Design, synthesis and evaluation of novel 2-amino-3-(naphth-2-yl)propanoic acid derivatives as potent inhibitors of platelet aggregation

Article abstract of DOI:10.1016/j.ejmech.2016.09.032

Based upon LX2421, a previously identified antiplatelet aggregation agent, a series of novel 2-amino-3-(naphth-2-yl)propanoic acid derivatives were designed, synthesized and evaluated. Among them, compounds LX14 and LX25 were identified as promising antiplatelet aggregation agents. The in?vitro biologic study demonstrated that LX14 can block platelet aggregation induced by four different inducers and displays comparable potency in inhibiting GPIIb/IIIa receptor in comparison with Tirofiban. In addition, LX14 has much lower risk of bleeding than Tirofiban and shows significant antithrombotic activity in?vivo. Taking together, the results indicated that LX14 is a promising GPIIb/IIIa receptor antagonist against platelet aggregation worthy of further evaluation.

Full text of DOI:10.1016/j.ejmech.2016.09.032

Accepted Manuscript
Design, synthesis and evaluation of novel 2-amino-3-(naphth-2-yl)propanoic acid
derivatives as potent inhibitors of platelet aggregation
Zhouling Xie, Benimana Oscar, Lulu Zhao, Xue Ding, Chen Cao, Sen Feng, Han Li,
Chen Pan, Ziyi Bian, Yang Li, Weijun Wang, Yi Kong, Zhiyu Li
PII:
S0223-5234(16)30762-0
10.1016/j.ejmech.2016.09.032
EJMECH 8898
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 May 2016
Revised Date: 4 September 2016
Accepted Date: 9 September 2016
Please cite this article as: Z. Xie, B. Oscar, L. Zhao, X. Ding, C. Cao, S. Feng, H. Li, C. Pan, Z. Bian, Y.
Li, W. Wang, Y. Kong, Z. Li, Design, synthesis and evaluation of novel 2-amino-3-(naphth-2-yl)propanoic
acid derivatives as potent inhibitors of platelet aggregation, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.09.032.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of novel 2-amino-3-(naphth-2-yl)propanoic acid derivatives as
potent inhibitors of platelet aggregation
Zhouling Xiea, Benimana Oscara, Lulu Zhaoa, Xue Dingb, Chen Caoa, Sen Fenga, Han Lia, Chen Pana,
Ziyi Biana, Yang Lia, Weijun Wang a, Yi Kongb**, Zhiyu Lia*
aJiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing
21009, China
bSchool of Life Science & Technology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
210009, P.R.China
E-mail addresses: zhiyuli@cpu.edu.cn (Z. Li), yikong668@163.com (Y. Kong)
Abstract: Based upon LX2421, a previously identified antiplatelet aggregation agent, a series of novel
2-amino-3-(naphth-2-yl)propanoic acid derivatives were designed, synthesized and evaluated. Among
them, compounds LX14 and LX25 were identified as promising antiplatelet aggregation agents. The in
vitro biologic study demonstrated that LX14 can block platelet aggregation induced by four different
inducers and displays comparable potency in inhibiting GPIIb/IIIa receptor in comparison with
Tirofiban. In addition, LX14 has much lower risk of bleeding than Tirofiban and shows significant
antithrombotic activity in vivo. Taking together, the results indicated that LX14 is a promising
GPIIb/IIIa receptor antagonist against platelet aggregation worthy of further evaluation.
Key words: antiplatelet aggregation, GPIIb/IIIa receptor, inhibitor, naphthalene derivatives,
antithrombotic activity, bleeding risk
1. Introduction
Coronary artery thrombosis diseases, such as myocardial infarction, acute coronary syndrome,
and pulmonary embolism, represent the most frequent cause of mortality and morbidity worldwide
[1-3]. Platelets are primary components for the development of thrombotic disorders. They can be
activated by a number of platelet inducers, including Von Willebrand factor, collagens, fibronectin,
vitronectin (VIN), adenosine 5′-diphosphate (ADP), thrombin, platelet activating factor (PAF), and
epinephrine (EPN), which are released to arterial circulation on account of plaque rupture [4, 5].
Activated platelets also can produce activating molecules, such as like thromboxane A2 (TXA2) and
ADP, further causing platelets shape change, secretion and increasing of the expression of GPIIb/IIIa
receptor [6-9].
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After platelet activation, GPIIb/IIIa receptors are exposed and activated to combine with the
Arg-Gly-Asp (RGD) sequence of fibrinogen [10, 11]. One molecule of fibrinogen can bind to several
platelets and one platelet can also combine with multiple fibrinogens [12]. Ultimately, this so called
“cross-linking effect” leads to formation of thrombus which overwhelms the luminal area of the vessel,
and causes various thrombotic diseases. Activation of GPIIb/IIIa receptor is the final common pathway
of platelet aggregation regardless of the initiating inducer molecules, thus blocking the platelet
GPIIb/IIIa receptor could effectively inhibit platelet aggregation (Figure 1) [13, 14].
Currently, three classes of antiplatelet aggregation drugs are frequently used in clinic: 1) aspirin,
which blocks the production of the platelet activating molecule TXA2 by inhibiting the cyclooxygenase
pathway [15]; 2) clopidogrel and prasugrel, which function as antagonists of the cell-surface ADP
receptor P2Y12 [16] and 3) the GPIIb/IIIa receptor antagonists, such as Abciximab, Eptifibatide and
Tirofiban [17]. In clinical application the efficacy of aspirin and P2Y12 receptors is limited by their
restricted spectrum of antiplatelet activity, because platelets can be activated by several other inducers.
This promotes the development of potent inhibitors of platelet aggregation based on the inhibition of
GPIIb/IIIa [18]. Among the GPIIa/IIIb receptor antagonists, Abciximab is a non-competitive antagonist
of GPIIb/IIIa which also interacts with two other intergrins αvβ3 and αMβ2, while Eptifibatide and
Tirofiban mimicking the RGD sequence of fibrinogen are selective and competitive antagonists of
GPIIb/IIIa [19]. The GPIIb/IIIa inhibitors have proven effectiveness in reducing the risk of
periprocedural myocardial infraction (PMI) and urgently target vessel revascularization during
catheterization. However, they also have several limitations, such as the risk of bleeding and the
induction of thrombocytopenia in some patients [20], therefore, novel GPIIb/IIIa inhibitors with
reduced risks are urgently needed.
In our previous efforts to discover novel antiplatelet agents, a series of tryptophan derivatives
were designed and synthesized based on a three mer peptide pENW derived from Agkistrodon acutus
Guenther venom [21, 22]. Among them, LX2421 was the most potent compound which inhibits platelet
aggregation induced by all of the four agonists (ADP, Thrombin, U46619 and Collagen). It has an IC50
value of 24.8 ꢀM in an assay using ADP-induced rabbit platelet-rich plasma and shows moderate
GPIIb/IIIa receptor inhibitory activity with an IC50 value of 5.4 ꢀM, as potent as the known GPIIb/IIIa
receptor antagonist, RGDs [23]. In addition, LX2421 has much lower bleeding risk than Tirofiban and
remarkable antithrombotic activity in arterial and venous thrombosis models in rats. Overall, LX2421
is a promising lead for continuous study. In this paper, we reported the design, synthesis and evaluation
of a novel series of 2-amino-3-(naphth-2-yl)propanoic acid derivatives with the purpose to improve
further the antiplatelet aggregation activity and to reduce the bleeding risk by structural modifications
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of LX2421.
2. Result and Discussion
2.1 Design and Structure-Activity Relationship
In order to explore the binding mode of LX2421 to GPIIb/IIIa, we have performed molecular
docking for its interaction with GPIIb/IIIa using the crystal structure of GPIIb/IIIa in complex with
Tirofiban (PDB CODE: 2VDM, Figure 2). Our results indicated that there are multiple interactions
between LX2421 and GPIIb/IIIa. The 4-piperidyl group in LX2421 has a salt bridge interaction with
the carboxyl group of the Asp224 residue in GPIIb subunit, while the carboxyl group forms several
hydrogen bonds with Ser123, Asn215 residues and also has interaction with the MIDAS Mg2+ ion of
GPIIIa. The indole ring has π-π interaction with the phenyl ring of Tyr190. To fully understand the
binding mode and key interactions of LX2421 and GPIIb/IIIa, molecular dynamics (MD) simulations
were carried out. The binding interactions between LX2421 and GPIIb/IIIa were investigated along the
40 ns MD trajectories. The occupancies (˃ 40%) of each binding interaction were displayed in Figure
2. The result indicated that π-π interaction between the indole ring and Tyr190 showed strong and
stable binding. It make a significant contribution to the inhibitory activity of LX2421 to GPIIb/IIIa
(detailed information about the MD simulation can be found in the Supporting Information).
To explore further of this π-π interaction, we designed LX1-LX3 in which a naphthalene ring was
used to replace the indole ring in LX2421 (Figure 3). These three compounds vary only in the length
of the linker between the naphthyl ring and the piperidine. To our disappointed, none of these
compounds inhibit platelet aggregation induced by ADP in human platelet-rich plasma (more precise
than the rabbit platelet-rich plasma) at 100 ꢀM (Table 1). However surprisingly, LX4-LX6 in which a
piperizine ring was used to replace the piperidine ring in LX1-LX3 have much improved activity in
inhibiting platelet aggregation, indicating that piperazine is more favorable in this scaffold. Among
these three compounds, LX5 which contains a three CH2 linker is the most potent one with an IC50
value of 0.37 ꢀM, being 60 folds more potent than two CH2 containing LX4 and 90 folds more potent
than four CH2 containing LX6, thus LX5 was selected as a new lead for the subsequent study.
Using LX5 as the lead compound, at first we explored the hydrophobic group on sulfonamide by
designing compounds LX14-LX18. Our results indicated that removing the bromo atom (LX14, IC50=
0.18 ꢀM) or replacing it with other electron withdrawn groups, such as fluoro atom (LX15, IC50= 0.28
ꢀM) or nitro group (LX16, IC50= 0.20 ꢀM) can only slightly influence the antiplatelet activity, but
introducing an electron donated methoxy group to the para position of the phenyl ring decreases the
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activity by about 8 folds, indicating that the electronic property of the phenyl ring may have influence
to the activity. Replacing the phenyl group in LX14 with a butyl group (LX18, IC50= 0.52 ꢀM)
decreases the activity by about 3 folds, suggesting phenyl ring is more favorable at this site.
We have also designed a series of compounds by replacing the sulfonamide group with an amide
group by designing compounds LX7-LX13. Our results indicated that amide containing compounds are
dramatically less potent than their sulfonamide containing analogues. For example, LX7 shows an IC50
value of 7.9 µM, being more than 40 times less potent than LX14, indicating the importance of the
sulfonamide structure for the activity.
Our results have indicated that the piperizine group is critical to the antiplatelet activity. Therefore,
in order to explore the influence of the basic group, we designed a series of benzamidine containing
compounds (Table 3), as benzamidine has been frequently used in GPIIb/IIIa antagonists in many
published literatures. Both the sulfonamide and the amide containing scaffolds were used in this series.
To our delight, at 50 µM several compounds, including LX24, LX25, LX26, LX30 can reach >99.5%
of inhibition, indicating benzamidine can also be used as the base group in our new scaffold.
Interestingly, unlike the piperizine containing compounds, sulfonamide containing compounds don’t
show dramatically more potent activity than the amide containing compounds in this series.
LX14 and LX25 are two of the most potent inhibitors of platelet aggregation in our two new
series of compounds with IC50 values of 0.18 ꢀM and 0.84 ꢀM, respectively. Therefore these two
compounds are selected for further evaluation. It is noticeable that LX14 is almost as potent as
Tirofiban.
2.2 The biologic assay and docking study
Besides ADP, platelet aggregation can also be activated by other inducers. Therefore, LX14 and
LX25 have also been evaluated for their antiplatelet aggregation activity induced by collagen, collagen
and thrombin together with ADP. As shown in Figure 4 and Table 4, LX14 and LX25 can significantly
inhibit platelet aggregation induced by ADP (20 ꢀM), collagen (1 ꢀg/ml), thrombin (0.26 U/ml) and
U46619 (2 ꢀM) with the IC50 values of 0.18, 1.32, 0.53, 1.01 ꢀM and 0.84, 27.7, 22.9, 23.6 ꢀM
respectively, suggesting that our compounds LX14 and LX25 can block the common pathway of
platelet aggregation by inhibiting the GPIIb/IIIa receptor. Interestingly, our compounds are more potent
in inhibiting platelet aggregation induced by ADP than those induced by other three inducers.
LX14 and LX25 have been evaluated for their binding affinity to GPIIb/IIIa receptor in the
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fibrinogen/GPIIb/IIIa enzyme-linked immunosorbent assay (ELISA) in comparison with Tirofiban
(Figure 5). Compounds LX14 and LX25 can potently block the binding of GPIIb/IIIa to the fibrinogen
in a concentration-dependent manner with the IC50 values of 0.08 and 1.33 ꢀM respectively, with LX14
being only two-fold less potent than Tirofiban, consistent to its activity in the inhibition of the
antiplatelet aggregation. Thus, LX14 represents a promising GPIIb/IIIa receptor antagonist against
platelet aggregation.
In order to probe the binding modes of compounds LX14 and LX25 with GPIIb/IIIa, we
performed molecular docking study using CDOCKER in Discovery studio 3.0 in comparison with
Tirofiban (Figure 6). The result indicated that LX14 binds to the same pocket as Tirofiban. LX14 has
hydrogen bond interactions with Ser123 and Asn215 residues and salt bridge interaction with the
MIDAS Mg2+ ion of GPIIIa as Tirofiban. Also similar to Tirofiban, the sulfonamido group in LX14
forms hydrogen bonds with Arg214 and Asn215 residues, and the 4-piperizyl group formed a slat
bridge with Asp224 residue, while the naphthalene of LX14 exhibits π-π interaction with the phenyl
ring of Tyr190. The interaction between LX25 and GPIIb/IIIa is similar to that between LX14 and
GPIIb/IIIa, however, the sulfonamido group in LX25 doesn’t have hydrogen bond interactions with
Arg214 and Asn215 residues due to the spatial shift of the amide bond, maybe this is the reason for its
less potent binding affinity to GPIIb/IIIa than LX14.
The risk of bleeding is one critical limitation for GPIIb/IIIa inhibitors in the preventing formation
of thrombus. Therefore, LX14 has been evaluated for the bleeding times in vivo in comparison with
Tirofiban. As shown in Figure 7, treatment with 3 mg/kg, 15 mg/kg and 30 mg/kg of LX14, the
bleeding times are approximately 6.72±1.23, 8.63±2.14 and 9.28±3.21 min (n= 6) respectively. While
only with 1 mg/kg of Tirofiban, the bleeding times are 16.8±2.33 min (n= 6). This result indicated that
LX14 has much lower bleeding risk than Tirofiban, representing a safer GPIIb/IIIa inhibitor against
platelet aggregation.
LX14 has also been evaluated for its in vivo antithrombotic activity in comparison with Tirofiban
in rat arterio-venous shunt model (Figure 8). Our results indicated that LX14 significantly decreases
the thrombus weight in a dose-dependent manner. At 10 mg/kg, it reduces the experimental thrombosis
by 40.0±5.8% (n= 6), as effective as Tirofiban at 1 mg/kg (37.1±4.7%), further confirming that it is a
promising antiplatelet agent.
2.3 Chemistry
Compound 1 was prepared by a described method reported by A. V. Marco, R. Frank et al [24].
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Reactions of compound 1 with various acyl chloride or sulfonyl chloride afforded 2-15 which were
further hydrolyzed with LiOH to yield intermediates 16-29 as shown in scheme 1. Then, 16-29 were
reacted with 4-piperidyl contained intermediates to provide LX1-LX3 with Boc cleavage. Methylation
of 16-29 with SOCl2/MeOH yielded our critical intermediates 30-43. Compounds 43-57 were further
obtained by treatment with the corresponding dibromide, which were reacted with Boc-paperazine to
give 58-72. Finally, LX4-LX18 were achieved by hydrolyzation of methyl ester and deprotection of
Boc. Additionally, 30-43 were refluxed with 4-cyano benzylbromide in dry acetone/K2CO3 to give
73-84 which were reacted with HCl/MeOH and NH3/MeOH by Pinner reaction and hydrolyzed to
afford LX19-LX30.
3. Conclusion
In summary, we identified compound LX5 as a novel lead by structure modification of LX2421.
The antiplatelet aggregation activity of it was more potent than LX2421 with the IC50 value of 7.9 ꢀM.
Further optimization, a novel series of compounds were designed and synthesized by exploring the
impact of the basic group and the functional group on the α-amino group on the antiplatelet aggregation
activity. Our effort led to the hit of LX14 and LX25, showing remarkably antiplatelet aggregation
activity, especially LX14, almost equally high potency as Tirofiban. Further study, LX14 and LX25
could inhibit the platelet aggregation induced by four different agonists, LX14 displayed comparable
ability as Tirofiban in inhibiting the GPIIb/IIIa receptor (IC50= 0.08 ꢀM). At last, LX14 was selected
to perform in vivo biologic evaluation in comparison with Tirofiban. The lower bleeding time and
significant antithrombotic activity were observed. Above all, LX14 could be a safer and promising
antiplatelet aggregation agent for further study.
4. Experimental procedures
4.1 Chemistry
All reagents were from commercial sources. With tetramethylsilane (TMS) as internal standard,
1
the H-NMR and 13C-NMR were recorded on Bruker AV-300 apparatus by using deuterated solvents.
HR-MS was collected on Agilent technologies 6520 Accurate-Mass Q-TOF LC/MS instruments. Every
targeted compound was purified via silica gel (60Å, 70-230 mesh) column chromatography. Melting
points were measured by XT-4 melting point apparatus. The purity (≥95%) of final compounds is
verified by the HPLC study performed on Agilent C18 (4.6 mm×150 mm, 3.5 ꢀm) column using a
mixture of solvent methanol/water at the flow rate of 0.5 mL/min and peak detection at 254 nm under
UV.
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(±)-2-Amino-3-(6-hydroxy-2-naphthyl)propanoic acid hydrobromide (2·HBr) 1 was synthesized
by a known method established within the literature.
4.1.1 Typical procedure for the preparation of 2~15
To the mixture of (±)-2-Amino-3-(6-hydroxy-2-naphthyl)propanoic acid hydrobromide (2·HBr) 1
(4.30 mmol) in acetone (20 mL) and water (10 mL), K2CO3 (13.0 mmol) was added, followed by
stirring at room temperature for 1 h, then substituted acyl chloride (5.60 mmol) was dropped slowly at
0℃, and the mixture was kept under stirring for 10 h at 65℃. After the reaction was completed,
monitored by TLC, the solvent was removed under reduced pressure, and the resulting residue was
dissolved in H2O (10 mL), acidified to pH 2-3 with 1 M HCl, then extracted with AcOEt (3×15mL).
The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The
crude residue was purified by flash column chromatography (petroleum ether/AcOEt 1:1 ) to give the
corresponding product.
4.1.2 Typical procedure for the preparation of 16~29
To the solution of the intermediate 2~15 (3.80 mmol) in MeOH/Acetone/H2O (30 mL,
v:v:v=1:1:1), LiOH•H2O (11.0 mmol) was added. The reaction mixture was stirred for 8 h at 50℃, and
TLC analysis indicated that the reaction was completed. The solvent was removed under reduced
pressure, and the resulting slurry was taken up in H2O, acidified to pH 2-3 with 1 M HCl. The
precipitate was filtered and dried to afford the corresponding compound.
4.1.3 Typical procedure for the preparation of LX1~LX3
1-Boc-4-(methylsulfonyl)oxy)propyl) piperidine or 1-Boc-4-(2-((methylsulfonyl)oxy)propyl)
piperidine
or
1-Boc-4-(2-((methylsulfonyl)oxy)propyl)piperidine
or
1-Boc-4-(3-((methylsulfonyl)oxy)propyl) piperidine or 1-Boc-4-(4-((methylsulfonyl)oxy)butyl)
piperidine (2.20 mmol) was added to a solution of the amino-substituted derivatives of
(±)-2-Amino-3-(6-hydroxy-2-naphthyl)propanoic acid hydrobromide (2·HBr) (16~29,1.70 mmol) in 15
mL of dry DMF, then dry K2CO3 power (5.10 mmol) was added. The mixture was stirred at 80 ℃ for
12 h under N2, and TLC analysis indicated the reaction was complete. The mixture was filtered, and the
filtrate was concentrated in vacuo. The crude residue was purified by flash column chromatography
and eluted with 2:1 petroleum ether/AcOEt to give the corresponding intermediate. The intermediate
was dissolved in 5 ml AcOEt. Pass the dry HCl gas at 0℃ through the system and the solution was
stirred and monitored by TLC. After completion, the solvent was removed in vacuo and the residue was
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dissolved in methanol. The solution was concentrated in vacuo to give the corresponding compound
LX1~LX3.
4.1.3.1 (±)-2-((4-bromophenyl)sulfonamido)-3-(6-(piperidin-4-ylmethoxy)naphthalen-2-yl) propanoic
acid·HCl（LX1）
It was obtained as a light yellow solid in 35% yield. m.p. 134-136℃; HRMS (ESI): m/z, calculated for
1
C25H27BrN2O5S, 547.0902 (M+H)+, found 547.0913; H-NMR (300MHz, DMSO-d6):
δ 9.79 (br, 1H,
HCl), 9.01 (s, 1H, piperidyl-NH), 8.73 (d, 1H, J= 8.7 Hz, -SO2-NH-CH-), 7.63~7.06 (m, 10H, Ar-H),
4.11~4.01 (m, 1H, -CH2-CH-NH-), 3.67 (t, 2H, J= 6.2 Hz, -CH2-O-), 3.13~2.67 (m, 6H,
piperidyl-CH2-N-CH2- and -CH2-CH2-NH-), 1.91~1.15 (m, 5H, Piperidine-H); 13C-NMR (75MHz,
DMSO-d6): δ 170.7, 155.1, 139.8, 133.4, 131.7, 130.0, 128.7, 128.0, 127.4, 126.0, 125.9, 118.7, 108.4,
73.1, 67.7, 57.4, 42.2, 37.7, 36.4, 32.8, 32.2, 30.7, 25.7, 24.5 ppm.
4.1.3.2 (±)-2-((4-bromophenyl)sulfonamido)-3-(6-(2-(piperidin-4-yl)ethoxy)naphthalen-2-yl)propanoic
acid·HCl（LX2）
It was obtained as a light yellow solid in 40% yield. m.p. 152-154℃; HRMS (ESI): m/z, calculated for
1
C26H29BrN2O5S, 561.1059 (M+H)+, found 561.1046; H-NMR (300MHz, DMSO-d6):
δ 9.01~8.72 (br,
2H, piperidyl-NH and HCl), 8.70 (d, 1H, J= 8.8 Hz, -SO2-NH-), 7.60~7.04 (m, 10H, Ar-H), 4.01~3.98
(m, 1H, -CH2-CH-NH-), 3.79~3.77 (m, 2H, -CH2-O-), 3.15~2.64 (m, 6H, piperidyl-CH2-N-CH2- and
-CH2-CH2-NH-), 1.59~1.10 (m, 7H, Piperidine and -CH2-CH2-O-); 13C-NMR (75MHz, DMSO-d6): δ
170.7, 155.1, 147.0, 139.9, 133.4, 131.7, 130.0, 128.7, 128.1, 127.4, 125.9, 124.2, 123.1, 122.6, 118.7,
108.4, 62.0, 61.3, 43.0, 42.7, 34.1, 33.6, 31.1, 30.3, 29.3, 28.6 ppm.
4.1.3.3(±)-2-((4-bromophenyl)sulfonamido)-3-(6-(3-(piperidin-4-yl)propoxy)naphthalen-2-yl)propanoi
c acid·HCl（LX3）
It was obtained as a light yellow solid in 43% yield. m.p. 146-148℃; HRMS (ESI): m/z, calculated for
1
C27H31BrN2O5S, 575.1215 (M+H)+, found 575.1215; H-NMR (300MHz, DMSO-d6):
δ 9.00~8.69 (s,
1H, piperidyl-NH), 8.69 (d, 1H, J= 8.9 Hz, -SO2-NH-), 8.67~8.45 (br, 1H, HCl), 7.59~7.01 (m, 10H,
Ar-H), 4.00 (m, 1H, -CH2-CH-NH-), 3.98~3.68 (t, 2H, J= 5.8 Hz, -CH2-O-), 3.17~2.69 (m, 6H,
piperidyl-CH2-N-CH2- and -CH2-CH2-NH-), 1.55~0.91 (m, 9H, Piperidine and -CH2-CH2-CH2-O-);
13C-NMR (75MHz, DMSO-d6): δ 170.7, 155.2, 139.9, 133.4, 131.7, 129.9, 128.7, 128.1, 127.4, 125.9,
118.7, 108.4, 64.5, 57.4, 42.9, 40.2, 38.5, 37.8, 32.4, 31.7, 31.3, 31.0, 29.7, 28.9, 28.0, 24.6 ppm.
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4.1.4 Typical procedure for the preparation of 30~43
To the stirred solution of the intermediate 16~29 (49.00 mmol) in methanol (160 mL) at 0℃,
sulfoxide chloride (98.00 mmol) was added dropwise, and stirred at 50℃ overnight. After the reaction,
monitored by TLC, was complete, the mixture was concentrated in vacuo. The corresponding methyl
intermediate was obtained by column chromatography.
4.1.5 Typical procedure for the preparation of 43~57
To a solution of intermediate 30~43 (22.03 mmol) in acetonitrile (70 mL), 1,2-dibromoethane or
1,3-dibromopropane or 1,4-dibromobutane (44.06 mmol), K2CO3 (66.09 mmol) were added. The
mixture was stirred at 60℃ for 48 h. The product was filtered, and the filtrate was concentrated in
vacuo. The crude residue was purified by flash column chromatography and eluted with 5:1 petroleum
ether/AcOEt to give the corresponding product.
4.1.6 Typical procedure for the preparation of 58~72
To the solution of the intermediate 43~57 in acetonitrile (40 mL), 1-Boc-piperazine (20.40 mmol)
and K2CO3 (20.40 mmol) were added. The mixture was stirred at 60℃ for 48 h. The reaction was
filtered, and the filtrate was concentrated in vacuo. The crude residue was purified by flash column
chromatography and eluted with 8:1 petroleum ether/AcOEt to give the corresponding product.
4.1.7 Typical procedure for the preparation of LX4~LX18
To the solution of the intermediate 58~72 (2.30 mmol) in MeOH/THF/H2O (15 mL, v:v:v=1:1:1),
LiOH•H2O (6.90 mmol) was added. The reaction mixture was stirred at room temperature for 2 h, and
TLC analysis indicated that the reaction was completed. The solvent was removed under reduced
pressure, and the resulting slurry was taken up in H2O, acidified to pH 2-3 with 1 M HCl. The
precipitate was filtered and dried to afford the corresponding compound with Boc-piperazine. Then the
intermediates (1.50 mmol) were dissolved in 5 ml AcOEt. Pass the dry HCl gas at 0℃ through the
system and the solution was stirred and monitored by TLC. After completion, the solvent was removed
in vacuo and the residue was dissolved in methanol. The solution was concentrated in vacuo to give the
corresponding compound.
4.1.7.1 (±)-2-((4-bromophenyl)sulfonamido)-3-(6-(2-(piperazin-1-yl)ethoxy)naphthalen-2-yl)propanoic
acid·HCl（LX4）
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It was obtained as a white solid in 41% yield. m.p. 153-155℃; HRMS (ESI): m/z, calculated for
1
C25H28BrN3O5S, 562.1006 (M+H)+, found 562.1033; H-NMR (300MHz, DMSO-d6):
δ 12.3 (s, 1H,
-COOH), 9.84 (br, 2H, piperidyl-NH and HCl), 8.52~8.49 (d, 1H, J= 9.0 Hz, -SO2-NH-), 7.71~7.22 (m,
10H, Ar-H), 4.56 (t, 2H, J= 6.0 Hz, -CH2-O-), 4.01~3.94 (m, 1H, -CH2-CH-NH-), 3.68~3.50 (m, 10H,
Piperazine and -CH2-CH2-O-), 3.02~2.73 (m, 2H, -CH2-CH-NH-); 13C-NMR (75MHz, DMSO-d6): δ
172.2, 155.0, 140.2, 132.8, 132.2, 131.4, 128.9, 128.5, 127.9, 127.8, 127.5, 126.5, 125.6, 118.5, 107.1,
62.4, 57.6, 54.5, 48.4, 37.6, 31.1, 29.8, 21.0 ppm.
4.1.7.2(±)-2-((4-bromophenyl)sulfonamido)-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propanoi
c acid·HCl（LX5）
It was obtained as a white solid in 45% yield. m.p. 171-173℃; HRMS (ESI): m/z, calculated for
1
C26H30BrN3O5S, 576.1062 (M+H)+, found 576.1078; H-NMR (300MHz, DMSO-d6):
δ 12.3 (s, 1H,
-COOH), 9.85 (br, 2H, piperidyl-NH and HCl), 8.78 (d, 1H, J= 9.0 Hz, -SO2-NH-), 7.79~7.10 (m, 10H,
Ar-H), 4.20 (t, 2H, J= 5.5 Hz, -CH2-O-), 4.04~3.98 (m, 1H, -CH2-CH-NH-), 3.73~3.49 (m, 10H,
Piperazine and -CH2-CH2-CH2-O-), 3.13~2.77 (m, 2H, -CH2-CH-NH-), 2.30~2.27 (m, 2H,
-CH2-CH2-CH2-O-); 13C-NMR (75MHz, DMSO-d6): δ 172.2, 155.8, 140.2, 133.0, 131.9, 131.4, 128.8,
128.3, 127.8, 127.5, 126.5, 125.5, 118.5, 106.7, 64.8, 57.6, 53.1, 47.7, 37.6, 31.1, 29.8, 29.2, 23.2 ppm.
4.1.7.3 (±)-2-((4-bromophenyl)sulfonamido)-3-(6-(4-(piperazin-1-yl)butoxy)naphthalen-2-yl)propanoic
acid·HCl（LX6）
It was obtained as a white solid in 53% yield. m.p. 169-171℃; HRMS (ESI): m/z, calculated for
1
C27H32BrN3O5S, 590.1319 (M+H)+, found 590.1347; H-NMR (300MHz, DMSO-d6):
δ 12.0 (s, 1H,
-COOH), 10.1 (br, 2H, piperidyl-NH and HCl), 8.80 (d, 1H, J= 9.0 Hz, -SO2-NH-), 7.66~7.13 (m, 10H,
Ar-H), 4.13~4.11 (m, 2H, -CH2-O-), 3.99~3.97 (m, 1H, -CH2-CH-NH-), 3.90~3.40 (m, 10H,
Piperazine and -CH2-CH2-CH2-CH2-O-), 3.219~3.08 (m, 2H, -CH2-CH-NH-), 1.91~1.87 (m, 4H,
-CH2-CH2-CH2-CH2-O-); 13C-NMR (75MHz, DMSO-d6): δ 172.2, 156.1, 140.2, 133.0, 131.8, 131.4,
128.7, 128.2, 127.8, 127.8, 127.4, 126.4, 125.5, 118.6, 106.6, 66.8, 57.6, 55.3, 47.6, 37.6, 25.8, 20.0
ppm.
4.1.7.4 (±)-2-benzamido-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propanoic acid·HCl（LX7）
It was obtained as a white solid in 45% yield. m.p. 171-173℃; HRMS (ESI): m/z, calculated for
1
C27H32N3O4, 462.2293 (M+H)+, found 462.2230; H-NMR (300MHz, DMSO-d6):
δ 9.66 (br, 2H,
piperidyl-NH and HCl), 8.78 (d, 1H, J= 8.3 Hz, -CO-NH-), 7.79~7.10 (m, 11H, Ar-H), 4.72~4.70 (m,
10

ACCEPTED MANUSCRIPT
1H, -CH2-CH-NH-), 4.15 (t, 2H, J= 6.1 Hz, -CH2-O-), 3.71~3.30 (m, 10H, Piperazine and
-CH2-CH2-CH2-O-), 3.29~3.14 (m, 2H, -CH2-CH-NH-), 2.24~2.22 (m, 2H, -CH2-CH2-CH2-O-);
13C-NMR (75MHz, DMSO-d6): δ 173.0, 166.5, 155.8, 133.8, 133.6, 133.4, 132.9, 132.8, 131.4, 128.8,
128.4, 128.1, 127.3, 127.2, 127.1, 126.5, 118.5, 106.8, 64.8, 54.4, 53.1, 47.7, 38.6, 36.6, 29.2, 28.9,
14.0 ppm.
4.1.7.5(±)-2-(3-fluorobenzamido)-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propanoic acid·HCl
（
LX8）
It was obtained as a white solid in 63% yield. m.p. 161-163℃; HRMS (ESI): m/z, calculated for
1
C27H31FN3O4, 480.2199 (M+H)+, found 480.2125; H-NMR (300MHz, DMSO-d6):
δ 11.9 (s, 1H,
-COOH), 9.76 (br, 2H, piperidyl-NH and HCl), 8.91 (d, 1H, J= 8.3 Hz, -CO-NH-), 7.75~7.10 (m, 10H,
Ar-H), 4.72~4.67 (m, 1H, -CH2-CH-NH-), 4.15 (t, 2H, J= 5.6 Hz, -CH2-O-), 3.70~3.42 (m, 10H,
Piperazine and -CH2-CH2-CH2-O-), 3.29~3.14 (m, 2H, -CH2-CH-NH-), 2.22~1.90 (m, 2H,
-CH2-CH2-CH2-O-); 13C-NMR (75MHz, DMSO-d6): δ 172.8, 165.0 (J= 367 Hz), 163.4, 160.1, 155.8,
136.1, 133.3, 132.9, 132.8, 130.4, 128.8, 128.4, 128.0, 127.1, 126.5, 123.5, 118.5, 118.3, 118.0, 114.2,
106.8, 64.9, 54.4, 53.1, 47.7, 36.2, 23.2, 13.9 ppm.
4.1.7.6(±)-2-(4-fluorobenzamido)-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propanoic acid·HCl
（
LX9）
It was obtained as a white solid in 65% yield. m.p. 177-179℃; HRMS (ESI): m/z, calculated for
1
C27H31FN3O4, 480.2199 (M+H)+, found 480.2134; H-NMR (300MHz, DMSO-d6):
δ 11.7 (s, 1H,
-COOH), 9.56 (br, 2H, piperidyl-NH and HCl), 8.83 (d, 1H, J= 8.0 Hz, -CO-NH-), 7.88~7.10 (m, 10H,
Ar-H), 4.69 (m, 1H, -CH2-CH-NH-), 4.16 (t, 2H, J= 5.9 Hz, -CH2-O-), 3.81~3.30 (m, 10H, Piperazine
and -CH2-CH2-CH2-O-), 3.21~3.13 (m, 2H, -CH2-CH-NH-), 2.21~2.20 (m, 2H, -CH2-CH2-CH2-O-);
13C-NMR (75MHz, DMSO-d6): δ 173.1, 165.5 (J= 247 Hz), 165.2, 162.2, 155.7, 133.4, 132.8, 130.2,
130.0, 129.8, 128.9, 128.4, 128.1, 127.1, 126.5, 118.5, 115.3, 115.0, 106.6, 64.8, 54.3, 53.1, 47.8, 36.2
ppm.
4.1.7.7(±)-2-(3,5-dichlorobenzamido)-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propanoic
acid·HCl（LX10）
It was obtained as a light yellow solid in 53% yield. m.p. 149-151℃; HRMS (ESI): m/z, calculated for
1
C27H30Cl2N3O4, 530.1413 (M+H)+, found 530.1433; H-NMR (300MHz, DMSO-d6):
δ 11.8 (s, 1H,
-COOH), 9.63 (br, 2H, piperidyl-NH and HCl), 9.04 (d, 1H, J= 8.0 Hz, -CO-NH-), 7.76~7.09 (m, 9H,
11

ACCEPTED MANUSCRIPT
Ar-H), 4.65 (m, 1H, -CH2-CH-NH-), 4.13 (t, 2H, J= 5.8 Hz, -CH2-O-), 3.80~3.30 (m, 10H, Piperazine
and -CH2-CH2-CH2-O-), 3.25~3.09 (m, 2H, -CH2-CH-NH-), 2.22~2.20 (m, 2H, -CH2-CH2-CH2-O-);
13C-NMR (75MHz, DMSO-d6): δ 172.7, 163.5, 155.8, 136.8, 134.2, 133.1, 132.9, 130.8, 128.8, 128.4,
128.0, 127.2, 126.5, 126.1, 118.6, 106.6, 64.8, 54.4, 53.1, 47.8, 38.6, 36.2, 23.2 ppm.
4.1.7.8 (±)-2-(4-nitrobenzamido)-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propanoic acid·HCl
（
LX11）
It was obtained as a yellow solid in 60% yield. m.p. 144-146℃; HRMS (ESI): m/z, calculated for
1
C27H31N4O6, 507.2044 (M+H)+, found 507.2060; H-NMR (300MHz, DMSO-d6):
δ 10.0 (br, 2H,
piperidyl-NH and HCl), 9.28 (d, 1H, J= 7.7 Hz, -CO-NH-), 8.18~6.98 (m, 10H, Ar-H), 4.62 (m, 1H,
-CH2-CH-NH-), 3.97 (t, 2H, J= 6.0 Hz, -CH2-O-), 3.43~3.30 (m, 10H, Piperazine and
-CH2-CH2-CH2-O-), 3.21~3.14 (m, 2H, -CH2-CH-NH-), 2.15~2.11 (m, 2H, -CH2-CH2-CH2-O-);
13C-NMR (75MHz, DMSO-d6): δ 171.5, 162.5, 157.1, 153.2, 138.5, 134.1, 133.5, 130.6, 127.4, 127.0,
126.8, 126.6, 126.3, 124.5, 123.5, 123.0, 122.4, 122.6, 114.1, 106.2, 106.1, 102.4, 55.2, 52.1, 44.2, 41.3
ppm.
4.1.7.9(±)-2-(4-methoxybenzamido)-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propanoic
acid·HCl（LX12）
It was obtained as a light yellow solid in 53% yield. m.p. 166-168℃; HRMS (ESI): m/z, calculated for
1
C28H34N3O5, 492.2398 (M+H)+, found 492.2312; H-NMR (300MHz, DMSO-d6):
δ 9.55 (s, 2H,
piperidyl-NH and HCl), 8.62 (d, 1H, J= 8.3 Hz, -CO-NH-), 7.79~6.94 (m, 10H, Ar-H), 4.67~4.64 (m,
1H, -CH2-CH-NH-), 4.15 (t, 2H, J= 5.8 Hz, -CH2-O-), 3.74 (s, 3H, CH3-O-), 3.44~3.31 (m, 10H,
Piperazine and -CH2-CH2-CH2-O-), 3.28~3.13 (m, 2H, -CH2-CH-NH-), 2.24~2.21 (m, 2H,
-CH2-CH2-CH2-O-); 13C-NMR (75MHz, DMSO-d6): δ 173.3, 165.7, 161.6, 155.7, 133.5, 132.8, 129.1,
128.9, 128.4, 128.1, 127.1, 126.4, 126.0, 118.5, 113.3, 106.6, 64.7, 59.7, 55.2, 54.2, 53.0, 47.8, 36.2,
31.3, 30.5, 29.6, 23.1, 14.0 ppm.
4.1.7.10 (±)-2-butyramido-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propanoic acid·HC（l LX13）
It was obtained as a light yellow solid in 33% yield. m.p. 155-157℃; HRMS (ESI): m/z, calculated for
1
C24H34N3O4, 428.2549 (M+H)+, found 428.2551; H-NMR (300MHz, DMSO-d6):
δ 11.9 (s, 1H,
-COOH), 9.76 (br, 2H, piperidyl-NH and HCl), 8.15 (d, 1H, J= 8.1 Hz, -CO-NH-), 7.72~7.09 (m, 6H,
Ar-H), 4.47 (m, 1H, -CH2-CH-NH-), 4.15 (t, 2H, J= 5.8 Hz, -CH2-O-), 3.70~3.42 (m, 10H, Piperazine
and -CH2-CH2-CH2-O-), 3.17~2.92 (m, 2H, -CH2-CH-NH-), 2.24~2.22 (m, 2H, -CH2-CH2-CH2-O-),
12

ACCEPTED MANUSCRIPT
2.00~1.87 (m, 2H, -CH2-CH2-CH3), 1.42~1.29 (m, 2H, -CH2-CH2-CH3), 0.65 (t, 3H, J= 7.1 Hz,
-CH2-CH2-CH3); 13C-NMR (75MHz, DMSO-d6): δ 173.2, 171.9, 155.7, 133.0, 132.8, 128.9, 128.4,
128.0, 127.2, 126.4, 118.5, 106.6, 64.8, 53.3, 53.0, 47.7, 36.9, 36.7, 30.8, 23.1, 18.5, 13.5, 13.3 ppm.
4.1.7.11(±)-2-(phenylsulfonamido)-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propanoic
acid·HCl（LX14）
It was obtained as a light yellow solid in 43% yield. m.p. 174-176℃; HRMS (ESI): m/z, calculated for
1
C26H32N3O5S, 498.2063 (M+H)+, found 498.2046; H-NMR (300MHz, DMSO-d6):
δ 12.1 (s, 1H,
-COOH), 9.89 (br, 2H, HCl and piperazine-NH), 8.32 (d, 1H, J= 9.06 Hz, -SO2-NH-), 7.67~7.09 (m,
11H, Ar-H), 4.16 (t, 2H, J= 5.8 Hz, -CH2-O-), 3.95~3.91 (m, 1H, -CH2-CH-NH-), 3.71~3.07 (m, 10H,
Piperazine and -CH2-CH2-CH2-O-), 3.07~2.76 (m, 2H, -CH2-CH-NH-), 2.46~2.22 (m, 2H,
-CH2-CH2-CH2-O-); 13C-NMR (75MHz, DMSO-d6): δ 172.2, 155.8, 140.9, 133.0, 131.9, 131.8, 128.9,
128.7, 128.5, 128.3, 128.1, 127.9, 127.4, 127.2, 126.5, 126.1, 126.0, 118.4, 106.6, 64.8, 57.4, 53.0, 47.7,
37.7, 23.2 ppm.
4.1.7.12(±)-2-((4-fluorophenyl)sulfonamido)-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propano
ic acid·HCl（LX15）
It was obtained as a light yellow solid in 38% yield. m.p. 159-161℃; HRMS (ESI): m/z, calculated for
1
C26H31FN3O5S, 516.1968 (M+H)+, found 516.1953; H-NMR (300MHz, DMSO-d6):
δ 11.9 (s, 1H,
-COOH), 9.70 (br, 2H, HCl and piperazine-NH), 8.42 (d, 1H, J= 9.1 Hz, -SO2-NH-), 7.67~6.97 (m,
10H, Ar-H), 4.15 (t, 2H, J= 5.8 Hz, -CH2-O-), 3.99~3.90 (m, 1H, -CH2-CH-NH-), 3.72~3.30 (m, 10H,
Piperazine and -CH2-CH2-CH2-O-), 3.10~3.04 (m, 2H, -CH2-CH-NH-), 2.30~2.25 (m, 2H,
-CH2-CH2-CH2-O-); 13C-NMR (75MHz, DMSO-d6): δ 173.5, 165.3 (J= 150 Hz), 162.2, 163.3, 151.1,
131.7, 128.7, 128.6, 128.4, 128.1, 127.4, 127.0, 126.4, 125.1, 124.5, 119.1, 111.3, 112.0, 110.8, 109.6,
102.0, 67.1, 55.8, 54.7, 48.0 ppm.
4.1.7.13(±)-2-((4-methoxyphenyl)sulfonamido)-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propa
noic acid·HCl（LX17）
It was obtained as a light yellow solid in 40% yield. m.p. 158-160℃; HRMS (ESI): m/z, calculated for
C27H34N3O6S, 528.2168 (M+H)+, found 528.2151; 1H-NMR (300MHz, DMSO-d6):
9.90 (br, 2H, HCl
δ
and piperazine-NH), 8.14 (d, 1H, J= 8.9 Hz, -SO2-NH-), 7.64~6.66 (m, 10H, Ar-H), 4.19~4.11 (m, 2H,
-CH2-O-), 3.87~3.85 (m, 1H, -CH2-CH-NH-), 3.66~3.30 (m, 13H, Piperazine and -CH2-CH2-CH2-O-
and CH3-O-), 3.00~2.46 (m, 2H, -CH2-CH-NH-), 2.25~2.22 (m, 2H, -CH2-CH2-CH2-O-); 13C-NMR
13

ACCEPTED MANUSCRIPT
(75MHz, DMSO-d6): δ 171.1, 170.8, 155.3, 152.1, 151.8, 151.3, 150.1, 136.1, 135.2, 134.1, 133.5,
131.8, 127.1, 126.8, 126.3, 126.1 125.3, 122.2, 121.0, 120.5, 67.8, 66.5, 45.2, 43.5 ppm.
4.1.7.14(±)-2-(butylsulfonamido)-3-(6-(3-(piperazin-1-yl)propoxy)naphthalen-2-yl)propanoic acid·HCl
（
LX18）
It was obtained as a light yellow solid in 33% yield. m.p. 167-169℃; HRMS (ESI): m/z, calculated for
1
C24H36N3O5S, 478.2376 (M+H)+, found 478.2361; H-NMR (300MHz, DMSO-d6):
δ 11.9 (s, 1H,
-COOH), 9.70 (s, 2H, piperidyl-NH and HCl), 7.79~7.14 (m, 6H, Ar-H), 4.20 (m, 1H, -CH2-CH-NH-),
4.11 (m, 2H, -CH2-O-), 3.45~3.37 (m, 10H, Piperazine and -CH2-CH2-CH2-O-), 3.26~2.89 (m, 2H,
-CH2-CH-NH-), 2.26~2.23 (m, 2H, -CH2-CH2-CH2-O-), 1.27~1.24 (m, 2H, -CH2-CH2-CH2-CH3),
1.18~1.15 (m, 2H, -CH2-CH2-CH2-CH3), 0.96~0.87 (m, 2H, -CH2-CH2-CH2-CH3), 0.55 (t, 3H, J=
7.5Hz, -CH2-CH2-CH2-CH3); 13C-NMR (75MHz, DMSO-d6): δ 173.1, 155.9, 133.0, 132.6, 129.2,
128.9, 128.3, 128.1, 127.7, 126.5, 118.6, 106.7, 64.9, 57.5, 53.1, 52.2, 47.8, 38.5, 37.7, 24.8, 23.4, 20.5,
13.1 ppm.
4.1.8 Typical procedure for the preparation of 73~84
To the solution of the intermediate 30~43 (10.20 mmol) in acetonitrile (40 mL), 4-cyanobenzyl
bromide (15.30 mmol) and K2CO3 (20.40 mmol) were added. The mixture was refluxed overnight. The
reaction was filtered, and the filtrate was concentrated in vacuo. The crude residue was purified by
flash column chromatography and eluted with 1:1 petroleum ether/AcOEt to give the corresponding
product.
4.1.9 Typical procedure for the preparation of LX19~LX30
The intermediate 73~84 (2.30 mmol) was dissolved in dry MeOH (3 mL), Pass the dry HCl gas at
0℃ through the system and the solution was stirred for 8h and monitored by TLC. After completion,
the solvent was removed in vacuo and the residue was dissolved in dry methanol (3ml) again. Pass the
dry NH3 gas through the system at room temperature and the solution was stirred for 6h and monitored
by TLC. After completion, the solvent was removed in vacuo and the residue was dissolved in AcOEt.
Then, the crude residue was purified by flash column chromatography and eluted with 10:1 AcOEt
/MeOH to give the corresponding intermediate.
To the solution of the intermediate (1.30 mmol) in MeOH/THF/H2O (3 mL, v:v:v=1:1:1),
LiOH•H2O (2.60 mmol) was added. The reaction mixture was stirred at room temperature overnight.
14

ACCEPTED MANUSCRIPT
After the reaction, monitored by TLC, was completed, the solvent was removed under reduced pressure,
and the mixture was diluted in H2O, acidified to pH 2-3 with 1 M HCl. The precipitate was filtered and
dried to afford the corresponding compound.
4.1.9.1(±)-2-benzamido-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)propanoic acid·HC（l LX19）
It was obtained as a grey solid in 45% yield. m.p. 181-184℃; HRMS (ESI): m/z, calculated for
C28H25N3O4, 468.2012 (M+H)+, found 468.2006; 1H-NMR (300MHz, DMSO-d6):
9.65 (br, 1H, HCl),
δ
8.56 (d, 1H, J= 8.3 Hz, -CO-NH-), 7.79~7.00 (m, 18H, Ar-H and NH =C-NH2), 5.51 (m, 1H,
-CH2-CH-NH-), 4.73~4.59 (m, 2H, -CH2-O-), 3.25~3.00 (m, 2H, -CH2-CH-NH-); 13C-NMR (75MHz,
DMSO-d6): δ 173.3, 166.0, 165.4, 162.2, 162.0, 154.8, 148.8, 134.0, 133.1, 132.6, 131.1, 128.7, 128.1,
128.0, 127.9, 127.8, 127.5, 127.4, 127.3, 127.2, 127.1, 126.4, 125.6, 118.5, 108.4, 62.1, 54.9, 37.2 ppm.
4.1.9.2(±)-2-(3-fluorobenzamido)-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)propanoic
acid·HCl（LX20）
It was obtained as a light yellow solid in 41% yield. m.p. 182-185℃; HRMS (ESI): m/z, calculated for
C28H24FN3O4, 486.1954 (M+H)+, found 486.1940; 1H-NMR (300MHz, DMSO-d6):
9.70~9.23 (m, 3H,
δ
NH=C-NH2), 8.72 (d, 1H, J= 6.1 Hz, -CO-NH-), 7.87~7.16 (m, 14H, Ar-H), 5.31~5.21 (m, 2H,
-CH2-O-), 4.63 (m, 1H, -CH2-CH-NH-), 3.30~30.1 (m, 2H, -CH2-CH-NH-); 13C-NMR (75MHz,
DMSO-d6): δ 173.4, 167.5 (J= 210 Hz), 165.6, 164.7, 155.7, 143.0, 140.1, 136.5, 133.9, 133.7, 132.7,
130.4, 130.2, 129.0, 128.5, 128.0, 127.6, 126.4, 123.5, 118.6, 118.2, 117.9, 114.3, 113.8, 107.3, 68.2,
55.0, 37.2 ppm.
4.1.9.3(±)-2-(4-fluorobenzamido)-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)propanoic
acid·HCl（LX21）
It was obtained as a red solid in 48% yield. m.p. 183-184℃; HRMS (ESI): m/z, calculated for
C28H24FN3O4, 486.1966 (M+H)+, found 486.1963; 1H-NMR (300MHz, DMSO-d6):
9.43~9.34 (m, 3H,
δ
NH=C-NH2), 8.85~8.73 (d, 1H, J= 8.5 Hz, -CO-NH-), 7.89~7.16 (m, 14H, Ar-H), 5.30~5.21(m, 2H,
-CH2-O-), 4.68~4.55 (m, 1H, -CH2-CH-NH-), 3.29~3.13 (m, 2H, -CH2-CH-NH-); 13C-NMR (75MHz,
DMSO-d6): δ 173.3, 167.6 (J= 405 Hz), 165.2, 162.2, 155.4, 143.1, 134.0, 133.6, 132.7, 130.5, 129.9,
129.4, 129.0, 128.5, 128.3, 127.6, 127.2, 126.5, 118.3, 115.2, 114.9, 107.3, 68.6, 68.2, 55.1, 54.4, 37.2,
36.3 ppm.
4.1.9.4(±)-2-(4-nitrobenzamido)-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)propanoic
15

ACCEPTED MANUSCRIPT
acid·HCl（LX22）
It was obtained as a light yellow solid in 35% yield. m.p. 178-179℃; HRMS (ESI): m/z, calculated for
C28H24N4O6, 513.1789 (M+H)+, found 513.1797; 1H-NMR (300MHz, DMSO-d6):
10.2~9.22
δ
(NH=C-NH2), 8.86 (d, 1H, J= 7.4 Hz, -CO-NH-), 8.26~7.16 (m, 17H, Ar-H), 5.30~5.21 (m, 2H,
-CH2-O-), 4.61 (m, 1H, -CH2-CH-NH-), 3.28~3.10 (m, 2H, -CH2-CH-NH-); 13C-NMR (75MHz,
DMSO-d6): δ 174.1, 172.9, 167.5, 165.6, 164.4, 155.6, 148.9, 148.8, 142.9, 140.1, 134.2, 133.8, 133.6,
132.7, 129.0, 128.8, 128.3, 128.1, 127.7, 127.1, 126.3, 123.3, 118.5, 107.2, 68.6, 55.9, 55.4, 55.2 ppm.
4.1.9.5(±)-2-(4-methoxybenzamido)-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)propanoic
acid·HCl（LX23）
It was obtained as a yellow solid in 46% yield. m.p. 184-187℃; HRMS (ESI): m/z, calculated for
C29H27N3O5, 498.2021 (M+H)+, found 498.2063; 1H-NMR (300MHz, DMSO-d6):
7.99~6.87 (m, 17H,
δ
Ar-H and NH=C-NH2), 5.19 (m, 2H, -CH2-O-), 4.51 (m, 1H, -CH2-CH-NH-), 3.73 (m, 2H, CH3-O-),
3.20~3.10 (m, 2H, -CH2-CH-NH-); 13C-NMR (75MHz, DMSO-d6): δ 167.6, 165.0, 161.3, 155.4, 140.2,
134.7, 133.6, 132.5, 129.1, 128.8, 128.7, 128.4, 128.2, 127.9, 127.6, 127.4, 127.1, 126.9, 126.0, 118.4,
113.3, 107.0, 68.6, 55.7, 55.2 ppm.
4.1.9.6(±)-2-butyramido-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)propanoic
LX24）
acid·HCl
（
It was obtained as a yellow solid in 40% yield. m.p. 188-190℃; HRMS (ESI): m/z, calculated for
1
C25H27N3O4, 434.2281 (M+H)+, found 434.2270; H-NMR (300MHz, DMSO-d6):
δ 12.6 (s, 1H,
-COOH), 9.42~9.24 (m, 3H, NH=C-NH2), 8.20 (d, 1H, J= 8.1 Hz, -CO-NH-), 7.80~7.04 (m, 10H,
Ar-H), 5.35~5.26 (m, 2H, -CH2-O-), 4.48 (m, 1H, -CH2-CH-NH-), 3.20~2.92 (m, 2H, -CH2-CH-NH-),
2.00 (m, 2H, -CH2-CH2-CH3), 1.98~1.34 (m, 2H, -CH2-CH2-CH3), 0.68 (t, 2H, J= 7.1 Hz,
-CH2-CH2-CH3); 13C-NMR (75MHz, DMSO-d6): δ 173.2, 172.0, 167.5, 165.4, 155.7, 155.4, 143.1,
140.1, 133.6, 133.2, 132.8, 129.0, 128.5, 128.2, 128.1, 127.6, 126.4, 118.6, 107.2, 107.1, 68.6, 68.2,
53.3, 18.5, 13.3 ppm.
4.1.9.7(±)-2-(phenylsulfonamido)-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)propanoic
acid·HCl（LX25）
It was obtained as a light yellow solid in 48% yield. m.p. 182-185℃; HRMS (ESI): m/z, calculated for
1
C27H25N3O5S, 504.1633 (M+H)+, found 504.1656; H-NMR (300MHz, DMSO-d6):
δ 12.8 (s, 1H,
16

ACCEPTED MANUSCRIPT
-COOH), 9.38~9.17 (m, 3H, NH=C-NH2), 8.34~8.30 (d, 1H, J= 9.2 Hz, -SO2-NH-), 7.89~7.19 (m, 14H,
Ar-H), 5.35 (m, 2H, -CH2-O-), 3.91~3.89 (m, 1H, -CH2-CH-NH-), 2.83~2.77 (m, 2H, -CH2-CH-NH-);
13C-NMR (75MHz, DMSO-d6): δ 172.2, 165.4, 155.5, 143.2, 140.9, 132.9, 132.4, 132.1, 131.8, 129.1,
128.5, 128.4, 128.3, 128.0, 127.6, 127.5, 127.3, 127.2, 126.5, 126.0, 118.6, 107.2, 68.2, 57.4 ppm.
4.1.9.8(±)-2-((4-methylphenyl)sulfonamido)-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)propa
noic acid·HCl（LX26）
It was obtained as a yellow solid in 42% yield. m.p. 177-180℃; HRMS (ESI): m/z, calculated for
1
C28H27N3O5S, 518.1823 (M+H)+, found 518.1818; H-NMR (300MHz, DMSO-d6): δ 9.71 (br, 1H,
HCl), 9.35 (d, 1H, J= 6.1 Hz, -SO2-NH-), 8.02~6.98 (m, 17H, Ar-H and NH=C-NH2), 5.37~5.28 (m,
2H, -CH2-O-), 4.68~4.61 (m, 1H, -CH2-CH-NH-), 3.10~2.70 (m, 2H, -CH2-CH-NH-), 2.20 (s, 3H,
CH3-); 13C-NMR (75MHz, DMSO-d6): δ 172.5, 167.5, 155.4, 143.1, 141.9, 137.9, 132.9, 132.3, 129.4,
128.9, 128.4, 128.3, 128.2, 127.6, 127.4, 127.2, 126.3, 126.0, 118.5, 57.5, 68.4, 58.3, 36.1, 20.9, 20.8
ppm.
4.1.9.9(±)-2-((4-methoxyphenyl)sulfonamido)-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)prop
anoic acid·HCl（LX27）
It was obtained as a light yellow solid in 33% yield. m.p. 178-181℃; HRMS (ESI): m/z, calculated for
1
C28H27N3O6S, 534.1728 (M+H)+, found 534.1704; H-NMR (300MHz, DMSO-d6):
δ 12.8 (s, 1H,
-COOH), 9.45~9.32 (m, 3H, NH=C-NH2), 9.20 (br, 1H, HCl), 8.17 (d, 1H, J= 8.0 Hz, -SO2-NH-),
8.19~6.73 (m, 14H, Ar-H), 5.44~5.28 (m, 2H, -CH2-O-), 4.81~4.36 (m, 1H, -CH2-CH-NH-), 3.89 (s,
3H, CH3-O-), 3.11~2.82 (m, 2H, -CH2-CH-NH-); 13C-NMR (75MHz, DMSO-d6): δ 172.4, 167.5, 165.4,
161.6, 155.8, 143.1, 140.1, 132.8, 132.2, 129.1, 128.4, 128.3, 128.2, 127.6, 127.4, 126.5, 126.3, 118.3,
113.6, 113.4, 107.1, 107.0, 68.7, 68.2, 57.7, 57.4, 55.3, 55.2 ppm.
4.1.9.10(±)-2-((4-nitrophenyl)sulfonamido)-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)propan
oic acid·HCl（LX28）
It was obtained as a light yellow solid in 38% yield. m.p. 188-191℃; HRMS (ESI): m/z, calculated for
1
C27H24N4O7S, 549.1560 (M+H)+, found 549.1568; H-NMR ( 300MHz, DMSO-d6): δ 9.38~9.26 (m,
4H, NH=C-NH2 and –SO2-NH-), 8.15~7.00 (m, 14H, Ar-H), 5.35~5.13 (m, 2H, -CH2-O-), 4.77~4.66
(m, 1H, -CH2-CH-NH-), 3.25~2.98 (m, 2H, -CH2-CH-NH-); 13C-NMR (75MHz, DMSO-d6): δ 170.8,
165.1, 155.1, 144.5, 143.6, 140.1, 133.3, 130.5, 129.1, 128.7, 128.6, 128.4, 128.2, 127.9, 127.7, 127.3,
127.1, 126.5, 126.0, 124.0, 123.9, 123.4, 118.6, 108.3, 61.6, 55.9, 18.5 ppm.
17

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4.1.9.11(±)-2-((4-bromophenyl)sulfonamido)-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)propa
noic acid·HCl（LX29）
It was obtained as a yellow solid in 42% yield. m.p. 184-186℃; HRMS (ESI): m/z, calculated for
1
C27H24BrN3O5S, 582.0811 (M+H)+, found 582.0832; H-NMR (300MHz, DMSO-d6): δ 9.47~9.31 (m,
4H, NH=C-NH2 and –SO2-NH-), 7.90~7.04 (m, 14H, Ar-H), 5.34~5.25 (m, 2H, -CH2-O-), 4.80~4.55
(m, 1H, -CH2-CH-NH-), 3.05~2.82 (m, 2H, -CH2-CH-NH-); 13C-NMR (75MHz, DMSO-d6): δ 173.5,
172.2, 151.1, 131.7, 127.4, 124.5, 112.0, 109.6, 102.0, 63.1, 52.8, 51.7, 49.0, 41.9, 36.8, 27.1, 18.5,
13.4 ppm.
4.1.9.12(±)-2-(butylsulfonamido)-3-(6-((4-carbamimidoylbenzyl)oxy)naphthalen-2-yl)propanoic
acid·HCl（LX30）
It was obtained as a light yellow solid in 43% yield. m.p. 197-199℃; HRMS (ESI): m/z, calculated for
C25H29N3O5S, 484.1938 (M+H)+, found 484.1948; 1H-NMR (300MHz, DMSO-d6): δ 9.47~9.31 (m, 4H,
NH=C-NH2 and –SO2-NH-), 7.92~7.20 (m, 14H, Ar-H), 5.36~5.27 (m, 2H, -CH2-O-), 4.77~4.45 (m,
1H, -CH2-CH-NH-), 3.05~2.82 (m, 2H, -CH2-CH-NH-), 1.63~1.40 (m, 2H, -CH2-CH2-CH2-CH3),
1.27~1.08 (m, 2H, -CH2-CH2-CH2-CH3), 1.01~0.81 (m, 2H, -CH2-CH2-CH2-CH3), 0.58~0.56 (m, 2H,
-CH2-CH2-CH2-CH3); 13C-NMR (75MHz, DMSO-d6): δ 175.0, 173.2, 167.7, 165.8, 155.5, 142.8, 134.3,
132.8, 129.0, 127.6, 126.4, 118.6, 107.3, 68.6, 65.3, 59.2, 58.0, 52.0, 50.7, 48.6, 31.0, 27.8, 24.9, 20.7,
13.2 ppm.
4.2 Biology
All animal experiments were handled according to the Animal Ethics Committees of the Institute
of Materia Medica, Chinese Academy of Medical Sciences. The animal protocol was approved by and
carried out according to the Institutional Animal Care and use Committee of China Pharmaceutical
University.
Antiplatelet aggregation assays
Platelet aggregation was measured at 37°C in a platelet aggregometer (PRECILBY-NJ4，Pu
Lisheng Corp.; Beijing, China) performed in Human platelet-rich plasma (PRP) was obtained by
purchasing apheresis platelets from Jiangsu Province Blood Center. Platelet poor plasma (PPP) was
prepared by further centrifugation at 3000r/min for 10 min, removed with a pipet and used in the
reference cell of the aggregometer. Test substances dissolved in saline or 0.2% aqueous DMSO were
18

ACCEPTED MANUSCRIPT
added to PRP to a final volume of 300ul and allowed to incubate for 1 min followed by the addition of
an inducer (10 µM ADP, 1 U/ml thrombin, 6 ꢀM U46619 or 9 ꢀg/ml collagen) to initiate aggregation.
Maximal platelet aggregation (MPA) was defined as the maximal percent increase in light transmission
after addition of ADP related to the light transmission in PPP. The maximal gradient of platelet
aggregation was defined as the maximal percent increase within the first 5 min. The data were obtained
in triplicates.
ꢀꢁꢂꢃꢄꢅꢆꢇꢄꢈꢉꢀꢁꢂꢊꢋꢌꢍꢈꢎ
Inhibition of antiplatelet aggregation =
× 100% (PAR: platelet aggregation ratio)
ꢀꢁꢂꢃꢄꢅꢆꢇꢄꢈ
Calculation of IC50
For each series of experiments in which the compounds were tested in, at least four concentrations
were chosen and a percentage inhibition-concentration curve was derived. From this curve the IC50
value was calculated as the concentration of inhibitor causing a 50% inhibition of the aggregation using
SPSS software.
Fibrinogen/GPIIb/IIIa enzyme-linked immunosorbent assay
Tissue culture-treated flat-bottom polystyrene 96-wells plates were coated with fibrinogen (10
µg/ml), diluted in 0.1 M sodium carbonate, pH 9.5 at 4℃ overnight. After unbound proteins were
removed by washing with TACTS (20 mM Tris, 0.15 M NaCl, pH 7.5, 2 mM CaCl2, 0.05% Tween 20)
wells were blocked with 1% BSA in TACTS for 1 h. The purified human platelet GPIIb/IIIa (50ꢀl, 20
ꢀg/ml in TACTS) (Enzyme Research Laboratories, Southbend, IN, USA), diluted in TACTS containing
0.5% BSA, were added to the wells; then, Samples were added. After 2 h incubation, wells were
washed three times with TACTS followed by addition of goat antihuman integrin β3 antibody (1:2,000,
CBL479; Millipore, Bedford, MA, USA) in TACTS containing 0.5% BSA. Following 1h incubation at
37°C, wells were washed (TACTS) and anti-goat IgG conjugated alkaline phosphatase (1:1,000, A3562;
Sigma) in TACTS containing 0.5% BSA was added to the wells. Before adding the stabilized
p-nitrophenyl phosphate liquid substrate (N7653; Sigma), wells were washed three times with TACTS.
After 30 min of substrate conversion, the reaction was stopped with 3 M NaOH, and absorbance was
read at 405 nm using a Thermomax microplate reader (Molecular Devices). Net specific binding was
obtained by subtracting optical density values from wells coated only with BSA from the total binding
measured as described above. Binding was not detected in the absence of GPIIb/IIIa. All experiments
were performed in triplicate.
Bleeding time assay
19

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The bleeding time was measured by a tail transection method. Briefly, ICR mice pretreated with 3
mg/kg, 1 mg/kg, 0.3 mg/kg LX14, 1 mg/kg Tirofiban or saline 30 min before the experiments, then,
mice were anesthetized intraperitoneal (i.p.) administration of 5% chloral hydrate and placed on a
heating pad to maintain a constant body temperature of 37℃. A 3-mm piece of the tail-tip was cut off
with a sharp scalpel. The tail was immersed into physiologic saline solution preheated to 37℃ and the
time until cessation of bleeding for more than 5s was recorded.
In vivo antithrombotic study by rat arteriovenous shunt thrombosis model
SD rats (both sexes，180~220 g) were used for as rat arteriovenous shunt thrombosis model
according to the method. Rats were treated by tail vein injection administration of 1 mg/kg Tirofiban
and 0.3 mg/kg, 1 mg/kg and 3 mg/kg LX14. After 15 min administration of samples by tail vein
injection, Rats were anesthetized by intraperitoneal (i.p.) administration of 10% chloral hydrate (3
ml/kg). The left jugular vein and right common carotid artery were isolated and catheterised by a shunt
catheter (American Health & Medical Supply International Corp., Scarsdale, NY, USA). This catheter
is composed of three parts including two 4 cm long polyethylene (PE) 60 catheters, which were
introduced into the blood vessels, and a 12 cm long PE160 catheter that is in the middle of the two
PE60 catheters. A rough silk thread (10 cm in length) was in the middle PE160 catheter to induce
thrombosis. The shunt was opened for 20 min and then closed. Silk strings were removed from the
middle PE160 catheter; Then, the dry weight of thrombus was weighed by drying the thrombus at 60℃
for 20 min and removing the cotton thread weigh.
4.3 Docking studies
The binding modes for the ligands to GPIIb/IIIa were generated by CDOCKER in Discovery
Studio 3.0 (Accelrys Software Inc.). In CDOCKER, random ligand conformations were generated
through molecular dynamics, and a variable number of translations/rotations were applied to each
conformation to generate low-energy orientations of the ligand within the active site of rigid receptor.
Final ligand conformations were sorted by CHARMm energy (interaction energy plus ligand strain).
The crystal structures of GPIIb/IIIa (PDB entry code: 2VDM) were extracted from the Protein
Database. All ligands were docked in all possible stereoisomeric forms in an active site located sphere
with 12Å radius for GPIIb/IIIa, which was generated with the CreateSphere function around the
subsequently removed crystal structure ligand. A total of 30 dockings for each ligand were performed,
and the conformers with the lowest CHARMm energy were chosen for interpreting the docking results.
4.4 Molecular Dynamic Simulations
20

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MD simulation of GPIIb/IIIa receptor bound to molecular LX2421 was performed using PMEMD
module of AMBER 12 with ff99SB modifications [25] of the Cornell et al. force field [26]. TIP3P
water molecules were applied to solvate the complex, extending 12 Å away from the protein atom. The
counterions were added to the solvent to keep the system neutral. The geometry of the system was
minimized before MD simulation. The whole system was heated from 0 to 300 K running 50 ps
molecular dynamics with position restraints at constant volume. Subsequent isothermal isobaric
ensemble (NPT)-MD was performed for 500 ps to adjust the solvent density followed by 500 ps of
constant pressure equilibration at 300K without constraints to relax the system. The production
dynamics at constant pressure achieved lengths of 40 ns of which snapshots saved at 20 ps intervals.
The “ptraj” tool in Amber 12 was used to analyze the time-dependence of the RMSD of the Cα atoms.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (81273375),
China Pharmaceutical University. The authors declare no other conflicts of interest.
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Figure caption
Fig. 1. The mechanism of platelet aggregation and some representing drugs of antiplatelet aggregation.
Fig. 2. (A) The docking model of LX2421 with GPIIb/IIIa receptor, the figure was prepared by Discovery Studio 3.0. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.). (B) The key
interactions formed between LX2421 and GPIIb/IIIa along the MD trajectories. The key interactions of which occupancies were
more than 40% were considered and shown in the figure. The key interactions are colored as red (occupancy greater than 80%),
purple (occupancy of 60−80%), blue (occupancy of 40−60%).
Fig. 3. The identification of novel compound LX5.
Fig. 4. The activity of LX14 (A, B, C) and LX25 (D, E, F) for inhibiting Collagen (1ꢀg/ml) Thrombin (0.26 U/ml) and U46619
(2 ꢀM)-induced human platelet aggregation. The activity was shown as an inhibition ratio to control group. Data are expressed as
mean ± SD (n=3).
Fig. 5. The GPIIb/IIIa receptor inhibitory activities of LX14 and LX25 in comparison with Tirofiban in the fibrinogen/GPIIb/IIIa
enzyme-linked immunosorbent assay. Data are expressed as mean ± SD (n=3).
Fig. 6. Docking mode of LX14 (A) and LX25 (B) binding to GPIIb/IIIa receptor in comparison with Tirfiban, figure A:
compound LX14 (purple, bold), Tirofbian (brown, thin); figure B: compound LX25 (blue). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.).
Fig. 7. Bleeding time induced by intravenous injection of compound LX14 (3, 15, and 30 mg/kg) and Tirofiban (1 mg/kg). The
accumulated bleeding time was recorded within 20 min. Data are expressed as mean ± SD (n= 6). *P < 0.05, ****P < 0.0001
compared with control group, #P < 0.01 compared with Tirofiban-treated group.
Fig. 8. The antithrombotic activities of compound LX14 and Tirofiban in vivo in the rat arteriovenous shunt thrombosis model.
Three doses were tested for LX14 (1, 5 and 10 mg/kg), and one dose (1 mg/kg) was tested for Tirofiban. Data are expressed as
mean ± SD (n= 6). *P < 0.05, ****P < 0.0001 compared with control group.
24

ACCEPTED MANUSCRIPT
Scheme caption
Scheme 1. Conditions and reagents: a) RCl , K2CO3, acetone/water (v:v=1:2), 65℃, overnight, 80-88%; b) LiOH, acetone/
methanol/water (v:v:v=1:1:1), 50 ℃ , overnight, 75-89%; c) 1-Boc-4-(methylsulfonyl)oxy)propyl)piperidine
1-Boc-4-(2-((methylsulfonyl)oxy)propyl)piperidine or 1-Boc-4-(2-((methylsulfonyl)oxy)propyl)piperidine
or
or
1-Boc-4-(3-((methylsulfonyl)oxy)propyl)piperidine or 1-Boc-4-(4-((methylsulfonyl)oxy)butyl)piperidine, K2CO3, DMF, N2,
80℃, 12 h, 42-48%; d) HCl (g), AcOEt, -5 ℃, 4h, 82-90%; e) SOCl2 , methanol, 50 ℃, 6 h, 95%; f) Br(CH2)nBr, K2CO3, dry
acetonitrile, 60℃, 48 h, 35-50%; g) N-Boc-piperazine, K2CO3, dry acetonitrile, 60℃, 48 h, 30-60%; h) LiOH•H2O,
MeOH/THF/H2O (v:v:v=1:1:1), r.t., 5 h, 40-55%; i) HCl(g), AcOEt, 0℃, 4 h, 90-95%. j) 4-CNPh-CH2Br (1.5 equiv.)/K2CO3 (2.0
equiv.), dry acetonitrile, reflux, overnight, 60%~70%; k) HCl/MeOH, NH3/MeOH, -5℃, overnight, 45%~50%; l) LiOH•H2O,
MeOH/THF/H2O (v:v:v=1:1:1), r.t., overnight, 65-68%.
25

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Table 1 Antiplatelet aggregation activity of naphthalene derivatives LX1-LX6 designed from LX2421.
Compound
LX1
R1
R2
n
Inhibition%/50µMa
IC50(µM)b
0
<5.00
>100
LX2
LX3
LX4
LX5
LX6
2421
1
2
1
2
3
<5.00
>100
>100
<5.00
97.3±1.6
99.1±2.3
91.2±2.4
38.1±1.8
12.8±1.04
0.37±0.03
18.8±1.12
NDc
a In vitro inhibition of ADP-induced (20 µM) human platelet aggregation; See the Experimental Section
for details; the data represent the mean of at least three independent determinations.
b
In vitro inhibition of ADP-induced (20 µM) human platelet aggregation; See the Experimental
Section for details; Values are means from two to three independent dose–response curves.
c ND means not determined.

ACCEPTED MANUSCRIPT
Table 2 Antiplatelet aggregation activity of naphthalene derivatives LX19-LX30 designed from LX5.
Compound
LX7
R
IC50(µM)a
Compound
LX13
R
IC50(µM)a
7.90±2.11
5.40±0.04
LX8
6.38±1.05
LX14
0.18±0.07
LX9
7.60±1.54
LX15
LX16
0.28±1.12
0.20±0.08
LX10
24.60±1.98
LX11
LX12
3.40±0.06
12.70±1.62
NDb
LX17
LX18
1.65±1.01
0.52±0.04
0.09±0.03
LX2421
Tirofiban
a In vitro inhibition of ADP-induced (20 µM) human platelet aggregation; See the Experimental Section
for details; Values are means from two to three independent dose–response curves.
b ND means not determined.

ACCEPTED MANUSCRIPT
Table 3 Antiplatelet aggregation activity of naphthalene derivatives LX19-LX30 designed from LX5.
Cpd
R
Inhibition%/
IC50(µM)a
Cpd
R
Inhibition%/ IC50(µM)a
50µMb
50µMb
LX19
LX20
LX21
LX22
LX23
LX24
LX2421
49.4±2.3
49.7±0.8
23.3±4.1
>99.5
NDc
ND
LX25
LX26
>99.5
>99.5
0.84±0.09
4.01±2.31
ND
ND
LX27
21.5±1.5
62.9±2.7
50.2±3.4
>99.5
2.10±1.22
ND
LX28
ND
32.4±3.9
>99.5
LX29
ND
1.08±0.06
ND
LX30
3.29±1.07
0.09±0.03
Tirofiban
a In vitro inhibition of ADP-induced (20 µM) human platelet aggregation; See the Experimental Section
for details; Values are means from two to three independent dose–response curves.
b
In vitro inhibition of ADP-induced (20 µM) human platelet aggregation; See the Experimental
Section for details; The data represent the mean of at least three independent determinations.
c ND means not determined.