Synthesis of novel spiro-tetrahydroquinoline derivatives and evaluation of their pharmacological effects on wound healing

Article abstract of DOI:10.3390/ijms22126251

A highly diastereoselective method for the synthesis of novel spiro-tetrahydroquinoline derivatives is reported here, using a one-pot reaction method. All compounds were characterized by¹ H-nuclear magnetic resonance (NMR) and mass spectroscopy

Full text of DOI:10.3390/ijms22126251

International Journal of
Molecular Sciences
Article
Synthesis of Novel Spiro-Tetrahydroquinoline Derivatives and
Evaluation of Their Pharmacological Effects on Wound Healing
Yan-Cheng Liou 1,†, Yan-An Lin 2,†, Ke Wang 2, Juan-Cheng Yang 3, Yeong-Jiunn Jang 3,
*
, Wenwei Lin 1
and Yang-Chang Wu 2,3,4,
*
1
2
3
4
Department of Chemistry, National Taiwan Normal University, Taipei 116, Taiwan;
s89218456@gmail.com (Y.-C.L.); wenweilin@ntnu.edc.tw (W.L.)
Graduate Institute of Integrated Medicine, China Medical University, Taichung 404, Taiwan;
riinryxc@gmail.com (Y.-A.L.); waker603@163.com (K.W.)
Chinese Medicine Research and Development Center, China Medical University Hospital,
Taichung 404, Taiwan; qq9113054@gmail.com
Department of Medical Laboratory Science and Biotechnology, College of Medical and Health Science,
Asia University, Taichung 41354, Taiwan
*
Correspondence: jinrain.tw@gmail.com (Y.-J.J.), yachwu@gmail.com (Y.-C.W.);
Tel.: +886-4-22053366 (ext. 3605) (Y.-C.W.)
†
These authors contributed equally to this work.
Abstract: A highly diastereoselective method for the synthesis of novel spiro-tetrahydroquinoline
derivatives is reported here, using a one-pot reaction method. All compounds were characterized
by 1H-nuclear magnetic resonance (NMR) and mass spectroscopy, and their stereo configurations
were confirmed by X-ray analysis. These activities of these derivatives were then tested in human
keratocyte cells. The responses of cells to treatment with selected compounds were studied using
scratch analysis, and the compounds were tested in a mouse excision wound model. Three of the
derivatives demonstrated significant wound-healing activities.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Liou, Y.-C.; Lin, Y.-A.;
Wang, K.; Yang, J.-C.; Jang, Y.-J.;
Lin, W.; Wu, Y.-C. Synthesis of Novel
Spiro-Tetrahydroquinoline
Derivatives and Evaluation of Their
Pharmacological Effects on Wound
Keywords: tetrahydroquinoline; 1,3-indandione; spiro-tetrahydroquinoline; one-pot reaction; wound
healing
Healing. Int. J. Mol. Sci. 2021, 22, 6251.
https://doi.org/10.3390/
ijms22126251
1. Introduction
Academic Editor: István Szatmári
Skin is the largest organ and the first defense to protect the human body [1,2]. It can
be damaged by trauma, burns, skin diseases and so on. Severe skin trauma can impose
physical, psychological, and economic burdens on patients. The wound healing process
involves the coordination of many distinct but overlapping physiological spaces, compris-
Received: 6 May 2021
Accepted: 7 June 2021
Published: 10 June 2021
ing hemostasis, inflammation, epithelial cell proliferation, and tissue remodeling [
elderly or diabetic patients, the risk of wound infection increased due to vascular aging
and the lower tissue repair capability, which may eventually lead to chronic wounds [ ].
Therefore, wound healing is one of the hot topics in skin surgery. At present, few drugs
have been found with substantial abilities of promoting wound healing [ ]. Actually, the
quality of wound regeneration mainly depends on the efficiency of wound care [ ]. In this
3–5]. In
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
6
7
8
study, we synthesized a series of novel spiro-tetrahydroquinoline derivatives and compared
their effects in wound healing in human epidermal cells and animal models.
Quinoline derivatives have attracted both synthetic and biological chemists because
of their diverse chemical and pharmacological properties, such as anticancer, antimy-
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
cobacterial, anticonvulsant, anti-inflammatory and anti-cardiovascular diseases [
9]. Some
quinoline derivatives were found to have wound healing activity [10 13]. Tetrahydroquino-
–
line derivatives, which are the reduced form of quinolines, were used as antibacterials,
antitumor and anti-HIV agents [14]. However, there are very few reports about tetrahydro-
quinoline on wound healing even though their structures are so similar to each other [15]. It
Int. J. Mol. Sci. 2021, 22, 6251. https://doi.org/10.3390/ijms22126251
https://www.mdpi.com/journal/ijms

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is a curious question of whether tetrahydroquinoline will deliver similar effects on wound
healing or not.
The natural pharmacophore, spiro-1,3-indandione, has attracted considerable attention
due to its diverse biological activities [16–18]. Fredericamycin A was isolated from a
fermentation broth of the strain Streptomyces griseus and reported to demonstrate antibiotic
properties [19]. Synthetic studies were also performed to obtain additional 1,3-indandione
derivatives, which demonstrated antitumor and antibiotic activities [20,21]. In recent
research, the spiro-1,3-indandione moiety has been considered to represent a valuable
functional group for medicinal chemistry [22]. However, the synthesis of carbon spiro-
1,3-indandiones remains a challenge [23,24]. Yan et al. previously synthesized spiro-
1,3-indandiones from cyclic azomethine imine and 2-arylideneindene-1,3-dione via 1,3-
dipolar cycloaddition [25]. Enders et al. used 1,4-dithiane-2,5-diol and 2-arylideneindene-
1,3-dione as the starting materials to obtain spiro-1,3-indandione, using a squaramide-
catalyzed sulfa-Michael/aldol domino reaction [26]. As a starting material that is commonly
used for the generation of spiro-1,3-indandiones, 2-arylidene-1,3-indandiones were the
focus of our recent study. In our previous research, 2-arylidene-1,3-indandiones were
used to obtain enantioselective spiro-nitrocyclopropanes, which contained the spiro-1,3-
indandione skeleton, catalyzed by cinchona alkoloid derivatives [27]. Indanedione-fused
2,6-disubstituted spiro-cyclohexanones have also been studied [28].
Recently, our team has focused on the wound-healing activities of natural prod-
ucts [29]. As tetrahydroquinoline and 1,3-indandione are both widely studied as phar-
macophores in medicinal chemistry, we considered whether the synthesis of hybrids of
tetrahydroquinoline and spiro-indandione residues could result in any meaningful active
compounds. Song and Du have synthesized highly functionalized spirothiazolidinone
tetrahydroquinolines via a squaramide-catalyzed cascade reaction [30]. Therefore, we
synthesized spiro-tetrahydroquinolines compounds with a similar method for further study,
focusing particularly on their wound-healing capabilities.
2. Results
The ortho-N-sulfonated aminophenyl α,β-unsaturated ketone 1a and 2-benzylidene-1,3-
indandione 2a were used as model substrates for the synthesis of spiro-tetrahydroqunioline
derivative 3a via an aza-Michael/Michael reaction (Table 1). Initially, the reaction of 1a
with 2a in the presence of DABCO furnished the desired product 3a in 71% yield at 30 ◦C
in p-xylene (Table 1, entry 1). Examination of different tertiary amines such as DMAP, Et3N
and DIPEA as catalysts did not improve the yield of the product 3a (Table 1, entries 2–4). It
is worthy to note that all the catalysts furnished the product 3a in excellent diastereomeric
ratio (>20:1). Furthermore, various solvents like toluene, CH2Cl2, THF, EtOAc and Et2O
were screened to optimize the reaction conditions (Table 1, entries 5–10). The results proved
that aprotic solvents are the best for the reaction conditions such as toluene and CH2Cl2
(Table 1, entries 5 and 6). Considering the solubility of the starting materials 1 and 2, we
chose to test the CH2Cl2 as optimized solvent for further screening of reaction. When the
reaction was carried out at 0 ◦C, the yield of the desired product 3a was improved to 97%,
but the reaction required longer times (38 h) compared to 30 ◦C (Table 1, entries 6 and 11).
The reaction conditions were further evaluated by the catalyst loading and concentration
of the solvent, which indicated that 5 mol% of DABCO was sufficient for the completion
of the reaction in CH2Cl2 (1 mL) (Table 1, entries 12 and 13). The reaction conditions in
Table 1, entry 13 were selected as the optimal conditions for further studies. The products
were purified by the recrystallization, and identified by 1H-NMR, 13C-NMR and HRMS.
The stereo configuration of the compound 3a was determined by single crystal X-ray
diffraction analysis as shown in Figure 1 [31].

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Table 1. Optimization of conditions of the aza-Michael/Michael reaction a.
◦
Yield (%) b,c
Entry
Cat.
Solvent
Temp. ( C)
Time (h)
1
2
3
4
5
6
7
8
9
DABCO
DMAP
NEt3
p-Xylene
p-Xylene
p-Xylene
p-Xylene
Toluene
CH2Cl2
THF
EtOAc
MeCN
Et2O
30
30
30
30
30
30
30
30
30
30
0
12
12
24
24
12
12
12
12
12
12
38
12
12
71
47
57
17
91
96
77
88
87
11
97
90
92
DIPEA
DABCO
DABCO
DABCO
DABCO
DABCO
DABCO
DABCO
DABCO
DABCO
10
11
CH2Cl2
CH2Cl2
CH2Cl2
12 d,e
13 d,f
a
30
30
b
Unless noted, all reactions were carried out with 1a (0.1 mmol), 2a (0.1 mmol), and catalyst (20 mol%) in 0.5 mL solvent. Yield and
c
d
diastereomeric ratio were determined by 400 MHz NMR with Ph3CH as an internal standard. Diastereomeric ratio was >20:1. 1.0 mL
solvent was used. 10 mol% of catalyst was used. 5 mol% of catalyst was used.
e
f
Figure 1. The single crystal X-ray diffraction of 3a.
With the optimal condition in hand, the substrate scope was further investigated.
In general, all the substrates and with different electron-withdrawing and electron-
1
2
donating R1 and R2 substituents were provided the desired spiro-tetrahydroquinolines
in good to excellent yields (Table 2). At first, different indandione derivatives of R2
substituents were tested with 1a. We noticed significant steric and electronic effects of
the R2 substituent in the reaction outcome. Substrates with meta- and para-bromo groups
as R2 substituents reacted well with 1a to afford the spiro-tetrahydroquinoline products
3c and 3d in up to 83% yields within 3 h, whereas the substrate with R2 as ortho-bromo
substituent was less reactive and the desired product 3b was obtained in 71% yield in
longer reaction times (12 h). It clearly indicates that the rate of the reaction is reduced by the
sterically hindered substituents. We also found that the reaction rate also depends on the
electronic properties of the R2 substituents. For example, the substrates bearing electron-
withdrawing groups 2d–2g subjected with 1a, furnished the corresponding products 3d–3g
in high yields (81–85%) within 3 h (Table 1, entries 4–7), but the substrate bearing an
electron-donating group such as 4-OMePh as R2 substituent 2h resulted in the desired

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product 3h in only moderate yield (68%), when prolonging the reaction time up to 29 h
(Table 1, entry 8). In contrast, when substrates with heteroaryl (furyl or thienyl) groups 2i
and 2j were employed, the reaction could not proceed to provide the products even after
24 h (Table 1, entries 9 and 10).
Table 2. Effect of different substituents on reaction rate a.
Yield (%) b
Entry
R1
R2
Time (h)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Ph (1a)
Ph (1a)
Ph (1a)
Ph (1a)
Ph (1a)
Ph (1a)
Ph (1a)
Ph (1a)
Ph (1a)
Ph (2a)
2-BrPh (2b)
3-BrPh (2c)
4-BrPh (2d)
4-ClPh (2e)
4-CNPh (2f)
4-NO2Ph (2g)
4-OMePh (2h)
Furyl (2i)
Thienyl (2j)
Ph (2a)
12
24
3
3
3
3
3
29
24
24
3
3
3
12
24
6
92 (3a)
71 (3b)
81 (3c)
83 (3d)
86 (3e)
81 (3f)
82 (3g)
68 (3h)
N.R.
Ph (1a)
H (1b)
N.R.
95 (3i)
93 (3j)
96 (3k)
89 (3l)
N.R.
4-BrPh (1c)
4-NO2Ph (1d)
4-OMePh (1e)
OEt (1f)
2-Naph (1g)
Ph (2a)
Ph (2a)
Ph (2a)
Ph (2a)
Ph (2a)
85 (3m)
a Unless noted, all reactions were carried out with
1 (0.2 mmol), 2 (0.2 mmol) and CH2Cl2
(1 mL) was used. b Yield of the product 3 was recrystallized from the ethanol and hexane,
dr > 20:1 (3a–3m).
Furthermore, different R1 substituents of
to prepare the desired spiro-tetrahydroquinolines
1
were also tested in the reaction conditions
. Delightfully, the substrate having
3
aldehyde (R1 = H) group reacted well with 2a to afford the corresponding product 3i in
95% yield within 3 h (Table 1, entry 11). As similar as R2, substrates bearing electron-
withdrawing R1 groups (1c and 1d) more efficiently furnished the desired products 3j and
3k compared to the electron-donating group such as 1e (Table 1, entries 12–14). When
substrate with ester group 1f was employed as the reactant, the corresponding product
could not be found in the reaction (Table 1, entry 15). It could be understood that the
second Michael addition was not efficient when the electron-rich ester is present rather
than an aldehyde or ketone. In addition, 2-naphthyl group of 1g also furnished the product
3m in 85% yield in 6 h (Table 1, entry 16).
The results of the MTT test revealed the cell viability of human keratinocyte cells
(HaCaT) treated with the 13 derived compounds as shown in Table 3. The survival
rates of cells treated with 3b were approximately 80% at all treatment concentrations
(Supplementary Figure S1A). Cells treated with 3c displayed a survival rate of 80% at
concentrations of 6.25 and 12.5
to or greater than 25 M (Supplementary Figure S1B). Cells treated with 3i displayed
low survival rates at concentrations greater than 25 M (Supplementary Figure S1C).
µM, but the growth was inhibited at concentrations equal
µ
µ
Cells treated with 3l displayed a survival rate of approximately 80% at all concentrations
(Supplementary Figure S1D). The survival rates of cells treated with 3m at concentrations
of 6.25 and 12.5 µM were 80% and 60%, respectively. Compared with the other tested

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compounds, the five compounds described above had little effect on HaCaT cell growth
(Supplementary Figure S1), whereas the remaining compounds displayed the strong in-
hibition of HaCaT cell growth. The results of the remaining samples can be found in the
supplementary materials. Therefore, we selected the five compounds with limited effects
on cell viability for use in subsequent experiments.
Table 3. In vitro cytotoxicity data of sample.
Cell Viability after 24 Hours (% Cell Viability) a
Compounds
6.25
12.5
25
50
100
3a
3b
3c
3d
3e
3f
3g
3h
3i
51.85 ± 1.52
91.58 ± 3.00
92.63 ± 1.89
71.53 ± 3.02
59.63 ± 3.10
43.08 ± 2.68
49.53 ± 0.63
73.02 ± 3.04
87.90 ± 5.68
40.36 ± 1.71
20.09 ± 0.64
87.31 ± 0.95
84.42 ± 1.69
46.53 ± 1.73
89.56 ± 4.50
75.38 ± 3.36
55.22 ± 4.37
51.38 ± 3.10
38.78 ± 3.54
43.68 ± 1.41
65.79 ± 0.77
61.38 ± 6.78
26.33 ± 0.92
7.05 ± 0.17
84.57 ± 1.78
70.56 ± 1.24
45.02 ± 2.07
86.63 ± 3.02
66.05 ± 0.57
45.98 ± 0.63
47.06 ± 1.18
39.22 ± 3.23
40.21 ± 1.04
62.66 ± 0.96
18.21 ± 4.26
19.02 ± 0.40
7.15 ± 0.07
82.97 ± 1.22
59.69 ± 2.02
39.66 ± 0.94
80.75 ± 2.61
54.61 ± 2.84
41.35 ± 2.36
42.13 ± 1.18
35.45 ± 2.68
38.91 ± 0.60
52.92 ± 0.76
14.23 ± 1.10
15.73 ± 0.47
7.03 ± 0.15
77.60 ± 1.43
47.18 ± 1.34
36.15 ± 0.91
75.33 ± 2.27
44.72 ± 4.14
35.24 ± 0.86
37.55 ± 1.78
27.08 ± 6.40
33.18 ± 1.32
43.04 ± 1.19
14.10 ± 1.69
11.45 ± 0.11
7.09 ± 0.13
76.15 ± 2.13
35.47 ± 0.28
3j
3k
3l
3m
a Inhibition of cell growth by the listed compounds was determined using the MTT assay.
HaCaT are human keratinocyte cell line.
Based on the results of the cell viability experiments, the activities of the five selected
compounds were evaluated in the scratch assay. The results shown in Figure 2 indicated
that the wounds healed gradually healed within 15–18 h when the tested drugs were added
to the culture medium. 3b had a significant effect on wound healing at the concentration
of 25
to 50
µ
M, but no significant effect was observed when the concentration was increased
M (Figure 3A). 3c had an effect on wound healing at concentrations of 12.5 and
µ
25
and 25
were observed for low concentration (6.25
µ
M (Figure 3B). 3i had a significant effect on wound healing at concentrations of 12.5
M. Compared with the control groups, no significant effects on wound healing
M) or high concentration (50 M) treatment
µ
µ
µ
with either 3c or 3i (Figure 3C). 3l and 3m displayed no significant effects on the promotion
of wound healing at any of the tested concentrations and were found to have inhibitory
effects (Figure 3D,E). The optimal concentration data based on these results is shown in
Figure 3F, and the compounds 3b, 3c, and 3i were selected for further study in an animal
model of wound closure.
Based on the results obtained from the scratch assay, wound-healing tests in mice were
performed to examine the effects of wound treatment using 3b 3c, and 3i (Figure 4A).
The control group was observed to shed the scab on the 9th day, and the wound repair
was completed on the 13th day. In mice treated with 3b at a concentration of 50 M,
the scab fell off on the 12th day, and the wound repair was completed on the 13th day
(Figure 4B). Treatment with 6.25 M of 3c resulted in the shedding of the pupae on the
9th day, but the wound was not completely repaired by the 13th day (Figure 4C). When
a 25 M concentration of 3i was applied, the pupae were shed on the 11th day, and the
wound was repaired. The wound healing was completed by the 11th day, and the pupae
were shed on the 7th day when the concentration of 3i was increased to 50 M (Figure 4D).
We compared the optimal concentrations of 3b 3c, and 3i treatment based on the results of
,
µ
µ
µ
µ
,
the mouse wound healing model. 3i was demonstrated to have the best effect on wound
healing at a concentration of 50 µM (Figure 4E).

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Figure 2. Representative images showing wound closures in HaCaT cells 0, 15, 18, and 24 h after
wounding. Compounds 3b
,
3c,
3i
, 3l, and 3m were added to the medium to test their effects on
wound healing (magnification: 4
are displayed in this figure. The remaining groups are shown in the supplementary materials.
×
objective). The groups with the strongest effects on cell migration
Figure 3. The migration of HaCaT cells treated with various doses of the derived compounds.
Cells were treated with (
A
)
3b, (B
)
3c, (C)
3i, (D) 3l, and (E) 3m at different concentrations for
24 h and subjected to the scratch assay. (F) Comparisons of the five compounds at their respective
best concentrations after 24-h treatment in HaCaT cells subjected to scratch assay, expressed as a
percentage of control (set as 100%). * p < 0.01, ** p < 0.001.

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Figure 4.
3i. Representative pictures of the wounds are shown at days 0, 3, 5, 7, 9, 11, and 13. Wound closure progression during
treatment with ( 3b, ( 3c, and ( 3i. ( ) Comparison of the effects of the optimal concentrations of 3b 3c, and 3i on
wound healing. 3b 3c, and 3i are arranged in accordance with the specific concentration of each compound that was used
(A) The closure of mouse wounds treated with empty fibrin gel (control) or fibrin gel containing 3b, 3c, and
B)
C
)
D)
E
,
,
in a gel applied to wounds in mice once per day for a total of 13 days.
3. Discussion
The plausible mechanism of the reaction is depicted in Scheme 1. Initially, the chalcone
derivative is deprotonated by tertiary amine, yielding a nitrogen-nucleophile which
would attack 2-arylidene-1,3-indandiones to generate aza-Michael adduct II, through
a first Michael addition (Scheme 1). The second intramolecular Michael addition upon
1
I
2

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II to provide III with both R1 and R2 in the same side and subsequent protonation and
enolization would result in the spiro-tetrahydroquinolines 3 in high yields.
Scheme 1. Proposed mechanism of synthesizing spiro-tetrahydroquinolines by one-pot reaction.
As shown in Scheme 1, the transition states have two isomers (syn- and anti-isomers)
that can interchange with each other. In the piperidine ring, if the green hydrogen atom
heads upward (syn-isomer), the steric effect of the sulfonamide and enone moiety was less
impact, and compound 3 was more easily formed. On the contrary, green enone moiety
heads upward in the anti-isomer which shows more steric effect and makes it more difficult
to form 30.
In previous studies that have examined wound healing, most researchers have focused
on natural products, such as extracts and extensions of Chinese herbal medicines or marine
natural products. Only a few studies have examined the effects of synthetic chemicals
on wound-healing outcomes. In this study, we explored the efficacy of wound healing
by studying synthetic compounds and their related skeletal extensions. All compounds
were preliminarily studied in human epidermal cells (HaCaT). Compound 3i was selected
because, at low concentrations, the survival rate of cells was over 80%. Compounds 3b
,
3c 3k, and 3m were shown to have less effect on the growth of HaCaT cells, based on the
,
results of the MTT assay (Supplementary Figure S1). We performed a scratch analysis and
observed that compound 3i has positive effects on wound healing. In the MTT assay, 3i
displayed the significant inhibition of cell growth at the 25
different outcomes observed for these two experiments, we considered whether 3i could
be effective in animal models. Compared with 3b and 3c 3i was demonstrated to represent
µM concentration. Due to the
,
the best compound for healing wounds on mouse skin. The wound was completely healed
by the 11th day in mice treated with 3i. Based on the results of the MTT and wound-healing
assay, 3i is thought to promote wound healing by promoting cell proliferation. In summary,

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we hope that the synthesis of spiro-tetrahydroqunioline derivatives might provide a new
method for identifying chemicals for application in future wound-healing research.
4. Materials and Methods
All chemicals were analytical grade, purchased from Sigma-Aldrich (St. Louis, MO,
USA), Alfa Aesar (Ward Hill, MA, USA), and Merck (Darmstadt, Germany). The purity of
compounds was determined by TLC plates coated with Merck Silica gel 60 F254 (0.2 mm).
Spots were observed under UV lamp or stained by dyeing agent. Infrared Spectroscopy
(IR) were recorded on JASCOF/IR-5300 (Easton, MD, USA), polystyrene was used as
1
internal standard to mark 1601 cm−1. H-NMR and 13C-NMR spectra were recorded on
Bruker Avance 400 MHz spectrophotometer (Billerica, MA, USA). In 1H-NMR, CDCl3 was
used as d-solvent, TMS as internal standard to mark 0 ppm. The definition of splitting
term: singlet (s), doublet (d), triplet (t), qutratet (q), multiplet (m), coupling constant (J). In
13C-NMR, chloroform was used as internal standard to mark 77.0 ppm. Mass Spectroscopy
(MS) and High-Resolution Mass Spectroscopy (HRMS) were recorded on JMS-700 (JEOL),
(Tokyo, Japan), double focusing mass spectrometer (FAB and EI), Applied Biosystems
4800 Proteomics Analyze (MALDI) (Foster City, CA, USA) or Waters (Milford, MA, USA)
LCT Premier XE (ESI). X-ray spectra were recorded on Enraf-Nonius FR590 and Non-
ious CAD4 Kappa Axis XRD (Bruker). Dulbecco’s Modified Eagle Medium (DMEM) and
Penicillin-Streptomycin were purchased from Gibco (Waltham, MA, USA). Fetal Bovine
Serum (FBS) was purchased from HyClone (Marlborough, MA, USA). Methythiazolyltetra-
zolium (MTT), Polyethylene glycol 400 (PEG 400), and Polyethylene glycol 4000 (PEG 4000)
were purchased from Sigma-Aldrich. Culture insert for migration assay was purchased
from ibidi (Planegg, Germany). Anesthesia for animal test (Zoletil) was purchased from
Virbac (Carros, France). Fluorescence interpretation and image analysis were collected
using CytationTM 5 Cell Imaging Multi-Mode Reader, BioTek (Winooski, VT, USA).
4.1. Ortho-N-Protected Aminophenyl α,β-Unsaturated Ketones (1)
2-aminobenzyl alcohol (20 mmol, 2.46 g) was dissolved in 100 mL DCM, and benzene-
sulfonyl chloride (22 mmol, 4.18 g) and 1 mL pyridine were added. The mixture was stirred
for 12 h at r.t. The solvent was removed under reduced pressure. The residue 4 was not
purified and dissolved in 50 mL DCM. Pyridinium chlorochromate (30 mmol, 6.46 g) was
added and the resulting solution was stirred at r.t. for 4 h. The reaction mixture was filtered
by Celite 545 and washed by DCM. After solvent was removed under reduced pressure,
the residue was purified by flash chromatography (DCM: Hexanes = 2:1) to give 5, yield
97%.
5 (3 mmol, 0.83 g) was dissolved in 15 mL toluene, 6 (3.3 mmol) was added. The
mixture was stirred at 80 ◦C for 12 h. The solvent was removed under reduced pressure,
residue was purified by flash chromatography to yield 1 as shown in Scheme 2.
Scheme 2. The common synthesis of compound 1.

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(E)-N-(2-(3-oxo-3-phenylprop-1-en-1-yl)phenyl)benzenesulfonamide (1a)
(mp 147.3–148.2 ◦C):
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 7.93 (d, 2H, J = 8.2 Hz), 7.74 (d, 1H,
J = 15.8 Hz), 7.71–7.66 (m, 2H),7.65–7.55 (m, 3H), 7.52–7.45 (m, 3H), 7.39 (t, 1H, J = 7.8 Hz),
7.36–7.21 (m, 4H), 7.20 (d, 1H, J = 15.8 Hz).
13C-NMR (100 MHz, CDCl3, 25 ◦C) δ/ppm: 190.21, 139.1, 138.8, 137.6, 135.2, 133.1,
132.9, 131.1, 131.0, 129.0, 128.63, 128.60, 127.9, 127.3, 127.2, 127.1, 124.2.
HRMS (ESI) for C21H18NO3S, [M + H]+ (364.1002), found 364.1006.
IR (KBr) v (cm−1): 3236, 3063, 2821, 1655, 1602, 1573, 1333, 1218, 1165, 1091, 734.
(E)-N-(2-(3-oxoprop-1-en-1-yl)phenyl)benzenesulfonamide (1b).
mp: 137.0–137.6 ◦C.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 9.50 (d, 1H, J = 7.6 Hz), 7.71 (d, 2H,
J = 7.8 Hz), 7.64–7.54 (m, 3H), 7.46 (t, 2H, J = 7.8 Hz), 7.38–7.30 (m, 2H), 7.16–7.07 (m, 2H),
6.54 (dd, 1H, J1 = 15.8 Hz, J2 = 7.8 Hz).◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 193.8, 147.2, 138.5, 134.5, 133.4, 131.7,
131.4, 129.9, 129.2, 128.3, 128.0, 127.4, 127.3.
IR (KBr) v (cm−1): 3237, 3063, 2828, 1671, 1623, 1448, 1331, 1160, 1132, 1091, 972, 758.
HRMS (ESI) for C15H13NO3SNa, [M + Na] + (310.0508), found 310.0517.
(E)-N-(2-(3-(4-bromophenyl)-3-oxoprop-1-en-1-yl)phenyl)benzenesulfonamide (1c).
mp: 137.0–137.6 ◦C.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 7.82–7.72 (m, 3H), 7.70 (d, 2H, J = 7.8 Hz),
7.63–7.58 (m, 3H), 7.55 (s, 1H), 7.45–7.26 (m, 6H), 7.16 (d, 1H, J = 15.8 Hz).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 189.2, 139.8, 138.9, 136.3, 135.2, 133.0,
131.9, 131.3, 131.0, 130.1, 129.0, 128.2, 127.8, 127.3, 127.2, 123.7.
IR (KBr) v (cm−1): 3236, 3072, 2835, 1654, 1598, 1397, 1330, 1216, 1164, 1091, 758.
HRMS (ESI) for C21H17NO3S79Br, [M + H]+ (442.0107), found 442.0111.
HRMS (ESI) for C21H17NO3S81Br, [M + H]+ (444.0087), found 442.0093.
(E)-N-(2-(3-(4-nitrophenyl)-3-oxoprop-1-en-1-yl)phenyl)benzenesulfonamide (1d).
mp: 185.4–185.7 ◦C.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.37 (d, 2H, J = 8.8 Hz), 8.13 (d, 2H,
J = 8.8 Hz), 7.81 (d, 1H, J = 15.8 Hz), 7.75–7.68 (m, 3H), 7.54–7.34 (m, 6H), 7.27–7.24 (m, 1H),
6.73 (s, 1H).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 189.0, 150.2, 142.5, 141.1, 138.7, 135.1,
133.2, 131.7, 131.0, 129.6, 129.2, 127.7, 127.4, 127.3, 123.9.
IR (KBr) v (cm−1): 3253, 3068, 2926, 1664, 1597, 1523, 1329, 1212, 1162, 1090, 734.
HRMS (ESI) for C21H17N2O5S, [M + H]+ (409.0853), found 409.0856.
(E)-N-(2-(3-(4-methoxyphenyl)-3-oxoprop-1-en-1-yl)phenyl)benzenesulfonamide (1e).
mp: 171.8–172.0 ◦C.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 7.95 (d, 2H, J = 7.8 Hz), 7.83 (s, 1H), 7.75
(d, 1H, J = 15.8 Hz), 7.71–7.65 (m, 2H), 7.59–7.50 (m, 2H), 7.39 (t, 1H, J = 7.8 Hz), 7.35–7.23
(m, 4H), 7.18 (d, 1H, J = 15.8 Hz), 6.95 (d, 2H, J = 7.8 Hz), 3.88 (s, 3H).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 188.4, 163.6, 138.9, 138.3, 135.1, 132.9,
131.1, 131.0, 130.9, 130.5, 129.0, 127.8, 127.2, 127.1, 124.2, 113.9, 55.5.
IR (KBr)
v
(cm−1): 3182, 3068, 2840, 1651, 1603, 1335, 1263, 1223, 1167, 1091, 1022, 760.
HRMS (ESI) for C22H20NO4S, [M + H]+ (394.1108), found 394.1114.
Ethyl-(E)-3-(2-(phenylsulfonamido)phenyl)acrylate (1f).
mp: 125.1–125.6 ◦C.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 7.68 (d, 2H, J = 8.0 Hz), 7.56 (d, 1H,
J = 15.8 Hz), 7.50 (t, 1H, J = 7.6 Hz), 7.47–7.42 (m, 2H), 7.42–7.33 (m, 3H), 7.27–7.21 (m, 2H),
6.12 (d, 1H, J = 15.8 Hz), 4.23 (q, 2H, J = 7.2 Hz), 1.32 (t, 3H, J = 7.2 Hz).
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 166.5, 138.8, 134.5, 133.0, 130.9, 130.4,
◦
129.0, 127.5, 127.3, 127.2, 127.0, 120.7, 60.8, 14.2.
IR (KBr)
(ESI) for C17H17NO4SNa, [M + Na]+ (354.0770), found 354.0775.
v
(cm−1): 3244, 3068, 2983, 1692, 1635, 1448, 1320, 1166, 1091, 979, 762. HRMS

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(E)-N-(2-(3-(naphthalen-2-yl)-3-oxoprop-1-en-1-yl)phenyl)benzenesulfonamide (1g).
mp: 161.2–161.5 ◦C.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.45 (s, 1H), 8.02–7.94 (m, 2H), 7.91–7.84
(m, 2H), 7.77 (d, 1H, J = 15.8 Hz), 7.73–7.67 (m, 2H), 7.67–7.53 (m, 3H), 7.51–7.43 (m, 2H),
7.43–7.25 (m, 6H).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 190.0, 139.0, 138.9, 135.6, 135.2, 134.9,
133.0, 132.5, 131.2, 131.1, 130.4, 129.6, 129.1, 128.7, 128.6, 127.8, 127.3, 127.2, 126.9, 124.5, 124.4.
IR (KBr) v (cm−1): 3213, 3063, 2813, 1653, 1626, 1598, 1485, 1327, 1165, 1091, 759.
HRMS (ESI) for C25H20NO3S, [M + H]+ (414.1158), found 414.1164.
4.2. 2-Arylidene-1,3-Indandiones (2)
A solution of the mixture of 1,3-indandione (5 mmol, 0.73 g), aldehyde (5.5 mmol) and
L-proline (1.5 mmol, 0.17 g) in methanol (90 mL) was stirred for 12 h at r.t. The solvent was
removed under reduced pressure, and the residue was washed by methanol. The solid
was purified by flash chromatography as shown in Scheme 3.
2
Scheme 3. The common synthesis of compound 2.
4.3. Spiro-Tetrahydroquinoline (3)
Compounds
1 (0.2 mmol) and 2 (0.2 mmol) were dissolved in 1 mL DCM, 1.2 mg
◦
DABCO (5 mol%) was added as catalysis. The reaction was sealed and stirred at 30 C. After
the reaction completed, the mixture was quenched with 1 N hydrochloric acid aqueous
solution and extracted with DCM. The organic layer was washed with water, dried over
MgSO4, filtered, re-crystallized in ethanol and hexane.
◦
3a (110.0 mg, yield 92%, mp 243–244 C): 1a (72.7 mg, 0.2 mmol) and 2a (46.9 mg,
0.2 mmol) as starting material, reacted for 12 h.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.03 (d, 1H, J = 7.8 Hz), 7.92 (d, 1H,
J = 7.6 Hz), 7.78–7.69 (m, 3H), 7.69–7.60 (m, 3H), 7.59–7.44 (m, 6H), 7.37 (t, 2H, J = 7.8 Hz),
7.20 (t, 1H, J = 7.6 Hz), 7.07–6.94 (m, 5H), 6.68 (d, 1H, J = 8.0 Hz), 5.87 (s, 1H), 3.05 (d, 1H,
J = 11 Hz), 2.95 (dd, 1H, J1 = 16.4 Hz, J2◦= 11 Hz), 2.34 (dd, 1H, J1 = 16.4 Hz, J2 = 2.0 Hz).
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 201.2, 197.2, 195.1, 142.7, 141.3, 138.7,
137.6, 137.3, 136.5, 135.9, 135.8, 133.5, 133.4, 133.3, 129.2, 128.7, 128.1, 127.9, 127.8, 127.7,
127.5, 127.1, 126.8, 125.3, 123.03, 122.98, 66.1, 66.0, 37.3, 36.3.
HRMS (ESI) for C37H27NO5S, [M + Na]+ (620.1508), found 620.1509.
IR (KBr) v (cm−1): 3440, 1741, 1705, 14◦48, 1356, 1250, 1169, 1093, 753, 582.
3b (96.1 mg, yield 71%, mp 229–230 C): 1a (72.7 mg, 0.2 mmol) and 2b (62.3 mg,
0.2 mmol) as starting material, reacted for 24 h.
◦
1H-NMR (400 MHz, CDCl3, 25 C) δ/ppm: 7.97 (d, 1H, J = 7.6 Hz), 7.92 (dd, 1H,
J1 = 7.6 Hz, J2 = 1.2Hz), 7.84–7.79 (m, 2H), 7.76(td, 1H, J1 = 7.2 Hz, J2 = 0.8 Hz), 7.71–7.67
(m, 2H), 7.64 (td, 1H, J1 = 7.6 Hz, J2 = 0.8 Hz), 7.62–7.55 (m, 3H), 7.52 (t, 1H, J = 7.6 Hz), 7.47
(t, 1H, J = 7.6 Hz), 7.43–7.34 (m, 3H), 7.28–7.20 (m, 2H), 7.14–7.06 (m, 2H), 6.89–6.82 (m, 1H),
6.77 (d, 1H, J = 7.8 Hz), 6.29 (s, 1H), 3.14–3.00 (m, 2H), 2.33 (d, 1H, J = 16 Hz).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 199.9, 197.7, 195.2, 142.4, 141.7, 138.6,
137.9, 137.0, 136.3, 135.9, 134.1, 133.4, 132.0, 131.5, 129.4, 129.0, 128.6, 128.04, 127.92, 127.7,
127.4, 127.2, 127.0, 125.6, 123.4, 122.7, 121.7, 66.1, 63.3, 36.3, 36.0.
HRMS (ESI) for C37H26Br79NO5S, [M + Na]+ (698.0607), found 698.0612.
HRMS (ESI) for C37H26Br81NO5S, [M + Na]+ (700.0587), found 700.0599.

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IR (KBr) v (cm−1): 3066, 1742, 1707, 1594, 1448, 1358, 1251, 1171, 1090, 949, 754.
◦
3c (109.6 mg, yield 81%, mp 254–255 C): 1a (72.7 mg, 0.2 mmol) and 2c (62.3 mg,
0.2 mmol) as starting material, reacted for 3 h.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.03 (d, 1H, J = 7.8 Hz), 7.95 (d, 1H,
J = 7.8 Hz), 7.79 (t, 1H, J = 7.4 Hz), 7.76–7.67 (m, 3H), 7.65–7.46 (m, 8H), 7.37 (t, 2H,
J = 7.4 Hz), 7.21 (t, 1H, J = 7.4 Hz), 7.17–7.08 (m, 2H), 7.04 (d, 1H, J = 7.4 Hz), 6.92 (t, 1H,
J = 7.6 Hz), 6.66 (d, 1H, J = 7.6 Hz), 5.80 (s, 1H), 3.08–2.87 (m, 2H), 2.33 (d, 1H, J = 16.0 Hz).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 200.9, 196.8, 194.9, 142.6, 141.2, 141.1,
137.3, 137.1, 136.7, 136.1, 135.8, 133.5, 133.3, 130.8, 129.80, 129.77, 129.30, 128.7, 128.0, 127.9,
127.7, 127.1, 126.9, 125.6, 125.3, 123.2, 123.1, 122.1, 65.8, 65.3, 37.4, 36.3.
HRMS (ESI) for C37H26Br79NO5S, [M + Na]+ (698.0607), found 698.0613.
HRMS (ESI) for C37H26Br81NO5S, [M + Na]+ (700.0587), found 700.0601.
IR (KBr) v (cm−1): 3064, 1741, 1707, 1594, 1448, 1357, 1250, 1169, 1090, 915, 732.
◦
3d (112.3 mg, yield 83%, mp 249–250 C): 1a (72.7 mg, 0.2 mmol) and 2d (62.3 mg,
0.2 mmol) as starting material, reacted for 3 h.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.01 (d, 1H, J = 8.0 Hz), 7.92 (d, 1H,
J = 7.6 Hz), 7.80 (t, 1H, J = 7.6 Hz), 7.75–7.66 (m, 3H), 7.64–7.44 (m, 8H), 7.36 (t, 2H,
J = 7.6 Hz), 7.20 (t, 1H, J = 7.6 Hz), 7.15 (d, 2H, J = 8.4 Hz), 6.95 (d, 2H, J = 8.4 Hz), 6.65 (d,
1H, J = 7.4 Hz), 5.83 (s, 1H), 3.02 (d, 1H, J = 11 Hz), 2.92 (dd, 1H, J1 = 16.4 Hz, J2 = 11 Hz),
2.32 (dd, 1H, J1 = 16.4 Hz, J2 = 1.8 Hz).◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 200.8, 197.0, 194.9, 142.5, 141.2, 138.0,
137.4, 137.2, 136.8, 136.2, 135.8, 133.45, 133.40, 133.2, 131.3, 129.3, 128.7, 128.6, 127.9, 127.8,
127.6, 127.1, 126.9125.3, 123.3, 123.1, 121.6, 65.7, 65.3, 37.6, 36.2.
HRMS (ESI) for C37H26Br79NO5S, [M + Na]+ (698.0607), found 698.0610.
HRMS (ESI) for C37H26Br81NO5S, [M + Na]+ (700.0587), found 700.0598.
IR (KBr) v (cm−1): 3068, 1741, 1707, 1594, 1488, 1356, 1250, 1169, 1073, 753.
◦
3e (108.7 mg, yield 86%, mp 253–254 C): 1a (72.7 mg, 0.2 mmol) and 2e (53.7 mg,
0.2 mmol) as starting material, reacted for 3 h.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.01 (d, 1H, J = 7.8 Hz), 7.92 (d, 1H,
J = 7.8 Hz), 7.78 (td, 1H, J1 = 7.6 Hz, J2 = 0.8 Hz), 7.75–7.67 (m, 3H), 7.65–7.44 (m, 8H), 7.36
(t, 2H, J = 7.6 Hz), 7.20 (t, 1H, J = 7.6 Hz), 7.04–6.96 (m, 4H), 6.66 (d, 1H, J = 7.6 Hz), 5.84 (s,
1H), 3.02 (d, 1H, J = 11 Hz), 2.93 (dd, 1H, J1 = 16.4 Hz, J2 = 11 Hz), 2.32 (dd, 1H, J1 = 16.4 Hz,
J2 = 1.6 Hz).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 200.9, 197.0, 194.9, 142.6, 141.2, 137.5,
137.4, 137.2, 136.7, 136.1, 135.8, 133.5, 133.4, 133.2, 129.3, 128.7, 128.4, 128.3, 127.9, 127.8,
127.6127.1, 126.9, 125.3, 123.3, 123.1, 65.8, 65.3, 37.5, 36.2.
HRMS (ESI) for C37H26Cl35NO5S, [M + Na]+ (654.1112), found 654.1118.
HRMS (ESI) for C37H26Cl37NO5S, [M + Na]+ (655.1146), found 656.1104.
IR (KBr) v (cm−1): 3112, 1739, 1707, 1583, 1355, 1250, 1170, 752.
◦
3f (100.9 mg, yield 81%, mp 239–240 C): 1a (72.7 mg, 0.2 mmol) and 2f (51.9 mg,
0.2 mmol) as starting material, reacted for 3 h.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.04 (d, 1H, J = 8.0 Hz), 7.94 (d, 1H,
J = 7.8 Hz), 7.81 (t, 1H, J = 7.4Hz), 7.74 (t, 1H, J = 7.6 Hz), 7.70 (d, 2H, J = 7.6 Hz), 7.65–7.58
(m, 3H), 7.58–7.46 (m, 5H), 7.42–7.30 (m, 4H), 7.24–7.16 (m, 3H), 6.65 (d, 1H, J = 7.8 Hz),
5.91 (s, 1H), 3.03 (d, 1H, J = 11 Hz), 2.93 (dd, 1H, J1 = 16.4 Hz, J2 = 11 Hz), 2.32 (dd, 1H,
J1 = 16.4 Hz, J2 = 1.6 Hz).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 200.4, 196.7, 194.7, 144.3, 142.3, 141.1,
137.1, 136.99, 136.96, 136.4, 135.7, 133.6, 133.5, 132.9, 132.0, 129.4, 128.7, 128.1, 127.8, 127.6,
127.5, 127.1, 125.3, 123.29, 123.23, 118.3, 111.5, 65.6, 65.2,37.7, 36.1.
HRMS (ESI) for C38H26N2O5S, [M + Na]+ (645.1455), found 645.1459.
IR (KBr) vv (cm−1): 3485, 2356, 1736, 1707, 1360, 1250, 1170, 749, 581.
◦
3g (105.4 mg, yield 82%, mp 247–248 C): 1a (72.7 mg, 0.2 mmol) and 2g (55.9 mg,
0.2 mmol) as starting material, reacted for 3 h.

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1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.06 (d, 1H, J = 8.0 Hz), 7.95 (d, 1H,
J = 7.6 Hz), 7.91 (d, 2H, J = 8.6Hz), 7.81 (t, 1H, J = 7.4 Hz), 7.76–7.67 (m, 3H), 7.65–7.58 (m,
3H), 7.58–7.47 (m, 5H), 7.36 (t, 2H, J = 7.8 Hz), 7.31–7.25 (m, 2H), 7.22 (t, 1H, J = 7.6 Hz), 6.66
(d, 1H, J = 7.6 Hz), 5.97 (s, 1H), 3.04 (d, 1H, J = 11 Hz), 2.94 (dd, 1H, J1 = 16.4 Hz, J2 = 11 Hz),
2.33 (dd, 1H, J1 = 16.4 Hz, J2 = 1.8 Hz).◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 200.3196.5, 194.6, 1478.1, 146.3, 142.2,
141.0, 137.1, 137.0, 136.9, 136.5, 135.7, 133.6, 133.5, 132.8, 129.4, 128.7, 128.1, 127.9, 127.8,
127.5, 127.1, 125.3, 123.4, 123.31, 123.26, 65.6, 64.9, 37.8, 36.1.
HRMS (ESI) for C37H26N2O7S, [M + Na] + (665.1353), found 665.1359.
IR (KBr) v (cm−1): 3068, 1743, 1708, 15◦98, 1522, 1340, 1250, 1169, 1088, 689.
3h (85.4 mg, yield 68%, mp 241–242 C): 1a (72.7 mg, 0.2 mmol) and 2h (52.9 mg,
0.2 mmol) as starting material, reacted for 29 h.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.00 (dd, 1H, J1 = 8.0 Hz, J2 = 1.2 Hz), 7.91
(d, 1H, J = 7.6 Hz), 7.75 (td, 1H, J1 = 7.2 Hz, J2 = 1.2 Hz), 7.72–7.60 (m, 5H), 7.59–7.43 (m, 6H),
7.36 (t, 2H, J = 7.6 Hz), 7.19 (td, 1H, J1 = 7.8 Hz, J2 = 1.2 Hz), 6.96 (d, 2H, J = 8.6 Hz), 6.67 (d,
1H, J = 7.6 Hz), 6.54 (d, 2H, J = 8.6 Hz), 5.83 (s, 1H), 3.61 (s, 3H), 3.04 (d, 1H, J = 11 Hz), 2.93
(dd, 1H, J1 = 16.4 Hz, J2 = 11 Hz), 2.33 (dd, 1H, J1 = 16.4 Hz, J2 = 2.0 Hz).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 201.3, 197.3, 195.1, 158.7, 142.8, 141.3,
137.6, 137.4, 136.5, 135.85, 135.8 3, 133.6, 133.4, 133.2, 131.0, 129.2, 128.6, 128.0, 127.9, 127.74,
127.68, 127.1, 126.7, 125.3, 123.1, 123.0, 113.5, 66.1, 65.7, 55.0, 37.3, 36.3.
HRMS (ESI) for C38H29NO6S, [M + Na]+ (650.1608), found 650.1611.
IR (KBr) v (cm−1): 3068, 1739, 1706, 1511, 1353, 1249, 1172, 1086, 720.
◦
3i (99.1 mg, yield 95%, mp 204–205 C): 1c (54.5 mg, 0.2 mmol) and 2a (46.9 mg,
0.2 mmol) as starting material, reacted for 3 h.
◦
1H-NMR (400 MHz, CDCl3, 25 C) δ/ppm: 9.13 (d, 1H, J = 2.4 Hz), 8.04 (dd, 1H,
J1 = 8.0 Hz, J2 = 0.8 Hz), 7.92 (d, 1H, J = 7.6 Hz), 7.75 (td, 1H, J1 = 7.6 Hz, J2 = 0.8 Hz),
7.73–7.68 (m, 2H), 7.67–7.61 (m, 2H), 7.57–7.51 (m, 3H), 7.41 (d, 1H, J = 7.6 Hz), 7.30 (td, 1H,
J1 = 7.6 Hz, J2 = 0.8 Hz), 7.04–6.96 (m, 5H), 6.68 (d, 1H, J = 7.8 Hz), 5.91 (s, 1H), 2.61 (dd, 1H,
J1 = 11.8 Hz, J2 = 2.4 Hz), 2.25 (ddd, 1H, J1 = 17.4 Hz, J2 = 11.8 Hz, J3 = 2.4 Hz), 1.83(dd, 1H,
J1 = 17.4 Hz, J2 = 2.4 Hz).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 200.9, 198.1, 196.5, 124.6, 141.1, 138.4,
137.7, 137.4, 136.6, 136.0, 133.2, 133.0, 129.1, 128.4, 128.3, 128.2, 127.6, 127.3, 127.1, 126.7,
125.0, 123.1, 123.0, 65.9, 65.8, 40.9, 36.2.
HRMS (ESI) for C31H23NO5S, [M + Na]+ (544.1189), found 544.1194.
IR (KBr) v(cm−1): 3059, 2861, 1722, 1710, 1585, 1357, 1246, 1169, 1083, 704.
◦
3j (125.9 mg, yield 93%, mp 265–266 C): 1c (88.5 mg, 0.2 mmol) and 2a (46.9 mg,
0.2 mmol) as starting material, reacted for 3 h.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.03 (d, 1H, J = 8.0 Hz), 7.91 (d, 1H,
J = 7.4 Hz), 7.74 (t, 1H, J = 7.6 Hz), 7.70–7.62 (m, 3H,), 7.58–7.42 (m, 9H), 7.21 (t, 1H,
J = 7.6 Hz), 7.07–6.91(m, 5H), 6.68(d, 1H, J = 7.6 Hz), 5.84 (s, 1H), 3.01 (d, 1H, J = 11.2 Hz),
2.87 (dd, 1H, J1 = 16.8 Hz, J2 = 11.2 Hz), 2.31 (dd, 1H, J1 = 16.8 Hz, J2 = 2.0 Hz).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 201.2, 197.1, 194.3, 142.8, 141.2, 138.6,
137.6, 137.3, 136.5, 135.9, 134.4, 133.3, 133.2, 132.0, 129.4, 129.1, 128.7, 128.1, 127.9, 127.8,
127.6, 127.1, 126.8, 125.2, 123.06, 123.00, 66.2, 66.0, 37.3, 36.4.
HRMS (ESI) for C37H26BrNO5S, [M + H]+ (676.0788), found 676.0783.
IR (KBr) v (cm−1): 3064, 1741, 1706, 1585, 1357, 1250, 1170, 1071, 757, 582.
◦
3k (123.4 mg, yield 96%, mp 269–270 C): 1d (81.7 mg, 0.2 mmol) and 2a (46.9 mg,
0.2 mmol) as starting material, reacted for 3 h.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.21 (d, 2H, J = 8.8 Hz), 8.04 (d, 1H,
J = 7.8 Hz), 7.93 (d, 1H, J = 7.6 Hz), 7.83–7.74 (m, 3H), 7.74–7.64 (m, 3H), 7.61–7.43 (m, 5H),
7.22 (t, 1H, J = 7.4 Hz), 7.03–6.95 (m, 5H), 6.60 (d, 1H, J = 7.6 Hz), 5.84 (s, 1H), 3.08–2.94 (m,
2H), 2.39 (d, 1H, 15.6 Hz).

Int. J. Mol. Sci. 2021, 22, 6251
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◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 201.2, 197.0, 193.8, 150.5, 142.8, 141.2,
140.1, 138.5, 137.8, 137.4, 136.7, 136.0, 133.2, 133.0, 129.2, 129.0, 128.2, 128.10, 127.98, 127.7,
127.2, 126.82, 126.79, 124.9, 123.9, 123.2, 123.1, 66.3, 66.0, 37.1, 36.9.
HRMS (ESI) for C37H26N2O7S, [M + H]+ (643.1533), found 643.1588.
IR (KBr) v (cm−1): 3429, 3075, 1706, 1594, 1524, 1346, 1251, 1172, 1089, 735, 558.
◦
3l (111.8 mg, yield 89%, mp 219–220 C): 1e (78.7 mg, 0.2 mmol) and 2a (46.9 mg,
0.2 mmol) as starting material, reacted for 12 h.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.02 (d, 1H, J = 8.2 Hz), 7.91 (d, 1H,
J = 7.4 Hz), 7.75 (t, 1H, J = 7.4 Hz), 7.71 (d, 2H, J = 7.8 Hz), 7.66 (t, 1H, J = 7.4 Hz), 7.61 (d,
2H, J = 8.8 Hz), 7.58–7.43 (m, 5H), 7.20 (t, 1H, J = 7.6 Hz), 7.06–6.94 (m, 5H), 6.83 (d, 2H,
J = 8.8 Hz), 6.70 (d, 1H, J = 7.8 Hz), 5.86 (s, 1H), 3.83 (s, 3H), 3.05 (d, 1H, J = 11.2 Hz), 2.88
(dd, 1H, J1 = 16.6 Hz, J2 = 11.2 Hz), 2.29 (dd, 1H, J1 = 16.6 Hz, J2 = 2.0 Hz).
13C-NMR (100 MHz, CDCl3, 25 ◦C) δ/ppm: 201.3, 197.3, 193.6, 163.7, 142.7141.3,
138.8, 137.6, 137.3, 136.4, 135.8, 133.7, 133.2, 130.2, 129.2, 128.9, 128.1, 127.8, 127.7, 127.5,
127.1, 126.8, 125.4, 123.0, 123.1, 113.8, 66.1, 66.0, 55.5, 37.5, 36.0.
HRMS (ESI) for C38H29NO6S, [M + H]+ (628.1788), found 628.1800.
IR (KBr) v (cm−1): 3430, 3066, 1706, 1685, 1599, 1354, 1251, 1169, 735, 582.
3m (110.2 mg, yield 85%, mp 250–251 ◦C): 1g (82.7 mg, 0.2 mmol) and 2a (46.9 mg,
0.2 mmol) as starting material, reacted for 6 h.
1H-NMR (400 MHz, CDCl3, 25 ◦C) δ/ppm: 8.08 (s, 1H), 8.04 (d, 1H, J = 7.6 Hz), 7.93
(d, 1H, J = 7.6 Hz), 7.89 (d, 1H, J = 7.8 Hz), 7.81 (dd, 2H, J1 = 12.0 Hz, J2 = 8.2 Hz), 7.76–7.67
(m, 2H), 7.64–7.50 (m, 5H), 7.50–7.43 (m, 2H), 7.28–7.19 (m, 3H), 7.14 (t, 1H, J = 7.4 Hz), 7.06
(d, 1H, J = 6.6 Hz), 7.02–6.89 (m, 4H), 5.89 (s, 1H), 3.10 (d, 1H, J = 11.2 Hz), 3.01 (dd, 1H,
J1 = 15.6 Hz, J2 = 11.2 Hz), 2.54 (dd, 1H, J1 = 15.6 Hz, J2 = 2.0 Hz).
◦
13C-NMR (100 MHz, CDCl3, 25 C) δ/ppm: 201.2, 197.0, 195.5, 142.7, 141.2, 138.7,
137.4, 137.2, 136.5, 135.9, 135.5, 133.5, 132.9, 132.8, 132.2, 129.7, 129.5, 128.9, 128.7, 128.5,
128.0, 127.8, 127.6, 127.5, 126.81, 126.77, 126.67, 125.4, 123.5, 122.9, 66.1, 66.0, 37.8, 36.6.
HRMS (ESI) for C41H29NO5S, [M + H]+ (648.1839), found 648.1846.
IR (KBr)
733, 582.
v
(cm−1): 3430, 3063, 1741, 1706, 1682, 1593, 1356, 1251, 1170, 1091, 755,
4.4. Cell Culture
HaCaT cells which were kindly provided by Prof. Hung-Rong Yen (Integration
of Traditional Chinese-Western Medicine Research Institute, China Medical University
Hospital, Taiwan) were maintained in a high-glucose Dulbecco’s modified Eagle medium
(DMEM), which was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin
and streptomycin. Cells were cultured at 37 ◦C in a humidified environment of 5% CO2.
Then, 0.25% Trypsin-EDTA was used to vaccinate when the amount of cultured cells
reached 1 × 106 [32].
4.5. Cell Viability Assay
Cytotoxicity was analyzed using the MTT assay, with slight modifications. Briefly, Ha-
CaT cells were seeded in 96-well plates to allow for overnight adhesion. The following day,
cells were treated with various concentrations (0, 6.25, 12.5, 25, 50, and 100
µ
M) of each com-
L/mL) was added to each well and incubated
L) was added to dissolve the formazan
pound for 2◦4 h. After treatment, MTT (100
µ
for 1 h at 37 C under 5% CO2, and then DMSO (100
µ
in the cells. The absorbance at 575 nm was measured using a microplate spectrophotometer.
The results are expressed as a percentage of the corresponding controls [33].
4.6. Scratch Assay
HaCaT cells using a culture-insert (
µ-Dish35mm, high, BLOSSOM BIOTECHNOLO-
GIES INC, Taipei, Taiwan) seeded in the precoated 24-well plates each compartment of
the◦insert filled with 100
µ
L of cell suspension (about 2
×
105 cells/chamber) for 24 h at
37 C and 5% CO2. The cell debris was removed by washing with PBS when the cells

Int. J. Mol. Sci. 2021, 22, 6251
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grow to 80% confluence. Serum medium (control) and medium containing each tested
compound (6.25, 12.5, 25, 50, and 100
µ
M) were added to corresponding wells, and the cells
were cultured with serum supplemented DMEM for 36 h at 37 ◦C with 5% CO2 [34
,
35].
All scratch assays were conducted in triplicate. The areas of cell migration were analyzed
using ImageJ software.
The wound closures were evaluated using the following formula:
Wound closure (%) = 100 − [(sample area/control area) × 100].
4.7. Splints
The outer diameter, inner diameter, and thickness of round silicone splints were 26,
16, and 500–600 mm, respectively. All procedures and instruments were conducted and
prepared, respectively, under aseptic conditions, which were maintained using autoclaves,
ethylene oxide gas, 70% ethanol, and povidone-iodine. Mice were anesthetized with
pentobarbital. An electric razor was used to remove the back hair, and a circular mark
(1.0 cm diameter) was placed on the center of the lumbar area; this section of skin was
totally excised using scissors. A splint was inserted beneath the skin near the wound defect
and attached to the fascia with 6-stitch ligations. The splint was then fixed to the skin with
surgical silk thread (6 stitches at regular intervals) [36,37].
4.8. Preparation of Sample-Containing PEG-Based Ointment
PEG 400 Da (100 mg) and PEG 4 kDa (20 mg) were mixed at a ratio of 5:1. The
mixture was then heated to above 85 ◦C until it became a clear solution. The prepared
compound (1 mg) was added to the solution before the solution was allowed to cool to
room temperature, forming a gel [38].
4.9. Animal Experiments
Compounds were tested in 8-week-old wild-type male C57BL/6 mice (BioLASCO
Taiwan Co., Ltd., Taipei, Taiwan). The mice were artificially wounded using splints.
The control gel or the compound-containing gel was applied daily and photographed to
measure the wound area for 13 days. The mice were sacrificed by CO2 exposure, and the
tissues at the location of the silicone ring were removed for subsequent analysis.
4.10. Statistical Analysis
Analyses were performed in triplicate, and the results were expressed as mean
±
SD.
Analysis of variance (ANOVA) was conducted, followed by Dunnett’s post hoc test, to
determine significant (p < 0.05) differences. Statistical analyses were performed using
GraphPad Prism v8.0 (GraphPad Software, San Diego, CA, USA).
5. Conclusions
In this article, we developed a one-pot reaction method that requires only a small
amount of catalyst to obtain nonselective isomers with high selectivity. In addition, the
experimental procedure is simple and only requires extraction and recrystallization. The
reaction was performed at room temperature and had a good yield. This reaction will
be studied in our further research by using different catalysts. We hope that the synthe-
sis strategy described in this article can be widely applied to various starting materials
with diverse substituents. We also hope that the synthesis of spiro-tetrahydroqunioline
derivatives could provide a new method for identifying chemicals that can be applied to
wound-healing research. Further structural modifications and biological evaluations are
ongoing, and the results will be reported in due course.
Supplementary Materials: Supplementary materials can be found at https://www.mdpi.com/
article/10.3390/ijms22126251/s1.

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Author Contributions: Methodology, conceptualization and design of study Y.-C.L. and Y.-A.L.
writing—original draft preparation, K.W.; data curation, W.L. and J.-C.Y.; writing—review and
editing, Y.-J.J. and K.W.; project administration and funding acquisition, W.L., Y.-C.W., J.-C.Y. and
Y.-J.J. All authors have read and agreed to the published version of the manuscript.
Funding: This work is supported by the Ministry of Science and Technology, Taiwan (MOST 107-
2628-M-003-001-MY3, MOST 109-2320-B-039-063-, MOST 107-2320-B-039-017-MY3, and MOST 108-
2113-M-039-005-) and China Medical University, Taiwan (CMU108-MF-96) for financial support.
Institutional Review Board Statement: The Institutional Animal Care (IACUC) of China Medical
University has approved this specific protocols used in this study (approval number: CMUIACUC-
2018-221; date: 19 January 2018).
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
TLC
NMR
thin layer chromatography
nuclear magnetic resonance
DABCO 1:4-diazabicyclo[2.2.2]octane
DMAP
Et3N
DCM
Et2O
THF
EA
4-dimethylaminopyridine
triethylamine
dichloromethane
ether
tetrahydrofuran
ethyl acetate
MeCN
r.t.
Mp
acetonitrile
room temperature
melting point
FBS
MTT
PEG
fetal bovine serum
methythiazolyltetrazolium
polyethylene glycol
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