Synthesis of andrographolide analogues and their neuroprotection and neurite outgrowth-promoting activities

Article abstract of DOI:10.1016/j.bmc.2019.04.025

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease and remains incurable. Currently, neuronal injury and synapse loss have been considered to be main features in the pathophysiology of AD. Thus, modulation of neuronal survival and neurite outgrowth may represent an efficient strategy for the treatment of AD. Based on the isolates from the traditional medicine Andrographis paniculata, a series of andrographolide analogues were prepared and evaluated for the neuroprotection and neurotrophic activity. Two compounds (3 and 12) could effectively inhibit LPS-induced NO production and iNOS expression as well as proinflammatory cytokines TNF-α and IL-6. Moreover, pretreatment with 3 and 12 could protect neurons against microglia-mediated neurotoxicity. Further, H₂O₂ ^? and 6-OHDA induced neurotoxicity in PC12 cells were also attenuated by the novel 12. Our next study indicated that compounds 1, 4 and 10 promoted NGF-induced neurite outgrowth in PC12 cells, with 10 the most potent. To clarify the underlying mechanisms of active compounds (3, 10 and 12), system pharmacology was employed. The results revealed that muscarinic acetylcholine receptors (mAChRs) may be the main targets of 12 against AD, while thyroid hormone signaling pathway was involved in the mechanisms of 10. These study point to the therapeutic potential of andrographolide analogues against AD.

Full text of DOI:10.1016/j.bmc.2019.04.025

Accepted Manuscript
Synthesis of andrographolide analogues and their neuroprotection and neurite
outgrowth-promoting activities
Yuanzhen Xu, Hongbo Wei, Jianping Wang, Weiwei Wang, Jinming Gao
PII:
S0968-0896(19)30042-2
https://doi.org/10.1016/j.bmc.2019.04.025
BMC 14879
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
8 January 2019
12 April 2019
16 April 2019
Please cite this article as: Xu, Y., Wei, H., Wang, J., Wang, W., Gao, J., Synthesis of andrographolide analogues
and their neuroprotection and neurite outgrowth-promoting activities, Bioorganic & Medicinal Chemistry (2019),
doi: https://doi.org/10.1016/j.bmc.2019.04.025
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Synthesis of andrographolide analogues and their neuroprotection
and neurite outgrowth-promoting activities
Yuanzhen Xu1, Hongbo Wei1, Jianping Wang, Weiwei Wang, Jinming Gao*
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest
A&F University, Yangling 712100, China
* Corresponding author: Jinming Gao, Shaanxi Key Laboratory of Natural Products & Chemical
Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China. E-
mail address: jinminggao@nwsuaf.edu.cn; Tel: 86-29-87092335.
1 These authors contributed equally to this work.
Abstract
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease and
remains incurable. Currently, neuronal injury and synapse loss have been considered
to be main features in the pathophysiology of AD. Thus, modulation of neuronal
survival and neurite outgrowth may represent an efficient strategy for the treatment of
AD. Based on the isolates from the traditional medicine Andrographis paniculata, a
series of andrographolide analogues were prepared and evaluated for the
neuroprotection and neurotrophic activity. Two compounds (3 and 12) could
effectively inhibit LPS-induced NO production and iNOS expression as well as
proinflammatory cytokines TNF-α and IL-6. Moreover, pretreatment with 3 and 12
could protect neurons against microglia-mediated neurotoxicity. Further, H2O2- and 6-
OHDA induced neurotoxicity in PC12 cells were also attenuated by the novel 12. Our
next study indicated that compounds 1, 4 and 10 promoted NGF-induced neurite
outgrowth in PC12 cells, with 10 the most potent. To clarify the underlying
mechanisms of active compounds (3, 10 and 12), system pharmacology was
employed. The results revealed that muscarinic acetylcholine receptors (mAChRs)
may be the main targets of 12 against AD, while thyroid hormone signaling pathway

was involved in the mechanisms of 10. These study point to the therapeutic potential
of andrographolide analogues against AD.
Keywords:
Andrographolide;
Neuroinflammation;
Oxidative stress;
Neurite outgrowth;
mAChRs;
Alzheimer’s disease
Abbreviations: LPS, lipopolysaccharide; NO, nitric oxide; iNOS, inducible nitric
oxide synthase; TNF-α, tumor necrosis factor alpha; IL-6, interleukin-6; H2O2,
hydrogen peroxide; 6-OHDA, 6-hydroxydopamine; ROS, reactive oxygen species; IL-
1β, interleukin-1β; COX-2, cyclooxygenase; HO-1, heme oxygenase-1; NGF, nerve
growth factor.
1. Introduction
Dementia is frequent in people aged 65 years and over and the number is
increasing at an alarming rate, making it a major public health concern. Alzheimer's
disease (AD) is the most common form of dementia worldwide and the number of
people with AD was estimated to be 5.7 million in the USA alone in 2016. By 2050,
this is projected to increase to 13.8 million.1 Clinically, AD causes progressive
impairments in cognitive functions and behavioral disturbances throughout the course
of the disease. However, there are no clinically effective approaches to slow or halt
the progression of AD at the present time.
The major neuropathological hallmarks of AD consist of amyloid plaques
deposits and neurofibrillary tangles (NFTs) accumulation.2 The senile plaques are
mainly formed by beta-amyloid (Aβ) derived from amyloid precursor protein (APP).
Compelling evidence indicated that neuroinflammation induced by activated
microglia play a significant role in the pathogenesis of AD.3 In the presence of

invading pathogens such as Aβ, microglia cells provide factors for amyloid
phagocytosis. However, chronic microglial activation could lead to the accumulation
of senile plaques rather than clearance, which would further induce microglial
activation and finally contribute to Aβ-induced neurodegeneration.4 Moreover, Aβ
deposition have been reported to interact with membrane receptors and finally lead to
synaptic dysfunction and neuronal loss.5 Tau, a microtubule-associated protein,
regulates axonal growth and neuronal development. Under pathological conditions,
hyperphosphorylated tau protein aggregate into NFTs and ultimately result in loss of
structural integrity of neurons.6 In addition, neurons are vulnerable to oxidative stress
due to the very high dioxygen consumption of the brain and relative paucity of
antioxidants and related enzymes,7 which could induce oxidative cell damage and cell
death. Acturally, Aβ deposition, NFTs, neuroinflammation and aging are contributing
factors to increased oxidative stress in AD. Thus, neuronal and synapse loss may be
the two main features in the pathophysiology of AD and this makes compounds
targeting neuronal survival and neurite outgrowth efficient therapeutic agents in the
therapeutic prevention and treatment of AD8-11
.
The herb Andrographis paniculata Nees, known as ‘king of bitters’, is
extensively used in the traditional medicines of India, China and other Asian countries
due to its key role in treating dysentery, infections, inflammation, laryngitis and
influenza.12,13 Extracts of the plant and their constituents were also reported to possess
a wide spectrum of biological activities. For example, ent-labdane diterpenoids from
A. paniculata such as andrographolide,14 14-deoxyandrographolide15 and 14-deoxy-
11,12-didehydroandrographolide,16 had been shown to exert anti-viral,
hepatoprotective and cardiovascular effects. Moreover, various semi-synthetic
analogues of andrographolide had been developed for searching more potent leading
compounds relative to anticancer, anti-HIV, anti-influenza virus, α-glucosidase
inhibitory activity and antidyslipidemic. However, there is no report on the
neuroprotection and neurotrophic activity. In light of previous study, a series of
andrographolide analogues were prepared based on the structures of isolates from A.

paniculata. The neuroprotection against microglia-mediated inflammatory injuries
and oxidative damage as well as neurite outgrowth promoting effects were also
evaluated. Finally, the underlying targets were also predicted by system
pharmacology approach. Our study will highlight the potential function of
andrographolide analogues against AD.
2. Results
2.1. Chemistry
Compounds 1(≥ 98%) and 2 (≥ 98%) were purchased from Shanghai Yuanye
Biotechnology Co. Ltd. The synthesis of compounds 3-11 had been reported and the
general synthetic routes are illustrated in Scheme 1. Compound 3 was obtained by
elimination of the C-14 hydroxyl and subsequent acetylation.17 Andrographolide was
converted to 14-deoxy-11,12-didehydroandrographolide (4) by refluxing in toluene
and catalytic amounts of Al2O3.18 Acetylation of the resulting 4 at C-3 and C-19 in the
presence of Ac2O at room tempreture (rt) afforded the di-acetylsilane product 5.
Compounds 6 and 8 were synthesized from andrographolide using sodium
borohydride (NaBH4) and Amberlyst-15.19 The further acetylation of 8 led to
derivative 9. The triacethylandrographolide was obtained from andrographolide and
the selective reduction by NaBH4 in MeOH resulted in the generation of 7,20 after
which the de-protection was carried out in 10% MeOH-HCl to furnish the hydrolysis
product 10. Andrographolide reacted with cons. hydrochloric acid (conc. HCl) at rt to
give 1121, which was subjected to the acetylation to afford compound 12. To
determine the absolute configuration of C12 of 12, we measured its electronic CD
spectrum in acetonitrile (1 mg/ml, Fig. 1). The experimental curve of 12 indicated the
positive Cotton effect (CE) at 215 nm (Δε +1.88) and the negative CE at 195 nm (Δε -
3.39), which are in good agreement with the calculated ECD one of 12S-12 by the
time-dependent density functional theory (TDDFT) method. Thus, the absolute
configuration of C12 was assigned as S.

Scheme 1. Reagents and conditions: (a)Ac2O, pyridine, rt, 12 h, 29%; (b) Al2O3, pyridine, toluene,
reflux, 16 h, 22%; (c) Ac2O, ZnCl2, rt, 4 h, 90%; (d) NaBH4, Amberlyst-15, THF, rt, 2 h, total
yields of 6 and 8 = 42%; (e) Ac2O, ZnCl2, rt, 4 h, 80%; (f) Ac2O, ZnCl2, rt, 4 h, 90%; (g) NaBH4,
MeOH, rt, 1 h, 64%; (h) 10% MeOH-HCl, rt, 1.5 h, 21.6% (i) conc. HCl, rt, 4.5 h, 88%; (j) Ac2O,
ZnCl2, rt, 4 h, 92%.

Fig. 1. Experimental ECD of 12 in MeCN and theoretical spectra of 12S-12 and 12R-12.
2.2. Effect of 1-12 on LPS-induced NO production
All the prepared compounds displayed no significant cytotoxicity (Data not
shown) and were initially screened against the production of NO in LPS-induced BV-
2 cells at 20 μM (Fig.2). Loss of activity was observed for compounds 1, 2, 4, 6, 8, 10
and 11, clearly indicating that the conjugated Δ12(13) double bond and the hydroxyl at
C-14 is critical to inhibit NO production. This is probably due to the Michael addition
between Δ12(13) exocylic double bond and the nucleophilic Cys62 of NF-κB p5017 as
well as the hydrogen bonds formation between the hydroxyl group at C-14 and other
residues. Moreover, the corresponding acetyl derivatives 3, 5, 7, 9 and 12 with greater
log P (Table 1) were found to be more active compared to 2, 4, 6, 8 and 11,
respectively, suggesting that improvement of lipophilicity would lead to an
increasement in inhibitory effects. Among these compounds, only two compounds 3
and 12 with the % inhibition as 78.0% and 95.7%, respectively, were more potent
than andrographolide (76.7%).

Fig. 2. Effect of 1-12 and andrographolide (Andro.) on LPS-induced NO production. BV-2 cells
were treated with 1-12 and andrographolide in the presence of LPS (1 μg/ml) for 24 h and NO
concentration were detected by Griess reagents. The data were presented as the mean ± S.D. of
three independent experiments. *P < 0.05 versus control; **P < 0.01 versus control; &&P < 0.001
versus control.
Table 1 Physicochemical properties of compounds 1-12.
Compound MWa
LogPb
3.07
3.28
3.87
3.23
3.83
3.43
Compound MWa
LogPb
4.02
2.54
3.71
2.9
1
480.3
332.4
416.5
332.4
416.5
334.5
7
8
418.5
352.4
478.6
334.5
350.5
476.6
2
3
9
4
10
11
12
5
2.16
2.96
6
a MW, molecular weight.
b log P, partition coefficient measured by http://www.sioc-ccbg.ac.cn/?p=42&software=xlogp322
2.3. Effect of 3 and 12 on LPS-induced NO production and the expression of iNOS
As compounds 3 and 12 showed better inhibition, we then selected these two
compounds for the following studies. Quercetin was used as the positive control. As
shown in Fig. 3A, the NO inhibition rate was concentration-dependently increased by
treatment with 3 or 12. Moreover, NO production could be suppressed about 34.4%
by 12 at 1 μM, more potent than that of quercetin. In addition, the expression of iNOS

relative to the production of NO was also investigated by immunocytochemical
analysis and RT-PCR. Our results showed that LPS-induced expression of iNOS were
markly attenuated by 3 or 12 especially at 20 μM (Fig. 3B and 3C).
Fig. 3. Effect of 3 and 12 on LPS-induced NO production and the expression of iNOS. (A) After
treatment with 3 or 12 and LPS for 24 h, the supernatant were collected and detected for NO
concentration. (B) After treatment, the total RNA was prepared and the level of iNOS mRNA
were measured using RT-PCR. (C) The expression of iNOS was also evaluated by the
fluorescence microscopy. *P < 0.05 versus control; **P < 0.01 versus control; &&P < 0.001
versus control.
2.4. Effect of 3 and 12 on LPS-induced pro-inflammatory factors TNF-α and IL-6
As illustrated in Fig. 4, TNF-a (Fig. 4A) and IL-6 (Fig. 4B) release into culture
medium were significantly increased in the LPS-induced microglia group. However,
the increased levels were remarkablely attenuated by both tested compounds in a dose
dependent manner, especially at 20 μM.

Fig. 4. Effect of compounds 3 and 12 on LPS-induced production of TNF-α and IL-6. BV-2 cells
were cultured with tested compounds and LPS for 6 h (TNF-α) or 24 h (IL-6) and then cultured
medium was detected for TNF-α (A) and IL-6 (B) concentrations. *P < 0.05 versus control;
**P < 0.01 versus control; &&P < 0.001 versus control.
2.5. Neuroprotection of 3 and 12 against microglia-mediated damage in PC12
neurons
As is well known, cytotoxic inflammatory mediators released from over-
activated microglia contributed to progressive neuronal damage. Thus, we next
investigate whether 3 and 12 could protect PC12 neurons against microglia-mediated
damage. As shown in Fig. 5A and 5B, a 40 % decrease in cell viability was observed
in PC12 cells exposed to conditioned medium from LPS treatment. And this decrease
were reversed by microglia-conditioned medium (CM) pretreated with 3 and 12.
However, post-treatment by 3 and 12 seemed to be of no important effect on cell
viability (Fig. 5C). Moreover, 12 could also protect PC12 cells in a co-culture
experimental model (Fig. 5B).

Fig.5. Neuroprotection of compounds 3 and 12 against microglia-mediated neuronal damage.
After treatment with microglia-conditioned medium pretreated with 3 (A) or 12 (B) for 24 h,
cell survival rate of PC12 cells was determined by MTT assay. PC12 cells were co-cultured
with BV-2 cells in the presence of 3 (A) or 12 (B) and LPS for 48 h and then the cell viability
was also evaluated by MTT assay. PC12 cells treated with CMC or CML and tested
compounds were selected as control (C). *P < 0.05 versus control; **P < 0.01 versus control;
&&P < 0.001 versus control.
2.6. Effect of compounds 1-12 on H2O2- and 6-OHDA induced damage in PC12 cells
To evaluate the neuroprotection of compounds 1-12 against oxidative stress,
H2O2 and 6-OHDA were used as oxidative stress inducers. The cell viability of PC12
cells exposed to 0.7 mM H2O2 and 0.9 mM 6-OHDA for 24 h were decreased to 64%
and 57% of controls, respectively (Fig. 6A and 6B). Among the tested compounds,
only compounds 11 and 12 are capable of rescuing the cell injury. It was shown that
the protective effect of 11 was observed at both concentrations, whereas the most
potent concentration was 10 μM (Fig. 6C and 6D). It is probably due to the synergy
toxic effect of 11 and oxidative stress inducers at concentrations greater than 10 μM.
Moreover, neurocytoprotective activity of 12 was only observed at 20 μM (Fig. 6E
and 6F).

Fig. 6. Effect of compounds 1-12 on H2O2- and 6-OHDA-induced decrease of cell viability in
PC12 cells. The cells were incubated with tested compounds at the indicated concentration for 24
h and followed by H2O2- and 6-OHDA-insult. The cell viability was measured by MTT assay. *P
< 0.05 versus control; **P < 0.01 versus control; &&P < 0.001 versus control.
2.7. Effect of compounds 1-12 on the neurite outgrowth of PC12 cells
The PC12 cell line was used as a cellular model for studies of neuronal
differentiation. Cell viability was first evaluated by the MTT assay and compound 12
exhibited strong toxic effects at 20 μM (Data not shown). Thus, we initially examined
the effect of compounds 1-11 at 20 μM on the neurotrophic activity of NGF. As
shown in Fig. 7A, andrographolide in combination with NGF (20 ng/ml) exerted a
moderate increase in the percentage of neurite-bearing cells. However, the derivatives
2, 3, 5-9 and 11 failed to influence neuronal differentiation, indicating that the
removal of C-14 hydroxyl (2 and 3), acetylation (such as 4 and 5) and reduction (8) is
unfavourable to the neurotrophic activity. Moreover, no differentiation effect was
observed in PC12 cells treated with 12 at 5 and 10 μM (Data not shown). Compound
4, 14-deoxy-11,12- didehydroandrographolide, increased the percentage of neurite
bearing cells up to 16.1± 1.3% compared to NGF-treated PC-12 cells (12.0%), while
the effect was abolished by the reduction of double bond Δ11(12) (6). Interestingly, the
isomerization of the exocyclic double bond Δ8(17) in 6 to the endocyclic double bond
Δ8(9) (10) could obviously enhance the activity of NGF-mediated neurite outgrowth of
PC12 cells. Furthermore, our results showed that compounds 1, 4 and 10 could induce

the formation of neurites in a concentration-dependent manner (Fig. 7B, 7C and 7D).
Notably, the percentage of neurite-bearing cells for cells treated with 1, 4 and 10 at 20
μM reached to 21.5 ± 1.1%, 18.6 ± 3.4% and 31.2 ± 2.8%, respectively (Fig. 7B-7E).
In addition, the derivatives 1, 4 and 10 showed promoting effects on neurite
outgrowth of PC12 cells in the absence of NGF.
Fig.7. Effect of compounds 1-11 on the NGF-induced neurite outgrowth in PC12 cells. (A) PC12
cells were incubated with tested compounds at 20 μM in the presence of NGF for 48 h. Then, cells
were photographed under a light microscopy (10 ×) and counted in five views. PC12 cells were
exposed to compounds 1 (B), 4 (C) or 10 (D) at 1, 5, 10 and 20 μM in the presence or absence of
NGF for 48 h and the percentage of neurite-bearing cells was examined. (E) After treatment with

1, 4 and 10 at 20 μM, the cells were stained by methylene blue and the morphological
characteristics were observed under a light microscopy (10 ×). *P < 0.05 versus control; **P <
0.01 versus control; &&P < 0.001 versus control.
2.8. Target proteins of compounds 3, 10 and 12 related to AD
In the present study, 10, 13 and 18 candidate genes were identified for
compounds 3, 10 and 12, respectively (Supporting information Table S1). Moreover,
the potential targets related to AD were screened. Notably, no target of 3 was detected
from the database. Compound 12 has the potential to act on 7 targets including
CHRM1, CHRM2, CHRM3, CHRM4, PTGS1, PTGS2 and MAP2K1. The further
gene ontology (GO) enrichment analysis (Fig. 8A) of potential target proteins of 12
was performed in DAVID (P-value ≤ 0.05). The biological processes of the target
proteins were mainly involved in regulation G-protein coupled acetylcholine receptor
signaling pathway, synaptic transmission, smooth muscle contraction, saliva
secretion, locomotion, cyclooxygenase pathway, nervous system development,
prostaglandin biosynthetic process, signal transduction, cell proliferation, blood
pressure, movement of cell or subcellular component, cellular protein modification
process, G-protein coupled receptor signaling pathway and response to oxidative
stress. As to the cellular components’ distribution, the target proteins were mainly
distributed in synapse, cell junction, plasma membrane and organelle membrane.
Additionally, we performed KEGG pathway enrichment analysis by DAVID and a
target protein-pathway network was constructed (Fig. 8B). As a result, 8 canonical
pathways related to AD were generated such as cholinergic synapse, neuroactive
ligand-receptor interaction and serotonergic synapse. However, only 4 AD-targets of
10 including CYP19A1, ESR1, HDAC2 and MAPT were identified and the pathway
of these targets was closely associated with thyroid hormone signaling pathway
(Table 2).

Fig. 8. (A) GO enrichment analysis of the AD-targets of 12. Biological process (blue), cellular
component (green), and molecular function (yellow). (B) Target-Pathway networks of 12. The red
and light blue nodes are potential targets and pathways, respectively.
Table 2 GO enrichment analysis and KEGG pathway enrichment analysis of the AD-related
targets of 10.
Category
Term
P Value
0.003
Genes
ESR1,
Biological Processes (1) androgen metabolic process;
CYP19A1
HDAC2, ESR1
(2) negative regulation of sequence -
specific DNA binding transcription
factor activity;
0.011
(3) chromatin remodeling;
0.015
0.031
0.001
HDAC2, ESR1
HDAC2, ESR1
HDAC2,
Cellular Components nuclear chromatin
Molecular Functions (1) enzyme binding
MAPT, ESR1
HDAC2, ESR1
HDAC2, ESR1
(2) transcription factor binding
0.049
0.049
KEGG-Pathway
Thyroid hormone signaling pathway

3. Discussion
The herb Andrographis paniculata is extensively used in traditional systems of
medicines in India, China and other Asian countries. Andrographolide, the major
labdane diterpenoidal constituent of A. paniculata, are reported to possess a broad
range of biological activities. Our group also evaluated the neuroprotection of
andrographolide against microglia-mediated inflammatory injuries and oxidative
damage (under review). According to the previous reports, there is no systematic
attempt made to explore the therapeutic potential of andrographolide analogues on
AD. Based on the structures of the metabolites isolated from A. paniculata,
compounds 1-12 were synthesized.
Microglia, the resident macrophages of the central nervous system (CNS),
comprised around 10% of the total CNS population. In fact, microglial cells are a
“double-edged sword” based on their level of activation. In response to infection or
injury, microglial cells are quickly activated and migrate to the site of injury to exert
neuroprotection.23 However, chronic microglial activation could release
proinflammatory cytokines (NO, ROS, TNF-α, IL-β, IL-6, COX-2) and ultimately
damage brain tissue in AD.24 Because NO is one of the main proinflammatory
mediators, we first examined the NO inhibitory effects of 1-12 using LPS-induced
BV-2 cells as a microglia-mediated neuroinflammation model. The results showed
that NO were severely inhibited by compounds 3 and 12. We also observed that 3 and
12 could suppress LPS-induced increase in mRNA levels of iNOS as well as
production of pro-inflammatory mediators TNF-α and IL-6. Moreover,
proinflammatory cytokines released from chronic microglial activation could further
elicit a wide spectrum of inflammatory reactions and finally result in neuronal toxicity
and death. To explore whether 3 and 12 could protect neurons from microglia-
mediated neuronal damage, conditioned medium were prepared and then a microglia-
neuron co-culture system was established. Our findings showed that these two
compounds could increase the cell viability of neurons not via direct neuroprotection
but inhibition of microglia-mediated inflammatory injuries. Unfortunately, 3 could

not afford neuroprotection in the co-culture system.
Oxidative stress refers to a condition that the oxidative system overwhelms the
antioxidative defense system. Due to the very high dioxygen consumption of the brain
and relative paucity of antioxidants and related enzymes,7 the brain is particularly
sensitive to oxidative damage. In fact, oxidative damage to biomolecules including
nucleic acid, protein, and lipids are extensively reported in AD.25 H2O2 could be
readily converted into highly toxic hydroxyl radical and further induce damage to
biomolecules. 6-OHDA induces selective neurotoxin for catecholaminergic neurons
through ROS generation.26 We next evaluated the neuroprotection of 1-12 against
H2O2- and 6-OHDA-induced damage in PC12 cells. However, the majority of the
andrographolide analogues showed no obvious neurocytoprotective activity. Nuclear
factor (erythroid-derived 2)-like 2 (Nrf2) is one of the critical transcription factors
modulating brain redox homeostasis and regulated by the ubiquitin ligase adaptor
Kelch-like ECH-associated protein(Keap)-1. Our previous results demonstrated that
andrographolide could activate Nrf2 and up-regulate the expression of downstream
factors-HO-1 (under review). The removal of C-14 hydroxyl and double bond Δ12(13)
may disturb the interaction of andrographolide and Keap-127 and finally led to the loss
of activity.
AD is characterized by the loss of synaptic connections and the death of neurons,
especially in hippocampus.28 Neurotrophic factors, such as NGF, could promote
neuritogenesis and neuronal survival and had been considered as potential therapeutic
agents for the treatment of AD. Due to the delivery issues and side effects,
identification of small molecules mimicing or enhancing the function of NGF is still
an urgent need. Therefore, neurotrophic actions of 1-12 in NGF-induced PC12 cells
were also determined. It is encouraging that andrographolide as well as 1, 4 and 10
could exert a pronounced increase in neurite-bearing cells. Actually, these four
compounds were natural compounds from A. paniculata and HPLC analysis of extract
showed > 30.0% (w/w) andrographolide, > 1.0% (w/w) 1 (neoandrographolide) and
≤5.0% (w/w) 4 (14-deoxy-11,12-didehydroandrographolide),29 suggesting the
potential of A. paniculata for the treatment of AD.

The emergence of systems pharmacology provided a powerful tool to decipher
complex interactions between drugs and biological systems. In order to explore the
potential mechanism of compounds 3, 10 and 12 against AD, network pharmacology
were used. First, the potential target proteins were identified and then the pathological
targets of AD were screened. As results, 4 and 7 AD-targets were obtained from 10
and 12, while no AD-targets found from 3. Resultant 4 core targets of 10 were mainly
involved in thyroid hormone signaling pathway. Further enrichment analysis of 7
target proteins of 12 demonstrated that cholinergic synapse, regulation of actin
cytoskeleton, neuroactive ligand-receptor interaction, serotonergic synapse, calcium,
cAMP and PI3K-Akt signaling pathway may be the important pathways regulated by
12. The majority of these pathways were closely related to muscarinic acetylcholine
receptors (mAChRs) such as CHRM1 and CHRM2. mAChRs are G-protein coupled
receptors (GPCRs) and specific mAChR subtypes were located in specific brain
regions. For example, the levels of M3 and M2 were found to be lower in the
hippocampus than that of M1 and M4.30 The previous reports have pointed out that
the level of mAChR expression is changed in AD and searching for selective mAChR
activators and antagonists has been considered as an effcient strategy in drug
discovery for AD.31 COX-1 and COX-2 were encoded by PTGS1 and PTGS2,
respectively. COX-2 could be induced by activated microglia which is involved in
chronic neuroninflammation and may contribute to the pathological changes in AD.32
4. Conclusion
In summary, 12 andrographolide analogues were obtained and this is the first
report on their neuroprotection and neurotrophic activity. The preliminary results
showed that compounds 3 and 12 could exert anti-inflammatory activity in microglial
cells and protect neuronal cells through suppression of pro-inflammatory mediator
production. Notebly, the novel compound 12 could also protect neuronal cells against
oxidative damage. Moreover, compounds 1, 4 and 10 could potentiate NGF-induced
neurite outgrowth. The potential mechanism of compounds 3, 10 and 12 against AD
were also explored by network pharmacology. We are currently attempting to
elucidate the detailed mechanisms of the most potent analogue.

5. Materials and methods
5.1. Chemicals and reagents
Fetal bovine serum (FBS) was from Hyclone (Logan, UT, USA). Dulbecco's
modified Eagle's medium (DMEM) and horse serum (HS) were purchased from
Gibco Ltd. (Grand Island, NY, USA). LPS from Escherichia coli 0111:B4, methylene
blue and poly-L-lysine (PLL) solution were obtained from Sigma-Aldrich Co. (St.
Louis, MO). MTT was from Solarbio Technology Company (Beijing, China). NO
assay kit was from Beyotime Institute of Biotechnology (Jiangsu, China). TNF-α and
IL-6 enzyme-linked immunosorbent assay (ELISA) kits were supplied by Boster
Biological Technology (Wuhan, China). RevertAid First Strand cDNA Synthesis kit
and TRIzol™ reagent were purchased from Thermo Fisher Scientific. Rabbit
polyclonal antibody against iNOS was purchased from Proteintech (Wuhan, China).
6-OHDA was supplied by Santa Cruz Biotechnology (Santa Cruz, CA). The mouse
7S NGF was supplied by Wuhan Hitech Biological Pharma Co. LTD (Wuhan, China).
All the chemicals and reagents were of analytical grade.
5.2. Synthesis and characterization of compounds
All progress of the reactions was monitored by analytical thin layer
chromatography (TLC) on silica gel under UV light at 254 nm. All compounds were
purified by column chromatography packed with silica gel (200-300 mesh) purchased
from Qingdao Haiyang Chemical Co. Ltd. 1H and 13C NMR spectra of new compound
12 were recorded on a Bruker Avance III spectrometer (500 and 125 MHz for 1H and
13C NMR, respectively) with tetramethylsilane (TMS) as an internal standard. HRMS
(ESI) were acquired on an AB Triple TOF® 4600 mass spectrometer (ABSCIEX,
Redwood City, CA, USA).
General procedure for preparation of compound 12: A mixture of catalytic zinc
chloride (ZnCl2, 13 mg), acetic anhydride (Ac2O, 0.5 ml) and compound 11 (55 mg)
were stirred at room temperature for 3.5 h. After completion, the reaction mixture was
diluted with dichloromethane (DCM) and washed with saturated aqueous Na2CO3.
The combined organic phase was dried over anhydrous Na2SO4 and filtered. After
evaporated under reduced pressure, the crude product was purified by column

chromatography on silica gel (petroleum ether/ethyl acetate = 1/2) to afford 12 as a
1
white oil (68.6 mg). Yield: 92%. H NMR (500 MHz, CDCl3) δ 7.27 (1H, s), 5.75
(1H, m), 4.80 (2H, m), 4.60 (1H, d, J = 10 Hz), 4.36 (1H, d, J = 10 Hz), 4.19 (1H, d, J
= 15 Hz), 2.65 (1H, m), 2.50 (1H, m), 2.09 (3H, s), 2.04 (6H, s), 2.01 (3H, m), 1.80-
1.75 (2H, m), 1.74-1.69 (2H, m), 1.55 (3H, s), 1.52 (1H, m), 1.35 (1H, d, J = 15 Hz),
13
1.03 (3H, s), 0.98 (3H, s); C NMR (125 MHz, CDCl3) δ 171.22, 170.71, 170.28,
169.47, 145.45, 134.14, 133.55, 130.61, 79.74, 69.90, 68.86, 64.99, 51.46, 40.81,
37.93, 35.01, 34.00, 31.25, 23.93, 22.49, 20.95, 20.86, 20.82, 20.42, 20.30, 19.46;
+
HRMS (ESI) m/z calcd for C26H37O8 (M+H)+ 477.24829, found 477.24841.
5.3. Computational section
The calculations were performed according to our previous reported protocol.33
A preliminary conformational search was performed in Conflex6.7 using MMFF94s
forcefield. Conformers were saved and further optimized using the density functional
theory (DFT) method and CPCM solvent model at B3LYP/6-31+G(d,p) level in
Gaussian 09 software package.34 Frequency was calculated at the same level of theory
to check optimized results. The stable conformers with populations greater than 1%
and without imaginary frequencies were submitted to ECD calculation by the TDDFT
using cam-B3LYP/TZVP method associated with CPCM solvent model in MeCN.
The excitation energies (E), oscillator strength (f), rotatory strength in velocity form
(Rvel), and rotatory strength in length form (Rlen) of the lowest 32 excited states
were calculated. ECD spectra of different conformers were summated in SpecDis
according to their Boltzmann-calculated distributions.35 The wavelength were
corrected according to the calculated UV and experimental UV spectra.
5.4. NO determination
Murine BV-2 microglia cell line was purchased from Cell Resource Centre of
Peking Union Medical College (Beijing, China) and cultured in DMEM containing
10% FBS (1% penicillin/streptomycin) at 37°C in an atmosphere of 5% CO2.
Accumulation of NO in microglial culture was determined by a NO assay kit based on
the Griess reaction. Briefly, 50 μl of BV-2 culture supernatant were collected and an

equal volume of Griess reagentsⅠ andⅡ was added. Then, the absorbance was
measured at 540 nm. Nitrite concentrations were calculated from a sodium nitrite
standard curve prepared in cell culture medium. NO inhibition (%) =1- (sample OD -
control OD)/(LPS OD - control OD)×100%.
5.5. Immunofluorescence
BV-2 cells (1.2 × 105 cells/ml) were cultured on the climbing slices coated with
PLL in 24-well plates. After treatments in the presence or absence of LPS (1 μg/ml),
the cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized
with 0.5% Triton X-100 for 20 min at room temperature. Nonspecific sites were
blocked with 10% normal goat serum for 30 min, after which the primary rabbit
◦
polyclonal anti-iNOS (1:50) were added in a humidified chamber overnight at 4 C.
The cells were washed with Tris-buffered saline containing 0.05% (v/v) Tween-20
(TBS-T) and incubated with the secondary TRITC-conjugated goat anti-rabbit IgG
antibody diluted to 1:64 in PBS-T at 37◦C for 2 h. Following washing, the nucleus
was stained by 4'-6-diamidino-2-phenylindole (DAPI) and analyzed by the
fluorescence microscopy (OLYMPUS, Japan).
5.6. Reverse transcription-polymerase chain reaction (RT-PCR)
BV-2 cells, cultured in 6-well plates, were treated with LPS and/or compounds 3
or 12 for 24 h. Total RNA was extracted with TRIzol reagent according to the
manufacturer's instruction. cDNA was synthesized using RevertAid First Strand
cDNA Synthesis kit. PCR amplification using primer sets specific for inducible nitric
oxide synthase (iNOS) from the cDNA was carried out at 94 °C for 3 min, 94 °C for
30 s, 60 °C for 30 s, 72 °C for 35 s, and repeated 25 cycles. Primer sequences used in
this study were as follows: for iNOS: forward 5'-CCC-TTC-CGA-AGT-TTC-TGG-
CAG-CAG-C-3' and reverse 5'-GGC-TGT- CAG-AGC-CTC-GTG-GCT-TTG-G-3'36;
for GAPDH: forward 5'-AAC- TTT-GGC-ATT-GTG-GAA-GG-3' and reverse 5'-
GGA-TGC-AGG-GAT-GAT- GTT-CT-3'. The relative expression of iNOS was
evaluated using GAPDH as an internal control.
5.7. Measurement of TNF-α and IL-6
BV-2 cells were treated with tested compounds in the absence or presence of

LPS (1 μg/ml) for 6 h (TNF-α) or 24 h (IL-6). The level of TNF-α and IL-6 in the
culture media were quantified by ELISA kit according to the manufacturer's
instructions.
5.8. Determination of neuroprotection
Microglia-conditioned medium: BV-2 cells (3.0 × 105 cells/ml) were seeded in
6-well plates and followed by incubation with 3 or 12 in the presence of LPS (1
μg/ml) for 24 h. The supernatants were centrifuged and collected as microglia-
conditioned medium (CM). Moreover, the supernatants from treatment with 0.1%
DMSO or LPS were collected to be used as CMC and CML, respectively. PC12 cells
(2.5 × 105 cells/ml) were seeded in PLL-coated 96-well plates and treated with CM,
CMC+compounds or CML + compounds for 24 h. Then MTT assay was used to
evaluate the effect on the viability.
Coculture system: The BV-2 cells (2.0 × 105 cells/ml) were seeded in transwell
inserts with 0.4 mm pore size (Corning, Inc., Corning, NY, USA). PC12 cells were
cultured at a density of 1.0 × 105/ml in 24-well culture plates. On the following day,
BV-2 cells were cultured in fresh medium containing tested compounds with LPS and
the inserts were then moved into the 24-well plates to coculture for another 48 h. The
cell viability of PC12 cells was measured by MTT assay.
5.9. Measurement of neurite outgrowth in PC-12 cells
Rat pheochromocytoma PC12 cell line was purchased from the Shanghai
Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences), and
maintained in a humidified atmosphere containing 5% CO2 at 37°C using DMEM
supplemented with 10% heat-inactivated HS and 5% FBS (1%
penicillin/streptomycin). PC-12 cells were seeded into 24-well plates coated with PLL
at a density of 1.5 × 104/ml. After replacement with fresh medium containing 2% HS
and 1% FBS for 16 h, cells were treated with tested compounds and NGF (20 ng/ml)
for another 48 h. Then, PC12 cells were photographed under a light microscopy and
the cells bearing neurites greater than or equal to the length of the cell body in five
views were counted. The data was expressed as a percentage of the total cell number.
After treatment, the cells were fixed with 4% paraformaldehyde for 20 min and

washed with PBS. Then, 0.1 M borate buffer was subsequently added for another 5
min at room temperature, after which PC12 cells were stained with methylene blue
solution (0.1% in 0.1 M borate buffer) for 2 h. Morphological analysis of neurite-
bearing cells were performed under a light microscopy.
5.10. MTT assay
PC12 cells (1.5 × 105 cells/ml), cultured in PLL-coated 96-well plates, were
incubated with tested compounds in fresh medium containing 2% HS and 1% FBS for
24 h at 37 °C. After replacement with fresh medium containing 0.7 mM H2O2 or 0.9
mM 6-OHDA for 24 h, MTT (5 mg/ml) was added and incubated for another 4 h.
After the solubilization of purple formazan in DMSO, the optical density of
absorbance was measured at 570 nm.
5.11. Targets prediction
Network pharmacology was carried out to identify the potential targets of 3, 10
and 12. Firstly, the biological targets of these three compounds were explored by
searching Similarity Ensemble Approach (SEA, http://sea.bkslab.org/),37 Swiss Target
Prediction (http://www.swisstargetprediction.ch/),38 STITCH (http://stitch.embl.de/)39
and BATMAN-TCM (http://bionet.ncpsb.org/batman-tcm/).40 Only “Homo sapiens”
target were selected. Secondly, all targets obtained from the previous step are sent to
Comparative Toxicogenomics Database (http://ctdbase.org/),41 Therapeutic Target
Database (TTD, http://bidd.nus.edu.sg/group/cjttd/),42 GeneCards: The human gene
database
(https://www.genecards.org/),43 Pharmacogenomics Knowledgebase
(https://www.pharmgkb.org/)44 and Molecule Annotation System 3.0 (MAS 3.0,
http://bioinfo.capitalbio.com/mas3/) to screen out potential targets related to AD.
5.12. Network construction
To investigate the possible mechanism of drug action, Target-Pathway network
(T-P network) was constructed and visualized using Cytoscape ver. 3.7.0
(https://cytoscape.org/).45 The Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway information of targets were extracted from Database for Annotation,
Visualization
and
Integrated
Discovery
(DAVID)
ver.
6.8
(https://david.ncifcrf.gov/summary.jsp).46,47 Gene Ontology (GO) enrichment analysis

was also carried out by linking the targets to DAVID.
5.13. Statistical analysis
All results above were presented as means ± S.D. and analyzed by one-way
ANOVA following by Dunnett’s multi-comparison test using SPSS program (version
12.0).
Acknowledgments
This work was financially supported by a grant from the National Natural Science
Funds (No. 81602994) and the Scientific Startup Foundation for Doctors of Northwest
A&F University (No. 2452015353).
Conflict of interest
The authors declare no conflict of interest.
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1. This is the first report on neuroprotection and neurotrophic activity of andrographolide
analogues.
2. Compound 12 was first synthesized and showed potent anti-neuroinflammatory and
neuroprotective properties.
3. Natural compounds 1, 4 and 10 were first found to be of neurite outgrowth-promoting activities.
4. Muscarinic acetylcholine receptors (mAChRs) may be the main targets of 12 against AD.