Kevin G. Pinney

Combretastatin family member OXI4503 induces tumor vascular collapse through the induction of endothelial apoptosis

The mechanism of tumor cell killing by OXI4503 was investigated by studying vascular functional and morphological changes post drug administration. SCID mice bearing MHEC5‐T hemangioendothelioma were given a single dose of OXI4503 at 100 mg/kg. Tumor blood flow, measured by microsphere fluorescence, was reduced by 50% at 1 hr, and reached a maximum level 6–24 hr post drug treatment. Tumor vascular permeability, measured by Evans blue and hemoglobin, increased significantly from 3 hr and peaked at 18 hr. The elevated tumor vessel permeability was accompanied by an increase in vascular endothelial growth factor (VEGF) from 1 hr post drug treatment. Immunohistochemical staining for CD31 and laminin showed that tumor blood vessels were affected as early as 3 hr but more prominent from 6 hr. From 12 hr, the vessel structure was completely destroyed. Histopathological and double immunohistochemical staining showed morphological change and induction of apoptosis in endothelial cells at 1–3 hr, followed by tumor cell necrosis from 6–72 hr. There were no statistically significant changes of Evans blue and hemoglobin contents in liver tissue over the time course. These results suggest that OXI4503 selectively targets tumor blood vessels, and induces blood flow shutdown while it enhances tumor blood vessel permeability. The early induction of endothelial cell apoptosis leads to functional changes of tumor blood vessels and finally to the collapse of tumor vasculature, resulting in massive tumor cell necrosis. The time course of the tumor vascular response observed with OXI4503 treatment supports this drug for development as a stand alone therapy, and also lends support for the use of the drug in combination with other cancer therapies.

A perspective on vascular disrupting agents that interact with tubulin: preclinical tumor imaging and biological assessment

The tumor microenvironment provides a rich source of potential targets for selective therapeutic intervention with properly designed anticancer agents. Significant physiological differences exist between the microvessels that nourish tumors and those that supply healthy tissue. Selective drug-mediated damage of these tortuous and chaotic microvessels starves a tumor of necessary nutrients and oxygen and eventually leads to massive tumor necrosis. Vascular targeting strategies in oncology are divided into two separate groups: angiogenesis inhibiting agents (AIAs) and vascular disrupting agents (VDAs). The mechanisms of action between these two classes of compounds are profoundly distinct. The AIAs inhibit the actual formation of new vessels, while the VDAs damage and/or destroy existing tumor vasculature. One subset of small-molecule VDAs functions by inhibiting the assembly of tubulin into microtubules, thus causing morphology changes to the endothelial cells lining the tumor vasculature, triggered by a cascade of cell signaling events. Ultimately this results in catastrophic damage to the vessels feeding the tumor. The rapid emergence and subsequent development of the VDA field over the past decade has led to the establishment of a synergistic combination of preclinical state-of-the-art tumor imaging and biological evaluation strategies that are often indicative of future clinical efficacy for a given VDA. This review focuses on an integration of the appropriate biochemical and biological tools necessary to assess (preclinically) new small-molecule, tubulin active VDAs for their potential to be clinically effective anticancer agents.

Design, synthesis and biological evaluation of dihydronaphthalene and benzosuberene analogs of the combretastatins as inhibitors of tubulin polymerization in cancer chemotherapy

A novel series of dihydronaphthalene and benzosuberene analogs bearing structural similarity to the combretastatins in terms of 1,2-diarylethene, trimethoxyphenyl, and biaryl functionality has been synthesized. The compounds have been evaluated in regard to their ability to inhibit tubulin assembly and for their cytotoxicity against selected human cancer cell lines. From this series of compounds, benzosuberene analogs 2 and 4 inhibited tubulin assembly at concentrations comparable to that of combretastatin A-4 (CA4) and combretastatin A-1 (CA1). Furthermore, analog 4 demonstrated remarkable cytotoxicity against the three human cancer cell lines evaluated (for example GI(50)=0.0000032 microM against DU-145 prostate carcinoma).

Design, synthesis, and biological evaluation of potent thiosemicarbazone based cathepsin L inhibitors

Abstract A small library of 36 functionalized benzophenone thiosemicarbazone analogs has been prepared by chemical synthesis and evaluated for their ability to inhibit the cysteine proteases cathepsin L and cathepsin B. Inhibitors of cathepsins L and B have the potential to limit or arrest cancer metastasis. The six most active inhibitors of cathepsin L (IC50 <85nM) in this series incorporate a meta-bromo substituent in one aryl ring along with a variety of functional groups in the second aryl ring. These six analogs are selective for their inhibition of cathepsin L versus cathepsin B (IC50 >10,000nM). The most active analog in the series, 3-bromophenyl-2′-fluorophenyl thiosemicarbazone 1, also efficiently inhibits cell invasion of the DU-145 human prostate cancer cell line.

Synthesis and biological evaluation of indole-based, anti-cancer agents inspired by the vascular disrupting agent 2-(3′-hydroxy-4′-methoxyphenyl)-3-(3″,4″,5″-trimethoxybenzoyl)-6-methoxyindole (OXi8006)

The discovery of a 2-aryl-3-aroyl indole-based small-molecule inhibitor of tubulin assembly (referred to as OXi8006) inspired the design, synthesis, and biological evaluation of a series of diversely functionalized analogues. In the majority of examples, the pendant 2-aryl ring contained a 3-hydroxy-4-methoxy substitution pattern, and the fused aryl ring featured a 6-methoxy group. Most of the variability was in the 3-aroyl moiety, which was modified to incorporate methoxy (33-36), nitro (25-27), halogen (28-29), trifluoromethyl (30), or trifluoromethoxy (31-32) functionalities. In two analogues (34 and 36), the methoxy substitution pattern in the fused aryl ring varied, while in another derivative (35) the phenolic moiety was translocated from the pendant 2-aryl ring to position-7 of the fused aryl ring. Each of the compounds were evaluated for their cytotoxicity (in vitro) against the SK-OV-3 (ovarian), NCI-H460 (lung), and DU-145 (prostate) human cancer cell lines and for their ability to inhibit tubulin assembly. Four of the compounds (30, 31, 35, 36) proved to be potent inhibitors of tubulin assembly (IC50 <5μM), and three of these compounds (31, 35, 36) were strongly cytotoxic against the three cancer cell lines. The most active compound (36) in this series, which incorporated a methoxy group at position-7, was comparable in terms of inhibition of tubulin assembly and cytotoxicity to the lead compound OXi8006.

Functionalized benzophenone, thiophene, pyridine, and fluorene thiosemicarbazone derivatives as inhibitors of cathepsin L

A series of thiosemicarbazone analogs based on the benzophenone, thiophene, pyridine, and fluorene molecular frameworks has been prepared by chemical synthesis and evaluated as small-molecule inhibitors of the cysteine proteases cathepsin L and cathepsin B. The two most potent inhibitors of cathepsin L in this series (IC(50)<135 nM) are brominated-benzophenone thiosemicarbazone analogs that are further functionalized with a phenolic moiety (2 and 6). In addition, a bromo-benzophenone thiosemicarbazone acetyl derivative (3) is also strongly inhibitory against cathepsin L (IC(50)=150.8 nM). Bromine substitution in the thiophene series results in compounds that demonstrate only moderate inhibition of cathepsin L. The two most active analogs in the benzophenone thiosemicarbazone series are highly selective for their inhibition of cathepsin L versus cathepsin B.

Synthesis of a 2-aryl-3-aroyl indole salt (OXi8007) resembling combretastatin A-4 with application as a vascular disrupting agent

The natural products colchicine and combretastatin A-4 are potent inhibitors of tubulin assembly, and they have inspired the design and synthesis of a large number of small-molecule, potential anticancer agents. The indole-based molecular scaffold is prominent among these SAR modifications, leading to a rapidly increasing number of agents. The water-soluble phosphate prodrug 33 (OXi8007) of 2-aryl-3-aroylindole-based phenol 8 (OXi8006) was prepared by chemical synthesis and found to be strongly cytotoxic against selected human cancer cell lines (GI₅₀ = 36 nM against DU-145 cells, for example). The free phenol, 8 (OXi8006), was a strong inhibitor (IC₅₀ = 1.1 μM) of tubulin assembly. The corresponding phosphate prodrug 33 (OXi8007) also demonstrated pronounced interference with tumor vasculature in a preliminary in vivo study utilizing a SCID mouse model bearing an orthotopic PC-3 (prostate) tumor as imaged by color Doppler ultrasound. The combination of these results provides evidence that the indole-based phosphate prodrug 33 (OXi8007) functions as a vascular disrupting agent that may prove useful for the treatment of cancer.

Synthesis, in vitro, and in vivo evaluation of phosphate ester derivatives of combretastatin A-4

Combretastatin A-4 disodiumphosphate (CA4P), a prodrug formulation of the natural product combretastatin A-4 (CA4), is currently in clinical investigation for the treatment of cancer. In vivo, CA4P is rapidly enzymatically converted to CA4, a potent inhibitor of tubulin polymerization (IC(50)=1-2 microM), and rapidly causes bloodflow shutdown in tumor tissues. A variety of alkyl and aryl di- and triesters of CA4P have been synthesized and evaluated as potential CA4 prodrugs and/or stable CA4P analogues.

Synthesis of structurally diverse benzosuberene analogues and their biological evaluation as anti-cancer agents

Diversely functionalized, fused aryl-alkyl ring systems hold a prominent position as well-established molecular frameworks for a variety of anti-cancer agents. The benzosuberene (6,7 fused, also referred to as dihydro-5H-benzo[7]annulene and benzocycloheptene) ring system has emerged as a valuable molecular core component for the development of inhibitors of tubulin assembly, which function as antiproliferative anti-cancer agents and, in certain cases, as vascular disrupting agents (VDAs). Both a phenolic-based analogue (known as KGP18, compound 39) and its corresponding amine-based congener (referred to as KGP156, compound 45), which demonstrate strong inhibition of tubulin assembly (low micromolar range) and potent cytotoxicity (picomolar range for KGP18 and nanomolar range for KGP156) are noteworthy examples of such benzosuberene-based compounds. In order to extend the structure-activity relationship (SAR) knowledge base related to benzosuberene anti-cancer agents, a series of eleven analogues (including KGP18) were prepared in which the methoxylation pattern on the pendant aryl ring as well as functional group incorporation on the fused aryl ring were varied. The synthetic approach to these compounds featured a sequential Wittig olefination, reduction, Eatons reagent-mediated cyclization strategy to achieve the core benzosuberone intermediate, and represented a higher-yielding synthesis of KGP18 (which we prepared previously through a ring-expansion strategy). Incorporation of a fluorine or chlorine atom at the 1-position of the fused aryl ring or replacement of one of the methoxy groups with hydrogen (on the pendant aryl ring of KGP18) led to benzosuberene analogues that were both strongly inhibitory against tubulin assembly (IC50 approximately 1.0 μM) and strongly cytotoxic against selected human cancer cell lines (for example, GI50=5.47 nM against NCI-H460 cells with fluoro-benzosuberene analogue 37). A water-soluble phosphate prodrug salt of KGP18 (referred to as KGP265, compound 44) and a water-soluble serinamide salt (compound 48) of KGP156 were also synthesized and evaluated in this study.

Initial evaluation of the antitumour activity of KGP94, a functionalized benzophenone thiosemicarbazone inhibitor of cathepsin L

Kinetic analysis of the mode of inhibition of cathepsin L by KGP94, a lead compound from a privileged library of functionalized benzophenone thiosemicarbazone derivatives, demonstrated that it is a time-dependent, reversible, and competitive inhibitor of the enzyme. These results are consistent with the formation of a transient covalent bond, and are supported by molecular modeling that places the thiocarbonyl of the inhibitor in proximity to the thiolate moiety of the enzyme active site Cys25. KGP94 significantly decreased the activity of cathepsin L toward human type I collagen, and impeded both migration and invasion of MDA-MB-231 human breast cancer cells. Growth retardation was achieved in vivo against both recently implanted and established tumours using a C3H mouse mammary carcinoma model.