Oligothiophene compounds inhibit the membrane fusion between H5N1 avian influenza virus and the endosome of host cell

Article abstract of DOI:10.1016/j.ejmech.2017.02.040

Hemagglutinin (HA) which is essential for influenza viral infection and replication has become a target for the design of anti-influenza drugs. A novel series of oligothiophene compounds focused on the target were synthesized as specific inhibitors agains

Full text of DOI:10.1016/j.ejmech.2017.02.040

Accepted Manuscript
Oligothiophene compounds inhibit the membrane fusion between H5N1 avian
influenza virus and the endosome of host cell
Zhibo Zhu, Zhili Yao, Xiantian Shen, Zhipeng Chen, Xiangtao Liu, Jon R. Parquette,
Shuwen Liu
PII:
S0223-5234(17)30113-7
10.1016/j.ejmech.2017.02.040
EJMECH 9236
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 November 2016
Revised Date: 14 February 2017
Accepted Date: 15 February 2017
Please cite this article as: Z. Zhu, Z. Yao, X. Shen, Z. Chen, X. Liu, J.R. Parquette, S. Liu,
Oligothiophene compounds inhibit the membrane fusion between H5N1 avian influenza virus
and the endosome of host cell, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.02.040.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
3sf against H5N1 influenza pseudovirus with an IC50 of 0.029 µM，
against H1N1 influenza virus MDCK with an IC50 of 1.240 µM.

ACCEPTED MANUSCRIPT
Oligothiophene compounds inhibit the membrane fusion between H5N1
avian influenza virus and the endosome of host cell
Zhibo Zhu1, 2, #, Zhili Yao2, #, Xiantian Shen1#, Zhipeng Chen1, Xiangtao Liu3, Jon R.
Parquette2*, Shuwen Liu1*
1School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515,
China
2Department of Chemistry, The Ohio State University, 100 W. 18th Ave. Columbus, Ohio
43210,USA
3Department of Psychiatry, University of Iowa, 200 Hawkins Dr., Iowa City, IA 52242,
USA
*Corresponding to: S. Liu, School of Pharmaceutical Sciences, Southern Medical
University, 1838 Guangzhou Avenue North, Guangzhou 510515, China. Tel: 86 (20)
6164-8538; Fax: 86 (20) 6164-8655. E-mail: liusw@smu.edu.cn
Jon R. Parquette, Phone: 614-292-5886. E-mail: parquett@chemistry.ohio-state.edu
# These authors contributed equally
1

ACCEPTED MANUSCRIPT
Abstract:
Hemagglutinin (HA) which is essential for influenza viral infection and replication has
become a target for the design of anti-influenza drugs. A novel series of oligothiophene
compounds focused on the target were synthesized as specific inhibitors against the H5
subtype of influenza A viruses because oligothiophene has stronger π–π interactions with
residues F1102 and M241 of HA2 side chains. Oligothiophene compounds were designed
and synthesized by a series of alkylation, azidation, amination and amidation reactions.
The entry inhibitory activities of those compounds were tested at a cellular level against
H5N1 influenza pseudovirus. Compound 3sf was revealed as the most active inhibitor in
this series with an IC50 of 0.029 µM. The activity of 3sf is almost 1000 times that of the
positive reference compound (CL-385319). A structure-activity analysis of these
compounds demonstrated that the size of the oligothiophene compounds was very
important for the inhibitory activity. Four compounds (3sk, 3sf, 3sc and 4sc) of strong
inhibitiory activity against H5N1 influenza pseudovirus were assessed against H1N1
influenza virus MDCK. They also showed strong inhibitiory activity with IC50s of 3.292
µM, 1.240 µM, 1.119 µM and 0.768 µM, respectively.
Keywords: oligothiophene compounds, H5N1 influenza virus, hemagglutinin(HA),
fusion inhibitor
2

ACCEPTED MANUSCRIPT
1. Introduction
The H5N1 avian influenza A viruses, which spreads quickly by transferring viral
particles into host cells is a serious threat to human health in the world.1 Membrane
fusion is an important step needed for the entry of the H5N1 avian influenza A viruses
into host cells.2 The fusion step is mediated by influenza virus hemagglutinin (HA),
which is the viral envelope protein (ENV) organized as a trimer; each monomer consists
of HA1 and HA2 subunits, linked by a single disulfide bond.3 HA1 separates from HA2,
which then turns inside out when the HA1:HA2 trimer encounters low pH in endosome
after endocytosis. In detail, the hemagglutinin conformational changes are triggered
following the attachment of the trimer to the cell surface, which is mediated by the
interaction of HA1 with its receptor at the cell surface. The HA2 protein undergoes an
irreversible conformation change from its metastable pre-fusion conformation to a
low-pH hairpin structure. The N terminus of HA2 inserts into a pocket , which is created
by a splaying apart from each other of the C termini of the HA2 coiled-coil helices.4-6
The resulting extrusion of HA2 toward the endosomal membrane promotes the fusion of
the viral and endosomal membranes.
Inhibition of these conformational changes could be an efficient means of blocking
infection. Influenza A virus infection can be suppressed by multivalent sialylation of
β-thio-glycoclusters.7 Several small molecules have been found to be fusion inhibitors of
the influenza A virus.8-11 We previously found that CL-385319 could inhibit H5N1
influenza virus infection by blocking viral entry.12 It was found that the recognition and
binding of CL-385319 to HA proceeds by a process of ‘‘induced fit’’ and the binding
3

ACCEPTED MANUSCRIPT
pocket is formed during their interaction. Those residues constituted the binding cavity:
V482, F1102, M241, E1052, R1062, E1032, T1072 and K512. Occupation of this pocket by
CL-385319 could stabilize the neutral pH structure of hemagglutinin, thus inhibiting the
conformational rearrangements required for membrane fusion.12 A novel series of
derivatives focused on the target pocket were synthesized as specific inhibitors against
the H5 subtype of influenza A viruses.10 We also found that molecule 1L was the most
active among these derivatives because the thiophene chromophore of molecule 1L has
stronger π–π interactions with the side chains of residues F1102 and M241 compared with
molecule CL-385319 according to a variety of theoretical calculations including docking,
molecular dynamics simulations, free energy calculations and quantum calculations.13,14
In this study, we concentrated on designing and synthesizing oligothiophene derivatives
since thiophene could act strongly on some residues of the cavity. We also evaluated the
activities of all synthesized compounds against H5N1 avian influenza A viruses.
2. Chemistry
All the reactions were prepared under an atmosphere of dry N2. Tetrahydronfuran
(THF) and diethylether were dried over sodium/benzophenone, while the methanol was
dried by distillation from CaF2. To generate aminoalkyl thiophene derivatives, a general
and scalable synthesis of the requisite oligothiophenes precursors were developed
(Scheme 1).15-17 2,5-dibromo thiophene/5,5’-dibromo-2,2’-bithiophene were subjected to
the Grignard reagent of thiophen-2-ylmagnesium bromide catalyzed by Ni(dppp)Cl2 in
the freshly distilled ether respectively to achieve terthiophene 3/tetrathiophene 4 as
4

ACCEPTED MANUSCRIPT
products. 5,5’-dibromo-2,2’-bithiophene was previously obtained through the
bromination of 2,2’-bithiophene by NBS in DMF.18 Subsequent alkylations of the
oligothiophenes were carried out by the treatment with 4 equivalents of n-butyl lithium in
hexane at -78 ℃ in dry THF, followed by the addition of 4 equivalents of corresponding
dibromoalkanes to achieve the monoalkylation products (3sd, 3sa, 4sa, 4sd) and
dialkylation products (2sa, 3si, 3sg, 4sg) at one pot.19 Based on the above experimental
results, the alkylation reactions with 1,4-dibromobutane yielded dialkyl-oligothiophenes
as major products and monoalkyl-oligothiophenes as minor products with a ratio around
2:1. In contrast, alkylation of 1,3-dibromopropane provided a 1:2 ratio of di/mono
products. However alkylation to monothiophene yielded only monoalkylation product.
At the same time, we also tried the alkylation reactions with 1,2-dibromoethane; however
this ethylation does not work.
The bromoalkylthiophenes and bisbromoalkylthiophenes were then treated with 2
equivalents and 4 equivalents of the NaN3, respectively, in DMF at 90 ℃ to give
azidoalkylthiophene (3se, 3sb, 4sb, 4se) and bisazidoalkylthiophene (3sj, 4sh) as
products, followed by hydrogenation over 20 wt% Pd/C-H2 (50 Psi) in degased 1:1
MeOH/THF, affording aminoalkylthiophene and bisaminoalkylthiophene (1sc, 2sb, 2sc,
2sd, 3sc, 3sf, 3sh, 3sk, 4sc, 4sf, 4si). The amino thiophene derivatives were then coupled
to 3-fluoro-5-(trifluoromethyl) benzoic acid by the treatment of 1 equivalent of
N,N’-diisopropylcarbodiimide (DIC), hydroxybenzotriazole (HOBT) in DMF providing
F4-thiophene derivatives (1sa, 1sd, 1se, 2se, 2sf, 3sm, 3sl). While
bisaminoalkylthiophenes were subjected to 2 equivalents of DIC/HOBT, and
5

ACCEPTED MANUSCRIPT
3-fluoro-5-(trifluoromethyl)benzoic acid in DMF providing F4-thiophene-F4 derivatives
(1sf, 2sg, 2sh, 2si, 2sj, 3sn, 3so) as products. For alkylation of monothiophene, the
reactions with 1,4-dibromobutane only yielded mono alkyl products in the first step. A
second alkylation of 2-(4-bromobutyl) thiophene yielded 2,5-bis(4-bromobutyl)
-thiophene as final product (1sb). The difference in structures of thiophene derivatives
does not affect yields of following azido and hydrogenation reactions.
S
Br
Br
S
S
S
Mg
S
S
3
Br
S
Mg Br
Et2O Reflux 3h
Ni(dppp)Cl2
Et2O Reflux 12h
1a
1b
Terthiophene
S
Br
S
S
Br
S
S
NBS
S
Mg Br
S
S
DMF overnight
Ni(dppp)Cl2
Et2O Reflux 12h
2a
2b
4
Tetrathiophene
Scheme 1. Synthesis of oligothiophenes
Scheme 2. Synthesis of oligothiophenes derivatives
3. Results and discussion
The compounds designed and synthesized are listed with the IC50 values for inhibition
of H5N1 pseudovirus infection in Table 1. All compounds were evaluated for inhibitory
6

ACCEPTED MANUSCRIPT
activity against the entry of H5N1 influenza virus.10, 12 Several compounds exhibited
stronger inhibitory activities against the infection of H5N1 virus compared with
CL-385319. The most active compound was 3sf with IC50 of 29 nM (Table 1).
3.1 The activity of oligothiophene as virus entry inhibitor.
CL-385319 inhibits the infection6,8 by H1-, H2-, H3- and H5-typed influenza A viruses
by interfering with the fusogenic function of the viral hemagglutinin which consists of
HA1 and HA2. CL-385319 is thought to distinguish6,12 and bind to the cavity of H5N1
HA2 stem region, which contains V482, F1102, M241, E1052, R1062, E1032, T1072 and
K512. The recognition and binding of CL-385319 to HA proceeds by a process of
‘‘induced fit’’, whereby the binding pocket is formed during the course of this interaction.
Occupation of this pocket by CL-385319 may stabilize the neutral pH structure of
hemagglutinin, thus inhibiting the conformational rearrangements required for membrane
fusion6. Thiophene has stronger π–π interactions with the side chains of residues F1102
and M241 which are crucial for conformational changes in the HA2 protein13 and for
membrane fusion compared with piperidine of molecule CL-385319 by a variety of
theoretical calculations, including docking, molecular dynamics simulations, free energy
calculations, as well as quantum calculations. In this study, a lot of oligothiophene
compounds are more active than CL-385319 (see Figure 2 and Table 1) and the highest
activity of 3sf is almost as 1000 times as CL-385319. Docking study was performed
between 3sf and HA according to reference 6. 3sf also binds to the cavity of H5N1 HA2
stem region (see Figure 1). Two thiophenes involve in hydrophobic interaction with
residues M241, L22, F1102. Aromatic interaction takes place between one of thiophenes
7

ACCEPTED MANUSCRIPT
and G472. Salt bridger forms between E251 and ammonium group of 3sf which is a
stronger interaction. So 3sf is more active than CL-385319.
A
B
Figure 1 Mode of Interaction between 3sf and HA
Table 1. Inhibitory activities of compounds against H5N1 pseudovirus
F
O
F
F
F
F
N
N
N
H
N
F
F
H
S
H
F
S
N
F
F
S
n2
n1
R
R
n
O
O
F
F
n = 1,2,3,4
n1=0, 3, 4; n2=3,4
1 sa
CL-385319
1L
Compound
n
R
IC50(µM)
CC50(µM)
n2
n1
1L
0.22 ± 0.11
ND
10.580±0.468
1373.002±99.185
CL-385319
8

ACCEPTED MANUSCRIPT
6.414 ±0.095
239.378 ±14.086
ND
1sa
1sb
1sc
1sd
1se
1sf
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
4
4
0
0
4
3
3
0
4
0
0
3
4
3
3
0
0
0
0
0
0
3
3
4
4
3
4
4
3
3
4
4
3
4
3
4
3
3
3
3
3
4
4
4
3
3
bromo
NA
amino
NA
ND
3-fluro-5-(trifluromethyl)-benzamide
7.236±0.355
4.733 ±0.474
NA
40.16±6.24
43.541 ±9.677
ND
3-fluro-5-(trifluromethyl)-benzamide
3-fluro-5-(trifluromethyl)-benzamide
bromo
NA
ND
2sa
2sb
2sc
2sd
2se
2sf
amino
NA
ND
amino
NA
ND
amino
0.0819±0.004
42.090±5.557
19.763 ±2.609
NA
26.472±5.944
46.735 ±7.990
27.354±3.788
ND
3-fluro-5-(trifluromethyl)-benzamide
3-fluro-5-(trifluromethyl)-benzamide
3-fluro-5-(trifluromethyl)-benzamide
2sg
2sh
2si
3-fluro-5-(trifluromethyl)-benzamide
NA
ND
2-chloro-benzamide
NA
ND
3,5-dihydroxy-benzamide
NA
ND
2sj
bromo
azido
12.538±1.869
23.034±2.873
2.875 0.569
11.399±0.791
11.084±1.868
0.029±0.002
6.641±0.734
4.151±0.564
35.322±5.433
>200
3sa
3sb
3sc
3sd
3se
3sf
amino
bromo
azido
18.858 ±1.304
23.760 ±0.958
>200
amino
bromo
amino
13.269±0.510
>200
3sg
3sh
>200
9

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
0
0
3
4
0
0
0
0
0
0
4
4
4
4
4
4
3
4
3
4
3
3
3
4
4
4
4
4
4
bromo
0.696±0.080
>200
ND
3si
3sj
azido
NA
amino
0.037±0.002
12.977 ±1.700
ND
3sk
3sl
3-fluro-5-(trifluromethyl)-benzamide
NA
3-fluro-5-(trifluromethyl)-benzamide
NA
ND
3sm
3sn
3so
4sa
4sb
4sc
4sd
4se
4sf
3-fluro-5-(trifluromethyl)-benzamide
NA
ND
3-fluro-5-(trifluromethyl)-benzamide
NA
ND
bromo
azido
74.117±14.132
>200
>100
>200
ND
46.053 ±3.830
amino
bromo
azido
0.171±0.007
NA
NA
ND
amino
bromo
azido
NA
ND
NA
ND
4sg
4sh
4si
NA
ND
Amino
NA
ND
NA is no activity. ND is not determined .
3.2 The structure - activity relationship of oligothiophene compounds.
From Table 1, the antiviral activities of compounds 1sa, 1sd and 1se were a little
higher than the positive control drug CL-385319, which suggested that the incorporation
of 3-fluoro-5-(trifluoromethyl)benzamide segment and thiophene group to the basic
structure was essential for the antiviral activities in the series of monothiophenes.
Introduction of additional functional groups, such as 3-fluoro-5-(trifluoromethyl)
benzamide segments did not result in enhanced activity.
10

ACCEPTED MANUSCRIPT
Furthermore, the length of spacer between thiophene group and
3-fluoro-5-(trifluoromethyl)benzamide did not significantly affect the antiviral activities.
It was found that the replacement of –N3 with –NH2 in R substituent increases the
activity (3sf > 3se; 4sc > 4sb) and the replacement of –Br with –N3 in R substituent
demonstrated the same results (3se > 3sd; 4sb > 4sa). Similarly, the addition of an –NH2
group was more effective than an azido function, which was more effetive than a bromo
substitutuent for the series of terthiophene and tetrathiophene. The structures of 2sc and
2sd are very similar, in which 2sd has another alkyl substituent in 5’ position of the
thiophene ring, while 2sc doesn’t have. Although 2sc and 2sd were differentiated only by
the presence of 5’ alkyl substituent in 2sd, 2sd was much more active than 2sc, indicating
that 2, 5’-disubstitution of the thiophene ring was the most important structural factor. For
dithiophene and terthiophene based virus entry inhibitors with same functional groups
(-NH2, N3, Br), compounds with four carbons alkyl chain exhibits higher activities than
the conterparts with three carbons (3sf > 3sc, 3sk > 3sh, 3si > 3sg, 3se>3sb, 2sd > 2sb).
Figure 2. The inhibitory activity of CL-385319 and 3sf against A/Thailand/Kan353/2004
(H5N1) pseudovirus. The samples were tested in triplicate and the data were presented in
mean ± SD. This experiment was repeated three times with similar results.
11

ACCEPTED MANUSCRIPT
3.3 Inhibition activity against H1N1 influenza virus MDCK.
H5 subtype is the highly pathogenic virus. H1 and H5 are in the same clade.20 H5N1
(strain A/Thailand/ Kan353/2004) and H1N1 (A/PuertoRico/8/1934) share 58.76%
sequence identity (see supporting information). In order to assess inhibitiory activity
against H5N1 influenza virus, we screened inhibition activity of some compounds (3sk,
3sf, 3sc and 4sc) against H1N1 influenza virus MDCK (see table 2), which are
powerfully active against H5N1 influenza pseudovirus (see table 1). They were all
effective in inhibiting the infection of H1N1 influenza A virus (A/PuertoRico/8/1934).
Compound 4sc was the most active inhibitor in this series with an IC50 of 0.768 µM. It is
interesting that compound 4sc is more active than compound 3sc. Thus compound 4sc is
safer than any other three compounds.
Table 2. Inhibition activity against H1N1 influenza virus MDCK of some compounds.
Compound
CL-385319
IC50(µM)
72.0±10.688
CC50(µM)
1373.002±99.185
12.977 ±1.700
13.269±0.510
18.858 ±1.304
>200
3sk
3sf
3sc
4sc
3.292±0.594
1.240±0.156
1.119±0.381
0.768±0.245
3. Conclusions
A novel series of oligothiophene compounds were synthesized as specific virus entry
inhibitors against the H5 subtype of influenza A viruses to explore how the enhanced
interactions with the side chains of residues F1102 and M241.expected for thiophene
12

ACCEPTED MANUSCRIPT
would impact activity. Compound 3sf was identified as the most active inhibitor in this
series with an IC50 of 0.029 µM, which validated this design model. The
structure-activity relationships analysis of these compounds showed that the size of the
oligothiophene compounds was also very important for activity. This work suggested that
compounds inhibited conformational changes of hemagglutinin (HA) could be an
efficient way of blocking influenza virus infection. A cavity of H5N1 HA2 stem region
which contained V482, F1102, M241, E1052, R1062, E1032, T1072 and K512 could be a
target to design new influenza virus entry inhibitors. Four compounds (3sk, 3sf, 3sc and
4sc) of strong inhibitiory activity against H5N1 influenza pseudovirus also strongly
inhibite H1N1 influenza virus MDCK. In the future, we will study further on the
mechanism of entry inhibition.
4. Experimental protocols
4.1 Chemical material and equipment.
Reagents for synthesis were obtained commmercially, unless otherwise noted. All
reactions were operated under nitrogen atmosphere. Tetrahydrofuran (THF) was dried by
distillation from sodium. Flash chromatography was performed on silica gel 60 (230-240
mesh, 60Å) with the indicated eluents. 1H NMR was recorded at 400 or 500 MHz and 13C
NMR at 100 or 125 MHz on a Bruker DPX-400 or DPX-500 instrument. Hydrogenation
was carried out with Dayton 5K906C hydrogenator. Mass spectra were recorded with
Bruker MicroTof. Final products were dissolved in MeOH/MeCN purified using
reverse-phase HPLC (C-18 column, H2O/CH3CN (10/90 to 100/0 over 25 minutes, 100/0
13

ACCEPTED MANUSCRIPT
to 100/0 from 25 min to 40 min (0.1 % TFA) ).
4.2 Cell lines, plasmids and materials.
Madin–Darby Canine Kidney (MDCK) cells and 293T cells were obtained from the
American Type Culture Collection (ATCC). Dulbecco’s modified Eagle’s medium
(DMEM) were obtained from Life technology; fetal calf serum (FCS) was supplied from
Gemini Bio-Products; HA plasmid expressing HA of the H5 subtype strain A/Thailand/
Kan353/2004 (H5N1) strain was kindly provided by Frank Kirchhoff (University of Ulm,
Germany). Cell culture lysis reagent and luciferase assay substrate were bought from
Promega.
4.3 Chemistry procedures.
General procedure for synthesis of 5, 5’-dibromo-2, 2’-bithiophene. To a solution of 2,
2’-bithiophene (12 mmol, 2 g) in anhydrous DMF (30ml), NBS (24 mmol, 4.28 g) was
added dropwisely (cooling with ice-water during addition of NBS solution) and stirred
for 3h. The reaction mixture was then poured into 100 ml of ice water and the beige solid
1
was separated by vacuum filtration.
The crude was purified with column
chromatography (silica gel 100-200 mesh), using hexane as eluent to yield yellow solid
as product (3.6 g, yield 90%).
General procedure for synthesis of 2,2':5',2''-terthiophene (3): An established
procedure was used for the synthesis of 2,2':5',2''-terthiophene 3. Mg turnings (0.20 g,
7.4 mmol) was added to a flame dried 100-mL two-necked flask containing
2-bromothiophene (1.1g, 6.4 mmol) in 10 mL anhydrous Ether at room temperature
under N2. The solution mixture was stirred vigorously and heated to reflux for 3h. After
14

ACCEPTED MANUSCRIPT
cooling to room temperature the Grignard reagent of 2-bromothiophene (6.4 mmol) was
added slowly to a solution of 2,5-dibromothiophene (0.74g, 3.1 mmol) catalyzed with
Ni(dppp)Cl2 ( 14mg, 0.026 mmol) in 20 mL anhydrous Ether and the solution was
refluxing 14h. After cooling to temperature, the reaction was quenched with water and
acidified with 6M HCl to pH 4-5. The solution was then extracted with CHCl3 (5 × 20
mL). The combined organic solvent was washed with brine, dried over Na2SO4 and
evaporated to dryness.2 The crude was purified with column chromatography (silica gel
100-200 mesh), using hexane/DCM as eluent to yield bright yellow solid identified as
tetrathiophene. Yield 0.77 g, 50%.
General procedure for synthesis of 2,2':5',2'':5'',2'''-tetrathiophene (4): An
established procedure was used for the synthesis of 2,2':5',2'':5'',2'''-Tetrathiophene 4. Mg
turnings (0.40 g, 14.7 mmol) was added to a flame dried 100-mL two-necked flask
containing 2-bromothiophene (2.1g, 12.7 mmol) in 20 mL anhydrous Ether at room
temperature under N2. The solution mixture was stirred vigorously and heated to reflux
for 3h. After cooling to room temperature the Grignard reagent of 2-bromothiophene
(12.7 mmol) was added slowly to a solution of 5,5'-dibromo-2,2'-bithiophene (2.0g, 6.2
mmol) catalyzed with Ni(dppp)Cl2 ( 28mg, 0.052 mmol) in 20 mL anhydrous Ether and
the solution was refluxing 14h. After cooling to temperature, the reaction was quenched
with water and acidified with 6M HCl to pH 4-5. The solution was then extracted with
CHCl3 (5 × 20 mL). The combined organic solvent was washed with brine, dried over
Na2SO4 and evaporated to dryness.2 The crude was purified with column chromatography
(silica gel 100-200 mesh), using hexane/DCM as eluent to yield bright yellow solid
15

ACCEPTED MANUSCRIPT
identified as tetrathiophene. Yield 1.98 g, 47%.
General procedure for synthesis of bromoalkylthiophene and bisbromoalkylthiophene:
To a solution containing 1 mmol of oligothiophene in freshly distilled THF (0.15 mol/L)
under N2, 4 equivalents of a 2.6 M solution of n-butyl lithium in hexane was added at
-78 ℃ drop wisely. The solution was stirred at -78 ℃ for 1 h, and then 4 equivalents of
dibromoalkane was added to the solution. The reaction was then allowed to warm to
room temperature and stirred 12h. The mixture was quenched with 10 mL water and
extracted with ether. The combined organic layer was washed with a saturated NaCl
solution, dried over MgS04, and concentrated under reduced pressure. Column
chromatography of the residue (hexane / DCM 10:1 to 5:1; Rf 0.3 and 0.5 with hexane /
DCM 5:1) afforded pure bromo-alkylthiophene (1sb, 3sd, 3sa, 4sa, 4sd, 2sa, 3si, 3sg,
4sg).14
General procedure for synthesis of azido-alkylthiophene and bis-azido-alkylthiophene:
Anhydrous DMF solution (2 mL) of 2 to 4 equivalents sodium azide
and
bromoalkylthiophene or bisbromoalkylthiophene (1 mmol) was stirred in round bottom
flask under N2 at 80 °C for 12h. Then water (10 mL) was added. The mixture was then
extracted with dichloromethane (3 × 10 mL).4 The organic layer was collected and then
washed with a saturated NaCl solution, dried over MgSO4, and concentrated under
reduced pressure. Column chromatography of the residue (hexane / DCM 5:1; Rf 0.3-0.4
with hexane/DCM 5:1) afforded pure Azido-alkylthiophene (3se, 3sb, 4sb, 4se) and pure
bis-Azido-alkylthiophene (3sj, 4sh).
General procedure for synthesis of aminoalkylthiophene or bisaminoalkylthiophene:
16

ACCEPTED MANUSCRIPT
To a solution of 10 mL N2 degassed distilled THF/Methanol 1:1, 1 mmol of
azido-alkylthiophene and 10% wt/wt palladium on activated carbon (10%) was added.
The reaction was stirred under 50 psi of H2 for 2 days. The mixture was then filtered, and
the solution was evaporated under reduced pressure to give pure aminoalkylthiophene.
General procedure for synthesis of 3-fluoro-5-(trifluoromethyl) benzoic acid
-Thiophene and 3-fluoro-5-(trifluoromethyl) benzoic acid -Thiophene-3-fluoro
-5-(trifluoromethyl) derivatives: To
a
solution of aminoalkylthiophene or
bisaminoalkylthiophene in DMF (10 mL), 1 equivalent of I’-diisopropylcarbodiimide
(DIC), hydroxybenzotriazole (HOBT), and 3-fluoro-5-(trifluoromethyl) benzoic acid
(1mmol amino group for one equivalent) was added and stirred 12h. The solution was
then evaporated under reduced pressure. The crude was purified with column
chromatography (silica gel 100-200 mesh), using hexane/DCM as eluent to yield
F4-Thiophene derivatives (1sa, 1sd, 1se, 2se, 2sf, 3sm, 3sl) and F4-Thiophene-F4
derivatives (1sf, 2sh, 2si, 2sj, 2sg, 3sn, 3so) as final products. Yield 60%~70%.
4.4 Measurement of the inhibitory activity against H5N1 pseudovirus.
MDCK cells and 293T cells were grown in Dulbecco’s Modified Eagle Medium
(DMEM, Gibco) containing glutamine, supplemented with 10% fetal calf serum (FCS).
The H5N1 pseudoviruses were prepared by transfecting HA plasmid from the H5 subtype
strain A/Thailand/ Kan353/2004(H5N1) strain and the NA plasmid from the N1 subtype
strain A/Thailand/Kan353/2004. Briefly, 293T cells (70-80% confluent) were
co-transfected with 1 µg HA plasmid, 1 µg NA plasmid and 3 µg HIV backbone plasmid
(pNL4-3.luc.R_E_) into six-well plate with polyethylenimine (PEI)5. Forty-eight hours
17

ACCEPTED MANUSCRIPT
after transfection, the culture supernatants were harvested and centrifuged at 2000 g for
10 min. Aliquots were stored at -80 °C. For measuring the inhibitory activities of test
compounds, MDCK cells (104/well) were seeded in 96-well plates and grown overnight.
Tested compounds at indicated concentrations were incubated with pseudotyped particles
for 30 min at 37 °C. Subsequently, the virus-compound mixture was transferred to the
cells and incubated for an additional 48 h. Cells were washed with phosphate buffer
saline (PBS) and lysed with luciferase cell culture lysis reagent (Promega, Madison, WI).
Aliquots of cell lysates were transferred to 96-well flat bottom luminometer plates
(Costar), followed by the addition of luciferase assay substrate (Promega). The luciferase
activity was measured in a microplate luminometer (Genios Pro, Tecan, US). As a
negative control, VSV-G pseudotyped particles were incubated with the tested compound
instead of H5N1 pseudovirus.
4.5 Screening inhibition activity against H1N1 influenza virus MDCK.
cells were plated at a density of 2.5×104 cells per well in 96-well plates and incubated
for 24 h at 37 °C in 5% CO2. Influenza A/PuertoRico/8/1934 (H1N1) was pretreated at
37 °C for 1h with 7 concentrations of 2-fold serial dilutions of test compounds starting at
8-200 µM. Then cells were washed twice with PBS and infected with the mixture of
virus-compound for 1h, thereafter refreshed and incubated with indicated concentration
of test compounds in DMEM containing 1 µg/ml TPCK-treated trypsin, at 37 °C for 48 h.
The progeny production in supernatant was measured by the modified neuraminidase
activity (NA) assay. Briefly for NA activity assay, the supernatants were transferred to 96
black
well
plates
and
incubated
with
20
µM
18

ACCEPTED MANUSCRIPT
2-(4-Methylumbelliferyl-a-D-N-acetylneuraminic acid sodium salt (MUNANA, Sigma,
cat. No M8639), dissolved in 33 mM 2-[N-morpholino]ethanesulfonic acid (pH 6.5) and
4 mM CaCl2, at 37 °C for 1 h. The reaction was terminated by adding 0.14 M NaOH in
83% ethanol. The fluorescence intensity was measured at an excitation wavelength of
340 nm and an emission wavelength of 535 nm using an ELISA reader (Genios Pro,
Tecan, US). The IC50 (the concentration of an agent causing 50% inhibition of progeny
virus production) values were calculated as described previously [Reed, L.J., Muench, H.,
1938. A simple method of estimating fifty percent endpoint. Am. J. Hyg. 27, 493-497].
CL-385319 is used as a positive control.
4.6 Cytotoxicity assay
The cytotoxicity of compounds for MDCK cells was measured using the MTT assay.
Briefly, 100 µl of MDCK cells (5×103/well) was seeded in a 96-well cell culture plate.
On the second day, the medium was changed to a fresh one. Then 100 µl of test extract
diluted in culture media was added. After incubation at 37 °C for 2 days, 10 µl of MTT
solution (5 mg/ml) was added, followed by an incubation of 4 h. The absorbance at 570
nm was measured with an ELISA reader (Genios Pro, Tecan, US). The CC50 (the
concentration of an agent causing 50% cytotoxicity) values were calculated using the
Calcusyn software.
4.7 Characterization.
3-fluoro-5-(trifluoromethyl)-N-(4-(5-(thio-phene-2-yl)thio
-phen-2-yl)butyl)benzamide (2sf). 345 mg of 2sf as yellow solid, Yield 81%. Purity
19

ACCEPTED MANUSCRIPT
1
96.67%. Mp 87-89 ℃. H NMR (CDCl3, 400 MHz) δ:1.68−1.71 (m, 2H), 1.73−1.76
(m, 2H), 2.82−2.86 (m, 2H), 3.46−3.50 (m, 2H), 6.11 ( s, 1H), 6.67−6.68 (d, 1H, J = 3.2
Hz), 6.96−6.97 (m, 2H), 7.06−7.07 (d, 1H, J = 2.4 Hz), 7.14−7.15 (d, 1H, J = 5.2 Hz),
13
7.42−7.44 (d, 1H, J = 8 Hz), 7.64−7.66 (d, 1H, J = 8.4 Hz), 7.75(s, 1H) , C NMR
(DMSO-D6, 125 MHz) δ: 28.9, 29.0, 40.2, 115.6, 115.8, 117.9, 118.1, 119.4, 121.9, 123.2,
123.6, 124.1, 125.3, 127.8, 132.9, 133.2, 133.3, 135.4, 137.8, 138.1, 138.2, 144.1, 161.6,
163.6, 164.9; EI(+)-HRMS m/z , calcd for C20H17F4NOS2 (M + Na) 450.0579, found
450.0579.
2-(4-bromobutyl)-5-(thiophen-2-yl)thiophene. 164.5 mg of product as yellow liquid,
1
yield 55%. Purity 95.56%. H NMR (CDCl3, 400 MHz) δ:1.81−1.84 (m, 2H ),
1.91−1.95 (m, 2H), 2.80−2.84 (m, 2H), 3.40−3.43 (m, 2H), 6.67- 6.68 ( d, 1H), 6.96−6.98
13
(m, 2H), 7.08−7.09 (d, 1H, J = 3.6 Hz), 7.14−7.16 (d, 1H, J = 6 Hz), C NMR (CDCl3,
125 MHz) δ: 29.5, 30.3, 32.3, 33.6, 123.5, 123.8, 124.2, 125.4, 128.0, 135.5, 138.1, 144.3;
EI(+)-HRMS m/z , calcd for C12H13BrS2 (M + Na) 322.9537, found 322.9534.
2-(4-azidobutyl)-5-(thiophen-2-yl)thiophene. 250 mg of product as yellow liquid, yield
95%. Purity 96.67%.. 1H NMR (CDCl3, 400 MHz) δ:1.64−1.68 (m, 2H), 1.74−1.78 (m,
2H), 2.80−2.83 (m, 2H), 3.27−3.31 (m, 2H), 6.66−6.67 (d, 1H, J = 3.6 Hz), 6.96−7.01 (m,
13
2H), 7.07−7.08 (d, 1H, J = 3.2 Hz), 7.14−7.16 (d, 1H, J = 6 Hz), C NMR (DMSO-D6,
125 MHz) δ: 28.9, 29.3, 30.3, 51.9, 123.9, 124.1, 124.6, 125.8, 128.4, 135.9, 138.4, 144.7;
EI(+)-HRMS m/z , calcd for C12H13N3S2 (M + Na) 286.0442, found 286.0443.
4-(5-(thiophen-2-yl)thiophen-2-yl)butan-1-amine (2sc). 225 mg of 2sc as Yellow
1
solid, yield 95%. Purity 98.90%. Mp 58-59 ℃. H NMR (CDCl3, 400 MHz)
20

ACCEPTED MANUSCRIPT
δ:1.50−1.56 (m, 4H), 1.69−1.73 (m, 2H), 2.70−2.73 (m, 2H), 2.78−2.81 (m, 2H),
6.66−6.67 (d, 1H, J = 3.6 Hz), 6.95−6.98 (m, 2H), 7.06−7.07 (d, 1H, J = 2.8 Hz),
13
7.13−7.15 (d, 1H, J =5. 6 Hz), C NMR (CDCl3, 125 MHz) δ: 28.9, 30.0, 33.1, 41.9,
123.1, 123.5, 123.8, 124.9, 127.7, 134.9, 137.9, 144.8; EI(+)-HRMS m/z , calcd for
C12H15NS2 (M + 1) 238.0718, found 238.0718.
3-fluoro-5-(trifluoromethyl)-N-(2-(pyrrolidin-1-yl)-2-(thiophen-2-yl)ethyl)benzamid
1
e (1sa). 367 mg of 1sa as yellow solid, yield 95%. Purity 97.86%. Mp 117−118. ℃. H
NMR (CDCl3, 400 MHz) δ: 1.77−1.78 (s, 4H), 2.61−2.62 (m, 4H), 3.61−3.68 (m, 1H),
3.91−3.98 (m, 2H), 6.59 (s, 1H), 6.96−6.97 (m, 1H), 6.97−7.01 (m, 1H), 7.27−7.29 (m,
1H), 7.43−7.45 (d, 1H, J = 8 Hz), 7.59−7.61 (d, 1H, J = 8.2 Hz), 7.74 (s, 1H), 13C NMR
(CDCl3, 125 MHz) δ: 23.6, 44.7, 51.4, 62.5, 115.7, 115.9, 117.9, 118.0, 119.9, 125.4,
126.0, 126.6, 133.1, 138.3, 142.7, 161.7, 163.7, 164.9; EI(+)-HRMS m/z , calcd for
C18H18F4N2OS (M)387.1143, found 387.1148.
2,5-bis(4-bromobutyl)thiophene (1sb). 157 mg of 1sb as white solid, yield 45%. Purity
1
98.87%. Mp 67−69 ℃. H NMR (CDCl3, 400 MHz) δ: 1.78−1.84 (m, 4H), 1.89−1.94
13
(m, 4H), 2.77− 2.81 (m, 4H), 3.41−3.44 (m, 4H), 6.58 (s, 2H), C NMR (CDCl3, 125
MHz) δ: 29.4, 30.3, 32.2, 33.6, 124.1, 142.6; EI(+)-HRMS m/z , calcd for C12H18Br2S
(M+23)374.9388, found 374.9392.
N,N'-(thiophene-2,5-diylbis(butane-4,1-diyl))bis(3-fluoro-5-(trifluoromethyl)benzam
1
ide) (1sf). 515 mg of 1sf as yellow solid, yield 85%. Purity 98.28%. Mp 87−89. ℃. H
NMR (CDCl3, 400 MHz) δ: 1.63−1.68 (m, 4H), 1.72−1.77 (m, 4H), 2.81− 2.84 (m, 4H),
3.44−3.48 (m, 4H), 6.31 (s, 2H), 6.56−6.58 (d, 2H, J = 7.2 Hz), 7.42−7.44 (d, 2H, J = 8.4
21

ACCEPTED MANUSCRIPT
13
Hz), 7.63−7.65 (d, 2H, J = 8.4 Hz), 7.53 (s, 2H), C NMR (CDCl3, 125 MHz) δ: 28.9,
29.0, 29.8, 40.4, 115.6, 115.9, 117.9, 118.2, 119.6, 119.7, 121.9, 124.2, 133.0, 133.2,
138.3, 138.4, 142.7, 161.6, 163.6, 165.1; EI(+)-HRMS m/z , calcd for C28H26F8N2O2S
(M+23)629.1497, found 629.1479.
3-fluoro-5-(trifluoromethyl)-N-(3-(thiophen-2-yl)propyl) benzamide (1sd). 264 mg of
1
1sd as yellow liquid, yield 80%. Purity 96.21%. H NMR (CDCl3, 400 MHz) δ:
2,00−2.07 (m, 2H), 2.94−2.98 (m, 2H), 3.52− 3.57 (m, 2H), 6.27 (s, 1H), 6.92−6.94 (d,
1H, J = 8.0 Hz), 7.13−7.14 (d, 1H, J = 5.2 Hz), 7.42−7.44 (d, 1H, J = 7.6 Hz), 7.58−7.59
(d, 1H, J = 8.4 Hz), 7.66 (s, 1H), 13C NMR (CDCl3, 125 MHz) δ: 28.0, 31.4, 40.3, 115.6,
115.7, 115.8, 115.9, 118.0, 118.2, 119.5, 119.5, 123.8, 124.8, 127.3, 138.1, 138.2, 144.3,
161.7, 163.7, 164.9; EI(+)-HRMS m/z , calcd for C15H13F4NOS (M+23)354.0538,
found354.0546.
3-fluoro-5-(trifluoromethyl)-N-(4-(thiophen-2-yl)butyl) benzamide (1se). 289 mg of
1
1se as yellow liquid, yield 84%. Purity 95.65%. H NMR (CDCl3, 400 MHz) δ:
1.66−1.68 (m, 2H), 1.73−1.82 (m, 2H), 2.88− 2.92 (m, 2H), 3.47−3.52 (m, 2H), 6.10 (s,
1H), 6.79−6.80 (m, 1H), 6.90−6.93 (m, 1H), 7.11−7.13 (d, 1H, J = 6.4 Hz), 7.44−7.46 (d,
1H, J = 8.0 Hz), 7.65−7.67 (d, 1H, J = 8.4 Hz), 7.76 (s, 1H), 13C NMR (CDCl3, 125 MHz)
δ: 29.0, 29.2, 29.6, 115.7, 115.9, 118.0, 118.2, 119.5, 123.3, 124.6, 127.0, 133.0, 133.1,
133.3, 138.2, 138.3, 144.8, 161.7, 163.7, 164.9; EI(+)-HRMS m/z , calcd for
C16H15F4NOS (M+23)368.0729, found 369.0733.
3-fluoro-5-(trifluoromethyl)-N-(4-(5-(thiophene-2-yl)-thiophen-2-yl)butyl)benzamid
1
e (3sm). 407 mg of 3sm as yellow solid, yield 80%. Purity 98.53%. Mp 115−117℃. H
22

ACCEPTED MANUSCRIPT
NMR (CDCl3, 400 MHz) δ:1.69−1.73 (m, 2H), 1.73−1.78 (m, 2H), 2.84−2.87 (m, 2H),
3.49−3.50 (m, 2H), 6.09 ( s, 1H), 6.68−6.69 (d, 1H, J = 3.6 Hz), 6.96−7.01 (m, 1H),
7.03−7.04 (d, 1H, J = 4 Hz), 7.13−7.14 (d, 1H, J = 4.4 Hz), 7.18−7.19 (m, 1H),
7.13−7.14 (d, 1H, J = 4.4 Hz), 7.43−7.45 (d, 1H, J = 8 Hz), 7.65−7.69 (d, 1H, J = 8.8 Hz),
13
7.76(s, 1H) , C NMR (DMSO-D6, 125 MHz) δ: 29.1, 29.2, 30.1, 40.4, 115.9, 118.3,
119.7, 123.7, 123.9, 124.6, 125.6, 128.2, 135.3, 136.1, 136.8, 137.5, 138.3, 144.6, 161.8,
163.8, 165.1; EI(+)-HRMS m/z , calcd for C24H19F4NOS3 (2M + Na) 1041.1022, found
1041.1053.
3-fluoro-5-(trifluoromethyl)-N-(3-(5-(thiophene-2-yl)thiophen-2-yl)propyl)benzamid
1
e (2se). 309 mg of 2se as yellow solid, yield 75%. Purity 95.25%. Mp 50-52 ℃. H
NMR (CDCl3, 400 MHz) δ:2.02−2.09 (m, 2H), 2.91−2.95 (m, 2H), 3.55−3.60 (m, 2H),
6.15 ( s, 1H), 6.74−6.75 (d, 1H, J = 3.2 Hz), 6.98−7.00 (m, 2H), 7.07−7.08 (d, 1H, J =
3.6 Hz), 7.17−7.18 (d, 1H, J = 5.6 Hz), 7.42−7.44 (d, 1H, J = 8 Hz), 7.59−7.61 (d, 1H, J
13
= 8.4 Hz), 7.74(s, 1H) , C NMR (CDCl3, 125 MHz) δ: 28.1, 31.3, 40.2, 115.7, 115.8,
115.9, 117.9, 118.1, 119.6, 123.5, 123.7, 124.3, 125.6, 127.9, 135.8, 137.6, 138.0, 143.5,
161.6, 163.6, 165.0; EI(+)-HRMS m/z , calcd for C19H15F4NOS2 (M + Na) 436.0420,
found 436.0423.
N,N'-([2,2'-bithiophene]-5,5'-diylbis(propane-3,1-diyl))bis-(3,5-dihydroxybenzamide)
1
(2sj). 414 mg of 2sj as yellow solid, yield 75%. Purity 99.31%. Mp 118−120 ℃. H
NMR (CDCl3, 400 MHz) δ: 0.99−1.01 (d, 2H, J = 6.4 Hz), 1.82−1.85 (m, 4H), 2.78−
2.82 (m, 4H), 3.25−3.26 (m, 4H), 6.34 (s, 2H), 6.66−6.67 (d, 4H, J = 1.6 Hz), 6.80−6.81
(d, 2H, J = 4.0 Hz), 7.00−7.01 (d, 2H, J = 3.2 Hz), 7.63−7.65 (d, 2H, J = 8.4 Hz), 8.27 (s,
23

ACCEPTED MANUSCRIPT
13
2H), 9.40 (s, 4H), C NMR (CDCl3, 125 MHz) δ: 23.3, 26.9, 30.9, 54.9, 104.9, 105.4,
122.9, 125.5, 134.5, 136.9, 143.5, 158.2, 166.5; EI(+)-HRMS m/z , calcd for
C28H28N2O6S2 (M+23)575.1285, found575.1281.
N,N'-([2,2'-bithiophene]-5,5'-diylbis(propane-3,1-diyl))bis
-(2-chlorobenzamide)
1
(2si). 361 mg of 2si as yellow solid, yield 65%. Purity 95.38%. Mp 144−145 ℃. H
NMR (CDCl3, 400 MHz) δ: 1.82−1.89 (d, 4H, J = 28.0 Hz), 2.84−2.88 (m, 4H), 3.26−
3.32 (m, 4H), 6.81−6.82 (d, 2H, J = 3.2 Hz), 7.02−7.03 (d, 2H, J = 3.6 Hz), 7.36−7.45 (m,
13
6H), 7.49−7.51 (d, 2H, J = 7.6 Hz), 8.49 (s, 2H), C NMR (CDCl3, 125 MHz) δ: 26.8,
30.8, 38.2, 123.0, 125.6, 127.0, 128.7, 129.5, 129.7, 130.5, 134.5, 137.2, 143.4;
EI(+)-HRMS m/z , calcd for C28H26Cl2N2O2S2 (M+23)579.0699, found579.0705.
5,5'-bis(3-bromopropyl)-2,2'-bithiophene (2sa). 170 mg of 2sa. as yellow solid, yield
42%. Purity 97.38%. Mp 97−99 ℃. 1H NMR (CDCl3, 400 MHz) δ: 2.17−2.24 (m, 4H),
2.96− 2.99 (m, 4H), 3.43−3.47 (m, 4H), 6.71−6.72 (d, 2H, J = 3.6 Hz), 6.91−6.92 (d, 2H,
J = 3.6 Hz), 13C NMR (CDCl3, 125 MHz) δ: 28.5, 32.7, 34.3, 123.6, 124.0, 126.1, 135.6,
136.3, 142.7; EI(+)-HRMS m/z , calcd for C14H16Br2S2 (M+23)428.8952, found
428.8952.
N,N'-([2,2'-bithiophene]-5,5'-diylbis(propane-3,1-diyl))bis
(3-fluoro-5-(trifluoromethyl)benzamide) (2sg). 330 mg of 2sg as yellow solid, yield
1
50%. Purity 97.94%. Mp 150−152 ℃. H NMR (CDCl3, 400 MHz) δ: 1.88−1.92 (m,
4H), 2.82− 2.86 (m, 4H), 3.31−3.38 (m, 4H), 6.81−6.82 (d, 2H, J = 3.2 Hz), 7.00−7.01 (d,
13
2H, J = 2.8 Hz), 7.88−7.90 (d, 2H, J = 9.6 Hz), 8.05 (s, 2H), 8.81−8.83 (m, 2H), C
NMR (CDCl3, 125 MHz) δ: 26.9, 30.6, 38.9, 115.3, 115.5, 118.4, 118.5, 120.0, 122.0,
24

ACCEPTED MANUSCRIPT
122.9, 124.2, 125.6, 130.9, 130.0, 131.1, 131.2, 134.5, 137.9, 138.1, 143.4, 160.9, 162.9,
163.4; EI(+)-HRMS m/z , calcd for C30H24F8N2O2S2 (M+23)683.1045, found683.1043.
N,N'-([2,2'-bithiophene]-5,5'-diylbis(butane-4,1-diyl))bis-(3-fluoro-5-(trifluoromethy
l)benzamide) (2sh). 340 mg of 2sh as yellow solid, yield 50%. Purity 98.28%. Mp
1
152−154 ℃. H NMR (CDCl3, 400 MHz) δ: 1.57−1.61 (m, 4H), 1.65−1.69 (m, 4H),
2.78− 2.82 (m, 4H), 3.29−3.34 (m, 4H), 6.76−6.77 (d, 2H, J = 3.6 Hz), 6.97−6.98 (d, 2H,
13
J = 3.6 Hz), 7.87−7.89 (d, 2H, J = 8.4 Hz), 8.05 (s, 2H), 8.78−8.80 (m, 2H), C NMR
(CDCl3, 125 MHz) δ: 28.2, 28.4, 28.9, 39.0, 115.3, 115.5, 118.3, 120.0, 122.0, 122.8,
124.2, 125.4, 130.9, 131.2, 134.4, 138.0, 138.1, 143.8, 160.9, 162.9, 163.3;
EI(+)-HRMS m/z , calcd for C32H28F8N2O2S2 (M+23)711.1357, found711.1356.
2-(5-(3-bromopropyl)thiophen-2-yl)-5-(thiophen-2-yl)thio-phen (3sa). 351 mg of 3sa
as yellow solid, yield 90%. Purity 95.68%. Mp 95−96 ℃. 1H NMR (CDCl3, 400
MHz) δ:2.17−2.24 (m, 2H), 2.96−2.99 (m, 2H), 3.43−3.46 (m, 2H), 6.72−6.74 (d, 1H, J
= 7.2 Hz), 6.95−7.00 (m, 3H), 7.14−7.15 (d, 1H, J = 5.2 Hz), 7.19−7.20 (d, 2H, J = 8
13
Hz) , C NMR (DMSO-D6, 125 MHz) δ: 28.5, 32.7, 34.3, 116.8, 123.6, 123.7, 123.8,
123.9, 124.0, 124.5, 124.6, 126.1, 128.1, 135.5, 136.0, 136.3, 136.6, 137.4, 142.7;
EI(+)-HRMS m/z , calcd for C15H13BrS3 (M + Na) 390.9255, found 390.9254.
2-(5-(3-azidopropyl)thiophen-2-yl)-5-(thiophen-2-yl) thiophene (3sb). 321 mg of 3sb
as yellow solid, yield 95%. Purity 95.68%. Mp 121−122 ℃. 1H NMR (CDCl3, 400 MHz)
δ:1.95−1.99 (m, 2H), 2.89−2.93 (m, 2H), 3.35−3.39 (m, 2H), 6.72−6.73 (d, 1H, J = 3.2
Hz), 6.97−7.02 (m, 3H), 7.06−7.07 (d, 1H, J = 3.6 Hz), 7.16−7.17 (d, 1H, J = 2.8 Hz),
13
7.21−7.22 (d, 1H, J = 4.4 Hz) , C NMR (DMSO-D6, 125 MHz) δ: 27.3, 30.8, 50.5,
25

ACCEPTED MANUSCRIPT
123.7,123.8, 124.0, 124.5, 124.6, 125.9, 128.1, 135.4, 136.1, 136.3, 136.6, 137.4, 143.2;
EI(+)-HRMS m/z , calcd for C15H13N3 (M + Na) 354.0164, found 354.0157.
3-fluoro-5-(trifluoromethyl)-N-(3-(5-(5-(thiophen-2-yl)thio-phen-2-yl)thiophen-2-yl)
propyl)benzamide (3sl). 198 mg of 3sl as white solid, yield 40%. Purity 97.02%. Mp
1
144−146 ℃. H NMR (CDCl3, 400 MHz) δ: 2.02−2.09 (m, 2H), 2.92−2.96 (m, 2H),
3.55−3.60 (m, 2H), 6.13 (s, 1H), 6.75−6.76 (d, 2H, J = 3.2 Hz), 6.96−6.98 (m, 1H),
7.01−7.02 (d, 1H, J = 4.0 Hz), 7.15−7.16 (d, 1H, J = 3.2 Hz), 7.20−7.21 (d, 1H, J = 4.8
Hz), 7.42−7.44 (d, 1H, J = 8.0 Hz), 7.59−7.62 (d, 1H, J = 8.8 Hz), 7.74 (s, 2H), 13C NMR
(CDCl3, 125 MHz) δ: 28.1, 31.3, 40.1, 117.9, 118.1, 119.6, 123.6, 123.8, 124.0, 124.4,
124.6, 125.7, 128.1, 135.5, 136.1, 136.4, 137.3, 138.1, 143.7, 163.7, 164.9; EI(+)-HRMS
m/z , calcd for C23H17F4NOS3 (M+23)518.0292, found518.0300.
N,N'-([2,2':5',2''-terthiophene]-5,5''-diylbis(butane-3,1-diyl))bis(3-fluoro-5-(trifluoro
methyl)benzamide) (3so). 539 mg of 3so as yellow solid, yield 70%. Purity 98.48%. Mp
162−164 ℃. 1H NMR (DMSO-d6, 400 MHz) δ: 1.70−1.72 (m, 4H), 1.74−1.77 (m, 4H),
2.83− 2.85 (m, 4H), 3.48−3.50 (m, 4H), 6.67−6.68 (d, 2H, J = 3.6 Hz), 6.94 (s, 4H),
13
7.43−7.45 (d, 2H, J = 7.6Hz), 7.64−7.67 (d, 2H, J = 8.4 Hz), 7.75 (s, 2H), C NMR
(DMSO-d6, 125 MHz) δ: 28.2, 28.4, 28.9, 39.2, 115.5, 118.3, 118.5, 120.0, 122.0, 123.7,
124.0, 125.7, 133.6, 135.1, 138.0, 138.1, 144.6, 160.9, 162.9, 163.3; EI(+)-HRMS m/z ,
calcd for C36H30F8N2O2S3 (M+23)793.1228, found798.1234.
2,5-bis(5-(3-bromopropyl)thiophen-2-yl)thiophene (3sg). 413 mg of 3sg as yellow
1
solid, yield 85%. Purity 95.37%. Mp 112−114 ℃. H NMR (CDCl3, 400 MHz)
δ:2.17−2.22 (m, 4H), 2.96−2.99 (m, 4H), 3.43−3.47 (m, 4H), 6.71−6.72 (d, 2H, J = 3.6
26

ACCEPTED MANUSCRIPT
Hz), 6.91−6.92 (d, 4H, J = 3.6 Hz) , 13C NMR (DMSO-D6, 125 MHz) δ: 28.5, 32.7, 34.3,
123.6, 123.9, 126.1, 135.6, 136.3, 142.7; EI(+)-HRMS m/z , calcd for C18H18Br2S3 (M
+ Na) 510.8829, found 510.8834.
N,
N'-([2,2':5',2''-terthiophene]-5,5''-diylbis(propane-3,1-diyl))bis(3-fluoro-5-(trifluoro
methyl)benzamide) (3sn). 549 mg of 3sn as yellow solid, yield 74%. Purity 96.94%. Mp
140−142 ℃. 1H NMR (DMSO-d6, 400 MHz) δ: 1.87−1.94 (m, 4H), 2.84−2.88 (m, 4H),
3.34−3.38 (m, 4H), 6.85−6.86 (d, 2H, J = 3.2 Hz), 7.11−7.13 (m, 4H), 7.87−7.89 (d, 2H,
J = 8.4 Hz), 7.96−7.99 (d, 2H, J = 9.2 Hz) , 8.06 (s, 2H), 8.83 (s, 2H), 13C NMR
(DMSO-d6, 125 MHz) δ: 26.9, 30.6, 39.2, 115.3, 115.5, 118.4, 118.5, 120.0, 122.0, 123.8,
124.1, 125.8, 130.8, 130.9, 131.2, 131.2, 133.7, 135.1, 138.0, 138.1, 144.2, 160.9, 162.9,
163.4; EI(+)-HRMS m/z , calcd for C34H26F8N2O2S3 (M+23)765.0917, found765.0920.
2-(4-bromobutyl)-5-(5-(5-(4-bromobutyl)thiophen-2-yl)thio-phen-2-yl)thiophene
1
(3si). 262 mg of 3si as yellow solid, yield 50%. Purity 96.61%. Mp 103−105 ℃. H
NMR (CDCl3, 400 MHz) δ: 1.81−1.86 (m, 4H), 1.92−1.99 (m, 4H), 2.82− 2.86 (m, 4H),
3.42−3.45 (m, 4H), 6.69−6.70 (d, 2H, J = 3.6 Hz), 6.96−6.97 (d, 2H, J = 3.6 Hz), 6.98 (s,
13
2H), C NMR (CDCl3, 125 MHz) δ: 29.5, 30.2, 32.1, 33.5, 123.5, 123.9, 125.4, 135.2,
136.3, 144.2;
found538.9142.
EI(+)-HRMS m/z , calcd for C20H22Br2S3(M+23)538.9169,
2-(4-azidobutyl)-5-(5-(5-(4-azidobutyl)thiophen-2-yl) thioph-en-2-yl)thiophene (3sj).
1
398 mg of 3sj as yellow solid, yield 90%. Purity 97.71%. Mp 78-79 ℃. H NMR
(CDCl3, 400 MHz) δ: 1.65−1.72 (m, 4H), 1.76−1.82 (m, 4H), 2.82− 2.86 (m, 4H),
27

ACCEPTED MANUSCRIPT
13
3.30−3.33 (m, 4H), 6.69−6.70 (d, 2H, J = 3.6 Hz), 6.96−6.97 (d, 4H, J = 3.6 Hz), C
NMR (CDCl3, 125 MHz) δ: 28.4, 28.8, 29.8, 51.4, 123.5, 123.8, 125.4, 135.2, 136.3,
144.3; EI(+)-HRMS m/z , calcd for C20H22N6S3 (M+23)465.0956, found465.0960
4-5-(5-(5-(4-aminobutyl)thiophen-2-yl)thiophen-2-yl)thio-phene-2-yl)butan-1-amine
1
(3sk). 385 mg of 3sk as yellow solid, yield 99%. Purity 98.80%. Mp 118-119 ℃. H
NMR (CDCl3, 400 MHz) δ: 1.42−1.47 (m, 4H), 1.64−1.69 (m, 4H), 2.54− 2.60 (m, 4H),
13
2.79−2.83 (m, 4H), 6.84−6.85 (d, 2H, J = 3.2 Hz), 7.14−7.17 (d, 4H, J = 10.8 Hz), C
NMR (CDCl3, 125 MHz) δ: 28.1, 28.9, 32.3, 40.9, 123.4, 123.7, 125.1, 133.2, 135.0,
144.8; EI(+)-HRMS m/z , calcd for C20H26N2S3 (M+1)391.1322, found391.1330
2-(4-bromobutyl)-5-(5-(thiophen-2-yl)thiophen-2-yl) thiophene (3sd). 112 mg of 3sd
1
as yellow solid, yield 40%. Purity 95.23%. Mp 60-62 ℃. H NMR (CDCl3, 400 MHz)
δ: 1.83−1.89 (m, 2H), 1.92−1.97 (m, 2H), 2.79−2.86 (m, 2H), 3.42−3.45 (m, 2H),
6.69−6.70 (d, 1H, J = 7.8 Hz), 6.96−7.02 (d, 4H, J = 22.4 Hz), 7.15−7.16 (d, 1H, J = 2.8
Hz), 7.20−7.21 (d, 1H, J = 5.2 Hz), 13C NMR (CDCl3, 125 MHz) δ: 29.5, 30.2, 32.1, 33.5,
123.7, 123.8, 123.9, 124.5, 125.5, 128.1, 135.1, 135.9, 136.8, 137.5, 144.4; EI(+)-HRMS
m/z , calcd for C16H15BrS3(M+1)382.9584, found382.9592.
2-(4-azidobutyl)-5-(5-(thiophen-2-yl)thiophen-2-yl)thioph-ene (3sb). 327 mg of 3sb as
yellow solid, yield 95%. Purity 99.02%. Mp 63-65 ℃. 1H NMR (CDCl3, 400 MHz) δ:
1.66−1.72 (m, 2H), 1.75−1.82 (m, 2H), 2.83−2.86 (m, 2H), 3.30−3.33 (m, 2H), 6.69−6.71
(d, 1H, J = 3.2 Hz), 6.98−7.03 (d, 3H, J = 17.6 Hz), 7.05−7.07 (d, 1H, J = 3.6 Hz),
13
7.16−7.17 (d, 1H, J = 3.2 Hz), 7.20−7.22 (d, 1H, J = 5.2 Hz), C NMR (CDCl3, 125
MHz) δ: 28.4, 28.8, 29.9, 51.4, 123.6, 123.8, 123.9, 124.5, 124.6, 125.4, 128.1, 135.1,
28