Design, synthesis and biological evaluation of novel (E)-N-phenyl-4-(pyridine-acylhydrazone) benzamide derivatives as potential antitumor agents for the treatment of multiple myeloma (MM)

Article abstract of DOI:10.1016/j.bioorg.2020.104189

A series of novel (E)-N-phenyl-4-(pyridine-acylhydrazone) benzamide derivatives were designed, synthesized, and evaluated for their anti-proliferative activity against two different human cancer cell lines and one human normal cell line. Compound 8b had t

Full text of DOI:10.1016/j.bioorg.2020.104189

Bioorganic Chemistry 103 (2020) 104189
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of novel (E)-N-phenyl-4-
(
pyridine-acylhydrazone) benzamide derivatives as potential antitumor
agents for the treatment of multiple myeloma (MM)
a,b,1
a
1,a
a
a
a
a
Xin-yang Li
, Shuai Li , Guo-qing Lu , De-pu Wang , Kai-li Liu , Xin-hua Qian ,
a,⁎
Wen-han Xue , Fan-hao Meng
a
b
School of Pharmacy, China Medical University, 77 Puhe Road, North New Area, Shenyang 110122, China
Department of Pharmacy, Shengjing Hospital of China Medical University, Shenyang 110122, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel (E)-N-phenyl-4-(pyridine-acylhydrazone) benzamide derivatives were designed, synthesized,
and evaluated for their anti-proliferative activity against two different human cancer cell lines and one human
normal cell line. Compound 8b had the best anti-proliferative activity (IC50 = 0.12 ± 0.09 μM, RPMI8226
cells) than the other compounds. And compound 8b had lower toxicity than imatinib. Flow cytometry analysis
showed that compound 8b could arrest the cell cycle at the G0/G1 phase, and induce apoptosis of RPMI8226
cells by promoting mitochondrial ROS release, thereby effectively inhibiting cell proliferation. Our findings
provided a promising lead compound 8b for further structural optimization and will be instructive for the
discovery of more potent antitumor drugs with high selectivity and low toxicity.
Antitumor
Multiple myeloma (MM)
Apoptosis
1
. Introduction
Multiple myeloma (MM) is a malignancy of terminally differ-
common hematological malignancy, as well as gastrointestinal stromal
tumors (GISTs). It should be pointed out that recent clinical investiga-
tions provided evidence that imatinib could effectively inhibit multiple
myeloma cell proliferation and potentiates the effect of common anti-
myeloma agents [4–6]. At the same time, sorafenib, as an effective
antitumor drug, has a similar structure to imatinib. They all possess an
aromatic-heterocyclic pyridine structure, and a diphenyl skeleton
linked by a bridge group of amide or urea group (Fig. 1).
entiated plasma cells characterized by aberrant proliferation of neo-
plastic plasma cells that secrete monoclonal immunoglobulins [1].
Multiple myeloma represents a cancer type that is the second most
common haematological malignancy, accounting for approximately 1%
of cancers worldwide. Despite the clinical advances of patients with
multiple myeloma has improved considerably in the past decades,
particularly the introduction of immunomodulatory drugs (Thalido-
mide and Lenalidomide) and proteasome inhibitors (Bortezomib) has
revolutionized the therapeutic management [2]. Unfortunately, there is
no cure for patients with multiple myeloma even after therapy with
stem cell transplantation. The vast majority of patients eventually de-
velop almost-inevitable resistance and relapse [3]. Therefore, the dis-
covery of novel drugs with a safer profile remains an urgent need in
clinical multiple myeloma management.
Molecular hybridization of multiple pharmacophores for the de-
velopment of a single molecule with enhanced biological activities has
become an attractive area in medicinal chemistry [7]. It was of interest
to combine pyridine core with N-phenyl benzamide fragment through a
linker bridge to develop more potent antitumor agents. As an essential
class of biologically privileged structure, N-acylhydrazone scaffold
(-CO-NH-N = CH-) has been one of the most ubiquitous pharmaco-
phores in medicinal chemistry due to their wide spectrum of biological
properties such as anti-inflammatory [8], antimicrobial [9], antifungal
[10], antiviral [11] and anticancer [12,13] activities. It’s worthy of
attention that some acylhydrazone derivatives have been highlighted as
potential antitumor agents [14–16]. The versatility of N-acylhy-
drazones in medicinal chemistry is based on their ease of synthesis, as
In the course of identifying various chemical fragments that could
serve as a scaffold for inhibiting multiple myeloma cell proliferation,
our programs began with the first-generation Bcr-Abl tyrosine-kinase
(
TK) inhibitor, imatinib (Gleevec, STI571). It was approved for clinical
treatment of most cases of chronic myeloid leukemia (CML), another
they are usually produced by a condensation reaction between
⁎
Corresponding author.
E-mail address: fhmeng@cmu.edu.cn (F.-h. Meng).
1
These two authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.104189
Received 16 April 2020; Received in revised form 6 July 2020; Accepted 8 August 2020
Available online 26 August 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.

X.-y. Li, et al.
Bioorganic Chemistry 103 (2020) 104189
Fig. 1. Structures of imatinib, sorafenib and target compounds.
1
aldehydes or ketones with hydrazides [17].
The H NMR spectra of all target compounds 7a-9g characterized by
the presence of a single signal at δ 12.15–12.30 ppm (1H) corre-
sponding to the proton of amide (-CO-NH-) on the N-acylhydrazone and
a singlet signal at δ 10.22–10.71 ppm (1H) corresponding to the proton
of amide (-CO-NH-). Moreover, a characteristic singlet signal was de-
tected at about δ 8.52–8.74 ppm (1H) corresponding to the proton of
imino (-N = CH-), which is evidence for the formation of N-acylhy-
Considering the above impressive biological properties of N-acyl-
hydrazone scaffold, aromatic-heterocyclic pyridine, and diphenyl
amide moiety, we are interested to know the combined effect of these
moieties in a single molecular framework. Hence, we would like to
synthesize these hybrid molecules by combining the pyridine ring with
the diphenyl amide moiety through an N-acylhydrazone bridge. In this
study, a series of (E)-N-phenyl-4-(pyridine-acylhydrazone) benzamide
derivatives were synthesized and reported for the first time. At the same
time, there in vitro antitumor activity against different cells and cyto-
toxicity was evaluated. The ultimate goal was to identify novel and
more effective antitumor drugs with excellent bioavailability and low
toxicity.
1
3
drazone scaffold. Next, the analysis of C NMR depicts the presence of
carbonyl carbon (C]O) of N-acylhydrazone and amide at
δ
164.94–165.80 ppm and 161.05–162.38 ppm, respectively.
Because of the imino bond (-N = CH-) of N-acylhydrazone, all
target compounds could exist as the E or Z isomer or a mixture of both.
To confirm the stereochemistry of the obtained isomer, compound 8b
was further selected to undergo NOESY experiments. The results
(Fig. 2) showed that an evident NOE signal was observed between the
2
. Results and discussion
H
1
(-N = CH-, δ = 8.78 ppm) and H (-NH-N=, δ = 12.16 ppm) in the
2
2.1. Chemistry
E isomer (E-8b), which should not be observed in the putative Z isomer
due to the more considerable intramolecular HeH distance (Z-8b).
Thus, the target compounds 7a-9g were unequivocally confirmed as the
E isomer [20].
The preparation of target compounds 7a-9g was illustrated in
Scheme 1. The commercially available material 4-formylbenzoic acid
reacted with thionyl chloride (SOCl
2
) in chloroform (CHCl ) at 55 °C for
3
4
–6 h in the presence of the catalytic amount of DMF to provide 4-
2.2. Biological evaluation
formylbenzoyl chloride 2, which were then used directly in the next
step. Subsequently, the key intermediates 3a-3g were generated
through the reaction of 4-formylbenzoyl chloride 2 with various ani-
2.2.1. MTT assay and SAR analysis.
In order to verify the biological activity of all the synthesized
compounds 7a-9g against multiple myeloma cells, these compounds
were evaluated by in vitro anti-proliferative assays against two human
multiple myeloma cell lines: U266 and RPMI8226 by the modified MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay
using imatinib as a positive control. The preliminary experimental re-
sults were summarized in Table 1.
lines in dichloromethane (CH
2
Cl
2
) using triethylamine (Et N) as a base
3
at room temperature for overnight [18] and were further purified by
recrystallization with ethyl acetate. Finally, the target compounds 7a-
9
g were successfully obtained via the reaction of intermediates 3a-3g
and corresponding picolinohydrazides (4, 5 and 6) in the presence of
the catalytic amount of acetic acid in ethanol under reflux condition for
3
–6 h in a satisfactory yield (71–86%) according to the reported
From the results of the MTT assay, we found that compound 8b had
the best anti-proliferative activity (IC50 = 0.12 ± 0.09 μM) than the
other compounds, which was excellent for RPMI8226 cells. When R was
method [19]. The experimental and post-treatment methods had de-
scribed explicitly in the general methods.
2

X.-y. Li, et al.
Bioorganic Chemistry 103 (2020) 104189
Scheme 1. Reagents and conditions: (a) SOCl
2
, CHCl
3
, DMF, 55 °C, 4–6 h; (b) appropriate anilines, Et
3
N, CH
2
Cl
2
, 39 °C, 12 h, 65%-81%; (c) EtOH, CH COOH, 80 °C,
3
3
–6 h, 70%-86%.
replaced by H, compound 8a had better anti-proliferative activity than
compound 7a and compound 9a. When R was replaced by -CH or
literature reports, most anticancer drugs could achieve anti-pro-
liferative effects by cell cycle arrest [21,22]. To further investigate
whether the inhibitory effect of the compound 8b on cell viability was
associated with cell cycle, we used flow cytometry to analyze the cell
cycle. RPMI8226 cells were treated with different concentrations of the
compound 8b (0, 0.05, 0.10, 0.20 μM) for 24 h, stained with propidium
iodide (PI), and then analyzed for cell cycle distribution by flow cyto-
metry. The results of flow cytometry showed that with the increase of
concentration, the percentage of the cells of the G0/G1 phase increased
significantly, and the S phase and G2/M phase decreased significantly
(Fig. 3. B).
3
-
OCH , compound 8b or compound 8c had better anti-proliferative
3
activity than others in the target compounds 8a-8g. When R was re-
placed by F, Cl, or Br, the anti-proliferative activity of compounds 8d-8f
was better than that of compounds 7d-7f and 9d-9f. At the same time,
we found that when R was replaced by 4-Cl-3-CF , the effect of the
3
compounds on RPMI8226 cells was significantly better than that of
U266 cells.
Besides, to further verify the safety of the target compounds 7a-9g,
we tested these compounds on human umbilical vein endothelial cells
(
HUVEC) in comparison with the reference drug imatinib. This is also
These results implied that compound 8b could cause cancer cell
cycle arrest in the G0/G1 phase, thereby inhibiting the proliferation of
RPMI8226 cells. The result is significantly different (P < 0.001).
shown in Table 1. As presented in Table 1, we found that the IC50 values
of all target compounds are higher than imatinib for HUVEC cells. The
results showed that the target compounds were safer and less toxic than
imatinib.
2
.2.3. Compound 8b could induce apoptosis to further inhibit cell
proliferation.
2
.2.2. Compound 8b could arrest cell cycle in the G0/G1phase.
Apoptosis is the process of programmed cell death that may occur in
MTT assay results showed that compound 8b had the most potent
multicellular organisms. Apoptosis plays a pivotal role in the patho-
genesis of many diseases, such as cancer. Apoptosis is an essential
anti-proliferative activity on RPMI8226 cells. And according to related
Fig. 2. NOESY of the representative compound 8b.
3

X.-y. Li, et al.
Bioorganic Chemistry 103 (2020) 104189
Table 1
phenyl-4-(pyridine-acylhydrazone) benzamide derivatives by a facile
method. Furthermore, these compounds were screened for anti-pro-
liferative activity against U266 and RPMI8226 cell lines by using MTT
assay. We found that compounds 8b exhibited excellent anti-pro-
liferative activity for RPMI8226 cells. And, compound 8b had lower
toxicity than imatinib. Flow cytometry analysis showed that compound
IC50 value of compounds and imatinib against U266, RPMI8226, and HUVEC
cell lines.
IC50a
Compound
U266
RPMI8226
HUVEC
8
b could obviously arrest the cell cycle of the G0/G1 phase, and induce
7
7
7
7
7
7
7
8
8
8
8
8
8
8
9
9
9
9
9
9
9
a
b
c
d
e
f
7.88 ± 0.96
4.64 ± 0.57
14.22 ± 0.59
9.80 ± 0.70
–
3.41 ± 0.62
18.06 ± 0.81
7.26 ± 0.87
6.60 ± 0.72
4.16 ± 0.73
4.22 ± 0.52
0.37 ± 0.10
0.96 ± 0.10
0.12 ± 0.09
2.01 ± 0.52
2.83 ± 0.79
4.98 ± 0.27
5.19 ± 0.51
6.45 ± 0.32
6.50 ± 0.95
13.42 ± 0.37
3.82 ± 0.89
–
> 50
the death of RPMI8226 cells by promoting the release of ROS from the
mitochondria of RPMI8226 cells, and finally achieve the anti-tumor
effect. From the design strategy of compounds, we knew that target
compounds were highly similar to imatinib and sorafenib. Therefore,
we preliminarily speculate that compound 8b is also a multi-target
kinase inhibitor, especially has a significant inhibitory effect on c-Kit.
At the same time, we are in the process of verifying the target of
compound 8b and in vivo biological evaluation experiments.
8.86 ± 0.95
> 50
> 50
> 50
13.96 ± 0.90
5.06 ± 0.89
4.17 ± 0.85
3.81 ± 0.74
5.85 ± 0.85
5.68 ± 0.76
4.71 ± 0.55
4.20 ± 0.57
5.78 ± 0.18
4.96 ± 0.34
6.41 ± 0.76
–
45.53 ± 5.67
24.54 ± 0.85
10.43 ± 1.06
12.09 ± 2.47
> 50
g
a
b
c
d
e
f
> 50
Taken together, this study provided a good starting point for the
development of novel potent anticancer agents with high selectivity and
low toxicity. Also, this study provided a good basis for later in vivo
evaluation experiments and target determination of compound 8b.
> 50
> 50
g
a
b
c
d
e
f
> 50
> 50
> 50
13.65 ± 2.57
> 50
16.44 ± 1.33
6.42 ± 0.56
6.86 ± 0.85
4.63 ± 0.70
1.73 ± 0.43
4.75 ± 0.15
3.87 ± 0.93
2.76 ± 0.46
1.09 ± 0.13
> 50
4. Experimental procedures
> 50
g
> 50
4.1. General methods
Imatinib
8.16 ± 1.63
a
All chemical reagents and solvents were purchased from commer-
IC50 values were used to express inhibition effect (IC50 = Mean ± SD).
cial sources, unless specified otherwise, and used without further pur-
ification.
From MTT assay after 24 h of treatment; the values were average from at least 3
independent experiments.
Melting points were determined on a capillary electrothermal
melting point apparatus and are uncorrected. Reaction progress was
monitored by thin-layer chromatography (TLC) using GF254 silica gel
from Qingdao Ocean Chemicals (Qingdao, Shandong, China) with
fluorescent indicator on glass plates at 254 nm and visualized by UV
pathway for cell death and is critical for maintaining cell homeostasis.
Induction of apoptosis is an essential strategy for tumor suppression.
Many studies have shown that when anticancer drugs cause cell cycle
arrest, it will further trigger the process of apoptosis [23,24]. To verify
that whether compound 8b eventually caused apoptosis when causing
cycle arrest, flow cytometry analysis was performed.
1
13
light. H NMR and C NMR spectra were recorded at ambient tem-
1
13
perature on a Bruker AMX500 ( H at 600 MHz and C at 150 MHz)
magnetic resonance spectrometer. All NMR spectra were recorded using
Flow cytometry showed that the apoptotic rate increased as the
concentration of the compound 8b increased (Fig. 4). And, the early
apoptotic rate and the late apoptotic rate up-regulated significantly.
The trend of early apoptotic rate was more significant than the late
apoptotic rate.
DMSO‑d as the solvent, and chemical shifts are reported in ppm (parts
6
per million) relative to tetramethyl silane (TMS) as an internal stan-
dard. The high-resolution mass spectra (HRMS) were measured by
Bruker micro TOF-Q instrument.
This result indicated that the compound 8b was able to finally in-
hibit cell proliferation by inducing apoptosis in RPMI8226 cells. The
result is significantly different (P < 0.001).
4.1.1. General procedure for the synthesis of the key intermediates (3a-3g)
To a solution of 4-formylbenzoic acid (3 g, 19.98 mmol) in
chloroform (CHCl
3
) (100 mL) were added thionyl chloride (SOCl )
2
2
.2.4. Compound 8b induced ROS generation in RPMI8226 cells.
(
3.57 g, 29.97 mmol) and a catalytic amount of DMF. The reaction
In the above study, it was detected by flow cytometry that com-
mixture was stirred at 55 °C for 5–7 h. After completion of the reaction
as determined by TLC, the solvent was evaporated in vacuo to afford 4-
formylbenzoyl chloride (2), as pale-yellow oil, which was used directly
in the next step. Next, to a solution of different substituted anilines
pound 8b could cause cell cycle arrest and induce apoptosis. Many
studies have shown that reactive oxygen species (ROS), namely oxygen
free radicals and their derivatives, are closely related to apoptosis
[
25,26]. ROS are mainly produced in mitochondria. When mitochon-
(
19.98 mmol) and triethylamine (Et
Cl
mylbenzoyl chloride (2) in CH
3
N) (29.97 mmol) in di-
drial ROS is released in large amounts, it will cause intrinsic apoptosis
of cells.
chloromethane (CH
2
2
)
(100 mL) were added dropwise 4-for-
Cl (20 mL) in an ice bath. Then the
2
2
In this study, we used DCFH-DA as a probe to detect changes in
intracellular ROS content. As shown in Fig. 5, after compound 8b
treatment, as the compound concentration increased, ROS production
in RPMI8226 cells increased. And compared with the control group, the
DCF fluorescence in the compound 8b treatment group increased sig-
nificantly.
reaction mixture was stirred at 39 °C for 12 h. After completion of the
reaction as determined by TLC, the solvent was evaporated in a va-
cuum. The residue was dissolved in methanol (30 mL) and poured into
water (150 mL) followed by adding hydrochloric acid (37%) to adjust
to pH = 5–6. The resulting mixture was extracted with ethyl acetate
(
50 mL × 3). The combined organic layers were washed with brine,
These results indicated that compound 8b could induce apoptosis by
promoting RPMI8226 cell release of ROS. The results were significantly
different (P < 0.001).
dried over sodium sulfate anhydrous, and concentrated in vacuo to give
crude products. A little ethyl acetate solution was added to the crude
product of compound 3a-3g, and the temperature was slowly raised to
5
5 °C. After the crude product was completely dissolved, the reaction
3
. Conclusions
flask was placed at room temperature to precipitate compound 3a-3g of
pure product. At this time, all impurities will be dissolved in ethyl
acetate.
In summary, we had successfully synthesized a series of novel (E)-N-
4

X.-y. Li, et al.
Bioorganic Chemistry 103 (2020) 104189
Fig. 3. Compound 8b could cause cell cycle arrest in RPMI8226 cells. (A): Effect of the compound 8b on cell cycle progression of RPMI8226 cells was evidenced by PI
staining and FACS analysis. (B): Percentage of cells in each period after treatment of RPMI8226 cells with different concentrations of the compound 8b. Three
independent experiments were performed, each column represented the mean ± SD of triplicate determinations (*P < 0.05; **P < 0.01 and ***P < 0.001.
Compound 8b-treated group VS control).
4
4
4
.1.2. 4-formyl-N-phenylbenzamide (3a)
4.1.5. N-(4-fluorophenyl)-4-formylbenzamide (3d)
-
-
A white powder, yield: 75%. ESI-MS [M−H] m/z: 224.09.
A white powder, yield: 81%. ESI-MS [M−H] m/z: 242.07.
.1.3. 4-formyl-N-(p-tolyl)benzamide (3b)
4.1.6. N-(4-chlorophenyl)-4-formylbenzamide (3e)
-
-
A white powder, yield: 72%. ESI-MS [M−H] m/z: 238.12.
A white powder, yield: 78%. ESI-MS [M−H] m/z: 258.04.
.1.4. 4-formyl-N-(4-methoxyphenyl)benzamide (3c)
4.1.7. N-(4-bromophenyl)-4-formylbenzamide (3f)
-
-
A white powder, yield: 76%. ESI-MS [M−H] m/z: 254.09.
A white powder, yield: 73%. ESI-MS [M−H] m/z: 302.02.
5

X.-y. Li, et al.
Bioorganic Chemistry 103 (2020) 104189
Fig. 4. Compound 8b could induce apop-
tosis in RPMI8226 cells. (A): The effect of
compound 8b on RPMI8226 cells death
was evidenced by Annexin V-FITC/PI
double staining and FACS analysis.
Apoptotic cells were Annexin V [+] and PI
[
[
-], late apoptotic cells were Annexin V
+] and PI [+], necrotic cells were
Annexin V [-] and PI [+] and living cells
were Annexin
V [-] and PI [-]. (B):
Percentage of apoptotic cells after treat-
ment of RPMI8226 cells with different
concentrations of the compound 8b. Three
independent experiments were performed,
each column represented the mean ± SD
of triplicate determinations (*P
<
0.05;
**P
<
0.01 and ***P
<
0.001.
Compound 8b-treated group VS control).
4
.1.8. N-(4-chloro-3-(trifluoromethyl)phenyl)-4-formylbenzamide (3g)
(600 MHz, DMSO‑d
6
) δ 12.21 (s, 1H), 10.34 (s, 1H), 8.81 (dd,
-
A white solid, yield: 65%. ESI-MS [M−H] m/z: 326.08.
J = 4.5, 1.4 Hz, 2H), 8.55 (s, 1H), 8.06 (d, J = 8.3 Hz, 2H), 7.91 (d,
J = 8.3 Hz, 2H), 7.85 (dd, J = 4.5, 1.4 Hz, 2H), 7.80 (d, J = 7.8 Hz,
1
3
2
H), 7.36 (t, J = 7.9 Hz, 2H), 7.11 (t, J = 7.4 Hz, 1H). C NMR
4
.1.9. General procedure for the synthesis of the target compounds (7a-9g)
To a solution of intermediates (3a-3g) (0.01 mol) in ethanol (25 mL)
(
150 MHz, DMSO‑d
6
) δ 165.41, 162.30, 150.86 (2C), 148.46, 140.84,
1
1
39.53, 137.34, 136.69, 129.11 (2C), 128.74 (2C), 127.61 (2C),
were added corresponding picolinohydrazides (4, 5, and 6) (0.012 mol,
+
24.27, 122.03 (2C), 120.92 (2C). HRMS (ESI) (m/z): [M + H]
1
.43 mL) and the catalytic amount of acetic acid. The reaction mixture
calcd for C20
H
17
4
N O
2
[M + H]+ 345.1346, found: 345.1350.
was stirred under reflux at 80 °C for 3–6 h. After cooling to room
temperature, the precipitate thus formed was collected by filtration,
washed with ethanol, and dried to yield pure target compounds 7a-9g
in moderate to excellent yields.
4
.1.9.2. (E)-4-((2-isonicotinoylhydrazono)methyl)-N-(p-tolyl)benzamide
1
(
7b). A white powder, yield: 75%. Mp: 261.8–261.6 °C. H NMR
(
600 MHz, DMSO‑d ) δ 12.20 (s, 1H), 10.26 (s, 1H), 8.80 (dd,
6
4
.1.9.1. (E)-4-((2-isonicotinoylhydrazono)methyl)-N-phenylbenzamide
J = 4.5, 1.5 Hz, 2H), 8.54 (s, 1H), 8.04 (d, J = 8.3 Hz, 2H), 7.90 (d,
J = 8.3 Hz, 2H), 7.84 (dd, J = 4.5, 1.5 Hz, 2H), 7.67 (d, J = 8.3 Hz,
1
(
7a). A white powder, yield: 78%. Mp: 257.8–259.6 °C. H NMR
6

X.-y. Li, et al.
Bioorganic Chemistry 103 (2020) 104189
Fig. 5. Compound 8b could induce apoptosis by promoting RPMI8226 cells release of ROS. RPMI8226 cells were treated with compound 8b (0, 0.05, 0.10, 0.20 µM)
for 24 h. The cells were harvested and stained with DCFH-DA and analyzed by FACS. Data are expressed as the mean ± SD (n = 3). *P < 0.05; **P < 0.01 and
***P < 0.001. Compound 8b-treated group VS control.
1
3
13
2
H), 7.16 (d, J = 8.3 Hz, 2H), 2.28 (s, 3H). C NMR (150 MHz,
) δ 165.18, 162.29, 150.85 (2C), 148.48, 140.84, 137.24,
3H). C NMR (150 MHz, DMSO‑d
6
) δ 164.94, 162.29, 156.12, 150.85
DMSO‑d
6
(2C), 148.50, 140.85, 137.18, 136.78, 132.58, 128.62 (2C), 127.59
1
37.00, 136.77, 133.24, 129.49 (2C), 128.68 (2C), 127.60 (2C), 122.03
(2C), 122.53 (2C), 122.03 (2C), 114.24 (2C), 55.66. HRMS (ESI) (m/z):
+
+
[M + H]+ 375.1452, found:
(
2C), 120.94 (2C), 20.98. HRMS (ESI) (m/z): [M + H] calcd for
[M + H] calcd for C21
H
19
N
4 3
O
+
C
21
H
19
N
4
O
2
[M + H] 359.1503, found: 359.1506.
375.1447.
4
.1.9.3. (E)-4-((2-isonicotinoylhydrazono)methyl)-N-(4-methoxyphenyl)
4.1.9.4. (E)-N-(4-fluorophenyl)-4-((2-isonicotinoylhydrazono)methyl)
1
benzamide (7c). A light-yellow powder, yield: 84%. Mp:
benzamide (7d). A white powder, yield: 82%. Mp: 260.8–261.2 °C. H
1
2
63.7–264.6 °C. H NMR (600 MHz, DMSO‑d
6
) δ 12.20 (s, 1H), 10.22
NMR (600 MHz, DMSO‑d ) δ 12.21 (s, 1H), 10.40 (s, 1H), 8.80 (d,
6
(
s, 1H), 8.80 (dd, J = 4.5, 1.5 Hz, 2H), 8.54 (s, 1H), 8.05 (d,
J = 8.2 Hz, 2H), 7.89 (d, J = 8.3 Hz, 2H), 7.84 (dd, J = 4.5,
.5 Hz, 2H), 7.69 (d, J = 8.9 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 3.75 (s,
J = 5.5 Hz, 2H), 8.54 (s, 1H), 8.05 (d, J = 8.1 Hz, 2H), 7.90 (d,
J = 8.1 Hz, 2H), 7.85 (d, J = 5.6 Hz, 2H), 7.81 (dd, J = 8.7, 5.1 Hz,
1
2H), 7.20 (t, J = 8.8 Hz, 2H). 13C NMR (150 MHz, DMSO‑d
) δ 165.31,
6
7

X.-y. Li, et al.
Bioorganic Chemistry 103 (2020) 104189
1
1
62.31, 159.64, 158.05, 150.84 (2C), 148.45, 140.83, 137.39, 136.49,
264.4–266.7 °C. 1H NMR (600 MHz, DMSO‑d
) δ 12.15 (s, 1H), 10.23
6
35.88 (d, J = 1.9 Hz), 128.70 (2C), 127.62 (2C), 122.76 (d,
(s, 1H), 9.10 (s, 1H), 8.78 (d, J = 3.8 Hz, 1H), 8.52 (s, 1H), 8.28 (d,
J = 7.9 Hz, 1H), 8.05 (d, J = 8.1 Hz, 2H), 7.89 (d, J = 8.1 Hz, 2H),
7.70 (d, J = 8.8 Hz, 2H), 7.58 (dd, J = 7.7, 4.9 Hz, 1H), 6.94 (d,
J = 8.0 Hz) (2C), 122.04 (2C), 115.67 (d, J = 22.0 Hz) (2C). HRMS
+
+
(
ESI) (m/z): [M + H] calcd for C20
H
16FN
4
O
2
[M + H] 363.1252,
1
3
found: 363.1243.
J = 8.9 Hz, 2H), 3.75 (s, 3H). C NMR (150 MHz, DMSO‑d ) δ 164.96,
6
1
62.37, 156.12, 152.86, 149.12, 147.91, 137.30, 136.68, 135.99,
4
.1.9.5. (E)-N-(4-chlorophenyl)-4-((2-isonicotinoylhydrazono)methyl)
132.59, 129.58, 128.61 (2C), 127.52 (2C), 124.10, 122.54 (2C),
1
+
benzamide (7e). A white powder, yield: 77%. Mp: 270.3–271.6 °C.
NMR (600 MHz, DMSO‑d ) δ 12.21 (s, 1H), 10.46 (s, 1H), 8.80 (dd,
J = 4.5, 1.5 Hz, 2H), 8.54 (s, 1H), 8.05 (d, J = 8.3 Hz, 2H), 7.91 (d,
H
114.24 (2C), 55.65. HRMS (ESI) (m/z): [M
+
H]
calcd for
[M + H]+ 375.1452, found: 375.1458.
6
C
21
H
19
N
4
O
3
1
3
J = 8.3 Hz, 2H), 7.85–7.82 (m, 4H), 7.42 (d, J = 8.8 Hz, 2H). C NMR
150 MHz, DMSO‑d ) δ 165.50, 162.30, 150.85 (2C), 148.40, 140.83,
4.1.9.11. (E)-N-(4-fluorophenyl)-4-((2-nicotinoylhydrazono)methyl)
1
(
6
benzamide (8d). A white powder, yield: 84%. Mp: 261.8–263.0 °C. H
1
38.51, 137.49, 136.38, 129.03 (2C), 128.77 (2C), 127.88, 127.64
NMR (600 MHz, DMSO‑d ) δ 12.16 (s, 1H), 10.40 (s, 1H), 9.10 (d,
6
+
(
2C), 122.40 (2C), 122.03 (2C). HRMS (ESI) (m/z): [M + H] calcd for
J = 1.5 Hz, 1H), 8.78 (dd, J = 4.7, 1.2 Hz, 1H), 8.52 (s, 1H), 8.28 (d,
+
C
20
H
16ClN
4
O
2
[M + H] 379.0956, found: 379.0950.
J = 7.9 Hz, 1H), 8.05 (d, J = 8.2 Hz, 2H), 7.90 (d, J = 8.2 Hz, 2H),
7
.81 (dd, J = 8.9, 5.0 Hz, 2H), 7.58 (dd, J = 7.7, 4.9 Hz, 1H), 7.20 (t,
13
4
.1.9.6. (E)-N-(4-bromophenyl)-4-((2-isonicotinoylhydrazono)methyl)
J = 8.8 Hz, 2H). C NMR (150 MHz, DMSO‑d ) δ 165.32, 162.37,
6
1
benzamide (7f). A white powder, yield: 83%. Mp: 278.7–279.3 °C.
H
159.63, 158.04, 152.87, 149.12, 147.84, 137.51, 136.39, 135.99,
NMR (600 MHz, DMSO‑d ) δ 12.21 (s, 1H), 10.46 (s, 1H), 8.80 (d,
6
135.90, 129.57, 128.69 (2C), 127.55 (2C), 124.10, 122.75 (d,
J = 5.4 Hz, 2H), 8.54 (s, 1H), 8.05 (d, J = 8.1 Hz, 2H), 7.90 (d,
J = 7.8 Hz), 115.68 (d, J = 22.4 Hz) (2C). HRMS (ESI) (m/z):
+
[M + H]+ 363.1252, found:
J = 8.1 Hz, 2H), 7.84 (d, J = 5.4 Hz, 2H), 7.78 (d, J = 8.7 Hz, 2H),
[M + H] calcd for C20
H
16FN
O
4 2
1
3
7
.55 (d, J = 8.6 Hz, 2H). C NMR (150 MHz, DMSO‑d ) δ 165.51,
6
363.1262.
1
62.31, 150.85 (2C), 148.41, 140.83, 138.94, 137.50, 136.37, 131.94
(
2C), 128.77 (2C), 127.64 (2C), 122.77 (2C), 122.03 (2C), 115.97.
4.1.9.12. (E)-N-(4-chlorophenyl)-4-((2-nicotinoylhydrazono)methyl)
+
[M + H]+
1
HRMS (ESI) (m/z): [M + H] calcd for C20
H16BrN
4
O
2
benzamide (8e). A white powder, yield: 78%. Mp: 265.9–267.1 °C. H
4
23.0451 or 425.0431, found: 423.0444.
NMR (600 MHz, DMSO‑d ) δ 12.17 (s, 1H), 10.46 (s, 1H), 9.10 (d,
6
J = 1.2 Hz, 1H), 8.78 (d, J = 3.7 Hz, 1H), 8.52 (s, 1H), 8.28 (d,
J = 7.9 Hz, 1H), 8.05 (d, J = 8.2 Hz, 2H), 7.90 (d, J = 8.2 Hz, 2H),
4
.1.9.7. (E)-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-((2-
isonicotinoylhydrazono)methyl)benzamide (7g). A white powder, yield:
7.84 (d, J = 8.8 Hz, 2H), 7.58 (dd, J = 7.7, 4.9 Hz, 1H), 7.42 (d,
1
13
7
6%. Mp: 25.4–276.1 °C. H NMR (600 MHz, DMSO‑d
6
) δ 12.22 (s, 1H),
J = 8.7 Hz, 2H). C NMR (150 MHz, DMSO‑d
6
) δ 165.50, 162.37,
1
0.71 (s, 1H), 8.80 (d, J = 5.8 Hz, 2H), 8.54 (s, 1H), 8.37 (d,
152.87, 149.12, 147.81, 138.53, 137.60, 136.27, 135.99, 129.56,
129.01 (2C), 128.75 (2C), 127.87, 127.56 (2C), 124.10, 122.40 (2C).
J = 2.3 Hz, 1H), 8.12 (dd, J = 8.7, 2.2 Hz, 1H), 8.07 (d,
J = 8.3 Hz, 2H), 7.92 (d, J = 8.3 Hz, 2H), 7.84 (d, J = 5.9 Hz, 2H),
+
+
HRMS (ESI) (m/z): [M + H] calcd for C20
H16ClN
4
O [M + H]
2
1
3
7
1
1
1
.70 (d, J = 8.8 Hz, 1H). C NMR (150 MHz, DMSO‑d
6
) δ 165.77,
379.0956, found: 379.0958.
62.30, 150.83 (2C), 148.30, 140.79, 139.06, 137.80, 135.78, 132.47,
28.81 (2C), 127.68 (2C), 127.12 (d, J = 30.7 Hz), 125.42, 124.88,
24.15, 122.02 (2C), 119.46 (d, J = 5.4 Hz). HRMS (ESI) (m/z):
4.1.9.13. (E)-N-(4-bromophenyl)-4-((2-nicotinoylhydrazono)methyl)
1
benzamide (8f). A white solid, yield: 81%. Mp: 270.4–271.6 °C.
H
+
+
[
M + H] calcd for C21
H
15ClF
3
N
4
O
2
[M + H] 447.0830, found:
NMR (600 MHz, DMSO‑d
6
) δ 12.15 (s, 1H), 10.46 (s, 1H), 9.09 (d,
4
47.0835.
J = 1.4 Hz, 1H), 8.78 (dd, J = 4.6, 1.1 Hz, 1H), 8.52 (s, 1H), 8.28 (d,
J = 7.9 Hz, 1H), 8.04 (d, J = 8.2 Hz, 2H), 7.90 (d, J = 8.2 Hz, 2H),
4
.1.9.8. (E)-4-((2-nicotinoylhydrazono)methyl)-N-phenylbenzamide
7.78 (d, J = 8.8 Hz, 2H), 7.58 (dd, J = 7.7, 4.9 Hz, 1H), 7.55 (d,
1
13
(
8a). A white powder, yield: 73%. Mp: 259.9–260.5 °C. H NMR
J = 8.7 Hz, 2H). C NMR (150 MHz, DMSO‑d
6
) δ 165.52, 162.38,
(
600 MHz, DMSO‑d
6
) δ 12.16 (s, 1H), 10.34 (s, 1H), 9.10 (s, 1H),
152.88, 149.11, 147.82, 138.94, 137.61, 136.26, 136.00, 131.93 (2C),
8
.78 (d, J = 3.8 Hz, 1H), 8.53 (s, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.06 (d,
129.56, 128.75 (2C), 127.56 (2C), 124.11, 122.77 (2C), 115.96. HRMS
+
[M + H]+ 423.0451 or
J = 8.1 Hz, 2H), 7.90 (d, J = 8.1 Hz, 2H), 7.80 (d, J = 7.9 Hz, 2H),
(ESI) (m/z): [M + H] calcd for C20
H
16BrN
O
4 2
7
.59 (dd, J = 7.7, 4.9 Hz, 1H), 7.36 (t, J = 7.8 Hz, 2H), 7.11 (t,
425.0431, found: 425.0444.
1
3
J = 7.3 Hz, 1H). C NMR (150 MHz, DMSO‑d
6
) δ 165.42, 162.38,
1
1
52.87, 149.11, 147.87, 139.54, 137.45, 136.59, 136.00, 129.57,
4.1.9.14. (E)-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-((2-
29.10 (2C), 128.72 (2C), 127.54 (2C), 124.26, 124.12, 120.93 (2C).
nicotinoylhydrazono)methyl)benzamide (8g). A white powder, yield:
+
1
HRMS (ESI) (m/z): [M
+
H]
calcd for
C
20
H
17
4
N O
2
70%. Mp: 271.2–272.7 °C. H NMR (600 MHz, DMSO‑d
6
) δ 12.17 (s,
+
[
M + H] 345.1346, found: 345.1345.
1H), 10.71 (s, 1H), 9.09 (d, J = 1.2 Hz, 1H), 8.78 (d, J = 3.7 Hz, 1H),
8
.52 (s, 1H), 8.37 (d, J = 2.0 Hz, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.13
4
.1.9.9. (E)-4-((2-nicotinoylhydrazono)methyl)-N-(p-tolyl)benzamide
(dd, J = 8.7, 1.8 Hz, 1H), 8.07 (d, J = 8.2 Hz, 2H), 7.92 (d, J = 8.2 Hz,
1
13
(
8b). A white powder, yield: 71%. Mp: 264.3–265.8 °C. H NMR
2H), 7.71 (d, J = 8.8 Hz, 1H), 7.58 (dd, J = 7.7, 4.9 Hz, 1H). C NMR
(
600 MHz, DMSO‑d
J = 1.4 Hz, 1H), 8.78 (dd, J = 4.7, 1.1 Hz, 1H), 8.52 (s, 1H), 8.28
d, J = 7.9 Hz, 1H), 8.04 (d, J = 8.2 Hz, 2H), 7.89 (d, J = 8.2 Hz, 2H),
.67 (d, J = 8.2 Hz, 2H), 7.59 (dd, J = 7.7, 4.9 Hz, 1H), 7.16 (d,
6
) δ 12.16 (s, 1H), 10.26 (s, 1H), 9.09 (d,
(150 MHz, DMSO‑d ) δ 165.80, 162.37, 152.88, 149.11, 147.71,
6
139.07, 137.92, 135.99, 135.69, 132.49, 129.54, 128.81 (2C), 127.61
(
(2C), 127.12 (d, J = 30.6 Hz), 125.44, 124.87, 124.10, 122.35, 119.47
+
7
(d,
J
=
5.3 Hz). HRMS (ESI) (m/z): [M
+
H]
calcd for
1
3
+
J = 8.2 Hz, 2H), 2.28 (s, 3H). C NMR (150 MHz, DMSO‑d
6
) δ 165.19,
C
21
H
15ClF
3
N
4
O
2
[M + H] 447.0830, found: 447.0841.
1
1
1
62.35, 152.87, 149.11, 147.88, 137.36, 137.01, 136.66, 135.99,
33.22, 129.58, 129.49 (2C), 128.67 (2C), 127.52 (2C), 124.12,
4.1.9.15. (E)-N-phenyl-4-((2-picolinoylhydrazono)methyl)benzamide
+
1
20.93 (2C), 20.98. HRMS (ESI) (m/z): [M
+
H]
calcd for
(9a). A white powder, yield: 86%. Mp: 255.8–257.1 °C. H NMR
+
C
21
H
19
N
4
O
2
[M + H] 359.1503, found: 359.1507.
(600 MHz, DMSO‑d ) δ 12.30 (s, 1H), 10.34 (s, 1H), 8.74 (s, 1H),
6
8
.72 (d, J = 4.4 Hz, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.08–8.04 (m, 3H),
4
.1.9.10. (E)-N-(4-methoxyphenyl)-4-((2-nicotinoylhydrazono)methyl)
7.87 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 7.9 Hz, 2H), 7.67 (dd, J = 6.9,
1
3
benzamide (8c). A light-yellow powder, yield: 79%. Mp:
5.2 Hz, 1H), 7.36 (t, J = 7.8 Hz, 2H), 7.11 (t, J = 7.3 Hz, 1H). C NMR
8

X.-y. Li, et al.
Bioorganic Chemistry 103 (2020) 104189
(
150 MHz, DMSO‑d
6
) δ 165.42, 161.08, 149.92, 149.00, 148.65,
72%. Mp: 274.4–275.3 °C. 1H NMR (600 MHz, DMSO‑d
) δ 12.30 (s,
6
1
1
39.55, 138.52, 137.69, 136.48, 129.09 (2C), 128.73 (2C), 127.58,
1H), 10.71 (s, 1H), 8.75–8.71 (m, 2H), 8.37 (d, J = 2.4 Hz, 1H),
27.50 (2C), 124.25, 123.26, 120.95 (2C). HRMS (ESI) (m/z):
8.16–8.11 (m, 2H), 8.08–8.04 (m, 3H), 7.88 (d, J = 8.4 Hz, 2H),
+
+
13
[
M + H] calcd for C20
H
17
N
4
O
2
[M + H] 345.1346, found:
7.72–7.66 (m, 2H). C NMR (150 MHz, DMSO‑d
6
) δ 165.78, 161.07,
3
45.1359.
149.90, 148.98, 148.48, 139.08, 138.50, 138.15, 135.56, 132.46,
1
28.81 (2C), 127.56 (2C), 127.11 (d, J = 30.6 Hz), 125.43, 124.86,
4
.1.9.16. (E)-4-((2-picolinoylhydrazono)methyl)-N-(p-tolyl)benzamide
124.15, 123.26, 122.34, 119.48 (q, J = 5.4 Hz). HRMS (ESI) (m/z):
1
+
[M + H]+ 447.0830, found:
(
9b). A white powder, yield: 76%. Mp: 260.1–261.7 °C. H NMR
[M + H] calcd for C21
447.0841.
H15ClF
3
N
4 2
O
(
600 MHz, DMSO‑d
J = 6.1 Hz, 2H), 8.16 (d, J = 7.7 Hz, 1H), 8.06 (dd, J = 18.8,
.7 Hz, 3H), 7.86 (d, J = 8.2 Hz, 2H), 7.68 (t, J = 6.7 Hz, 3H), 7.16 (d,
6
) δ 12.29 (s, 1H), 10.26 (s, 1H), 8.73 (d,
4
4.2. Cell culture and grouping
1
3
J = 8.2 Hz, 2H), 2.28 (s, 3H). C NMR (150 MHz, DMSO‑d
6
) δ 165.19,
1
1
1
61.06, 149.94, 149.00, 148.65, 138.53, 137.59, 137.02, 136.55,
We purchased the human myeloma cell line U266, human multiple
myeloma cell line RPMI8226, and human umbilical vein endothelial
cells HUVEC from the American Type Culture Collection (ATCC,
Manassas, VA, USA). We then diluted them with Roswell Park Memorial
Institute (RPMI) 1640 culture medium (Beijing Thermo Fisher Scientific
Company, Beijing, China). We used RPMI 1640 nutrient solution to
suspend these cells. The solution contained 100 µg/mL penicillin, 10%
heat-inactivated fetal calf serum, and 100 µg/mL streptomycin. Then,
33.21, 129.49 (2C), 128.67 (2C), 127.58, 127.48 (2C), 123.26,
+
20.95 (2C), 20.98. HRMS (ESI) (m/z): [M
+
H]
calcd for
+
C
21
H
19
N
4
O
2
[M + H] 359.1503, found: 359.1511.
4
.1.9.17. (E)-N-(4-methoxyphenyl)-4-((2-picolinoylhydrazono)methyl)
1
benzamide (9c). A white powder, yield: 71%. Mp: 258.4–259.9 °C.
H
NMR (600 MHz, DMSO‑d ) δ 12.29 (s, 1H), 10.22 (s, 1H), 8.73 (dt,
6
J = 4.6, 1.8 Hz, 2H), 8.15 (dt, J = 7.9, 1.0 Hz, 1H), 8.06 (ddd,
J = 12.8, 9.4, 5.0 Hz, 3H), 7.85 (d, J = 8.4 Hz, 2H), 7.71–7.67 (m, 3H),
the cells will be cultured in an incubator at 37 °C, 5% CO
rated humidity (Memmert Company, Germany).
2
, and satu-
1
3
6
.96–6.94 (m, 1H), 6.94–6.92 (m, 1H), 3.75 (s, 3H).
C NMR
(
150 MHz, DMSO‑d
6
) δ 164.95, 161.05, 156.11, 149.94, 149.01,
4.3. MTT assay
1
48.66, 138.54, 137.52, 136.56, 132.60, 128.62 (2C), 127.59, 127.47
(
2C), 123.26, 122.53 (2C), 114.24 (2C), 55.66. HRMS (ESI) (m/z):
This experimental operation mainly refered to our previous research
reports. [27,28]
+
+
[
M + H] calcd for C21
H
19
N
4
O
3
[M + H] 375.1452, found:
3
75.1463.
4.4. Cell cycle analysis
4
.1.9.18. (E)-N-(4-fluorophenyl)-4-((2-picolinoylhydrazono)methyl)
1
benzamide (9d). A white powder, yield: 85%. Mp: 262.4–263.7 °C.
H
For the cell cycle analysis, RPMI8226 cells were cultured in 6 well
5
NMR (600 MHz, DMSO‑d
6
) δ 12.29 (s, 1H), 10.39 (s, 1H), 8.75–8.71 (m,
H), 8.16 (d, J = 7.8 Hz, 1H), 8.08–8.04 (m, 3H), 7.87 (d, J = 8.3 Hz,
H), 7.83–7.80 (m, 2H), 7.70–7.66 (m, 1H), 7.20 (t, J = 8.9 Hz, 2H).
plates at the density of 5 × 10 /mL at a volume of 2 mL. The cells were
treated with 0, 0.05, 0.10, 0.20 μM compounds or DMSO control for
24 h. Then the cells were harvested and resuspended in PBS, which
containing 70% ethanol for fixation at 4 °C overnight. Then, the cells
were washed by PBS to remove the ethanol and stained by PI/Rnase
Staining Buffer (BD pharming) for 15 min.
2
2
1
3
C NMR (150 MHz, DMSO‑d
6
) δ 165.32, 161.07, 159.63, 158.04,
1
49.92, 149.00, 148.62, 138.52, 137.74, 136.28, 135.90 (d,
J = 2.4 Hz), 128.70 (2C), 127.54 (d, J = 10.7 Hz) (2C), 123.26,
1
22.76 (d, J = 7.8 Hz) (2C), 115.67 (d, J = 22.0 Hz) (2C). HRMS (ESI)
+
+
(
m/z): [M + H] calcd for C20
H16FN
4
O
2
[M + H] 363.1252, found:
4.5. Annexin V/propidium iodide (PI) staining
3
63.1263.
5
RPMI8226 cells (3 × 10 cells/mL) were seeded in 6 well plates and
4
.1.9.19. (E)-N-(4-chlorophenyl)-4-((2-picolinoylhydrazono)methyl)
treated with compounds at the different concentrations for 24 h. The
cells were then harvested by trypsinization and washed twice with cold
PBS. After centrifugation and removal of the supernatants, cells were
resuspended in 500 μL of 1 × binding buffer, which was then added to
5 μL of annexin V-FITC and incubated at room temperature for 15 min.
After adding 10 μL of PI, the cells were incubated at room temperature
for another 15 min in the dark. A flow cytometer analyzed the stained
cells. All experiments were performed three times.
1
benzamide (9e). A white powder, yield: 81%. Mp: 260.9–261.5 °C.
H
NMR (600 MHz, DMSO‑d ) δ 12.30 (s, 1H), 10.46 (s, 1H), 8.73 (d,
6
J = 5.8 Hz, 2H), 8.15 (d, J = 7.8 Hz, 1H), 8.09–8.03 (m, 3H), 7.87 (d,
J = 8.3 Hz, 2H), 7.84 (d, J = 8.8 Hz, 2H), 7.68 (dd, J = 7.1, 5.1 Hz,
1
H), 7.42 (d, J = 8.8 Hz, 2H).
13
C NMR (150 MHz, DMSO‑d ) δ 165.50, 161.07, 149.92, 149.01,
6
1
1
48.57, 138.53, 137.84, 136.16, 129.02 (2C), 128.76 (2C), 127.86,
27.59, 127.52 (2C), 123.27, 122.41 (2C). HRMS (ESI) (m/z):
+
+
[
M + H] calcd for C20
H16ClN
4
O
2
[M + H] 379.0956, found:
4.6. Intracellular ROS detection
3
79.0968.
5
RPMI8226 cells (3 × 10 cells/mL) were seeded in 6 well plates and
4
.1.9.20. (E)-N-(4-bromophenyl)-4-((2-picolinoylhydrazono)methyl)
treated with different concentrations of compounds for 24 h. Then re-
place DCFH-DA with a serum-free medium at 1:1000 to make the final
concentration 10 μmol/L. RPMI8226 cells were collected and sus-
1
benzamide (9f). A white powder, yield: 79%. Mp: 273.2–274.8 °C.
H
NMR (600 MHz, DMSO) δ 12.30 (s, 1H), 10.45 (s, 1H), 8.74–8.72 (m,
5
2
2
H), 8.15 (d, J = 7.8 Hz, 1H), 8.08–8.04 (m, 3H), 7.87 (d, J = 8.3 Hz,
H), 7.78 (d, J = 8.9 Hz, 2H), 7.68 (ddd, J = 7.5, 4.7, 1.1 Hz, 1H), 7.55
pended in reconstituted DCFH-DA at a cell concentration of 1 × 10 /
mL and extended in a 37 °C cell incubator for 20 min. Invert the mixture
every 3–5 min to make the DCFH-DA and the cells fully contact. The
cells were washed three times with serum-free cell culture medium to
remove the DCFH-DA that did not fully enter the cells. Then use flow
cytometry to detect ROS levels within 20 min.
1
3
(
d, J = 8.8 Hz, 2H). C NMR (150 MHz, DMSO‑d
6
) δ 165.52, 161.07,
1
1
49.92, 149.01, 148.57, 138.96, 138.54, 137.85, 136.15, 131.94 (2C),
28.77 (2C), 127.60, 127.52 (2C), 123.27, 122.78 (2C), 115.95. HRMS
+
+
(
ESI) (m/z): [M + H] calcd for C20
H
4
16BrN O
2
[M + H] 423.0451 or
4
25.0431, found: 425.0445.
4.7. Statistical analysis
4
.1.9.21. (E)-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-((2-
picolinoylhydrazono)methyl)benzamide (9g). A white powder, yield:
Data were analyzed using GraphPad Prism 5 (GraphPad Software;
9

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Bioorganic Chemistry 103 (2020) 104189
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iance (ANOVA) was used to determine comparisons between groups.
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square tests to compare them. P < 0.05 was considered to be statis-
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