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. A new synthetic method using an oxetane-containing tertiary alcohol subjected to Lewis acid catalyzed conditions led to successful Friedel-Crafts alkylation and yielded fourteen new oxetane-containing indole-based molecules. This synthetic approach represents the first method to successfully install an oxetane ring at the 3-position of a 2-aryl-indole system. Several analogues showed potent cytotoxicity (micromolar GI50 values) against human breast cancer cell lines (MCF-7 and MDA-MB-231) and a pancreatic cancer cell line (PANC-1), although they proved to be ineffective as inhibitors of tubulin polymerization. Molecular docking studies comparing colchicine with the OXi8006-oxetane analogue 5m provided a rationale for the differential interaction of these molecules with the colchicine site on the tubulin heterodimer.

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. Thus, local damage generates massive amplification and tumor destruction. The tumor vasculature is readily accessed and potentially a common target irrespective of disease site in the body. Development of a therapeutic approach and particularly next generation agents benefits from effective non-invasive assays. Imaging technologies offer varying degrees of sophistication and ease of implementation. This review considers technological strengths and weaknesses with examples from our own laboratory. Methods reveal vascular extent and patency, as well as insights into tissue viability, proliferation and necrosis. Spatiotemporal resolution ranges from cellular microscopy to single slice tomography and full three-dimensional views of whole tumors and measurements can be sufficiently rapid to reveal acute changes or long-term outcomes. Since imaging is non-invasive, each tumor may serve as its own control making investigations particularly efficient and rigorous. The concept of tumor vascular disruption was proposed over 30 years ago and it remains an active area of research.

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.

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. (3,5-Dibromophenyl)-(3-hydroxyphenyl) ketone thiosemicarbazone (22), with an IC50 value of 202nM, exhibited similar inhibitory activity against cathepsin L compared to KGP94 (IC50=189nM). Due to limited aqueous solubility of KGP94, a water-soluble phosphate salt (KGP420) was prepared in order to facilitate future in vivo studies. Enzymatic hydrolysis with alkaline phosphatase (ALP) demonstrated that the phosphate prodrug, KGP420, was readily converted to the parent compound, KGP94.

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. The amino-based parent anticancer agents 7, 8, 32 (also referred to as KGP05) and 33 (also referred to as KGP156) demonstrated potent cytotoxicity (GI50=0.11-40nM) across all evaluated cell lines, and they were strong inhibitors of tubulin polymerization (IC50=0.62-1.5µM). The various prodrug conjugates and their corresponding salts were investigated for cleavage by the enzyme leucine aminopeptidase (LAP). Four of the glycine water-soluble AAPCs (16, 18, 44 and 45) showed quantitative cleavage by LAP, resulting in the release of the highly cytotoxic parent drug, whereas partial cleavage (<10-90%) was observed for other prodrugs (15, 17, 24, 38 and 39). Eight of the nineteen AAPCs (13-16, 42-45) showed significant cytotoxicity against selected human cancer cell lines. The previously reported CA1-diamine analog and its corresponding hydrochloride salt (8 and 10, respectively) caused extensive disruption (at a concentration of 1.0µM) of human umbilical vein endothelial cells growing in a two-dimensional tubular network on matrigel. In addition, compound 10 exhibited pronounced reduction in bioluminescence (greater than 95% compared to saline control) in a tumor bearing (MDA-MB-231-luc) SCID mouse model 2h post treatment (80mg/kg), with similar results observed upon treatment (15mg/kg) with the glycine amino-dihydronaphthalene AAPC (compound 44). Collectively, these results support the further pre-clinical development of the most active members of this structurally diverse collection of water-soluble prodrugs as promising anticancer agents functioning through a mechanism involving vascular disruption.

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. A series of twenty-two new benzosuberene-based analogues of KGP18 was designed and synthesized. These compounds vary in their methoxylation pattern and separately incorporate trifluoromethyl groups around the pendant aryl ring for the evaluation of the effect of functional group modifications on the fused six-membered aromatic ring. In addition, the 8,9-saturated congener of KGP18 has been synthesized to assess the necessity of unsaturation at the carbon atom bearing the pendant aryl ring. Six of the molecules from this benzosuberene-series of compounds were active (IC50 < 5 lM) as inhibitors of tubulin polymerization while four analogues were comparable (IC50 approximately 1 lM) in their tubulin inhibitory activity to CA4 and KGP18. The potency of a bis-trifluoromethyl analogue 74 and the unsaturated KGP18 derivative 73 as inhibitors of tubulin assembly along with their moderate cytotoxicity suggested the potential utility of these compounds as vascular disrupting agents (VDAs) to selectively target microvessels feeding tumors. Accordingly, water-soluble and DMSO-soluble phosphate prodrug salts of each were synthesized for preliminary in vivo studies to assess their potential efficacy as VDAs.

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 9.9 nM, 14.4 nM, and 8.1 nM, respectively. The benzoylbenzophenone thiosemicarbazone analogues evaluated were selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10 µM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5 µM. The most active cathepsin L inhibitors from this benzoylbenzophenone thiosemicarbazone series (1, 8, and 32) displayed low cytotoxicity toward normal primary cells [in this case human umbilical vein endothelial cells (HUVECs)]. In an initial in vivo study, 3-benzoylbenzophenone thiosemicarbazone (1) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L. Active members of this structurally diverse group of benzoylbenzophenone thiosemicarbazone cathepsin L inhibitors show promise as potential anti-metastatic, pre-clinical drug candidates.

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). Our previous studies led to the discovery and development of a 2-aryl-3-aroyl-indole analogue (OXi8006) that inhibited tubulin polymerization and demonstrated low nM IC50 values against a variety of human cancer cell lines. A water-soluble phosphate prodrug salt (OXi8007), synthesized from OXi8006, displayed promising vascular disrupting activity in mouse models of cancer. To further extend structure-activity relationship correlations, a series of 6-aryl-3-aroyl-indole analogues was synthesized and evaluated for their inhibition of tubulin polymerization and cytotoxicity against human cancer cell lines. Several structurally diverse molecules in this small library were strong inhibitors of tubulin polymerization and of MCF-7 and MDA-MB-231 human breast cancer cells. One of the most promising analogues (KGP591) caused significant G2/M arrest of MDA-MB-231 cells, disrupted microtubule structure and cell morphology in MDA-MB-231 cells, and demonstrated significant inhibition of MDA-MB-231 cell migration in a wound healing (scratch) assay. A phosphate prodrug salt, KGP618, synthesized from its parent phenolic precursor, KGP591, demonstrated significant reduction in bioluminescence signal when evaluated in vivo against an orthotopic model of kidney cancer (RENCA-luc) in BALB/c mice, indicative of VDA efficacy. The most active compounds from this series offer promise as anticancer therapeutic agents.

Probing the specificity of gamma-glutamylamine cyclotransferase: an enzyme involved in the metabolism of transglutaminase-catalyzed protein crosslinks.

γ-Glutamylamine cyclotransferase (gGACT) catalyzes the intramolecular cyclization of a variety of L-γ-glutamylamines producing 5-oxo-L-proline and free amines. Its substrate specificity implicates it in the downstream metabolism of transglutaminase products, and is distinct from that of γ-glutamyl cyclotransferase which acts on L-γ-glutamyl amino acids. To elucidate the mechanism by which gGACT distinguishes between L-γ-glutamylamine and amino acid substrates, the specificity of the rabbit kidney enzyme for the amide region of substrates was probed through the kinetic analysis of a series of L-γ-glutamylamines. The isodipeptide N(ε)-(L-γ-glutamyl)-L-lysine 1 was used as a reference. The kinetic constants of the L-γ-glutamyl derivative of n-butylamine 7, were nearly identical to those of 1. Introduction of a methyl or carboxylate group on the carbon adjacent to the side-chain amide nitrogen in L-γ-glutamylamine substrates resulted in a dramatic decrease in substrate properties for gGACT thus providing an explanation of why gGACT does not act on L-γ-glutamyl amino acids except for L-γ-glutamylglycine. Placement of substituents on carbons further removed from the side-chain amide nitrogen in L-γ-glutamylamines restored activity for gGACT, and L-γ-glutamylneohexylamine 19 had a higher specificity constant (k(cat) /K(m)) than 1. gGACT did not exhibit any stereospecificity in the amide region of L-γ-glutamylamine substrates. In addition, analogues (26-30) with heteroatom substitutions for the γ methylene position of the L-γ-glutamyl moiety were examined. Several thiocarbamoyl derivatives of L-cysteine (28-30) were excellent substrates for gGACT.

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. Herein we report an important extension of this work designed to explore fused aryl-alkyl ring molecular systems that feature nitrogen atom incorporation (dihydroquinoline-based) and carbon atom exclusivity (tetrahydronaphthalene-based). In addition, analogues that contain oxygen (chromanone-based), sulfur (thiochroman-based), sulfoxide, and sulfone functionalization have been prepared in order to further investigate the structure-activity relationship aspects associated with these compounds and their ability to inhibit cathepsins L and B. From this small-library of 30 compounds, five were found to be strongly inhibitory (IC50 <500 nM) against cathepsin L with the most active compound (7-bromodihydroquinoline thiosemicarbazone 48) demonstrating an IC50=164 nM. All of the compounds evaluated were inactive (IC50 >10,000 nM) as inhibitors of cathepsin B, thus establishing a high degree (>20-fold) of selectivity (cathepsin L vs. cathepsin B) for the most active cathepsin L inhibitors in this series.

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.

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.

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. At low nanomolar concentrations OXi6196 caused G2/M cell cycle arrest and apoptosis in MDA-MB-231 breast cancer cells and monolayers of rapidly growing HUVECs underwent concentration-dependent changes in their morphology. Loss of the microtubule structure and increased bundling of filamentous actin into stress fibers followed by cell collapse, rounding and blebbing was observed. OXi6196 (100 nM) disrupted capillary-like endothelial networks pre-established with HUVECs on Matrigel®. When prodrug OXi6197 was administered to mice bearing orthotopic MDA-MB-231-luc tumors, dynamic bioluminescence imaging (BLI) revealed dose-dependent vascular shutdown with >80% signal loss within 2 h at doses ≥30 mg/kg and >90% shutdown after 6 h for doses ≥35 mg/kg, which remained depressed by at least 70% after 24 h. Twice weekly treatment with prodrug OXi6197 (20 mg/kg) caused a significant tumor growth delay, but no overall survival benefit. Similar efficacy was observed for the first time in orthotopic RENCA-luc tumors, which showed massive hemorrhage and necrosis after 24 h. Twice weekly dosing with prodrug OXi6197 (35 mg/kg) caused tumor growth delay in most orthotopic RENCA tumors. Immunohistochemistry revealed extensive necrosis, though with surviving peripheral tissues. These results demonstrate effective vascular disruption at doses comparable to the most effective vascular-disrupting agents (VDAs) suggesting opportunities for further development.

Regioselective synthesis of water-soluble monophosphate derivatives of combretastatin A-1.

The natural products combretastatin A-4 (CA4) and combretastatin A-1 (CA1) are potent cancer vascular disrupting agents and inhibitors of tubulin assembly (IC50 = 1-2 µM). The phosphorylated prodrugs CA4P and CA1P are undergoing human clinical trials against cancer. CA1 is unique due to its incorporation of a vicinal phenol, which has afforded the opportunity to prepare both diphosphate and regioisomeric monophosphate derivatives. Here, we describe the first synthetic routes suitable for the regiospecific preparation of the CA1-monophosphates CA1MPA (8a/b) and CA1MPB (4a/b). The essential regiochemistry necessary to distinguish between the two vicinal phenolic groups was accomplished with a tosyl protecting group strategy. Each of the four monophosphate analogues (including Z and E isomers) demonstrated in vitro cytotoxicity against selected human cancer cell lines comparable to their corresponding diphosphate congeners. Furthermore, Z-CA1MPA (8a) and Z-CA1MPB (4a) were inactive as inhibitors of tubulin assembly (IC50 > 40 µM), as anticipated in this pure protein assay.

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). In syngeneic orthotopic 4T1-luc breast and RENCA-luc kidney tumors, dynamic BLI and oxygen enhanced multispectral optoacoustic tomography (OE-MSOT) were used to compare vascular shutdown following the administration of KGP265 (7.5 mg/kg). The BLI signal and vascular oxygenation response (ΔsO2) to a gas breathing challenge were both significantly reduced within 2 h, indicating vascular disruption, which continued over 24 h. A correlative histology confirmed increased necrosis and hemorrhage. Twice-weekly doses of KGP265 caused significant growth delay in both MDA-MB-231 and 4T1 breast tumors, with no obvious systemic toxicity. A combination with carboplatin produced significantly greater tumor growth delay than carboplatin alone, though significant carboplatin-associated toxicity was observed (whole-body weight loss). KGP265 was found to be effective at low concentrations, generating long-term vascular shutdown and tumor growth delay, thus providing strong rationale for further development, particularly in combination therapies.

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.

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.

Regio- and stereospecific synthesis of mono-beta-d-glucuronic acid derivatives of combretastatin A-1.

Synthetic routes have been established for the preparation of regio- and stereoisomerically pure samples of the mono-beta-d-glucuronic acid derivatives of combretastatin A-1, referred to as CA1G1 (5a) and CA1G2 (6a). Judicious choice of protecting groups for the catechol ring was required for the regiospecific introduction of the glucuronic acid moiety. The tosyl group proved advantageous in this regard. The two monoglucuronic acid analogues demonstrate low cytotoxicity (compared to CA1, 2) against selected human cancer cell lines, with CA1G1 being slightly more potent than CA1G2.

Application of the McMurry coupling reaction in the synthesis of tri- and tetra-arylethylene analogues as potential cancer chemotherapeutic agents.

Structural redesign of selected non-steroidal estrogen receptor binding compounds has previously been successful in the discovery of new inhibitors of tubulin assembly. Accordingly, tetra-substituted alkene analogues (21-30) were designed based in part on combinations of the structural and electronic components of tamoxifen and combretastatin A-4 (CA4). The McMurry coupling reaction was used as the key synthetic step in the preparation of these tri- and tetra-arylethylene analogues. The structural assignment of E, Z isomers was determined on the basis of 2D-NOESY experiments. The ability of these compounds to inhibit tubulin polymerization and cell growth in selected human cancer cell lines was evaluated. Although the compounds were found to be less potent than CA4, these analogues significantly advance the known structure-activity relationship associated with the colchicine binding site on beta-tubulin.