Discovery of thiosemicarbazone-containing compounds with potent anti-proliferation activity against drug-resistant K562/A02 cells

Article abstract of DOI:10.1016/j.bmcl.2020.127638

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a major obstacle to successful chemotherapy for leukemia. In this study, a series of thiosemicarbazone-containing compounds (4a-b, 7a-q) were synthesized. Biological evaluation showed that the m

Full text of DOI:10.1016/j.bmcl.2020.127638

Journal Pre-proofs
Discovery of thiosemicarbazone-containing compounds with potent anti-pro‐
liferation activity against drug-Resistant K562/A02 Cells
Xiaoke Gu, Xin Li, Mingyu Guan, Chunyu Jiang, Qinghua Song, Nan Sun,
Yueting Zou, Qingqing Zhou, Jing Chen, Jingying Qiu
PII:
S0960-894X(20)30749-6
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127638
BMCL 127638
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
18 August 2020
2 October 2020
18 October 2020
Please cite this article as: Gu, X., Li, X., Guan, M., Jiang, C., Song, Q., Sun, N., Zou, Y., Zhou, Q., Chen, J., Qiu,
J., Discovery of thiosemicarbazone-containing compounds with potent anti-proliferation activity against drug-
Resistant K562/A02 Cells, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2020.127638
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Discovery of thiosemicarbazone-containing compounds with potent anti-proliferation
activity against drug-Resistant K562/A02 Cells
Xiaoke Gua, #, *, Xin Lia, #, Mingyu Guana, Chunyu Jianga, Qinghua Songa, Nan Suna, Yueting,
Zoub, Qingqing Zhoub, Jing Chenb, Jingying Qiua, b, 
a
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical
University, Xuzhou 221004, People’s Republic of China
b Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical University, Xuzhou
221004, People’s Republic of China
Abstract
P-glycoprotein (P-gp)-meidated muthidrug resistance (MDR) is a major obstacle to successful
chemotherapy for leukemia. In this study, a series of thiosemicarbazone-containing compounds (4a-
b, 7a-q) were synthesized. Biological evaluation showed that the most active compound 7e
displayed potent anti-leukemia activity against P-gp overexpressing drug-resistant K562/A02 cells,
with an IC50 value of 0.44 μM. Notably, compound 7e exhibited a selective killing effect on
K562/A02 cells by dose-dependently increasing the intracellular levels of reactive oxygen species
(ROS), thus exerting a potential collateral sensitivity (CS)-promoting effect in vitro. Moreover,
compound 7e could inhibit HDAC1 and HDAC6, and induce the apoptosis of K562/A02 cells by
increasing the expression of Bax, decreasing Bcl-2 protein level, and promoting the cleavage of
caspase-3 and PARP, respectively. Overall, 7e may be a potential anti-cancer agent against drug-
resistant myelogenous leukemia.
Keywords: Multidrug resistance; P-glycoprotein; Thiosemicarbazones; Collateral sensitivity;
HDACs inhibitor.
 Corresponding author. Tel.: +86-516-83262138; fax: +86-516-83262630; e-mail: jingyqiu@xzhmu.edu.cn (J. Q.)
Tel.: +86-516-83262137; fax: +86-516-83262137; e-mail: gu_xk@xzhmu.edu.cn (X. G.)
 These two authors contributed equally to this work.
1

Multidrug resistance (MDR) is one of the leading obstacle to successful chemotherapy for
leukemia.1-3 Therefore, it is of great interest to search for novel, effective MDR-reversing antitumor
agents to overcome drug resistant leukemia. Molecularly, the predominant cause of MDR is the
overexpression of ATP-binding cassette (ABC) transporters on the plasma membrane of cancer cells,
which could increase the efflux of anti-cancer drugs with diverse structure and action mechanisms.4,
5 Among these transporters, P-glycoprotein (P-gp) is the most well-studied and significant one.6, 7
Thus, development of P-gp inhibitors/modulators is regarded as a promising approach to overcome
MDR in cancer treatment.
During the past few decades, three generations of P-gp inhibitors have been developed. However,
the low selectivity, poor potency and/or pharmacokinetic interaction significant limit their clinical
application.8 So far, there is no inhibitor available in clinic. Accordingly, alternative strategies are
urgently needed to conquer P-gp-medicated MDR. Notably, recent research showed that P-gp
overexpressing drug-resistant cancer cells could be hypersensitive to drugs with a particular
structure, a phenomenon called collateral sensitivity (CS). As more and more compounds were
found to selectively kill the resistant cancer cells, CS conveyed by P-gp represents a novel and
attractive strategy to conquer P-gp-mediated MDR.9, 10
Recently, compounds with a thiosemicarbazone moiety (ie. Dp44mT and Bp44mT, Figure 1)
have been reported to selectively kill resistant cancer cells11, 12, thus overcoming P-gp mediated
MDR by targeting CS. More importantly, thiosemicarbazones possessed diverse biological activities
and several thiosemicarbazones have been investigated in clinical trials to treat malignancies
including leukemia.13 Keeping these in mind and considering the role of histone deacetylases
(HDACs) in leukemogenesis and the promising potentials of HDACs inhibitors for the treatment of
leukemia14-16, we hypothesized that thiosemicarbazones scaffold hybridized with pharmacophore of
HDACs inhibitors might be benefical for the anti-cancer activity against drug-resistant cells.
Therefore, in this paper, we designed and synthesized nineteen thiosemicarbazone-containing
compounds with a similar linker and ZBG of vorinostat (SAHA), a well-known HDACs inhibitor
(Figure 1), and susequently determined their anti-proliferation effect and preliminary mechanisms
on P-gp overexpressing drug-resistant leukemic K562/A02 cells.
2

Figure 1. Chemical structures of Bp44mT, Dp44mT, SAHA and the target compounds
The synthetic procedures of the target compounds 4a and 4b were depicted in Scheme 1. Briefly,
methyl 6-aminohexanoate hydrochloride was treated with carbon disulphide and p-Toluenesulfonyl
chloride to give the isothiocyanate intermediate 1, which was then reacted with hydrazine
monohydrate to obtain thiosemicarbazide intermediate 2. Intermediate 2 was subsequently
condensed with dipyridin-2-yl methanone or 2-benzoylpyridine to prepare intermediates 3a-b,
respectively. The crude products 3a-b were finally aminolized with hydroxylamine hydrochloride
to give the target compounds 4a-b, respectively.
Scheme 1. Synthetic route of target compounds 4a and 4b. Reagents and conditions: a) Carbon disulphide, p-
Toluenesulfonyl chloride, Triethylamine, Tetrahydrofuran, 0 ℃-rt, 1.5 h; b) Hydrazine monohydrate, MeOH, -10 ℃,
2 h; c) dipyridin-2-yl methanone or 2-benzoylpyridine, MeOH, rt, 5 h; d) NH2OH.HCl, KOH, MeOH, -10 ℃, 4 h.
The synthetic procedures of the target compounds 7a-q were depicted in Scheme 2. Briefly,
acetophenone, benzophenone or their derivatives were condensed with thiosemicarbazide to give
intermediates 5a-n, which were subsequently reacted with ethyl 6-bromohexanoate or ethyl 4-
3

bromobutyrate to obtain intermediates 6a-q, respectively. The crude products 6a-q were finally
aminolized with hydroxylamine hydrochloride to give the target compounds 7a-q, respectively.
Scheme 2. Synthetic route of target compounds 7a-q. Reagents and conditions: a) Thiosemicarbazide, MeOH,
100 ℃, 8 h; b) NaH, 1,4-Dioxane, Ethyl 6-bromohexanoate/Ethyl 4-bromobutyrate, reflux, 10 h; c) EtOH,
NH2OH.HCl, KOH, -10 ℃, 4 h.
It is known that P-gp-mediated MDR is a major obstacle to successful chemotherapy for leukemia.
Therefore, we determined the anti-proliferation effect of the target compounds on P-gp
overexpressing drug-resistant leukemic K562/A02 cells as well as parental sensitive K562 cells by
MTT assay.17 Anti-cancer drug adriamycin (ADR), HDACs inhibitor SAHA and Bcr-Abl tyrosine
kinase inhibitor imatinib were served as positive controls. Compound Dp44mT was selected as the
lead control. In addition, we calculated the relative resistance (RR) index of these compounds
according to the previous methods in literatures10, 18, which could be used to preliminarily estimate
the potential of CS-inducing effect. Generally, RR > 1 means MDR cells are resistant to the
compound. RR < 1 indicates the compound could more effectively kill MDR cells than parental
sensitive cells. RR ≤ 0.5 suggests the compound might be a CS-promoting agent.
Table 2. Anti-proliferation activity of target compounds against drug resistant K562/A02 and sensitive K562 cells
a IC50 (μM)
K562 K562/A02
IC50 (μM)
K562/A02
Comp.
b RR
Comp.
K562
RR
4

2.87 ± 0.25
2.14 ± 0.24
3.81 ± 0.40
3.51 ± 0.38
4.34 ± 0.39
4.63 ± 0.51
2.18 ± 0.18
2.03 ± 0.23
3.64 ± 0.37
4.22 ± 0.46
3.15 ± 0.27
6.01 ± 0.68
0.76
0.95
0.96
1.20
0.73
1.30
2.33 ± 0.24
1.63 ± 0.14
2.89 ± 0.30
3.56 ± 0.34
> 10
1.31 ± 0.13
0.94± 0.092
4.53 ± 0.51
2.91 ± 0.31
> 10
0.56
0.58
1.57
0.82
-
4a
4b
7a
7b
7c
7k
7l
7m
7n
7o
> 10
> 10
> 10
> 10
-
-
7d
7e
7f
7p
7q
0.96± 0.075 0.44± 0.039
0.46
0.98
0.54
1.91 ± 0.22
1.89 ± 0.20
1.26 ± 0.11
1.40 ± 0.13
3.84 ± 0.43
1.89 ± 0.17
1.02± 0.093
0.52± 0.049
0.89± 0.072
3.57 ± 0.28
1.35 ± 0.12
3.13 ± 0.35
2.32
SAHA
3.43 ± 0.40
0.83 ± 0.095
0.61 ± 0.057
3.02 ± 0.29
23.42 ± 4.73
0.98 ± 0.079
0.88
28.21
1.61
7g
7h
7i
Dp44mT
ADR
0.41
0.64
0.93
Imatinib
7j
a IC50 values are expressed as means ± SD of triplicate experiments. b Relative resistance (RR) index: RR = IC50
(corresponding resistant cells)/IC50(parental cells).
As shown in Table 1, P-gp overexpressing K562/A02 cells were resistant to ADR, SAHA and
Imatinib, for their RR values were 28.21, 2.32 and 1.61, respectively. In sharp contrast, K562/A02
cells were sensitive to most of the target compounds, especially compounds 7e, 7g, 7h, 7i and 7l
with RR values far below 1. The RR value of lead compound Dp44mT was 0.88, indicating that
Dp44mT displayed a clear selective killing effect on K562/A02 cells, however, it did not exhibit a
CS-promoting effect under our experimental conditions. In addition, thiosemicarbazones scaffold
hybridized with the pharmacophore of HDACs inhibitors was indeed benefical for the anti-cancer
activity against drug resistat cells (4a vs. Dp44mT). Compounds with a methyl and phenyl
substituents on the carbon atom of imine generally exhibited more potent anti-proliferation activity
than those with two aromatic substituents. Compound with a five-carbon chain linked to the nitrogen
atom of thiosemicarbazide moiety and the carbon atom of amid bond showed more potent anti-
proliferation activity than the compound with a three-carbon chain (7l vs.7o; 7m vs.7p; 7n vs.7q).
Notably, among the target compounds, 7e and 7h were found to most potently inhibit the
proliferation of K562/A02 cells with IC50 values of 0.44 μM and 0.52 μM, respectively, which were
5

much lower than that of positive control ADR (with an IC50 value of 23.42 μM), SAHA (with an
IC50 value of 3.13 μM), Imatinib (with an IC50 value of 0.98 μM), and lead control Dp44mT (with
an IC50 value of 3.02 μM). More importantly, their RR values were 0.46 and 0.41, respectively,
suggesting their potential CS-inducing ability in drug-resistant K562/A02 cells.
20
As mentioned above, HDACs act a crucial role in leukemogenesis.19,
Therefore, we
investigated the inhibitory effect of the active compounds 7e, 7g, 7h and 7l on HDACs activity
using recombinant human HDAC1, HDAC6 and HDAC8 enzymes according to previous
methods.21 HDACs inhibitor SAHA and Dp44mT were served as positive and lead control,
respectively.
Data showed that (Table 3), lead compound Dp44mT displayed little effect on HDACs activity.
As expected, compounds 7e, 7g, 7h and 7l with a similar pharmacophore of HDACs inhibitor SAHA,
could significantly inhibit HDAC1 and HDAC6 activity, but did not inhibit HDAC8 activity. Among
them, 7e was the most potent one, with IC50 values of 38.57 nM and 34.60 nM against HDAC1 and
HDAC6, respectively, which were comparable to those of the positive control SAHA.
Table 3. Inhibitory effect of compounds 7e, 7g, 7h and 7l on HDAC1, 6 and 8 activity.
a IC50 (nM)
Compounds
HDAC1
HDAC6
HDAC8
> 5000
> 5000
> 5000
38.57 ± 3.08
69.25 ± 7.24
54.62 ± 6.01
34.60 ± 2.83
62.86 ± 6.99
37.73 ± 4.15
7e
7g
7h
71.10 ± 6.96
> 5000
46.82 ± 5.57
> 5000
> 5000
> 5000
> 5000
7l
Dp44mT
SAHA
37.7 ± 3.7
33.1 ± 2.9
a Data are expressed as mean ± S.D. of three independent experiments
Based on the results of above experiments, the most active compound 7e, with a selective killing
activity and potential CS-inducing effect on K562/A02 cells, was selected to investigate the
potential mechanisms underlying the action. Considering that CS may be associated with the
6

increased ROS level in cells overexpressing ABC transporters 9, 22, we therefore determined the
intracellular ROS levels in K562/A02 and K562 cells treated with 7e by DCFHA-DA assay.23, 24
Results showed that, after treatment with 7e, the intracellular ROS in P-gp overexpressing
K562/A02 cells was significantly increased in a dose-related manner (Fig. 2A, blue bar). In sharp
contrast, the ROS levels in parental sensitive K562 cells were not changed obviously (Fig. 2A, red
bar). Moreover, the intracellular ROS levels in K562/A02 and the cytotoxicity of 7e against
K562/A02 cells were dramatically attenuated in the presence of non-toxic ROS scavenger Nacetyl-
cysteine (NAC) (Figure 2). In addition, we also found that 7e exhibited little effect on P-gp efflux
function and expression (data not shown). These results indicated that the selectively killing activity
and potential CS-inducing effect of 7e against drug resistant K562/A02 cells might be achieved via
increasing the intracellular ROS in K562/A02 cells, at least partly.
Figure 2. ROS levels in K562/A02 and K562 cells treated with 7e (A) and effect of ROS on the anti-proliferation
activity of 7e against K562/A02 cells (B). (A) ROS levels in K562 (red bar) and K562/A02 (blue bar) cells treated
with 7e were determined by DCFHA-DA assay. **P < 0.01 of compound treated group vs. control group (blue bar).
##P < 0.01 of NAC + 7e (3.0 μM) group vs. 7e (3.0 μM) group in K562/A02 cells (blue bar). (B) The viability of
K562/A02 cells treated with 7e (3.0 μM) for 24 h was determined by MTT assay. In case of inhibition, K562/A02
cells were incubated with 7e (3.0 μM) in the presence of 10 mM ROS scavenger NAC. **P < 0.01 of NAC + 7e (3.0
μM) group vs. the 7e (3.0 μM) group. For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.
Subsequently, the promoting effect of 7e on cell apoptosis, a major mechanism of anti-
proliferation, was determined by Annexin V/PI assay in conjunction with flow cytometry.25, 26 Data
7

showed that, after treatment with 7e (1.0 or 3.0 μM) for 48 h, apoptotic K562/A02 cells at early
(Annexin-V+/PI−) and late (Annexin-V+/PI+) stages were obviously increased to 68.35% and
75.97%, respectively, suggesting the apoptosis-promoting effect on P-gp overexpressing K562/A02
cells.
Figure 3. Effect of 7e on K562/A02 cells apoptosis. (A) K562/A02 cells were treated with or without 7e (1.0 or
3.0 μM) for 48 h, then cells were collected and stained with Annexin V/PI to analyze early apoptotic (Annexin-
V+/PI−) and late apoptotic (Annexin-V+/PI−) cell fractions using flow cytometry; (B) The percentage of cell
distribution.
To further confirm the apoptosis-inducing effect on K562/A02 cells as well as the inhibitory effect
of 7e on HDACs activity, the expression of key apoptotic proteins, including Bax, Bcl-2 27, and the
cleavage states of caspase-3 and PARP (apoptosis markers) and Ac-H3, Ac-H4 (well-known
substrates of HDAC1, 2 and 3) in K562/A02 cells treated with 7e were measured by western blot
assay, respectively.
It was shown that, compared to the control group, 7e could obviously increase the expression of
pro-apoptotic Bax while reducing anti-apoptotic Bcl-2 protein expression in a dose-dependent
manner (Figure 4). Meanwhile, 7e could also dose-dependently promote the cleavage of caspase-3
and PARP. In addition, after treatment with 7e, the expression of Ac-H3 and Ac-H4 in K562/A02
cells was also significantly increased. These results confirmed that 7e could indeed induce the
K562/A02 cells apoptosis and inhibit HDACs activity.
8

Figure 4. Effect of 7e on apoptosis-related proteins and Ac-H3, Ac-H4 expression. (A) K562/A02 cells were
incubated with or without 7e (1.0 or 3.0 μM) for 48 h, and then harvested and lysed to detect the expression of Bax,
Bcl-2, Cleaved Caspase3 and Cleaved PARP and Ac-H3, Ac-H4 by western blotting assay.; (B) Density ratios of
Bax, Bcl-2, Cleaved Caspase3 and Cleaved PARP, and Ac-H3, Ac-H4 to GADPH.
In conclusion, nineteen thiosemicarbazone-containing compounds were synthesized as novel
anti-leukemia agents against P-gp overexpressing drug-resistant K562/A02 cells. Among them,
compound 7e exhibited a selective killing activity against P-gp overexpressing drug-resistant
leukemic K562/A02 cells, with an IC50 value of 0.44 μM. Preliminary mechanism studies indicated
that compound 7e could dose-dependently increase the ROS levels in K562/A02 cells, thus
selectively killing K562/A02 cells and exerting a potential CS-inducing effect. In addition,
compound 7e exhibited an inhibitory effect on HDAC1 and HDAC6, and could dose-dependently
upregulate the expression of Bax, decrease Bcl-2 protein level and promote the cleavage of caspase-
3 and PARP, thus inducing K562/A02 cells apoptosis. Therefore, compound 7e might be a promising
lead to develop novel anti-cancer agents against P-gp overexpressing leukemic cells.
Acknowledgements
This work was supported by the grants from the Natural Science Foundation of Jiangsu province
(No. BK20171179 and BK20181151), the Six Talent Peaks Project in Jiangsu Province (Grant No.
2017-YY-039), Jiangsu Provincial 333 High-level Talents Cultivation Project (BRA201927), the
Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX20_2506)
and Jiangsu Training Programs of Innovation and Entrepreneurship for Undergraduates (Grant No.
9

201810313083X). We thank to Public Experimental Research Center of Xuzhou Medical University
for providing chemical structure analysis.
Supporting Information
Supplementary data (compound synthesis, spectroscopic characterization, representative spectra
and pharmacological assays) to this article can be found online.
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13

Highlights
1. Thiosemicarbazone-containing compounds were prepared as novel anti-leukemia
agents.
2. 7e showed potent anti-leukemia effect on P-gp overexpressing drug resistant
K562/A02 cells.
3. 7e could increase ROS levels in K562/A02 cells, thus inducing a collateral
sensitivity effect.
4. 7e could regulate apoptosis-related proteins expression in drug resistant K562/A02
cells.
5. 7e displayed potent inhibitory effect on HDAC1/6 activity in drug resistant
K562/A02 cells..
14

Compound 7e could more potently inhibit the proliferation of P-gp overexpressing K562/A02 cells
than HDACs inhibitor SAHA.
15