 4. Plasma parmacokinetic parameters of 3bgF upon oral administration

Tissue distribution profile of 3bgF showed that it distributes higher amount of compound 3bg in tumor (Cmax 9.39±1.71 µg/mL) in comparison to liver (Cmax 6.43±1.01 µg/mL), kidney (Cmax 3.36±2.110 µg/mL) and heart (Cmax 1.67±0.36 µg/mL). Higher distribution of drug in the tumor is attributed to the particle size-based passive targeting in addition to active targeting through enhanced permeation and retention effect. Higher distribution of

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compound 3bg in tumor directly beneficial in the reduction of dose as well as reduce

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adverse effects oriented through the non-specific distribution of the drug (Fig. 9C).

Fig. 9. (A) Comparison of free drug release behavior of developed formulation 3bgF and pure compound 3bg suspension. (B) Plasma concentration versus time profile of 3bgF after oral administration. (C) Tissue distribution profiles of compound 3bg after oral administration of 3bgF. The concentration of free 3bg is measured in the tumor, kidney, liver and heart tissue from animal (n=3) at different time points.

2.2.18. In vivo anti-tumor activity of 3bgF in syngeneic LA-7 rat mammary tumor model The 3bgF was administered orally to the tumor-bearing adult SD rats into two different dose group of 10 mg/kg bd. wt. (n=5) and 20 mg/kg body weight (n=5) for 30 consecutive days. The body weight of the animals showed normal gain possibly suggesting lack of any

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acute adverse effects (Fig. 10D and E). Any major alteration in gross body weight is an important preliminary indication of toxicity or side effects of administered agent [57, 58]. In addition, there were also no significant changes in mean absolute and relative total weight of major vital organs measured at the end of the experiment (Fig. 10B and C). This

administration with dose as high as 20 mg/kg body weight.

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grossly supports that 3bgF did not induce major organ toxicity up to 30 days of oral

Treatment of LA-7 induced syngeneic mammary tumor-bearing animals with oral gavage of 3bgF for a period of 30 days caused significant reduction in mean in situ tumor volume

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and mean ex situ tumor weight at the time of sacrifice. This data suggest that 3bgF showed

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tumor regressing activity in vivo similar to in vitro activity.

Fig. 10. (A) Pictorial representation of tumor size in situ and ex-situ in rat LA-7 syngenic mammary tumor model with 30 days of oral administration of vehicle and 3bgF (10 and 20 mg/kg). Oral administration of 3bgF does not disrupt gross bd. wt. and major organ weight. Absolute organ weight (B), mean relative organ weight (C), mean bd. wt. (D), mean bd. wt. gain (E). 3bgF induces loss of mean tumor weight (F), mean relative tumor weight (G) and tumor volume (H), (n=5). Statistical analysis of each parameter for 3bgF treated groups was compared with non-treated groups using one-way ANOVA (non-parametric) with Newman-Keuls post-hoc test. The difference was considered statistically significant if *P<0.05, **P<0.01 and ***P<0.001 vs control.

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3. Discussion A series of new carbazole derivatives were synthesized through semi-synthetic modification of natural koenimibine and koenidine and evaluated for anti-cancer activity. Among all, 3bg exhibited significant activity against MDA-MB-231 cells and selected as

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most potent lead. The SEDDS formulation of compound 3bg was developed with the rational to improve the oral bioavailability and passive targeting to the tumor. Optimized 3bgF showed mean particle size diameter of 453.94±70.85 nm with acceptable PDI 0.4670±0.103 and zeta potential of -29.6±1.61 mV. The higher zeta potential of developed

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formulation reflects the higher stability of the formulation. Drug release study demonstrated that 3bgF showed sustained manner release which may be attributed to the

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fact that the drug encapsulated in the core of lipid vesicle constrains the release of drug. Moreover, similar release behavior from SEDDS is also reported earlier which support our findings. Tissue distribution of 3bgF performed into breast tumor rat model for assessment of our hypothesis of passive targeting and EPR (enhanced permeability and retention) effect of the developed formulation. 3bgF showed 1.46, 2.79 and 5.62-fold higher Cmax in the tumor as compared to liver, kidney and heart, respectively. Higher Cmax of 3bgF in the

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tumor in comparison to other organs endorsed to particulate based passive targeting and EPR effect. Moreover, tissue distribution result is also supported by in vivo anti-cancer study results. In longer duration with peak plasma concentration of 3.139 µg/mL with plasma half-life of 7.6996 h and improved tumor tissue bioavailability. 3bg and its

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formulation 3bgF induced cell cycle arrest at G2/M phase, loss of MMP, and decrease in ROS generation and caspase-3 dependent apoptosis in MDA-MB-231 cells. 3bg at 8 µM induced G2/M arrest (10.3%) significantly (p<0.05) as compared to the control which is

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5.4% and resulted in the increase in sub-diploid population by 4.0%. Similarly, 3bgF cause G2/M arrest (20%) significantly (p<0.01) at a higher dose of 2.5 µM as compare to control 9.2% and causes S phase reduction. Cancer cells have generally increased ROS levels due to increased metabolic activity and defective mitochondrial function [59]. 3bg and its formulation 3bgF decreased ROS level in a dose-dependent manner. The activation of apoptotic responses is an important strategy in the treatment of cancer [60]. Drugs and treatment strategies that can reinstate the apoptotic signaling pathways or induce apoptosis have the potential to reduce cancer cells. Similar to MTT assay, in apoptosis assay also, 3bg showed induction of MDA-MB-231 total cell death population 17.6% (p<0.01) at 1 µM, 20.2% (p<0.01) at 5.7 µM, and 21.4% (p<0.01) at 8 µM, which

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were higher than 16.1% vehicle-treated groups. Concurrently, 3bgF showed induction of MDA-MB-231 total cell death population 18.8% (p<0.01) at 0.5 µM, 21.9% (p<0.01) at 1 µM, and 22.4% (p<0.01) at 2.5 µM, which were higher than 16.1% vehicle treated groups. During the onset of the apoptotic signal into the cell, there is a modification in the

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permeability of mitochondrial membranes, which leads to the translocation of apoptogenic protein cytochrome c into the cytoplasm, which then activates death driving proteolytic proteins known as caspases [61]. It requires permeabilization of the outer mitochondrial membrane that proceeds in the absence of caspase activity. It is connected with a loss of

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outer mitochondrial membrane potential (∆ψM) [62], and this process is controlled by both pro and anti-apoptotic proteins. Compounds 3bg and its formulation 3bgF induced mitochondrial depolarization which led to the apoptosis through the intrinsic pathway.

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Compound 3bg induced down-regulation of Bcl-2 and up-regulation of Bax in a dosedependent manner. These results clearly showed that compound 3bg exhibited apoptosis via the intrinsic pathway. Activation of the caspase-3 pathway is a hallmark feature of apoptosis [63]. Our results showed cleaved caspase-3 by treatment of compound 3bg which further validated pro-apoptotic nature of 3bg.

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In the search of a possible target of 3bg, we performed DNA binding assay and molecular docking with crystal structure of tubulin. 3bg does not bind to DNA in gel shift assay. However, in silico studies showed a possibility of intermolecular interaction of 3bg with tubulin. This was further supported by in vitro experimental validation that showed 3bg

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increased tubulin polymerization similar to standard microtubule stabilizing agents like paclitaxel. 3bg also inhibited activation of Akt and mTOR in MDA-MB-231 cells. Clinical response of microtubule inhibitor is known to be influenced by status of Akt in tumor [64].

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It is reported that phospho-Akt (Ser473) status determines paclitaxel therapy response in breast cancer patients [65]. PI3K/Akt pathway is known to regulate cell division and cellular ploidy [66]. Later, it has also been proposed that PI3K/Akt pathway regulate mitotic spindle formation and organization by influencing microtubule dynamics via GSK3 [67]. On the other hand, paclitaxel is reported to activate Akt and its downstream cascades in ovarian cancer cells critical for their sensitivity towards paclitaxel [68]. In contrast, here we observed that 3bg could interact with tubulin in silico and increase tubulin polymerization like paclitaxel under in vitro condition, however, downregulates Akt and mTOR activation. Akt and mTOR are also known to regulate mitochondrial physiology [69] and that’s how probably their downregulation by compound 3bg brings

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about mitochondrial-mediated intrinsic apoptosis in MDA-MB-231 cells. Similarly, low level of activated Akt and mTOR also synergies cell cycle arrest induced by compound 3bg [70, 71]. However, it is not clear, how the microtubule stabilization induced by compound 3bg is linked to down-regulation of mTOR and Akt. The in silico molecular

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docking studies indicated the possibility of direct interaction of 3bg with tubulin and therefore it may be presumed that mTOR and Akt downregulation is downstream event of microtubule stabilization or bundling. However, none of the earlier studies supports this and thus, further detailed cell signaling studies in knockdown/knockout conditions would

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definitely be required to derive such a conclusion.

Survival factors can repress apoptosis in a transcription-independent way by activating the serine/threonine kinase Akt, which afterward phosphorylates and inactivates components

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of the apoptotic machinery, containing caspase 9 and BAD [72] and promotes cell survival. Akt phosphorylation usually employed for cell survival and apoptosis. Akt possesses oncogenic and anti-apoptotic activities and is a serine/threonine protein kinase [73]. Phosphatidylinositol 3-kinase generates phosphatidylinositol (3,4,5)-trisphosphate, which activates Akt [74]. Akt, in turn, phosphorylates a range of proteins, including several

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correlated with cell death pathways such as BAD, NF-kB, MDM2, CREB, and Forkhead, leading to reduced apoptotic cell death [75]. Akt endorses cell survival by suppressing apoptosis, and its phosphorylation has been considered a significant factor. Akt deactivation characterizes both caspase-dependent and independent cell death. Akt protein

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was down-regulated during apoptosis. The down-regulation was inhibited by a caspase inhibitor, signifying that Akt was cleaved by caspases during apoptosis [76]. We have shown that compound 3bg induced apoptosis was caspase dependent. When MDA-MB-

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231 cells treated with compound 3bg in the presence of pan caspase inhibitor z-VADFMK, total percentage of apoptotic cells decrease to 5.3% (p<0.001) from 10.7% (absence of pan caspase inhibitor). Similar results were also found with formulation 3bgF in which total percentage of apoptotic cells decrease to 5.9% (p<0.01) in pan caspase inhibitor zVAD-FMK treated group from 9.5% (absence of pan caspase inhibitor). Here we can assume that decreased activation of Akt may lead to caspase dependent apoptosis. The formulation 3bgF significantly inhibited tumor growth in LA-7-induced syngeneic mammary tumors in SD rats with both 10 and 20 mg/kg body weight at the oral administration for 30 consecutive days. This not only supports adequate tumor tissue distribution of 3bgF, but also validates optimal retaining of in vitro observed activity of

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3bg and 3bgF in more complex animal model system. Overall, all the findings comprehensively establish that pyranocarbazole derivatives has significant prospect as anti-cancer leads with potent in vitro and in vivo activity.

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4. Experimental section 4.1. Chemistry 4.1.1. General

All reagents were purchased from commercial sources and used without further

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purification. Reactions were monitored by thin-layer chromatography (TLC) on silica gel plates with fluorescence F254. The melting point was recorded on a capillary melting point apparatus and was uncorrected. The IR spectra were recorded using an FT-IR

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spectrophotometer, and values are reported in cm−1. 1H NMR spectra were recorded using a Bruker Avance (300 and 400 MHz), using CDCl3 and DMSO-d6 as solvents at 25 °C, and the chemical shifts are expressed in δ (ppm) relative to the TMS peak.

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spectra were recorded in on a 75 and 100 MHz Bruker Avance spectrometer using CDCl3 and CDCl3+DMSO-d6 as solvents. The multiplicities of NMR signals were assigned as

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singlet (s), doublet (d), triplet (t), multiplet (m), and broad (br). The ESI-HRMS data were acquired on a Q-TOF mass spectrometer. The purification of compounds was performed by using silica gel 60−120 mesh column chromatography using EtOAc-n-hexane as an

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eluent. All tested compounds were ≥95% pure by HPLC.

4.1.2. Experimental procedure for the alkylation reaction

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Benzyl halides (2, 1.1 equiv.) was added drop-wise to a solution of compound 1a or 1b (100 mg, 1 equiv.) and Cs2CO3 (1.5 equiv.) in a 10 mL of round bottom flask contains 2 mL DMF. The mixture was stirred for 12 h at room temperature. After the completion of reaction (TLC) monitored by TLC, the reaction mixture was poured in 15 mL of water and extracted with (10 x 3) mL of EtOAc. The organic layer was washed with 10 mL of brine solution and dried over anhydrous Na2SO4 and evaporated under reduced pressure to give the crude mass. Purification for most of the compounds was performed by washing with HPLC grade n-hexane followed by crystallization in the mixture of EtOAc and n-hexane. Some of the compounds were purified by column chromatography using 60-120 mesh

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silica gel and ethyl acetate: hexane (2: 8) as an eluent to give the final compound 3 as white solid in good to excellent yield. 4.1.3. Experimental procedure for iodination reaction To a solution of compound 3af in DCM (5 mL) was added iodine (1.5 equiv.) at room

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temperature and continue stirred for 24 h. After the completion of reaction, monitored by TLC, the reaction mixture was poured in 10 mL of aqueous solution Na2S2O3.5H2O (hypo solution). The separated organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure to give the crude mass. The compound was purified by column

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chromatography using 60-120 mesh silica gel and ethyl acetate: hexane (2: 8) as an eluent to give the final compound 4bf as a white solid in 48% yield.

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4.1.4. Experimental procedure for oxidation reaction

DDQ (1.5 equiv.) was added to a solution of compound 1a or 1b (100 mg, 1 equiv.) in 10 mL of MeOH: H2O (10:1). The reaction mixture was stirred for 20 h at room temperature. After the completion of reaction monitored by TLC, solvent was evaporated under reduced pressure. The residue was extracted with EtOAc and water, dried over anhydrous Na2SO4 and evaporated under reduced pressure to give the crude mass. Purification by column

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chromatography using 60-120 mesh silica gel and ethyl acetate: hexane (3: 7) as an eluent to gave the yellow color solid products (5) in good yields. 4.1.4.1. 11-Benzyl-8-methoxy-3,3,5-trimethyl-3,11-dihydropyrano[3,2-a]carbazole (3aa)

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White solid; yield = 86%; mp = 179-181 °C; 1H NMR (400 MHz, CDCl3): δH = 7.72 (s, 1H), 7.47 (d, J = 2.8 Hz, 1H), 7.32-7.23 (m, 3H), 7.14 (d, J = 6.8 Hz, 2H), 7.10 (d, J = 8.4 Hz, 1H), 6.93 (dd, J = 8.8, 2.4 Hz, 1H), 6.66 (d, J = 9.6 Hz, 1H), 5.56 (s, 2H), 5.49 (d, J = 9.6 Hz, 1H), 3.91 (s, 3H), 2.34 (s, 3H), 1.44 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δC = 154.2, 151.0, 138.2, 136.7, 136.6, 129.0, 128.5, 127.3, 125.8, 123.8, 121.3, 118.6, 118.5, 117.1, 113.1, 109.3, 105.8, 102.6, 74.8, 56.1, 49.1, 27.1, 16.3 ppm; IR (KBr): v 3409, 3019, 1635, 1523, 1384, 1154, 758, 669 cm-1; HRMS (ESI): Calcd for C26H26NO2 [M + H]+ 384.1958, Found: 384.1960. 4.1.4.2. 8-Methoxy-3,3,5-trimethyl-11-(3-methylbenzyl)-3,11-dihydropyrano[3,2a]carbazole (3ab) White solid; yield = 90%; mp = 148-150 °C; 1H NMR (400 MHz, CDCl3): δH = 7.72 (s, 1H), 7.47 (s, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.10-7.05 (m, 2H), 6.98-6.93 (m, 3H), 6.67 (d, J = 9.6 Hz, 1H), 5.52 (s, 2H), 5.50 (d, J = 10 Hz, 1H), 3.91 (s, 3H), 2.35 (s, 3H), 2.27 (s, 3H), 1.44 (s, 6H) ppm; 13C NMR (75 MHz, CDCl3): δC = 154.1, 151.0, 138.7, 138.2, 136.8, 136.7, 128.9, 128.4, 128.1, 126.4, 123.8, 122.9, 121.3, 118.7, 118.4, 117.1, 113.1, 109.4, 105.8, 102.5, 74.8, 56.1, 49.2, 27.1, 21.5, 16.3 ppm; IR (KBr): v 3404, 3019, 1636,

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White solid; yield = 88%; mp = 173-175 °C; 1H NMR (400 MHz, CDCl3): δH = 7.71 (s, 1H), 7.46 (d, J = 2.4 Hz, 1H), 7.08 (d, J = 8.8 Hz, 1H), 6.96-6.88 (m, 2H), 6.72-6.63 (m, 2H), 6.56 (d, J = 10 Hz, 1H), 5.55-5.52 (d, J = 10.8 Hz, 3H, overlapped signals), 3.91 (s, 3H), 2.34 (s, 3H), 1.43 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δC = 162.5 (dd, 247, 12 Hz), 159.8 (dd, J = 247, 12 Hz), 154.4, 151.1, 136.4, 136.3, 129.1, 128.6 (dd, J = 9, 6 Hz), 124.0, 121.5, 121.2 (dd, J = 15, 4 Hz), 118.9, 118.1, 117.3, 113.3, 111.9 (dd, J = 21, 3 Hz), 109.1, 105.9, 104.0 (t, J = 25 Hz), 102.7, 75.0, 56.2, 43.0 (d, J = 5 Hz), 27.1, 16.4; IR (KBr): v 3419, 3021, 1489, 1215, 761 cm-1; HRMS (ESI): Calcd for C26H24F2NO2 [M + H]+ 420.1770, Found: 420.1767.

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4.1.4.4. 11-(2,5-Difluorobenzyl)-8-methoxy-3,3,5-trimethyl-3,11-dihydropyrano[3,2a]carbazole (3ad)

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White solid; yield = 81%; mp = 159-161 °C; 1H NMR (400 MHz, CDCl3): δH = 7.71 (s, 1H), 7.47 (d, J = 2 Hz, 1H), 7.13-7.07 (m, 2H), 6.97-6.89 (m, 2H), 6.55 (d, J = 10 Hz, 1H), 6.48-6.46 (m, 1H), 5.56 (s, 2H), 5.54 (d, J = 9.6 Hz, 1H) 3.91 (s, 3H), 2.34 (s, 3H), 1.44 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δC = 159.3 (d, J = 243 Hz), 155.7 (d, J = 241 Hz), 151.1, 136.3, 136.2, 129.1, 127.4 (dd, J = 17, 7 Hz), 124.1, 121.5, 119.0, 118.0, 117.4, 116.5 (dd, J = 23, 8 Hz), 115.4 (dd, J = 24, 8 Hz), 114.5 (dd, J = 26, 4 Hz), 113.3, 109.0, 105.8, 102.8, 75.0, 56.2, 43.4 (d, J = 4 Hz), 27.1, 16.4 ppm; IR (KBr): v 3429, 3019, 1493, 1215, 769 cm-1; HRMS (ESI): Calcd for C26H24F2NO2 [M + H]+ 420.1770, Found: 420.1761.

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4.1.4.5. 11-(2-Bromobenzyl)-8-methoxy-3,3,5-trimethyl-3,11-dihydropyrano[3,2a]carbazole (3ae)

AC C

White solid; yield = 79%; mp = 170-172 °C; 1H NMR (400 MHz, CDCl3): δH = 7.71 (s, 1H), 7.64 (dd, J = 1.2, 7.6 Hz, 1H), 7.47 (d, J = 2.4 Hz, 1H), 7.15-7.09 (m, 2H), 7.08-7.04 (m, 1H), 6.93 (dd, J = 2.4, 8.8 Hz, 1H), 6.68-6.66 (m, 1H), 6.43 (d, J = 10 Hz, 1H), 5.53 (s, 2H), 5.49 (d, J = 10 Hz, 1H), 3.90 (s, 3H), 2.33 (s, 3H), 1.42 (s, 6H) ppm; 13C NMR (75 MHz, CDCl3): δC = 154.2, 150.9, 136.8, 136.3, 136.1, 132.8, 128.98, 128.91, 128.0, 127.6, 123.8, 121.5, 121.3, 118.7, 118.0, 117.1, 113.1, 109.0, 105.8, 102.6, 74.9, 56.0, 49.7, 27.0, 16.3 ppm; IR (KBr): v 3409, 3019, 1633, 1384, 1215, 759, 619 cm-1; HRMS (ESI): Calcd for C26H25BrNO2 [M + H]+ 462.1063, Found: 462.1052. 4.1.4.6. 8-Methoxy-3,3,5-trimethyl-11-(prop-2-yn-1-yl)-3,11-dihydropyrano[3,2a]carbazole (3af) White solid; yield = 80%; mp = 194-196 °C; 1H NMR (400 MHz, CDCl3): δH = 7.65 (s, 1H), 7.41 (d, J = 2.4 Hz, 1H), 7.27 (d, J = 10 Hz, 1H), 7.16 (d, J = 9.6 Hz, 1H), 7.01 (dd, J = 2.4, 8.8 Hz, 1H), 5.71 (d, J = 9.6 Hz, 1H), 5.02 (d, J = 2.4 Hz, 2H), 3.90 (s, 3H), 2.352.33 (m, 4H), 1.49 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δC = 154.4, 151.1, 136.0,

ACCEPTED MANUSCRIPT

128.9, 124.2, 121.4, 118.9, 118.8, 117.3, 113.1, 109.1, 106.1, 102.8, 79.3, 75.1, 73.0, 56.2, 35.7, 27.2, 16.3 ppm; IR (KBr): v 3408, 3019, 1637, 1384, 1215, 759 cm-1; HRMS (ESI): Calcd for C22H22NO2 [M + H]+ 332.1645, Found: 332.1650. 4.1.4.7. (3ba)

11-Benzyl-8,9-dimethoxy-3,3,5-trimethyl-3,11-dihydropyrano[3,2-a]carbazole

SC

RI PT

White solid; yield = 86%; mp = 184-186 °C; 1H NMR (400 MHz, CDCl3): δH = 7.64 (s, 1H), 7.44 (s, 1H), 7.32-7.25 (m, 3H), 7.14 (d, J = 7.2 Hz, 2H), 6.70 (s, 1H), 6.63 (d, J = 9.6 Hz, 1H), 5.55 (s, 2H), 5.48 (d, J = 10 Hz, 1H), 3.99 (s, 3H), 3.87 (s, 3H), 2.33 (s, 3H), 1.42 (s, 6H) ppm; 13C NMR (75 MHz, CDCl3): δC = 149.7, 148.4, 144.5, 138.1, 136.3, 135.5, 129.0, 128.6, 127.3, 125.7, 120.3, 118.6, 118.5, 117.4, 115.4, 105.9, 102.1, 92.9, 74.6, 56.6, 56.3, 49.1, 26.9, 16.2 ppm; IR (KBr): v 3402, 3019, 1654, 1384, 1215, 769 cm1 ; HRMS (ESI): Calcd for C27H28NO3 [M + H]+ 414.2064, Found: 414.2066.

M AN U

4.1.4.8. 8,9-Dimethoxy-3,3,5-trimethyl-11-(3-methylbenzyl)-3,11-dihydropyrano[3,2a]carbazole (3bb)

TE D

White solid; yield = 83%; mp = 156-158 °C; 1H NMR (400 MHz, CDCl3): δH = 7.64 (s, 1H), 7.44 (s, 1H), 7.20 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 7.2 Hz, 1H), 6.97 (s, 1H), 6.95 (d, J = 8 Hz, 1H), 6.71 (s, 1H), 6.64 (d, J = 9.6 Hz, 1H), 5.51 (s, 2H), 5.49 (d, J = 10 Hz, 1H), 3.99 (s, 3H), 3.87 (s, 3H), 2.34 (s, 3H), 2.28 (s, 3H), 1.43 (s, 6H) ppm; 13C NMR (75 MHz, CDCl3): δC = 149.7, 148.5, 144.5, 138.8, 138.1, 136.4, 135.7, 128.9, 128.7, 128.2, 126.3, 122.9, 120.3, 118.8, 118.5, 117.5, 115.4, 106.0, 102.1, 93.1, 74.6, 56.7, 56.4, 49.1, 27.0, 21.5, 16.3 ppm; IR (KBr): v 3428, 3019, 1637, 1385, 1215, 758 cm-1; HRMS (ESI): Calcd for C27H30NO3 [M + H]+ 428.2220, Found: 428.2220. 4.1.4.9. 11-(2-Bromobenzyl)-8,9-dimethoxy-3,3,5-trimethyl-3,11-dihydropyrano[3,2a]carbazole (3be)

AC C

EP

White solid; yield = 88%; mp = 180-183 °C; 1H NMR (400 MHz, CDCl3): δH = 7.66 and 7.64 (two s, 2H, overlapped), 7.45 (s, 1H), 7.16-7.08 (m, 2H), 6.68 and 6.66 (two s, 2H, overlapped), 6.43 (d, J = 7.2 Hz, 1H), 5.52 (s, 2H), 5.50 (overlapped d, 1H), 3.99 (s, 3H), 3.88 (s, 3H), 2.33 (s, 3H), 1.41 (s, 6H) ppm; 13C NMR (75 MHz, CDCl3): δC = 149.7, 148.5, 144.6, 136.7, 135.9, 135.0, 132.8, 129.1, 129.0, 128.1, 127.6, 121.5, 120.3, 118.7, 118.0, 117.5, 115.3, 105.9, 102.1, 92.7, 74.7, 56.6, 56.4, 49.7, 26.9, 16.3 ppm; IR (KBr): v 3402, 3019, 1384, 769 cm-1; HRMS (ESI): Calcd for C27H27BrNO3 [M + H]+ 492.1169, Found: 492.1167. 4.1.4.10. 8,9-Dimethoxy-3,3,5-trimethyl-11-(prop-2-yn-1-yl)-3,11-dihydropyrano[3,2a]carbazole (3bf) White solid; yield = 83%; mp = 161-163 °C; 1H NMR (400 MHz, CDCl3): δH = 7.57 (s, 1H), 7.38 (s, 1H), 7.16 (d, J = 9.6 Hz, 1H), 6.89 (s, 1H), 5.72 (d, J = 9.6 Hz, 1H), 5.01 (d, J = 2.4 Hz, 2H), 4.01 (s, 3H), 3.98 (s, 3H), 2.37 (s, 1H), 2.32 (s, 3H), 1.49 (s, 6H) ppm; 13C NMR (75 MHz, CDCl3): δC = 149.9, 148.5, 144.7, 135.6, 135.1, 129.1, 120.4, 118.9, 118.8, 117.7, 115.7, 106.2, 102.3, 93.0, 79.2, 74.9, 73.2, 56.7, 56.5, 35.8, 27.1, 16.3; IR

ACCEPTED MANUSCRIPT (KBr): v 3406, 3021, 1388, 1215, 762 cm-1; HRMS (ESI): Calcd for C23H24NO3 [M + H]+ 362.1751, Found: 362.1754. 4.1.4.11. (E)-11-(2,3-Diiodoallyl)-8,9-dimethoxy-3,3,5-trimethyl-3,11-dihydropyrano[3,2a]carbazole (4bf)

SC

RI PT

White solid; yield = 48%; mp = 183-185; 1H NMR (400 MHz, CDCl3): δH = 7.60 (s, 1H), 7.40 (s, 1H), 7.27 (t, J = 1.6 Hz, 1H), 6.86 (d, J = 9.6 Hz, 1H), 6.75 (s, 1H), 5.70 (d, J = 9.6 Hz, 1H), 5.06 (d, J = 2 Hz, 2H), 3.99 and 3.98 (s, 6H), 2.33 (s, 3H), 1.48 (s, 6H) ppm; 13 C NMR (75 MHz, CDCl3): δC = 149.8, 148.4, 144.8, 135.7, 135.2, 129.0, 120.4, 119.0, 118.9, 117.8, 115.9, 106.2, 102.2, 102.1, 94.0, 79.4, 74.7, 56.59, 56.55, 55.9, 26.9, 16.2 ppm; IR (KBr): v 3398, 3019, 1633, 1588, 1384, 1321, 1116, 767, 669 cm-1; HRMS (ESI): Calcd for C23H24I2NO3 [M + H]+ 615.9840, Found: 615.9815. 4.1.4.12. 11-(3-Chlorobenzyl)-8,9-dimethoxy-3,3,5-trimethyl-3,11-dihydropyrano[3,2a]carbazole (3bg)

11-Allyl-8,9-dimethoxy-3,3,5-trimethyl-3,11-dihydropyrano[3,2-a]carbazole

TE D

4.1.4.13. (3bh)

M AN U

White solid; yield = 82%; mp = 167-169 °C; 1H NMR (400 MHz, CDCl3): δH = 7.64 (s, 1H), 7.44 (s, 1H), 7.24-7.23 (d, J = 6 Hz, 2H), 7.19 (s, 1H), 6.98 (d, J = 6 Hz, 1H), 6.66 (s, 1H), 6.57 (d, J = 10 Hz, 1H), 5.53-5.50 (m, 3H), 3.99 (s, 3H), 3.88 (s, 3H), 2.33 (s, 3H), 1.43 (s, 6H) ppm; 13C NMR (75 MHz, CDCl3): δC = 149.8, 148.5, 144.7, 140.3, 136.1, 135.4, 135.1, 130.4, 129.0, 127.7, 126.0, 124.0, 120.5, 118.8, 118.4, 117.6, 115.6, 105.9, 102.2, 92.9, 74.7, 56.7, 56.4, 48.7, 27.0, 16.3; IR (KBr): v 3401, 3019, 2400, 1384, 1215, 758 cm-1; HRMS (ESI): Calcd for C27H27ClNO3 [M + H]+ 448.1674, Found: 448.1677.

AC C

EP

White solid; yield = 86%; mp = 116-118 °C; 1H NMR (400 MHz, CDCl3): δH = 7.60 (s, 1H), 7.40 (s, 1H), 6.89 (d, J = 9.6 Hz, 1H), 6.75 (s, 1H), 6.17-6.10 (m, 1H), 5.63 (d, J = 10 Hz, 1H), 5.24 (d, J = 10.4 Hz, 1H), 5.03 (d, J = 17.2 Hz, 1H), 4.91 (s, 2H), 3.98 and 3.96 (two s, 6H), 2.33 (s, 3H), 1.47 (s, 6H) ppm; 13C NMR (75 MHz, CDCl3): δC = 149.7, 148.4, 144.4, 136.1, 135.6, 135.5, 128.5, 120.3, 118.8, 118.3, 117.4, 116.6, 115.4, 105.9, 102.1, 93.0, 74.7, 56.7, 56.4, 47.8, 27.1, 16.3 ppm; IR (KBr): v 3416, 3019, 1626, 1384, 1258, 758 cm-1; HRMS (ESI): Calcd for C23H26NO3 [M + H]+ 364.1907, Found: 364.1910. 4.1.4.14. 8,9-Dimethoxy-11-(3-methoxybenzyl)-3,3,5-trimethyl-3,11-dihydropyrano[3,2a]carbazole (3bi) White solid; yield = 80%; mp = 152-154 °C; 1H NMR (400 MHz, CDCl3): δH = 7.63 (s, 1H), 7.43 (s, 1H), 7.23 (t, J = 7.9 Hz, 1H), 6.80 (dd, J = 8.2, 2.2 Hz, 1H), 6.72 (br d, 3H, overlapped signals), 6.65 (d, J = 9.8 Hz, 1H), 5.50 (br d, 3H, overlapped signals), 3.99 (s, 3H), 3.87 (s, 3H), 3.71 (s, 3H), 2.33 (s, 3H), 1.43 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δC = 160.3, 149.8, 148.5, 144.6, 140.0, 136.5, 135.7, 130.2, 128.8, 120.4, 118.8, 118.6, 118.1, 117.6, 115.5, 112.6, 111.7, 106.1, 102.2, 93.1, 74.7, 56.8, 56.4, 55.3, 49.2, 27.1, 16.4 ppm; IR (KBr): v 3398, 3019, 1633, 1588, 1384, 1321, 1116, 767, 669 cm-1; HRMS (ESI): Calcd for C28H30NO4 [M + H]+ 444.2169, Found: 444.2166.

ACCEPTED MANUSCRIPT

4.1.4.15. 11-(2,5-Dichlorobenzyl)-8,9-dimethoxy-3,3,5-trimethyl-3,11-dihydropyrano[3,2a]carbazole (3bj)

8-Methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazole-5-carbaldehyde

SC

4.1.4.16. (5a)

RI PT

White solid; yield = 85%; mp = 212-214 °C; 1H NMR (400 MHz, CDCl3): δH = 7.64 (s, 1H), 7.50 (d, J = 2.0 Hz, 1H), 7.44 (s, 1H), 7.04 (dd, J = 8.4, 2.0 Hz, 1H), 6.62 (d, J = 8.7 Hz, 2H), 6.40 (d, J = 9.9 Hz, 1H), 5.52 (t, J = 5.7 Hz, 3H, overlapped signals), 3.99 (s, 3H), 3.89 (s, 3H), 2.33 (s, 3H), 1.42 (s, 3H) ppm; 13C NMR (100 MHz, CDCl3): δC = 149.9, 148.7, 144.9, 135.9, 135.1, 134.1, 134.0, 132.4, 129.6, 129.4, 128.5, 127.9, 120.5, 119.0, 117.9, 117.6, 115.6, 106.0, 102.3, 92.7, 74.8, 56.7, 56.5, 46.9, 27.1, 16.4 ppm; IR (KBr): v 3388, 3017, 1631, 1584, 1380, 1116, 768, 669 cm-1; HRMS (ESI): Calcd for C27H26Cl2NO3 [M + H]+ 482.1284, Found: 482.1317.

8,9-Dimethoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazole-5-

TE D

4.1.4.17. carbaldehyde (5b)

M AN U

Yellow solid; yield = 55%; mp = 202-204 °C; lit. mp = 203-204 °C [77]; 1H NMR (300 MHz, CDCl3): δH = 10.30 (s, 1H), 8.19 (s, 2H), 7.09 (d, J = 9 Hz, 1H), 6.80 (d, J = 8.7 Hz, 1H), 6.44 (d, J = 9.9 Hz, 1H), 5.58 (d, J = 9.9 Hz, 1H), 5.11 (s, 1H), 3.70 (s, 3H), 1.36 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δC = 189.5, 154.9, 154.8, 141.2, 134.9, 129.9, 124.8, 119.9, 118.46, 118.40, 116.4, 114.8, 111.6, 104.3, 103.3, 77.4, 56.0, 27.8 ppm; IR (KBr): v 3424, 3019, 1640, 1385, 1215, 758, 669 cm-1; HRMS (ESI): Calcd for C19H18NO3 [M + H]+ 308.1281, Found: 308.1281.

EP

Yellow solid; yield = 57%; mp = 247-248 °C; 1H NMR (400 MHz, CDCl3): δH = 10.29 (s, 1H), 8.14 (s, 1H), 7.34 (s, 1H), 6.86 (s, 1H), 6.64 (d, J = 9.8 Hz, 1H), 5.65 (d, J = 9.8 Hz, 1H), 3.86 and 3.84 (d, J = 8.2 Hz, 3H), 3.70 (s, 3H), 1.42 (s, 6H) ppm; 13C NMR (100 MHz, DMSO-d6+CDCl3): δC = 187.8, 152.7, 148.8, 144.3, 140.3, 135.3, 129.1, 118.0, 117.77, 117.70, 117.1, 116.9, 115.1, 103.8, 103.0, 95.0, 76.5, 56.0, 55.6, 48.7, 27.1 ppm; IR (KBr): v 3400, 3019, 1637, 1384, 1117, 759, 669 cm-1; HRMS (ESI): Calcd for C20H20NO4 [M + H]+ 338.1387, Found: 338.1393.

AC C

4.1.4.18. 11-Benzyl-8-methoxy-3,3-dimethyl-3,11-dihydropyrano[3,2-a]carbazole-5carbaldehyde (6aa) White solid; yield = 88%; mp = 161-163 °C; 1H NMR (400 MHz, CDCl3): δH = 10.49 (s, 1H), 8.46 (s, 1H), 7.53 (d, J = 2.4 Hz, 1H), 7.35-7.26 (m, 3H), 7.12 (t, J = 8.9 Hz, 3H), 6.98 (dd, J = 8.8, 2.5 Hz, 1H), 6.64 (d, J = 9.9 Hz, 1H), 5.58 (t, J = 4.9, 3H, overlapped signals), 3.91 (s, 3H), 1.50 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δC = 189.4, 155.8, 155.3, 141.7, 137.3, 137.1, 129.3, 127.8, 125.7, 124.4, 120.0, 118.7, 118.5, 117.8, 114.8, 110.0, 105.5, 103.2, 76.2, 56.1, 49.2, 27.1 ppm; IR (KBr): v 3398, 3019, 1633, 1588, 1384, 1321, 1116, 767, 669 cm-1; HRMS (ESI): Calcd for C26H24NO3 [M + H]+ 398.1751, Found: 398.1755. 4.1.4.19. 11-(3-Chlorobenzyl)-8,9-dimethoxy-3,3-dimethyl-3,11-dihydropyrano[3,2a]carbazole-5-carbaldehyde (6bg)

ACCEPTED MANUSCRIPT

RI PT

White solid; yield = 90%; mp = 136-138 °C; 1H NMR (400 MHz, CDCl3): δH = 10.49 (s, 1H), 8.37 (s, 1H), 7.51 (s, 1H), 7.28-7.26 (m, 2H, overlapped signals), 7.19 (s, 1H), 6.98 (dd, J = 4.9, 2.2 Hz, 1H), 6.67 (s, 1H), 6.56 (d, J = 9.9 Hz, 1H), 5.61 (d, J = 9.9 Hz, 1H), 5.54 (s, 2H), 3.99 (s, 3H), 3.88 (s, 3H), 1.49 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δC = 189.5, 154.9, 149.6, 145.8, 140.7, 139.2, 136.9, 135.4, 130.7, 129.7, 128.2, 125.9, 123.9, 119.1, 118.8, 117.5, 115.9, 105.7, 102.6, 93.3, 76.1, 56.6, 56.5, 48.8, 29.8, 27.0 ppm; IR (KBr): v 3389, 3017, 1637, 1578, 1374, 1110, 769, 668 cm-1; HRMS (ESI): Calcd for C27H25ClNO4 [M + H]+ 462.1467, Found: 462.1470. 4.1.4.20. 8,9-Dimethoxy-11-(3-methoxybenzyl)-3,3-dimethyl-3,11-dihydropyrano[3,2a]carbazole-5-carbaldehyde (6bi)

4.2. Biology 4.2.1. Cells and cell culture conditions

M AN U

SC

White solid; yield = 81%; mp = 174-176 °C; 1H NMR (400 MHz, CDCl3): δH = 10.49 (s, 1H), 8.36 (s, 1H), 7.50 (s, 1H), 7.26 (t, J = 7.9 Hz, 1H), 6.83 (dd, J = 8.2, 2.2 Hz, 1H), 6.73-6.69 (m, 3H), 6.63 (d, J = 9.9 Hz, 1H), 5.59 (d, J = 9.9 Hz, 1H), 5.53 (s, 2H), 3.99 (s, 3H), 3.87 (s, 3H), 3.73 (s, 3H), 1.49 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δC = 189.6, 160.5, 154.8, 149.5, 145.6, 141.0, 138.8, 137.2, 130.5, 129.4, 119.1, 118.7, 118.6, 117.9, 117.8, 115.8, 112.8, 111.8, 105.7, 102.6, 93.4, 76.0, 56.6, 56.5, 55.3, 49.2, 27.0 ppm; IR (KBr): v 3395, 3018, 1635, 1583, 1384, 1117, 768, 668 cm-1; HRMS (ESI): Calcd for C28H28NO5 [M + H]+ 458.1962, Found: 458.1931.

TE D

Breast cancer cells (MCF-7 and MDA-MB-231), DLD-1, A549, DU145, PC3, HepG2 and HEK-293 cells were obtained from ATCC, USA and maintained in the laboratory. MCF-7, MDA-MB-231, HepG2, and HEK-293 were maintained in DMEM. DLD-1, A549, DU145

EP

and PC3 were maintained in RPMI with supplementation of 10% fetal bovine serum (FBS), MCF-10A in DMEM phenol red supplemented with 10% horse serum, 100 ƞg/ml cholera toxin 20 ƞg/ml epidermal growth factor, 500 ƞg/ml hydrocortisone, 10 µg/ml

AC C

insulin, LA-7 cells were maintained in DMEM high glucose supplemented with 5µg/ml bovine insulin, 50 ƞM hydrocorticosone, 10% FBS and 1% antibiotic in humidified incubator at 37ºC with 5% CO2 [36]. 4.2.2. MTT assay

The anti-cancer activities of carbazoles derivatives were determined using MTT reduction assay. Cells were seeded at a density of 10,000 in DMEM supplemented with 10% FBS in 96 well plate and allow to attach overnight. 10 mM stock solution of the compounds were prepared in DMSO and further diluted to different micromolar ranges in DMEM media containing in 0.5% FBS. Cells were treated with various concentrations of compounds for 24 h. At the end of incubation, 5 mg/mL of MTT was added and the plates were further

ACCEPTED MANUSCRIPT

incubated for 3 h in dark condition. Supernatant from each well was carefully removed and formazon crystals were dissolved in DMSO and absorbance at 540 ηM wavelength by spectrometer was recorded. Percentage of inhibition of cell growth was calculated with the formula given below: -

Mean O.D. of control

x 100

RI PT

% cell inhibition = Mean O.D. of control – Mean O.D. of the treated

Percent cell inhibition was plotted against various concentrations of each compound and IC50 concentration of test compounds were calculated using Graphpad Prism software [78].

SC

4.2.3. Morphological analysis of cells treated with compound 3bg and 3bgF

Cells were seeded at a density of 5×104 cells/well in 6-well plate and incubated for 24 h.

M AN U

Then, the medium was replaced, and cells were treated with desired concentrations of compound 3bg and its optimized formulation 3bgF for 24 h. For untreated control group in the experiment, cells were treated with vehicle (0.001% DMSO in culture medium). After the period of treatment, random fields of cells from each group were captured under bright field inverted microscope (Leica, Germany) [79].

TE D

4.2.4. Colony formation assay

The colony formation assay has been the gold standard for determining the effects of cytotoxic agents on cancer cell growth in vitro [42]. 1×103 cells/well was plated in 6 well plate. After 24 h incubation cells were treated with 3bg and 3bgF and further incubated for

EP

24 h. Then media along with test compound were washed with PBS and incubated in DMEM media for 7 to 10 days. At the end of incubation period, the media were removed

AC C

and cells were fixed with methanol and stained with 0.4% crystal violet and image was captured.

4.2.5. Cell cycle analysis assay Cell cycle distribution analysis was performed in MDA-MB-231 cells employing flow cytometry. Cells were plated in 6 well plate in the density of 1×105 cells /well and allowed to adhere for 24 h. Treatment of 3bg and 3bgF was given for pre-determined time points. Further cells were trypsinized, washed with PBS and fixed in pre-cooled 70% alcohol for overnight in -20 ºC. Fixed cells were centrifuged in swing bucket type centrifuge and treated with DNA extraction buffer for 15 min. Washing of the cells was done with PBS and cells were treated with RNase A (100 µg/mL), followed by propidium iodide (50

ACCEPTED MANUSCRIPT

µg/mL) staining for 30 min at room temperature. Cell cycle status was analysed using FACS Calibur flow cytometer (Becton- Dickinson, San Jose, CA, USA) and analysed with CellQuest software [80]. 4.2.6. Apoptosis detection

RI PT

The apoptosis induced by 3bg and 3bgF was determined using Annexin-V-FITC-PI apoptosis detection kit (Sigma, USA) as per manufacturer’s instructions. MDA-MB-231 Cells were seeded in six well plates (1×105 cells/well) and after attaining morphology, treatment was given. After incubation, cells were mildly trypsinized and centrifuged. The

SC

cell pallets were washed with PBS, resuspended in 500 µL DNA binding buffer and was stained with Annexin V-FITC and propidium iodide as per manufacturer instructions. Live,

M AN U

apoptotic, and necrotic cell populations were using flow cytometer (FACS Calibur, Becton- Dickinson, San Jose, CA, USA) [81]. 4.2.7. Intracellular ROS measurement

For determination of the intracellular accumulation of ROS (reactive oxygen species), the 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) method was used. DCFH-DA crosses the cell membrane, subsequently undergoes deacetylation by intracellular esterases.

TE D

The deacetylated 2’,7,-dichlorodihydrofluorescein (DCFH) reacts with intracellular hydrogen peroxide or other oxidizing ROS to give the fluorescent2’,7,-dichlorofluorescein (DCF). After treatment of 3bg and 3bgF at IC50 and above IC50 in MDA-MB-231cells respectively for 24 h, DCFH-DA at a final concentration of 2 mg/mL was added after

EP

trypsinization and fixing with 1 mL methanol. After incubation for 30 min at 37 °C, cells

[82].

AC C

were washed with PBS. Intracellular ROS accumulation was measured by flow cytometry

4.2.8. Mitochondrial membrane potential (MMP) analysis MMP was determined using JC-1 dye (Molecular probe). MDA-MB-231 cells were cultured and treated with 3bg and 3bgF in 6-well plates for 24h and were rinsed with PBS twice, stained with 1 mL culture medium containing 5 mmol/L JC-1 for 30 min at 37 °C after their respective exposure times. Cells were rinsed with ice-cold PBS twice, resuspended in 300 µL ice-cooled PBS, and instantly assessed for red and green fluorescence with flow cytometry[83]. 4.2.9. Western blotting

ACCEPTED MANUSCRIPT

Cells were grown in T-25 flasks, and treated with test compound 3bg in pre-defined concentrations as well as time intervals. After incubation, cells were washed with ice-cold PBS and then lysed in RIPA buffer. Lysates containing equal amount of protein were electrophoresed and transferred to PVDF membrane (Millipore, USA), probed with

RI PT

appropriate primary antibodies [1:1000 dilution for mTOR, p-Akt (Ser473), Akt, p-mTOR (Ser2448), cleaved caspase 3, bax, bcl2 and β-actin, 1:5000 dilution for β-actin,]. Blots were reacted for 1 h with 1:10,000 HRP-conjugated anti-mouse and anti-rabbit IgG (Cell Signaling Technology). Finally, blots were developed using ECL solution (ImmobilonTM

SC

western, Millipore USA) and scanned by gel documentation system (GE Image quanta) [81].

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4.2.10. Tubulin polymerization assay

Tubulin polymerization experiment was done as per reported method using ‘assay kit’ from Cytoskeleton, USA. In brief, tubulin protein (3 mg/mL) in tubulin polymerization buffer (80 mM PIPES, pH 6.9, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP and 15% glycerol) was placed in pre-warmed half area 96-well microtiter plates at 37 °C in the presence of potent compound 3bg with variable concentrations. All samples were mixed

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well and polymerization was monitored kinetically at 340 nm at every min for 1 h using Spectramax plate reader. Paclitaxel was used as standard stabilizer of tubulin polymerization [82].

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4.2.11. Molecular docking

5JCO and 1TUB structures were retrieved from the online database (RCSB) [84]. The docking study was performed on the Human neuronal tubulin structure (5JCO). The

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docking was done using Autodock Vina software [49]. Figures were prepared with the help of Maestro 10.4 (EPSRC UK National Service for Computational Chemistry Software, London, UK).

4.2.12. Formulation development Compound 3bg was insoluble in aqueous solution and therefore SEDDS containing compound 3bg have been successfully prepared to improve its solubility as well as bioavailability [85]. 2 mg of 3bg weighs accurately and dissolved in chloroform. An optimized amount of PEG 400 added slowly in above solution under stirring at 1000 rpm at a constant temperature of 40 ºC. Lauroglycol 90 followed by labrasol was slowly added in above solution under stirring. Finally triple distilled water was added to above solution

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to make up volume up to 1 mL. Chloroform was removed from final solution by heating at 40 ºC with constant stirring at 1000 rpm for overnight. Size, PDI and zeta potential measurements were performed by photon correlation spectroscopy, with a Malvern zetasizer (Nano ZS, Malvern Instruments, UK). For size and PDI measurements,

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lyophilized nanoparticles were dispersed with Milli-Q water and measured for a minimum of 120s. Zeta potential was assessed by measuring electrophoretic mobility of the particle employing a laser based multiple angle electrophoresis analyzer. 4.2.13. Drug release study

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Compound 3bg release from 3bgF and compound 3bg suspension in PBS was conducted in USP paddle type dissolution apparatus (Labindia 2000, India) by equilibrium dialysis

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membrane method [86]. Specified amount of 3bgF and compound 3bg were hermetically sealed in activated dialysis sack. Phosphate-buffer (pH 7.4) contains 1% tween 80 at 37 ºC was used as dissolution media stirred at 100 rpm. Tween-80 was employed to provide solubility for 3bg in aqueous phase. Samples were collected at predetermined time points and media was replenished after each sampling to maintain sink condition [86]. 4.2.14. Oral plasma kinetics

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To explore the plasma pharmacokinetic behavior of optimized formulation (3bgF) of compound 3bg, we performed a time dependent study employing SD rats (150-170 gm). Rats (n=3) were kept on standard diet and water. The 3bgF was administered orally at dose

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10 mg/kg. Serial blood samples were collected in heparinised tubes at predestined time points (0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h). The plasma was separated from blood by centrifugation (3000 × g for 10 min) and samples were stored at -80 °C prior to analysis.

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Drug estimation from plasma sample was performed by protein precipitation method followed by chromatographic method discussed previously [87]. 4.2.15. Tissue distribution profile Tissue distribution study of compound 3bg was performed on tumor induced SD rat (n=3), weight = 150-170 gm. Animals weighing about 150-170 gm were divided into five groups (n=3). Developed formulation (3bgF) at dose equivalent to 10 mg/kg 3bg (3bgF) was administered orally to all groups. Animals were sacrificed at different time interval (0.5, 4, 12, and 24 and 48 h) by cervical dislocation ethically while harvesting major tissues such as tumor, kidney, liver and heart for study. All samples were stored at -80ºC (Thermo Scientific SLT-21V-40D41 Upright Freezer, USA) till analysis. Tissue samples were

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thawed at room temperature and homogenized with three fold volume of PBS pH 7.4. Accurately measured 200 µL of tissue homogenate was taken and mixed with 400 µL methanol for protein precipitation and vortexed for 10 min, followed by evaporation under reduced pressure (Buchi Rotary Evaporators, Mumbai, India). Then 800 µL of methanol

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was added and kept for incubation. After incubation the samples were centrifuged at 12,000 rpm for 10 min (Eppendorf Centrifuge 5415R, thermofisher, Massachusetts, United States) and the supernatant after filtration (0.45µm) was subjected to HPLC analysis (LC10ATvp, Shimadzu, Tokyo, Japan) [88].

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4.2.16. In vivo study

Animal study was conducted with prior approval from the Institutional Animal Ethics

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Committee (IAEC), CSIR-CDRI, Lucknow, India. Adult SD rats were used to develop LA7 syngenic mammary tumor model. Briefly, LA-7 (6 × 106) cells were transplanted onto the mammary fat pad of female SD rats. After 10 days of cell implantation, when tumors were measurable, animals were randomly divided into three groups (n=5). 3bgF was orally administered at a dose of 10 and 20 mg/kg bd. wt. per day. Control group animals only received vehicles orally. During the period of study, bd. wt. and tumor size was measured

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on 5th day of interval. The tumor volume was calculated using formula, V = [(Length) × (Width)2]/2. Tumor volumes of treated groups were compared with vehicle control group [82].

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4.2.17. Statistical analysis

Data from at least three independent experiments are expressed as mean ± SEM. The comparisons were made between controls and treated group. Statistical comparison of

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more than two groups was performed using one-way ANOVA followed by a NewmanKeuls test. P-values if significant P < 0.05 (*), highly significant if P < 0.01 (**), and very highly significant if P < 0.001 (***). Statistical analysis was undertaken using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Conflict of interest The authors declare no conflict of interest. Author contributions O.P.S.P. and A.A. contributed equally to this work. P.P.Y. and R.K. conceived of the study and participated in the design and coordination and helped to draft the manuscript. O.P.S.P.

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and D.S. isolated the parent compounds in major amount and synthesized a series of new derivatives of carbazoles alkaloids. A.A. performed the entire biological assay. P.K.S. and M.K.C. developed formulation, performed phramacokinetics study like drug release and tissue distribution. S.S.K. performed docking studies. All authors read and approved the

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final draft. Acknowledgements

O.P.S.P. and S.S.K. are thankful to UGC and A.A. is thankful to CSIR, New Delhi, India for financial assistance. Authors acknowledge Mr. Anoop K. Srivastava for technical

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support, Mr. A. L. Vishwakarma for flow cytometric analysis, Mr. Rajakrishnan Purshottam for HPLC purity analysis of compounds and SAIF-CDRI, Lucknow, India, for

Appendix A. Supplementary data

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providing spectral and analytical data. This is CDRI communication no. xxxx.

Supplementary data (1H, 13C and HPLC purity profile of compounds) to this article can be found online at http://doi.org/..................... References

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New pyranocarbazole derivatives were synthesized from Koenimbine and Koenidine NPs. 3bg and its formulation 3bgF show significant activity against MDA-MB-231 cells.

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3bgF shows higher bioavailability at a tumor site compared to other vital organs.

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3bgF significantly reduces tumor growth in syngenic rat LA-7 mammary tumor

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