We synthesized a series of enediyne containing Combretastatin A-4 analogues and evaluated the growth inhibitory activity of analogues against two human hepatoma cell lines (Hep G2, Hep 3B), two breast cancer cell lines (MDA-MB-231, MCF-7) and human lung cancer cell line (A-549). It was found that compounds 39a, 39d, 39e, 39f and 40a produced growth inhibitory activity for the five human cancer cell lines. Among them, compound 40a showed the best inhibitory activity (IC50 = 0.8 £gM for A-549 and IC50 = 8.53 £gM for Hep 3B) which were comparable to the Combretastatin A-4 (IC50 = 1.57 £gM for A-549 and IC50 = 14.32 £gM for Hep 3B).

A total of 27 new enediyne analogs of combretastatin A-4 (CA-4), with different substituents (Cl, Br, OCH3, SCH3 or N(CH3)2 ) of the aryl A-ring, were designed, synthesized, and evaluated for their growth inhibition activity against human tumor cell lines. Among them, compound 14a showed most significant inhibition activity against Hep3B (0.22 £gM), A-549 (0.49 £gM) and MCF-7 (0.22 £gM), and these activities were greater than CA-4. The enediyne analog 14a may be consider as a new drug candidate worthy of further investigation and development as a potential antitumor agent.

We designed a new series of enediyne and their derivatives, and evaluating for their growth inhibition activity against human tumor cell lines. This dissertation is composed of two parts. First, compounds 6c, 6e, 6g and 7g displayed good growth inhibition activity against A549 (non-small-cell lung cancer), AGS (human stomach adenocarcinoma), PC-3 (prostate cancer), BT483 (breast carcinomas), HeLa (human cervical epithelioid carcinoma), OVCA (ovarian cancer cell line), SKHep (hepatocellular carcinoma), H460 (human lung cancer cell line) and SW620 (Human Colorectal Cancer Cell Line), especially compound 7g is better, and its average IC50 is 8.79£gM. Second, compounds 25a and 26 displayed better growth inhibition activity against HeLa (human cervical epithelioid carcinoma), OVCA (ovarian cancer cell line), AGS (human stomach adenocarcinoma) and H460 (human lung cancer cell line), and their average IC50 are10.82 and 11.08 £gM.

A series of anilines and enediynes containing Combretastatin A-4 analogues were synthesized. The growth inhibitory activity of these analogues were evaluated against three human cancer cell lines including human hepatoma (Hep G2), breast cancer (MBA-MB231) and lung cancer (A-549) cell lines. The IC50 values of the synthesized compounds are between 10-5-10-7 £gM. We found that compounds 11e, 12e and 13e were observed good growth inhibitory activity for the three human cancer cell lines, especially for A549 (IC50 = 0.22, 0.40 and 0.16 £gM respectively).

Cancer constitutes a terrible burden on modern society. In the United States there are an estimated 1,658,370 new cancer diagnoses resulting in 589,430 deaths in 2015 alone.[1] An estimated 41,170 of these cases will be diagnosed right here in Virginia. With new cancer patients comes the expanding demand for new treatments. As we all know, many modern chemotherapeutics cause adverse reactions to patients. This is because the toxic nature of these therapies often affects normal tissue alongside the tumors that are infesting the body. Therefore, researching novel ways to make chemotherapeutics selective for cancer, while leaving healthy tissue unscathed, is of paramount importance. There are a few ways in which we have approached cancer-specific chemotherapeutics. Through the use of light controlled toxicity and drug release and the targeting of tumor phenotypes such as overexpressed proteins and the Warburg effect, we begin to tackle the problem of non-specificity of current chemotherapeutics. Combretastatin A-4 (CA4) is highly potent anticancer drug that acts as an inhibitor of tubulin polymerization.[2, 3] The core of the CA4 structure contains a cis-stilbene, and it is known that the trans isomer is significantly less potent. We prepared an azobenzene analog of CA4 (Azo-CA4) that shows 13-35 fold enhancement in potency upon external irradiation. GI50 values in the light were in the mid nM range. Due to its ability to thermally revert to the less toxic trans form, Azo-CA4 also has the ability to automatically turn its activity off with time. Therefore, this work establishes a novel strategy for switchable potency for cancer treatment. Doxorubicin (Adriamycin) is an anthracycline type of chemotherapeutic that intercalates double-stranded DNA.[4] Although this drug has played a huge role in the treatment of cancer, its usefulness declines in cases of cancer recurrence because of the impact this drug has on the cardiovascular system. Therefore, we prepared this drug as a cell impermeable conjugate that gains penetrability through the use of external radiation.[5] Folate receptor alpha (FRα) is overexpressed in a variety of cancer cells and accepts folic acid as a natural ligand.[6] Therefore, conjugation of drugs to folic acid introduces a promising way to bring these drugs to cancer cells with greater specificity. We took this concept one step further with the introduction of a photo-labile linker, connecting doxorubicin to folic acid, which offers dual-specificity through ligand targeting and light activation. Finally, many cancer cells produce adenosine triphosphate, the energy currency of a cell, through an abnormal upregulation of glycolysis.[7] This pathway results in a larger-than-normal production of lactic acid and lowers the pH of cancer cells through a phenomenon known as the Warburg Effect. We hypothesized that through the use of L-canavanine, an L-arginine analog, we could construct short peptides that would gain cell permeability in a low pH environment. Attaching a cargo to these peptides, such as doxorubicin will ultimately allow for targeting the low pH extracellular environment of cancer cells. Through the use of these strategies we have furthered the fight against cancer. Targeting cancer by taking advantage of its phenotypes or through the use of light is vital in reducing negative side-effects of current chemotherapeutics. The novel technologies offered above bring us a step closer to side-effect free treatment of cancer patients.

Resveratrol has been reported as a potential anticancer agent but cannot be used as an antitumor drug due to its chemical and metabolic instability. We have designed and synthesized 184 novel compounds related to resveratrol in an attempt to produce more potent and drug-like molecules. We have identified a tetrazole analog of resveratrol, ST-145(a) as a lead anticancer agent from the resveratrol analog series of compounds with a GI50 value of less than 10nM against almost all the human cancer cell lines in the National Cancer Institute’s screening panel. In a separate study, we tested the hypothesis that the limited bioavailability of resveratrol, can be improved by synthesizing analogs which would be glucuronidated at a lower rate than resveratrol itself. We demonstrated that ST-05 and ST-12(a) exhibit lower glucuronidation profiles when compared to resveratrol and that these synthesized stilbenoids likely represent useful scaffolds for the design of efficacious resveratrol analogs. We have also initiated a new discovery program to identify selective CB1 and CB2 receptor ligands from a library of novel stilbene scaffolds structurally related to the resveratrol molecule. From the screened resveratrol analogs, two compounds were identified as selective CB2 and CB1 ligands. Compound ST-179 had 47-fold selectivity for CB2 (Ki = 284 nM) compared to CB1, while compound ST-160 was 2-fold selective for CB1 (Ki = 400 nM) compared to the CB2 receptor. These structural analogs have the potential for development as novel cannabinoid therapeutics for treatment of obesity and/or drug dependency. Combretastatin A4 (CA-4) is one of the most potent antiangiogenic and antimitotic agents of natural origin. However, CA-4 suffers from chemical instability due to cis-trans isomerism in solution. To circumvent this problem, we have developed a facile procedure for the synthesis of novel 4,5-diaryl-2H-1,2,3-triazoles as CA-4 analogs to constrain the molecule to its cis-configuration. Twenty three triazoles were prepared as CA-4 analogs and submitted for anticancer screening. Among these CA-4 analogs, ST-467 and ST-145(b) can be considered as lead anticancer agents from this series, and further investigation against various cancer cell types in vivo with this class of compound may provide novel therapeutic avenues for treatment.

Purpose: Combretastatin A4 phosphate (CA4P) and its structural analog, combretastatin A1 phosphate (CA1P), are soluble prodrugs capable of interacting with tubulin and causing rapid vascular shutdown within tumors. CA4P has completed Phase I clinical trials, but recent preclinical studies have shown that CA1P displays a greater antitumor effect than the combretastatin A4 (CA4) analog at equal doses. The aim of this study, therefore, is to compare pharmacokinetics and metabolism of the two compounds to determine whether pharmacokinetics plays a role in their differential activity. Experimental Design: NMRI mice bearing MAC29 tumors received injection with either CA4P or CA1P at a therapeutic dose of 150 mg·kg-1, and profiles of both compounds and their metabolites analyzed by a sensitive and specific liquid chromatography/mass spectroscopy method. Results: The metabolic profile of both compounds is complex, with up to 14 metabolites being detected for combretastatin A1 (CA1) in the plasma. Many of these metabolites have been identified by liquid chromatography/mass spectroscopy. Initial studies, however, focused on the active components CA4 and CA1, where plasma and tumor areas under the curve were 18.4 and 60.1 µg·h·ml-1 for CA4, and 10.4 and 13.1 µg·h·ml-1 for CA1, respectively. In vitro metabolic comparisons of the two compounds strongly suggest that CA1 is metabolized to a more reactive species than the CA4. Conclusions: Although in vitro studies suggest that variable rates of tumor-specific prodrug dephosphorylation may explain these differences in pharmacokinetics profiles, the improved antitumor activity and altered pharmacokinetic profile of CA1 may be due to the formation of a more reactive metabolite.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A fenstatina, quimicamente designada de 4-metoxifenil-3,4,5-trimetoxifenilmetanona, denominada RR07, Ã uma bisarilcetona de esqueleto estilbenÃide que pode ser obtida a partir da combretastatina A4, com reconhecida atividade citotÃxica, por meio da oxidaÃÃo de Jacobsen. Dentre as muitas atividades desencadeadas pelos estilbenos destacam-se atividade antiangiogÃnica, citotÃxica e antitumoral. Para avaliar o seu potencial antineoplÃsico, um estudo farmacolÃgico de suas propriedades anticÃncer foi realizado em vÃrios modelos biolÃgicos. Utilizando o ensaio do MTT foi feito um estudo comparativo da citotoxicidade de molÃculas estilbenÃides com estruturas relacionadas ao composto RR07, onde foi observado que a presenÃa do anel A (3,4,5-trimetoxifenil) Ã essencial para a atividade citotÃxica da molÃcula. Determinou-se inicialmente a atividade citotÃxica, frente a 13 linhagens tumorais pelo ensaio de reduÃÃo do MTT sendo testados 11 compostos, (RR01, RR02, RR03, RR04, RR05, RR06, RR07, RR08, RR09, RR10, RR11), onde o estilbenÃide RR07 destacou-se como um dos mais citotÃxicos apresentando valores de CI50 que variaram de 0,009 a 17,49 M. Posteriormente, os estudos de mecanismo de aÃÃo com o RR07 nas concentraÃÃes de 0,25; 0,50; 1,00; 2,00 e 4,00 M, revelaram reduÃÃo, de forma concentraÃÃo-dependente, na viabilidade celular pelos mÃtodos do azul de tripan e de BrdU (bromodeoxiuridina). As anÃlises morfolÃgicas feitas por hematoxilina/eosina mostraram nÃcleos metafÃsicos, onde no ensaio de incorporaÃÃo por brometo de etÃdio/laranja de acridina evidenciou-se morte celular por apoptose nas concentraÃÃes de 0,25 e 0,50 M, com cÃlulas em necrose nas concentraÃÃes de 1,00; 2,00 e 4,00 M, destacando-se alteraÃÃes como desintegraÃÃo membranar e picnose nuclear, nas concentraÃÃes de 0,25 M e 1,00 M, respectivamente. Nos ensaios por citometria de fluxo foi observado fragmentaÃÃo do DNA com parada de ciclo na fase G2/M a partir da concentraÃÃo 0,25 M. A anÃlise pelo ensaio do cometa, para o RR07 revelou atividade genotÃxica do tipo concentraÃÃo-dependente entre cÃlulas mononucleadas de sangue perifÃrico (PBMC), principalmente em 2,00 e 4,00 M. A avaliaÃÃo do RR07 no ensaio com tubulina isolada revelou inibiÃÃo na polimerizaÃÃo da mesma em uma concentraÃÃo de 10 M. A anÃlise antitumoral evidenciou inibiÃÃo de 30,9% e 48,19%, respectivamente, para as doses de 20 e 40 mg/kg/dia de RR07 por via intraperitoneal (ip), e 55,68% de inibiÃÃo para a associaÃÃo de 10 mg/kg/dia de 5-fluorouracil com 20 mg/kg/dia de RR07 (ip), em camundongos transplantados com Sarcoma 180, onde se verificou reduÃÃo no crescimento tumoral e alteraÃÃes renais iniciais e reversÃveis. Desta forma, a fenstatina, RR07, apresenta-se como uma proposta de ferramenta farmacolÃgica Ãtil para a pesquisa de novos derivados. / The phenstatin, chemically known as 4-methoxyphenyl-3,4,5-trimetoxiphenylmethane, was called in the present study as RR07. This compound is a bisarylketone with stilbenoid skeleton obtained from combretastatin A4, with recognized cytotoxic and antineoplasic activities. Then, a detailed study of RR07 anticancer properties was conducted in several biological models. The cytotoxic activity of eleven compounds (RR01, RR02, RR03, RR04, RR05, RR06, RR07, RR08, RR09, RR10, RR11) was determined against thirteen tumor cell lines by MTT assay. Structure-activity relationship pointed out that the presence of ring A (3,4,5-trimethoxiphenyl) is essential for the cytotoxic potential. The RR07 was one of the most cytotoxic compounds, showing IC50 values ranging from 0.009 to 17.49 ÂM. Investigations on mechanism of action (0.25, 0.50, 1.00, 2.00 and 4.00 ÂM) showed a concentration-dependent manner cell viability reduction (trypan blue dye exclusion test) and proliferation decreasing (BrdU assay). Morphological alterations determined by hematoxylin/eosin and acridine orange/ethidium bromide fluorescent double staining methods indicated an increased number of apoptotic cells at lowest concentrations (0.25 and 0.50 ÂM) and necrotic cells at higher concentrations (1.00, 2.00 and 4.00 ÂM). Flow cytometry analyses showed that RR07 induced DNA fragmentation and cell cycle arrest at G2/M starting at 0.25 ÂM. In the comet assay performed with human peripheral blood mononuclear cells (PBMC), RR07 caused DNA strand breaks only at higher concentrations (2.00 and 4.00 ÂM). Experiments with isolated tubulin, RR07 also induced tubulin polymerization inhibition in a concentration of 10 μM. In vivo antitumor experiments showed that Sarcoma 180 transplanted-mice treated with RR07 intraperitoneally (ip) presented a tumor growth inhibition ratios of 30.9% and 48.19% (20 mg/kg/day and 40 mg/kg/day, respectively) and inhibition of 55.68% in associated treatment (5-Fluoruracil 10 mg/kg/day + RR07 20 mg/kg/day). Histopathological analyses of kidneys, spleen and livers revealed incipient and reversible alterations. In summary, RR07 can be considered as a pharmacological useful tool as well as a lead molecule to obtain novel compounds with low toxicity and promising antitumor properties.

The goal of this research is to study how the cholesterol content in liposomal formulations affects the anticancer activity (e.g., cell growth suppression) of combretastatin A4 phosphate (CA4P). CA4P is a powerful antivascular agent currently under clinical trials for treating solid tumors. Liposomal CA4P has several advantages over free CA4P, including the reduced toxicities and the increased overall drug efficacy. In this thesis work, I have demonstrated that the proliferation of breast cancer MCF-7 cells varies with the cholesterol mole fraction in the formulation of liposomal CA4P in a biphasic manner, displaying a local minimum at the critical sterol mole fractions (Cr) for maximal superlattice formation. Cell proliferation was monitored using a fluorescence-based assay. Since cholesterol content determines membrane lateral organization, my results imply that membrane lateral organization plays an important role in regulating the anti-cancer activity of liposomal CA4P. This finding provides a new concept in the rational design of liposomal anti-cancer drugs. More than 20 anticancer drug formulations are in the market or under clinical trials. Most of them include cholesterol as a major component. My present study indicates that cholesterol is not just serving as a vesicle stabilizing agent, but also modulates the activity of liposomal drugs. The principle learned from CA4P can be extended to other liposomal anti-cancer drugs. This study is also significant from the membrane biophysics point of view. The data provide additional support for the sterol superlattice model and illustrate that the concept of sterol superlattice can be applied to biotechnology development. / Biochemistry

No / Purpose: Combretastatin A4 phosphate (CA4P) and its structural analog, combretastatin A1 phosphate (CA1P), are soluble prodrugs capable of interacting with tubulin and causing rapid vascular shutdown within tumors. CA4P has completed Phase I clinical trials, but recent preclinical studies have shown that CA1P displays a greater antitumor effect than the combretastatin A4 (CA4) analog at equal doses. The aim of this study, therefore, is to compare pharmacokinetics and metabolism of the two compounds to determine whether pharmacokinetics plays a role in their differential activity. Experimental Design: NMRI mice bearing MAC29 tumors received injection with either CA4P or CA1P at a therapeutic dose of 150 mg/kg-1 , and profiles of both compounds and their metabolites analyzed by a sensitive and specific liquid chromatography/mass spectroscopy method. Results: The metabolic profile of both compounds is complex, with up to 14 metabolites being detected for combretastatin A1 (CA1) in the plasma. Many of these metabolites have been identified by liquid chromatography/mass spectroscopy. Initial studies, however, focused on the active components CA4 and CA1, where plasma and tumor areas under the curve were 18.4 and 60.1 microgram/h/ml-1 for CA4, and 10.4 and 13.1 microgram/h/ml-1 for CA1, respectively. In vitro metabolic comparisons of the two compounds strongly suggest that CA1 is metabolized to a more reactive species than the CA4. Conclusions: Although in vitro studies suggest that variable rates of tumor-specific prodrug dephosphorylation may explain these differences in pharmacokinetics profiles, the improved antitumor activity and altered pharmacokinetic profile of CA1 may be due to the formation of a more reactive metabolite.