Bioreductively Activatable Prodrug Conjugates of Combretastatin A-1 and Combretastatin A-4 as Anticancer Agents Targeted toward Tumor-Associated Hypoxia.

The natural products combretastatin A-1 (CA1) and combretastatin A-4 (CA4) function as potent inhibitors of tubulin polymerization and as selective vascular disrupting agents (VDAs) in tumors. Bioreductively activatable prodrug conjugates (BAPCs) can enhance selectivity by serving as substrates for reductase enzymes specifically in hypoxic regions of tumors. A series of CA1-BAPCs incorporating nor-methyl, mono-methyl, and gem-dimethyl nitrothiophene triggers were synthesized together with corresponding CA4-BAPCs, previously reported by Davis (Mol. Cancer Ther. 2006, 5 (11), 2886), for comparison. The CA4-gem-dimethylnitrothiophene BAPC 45 proved exemplary in comparison to its nor-methyl 43 and mono-methyl 44 congeners. It was stable in phosphate buffer (pH 7.4, 24 h), was cleaved (25%, 90 min) by NADPH-cytochrome P450 oxidoreductase (POR), was inactive (desirable prodrug attribute) as an inhibitor of tubulin polymerization (IC50 > 20 µM), and demonstrated hypoxia-selective activation in the A549 cell line [hypoxia cytotoxicity ratio (HCR) = 41.5]. The related CA1-gem-dimethylnitrothiophene BAPC 41 was also promising (HCR = 12.5) with complete cleavage (90 min) upon treatment with POR. In a preliminary in vivo dynamic bioluminescence imaging study, BAPC 45 (180 mg/kg, ip) induced a decrease (within 4 h) in light emission in a 4T1 syngeneic mouse breast tumor model, implying activation and vascular disruption.

The vascular disrupting activity of OXi8006 in endothelial cells and its phosphate prodrug OXi8007 in breast tumor xenografts.

This study describes the vascular disrupting ability and the mechanism of action of the indole-based tubulin-binding compound, OXi8006, and its water-soluble phosphate prodrug OXi8007. Treatment of rapidly proliferating human umbilical vein endothelial cells (HUVECs), used as a model for the tumor vasculature, with OXi8006 or OXi8007, caused potent microtubule disruption followed by extensive reorganization of the cytoskeletal network. The mechanism of action involved an increase in focal adhesion formation associated with an increase in phosphorylation of both non-muscle myosin light chain and focal adhesion kinase. These effects were dramatically diminished by an inhibitor of RhoA kinase, a downstream effector of RhoA. Cell cycle blockade at G2/M and cytotoxicity toward rapidly proliferating HUVECs were also observed. Capillary-like networks of HUVECs were disrupted by the action of both OXi8006 and OXi8007. The prodrug OXi8007 exhibited potent and rapid dose-dependent antivascular activity assessed by dynamic bioluminescence imaging (BLI) in an MDA-MB-231-luc breast cancer xenograft mouse model. By 6 hours post treatment, over 93% of the BLI signal was abolished with only a slight recovery at 24 hours. These findings were confirmed by histology. The results from this study demonstrate that OXi8007 is a potent vascular disrupting agent acting through an anti-microtubule mechanism involving RhoA.

Evaluation of tumor ischemia in response to an indole-based vascular disrupting agent using BLI and (19)F MRI.

Vascular disrupting agents (VDAs) have been proposed as an effective broad spectrum approach to cancer therapy, by inducing ischemia leading to hypoxia and cell death. A novel VDA (OXi8007) was recently reported to show rapid acute selective shutdown of tumor vasculature based on color-Doppler ultrasound. We have now expanded investigations to noninvasively assess perfusion and hypoxiation of orthotopic human MDA-MB-231/luc breast tumor xenografts following the administration of OXi8007 based on dynamic bioluminescence imaging (BLI) and magnetic resonance imaging (MRI). BLI showed significantly lower signal four hours after the administration of OXi8007, which was very similar to the response to combretastatin A-4P (CA4P), but the effect lasted considerably longer, with the BLI signal remaining depressed at 72 hrs. Meanwhile, control tumors exhibited minimal change. Oximetry used (19)F MRI of the reporter molecule hexafluorobenzene and FREDOM (Fluorocarbon Relaxometry using Echo Planar Imaging for Dynamic Oxygen Mapping) to assess pO2 distributions during air and oxygen breathing. pO2 decreased significantly upon the administration of OXi8007 during oxygen breathing (from 122 ± 64 to 34 ± 20 Torr), with further decrease upon switching the gas to air (pO2 = 17 ± 9 Torr). pO2 maps indicated intra-tumor heterogeneity in response to OXi8007, though ultimately all tumor regions became hypoxic. Both BLI and FREDOM showed the efficacy of OXi8007. The pO2 changes measured by FREDOM may be crucial for future study of combined therapy.

Mechanistic Considerations in the Synthesis of 2-Aryl-Indole Analogues under Bischler-Mohlau Conditions.

Mechanistic insight into the pathway of the Bischler-Mohlau indole formation reaction is provided by isotopic labeling utilizing judicious incorporation of a 13C atom within the α-bromoacetophenone analogue reactant. The resulting rearranged 2-aryl indole, isolated as the major product, located the 13C isotope label at the methine carbon of the fused five-membered heterocyclic ring, which suggested that the mechanistic pathway of cyclization, in this specific example, required two equivalents of the aniline analogue reactant partner and proceeded through an imine intermediate rather than by direct formation of the corresponding 3-aryl indole accompanied by a concomitant 1,2-aryl shift rearrangement.

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, Eaton's 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.

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 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.

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

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<85 nM) 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>10,000 nM). 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.