Drug-Linker Constructs Bearing Unique Dual-Mechanism Tubulin Binding Payloads Tethered through Cleavable and Non-Cleavable Linkers.

Antibody-drug conjugates (ADCs) have advanced as a mainstay among the most promising cancer therapeutics, offering enhanced antigen targeting and encompassing wide diversity in their linker and payload components. Small-molecule inhibitors of tubulin polymerization have found success as payloads in FDA approved ADCs and represent further promise in next-generation, pre-clinical and developmental ADCs. Unique dual-mechanism payloads (previously designed and synthesized in our laboratories) function as both potent antiproliferative agents and promising vascular disrupting agents capable of imparting selective and effective damage to tumor-associated microvessels.

Design, Synthesis and Biological Evaluation of 2-Phenyl Indole Analogues of OXi8006 as Colchicine Site Inhibitors of Tubulin Polymerization and Vascular Disrupting Agents.

Inhibitors of tubulin polymerization represent a promising therapeutic approach for the treatment of solid tumors. Molecules that bind to the colchicine site are of interest as they can function with a dual mechanism of action as both potent antiproliferative agents and tumor-selective vascular disrupting agents (VDAs). One such example is a 2-aryl-3-aroyl-indole molecule (OXi8006) from our laboratory that demonstrates potent inhibition of tubulin polymerization and strong antiproliferative activity (cytotoxicity) against a variety of human cancer cell lines.

Synthesis and biological evaluation of structurally diverse 6-aryl-3-aroyl-indole analogues as inhibitors of tubulin polymerization.

The synthesis and evaluation of small-molecule inhibitors of tubulin polymerization remains a promising approach for the development of new therapeutic agents for cancer treatment. The natural products colchicine and combretastatin A-4 (CA4) inspired significant drug discovery campaigns targeting the colchicine site located on the beta-subunit of the tubulin heterodimer, but so far these efforts have not yielded an approved drug for cancer treatment in human patients. Interest in the colchicine site was enhanced by the discovery that a subset of colchicine site agents demonstrated dual functionality as both potent antiproliferative agents and effective vascular disrupting agents (VDAs).

Structure Guided Design, Synthesis, and Biological Evaluation of Oxetane-Containing Indole Analogues.

The oxetane functional group offers a variety of potential advantages when incorporated within appropriate therapeutic agents as a ketone surrogate. OXi8006, a 2-aryl-3-aroyl-indole analogue, functions as a small-molecule inhibitor of tubulin polymerization that has a dual mechanism of action as both an antiproliferative agent and a tumor-selective vascular disrupting agent. Replacement of the bridging ketone moiety in OXi8006 with an oxetane functional group has expanded structure activity relationship (SAR) knowledge and provided insights regarding oxetane incorporation within this class of molecules.

Demonstrating Tumor Vascular Disrupting Activity of the Small-Molecule Dihydronaphthalene Tubulin-Binding Agent OXi6196 as a Potential Therapeutic for Cancer Treatment.

The vascular disrupting activity of a promising tubulin-binding agent (OXi6196) was demonstrated in mice in MDA-MB-231 human breast tumor xenografts growing orthotopically in mammary fat pad and syngeneic RENCA kidney tumors growing orthotopically in the kidney. To enhance water solubility, OXi6196, was derivatized as its corresponding phosphate prodrug salt OXi6197, facilitating effective delivery. OXi6197 is stable in water, but rapidly releases OXi6196 in the presence of alkaline phosphatase.

Imaging-Guided Evaluation of the Novel Small-Molecule Benzosuberene Tubulin-Binding Agent KGP265 as a Potential Therapeutic Agent for Cancer Treatment.

The selective disruption of tumor-associated vasculature represents an attractive therapeutic approach. We have undertaken the first in vivo evaluation of KGP265, a water-soluble prodrug of a benzosuberene-based tubulin-binding agent, and found promising vascular-disrupting activity in three distinct tumor types. Dose escalation in orthotopic MDA-MB-231-luc breast tumor xenografts in mice indicated that higher doses produced more effective vascular shutdown, as revealed by dynamic bioluminescence imaging (BLI).

Non-Invasive Evaluation of Acute Effects of Tubulin Binding Agents: A Review of Imaging Vascular Disruption in Tumors.

Tumor vasculature proliferates rapidly, generally lacks pericyte coverage, and is uniquely fragile making it an attractive therapeutic target. A subset of small-molecule tubulin binding agents cause disaggregation of the endothelial cytoskeleton leading to enhanced vascular permeability generating increased interstitial pressure. The resulting vascular collapse and ischemia cause downstream hypoxia, ultimately leading to cell death and necrosis.

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 methyl, -methyl, and dimethyl nitrothiophene triggers were synthesized together with corresponding CA4-BAPCs, previously reported by Davis ( , 5 (11), 2886), for comparison.

Synthesis and biological evaluation of structurally diverse α-conformationally restricted chalcones and related analogues.

Numerous members of the combretastatin and chalcone families of natural products function as inhibitors of tubulin polymerization through a binding interaction at the colchicine site on β-tubulin. These molecular scaffolds inspired the development of many structurally modified derivatives and analogues as promising anticancer agents. A productive design blueprint that involved molecular hybridization of the pharmacophore moieties of combretastatin A-4 () and the chalcones led to the discovery of two promising lead molecules referred to as and .

Structure Guided Design, Synthesis, and Biological Evaluation of Novel Benzosuberene Analogues as Inhibitors of Tubulin Polymerization.

A promising design paradigm for small-molecule inhibitors of tubulin polymerization that bind to the colchicine site draws structural inspiration from the natural products colchicine and combretastatin A-4 (CA4). Our previous studies with benzocycloalkenyl and heteroaromatic ring systems yielded promising inhibitors with dihydronaphthalene and benzosuberene analogues featuring phenolic (KGP03 and KGP18) and aniline (KGP05 and KGP156) congeners emerging as lead agents. These molecules demonstrated dual mechanism of action, functioning both as potent vascular disrupting agents (VDAs) and as highly cytotoxic anticancer agents.

Synthesis of dihydronaphthalene analogues inspired by combretastatin A-4 and their biological evaluation as anticancer agents.

The natural products colchicine and combretastatin A-4 () have provided inspiration for the discovery and development of a wide array of derivatives and analogues that inhibit tubulin polymerization through a binding interaction at the colchicine site on β-tubulin. A water-soluble phosphate prodrug salt of (referred to as ) has demonstrated the ability to selectively damage tumor-associated vasculature and ushered in a new class of developmental anticancer agents known as vascular disrupting agents (VDAs). Through a long-term program of structure activity relationship (SAR) driven inquiry, we discovered that the dihydronaphthalene molecular scaffold provided access to small-molecule inhibitors of tubulin polymerization.

Synthesis and Biological Evaluation of Benzocyclooctene-based and Indene-based Anticancer Agents that Function as Inhibitors of Tubulin Polymerization.

The natural products colchicine and combretastatin A-4 () have been inspirational for the design and synthesis of structurally related analogues and spin-off compounds as inhibitors of tubulin polymerization. The discovery that a water-soluble phosphate prodrug salt of (referred to as ) is capable of imparting profound and selective damage to tumor-associated blood vessels paved the way for the development of a new therapeutic approach for cancer treatment utilizing small-molecule inhibitors of tubulin polymerization that also act as vascular disrupting agents (VDAs). Combination of salient structural features associated with colchicine and led to the design and synthesis of a variety of fused aryl-cycloalkyl and aryl-heterocyclic compounds that function as inhibitors of tubulin polymerization.

Synthesis and biological evaluation of a water-soluble phosphate prodrug salt and structural analogues of KGP94, a lead inhibitor of cathepsin L.

The magnitude of expression of cathepsin L, often upregulated in the tumor microenvironment, correlates with the invasive and metastatic nature of certain tumors. Inhibition of cathepsin L represents an emerging strategy for the treatment of metastatic cancer. A potent, small-molecule inhibitor (referred to as KGP94) of cathepsin L, and new KGP94 analogues were synthesized.

Design, synthesis, and biological evaluation of water-soluble amino acid prodrug conjugates derived from combretastatin, dihydronaphthalene, and benzosuberene-based parent vascular disrupting agents.

Targeting tumor vasculature represents an intriguing therapeutic strategy in the treatment of cancer. In an effort to discover new vascular disrupting agents with improved water solubility and potentially greater bioavailability, various amino acid prodrug conjugates (AAPCs) of potent amino combretastatin, amino dihydronaphthalene, and amino benzosuberene analogs were synthesized along with their corresponding water-soluble hydrochloride salts. These compounds were evaluated for their ability to inhibit tubulin polymerization and for their cytotoxicity against selected human cancer cell lines.

Structural interrogation of benzosuberene-based inhibitors of tubulin polymerization.

The discovery of 3-methoxy-9-(30,40,50-trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-ol (a benzosuberene-based analogue referred to as KGP18) was originally inspired by the natural products colchicine and combretastatin A-4 (CA4). The relative structural simplicity and ease of synthesis of KGP18, coupled with its potent biological activity as an inhibitor of tubulin polymerization and its cytotoxicity (in vitro) against human cancer cell lines, has resulted in studies focused on new analogue design and synthesis. Our goal was to probe the relationship of structure to function in this class of anticancer agents.

Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L.

Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from studies on benzophenone thiosemicarbazone cathepsin inhibitors, a series of fourteen benzoylbenzophenone thiosemicarbazone analogues were designed, synthesized, and evaluated for their inhibitory activity against cathepsins L and B. Thiosemicarbazone inhibitors 3-benzoylbenzophenone thiosemicarbazone 1, 1,3-bis(4-fluorobenzoyl)benzene thiosemicarbazone 8, and 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32 displayed the greatest potency against cathepsin L with low IC50 values of 9.

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.

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).

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.

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).

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.

An Amino-Benzosuberene Analogue That Inhibits Tubulin Assembly and Demonstrates Remarkable Cytotoxicity.

The recent discovery of a small-molecule benzosuberene-based phenol that demonstrates remarkable picomolar cytotoxicity against selected human cancer cell lines and strongly inhibits tubulin polymerization (1-2 µM) inspired the design and synthesis of a variety of new, structurally diverse benzosuberene derivatives. An efficient synthetic route to functionalized benzosuberenes was developed. This methodology utilized a Wittig reaction, followed by a selective alkene reduction and ring-closing cyclization to form the core benzosuberone structure.

Small-molecule inhibitors of cathepsin L incorporating functionalized ring-fused molecular frameworks.

Cathepsin L is a cysteine protease that is upregulated in a variety of malignant tumors and plays a significant role in cancer cell invasion and migration. It is an attractive target for the development of small-molecule inhibitors, which may prove beneficial as treatment agents to limit or arrest cancer metastasis. We have previously identified a structurally diverse series of thiosemicarbazone-based inhibitors that incorporate the benzophenone and thiochromanone molecular scaffolds.

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.