Ultrasonic-assisted synthesis of 1,4-disubstituted 1,2,3-triazoles via various terminal acetylenes and azide and their quorum sensing inhibition

Article abstract of DOI:10.1016/j.ultsonch.2016.12.011

An efficient synthesis of 1,4-disubstituted 1,2,3-triazole derivatives was studied. 1,4-Disubstituted 1,2,3-triazoles containing isoxazole and thymidine structures were synthesized in 84–96% yields starting from various terminal isoxazole ether alkynes and β-thymidine azide derivatives via a 1,3-dispolar cycloaddition using copper acetate, sodium ascorbate as the catalyst under ultrasonic assisted condition. All the target compounds were characterized by HRMS, FT-IR, ¹H NMR and ¹³C NMR spectroscopy. Furthermore, the quorum sensing inhibitory activities of synthesized compounds were evaluated with Chromobacterium violaceum (C. Violaceum CV026) based on their inhibition of violacein production, with compound C₁₀-HSL as a positive control. The compounds 8a, 8c and 8f exhibited considerable levels of inhibitory activity against violacein production, and IC₅₀ values were 217?±?19, 223?±?20 and 42.8?±?4.5?μM, respectively, which highlighted the potential of these compounds as lead structures for further research towards the development of novel QS inhibitors.

Full text of DOI:10.1016/j.ultsonch.2016.12.011

Accepted Manuscript
Ultrasonic-assisted synthesis of 1,4-disubstituted 1,2,3-triazoles via various ter-
minal acetylenes and azide and their quorum sensing inhibition
Da-wei Zhang, Yu-min Zhang, Jing Li, Tian-qi Zhao, Qiang Gu, Feng Lin
PII:
S1350-4177(16)30450-3
DOI:
http://dx.doi.org/10.1016/j.ultsonch.2016.12.011
ULTSON 3466
Reference:
To appear in:
Ultrasonics Sonochemistry
Received Date:
Revised Date:
Accepted Date:
25 October 2016
7 December 2016
8 December 2016
Please cite this article as: D-w. Zhang, Y-m. Zhang, J. Li, T-q. Zhao, Q. Gu, F. Lin, Ultrasonic-assisted synthesis
of 1,4-disubstituted 1,2,3-triazoles via various terminal acetylenes and azide and their quorum sensing inhibition,
Ultrasonics Sonochemistry (2016), doi: http://dx.doi.org/10.1016/j.ultsonch.2016.12.011
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Graphical Abstract:
Ultrasonic-assisted synthesis of 1,4-disubstituted 1,2,3-triazoles via various
terminal acetylenes and azide and their quorum sensing inhibition
1
Da-wei Zhang , Yu-min Zhang , Jing Li , Tian-qi Zhao , Qiang Gu *, Feng Lin *
1
1
1
1
2
1
College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun, 130012, P. R. China
2
College of Life Sciences, Jilin University, 2699 Qianjin Street, Changchun, 130012, P. R. China
*
*
Corresponding Author: Professor Qiang Gu
Corresponding Author: Professor Feng Lin
Tel: (+86)-431-85168470 (3)
Fax: (+86)-431-85168441
E-mail: guq@jlu.edu.cn
E-mail: linfeng@jlu.edu.cn.
An efficient synthesis of 1,4-disubstituted 1,2,3-triazole derivatives was developed via
1
,3-dispolar cycloaddition starting from various terminal isoxazole ether alkynes and β-thymidine
azides under ultrasonic assisted conditions in satisfactory yields. The compounds 8a, 8c and 8f
exhibited considerable levels of inhibitory activity against violacein production, which
highlighted the potential of these compounds as lead structures for further research towards the
development of novel QS inhibitors.
1

Ultrasonic-assisted synthesis of 1,4-disubstituted 1,2,3-triazoles via various
terminal acetylenes and azide and their quorum sensing inhibition
1
Da-wei Zhang , Yu-min Zhang , Jing Li , Tian-qi Zhao , Qiang Gu *, Feng Lin *
1
1
1
1
2
1
College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun, 130012, P. R. China
2
College of Life Sciences, Jilin University, 2699 Qianjin Street, Changchun, 130012, P. R. China
*
*
Corresponding Author: Professor Qiang Gu
Corresponding Author: Professor Feng Lin
Tel: (+86)-431-85168470 (3)
Fax: (+86)-431-85168441
E-mail: guq@jlu.edu.cn
E-mail: linfeng@jlu.edu.cn.
Abstract
An efficient synthesis of 1,4-disubstituted 1,2,3-triazole derivatives was studied.
1
,4-Disubstituted 1,2,3-triazoles containing isoxazole and thymidine structures were synthesized
in 84–96% yields starting from various terminal isoxazole ether alkynes and β-thymidine azide
derivatives via a 1,3-dispolar cycloaddition using copper acetate, sodium ascorbate as the catalyst
under ultrasonic assisted condition. All the target compounds were characterized by HRMS,
1
13
FT-IR, H NMR and C NMR spectroscopy. Furthermore, the quorum sensing inhibitory
activities of synthesized compounds were evaluated with Chromobacterium violaceum (C.
Violaceum CV026) based on their inhibition of violacein production, with compound C10-HSL as
a positive control. The compounds 8a, 8c and 8f exhibited considerable levels of inhibitory
activity against violacein production, and IC50 values were 217±19, 223±20 and 42.8±4.5 µM,
respectively, which highlighted the potential of these compounds as lead structures for further
research towards the development of novel QS inhibitors.
Keywords 1,2,3-Triazole; Isoxazole; Thymidine; 1,3-Dispolar cycloaddition; Ultrasonic heating;
Quorum sensing
2

1
. Introduction
Quorum sensing (QS) is one of cell communication mechanisms involving the generation,
release and detection of small signaling molecules which can activate specific receptors
associated with transcription signals that are responsible for controlling a variety of different
biochemical processes.[1–3] QS can effectively regulate some important phenotypes that are
mostly relevant to antibiotics resistance[4–6], such as the bioluminescence, virulence expression
and formation of biofilm and so on. In recent years, much more bacterial strains showed
resistance to antibiotics, and it is urgent to find an effective solution. QS inhibitors are new target
agents, which can provide insights into bacterial signaling processes from fundamental and
applied perspectives, conducive to the discovery of novel antibacterial strategy and antimicrobial
agents.[7–9] It was reported that the amide structure of the pyrimidine ring has a potential quorum
sensing inhibitory effect. β-Thymidine derivatives contain lactam structure are expected to
possess good quorum sensing inhibitory effect to Gram negative bacteria.[10,11]
Thymidine derivatives are more widely used in the world of organic heterocyclic
compounds,[12,13] in particular, the amide structure of the pyrimidine ring has a potential quorum
sensing inhibitory effect. They are concerned by people because they exhibit good biological and
pharmaceutical
activity[14,15]
etc..
Several
2',3'-dideoxynucleosides,
including
3
'-azido-3'-deoxythymidine (AZT, zidovudine), have been used as antiretroviral drugs for the
treatment of acquired immunodeficiency syndrome (AIDS).[16,17] In addition, some nucleoside
derivatives also have strong resistance to some tumour cells,[18,19] bacteria[20,21] and so on.
Isoxazoles and 1,2,3-triazoles with better anti-bacterial,[22,23] anti-cancer,[24,25] antiviral,[26,27]
analgesic and anti-inflammatory[28,29] are also important heterocyclic skeleton for synthesizing a
drug molecule, and have been widely used in clinical practice.[30,31] Therefore, the study of the
synthetical method and biological activity of heterocyclic compounds containing thymidine,
isoxazole and 1,2,3-triazole has always been a search realm with vitality in the organic chemistry
and medical chemistry. The structural features and physiochemical properties of their fragment
are of great interest in drug design and discovery.
It is known that 1,3-dipolar cycloaddition reaction occurred between 1,3-dipole body and
olefins, alkynes or corresponding derivatives. Nitrile oxides and azides as the most common
3

1
,3-dipolar body were important precursor for synthesizing isoxazole and 1,2,3-triazole
derivatives.[32–35] The most widely used method to build the 1,2,3-triazole system is the
,3-dipolar cycloaddition of alkynes with azides, which is the famous Click reaction.[36]
However, the major disadvantage of this method is the parallel formation of 1,4 and
,5-regioisomers. Early as 2002, it was reported that Husigen-Click reaction, which
copper(I)-catalyzed alkyne-azide independently conducted 1,3-dipolar cycloaddition reaction
CuAAC), was able to exclusively obtain 1,4-product with a good regioselectivity.[37]
Successively, ruthenium-catalyzed method was reported for the regioselective synthesis of the
,5-product.[38] However, the synthesis of isoxazole-thymidine derivatives has not been reported
1
1
(
1
using copper(I) as a catalyst. With the development of technology, several improved methods
including microwave and ultrasonic assisted method were reported to increase the yield of the
cycloaddition.[39,40] Ultrasonic-assisted organic synthesis is a powerful technique that is widely
being used in organic synthesis reaction. The notable features of the ultrasound approach are
enhanced reaction rate, formation of pure product in high yield, and easier manipulation. In this
work, we reported the cyclization between 3-aryl-5-((prop-2-yn-1-yloxy)methyl) isoxazoles and
1
1
-(4-azido-5-(azidomethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione
or
-(4-azido-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione using
the CuAAC reaction under conventional heating and ultrasonic-assisted condition. The structures
1
13
of the synthesized compounds were characterized by HRMS, FT-IR, H and C NMR
spectroscopy. Herein, we combined 1,2,3-trizole and isoxazole into thymidine derivative structure
get novel target molecules, further evaluated their QS to Gram negative bacteria. It suggested that
4
-substituted group of phenyl on isoxazole had important effect on antibacterial and antimicrobial
agents.
2
2
. Results and discussion
.1 Synthesis
It is known that isoxazole is a versatile scaffold for the synthesis of varieties of complex
natural products, and functionalized isoxazole derivatives are active pharmacophores in many
pharmacologically important molecules.[41,42] In the present work, 3-substituted
4

phenyl-5-((prop-2-yn-1-yloxy)methyl)isoxazoles (4) were synthesized in three steps starting form
substituted benzaldehyde (Scheme 1, Table 1) according to the literature.[43,44] In the process for
synthesizing 3-substituted phenylisoxazol-5-yl)methanols (3), 1,3-dipolar cycloaddition reaction
was catalyzed using ZnCl
2
as a catalyst by one-pot method under ultrasound-assisted with
8–88% yields, which resulted in the yield of the synthesized desired products enhancing
2–29% compared with conventional method. A series of compounds 4 were prepared by
5
1
employing intermediate 3 and propargyl bromide under NaH with 68–96% yields at room
temperature condition, and the influence of different substituents on the yield was investigated.
Scheme 1. The route for synthesizing 3-substituted phenyl-5-((prop-2-yn-1-yloxy)methyl)isoxazoles 4a-l from
aromatic aldehyde 1a-l. Reagents and conditions: (i) NaOH aq. (6mol/L), EtOH, reflux. (ii) a) NCS, DMF. b)
o
o
2 3
Propargyl alcohol, ZnCl , Et N, U.S. 0 C–r.t.. (iii) NaH, propargyl bromide, THF, 0 C–r.t..
Table 1. 3-substituted phenyl-5-((prop-2-yn-1-yloxy)methyl)isoxazoles yields under different substituent
a
Entry
Alcohol
3a
R
C
Time / h
Product
4a
Yield / %
80
1
2
3
4
5
6
7
8
9
6
H
5
4
5
6
5
6
5
8
5
6
8
4
3b
3c
2-Cl-C
4-Cl-C
6
6
H
4
4b
4c
83
H
4
90
3d
3e
2-MeO-C
6
6
H
4
4e
82
4-MeO-C
H
4
4f
96
3f
4-Me-C
4-tBu-C
4-F-C
4-Br-C
3-NO -C
styryl
6
H
4
4d
4g
83
3g
6
H
4
85
3h
3i
6
H
4
4h
4i
97
6
H
4
86
1
0
1
3j
2
6
H
4
4j
93
1
3k
4k
98
5

1
2
3l
2-pyridyl
4
4l
97
a
Isolated product yields.
Scheme 2. Synthesis of 1,2,3-triazole derivatives 7a-l and 8a-g starting from various isoxazole ether alkynes
and thymidine azide derivatives. Reagents and conditions: (i) Cu(OAc) ·H O, sodium ascorbate(NaAsc),
2
2
o
2
i-PrOH-H O (2:1, v/v), 45 C.
Sequentially, the synthesis of 5-((4-(((3-(4-chlorophenyl)isoxazol-5-yl)methoxy)methyl)-1H-
,2,3-triazol-1-yl)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione
1
(
7c) were explored by employing 3-(4-chlorophenyl)-5-((prop-2-yn-1-yloxy)methyl)isoxazole (4c)
and β-thymidine azide derivatives (5) in the presence of copper(I)(Scheme 2). In order to
optimize the reaction conditions, we screened the different heating mode and catalyst, a variety of
2
H O-organic solvents and examined their influence on reaction time and yield of the obtained
target product. The results are summarized in Table 2.
a
Table 2. Effect of various solvents on the model reaction
6

o
Temp. / C
Entry Cu source Solvent
Traditional heating
Ultrasonic radiation
b
Time/h Yield /% Time/min Yield /%
b
1
2
3
4
5
6
7
8
9
None
i-PrOH/H
i-PrOH/H
i-PrOH/H
i-PrOH/H
i-PrOH/H
i-PrOH/H
i-PrOH/H
i-PrOH/H
i-PrOH/H
i-PrOH/H
i-PrOH
2
O (2:1)
O (2:1)
O (2:1)
O (2:1)
O (2:1)
O (2:1)
O (2:1)
O (2:1)
O (1:1)
O (3:1)
45
45
45
45
45
35
r.t
8
10
8
N.D.
73
20
15
10
10
10
10
10
10
10
10
20
20
10
10
10
10
N.D.
82
CuCl
2
2
CuSO
4
2
77
88
Cu(OAc)
2
2
8
78
92
c
CuI
2
8
74
85
Cu(OAc)
Cu(OAc)
Cu(OAc)
Cu(OAc)
Cu(OAc)
Cu(OAc)
Cu(OAc)
Cu(OAc)
Cu(OAc)
Cu(OAc)
Cu(OAc)
2
2
2
2
2
2
2
2
2
2
2
2
8
58
70
2
8
35
56
2
55
45
45
45
45
45
45
45
45
7
78
90
2
8
68
84
1
0
2
8
72
87
1
1
2
3
4
5
6
24
24
8
46
58
1
1
1
1
1
H
2
O
trace
76
trace
81
THF/H
DMSO/H
DMF/H O (2:1)
MeCN/H O (2:1)
2
O (2:1)
2
O (2:1)
4
85
91
2
4
81
89
2
11
56
79
a
The reaction was conducted with alkyne (1.2mmol), azide-thymidine (1mmol), Cu source (0.1mmol),
Na-ascorbate (0.2mmol). N.D. (not detected).
b
Isolated product yields.
c
No Na-ascorbate was added.
As shown in Table 2, the desired 7c was successfully synthesized with 56–92% and 35–85%
yields in the presence of organic solvents and catalyst under ultrasonic radiation and traditional
7

heating, respectively. Exhilaratingly, ultrasonic radiation as an efficient and convenient process,
could shorten reaction time(20 h→20 min; 10 h→10 min and 4 h→10 min). Also, the yields of
the obtained target product were enhanced 6–21% compared with traditional heating. Under the
other identical reaction conditions, no desired product was obtained in the absence of catalyst.
When copper acetate was used as in situ generation of copper(I) ion with sodium ascorbate as a
catalyst, the yield of the obtained 7c was higher than that of copper sulfate, copper chloride and
directly loading copper(I) iodide (Table 2, entries 2–5). However, when the reaction temperature
o
was decreased to room temperature and 35 C, respectively, the yield of the obtained 7c
respectively decreased to 35% and 70% (traditional heating) and 56% and 70% (ultrasonic
o
radiation)(Table 2, entries 4, 6 and 7). Sequentially raising the reaction temperature to 55 C, the
yield of the obtained target product was remained (78% and 90% under the different heating
mode)(Table 2, entry 8). Besides, the solvent played a significant role in progress of reaction.
Among the examined solvents, when i-PrOH and water were used alone, the yields of 46%
(
conventional heating), 58% (ultrasonic heating) and trace for the model reaction were obtained
after 24h, respectively (Table 2, entries 11 and 12). The low yield obtained for the corresponding
triazole derivatives using pure water as a solvent is attributed to lack of organic material
2 2 2
solubility in water. The mixed solvents of i-PrOH–H O, DMF–H O and DMSO–H O (2:1, V/V)
all afforded the better yield (Table 2, entries 4, 14 and 15). Considering economy and
environment, the confirmed optimal synthesis condition was copper acetate, sodium ascorbate as
o
O mixture (2:1, V/V), under ultrasound-assisted at 45 C for 10min.
the catalyst, i-PrOH‒H
2
Table 3 Synthesis and yields of 1,2,3-triazole derivatives 7a-l under the traditional heating and ultrasonic
radiation, respectively.
Entry Alkyne
R
Product
Traditional heating
Ultrasonic radiation
a
Yield /%
a
Time/min Yield /%
Time/h
8

1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
4f
C
6
H
5
7a
7b
7c
7d
7e
7f
7
8
75
78
78
70
84
82
68
71
85
74
72
66
10
10
10
10
10
10
10
10
10
10
10
15
90
93
92
95
95
94
85
96
94
89
89
84
2-Cl-C
6
6
H
4
4-Cl-C
H
4
8
4-MeO-C
4-Me-C
4-tBu-C
3-NO -C
2-MeO-C
6
H
4
10
8
6
H
4
6
H
4
8
4g
4h
4i
2
6
H
4
7g
7h
7i
8
6
H
4
10
8
4-F-C
6
H
4
1
0
4j
4-Br-C
6
H
4
7j
8
11
4k
4l
styryl
7k
7l
9
1
2
2-pyridyl
9
a
Isolated product yields.
Having been optimized the reaction conditions for the model system (Table 2, entry 4) was
chosen to explore the scope and limitations of this protocol (Table 3). As shown in Table 3, all
scanned reactants afforded the corresponding 1,2,3-triazole derivatives under conventional
heating and ultrasonic heating in 66–85% and 84–96% yields, respectively. The results indicated
that the good yields were obtained when benzene ring of R on terminal alkynes was substituted
by ortho and para directing group (Table 3, entries 1–6 and 8–11). However, when benzene ring
2
of R on terminal alkynes was substituted by meta-directing group (–NO and pyridyl etc), the
yields of the synthesized compounds were lower (Table 3, entries 7 and 12). This is because the
meta-directing group could lead to the electron cloud density of isoxazole terminal alkynes
decreased.
Table 4 Synthesis and yields of 1,2,3-triazole derivatives 8a-g under the traditional condition and ultrasonic
radiation, respectively.
9

Entry
Alkyne
R
C
Product
Traditional heating
Ultrasonic radiation
a
Yield /% Time /min Yield /%
a
Time /h
1
2
3
4
5
6
7
4a
4b
4c
4d
4e
4f
6
H
5
8a
8b
8c
8d
8e
8f
8
9
76
82
79
71
83
77
68
10
10
10
10
10
10
10
89
92
91
96
94
90
87
2-Cl-C
6
6
H
4
4-Cl-C
H
4
9
4-MeO-C
4-Me-C
4-tBu-C
3-NO -C
6
H
4
13
9
6
H
4
6
H
4
9
4g
2
6
H
4
8g
8
a
Isolated product yields.
It is known that several 2',3'-dideoxynucleosides, including 3'-azido-3'-deoxythymidine (AZT,
zidovudine) (6) (Scheme 2), have been used as antiretroviral drugs for the treatment of acquired
immunodeficiency syndrome (AIDS).[45] In addition, there are some examples of application of
AZT as an antitumor agent.[46,47] Triazole is versatile scaffold for the synthesis of a wide variety
of complex natural products, and functionalized triazole derivatives are active pharmacophores in
many pharmacologically important molecules.[48,49] Next, we successfully synthesized seven
novel 1,2,3-triazole derivatives containing isoxazole ring and deoxythymidine in 68–83% and
8
7–96% yields starting from isoxazole terminal alkynes (4a–g) and AZT using the above
obtained optimal reaction conditions under conventional heating and ultrasonic heating,
respectively (Table 4). The effect of substituents on the yield was similar to that of compounds
7
a–l.
The scope of this protocol was examined by its application to between various isoxazole
terminal alkynes and β-thymidine azide. The target compounds 7a–l and 8a–g were successfully
1
0

prepared in 66%–85% (traditional heating), 84%–96% (ultrasonic radiation) and 68%–83%
traditional heating), 87%–96% (ultrasonic radiation) yields, respectively (Table 3 and Table 4).
(
1 13
The structures of compound 7a–l and 8a–g were confirmed by FT-IR, H and C NMR and
HRMS spectra analysis. The –CH –O–CH – protons and =CH– protons of 1,2,3-triazole ring
2
2
exhibited resonances at δ 4.64–4.79 ppm and δ 7.80–8.67 ppm using DMSO as solvent, while the
resonances for the corresponding –CH –O–CH – and =CH– carbon atom were observed peaks at
2
2
δ 61.78–64.18 ppm and δ 125.33–128.57 ppm, respectively.
A plausible mechanism for the preparation of 1,2,3-tiazole derivatives via 1,3-dispolar
cycloaddition of various terminal isoxazole ether alkynes and β-thymidine azide derivatives using
copper acetate, NaAsc as the catalyst under ultrasonic irradiation was suggested according to the
reference[37,50,51] (Scheme 3). Firstly, copper(I) was in situ generated by redox reaction of
copper(II) and NaAsc, and subsequently attacked terminal alkynes to form intermediate A. Then,
the formation of intermediate C was achieved by the nucleophilic addition of azide and
intermediate A with the formation of intermediate B-1 and B-2. Finally, the target 1,2,3-triazole
products were obtained by the removal of copper coordination compound from intermediate C.
Herein, ultrasound accelerated the reaction rate and provided a certain amount of energy in the
whole process of reaction.
Scheme 3. Proposed mechanism for the synthesis of 1,2,3-triazole derivatives under ultrasonic assisted
2
.2 Quorum Sensing Inhibition
The QS inhibitory activities of the target compounds 7a–l and 8a–g were evaluated with C.
11

Violaceum CV026 based on their inhibition of violacein production, with compound C10-HSL
being used as a positive control. The IC50 value for each compound has been summarized in
Table 5.
Table 5 IC50 of compounds for inhibiting violacein production
a
Entry
Compound
R
IC50 (µM)
0.124±0.014
>256
1
2
3
4
5
6
7
8
9
C
10-HSL
7a
7b
7c
C
6
H
5
2-Cl-C
6
6
H
4
>256
4-Cl-C
H
4
>256
7d
7e
4-MeO-C
4-Me-C
4-tBu-C
3-NO -C
2-MeO-C
6
H
4
>256
6
H
4
>256
7f
6
H
4
>256
7g
7h
7i
2
6
H
4
>256
6
H
4
>256
b
1
0
1
4-F-C
6
H
4
NA
b
1
7j
4-Br-C
6
H
4
NA
b
1
1
1
1
1
1
1
1
2
2
3
4
5
6
7
8
9
0
7k
7l
styryl
NA
b
2-pyridyl
NA
8a
8b
8c
C
6
H
5
217±19
>256
2-Cl-C
6
6
H
4
4-Cl-C
H
4
223±20
>256
8d
8e
4-MeO-C
4-Me-C
4-tBu-C
3-NO -C
6
H
4
6
H
4
>256
8f
6
H
4
42.8±4.5
>256
8g
2
6
H
4
a
Half maximal inhibitory concentration.
NA means no significant inhibition.
b
C. violaceum is a Gram-negative bacteria and the production of virulence factors, such as
violacein production, is regulated in a cell density-dependent manner known as QS. The
1
2

autoinducer in QS of C. violaceum is N-decanoyl-L-Homoserine lactone (C10-HSL). C.
Violaceum CV026 has been widely used as a model for screen novel QS inhibitors for rapid and
easy detection method. To the best of our knowledge, C10-HSL and Chloro Lactone (CL) are the
best inhibitors from violacein production. Thus, C10-HSL is used as positive control in this
study.
As shown in Table 5, the synthesized compounds have certain inhibitory activity against
violacein production except compound 7i, 7j, 7k, 7l. In particular, compounds 8a, 8c and 8f
exhibited considerable levels of inhibitory activity with IC50 values of 217±19, 223±20 and
4
2.8±4.5 µM, respectively. This maybe is because phenyl and substituted phenyl consisted in a
specific interaction to the LuxR protein.[52] For the LuxR protein, the acknowledged model was
that dimerization requested the preliminary binding of acyl-HSL.[53] It could deduce that the
inhibitory activity of the synthesized compound might result from the interaction with one or
several aromatic amino acid of the LuxR protein which contains the HSL binding site or in the
dimerization domain or both. Besides, the introduction of an aryl group at the R position provided
enhanced levels of inhibitory activity relative to the alkyl groups, as compound 8e and 8f. When
the group at the R position was an alkyl group, the activity was dependent on the length of the
chain, with a longer chain length providing the highest level of inhibitory activity among all these
compounds.[54] Thus, compound 8f showed good QS inhibition activity in this paper comparing
with 8e. Methyl and chlorine are bioisostere, when they are substituted in the para of benzene
ring, chlorine substituted benzene had inhibition activity. For chlorine is hydrophily group, which
is important for inhibition activity.[52,54] In a word, the study suggested that the potential of these
compounds could be used a lead structure for further research towards the development of novel
QS inhibitors.
3
. Conclusions
In conclusion, a convenient, efficient method for the synthesis of 1,4-disubstituted
,2,3-triazoles containing isoxazole and thymidine structures using copper acetate, sodium
1
ascorbate as the catalyst under ultrasonic assisted has been presented. Ultrasonic heating can
1
3

significantly shorten reaction time and increased yields of the products (9‒25%) compared with
conventional
-((2R,4S,5R)-4-hydroxy-5-((4-(((3-arylisoxazol-5-yl)methoxy)methyl)-1H-1,2,3-
triazol-1-yl)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione
heating.
Twelve
novel
1
and
seven
1
1
8
-((2R,4S,5S)-5-(hydroxymethyl)-4-(4-(((3-arylisoxazol-5-yl)methoxy)methyl)-1H-1,2,3-triazol-
-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione were obtained in 84–96% and
7–96% yields, respectively. The quorum sensing inhibitory activities of synthesized compounds
were evaluated with CV026 based on their inhibition of violacein production, with compound
10-HSL being used as a positive control. The compounds 8a, 8c and 8f exhibited considerable
C
levels of inhibitory activity against violacein production, and IC50 values were 217±19, 223±20
and 42.8±4.5 µM, respectively. These compounds have potential application prospect as lead
structures towards the development of novel QS inhibitors.
4
. Experimental
4
.1 General Remarks
Various substituted benzaldehydes were of analytical-reagent grade from Aladdin reagent Co.
(
China) and used without further purification. The other solvents and reagents used were supplied
by Tianjin Tiantai Chemical Co. Ltd (China) and Beijing Chemical Plant (China).
N-decanoyl-L-Homoserine lactone (C10-HSL) was obtained from Cayman Chemical. All melting
1
points were determined on an XT−4 melting point apparatus (China) and were uncorrected. H
1
3
and C NMR spectra were measured using a Bruker AVANCE-500 NMR (Germany)
spectrometer and with TMS as an internal standard. The chemical shift is given in δ relative to
TMS. MS was collected using an Agilent 1290-micrOTOF Q II (USA) spectrometer, respectively.
FT-IR spectra were obtained as KBr pellets using an IRAffinity-1 instrument (Shimadzu, Japan)
–
in the range of 500–4000 cm . An KQ2200E-type ultrasonic reactor (100 W) (China) with a
1
thermometer for ultrasonic application was used in experiments.
4
.2 General synthesis process for 3-substituted phenyl-5-prop-2-ynyloxymethyl-isoxazoles (4a-l)
In 100 mL three-necked flask, (3-substituted phenylisoxazol-5-yl)methanols (3a-l) (5 mmol)
1
4

was poured into a stirred mixture of sodium hydride (15 mmol) and anhydrous THF (10 mL)
previously cooled in glacial bath. Propargyl bromide (6 mmol, 0.47 mL) was added, and the
o
mixture was stirred at room temperature (20–25 C) until the reaction was over by TLC
monitoring. The slurry was filtrated by sabouraud funnel. Filtrate was evaporated under a vacuum
to provide the crude product which was purified by column chromatography (silica gel, 200–300
r
mesh) using petroleum ether/ethyl acetate (ϕ = 4 : 1) to furnish the desired product 4a-l in
6
8−96% yield.
4
.3 General synthesis process for 1,4-disubstituted 1,2,3-triazole derivatives (7a-l and 8a-g)
Traditional heating method: 3-Substituted phenyl-5-prop-2-ynyloxymethyl-isoxazoles (4a-l)
(
2
1.2 mmol), and β-thymidine azide derivatives (1 mmol) were dissolved into i-PrOH/H O=2/1 (6
mL) in 50 mL three-necked flask. After dissolving, CuOAc (10% mmol), H
2
O (3 mL) and
o
sodium ascorbate (20% mmol) was added dropwise, and the mixture was stirred at 45 C until the
reaction was over by TLC monitoring. After the reaction was over, the slurry was extracted with
ethyl acetate. The organic layers were dried over anhydrous sodium sulfate, filtrated and then
evaporated under a vacuum to provide the crude product which was purified by column
chromatography (silica gel, 200-300 mesh) using ethyl acetate/ methanol (ϕ
r
= 20 : 1) to furnish
the product. The yields of obtained 1,4-disubstituted 1,2,3-triazole derivatives 7a-l and 8a-g were
6
6%–85% and 68%–83%, respectively.
Ultrasonic radiation method: 3-Substituted phenyl-5-prop-2-ynyloxymethyl-isoxazoles (4a-l)
(
1.2 mmol), and β-thymidine azide derivatives (1 mmol) were dissolved into i-PrOH/H
2
O=2/1 (6
mL) in 50 mL three-necked flask. After dissolving, CuOAc (10% mmol), H
2
O (3 mL) and
sodium ascorbate (20% mmol) was added dropwise to the reaction mixture. The reaction mixture
o
was submitted to ultrasound irradiation and stirring condition at 45 C (the solution become clear),
respectively, and the process was monitored by TLC. After the reaction was over, the slurry was
extracted with ethyl acetate. The organic layers were dried over anhydrous sodium sulfate,
filtrated and then evaporated under a vacuum to provide the crude product which was purified by
column chromatography (silica gel, 200-300 mesh) using ethyl acetate/ methanol (ϕ
furnish the product. The yields of obtained 1,4-disubstituted 1,2,3-triazole derivatives 7a-l and
a-g were 84%–96% and 87%–96%, respectively.
r
= 20 : 1) to
8
1
5

4
.4. Representative spectral data
4
1
.4.1. 1-((2R,4S,5R)-4-hydroxy-5-((4-(((3-phenylisoxazol-5-yl)methoxy)methyl)-1H-1,2,3-triazol-
-yl)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7a)
o
–1
Yield: 90%, orange solid, mp 200–201 C. IR (KBr) (ν/cm ) 3746, 3391, 3178, 3117, 3067,
2
6
7
4
947, 2924, 1721, 1655, 1577, 1507, 1474, 1439, 1366, 1273, 1204, 1096, 1040, 907, 860, 768,
1
91, 610; H NMR (500 MHz, DMSO) δ ppm: 11.31 (s, 1H), 8.17 (s, 1H), 7.89 – 7.86 (m, 2H),
.54 – 7.50 (m, 3H), 7.34 (d, J = 1.2 Hz, 1H), 7.07 (s, 1H), 6.17 (t, J = 7.0 Hz, 1H), 5.50 (d, J =
.4 Hz, 1H), 4.75 – 4.70 (m, 3H), 4.66 – 4.60 (m, 3H), 4.29 (td, J = 7.8, 4.0 Hz, 1H), 4.11 – 4.07
1
m, 1H), 2.21 – 2.14 (m, 1H), 2.10 (ddd, J = 13.5, 6.5, 3.8 Hz, 1H), 1.79 (d, J = 1.0 Hz, 3H); C
3
(
NMR (126 MHz, DMSO) δ ppm: 170.14, 164.09, 162.29, 150.85, 143.74, 136.50, 130.71, 129.58,
1
28.91, 127.08, 125.49, 110.31, 102.27, 84.51, 84.40, 71.20, 63.71, 62.48, 51.64, 38.37, 12.50;
+
[M+H] 481.1836, found 481.1863.
HRMS (EI): calcd for C23
H
24
N
6
O
6
4
1
.4.2. 1-((2R,4S,5R)-5-((4-(((3-(2-chlorophenyl)isoxazol-5-yl)methoxy)methyl)-1H-1,2,3-triazol-
-yl)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7b)
o
–1
Yield: 93%, light yellow solid, mp 226–228 C. IR (KBr) (ν/cm ) 3750, 3391, 3178, 3110,
3
059, 2947, 2924, 1717, 1655, 1512, 1474, 1436, 1369, 1273, 1204, 1099, 1045, 957, 764, 610;
1
H NMR (500 MHz, DMSO) δ ppm: 11.31 (s, 1H), 8.17 (s, 1H), 7.70 (dd, J = 7.6, 1.8 Hz, 1H),
7
.64 (dd, J = 8.0, 1.2 Hz, 1H), 7.55 (td, J = 7.8, 1.8 Hz, 1H), 7.49 (td, J = 7.5, 1.3 Hz, 1H), 7.33
(
d, J = 1.2 Hz, 1H), 6.94 (s, 1H), 6.16 (t, J = 7.0 Hz, 1H), 5.50 (d, J = 4.4 Hz, 1H), 4.75 (s, 2H),
4
4
.74 – 4.70 (m, 1H), 4.66 (s, 2H), 4.63 (dd, J = 14.3, 7.4 Hz, 1H), 4.28 (dt, J = 7.9, 3.9 Hz, 1H),
.10 – 4.07 (m, 1H), 2.20 – 2.13 (m, 1H), 2.09 (ddd, J = 13.5, 6.5, 3.8 Hz, 1H), 1.78 (d, J = 1.0
1
3
Hz, 3H); C NMR (126 MHz, DMSO) δ ppm: 169.53, 164.09, 160.93, 150.84, 143.72, 136.48,
1
5
4
1
32.29, 132.08, 131.53, 130.83, 128.15, 125.50, 110.31, 105.24, 84.50, 84.39, 71.19, 63.78, 62.36,
+
[M+H] 515.1446, found 515.1466.
1.63, 38.38, 12.50; HRMS (EI): calcd for C23
H23ClN
6
O
6
.4.3. 1-((2R,4S,5R)-5-((4-(((3-(4-chlorophenyl)isoxazol-5-yl)methoxy)methyl)-1H-1,2,3-triazol-
-yl)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7c)
o
–1
Yield: 92%, gray solid, mp 223–225 C. IR (KBr) (ν/cm ) 3746, 3383, 3178, 3113, 3063, 2951,
1
924, 1721, 1655, 1524, 1474, 1435, 1369, 1273, 1204, 1096, 1042, 980, 845, 652, 610; H NMR
2
(
500 MHz, DMSO) δ ppm: 11.31 (s, 1H), 8.16 (s, 1H), 7.91 (t, J = 2.1 Hz, 1H), 7.91 – 7.89 (m,
1
6

1
H), 7.60 (d, J = 1.9 Hz, 1H), 7.58 (t, J = 2.1 Hz, 1H), 7.34 (d, J = 1.0 Hz, 1H), 7.10 (s, 1H), 6.16
t, J = 7.0 Hz, 1H), 5.50 (d, J = 4.4 Hz, 1H), 4.74 – 4.70 (m, 3H), 4.66 – 4.60 (m, 3H), 4.28 (td, J
7.8, 4.0 Hz, 1H), 4.10 – 4.06 (m, 1H), 2.17 (dt, J = 13.7, 6.9 Hz, 1H), 2.10 (ddd, J = 13.5, 6.4,
(
=
1
3
3
1
8
5
4
1
.8 Hz, 1H), 1.78 (d, J = 0.8 Hz, 3H); C NMR (126 MHz, DMSO) δ ppm: 170.44, 164.10,
61.38, 150.85, 143.73, 136.50, 135.40, 129.69, 128.88, 127.78, 125.50, 110.31, 102.30, 84.50,
+
4.39, 71.19, 63.71, 62.46, 51.63, 38.36, 12.51; HRMS (EI): calcd for C23
15.1446, found 515.1462.
6 6
H23ClN O [M+H]
.4.4. 1-((2R,4S,5R)-4-hydroxy-5-((4-(((3-(4-methoxyphenyl)isoxazol-5-yl)methoxy)methyl)-1H-
,2,3-triazol-1-yl)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7d)
o
–1
Yield: 95%, white solid, mp 138–139 C. IR (KBr) (ν/cm ) 3750, 3379, 3178, 3113, 3059,
1
952, 2928, 1717, 1655, 1524, 1474, 1431, 1366, 1273, 1204, 1096, 1042, 957, 837, 652, 606; H
2
NMR (500 MHz, DMSO) δ ppm: 11.33 (s, 1H), 8.17 (s, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.35 (s,
1
4
1
1
1
H), 7.06 (d, J = 8.8 Hz, 2H), 7.01 (s, 1H), 6.17 (t, J = 7.0 Hz, 1H), 4.75 – 4.69 (m, 3H), 4.66 –
.60 (m, 3H), 4.31 – 4.25 (m, 1H), 4.11 – 4.06 (m, 1H), 3.82 (s, 3H), 2.18 (dt, J = 13.8, 6.9 Hz,
1
3
H), 2.10 (ddd, J = 13.4, 6.3, 3.8 Hz, 1H), 1.79 (s, 3H); C NMR (126 MHz, DMSO) δ ppm:
69.78, 164.12, 161.88, 161.20, 150.86, 143.75, 136.50, 128.57, 125.48, 121.30, 114.97, 110.31,
02.02, 84.52, 84.40, 71.20, 63.69, 62.50, 55.77, 51.64, 38.37, 12.51; HRMS (EI): calcd for
+
[M+H] 511.1941, found 511.1958.
C
24
H
26
N
6
O
7
4
.4.5.
1-((2R,4S,5R)-4-hydroxy-5-((4-(((3-(p-tolyl)isoxazol-5-yl)methoxy)methyl)-1H-1,2,3-
triazol-1-yl)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7e)
o
–1
Yield: 95%, orange solid, mp 223–225 C. IR (KBr) (ν/cm ) 3750, 3379, 3178, 3113, 3063,
1
950, 2924, 1721, 1655, 1512, 1477, 1437, 1366, 1273, 1204, 1096, 1042, 980, 822, 652, 610; H
2
NMR (500 MHz, DMSO) δ ppm: 11.31 (s, 1H), 8.16 (s, 1H), 7.78 – 7.75 (m, 2H), 7.33 (dd, J =
9
3
2
.5, 4.5 Hz, 3H), 7.03 (s, 1H), 6.16 (t, J = 7.0 Hz, 1H), 5.50 (d, J = 4.4 Hz, 1H), 4.75 – 4.70 (m,
H), 4.66 – 4.60 (m, 3H), 4.28 (td, J = 7.8, 3.9 Hz, 1H), 4.11 – 4.07 (m, 1H), 2.36 (s, 3H), 2.20 –
13
.14 (m, 1H), 2.10 (ddd, J = 13.5, 6.4, 3.8 Hz, 1H), 1.79 (d, J = 1.0 Hz, 3H); C NMR (126 MHz,
DMSO) δ ppm: 169.94, 164.09, 162.18, 150.85, 143.75, 140.41, 136.50, 130.12, 126.98, 126.12,
1
25.48, 110.31, 102.16, 84.51, 84.40, 71.20, 63.69, 62.48, 51.63, 38.36, 21.42, 12.50; HRMS (EI):
+
[M+H] 495.1992, found 495.2024.
calcd for C24
H
26
N
6
O
6
1
7

4
.4.6.
1-((2R,4S,5R)-5-((4-(((3-(4-(tert-butyl)phenyl)isoxazol-5-yl)methoxy)methyl)-1H-1,2,3-
triazol-1-yl)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7f)
o
–1
Yield: 94%, light yellow solid, mp 195–197 C. IR (KBr) (ν/cm ) 3746, 3383, 3173, 3110,
3
8
2
059, 2952, 2924, 2855, 1717, 1659, 1512, 1474, 1435, 1366, 1273, 1204, 1099, 1045, 980, 907,
1
41, 606; H NMR (500 MHz, DMSO) δ ppm: 11.33 (s, 1H), 8.17 (s, 1H), 7.80 (d, J = 8.5 Hz,
H), 7.53 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 1.0 Hz, 1H), 7.03 (s, 1H), 6.17 (t, J = 7.0 Hz, 1H), 5.52
(
d, J = 4.4 Hz, 1H), 4.76 – 4.69 (m, 3H), 4.67 – 4.60 (m, 3H), 4.28 (td, J = 7.5, 3.6 Hz, 1H), 4.11
4.06 (m, 1H), 2.18 (dt, J = 13.8, 6.8 Hz, 1H), 2.09 (ddd, J = 13.5, 6.4, 3.8 Hz, 1H), 1.79 (d, J =
–
0
1
6
5
4
1
1
3
.8 Hz, 3H), 1.31 (s, 9H); C NMR (126 MHz, DMSO) δ ppm: 169.93, 164.09, 162.11, 153.38,
50.84, 143.74, 136.50, 126.86, 126.34, 126.15, 125.48, 110.30, 102.20, 84.51, 84.40, 71.20,
+
3.72, 62.50, 51.63, 38.37, 35.04, 31.41, 12.50; HRMS (EI): calcd for C27
37.2462, found 537.2483.
32 6 6
H N O [M+H]
.4.7.
1-((2R,4S,5R)-4-hydroxy-5-((4-(((3-(3-nitrophenyl)isoxazol-5-yl)methoxy)methyl)-1H-
,2,3-triazol-1-yl)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7g)
o
–1
Yield: 85%, light yellow solid, mp 191–193 C. IR (KBr) (ν/cm ) 3750, 3383, 3179, 3113,
3
7
063, 2954, 2928, 1721, 1655, 1512, 1477, 1435, 1350, 1273, 1204, 1096, 1042, 910, 864, 806,
1
45, 696, 610; H NMR (500 MHz, DMSO) δ ppm: 11.32 (s, 1H), 8.68 – 8.64 (m, 1H), 8.36 (td, J
=
8.2, 4.7 Hz, 2H), 8.17 (s, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.35 (s, 1H), 7.31 (s, 1H), 6.16 (t, J =
7
3
6
1
8
.0 Hz, 1H), 5.52 (d, J = 4.3 Hz, 1H), 4.77 – 4.70 (m, 3H), 4.69 – 4.60 (m, 3H), 4.28 (dt, J = 7.7,
.9 Hz, 1H), 4.09 (dt, J = 7.8, 4.1 Hz, 1H), 2.18 (dt, J = 13.7, 6.9 Hz, 1H), 2.10 (ddd, J = 13.4,
1
3
.4, 3.8 Hz, 1H), 1.79 (s, 3H); C NMR (126 MHz, DMSO) δ ppm: 170.99, 164.09, 160.86,
50.84, 148.82, 143.72, 136.50, 133.41, 131.36, 130.47, 125.50, 125.33, 121.53, 110.31, 102.59,
23 7 8
4.52, 84.40, 71.20, 63.77, 62.47, 51.65, 38.37, 12.50; HRMS (EI): calcd for C23H N O
+
M+H] 526.1686, found 526.1655.
[
4
1
.4.8. 1-((2R,4S,5R)-4-hydroxy-5-((4-(((3-(2-methoxyphenyl)isoxazol-5-yl)methoxy)methyl)-1H-
,2,3-triazol-1-yl)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7h)
o
–1
Yield: 96%, white solid, mp 138–139 C. IR (KBr) (ν/cm ) 3750, 3383, 3178, 3110, 3059,
2
6
1
954, 2928, 1721, 1655, 1511, 1474, 1435, 1366, 1273, 1204, 1096, 1052, 1018, 957, 860, 752,
1
10; H NMR (500 MHz, DMSO) δ ppm: 11.33 (s, 1H), 8.17 (s, 1H), 7.74 (dd, J = 7.6, 1.5 Hz,
H), 7.51 – 7.46 (m, 1H), 7.34 (d, J = 0.8 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.08 – 7.04 (m, 1H),
1
8

6
.88 (s, 1H), 6.16 (t, J = 7.0 Hz, 1H), 5.51 (d, J = 4.3 Hz, 1H), 4.75 – 4.70 (m, 3H), 4.66 – 4.60
(
m, 3H), 4.28 (dt, J = 7.6, 3.8 Hz, 1H), 4.08 (dt, J = 7.6, 4.0 Hz, 1H), 3.87 (s, 3H), 2.17 (dt, J =
1
3.7, 6.9 Hz, 1H), 2.09 (ddd, J = 13.5, 6.4, 3.8 Hz, 1H), 1.79 (s, 3H); C NMR (126 MHz,
3
1
DMSO) δ ppm: 168.84, 164.09, 159.88, 157.41, 150.85, 143.78, 136.48, 132.08, 129.31, 125.47,
1
1
4
1
21.19, 117.54, 112.70, 110.31, 105.35, 84.50, 84.40, 71.19, 63.71, 62.42, 56.17, 51.62, 38.37,
+
[M+H] 511.1941, found 511.1933.
2.50; HRMS (EI): calcd for C24
H
26
N
6
O
7
.4.9. 1-((2R,4S,5R)-5-((4-(((3-(4-fluorophenyl)isoxazol-5-yl)methoxy)methyl)-1H-1,2,3-triazol-
-yl)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7i)
o
–1
Yield: 94%, orange solid, mp 212–214 C. IR (KBr) (ν/cm ) 3746, 3383, 3173, 3113, 3063,
1
955, 2924, 1721, 1655, 1524, 1474, 1435, 1369, 1273, 1227, 1096, 1042, 980, 845, 652, 610; H
2
NMR (500 MHz, DMSO) δ ppm: 11.32 (s, 1H), 8.17 (s, 1H), 7.94 (dd, J = 8.8, 5.5 Hz, 2H), 7.40
–
3
6
7.32 (m, 3H), 7.08 (s, 1H), 6.16 (t, J = 7.0 Hz, 1H), 5.51 (d, J = 4.4 Hz, 1H), 4.72 (q, J = 4.2 Hz,
H), 4.67 – 4.59 (m, 3H), 4.28 (td, J = 7.5, 3.7 Hz, 1H), 4.11 – 4.06 (m, 1H), 2.18 (dt, J = 13.7,
1
3
.9 Hz, 1H), 2.10 (ddd, J = 13.5, 6.4, 3.8 Hz, 1H), 1.78 (s, 3H); C NMR (126 MHz, DMSO) δ
ppm: 170.28, 164.65, 164.09, 162.68, 161.42, 150.85, 143.74, 136.50, 129.47, 129.40, 125.49,
1
16.72, 116.54, 110.31, 102.26, 84.52, 84.40, 71.20, 63.72, 62.48, 51.64, 38.37, 12.50; HRMS
+
[M+H] 499.1741, found 499.1721.
(
EI): calcd for C23
H23FN
6
O
6
4
1
.4.10. 1-((2R,4S,5R)-5-((4-(((3-(4-bromophenyl)isoxazol-5-yl)methoxy)methyl)-1H-1,2,3-triazol-
-yl)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7j)
o
–1
Yield: 89%, yellow solid, mp 232–234 C. IR (KBr) (ν/cm ) 3750, 3379, 3173, 3113, 3063,
1
950, 2924, 1721, 1655, 1512, 1477, 1427, 1369, 1273, 1204, 1096, 1041, 977, 822, 652, 610; H
2
NMR (500 MHz, DMSO) δ ppm: 11.33 (s, 1H), 8.17 (s, 1H), 7.84 (d, J = 8.5 Hz, 2H), 7.79 –
7
3
1
.69 (m, 2H), 7.35 (s, 1H), 7.11 (s, 1H), 6.17 (t, J = 7.0 Hz, 1H), 5.54 (s, 1H), 4.73 (q, J = 4.7 Hz,
H), 4.68 – 4.57 (m, 3H), 4.28 (d, J = 2.1 Hz, 1H), 4.13 – 4.04 (m, 1H), 2.18 (dt, J = 13.7, 6.8 Hz,
1
H), 2.10 (ddd, J = 13.4, 6.4, 3.9 Hz, 1H), 1.77 (d, J = 16.4 Hz, 3H); C NMR (126 MHz,
3
DMSO) δ ppm: 170.47, 164.11, 161.47, 150.86, 143.71, 136.51, 132.61, 129.10, 128.13, 125.50,
1
24.14, 110.31, 102.27, 84.51, 84.40, 71.19, 63.72, 62.46, 51.64, 38.37, 12.51; HRMS (EI): calcd
+
[M+H] 559.0941, found 561.0887.
for C23
H23BrN
6
O
6
4
.4.11. 1-((2R,4S,5R)-4-hydroxy-5-((4-(((3-((E)-styryl)isoxazol-5-yl)methoxy)methyl)-1H-1,2,3-
triazol-1-yl)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7k)
1
9

o
–1
Yield: 89%, orange solid, mp 206–208 C. IR (KBr) (ν/cm ) 3746, 3507, 3416, 3173, 3135,
3
7
7
5
4
3
1
6
5
4
3
065, 3021, 2957, 2924, 1713, 1639, 1466, 1436, 1369, 1281, 1240, 1142, 1065, 964, 895, 810,
1
56, 694, 640, 579; H NMR (500 MHz, DMSO) δ ppm: 11.32 (s, 1H), 8.17 (s, 1H), 7.67 (d, J =
.3 Hz, 2H), 7.48 – 7.34 (m, 5H), 7.24 (d, J = 16.5 Hz, 1H), 6.96 (s, 1H), 6.17 (t, J = 7.0 Hz, 1H),
.52 (d, J = 4.3 Hz, 1H), 4.76 – 4.67 (m, 3H), 4.67 – 4.60 (m, 3H), 4.29 (td, J = 7.6, 3.8 Hz, 1H),
.12 – 4.06 (m, 1H), 2.18 (dt, J = 13.8, 6.9 Hz, 1H), 2.10 (ddd, J = 13.5, 6.4, 3.8 Hz, 1H), 1.79 (s,
1
3
H); C NMR (126 MHz, DMSO) δ ppm: 169.22, 164.10, 162.05, 150.85, 143.77, 137.00,
36.51, 136.09, 129.42, 129.31, 127.59, 125.47, 116.02, 110.32, 101.35, 84.52, 84.40, 71.21,
+
[M+H] 507.1992, found
3.68, 62.49, 51.64, 38.36, 12.51; HRMS (EI): calcd for C25
07.1974.
H
26
N
6
O
6
.4.12. 1-((2R,4S,5R)-4-hydroxy-5-((4-(((3-(pyridin-2-yl)isoxazol-5-yl)methoxy)methyl)-1H-1,2,
-triazol-1-yl)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7l)
o
–1
Yield: 84%, white solid, mp 178–179 C. IR (KBr) (ν/cm ) 3750, 3391, 3173, 3113, 3055,
1
950, 2924, 1721, 1655, 1512, 1477, 1403, 1371, 1273, 1204, 1096, 918, 864, 787, 610; H NMR
2
(
500 MHz, DMSO) δ ppm: 11.32 (s, 1H), 8.72 (d, J = 4.8 Hz, 1H), 8.18 (s, 1H), 8.03 (d, J = 7.8
Hz, 1H), 7.97 (td, J = 7.8, 1.7 Hz, 1H), 7.55 – 7.51 (m, 1H), 7.34 (s, 1H), 7.05 (s, 1H), 6.17 (t, J =
7
3
1
1
6
4
4
3
.0 Hz, 1H), 5.51 (d, J = 4.3 Hz, 1H), 4.76 – 4.70 (m, 3H), 4.67 – 4.60 (m, 3H), 4.28 (dt, J = 7.6,
.9 Hz, 1H), 4.12 – 4.06 (m, 1H), 2.17 (dt, J = 13.8, 6.9 Hz, 1H), 2.09 (ddd, J = 13.4, 6.3, 3.8 Hz,
13
H), 1.78 (s, 3H); C NMR (126 MHz, DMSO) δ ppm: 170.41, 164.09, 163.23, 150.84, 150.43,
47.98, 143.75, 137.99, 136.48, 125.58, 125.49, 121.85, 110.31, 102.82, 84.51, 84.40, 71.20,
+
[M+H] 482.1788, found
3.69, 62.32, 51.63, 38.38, 12.50; HRMS (EI): calcd for C22
82.1783.
H
23
N
7
O
6
.4.13. 1-((2R,4S,5S)-5-(hydroxymethyl)-4-(4-(((3-phenylisoxazol-5-yl)methoxy)methyl)-1H-1,2,
-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8a)
o
–1
Yield: 89%, white solid, mp 132–134 C. IR (KBr) (ν/cm ) 3746, 3447, 3229, 3116, 3071,
3
008, 2957, 2928, 1694, 1607, 1466, 1404, 1365, 1277, 1206, 1076, 976, 930, 791, 768, 691, 610;
1
H NMR (500 MHz, DMSO) δ ppm: 11.35 (s, 1H), 8.37 (s, 1H), 7.90 – 7.87 (m, 2H), 7.82 (d, J =
1
.2 Hz, 1H), 7.53 – 7.51 (m, 2H), 7.08 (s, 1H), 6.42 (t, J = 6.6 Hz, 1H), 5.38 (dt, J = 8.7, 5.4 Hz,
H), 5.28 (t, J = 5.2 Hz, 1H), 4.74 (s, 2H), 4.69 (s, 2H), 4.28 (q, J = 7.1 Hz, 1H), 4.24 – 4.20 (m,
H), 3.70 (ddd, J = 11.9, 5.2, 3.5 Hz, 1H), 3.62 (ddd, J = 12.1, 5.1, 3.9 Hz, 1H), 2.74 (ddd, J =
1
1
2
0

1
3.8, 6.7, 5.5 Hz, 1H), 2.68 – 2.61 (m, 1H), 1.81 (d, J = 1.0 Hz, 3H); C NMR (126 MHz,
3
1
DMSO) δ ppm: 170.16, 164.18, 162.28, 150.89, 143.96, 136.69, 131.95, 130.72, 129.58, 129.10,
1
1
4
1
28.90, 127.08, 124.41, 110.08, 102.29, 84.91, 84.34, 63.78, 62.53, 61.78, 61.18, 59.67, 37.61,
+
[M+H] 481.1836, found 481.1814.
2.72; HRMS (EI): calcd for C23
H
24
N
6
O
6
.4.14. 1-((2R,4S,5S)-4-(4-(((3-(2-chlorophenyl)isoxazol-5-yl)methoxy)methyl)-1H-1,2,3-triazol-
-yl)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8b)
o
–1
Yield: 92%, yellow solid, mp 120–122 C. IR (KBr) (ν/cm ) 3746, 3429, 3240, 3174, 3116,
1
067, 2957, 2924, 1732, 1686, 1607, 1454, 1409, 1362, 1265, 1096, 1045, 972, 756, 621, 571; H
3
NMR (500 MHz, CDCl
3
) δ ppm: 9.13 (s, 1H), 7.80 (s, 1H), 7.70 (dd, J= 7.5, 1.7 Hz, 1H), 7.50 –
7
1
1
.46 (m, 1H), 7.43 (s, 1H), 7.38 (tdd, J= 15.0, 10.6, 4.6 Hz, 2H), 6.77 (s, 1H), 6.22 (t, J= 6.5 Hz,
H), 5.47 (dd, J= 13.1, 5.4 Hz, 1H), 4.79 (s, 2H), 4.77 (s, 2H), 4.46 – 4.42 (m, 1H), 4.00 (d, J=
1
3
0.8 Hz, 1H), 3.79 (d, J= 10.9 Hz, 1H), 2.96 (qd, J= 14.1, 6.7 Hz, 2H), 1.91 (s, 3H); C NMR
(
126 MHz, CDCl
3
) δ ppm: 168.32, 163.62, 161.07, 150.41, 144.52, 137.89, 132.86, 131.02,
1
3
4
1
30.94, 130.45, 128.00, 127.17, 123.19, 111.32, 104.93, 88.90, 85.23, 64.18, 63.09, 61.64, 59.51,
+
[M+H] 515.1446, found 515.1424.
7.45, 12.42; HRMS (EI): calcd for C23
H23ClN
6
O
6
.4.15. 1-((2R,4S,5S)-4-(4-(((3-(4-chlorophenyl)isoxazol-5-yl)methoxy)methyl)-1H-1,2,3-triazol-
-yl)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8c)
o
–1
Yield: 91%, yellow solid, mp 78–80 C. IR (KBr) (ν/cm ) 3746, 3397, 3132, 3063, 2924, 2867,
1
697, 1607, 1512, 1474, 1427, 1366, 1273, 1092, 1045, 949, 907, 833, 610; H NMR (600 MHz,
1
DMSO) δ ppm: 11.36 (s, 1H), 8.36 (s, 1H), 7.92 – 7.91 (m, 1H), 7.91 – 7.90 (m, 1H), 7.82 (d, J =
1
–
.2 Hz, 1H), 7.61 – 7.60 (m, 1H), 7.59 – 7.58 (m, 1H), 7.12 (s, 1H), 6.42 (t, J = 6.6 Hz, 1H), 5.39
5.35 (m, 1H), 5.28 (t, J = 5.2 Hz, 1H), 4.74 (s, 2H), 4.68 (s, 2H), 4.23 – 4.20 (m, 1H), 3.69 (ddd,
J = 12.0, 5.2, 3.5 Hz, 1H), 3.64 – 3.60 (m, 1H), 2.75 – 2.70 (m, 1H), 2.67 – 2.61 (m, 1H), 1.81 (d,
1
3
J = 1.0 Hz, 3H); C NMR (151 MHz, DMSO) δ ppm: 170.47, 164.19, 161.38, 150.90, 143.95,
1
3
4
36.69, 135.42, 129.70, 128.88, 127.77, 110.10, 102.33, 84.90, 84.34, 63.79, 62.51, 61.18, 59.69,
+
[M+H] 515.1446, found 515.1410.
7.61, 12.73; HRMS (EI): calcd for C23
.4.16.
H23ClN
6
O
6
1-((2R,4S,5S)-5-(hydroxymethyl)-4-(4-(((3-(4-methoxyphenyl)isoxazol-5-yl)methoxy)
methyl)-1H-1,2,3-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8d)
o
–1
Yield: 96%, white solid, mp 126–128 C. IR (KBr) (ν/cm ) 3746, 3447, 3225, 3129, 3075,
3
008, 2928, 2874, 1701, 1612, 1528, 1460, 1435, 1365, 1254, 1177, 1080, 1022, 930, 814, 747,
2
1

1
6
2
52, 601; H NMR (500 MHz, DMSO) δ ppm: 11.38 (s, 1H), 8.37 (s, 1H), 7.82 (d, J= 2.0 Hz,
H), 7.82 – 7.80 (m, 1H), 7.08 – 7.06 (m, 1H), 7.06 – 7.05 (m, 1H), 7.02 (s, 1H), 6.42 (t, J= 6.6
Hz, 1H), 5.37 (dt, J= 8.7, 5.4 Hz, 1H), 5.30 (t, J= 5.2 Hz, 1H), 4.72 (s, 2H), 4.68 (s, 2H), 4.23 –
.19 (m, 1H), 3.82 (s, 3H), 3.69 (ddd, J= 11.9, 5.2, 3.5 Hz, 1H), 3.64 – 3.59 (m, 1H), 2.76 – 2.70
4
1
3
(
m, 1H), 2.67 – 2.60 (m, 1H), 1.81 (d, J= 0.7 Hz, 3H); C NMR (126 MHz, DMSO) δ ppm:
1
1
69.80, 164.18, 161.87, 161.20, 150.92, 143.98, 136.69, 128.57, 124.40, 121.27, 114.97, 110.08,
02.03, 84.90, 84.34, 63.77, 62.55, 61.18, 59.67, 55.77, 37.61, 12.72; HRMS (EI): calcd for
+
[M+H] 511.1941, found 511.1918.
C
24
H
26
N
6
O
7
4
1
.4.17.
1-((2R,4S,5S)-5-(hydroxymethyl)-4-(4-(((3-(p-tolyl)isoxazol-5-yl)methoxy)methyl)-1H-
,2,3-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8e)
o
–1
Yield: 94%, white solid, mp 149–151 C. IR (KBr) (ν/cm ) 3746, 3435, 3225, 3129, 3071,
1
008, 2928, 2874, 1701, 1612, 1531, 1458, 1429, 1371, 1277, 1080, 1052, 934, 814, 664, 608; H
3
NMR (600 MHz, DMSO) δ ppm: 11.36 (s, 1H), 8.37 (s, 1H), 7.82 (d, J = 1.1 Hz, 1H), 7.78 –
.75 (m, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.05 – 7.02 (m, 1H), 6.42 (t, J = 6.6 Hz, 1H), 5.36 (ddd, J
7
=
41.9, 22.5, 8.5 Hz, 2H), 4.73 (s, 2H), 4.68 (s, 2H), 4.21 (dt, J = 5.5, 3.7 Hz, 1H), 3.69 (dd, J =
1
2
1
8
2.1, 3.4 Hz, 1H), 3.62 (dd, J = 12.1, 3.7 Hz, 1H), 2.73 (ddd, J = 15.1, 6.7, 5.6 Hz, 1H), 2.67 –
1
3
.61 (m, 1H), 2.36 (s, 3H), 1.81 (d, J = 0.9 Hz, 3H); C NMR (151 MHz, DMSO) δ ppm: 169.97,
64.20, 162.18, 150.90, 143.97, 140.44, 136.70, 130.13, 126.98, 126.10, 110.09, 102.18, 84.91,
26 6 6
4.33, 63.77, 62.53, 61.17, 59.67, 37.61, 21.43, 12.73; HRMS (EI): calcd for C24H N O
+
M+H] 495.1992, found 495.1975.
[
4
.4.18.
1-((2R,4S,5S)-4-(4-(((3-(4-(tert-butyl)phenyl)isoxazol-5-yl)methoxy)methyl)-1H-1,2,3-
triazol-1-yl)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8f)
o
–1
Yield: 90%, light yellow solid, mp 107–109 C. IR (KBr) (ν/cm ) 3734, 3435, 3180, 3135,
3
6
7
1
4
1
063, 2959, 2924, 2868, 1697, 1563, 1470, 1429, 1362, 1269, 1096, 1045, 903, 836, 774, 691,
1
14; H NMR (500 MHz, DMSO) δ ppm: 11.38 (s, 1H), 8.38 (s, 1H), 7.82 (d, J= 1.1 Hz, 1H),
.81 (d, J= 1.8 Hz, 1H), 7.80 – 7.78 (m, 1H), 7.55 – 7.53 (m, 1H), 7.53 – 7.52 (m, 1H), 7.04 (s,
H), 6.43 (t, J= 6.6 Hz, 1H), 5.38 (dt, J= 8.6, 5.4 Hz, 1H), 5.30 (t, J= 5.2 Hz, 1H), 4.73 (s, 2H),
.68 (s, 2H), 4.21 (dd, J= 8.9, 3.6 Hz, 1H), 3.69 (ddd, J= 11.9, 5.1, 3.5 Hz, 1H), 3.64 – 3.59 (m,
1
H), 2.76 – 2.69 (m, 1H), 2.67 – 2.60 (m, 1H), 1.81 (d, J= 0.9 Hz, 3H), 1.31 (s, 9H); C NMR
3
(
126 MHz, DMSO) δ ppm: 169.96, 164.18, 162.11, 153.40, 150.89, 143.97, 136.69, 126.86,
2
2

1
3
4
1
26.35, 126.14, 124.41, 110.09, 102.23, 84.91, 84.34, 63.81, 62.56, 61.19, 59.68, 37.61, 35.04,
+
[M+H] 537.2462, found 537.2429.
1.40, 12.72; HRMS (EI): calcd for C27
H
32
N
6
O
6
.4.19. 1-((2R,4S,5S)-5-(hydroxymethyl)-4-(4-(((3-(3-nitrophenyl)isoxazol-5-yl)methoxy)methyl)-
H-1,2,3-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8g)
o
–1
Yield: 87%, light yellow solid, mp 96–98 C. IR (KBr) (ν/cm ) 3750, 3653, 3266, 3152, 3074,
1
928, 2874, 1697, 1531, 1458, 1346, 1269, 1048, 999, 945, 903, 810, 745, 694, 594; H NMR
2
(
(
500 MHz, DMSO) δ ppm: 11.35 (s, 1H), 8.67 – 8.65 (m, 1H), 8.38 – 8.34 (m, 3H), 7.86 – 7.81
m, 2H), 7.32 (s, 1H), 6.42 (t, J = 6.6 Hz, 1H), 5.38 (dt, J = 8.6, 5.4 Hz, 1H), 5.28 (t, J = 5.2 Hz,
1
3
H), 4.78 (s, 2H), 4.71 (s, 2H), 4.23 – 4.20 (m, 1H), 3.70 (ddd, J = 12.0, 5.2, 3.5 Hz, 1H), 3.65 –
1
3
.60 (m, 1H), 2.77 – 2.71 (m, 1H), 2.68 – 2.61 (m, 1H), 1.81 (d, J = 1.0 Hz, 3H). C NMR (126
MHz, DMSO) δ ppm: 171.02, 164.17, 160.86, 150.89, 148.82, 143.94, 136.68, 133.41, 131.38,
1
1
4
30.46, 125.34, 124.43, 121.54, 110.09, 102.62, 84.91, 84.36, 63.84, 62.53, 61.19, 59.70, 37.61,
+
2.71; HRMS (EI): calcd for C23
.5 Evaluation of the biological activity
In preliminary color screens for QS activity, we used the biomonitor CV026 for short-chain
H
23
N
7
O
8
[M+H] 526.1686, found 526.1670.
AHL inhibitor detection. CV026 was cultured in LB broth with shaking at 170 rpm overnight.
C6-HSL (15 µL, 0.125 mM) was then added and the mixture was gently mixed with molten
semi-solid LB agar (5 mL). Then, the mixture was overlaid on the solid LB agar. After
solidification of the top layer, the synthesized compounds were spotted on the plate and the plates
o
were incubated overnight at 30 C. Finally, the inhibiting activities were determined by the
presence of white colonies/holes in a purple background of the plates.[55,56]
For the secondary screening of the compounds inhibited activities, CV026 were cultured in LB
broth and subsequently diluted and added into 12-well plates. The PBS solution of C6-HSL (15
µL, 0.125 mM) and different concentrations of synthesized compounds were added to the wells.
After incubation for 16–18 h, 1 mL of the culture was taken off each mixture and centrifuged at
1
2,000 g for 10 min. The supernatant was then discarded and DMSO (500 µL) was added to each
tube. Then the mixtures were centrifuged at 12,000 g for 10 min. Finally, 200 µL of the upper
layer was put into a 96-well plate, and the absorbance was read at 585 nm.[57] This is because the
maximum absorbance wavelength of violacein solution appears on 585 nm. Also, the absorbance
change showed the concentration changes of violacein. Thus, the QS inhibition effect of the
2
3

synthesized compound to Gram-negative bacteria was evaluated by detecting the absorbance
change at 585 nm. The expriments were carried out three times. The IC50 (half maximum
inhibitory concentration) of each compounds were calculated using Graphpad 6.0 software and
were defined to be the average of three determinations.
Acknowledgements
We are grateful to Mr C. Y. Wang for NMR spectra, Mr Z. L. Wei for MS spectra.
References and notes
[
[
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1] G.D. Geske, J.C. O’Nell, D.M. Miller, M.E. Mattmann, H.E. Blackwell, Modulation of bacterial quorum
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7] J.E. Kang, J.W. Han, B.J. Jeon, B.S. Kim, Efficacies of quorum sensing inhibitors, piericidin A
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11] Y. Igarashi, K. Yamamoto, T. Fukuda, A. Shojima, J. Nakayama, L. Carro, M.E. Trujillo, Arthroamide, a
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14] I.V. Daele, H. Munier-Lehmann, M. Froeyen, J. Balzarini, S.V. Calenbergh, Rational design of
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2
8

Highlights
ꢀ
ꢀ
ꢀ
An efficient and eco-friendly ultrasonic-assisted Click chemistry method for synthesizing
1
,2,3-tiazoles containing isoxazole and β-thymidine structures was developed.
The reaction conditions were optimized. Nineteen new target products were obtained in
8
4–96% yields.
The synthesized compounds have a significant quorum sensing inhibitory activities.
2
9