The synthesis, structure-toxicity relationship of cisplatin derivatives for the mechanism research of cisplatin-induced nephrotoxicity

Article abstract of DOI:10.1016/j.bmcl.2017.04.077

Cisplatin is a widely used antineoplastic drug, while its nephrotoxicity limits the clinical application. Although several mechanisms contributing to nephrotoxicity have been reported, the direct protein targets are unclear. Herein we reported the synthesis of 29 cisplatin derivatives and the structure-toxicity relationship (STR) of these compounds with MTT assay in human renal proximal tubule cells (HK-2) and pig kidney epithelial cells (LLC-PK1). To the best of our knowledge, this study represented the first report regarding the structure-toxicity relationship (STR) of cisplatin derivatives. The potency of biotin-pyridine conjugated derivative 3 met the requirement for target identification, and the preliminary chemical proteomics results suggested that it is a promising tool for further target identification of cisplatin-induced nephrotoxicity.

Full text of DOI:10.1016/j.bmcl.2017.04.077

Accepted Manuscript
The Synthesis, Structure-Toxicity Relationship of Cisplatin Derivatives for the
Mechanism Research of Cisplatin-induced Nephrotoxicity
Jing Hu, Tian-Ming Wu, Hong-Ze Li, Ze-Ping Zuo, Ying-Lan Zhao, Li Yang
PII:
S0960-894X(17)30454-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.077
BMCL 24930
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 January 2017
28 March 2017
25 April 2017
Please cite this article as: Hu, J., Wu, T-M., Li, H-Z., Zuo, Z-P., Zhao, Y-L., Yang, L., The Synthesis, Structure-
Toxicity Relationship of Cisplatin Derivatives for the Mechanism Research of Cisplatin-induced Nephrotoxicity,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.04.077
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The Synthesis, Structure-Toxicity Relationship of Cisplatin Derivatives for the
Mechanism Research of Cisplatin-induced Nephrotoxicity
Jing Hu, Tian-Ming Wu, Hong-Ze Li, Ze-Ping Zuo, Ying-Lan Zhao*, Li Yang*
Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation
Center, Chengdu 610041, China
*To whom correspondence should be addressed: Ying-Lan Zhao and Li Yang.
Tel: +86-28-85164063; Fax: +86-28-85164060, E-mail: zhaoyinglan@scu.edu.cn
(Ying-Lan Zhao), yangli@scu.edu.cn(Li Yang).
Abstract:
Cisplatin is a widely used antineoplastic drug, while its nephrotoxicity limits the
clinical application. Although several mechanisms contributing to nephrotoxicity have
been reported, the direct protein targets are unclear. Herein we reported the synthesis
of 29 cisplatin derivatives and the structure-toxicity relationship (STR) of these
compounds with MTT assay in human renal proximal tubule cells (HK-2) and pig
kidney epithelial cells (LLC-PK1). To the best of our knowledge, this study
represented the first report regarding the structure-toxicity relationship (STR) of
cisplatin derivatives. The potency of biotin-pyridine conjugated derivative 3 met the
requirement for target identification, and the preliminary chemical proteomics results
suggested that it is a promising tool for further target identification of
cisplatin-induced nephrotoxicity.
Keywords: Cisplatin, nephrotoxicity, Structure-toxicity relationship (STR), biotin
labeling, chemical proteomics, target identification
In recent years, target identification of small bioactive molecules received
increasing attention for its important role in both academic and pharmaceutical
- 1 -

5, 6
research.1-4 For instance, the target identification of thalidomide teratogenicity
offered a hint to the development of novel thalidomide drugs without teratogenic
activity.
Cisplatin (cis-diamminedichloroplatinum (II), CDDP) is one of the most
important anti-cancer drugs, which is widely used in the treatment of various solid
tumors, e.g., germ cell tumor, head and neck cancer, ovarian cancer and bladder
cancer. However, its clinical application is greatly hampered by its side effects,
including nephrotoxicity, neurotoxicity, ototoxicity, etc.7 Among them, nephrotoxicity
is the most severe adverse effect of cisplatin for clinic patients.8
After decades of investigation, several mechanisms contributing to
nephrotoxicity have been uncovered. DNA has conventionally been considered as the
target, as cisplatin can crosslink with DNA, thereby interfering with DNA repair and
causing DNA damage, and eventually inducing apoptosis in cancer cells.9 Some cell
death pathways were found to be involved in nephrotoxicity.10 Cisplatin can also be
transported into renal epithelial cells by some specific transporters, such as organic
cation transporters (OCT) and copper transporter 1 (Ctr1), contributing to the
accumulation of cisplatin in kidney cells.11 Cisplatin can be metabolized to a reactive
toxic thiol derivative through a biotransformation pathway that requires γ-glutamyl
transpeptidase, aminopeptidase and renal dipeptidase.12 Moreover, oxidative stress,
inflammation and immunity are believed to be involved in nephrotoxicity as well.13, 14
Although many mechanistic studies of cisplatin-induced neurotoxicity have been
reported previously, the direct protein targets are unclear. Among the frequently used
protocols for target identification, biotin is a widely used tag. Therefore, a
biotin-conjugated cisplatin derivative with comparable cisplatin-induced neurotoxicity
will be of great help for target identification. In this study, we synthesized for the first
time such a derivative as 3 (Scheme 2) on the basis of synthesis of a series of cisplatin
derivatives and demonstrated that 3 might be a powerful tool for target identification
based on the STR studies and pull-down assays.
As illustrated in Scheme 1 and Figure 1, 29 cisplatin derivatives were prepared in
parallel through the reaction of various ligands with K2PtCl4 by reported methods
- 2 -

(Scheme1).15,16
Scheme 1. Synthetic scheme for cisplatin derivatives. L denotes ligand.
Figure 1. Structure of cisplatin derivatives 1a-1m and 2a-2o
The renal epithelial toxicity of these compounds was evaluated in human renal
- 3 -

proximal tubule cells (HK-2) and pig kidney epithelial cells (LLC-PK1) by methyl
thiazolyl tetrazolium assay (MTT).17 The results are summarized in Table 1.
In the first round screening, compounds 1a (aliphatic amine), 1b (alicylic amine),
1c-1e (aniline) and 1f-1m (heteroarylic amine) were selected based on the
structure-diversity exploration of ligand. The toxicity study showed that compounds
1d, 1i and 1j exhibited comparable renal epithelial toxicity to cisplatin, indicating
pyridine is a promising ligand for further study, given the easy availability and
promising potency.
In the second round screening, the substitute effects on the pyridine ring were
first studied, with 2-aminopyridine (2a), unsubstituted pyridine (2b) or 3-bromide
pyridine (2c) to determine whether the bromine or amino group was required. It could
be seen in Table 1 that compound 2c exhibited comparable activity to cisplatin,
indicating that the bromine, but not the amino group, was essential for nephrotoxicity.
To further explore the effect of halogen position and halogen type on the pyridine
ring, 4 compounds (2d-2g) were synthesized. The results indicated that the position of
bromine greatly affected the activity of the compound, both the ortho- (2d) and para-
position (2e) reduced the potency. Replacement of the bromine on the pyridine ring
with fluorine (2f), or chloride (2g) groups yielded compounds with comparable
potency and without clear STR trends.
As the aim of present study was to tag cisplatin derivatives with biotin, the
compounds with different reactive groups, amino, hydroxyl and carboxyl attached to
pyridine were assessed (2h-2j). It was noted that the type of reactive group had great
impact on cell viability. Derivative with amino analogue (2i) was highly toxic to renal
epithelial cell lines, producing an extremely high level of cell death. However, cells
could tolerate very well the derivative with hydroxyl or carboxyl group (2h, 2j), and
compounds without bromine had little activity on the viability of these cells (2k-2m).
We also examined the antiproliferative effect of compounds with amino group
and different halogens in renal epithelial cell lines (2n, 2o),and the activity was
slightly reduced compared with their respective bromide analogues (2i).
- 4 -

Table 1. The nephrotoxicity profile of cisplatin and its analogues 1a-7a, 2a-2o
and 3.
IC50 (µM)
IC50 (µM)
Compounds
Compounds
(Ligand)
(Ligand)
HK-2 LLC-PK1
HK-2 LLC-PK1
17.0
55.2
15.8
7.75
2b (pyridine)
32.0
12.9
>100
24.4
Cisplatin
1a(ethylenediamine)
2c (3-Br Pyridine)
1b
> 100
>100
2d (2-Br pyridine)
64.0
37.2
(N-(2-Hydroxyethyl)piperazine)
1c (aniline)
36.6
20.9
>100
>100
16.6
43.1
2e (4-Br pyridine)
2f (3-F pyridine)
2g (3-Cl pyridine)
19.5
28.3
10.1
>100
36.1
9.40
1d (2-NH2 aniline)
1e (4-Cl-2-NH2 aniline)
2h (3-Br 5-OH
1f (2-NH2 thiazole)
>100
>100
78.4
28.0
>100
>100
37.0
8.19
64.0
19.8
>100
>100
26.2
pyridine)
2i (5-Br 3-NH2
1g (2-pyrimidinamine)
pyridine)
1h (2-pyrimidinamine
dimer)
2j (5-Br 3-COOH
>100
>100
pyridine)
1i (2-NH2-3-Br-5-methyl
pyridine)
2k (3-OH pyridine)
2l (3-NH2 pyridine)
33.1
32.0
1j (2-NH2-5-Br-pyridine)
1k (indole)
26.2
> 100
> 100
27.6
>100
> 100
>100
>100
41.6
2m (3-COOH pyridine) >100
2n (3-F 5-NH2 pyridine) 45.8
1l (benzothiazole)
2o (3-Cl 5-NH2
34.6
1m (HOBT)
> 100
> 100
49.4
36.3
pyridine)
2a (2-NH2 pyridine)
>100
8 （Ligand of 3）
>100
>100
3 (biotinylated 3-Br 5-NH2
pyridine)
53.2
44.8
a The cytotoxic effects of various compounds on HK-2 and LLC-PK1 cells are
determined by the MTT assay, and the results are expressed as the mean IC50
calculated from three independent experiments.
Based on the above STR studies, 2i was selected for biotin labeling. As shown in
Scheme 2, biotin-pyridine conjugated derivative 3 was synthesized via a 5-step
synthesis route using
4
as starting material. Biotin was bound to
3-amino-5-bromopyridine with a 6-carbon amide linker.18 Intermediates 5-7 and
18, 19
ligand 8 were prepared by reported methods
with modifications presented in
Scheme 2.20 23
-
- 5 -

Scheme 2. The synthesis of ligand 8 and cisplatin derivative 3
The activity (Table 1) of the biotinylated cisplatin derivative 3 met the
requirement for target identification. With derivatives 8 and 3, the preliminary
mechanism of cisplatin-induced nephrotoxicity was studied with the modified
pull-down assay following the listed protocol.24
Figure 2. Proteins bind to biotinylated platinum compound in HeLa cell nuclear extracts. HeLa
cell nuclear extracts were incubated with 3 (lane 1), 8 (lane 2) or DMSO (lane 3). 3
- 6 -

specific/cisplatin related bands are indicated with solid arrow heads.
As shown in Figure 2, there are three specific bands for 3, suggesting 3 has the
potential for target identification.
In conclusion, a series of platinum derivatives were prepared and all the
compounds were screened for cytotoxicity against HK-2 and LLC-PK1 cells. Among
all the tested compounds, 2i exhibited promising cytotoxicity and was used for
biotinylation; the biotinylated compound 3 was able to inhibit HK-2 cell growth and
bind some specific proteins. Based on the results of the present study, 3 may be
prospectively used in target exploration of cisplatin-induced nephrotoxicity.
Acknowledgments
This work was supported by Project of the National Natural Sciences Foundation of
China (81272459, 81322035) and Foundation for Young Scientist of Sichuan
Province (2014JQ0002) .
Supplementary data
The list of experimental details and spectroscopic characterization of 1-8 are available
online.
References and notes
1. Rix, U.; Superti-Furga, G. Nat. Chem. Biol. 2009, 5, 616.
2. Ziegler, S.; Pries, V.; Hedberg, C.; Waldmann, H. Angew. Chem. Int. Ed. Engl. 2013, 52, 2744.
3. Golkowski, M.; Vidadala, R. S.; Lombard, C. K.; Suh, H. W.; Maly, D. J.; Ong, S. E. J. Proteome Res.
2017, 16, 1216.
4. Fung, S. K.; Zou, T.; Cao, B.; Lee, P. Y.; Fung, Y. M.; Hu, D.; Lok, C. N.; Che, C. M. Angew. Chem. Int.
Ed. Engl. 2017, 56, 3892.
5. Ito, T.; Ando, H.; Suzuki, T.; Ogura, T.; Hotta, K.; Imamura, Y.; Yamaguchi, Y.; Handa, H. Science
2010, 327, 1345.
6. Fischer, E. S.; Bohm, K.; Lydeard, J. R.; Yang, H.; Stadler, M. B.; Cavadini, S.; Nagel, J.; Serluca, F.;
Acker, V.; Lingaraju, G. M.; Tichkule, R. B.; Schebesta, M.; Forrester, W. C.; Schirle, M.; Hassiepen, U.;
Ottl, J.; Hild, M.; Beckwith, R. E.; Harper, J. W.; Jenkins, J. L.; Thoma, N. H. Nature 2014, 512, 49.
7. Travis, L. B.; Fossa, S. D.; Sesso, H. D.; Frisina, R. D.; Herrmann, D. N.; Beard, C. J.; Feldman, D. R.;
Pagliaro, L. C.; Miller, R. C.; Vaughn, D. J.; Einhorn, L. H.; Cox, N. J.; Dolan, M. E. J. Natl. Cancer Inst.
- 7 -

2014, 106.
8. dos Santos, N. A.; Carvalho Rodrigues, M. A.; Martins, N. M.; dos Santos, A. C. Arch. Toxicol. 2012,
86, 1233.
9. Kelland, L. Nat. Rev. Cancer 2007, 7, 573.
10. Zhu, S.; Pabla, N.; Tang, C.; He, L.; Dong, Z. Arch. Toxicol. 2015, 89, 2197.
11. Harrach, S.; Ciarimboli, G. Front. Pharmacol. 2015, 6, 85.
12. Wainford, R. D.; Weaver, R. J.; Stewart, K. N.; Brown, P.; Hawksworth, G. M. Toxicology 2008, 249,
184.
13. Trujillo, J.; Chirino, Y. I.; Molina-Jijon, E.; Anderica-Romero, A. C.; Tapia, E.; Pedraza-Chaverri, J.
Redox Biol 2013, 1, 448.
14. Tadagavadi, R. K.; Reeves, W. B. J. Am. Soc. Nephrol. 2010, 21, 53.
15. Hoog, P.; Boldron, C.; Gamez, P.; Sliedregt-Bol, K.; Roland, I.; Pitie, M.; Kiss, R.; Meunier, B.;
Reedijk, J. J. Med. Chem. 2007, 50, 3148.
16. Typical procedure for the synthesis of cisplatin analogues.
The solution of L was added dropwise to the water solution of K2PtCl4 at constant stirring. After the
addition of the ligand, the solution was stirred for 2 days away from light. The precipitation was
filtered, washed several times with ethyl ether and dried in vacuum desiccator.
17. Briefly, cells (5000/well) were seeded in 96-well plates and cultured for 24 hours, followed by
treatment with the compounds for 48 h. 20 µL of 5 mg/mL MTT was added per well and incubated for
another 4 h at 37°C. Then the supernatant was removed and 150 µL/well DMSO was added. The
absorbance (OD) of each well was measured at 570 nm, using a Multiscan Mk3 elisa reader (Thermo
Scientific).
18. Wang, Z.; Gu, C.; Colby, T.; Shindo, T.; Balamurugan, R.; Waldmann, H.; Kaiser, M.; van der Hoorn,
R. A. Nat Chem Biol 2008, 4, 557.
19. Gros, L.; Lorente, S. O.; Jimenez, C. J.; Yardley, V.; Rattray, L.; Wharton, H.; Little, S.; Croft, S. L.;
Ruiz-Perez, L. M.; Gonzalez-Pacanowska, D.; Gilbert, I. H. J. Med. Chem. 2006, 49, 6094.
20. Thionyl chloride (1.4 equiv) was added dropwise to a stirred methanol suspension of amide
linker 4 (1 equiv) at 0 ℃. The resulting solution was stirred overnight at room temperature. The
solvent and thionyl chloride in excess were removed under reduced pressure.
21. HOBt (1.2 equiv), biotin (1.2 equiv), EDCI (1.2 equiv) and DIPEA (1 equiv) were dissolved in 100
mL DMF. Then added 5 (1 equiv) and DIPEA (3 equiv) in 50 mL DMF, and the solution was stirred
overnight at room temperature. The reaction was diluted with 600 mL DCM, then washed with
saturated solution of NaCl (3 × 80 mL), HCl 0.2 N (3 × 80 mL), saturated solution of NaHCO3 (1 × 80
mL), saturated solution of NaCl (1 × 80 mL). The solution was dried with magnesium sulfate and the
remaining solvents were removed under reduced pressure.
22. Lithium hydroxide (4 equiv) solution was added to 6 (1 equiv) dissolved in methanol. The solution
was heated to reflux overnight. The product 7 was precipitated as white solid. Then a hydrochloric
acid solution (6 N) was added until the pH was 5. The product was filtered, and dried under vacuum.
23. Thionyl chloride (20 equiv) was added dropwise to dry solid 7 (1 equiv) at 0 ℃. The solution was
stirred overnight at room temperature. The thionyl chloride in excess was removed under reduced
pressure. Then the product was dissolved in DCM and added dropwise to 3-amino-5-bromopyridine (1
equiv) in DCM at 0 ℃. The resulting mixture was stirred overnight at room temperature. The product
was filtered, washed with saturated solution of NaHCO3, and dried under vacuum.
24. HeLa cell nuclear extracts were prepared using KeyGEN nuclear and cytoplasmic protein
- 8 -

extraction kit according to the manuals. 200 μL nuclear extracts were incubated with 50 μM 3 (lane 1),
8 (lane 2) or same volume of DMSO (lane 3) on rotator at 4℃for several hours. Then the extracts were
incubated with 20 μL Pierce Streptavidin Agarose Resins(Thermo Fisher Scientific) on rotator at 4℃
overnight. The resins were washed twice with PBS and boiled in SDS-PAGE sample buffer. The samples
were used for the analysis by SDS-PAGE, followed by Coomassie blue staining.
- 9 -

Graphical abstract
The Synthesis, Structure-Toxicity Relationship of Cisplatin Derivatives for the
Mechanism Research of Cisplatin-induced Nephrotoxicity
Herein we reported the synthesis of 29 cisplatin derivatives and the structure-toxicity
relationship (STR) of these compounds with MTT assay. The potency of
biotin-pyridine conjugated derivative 3 met the requirement for target identification,
and the preliminary chemical proteomics results suggested that it is a promising tool
for further target identification of cisplatin-induced nephrotoxicity.
- 10 -