Biological activities of novel pyrazolyl hydroxamic acid derivatives against human lung cancer cell line A549

Article abstract of DOI:10.1016/j.ejmech.2014.06.065

We synthesized a series of novel pyrazolyl hydroxamic acid derivatives (4a-4l) and investigated their biological activities against human lung cancer cell line A549 in vitro to determine their mechanism of action. The results showed that the majority of d

Full text of DOI:10.1016/j.ejmech.2014.06.065

Accepted Manuscript
Biological activities of novel pyrazolyl hydroxamic acid derivatives against human lung
cancer cell line A549
Jin-Feng Zhang, Meng Li, Jun-Ying Miao, Bao-Xiang Zhao
PII:
S0223-5234(14)00594-7
10.1016/j.ejmech.2014.06.065
EJMECH 7108
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 May 2014
Revised Date: 26 June 2014
Accepted Date: 28 June 2014
Please cite this article as: J.-F. Zhang, M. Li, J.-Y. Miao, B.-X. Zhao, Biological activities of novel
pyrazolyl hydroxamic acid derivatives against human lung cancer cell line A549, European Journal of
Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.065.
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Biological activities of novel pyrazolyl hydroxamic acid derivatives
against human lung cancer cell line A549
a,c,†
b,†
a,
b,
Jin-Feng Zhang
, Meng Li , Jun-Ying Miao * , Bao-Xiang Zhao *
a
Institute of Developmental Biology, School of Life Science, Shandong University,
Jinan 250100, PR China
b
Institute of Organic Chemistry, School of Chemistry and Chemical Engineering,
Shandong University, Jinan 250100, PR China
c
School of Municipal and Environmental Engineering, Shandong Jianzhu University,
Jinan 250101, PR China
A B S T R A C T
We synthesized a series of novel pyrazolyl hydroxamic acid derivatives (4a-4l) and
investigated their biological activities against human lung cancer cell line A549 in
vitro to determine their mechanism of action. The results showed that the majority of
derivatives had inhibitory effects on the growth of A549 cancer cells in dose and
time-dependent manners, in which the compounds 4b, 4f, 4h and 4j (10ꢀM) exerted
more effective anti-proliferation activity. However, it should be noted that 4j may
result in necrosis at 10ꢀM. Furthermore, the three compounds 4b, 4f and 4h induced
cell cycle arrest at G1 phase and triggered autophagy, but could not obviously induce
*
Corresponding authors. Tel.: +86 531 88366425; fax: +86 531 88564464;
e-mail addresses: bxzhao@sdu.edu.cn; miaojy@sdu.edu.cn;
Equal contribution
†

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apoptosis and necrosis under the stimulatory condition. Therefore, the pyrazolyl
hydroxamic acid derivatives 4b, 4f and 4h can be used to investigate the regulatory
mechanism of autophagy and offer new approaches to the prevention of lung cancer.
Key words: Pyrazole; Hydroxamic acid; Lung cancer cell; Cell cycle; Autophagy.
1
. Introduction
Cancer is one of the important contributors to deaths worldwide, corresponding to
almost 1600 deaths per day in the United States. Lung cancer is a leading cause of
cancer death accounting for approximately 26% of all female and 28% of all male
cancer deaths in 2013 [1]. Among the anti-cancer strategies chemotherapy is available
with drugs/chemicals which interfere with cell division, inhibit tumor angiogenesis or
induce cancer cell death by various signaling pathways but have potential harm to
normal cells [2-4]. However, it should be noted that many types of cancer develop
resistance to chemotherapeutic drugs [5]. Therefore, the research of anti-cancer drugs
with better activity and less side effect is becoming increasingly active.
Autophagy is a conserved mechanism in eukaryotes by which long-lived proteins and
organelles were sequestrated, numerous autophagic vacuoles are formed and delivered
to lysosomes for degradation and recycling [6]. Increasing evidence has revealed that
autophagy plays a crucial role in many human physiological and pathological

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processes, including tumors [7], infection [8], aging [9] and cardiovascular diseases
[
10]. It has been suggested that autophagy deficiency by deletion of autophagy genes
has a positive effect on malignant transformation, indicating autophagy as a tumor
suppressor mechanism [11, 12]. On the other hand, several other studies revealed that
certain cancer cell types maintained the cellular functions by triggering autophagy
under stressful conditions such as hypoxia or anticancer agent treatment while
excessive autophagy could contribute to cell death [13, 14]. Therefore, autophagy has
pro-survival and pro-death functions in carcinogenesis depending on the specific cell
types and different stages of tumor progression though the dual roles of autophagy
remain controversial [15, 16]. Due to the impaired autophagy contributing to various
human disorders, many modulators of autophagy, such as Tat–beclin1 (a domain of
beclin 1 interacting with HIV-1 Nef) [17], spermidine [18], AZD8055 [19] and
AA005 (annonaceous acetogenin mimetic) [20], have been identified to date, which
are sufficient for enhancing autophagy and may provide promising therapeutic
interventions in many diseases especially cancer .
Hydroxamic acid derivatives have received much attention due to its therapeutic
application. Accumulating studies have proved that hydroxamic acid derivatives
scavenge metals and inhibit metallo-enzymes leading to numerous pharmacological
properties: antioxidation, antimicrobial, antitumor and anti-inflammation [21-23].
Hydroxamic acid-derived compounds trichostatin (TSA) and suberoylanilide
hydroxamic acid (SAHA), which have been applied in clinical trials for cancer
therapies, could bind to the catalytic site of histone deacetylase (HDAC) and directly
suppress HDAC activity resulting in cell cycle arrest, apoptosis, autophagy, tumor

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growth inhibition, invasion reduction and the sensitivity of anti-cancer therapy
enhancement
[24-26].
Furthermore,
a
series
of
substituted
5
-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acids were testified to be the inhibitors
of HDAC, among which 6j was more efficacious than SAHA in anti-proliferation
activity in vitro and in vivo [27].
Many pyrazole compounds have been characterized to possess significant anti-cancer
activity and alter a wide variety of essential cellular processes in cancer cells [28-34].
It is noteworthy that the pyrazole core structure has been demonstrated to be an
promising scaffold in medicinal chemistry as potent protein inhibitors such as HDAC
[
(
35], Aurora-A kinase [36], type I TGF-β receptor [37] or cyclin dependent kinases
CDKs) [28]. An overview has highlighted pyrazole and its derivatives for the
development of novel anticancer therapeutics [38]. Among the four
H-pyrazole-3-carboxamide derivatives, pym-5 exerted the highest DNA-binding
1
affinity, suggesting its interaction with DNA and subsequent DNA damage may
contribute to the antitumor activities on HCT116 and HepG2 cells [39]. Elena
Strocchi
et
al.
[32]
revealed
that
two
pyrazole
derivatives
and
N,1-bis(4-aminophenyl)-3-(2,3-dimethylphenyl)-1H-pyrazole-5-carboxamide
5
3
N ,1-bis(4-aminophenyl)-N -(2,3-dimethylphenyl)-1H-pyrazole-3,5-dicarboxamide
decreased the level of p-AKT and inhibited the PI3K/AKT/mTOR pathway, which
were responsible for cell growth inhibition, cell cycle arrest and apoptosis. Moreover,
Yong Xu et al. [40] identified 3-(trifluoromethyl)-1H-pyrazole-5-carboxamide as
potent pyruvate kinase M2 (hPKM2) activators. In our previous studies, we
synthesized different pyrazole derivatives and evaluated their bioactivities and
mechanisms
of
action.
MPD
(ethyl
3
-(o-chlorophenyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxylate) could inhibit
apoptosis of human umbilical vein endothelial cells (HUVECs) via integrin β4, ROS
and p53 signaling pathway when HUVECs were deprived of serum and basic
fibroblast growth factor 2 (bFGF-2) [41]. In addition, MPD promoted angiogenesis

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through elevating ROS levels and depressing interferon-inducible protein 10 (IP-10)
level [42]. Ethyl 1-(2’-hydroxy-3’-aroxypropyl)-3-aryl-1H-pyrazole-5-carboxylate
derivatives
hydrazone derivatives could suppress the growth and induce apoptosis of A549 cells
43, 44]. The complexes of copper (Cu) and salicylaldehyde pyrazole hydrazine
and
3-aryl-1-(4-tert-butylbenzyl)-1H-pyrazole-5-carbohydrazide
[
derivatives (Cu-SPHs) had the similar effects on H322 lung carcinoma cells and
HUVECs as well. The protein level of integrin β4 was enhanced by Cu-15 or Cu-16,
thus, integrin β4 was partly involved in apoptosis pathway [45, 46]. Ge et al. [47]
confirmed that Y-1494 phosphorylation of integrin β4 via fibroblast growth factor
receptor 1 (FGFR1) and its nuclear translocation, which increased the expression of
interleukin 8 (IL-8), prostaglandin-endoperoxide synthase 2 (PTGS2) and activating
transcription factor 3 (ATF3), contributed to the apoptosis in HUVECs by ethyl
1
-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3-(4-chlorophenyl)-1H-pyrazole-5-carboxy
late (ECPC).
Although many researches have shown the anti-cancer effectiveness of pyrazole
derivatives, there are few reports on the synthesis and biological evaluation of
pyrazolyl hydroxamic acids. Based fragment strategy, hence, in this study we
synthesized a series of novel pyrazolyl hydroxamic acid derivatives and investigated
their biological properties to screen bioactive chemicals targeting death of cancer
cells.
2
. Chemistry
2
.1.Synthesis of compounds 4a-4l

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The synthesis of novel pyrazolyl hydroxamic acid derivatives 4a-4l has been
accomplished as outlined in Scheme 1 starting from compounds 3 that can be
synthesized from 1 and the commercially available 2 as described in previous paper
[
48].
Scheme 1
2
.2.Single-crystal structural characterization by X-ray
The structures of 4a-4l were determined by IR, 1H NMR and mass spectroscopy.
Moreover, the structure was confirmed by the X-ray diffraction as shown in Fig. 1.
Figure 1
3
. Pharmacology
The cytotoxicity in vitro was carried out to investigate the biological effects of the
pyrazolyl hydroxamic acid derivatives (4a-4l) against A549 human lung cancer cells
by the SRB assay. Cells were treated with the compounds (4a-4l) at 1, 5 and 10 ꢀM
for 24h or 48 h and cell viability was calculated compared with the cells treated with
0
.1% DMSO (as control).

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4
. Results and Discussion
4
.1.Compounds 4a-4l inhibited the proliferation of A549 cancer cells
In the present work, we investigated the biological effects of a series of novel
pyrazolyl hydroxamic acid derivatives to determine their mechanism of action in
human lung cancer cell line A549. The cytotoxicity of the prepared compounds (4a-4l)
against A549 cells was first conducted using the SRB assay. As shown in Fig. 2, the
compounds 4b and 4f had obvious cytotoxicity (p<0.05, n = 3), whereas other
compounds had not so much effect at 1 ꢀM concentration (p>0.05, n = 3). The
majority of compounds at 5 and 10 ꢀM exhibited anti-proliferation effects on A549
cells (p<0.05 or 0.01, n = 3) except for compounds 4g and 4l (5 ꢀM). Consequently,
the pyrazolyl hydroxamic acid derivatives suppressed the growth of A549 cells in
dose- and time-dependent manners (data of 24 h not shown). Moreover, compounds
4
b, 4f, 4h and 4j exhibited more significant cytotoxicity and the inhibitory rate was
almost up to 70-80% equivalent to 5-FU. Our finding demonstrated that all tested
derivatives exhibited considerable cell growth inhibition at different concentrations (5
or 10 ꢀM), in which compounds 4b, 4f, 4h and 4j exerted optimal results and the
others modest anti-proliferation efficiency (Fig. 2), thereby IC of the compounds
5
0
were totally less than 10 ꢀM (Table 1). Therefore, we selected 5 and 10 ꢀM
concentration for the following investigation according to the results of cell viability.
Figure 2

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Table 1
4
.2. Structure–activity relationships
Comparing to our previous report [49], we found that the pyrazolyl hydroxamic acid
compounds exhibited more effects on the cell growth inhibition than corresponding
substituted pyrazole-5-carbohydrazide derivatives. Furthermore, the cytotoxic potency
was highly dependent, as expected, on the substitution types and patterns on the aryl
ring. Different substituents at the 4-position of 3-aryl ring can slightly alter the
cytotoxicities against the cancer cells tested. However, replacing the hydrogen at the
4
-position of 1-aryl ring with a bulkier tert-butyl group resulted in a significant
activity increasing, that is, the compounds with tert-butyl group 4b, 4f and 4j were
more effective to inhibit A549 cell growth, which was probably caused by favorable
steric interactions of the bulky hydrophobic alkyl group at 4-position of 1-aryl ring
with the binding site. It is interesting that the SAR is consistent with previous results
[
49].
4
.3. Effects of compounds 4a-4l on chromatin condensation and DNA fragmentation
of A549 cells
It is well known that apoptosis is typeⅠprogrammed cell death with the main

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characteristics of chromatin condensation and DNA fragment [50]. As a major
mechanism of cell death, apoptosis is involved in embryogenesis and response to
environmental stress [51, 52]. Additionally, the dysfunction of apoptosis has a close
relationship with tumorigenesis [53]. Therefore, we performed Hoechst 33258
staining of A549 cells to detect whether the prepared compounds induce cell apoptosis.
The results showed that the morphological changes associated with apoptosis
including chromatin condensation and nuclear fragmentation occurred only in a few
cells, but apoptotic bodies were not observed markedly compared with the control
(Fig. 3). Hence, the observations suggested that the compounds at 10 ꢀM
concentration did not prominently induce apoptosis in A549 cells.
Figure 3
4
.4. Effects of compounds 4a-4l on LDH activity of A549 cells
There is growing evidence that necrosis is an alternative mode of programmed cell
death and occurred when cells are exposed to extreme injury or other stimuli,
provoking an inflammatory response in the surrounding tissue [54]. For this reason,
necrosis is often avoided due to its detrimental effect. To detect whether the
compounds resulted in necrosis of A549 cells, LDH activity in cell culture medium
was measured. The result showed that there were no significant differences in LDH
activity between the control group and those treated with the compounds (10 ꢀM, 48

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h) (p>0.05, n = 3). However, LDH activity was elevated obviously in the culture
medium after treatment with compound 4j, indicating necrosis possibly be induced in
A549 cells by the compound (Fig. 4, p<0.05, n = 3). So the results indicated that it
was impossible for necrosis to be a mechanism of the anti-proliferative effects of most
compounds on A549 cancer cells.
Figure 4
4
.5. Acidic vesicles accumulated in A549 cells treated with compounds 4a-4l
We further examined whether acidic vesicles accumulated in A549 cells by AO
staining assay. Acidic vesicles prominently accumulated in A549 cells after treatment
with the compounds (10 ꢀM) especially 4e, 4f, 4g or 4h for 48 h (Fig. 5). This finding
demonstrated that the tested compounds promoted acidic vesicles accumulation and
autophagy probably occurred in A549 cells.
Figure 5
4
.6. Three compounds 4b, 4f and 4h induced autophagy
The microtubule-associated protein 1 light chain 3 (LC3) plays an essential role in
autophagic vacuole formation by LC3-I conjugation of phosphatidylethanolamine (PE)

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and conversion to LC3-II, the typical characteristic of autophagosome [55]. p62
(sequestosome 1) has been proved to bind directly to LC3A and –B through the LC3
interacting region (LIR). Subsequently, p62- and LC3-positive bodies are degraded by
autophagy. Therefore, p62 may also be used as an autophagic marker to monitor
autophagic flux [56]. According to our results, the majority of compounds led to
acidic vesicle formation implying autophagy occurrence in A549 cells, so LC3 and
p62 protein levels were detected by western blot. Although the compounds 4b, 4f, 4h
and 4j exerted the optimum inhibitory efficiency almost equivalent to 5-FU, it should
be taken in account that 4j obviously elevated LDH activity in the culture medium
indicating its possibility of inducing necrosis and inflammation. So we selected 4b, 4f
and 4h for the following investigation. It was observed that the conversion of LC3-I
to LC3-II increased and there were marked differences in LC3-II protein level and
LC3-II/LC3-I ratio between the control and the treated groups (Fig. 6, n = 3).
Moreover, the p62 level decreased indicating autophagic flux was intact when the
A549 cells were treated by the three compounds (Fig. 7, n = 3).Thus, the
compounds 4b, 4f and 4h effectively triggered autophagosome formation and
promoted autophagic flux in A549 cancer cells, which is consistent with the
observation of AO staining.
Figure 6
Figure 7

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4
.7. Three compounds 4b, 4f and 4h induced cell cycle arrest at G1 phase
In order to further determine whether cell cycle progression varies, the cell cycle
distribution of A549 cells was examined by FCM analysis. Compared to 54.50% cells
at G1 phase of the control group, the percentages of the cells at G1 phase in the
treated groups were 72.80%, 78.73% and 71.91% respectively, followed by the cell
population decrease at S and G2 phases (Fig. 8). Therefore, the results demonstrated
that the cell cycle arrested at G1 phase after incubation with the compounds 4b, 4f
and 4h for 48 h, which was thought to be critical for cell growth inhibition.
Figure 8
5
. Conclusion
In summary, we synthesized a series of novel pyrazolyl hydroxamic acid derivatives
4a-4l) and investigated their biological activities against human lung cancer cell line
(
A549 in vitro. The exciting observations revealed that the compounds 4a-4l (10µM)
remarkably inhibited the proliferation of A549 human lung cancer cells, in which 4b,
4
f, 4h and 4j showed better potencies equivalent to the reference compound 5-FU
10µM). However, the compound 4j may cause inflammation due to the effect of
necrosis. In addition, the compounds 4b, 4f and 4h induced cell cycle arrest at G1
(

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phase and triggered autophagy which might be detrimental to the cancer cells,
although a slight chromatin condensation and DNA fragmentation were observed.
Therefore, cell cycle arrest and autophagy were mainly responsible for the
anti-proliferation action of the three compounds under the stimulatory condition rather
than apoptosis and necrosis. The compounds 4b, 4f and 4h are effective autophagy
inducers and can be used to investigate the molecular mechanisms of autophagy
regulation and potentially applied for future cancer therapy.
6
. Experimental
6
.1. Reagents and apparatus
All reagents were of analytical grade or chemically pure. RPMI-1640 was purchased
from Gibco Co (Carlsbad, CA, USA). Bovine calf serum was obtained from
Hyclone Lab Inc. (USA). DMSO was supplied by Beijing Solaris Science ＆
Technology Company (Beijing, China). Acridine orange (AO) was purchased from
Shandong Chemical Industries (Jinan, China). Analytical TLC was performed on
silica gel 60 F254 plates (Merck KGaA). Melting points were determined on an XD-4
1
digital micromelting point apparatus and not corrected. H NMR spectra were
recorded on a Bruker Avance 300 (300 MHz) spectrometer, using CDCl or DMSO as
3
solvent and tetramethylsilane (TMS) as internal standard. IR spectra were recorded

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with an IR spectrophotometer Avtar 370 FT-IR (Termo Nicolet). HRMS spectra were
recorded on a LTQ Orbitrap Hybrid mass spectrograph.
6
.2. General procedure for the synthesis of pyrazolyl hydroxamic acids (4a-4l)
Hydroxylamine hydrochloride (834 mg, 6 mmol) was added to a solution of Na (460
mg, 20 mmol) in absolute methanol (15 ml) under nitrogen and kept stirring for 1 h.
The appropriate carboxylate (3a-3l, 2 mmol) was added to the prepared solution. The
mixture was heated at reflux for 10 to 30 min. The reaction was diluted with ethyl
acetate (50 ml) and saturated NaHCO solution (100 ml). The organic layer was
3
isolated and the aqueous was further extracted with ethyl acetate. The combined
organic layer was washed with brine, dried with anhydrous sodium sulphate, filtered,
and concentrated under reduced pressure to obtain the crude products. The products
were obtained from recrystallization with ethyl acetate/petroleum ether.
6
.3. Spectroscopy data of compounds 4a-4l
6
.3.1. 1-benzyl-N-hydroxy-3-phenyl-1H-pyrazole-5-carboxamide (4a)
o
Yield: 76.7%; white solid; mp 145-146 C. IR (KBr) ν: 3450 (OH), 3332 (NH), 1650
1
(
C=O). H NMR (DMSO, 400 MHz) δ = 5.76 (s, 2H, -CH -), 7.17-7.21 (m, 3H,
2
pyrazole-H and ArH), 7.26-7.27 (m, 1H, ArH), 7.30-7.35 (m, 3H, ArH), 7.43 (t, J =
.54 Hz, 2H, ArH), 7.76 (dd, J = 7.16, J = 1.2 Hz, 2H, ArH), 9.31 (s, 1H, -OH),
7
1
2

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1
3
1
1.36 (s, 1H, -NH). C NMR (DMSO, 100 M Hz) δ (ppm) = 54.14, 104.37, 125.56
(2C), 127.68 (2C), 127.88, 128.46, 128.92 (2C), 129.30 (2C), 132.84, 135.24, 138.26,
+
1
49.59, 157.82. HRMS: m/z [M+H] calcd for C H N O : 294.1243, found
1
7
16
3
2
2
94.1260.
6
.3.2. 1-(4-(tert-butyl) benzyl)-N-hydroxy-3-phenyl-1H-pyrazole-5-carboxamide (4b)
Yield: 76.8%; white solid; mp 128-129 oC. IR (KBr) ν: 3602 (OH), 3210 (NH), 1661
C=O). 1H NMR (DMSO, 400 MHz) δ = 1.24 (s, 9H, -CH3), 5.71 (s, 2H, -CH2-),
(
7
2
.14-7.16 (m, 3H, ArH and pyrazole-H), 7.32-7.35 (m, 3H, ArH), 7.43 (t, J = 7.58 Hz,
1
3
H, ArH), 7.75 (d, J = 7.32 Hz, 2H, ArH), 9.30 (s, 1H, -OH), 11.34 (s, 1H, -NH). C
NMR (DMSO, 100 M Hz) δ (ppm) = 31.55 (3C), 34.66, 53.77, 104.30, 125.54 (2C),
1
1
3
25.67 (2C), 127.53 (2C), 128.42, 129.29 (2C), 130.80, 132.88, 135.27, 149.51,
+
50.31, 157.63. HRMS: m/z [M+H] calcd for C H N O : 350.1869, found
2
1
24
3
2
50.1862.
6
.3.3. N-hydroxy-1-(4-nitrobenzyl)-3-phenyl-1H-pyrazole-5-carboxamide (4c)
o
Yield: 43.0%; light yellow solid; mp 167-168 C. IR (KBr) ν: 3346 (OH), 3157
1
(
NH)1665 (C=O). H NMR (DMSO, 400 MHz) δ = 5.90 (s, 2H, -CH -), 7.24 (s, 1H,
2
pyrazole-H), 7.35 (t, J = 7.32 Hz, 1H, ArH), 7.40-7.46 (m, 4H, ArH), 7.77 (d, J = 7.32
Hz, 2H, ArH), 8.20 (d, J = 8.68 Hz, 2H, ArH), 9.32 (s, 1H, -OH), 11.41 (s, 1H, -NH).
1
3
C NMR (DMSO, 100 M Hz) δ (ppm) = 53.83, 104.52, 124.16 (2C), 125.63 (2C),

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1
28.62 (3C), 129.33 (2C), 132.64, 135.36, 145.94, 147.29, 150.09, 157.55. HRMS
+
m/z [M+H] calcd for C H N O : 339.1093, found 339.1082.
1
7
15
4
4
6
.3.4. 1-((6-chloropyridin-3-yl)methyl)-N-hydroxy-3-phenyl-1H-pyrazole-5-carboxa
mide (4d)
o
Yield: 49.1%; white solid; mp 185-186 C. IR (KBr) ν: 3149 (OH + NH), 1657 (C=O).
1
H NMR (DMSO, 400 MHz) δ = 5.78 (s, 2H, -CH -), 7.20 (s, 1H, pyrazole-H), 7.35 (t,
2
1
H, J = 7.32 Hz, ArH), 7.44 (t, 2H, J = 7.58 Hz, ArH), 7.50 (d, 1H, J = 8.28 Hz,
pyridine-H), 7.69 (dd, 1H, J = 8.28, J = 2.44 Hz, pyridine-H), 7.76 (d, 2H, J = 7.52
1
2
Hz, ArH), 8.34 (d, 1H, J = 2.28 Hz, pyridine-H), 9.34 (s, 1H, -OH), 11.40 (s, 1H,
1
3
-
NH). C NMR (DMSO, 100 M Hz) δ (ppm) = 51.26, 104.52, 124.73, 125.62 (2C),
1
28.61, 129.32 (2C), 132.63, 133.34, 135.21, 139.38, 149.41, 149.93, 150.05, 157.61.
+
HRMS: m/z [M+H] calcd for C H ClN O : 329.0805, found 329.0820.
1
6
14
4
2
6
.3.5. 1-benzyl-3-(4-chlorophenyl)-N-hydroxy-1H-pyrazole-5-carboxamide (4e)
o
Yield: 82.0%; white solid; mp 174-175 C. IR (KBr) ν: 3250 (OH + NH), 1642 (C=O).
1
H NMR (DMSO, 400 MHz) δ = 5.75 (s, 2H, -CH -), 7.18-7.21 (m, 3H, ArH
2
andpyrazole-H), 7.26 (t, J = 7.22 Hz, 1H, ArH), 7.32 (t, J = 7.18 Hz, 2H, ArH), 7.49
(d, J = 8.48 Hz, 2H, ArH), 7.77 (d, J = 8.52 Hz, 2H, ArH), 9.33 (s, 1H, -OH), 11.33 (s,
1
3
1
1
H, -NH). C NMR (DMSO, 100 M Hz) δ (ppm) = 54.19, 104.52, 127.26 (2C),
27.73 (2C), 127.92, 128.93 (2C), 129.36 (2C), 131.74, 132.93, 135.54, 138.12,

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+
1
3
48.47, 157.69. HRMS: m/z [M+H] calcd for C H ClN O : 328.0853, found
17
15
3
2
28.0854.
6
.3.6. 1-(4-(tert-butyl)benzyl)-3-(4-chlorophenyl)-N-hydroxy-1H-pyrazole-5-carboxa
mide (4f)
o
Yield: 74.5%; white solid; mp 101-103 C. IR (KBr) ν: 3623 (OH), 3451 (NH), 1671
1
(
C=O). H NMR (DMSO, 400 MHz) δ = 1.24 (s, 9H, -CH ), 5.70 (s, 2H, -CH -), 7.15
3
2
(m, 3H, ArH and pyrazole-H), 7.33 (d, J = 8.32 Hz, 2H, ArH), 7.49 (d, J = 8.52 hz,
1
3
2
H, ArH), 7.77 (d, J = 8.52 Hz, 2H, ArH), 9.33 (s, 1H, -OH), 11.34 (s, 1H, -NH). C
NMR (DMSO, 100 M Hz) δ (ppm) = 31.54 (3C), 34.69, 53.83, 104.49, 125.70 (2C),
1
1
3
27.25 (2C), 127.57 (2C), 129.36 (2C), 131.75, 132.91, 135.11, 135.37, 148.39,
+
50.37, 157.74. HRMS: m/z [M+H] calcd for C H ClN O : 384.1479, found
21
23
3
2
84.1473.
6
.3.7. 3-(4-chlorophenyl)-N-hydroxy-1-(4-nitrobenzyl)-1H-pyrazole-5-carboxamide
4g)
(
o
Yield: 68.0%; white solid; mp 207-208 C. IR (KBr) ν: 3334 (OH), 3147 (NH), 1631
1
(
C=O). H NMR (DMSO, 400 MHz) δ = 5.90 (s, 2H, -CH -), 7.26 (s, 1H, pyrazole-H),
2
7
.41 (d, J = 8.80 Hz, 2H, ArH), 7.50 (dt, J = 10.88, J = 1.76 Hz, 2H, ArH), 7.78 (d,
1 2
J = 8.52 Hz, 2H, ArH), 8.20 (dt, J = 8.76, J = 2.08 Hz, 2H, ArH), 9.36 (s, 1H, -OH),
1
2
1
3
1
1.44 (s, 1H, -NH). C NMR (DMSO, 100 M Hz) δ (ppm) = 53.86, 104.67, 124.18
(2C), 127.33 (2C), 128.67 (2C), 129.40 (2C), 131.52, 133.12, 135.59, 145.77, 147.32,

ACCEPTED MANUSCRIPT
+
1
3
48.96, 157.47. HRMS: m/z [M+H] calcd for C H ClN O : 373.0704, found
17
14
4
4
73.0692.
6
.3.8. 3-(4-chlorophenyl)-1-((6-chloropyridin-3-yl) methyl)-N-hydroxy-1H-pyrazole
5-carboxamide (4h)
-
o
Yield: 67.1%; white solid; mp 218-219 C. IR (KBr) ν: 3155 (OH + NH), 1655 (C=O).
1
H NMR (DMSO, 400 MHz) δ = 5.77 (s, 2H, -CH -), 7.21 (s, 1H, pyrazole-H),
2
7
.49-7.51 (m, 3H, ArH and pyridine-H), 7.69 (dd, J = 8.28, J = 2.48 Hz, 1H,
1 2
pyridine-H), 7.78 (d, J = 8.52 Hz, 2H, ArH), 8.34 (d, J = 2.28 hz, 1H, pyridine-H),
1
3
9
1
1
3
.38 (s, 1H, -OH), 11.43 (s, 1H, -NH). C NMR (DMSO, 100 M Hz) δ (ppm) = 51.31,
04.67, 124.74, 127.33 (2C), 129.38 (2C), 131.52, 133.12, 133.20, 135.44, 139.42,
+
48.92, 149.46, 149.97, 157.65. HRMS: m/z [M+H] calcd for C H Cl N O :
16
13
2
4
2
63.0416, found 363.0422.
6
.3.9. 1-benzyl-N-hydroxy-3-(4-methoxyphenyl)-1H-pyrazole-5-carboxamide (4i)
o
Yield: 55.8%; white solid; mp 133-135 C. IR (KBr) ν: 3609 (OH), 3302 (NH), 1661
1
(
C=O). H NMR (DMSO, 400 MHz) δ = 3.78 (s, 3H, -CH ), 5.73 (s, 2H, -CH -), 6.99
3
2
(d, J = 8.8 Hz, 2H, ArH), 7.08 (s, 1H, pyrazole-H), 7.19 (d, J = 7.00 Hz, 2H, ArH),
7
.25 (t, J = 7.12 Hz, 1H, ArH), 7.31 (t, J = 7.20 Hz, 2H, ArH), 7.68 (d, J = 8.76 Hz,
1
3
2
H, ArH), 9.28 (s, 1H, -OH), 11.31 (s, 1H, -NH). C NMR (DMSO, 100 M Hz) δ
(ppm) = 54.00, 55.61, 103.76, 114.69 (2C), 125.49, 126.88 (2C), 127.65 (2C), 127.83,

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+
1
28.90 (2C), 135.09, 138.37, 149.51, 157.89, 159.61. HRMS: m/z [M+H] calcd for
C H N O : 324.1348, found 324.1342.
18
18
3
3
6
.3.10. 1-(4-(tert-butyl)benzyl)-N-hydroxy-3-(4-methoxyphenyl)-1H-pyrazole-5-carbo
xamide (4j)
o
Yield: 81.0%; white solid; mp 88-89 C. IR (KBr) ν: 3312 (OH), 3145 (NH), 1668
1
(
C=O). H NMR (DMSO, 400 MHz) δ = 1.24 (s, 9H, -CH ), 3.78 (s, 3H, -OCH ),
3
3
5
.68 (s, 2H, -CH -), 6.97-6.98 (m, 2H, ArH), 6.99-7.00 (m, 1H, ArH), 7.06 (s, 1H,
2
pyrazole-H), 7.14 (d, J = 8.32 Hz, 2H, ArH), 7.32-7.34 (m, 2H, ArH), 7.66-7.69 (m,
1
3
2
H, ArH), 9.28 (s, 1H, -OH), 11.31 (s, 1H, -NH). C NMR (DMSO, 100 M Hz) δ
(ppm) = 31.54 (3C), 34.64, 49.07, 53.66, 103.70, 114.67 (2C), 125.56, 125.64 (2C),
1
26.87 (2C), 127.52 (2C), 135.03, 135.39, 149.45, 150.26, 157.91, 159.95. HRMS:
+
m/z [M+H] calcd for C H N O : 380.1974, found 380.1974.
2
2
26
3
3
6
.3.11. N-hydroxy-3-(4-methoxyphenyl)-1-(4-nitrobenzyl)-1H-pyrazole-5-carboxamid
e (4k)
o
Yield: 50.8%; white solid; mp 173-174 C. IR (KBr) ν: 3371 (OH), 3137 (NH), 1683
1
(
C=O). H NMR (DMSO, 400 MHz) δ = 3.78 (s, 3H, -OCH ), 5.88 (s, 2H, -CH -),
3
2
7
.00 (dt, J = 8.84, J = 2.44 Hz, 2H, ArH), 7.15 (s, 1H, pyrazole-H), 7.39 (d, J = 8.72
1 2
Hz, 2H, ArH), 7.69 (dt, J = 8.76, J = 1.60 Hz, 2H, ArH), 8.20 (dt, J = 11.08, J =
1
2
1
2
1
3
1
.80 Hz, 2H, ArH), 9.31 (s, 1H, -OH), 11.39 (s, 1H, -NH). C NMR (DMSO, 100 M
Hz) δ (ppm) = 53.70, 55.62, 103.93, 114.72 (2C), 124.15 (2C), 125.29, 126.97 (2C),

ACCEPTED MANUSCRIPT
+
1
28.59 (2C), 135.21, 146.08, 147.26, 150.01, 157.63, 159.73. HRMS: m/z [M+H]
calcd for C H N O : 369.1199, found 369.1212.
1
8
17
4
5
6
.3.12. 1-((6-chloropyridin-3-yl)methyl)-N-hydroxy-3-(4-methoxyphenyl)-1H-pyrazole
5-carboxamide (4l)
-
o
Yield: 73.4%; white solid; mp 209-210 C. IR (KBr) ν: 3449 (OH), 3167 (NH), 1649
1
(
C=O). H NMR (DMSO, 400 MHz) δ = 3.78 (s, 3H, -OCH ), 7.00 (d, J = 8.8 Hz, 2H,
3
ArH), 7.10 (s, 1H, pyrazole-H), 7.50 (d, J = 8.24 Hz, 1H, pyridine-H), 7.66-7.69 (m,
3
H, ArH and pyridine-H), 8.32 (d, J = 2.28 Hz, 1H, pyridine-H), 9.33 (s, 1H, -OH),
1
3
1
1.38 (s, 1H, -NH). C NMR (DMSO, 100 M Hz) δ (ppm) = 51.13, 55.61, 103.93,
1
14.71 (2C), 124.71, 125.28, 126.97 (2C), 133.44, 135.06, 139.35, 149.38, 149.88,
+
1
49.98, 157.70, 159.72. HRMS: m/z [M+H] calcd for C H ClN O : 359.0911,
1
7
16
4
3
found 359.0893.
6
.4. X-ray Crystallography
Suitable single crystals of 4l for X-ray structural analysis were obtained by slow
evaporation of a solution of the solid in ethyl acetate at room temperature for 10 days.
The crystals with approximate dimensions of 0.08 mm × 0.10 mm × 0.08 mm for 4l
were mounted on a Bruker Smart Apex II CCD equipped with a graphite
monochromated MoKα radiation (λ = 0.71073 Ǻ) by using ϕ and ω scan modes and
the data were collected at 298(2) K. The structure of the crystal was solved by direct

ACCEPTED MANUSCRIPT
methods and refined by full-matrix least-squares techniques implemented in the
SHELXTL-97 crystallographic software [57]. The non-hydrogen atoms were refined
anisotropically. The hydrogen atoms bound to carbon were located by geometrical
calculations, with their position and thermal parameters being fixed during the
structure refinement. Molecular graphics were designed by using XP and DIAMOND
3
.2.
6
.5. Cell culture
Human lung cancer cell line A549 was cultured in RPMI-1640 medium supplemented
with 10% (v/v) bovine calf serum and 80 U/mL penicillin/streptomycin at 37 °C in 5%
CO Cells were seeded in 96-well plates or the indicated dishes at a density of 6250
2
.
-2
cells·cm for 24 h. Thereafter, the culture medium was changed into RPMI-1640
containing 1% bovine calf serum when treated with the compounds.
6
.6. Cell viability assay
Cells were plated in 96-well plates for 24 h and then treated with 0.1% DMSO (v/v, as
control), 5-fluorouracil (5-FU, as positive group) and the compounds (4a-4l) at
indicated concentrations (1, 5 and 10 µM) for 24 h and 48 h, respectively. Cell
viability was determined by Sulforhodamine B (SRB) assay as previously described

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[
58]. The light absorption was measured at λ = 540 nm using Tecan Infinite 200 PRO
microplate spectrophotometer (Tecan Austria GmbH).
6
.7. Hoechst 33258 staining
Cells were seeded into 24-well plates for 24 h before treated with 0.1% DMSO (as
control) or the compounds (4a-4l, 10 µM) for 48 h. Cells were washed with PBS and
fixed with 4% paraformaldehyde for 10 min at room temperature. Then cells were
-1
stained with Hoechst 33258 (2 ꢀg·mL ) for 20 min and photographed using an
Olympus BH-2 fluorescence microscope (Japan).
6
.8. LDH assay
Cell culture medium was collected after treatment with 0.1% DMSO (as control) or
the compounds (4a-4l) at the concentration of 10 µM for 48 h. The LDH assay was
performed using a Lactate Dehydrogenase (LDH) kit (Nanjing Jiancheng Co, China)
according to the manufacturer’s protocol.
6
.9. Acridine orange (AO) staining

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Cells were incubated with the compounds (4a-4l) at 10 µM for 48 h and stained with
-1
acridine orange (100 ꢀg·mL ) for 1 min at room temperature. The cells were
observed and photographed using a fluorescence microscope.
6
.10. Western blot analysis
The western blot procedure was performed as described previously [59]. Cells were
washed twice with PBS and lysed with 100 ꢀL lysis buffer (1% SDS in 25 mM
-1
Tris-HCl, pH 7.5, 4 mM EDTA, 100 mM NaCl, 1 mM PMSF, 10 mg·mL leupeptin
-1
and 10 mg·mL soybean trypsin inhibitor). Whole cell lysates were centrifuged at
1
2000×g for 15 min. Thereafter, the protein concentration was determined with
bicinchonininc acid (BCA) protein assay kit (Beyotime Co, China). The samples were
subjected to SDS-PAGE at 4°C for 2 h and transferred to PVDF membranes. After
blocked with 5% nonfat dry milk in PBS-t for 1 h at room temperature, the
membranes were incubated with anti-LC3 (Cell Signalling, Beverly, MA, USA) and
anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies overnight at
4
°C and then with HRP-conjugated secondary antibodies for 1 h at room temperature.
Proteins were detected by ECL procedures (Thermo Fisher Scientific, USA) and
analysed by ImageJ software.
6
.11. Flow cytometric (FCM) analysis of cell cycle distribution

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Briefly, after incubation with the compounds 4b, 4f and 4h (10 µM) for 48 h, both
adherent and floating cells were harvested, fixed in cold 70% (v/v) ethanol overnight
-1
at -20°C and then stained with a fluorescent dye propidium iodide (PI, 50 ꢀg·mL )
-1
containing 10 µg·mL RNase A for 1 h at 4 °C. Flow cytometric measurements and
DNA contents of cells were analysed using a FACS Calibur flow cytometry (Becton
Dickinson, USA). Cell cycle distribution was calculated by Modifit program.
6
.12. Statistical analysis
Data were presented as means ± SE from at least three separate experiments.
Statistical comparison was performed by Student’s t-test. Differences were considered
statistically significant when p-value was <0.05.
Acknowledgements
This study was supported by National Natural Science Foundation of China
(91313303 and 90813022).
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Legend of Figures
Figure 1 Crystal structure of compound 4l (CCDC 986431)
Figure 2 The viability of A549 cancer cells treated with the compounds (4a-4l
)
for 48 h. Cells were treated with the compounds (4a-4l) at 1ꢀM, 5ꢀM and 10
M for 48 h. The cytotoxicity against the A549 cells was conducted using the
SRB assay. 5-FU was used as positive control. Data are mean ± SEM. * <0.05,
<0.01 vs. control (Ctr), n = 3.
ꢀ
p
** p
Figure 3 Effects of the compounds (4a-4l) on chromatin condensation and DNA
fragmentation of A549 cells. Hoechst 33258 staining was performed after cells were
treated with the compounds (4a-4l) at 10 ꢀM for 48 h (magnification 200×). The
compounds at 10 ꢀM did not prominently induce apoptosis.
Figure 4 Effects of the compounds (4a-4l) on LDH activity of A549 cells. After
incubation with the compounds (4a-4l, 10 ꢀM) for 48 h, the cell culture medium was
collected. Data are mean ± SEM. * p<0.05 vs. control (Ctr), n = 3.
Figure 5 Acidic vesicles accumulated in A549 cells treated with the compounds
(4a-4l). Cells were subjected to 10 ꢀM compounds (4a-4l) for 48 h and acidic vesicles
were examined by AO staining assay (magnification 200×). The tested compounds