2010 - Boa.unimib.it
University of Milano - Bicocca Department of Biotechnology and Biosciences2010Alexandre OrsatoStudies on Tumor Drug TargetingSupported by the Programme Alβan,the European Union Programme ofHigh Level Scholarships for LatinAmericaScholarship no. E07D400786BR
Thesis SupervisorProf. Francesco NicotraFull Professor of Organic ChemistryUniversity of Milano-BicoccaDepartment of Bioscience and BiotechnologyItalyThesis Co-SupervisorBarbara La Ferla Ph.DResearcherUniversity of Milano-BicoccaDepartment of Bioscience and BiotechnologyItalyPh.D. School CoordinatorProf. Franca MorazzoniFull Professor of General and Inorganic ChemistryUniversity of Milano-BicoccaDepartment of Material ScienceItaly
ide,Peptidomimetics, Computer-based drug design, Synthesis, Akt,Phosphatidylinositol phosphate analoguesabstractTumor drug targeting is one of the most promising therapeuticstrategies in oncology. The aim of this PhD work was the study ofthe essential features required for the assembly of tumor targetingconjugates.This work was focused on the deveploment of ligandsfor the GRP receptor that should function as carrier molecules forthe targeting of tumor cells overexpressing this receptor. For thispurpose, non-peptide GRP mimetics were designed, using acomputer-based drug design technique, synthesized and tested.Two analogue compounds based on a bicyclic scaffold exerted anantagonist behaviour on the GRP receptor. Synthetic studies havebeen performed to optimize their production as well as biologicaltests to determine their potential as carrier molecules. Apart fromthe targeting moiety, we also studied the antineoplastic part oftumor targeting conjugates. Akt is a proto-oncogenic kinase thathas been associated to cancer development. Therefore, the Aktinhibitory activity of phosphatidylinositol phosphate analogueswas exploited. A small library of iminosugar-basedphosphatidylinositol phosphate analogues was designed andsynthesized. During the biological evaluation, target compoundsdisplayed low to moderate inhibitory activity for Akt, whichsuggests their feasibility for the development of new and morepotent Akt inhibitors.
CONTENTSABREVIATIONS1.2.3.STATE OF THE ART . 11.1.Tumor Drug Targeting . 41.2.Peptide Receptors as Tumor Targets . 61.3.Bombesin/Gastrin-Releasing Peptide Receptors . 131.4.Bombesin/GRP analogs – antagonists, agonists and tumor targeting conjugates . 171.5.Computer-aided design of peptidomimetics . 291.6.Akt kinase inhibitors as potential anticancer drugs . 35OBJECTIVES . 392.1.Design and synthesis of ligands for the GRP receptor . 402.2.Design and synthesis of iminosugar-based Akt inhibitors . 40RESULTS AND DISCUSSION . 413.1.3D pharmacophore template. 413.2.Scaffold hopping . 453.3.Synthesis of GRP mimetics . 513.3.1.Synthesis of the scaffold . 513.3.2.Design and synthesis of the building blocks . 523.3.3.Attachment of the pharmacophores to the scaffold . 553.3.4.Biological tests performed with compounds 16 and 17 . 603.3.5.Optimization of the synthesis of target compounds . 633.3.6.Synthesis of fluorescent GRP mimetics. 773.4.3.4.1.4.5.iiiDesign and synthesis of iminosugar-based potential Akt inhibitors . 80Preliminary biological evaluation of target compounds . 89CONCLUSIONS . 914.1.Design and synthesis of GRP mimetics . 914.2.Design and synthesis of iminosugar-based Akt inhibitors . 92EXPERIMENTAL PROCEDURES. 935.1.Computational studies . 935.2.Chemistry . 945.2.1.Synthesis of the scaffold . 94i
5.2.2.Synthesis of building blocks . 955.2.3.Synthesis of GRP mimetics . 965.2.4.Synthesis of iminosugar-based Akt inhibitors . 1065.3.6.iiBiological tests . 114BIBLIOGRAPHY. 117
lEthyl acetateAllyl ly-HisLeu-Met-NH2BCRPBreast cancer resistance proteinBnBenzylBNBombesinBoctert-butyl carbamateBombesin(6- Bombesin fragment from residue 6 to 1313)BSTFAN,O-bis(trimethylsilyl)trifluoro acetamideBuButyl groupBu2SnODibutyltin oxideCANCerium Ammonium NitrateCbzCarboxybenzyl carbamateCNSCentral nervous systemCpaChlorophenylalanineCSACamphorsulfonic acidCTComputer deddDouble doubletdddDouble doublet of doublets14des[Met ]Peptide with metionine14 deletedDIADDiisopropyl thyl 10-tetraacetic acidDPPADiphenylphosphoryl azideDTPADiethylene triamine pentaacetic acide.g. and i.e.For exampleiii
PIP2PH hylaminopropyl)carbodiimideEpidermal growth factorFibroblast growth factorPyroglutamic acidGastrin-Releasing PeptideC-terminal decapeptide fragment of Gastrin-releasing peptideHydrocinnamic acidHalf maximal inhibitory concentrationInsulin-like growth factorInterleukinInfraredCoupling constantDissociation constantKilodaltonDissociation constant for inhibitor bindingLowest binding energyLow-density lipoproteinLuteinizing hormone-releasing hormoneMultipletMean binding energyMultidrug resistance protein 1MethanolMinutesMultidrug resistance associated protein 1N-bromosuccinimideNorleucineNuclear magnetic resonanceN-iodosuccinimideNuclear overhauser effectHuman pancreatic carcinoma cell linePolyethylene glycolReference inhibitor of AktPhosphatidylinositol diphosphatePleckstrin homology domainProtein kinase BTriphenylphosphinePart per millionPaclitaxelPyridineRoom temperatureSingletSmall cell lung cancer
TpiTrtvs ψ-CH2NHN-succinimidyl-4-fluorobenzoatesingle photon emission computed tomographythiazolidine-4-carboxylic acidTetra-N-butylammonium fluorideTetra-N-butylammonium Tetramethylpiperidine-1-oxylTrifluoroacetic acidTransforming growth factorsTetrahydrofuranThin-layer chromatographyTrimethylsilyl ido[3,4-b]indol-3-carboxylic acidTritylVersusChemical ShiftReduced peptide bondv
Introduction"Schur, you remember our 'contract' not toleave me in the lurch when the time had come.Now it is nothing but torture and makes nosense."1. State of the art(SigmundFreud,afteryearsofsuffering from cancer of the jaw, he convincedhis personal physician to give him severallarge doses of morphine for the pain. He fellinto a coma and died the next day.)1Cancer is a generic term for a large group of diseases that can affect anypart of the body. Other terms used are malignant tumors and neoplasms. Onedefining feature of cancer is the rapid creation of abnormal cells that grow beyondtheir usual boundaries, and which can then invade adjoining parts of the body andspread to other organs. This process is referred to as metastasis. Metastases arethe major cause of death from cancer.2In the context of cell biology, cancer has a unique importance, and theemphasis given to cancer research has profoundly benefited a much wider area ofmedical knowledge than that of cancer alone. It is a disease in which individualmutant clones of cells begin by prospering at the expense of their neighbors, but inthe end destroy the whole cellular society.3The molecular mechanism of cancer development is a complex multi-stepprocess, which is still subject of intensive research and discussion by the scientificand medical fields.4 Carcinogenesis (the generation of cancer) is linked withmutagenesis (the production of a change in the DNA sequence), which is a clearcorrelation for three classes of agents: chemical carcinogens, ionizing radiationsand certain viruses. Susceptibility to the disease can also be inherited, asindividuals with an inherited genetic defect in the DNA repair mechanisms canaccumulate mutations at an elevated rate.3 In addition to these individual geneticfactors, malignant transformation is also strongly influenced by some molecularchanges associated with ageing.4The malignant transformation occurs on a time scale of months or years ina population of cells in the body. A single mutation is not enough to convert atypical healthy cell into a cancer cell that proliferates without restraint. Indeed, thegenesis of a cancer typically requires that several independent, rare mutational1
Studies on Tumor Drug Targetingevents occur in the lineage of one cell. Studies revealed that an individualmalignant cell generally harbors multiple mutations and that different combinationsof mutations are found in different forms of cancers.3Critical mutations involve genes known as proto-oncogenes and tumorsuppressor genes. Mutations that activate proto-oncogenes stimulate cells toincrease their number when they should not. Mutations that inactivate tumorsuppressor genes allow cells to proliferate without inhibition. Both classes ofgenes code for components of the pathways that regulate the social andproliferative behavior of the cells in the body – in particular , the mechanisms bywhich signals from a cell’s neighbors can impel it to divide, differentiate, or die.3The disease does not usually become apparent until several years after theinitial genetic lesion. During this long incubation period, the prospective cancercells undergo a succession of changes. Cells that descend from the initial mutantclone undergo further mutations that make them proliferate more rapidly. At eachstage, one cell acquires an additional mutation that gives it a selective advantageover its neighbors, making it better able to thrive in its harsh environment, with lowlevels of oxygen, scarce nutrients, and the natural barriers to growth presented bythe surrounding normal tissues. Mutations that help cells to increase in number arecritical for the development of cancer. These mutations generate either anincreased rate of cell division or the resistance to programmed cell death byapoptosis. These special properties include alterations in cell signaling pathways,enabling the cells in a tumor to ignore the signals from their environment thatnormally keep cell proliferation under tight control. In many cancers, changes thatblock the normal maturation of cells toward a non-dividing and terminallydifferentiated state play an important role. The multiple number of mutationsneeded for tumor development is due to the number of different regulatorysystems and barriers that must be disrupted by malignant cancer cell candidates.A powerful contribution to cancer progression is the genetic instability of cancercells, generated by defective correction and repair of DNA damage and trouble inmaintaining chromosome integrity, which increases the probability that cells willexperience a mutation that would lead toward malignancy.32
IntroductionDuring the formation of a neoplasia, dividing cells become no longerconfined to their original location, resulting in a slightly disordered tissue. Somelesions may progress to a more serious stage, where most of the original tissue isoccupied by undifferentiated dividing cells, which are usually highly variable in celland nuclear size and shape. At this stage, it is still easy to achieve a completecure by destroying or removing the abnormal tissue surgically. Without treatment,the abnormal tissue may simply persist and progress no further or may evenregress spontaneously; but in at least 30-40% of cases, progression will occur,giving rise to a frank invasive carcinoma – a malignant lesion where cells crossand destroy the tissue boundaries, invade the surrounding tissues, andmetastasize via the lymphatic vessels.3Metastasis is the most feared and least understood aspect of cancer. Tometastasize, a cancer cell must detach from the primary tumor, invade localtissues and vessels, survive and proliferate in an alien environment to generatenew colonies at distant sites. Metastatic cells need a whole range of aberrantproperties- in other words, new skills - to overcome several barriers andregulatory checkpoints during this process. Indeed, just one in a thousand,perhaps one in a million malignant cells manage to complete this sequence ofevents. Surgical cure becomes progressively more difficult as the invasive growthspreads, as well as localized irradiation. For this reason, early detection of cancerdevelopment enhances the chances to eradicate the disease.3Although rare events are required in the development of cancer cells, theincidence of the disease is becoming progressively higher in the present days.This fact is supported by the observation that carcinogenic environmental factorshave been linked to 75% of human cancers. Such factors are limited to societiesthat are affected by modern lifestyle issues such as tobacco use and pollutionresulting from industrialization. Consequently, every year millions of people learnfor the first time that they have cancer.5Cancer is one of the main causes of death worldwide. It is the secondleading cause of death in developed countries and the third in developingcountries. The disease accounted for an estimate 7.6 million deaths (around 12%worldwide) in 2008, with 1.7 million in Europe (around 19% of deaths).6 Deaths3
Studies on Tumor Drug Targetingfrom cancer worldwide are projected to continue rising, with an estimated 12million deaths in 2030. The main types of cancer leading to overall mortality are:lung, stomach, colorectal, liver and breast.2In addition to the human toll of cancer, the financial cost of cancer is substantial.The direct costs include payments and resources used for treatment, as well asthe costs of care and rehabilitation related to the illness. Data limitations do notallow estimating the worldwide economic costs of cancer. However, the costs ofcancer are staggering.71.1. Tumor Drug TargetingClinical cancer chemotherapy in the 20th century has been dominated bythe development of cytotoxic drugs, initiated by the accidental discovery of theanticancer properties of nitrogen mustard and the folic acid analog aminopterin inthe 1940’s.8 For more than five decades, chemotherapy has been the mainmodality for systemic treatment of advanced or metastatic cancers.9 With theadvances in the comprehension of cancer mechanisms, several cytotoxiccompounds with high potential to become antitumor drugs have been discoveredover the last decades. However, their success in turning into new cancertherapeutics has been hampered by their lack of selectivity for tumor cells. Most ofthe currently available cancer chemotherapeutic agents target DNA or theenzymes involved in DNA replication so that they can exploit the enhancedproliferative rate of cancer cells. Thus, these chemotherapeutic agents (andionizing radiations, which also target mainly DNA) do not have any selectivedestructive effect against cancer cells. They destroy all rapidly dividing cells,including normal dividing cells in vital tissues such as lympho-hematopoetictissues, gonads, hair follicles, and the lining epithelium of the gastrointestinal tractand mouth. The high toxicity of these drugs make them a threat for healthy cells.Their low chemotherapeutic index leads to severe generalized toxic effects whenused at dosages necessary to kill tumor cells.10 Undesirable side effects can be sodevastating to result in the death of the patient.4
IntroductionTherefore, enhancement of the selectivity of cytotoxic drugs for tumor cellsis a matter of extreme importance in oncology. The ultimate goal of cancer therapyis to develop agents that will selectively destroy cancer cells, sparing the normaltissues of the patient. One hundred years ago, after detecting the specificity ofantigen-antibody interactions, Paul Ehrlich created the concept of a “magic-bullet”for cancer therapy, consisting of a molecule selective for a targeted cancer cellthat should be linked to a highly toxic group. This concept became the basis oftumor drug targeting. The expectation has been that the cytotoxicity of theseconjugates will be focused onto tumor cells, sparing normal tissues. Quantitativelymore drug can thus be selectively delivered to the tumor site, to allow a higherdrug concentration in the target cells and avoid systemic toxicity. 10 In general, theaim of tumor drug targeting is to increase the efficacy and reduce the toxicity ofdrugs. The use of a targeting system attached to the drug could help considerablyto manipulate its body distribution and cellular disposition. This genuine idea madea major impact on medicine and chemistry, but remained essentially unexploredfor many decades.11Tumor targeted therapies need to take advantage of some properties ofcancer cells that distinguishes them from normal cells. The first approach to targetcytotoxic compounds directly to the cancer cells exploited tumor-associatedantigens present on their surface.3 More than 25 years ago, monoclonal antibodiesagainst these antigens became very popular as potential “magic bullets” to beused in cancer. However, this fascinating and simple principle turned out to bemuch more difficult to transpose into reality than expected, mainly because of theexcessive molecular mass ( 150KDa) of antibodies. It was only in the past fewyears that adequate drugs based on antibody or antibody fragments have becomecommercially available for diagnosis and therapy of cancer, in particular ofhematological neoplasias.12 A large number of tumor-associated antigens havebeen discovered in the last decades, paving the way for the development of theimmunotherapy of tumors.10 The approach was used for the construction ofmonoclonal antibodies-toxin conjugates called immunotoxins and other chimerictoxins.9 However, the efficacy of cancer immunotherapy with antibodies remainslimited not only by their large size, which compromises its infiltration into the entire5
Studies on Tumor Drug ificbindingtothereticuloendothelial system. This later property is particularly problematic if theantibody is used as a vehicle to deliver radionuclides, cytotoxic drugs, or toxins tothe tumor site, resulting in high toxicity to bone marrow, liver and spleen. 13Apart from antibodies, a number of other carriers with varying degrees oftumor specificity have been investigated for drug targeting to tumors. Theselectivity of most of these carriers is based on specific recognition of cell-surfaceassociated receptors that are found overexpressed and abundant on cancer cells.This group of carriers includes lectins, growth factors (epidermal growth factor[EGF], transforming growth factors [TGF], insulin-like growth factor [IGF], fibroblastgrowth factor [FGF], and transferring), cytokines (IL-2, IL-4, IL-6), hormones, lowdensity lipoproteins (LDL) and folic acid. Hormones that have been successfullyused for drug targeting include melonotropin, insulin, thyrotropin, atinandbombesin/gastrin-releasing peptide. Effective tumor inhibition by cytotoxic agent-linked hormones,cytokines, and transferrin have been demonstrated in human tumor xenograftedmodels especially when adequate numbers of high-affinity receptors are availableon tumor cells. Peptide-hormone receptors have also been successfully targetedby small peptide derivatives of the receptor-specific hormone and their analogs.101.2. Peptide Receptors as Tumor TargetsAn innovative and fascinating application is the use of small peptides todeliver cytotoxic drugs to tumors. One of the main reasons for the increasinginterest for peptides and peptide receptors in cancer is the possibility of receptortargeting, because the peptide receptors are often overexpressed in many primaryhuman cancers, in comparison to their expression in normal tissue adjacent to theneoplasm and/or in its normal tissue of origin. Thus, these receptors can be usedas binding sites for the selective delivery of cytotoxic drugs or radionuclides to thecancer cells.12This field of investigation, which appears to be a small niche in the verylarge oncology field, has gained increased interest in the past decade. The6
Introductiontargeting of overexpressed peptide receptors in tumors by small peptides hasbecome a very strong focus of interest for nuclear medicine. The internationalauthority on nuclear medicine, Dr. Henry Wagner, at the 100-year anniversary ofnuclear medicine, named the peptide approach in nuclear oncology as one of themost promising fields for the present decade.Gastroenterologists andendocrinologists are also attracted by the concept of peptide receptor targeting.12Regulatory peptides represent a group of different families of moleculesknown to act on multiple targets in the human body at extremely lowconcentrations. Targets of these peptides are
EGF Epidermal growth factor FGF Fibroblast growth factor . TLC Thin-layer chromatography TMSOTf Trimethylsilyl trifluoromethanesulfonate Tpi 2,3,4,9-tetrahydro-1H-pyrido . experience a mutation that would lead toward malignancy.3. Introduction 3 During the form
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