Synthesis, in vitro cytotoxicity and biological evaluation of twenty novel 1,3-benzenedisulfonyl piperazines as antiplatelet agents

Article abstract of DOI:10.1016/j.bmc.2021.116390

In order to discover antiplatelet drug with novel structure and expand our research scope, total twenty 1,3-benzenedisulfonyl piperazines, were designed and synthesized. These target compounds were divided into two series, namely 4-methoxy-1,3-benzenedisulfonyl piperazines of series 1 and 4-ethoxy-1,3-benzenedisulfonyl piperazines of series 2. With adenosine diphosphate (ADP), arachidonic acid (AA) and collagen as inducers, respectively, the Born turbidimetric method was used to screen the antiplatelet activity in vitro of all target compounds at a concentration of 1.3 μM, with aspirin and picotamide as positive control drugs. And of which, the activities of five compounds for collagen were higher than both picotamide and aspirin. In ADP or AA channel, compounds with an inhibition rate greater than 33% were selected, and their corresponding IC₅₀ values were obtained. According to the IC₅₀, the in vitro activity of one compound for ADP was higher than picotamide, and for AA, two compounds were higher than two positive control drugs and other two compounds only higher than or equal to aspirin. The preliminary analysis of the structure-activity relationship of the target compounds involved in this study was completed. Further, eight compounds exhibiting higher activity in one or two test channels, were subjected to cytotoxicity test on mouse fibroblasts (L929) by CCK-8 method. The in vitro cytotoxicity of most test compounds showed less than or same to control drug picotamide at 10 μM, but at the higher concentration of 100 μM, merely two compounds exhibited higher cell survival rate than that of picotamide. In addition, compound N¹,N³-di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-(m-tolyl)piperazine), which is delivery activity in the three test channels, and another compound N¹,N³–di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-(m-tolyl)piperazine), which has the lowest cytotoxic in vitro compound among series 1 and series 2, respectively, are found and selected for simulation analysis as two most likely to dock with the receptor P₂Y₁₂. Each of synthesized compounds in silico molecular property and ADME (absorption, distribution, metabolism and excretion) are predicted by using Molinspiration property engine v2018.10 and PreADMET online servers, respectively. Compared with other series of compounds in the previous stage, the two series compounds obtained after the introduction of piperazinyl have a similar in vitro activity.

Full text of DOI:10.1016/j.bmc.2021.116390

Bioorg. Med. Chem. 46 (2021) 116390
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, in vitro cytotoxicity and biological evaluation of twenty novel
1,3-benzenedisulfonyl piperazines as antiplatelet agents
,
b
,
,
,*
Xiu-jie Liua *, Yan Wanga c, Xiao Wanga, Zhi-hao Zhangd
a School of Chemistry and Chemical Engineering, Tianjin University of Technology, 300384 Tianjin, China
b Tianjin Key Laboratory of Drug Targeting and Bioimaging, Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion, School of Chemistry and
Chemical Engineering, Tianjin University of Technology, 300384 Tianjin, China
c Asymchem Laboratories (Tianjin) Co., Ltd., Tianjin 300457, China
d School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In order to discover antiplatelet drug with novel structure and expand our research scope, total twenty 1,3-ben-
zenedisulfonyl piperazines, were designed and synthesized. These target compounds were divided into two se-
ries, namely 4-methoxy-1,3-benzenedisulfonyl piperazines of series 1 and 4-ethoxy-1,3-benzenedisulfonyl
piperazines of series 2. With adenosine diphosphate (ADP), arachidonic acid (AA) and collagen as inducers,
respectively, the Born turbidimetric method was used to screen the antiplatelet activity in vitro of all target
N1,N3-dipiperazinyl-4-methoxy(ethoxy)-1,3-
benzenedisulfonamide
Synthesis
In vitro anti-platelet activity
Cytotoxicity
compounds at a concentration of 1.3 μM, with aspirin and picotamide as positive control drugs. And of which, the
Docking studies
activities of five compounds for collagen were higher than both picotamide and aspirin. In ADP or AA channel,
compounds with an inhibition rate greater than 33% were selected, and their corresponding IC50 values were
obtained. According to the IC50, the in vitro activity of one compound for ADP was higher than picotamide, and
for AA, two compounds were higher than two positive control drugs and other two compounds only higher than
or equal to aspirin. The preliminary analysis of the structure-activity relationship of the target compounds
involved in this study was completed. Further, eight compounds exhibiting higher activity in one or two test
channels, were subjected to cytotoxicity test on mouse fibroblasts (L929) by CCK-8 method. The in vitro cyto-
In silico ADME prediction
toxicity of most test compounds showed less than or same to control drug picotamide at 10 μM, but at the higher
concentration of 100 μM, merely two compounds exhibited higher cell survival rate than that of picotamide. In
addition, compound N1,N3-di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-(m-tolyl)piperazine), which is delivery
activity in the three test channels, and another compound N1,N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-
(m-tolyl)piperazine), which has the lowest cytotoxic in vitro compound among series 1 and series 2, respectively,
are found and selected for simulation analysis as two most likely to dock with the receptor P2Y12. Each of
synthesized compounds in silico molecular property and ADME (absorption, distribution, metabolism and
excretion) are predicted by using Molinspiration property engine v2018.10 and PreADMET online servers,
respectively. Compared with other series of compounds in the previous stage, the two series compounds obtained
after the introduction of piperazinyl have a similar in vitro activity.
1. Introduction
antiplatelet drug with higher activity, lower toxicity, and broader effects
has extremely high social & economic significance.5
With the acceleration of economic development and people’s lives,
the prevalence and mortality of thrombotic diseases have been
increasing.1–3 Among them, platelet is a key participant in athero-
thrombosis.4 Antiplatelet drugs occupy an important position in the
treatment of thrombotic diseases. So, the development of new
Picotamide (Fig. 1), as an anti-platelet aggregation drug, has a
certain broad spectrum and multiple mechanisms of action.6 The clinical
comparison studies in recent years have shown that picotamide signif-
icantly surpasses aspirin in the treatment of atherosclerosis and the
treatment of thrombosis in diabetic patients with cardiac and
* Corresponding authors at: School of Chemistry and Chemical Engineering, Tianjin University of Technology, 300384 Tianjin, China (X.-J. Liu); School of
Pharmacy, Nanjing Medical University, 211166 Nanjing, China (Z.-H. Zhang).
E-mail addresses: liuxiujiechem@163.com (X.-j. Liu), zhangzhihaochem@163.com (Z.-h. Zhang).
https://doi.org/10.1016/j.bmc.2021.116390
Received 13 June 2021; Received in revised form 23 August 2021; Accepted 25 August 2021
Available online 30 August 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

X.-j. Liu et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116390
cerebrovascular complications.7,8 Relevant departments in many coun-
tries pay more attention to and strengthen the research and development
of picotamide itself and its structural modification work,9–12 and 2NTX-
99 is one of the most successful.13 The piperazines has always been a
type of substituent that often appear in drug structure, which could
usually increase the water solubility of the drug, thereby improving the
bioavailability of the drug.14,15
property engine v2018.10 and PreADMET online servers, respectively.
2. Results and discussion
2.1. Chemistry
One kind of the key intermediates for the synthesis of target com-
pounds are different N-hydrocarbyl substituted piperazines 4a-j. Among
them, the synthetic pathway of the 4h-j self-synthesized was depicted in
Scheme 1. Using bis(2-hydroxyethyl)amine (1) as the starting material,
react firstly with SOCl2 to produce bis(2-chloroethylamine)
hydrochloride (2), and then continue to undergo two amination re-
actions with different aromatic amines (3h–j) to produce the key in-
termediate N-hydrocarbyl substituted piperazines (4h-j) according to
design needs: N-(2-methylphenyl)piperazine (4h, yield: 31.26%), N-(3-
methylphenyl)piperazine (4i, yield: 33.14%) and N-(4-methylphenyl)
piperazine (4j, yield: 71.90%). while other substituted piperazines 4a-g
used were commercially available.
Since 1999, by replacing two pyridine groups connected to the two
side chains at the 1,3-position of picotamide structure with two
substituted phenyl groups, our research group began to design and
synthesize novel structural analogs of picotamide.16–19 After screening
the in vitro antiplatelet activity of nearly one hundred 4-methoxy-1,3-
phthaloyl compounds obtained, it was found that eighteen compounds
had higher activity than that of picotamide. Next, according to the iso-
stere theory and similar research on a larger scale, substituting sulfonyl
for acyl, and further substituting 4-ethoxy for methoxy, two new series
of 4-methoxy-1,3-benzenedisulfonamides (Fig. 1) and 4-ethoxy-1,3-ben-
zenedisulfonamides (Fig. 1) were prepared.20,21 Compared with the
amide compounds prepared in the early stage, the sulfonamide com-
pounds developed in the later stage with lower synthesis cost and better
water solubility, which allows more choices of solvents for recrystalli-
zation and subsequent pharmacological research, and so that new
compounds with higher activity in almost the same proportions were
discovered.
Two synthetic routes and the structures of target compounds 7a-j of
series 1 and 10a-j of series 2 are listed in the Scheme 2. Regarding the
synthetic route of target compounds 7a-j of series 1, using anisole (5) as
starting material, react with chlorosulfonic acid and thionylchide to
produce intermediate 4-methoxybenzene-1,3-disulfonyl chloride (6) in
dichloromethane, continued with different N-hydrocarbyl piperidines
and undergo amination reaction in tetrahydrofuran to generate the
target compounds 7a-j.
In order to discover antiplatelet drugs with novel structures and
expand our research scope, this paper used piperazine groups with
better water solubility and higher polarity to replace the anilines, total
twenty 1,3-benzenedisulfonyl bis(1-hydrocarbyl piperazines), were
designed and synthesized, and were divided into two series, namely 4-
methoxy-1,3-benzenedisulfonyl bis(1-hydrocarbyl piperazine) of series
1 (7a-j) and 4-ethoxy-1,3-benzenedisulfonyl bis(1-hydrocarbyl pipera-
zine) of series 2 (10a-j). The chemical structures of each target com-
pounds have been confirmed by 1H NMR, 13C NMR, IR and ESI-MS. The
design idea and corresponding structures of target compounds were
given in Fig. 1. With adenosine diphosphate (ADP), arachidonic acid
(AA) or collagen as an inducer, respectively, two clinically mature an-
tiplatelet drugs aspirin and picotamide were selected as positive control
In addition to different one starting material phenethyl ether (8), the
synthetic method of target compounds 10a-j of series 2, is similar to
series 1. With phenethyl ether (8) as the starting material, 4-ethoxy-1,3-
phthaloyl chloride (9) is generated by the reaction of chlor-
osulfonylation, and then, the target compounds 10a-j are generated by
the reaction (9) of amination with different N-hydrocarbyl substituted
piperazines 4a-j.
Compared with the compounds obtained by introducing the phenyl
group in the previous stage, the water solubility of the compound syn-
thesized by introducing the piperazine group in this study has been
improved, so that the number of solvents that can be used for recrys-
tallization is increased.
drugs, and at the concentration of 1.3
μM, the Born turbidimetric
method was used to screen the anti-platelet activity in vitro of com-
pounds 7a-j and 10a-j. Eight compounds 7a, 7e, 7f, 7i, 10e, 10f, 10i and
10j exhibiting high antiplatelet activity in one or two test channels, were
subjected to cytotoxicity test on mouse fibroblasts (L929) by CCK-8
method. In addition, 7i and 10i, as representatives of series 1 and se-
ries 2, respectively, were selected for simulation analysis as two most
likely to dock with the receptor P2Y12. Each of synthesized compounds
in silico molecular property and ADME (absorption, distribution,
metabolism and excretion) were predicted by using Molinspiration
2.2. Biological assays
2.2.1. Evaluation of in vitro anti-platelet aggregation activity
After the target compounds are prepared, purified and structure
determined, the Born turbidimetric method22,23 is used to screen their
antiplatelet aggregation activity in vitro with ADP, AA or collagen as an
inducer, respectively. Of which, the determination of collagen channel is
limited to the study of the aggregation rate only at the concentration of
Fig. 1. Structure of picotamide and target compounds & the design idea.
2

X.-j. Liu et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116390
Scheme 1. Synthetic routes to intermediates of N-hydrocarbyl piperazines 4h-j.
Table 1
Anti-platelet aggregation activity in vitro data of compounds.
Compd.
Inhibition
rate/%
IC50
/
Inhibition
IC50
/
Inhibition
rate/%
(
μ
М)
rate/% (AA)
(μМ)
(ADP)
(ADP)
0.98
(AA)
(collagen)
7a
38.19% ±
0.03**##
24.27% ±
0.09*#
29.52% ±
0.14**
52.63% ±
0.17
7b
22.41% ±
42.64% ±
0.13**##
49.42% ±
0.11**#
0.04**
7c
28.38% ±
27.07% ±
0.04**
0.12*#
7d
27.91% ±
0.22**##
33.21% ±
0.07**##
27.97% ±
0.08*#
15.67% ±
20.41% ±
0.07
0.06**
7e
1.15
0.95
38.46% ±
0.13
0.33
0.48
22.79% ±
0.07
7f
40.64% ±
28.81% ±
0.14**##
27.59% ±
0.13**##
32.13% ±
0.09**##
54.24% ±
0.16**##
32.61% ±
0.08**##
29.53% ±
0.11**#
0.11**
7g
28.74% ±
0.11**##
38.94% ±
0.12**##
21.06% ±
0.06**##
30.37% ±
0.14**##
24.48% ±
0.05**##
28.84% ±
0.1*##
28.15% ±
0.13**
Scheme 2. Synthetic routes and structures of target compounds 7a-j of series 1
and 10a-j of series 2.
7h
36.88% ±
0.08
0.36
0.71
0.68
7i
33.74% ±
0.06**
1.3 μM. Through conversion, the corresponding inhibition rate is ob-
7j
34.14% ±
0.09**
tained. Among them, seven compounds with over 33% inhibition rate in
AA test channel and eight compounds with over 33% inhibition rate in
ADP test channel, respectively, and their corresponding IC50 values are
also obtained by calculation. And the positive drugs are picotamide and
aspirin.
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
Picotamide
Aspirin
18.12% ±
0.05**
29.90% ±
0.06**
41.70% ±
0.17**##
39.05% ±
0.11**##
26.95% ±
0.06*#
36.38% ±
0.80
0.91
24.93% ±
In vitro anti-platelet aggregation activities of target compounds are
measured using rabbit venous blood. Fresh venous blood is taken from
the ear vein of rabbits (weight: 2–3 kg, male), with 3.8% sodium citrate
as anticoagulant (9:1 by volume). Samples are centrifuged at 1000 rpm/
min for 10 min at room temperature to obtain the platelet-rich plasma
(PRP). The PRP supernatant is removed and the residue is centrifuged at
3000 rpm/min for 10 min at room temperature to obtain platelet-poor
plasma (PPP), which is used as the blank. Target compounds (1.3 µM)
are previously dissolved in dimethylsulfoxide (DMSO), and the solution
(5 µL) is added into PRP (200 µL) and incubated for 2 min. Aggregation
is induced by adding 20 µL ADP (5 mM), collagen (1 mg/mL) or AA (20
0.1*#
0.12**
40.07% ±
0.14*#
23.61% ±
0.07*
24.00% ±
20.14% ±
49.76% ±
0.12**##
50.28% ±
0.07**##
25.25% ±
0.07**##
41.59% ±
0.06**##
48.79% ±
0.07**##
28.50% ±
0.07**##
49.17% ±
0.13
0.08**#
0.10**
21.02% ±
0.08*#
25.81% ±
0.14**
25.52% ±
0.11**##
28.94% ±
0.14*#
23.08% ±
0.13**
33.08% ±
0.14*
0.65
0.43
0.26
0.34
0.43
50.27% ±
0.45
0.72
0.47
0.44
34.84% ±
0.15*
0.06*#
μ
M), repectively. DMSO (0.5% v/v) is used as negative control (ac-
43.61% ±
0.11*##
42.98% ±
0.09
cording to the pre-experiment, 5 µL DMSO showed no significant effect
on the platelet aggregation), and picotamide and aspirin are used as
standard drugs. When the inducer used is ADP or AA, the compounds
which have higher anti-platelet aggregation activity at the concentra-
45.49% ±
0.12
37.08% ±
0.16
46.30% ±
38.45% ±
49.12% ±
0.18
0.06
0.13
tion of 1.3 μM are diluted to 0.65 μM and 0.325 μM, and then repeat the
*
ρ
ρ
< 0.05 versus control, **
ρ < 0.01 versus control.
same operation and calculation method to get the corresponding IC50
value. The platelet aggregation inhibition rate (%) are calculated ac-
cording to the following formula:
#
< 0.05 versus picotamide, ##
ρ
< 0.01 versus picotamide.
and AA channels, target compounds with an inhibition rate greater than
33% are selected, corresponding IC50 values are also given in Table 1.
1. As illustrated in Table 1 and Fig. 2 for the platelet aggregation
induced by ADP, the order of the target compounds with obvious anti-
Inhibition% = (1 ꢀ D/S) × 100%
where D = platelet aggregation in the presence of test compounds, S =
platelet aggregation in the presence of solvent. The primary screening
platelet activity of series 2 at the concentration of 1.3
μM is: 10i
data for all compounds (1.3 μM) in vitro activity on antiplatelet aggre-
(50.27%) > aspirin (46.30%) > picotamide (45.49%) > 10j (43.61%) >
10d (40.07%) > 10c (36.38%). Among them, the inhibition rate of
gation of the synthesized compounds are given in Table 1. The Statistical
analysis is performed with ANOVA followed by Tukey’s test. In the ADP
3

X.-j. Liu et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116390
Fig. 2. The inhibition rate and IC50 of some target compounds of series 2 for ADP.
compound 10i is the highest. The order of the calculated IC50 values of
four potent compounds is: 10d (0.91 М) > 10c (0.80 М) > 10j (0.72
М) > picotamide (0.47 М) > 10i (0.45 М) > aspirin (0.44 М). There
position N of piperazine, has the lowest IC50 value and the highest in
vitro antiplatelet activity among both all test compounds and positive
control drugs. Further analysis the data of anti-platelet activity, the
preliminary speculations on the structure–activity relationship is
initially summarized as follows:
μ
μ
μ
μ
μ
μ
is a compound 10i with IC50 values lower than picotamide. The order of
the corresponding structure and activity relationship thus obtained is:
10i (N-3-CH3 Phenyl) > 10j (N-4-CH3 Phenyl) > 10c (N-CH(CH3)2) >
10d (N-Phenyl).
For the compound 7a-j of series 1 where the parent benzene ring is
connected to the methoxy group at the 4-position, the order of in vitro
anti-platelet activity of related compounds is: 7e (N-CH2Ph) > 7a (N-
CH3) > 7c (N-CH(CH3)2) > 7b (N-CH2CH3) > 7d (N-Ph). It is shown that
the steric hindrance of N-substituents has no obvious effect on the ac-
tivity of the compound. When the 4-position N is substituted by a phenyl
group, the order of activity after connecting different groups at the 2-po-
sition of the corresponding phenyl group is: 7h (2-CH3) > 7f (2-F) > 7g
(2-OCH3) > 7d (2-H). And the order of activity after connecting the
different positions of the corresponding phenyl group to the methyl
group is: 7h (2-CH3) > 7j (4-CH3) > 7i (3-CH3), when substituent is
methyl on the 2-position, the anti-platelet aggregation activity is
significantly higher.
From the structural point of view, compound 10i, that is N1,N3-di(4-
ethoxy-1,3-phenylenedisulfonyl)bis(1-(m-tolyl)piperazine), in which
introducing a 3-methylphenyl group at the 4-position N of the two side
chain piperazine rings, respectively. At this point, it seems that
increasing the steric hindrance of N-substituents could increase the anti-
platelet aggregation activity of target compounds. When a methyl
phenyl group is introduced into the 4-position N of two side chain
piperazine rings, the 3-position methyl group is more advantageous. The
in vitro activity order of the obtained methyl groups at different posi-
tions is: 10i (3-CH3) > 10j (4-CH3) > 10h (2-CH3).
Obviously, the compounds 10a-j of series 2 with 4-ethoxy structure
are generally stronger than the compounds 7a-j series 1 with 4-methoxy
structure in terms of in vitro antiplatelet activity. And in the latter, no
compound can reach or approach the inhibition rate of picotamide.
Since the in vitro antiplatelet activity of series 1 is generally low, the
structure–activity relationship has not been discussed here.
For the compound 10a-j of series 2, their order of in vitro activity of
related compounds is: 10b (N-CH2CH3) > 10c (N-CH(CH3)2) > 10d (N-
Ph) > 10e (N-CH2Ph) > 10a (N-CH3), which indicates that there is no
obvious law of influence of steric hindrance of N-substituents on the
anti-platelet aggregation activity of the compound. When the N-sub-
stituent is phenyl, by continuing to introduce a group at the 2-position of
the phenyl, the effect on the increase of platelet activity is not obvious
enough, but the methyl substitution seems to be more advantageous:
10h (2-CH3) > 10f (2-F) > 10d (2-H) > 10g (2-OCH3). And the order of
activity after connecting the different positions of the corresponding
phenyl group to the methyl group is: 10j (4-CH3) > 10i (3-CH3) > 10h
(2-CH3), that is para- position > meta- position > ortho- position.
3. According to the inhibition rate illustrated in Fig. 4 of collagen
channel, the in vitro antiplatelet activities of five compounds 7a, 7c, 7i,
10e and 10f are obviously higher than that of two control drugs and
2. For AA-induced platelet aggregation, based on the data of Table 1
and Fig. 3, three compounds 7e, 7f and 10j showed higher inhibitory
activity on platelet aggregation than both picotamide and aspirin. The
order of the corresponding inhibition rate of five main compounds and
two positive control drugs at a concentration of 1.3 μM is: 10j (42.98%)
> 7f (40.64%) > 7e (38.46%) > aspirin (38.45%) > picotamide
(37.08%) > 7h (36.88%) > 10i (34.84%). Their IC50 value calculated in
descending order is: 7f (0.48
7h (0.36
μ
М) > aspirin (0.43
μ
М), 10i (0.43
μ
М) >
μ
М) > picotamide (0.34
μ
М) > 7e (0.33
μ
М) > 10j (0.26
μ
М).
There are two compounds 7e and 10j with IC50 values lower than or
equivalent to that of picotamide. It has shown that compound 10j, that is
N1,N3-di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-(p-tolyl)piperazine),
which was prepared by introducing a 4-methylphenyl group at the 4-
other fifteen compounds at a concentration of 1.3 μM. The order of in-
hibition rate of the main compounds is: 7i (54.24%) > 7a (52.63%) >
10f (50.28%) > 10e (49.76%) > 7c (49.42%) > picotamide (49.17%) >
Fig. 3. The inhibition rate and IC50 of potent target compounds for AA.
4

X.-j. Liu et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116390
well and incubated for a further 30 min, and the absorbance at 450 nm
was measured on a microplate reader (Bio-TekFLx800 fluorescence
microplate reader). Table 2 gives relative cell viability (%) relative to
control wells at two concentrations tested.
The cell viability (%) is calculated by the following formula:
Cell viability(%) = Abs (test cell)/Abs (controlled cell) × 100%;
As it appears in the various pictures of Fig. S1, if a test compound or a
control drug has less toxic, the cells will be shuttle-shaped and adhere
well, and on the contrary, the cells will be round and no longer adhere to
the wall. Fig. 6 shows a comparison chart of in vitro cell viability tested
at two concentrations: the darker is at 10
M. When the test dose is 10
pounds 7a, 7e, 7f, 7i, 10e, 10f and 10i are comparable or lower than
that of picotamide, but when the test dose is 100 M, only the cell
μ
M, and the lighter is at 100
μ
μM, the cytotoxicity of seven test com-
μ
survival rate of two compounds 7f and 7i are higher than that of pico-
tamide. This means that at higher dose, compounds except 7f and 7i
showed higher cytotoxicity than that of picotamide. It is found that
compound 7i, that is N1,N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis
(1-(m-tolyl)piperazine), has lowest in vitro cytotoxicity among both
tested compounds and picotamide (see Fig. 5).
Fig. 4. The inhibition rate of potent target compounds for collagen.
aspirin (49.12%) > 10i (48.79%). From a structural point of view,
compound 7i, that is N1, N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis
(1-(m-tolyl)piperazine), in which introducing a 3-methylphenyl group at
the 4-position N of the two side chain piperazine rings, respectively, has
the highest in vitro antiplatelet activity. Further analysis the data of
antiplatelet activity, the preliminary speculations on the structur-
e–activity relationship as follows:
2.3. Docking studies
The crystal structure of the P2Y12 receptor is obtained from the RCSB
protein database (PDB code 4PXZ). The 3D structures of the two target
compounds 7i and 10i are drawn in mol2 format by ChemBio 3D.
Docking research with SYBYL, and we put the results of molecular
docking in Fig 6. The “prepare protein structure” tool in the SURFLEX-
DOCK module is used to repair the structure of P2Y12 receptor 4PXZ,
which is mainly divided into fellowing steps: Protein structure with
excess water molecules deleted is used for docking simulations; Extract
the ligand 2MeSADP to generate the interface bag; Add Essential
hydrogen to saturate terminal amino and carboxyl groups to complete
the reparation side chain amino acid residue; AMBER FF99 charge is
used to describe the electrostatic characteristics. For small molecule
compounds, the Tripos forcefield is used to optimize them, and the
Gasteiger-Huckel charge is used to calculate the electrostatic potential.
The energy convergence value and the maximum number of iterations in
the optimization process are 0.005 kcal/(mol⋅Å) and 1,000.
Regarding the effect of the substituents on piperidine N-4 on the in
vitro activity of the compound, for the compounds of series 1, the an-
tiplatelet aggregation activity of the compound with an aliphatic sub-
stituent is often higher than that of an aromatic substituent, and the
order of in vitro activity of related compounds is: 7a (N-CH3) > 7c (N-CH
(CH3)2) > 7b (N-CH2CH3) > 7e (N-CH2Ph) > 7d (N-Ph). And when the
N-substituent is phenyl, the introduction of a substituent at the 2-posi-
tion of the phenyl group often could increase the activity: 7h (2-CH3)
> 7f (2-F) > 7g (2-OCH3) > 7d (2-H).
For the compound of series 2 where the parent benzene ring is
connected to the ethoxy group at the 4-position, basis on the data in
Table 1, the order of in vitro activity of related compounds is: 10e (N-
CH2Ph) > 10b (N-CH2CH3) > 10c (N-CH(CH3)2) > 10a (N-CH3) > 10d
(N-Ph). It is shown that the steric hindrance of N-substituents has no
obvious effect on antiplatelet aggregation activity. When the N-substit-
uent is phenyl, continue to introduce a substituent on this phenyl, such
as methyl or fluorine at the 2-position, could increase the anti-platelet
aggregation activity: 10f (2-F) > 10h (2-CH3) > 10d (2-H) > 10g (2-
OCH3). And when the side chain phenyls’ substituent is methyl, the
order of activity when the methyl group is attached to different positions
of the benzene ring, the order of in vitro activity is: 10i (3-CH3) > 10h
(2-CH3) > 10j (4-CH3).
In order to determine if the target compound can interact with the
P2Y12 receptor site on the platelet membrane, due to compound N1,N3-di
(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-(m-tolyl)-piperazine), which is
delivery activity in the three test channels, and compound N1,N3–di(4-
Table 2
Cell survival rate of target compounds in vitro cytotoxicity on L929.
It was found in this study that compound 10i, 10j and 7i have the
highest in vitro platelet aggregation inhibition rate in a test channel of
ADP, AA or collagen in turn, respectively.
Compd.
Dose (μM)
Average Absorbance
Survival rate (%)
Blank
–
0.121
0.130
0.126
0.125
0.128
0.124
0.126
0.123
0.128
0.127
0.129
0.127
0.126
0.123
0.127
0.121
0.126
0.124
0.126
0.122
–
Control
Picotamide
–
–
10
55.56 ± 5.56
44.44 ± 6.79
77.78 ± 4.49
33.33 ± 3.33
55.56 ± 3.17
22.22 ± 2.22
77.78 ± 4.48
66.67 ± 6.67
85.18 ± 4.91
66.67 ± 3.84
55.56 ± 7.86
22.22 ± 4.44
70.37 ± 4.06
3.70 ± 0.74
56.56 ± 3.21
33.33 ± 3.33
51.82 ± 5.96
7.40 ± 0.74
2.2.2. Cytotoxicity test
100
10
Eight compounds 7a, 7e, 7f, 7i, 10e, 10f, 10i and 10j with lower or
close to the IC50 value of positive control drugs aspirin and picotamide in
one or two test channels of ADP, AA or collagen, are subjected to
cytotoxicity in vitro test on Mouse fibroblast cells (L929) by CCK-8
method.24,25
7a
100
10
7e
100
10
7f
100
10
Experiment is mainly divided into the following steps: L929 cells are
seeded in 96-well U-bottom plates at a density of 10,000 cells per well
and incubated at 37 ◦C for 24 h in culture medium. The cells with 100
mL of RPMI-1640 per well were allowed to attach for 24 h. Subse-
quently, the cells were then exposed to target compounds at a range of
concentrations at 37 ◦C for 48 h. Target compounds concentration of 10
7i
100
10
10e
10f
10i
10j
100
10
100
10
100
10
and 100
removed and replaced with 100
1640. Then, CCK-8 solution was added to the 96-well plates at 10
μ
M were added to L929 cells. After that, the medium was
M of fresh complete medium of RPMI-
M per
μ
100
μ
5

X.-j. Liu et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116390
Fig. 5. Cytotoxicity in vitro effect on L929 cells.
Fig. 6. a) Docking results for compound 7i. b) Docking results for compound 10i.
methoxy-1,3-phenylenedisulfonyl)bis(1-(m-tolyl)piperazine), which is
the lowest cytotoxic in vitro compound among series 1 and series 2,
respectively, both of them are selected to be docked with the receptor
P2Y12. The results of molecular docking show that the docking between
the two compounds and P2Y12 receptor is not ideal. Among them, the
docking score of the compound 10i is below one, and the docking score
of the compound 7i is even below zero. Compared with the previous
design compounds with a docking score of more than 5 point, the use of
piperazine groups instead of phenyl groups to introduce into the amide
structural analogues of picotamide obviously does not bind to the P2Y12
receptor. The carbon chain length does not change much, but the dif-
ference in aromaticity is obvious. The more precise reason that causes
the decrease in the binding degree of the target compounds to the P2Y12
receptor needs to be further studied and concluded.
Table 3
In silico prediction of molecular properties for new design compounds.
Code
Rule
MW
MiLogP
HBA
HBD
n
TPSA
<160
Å2
Volume
<500
≤5
≤10
≤5
violation
≤1
7a
432.57
460.62
488.68
556.71
584.76
592.69
616.76
584.76
584.76
584.76
446.60
474.65
502.70
570.74
598.79
606.72
630.79
589.79
589.79
589.79
526.64
0.33
1.08
1.68
3.73
3.13
3.96
3.74
4.53
4.58
4.62
0.71
1.46
2.05
4.10
3.50
4.33
4.12
4.90
4.95
5.00
8.07
9
9
9
9
9
9
11
9
9
9
9
9
9
9
9
9
11
9
9
9
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
1
1
1
2
1
1
1
0
0
1
1
1
1
2
1
1
1
2
90.47
90.47
90.47
90.47
90.47
90.47
108.94
90.47
90.47
90.47
90.47
90.47
90.47
90.47
90.47
90.47
108.94
90.47
90.47
90.47
67.43
369.44
403.04
436.22
479.14
512.74
489.00
530.23
512.26
512.26
512.26
386.24
419.85
453.02
495.94
529.54
505.80
547.03
529.06
529.06
529.06
491.60
7b
7c
7d
7e
7f
7g
7h
7i
7j
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
PN 113
2.3.1. In silico prediction of molecular properties
To predict if a chemical compound could be used orally, all newly
designed compounds and previous compound PN 113 (see Fig. 7) with
outstanding anti-platelet activity are analyzed according to Lipinski’s
“rule of five” using Molinspiration property engine v2018.10 (www.
molinspiration.com) online calculate properties.26 Table 3 gives the
results in silico prediction of molecular property.
As shown in Table 3, PN 113 is violated the Lipinski rule of five in
two places, and does not meet the requirements of the Lipinski rule of
five. With the introduction of piperazine in the side chain, the MiLogP
value of the newly designed target compounds is all less than 5, so that
target compounds except 7g and 10g only one violates or fully complies
with the Lipinski rule of five. The drug-likeness of the target compounds
is improved, it provides new vitality for the design of new antiplatelet
drugs.
2.3.2. In silico ADME prediction
Favorable ADME has been identified as the major cause of success for
Fig. 7. Structure of PN 113.
6

X.-j. Liu et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116390
new drug molecules development.27 Thus, with the objective of
increasing the success rate of newly designed compounds reaching
development, in silico ADMET prediction studies are performed using
PreADMET online server (http://www.bmdrc.org). Table 4 gives the in
silico ADME prediction results of eight compounds with better activities
in ADP, AA or collagen test channel, and in silico molecular property
prediction results of other twelve target compounds are given in
Table S1.
compound 10i (IC50(ADP): 0.45 μМ, IC50(AA): 0.43 μМ, Inhibition rate
(collagen): 48.79%) exhibits generally stronger in vitro antiplatelet ac-
tivity in the three test channels of ADP, AA and collagen.
Further, eight compounds 7a, 7e, 7f, 7i, 10e, 10f, 10i and 10j
exhibiting the high activity in one or two test channels, were subjected
to cytotoxicity test on L929 by CCK-8 method. And the result shows that
among them, the in vitro cytotoxicity of seven compounds 7a, 7e, 7f, 7i,
10e, 10f and 10i showed lower than or similar to the control drug
As shown in Table 4, eight compounds express > 90% HIA values,
which exhibit good permeation of across the membrane. The results of
Caco-2 cell permeability in vitro indicates that all compounds exhibit
moderate permeation. The MDCK cell permeability test in vitro shows
the permeation of eight target compounds < 25 nm/s, indicating low
permeability. The data of in vitro skin permeability shows that all newly
designed compounds exhibit negative permeability values, which in-
dicates that transdermal mode of administration is not the suitable
means to administer all newly designed compounds. In addition, newly
designed compounds are predicted to possess > 90% plasma protein
binding (PPB) except three compounds 7a, 7e and 10e, indicating
decrease excretion and increase half-life.
picotamide at 10 μM, while at the higher concentration of 100 μM,
merely two compounds 7f and 7i exhibited higher cell survival rate than
that of picotamide. And compound 7i has lowest in vitro cytotoxicity at
two test concentrations.
In addition, two compounds 7i and 10i, as representatives of series 1
and series 2, respectively, are selected for simulation analysis as two
most likely to dock with the receptor P2Y12. In silico molecular property
and ADME (absorption, distribution, metabolism and excretion) of all
synthesized compounds were predicted by using Molinspiration prop-
erty engine v2018.10 and PreADMET online servers, respectively.
Compared with other series of compounds obtained by introducing
the phenyl group in the previous stage, the water solubility of the
compound synthesized by introducing the piperazine group in this study
has been improved, so that the solvent that can be used for recrystalli-
zation is increased. Compared with previous work, the compounds of
two series 1 and series 2 of obtained after the introduction of piperazinyl
almost have the same proportions in vitro activity higher than control
drug. Most target compounds, including compound 7i and 10i, pre-
sented satisfactory drug-like characteristics and ADME properties. In
silico prediction data of molecular properties shown that the MiLogP of
newly synthesized compounds by introducing piperazine groups is
indeed significantly improved. The above conclusions are limited to the
scope and results of this research. More accurate conclusions might
obtain after more extensive and in-depth research.
In general, most newly designed compounds including compound 7i
and 10i present satisfactory drug-like characteristics and ADME
properties.
3. Conclusion
In order to discover antiplatelet drugs with novel structures and
expand our research scope, using anisole and phenylethyl ether as
starting materials, respectively, through chlorosulfonation and amina-
tion reaction, total twenty target compounds, including ten 4-methoxy-
1,3-benzenedisulfonyl bis(1-hydrocarbylpiperazines) 7a-j of series 1
and ten 4-ethoxy-1,3-benzenedisulfonyl bis(1-hydrocarbyl piperazines)
10a-j of series 2, are synthesized. And their chemical structure has been
confirmed by 1H NMR, 13C NMR, IR and ESI-MS.
4. Experiments
With aspirin and picotamide as positive control drugs, and with ADP,
AA or collagen as an inducer, respectively, the Born turbidimetric
method was used to screen the anti-platelet activity in vitro of all target
4.1. Chemistry
compounds 7a-j and 10a-j at a concentration of 1.3
μM. It was found in
All chemical reagents were purchased from Aladdin Industrial Cor-
poration (China), Tianjin Heng shan (China) and Energy Chemical
(China). And all chemical reagents used without further purification.
The biological reagents arachidonic acid (AA) and adenosine diphos-
phate (ADP) were purchased from Sigma and reagents of cell viability
were purchased from Beyotime Biotechnology (China). The melting
points were determined with a Kofler micro melting point apparatus
(uncorrected when used). Nuclear magnetic resonance (1H/13C NMR)
spectra were recorded on a Bruker 400 MHz spectrometers (Bruker,
Rheinstetten, Germany), TMS (0.05% v/v) as internal standard, pick
positions are illustrated in parts per million (d) in DMSO‑d6 solution and
coupling constant values (J) are given in Hertz. Signal multiplicities are
this study that compound 10i, 10j and 7i have the highest in vitro
platelet aggregation inhibition rate in a test channel of ADP, AA or
collagen in turn, respectively. And of which, the activity in vitro of five
compounds 7a, 7c, 7i, 10e and 10f for collagen was higher than both
picotamide and aspirin. In the ADP and AA test channels, compounds
with an inhibition rate greater than 33% were selected, and corre-
sponding IC50 values were obtained by calculation. According to the
results of IC50 value, the in vitro antiplatelet activity of one compound
10i for ADP was higher than picotamide. And for AA, two compounds 7e
and 10j were higher than both picotamide and aspirin, and two com-
pounds 7h and 10i only higher or equivalent than aspirin. In general,
Table 4
In silico ADME prediction for the title compounds.
Code
Absorption
Distribution
Rule
Human intestinal
absorption (%)
0–20 (poor)
20–70 (moderate)
70–100 (well)
99.29
In vitro Caco-2 cell
permeability (nm/s)
<4 (low)
4–70 (moderate)
>70 (high)
21.67
In vitro MDCK cell
In vitro skin permeability (log K p, cm/h)
In vitro PPB (%)
permeability (nm/s)
<25 (low)
>90 (strongly bound)
<90 (weakly bound)
25–500 (moderate)
>500 (high)
0.50
7a
ꢀ 3.91
ꢀ 1.81
ꢀ 2.37
ꢀ 1.89
ꢀ 1.72
ꢀ 2.32
ꢀ 1.80
ꢀ 1.77
ꢀ 1.72
16.99
7e
97.86
21.71
0.11
72.10
7f
97.86
21.70
0.04
100.00
100.00
70.87
7i
97.79
21.70
0.04
10e
10f
10i
10j
PN113
97.82
21.71
0.08
97.81
21.70
0.04
100.00
100.00
100.00
91.93
97.77
21.71
0.04
97.77
21.71
0.04
96.41
34.45
0.04
7

X.-j. Liu et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116390
reported by: s (singlet), d (doublet), t (triplet), q (quadruplet) and m
(multiplet). Thin-layer chromatography (TLC) was performed with
Merck silica gel plates and visualized with UV irradiation (254 nm).
High-resolution mass spectra (HR-MS) were recorded on an Agilent
6520B UPLC-Q-TOF mass spectrometer (Agilent Technologies, Santa
Clara, CA, USA). The IR spectra were taken by a PerkinElmer 843
spectrometer (KBr as diluent).
hydroxide solution and 30 mL water, respectively to obtain the crude
product. The crude product was recrystallized from ethanol to obtain
white solid powder (760 mg).
4.6.1. N1,N3-di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-
methylpiperazine) (7a)
Following the general procedure, compound 7a was synthesized
from 600 mg (6.0 mmol) of N-methylpiperazine, 1.0 g (3.0 mmol) in-
termediate (6). It was generated as white solid powder (760 mg). Yield
= 59%; m.p: 189–190 ℃ (Ethanol); IR (KBr, cmꢀ 1): 2937 (CH), 2793,
4.2. Synthesis of bis(2-chloroethyl) amine hydrochloride (2)
–
–
2361, 1591, 1488 (C C), 1457, 1395, 1348, 1290 (SO ), 1168, 1146
The corresponding (1) diethanolamine (5.3 g, 0.050 mol) and 25 mL
dichloromethane were added into a 250 mL three-necked flask with a
mechanical stirring bar, and a solution of thionyl chloride (24 g, 0.20
mol) in 20 mL dichloromethane (CH2Cl2) was added slowly at room
temperature. After the reaction mixture was stirred at room temperature
for 3 h, the solvent and excess thionyl chloride was concentrated under
vacuum to obtain crude product (pale yellow solid). Then the crude
product was washed with acetone to obtain white flake crystals (5.4 g),
which could be used directly in the next step without further purifica-
tion. Yield: 60%.
2
(SO2), 1066, 1012, 944, 733, 605, 557; 1H NMR (400 MHz, CDCl3) δ
8.27 (d, J = 2.3 Hz, 1H, 2-H), 7.90 (dd, J = 8.7, 2.3 Hz, 1H, 6-H), 7.11 (d,
J = 8.8 Hz, 1H, 5-H), 3.99 (s, 3H, OCH3), 3.30 (s, 4H, piperazine-H),
3.04 (s, 4H, piperazine-H), 2.48 (s, 8H, piperazine-H), 2.31 (s, 3H, N-
CH3), 2.27 (s, 3H, N-CH3); 13C NMR (101 MHz, CDCl3) δ 160.1 (4-C),
134.3, 131.6, 127.9, 127.8, 112.5, 56.7 (O-CH3), 54.8, 54.0, 46.0, 45.9
(N-CH3), 45.8 (N-CH3); HR ESI-MS (m/z): 433.1501 [M+H]+ found:
433.1579.
4.6.2. N1,N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-
ethylpiperazine) (7b)
4.3. Synthesis of N-(methyl-substituted phenyl) piperazine (4h-j)
Following the general procedure, compound 7b was synthesized
from 750 mg (6.0 mmol) of N-ethylpiperazine, 1.0 g (3.0 mmol) inter-
mediate (6). It was generated as white solid powder (630 mg). Yield =
46%; m.p: 181–182 ℃ (Ethanol); IR (KBr, cmꢀ 1): 2967, 2929 (CH),
The corresponding bis(2-chloroethyl) amine hydrochloride (3.0 g,
0.017 mol) and 50 mL n-butanol were added into a 250 mL three-necked
flask, and the reaction was allowed to raise to 130 ◦C. After the solid was
completely dissolved, a mixture of o-methylaniline (1.6 g, 0.015 mol)
was dissolved in 25 mL tert-butanol was added. This reaction mixture
was refluxed at 130 ◦C for 24 h. After the reaction was complete,
anhydrous sodium carbonate (1.6 g, 0.015 mol) was added to the re-
action system in batches. After TLC (developing solvent: petroleum
ether:ethyl acetate = 1:2) showed the end point of the reaction, cool
overnight, and white solid precipitates out. The obtained white solid was
dissolved in water (25 mL), treated with 5% sodium hydroxide solution
until the pH > 12, and the aqueous layer was extracted with ethyl ac-
etate (3 × 20 mL). Combine each of the organic phases and wash with
saturated saline solution. After drying with anhydrous sodium sulfate,
the reaction solution was filtered out, and concentrated under the
reduced pressure to obtain brown–red oil (830 mg). Yield: 31%.
Other two intermediates 4i and 4jwere prepared in the same manner.
–
2807, 2362, 1591, 1486 (C C), 1349, 1327 (SO ), 1289, 1260, 1168
–
2
(SO2), 1062, 1012, 949, 845, 738, 603, 556, 524; 1H NMR (400 MHz,
CDCl3) δ 8.28 (d, J = 2.2 Hz, 1H, 2-H), 7.90 (dd, J = 8.7, 2.2 Hz, 1H, 6-
H), 7.10 (d, J = 8.7 Hz, 1H, 5-H), 3.99 (s, 3H, OCH3), 3.31 (s, 4H,
piperazine-H), 3.05 (s, 4H, piperazine-H), 2.52 (s, 8H, piperazine-H),
2.45 (m, 2H, N-CH2CH3), 2.43–2.37 (m, 2H, N-CH2CH3), 1.08 (t, J =
7.2 Hz, 3H, N-CH2CH3), 1.03 (t, J = 7.2 Hz, 3H, N-CH2CH3); 13C NMR
(101 MHz, CDCl3) δ 160.1 (4-C), 134.3, 131.7, 127.6, 112.5, 56.7
(OCH3), 52.5, 52.2, 52.0 (N-CH2CH3), 51.8 (N-CH2CH3), 46.2, 46.1,
12.0 (N-CH2CH3), 12.0 (N-CH2CH3); HR ESI-MS (m/z): 461.1908
[M+H]+ found: 461.1892.
4.6.3. N1,N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-
isopropylpiperazine) (7c)
Following the general procedure, compound 7c was synthesized
from 840 mg (6.0 mmol) of N-isopropylpiperazine, 1.0 g (3.0 mmol)
intermediate (6). It was generated as white solid powder (410 mg).
Yield = 26%; m.p: 166–167 ℃ (Benzene-Petroleum Ether); IR (KBr,
4.4. Synthesis of intermediates 4-methoxybenzene-1,3-disulfonyl chloride
(6)
cmꢀ 1): 2965 (CH), 2814, 1591, 1485 (C C), 1350 (SO ), 1288, 1162
–
–
Intermediates (6) 4-methoxybenzene-1,3-disulfonyl chloride was
(SO2), 1063, 1011, 974, 943, 739, 604, 554; 1H NMR (4020 MHz, CDCl3)
δ 8.27 (d, J = 2.0 Hz, 1H, 2-H), 7.90 (dd, J = 8.7, 2.0 Hz, 1H, 6-H), 7.10
(d, J = 8.7 Hz, 1H, 5-H), 4.00 (s, 3H, OCH3), 3.31 (s, 4H, piperazine-H),
3.03 (s, 4H, piperazine-H), 2.74–2.65 (m, 2H, N-CH(CH3)2), 2.63 (s, 8H,
piperazine-H), 1.05 (d, J = 5.9 Hz, 6H, N-CH(CH3)2), 1.00 (d, J = 6.5 Hz,
6H, N-CH(CH3)2); 13C NMR (101 MHz, CDCl3) δ 160.2 (4-C), 134.2,
131.7, 128.4, 127.7, 112.6, 56.7 (OCH3), 54.7 (N-CH(CH3)2), 54.4 (N-
CH(CH3)2), 48.5, 47.8, 46.5, 46.4, 18.4 (2 × N-CH(CH3)2); HR ESI-MS
(m/z): 489.2127 [M+H]+ found: 489.2238.
synthesized according to the literature in 2015.20
4.5. Synthesis of intermediates 4-ethoxybenzene-1,3-disulfonyl chloride
(9)
Intermediates (9) 4-ethoxy-1,3-benzenedisulfonyl chloride was syn-
thesized according to the literature method in 2019.21
4.6. General procedure for synthesis of target compounds (Take N1,N3-di
(4-methoxy-1,3–phenylene-disulfonyl)bis(1-methylpiperazine) (7a) as an
example)
4.6.4. N1,N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-
phenylpiperazine) (7d)
Following the general procedure, compound 7d was synthesized
from 530 mg (3.3 mmol) of N-Phenylpiperazine, 500 mg (1.6 mmol)
intermediate (6). It was generated as white solid powder (490 mg).
Yield = 55%; m.p: 297–298 ℃ (N, N-Dimethylformamide-Water); IR
N-methylpiperazine (600 mg 6.0 mmol) was placed in a 100 mL
round bottom flask and 20 mL CH2Cl2 was added to dissolved it. Under
ice bath condition, slowly added dropwise a mixed solution of 4-
methoxy-1,3-benzenedisulfonyl chloride (1.0 g, 3.0 mmol) in 10 mL
CH2Cl2. During the reaction, 3–4 drops of triethylamine was added as an
acid binding agent. The reaction process and end point were monitored
by TLC (developing solvent: petroleum ether: ethyl acetate = 1:1) at
room temperature. Concentrate the reaction solution and evaporate the
solvent under reduced pressure. And wash it with 10 mL 5% sodium
(KBr, cmꢀ 1): 2924, 2854 (CH), 2362, 2336, 1594, 1489 (C C), 1455,
–
–
1393, 1342 (SO2), 1288, 1235, 1161, (SO2), 948, 740, 556; 1H NMR
(400 MHz, DMSO) δ 8.04 (d, J = 9.0 Hz, 2H, Ar-H), 7.52 (d, J = 9.1 Hz,
1H, Ar-H), 7.19 (t, J = 7.3 Hz, 4H, Ar-H), 6.90 (d, J = 7.0 Hz, 4H, Ar-H),
6.79 (t, J = 6.8 Hz, 2H, Ar-H), 4.02 (s, 3H, OCH3), 3.29 (s, 4H,
8

X.-j. Liu et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116390
piperazine-H), 3.21 (s, 4H, piperazine-H), 3.15 (s, 4H, piperazine-H),
3.04 (s, 4H, piperazine-H); 13C NMR (101 MHz, CDCl3) δ 159.6 (4-C),
150.5, 134.2, 131.6, 129.2, 129.2, 127.7, 127.3, 120.9, 116.9, 116.9,
113.4, 65.7 (OCH3), 49.8, 49.0, 46.0, 14.5; HR ESI-MS (m/z): 557.1892
[M+H]+ found: 557.1893.
600, 554; 1H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 1.9 Hz, 1H, 2-H),
8.00 (dd, J = 8.7, 1.9 Hz, 1H, 6-H), 7.25 (d, J = 8.8 Hz, 1H, 5-H),
7.21–7.15 (m, 4H, Ar-H), 7.02 (dd, J = 9.8, 7.7 Hz, 4H, Ar-H), 4.09 (s,
3H, OCH3), 3.46 (s, 4H, piperazine-H), 3.19 (s, 4H, piperazine-H),
3.04–2.92 (m, 8H, piperazine-H), 2.27 (s, 3H, Ph-CH3), 2.21 (s, 3H,
Ph-CH3); 13C NMR (101 MHz, CDCl3) δ 160.2 (4-C), 150.8 (1-C), 150.6
(3-C), 134.3, 132.8, 132.7, 131.6, 131.3, 128.2, 128.1, 126.9, 126.8,
124.1, 119.5, 119.4, 112.8, 56.8 (OCH3), 51.9, 51.2, 46.8, 46.6, 17.9 (2
× Ph-CH3); HR ESI-MS (m/z): 585.2196 [M+H]+ found: 585.2205.
4.6.5. N1,N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-
benzylpiperazine) (7e)
Following the general procedure, compound 7e was synthesized
from 580 mg (3.3 mmol) of N-benzylpiperazine, 500 mg (1.6 mmol)
intermediate (6). It was generated as white solid powder (540 mg).
Yield = 58%; m.p: 210–212 ℃ (Acetone); IR (KBr, cmꢀ 1): 2923 (CH),
4.6.9. N1,N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-(m-tolyl)
piperazine) (7i)
–
2361, 1646, 1588 (C C), 1458, 1396, 1357 (SO ), 1286, 1165 (SO ),
Following the general procedure, compound 7i was synthesized from
600 mg (3.4 mmol) of N-(3-methylphenyl)piperazine, 510 mg (1.7
mmol) intermediate (6). It was generated as white solid powder (500
mg). Yield = 50%; m.p: 284–285 ℃ (N, N-Dimethylformamide-Water);
–
2
1069, 941, 848, 735, 698, 600, 554; 1H NMR (4020 MHz, CDCl3) δ 8.26
(d, J = 2.2 Hz, 1H, 2-H), 7.90 (dd, J = 8.7, 2.2 Hz, 1H, 6-H), 7.28 (m,
10H, Ar-H), 7.11 (d, J = 8.7 Hz, 1H, 5-H), 4.00 (s, 3H, OCH3), 3.54 (s,
2H, N-CH2-), 3.49 (s, 2H, N-CH2-), 3.31 (s, 4H, piperazine-H), 3.03 (s,
4H, piperazine-H), 2.53 (s, 8H, piperazine-H); 13C NMR (101 MHz,
CDCl3) δ 160.1 (4-C), 137.5, 137.4, 134.3, 131.6, 129.3, 129.2, 128.5,
128.5, 127.9, 127.8, 127.5, 112.6, 62.8 (N-CH2-), 62.6 (N-CH2-), 56.7
(OCH3), 52.8, 52.1, 46.2, 46.1; HR ESI-MS (m/z): 585.2201 [M+H]+
found: 585.2205.
IR (KBr, cmꢀ 1): 2827 (CH), 2362, 1593 (C C), 1489, 1451, 1391, 1343
–
–
(SO2), 1250, 1161 (SO2), 1065, 1007, 954, 848, 778, 741, 694, 556; 1H
NMR (400 MHz, DMSO) δ 8.04 (d, J = 8.8 Hz, 2H, Ar-H), 7.52 (d, J = 8.5
Hz, 1H, Ar-H), 7.07 (t, J = 7.8 Hz, 2H, Ar-H), 6.75–6.66 (m, 4H, Ar-H),
6.62 (d, J = 6.7 Hz, 2H, Ar-H), 4.02 (s, 3H, OCH3), 3.28 (s, 4H,
piperazine-H), 3.19 (s, 4H, piperazine-H), 3.12 (s, 4H, piperazine-H),
3.03 (s, 4H, piperazine-H), 2.21 (s, 6H, Ph-CH3); 13C NMR (101 MHz,
DMSO) δ 160.5 (4-C), 150.7, 138.5, 130.6, 129.2, 126.7, 120.9, 117.2,
115.0, 113.7, 57.4 (OCH3), 49.0, 48.3, 46.2, 46.0, 21.7 (2 × Ph-CH3);
HR ESI-MS (m/z): 585.2198 [M+H]+ found: 585.2205.
4.6.6. N1,N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-(2-
fluorophenyl)piperazine) (7f)
Following the general procedure, compound 7f was synthesized from
590 mg (3.3 mmol) of N-(2-fluorophenyl)piperazine, 500 mg (1.6 mmol)
intermediate (6). It was generated as white solid powder (620 mg).
4.6.10. N1,N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-(p-tolyl)
piperazine) (7j)
Yield = 65%; m.p: 237–238 ℃ (Acetone); IR (KBr, cmꢀ 1): 3415, 3134
–
(=CH), 1591, 1502 (C C), 1484, 1397, 1351 (SO ), 1335, 1289, 1239,
Following the general procedure, compound 7j was synthesized from
1.6 g (9.3 mmol) of N-(4-methylphenyl)piperazine, 1.4 g (4.7 mmol)
intermediate (6). It was generated as white solid powder (990 mg).
Yield = 36%; m.p: 300–302 ℃ (N, N-Dimethylformamide-Water); IR
–
1158 (SO2), 948, 744, 556; 1H NMR (400 MHz, CD2Cl3) δ 8.36 (d, J = 2.2
Hz, 1H, 2-H), 7.98 (dd, J = 8.7, 2.3 Hz, 1H, 6-H), 7.18 (d, J = 8.8 Hz, 1H,
5-H), 7.11–7.03 (m, 3H, Ar-H), 6.98 (m, 5H, Ar-H), 4.06 (s, 3H, OCH3),
3.53–3.44 (m, 4H, piperazine-H), 3.21 (d, J = 4.6 Hz, 4H, piperazine-H),
3.16 (dd, J = 10.3, 6.1 Hz, 8H, piperazine-H); 13C NMR (101 MHz,
CDCl3) δ 160.2 (4-C), 157.0 (1-C), 154.6 (3-C), 139.2, 134.4, 131.8,
127.9, 127.9, 124.8, 124.8, 124.7, 123.6, 123.6, 119.6, 119.5, 116.5,
116.3, 112.8, 100.1, 56.8 (OCH3), 50.8, 50.0, 46.4, 46.3; HR ESI-MS (m/
z): 593.1700 [M+H]+ found: 593.1704.
(KBr, cmꢀ 1): 3446, 2362, 1591, 1516 (C C), 1484, 1392, 1343 (SO ),
–
–
2
1290, 1237, 1162 (SO2), 948, 741, 601, 541; 1H NMR (400 MHz, DMSO)
δ 8.04 (d, J = 8.1 Hz, 2H, Ar-H), 7.52 (d, J = 8.3 Hz, 1H, Ar-H), 7.00 (d, J
= 8.0 Hz, 4H, Ar-H), 6.80 (d, J = 8.3 Hz, 4H, Ar-H), 4.02 (s, 3H, OCH3),
3.27 (s, 4H, piperazine-H), 3.14 (s, 4H, piperazine-H), 3.07 (s, 4H,
piperazine-H), 3.03 (s, 4H, piperazine-H), 2.17 (s, 3H, Ph-CH3), 2.16 (s,
3H, Ph-CH3); 13C NMR (101 MHz, CDCl3) δ 160.0 (4-C), 150.6 (1-C),
150.4 (3-C), 134.2, 132.6, 132.6, 131.4, 131.1, 128.0, 127.9, 126.7,
126.7, 124.0, 123.9, 119.3, 119.3, 112.7, 56.6 (OCH3), 51.8, 51.0, 46.6,
46.5, 17.7 (2 × Ph-CH3); HR ESI-MS (m/z): 585.2211 [M+H]+ found:
585.2205.
4.6.7. N1,N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-(2-
methoxyphenyl)piperazine) (7g)
Following the general procedure, compound 7g was synthesized
from 880 mg (4.6 mmol) of N-(2-methoxyphenyl)piperazine, 700 mg
(2.3 mmol) intermediate (6). It was generated as white solid powder
(680 mg). Yield = 48%; m.p: 208–209 ℃ (Acetone); IR (KBr, cmꢀ 1):
4.6.11. N1,N3–di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-
methylpiperazine) (10a)
–
3129 (=CH), 2837 (CH), 1589, 1501 (C C), 1395 (SO ), 1242, 1165
–
2
(SO2), 1067, 1023, 948, 844, 744, 554; 1H NMR (400 MHz, CDCl3) δ
8.36 (d, J = 2.1 Hz, 1H, 2-H), 7.96 (dd, J = 8.7, 2.1 Hz, 1H, 6-H), 7.16 (d,
J = 8.8 Hz, 1H, 5-H), 7.08–7.00 (m, 2H, Ar-H), 6.93 (dd, J = 6.6, 3.6 Hz,
4H, Ar-H), 6.86 (t, J = 8.2 Hz, 2H, Ar-H), 4.04 (s, 3H, OCH3), 3.85 (s, 3H,
Ph-OCH3), 3.82 (s, 3H, Ph-OCH3), 3.48 (s, 4H, piperazine-H), 3.22 (s,
4H, piperazine-H), 3.13 (s, 8H, piperazine-H); 13C NMR (101 MHz,
CDCl3) δ 160.3 (4-C), 152.2, 140.5, 140.3, 134.4, 132.0, 127.8, 127.7,
123.9, 121.2, 121.1, 118.7, 118.6, 112.7, 111.3, 56.8 (OCH3), 55.5 (2 ×
Ph-OCH3), 50.8, 50.0, 46.5, 46.4; HR ESI-MS (m/z): 617.2104 [M+H]+
found: 617.2104.
Following the general procedure, compound 10a was synthesized
from 5.4 g (5.4 mmol) of N-methylpiperazine, 860 mg (2.7 mmol) in-
termediate (9). It was generated as white solid powder (570 mg). Yield
= 48%; m.p: 147–148 ℃ (Petroleum ether-Ethyl aceta); IR (KBr, cmꢀ 1):
–
2982, 2939 (CH), 2850, 2798, 1588 (C C), 1456, 1394, 1356, 1333,
–
1290 (SO2), 1172 (SO2), 1068, 943, 842, 787, 746, 603, 567, 520; 1
H
NMR (400 MHz, CDCl3) δ 8.26 (d, J = 2.3 Hz, 1H, 2-H), 7.86 (dd, J = 8.7,
2.3 Hz, 1H, 6-H), 7.08 (d, J = 8.8 Hz, 1H, 5-H), 4.22 (q, J = 7.0 Hz, 2H,
OCH2CH3), 3.31 (s, 4H, piperazine-H), 3.03 (s, 4H, piperazine-H), 2.47
(d, J = 4.6 Hz, 4H, piperazine-H), 2.45 (d, J = 4.7 Hz, 4H, piperazine-H),
2.30 (s, 3H, N-CH3), 2.26 (s, 3H, N-CH3), 1.52 (t, J = 7.0 Hz, 3H,
OCH2CH3); 13C NMR (101 MHz, CDCl3) δ 159.6 (4-C), 134.2, 131.7,
127.8, 127.4, 113.3, 65.8 (OCH2CH3), 54.8 (N-CH3), 54.0 (N-CH3), 46.1,
46.0, 46.0, 45.8, 14.6 (OCH2CH3); HR ESI-MS (m/z): 447.1758 [M+H]+
found: 447.1736.
4.6.8. N1,N3–di(4-methoxy-1,3-phenylenedisulfonyl)bis(1-(o-tolyl)
piperazine) (7h)
Following the general procedure, compound 7h was synthesized
from 550 mg (3.1 mmol) of N-(2-methylphenyl)piperazine, 470 mg (1.6
mmol) intermediate (6). It was generated as white solid powder (230
mg). Yield = 26%; m.p: 227–229 ℃ (N, N-Dimethylformamide-Water);
4.6.12. N1,N3–di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-ethylpiperazine)
(10b)
IR (KBr, cmꢀ 1): 3446, 2950, 2850 (CH), 1592 (C C), 1489, 1448, 1347
–
–
(SO2), 1285, 1260, 1226, 1162 (SO2), 1067, 1012, 951, 845, 767, 735,
Following the general procedure, compound 10b was synthesized
9

X.-j. Liu et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116390
from 360 mg (3.1 mmol) of N-ethylpiperazine, 500 mg (1.6 mmol) in-
termediate (9). It was generated as white solid powder (260 mg). Yield
= 35%; m.p: 170–171 ℃ (Ethanol); IR (KBr, cmꢀ 1): 3131 (=CH), 2811
4.6.16. N1,N3–di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-(2-
fluorophenyl)piperazine) (10f)
Following the general procedure, compound 10f was synthesized
from 560 mg (3.1 mmol) of N-(2-fluorophenyl)piperazine, 500 mg (1.6
mmol) intermediate (9). It was generated as white solid powder (420
–
(CH), 1587 (C C), 1401 (SO ), 1350, 1283, 1176, 1150 (SO ), 1107,
–
1062, 1032, 960, 840, 743, 598, 569, 520; 1H NMR (400 MHz,2CDCl3) δ
8.24 (d, J = 3.6 Hz, 1H, 2-H), 7.85 (dd, J = 9.2, 7.0 Hz, 1H, 6-H), 7.07 (d,
J = 8.7 Hz, 1H, 5-H), 4.19 (dd, J = 14.4, 7.5 Hz, 2H, OCH2CH3), 3.30 (s,
4H, piperazine-H), 3.02 (s, 4H, piperazine-H), 2.49 (s, 8H, piperazine-
H), 2.40 (dt, J = 14.1, 7.3 Hz, 4H, N-CH2CH3), 1.56–1.46 (m, 3H,
OCH2CH3), 1.10–0.95 (m, 6H, N-CH2CH3); 13C NMR (101 MHz, CDCl3) δ
159.7 (4-C), 134.2, 131.8, 127.7, 127.3, 113.3, 65.7 (OCH2CH3), 52.6,
52.2, 51.9, 51.8, 46.2 (2 × N-CH2CH3), 14.6 (O-CH2CH3), 12.0 (N-
CH2CH3); HR ESI-MS (m/z): 475.2068 [M+H]+ found: 475.2049.
2
mg). Yield = 44%; m.p: 178–179 ℃ (Ethanol); IR (KBr, cmꢀ 1): 3132
–
(=CH), 1616, 1589 (C C), 1499, 1401 (SO ), 1287, 1239, 1164 (SO ),
–
949, 732, 600, 571; 1H NMR (400 MHz, CDC2l3) δ 8.36 (d, J = 2.3 Hz, 12H,
2-H), 7.94 (dd, J = 8.7, 2.4 Hz, 1H, 6-H), 7.15 (d, J = 8.8 Hz, 1H, 5-H),
7.09 (dd, J = 7.2, 2.1 Hz, 1H), 7.07 (d, J = 2.0 Hz, 1H), 7.06–7.04 (m,
1H), 7.03–7.01 (m, 1H), 6.99 (dd, J = 5.0, 1.8 Hz, 2H), 6.97 (dd, J = 2.9,
1.5 Hz, 1H), 6.95–6.91 (m, 1H), 4.29 (q, J = 7.0 Hz, 2H, OCH2CH3),
3.56–3.44 (m, 4H, piperazine-H), 3.21 (d, J = 4.7 Hz, 4H, piperazine-H),
3.16 (dd, J = 10.2, 5.9 Hz, 8H, piperazine-H), 1.57 (t, J = 7.0 Hz, 3H,
OCH2CH3); 13C NMR (101 MHz, CDCl3) δ 159.8 (4-C), 157.0 (1-C), 154.6
(3-C), 139.3, 134.3, 131.8, 127.9, 127.5, 124.8, 124.7, 124.7, 123.6,
119.6, 119.4, 116.5, 116.3, 113.6, 65.9 (OCH2CH3), 50.8, 50.0, 46.4,
14.7 (OCH2CH3); HR ESI-MS (m/z): 607.1873 [M+H]+ found:
607.1860.
4.6.13. N1,N3–di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-
isopropylpiperazine) (10c)
Following the general procedure, compound 10c was synthesized
from 770 mg (6.0 mmol) of N-isopropylpiperazine, 1.0 g (3.0 mmol)
intermediate (9). It was generated as white solid powder (1.2 g). Yield =
77%; m.p: 175–176 ℃ (Ethanol); IR (KBr, cmꢀ 1): 3416, 3132 (=CH),
2968 (CH), 1588 (C C), 1470, 1397 (SO ), 1348, 1288, 1175 (SO ),
4.6.17. N1,N3–di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-(2-
methoxyphenyl)piperazine) (10g)
–
–
2
2
1174, 952, 743, 601, 568; 1H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 2.0
Hz, 1H, 2-H), 7.86 (dd, J = 8.7, 2.0 Hz, 1H, 6-H), 7.07 (d, J = 8.7 Hz, 1H,
5-H), 4.22 (q, J = 6.9 Hz, 2H, OCH2CH3), 3.30 (s, 4H, piperazine-H),
3.02 (s, 4H, piperazine-H), 2.72–2.64 (m, 2H, N-CH(CH3)2), 2.59 (s,
8H, piperazine-H), 1.53 (t, J = 6.9 Hz, 3H, OCH2CH3), 1.04 (d, J = 6.4
Hz, 6H, N-CH(CH3)2), 1.00 (d, J = 6.5 Hz, 6H, N-CH(CH3)2); 13C NMR
(101 MHz, CDCl3) δ 159.7 (4-C), 134.1, 131.8, 127.7, 127.4, 113.3, 65.7
(OCH2CH3), 54.6 (N-CH(CH3)2), 54.4 (N-CH(CH3)2), 48.5, 47.8, 46.5,
18.4 (N-CH(CH3)2), 14.6 (OCH2CH3); HR ESI-MS (m/z): 503.2383
[M+H]+ found: 503.2362.
Following the general procedure, compound 10g was synthesized
from 840 mg (4.4 mmol) of N-(2-methoxyphenyl)piperazine, 700 mg
(2.2 mmol) intermediate (9). It was generated as white solid powder
(550 mg). Yield = 40%; m.p: 208–209 ℃ (Acetone); IR (KBr, cmꢀ 1):
–
3131 (=CH), 1619, 1590 (C C), 1499, 1401 (SO ), 1294, 1243, 1164
–
2
(SO2), 949, 747, 575; 1H NMR (400 MHz, CDCl3) δ 8.36 (d, J = 1.7 Hz,
1H, 2-H), 7.93 (dd, J = 8.6, 1.7 Hz, 1H, 6-H), 7.12 (d, J = 8.7 Hz, 1H, 5-
H), 7.08–6.98 (m, 2H), 6.92 (d, J = 4.8 Hz, 4H), 6.86 (t, J = 8.0 Hz, 2H),
4.26 (q, J = 6.7 Hz, 2H, OCH2CH3), 3.85 (s, 3H, Ph-OCH3), 3.82 (s, 3H,
Ph-OCH3), 3.50 (s, 4H, piperazine-H), 3.22 (s, 4H, piperazine-H), 3.13
(s, 8H, piperazine-H), 1.56 (t, J = 6.9 Hz, 3H, OCH2CH3); 13C NMR (101
MHz, CDCl3) δ 159.9 (4-C), 152.3, 140.6, 140.4, 134.3, 132.0, 127.9,
127.5, 123.8, 121.2, 118.7, 118.6, 113.5, 111.4, 65.9 (OCH2CH3), 55.5
(2 × Ph-OCH3), 50.8, 50.1, 46.6, 46.5, 14.6 (OCH2CH3); HR ESI-MS (m/
z): 631.2279 [M+H]+ found: 631.2260.
4.6.14. N1,N3–di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-
phenylpiperazine) (10d)
Following the general procedure, compound 10d was synthesized
from 1.0 g (6.3 mmol) of N-phenylpiperazine, 1.0 g (3.0 mmol) inter-
mediate (9). It was generated as white solid powder (660 mg). Yield =
37%; m.p: 297–298 ℃ (N, N-Dimethylformamide-Water); IR (KBr,
cmꢀ 1): 3131 (=CH), 1595 (C C), 1401 (SO ), 1234, 1159 (SO ), 952,
4.6.18. N1,N3–di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-(o-tolyl)
piperazine) (10h)
–
–
2
739, 602, 555; 1H NMR (400 MHz, CDCl3) δ 8.36 (d, J = 2.3 Hz2, 1H, 2-
H), 7.94 (dd, J = 8.7, 2.3 Hz, 1H, 6-H), 7.28 (dt, J = 7.2, 5.6 Hz, 4H, Ar-
H), 7.14 (d, J = 8.8 Hz, 1H, 5-H), 6.91 (m, 6H, Ar-H), 4.27 (q, J = 7.0 Hz,
2H, OCH2CH3), 3.47 (s, 4H, piperazine-H), 3.25 (dd, J = 11.8, 6.7 Hz,
8H, piperazine-H), 3.20–3.12 (m, 4H, piperazine-H), 1.56 (t, J = 7.0 Hz,
3H, OCH2CH3); 13C NMR (101 MHz, CDCl3) δ 159.8 (4-C), 150.8 (1-C),
150.6 (3-C), 134.4, 131.7, 129.4, 127.8, 127.5, 121.0, 117.1, 117.1,
113.5, 65.9 (OCH2CH3), 50.0, 49.2, 46.2, 46.1, 14.6 (OCH2CH3); HR
ESI-MS (m/z): 571.2039 [M+H]+ found: 571.2049.
Following the general procedure, compound 10h was synthesized
from 580 mg (3.30 mmol) of N-(2-methylphenyl)piperazine, 500 mg
(1.6 mmol) intermediate (9). It was generated as white solid powder
(360 mg). Yield = 37%; m.p: 174–176 ℃ (Ethanol); IR (KBr, cmꢀ 1):
–
3131 (=CH), 1590, 1491 (C C), 1401, 1352 (SO ), 1293, 1260, 1225,
–
2
1166 (SO2), 948, 757, 575; 1H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 2.1
Hz, 1H, 2-H), 7.97 (dd, J = 8.7, 2.1 Hz, 1H, 6-H), 7.22–7.14 (m, 5H),
7.02 (dd, J = 9.0, 7.6 Hz, 4H), 4.32 (q, J = 6.9 Hz, 2H, OCH2CH3), 3.49
(s, 4H, piperazine-H), 3.18 (s, 4H, piperazine-H), 3.05–2.93 (m, 8H,
piperazine-H), 2.28 (s, 3H, Ph-CH3), 2.21 (s, 3H, Ph-CH3), 1.58 (t, J =
6.9 Hz, 3H, OCH2CH3); 13C NMR (101 MHz, CDCl3) δ 159.7 (4-C), 150.8
(1-C), 150.6 (3-C), 134.3, 132.8, 132.7, 131.7, 131.3, 128.2, 127.9,
126.9, 126.8, 124.1, 124.1, 119.5, 113.5, 65.8 (OCH2CH3), 52.0, 51.2,
46.8, 46.7, 17.9 (2 × Ph-CH3), 14.7 (OCH2CH3); HR ESI-MS (m/z):
599.2369 [M+H]+ found: 599.2362.
4.6.15. N1,N3–di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-
benzylpiperazine) (10e)
Following the general procedure, compound 10e was synthesized
from 660 mg (3.8 mmol) of N-benzylpiperazine, 600 mg (1.9 mmol)
intermediate (9). It was generated as white solid powder (510 mg).
Yield = 45%; m.p: 194–195 ℃ (Methanol); IR (KBr, cmꢀ 1): 3411, 3132
(=CH), 1619, 1401 (SO2), 1166 (SO2), 742, 619, 567, 477; 1H NMR
(400 MHz, CDCl3) δ 8.26 (d, J = 2.1 Hz, 1H, 2-H), 7.86 (dd, J = 8.7, 2.1
Hz, 1H, 6-H), 7.32 (dd, J = 9.9, 6.3 Hz, 6H, Ar-H), 7.24 (d, J = 7.2 Hz,
4H, Ar-H), 7.08 (d, J = 8.7 Hz, 1H, 5-H), 4.23 (q, J = 6.9 Hz, 2H,
OCH2CH3), 3.53 (s, 2H, N-CH2-Ph), 3.49 (s, 2H, N-CH2-Ph), 3.32 (s, 4H,
piperazine-H), 3.02 (s, 4H, piperazine-H), 2.52 (s, 8H, piperazine-H),
1.52 (t, J = 7.0 Hz, 3H, OCH2CH3); 13C NMR (101 MHz, CDCl3) δ
159.7 (4-C), 137.6, 134.2, 131.7, 129.3, 129.1, 128.5, 128.5, 127.9,
127.5, 127.4, 113.3, 65.8 (OCH2CH3), 62.9 (N-CH2-Ph), 62.6 (N-CH2-
Ph), 52.9, 52.1, 46.3, 14.6 (OCH2CH3); HR ESI-MS (m/z): 599.2385
[M+H]+ found: 599.2362.
4.6.19. N1,N3–di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-(m-tolyl)
piperazine) (10i)
Following the general procedure, compound 10i was synthesized
from 550 mg (3.1 mmol) of N-(3-methylphenyl)piperazine, 500 mg (1.6
mmol) intermediate (9). It was generated as white solid powder (440
mg). Yield = 46%; m.p: 219–220 ℃ (N, N-Dimethylformamide-Water);
IR (KBr, cmꢀ 1): 2922, 2848 (CH), 1591 (C C), 1453, 1391, 1343 (SO ),
–
–
2
1247, 1158 (SO2), 1022, 954, 837, 779, 739, 554; 1H NMR (400 MHz,
DMSO) δ 8.06 (d, J = 1.5 Hz, 1H, 2-H), 8.01 (dd, J = 8.7, 1.5 Hz, 1H, 6-
H), 7.50 (d, J = 8.8 Hz, 1H, 5-H), 7.07 (t, J = 7.7 Hz, 2H), 6.74–6.67 (m,
10

X.-j. Liu et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116390
4
5
Fuentes E, Moore-Carrasco R, de Andrade Paes AM, Trostchansky A. Role of Platelet
Activation and Oxidative Stress in the Evolution of Myocardial Infarction.
J Cardiovasc Pharmacol Ther. 2019;24:509–520.
4H), 6.61 (d, J = 7.1 Hz, 2H), 4.31 (q, J = 6.9 Hz, 2H, OCH2CH3), 3.29
(s, 4H, piperazine-H), 3.19 (s, 4H, piperazine-H), 3.13 (s, 4H,
piperazine-H), 3.02 (s, 4H, piperazine-H), 2.21 (s, 6H, 2 × Ph-CH3), 1.41
(t, J = 6.8 Hz, 3H, OCH2CH3); 13C NMR (101 MHz, DMSO) δ 160.0 (4-C),
151.0 (1-C), 150.8 (3-C), 138.6, 135.0, 130.8, 129.3, 126.6, 126.3,
121.0, 117.2, 117.2, 115.6, 113.8, 113.8, 66.1 (OCH2CH3), 49.0, 48.3,
46.3, 46.2, 21.8 (2 × Ph-CH3), 14.7 (OCH2CH3); HR ESI-MS (m/z):
599.2379 [M+H]+ found: 599.2362.
Fan M, Han M, Xia Y, et al. Design and Synthesis of Potent PAR-1 Antagonists Based
on Vorapaxar. Bioorg Med Chem Lett. 2020;30:127046. https://doi.org/10.1016/j.
bmcl.2020.127046.
6
7
Celestini A, Violi F. A review of picotamide in the reduction of cardiovascular events
in diabetic patients. Vasc Health Risk Manag. 2007;3:93–98.
Hennenberg M, Tamalunas A, Wang Y, et al. Inhibition of agonist-induced smooth
muscle contraction by picotamide in the male human lower urinary tract outflow
region. Eur J Pharmacol. 2017;803:39–47.
8
9
Sharma AN, Deyell JS, Sharma SN, Barseghian A. Role of and Recent Evidence for
Antiplatelet Therapy in Prevention of Cardiovascular Disease in Diabetes. Curr
Cardiol Rep. 2019;21. https://doi.org/10.1007/s11886-019-1168-y.
Xiu-jie L, Zhi-hao Z, Qing-song D, Xin C, Chao-qing W. Synthesis, in vitro cytotoxicity
and anti-platelet activity of new 1,3-bentzenedisulfonamides. Med Chem Res. 2019;
28:1864–1872.
4.6.20. N1,N3–di(4-ethoxy-1,3-phenylenedisulfonyl)bis(1-(p-tolyl)
piperazine) (10j)
Following the general procedure, compound 10j was synthesized
from 880 mg (5.0 mmol) of N-(4-methylphenyl)piperazine, 800 mg (2.5
mmol) intermediate (9). It was generated as white solid powder (400
mg). Yield = 27%; m.p: 225–226 ℃ (Acetone); IR (KBr, cmꢀ 1): 2985,
10 Wang Y, Wang X, Chen X, et al. Synthesis and in vitro anti-platelet aggregation
activities of 2-methoxy-5-arylamido-N-(pyridin-3-yl-methyl)benzamides. Arch Pharm
(Weinheim). 2019;352:1800257.
–
2919, 2823 (CH), 1589, 1515 (C C), 1475, 1453, 1392, 1347 (SO ),
–
2
11 Wang CQ, Wang Y, Deng QS, et al. Synthesis of 4-Methoxy-1,3-Benzenediolylhy-
drazones and Evaluation of Their Anti-Platelet Aggregation Activity. Iran J Pharm
Res. 2019;18:1803–1815.
1236. 1160 (SO2), 1061, 952, 814, 737, 601, 549; 1H NMR (400 MHz,
DMSO) δ 8.05 (d, J = 1.5 Hz, 1H, 2-H), 8.00 (dd, J = 9.0, 1.5 Hz, 1H, 6-
H), 7.49 (d, J = 8.8 Hz, 1H, 5-H), 7.00 (d, J = 8.2 Hz, 4H), 6.80 (d, J =
7.7 Hz, 4H), 4.31 (q, J = 6.9 Hz, 2H, OCH2CH3), 3.29 (s, 4H, piperazine-
H), 3.14 (s, 4H, piperazine-H), 3.08 (s, 4H, piperazine-H), 3.02 (s, 4H,
piperazine-H), 2.17 (s, 3H, Ph-CH3), 2.16 (s, 3H, Ph-CH3), 1.41 (t, J =
6.9 Hz, 3H, OCH2CH3); 13C NMR (101 MHz, DMSO) δ 159.9 (4-C), 148.7
(1-C), 148.5 (3-C), 134.9, 130.8, 129.8, 129.1, 126.7, 126.4, 116.8,
116.8, 115.5, 66.0 (OCH2CH3), 49.5, 48.7, 46.2, 46.1, 20.4 (2 × Ph-
CH3), 14.6 (OCH2CH3); HR ESI-MS (m/z): 599.2353 [M+H]+ found:
599.2362.
12 Zhang Z-H, Wang X, Wang C-Q, Liu X-J. Synthesis, In Vitro Antiplatelet Activity of 4-
Ethoxy-isophthalamides. ChemistrySelect. 2020;5:10163–10167.
13 Buccellati C, Sala A, Rossoni G, et al. Pharmacological characterization of 2NTX-99
[4- methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-
benzenedicarboxamide], a potential antiatherothrombotic agent with
antithromboxane and nitric oxide donor activity in platelet and vascular
preparations. J Pharmacol Exp Ther. 2006;317:830–837.
14 Ma W-F, Yang H-K, Hu M-J, et al. One-pot synthesis and antiproliferative activity of
novel 2,4-diaminopyrimidine derivatives bearing piperidine and piperazine moieties.
Eur J Med Chem. 2014;84:127–134.
˘
15 Saglık BN, Osmaniye D, Çevik UA, et al. Synthesis, characterization and carbonic
anhydrase I and II inhibitory evaluation of new sulfonamide derivatives bearing
dithiocarbamate. Eur J Med Chem. 2020;198:112392. https://doi.org/10.1016/j.
ejmech.2020.112392.
Declaration of Competing Interest
16 Liu X, Wang S, Zhang J, et al. Design, synthesis, and activities of novel derivatives of
isophthalamide and benzene-1,3-disulfonamide. Chem Res Chin U. 2006;22:356–359.
17 Liu XJ, Shi XX, Zhong YL, Liu N, Liu K. Design, synthesis and in vitro activities on
anti-platelet aggregation of 4-methoxybenzene-1,3-isophthalamides. Bioorg Med
Chem Lett. 2012;22:6591–6595.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
18 Liu X, Wang Y, Liu L, Chen G. Synthesis and in vitro activities on anti-platelet
aggregation of 4-methoxyisophthalamides. Med Chem Res. 2018;27:1971–1983.
19 Chen G, Wang C, Zhang Z, Liu X. Synthesis and in vitro activities on anti-platelet
aggregation of 4-methoxy-1,3-phthalamidesamides and benzenedisulfonamides. Med
Chem Res. 2019;28:1413–1424.
Acknowledgments
20 Li GA, Wang X, Meng X, et al. Design, synthesis of novel N, N’-bis-(halogenophenyl)-
4-methoxybenzene-1,3-disulfonamides and evaluation of their antiplatelet
aggregation activity. Acta Pharmaceutica Sinica. 2015;50:185–190.
21 Chen X, Liu X, Qiu K, Xu X, Li C, Wang Y. N, N’-disubstitutedphenyl-4-ethoxyl
benzene-1,3-disulfonamides: design, synthesis, and evaluation of anti-platelet
aggregation activity. Med Chem Res. 2019;28:1388–1401.
The authors are grateful to the National Science Foundation of China
(11341014), the Committee of Science and Technology of Tianjin
(15JCZDJC33100) and Shenyang Pharmaceutical University of China
for running platelet aggregation assays.
22 Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal.
Nature. 1962;194:927–929.
Appendix A. Supplementary material
23 Violi F, Ghiselli A, Iuliano L, et al. Inhibition bypicotamide of thromboxane
production in vitro and ex vivo. Eur J Clin Pharmacol. 1988;33:599–602.
24 Abe S, Yamaguchi H, Ochi H, et al. Characteristic biological effects of itraconazole on
L929 fibroblasts and their cell membrane. J Infect Chemother. 2000;6:35–40.
25 Xiong JW, Xiao H, Zhang ZX, Osumi M, Yamaguchi H. An experimental research on
different detection conditions between MTT and CCK-8. Acta Laser Biol Sin. 2007;16:
559–562.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116390.
References
26 Lipinski CA, Lombardo F, Dominy BW, et al. Experimental and computational
approaches to estimate solubility and permeability in drug discovery and
development settings. Adv Drug Deliv Rev. 2001;46:3–26.
1
2
3
Chen P, Ren S, Song H, et al. Synthesis and biological evaluation of BMS-986120 and
its deuterated derivatives as PAR4 antagonists. Bioorg Med Chem. 2019;27:116–124.
Townsend N, Wilson L, Bhatnagar P, et al. Cardiovascular disease in Europe 2016: an
epidemiological update. Eur Heart J. 2016;37:3232–3245.
27 Zhong F, Xing J, Li X, et al. (2018) Artificial intelligence in drug design. Sci China Life
Sci. 2018;61:1191–1204.
Lu Lu, Wu Z-Y, Li X, Han F. State-of-the-art: functional fluorescent probes for
bioimaging and pharmacological research. Acta Pharmacol Sin. 2019;40:717–723.
11