Keywords

17.1 Introduction

Wide range of solid tumors are often characterized with hypoxia which is a result of mismatch between oxygen delivery and consumption. Hypoxia has remarkable effect on tumor progression, for example, (1) selection of genotypes that favor hypoxia reoxygenation injury (TP53 mutations) and (2) change in gene expression that suppresses apoptosis and support autophagy (Wigerup et al. 2016). Hypoxia is also responsible for enhancing receptor tyrosine kinase-mediated signaling, angiogenesis, vasculogenesis, tumor invasion, and metastasis. Immune activity is also suppressed due to the hypoxia (Muz et al. 2015). In addition, hypoxia prompts generation of ROS that reduce the genomic stability and the downregulation of DNA repair pathways. Therefore, hypoxia often leads to the chemoresistance to the majority of the tumors (Begg and Tavassoli 2020). Therefore, an alternative strategy to overcome hypoxia is thus of paramount importance in the treatment of solid cancers. Strategic development of functional molecules is based on three parameters. (1) Anti-hypoxia agents like MnO2 or hemoglobin-based O2 carriers primarily reverse the hypoxia by generating O2 (Li et al. 2021a), although the oxygenation strategy is limited by cytotoxicity, lower blood circulation, and poor penetration within a tumor, (2) hypoxia-active nanoparticles/agents are activated only in hypoxic tumor microenvironment (Wang et al. 2019), and (3) hypoxia-targeting agents target biomarkers of tumor hypoxia to improve the efficacy of the existing chemotherapeutic drugs. Considering the enhanced level of HIF-1 and mTOR pathways in hypoxia, hypoxia-targeting agents were developed for treating hypoxia (Burroughs et al. 2013a). The preferred aerobic glycolysis in cancer cells rather than the oxidative phosphorylation pathway results in the accumulation of reducing agents and oxidoreductases (NADH or NADPH, β-glucuronidase, alkaline phosphatase nitroreductase, DT-diaphorase, and cytochrome P450 reductase), making the cancer cell microenvironment as reducing (Jiang 2017). The reducing enzymes typically can reduce hypoxia-active or bio-reductive prodrugs through one- or two-electron reductions. In normoxic cancer cells, back oxidation of the reduced form of the prodrug to the oxidized form is kinetically favored and hence may result into the poor efficacy of the prodrug. However, in a hypoxic condition, the enhanced lifetime of the radical anion intermediate or the reduced form of the prodrug facilitates the forward reactions and makes the prodrugs to be converted into the cytotoxic agent efficiently. The deferential reversibility of the reduction-oxidation reaction ensures the prodrug activation in hypoxia condition, resulting in hypoxia-selective cell death. Among the several types of bio-reductive prodrugs, the unique redox and kinetic properties of cobalt complexes make them potential tools for the development of hypoxia-selective prodrugs. Cobalt(III) complexes are kinetically inert, and the rate of ligand substitution reactions is significantly lower than the other first-row metal on the periodic table. In contrast, cobalt in +2 oxidation state is labile making the ligand substitution reaction much faster and facile. These differences in lability of the cobalt complexes between these two oxidation states have enabled the development of Co(III)-based prodrugs that undergo reduction in biological systems to form labile Co(II) complexes resulting in subsequent release of cytotoxic drugs and exhibit hypoxia-selective anticancer activity (Sharma et al. 2019; Renfrew 2014). There are several different classes of Co(III) complexes with the ultimate objective of selectively targeting hypoxic environments. The present book chapter illustrated recent advances on the development of Co(III)-based hypoxia-selective anticancer agents (Heffern et al. 2013).

17.1.1 Proteins Involved in Inducing Hypoxic Cancer Cells

There are several proteins which are responsible for these characteristics of hypoxic tumor cells, namely:

HIF-1α

HIF-1α is an oxygen concentration-responsive protein. HIF-1α acts as oxygen concentration indicator in the human cells (Masoud and Li 2015). Generally it has short lifetime inside the cell. It gets synthesized via phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways which are independent of oxygen concentration inside the cell (Vaupel et al. 2001). However, the degradation process of this protein is controlled by oxygen-dependent degradation domain (Tianchi et al. 2017). In normoxia condition, when oxygen concentration is normal (between 10 and 20%), then P402 and P564 amino acid residues of HIF-1α undergo hydroxylation in the presence of O2, 2-oxoglutarate, and active PHD (prolyl hydroxylation domain) (Lee et al. 2004a). HIF-1α gets identified by von Hippel-Lindau tumor suppressor gene and undergoes ubiquitination followed by proteasomal degradation by 26S proteasome (Ke et al. 2006) as both hydroxylation of P402/P564 needs sufficient amount of oxygen (Burroughs et al. 2013b). Hence in normoxia condition, HIF-1α gets metabolized, but deep-seated hypoxic tumor cells with poor concentration of oxygen lead to stabilization of HIF-1α (Lee et al. 2004b). Hypoxia-inducible factor-1α activates a number of cancer-causing gene, i.e., vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), angiopoeitin-1 (ANGPT1), etc., and promotes cell survival, cancer metathesis, tumor invasion, and angiogenesis (Hong et al. 2004). In hypoxic condition when physiological oxygen concentration is low, all of the above explained processes get stalled, which results in the increase of concentration of HIF-1α in cell cytosol that enters to the cell nucleus, where it binds with previously present HIF-1β and forms heterodimer HIF-1α (Marxsen et al. 2004). HIF-1 activates transcription of more than 60 hypoxia-responsive genes. These genes are generally associated with cancer encoding angiogenic factors, survival factors, glucose transporters, and glycolytic enzyme (Ziello et al. 2007).

VEGF

Vascular endothelial growth factor (VEGF) is induced in the hypoxic tumor cells by HIF-1α protein-dependent process. This protein plays an important role in increasing vascular permeability, elevation of interstitial fluid pressure, and endothelial proliferation. This further leads to angiogenesis. VEGF also inhibits dendritic cell maturation and induction of prostaglandins (Morfoisse et al. 2014).

GLUT-1

In hypoxic conditions HIF-1α protein is upregulated. The glucose transporter proteins (GLUTs) control the glucose metabolism into the cancer cells. Upon activation of the HIF-1α protein, the GLUT-1 protein gets overexpressed in the hypoxic tumor cells. This GLUT-1 protein generally shifts the glucose metabolism into the in the cancer cells toward glycolysis, and this process is associated with the formation of acidic byproducts of glycolysis pathway and lowering cancel cellular pH (Chung et al. 2009).

MMP

Matrix metalloproteases are overexpressed by the HIF-1α protein. The MMP proteins get accumulated in the extracellular part of the tumor cells. The MMP proteins destroy the epithelial barrier, and this phenomenon facilitates the angiogenesis and tumor migration (Kessenbrock et al. 2010).

17.1.2 Challenges of Hypoxic Tumor Treatment

All these processes discussed above are involved in the hypoxic tumor microenvironment and induce the special characteristics of the hypoxic environment. The high concentration of reducing agents inside the cancer cells makes them resistive to many conventional antiproliferative drugs as the antiproliferative drugs generally are associated with the generation of reactive oxygen species (ROS) or DNA-binding properties (Calman et al. 1980; Yokoyama et al. 2017; Perillo et al. 2020). The lack of oxygen in these hypoxic tumor cells makes the generation of ROS difficult. Also the DNA-binding drugs are generally metal complexes, where metal centers get reduced in the presence of high concentrations of reducing agents (Li et al. 2017). And the lower pH of the hypoxic tumor cells affects the activity of slightly basic anticancer drugs like doxorubicin, where doxorubicin is converted into a charged form in the low pH of the hypoxic cancer cells, and it results in a lower accumulation of doxorubicin in the cells (Gerweck et al. 1999; Raghunand and Gillies 2000). The solid exterior of the hypoxic tumor cells also makes them resistant to ionizing radiations. Hence, we require an effective strategy to overcome the abovementioned properties of hypoxic cancer cells for their treatment.

17.2 Bio-Reductive Prodrugs as Hypoxia-Selective Anticancer Agents

17.2.1 Organic Molecules as Bio-Reductive Hypoxia-Selective Anticancer Prodrugs

Several hypoxia-activated prodrugs can be used for a hypoxic cell-selective activity like tirapazamine, AQ4N, PR-104, apaziquone, evofosfamide, etc. These drugs can be selectively activated in the hypoxic media using high intracellular reducing atmosphere and cytochrome P450 oxidoreductase, and they exhibited cytotoxicity by pH-dependent DNA-damaging ability and downregulation of hypoxic proteins like HIF-1α, CA-IX, and VEGF. These bio-reductive compounds are currently in clinical trials for hypoxia-selective anticancer agents (Table 17.1).

Table 17.1 Organic hypoxia-selective anticancer agents and their mode of action
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17.2.2 Transition Metal Complexes as Bio-Reductive Hypoxia-Selective Anticancer Agents

Transition metal complexes also are emergent in hypoxia-selective therapy. Transition metal complexes provide a wide range of tunable properties like geometry, oxidation states, redox properties, etc. Hence generally these properties are now being exploited for hypoxia-selective treatment including hypoxia-selective drug delivery.

17.2.2.1 Platinum Complexes as Hypoxia-Selective Prodrugs

Among these transition metal complexes, Pt(IV) complexes are emergent for their bio-reductive properties. Pt(IV) complexes can get converted to Pt(II) by the high concentration of reducing agents present in the hypoxic tumor cells. This property of Pt(IV) complexes is used for hypoxia-selective delivery of anticancer drugs along with the DNA-binding ability of Pt(II) complexes which also attributes to the activity of the Pt(IV) complexes in hypoxia (Galsky et al. 2011; Wexselblatt and Gibson 2012; Vouillamoz-Lorenz et al. 2003). Brynzak et al. in 2016 explored the cytotoxic effect of satraplatin derivatives and satraplatin against the hypoxic cancer cell spheroids. The complexes exhibited cytotoxicity in the range of 6.4–0.20 μM but observed less drug localization into the tumor spheroids. The complexes get reduced in the hypoxic microenvironment into Pt(II) followed by release of cytotoxic agents (Brynzak et al. 2016). But the problem arises from the soft nature of the Pt(II) center, as they can get easily bound with intercellular sulfur-containing reducing agents like glutathione, and this factor drastically reduces the activity of the complexes, and in tandem, it may raise the heavy metal toxicity in the body.

17.2.2.2 Cobalt Complexes as Hypoxia-Selective Prodrugs

Here Co(III) complexes have emerged as an effective solution to overcome all these problems. Co is essential for our body as cobalamins. Co metabolism in the human body is also well explored. And Co(III) complexes are bio-reductive in nature, which makes them a suitable choice for hypoxia-selective chemotherapy. In the highly reducing environment of hypoxic tumor cells, the Co(III) center of the complex gets reduced to Co(II). The reduced Co(II) center readily releases a bidentate ligand into the hypoxic environment. This bidentate ligand is generally an anticancer drug. Hence Co(III) complexes themselves can act as hypoxia-selective prodrugs.

17.2.2.2.1 Ternary Co(III) Complexes with Bidentate Anticancer Agents

Tetradentate ternary N,N,N,N-based ligands like cyclams, cyclanes, and TPA provide a very good template for stable Co(III) complexes. The complexes can further be bound with bidentate cytotoxic agents for hypoxia-selective drug delivery under bio-deducible atmosphere. For a long time back, researchers have been utilizing these types of Co(III) complexes as drug delivery agents. In 2006 Bonnitcha et al. used X-ray absorption near-edge spectroscopy (XANEX) in cellulo and reported the release of drugs by Co(III) complexes in cellular media via the conversion of Co(III) to Co(II) (Bonnitcha et al. 2006).

17.2.2.2.1.1 Cyclen- and Cyclam-Based sp3 N,N,N,N Donor Ternary Co(III) Complexes

Among the N,N,N,N donor ligands, cyclam and cyclens consist of sp3 donor N atoms, and these complexes can exhibit bio-reductive properties under the hypoxic conditions. Ahn et al. in 2006 reported the synthesis of cyclen-based Co(III) complex with 8-hydroxyquinoline (1) and DNA minor groove alkylator ligand azaCBI (2). These two bidentate ligands are known for their DNA-binding properties. The 8-hydroxyquinoline and azaCBI were released from the complex in hypoxic conditions. As evident from the HPLC in human plasma, the release of ligands was also observed in hypoxic HT29 cell lines, but in A549 cells, cytochrome P450 reductase is overexpressed. There the release of the ligands was not so prominent. The complexes were also found to be releasing the cytotoxic agents in the presence of radiolytic radiations (Ahn et al. 2006). Lu et al. in 2011 reported N-alkylated cyclen Co(III) complexes of 1-(choromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo(3,2-f quinoline-5-ol) (3–8) as radiolytically activable prodrug. The seco-6-azaCBI-TMI is an azaCBI derivative, and it also works as DNA minor groove alkylator. The complexes are lipophilic due to cationic charges, and they can enter the cells via passive diffusion. The compounds also can be reduced by outer cellular Mito reductases, and it exhibits some promising radiolytically activable prodrug properties. The IC50 value of the complex in the normoxic condition in SKOV3 and HT29 cell lines are 2.26 and 2.4 μM, respectively, whereas in the hypoxic condition, the IC50 values are 0.5 and 0.4 μM (Lu et al. 2011). In 2012, Hambley et al. synthesized several cyclen-based complex of cobalt with different bidentate oxygen co-ligands including hydroxamic acid, β-diketone, and catechol (9–23). Hydroxamic acid complexes have a potential for targeting tumor under the acidic and hypoxic condition as the protonation of the acid changed the formal charge of metal which get reduced at a more positive reduction value under acidic conditions. These alter the characteristics of the complex cellular uptake. Under the alkaline condition, the hydroxamate forms dominate which increases the negative reduction potential allowing lower disassociation of ligand and lower cellular uptake (Bonnitcha et al. 2012). Chang et al. in 2013 reported several cyclen- and cyclam-based cross-bridged Co(III) complexes (24–26) with minor DNA groove alkylator bidentate ligand 8-hydroxyquinoline and azaCBI derivatives for their DNA alkylation properties to release them in the hypoxic cancer cell microenvironment and attain hypoxia-selective cytotoxicity. The complexes exhibited higher cytotoxicity in the hypoxic condition as compared to the free DNA alkylator ligand. For complex 25, it was 81–212 times more cytotoxic in hypoxia than 20% oxygen concentration in a series of 10 human tumor cell lines. But the complex did not exhibit significant cytotoxicity in the xenograft model of HT29 cell lines. Hence this result limits the pharmacological activity of the complex in vivo (Chang et al. 2013). He also reported cross-bridged cyclam-Co(III) complexes with cytotoxic bidentate ligands and observed a 200-fold greater potency of the complexes in hypoxia than that of normoxia.

Other sp3 4 N donor ligands like tren also are widely used for preparation of hypoxia-sensitive bio-reducible Co(III) complexes. Gopinathan et al. in 2014 reported two Co(III) complexes with trien and phen ligands (27–28). The complexes bind with CT-DNA as a groove finder as evident from UV-Vis, Fluorescence spectroscopy, and cyclic voltammetry as well as viscosity measurements. The complexes were found to be cytotoxic against human liver cancer cells. The complexes also exhibited antibacterial and antimicrobial activities in vitro (Gopinathan et al. 2014). Kozsup et al. in 2021 also reported 16 Co(III) complexes (29–37) with N,N,N,N donor ligands like tris and bidentate ligand flavonoids. The flavonoids are polyphenolic natural products that have antibacterial, antioxidant, antiviral, cardioprotective, and antitumor activities. The tren complexes were found to be more stable. The tren complex 32 also exhibited slight hypoxia selectivity with moderate cytotoxicity (Kozsup et al. 2021). Also 8-quinolinol cobalt(III) complexes (38–39) were synthesized by Ware et al. using a tetradentate cyclen as an auxiliary ligand. It showed that with the increase in charge density and hydrophilicity of the complex, their cellular uptake decreased and reduced the cytotoxicity. The complex shows quasi-reversible redox behavior at −440 and − 300 mV, but the cytotoxicity of the complexes has not been reported experimentally (Fig. 17.1).

Fig. 17.1
39 chemical structures. They exhibit varying bond formations of C o, N, O, and H, with the charges moving from positive to negative, from 1 through 39.

Structure of cyclen- and cyclam-based sp3 N,N,N,N donor ternary Co(III) complexes (1–39)

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17.2.2.2.1.2 Tpa- and Tren-Based sp2 N,N,N and sp3 N Donor Ternary Co(III) Complexes

Pyridine nitrogen are better sigma donors than that of other sp2 or sp3 nitrogen donor atoms. This is due to the fact that in pyridine nitrogen the lone pair over nitrogen atom resides in the orbital with higher percentage of p character. Hence there are several reports of pyridine containing sp2 N,N,N and sp3 N ligands like tpa and tren to form stable bio-reductive ternary Co(III) complexes.

Yamamoto et al. (2012) reported Co(III) ternary complexes (40–43) with coumarin fluorophore. The bidentate ancillary ligands are fluorescent mimics of hydroxamic acids. In hypoxic condition they are subjected to release the cytotoxic agents. Among the series of the compounds, complexes 42 and 12 exhibited the highest electron-withdrawing effects of hydroxamate moieties. The electronic-withdrawing groups of the nicotinic acid moieties decrease the stability of the Co(III) state. These complexes exhibited pH-sensitive delivery of fluorescent coumarin into the hypoxic and acidic tumor microenvironment (Yamamoto et al. 2012). That year, Bonnitcha et al. also reported the synthesis of several tpa- and tren-based complex of cobalt with different bidentate oxygen co-ligands including hydroxamic acid, β-diketone, and catechol (44–53) (Bonnitcha et al. 2012). The tren complexes have more negative reduction potential which is irreversible because of the interaction of the π-acceptor pyridyl rings with the metal center. The decrease in reduction potential increased solvolysis which further enhances the hindrance of waves for reoxidation. The tpa complex with the same ancillary ligand has a lesser negative reduction potential because of the σ interaction of amine with the metal centers and also observed greater reversibility due to π-back bonding interactions which decrease the ligand lability. The cytotoxicity of tpa complex with β-diketone is almost similar to the free ligand with IC50 values ranging from 74 μM to 66 μM in both hypoxic and normoxic conditions, while the tren complex shows a decrease in cytotoxicity indicating the masking of the ligand with tren ligand. Hydroxamic acid complexes have a potential for targeting tumor under the acidic and hypoxic condition as the protonation of the acid changed the formal charge of metal which get reduced at a more positive reduction value under acidic conditions. These alter the characteristics of the complex cellular uptake. Under the alkaline condition, the hydroxamate forms dominate which increases the negative reduction potential allowing lower disassociation of ligand and lower cellular uptake.

The cytotoxin curcumin is a natural anticancer drug isolated from Curcuma longa (turmeric) and has the potential to act as antiproliferative, antimetastatic, and antiangiogenic agent. Low solubility, rapid metabolism, and low bioavailability upon administration orally have posed a major problem in exploiting curcumin as anticancer drugs. In 2013, Renfrew et al. synthesized cobalt complexes with curcumin along with tris(2-methylpyridine)amine as ancillary ligands (54) which is found to increase the stability and solubility in aqueous medium. It also enhanced uptake and penetration in tumor hypoxic cells. The complexes showed one-electron reduction with IC50 value of around 30 μM against colorectal cancer cell line DLD-1. The study showed that the Co-TPA moiety didn’t show any cytotoxicity alone but upon release of cytotoxin curcumin by reduction of the metal center (Renfrew et al. 2013). O’Neill et al. in 2017 reported the synthesis and anticancer activity of four TPA-based Co(III) complexes (55–58) and their structure-activity relationship. The complexes were characterized by 1H, 13C, 59Co NMR, HRMS, IR, and UV-visible spectroscopy. The cyclic voltammetry of complex 55 exhibited a quasi-reversible redox character involving acetylacetone, but in a sustained reducing environment, the acetylacetone was lost from the complex, whereas 56 to 58 exhibited irreversible one-electron reduction character in the cyclic voltammetry, which concurs with the more facile release of acetylacetone ligand. The increase of carboxylic acid (-COOH) groups increases the stabilization of the complexes by inducing more electron density toward the pyridine moieties, thereby stabilizing Co(III) center. The relaxivity (r2/r1) ratio was also found to be in the range of 4.0–8.7. The complexes were found to be almost nontoxic against DLD-1 colorectal cancer cells as greater than 60% of cell viability at the highest concentrations. In 56 to 58, there is a decreased cellular uptake for the decrease in surface area to volume ratio. The complex TPA3 also exhibited an increase in MRI signal intensity in the inner cellular region but not in the outer region indicating the formation of Co(II) inside the cells (O’Neill et al. 2017). Buglyó et al. in 2017 reported 16 Co(III) complexes (59–75) with 4 N donor tripodal ligands with hydroxamate ligand derivatives. The cyclic voltammetry suggests that Co(III)abap complex possesses Co(III) to Co(II) reduction potential far below than that of the biological reduction range. From the structure-activity studies, they have also found that the +3 oxidation state is highly stabilized by the presence of a doubly protonated form of benzo hydroxamate. The in vitro studies also signify the release of hydroxamates in biological media (Buglyó et al. 2017). Kozsup et al. in 2021 also reported 16 Co(III) complexes (76–83) with N,N,N,N donor ligands like tpa, tren, and bidentate ligand flavonoids. The flavonoids are polyphenolic natural products that have antibacterial, antioxidant, antiviral, cardioprotective, and antitumor activities. The tren complexes were found to be more stable. Tren complex 83 also exhibited slight hypoxia selectivity with moderate cytotoxicity (Kozsup et al. 2021). Palmeira-Mello et al. in 2020 reported three Co(III)-based complexes: [Co(esc)(py2en)]ClO4·(CH3OH)2, [CoIII(esc)(TPA)]ClO4·3H2O, and [CoIII(bipy)2(esc)]ClO4·2.5H2O (84–86). In air argon and dioxygen conditions, Co(III) center gets reduced to Co(II) by biologically relevant reducing agents like ascorbic acid, cysteine, and glutathione. The reduction of the metal center is observed to be more facile in hypoxic conditions, and the rate of reduction is 86 > 85 > 84. Complex 86 exhibited cytotoxicity against HCT-116, but not against HT-29 or HEK293. Complex 85 was also reported to exhibit electrostatic DNA-binding abilities (Palmeira-Mello et al. 2020a). Cobalt complexes (87–91) with esculetin and different ancillary ligand py2en, TPA, and bipy have been synthesized. Esculetin (6,7-dihydroxycoumarin) is a derivative of coumarin which is present in plants and has antibacterial and anticancer activity. It was also found to inhibit angiogenesis-induced vascular endothelial growth factor (VEGF) and caspase-mediated apoptosis. All the complexes showed an irreversible reduction with a large ΔE value ranging from 0.30 V to 0.41 V. Since the complex reduction potential depended on the electron-donating potential of the ancillary ligand, there was an observed anodic shift with the highest in py2en complex followed by TPA and bipy complex. The reduction potential of py2en complex and bipy complex was −0.27 V and − 0.22 V which falls under the biological reduction window, while the complex 3D had −0.08 V which is outside the reduction window. Although py2en complex reduction potential is under a biological window, it could not be reduced by biological reductase. TPA and bipy complex showed a reduction by decreasing the absorbance at around 390 nm of all the reducing agents with more effective conversion or dissociation of esculetin in lower oxygen level shown by complex bipy complex in spite of its reduction potential outside the biological window. Cytotoxicity assay against HCT-116 and HT-29 cell lines showed that py2en and TPA complexes have IC50 > 100 μM in both normoxic and hypoxic conditions and bipy complex shows higher toxicity with IC50 value 31 μM in HCT-116 hypoxic cells only. All complexes showed electrostatic DNA interactions (Palmeira-mello et al. 2020b). Batista et al. synthesized another cobalt complex (92–94) with triazole ligand and ancillary ligands TPA, Py2en, and Py2enMe2. A class of triazole, i.e., carboxyamidotriazole, is a promising cancer therapy drug and has many pharmaceutical applications. The oxime moiety of the triazole ligand is bidentately linked to the metal providing the stability from immature reduction. The effect of the ancillary ligand has been shown effective in the reduction potential of the metal center. The replacement of pyridine and tertiary amine in TPA ligand by two secondary amines in py2en observed a cathodic shift, and again when it was replaced by methylated py2en, there was an observed anodic shift in the complex. The CH3 groups act as steric hindrance groups that keep the nitrogen atom away from the cobalt instead of increasing the basicity of amines through inductive effects. These increased the bond length, and hence the ability of N-atom for σ-donation readily decreased causing an anodic shift. The hypoxia selectivity of the complex has been demonstrated using ascorbic acid depleting in the presence of oxygen (Batista et al. 2018).

Melphalan is a derivative of amino acid phenylalanine anticancer drugs of the nitrogen mustard class. The toxicity of the drugs was shown by alkylation of DNA bases, rendering replication and transcription of DNA. The aziridinium form as an intermediate attacks the nucleic acid bases of DNA, and the availability of lone pair in the nitrogen mustard atom defined its reactivity. So in order to tune the reactivity of the nitrogen mustard family, a large alkyl or aryl group has been attached. In 2020, De Souza et al. synthesized a Co(III) L-phenylalanine complex with TPA, Py2en, Py2enMe, and bipy as ancillary ligands (95–97). The electron donor capability of the ligand has been observed by the cathodic shift in the potential with the py2en ligand the most negative attributed to its σ-donor ability of two pyridyl and two 20 amine group followed by TPA due to the presence of three pyridyl and one 30 amine group. For complex with Py2enMe, a positive cathodic shift is observed due to the presence of the CH3 group which causes the steric hindrance, and the bipy complex which has the weakest σ-donor and π-acceptor shows a positive reduction potential. ΔE value of all complexes ranges from 0.14 V to 0.20 V showing irreversibility reduction. Ascorbic acid reduction shows O2-dependent dissociation of L-phe at pH 7.4 by TPA, Py2en, and Py2enMe complex, while bipy complex showed independent disassociation unaffected by the presence of oxygen or change in pH (De Souza et al. 2020) (Fig. 17.2).

Fig. 17.2
22 chemical structures. 1 for 40 to 43, 1 each for 44 through 58, 1 for 59 through 75, 2 for the bha H and phebha H, and 1 for the N H 2 - N series. They exhibit varying bond formations involving R 2, R 1, N, O, N H, and C o.figure 2

Structure of tpa- and tren-based sp2 N,N,N and sp3 N donor ternary Co(III) complexes (40–97)

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17.2.2.2.1.3 Bimetallic N,N,N,N Donor Ternary Co(III) Complexes

Quinizarin is an anthracycline class of drugs that shows anticancer activity against breast, lung, and ovarian cancers as well as neuroblastomas and leukemias. Quinizarin is present in a variety of anthracycline which is a class of antineoplastic drugs as a redox-active moiety and known to inhibit the growth of bacteria and have antiproliferative activity. Hence there are few reports of quinizarin-based bimetallic Co(III) ternary complexes with both cyclen and tpa type of ligands. Kozsup et al. in 2020 synthesized cobalt(III) complexes (98–101) with quinizarin. Tris(2-aminoethyl)amine (tren) and tris(2-pyridylmethyl)amine (TPA) are taken as ancillary ligands. The two ligands show reversible reduction potential at −450 to −800 mV, and the sulfonated ligand shows less negative reduction potential by 150 mV assigned to the electron-withdrawing potential of the sulfur atom. Both the complexes showed affinity toward HSA (human serum albumin) in which quinSH3 ligand shows two binding sites on albumin. The complexes do not seem to have any affinity for HSA, but their reduction potential falls under the biological reduction window and has the potential to reduce under hypoxia conditions and initiate ROS generation (Kozsup et al. 2020a). The cytotoxic activity of both the complex was identified by Crlikova et al. in 2020 in HeLa cells, HCT-116 cells, MCF-7 cells, A2780 cells, and A2780 cisR. TPA is more potent than tren complex with IC50 ranging from 26.4 to 15 μM in all cell types. The TPA compound which is more lipophilic as compared to tren compound showed higher cellular accumulation in HCT-116 cells and showed greater DNA association by intercalative DNA-binding mode and increasing the rigidity of DNA, while TPA complex is proposed to bind with DNA by groove-binding mode. For antiproliferative efficiency, TPA complex was higher because of its redox potentials and its ability to form radicals that can cleave DNA by generating singlet oxygen via hydroxyl radicals (Crlikova et al. 2020). Kozsup et al. in 2020 reported four Co(III) complexes (102–105) with ternary N,N,N,N donor ligands like tris, tpa, and bidentate ligands quinH2 and quinSH3 (Fig. 17.3). The complexes exhibited binding affinity toward human serum albumin protein. The sulfonate complexes make the reduction potential of Co(III) center slightly more negative, and it also increases the binding affinity of the complexes toward HAS (Kozsup et al. 2020b).

Fig. 17.3
8 chemical structures. 1 each for 98 through 105. They exhibit varying bond formations involving C o, N H 2, O, N, and X with a positive charge. X = S O 2.

Structure of bimetallic N,N,N,N donor ternary Co(III) complexes (98–105)

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17.2.2.2.2 Bio-Reductive Co(III) Complexes with Acetylacetonate Ligands

Acetylacetonate type of ligands also provides stability to the cobalt(III) complexes, and utilizing this type of ligands, we can attach cytotoxic agents like nitrogen mustard for bio-reductive drug release. These types of complexes showed promising results against the hypoxic tumor cells which lower pH levels.

17.2.2.2.2.1 Binary Co(III) Complexes with Acetylacetonate and Nitrogen Mustard Ligands

Since 1942 nitrogen mustard compounds are known as very good alkylating agent. The labile Cl groups present in the nitrogen mustard compounds attribute to the cross-linking properties of the compound to DNA, and thereby they can prevent DNA replication and gradually lead to tumor cell death. Hence the release of nitrogen mustard compounds as cytotoxins in hypoxic tumor microenvironment is an effective strategy for hypoxic cancer treatment.

Ware et al. in (1991) showed that cobalt complexes with nitrogen mustard (106–109) could be hypoxia-selective cytotoxins. The cytotoxicity and selectivity of the compounds were evaluated in cell lines AA8 and UV4. The complexes showed similar cytotoxicity with the free ligand which showed that the toxicity was due to the release of the ligand after reduction. Acac derivatives as ancillary ligand with the chlorine substituent having higher reduction potential (−0.13 V) were shown to have large aerobic toxicity, while the complex with meacac ligand showed higher cytotoxicity in hypoxia conditions (Ware and Wilson 1991). Ware et al. in 1993 reported two types of Co(III) complexes with acac and nitrogen mustard ligands (110–113). For improvement of the cytotoxic efficacy of the complex, they also prepared a similar Co(III) complex with anticancer nitrogen mustard drug melphalan. The melphalan-based complex exhibited higher cytotoxicity, where IC50 values were ranging from 34 ± 0.8 to 1.36 ± 0.06 μM in AA8 cell line and subline UV4. These melphalan-based complexes exhibited the release of melphalan in a hypoxic environment, thus resulting to the higher cytotoxicity of the melphalan-based complexes (Ware et al. 1993). In 2000, Ware et al. also synthesized a tridentate nitrogen mustard complex with cobalt(III) (114–115) but found it to be less toxic than the bidentate nitrogen mustard which might be due to deactivation or masking of the mustard group in coordination with metal center (Ware et al. 2000) (Fig. 17.4).

Fig. 17.4
7 chemical structures. 1 each for 106 to 107 and 108 to 109, 1 each for 110 through 113, and 1 for 114 to 115. They exhibit varying bond formations involving R, O, C o, N, C l, N, H, N H 2, N 2 O, and X, with a positive charge. X = H, C l and R = H, M e.

Structure of binary Co(III) complexes with acetylacetonate and nitrogen mustard ligands (106–115)

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17.2.2.2.2.2 Binary Co(III) Complexes with Acetylacetonate and Other Bidentate Ligands

Thamilarasan et al. in 2016 reported three Co(III) complexes with acac ligand and bidentate ligands, bpy, en, 2-pic, and N3 (116–118). The complexes were found to be binding with CT-DNA and BSA. The complexes also exhibited pBR322 DNA cleavage in the presence of MPA, promoted by singlet oxygen generation. The complexes were also found to be cytotoxic against MCF-7 cell line (IC50 is in order 116 > 118 > 117) (Thamilarasan et al. 2016). Mathuber et al. in 2020 reported Co(III) acetylacetonate complex with tyrosine kinase inhibitor (119–122). Tyrosine kinase is a protein that triggers the loss of apoptosis in cancer cells. The methyl substitution of the complex increases the lipophilicity of the complex. The complexes exhibited significant stability in the blood plasma. In hypoxic conditions and higher concentrations of glutathione (GSH), the tyrosine kinase inhibitor gets detached from the complex and inhibits tyrosine kinase activity as evident from Western blot experiments. The complexes exhibited higher cytotoxicity in A431 cells at 0.1% oxygen concentration hypoxic environment (IC50 up to 7.2 μM) than normoxia (IC50 22.9). The cytotoxicity of the complex at 0.1% oxygen concentration in hypoxic environment is higher than the free tyrosine kinase inhibitor itself (Mathuber et al. 2020).

Ponatinib is known to inhibit tyrosine kinase which played an important role in cell growth, proliferation, or differentiation by catalyzing in the transfer of ATP. It targeted the Abelson kinase (ABL), FGF receptor (fibroblast growth factor), and PDGF receptor (platelet-derived growth factor). Ponatinib is a clinically approved and highly active anticancer drug. In 2021, Mathuber et al. synthesized a cobalt complex (123–124) with ponatinib using methyl acetone (meacac) and acetylacetone (acac) as ancillary ligands. The fluorescence emission wavelength of ponatinib at 470 nm was highly quenched in the complexes as the ligand-based fluorescence was difficult to observe due to the shorter lifetime of the triplet excited states (Fig. 17.5). The two complexes showed a single reduction irreversible cathodic peak which can be assigned to the reduction of Co(III) to Co(II). The cobalt meacac ponatinib complex was shown to have lower reduction potential implying more stability and slow release of ligand than their corresponding acac complex with ponatinib and erlotinib derivatives which the group synthesized previously for EGRF inhibitors. Due to the -CH2- spacer present in ponatinib, the ethylenediamine moiety is not in direct contact with quinazoline ring system, and hence the cathodic peak potential decreases which increases the stability of the complex in blood serum. The complexes show inhibition of FGFR- and ABL-dependent human cancer cells while only the acac complex exhibit toxicity in the leukemic K-562 model in vivo (Mathuber et al. 2021).

Fig. 17.5
8 chemical structures. 1 each for 116 through 122, and 1 for 123 to 124. They exhibit varying bond formations that involve C o, O, N, N 3, N H, N H 2, B r, R, and C.

Structure of binary Co(III) complexes with acetylacetonate and other bidentate ligands (117–124)

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17.2.2.2.3 Co(III) Complexes with Schiff Base Ligands

N,N donor-type Schiff base ligands can also form stable Co(III) complexes, but their main activity is generated from type-I photo-processes by hydroxyl radical generation. These types of compounds can be utilized against normoxic cancer cells. King et al. in 2017 reported nine Co(III) bis(thiosemicarbazone) complexes (125–133). The stability of the complexes in the phosphate buffer medium was found to be dependable on the axial ligand of the complexes. The stability trend was found to be NH3 > imidazole > benzylamine. The equatorial bis(thiosemicarbazone) ligand dictates the cellular uptake and the cytotoxicity of the complexes. The diacetyl bis(thiosemicarbazone) complexes exhibited higher cellular uptake than that of pyruvaldehyde bis(thiosemicarbazone) and glyoxal bis(thiosemicarbazone) complexes. In hypoxic conditions, the cytotoxicity trends were also found to be similar. But the difference in cellular uptake or cytotoxicity was not drastic (King et al. 2017). Garcia et al. in 2016 reported four Co(III) complexes with Schiff base ligands and bidentate cytotoxic ligands (134–136). The reduction potential of the complexes was in the biological redox window. Complex 135 exhibited a reduction by sodium dithionite followed by the release of the ligands. The complexes were also found to be binding with human serum albumin protein that suggests the facile transport of the complexes through blood (Garcia et al. 2017). In 2021 Gowdhami et al. reported two Schiff base Co(III) complexes (137–138) and reported their activity to inhibit the growth of A549 and MCF-7 cells. The complexes were reported to exhibit antiproliferative properties by the generation of ROS. The complexes also lead to express m-RNA and apoptotic genes, thereby triggering the apoptotic death of the cancer cells (Gowdhami et al. 2021). Cobalt(III) complexes derived from salen ligand (139) by areal oxidation with tert-butylated and trans-DACH ancillary ligands showed cytotoxic effects toward Burkitt lymphoma and leukemia cell lines (Fig. 17.6). The ligand didn’t have any toxicity, and the Co(III) salen complexes showed maximum apoptosis at 50 μM concentration, and also it showed a reduction of cell proliferation of the Burkitt lymphoma by 80%. The inhibition was proved to be caused by apoptosis activating the caspase-3 responsible for induced cell death and also decreasing the mitochondrial membrane potential. The compound showed a cytotoxic effect against Nalm6 cell (NDau) which is a daunorubicin resistance cell with IC50 value of less than 70 μM (Hopff et al. 2020).

Fig. 17.6
10 chemical structures. 3 for 125 to 133, 1 for 134 to 135, and 136 to 137, 2 for 138, 1 each for L, R, and a formation involving t-B u. They exhibit varying bond formations with C o, N, L, N H 2, N, H, N H 3, R, C H 3, O, R, C l, C I O 4, and C O 2. R = H, C l.

Structure of Co(III) complexes with Schiff base ligands (125–139)

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17.2.2.2.4 Co(III) Complex with Phenanthroline-Based Ligands

Sarkar et al. in 2021 reported a series of Co(III) complexes (140–145) with general molecular formulae [Co(B)2(L)]ClO4; here B is N,N donor phenanthroline-based ligand, and L is a catechol-based O,O donor ligand. The complexes exhibited d-d transition band around 700 nm with π- π* band at 403 nm. The complexes exhibited light-activated cytotoxicity against HeLa and MCF-7 cell lines. The complex 145 was also reported to exhibit light-activated DNA cleavage and generation of hydroxyl radical via type-I photo-redox process (Sarkar et al. 2021). Suntharalingam et al. synthesized a cobalt(III) cyclam complex (146–148) having nonsteroidal anti-inflammatory drug (NSAID)-bonded monodentate to it. NSAID is a type of drug which inhibits the production of prostaglandin which is an effector for inflammation (Fig. 17.7). The production of prostaglandins is mediated by cyclooxygenase COX-1 and COX-2. COX-2 is overly expressed in some cancer stem cells and plays a major role in the proliferation of the cell. Therefore inhibition of COX-2 has been exploited for cancer drug targeting. The cyclam complexes were less potent due to facile reduction of the metal center and immature release of NSAID ligand. For increased improvisation, they synthesized cobalt complexes with diflunisal bidentate NSAID ligand having salicylate moiety which stabilized the Co(III) center. The cytotoxicity of the complexes was observed in HMLER and HMLER-shEcad cell lines. IC50 value of all complexes ranges from 13.9 to 0.1 μM and 8.2 to 0.3 μM in both the cell lines. In breast cancer cell line MDA-MB-231, the complex shows the highest toxicity, and the complex with 1,10-phenanthroline releases diflunisal in the reducing environment and kills the cancer stem cells by damaging DNA and downregulation of COX-2 (Abe et al. 2018) (Table 17.2).

Fig. 17.7
9 chemical structures. 1 each for 140 through 149. They exhibit varying bond formations involving C o, O, and N with a positive charge.

Structure of Co(III) complex with phenanthroline-based ligands (140–148)

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Table 17.2 Cytotoxicity of Co(III)-based prodrugs against different cancer cell lines
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17.2.2.2.5 Other Types of Co(III) Complexes with Anticancer Activities

Ware et al. in 1991 reported the synthesis and hypoxia-selective cytotoxicity of Co(III) complex with molecular formulae [Co(Az)4(NO2)2]Br.2H2O.LiBr (149). The crystal structure and the NMR spectroscopy suggested the formation of symmetric complexes. Cyclic voltammetry proves the complexes were also converted to Co(II) in an irreversible manner (Ware et al. 1991). Hypoxic conditions in the complexes were also reported to release aziridine in a facile manner. Denny et al. in 1997 reported a series of Co(III) complexes with general formulae [Co(trop)2(L)]+, where trop is tropolonate anion and L is a hypoxia-selective cytotoxic agent (150–153). The complex possesses significantly higher reduction potentials than Co(III) (acac) complexes. The Co(III) (trop) complex with nitrogen mustard ligand possesses IC50 values similar to free nitrogen mustard ligand. But the complex 3-methyl acac exhibited significant hypoxia selectivity in AA8 and UV4 cell lines. The IC50 value ranged from 3100 μM to 4.6 μM, and the q ratio was found to be in the range of 0.7 to 47 (Ware et al. 1997). Gust et al. in 2004 reported three Co-alkyne complexes (154–155) and found that the complexes exhibit growth-inhibiting properties against LAMA-84, K-562, SD-1 leukemia, and U-937 lymphoma cells. The IC50 values were found to be in the range of 7.7 to 18.6 μM. The complexes were sensitive against LAMA-84 cell line but found to be insensitive against K-562 cells (Ott et al. 2003). Trofimov et al. in 2019 reported Co(II) complex of N-allylimidazole (155), and it was reported to exhibit anti-hypoxic effects against all types of acute hypoxia, and thereby it increases the lifetime of white nonlinear mice dosage. The complex had more anti-hypoxic properties than mexidol and hypoxen. Hence the complex is a suitable candidate for hypoxia-related clinical trials (Trofimov et al. 2019). Bryce et al. developed a fluorescence probe model for hypoxia cell target delivery by two cobalt(III) cyclam complexes with the fluorescent ligand anthraquinone-2-carboxylic acid and coumarin (157–158) (Fig. 17.8). The cytotoxicity of the complex was observed in DLD-1 colon cancer cells and found that the coumarin cyclam complex was more toxic with IC50 value 1.3 μM in normoxic and 1.9 μM in hypoxic conditions. Due to the presence of negatively charged oxygen donor in both the fluorophore, there is greater stabilization of the metal center with more negative reversible reduction potentials. These compounds are also able to release drugs at the pH of the cancer cells. The release of coumarin is not complete in hypoxia and the delayed release of the ligands enhanced the penetration of the compounds inside the cells in the reduced cellular environment (Kim et al. 2011).

Fig. 17.8
9 chemical structures. 1 for 149, 150 to 151, and 152 to 153, and 1 each for 154 through 158. They exhibit varying bond formations involving C o, B r, N O 2, N, R, N H 2, O, C l, and N H with a positive charge.

Structure of other types of Co(III) complexes with anticancer activities (149–158)

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17.3 Summary and Conclusion

Among all types of cancers, hypoxic tumor cells are specifically very difficult to treat because of their solid outer surface which prevents the permission of the radiation used for treatment. As these type of tumors are distantly localized from the blood vessels. Hence it is very difficult to supply chemotherapeutic drugs to this kind of cancer cell. Also, the lack of oxygen supply in these cells makes the activity of the chemotherapeutic drug more difficult as the chemotherapeutic drugs generally generate reactive oxygen species and activate the apoptotic mechanism in the cancer cells by inducing oxidative stress. And some chemotherapeutic drugs act as DNA binders, but the lack of oxygen adds the presence of reducing agents like glutathione and NADPH making this type of drug practically useless. Hence to treat this type of cancer, we require bio-activable single agents that can be activated in a lower pH environment. Several organic molecules are implemented for this kind of cancer cell, but the use of transition metal complexes gives us tunability and dual activity for our active agent against this type of cancer. Platinum complexes are also used for this type of cancer cell treatment. But the drawback is that platinum complexes get easily bound with sulfur-containing reducing agents like glutathione and lose their activity, and these cases generate a possibility of heavy metal toxicity in the patient’s body. To overcome this drawback, researchers have implemented Co(III) complexes as bio-reducible prodrugs; for two decades researchers have been developing Co(III) complexes for hypoxia-selective drug delivery. This type of complex provides an excellent template for attachment of bidentate donor ligands as drugs with N,O or O,O donor sites, and in the hypoxic condition, these bidentate ligands get released in the cancer cells and show their activity. Cobalt(III) complexes with ternary nitrogen-based donor ligands like cyclam, tpa, tpb, tren, etc. are frequently used for this type of metal complex. Along with them bidentate ligands like acetylacetone complexes also can release nitrogen mustard-like drugs. Other Co(III) complexes with Schiff base and phenanthroline-based ligands are also reported to exhibit hypoxia-selective activity. There are also reports of Co(III) phenanthroline-based complexes that absorb at 700 nm and can generate hydroxyl radical by inducing a photoinduced type-I process. This type of complex can further be modified for organ-selective drug delivery into the hypoxic cells or specific hypoxic cell targeting rather than normal cells. This way the Co(III) complexes can open a wide range of scope, which can be used to treat these types of hypoxic cancer cells. Hypoxia is also associated with several proteins like HIF-1α, MMP, VEGF, etc.; hence by exploiting the bio-reductive nature of Co(III) complexes, we can release the inhibitors for those proteins and other potent anticancer agents for hypoxia-selective treatment.