Rational design of anticancer platinum(IV) prodrugs

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Rational design of anticancer platinum(IV) prodrugs Shuren Zhanga, Xiaoyong Wangb,∗, Zijian Guoa,∗ a

State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, PR China State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, PR China ∗ Corresponding authors: e-mail address: [email protected]; [email protected] b

Contents 1. Introduction 2. PtIV complexes for overcoming cisplatin resistance 2.1 Intervening in “pre-target resistance” 2.2 Intervening in “on-target resistance” 2.3 Intervening in “post-target resistance” 2.4 Intervening in “off-target resistance” 2.5 Intervening in multiple stages 3. PtIV complexes for reducing systemic toxicities 3.1 PtIV complexes with tumor-targeted moieties 3.2 PtIV complexes attached to human serum albumin 4. PtIV complexes targeting tumor microenvironment 4.1 Targeting tumor-associated blood vessels 4.2 Interfering in tumor immune 5. Conclusions and perspectives Acknowledgments References

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Abstract Platinum-based anticancer drugs such as cisplatin play a crucial role in the treatment of various malignant tumors; however, their therapeutic effects are severely hampered by drug resistance and systemic toxicities. Recently, octahedral PtIV prodrugs have attracted much attention as the next generation of Pt-based anticancer drug candidates. PtIV complexes can be easily functionalized with some biologically innocent or active ligands to tune their pharmacological properties for overcoming the drawbacks of traditional PtII-based drugs. This review presents a comprehensive overview on the PtIV anticancer complexes that have been reported in the past decades from drug design perspectives. These complexes are classified into three categories based on their different design purposes, including to conquer cisplatin resistance, to ameliorate cisplatin toxicities, and to target tumor microenvironment. Representative examples of

Advances in Inorganic Chemistry ISSN 0898-8838 https://doi.org/10.1016/bs.adioch.2019.10.009

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each type are discussed in terms of design principle, chemical structure, cytotoxicity, mechanism of action, and possible limitations in medical application. In the end, future opportunities and challenges in this field are tentatively proposed. The information and views presented in this review are suggested to help generating new ideas and strategies to design and develop novel Pt-based anticancer drugs with high efficacy and low toxicity.

1. Introduction Platinum-based anticancer agents are the mainstay of clinical drugs for the treatment of various malignant tumors, especially testicular cancer.1 In fact, since the introduction of cisplatin into the treatment regimen of testicular cancer patients, cure rates for this disease have exceeded 95%.2 Platinum anticancer drugs represent one of the great success stories in the field of medicinal inorganic chemistry, highlighting the confluence of serendipity and rational design in drug development. In addition to cisplatin, two other Pt-based drugs, carboplatin and oxaliplatin, are approved worldwide for treating cancers (Fig. 1). Carboplatin received regulatory approval in 1988 and is primarily used to treat ovarian cancer. Oxaliplatin entered the clinic in 2002 and is used as a frontline drug for colorectal cancer in combination with 5-fluorouracil and folinic acid in a regimen referred to as “Folfox.”3 Several less common Pt-based drugs are approved in specific countries (Fig. 1). Nedaplatin is used in Japan for the treatment of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), esophageal cancer and head and neck cancers.4 Lobaplatin is used in China to treat inoperable metastatic breast cancer, chronic myelogenous leukemia (CML) and SCLC.5 Heptaplatin is approved in Korea for the treatment of gastric cancer.6 More recently, Miriplatin was approved in Japan for treating

Fig. 1 Chemical structures of clinically approved platinum anticancer drugs.

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hepatocellular carcinoma.7 Currently, Pt-based anticancer drugs are used in nearly 50% of all chemotherapeutic regimens administered in the clinical oncology treatment.8 Although Pt-based chemotherapy is quite successful in the clinic, there are still some major drawbacks including recurring drug resistance, doselimiting side effects and low bioavailability as well as the inability to administer orally.9 To overcome these drawbacks, many novel platinum complexes were developed such as trans oriented complexes, mono-functional complexes, and polynuclear complexes, which have been described in a recent review.10 Another popular strategy is the use of PtIV complexes, which are prodrugs of cisplatin or its derivatives. Actually, the anticancer potential of PtIV complexes has been recognized from half a century ago,11 but their clinical value was realized only in recent years. The physicochemical properties of PtIV complexes differ significantly from those of their PtII precursors, in that they have a kinetically inert d6 electronic configuration and a six coordinate octahedral geometry.12 Therefore, they can not only avoid diversion to off-target biological nucleophiles before reaching the purine bases in nuclear DNA, but also provide opportunities for fine tuning the kinetic stability, redox potential, lipophilicity and biological activity of the complexes through additional ligands.13 PtIV complexes are kinetically inert in blood plasma; while they could be reduced to the classical active PtII species in the hypoxic or reductive microenvironment of the tumors, which are expected to exert its therapeutic effect.14 Over the last two decades, numerous PtIV complexes have been reported as potential anticancer agents.15 In particular, tetraplatin, iproplatin, satraplatin and LA-12 have entered clinical trials (Fig. 2).16 Tetraplatin is one of the first PtIV agents to enter clinical trials.17 Although it can be rapidly reduced to its active PtII form in both tissue culture medium and undiluted

Fig. 2 Chemical structures of platinum(IV) agents that have undergone clinical trials.

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rat plasma, the severe neurotoxicity limited its further clinical application.18 Due to the presence of hydroxide axial ligands, iproplatin was found to be less prone to reduction by biological reductants than its analogue tetraplatin. However, it did not exhibit overwhelming activity as compared to that of cisplatin or carboplatin in clinical trials.19 The other two PtIV complexes, satraplatin and LA-12, adopting similar structures with suitable lipophilicity and stability for oral administration, are currently undergoing clinical trials.20 Satraplatin as the first reported oral platinum agent has undergone a variety of Phase I–III clinical trials for the treatment of several cancers. Although the complex exhibited better antitumor efficacy than that of cisplatin toward some tumors, it failed to be approved by the U.S. Food and Drug Administration (FDA), because the overall survival rate was not significantly improved.21 LA-12 with adamantylamine as a non-leaving amine group replacing the cyclohexylamine of satraplatin has completed Phase I clinical trial.22 Positive results have been obtained in vivo against A2780 cell line, which make the complex hopeful for further investigation in clinical trials.23 Although none of PtIV complexes has been used in the clinic, the initial success of these complexes has forcefully propelled the development of PtIV complexes for cancer treatment. All PtIV prodrugs bearing non-bioactive ligands failed in clinical trials; fortunately, PtIV complexes can be easily functionalized with bioactive molecules at the axial positions. PtIV prodrugs bearing bioactive axial ligands can release both PtII species and axial ligands simultaneously upon reduction, thus endowing them with “dual-threat” or “multi-threat” against cancer cells.24 Hence, researchers have turned to the development of PtIV complexes with bioactive molecules, and various multi-targeted PtIV complexes have been reported. In this review, we mainly focus on the design principles and strategies of PtIV complexes. According to the design goals, we divided them into three categories, including complexes for overcoming cisplatin resistance, for reducing systemic toxicities, and for targeting tumor microenvironment.

2. PtIV complexes for overcoming cisplatin resistance The mechanisms of tumor resistance to cisplatin have been extensively studied in recent years and were well summarized in another review.25 It is suggested that cisplatin resistance derives from multifactorial cellular alterations and can be classified as four stages based on functional and hierarchical parameters.26 They are (i) pre-target resistance, i.e., resistance before the

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binding of cisplatin to nuclear DNA; (ii) on-target resistance, i.e., resistance directly related to the DNA damage provoked by cisplatin; (iii) post-target resistance, i.e., resistance in the lethal signaling pathways triggered by DNA lesions; and (iv) off-target resistance, i.e., resistance influencing molecular circuitries that are not intimately associated with cisplatin-elicited DNA damage signals.27 Aiming at these stages, many PtIV complexes are designed and synthesized as well as investigated for overcoming the resistance to Pt-based anticancer drugs.

2.1 Intervening in “pre-target resistance” Before the binding of cisplatin to nuclear DNA (pre-target stage), multiple cellular factors may affect the drug uptake and metabolism in cancer cells, leading to reduced cellular accumulation of Pt and detoxification of Pt by thiol-containing biomolecules.28 These factors would result in fewer DNA lesions in the genomic DNA, increasing the survival chance of cancer cells. By tackling these factors, many PtIV complexes were developed for enhancing cytotoxicity and overcoming the drug resistance to traditional Pt-based drugs. Modification at the axial positions of PtIV complexes could finely tune their lipophilicity and solubility and then affect cellular uptake. Recently, a class of PtIV prodrugs of cisplatin with contrasting lipophilic and hydrophilic ligands were developed using a sequential acylation strategy.29 The stability, reduction rates, lipophilicity, aqueous solubility, anti-proliferative efficacies, and the correlations among the parameters of these complexes were investigated. The complexes with high lipophilicity result in better anti-proliferative effects in cisplatin-resistant ovarian cancer cell line A2780/cis. The resistance factor (RF) value (the IC50 value in cisplatinresistant cells to that in cisplatin-sensitive cells) is 10.6 for cisplatin, while the value decreases to 2.7 for complex 1 with the highest lipophilicity among the designed complexes (Fig. 3). Several PtIV prodrugs conjugating the

Fig. 3 Chemical structures of 1–4.

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lipophilic medium-chain fatty acid were synthesized with the same sense.30 Among them, complexes 2 and 3 markedly enhanced their lipophilicity, cellular accumulation, DNA-binding, and cytotoxicity as compared with cisplatin (Fig. 3). The RF value in A2780/A2780cisR cells is 4.9 for cisplatin, while those for 2 and 3 decreases to 0.7 and 1.1, respectively. Following a similar strategy, a PtIV prodrug of oxaliplatin (4), containing both lipophilic and hydrophilic axial ligands was also reported (Fig. 3).31 The overall results indicate that it could inhibit tumor growth in both orthotopic and xenograft tumor models in comparison to oxaliplatin. The RF values in A2780/ A2780-cisR for cisplatin, oxaliplatin and 4 are 15.53, 5.21, and 0.93, respectively. Cisplatin enters cancer cells mainly by two pathways including passive diffusion through the plasma membrane and active transport mediated by membrane proteins.2 Alterations in the expression level of membrane proteins have been associated with declined cellular accumulation as well as cisplatin resistance, both in preclinical models and in cancer patients.32 Therefore, the design of platinum complexes taken up by cancer cells via active transport holds promise in combating cisplatin resistance. Recently, complex 5, one of the most cytotoxic PtIV prodrugs to date, has been obtained by Zhu et al. (Fig. 4).33 This mono-carboxylated PtIV prodrug accumulated effectively and rapidly in cancer cells through a transportmediated process, quickly bound to DNA after activation, and induced p53-independent apoptosis within a short period of time. In A549 and A549cisR cells, the RF value is 4.3 for cisplatin, but is 1.8 for 5. The potential role of transporter(s) in the cellular accumulation of this PtIV prodrug broadens the strategy for designing more active PtIV prodrugs with the help of transporter-mediated cellular uptake, although the details of its active transport process require further exploration. An increase in glutathione (GSH) is found in cases of cisplatin resistance, and the reaction between aqueous cisplatin and GSH can induce detoxification of Pt.34 GSH-S-transferases (GSTs) represent a major group of

Fig. 4 Chemical structure of 5.

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detoxification enzymes, which catalyze the nucleophilic S-conjugation between the thiol group of GSH and electrophilic compounds.35 Innate and acquired resistance toward xenobiotic compounds, such as Pt-based drugs, have been associated with GST-catalyzed detoxification.36 Certain cell lines and tumors that show resistance to cisplatin have also demonstrated overexpression of GSTs.35 Over the past years, a series of Pt-based complexes coupled with GST inhibitory molecules were developed. For example, a PtIV complex 6 incorporating a cisplatin scaffold with two GSTs inhibitory ethacrynic acids was rationally designed to overcome the cisplatin resistance (Fig. 5).37 Complex 6 exhibited much lower IC50 values than cisplatin in a series of cisplatin-resistant breast MCF7 and T47D, lung A549 and colon HT29 cancer cells. GST P1-1 was chosen as the protein target in this study due to its importance in the mercapturic acid detoxification pathway. The results suggested that complex 6 binds to GST P1-1 at the dimer interface with a subsequent interaction between the ethacrynate ligands at both active sites. The Pt scaffold remains bound to the enzyme at the interface rather than to the anticipated DNA, which accounts for the strong and irreversible inhibition of enzymatic activity by 6.38 Even so, complex 6 did not readily release PtII species to exert a synergistic cytotoxic effect. Recently, a mono-functionalized PtIV complex 7 containing a hydroxido axial ligand was designed to enhance the H-bonding interactions with potential biological reductants, making it dissociate readily in an intracellular environment to yield a cytotoxic PtII derivative and a GST inhibitor (Fig. 5).39 Compared with 6 and cisplatin, complex 7 showed more potent anti-proliferative activity against cisplatin-sensitive and cisplatin-resistant cancer cells. Importantly, complex 7 was able to decrease tumor growth in vivo in a chicken embryo with significantly decreased toxicity relative to cisplatin, as evidenced by increased embryo survivals. Very recently, another PtIV complex 8 containing cisplatin and 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)-hexanol (NBDHEX), a stronger GSTs inhibitor than ethacrynic acid was reported

Fig. 5 Chemical structures of 6 and 7.

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Fig. 6 Chemical structures of 8 and 9.

(Fig. 6).40 Complex 8 showed more potent anticancer activity than cisplatin against all the tested cancer cells, especially toward cisplatin resistant A549/DDP cells, with a RF value of 0.37. Dhar et al. reported a unique dual-acting PtIV complex 9, a prodrug of cisplatin and pipobroman mimicking alkylating agent 6-BH (Fig. 6).41 It was constructed on a self-defense mechanism to protect the active drug components from early sequestration by elevated cellular thiols present in cisplatinresistant cancer cells. Complex 9 demonstrated a 20-fold higher cytotoxicity than cisplatin in cisplatin-resistant ovarian cancer cell line A2780/CP70. Detailed investigations on the mechanism of action suggested that complex 9 was able to induce decreases in cellular GSH, change mitochondrial bioenergetics, form mtDNA adducts, and ultimately lead to loss of mitochondrial mass in cisplatin-resistant cancer cells. Chromatin packages DNA into the microscopic space of the eukaryotic nucleus by wrapping DNA around core histone proteins, leading to a highly compact and tense structure,42 which may impede nuclear DNA platination to some extent. Histone deacetylases (HDACs) play an important regulatory role in the transcription of many genes through conformational changes of chromatin. Inhibition of HDACs can therefore dramatically affect chromatin structure and ultimately reprogram transcription.43 HDAC inhibitors result in hyper-acetylated histones, de-condensing chromatin to leave nuclear DNA in an open form, and making it more susceptible to DNAdamaging agents like cisplatin. In fact, some HDAC inhibitors have been used in combination with conventional chemotherapeutic agents to re-sensitize resistant cancer cells.44 Valproic acid (VA) is an established anti-epilepsy drug, which can inhibit HDACs, upregulate the cisplatinmediated DNA damage and enhance cytotoxicity to the cisplatin-resistant ovarian cancer cells.45 VA ligands were thus appended in the axial positions of a PtIV scaffold to develop a PtIV-VA conjugates (10, Fig. 7).46 The conjugate was then packaged into polyethylene glycol-polycaprolactone

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Fig. 7 Chemical structures of 10 and 11.

(PEG-PCL) nanoparticles or dispersed into a Tween 80 surfactant to facilitate its uptake into tumor cells. Both the complex and its nanoparticles showed substantially higher cytotoxicity than cisplatin across multiple cancer cell lines, even up to 12-times cytotoxic against A549 cells. More importantly, they displayed significant in vivo antitumor activity with decreased nephrotoxicity. The cytotoxicity of 10 was further tested on several tumor cell lines including four different highly malignant plural mesothelioma (MPM) cell lines.47 Notably, complex 10 demonstrated higher cytotoxicity than cisplatin against cisplatin-resistant MM98R cells, with its RF value decreasing from 6.1 for cisplatin to 1.0. A follow-up study gained a deeper insight into the mechanism of action of PtIV-HDAC inhibitor prodrugs with a view to optimizing the selectivity of the Pt framework and axial HDACinhibiting ligands.48 The study involved a library of PtIV derivatives of cisplatin, oxaliplatin and trans-[Pt(n-butylamine)(piperidino-piperidine)Cl2]+ incorporating either VA or another HDAC inhibitor, 4-phenyl-butyrate (PhB), in one or both axial positions. Among them, complex 11 was the most cytotoxic agent against several human cancer cell lines, roughly up to 115 times more cytotoxic than cisplatin in cisplatin-resistant ovarian cancer cell line A2780cisR (Fig. 7). The RF value for 11 in this pair of cells is only 0.85, while for cisplatin is up to 4.9. Additionally, all these conjugates demonstrated high HDAC inhibitory activity and intracellular DNA platination.

2.2 Intervening in “on-target resistance” The sensitivity of cancer cells to cisplatin is limited in the presence of a proficient DNA repair apparatus. Many cisplatin-resistant cell lines have been shown to have increased DNA repair capacity in comparison to sensitive counterparts.49 There are four major DNA repair pathways in cancer cells, including nucleotide excision repair (NER), base excision repair (BER),

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mismatch repair (MMR) and double-strand break repair. NER is the main mechanism responsible for repairing cisplatin-DNA adducts. An increased NER proficiency has been associated with cisplatin resistance in vitro, in murine models as well as in cohorts of cancer patients.50 Overexpression of NER components such as XPA, ERCC1, or ERCC1-XPF complex has been implicated in cisplatin-resistant tumors.51 The MMR system is believed to detect DNA adducts caused by cisplatin and engage in their repair, and if repair fail ultimately, it would transmit a pro-apoptotic signal. Accordingly, genes encoding MMR components such as mutS homolog 2 (MSH2) and mutL homolog 1 (MLH1) are frequently mutated or downregulated in the process of acquired cisplatin resistance.52 Loss of MMR with respect to cisplatin-DNA adducts therefore results in reduced apoptosis and, consequently, drug resistance. However, the clinical relevance of the loss of MMR to platinum-chemotherapy resistance is still controversial. Some data indicate a possible role in acquired drug resistance, whereas other data show no correlation with intrinsic resistance.53 Cisplatin-elicited DNA adducts can engender double-strand breaks (DSBs), which are normally repaired along with or shortly after DNA synthesis via homologous recombination (HR).54 Accordingly, HR-deficient neoplasms, such as those bearing loss-of-function mutations in the genes encoding breast cancer 1 (BRCA1) or breast cancer 2 (BRCA2), are generally more susceptible to the genotoxic effects of cisplatin than HR-proficient cancers of the same type. Moreover, the appearance of compensatory mutations in BRCA1 and BRCA2 that restore the functionality of HR has been shown to favor cisplatin resistance in breast carcinoma cells.55 In addition, other DNA damage repair proteins such as cyclin-dependent kinase 2 (CDK2), poly(ADPribose) polymerase 1 (PARP1) are related to cisplatin resistance to some extent.27,56 All these findings provide valuable insights into the development of rational approaches to circumvent cisplatin resistance, and several PtIV complexes targeting DNA damage repair are reported recently in succession.57–59 A PtIV anticancer prodrug 12 targeting NER by adding molecules to axial positions for blocking the interaction between ERCC1 and XPF has been synthesized (Fig. 8).57 Complex 12 showed a 34- and 88-fold higher cytotoxicity against cisplatin-resistant A2780cisR and A549cisR cells, respectively, than cisplatin. The RF values of cisplatin are 28.6 and 5.6 in A2780/A2780cisR and A549/A549cisR cells, and those of 12 are as low as 5.0 and 1.1, respectively. Besides, it induced higher levels of Pt-GG intra-strand crosslinks than cisplatin, suggesting that NER pathway

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Fig. 8 Chemical structures of 12 and 13.

was interrupted. The model of bifunctional PtIV prodrugs inhibiting NER provides a promising strategy to conquer the resistance to cisplatin. The CK2 inhibitor CX-4945 was also used as an axial ligand to design a IV Pt prodrug 13 (Fig. 8).58 This complex disrupted DNA damage response (DDR) through the suppression of CK2-phosphorylated MDC1, thereby combining the fork-head-associated domain of aprataxin with DNA DABs and finally inducing apoptosis in cancer cells. The complex exhibited high cytotoxicity toward a panel of human cancer cells, especially cisplatinresistant gastric cancer cell lines SGC7901/cDDP, and the RF value decreased from 2.42 for cisplatin to 0.78. BRCA1/2 are required for DNA DSBs repair mediated by homologous recombination (HR). Therefore, ovarian and breast cancers with mutations in these genes are hypersensitive to DNA cross-linking agents like cisplatin or carboplatin due to impaired HR, associated with a better survival rate. However, only a small fraction of tumors are BRCA-deficient and BRCA-proficient cancers are usually refractory to platinum agents owing to the efficient HR repair of DNA DSBs triggered by the drugs, which greatly restricts the therapeutic utility of platinum chemotherapy.55 Recently, we designed two RAD51-targeted PtIV-artesunate prodrugs to defeat cisplatin-refractory BRCA-proficient ovarian and breast cancers.59 The bi-artesunated complex 14 showed 13- and 11-fold enhancement in cytotoxicity compared with cisplatin against BRCA-proficient ovarian and breast cancer cells, respectively (Fig. 9). It entered cells efficiently and was reduced to PtII precursors with a concomitant release of artesunate. DNA DSBs induced by PtII species, reduced RAD51 expression, and

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Fig. 9 Chemical structure of 14.

inhibition of RAD51 foci triggered by artesunate were confirmed to occur inside Caov3 cells. The reduced formation of RAD51 foci would hinder HR to repair DNA DSBs, thereby potentiating the cytotoxicity of PtII species. This study demonstrates that introducing RAD51-targeted group(s) to PtIV prodrugs is a promising strategy for the treatment of platinumrefractory ovarian and breast cancers harboring proficient BRCA genes.

2.3 Intervening in “post-target resistance” Post-target resistance to cisplatin can result from a plethora of alterations including defects in the signal transduction pathways that normally elicit apoptosis in response to DNA damage as well as problems with the cell death executioner machinery itself.60 One of the most predominant mechanisms of the post-target resistance involves the inactivation of TP53 which occurs in approximately half of all human neoplasms.61 In addition, post-target cisplatin resistance appears to be significantly influenced by the expression level and functional status of BCL-2 family members and caspases that have a major role in the execution of apoptotic cell death. Many apoptosis-related proteins such as BCL-2, BCL-XL and MCL-1 are often upregulated in cisplatin-resistant tumors.62 In line with this notion, small molecules that antagonize the anti-apoptotic effects of BCL-2-like proteins (e.g., ABT263) have been intensively investigated in clinical trials, either as stand-alone anticancer interventions or in combination with various chemotherapeutics including cisplatin.63 Lippard et al. have reported the synthesis of two PtIV complexes conjugated with a vitamin E analog, α-tocopherol succinate (α-TOS), which inhibited anti-apoptotic proteins BCL-XL and BCL-2 proteins.64 One of the complexes exhibited impressive potency, 7–220 times greater than that of cisplatin or combinations of cisplatin and α-TOS, across several tumor cell

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Fig. 10 Chemical structure of 15.

Fig. 11 Chemical structure of 16.

lines (15, Fig. 10). Particularly, complex 15 showed 5.5-fold enhancement in cytotoxicity against cisplatin-resistant A2780/CP70 cells. The function of p53 has been correlated with cisplatin sensitivity in certain types of cancer cells.65 Thus, promoting p53 activation could be used as a strategy to increase the anticancer activity of cisplatin. The dual-targeting PtIV complex 16 carries not only the DNA-binding platinum warhead but also a small molecule fragment that can promote the p53 pathway (Fig. 11).66 Compared with cisplatin, complex 16 significantly increased the cytotoxicity in p53 wild-type but not in p53 null cells. Notably, it showed 32-fold higher cytotoxicity toward cisplatin-resistant A549cDDP cells, and the RF value declined from 6.8 for cisplatin to 0.8. Profound mechanistic studies revealed that complex 16 effectively entered cells, bound to genomic DNA, arrested the cell cycle at both the S and G2/M phases, induced p53 expression, and increased the apoptosis level.

2.4 Intervening in “off-target resistance” The susceptibility of cancer cells to cisplatin can also be limited by off-target mechanisms, that is, molecular circuitries that deliver compensatory survival signals even though they are not directly activated by cisplatin.26,27 For instance, overexpression of cyclooxygenases (COXs), enzymes that catalyze key steps in the biosynthesis of prostanoids, is implicated in cisplatin

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resistance.67 Overexpression of v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2), which is common in breast and ovarian carcinomas, has been suggested to promote cisplatin resistance.68 Several relatively unspecific adaptive responses to stress have been involved in cisplatin resistance. Such general responses include macro-autophagy and the so-called heat-shock response, that is, the adaptation of cells to increased temperatures and other conditions that promote protein unfolding.69 In addition, although cisplatin has been recognized as a DNA-damaging agent, only around 1% of intracellular Pt is bound to nuclear DNA, which implies that the great majority of intracellular platinum drug interact with other biomacromolecules or subcellular organelle. Among the non-DNA targets of platinum compounds, mitochondria, principal subcellular organelles responsible for energy metabolism and cell death, are closely relevant to cisplatin resistance of tumor cells, and have been proposed to be putative targets to modulate the sensitivity or resistance to cisplatin.70 In short, malignant cells can lose their sensitivity to cisplatin owing to a wide panel of molecular and functional alterations. This provides some new insights into the development of PtIV prodrugs to overcome cisplatin resistance. An asymmetric PtIV prodrug incorporating aspirin in one of its axial positions (17, Fig. 12) has been reported by two research groups independently.71 Aspirin is a highly effective nonsteroidal anti-inflammatory drug (NSAID) by inhibiting COXs, and it has been shown to prevent or alleviate cancers.72 The anticancer and anti-inflammatory properties of this compound were explored. It was found that complex 17 almost fully overcomes the drug resistance to cisplatin in cisplatin-resistant NSCLC cells A549R, with the RF value reducing from 5.38 for cisplatin to 1.11 for 17.

Fig. 12 Chemical structures of 17, 18, and 19.

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In addition, an in vivo assay demonstrated that 17 possesses higher antitumor efficacy with lower toxicity in comparison with cisplatin. Recently, complex 17 was further examined on preclinical mouse- and guinea pig-based models to prove its efficacy on the reduction side effects related to ototoxicity, locomotor activity, and clinical chemistry parameters.73 Complex 17 also showed high tolerability from the dose-dependent toxicity studies. More than 90% tumor reduction was observed in animals treated with 17 (14 mg/kg). In vitro studies demonstrated that when 17 is used in combination with a clinically relevant dose of radiation, its efficacy can be further improved. The properties of 17 highlighted its superior ability to reduce the toxicity, suggesting that the combination of cisplatin and an antiinflammatory drug in a single prodrug form could produce marked advantages in terms of efficacy and reduction of side effects. Afterward, numerous PtIV-NSAID conjugates were reported to enhance anticancer effect and overcome cisplatin resistance. For example, NSAIDs indomethacin and ibuprofen were introduced to PtIV center as axial ligands.74 Both prodrugs showed enhanced cytotoxicity toward cancer cell lines as compared with cisplatin. Particularly, PtIV-ibuprofen complex 18 exerted elevated cytotoxicity in cisplatin-refractory triple-negative breast cancer (TNBC) cell line MDA-MB-231, with an up to 400-fold lower IC50 values than that of cisplatin (Fig. 12). Recently, an asymmetric PtIV complex was formed via the conjugation of a tumor-targeting biotin and indomethacin (19, Fig. 12).75 The prodrug selectively accumulated in biotin receptor-overexpressed cancer cells and released both cisplatin and indomethacin moieties in cancer cells to synergistically inhibit tumor growth by damaging DNA and inhibiting COXs. Importantly, the RF value of cisplatin in SG-C7901/CDDP cells was 7.37, whereas that of the prodrug was as low as 0.29, indicating its excellent ability to overcome cisplatin resistance. Taking together, conjugating COXs inhibitors NSAIDs to PtIV prodrugs is able to effectively defeat the drug resistance to cisplatin in antitumor therapy. Mitochondria play important roles in the production of ATP and the intermediates needed for macromolecule biosynthesis and the activation of signaling pathways. Accumulating evidences suggest that mitochondrial bioenergetics, biosynthesis and signaling are required for tumorigenesis and chemo-resistance. Emerging studies have demonstrated that mitochondrial metabolism is potentially a fruitful arena for cancer therapy.76 A series of PtIV complexes targeting at mitochondrial metabolism were designed to enhance the antineoplastic efficacy and combat cisplatin resistance. Dhar and Lippard reported a potent PtIV prodrug mitaplatin comprised of cisplatin

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Fig. 13 Chemical structures of 20, 21, and 22.

and dichloroacetate (DCA) (20, Fig. 13).77 DCA is an inhibitor of glycolysis that activates oxidative phosphorylation (OXPHOS) through inhibiting pyruvate dehydrogenase kinases (PDK), thereby increasing the amount of pyruvate entering into mitochondria and promoting glucose oxidation rather than glycolysis.78 Complex 20 has displayed a dual-killing mode toward cancer cells, that is, damaging nuclear DNA by the platinum center and attacking mitochondria by the released DCA upon reduction. Its cytotoxicity in a variety of cancer cell lines is comparable to that of cisplatin. However, in cisplatin-resistant ovarian cancer cells A2780/CP70, complex 20 showed 1.8-fold lower IC50 value than cisplatin, and the RF values in A2780 and A2780/CP70 also decreased from 10.7 for cisplatin to 3.0 for 20. Later, it was found that 20 could induce more apoptosis in cisplatinresistant human epidermoid adenocarcinoma KB-CP 20 and hepatoma BEL 7404-CP 20 cells than cisplatin.79 DCA was also conjugated with a PtIV derivative of oxaliplatin to obtain complexes 21 and 22 (Fig. 13).80 These two complexes exhibited markedly higher cytotoxicity than that of oxaliplatin in cisplatin-resistant A2780/cisR cell lines, and the RF values are 1.5 and 1.0, respectively, while that for oxaliplatin is as high as 6.7. Recently, a series of mitochondria-targeted dual-action PtIV complexes with DCA ligands were designed to enhance the accumulation within the mitochondria and to maximize the effect of the PDK inhibitors.81 Complex 23 showed a marked improvement in vitro and in vivo activity as compare with cisplatin and overcome effectively cisplatin resistance in ovarian cancer cells (Fig. 14). Our group also reported a biotin and DCA modified PtIV prodrug 24, which enhanced the cellular uptake in HeLa cell lines expressing biotin receptor as compared with cisplatin and greatly suppressed the energy metabolism, including OXPHOS and glycolysis, staving the cancer cells to death (Fig. 14).82

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Fig. 14 Chemical structures of 23, 24, and 26.

Fig. 15 Chemical structure of 25.

Triphenylphosphine (TPP) has a remarkable activity to target mitochondria of cells due to its high lipophilic properties, delocalized positive charge, and appropriate size range83; so some TPP-modified PtIV complexes were developed to target mitochondrion as well as energy metabolism for cancer suppression. A hydrophobic mitochondria-targeted cisplatin prodrug 25 was constructed using a strain promoted alkyne–azide cycloaddition chemistry (Fig. 15).84 Complex 25 entered into the mitochondrial matrix to attack mtDNA and exhibited 16-fold lower IC50 value than cisplatin in cisplatin-resistant A2780/CP70 cells. In addition, an efficient delivery of 25 was achieved using a biocompatible polymeric nanoparticle based on

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biodegradable poly(lactic-co-glycolic acid)-block-poly-ethyleneglycol functionalized with a terminal TPP cation. The nano-entity has a remarkable activity to target mitochondria of cells, resulting in controlled release of cisplatin from 25 locally inside the mitochondrial matrix to attack mtDNA. Recently, we reported a di-TPP-tethered PtIV prodrug 26 (Fig. 14).85 It exhibited stronger disruptive effect on mitochondrial morphology, inhibited both mitochondria respiration and aerobic glycolysis to cut off the energy supply for tumor growth, thus showing an alternative cytostatic pathway for PtIV complexes. Several unspecific adaptive responses to stress have been implicated in cisplatin resistance.67 Heat-shock protein70 (HSP70) for example is a stress-inducible chaperone, which maintains protein homeostasis during normal cell growth but is upregulated and stabilizes its protein substrates until adverse conditions improve during a stress response. It is overexpressed in many cancers including colorectal cancer and is associated with cancer progression, poor prognosis and drug resistance.69,86 A subset of PtIV tumor penetrating peptide conjugates (27, 28) were designed to target the membrane-bound HSP70-positive (memHSP70+) phenotype in cancer cells (Fig. 16).87 The conjugates exhibit superior cytotoxicity as compared to oxaliplatin alone in Pt-resistant colorectal cancer cells with a relatively high memHSP70+ expression.

Fig. 16 Chemical structures of 27 and 28.

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2.5 Intervening in multiple stages In fact, cisplatin resistance is complicated and involved in several stages, targeting only one stage may not solve this problem effectively. Hence, the action of a PtIV complex specially targeting one mechanism of resistance may be limited in actual clinical use, and the development of complexes targeting different mechanisms of cisplatin resistance seems to be a promising direction. Therefore, many PtIV complexes with multi-action properties have been constructed to hit several therapeutic targets simultaneously. In contrast to the dual-action PtIV complexes, these complexes exert several biological activities at once to achieve different functions. Gibson et al. developed a quadruple-action PtIV prodrug 29 that simultaneously released four bioactive agents including cisplatin, DCA, PhB, and [Pt(1S,2S-diaminocyclohexane)(5,6-dimethyl-1,10-phenanthroline)]2+ (Pt56MeSS), in cancer cells (Fig. 17).88 DCA is an inhibitor of pyruvate dehydrogenase, PhB is an HDAC inhibitor, and Pt56Mess is a non-covalent DNA-binding cytotoxic agent. Complex 29 showed improved activity against both monolayer (2D) and spheroid (3D) cancer cells compared with cisplatin. Intriguingly, the complex exhibited 200- to 450-fold increased cytotoxicity relative to cisplatin toward KRAS-mutated pancreatic and colon cancer cells and displayed 40-fold selectivity to KRAS-mutated cells relative to noncancerous cells. Very recently, they reported triple-action PtIV prodrugs of cisplatin.89 The axial ligands include inhibitors of cyclooxygenase (COXi), HDAC (HDACi), or pyruvate dehydrogenase kinase (PDKi). These compounds were more cytotoxic than cisplatin and had lower resistance factors. The leading complex 30 (Fig. 17) showed up to 29-fold increased cytotoxicity in cisplatin-resistant ovarian cancer cell line

Fig. 17 Chemical structures of 29 and 30.

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C13* as compared with cisplatin, and the RF value is as low as 1.1 (10.1 for cisplatin). Therefore, by simultaneously targeting different resistance factors, the multitalented PtIV prodrugs have presented great potential to overcome cisplatin resistance derived from multifactorial cellular alterations. Although vast PtIV prodrugs have been synthesized to conquer cisplatin resistance, there are many problems to be solved. For example, most PtIV complexes mentioned above showed potential to overcome cisplatin resistance in monolayer cell models, however, the in vivo anti-tolerance potency of them is unclear. The complicated biological environment may influence the effectiveness of these PtIV complexes. Besides, the animal models for testing the action of defeating cisplatin resistance are lacking. Safety is another problem, which is largely overlooked in the drug design. To overcome cisplatin resistance and enhance cytotoxicity, PtIV complexes usually act in multi-pathways, which may boost the toxicity toward normal cells and cause undesired side effects.

3. PtIV complexes for reducing systemic toxicities In addition to inherent and acquired drug resistance, severe and doselimiting side effects also limit the clinical use of Pt-based chemotherapy.90 Actually, all cytotoxic chemotherapy drugs, including Pt-based drugs, display a range of severe side effects due to their poor selectivity for cancerous tissue over normal one. While these drugs are taken up by fast growing cancer cells, they are also taken up into other tissues that are fast growing. Fast growing tissues that can be affected by chemotherapy drugs include the mucous membranes of the mouth, throat, stomach and intestines, which lead to gastrointestinal toxicities, and bone marrow which can result in reduced white and red blood cell production. Hair follicles also comprise fast growing cells, implying that chemotherapy drugs can lead to the loss of hair (alopecia), as well as hair follicles in the ear (ototoxicity). Finally, chemotherapy drugs are commonly known to display nephrotoxicity, and hepatotoxicity. The nephrotoxicity arises because excretion of the drugs occurs in urine and the hepatotoxicity arises because the body tries to metabolize and detoxify the drugs in the liver, giving platinum agents the opportunity to be taken up by cells in both organs. It is noteworthy that the side effects experienced by patients treated with Pt-based drugs are prevalent, though the specific dose-limiting toxicity (DLT) is different for each drug. For cisplatin the DLT is nephrotoxicity, for carboplatin it is myelosuppression, and for oxaliplatin it is neurotoxicity.90

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In fact, only a small portion of the Pt administered to a patient reacts with nuclear DNA of cancer cells, and the reaction with off-target nucleophiles like serum proteins, especially human serum albumin, is also an important factor of toxic side effects induced by Pt-based drugs.25,91 The toxicity of a platinum complex is directly related to its reactivity. The bioreactivity of a platinum complex is largely determined by its labile groups. The more labile the group(s), the more reactive the complex and the more toxic side effects it produces. For example, the development of carboplatin was based directly on this structure-activity relationship. Replacement of the chloride ligands of cisplatin with the bis-carboxylate ligand of carboplatin results in a significantly reduced rate of aquation, and hence, a reduced level of toxicity when compared with cisplatin.2,10 Octahedral PtIV prodrugs are more inert to ligand substitution than the parent PtII complexes; therefore, they are often less toxic than their PtII precursors, and the two additional axial ligands provide an opportunity to attach tumor-targeted molecules to increase the specificity for tumor cells, thus reducing systemic toxicities ultimately.

3.1 PtIV complexes with tumor-targeted moieties Some receptors are overexpressed on the surface of cancer cells. Ligands that have specific interactions with these receptors have an affinity for cancer cells. Conjugating such ligands with the PtIV scaffold endows the prodrugs with the ability to enter cancer cells specifically. Thus, this strategy holds great potential to increase the selectivity of platinum agents to cancer cells and cellular accumulation, thereby improving cytotoxicity toward cancer cells and relieve toxicity toward normal cells. Both peptides, such as c(RGDfk) and chlorotoxin, and small molecules, such as biotin as well as glycose and its derivatives, have been conjugated with PtIV prodrugs.12,15b,92 These complexes show improved cytotoxicity in cancer cell lines that overexpress the corresponding receptors and decreased toxicity in normal cell lines as compared with cisplatin. A series of conjugates of a PtIV derivative of picoplatin with monomeric (31) and tetrameric (32) RGD-containing peptides were synthesized with the aim of exploiting their selectivity and high affinity for αVβ3 and αVβ5 integrins for targeted delivery to tumor cells overexpressing these receptors (Fig. 18).93 Complexes 31 and 32 exerted 2.6- and 20-fold higher cytotoxicity in SK-MEL-28 malignant melanoma cell line overexpressing these integrins than their PtII precursor picoplatin. However, the cytotoxicity

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Fig. 18 Chemical structures of 31 and 32.

of the conjugates was inhibited in CAPAN-1 pancreatic cancer cells and 1BR3G fibroblasts lacking αVβ3 and αVβ5 integrins. The results demonstrated the potential of these conjugates to improve anticancer potency and reduce side effects in normal cells. It has been proved that carbohydrate uptake of cancer cells significantly increased due to their unrestricted proliferation.94 Glucose transporter 1 (GLUT1), the most common glucose transporter, is widely overexpressed in many human cancers, and its overexpression is a prognostic indicator for cancers. Conjugating 2-deoxy glucose with therapeutic or diagnostic agents was successfully achieved as a GLUT-targeted drug for specific tumor delivery.95 Thus, a series of glycosylated PtIV prodrugs were designed and synthesized.96 The biological evaluations of these prodrugs indicated that the mannose conjugated PtIV complexes 33 and 34 exhibited a high potent cytotoxicity to cancer cells, especially to prostate cancer cell line LNCaP (Fig. 19). Furthermore, they showed low toxicity to the normal cell line 3T3, which again proved that tethering tumor-targeted ligands to PtIV center could improve the selectivity to cancer cells and decrease the toxicity to normal ones.

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Fig. 19 Chemical structures of 33 and 34.

Fig. 20 Chemical structures of 35 and 36.

Biotin is absorbed mainly through a sodium-dependent multi-vitamin transporter (SMVT), which is overexpressed in breast, lung, ovarian, and renal cancer cells.97 We recently reported two biotin-conjugated PtIV complexes 35 and 36, by tethering one or two biotin molecules to the axial positions of the PtIV scaffold, respectively (Fig. 20).98 The two PtIV complexes showed increased Pt accumulation in breast cancer cells and low accumulation of Pt in breast epithelial cells. Complex 35 showed 3.3-fold improved cytotoxicity against MDA-MB-231 breast cancer cells and 4.3-fold decreased toxicity against MCF-10A/vector human mammary epithelial cells as compared with cisplatin. Although the cancer-targeted PtIV complexes showed high selectivity in monolayer cell models, the in vivo anticancer potency of them is uncertain, as the complicated tumor environment may compromise the efficacy of these complexes. In addition, the targeting groups attached to PtIV complexes for receptor-mediated drug delivery must be extraordinarily sensitive because concentrations of biomolecules abnormally expressed in tumor tissues are very low. It should be noted that not all tumor cell types overexpress

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the same unique receptors, and the overexpressed receptors may also be present on normal tissues. Therefore, absolute targeting for cancer cells seems unattainable. In fact, many “tumor specific” target molecules such as receptors for biotin and integrins also exist in normal cells, which may pose the risk of off-target binding and undesired side effects.

3.2 PtIV complexes attached to human serum albumin A significant impediment to the clinical progression of PtIV prodrugs is premature reduction in the bloodstream, which declines their antitumor effect and causes undesired side effects.99 Lippard et al. presented a series of PtIV complexes designed to exploit the endogenous protein, human serum albumin (HSA), as a delivery device.100 Among them, the lead compound 37 exhibited excellent in vitro anticancer activity, showing 9–70 times higher activity than cisplatin in lung and ovarian cancer cell lines (Fig. 21). A strong non-covalent interaction between 37 and HSA permits the formation of Pt-HSA complex, which can act as a delivery vehicle for 37. This interaction with HSA protects 37 in the reducing environment of the blood and contributes to the stability of 37 in whole human blood. Complex 37 showed a half-life of 6.8 h in whole human blood, which is significantly longer than that of cisplatin (t1/2  20 min) or satraplatin (t1/2  6 min). This study provides new insights into overcoming the side effect of PtIV prodrugs or classical platinum-based drugs.

4. PtIV complexes targeting tumor microenvironment Conventional cancer therapies including chemotherapy and molecularly-targeted therapy are based on killing tumor cells or inhibiting tumor proliferation directly. In reality, cancer cells are not isolated in the

Fig. 21 Chemical structure of 37.

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human body, and the crosstalk between cancer cells and the surrounding microenvironment also plays an important role in cancer initiation and progression.94,101 Tumor microenvironments are highly flexible and their constant evolution facilitates tumor formation, metastasis and development of refractoriness to cancer therapy.102 Therefore, the rational design of platinum complexes targeting tumor microenvironment becomes an attractive approach for cancer treatment. Although the mechanism of cellular response to cisplatin has been well studied, the contribution of tumor microenvironment, including stromal cells, tumor-associated fibroblasts and immune cells, to the tumor growth and cisplatin activity is relatively unexplored.103 Because on one hand, the rapid growth of a tumor requires to get enough nutrients and oxygen from the surrounding microenvironment via angiogenesis, on the other hand, tumor cells also need to evade surveillance and chase from immune cells, we herein review the microenvironmenttargeted PtIV prodrugs from these two aspects.

4.1 Targeting tumor-associated blood vessels The large-scale growth of a tumor always requires an abundant blood supply. To obtain this blood supply, tumor cells can stimulate angiogenic factors to drive vascular growth by attracting and activating cells from within the microenvironment of the tumor.104 Therefore, many antiangiogenic PtIV prodrugs have been designed and developed in the past years. Lippard et al. conjugated peptides containing Arg-Gly-Asp (RGD) and Asn-GlyArg (NGR) to the axial position of PtIV complexes for targeting the tumor vasculature.105 RGD and NGR can be recognized by integrins αvβ3 and αvβ5 and the membrane-spanning surface protein aminopeptidase N, respectively, which are highly expressed in tumor-induced angiogenesis. Two phosphaplatins, a PtIV complex without a bioactive ligand, 38, and its PtII counterpart, targeting vascular endothelial growth factor (VEGF) receptor were reported (Fig. 22).106 They markedly inhibited both cell migration and tube formation in vitro and tumor vascularization in vivo, whereas cisplatin and carboplatin did not. Based on ATP binding competition assays, the researchers proposed that the antiangiogenic activity of these complexes is at least in part due to inhibition of the kinase domain of the growth factor receptor VEGFR-2. NMR studies suggest that sulfurcontaining protein side chains may be capable of forming metal-ligand complexes with the complexes through displacement of the Pt pyrophosphate ligand. Even though, the site(s) of platination in VEGFR-2 has not yet been

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Fig. 22 Chemical structures of 38 and 39.

identified. Recently, we developed a PtIV prodrug 39 with great potential for the therapy of metastatic triple-negative breast cancer (TNBC) through a synergistic action of anti-angiogenesis and anti-metastasis (Fig. 22).107 The mechanistic study indicates that complex 39 can disrupt the tube formation of HUV-EC cells effectively and reduce the intersegmental vessel (ISV) formation in zebrafish, which may cut off the supply of oxygen and nutrients and further kill tumor cells in situ and prevent tumor metastasis.

4.2 Interfering in tumor immune The loss of antigenicity and defects in antigen presentation of tumor cells enable them to escape from the immune system. Tumor cells also evade immune surveillance by suppressing the immune response via alteration of the tumor microenvironment. Attacking cancers by restoring and using the immune surveillance activity has been widely explored and is known as immunotherapy.108 Presently, attractive immunotherapy approaches mainly include chimeric antigen receptor (CAR) T-cell therapies, cancer vaccines, dendritic cell therapies, and immune checkpoint inhibitors. The common inhibitory immune checkpoints include programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin-domain containing-3 (TIM3), and indoleamine 2,3-dioxygenase (IDO).109 Targeted immune checkpoint therapy could increase the antitumor immune response by affecting T cells. In fact, cisplatin has been shown to promote the antigen-presenting ability of dendritic cells.110 A dose-dense regimen of cisplatin and paclitaxel reversed the immunosuppressive tumor microenvironment in platinumresistant ovarian cancer.111 These findings indicate that cisplatin participates

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in immune effector functions. In addition, clinical data have shown that immunotherapy can enhance the efficacy of common chemotherapeutics and are synergistic with radiation therapy.112 The IDO inhibitor methylthiohydantoin-tryptophan in combination with cisplatin regresses autochthonous murine breast tumors.112a Taking advantage of the potential synergy between platinum drugs and immunotherapy agents, many immuno-chemotherapeutic PtIV prodrugs were reported in recent years. A pioneering study of PtIV prodrugs with immune activity was carried out by Ang et al., who reported a series of immuno-chemotherapeutic PtIV prodrugs, which harbor formyl peptide receptor (FPR)-binding ligands at the axial position.113 The FPR-binding ligands activated innate immune effectors such as monocytes, dendritic cells, and natural killer cells, which enhanced the immune response. Peripheral blood mononuclear cells (PBMCs) pretreated with prodrugs that contain the WKYMVm peptide as the FPR-binding ligand showed higher cytotoxicity against MCF-7 and MDA-MB-231 cancer cells than PBMCs pretreated with cisplatin (40 and 41, Fig. 23). This effect might owe to the prodrug-enhanced secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) from the PBMCs. Intriguingly, the p53 mutant MDA-MB-231 cells that are resistant to cisplatin-induced and DNA damage-triggered cell death were sensitive to immune-mediated cytotoxicity. These observations indicate that multipronged immuno-chemotherapy is a promising strategy in the treatment of cisplatin-resistant tumors. An IDO inhibitor, PtIV-(D)-1-methyltryptophan conjugate, was obtained by introducing (D)-1-methyltryptophan (D-1-MT) in the axial position of a PtIV prodrug to boost T cells and in turn enhance the activity of cisplatin.114 The PtIV–D-1-MT conjugate 42 effectively inhibited IDO, blocked kynurenine production, and promoted T-cell proliferation (Fig. 24). Moreover, it showed high cytotoxicity against IDO-expressing A2780 and SKOV3 cells compared with cisplatin with 2- to 40-fold increased cytotoxicity. Impressively, it was able to induce cell death in both

Fig. 23 Chemical structures of 40 and 41.

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Fig. 24 Chemical structure of 42.

Fig. 25 Chemical structures of 43 and 44.

cisplatin-sensitive and -resistant ovarian cancer cells and showed no crossresistance with cisplatin. This work highlights the fact that immunotherapy can promote cisplatin-based chemotherapy in the cisplatin-refractory cancer cells. Besides IDO, tryptophan 2,3-dioxygenase (TDO) also belongs to immune checkpoints, and is expressed in many human cancers such as melanoma, colorectal, bladder, breast and lung cancers. TDO can catalyze the essential amino acid tryptophan (Trp) catabolism to N-formylkynurenine via the kynurenine (kyn) pathway.115 As a result, inadequate amount of tryptophan and accumulation of its production of kynurenine lead to anergy or apoptosis of T cells, thereby supporting formation and progression of tumor cells. Thus, blocking the expression of TDO is also a promising strategy to activate antitumor immunity.116 A series of PtIV complexes derived from the conjugation of PtII anticancer agents with an immune checkpoint TDO inhibitor were reported.117 These complexes not only showed significant cytotoxic effects on many cancer cell lines, but also enhanced antitumor immune response. Particularly, complex 43 displayed 35-fold more potency than cisplatin against TDO-overexpressed HepG-2 cancer cells (Fig. 25). Further study indicated that 43 could inhibit TDO via releasing a derivative of a TDO inhibitor and blocking production of

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kynurenine, resulting in T-cell activation and proliferation in vitro. More importantly, in vivo tests proved that it could promote the antitumor activity of cisplatin and suppress the expression of TDO as a potent immunemodulator. Elevated intra-tumoral immune infiltrate is associated with an improved prognosis in cancer of distinct origins.118 Recently a PtIV complex 44 based on cisplatin and the marketed drug tranilast was developed (Fig. 25).119 As compared in vitro to cisplatin, 44 showed increased cytotoxic activity against colon and lung cancer cells but decreased activity against immune cells. In addition, complex 44 was evaluated in tumor explants derived from colorectal cancer samples from patients subjected to intended curative surgery. Complex 44 induced strong intra-tumoral cytotoxic activity yet was associated with an elevated presence of immune cell infiltrate, suggesting a reduced cytotoxic activity against immune cells in colorectal cancer.

5. Conclusions and perspectives Cisplatin has been used in the clinic for more than 40 years. However, the severe systemic toxicities and acquired or inherent resistance largely limit its clinical performance. The more inert octahedral PtIV prodrugs represent a promising way to solve the drawbacks of cisplatin, as they can be modified with a wide range of ligands for targeting cancer cells, altering cytostatic pathways and combining with other therapeutic warheads. This review summarized recent progresses in the design and development of PtIV prodrugs according to three design aims: overcoming cisplatin resistance, reducing side effects and targeting tumor environment. Early reported dual- or multi-action PtIV prodrugs showed improved cytotoxicity, especially in cisplatin-resistant cancer cells, and they were often designed to mediate the related pathways inside cancer cells. With the increased knowledge of the influence of tumor microenvironment on the progress of tumors, cancer therapy paradigm has shifted from targeting tumor itself to tumor environment. Although some pioneering PtIV prodrugs targeting tumor microenvironment have been developed and showed synergistic effects, the rational design of microenvironment-targeted PtIV prodrugs is still a nascent area. Despite numerous PtIV complexes showed high cytotoxicity and unique action in monolayer cell models, the in vivo anticancer potency and mechanism of action of these prodrugs are unclear. Since the tumor environment may compromise the effectiveness of PtIV complexes, and their exact mechanism of action and the behavior in biological environment are

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not fully understood, the evaluation of anticancer effect and mechanism in vivo are highly desired. In the past years, many highly potent PtIV prodrugs were designed and synthesized, however, the data on their systemic toxicity are still scarce. Besides, little is known about their absorption, distribution, metabolism, and excretion properties in the human body. Hence, there is still much work to be done in the evaluation of systemic toxicity in vivo and the design of hypotoxic PtIV complexes.

Acknowledgments We acknowledge the financial support from the National Natural Science Foundation of China (Grants 21731004, 31570809, and 21877059), the National Basic Research Program of China (Grant 2015CB856300), and the Natural Science Foundation of Jiangsu Province (BK20150054).

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