Julie M. Locke

Cytotoxic and Anticancer Activities of Isatin and Its Derivatives: A Comprehensive Review from 2000-2008

Isatin (1H-indole-2,3-dione) and its derivatives demonstrate a diverse array of biological and pharmacological activities including anticonvulsant, antibacterial, antifungal, antiviral and anticancer properties. This broad spectrum of biochemical targets has been facilitated by the synthetic versatility of isatin, which has allowed the generation of a large number of structurally diverse derivatives including analogues derived from substitution of the aryl ring, and/or derivatisation of the isatin nitrogen and C2/C3 carbonyl moieties. The recent FDA approval of the oxindole sunitinib malate, as a kinase inhibitor for the treatment of advanced renal carcinoma and gastrointestinal stromal tumours, underscores the increasing interest in isatins as a new class of antineoplastic agents. In addition to potent kinase inhibition, the mechanism of action of other isatin derivatives includes the inhibition and/or modulation of proteases, translation initiation, neo-vascularisation and tubulin polymerisation. It was therefore the objective of this review to systematically evaluate the cytotoxic and anticancer properties of various substituted isatins and collate these findings to be used as a guide for future structure-activity relationship and mode of action studies. This is the first review to comprehensively discuss the in vitro and in vivo anticancer activities of isatin and its substituted derivatives.

N-Phenethyl and N-naphthylmethyl isatins and analogues as in vitro cytotoxic agents

A range of N-phenethyl, N-phenacyl, and N-(1- and 2-naphthylmethyl) derivatives of 5,7-dibromoisatin 2 were prepared by N-alkylation reactions. Their activity against human monocyte-like histiocytic lymphoma (U937), leukemia (Jurkat), and breast carcinoma (MDA-MB-231) cell lines was assessed. The results allowed further development of structure-activity relationships. The compound 5,7-dibromo-N-(1-naphthylmethyl)-1H-indole-2,3-dione 5a was the most potent against U937 cells with an IC(50) value of 0.19 microM.

Recent highlights in the development of isatin- based anticancer agents

Isatin (1H-indole-2,3-dione) and its derivatives are responsible for a broad spectrum of biological activities. Among these the cytotoxic and antineoplastic properties have been the most widely reported. The synthetic versatility of the isatin, due to its privileged scaffold, has led to the generation of a large number of structurally diverse derivatives which include analogues derived from either mono-, di-, and tri- substitution of the aryl ring A, and/or those obtained by derivatisation of the isatin nitrogen and C2/C3 carbonyl moieties. These compounds inhibit cancer cell proliferation and tumour growth via interaction with a variety of intracellular targets such as DNA, telomerase, tubulin, P-glycoprotein, protein kinases and phosphatases. Herein we review recent highlights in the development of isatin-based compounds as anticancer agents with a particular focus on the cytotoxicity and structure activity relationships.

Anti-cancer activity of an acid-labile N-alkylisatin conjugate targeting the transferrin receptor

We have previously reported a series of pH-sensitive imine-linked N-alkylisatin prodrugs that are stable at pH 7.4, but readily cleaved at pH 4.5. Herein, one of the most potent prodrugs, 5,7-dibromo-N-(p-methoxybenzyl)isatin (NAI), was functionalized with a para-phenylpropionic acid linker, and the resulting NAI-imine prodrug conjugated to transferrin (Tf) to form a NAI-imine-Tf conjugate. Cytotoxicity assays revealed the conjugate was equipotent to the free drug against MCF-7 breast cancer cells, with clear selectivity patterns based on TfR levels. These results suggest that this novel isatin-based cytotoxin conjugated to a tumor targeting protein via an acid-labile linker warrants further preclinical testing.

Synthesis and hydrolytic evaluation of acid-labile imine-linked cytotoxic isatin model systems

In this study a series of isatin-based, pH-sensitive aryl imine derivatives with differing aromatic substituents and substitution patterns were synthesised and their acid-catalysed hydrolysis evaluated. These derivatives were functionalised at the C3 carbonyl group of a potent N-substituted isatin cytotoxin and were stable at physiological pH but readily cleaved at pH 4.5. Observed rates of hydrolysis for the embedded imine-acid moiety were in the order para-phenylpropionic acid>phenylacetic acid (para>meta)>benzoic acid (meta>para). The ability to fine-tune hydrolysis rates in this way has potential implications for optimising imine linked, tumour targeting cytotoxin-protein conjugates.

Preclinical evaluation of novel, all-in-one formulations of 5-fluorouracil and folinic acid with reduced toxicity profiles.

5-Fluorouracil (5-FU) in combination with its synergistic biomodulator folinic acid maintains a pivotal position in cancer chemotherapy. However, clinical limitations such as phlebitis and catheter blockages persist with the administration of these drugs in combination, and are associated with reduced efficacy and/or quality of life for patients. We have reported earlier on the novel, all-in-one, pH neutral, parenteral 5-FU and folinic acid formulations (termed Fluorodex) incorporating &bgr;-cyclodextrins. Fluorodex maintains potency while overcoming the accepted incompatibility of 5-FU and folinic acid. We carried out toxicological, pharmacokinetic and biodistribution, and efficacy evaluations of Fluorodex compared with 5-FU:folinic acid using several administration routes and schedules in two rodent models. These were compared with the dose-matched sequential administration of 5-FU:folinic acid. Fluorodex showed bioequivalence to 5-FU:folinic acid as assessed by the tissue distribution and pharmacokinetic studies of 5-FU, but was generally better tolerated as determined by weight loss, hematological, and other clinical parameters. Compared with 5-FU:folinic acid, Fluorodex was also associated with reduced phlebitis using a rabbit ear vein model. Furthermore, using human carcinoma tumor models in mice, Fluorodex resulted in equivalent or improved efficacy profiles compared with 5-FU:folinic acid. In conclusion, these novel, all-in-one formulations represent a superior injectable form of 5-FU that allows codelivery of folinic acid. This should translate into improved patient tolerability with potential for enhanced efficacy.

The antiangiogenic properties of sulfated β-cyclodextrins in anticancer formulations incorporating 5-fluorouracil

Sulfated &bgr;-cyclodextrins (S-&bgr;-CDs) are useful excipients for improving the solubility of drugs. One such formulation incorporating 5-fluorouracil (5-FU), termed FD(S), showed improved efficacy over 5-FU alone in orthotopic carcinoma xenograft models. S-&bgr;-CDs have heparin-like anticoagulant properties, which may have contributed toward the improved antitumor effect of FD(S). S-&bgr;-CDs have also been reported to modify a number of processes involved in angiogenesis. Although the anticoagulant nature of S-&bgr;-CDs was established, the antiangiogenic properties of S-&bgr;-CDs within FD(S) were unknown. The effect of S-&bgr;-CD and FD(S) on the proliferation and migration of endothelial cells in live-cell kinetic assays, and the reorganization of human umbilical vein endothelial cells into tubule structures in vitro was assessed. The effects of S-&bgr;-CD on angiogenesis in vitro were validated ex vivo using the rat aorta ring assay and the chick embryo chorioallantoic membrane assay. S-&bgr;-CD does not alter proliferative endothelial cell sensitivity to 5-FU cytotoxicity. S-&bgr;-CD alone and within FD(S) significantly inhibited angiogenesis by impeding endothelial cell migration, resulting in the inhibition of tubule formation and hence new vasculature. In addition to the cytotoxic action of the drug 5-FU, therapeutic inhibition of angiogenesis by S-&bgr;-CDs within FD(S) could potentially limit local invasion and metastases. This has important implications for the exploitation of S-&bgr;-CDs for drug formulation improvements or for drug delivery of anticancer biologics.

Targeting Urokinase and the Transferrin Receptor with Novel, Anti-Mitotic N-Alkylisatin Cytotoxin Conjugates Causes Selective Cancer Cell Death and Reduces Tumor Growth

Tumor-specific delivery of ligand-directed prodrugs can increase the therapeutic window of chemotherapeutics by maintaining efficacy whilst decreasing toxic side effects. We have previously described a series of synthetic N-alkylated isatin cytotoxins that destabilize microtubules and induce apoptosis with 10-fold greater potency than conventional anti-mitotics in vitro. Here, we report the characterization, in vitro cytotoxicity and in vivo efficacy of a lead compound, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (N-AI) conjugated via an esterase-labile linker (N-AIE) to two proven targeting ligands, transferrin (Tf) and plasminogen activator inhibitor type 2 (PAI-2/serpinB2). N-AI was released from N-AIE and the targeting ligands Tf/PAI-2 in an esterase-dependent manner at 37 C and both Tf- and PAI-2-N-AIE conjugates were stable at physiological pH. Human cancer cell lines which vary in their expression levels of Tf receptor (TfR/CD71) and PAI-2 target, receptor bound urokinase (uPA) selectively internalized the conjugates. Tf-N-AIE was up to 24 times more active than the free drug and showed clear selectivity patterns based on TfR levels. PAI-2-N-AIE showed equivalent activity compared to the parent drug and strong selectivity patterns for uPA levels. In preliminary in vivo experiments, the PAI-2- and Tf-N-AIE conjugates were efficacious at 1/20(th) and 1/10(th) of the dose of the free N-AI, respectively, in a metastatic, orthotopic human breast tumor xenograft mouse model. Thus, this strategy specifically delivers and concentrates a novel class of isatin-based, tubulin destabilizing agents to tumors in vivo and warrants further detailed preclinical investigation.

Development and assessment of novel all-in-one parenteral formulations with integrated anticoagulant properties for the concomitant delivery of 5-fluorouracil and calcium folinate

5-Fluorouracil in combination with its biomodulator folinic acid maintains a pivotal position in current anticancer treatment regimens. However, limitations in clinical management persist with the administration of these drugs. These limitations are associated with the use of a high pH to maintain 5-fluorouracil in solution, resulting in high rates of phlebitis and catheter blockages. Herein, we describe and compare initial studies on novel all-in-one formulations of 5-fluorouracil and folinic acid incorporating either sulfated or hydroxypropyl &bgr;-cyclodextrins at physiological pH that potentially address these issues. All formulations markedly improved the stability of supersaturated solutions of 5-fluorouracil in the presence of folinic acid. In-vitro evaluation of the PC-3, HCT-116, MDA-MB-231, PC-14, and COLO-201 human carcinoma cell lines showed that all formulations exhibited equivalent or better cytotoxicity compared with cells exposed to 5-fluorouracil and folinic acid. Thus, these cyclodextrins do not compromise the cytotoxicity of 5-fluorouracil. Preliminary in-vivo dose tolerance profiles of the formulations were also equivalent to 5-fluorouracil and folinic acid administered separately. Furthermore, given the association between thrombosis and cancer, the potentially beneficial anticoagulant activity of the sulfated cyclodextrin-based formulations was also confirmed in vitro. Extended activated partial thromboplastin times and prothrombin times were observed for the sulfated cyclodextrins in human plasma both as individual compounds and as components of the formulations. In conclusion, these novel all-in-one formulations maintain the in-vitro potency while overcoming the accepted incompatibility of 5-fluorouracil and folinic acid, and represent improved injectable forms of 5-fluorouracil that may reduce phlebitis, catheter blockages, and thromboembolic events.

N-alkylated isatins evade P-gp mediated efflux and retain potency in MDR cancer cell lines

The search for novel anticancer therapeutics with the ability to overcome multi-drug resistance (MDR) mechanisms is of high priority. A class of molecules that show potential in overcoming MDR are the N-alkylated isatins. In particular 5,7-dibromo-N-alkylisatins are potent microtubule destabilizing agents that act to depolymerize microtubules, induce apoptosis and inhibit primary tumor growth in vivo. In this study we evaluated the ability of four dibrominated N-alkylisatin derivatives and the parent compound, 5,7-dibromoisatin, to circumvent MDR. All of the isatin-based compounds examined retained potency against the MDR cell lines; U937VbR and MES-SA/Dx5 and displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell lines. We show that one mechanism by which the isatin-based compounds overcome MDR is by circumventing P-glycoprotein (P-gp) mediated drug efflux. Thus, as the isatin-based compounds are not susceptible to extrusion from P-gp overexpressing tumor cells, they represent a promising alternative strategy as a stand-alone or combination therapy for treating MDR cancer.