Drug repurposing and rediscovery: Design, synthesis and preliminary biological evaluation of 1-arylamino-3-aryloxypropan-2-ols as anti-melanoma agents

Article abstract of DOI:10.1016/j.bmc.2020.115404

Malignant melanoma (MM) presents as the highest morbidity and mortality type in skin cancer. Herein, inspired by the previously reported anti-melanoma effect of propranolol, a widely applied β adrenergic receptor antagonist as cardiovascular drug, we set out to exploit its potential as anti-melanoma therapy based on the drug repurposing strategy. Structural optimization of propranolol yielded 5m, which exhibits dramatically improved potency on human melanoma cell growth (1.98–3.70 μM), compared to propranolol (59.5–75.8 μM). Further investigation demonstrated that 5m could inhibit colony formation of melanoma cell line (completely abolished at 2 μM for 5m, partially inhibited at 50 μM for propranolol), induce cell apoptosis and cell cycle arrest in the G₂/M phase (both observed at 1 μM). Preliminary mechanism study indicated that 5m could disrupt the cellular microtubule network, which suggested tubulin as a potential target. Docking study provided a structural insight into the interaction between 5m and tubulin. In summary, our study presents a drug repurposing case that redirects a cardiovascular agent to an anti-melanoma agent.

Full text of DOI:10.1016/j.bmc.2020.115404

Journal Pre-proofs
Drug repurposing and rediscovery: Design, synthesis and preliminary biologi-
cal evaluation of 1-arylamino-3-aryloxypropan-2-ols as anti-melanoma agents
Qi Chang, Jing Long, Liqing Hu, Zhuo Chen, Qianbin Li, Gaoyun Hu
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S0968-0896(20)30209-1
https://doi.org/10.1016/j.bmc.2020.115404
BMC 115404
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Bioorganic & Medicinal Chemistry
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28 December 2019
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28 February 2020
Please cite this article as: Q. Chang, J. Long, L. Hu, Z. Chen, Q. Li, G. Hu, Drug repurposing and rediscovery:
Design, synthesis and preliminary biological evaluation of 1-arylamino-3-aryloxypropan-2-ols as anti-melanoma
agents, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115404
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Drug repurposing and rediscovery: Design, synthesis and
preliminary biological evaluation of 1-arylamino-3-
aryloxypropan-2-ols as anti-melanoma agents
Qi Changa, Jing Longb, Liqing Hua, Zhuo Chena, Qianbin Lia, Gaoyun Hua,*
a
Department of Medicinal Chemistry, Xiangya School of Pharmaceutical Sciences, Central South
University, Changsha 410013, China
b Department of Dermatology, Xiangya Hospital, Central South University, Changsha 410008, China
Abstract
Malignant melanoma (MM) presents as the highest morbidity and mortality type in skin cancer. Herein,
inspired by the previously reported anti-melanoma effect of propranolol, a widely applied β adrenergic
receptor antagonist as cardiovascular drug, we set out to exploit its potential as anti-melanoma therapy
based on the drug repurposing strategy. Structural optimization of propranolol yielded 5m, which
exhibits dramatically improved potency on human melanoma cell growth (1.98 – 3.30 μM), compared to
propranolol (59.5 – 75.8 μM). Further investigation demonstrated that 5m could inhibit colony formation
of melanoma cell line (completely abolished at 2 μM for 5m, partially inhibited at 50 μM for propranolol),
induce cell apoptosis and cell cycle arrest in the G2/M phase (both observed at 1 μM). Preliminary
mechanism study indicated that 5m could disrupt the cellular microtubule network, which suggested
tubulin as a potential target. Docking study provided a structural insight into the interaction between 5m
and tubulin. In summary, our study presents a drug repurposing case that redirects a cardiovascular agent
to an anti-melanoma agent.

Graphical Abstract
O
N
O
N
H
H
OH
OH
N
H
Repurposing
Propranolol
5m
anti-melanoma agent
-adrenergic receptor antagonist
O
● Improved cell growth inhibition
● Cell migration inhibition
● Apoptosis induction
O
N
H
OH
O
● Cell cycle arrest
● Mircrotubule network disruption
● Predicted binding mode with tubulin
N
H
Carvedilol
Keywords
melanoma, drug repurposing, β adrenergic receptor antagonists, microtubule

1. Introduction
Malignant melanoma (MM) ranks the first global incidence and mortality in skin cancer with enormous
increasing occurrence over the past few decades. [1,2] In 2018, approximately 280,000 cases and 60,000
deaths of melanoma were reported worldwide. [1,3-7] Patients with late-stage MM is generally
associated with poor prognosis, the 5-year survival is <5%. [4-6] Currently, the main strategies for the
treatment of MM are surgery, chemotherapy and biological therapy, but the severe toxicity and recurrent
relapse are prevalent adverse effects, mainly due to the unselectively attachment to both normal and
tumor tissue and oncogene mutation. [7] In recent years, targeted therapy has been emerged as a
beneficial choice for patients of late-stage MM, which comprise small-molecule kinase inhibitors
(vemurafenib, trametinib, etc.) and monoclonal antibodies (ipilimumab, nivolumab, etc.) known as
immune checkpoint inhibitors. [4,8] Mutationally activated kinases in mitogen-activated protein kinase
(MAPK) pathway confer cancer cell unlimited proliferation propensity. [8-10] Thus, the development of
inhibitors against the signaling mediators, especially BRAF and MEK, has led to widely accepted clinical
benefit in the treatment of patients with MM. [9,10] However, multifactorial resistance (oncogene
mutation, immune escape, etc.) to these inhibitors often occurs in a few months (for vemurafenib at least
in half a year in 60% patients) which lead to therapeutic failure. [10] As alternative strategy, antibodies
that block the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or the programmed cell death 1
(PD-1) pathway have been proven to elicit long-lasting antitumor responses in patients with MM in recent
years. [11-13] These advances notwithstanding, yet about one-third patients relapse and mechanism of
required resistance are currently poorly understood, and immune-related side effects have been widely
reported [14,15]. In a word, these unmet needs for MM treatment prompted research efforts to the
development of novel therapy.
There is a broad consensus that new drug development is a long, expensive and complex process, which
costs of 2.6 billion USD and takes over 12 years on average. [16] New way for cutting down the
expenditure and speeding up the development course has been presented as an urgent and challenging
problem toward both academia and pharmaceutical industry. Drug repurposing, also known as drug
reprofiling or repositioning, has been emerged as an attractive approach for identifying new applications
of approved drugs or clinical candidates that beyond their original indications. [17] The example of
repurposing of thalidomide for multiple myeloma and erythema nodosum leprosum has substantially
proved the success of this proposition. [18]
Recently, accumulated studies demonstrated that β adrenergic receptor antagonists (β-blockers), a drug
category extensively applied for the treatment of arrhythmia, angina pectoris, and hypertension, exhibit
anticancer activities in vitro, in vivo, and in clinic. [19-22] A series of studies suggested the potency of
propranolol, a non-selective β-blocker in multiple cancers, including melanoma [23], prostate cancer [24],
non-small cell lung cancer (NSCLC) [25], breast cancer [26], and liver carcinoma [27]. Up to date, many
reported cancer treatments involving β-blockers were incidental use, and some cancer types have even
no response to β-blockers [28], thus the therapeutic benefit for these diseases is required to be elucidated,
further cohort studies are urgent. However, a great body of clinical studies of the efficacy of β-blockers
on melanoma was reported. [29] A cohort study published on JAMA Oncology indicated that the use of

propranolol (80 mg daily) from the time of diagnosis significantly delay the recurrence of melanoma,
and the risk for users was reduced about 80%. [30] Furthermore, two cohort study with 2.5- and 8-years
follow-up observed that only 3% and 30% patients treated with β-blockers showed melanoma
progression, respectively. These clinical data confirmed that the use of β-blockers significantly reduce
the recurrence and mortality in melanoma patients. [31,32] Moreover, propranolol could inhibit cell
proliferation, induce cell apoptosis and cell arrest in several melanoma cell lines and patient-derived
primary cells in vitro [33,34]. However, despite these considerable anticancer effects, the pronounced
activity of β-blockers like propranolol on melanoma cell proliferation required long-time treatment (72
h) and high dose (100 μM). [35] These distinctions observed between cellular and in vivo models
indicated undiscovered multiple mechanisms involving in the anticancer effects of this drug category.
In this study, we made our efforts to find a novel series of anti-melanoma agents repurposed from β-
blockers with improved potency, which led to the discovery of 5m. Further biological evaluation and
preliminary mechanistic investigation were also discussed.

2. Results and Discussion
2.1. Chemistry
Synthesis of target compounds is described in Scheme 1. To obtain the 1-arylamino-3-aryloxypropan-2-
ols, phenols reacted with epichlorohydrin via a nucleophilic substitution to yield corresponding 2-
(aryloxymethyl)oxiranes according to previous literature. [36,37] The racemic target compounds were
afforded by using excess amines to open the oxirane ring with modest yields (Table 2). To obtain the
asymmetric compounds, commercially available asymmetric epichlorohydrin were used to form the
corresponding intermediates, the reaction with amines giving the desired products. This procedure is
previously reported and asymmetric species has been characterized by X-ray crystallography. [37] To
afford the aryl-3-aryloxypropan-1-amines, stepwise nucleophilic substitutions were conducted starting
from phenols. The target compounds were obtained by reacting with excess amines at a moderate to good
yield (Table 2).
Scheme 1. Synthetic routes of target compounds
R1
R1
a
b
R2
Cl
R1 OH
O
O
O
O
OH
1a-1e
2
3a-3e
R1
5a-5o
R1
R2
1a, 3a
1b, 3b
1c, 3c
1d, 3d
1e, 3e
5a
5b
5c
5d
5e
5f
5g
5h
5i
naphthalen-1-yl
naphthalen-2-yl
9H-carbazol-4-yl
[1,1'-biphenyl]-4-yloxy
(3-(phenylamino)phenly
naphthalen-1-yl
naphthalen-2-yl
9H-carbazol-4-yl
9H-carbazol-4-yl
9H-carbazol-4-yl
9H-carbazol-4-yl
9H-carbazol-4-yl
[1,1'-biphenyl]-4-y
[1,1'-biphenyl]-4-yl
[1,1'-biphenyl]-4-yl
[1,1'-biphenyl]-4-yl
9H-carbazol-4-yl
2-(2-methoxyphenoxy)ethyl)amino
2-(2-methoxyphenoxy)ethyl)amino
2-methoxyphenyl)piperazin-1-yl
4-phenylpiperazin-1-yl
4-(pyridin-2-yl)piperazin-1-yl
([1,1'-biphenyl]-4-ylmethyl)amino
[1,1'-biphenyl]-4-ylamino
isopropylamino
2-(2-methoxyphenoxy)ethyl)amino
4-(pyridin-2-yl)piperazin-1-yl
4-phenylpiperazin-1-yl
5j
5k
5l
4-phenylpiperazin-1-yl
5m
5n
5o
(3-(phenylamino)phenly ([1,1'-biphenyl]-4-ylmethyl)amino
9H-carbazol-4-yl
9H-carbazol-4-yl
([1,1'-biphenyl]-4-ylmethyl)amino
2-(2-methoxyphenoxy)ethyl)amino
R1
O
R1
d
c
R1 OH
Br
Br
Br
O
R2
1a, 1b
6
7a, 7b
8a-8g
R1
9H-carbazol-4-yl
R1
R2
1a, 7a
1b, 7b
8a
8b
8c
8d
8e
8f
9H-carbazol-4-yl
9H-carbazol-4-yl
9H-carbazol-4-yl
9H-carbazol-4-yl
9H-carbazol-4-yl
2-(2-methoxyphenoxy)ethyl)amino
2-methoxyphenyl)piperazin-1-yl
4-phenylpiperazin-1-yl
4-(pyridin-2-yl)piperazin-1-yl
([1,1'-biphenyl]-4-ylmethyl)amino
(3-(phenylamino)phenly
(3-(phenylamino)phenly ([1,1'-biphenyl]-4-ylmethyl)amino
8g
9H-carbazol-4-yl
2-methylquinolin-8-amino
Reagents and conditions: a. NaOH (1 M), DMSO : H2O = 2 : 1 (V/V), RT, 2h; b. i-PrOH, reflux, 5h; c.
KI, K2CO3, CH3CN, RT, 2h; d. corresponding amines, CH3CN, reflux, 5h.
2.2. Biological Activity

2.2.1. Structure-Activity Analysis of β adrenergic receptor antagonists
Our investigation began with a brief screening of ten approved β adrenergic receptor antagonists (β-
blockers, propranolol, carvedilol, carazolol, atenolol, sotalol, bisoprolol, timolol, esmolol, metoprolol,
and labetalol). We determined their effects on the cell growth in three human malignant melanoma cell
lines (SK-MEL-5, SK-MEL-28, and A375) maintained in our laboratory using a 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
As shown in Table 1, seven β-blockers (atenolol, sotalol, bisoprolol, timolol, esmolol, metoprolol, and
labetalol) exhibited very low cell growth inhibition in the tested melanoma cell lines with IC50 values
more than 100 μM. Among tested drugs, naphthalene-containing propranolol showed cell growth
inhibition with IC50 values of 59.5 – 75.8 μM. Despite harboring an isopropyl, the introduction of
naphthalene covered the effect of this aliphatic group. To our surprise, our screening demonstrated that
carvedilol, an α1,β-blocker, is the most potent with IC50 values of 13.0 – 15.5 μM. Structurally, carbazole,
a widely reported pharmacophore in anticancer agents [38,39], displaced the naphthalene ring in
propranolol, and the isopropyl was replaced by a 2-(2-methoxyphenoxy)ethan-1-amine moiety.
Carazolol, another β-blocker, exhibited a moderate potency with IC50 values of 31.3 – 35.4 μM, which
fused with the carbazole core from carvedilol and the isopropyl from most β-blocker. These results
primarily indicated the significance of the aromaticity addiction into the molecule, so our medicinal
chemistry journey was initiated from the optimization of propranolol and carvedilol.
Table 1. Structures and cell growth inhibitory activities of β adrenergic receptor antagonists in
melanoma cell lines
IC50 (μM) in cell growth inhibition or cell growth
inhibition rate at 100μMa
Compound
SK-MEL-5
SK-MEL-28
A375
OH
H
N
O
Propranolol
Carvedilol
62.21 ± 0.54
75.82 ± 5.55
59.50 ± 4.25
O
O
OH
H
N
O
13.73 ± 0.20
33.74 ± 4.22
>100 (5.2%)
15.55 ± 0.11
31.28 ± 0.93
>100 (6.9%)
13.01 ± 0.78
35.42 ± 0.45
>100 (8.6%)
HN
OH
H
N
O
Carazolol
Atenolol
HN
OH
H
N
O
O
H2N

OH
H
N
O
Sotalol
>100 (2.1%)
>100 (14.8%)
>100 (7.4%)
>100 (6.4%)
>100 (15.2%)
>100 (28.3%)
S
N
O
H
OH
H
O
N
Bisoprolol
O
O
OH
H
N
O
N
S
Timolol
>100 (33.3%)
>100 (18.6%)
>100 (22.7%)
N
N
O
OH
H
N
O
Esmolol
>100 (31.4%)
>100 (38.8%)
>100 (25.8%)
>100 (30.0%)
>100 (22.8%)
>100 (26.3%)
O
O
OH
H
N
O
Metoprolol
O
OH
H
N
Labetalol
>100 (36.2%)
>100 (35.6%)
>100 (38.1%)
HO
O
NH2
a Data are presented as mean ± SD, n=3, from three independent experiments.
2.2.2. Antiproliferative Assay and SAR Study
Our structural optimization on propranolol and carvedilol was focused on three parts: the two aromatic
terminus moieties and the atomic linker. As described above, the changes of either sides to an aromatic
ring showed improved potency, thus we set out to find the discrepancy from the introduction of various
ring to the both sides. We first fixed the amino portion to find out good fragments at ether moiety, then
investigated another side. Moreover, the hydroxyl in the atomic linker in the propan-2-ol provided a
symmetric center, so we would also like to access the necessity to potency of the hydroxyl.
First, we merged the naphthalene ring from propranolol with the 2-(2-methoxyphenoxy)ethan-1-amine
moiety from carvedilol, which resulted in 5a with IC50 values of 10.1 – 18.9 μM in cell growth inhibition
being similar to carvedilol (Table 2). Then, the propan-2-ol linker was tethered to the β-position in the
naphthalene, which yielded 5b with somewhat decreased potency (18.5 – 21.1 μM). In this case,
naphthalene was not considered to be employed in the further investigation. Due to the relatively long
size of carvedilol, the ethan-1-amine moiety was rigidified and resulted in 5c, which harbored a N-
phenylpiperazine. The cell growth inhibition of 5c was elevated (13.6 – 14.1 μM) compared with
carvedilol. The removal of methylhydoxyl on the benzene ring yielded 5d (16.3 – 18.5 μM) with
attenuated potency. To investigate the effect of heterocycle at benzene region, introduction of N atom at
the site in methylhydoxyl resulted in 5e, which showed a further decreased potency (24.7 – 31.8 μM),
this might result from the negative effect of mitigated electron density compared with benzene ring. To

further increased the aromaticity, the N-phenylpiperazine was replaced by a diphenyl moiety, which
produced 5f. To our delight, 5f exhibited dramatically increased potency in cell growth inhibition with
IC50 values of 3.1 – 5.6 μM, which was about 15-fold more potent than carvedilol. Deletion of the benzyl
carbon obtained 5g with IC50 values of 12.4 – 13.9 μM, which indicated that the flexibility of diphenyl
moiety played an important role in cell growth inhibition. As an attractive pharmacophore, quinoline has
been widely used in anticancer agents [40], thus we employed a previously reported 2-methylquinolin-
8-amine moiety which afforded 5l (14.1 – 14.5 μM). However, 5l showed less potency than parent lead
carvedilol. As diphenyl has been appeared as a perfect fragment, it was moved to the ester region on the
other side of the propan-2-ol linker. We still started from the isopropyl in carazolol to investigate the
importance of diphenyl which led to 5h-5k. As expected, these compounds exhibited elevated cellular
potency ranging from 5.9 to 28.2 μM (Table 2), but showed negligible improvement. Compound 5f has
a planner carbazole ring, we want to know whether the open ring could make difference. Gratifyingly,
5m appeared a 3-fold increased potency compared with 5f in cell growth inhibition with IC50 values of
2.0 – 3.7 μM. Two asymmetric compounds (5n and 5o) with (S)-configuration were prepared according
to previously reported procedure [37]. However, their potency had no obvious distinction compared with
racemic mixture (Table 2).
Next, we investigated the role of hydroxyl in our series of compounds, selected counterparts were
obtained. The removal of the hydroxyl in carvedilol resulted in 8a (IC50 = 6.2 – 6.9 μM) showed 2-fold
increased potency. Other selected non-hydroxyl products were also obtained. The potency of 8b (IC50
=
10.8 – 13.7 μM) had no discrepancy compared with 5c. The two couples of counterparts (8c and 8d) with
just one N atom distinction exhibited slightly improved activity. Carbazole ring and ring-open
counterparts (8e and 8f) showed similar potency. In this section, the hydroxyl could have no significant
effect on cell growth inhibition. However, the maintained hydroxy may provide more drug-like
physiochemical properties and addicted interactions with the biological targets of the compounds. To
summarize, the introduction of diphenylamine and diphenyl to the two terminuses dramatically improve
the cellular potency, which proved the benefit of aromaticity addition into the molecule. Besides, the
hydroxyl group which is necessary for β adrenergic receptor antagonism has negligible impact on the
anti-melanoma activity.
Table 2. Structures and cell growth inhibitory activities of target compounds in melanoma cell
lines
IC50 (μM) in cell growth inhibitiona
Compound
Yield (%)
SK-MEL-5
SK-MEL-28
A375
O
O
O
N
H
OH
5a
5b
86
10.13 ± 0.85
13.45 ± 0.84
18.87 ± 0.47
O
O
N
H
OH
O
82
18.54 ± 0.76
19.71 ± 0.48
21.09 ± 0.14

O
O
O
N
N
N
OH
OH
OH
N
O
5c
5d
5e
5f
72
80
81
75
13.58 ± 0.27
17.02 ± 0.15
26.05 ± 0.66
3.14 ± 0.15
14.07 ± 1.07
18.53 ± 0.61
24.70 ± 1.80
3.80 ± 0.06
13.90 ± 0.67
16.33 ± 0.31
31.76 ± 1.84
5.56 ± 0.13
N
H
N
N
N
H
N
N
H
O
N
H
OH
N
H
O
N
5g
5h
5i
73
69
78
81
75
13.86 ± 0.17
12.01 ± 0.39
5.86 ± 0.06
21.22 ± 2.13
12.21 ± 1.98
12.45 ± 0.05
11.88 ± 0.60
6.01 ± 0.22
25.01 ± 1.33
15.33 ± 0.83
13.48 ± 0.06
10.98 ± 0.91
8.37 ± 0.22
28.23 ± 0.95
14.28 ± 1.02
H
OH
N
H
O
N
H
OH
O
O
O
N
H
OH
O
N
OH
N
N
5j
O
N
OH
N
5k
O
N
H
5l
5m
5n
72
71
74
14.50 ± 0.39
3.70 ± 0.17
2.69 ± 0.18
14.25 ± 0.09
3.30 ± 0.06
4.99 ± 0.15
14.13 ± 0.40
1.98 ± 0.10
5.22 ± 0.07
OH
N
N
H
O
N
H
OH
N
H
O
N
H
OH
N
H

O
O
O
O
N
H
OH
5o
8a
8b
8c
8d
8e
8f
81
83
81
85
82
76
70
72
7.04 ± 0.93
6.18 ± 0.17
13.69 ± 1.18
36.86 ± 2.13
11.48 ± 1.26
4.28 ± 0.05
4.72 ± 0.08
16.38 ± 0.14
10.52 ± 0.33
6.66 ± 0.56
11.44 ± 0.63
19.15 ± 0.42
15.54 ± 0.36
4.39 ± 0.34
3.80 ± 0.17
19.84 ± 0.67
14.74 ± 0.16
6.88 ± 0.16
10.76 ± 0.33
22.60 ± 2.66
13.88 ± 0.47
6.59 ± 0.90
2.31 ± 0.10
22.27 ± 0.17
N
H
O
O
N
H
N
H
O
N
N
N
O
H
O
O
N
N
N
N
H
N
N
N
H
O
N
H
N
H
O
N
H
N
H
O
N
H
8g
N
N
H
a Data are presented as mean ± SD, n=3, from three independent experiments.
We set out to evaluated the comprehensive anti-melanoma effects of our promising compounds (5f, 5m,
8e, and 8f). Firstly, we investigated the inhibition on colony formation of these compounds, which
detailed the activity on cell growth. Potent compounds were subjected to evaluated their activity on cell
apoptosis and cell cycle, and immunofluorescent staining of tubulin was conducted to discuss the effect
on cellular microtubule networks of our compound.
2.2.3. Colony Formation Assay
To access the effect of our compounds on colony formation, the human melanoma SK-MEL-28 cells
were employed. As shown in Figure 1, the lead propranolol and carvedilol was tested, and cell growth
was inhibited at a high level. We further investigated 5f, 5m, 8e, and 8f, cells were treated with the same
dilution series and counted. Compound 5m showed the most potent one, colony formation was
completely abolished at the concentration of 2 μM and above.

Figure 1. Effects on colony formation in SK-MEL-28 malignant melanoma cell lines. (A) Effects of
propranolol and carvedilol on colony formation required relative high concentrations; (B – E) Effects of
5f, 8e, 8f and 5m on colony formation at 1, 2, 3, 5 and 10 μM; (F) Quantified colony fractions compared
with control group.
2.2.4. Wound Healing Assay
Malignant melanoma presents as a metastatic disease with poor prognosis. Hydroxyl compounds 5m and
5f were selected to evaluate their effect on cell migration via wound healing assay. As shown in Figure
2, compared with 5f, 5m exhibited significant inhibition on migration even at 1 μM, so 5m was subjected
to further biological evaluations.

Figure 2. Compound 8f and 5m inhibit cell migration of SK-MEL-28 malignant melanoma cell lines.
(A) Diagrams of effects of 8f and 5m on cell migration at 1 μM and 2 μM for 48 h (scale bar = 1000
μm); (B) Quantified wound width change compared with control group.
2.2.5. Apoptotic Assay
To investigate whether the cell growth inhibition of 5m was due to the effects on cell apoptosis, a dual
Annexin V-FITC/PI staining was performed, colchicine was used as positive control. As shown in Figure
3, compared with vehicle, 5m induced cell apoptosis in a concentration-dependent manner. Furthermore,
5m at concentration of 2 μM dramatically induced equivalent cell apoptosis to positive control.

Figure 3. Compound 5m induce cell apoptosis in SK-MEL-28 malignant melanoma cell lines. (A)
Fluorescence vision of Annexin V-FITC, PI, and DAPI staining after treatment of vehicle, colchicine (5
nM), or 5m (1 μM or 2 μM) for 24 h, respectively (scale bar = 200 μm); (B) Quantified result of the
fraction of apoptotic cells.
2.2.6. Cell cycle Assay
To evaluate whether the cell growth inhibition of 5m was resulted from cell cycle arrest, we conducted
a Hochest 33324 staining, colchicine was used as positive control. As shown in Figure 4, comparing the
fluorescence histograms of cells treated with colchicine or 5m to the vehicle group, the relative DNA
number was concentrated at 20000 to 25000 intervals in a dose-dependent manner, which indicated that
5m exhibited a G2/M phase arrest being similar to colchicine.
Figure 4. Compounds induce cell cycle arrest in SK-MEL-28 malignant melanoma cell lines. Above:
Fluorescence vision of Hochest 33324 dye staining after treatment of colchicine (5 nM), or 5m (1 or 2
μM) for 12h, respectively (scale bar = 200 μm). Below: Quantified histogram of the DNA number
distribution.
2.2.7. Immunofluorescent Staining

It’s well known that cell growth and differentiation involving the dynamics of cellular microtubule
network. To investigated whether the effects of cell apoptosis and cell cycle arrest were due to the
disruption of microtubule, 5m was subjected to an immunofluorescent staining. [40] As shown in Figure
5, the microtubule network of control group showed normal arrangement with fibrous and slim
microtubule surrounding by nucleus. After exposed to colchicine or 5m at virous concentrations, the
microtubule network was disrupted, which could finally lead to cell apoptosis and cell arrest. The result
indicated that tubulin might be a potential target of 5m.
Figure 5. Effect of 5m on cellular microtubule network (scale bar = 100 μm). SK-MEL-28 cells were
treated with 0.1% DMSO as control group, colchicine (5 nM), and 5m (1 μM and 2 μM), then the cells
were fixed and stained with anti-α-tubulin antibody, Alexa Fluor 488 dye (red), and DAPI (blue). Cells
with disordered microtubule network were depicted by white arrows.
2.3. Molecular Docking
To obtain a structural insight into the interaction between our compounds and tubulin and discuss their
structure – activity relationships further, molecular docking studies was performed. The crystal structure
of tubulin in complex with a reported carbazole-containing small molecule (PDB code 6h9b) was chosen
as a docking protein model. As shown in Figure 6A&B, 5m adopts a conformation between the β-tubulin
and α-tubulin heterodimers, which is known as colchicine binding site. Detailed interactions are depicted
in Figure 6C, the diphenyl moiety is buried in a hydrophobic pocket of β-tubulin, surrounded in by
strands S8, helix H7 and T7 loop, the diphenylamine moiety is exposed toward the α-tubulin T5 loop,
the diphenyl moiety makes van der Waals contacts with tubulin. As for the propan-2-ol linker, the
hydroxyl forms a H-bond with the side chain of Val351, the amine N atom forms H-bond with the side
chain of Lys352 and Asn349. These H-bonding interactions might facilitate the tight binding into the β-
tubulin and α-tubulin interface, especially the β-tubulin domain.

In terms of the hydroxyl-removed counterpart 8f, the whole molecule adopts a similar location and
orientation with 5m, as the diphenyl moiety inserts into the box composed of strands S8, helix H7 and
T7 loop, while the diphenylamine moiety faced to α-tubulin domains. However, the lost H-bonding could
attenuate the intense interaction (Figure 6D), which is in accordance with the cellular potency. Regarding
the carbazole counterparts, different orientations are observed (Figure 6E&F). The carbazole ring in
these compounds is buried into the strands S8, helix H7 and T7 loop, the diphenyl faces to α-tubulin
domains which appears an inverse orientation. For compound 5f, the critical interaction including the H-
bond between hydroxyl O atom and Asn349, amine N atom and Lys352, Asn349. In addition, Leu255
forms a H-pi interaction with carbazole ring, which facilitates the bonding in this site. The removal of
hydroxyl resulted in 8f, whose carbazole ring comes outside of the box consisted of strands S8, helix H7
and T7 loop. These observations give us a clear structural basis in agreement with their cellular potency.

Figure 6. Putative binding mode for 5m, 8f, 5f, and 8e bound to tubulin (PDB code 6h9b). (A) Overall
view of the binding site for 5m in the complex; (B) Molecular surface map for 5m in the complex; (C –
F) Critical interactions for 5m, 8f, 5f, and 8e bound to tubulin.
3. Conclusions
To our knowledge, this work first presents a drug repurposing and rediscovery journey from
cardiovascular agents to anti-melanoma agents. Inspired by clinical and preclinical evidences, structural
optimization was started with propranolol and carvedilol, two well-known β-blockers. Cell proliferative
activity-based evaluation led to the discovery of 5m, which exhibited significantly improved potency.
Further biological evaluation indicated that 5m could inhibit colony formation, migration, induce cell
apoptosis and G2/M phase arrest in melanoma cell line. It could disrupt the cellular microtubule network,
which demonstrated that tubulin might be a potential target. Molecular docking studies gave a structural
insight into the interaction between 5m and tubulin. However, the detailed mechanism of action and
target warranted to be further investigated, which is ongoing in our group. Together, this study provided
a primary drug repurposing case.
4. Experimental Section
4.1. Chemistry
4.1.1. General Information
Unless otherwise indicated, all commercially available reagents were used without further purification.
Solvents were dried through routine protocols. Column chromatography was carried out on 200–300

mesh silica gel (Qingdao Haiyang Chemical, China). Reactions were monitored by thin-layer
chromatography on 0.25 mm silica gel plates (GF254) (Huanghai, China) and visualized under UV light.
Melting point was determined on a WRS-2 melting apparatus (Shenguang, China). Purity of all target
compounds was ≥96% as determined by HPLC (Shimadzu Labsolutions, UV detection at λ=254 nm)
analysis on the Agilent C18 column (4.6×150 mm, 5 μm). 1H NMR and 13C NMR spectra were recorded
with a Bruker Avance-III spectrometer (Bruker Company, Germany) in the indicated solvents (CDCl3 or
DMSO-d6, TMS as internal standard). The chemical shifts (δ values) are reported in ppm and corrected
to the signal of the deuterated solvents (7.26 ppm (1H NMR) and 77.16 ppm (13C NMR) for CDCl3; 2.50
ppm (1H NMR) and 39.52 ppm (13C NMR) for (CD3)2SO; and 3.31 ppm (1H NMR) and 49.00 ppm (13C
NMR) for CD3OD). Peak multiplicities abbreviations are used as follows: s (singlet), d (doublet), dd
(doublet of doublets), t (triplet), td (triplet of doublets), q (quartet), m (multiplet), br (broadened).
Coupling constants are reported in hertz (Hz). High resolution mass spectra (HRMS) were measured on
a Finnigan MAT 95 spectrometer (Finnigan, Germany).
4.1.2. General procedure (A) for the synthesis of 2-(aryloxymethyl)oxiranes
Commercially unavailable 2-(aryloxymethyl)oxiranes were synthesized by following procedure as
previously reported. To a solution of corresponding phenols in dimethyl sulfoxide (DMSO) at room
temperature, NaOH aqueous solution (1 M, 1.2 equiv) was added, the volume ratio of solvent is DMSO :
H2O = 2 : 1, the mixture was stirred for 10 min and epichlorohydrin (1.5 equiv) was added dropwise
within 10 min, the reaction mixture was allowed to stirred for further 2 h. AcOEt was added to the mixture,
which was washed by brine for 3 times. The solvents were evaporated under vacuum, and the crude
residue was purified by column chromatography on silica gel.
4.1.3. General procedure (B) for the synthesis of 3-bromopropoxyaryl
In general, a solution of corresponding phenols (5 mmol, 1 equiv), 1,3-dibromopropane (15 mmol, 3
equiv), and KOH (7.5 mmol, 1.5 equiv) were dissolved in 30 mL CH3CN was stirred at room temperature
overnight. Then, the solvent was removed under reduced pressure, the crude residue was purified by
column chromatography on silica gel.
4.1.4. General procedure (C) for the synthesis of 1-arylamino-3-aryloxypropan-2-ols
Corresponding 2-(aryloxymethyl)oxiranes (5 mmol, 1 equiv) and amines (7.5 mmol, 1.5 equiv) were
dissolved in 30 mL i-PrOH. The reaction was purged with argon 3 times and stirred at reflux for 5 h, and
then the mixture was cooled to room temperature and added AcOEt. After washing with brine 3 times,
the organic layer was dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. Purification of the
crude residue by column chromatography on silica gel yielded target compounds.
4.1.5. General procedure (D) for the synthesis of aryl-3-aryloxypropan-1-amines
Corresponding 3-bromopropoxyaryl (5 mmol, 1 equiv) and amines (7.5 mmol, 1.5 equiv) was dissolved

in 30 mL CH3CN. The reaction was purged with argon 3 times and stirred at reflux for 5 h, and then the
mixture was cooled to room temperature and added AcOEt. After washing with brine 3 times, the organic
layer was dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. Purification of the crude
residue by column chromatography on silica gel afforded target compounds.
1-((2-(2-methoxyphenoxy)ethyl)amino)-3-(naphthalen-1-yloxy)propan-2-ol (5a).
1
Compound was prepared according to procedure C. White solid, yield 86%, m.p. 109.7-111.2°C. H
NMR (500 MHz, DMSO-d6) δ 8.30 – 8.22 (m, 1H), 7.90 – 7.83 (m, 1H), 7.57 – 7.50 (m, 1H), 7.50 –
7.44 (m, 2H), 7.40 (t, J = 7.9 Hz, 1H), 7.01 – 6.93 (m, 3H), 6.88 (dtd, J = 18.5, 7.4, 1.8 Hz, 2H), 5.19 (s,
1H), 4.17 – 4.11 (m, 1H), 4.08 (q, J = 4.1, 2.8 Hz, 2H), 4.02 (t, J = 5.6 Hz, 2H), 3.73 (s, 3H), 2.94 (t, J =
5.6 Hz, 2H), 2.88 (dd, J = 11.8, 4.0 Hz, 1H), 2.79 (dd, J = 11.8, 6.3 Hz, 1H), 2.06 (s, 1H); 13C NMR (126
MHz, DMSO) δ 154.6, 149.6, 148.5, 134.5, 127.8, 126.9, 126.7, 125.6, 125.5, 122.3, 121.5, 121.2, 120.3,
114.0, 112.6, 105.6, 71.3, 68.9, 68.8, 55.9, 52.9, 49.0. HRMS-ESI (m/z) calcd for C22H26NO4+ [M+H]+
368.1856, found 368.1867.
1-((2-(2-methoxyphenoxy)ethyl)amino)-3-(naphthalen-2-yloxy)propan-2-ol (5b).
Compound was prepared according to procedure C. White solid, yield 82%, m.p. 83.1-86.5°C. 1H NMR
(500 MHz, Chloroform-d) δ 7.82 – 7.70 (m, 3H), 7.51 – 7.41 (m, 1H), 7.36 (ddd, J = 8.1, 6.9, 1.1 Hz,
1H), 7.22 – 7.16 (m, 2H), 7.02 – 6.88 (m, 4H), 4.18 (t, J = 5.2 Hz, 3H), 4.14 (d, J = 4.4 Hz, 2H), 3.87 (s,
3H), 3.13 (t, J = 5.1 Hz, 2H), 3.03 (dd, J = 12.3, 3.7 Hz, 1H), 2.93 (dd, J = 12.3, 7.6 Hz, 1H); 13C NMR
(126 MHz, CDCl3) δ 156.6, 149.7, 148.2, 134.5, 129.4, 129.1, 127.6, 126.8, 126.4, 123.7, 121.7, 120.9,
+
118.8, 114.1, 111.8, 106.8, 70.4, 68.8, 68.2, 55.8, 51.6, 48.7. HRMS-ESI (m/z) calcd for C22H26NO4
[M+H]+ 368.1856, found 367.1866.
1-((9H-carbazol-4-yl)oxy)-3-(4-(2-methoxyphenyl)piperazin-1-yl)propan-2-ol (5c).
1
Compound was prepared according to procedure C. White solid, yield 72%, m.p. 162.6-163.4°C. H
NMR (500 MHz, DMSO-d6) δ 11.34 – 11.17 (m, 1H), 8.35 – 8.22 (m, 1H), 7.46 (td, J = 7.8, 4.3 Hz, 1H),
7.35 (td, J = 7.5, 3.0 Hz, 1H), 7.30 (tt, J = 8.0, 2.5 Hz, 1H), 7.16 (dq, J = 12.1, 7.6, 5.5 Hz, 1H), 7.08 (td,
J = 8.0, 4.6 Hz, 1H), 7.00 – 6.89 (m, 2H), 6.89 – 6.79 (m, 2H), 6.71 (dd, J = 8.0, 1.6 Hz, 1H), 5.03 (dt, J
= 6.7, 4.2 Hz, 1H), 4.22 (dt, J = 7.0, 3.2 Hz, 2H), 4.16 (tt, J = 7.2, 2.8 Hz, 1H), 3.77 (d, J = 3.3 Hz, 3H),
3.38 (d, J = 14.7 Hz, 1H), 3.34 (s, 1H), 2.80 – 2.53 (m, 6H); 13C NMR (126 MHz, DMSO) δ 155.5, 152.4,
141.7, 141.6, 139.4, 127.0, 125.0, 123.0, 122.8, 122.2, 121.3, 119.0, 118.3, 112.3, 112.1, 110.8, 104.3,
+
100.9, 71.3, 67.2, 61.9, 55.8, 54.3, 50.6. HRMS-ESI (m/z) calcd for C26H30N3O3 [M+H]+ 432.2282,
found 432.2293.
1-((9H-carbazol-4-yl)oxy)-3-(4-phenylpiperazin-1-yl)propan-2-ol (5d).
Compound was prepared according to procedure C. White solid, yield 80%, m.p. 70.6-73.3°C. 1H NMR
(500 MHz, Chloroform-d) δ 8.32 (d, J = 7.8 Hz, 1H), 8.11 (s, 1H), 7.48 – 7.38 (m, 2H), 7.36 (t, J = 8.0

Hz, 1H), 7.31 (t, J = 7.5 Hz, 2H), 7.28 – 7.25 (m, 1H), 7.09 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 8.1 Hz, 2H),
6.90 (t, J = 7.3 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H), 4.46 – 4.32 (m, 2H), 4.29 (dd, J = 9.2, 4.8 Hz, 1H),
3.60 (s, 1H), 3.28 (tt, J = 11.5, 5.8 Hz, 4H), 2.94 (dt, J = 11.0, 4.2 Hz, 2H), 2.87 – 2.77 (m, 2H), 2.73 (dt,
J = 7.6, 4.0 Hz, 2H); 13C NMR (126 MHz, DMSO) δ 155.5, 151.5, 141.6, 139.4, 129.4, 127.0, 125.0,
123.0, 122.2, 119.2, 119.0, 115.8, 112.1, 110.8, 104.3, 100.9, 71.2, 67.3, 61.8, 54.0, 48.7. HRMS-ESI
(m/z) calcd for C24H27N4O2+ [M+H]+ 402.2176, found 402.2188.
1-((9H-carbazol-4-yl)oxy)-3-(4-(pyridin-2-yl)piperazin-1-yl)propan-2-ol (5e).
1
Compound was prepared according to procedure C. White solid, yield 81%, m.p. 170.7-173.4°C. H
NMR (500 MHz, Chloroform-d) δ 8.30 (d, J = 7.8 Hz, 1H), 8.23 (dd, J = 5.1, 1.7 Hz, 1H), 8.20 (s, 1H),
7.57 – 7.49 (m, 1H), 7.42 (d, J = 6.1 Hz, 2H), 7.35 (t, J = 8.0 Hz, 1H), 7.31 – 7.23 (m, 1H), 7.08 (d, J =
8.0 Hz, 1H), 6.69 (dt, J = 12.0, 7.6 Hz, 3H), 5.32 (s, 0H), 4.41 (dq, J = 9.4, 4.9 Hz, 1H), 4.35 (dd, J =
9.5, 5.0 Hz, 1H), 4.27 (dd, J = 9.5, 5.1 Hz, 1H), 3.64 (tt, J = 12.6, 9.0 Hz, 4H), 2.88 (ddt, J = 10.4, 7.9,
3.8 Hz, 2H), 2.81 (t, J = 6.8 Hz, 2H), 2.68 (ddd, J = 10.8, 6.4, 3.6 Hz, 2H); 13C NMR (126 MHz, CDCl3)
δ 159.4, 155.2, 148.0, 141.0, 138.8, 137.6, 126.7, 125.0, 122.9, 122.5, 119.6, 113.5, 112.7, 110.1, 107.2,
+
103.9, 101.2, 70.3, 65.9, 61.2, 53.2, 45.3. HRMS-ESI (m/z) calcd for C24H27N4O2 [M+H]+ 403.2129,
found 403.2139.
1-((9H-carbazol-4-yl)oxy)-3-(([1,1'-biphenyl]-4-ylmethyl)amino)propan-2-ol (5f).
1
Compound was prepared according to procedure C. White solid, yield 75%, m.p. 145.7-148.4°C. H
NMR (500 MHz, DMSO-d6) δ 11.24 (s, 1H), 8.15 (d, J = 7.7 Hz, 1H), 7.64 – 7.59 (m, 2H), 7.59 – 7.53
(m, 2H), 7.53 – 7.39 (m, 5H), 7.38 – 7.32 (m, 1H), 7.32 – 7.25 (m, 2H), 7.08 (td, J = 7.9, 2.1 Hz, 2H),
6.69 (d, J = 7.9 Hz, 1H), 5.14 (d, J = 4.6 Hz, 1H), 4.23 – 4.11 (m, 3H), 3.83 (s, 2H), 2.88 (dd, J = 11.9,
4.3 Hz, 1H), 2.77 (dd, J = 11.8, 6.4 Hz, 1H); 13C NMR (126 MHz, DMSO) δ 155.4, 141.5, 140.7, 140.6,
139.3, 138.8, 129.3, 128.9, 127.6, 127.0, 126.9, 126.9, 124.9, 122.9, 122.2, 119.0, 112.0, 110.8, 70.9,
68.9, 53.1, 52.4. HRMS-ESI (m/z) calcd for C28H27N2O2+ [M+H]+ 423.2067, found 423.2072.
1-((9H-carbazol-4-yl)oxy)-3-([1,1'-biphenyl]-4-ylamino)propan-2-ol (5g).
1
Compound was prepared according to procedure C. White solid, yield 73%, m.p. 184.7-186.3°C. H
NMR (500 MHz, DMSO-d6) δ 11.26 (s, 1H), 8.28 (d, J = 7.6 Hz, 1H), 7.59 – 7.49 (m, 2H), 7.46 (d, J =
8.1 Hz, 1H), 7.43 – 7.32 (m, 5H), 7.30 (t, J = 8.0 Hz, 1H), 7.25 – 7.18 (m, 1H), 7.18 – 7.11 (m, 1H), 7.08
(d, J = 8.1 Hz, 1H), 6.80 – 6.73 (m, 2H), 6.70 (d, J = 7.9 Hz, 1H), 5.92 (t, J = 6.0 Hz, 1H), 5.36 (d, J =
4.4 Hz, 1H), 4.35 – 4.14 (m, 3H), 3.54 – 3.45 (m, 1H), 3.32 – 3.26 (m, 1H); 13C NMR (126 MHz, CDCl3)
δ 156.9, 154.8, 147.8, 147.3, 141.1, 138.7, 128.7, 128.1, 126.7, 126.4, 126.2, 125.2, 122.8, 122.4, 119.9,
+
113.7, 110.2, 104.2, 101.3, 70.2, 69.0, 47.1, 29.7. HRMS-ESI (m/z) calcd for C27H25N2O2 [M+H]+
409.1911, found 409.1922.
1-([1,1'-biphenyl]-4-yloxy)-3-(isopropylamino)propan-2-ol (5h).

1
Compound was prepared according to procedure C. White solid, yield 69%, m.p. 111.3-113.0°C. H
NMR (500 MHz, DMSO-d6) δ 7.60 (t, J = 8.8 Hz, 4H), 7.43 (t, J = 7.5 Hz, 2H), 7.31 (t, J = 7.3 Hz, 1H),
7.03 (d, J = 8.2 Hz, 2H), 5.01 (s, 1H), 4.00 (dd, J = 9.6, 4.5 Hz, 1H), 3.91 (dd, J = 9.7, 5.9 Hz, 1H), 3.87
(q, J = 5.5 Hz, 1H), 2.83 – 2.63 (m, 2H), 2.63 – 2.54 (m, 1H), 1.23 (d, J = 4.3 Hz, 1H), 0.99 (dd, J = 6.1,
2.6 Hz, 6H); 13C NMR (126 MHz, DMSO) δ 158.8, 140.3, 132.9, 129.3, 128.2, 126.6, 115.4, 71.4, 68.8,
50.5, 48.7, 23.4. HRMS-ESI (m/z) calcd for C18H24NO2+ [M+H]+ 286.1802, found 286.1811.
1-([1,1'-biphenyl]-4-yloxy)-3-((2-(2-methoxyphenoxy)ethyl)amino)propan-2-ol (5i).
1
Compound was prepared according to procedure C. White solid, yield 78%, m.p. 114.8-115.3°C. H
NMR (500 MHz, DMSO-d6) δ 7.60 (ddd, J = 10.9, 8.0, 1.8 Hz, 4H), 7.43 (t, J = 7.8 Hz, 2H), 7.34 – 7.28
(m, 1H), 7.06 – 7.00 (m, 2H), 6.97 (ddd, J = 7.8, 5.8, 2.0 Hz, 2H), 6.88 (dtd, J = 16.7, 7.4, 1.8 Hz, 2H),
5.08 (d, J = 4.3 Hz, 1H), 4.01 (q, J = 4.9, 4.1 Hz, 3H), 3.93 (dq, J = 9.4, 5.5, 5.1 Hz, 2H), 3.75 (s, 3H),
2.91 (t, J = 5.6 Hz, 2H), 2.78 (dd, J = 11.8, 4.1 Hz, 1H), 2.68 (dd, J = 11.8, 6.3 Hz, 1H), 2.09 (s, 1H);
13C NMR (126 MHz, DMSO) δ 158.8, 149.6, 148.5, 140.3, 132.9, 129.3, 128.2, 127.2, 126.6, 121.5,
+
121.2, 115.4, 114.1, 112.7, 71.2, 68.8, 68.7, 55.9, 52.9, 48.9. HRMS-ESI (m/z) calcd for C24H28NO4
[M+H]+ 394.2013, found 394.2028.
1-([1,1'-biphenyl]-4-yloxy)-3-(4-(pyridin-2-yl)piperazin-1-yl)propan-2-ol (5j).
1
Compound was prepared according to procedure C. White solid, yield 81%, m.p. 155.5-157.2°C. H
NMR (500 MHz, DMSO-d6) δ 11.24 (s, 1H), 8.22 (d, J = 7.5 Hz, 1H), 7.44 (dd, J = 8.2, 2.7 Hz, 1H),
7.37 – 7.30 (m, 1H), 7.28 (dt, J = 7.9, 3.9 Hz, 1H), 7.12 (td, J = 7.5, 2.9 Hz, 1H), 7.06 (dd, J = 8.0, 2.7
Hz, 1H), 6.94 (dt, J = 5.9, 2.8 Hz, 2H), 6.91 – 6.78 (m, 2H), 6.68 (dd, J = 8.0, 3.3 Hz, 1H), 5.19 (s, 1H),
4.16 (tq, J = 15.9, 4.8 Hz, 3H), 4.02 (q, J = 5.1 Hz, 2H), 3.72 (d, J = 3.6 Hz, 3H), 2.95 (dt, J = 9.1, 4.5
Hz, 3H), 2.90 – 2.79 (m, 1H); 13C NMR (126 MHz, DMSO) δ 159.5, 158.9, 148.0, 140.3, 137.9, 132.9,
129.3, 128.2, 127.2, 126.6, 115.4, 113.4, 107.5, 71.6, 67.1, 61.6, 53.8, 45.2. HRMS-ESI (m/z) calcd for
C24H28N3O2+ [M+H]+ 390.2167, found 390.2190.
1-([1,1'-biphenyl]-4-yloxy)-3-(4-phenylpiperazin-1-yl)propan-2-ol (5k).
1
Compound was prepared according to procedure C. White solid, yield 75%, m.p. 184.7-187.6°C. H
NMR (500 MHz, DMSO-d6) δ 7.61 (td, J = 7.3, 6.8, 1.8 Hz, 4H), 7.43 (t, J = 7.7 Hz, 2H), 7.33 – 7.28
(m, 1H), 7.25 – 7.17 (m, 2H), 7.07 – 7.02 (m, 2H), 6.97 – 6.88 (m, 2H), 6.80 – 6.73 (m, 1H), 4.95 (d, J
= 4.7 Hz, 1H), 4.05 (td, J = 10.5, 9.9, 4.5 Hz, 2H), 3.94 (dd, J = 9.2, 5.4 Hz, 1H), 3.13 (t, J = 5.0 Hz,
4H), 2.62 (qt, J = 10.9, 4.8 Hz, 4H), 2.57 – 2.52 (m, 1H), 2.44 (dd, J = 12.6, 6.2 Hz, 1H); 13C NMR (126
MHz, DMSO) δ 158.9, 151.5, 140.3, 132.9, 129.4, 129.3, 128.2, 127.2, 126.6, 119.2, 115.8, 115.4, 71.6,
67.1, 61.5, 54.0, 48.7. HRMS-ESI (m/z) calcd for C25H28N2O2+ [M+H]+ 389.2224, found 389.2232.
1-((9H-carbazol-4-yl)oxy)-3-((2-methylquinolin-8-yl)amino)propan-2-ol (5l).
1
Compound was prepared according to procedure C. White solid, yield 72%, m.p. 100.1-103.7°C. H

NMR (500 MHz, Chloroform-d) δ 8.35 (d, J = 7.8 Hz, 1H), 8.15 (s, 1H), 7.98 (d, J = 8.3 Hz, 1H), 7.43
(d, J = 4.0 Hz, 2H), 7.38 – 7.34 (m, 1H), 7.34 – 7.31 (m, 1H), 7.31 – 7.24 (m, 2H), 7.14 – 7.02 (m, 2H),
6.86 (d, J = 7.6 Hz, 1H), 6.70 (d, J = 7.9 Hz, 1H), 6.67 – 6.45 (m, 1H), 4.62 (dt, J = 9.9, 4.9 Hz, 1H),
4.41 (d, J = 5.2 Hz, 2H), 3.84 (dd, J = 13.6, 4.7 Hz, 1H), 3.72 (dd, J = 13.6, 7.2 Hz, 1H), 3.05 – 2.77 (m,
1H), 2.72 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 155.6, 155.4, 144.4, 141.6, 139.4, 137.3, 136.6, 127.2,
127.0, 126.8, 125.0, 122.7, 122.2, 121.0, 119.1, 113.6, 111.9, 110.9, 104.7, 104.3, 100.9, 66.1, 40.5, 29.1,
25.1. HRMS-ESI (m/z) calcd for C25H24N3O2+ [M+H]+ 398.1863, found 398.1877.
1-(([1,1'-biphenyl]-4-ylmethyl)amino)-3-(3-(phenylamino)phenoxy)propan-2-ol (5m).
1
Compound was prepared according to procedure C. White solid, yield 71%, m.p. 102.8-104.2°C. H
NMR (500 MHz, Chloroform-d) δ 7.65 – 7.59 (m, 3H), 7.58 (d, J = 2.0 Hz, 1H), 7.46 (t, J = 7.7 Hz, 2H),
7.42 (d, J = 8.0 Hz, 2H), 7.40 – 7.34 (m, 1H), 7.33 – 7.28 (m, 2H), 7.18 (t, J = 8.4 Hz, 1H), 7.14 – 7.03
(m, 2H), 7.03 – 6.92 (m, 1H), 6.74 – 6.59 (m, 2H), 6.55 – 6.44 (m, 1H), 5.72 (s, 1H), 4.11 (dq, J = 8.9,
4.9 Hz, 1H), 3.99 (d, J = 5.2 Hz, 2H), 3.95 – 3.85 (m, 2H), 2.94 (dd, J = 12.2, 3.9 Hz, 1H), 2.84 (dd, J =
12.2, 7.7 Hz, 1H); 13C NMR (126 MHz, CDCl3) δ 159.8, 144.7, 142.6, 140.9, 140.1, 139.0, 130.1, 129.4,
128.8, 128.6, 127.2, 127.2, 127.1, 121.4, 118.5, 110.5, 106.6, 103.8, 70.4, 68.5, 53.5, 51.2. HRMS-ESI
(m/z) calcd for C28H29N2O2+ [M+H]+ 425.2224, found 425.2234.
(S)-1-((9H-carbazol-4-yl)oxy)-3-(([1,1'-biphenyl]-4-ylmethyl)amino)propan-2-ol (5n).
1
Compound was prepared according to procedure C. White solid, yield 74%, m.p. 145.8-147.5°C. H
NMR (500 MHz, DMSO-d6) δ 11.25 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 7.64 – 7.59 (m, 2H), 7.56 (d, J =
7.9 Hz, 2H), 7.52 – 7.38 (m, 5H), 7.35 (t, J = 7.3 Hz, 1H), 7.30 (td, J = 7.8, 5.3 Hz, 2H), 7.08 (t, J = 8.3
Hz, 2H), 6.70 (d, J = 7.9 Hz, 1H), 5.15 (d, J = 17.6 Hz, 1H), 4.30 – 4.10 (m, 3H), 3.83 (s, 2H), 2.89 (dd,
J = 11.9, 4.3 Hz, 1H), 2.78 (dd, J = 11.8, 6.3 Hz, 1H); 13C NMR (126 MHz, DMSO) δ 155.4, 141.5,
140.6, 140.5, 139.3, 138.9, 129.3, 128.9, 127.6, 127.0, 126.9, 126.9, 124.9, 122.9, 122.2, 119.0, 112.0,
110.8, 104.3, 100.9, 70.9, 68.9, 53.1, 52.4. HRMS-ESI (m/z) calcd for C28H27N2O2+ [M+H]+ 432.2067,
found 432.2081.
(S)-1-((9H-carbazol-4-yl)oxy)-3-((2-(2-methoxyphenoxy)ethyl)amino)propan-2-ol (5o).
Compound was prepared according to procedure C. White solid, yield 81%, m.p. 117.5-120.4°C. 1H
NMR (500 MHz, DMSO-d6) δ 11.27 (s, 1H), 8.23 (d, J = 7.7 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.38 –
7.31 (m, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.13 (t, J = 7.4 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.95 (dt, J = 7.8,
2.0 Hz, 2H), 6.89 (td, J = 7.6, 1.8 Hz, 1H), 6.85 (td, J = 7.6, 1.7 Hz, 1H), 6.69 (d, J = 7.9 Hz, 1H), 4.17
(qt, J = 9.2, 4.9 Hz, 3H), 4.03 (t, J = 5.6 Hz, 2H), 3.73 (s, 3H), 2.98 (d, J = 5.3 Hz, 2H), 2.95 (d, J = 4.9
Hz, 1H), 2.86 (dd, J = 11.9, 6.6 Hz, 1H); 13C NMR (126 MHz, DMSO) δ 155.4, 149.6, 148.5, 141.6,
139.3, 127.0, 125.0, 122.9, 122.2, 121.5, 121.2, 119.0, 114.1, 112.6, 112.0, 110.8, 104.3, 100.9, 70.9,
68.8, 68.7, 55.9, 53.0, 48.9. HRMS-ESI (m/z) calcd for C24H27N2O4+ [M+H]+ 407.1965, found 407.1975.
3-((9H-carbazol-4-yl)oxy)-N-(2-(2-methoxyphenoxy)ethyl)propan-1-amine (8a).

1
Compound was prepared according to procedure D. White solid, yield 83%, m.p. 132.8-135.8°C. H
NMR (500 MHz, DMSO-d6) δ 11.25 (s, 1H), 8.16 (d, J = 7.7 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.31 (dt,
J = 18.9, 7.7 Hz, 2H), 7.13 (t, J = 7.5 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.95 (dd, J = 7.9, 1.5 Hz, 2H),
6.90 (t, J = 7.5 Hz, 1H), 6.86 (dd, J = 7.6, 1.6 Hz, 1H), 6.70 (d, J = 7.9 Hz, 1H), 4.28 (s, 1H), 4.03 (s,
1H), 3.72 (s, 3H), 3.07 – 2.85 (m, 3H), 2.09 (s, 1H); 13C NMR (126 MHz, DMSO) δ 155.4, 149.7, 148.4,
141.6, 139.4, 127.0, 125.0, 122.6, 122.2, 121.6, 121.2, 119.1, 114.3, 112.7, 111.9, 110.9, 104.2, 100.9,
+
68.5, 66.1, 55.9, 48.6, 46.5, 29.7.HRMS-ESI (m/z) calcd for C24H27N2O3 [M+H]+ 391.2016, found
391.2028.
4-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propoxy)-9H-carbazole (8b).
1
Compound was prepared according to procedure D. White solid, yield 81%, m.p. 153.8-156.5°C. H
NMR (500 MHz, DMSO-d6) δ 11.25 (s, 1H), 8.18 (d, J = 7.7 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.34 (t,
J = 7.6 Hz, 1H), 7.30 (t, J = 7.9 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.93 (d, J =
5.3 Hz, 2H), 6.87 (d, J = 6.3 Hz, 2H), 6.70 (d, J = 7.9 Hz, 1H), 4.27 (t, J = 6.1 Hz, 2H), 3.78 (s, 3H), 2.99
(s, 4H), 2.65 (t, J = 7.1 Hz, 2H), 2.59 (s, 3H), 2.11 (p, J = 6.6 Hz, 2H); 13C NMR (126 MHz, DMSO) δ
155.4, 152.5, 141.8, 141.6, 139.4, 127.0, 125.0, 122.8, 122.6, 122.2, 121.3, 119.1, 118.4, 112.4, 112.0,
110.9, 104.2, 100.9, 66.3, 55.8, 55.3, 53.7, 50.6, 26.9. HRMS-ESI (m/z) calcd for C26H30N3O2+ [M+H]+
416.2333, found 416.2341.
4-(3-(4-phenylpiperazin-1-yl)propoxy)-9H-carbazole (8c).
1
Compound was prepared according to procedure D. White solid, yield 85%, m.p. 138.1-140.4°C. H
NMR (500 MHz, Chloroform-d) δ 8.33 (d, J = 7.8 Hz, 1H), 8.11 (s, 1H), 7.42 (q, J = 8.2 Hz, 2H), 7.35
(t, J = 8.0 Hz, 1H), 7.31 (d, J = 7.7 Hz, 2H), 7.26 (d, J = 6.9 Hz, 2H), 7.07 (d, J = 8.0 Hz, 1H), 6.98 (d,
J = 8.1 Hz, 2H), 6.90 (t, J = 7.3 Hz, 1H), 6.71 (d, J = 7.9 Hz, 1H), 4.35 (t, J = 6.1 Hz, 2H), 2.82 (t, J =
7.4 Hz, 2H), 2.77 (s, 3H), 2.29 (p, J = 6.6 Hz, 2H); 13C NMR (126 MHz, DMSO) δ 155.4, 151.5, 141.6,
139.4, 129.4, 127.0, 125.0, 122.6, 122.2, 119.2, 119.1, 115.8, 111.9, 110.9, 104.2, 100.9, 71.2, 66.2, 55.2,
53.4, 48.7. HRMS-ESI (m/z) calcd for C25H28N3O+ [M+H]+ 386.2227, found 386.2241.
4-(3-(4-(pyridin-2-yl)piperazin-1-yl)propoxy)-9H-carbazole (8d).
1
Compound was prepared according to procedure D. White solid, yield 82%, m.p. 120.4-121.8°C. H
NMR (500 MHz, Chloroform-d) δ 8.33 (d, J = 7.8 Hz, 1H), 8.23 (dd, J = 5.0, 1.8 Hz, 1H), 8.18 (s, 1H),
7.55 – 7.47 (m, 1H), 7.42 (d, J = 6.7 Hz, 2H), 7.35 (t, J = 8.0 Hz, 1H), 7.28 (d, J = 3.3 Hz, 1H), 4.34 (s,
1H), 3.63 (t, J = 5.0 Hz, 4H), 2.79 (t, J = 7.5 Hz, 2H), 2.69 (t, J = 5.0 Hz, 4H), 2.27 (p, J = 6.5 Hz, 2H);
13C NMR (126 MHz, CDCl3) δ 159.5, 155.5, 148.0, 140.9, 138.7, 137.5, 126.7, 124.9, 123.0, 122.7,
119.6, 113.4, 112.6, 110.0, 107.1, 103.4, 101.1, 66.2, 55.6, 53.2, 45.2, 26.9. HRMS-ESI (m/z) calcd for
C24H27N4O+ [M+H]+ 387.2179, found 387.2185.
3-((9H-carbazol-4-yl)oxy)-N-([1,1'-biphenyl]-4-ylmethyl)propan-1-amine (8e).

1
Compound was prepared according to procedure D. White solid, yield 76%, m.p. 242.6-244.4°C. H
NMR (500 MHz, Chloroform-d) δ 8.27 (d, J = 7.8 Hz, 2H), 8.10 (s, 2H), 7.55 (dd, J = 14.7, 7.8 Hz, 8H),
7.51 – 7.29 (m, 17H), 7.20 (d, J = 7.3 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H), 6.67 (d, J = 7.9 Hz, 2H), 4.34
(t, J = 6.0 Hz, 4H), 3.95 (s, 4H), 3.09 (t, J = 7.0 Hz, 4H), 2.30 (q, J = 6.5 Hz, 4H); 13C NMR (126 MHz,
CDCl3) δ 155.4, 140.9, 140.8, 140.2, 138.7, 128.9, 128.7, 127.2, 127.2, 127.1, 126.7, 124.9, 123.0, 122.6,
119.7, 112.6, 109.9, 103.5, 101.1, 66.0, 53.3, 46.2, 29.7, 29.4. HRMS-ESI (m/z) calcd for C28H27N2O+
[M+H]+ 407.2118, found 407.2134.
3-(3-(([1,1'-biphenyl]-4-ylmethyl)amino)propoxy)-N-phenylaniline (8f).
1
Compound was prepared according to procedure D. White solid, yield 70%, m.p. 198.8-200.9°C. H
NMR (500 MHz, DMSO-d6) δ 8.81 (s, 2H), 8.20 (s, 1H), 7.76 (d, J = 8.2 Hz, 2H), 7.73 – 7.67 (m, 2H),
7.61 (d, J = 8.1 Hz, 2H), 7.49 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 7.4 Hz, 1H), 7.24 (t, J = 7.8 Hz, 2H), 7.13
(t, J = 8.1 Hz, 1H), 7.08 (d, J = 7.9 Hz, 2H), 6.84 (t, J = 7.3 Hz, 1H), 6.40 (dd, J = 8.1, 2.3 Hz, 1H), 4.24
(s, 2H), 4.03 (t, J = 5.9 Hz, 2H), 3.13 (t, J = 7.7 Hz, 2H), 2.11 (dq, J = 12.4, 6.3 Hz, 2H); 13C NMR (126
MHz, DMSO) δ 160.1, 145.2, 143.9, 140.5, 140.4, 138.9, 130.3, 129.6, 129.3, 129.0, 127.7, 127.0, 126.9,
120.3, 117.7, 109.5, 106.1, 103.2, 66.1, 53.0, 45.8, 29.6. HRMS-ESI (m/z) calcd for C28H29N2O+ [M+H]+
409.2274, found 409.2287.
N-(3-((9H-carbazol-4-yl)oxy)propyl)-2-methylquinolin-8-amine (8g).
1
Compound was prepared according to procedure D. Yellow solid, yield 72%, m.p. 161.0-163.3°C. H
NMR (500 MHz, DMSO-d6) δ 11.27 (s, 1H), 8.25 (d, J = 7.7 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.46 (d,
J = 8.1 Hz, 1H), 7.39 – 7.35 (m, 1H), 7.35 – 7.32 (m, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.25 (t, J = 7.9 Hz,
1H), 7.15 – 7.11 (m, 1H), 7.08 (d, J = 8.0 Hz, 1H), 7.02 (s, 1H), 6.73 (t, J = 7.3 Hz, 2H), 6.53 (t, J = 6.0
Hz, 1H), 4.36 (t, J = 5.9 Hz, 2H), 3.62 (q, J = 6.7 Hz, 2H), 2.58 (s, 3H), 2.33 (p, J = 6.3 Hz, 2H); 13
C
NMR (126 MHz, DMSO) δ 156.0, 155.0, 144.2, 141.0, 138.8, 137.8, 136.2, 126.7, 125.1, 122.9, 122.5,
122.3, 119.8, 114.6, 112.7, 110.1, 105.5, 104.1, 101.3, 70.1, 69.2, 46.8, 31.6, 25.2, 22.7. HRMS-ESI (m/z)
calcd for C25H24N3O+ [M+H]+ 382.1914, found 382.1923.
4.2. Biological Activity
4.2.1. Cell Culture
The human melanoma cell lines SK-MEL-5, SK-MEL-28 and A375 were purchased from ATCC
(Manassas VA, USA). Cells were cultured in high glucose Dulbecco's modified Eagle medium (DMEM,
Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), penicillin (100 U/mL) and
streptomycin (100μg/mL) (Solarbio, China), and incubated at 37°C in 5% CO2 humidified incubator.
4.2.2. Cell viability assay
The human melanoma cells (SK-MEL-28, SK-MEL-5 and A375) were seeded in a 96-well plate with a

density of 5×103 cells per well and cultured at 37°C in 5% CO2 humidified incubator for 24 h prior to
the experiment. After the medium was removed, 100 μL fresh complete medium containing various
concentrations of compound were added in each well and continued to incubated for 48 h. The proportion
of DMSO from parent solution of compound in the medium did not exceed 0.1%. Subsequently, 10 μL
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL in PBS,
neoFroxx, Germany) was added in each well and incubated for further 4 h. The supernatant was removed
and 150 μL DMSO was added in each well to dissolve the formazan crystal. Optical density was
measured at a wavelength of 490 nm on Cytation 5 Cell Imaging Multimode Reader (BioTek, USA).
Data were analyzed by GraphPad Prism 7. All experiments were performed in triplicate and repeated
three times.
4.2.3. Colony formation assay
The human melanoma SK-MEL-28 cells were seeded in six-well tissue culture plates with a density of
1000 cells per well and maintained in regular culture media. After 24 h, the cells were treated with
compounds at indicated concentrations. The culture media with the compounds were changed every 72
h. At the end of two weeks, the wells were washed twice with PBS and 2 mL of 0.05% crystal violet
staining buffer was added to each well and incubated for 30 min. The wells were then washed with PBS
for 5 min three times and allowed to dry. Photos were then taken, visible colonies were counted, and
surviving fractions were calculated. Experiments were performed in triplicate, and the surviving fractions
were analyzed using GraphPad Prism 5 software package. The error bars represent standard deviation.
4.2.4. Cell migration assay
The human melanoma SK-MEL-28 cells were seeded in a 6-well plate with a density of 3×105 cells per
well and cultured at 37°C in 5% CO2 humidified incubator to 95% confluence. Cells were scratched with
a 200 μL pipette tip and washed with PBS three times. 2 mL fresh medium containing the indicated
concentrations of compound were added and continued to incubated for different time points. The width
of the wounds at the same fields were quantified using Gene 5TM software coordinated with Cytation 5
Cell Imaging Multimode Reader (BioTek, USA). Relative wound (%) were determined to evaluated cell
migration.
4.2.5. Cell apoptosis assay
The human melanoma SK-MEL-28 cells were seeded in a 24-well plate with a density of 1×104 cells per
well and cultured at 37°C in 5% CO2 humidified incubator overnight, and treated with 1 mL fresh
complete medium containing the indicated concentrations of compound and continued to incubated for
24 h. Cells were stained using the Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, China)
according to the manufacturer’s recommendation. The fluorescence of stained cells were detected and
counted on a Cytation 5 Cell Imaging Multimode Reader (BioTek, USA), the single Annexin V-FITC-
positive and dual positive cells were recognized as apoptosis.

4.2.6. Cell cycle assay
The human melanoma SK-MEL-28 cells were seeded in a 24-well plate with a density of 1×104 cells per
well and cultured at 37°C in 5% CO2 humidified incubator overnight, and treated with 1 mL fresh
complete medium containing the indicated concentrations of compound and continued to incubated for
24 h. Cells were stained using Hochest 33324 dye and the fluorescence were detected on a Cytation 5
Cell Imaging Multimode Reader (BioTek, USA), the density of fluorescence was correlated with the
DNA number. The fluorescence distribution histograms were output using the coordinated Gene5TM
software.
4.2.7. Microtubule immunofluorescent staining
The human melanoma SK-MEL-28 cells were seeded into six-well plates and then treated with vehicle
control 0.1% DMSO or 24d. The cells were fixed with 4% paraformaldehyde, followed by 0.1% Triton
X-100, then washed with PBS for 3 times. After blocking for 20 min by adding 5% BSA at room
temperature, cells were incubated with an anti-α-tubulin monoclonal antibody at 4 °C overnight. Then,
the cells were washed three times by PBS following staining by the fluorescence secondary antibody and
labeling of nuclei by 4,6-diamidino-2-phenylindole (DAPI). Cells were finally visualized on a
fluorescence microscope (BioTek, USA).
4.3. Molecular Docking
All chemical structures were drawn in ChemDraw Professional 17.0 and saved as sdf. files. The
molecules were prepared by MOE 2018 to obtain the low energy conformations. The structure of tubulin
complex (PDB code 6h9b) was downloaded from the Protein Data Bank, and prepared using the protein
preparation module. Molecular docking was performed using the flexible docking wizard and the results
were ranked with the scoring function. The best docking pose of complex was selected to be analyzed.
Supporting Information
1H NMR, 13C NMR, HPLC and HRMS spectra for target compounds characterization (PDF).
Author Information
Corresponding Author
*E-mail: hugaoyun@csu.edu.cn. Phone: +81-0731-82650370. Fax: +81-0731-82650370.
ORCID

Qi Chang: 0000-0003-0012-3863
Liqing Hu: 0000-0003-3592-488X
Qianbin Li: 0000-0003-4522-3067
Author Contribution
The manuscript was written through contributions of all authors. All authors have given approval to the
final version of the manuscript.
Notes
The authors declare no competing financial interest.
Acknowledgements
This work was supported by the Natural Science Foundation of China (No. 81573287, 81773640 and
81803373) and the Innovation Project for Postgraduate of Central South University (No. 2018zzts868 to
Q.C.). We thank the NMR Laboratory of Advanced Research Center of Central South University for the
support of compound characterization. Q.C. genuinely thank his parents for their priceless support.
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