Synthesis of some new acylhydrazone compounds containing the 1,2,4-triazole structure and their neuritogenic activities in Neuro-2a cells

Article abstract of DOI:10.1039/d0ra02880k

In the present study, a novel series of acylhydrazone compounds (A0-A10) with the structure of 1,2,4-triazole have been designed and synthesized. In addition, all the synthesized compounds have been evaluated for neuritogenic activity in mouse neuroblastoma (Neuro-2a) cells. Notably, we found that one of these 11 acylhydrazone compounds, compoundA5(2-(4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-ylthio)-N′-(2-hydroxybenzylidene)-acetohydrazide) displays excellent neuritogenic activity. Moreover, our present study revealed that compoundA5had the ability to induce neurite outgrowth through the PI3K/Akt and MEK-ERK signaling pathway in Neuro-2a cells. These findings suggest that compoundA5might exert neuritogenic effects and thus may be useful for the treatment of neural repair and regeneration.

Full text of DOI:10.1039/d0ra02880k

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Synthesis of some new acylhydrazone compounds
containing the 1,2,4-triazole structure and their
neuritogenic activities in Neuro-2a cells†
Cite this: RSC Adv., 2020, 10, 18927
c
b
b
b
Xia Jiang,ab Genyun Tang, Jie Yang, Jiacheng Ding, Hongwei Lin
*
a
*
and Xiaoliang Xiang
In the present study, a novel series of acylhydrazone compounds (A0–A10) with the structure of 1,2,4-
triazole have been designed and synthesized. In addition, all the synthesized compounds have been
evaluated for neuritogenic activity in mouse neuroblastoma (Neuro-2a) cells. Notably, we found that one
of these 11 acylhydrazone compounds, compound A5 (2-(4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-
ylthio)-N0-(2-hydroxybenzylidene)-acetohydrazide) displays excellent neuritogenic activity. Moreover, our
present study revealed that compound A5 had the ability to induce neurite outgrowth through the PI3K/
Akt and MEK-ERK signaling pathway in Neuro-2a cells. These findings suggest that compound A5 might
exert neuritogenic effects and thus may be useful for the treatment of neural repair and regeneration.
Received 1st April 2020
Accepted 5th May 2020
DOI: 10.1039/d0ra02880k
rsc.li/rsc-advances
superior performance in various aspects, making it a promising
therapeutic drug and functional material.9,10 In recent years,
1. Introduction
researchers have found that acylhydrazone compounds exhibit
many benecial biological activities, such as anti-bacterial,11–13
anti-inammatory,14–16 anti-viral17,18 and anti-tumor activi-
ties.19–21 However, there are currently few studies on the neu-
roactivity of acylhydrazone compounds.
In the present study, some new acylhydrazone compounds
containing 1,2,4-triazole structure were synthesized, and their
neurite outgrowth-promoting activities were investigated in the
Neuro-2a cells. This cell line is commonly used as an in vitro
model system to study neuronal differentiation, neurite
outgrowth and neurotoxicological studies.
The functions of the nervous system are mediated by neural
circuits that are formed during brain development.1 Neurite
outgrowth is a critical cellular process underlying neural circuit
formation.2 It is well known that abnormalities of this process
are associated with some neurodegenerative diseases. For
instance, neurite atrophy is one of the typical symptoms in early
stages of Alzheimer's and Parkinson's diseases.3–5 Furthermore,
neurite loss is one of the cardinal features of neuronal injury.6
Therefore, it has been suggested that promoting neurite
outgrowth is important when repairing nerve system damage,
and it is necessary for the recovery of neuronal functions. So far,
various compounds derived from bioactive natural products or
chemical synthesis with potential neurite outgrowth have been
studied for neuroregeneration.7,8
2. Results and discussion
2.1 Synthesis of acylhydrazone compounds
Acylhydrazone Schiff base was synthesized by the conden-
sation of hydrazide with aldehyde or ketone, it has strong
coordination ability, a wide spectrum of biological activities and
The compound A0–A10 were designed and synthesized via the
routes as shown in Scheme 1. Initially, isoniazide and potas-
sium hydroxide were dissolved in ethanol, adding carbon
disulde to obtained potassium 2-isonicotinoylhydrazine-
carbodithioate, which reacted with hydrazine hydrate under
143 ꢀC to afford4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol
(A0). Secondly, ethyl 2-(4-amino-5-(pyridin-4-yl)-4H-pyrazol-3-
ylthio) acetate (A1) was synthesized by reaction of compound
aKey Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of
Hunan Province, College of Biological and Food Engineering, Huaihua University,
Huaihua 418008, P. R. China. E-mail: jiangxia285333006@163.com;
xiangxiaoliang225@163.com
bHunan Engineering Laboratory for Preparation Technology of Polyvinyl Alcohol (PVA)
Fiber Material, College of Chemistry and Materials Engineering, Huaihua University, A0 and ethyl chloroacetate in sodium hydroxide solution.
Huaihua 418008, P. R. China. E-mail: 1520224252@qq.com; 1471157054@qq.com;
Thirdly, hydrazine hydrate and compound A1 were reuxed in
methanol to obtain 2-(4-amino-5-(pyridin-4-yl)-4H-pyrazol-3-
linhongwei1968@163.com
cSchool of Medicine, Hunan Provincial Key Laboratory of Dong Medicine, Hunan
ylthio) acetohydrazide (A2). Finally, compound A2 was reacted
University of Medicine, Huaihua, Hunan 418000, P. R. China. E-mail:
with different structures of aldehydes to gain the target
genyun1983311@126.com
compounds A3–A10. Structural explanations of the nal
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra02880k
compounds were performed with IR, 1H-NMR, 13C-NMR and
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Scheme 1 The synthetic routes to the target compounds (A0–A10).
GC-MS. In the IR spectra of the compounds, stretching in Fig. 1B, the compounds A0, A1, A2, A3, A4, A5, A7 and A8
absorptions around 3300 cmꢁ1 proved N–H bone of amino in showed that the dependence of cell viability on concentration is
the triazole and amide. Signals belonging to carbonyl (C]O) clearly noticed. Cytotoxic effects according to ISO 10993-5
function appeared at 1651–1764 cmꢁ1 (Amide I band), the standard,22 if cell viability was above 80% are considered as non-
absorption peak of another band of amide appeared near cytotoxic; weak (60 to 80%); moderate (40 to 60%) and below
1265 cmꢁ1. The absorption at about 1529 to 1608 cmꢁ1 was 40% strong cytotoxicity, respectively. Among the tested
registered for C]N bonds. The out of plane bending belonging compounds, A0, A1, A2, A3, A4, A5 and A8 showed no cytotoxic
to substituted benzene was determined at 794–833 cmꢁ1. In the effect on Neuro-2a cells at 5 mM, whereas, compounds A7, A9
1H-NMR spectra, protons belonging to amide (CONH–) were and A10 showed weak cytotoxicity. Notably, compound A6
observed as singlet peaks at 9.91 ppm and 11.99 ppm. Protons exhibited strong cytotoxicity at this concentration. Therefore,
of methylene bridge (–SCH2) were recorded as singlet peaks a concentration of 5 mM was used in subsequent experiments.
between 4.10 ppm and 4.50 ppm. Amino groups on the triazole
gave peaks between 6.30 ppm and 6.89 ppm. Protons of carbon–
nitrogen double bond in hydrazone (–CONHN]CH–) were
recorded as singlet peaks between 7.57 ppm and 8.82 ppm. The
target compounds A3–A10 had E/Z geometric isomers and cis–
trans conformation isomers, so in the absence of coupling, the
proton signal peaks of CONH, SCH2, NH2 and N]CH showed
splitting. In 13C-NMR, carbons of methylene bridge (–SCH2)
were recorded around 34.5 ppm. All aromatic carbons gave
peaks from 110 ppm to 159 ppm. C3 of 1,2,4-triazole ring were
recorded around 166 ppm and carbonyl group gave peaks over
168 ppm. In the GC-Ms spectra, all masses were matched well
with the expected M + H values.
2.3 Effects of acylhydrazone compounds on neuronal
differentiation of Neuro-2a cells
To investigate the inuences of acylhydrazone compounds on
neuronal differentiation. Neuro-2a cells were cultured in a low-
serum (0.5% FBS) medium and treated with compounds A0–
A10 (5 mM) for 48 h. As shown in Fig. 2A, morphological changes
were captured with phase contrast microscope. The differentia-
tion rate and the longest length of neurite of each cell have been
analyzed. As shown in Fig. 2B, only compound A5 apparently
induced Neuro-2a cells differentiation rate aer 48 h treatment,
whereas others were not. Fig. 2C shows that in addition to
compound A5, compound A7 also signicantly promoted neurite
outgrowth.
2.2. Cytotoxic effects of acylhydrazone compounds on
Neuro-2a cells
2.4 Effects of compound A5 on neuronal differentiation of
Neuro-2a cells
In addition to examination of the effects of acylhydrazone
compounds on cell cytotoxicity, cell viability was determined by As we revealed that compound A5 had the neuritogenic activity,
MTT assay (Fig. 1A). For this experiment, cells were seeded in we then examined in detail the effect of compound A5 on
wells of 96-well plate and maintained in culture medium for neurite outgrowth in Neuro-2a cells. The cells were treated with
24 h. The cells were then treated with test compounds (5, 10, 20, compound A5 at the concentration of 1–10 mM and cultured in
50 mM concentration of each compound) for 24 h. As indicated differentiation medium for 48 h. As a positive control, retinoic
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Fig. 1 Cytotoxic effect of test compounds. (A) Chemical structures of compounds A0–A10. (B) Effect of test compounds on the viability of
Neuro-2a cells.Neuro-2a cells treated with test compounds (compounds A0–A10 at different concentration (5–50 mM)). The results represent
the mean ꢂ SEM. (n ¼ 5). *P < 0.05, **P < 0.01, ***P < 0.001, indicated compound vs. DMSO, one-way analysis of variance (ANOVA) followed by
Tukey's test.
acid (RA) was used at the concentration of 10 mM. Fig. 3A shows classical MAPK pathways revealed that c-Jun amino-terminal
the untreated cells (DMSO) having a round shape with few kinases (JNK) was not affected by compound A5. Besides up-
neurites and the RA-treated cells apparently displaying long regulating ERK1/2 and p38 pathways, the Akt pathway was
neurites. Here, we compared the effect of the differentiation of slightly induced by A5.
Neuro-2a cells induced by A5 and RA. Notably, all aspects we
To further determine the important signaling molecules that
examined, including differentiation rate (Fig. 3B) and the are required for compound A5 promoted neurite outgrowth, we
longest neurite length (Fig. 3C), A5 exhibited stronger activities took advantage of a series of pharmacological inhibitors,
as RA did. Moreover, compound A5 promoted neurite including LY294002 (PI3K inhibitor), U0126 (MEK1/2 inhibitor),
outgrowth in a concentration-dependent manner.
BIRB795 (p38 inhibitor) and SP600125 (JNK inhibitor). We
preincubated Neuro-2a cells with each of these inhibitors for 1 h
before the treatment of compound A5, and the differentiation
rate and the longest neurite length were examined (Fig. 5A).
Consistent with the western blot analysis that ERK and Akt were
activated by compound A5, the activities of ERK upstream
kinases MEKs (MAPK/ERK kinase) and the Akt upstream kinase
PI3K were both essentially required for compound A5 induced
neurite outgrowth (Fig. 5B and C). By contrast, the activity of
p38 was not important for compound A5's effect on promoting
neurite outgrowth (Fig. 5B and C). Interestingly, although A5
2.5 Signaling pathway involved in compound A5-induced
neurite outgrowth from Neuro2a cells
To understand what signaling pathways functioning in the
compound A5-induced neurite outgrowth, we treated Neuro-2a
cells with A5 for different time points (0–240 min) and exam-
ined the levels of activated/phosphorylated forms of different
signaling molecules (Fig. 4). Notably, compound A5 markedly
activated extracellular signal regulated kinases 1/2 (ERK1/2) and
p38 at 30 min treatment and thereaer. Examination of another
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Fig. 2 Effect of Acylhydrazone compounds on the differentiation of Neuro-2a cells. (A) Neuro-2a cells were treated with different compounds
(5 mM) for 48 h. Scale bar, 50 mm. The differentiation rate (B) and the longestl neurite length of each differentiated cell (C) were analyzed. At least
400 cells/group were analyzed in each experiment (n ¼ 3), and values were presented as mean ꢂ SEM. ***P < 0.001, indicated compound vs.
DMSO, one-way analysis of variance (ANOVA) followed by Tukey's test.
does not affect the activity of JNK, but inhibition of JNK activity by the method previously reported.23,24 The structures of the
can offset the promotion of A5 on neurite growth (Fig. 5B and synthesised compounds were proved by 1H-NMR, 13C-NMR and
C). These results collectively suggest that compound A5 induces GC-MS. The 1H-NMR and 13C-NMR spectra were obtained in
neuronal differentiation and neurite extension through activa- DMSO-d6 by a Bruker Avance 400 MHz NMR spectrometer at
tion of MEK-ERK and PI3K-Akt pathways, although the identity 400 MHz and 100 MHz, respectively. The FT-IR spectra was
of the direct molecular target of A5 awaits further investigation. recorded under ambient conditions in the wave number range
of 4000–400 cmꢁ1 using an FT-IR Nicolet (Is5) spectrometer. GC-
MS studies were performed on a GCMS-QP 2010 Plus (Shi-
madzu, Tokyo, Japan). Chemical purities of the compounds
3. Materials and methods
3.1 Chemistry
were checked by TLC applications performed on silica gel
60F254. The 1H-NMR, 13C-NMR and GC-MS spectra of the
compounds available in ESI.†
All chemicals in this study were purchased from Shanghai
Aladdin Reagent Company. A series of new acylhydrazone
compounds containing 1,2,4-triazole (A0–A10) were synthesized
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dissolved. The reaction mixture was cooled to room tempera-
ture. Then, isoniazid (10.04 g, 73.2 mmol) was added into the
reaction mixture and 6 mL carbon disulde (CS2) was slowly
added dropwise. The mixture was stirred at room temperature
for 10 h. The precipitated product was ltered and dried in
vacuo to get the intermediate as a yellow powder.
Step 2: preparation of 4-amino-5-(pyridin-4-yl)-4H-pyrazole-3-
thiol (A0). Potassium 2-isonicotinoylhydrazinecarbodithioate
(12.99 g, 0.05 mol) was reuxed with 80% hydrazine hydrate (20
mL, 0.41 mol) in oil bath at 143 ꢀC for 4 h. The obtained product
was mixed with 20 mL distilled-ice water, and then adjust pH ¼
5 with concentrated hydrochloric acid. The obtained residue
was ltered and dried in vacuo to get the compound A0 as white
outgrowth. Neuro-2a cells were treated with compound A5 at solid (yield, 77.6%). H-NMR (DMSO-d6, 400 MHz) d: 14.14 (s,
Fig. 3 Effect of compound A5 at different concentration on neurite
1
concentrations from 1 to 10 mM for 48 h. (A) Representative images of
Neuro-2a cells after treatment with compound A5. Scale bar, 50 mm.
The differentiation rate (B) and the longest neurite length of each
differentiated cell (C) were analyzed. At least 400 cells/group were
1H, –SH); 8.75–8.77 (d, J ¼ 8, 2H, PyH); 8.02–8.03 (d, J ¼ 4, 2H,
PyH); 5.85 (s, 2H, NH2). 13C-NMR (DMSO-d6, 100 MHz):168.08,
151.0, 150.59, 147.79, 133.40, 123.21, 121.05 MS (ESI) m/z: 193
(M+).
analyzed in each experiment (n ¼ 3), and values were presented as
mean ꢂ SEM. *P < 0.05, **P < 0.01, ***P < 0.001, indicated compound
vs. DMSO, one-way ANOVA followed by Tukey's test.
3.2.2 Synthesis procedure of ethyl 2-(4-amino-5-(pyridin-4-
yl)-4H-pyrazol-3-ylthio) acetate 9 (A1). NaOH (1.93 g, 0.048 mol)
was resolved in 45 mL distilled water. An amount of 4.89 g
(0.025 mol) compound A0 was added. Then ethyl chloroacetate
(5.60 mL, 0.045 mol) was slowly added dropwise. The mixture
ꢀ
was stirred at 50 C for 4 h. The mixture was cooled to room
temperature, a precipitated product was crystallized, ltered
and dried to give compound A1 as light-pink solid (yield,
40.9%). 1H-NMR (DMSO-d6, 400 MHz) d: 8.73–8.74 (d, J ¼ 4, 2H,
PyH); 7.98–7.99 (d, J ¼ 4, 2H, PyH); 6.325 (s, 2H, NH2); 4.12–4.16
(m, 4H, CH2); 1.18–1.12 (m, 3H, CH3). 13C-NMR (DMSO-d6, 100
MHz):168.45, 154.82, 152.57, 150.57, 134.32, 121.75, 61.73,
33.53, 14.46. MS (ESI) m/z: 279 (M+).
3.2.3 Synthesis procedure of2-(4-amino-5-(pyridin-4-yl)-4H-
pyrazol-3-ylthio)acetohydrazide (A2). Compound A1 (1.45 g, 5.2
mmol) was resolved in 35 mL anhydrous ethanol. An amount of
2.0 mL (0.41 mol) hydrazine hydrate (80%) was added. The
ꢀ
solution was stirred and reuxed at 80 C for 4 h. The mixture
was cooled to room temperature, a precipitated product was
crystallized, ltered and dried to give compound A2 as white
solid (yield, 85.1%). 1H-NMR (DMSO-d6, 400 MHz) d: 9.34 (s,
1H, CONH); 8.73–8.74 (d, J ¼ 4, 2H, PyH); 8.0–8.02 (d, J ¼ 8, 2H,
PyH); 6.30 (s, 2H, –NH2); 4.32 (s, 2H, –NNH2), 3.88 (s, 2H,
–SCH2). 13C-NMR (DMSO-d6, 100 MHz):166.94, 154.93, 152.43,
150.57, 134.38, 121.79, 34.25. MS (ESI) m/z: 234.
3.2.4 Synthesis procedure of acylhydrazone derivatives
(A3–A10)
Fig. 4 Compound A5 increases phosphorylation levels of Akt, ERK1/2
and p38 in Neuro-2a cells. Compound A5 (5 mM) was added in Neuro-
2a cells for indicated time points (0–240 min). Cells lysates were
subjected to western blot analysis for the phosphorylated and total
forms of different signaling molecules.
2-(4-Amino-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-ylthio)-N0-(3-phe-
nylallylidene) acetohydrazide (A3). Compound A2 (0.21 g, 0.78
mmol) was resolved in 70 mL anhydrous ethanol. Then, cin-
namaldehyde (0.18 mL, 1.43 mmol) was slowly added dropwise.
The solution was stirred and reuxed at 67 ꢀC for 4 h. The
reaction mixture obtained was a clear and transparent solution.
The mixture was cooled to room temperature and a solid
3.2 Synthesis
3.2.1 Synthesis procedure of 4-amino-5-(pyridin-4-yl)-4H- separates out. Separated solid was ltered, dried and recrys-
pyrazole-3-thiol (A0)
tallised by absolute ethanol to give compound A3 as white
Step 1: preparation of potassium 2-isonicotinoylhydrazine- crystal (yield, 38.1%). 1H-NMR (DMSO-d6, 400 MHz) d:11.65
carbodithioate. KOH (7.80 g, 0.139 mmol) was added into (cis), 11.56 (trans) (s, 1H, CONH); 8.70–8.74 (m, 2H, PyH); 7.99–
absolute ethanol (150 mL) and stirred with heating until it 8.01 (d, J ¼ 8, 2H, PyH); 7.57–7.59 (d, J ¼ 8, 1H, N]CH); 7.37–
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Fig. 5 Activation of MEK-ERK, PI3K-Akt and JNK1/2/3 signaling pathway is required for compound A5 induced neurite outgrowth. Neuro-2a
cells were pretreated with different inhibitors, including PI3K inhibitor (LY290042, 10 mM), MEK inhibitor (U0126, 10 mM), p38 inhibitor (BIRB796,
10 mM) or JNK inhibitor (SP600125, 10 mM) for 1 h, followed by compound A5 treatment (5 mM) for 48 h. (A) Neurites were visualized by inverted
phase contrast microscope. Cell differentiation rate (B) and the longest length of neurites per differentiated cell (C) were quantifified. At least 400
cells/group were analyzed in each experiment (n ¼ 3), and values were presented as mean ꢂ SEM. ***P < 0.001, DMSO or inhibitor and
compound A5 cotreatment vs. compound A5 single treatment.
7.39 (d, J ¼ 8, 2H, –CH]CH); 7.35 (m, 3H, Ph–H); 7.33 (m, 2H, 7.99 (s, 2H, PyH); 7.64–7.66 (m, 1H, thiophen–H); 7.45–7.48 (m,
Ph–H); 6.32 (s, 2H, –NH2); 4.01 (cis), 4.41 (trans) (s, 2H, –SCH2). 1H, thiophen–H); 7.11–7.12 (s, 1H, thiophen–H); 6.31–6.33 (d, J ¼
trans/cis: 5/4; 13C-NMR (DMSO-d6, 100 MHz) d: 169.05, 164.03, 8, 2H, –NH2) 4.07(cis), 4.22 (trans) (s, 2H, –SCH2); trans/cis: 10/11;
155.10, 155.02, 152.52, 152.41, 150.58, 150.55, 149.51, 146.87, 13C-NMR (DMSO-d6, 100 MHz) d:168.96, 164.02, 155.15, 155.05,
139.84, 139.55, 136.27, 134.39, 129.31, 129.26, 127.57, 127.53, 152.51, 152.41, 150.57, 150.54, 142.73, 139.42, 139.21, 139.07,
125.77, 125.45, 121.77, 4.69 (trans), 34.63 (cis). MS (ESI) m/z: 134.41, 134.34, 131.65, 131.07, 129.53, 129.07, 128.39, 128.33,
380.0 (M+) IR (KBr) n: 3332, 3201, 1764, 1529.3.
121.78, 34.70 (trans), 34.30 (cis). MS (ESI) m/z: 359.0 (M+) IR (KBr)
The syntheses of compounds A4–A10 were carried out by the n: 3329, 3039, 2930, 1651, 1543, 1265.
same procedure as described for the preparation of A3.
2-(4-Amino-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-ylthio)-N0-(2-
2-(4-Amino-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-ylthio)-N0-(thiophen- hydroxybenzylidene)-acetohydrazide (A5). White ake solid
2-ylmethylene)acetohydrazide (A4). White crystal (yield, 28.41%). (yield, 78.5%). 1H-NMR (DMSO-d6, 400 MHz) d: 12.05 (cis),
1H-NMR (DMSO-d6, 400 MHz) d:11.64 (trans), 11.72 (cis) (s, 1H, 11.67 (trans) (s, 1H, –ph–OH); 11.02 (trans), 10.11 (cis) (s, 1H,
CONH); 8.73 (s, 2H, PyH); 8.42 (trans), 8.21 (cis) (s, 1H, N]CH); CONH); 8.77 (s, 2H, PyH), 8.03 (s, 2H, PyH); 8.46 (cis), 8.37
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2-(4-Amino-5-(pyridin-4-yl)-4H-pyrazol-3-ylthio)-N0-benzylide-
neacetohydrazide (A10). White powder (yield, 20.13%). H-NMR
(trans) (s, 1H, N]CH); 7.26–7.33 (m, 2H, Ph–H), 7.59–7.60 (d,
J ¼ 4, 1H, Ph–H), 7.72–7.74 (d, J ¼ 8, 1H, Ph–H); 6.37–6.39 (d,
J ¼ 8, trans), 6.89–6.96 (m, cis) (2H, –NH2); 4.52 (trans), 4.14
(cis) (s, 2H, –SCH2). trans/cis: 15/16. 13C-NMR (DMSO-d6, 100
MHz) d: 169.03, 164.08, 157.74, 156.88, 155. 19, 155.14,
152.57, 152.44, 150.61, 150.58, 147.57, 141.69, 134.41,
134.33, 131.98, 131.73, 129.61, 126.71, 121.79, 120.50,
119.90, 119.85, 119.11, 116.82, 116.61, 34.52 (trans), 34.33
(cis). MS (ESI) m/z: 369.0 (M+) IR (KBr) n: 3425, 3309, 3202,
2930, 1666, 1582, 1265.
1
(DMSO-d6, 400 MHz) d: 11.77 (cis), 11.66 (trans) (s, 1H,
CONH); 8.73 (s, 2H, PyH); 8.21 (s, 1H, N]CH); 7.99–8.03 (d, J ¼
16, 2H, PyH); 7.69 (s, 2H, Ph–H); 7.42 (s, 3H, Ph–H); 6.32 (s, 2H,
–NH2); 4.09 (cis), 4.50 (trans) (s, 2H, –SCH2); trans/cis: 8/5; 13C-
NMR (DMSO-d6, 100 MHz) d: 169.38, 164.20, 155.08, 152.51,
152.41, 150.53, 147.56, 144.27, 134.41, 130.62, 130.43, 129.27,
127.59, 127.34, 121.75, 34.74 (trans), 34.46 (cis). MS (ESI) m/z:
352.0 (M+) IR (KBr) n: 3250, 3035, 1662, 1552, 1307.
2-(4-Amino-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-ylthio)-N0-(naphthalen-
1
1-ylmethylene)acetohydrazide (A6). White crystal (yield, 79.2%). H-
3.3 Cell culture
NMR (DMSO-d6, 400 MHz) d: 11.87 (cis), 11.72 (trans) (s, 1H,
CONH); 8.72–8.82 (m, 4H, PyH); 8.59–8.62 (d, J ¼ 12, 1H, N]CH);
7.96–7.98 (m, 4H, naphthalene–H); 7.60–7.64 (m, 3H, naphthalene–
H); 6.33–6.35 (d, J ¼ 8, 2H, –NH2); 4.15 (cis), 4.58 (trans) (s, 2H,
–SCH2). trans/cis: 5/4; 13C-NMR (DMSO-d6, 100 MHz) d: 169.37,
164.32, 155.13, 152.47, 150.52, 147.69, 143.90, 134.39, 133.95,
131.16, 130.89, 130.60, 129.75, 129.70, 129.30, 129.25, 128.70,
127.81, 127.47, 126.71, 125.96, 124.96, 124.07, 121.82, 121.75, 34.89
(trans), 34.63 (cis). MS (ESI) m/z: 403.0 (M+) IR (KBr) n: 3320, 3089,
2930, 1685, 1593, 1357.
2-(4-Amino-5-(pyridin-4-yl)-4H-pyrazol-3-ylthio)-N0-(5-bromo-2-
hydroxybenzylidene)acetohydrazide (A7). White powder (yield,
32.38%). 1H-NMR (DMSO-d6, 400 MHz) d: 12.06 (cis), 11.66
(trans) (s, 1H, –ph–OH); 11.04 (cis), 10.37 (trans) (s, 1H, CONH);
8.73 (s, 2H, PyH); 8.39 (cis), 8.25 (trans) (s, 1H, N]CH); 8.0 (s,
2H, PyH); 7.43–7.82 (m, 3H, Ph–H); 6.85 (cis), 6.32 (trans) (s, 2H,
–NH2); 4.10 (cis), 4.49 (trans) (s, 2H, –SCH2). trans/cis: 20/19; 13C-
NMR (DMSO-d6, 100 MHz) d: 169.24, 164.28, 155.03, 152.55,
152.39, 150.57, 150.51, 145.21, 139.72, 134.40, 134.12, 133.90,
133.70, 130.70, 128.24, 122.95, 121.77, 121.66, 119.10, 118.87,
111.32, 110.94, 34.45 (trans), 34.41 (cis). MS (ESI) m/z: 449.0 (M+)
IR (KBr) n: 3340, 3279, 2970, 1681, 1550, 1357.
2-(4-Amino-5-(pyridin-4-yl)-4H-pyrazol-3-ylthio)-N0-(pyridin-2-
ylmethylene)acetohydrazide (A8). White powder (yield, 85.1%).
1H-NMR (DMSO-d6, 400 MHz) d: 11.99 (cis), 11.85 (trans) (s, 1H,
CONH); 8.71–8.74 (m, 2H, PyH); 8.58–8.61 (m, 1H, PyH); 8.22
(cis), 8.05 (trans) (s, 1H, N]CH); 7.81–7.97 (m, 4H, PyH); 7.37–
7.44 (m, 1H, PyH); 6.34 (s, 2H, –NH2) 4.12 (cis), 4.51 (trans) (s, 2H,
–SCH2); trans/cis: 8/5; 13C-NMR (DMSO-d6, 100 MHz) d: 172.48,
169.64, 164.54, 155.06, 154.85, 153.39, 153.19, 152.54, 152.43,
150.48, 149.94, 147.78, 144.64, 137.37, 137.29, 134.40, 124.97,
124.79, 121.75, 120.46, 120.30, 34.67 (trans), 34.24 (cis). MS (ESI)
m/z: 354.0 (M+) IR (KBr) n: 3340, 3197, 2930, 1689, 1608, 1377.
2-(4-Amino-5-(pyridin-4-yl)-4H-pyrazol-3-ylthio)-N0-(4-hydroxy-
Mouse neuroblastoma Neuro-2a cell line was obtained from
ATCC (Manassas, VA, United States) and cultured in MEM
medium supplemented with 1% penicillin/streptomycin and
10% heat-inactivated fetal bovine serum at 37 C in a humidi-
ed 5% CO2 atmosphere. Cells were passaged every 3–4 days.
ꢀ
3.4 Cell viability assay
Cell viability was determined by MTT reduction assay. For assay,
5 ꢃ 103 cells were plated in 96-well microtiter plates and grown
for 24 h. Aerwards cells were treated with different concen-
trations of test compounds (5, 10, 20 and 50 mM). Aer 24 h
incubation, 10 mL of stock MTT (5 mg mLꢁ1) was added to each
well and further incubated for four hours, and then the medium
in the wells was removed and 200 mL DMSO was added to each
well. The absorbance was measured by a microplate reader at
570 nm.Cell viability was shown relative to the control in
a graph.
3.5 Neurite outgrowth-promoting assay
For neurite outgrowth assay, Neuro-2a cells were seeded into 24-
well plates at a density of 1 ꢃ 104 cells per mL and grown for
24 h. Aer 24 h incubation, the culture medium was replaced
with differentiation medium (MEM supplied with 0.5% FBS and
1% penicillin/streptomycin) containing different test
compounds for 24 h. Neuro-2a cells were captured and counted
under a phase contrast microscope. Neurites were dened as
a protrusion with lengths longer than one diameter of the cell
body. The neurite length of each cell was measured by Image J
soware.
3.6 Protein extraction and western blot analysis
Neuro-2a cells were seeded in 35 mm dish (6 ꢃ 104/dish) over-
benzylidene)acetohydrazide (A9). White powder (yield, 60.2%). night and then incubated with compound A5 for different time
1H-NMR (DMSO-d6, 400 MHz) d: 11.55 (cis), 11.46 (trans) (s, 1H, (0–240 min) at 37 ꢀC. Cells were lysed in RIPA buffer containing
–ph–OH); 9.91 (cis), 9.88 (trans) (s, 1H, CONH); 8.73 (s, 2H, PyH), protease and phosphatase inhibitors and whole cell lysates were
7.98–8.0 (d, J ¼ 8, 2H, PyH); 8.09 (cis), 7.92 (trans) (s, 1H, N] quantied using a BCA protein assay kit according to the
CH); 7.50–7.53 (d, J ¼ 12, 2H, Ph–H); 6.80–6.82 (d, J ¼ 8, 2H, Ph– manufacturer's instructions. Those cell lysates were subse-
H); 6.31–6.32 (s, 2H, –NH2); 4.06 (cis), 4.46 (trans) (s, 2H, –SCH2); quently separated by SDS-PAGE, and transferred to PVDF
trans/cis: 10/6; 13C-NMR (DMSO-d6, 100 MHz) d: 168.99, 163.81, membranes. The membranes were probed with primary anti-
159.96, 159.78, 155.09, 152.40, 150.53, 147.92, 144.62, 134.42, body, and then subsequently with secondary antibody, followed
129.38, 129.08, 125.43, 121.77, 116.16, 34.82 (trans), 34.62 (cis). by electrochemiluminescence (ECL) detection. The following
MS (ESI) m/z: 369.0 (M+) IR (KBr) n: 3437, 3321, 3190, 1651, 1582, primary antibodies were used: P-Akt (Ser473) (#AA329), Akt
1265.
(#AA326), P-ERK1/2 (#AF1891), ERK1/2 (#AF1051), P-p38
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(#AM063), p38 (#AF1111), P-JNK1/2/3 (#AF1762), JNK1/2/3
(#AF1048) and b-actin (#AA128) antibodies. The antibodies
and secondary antibody (#A0216, #A0208) were all purchased
from Beyotime (Jiangsu, China)
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3.7 Statistical analysis
The results are expressed as the mean ꢂ standard error of the
mean (SEM). Data were subjected to Student's t test or oneway
analysis of variance (ANOVA) followed by Tukey's test to assess
the differences between the relevant control and each experi-
mental group. A value of P < 0.05 was considered statistically
signicant.
4. Conclusions
In this work, several acylhydrazone derivatives (A0–A10) have
been designed and synthesized, among them two newly
synthesized compounds (A6, A8). All of the compounds were
evaluated for neuritogenic activity in Neuro-2a cells. The phar-
macological results indicated that compound A5 exhibit highly
potent neuritogenic activity through the PI3K/Akt and MEK-ERK
signaling pathway in vitro. The above results suggested that
compound A5 hold high potential use for treating neural injury
and neurodegenerative diseases.
Conflicts of interest
The authors declare that they have no conict interests.
Acknowledgements
This work was nancially supported by the Research Founda-
tion of Education Department of Hunan Province, China (No.
17A66, No. 17B208), and the National Natural Science Foun-
dation of China (No. 31900705, No. 81703821), as well as the
Foundation of Hunan Double First-rate Discipline Construction
Projects of Bioengineering (No. YYZW2019-04).
14 Y. K. da Silva, C. V. Augusto, M. L. de Castro Barbosa,
G. M. de Albuquerque Melo, A. C. de Queiroz, T. de Lima
Matos Freire Dias, W. B. Junior, E. J. Barreiro, L. M. Lima
and
M.
S.
Alexandre-Moreira,
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