Novel hybrids of 3-n-butylphthalide and edaravone: Design, synthesis and evaluations as potential anti-ischemic stroke agents

Article abstract of DOI:10.1016/j.bmcl.2015.06.090

Abstract Fourteen hybrids (10a-g, 11a-g) of 3-n-butylphthalide (NBP) and edaravone (Eda) analogues have been designed and synthesized as potential anti-ischemic stroke agents. In vitro biological studies showed that compounds 10d and 10g exhibited more potent anti-platelet aggregation than ticlopidine (Ticlid), aspirin (ASP) and NBP. Compound 10g more significantly prevented H₂O₂-mediated neuronal cell (PC12) death than NBP, Eda or NBP together with Eda. Meanwhile, 10g also possessed potent radical scavenging effects on hydroxyl radical (·OH) and superoxide anion radical (·O₂^-). Our findings may provide new insights into the development of these hybrids, like 10g, for the intervention of ischemic stroke.

Full text of DOI:10.1016/j.bmcl.2015.06.090

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel hybrids of 3-n-butylphthalide and edaravone: Design,
synthesis and evaluations as potential anti-ischemic stroke agents
a,b,c,
Xiao Sheng a,b,c,e, , Kai Hua a,d, , Chunyu Yang a,b,c, Xiaoli Wang a,b,c, Hui Ji a,d, , Jinyi Xu
,
⇑
⇑
Zhangjian Huang a,b,c, , Yihua Zhang a,b,c
⇑
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
b Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China Pharmaceutical University, Nanjing 210009, PR China
c Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
d Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, PR China
e Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Fourteen hybrids (10a–g, 11a–g) of 3-n-butylphthalide (NBP) and edaravone (Eda) analogues have been
designed and synthesized as potential anti-ischemic stroke agents. In vitro biological studies showed that
compounds 10d and 10g exhibited more potent anti-platelet aggregation than ticlopidine (Ticlid), aspirin
(ASP) and NBP. Compound 10g more significantly prevented H2O2-mediated neuronal cell (PC12) death
than NBP, Eda or NBP together with Eda. Meanwhile, 10g also possessed potent radical scavenging effects
on hydroxyl radical (ÅOH) and superoxide anion radical (ÅOÀ2 ). Our findings may provide new insights into
the development of these hybrids, like 10g, for the intervention of ischemic stroke.
Received 24 January 2015
Revised 10 June 2015
Accepted 25 June 2015
Available online xxxx
Keywords:
3-n-Butylphthalide (NBP)
Edaravone (Eda)
Ó 2015 Elsevier Ltd. All rights reserved.
Anti-platelet aggregation
Antioxidant activity
Anti-ischemic stroke
Stroke, is one of the common cardiovascular and cerebrovascu-
lar diseases, and has become a chief cause of adult disability and
the second cause of death worldwide.1 Among strokes, ischemic -
stroke occupies approximately 80% of all, seriously harming to
human health.2 It is reported that platelet-mediated thrombosis
can cause blood vessel blockage,3 leading to interrupted blood sup-
ply to the brain, thereby causing the brain tissue energy depletion,
metabolic dysfunction, and ischemic brain damage.4
Currently, therapeutic strategies for ischemic stroke include
two aspects: (1) preventing and/or correcting the inducing factors
of cerebral ischemia; (2) recovering brain perfusion after ischemia
to improve cerebral blood and to block ischemic injury cascade,
thus, preventing neuronal damage. In view of the complicated
pathological mechanisms of ischemic stroke, the drugs with
different mechanisms, rather than a single antiplatelet activity,
are needed. In this regard, a hybrid multitargeting prodrug that
will release each component in vivo, to furnish the desired
pharmacological effects at the same prodrug dose, warrants further
exploration, especially in the development of anti-ischemic stroke
drugs.
Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) (Fig. 1) is a
novel free radical scavenger, and was approved by the Japanese
Ministry of Health in 2001, mainly for the treatment of ischemic
stroke. Edaravone exerts neuroprotective effects through scaveng-
ing free radicals, inhibiting lipid peroxidation and oxidative dam-
age to brain cells, endothelial cells and nerve cells, reducing
cerebral ischemia and edema, and thus decreasing tissue damage.
Furthermore, edaravone can also effectively improve neurological
deficits caused by the acute cerebral infarction.5
It is demonstrated that edaravone has three tautomeric forms:
the amine (Eda), keto (1), and enol (2) forms. Edaravone exerts free
radical scavenging effects through a one-electron transfer mecha-
nism. The pyrazolone ring enol (2) loses a proton to form pyra-
zolone ring conjugated anionic form (3). Subsequently, the anion
3 transfers an electron to free radicals, such as peroxyl (LOOÁ)
and hydroxyl radicals (ÅOH), to furnish a radical intermediate
⇑
Corresponding authors. Tel.: +86 25 86021369; fax: +86 25 86635503 (H.J.);
tel.: +86 25 83271445; fax: +86 25 83302827 (J.X.); tel.: +86 25 83271072; fax: +86
25 83271015 (Z.H.).
(3A), which finally forms
a stable oxidation product (OPB:
2-oxo-3-(phenylhydrazono)-butanoic acid), generating free radical
scavenging effects (Fig. 1).6
E-mail addresses: huijicpu@163.com (H. Ji), jinyixu@china.com (J. Xu),
cpudahuang@163.com (Z. Huang).
The racemic 3-n-butylphthalide (NBP) (Fig. 2), as a natural drug
extracted from celery seed, was approved for the treatment of
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2015.06.090
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sheng, X.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.090

2
X. Sheng et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
R2
N
N
N
O
O
NH
OH
HN
N
O
N
N
O
2
Edaravone
1
O
R1
H
H
O
O
O
R2
N
OH
R2
N
O
LOO
HO
N
N
OH
N
N
O
LO
OH
O
3
R1
R1
HPBA
10a-g,11a-g
LOO
LO
OH
Figure 3. Rationale for the design of hybrids 10a–g and 11a–g.
NH
O
OH
N
N
O
N
O
ongoing program, the hydroxyl group of enol tautomer of
edaravone or its trifluoromethyl analogue was coupled with the
carboxylic acid group of HPBA to form a group of hybrids (10a–g
and 11a–g, Fig. 3). We hypothesized that these hybrids, under
esterase-induced hydrolysis in vivo, could spontaneously generate
edaravone or edaravone analogues and HPBA, which forms
3A
OPB
Figure 1. Edaravone and its two tautomeric forms, as well as a one-electron
transfer mechanism underlying its free radical scavenging activity.
ischemic stroke by State Food and Drug Administration (SFDA) of
China in 2002. It has been reported that NBP can have effects on
multiple points of pathological processes in ischemic stroke, pos-
sessing a variety of biological activities such as anti-platelet aggre-
gation, anti-thrombosis, improving cerebral microcirculation and
reducing infarct volume.7 HPBA (Fig. 2) is the ring-opening deriva-
tive of NBP, could cyclize to NBP in vivo.8 Recently, our research
group has designed and synthesized several groups of hybrids of
HPBA with other small active molecules, and some hybrids such
as (R/S)-ZJM-289 and compound 4 (Fig. 2) possess more potent
therapeutic efficacy for intervention of ischemic stroke than
NBP.9–11
Given that edaravone and NBP have been clinically used for the
treatment of ischemic stroke via different mechanisms, it is there-
fore of interest to determine whether coupling edaravone and NBP
would provide a hitherto unknown class of hybrids that possess
potent and synergetic anti-ischemic stroke activity. As part of this
NBP after a cyclization reaction, generating complementary
anti-ischemic stroke effects. Herein, we described the synthesis
and biological evaluations for these hybrids.
The synthesis of the target compounds 10a–g and 11a–g was
depicted in Scheme 1. Edaravone derivatives 6a–g and 7a–g were
obtained in 50–70% yields from the reactions of substituted phenyl
hydrazine hydrochloride with acetylacetic ether or trifluoroacetyl
ethyl acetate in the presence of sodium acetate under reflux.
Starting from the reaction of 2-formylbenzoic acid 8 with
Grignard reagent n-BuMgBr, the consequent acidification gave
NBP in 85% yield. Subsequent ring-opening of NBP in the presence
of sodium hydroxide produced HPBA in 92% yield. Without further
purification, the hydroxyl at the side chain of HPBA was reacted
with acetyl chloride, providing intermediated 9 in the yield of
77%. Compound 9 was reacted with oxalyl chloride in anhydrous
dichloromethane to give the intermediate acyl chloride, followed
by the esterification with edaravone derivatives in the presence
of triethylamine to offer target compounds 10a–g and 11a–g in
43–60% yields. All target compounds were purified by column
chromatography and their structures were characterized by IR,
MS, 1H NMR, 13C NMR and HR-MS.12,13
OH
The inhibitory effects of target compounds on adenosine
diphosphate (ADP)- or arachidonic acid (AA)-induced platelet
aggregation in vitro were evaluated using Born’s turbidimetric
method, and ticlopidine hydrochloride (Ticlid) and aspirin (ASP)
were used as positive controls (Table 1).14,15 As comparison, the
inhibitory effects of NBP, Eda or combination administration of
NBP together with Eda groups were also investigated. The assay
was repeated six times, and data were calculated and expressed
as the inhibition rate of platelet aggregation.11,16
As shown in Table 1, for inhibiting the ADP-induced platelet
aggregation, 10c (30.17%), 10d (35.20%), 10e (29.33%), 10f
(33.87%), 10g (31.80%), 11a (26.93%), 11b (25.48%) and
11d (31.24%) showed more potent inhibitory effect than Ticlid
(11.53%), NBP (22.95%), Eda (24.77%) and NBP together with Eda
(28.06%). And 10d, 10g inhibited the AA-induced platelet
aggregation with inhibition rates of 46.11% and 59.87%, respec-
tively, significantly superior to all reference compounds ASP
(16.16%), NBP (14.10%), Eda (25.47%) and NBP together with Eda
(42.45%) in vitro.
O
OH
O
O
HPBA
NBP
O
O
N
N
H
O
O
O
N
HCl
O
O
O
O
O
O
O
O
H
N
O
O
O
(CH2)4ONO2
O
N
(R)-ZJM289
(S)-ZJM289
4
Figure 2. Structrues of NBP, HPBA, (R/S)-ZJM289 and 4.
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X. Sheng et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
NHNH2
HCl
6a R1 = H, R2 = CH3
7a R1 = H, R2 = CF3
O
6b R1 = 4-CH3, R2 = CH3 7b R1 = 4-CH3, R2 = CF3
a
6c R1 = 4-OCH3, R2 = CH3 7c R1 = 4-OCH3, R2 = CF3
N
R1
R1
5a- g
R1 = 3-Cl, R2 = CH3
R1 = 4-Cl, R2 = CH3
R1 = 4-NO2, R2 = CH3
R1 = 4-F, R2 = CH3
R1 = 3-Cl, R2 = CF3
R1 = 4-Cl, R2 = CF3
R1 = 4-NO2, R2 = CF3
R1 = 4-F, R2 = CF3
N
R2
6d
6e
6f
7d
7e
7f
6a-g, 7a-g
6g
7g
O
O
O
OH
OH
b
d
c
H
OH
O
OH
O
O
O
O
9
8
NBP
HPBA
O
O
10a R1 = H, R2 = CH3
11a R1 = H, R2 = CF3
O
10b R1 = 4-CH3, R2 = CH3 11b R1 = 4-CH3, R2 = CF3
10c R1 = 4-OCH3, R2 = CH3 11c R1 = 4-OCH3, R2 = CF3
e
R2
R1 = 3-Cl, R2 = CH3
R1 = 4-Cl, R2 = CH3
R1 = 4-NO2, R2 = CH3
R1 = 4-F, R2 = CH3
R1 = 3-Cl, R2 = CF3
R1 = 4-Cl, R2 = CF3
R1 = 4-NO2, R2 = CF3
R1 = 4-F, R2 = CF3
10d
10e
10f
10g
11d
11e
11f
11g
N
N
O
R1
10a-g, 11a-g
Scheme 1. Synthetic routes of the target compounds 10a–g and 11a–g. Reagents and conditions: (a) AcONa, AcOH, reflux, N2, 5–10 h; (b) (i) n-BuMgBr, Et2O, À5 to 0 °C, 5 h;
(ii) 1 M HCl, rt, 0.5 h; (c) (i)NaOH, CH3OH–H2O, 50 °C, 0.5 h; (ii) 1 M HCl, À10 to 0 °C; (d) CH3COCl, Et3N, DMAP, dry CH2Cl2, À10 °C, 7 h; (e) (i) (COCl)2, dry CH2Cl2, rt, 8 h; (ii)
6a–g or 7a–g, Et3N, dry CH2Cl2, 0 °C–rt, 4–6 h.
Table 1
Inhibitory effects of 10a–g and 11a–g (0.1 mM) on ADP- or AA-induced platelet
ꢀ
aggregation in vitro (n = 6, x s)
Compound
ADP (%)
AA (%)
Ticlopidine
Aspirin
NBP
Eda
NBP + Eda
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
11e
11f
11.53 3.20
16.16 3.16
14.10 3.17
25.47 4.05
42.45 4.87
15.39 5.50
20.49 5.12
29.36 4.90
46.11 4.26
40.32 3.47
39.61 4.86
59.87 6.03***
29.29 3.81
40.67 4.54
37.66 5.86
21.21 3.88
26.58 2.91
21.30 1.31
27.52 4.23
22.95 2.45
24.77 3.60
28.06 1.82
23.01 2.89
21.32 2.81
30.17 3.79
35.20 4.10***
29.33 1.79
33.87 4.11***
31.80 4.59***
26.93 3.85
25.48 3.56
19.88 2.22
31.24 3.10
23.22 3.22
15.43 1.66
13.60 2.55
Figure 4. Protective effects of the target compounds on nerve cells damage in vitro.
PC12 cells were pre-treated with vehicle, all positive controls (10 M) or target
compounds (10 M) for 1 h, and then co-cultured with H2O2 (800 M) for 3 h. The
l
11g
l
l
PRP were preincubated with tested compounds (0.1 mM) at 37 °C for 5 min fol-
lowed by the addition of ADP (10 M) or AA (1 mM). Data are expressed as
mean SD of each group (n = 6) and analyzed by one-way analysis of variance
cell viability was determined by MTT assay. Results were obtained from indepen-
dent experiments and were expressed as mean SD (n = 6). ⁄P <0.05 versus NBP
group; ⁄⁄P <0.01 versus NBP group; ⁄⁄⁄P <0.001 versus NBP group; ###P <0.001
versus NBP + Eda group.
l
(ANOVA) followed by post hoc Tukey test.
***
P<0.05 versus NBP + Eda.
As shown in Figure 4, the protective effects of 10b (86.02%), 10f
(85.22%), 10g (93.33%), 11e (82.95%) and 11f (87.31%) were supe-
rior to those of all positive controls, Ticlid (67.22%), ASP (77.37%),
NBP (71.01%), Eda (76.89%) and NBP together with Eda (76.60%)
at the same concentration, and 10g exhibited the greatest
protective effects in all test compounds. All these results supported
us in selecting 10g as the candidate compound for further
investigation.
Since these hybrids are the precursors of edaravone or its triflu-
oromethyl analogue, which act as radical scavengers generating
protection activity,17 we established the hydrogen peroxide
induced PC12 cells injury model in vitro, and preliminarily investi-
gated the protective effects of all target compounds on nerve cells
injury caused by the treatment of H2O2, using Ticlid, ASP, NBP, Eda
and NBP together with Eda as positive controls.18,19
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4
X. Sheng et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Given that 10g is composed of two moieties, NBP’s precursor 9
Furthermore, we evaluated the hydroxyl radical (ÅOH) and
superoxide anion radical (ÅO2À) scavenging activity of 10g in vitro
using Griess assay and NBT assay, respectively.20,21 It was found
that the ÅOH scavenging activity of 10g (65.5100 U/mL) was
significantly superior to that of Ticlid (15.0616 U/mL), ASP
(30.6956 U/mL), NBP (16.2026 U/mL), Eda (25.2753 U/mL) and
NBP together with Eda (36.8347 U/mL) at the same concentration
and edaravone’s precursor 6g, we investigated their individual
contribution to the overall inhibitory activity against ADP-induced
or AA-induced platelet aggregation and protective effects on nerve
cells damage caused by the treatment of H2O2 in vitro.
It is shown that compounds 6g, 9 or 6g together with 9 all dis-
played moderate anti-platelet aggregation activity (inhibition rates
are 13.46% and 24.34%, 17.73% and 27.73%, 16.21% and 25.03%, in
two respective models), but less than 10g (31.80% and 59.87%
Fig. 5A and B). Similarly, 10g exhibited better nerve cell protective
activity (cell viability percentages are 93.33% and 88.06% at the
of 10 l
M. (Fig. 5D) The ÅO2À scavenging activity of 10g
(141.4900 U/L) had an advantage over that of Ticlid (26.9592 U/L),
ASP (79.3793 U/L), NBP (36.7429 U/L) and Eda (97.6942 U/L), and
was comparable to that of NBP together with Eda (140.7870 U/L)
Å
dose of 10
lM and 1
l
M, respectively, Fig. 5C) than its moiety
at the same concentration of 10
l
M. (Fig. 5E). Notably, the OH or
compounds 6g, 9 or 6g together with 9. These data suggested
that the improved inhibitory activity against ADP-induced or
AA-induced platelet aggregation and protective effect against
H2O2-induced cell damage in vitro may derive from the synergistic
effects of its two moieties.
ÅOÀ2 scavenging effect of 6g (31.7447 U/mL or 117.5790 U/L), 9
(17.6015 U/mL or 56.1954 U/L), and 6g together with
9
(44.0229 U/mL or 133.1410 U/L) were all less than 10g at the same
dosage. These may also suggest that the two moieties have syner-
gistic effects on scavenging the ÅOH or ÅO2À free radical in vitro.
Figure 5. (A) Inhibition of ADP-induced rabbit platelet aggregation by selected compounds in vitro. (B) Inhibition of AA-induced rabbit platelet aggregation by selected
compounds in vitro. Rabbit platelet suspensions were pre-incubated with tested compounds (0.1 mM) at 37 °C for 5 min followed by addition of ADP (10 M) or AA (l mM)
was added and incubated with the drug-platelet suspension in the indicated group. (C) Protective effects of selected compounds on nerve cells damage in vitro. PC12 cells
l
were pre-treated with model, all positive controls(10
l
M), selected compounds (10
l
M) and compound 10g (1–10
l
M) for 1 h, and then co-cultured with H2O2 (800
l
M) for
Å
3 h. The cell viability was determined by MTT assay. (D) OH scavenging effects of selected compounds in PC12 cells. PC12 cells were pre-treated with all positive controls
(10 lM), selected compounds (10 lM) and compound 10g (1–10 lM) for 1 h, then co-cultured with 1 mM H2O2/20 l
M Fe2+ (pH = 7.4). The reduced activity of ÅOH was
detected by Griess assay. (E)ÁÅO2À scavenging effects of selected compounds in PC12 cells. PC12 cells were pre-treated with all positive controls (10
l
M), selected compounds
M NBT at the same concentration. The reduced activity of OÀ2 was
Å
(10
lM) and compound 10g (1–10
lM) for 1 h, then co-cultured with DMSO, 468
l
M NADH and 300
l
detected by NBT assay. Data are expressed as mean SD of each group (n = 6) for six separate experiments. Data was analysed by one-way analysis of variance (ANOVA)
followed by post hoc Turkey test. &&P <0.01 versus NBP + Eda group, &&&P <0.001 versus NBP + Eda group, ⁄⁄P <0.01 versus compound 9, ⁄⁄⁄P <0.001 versus compound 9,
###P <0.001 versus 6g + 9 group.
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X. Sheng et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Iwasaki, Y. Brain Res. 2004, 1029, 200; (c) Watanabe, K.; Morinaka, Y.; Iseki, K.;
Watanabe, T.; Yuki, S.; Nishi, H. Redox Rep. 2003, 8, 151; (d) Higashi, Y.; Jitsuiki,
D.; Chayama, K.; Yoshizumi, M. Recent Pat. Cardiovasc. Drug Discov. 2006, 1, 85;
(e) Yoshida, H.; Yanai, H.; Namiki, Y.; Fukatsu-Sasaki, K.; Furutani, N.; Tada, N.
CNS Drug Rev. 2006, 12, 9; (f) Nakamura, T.; Kuroda, Y.; Yamashita, S.; Zhang,
X.; Miyamoto, O.; Tamiya, T.; Nagao, S.; Xi, G. H.; Keep, R. F.; Itano, T. Stroke
2008, 39, 463.
Among target compounds 10a–g, the substituent on the phenyl
ring in edaravone moiety affected the inhibitory activity of platelet
aggregation, and electron withdrawing substituted compounds
were more active than electron donating groups. As to 11a–g, the
relationship between electronic effect of substitution and
inhibitory activity of platelet aggregation is contrary to 10a–g.
Interestingly, when the phenyl ring of edaravone derivatives sub-
stituted with electron donating groups, the inhibitory activity of
platelet aggregation of the target compounds 11a–c with 3-triflu-
oromethyl pyrazolone moiety was also higher than 10a–c with
3-methyl pyrazolone. Meanwhile, when the phenyl ring of edar-
avone derivatives substituted with electron withdrawing groups,
such as chlorine atom, fluorine atom, and nitro group, the inhibi-
tory activity of platelet aggregation of the target compounds
10d–g with 3-methyl pyrazolone moiety is much higher than that
of 11d–g. However, the antioxidant activity did not show the same
rules, the target compounds showed varying degrees of antioxi-
dant activity, and the anti-platelet aggregation and antioxidant
activity of 10g is optimal in all. From these results we can speculate
that those various substituents may have different abilities to
modulate the structure, stability, metabolism and penetrability of
the hybrids, affecting the releasing of NBP and edaravone ana-
logues, and leading to varied bioactivities of the hybrids.
Altogether, a series of novel hybrids of edaravone analogues and
NBP ring-opening compound were designed and synthesized in the
present study. Among them, 10d, 10g have stronger inhibitory
effects on ADP- induced and AA-induced platelet aggregation than
Ticlid, ASP, NBP, Eda and NBP together with Eda in vitro.
Furthermore, we also found that 10g exhibited a potent inhibitory
activity against the H2O2-mediated cytotoxicity in PC12 cells and
could scavenge free radical (ÅOH) and (ÅOÀ2 ), a major contributor
to secondary brain injury induced by reperfusion after occlusion
of brain vessels, which is more potent than those of NBP, Eda, 6g,
9, even than the co-administration of two active fragment 6g and
9 in further study. Notably, 10g has an ester bond between its
two fragments, which may make it has a better liposolubility to
enter into cells easily or the breaking of the ester bond may take
a long time so as to prolong the duration of action. All these sug-
gest that 10g may undergo rapid hydrolysis by esterases to yield
edaravone analogue 6g and NBP ring-opening derivative 9, which
can undergo a conversion to produce NBP. So it would exhibit edar-
avone-dependent and NBP-dependent activities to exert synergis-
tic effect and to enhance its therapeutic potency. In summary,
10g may be a potential agent for the intervention of ischemic
stroke and such hybrids based on edaravone and NBP ring-opening
derivatives may represent a novel class of candidates for the
treatment of ischemic stroke.
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Zhang, Y. H. Org. Biomol. Chem. 2011, 9, 5670.
10. Wang, X. L.; Zhao, Q.; Wang, X. L.; Li, T. T.; Lai, Y. S.; Peng, S. X.; Ji, H.; Xu, J. Y.;
Zhang, Y. H. Org. Biomol. Chem. 2012, 10, 9030.
11. Wang, X. L.; Wang, L. N.; Li, T. T.; Huang, Z. J.; Lai, Y. S.; Ji, H.; Wan, X. L.; Xu, J.
Y.; Tian, J. D.; Zhang, Y. H. J. Med. Chem. 2013, 56, 3078.
12. General procedure for the synthesis of the target compounds 10a–g and 11a–
g: acetylacetic ether and trifluoroacetyl ethyl acetate (25 mmol) were added to
a mixture solution of 5a–g (25 mmol) and sodium acetate (26 mmol) in acetic
acid (20 mL). The reaction mixture was stirred at reflux temperature for 5–
10 h. After cooling, the mixture was added to a saturated solution of NaHCO3
until its pH value is adjusted to 7 and extracted with ethyl acetate for three
times. The combined organic layer was then dried, filtered, concentrated and
the residue was purified by column chromatography (PE/EtOAc = 6:1, v/v) to
give light yellow solid 6a–g and 7a–g in 50–70% yield. To a solution of 9
(4.7 mmol) in anhydrous dichloromethane (20 mL), oxalyl chloride
(6.24 mmol) was added and stirred at room temperature for 8 h, then
solvent of the mixture was evaporated to obtain the corresponding acyl
chloride. A solution of 6a–g and 7a–g (7.2 mmol), triethylamine (9.6 mmol) in
anhydrous dichloromethane (20 mL) was stirred for 10 min under 0 °C. Then
the acyl chloride of 9 obtained above in anhydrous dichloromethane (20 mL)
was added dropwise to the solution, the reaction mixture was stirred at room
temperature for 4–6 h and then poured into water, extracted with ethyl acetate
for three times, the combined organic phase was then dried, filtered, and
evaporated to dryness. The residue was purified by column chromatography
(PE/EtOAc = 10:1, v/v) to give pure 10a–g and 11a–g in 43–60% yield.
13. Analytical data for 10g: mp 67–69 °C. ESI-MS m/z: 425 [M+H]+; 447 [M+Na]+.
IR (cmÀ1, KBr): 3076, 2957, 2923, 2856, 1752, 1731, 1601, 1546, 1417, 1390,
1146, 1096, 939 783, 750, 661. 1HNMR (CDCl3, 300 MHz, d): 0.87 (t, 3H, CH3,
J = 7.2 Hz), 1.26–1.41 (m, 4H, 2 Â CH2), 1.69–1.85 (m, 2H, CH2), 2.07 (s, 3H,
CH3), 2.35 (s, 3H, CH3), 6.25 (s, 1H, C@CH), 6.44–6.49 (m, 1H, CH), 7.07–7.14
(m, 2H, ArH), 7.31–7.36 (m, 1H, ArH), 7.52–7.62 (m, 4H, ArH), 7.91 (d, 1H, ArH,
J = 7.8 Hz). 13C NMR (CDCl3,75 MHz, d):170.2, 163.2, 161.7, 159.9, 149.1, 145.6,
144.4, 133.7, 130.5, 127.4, 126.5, 125.5, 125.3, 125.2, 116.1, 115.8, 95.9, 72.6,
36.3, 27.9, 22.3, 21.1, 14.4, 13.9. HR-MS (ESI) for C24H25FN2O4 ([M+Na]+) calcd
447.1696, found: 447.1709.
14. Born, G. V.; Cross, M. J. J. Physiol. 1963, 168, 178.
15. Jacobson, A. K. Best Pract. Res. Clin. Haematol. 2004, 17, 55.
16. Blood samples were withdrawn from rabbit carotid artery and mixed with 3.8%
trisodium citrate (9:1 v/v), followed by centrifuging at 500 rpm for 10 min. The
supernatants were collected and used as platelet rich plasma (PRP). Additional
samples were centrifuged at 3000 rpm for 10 min and the supernatants were
collected as platelet poor plasma (PPP). The effect of individual compounds on
the ADP-induced and AA-induced platelet aggregation was measured by Born’s
turbidimetric method using a Platelet-Aggregometer (LG-PABER-I Platelet-
Acknowledgements
This study was financially supported by Grants from the
National Natural Science Foundation of China (General Program,
No. 81373419), the Ph.D. Programs Foundation of Ministry of
Education of China, China Pharmaceutical University (No.
20130096110005) and the Project Program of State Key
Laboratory of Natural Medicines, China Pharmaceutical
University (No. SKLNMZZYQ201301).
Aggregometer, Beijing). Briefly, PRP (240
vehicle, different concentrations of all positive drugs and all individual
compounds for 5 min and exposed to 10 of ADP or 1.0 mM of AA
lL) was pre-treated in duplicate with
lM
incubated at 37 °C for 5 min, respectively. The formation of platelet
aggregation was monitored longitudinally by optical density. Compounds
under study or vehicle alone were added to the PRP samples 5 min before
addition of the aggregating agent. The antiplatelet aggregatory activity of all
positive control and all individual compounds was evaluated as percent
inhibition of platelet aggregation compared to positive controls that had been
pre-treated with vehicle alone and exposed to the inducer samples.
17. Kamida, T.; Fujiki, M.; Ooba, H.; Anan, M.; Abe, T.; Kobayashi, H. Seizure 2009,
18, 71.
References and notes
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19. PC12 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% heat inactivated horse serum (Hyclone), 5% fetal
bovine serum (GIBCO), 1.0 mM sodium pyruvate, 100 U/mL penicillin, and
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100 lg/mL streptomycin at 37 °C in a 5% CO2 atmosphere (Thermo Scientific,
3110, OH, U.S.). During the exponential phase of growth, PC12 cells
(20,000 cells/well) were cultured in 96-well plates that had been coated with
Please cite this article in press as: Sheng, X.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.090

6
X. Sheng et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
-lysine for 24 h. When the concentration of H2O2 is 800 mM in 3 h, the of 10g for 1 h. Then we exposed them to 1 mM H2O2/20 l
M Fe2+ (pH = 7.4).
poly-
L
cell survival rate was 50.85%, so we choose this concentration as the best
concentration of damage. The cells were treated in triplicate with different
After replacement with fresh medium, the cells of every couple was collected
in 0.5 mL of phosphate buffer (pH = 7.4), we detected the reduced activity of
ÅOH by Griess assay. On the other hand, 0.5 mL every test tube was added
concentrations (1–10
800 M H2O2 for 3 h. After replacement with fresh medium, the cells were
incubated for 4 h and the cell viability was determined by MTT.
lM) of the tested compounds for 1 h and exposed to
l
2.0 mL of DMSO, 2.0 mL of 468 lm NADH, 1.0 mL of 300 lm NBT at the same
Å
concentration. Then we detected the reduced activity of OÀ2 by DMSO, which
was used as solvent for completely solubilizing the formazan compounds
formed in the original NBT assay. The inhibition effect of ÅO2À or ÅOH scavenging
(U/L or U/mL) was calculated. Results were expressed as the mean standard
deviation (SD) of triplicate determinations.
20. Bal-Demirci, T.; Sahin, M.; Ozyurek, M.; Kondakcı, E.; Ulkuseven, B.
Spectrochim. Acta A Mol. Biomol. Spectrosc. 2014, 126, 317.
21. PC12 cells were grown as the same as before, and the cells were treated in
triplicate with all referential controls and different concentrations (1–10 lM)
Please cite this article in press as: Sheng, X.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.090