Structural optimization and neurotrophic activity evaluation of neurotrophic gentiside derivatives

Article abstract of DOI:10.1016/j.bmcl.2019.126685

C14 alkyl benzoate ABG001, derived from naturally occurring gentisides, was reported to exhibit neurotrophic activity which is similar to NGF (Nerve Growth Factor). In this research, ABG001 was modified by the strategy of isosteric replacement and conformational restriction with the purpose of improving the bioactivity. The cellular neurotrophic activity of those ABG001 derivatives were evaluated, among which 3-hydroxyquinolin-2-(1H)-one A3 and 4-decylphenol ester B7 displayed much better neurotrophic activity compared with ABG001, which highlights the potential of those novel scaffolds for future neurotrophic agent development.

Full text of DOI:10.1016/j.bmcl.2019.126685

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Structural optimization and neurotrophic activity evaluation of
neurotrophic gentiside derivatives
Zhenkang Wang, Chunhua Ma, Yujie Wang, Qiang Xiao,
Chenghui Xu, Yingxia Li
PII:
S0960-894X(19)30635-3
DOI:
https://doi.org/10.1016/j.bmcl.2019.126685
BMCL 126685
Reference:
To appear in:
Received date:
Revised date:
Accepted date:
16 June 2019
10 September 2019
12 September 2019
Please cite this article as: Z. Wang, C. Ma, Y. Wang, et al., Structural optimization
and neurotrophic activity evaluation of neurotrophic gentiside derivatives, (2019),
https://doi.org/10.1016/j.bmcl.2019.126685
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Structural optimization and neurotrophic activity evaluation of
neurotrophic gentiside derivatives
Zhenkang Wanga,1, Chunhua Mab,1, Yujie Wanga, Qiang Xiaoa,
Chenghui Xuc, Yingxia Lia,*
a
b
School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
School of Chemistry and Chemical Engineering, Henan Normal University, Collaborative Innovation Center of Henan
Province for Green Manufacturing of Fine Chemicals, Key Laboratory of Green Chemical Media and Reactions of Ministry of
Education, Xinxiang 453007, China
c
Division of Antitumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi
Road, Shanghai 201203, China.
ABSTRACT
C14 alkyl benzoate ABG001, derived from naturally occurring gentisides, was reported to exhibit neurotrophic activity
which is similar to NGF (Nerve Growth Factor). In this research, ABG001 was modified by the strategy of
isosteric replacement and conformational restriction with the purpose of improving the bioactivity. The cellular
neurotrophic activity of those ABG001 derivatives were evaluated, among which 3-hydroxyquinolin-2-(1H)-one A3 and
4-decylphenol ester B7 displayed much better neurotrophic activity compared with ABG001, which highlights the
potential of those novel scaffolds for future neurotrophic agent development.
Key Words: Gentisides, neurotrophic activity, isosteric replacement, conformational restriction, PC 12 cells
* Corresponding author: Yingxia Li Tel.: +86 21 51980127.
E-mail addresse: liyx417@fudan.edu.cn.
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1 These authors contributed equally to this work.
Neurodegenerative diseases, which includes Alzheimer’s disease, Parkinson’s disease, Huntington’s
disease, and amyotrophic lateral sclerosis, are characterized by nervous system dysfunction resulting from
progressive neuronal degeneration.1 Additionally, spinal cord injury is a damage to the spinal cord resulting
from trauma or disease.2 About 50 million people in the world are suffering from neurodegenerative diseases
or spinal cord injury, which costs over 600 billion dollars of annual healthcare budget.3 The currently
available therapies, which mainly relieve disease symptoms and control the damage, are insufficient to
eradicate these diseases. It is well-known that promoting the survival and regeneration of neurons within the
adult nervous system would be an ideal preventive and therapeutic strategy. 4 Nevertheless, such therapeutic
approaches have not yet been successfully developed currently.
Neurotrophins are endogenous proteins that regulate neuron survival, development, and function.5
Nerve growth factor (NGF), which acts as a prototypical neurotrophin, is essential for the development and
survival of neuron cells, maintenance of synaptic connection and plasticity, and prevention of aging in the
nervous system.6 Nonetheless, the suboptimal pharmacokinetic properties of NGF restrict its clinical use
such as poor in vivo stability and limited penetration of the blood-brain barrier owing to its large molecular
size.7 However, low-molecular weight compounds that can enhance or mimic the neurotrophic ability of
NGF without those foregoing protein-based limitations have shown better clinical applications.8 Over the
past several decades, a lot of neurotrophic compounds were reported (Figure 1), for example, caffeic acid
amide analogues synthesized by Omidreza Firuzi showed strong neurotrophic activity;9 RC-33 could
enhance the NGF-mediated neurite outgrowth by selectively activating σ1 receptors;10 Verb-5 potentiated
NGF-induced neurite outgrowth of PC-12D cell;11 Sar-6 modified from Sarcodonin G exhibited excellent
activity to promote NGF-induced neurite outgrowth;12 Ribisin C was found to enhance the neurite outgrowth
in the presence of NGF;13 Piperodione isolated from the fruits of P. retrofractum promoted the neurite
outgrowth of NGF.14 It's worth mentioning that professor Jin-Ming Gao from Northwest A&F University is
devoted to discovering neurotrophic natural products from native microbial and plant resources for decades,
many natural compounds bearing fascinating chemical structures have showed good neurotrophic activity,
such as Striatoids A−F, Cyafricanins G, Qinbunamides A-C and Neocyathins T. 15
As listed above, most of these compounds exhibit neurotrophic activity in a NGF dependent manner,
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and compounds with significant independent neuritogenic activity were rarely reported.
Figure 1. The structure of reported neurotrophic compounds
Gentisides isolated from Chinese medicine Gentiana Rigescens showed potent neurotrophic activity.16
And further structural simplification led to compound ABG001 with neurotrophic activity equivalent to
NGF.17 As shown in Figure 2, ABG001 is a long-chain alkyl benzoate, and can be divided into three parts:
2,3-dihydroxy phenyl, ester group and a long alkyl chain. In this paper, ABG001 was used as lead compound
for structural optimization to explore novel NGF mimics as shown in Figure 2. The 2,3-dihydroxy phenyl
group is unstable in vivo and easily oxidized to quinone which is detrimental for human body. The
corresponding bioisosteres of 2,3-dihydroxy phenyl group was explored, such as pyrazolyl, 2-aminothiazole,
1H-benzo[d]imidazole, and quinoline-2,3-diol group, which could retain hydrogen bonds interactions and
aromatic annulus (Figure 2, Table 1). Besides the long flexible alkyl chain may not be favorable for the
binding with the target due to the entropy cost, therefore the proper restrictions to the long flexible chain
could potentially improve the binding affinity. We introduced sterically equivalent 1,4-phenylene group and
1,1'-biphenyl group, which are effective templates for conformational restriction,18 to replace four or eight
methylene units of the long alkyl chain in ABG001 and investigated the effects of distance (m) between
newly incorporated group and ester group and length of the tail alkyl chain (n) on the neurotrophic activity
(Figure 2, Table 2 and Table 3).
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Figure 2. The design guideline of gentiside derivatives A1-A4, B1-B11 and C1-C4.
Table 1 The structures of gentiside derivatives A1-A4
Compound
A1
A
R
(CH2)13CH3
(CH2)13CH3
(CH2)13CH3
A2
A3
(CH2)13CH3
A4
Table 2 The structures of gentiside derivatives B1-B11
Compound
m
R
H
H
0
1
B1
B2
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2
3
H
B3
B4
H
H
4
B5
10
0
H
B6
(CH2)9CH3
(CH2)8CH3
(CH2)9CH3
CH=CH(CH2)6CH3
CH=CH(CH2)7CH3
B7
1
B8
1
B9
1
B10
B11
1
Table 3 The structures of gentiside derivatives C1-C4
Compound
m
0
R
H
C1
C2
C3
C4
1
H
0
(CH2)5CH3
(CH2)4CH3
1
The target compounds were synthesized efficiently via the route outlined in Scheme 1-4. As shown in
Scheme 1, The esterification reaction of propiolic acid with 1-tetradecyl alcohol in the presence of a
catalytic amount of p-toluenesulfonic acid gave alkynes 1, which reacted with trimethylsilyldiazomethane at
room temperature to afford the pyrazolyl derivate A1. The A2 were synthesized by a series of reactions as
shown in the Scheme 2. Intermediate 2 was synthesized by cyclizing ethyl bromopyruvate and thiourea in
ethanol at 78 °C, then was converted to 2-aminothiazole derivate A2 by t-butoxycarbonyl protection, ester
hydrolysis, followed by Yamaguchi esterification and deprotection of amino protecting group in order. As
depicted in Scheme 3, Isatin was reacted with tetradecyl 2-diazoacetate in the presence of diethylamine, and
the 3-hydroxyquinolin-2-(1H)-one derivative A3 was obtained via subsequent Eistert ring expansion of the
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crude adducts after dilute aqueous acidic treatment. 1H-benzo[d]imidazole-7-carboxylic acid afforded
desired compound A4 using the N, N-dicyclohexylcarbodiimide as the dehydration agent (not shown in the
scheme). The transformation of phenol followed by esterification, Fries rearrangement and ketone carbonyl
reduction resulted in para alkyl-substitution phenol 7. Commercially available methyl 4-formylbenzoate can
react with octyltriphenylphosphonium bromide and nonyltriphenylphosphonium bromide respectively under
alkaline conditions to get styrene 8a and 8b, followed by ester reduction with lithium aluminium hydride to
further obtain 9a and 9b. The previous intermediates were carried on hydrogenolysis with 10% palladium on
carbon to get the benzyl alcohol 10a and 10b in quantitative yield. The 1,1'-biphenyl derivates 11a and 11b
are obtained by Suzuki cross-coupling reaction using corresponding phenyl bromide and phenylboronic acid.
Finally, the target products B1-B11 and C1-C4 were obtained successively by esterification of
2,3-dihydroxybenzoic acid with the corresponding phenols or alcohols in dry acetonitrile using N,
N-dicyclohexylcarbodiimide as condensation agent.
Scheme 1. Reagents and conditions: (a) 1-tetradecanol, p-toluenesulfonic acid, toluene, reflux, overnight, 84%; (b)
trimethylsilyldiazomethane, n-hexane, rt, 2 h, 51%.
Scheme 2. Reagents and Conditions: (a) EtOH, 78°C, 4 h, 91%; (b) (Boc)2O, pyridine, reflux, 6 h, 54%; (c) 1M
LiOH, THF, rt, 4 h; (d) 2,4,6-trichlorobenzoyl chloride, DIPEA, 1-tetradecanol, DMAP, THF, 0°C, 1.5 h, rt, 1 h ,
73%; (e) TFA/DCM = 4:1, rt, 4 h, 90%.
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Scheme 3. Reagents and conditions: (a) Et2NH, ethanol, rt, 72 h; (b) 1N HCl, rt, 2 h, 40%.
Scheme 4. Reagents and conditions: (a) AlCl3, 130 °C, 1 h; (b) NH2-NH2, DEG, KOH, 250 °C, 2 h, 190 °C, 4 h,
82%; (c) ylide agents, n-BuLi, THF, -20 °C, 1 h, rt, 2 h, 53% for 8a, 59% for 8b ; (d) LiAlH4, THF, 0 °C, 2 h,
94% for 9a and 9b; (e) Pd/C, H2, MeOH, rt, overnight; (f) Pd(PPh3)4, K2CO3, DMF, EtOH, H2O, 90 ℃, overnight,
86% for 11a and 67% for 11b; (g) DCC, DMAP, CH3CN, 50 °C, 1 h, 65 °C, 3 h, 37%-69%.
Undifferentiated rat pheochromocytoma cell (PC-12 cell), which can be induced to differentiate into
sympathetic-like neurons after exposure to NGF, is a useful model system to evaluate the neurotrophic
activity of compounds in vitro.19 The effects of the target compounds on neurite outgrowth of
undifferentiated PC-12 cells were evaluated by morphological observations and quantitative analysis of
percentage of neurite-bearing cells.
The effect of different bioisosteres (A1-A4) on the neurotrophic activity was first investigated. As
depicted in Figure 3, the 2,3-dihydroxy phenyl group replaced by pyrazolyl heterocycle (A1),
2-aminothiazole heterocycle (A2) or 1H-benzo[d]imidazole heterocycle (A4) are detrimental to the
neurotrophic activity, while 3-hydroxyquinolin-2-(1H)-one scaffold (A3) could improve the activity.
Next, we evaluated the effect of the distance (m) between newly incorporated phenyl group and ester
group (B1-B6) on the neurotrophic activity. As Figure 3 shows, the phenolic ester B1 (m=0) and benzyl
ester B2 (m=1) displayed much better neurotrophic activity, and longer distances led to impaired activity
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(B3-B6), suggesting that the distance (m) is of critical importance to the neurotrophic activity. Using B1 and
B2 as templates, to determine the the impact of the length of alkyl chain (n) on the activity, we introduced
1,4-phenylene group to replace the four methylene units in the alkyl chain of ABG001 to obtain compounds
B7 and B8. To our delight, the two compounds exhibited remarkably increased activity compared with
ABG001. While further increasing the alkyl chain by one carbon (B9) resulted in slight reduction in potency
compared with B8. When double bonds are introduced into the alkyl chain of B8 and B9 , the activity
decreases considerably compared with saturated carbon chains (B10 vs B8, B11 vs B9). After that we replace
the eight methylene units in the long fat chain with a biphenyl group to further enhance the rigidity of the
compound and also evaluated the effect of the distance (m) between newly incorporated group and ester
group on the neurotrophic activity (C1-C4). Unfortunately, the neurotrophic activity of all compounds in this
series was significantly decreased compared with the corresponding phenyl substituted compounds ( C1 vs
B1, C2 vs B2, C3 vs B7 and C4 vs B8).
Particularly, 3-hydroxyquinolin-2-(1H)-one derivative A3, phenol ester B7 and benzyl ester B8
displayed even better activity than lead compound ABG001 (Figure 3). To the best of our knowledge,
compounds bearing quinoline motif was first reported to have neurotrophic activity. Figure 4 shows the
morphological changes of PC12 cells after treatment with our artificial compounds bearing good bioactivity
(A3, B7 and B8) in comparison with the solvent control (0.5% DMSO), and the positive control (NGF and
ABG001 ). Control cells (without any test compound) showed very few short neurite outgrowth. When
treated with A3 (1 μM ), B7 (1 μM ) and B8 (1 μM ), the cells extended long multipolar neurite outgrowths
48 h after treatment, which were similar to those produced following treatment with NGF (40 ng/mL) and
ABG001 (1 μM ).
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Figure 3. Effects of compounds A1-A4, B1-B11 and C1-C4 on neurite outgrowth in PC12 cells. Undifferentiated
PC12 cells were treated with Con (0.5% DMSO), NGF (40 ng/mL), ABG001(1 μM), A1-A4 (1 μM), B1-B6 (30
μM), B7-B11 (1 μM) and C1-C4 (10 uM) for 48 h to evaluate their neurotrophic effects by using phase-contrast
microscope. Five images were selected randomly under a microscope for each well. At least 100 cells in each of
five randomly separated fields were scored and the proportion of cells with neuritis greater than or equal to the
length of one cell body were positive for neurite outgrowth, and expressed as a percentage of the total cell number
in five fields (Y-axis). Experiments were repeated at least three times. Significant differences between each groups
were tested by ANOVA, followed by two-tailed multiple t-tests using SPSS biostatistics software (IBM; Armonk,
NY, U.S.A.), values represent the mean ± SE (n =3). ** p < 0.01 compared with control.
Control
NGF
ABG001
A3
B7
B8
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Figure 4. Photomicrographs of PC12 cells under a phase-contrast microscope (the magnification is ×400) 48 h
after treatment with 0.5% DMSO (Control), NGF (40 ng/mL), ABG001 (1μM), A3 (1μM), B7 (1μM), or B8
(1μM).
Sequentially, the dose-response test and the cell viability test of the three promissing compounds (A3,
B7 and B8) in PC12 cells were executed. As can be seen from the cell viability curve (Figure 5),
considerable toxicity to PC-12 cells was detected when concentrations of these compounds were above 5 μM,
also obviously A3 and ABG001 showed the higher toxicity when tested in high concentrations (10 μM)
compared with B7 and B8. It determined that the optimal concentration should not exceed the 1 μM. Besides
the three compounds showed dose-dependent activity when evaluated in the concentration range of 0.1 to 1
μM (Figure 6).
Figure 5. PC-12 cells survival rate of A3, B7, B8 and ABG001 48 h after treatment. ** p < 0.01 compared with
control (0.5% DMSO).
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Figure 6. Dose-dependent curve of the NGF-mimicking activity of A3, B7, B8 and ABG001 48 h after treatment.
** p < 0.01 compared with control (0.5% DMSO).
In summary, one series of neurotrophic gentiside derivatives with isosteric replacement or the long
alkyl chain optimization of the lead compound ABG001 were reported. 3-hydroxyquinolin-2-(1H)-one
scaffold with excellent neurotrophic activity is first reported. The most potent compounds A3 and B7
exhibited better neurotrophic activity profiles than ABG001, which could be served as new leads for further
exploration in the future neuritogenic agent development. Our research also highlight the importance of
employing isosteric replacement and conformation restriction strategy to the lead optimization in drug
discovery.
Acknowledgements
We gratefully acknowledge the financial support from National Natural Science Foundation of China (Grant
No. 81773576).
Supplementary data
Supplementary data is available on the publishers’ web site along with the published article.
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