Design, synthesis, and biological activity evaluation of BACE1 inhibitors with antioxidant activity

Article abstract of DOI:10.1002/ddr.21585

The proteolytic enzyme β-secretase (BACE1) plays a central role in the synthesis of the pathogenic β-amyloid peptides (Aβ) in Alzheimer's disease (AD), antioxidants could attenuate the AD syndrome and prevent the disease progression. In this study, BACE1 inhibitors (D1–D18) with free radical-scavenging activities were synthesized by molecular hybridization of 2-aminopyridine with natural antioxidants. The biological activity evaluation showed that D1 had obvious inhibitory activity against BACE1, and strong antioxidant activity in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS^+?) assay, which could be used as a lead compound for further study.

Full text of DOI:10.1002/ddr.21585

Received: 12 April 2019
DOI: 10.1002/ddr.21585
Revised: 6 July 2019
Accepted: 24 July 2019
R E S E A R C H A R T I C L E
Design, synthesis, and biological activity evaluation of BACE1
inhibitors with antioxidant activity
He-Min Li | Shao-Peng Yu | Tian-Yuan Fan | Yue Zhong | Ting Gu |
Wen-Yu Wu | Chao Zhao | Zhi Chen | Min Chen | Nian-Guang Li
Xiao-Long Wang
|
Department of Medicinal Chemistry, Nanjing University of Chinese Medicine, Nanjing, China
Correspondence
Abstract
Nian-Guang Li and Xiao-Long Wang,
Department of Medicinal Chemistry, Nanjing
The proteolytic enzyme β-secretase (BACE1) plays a central role in the synthesis of the
pathogenic β-amyloid peptides (Aβ) in Alzheimer's disease (AD), antioxidants could
attenuate the AD syndrome and prevent the disease progression. In this study, BACE1
inhibitors (D1–D18) with free radical-scavenging activities were synthesized by molec-
ular hybridization of 2-aminopyridine with natural antioxidants. The biological activity
evaluation showed that D1 had obvious inhibitory activity against BACE1, and strong
antioxidant activity in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,20-azinobis-
(3-ethylbenzthiazoline-6-sulphonate) (ABTS+•) assay, which could be used as a lead
compound for further study.
University of Chinese Medicine, Nanjing,
Jiangsu 210023, China.
Email: linianguang@njutcm.edu.cn (N.-G. L.)
and
Email: gregwang@njucm.edu.cn (X.-L. W.)
Funding information
Key Laboratory of Therapeutic Material of
Chinese Medicine, Jiangsu Province; Natural
Science Foundation of Jiangsu Province,
Grant/Award Number: BK20151563; Program
for Excellent Talents in School of Pharmacy of
Nanjing University of Chinese Medicine,
Grant/Award Number: 15ZYXET-1; Project
Funded by the Flagship Major Development of
Jiangsu Higher Education Institutions, Grant/
Award Number: PPZY2015A070; Project
Funded by the Priority Academic Program
Development of Jiangsu Higher Education
Institutions; State Key Laboratory Cultivation
Base for TCM Quality and Efficacy, Nanjing
University of Chinese Medicine
K E Y W O R D S
Alzheimer's disease, antioxidant activity, BACE1, molecular docking, molecular hybridization
fragment (Vassar, Kovacs, Yan, & Wong, 2009). Subsequently, intra-
1
| INTRODUCTION
membrane processing of the former fragment C99 by γ-secretase
affords Aβ (Sisodia & St George-Hyslop, 2002). Most Aβ peptides end
at residue 40 but about 5–10% terminates at residue 42, which is a
major actor in the pathogenesis of AD. The studies with knockout mice
in the absence of BACE1 showed that both wild type and mutant forms
of APP failed to produce Aβ, they were in good health and did not show
any side effects of BACE1 deficiency, thus evinced the key role of
BACE1 in APP processing (Luo et al., 2001) and further confirmed that
BACE1 was very important as a target to therapeutically intervent AD.
The research on BACE1 inhibitors was initiated from the substrate-
based design of transition-state compounds, the cleavable amide bond
of APP was replaced by a stable mimetic of the putative transition-state
structure (Evin, Lessene, & Wilkins, 2011). Thus some peptidomimetic
For older people, Alzheimer's disease (AD) is the main reason to cause
dementia, it consists a progressive and chronic neurodegenerative disor-
der (Selkoe, 2001). The pathological features of AD include two kinds of
lesions in the patient brain, the first kind is the extracellular accumulation
of amyloid plaques which is composed of the β-amyloid (Aβ) peptide, and
the second kind is the intracellular neurofibrillary tangles which is pro-
duced from the aggregation of hyperphosphorylated microtubule-
associated tau protein (V. M. Lee, Balin, Otvos Jr, & Trojanowski, 1991).
The β-secretase, also called as beta-site amyloid precursor protein
(APP) cleaving enzyme 1 (BACE1), is an enzyme that initiates Aβ forma-
tion by hydrolysis of the APP extracellular domain, thus generates the
membrane bound C-terminal fragment C99 and the N-terminal Aβ
Drug Dev Res. 2019;1–9.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

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LI ET AL.
FIGURE 1 The chemical structures of
LY2811376, LY2886721, and MK-8931
FIGURE 2 The chemical structures of some compounds with 2-aminopyridine scaffold, and the design of BACE1 inhibitors (D1–D18) bearing
2-aminopyridine and natural antioxidants
inhibitors have been designed, such as hydroxyethylen and statine-
based agents, these compounds exhibited potent in vitro subnanomolar
activities. However, some peptidic inhibitors suffered from poor phar-
macological properties, because they had unfavorable physicochemical
characteristics and poor brain penetration (Ghosh, Brindisi, & Tang,
2012; Hom et al., 2003; Hong et al., 2000). So the study to develop
potent non-peptidic small-molecule BACE1 inhibitors has become the
focus of many academic and pharmaceutical researchers (Albert, 2009),
and some candidates have been put into clinical studies.
neutralize the oxidative substance. In the early AD stage, the oxidative
stress could initiate the Aβ aggregation and tau protein hyper-
phosphorylation (Gomes et al., 2018). Many evidences have proved that
the antioxidants could attenuate the AD syndrome, and prevent the disease
progression (Gomes et al., 2018). Thus, drugs with specifically antioxidant
activity might be useful for either the prevention or the treatment of AD.
So in this manuscript, we designed and synthesized a series of
BACE1 inhibitors with antioxidant activities. Recently, BACE1 inhibi-
tors bearing 2-aminopyridine scaffold have received great attention
because they had a superior brain penetration. Wyeth (Malamas et al.,
2010) identified compound W44 (Figure 2) as a potent BACE1 inhibi-
tor (IC50 = 0.1 μM) demonstrating high brain permeability with a brain
to plasma ratio of 1.1. Schering-Plough (Y. S. Wang et al., 2010) opti-
mized the 2-aminopyridine and led series to more potent small-
molecule inhibitors of BACE-1, SP5 (Figure 2) inhibited BACE-1 with
an IC50 value of 250 μM in the BACE-1 homogeneous time-resolved
fluorescence (HTRF) assay. Konno, Sato, Saito, Sakamoto, and Akaji
(2015) prepared a variety of aminopyridine derivatives, and found that
6-alkyl-2-aminopyridine K30 (Figure 2) exhibited the greatest inhibi-
tory activity when it was screened for rBACE1 inhibition at a 2.0 mM
concentration (56% inhibition). The X-ray structure crystal studies
showed that the 2-aminopyridine interacted with the catalytic acids
The first generation of orally active BACE1 inhibitor BI1181181 (its
chemical structure has not been publicly disclosed), failed in Phase I trials
because of low oral bioavailability and low blood–brain barrier penetration.
Then the second-generation BACE1 inhibitors LY2811376 (Phase I)
(Figure 1), LY2886721 (Phase II) (Figure 1), and RG7129 (Phase I; its chem-
ical structure has not been publicly disclosed) also failed in clinical trials
because they had liver toxicity. Although the third-generation BACE1
inhibitor MK-8931 (Figure 1) reduces CNS Aβ in animal models and in AD
patients, however, Merck has stopped the clinical trial in mild-to-moderate
AD patients because it showed minor efficacy (Hung & Fu, 2017).
In recent years, more attention has been paid to oxidative stress
because of its role in AD (H. A. Lee et al., 2018). Several studies indicated
that the antioxidant defense system in some elderly people lost its ability to

LI ET AL.
3
SCHEME 1 Reagents and conditions: (a) o-phthalic anhydride (2) (1.0 equiv.), CH3COOH, reflux, 5 hr, 95.2%; (b) NBS (1.1 equiv.), AIBN (0.1
equiv.), benzene, 85ꢀC, 5 hr, 4 (46.7%), 5 (21.3%); (c) phthalimide potassium salt (1 equiv.), DMF, 160ꢀC for 4 hr, room temperature overnight,
90.3%; (d) HCl, reflux, 20 hr, 93.7%; (e) acid, EDCI (1.2 equiv.), HOBT (1.2 equiv.), DIPEA (4.0 equiv.), room temperature overnight, 78.8–89.6%.
EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; HOBT, 1-hydroxybenzotriazole; DIPEA, N,N-diisopropylethylamine
Asp32 and Asp228 in BACE1 enzyme via a hydrogen-bounding net-
work (Cole et al., 2006). Furthermore, some natural antioxidants
including hydroxycinnamic acid derivatives, ferulic acid (Gay et al.,
conditions using flame-dried glassware within a nitrogen atmosphere in
dry, freshly distilled solvents, unless otherwise stated. Yields referred to
chromatographical seperation, unless otherwise noted. Reactions were
monitored by thin-layer chromatography (TLC) carried out on
0.15–0.20 mm Yantai silica gel plates (RSGF 254; Yantai Chemical Indus-
try Research Institute, Yantai, Shandong, China) using ultraviolet light as
the visualizing agent. Chromatography was performed on Qingdao silica
gel (160–200 mesh) (Qingdao Banke separation materials Company Lim-
ited, Qingdao, Shandong, China) using ethyl acetate in petroleum ether
and methanol in dichloromethane as the eluting solvent. Melting points
(mp) were measured on a WRS-1B apparatus and were uncorrected. 1H
Nuclear Magnetic Resonance (NMR) spectra were obtained using a Bruker
AV-400 (400 MHz; Bruker Corporation, Germany). Chemical shifts were
recorded in parts per million (ppm) downfield from tetramethylsilane. J-
values were given in Hertz (Hz). Abbreviations used were singlet (s), dou-
blet (d), triplet (t), quartet (q), broad (b), and multiplet (m). Electrospray ioni-
zation tandem mass spectrometry (ESI-MS) was recorded on a Waters
Synapt high definition mass spectrometry spectrometer.
ꢀ
2018), sinapic acid (Tanska, Mikołajczak, & Konopka, 2018), and
caffeic acid (J. Liu et al., 2018) have been shown to scavenge radicals
effectively and to repair the oxidizing OH adducts of DNA via elec-
tron transfer reactions. So in this study, BACE1 inhibitors (D1–D18)
(Figure 2) bearing 2-aminopyridine with natural antioxidants were
designed and synthesized by molecular hybridization.
2
| MATERIALS AND METHODS
2.1 | Synthesis
The compounds were prepared according to Scheme 1. Treatment of
aminopyridine 1 with o-phthalic anhydride (2) produced imide 3, which
was brominated by N-bromosuccinimide (NBS) to afford two benzyl
bromides 4 and 5. In order to improve the yield of important interme-
diate 4, we subsequently optimized this reaction condition by chang-
ing the ratio of NBS and azodiisobutyronitrile (AIBN), and we found
that the best result for the synthesis of 4 as the main product was that
the ratio of NBS and AIBN should be 1.1 equiv. and 0.1 equiv., which
afforded 4 and 5 in 46.7 and 21.3%, respectively. Then the reaction of
4 with phthalimide potassium salt afforded 6 which could produce
amine hydrochloride 7 after hydrolysis with hydrochloric acid. At last,
the target compounds D1–D18 were obtained by reaction of 7 and
substituted acids using 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), and
N,N-diisopropylethylamine (DIPEA) as condensation agents.
3.1 | 2-(6-Methylpyridin-2-yl)isoindoline-
1,3-dione (3)
6-Methylpyridin-2-amine (1) (5.00 g, 46.30 mmol) and phthalic anhy-
dride (2) (6.85 g, 46.30 mmol, 1.0 equiv.) were added into glacial acetic
acid (20 ml), then the solution was refluxed for 5 hr under the nitrogen.
After the reaction was complete by TLC analysis, it was poured into
cold water (200 ml), the solid appeared was filtered, washed with water,
and then dried under vacuum to afford 3 as white solid (10.49 g, 95.2%
yield). mp 193–194ꢀC. 1H NMR (400 MHz, CDCl3) δ 2.65 (s, 3H), 7.26
(dd, J1 = 7.8 Hz, J2 = 10.8 Hz, 2H), 7.77–7.83 (m, 3H), 7.99 (dd,
J1 = 3.0 Hz, J2 = 5.5 Hz, 2H). ESI-MS: m/z 239 [M + H]+, 261 [M + Na]+.
3
| EXPERIMENTAL
Reagents and solvents were used without further purification unless oth-
erwise specified. Air- and moisture-sensitive liquids and solutions were
transferred via syringe or stainless steel cannula. Organic solutions were
concentrated below 45ꢀC at approximately 20 mmHg by rotary evapora-
tion. All nonaqueous reactions were carried out under anhydrous
3.2 | 2-(6-(Bromomethyl)pyridin-2-yl)isoindoline-
1,3-dione (4)
3 (5.00 g, 21.01 mmol) was added into a solution of AIBN (344.56 mg,
2.101 mmol, 0.1 equiv.) in N,N-dimethylformamide (DMF) (90 ml),

4
LI ET AL.
NBS (4.114 g, 23.11 mmol, 1.1 equiv.) was then added into this solu-
tion in three parts every 30 min, after this mixture was refluxed for
5 hr, it was concentrated and then purified by column chromatogra-
phy using 10% ethyl acetate in petroleum ether as eluent to afford
4 (3.10 g, 46.7%) as white solid. mp 177–179ꢀC. 1H NMR (400 MHz,
CDCl3) δ 4.62 (s, 2H), 7.38 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 7.3 Hz, 1H),
7.84 (dd, J1 = 3.1 Hz, J2 = 5.5 Hz, 2H), 7.93 (t, 1H), 8.00 (dd,
1H), 7.32–7.38 (m, 2H), 8.36 (t, 1H). ESI-MS: m/z 300 [M + H]+,
322 [M + Na]+.
3.5.2 | N-((6-aminopyridin-2-yl)methyl)
cinnamamide (D2)
Yield 78.8%, white solid. mp 140–142ꢀC. 1H NMR (400 MHz, DMSO-
d6) δ 4.25 (d, J = 8.0 Hz, 2H), 5.91 (s, 2H), 6.32 (d, J = 8.0 Hz, 1H), 6.41
(d, J = 8.0 Hz, 1H), 6.75 (d, J = 16.0 Hz, 1H), 7.31–7.35 (m, 1H),
7.38–7.49 (m, 4H), 7.57–7.59 (m, 2H), 8.54 (t, 1H). ESI-MS: m/z
254 [M + H]+, 276 [M + Na]+.
J1
239 [M + Na]+.
= 3.1 Hz, J2 =
5.5 Hz, 2H). ESI-MS: m/z 317 [M + H]+,
3.3 | 2-(2-(1,3-dioxoisoindolin-2-yl)methyl)
isoindoline-1,3-dione (6)
3.5.3 | (E)-N-((6-aminopyridin-2-yl)methyl)-
3-(3,4-dihydroxyphenyl)acrylamide (D3)
Phthalimide potassium salt (2.93 g, 15.82 mmol, 1.0 equiv.) was added
into a solution of 4 (5.00 g, 15.82 mmol) in DMF (50 ml), after this
mixture was refluxed for 4 hr, it was poured into cold water (500 ml),
the solid appeared was filtered, dried, and then purified by column
chromatography using 2% methanol in dichloromethane as eluent to
afford 6 (5.47 g, 90.3%) as yellow solid. mp 114–116ꢀC. 1H NMR
(400 MHz, CDCl3) δ 4.85 (s, 2H), 6.38 (d, J = 8.0 Hz, 1H), 6.59
(d, J = 8.0 Hz, 1H), 7.35–7.39 (m, 1H), 7.71–7.82 (m, 4H), 7.88–7.97
(m, 4H). ESI-MS: m/z 384 [M + H]+, 406 [M + Na]+.
Yield 83.5%, white solid. mp 195–197ꢀC. 1H NMR (400 MHz, DMSO-
d6) δ 4.23 (s, 2H), 5.89 (s, 2H), 6.31 (d, J = 8.0 Hz, 1H), 6.39–6.44 (m,
2H), 6.72 (d, J = 8.0 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.95 (s, 1H),
7.23–7.35 (m, 2H), 8.41 (s, 1H). ESI-MS: m/z 286 [M + H]+,
308 [M + Na]+.
3.5.4 | (E)-N-((6-aminopyridin-2-yl)methyl)-
3-(3-hydroxy-4-methoxyphenyl)acrylamide (D4)
3.4 | 6-(Aminomethyl)pyridin-2-amine (7)
Yield 85.8%, white solid. mp 214–216ꢀC. 1H NMR (400 MHz,
DMSO-d6) δ 3.80 (s, 3H), 4.23 (s, 2H), 5.91 (s, 2H), 6.32 (d,
J = 8.0 Hz, 1H), 6.41 (d, J = 16.0 Hz, 1H), 6.51 (d, J = 16.0 Hz, 1H),
6.93–7.00 (m, 3H), 7.30–7.36 (m, 2H), 8.43 (t, 1H), 9.19 (s, 1H). ESI-
MS: m/z 300 [M + H]+, 322 [M + Na]+.
6
(5.00 g, 13.05 mmol) was added into 40 ml concentrated
hydrochloric acid, after the mixture was refluxed for 20 hr, it was fil-
tered and washed with water, then the crude product was rec-
rystallized from ethanol to afford 7 (1.50 g, 93.7%) as yellow solid. mp
201–203ꢀC. 1H NMR (400 MHz, D2O) δ 4.25 (s, 2H), 6.89 (d, J = 8.0 Hz,
1H), 6.98 (d, J = 9.0 Hz, 1H), 7.85 (t, 1H). ESI-MS: m/z 124 [M + H]+,
146 [M + Na]+.
3.5.5 | (E)-N-((6-aminopyridin-2-yl)methyl)-
3-(3,5-dihydroxy-4-methoxyphenyl)acrylamide (D5)
Yield 82.1%, white solid. mp 97–99ꢀC. 1H NMR (400 MHz, DMSO-d6)
δ 3.80 (s, 6H), 4.26 (s, 2H), 6.06 (s, 2H), 6.35 (d, J = 8.0 Hz, 1H), 6.42
(d, J = 8.0 Hz, 1H), 6.63 (d, J = 16.0 Hz, 1H), 6.88 (s, 2H), 7.35–7.39
(m, 2H), 8.41 (t, 1H), 8.84 (s, 1H). ESI-MS: m/z 316 [M + H]+,
338 [M + Na]+.
3.5 | Synthesis of D1–D18
The natural acid (0.52 mmol) was added into a solution of HOBT
(84 mg, 0.62 mmol, 1.2 equiv.), EDCI (119 mg, 0.62 mmol, 1.2 equiv.),
DIPEA (343 μL, 2.08 mmol, 4.0 equiv.) in 10 ml DMF at 0ꢀC, 15 min
later, 7 (76 mg, 0.62 mmol, 1.2 equiv.) was added into this solution.
After the mixture was reacted at 25ꢀC for 12 hr, it was poured into
100 ml cold water, and then extracted with ethyl acetate (30 ml × 4).
The ethyl acetate layer was washed with saturated salt water, dried
over anhydrous sodium sulfate, concentrated and purified by column
chromatography using 10% ethyl acetate in petroleum ether as eluent
to afford D1–D18 (yields 78.8–89.6%).
3.5.6 | (E)-N-((6-aminopyridin-2-yl)methyl)-
3-(4-hydroxyphenyl)acrylamide (D6)
Yield 89.6%, white solid. mp 70–72ꢀC. 1H NMR (400 MHz, DMSO-d6)
δ 4.24 (s, 2H), 5.90 (s, 2H), 6.31 (d, J = 8.0 Hz, 1H), 6.40 (d, J = 8.0 Hz,
1H), 6.53 (d, J = 16.0 Hz, 1H), 6.80 (d, J = 8.0 Hz, 2H), 7.33 (d,
J = 8.0 Hz, 2H), 7.40 (d, J = 12.2 Hz, 2H), 8.42 (t, 1H), 9.97 (s, 1H).
ESI-MS: m/z 270 [M + H]+, 292 [M + Na]+.
3.5.1 | (E)-N-((6-Aminopyridin-2-yl)methyl)-
3-(4-hydroxy-3-methoxyphenyl)acrylamide (D1)
3.5.7 | (E)-N-((6-aminopyridin-2-yl)methyl)-
3-(2-hydroxyphenyl)acrylamide (D7)
Yield 81.2%, white solid. mp 97–99ꢀC. 1H NMR (400 MHz, dimethyl
sulfoxide (DMSO)-d6) δ 3.81 (s, 3H), 4.23 (d, J = 8.0 Hz, 2H), 5.89 (s,
2H), 6.31 (d, J = 8.0 Hz, 1H), 6.40 (d, J = 8.0 Hz, 1H), 6.57 (d,
J = 16.6 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 7.01–7.03 (m, 1H), 7.14 (s,
Yield 80.3%, white solid. mp 85–87ꢀC. 1H NMR (400 MHz, DMSO-d6)
δ 4.24 (d, J = 8.0 Hz, 2H), 5.91 (s, 2H), 6.31 (d, J = 8.0 Hz, 1H), 6.40 (d,

LI ET AL.
5
J = 8.0 Hz, 1H), 6.74–6.91 (m, 3H), 7.19 (t, 1H), 7.33 (t, 1H), 7.44 (d,
J = 8.0 Hz, 1H), 7.67 (d, J = 16.0 Hz, 1H), 8.50 (t, 1H), 10.07 (s, 1H).
ESI-MS: m/z 270 [M + H]+, 292 [M + Na]+.
1H), 6.38 (d, J = 8.0 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 7.30 (t, 1H), 7.38
(d, J = 8.0 Hz, 1H), 7.43 (s, 1H), 8.62 (t, 1H) ESI-MS: m/z 274 [M
+ H]+, 296 [M + Na]+.
3.5.8 | N-((6-aminopyridin-2-yl)methyl)-
3.5.14 | N-((6-aminopyridin-2-yl)methyl)-
4-hydroxybenzamide (D8)
4-fluorobenzamide (D14)
Yield 88.1%, white solid. mp 200–202ꢀC. 1H NMR (400 MHz, DMSO-
d6) δ 4.30 (d, J = 8.0 Hz, 2H), 5.89 (s, 2H), 6.29 (d, J = 8.0 Hz, 1H), 6.38
(d, J = 8.0 Hz, 1H), 6.81 (d, J = 8.0 Hz, 2H), 7.29–7.33 (m, 1H), 7.78 (d,
J = 8.0 Hz, 2H), 8.67 (t, 1H), 9.99 (s, 1H). ESI-MS: m/z 244 [M + H]+,
266 [M + Na]+.
Yield 82.8%, white solid. mp 158–160ꢀC. 1H NMR (400 MHz, DMSO-
d6) δ 4.33 (d, J = 8.0 Hz, 2H), 5.93 (s, 2H), 6.31 (d, J = 8.0 Hz, 1H), 6.40
(d, J = 8.0 Hz, 1H), 7.32 (t, 3H), 7.99 (t, 2H), 9.00 (t, 1H). ESI-MS: m/z
246 [M + H]+, 268 [M + Na]+.
3.5.15 | N-((6-aminopyridin-2-yl)methyl)-
3.5.9 | N-((6-aminopyridin-2-yl)methyl)
3-bromobenzamide (D15)
benzamide (D9)
Yield 84.4%, white solid. mp 162–164ꢀC. 1H NMR (400 MHz, DMSO-
d6) δ 4.31 (d, J = 8.0 Hz, 2H), 5.91 (s, 2H), 6.31 (d, J = 8.0 Hz, 1H), 6.41
(d, J = 8.0 Hz, 1H), 7.30–7.34 (m, 1H), 7.45–7.48 (m, 1H), 7.76 (d,
J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 8.10 (s, 1H), 9.10 (t, 1H). ESI-
MS: m/z 306 [M + H]+, 328 [M + Na]+.
Yield 82.7%, white solid. mp 142–144ꢀC. 1H NMR (400 MHz, DMSO-
d6) δ 4.34 (d, J = 8.0 Hz, 2H), 5.90 (s, 2H), 6.30 (d, J = 8.0 Hz, 1H), 6.41
(d, J = 8.0 Hz, 1H), 7.30–7.34 (m, 1H), 7.47–7.55 (m, 3H), 7.92 (d,
J
=
8.0 Hz, 2H), 8.95 (t, 1H). ESI-MS: m/z 228 [M + H]+,
260 [M + Na]+.
3.5.16 | N-((6-aminopyridin-2-yl)methyl)-
3.5.10 | N-((6-aminopyridin-2-yl)methyl)-
2-chlorobenzamide (D16)
4-methoxybenzamide (D10)
Yield 87.3%, white solid. mp 161–163ꢀC. 1H NMR (400 MHz, DMSO-
d6) δ 4.30 (d, J = 8.0 Hz, 2H), 5.92 (s, 2H), 6.32 (d, J = 8.0 Hz, 1H), 6.54
(d, J = 8.0 Hz, 1H), 7.35–7.50 (m, 4H), 7.90 (t, 1H), 8.89 (t, 1H). ESI-
MS: m/z 262 [M + H]+, 284 [M + Na]+.
Yield 80.9%, white solid. mp 178–180ꢀC. 1H NMR (400 MHz, DMSO-
d6) δ 3.82 (s, 3H), 4.32 (d, J = 8.0 Hz, 2H), 5.87 (s, 2H), 6.30 (d,
J = 8.0 Hz, 1H), 6.39 (d, J = 8.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 2H), 7.31
(t, 1H), 7.91 (d, J = 8.0 Hz, 2H), 8.79 (t, 1H). ESI-MS: m/z 258 [M
+ H]+, 280 [M + Na]+.
3.5.17 | N-((6-aminopyridin-2-yl)methyl)-2-hydroxy-
2-phenylacetamide (D17)
3.5.11 | N-((6-aminopyridin-2-yl)methyl)-
2-hydroxybenzamide (D11)
Yield 84.2%, white solid. mp 78–80ꢀC. 1H NMR (400 MHz, DMSO-d6)
δ 4.14 (t, 2H), 4.99 (s, 1H), 5.88 (s, 2H), 6.29 (d, J = 8.0 Hz, 2H),
7.25–7.44 (m, 4H), 7.45 (d, J = 8.0 Hz, 2H), 8.37 (t, 1H). ESI-MS: m/z
258 [M + H]+, 280 [M + Na]+.
Yield 79.5%, white solid. mp 169–171ꢀC. 1H NMR (400 MHz, DMSO-
d6) δ 4.37 (d, J = 8.0 Hz, 2H), 5.91 (s, 2H), 6.32 (d, J = 8.0 Hz, 1H), 6.43
(d, J = 8.0 Hz, 1H), 6.89–6.93 (m, 2H), 7.33 (t, 1H), 7.42 (t, 1H), 7.93
(d, J = 8.0 Hz, 1H), 9.27 (t, 1H). ESI-MS: m/z 244 [M + H]+,
266 [M + Na]+.
3.5.18 | N-((6-aminopyridin-2-yl)methyl)-
2-(2-hydroxyphenyl)acetamide (D18)
Yield 80.6%, white solid. mp 168–170ꢀC. 1H NMR (400 MHz, DMSO-
d6) δ 3.48 (s, 2H), 4.12 (d, J = 8.0 Hz, 2H), 5.88 (s, 2H), 6.29 (d, J = 8.0 Hz,
1H), 6.39 (d, J = 8.0 Hz, 1H), 6.75 (t, 1H), 6.81 (d, J = 8.0 Hz, 1H),
7.11–7.28 (m, 2H), 7.31 (t, 1H), 8.37 (t, 1H), 9.72 (s, 1H). ESI-MS: m/z
258 [M + H]+, 280 [M + Na]+.
3.5.12 | N-((6-aminopyridin-2-yl)methyl)-
3,4-dihydroxybenzamide (D12)
Yield 83.9%, white solid. mp 116–118ꢀC. 1H NMR (400 MHz, DMSO-
d6) δ 4.27 (d, J = 8.0 Hz, 2H), 5.88 (s, 2H), 6.29 (d, J = 8.0 Hz, 1H), 6.36
(d, J = 8.0 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 7.25–7.33 (m, 3H), 8.60 (t,
1H). ESI-MS: m/z 260 [M + H]+, 282 [M + Na]+.
4
| BIOLOGICAL SCREENING
3.5.13 | N-((6-aminopyridin-2-yl)methyl)-4-hydroxy-
4.1 | BACE1 inhibitory activity assay
3-methoxybenzamide (D13)
The enzyme inhibition assay was evaluated according to the manufac-
turer instructions (Al-Tel et al., 2011) using BACE1 fluorescence reso-
nance energy transfer assay kit, Red (P2985, Thermo fisher).
Yield 85.3%, white solid. mp 88–90ꢀC. 1H NMR (400 MHz, DMSO-d6)
δ 3.79 (s, 3H), 4.30 (d, J = 8.0 Hz, 2H), 5.87 (s, 2H), 6.29 (d, J = 8.0 Hz,

6
LI ET AL.
Epigallocatechin gallate (EGCG) was used as a positive control. The
BACE1 enzyme (purified baculovirus-expressed enzyme) was diluted
with the assay buffer (50 mM sodium acetate, pH 4.5) to make a 3×
working solution (1 Unit/ml). The peptide substrate (Rh-EVNLDAEFK-
Quencher) was also diluted with the same assay buffer to provide the
3× stock solution (750 nM). The inhibitor stock solutions in DMSO
were diluted with assay buffer to provide 3× solution of test com-
pounds at different concentrations. The 3× solution of BACE1
enzyme (10 μl) and each inhibitor sample (10 μl) were placed in the
96-well plate and gently mixed. The substrate 3× solution (10 μl) was
then added to this mixture in each well to start the reaction, and the
final reaction volume was 30 μl (the concentration of DMSO in each
sample and control wells was less than 4%). The reaction mixtures
were incubated at 25ꢀC for 90 min in the dark and then the reaction
was stopped with 10 μL of 2.5 mM sodium acetate. Fluorescence was
monitored at 545 nm (excitation wavelength) and 585 nm (emission
wavelength). The percentage of enzyme inhibition for each concentra-
tion of test compound was calculated compared to maximum enzyme
activity wells (containing substrate plus enzyme) and baseline wells
(containing substrate). IC50 values (concentration of the compound
that induces 50% inhibition of enzymatic activity) were calculated
with the CurveExpert software version 1.34 for Windows.
A0] × 100%, where A0 was the absorbance of the control (without
sample), A1 was the absorbance in the presence of the sample, and A2
was the absorbance without ABTS+•
.
4.4 | Docking studies
Compound D1 was docked into the crystal structures of BACE
1 (PDB ID: 3MSL) by using Discovery Studio 2.5. Standard precession
with standard deviation (SD) was used to dock D1. Optimization was
performed on the following conditions: CHARMm for force field set-
ting, an active site radius was 10.0 Å. Physicochemical properties and
their scoring functions (a combination of interaction energy, hydrogen
bonding, and electrostatic interactions) were used to select the
final pose.
5
| RESULTS AND DISCUSSION
5.1 | BACE1 inhibiting activity
Epigallocatechin gallate has been proved to be a BACE1 inhibitor with
its IC50 value was 1.6 μmol L−1 (Jeon, Bae, Seong, & Song, 2003). So
in this research, EGEG was used as positive control to evaluate the
BACE1 inhibiting activity, and the results were shown in Table 1.
From the results, we could see that D1–D7 had potent inhibitory
activity against BACE1, compared to EGCG with its IC50 was
1.18 μM. For compounds D2, D3, D6, and D7 with benzene ring or
hydroxyl substituted benzene ring that were in the R substituent, the
4.2 | 1,1-Diphenyl-2-picrylhydrazyl radical-
scavenging activity assay
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity
assay was measured according to the method (H. Wang, Gao,
Zhou, Cai, & Yao, 2008) with a few modifications. The sample
(4–250 μmol/L, 100 μl) which was dissolved in ethanol was added to
ethanol solution (100 μl) containing DPPH radicals (80 μmol/L) in
96-well plates. The mixture was shaken and left at room temperature
for 30 min in the dark, and the absorption was measured at 517 nm,
the Trolox was used as the positive control. A lower absorbance rep-
resents a higher DPPH scavenging activity. The percentage scaveng-
ing effect was calculated as scavenging rate (%) = [1 − (A1 − A2)/
A0] × 100%, where A0 was the absorbance of the control (without
sample), A1 was the absorbance in the presence of the sample, and A2
was the absorbance without DPPH.
inhibition rates were less than 50% in the concentration of 2 μmol L−1
.
Interestingly, for compounds D1, D4, and D5 in which the methoxy
group was introduced, the inhibitory activities against BACE1 were
improved with their inhibition rates were all above 50%, and the IC50
s
for these three compounds were 1.80, 2.07, and 1.96 μM, respec-
tively, this information indicated that introduction of methoxy group
was very important for the inhibitory activities against BACE1. When
the vinyl group was removed, the obtained compounds D8–D13 and
D18 lost their inhibitory activities dramatically with their inhibition
rates were all below 10%, this information confirmed that cinnamyl
group in the side chain was very important to inhibit BACE1. Unfortu-
nately, compounds D14–D17 with no phenolic hydroxyl group was
substituted in the benzene ring were inactive in this research, these
results indicated that the phenolic hydroxyl group in the side chain
contributed to the BACE1 inhibitory activities.
4.3 | 2,20-Azinobis-(3-ethylbenzthiazoline-
6-sulphonate) radical-scavenging activity assay
The 2,20-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS+•
)
5.2 | Antioxidant activities
radical-scavenging activity assay was measured according to a litera-
ture procedure (Z. Q. Liu, 2010). K2S2O8 (2 mmol/L) and ABTS
(7 mmol/L) were dissolved and mixed in deionized water, reacting at
room temperature for 12–16 hr. The mixture (ABTS+• stock solution)
was then diluted by phosphate buffer solution to give an absorbance
at 734 nm near 0.7, defined as the reference absorbance (A0). A0
decreased to a stable value (A1) when ABTS+• (100 μl) was mixed with
the sample (1–62.5 μmol/L, 100 μl) for 6 min. The percentage scav-
enging effect was calculated as scavenging rate (%) = [1 − (A1 − A2)/
The in vitro antioxidant activities of these compounds were assessed
by DPPH and ABTS+• assays. DPPH and ABTS+• (both stable radicals)
were widely used to evaluate the antioxidant capacity of complex
mixtures and individual compounds (Z. Q. Liu, 2010). The in vitro anti-
oxidant activity of the target compounds in comparison with Trolox
was evaluated by DPPH and ABTS+• assays. The IC50 value was
defined as the concentration of sample that causes 50% loss of the
radical. The data obtained were summarized in Table 1. The IC50s for

LI ET AL.
7
TABLE 1 The inhibitory activity against BACE1 and antioxidant activities of the target compounds D1–D18
H
R
N
N
NH2
D1-D18
O
Inhibition rate (%)
Activity
DPPH assay
ABTS assay
Compd.
EGCG
D1
R
(at 2 μm)
(IC50, μM)
1.18 0.41
1.80 0.22
Compd.
Trolox
D1
IC50 (μM)
IC50 (μM)
56.41 0.47
53.22 1.19
21.53
67.85
26.83
27.14
H3CO
HO
D2
D3
43.52 3.59
47.53 3.34
ND
ND
D2
D3
ND
ND
18.67
24.83
HO
HO
D4
D5
HO
H3CO
51.61 2.50
52.61 1.71
2.07 0.51
1.96 0.60
D4
D5
ND
27.79
23.76
HO
53.64
H3CO
HO
D6
D7
47.29 0.50
40.18 3.22
ND
ND
D6
D7
ND
ND
45.82
40.11
HO
OH
D8
9.03 2.24
6.54 1.76
4.07 2.34
ND
ND
ND
D8
ND
ND
ND
83.45.
ND
HO
D9
D9
D10
D10
ND
H3CO
OH
D11
D12
D13
3.12 3.04
4.79 0.51
4.98 3.06
ND
ND
ND
D11
D12
D13
ND
72.91
35.58
40.63
35.21
ND
HO
HO
H3CO
HO
D14
D15
0
0
ND
ND
D14
D15
ND
ND
ND
ND
F
Br
D16
D17
0
0
ND
ND
D16
D17
ND
ND
ND
ND
Cl
OH
(Continues)

8
LI ET AL.
TABLE 1 (Continued)
H
N
R
N
NH2
D1-D18
O
Inhibition rate (%)
Activity
DPPH assay
ABTS assay
Compd.
D18
R
(at 2 μm)
(IC50, μM)
Compd.
D18
IC50 (μM)
IC50 (μM)
1.80 2.29
ND
ND
27.56
OH
Abbreviations: ABTS, 2,20-azinobis-(3-ethylbenzthiazoline-6-sulphonate); DPPH, 1,1-diphenyl-2-picrylhydrazyl; EGCG, epigallocatechin gallate; ND, not
determined.
FIGURE 3 Molecular docking studies of compound D1 with BACE 1 protein
Trolox in DPPH and ABTS+• assays were 21.53 and 26.83 μM. Only
one phenolic hydroxyl group substituted compounds showed good
antioxidant activity in ABTS+• assay, for example, the IC50s for D1,
D4, D6, D7, D8, D11, D13, and D18 were 27.14, 27.79, 45.82, 40.11,
83.45, 72.91, 40.63, and 27.56 μM, respectively. When the two phe-
nolic hydroxyl groups were introduced into the benzene ring, the anti-
oxidant activities were enhanced. For example, the IC50s for D3 in
DPPH and ABTS+• assays were 18.67 and 24.83 μM, the IC50s for D5
the aminopyridine moiety made one hydrogen bond with Asp32. Fur-
thermore, the amide bond in the side chain also formed two hydrogen
bonds with the active sites, the carbonyl group formed one hydrogen
bond with Gln73, and the amino group formed one hydrogen with
Thr231. These actions might verify that D1 had potent inhibitory
activity against BACE1.
6
| CONCLUSIONS
in DPPH and ABTS+• assays were 53.64 and 23.76 μM, and the IC50
s
for D12 in DPPH and ABTS+• assays were 35.21 and 35.58 μM. This
information confirmed that the phenolic hydroxyl group was very
important for antioxidant activities.
In summary, we have synthesized new class of BACE1 inhibitors with
antioxidant activities by molecular hybridization of the 2-aminopyridine
with some antioxidant natural products. The aminopyridine could form
hydrogen bonds with Asp32 and Asp228, and the amide bond in the
side chain also formed two hydrogen bonds with Gln73 and Thr231.
Furthermore, the phenolic hydroxyl group in the side chain contributed
to the antioxidant activities. These information were very important to
design and synthesize BACE1 inhibitors with good antioxidant activities
for the treatment of AD.
5.3 | Molecular docking studies
Considering that compound D1 exhibited the most potent BACE1
inhibitory activity with its IC50 was 1.80 μM, and good antioxidant
activities with its IC50s in DPPH and ABTS+• assays were 67.85 and
27.14 μM, so D1 was selected for molecular docking studies. As
shown in Figure 3, compound D1 could extend deeply into the cavity
of BACE1, and combine excellently with hydrophilic surface and
hydrophobic surface (Figure 3), thus contributing to the ligand affinity.
As expected, the amino group in the aminopyridine moiety made two
hydrogen bonds with Asp32 and Asp228, and the nitrogen atom in
ACKNOWLEDGMENTS
This work was financially supported by Natural Science Foundation of
Jiangsu Province (BK20151563), Program for Excellent Talents in

LI ET AL.
9
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evaluation of aminopyridine derivatives as potential BACE1 inhibitors.
Bioorganic & Medicinal Chemistry Letters, 25, 5127–5132.
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School of Pharmacy of Nanjing University of Chinese Medicine
(15ZYXET-1), Project Funded by the Priority Academic Program Devel-
opment of Jiangsu Higher Education Institutions, Project Funded by the
Flagship Major Development of Jiangsu Higher Education Institutions
(PPZY2015A070), Key Laboratory of Therapeutic Material of Chinese
Medicine, Jiangsu Province, State Key Laboratory Cultivation Base for
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ORCID
Nian-Guang Li
https://orcid.org/0000-0001-9107-6956
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Design, synthesis, and biological activity evaluation of BACE1
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