Design and synthesis of novel senkyunolide analogues as neuroprotective agents

Article abstract of DOI:10.1016/j.bmcl.2018.01.018

A class of senkyunolide analogues bearing benzofuranone fragment were designed, synthesized and evaluated for their neuroprotective effect in models of oxygen glucose deprivation (OGD) and oxidative stress. All tested compounds showed neuroprotection profile based on the cell viability assay. In particular, derivatives 1f–1i possessing furoxan-based nitric oxide releasing functionality exhibited significant biological activities in OGD models. More importantly, compound 1g containing short linker with furoxan displayed the most potent neuroprotection at the concentration of 100 μM (cell survival up to 145.2%). Besides, 1g also showed the middle level neuroprotective effect in model of oxidative stress.

Full text of DOI:10.1016/j.bmcl.2018.01.018

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel senkyunolide analogues
as neuroprotective agents
Yuanying Fang a,c, Rikang Wang a,c, Qi Wang a, Yongbing Sun a, Saisai Xie a,
Zunhua Yang b, , Min Li b, Yi Jin a, , Shilin Yang a
⇑
⇑
a National Engineering Research Center for Manufacturing Technology of TCM Solid Preparation, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China
b College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A class of senkyunolide analogues bearing benzofuranone fragment were designed, synthesized and eval-
uated for their neuroprotective effect in models of oxygen glucose deprivation (OGD) and oxidative
stress. All tested compounds showed neuroprotection profile based on the cell viability assay. In partic-
ular, derivatives 1f–1i possessing furoxan-based nitric oxide releasing functionality exhibited significant
biological activities in OGD models. More importantly, compound 1g containing short linker with furoxan
Received 22 November 2017
Revised 7 January 2018
Accepted 12 January 2018
Available online xxxx
displayed the most potent neuroprotection at the concentration of 100
Besides, 1g also showed the middle level neuroprotective effect in model of oxidative stress.
Ó 2018 Published by Elsevier Ltd.
lM (cell survival up to 145.2%).
Keywords:
Senkyunolide analogues
Neuroprotective agents
Acute cerebral ischemia
Furoxan
Cerebrovascular disorders constitute a serious public health
problem worldwide, and among them, acute cerebral ischemia
(ischemia stroke) represents one of the leading causes of death
and neurological disability.1 Although the pathological mechanism
of acute cerebral ischemia is complex and not fully disclosed, sig-
nificant progress has been made to describe the several patholog-
ical features that are in common with other neurodegenerative
disorders.2 Currently, there are two conventional treatment strate-
gies for acute cerebral ischemia. One is using neuroprotective drug,
which aims on blocking cascade of neuronal death to preserve and
recover the neurological function in the ischemic regions. Another
one is treatment of ischemia stroke by preventing or reducing vas-
cular thrombosis, and dilating vessels that leads to supply oxygen
and blood on the ischemic area.3,4
Ligusticum chuanxiong Hort. (Umbelliferae) is one of the most
commonly used traditional Chinese medicinal herb for treating
cardio-cerebrovascular diseases such as angina pectoris, coronary
disease, ischemic encephalopathy and hyperviscosity syndrome.5–9
Several abundant components were isolated from Ligusticum
chuanxiong Hort. including various types of phthalide derivatives,
such as ligustilide and senkyunolide (Fig. 1), which were reported
to possess potent biological activities for treatment of ischemic
diseases via improving cerebral blood flow, inhibiting thrombosis
and preserving nerve cells function.5,10,11 However, because of
the instability and short half-life of those compounds, it’s unprac-
tical to develop them directly as clinical drugs. Butylphthalide (3-
n-butylphthalide or NBP), a stable analogue of senkyunolide A, was
approved in China for the treatment of cerebral ischemia in 2004.4
Nitric oxide (NO) has attracted a tremendous interest in a broad
field of basic and applied research as one of the most significant
physiological signalling molecule in the body.12 NO could diffuse
into vascular smooth muscle cells and react with the iron of soluble
guanylate cyclase, leading to relaxation of the smooth muscle cells
and dilation of blood vessels.13 Therefore, a series of novel
senkyunolide analogues containing benzofuranone framework
and furoxan fragments as NO donors were designed and synthe-
sized. Biological evaluation of these compounds was carried out
including neuroprotective effect in oxygen glucose deprivation
(OGD) model against acute cerebral ischemia and antioxidant
activity in oxidative stress model. It was envisaged that these
derivatives could cause significant pharmacological change.
Twelve final compounds (1a–1l) were designed in three various
series. Compounds 1a–1e are benzofuran-3-one derivatives with
different methoxyl substitution in benzene ring and butylidene
group or reduced butyl at 2-position. Compounds 1f–1i are
5-hydroxyl benzofuran-3-one derivatives connected with furoxan
moiety via ether linkages and succinate diester. Compounds
1j–1l are benzofuran-2-one derivatives.
⇑
Corresponding authors.
E-mail addresses: joshyyy@126.com (Z. Yang), yinghua616909@hotmail.com
(Y. Jin).
c
The authors contribute equally to this work.
https://doi.org/10.1016/j.bmcl.2018.01.018
0960-894X/Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Fang Y., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.018

2
Y. Fang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
(II) bromide to afford bromides 2a–2c, which were treated with
KF to give benzofuranones 3a–3c.14,15 Condensation of intermedi-
ates 3a–3c with butyraldehyde gave target compounds 1a–1c with
E-isomer as the major product (85%–95%) according to the chemi-
cal shifts of olefinic proton in 1H NMR spectra and reported data.16
Catalytic hydrogenation of 1b and 1c using Pd/C in H2 afforded
butyl compounds 1d and 1e.
As shown in Scheme 2, the synthetic precursor 6 of furoxan
moiety was prepared from thiophenol according to the precedent
literatures.17–19 The diphenylsulfonylfuroxan 6 was treated with
kinds of diols and 25% NaOH aqueous solution at room tempera-
ture yielded the monoalchohols 7f–7i with linker of different
length, which were reacted with succinic anhydride in DMF to give
the NO-donor fragments 8f–8i. Compound 7i was a mixture
including E and Z form due to 2-butene-1,4-diol containing cis-
and trans-isomers.
5-Hydroxyl benzofuran-3-one 10 was obtained using the simi-
lar procedure with compounds 1a. Esterification between 10 and
NO-donors 8f–8i with EDCI and HOBt furnished the key intermedi-
ates 11f–11i, which was coupled with butyraldehyde to afford final
compounds 1f–1i (Scheme 3).
The substituted acetophenones were explored to generated
compounds 1j–1l, which were shown in Scheme 4.20–23 Will-
gerodt-Kindler reaction of the starting 12j–12l with morpholine
and sulphur formed thio-morpholide adduct following by
hydrolysis to afford acid 13j–13l. Cyclization of 13j–13l in the
presence of phosphorus oxychloride gave lactone 14j–14l. Reac-
tion of 14j–14l and butyraldehyde with KOH yielded target
compounds 1j–1l with E-isomer as the major product based
on 1H NMR spectra.
Fig. 1. The structures of senkyunolide and butylphthalide.
R1
R1
R1
R2
OH
O
R2
R2
OH
O
O
a
b
Br
O
3a-3c
2a-2c
R1
R1
R2
R2
O
c
d
O
O
O
1a 1 = H; R2 = H
: R
: R
1d 1 = H; R2 = OCH3
1b 1 = H; R2 = OCH3
: R
1e 1 = OCH3; R2 = OCH3
: R
: R1 = OCH3; R2 = OCH3
1c
Scheme 1. Reagents and conditions: (a) CuBr2, EtOAc, CHCl3, 65 °C, overnight; (b)
KF, DMF, R.T., overnight; (c) butyraldehyde, Al2O3, CH2Cl2, R.T., 0.5 h; (d) Pd/C, H2,
MeOH, R.T., 2 h.
Compounds 1a–1e were generated from substituted acetophe-
nones following the procedures and conditions as shown in
Scheme 1. The starting acetophenones were reacted with copper
O
S
O
S
O
S
OO
SH
S
COOH
a
c
b
COOH
O
N
N
O
O
6
4
5
O
O
S
O
S
OH
O
O
O
O
O
O R
OH
d
e
O R O
N
N
N
N
O
O
R
7f
8f
CH2CH2CH2CH2
7g
8g
8h
8i
CH2CH2CH2
CH2CH2CH(CH3)
7h
7i
CH2CH CHCH2
Scheme 2. Reagents and conditions: (a) ClCH2COOH, NaOH, reflux, 2 h; (b) 30% H2O2, CH3COOH, R.T., 2.5 h; (c) fuming HNO3, 90 °C, 1.5 h; (d) Diol, 25% NaOH aq., THF, R.T., 3
h; (e) succinic anhydride, DMAP, DMF, 85 °C, 6 h.
OH
O
OH
O
O
a
b
c
HO
HO
Br
HO
O
O
9
10
O
O
O
O
O
S
O
S
O
O
O
O
O
O
O
O
O
R
O
R
d
O
N
N
O
N
N
O
O
O
R
11f
1f
CH2CH2CH2CH2
11g
1g
1h
1i
CH2CH2CH2
CH2CH2CH(CH3)
11h
11i
CH2CH CHCH2
Scheme 3. Reagents and conditions: (a) CuBr2, EtOAc, CHCl3, 65 °C, overnight; (b) KF, DMF, R.T., overnight; (c) 8f–8i, EDCI, HOBt, DIPEA, CH2Cl2, R.T., overnight; (d)
butyraldehyde, Al2O3, CH2Cl2, R.T., 0.5 h.
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Y. Fang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
R5
R6
O
R5
R6
OH
O
R5
R6
OH
COOH
R5
R6
O
a
b
c
O
O
1j: R5 = H; R6 = H
1k: R5 = H; R6 = OMe
1l: R5 = OMe; R6 = H
13j-13l
12j-12l
14j-14l
Scheme 4. Reagents and conditions: (a) S, morpholine, TsOH, 3 h, 125 °C, then 20%NaOH, TBAI, 100 °C, overnight; (b) POCl3, CH2Cl2, R.T., overnight, under N2; (c)
butyraldehyde, KOH, ethanol, R.T., 3 h, under N2.
We investigated the neuroprotective effects of analogues 1a–1l
in acute cerebral ischemia models of OGD using MTT assay.24 Feru-
lic acid, a phytochemical antioxidant, was selected as positive con-
trol. The hippocampal neuronal cell line, HT-22 were divided to
blank control, OGD model and test group. The model group got a
cell viability of 57.5%. The test group were treated with ferulic acid
or senkyunolide analogues 1a–1l in 5 concentrations (the dosage of
ferulic acid is 10 times of tested compounds respectively) and the
results of cell viability were illustrated in Figs. 2–5. Ferulic acid
gave best neuroprotective effect at maximal concentration (1000
lM). All compounds showed the neuroprotective profiles in the
OGD models in a concentration-dependent manner largely. Among
Fig. 2. Neuroprotective effect of ferulic acid and compounds 1a–1c toward the OGD model of HT-22 cells. Cell viability was evaluated by MTT assay; ##P < .01 versus control
group, *P < .05 compared to the model group; Assay was done in triplicate.
Fig. 3. Neuroprotective effect of ferulic acid and compounds 1d–1f toward the OGD model of HT-22 cells. Cell viability was evaluated by MTT assay; ##P < .01 versus control
group, *P < .05 compared to the model group, **P < .01 compared to the model group; Assay was done in triplicate.
Please cite this article in press as: Fang Y., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.018

4
Y. Fang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 4. Neuroprotective effect of ferulic acid and compounds 1g–1i toward the OGD model of HT-22 cells. Cell viability was evaluated by MTT assay; ##P < .01 versus control
group, *P < .05 compared to the model group, **P < .01 compared to the model group; Assay was done in triplicate.
Fig. 5. Neuroprotective effect of ferulic acid and compounds 1j–1l toward the OGD model of HT-22 cells. Cell viability was evaluated by MTT assay; ##P < .01 versus control
group, *P < .05 compared to the model group; Assay was done in triplicate.
them, analogues (1j–1l) bearing benzofuran-2-one fragment dis-
played lightly better biological activities than compounds with
benzofuran-3-one (1a–1e). Compound 1a without any substituted
group in benzofuranone ring exhibited stronger neuroprotection
comparing with derivatives 1b and 1c. Reduction of butylidene
with butyl had no effect on the cell viability (1d vs. 1b; 1e vs.
1c). Surprisingly, analogues 1f–1i containing NO-donated group
(furoxans) displayed the statistically significant neuroprotective
effect based on the cell survival. In addition, compound 1g bearing
8g moiety revealed the most potent neuroprotection at the concen-
tration of 100 lM (Fig. 4). These results indicated that NO releasing
fragment was favorable to bioactivity and the shorter linker of
furoxans could increase the neuroprotective effect.
Next, we were interested in assessing the antioxidant effects
with the selected compounds 1b, 1f and 1g. They were evaluated
the cell viability against H2O2 induced oxidative damage using
MTT assay and the biological results were shown in Fig. 6. They
presented the neuroprotective effects at all concentrations of 1,
3, 10, 30, 100 M and shown the very close cell survival values
in the oxidative stress model though 1g exhibited the most potent
l
neuroprotection in the ODG model.
In summary, a series of novel senkyunolide analogues bearing
benzofuranone were designed and synthesized. Besides, introduc-
tion of furoxan moieties as NO donors were also explored in this
study. All compounds were evaluated their neuroprotective effect
toward OGD model and 3 of them for oxidative stress model by
MTT assay. As a result, all derivatives exhibited neuroprotection
profiles according to cell survival values. And analogues (1j–1l)
bearing benzofuran-2-one fragment displayed slightly better activ-
ities than compounds with benzofuran-3-one (1a–1e) in OGD
model. It is exciting that compounds 1f–1i possessing NO-donated
group (furoxans) showed the statistically significant neuroprotec-
tive effect compared with other two series based on the cell sur-
vival values in OGD model. More importantly, compound 1g
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Y. Fang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
2. Jellinger KA. General aspects of neurodegeneration. J Neural Transm Suppl.
2003;65:101–144.
3. del Zoppo GJ. Stroke and neurovascular protection.
2006;354:553–555.
N Engl J Med.
4. Zhang W, Song JK, Du LD, Zhang X, Du GH. Advances in pharmacologic
treatment of acute cerebral ischemia. Acta Neuropharmacol. 2014;4:50–58.
5. Chan SSK, Cheng TY, Lin G. Relaxation effects of ligustilide and senkyunolide A,
two main constituents of Ligusticum chuanxiong, in rat isolated aorta.
Ethnopharmacol. 2007;111:677–680.
J
6. Xiong YK, Lin X, Liang S, Yu Z, Du Y, Feng Y. HPLC determination of senkyunolide
in mice plasma and its pharmacokinetics. Chin Pharm Anal.
2013;33:371–375.
I
J
7. Sun YM, Lou JT, Huang GQ. Clinical study on sodium ferulate for intracerebral
hemorrhage in early stage. Chin J Chin Mater Med. 2008;33:2545–2548.
8. Zhao QZ, Liu YL, Zhang H, Bo AH. Effects of ligustrazine on hippocampal neurons
expression of Bax mRNA in cerebral ischemia-reperfusion in rats. Lishizhen Med
Mater Med Res. 2011;22:435–436.
9. Zhang JQ, Zhao XQ, Ji XM, et al. The effects of ligustrazine against cerebral
ischemia/reperfusion injury in rats. Clin Misdiagn Misther. 2011;24:41–43.
10. Lin H. Protective effect of senkyunolide A against rats cerebral ischemia-
reperfusion injury. J. North Pharmacy. 2016;13:114–115.
11. Zhang LJ, Liu JY, Yao C, Tai ZG, Sun LP, Gao S. Research progress in
pharmacological activities of senkyunolides. Chin Pharm J. 2015;50:1081–1084.
12. Carpenter AW, Schoenfisch MH. Nitric oxide release: part II. Therapeutic
applications. Chem Soc Rev. 2012;41:3742–3752.
13. Ferrari CKB, Franca EL, Honorio-Franca AC. Nitric oxide, health and disease. J
Appl Biomed. 2009;7:163–173.
Fig. 6. Neuroprotective effect of compounds 1b, 1f and 1g toward impaired HT-22
cells induced by H2O2. Cell viability was evaluated by MTT assay; ##P < .01 versus
control group, *P < .05 compared to the model group; Assay was done in triplicate.
14. Black DS, Kumar N, McConnell DB. Synthesis of tethered indoles in the search
for conformationally controlled calixindoles: an indole 3-substituent tether.
Tetrahedron. 2001;57:2203–2211.
bearing 8g moiety revealed the most potent neuroprotection at the
15. Wallez V, Durieux-Poissonnier S, Chavatte P, et al. Synthesis and structure-
affinity-activity relationships of novel benzofuran derivatives as MT2
melatonin receptor selective ligands. J Med Chem. 2002;45:2788–2800.
16. Lee CY, Chew EH, Go ML. Functionalized aurones as inducers of NAD(P)H:
concentration of 100 lM. These results proved that NO-donated
fragment was beneficial for biological activity and encouraged us
to discover the novel neuroprotection agents based on natural
product. The follow-up investigation on other acute cerebral ische-
mia cell models and animal models are in progress and the results
will be reported in due time.
quinone oxidoreductase
1 that activate AhR/XRE and Nrf2/ARE signaling
pathways: synthesis, evaluation and SAR. Eur J Med Chem. 2010;45:2957–2971.
17. Li R, Zhang Y, Ji H, Yu X, Peng S. Synthesis and anti-inflammatory activity of
benzenesulfonylfuroxan-coupled diclofenac. Acta Pharm Sin. 2001;36:821–826.
18. Kelley JL, Mclean EW, Williard KF. Synthesis of bis(arylsuh’onyl)furoxans from
aryl nitromethyl sulfones. J Heterocycl Chem. 1977;14:1415–1416.
19. Fang Y, Wang R, He M, et al. Bioorg Med Chem Lett. 2017;27:98–101.
20. Venkateswarlu S, Panchagnula GK, Guraiah MB, Subbaraju GV. Isoaurostatin:
total synthesis and structural revision. Tetrahedron. 2005;61:3013–3017.
21. Venkateswarlu S, Panchagnula GK, Guraiah MB, Subbaraju GV. Isoaurones:
synthesis and stereochemical assignments of geometrical isomers. Tetrahedron.
2006;62:9855–9860.
Acknowledgments
This work was supported by National Natural Science Founda-
tion of China (81460526, 81560662), Jiangxi Provincial Depart-
ment of Science and Technology (20171BAB205103), Hongcheng
Haiou Plan of Nanchang City (Zunhua Yang, 2017) and Health
and Family Planning Commission of Jiangxi Province (2016A013).
22. Mujahid Alam M, Adapa SR.
A facile synthesis of phenylacetic acids via
Willgerodt-Kindler reaction under PTC Condition. Synth Commun.
2003;33:59–63.
23. E1-Sayrafi S, Rayyan S. Intramolecular acylation of aryl- and aroyl-aliphatic
acids by the action of pyrophosphoryl chloride and phosphorus oxychloride.
Molecules. 2001;6:279–286.
References
24. Shen JH, He J, Wei S. Effect of corosolic acid on oxygen and glucose deprivation
1. Tenti G, Parada E, Leon R, et al. New 5-unsubstituted dihydropyridines with
improved CaV1.3 selectivity as potential neuroprotective agents against
ischemic injury. J Med Chem. 2014;57:4313–4323.
injury in rat pheochromocytoma PC12 cell. Chin
J
Clin Pharmacol.
2010;26:932–935.
Please cite this article in press as: Fang Y., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.018