Design, synthesis and evaluation of PD176252 analogues for ameliorating cisplatin-induced nephrotoxicity

Article abstract of DOI:10.1039/c8md00632f

Cisplatin is a clinical chemotherapy drug for cancers; however, its remarkably high kidney toxicity and other toxicities pose a danger to patients. As the small molecule inhibitor of GRPR, PD176252 can inhibit the growth and proliferation of various cancer cells, but the characteristics of high toxicity and poor water solubility has limited its use as a drug. When we studied PD176252 for the reduction of toxicity of cisplatin, we modified its structure to synthesize 16 analogues. Surprisingly, the analogues showed reduced cisplatin-induced renal toxicity, and unlike PD176252, the analogues 5d and 5m were almost non-toxic to the normal HK2 cells. Furthermore, the analogue 5d and PD176252 were subjected to cisplatin-induced inflammatory response in vitro. The results showed that 5d was able to better prevent this condition by effectively inhibiting its inflammatory response. Thus, this study will help in clinically reducing the side effects of cisplatin.

Full text of DOI:10.1039/c8md00632f

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RESEARCH ARTICLE
Design, synthesis and evaluation of PD176252
analogues for ameliorating cisplatin-induced
Cite this: DOI: 10.1039/c8md00632f
nephrotoxicity†
Sen Yao, a Biao Wei,b Mingjun Yu,a Xiaoming Meng,b
a
ac
*
Meng He and Risheng Yao
Cisplatin is a clinical chemotherapy drug for cancers; however, its remarkably high kidney toxicity and other
toxicities pose a danger to patients. As the small molecule inhibitor of GRPR, PD176252 can inhibit the
growth and proliferation of various cancer cells, but the characteristics of high toxicity and poor water sol-
ubility has limited its use as a drug. When we studied PD176252 for the reduction of toxicity of cisplatin, we
modified its structure to synthesize 16 analogues. Surprisingly, the analogues showed reduced cisplatin-
induced renal toxicity, and unlike PD176252, the analogues 5d and 5m were almost non-toxic to the nor-
mal HK2 cells. Furthermore, the analogue 5d and PD176252 were subjected to cisplatin-induced inflamma-
tory response in vitro. The results showed that 5d was able to better prevent this condition by effectively
inhibiting its inflammatory response. Thus, this study will help in clinically reducing the side effects of
cisplatin.
Received 29th December 2018,
Accepted 19th March 2019
DOI: 10.1039/c8md00632f
rsc.li/medchemcomm
important markers for the diagnosis of renal toxicity in vitro
and in vivo.17–19
Introduction
Cisplatin, one of the main pillars of clinical chemotherapy
drugs, is widely used to treat many kinds of human can-
cers, including lung, head and neck, breast, bladder, ovar-
ian, and prostate cancers.1,2 The complications of cisplatin
have been gradually discovered. In particular, kidney toxicity
has been a main side effect in cancer patients undergoing
cisplatin therapy.3,4 Furthermore, a high dose or prolonged
use of cisplatin is prone to cause kidney failure5–7 and even
death.8,9
At present, it has been found that cisplatin-induced kid-
ney inflammation is one of the important factors of renal
toxicity.10–12 In addition, the inhibition of this inflammatory
response could significantly reduce kidney toxicity.13–15
Researchers have demonstrated that monocyte chemotactic
protein (MCP-1), inflammatory cytokine (IL-6), and
proinflammatory cytokine TNF-α16 play an important role in
renal toxicity induced by cisplatin. Thus, they can be used as
Bombesin/gastrin-releasing peptide receptor (GRPR),20–23
which is a G protein-coupled receptor,24 is involved in the
regulation of the release of inflammatory factors and plays a
role in inflammatory diseases.22,25 In recent studies, GRPR
inhibitors have been known to ameliorate some diseases by
inhibiting the inflammatory response.26,27
PD176252 is a nonpeptide GRPR antagonist28,29 that can
inhibit the growth and proliferation of various cancer cells,
including head and neck cancer30 and lung cancer,21,31 but it
was not studied in the field of inflammatory diseases. In this
study, PD176252 has been used as a subject to ameliorate
cisplatin-induced nephrotoxicity. The result was pleasing
with PD176252 effectively inhibiting the cisplatin-induced ap-
optosis of human renal proximal tubular cells (HK-2 cells).
However, the characteristics of high cytotoxicity and poor wa-
ter solubility of PD176252 have limited its application and
development as a medicine.
Subsequently,
a series of PD176252 analogues were
designed and synthesized. 16 analogues were efficiently syn-
thesized and evaluated for their activity of inhibition of
cisplatin-induced apoptosis of human renal proximal tubular
cells. To our delight, many of the analogues could reduce
cisplatin-induced renal toxicity, and the more active ana-
logues did not cause toxicity to the normal cells in HK2 cells.
In further studies, analogues 5d, 5m, and PD176252 were
a School of Food and Biological Engineering, Hefei University of Technology, Hefei
230000, China. E-mail: rishengyao@163.com
b School of Pharmacy, Anhui Medical University, Hefei, 230032, China
c Engineering Research Center of Bioprocess, Ministry of Education, PRC, Hefei
University of Technology, Hefei 230009, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8md00632f
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Table 1 The structure of the compounds
studied as compounds for the inhibition of cisplatin-induced
inflammatory responses in HK2 cells in vitro.
Compound
R1
R2
R3
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
NO2
NO2
CH3
CH3
F
Phenyl
3-Indolyl
p-Hydroxyphenyl
Phenyl
p-Hydroxyphenyl
Phenyl
3-Indolyl
3-Indolyl
Phenyl
3-Indolyl
p-Hydroxyphenyl
Phenyl
p-Hydroxyphenyl
Phenyl
3-Indolyl
3-Indolyl
Imidazole
Imidazole
Imidazole
Imidazole
Imidazole
Imidazole
Imidazole
Imidazole
Morpholine
Morpholine
Morpholine
Morpholine
Morpholine
Morpholine
Morpholine
Morpholine
Results and discussion
Design and chemistry
F
F
As shown in Fig. 1, the PD176252 structure consisted of three
fragments. In order to reduce the cytotoxicity and improve
water solubility, the left part was replaced by phenylacetic
acid derivatives, which have commonly been used as frag-
ments in various lead compounds32 and drugs with anti-
inflammatory activity such as benzylpenicillin and diclofenac
sodium. Furthermore, natural amino acids containing L-
phenylalanine, L-tyrosine or L-tryptophan were inserted as an
intermediate part. Last but not the least, the unique structure
of the morpholine ring and imidazole ring with the desired
electron-rich feature is conducive to derivatives that readily
bind to receptors and various enzymes in biological systems
via a variety of weak interactions and exhibit a wide range of
biological activities.33,34 The structure of the compounds
used in this study is shown in Table 1.
The procedure for the synthesis of the target com-
pounds 5a–p is illustrated in Scheme 1. Phenylacetic acid
derivatives and the amino acid methyl ester hydrochloride
were reacted to form chemical intermediates, and the in-
termediates were subsequently hydrolyzed. Then, the com-
pounds 4a–h and 2-morpholinoethan-1-amine (or 2-(1H-
imidazol-1-yl)ethan-1-amine) were converted to the target
compounds 5a–p (Table 1). The results obtained confirm
that the synthesis route was fast and efficient. The synthe-
sis route of the compounds used in this study is shown
in Scheme 1.
CF3
NO2
NO2
CH3
CH3
F
F
F
CF3
Activity of ameliorating cisplatin-induced nephrotoxicity
The effect of the synthesized compounds on the cell viability
of HK2 cells was evaluated by the MTT assay. The HK2 cells
were treated with cisplatin alone or be treated with a combi-
nation of the compound and cisplatin. The results are shown
in Fig. 2. The lethality of cisplatin was detected in the normal
HK2 cells; nevertheless, a majority of analogues synthesized
in this work showed an inhibitory effect on cisplatin-induced
cell death. In particular, 5d and 5m obviously enhanced the
survival rate compared with PD176252 and increased it by
more than 40% and 30%, respectively, compared with the
control group of cisplatin. A dose–response study of the ana-
logues 5d and 5m, which was performed on HK2 cells, fur-
ther demonstrated that the analogues exhibited more
Fig. 1 The general design strategy.
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Scheme 1 Synthesis of compounds 5a–p. Reagents and conditions: (i) EDCI, HoBt, TEA, DCM, rt., 24 h; (ii) Na2CO3/CH3OH, 3–5 h; HCl/H2O; (iii)
EDCI, HoBt, TEA, DCM, rt., 24 h.
powerful potency than PD176252 and maintained the level of
protection in cells treated with cisplatin in a concentration-
dependent manner (Fig. 3).
concentrations of 5d, 5m and PD176252 on the viability of
HK2 cells by the MTT assay was used to study cell cytotoxicity
(Fig. 4). PD176252 reduced the viability of HK2 cells in a
dose-dependent manner, while 5d and 5m did not show any
significant cytotoxicity. This result was obtained by using a
low concentration to about 60 times the concentration of the
dosage. In this condition, the analogues 5d and 5m modified
by the PD176252 structure can indeed provide better safety
than the control.
It is not hard to find that the methyl group, as an
electron-donating group, gives better protection. However,
the analogues become more active after the indole ring is re-
placed by a benzene ring or p-hydroxyphenyl moiety. More-
over, good results can be obtained while using 1-ethyl-1H-
imidazole or 4-ethylmorpholine, both of which are simple and
facile to obtain, instead of the rightmost fragment of
PD176252.
Anti-inflammatory activity by real-time PCR
Proinflammatory mediators (TNF-α, IL-6 and MCP-1) play im-
portant roles in cisplatin-induced cell injury. Their expression
level in HK2 cells is a crucial indicator for the degree of
Cytotoxicity assay
During the discovery of a drug or a lead compound, there is
a high safety requirement for targets; therefore, this protec-
tion must be considered. In this case, the effect of different
Fig. 2 Effect of compounds on cell viability with cisplatin treatment.
Compounds and PD176252 (4 μM) restored cell viability in cisplatin-
treated HK2 cells (ATCC, USA). HK2 cells were pre-cultured for 24 h,
the cells were then treated with the indicated concentrations of com-
pounds for 6 h, and then exposed to 20 μM cisplatin for 24 h. The re-
sults are shown as means SD (n = 3) for at least three independent
experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the
control group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the
cisplatin-stimulated group. CIS, cisplatin; compounds, 5a–p; PD, PD
176252.
Fig. 3 Effect of different concentrations of 5d or 5m on cell
viability with cisplatin treatment. 5d and 5m restored cell viability in
cisplatin-treated HK2 cells (ATCC, USA). HK2 cells were pre-cultured
for 24 h, the cells were then treated with the indicated concentra-
tions of compounds for 6 h, and then exposed to 20 μM cisplatin
for 24 h. The results are shown as means
three independent experiments. *p 0.05, **p
0.001 compared with the control group; #p < 0.05, ##p < 0.01,
SD (n = 3) of at least
<
<
0.01, ***p
<
###p
cisplatin.
<
0.001 compared with cisplatin-stimulated group. CIS,
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nephrotoxicity.11 To assess whether 5d, 5m and PD176252 re-
duced kidney damage by the inhibition of inflammatory re-
sponses, the mRNA expression of TNF-α, IL-6 and MCP-1
was examined in HK2 cells using real-time PCR. Cisplatin
used alone significantly increased the expression of in-
flammatory response compared with normal saline-treated
cells, which is shown in Fig. 5, and the phenomenon is
the same as that reported in the literature.35–38 Addition-
ally, real-time PCR showed that 5d, 5m and PD176252
suppressed the levels of TNF-α, IL-6 and MCP-1. Perhaps,
an important reason for the effective protection of 5d and
5m in HK2 cells with cisplatin is that the gene transcrip-
tion and mRNA stabilization of proinflammatory mediators
were interdicted.
Fig.
4 Cytotoxicity of different concentrations of 5d, 5m or PD
Anti-inflammatory activity by enzyme-linked immuno-sorbent
assay (ELISA)
176252 on HK2 cells. 5d and 5m reduce the viability of HK2 cells
(ATCC, USA). HK2 cells were pre-cultured for 24 h, the cells were then
treated with the indicated concentrations of compounds for 24 h. The
results are shown as means SD (n = 3) of at least three independent
experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with con-
trol group.
The anti-inflammatory effect of the synthesized compounds
was further evaluated by ELISA analysis. From the results in
Fig. 6, it can be seen that 5d and PD176252 showed
Fig. 5 Effect of 5d or 5m for inflammatory response in HK2 cells with cisplatin treatment. Real-time PCR in HK2 cells (ATCC, USA). The results
demonstrate that the treatment of compounds reduced cisplatin-induced mRNA levels of TNF-α, IL-6, and MCP-1. Data represent the mean
SEM for 3–4 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control. #p < 0.05, ##p < 0.01, ###p < 0.001 com-
pared to the cisplatin-treated group. CIS, cisplatin; PD, PD176252.
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Fig. 6 Effect of 5d or 5m for inflammatory response in HK2 cells with cisplatin treatment. ELISA in HK2 cells (ATCC, USA). The results demonstrate
that the treatment of compounds reduced cisplatin-induced mRNA levels of TNF-α, IL-6, and MCP-1. Data represent the mean SEM for 3–4 in-
dependent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control. #p < 0.05, ##p < 0.01, ###p < 0.001 compared to
cisplatin-treated group. CIS, cisplatin; PD, PD 176252.
significant anti-inflammatory activity as evidenced by the de-
creased MCP-1, IL-6, and TNF-α expression levels. Moreover,
5d was significantly effective than the PD agent in the abate-
ment of TNF-α and MCP-1 levels.
Fig. 7 Effect of 5d and PD 176252 on cisplatin-induced phosphorylation of NF-κB p65. Results of western blot and quantitative data indicated that
5d and PD176252 had an effect on the phosphorylation of p65 for cisplatin-treated HK2 cells (ATCC, USA). Data represent the mean SEM for 3–4
independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control. #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the
cisplatin-treated group. CIS, cisplatin; PD, PD 176252.
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kines.39 The IκB protein is phosphorylated to turn on tran-
scriptions of inflammatory genes when cisplatin stimulates
the activation of NF-κB. Herein, in Fig. 7, the result of west-
ern blots and quantitative data showed that compound 5d
significantly blocked the cisplatin-induced phosphorylation
of NF-κB p65. It is a possible mechanism by which com-
pound 5d alleviated the cisplatin-induced inflammation. It is
noteworthy that PD176252 displayed a certain level of toxic-
ity, i.e., it gently induced p65 phosphorylation; however,
PD176252 significantly reduced the phosphorylation level of
NF-κB when cisplatin was added.
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Conclusions
In the in vitro experiments, PD176252 analogues, 5d and 5m,
possessed better activity in alleviating cisplatin-induced
nephrotoxicity through screening for a higher activity. A
dose–response study of 5d and 5m demonstrated that they
exhibited effective protection in a concentration-dependent
manner. It is worth noting that the synthesized analogues
have very low cytotoxicity. We further identified that 5d and
5m suppressed the inflammatory response via a NF-κB-
dependent mechanism. In conclusion, this study demon-
strated that compound 5d protects against cisplatin-induced
cell injury and inflammation. It may be further explored as a
preventive agent for cancer patients treated with cisplatin.
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Conflicts of interest
The authors declare no conflict of interest associated with
this manuscript.
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Acknowledgements
This work was supported in part by Key Program of Natural
Science Research by Education Department of Anhui Province
of China (KJ2017A707).
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