Design and evaluation of Nrf2 activators with 1,3,4-oxa/thiadiazole core as neuro-protective agents against oxidative stress in PC-12 cells

Article abstract of DOI:10.1016/j.bmcl.2019.126853

Oxidative stress plays vital roles in virous neurodegenerative diseases including Alzheimer's disease. Activation of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), the key regulator of oxidative stress, may provide a new ther

Full text of DOI:10.1016/j.bmcl.2019.126853

Journal Pre-proofs
Design and Evaluation of Nrf2 Activators with 1,3,4-Oxa/thiadiazole Core as
Neuro-protective Agents against Oxidative Stress in PC-12 Cells
Hongzhi Lin, Yuting Qiao, Hongyu Yang, Qi Li, Yao Chen, Wei Qu, Wenyuan
Liu, Feng Feng, Haopeng Sun
PII:
S0960-894X(19)30827-3
DOI:
https://doi.org/10.1016/j.bmcl.2019.126853
BMCL 126853
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Bioorganic & Medicinal Chemistry Letters
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2 November 2019
21 November 2019
Please cite this article as: Lin, H., Qiao, Y., Yang, H., Li, Q., Chen, Y., Qu, W., Liu, W., Feng, F., Sun, H., Design
and Evaluation of Nrf2 Activators with 1,3,4-Oxa/thiadiazole Core as Neuro-protective Agents against Oxidative
Stress in PC-12 Cells, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.126853
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Design and Evaluation of Nrf2 Activators with 1,3,4-Oxa/thiadiazole Core as Neuro-protective
Agents against Oxidative Stress in PC-12 Cells
Hongzhi Lin a, Yuting Qiao a, Hongyu Yang a, Qi Li a, Yao Chen c, Wei Qu d, Wenyuan Liu e,
Feng Feng b, *, Haopeng Sun a, b, *
a
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, 211198,
People’s Republic of China
b
Jiangsu Food and Pharmaceutical Science College, Huaian, 223003, People’s Republic of
China
c School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China;
d
Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing,
211198, People’s Republic of China
e
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing, 210009,
People’s Republic of China
Corresponding Authors: sunhaopeng@163.com (H. P. Sun); fengsunlight@163.com (F. Feng)
Abstract
Oxidative stress plays vital roles in virous neurodegenerative diseases including
Alzheimer’s disease. Activation of the transcription factor Nrf2 (nuclear factor erythroid 2-
related factor 2), the key regulator of oxidative stress, may provide a new therapeutic strategy
for these diseases. Herein we synthesized and evaluated a series of 1,3,4-oxa/thiadiazole core
Nrf2 activators as neuroprotective agents. The representative compound 8 exhibited
cytoprotective and Nrf2 activation effects in a neuron-like PC-12 cells. Additionally, compound
8 showed good membrane permeability, indicating this compound could penetrate blood-brain
barrier (BBB) to reach central nervous system (CNS) as a neuro-protective agent. These results
indicated that these Nrf2 activators with 1,3,4-oxa/thiadiazole core could serve as a new
chemotype against oxidative stress in neurodegenerative diseases.
Key word: Nrf2 activator; neuro-protective agent; oxidative stress.
1

Alzheimer’s disease (AD) is one of the severest health problems around the world1. This
neurodegenerative disease is the most common cause of dementia. With the increase of the
population of old people, the number of AD patients will escalate rapidly in the coming years.
2, 3. Only five drugs have been proved by Food and Drug Administration (FDA) for the treatment
of AD, including rivastigmine, galantamine, donepezil, memantine, and tacrine (tacrine is now
discontinued in the United States). But the effectiveness of these drugs is temporary and varies
from person to person. Considering the huge social burden caused by AD, new drugs are
urgently needed to treat AD. So far, the etiology of AD has not been well understood. A number
of theories of AD pathogenesis are proposed, including cholinergic dysfunction4, amyloid
cascade5, hyperphosphorylation of tau-protein6, cell cycle hypothesis7, brain-derived
neurotrophic factor hypothesis8, and oxidative stress related free radical formation9, metal
dyshomeostasis10, and mitochondrial dysfunction hypotheses11. However, none of the
pharmacologic treatments alleviates or prevents the progression of AD.
The relationship between AD and oxidative stress is still obscure. It is difficult to assess
that oxidative stress is a cause, a consequence, or just an independent biomarker of AD12.
Nevertheless, increasing number of studies demonstrate that oxidative stress plays vital roles
in the progression of AD. In the brain tissues of AD patients, high level of reactive oxygen
species (ROS) directly leads to a range of cellular damages. Nuclear factor erythroid 2-related
factor 2 (Nrf2) is the key regulator of cellular antioxidative process. Under normal condition,
the newly synthetic Nrf2 proteins are quickly ubiquitylated by Kelch-like ECH-associated
protein 1 (Keap1) and then are degenerated through ubiquitin-proteasome pathway. Under
stress condition, Nrf2 translocates into nucleus and binds with the antioxidant response element
(ARE) to induce the expression of its downstream genes13, leading to the activation of more
than 100 antioxidative proteins, including antioxidant proteins, phase I and II detoxification
enzymes, transport proteins, and some transcription factors14, 15. Thus, activating Nrf2-ARE
pathway could systemically alleviate oxidative stress and Nrf2 is regarded as an attractive
therapeutic target for neurodegenerative diseases16.
Small molecular Nrf2 activators are generally classified into two distinct groups17-19: 1,
electrophilic activators that can covalently modify Keap120-23; 2, activators interfere Keap1-
2

Nrf2 protein-protein interaction (PPI)24, 25. So far, most of the preclinical and clinical studies of
Nrf2 activator are focus on cancer chemoprevention and inflammatory diseases in lung or
kidney. However, the use of Nrf2 activators in central nervous system is limited by the potential
long-term toxicity or poor pharmacokinetics properties18, 26
.
Previously, we identified a series of compounds with 1,2,4-oxadiazole core as Nrf2
activators27, 28. The most potent activator, compound 1 (figure 1), upregulated gene and protein
level of Nrf2 as well as its downstream genes, including NQO1, GCLM, and HO-1, in vivo.
Moreover, it remarkably suppressed inflammatory symptom in the LPS-challenged mouse
model and the DSS-induced murine colitis27, 29. However, the low aqueous solubility of
compound 1 limits its further development. In this study, on the basis of compound 1, we
synthesized and evaluated a series of compounds with 1,3,4-oxadiazole or 1,3,4-thiadiazole
core. These compounds displayed similar Nrf2 inductive activities compared with the activators
with 1,2,4-oxadiazole core. More importantly, replacing 1,2,4-oxadiazole moiety by 1,3,4-
oxadiazole led to increased aqueous solubility and lower lipophilicity. Moreover, we explored
the potential neuro-protective effects of these 1,3,4 oxa/thiadiazole core Nrf2 activators in PC-
12 cells. The results showed that the most potent compound 8 can upregulate the mRNA and
protein levels of Nrf2 and its downstream genes and protect PC-12 cells against the H2O2-
induced damage.
The previous SAR studies about the ring A and C can be briefly descripted as: 1, on ring
A, electron-withdrawing groups can improve the ARE-induced activity; 2, para-substitution on
ring A is more preferable than meta- and ortho-substitutions27; 3, for the ring C, introduction
of electron-donating groups enhances the ARE-induced activity30. However, the significance
of the B ring, not only for the Nrf2 activation, but also for its effects on the physicochemical
properties, has not been fully discussed before. Comparing with 1,2,4-oxadiazole, 1,3,4-
oxadiazole isomer shows an order of magnitude lower lipophilicity (log D) and higher aqueous
solubility31. To further improve the solubility of the 1,2,4-oxadiazole core Nrf2 activators,
especially with the aim to optimize their BBB permeability, in the present study, we synthesized
and evaluated a series of compounds bearing 1,3,4-oxa/thiadiazole core.
Derivatives were synthesized through two steps. Firstly, ethyl benzimidazole-5-
carboxylate was treated with hydrazine hydrate and refluxed in ethanol for 48 hours to give
3

benzimidazole-5-carbohydrazide (2). Then, the target compounds 3-12 were cyclized from 2
with substituted benzoic acid. The ARE inductivities of compound 3-12 were evaluated by
luciferase reporter assay in HepG2-ARE-C8 cells27. As shown in table 1, most of the
compounds with 1,3,4-oxadiazole core exhibited ARE inductivity at all tested concentrations.
Compared to compound 3, the nitro and n-butyl substituted compounds (9 and 8) displayed
stronger Nrf2 induced activities. Compounds with halogen substituents in ring A (4, 5, and 6)
exhibited similar activities with compound 3. However, under the high concentrations (40 μM
and 80 μM), the ARE inductivity of the bromo substituted compound 4 went through a
downward turn. It is noteworthy that similar activity-declining results have been found during
the SAR study of chlorine and bromine substituted Nrf2 activators with 1,2,4-oxadiazole core
27, 30
(figure. S1). Compounds with electron-donating group (7, 11, and 12) showed reduced
ARE inductivities. The results indicated that the electron-withdrawing group was more
preferable for this series of Nrf2 activators. However, compound 10 with electron-withdrawing
cyano group and compound 8 with electron-donating n-butyl group were two exceptions.
Compound 10 just maintained 2~3-fold ARE inductivities under all test concentration.
Similarly, the cyano-substituent 1,2,4-oxadiazole core Nrf2 activators did not exhibit good
ARE inductivities, too27. Compound 8 exhibited a strong inductivity, with 20.05 ± 2.40-fold
upregulation of the ARE level at 40 μM. Overall, the 1,3,4-oxadiazole core Nrf2 activators
displayed slightly less ARE inductivities than the corresponding 1,2,4-oxadiazole core
activators. However, compound 8 displayed closed ARE inductivity with compound 1 in a
preliminary screening (Figure S2). Meanwhile, compounds with 1,3,4-oxadiazole core shared
similar SAR features with 1,2,4-oxadiazole core Nrf2 activators. Thus, we suggested that the
replacement of five-member ring B was tolerable in structural optimization of physicochemical
properties of the Nrf2 activators.
In order to verify this result, we further synthesized compounds with 1,3,4-thiadiazole core
(13 - 16). Their Nrf2 inductivities were also evaluated by luciferase reporter assay. As shown
in table 1, Compound 13 (unsubstituted), 14 (p-fluorine substituent), and 16 (p-nitrogen
substituent) shown slightly reduced Nrf2 inductivities comparing with 1,2,4- and 1,3,4-
oxadiazole core compounds. But the n-butyl substituted compound 15 just exhibited very
limited activity. As a cell-based assay, the luciferase report gene assay is always affected by
4

the status of the tested cells, especially under high induced-folds. However, in this work, we
believed the large errors may be caused by two additional reasons: 1, cytotoxicity under high
concentration of the compounds may affect the cell condition. The potential toxicity of nitro-
substituted compounds was found in this work and previous study. The large errors of
compound 9 and 16 may be caused by their potential cytotoxicity. 2, the low solubility of
several compounds under high concentration may be responsible for the errors, too.
In the previous studies, the Nrf2 activators with 1,2,4-oxadiazole core ameliorated the
oxidative stress both in vitro and in vivo 27-30. However, their biological effects on neurocytes
have not been elucidated. In order to explore the potentially protective effect of these
compounds on central nervous system (CNS), several representative compounds were
evaluated in a neuron-like cell line PC-12. Firstly, the cytotoxicity of representative compounds
8, 9, 14, and 16 toward PC-12 cells were determined by MTT assay. As shown in figure 2, all
the four compounds did not display significant cytotoxicity to the cells. The IC50 values of
compounds 8 and 9 (1,3,4-oxadiazole core) were greater than 100 μM, while the IC50 values of
compounds 14 and 16 (1,3,4-thiadiazole core) were above 500 μM. Additionally, all the four
compounds did not show remarkable cytotoxicity against other neuron-like SH-SY5Y cells at
100 µM (figure S3). Next, we determined the protective effects of the four compounds against
the H2O2-induced damage in PC-12 cells, a well-established cellular model of
neurodegenerative disorders20, 32. PC-12 cells were pretreated with compounds for 6 hours and
subsequently were treated with medium that contained 1000 μM H2O2 for additional 12h. As
shown in figure 3, under the low concentration (5 μM), the two nitro-contain compounds, 9 and
16, displayed good protective effects against H2O2-induced damage. However, under the higher
concentrations (10 and 20 μM), the protective effects of the two compounds vanished. This
result indicated the nitro group may lead to potential cytotoxicity under oxidative stress
condition. Two other tested compounds 8 and 14, by contract, did not display obvious
cytoprotective activity in 5 μM. Nevertheless, the two compounds gave excellent performance
under the high concentrations (10 and 20 µM). Therefore, compound 8 was selected for further
mechanism investigation.
With the aim of confirming the Nrf2 inductivity of 8 in PC-12 cells, we further examined
the concentration-dependent effect of 8 on the mRNA level of Nrf2 and its downstream genes,
5

NQO1 and HO-1, by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. As
shown in figure 4, compound 8 can dose-dependent increase the expressions of the three genes.
Then we evaluated the protein levels of Nrf2, NQO1 and HO-1 in PC-12 cell after the treatment
of compound 8. As shown in figure 5, compound 8 led to moderate up-regulation of these
proteins in a dose-dependent manner. Taken together, these results exhibited that compound 8
can activate the Nrf2-ARE pathway in both mRNA level and protein level in PC-12 cells. And
these results were consistent with previously published results using compound 1 in HCT116
cells27.
Membrane permeability is a significant property for CNS drugs33, 34. In an effort to identify
the membrane permeability of the representative compound 8, we determined its permeability
(Pe value) via parallel artificial membrane permeability assay (PAMPA)35. Five known
compounds were also involved as internal standards (table S1). Generally, a compound with a
Pe value greater than 4 × 10−6 cm/s can be regarded to be able to penetrate blood-brain barrier
(BBB)35. In the PAMPA experiment, 8 displayed good membrane permeability, with the Pe
value of 12.35 × 10−6 cm/s, indicating this compound could reach CNS. With the aim of
evaluating the effect of rearranging 1,2,4-oxadiazole to 1,3,4-oxadiazole, we determined some
physicochemical properties of compound 5 which had same structural moieties of ring A and
C with compound 1. The LogD7.4 value and intrinsic aqueous solubility of compound 5 were
determined on a Gemini profiler instrument (pION) by the “goldstandard” Avdeef-Bucher
potentiometric titration method27, 30. In comparison with 127, compound 5 exhibited a lower
LogD7.4 value (2.89 vs 3.37) and an improved intrinsic solubility (102.50 μM vs 66.90 μM).
We inferred that the greater dipole moment and polarity of 1,3,4-oxadiazole toward 1,2,4-
oxadiazole was the reason for the lower lipophilicity and increased solubility31.
In summary, a series of Nrf2 activators with 1,3,4-oxa/thiadiazole core were designed and
synthesized, and the representative compound 8 displayed cytoprotective and Nrf2 activation
effects in a neuron-like PC-12 cells. Additionally, compound 8 showed good membrane
permeability, indicating this compound could reach CNS as a neuro-protective agent. Moreover,
these results deepen our understanding of the SAR study on this series of Nrf2 activators. While
the modification of the five-member heterocycle (ring B) of this series of compounds was not
previously discussed27, 30, this study reveals that replacing ring B by other five-member ring is
6

tolerable in maintaining Nrf2 inductive activity. More importantly, the 1,3,4-oxadiazole core
resulted in improved solubility and lower lipophilicity. Altogether, these results provide a new
chemical tool to evaluate the therapeutic effect of Nrf2 activation in neurodegenerative diseases.
Acknowledgement
We gratefully thank the support from the grants (Nos. 81573281, 81872728, and 81830105) of
National Natural Science Foundation of China. We also thank the support from Double First-
Class" initiative Innovation team project of China Pharmaceutical University (Nos.
CPU2018GF11 and CPU2018GY34).
7

N
N
C
N
C
O
ring B replacement
N
A
N
A
X
N
N
N
B
B
R
F
H
H
1
Nrf2 activator with 1,2,4-oxadiazole core ( )
Nrf2 activator with 1,3,4-oxa/thiadiazole core
Figure 1. The molecular design strategy of the target compounds.
Figure 2. Cytotoxicity evaluation of representative compounds (8, 9, 14, and 16) in PC-12 cells.
The cells were plated in a 96-well plate for 12 h and subsequently treated with different
concentrations (1, 5, 10, 50, 100, and 500 µM) of the selected derivatives for another 24 h. The
cell viability was measured by the MTT assay. All data represent the mean ± SEM of three
independent experiments.
8

Figure 3. Protective effects of representative compounds against H2O2-induced damage in PC-
12 cells. Cell viability was determined using the MTT assay. The viability of untreated cells is
defined as 100%. All data represent the mean ± SD of three independent experiments.
O
O
N
N
(a)
(b) or (c)
H2N
N
N
O
N
N
X
H
R
N
H
N
H
N
H
3
16
-
2
Scheme 1. Synthetic Route of the 1,3,4-oxadiazole and 1,3,4-thiodiazole core derivatives.
Reagents and conditions: (a) hydrazine hydrate, EtOH, reflux, 48h; (b) substituted benzoic acid,
CDI, THF, POCl3, reflux, 5h; (c) substituted benzoic acid, CDI, P2S5, DMF. 6h.
9

Figure 4. Quantitative real-time PCR analysis of Nrf2, NQO1, and HO-1 in PC-12 cells. All
genes transcription levels were determined after treatment with different concentrations of 8
for 12 h.
Figure 5. Dose-dependent increase of Nrf2 and its downstream protein NQO1 and HO-1 after
treatment with 8. PC-12 cells were treated with 8 at different concentrations (0.1 µM, 1 µM, 10
µM, 20 µM, 40 µM) for 12 h. GAPDH was determined as the loading control.
10

Table 1, Nrf2 inductivity of compound 3 to 16 in Luciferase reporter assay.
ARE-inducing activity in luciferase reporter assaya
cpd.
X
R
0.1 M
2.49 ± 0.40
1.70 ± 0.40
1.45 ± 0.10
1.58 ± 0.03
2.05 ± 0.55
0.90 ± 0.05
1.80 ± 0.50
1.50 ± 0.45
1.40 ± 0.00
1.40 ± 0.35
1.64 ± 0.55
1.70 ± 0.05
1.60 ± 0.40
1.08 ± 0.18
1 M
10.23 ± 0.66
2.35 ± 0.95
2.38 ± 0.33
1.28 ± 0.08
4.85 ± 0.60
1.60 ± 0.30
6.73 ± 0.88
1.90 ± 0.35
1.65 ± 0.80
1.45 ± 0.15
1.72 ± 0.08
2.03 ± 0.23
3.25 ± 0.95
2.33 ± 0.23
10 M
14.21± 0.66
6.70 ± 0.95
1.35 ± 0.15
2.60 ± 0.25
7.98 ± 1.53
1.58 ± 0.13
14.75 ± 0.80
7.68 ± 0.37
1.33 ± 0.73
3.25 ± 0.80
1.72 ± 0.53
8.95 ± 3.15
13.10 ± 2.15
2.30 ± 0.45
20 M
27.28 ± 1.26
9.15 ± 2.25
1.65 ± 0.70
6.83 ± 0.68
8.08 ± 0.03
3.15 ± 0.30
15.88 ± 0.07
12.23 ± 3.48
2.35 ± 0.55
3.35 ± 1.25
1.83 ± 0.50
8.18 ± 0.18
14.90 ± 2.15
1.68 ± 0.03
40 M
40.81 ± 0.22
13.48 ± 0.67
1.75 ± 0.10
8.73 ± 1.53
6.23 ± 0.52
5.18 ± 0.63
24.58 ± 1.18
20.05 ± 2.40
1.78 ± 0.18
4.95 ± 1.40
2.41 ± 1.02
13.83 ± 4.88
22.30 ± 0.95
1.33 ± 0.03
80 M
44.40 ± 0.89
19.10 ± 1.65
1.63 ± 0.02
24.40 ± 1.10
7.98 ± 0.28
9.10 ± 1.60
19.55 ± 0.45
33.83 ± 0.93
2.83 ± 1.18
6.60 ± 1.80
7.50 ± 2.35
15.70 ± 2.45
9.90 ± 0.80
1.43 ± 0.23
1
3
-
-
O
O
O
O
O
O
O
O
O
O
S
H
4
3-F
5
4-F
6
4-Br
4-Ph
4-n-Bu
4-NO2
4-CN
4-Me
3-OMe
H
7
8
9
10
11
12
13
14
15
S
4-F
S
4-n-Bu
11

16
S
-
4-NO2
-
2.20 ± 0.20
1.68 ± 0.38
5.75 ± 0.40
1.70 ± 0.40
19.45 ± 5.45
ND
17.43 ± 3.48
ND
22.18 ± 5.48
5.10 ± 1.05
24.63 ± 3.78
4.73 ± 0.58
t-BHQ
aThe inductivity of the compound is calculated compared to the blank control, and data are presented as mean ± SD of two separate experiments.
12

References
1. Crane PK, Trittschuh E, Mukherjee S, et al. Alzheimers Dement. 2017;13: 1307-1316.
2. Alzheimer's & Dementia. 2018;14: 367-429.
3. Ferri CP, Prince M, Brayne C, et al. Lancet. 2005;366: 2112-2117.
4. Francis PT, Palmer AM, Snape M, Wilcock GK. J Neurol Neurosur Ps 1999;66: 137-147.
5. Karran E, Mercken M, De Strooper B. Nat Rev Drug Discov.. 2011;10: 698-U1600.
6. Ballatore C, Lee VMY, Trojanowski JQ. Nat Rev Neurosci. 2007;8: 663-672.
7. Webber KM, Raina AK, Marlatt MW, et al. Mech Ageing Dev. 2005;126: 1019-1025.
8. Tapia-Arancibia L, Aliaga E, Silhol M, Arancibia S. Brain Res Rev. 2008;59: 201-220.
9. Dumont M, Beal MF. Free Radic Biol Med. 2011;51: 1014-1026.
10. Jomova K, Valko M. Toxicology. 2011;283: 65-87.
11. Devi L, Anandatheerthavarada HK. Biochim Biophys Acta. 2010;1802: 11-19.
12. Pohanka M. Bratisl med J. 2018;119: 535-543.
13. Zhang DD. Drug Metab Rev.2006;38: 769-789.
14. Kobayashi M, Yamamoto M. Antioxid Redox Sign. 2005;7: 385-394.
15. Itoh K, Chiba T, Takahashi S, et al. Biochem Bioph Res Co. 1997;236: 313-322.
16. Lu MC, Ji JA, Jiang ZY, You QD. Med Res Rev. 2016;36: 924-963.
17. Sun H, Zhu J, Lin H, Gu K, Feng F. Expert Opin Ther Pat. 2017;27: 763-785.
18. Wilson AJ, Kerns JK, Callahan JF, Moody CJ. J Med Chem. 2013;56: 7463-7476.
19. Magesh S, Chen Y, Hu L. Med Res Rev. 2012;32: 687-726.
20. Peng S, Zhang B, Meng X, Yao J, Fang J. J Med Chem. 2015;58: 5242-5255.
21. Yao J, Zhang B, Ge C, Peng S, Fang J. J Agric Food Chem. 2015;63: 1521-1531.
22. Peng S, Zhang B, Yao J, Duan D, Fang J. Food Funct. 2015;6: 2091-2100.
23. Peng S, Hou Y, Yao J, Fang J. Food Funct. 2017;8: 997-1007.
24. Jiang ZY, Lu MC, Xu LL, et al. J Med Chem. 2014;57: 2736-2745.
25. Lu MC, Zhang X, Wu F, et al. J Med Chem. 2019;62: 6796-6813.
26. Pallesen JS, Tran KT, Bach A. J Med Chem. 2018;61: 8088-8103.
27. Xu LL, Zhu JF, Xu XL, et al. J Med Chem. 2015;58: 5419-5436.
28. Xu LL, Zhang X, Jiang ZY, You QD. Bioorg Med Chem. 2016;24: 3540-3547.
29. Xu LL, Liu T, Wang L, et al. Oxid Med Cell Longev. 2018;2018: 3271617.
30. Xu LL, Wu YF, Wang L, et al. Eur J Med Chem. 2018;157: 1376-1394.
31. Bostrom J, Hogner A, Llinas A, Wellner E, Plowright AT. J Med Chem. 2012;55: 1817-
1830.
32. Ning X, Guo Y, Wang X, et al. J Med Chem. 2014;57: 4302-4312.
33. Pardridge WM. Expert Opin Investig Drugs. 2019;8: 1-4.
34. Di L, Rong H, Feng B. J Med Chem. 2013;56: 2-12.
35. Di L, Kerns EH, Fan K, McConnell OJ, Carter GT. Eur J Med Chem. 2003;38: 223-232.
Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
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