Accepted Manuscript

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

Zhiyong Wen , Jingwen Xu , Zhiwei Wang , Huan Qi , Qile Xu , Zhaoshi Bai , QianZhang , Kai Bao , Yingling Wu , Weige Zhang

PII: S0223-5234(14)01046-0

DOI: 10.1016/j.ejmech.2014.11.024

Reference: EJMECH 7517

To appear in: European Journal of Medicinal Chemistry

Received Date: 11 August 2014

Revised Date: 9 November 2014

Accepted Date: 11 November 2014

Please cite this article as: Z. Wen, J. Xu, Z. Wang, H. Qi, Q. Xu, Z. Bai, Q. Zhang, K. Bao, Y. Wu,W. Zhang, 3-(3,4,5-Trimethoxyphenylselenyl)-1H-indoles and their selenoxides as combretastatinA-4 analogs: Microwave-assisted synthesis and biological evaluation, European Journal of MedicinalChemistry (2014), doi: 10.1016/j.ejmech.2014.11.024.

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A microwave-assisted procedure for the effective synthesis of a series of

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

against cancer cell proliferation and tubulin polymerization.

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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

Zhiyong Wena, Jingwen Xub, Zhiwei Wanga, Huan Qib, Qile Xua, Zhaoshi Baib, Qian Zhanga

Kai Baoa,c, Yingling Wub,*, Weige Zhanga,*

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 IC50 values. 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.

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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

G2/M phase of the cell cycle, causing mitotic catastrophe and, ultimately, apoptotic cell death [3,

4]. However, poor bioavailability and water solubility prevent 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

exhibit potent antitumor activity 6-10. Briefly, these successful structural modifications include the

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

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

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

bioisosteres.

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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

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synthesized by the reaction of 2-methyl-1H-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

38% 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

30 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

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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 SeO2 to afford the

corresponding aldehydes; after workup, those aldehydes were reduced with NaBH4 to yield the

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% H2O2 in THF at room

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% (IC50) following incubation in culture

medium for 72 h are displayed in Table 3. The IC50 values obtained for CA-4 (1) in this assay are

11.4, 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 IC50s against all tested cells, comparable

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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 d), while their

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

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 f. In particular, N1-methyl selenide 12n exhibited superior

antiproliferative activity against SGC7901 and HT1080 cells than positive control CA-4 (1),

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 k

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

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

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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 IC50 values were found to be 6339.8 and 438.4 nM,

respectively. It was noticeable that IC50 values were 90- to 2640-fold higher than that against

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

more selective antiproliferative activity than 13a.

3.2. Tubulin polymerization

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

the CA-4 (1) analogs, on the inhibitory of tubulin assembly were evaluated in vitro. CA-4

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

shown in Fig. 3, both 13a and CA-4 (1) inhibited tubulin polymerization in a

dose-dependent manner. At 0.5 µΜ, 13a and CA-4 (1) exhibited moderate inhibitory

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

noting that the IC50 of 13a was 1.648 µM, which is close to that of the positive control CA-4

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

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

tubulin by 47.2%, when the concentration rose to 10 µΜ 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

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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 (1) on the microtubule network,

suggesting that 13a is most likely targeting tubulin.

(Figure 5. should be listed here)

3.4．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 (1) was also docked in the colchicine binding site on tubulin. The most

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 (1), which may be extremely

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)

4. Conclusion

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

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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

AVANCE 300 (1H, 300 MHz; 13C, 100 MHz), Bruker AVANCE 400 (1H, 400 MHz; 13C, 100

MHz) or Bruker AVANCE 600 (1H, 600 MHz; 13C, 150 MHz), in CDCl3 or DMSO-d6 (internal

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

5.1.1. 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

95% 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 CH2Cl2 and concerntrated in

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

188-190 °C; 1H NMR (CDCl3 400 MHz) δH 8.39 (1H, br s, NH), 7.66 (2H, d, J 7.6 Hz, 2×ArH),

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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

(0.14 g, 58%); mp 185-187 °C; 1H NMR (CDCl3 400 MHz) δH 8.26 (1H, br s, NH), 7.60 (1H, dd,

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, OCH3). MS (ESI, neg) 240.0 [M-H]-.

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 NaHCO3 solution (40 mL), and

extracted with CH2Cl2 (3×40 mL), The combined organic layers were dried over anhydrous

Na2SO4, and concentrated in vacuo. The crude product was purified by column chromatography

(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%); 1H NMR (CDCl3

400 MHz) δH 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

(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,

NCH3). MS (ESI, pos) 131.2 [M]+.

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; 1H

NMR (CDCl3 400 MHz) δH 7.45 (1H, d, J 1.7 Hz, ArH), 7.12 (1H, d, J 8.6 Hz, ArH), 7.07 (1H,

dd, J 8.6, 1.7 Hz, ArH), 6.12 (1H, br s, ArH), 3.61 (3H, s, NCH3), 2.39 (3H, s, CH3). MS (ESI,

pos) 179.1 [M]+.

5.1.5. 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; 1H

NMR (CDCl3 400 MHz) δH 7.12 (1H, d, J 8.8 Hz, ArH), 7.00 (1H, d, J 2.4 Hz, ArH), 6.80 (1H,

dd, J 8.8, 2.4 Hz, ArH), 5.33 (1H, br s, ArH), 3.83 (3H, s, OCH3), 3.61 (3H, s, NCH3), 2.39 (3H, s,

CH3). MS (ESI, pos) 175.2 [M]+.

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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

using the same manner as for 10l, as a colourless oil (0.94 g, 98% ); 1H NMR (CDCl3 400 MHz)

δH 7.65 (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, NCH2). MS (ESI) 225.2 [M]+.

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 N2 atmosphere. After the initial

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 Na2Se2 was then ready for further use. 3,4,5-trimethoxyaniline (6.00 g, 32.75 mmol)

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

NaNO2 (2.26 g, 32.75 mmol) in water (10 mL) was added via syringe over 15 minutes under

stirring. The solution was stirred for a further 30 minutes at 0 °C and

3,4,5-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

3,4,5-trimethoxybenzenediazonium chloride was added drop wise over 15 minutes. After addition,

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

and the residue washed with CH2Cl2 (30 mL). The mixture was transferred to a separatory funnel,

and the organic layer was separated. The aqueous layer was extracted with CH2Cl2 (3×50 mL). The

combined organic layer was washed with brine (50 mL) and dried over anhydrous Na2SO4, finally

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

2.60 g, as a yellow solid, without further separation. Mp 139-140 °C; 1H NMR (CDCl3 400 MHz)

δH 6.48 (4H, s, 4×ArH), 3.83 (6H, s, 2×OCH3), 3.81 (12H, s, 4×OCH3). 13C NMR (100MHz,

CDCl3): δC 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)

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m/z: 492.9 [M+H] +, 514.8 [M+Na] +; HRMS (ESI):calc. for C18H22NaO6Se2 [M+Na]+ 516.9639,

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 I2 (1 mol%, 0.0001 g/mL in CH3CN) was placed into the microwave cavity (closed

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 CH2Cl2.

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

138-140°C; 1H NMR (300 MHz, CDCl3): δH 8.33 (1H, s, NH), 7.58 (1H, dd, J 7.3, 1.4Hz, ArH),

7.33 (1H, m, ArH), 7.15 (2H, m, 2×ArH), 6.43 (2H, s, 2×ArH), 3.76 (3H, s, OCH3), 3.64 (6H, s,

2×OCH3), 2.58 (3H, s, CH3); 13C NMR (100MHz, CDCl3): δC 153.5 (2×C), 140.7, 136.07, 135.7,

131.1, 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)

375.9 [M-H]-, 411.8 [M+Cl]-; HRMS (ESI): m/z, calcd. For C18H19NNaO3Se [M+Na]+ 400.0422,

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

130-134°C; 1H NMR (400 MHz, CDCl3): δH 8.57 (1H, s, NH), 7.66 (1H, d, J 7.8Hz, ArH), 7.49

(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),

6.51 (2H, s, 2×ArH), 3.76 (3H, s, OCH3), 3.65 (6H, s, 2×OCH3); 13C NMR (100MHz, CDCl3): δC

153.5 (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,

60.8, 56.0 (2×C). HRMS (ESI): m/z, calcd. For C17H17NNaO3Se [M+Na]+ 386.0266, found

386.0275.

5.1.8.3. 2-Phenyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12c).

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Compound 12c was obtained from 10c and 11 as a pale yellow oil (0.13 g, 50%); 1H NMR

(300 MHz, CDCl3): δH 8.66 (1H, s, NH), 7.77 (2H, dd, J 8.2, 1.4 Hz, 2×ArH), 7.71 (1H, d, J 7.7

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

(3H, s, OCH3), 3.61 (6H, s, 2×OCH3); 13C NMR (100MHz, CDCl3): δC 153.5 (2×C), 142.0, 136.2,

136.1, 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),

105.3, 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 C23H21NNaO3Se [M+Na]+ 462.0579, found 462.0581.

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

177-180 °C; 1H NMR (300 MHz, CDCl3): δH 8.71 (1H, s, NH), 7.69 (1H, d, J 7.6 Hz, ArH), 7.54

(1H, dd, JH, F 12.3, 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, OCH3), 3.75 (3H, s,

OCH3), 3.61 (6H, s, 2×OCH3); 13C NMR (100MHz, CDCl3): δC 153.6 (2×C), 152.1 (d, JC, F 244.1

Hz), 148.0 (d, JC, F 10.3 Hz), 140.6 (d, JC, F 1.8Hz), 136.3, 136.0, 132.0, 128.1, 125.3 (d, JC, F

7.3Hz), 124.6 (d, JC, F 3.3Hz), 123.4, 121.2, 120.8, 116.3 (d, JC, F 20.8 Hz), 116.2, 113.4 (d, JC, F

1.8 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 C24H22FNNaO4Se [M+Na]+ 510.0590,

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

122-125 °C; 1H NMR (300 MHz, CDCl3): δH 8.49 (1H, s, NH), 7.22 (2H, m, 2×ArH), 6.89 (1H,

m, ArH), 6.42 (2H, s, 2×ArH), 3.77 (3H, s, OCH3), 3.65 (6H, s, 2×OCH3), 2.55 (3H, s, CH3); 13C

NMR (100MHz, CDCl3): δC 158.6 (d, JC, F 234.5Hz), 153.6 (2×C), 142.7, 136.3, 132.1, 128.0,

123.7, 111.2 (d, JC, F 10.3 Hz), 110.2 (d, JC, F 26.4 Hz), 105.8 (2×C), 104.9 (d, JC, F 24.9 Hz), 96.8

(d, JC, F 4.4 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 C18H18FNNaO3Se [M+Na]+

418.0328, found 418.0326.

5.1.8.6. 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

ºC; 1H NMR (300 MHz, CDCl3): δH 8.41 (1H, s, NH), 7.56 (1H, d, J 1.9 Hz, ArH), 7.24 (1H, d, J

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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, OCH3), 3.67

(6H, s, 2×OCH3), 2.56 (3H, s, CH3); 13C NMR (100MHz, CDCl3): δC 153.6 (2×C), 142.4, 136.3,

134.1, 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 C18H18ClNNaO3Se [M+Na]+ 434.0033,

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

ºC; 1H NMR (300 MHz, CDCl3): δH 8.44 (1H, s, NH), 7.72 (1H, s, ArH), 7.26 (1H, dd, J 8.6, 1.7

Hz, ArH), 7.19 (1H, d, J 8.6 Hz, ArH), 6.41 (2H, s, 2×ArH), 3.77 (3H, s, OCH3), 3.67 (6H, s,

2×OCH3), 2.57 (3H, s, CH3); 13C NMR (100MHz, CDCl3): δC 153.6 (2×C), 142.2, 136.3, 134.4,

133.0, 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)

456.0 [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 C18H18BrNNa O3Se [M+Na]+ 477.9527, found 477.9526.

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

145-148 ºC; 1H NMR (300 MHz, CDCl3): δH 8.42 (1H, s, NH), 7.35 (1H, d, J 5.9 Hz, ArH), 7.31

(1H, d, J 9.4Hz, ArH), 6.39 (2H, s, 2×ArH), 3.77 (3H, s, OCH3), 3.67 (6H, s, 2×OCH3), 2.56 (3H,

s, CH3); 13C NMR (100MHz, CDCl3): δC 153.8 (d, JC, F 238.8 Hz), 153.6 (2×C), 143.2, 136.4,

131.8, 130.6 (d, JC, F 9.3 Hz), 127.6, 115.5 (d, JC, F 21.0 Hz), 111.8, 106.1, 105.9 (2×C), 105.2,

97.0, 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 C18H17ClFNNaO3Se [M+Na]+ 451.9938, found 45.9938.

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

117-120ºC; 1H NMR (300 MHz, CDCl3): δH 8.23 (1H, s, NH), 7.37 (1H, s, ArH), 7.21 (1H, d, J

8.2Hz, ArH), 6.70 (1H, dd, J 8.2, 1.4 Hz, ArH), 6.43 (2H, s, 2×ArH), 3.76 (3H, s, OCH3), 3.65

(6H, s, 2×OCH3), 2.56 (3H, s, CH3), 2.42 (3H, s, CH3); 13C NMR (100MHz, CDCl3): δC 153.5

(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 C19H21NNaO3Se [M+Na]+ 414.0579, found 414.0594.

5.1.8.10. 5-Methoxy-2-methyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole(12j).

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

108-114 ºC; 1H NMR (300 MHz, CDCl3): δH 8.30 (1H, s, NH), 7.21 (1H, d, J 8.7Hz, ArH), 7.04

(1H, d, J 2.3 Hz, ArH), 6.82 (1H, m, ArH), 6.43 (2H, s, 2×ArH), 3.81 (3H, s, OCH3), 3.76 (3H, s,

OCH3), 3.65 (6H, s, 2×OCH3), 2.54 (3H, s, CH3); 13C NMR (100MHz, CDCl3): δC 155.0, 153.6

(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),

55.9, 13.3. MS (ESI, neg) 405.9 [M-H]-; HRMS (ESI): m/z, calcd. For C19H21NNaO4Se [M+Na]+

430.0528, found 430.0544.

5.1.8.11. 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;

1H NMR (400 MHz, CDCl3): δH 8.15 (1H, s, NH), 7.12 (1H, d, J 8.4 Hz, ArH), 6.86 (1H, d, J 1.7

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,

s, 2×OCH3), 2.52 (3H, s, CH3); 13C NMR (100MHz, DMSO-d6): δC 153.7 (2×C), 142.6, 136.8,

136.2, 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 C18H21N2O3Se

[M+H] + 393.0712, found 393.0717.

5.1.8.12. 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%); 1H NMR

(400 MHz, CDCl3): δH 7.66 (1H, d, J 7.9 Hz, ArH), 7.36 (1H, d, J 7.9 Hz, ArH), 7.33 (1H, s,

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),

3.76 (3H, s, OCH3), 3.65 (6H, s, 2×OCH3); 13C NMR (100MHz, CDCl3): δC 153.4 (2×C), 137.3,

136.3, 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 C18H19NNaO3Se [M+Na]+ 400.0422, found 400.0440.

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

ºC; 1H NMR (400 MHz, CDCl3): δH 7.58 (1H, d, J 1.3 Hz, ArH), 7.20 (1H, d, J 8.6 Hz, ArH), 7.14

(1H, dd, J 8.6, 1.3 Hz, ArH), 6.41 (2H, s, 2×ArH), 3.76 (3H, s, NCH3), 3.74 (3H, s, OCH3), 3.67

(6H, s, 2×OCH3), 2.57 (3H, s, CH3); 13C NMR (100MHz, CDCl3): δC 153.7 (2×C), 143.8, 136.4,

135.7, 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 C19H20ClNNaO3Se [M+Na]+ 448.0189, found 448.0175.

5.1.8.14. 5-Methoxy-1,2-dimethyl-3-((3,4,5-trimethoxyphenyl)selenyl)-1H-indole (12n).

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

127-130 ºC 1H NMR (400 MHz, DMSO-d6): δH 7.41 (1H, d, J 8.6 Hz, ArH), 6.90 (1H, d, J 2.4

Hz, ArH), 6.80 (1H, dd, J 8.6, 2.4 Hz, ArH), 6.45 (2H, s, 2×ArH), 3.76 (3H, s, OCH3), 3.73 (3H,

s, OCH3), 3.58 (6H, s, 2×OCH3), 3.57 (3H, s, NCH3), 2.53 (3H, s, CH3); 13C NMR (100MHz,

DMSO-d6): δC 154.8, 153.7 (2×C), 143.9, 136.3, 132.5, 131.0, 128.5, 111.4, 111.2, 106.2 (2×C),

101.1, 94.2, 60.4, 56.5, 56.3 (2×C), 55.8, 12.2. HRMS (ESI): m/z, calcd. For C20H24NO4Se

[M+H] + 422.0865, found 422.0879.

5.1.8.15. 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%); 1H NMR

(600 MHz, CDCl3): δH 7.63 (1H, d, J 7.8 Hz, ArH), 7.40 (1H, s, ArH), 7.32 (1H, d, J 8.2 Hz,

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

8.6 Hz, 2×ArH), 6.49 (2H, s, 2×ArH), 5.34 (2H, s, NCH2), 3.76 (3H, s, OCH3), 3.63 (6H, s,

2×OCH3); 13C NMR (100MHz, CDCl3): δC 162.3 (d, JC, F 246.1 Hz), 153.5 (2×C), 136.8, 136.4,

134.6, 132.6 (d, JC, F 3.2 Hz), 130.8, 128.6, 128.5, 128.1, 122.9, 120.7 (d, JC, F 14.4 Hz), 115.9,

115.7, 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 C24H22FNNaO3Se [M+Na]+ 494.0641, 494.0635.

5.1.8.16. (3-(3,4,5-Trimethoxyphenylselenyl)-1H-indol-2-yl)methanol (12p).

Similar to the reported method,21 to a stirred solution of 12a (0.15 g, 0.4 mmol) in

1,4-dioxane (15 mL) was added SeO2 (0.22 g, 1.99 mmol) and the reaction mixture was refluxed

under a N2 atmosphere for 6 hours. The reaction mixture was filtered and concentrated under

reduced pressure. NaBH4 (0.03 g, 0.80 mmol) was added to the residue in EtOH (5 mL), and the

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 CH2Cl2 (3×20 mL). The combined

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

afford 12p as a brown oil (0.064 g, 41%), 1H NMR (300 MHz, CDCl3): δH 8.99 (1H, s, NH),

7.64 (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),

6.44 (2H, s, 2×ArH), 4.99 (2H, s, CH2), 3.75 (3H, s, OCH3), 3.63 (6H, s, 2×OCH3); 13C NMR

(100MHz, CDCl3): δC 153.5 (2×C), 142.3, 136.3, 136.0, 130.7, 128.1, 123.0, 120.8, 120.2, 111.4,

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106.0 (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 C18H19NNaO4Se [M+Na]+ 416.0372, found 416.0373.

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

a light yellow oil (0.06 g, 33%); 1H NMR (400 MHz, CDCl3): δH 9.09 (1H, s, NH), 7.62 (1H, s,

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

(2H, s, CH2), 3.76 (3H, s, OCH3), 3.65 (6H, s, 2×OCH3); 13C NMR (100MHz, CDCl3): δC 153.6

(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,

56.1 (2×C). HRMS (ESI): m/z, calcd. For C18H18ClNNaO4Se [M+Na]+ 449.9982, found 449.9977.

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

Similar to a reported method,22 a mixture of 3-(trimethoxyphenylselenyl)-1H-indoles (0.6

mmol) and 30% H2O2 (10 d) in THF (10 mL) was stirred at room temperature for 5-12 hours until

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 CH2Cl2/CH3OH

(v/v= 10:1).

5.1.9.1. 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; 1H

NMR (400 MHz, DMSO-d6): δH 11.73 (1H, s, NH), 7.33 (2H, d, J 8.9 Hz, 2×ArH), 7.11 (2H, s,

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×OCH3), 3.66 (3H,

s, OCH3), 2.63 (3H, s, CH3);13C NMR (100MHz, DMSO-d6): δC 153.9 (2×C), 141.0, 139.3, 136.8,

136.1, 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)

394.1 [M+H]+, 416.1 [M+Na]+, 432.0 [M+K]+; HRMS (ESI): m/z, calcd. For C18H20NO4Se

[M+H] + 394.0552, found 394.0550.

5.1.9.2. 3-(3,4,5-Trimethoxyphenylseleninyl)-1H-indole (13b).

Compound 13b was obtained from 10b as a brown oil (0.20 g, 88%); 1H NMR (300 MHz,

DMSO-d6): δH 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

(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,

ArH), 3.77 (6H, s, 2×OCH3), 3.67 (3H, s, OCH3); 13C NMR (100MHz, CDCl3): δC 153.4(2×C),

136.5, 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

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(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 C17H18NO4Se [M+H]+ 380.0396, found 380.381.

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; 1H

NMR (300 MHz, CDCl3): δH 11.37 (1H, s, NH), 7.34 (4H, m, ArH), 7.22 (1H, d, J 7.1 Hz, ArH),

7.07 (3H, m, ArH), 6.96 (1H, d, J 7.5 Hz, ArH), 6.93 (2H, s, 2×ArH), 3.84 (3H, s, OCH3), 3.73

(6H, s, 2×OCH3); 13C NMR (100MHz, DMSO-d6): δC 153.9 (2×C), 143.2, 139.4, 136.9, 136.4,

130.9, 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,

60.6, 56.6 (2×C). MS (ESI) 456.0 [M+H]+; HRMS (ESI): m/z, calcd. For C23H22NO4Se [M+H]+

456.0709, found 456.0710.

5.1.9.4. 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; 1H

NMR (300 MHz, CDCl3): δH 12.16 (1H, s, NH), 7.38 (1H, d, J 8.1 Hz, ArH), 7.22 (2H, d, J 7.1

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),

6.60 (1H, d, J 7.8 Hz, ArH), 5.99 (1H, t, J 8.1, 7.8 Hz, ArH), 3.84 (3H, s, OCH3), 3.76 (6H, s,

2×OCH3), 3.71 (3H, s, OCH3); 13C NMR (100MHz, DMSO-d6): δC 153.8 (2×C), 152.09 (d, JC,F

245.2) 148.5 (d, JC,F 10.5 Hz), 141.9, 139.4, 136.5, 136.1, 127.5, 126.5 (d, JC,F 3.0 Hz), 123.6 (d,

JC,F 7.5 Hz), 123.2, 121.1, 119.7, 117.3 (d, JC,F 19.8 Hz), 114.6, 112.5, 110.7, 104.1 (2×C), 60.6,

56.7, 56.6 (2×C). MS (ESI, neg) 502.0 [M-H]-; HRMS (ESI): m/z, calcd. For C24H23FNO5Se

[M+H] + 504.0720, found 504.0740.

5.1.9.5. 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; 1H

NMR (300 MHz, CDCl3): δH 11.55 (1H, s, NH), 7.07 (1H, m, ArH), 6.96 (2H, s, 2×ArH), 6.74

(2H, m, ArH), 3.89 (3H, s, OCH3), 3.81 (6H, s, 2×OCH3), 2.08 (3H, s, CH3); 13C NMR (100MHz,

CDCl3): δC 158.2 (d, JC, F 237.1 Hz), 154.2 (2×C), 153.7 (d, JC, F 38.8 Hz), 144.0, 140.2, 132.8,

132.6, 126.4 (d, JC, F 11.8 Hz), 112.7 (d, JC, F 9.3 Hz), 110.6 (d, JC, F 26.1 Hz), 103.7 (2×C), 102.7,

61.1, 56.5 (2×C), 12.1. MS (ESI, neg) 409.9 [M-H]-; HRMS (ESI): m/z, calcd. For C18H19FNO4Se

[M+H] + 412.0458, found 412.0448.

5.1.9.6. 5-Chloro-2-methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13f).

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Compound 13f was obtained from 10f as a off-white solid (0.13 g, 50%); mp 113-115 ºC; 1H

NMR (300 MHz, CDCl3): δH 11.20 (1H, s, NH), 7.14 (1H, d, J 1.7 Hz, ArH), 7.10 (1H, d, J 8.7

Hz, ArH), 6.98 (1H, dd, J 8.7 Hz, 1.7 Hz, ArH), 6.97 (2H, s, 2×ArH), 3.89 (3H, s, OCH3), 3.83

(6H, s, 2×OCH3), 2.12 (3H, s, CH3); 13C NMR (100MHz, CDCl3): δC 154.3 (2×C), 143.6, 140.4,

134.6, 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 C18H19ClNO4Se [M+H]+ 428.0162, found 428.0160.

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; 1H

NMR (300 MHz, CDCl3): δH 10.81 (1H, s, NH), 7.33 (1H, s, ArH), 7.11 (2H, m, ArH), 6.98 (2H,

s, 2×ArH), 3.89 (3H, s, OCH3), 3.84 (6H, s, 2×OCH3), 2.17 (3H, s, CH3); 13C NMR (100MHz,

CDCl3): δC 153.6 (2×C), 142.2, 136.4, 134.4, 133.0, 125.0, 124.9, 122.3, 120.1, 114.1, 112.1,

112.0, 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 C18H19BrNO4Se [M+H]+ 471.9657, found

471.9659.

5.1.9.8. 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; 1H

NMR (300 MHz, CDCl3): δH 11.63 (1H, s, NH), 7.15 (1H, d, J 5.7 Hz, ArH), 6.95 (2H, s, 2×ArH),

6.81 (1H, s, J 9.7 Hz, ArH), 3.89 (3H, s, OCH3), 3.84 (6H, s, 2×OCH3), 2.18 (3H, s, CH3); 13C

NMR (100MHz, CDCl3): δC 153.7 (d, JC, F 239.6 Hz), 154.3 (2×C), 144.3, 140.4, 132.4 (d, JC, F

22.3 Hz), 124.8 (d, JC, F 10.4 Hz), 116.3 (d, JC, F 20.8 Hz), 113.1, 111.9 (d, JC, F = 11.9 Hz), 105.9,

104.8 (d, JC, F 25.3 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

C18H18ClFNO4Se [M+H]+ 446.0068, found 446.0088.

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%); 1H NMR (600 MHz,

CDCl3): δH 11.03 (1H, s, NH), 7.10 (1H, d, J 8.3 Hz, ArH), 7.02 (1H, s, ArH), 6.99 (2H, s,

2×ArH), 6.85 (1H, d, J 8.3 Hz, ArH), 3.87 (3H, s, OCH3), 3.80 (6H, s, 2×OCH3), 2.25 (3H, s,

CH3), 2.05 (3H, s, CH3); 13C NMR (100MHz, DMSO-d6): δC 153.9 (2×C), 140.8, 139.3, 136.7,

134.3, 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

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(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 C19H22NO4Se [M+H]+ 408.0709, found 408.0721.

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%); 1H NMR (300 MHz,

CDCl3): δH 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,

ArH), 7.06 (2H, s, ArH), 3.87 (3H, s, OCH3), 3.86 (6H, s, 2OCH3), 3.82 (3H, s, NCH3); 13C NMR

(100MHz, DMSO-d6): δC 153.6 (2×C), 139.1, 137.6, 136.7, 132.9, 125.7, 122.7, 120.8, 119.4,

112.9, 110.0, 103.4 (2×C), 60.3, 56.3 (2×C), 33.1. MS (ESI) 394.0 [M+H]+, 416.0 [M+Na]+,

432.0 [M+K]+; HRMS (ESI): m/z, calcd. For C18H20NO4Se [M+H]+ 394.0552, found 394.0563.

5.1.9.11. 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 ; 1H

NMR (400 MHz, CDCl3): δH 7.40 (1H, d, J 1.8 Hz, ArH), 7.19 (1H, d, J 8.7 Hz, ArH), 7.13 (1H,

dd, J 8.7, 1.8 Hz, ArH), 7.01 (2H, s, 2×ArH), 3.87 (3H, s, OCH3), 3.86 (6H, s, 2×OCH3), 3.46

(3H, s, NCH3), 2.63 (3H, s, CH3); 13C NMR (100MHz, CDCl3): δC 154.1 (2×C), 142.7, 140.1,

135.6, 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 C19H21ClNO4Se [M+H]+ 442.0319, found 442.0343.

5.1.9.12. 1-(4'-Fluorobenzyl)-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13l).

Compound 13l was obtained from 10o as a white solid (0.19 g, 64%); mp 147-149 ºC ; 1H

NMR (400 MHz, CDCl3): δH 7.61 (1H, d, J 8.0 Hz, ArH), 7.42 (1H, s, ArH), 7.30 (1H, d, J 8.2

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, NCH2), 3.87 (3H, s, OCH3), 3.85 (6H, s, 2×OCH3);

13C NMR (100MHz, CDCl3): δC 162.5 (d, JC, F 247.0 Hz), 154.0 (2×C), 140.1, 137.1, 135.3, 131.6

(d, JC, F 3.4 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 C24H23FNO4Se [M+H]+ 488.0771, found 488.0772.

5.2 Biochemistry methods

5.2.1 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,

cells were seeded into 96-well plates at a density of 1-3×104/well (depends on the cell growth rate),

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

CO2, the drug containing medium was removed and replaced by 100 µL fresh medium with 5

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

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

purple formazan crystals were dissolved and the OD490 was determined using a microplate reader

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

control. The 50% inhibitory concentration (IC50) was defined as the concentration that reduced the

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 MgCl2, 0.5 mM EGTA, 1 mM GTP, 20% (v/v) glycerol) and added

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 calculation while IC50 values were calculated by

using GraphPad Prism software.

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

10 µΜ using the methods similar to 13a.

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

at 1×104 per well on a 24 well plate and grown for 24 h. Cells were treated with vehicle, 30 nM of

13a 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

0.1 % (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

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(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

100 µm).

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

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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. NaBH4, EtOH, r.t.

Scheme 2. The synthesis of compounds 13a-l. Reagents and conditions: a) H2O2, THF, rt.

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

Entry Heating Mode Temp

(ºC) Time

Power

(W)

Yield

(%)a

1 oil bath - 80 24 h - 27

2 oil bath - 80 36 h - 35

3 oil bath - 80 48 h - 47

4 MWb OVc 80 10 min 150 38

5 MW OV 80 30 min 150 69

6 MW CVd 80 30 min 150 72

7 MW CV 100 10 min 200 42

8 MW CV 100 15 min 200 61

9 MW CV 100 30 min 200 71

10 MW CV 100 60 min 200 73 a isolated yield. b MW: microwave. c OV: open vessel. d CV: closed vessel.

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

Entry Indole R1 R2 R3 Product Yielda (%)

1 10b H H H 12b 60

2 10c H phenyl H 12c 50

3 10d H 3'-fluoro-4'-mehoxyphenyl H 12d 45

4 10e H Me 5-F 12e 63

5 10f H Me 5-Cl 12f 79

6 10g H Me 5-Br 12g 41

7 10h H Me 5-F, 6-Cl 12h 63

8 10i H Me 5-Me 12i 80

9 10j H Me 5-OMe 12j 84

10 10k H Me 5-NH2 12k 29

11 10l Me H H 12l 60

12 10m Me Me 5-Cl 12m 71

13 10n Me Me 5-OMe 12n 48

14 10o 4'-fluorobenzyl H H 12o 62 a isolated yield.

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

three human cancer cell lines.

Compouns IC50

a (nM± SD)

SGC7901 KB HT1080

12a 220.8± 10.1 215.2± 12.1 531.5± 25.0

12b 135.3± 5.2 173.9± 41.2 599.0± 46.5

12c 1148.3± 50.0 517.7± 20.0 1516.7± 320.5

12d 1457.8± 78.6 1162.6± 102.1 545.1± 70.8

12e 185.9± 20.3 172.7± 60.2 501.6± 45.1

12f 266.4± 30.2 >10000 >10000

12g 223.2± 10.5 >10000 2912.8± 310.0

12h 1966.1± 105.4 693.9± 70.0 104.4± 15.6

12i 143.1± 26.2 238.0± 20.2 110.2± 10.0

12j 48.4± 3.5 53.8± 3.2 37.8± 3.3

12k 40.9± 2.7 15.3± 2.1 84.3± 2.2

12l 42.5± 3.3 15.9± 1.5 162.1± 10.0

12m 14.1± 2.1 68.3± 4.4 98.9± 5.0

12n 9.5± 0.8 69.0± 2.0 2.4± 0.5

12o >10000 >10000 >10000

12p 86.9± 6.5 36.8± 1.0 188.6± 40.3

12q 53.9± 3.6 18.7± 2.2 21.0± 1.3

13a 12.3± 1.6 13.5± 1.5 25.1± 2.0

13b 51.5± 6.2 39.5± 5.3 112.1± 12.8

13c 1178.0± 55.1 765.7± 51.4 1379.3± 240.3

13d 3371.7± 120.3 5586.4± 130.0 3731.3± 150.2

13e 143.6± 47.3 438.7± 20.1 273.0± 61.3

13f 30.4± 4.2 63.7± 4.4 33.2± 1.7

13g 2083.0± 156.2 890.2± 86.6 92.1± 4.6

13h 34.0± 2.3 117.3± 12.2 78.4± 6.9

13i 40.2± 1.8 79.1± 2.4 48.0± 2.3

13j 2159.9± 220.3 991.3± 70.0 565.6± 50.0

13k 88.5± 5.5 11.3± 1.1 158.8± 20.0

13l >10000 >10000 >10000

CA-4 (1) 11.4± 1.2 4.1± 0.7 10.9± 1.8 aIC50, expressed as the concentration of drug inhibiting cell growth by 50%. Data are expressed as

means± SDs (standard deviations) from at least three independent experiments.

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Highlights

1. 3-(3,4,5-Trimethoxyphenylselenyl)-1H-indoles were designed as novel analogs of CA-4.

2. A microwave-assisted progress for the synthesis of 3-arylselenylindoles was developed.

3. Most of the analogs showed potent antitumor activity; some showed nanomolar IC50s.

4. Compound 13a inhibited tubulin polymerization and disrupted microtubule dynamics.

5. Compound 13a exhibited a binding mode similar to that of CA-4.

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Supporting Information

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

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

evaluation

Zhiyong Wena, Jingwen Xub, Zhiwei Wanga, Huan Qib, Qile Xua, Zhaoshi Baib, Qian Zhanga,

Kai Baoa,c, Yingling Wub,*, Weige Zhanga,*

1H NMR and 13C NMR spectra of the starting materials

2-phenyl-1H-indole (10c)

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

1-methyl-1H-indole (10l)

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5-chloro-1,2-dimethyl-1H-indole (10m)

5-methoxy-1,2-dimethyl-1H-indole (10n)

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1-(4'-fluorobenzyl)-1H-indole (10o)

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

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1H NMR and 13C NMR spectra of the products

2-methyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12a)

NH

Se

MeO

MeOOMe

Me

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3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12b)

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2-phenyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12c)

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2-(3'-fluoro-4'-methoxyphenyl)-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12d)

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5-fluoro-2-methyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12e)

NH

Se

MeO

MeOOMe

Me

F

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5-chloro-2-methyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12f)

NH

Se

MeO

MeOOMe

Me

Cl

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5-bromo-2-methyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12g)

NH

Se

MeO

MeOOMe

Me

Br

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6-chloro-5-fluoro-2-methyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12h)

NH

Se

MeO

MeOOMe

Me

F

Cl

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2,5-dimethyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12i)

NH

Se

MeO

MeOOMe

Me

Me

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5-methoxy-2-methyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12j)

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2-methyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indol-5-amine (12k)

NH

Se

MeO

MeOOMe

H2N

Me

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1-methyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12l)

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5-chloro-1,2-dimethyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12m)

N

Se

MeO

MeOOMe

Me

Cl

Me

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5-methoxy-1,2-dimethyl-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12n)

N

Se

MeO

MeOOMe

MeO

Me

Me

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1-(4'-fluorobenzyl)-3-(3,4,5-trimethoxyphenylselenyl)-1H-indole (12o)

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(3-(3,4,5-trimethoxyphenylselenyl)-1H-indol-2-yl)methanol (12p)

NH

Se

MeO

MeOOMe

CH2OH

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

NH

Se

MeO

MeOOMe

CH2OH

Cl

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2-methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13a)

NH

Se

MeO

MeOOMe

Me

O

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3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13b)

NH

Se

MeO

MeOOMe

O

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2-phenyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13c)

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2-(3'-fluoro-4'-methoxyphenyl)-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13d)

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5-fluoro-2-methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13e)

NH

Se

MeO

MeOOMe

Me

F

O

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5-chloro-2-methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13f)

NH

Se

MeO

MeOOMe

Me

Cl

O

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5-bromo-2-methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13g)

NH

Se

MeO

MeOOMe

Me

Br

O

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

NH

Se

MeO

MeOOMe

Me

F

Cl

O

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2,5-dimethyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13i)

NH

Se

MeO

MeOOMe

Me

Me

O

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1-methyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13j)

N

Se

MeO

MeOOMe

Me

O

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5-chloro-1,2-dimethyl-3-(3,4,5-trimethoxyphenylseleninyl)-1H-indole (13k)

N

Se

MeO

MeOOMe

Cl

Me

Me

O

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