DOI: 10.1002/cmdc.201300249

cis-[PtII(1R,2R-DACH)(3-acetoxy-1,1-cyclobutanedicarboxylato)] , a Water-Soluble, Oxalate-Freeand Stable Analogue of Oxaliplatin: Synthesis,Characterization, and Biological EvaluationsWeiping Liu,[a] Qingsong Ye,[a] Jing Jiang,[a] Liguang Lou,*[b] Yongping Xu,[b] Chengying Xie,[b]

and Mingjin Xie[c]

Oxaliplatin, developed by Kidani et al. ,[1] is a third-generationplatinum antitumor drug following cisplatin and carboplatin,and has been used worldwide in combination chemotherapeu-tic treatments of metastatic colorectal cancer.[2–4] Oxaliplatin isalso currently being explored for its potential as a treatmentoption after failure of cisplatin or carboplatin therapy, owing toits activity in cisplatin-resistant tumor models.[5] Oxaliplatin dif-fers from carboplatin importantly in that two ammine groupsof the latter are replaced by (1R,2R)-diaminocyclohexane(DACH), which is largely credited for the unique anticancerproperties of oxaliplatin. However, the efficacy of oxaliplatin is

often compromised due to the substantial risk of severe toxici-ties; in particular, neurotoxicity was found to be a dose-limit-ing liability, and various clinical approaches failed in the pre-vention of neurotoxicity withthis drug.[6] It has been demon-strated that oxalate, the leavinggroup as well as one of the me-tabolites of oxaliplatin, is respon-sible for neurotoxicity via deple-tion of cellular Ca2 + and/or Mg2 +

ion levels, as C2O42� is able to

form the respective insolublesalts CaC2O4 and MgC2O4.[7, 8]

Therefore, much effort has beendevoted to the development ofnew oxaliplatin analogues by

substituting the oxalate group with other leaving groups suchas malonate and its derivatives, 1,1-cyclobutanedicarboxylateand its derivatives, 2-hydropropanote, glycolate, pyruvate, andisocitrate.[9–14] Unfortunately most of the resulting complexesdid not have sufficient water solubility and/or stability for fur-ther studies, and few displayed anticancer activity superior orsimilar to that of oxaliplatin. Herein we present a new water-soluble, oxalate-free and stable derivative 1 that still exhibitshigh antitumor activity, but lower toxicity than oxaliplatin.

Complex 1 was synthesized by following the route illustrat-ed in Scheme 1. The preparation of 2 was described in our pre-vious work.[15, 16] Acetylation of 2 with acetyl chloride gave riseto 3, which was converted into the insoluble Ag salt 4 as an in-termediate. The formation of 3 was confirmed by elementalanalysis, 1H NMR, and 13C NNR (see Supporting Information).Two protons of CH2 split into two bands near d= 2.76 and2.44 ppm, due to different spatial orientations on a chair con-figuration of the cyclobutane ring after the introduction ofOCOCH3 at the 3-position. The carbonyl and methyl groupcarbon signals appeared at d= 170 and 20.4 ppm, respectively.

cis-[Pt(DACH)I2] 5 was prepared according the conventionalmethod[17] from commercially available potassium tetrachloro-platinate(II) as the starting material. The quantitative reactionin water of 4 with 5 yielded the desired complex 1 plus silver(I)iodide, which was separated out by filtration. The filtrate wascondensed at 45 8C under reduced pressure to produce 1 asa white product, followed by re-crystallization.

Complex 1 was fully characterized by elemental analysis,FABMS, IR, and 1H and 13C NNR as well as X-ray crystallography.All the data (Supporting Information) agree well with the

Scheme 1. The synthetic route for complex 1. Reagents and conditions : a) CH3COCl, 40–45 8C, 4 h; b) AgNO3,NaHCO3, 20–25 8C, 2 h; c) KI, dark, RT, 40 min; d) (1R,2R)-DACH, 94 %, RT, 2 h; e) 40–45 8C, 48 h.

[a] Prof. Dr. W. Liu, Dr. Q. Ye, Dr. J. JiangState Key Lab for Platinum Group MetalsKunming Institute of Precious Metals, 650106 Yunnan (China)

[b] Prof. Dr. L. Lou, Dr. Y. Xu, Dr. C. XieShanghai Institute of Materia MedicaChinese Academy of Science, 201203 Shanghai (China)E-mail : liguanglou1@126.com

[c] Prof. Dr. M. XieDepartment of Chemistry, Yunnan University, 650091 Yunnan (China)

Supporting information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cmdc.201300249.

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expected structure. The composition is in good agreementwith the calculated values based on the molecular formulaC14H22O6N2Pt. A peak of 100 % relative intensity develops atm/z 510, corresponding to [M]+ . It has three typical protonatedmolecular ion peaks reflecting the platinum isotopes: 194Pt(33 %), 195Pt (34 %), and 196Pt (25 %). The characteristic absorp-tion bands arising from NH2, C=O, and COO� groups are appar-ent at 3209, 1738, and 1646 cm�1, respectively. The coordina-tion of platinum with nitrogen and oxygen is supported by theabsorption bands at 482 and 439 cm�1. The nas(COO)�ns(COO)

value is >200 cm�1, suggesting that COO� acts as a monoden-tate ligand.[18] 1H NMR is consistent with the protons of com-plex 1 both in terms of chemical shifts and integration. All the13C NMR signals can be assigned to the corresponding carbonatoms in the complex molecule. The coordination of COO� toplatinum is accompanied by an upfield shift of the carbon res-onances from d= 172–176 ppm. Crystallographic studies wereconducted[19] in order to further elucidate the structure of com-plex 1.

The structure, as determined from single-crystal X-ray diffrac-tion data, is shown in Figure 1. The PtII atom is coordinated ona slightly distorted square by two nitrogen atoms of oneDACH molecule and two oxygen atoms of a 3-acetoxy-1,1-cy-clobutanedicarboxylate anion with the metal 0.044 � out of

the mean plane defined by the four donor atoms. The averagePt�N and Pt�O bond lengths are 2.02 and 2.00 �, respectively,while the N–Pt–N and O–Pt–O angles are 85.5(6)8 and 89.4(5)8,respectively; all of these agree with the data reported foroxaliplatin and carboplatin (Table 1).[20, 21] The six-memberedchelate ring formed between PtII

and 3-acetoxy-1,1-cyclobutane-dicarboxylate has a boat confor-mation, while the cyclobutanering adopts a chair configurationand is nearly perpendicular tothe PtII coordination plane.CCDC 903682 contains the sup-plementary crystallographic datafor this paper. These data canbe obtained free of charge from

The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Complex 1 is stable in water, as there is no apparent changein 1H NMR data for 1 in D2O (see Supporting Information)within 72 h at 25 or 37 8C; more importantly, it has an aqueoussolubility of 8 mg mL�1 in water at room temperature, sufficientfor further development and nearly equal to that of oxaliplatin(7.8 mg mL�1) ; [a]D

25 : + 56.48 (c = 6 mg mL�1 in water).The in vitro anticancer activity of 1 was compared with that

of the parent oxaliplatin in six human cancer cell lines repre-senting five tumor entities: non-small-cell lung carcinoma(A549/ATCC), gastric carcinoma (SGC-7901), ovarian carcinoma(SK-OV-3, OVCR/8), colon carcinoma (HT-29), and breast carci-noma (MCF-7/ADR). Among them, OVCR/8 is insensitive to car-boplatin, whereas MCF-7/ADR is relatively resistant to manychemotherapeutic agents including cisplatin and carboplatin.Carboplatin was also selected for comparison, as it has a leav-ing group similar to that of 1. Cell viability was determined bymeans of the SRB protocol after 72 h of drug treatment, andthe results are listed in Table 2. Compared with oxaliplatin,1 shows equal potency in HT-29 cells and slightly lower cyto-toxic potency in A549/ATCC, SGC-7901, SK-OV-3, OVCR/8, andMCF-7/ADR cells; however, its overall activity is much greaterthan that of carboplatin. Moreover, as expected, 1 exhibits sig-nificant activity against OVCR/8 and MCF-7/ADR cells, implyingits potential to overcome cross-resistance in certain tumors.

The antitumor activity of 1 and oxaliplatin was compared inmurine S180 sarcoma by following a well-establishedmethod.[22] Animal experiments were conducted in accordancewith the Institutional Animal Care and Use Committee guide-lines of Shanghai Institute of Materia Medica, Chinese Acade-my of Sciences. As evident from Table 3, 1 displays high antitu-mor activity in a dose-dependent manner with a 42.1 % inhibi-tion rate of tumor growth (p<0.01) at 60 mg kg�1, superior to9 mg kg�1 oxaliplatin.

Figure 1. Crystal structure of complex 1.

Table 2. In vitro anticancer activity of complex 1.

Complex IC50 [mm][a]

A549/ATCC SGC-7901 HT-29 SK-OV-3 OVCR/8 MCF-7/ADR

1 2.56�0.27 4.38�0.37 2.01�0.21 27.2�3.2 9.96�1.3 15.0�1.2oxaliplatin 1.14�0.21 3.33�0.27 2.09�0.19 14.4�1.2 5.71�0.45 10.4�1.3

carboplatin 41.9�3.5 26.8�2.1 68.1�7.6 38.7�4.5 259�17 154�18

[a] Values are the mean�SD of n = 3 experiments, each performed in triplicate; t = 72 h.

Table 1. Selected bond lengths and angles for complex 1.

Bond l [�] Bond ] [8]

Pt1�O3 2.014(12) N1�Pt1�N2 85.5(6)Pt1�N1 1.952(15) N2�Pt1�O3 90.5(5)Pt1�O1 2.021(14) N2�Pt1�O1 177.4(6)Pt1�N2 2.014(15) N1�Pt1�O3 176.0(6)

N1�Pt1�O1 94.6(6)O3�Pt1�O1 89.4(5)

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In conclusion, a new water-soluble and oxalate-free deriva-tive of the clinically established anticancer drug oxaliplatin, cis-[PtII(1R,2R-DACH)(3-acetoxy-1,1-cyclobutanedicarboxylato)] , wassynthesized and characterized by elemental analysis, FABMS,FTIR, and 1H and 13C NMR, along with single-crystal X-ray dif-fraction. It was evaluated for cytotoxicity against six humancancer cell lines and for antitumor activity in a murine S180sarcoma model. The results show that despite its cytotoxic po-tency being slightly lower than that of oxaliplatin, complex1 exhibits high antitumor activity and is less toxic, a promisingbehavior that warrants further research and development.

Acknowledgements

The authors are grateful to the National and Yunnan ProvincialScience Foundation of P. R. China (Grant No. 21161010) and theChina National Research and Development Program (Grant No.2012BAE06B08) for financial support.

Keywords: antitumor agents · biological activity · oxaliplatin ·platinum · synthesis

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Received: June 1, 2013Published online on July 3, 2013

Table 3. In vivo antitumor activity of 1 and oxaliplatin in KM mice with Sarcoma 180.[a]

Group Dose [mg kg�1] Number of Mice Mean body weight [g] Tumor weight [g] Inhibition [%]D1 D8 D1 D8 mean�SD

control vehicle 10 10 23.60 34.40 2.15�0.341 60 7 7 23.57 29.93 1.24�0.25 42.1**

120 7 6 23.64 21.33 1.01�0.26 53.2**oxaliplatin 9 7 7 23.62 27.86 1.45�0.37 32.5**

[a] Tumor-bearing mice were i.p. administered 1, oxaliplatin, or vehicle on days 1, 4, and 7; **p<0.01 vs. con-trol.

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