Discovery of 4-(3,5-dimethoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline as a novel c-myc inhibitor against colorectal cancer in vitro and in vivo

Discovery

-(3,5-dimethoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline as

of

a

4

novel c-Myc inhibitor against colorectal cancer in vitro and in vivo

Graphical Abstract:

The 4-(3,5-dimethoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline (42) as

a novel potent c-Myc inhibitor was identified. The results demonstrated that 42 showed good

binding affinity to c-Myc/MAX dimerization and DNA binding, downregulation of c-Myc

expression level, and exhibited excellent antitumor activity in vitro and vivo with low toxicity.

Discovery

-(3,5-dimethoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline as

novel c-Myc inhibitor against colorectal cancer in vitro and in vivo

of

a

4

a, 1

a

b

a

Jie Yang , Caoguo Cao , Dan Luo , Suke Lan , Meng luo , Huifang Shan , Xinyu Ma ,

a

a, *

Yuanyuan Liu , Su Yu , Xinxin Zhong , Rui Li

a

State Key Laboratory of Biotherapy, Collaborative Innovation Center of Biotherapy and Cancer Center, West

China Hospital of Sichuan University, Chengdu, 610041, China

b

College of Chemistry & Environment Protection Engineering, Southwest Minzu University, Chengdu, 610041,

China

E-mail address: lirui@scu.edu.cn

*

Corresponding author

1

These two authors contribute equally to this work.

Abstract： Proto-oncogene c-Myc plays an essential role in the development of colorectal cancer

(CRC), since downregulation of c-Myc inhibits intestinal polyposis, which is the most cardinal

pathological change in the development of CRC. Herein, series of novel

a

phenoxy-N-phenylaniline derivatives were designed and synthesized. The cytotoxicity activities of

all the derivatives were measured by MTT assay in different colon cancer cells,

4

-(3,5-dimethoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline (42) was

discovered, the lead compound 42 with excellent cytotoxicity activity of IC = 0.32 μM, IC =

5

0

50

0

.51 μM , in HT29 and HCT 15 cells, respectively. Compound 42 had a good inhibitory activity of

c-Myc/MAX dimerization and DNA binding. Besides, compound 42 could effectively induce

apoptosis and induced G2/M arrest in low concentration and G0/G1 arrest in high concentration to

prevent the proliferation and differentiation in colon cancer cells. Western blot analysis confirmed

the 42 strongly down-regulated expression of c-Myc. Furthermore, during 30 days treatment 42

exhibited excellent efficacy in HT29 tumor xenograft model without causing significant weight

loss and toxicity. Consequently, 42 could be a promising drug candidate for CRC therapy.

Key words: colorectal cancer, c-Myc inhibitor, anti-CRC

1

. INRODUCTION

Globally, colorectal cancer (CRC) is the second leading cause of cancer death, and the second

most common cancer in women and the third most common in men. According to Globocan[1],

the global prevalence of colorectal cancer in 2018 was estimated at over 6.3 million, with 1.8

million newly diagnosed and caused 880 792 deaths. The World Economic Forum estimated the

total costs associated with colorectal cancer in 2010 to be approximately USD 31.6 billion, CRC

resulted in a significant economic burden on patients and society[2-4]. Even though an increasing

number of potentially curative treatments including chemotherapy agents, surgery, radiotherapy

and molecular targeting therapies have been developed for CRC, the clinical prognosis still remain

unsatisfactory, especially as CRC patients with metastases[5-8]. Furthermore, tumor heterogeneity

leads to a significant consequence of resistant responses to chemotherapies and targeted therapies.

The current first-line drugs for colorectal cancer patients, such as irinotecan, 5-Fluorouracil and its

derived prodrugs, platinum drugs, etc[7, 8]. However, all of them have poor clinical efficacy, poor

patient prognosis and serious drug resistance. Therefore, seeking for a new and effective drug is an

enormous challenge for the therapy of CRC in the future.

C-Myc is the member of the Myc proto-oncogene family, a frequent genetic abnormality seen in

CRC is the elevated expression of c-Myc, and the importance of c-Myc expression in CRC has

been demonstrated in both studies of transgenic mice and clinical research, including tumor

growth, replication, metabolism, differentiation, and apoptosis[9-14]. Downregulation of c-Myc

can inhibit intestinal polyposis, which is the most important lesion in the process of colon

cancer[10, 15, 16]. Thus, c-Myc inhibitors could help to suppress the initiation and progression of

tumors. Recently, lots of c-Myc inhibitors were discovered including Myc-Max protein-protein

interactions[17-19], such as 10058-F4, 10074-G5, SF-4-017, NUCC-0176242, or Myc-Max

protein-DNA binding interactions[20, 21], such as Mycro 3, KJ Pyr 9, or binding to c-Myc

G-quadruplex(G4) stabilizers[22, 23], such as Indenoisoquinoline 5, IZCZ-3, etc. Despite plenty

of c-Myc inhibitors (shown in Figure 1) reported, there is no anti-Myc drug clinically available.

Moreover, many of the known c-Myc inhibitors are not suitable for clinical studies because of low

potency or poor antitumor activity in vivo[24, 25]. Thus, discovery of more novel c-Myc inhibitors,

particularly with novel scaffolds is still necessary at present.

The c-Myc protein is the member of intrinsically disordered (ID) proteins[26, 27], which is

characterized by a lack of stable structure and extensive backbone flexibility[28-30]. It is a

relevant yet challenging target for drug discovery[31-33]. In an effort to seek novel c-Myc

inhibitors, c-Myc–Max interactions inhibitors are most valuable for clinical research[34-37]. We

initially studied the core structures of these small molecular inhibitors, and found most of these

inhibitors contained a long molecular structure, because of the c-Myc-Max dimer interface was a

left-handed, parallel and four-helix bundle, with each monomer consisted of two R-helices

separated by an annulus[28, 38].

To identify novel inhibitors, biphenyl group[39-41] and diphenylamine group[42-46] were

found in a great many of antitumor drugs with good biological activities, we designed by

incorporation of the core structure a long molecular composed of three benzene rings. Therefore,

we synthesized a series of fused 3(or4)-(phenoxy)-N-phenylaniline derivatives. In this study, we

discovered a novel c-Myc inhibitor compound 42 that potently decreased the expression of c-Myc

protein verified by western blot. In vitro antiproliferative activity showed that compound 42

showed significant cytotoxicity in several colon cancer cells, especially in HT29 cells (IC = 0.32

5

0

μM) and HCT15 cells (IC = 0.51 μM). The lead compound 42 can induce apoptosis for HT29

5

0

and HCT15 cell lines and significantly induce G0/G1 arrest in high concentration and G2/M in

low concentration. In vivo HT29 xenograft model study showed that compound 42 could

significantly inhibit tumor growth without obvious toxicity.

On the basis of these findings, in vivo administration of compound 42 as c-Myc inhibitor for the

treatment of colorectal cancer resulted in good anti-tumor activity and low toxicity. Thus,

compound 42 represents a new class of c-Myc inhibitors as potential clinical agents against CRC.

2

. RESULTS AND DISCUSSION

2

.1. Design and synthesis of N-phenylaniline derivatives

With considering the good anti-tumor activities of these c-Myc/MAX inhibitors, we studied the

core group of these inhibitors. Regarding the specificity of c-Myc/MAX protein binding[28, 34,

7], we designed and synthesized N-phenylaniline derivatives with skeletal transition based on the

3

original small molecule c-Myc/MAX inhibitors. A great number of studies on the structural

modifications based on the N-phenylaniline, and the incorporation with the N-phenylaniline may

enhance biological activities significantly[47-51]. However, there were few studies on small

molecular inhibitors of c-Myc. In order to design N-phenylaniline derivatives with a better

biological activity and novelty, we designed and synthesized 3(or4)-(phenoxy)-N-phenylaniline

derivatives shown in Scheme1.

The chemistry efforts to obtain 42 and a series of analogues are outlined in Schemes 2-3[52-55].

First, we synthesized 4-fluoro-2,6-dimethoxybenzaldehyde(3) by Vilsmeier-Haack reaction[52]

with commercial compound 2. Then, 3-(phenylamino)phenol(5) was prepared by 3-aminophenol

and bromobenzene via Buchwald–Hartwig coupling reaction[53]. Subsequently, in the

construction of the key intermediates 6a-6d, we screened out a mild reaction condition, compound

5

, K CO and 4-Fluorobenzaldehyde with different substituents under argon atmosphere in

2 3

o

anhydrous DMSO was stirred at 100 C for 3 h, the yields of desired compound were 70%-80%.

Finally, these intermediates were stirred with different amines in the presence of sodium

o

triacetoxyborohydride, under argon atmosphere in anhydrous DMF at 80 C, to afford final

compounds 7-38 with 45%-60% yields, as indicated in Scheme 1. Analogously, compounds 42-45

were synthesized following the synthetic route depicted in Scheme 2.

2

.2. In vitro cell viability

In order to assess the cytotoxicity of these final products, the cytotoxicity on HCT116, HCT15,

HT29, DLD-1, SW620 were tested by MTT[56-58] and the results achieved are shown in Table 1.

Cisplatin and 5-Fluorouracil were effective drugs used in the clinical treatment of CRC[7, 8], so

we chose them as positive drugs in the MTT test.

Firstly, we studied compounds 7-16 with R5 group bearing different aliphatic alkylamine

substituents. R5 appendage carrying hydrophobic groups showed anti-proliferation activity against

HT29, HCT15, DLD-1 cancer cell lines. Furthermore, we found that compound 22 showed an

outstanding antiproliferative activity in five CRC lines. To further investigate the activity

relationship (SAR) of R5 with extended chain length, the R5 groups were replaced by various

substituents of 4-phenylethylamine groups. Among these molecules (27-32), the compound 32

exhibited excellent activities in HT29, HCT15, HCT116, DLD-1 cell lines with IC₅₀values of

1

.02 μM, 1.25 μM, 0.98 μM, 1.12 μM, respectively. Next, we discussed the effects of the

R5-phenyl with methoxyl at C2-position and C3-position. As a result, the activities of the

compounds were not increased significantly. Even though, we altered the linker length of R5

group and the pattern substitution of methoxyl groups in Region � , but no more active

compounds were found.

Intriguingly, we changed the relative position of the aniline moiety relative to the oxygen that

went from relative position 1,3 to 1,4 within Region � , and discussed the position of methoxyl

substitution in Region � , compound 42 showed an extraordinary anti-tumor activity against HT29

with IC₅₀value of 0.32 μM (As shown in Figure 2A), 3 fold than that of 32 and 31 fold than

positive drugs(Cisplatin: IC = 10.75 μM, 5-Fluorouracil: IC = 13.37 μM).

5

0

50

Moreover, we analysed the toxicity of compound 42 against the human non-tumor liver cell line

LO2) by MTT assay. The results shown in Figure 2B, revealed that the cytotoxicity of compound

(

4

2 was significantly lower than that of positive drugs Cisplatin and 5-Fluorouracil, thus proving a

good in vitro safety for compound 42.

2

.3 Inhibition of compounds with c-Myc/MAX dimerization and DNA complex

To identified interaction of synthetic compounds and c-Myc/MAX dimerization was determined

by Enzyme-Linked Immunosorbent Assay (ELISA)[37, 59]. The c-Myc/MAX inhibitor

sAJM589[60] was used as positive control in ELISA test. As shown in Table 2, molecules with R5

group bearing aliphatic alkylamine analogues caused poor inhibition of c-Myc/MAX dimerization,

suggesting that the substitutions of R5 group were worthy of further optimization. Subsequent

efforts were focused on the substitutions of aromatic analogues of R5 group. Compared with

compound 32 substituted with a 2-(4-methoxyphenyl)ethan-1-amine of R5 group showed a

dramatic increase inhibitory activity of c-Myc/MAX dimerization. The reason may be that when

2

-(4-methoxyphenyl)ethan-1-amine introduced into the molecule, which enhanced hydrophobic

interactions of c-Myc/MAX dimerization. Next, we continued to modify the relative position 1,3

to 1,4 within Region � , compound 42 showed increased inhibitory potency when compared to

that of compound 32. Among all the molecules, compound 42 showed significant inhibitory

activity of c-Myc/MAX dimerization with an IC₅₀value of 63 μM, and positive agent sAJM589

with an IC value of 36 μM.

5

0

In addition, compounds interfered with c-Myc/Max/DNA binding behavior was identified by

Electrophoretic Mobility-Shift Assay (EMSA)[37, 56, 61, 62]. As shown in Table 3, the different

binding inhibition rates were displayed, DMSO as a vehicle control, and among these molecules,

compound 42 was demonstrated excellent potent disruption activity of the c-Myc/Max/DNA

complex. In Figrue S1(Supporting Information, Figure S1), the data showed compound 42 could

interfered with c-Myc/Max binding DNA in a concentration-dependent manner, when the

concentration of 42 was increased to 40 μM, almost complete inhibition of c-Myc/Max/DNA

binding.

2

.4 Molecular docking study

To further investigate the possible binding mode of 42 with c-Myc/Max, the protein crystal

structure of Myc/Max (PDB ID:1NKP)[28] was used in this study. As illustrated in Figure 3, the

predicted binding mode and the detailed interaction of compound 42 with Myc/Max were

elucidated. Compound 42 could tightly bind to the basic/helix-loop-helix/leucine zipper (bHLHZ)

domains of Myc-Max. It was clearly seen that NH in N-phenylaniline (Region � ) segment and

Ala237 formed hydrogen bond, and N-phenylaniline interacted with ILe242, arg226, His223 via

hydrophobic features, respectively. Besides, two methoxy groups of Region � segment interacted

with Arg913 by hydrogen bond. More importantly, NH in Region � segment and Arg913 formed

an additional hydrogen bond. Furthermore, the phenyl group in Region � among Lys939, Lys918,

Arg914 formed hydrophobic interaction, respectively. Additionally, the whole molecule located in

Myc-Max dimer interface and protein-DNA interface, which indicated that the binding stability of

the molecule and Myc-Max proteins that could disrupt c-Myc/Max/DNA complex formation. All

characteristics of molecular binding mode could prefigure the significant cytotoxicity activity of

compound 42.

2

.5. Colony formation assay of compound 42

Plate clone formation test[61] of compound 42 was indicated in Figure 4, HT29 cells and

o

HCT15 cells were treated with compound 42, then incubated overnight at 37 C for 15days. The

colony formation rate of HCT15 cells and HT29 cells decreased dramatically in the treatment

groups. It can be appreciated in Figure 4, that when the concentration of 42 was increased to 1.25

μM, almost no colony formation was observed in HT29 cells. Similarity, when the concentration

of 42 reached 2.5 μM in HCT15 cells, almost complete inhibition of colony formation was

observed. As was expected, the results of plate colony formation corresponded with the results of

MTT assay.

2

.6. Flow cytometry of compound 42.

To investigate the effect of the cell cycle distribution and the cell apoptosis rate by compound

2 HT29 and HCT15 cells were treated at different concentrations of 42 for 24 h[61]. As

4

displayed in Figure 5A, cell cycle progression was significantly arrested at G0/G1 phase both in

HT29 and HCT15 cells, and the differentiation of the cell cycle distribution was evidently in a

dose-dependent manner with compound 42. The induction of cell cycle arrest inhibited cell

proliferation that reflected their capabilities to disrupt binding to the c-Myc/Max/DNA complex.

As represented in Figure 5B, 42 was found to significantly affect HT29 and HCT15 cells

apoptosis in a dose-dependent manner with different concentration. Compound 42, at a

concentration of 10 μM, causes apoptosis of HT29 and HCT15 cells after 24 h, demonstrating that

the inhibition ofc-Myc in proliferating cells was splendid.

2

.7. Western blot assay of compound 42

The ability of 42 to inhibit c-Myc expression was detected by western blot [62]. The results

showed that 42 decreased the expression level of c-Myc protein both in HT29 cells and HCT15

cells. As shown in Figure 6, it was clearly seen that downregulation of c-Myc protein was

achieved in a dose-dependent manner with compound 42. These results suggest that compound 42

might target the c-Myc/Max in HT29 cells and HCT15 cells, thus presumably down-regulating its

expression.

2

.8. In vivo effects of 42 on HT29 tumor xenograft model

The in vitro promising results achieved with compound 42 prompted us to study the in vivo[61,

6

3] activity of this compound. Female BALB/c nude mice (6-7 weeks old) bearing HT29

xenograft tumors were treated with 42 by oral administration for 30 days. As illustrated in Figure

, compound 42 remarkably inhibited tumor growth in a dose-dependent manner. Tumor growth

7

inhibitions (TGIs) of 71.58%, 61.21%, 55.17% were observed in the HT29 xenograft model at

dose of 120mg/kg, 80mg/kg, 40mg/kg, respectively. 5-Fluorouracil was used as positive control at

dose of 30mg/kg, the mice body weight was significantly reduced after 10 days of treatment. On

the contrary, during 30 days treatment with 42 exhibited excellent anti-tumor efficacy without

causing significant weight loss and toxicity.

3

. CONCLUSIONS

In this investigation, a series of N-phenylaniline derivatives were designed and synthesized, and

the in vitro cell viability against CRC lines was evaluated. Among these synthetic molecules,

derivative 42 showed good in vitro antiproliferative activities against HT29, HCT15, HCT16 with

IC50 values of 0.32 μM, 0.51 μM, 0.82 μM, respectively. Additionally, SAR study presented that

the junction of Region Ⅱ with Region Ⅱ to C-4 position, and Region Ⅱ connected to

N-(2,6-dimethoxybenzyl)-2-(4-methoxyphenyl)ethan-1-amine might make an enormous

contribution to the biological activity. Besides, EMSA binding ability test and molecular docking

study demonstrated that 42 exhibit an excellent binding affinity to c-Myc/Max via its unique

structural features. The target compound 42 showed explicit induced apoptosis for HT29 and

HCT15 in a dose-dependent manner, and cell cycle arrest at the G0/G1 phase on HT29 and

HCT15 cells. Furthermore, western blot analysis showed down-regulation of the expression of

c-Myc in HT29 and HCT15 cells by compound 42, which could indicate that compound 42 would

be inhibiting Myc/MAX interaction. More importantly, we demonstrated that compound 42

revealed eminent antitumor ability in vivo on HT29 tumor xenograft model with generating no

significant weight loss and toxicity. All of the studies illustrate here support 42 is a promising

novel drug candidate for therapy of CRC.

4

. EXPERIMENTAL SECTION

.1 Chemistry. All reagents and solvents were obtained from standard suppliers and used without

further purification. Anhydrous solvents were dried and purified by standard chemical reagent

1

13

purification. H (400 MHz) and C NMR (101 MHz) spectra were recorded on a Bruker AV-400

o

spectrometer at 25 C with CDCl . Chemical shifts are represented in δ(ppm) values with

3

tetramethylsilane (TMS) as an internal standard. Low-resolution and high-resolution ESI−MS was

taken on a Bruker Amjzon mass spectrometer. All reactions were measured by thin-layer

chromatography (TLC) under ultraviolet light (254nm or 365nm) and purified by Silica Gel

Column Chromatography (300−400 mesh). All the final products were tested by the Waters e2695

HPLC system, the purity of the final compounds was over 95% for biological testing.

Synthesis

of

4-fluoro-2,6-dimethoxybenzaldehyde

(3).

To

a

solution

of

1

-fluoro-3,5-dimethoxybenzene 2 (10 g, 64.06 mmol) under argon atmosphere in anhydrous DMF

o

(20 mL) was stirred at 0 C for 20 min, POCl (11.77 g, 76.87 mmol) was added in 15min, the

3

o

reaction mixture was stirred at room temperature for 30 min, then heated at 60 C for 4 h. The

reaction was completed, allowed to cool to room temperature, poured into ice-water (400 mL).The

aqueous phase was adjusted to pH=9-10 by 4N NaOH, extracted with ethyl acetate (2 x 400 mL),

combined organic phases and washed with brine, dried over anhydrous MgSO . The organic layer

4

was removed by rotary evaporation in vacuo, purified by column chromatography to afford 7.35g

1

white solid product. Yield 62.30%. H NMR (400 MHz, CDCl ) δ 10.44 (s, 1H), 6.77 (s, 2H), 3.91

3

+

(

s, 6H). ESI-MS: mass calcd for [M + H] (C H FO ) 185.05;found m/z, 185.06.

9

3

Synthesis of 3-(phenylamino)phenol (5). A mixture of 3-aminophenol 3 (5 g, 45.82 mmol) and

bromobenzene (8.63 g, 54.98 mmol), sodium tert-butoxide (1.32 g, 137.46 mmol), BrettPhos

Palladacycle (182.78 mg, 0.23 mmol, 0.5 mmol %) under argon atmosphere in anhydrous

o

1

,4-dioxane 25 mL was stirred at 90 C for 2 h. The reaction was completed monitored by TLC.

The reaction mixture was concentrated in vacuo, added 500 mL water, extracted with ethyl acetate

2 x 400 mL), combined organic phases and washed with brine, dried over anhydrous MgSO . The

(

4

organic layer was concentrated in vacuo and purified by silica gel chromatography to afford 4.31 g

1

brown solid. Yield 86.01%. H NMR (400 MHz, CDCl ) δ 7.28 (dd, J = 7.0, 1.5 Hz, 1H), 7.25 (s,

3

1

H), 7.14 – 7.06 (m, 3H), 6.95 (dd, J = 10.5, 4.2 Hz, 1H), 6.64 – 6.58 (m, 1H), 6.56 (t, J = 2.2 Hz,

1

H), 6.37 (ddd, J = 8.0, 2.3, 0.7 Hz, 1H), 5.68 (s, 1H), 4.71 (s, 1H). ESI-MS: mass calcd for [M +

+

H] (C H NO) 186.08; found m/z, 186.09.

12

11

General procedure A for the synthesis of 6a-6d. To a solution of 3-(phenylamino)phenol 5 (3 g,

6.20 mmol) and K CO (6.72 g, 48.59 mmol), 4-Fluorobenzaldehyde with different substituents

1

2

3

o

(16.20 mmol) under argon atmosphere in anhydrous DMSO(15 mL) was stirred at 100 C for 3 h

and monitored by TLC. The reaction was quenched by cold water (100 mL), extracted with DCM,

dried over anhydrous MgSO , concentrated in vacuo to get crude products, and purified by silica

4

gel chromatography to get desired compounds.

2

,6-dimethoxy-4-(3-(phenylamino)phenoxy)benzaldehyde (6a). The title compound was prepared

from 5 and 4-fluoro-2,6-dimethoxybenzaldehyde following the general procedure of A. Yield:

1

8

1

2

5.21%, brown solid. H NMR (400 MHz, CDCl ) δ 10.30 (s, 1H), 7.25 – 7.20 (m, 2H), 7.17 (s,

3

H), 7.07 – 7.02 (m, 2H), 6.92 (t, J = 7.4 Hz, 1H), 6.80 (ddd, J = 8.1, 2.2, 0.8 Hz, 1H), 6.73 (t, J =

.2 Hz, 1H), 6.53 (ddd, J = 8.1, 2.3, 0.8 Hz, 1H), 6.10 (s, 2H), 3.74 (s, 6H). ESI-MS: mass calcd

+

for [M + H] (C H NO ) 350.13; found m/z, 350.23.

2

1

19

4

2

-methoxy-4-(3-(phenylamino)phenoxy)benzaldehyde (6b). The title compound was prepared

from 5 and 4-fluoro-2-methoxybenzaldehyde following the general procedure of A. Yield: 82.35%,

1

white solid. H NMR (400 MHz, CDCl ) δ 10.31 (s, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.30 – 7.23 (m,

3

H), 7.09 (d, J = 7.6 Hz, 2H), 6.96 (t, J = 7.4 Hz, 1H), 6.87 (dd, J = 8.1, 1.6 Hz, 1H), 6.78 (t, J =

+

2

.1 Hz, 1H), 6.62 – 6.52 (m, 3H), 5.90 (s, 1H), 3.85 (s, 3H). ESI-MS: mass calcd for [M + H]

(

C H NO ) 320.12; found m/z, 320.13.

20 17 3

3

-methoxy-4-(3-(phenylamino)phenoxy)benzaldehyde (6c). The title compound was prepared

from 5 and 4-fluoro-3-methoxybenzaldehyde following the general procedure of A. Yield: 78.82%,

1

colorless oil. H NMR (400 MHz, CDCl ) H NMR (400 MHz, CDCl ) δ 9.89 (s, 1H), 7.51 (d, J =

3

1

.7 Hz, 1H), 7.39 (dd, J = 8.2, 1.8 Hz, 1H), 7.29 – 7.22 (m, 3H), 7.08 (d, J = 7.8 Hz, 2H), 6.97 (dd,

J = 15.6, 7.8 Hz, 2H), 6.83 (dd, J = 8.1, 2.0 Hz, 1H), 6.77 (t, J = 2.1 Hz, 1H), 6.56 (dd, J = 8.1, 2.2

+

Hz, 1H), 3.96 (s, 3H). ESI-MS: mass calcd for [M + H] (C H NO ) 320.12; found m/z, 320.12.

2

0

17

3

4

-(3-(phenylamino)phenoxy)benzaldehyde (6d). The title compound was prepared from 4 and

1

4

-fluorobenzaldehyde following the general procedure of A. Yield: 73.55%, colorless oil. H NMR

(

400 MHz, CDCl ) δ 9.92 (s, 1H), 7.88 – 7.82 (m, 2H), 7.31 – 7.25 (m, 3H), 7.09 (ddd, J = 7.9,

3

4

6

2

.8, 1.8 Hz, 4H), 6.97 (dd, J = 10.5, 4.2 Hz, 1H), 6.90 – 6.85 (m, 1H), 6.78 (t, J = 2.2 Hz, 1H),

+

.59 (ddd, J = 8.1, 2.2, 0.7 Hz, 1H), 5.79 (s, 1H). ESI-MS: mass calcd for [M + H] (C H NO )

1

9

15

2

90.11; found m/z, 290.12.

General procedure B for the synthesis of 7-38. To a solution of 6(a-d) (0.30mmol) , sodium

triacetoxyborohydride (127 mg, 0.60 mmol), different amines (0.36mmol), added acetic acid

o

(

0.03mmol) under argon atmosphere in anhydrous DMF(2 mL) was stirred at 80 C for over

night and monitored by TLC. The reaction was quenched by cold water (100 mL), extracted with

ethyl acetate, dried over anhydrous MgSO , concentrated in vacuo to get crude products, and

4

purified by flash column chromatography to get desired compounds.

3

-(3,5-dimethoxy-4-((methylamino)methyl)phenoxy)-N-phenylaniline (7). The title compound

was prepared from 6a and methylamine hydrochloride following the general procedure of B. Yield:

1

5

2.50%, brown solid. H NMR (400 MHz, CDCl ) δ 7.28 (d, J = 7.8 Hz, 2H), 7.22 (t, J = 8.1 Hz,

3

1

H), 7.10 (d, J = 7.9 Hz, 2H), 6.96 (t, J = 7.3 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.73 (s, 1H), 6.56

1

3

(d, J = 7.9 Hz, 1H), 6.23 (s, 2H), 4.21 (s, 2H), 3.81 (s, 6H), 2.54 (s, 3H). C NMR (101 MHz,

CDCl ) δ 160.42, 160.03, 157.26, 145.23, 142.27, 130.47, 129.41, 121.76, 118.68, 112.52, 111.12,

3

+

1

3

07.78, 101.09, 94.70, 56.01, 40.47, 31.19. ESI-MS: mass calcd for [M + H] (C H N O )

2

24

2

3

65.18; found m/z, 365.19. Purity 95.1% by HPLC.

-(4-((cyclopropylamino)methyl)-3,5-dimethoxyphenoxy)-N-phenylaniline

(8). The title

compound was prepared from 6a and cyclopropanamine following the general procedure of B.

1

Yield: 61.05%, brown solid. H NMR (400 MHz, CDCl ) δ 7.25 (d, J = 7.2 Hz, 2H), 7.19 (t, J =

3

8

.1 Hz, 1H), 7.09 (d, J = 7.6 Hz, 2H), 6.95 (t, J = 7.4 Hz, 1H), 6.80 (dd, J = 8.0, 2.0 Hz, 1H), 6.71

(

t, J = 2.2 Hz, 1H), 6.54 (dd, J = 8.1, 2.2 Hz, 1H), 6.26 (s, 2H), 5.81 (s, 1H), 4.02 (s, 2H), 3.77 (s,

1

3

6

H), 2.20 (tt, J = 7.2, 3.7 Hz, 1H), 0.71 (m, 2H), 0.52 (q, J = 6.6 Hz, 2H). C NMR (101 MHz,

CDCl ) δ 159.82, 145.11, 142.36, 130.39, 129.38, 121.64, 118.56, 112.16, 110.77, 107.44, 95.18,

3

+

5

3

5.92, 40.47, 29.37, 4.40. ESI-MS: mass calcd for [M + H] (C H N O ) 391.19; found m/z,

2

4

26

2

3

91.20. Purity 96.7% by HPLC.

-(4-((isopropylamino)methyl)-3,5-dimethoxyphenoxy)-N-phenylaniline (9). The title compound

was prepared from 6a and propan-2-amine following the general procedure of B. Yield: 52.36%,

1

brown solid. H NMR (400 MHz, CDCl ) δ 7.29 (s, 2H), 7.25 (s, 1H), 7.20 (t, J = 8.1 Hz, 1H),

3

7

.10 (d, J = 7.7 Hz, 2H), 6.95 (t, J = 7.3 Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H), 6.70 (d, J = 2.0 Hz, 1H),

.55 (dd, J = 8.1, 1.7 Hz, 1H), 6.25 (s, 2H), 5.92 (s, 1H), 4.02 (s, 2H), 3.77 (s, 6H), 2.95 (dt, J =

6

1

3

1

2.9, 6.4 Hz, 1H), 1.23 (d, J = 6.4 Hz, 6H). C NMR (101 MHz, CDCl ) δ 159.57, 157.93, 145.09,

3

1

42.36, 130.38, 129.38, 121.64, 118.55, 112.05, 110.67, 107.33, 95.42, 55.88, 46.94, 40.29, 37.81,

+

2

3.17. ESI-MS: mass calcd for [M + H] (C H N O ) 393.21; found m/z, 393.21. Purity 98.1%

2

4

28

2

3

by HPLC.

3

-(4-((dimethylamino)methyl)-3,5-dimethoxyphenoxy)-N-phenylaniline

(10).

The

title

compound was prepared from 6a and dimethylamine following the general procedure of B. Yield:

1

4

1

2

1

5.77%, brown solid. H NMR (400 MHz, CDCl ) δ 7.31 – 7.27 (m, 2H), 7.23 (d, J = 8.1 Hz,

3

H), 7.11 (d, J = 7.6 Hz, 2H), 6.97 (t, J = 7.3 Hz, 1H), 6.86 (dd, J = 8.1, 1.9 Hz, 1H), 6.76 (t, J =

.2 Hz, 1H), 6.58 (dd, J = 8.0, 2.1 Hz, 1H), 6.25 (s, 2H), 5.89 (s, 1H), 4.22 (s, 2H), 3.81 (s, 6H),

1

3

.72 (s, 6H). C NMR (101 MHz, CDCl ) δ 161.35, 160.49, 156.76, 145.32, 142.18, 130.58,

3

29.43, 121.90, 118.78, 112.95, 111.44, 108.02, 99.54, 94.50, 56.04, 49.03, 42.40, 29.70. ESI-MS:

+

mass calcd for [M + H] (C H N O ) 379.19; found m/z, 379.20. Purity 95.3% by HPLC.

2

3

26

2

3

2

,2'-((2,6-dimethoxy-4-(3-(phenylamino)phenoxy)benzyl)azanediyl)bis(ethan-1-ol) (11). The

title compound was prepared from 6a and 2,2'-azanediylbis(ethan-1-ol) following the general

1

procedure of B. Yield: 56.15%, yellow oil. H NMR (400 MHz, CDCl ) δ 7.29 (d, J = 7.8 Hz, 1H),

3

7

.23 (d, J = 8.1 Hz, 1H), 7.11 (d, J = 7.9 Hz, 1H), 6.97 (t, J = 7.3 Hz, 1H), 6.86 (dd, J = 8.2, 1.5

Hz, 1H), 6.76 (s, 1H), 6.58 (dd, J = 7.9, 1.6 Hz, 1H), 6.25 (s, 1H), 5.88 (s, 1H), 4.35 (s, 1H), 3.99

13

(

s, 2H), 3.81 (s, 3H), 3.28 (s, 2H). C NMR (101 MHz, CDCl ) δ 160.02, 130.58, 129.41, 121.84,

3

1

18.76, 112.88, 111.44, 108.05, 94.65, 56.15, 56.10, 55.35, 47.58. ESI-MS: mass calcd for [M +

+

H] (C H N O ) 439.22; found m/z, 439.23. Purity 97.2% by HPLC.

25

30

2

5

N-(2-((2,6-dimethoxy-4-(3-(phenylamino)phenoxy)benzyl)amino)ethyl)acetamide (12). The title

compound was prepared from 6a and N-(2-aminoethyl)acetamide following the general procedure

1

of B. Yield: 61.42 %, brown oil. H NMR (400 MHz, CDCl ) δ 7.27 (d, J = 7.0 Hz, 2H), 7.20 (t, J

3

=

8.1 Hz, 1H), 7.13 – 7.08 (m, 2H), 7.00 (s, 1H), 6.94 (t, J = 7.3 Hz, 1H), 6.83 (dd, J = 8.1, 1.5 Hz,

1

3

H), 6.64 (t, J = 2.2 Hz, 1H), 6.55 (dd, J = 8.1, 1.6 Hz, 1H), 6.26 (s, 2H), 6.08 (s, 1H), 3.92 (s, 2H),

1

3

.77 (s, 6H), 3.39 (d, J = 5.3 Hz, 2H), 2.84 – 2.73 (m, 2H), 1.97 (s, 3H). C NMR (101 MHz,

CDCl ) δ 170.65, 159.51, 158.60, 158.02, 145.15, 142.43, 130.37, 129.36, 121.57, 118.51, 111.85,

3

+

110.59, 107.22, 95.44, 55.88, 46.94, 40.29, 37.81, 23.17. ESI-MS: mass calcd for [M + H]

(

C H N O ) 436.22; found m/z, 436.23. Purity 98.1% by HPLC.

25

29

3

4

2

-(4-(2,6-dimethoxy-4-(3-(phenylamino)phenoxy)benzyl)piperazin-1-yl)ethan-1-ol (13). The title

compound was prepared from 6a and 2-(piperazin-1-yl)ethan-1-ol following the general procedure

1

of B. Yield: 53.18%, brown oil. H NMR (400 MHz, CDCl ) δ 7.28 (dd, J = 7.0, 1.4 Hz, 2H), 7.23

3

(t, J = 8.1 Hz, 1H), 7.14 – 7.08 (m, 2H), 6.96 (t, J = 7.4 Hz, 1H), 6.84 (dd, J = 8.1, 1.5 Hz, 1H),

6

3

.75 (t, J = 2.2 Hz, 1H), 6.57 (dd, J = 8.1, 1.6 Hz, 1H), 6.25 (s, 2H), 5.88 (s, 1H), 4.08 (s, 2H),

1

3

.76 (s, 6H), 3.70 – 3.65 (m, 2H), 2.93 (s, 7H), 2.72 – 2.66 (m, 2H). C NMR (101 MHz, CDCl )

3

δ 160.54, 157.23, 145.23, 142.25, 130.49, 129.41, 121.81, 118.69, 112.62, 111.18, 107.79, 99.99,

+

9

4.89, 59.16, 57.64, 55.93, 50.55, 48.17. ESI-MS: mass calcd for [M + H] (C H N O ) 464.25;

2

7

33

3

4

found m/z, 464.26. Purity 96.5% by HPLC.

3

-(4-((4-ethylpiperazin-1-yl)methyl)-3,5-dimethoxyphenoxy)-N-phenylaniline (14). The title

compound was prepared from 6a and 1-ethylpiperazine following the general procedure of B.

1

Yield: 62.33%, brown solid. H NMR (400 MHz, CDCl ) δ 7.29 (s, 1H), 7.25 (s, 1H), 7.20 (t, J =

3

8

1

=

.1 Hz, 1H), 7.10 (d, J = 8.0 Hz, 2H), 6.95 (t, J = 7.3 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 6.71 (s,

H), 6.55 (dd, J = 8.1, 1.9 Hz, 1H), 6.24 (s, 2H), 5.88 (s, 1H), 3.84 (s, 2H), 3.73 (s, 6H), 2.72 (d, J

1

3

46.0 Hz, 8H), 2.50 – 2.44 (m, 2H), 1.10 (t, J = 7.1 Hz, 3H). C NMR (101 MHz, CDCl ) δ

3

1

60.30, 158.01, 145.04, 142.40, 130.34, 129.38, 121.61, 118.55, 112.07, 110.73, 107.38, 95.41,

+

5

5.82, 52.10, 51.92, 51.50, 48.48, 11.67. ESI-MS: mass calcd for [M + H] (C H N O ) 448.25;

2

7

33

3

found m/z, 448.25. Purity 95.4% by HPLC.

(1s,3s)-N-(2,6-dimethoxy-4-(3-(phenylamino)phenoxy)benzyl)adamantan-1-amine (15). The

title compound was prepared from 6a and amantadine following the general procedure of B. Yield:

1

4

1.27%, yellow solid. H NMR (400 MHz, CDCl ) δ 7.28 (s, 1H), 7.24 (s, 1H), 7.21 (d, J = 8.1 Hz,

3

1

H), 7.12 (d, J = 7.7 Hz, 2H), 6.94 (t, J = 7.3 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.69 (s, 1H), 6.55

(

dd, J = 8.0, 1.6 Hz, 1H), 6.23 (d, J = 8.1 Hz, 2H), 4.16 (s, 2H), 3.82 (s, 6H), 2.13 (s, 3H), 2.00 (s,

1

3

6

1

2

H), 1.64 (s, 6H). C NMR (101 MHz, CDCl ) δ 159.91, 159.42, 157.66, 145.22, 142.36, 130.38,

3

29.34, 121.57, 118.62, 111.91, 110.82, 107.57, 102.98, 95.45, 58.30, 56.16, 38.68, 35.58, 33.64,

+

9.11. ESI-MS: mass calcd for [M + H] (C H N O ) 485.27; found m/z, 485.28. Purity 97.3%

3

1

36

2

3

by HPLC.

2,6-dimethoxy-4-(3-(phenylamino)phenoxy)phenyl)methanol (16). The title compound was

(

prepared from 6a (50 mg, 0.142mmol) and NaBH (2 mg, 0.057 mmol) stirred in MeOH at room

4

room temperature for 2min. The reaction was concentrated in vacuo to get crude products, and

1

purified by silica gel chromatography to get 35. Yield: 65.25 %, colorless oil. H NMR (400 MHz,

CDCl ) δ 7.28 – 7.24 (m, 2H), 7.19 (t, J = 8.1 Hz, 1H), 7.08 (d, J = 7.6 Hz, 2H), 6.96 (d, J = 7.3

3

Hz, 1H), 6.79 (dd, J = 8.0, 1.9 Hz, 1H), 6.72 (t, J = 2.2 Hz, 1H), 6.54 (dd, J = 8.1, 1.8 Hz, 1H),

+

6

.26 (s, 2H), 5.78 (s, 1H), 4.73 (s, 2H), 3.77 (s, 6H). ESI-MS: mass calcd for [M + H]

(

C H NO ) 352.15; found m/z, 352.16. Purity 96.9% by HPLC.

21

4

3

-(4-((benzylamino)methyl)-3,5-dimethoxyphenoxy)-N-phenylaniline (17). The title compound

was prepared from 6a and phenylmethanamine following the general procedure of B. Yield:

1

5

8.02%, brown solid. H NMR (400 MHz, CDCl ) δ 7.31 (dt, J = 9.7, 4.2 Hz, 5H), 7.23 (d, J = 6.4

3

Hz, 2H), 7.19 (t, J = 8.1 Hz, 1H), 7.08 (d, J = 7.8 Hz, 2H), 6.94 (t, J = 7.3 Hz, 1H), 6.78 (dd, J =

8

.0, 1.6 Hz, 1H), 6.72 (t, J = 2.1 Hz, 1H), 6.54 (dd, J = 8.1, 2.0 Hz, 1H), 6.26 (s, 2H), 5.75 (s, 1H),

1

3

.84 (s, 2H), 3.76 (s, 2H), 3.73 (s, 6H). C NMR (101 MHz, CDCl ) δ 159.45, 158.45, 157.27,

3

1

44.94, 142.43, 140.86, 130.30, 129.39, 128.20, 126.67, 121.62, 118.50, 111.91, 111.74, 110.49,

+

1

07.19, 95.62, 55.73, 53.12, 41.05. ESI-MS: mass calcd for [M + H] (C H N O ) 441.21; found

2

8

28

2

3

m/z, 441.21. Purity 95.6% by HPLC.

3

-(4-(((4-fluorobenzyl)amino)methyl)-3,5-dimethoxyphenoxy)-N-phenylaniline (18). The title

compound was prepared from 6a and (4-fluorophenyl)methanamine following the general

1

procedure of B. Yield: 53.72%, yellow solid. H NMR (400 MHz, CDCl ) δ 7.32 – 7.23 (m, 4H),

3

7

1

3

1

.18 (t, J = 8.1 Hz, 1H), 7.07 (d, J = 7.7 Hz, 2H), 7.02 – 6.91 (m, 3H), 6.77 (dd, J = 8.1, 1.4 Hz,

H), 6.71 (t, J = 2.0 Hz, 1H), 6.53 (dd, J = 8.1, 1.7 Hz, 1H), 6.25 (s, 2H), 5.80 (s, 1H), 3.82 (s, 2H),

1

3

.72 (s, 8H). C NMR (101 MHz, CDCl ) δ 163.03, 160.61, 159.44, 158.39, 157.37, 144.99,

3

42.46, 136.61, 136.58, 130.32, 129.75, 129.67, 129.59, 129.39, 121.60, 121.25, 118.50, 115.01,

1

14.80, 111.97, 111.59, 110.50, 107.23, 95.59, 55.73, 52.33, 40.92. ESI-MS: mass calcd for [M +

+

H] (C H FN O ) 459.20; found m/z, 459.21. Purity 98.1% by HPLC.

28

27

2

3

-(4-(((4-chlorobenzyl)amino)methyl)-3,5-dimethoxyphenoxy)-N-phenylaniline (19). The title

compound was prepared from 6a and (4-chlorophenyl)methanamine following the general

1

procedure of B. Yield: 58.87%, brown solid. H NMR (400 MHz, CDCl ) δ 7.27 – 7.22 (m, 6H),

3

7

.19 (d, J = 8.1 Hz, 1H), 7.10 – 7.05 (m, 2H), 6.93 (t, J = 7.4 Hz, 1H), 6.80 – 6.75 (m, 1H), 6.71 (t,

1

3

J = 2.2 Hz, 1H), 6.56 – 6.51 (m, 1H), 6.25 (s, 2H), 5.80 (s, 1H), 3.81 (s, 2H), 3.72 (s, 8H). C

NMR (101 MHz, CDCl ) δ 159.44, 158.34, 157.46, 144.99, 142.45, 139.26, 132.34, 130.33,

3

1

29.58, 129.39, 128.28, 121.61, 118.51, 112.01, 111.35, 110.53, 107.26, 95.54, 55.73, 52.25,

+

4

0.86. ESI-MS: mass calcd for [M + H] (C H ClN O ) 475.17; found m/z, 475.17. Purity 97.1%

2

8

27

2

3

by HPLC.

3

-(4-(((4-bromobenzyl)amino)methyl)-3,5-dimethoxyphenoxy)-N-phenylaniline (20). The title

compound was prepared from 6a and (4-bromophenyl)methanamine following the general

1

procedure of B. Yield: 61.25%, brown solid. H NMR (400 MHz, CDCl ) δ 7.42 (d, J = 8.3 Hz,

3

2

6

1

5

H), 7.26 – 7.17 (m, 5H), 7.08 (d, J = 7.6 Hz, 2H), 6.94 (t, J = 7.4 Hz, 1H), 6.80 – 6.76 (m, 1H),

.72 (t, J = 2.2 Hz, 1H), 6.56 – 6.52 (m, 1H), 6.25 (s, 2H), 5.77 (s, 1H), 3.81 (s, 2H), 3.72 (d, J =

1

3

.5 Hz, 8H). C NMR (101 MHz, CDCl ) δ 159.43, 158.33, 157.46, 144.97, 142.44, 139.79,

3

31.23, 130.33, 129.96, 129.39, 121.63, 120.42, 118.51, 112.01, 111.35, 110.55, 107.26, 95.54,

+

5.73, 52.28, 40.85. ESI-MS: mass calcd for [M + H] (C H BrN O ) 519.12, 521.12; found m/z,

2

8

27

2

3

5

19.13, 521.13 . Purity 96.8% by HPLC.

4

-(((2,6-dimethoxy-4-(3-(phenylamino)phenoxy)benzyl)amino)methyl)phenol (21). The title

compound was prepared from 6a and 4-(aminomethyl)phenol following the general procedure of

1

B. Yield: 48.73%, brown oil. H NMR (400 MHz, CDCl ) δ 7.26 – 7.22 (m, 2H), 7.18 (t, J = 8.1

3

Hz, 1H), 7.07 (dd, J = 8.5, 1.0 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 6.93 (t, J = 7.3 Hz, 1H), 6.81 –

6

3

1

9

4

3

.77 (m, 1H), 6.69 (t, J = 2.2 Hz, 1H), 6.58 – 6.51 (m, 3H), 6.23 (s, 2H), 5.84 (s, 1H), 3.89 (s, 2H),

1

3

.70 (s, 6H), 3.62 (s, 2H). C NMR (101 MHz, CDCl ) δ 159.46, 158.21, 157.75, 156.24, 145.01,

3

42.45, 130.33, 129.97, 129.71, 129.37, 121.60, 118.51, 115.76, 111.97, 110.61, 110.16, 107.34,

+

5.46, 55.74, 52.09, 40.74. ESI-MS: mass calcd for [M + H] (C H N O ) 457.20; found m/z,

2

8

28

2

4

57.21. Purity 97.3% by HPLC.

-(3,5-dimethoxy-4-(((4-methoxybenzyl)amino)methyl)phenoxy)-N-phenylaniline (22). The title

compound was prepared from 6a and (4-methoxyphenyl)methanamine following the general

1

procedure of B. Yield: 55.61%, brown solid. H NMR (400 MHz, CDCl ) δ 7.35 (d, J = 8.5 Hz,

3

2

–

4

1

H), 7.26 (s, 2H), 7.20 (t, J = 8.1 Hz, 1H), 7.10 (d, J = 7.7 Hz, 2H), 6.95 (t, J = 7.3 Hz, 1H), 6.88

6.83 (m, 3H), 6.66 (t, J = 2.1 Hz, 1H), 6.53 (dd, J = 8.0, 1.9 Hz, 1H), 6.20 (s, 2H), 5.98 (s, 1H),

13

.07 (s, 2H), 3.91 (s, 2H), 3.77 (s, 3H), 3.73 (s, 6H). C NMR (101 MHz, CDCl ) δ 159.85,

3

59.65, 157.58, 145.19, 142.35, 131.06, 130.41, 129.39, 124.88, 121.68, 118.60, 114.11, 112.13,

+

1

4

3

10.86, 107.53, 95.06, 55.90, 55.29, 49.65, 39.00. ESI-MS: mass calcd for [M + H] (C H N O )

2

9

30

2

4

71.22; found m/z, 471.22. Purity 96.2% by HPLC.

-(3,5-dimethoxy-4-(((4-methylbenzyl)amino)methyl)phenoxy)-N-phenylaniline (23). The title

compound was prepared from 6a and p-tolylmethanamine following the general procedure of B.

1

Yield: 52.72%, brown solid. H NMR (400 MHz, CDCl ) δ 7.21 – 7.18 (m, 2H), 7.15 (dd, J = 10.1,

3

.5 Hz, 3H), 7.11 (t, J = 8.1 Hz, 1H), 7.04 (d, J = 7.9 Hz, 2H), 7.01 (dd, J = 8.5, 1.0 Hz, 2H), 6.86

(

dd, J = 10.5, 4.2 Hz, 1H), 6.72 (dd, J = 8.1, 1.5 Hz, 1H), 6.64 (t, J = 2.2 Hz, 1H), 6.48 – 6.44 (m,

1

3

1

H), 6.17 (s, 2H), 5.74 (s, 1H), 3.80 (s, 2H), 3.68 (s, 2H), 3.65 (s, 6H), 2.25 (s, 3H). C NMR

(

101 MHz, CDCl ) δ 159.49, 158.27, 157.70, 145.00, 142.43, 136.62, 136.36, 130.31, 129.38,

3

1

29.00, 128.39, 121.61, 118.52, 111.97, 110.55, 110.29, 107.28, 95.50, 55.75, 52.35, 40.73, 21.11.

+

ESI-MS: mass calcd for [M + H] (C H N O ) 455.23; found m/z, 455.24. Purity 98.6% by

2

9

30

2

3

HPLC.

-(4-(((benzo[d][1,3]dioxol-5-ylmethyl)amino)methyl)-3,5-dimethoxyphenoxy)-N-phenylaniline

3

(

24). The title compound was prepared from 6a and benzo[d][1,3]dioxol-5-ylmethanamine

1

following the general procedure of B. Yield: 58.36%, brown solid. H NMR (400 MHz, CDCl ) δ

3

7

.28 (d, J = 8.5 Hz, 2H), 7.20 (t, J = 8.1 Hz, 1H), 7.10 (d, J = 7.6 Hz, 2H), 6.99 (s, 1H), 6.95 (t, J

7.4 Hz, 1H), 6.90 (d, J = 7.9 Hz, 1H), 6.84 (dd, J = 8.1, 1.6 Hz, 1H), 6.74 (d, J = 7.9 Hz, 1H),

.67 (t, J = 2.2 Hz, 1H), 6.52 (dd, J = 8.0, 1.7 Hz, 1H), 6.19 (s, 2H), 5.92 (s, 2H), 4.09 (s, 2H),

=

6

3

1

3

.90 (s, 2H), 3.75 (s, 6H). C NMR (101 MHz, CDCl ) δ 160.08, 159.67, 157.35, 148.17, 147.94,

3

45.21, 142.31, 130.44, 129.41, 125.31, 123.86, 121.74, 118.64, 112.32, 111.01, 110.18, 108.43,

+

07.69, 102.21, 101.31, 94.87, 55.95, 49.76, 38.85. ESI-MS: mass calcd for [M + H]

(

C H N O ) 485.20; found m/z, 485.20. Purity 95.4% by HPLC.

29 28 2 5

(R)-3-(3,5-dimethoxy-4-(((1-phenylethyl)amino)methyl)phenoxy)-N-phenylaniline (25). The title

compound was prepared from 6a and (R)-1-phenylethan-1-amine following the general procedure

1

of B. Yield: 51.83 %, brown solid. H NMR (400 MHz, CDCl ) δ 7.51 – 7.45 (m, 2H), 7.32 (ddd, J

3

=

9.4, 6.6, 1.9 Hz, 4H), 7.24 (t, J = 1.9 Hz, 1H), 7.17 (t, J = 8.1 Hz, 1H), 7.09 (dd, J = 8.5, 1.0 Hz,

2

H), 6.95 (t, J = 7.3 Hz, 1H), 6.81 (dd, J = 8.1, 1.5 Hz, 1H), 6.67 (t, J = 2.2 Hz, 1H), 6.50 (dd, J =

8

6

1

5

4

.1, 1.6 Hz, 1H), 6.16 (s, 2H), 5.92 (s, 1H), 3.98 (d, J = 6.7 Hz, 1H), 3.97 – 3.92 (m, 2H), 3.71 (s,

1

3

H), 1.64 (d, J = 6.8 Hz, 3H). C NMR (101 MHz, CDCl ) δ 159.64, 159.27, 157.75, 145.10,

3

42.40, 130.33, 129.39, 128.70, 128.30, 127.57, 121.62, 118.54, 112.10, 110.75, 107.49, 95.04,

+

7.98, 55.80, 38.52, 21.10. ESI-MS: mass calcd for [M + H] (C H N O ) 455.23; found m/z,

2

9

30

2

3

55.23. Purity 97.7% by HPLC.

(R)-3-(3,5-dimethoxy-4-(((1-(4-methoxyphenyl)ethyl)amino)methyl)phenoxy)-N-phenylaniline

(

26). The title compound was prepared from 6a and (R)-1-(4-methoxyphenyl)ethan-1-amine

1

following the general procedure of B. Yield: 50.07 %, brown solid. H NMR (400 MHz, CDCl ) δ

3

7

.30 – 7.25 (m, 4H), 7.18 (t, J = 8.1 Hz, 1H), 7.09 (d, J = 7.7 Hz, 2H), 6.94 (t, J = 7.3 Hz, 1H),

.86 (d, J = 8.6 Hz, 2H), 6.79 (dd, J = 8.1, 1.4 Hz, 1H), 6.69 (t, J = 2.1 Hz, 1H), 6.53 (dd, J = 8.1,

.6 Hz, 1H), 6.23 (s, 2H), 5.79 (s, 1H), 3.80 (s, 3H), 3.71 (s, 6H), 3.69 (s, 2H), 1.34 (d, J = 6.6 Hz,

6

1

13

3

H). C NMR (101 MHz, CDCl ) δ 159.40, 158.47, 158.46, 157.24, 144.94, 142.46, 130.28,

3

1

29.38, 127.86, 121.59, 118.49, 113.57, 111.81, 110.47, 107.16, 95.65, 56.83, 55.68, 55.28, 39.50,

+

2

4.26. ESI-MS: mass calcd for [M + H] (C H N O ) 485.24; found m/z, 485.25. Purity 98.3%

3

0

32

2

4

by HPLC.

3

-(3,5-dimethoxy-4-((phenethylamino)methyl)phenoxy)-N-phenylaniline

(27).

The

title

compound was prepared from 6a and 2-phenylethan-1-amine following the general procedure of B.

1

Yield: 61.09%, brown solid. H NMR (400 MHz, CDCl ) δ 7.28 (d, J = 4.6 Hz, 3H), 7.22 (dd, J =

3

7

.6, 2.9 Hz, 2H), 7.17 – 7.12 (m, 2H), 7.09 (d, J = 7.6 Hz, 2H), 6.96 (t, J = 7.4 Hz, 1H), 6.83 (dd,

J = 8.1, 1.5 Hz, 1H), 6.71 (t, J = 2.2 Hz, 1H), 6.53 (dd, J = 8.1, 1.7 Hz, 1H), 6.18 (s, 2H), 5.85 (s,

1

3

1

H), 4.19 (s, 2H), 3.65 (s, 6H), 3.11 (d, J = 6.2 Hz, 2H), 3.06 (d, J = 6.2 Hz, 2H). C NMR (101

MHz, CDCl ) δ 159.92, 159.62, 157.36, 145.21, 142.24, 136.87, 130.48, 129.42, 128.92, 128.90,

3

1

27.05, 121.83, 118.69, 112.52, 110.96, 107.59, 94.94, 55.88, 47.27, 40.17, 32.62. ESI-MS: mass

+

calcd for [M + H] (C H N O ) 455.23; found m/z, 455.24. Purity 96.5% by HPLC.

2

9

30

2

3

-(4-(((4-chlorophenethyl)amino)methyl)-3,5-dimethoxyphenoxy)-N-phenylaniline (28). The

title compound was prepared from 6a and 2-(4-chlorophenyl)ethan-1-amine following the general

1

procedure of B. Yield: 53.57%, brown solid. H NMR (400 MHz, CDCl ) δ 7.29 (s, 2H), 7.25 (d,

3

J = 2.5 Hz, 2H), 7.21 (d, J = 8.1 Hz, 1H), 7.09 (dd, J = 7.5, 5.3 Hz, 4H), 6.96 (t, J = 7.3 Hz, 1H),

6

.83 (dd, J = 8.1, 2.1 Hz, 1H), 6.71 (t, J = 2.1 Hz, 1H), 6.53 (dd, J = 8.1, 2.2 Hz, 1H), 6.19 (s, 2H),

1

3

5

.86 (s, 1H), 4.17 (s, 2H), 3.67 (s, 6H), 3.07 (d, J = 6.4 Hz, 2H), 3.02 (d, J = 6.4 Hz, 2H). C

NMR (101 MHz, CDCl ) δ 160.43, 159.67, 157.11, 145.27, 142.19, 134.94, 133.15, 130.55,

3

1

30.30, 129.44, 129.10, 121.89, 112.72, 111.03, 107.69, 94.81, 55.95, 46.78, 40.05, 31.53.

+

ESI-MS: mass calcd for [M + H] (C H ClN O ) 489.19; found m/z, 489.20. Purity 98.5% by

29

2

3

HPLC.

-(4-(((4-fluorophenethyl)amino)methyl)-3,5-dimethoxyphenoxy)-N-phenylaniline (29). The

3

title compound was prepared from 6a and 2-(4-fluorophenyl)ethan-1-amine following the general

1

procedure of B. Yield: 48.28%, yellow solid. H NMR (400 MHz, CDCl ) δ 7.25 (t, J = 7.9 Hz,

3

2

H), 7.20 – 7.06 (m, 5H), 6.99 – 6.90 (m, 3H), 6.77 (dd, J = 8.0, 1.5 Hz, 1H), 6.70 (t, J = 2.0 Hz,

H), 6.52 (dd, J = 8.1, 1.7 Hz, 1H), 6.23 (s, 2H), 5.81 (s, 1H), 3.81 (s, 2H), 3.67 (s, 6H), 2.80 (dd,

1

3

J = 11.0, 5.3 Hz, 4H). C NMR (101 MHz, CDCl ) δ 162.60, 160.18, 159.34, 158.41, 157.28,

3

1

44.98, 142.46, 136.02, 135.99, 130.30, 130.14, 130.07, 129.38, 121.59, 118.49, 115.12, 114.92,

1

11.94, 111.39, 110.42, 107.17, 95.56, 55.66, 50.15, 41.15, 35.31. ESI-MS: mass calcd for [M +

+

H] (C H FN O ) 473.22; found m/z, 473.23. Purity 97.1% by HPLC.

29

2

3

-(3,5-dimethoxy-4-(((4-methylphenethyl)amino)methyl)phenoxy)-N-phenylaniline (30). The

title compound was prepared from 6a and 2-(p-tolyl)ethan-1-amine following the general

1

procedure of B. Yield: 55.37%, yellow oil. H NMR (400 MHz, CDCl ) H NMR (400 MHz,

3

CDCl ) δ 7.26 (s, 1H), 7.23 (d, J = 5.4 Hz, 1H), 7.17 (t, J = 8.1 Hz, 1H), 7.10 – 7.03 (m, 6H), 6.93

3

(t, J = 7.3 Hz, 1H), 6.77 (dd, J = 8.0, 1.6 Hz, 1H), 6.70 (t, J = 2.2 Hz, 1H), 6.52 (dd, J = 8.1, 1.8

Hz, 1H), 6.22 (s, 2H), 5.89 (s, 1H), 3.86 (s, 2H), 3.65 (s, 6H), 2.83 (dd, J = 10.6, 5.3 Hz, 4H), 2.29

13

(

s, 3H). C NMR (101 MHz, CDCl ) δ 159.43, 158.29, 157.67, 145.06, 142.48, 136.73, 135.53,

3

1

4

30.31, 129.38, 129.09, 128.68, 121.58, 118.52, 111.98, 110.47, 110.08, 107.24, 95.47, 55.68,

+

9.79, 40.94, 35.14, 21.02. ESI-MS: mass calcd for [M + H] (C H N O ) 469.24; found m/z,

3

0

32

2

3

69.25. Purity 97.4% by HPLC.

-(2-((2,6-dimethoxy-4-(3-(phenylamino)phenoxy)benzyl)amino)ethyl)phenol (31). The title

compound was prepared from 6a and 4-(2-aminoethyl)phenol following the general procedure of

1

B. Yield: 51.03 %, brown oil. H NMR (400 MHz, MeOD) δ 7.21 (td, J = 8.2, 4.6 Hz, 3H), 7.11 –

7

.04 (m, 4H), 6.89 – 6.84 (m, 2H), 6.77 (s, 1H), 6.74 (dd, J = 4.4, 2.2 Hz, 2H), 6.51 – 6.47 (m,

1

3

1

H), 6.36 (s, 2H), 4.21 (s, 2H), 3.79 (s, 6H), 3.17 – 3.12 (m, 2H), 2.94 – 2.87 (m, 2H). C NMR

(

101 MHz, CDCl ) δ 165.12, 163.81, 160.91, 160.36, 150.01, 146.90, 134.04, 133.31, 132.73,

3

1

30.79, 124.57, 121.86, 119.31, 116.20, 114.04, 110.78, 105.23, 98.17, 59.12, 43.41, 34.69.

+

ESI-MS: mass calcd for [M + H] (C H N O ) 471.22; found m/z, 471.23. Purity 95.8% by

2

9

30

2

4

HPLC.

-(3,5-dimethoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline (32). The

3

title compound was prepared from 6a and 2-(4-methoxyphenyl)ethan-1-amine following the

1

general procedure of B. Yield: 58.95 %, brown solid. H NMR (400 MHz, CDCl ) δ 7.25 (t, J =

3

7

–

3

.9 Hz, 2H), 7.18 (t, J = 8.1 Hz, 1H), 7.08 (dd, J = 8.5, 2.1 Hz, 4H), 6.94 (t, J = 7.3 Hz, 1H), 6.85

6.76 (m, 3H), 6.70 (d, J = 1.9 Hz, 1H), 6.52 (dd, J = 8.1, 2.1 Hz, 1H), 6.22 (s, 2H), 5.85 (s, 1H),

1

3

.84 (s, 2H), 3.77 (s, 3H), 3.67 (s, 6H), 2.83 (d, J = 5.8 Hz, 2H), 2.79 (d, J = 6.0 Hz, 2H). C

NMR (101 MHz, CDCl ) δ 160.25, 159.73, 158.67, 157.23, 145.26, 142.26, 130.48, 129.92,

3

1

29.41, 128.46, 121.78, 118.68, 114.29, 112.52, 111.00, 107.67, 101.40, 94.81, 55.91, 55.29,

+

4

7.02, 39.72, 31.23. ESI-MS: mass calcd for [M + H] (C H N O ) 485.24; found m/z, 484.25.

3

0

32

2

4

Purity 96.9% by HPLC.

3

-(3,5-dimethoxy-4-(((2-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline (33). The

title compound was prepared from 6a and 2-(2-methoxyphenyl)ethan-1-amine following the

1

general procedure of B. Yield: 47.64 %, brown solid. H NMR (400 MHz, CDCl ) δ 7.25 (t, J =

3

7

3

1

.9 Hz, 2H), 7.20 – 7.11 (m, 3H), 7.08 (d, J = 7.6 Hz, 2H), 6.94 (t, J = 7.3 Hz, 1H), 6.88 – 6.76 (m,

H), 6.70 (t, J = 2.1 Hz, 1H), 6.51 (dd, J = 8.1, 1.7 Hz, 1H), 6.22 (s, 2H), 5.83 (s, 1H), 3.87 (s, 2H),

1

3

.75 (s, 3H), 3.67 (s, 6H), 2.87 (s, 4H). C NMR (101 MHz, CDCl ) δ 159.40, 158.37, 157.68,

3

57.45, 145.01, 142.45, 130.42, 130.29, 129.38, 128.29, 127.38, 121.59, 120.40, 118.52, 111.92,

1

10.40, 110.38, 107.17, 95.52, 55.70, 55.26, 48.33, 41.06, 30.36. ESI-MS: mass calcd for [M +

+

H] (C H N O ) 485.24; found m/z, 484.25. Purity 97.5% by HPLC.

30

32

2

4

3

-(3,5-dimethoxy-4-(((3-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline (34). The

title compound was prepared from 6a and 2-(3-methoxyphenyl)ethan-1-amine following the

1

general procedure of B. Yield: 52.11 %, brown solid. H NMR (400 MHz, CDCl ) δ 7.24 (t, J =

3

7

.9 Hz, 2H), 7.17 (t, J = 8.0 Hz, 2H), 7.07 (d, J = 7.7 Hz, 2H), 6.93 (t, J = 7.3 Hz, 1H), 6.81 – 6.67

(

m, 5H), 6.52 (dd, J = 8.1, 1.8 Hz, 1H), 6.22 (s, 2H), 5.87 (s, 1H), 3.83 (s, 2H), 3.76 (s, 3H), 3.66

1

3

s, 6H), 2.83 (dd, J = 15.7, 5.8 Hz, 4H). C NMR (101 MHz, CDCl ) δ 159.68, 159.38, 158.42,

3

1

57.33, 145.01, 142.49, 141.90, 130.29, 129.38, 129.31, 121.55, 121.22, 118.48, 114.43, 111.87,

1

11.42, 111.17, 110.41, 107.16, 95.56, 55.66, 55.14, 49.87, 41.13, 36.07. ESI-MS: mass calcd for

+

[M + H] (C H N O ) 485.24; found m/z, 484.25. Purity 98.1% by HPLC.

30 32 2 4

3

-(3,5-dimethoxy-4-(((3-(4-methoxyphenyl)propyl)amino)methyl)phenoxy)-N-phenylaniline

(

35). The title compound was prepared from 6a and 3-(4-methoxyphenyl)propan-1-amine

1

following the general procedure of B. Yield: 55.61 %, brown oil. H NMR (400 MHz, CDCl ) δ

3

7

6

3

.28 – 7.22 (m, 2H), 7.18 (t, J = 8.1 Hz, 1H), 7.11 – 7.05 (m, 4H), 6.93 (t, J = 7.3 Hz, 1H), 6.83 –

.77 (m, 3H), 6.70 (t, J = 2.2 Hz, 1H), 6.56 – 6.51 (m, 1H), 6.24 (s, 2H), 5.87 (s, 1H), 3.83 (s, 2H),

1

3

.76 (s, 3H), 3.73 (s, 6H), 2.60 (dt, J = 24.7, 7.5 Hz, 4H), 1.88 – 1.77 (m, 2H). C NMR (101

MHz, CDCl ) δ 159.47, 158.32, 157.72, 157.59, 145.03, 142.49, 134.26, 130.31, 129.38, 129.29,

3

1

21.56, 118.50, 113.75, 111.94, 110.60, 110.49, 107.26, 95.52, 55.77, 55.27, 48.19, 40.99, 32.66,

+

3

1.33. ESI-MS: mass calcd for [M + H] (C H N O ) 499.25; found m/z, 499.26. Purity 98.6%

3

1

34

2

4

by HPLC.

3

-(3-methoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline (36). The title

compound was prepared from 6b and 2-(4-methoxyphenyl)ethan-1-amine following the general

1

procedure of B. Yield: 50.09 %, brown solid. H NMR (400 MHz, CDCl ) δ 7.24 (dd, J = 11.2, 4.6

3

Hz, 2H), 7.16 (t, J = 8.1 Hz, 1H), 7.12 (d, J = 8.1 Hz, 1H), 7.10 – 7.04 (m, 4H), 6.92 (t, J = 7.3 Hz,

1

H), 6.84 – 6.79 (m, 2H), 6.79 – 6.75 (m, 1H), 6.69 (t, J = 2.2 Hz, 1H), 6.56 (d, J = 2.2 Hz, 1H),

6

.51 (ddd, J = 7.6, 5.6, 2.0 Hz, 2H), 5.83 (s, 1H), 3.76 (s, 5H), 3.67 (s, 3H), 2.84 (dd, J = 10.9, 4.2

1

3

Hz, 2H), 2.77 (dd, J = 10.7, 3.9 Hz, 2H). C NMR (101 MHz, CDCl ) δ 158.71, 158.44, 158.06,

3

1

57.29, 144.99, 142.49, 131.96, 130.64, 130.31, 129.69, 129.38, 122.74, 121.54, 118.50, 113.90,

1

11.96, 110.61, 110.32, 107.37, 102.46, 55.34, 55.29, 50.33, 48.72, 35.08. ESI-MS: mass calcd for

+

[M + H] (C H N O ) 455.23; found m/z, 455.23. Purity 95.4% by HPLC.

29 30 2 3

3

-(2-methoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline (37). The title

compound was prepared from 6c and 2-(4-methoxyphenyl)ethan-1-amine following the general

1

procedure of B. Yield: 55.62 %, brown oil. H NMR (400 MHz, CDCl ) δ 7.24 – 7.20 (m, 2H),

3

7

.12 (t, J = 8.2 Hz, 3H), 7.06 – 7.02 (m, 2H), 6.96 – 6.88 (m, 3H), 6.85 – 6.78 (m, 3H), 6.71 (dd, J

8.0, 1.5 Hz, 1H), 6.64 (t, J = 2.2 Hz, 1H), 6.45 (dd, J = 7.9, 2.0 Hz, 1H), 5.76 (s, 1H), 3.80 (s,

=

1

3

1

H), 3.76 (s, 5H), 2.88 (t, J = 6.8 Hz, 2H), 2.78 (t, J = 6.9 Hz, 2H). C NMR (101 MHz, CDCl ) δ

3

59.21, 158.13, 151.46, 144.70, 143.78, 142.69, 136.86, 131.86, 130.09, 129.68, 129.32, 121.28,

21.08, 120.63, 118.30, 113.98, 112.67, 111.45, 109.28, 106.14, 56.02, 55.29, 53.54, 50.62, 35.21.

+

ESI-MS: mass calcd for [M + H] (C H N O ) 455.23; found m/z, 455.24. Purity 97.3% by

2

9

30

2

3

HPLC.

-(4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline (38). The title compound

3

was prepared from 6d and 2-(4-methoxyphenyl)ethan-1-amine following the general procedure of

1

B. Yield: 53.78 %, brown oil. H NMR (400 MHz, CDCl ) δ 7.25 – 7.19 (m, 4H), 7.14 (t, J = 8.1

3

Hz, 1H), 7.09 (d, J = 8.6 Hz, 2H), 7.03 (dd, J = 8.5, 1.0 Hz, 2H), 6.98 – 6.93 (m, 2H), 6.90 (dd, J

=

10.5, 4.2 Hz, 1H), 6.84 – 6.79 (m, 2H), 6.76 – 6.72 (m, 1H), 6.67 (t, J = 2.2 Hz, 1H), 6.51 – 6.47

(

m, 1H), 5.79 (s, 1H), 3.74 (s, 3H), 3.73 (s, 2H), 2.85 (dd, J = 10.8, 3.8 Hz, 2H), 2.74 (t, J = 7.0

1

3

Hz, 2H). C NMR (101 MHz, CDCl ) δ 158.64, 158.12, 156.01, 145.01, 142.56, 135.30, 132.06,

3

1

30.35, 129.71, 129.54, 129.42, 121.53, 119.11, 118.52, 114.01, 111.96, 110.70, 107.49, 55.31,

+

5

3.32, 50.76, 35.41. ESI-MS: mass calcd for [M + H] (C H N O ) 425.22; found m/z, 425.23.

2

8

28

2

Purity 98.2% by HPLC.

Synthesis of 4-(phenylamino)phenol (40). The title compound was prepared from 4-aminophenol

1

and bromobenzene following procedure the same as compound 5. Yield: 82.37 %, brown solid. H

NMR (400 MHz, CDCl ) δ 7.24 – 7.18 (m, 2H), 7.04 – 6.99 (m, 2H), 6.93 – 6.86 (m, 2H), 6.83 (t,

3

+

J = 7.3 Hz, 1H), 6.80 – 6.74 (m, 2H), 5.46 (s, 1H), 4.64 (s, 1H). ESI-MS: mass calcd for [M + H]

(

C H NO) 186.08; found m/z, 186.09.

12 11

Synthesis of 2,6-dimethoxy-4-(4-(phenylamino)phenoxy)benzaldehyde (41). The title compound

was prepared from 40 and 4-fluoro-2,6-dimethoxybenzaldehyde following general procedure A.

1

Yeid:76.55%, colorless oil. H NMR (400 MHz, CDCl ) δ 10.32 (s, 1H), 7.26 – 7.21 (m, 2H), 7.07

3

–

7.02 (m, 4H), 6.96 (d, J = 8.8 Hz, 2H), 6.93 (t, J = 7.4 Hz, 1H), 6.12 (s, 2H), 3.72 (s, 6H).

+

ESI-MS: mass calcd for [M + H] (C H NO ) 350.13; found m/z, 350.14.

2

1

19

4

-(3,5-dimethoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline (42). The

title compound was prepared from 41 and 2-(4-methoxyphenyl)ethan-1-amine following the

1

general procedure of B. Yield: 56.35 %, brown oil. H NMR (400 MHz, CDCl ) δ 7.25 (t, J = 7.8

3

Hz, 2H), 7.08 (t, J = 8.3 Hz, 4H), 7.01 (d, J = 8.1 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 6.90 (t, J =

7

.3 Hz, 1H), 6.81 (d, J = 8.5 Hz, 2H), 6.17 (s, 2H), 5.68 (s, 1H), 3.84 (s, 2H), 3.77 (s, 3H), 3.66 (s,

1

3

6

H), 2.81 (dd, J = 13.1, 5.6 Hz, 4H). C NMR (101 MHz, CDCl ) δ 159.38, 158.75, 157.99,

3

1

50.95, 143.82, 138.93, 132.05, 129.69, 129.40, 120.63, 120.28, 120.09, 116.99, 113.82, 109.89,

+

9

4.39, 55.66, 55.28, 49.97, 41.03, 34.86. ESI-MS: mass calcd for [M + H] (C H N O ) 485.24;

3

0

32

2

4

found m/z, 485.25. Purity 98.6% by HPLC.

4

-(3,5-dimethoxy-4-(((3-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline(43). The

title compound was prepared from 41 and 2-(3-methoxyphenyl)ethan-1-amine following the

1

general procedure of B. Yield: 51.21 %, brown oil. H NMR (400 MHz, CDCl ) δ 7.28 – 7.25 (m,

3

1

H), 7.24 (dd, J = 5.7, 3.8 Hz, 1H), 7.21 – 7.16 (m, 1H), 7.09 – 7.05 (m, 2H), 7.01 (dd, J = 8.6, 1.0

Hz, 2H), 6.98 – 6.93 (m, 2H), 6.90 (t, J = 7.3 Hz, 1H), 6.79 – 6.71 (m, 3H), 6.17 (s, 2H), 5.66 (s,

1

3

1

H), 3.85 (s, 2H), 3.77 (s, 3H), 3.65 (d, J = 7.9 Hz, 6H), 2.85 (dd, J = 12.5, 5.6 Hz, 4H). C NMR

(

101 MHz, CDCl ) δ 161.02, 159.93, 159.71, 149.71, 143.49, 139.68, 138.95, 129.77, 129.42,

3

1

21.13, 120.95, 120.90, 119.69, 117.32, 114.23, 112.53, 93.70, 55.82, 55.22, 47.29, 40.00, 32.87.

+

ESI-MS: mass calcd for [M + H] (C H N O ) 485.24; found m/z, 485.25. Purity 95.8% by

3

0

32

2

4

HPLC.

-(3,5-dimethoxy-4-(((2-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline(44). The

4

title compound was prepared from 41 and 2-(2-methoxyphenyl)ethan-1-amine following the

1

general procedure of B. Yield: 47.38 %, brown oil. H NMR (400 MHz, CDCl ) δ 7.19 (d, J = 7.2

3

Hz, 1H), 7.16 (s, 1H), 7.12 – 7.05 (m, 2H), 7.02 – 6.98 (m, 2H), 6.94 (dd, J = 8.5, 0.9 Hz, 2H),

6

.90 – 6.85 (m, 2H), 6.84 (d, J = 7.3 Hz, 1H), 6.80 – 6.77 (m, 1H), 6.75 (d, J = 8.2 Hz, 1H), 6.10

1

3

(s, 2H), 5.60 (s, 1H), 3.79 (s, 2H), 3.70 (s, 3H), 3.59 (s, 6H), 2.79 (s, 4H). C NMR (101 MHz,

CDCl ) δ 160.92, 159.60, 157.50, 149.69, 143.45, 139.75, 131.18, 129.42, 128.49, 125.30, 120.93,

3

1

20.86, 120.83, 119.65, 117.36, 110.48, 93.75, 55.82, 55.20, 45.68, 40.21, 28.44. ESI-MS: mass

+

calcd for [M + H] (C H N O ) 485.24; found m/z, 485.25. Purity 96.1% by HPLC.

3

0

32

2

4

-(3,5-dimethoxy-4-(((3-(4-methoxyphenyl)propyl)amino)methyl)phenoxy)-N-phenylaniline(45).

The title compound was prepared from 41 and 3-(4-methoxyphenyl)propan-1-amine following the

1

general procedure of B. Yield: 53.13 %, brown oil. H NMR (400 MHz, CDCl ) δ 7.28 (s, 1H),

3

7

.24 (s, 1H), 7.10 – 7.06 (m, 3H), 7.05 – 7.01 (m, 3H), 6.98 – 6.94 (m, 2H), 6.90 (dd, J = 11.6, 4.1

Hz, 1H), 6.81 – 6.77 (m, 2H), 6.18 (s, 2H), 5.69 (s, 1H), 3.94 (s, 2H), 3.76 (s, 3H), 3.73 (s, 6H),

1

3

2

1

.71 – 2.66 (m, 2H), 2.59 – 2.54 (m, 2H), 1.97 – 1.90 (m, 2H). C NMR (101 MHz, CDCl3) δ

60.89, 159.85, 157.92, 149.86, 143.53, 139.59, 132.78, 129.42, 129.29, 120.90, 120.87, 119.76,

+

1

17.28, 113.83, 93.71, 55.85, 55.24, 45.80, 39.55, 32.16, 28.18. ESI-MS: mass calcd for [M + H]

(

C H N O ) 499.25; found m/z, 499.26. Purity 95.8% by HPLC.

31 34 2 4

4

.2. Biological Assays

4

.2.1 Cell culture. All the cell licenced in this investigation were obtained from the American Type

o

Culture Collection (Manassas, VA, USA). Cells were cultured at 37 C with 5% CO in RPMI 1640,

2

L-15 Medium or DMEM, supplemented with 10%(v/v) fetal bovine serum (Gibco) and 1%(v/v)

penicillin-streptomycin (HyClone).

4

.2.2 Cell viability assay. Evaluation of cytotoxicity was performed using the MTT assay. Cells were

3

seeded at a cell density of 2-4 x10 cells/0.1 mL/well in a 96-well plate. The compounds were added 24

h plating at the indicated concentrations. After treatment for 72 h, a volume of 20 μL of MTT solution

o

(

5 mg/mL) was added to each well and incubated for additional 2-4 h incubation at 37 C. Then the

medium was discarded, and the formazan salt was dissolved with 150 μL DMSO for10-15 min. The

absorbance values (OD) of the 96-well plate was measured at 570 nm using a Spectra MAX M5

microplate spectrophotometer (Molecular Devices, CA, USA). The IC₅₀values were the means of at

least three independent experiments and calculated by GraphPad Prism5 software.

4

.2.3 Purification of c-Myc and Max. Human Myc bHLHZip domain (residues 353-439), and Max(S)

(

151 amino acids residues) were expressed in Escherichia coli strain BL21DE3 an N-terminal

hexa-histidine (His 6) tag proteins by backbone vector pET151D/-TOPO. Bacterial culture and protein

o

purification were conducted by previous published methods[61, 62], Bacteria were cultured at 37 C,

2

25 rpm for overnight and for another 20

h

to express proteins in 0.8 mM

isopropyl-L-thio-B-D-galactopyranoside (IPTG, Sigma). Then, cultures were harvested and lysed in a

buffer (8 M urea, 100 mM NaH PO , and 10 mM Tris), proteins were purified by NTA-Ni-agarose

2

4

chromatography columns (Qiagen, Inc. Chatsworth) and dialyzed in storage buffer(TrisHCl 50 mM, pH

.5, NaCl 150 mM, and 30% glycerol). Proteins were quantified by using Nanodrop and utilized in

Enzyme-Linked Immunosorbent Assay (ELISA) and Electrophoretic Mobility-Shift Assay (EMSA).

6

4

.2.4 Enzyme-Linked Immunosorbent Assay. Purified Myc bHLHZip domain (5.5 μg, 50 μL/well, in 1

x buffer: 10mM EDTA, 1 × PBS (pH = 7.0), 500 mM KCl, 30 mM MgCl , 5% glycerol, 0.1% NP40,

2

o

1

.5mM DTT) were coated on an ELISA plate (Costar) at 4 C for 24 h. The plates were rinsed with 1 x

buffer for three times and blocked with 200 μL (per well) of blocking buffer (3% BSA, 0.5 x buffer) for

o

2

h at 37 C. Then rinsing three times with 1 x buffer for a total 40min, 100 μL of 1 x buffer containing

Max (6.5 μg/per well) and different concentrations of compounds (10 μM, 30 μM, 60 μM, 90 μM, 120

μM, 150 μM.) were added to the wells, DMSO as vehicle control, c-Myc/MAX inhibitor sAJM589 as

positive control. All test mixtures contained 5% DMSO. The plates were incubated for 1 h at 37°C and

3

0 min at 23°C, wells were washed four times with 0.5 x buffer and incubated with rabbit anti-c-Myc

antibody (ab39688, Abcam) for 1 h at 37°C. Then washed three times with 1 x buffer, 100 μL of the

same buffer containing a secondary HRP goat anti-rabbit IgG (ab205718, Abcam) was added and

incubated for 1.5 hour at 37°C, tetramethylbenzidine (TMB, Sigma)was added to measure enzyme

activity, and the absorption at 450 nm was detected by Spectra MAX M5 microplate spectrophotometer

(

Molecular Devices, CA, USA). Data was analyzed by GraphPad Prism5 software.

4

5

.2.5 Electrophoretic Mobility-Shift Assay. EMSA were prepared as previously reported methods[24,

6], the double-stranded DNA biotinylated oligonucleotide (5-CACCCGGTCACGTGGCCTACAC-3,

0 nM) containing c-Myc/Max dimers binding site and purified proteins (Myc/Max complex, 50 nM)

were performed. Binding reaction buffer contained 10mM EDTA, 1 × PBS (pH = 7.0), 500 mM KCl,

0 mM MgCl , 5% glycerol, 0.1% NP40, 1.5mM DTT, 10%DMSO. Compounds were dissolved in

3

2

DMSO for test, DMSO as control. Proteins interaction complex and 20 μM compounds were prepared

for 1.5 h at room temperature, then added the DNA oligo for further 0.5 h. Data were detected by

BioRad FX molecular imager (BioRad, CA) and analyzed by Image J (Bio-Rad, USA).

4

.2.6 Molecular docking study. To apprehend the potential binding modes of 42 in c-Myc/Max,

molecular docking analysis was performed using Discovery Studio 3.5(DS 3.5, Accelrys Inc., San

Diego, CA, USA). The crystal structure of the Myc-Max (PDB ID:1NKP) was obtained from the

RCSB Protein Data Bank. The crystal structure of the c-Myc/Max domain were removed water

molecules and hydrogen atoms were added to the protein. In the course of docking, the binding site was

set 15 Å, and the other docking parameters were set as default value.

4

.2.7 Flow cytometry. Flow cytometry (FCM) was used to detect the cell cycle distribution and the cell

apoptosis rate induced by compound 42. HT29 cells and HCT15 cells were seeded in a 6-well plate at

5

3

×10 cells per well and incubated for 24 h, and treated with compound 42 for 24 h respectively. Cells

were harvested and fixed with ice-cold 70% ethanol at 4 ºC for 12 h, and then ethanol was removed and

the cells were washed with cold PBS. After that, the cells were detached from the plate using

trypsinization, collected by centrifugation at 1000 rpm for 5min. Then the cells were resuspended in

binding buffer and stained by Cell Cycle Detection Kit (keygentec, KGA512) and Annexin V-FITC/PI

double-labeling (keygentec, KGA107). The resulting samples were analyzed by flow cytometry using

an ACEA NovoCyte Advanteon (ACEA Biosciences Inc, USA).

4

.2.8 Western blot analysis. Cells were seeded in 60 × 15 mm dishes overnight prior to treatment and

incubated with the indicated doses of compound 42 for 24 h at 37 °C.Then, cell pellets were collected

and resuspended withNP40 lysis buffer (Beyotime), which was added with extra-proteasome inhibitor

PMSF and phosphatase inhibitor Cocktail(Sigma). Whole-cell protein lysates were incubated on ice for

3

0 min and centrifuged at 12000 rpm and 4 °C for 20 min. The supernatants were determined using the

BCA ProteinAssay Kit (Solarbio PC0020). The samples were separated by SDS-PAGE (sodium

dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to polyvinylidene fluoride

membranes (Millipore). The membranes were incubated at 4 °C overnight with the primary antibodies

in 5% BSA/TBST buffer with gentle shaking. After washing, the membranes were incubated for 1 h at

room temperature with appropriate peroxidase-conjugated secondary antibodies (anti-IgG-HRP1:5000,

CST) and washed again, protein bands were visualized using enhanced ECL western blotting detection

reagents (Millipore) followed by capturing photos with chemiluminescence system. Antibody

information: c-Myc Antibody (1/1000dilution, CST#9402), β-actin (1/1000dilution, CST#4970).

4

.2.9 Colony formation assay. To test the survival of cells treated with compound 42, HT29 cells and

o

HCT15 cells (500 cells/well) were plated in a 6-well plate and incubated overnight at 37 C, followed

by the indicated doses of compound 42 (0 - 5.0 μM, DMSO as control) for 15 days with fresh medium.

The cell culture was terminated when the cell formed colony was observed, then the supernatant was

removed and washed with PBS buffer solution for two times, then it was fixed with 4%

polyformaldehyde for 15 min, before the solution was abandoned. Colonies were fixed with 4%

paraformaldehyde and stained with a 0.5% crystal violet solution for 15 min, then the crystal violet

solution was removed and PBS buffer solution was used to scour off the dyeing liquor. The data were

expressed from three independent experiments. The number of colonies in treated cultures was

expressed as a percentage of the control cultures.

4

.2.10 In Vivo Study. Animal studies were conducted under the approval of the Experimental Animal

Management Committee of Sichuan University. The female BALB/c nude mice were purchased

6

(

Beijing HFK Bioscience Co. ltd., Beijing, China). HT29 cells (8× 10 ) were injected subcutaneously

into 6−7 weeks old female BALB/c mice. Once the average tumor volume grew to approximately 100

3

mm , the mice were divided into five groups randomly (n=5, each group). The mice were treated daily

for 30 days by via oral gavage administration with vehicle control(10%DMSO, 1%Tween-80 and

saline), 42(40mg/kg, 80mg/kg, 120mg/kg dissolved in 10%DMSO, 1%Tween-80 and saline),

5

-Fluorouracil (positive control, 30mg/kg dissolved in 10%DMSO, 1%Tween-80 and saline) .Tumor

size and body weights were determined every two days. The tumor volume was measured by Vernier

2

calipers and calculated as [0.5 x shortest diameter x longest diameter]. Inhibition rate of tumor growth

was calculated using the following formula: 100 × {1 − [(tumor volume final − tumor volume initial)

for treated group]/ [(tumor volume final − tumor volume initial) for the vehicle-treated group]}.

4

.3 Statistical analysis. Data were analyzed by using descriptive statistics, single factor analysis of

variance (ANOVA), and presented as mean values ± standard deviation (SD). Statistical analysis of all

results (mean ± SEM) was evaluated by unpaired t test: *p < 0.05; **p < 0.01; ***p < 0.001, NS (no

significance): p > 0.05.

NOTES

The authors declare no competing interest.

ACKNOWLEDGMENTS

This work was supported by National Natural Science Foundation of China (grants 81773195 and

8

1472780).

Scheme 1. Design of series derivatives based on c-Myc inhibitors.

O

H

F

O

F

2

3

R1

R4

^R1

H

H2N

OH

N

OH

N

O

R₂

O

N

O

R2

H

^R5

R3

R₃

R₄

4

5

6

7-38

6a R1=R3=H, R2=R4=OCH3

6b R1=R3=R4=H, R2=OCH3

6c R2=R3=R4=H, R1=OCH3

6d R1=R2= R3=R4=H

1 3 2

7-35 R =R =H, R =R₄=OCH₃

36

37

38

1 3 4

R =R =R =H, R₂=OCH₃

R =R =R =H, R₁=OCH₃

2

3

4

R =R = R =R₄=H

1

2

3

7

-35, R5=:

R5=

OH

O

Cl

Br

H

N

13 R5=

N

19 R5=

20 R5=

21 R5=

H

25 R5=

7

N

31 R₅=

N

H

OH

O

H

N

H

N

8

9

R5=

14 R5=

15 R5=

26 R5=

27 R5=

32 R5=

33 R5=

N

H

OH

O

H

N

H

N

H

N

H

Cl

N

H

N

1

0

1

R5=

16 R5=

17 R5=

OH

22 R5=

23 R5=

28 R5=

29 R5=

N

34 R5=

35 R5=

H

N

H

O

CH3

O

F

O

OH

H

N

H

1

R5=

N

O

H

F

^C^H3

O

H

N

12

R5=

N

18 R5=

24 R5=

H

N

30 R5=

N

H

O

36-38, R5=:

N

H

Scheme 2. Synthetic Routes for Compounds 7-38

o

Reagents and conditions: (Ⅱ ) POCl , DMF, 0 C to 60 C, 4 h. 62%; (Ⅱ ) bromobenzene, t-BuONa, BrettPhos

3

o

Palladacycle, 1,4-dioxane, 90 C, 2h. 86%; (Ⅱ ) substituted 4-fluorobenzaldehydehyde, K CO , DMSO, 100 C, 3 h.

2

3

o

7

3%-85%; (Ⅱ ) Amine, NaBH(AcO) , AcOH, DMF, 80 C, overnight. 45%-60%.

3

Scheme 3. Synthetic Routes for Compounds 42-45

Reagents and conditions: (Ⅱ ) bromobenzene, t-BuONa, BrettPhos Palladacycle, 1,4-dioxane, 90 C, 2h. 82%; (Ⅱ )

o

1

-Fluoro-3,5-dimethoxybnzene, K CO , DMSO, 100 C, 3 h. 76%; (Ⅱ ) Amine, NaBH(AcO) , AcOH, DMF, 80 C,

2 3 3

overnight. 47%-56%.

Table 1. In vitro antiproliferative activities

a

Compound

IC (μM)

50

Cell lines

HCT116

HT29

HCT15

8.93±0.62

DLD-1

SW620

7

8

9

7.43±0.81

>20

11.87±1.23

>20

15.04±1.52

>20

12.28±1.17 6.14±0.56

>20

1

0

15.61±1.02 12.86±0.95

>20

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

2

3

4

5

>20

8.97±0.49

5.09±0.33

6.43±0.57

5.09±0.43

6.51±0.18

>20

1

2

3

4

3.96±0.17

>20

18.2±1.82

>20

6.01±0.32

>20

11.93±1.07

>20

3.53±0.07

>20

7.37±0.67

18.13±2.21

5.22±0.16

2.65±0.72

3.74±0.42

3.52±0.50

2.69±0.15

1.91±0.23

6.08±0.82

8.57±1.36

8.57±0.95

3.50±0.56

6.77±0.49

3.40±0.13

2.87±0.35

1.86±0.09

4.03±0.26

1.12±0.05

2.32±0.29

2.35±0.12

2.54±0.06

4.06±0.28

5.92±0.19

4.65±0.06

1.31±0.26

5.47±0.08

3.59±0.27

4.65±0.22

15.22±2.16

11.40±1.32

>20

13.88±2.01 12.13±1.36

>20

3.34±0.67

2.75±0.33

3.35±0.14

3.06±0.38

2.18±0.12

1.89±0.14

3.88±0.18

8.50±0.62

5.12±0.58

1.63±0.04

6.06±0.47

2.82±0.28

2.52±0.18

1.25±0.06

2.93±0.08

1.02±0.21

2.12±0.38

2.33±0.31

3.32±0.21

3.97±0.08

2.75±0.25

6.89±0.14

0.32±0.05

3.62±0.14

2.21±0.19

6.89±0.36

5.02±0.23

3.64±0.25

4.85±0.29

5.41±0.46

3.43±0.28

2.65±0.12

5.40±0.39

8.93±0.83

7.55±0.77

4.69±0.24

6.14±0.30

3.76±0.12

2.34±0.09

2.88±0.16

6.14±0.39

1.25±0.13

1.19±0.34

1.29±0.06

0.73±0.18

1.02±0.05

3.35±0.51

2.18±0.12

0.51±0.09

1.65±0.17

3.77±0.24

2.18±0.13

4.07±0.82

4.22±0.38

3.31±0.36

6.23±0.31

3.36±0.39

2.62±0.55

13.81±1.95

>20

7.49±0.54

6.77±0.47

6.34±0.87

11.21±0.11

7.94±0.92

6.97±0.46

11.79±2.28

>20

18.67±1.82

8.25±0.37

11.62±1.39

10.04±0.72

8.95±0.14

5.77±0.41

12.70±1.82

6.23±0.43

3.65±0.12

3.93±0.09

6.40±0.68

7.84±0.06

10.96±1.33

14.60±1.85

3.78±0.54

6.12±0.31

8.76±0.51

14.60±2.11

17.35±1.62

15.29±1.48

2.44±0.18

13.63±0.93

5.40±0.39

3.19±0.13

3.32±0.22

15.73±2.15

0.98±0.12

3.40±0.18

4.02±0.24

2.85±0.13

2.67±0.51

3.11±0.04

3.99±0.27

0.82±0.32

3.91±0.26

4.1±0.16

3.23±0.08

13.78±1.21

Cisplatin

10.75±0.97 8.92±0.32

5

-Fluorouracil 13.37±1.08 10.86±0.91 12.31±1.51

a

IC50, the mean ± SD value of triplicate measurement, inhibitory activity was assayed by treatment with

substances for 72 h.

Table 2. The compounds binding inhibition of Myc/Max dimerization confirmed by ELISA

a

Compound

ELISA IC (μM)

Compound

ELISA IC (μM)

5

0

50

7

8

9

>150

26

27

108±25

96±18

92±20

95±25

87±12

83±14

69±15

72±19

78±16

147±21

102±17

107±14

116±15

63±13

75±16

147±21

72±15

36±9

>150

28

1

0

1

>150

29

1

>150

30

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

>150

31

>150

32

>150

33

147±21

>150

34

35

125±15

137±20

123±18

129±17

123±16

86±15

119±20

105±18

116±22

36

37

38

42

43

44

45

sAJM589

a

IC50, the mean ± SD value of triplicate measurement.

Table 3. The compounds binding inhibition rates at 20 μM detected by EMSA

a

Compound

Relative inhibition (%)

Compound

Relative inhibition (%)

7

8

9

2.5

1.7

26

27

36.1

45.1

43.7

40.5

46.2

51.7

75.1

67.4

60.8

63.4

42.1

42.3

35.6

83.2

65.2

57.3

70.6

0

b

N/A

28

1

0

1

3.2

29

1

6.1

30

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

10.2

N/A

8.9

31

32

33

12.6

N/A

26.4

21.2

25.7

29.3

31.8

48.7

32.4

42.1

30.5

34

35

36

37

38

42

43

44

45

DMSO

a

Relative inhibition was calculated by the ratio of experimental group and control (DMSO)

Not tested

b

All data represent three independent experiments.

N

O

N

H

N

O

N

O

N

NO

2

HN

NO

2

SH

O

S

SF-4-017

1

0058-F4

10074-G5

O

NO

2

H N

N

NH

O

F

O

OH

N

NH

N

O

F

Cl

O

F

O

NUCC-0176242

Mycro 3

KJ Pyr 9

O

N

O

N

O

N

NH

F

O

N

Indenoisoquinoline 5

IZCZ-3

Figure 1. Chemical structures of previously reported c-Myc inhibitors.

Figure 2. The effect of compound 42 on CRC cells and normal liver cell LO2 viability. (A) The compound 42

with IC50 in HT29, HCT15, HCT116, DLD-1, respectively. (B) Toxicities of 42, 5-Fluorouracil, and Cisplatin

against LO2. The data were expressed from three independent experiments (N = 3 per group, mean ± SD).

Figure 3. (A) Predicted binding modes of 42(green) with key amino acid residues of c-Myc/Max. (B) Predicted

binding modes of 42 in c-Myc/Max dimer face. (C) In silico binding mode of 42 complex with Myc-Max-DNA.

(PDB code: 1NKP).

Figure 4. Colony formation assay of 42 in HT29, HCT15. The colony clusters were detected after 15days

treatment with 42.

A

B

Figure 5. Flow cytometry of compound 42. (A) Cells were cultured with 42 from 0 to 5 μM for 24 h, the cell

cycle distribution was analyzed by flow cytometry after propidium iodide staining. (B) Cells were treated with

indicated concentrations of compound 42 for 24 h, and the level of apoptosis was evaluated by FITC-Annexin V/PI

and analysis of apoptotic cells analyzed by flow cytometry. Data shown were from three independent experiments

(N = 3 per group, mean ± SD). *P < 0.05 vs DMSO, **P < 0.01 vs DMSO; ***P < 0.001 vs DMSO.

Figure 6. Effects of 42 on c-Myc expression was determined by Western blotting. Cells were treated with

indicated dose of 42 for 24h. All of the experiments were repeated three independent times

Figure 7. In vivo efficacy of 42 in HT29 tumor xenograft model (N = 5 per group, mean ± SD). (A) Growth

inhibitory effect of 42 treated on HT29 xenograft tumor in female BALB/c nude mice. (B) Average body weights

of the mice in each group during 30days treatment. (C) Tumor weights of the mice in each group when the

treatment ended. **P < 0.01 vs DMSO; ***P < 0.001 vs DMSO.

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Article Doi

Discovery of 4-(3,5-dimethoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline as a novel c-myc inhibitor against colorectal cancer in vitro and in vivo

DOI: 10.1016/j.ejmech.2020.112336

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