Synthesis and Anti-neuroinflammatory Activity of Lactone Benzoyl Hydrazine and 2-nitro-1-phenyl-1H-Indole Derivatives as p38α MAPK Inhibitors

Article abstract of DOI:10.1111/cbdd.12581

Inhibition of p38 mitogen-activated protein kinases (MAPKs) would allow significant modulation of the neuroinflammation condition associated with Alzheimer's disease (AD). Inspired from the pharmacophore of natural NF-κB and p38α MAPK inhibitor 5,6-dehydrokawain and p38α MAPK inhibitors 1a, 1-pyrazolyl-3-(4-((2-anilinopyrimidin-4-yl)oxy)napththalen-1-yl)ureas, and 1b, a class of indole-pyrimidinyl compounds which were patented respectively, we designed, de novo synthesized, and evaluated two kinds of novel series of lactone benzoyl hydrazine derivatives and 2-nitro-1-phenyl-1H-indole derivatives in an effort to develop pharmacologically tractable agents to alleviate the progression of AD. Fourteen of the seventeen synthesized compounds exhibit significant inhibitory effect on the nitric oxide (NO) production induced by lipopolysaccharide (LPS)-induced microglia activation with IC₅₀ less than the control 5,6-dehydrokawain. Notably, compound 27, 6-methoxy-2-nitro-1-(1H-1, 2, 3-triazol-1-yl)-1H-indole, with IC₅₀ values of 1.6 μm can markedly inhibit p38α MAPK and NO release in BV-2 microglial cells. The molecular dynamic (MD) simulations demonstrate that compound 27 inhibits p38α MAPK through binding to the Glu71 and Asp168 residues. Moreover, in vitro study shows that all compounds can easily cross the blood-brain barrier (BBB) and did not exhibit any acute cellular toxicity checked by MTT assay. These investigations provide promising chemical lead candidate as anti-neuroinflammatory agents for AD. We designed, synthesized and evaluated two series lactone benzoyl hydrazine and 2-nitro-1-phenyl-1H-Indole derivatives for novel promising chemical lead p38α MAPK inhibitors as anti-neuroinflammatory agents in fighting against Alzheimer's diseases.

Full text of DOI:10.1111/cbdd.12581

Received Date : 04-Feb-2015
Revised Date : 08-Apr-2015
Accepted Date : 27-Apr-2015
Article type : Research Article
Synthesis and Anti-neuroinflammatory Activity of Lactone Benzoyl Hydrazine and 2-
nitro-1-phenyl-1H-Indole Derivatives as p38α MAPK Inhibitors
1
2
1
2
2
2*
1*
Bao Cheng , Yongsheng Lin , Ming Kuang , Sai Fang , Qiong Gu , Jun Xu , Laiyou Wang
1
. Guangdong Metabolic Diseases Research Center of Integrated Chinese and Western
Medicine, Guangdong TCM Key Laboratory against Metabolic Diseases, Guangdong
Pharmaceutical University, Guangzhou Higher Education Mega Centre, Guangzhou
5
10006, PR China
2
. Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-
Sen University, Guangzhou 510006, People’s Republic of China
*
0
Corresponding author: Jun Xu, junxu@biochemomes.com; Laiyou Wang, Phone number:
086-20-39352607, Fax: 0086-20-39352606, E-mail: laiyouwang@hotmail.com
Suggested short running title:
Synthesis and biological activity of novel p38α Inhibitors
Key words: Neuroinflammation, 5,6-dehydrokawain, p38α MAPK, 2-nitro-1-phenyl-1H-
indole derivatives, nitric oxide, molecular dynamics
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12581
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Abstract
Inhibition of p38 mitogen-activated protein kinases (MAPKs) would allow significant
modulation of the neuroinflammation condition associated with Alzheimer’s disease (AD).
Inspired from the pharmacophore of natural NF-κB and p38α MAPK inhibitor 5,6-
dehydrokawain and p38α MAPK inhibitors 1a,1-pyrazolyl-3- (4- ((2 -anilinopyrimidin- 4 - yl)
oxy) napththalen- 1- yl) ureas, and 1b, a class of indole-pyrimidinyl compounds which were
patented respectively, we designed, de novo synthesized and evaluated two kinds of novel
series of lactone benzoyl hydrazine derivatives and 2-nitro-1-phenyl-1H-indole derivatives in
an effort to develop pharmacologically tractable agents to alleviate the progression of AD.
Fourteen out of the seventeen synthesized compounds exhibit significant inhibitory effect on
the nitric oxide (NO) production induced by lipopolysaccharide (LPS)-induced microglia
activation with IC50 less than the control 5,6-dehydrokawain. Notably, compound 27, 6-
methoxy-2-nitro-1-(1H-1, 2, 3-triazol-1-yl)-1H-indole, with IC50 values of 1.6 μM can
markedly inhibit p38α MAPK and NO release in BV-2 microglial cells. The molecular
dynamic (MD) simulations demonstrate that compound 27 inhibits p38α MAPK through
binding to the Glu71 and Asp168 residue. Moreover, in vitro study shows that all compounds
can easily cross the blood−brain barrier (BBB) and did not exhibit any acute cellular toxicity
checked by MTT assay. These investigations provide promising chemical lead candidate as
anti-neuroinflammatory agents for AD.
Key words: Neuroinflammation, nitric oxide (NO), p38α MAPK, 2-nitro-1-phenyl-1H-
indole derivatives, nitric oxide, molecular dynamics
Introduction
Alzheimer's disease (AD) is the most common form of neurodegenerative senile dementia
among the elderly, characterized by memory loss and progressive impairment of cognitive
functions (1). Available evidence suggests that excessive microglial activation and thereof
up-regulation of proinflammatory cytokine production play a previously underappreciated
role in the pathophysiology of AD involved in neuroinflammation (2-3). Meanwhile, the
production and secretion of proinflammatory cytokines and various oxidative stress
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molecules such as nitric oxide (NO) and TNF-α could be modulated by different cellular
signalling pathways especially including nuclear factor kappa B (NF-kB) and mitogen-
activated protein kinases (MAPK) (4-5). MAPKs are intracellular enzymes which allow it
possible for cells to respond to stimuli from the extracellular environment, which constitute a
family of specific serine/threonine kinases including extracellular signal-regulated kinases
(
ERK1-5), c-Jun N-terminal kinases (JNK1-3) and p38 isoforms (p38α, p38β, p38δ, p38γ) (6-
). Among them, p38α MAPK represent one of the most heralded classes of target for the
7
treatment of inflammation in the past decade. However, due to the repeated negative reports
and the discontinuation related to adverse events in the clinical trial for the beleaguered
development of p38α MAPK inhibitors, the non-toxic effective lead compound to treat
neuroinflammation are continually pursued (8).
5
,6-dehydrokawain (Figure 1) was reported to be a novel NF-κB and p38α MAPK inhibitor
which can inhibit the release of NO and TNF-α as well as exhibiting antioxidant activities (9-
0). Recently, it was reported that compound 1a and its derivatives as inhibitor against LPS-
1
induced neutrophil accumulation in an animal model by RespiVert Ltd, indicating
amendatory activity duration and therapeutic index (11). Follow-up studies by Confluence
Life Sciences, Inc showed that 1b indole-pyrimidinyl and its derivatives furtherly enhanced
potency and ameliorative safety features in preclinical and human clinical setting experiments
compared to the reported classical p38α inhibitors (11). After analysing the potential
pharmacophore group of the above chemicals 5,6-dehydrokawain, compounds 1a and 1b, we
designed, synthesized and evaluated the fused chimera compound series lactone benzoyl
hydrazine derivatives and 2-nitro-1-phenyl-1H-indole derivatives. The identification of novel
p38α inhibitors would provide a valuable tool for anti-AD drug discovery and further
understanding of the role of MAPKs in neuroinflammation.
Materials and Methods
Synthesis
A focused library of seventeen novel lactone benzoyl hydrazine derivatives and 2-nitro-1-
phenyl-1H-indole derivatives (5-15 and 22-27) were synthesized (Scheme 1, Scheme 2 and
1
13
Scheme 3) and evaluated. The spectroscopic data ( H NMR, C NMR, MS and HRESIMS)
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were consistent with the proposed structures and were well supported. LC-MS of all
compounds was recorded.
All chemicals used were reagent grade. The solvents were distilled prior to use. Reactions
were monitored by thin-layer chromatography (TLC) and visualized with UV light or iodine
vapour. The usual work-up included washing with brine and drying the organic phase with
sodium sulphate before concentration in vacuum. Final compounds were typically purified by
flash chromatography on silica gel or by preparative thin-layer chromatography. The purity
of compounds2-27 (higher than 95%) was confirmed through HPLC (Shimadzu-SDP-M20A,
TC-C18 column (4.6 mm × 250mm, 5 μm), and the compounds eluted with methanol/water
1
(
0.1%TFA), 70:30, at a flow rate of 0.5 mL/min). H NMR spectra were recorded on a Bruker
4
00 spectrometer with TMS as an internal reference in the indicated solvent at room
temperature. Chemical shifts were reported in ppm. Mass spectra in the electrospray
ionization (ESI) mode were obtained using a Q-TOF Micro system mass spectrometer.
Compounds 2-4 were synthesized according to the literature reported (12-13).
4
-Methoxy-6-methyl-2H-pyran-2-one (2)
1
White solid, yield: 86%; R (Petroleum ether: EtOAc, 4:1 v/v): 0.30; H NMR (400 MHz,
f
CDCl
3
) δ 5.71 (s, J = 0.8 Hz, 1H), 5.34 (s, J = 2.2 Hz, 1H), 3.73 (s, J = 2.5 Hz, 3H), 2.14 (s, J
=
2.1 Hz, 3H). HPLC purity: 98.7%.
4-Methoxy-2-oxo-2H-pyran-6-carbaldehyde (3)
1
Yellowish solid, yield: 91%; R
CDCl ) δ 5.70 (d, J = 0.8 Hz, 1H), 5.33 (d, J = 2.2 Hz, 1H), 3.72 (d, J = 2.5 Hz, 3H). HPLC
purity: 99.3%.
f
(Petroleum ether: EtOAc, 2:1 v/v): 0.33; H NMR (400 MHz,
3
(E)-3-(4-methoxy-2-oxo-2H-pyran-6-yl)acrylic acid (4)
1
Yellow solid, yield: 61%; R
f
(DCM: EtOAc, 4:1 v/v): 0.31; H NMR (400 MHz, DMSO-d
6
) δ
7
3
1
.23 (dd, J = 15.7, 2.6 Hz, 1H), 6.71 (s, 1H), 6.41 (dd, J = 15.7, 2.8 Hz, 1H), 5.79 (s, 1H),
13
.83 (d, J = 10.5 Hz, 3H). C NMR (100 MHz, DMSO-d
6
) δ 169.9, 166.3, 162.0, 155.3,
33.9, 124.1, 106.3, 91.2, 56.6. HPLC purity: 98.1%.
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General procedure for the preparation of lactone benzoyl hydrazine derivatives (5-15)
AcOH (1 mL) was added dropwise into a mixture of 4-methoxy-2-oxo-2H-pyran-6-
carbaldehyde (3) (1 equiv) (12) and the corresponding benzohydrazide or 5-
(
dimethylamino)naphthalene-1-sulfonohydrazide (1 equiv) in EtOH (19 mL), then the
reaction mixture was refluxed and monitored by TLC until the reaction completion. The
mixture was concentrated in vacuum. Compounds 5-13 were obtained in yields varied from
6
1% to 76%. And, (E)-3-(4-methoxy-2-oxo-2H-pyran-6-yl) acrylic acid (4) (1 equiv) (13)
and two drops of piperidine were added to corresponding benzohydrazide (1 equiv) in
pyridine (5 mL). The mixture was refluxed for 3-4 h and evaporated to remove pyridine. The
residue was further purified by column chromatography to afford 14-15 (64-68%).
(E)-N'-((4-methoxy-2-oxo-2 H-pyran-6-yl)methylene)benzohydrazide (5)
1
White solid, yield: 82%; R
f
(DCM: EtOAc, 8:1 v/v): 0.25; H NMR (400 MHz, DMSO-d
6
) δ
1
2.21 (s, 1H), 8.10 (s, 1H), 7.91 (d, J = 6.4 Hz, 2H), 7.61 (dd, J = 14.1, 7.2 Hz, 1H), 7.54 (t, J
13
=
6.9 Hz, 1H), 6.66 (s, 1H), 5.78 (s, 1H), 3.86 (s, 2H). C NMR (100 MHz, DMSO-d
6
) δ
1
70.0, 163.5, 162.2, 155.2, 139.1, 132.7, 132.1,132.1, 128.5, 127.8, 127.7, 104.5, 90.7, 56.6.
+
-
12 2 4
LC MS (ESI) m/z: 273.1 [M + H] . HRMS (ESI): m/z calcd for C14H N O [M − H] ,
−
2
71.0724; found, 271.0721. HPLC purity: 97.8%.
(E)-2-fluoro-N'-((4-methoxy-2-oxo-2H-pyran-6-yl)methylene) benzo hydrazide (6)
1
White solid, yield: 74%; R
f
(DCM: EtOAc, 8:1 v/v): 0.28; H NMR (400 MHz, DMSO-d
6
) δ
1
2.22 (s, 1H), 7.97 (s, 1H), 7.72 – 7.66 (m, 1H), 7.64 – 7.59 (m, 1H), 7.36 (dd, J = 16.9, 9.0
13
Hz, 2H), 6.69 (s, 1H), 5.80 (s, 1H), 3.86 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 170.0,
6
1
62.2,160.9, 154.9, 139.4,133.3, 133.2, 130.2, 124.7, 116.4, 116.1, 105.0, 90.9, 56.6. LC MS
−
+
-
(
ESI) m/z: 291.1 [M + H] . HRMS (ESI): m/z calcd for C H N O F [M − H] , 289.0630;
14 11 2 4
found, 289.0625. HPLC purity: 99.2%.
(E)-3-fluoro-N'-((4-methoxy-2-oxo-2H-pyran-6-yl)methylene)benzo hydrazide (7)
1
White solid, yield: 76%; R
f
(DCM: EtOAc, 8:1 v/v): 0.30; H NMR (400 MHz, DMSO-d
6
) δ
1
2.23 (s, 1H), 8.07 (s, 1H), 7.76 (d, J = 6.7 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.59 (d, J = 6.2
13
Hz, 1H), 7.47 (d, J = 7.4 Hz, 1H), 6.68 (s, 1H), 5.76 (d, J = 16.2 Hz, 1H), 3.85 (s, 3H). C
NMR (100 MHz, DMSO-d ) δ 170.0, 162.2, 160.6, 155.0, 139.6, 135.1, 130.7, 124.0, 119.0,
6
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+
1
14.7, 114.4, 104.9, 90.8, 56.6. LC MS (ESI) m/z: 291.1 [M + H] . HRMS (ESI): m/z calcd
−
-
for C14
H
11
N
2
O
4
F [M − H] , 289.0630; found, 289.0624. HPLC purity: 96.4%.
(E)-4-fluoro-N'-((4-methoxy-2-oxo-2H-pyran-6-yl) methylene) benzo hydrazide (8)
1
White solid, yield: 61%; R
f
(DCM: EtOAc, 8:1 v/v): 0.33; H NMR (400 MHz, DMSO-d
6
) δ
1
3
1
2.25 (s, 1H), 8.04 (d, J = 32.9 Hz, 3H), 7.39 (t, J = 8.4 Hz, 2H), 6.69 (s, 1H), 5.80 (s, 1H),
1
3
.86 (s, 3H). C NMR (100 MHz, DMSO-d
6
) δ 170.6, 162.8, 155.6, 139.6, 131.1, 131.1,
+
31.0, 129.7, 116.2, 116.2, 116.0, 105.3, 91.2, 57.2. LC MS (ESI) m/z: 291.1 [M + H] .
−
-
HRMS (ESI): m/z calcd for C14
7.0%.
H
11
N
2
O
4
F [M − H] , 289.0630; found, 289.0625. HPLC purity:
9
(E)-N'-((4-methoxy-2-oxo-2H-pyran-6-yl)methylene)-4-(trifluoromethoxy)benzohydrazide
(9)
1
White solid, yield: 68%; R
f
(DCM: EtOAc, 8:1 v/v): 0.28; H NMR (400 MHz, DMSO-d6) δ
1
2.28 (s, 1H), 8.15 – 7.91 (m, 3H), 7.54 (d, J = 8 .1 Hz, 2H), 6.69 (s, 1H), 5.78 (s, 1H), 3.85
1
3
(
s, 3H). C NMR (100 MHz, DMSO-d ) δ 170.0, 162.2,162.3, 155.0, 150.8, 139.6, 139.5,
6
+
1
31.8, 130.2, 121.2, 120.8, 118.6, 104.9, 90.8, 56.6. LC MS (ESI) m/z: 357.1 [M + H] .
−
-
HRMS (ESI): m/z calcd for C H N O F [M − H] , 355.0544; found, 355.0547. HPLC
1
5
11
2
5 3
purity: 96.7%.
(E)-N'-((4-methoxy-2-oxo-2H-pyran-6-yl)methylene)benzo[d][1,3]dioxole-5-carbohydrazide
(10)
1
White solid, yield: 74%; R
f
(DCM: EtOAc, 8:1 v/v): 0.27; H NMR (400 MHz, DMSO-d
6
) δ
1
2.05 (s, 1H), 8.06 (s, 1H), 7.53 (d, J = 6.6 Hz, 1H), 7.45 (d, J = 1.5 Hz, 1H), 7.07 (d, J = 8.2
13
Hz, 1H), 6.65 (s, 1H), 6.14 (s, 2H), 5.78 (d, J = 2.1 Hz, 1H), 3.86 (s, 3H). C NMR (100
MHz, DMSO-d
6
) δ 170.1, 162.2, 155.2, 150.5, 147.5, 138.6, 138.5, 126.4, 123.2, 108.1,
+
1
07.7, 104.3, 101.9, 90.6, 56.6. LC MS (ESI) m/z: 317.1 [M + H] . HRMS (ESI): m/z calcd
−
-
12 2 6
for C15H N O [M − H] , 315.0623; found, 315.0619. HPLC purity: 96.4%.
(E)-4-(diethylamino)-N'-((4-methoxy-2-oxo-2H-pyran-6-yl) methylene) benzohydrazide (11)
1
White solid, yield: 68%; R (DCM: EtOAc, 8:1 v/v): 0.31; H NMR (400 MHz, DMSO-d ) δ
f
6
1
1.88 (s, 1H), 8.06 (s, 1H), 7.79 (d, J = 9.0 Hz, 2H), 6.71 (d, J = 9.1 Hz, 2H), 6.59 (d, J = 2.2
Hz, 1H), 5.76 (d, J = 2.2 Hz, 1H), 3.86 (s, 3H), 3.41 (q, J = 7.0 Hz, 4H), 1.12 (t, J = 7.0 Hz,
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1
3
6
1
H). C NMR (100 MHz, DMSO-d
6
) δ 170.2, 162.3, 155.7, 150.2, 137.0, 137.0, 136.9 129.8,
17.7, 110.2, 110.2, 103.5, 90.2, 56.6, 43.7, 43.7, 12.3, 12.3. LC MS (ESI) m/z: 344.1 [M +
−
+
-
21 3 4
H] . HRMS (ESI): m/z calcd for C18H N O [M − H] , 342.1459; found, 342.1447. HPLC
purity: 98.2%.
(E)-N'-((4-methoxy-2-oxo-2H-pyran-6-yl)methylene)-4-nitrobenzo hydrazide (12)
1
White solid, yield: 72%; R
f
(DCM: EtOAc, 8:1 v/v): 0.37; H NMR (400 MHz, DMSO-d
6
) δ
1
7
2.50 (s, 1H), 8.75 (s, 1H), 8.46 (d, J = 7.8 Hz, 1H), 8.37 (d, J = 7.3 Hz, 1H), 8.11 (s, 1H),
13
.86 (t, J = 7.8 Hz, 1H), 6.71 (s, 1H), 5.81 (s, 1H), 3.87 (s, 3H). C NMR (100 MHz,
DMSO-d ) δ 169.9, 162.1, 161.4, 154.8, 147.8, 140.1, 134.2, 134.1, 130.4, 126.7, 122.4,
6
+
1
05.2, 90.9, 56.6. LC MS (ESI) m/z: 318.1 [M + H] . HRMS (ESI): m/z calcd for
−
-
C H N O [M − H] , 316.0575; found, 316.0571. HPLC purity: 99.1%.
14
11
3
6
(E)-5-(dimethylamino)-N'-((4-methoxy-2-oxo-2H-pyran-6-yl) methylene) naphthalene-1-
sulfonohydrazide (13)
1
Yellow solid, yield: 81%; R (DCM: EtOAc, 8:1 v/v): 0.41; H NMR (400 MHz, CDCl ) δ
f
3
1
1
2
0.26 (s, 1H), 8.54 (d, J = 8.5 Hz, 1H), 8.42 (d, J = 8.7 Hz, 1H), 8.36 (dd, J = 7.4, 1.2 Hz,
H), 7.60 (s, 1H), 7.52 (ddd, J = 8.4, 7.5, 5.7 Hz, 2H), 7.14 (d, J = 7.4 Hz, 1H), 6.34 (d, J =
1
3
.2 Hz, 1H), 5.54 (d, J = 2.2 Hz, 1H), 3.80 (s, 3H), 2.85 (s, 6H). C NMR (100 MHz, CDCl )
3
δ 169.9, 163.0, 154.5, 150.8, 135.8, 132.7, 130.3, 129.8, 128.8, 128.8, 127.4, 122.3, 118.3,
+
1
14.4, 99.5, 89.2, 55.3, 44.4, 44.4. LC MS (ESI) m/z: 402.1 [M + H] . HRMS (ESI): m/z
−
+
calcd for C19
H
19
N
3
O
5
S [M + H] , 402.1118; found, 402.1122. HPLC purity: 98.3%.
(E)-N'-(3-(4-methoxy-2-oxo-2H-pyran-6-yl)acryloyl)benzohydrazide (14)
1
White solid, yield: 64%; R (DCM: EtOAc, 8:1 v/v): 0.31; H NMR (400 MHz, DMSO-d ) δ
f
6
1
0.59 (s, 1H), 10.45 (s, 1H), 7.90 (d, J = 7.1 Hz, 2H), 7.60 (t, J = 7.3 Hz, 1H), 7.51 (t, J = 7.5
Hz, 2H), 7.25 (d, J = 15.4 Hz, 1H), 6.85 (d, J = 15.5 Hz, 1H), 6.70 (d, J = 2.1 Hz, 1H), 5.79
1
3
(
d, J = 2.2 Hz, 1H), 3.85 (s, 3H). C NMR (100 MHz, DMSO-d
6
) δ 170.1, 165.2, 163.0,
1
62.1, 155.7, 132.2, 131.9, 131.0, 128.4, 128.4, 127.4, 127.4, 124.3, 105.9, 90.9, 56.6. LC
−
+
-
MS (ESI) m/z: 315.1 [M + H] . HRMS (ESI): m/z calcd for C16
H
14
N
2 5
O [M − H] , 313.0830;
found, 313.0827. HPLC purity: 97.3%.
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(E)-2-fluoro-N'-(3-(4-methoxy-2-oxo-2H-pyran-6-yl)acryloyl)benzo hydrazide (15)
1
White solid, yield: 68%; R
f
(DCM: EtOAc, 8:1 v/v): 0.34; H NMR (400 MHz, DMSO-d
6
) δ
1
0.57 (d, J = 1.8 Hz, 1H), 10.47 (s, 1H), 7.66 (td, J = 7.5, 1.7 Hz, 1H), 7.60 (dd, J = 8.2, 2.0
Hz, 1H), 7.35 – 7.30 (m, 2H), 7.25 (d, J = 15.4 Hz, 1H), 6.82 (d, J = 15.5 Hz, 1H), 6.70 (d, J
13
=
2.1 Hz, 1H), 5.79 (d, J = 2.2 Hz, 1H), 3.85 (s, 3H). C NMR (100 MHz, DMSO-d
6
) δ
1
1
70.1, 162.5, 162.1, 160.5, 158.0, 155.7, 133.1, 131.1, 130.1, 124.6, 124.2, 122.0, 116.3,
+
06.0, 90.9, 56.6. LC MS (ESI) m/z: 333.1 [M + H] . HRMS (ESI): m/z calcd for
−
-
C
16
H
13
N
2
O
5
F [M − H] , 331.0632; found, 331.0625. HPLC purity: 95.8%.
General procedure for the preparation of 2-nitro-1-phenyl-1H-indole derivatives (22-27)
A mixture of corresponding 2-nitrobenzofuran (1 equiv) and the corresponding aniline or 1H-
1
,2,3-triazol-1-amine (1 equiv) in NaOH (0.2 mol/L, 3 mL) was added dropwise. Then the
reaction mixture was refluxed and monitored by TLC until the reaction completion. The
reaction mixture was neutralized with 10% HCl. Dichloromethane (10 mL) was then added,
organic layer was separated and washed with H
2
O (10 mL) and brine (10 mL), dried over
anhydrous sodium sulphate and concentrated in vacuum. The yields were varied from 41% to
6
2
8%.
-Nitrobenzofuran (19) (14)
1
Yellow solid, yield: 90%; R (Petroleum ether: EtOAc, 4:1 v/v): 0.37; H NMR (400 MHz,
f
CDCl
3
) δ 7.34 (dd, J = 11.8, 4.3 Hz, 2H), 7.08 – 7.02 (m, 2H), 6.00 (d, J = 1.0 Hz, 1H). LC-
+
MS (ESI) m/z: 163.1 [M + H] . HPLC purity: 99.4%.
6
-Methoxy-2-nitrobenzofuran (20) (14)
1
Brown solid, yield: 86%; R
f
(Petroleum ether: EtOAc, 4:1 v/v): 0.34; H NMR (400 MHz,
13
CDCl ) δ 7.68 – 7.55 (m, 2H), 7.09 – 6.98 (m, 2H), 3.90 (s, 3H). C NMR (100 MHz, CDCl
3
3
)
δ 162.4, 155.0, 124.4, 124.4, 119.0, 116.1, 108.1, 95.6, 55.9. LC-MS (ESI) m/z: 194.1 [M +
+
H] . HPLC purity: 98.9%.
5
-Methoxy-2-nitrobenzofuran (21) (14)
1
Brown solid, yield: 83%; R
CDCl ) δ 7.60 (d, J = 0.7 Hz, 1H), 7.50 (d, J = 9.2 Hz, 1H), 7.20 (dd, J = 9.2, 2.6 Hz, 1H),
.12 (d, J = 2.6 Hz, 1H), 3.87 (s, 3H). C NMR (100 MHz, CDCl
f
(Petroleum ether: EtOAc, 4:1 v/v): 0.36; H NMR (400 MHz,
3
13
7
3
) δ 157.51, 148.4, 126.4,
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+
1
9
20.4, 120.4, 113.5, 107.3, 104.3, 55.9. LC-MS (ESI) m/z: 194.1 [M + H] . HPLC purity:
7.9%.
4-(2-Nitro-1H-indol-1-yl)benzohydrazide (22)
1
Yellow solid, yield: 68%; R
f
(DCM: MeOH, 49:1 v/v): 0.25; H NMR (400 MHz, DMSO-d
6
)
δ 12.31 (s, 1H), 12.06 (s, 1H), 7.76 (d, J = 7.5 Hz, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.55 (t, J =
.3 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 6.67 (d, J = 8.6 Hz, 2H), 6.06
7
1
3
(
s, 2H), 3.34 (s, 5H). C NMR (100 MHz, DMSO-d
6
) δ 162.4, 156.6, 153.2, 148.8, 134.0,
1
32.0, 129.3, 124.5, 121.6, 121.2, 117.9, 113.0, 113.0, 111.5. LC-MS (ESI) m/z: 298.1 [M +
+
+
12 4 3
H] . HRMS (ESI): m/z calcd for C15H N O [M + H] , 298.0934; found, 298.0929. HPLC
purity: 95.7%.
4-(5-Methoxy-2-nitro-1H-indol-1-yl)-N,N-dimethylaniline (23)
1
Yellow solid, yield:41%; R
f
(DCM: MeOH, 49:1 v/v): 0.31; H NMR (400 MHz, CDCl
3
) δ
1
6
4.40 (s, 1H), 7.34 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 9.0 Hz, 2H), 6.68 (d, J = 9.0 Hz, 2H),
1
3
.60 (d, J = 2.3 Hz, 1H), 6.42 (dd, J = 8.8, 2.3 Hz, 1H), 3.78 (s, 3H), 2.95 (s, 6H). C NMR
(
100 MHz, CDCl
3
) δ 165.4, 164.0, 155.8, 149.5, 126.5, 122.7, 122.7, 112.4, 112.4, 111.7,
+
1
11.0, 96.8, 55.9, 40.5, 40.5. LC MS (ESI) m/z: 312.1 [M + H] . HRMS (ESI): m/z calcd for
−
+
C
17
H
17
N
3
O
3
[M + H] , 312.1343; found, 312.1337. HPLC purity: 96.3%.
4-(6-Methoxy-2-nitro-1H-indol-1-yl)benzohydrazide (24)
1
Brown solid, yield:45%; R
f 5
(DCM: MeOH, 49:1 v/v): 0.28; H NMR (400 MHz, Pyridine-d )
δ 14.60 (s, 1H), 12.69 (s, 1H), 8.07 (d, J = 8.2 Hz, 2H), 7.82 (d, J = 8.5 Hz, 1H), 6.89 (s, 1H),
13
6
.81 (d, J = 8.5 Hz, 1H), 6.74 (d, J = 8.3 Hz, 2H), 6.52 (s, 2H), 3.69 (s, 3H). C NMR (100
MHz, Pyridine-d
5
) δ 165.6, 161.3, 155.6, 152.5, 151.3, 136.9, 132.1, 124.9, 124.7, 122.7,
+
1
17.3, 115.8, 113.6, 113.6, 99.5, 57.8. LC MS (ESI) m/z: 327.1 [M + H] . HRMS (ESI): m/z
−
+
calcd for C16
H
4
14NO [M + H] , 327.1031; found, 327.1027. HPLC purity: 96.8%.
4-(5-Methoxy-2-nitro-1H-indol-1-yl) benzohydrazide (25)
1
Brown solid, yield:43%; R
f 5
(DCM: MeOH, 49:1 v/v): 0.30; H NMR (400 MHz, Pyridine-d )
δ 14.58 (s, 1H), 12.79 (s, 1H), 8.06 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 2.6 Hz, 1H), 7.17 (d, J =
8
3
.8 Hz, 1H), 7.04 (dd, J = 8.9, 2.7 Hz, 1H), 6.74 (d, J = 8.6 Hz, 2H), 5.03 (s, 2H), 3.64 (s,
13
H). C NMR (100 MHz, Pyridine-d
5
) δ 165.8, 159.1, 155.8, 154.1, 152.8, 137.4, 134.4,
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1
32.2, 122.4, 121.7, 115.8, 115.8, 115.7, 114.4, 107.0, 57.8. LC-MS (ESI) m/z: 327.1 [M +
+
+
H] . HRMS (ESI): m/z calcd for C H N O [M + H] , 327.1031; found, 327.1026. Purity:
16
14
4
4
9
6.8% (by HPLC). HPLC purity: 96.2%.
-Methoxy-2-nitro-1-(1H-1,2,3-triazol-1-yl)-1H-indole (26)
5
1
Brown solid, yield: 52%; R
δ 15.12 (s, 1H), 7.38 – 7.25 (m, 3H), 7.24 (dd, J = 7.8, 1.7 Hz, 2H), 3.68 (s, 3H). C NMR
100 MHz, Pyridine-d ) δ 180.2, 159.2, 156.6, 148.6, 131.3, 129.0, 126.7, 122.3, 114.1, 106.0,
f 5
(DCM: MeOH, 49:1 v/v): 0.20; H NMR (400 MHz, Pyridine-d )
13
(
5
+
+
5
2
5.7. LC MS (ESI) m/z: 260.1 [M + H] . HRMS (ESI): m/z calcd for C11
H
9
N
5
O
3
[M + H] ,
−
60.0721; found, 260.0716. HPLC purity: 97.3%.
6-Methoxy-2-nitro-1-(1H-1,2,3-triazol-1-yl)-1H-indole (27)
1
Brown solid, yield: 45%; R
f 5
(DCM: MeOH, 49:1 v/v): 0.21; H NMR (400 MHz, Pyridine-d )
δ 14.95 (s, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.56 (s, 2H), 6.85 (d, J = 2.1 Hz, 1H), 6.75 (dd, J =
1
3
8
1
.6, 2.1 Hz, 1H), 3.73 (d, J = 6.5 Hz, 3H). C NMR (100 MHz, Pyridine -d
5
) δ 179.1, 168.3,
67.1, 148.4, 125.8, 123.4, 122.9, 115.2, 112.6, 97.3, 56.0. LC MS (ESI) m/z: 260.1 [M +
−
+
+
9 5 3
H] . HRMS (ESI): m/z calcd for C11H N O [M + H] , 260.0721; found, 260.0714. HPLC
purity: 96.8%.
Biological Activity
Cell culture and viability assay
BV-2 microglial cells were obtained from Southern Medical University (SMU) Cell Bank
4
(
Guangzhou, People’s Republic of China). Cells were plated into a 96-well plate (1×10
cells/well). After 48 h, they were pre-treated with compounds for 60 min and stimulated with
μg/mL LPS for another 24 h. The cell viability of the cultured cells was assessed by MTT
1
assay. Briefly, BV-2 cells were incubated with 20 μL MTT solution (0.25 mg/mL in medium)
for 4 h keep in dark place, and then the supernatants were removed and residues were
dissolved in 120 μL DMSO. The absorbance was detected at 492 nm using a microplate
reader (Molecular Devices, USA) and analyzed using a SoftMax Pro 5 software (Molecular
Devices, USA). Survival rate of cells was calculated via the following formula: cell survival
rate (%) = (OD sample-OD blank)/(OD control-OD blank) × 100 %.
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Nitrite assay
The NO concentration was measured by the Griess reaction. Briefly, BV-2 cells were treated
with LPS (1.0 μg/mL) and compounds (gradient concentrations 100, 50, 25, 10, 5, 2.5, 1, 0.5
μM for 5-15, and 60, 30, 15, 7.5, 3.75, 1.87, 0.94, 0.47 μM for 22-27 and 5,6-dehydrokawain)
for 24 h. After that, 50 μL of culture supernatant was allowed to react with 50 μL of Griess
reagent (1% sulfanilamide, 0.1% N-1-naphthylethylenediamine dihydrochloride in 5%
phosphoric acid) for 10 min at RT in the dark. Then, the optical density (150 μL per well)
was measured at 540 nm using a microplate reader (Molecular Devices, USA). Sodium nitrite
was used as a standard to calculate the nitrite concentration. Inhibition (%) = (1 − (ALPS+sample
−
Auntreated)/(ALPS − Auntreated) ) × 100. The experiments were performed in triplicates and the
IC50 data are expressed as the mean ± standard deviation (SD) values through processing of
GraphPad Prism 5.
In vitro blood−brain barrier (BBB) permeation assay
To evaluate the permeability of synthesized compounds across BBB which is necessary to a
candidate for anti-neuroinflammation, the parallel artificial membrane permeation assay of
the blood-brain barrier (PAMPA-BBB) was performed according to the previous report
described by Di et al.(15), and we followed the method established by Lu et al in School of
Pharmaceutical Sciences, Sun Yat-sen University (16).
Western blot analysis
To determine p38α MAPK, BV-2 microglial cells was pretreated with 27 (10 μM) for 1 h,
and then exposed to LPS for 24 h. To make whole cell lysates, cells were washed with ice-
cold PBS and lysed for 10 min in RIPA lysis buffer (50 mM Tris-Cl, pH 7.4, 150mM NaCl,
0
.1%SDS, 1% NP-40, 1% sodiumdeoxycholate, 1mM phenylmethyl-sulfonylfluoride, 1μg/ml
pepstatin, 1 μg/ml leupeptin, and 1 μg/ml aprotinin). The protein concentration in the
supernatant of the lysate was measured by BCA protein assay (Beyotime, China). Equal
amounts (60 μg) of proteins were separated electrophoretically by SDS–PAGE and then
transferred to 0.45μM polyvi- nylidene fluoride membrane (PVDF: Millipore, Bedford, MA).
The membranes were soaked in blocking buffer (5% skimmed milk or BSA) and then
incubated overnight with primary antibodies, followed by horseradish peroxidase conjugated
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secondary antibodies. Following three washes in Tris-buffered saline-Tween 20 (TBST),
immunoreactive bands were visualized by the enhanced chemiluminescence (ECL).
Molecular dynamics (MD) stimulations for binding mechanism
Due to a good crystallographic resolution (1.8 Å), 3GC7 was selected from Protein Data
Bank. The initial structures of 27 were optimized using Amber12: EHT (17), and the Powell
method was used for energy minimization via default parameters in Discovery Studio 3.5. In
this work, docking calculations were performed using MOE 2013.8 via default parameters,
and evaluated the binding energies between ligand and receptor. All docked poses of 27 were
clustered using a tolerance of 2 Å for root mean square deviation (RMSD), and were ranked
based on binding docking energies. The lowest energy conformation in the most populated
cluster was chosen for MD simulations, which were carried out using the AMBER 12
software package on graphics processing units (GPUs), with constant number, pressure, and
temperature (NPT), and periodic boundary condition (18). The AMBER 12: EHT force field
(
19) was applied for peptides. Parameters for 27 were obtained from the ANTECHAMBER
module using the Generalized Amber force field (GAFF). The partial atomic charges for the
ligand atoms were assigned using RESP charge-fitting procedure, with input from Hartree–
Fock calculations at the 6-31G* level through use of the Gaussian03 program (20). During
the simulations, the pressure and the temperature were coupled to 1 bar, with an anisotropic
coupling time of 1.0 ps, and kept at 300 K with a coupling time of 0.1 ps. The coordinates of
the whole 20 ns MD run were saved every 2 ps.
Results and Discussions
The lactone benzoyl hydrazine derivatives and 2-nitro-1-phenyl-1H-indole derivatives were
synthesized according to the approaches illustrated in Schemes 1, 2 and 3. Initially,
commercially available 4-hydroxy-6-methyl-2H-pyran-2-one (50mmol, 6.3g) (1) was reacted
with K
2
CO
3
(5eq) and (CH
3
)
2
SO
4
(1eq), at room temperature in acetone for overnight to give
(6eq) were dissolved in dry dioxane
2
(yield: 85%). Synthesis of 3 under nitrogen, 2 and SeO
2
(200mL), inside a sealed tube. The mixture was heated up to160 ℃ for 5 h. Compound 3
(1mmol, 154.1mg) was reacted with different benzohydrazide derivatives (1eq) and 5-
(dimethylamino) naphthalene-1-sulfonohydrazide (1eq) in the presence EtOH (5mL) and
AcOH (1eq) to provide the target compounds 5-13 shown in scheme 1 (yield: 61-76%),
which combine the lactone and benzamido moities. And 3 (10mmol, 1.5g) was reacted with
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malonic acid (1eq) in pyridine (10mL) and piperidine (4 drops) at r.t. provided (E)-3-(4-
methoxy-2-oxo-2H-pyran-6-yl)acrylic acid yield: 61%). And 3 (1mmol, 154.1mg) was
reacted with benzohydrazide (1eq) or 2-fluorobenzohydrazide (1eq) to provide the target
compounds 14-15 shown in scheme 2 yield: 64-68%). Then, commercially available
(
(
bromonitromethane (20mmol, 2.8g) were reacted with 2-hydroxybenzaldehyde (1eq), 2-
hydroxy-5-methoxybenzaldehyde (1eq) and 2-hydroxy-4-methoxybenzaldehyde (1eq) to
give compounds 19-21, then in acetic anhydride (30mL) was refluxed gave the 2-
nitrobenzofuran derivatives 19-21 shown in scheme 3 (yield: 83-90%). Compounds 19-21
(2mmol) were reacted with different aniline derivatives (1eq) in the presence of NaOH (0.2
mol/L, 3 mL) to provide the targets compounds 22-25 (yield: 41-68%). Similarly, target
compounds 26 and 27 (yield: 45-52%) shown in scheme 3, were obtained by the same
reaction of 20 and 21 with 1H-1,2,3-triazol-1-amine.
All the seventeen synthesized compounds didn’t show significant cytotoxicity at the tested
concentrations in BV2 cell lines. Therefore, the tested concentrations are suitable for the
subsequent experiments. Significantly, compounds 13, 26 and 27 exhibited pronounced
inhibition on the NO production with IC50 values of 14.1, 2.4 and 1.6 μM, respectively, being
more active than the positive control, 5,6-dehydrokawain (IC50 = 50.1 μM) as shown in
Table1. Out of these three compounds, the compounds 5, 6, 9, 11, 22, and 23 efficiently
assuaged anti-neuroinflammation in comparison to 5,6-dehydrokawain. Unlike compounds 7,
8
, 10, 12, 14, 15, 24 and 25 were neither cytotoxic nor anti-neuroinflammatory in nature.
The lead discovery process for drugs that target the central nervous system suffers from a
very high rate of failure due to the blockade of the BBB. Using a PAMPA to assess the BBB
permeability has shown to be a high throughput way to compare new candidates with
currently controls via their effective permeability values (21). We compared the permeability
of 13 commercial drugs with reported values to validate the assay system (see supplementary
data, Table S1). A plot of the experimental data versus the reported values produced a good
2
linear correlation, P
e
(exp) = 1.4574 P
e
(lit.) −1.0773 (R = 0.9427) (Figure S1). From this
equation and considering the limit established by Di et al, compounds with permeability
−6
greater than 4.7×10 cm/s (Table S2) could be regarded as enabling them to cross the blood-
brain barrier (16). On the basis of the measured permeability, 27 could cross the BBB. Due to
the highest activity for inhibiting the NO production and drug-like physicochemical
characteristics, compound 27 was chosen for further evaluation.
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LPS usually mimics as inflammatory trigger following binding to TLR4 that activate MAPK
to induce the cytokine expression (22). To understand whether the anti-inflammatory
activities of the compound 27 are mediated through the p38α MAPK pathway, we examined
the effects of 27 on LPS-induced phosphorylations of p38 MAPK by western-blot analysis.
As shown in Figure 2, LPS-induced p38α-phosphorylation (p-p38α) was significantly
inhibited by 27.
In order to explore the possible interaction mechanism between p38α and compound 27, 20
ns MD simulations for the p38α and MAPK-27 complex were performed. Results
demonstrated that compound 27 stabilized p38α MAPK secondary structure. Initially, 27
bound to p38α MAPK at its C-terminus, and formed one hydrogen bond at residue Asp168
(Fig. 3A), which is the important member of the known DFG motif (Asp168 (D), Phe169 (F)
and Gly170 (G)) in the activation region as well as the catalytic site of adenosine triphosphate
for competitive inhibitor (23). After 20 ns MD simulations in water, two hydrogen bonds at
residues Glu71 and Asp168, and forms two arene-H at residues Lys53 and Phe169 (Fig. 3B),
2
7 is more deeply docked in the pocket. This complex of p38α MAPK-27 is energetically
more stable. The RMSD evolution curves (Fig. 3C) indicated the complex became stable
after 2 ns.
Conclusion
Inspired from the core pieces of reported active compound, we have successfully synthesized
a series of lactone benzoyl hydrazine derivatives and 2-nitro-1-phenyl-1H-indole derivatives
with various substitution groups. Three compounds 13, 26 and 27 were found to be potent
and 27 exhibited the strongest activity for anti-inflammation toward p38α MAPK inhibition
in the cell-based assay. Moreover, these compounds could cross the BBB in vitro. Therefore,
these compounds especially 27 can possibly lead to an efficient agent in the fight against the
Alzheimer’s disease. So far, we will introduce more functional groups, heterocycle of 26 and
2
7, such as pyrrole, pyridine and pyrazine instead of the triazol ring so that we could gain
more insights into better active compounds and their structure-activity relationships.
Acknowledgements: This work was financially supported by the National Natural Science
Foundation of China (No. 81102502 and 81473138), Natural Science Foundation of
Guangdong Province, China (No. S2013010015021) and the National High-tech R&D
Program of China (863 Program) (2012AA020307).
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Conflict of interest statement: All authors declare no financial/commercial conflicts of
interest.
1
13
Supplementary data: Full experimental details and spectroscopic data ( H and C NMR
spectra) for compounds can be found in the Supplementary Data section of this article’s Web
page.
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Legends for the Figures and Schemes
Figure 1: Drug design for two series of lactone benzoyl hydrazine derivatives and 2-nitro-1-
phenyl-1H-indole derivatives.
Figure 2: Effects of compound 27 on the p38α MAPK in LPS-stimulated BV-2 cells. Cells
was stimulated with LPS (1 μg/ml) for 1h and then treated with 27 (10μM) for 24h. Cell
lysates were analysed by western blot. And GAPDH was used as internal control. Figure 2a
shows the representative blots and Figure 2b shows the protein band intensity value ratio
##
**
based on three independent experiments with statistical data. p < 0.01 p < 0.05.
Figure 3: A Molecular dynamics (MD) simulations demonstrated that compound 27
stabilized the p38α MAPK. (A): The 27 binding mode before MD simulations. (B): The 27
binding mode after 20 ns MD simulations. (C): p38α MAPK backbone atoms stabilization is
confirmed by the RMSD curves. Red curve indicates that the backbone atoms in p38α MAPK
domain are stable after 2 ns simulations.
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Scheme 1: Synthesis of 5-13. Reagents and conditions: (a) K
2
CO
3 3 2 4
, Acetone, (CH ) SO , r.t.,
overnight, yield: 85%. (b) SeO
2
, dioxane, 160℃，4h, yield: 91%. (c) AcOH, EtOH, reflux,
overnight, yield: 61-76%.
Scheme 2: Synthesis of 14-15. Reagents and conditions: (d) pyridine, malonic acid,
piperidine, r.t., overnight, yield: 61%. (e) sulfur dichloride, triethylamine, chloroform, reflux,
2
h, yield: 64-68%.
2 2 2 3
Scheme 3: Synthesis of 22-27. Reagents and conditions: (f) BrCH NO , K CO , Acetone, r.t.,
6
h; Acetic anhydride, reflux, 6h, yield: 83-90%. (g) NaOH, 90℃, 4h, yield: 41-68%.
Table1. Effects of lactone benzoyl hydrazine derivatives and 2-nitro-1-phenyl-1H-indole
derivatives on LPS-induced NO production in BV-2 cells.
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Table 1:
Compound
a
a
IC50
Compound
14
IC50
5
6
7
8
9
4
4
5
5
4
8
4
1.7±2.8
7.5±1.3
5.3±5.1
0.1±0.5
7.4±2.1
9.1±2.3
82.3±1.4
54.7±1.9
18.6±2.7
15
22
23
39.8±0.4
24
>60
1
0
25
>60
11
26
27
2.9±4.1
2.4±1.1
1
1
2
3
>100
1
.6±0.7
5,6-dehydrokawain
1
4.1±0.8
50.1±1.8
a
Concentration (μM) for 50% inhibition of NO release in BV-2 cells. The IC50 values are the mean ± SEM for at least three experimental
determinations.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.