3-(3,4,5-Trimethoxyphenylselenyl)-1 H -indoles and their selenoxides as combretastatin A-4 analogs: Microwave-assisted synthesis and biological evaluation

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Graphical Abstract

microwave-assisted

-(3,4,5-trimethoxyphenylselenyl)-1H-indoles is reported. The target compounds showed potent in vitro activity

against cancer cell proliferation and tubulin polymerization.

A

procedure

for

the

effective

synthesis

of

a

series

of

3

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3

-(3,4,5-Trimethoxyphenylselenyl)-1H-indoles and their selenoxides as

combretastatin A-4 analogs: Microwave-assisted synthesis and biological

evaluation

a

b

a

b

a

b

a

Zhiyong Wen , Jingwen Xu , Zhiwei Wang , Huan Qi , Qile Xu , Zhaoshi Bai , Qian Zhang

a,c

b,*

a,*

Kai Bao , Yingling Wu , Weige Zhang

a

Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang

Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, China;

b

Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District,

Shenyang 110016, China;

c

Division of Hematology/ Oncology, Department of Medicine, Beth Israel Deaconess Medical Center and

Harvard Medical School, Boston, MA 02215, USA

*

Corresponding author. Tel./ fax: +86 24 23986422 (W. Zhang) ; +86 24 23986278(Y. Wu);

e-mail: zhangweige2000@sina.com (W. Zhang); yingliang_1016@163.com (Y. Wu).

Abstract

A series of 3-(3,4,5-trimethoxyphenylselenyl)-1H-indoles and their selenoxides were

designed as a new class of combretastatin A-4 (CA-4) analogs. The B ring and the cis double bond

of CA-4 were replaced by an indole moiety and selenium atom, respectively. A facile and efficient

microwave-assisted synthesis of 3-arylselenylindoles was developed to prepare the target

compounds, which were then evaluated for antiproliferative activity against three human cancer

cell lines using an MTT assay. Most of these compounds exhibited significant antiproliferative

activity, with some showing nanomolar IC₅₀values. Tubulin polymerization and immunostaining

experiments revealed that 13a potently inhibited tubulin polymerization and disrupted tubulin

microtubule dynamics in a similar manner to CA-4. Docking studies demonstrated that 13a adopts

an orientation similar to that of CA-4 at the colchicine binding site on tubulin.

Keywords: Antiproliferative, Combretastatin A-4, Selenium, Indole, Tubulin polymerization

inhibitors, Microwave-assisted synthesis.

1

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1

. Introduction

Microtubules are key components of the cytoskeleton of eukaryotic cells and are involved in

numerous cellular functions, including motility, division and vesicle transport [1]. The assembly

of α- and β-tubulin heterodimers and the disassembly of the polymeric form are dynamic

processes that lead to the formation of microtubules [2]. The disruption of these dynamic

processes blocks the cell division machinery at mitosis and results in cell death [1].

Combretastatin A-4 (CA-4, 1) is a natural product from the African willow tree, Combretum

caffrum, which disrupts microtubules by binding to the colchicine binding site and arresting the

G /M phase of the cell cycle, causing mitotic catastrophe and, ultimately, apoptotic cell death [3,

2

4

]. However, poor bioavailability and water solubilityprevent the development of CA-4 (1) as an

antitumor drug [5]. Combretastatin A-4 phosphate (CA-4P, 2), a pro-drug of CA-4 (1), exhibits

increased water solubility and has entered phase II/III clinical trials as a potential

vascular-targeting agent [6]. Owing to its potent biological activities and relatively simple

chemical structure, CA-4 (1) has attracted significant research attention. A wide range of analogs

of CA-4 (1) has been identified as colchicine binding site inhibitors (CBSIs) with potent antitumor

activity. The structure-activity relationships (SAR) of CA-4 (1) indicate that a cis-configuration of

the olefinic bridge, the presence of 3,4,5-trimethoxy groups on ring A, and a non-coplanar B ring

with a para-methoxy group are required for anti-tubulin activity. Because modifications of ring A

reduce the bioactivity in most analogs of CA-4 (1), ring B and the olefinic bridge offer better

prospects for modification.

(Figure 1. should be listed here)

Indoles, which are important components of the pharmacophore in many anticancer drugs,

are also found in many tubulin polymerization inhibitors [6]. A number of analogs of CA-4 (1)

substitute an indole ring or bioisosteres (e.g., benzofuran and benzothiophene) for ring B and

6

-10

exhibit potent antitumor activity

replacement of the cis double bond of CA-4 (1) with one-atom bridges. For example, carbonyl [4,

, 8] (e.g., 3, 4, 5, 9), methylene [9] (e.g., 6), sulfur [9] (e.g., 7), and amino [10] (e.g., 8) groups

. Briefly, these successful structural modifications include the

7

have been used to link the 3,4,5-trimethoxybenzyl ring A to positions 2 or 3 of indoles or

bioisosteres.

2

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Selenium, which is an essential trace element for human nutrition, occurs in the active sites

of a number of enzymes. Many diseases, such as cancer, arthritis and heart disease, are related to

selenium deficiency [11]. Some organoselenium compounds are bioactive and have demonstrated

versatility and utility in biochemistry and pharmacology. A feasible way for the development of

organoselenium compounds as antitumor agents is to replace the sulfur atom of sulfur-containing

compounds possessing antitumor activity with selenium atom, for instance 6-selenoguanine, a

analog of 6-thioguanine with significant antitumor activity [12].

(Figure 2. should be listed here)

As discussed above, sulfur atoms and carbonyl, amino, and methylene linkers permit a viable

binding conformation for the resulting bioactive molecules. It is reasonable to expect selenium

atom to play a similar role, because it is a bioisostere of these structural units. There has been no

prior report of the biological evaluation of 3-arylselenylindoles for antitumor activity. With the

aim of developing more efficient analogs of CA-4 (1),

a

series of

3

-(3,4,5-trimethoxyphenylselenyl)-1H-indoles and selenoxides were designed, in which the oleﬁn

and B ring of CA-4 (1) were replaced with selenium and indoles, respectively. (Fig. 2). The effect

of diverse substituents at the N1, C2 and C5/6 positions of the indole ring and of changing the

valence of the selenium bridge were evaluated. Additionally, a simple and efficient

microwave-assisted method for synthesizing 3-(3,4,5-trimethoxyphenylselenyl)-1H-indoles is

reported here.

2

. Chemistry

Most methods for the synthesis of 3-selenylindoles involve the direct selenenylation of the

indole nucleus with various electrophilic phenylselenium reagents, such as

N-phenylselenophthalimide (N-PSP) and N-phenylselenosuccinimide (N-PSS) [13, 14].

However, these methods are limited by the instability of phenylselenium reagents and by their

complex synthetic protocols. Fang et al. reported a convenient and efficient synthesis of

3

-arylselenylindoles via iron-catalyzed selenenylation of indoles with diselanes and iodine [15].

Although this method delivers 3-selenylindoles with ease, it suffers from long reaction times.

The 3-selenenylation of indole 10a to afford 12a was investigated to optimize the

conditions; the results are summarized in Table 1. Following Fang’s method, 12a was

3

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synthesized

by

the

reaction

of

2-methyl-1

H

-indole

(

10a

)

with

1

,2-bis-(3,4,5-trimethoxyphenyl)diselenide (11), which was prepared following previously

reported methods [16, 17] and used without purification in the presence of 20 mol % FeCl₃

and 1 mol % iodine. As shown in entries 1-3, 12a was isolated in only 47% yield when the

reaction was refluxed for 48 h.

Microwave irradiation offers many advantages, such as rate enhancements and higher yields,

over conventional heating and has become a popular technique that is widely used in organic

synthesis today [18]. When microwave irradiation was applied to the synthesis of selenide 12a,

we noted that the 3-selenylation of indoles occurred at significantly faster rates. When the reaction

mixture was heated to 80 ºC for 10 min by microwave irradiation at 150 W, 12a was obtained in

3

8% yield (entry 4), which is comparable to the results under conventional heating to the same

temperature for 36 h (entry 2). A clear improvement in yield was observed when the reaction time

was prolonged to 30 min (entry 5). Compared to entries 5 and 6, pressure did not improve the

reaction: open and closed vessels gave similar results. Raising the temperature to 100 ºC in a

closed vessel increased the reaction rate by a small amount (entries 4 and 7). However, it had very

little effect on yield (entries 5 and 9); similarly, further prolonging the reaction time to 60 min at

this temperature had no significant impact (entries 9 and 10). The best results were observed when

the reaction was carried out under microwave irradiation at 150 W in a closed vessel at 80 ºC for

3

0 min (entry 6).

(Table 1. should be listed here)

We next applied the optimized conditions to the synthesis of selenides 12b-o, and the

corresponding results are summarized in Table 2. Starting materials 10a, b and e-k were obtained

commercially. Indoles 10c, d and l-o were prepared as previously reported [19]. Compared with

C2-methylated substrate 10a (entry 6, Table 1), C2-unsubstituted or C2-aryl substituted indoles

gave slightly lower yields (entries 1, 2 and 3), indicating that the C2 positions of the indole ring

tolerates different groups. When the C5/6 positions of the indole ring were substituted with

electron-donating (e.g., methyl, methoxy) or electron-withdrawing (e.g., halogen) groups in

addition to the C2-methyl substituent, the reactions proceeded expeditiously in moderate to good

isolated yields, ranging from 41 to 84% (entries 4-9). The C2-methyl, C5-methoxy-substituted

compound 10j gave the best yield (entry 9). However, the C2-methyl C5-amino-substituted 10k

4

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gave unsatisfactory results (entry 10), with no significant improvement in yield, even after 60 min

(results not shown). Remarkably, steric bulk at the N1 position had very little influence on the

reaction: all N1-methyl (10l, m and n) and N1-benzyl (10o) substituted substrates gave acceptable

yields (entries 11, 12, 13 and 14).

(Table 2. should be listed here)

Compounds 12p and 12q were synthesized using a method similar to that reported previously

(

Scheme 1) [20].Briefly, C2-methyl substituted 12a and 12f were oxidized with SeO to afford the

2

corresponding aldehydes; after workup, those aldehydes were reduced with NaBH to yield the

4

corresponding C2-hydroxymethyl selenides 12p and 12q.

(Scheme 1. should be listed here)

Selenoxides 13a-l were synthesized using conditions similar to a previous method (Scheme

3

) [21]. Various selenides (12a-i, l, m and o) were treated with 30% H O in THF at room

2 2

temperature for 5 to 12 h to yield selenoxides 13a-l at 31-88%.

(Scheme 2. should be listed here)

3

. Biochemical studies and discussion

3

.1. Antiproliferative activity assay

In vitro antiproliferative activity against three human cancer cell lines, including gastric

adenocarcinoma (SGC7901), oral carcinoma (KB) and fibrosarcoma (HT1080), was determined

using an MTT (tetrazolium) assay. CA-4 (1) was employed as a positive control. The drug

concentrations required to inhibit cell growth by 50% (IC ) following incubation in culture

5

0

medium for 72 h are displayed in Table 3. The IC values obtained for CA-4 (1) in this assay are

5

0

1

1.4, 4.1 and 10.9 nM for SGC7901, KB and HT1080, respectively. These results are in good

agreement with previously reported values for CA-4 (1) using the MTT assay [22, 23].

(Table 3. should be listed here)

From the results in Table 3, it is evident that almost all target compounds displayed

moderate to potent antiproliferative activity. First, the effects of various groups at the C2

position of the indole ring upon the growth inhibition of the three cancer cell lines were

investigated. The C2-methyl substituted selenide 12a exhibited significant activity. The

corresponding selenoxide 13a displayed nanomolar IC s against all tested cells, comparable

5

0

5

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to CA-4 (

1), and was thus one of the most active compounds in terms of antiproliferative

activity. Replacing the C2-methyl group of selenide 12a with larger groups, such as phenyl

or 4-fluoro-3-methoxyphenyl groups, slightly reduced the activity (12c and ), while their

corresponding selenoxides 13c and were significantly less potent than 13a. The removal of

d

the C2-methyl group from selenoxide 13a tended to reduce the potency (see the

C2-unsubstituted selenoxide 13b) as well. The results indicate that the presence of small

groups, such as methyl groups, at C2 is essential for potent antiproliferative activity; thus,

C2-methyl substitution was retained for further research.

Using methyl substituents at the C2 position, next, we investigated the effect of

substitution at the C5/6 positions of the indole ring. Of all C5-halogen substituted selenides

(

e.g., F, Cl, Br), only the ﬂuoro-substituted selenide 12e showed similar potency to 12a.

Further, of the selenoxides, only C5-chloro-substituted 13f showed significant activity

against all tested cells. Compared to the C5-unsubstituted 12a and 13a, dihalogenation at

C5/6 (as in selenide 12h and selenoxide 13h) also tended to slightly reduce the potency of

the compound. In contrast, the introduction of electron-donating groups, such as methyl,

methoxy and amino groups, at C5 of selenide 12a led to the significantly more potent

selenides 12i, 12j and 12k, respectively. Generally, the nature of the substituents at the C5/6

positions of the indole rings significantly influenced the biological activity.

Compounds with N1 substituents were also evaluated. Selenides 12l and

m, which

possess a methyl group at N1, were more potent than the corresponding N1-unsubstituted

selenides 12b and . In particular, N1-methyl selenide 12n exhibited superior

antiproliferative activity against SGC7901 and HT1080 cells than positive control CA-4 ( ),

with IC₅₀= 9.5± 0.8 and 2.4± 0.5 nM, respectively. However, these results are in sharp

contrast with those of the corresponding selenoxides: N1-methylated selenoxides 13j and

were less potent than the corresponding unsubstituted selenoxides 13b and . Additionally, a

f

1

k

f

benzyl group at N1tended to almost eliminate the activity, such as that in selenide 12o and

selenoxide 13l. These data indicate that a large group at N1 was not tolerated in this series.

Of particular interest are the selenides 12p and

q, in which the C2-methyl group was

replaced with a hydroxymethyl group (12a and

f), resulting in further improvements in

6

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potency. Selenide 12q was one of the most active compounds against all three tested cell

lines, similar to CA-4 (1), indicating that a C2-hydroxymethyl group contributes to activity.

The therapeutic window is of great significance for anticancer agents. For further

understanding the cytotoxic potential of these target compounds for normal human

cells

， two of the most active compounds, 12n and 13a, were assayed in vitro against L929

cell, a normal fibroblast cell, and the IC values were found to be 6339.8 and 438.4 nM,

5

0

respectively. It was noticeable that IC values were 90- to 2640-fold higher than that against

5

0

the tested tumor cells for 12n, and 18- to 36-fold higher for 13a, thus compound 12n showed

more selective antiproliferative activity than 13a

.2. Tubulin polymerization

Based on these results, the effects of 12n and 13a, which were two of the most active of

the CA-4 ( ) analogs, on the inhibitory of tubulin assembly were evaluated in vitro. CA-4

) and paclitaxel (PAC) were employed as negative and positive controls, respectively. As

shown in Fig. 3, both 13a and CA-4 ( ) inhibited tubulin polymerization in a

dose-dependent manner. At 0.5 ꢀΜ, 13a and CA-4 ( ) exhibited moderate inhibitory

activity; at 1-4 ꢀΜ, both 13a and CA-4 ( ) strongly inhibited tubulin assembly. It is worth

noting that the IC of 13a was 1.648 ꢀM, which is close to that of the positive control CA-4

.

3

1

(

1

5

0

(1) (0.924 ꢀM). Simultaneouly, 12n showed a closely tubulin inhibitory potency to the one

observed with 13a. At the concentration of 3.3 ꢀΜ 12n inhibited the polymerization of

tubulin by 47.2%, when the concentration rose to 10 ꢀΜ the inhibition increased to 73.0%.

In contrast, PAC enhanced the rate of tubulin polymerization compared with untreated cells.

An excellent correlation was observed between the antiproliferative activity and inhibition

of tubulin polymerization for 12n and 13a, indicating that the molecular target of this series

of combretastatin A-4 analogs is most likely tubulin.

(

Figure 3. should be listed here)

Figure 4. should be listed here)

(

3

.3. Immunofluorescence studies

To confirm our finding that 13a strongly inhibited tubulin polymerization, the effect of

compound 13a on cellular microtubule networks was examined using immunofluorescence;

CA-4 (

1) was also tested for comparison. As shown in Fig. 4, the microtubule structures in

7

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control HT-1080 cells were homogenous, linear and well organized. However, cells exposed

to either 20 nM CA-4 (1) or 30 nM 13a for 24 h exhibited the complete inhibition of

microtubule formation, which is consistent with the presence of depolymerized

microtubules. Furthermore, multinucleated cells (mitotic catastrophe) were also observed.

These results confirm that 13a exerts similar effects to CA-4 (

suggesting that 13a is most likely targeting tubulin.

1) on the microtubule network,

(

Figure 5. should be listed here)

.4 Docking study

To investigate the possible binding of target compounds to the colchicine site of

tubulin, a docking study was carried out on the most potent derivative in this series, 13a. For

comparison, CA-4 ( ) was also docked in the colchicine binding site on tubulin. The most

3

．

1

probable orientation of compound 13a in the colchicine binding site (PDB ID: 1SA0) [24]

was determined by docking, using Discovery studio 2.5. program. The docking study (Fig.

5) clearly indicated that compound 13a coincides closely with CA-4 (

1) in the tubulin

binding site; 13a adopts a twisted geometry similar to CA-4 ( ), which may be extremely

1

significant for its bioactivity. The oxygen atom of the 4'-methoxy group of 13a established a

critical strong hydrogen bond with the sulfhydryl group of Cys-241, which was also

observed for CA-4 (1) as previously reported. The results of this docking study are in good

agreement with the potent antiproliferative activity of 13a and its ability to inhibit tubulin

polymerization.

(

Figure 6. should be listed here)

. Conclusion

We designed a set of 3-(3,4,5-trimethoxyphenylselenyl)-1

4

H

-indoles and selenoxides as

a new class of combretastatin A-4 analogs, while the structure of those target compounds are

unique as they all containing selenium within the core structure. The

3

-(3,4,5-trimethoxyphenylselenyl)-1

H-indoles were effectively synthesized under

microwave irradiation. The replacement of the B ring and the cis oleﬁnic core of CA-4 (

1)

with an indole moiety and selenium atom, respectively, maintained or slightly improved the

antiproliferative activity of the compound. The results of in vitro tubulin polymerization and

8

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immunofluorescence experiments and in silico docking are in good agreement with the

antiproliferative activity results for 13a, indicating that the inhibition of tubulin assembly is

the most likely mechanism of action for this series of compounds. These compounds deserve

further investigation.

5

. Experimental section

General: All reagents and solvents were commercially available and were used without

further purification unless otherwise indicated. NMR spectra were performed on a Bruker

1

13

1

13

AVANCE 300 ( H, 300 MHz; C, 100 MHz), Bruker AVANCE 400 ( H, 400 MHz; C, 100

1

13

MHz) or Bruker AVANCE 600 ( H, 600 MHz; C, 150 MHz), in CDCl or DMSO-d (internal

3

6

standard tetramethylsilane). Mass spectra (MS) were measured on an Agilent 1100-sl mass

spectrometer with an electrospray ionization source from Agilent Co., Ltd. High resolution

accurate mass determinations (HRMS) for all final target compounds were obtained on a Bruker

Micromass Time of Flight mass spectrometer equipped with electrospray ionisation (ESI). Melting

points were measured by a X-4 Micro-Melting point detector form Beijing Tech Instrument Co.,

Ltd, without correction. The microwave reactions were performed on a discover-sp single mode

microwave reactor from CEM Corporation. The percentage of diselenide (11) was determined by

using a Shimadzu HPLC-LC-20AT with a Shimadzu SPD-20A UV detector while 75% methanol

in water was used as mobile elution.

5

.1. Chemistry

.1.1. 2-Phenyl-1H-indole (10c).

Following the previously reported methods [20], a mixture of phenylhydrazine hydrochloride

(

0.2 g, 1.38 mmol), acetophenone (0.17 g, 1.38mmol) and sodium acetate (0.11 g, 1.38 mmol) in

5% ethanol (5 mL) was heated by a microwave irradiation at 250 W, 80 °C, for 5 min, the

progress of the reaction was monitored by TLC, filtered, washed with CH Cl and concerntrated in

9

2

vacuo to give the hydrazone, then PPA (3.74 g, 11.06 mmol) was added. The mixture was stirred

at 110 °C for 30 min and then quenched on ice-water. The solid was filtered washed with water,

and dried. The crude product was purified by chromatographic column using silica gel (200- 300

mesh) and petroleum ether/AcOEt (v/v= 10:1) to give 10c as a white solid (0.12 g, 44%); mp

1

88-190 °C; H NMR (CDCl 400 MHz) δ 8.39 (1H, br s, NH), 7.66 (2H, d, J 7.6 Hz, 2×ArH),

3

H

9

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7

.62 (1H, d, J 7.9 Hz, ArH), 7.43 (2H, d, J 7.6 Hz, 2×ArH), 7.40 (1H, d, J 5.7 Hz, ArH), 7.31 (1H,

t, J 7.5 Hz, ArH), 7.19 (1H, m, ArH), 7.12 (1H, t, J 7.5 Hz, ArH),6.80 (1H, br s , ArH) MS (ESI,

-

neg) 191.8 [M-H] .

5

.1.2. 2-(3'-fluoro-4'-methoxyphenyl)-1H-indole (10d).

Indole 10d was obtained from 1-(3-fluoro-4-methoxyphenyl)ethanone as a light yellow solid

1

(

0.14 g, 58%); mp 185-187 °C; H NMR (CDCl 400 MHz) δ 8.26 (1H, br s, NH), 7.60 (1H, dd,

3

H

J 7.69, 0.49 Hz), 7.36 (3H, m, 3×ArH), 7.18 (1H, m, ArH), 7.11 (1H, m, ArH), 7.00 (1H, m, ArH),

-

6

.71 (1H, m, ArH), 3.93 (3H, s, OCH ). MS (ESI, neg) 240.0 [M-H] .

3

General procedure for synthesis of indoles 10l-10n. To a stirred solution of substituted indole (4.25

mmol) in acetone (10 mL) was slowly added potassium tert-butanolate (6.38 mmol) and dimethyl

sulphate (8.5 mmol) or 1-(chloromethyl)-4-fluorobenzene (8.5 mmol) at room temperature for 1

hour, then the reaction mixture was poured into saturated aqueous NaHCO solution (40 mL), and

3

extracted with CH Cl (3×40 mL), The combined organic layers were dried over anhydrous

2

Na SO , and concentrated in vacuo. The crude product was purified by column chromatography

2

4

(petroleum ether /AcOEt).

5

.1.3. 1-Methyl-1H-indole (10l).

Compound (10l) was obtained from 10b as a colourless oil (0.52 g, 94%); H NMR (CDCl

1

3

4

00 MHz) δ 7.62 (1H, d, J 7.9 Hz, ArH), 7.32 (1H, d, J 7.9 Hz, ArH), 7.22 (1H, m, ArH), 7.10

H

(1H, t, J 7.3 Hz, ArH), 7.04 (1H, d, J 1.7 Hz, ArH), 6.48 (1H, d, J 2.1 Hz, ArH), 3.77 (3H, s,

+

NCH ). MS (ESI, pos) 131.2 [M] .

3

5

.1.4. 5-Chloro-1,2-dimethyl-1H-indole (10m).

Compound 10m was obtained from 10f as a white solid (0.71 g, 93% ); mp 56-58 °C; H

NMR (CDCl 400 MHz) δ 7.45 (1H, d, J 1.7 Hz, ArH), 7.12 (1H, d, J 8.6 Hz, ArH), 7.07 (1H,

1

3

H

dd, J 8.6, 1.7 Hz, ArH), 6.12 (1H, br s, ArH), 3.61 (3H, s, NCH ), 2.39 (3H, s, CH ). MS (ESI,

3

+

pos) 179.1 [M] .

.1.5. 5-Methoxy-1,2-dimethyl-1H-indole (10n).

Compound 10n was obtained from 10j as a white solid (0.66 g, 89% ); mp 59-62 °C; H

NMR (CDCl 400 MHz) δ 7.12 (1H, d, J 8.8 Hz, ArH), 7.00 (1H, d, J 2.4 Hz, ArH), 6.80 (1H,

5

1

3

H

dd, J 8.8, 2.4 Hz, ArH), 5.33 (1H, br s, ArH), 3.83 (3H, s, OCH ), 3.61 (3H, s, NCH ), 2.39 (3H, s,

3

+

CH ). MS (ESI, pos) 175.2 [M] .

3

1

0

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5

.1.6. 1-(4'-Fluorobenzyl)-1H-indole (10o).

Compound 10o was prepared from 10b, 1-(chloromethyl)-4-fluorobenzene and KOH in DMF

1

using the same manner as for 10l, as a colourless oil (0.94 g, 98% ); H NMR (CDCl 400 MHz)

3

δ_H7.65 (1H, d, J 7.7 Hz, ArH), 7.24 (1H, s, J 8.2 Hz, ArH), 7.17 (1H, m, ArH), 7.12 (1H, dd, J

7

.7, 1.2 Hz, ArH), 7.09 (1H, d, J 3.0 Hz, ArH), 7.04 (2H, m, 2×ArH), 6.95 (2H, m, 2×ArH), 6.55

+

(

1H, dd, J 3.0, 0.50 Hz, ArH), 5.25 (2H, s, NCH ). MS (ESI) 225.2 [M] .

2

5

.1.7. 1,2-Bis-(3,4,5-trimethoxyphenyl)diselenide (11).

Following the previously reported methods [17, 18], sodium borohydride (2.48 g, 65.50

mmol) in 20mL of water was added with magnetic stirring to 2.59 g (32.75 mmol) of selenium

suspended in 50 mL of water at room temperature under a N atmosphere. After the initial

2

vigorous reaction had subsided (10 min), 1 additional equiv. of selenium (2.59 g, 32.75 mmol)

was added. The mixture was stirred and warmed to 60 °C for 2 hours. The brownish red aqueous

solution of Na Se was then ready for further use. 3,4,5-trimethoxyaniline (6.00 g, 32.75 mmol)

2

was dissolved in conc. HCl (8.4 mL) and 50 mL water, and cooled with an ice bath. A solution of

NaNO (2.26 g, 32.75 mmol) in water (10 mL) was added via syringe over 15 minutes under

2

stirring. The solution was stirred for

,4,5-trimethoxybenzenediazonium chloride was prepared. NaOH (7.86 g, 196.50 mmol) in 15

mL water was added to the solution of sodium diselenide at 0 °C, then the

,4,5-trimethoxybenzenediazonium chloride was added drop wise over 15 minutes. After addition,

a

further 30 minutes at

0

°C and

3

the solution was warmed to room temperature and stirred for a further 2 hours, and was filtered

and the residue washed with CH Cl (30 mL). The mixture was transferred to a separatory funnel,

2

and the organic layer was separated. The aqueous layer was extracted with CH Cl (3×50 mL). The

2

combined organic layer was washed with brine (50 mL) and dried over anhydrous Na SO , finally

2

4

concentrated under reduced pressure. The purification was by chromatographic column using

silica gel (200- 300 mesh) and petroleum ether/AcOEt (v/v= 4:1) to afford a mixture of

1

2

,2-bis-(3,4,5-trimethoxyphenyl)diselenide with 1,1'-selenobis(3,4,5-trimethoxybenzene) (1:1.6)

1

.60 g, as a yellow solid, without further separation. Mp 139-140 °C; H NMR (CDCl 400 MHz)

3

1

3

δ 6.48 (4H, s, 4×ArH), 3.83 (6H, s, 2×OCH ), 3.81 (12H, s, 4×OCH ). C NMR (100MHz,

H

3

CDCl ): δ 153.2 (4×C), 138.4 (2×C), 125.3(2×C), 110.2 (4×C), 60.9 (2×C), 56.2 (4×C). MS (EI)

3

C

1

ACCEPTED MANUSCRIPT

+

m/z: 492.9 [M+H] , 514.8 [M+Na] ; HRMS (ESI):calc. for C H NaO Se [M+Na] 516.9639,

1

8

22

6

2

found 516.9679.

5

.1.8. General procedure for synthesis of 3-(Trimethoxyphenylselenyl)-1H-indoles 12a-12o.

Similar to the repored method [19], a mixture of the appropriate indole (0.6 mmol),

1

,2-bis-(3,4,5-trimethoxyphenyl)diselenide

(0.35

mmol,

0.45

g

mixture

of

1

,2-bis-(3,4,5-trimethoxyphenyl)diselenide with 1,1'-selenobis(3,4,5-trimethoxybenzene)), FeCl₃

(

20 mol%) and I (1 mol%, 0.0001 g/mL in CH CN) was placed into the microwave cavity (closed

2

3

vessel mode). Microwave irradiation at 150 W was used, the temperature being ramped from 25

C to 80 °C. Once 80 °C was reached, taking about 1 min, the reaction mixture was held at this

°

temperature for 30 min while stirring, until complete consumption of the starting material, as

monitored by TLC. After the evaporation of the solvent, the residual crude product was purified

by column chromatography on silica gel (200- 300 mesh) with petroleum ether/AcOEt (v/v= 5:1)

or pure CH Cl .

2

5

.1.8.1. 2-Methyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12a).

Compound 12a was obtained from 10a and 11 as a light yellow solid ꢀ0.16 g, 72%ꢁ, mp

1

38-140°C; H NMR (300 MHz, CDCl ): δ 8.33 (1H, s, NH), 7.58 (1H, dd, J 7.3, 1.4Hz, ArH),

3

H

7

.33 (1H, m, ArH), 7.15 (2H, m, 2×ArH), 6.43 (2H, s, 2×ArH), 3.76 (3H, s, OCH ), 3.64 (6H, s,

3

1

3

2

×OCH ), 2.58 (3H, s, CH ); C NMR (100MHz, CDCl ): δ 153.5 (2×C), 140.7, 136.07, 135.7,

3 3 3 C

1

31.1, 128.3, 122.1, 120.6, 119.7, 110.5, 105.7 (2×C), 96.6, 60.8, 60.0 (2×C), 13.2. MS (ESI)

-

+

3

75.9 [M-H] , 411.8 [M+Cl] ; HRMS (ESI): m/z, calcd. For C H NNaO Se [M+Na] 400.0422,

18 19 3

found 400.0426.

5

.1.8.2. 3-((3,4,5-Trimethoxyphenyl)selenyl)-1H-indole (12b).

Compound 12b was obtained from 10b and 11 as a white solid (0.13 g, 60% ), mp

1

30-134°C; H NMR (400 MHz, CDCl ): δ 8.57 (1H, s, NH), 7.66 (1H, d, J 7.8Hz, ArH), 7.49

3

H

(1H, d, J 1.7 Hz, ArH), 7.43 (1H, d, J 7.8Hz, ArH), 7.25 (1H, m, ArH), 7.18 (1H, t, J 7.4Hz, ArH),

1

3

6

1

6

3

5

.51 (2H, s, 2×ArH), 3.76 (3H, s, OCH ), 3.65 (6H, s, 2×OCH ); C NMR (100MHz, CDCl ): δ

3 3 3 C

53.5 (2×C), 136.4, 131.2, 129.9, 128.1, 122.9, 120.8, 120.3, 111.5, 106.3 (2×C), 105.3, 98.5,

+

0.8, 56.0 (2×C). HRMS (ESI): m/z, calcd. For C H NNaO Se [M+Na] 386.0266, found

1

7

17

3

86.0275.

.1.8.3. 2-Phenyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12c).

1

2

ACCEPTED MANUSCRIPT

1

Compound 12c was obtained from 10c and 11 as a pale yellow oil (0.13 g, 50%); H NMR

300 MHz, CDCl ): δ 8.66 (1H, s, NH), 7.77 (2H, dd, J 8.2, 1.4 Hz, 2×ArH), 7.71 (1H, d, J 7.7

(

3

H

Hz, ArH), 7.43 (4H, m, 4×ArH), 7.27 (1H, m, ArH), 7.19 (1H, m, ArH), 6.45 (2H, s, 2×ArH), 3.75

1

3

(

3H, s, OCH ), 3.61 (6H, s, 2×OCH ); C NMR (100MHz, CDCl ): δ 153.5 (2×C), 142.0, 136.2,

3 3 3 C

1

36.1, 132.1, 132.0, 128.7, 128.7, 128.6, 128.2, 123.7, 123.3, 121.1, 120.9, 111.1, 106.0 (2×C),

+

05.3, 96.5, 60.8, 56.0 (2×C). MS (ESI) 440.0 [M+H] , 462.0 [M+Na] , 901.0 [2M+Na] ; HRMS

+

(

ESI): m/z, calcd. For C H NNaO Se [M+Na] 462.0579, found 462.0581.

2

3

21

3

5

.1.8.4. 2-(3'-Fluoro-4'-methoxyphenyl)-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12d).

Compound 12d was obtained from 10d and 11 as a pale brown solid (0.13 g, 45%); mp

1

77-180 °C; H NMR (300 MHz, CDCl ): δ 8.71 (1H, s, NH), 7.69 (1H, d, J 7.6 Hz, ArH), 7.54

3

H

(1H, dd, J_H_,_F12.3, J 2.3 Hz, ArH), 7.47 (1H, m, ArH), 7.39 (1H, d, J 7.6 Hz, ArH), 7.21 (2H, m,

2

×ArH), 7.00 (1H, t, J 8.7 Hz, ArH), 6.45 (2H, s, 2×ArH), 3.91 (3H, s, OCH ), 3.75 (3H, s,

3

1

3

OCH ), 3.61 (6H, s, 2×OCH ); C NMR (100MHz, CDCl ): δC 153.6 (2×C), 152.1 (d, J 244.1

3

C, F

Hz), 148.0 (d, J_C_,_F10.3 Hz), 140.6 (d, J_C_,_F1.8Hz), 136.3, 136.0, 132.0, 128.1, 125.3 (d, J_C_,_F

7

1

.3Hz), 124.6 (d, J_C_,_F3.3Hz), 123.4, 121.2, 120.8, 116.3 (d, J_C_,_F20.8 Hz), 116.2, 113.4 (d, J_C_,_F

+

.8 Hz), 111.0, 106.0 (2×C), 96.4, 60.8, 56.3, 56.0 (2×C). MS (ESI) 488.0 [M+H] , 510.0

+

[

M+Na] , 526.0 [M+K] ; HRMS (ESI): m/z, calcd. For C H FNNaO Se [M+Na] 510.0590,

24 22 4

found 510.0567.

5

.1.8.5. 5-Fluoro-2-methyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12e).

Compound 12e was obtained from 10e and 11 as a pale yellow solid (0.15 g, 63%); mp

1

22-125 °C; H NMR (300 MHz, CDCl ): δ 8.49 (1H, s, NH), 7.22 (2H, m, 2×ArH), 6.89 (1H,

3

H

1

3

m, ArH), 6.42 (2H, s, 2×ArH), 3.77 (3H, s, OCH ), 3.65 (6H, s, 2×OCH ), 2.55 (3H, s, CH ); C

3

NMR (100MHz, CDCl ): δ 158.6 (d, J 234.5Hz), 153.6 (2×C), 142.7, 136.3, 132.1, 128.0,

C, F

3

C

1

23.7, 111.2 (d, J_C_,_F10.3 Hz), 110.2 (d, J_C_,_F26.4 Hz), 105.8 (2×C), 104.9 (d, J_C_,_F24.9 Hz), 96.8

+

(

d, J_C_,_F4.4 Hz), 60.8, 56.0 (2×C), 13.6. MS (ESI, pos) 396.1 [M+H] , 418.0 [M+Na] , MS (ESI,

-

+

neg) 393.9 [M-H] , 429.8 [M+Cl] ; HRMS (ESI): m/z, calcd. For C H FNNaO Se [M+Na]

1

8

18

3

4

18.0328, found 418.0326.

.1.8.6. 5-Chloro-2-methyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12f).

Compound 12f was obtained from 10f and 11 as a off-white solid (0.19 g, 79%); mp 155-157

5

1

ºC; H NMR (300 MHz, CDCl ): δ 8.41 (1H, s, NH), 7.56 (1H, d, J 1.9 Hz, ArH), 7.24 (1H, d, J

3

H

1

3

ACCEPTED MANUSCRIPT

8

.6 Hz, ArH), 7.12 (1H, dd, J 8.6, 1.9 Hz, ArH), 6.41 (2H, s, 2×ArH), 3.77 (3H, s, OCH ), 3.67

3

1

3

(

6H, s, 2×OCH ), 2.56 (3H, s, CH ); C NMR (100MHz, CDCl ): δ 153.6 (2×C), 142.4, 136.3,

3 3 3 C

1

34.1, 132.5, 127.9, 126.5, 122.4, 119.2, 111.6, 105.8 (2×C), 96.5, 60.8, 56.1 (2×C), 13.3. MS

-

+

(

ESI, neg) 409.8 [M-H] ; HRMS (ESI): m/z, calcd. For C H ClNNaO Se [M+Na] 434.0033,

18 18 3

found 434.0016.

5

.1.8.7. 5-Bromo-2-methyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12g).

Compound 12g was obtained from 10g and 11 as a yellow solid (0.11 g, 41%); mp 174-177

1

ºC; H NMR (300 MHz, CDCl ): δ 8.44 (1H, s, NH), 7.72 (1H, s, ArH), 7.26 (1H, dd, J 8.6, 1.7

3

H

Hz, ArH), 7.19 (1H, d, J 8.6 Hz, ArH), 6.41 (2H, s, 2×ArH), 3.77 (3H, s, OCH ), 3.67 (6H, s,

3

1

3

2

1

4

×OCH ), 2.57 (3H, s, CH ); C NMR (100MHz, CDCl ): δ 153.6 (2×C), 142.2, 136.3, 134.4,

3 3 3 C

33.0, 127.9, 125.0, 122.3, 114.0, 112.1, 105.9 (2×C), 96.4, 60.8, 56.1 (2×C), 13.3. MS (ESI, pos)

+

-

56.0 [M+H] , 478.0 [M+Na] , 494.0 [M+K] ; MS (ESI, neg) 453.8 [M-H] , 489.7 [M+Cl] ;

+

HRMS (ESI): m/z, calcd. For C H BrNNa O Se [M+Na] 477.9527, found 477.9526.

1

8

18

3

5

.1.8.8. 6-Chloro-5-fluoro-2-methyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12h).

Compound 12h was obtained from 10h and 11 as a light yellow solid (0.16 g, 63%); mp

1

45-148 ºC; H NMR (300 MHz, CDCl ): δ 8.42 (1H, s, NH), 7.35 (1H, d, J 5.9 Hz, ArH), 7.31

3

H

(

1H, d, J 9.4Hz, ArH), 6.39 (2H, s, 2×ArH), 3.77 (3H, s, OCH ), 3.67 (6H, s, 2×OCH ), 2.56 (3H,

3

1

3

s, CH₃); C NMR (100MHz, CDCl ): δ 153.8 (d, J 238.8 Hz), 153.6 (2×C), 143.2, 136.4,

C, F

3

C

1

9

31.8, 130.6 (d, J_C_,_F9.3 Hz), 127.6, 115.5 (d, J_C_,_F21.0 Hz), 111.8, 106.1, 105.9 (2×C), 105.2,

+

7.0, 60.8, 56.1(2×C), 13.4. MS (ESI, pos) 430.1 [M+H] , 452.1 [M+Na] ; MS (ESI, neg) 427.9

-

+

[

M-H] ; HRMS (ESI): m/z, calcd. For C H ClFNNaO Se [M+Na] 451.9938, found 45.9938.

18 17 3

5

.1.8.9. 2,5-Dimethyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12i).

Compound 12i was obtained from 10i and 11 as a light yellow solid (0.19 g, 80%); mp

1

8

17-120ºC; H NMR (300 MHz, CDCl ): δ 8.23 (1H, s, NH), 7.37 (1H, s, ArH), 7.21 (1H, d, J

3

H

.2Hz, ArH), 6.70 (1H, dd, J 8.2, 1.4 Hz, ArH), 6.43 (2H, s, 2×ArH), 3.76 (3H, s, OCH ), 3.65

3

1

3

(

6H, s, 2×OCH ), 2.56 (3H, s, CH ), 2.42 (3H, s, CH ); C NMR (100MHz, CDCl ): δ 153.5

3 3 3 3 C

2×C), 140.8, 136.0, 134.0, 131.3, 129.9, 128.4, 123.6, 119.3, 110.1, 105.5 (2×C), 95.9, 60.7, 56.0

+

-

2×C), 21.4, 13.2. MS (ESI, pos) 392.2 [M+H] , 414.2 [M+Na] ; MS (ESI, neg) 390.0 [M-H] ;

+

HRMS (ESI): m/z, calcd. For C H NNaO Se [M+Na] 414.0579, found 414.0594.

1

9

21

3

5

.1.8.10. 5-Methoxy-2-methyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole(12j).

1

4

ACCEPTED MANUSCRIPT

Compound 12j was obtained from 10j and 11 as a light yellow solid (0.21 g, 85%); mp

1

08-114 ºC; H NMR (300 MHz, CDCl ): δ 8.30 (1H, s, NH), 7.21 (1H, d, J 8.7Hz, ArH), 7.04

3

H

(

1H, d, J 2.3 Hz, ArH), 6.82 (1H, m, ArH), 6.43 (2H, s, 2×ArH), 3.81 (3H, s, OCH ), 3.76 (3H, s,

3

1

3

OCH ), 3.65 (6H, s, 2×OCH ), 2.54 (3H, s, CH ); C NMR (100MHz, CDCl ): δ 155.0, 153.6

3

C

(2×C), 141.4, 136.1, 131.9, 130.6, 128.4, 112.1, 111.4, 105.6, 101.6 (2×C), 96.3, 60.8, 56.0 (2×C),

-

+

5

4

5

5.9, 13.3. MS (ESI, neg) 405.9 [M-H] ; HRMS (ESI): m/z, calcd. For C H NNaO Se [M+Na]

19 21 4

30.0528, found 430.0544.

.1.8.11. 2-Methyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indol-5-amine (12k).

Compound 12k was obtained from 10k and 11 as a brown solid (0.07 g, 29%); mp 70-72 ºC;

H NMR (400 MHz, CDCl ): δ 8.15 (1H, s, NH), 7.12 (1H, d, J 8.4 Hz, ArH), 6.86 (1H, d, J 1.7

1

3

H

Hz, ArH), 6.62 (1H, dd, J 8.4, 1.7 Hz, ArH), 6.41 (2H, s, 2×ArH), 3.76 (3H, s, OCH3), 3.66 (6H,

1

3

s, 2×OCH3), 2.52 (3H, s, CH3); C NMR (100MHz, DMSO-d ): δ 153.7 (2×C), 142.6, 136.8,

6

C

1

36.2, 131.9, 131.6, 128.7, 113.5, 112.0, 106.0, 105.8 (2×C), 93.6, 60.4, 56.3 (2×C), 13.2. MS

+

(

ESI) 392.9 [M+H] , 314.9 [M+Na] , 430.9 [M+K] ; HRMS (ESI): m/z, calcd. For C H N O Se

18 21 2 3

+

[

M+H] 393.0712, found 393.0717.

.1.8.12. 1-Methyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12l).

Compound 12l was obtained from 10l and 11 as a a light yellow oil (0.14 g, 60%); H NMR

400 MHz, CDCl ): δ 7.66 (1H, d, J 7.9 Hz, ArH), 7.36 (1H, d, J 7.9 Hz, ArH), 7.33 (1H, s,

5

1

(

3

H

ArH), 7.28 (1H, t, m, ArH), 7.18 (1H, t, J 7.4 Hz, ArH), 6.52 (2H, s , 2×ArH), 3.84 (3H, s, NCH₃),

1

3

1

.76 (3H, s, OCH ), 3.65 (6H, s, 2×OCH ); C NMR (100MHz, CDCl ): δ 153.4 (2×C), 137.3,

3 3 3 C

36.3, 135.3, 130.5, 128.2, 122.4, 120.3, 120.3, 109.5, 106.3 (2×C), 96.4, 60.7, 56.0 (2×C), 33.0.

+

HRMS (ESI): m/z, calcd. For C H NNaO Se [M+Na] 400.0422, found 400.0440.

1

8

19

3

5

.1.8.13. 5-Chloro-1,2-dimethyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole(12m).

Compound 12m was obtained from 10m and 11 as a yellow solid (0.18 g, 71%); mp 95-98

1

ºC; H NMR (400 MHz, CDCl ): δ 7.58 (1H, d, J 1.3 Hz, ArH), 7.20 (1H, d, J 8.6 Hz, ArH), 7.14

3

H

(

1H, dd, J 8.6, 1.3 Hz, ArH), 6.41 (2H, s, 2×ArH), 3.76 (3H, s, NCH ), 3.74 (3H, s, OCH ), 3.67

3 3

1

3

(

6H, s, 2×OCH ), 2.57 (3H, s, CH ); C NMR (100MHz, CDCl ): δ 153.7 (2×C), 143.8, 136.4,

3 3 3 C

1

35.7, 131.7, 128.1, 126.3, 122.0, 119.2, 110.1, 106.0, 105.2, 95.5, 60.8, 56.1 (2×C), 30.7, 12.2.

+

HRMS (ESI): m/z, calcd. For C H ClNNaO Se [M+Na] 448.0189, found 448.0175.

1

9

20

3

5

.1.8.14. 5-Methoxy-1,2-dimethyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12n).

1

5

ACCEPTED MANUSCRIPT

Compound 12n was obtained from 10n and 11 as a light yellow solid (0.12 g, 48%); mp

1

27-130 ºCꢂ H NMR (400 MHz, DMSO-d ): δ 7.41 (1H, d, J 8.6 Hz, ArH), 6.90 (1H, d, J 2.4

6

H

Hz, ArH), 6.80 (1H, dd, J 8.6, 2.4 Hz, ArH), 6.45 (2H, s, 2×ArH), 3.76 (3H, s, OCH ), 3.73 (3H,

3

1

3

s, OCH ), 3.58 (6H, s, 2×OCH ), 3.57 (3H, s, NCH ), 2.53 (3H, s, CH ); C NMR (100MHz,

3

DMSO-d ): δ 154.8, 153.7 (2×C), 143.9, 136.3, 132.5, 131.0, 128.5, 111.4, 111.2, 106.2 (2×C),

6

C

1

01.1, 94.2, 60.4, 56.5, 56.3 (2×C), 55.8, 12.2. HRMS (ESI): m/z, calcd. For C H NO Se

20 24 4

+

[

M+H] 422.0865, found 422.0879.

.1.8.15. 1-(4 -Fluorobenzyl)-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12o).

Compound 12o was obtained from 10o and 11 as a light yellow oil (0.17 g, 62%); H NMR

600 MHz, CDCl ): δ 7.63 (1H, d, J 7.8 Hz, ArH), 7.40 (1H, s, ArH), 7.32 (1H, d, J 8.2 Hz,

5

'

1

(

3

H

ArH), 7.24 (1H, t, J 7.6 Hz, ArH), 7.19 (1H, t, J 7.6 Hz, ArH), 7.12 (2H, m, 2×ArH), 6.99 (2H, t, J

8

2

1

.6 Hz, 2×ArH), 6.49 (2H, s, 2×ArH), 5.34 (2H, s, NCH ), 3.76 (3H, s, OCH ), 3.63 (6H, s,

2

3

1

3

×OCH₃); C NMR (100MHz, CDCl ): δ 162.3 (d, J 246.1 Hz), 153.5 (2×C), 136.8, 136.4,

3

C

C, F

34.6, 132.6 (d, J_C_,_F3.2 Hz), 130.8, 128.6, 128.5, 128.1, 122.9, 120.7 (d, J_C_,_F14.4 Hz), 115.9,

+

15.7, 110.0, 106.1 (2×C), 105.2, 97.7, 60.8, 56.0 (2×C), 49.7. MS (ESI) 472.0 [M+H] , 965.2

+

[

2M+Na] ; HRMS (ESI): m/z, calcd. For C H FNNaO Se [M+Na] 494.0641, 494.0635.

24 22 3

5

.1.8.16. (3-(3,4,5-Trimethoxyphenylselenyl)-1H-indol-2-yl)methanol (12p).

21

Similar to the reported method, to a stirred solution of 12a (0.15 g, 0.4 mmol) in

1

,4-dioxane (15 mL) was added SeO (0.22 g, 1.99 mmol) and the reaction mixture was refluxed

2

under a N atmosphere for 6 hours. The reaction mixture was filtered and concentrated under

2

reduced pressure. NaBH (0.03 g, 0.80 mmol) was added to the residue in EtOH (5 mL), and the

4

mixture was stirred at room temperature for 1 hour. The reaction was quenched by the addition of

HCl (1 mol/L), and the resulting residue was extracted with CH Cl (3×20 mL). The combined

2

organic extracts were dried, filtered, and concentrated, and the crude residue was purified by

column chromatography on silica gel (200- 300 mesh) with petroleum ether/AcOEt (v/v= 2:1) to

1

afford 12p as a brown oil (0.064 g, 41%), H NMR (300 MHz, CDCl ): δ 8.99 (1H, s, NH),

3

H

7

6

.64 (1H, d, J 7.7 Hz, ArH), 7.38 (1H, d, J 7.8 Hz, ArH), 7.22 (1H, m, ArH), 7.16 (1H, m, ArH),

1

3

.44 (2H, s, 2×ArH), 4.99 (2H, s, CH ), 3.75 (3H, s, OCH3), 3.63 (6H, s, 2×OCH3); C NMR

2

(

100MHz, CDCl ): δ 153.5 (2×C), 142.3, 136.3, 136.0, 130.7, 128.1, 123.0, 120.8, 120.2, 111.4,

3 C

1

6

ACCEPTED MANUSCRIPT

-

1

06.0 (2×C), 96.0, 60.8, 57.6, 56.0 (2×C). MS (ESI, neg) 392.0 [M-H] , 427.9 [M+Cl] ; HRMS

+

(

ESI): m/z, calcd. For C H NNaO Se [M+Na] 416.0372, found 416.0373.

1

8

19

4

5

.1.8.17. (5-Chloro-3-(3,4,5-trimethoxyphenylselenyl)-1H-indol-2-yl)methanol (12q).

Compound 12q was prepared from 12f (0.16 g, 0.4 mmol) in the same manner as for 12p, as

1

a light yellow oil (0.06 g, 33%); H NMR (400 MHz, CDCl ): δ 9.09 (1H, s, NH), 7.62 (1H, s,

3

H

ArH), 7.31 (1H, d, J 8.5 Hz, ArH), 7.17 (1H, dd, J 8.5, 1.3 Hz, ArH), 6.41 (2H, s, 2×ArH), 4.99

1

3

(

2H, s, CH ), 3.76 (3H, s, OCH ), 3.65 (6H, s, 2×OCH ); C NMR (100MHz, CDCl ): δ 153.6

2 3 3 3 C

(2×C), 143.8, 134.2, 132.0, 127.5, 126.7, 123.3, 119.6, 112.4, 110.1, 106.0 (2×C), 95.4, 60.8, 57.7,

+

5

6.1 (2×C). HRMS (ESI): m/z, calcd. For C H ClNNaO Se [M+Na] 449.9982, found 449.9977.

1

8

18

4

5

.1.9. General procedure for synthesis of compounds 13k-13l.

2

Similar to a reported method, a mixture of 3-(trimethoxyphenylselenyl)-1H-indoles (0.6

mmol) and 30% H O (10 d) in THF (10 mL) was stirred at room temperature for 5-12 hours until

2

complete consumption of the starting material, as monitored by TLC. After the evaporation of the

solvent, the residual crude product was purified by flash chromatography with CH Cl /CH OH

2

3

(

v/v= 10:1).

.1.9.1. 2-Methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13a).

Compound 13a was obtained from 10a as a off-white solid (0.09 g, 39%), mp 98-101ºC; H

NMR (400 MHz, DMSO-d ): δ 11.73 (1H, s, NH), 7.33 (2H, d, J 8.9 Hz, 2×ArH), 7.11 (2H, s,

5

1

6

H

2

×ArH), 7.05 (1H, t, J 7.6 Hz, ArH), 6.92 (1H, t, J 7.6 Hz, ArH), 3.76 (6H, s, 2×OCH ), 3.66 (3H,

3

1

3

s, OCH ), 2.63 (3H, s, CH ); C NMR (100MHz, DMSO-d ): δ 153.9 (2×C), 141.0, 139.3, 136.8,

3

6

C

1

3

36.1, 126.6, 122.1, 120.6, 118.9, 111.9, 109.9, 103.8 (2×C), 60.6, 56.7 (2×C), 12.8. MS (ESI)

94.1 [M+H]+, 416.1 [M+Na]+, 432.0 [M+K]+; HRMS (ESI): m/z, calcd. For C H NO Se

1

8

20

4

+

[

M+H] 394.0552, found 394.0550.

.1.9.2. 3-(3,4,5-Trimethoxyphenylseleninyl)-1H-indole (13b).

Compound 13b was obtained from 10b as a brown oil (0.20 g, 88%); H NMR (300 MHz,

DMSO-d ): δ 11.78 (1H, s, NH), 7.81 (1H, s, J 2.0 Hz, ArH), 7.57 (1H, d, J 8.1 Hz, ArH), 7.45

5

1

6

H

(1H, d, J 8.1 Hz, ArH), 7.16 (2H, s, 2×ArH), 7.13 (1H, d, J 7.9 Hz, ArH), 7.02 (1H, t, J 7.9 Hz,

1

3

ArH), 3.77 (6H, s, 2×OCH ), 3.67 (3H, s, OCH ); C NMR (100MHz, CDCl ): δ 153.4(2×C),

3

C

1

36.5, 136.3, 131.2, 129.9, 128.2, 122.9, 120.8, 120.2, 111.5, 106.3 (2×C), 103.1, 60.8, 56.1

1

7

ACCEPTED MANUSCRIPT

+

-

(

2×C). MS (ESI, pos) 380.0 [M+H] , 402.0 [M+Na] ; MS (ESI, neg) 377.8 [M-H] ; HRMS (ESI):

+

m/z, calcd. For C H NO Se [M+H] 380.0396, found 380.381.

17

18

4

5

.1.9.3. 2-Phenyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13c).

Compound 13c was obtained from 10c as a white solid (0.09 g, 33%); mp 101-104 ºC; H

NMR (300 MHz, CDCl ): δ 11.37 (1H, s, NH), 7.34 (4H, m, ArH), 7.22 (1H, d, J 7.1 Hz, ArH),

1

3

H

7

.07 (3H, m, ArH), 6.96 (1H, d, J 7.5 Hz, ArH), 6.93 (2H, s, 2×ArH), 3.84 (3H, s, OCH ), 3.73

3

1

3

(

6H, s, 2×OCH₃); C NMR (100MHz, DMSO-d ): δ 153.9 (2×C), 143.2, 139.4, 136.9, 136.4,

6 C

1

6

4

5

30.9, 129.9, 129.8 (2×C), 129.5 (2×C), 129.4, 127.2, 125.4, 123.3, 121.1, 119.9, 112.6, 104.0,

+

0.6, 56.6 (2×C). MS (ESI) 456.0 [M+H]+; HRMS (ESI): m/z, calcd. For C H NO Se [M+H]

2

3

22

4

56.0709, found 456.0710.

.1.9.4. 2-(3 -Fluoro-4 -methoxyphenyl)-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13d).

Compound 13d was obtained from 10d as a white solid (0.13 g, 44%); mp 147-149 ºC;

NMR (300 MHz, CDCl ): δ 12.16 (1H, s, NH), 7.38 (1H, d, J 8.1 Hz, ArH), 7.22 (2H, d, J 7.1

'

1

H

3

H

Hz, 2×ArH), 7.07 (1H, t, J 7.8, 7.1 Hz, ArH), 6.96 (1H, d, J 7.5 Hz, ArH), 6.92 (2H, s, 2×ArH),

6

2

.60 (1H, d, J 7.8 Hz, ArH), 5.99 (1H, t, J 8.1, 7.8 Hz, ArH), 3.84 (3H, s, OCH ), 3.76 (6H, s,

3

1

3

×OCH ), 3.71 (3H, s, OCH );

C NMR (100MHz, DMSO-d ): δ 153.8 (2×C), 152.09 (d, J

6 C C,F

3

45.2)ꢃ148.5 (d, J_C_,_F10.5 Hz), 141.9, 139.4, 136.5, 136.1, 127.5, 126.5 (d, J_C_,_F3.0 Hz), 123.6 (d,

J_C_,_F7.5 Hz), 123.2, 121.1, 119.7, 117.3 (d, J_C_,_F19.8 Hz), 114.6, 112.5, 110.7, 104.1 (2×C), 60.6,

-

5

6.7, 56.6 (2×C). MS (ESI, neg) 502.0 [M-H] ; HRMS (ESI): m/z, calcd. For C H FNO Se

2

4

23

5

+

[

M+H] 504.0720, found 504.0740.

.1.9.5. 5-Fluoro-2-methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13e).

Compound 13e was obtained from 10e as a white solid (0.11 g, 43%); mp 112-113 ºC; H

NMR (300 MHz, CDCl ): δ 11.55 (1H, s, NH), 7.07 (1H, m, ArH), 6.96 (2H, s, 2×ArH), 6.74

5

1

3

H

1

3

(

2H, m, ArH), 3.89 (3H, s, OCH ), 3.81 (6H, s, 2×OCH ), 2.08 (3H, s, CH ); C NMR (100MHz,

3 3 3

CDCl ): δ 158.2 (d, J 237.1 Hz), 154.2 (2×C), 153.7 (d, J_C_,_F38.8 Hz), 144.0, 140.2, 132.8,

C, F

3

C

1

6

32.6, 126.4 (d, J_C_,_F11.8 Hz), 112.7 (d, J_C_,_F9.3 Hz), 110.6 (d, J_C_,_F26.1 Hz), 103.7 (2×C), 102.7,

-

1.1, 56.5 (2×C), 12.1. MS (ESI, neg) 409.9 [M-H] ; HRMS (ESI): m/z, calcd. For C H FNO Se

1

8

19

4

+

[

M+H] 412.0458, found 412.0448.

5

.1.9.6. 5-Chloro-2-methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13f).

1

8

ACCEPTED MANUSCRIPT

1

Compound 13f was obtained from 10f as a off-white solid (0.13 g, 50%); mp 113-115 ºC; H

NMR (300 MHz, CDCl ): δ 11.20 (1H, s, NH), 7.14 (1H, d, J 1.7 Hz, ArH), 7.10 (1H, d, J 8.7

3

H

Hz, ArH), 6.98 (1H, dd, J 8.7 Hz, 1.7 Hz, ArH), 6.97 (2H, s, 2×ArH), 3.89 (3H, s, OCH ), 3.83

3

1

3

(

6H, s, 2×OCH ), 2.12 (3H, s, CH ); C NMR (100MHz, CDCl ): δ 154.3 (2×C), 143.6, 140.4,

3 3 3 C

1

34.6, 132.6, 127.0, 126.7, 122.7, 118.0, 112.9, 103.8 (2×C), 102.8, 61.1, 56.6 (2×C), 12.2. MS

+

-

(

ESI, pos) 428.0 [M+H] , 450.0 [M+Na] ; MS (ESI, neg) 425.8 [M-H] ; HRMS (ESI): m/z, calcd.

+

For C H ClNO Se [M+H] 428.0162, found 428.0160.

18

19

4

5

.1.9.7. 5-Bromo-2-methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13g).

Compound 13g was obtained from 10g as a off-white solid (0.12 g, 41%); mp 115-117 ºC; H

NMR (300 MHz, CDCl ): δ 10.81 (1H, s, NH), 7.33 (1H, s, ArH), 7.11 (2H, m, ArH), 6.98 (2H,

1

3

H

1

3

s, 2×ArH), 3.89 (3H, s, OCH ), 3.84 (6H, s, 2×OCH ), 2.17 (3H, s, CH ); C NMR (100MHz,

3

CDCl ): δ 153.6 (2×C), 142.2, 136.4, 134.4, 133.0, 125.0, 124.9, 122.3, 120.1, 114.1, 112.1,

3

C

+

1

12.0, 105.9, 60.8, 56.1 (2×C), 13.3. MS (ESI, pos) 471.9 [M+H] , 493.9 [M+Na] ; MS (ESI,

-

+

neg) 469.8 [M-H] ; HRMS (ESI): m/z, calcd. For C H BrNO Se [M+H] 471.9657, found

18

19

4

71.9659.

.1.9.8. 6-Chloro-5-fluoro-2-methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13h).

Compound 13h was obtained from 10h as a light red solid (0.09 g, 32%); mp 185-188 ºC; H

NMR (300 MHz, CDCl ): δ 11.63 (1H, s, NH), 7.15 (1H, d, J 5.7 Hz, ArH), 6.95 (2H, s, 2×ArH),

5

1

3

H

1

3

6

.81 (1H, s, J 9.7 Hz, ArH), 3.89 (3H, s, OCH ), 3.84 (6H, s, 2×OCH ), 2.18 (3H, s, CH ); C

3 3 3

NMR (100MHz, CDCl ): δ 153.7 (d, J 239.6 Hz), 154.3 (2×C), 144.3, 140.4, 132.4 (d, J_C_,_F

3

C

C, F

2

2.3 Hz), 124.8 (d, J_C_,_F10.4 Hz), 116.3 (d, J_C_,_F20.8 Hz), 113.1, 111.9 (d, J_C_,_F= 11.9 Hz), 105.9,

+

1

04.8 (d, J_C_,_F25.3 Hz), 103.6 (2×C), 61.1, 56.6 (2×C), 12.3. MS (ESI, pos) 446.0 [M+H] , 468.0

+

-

[

M+Na] , 483.9 [M+K] ; MS (ESI, neg) 443.8 [M-H] ; HRMS (ESI): m/z, calcd. For

+

C H ClFNO Se [M+H] 446.0068, found 446.0088.

1

8

18

4

5

.1.9.9. 2,5-Dimethyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13i).

Compound 13i was obtained from 10i as a yellow oil (0.09 g, 37%); H NMR (600 MHz,

CDCl ): δ 11.03 (1H, s, NH), 7.10 (1H, d, J 8.3 Hz, ArH), 7.02 (1H, s, ArH), 6.99 (2H, s,

1

3

H

2

×ArH), 6.85 (1H, d, J 8.3 Hz, ArH), 3.87 (3H, s, OCH ), 3.80 (6H, s, 2×OCH ), 2.25 (3H, s,

3 3

1

3

CH ), 2.05 (3H, s, CH ); C NMR (100MHz, DMSO-d ): δ 153.9 (2×C), 140.8, 139.3, 136.7,

3

6

C

1

34.3, 129.0, 126.9, 123.5, 118.7, 111.5, 109.3, 103.9 (2×C), 60.6, 56.6 (2×C), 21.6, 12.8. MS

1

9

ACCEPTED MANUSCRIPT

+

-

(

ESI, pos) 408.2 [M+H] , 430.2 [M+Na] , 446.2 [M+K] ; MS (ESI, neg) 406.0 [M-H] ; HRMS

+

(

ESI): m/z, calcd. For C H NO Se [M+H] 408.0709, found 408.0721.

1

9

22

4

5

.1.9.10. 1-Methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13j).

Compound 13j was obtained from 10l as a colorless oil (0.21 g, 88%); H NMR (300 MHz,

CDCl ): δ 7.59 (1H, d, J 8.2 Hz), 7.35 (1H, s, ArH), 7.34 (2H, m, ArH), 7.17 (1H, t, J 7.6 Hz,

1

3

H

1

3

ArH), 7.06 (2H, s, ArH), 3.87 (3H, s, OCH ), 3.86 (6H, s, 2OCH ), 3.82 (3H, s, NCH ); C NMR

3

(

100MHz, DMSO-d ): δ 153.6 (2×C), 139.1, 137.6, 136.7, 132.9, 125.7, 122.7, 120.8, 119.4,

6 C

1

4

5

12.9, 110.0, 103.4 (2×C), 60.3, 56.3 (2×C), 33.1. MS (ESI) 394.0 [M+H]+, 416.0 [M+Na]+,

+

32.0 [M+K]+; HRMS (ESI): m/z, calcd. For C H NO Se [M+H] 394.0552, found 394.0563.

1

8

20

4

.1.9.11. 5-Chloro-1,2-dimethyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13k).

Compound 13k was obtained from 10m as a white solid (0.10 g, 37%); mp 90-93 ºC ; H

NMR (400 MHz, CDCl ): δ 7.40 (1H, d, J 1.8 Hz, ArH), 7.19 (1H, d, J 8.7 Hz, ArH), 7.13 (1H,

1

3

H

dd, J 8.7, 1.8 Hz, ArH), 7.01 (2H, s, 2×ArH), 3.87 (3H, s, OCH ), 3.86 (6H, s, 2×OCH ), 3.46

3

1

3

(

3H, s, NCH ), 2.63 (3H, s, CH ); C NMR (100MHz, CDCl ): δ 154.1 (2×C), 142.7, 140.1,

3 3 3 C

1

35.6, 134.8, 127.1, 126.8, 122.8, 118.7, 110.5, 103.6 (2×C), 103.1, 60.0, 56.6 (2×C), 30.1, 11.8.

+

HRMS (ESI): m/z, calcd. For C H ClNO Se [M+H] 442.0319, found 442.0343.

1

9

21

4

5

.1.9.12. 1-(4

Compound 13l was obtained from 10o as a white solid (0.19 g, 64%); mp 147-149 ºC ; H

NMR (400 MHz, CDCl ): δ 7.61 (1H, d, J 8.0 Hz, ArH), 7.42 (1H, s, ArH), 7.30 (1H, d, J 8.2

'-Fluorobenzyl)-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13l).

1

3

H

Hz, ArH), 7.23 (1H, dd, J 8.0, 1.1 Hz, ArH), 7.16 (1H, m, ArH), 7.11 (2H, m, 2×ArH), 7.04 (2H,

s, 2×ArH), 7.00 (2H, m, 2×ArH), 5.29 (2H, s, NCH ), 3.87 (3H, s, OCH ), 3.85 (6H, s, 2×OCH );

2

3

1

3

C NMR (100MHz, CDCl ): δ 162.5 (d, J 247.0 Hz), 154.0 (2×C), 140.1, 137.1, 135.3, 131.6

3

C

C, F

(d, J_C_,_F3.4 Hz), 131.3, 128.9, 128.8, 126.3, 123.6, 121.7, 119.8, 116.1, 115.9, 114.1, 110.6, 103.6

+

(

2×C), 61.0, 56.4 (2×C), 50.1. MS (ESI) 488.0 [M+H] , 975.0 [2M+H] ; HRMS (ESI): m/z, calcd.

+

For C H FNO Se [M+H] 488.0771, found 488.0772.

24

23

4

5

.2 Biochemistry methods

.2.1 Antiproliferative activity assay

The in vitro antiproliferative activity assay of CA-4 (1) and all the target compounds were

determined by MTT (Sigma) assay, which were carried out as previously published [25]. Briefly,

4

cells were seeded into 96-well plates at a density of 1-3×10 /well (depends on the cell growth rate),

2

0

ACCEPTED MANUSCRIPT

2

4 h later, triplicate wells were treated with media and agents. After 72 h incubated at 37 °C in 5%

CO , the drug containing medium was removed and replaced by 100 ꢀL fresh medium with 5

2

mg/mL MTT solution. After 4 h incubation, the medium with MTT was removed and 100 ꢀL

dimethyl sulfoxide (DMSO) was added to each well. The plates were gently agitated until the

purple formazan crystals were dissolved and the OD₄₉₀was determined using a microplate reader

(MK3, Thermo, Germany). The data was calculated and plotted as percent viability compared to

control. The 50% inhibitory concentration (IC ) was defined as the concentration that reduced the

5

0

absorbance of the untreated wells by 50% of the vehicle in the MTT assay.

Cytotoxic potential of 12n and 13a against L929 cell were carried out in the similar way.

5

.2.2 Tubulin polymerisation study

The effect of compounds 13a and CA-4 (1) on the polymerization of tubulin was determined

by employing a fluorescence-based tubulin polymerization assay kit (Cytoskeleton-Cat.#

BK011P) according to the manufacturer’s protocol. Tubulin was re-suspended in ice cold G-PEM

buffer (80 mM PIPES, 2 mM MgCl , 0.5 mM EGTA, 1 mM GTP, 20% (v/v) glycerol) and added

2

to wells on a 96 well plate containing the designated concentration of 13a, CA-4 (1) or vehicle.

Samples were mixed well and tubulin assembly was monitored (emission wavelength is 450 nm;

excitation wavelength is 360 nm) at 1 min intervals for 90 min at 37 °C using a plate reader

(

FASCalibur, BD Biosciences, USA) and the increasing absorbance was turned into the extent of

polymerization of tubulin (%) by using computer calculationꢃwhile IC values were calculated by

5

0

using GraphPad Prism software.

The inhibitory of tubulin for compound 12n was determined at the concenrtration of 3.3 and

0 ꢀΜ using the methods similar to 13a.

1

5

.2.3Immunofluorescence studies

Immunofluorescence studies were carried out to detect microtubule associated tubulin protein

after exposure to CA-4 (1) and the selected compounds 13a [25]. The HT-1080 cells were seeded

4

at 1×10 per well on a 24 well plate and grown for 24 h. Cells were treated with vehicle, 30 nM of

1

3a or 20 nM of CA-4 (1) for 48 h. The control and treated cells were fixed with 4 %

formaldehyde in PBS for 30 min at -20 °C, then washed twice with PBS and permeabilized with

.1 % (v/v) Triton X-100 in PBS for 5 min. Then, the cells were blocked with 5 % bovine serum

albumin (BSA) in PBS for 10 min. The primary α-tubulin antibody (Santa Cruz, CA) was diluted

0

2

1

ACCEPTED MANUSCRIPT

(1:100) with 2% BSA in PBS and incubated overnight at 4 °C. The cells were washed with PBS to

remove unbound primary antibody and then cells were incubated with FITC-conjugated

anti-mouse secondary antibody, diluted (1:1000) with 2% BSA in PBS, for 3 h at 37 °C. The cells

were washed with PBS to remove unbound secondary antibody, nucleus was stained with 4,

6

-diamino-2-phenolindol dihydrochloride (DAPI) and then, immunofluorescence was detected

using a fluorescence microscope (Olympus, Tokyo, Japan).

5

.3 Docking study

Docking study was performed with Discovery studio 2.5 program. The structures of

compound 13a and CA-4 (1) were built using Chemdraw 11.0 program, followed by changed the

force-field, added hydrogen atoms and minimized their energy using Discovery studio 2.5

program, thus those two molecules were converted into their 3D structures, and the ligands were

obtained. The X-ray structure of tubulin complex with DAMA-colchicine was downloaded from

the Protein Data Bank (PDB: 1sa0) [24]. Only subunits A and B (the colchicine binding site) were

conserved while other subunits were removed. Hydrogen atoms were added to the protein and the

energy was minimizd using Discovery studio 2.5 program, over the refined model, the active site

of tubulin was defined around the crystallized colchicine, then the colchicine was removed form

the X-ray structure, and the receptor were prepared. Finally, the obtained ligands 13a and CA-4

(1) were docked into the colchicine binding site and the docking simulations were performed

using Discovery studio 2.5 program.

Acknowledgements

We gratefully acknowledge the National S&T Major Project (2012ZX09103101-060), the

National Natural Science Foundation of China (30973614), and the Natural Science Foundation of

Liaoning Province (2013010434-401) for generous financial support. This work was also

supported by Program for Innovative Research Team of the Ministry of Education and Program

for Liaoning Innovative Research Team in University.

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Legends (Figure and table captions)

Figure 1. The structures of CA-4 (1), CA-4P (2) and their analogs

Figure 2. The rational design of target compounds

Figure 3. The effects of PAC and CA-4 (1) on the inhibition of tubulin polymerization.

Employing a fluorescence-based tubulin polymerization assay kit (Cytoskeleton-Cat.# BK011P),

the effects of control (DMSO), PAC and CA-4 (1) at designed concentration on tubulin assembly

were monitored (emission wavelength is 450 nm; excitation wavelength is 360 nm) at 1 min

intervals for 90 min at 37 °C using a plate reader (FASCalibur, BD Biosciences, USA) and the

increasing absorbance was turned into the extent of polymerization of tubulin (%) by using

computer calculation.

Figure 4. The effects of 13a on the inhibition of tubulin polymerization. Employing a

fluorescence-based tubulin polymerization assay kit (Cytoskeleton-Cat.# BK011P), the effects of

control (DMSO) and 13a at designed concentration on tubulin assembly were monitored (emission

wavelength is 450 nm; excitation wavelength is 360 nm) at 1 min intervals for 90 min at 37 °C

using a plate reader (FASCalibur, BD Biosciences, USA) and the increasing absorbance was

turned into the extent of polymerization of tubulin (%) by using computer calculation.

Figure 5. The effect of various compounds on the microtubule network: HT-1080 cells were

treated with either 13a (30 nM) or CA-4 (1, 20 nM) for 12 h. After incubation, the cells were fixed

and stained with monoclonal α-tubulin antibody (green). DAPI (blue) was used for nuclear

counterstaining. The cellular microtubules were observed by fluorescence microscopy (bar scale

1

00 ꢀm).

Figure 6. The docked poses of 13a (green) and CA-4 (1, purple) in the tubulin binding site.

Table 1. The optimization of conditions for the preparation of compound 12a

2

5

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Table 2. The microwave synthesis of 3-(trimethoxyphenylselenyl)-1H-indoles 12b-o

Table 3. The in vitro antiproliferative activity of the synthesized compounds (12a-13l) against

three human cancer cell lines.

Scheme 1. The synthesis of compounds 12p and 12q. Reagents and conditions: a) i. SeO₂,

1

,4-dioxane, reflux; ii. NaBH , EtOH, r.t.

4

Scheme 2. The synthesis of compounds 13a-l. Reagents and conditions: a) H O , THF, rt.

2

6

ACCEPTED MANUSCRIPT

Table 1. The optimization of conditions for the preparation of compound 12a

Temp

ºC)

Power

Yield

Entry

Heating

Mode

Time

a

(

(W)

-

(%)

1

2

3

4

5

6

7

8

9

oil bath

-

80

24 h

27

35

47

38

69

72

42

61

71

73

36 h

-

80

48 h

-

b

c

MW

OV

80

10 min

30 min

10 min

15 min

30 min

60 min

150

200

MW

OV

80

d

CV

80

CV

100

1

0

CV

c

a

b

d

isolated yield. MW: microwave. OV: open vessel. CV: closed vessel.

ACCEPTED MANUSCRIPT

Table 2. The microwave synthesis of 3-(trimethoxyphenylselenyl)-1H-indoles 12b-o

a

Entry Indole

R₁

R₂

R₃

H

Product Yield (%)

1

2

3

4

5

6

7

8

9

10b

10c

10d

10e

10f

H

12b

12c

12d

12e

12f

60

50

45

63

79

41

63

80

84

29

60

71

48

62

H

phenyl

H

3'-fluoro-4'-mehoxyphenyl

H

Me

H

5-F

H

5-Cl

5-Br

5-F, 6-Cl

5-Me

5-OMe

5-NH2

H

10g

10h

10i

H

12g

12h

12i

H

10j

H

12j

1

0

10k

10l

H

12k

12l

1

2

3

4

Me

1

10m

10n

10o

Me

H

5-Cl

5-OMe

H

12m

12n

12o

Me

4'-fluorobenzyl

a

isolated yield.

Article Doi

3-(3,4,5-Trimethoxyphenylselenyl)-1 H -indoles and their selenoxides as combretastatin A-4 analogs: Microwave-assisted synthesis and biological evaluation

DOI: 10.1016/j.ejmech.2014.11.024

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Authors:

Article abstract of DOI:10.1016/j.ejmech.2014.11.024

Full text of DOI:10.1016/j.ejmech.2014.11.024

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