Design, synthesis and biological evaluation of novel acridine and quinoline derivatives as tubulin polymerization inhibitors with anticancer activities

DOI: 10.1016/j.bmc.2021.116376

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Bioorganic and Medicinal Chemistry (2021)

Update date:2022-08-04

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Authors:

Ren, Yichang

Ruan, Yong

Cheng, Binbin

Li, Ling

Liu, Jin

Fang, Yuyu

Chen, Jianjun

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Article abstract of DOI:10.1016/j.bmc.2021.116376

A series of acridine and quinoline derivatives were designed and synthesized based on our previous work as novel tubulin inhibitors targeting the colchicine binding site. Among them, compound 3b exhibited the highest antiproliferative activity with an IC₅₀ of 261 nM against HepG-2 cells (the most sensitive cell line). In addition, compound 3b was able to suppress the formation of HepG-2 colonies. Mechanism studies revealed that compound 3b effectively inhibited tubulin polymerization in vitro and disrupted microtubule dynamics in HepG-2 cells. Furthermore, compound 3b inhibited the migration of cancer cells in a dose dependent manner. Moreover, compound 3b induced cell cycle arrest in G2/M phase and led to cell apoptosis. Finally, docking studies demonstrated that compound 3b fitted nicely in the colchicine binding site of tubulin and overlapped well with CA-4. Collectively, these results suggested that compound 3b represents a novel tubulin inhibitor deserving further investigation.

Full text of DOI:10.1016/j.bmc.2021.116376

Bioorg. Med. Chem. 46 (2021) 116376

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Design, synthesis and biological evaluation of novel acridine and quinoline

derivatives as tubulin polymerization inhibitors with anticancer activities

Yichang Ren^a, Yong Ruan^a, Binbin Cheng^b, Ling Li^a, Jin Liu^a, Yuyu Fang^c, Jianjun Chen^a

^aSchool of Pharmaceutical Sciences, Guangdong Provincial Key Laboratory of New Drug Screening, Southern Medical University, Guangzhou 510515, China

^bSchool of Medicine, Hubei Polytechnic University, Huangshi 435003, China

^cDepartment of Nephrology, First People’s Hospital of Pingjiang County, Yueyang 414500, China

A R T I C L E I N F O

A B S T R A C T

Keywords:

A series of acridine and quinoline derivatives were designed and synthesized based on our previous work as novel

tubulin inhibitors targeting the colchicine binding site. Among them, compound 3b exhibited the highest anti-

proliferative activity with an IC₅₀of 261 nM against HepG-2 cells (the most sensitive cell line). In addition,

compound 3b was able to suppress the formation of HepG-2 colonies. Mechanism studies revealed that com-

pound 3b effectively inhibited tubulin polymerization in vitro and disrupted microtubule dynamics in HepG-2

cells. Furthermore, compound 3b inhibited the migration of cancer cells in a dose dependent manner. More-

over, compound 3b induced cell cycle arrest in G2/M phase and led to cell apoptosis. Finally, docking studies

demonstrated that compound 3b ﬁtted nicely in the colchicine binding site of tubulin and overlapped well with

CA-4. Collectively, these results suggested that compound 3b represents a novel tubulin inhibitor deserving

further investigation.

Acridine and quinoline derivatives

Tubulin inhibitor

Colchicine binding site

Apoptosis

Anticancer agents

1. Introduction

ABI,¹⁵SMART,¹⁹PAT,²⁰SAIs,²¹benzofused ﬁve membered heterocyclic

compounds (a8,²²a9²²and a10²³), acridane (b1)²⁴and quinolone (b2,

Microtubules, consisting of

/β-tubulin heterodimers, are involved

b3, b4) derivatives.^25–27In particular, the acridane (b1) and quinoline

analogs (e.g. b2, b3, b4) were designed to mimic CA-4, a naturally

occurring and highly potent CBSI that is currently undergoing multiple

clinical trials for cancer treatment. However, the development of CA-4

has been hampered by its instability due to the presence of the cis-

double bond which can be converted to the inactive trans-conformation

under in vivo conditions. The acridane and quinoline analogs were

designed to avoid the deactivating cis–trans conversion of CA-4. In the

acridane derivatives (b1), the 3,4,5-trimethoxyphenyl (TMP) group was

retained, while in the quinoline analogs (b2, b3, b4), the TMP moiety

was replaced by the quinoline group.

in many important cellular processes like mitosis, cell division, prolif-

eration, cell shape maintenance, cell signaling and intracellular trans-

port.¹Microtubule targeting agents (MTAs) have been generally

considered to be an effective treatment for cancer.²MTAs exert their

anticancer activity by binding to one of the six binding sites on tubulin,

including the paclitaxel site,³vinca site,⁴laulimalide site,⁵pironetin

site,⁶maytansine site⁷and colchicine site,⁸thus disrupting the dynamics

of microtubules,⁹To date, only MTAs targeting the paclitaxel site or

vinca site (e.g. taxanes and vinca alkaloids) have been approved for

clinical uses.¹⁰However, several disadvantages (e.g. structural

complexity, low water solubility and multidrug resistance) have hin-

dered the clinical applications of existing MTAs.^11,12On the other hand,

MTAs that bind to the colchicine binding site (also called CBSIs) have the

potential to overcome the drawbacks associated with taxanes and vinca

alkaloids, owing to their structural simplicity, non-substrate nature for

the multidrug resistance protein 1 (P-glycoprotein) and reasonable

physicochemical properties.^13–15Therefore, the search for CBSIs has

been intensiﬁed in the past two decades with numerous novel scaffolds

being discovered (Fig. 1), for example colchicine,¹⁶CA-4,¹⁷CA-4P,¹⁸

We have previously reported the discovery of several classes of TMP-

based tubulin inhibitors (e.g. ABI, SMART, PAT, SAIs, acridane analog

b1) as potent anticancer agents.^{15,19–21,24}In a continuation of our

research interest in tubulin inhibitors, we designed several series of

acridine and quinoline derivatives based on the following rationales

(Fig. 2): 1) substituting the acridane system A of compound b1 (reported

by us before²⁴) with an acridine moiety, or the quinazoline system A of

compound b4 with various aryl rings (e.g. quinoline, pyrimidine, pyri-

dine); 2) replacing the phenyl B ring and its substituents (R₁, R₂, R₃)

* Corresponding author.

E-mail address: jchen21@smu.edu.cn (J. Chen).

https://doi.org/10.1016/j.bmc.2021.116376

Received 22 June 2021; Received in revised form 7 August 2021; Accepted 11 August 2021

Available online 23 August 2021

Y. Ren et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116376

with other pharmacophores such as TMP moiety which has been

generally considered to be essential for interaction with the colchicine

binding site of tubulin; 3) exchanging the methyl moiety at R₄position

with a bulkier group (e.g. butyl) to explore the effects of substituent size

on activity. Herein, we describe their synthesis and in vitro anticancer

activities of the quinolone derivatives as detailed below.

Compound 3b displayed the strongest antiproliferative activities against

HepG-2 cells, the most sensitive cell line, with an IC₅₀value of 261 nM.

For series 1 compounds (2a-e, 3a-e, 4a-e), when R₁was a 3,4,5-trime-

thoxyphenyl or 4-methoxythenyl group, the compounds were gener-

ally more potent than that of the corresponding analogs with a phenyl,

4-cyanophenyl or 3-chloro-4-ﬂuorophenyl group at R₁position (e.g. IC₅₀

of 261 nM–10 μM for 2a-4a, 2b-4b vs IC₅₀of 16 μM–45 μM for 2c-4c,

2. Chemistry

2d-4d, 2e-4e). These results suggest that 3,4,5-trimethoxyphenyl and 4-

methoxythenyl groups were optimal for antiproliferative activity.

Inspired by this, series 2 compounds (6a-11a, 6b-11b) and series 3

compounds (6c-11c, 6d-11e, 6e-11e) were synthesized. For series 1 and

series 3 compounds, when R₁was a 4-methoxyphenyl moiety, com-

pounds with a methyl substituent at R₂position displayed higher po-

tency than the ones with a hydrogen or n-butyl substitution (e.g. 2b-4b,

The synthesis of series 1 compounds (2a-e, 3a-e, 4a-e) was illus-

trated in Scheme 1. Under Chan-Lam reaction condition, 5-aminoquino-

line was coupled with a variety of arylboronic acids to produce

compounds 2a-e. Intermediates 2a-e were reacted with methyl iodide or

1-iodobutane in the presence of sodium hydride to smoothly afford

compounds 3a-e or 4a-e, respectively.

–

H). Among them,

6c-8c, 6d-8d, 6e-8e; CH₃

CH₂CH₂CH₂CH₃

Series 2 (6a-11a, 6b-11b) and series 3 compounds (6c-11c, 6d-11e,

6e-11e) were prepared following the synthetic route outlined in Scheme

2. First, aryl chloride compounds 5a-e were coupled with 4-methoxyani-

line in anhydrous ethanol (EtOH) with catalytic amount of HCl (conc.) to

give intermediates 6a-e. Intermediates 6a-e were methylated with

methyl iodide or butylated with 1-iodobutane in the presence of sodium

hydride to generate compounds 7a-e or 8a-e, respectively. Similarly,

compounds 5a-e were reacted with 3,4,5-trimethoxyaniline to give in-

termediates 9a-e, followed by methylation with methyl iodide or

butylation with 1-iodobutane to yield compounds 10a-e or 11a-e,

respectively.

compound 3b exhibited the highest activity with an average IC₅₀value

of 0.87 μM against the four cancer cell lines. However, when R₁was a

3,4,5-trimethoxyphenyl group, compounds without substituents at R₂

position (R₂= hydrogen) exhibited better activity than the ones with

–

methyl or n-butyl moiety (e.g. 2a-4a, 9c-11c, 9d-11d, 9e-11e; H >

–

CH₃> CH₂CH₂CH₂CH₃). Among them, compound 2a demonstrated

the highest activity with an average IC₅₀value of 1.50 M against the

four cancer cell lines. For series 2 compounds, when R₁was a 4-methox-

yphenyl group, compounds without substituents at R₂position (R₂

hydrogen) showed higher potency than that with a methyl or n-butyl

–

CH₂CH₂CH₂CH₃).

substitution (e.g. 6a-8a, 6b-8b; H > CH₃

When R₁was a 3,4,5-trimethoxyphenyl group, compounds a methyl

substituent at R₂position exhibited better activity than that with a

3. Biological evaluation

–

CH₃

hydrogen or n-butyl group (e.g. 9a-11a, 9b-11b;

–

H).

3.1. In vitro antiproliferative activities

CH₂CH₂CH₂CH₃

The in vitro cytotoxicity of the newly synthesized compounds 2a-e,

3a-e, 4a-e, 6a-e, 7a-e, 8a-e, 9a-e, 10a-e and 11a-e were evaluated

against four cancer cell lines (B16-F10, HepG-2, Hela and MCF-7) using

MTT assay with CA-4 as positive control. As shown in Tables 1 and 2, a

majority of the compounds (e.g. 2a-4a, 2b-4b, 6a-11a, 6b-11b, 6c-9c,

7e-9e) exhibited moderate to high antiproliferative activities with IC₅₀

3.2. Compound 3b inhibited the viability and colony formation of cancer

cells

To further validate the in vitro antiproliferative activities, the most

potent compound 3b was chosen for an additional MTT and a colony

formation assay.^28,29As shown in Fig. 3 (A-B), compound 3b signiﬁ-

cantly reduced the viability of B16-F10, HepG-2, Hela and MCF-7 cancer

values in the low micromolar to high nanomolar range (261 nM–10 μM).

Fig. 1. Structures of reported CBSIs.

Y. Ren et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116376

cells in a dose- and time-dependent manner. In addition, compound 3b

was able to inhibit the formation of HepG-2 colonies in a dose-

dependent manner, with the colony-forming ability of HepG-2 cells

When cells were exposed to increasing concentrations of compound 3b

(0.5, 1.0 and 5.0 μM), the percentages of cells arrested in G2/M phase

were increased from 17.46% to 32.13%. In contrast, only 9.27% of the

cells were arrested in G2/M phase in the control group.

being totally suppressed after 24 h treatment with 1.0 μM of compound

3b (Fig. 3C, 3D).

3.7. Analysis of cancer cell apoptosis

3.3. In vitro tubulin polymerization assay

Studies have shown that tubulin polymerization inhibitors are able

to induce cellular apoptosis,²³therefore, we assessed the apoptotic ef-

fects of compound 3b against HepG-2 cells using an annexin V-FITC and

propidium iodide (PI) assay. As shown in Figure 8, when HepG-2 cells

were treated with increasing concentrations of compound 3b (0.5, 1.0

To explore the mechanism of action, we investigated the inhibitory

effects of compound 3b on tubulin polymerization with CA-4 as a pos-

itive control. As exhibited in Fig. 4, compound 3b inhibited tubulin

polymerization with an IC₅₀of 12.38 ± 1.12

μM (Fig. 4A), which was

weaker than that of CA-4 (IC₅₀= 1.84 ± 0.41

μM, Fig. 4B). Furthermore,

and 5.0

μM) for 48 h, the total proportion of apoptotic cells were

compound 3b inhibited tubulin polymerization in a concentration-

dependent manner (Fig. 4C).

increased signiﬁcantly compared with the control. The results suggested

that compound 3b was able to cause the apoptosis of HepG-2 cells

efﬁciently.

3.4. Immunoﬂuorescence staining study

4. Docking studies of compound 3b at colchicine binding site of

tubulin

It is well known that microtubule dynamics play a vital role in cancer

cell growth. Therefore, compound 3b was evaluated for its ability to

disrupt microtubule dynamics by an immunoﬂuorescence staining assay

in HepG-2 cells. As shown in Fig. 5A, the microtubule networks dis-

played a normal arrangement with slim and ﬁbrous microtubules

(green) wrapped around the cell nucleus (blue) in the control cells.

However, the microtubule networks were disrupted in the cells treated

To investigate the potential binding interaction of compound 3b

with the tubulin, molecular docking studies were performed by

employing Schrodinger Molecular Modeling Suite 2011 (Schrodinger

LLC, New York, NY) and the tubulin crystal structure (PDB code: 5lyj).

As shown in Figure 9, compound 3b ﬁtted nicely to the colchicine site,

sharing the same pocket and overlapping well with CA-4. There was an

aromatic hydrogen bond (H-Bond) formed between CA-4 and the

with compound 3b (0.1, 0.5 and 1.0 μM) for 12 h (Fig. 5B, C, D). These

results indicated that compound 3b could induce the collapse of the

microtubule networks in a dose-dependent manner, further conﬁrming

that compound 3b could interfere with microtubule dynamics.

–

O of Thr-179 in tubulin. Similarly, two aromatic H-Bonds were

–

observed between compound 3b and tubulin (

C O of Thr-179,

–

O of Ala-317). The docking results suggest that compound 3b

–

3.5. Inhibition of cancer cell migration

bound to the colchicine site of tubulin resembling the binding mode of

CA-4.

Tumor cells are able to migrate to distant organs, leading to tumor

metastasis. We have previously reported that tubulin polymerization

inhibitors could inhibit cancer cell migration.²²Therefore, we per-

formed a wound healing assay to study the anti-migratory effects of

compound 3b against cancer cells. When HepG-2 cells were exposed to

5. Conclusion

In this work, we designed and synthesized a series of novel acridine

and quinoline derivatives as tubulin polymerization inhibitors targeting

the colchicine binding site. The target compounds were designed based

on our previously reported acridane-based tubulin polymerization in-

hibitor b1 and the qunoline compound b4. The newly synthesized

compounds showed moderate to high antiproliferative potency against a

panel of cancer cell lines. Among them, compound 3b exhibited the

highest antiproliferative activity with an IC₅₀value of 261 nM in HepG-2

cells, the most sensitive cell line. It also signiﬁcantly suppressed the

formation of HepG-2 colonies. Mechanism studies revealed that com-

compound 3b (0.1, 0.5 and 1.0 μM) for 24 h, the wound closure was

suppressed (Fig. 6) in a concentration-dependent manner, indicating

that compound 3b was able to inhibit the migration of cancer cells

effectively.

3.6. Cell cycle effects

Knowing that tubulin polymerization inhibitors are able to interfere

with cell division, we measured the effects of compound 3b on the cell

cycle of HepG-2 cells by ﬂow cytometry. As depicted in Figure 7, com-

pound 3b dose-dependently induced cell cycle arrest at G2/M phase.

pound 3b effectively inhibited tubulin polymerization in vitro (IC₅₀

11.7 M) and disrupted microtubule dynamics. Further, compound 3b

inhibited the migration of HepG-2 cancer cells in a dose dependent

Fig. 2. Design of target compounds based on our previous work.

Y. Ren et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116376

Scheme 1. Synthesis of series 1 compounds (2a-e, 3a-e, 4a-e). Reagents and reaction conditions: (a) Cu(OAc)₂, Pyr, DCM, r.t.; (b) NaH, DMF.

Scheme 2. Synthesis of series 2 (6a-11a, 6b-11b) and series 3 compounds (6c-11c, 6d-11e, 6e-11e). Reagents and reaction conditions: (a) EtOH, HCl, reﬂux; (b)

NaH, DMF.

Table 1

In vitro antiproliferative activities of series 1 compounds against four cancer cell lines.^a

IC₅₀(μM)

Structure

Compound

R₁

R₂

B16-F10

HepG-2

Hela

MCF-7

3,4,5-trimethoxyphenyl

4-methoxyphenyl

phenyl

1.279 0.311

6.584 ± 1.195

7.651 ± 0.597

4.567 ± 0.436

0.563 0.099

2.433 ± 0.108

28.61 ± 2.19

18.06 ± 1.94

27.06 ± 3.08

40.37 ± 3.39

25.16 ± 3.41

33.07 ± 3.91

38.13 ± 4.86

25.52 ± 0.85

29.49 ± 3.28

0.783 0.065

5.237 ± 0.821

6.997 ± 0.736

3.794 ± 0.265

0.261 0.025

1.366 ± 0.083

26.78 ± 3.06

16.03 ± 2.70

22.14 ± 4.12

37.51 ± 4.76

28.69 ± 2.18

30.14 ± 2.36

32.07 ± 1.01

20.12 ± 1.84

26.71 ± 2.17

2.801 0.614

8.267 ± 0.809

9.286 ± 1.158

4.163 ± 0.347

2.047 0.168

3.062 ± 0.156

30.72 ± 1.57

23.99 ± 2.97

29.90 ± 2.65

43.18 ± 6.03

36.48 ± 1.05

42.87 ± 5.22

44.92 ± 6.18

30.87 ± 1.22

32.14 ± 1.63

1.141 0.251

6.360 ± 0.374

8.327 ± 1.023

4.643 ± 0.517

0.609 0.062

3.776 ± 0.249

26.39 ± 4.28

20.37 ± 3.26

19.97 ± 2.71

35.63 ± 5.11

30.91 ± 3.87

29.74 ± 3.02

36.37 ± 2.54

23.64 ± 0.96

33.77 ± 4.19

Methyl

n-Butyl

Methyl

n-Butyl

phenyl

Methyl

n-Butyl

phenyl

4-cyanophenyl

Methyl

n-Butyl

4-cyanophenyl

3-chloro-4-ﬂuorophenyl

Methyl

n-Butyl

CA-4

0.014 ± 0.003

0.010 ± 0.002

0.023 ± 0.005

0.019 ± 0.007

Cells were exposed to different concentrations of compounds for 48 h prior to determination of cell viability by the MTT assay. IC₅₀values are presented as mean

+/- standard deviation from three independent experiments.

manner. Moreover, compound 3b induced cell cycle arrest in G2/M

phase and led to cell apoptosis. Finally, docking studies demonstrated

that compound 3b ﬁtted nicely in the colchicine binding site of tubulin

and overlapped well with CA-4, which may explain the high activity of

3b. Collectively, these results suggested that compound 3b represents a

novel tubulin inhibitor deserving further investigation.

Y. Ren et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116376

Table 2

In vitro antiproliferative activities of series 2 and series 3 compounds against four cancer cell lines.^a

IC₅₀(μM)

Structure

Compound

R₁

R₂

B16-F10

HepG-2

Hela

MCF-7

4-methoxyphenyl

2.135 0.134

5.781 ± 0.338

6.451 ± 0.580

6.537 ± 2.312

1.394 0.075

4.680 ± 1.028

1.473 0.077

4.177 ± 0.512

3.440 ± 0.451

4.455 ± 0.085

1.810 0.036

3.546 ± 0.873

3.667 0.314

6.492 ± 0.934

8.651 ± 0.778

6.799 ± 0.647

2.697 0.184

5.304 ± 1.087

1.228 0.060

5.175 ± 1.213

7.113 ± 1.096

5.674 ± 1.610

2.027 0.249

4.442 ± 0.315

4-methoxyphenyl

Methyl

n-Butyl

4-methoxyphenyl

3,4,5-trimethoxyphenyl

10a

11a

Methyl

n-Butyl

4-methoxyphenyl

4.983 ± 0.371

8.047 ± 0.364

9.037 ± 0.841

6.974 ± 0.456

3.787 ± 0.218

4.663 ± 0.636

3.187 ± 0.574

7.236 ± 0.833

8.162 ± 0.624

5.231 ± 0.149

2.832 ± 0.387

3.503 ± 0.250

7.200 ± 0.946

8.954 ± 0.559

9.821 ± 0.394

8.073 ± 0.517

4.009 ± 0.105

6.079 ± 0.317

5.167 ± 0.670

7.773 ± 0.741

9.057 ± 0.418

6.602 ± 0.619

3.132 ± 0.373

4.864 ± 0.566

4-methoxyphenyl

Methyl

n-Butyl

4-methoxyphenyl

3,4,5-trimethoxyphenyl

10b

11b

Methyl

n-Butyl

4-methoxyphenyl

8.319 ± 0.956

1.384 ± 0.057

6.840 ± 1.063

7.218 ± 1.308

13.34 ± 5.91

14.65 ± 2.40

6.982 ± 0.384

0.939 ± 0.037

5.691 ± 0.627

6.823 ± 1.050

11.37 ± 2.48

15.44 ± 1.28

8.788 ± 1.351

2.671 ± 0.148

6.473 ± 0.889

8.647 ± 0.547

16.15 ± 4.81

17.32 ± 2.54

9.687 ± 0.846

4.422 ± 0.341

7.118 ± 0.708

9.271 ± 1.068

14.34 ± 3.54

16.39 ± 1.98

4-methoxyphenyl

Methyl

n-Butyl

4-methoxyphenyl

3,4,5-trimethoxyphenyl

10c

11c

Methyl

n-Butyl

4-methoxyphenyl

16.65 ± 0.87

15.37 ± 0.68

20.17 ± 1.79

18.95 ± 1.22

22.51 ± 0.87

18.37 ± 1.38

14.85 ± 1.56

15.48 ± 1.10

18.73 ± 1.46

11.38 ± 0.25

18.61 ± 2.13

15.61 ± 1.17

20.97 ± 3.54

18.65 ± 2.13

19.71 ± 1.32

16.07 ± 2.03

14.05 ± 0.95

16.83 ± 1.30

21.09 ± 1.71

13.27 ± 0.99

22.46 ± 1.80

13.27 ± 0.82

15.35 ± 2.01

17.34 ± 0.97

4-methoxyphenyl

Methyl

n-Butyl

4-methoxyphenyl

3,4,5-trimethoxyphenyl

10d

11d

Methyl

n-Butyl

4-methoxyphenyl

10.62 ± 1.03

4.058 ± 0.259

9.064 ± 0.841

9.997 ± 0.623

15.37 ± 1.03

19.34 ± 1.19

6.780 ± 0.543

1.396 ± 0.039

6.711 ± 0.648

8.183 ± 0.276

12.68 ± 1.48

16.77 ± 0.85

13.62 ± 1.24

6.121 ± 0.211

8.608 ± 0.470

10.60 ± 0.34

16.20 ± 1.08

16.98 ± 1.70

12.46 ± 0.88

5.322 ± 0.315

9.381 ± 0.813

11.49 ± 0.73

14.53 ± 2.04

20.18 ± 0.37

4-methoxyphenyl

Methyl

n-Butyl

4-methoxyphenyl

3,4,5-trimethoxyphenyl

10e

11e

Methyl

n-Butyl

CA-4

0.014 ± 0.003

0.010 ± 0.002

0.023 ± 0.005

0.019 ± 0.007

Cells were exposed to different concentrations of compounds for 48 h prior to determination of cell viability by the MTT assay. IC₅₀values are presented as mean

+/- standard deviation from three independent experiments.

Fig. 3. Growth inhibitory effects of compound 3b on

cancer cells. Four cancer cell lines (B16-F10, HepG-2,

Hela and MCF-7) were treated with: (A) the indicated

concentration of compound 3b for 48 h (left panel);

or (B) compound 3b (0.5 μM) for the indicated time

(right panel). Cell viability was evaluated by MTT

assay; (C) The number of colonies was counted after

treating HepG-2 cells with the indicated concentra-

tion of compound 3b for 24 h; (D) Histograms

display the colony-forming units after treatment with

compound 3b for 24 h. (n = 3, **P < 0.05 versus

DMSO control).

Y. Ren et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116376

Fig. 4. Inhibition of tubulin polymerization in vitro. (A) The inhibition of tubulin polymerization by compound 3b. (B) The inhibition of tubulin polymerization by

CA-4. (C) Compound 3b showed a concentration-dependent inhibition of tubulin polymerization. Data are expressed as mean +/- standard deviation from three

independent experiments.

Fig. 5. Effects of compound 3b on microtubules. HepG-2 cells were treated with vehicle control (0.1% DMSO) (A), or compound 3b: 0.1 μM (B); 0.5 μM (C); 1.0 μM

(D) for 12 h. Microtubules were visualized with an anti-β-tubulin antibody (green), and the cell nucleus was visualized with DAPI (blue). The detection of the ﬁxed

and stained cells was performed by LSM 880 laser confocal microscope (Carl Zeiss, Germany).

Fig. 6. Effects of compound 3b on HepG-2 cancer cell migration. (A) Representative images of wound area in a wound healing assay. Images were obtained at 0 h

and 24 h after HepG-2 cells being treated with 0, 0.1, 0.5 and 1.0

μM of compound 3b; (B) Histograms displayed the length of the scratches at 0 h and 24 h. (n = 3,

**P < 0.05 versus DMSO control).

Y. Ren et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116376

Fig. 7. Effects of compound 3b on the cell cycle of HepG-2 cells. (A) Compound 3b induced G2/M arrest in HepG-2 cells. Cells were exposed to increasing con-

centrations of compound 3b (0.5, 1.0 and 5.0 μM) for 48 h; (B) Histograms displayed the percentages of cell cycle distribution after treatment with compound 3b.

Data are expressed as mean +/- standard deviation from three independent experiments.

Fig. 8. Effects of compound 3b on HepG-2 cell apoptosis. (A) Compound 3b induced apoptosis in HepG-2 cells. Cells were incubated with varying concentrations of

compound 3b (0.5, 1.0 and 5.0 μM) for 48 h. The percentages of cells in each stage of cell apoptosis were quantiﬁed by ﬂow cytometry: necrosis cells (upper left

quadrant); late apoptotic cells (upper right quadrant); live cells (bottom left quadrant); and early apoptotic cells (bottom right quadrant); (B) Histograms displayed

the percentages of cell cycle distribution after treatment with compound 3b. Data are expressed as mean +/- standard deviation from three independent experiments.

Fig. 9. Molecular modeling. (A) Docking of compound 3b (yellow) into the colchicine binding site of tubulin (PDB code: 5lyj) and overlapping with CA-4 (green). (B)

A 90^◦rotation of (A). Oxygen and nitrogen atoms are colored red and blue, respectively.

Y. Ren et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116376

6. Experimental section

6.1.7. N-(3-chloro-4-ﬂuorophenyl)quinolin-5-amine (2e) C₁₅H₁₀ClFN₂

Yellow solid; yield: 42.3%. ¹H NMR (400 MHz, CDCl₃) δ 8.97–8.91

(m, 1H), 8.32 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.64 (t, J =

8.0 Hz, 1H), 7.39 (dd, J = 8.5, 4.2 Hz, 1H), 7.32 (d, J = 7.5 Hz, 1H), 7.03

(t, J = 8.7 Hz, 1H), 6.96 (dd, J = 6.1, 2.5 Hz, 1H), 6.78 (dt, J = 8.5, 3.1

Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 154.36, 151.95, 150.28, 148.86,

141.43, 139.01, 131.30, 130.15, 124.23, 121.70, 119.50, 117.37,

117.33, 117.11, 116.89. ESI-MS m/z 273.04, 275.04 (3:1) [M+H]⁺.

6.1. Chemistry

6.1.1. General methods

All chemicals and reagents were acquired from commercial suppliers

and used without further puriﬁcation. The control compound Com-

bretastatin A4 (CA-4) was acquired from InvivoChem (Libertyville, IL

60048, USA). Column chromatography were performed on commer-

cially available silica gel (200–300 mesh) under pressure. ¹H- and ¹³

6.1.8. General procedures of compounds 3a-e

NMR spectra were recorded on a Bruker Avance at 400 MHz, using

CDCl₃or DMSO‑d₆as solvent and tetramethylsilane (TMS) as internal

reference. Chemical shifts were reported in parts per million (ppm).

Multiplicities were reported as follows: singlet (s), doublet (d), triplet

(t), quartet (q), multiplet (m), broad (br), doublet of doublets (dd),

doublet of triplets (dt), doublet of quartets (dq). Mass spectrometry (MS)

was obtained on an APITF-5000 mass spectrometer with electron spray

ionization (ESI) as the ion source. The purity of target compounds were

estimated by reversed phase C18 HPLC. The major peak area of each

tested compound was ≥ 95% of the combined total peak area.

To a solution of 2a-e (1.0 equiv) in DMF cooled at 0 ^◦C was added

sodium hydride (2.0 equiv), followed by methyl iodide (2.0 equiv). The

mixture was stirred at 0 ^◦C for 1 h. Then the mixture was allowed to

warm to room temperature and stirred for another 2 h. Finally, the re-

action mixture was quenched with water. After extracted with ethyl

acetate, the combined organic layer was washed with brine, dried over

anhydrous Na₂SO₄and concentrated in vacuo to provide the crude

product, which was subjected to silica gel column chromatography

eluted with petroleum and ethyl acetate to give compounds 3a-e.

6.1.9. N-methyl-N-(3,4,5-trimethoxyphenyl)quinolin-5-amine (3a)

6.1.2. General procedures of compounds 2a-e

₁₉H₂₀N₂O₃

To a solution of 5-aminoquinoline (1) (100 mg, 0.694

mol) in 5 mL

Yellow solid; yield: 91.2%. ¹H NMR (400 MHz, CDCl₃) δ 8.93 (dd, J

dichloromethane was added arylboronic acid (0.832 mmol), pyridine

(2.08 mmol, 3.0 equiv) and Cu(OAc)₂(0.139 mmol, 0.2 equiv). The

mixture was stirred at room temperature for overnight. Then the solvent

was removed in vacuo. After that the residue was diluted by water, and

extracted with ethyl acetate. The combined organic layer was then

washed with brine, dried over anhydrous Na₂SO₄and concentrated in

vacuo to provide the crude product, which was subjected to silica gel

column chromatography eluted with petroleum and ethyl acetate to give

compounds 2a-e.

= 2.9, 0.8 Hz, 1H), 8.23 (d, J = 8.7 Hz, 1H), 8.04 (d, J = 9.0 Hz, 1H),

7.74 (t, J = 8.0 Hz, 1H), 7.42 (d, J = 7.3 Hz, 1H), 7.38–7.35 (m, 1H),

5.85 (s, 2H), 3.77 (s, 3H), 3.66 (s, 6H), 3.39 (s, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 162.30, 153.83, 147.28, 146.66, 141.74, 135.02, 131.61,

129.90, 121.86, 121.15, 118.79, 112.28, 93.08, 61.20, 56.23, 41.25.

ESI-MS m/z 325.15 [M+H]⁺.

6.1.10. N-(4-methoxyphenyl)-N-methylquinolin-5-amine (3b) C₁₇H₁₆N₂O

Yellow solid; yield: 88.6%. ¹H NMR (400 MHz, CDCl₃) δ 8.89 (d, J =

3.1 Hz, 1H), 8.25 (d, J = 8.5 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.71 (t, J

= 8.0 Hz, 1H), 7.35 (d, J = 7.4 Hz, 1H), 7.30 (dd, J = 8.5, 4.2 Hz, 1H),

6.76 (d, J = 9.0 Hz, 2H), 6.65 (d, J = 9.0 Hz, 2H), 3.74 (s, 3H), 3.37 (s,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 153.17, 150.51, 149.77, 146.71,

145.02, 132.94, 129.95, 126.83, 126.06, 123.13, 120.97, 117.49,

114.73, 55.79, 41.82. ESI-MS m/z 265.16 [M+H]⁺.

6.1.3. N-(3,4,5-trimethoxyphenyl)quinolin-5-amine (2a) C₁₈H₁₈N₂O₃

Yellow solid; yield: 46.7%. ¹H NMR (400 MHz, CDCl₃) δ 8.95–8.90

(m, 1H), 8.37 (d, J = 8.5 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.61 (t, J =

8.0 Hz, 1H), 7.35 (dd, J = 13.8, 6.1 Hz, 2H), 6.24 (s, 2H), 3.82 (s, 3H),

3.75 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 116.48, 112.97, 111.76,

102.92, 102.07, 93.40, 93.11, 92.26, 86.26, 85.01, 82.82, 78.10, 58.60,

23.57, 18.57. ESI-MS m/z 311.13 [M+H]⁺.

6.1.11. N-methyl-N-phenylquinolin-5-amine (3c) C₁₆H₁₄N₂

Yellow solid; yield: 89.2%. ¹H NMR (400 MHz, CDCl₃) δ 8.94–8.89

(m, 1H), 8.20 (d, J = 8.5 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.74 (t, J =

7.9 Hz, 1H), 7.43 (d, J = 7.4 Hz, 1H), 7.33 (dd, J = 8.5, 4.2 Hz, 1H), 7.17

(t, J = 7.8 Hz, 2H), 6.77 (t, J = 7.3 Hz, 1H), 6.62 (d, J = 8.4 Hz, 2H), 3.41

(s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 150.73, 150.17, 149.81, 145.66,

132.64, 130.09, 129.19, 127.97, 126.59, 125.28, 121.36, 118.21,

114.37, 40.76. ESI-MS m/z 235.11 [M+H]⁺.

6.1.4. N-(4-methoxyphenyl)quinolin-5-amine (2b) C₁₆H₁₄N₂O

Yellow solid; yield: 55.1%. ¹H NMR (400 MHz, CDCl₃) δ 8.91 (dd, J

= 4.1, 1.3 Hz, 1H), 8.35 (d, J = 8.5 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H),

7.55 (t, J = 8.0 Hz, 1H), 7.37 (dd, J = 8.5, 4.2 Hz, 1H), 7.12 (d, J = 7.6

Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 3.81 (s, 3H).

¹³C NMR (101 MHz, CDCl₃) δ 155.53, 150.20, 149.18, 141.40, 136.52,

130.40, 130.07, 122.14, 121.80, 121.24, 120.06, 114.95, 112.19, 55.69.

ESI-MS m/z 251.09 [M+H]⁺.

6.1.12. N-(4-cyanophenyl)-N-methylquinolin-5-amine (3d) C₁₇H₁₃N₃

Yellow solid; yield: 87.5%. ¹H NMR (400 MHz, CDCl₃) δ 8.97 (d, J =

2.9 Hz, 1H), 8.15 (d, J = 8.6 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.83–7.76

(m, 1H), 7.45 (d, J = 7.3 Hz, 1H), 7.43–7.37 (m, 3H), 6.54 (d, J = 8.7 Hz,

2H), 3.45 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 152.58, 151.18, 149.88,

143.14, 133.62, 131.70, 130.15, 129.66, 126.31, 126.03, 122.01,

120.28, 113.04, 99.69, 40.45. ESI-MS m/z 260.10 [M+H]⁺.

6.1.5. N-phenylquinolin-5-amine (2c) C₁₅H₁₂N₂

Yellow solid; yield: 45.8%. ¹H NMR (400 MHz, CDCl₃) δ 8.92 (dd, J

= 4.1, 1.5 Hz, 1H), 8.36 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H),

7.62 (t, J = 8.0 Hz, 1H), 7.42–7.34 (m, 2H), 7.30–7.25 (m, 2H), 6.95 (dd,

J = 11.8, 7.6 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 150.58, 149.46,

144.59, 139.22, 130.89, 129.82, 129.58, 124.39, 123.14, 121.15,

120.53, 117.68, 116.48. ESI-MS m/z 221.08 [M+H]⁺.

6.1.13. N-(3-chloro-4-ﬂuorophenyl)-N-methylquinolin-5-amine (3e)

₁₆H₁₂ClFN₂

6.1.6. N-(4-cyanophenyl)quinolin-5-amine (2d) C₁₆H₁₁N₃

Yellow solid; yield: 48.6%. ¹H NMR (400 MHz, CDCl₃) δ 8.97 (s, 1H),

8.28 (d, J = 8.5 Hz, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.72 (t, J = 8.0 Hz,

1H), 7.50 (d, J = 7.4 Hz, 1H), 7.46 (d, J = 8.7 Hz, 2H), 7.41 (dd, J = 8.5,

4.1 Hz, 1H), 6.79 (d, J = 8.7 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ

150.92, 149.79, 149.33, 136.05, 133.95, 131.31, 129.70, 127.61,

124.89, 121.95, 121.36, 119.90, 114.91, 101.72. ESI-MS m/z 246.09

[M+H]⁺.

Yellow solid; yield: 81.8%. ¹H NMR (400 MHz, CDCl₃) δ 8.98–8.92

(m, 1H), 8.17 (d, J = 8.5 Hz, 1H), 8.09 (d, J = 8.5 Hz, 1H), 7.76 (t, J =

8.0 Hz, 1H), 7.44–7.36 (m, 2H), 6.91 (t, J = 8.9 Hz, 1H), 6.65 (dd, J =

6.0, 3.0 Hz, 1H), 6.39 (dt, J = 8.9, 3.3 Hz, 1H), 3.37 (s, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 150.71, 149.50, 147.15, 145.02, 132.56, 130.31,

128.20, 126.30, 125.40, 121.63, 116.89, 116.68, 115.58, 113.82,

113.76, 41.18. ESI-MS m/z 287.06, 289.06 (3:1) [M+H]⁺.

Y. Ren et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116376

6.1.14. General procedures of compounds 4a-e

6.1.20. General procedures of compounds 6a-e

To a solution of 2a-e (1.0 equiv) in DMF cooled at 0 ^◦C was added

sodium hydride (2.0 equiv), followed by 1-iodobutane (2.0 equiv). The

mixture was stirred at 0 ^◦C for 1 h. Then the mixture was allowed to

warm to room temperature and stirred for another 2 h. Finally, the re-

action mixture was quenched with water. After extracted with ethyl

acetate, the combined organic layer was washed with brine, dried over

anhydrous Na₂SO₄and concentrated in vacuo to provide the crude

product, which was subjected to silica gel column chromatography

eluted with petroleum and ethyl acetate to give compounds 4a-e.

To a solution of 5a-e (1.0 equiv) and 4-methoxyaniline (1.2 equiv) in

anhydrous ethanol was added catalytic amount of HCl (conc.). The

mixture was stirred and reﬂuxed for 4 h. Then the precipitate was

collected by ﬁltration, washed with cold ethanol, Finally, the solid was

dried at 70 ^◦C to remove the residual ethanol, provided compound 6a-e.

6.1.21. N-(4-methoxyphenyl)acridin-9-amine (6a) C₂₀H₁₆N₂O

Orange solid; yield: 91.5%. ¹H NMR (400 MHz, DMSO) δ 8.22 (d, J =

8.7 Hz, 2H), 8.08 (d, J = 8.5 Hz, 2H), 7.91 (t, J = 7.6 Hz, 2H), 7.35 (t, J

= 8.0 Hz, 4H), 7.05 (d, J = 8.9 Hz, 2H), 3.81 (s, 3H). ¹³C NMR (101 MHz,

DMSO) δ 158.28, 155.01, 140.14, 134.79, 125.98, 125.75, 123.31,

119.05, 115.10, 113.46, 55.46. ESI-MS m/z 301.11 [M+H]⁺.

6.1.15. N-butyl-N-(3,4,5-trimethoxyphenyl)quinolin-5-amine (4a)

₂₂H₂₆N₂O₃

Yellow solid; yield: 86.9%. ¹H NMR (400 MHz, CDCl₃) δ 8.91 (dd, J

= 4.1, 1.4 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H),

7.75 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 7.3 Hz, 1H), 7.34 (dd, J = 8.5, 4.2

Hz, 1H), 5.78 (s, 2H), 3.78–3.70 (m, 5H), 3.63 (s, 6H), 1.72–1.66 (m,

2H), 1.38–1.33 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 153.84, 150.72, 149.87, 146.43, 143.98, 132.81, 130.74,

129.91, 128.07, 126.97, 126.40, 121.33, 92.77, 61.20, 56.11, 53.30,

30.30, 20.47, 14.04. ESI-MS m/z 367.20 [M+H]⁺.

6.1.22. 6-chloro-2-methoxy-N-(4-methoxyphenyl)acridin-9-amine (6b)

₂₁H₁₇ClN₂O₂

Orange solid; yield: 94.4%. ¹H NMR (400 MHz, DMSO) δ 8.00 (dd, J

= 16.1, 9.7 Hz, 3H), 7.71 (d, J = 8.6 Hz, 2H), 7.41 (t, J = 9.0 Hz, 3H),

7.11 (d, J = 8.8 Hz, 2H), 3.82 (s, 3H), 3.73 (s, 3H). ¹³C NMR (101 MHz,

DMSO) δ 158.45, 155.60, 153.52, 139.86, 138.74, 135.65, 133.23,

128.12, 127.79, 126.41, 123.78, 120.96, 117.83, 115.16, 114.75,

111.37, 103.56, 55.75, 55.53. ESI-MS m/z 365.07, 367.09 (3:1)

[M+H]⁺.

6.1.16. N-butyl-N-(4-methoxyphenyl)quinolin-5-amine (4b) C₂₀H₂₂N₂O

Yellow solid; yield: 91.2%. ¹H NMR (400 MHz, CDCl₃) δ 8.88 (d, J =

4.0 Hz, 1H), 8.21 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H), 7.72 (t, J

= 7.9 Hz, 1H), 7.39 (d, J = 7.4 Hz, 1H), 7.30–7.26 (m, 1H), 6.73 (d, J =

9.0 Hz, 2H), 6.57 (d, J = 9.0 Hz, 2H), 3.75–3.68 (m, 5H), 1.69–1.64 (m,

2H), 1.36 (d, J = 7.9 Hz, 2H), 0.89 (d, J = 7.4 Hz, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 152.69, 150.44, 149.93, 144.98, 144.41, 133.08, 129.91,

127.07, 126.82, 124.90, 121.01, 117.11, 114.78, 55.81, 53.76, 30.33,

20.52, 14.06. ESI-MS m/z 307.16 [M+H]⁺.

6.1.23. 7-chloro-N-(4-methoxyphenyl)quinolin-4-amine (6c)

₁₆H₁₃ClN₂O

Yellow solid; yield: 87.7%. ¹H NMR (400 MHz, DMSO) δ 8.84 (d, J =

9.1 Hz, 1H), 8.47 (d, J = 7.0 Hz, 1H), 8.14 (d, J = 1.5 Hz, 1H), 7.84 (d, J

= 9.0 Hz, 1H), 7.39 (d, J = 8.8 Hz, 2H), 7.12 (d, J = 8.8 Hz, 2H), 6.65 (d,

J = 7.0 Hz, 1H), 3.82 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 158.44,

155.25, 142.99, 139.00, 138.21, 129.42, 127.09, 126.22, 119.05,

115.71, 115.09, 99.94, 55.45. ESI-MS m/z 285.03, 287.04 (3:1)

[M+H]⁺.

6.1.17. N-butyl-N-phenylquinolin-5-amine (4c) C₁₉H₂₀N₂

Yellow solid; yield: 81.3%. ¹H NMR (400 MHz, CDCl₃) δ 8.91 (d, J =

4.0 Hz, 1H), 8.16 (d, J = 8.5 Hz, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.76 (t, J

= 7.9 Hz, 1H), 7.45 (d, J = 7.4 Hz, 1H), 7.31 (dd, J = 8.5, 4.2 Hz, 1H),

7.13 (t, J = 7.9 Hz, 2H), 6.72 (t, J = 7.2 Hz, 1H), 6.55 (d, J = 8.3 Hz, 2H),

3.79–3.71 (m, 2H), 1.70 (dd, J = 15.4, 7.8 Hz, 2H), 1.38–1.32 (m, 2H),

0.91 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 150.67, 149.97,

149.55, 143.97, 132.80, 130.06, 129.25, 128.17, 127.09, 126.78,

121.37, 117.61, 113.99, 52.98, 30.16, 20.44, 14.04. ESI-MS m/z 277.16

[M+H]⁺.

6.1.24. N-(4-methoxyphenyl)-2-methylpyridin-4-amine (6d) C₁₃H₁₄N₂O

White solid; yield: 84.0%. ¹H NMR (400 MHz, DMSO) δ 8.09 (d, J =

7.0 Hz, 1H), 7.24 (d, J = 8.8 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H), 6.89 (s,

1H), 6.80 (s, 1H), 3.78 (s, 3H), 2.45 (s, 3H). ¹³C NMR (101 MHz, DMSO)

δ 157.60, 156.76, 151.33, 139.68, 129.82, 125.47, 115.00, 106.79,

99.52, 55.39, 18.81. ESI-MS m/z 215.07 [M+H]⁺.

6.1.25. N-(4-methoxyphenyl)-2-methylpyrimidin-4-amine (6e)

₁₂H₁₃N₃O

6.1.18. N-butyl-N-(4-cyanophenyl)quinolin-5-amine (4d) C₂₀H₁₉N₃

Yellow solid; yield: 87.4%. ¹H NMR (400 MHz, CDCl₃) δ 8.95 (d, J =

3.5 Hz, 1H), 8.15 (d, J = 8.5 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.80 (d, J

= 7.8 Hz, 1H), 7.44 (d, J = 7.3 Hz, 1H), 7.40–7.32 (m, 3H), 6.47 (d, J =

8.7 Hz, 2H), 3.74 (s, 2H), 1.71 (dt, J = 15.7, 7.8 Hz, 2H), 1.35 (dd, J =

15.0, 7.4 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

152.01, 151.11, 149.99, 141.66, 133.66, 131.79, 130.06, 129.73,

127.34, 126.29, 121.97, 120.34, 112.81, 99.10, 52.81, 29.70, 20.27,

13.93. ESI-MS m/z 302.15 [M+H]⁺.

White solid; yield: 86.7%. ¹H NMR (400 MHz, DMSO) δ 8.21 (d, J =

7.2 Hz, 1H), 7.57 (s, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 41.3 Hz,

1H), 3.77 (s, 3H), 2.55 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 162.11,

156.97, 142.51, 130.19, 123.49, 114.20, 105.01, 55.32, 21.73. ESI-MS

m/z 216.07 [M+H]⁺.

6.1.26. General procedures of compounds 7a-e

To a solution of 6a-e (1.0 equiv) in DMF cooled at 0 ^◦C was added

sodium hydride (2.0 equiv), followed by methyl iodide (2.0 equiv). The

mixture was stirred at 0 ^◦C for 1 h. Then the mixture was allowed to

warm to room temperature and stirred for another 2 h. Finally, the re-

action mixture was quenched with water. After extracted with ethyl

acetate, the combined organic layer was washed with brine, dried over

anhydrous Na₂SO₄and concentrated in vacuo to provide the crude

product, which was subjected to silica gel column chromatography

eluted with petroleum and ethyl acetate to give compounds 7a-e.

6.1.19. N-butyl-N-(3-chloro-4-ﬂuorophenyl)quinolin-5-amine (4e)

₁₉H₁₈ClFN₂

Yellow solid; yield: 89.6%. ¹H NMR (400 MHz, CDCl₃) δ 8.96–8.91

(m, 1H), 8.11 (t, J = 7.6 Hz, 2H), 7.77 (t, J = 8.0 Hz, 1H), 7.43 (d, J =

7.3 Hz, 1H), 7.36 (dd, J = 8.5, 4.2 Hz, 1H), 6.87 (t, J = 8.9 Hz, 1H), 6.55

(dd, J = 6.1, 3.0 Hz, 1H), 6.31 (dt, J = 9.0, 3.4 Hz, 1H), 3.73–3.66 (m,

2H), 1.67 (dd, J = 15.5, 7.5 Hz, 2H), 1.33 (t, J = 7.5 Hz, 2H), 0.91 (t, J =

7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 152.34, 150.73, 149.81,

146.57, 143.26, 132.53, 130.16, 128.51, 126.80, 121.63, 116.91,

116.69, 115.19, 113.39, 113.33, 53.34, 29.99, 20.39, 13.99. ESI-MS m/z

329.10, 331.11 (3:1) [M+H]⁺.

6.1.27. N-(4-methoxyphenyl)-N-methylacridin-9-amine (7a) C₂₁H₁₈N₂O

Orange solid; yield: 91.9%. ¹H NMR (400 MHz, CDCl₃) δ 8.30 (d, J =

8.8 Hz, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.80–7.73 (m, 1H), 7.48–7.41 (m,

1H), 6.79 (t, J = 7.2 Hz, 2H), 6.80–6.73 (m, 2H), 6.64–6.58 (m, 2H),

6.54–6.49 (m, 2H), 3.74 (d, J = 5.8 Hz, 3H), 2.80 (s, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 155.87, 152.30, 152.20, 150.86, 150.71, 144.05, 143.82,

Y. Ren et al.

Bioorganic & Medicinal Chemistry 46 (2021) 116376

135.21, 130.36, 130.32, 126.15, 125.17, 124.54, 115.03, 113.98,

1.92–1.82 (m, 2H), 1.55 (dq, J = 14.6, 7.3 Hz, 2H), 1.08 (t, J = 7.3 Hz,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 158.55, 152.10, 147.18, 146.83,

143.02, 138.51, 135.53, 131.56, 128.35, 128.31, 126.62, 125.76,

125.41, 122.96, 115.05, 114.17, 99.96, 55.98, 55.87, 50.54, 31.94,

20.46, 14.06. ESI-MS m/z 421.17, 423.17 (3:1) [M+H]⁺.

113.76, 96.98, 55.97, 40.40. ESI-MS m/z 315.23 [M+H]⁺.

6.1.28. 6-chloro-2-methoxy-N-(4-methoxyphenyl)-N-methylacridin-9-

amine (7b) C₂₂H₁₉ClN₂O₂

Orange solid; yield: 89.8%. ¹H NMR (400 MHz, CDCl₃) δ 8.24 (d, J =

1.4 Hz, 1H), 8.13 (d, J = 9.4 Hz, 1H), 7.82 (d, J = 9.2 Hz, 1H), 7.45 (dd,

J = 9.4, 2.6 Hz, 1H), 7.34 (dd, J = 9.2, 1.6 Hz, 1H), 7.04 (d, J = 2.6 Hz,

1H), 6.77 (d, J = 8.7 Hz, 2H), 6.53 (d, J = 8.7 Hz, 2H), 3.78 (s, 3H), 3.73

(s, 3H), 3.51 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 157.87, 152.48,

149.07, 148.76, 148.53, 143.68, 135.08, 131.91, 128.80, 127.49,

126.08, 126.04, 125.58, 123.65, 115.09, 114.19, 99.65, 55.82, 55.63,

39.81. ESI-MS m/z 379.21, 381.20 (3:1) [M+H]⁺.

6.1.35. N-butyl-7-chloro-N-(4-methoxyphenyl)quinolin-4-amine (8c)

₂₀H₂₁ClN₂O

Yellow solid; yield: 83.2%. ¹H NMR (400 MHz, CDCl₃) δ 8.70 (d, J =

5.0 Hz, 1H), 7.97 (s, 1H), 7.49 (d, J = 9.1 Hz, 1H), 7.09 (d, J = 9.1 Hz,

1H), 6.96 (d, J = 5.0 Hz, 1H), 6.88 (dd, J = 9.0, 2.3 Hz, 2H), 6.79 (dd, J

= 9.0, 2.3 Hz, 2H), 3.84–3.78 (m, 2H), 3.78 (d, J = 3.6 Hz, 3H),

1.73–1.63 (m, 2H), 1.41 (dt, J = 14.5, 7.4 Hz, 2H), 0.93 (t, J = 7.3 Hz,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 156.08, 152.89, 151.62, 150.76,

142.95, 134.69, 128.57, 126.93, 125.65, 124.49, 122.15, 115.00,

111.04, 55.61, 55.06, 29.39, 20.54, 14.02. ESI-MS m/z 341.21, 343.18

(3:1) [M+H]⁺.

6.1.29. 7-chloro-N-(4-methoxyphenyl)-N-methylquinolin-4-amine (7c)

₁₇H₁₅ClN₂O

Yellow solid; yield: 88.0%. ¹H NMR (400 MHz, CDCl₃) δ 8.73 (d, J =

5.1 Hz, 1H), 8.00 (d, J = 2.0 Hz, 1H), 7.49 (d, J = 9.1 Hz, 1H), 7.12 (dd,

J = 9.1, 2.1 Hz, 1H), 6.97–6.91 (m, 3H), 6.84–6.79 (m, 2H), 3.78 (s, 3H),

3.43 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 156.36, 153.93, 151.86,

150.48, 143.90, 134.75, 128.62, 126.88, 125.74, 124.26, 121.57,

115.09, 110.27, 55.64, 43.49. ESI-MS m/z 299.14, 301.15 (3:1)

[M+H]⁺.

6.1.36. N-butyl-N-(4-methoxyphenyl)-2-methylpyridin-4-amine (8d)

₁₇H₂₂N₂O

White solid; yield: 68.1%. ¹H NMR (400 MHz, CDCl₃) δ 8.00 (d, J =

6.6 Hz, 1H), 7.08 (d, J = 8.9 Hz, 2H), 6.99 (d, J = 8.9 Hz, 2H), 6.39 (dd,

J = 6.6, 2.3 Hz, 1H), 6.27 (s, 1H), 3.85 (s, 3H), 3.71–3.58 (m, 2H), 2.50

(s, 3H), 1.63 (dd, J = 15.4, 7.9 Hz, 2H), 1.39–1.33 (m, 2H), 0.93 (t, J =

7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.12, 156.31, 154.47,

143.84, 136.18, 128.97, 115.81, 107.11, 105.78, 55.70, 52.60, 29.13,

22.13, 20.17, 13.91. ESI-MS m/z 271.24 [M+H]⁺.

6.1.30. N-(4-methoxyphenyl)-N,2-dimethylpyridin-4-amine (7d)

₁₄H₁₆N₂O

White solid; yield: 27.6%. ¹H NMR (400 MHz, CDCl₃) δ 7.95 (d, J =

5.9 Hz, 1H), 7.06 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 6.42 (d, J

= 5.3 Hz, 1H), 6.35 (s, 1H), 3.79 (s, 3H), 3.33 (s, 3H), 2.50 (s, 3H). ¹³

6.1.37. N-butyl-N-(4-methoxyphenyl)-2-methylpyrimidin-4-amine (8e)

NMR (101 MHz, CDCl₃) δ 159.05, 157.06, 153.37, 142.02, 136.97,

127.82, 115.72, 107.09, 105.92, 55.61, 40.51, 21.11. ESI-MS m/z

229.16 [M+H]⁺.

₁₆H₂₁N₃O

White solid; yield: 72.4%. ¹H NMR (400 MHz, CDCl₃) δ 7.87 (d, J =

6.2 Hz, 1H), 7.08 (d, J = 8.9 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 5.84 (d, J

= 6.2 Hz, 1H), 3.96–3.87 (m, 2H), 3.82 (s, 3H), 2.51 (s, 3H), 1.60–1.49

6.1.31. N-(4-methoxyphenyl)-N,2-dimethylpyrimidin-4-amine (7e)

(m, 2H), 1.33 (dt, J = 14.8, 7.4 Hz, 2H), 0.90 (t, J = 7.3 Hz, 3H). ¹³

₁₃H₁₅N₃O

NMR (101 MHz, CDCl₃) δ 167.21, 162.49, 158.68, 154.47, 135.77,

129.51, 115.32, 101.78, 55.56, 49.10, 29.86, 26.28, 20.07, 13.96. ESI-

MS m/z 272.22 [M+H]⁺.

White solid; yield: 19.8%. ¹H NMR (400 MHz, CDCl₃) δ 7.92 (d, J =

6.1 Hz, 1H), 7.14 (d, J = 8.9 Hz, 2H), 6.97 (d, J = 8.9 Hz, 2H), 6.03 (d, J

= 6.3 Hz, 1H), 3.84 (s, 3H), 3.45 (s, 3H), 2.58 (s, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 167.45, 162.65, 158.60, 154.55, 137.40, 128.43, 115.37,

101.95, 55.65, 37.89, 26.35. ESI-MS m/z 230.16 [M+H]⁺.

6.1.38. General procedures of compounds 9a-e

To a solution of 5a-e (1.0 equiv) and 3,4,5-trimethoxyaniline (1.2

equiv) in anhydrous ethanol was added catalytic amount of HCl (conc.).

The mixture was stirred and reﬂuxed for 4 h. Then the precipitate was

collected by ﬁltration, washed with cold ethanol, and the solid was dried

at 70 ^◦C to provide compound 9a-e.

6.1.32. General procedures of compounds 8a-e