Design, synthesis and biological evaluation of novel osthole-based derivatives as potential neuroprotective agents

Article abstract of DOI:10.1016/j.bmcl.2020.127633

A total of 26 compounds based on osthole skeleton were designed, synthesized. Their cytoprotective abilities of antioxidation, anti-inflammation and Aβ₄₂(Amyloid β-protein 42)-induced neurotoxicity were evaluated by MTT assays. Mechanism of the action of selected compounds were investigated by molecular docking. AlogP, logS and blood–brain barrier (BBB) permeability of all these compounds were simulated by admetSAR. Most of the compounds showed better antioxidative and anti-inflammatory activities compared with osthole, especially OST7 and OST17. The compound OST7 showed relative high activity in neuroprotection against H₂O₂ (45.7 ± 5.5%), oxygen glucose deprivation (64.6 ± 4.8%) and Aβ₄₂ (61.4 ± 5.2%) at a low concentration of 10 μM. EC₅₀ of selected compounds were measured in both H₂O₂ and OGD induced cytotoxicity models. Moreover, NO inhibiting ability of OST17(50.4 ± 7.1%) already surpassed the positive drug indomethacin. The structure activity relationship study indicated that introduction of piperazine group, tetrahydropyrrole group and aromatic amine group might be beneficial for enhancement of osthole neuroprotective properties. Molecular docking explained that the reason OST7 exhibited relatively stronger neuroprotection against Aβ because of the greater area of interactions between molecule and target protein. OST7 and OST17 both provided novel methods to investigate osthole as anti-AD drugs.

Full text of DOI:10.1016/j.bmcl.2020.127633

Journal Pre-proofs
Design, synthesis and biological evaluation of novel osthole-based derivatives
as potential neuroprotective agents
Li Zhang, Yuhang Wu, Guixiang Yang, Haixian Gan, Dayong Sang, Jiye
Zhou, Lin Su, Rui Wang, Lei Ma
PII:
S0960-894X(20)30744-7
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127633
BMCL 127633
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Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
25 August 2020
13 October 2020
16 October 2020
Please cite this article as: Zhang, L., Wu, Y., Yang, G., Gan, H., Sang, D., Zhou, J., Su, L., Wang, R., Ma, L.,
Design, synthesis and biological evaluation of novel osthole-based derivatives as potential neuroprotective
agents, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127633
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Design, synthesis and biological evaluation of novel osthole-based
derivatives as potential neuroprotective agents
Li Zhang a, †, Yuhang Wu a, †, Guixiang Yang a, †, Haixian Gan a, Dayong Sang b, Jiye
Zhoua, Lin Sua, Rui Wang a, *, Lei Ma a, *
a
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China
University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
b College of Chemical Engineering and Pharmacy, Jingchu University of Technology,
Jingmen, Hubei 448000, China
†These authors contributed equally to this work.
* Corresponding authors. Tel.: +86 2164253691; Fax: +86 2164253691.
E-mail addresses: malei@ecust.edu.cn (L. Ma), ruiwang@ecust.edu.cn (R. Wang)

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Abstract:
A total of 26 compounds based on osthole skeleton were designed, synthesized.
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Their cytoprotective abilities of antioxidation, anti-inflammation and Aβ42（Amyloid
β-protein 42）-induced neurotoxicity were evaluated by MTT assays. Mechanism of
the action of selected compounds were investigated by molecular docking. AlogP, logS
and blood-brain barrier (BBB) permeability of all these compounds were simulated by
admetSAR. Most of the compounds showed better antioxidative and anti-inflammatory
activities compared with osthole, especially OST7 and OST17. The compound OST7
showed relative high activity in neuroprotection against H2O2 (45.7 ± 5.5 %), oxygen
glucose deprivation (64.6 ± 4.8 %) and Aβ42 (61.4 ± 5.2 %) at a low concentration of
10 μM. EC50 of selected compounds were measured in both H2O2 and OGD induced
cytotoxicity models. Moreover, NO inhibiting ability of OST17(50.4 ± 7.1 %) already
surpassed the positive drug indomethacin. The structure activity relationship study
indicated that introduction of piperazine group, tetrahydropyrrole group and aromatic
amine group might be beneficial for enhancement of osthole neuroprotective properties.
Molecular docking explained that the reason OST7 exhibited relatively stronger
neuroprotection against Aβ because of the greater area of interactions between
molecule and target protein. OST7 and OST17 both provided novel methods to
investigate osthole as anti-AD drugs.
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Keywords: Osthole; Neuroprotection; Antioxidant; Anti-inflammation; Amyloid β-
protein, Molecular docking.

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Alzheimer’s disease, also referred to as AD, is a neurodegenerative disease, which is
usually caused by the chronic damages of neurons and worsens over time.1,2 The most
common initial symptom is memory deficit.1 As the disease progresses, other
symptoms may include but not limited to oral presentation difficulties, mobility
problems, mood swings and disorientation.1,3 Progressive loss of physiological
functions may eventually lead to death.4 According to recent research, AD is the sixth
leading cause of death in the United States and the fifth leading cause of death among
people 65 and older.5 Unless significant breakthroughs in medicine are made, the
number of people 65 and older with AD could rise from 5.5 million to 13.8 million by
2050 (almost as 3 times as current) by predictions. Meanwhile, total cost for medical
care and long-term care are expected to exceed $1.1 trillion.6 Thus, there is an urgent
need for a better therapeutic strategy for AD.
The pathology of AD still remains uncertain.7 Mainstream hypotheses include
amyloid hypothesis, cholinergic hypothesis, inflammatory hypothesis, etc.8-10 On the
one hand, Excess ROS, including hydroxyl radical (·OH), hydrogen peroxide (H2O2),
superoxide radical (·O−2), can cause damages to mitochondrial membrane and the DNA
strands which maintain the normal cytofunctions.11 On the other hand, severe
inflammatory responses happened in the nidus. 12 Any factor that can cause tissue and
cell damage can cause inflammation, and these factors are collectively called
inflammatory mediators. Study showed almost all AD patients were accompanied by
varying degrees of amyloid β-protein (Aβ) plaques accumulation in brain.13 Another
research showed the synthesis and release of multiple inflammatory mediators,

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including interleukin and tumor necrosis factor (TNF), involved by Aβ,14 illustrating
the aggregation of Aβ promoted the magnitude of inflammatory responses and then
added to condition aggravation. Molecular design strategy should be adapted to these
mechanisms.
Osthole, one kind of furanocoumarins isolated from Cnidium monnieri (L.) Cuss.,
was reported to have extensive pharmacological activities, such as anti-inflammatory
and antioxidant.15 A recent research indicated osthole showed activities in
neuroprotection against oxygen and glucose deprivation (OGD) by regulating the
expression of mitogen-activated protein kinase (MAPK) proteins.16 Another research
showed Aβ levels can be reduced by osthole through up-regulation of miRNA-107.17
These all revealed that osthole is a promising skeleton for developing anti-Alzheimer’s
drugs.
In the work presented herein, the design and synthesis of osthole derivatives were
described. In our previous work, we designed sarsasapogenin and diosgenin carbamate
derivatives as antioxidants. Phenyl carbamate as potent inflammation inhibitors are
reported in neuroprotective drug design.18 Thus, 4 new compounds were prepared
OST1-OST4 (Scheme 1). We also noticed the introduction of aromatic amine and
piperazine were frequently applied as antioxidants and anti-inflammatory agents,19-22
so we designed other 14 new compounds OST5-OST18 (Scheme 2, Scheme 3). In
order for further exploration of promising skeletons for neuroprotection, we
synthesized columbianetin (CBN) from osthole. CBN shows an activity in anti-
inflammatory properties but there have been no reports of its neuroprotection yet.23-25

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Resveratrol, a widely known nature product possesses both anti-inflammation and
antioxidation,26 contains the structure of toluylene. Keep this in mind, we synthesized
8 new compounds OST19-OST26 (Scheme 4) based on CBN. All synthesis strategy
was show in Figure 1.
Cys, NaH
HO
O
O
O
O
O
DMF
Osthole
M1
O
O
N
R1-NH-R1'
R1
O
O
O
R1''
O
O
O
DIPEA, BTC
R1'
R1
R1''
N
OST1-OST4
R1'
H
N
N
H
N
H
N
H
R=
O
OST1
OST2
OST4
OST3
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Scheme 1. Synthesis of OST1-OST4
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R2
N
ClCOCH2Cl
HO
O
O
R2-NH-R2'
R2'
Cl
+
Toluene/10%Na2CO3
O
M1
R2
N
R2''
K2CO3
R2'
O
O
O
O
O
O
ACN, 50 oC
O
O
OST5-OST10
N
N
N
R2''=
N
N
HN
R2
N
Boc
R2''
OST6
OST7
OST5
R2'
N
N
N
O
OST8
OST9
OST10
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Scheme 2. Synthesis of OST5-OST10
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O
N
Cl
H
HN
K2CO3
N
O
O
O
ACN, 50oC
N
O
R3''
R3''
M2
OST11-OST14
+
HO
O
O
O
Cl
HN
H
N
O
M1
K2CO3
O
O
ACN, 50oC
O
R3''
R3''
M3
OST15-OST18
N
N
N
N
R3''=
HN
O
OST11, OST15
OST12, OST16
OST13, OST17
OST14, OST18
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Scheme 3. Synthesis of OST11-OST18
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MeCN, CuBr2
NBS, rt
m-CPBA, K2CO3
HO
O
O
O
O
O
DCM, rt
HO
M1
M4
R4
Br
O
OH
OH
B
R
O
O
O
O
O
K2CO3, Pd(PPh3)4, 80oC
HO
HO
OST19-OST26
M5
OST19
OST23
OST20
OST24
OST21
OST25
OST22
OST26
H;
4-F;
4-CH3;
3-F;
4-OCH3;
2-F;
3-OCH3;
4-CF3
R4=
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Scheme 4. Synthesis of OST19-OST26

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O
O
O
HO
O
O
O
O
O
OH
CBN
M1
Osthole
R3
R2'
O
R1'
N
R2
O
O
O
N
O
O
O
O
O
O
R1
O
OH
OST1-OST4
OST5-OST18
OST19-OST26
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Figure 1. The synthesis strategy of target compounds.
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Furanocoumarins including osthole are known to be relatively less soluble in water,27
which hampers their druggability for oral drug. To achieve optimal brain penetration
for CNS-targeted drugs, relatively high lipophilicity is preferred. Thus, AlogP, logS
and blood-brain barrier (BBB) permeability of all these compounds were simulated by
admetSAR (http://lmmd.ecust.edu.cn/admetsar2/, developed by Yang from ECUST).
The results were listed in Table 1. As we can see, all these compounds exhibited a better
BBB permeability than osthole in simulation. The introduction of nonpolar groups
enhanced the hydrophobicity of osthole. Unsubstituted at one end of piperazine showed
the highest value (OST5: -3.160, OST11: -3.309, OST15: -3.329 ) compared with other
similar derivatives. Illustrated that the solubility can be improved by introducing basic
amines. The ALogP value were all less than 5, which in accordance with Lipinski's
Rule of Five.28

BBB permeant
probability
0.7675
Compd.
ALogP
LogS
osthole
OST1
3.31
4.05
4.72
4.90
4.60
2.11
2.84
3.76
2.54
3.30
3.69
3.12
3.55
4.31
4.70
3.73
4.16
4.92
5.31
3.53
3.84
3.54
3.54
3.67
3.67
3.67
4.55
-4.372
-4.117
-4.153
-3.862
-3.660
-3.160
-3.674
-3.581
-3.247
-3.132
-3.247
-3.309
-3.528
-3.382
-3.528
-3.239
-3.556
-3.446
-3.556
-3.935
-4.164
-4.110
-4.110
-4.388
-4.388
-4.388
-4.503
0.9221
0.9510
0.9221
0.9237
0.9459
0.9729
0.9736
0.9756
0.9788
0.9756
0.9074
0.9564
0.9536
0.9564
0.9165
0.9627
0.9606
0.9627
0.8794
0.8794
0.8380
0.8380
0.9696
0.9696
0.9696
0.9631
OST2
OST3
OST4
OST5
OST6
OST7
OST8
OST9
OST10
OST11
OST12
OST13
OST14
OST15
OST16
OST17
OST18
OST19
OST20
OST21
OST22
OST23
OST24
OST25
OST26
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Table 1. Physicochemical properties of compounds in silico including BBB permeant
probability, ALogP and LogS
In order to evaluate the neuroprotective activity of osthole derivatives, cell viabilities

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of OST1-OST26 against oxidative stress induced by H2O2 in SH-SY5Y cells were
carried out by MTT assay. As shown in Table 2, most tested compounds could exhibit
cytoprotective properties. Carbamates have been frequently applied in cytoprotection,
but unfortunately, direct introduction of carbamate groups into osthole as OST1-4 did
not bring a significant enhancement of cell viabilities. In the comparison between
OST5-7 and OST1-4 we found that introducing piperazines into osthole increased cell
viabilities more than introducing carbamates. Compound OST7, which possesses both
carbamate and piperazine structure, was superior to other piperazine derivatives (OST5,
OST6) in cytoprotection. This result accord with previous experiences and it
demonstrated that the introduction position of carbamates could bring wide differences
in bioactivities. When the piperazine group was replaced by morpholine and piperidine,
one compound obtained (OST8) exhibited a similar antioxidative ability due to the
similar electron density but the other derivative (OST10) did not give a satisfactory
result. Interestingly, when the piperidine group was replaced by tetrahydropyrrole,
which makes the ring smaller, the obtained OST9 showed a better cytoprotective ability.
Comparing between OST11-14 or OST15-18 revealed same situation that the
introduction of piperazine and morpholine could both increase cell viabilities, and so
do the tetrahydropyrrole. Comparing between OST5, OST11, OST15; OST8, OST12,
OST16; OST9, OST17 or OST10, OST14, OST18 (we can temporarily divide them
into 4 series) revealed aromatic amine did work as groups that enhanced cell viabilities.
Pyridinamines (OST11-14) added more cytoprotective properties to osthole than
phenyl amines (OST15-18). This might be relevant to electron cloud distributions of

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aromatic rings. When it comes to the designed novel skeleton, para substituted benzene
ring, with weather electron-donating groups (OST20, OST21) or electron-withdrawing
groups (OST23, OST26), did not display satisfactory activities. However, ortho or
meta substituted benzene ring could both increase the cytoprotective activities when
compared to compound OST19. In summary, 18 compounds (OST4-9, OST11-17,
OST19 and OST22-25) exhibited good cytoprotective abilities against H2O2 were
selected for further studies.
Neuroprotection (%)
Neuroprotection (%)
(10μM)
(10μM)
Compd.
Compd.
H2O2
OGD
H2O2
OGD
1 mM:
1 mM:
NAC
Osthole
OST1
OST2
OST3
OST4
OST5
OST6
OST7
OST8
OST9
OST10
OST11
OST12
OST13
OST14
OST15
OST16
OST17
OST18
OST19
OST20
OST21
OST22
OST23
OST24
OST25
OST26
45.2 ± 4.5
32.7 ± 3.6
22.5 ± 1.9
39.5 ± 1.9
47.7 ± 2.1
12.5 ± 1.8
19.7 ± 2.5
18.2 ± 2.3
NA
58.2 ± 1.7
16.8 ± 2.1
8.4 ± 2.5
13.2 ± 1.2
63.4 ± 7.3
-
37.1 ± 2.5
84.3 ± 6.7
23.2 ± 1.7
NA
28.6 ± 2.3
-
13.0 ± 1.6
NA
-
-
28.7 ± 2.6
29.2 ± 3.2
33.7 ± 2.9
45.7 ± 5.5
31.5 ± 1.6
28.0 ± 2.3
5.5 ± 3.3
44.5 ± 1.6
43.5 ± 3.3
35.0 ± 4.4
28.2 ± 2.1
48.0 ± 3.7
64.6 ± 4.8
23.2 ± 4.2
43.6 ± 7.5
-
23.2 ± 3.3
-
-
31.0 ± 1.6
21.3 ± 1.5
29.0 ± 1.1
36.5 ± 2.8
NA
42.8 ± 1.5
23.5 ± 4.1
41.2 ± 4.2
37.2 ± 4.1
-
49.4 ± 5.1
51.4 ± 4.1

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Table 2. Neuroprotection of compounds against H2O2 and OGD induced cytotoxity in
SH-SY5Y cells
Compounds selected above were tested for their neuroprotection against oxygen
glucose deprivation (OGD)-induced cell damage in SH-SY5Y cells. OGD model is also
used to evaluate the cytoprotection of drugs against oxidative stress damage. The results
were also listed in Table 2. Trends of cell viabilities were similar as in H2O2 model
besides OST14~16. Pyridinamine structure seems more effective than phenyl amine
structure in OGD model. But introducing pyridinamine could bring strong cytotoxicity
during MTT assay (OST11, OST13). Thus, OST4, OST6, OST7, OST8, OST9,
OST12, OST17, OST22, OST24, OST25 were selected for next evaluation.
EC50 value of each compound was defined as the concentration that caused 50 %
protective effect compare to the control group. Data listed in Table 3 are averages of
three independent experiments, and expressed as mean ± SEM. As we can see, most
compounds showed cytoprotective effects at low concentrations. OST7 exhibited 50 %
cytoprotective effect at 10.27 ± 0.54 and 3.27 ± 0.21 μM against H2O2 and OGD
induced cytotoxity respectively in SH-SY5Y cells. We also obtained OST12 and
OST17 that also exerted notable cytoprotective effects, with EC50 values of 16.61 ±
1.16 and 11.93 ± 1.36 μM against H2O2 induced cytotoxity and 9.05 ± 1.49 and 5.62 ±
0.65 μM against OGD induced cytotoxity. OST4, OST9 and OST24 showed higher
values than osthole against H2O2. Especially OST4 (> 100 μM), demonstrated again
that introducing carbamates in this position could not bring a satisfactory bioactivity.
OST8 and OST25 showed higher EC50 values than osthole against OGD, indicated that

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though there are similarities in structure, introduction of piperazine can be more
efficient than morpholine in cytoprotection.
Compd.
Osthole
OST4
EC50 (H2O2) (μM)
33.26 ± 0.25
> 100
EC50 (OGD) (μM)
26.45 ± 1.35
23.29 ± 0.56
12.46 ± 1.08
3.27 ± 0.21
> 100
OST6
29.84 ± 1.81
10.27 ± 0.54
27.52 ± 1.23
67.32 ± 2.21
16.61 ± 1.16
11.93 ± 1.36
30.37 ± 2.27
75.91 ± 2.38
32.34 ± 1.22
OST7
OST8
OST9
15.29 ± 1.67
9.05 ± 1.49
5.62 ± 0.65
13.28 ± 1.16
18.84 ± 2.76
40.05 ± 1.92
OST12
OST17
OST22
OST24
OST25
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Table 3. EC50 values of selected compounds against H2O2 and OGD induced
cytotoxity in SH-SY5Y cells
Inhibitory activities on LPS-induced NO production in RAW264.7 cell lines of the
ten compounds mentioned above were illustrated in Table 4. OST17 displayed better
NO inhibitory ability than other selected derivatives. Compounds containing piperazine
(OST6, OST7) and tetrahydropyrrole (OST9, OST17) group significantly inhibited
NO production than compounds containing morpholine group (OST8, OST12).
Though OST22, OST24, OST25 did not offer satisfactory results in NO inhibition

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when compared to other derivatives, modification as OST22 did improve its original
cytoprotective properties. It is worthwhile to further study columbianetin skeletons in
neuroprotection.
NO production (%)
Compd.
(10μM)
Control
Indometacin
Osthole
OST4
10.2 ± 0.8
53.2 ± 6.2
85.6 ± 13.3
77.4 ± 5.6
72.3 ± 5.6
62.8 ± 6.1
74.1 ± 11.3
75.4 ± 8.6
80.3 ± 9.1
50.4 ± 7.1
73.3 ± 6.5
106.2 ± 12.7
85.2 ± 8.3
OST6
OST7
OST8
OST9
OST12
OST17
OST22
OST24
OST25
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Table 4. NO production activity of compounds against LPS induced RAW264.7 cells
Aβ is generated from the continuous processing of β-protease and γ-protease.29 C-
terminus of Aβ resulting from the processing of γ-protease, which digests at the
transmembrane region of amyloid precursor protein (APP) and can produce a large
number of 39~43 amino acid residue subtypes.30 The most common residue subtypes
are Aβ40 and Aβ42. Among the two, Aβ40 is seen more commonly, but Aβ42 relevant

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more to the AD.31 Cell viabilities of OST7 and OST17 against PC12 cells induced by
Aβ42 were performed by MTT assay were illustrated in Table 5. OST7 displayed
stronger neuroprotective activities at a low concentration of 1 μM and 10 μM.
Neuroprotection (%)
Compd.
Concentration (μM)
(10μM)
1
10
30
1
NA
Osthole
32.6 ± 2.2
37.1 ± 3.6
55.2 ± 4.3
61.4 ± 5.2
47.8 ± 4.8
46.3 ± 3.9
OST7
10
1
OST17
10
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Table 5. Neuroprotection of compounds against Aβ42 induced cytotoxity in SH-SY5Y
cells
Action mechanisms of OST7 and OST17 have been explored by computer modeling
at the same time. Autodock vina was applied to calculate the possible binding method
and affinity between Aβ42 with OST7 and OST17.32 The 3D structure of Aβ42 (1IYT)
used was downloaded from the Protein Data Bank. As is observed in Figure 2, OST7
binds to C-terminus region of the peptide in L-shape while OST17 in U-shape binding
gesture. OST7 could form interactions with residues LEU34, GLY25, VAL24, ALA21
and PHE20. OST17 could also form in a same distance with MET35, LEU34, ILE31,
GLY25, VAL24 ALA21 and PHE20. During the aggregation of Aβ42, a salt bridge is
formed between K28 and E22 or D23 in the conformational change process.33 An
interaction with GLY25, VAL24 ALA21 and PHE20 might interrupt the production of

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salt bridge, weaken the contraction between K28 and E22. The carbonyl group,
isopentenyl group, and 1-acyl-4-boc piperazine structure can make OST7 transform
into a three-jaw configuration to better accommodate the surface of the α-helix of Aβ42.
As a result, the strong intermolecular interactions between OST7 and Aβ42 in the E22-
K28 region would make this compound a promising molecule with potential Aβ42 anti-
aggregation activity.
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(A)
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(B)
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Figure 2. Predicted binding modes of compounds OST7 (A) and OST17 (B) with
Aβ42
In conclusion, A total of 26 compounds based on osthole were designed, synthesized.
Their cytoprotective abilities of H2O2-induced, oxygen glucose deprivation-induced

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neurotoxicity, LPS-induced NO production and Aβ42-induced neurotoxicity were
evaluated by MTT assays. Mechanism of the action of OST7 and OST17 were
investigated by molecular docking. The structure activity relationship study indicated
that introduction of piperazine-substituted carbamate and aromatic amine with
tetrahydropyrrole into osthole can both increase its cytoprotective properties against
H2O2 and OGD. Introduction of aromatic amine with tetrahydropyrrole structure as
OST17 added more NO inhibitive abilities to osthole. And molecular docking
explained that the reason OST7 exhibited stronger neuroprotection against Aβ because
of the greater area of interactions between molecule and target protein. OST7 and
OST17 both provided novel methods to investigate osthole as anti-AD drugs.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
(Grants 81973196 and 81673318), the 111 Project (Grant BP0719034) and the Natural
Science Foundation of Shanghai (Grant 19ZR1413900).
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Conflict of interest
We declare that we have no conflict of interest.
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Twenty-six osthloe derivatives were designed and synthesized.
Osthloe and its derivatives were investigated to evaluate the neuroprotective
activity in four cell models.

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Molecular docking studies were further performed to explain possible binding
poses to Aβ42 for the most promising analogs.
Compounds OST7 and OST17 showed better activities and provided novel
methods to investigate osthole as anti-AD drugs.