Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors with potent antiproliferative activity

RESEARCH ARTICLE

Synthesis and bioevaluation of N,4-diaryl-1,3-

thiazole-2-amines as tubulin inhibitors with

potent antiproliferative activity

Maolin Sun^1☯, Qile Xu^1☯, Jingwen Xu², Yue Wu¹, Yueting Wang¹, Daiying Zuo², Qi Guan¹,

Kai Bao^1,3, Jian Wang¹*, Yingliang Wu², Weige Zhang¹*

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

Pharmaceutical University, Shenyang, China, 2 Department of Pharmacology, Shenyang Pharmaceutical

University, Shenyang, China, 3 Gordon Center for Medical Imaging, Division of Nuclear Medicine and

Molecular Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School,

Boston, Massachusetts, United States of America

a 1111111111

☯ These authors contributed equally to this work.

* zhangweige2000@sina.com (WZ); jianwang@syphu.edu.cn (JW)

Abstract

OPEN ACCESS

A series of N,4-diaryl-1,3-thiazole-2-amines containing three aromatic rings with an amino

linker were designed and synthesized as tubulin inhibitors and evaluated for their antiproli-

ferative activity in three human cancer cell lines. Most of the target compounds displayed

moderate antiproliferative activity, and N-(2,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-1,3-

thiazol-2-amine (10s) was determined to be the most potent compound. Tubulin polymeriza-

tion and immunostaining experiments revealed that 10s potently inhibited tubulin polymeri-

zation and disrupted tubulin microtubule dynamics in a manner similar to CA-4. Moreover,

10s effectively induced SGC-7901 cell cycle arrest at the G₂/M phase in both concentration-

and time-dependent manners. The molecular docking results revealed that 10s could bind

to the colchicine binding site of tubulin.

Citation: Sun M, Xu Q, Xu J, Wu Y, Wang Y, Zuo D,

et al. (2017) Synthesis and bioevaluation of N,4-

diaryl-1,3-thiazole-2-amines as tubulin inhibitors

with potent antiproliferative activity. PLoS ONE 12

(3): e0174006. https://doi.org/10.1371/journal.

pone.0174006

Editor: Sandra B. Gabelli, Johns Hopkins University

School of Medicine, UNITED STATES

Received: July 23, 2016

Accepted: March 1, 2017

Published: March 23, 2017

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Introduction

Microtubules are key structural components in eukaryotic cells and play a crucial role in a

number of cellular functions, including regulation of motility, cell division, organelle trans-

port, maintenance of cell morphology and signal transduction [1,2]. Tubulin-binding drugs

disrupt microtubule/tubulin dynamics by binding to distinct sites such as taxol, vinca alkaloid

and colchicine binding sites and arrest cells during mitosis, leading to cell death [3–5]. In

contrast to macromolecular paclitaxel and vinblastine, most novel colchicine binding site

inhibitors (CBSIs) have high potency, relatively simple and diverse chemical structures for

optimization, and selective toxicity toward tumor vasculature [6]. Given the success of pacli-

taxel and vinblastine, research efforts have been focused on developing CBSIs for cancer treat-

ment, and a number of effective CBSIs are currently being investigated in clinical studies [7].

Some representative CBSIs include the natural products colchicine (1), combretastatin A-4

(CA-4, 2) and nocodazole (3), shown in Fig 1A [8–12]. Phenstatin (4) is a CA-4 analog in

which the double bond of CA-4 is replaced with a carbonyl linker [13]. The common structural

Data Availability Statement: All relevant data are

within the paper and its Supporting Information

file.

Funding: This work was supported by the the

National Natural Science Foundation of China

(81502932), State Key Laboratory of Natural

Medicines and Active Substance (No.

GTZK201603), the Programme for Innovative

Research Team of the Ministry of Education and

the Programme for Liaoning Innovative Research

Team in University. The funders had no role in

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

study design, data collection and analysis, decision

to publish, or preparation of the manuscript.

Competing interests: The authors have declared

that no competing interests exist.

Fig 1. (A) Structures of known tubulin inhibitors. (B) Superposition of the skeletal structure of target

compounds 10a-m and SMART. All density functional theory calculations and geometry optimizations were

performed using the Gaussian 09 software.

https://doi.org/10.1371/journal.pone.0174006.g001

features of CBSIs include the 3,4,5-Trimethoxyphenyl unit and the appropriate distance and

certain dihedral angle between the aromatic rings. These were proposed as important struc-

tural features that are responsible for their antiproliferative and antitubulin activities [6].

The CBSIs 4-substituted methoxybenzoyl-aryl-thiazole (SMART, 5), 2-aryl-4-benzoyl-

imidazole (ABI, 6), 4-aryl-2-benzoyl-imidazoles (RABI, 7) and 3-aroyl-1-arylpyrrole (ARAP,

8) are the representative synthetic CBSIs that exhibit potent antiproliferative activity (Fig 1A)

[14–17]. Furthermore, a set of oxadiazole CBSIs with an oxime linker between the A-ring and

the B-ring was previously reported by our group (9, Fig 1A) [18]. The structures of these

CBSIs feature three aromatic rings (A-, B- and C- rings) and an sp²hybridized linker between

the A-ring and B-ring (Fig 1A).

2-Aminothiazoles and derivatives are seen in many bioactive scaffolds and exhibit a variety

of biological properties, including anticancer activity [19]. Furthermore, CBSIs containing an

sp³amino linker have also been reported [20]. In our continued research into novel CBSIs [18,

21, 22], we hypothesized that the sp²linker between the A- and B-rings could be replaced by a

sp³amino linker and that the substitutions on the A- and C-rings could be different groups.

As expected from calculations using the Gaussian 09 software, the skeletal structure of target

compounds superimposed well with that of SMART (Fig 1B). In this study, we report the veri-

fication of our hypothesis through the synthesis and biological evaluation of the designed tar-

get CBSIs (Table 1). In addition, the preliminary structure-activity relationships and the

mechanism of the representative compound 10s are investigated and determined.

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

Table 1. Molecular structures of compounds 10a-v.

Structures

10a

10b

10c

10d

10e

10f

R₁

R₂

R₃

R₄

R₅

H

R₆

H

R₇

R₈

H

R₉

H

OCH₃

H

OCH₃

H

OCH₃

H

CH₃

H

OCH₃

F

H

Cl

H

Br

H

NO₂

H

10g

10h

10i

H

OCH₃

F

OCH₃

F

H

NO₂

NH₂

OBn

OH

F

H

10j

H

10k

10l

H

10m

10n

10o

10p

10q

10r

H

OCH₃

CH₃

H

OCH₃

CH₃

H

OCH₃

H

OCH₃

NO₂

NH₂

H

CH₃

H

CH₃

H

OCH₃

H

10s

10t

H

OCH₃

H

OCH₃

H

10u

10v

H

CH₃

Ac

H

https://doi.org/10.1371/journal.pone.0174006.t001

Results and discussion

Chemistry

The target compounds contained a 2-aminothiazole (2-AT) scaffold that has been widely used

by medicinal chemists in drug discovery research. Few drugs containing a 2-AT core (e.g., mir-

abegron, riluzole, cefotaxime and meloxicam) have been launched in the market [23]. In our

designed compounds, the amino group played a vital role in maintaining the spatial configura-

tion and biological activity of target compounds. Target compounds were prepared as outlined

in Fig 2. In brief, the condensation of commercially aniline derivatives 11 with carbon disulfide

in the presence of triethylamine gave the corresponding dithiocarbamatets 12 at room temper-

ature at an 80–96% yield [24]. Subsequently, dithiocarbamates 12 were used for the synthesis

of aryl thioamides 14 using an efficient one-pot method [25]. More speculatively, dithiocarba-

mates 12 in ethyl acetate were incubated with triethylamine at 0˚C. Iodine was then added

pinch-wise over a period of 15 minutes to yield phenylisothiocyanates 13, which were then

reacted with ammonium hydroxide to obtain the key intermediates aryl thioureas 14 at room

temperature. Alternatively, the commercially available starting acetophenones 15 were sub-

jected to α-bromination with copper dibromide in refluxing chloroform-ethyl acetate to pro-

duce α-bromoacetophenones 16 at 75–95% yield. Finally, α-bromoacetophenones 16 were

reacted with aryl thioureas 14 to generate the desired compounds 10a-t in ethanol within 5

min under microwave irradiation (150 W, 80˚C) in the absence of catalysts [26]. The com-

pound 10u was obtained by a simple reaction of the compound 10s with methyl iodide in

anhydrous DMF, and the compound 10v was obtained by an acetylation reaction of com-

pound 10s with acetic anhydride.

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

Fig 2. Reagents and conditions. (i) CS₂, Et₃N, Et₂O, 25˚C; (ii) I₂, Et₃N, EtOAc, 0˚C; (iii) NH₃ÁH₂O; (iv)

CuBr₂, CHCl₃, EtOAc; (v) EtOH, MW 80˚C; (vi) 1) CH₃I, DMF, 25˚C or 2) Ac₂O, 50˚C.

https://doi.org/10.1371/journal.pone.0174006.g002

Biological studies

In vitro antiproliferative activity. Gastric cancer and lung cancers are the two most com-

mon cancers and the two most frequent causes of cancer-related deaths [27]. HT-1080 is a

fibrosarcoma cell line which has been used extensively in biomedical research [28]. So we

chose these three human cancer cell lines (gastric adenocarcinoma SGC-7901 cells, lung ade-

nocarcinoma A549 cells and fibrosarcoma HT-1080 cells) for antiproliferative activity. To

evaluate the ability of various 2-aminothiazole derivatives to inhibit cancer cell growth, the

target compounds 10a-v, reference compounds CA-4 (2), nocodazole (3) and SMART (5)

were screened for antiproliferative activity in three human cancer cell lines using a standard

MTT assay (Table 2). The reported IC₅₀values are the average of at least three independent

experiments. As illustrated in Table 2, most of the target compounds showed moderate anti-

proliferative activities with potencies in the μM range. Comparison of the IC₅₀values of the

corresponding compounds 10a-m with 3,4,5-trimethoxy (R₂, R₃, R₄= OMe) substitution

revealed that only the 3-hydroxy-4-methoxy-substituted compound 10l significantly inhibited

growth of the HT-1080 cell line at sub-micromolar concentrations. The target compounds

contain halide substituents that has been widely used by medicinal chemists in CBSI research,

so we synthesis these compounds to evaluate the potential antiproliferative activities. Unex-

pectedly, we found that these compounds (10c, 10d, 10e, 10h, 10m) showed only moderate

antiproliferative activities. Interestingly, the most significant enhancement of antiproliferative

activity was observed in the amino-linked compounds where the A ring accommodates the

2,4-dimethoxy substitutions. N-(2,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-1,3-thiazol-

2-amine 10s exhibited the most potent antiproliferative activity, with IC₅₀values between 0.36

and 0.86 μM in the three cancer cell lines. However, the introduction of a methyl (10u) or ace-

tyl (10v) group at the N position of the 2-aminothiazole skeleton resulted in reduced activity

compared to the corresponding compound (10s).

Tubulin polymerization. CBSIs exert their biological activity by inhibiting tubulin assem-

bly. To confirm that the antiproliferative activities of these compounds were related to the

microtubule system, the most active compound 10s was evaluated for tubulin polymerization

inhibition compared to the positive control CA-4 and the negative control paclitaxel. As

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

Table 2. Antiproliferative activity of compounds 10a-v, CA-4 (2), Nocodazole (3) and SMART (5).

Antiproliferative activity (IC₅₀± SD, μM) ^a

A549

Compounds

SGC-7901

1.12 ± 0.13

7.72 ± 0.26

27.7 ± 4.9

8.22 ± 0.17

2.30 ± 0.32

>100

HT-1080

1.17 ± 0.19

26.8 ± 1.2

10a

10b

1.32 ± 0.12

26.8 ± 2.2

10c

16.1 ± 1.1

7.40 ± 0.42

1.26 ± 0.25

6.61 ± 0.18

>100

10d

1.80 ± 0.19

3.18 ± 0.16

23.6 ± 2.3

10e

10f

10g

4.89 ± 0.15

4.92 ± 0.12

9.11 ± 0.62

1.17 ± 0.17

13.0 ± 2.1

1.36 ± 0.09

10.1 ± 1.1

0.82 ± 0.06

19.5 ± 2.4

9.89 ± 0.18

14.8 ± 1.5

1.6 ± 2.2

5.36 ± 0.16

6.17 ± 0.32

9.25 ± 0.33

6.54 ± 0.16

13.1 ± 1.1

6.94 ± 0.14

5.48 ± 0.21

6.24 ± 0.24

4.69 ± 0.62

5.01 ± 0.18

0.77 ± 0.11

8.46 ± 0.17

3.36 ± 0.11

12.7 ± 1.9

10h

10i

10j

10k

10l

3.21 ± 0.17

26.4 ± 1.3

10m

10n

9.62 ± 0.17

17.7 ± 1.6

10o

10p

12.8 ± 1.3

25.8 ± 3.5

10q

10.9 ± 3.2

4.89 ± 0.25

1.95 ± 0.13

0.58 ± 0.13

2.46 ± 0.17

9.39 ± 0.21

56.6 ± 1.0

10r

1.82 ± 0.12

0.86± 0.07

10s

0.36 ± 0.19

10.1 ± 1.0

28.2 ± 1.9

23.2 ± 0.11

0.025 ± 0.012

0.24 ± 0.07

0.045 ± 0.013

10t

11.4 ± 1.3

10u

19.0 ± 2.0

10v

35.7 ± 0.08

0.019 ± 0.011

0.15 ± 0.09

0.059 ± 0.012

CA-4 (2)^b

Nocodazole (3)^b

SMART (5)^b

0.032 ± 0.006

0.12 ± 0.08

0.032 ± 0.009

^aIC₅₀: Concentration of the compound (μM) that resulted in 50% cell growth inhibition after 72 h of drug exposure, as determined by the MTT assay. Each

experiment was carried out in triplicate.

^bUsed as a positive control.

https://doi.org/10.1371/journal.pone.0174006.t002

shown in Fig 3, both CA-4 and 10s inhibited tubulin assembly, and compound 10s inhibited

tubulin polymerization in a dose-dependent manner. However, compound 10s displayed less

active antitubulin activity than did CA-4 (IC₅₀= 26.8 μM and 0.64 μM, respectively). In con-

trast, paclitaxel enhanced the rate of tubulin polymerization compared with untreated cells.

These results suggested that compound 10s interfered with microtubule polymerization.

Analysis of immunofluorescence staining. Colchicine binding site inhibitors could

inhibit tubulin assembly and suppress microtubule formation. With the goal of ascertaining

the mechanism of action of compound 10s, we decided to use a 0.72 μM drug concentration

for the cell microtubule organization studies. As shown in Fig 4, the fibrous microtubules

(green) and the cell nucleus (blue) were visualized upon the immunofluorescence staining of

SGC-7901 cells. These results implied that 10s and CA-4 induced significant defects in tubulin

assembly in the treated cells, leading to the formation of abnormal mitotic spindles in compar-

ison with the control. These results further confirmed that tubulin was the molecular target for

compound 10s.

Cell cycle study. Because tubulin inhibitors can arrest dividing cells in the G2/M phase of

the cell cycle, the effect of 10s on the cell cycle progression of SGC-7901 cells was investigated

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

Fig 3. Effects of 10s on tubulin polymerization. Tubulin was pre-incubated for 1 min with 10s at 2 μM,

10 μM, 25 μM and 50 μM, CA-4 at 0.5 μM, paclitaxel at 5 μM or vehicle DMSO (control) at room temperature

before GTP was added to start the tubulin polymerization reactions. The reaction was monitored at 37˚C.

https://doi.org/10.1371/journal.pone.0174006.g003

by flow cytometry analysis. As demonstrated in Fig 5A, 10s caused a dramatic increase in G2/

M phase cell counts in a concentration-dependent manner. Furthermore, treatment of SGC-

7901 cells with 10s (0.72 μM) or CA-4 (0.05 μM) from 6 to 24 h induced a dramatic increase in

Fig 4. Immunostaining of tubulin assembly in SGC-7901 cells. SGC-7901 cells were treated with 10s

(0.72 μM) for 48 h. Tubulin assembly was stained with FITC (left panel) and DAPI (middle panel). A merge of

the two panels is shown on the right. Images were taken using a confocal microscope.

https://doi.org/10.1371/journal.pone.0174006.g004

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

Fig 5. (A) 10s caused G2/M phase arrest in a concentration-dependent manner in SGC-7901 cells. Cells

were treated with 10s (0.18, 0.36, 0.72 μM) for 24 h. (B) 10s and CA-4 induced G2/M phase arrest in a time-

dependent manner. SGC-7901 cells were treated with 10s (0.72 μM) or CA-4 (0.05 μM) for 6, 12 and 24 h.

https://doi.org/10.1371/journal.pone.0174006.g005

the number of cells in the G2/M phase in a time-dependent manner. These results further

demonstrated that 10s was a tubulin inhibitor.

Molecular modeling. Molecular modeling studies were performed to investigate the

potential binding ability of the most active compound 10s and a moderate compound 10u to

the colchicine binding site of α- and β-tubulin. The molecular modeling study was performed

using the CDOCKER protocol in Discovery Studio 3.0 with the default settings to predict the

binding mode of our ligand (PDB: 3HKD). These studies showed that 10s, 10u and SMART

can occupy the colchicine binding site of tubulin (Fig 6). The trimethoxyphenyl moiety of

SMART and the dimethoxyphenyl moiety of 10s and 10u in the A-rings extended toward the

Fig 6. (A). The binding mode of compound 10s (violet), 10u (cyan) and SMART (green) in the colchicine binding site of tubulin. (B).Overlay of 10s in the

binding site. Hydrogen bonds are shown using green dashed lines and the distance is less than 3 Å. (C).Overlay of 10u in the binding site.

https://doi.org/10.1371/journal.pone.0174006.g006

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

Fig 7. We used a linear fitting curve to compare the predicted pIC50 values (modeled based on a

known dataset of 21 defined compounds in SGC-7901 cells) with the experimental pIC50 values.

y = 0.992x + 0.007, R – square = 0.992.

https://doi.org/10.1371/journal.pone.0174006.g007

α- and β-tubulin interfaces. Several amino acids of β-tubulin formed hydrophobic interactions

with compounds 10s and 10u. The thiol group of Cys β241 formed a hydrogen bond with the

nitrogen atom of the amino linker and the methoxy oxygen atom in ring A of 10s formed a

hydrogen bond with the hydroxyl group of Thr α179. However, the thiol group of Cys β241

could not form a hydrogen bond with the nitrogen atom of 10u due to the methyl group on

nitrogen atom. This may be the reason that the activities of 10u was less active than 10s. The

docking study results revealed that 10s could bind the colchicine binding site of tubulin.

QSAR model. By using the Create 3D QSAR protocol in Discovery Studio 3.0, twenty-

one synthesized compounds with definite IC₅₀values against SGC-7901 were selected as the

model dataset. By convention, we used the pIC₅₀scale (−log IC₅₀), in which higher values indi-

cate exponentially greater potency, to measure inhibitory activity. The training set and testing

set were chosen by the Diverse Molecules method in Discovery Studio 3.0. To ensure a good

alignment, we chose the alignment conformation of each molecule with the lowest energy in

the docked results of the CDOCKER protocol. We applied the alignment of the substructure

10a before building the QSAR model. The correlation coefficient r²between the observed

activity of the testing set and the training set was found to be 0.992; therefore, the QSAR

model that we built was acceptable (greater than 0.5, Fig 7). Additionally, the molecules that

aligned with the iso-surfaces of the 3D-QSAR model (Organic & Biomolecular Chemistry

Page 10 of 349 coefficients) on Van der Waals grids (Fig 8A) and electrostatic potential grids

(Fig 8B) were listed. Our electrostatic map indicated red contours around regions where high

electron density (negative charge) was expected to increase activity, and blue contours repre-

sented areas where low electron density (partial positive charge) was expected to increase activ-

ity. Similarly, our steric map indicated areas where steric bulk was predicted to increase

(green) or decrease (yellow) activity. It was widely acceptable that a better inhibitor based on

the 3D QSAR model should have stronger Van der Waals attractions in the green areas and a

polar group in the blue electrostatic potential areas (which were dominant close to the skele-

ton). Thus, this promising model would provide a guideline to design and optimize more

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

Fig 8. (A). 3D-QSAR model coefficients on electrostatic potential grids. Blue represents positive coefficients;

red represents negative coefficients. (B). 3D-QSAR model coefficients on Van der Waals grids. Green

represents positive coefficients; yellow represents negative coefficients. (For interpretation of the references

to color in this figure legend, the reader is referred to the web version of this article.)

https://doi.org/10.1371/journal.pone.0174006.g008

effective tubulin inhibitors based on the N,4-diaryl-1,3-thiazole-2-amines skeleton and pave

the way for us to further study these compounds in the future.

Conclusions

In summary, we designed and synthesized a series of N,4-diaryl-1,3-thiazole-2-amines containing

three aromatic rings with an amino linker as novel CBSIs. Most of the target compounds showed

moderate anti-proliferative activity in different cancer cells lines with potencies in the μM range.

Among them, N-(2,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-1,3-thiazol-2-amine, 10s, was

found to be the most potent compound with IC₅₀values at the submicromolar level in three

human cancer cell lines. Tubulin polymerization and immunostaining experiments revealed that

10s potently inhibited tubulin polymerization and disrupted tubulin microtubule dynamics in a

manner similar to CA-4. Moreover, 10s effectively induced SGC-7901 cell cycle arrest at the

G₂/M phase in both concentration- and time-dependent manners. Molecular docking results

revealed that 10s could bind the colchicine binding site of tubulin. Taken together, the present

study indicates that the utilization of an sp³amino linker to replace the sp²carbonyl linker can be

one of the future directions of novel CBSI synthesis and is deserving of further study. Investigation

of the modification of these novel CBSIs and mechanistic studies for anticancer properties are

underway in our lab. We will continue to optimize and refine the modification of these com-

pounds and will then select one or two active compounds for in vivo testing.

Experimental section

Chemistry

All solvents and chemical materials were obtained from commercially available sources and

were used without purification. The microwave reactions were performed on a discover-sp

single mode microwave reactor from CEM Corporation. The progress of reactions was moni-

tored by TLC using silica gel plates under UV light. NMR spectra were recorded on a Bruker

AVANCE 400, or 600 spectrometer (¹H, 400 MHz, 600 MHz; ¹³C, 100 MHz, 150 MHz), in

CDCl₃or DMSO-d₆(TMS was used as an internal standard). Chemical shifts are expressed as

parts per million downfield from tetramethylsilane. Mass spectra (MS) were measured on an

Agilent 1100-sl mass spectrometer with an electrospray ionization source. Melting points were

measured on a hot stage microscope (X-4, Beijing Taike Ltd.) and are uncorrected.

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

General synthetic procedures for N,4-diaryl-1,3-thiazole-2-amines 10a-v. The α-bro-

moketones 16 (1.0 mmol) and aryl thioamides 14 (1.0 mmol) were dissolved in ethanol (5

mL). The mixture was heated to 80˚C for 5 min under microwave irradiation (discover-sp,

CEM Corporation, 150 W). After the reaction was completed, the mixture was poured into

water and extracted with methylene chloride. The organic layer was washed with deionized

water, dried over anhydrous sodium sulfate, and filtered, and the solvent was then evaporated

under reduced pressure to yield compounds 10a-t. The compound 10u was obtained by a sim-

ple reaction of the compound 10s with methyl iodide in anhydrous DMF, and the compound

10v was obtained by an acetylation reaction of compound 10s with acetic anhydride.

N-(3,4,5-trimethoxyphenyl)-4-(4-methyphenyl)-1,3-thiazol-2-amine (10a). Yellow solid;

yield: 81%; M.p.: 179–181˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.75 (d, J = 7.9 Hz, 2H), 7.19

(d, J = 7.9 Hz, 2H), 6.76 (s, 1H), 6.59 (s, 2H), 3.80 (s, 3H), 3.71 (s, 6H), 2.36 (s, 3H); ¹³C-NMR

(150 MHz, CDCl₃): δ 165.6, 153.6 (2C), 151.0, 137.8, 136.7, 133.6, 131.7, 129.3 (2C), 126.0

(2C), 100.5 (2C), 96.2, 60.9, 55.8 (2C), 21.1; ESI-MS: m/z = 357.2 [M+H]⁺, 379.1 [M+Na]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(4-methoxyphenyl)-1,3-thiazol-2-amine (10b). Brown solid;

yield: 79%; M.p.: 65–67˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.77 (d, J = 8.7 Hz, 2H), 6.90 (d,

J = 8.7 Hz, 2H), 6.66 (s, 1H), 6.62 (s, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 3.77 (s, 6H); ¹³C-NMR (150

MHz, CDCl₃): δ 165.4, 159.4, 153.6 (2C), 150.6, 136.6, 133.7, 127.3 (2C), 127.2, 114.0 (2C),

99.5, 96.3 (2C), 60.9, 55.9 (2C), 55.2; ESI-MS: m/z = 373.2 [M+H]⁺, 395.1 [M+Na]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(4-fluorophenyl)-1,3-thiazol-2-amine (10c). Pale brown solid;

yield: 70%; M.p.: 134–136˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.80 (m, 2H), 7.06 (m, 2H), 6.73

(s, 1H), 6.64 (s, 2H), 3.82 (s, 3H), 3.81 (s, 6H); ¹³C-NMR (150 MHz, CDCl₃): δ 165.5, 162.5

(d, J = 248.0 Hz), 153.7 (2C), 150.0, 136.4, 133.9, 130.6 (d, J = 3.0 Hz), 127.7 (d, J = 8.0 Hz, 2C),

115.5 (d, J = 21.7 Hz, 2C), 101.0, 96.5 (2C), 61.0, 56.0 (2C); ESI-MS: m/z = 361.2 [M+H]⁺,

383.1 [M+Na]⁺, 399.1 [M+K]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(4-chlorophenyl)-1,3-thiazol-2-amine (10d). Pale yellow solid;

yield: 77%; M.p.: 149–151˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.77 (d, J = 8.5 Hz, 2H), 7.34

(d, J = 8.5 Hz, 2H), 6.79 (s, 1H), 6.61 (s, 2H), 3.82 (s, 3H), 3.77 (s, 6H); ¹³C-NMR (150 MHz,

CDCl₃): δ 165.7, 153.7 (2C), 149.9, 136.4, 133.9, 133.6, 132.9, 128.8 (2C), 127.2 (2C), 101.8,

96.5 (2C), 61.0, 55.9 (2C); ESI-MS: m/z = 377.1 [M+H]⁺, 399.1 [M+Na]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(4-bromophenyl)-1,3-thiazol-2-amine (10e). Yellow solid;

yield: 82%; M.p.: 144–146˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.69 (d, J = 8.4 Hz, 2H), 7.51 (d,

J = 8.4 Hz, 2H), 6.79 (s, 1H), 6.65 (s, 2H), 3.83 (s, 9H); ¹³C-NMR (150 MHz, CDCl₃): δ 165.6,

153.7 (2C), 149.9, 136.3, 134.0, 133.2, 131.7 (2C), 127.5 (2C), 121.8, 101.9, 96.6 (2C), 61.0, 56.0

(2C); ESI-MS: m/z = 421.0 [M+H]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(4-nitrophenyl)-1,3-thiazol-2-amine (10f). Orange solid; yield:

73%; M.p.: 98–99˚C; ¹H-NMR (600 MHz, CDCl₃): δ 8.32 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0

Hz, 2H), 6.36 (s, 2H), 5.99 (s, 1H), 3.84 (s, 9H); ¹³C-NMR (150 MHz, CDCl₃): δ 160.1, 153.8

(2C), 147.9, 138.4, 137.5, 133.9, 129.1 (2C), 126.4, 124.1 (2C), 98.7, 98.3 (2C), 60.9, 55.9 (2C);

ESI-MS: m/z = 388.2 [M+H]⁺, 410.2 [M+Na]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(3,4-dimethoxyphenyl)-1,3-thiazol-2-amine (10g). Brown

solid; yield: 75%; M.p.: 54–56˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.38 (m, 2H), 6.88 (d, J = 8.7

Hz, 1H), 6.67 (s, 1H), 6.66 (s, 2H), 3.92 (s, 3H), 3.90 (s, 3H), 3.82 (s, 9H); ¹³C-NMR (150 MHz,

CDCl₃): δ 165.4, 153.7 (2C), 150.1, 149.1, 148.9, 136.2, 134.0, 127.0, 118.5, 111.2, 109.3, 99.7,

96.5 (2C), 60.9, 56.0 (2C), 55.9, 55.8; ESI-MS: m/z = 403.2 [M+H]⁺, 425.1 [M+Na]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(3-fluoro-4-methoxyphenyl)-1,3-thiazol-2-amine (10h). Brown

red solid; yield: 76%; M.p.: 148–150˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.56 (s, 1H), 7.54 (s, 1H),

6.93–6.96 (m, 1H), 6.67 (s, 1H), 6.65 (s, 2H), 3.90 (s, 3H), 3.82 (s, 3H), 3.80 (s, 6H); ¹³C-NMR

(150 MHz, CDCl₃): δ 165.5, 153.6 (2C), 152.3 (d, J = 245.0 Hz), 149.6, 147.4 (d, J = 10.8 Hz),

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

136.4, 133.8, 127.9 (d, J = 6.9 Hz), 121.8 (d, J = 2.2 Hz), 113.8 (d, J = 19.7 Hz), 113.3, 100.6, 96.5

(2C), 60.9, 56.2, 55.9 (2C); ESI-MS: m/z = 391.2 [M+H]⁺, 413.2 [M+Na]⁺, 429.1 [M+K]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(3-nitro-4-methoxyphenyl)-1,3-thiazol-2-amine (10i). Yellow

solid; yield: 84%; M.p.: 173-174˚C; ¹H-NMR (600 MHz, CDCl₃): δ 8.35 (d, J = 1.5 Hz, 1H),

7.94 (dd, J = 8.7 Hz, J = 1.5Hz, 2H), 7.08 (d, J = 8.7 Hz, 1H), 6.76 (s, 1H), 6.75 (s, 2H), 3.97 (s,

3H), 3.86 (s, 6H), 3.83 (s, 3H); ¹³C-NMR (150 MHz, CDCl₃): δ 165.2, 153.7 (2C), 152.4, 148.5,

139.4, 136.3, 133.9, 131.2, 127.4, 123.4, 113.6, 101.6, 96.3 (2C), 61.0, 56.6, 56.0 (2C); ESI-MS:

m/z = 418.2 [M+H]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(3-amino-4-methoxyphenyl)-1,3-thiazol-2-amine (10j). Yellow

solid; yield: 65%; M.p.: 166–167˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.20 (m, 2H), 6.79 (d,

J = 8.1 Hz, 1H), 6.62 (s, 2H), 6.61 (s, 1H), 3.87 (s, 3H), 3.81 (s, 3H), 3.79 (s, 6H); ¹³C-NMR (150

MHz, CDCl₃): δ 165.4, 153.6 (2C), 151.0, 147.3, 136.6, 136.1, 133.7, 127.5, 116.5, 112.7, 110.3,

99.5, 96.4 (2C), 60.9, 55.9 (2C), 55.5; ESI-MS: m/z = 388.2 [M+H]⁺, 410.2 [M+Na]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(3-benzyloxy-4-methoxyphenyl)-1,3-thiazol-2-amine (10k).

Red solid; yield: 74%; M.p.: 77–78˚C; ¹H-NMR (600 MHz, DMSO-d₆): δ 10.20 (s, 1H), 7.61 (d,

J = 2.0 Hz, 1H), 7.51 (dd, J = 8.3 Hz, J = 2.0 Hz, 1H), 7.49 (d, J = 7.3 Hz, 2H), 7.41 (m, 2H), 7.35

(d, J = 7.3 Hz, 1H), 7.19 (s, 1H), 7.16 (s, 2H), 7.03 (d, J = 8.3Hz, 1H), 5.12 (s, 2H), 3.82 (s, 6H),

3.80 (s, 3H), 3.63 (s, 3H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 163.3, 153.4 (2C), 150.3, 149.3,

148.2, 137.9, 137.4, 132.2, 128.8 (2C), 128.3 (2C), 128.0, 119.0, 112.6, 111.6, 111.6, 101.3, 95.0

(2C), 70.4, 60.5, 56.0, 56.0 (2C); ESI-MS: m/z = 479.2 [M+H]⁺, 501.1 [M+Na]⁺, 517.1 [M+K]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-1,3-thiazol-2-amine (10l). Red

solid; yield: 73%; M.p.: 98–100˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.37 (d, J = 1.9 Hz, 1H), 7.36

(dd, J = 8.3 Hz, J = 1.9 Hz, 2H), 6.85 (d, J = 8.3 Hz, 1H), 6.65 (s, 2H), 6.64 (s, 1H), 3.90 (s, 3H),

3.82 (s, 6H), 3.82 (s, 3H); ¹³C-NMR (150 MHz, CDCl₃): δ 165.3, 153.6 (2C), 150.5, 146.6,

145.6, 136.6, 133.7, 128.0, 118.1, 112.3, 110.7, 100.0, 96.4 (2C), 60.9, 56.0 (2C), 55.9; ESI-MS:

m/z = 389.1 [M+H]⁺, 777.3 [2M+H]⁺, 799.3 [2M+Na]⁺.

N-(3,4,5-trimethoxyphenyl)-4-(3,4-difluorophenyl)-1,3-thiazol-2-amine (10m). Yellow solid;

yield: 64%; M.p.: 153-155˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.70-7.71 (m, 1H), 7.62-7.65 (m,

1H), 7.14-7.18 (m, 1H), 6.74 (s, 1H), 6.70 (s, 2H), 3.86 (s, 6H), 3.83 (s, 3H); ¹³C-NMR (150 MHz,

CDCl₃): δ 165.3, 153.7 (2C), 149.0, 136.3, 134.0, 132.2, 130.9, 128.8, 121.8, 117.4 (d, J = 16.8 Hz),

115.0 (d, J = 18.9 Hz), 102.0, 96.5 (2C), 61.0, 56.0 (2C); ESI-MS: m/z = 379.1 [M+H]⁺.

N-(3,5-dimethoxyphenyl)-4-(4-methoxyphenyl)-1,3-thiazol-2-amine (10n). Pale yellow solid;

yield: 63%; M.p.: 150–151˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.79 (d, J = 8.7 Hz, 2H), 6.91 (d,

J = 8.7 Hz, 2H), 6.71 (s, 1H), 6.51 (d, J = 2.1 Hz, 1H), 6.15 (m, 1H), 3.82 (s, 3H), 3.70 (s, 6H);

¹³C-NMR (150 MHz, CDCl₃): δ 164.5, 161.4 (2C), 159.4, 150.6, 142.1, 130.2, 127.3 (2C), 114.0

(2C), 100.1, 96.2 (2C), 95.1, 55.2, 55.2 (2C); ESI-MS: m/z = 343.2 [M+H]⁺, 365.2 [M+Na]⁺.

N-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,3-thiazol-2-amine (10o). Khaki solid;

yield: 66%; M.p.: 66–68˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.20 (d, J = 8.8 Hz, 2H), 7.02 (s,

2H), 6.82 (d, J = 8.8 Hz, 2H), 6.63 (s, 1H), 3.84 (s, 6H), 3.83 (s, 3H), 3.77 (s, 3H); ¹³C-NMR

(150 MHz, CDCl₃): δ 167.4, 156.3, 153.2 (2C), 137.9, 133.6, 130.3, 121.9 (2C), 114.7, 114.5

(2C), 103.4 (2C), 100.5, 60.8, 56.0 (2C), 55.4; ESI-MS: m/z = 373.2 [M+H]⁺, 395.1 [M+Na]⁺.

N-(3,4-dimethphenyl)-4-(3-nitro-4-methoxyphenyl)-1,3-thiazol-2-amine (10p). Red solid;

yield: 63%; M.p.: 166–167˚C; ¹H-NMR (600 MHz, CDCl₃): δ 8.27 (s, 1H), 7.94 (d, J = 8.7 Hz,

1H), 7.08 (s, 2H), 7.01 (m, 2H), 6.70 (s, 1H), 3.93 (s, 3H), 2.22 (s, 3H), 2.20 (s, 3H); ¹³C-NMR

(150 MHz, CDCl₃): δ 166.3, 152.2, 148.4, 139.4, 137.8, 137.8, 132.1, 131.6, 130.4, 127.5, 123.2,

120.7, 116.5, 113.5, 101.4, 56.5, 19.9, 19.0; ESI-MS: m/z = 356.1 [M+H]⁺, 378.1 [M+Na]⁺.

N-(3,4-dimethphenyl)-4-(3-amino-4-methoxyphenyl)-1,3-thiazol-2-amine (10q). Brown

solid; yield: 68%; M.p.: 146–148˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.18 (s, 1H), 7.16 (m, 1H),

7.09 (m, 2H), 7.06 (s, 1H), 6.76 (d, J = 8.8 Hz, 1H), 6.55 (s, 1H), 3.85 (s, 3H), 2.23 (s, 3H), 2.22

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

(s, 3H); ¹³C-NMR (150 MHz, CDCl₃): δ 165.8, 150.4, 147.3, 138.0, 137.7, 136.1, 131.7, 130.3,

127.3, 120.5, 116.5, 116.3, 112.9, 110.3, 99.2, 55.5, 19.9, 19.0; ESI-MS: m/z = 326.1 [M+H]⁺,

348.1 [M+Na]⁺, 364.0 [M+K]⁺.

N-(2,4-dimethoxyphenyl)-4-(4-methphenyl)-1,3-thiazol-2-amine (10r). Brown solid; yield:

75%; M.p.: 65–66˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.91 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.0 Hz,

2H), 7.21 (d, J = 8.0 Hz, 2H), 6.71 (s, 1H), 6.54 (m, 2H), 3.87 (s, 3H), 3.82 (s, 3H) 2.38 (s, 3H);

¹³C-NMR (150 MHz, CDCl₃): δ 165.1, 155.8, 151.1, 149.6, 137.5, 131.8, 129.2 (2C), 125.9 (2C),

123.7, 118.6, 103.7, 100.4, 99.1, 55.7, 55.5, 21.2; ESI-MS: m/z = 327.2 [M+H]⁺, 349.1 [M+Na]⁺.

N-(2,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-1,3-thiazol-2-amine (10s). Brown red solid;

yield: 80%; M.p.: 98–99˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.92 (d, J = 8.4 Hz, 1H), 7.79 (d,

J = 8.7 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 6.62 (s, 1H), 6.53 (m, 2H), 3.85 (s, 3H), 3.83 (s, 3H),

3.81 (s, 3H); ¹³C-NMR (150 MHz, CDCl₃): δ 165.0, 159.2, 155.7, 151.0, 149.5, 127.7, 127.3

(2C), 123.8, 118.5, 113.8 (2C), 103.7, 99.4, 99.1, 55.6, 55.5, 55.2; ESI-MS: m/z = 343.2 [M+H]⁺,

365.1 [M+Na]⁺, 381.1 [M+K]⁺.

N-(2,4-dimethoxyphenyl)-4-(2,4-dimethoxyphenyl)-1,3-thiazol-2-amine (10t). Brown solid;

yield: 65%; M.p.: 151–153˚C; ¹H-NMR (600 MHz, CDCl₃): δ 8.13 (d, J = 8.4 Hz, 1H), 7.95 (d,

J = 8.5 Hz, 1H), 7.13 (s, 1H), 6.59 (dd, J = 8.5 Hz, J = 1.9 Hz, 1H), 6.53 (m, 3H), 3.91 (s, 3H),

3.86 (s, 3H), 3.84 (s, 3H), 3.81 (s, 3H); ¹³C-NMR (150 MHz, CDCl₃): δ 163.1, 160.0, 157.9,

155.5, 149.3, 146.8, 130.7, 124.0, 118.2, 116.8, 104.5, 104.0, 103.7, 99.0, 98.6, 55.6, 55.5, 55.3,

55.3; ESI-MS: m/z = 373.2 [M+H]⁺, 395.1 [M+Na]⁺.

N-(2,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-N-methyl-1,3-thiazol-2-amine (10u). NaH

(5.4 mmol) was added to a stirred solution of 10s (2.8 mmol) in DMF (5 mL), and the mixture

was stirred at 25˚C for 2 min. MeI (2.8 mmol) was added dropwise, and the mixture was stir-

red at 25˚C for 1 h. The reaction was quenched with saturated aqueous NH₄Cl solution (15

mL), and the mixture was extracted with EtOAc (3 × 15 mL). The combined organic fraction

was dried and the solvent was evaporated. The crude solid was purified by column chromatog-

raphy to yield 10u. White solid; yield: 53%; M.p.: 126–128˚C; ¹H-NMR (600 MHz, CDCl₃): δ

7.81 (d, J = 8.8 Hz, 2H), 7.27 (s, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.58 (d, J = 2.6 Hz, 1H), 6.52 (dd,

J = 8.6 Hz, J = 2.6 Hz, 1H), 6.48 (s, 1H), 3.85 (s, 3H), 3.84 (s, 3H) 3.80 (s, 3H), 1.27 (s, 3H);

¹³C-NMR (150 MHz, CDCl₃): δ 171.6, 160.4, 158.9, 156.9, 151.2, 130.1, 128.4, 127.7, 127.2

(2C), 113.7 (2C), 104.7, 100.2, 99.7, 55.6, 55.5, 55.2, 39.3; ESI-MS: m/z = 357.2 [M+H]⁺, 379.1

[M+Na]⁺, 395.1 [M+K]⁺.

N-(2,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-N-acetyl-1,3-thiazol-2-amine (10v). A flask

immersed in a room temperature oil bath was charged with 10s (2 mmol) and Ac₂O (10 mL)

and heated to 50˚C. After completion of the reaction, the reaction was quenched upon addi-

tion of 50 g of ice. A yellow precipitate formed, which was filtered and washed with H₂O. The

crude solid was purified by column chromatography to give pure 10v. Yellow solid; yield: 54%;

M.p.: 146–148˚C; ¹H-NMR (600 MHz, CDCl₃): δ 7.58 (d, J = 8.8 Hz, 2H), 7.22 (d, J = 9.2 Hz,

1H), 7.03 (s, 1H), 6.82 (d, J = 8.8 Hz, 2H), 6.60 (m, 2H), 3.89 (s, 3H), 3.77 (s, 3H), 3.74 (s, 3H),

2.06 (s, 3H); ¹³C-NMR (150 MHz, CDCl₃): δ 171.0, 161.2, 159.1, 155.9, 148.8, 130.7, 127.8,

127.1 (2C), 122.2, 113.7 (2C), 106.5, 104.8, 99.6, 55.7, 55.5, 55.2, 22.9; ESI-MS: m/z = 385.2

[M +H]⁺, 407.1 [M+Na]⁺.

Biology

MTT assay: The SGC-7901, A549 and HT-1080 cell lines were purchased from the American

Type Culture Collection (ATCC, Manassas, VA, USA). The in vitro antiproliferative activities

of CA-4 (2), Nocodazole (3) and all of the target compounds were determined with an MTT

assay following a previously reported method [21,22].

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

Tubulin assembly assay: The effect of 10s on the polymerization of purified brain tubulin

was determined using a fluorescence-based tubulin polymerization assay kit (Cat. #BK011P,

Cytoskeleton, Inc., USA) following a previously reported method [22].

Immunofluorescence assay: Immunostaining was performed to detect the microtubule-

associated tubulin protein after exposure to CA-4 (2) or the investigated compound 10s fol-

lowing a previously reported method [21,22].

Cell cycle analysis: SGC-7901 cells (8 × 10⁴cells) were incubated with various concentra-

tions of CA-4 (2), 10s or 0.05% DMSO for the indicated times. The cells were collected by

centrifugation, washed with PBS and fixed in ice-cold 70% ethanol. The fixed cells were

harvested by centrifugation and resuspended in 500 μl of PBS containing 1 mg/mL RNase.

After 30 min of incubation at 37˚C, the cells were stained with 50 mg/mL propidium iodide

(PI) at 4˚C in the dark for 30 min. The samples were then analyzed by FACScan flow cytome-

try (Becton-Dickinson, Franklin Lakes, NJ, USA). The experiments were repeated at least

three times.

Molecular modeling: The molecular modeling studies were performed using Accelrys Dis-

covery Studio 3.0. The crystal structure of tubulin complexed with DAMA-colchicine (PDB:

3HKD) was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb). In the

docking process, the protein protocol was prepared via several operations, including the stan-

dardization of atom names, insertion of missing atoms in residues and removal of alternate

conformations, insertion of missing loop regions based on SEQRES data, optimization of

short and medium sized loop regions with the Looper Algorithm, minimization of remaining

loop regions, calculation of pK, and protonation of the structure. The receptor model was then

typed with the CHARMm force field, and a binding sphere with a radius of 9.0 Å was defined

with the original ligand (DAMA-colchicine) as the binding site. The 10s was drawn with

Chemdraw and fully minimized using the CHARMm force field. Finally, 10s was docked into

the binding site using the CDOCKER protocol with the default settings.

QSAR Model: In the model, 80% of the target compounds were utilized as a training set for

QSAR modeling. The remaining 20% were chosen as a test subset using the same protocol.

The literature-based inhibitory activity values of the known compounds [IC₅₀of SGC-7901

cells] were initially converted into the minus logarithmic scale [pIC₅₀] and then used for subse-

quent QSAR analysis as the response variable. In Discovery Studio, the CHARMm force field

was applied and the electrostatic potential together with the Van der Waals potential were

treated as separate terms. As the electrostatic potential probe, A+ le point change was used

while distance-dependent dielectric constant was used to mimic the solvent effect. A carbon

atom with a radius of 1.73 Å was used as a probe for the Van der Waals potential. A Partial

Least-Squares (PLS) model was built using energy grids as descriptors. QSAR models were

built by using the Create 3D QSAR Model protocol in Discovery Studio 3.0.

Supporting information

S1 File. (1). Synthesis. 1. General synthetic procedures for aryl thioamides 14. 2. General syn-

thetic procedures for α-bromoacetophenones 16. (2). Contents: 1H-NMR and 13C-NMR

spectra of all target compounds.

(PDF)

Acknowledgments

This work was supported by the Programme for Innovative Research Team of the Ministry of

Education and the Programme for Liaoning Innovative Research Team in University.

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

Author Contributions

Conceptualization: MS QX WZ.

Data curation: MS QX JX.

Formal analysis: MS QX WZ.

Funding acquisition: WZ.

Investigation: MS QX WZ.

Methodology: MS QX JX Yue Wu Y. Wang.

Project administration: WZ.

Resources: MS QX WZ.

Software: QX Yue Wu JW.

Supervision: KB WZ.

Validation: DZ QG KB JW Yingliang Wu.

Visualization: MS QX KB WZ.

Writing – original draft: QX.

Writing – review & editing: MS QX KB WZ.

References

1. Stephens RE, Edds KT. Microtubules: structure, chemistry, and function. Physiol Rev. 1976; 56(4):

709–777. PMID: 790425

2. Etienne-Manneville S. From signaling pathways to microtubule dynamics: the key players. Curr Opin

Cell Biol. 2010; 22(1): 104–111. https://doi.org/10.1016/j.ceb.2009.11.008 PMID: 20031384

3. Kavallaris M. Microtubules and resistance to tubulin-binding agents. Nat Rev Cancer. 2010; 10(3):

194–204. https://doi.org/10.1038/nrc2803 PMID: 20147901

4. Dumontet C, Jordan MA. Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat Rev

Drug Discov. 2010; 9(10): 790–803. https://doi.org/10.1038/nrd3253 PMID: 20885410

5. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer. 2004; 4(4): 253–

265. https://doi.org/10.1038/nrc1317 PMID: 15057285

6. Dong M, Liu F, Zhou H, Zhai S, Yan B. Novel Natural Product- and Privileged Scaffold-Based Tubulin

Inhibitors Targeting the Colchicine Binding Site. Molecules. 2016; 21(10): 1375–1401.

7. Carlson RO. New tubulin targeting agents currently in clinical development. Expert Opin. Expert Opin

Investig Drugs. 2008; 17(5): 707–722. https://doi.org/10.1517/13543784.17.5.707 PMID: 18447597

8. Graening T, Schmalz HG. Total syntheses of colchicine in comparison: a journey through 50 years of

synthetic organic chemistry. Angew Chem Int Ed Engl. 2004; 43(25): 3230–3256. https://doi.org/10.

1002/anie.200300615 PMID: 15213947

9. Pettit GR, Singh SB, Niven ML, Hamel E, Schmidt JM. Isolation, structure, and synthesis of combretas-

tatins A-1 and B-1, potent new inhibitors of microtubule assembly, derived from Combretum caffrum. J

Nat Prod. 1987; 50(1): 119–131. PMID: 3598594

10. Pettit GR, Singh SB, Hamel E, Lin CM, Alberts DS, Garcia-Kendall D. Isolation and structure of the

strong cell growth and tubulin inhibitor combretastatin A-4. Experientia. 1989; 45(2): 209–211. PMID:

2920809

11. Chaudhary A, Pandeya SN, Kumar P, Sharma PP, Gupta S, Soni N, et al. Combretastatin a-4 analogs

as anticancer agents. Mini Rev Med Chem. 2007; 7(12): 1186–1205. PMID: 18220974

12. Attia SM. Molecular cytogenetic evaluation of the mechanism of genotoxic potential of amsacrine and

nocodazole in mouse bone marrow cells. J Appl Toxicol. 2013; 33(6): 426–433. https://doi.org/10.1002/

jat.1753 PMID: 22081495

PLOS ONE | https://doi.org/10.1371/journal.pone.0174006 March 23, 2017

14 / 15

Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors

13. Pettit GR, Toki B, Herald DL, Verdier-Pinard P, Boyd MR, Hamel E, et al. Antineoplastic agents. 379.

Synthesis of phenstatin phosphate. J Med Chem. 1998; 41(10): 1688–1695. https://doi.org/10.1021/

jm970644q PMID: 9572894

14. Lu Y, Li CM, Wang Z, Ross CR, Chen J, Dalton JT, et al. Discovery of 4-substituted methoxybenzoyl-

aryl-thiazole as novel anticancer agents: synthesis, biological evaluation, and structure-activity relation-

ships. J Med Chem. 2009; 52(6): 1701–1711. https://doi.org/10.1021/jm801449a PMID: 19243174

15. Chen J, Wang Z, Li CM, Lu Y, Vaddady PK, Meibohm B, et al. Discovery of novel 2-aryl-4-benzoyl-imid-

azoles targeting the colchicines binding site in tubulin as potential anticancer agents. J Med Chem.

2010; 53(20): 7414–7427. https://doi.org/10.1021/jm100884b PMID: 20919720

16. Xiao M, Ahn S, Wang J, Chen J, Miller DD, Dalton JT, et al. Discovery of 4-Aryl-2-benzoyl-imidazoles

as tubulin polymerization inhibitor with potent antiproliferative properties. J Med Chem. 2013; 56(8):

3318–3329. https://doi.org/10.1021/jm4001117 PMID: 23547728

17. La Regina G, Bai R, Coluccia A, Famiglini V, Pelliccia S, Passacantilli S, Mazzoccoli C, et al. New Pyr-

role Derivatives with Potent Tubulin Polymerization Inhibiting Activity As Anticancer Agents Including

Hedgehog-Dependent Cancer. J Med Chem. 2014; 57(15): 6531−6552. https://doi.org/10.1021/

jm500561a PMID: 25025991

18. Guan Q, Feng D, Bai Z, Cui Y, Zuo D, Zhai M, et al. Microwave-assisted synthesis, molecular docking

and antiproliferative activity of (3/5-aryl-1,2,4-oxadiazole-5/3-yl)(3,4,5-trimethoxyphenyl)methanone

oxime derivatives. Med Chem Comm. 2015; 6(8): 1484–1493.

19. Kim KS, Kimball SD, Misra RN, Rawlins DB, Hunt JT, Xiao HY, et al. Discovery of Aminothiazole Inhibi-

tors of Cyclin-Dependent Kinase 2: Synthesis, X-ray Crystallographic Analysis, and Biological Activities.

J. Med. Chem. 2002; 45(18): 3905–3927. PMID: 12190313

20. Wang XF, Ohkoshi E, Wang SB, Hamel E, Bastow KF, Morris-Natschke SL, et al. Synthesis and biolog-

ical evaluation of N-alkyl-N-(4-methoxyphenyl)pyridin-2-amines as a new class of tubulin polymerization

inhibitors. Bioorg. Med. Chem. 2013; 21(3): 632–642. https://doi.org/10.1016/j.bmc.2012.11.047

PMID: 23274123

21. Guan Q, Yang F, Guo D, Xu J, Jiang M, Liu C, et al. Synthesis and biological evaluation of novel 3,4-dia-

ryl-1,2,5-selenadiazol analogues of combretastatin A-4. Eur J Med Chem. 2014; 87: 1–9. https://doi.

org/10.1016/j.ejmech.2014.09.046 PMID: 25233100

22. Xu Q, Qi H, Sun M, Zuo D, Jiang X, Wen Z; et al. Synthesis and Biological Evaluation of 3-Alkyl-1,5-Dia-

ryl-1H-Pyrazoles as Rigid Analogues of Combretastatin A-4 with Potent Antiproliferative Activity. PLoS

One. 2015; 10(6): e0128710. https://doi.org/10.1371/journal.pone.0128710 PMID: 26061410

23. Das D, Sikdar P, Bairagi M. Recent developments of 2-aminothiazoles in medicinal chemistry. Eur J

Med Chem. 2016; 109: 89–98. https://doi.org/10.1016/j.ejmech.2015.12.022 PMID: 26771245

24. Taguchi M, Kondo H, Inoue Y, Kawahata Y, Jinbo Y, Sakamoto F, et al. Synthesis and antibacterial

activity of new tetracyclic quinolone antibacterials. J Med Chem. 1992; 35(1): 94–99. PMID: 1310116

25. Jiang Z, Chen Y, Yang C, Cao Y, Tao Y, Qin J, et al. A fully diarylmethylene-bridged triphenylamine

derivative as novel host for highly efficient green phosphorescent OLEDs. Org Lett. 2009; 11(7): 1503–

1506. https://doi.org/10.1021/ol9001152 PMID: 19256517

26. Kabalka GW, Mereddy AR. Microwave promoted synthesis of functionalized 2-aminothiazoles. Tetrahe-

dron Letters. 2006; 47: 5171–5172.

27. Sato T, Tomaru K, Koide T, Masuda M, Yamamoto M, Miyazawa N; et al. Synchronous lung and gastric

cancers successfully treated with carboplatin and pemetrexed: a case report. J Med Case Rep. 2012;

6: 266. https://doi.org/10.1186/1752-1947-6-266 PMID: 22938085

28. Rasheed S, Nelson-Rees WA, Toth EM, Arnstein P, Gardner MB. Characterization of a newly derived

human sarcoma cell line (HT-1080). Cancer. 1974; 33(4): 1027–1033. PMID: 4132053

PLOS ONE | https://doi.org/10.1371/journal.pone.0174006 March 23, 2017

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Synthesis and bioevaluation of N,4-diaryl-1,3-thiazole-2-amines as tubulin inhibitors with potent antiproliferative activity

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