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WO1999064046A1 - Agonistes polyvalents, agonistes partiels, agonistes inverses et antagonistes des recepteurs 5-ht¿3? - Google Patents

Agonistes polyvalents, agonistes partiels, agonistes inverses et antagonistes des recepteurs 5-ht¿3? Download PDF

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Publication number
WO1999064046A1
WO1999064046A1 PCT/US1999/012768 US9912768W WO9964046A1 WO 1999064046 A1 WO1999064046 A1 WO 1999064046A1 US 9912768 W US9912768 W US 9912768W WO 9964046 A1 WO9964046 A1 WO 9964046A1
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ligand
substituted
linker
ligands
linkers
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PCT/US1999/012768
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WO1999064046A9 (fr
WO1999064046A8 (fr
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Guang Yang
Susan Meier-Davis
John H. Griffin
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Advanced Medicine, Inc.
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Priority to EP99928450A priority Critical patent/EP1085888A1/fr
Priority to CA002321191A priority patent/CA2321191A1/fr
Priority to AU45514/99A priority patent/AU4551499A/en
Publication of WO1999064046A1 publication Critical patent/WO1999064046A1/fr
Publication of WO1999064046A9 publication Critical patent/WO1999064046A9/fr
Publication of WO1999064046A8 publication Critical patent/WO1999064046A8/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G01N33/942—Serotonin, i.e. 5-hydroxy-tryptamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/301—Anxiety or phobic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/304—Mood disorders, e.g. bipolar, depression

Definitions

  • This invention relates to novel therapeutic agents which bind to mammalian receptors and modulate their activity. More particularly, the invention relates to novel therapeutic agents that bind to and modulate the in vivo activity of 5-HT 3 receptors in mammals by acting as multi-binding compounds.
  • the therapeutic agents or multi-binding compounds described herein comprise at least two ligands connected by a linker or linkers, wherein the ligands in their monovalent state bind to and/or are capable of modulating the activity of the 5-HT 3 receptor.
  • the linking moiety is chosen such that the multi-binding compounds so constructed demonstrate increased biological activity as compared to individual units of the ligand.
  • the invention also relates to methods of using such compounds, to methods of preparing such compounds and to pharmaceutical compositions containing them.
  • this invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a multi-binding compound of this invention.
  • the multi-binding compounds are useful as affinity resins for affinity chromatography.
  • the compounds of the invention may be used as a tool in immunoprecipitation.
  • the compounds may be used to identify a receptor in vitro for example in microscopy, electrophoresis and chromatography.
  • Chevallier, B "The Control of Acute Cisplatin-induced Emesis - a Comparative Study of Granisetron and a Combination Regimen of High-dose Metoclopramide and Dexamethasone", Br. J. Cancer
  • a receptor is a biological structure with one or more binding domains that reversibly complexes with one or more ligands, where that complexation has biological consequences.
  • Receptors can exist entirely outside the cell (extracellular receptors), within the cell membrane (but presenting sections of the receptor to the extracellular milieu and cytosol), or entirely within the cell (intracellular receptors). They may also function independently of a cell (e.g. , clot formation). Receptors within the cell membrane allow a cell to communicate with the space outside of its boundaries (i.e. , signaling) as well as to function in the transport of molecules and ions into and out of the cell.
  • a ligand is a binding partner for a specific receptor or family of receptors.
  • a ligand may be the endogenous ligand for the receptor or alternatively may be a synthetic ligand for the receptor such as a drug, a drug candidate or a pharmacological tool.
  • the G-proteins when activated, affect a wide range of downstream effector systems both positively and negatively (e.g. , ion channels, protein kinase cascades, transcription, transmigration of adhesion proteins, and the like).
  • the ligands that bind to G-protein cellular receptors may be specifically classified as follows:
  • Antagonist- ligands that when bound inhibit or prevent the activity arising from a natural ligand binding to the receptor.
  • Antagonists may be of the surmountable class (results in the parallel displacement of the dose-response curve of the agonist to the right in a dose dependent fashion without reducing the maximal response for the agonist) or insurmountable class (results in depression of the maximal response for a given agonist with or without the parallel shift);
  • G-protein cellular receptors There are four fundamental measurable properties that pertain to the interaction of a ligand with its receptor including G-protein cellular receptors:
  • an activating ligand or agonist
  • an inhibiting ligand has affinity for the receptor, and their efficacy lies in their ability to effectively block agonism of the receptor.
  • Selectivity defines the ratios of affinities or the ratios of efficacies of a given ligand compared across two receptors. It is the selectivity of a specific drug that provides the required biological profile. For example, in certain therapeutic settings, it is currently thought that a highly selective drug may be preferred (e.g. , Losartan (Cozaar), an antihypertensive, is a highly selective antagonist for the ATI receptor). In contrast, it is considered that a drug with a broad spectrum of receptor activity may be preferred in other therapeutic settings.
  • a highly selective drug may be preferred (e.g. , Losartan (Cozaar), an antihypertensive, is a highly selective antagonist for the ATI receptor).
  • Losartan Losartan
  • a drug with a broad spectrum of receptor activity may be preferred in other therapeutic settings.
  • ligands targeting receptors, including G-protein receptors
  • G-protein receptors have clinical shortcomings identified by one or more of low efficacy, low affinity, poor safety profile, lack of selectivity or overselectivity for the intended receptor, and suboptimal duration of action and onset of action. Accordingly, it would be beneficial to develop ligands that have improved affinity, efficacy, selectivity, onset of action and duration of action.
  • An increase in ligand affinity to the target receptor may contribute to reducing the dose of ligand required to induce the desired therapeutic effect.
  • a reduction in ligand affinity will remove activity and may contribute to the selectivity profile for a ligand.
  • Efficacy of ligand at a target receptor can lead to a reduction in the dose required to mediate the desired therapeutic effect. This increase in efficacy may arise from an improved positive functional response of the ligand or a change from a partial to full agonist profile. Reduced efficacy of a full agonist to a partial agonist may provide clinical benefit by modulating the biological response. Further, antagonists have efficacy at inhibiting the agonism of the receptor.
  • a decrease in the selectivity of the ligand may also be desired.
  • the angiotensin II endogenous ligand activates both the ATI and AT2 receptor subtypes.
  • ⁇ 2 adrenergic agonists such as albuterol have a relatively short duration of action of approximately 3-4 hours and an increase in duration of action would simplify the dosing regimen required to administer this drug (ligand).
  • Desensitization is best defined as the variety of processes by which the functional interaction of the receptor with its G-protein are influenced. These processes lead ultimately to a reduction in cellular response to the activating agonist. Such phenomena are most often observed during prolonged stimulation of the receptor.
  • the two main pathways for receptor desensitization are reduction in receptor density or changes in receptor structure by phosphorylation mechanisms.
  • Receptor density is altered by receptor sequestration. This is a reversible process that is observable within minutes and is a dynamic sorting of receptors with receptors being cycled to and from the membrane. On the other hand, receptor down-regulation is generally slower, in the order of hours, and is irreversible, involving destruction of the receptor. Finally, receptor density may be affected by an alteration in the rate of synthesis. For example, the rates of ⁇ 2 mRNA synthesis and degradation are controlled by levels of c-AMP within the cell. Alternatively, receptor desensitization may occur through changes in receptor structure, such as receptor phosphorylation.
  • agonist induced activation of the ⁇ 2 -adrenergic receptor which is positively coupled to adenylate cyclase through Gs, results in an elevation in an increase in the levels of c-AMP and an increase in the activity of protein kinase A.
  • This kinase can readily phosphorylate the consensus site in the third intracellular loop.
  • the phosphorylated ⁇ 2 -adrenergic receptor exhibits significantly reduced coupling to Gs.
  • the G-protein coupled receptor kinases GRK
  • GRK G-protein coupled receptor kinases
  • Receptor oligomerization also plays a role in receptor function. This is best exemplified in the area of growth receptors that are known to act functionally and structurally as dimers, e.g. , EGF-R and interferon receptor. It is also known that dimerization is involved in the functioning of the steroid receptor. Preliminary evidence is beginning to appear on the importance of oligomerization in G-protein coupling and signaling. It is proposed that receptor oligomerization may play a role in different receptor functions such as mediating coupling of the G-protein or receptor internalization.
  • 5-HT 3 receptors are ligand-gated ion channels which are located on neurons in many systems in the periphery and in the brain. 6
  • the 5-HT 3 receptor is unique in that it belongs to the superfamily of ion-channel receptors 14 and is a ligand-gated ion channel that is permeant to Na + , K + , Ca 2+ and other cations. 6
  • Another family of receptors is the ligand-gated ion channel receptors.
  • membrane bound cell surface receptors are composed of multiple subunits, typically 5 subunits, which may be the same or different.
  • Ligands which bind to this receptor and therapeutic indications include:
  • Vomiting results from an intricate series of physiological events mediated by humoral factors and afferent fibers, and both inhibition and excitation of somatic visceral musculature that are ultimately coordinated by the vomiting center in the brain.
  • the emetic (vomiting) center is a nucleus of cells located in the medulla and is the motor center responsible for the coordination of emesis. 4
  • 5-HT 5 -hydroxy - tryptamine
  • 5-HT 3 receptor antagonists do not prevent the release of serotonin but instead create a blockade that prevents serotonin from binding to the 5-HT 3 receptors.
  • the binding of the antagonist to the receptor thereby prevents transmission of impulses that initiate nausea and vomiting.
  • Other receptors associated with nausea and vomiting include histamine (HI), dopamine (D2), muscarinic (Ml), acetylcholine. noradrenaline and endorphin. 8
  • 5-HT 3 receptor antagonists are used to treat chemotherapy induced emesis.
  • these antagonists include ondansetron (Zofran * , Glaxo Wellcome), granisetron (Kytrif, SmithKline), dolasetron (Anzemet * , Hoechst Marion Roussel), tropisetron (Sandoz), itasetron (currently in Phase III testing, Boehringer Ingelheim) and mirtazipine.
  • These can be used either alone as a single agent, in combination, or in a combination cocktail with dexamethasone, a corticosteroid.
  • the compounds can also be used in combination with NK1 receptor antagonists. Currently, the use of these compounds does not eliminate chemotherapy induced emesis in all patients.
  • novel ligands having desired potency and therapeutic effect for the 5-HT 3 receptor would be particularly desirable in order to further inhibit emesis, especially chemotherapy induced emesis, in mammalian patients.
  • Such novel ligands would preferably achieve the desired potency and therapeutic effect by modulating one or more of the ligand's properties as to efficacy, affinity, safety profile, selectivity, duration of action and/or onset of action.
  • This invention is directed, in part, to novel multi-binding compounds that bind 5HT 3 receptors and consequently these compounds can be used to treat conditions mediated by 5HT 3 receptors such as chemotherapy induced and radiation induced emesis, anxiety, schizophrenia, drug withdrawal and cognitive disorders.
  • this invention is directed to a multi-binding compound and salts thereof comprising 2 to 10 ligands, which may be the same or different and which are covalently attached to a linker or linkers which may be the same of different, at least one of the ligands comprising a ligand domain capable of binding to a 5-HT 3 receptor.
  • at least two and more preferably each of the ligands comprises a ligand domain capable of binding to a 5HT 3 receptor. Since the 5-HT 3 receptor is a pentameric construct, the most preferred compounds include between 2 and 5 ligands . More preferably, each of the ligands independently comprises an agonist, partial agonist, inverse agonist or antagonist of the 5HT 3 receptors.
  • the multi-binding compounds of this invention are preferably represented by formula I:
  • each L is independently selected from ligands comprising a ligand domain capable of binding to a 5-HT 3 receptor;
  • X is independently a linker;
  • p is an integer of from 2 to 10;
  • q is an integer of from 1 to 20; and pharmaceutically acceptable salts thereof.
  • q is less than/?.
  • the ligands comprise a ligand domain capable of binding to one or more 5-HT 3 receptors which modulate chemotherapy induced and radiation induced emesis, anxiety, schizophrenia, drug withdrawal and cognitive disorders in mammals. More preferably, the ligands are selected from the group consisting of ondansetron, granisetron, tropisetron, dolasetron, mirtazapine and itasetron. However, in one embodiment, at least one of the ligands in the multi-binding compound is a corticosteroid.
  • this invention provides a multibinding compound of formula II:
  • each L' is independently a ligand comprising a ligand binding site for the the 5HT 3 receptors, and is preferably an agonist, partial agonist, inverse agonist or antagonist of the 5HT 3 receptors and X' is a linker; and pharmaceutically-acceptable salts thereof.
  • each ligand, L' is independently selected from the group consisting of ondansetron, granisetron, tropisetron, dolasetron, mirtazapine and itasetron;
  • X' is a linker; and pharmaceutically-acceptable salts thereof.
  • each linker i.e. , X or X'
  • each linker independently has the formula: -X a -Z-(Y a -Z) m -Y b -Z-X a - wherein m is an integer of from 0 to 20;
  • X at each separate occurrence is selected from the group consisting of -O-, -S-, -NR-, -C(O)-, -C(O)O-, -C(O)NR-, -C(S), -C(S)O-, -C(S)NR- or a covalent bond where R is as defined below;
  • Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;
  • this invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of a multi-binding compound, or a pharmaceutically acceptable salt thereof, comprising 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers which may be the same or different, at least one of the ligands comprising a ligand domain capable of binding to one or more 5-HT 3 receptors.
  • the compound is a compound of formula I or II.
  • this invention is directed to a method for treating or preventing disorders mediated by 5-HT 3 receptors, such as chemotherapy induced and radiation induced emesis, anxiety, and schizophrenia and cognitive disorders or ameliorating the effects of drug withdrawal in a mammal
  • a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a multi-binding compound, or a pharmaceutically acceptable salt thereof, comprising 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers which may be the same or different, at least two of said ligands comprising a ligand domain capable of binding to a 5- HT 3 receptor.
  • the pharmaceutical composition comprises a pharmaceutically acceptable excipient and a multi-binding compound represented by formulas I or II as described above.
  • the ligand is selected from the group consisting of ondansetron, granisetron, tropisetron, dolasetron, mirtazapine and itasetron.
  • at least one of the ligands in said multi- binding compound is a corticosteroid.
  • This invention is also directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties with respect to the 5HT 3 receptors.
  • the diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage.
  • the library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarizabihty and/or polarization.
  • the library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.
  • This invention is also directed to libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties with respect to the 5HT 3 receptors.
  • These libraries are prepared via the methods described above and permit the rapid and efficient evaluation of what molecular constraints impart multibinding properties to a ligand or a class of ligands targeting the 5HT 3 receptors.
  • this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties with respect to the 5HT 3 receptors which method comprises:
  • each linker in the library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
  • this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties which method comprises:
  • each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
  • the preparation of the multimeric ligand compound library is achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands identified in (a) with the linkers identified in (b).
  • Sequential addition is preferred when a mixture of different ligands is employed to ensure heteromeric or multimeric compounds are prepared. Concurrent addition of the ligands occurs when at least a portion of the multimer comounds prepared are homomultimeric compounds.
  • the assay protocols recited in (d) can be conducted on the multimeric ligand compound library produced in (c) above, or preferably, each member of the library is isolated by preparative liquid chromatography mass spectrometry (LCMS).
  • LCMS preparative liquid chromatography mass spectrometry
  • this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties which library is prepared by the method comprising:
  • each linker in the library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
  • this invention is directed to a library of multimeric ligand compounds which bind to the 5HT 3 receptors which may possess multivalent properties which library is prepared by the method comprising:
  • each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
  • the library of linkers employed in either the methods or the library aspects of this invention is selected from the group comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarizabihty and/or polarization and amphiphilic linkers.
  • each of the linkers in the linker library may comprise linkers of different chain length and/or having different complementary reactive groups. Such linker lengths can preferably range from about 2 to 100A.
  • the ligand or mixture of ligands is selected to have reactive functionality at different sites on the ligands in order to provide for a range of orientations of the ligand on the multimeric ligand compounds.
  • reactive functionality includes, by way of example, carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, pseudohalides isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates and precursors thereof. It is understood, of course, that the reactive functionality on the ligand is selected to be complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.
  • the multimeric ligand compound is homomeric (i.e. , each of the ligands is the same, although it may be attached at different points) or heteromeric (i.e. , at least one of the ligands is different from the other ligands).
  • this invention provides for an iterative process for rationally evaluating what molecular constraints impart multibinding properties to a class of multimeric compounds or ligands targeting the 5HT 3 receptors.
  • this method aspect is directed to a method for identifying multimeric ligand compounds possessing multibinding properties with respect to the 5HT 3 receptors which method comprises:
  • steps (f) and (f) are repeated at least two times, more preferably at from 2-50 times, even more preferably from 3 to 50 times, and still more preferably at least 5-50 times.
  • Figures 1-4 illustrate reaction schemes for preparing multi-binding compounds of this invention.
  • Figure 5 illustrates examples of multibinding compounds comprising 2 ligands attached in different forms to a linker.
  • Figure 6 illustrates examples of multibinding compounds comprising 3 ligands attached in different forms to a linker.
  • Figure 7 illustrates examples of multibinding compounds comprising 4 ligands attached in different forms to a linker.
  • Figure 8 illustrates examples of multibinding compounds comprising 5-10 ligands attached in different forms to a linker.
  • Ligand (drug) interactions with cellular receptors are controlled by molecular interaction/recognition between the ligand and the receptor. In turn, such interaction can result in modulation or disruption of the biological processes/functions of these receptors and, in some cases, leads to cell death. Accordingly, when cellular receptors mediate mammalian pathologic conditions, interactions of the ligand with the cellular receptor can be used to treat these conditions.
  • mammalian 5-HT 3 receptors which are known to modulate emetic conditions in mammals; particularly chemotherapy induced emetic conditions.
  • this invention is directed, in part, to multi-binding compounds that bind 5-HT 3 receptors.
  • affinity and specificity of the 5-HT receptor and a ligand thereto are dependent upon the complementarity of molecular binding surfaces and the energetic costs of complexation. "Affinity” is sometimes quantified by the equilibrium constant of complex formation. Specificity relates to the difference in affinity between the same ligand binding to different ligand binding sites on the cellular receptor.
  • the multi-binding compounds of this invention are capable of acting as multi-binding agents and the surprising activity of these compounds arises at least in part from their ability to bind in a multivalent manner with mammalian 5-HT 3 receptors.
  • the ligands in the multibinding compounds comprise agonists, partial agonists, inverse agonists or antagonists of the 5HT 3 receptors.
  • the following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.
  • alkyl refers to a monoradical branched or unbranched saturated hydrocarbon chain preferably having from 1 to 40 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, ⁇ -propyl, tso-propyl, ⁇ -butyl, iso- butyl, fl-hexyl, ⁇ -decyl, tetradecyl, and the like.
  • substituted alkyl refers to an alkyl group as defined above, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,
  • alkylene refers to a diradical of a branched or unbranched saturated hydrocarbon chain, preferably having from 1 to 40 carbon atoms, more preferably 1 to 10 carbon atoms and even more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methylene (-CH 2 -), ethylene
  • substituted alkylene refers to an alkylene group, as defined above, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioal
  • substituted alkylene groups include those where 2 substituents on the alkylene group are fused to form one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fused to the alkylene group.
  • fused groups contain from 1 to 3 fused ring structures.
  • alkaryl refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein. Such alkaryl groups are exemplified by benzyl, phenethyl and the like.
  • alkoxy refers to the groups alkyl-O-, alkenyl-O-, cycloalkyl-O- , cycloalkenyl-O-, and alkynyl-O-, where alkyl, alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.
  • Preferred alkoxy groups are alkyl-O- and include, by way of example, methoxy, ethoxy, n-propoxy, /s ⁇ -propoxy, «-butoxy, tert-butoxy, sec-butoxy, ⁇ -pentoxy, ⁇ -hexoxy, 1 ,2-dimethylbutoxy, and the like.
  • substituted alkoxy refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.
  • alkylalkoxy refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl and substituted alkylene- O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.
  • Preferred alkylalkoxy groups are alkylene-O-alkyl and include, by way of example, methylenemethoxy (-CH 2 OCH 3 ), ethylenemethoxy (-CH 2 CH 2 OCH 3 ), n-propylene-wo-propoxy (-CH 2 CH 2 CH 2 OCH(CH 3 ) 2 ), methylene-t-butoxy (-CH 2 -O-C(CH 3 ) 3 ) and the like.
  • alkylthioalkoxy refers to the group -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene - S-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.
  • Preferred alkylthioalkoxy groups are alkylene-S- alkyl and include, by way of example, methylenethiomethoxy (-CH 2 SCH 3 ), ethylenethiomethoxy (-CH 2 CH 2 SCH 3 ), n-propylene- r ⁇ -thiopropoxy (-CH 2 CH 2 CH 2 SCH(CH 3 ) 2 ), methylene-t-thiobutoxy (-CH 2 SC(CH 3 ) 3 ) and the like.
  • alkenyl refers to a monoradical of a branched or unbranched unsaturated hydrocarbon group preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of vinyl unsaturation.
  • substituted alkenyl refers to an alkenyl group as defined above having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino
  • alkenylene refers to a diradical of a branched or unbranched unsaturated hydrocarbon group preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of vinyl unsaturation.
  • substituted alkenylene refers to an alkenylene group as defined above having from 1 to 5 substituents, and preferably from 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamin
  • substituted alkenylene groups include those where 2 substituents on the alkenylene group are fused to form one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fused to the alkenylene group.
  • alkynyl refers to a monoradical of an unsaturated hydrocarbon preferably having from 2 to 40 carbon atoms, more preferably 2 to 20 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of acetylene (triple bond) unsaturation.
  • Preferred alkynyl groups include ethynyl (-C ⁇ CH), propargyl (-CH 2 C ⁇ CH) and the like.
  • substituted alkynyl refers to an alkynyl group as defined above having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxya
  • alkynylene refers to a diradical of an unsaturated hydrocarbon preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of acetylene (triple bond) unsaturation.
  • Preferred alkynylene groups include ethynylene (-C ⁇ C-). propargylene (-CH 2 C ⁇ C-) and the like.
  • substituted alkynylene refers to an alkynylene group as defined above having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxya
  • acyl refers to the groups HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)- and heterocyclic- C(O)- where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.
  • acylamino or “aminocarbonyl” refers to the group -C(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, heterocyclic or where both R groups are joined to form a heterocyclic group (e.g. , morpholino) wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
  • aminoacyl refers to the group -NRC(O)R where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
  • aminoacyloxy or “alkoxycarbonylamino” refers to the group
  • each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
  • acyloxy refers to the groups alkyl-C(O)O-, substituted alkyl- C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclic-C(O)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • aryl refers to an unsaturated aromatic carbocyclic group of from 6 to 20 carbon atoms having a single ring (e.g. , phenyl) or multiple condensed (fused) rings (e.g. , naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the like.
  • such aryl groups can optionally be substituted with from 1 to 5 substituents, preferably 1 to 3 substituents, selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryl
  • aryloxy refers to the group aryl-O- wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.
  • arylene refers to the diradical derived from aryl (including substituted aryl) as defined above and is exemplified by 1 ,2-phenylene, 1,3- phenylene, 1 ,4-phenylene, 1 ,2-naphthylene and the like.
  • amino refers to the group -NH 2 .
  • substituted amino refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic provided that both R's are not hydrogen.
  • cycloalkyl refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings.
  • Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
  • substituted cycloalkyl refers to cycloalkyl groups having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino
  • cycloalkenyl refers to cyclic alkenyl groups of from 4 to 20 carbon atoms having a single cyclic ring and at least one point of internal unsaturation.
  • suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.
  • substituted cycloalkenyl refers to cycloalkenyl groups having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxya
  • halo or halogen refers to fluoro, chloro, bromo and iodo.
  • heteroaryl refers to an aromatic group of from 1 to 15 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring) .
  • heteroaryl groups can be optionally substituted with 1 to 5 substituents, preferably 1 to 3 substituents, selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy
  • Preferred aryl substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.
  • Such heteroaryl groups can have a single ring (e.g. , pyridyl or furyl) or multiple condensed rings (e.g. , indolizinyl or benzothienyl).
  • Preferred heteroaryls include pyridyl, pyrrolyl and furyl.
  • heteroaryloxy refers to the group heteroaryl-O-.
  • heteroarylene refers to the diradical group derived from heteroaryl (including substituted heteroaryl), as defined above, and is exemplified by the groups 2,6-pyridylene, 2,4-pyridiylene, 1 ,2-quinolinylene, 1 ,8- quinolinylene, 1 ,4-benzofuranylene, 2,5-pyridnylene, 2,5-indolenyl and the like.
  • heterocycle or “heterocyclic” refers to a monoradical saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 40 carbon atoms and from 1 to 10 hetero atoms, preferably 1 to 4 heteroatoms, selected from nitrogen, sulfur, phosphorus, and/or oxygen within the ring.
  • heterocyclic groups can be optionally substituted with 1 to 5, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro
  • nitrogen heterocycles and heteroaryls include, but are not limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, morpholino, piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containing
  • a preferred class of heterocyclics include “crown compounds” which refers to a specific class of heterocyclic compounds having one or more repeating units of the formula [-(CH 2 -) m Y-] where m is > 2, and Y at each separate occurrence can be O, N. S or P.
  • Examples of crown compounds include, by way of example only, [-(CH 2 ) 3 -NH-] 3 , [-((CH 2 ) 2 -O) 4 -((CH 2 ) 2 -NH) 2 ] and the like. Typically such crown compounds can have from 4 to 10 heteroatoms and 8 to 40 carbon atoms.
  • heterocyclooxy refers to the group heterocyclic-O-.
  • thioheterocyclooxy refers to the group heterocyclic-S-.
  • heterocyclene refers to the diradical group formed from a heterocycle, as defined herein, and is exemplified by the groups 2,6-morpholino, 2,5-morpholino and the like.
  • oxy acylamino or “aminocarbonyloxy” refers to the group -OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
  • spiro-attached cycloalkyl group refers to a cycloalkyl group attached to another ring via one carbon atom common to both rings.
  • thiol refers to the group -SH.
  • thioalkoxy refers to the group -S-alkyl.
  • substituted thioalkoxy refers to the group -S-substituted alkyl.
  • thioaryloxy refers to the group aryl-S- wherein the aryl group is as defined above including optionally substituted aryl groups also defined above.
  • heteroaryloxy refers to the group heteroaryl-S- wherein the heteroaryl group is as defined above including optionally substituted aryl groups as also defined above.
  • the ligands and linkers which comprise the multibinding agents of the invention and the multibinding compounds themselves may have various stereoisomeric forms, including enantiomers and diastereomers. It is to be understood that the invention contemplates all possible stereoisomeric forms of multibinding compounds, and mixtures thereof.
  • pharmaceutically-acceptable salt refers to salts which retain the biological effectiveness and properties of the multibinding compounds of this invention and which are not biologically or otherwise undesirable.
  • the multibinding compounds of this invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
  • Salts derived from inorganic bases include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as alkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines, di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl) amines, tri(substituted alkenyl) amines, cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted cycloalkyl amines, substituted cycloalkyl amines, substituted
  • Suitable amines include, by way of example only, isopropylamine, trimethyl amine, diethyl amine, tri( sopropyl) amine, tri( ⁇ - propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like.
  • carboxylic acid derivatives would be useful in the practice of this invention, for example, carboxylic acid amides, including carboxamides, lower alkyl carboxamides, dialkyl carboxamides, and the like.
  • Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, /?-toluene-sulfonic acid, salicylic acid, and the like.
  • pharmaceutically-acceptable cation refers to the cation of a pharmaceutically-acceptable salt .
  • protecting group refers to any group which when bound to one or more hydroxyl, thiol, amino or carboxyl groups of the compounds (including intermediates thereof) prevents reactions from occurring at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, thiol, amino or carboxyl group.
  • removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t- butyl-diphenylsilyl and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.
  • substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t- butyl-diphenylsilyl and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.
  • Preferred removable thiol blocking groups include disulfide groups, acyl groups, benzyl groups, and the like.
  • Preferred removable amino blocking groups include conventional substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), fluorenylmethoxycarbonyl (FMOC), allyloxycarbonyl (ALOC), and the like which can be removed by conventional conditions compatible with the nature of the product.
  • Preferred carboxyl protecting groups include esters such as methyl, ethyl, propyl, t-butyl etc. which can be removed by mild conditions compatible with the nature of the product.
  • ligand denotes a compound that is a binding partner for the 5HT 3 receptors and is bound thereto by complementarity.
  • the ligand is preferably an agonist, partial agonist, inverse agonist or antagonist of the 5HT 3 receptors.
  • the specific region or regions of the ligand that is (are) recognized by the receptor is designated as the "ligand domain” .
  • a ligand may be either capable of binding to a receptor by itself, or may require the presence of one or more non-ligand components for binding (e.g., Ca +2 , Mg +2 or a water molecule is required for the binding of a ligand to various ligand binding sites).
  • ligands and linkers which comprise the multibinding agents of the invention and the multibinding compounds themselves may have various stereoisomeric forms, including enantiomers and diastereomers. It is to be understood that the invention contemplates all possible stereoisomeric forms of multibinding compounds and mixtures thereof.
  • ligands useful in this invention are described herein, and specifically include mirtazapine, granisetron, ondansetron, paroxetine (binds all 5- HT receptor subtypes), tropisetron, dolasetron, and itasetron and analogs thereof.
  • ligand structure that are not essential for specific molecular recognition and binding activity may be varied substantially, replaced or substituted with unrelated structures (for example, with ancillary groups as defined below) and, in some cases, omitted entirely without affecting the binding interaction.
  • the primary requirement for a ligand is that it has a ligand domain as defined above.
  • ligand is not intended to be limited to compounds known to be useful in binding to 5HT 3 receptors, (e.g. , known drugs).
  • ligand can equally apply to a molecule that is not normally associated with receptor binding properties.
  • ligands that exhibit marginal activity or lack useful activity as monomers can be highly active as multivalent compounds because of the benefits conferred by multivalency.
  • ligands useful in this invention are ligands directed to other receptors which modulate chemotherapy induced emesis such as the dopamine (D2), histamine (HI) and muscarinic (Ml).
  • D2 antagonists useful in this invention include phenothiazines, such as chlo ⁇ romazine, perphenazine, prochlorperazine, promethazine, thiethylperazine, triflupromazine; benzimidazole derivatives such as domperidon; and butyrophenones such as haloperidol and droperidol.
  • Substituted benzamides such as metoclopramide, trimethobenzamide and metopimazine are both D2 and 5-HT 3 antagonists.
  • Combinations of ligands to the 5-HT 3 and the D2 receptors may also be made as multi-binding compounds.
  • Antagonists to HI (histamine) receptor currently thought to be minimally effective in reducing chemotherapy induced emesis, such as diphenhydramine and meclizine, may also be used in this invention.
  • multibinding compound or agent refers to a compound that is capable of multivalency, as defined below, and which has 2-10 ligands which comprise a ligand binding domain which is capable of binding to one or more 5HT 3 receptors, and which is covalently bound to one or more linkers which may be the same or different.
  • Multibinding compounds provide a biological and/or therapeutic effect greater than the aggregate of unlinked ligands equivalent thereto which are made available for binding. That is to say that the biological and/or therapeutic effect of the ligands attached to the multibinding compound is greater than that achieved by the same amount of unlinked ligands made available for binding to the ligand binding sites (receptors).
  • the phrase "increased biological or therapeutic effect” includes, for example: increased affinity, increased selectivity for target, increased specificity for target, increased potency, increased efficacy, decreased toxicity, improved duration of activity or action, decreased side effects, increased therapeutic index, improved bioavailibity, improved pharmacokinetics, improved activity spectrum, and the like.
  • the multibinding compounds of this invention will exhibit at least one and preferably more than one of the above-mentioned effects.
  • Emesis is the act or instance of vomiting.
  • An “emetic agent” is an agent that induces vomiting. Emesis also includes nausea.
  • emetic agents include compounds used in chemotherapy and radiation therapy.
  • “Chemotherapy induced emesis” refers to emetic episodes which are induced by exposure to chemotherapy and/or anti-cancer treatments such as treatment with, for example, cisplatin, adriamycin, apomorphine, cyclohexamide, cyclophosphamide, copper sulphate, ipecacuanha, mustine and radiation, among others.
  • Chemotherapy induced emetic episodes may be acute or may be delayed up to several days. There are generally three types of chemotherapy induced emesis seen clinically: acute, delayed and anticipatory (conditioned). 7 Acute chemotherapy induced nausea and vomiting generally is considered to be that which occurs within the first 24 hours following drug administration.
  • Delayed emesis is generally defined as emetic episodes starting about 24 hours or more following the last treatment. 7 This can be a serious complication for patients, as it can be protracted and severe and there are very few good treatment options for its prevention. 4 Conditioned or anticipatory emesis results from poor control of acute or delayed emesis. It is typically associated with anxiety prior to the next dose of chemotherapy, followed by nausea or vomiting before, during or possibly after the administration of chemotherapy. 7
  • potency refers to the minimum concentration at which a ligand is able to achieve a desirable biological or therapeutic effect.
  • the potency of a ligand is typically proportional to its affinity for its ligand binding site. In some cases, the potency may be non-linearly correlated with its affinity.
  • the dose-response curve of each is determined under identical test conditions (e.g. , in an in vitro or in vivo assay, in an appropriate animal model). The finding that the multibinding agent produces an equivalent biological or therapeutic effect at a lower concentration than the aggregate unlinked ligand is indicative of enhanced potency.
  • univalency refers to a single binding interaction between one ligand as defined herein with one ligand binding site as defined herein. It should be noted that a compound having multiple copies of a ligand (or ligands) exhibit univalency when only one ligand is interacting with a ligand binding site. Examples of univalent interactions are depicted below.
  • multivalency refers to the concurrent binding of from 2 to 10 linked ligands (which may be the same or different) and two or more corresponding receptors (ligand binding sites) on one or more receptors which may be the same or different.
  • selectivity is a measure of the binding preferences of a ligand for different ligand binding sites (receptors).
  • the selectivity of a ligand with respect to its target ligand binding site relative to another ligand binding site is given by the ratio of the respective values of K d (i.e. , the dissociation constants for each ligand-receptor complex) or, in cases where a biological effect is observed below the K d , the ratio of the respective EC 50 's (i.e. , the concentrations that produce 50% of the maximum response for the ligand interacting with the two distinct ligand binding sites (receptors)).
  • ligand binding site denotes the site on the 5HT 3 receptors that recognizes a ligand domain and provides a binding partner for the ligand.
  • the ligand binding site may be defined by monomeric or multimeric structures. This interaction may be capable of producing a unique biological effect, for example, agonism, antagonism, modulatory effects, may maintain an ongoing biological event, and the like. However, in one embodiment, the ligand(s) merely bind to a ligand binding site and do not have agonistic or antagonistic activity.
  • Ligands which are full agonists are ligands which when bound trigger the maximum activity seen by the natural ligands.
  • Ligands which are partial agonists are ligands which when bound trigger sub-maximum activity.
  • Ligands which are antagonists are ligands that when bound, inhibit or prevent the activity arising from a natural ligand binding to the receptor.
  • Antagonists may be of the surmountable class (results in the parallel displacement of the dose-response curve of the agonist to the right in a dose dependent fashion without reducing the maximal response for the agonist) or insurmountable class (results in depression of the maximal response for a given agonist with or without the parallel shift).
  • Ligands which are inverse agonists are ligands that, when bound, decrease the basal activity of the unbound receptor or which provide an activity opposite of the natural agonist.
  • Ligands have measurable properties that relate to the interaction of the ligand and the receptor. These include the affinity of the ligand for the receptor, which relates to the energetics of the binding, the efficacy of the ligand for the receptor, which relates to the functional downstream activity of the ligand, the kinetics of the ligand for the receptor, which defines the onset of action and the duration of action, and the desensitization of the receptor for the ligand. Selectivity defines the ratio of the affinity and/or efficacy of a ligand across two receptors.
  • modulatory effect refers to the ability of the ligand to change the activity of an agonist or antagonist through binding to a ligand binding site. It is a combination of these properties which provides the foundation for defining the nature of the functional response.
  • the ligand binding sites of the receptor that participate in biological multivalent binding interactions are constrained to varying degrees by their intra- and inter-molecular associations (e.g., such macromolecular structures may be covalently joined to a single structure, noncovalently associated in a multimeric structure, embedded in a membrane or polymeric matrix, and so on) and therefore have less translational and rotational freedom than if the same structures were present as monomers in solution.
  • inert organic solvent or “inert solvent” means a solvent which is inert under the conditions of the reaction being described in conjunction therewith including, by way of example only, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform, methylene chloride, diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol, ethanol, propanol, isopropanol, t-butanol, dioxane, pyridine, and the like.
  • the solvents used in the reactions described herein are inert solvents.
  • the "5-HT 3 receptor” is a serotonin receptor and plays a role in the mechanisms of nausea and emesis.
  • 5-HT 3 receptors are located on neurons in many systems in the periphery and in the brain.
  • the 5-HT 3 receptor is unique in that it belongs to the superfamily of ion-channel receptors 14 and is a ligand-gated ion channel that is permeant to Na + , K + , Ca 2+ and other cations.
  • 6 5-HT 3 receptor antagonists are effective in the control of chemotherapy induced emesis in mammals. They are also promising in the control of central nervous system conditions such as anxiety, schizophrenia, drug withdrawal and cognitive disorders. 14
  • the 5-HT 3 receptors that participate in biological multivalent binding interactions are constrained to varying degrees by their intra- and intermolecular associations (e.g. cellular receptors may be covalently joined in a single structure, noncovalently associated in a multimeric structure, embedded in a membrane or polymeric matrix and so on) and therefore have less translational and rotational freedom than if the same cellular receptors were present as monomers in solution.
  • the 5-HT 3 receptors are generally pentameric structures, formed by association of five identical subunits.
  • treatment refers to any treatment of a pathologic condition in a mammal, particularly a human, and includes: (i) preventing the pathologic condition from occurring in a subject which may be predisposed to the condition but has not yet been diagnosed with the condition and, accordingly, the treatment constitutes prophylactic treatment for the disease condition;
  • pathologic condition which is modulated by treatment with a ligand covers all disease states (i.e. , pathologic conditions) which are generally acknowledged in the art to be usefully treated with a ligand for the 5HT 3 receptors in general, and those disease states which have been found to be usefully treated by a specific multibinding compound of our invention.
  • disease states include, by way of example only, chemotherapy induced and radiation induced emesis, anxiety, schizophrenia, drug withdrawal and cognitive disorders.
  • therapeutically effective amount refers to that amount of multibinding compound which is sufficient to effect treatment, as defined above, when administered to a mammal in need of such treatment.
  • the therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • linker refers to a group or groups that covalently links from 2 to 10 ligands (as identified above) in a manner that provides for a compound capable of multivalency, for example, when in the presence of at least one cellular receptor having 2 or more ligand binding sites.
  • the linker is a ligand -orienting entity that permits attachment of multiple copies of a ligand (which may be the same or different) thereto. In some cases, the linker may itself be biologically active.
  • linker does not, however, extend to cover solid inert supports such as beads, glass particles, fibers, and the like. But it is understood that the multibinding compounds of this invention can be attached to a solid support if desired. For example, such attachment to solid supports can be made for use in separation and purification processes and similar applications.
  • library refers to at least 3, preferably from 10 2 to 10 9 and more preferably from 10 2 to 10 4 multimeric compounds. Preferably, these compounds are prepared as a multiplicity of compounds in a single solution or reaction mixture which permits facile synthesis thereof.
  • the library of multimeric compounds can be directly assayed for multibinding properties.
  • each member of the library of multimeric compounds is first isolated and, optionally, characterized. This member is then assayed for multibinding properties.
  • selection refers to a set of multimeric compounds which are prepared either sequentially or concurrently (e.g. , combinatorially).
  • the collection comprises at least 2 members; preferably from 2 to 10 9 members and still more preferably from 10 to 10 4 members.
  • multimeric compound refers to compounds comprising from 2 to 10 ligands covalently connected through at least one linker which compounds may or may not possess multibinding properties (as defined herein).
  • pseudohalide refers to functional groups which react in displacement reactions in a manner similar to a halogen.
  • Such functional groups include, by way of example, mesyl, tosyl, azido and cyano groups.
  • linker or linkers that joins the ligands presents these ligands to the array of available ligand binding sites. Beyond presenting these ligands for multivalent interactions with ligand binding sites, the linker or linkers spatially constrains these interactions to occur within dimensions defined by the linker or linkers.
  • structural features of the linker valency, geometry, orientation, size, flexibility, chemical composition, etc. are features of multibinding agents that play an important role in determining their activities.
  • the linkers used in this invention are selected to allow multivalent binding of ligands to the ligand binding sites of 5HT 3 receptors, wherever such sites are located on the receptor structure, whether such sites are located interiorly, both interiorly and on the periphery of the molecule, or at any intermediate position thereof.
  • the distance between the nearest neighboring ligand domains is preferably in the range of about 2 A to about 100A, more preferably in the range of about 10A to about 50A.
  • the linker lengths are in the range of about 3 A to about 10A.
  • the ligands are covalently attached to the linker or linkers using conventional chemical techniques providing for covalent linkage of the ligand to the linker or linkers.
  • Reaction chemistries resulting in such linkages are well known in the art and involve the use of complementary functional groups on the linker and ligand.
  • the complementary functional groups on the linker are selected relative to the functional groups available on the ligand for bonding or which can be introduced onto the ligand for bonding. Again, such complementary functional groups are well known in the art.
  • reaction between a carboxylic acid of either the linker or the ligand and a primary or secondary amine of the ligand or the linker in the presence of suitable, well-known activating agents results in formation of an amide bond covalently linking the ligand to the linker; reaction between an amine group of either the linker or the ligand and a sulfonyl halide of the ligand or the linker results in formation of a sulfonamide bond covalently linking the ligand to the linker; and reaction between an alcohol or phenol group of either the linker or the ligand and an alkyl or aryl halide of the ligand or the linker results in formation of an ether bond covalently linking the ligand to the linker.
  • Table I illustrates numerous complementary reactive groups and the resulting bonds formed by reaction there between.
  • the linker is attached to the ligand at a position that retains ligand domain- ligand binding site interaction and specifically which permits the ligand domain of the ligand to orient itself to bind to the ligand binding site. Such positions and synthetic protocols for linkage are well known in the art.
  • the term linker embraces everything that is not considered to be part of the ligand.
  • the relative orientation in which the ligand domains are displayed derives from the particular point or points of attachment of the ligands to the linker, and on the framework geometry.
  • the determination of where acceptable substitutions can be made on a ligand is typically based on prior knowledge of structure-activity relationships (SAR) of the ligand and/or congeners and/or structural information about ligand-receptor complexes (e.g. , X-ray crystallography, NMR, and the like).
  • SAR structure-activity relationships
  • ligand-receptor complexes e.g. , X-ray crystallography, NMR, and the like.
  • Such positions and the synthetic methods for covalent attachment are well known in the art.
  • the univalent linker-ligand conjugate may be tested for retention of activity in the relevant assay.
  • the multibinding agent is a bivalent compound, e.g. , two ligands which are covalently linked to linker X.
  • the linker when covalently attached to multiple copies of the ligands, provides a biocompatible, substantially non-immunogenic multibinding compound.
  • the biological activity of the multibinding compound is highly sensitive to the valency, geometry, composition, size, flexibility or rigidity, position of atachment, etc. of the linker and, in turn, on the overall structure of the multibinding compound, as well as the presence or absence of anionic or cationic charge, the relative hydrophobicity/hydrophilicity of the linker, and the like on the linker. Accordingly, the linker is preferably chosen to maximize the biological activity of the multibinding compound.
  • the linker may be chosen from any organic molecule construct that orients two or more ligands to their ligand binding sites to permit multivalency.
  • the linker can be considered as a "framework" on which the ligands are arranged in order to bring about the desired ligand-orienting result, and thus produce a multibinding compound.
  • different orientations can be achieved by including in the framework groups containing mono- or polycyclic groups, including aryl and/or heteroaryl groups, or structures incorporating one or more carbon-carbon multiple bonds (alkenyl, alkenylene, alkynyl or alkynylene groups).
  • Other groups can also include oligomers and polymers which are branched- or straight-chain species.
  • rigidity is imparted by the presence of cyclic groups (e.g. , aryl, heteroaryl, cycloalkyl, heterocyclic, etc.).
  • the ring is a six, nine, ten, twelve, fifteen and eighteen member ring.
  • the ring is an aromatic ring such as, for example, phenyl or naphthyl.
  • the hydrophobic/hydrophilic characteristics of the linker as well as the presence or absence of charged moieties can readily be controlled by the skilled artisan.
  • the hydrophobic nature of a linker derived from hexamethylene diamine (H 2 N(CH 2 ) 6 NH 2 ) or related polyamines can be modified to be substantially more hydrophilic by replacing the alkylene group with a poly(oxyalkylene) group such as found in the commercially available "Jeffamines" .
  • the ability of the compounds to cross the blood/brain barrier can be controlled. This can be important when one wishes to maximize or minimize CNS effects.
  • core structures other than those shown here can be used for determining the optimal framework display orientation of the ligands.
  • the process may require the use of multiple copies of the same central core structure or combinations of different types of display cores.
  • the above-described process can be extended to trimers and compounds of higher valency.
  • the physical properties of the linker can be optimized by varying the chemical composition thereof.
  • the composition of the linker can be varied in numerous ways to achieve the desired physical properties for the multibinding compound.
  • linkers include aliphatic moieties, aromatic moieties, steroidal moieties, peptides, and the like. Specific examples are peptides or polyamides, hydrocarbons, aromatic groups, ethers, lipids, cationic or anionic groups, or a combination thereof.
  • linker can be modified by the addition or insertion of ancillary groups into or onto the linker, for example, to change the solubility of the multibinding compound (in water, fats, lipids, biological fluids, etc.), hydrophobicity, hydrophilicity, linker flexibility, antigenicity, stability, and the like.
  • the introduction of one or more poly(ethylene glycol) (PEG) groups onto or into the linker enhances the hydrophilicity and water solubility of the multibinding compound, increases both molecular weight and molecular size and, depending on the nature of the unPEGylated linker, may increase the in vivo retention time. Further PEG may decrease antigenicity and potentially enhances the overall rigidity of the linker.
  • PEG poly(ethylene glycol)
  • Ancillary groups which enhance the water solubility /hydrophilicity of the linker and, accordingly, the resulting multibinding compounds are useful in practicing this invention.
  • ancillary groups such as, for example, small repeating units of ethylene glycols, alcohols, polyols (e.g. , glycerin, glycerol propoxylate, saccharides, including mono-, oligosaccharides, etc.), carboxylates (e.g. , small repeating units of glutamic acid, acrylic acid, etc.), amines (e.g.
  • the ancillary group used to improve water solubility /hydrophilicity will be a polyether .
  • lipophilic ancillary groups within the structure of the linker to enhance the lipophilicity and/or hydrophobicity of the multibinding compounds described herein is also within the scope of this invention.
  • Lipophilic groups useful with the linkers of this invention include, by way of example only, aryl and heteroaryl groups which, as above, may be either unsubstituted or substituted with other groups, but are at least substituted with a group which allows their covalent attachment to the linker.
  • Other lipophilic groups useful with the linkers of this invention include fatty acid derivatives which do not form bilayers in aqueous medium until higher concentrations are reached.
  • lipid refers to any fatty acid derivative that is capable of forming a bilayer or a micelle such that a hydrophobic portion of the lipid material orients toward the bilayer while a hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro and other like groups well known in the art.
  • Hydrophobicity could be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups of up to 20 carbon atoms and such groups substituted by one or more aryl, heteroaryl, cycloalkyl, and/or heterocyclic group(s).
  • Preferred lipids are phosphglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl-ethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoyl-phosphatidylcholine or dilinoleoylphosphatidylcholine could be used.
  • lipid Other compounds lacking phosphorus, such as sphingolipid and glycosphingolipid families are also within the group designated as lipid. Additionally, the amphipathic lipids described above may be mixed with other lipids including triglycerides and sterols.
  • the flexibility of the linker can be manipulated by the inclusion of ancillary groups which are bulky and/or rigid.
  • the presence of bulky or rigid groups can hinder free rotation about bonds in the linker or bonds between the linker and the ancillary group(s) or bonds between the linker and the functional groups.
  • Rigid groups can include, for example, those groups whose conformational lability is restrained by the presence of rings and/or multiple bonds within the group, for example, aryl, heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups.
  • Other groups which can impart rigidity include polypeptide groups such as oligo- or polyproline chains.
  • Rigidity may also be imparted by internal hydrogen bonding or by hydrophobic collapse.
  • Bulky groups can include, for example, large atoms, ions (e.g. , iodine, sulfur, metal ions, etc.) or groups containing large atoms, polycyclic groups, including aromatic groups, non-aromatic groups and structures incorporating one or more carbon-carbon multiple bonds (i.e. , alkenes and alkynes). Bulky groups can also include oligomers and polymers which are branched- or straight-chain species. Species that are branched are expected to increase the rigidity of the structure more per unit molecular weight gain than are straight-chain species.
  • rigidity is imparted by the presence of cyclic groups (e.g. , aryl, heteroaryl, cycloalkyl, heterocyclic, etc.).
  • the linker comprises one or more six-membered rings.
  • the ring is an aryl group such as, for example, phenyl or naphthyl.
  • Rigidity can also be imparted electrostatically.
  • the ancillary groups are either positively or negatively charged, the similarly charged ancillary groups will force the presenter linker into a configuration affording the maximum distance between each of the like charges.
  • the energetic cost of bringing the like- charged groups closer to each other will tend to hold the linker in a configuration that maintains the separation between the like-charged ancillary groups.
  • Further ancillary groups bearing opposite charges will tend to be attracted to their oppositely charged counterparts and potentially may enter into both inter- and intramolecular ionic bonds . This non-covalent mechanism will tend to hold the linker into a conformation which allows bonding between the oppositely charged groups.
  • ancillary groups which are charged, or alternatively, bear a latent charge when deprotected, following addition to the linker, include deprotectation of a carboxyl, hydroxyl, thiol or amino group by a change in pH, oxidation, reduction or other mechanisms known to those skilled in the art which result in removal of the protecting group, is within the scope of this invention.
  • the multibinding compounds described herein comprise 2-10 ligands attached to a linker that links the ligands in such a manner that they are presented to the receptor for multivalent interactions with ligand binding sites thereon/ therein.
  • the linker spatially constrains these interactions to occur within dimensions defined by the linker. This and other factors increases the biological activity of the multibinding compound as compared to the same number of ligands made available in monobinding form.
  • the compounds of this invention are preferably represented by the empirical formula (L) p (X) q where L, X, p and q are as defined above.
  • L, X, p and q are as defined above.
  • the linker may be considered as a framework to which ligands are attached.
  • the ligands can be attached at any suitable position on this framework, for example, at the termini of a linear chain or at any intermediate position.
  • the simplest and most preferred multibinding compound is a bivalent compound which can be represented as L-X-L, where each L is independently a ligand which may be the same or different and each X is independently the linker. Examples of such bivalent compounds are provided in Figure 5, where each shaded circle represents a ligand.
  • a trivalent compound could also be represented in a linear fashion, i.e. , as a sequence of repeated units L-X-L-X-L, in which L is a ligand and is the same or different at each occurrence, as can X.
  • a trimer can also be a radial multibinding compound comprising three ligands attached to a central core, and thus represented as (L) 3 X, where the linker X could include, for example, an aryl or cycloalkyl group.
  • Illustrations of trivalent and tetravalent compounds of this invention are found in Figures 6 and 7 respectively where, again, the shaded circles represent ligands. Tetravalent compounds can be represented in a linear array, e.g. ,
  • X and L are as defined herein.
  • X and L could be represented as an alkyl, aryl or cycloalkyl derivative as above with four (4) ligands attached to the core linker.
  • a preferred linker may be represented by the following formula:
  • m is an integer of from 0 to 20;
  • X a at each separate occurrence is selected from the group consisting of -O-, -S-, -NR-, -C(O)-, -C(O)O-, -C(O)NR-, -C(S), -C(S)O-, -C(S)NR- or a covalent bond where R is as defined below;
  • Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;
  • Y a and Y b at each separate occurrence are selected from the group consisting of.
  • n 0, 1 or 2;
  • R, R' and R" at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic.
  • the linker moiety can be optionally substituted at any atom therein by one or more alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic group.
  • the linker i.e. , X or X'
  • Table II is selected those shown in Table II:
  • the linker i.e. , X or X'
  • the linker has the formula:
  • each R is independently selected from the group consisting of a covalent bond, alkylene, substituted alkylene and arylene; each R b is independently selected from the group consisting of hydrogen, alkyl and substituted alkyl; and ⁇ ' is an integer ranging from 1 to about 20.
  • linker when used in combination with the term “multibinding compound” includes both a covalently contiguous single linker (e.g. , L-X-L) and multiple covalently non-contiguous linkers (L-X-L-X-L) within the multibinding compound.
  • Any compound which is comprises a ligand domain capable of binding to a 5HT 3 receptor, preferably, which is an agonist, partial agonist, inverse agonist or antagonist of the 5HT 3 receptors, and which can be covalently linked to a linker can be used as a ligand to prepare the compounds described herein.
  • ligands are well known to those of skill in the art.
  • 5HT 3 ligands which can be used as ligands to prepare the compounds of the present invention include mirtazapine, granisetron, ondansetron, paroxetine (binds all 5-HT receptor subtypes), tropisetron, dolasetron, and itasetron. and analogs thereof. Analogs (for these and other known compounds which bind to the 5HT 3 receptors) which can be used include alkylated, acylated, carboxylated, amidated, sulfonated, phosphorylated, aminated, hydroxylated, thiolated, and halogenated derivatives. Examples of some of these ligands are shown below:
  • Ligands which bind to 5HT 3 receptors are well-known in the art and can be readily prepared using art- recognized starting materials, reagents and reaction conditions.
  • the following patents and publications disclose compounds, intermediates and procedures useful in the preparation of ligands which bind to 5HT 3 receptors:
  • the multibinding compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e. , reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
  • protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions.
  • the choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991 , and references cited therein.
  • any compound which is an agonist, partial agonist, inverse agonist or antagonist of the 5HT 3 receptors, preferably antagonists of the 5HT 3 receptors, can be used as a ligand in this invention.
  • numerous such receptor agonists, partial agonists, inverse agonists and antagonists are known in the art and any of these known compounds or derivatives thereof may be employed as ligands in this invention.
  • a compound selected for use as a ligand will have at least one functional group, such as an amino, hydroxyl, thiol or carboxyl group and the like, which allows the compound to be readily coupled to the linker.
  • Compounds having such functionality are either known in the art or can be prepared by routine modification of known compounds using conventional reagents and procedures.
  • the patents and publications set forth above provide numerous examples of suitably functionalized agonists, partial agonists and antagonists of the 5HT 3 receptors, and intermediates thereof, which may be used as ligands in this invention.
  • the ligands can be covalently attached to the linker through any available position on the ligands, provided that when the ligands are attached to the linker, at least one of the ligands retains its ability to bind to the 5HT 3 receptors.
  • Certain sites of attachment of the linker to the ligand are preferred based on known structure-activity relationships.
  • the linker is attached to a site on the ligand where structure-activity studies show that a wide variety of substituents are tolerated without loss of receptor activity.
  • Ligand precursors for example, ligands containing a leaving group or a nucleophilic group
  • linker precursor containing a nucleophilic group or a leaving group
  • ligand precursors with a halide, tosylate, or other leaving group can be readily coupled to a linker precursor containing two nucleophilic groups, for example, amine groups, to form a dimer.
  • the leaving group employed in this reaction may be any conventional leaving group including, by way of example, a halogen such as chloro, bromo or iodo. or a sulfonate group such as tosyl, mesyl and the like.
  • nucleophilic group is a phenol
  • any base which effectively deprotonates the phenolic hydroxyl group may be used, including, by way of illustration, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydride, sodium hydroxide, potassium hydroxide, sodium ethoxide, triethylamine, diisopropylethylamine and the like.
  • Nucleophilic substition reactions are typically conducted in an inert diluent, such as tetrahydrofuran, N,N-dimethylformamide, NN-dimethylacetamide, acetone, 2- butanone, l-methyl-2-pyrrolidinone and the like. After the reaction is complete, the dimer is typically isolated using conventional procedures, such as extraction, filtration, chromatography and the like.
  • dimers with a hydrophilic linker can be formed using a ligand precursor containing nucleophilic groups and a a polyoxyethylene containing leaving groups, for example, poly(oxyethylene) dibromide (where the number of oxyethylene units is typically an integer from 1 to about 20).
  • a ligand precursor containing nucleophilic groups and a a polyoxyethylene containing leaving groups for example, poly(oxyethylene) dibromide (where the number of oxyethylene units is typically an integer from 1 to about 20).
  • two molar equivalents of the ligand precursor are reacted with one molar equivalent of the poly(oxyethylene) dibromide in the presence of excess potassium carbonate to afford a dimer.
  • This reaction is typically conducted in N,N-dimethylformamide at a temperature ranging from about 25 °C to about 100°C for about 6 to about 48 hours.
  • the linker connecting the ligands may be prepared in several steps. Specifically, a ligand precursor can first be coupled to an "adapter" , i.e., a bifunctional group having a leaving group at one end and another functional group at the other end which allows the adapter to be coupled to a intermediate linker group. In some cases, the functional group used to couple to the intermediate linker is temporarily masked with a protecting group ("PG").
  • PG protecting group
  • adapters include, by way of illustration, tert-butyl bromoacetate, 1- Fmoc-2-bromoethylamine, l-trityl-2-bromoethanethiol, 4-iodobenzyl bromide, propargyl bromide and the like.
  • Ligand precursors can be coupled with adapters which include both leaving groups and protecting groups to form protected intermediates.
  • the leaving group employed in this reaction may be any conventional leaving group including, by way of example, a halogen such as chloro, bromo or iodo, or a sulfonate group such as tosyl, mesyl and the like.
  • any conventional protecting group may be employed including, by way of example, esters such as the methyl, tert- butyl, benzyl (“Bn”) and 9-fluorenylmethyl (“Fm”) esters.
  • Protected intermediates can then be deprotected using conventional procedures and reagents to afford deprotected intermediates.
  • tert- butyl esters are readily hydrolyzed with 95 % trifluoroacetic acid in dichloromethane; methyl ester can be hydrolyzed with lithium hydroxide in tetrahydrofuran water; benzyl esters can be removed by hydrogenolysis in the presence of a catalyst, such as palladium on carbon; and 9-fluorenylmethyl esters are readily cleaved using 20% piperidine in DMF.
  • a catalyst such as palladium on carbon
  • 9-fluorenylmethyl esters are readily cleaved using 20% piperidine in DMF.
  • other well-known protecting groups and deprotecting procedures may be employed in these reactions to form deprotected intermediates.
  • ligand precursors having an adapter with an amine functional group can be prepared.
  • Ligand precursors can be coupled with adapters which include leaving groups and protected amine groups to afford protected intermediates.
  • the leaving group employed in this reaction may be any conventional leaving group.
  • any conventional amine protecting group may be employed including, by way of example, trityl, tert-butoxycarbonyl ("Boc”), benzyloxycarbonyl (“CBZ”) and 9-fluorenylmethoxy-carbonyl (“Fmoc”).
  • a trityl group is readily removed using hydrogen chloride in acetone; a Boc group is removed using 95% trifluoroacetic acid in dichloromethane; a CBZ group can be removed by hydrogenolysis in the presence of a catalyst, such as palladium on carbon; and a 9-fluorenylmethoxycarbonyl group is readily cleaved using 20% piperidine in DMF to afford the deblocked amine.
  • a catalyst such as palladium on carbon
  • a 9-fluorenylmethoxycarbonyl group is readily cleaved using 20% piperidine in DMF to afford the deblocked amine.
  • Other well-known amine protecting groups and deprotecting procedures may be employed in these reactions to form amine -containing intermediates and related compounds.
  • Ligand precursors having an adapter can be readily coupled to intermediate linkers having complementary functional groups to form multibinding compounds as described herein.
  • the coupling reaction typically employs a conventional peptide coupling reagent and is conducted under conventional coupling reaction conditions, typically in the presence of a trialkylamine, such as ethyldiisopropylamine.
  • Suitable coupling reagents for use in this reaction include, by way of example, carbodiimides, such as ethyl-3-(3-dimethylamino)propylcarboiimide (EDC), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and the like, and other well-known coupling reagents, such as N,N' -carbonyldiimidazole, 2-ethoxy-l-ethoxycarbonyl- 1,2-dihydroquinoline (EEDQ), benzotriazol-1-yloxy- tris(dimethylamino)phosphonium hexafluorophosphate (BOP), O-(7- azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and the like.
  • carbodiimides such as ethyl-3
  • this coupling reaction is conducted at a temperature ranging from about 0°C to about 60 °C for about 1 to about 72 hours in an inert diluent, such as THF, to afford the dimer.
  • an inert diluent such as THF
  • the multibinding compounds described herein can also be prepared using a wide variety of other synthetic reactions and reagents.
  • ligand precursors having aryliodide, carboxylic acid, amine and boronic acid functional groups can be prepared.
  • Hydroxymethyl pyrrole can be readily coupled under Mitsunobu reaction conditions to various phenols to provide, after deprotection, functionalized intermediates.
  • the Mitsunobu reaction is typically conducted by reacting hydroxymethyl pyrrole and the appropriate phenol using diethyl azodicarboxylate (DEAD) and triphenylphosphine at ambient temperature for about 48 hours. Deprotection, if necessary, using conventional procedures and reagents then affords the functionalized intermediates.
  • DEAD diethyl azodicarboxylate
  • triphenylphosphine triphenylphosphine
  • aryliodide intermediates can be coupled with bis-boronic acid linkers to provide dimers.
  • this reaction is conducted by contacting two molar equivalents of the aryliodide and one molar equivalent of the bis-boronic acid in the presence of tetrakis(triphenylphosphine)palladium(0), sodium carbonate and water in refluxing toluene.
  • Aryliodide intermediates can also be coupled with acrylate intermediates or alkyne intermediate to afford dimers. These reactions are typically conducted by contacting two molar equivalents of aryliodide intermediates with one molar equivalent of either acrylates or alkynes in the presence of dichlorobis(triphenylphosphine)palladium (II), copper (I) iodide and diisopropylethylamine in N,N-dimethylformamide to afford the respective dimers.
  • Hibert, et al. 24 assessed the low-energy conformations to define a pharmacophore and receptor map that may account for the activity of ondansetron, metoclopramide and some tropane -based esters and amides.
  • the basic pharmacophore consists essentially of a carbonyl group coplanar to an aromatic ring and a basic center with the relative positions and dimensions illustrated below:
  • linking acyl functional group - can be substituted by other hydrogen bond acceptor analogous to a carbonyl group
  • bivalent 5-HT 3 ligands can be prepared.
  • such compounds selected for use as a ligand will have at least one functional group, such as an amino, thiol, hydroxyl or carboxyl group and the like, which allows the compound to be readily coupled to another ligand via a suitable linker.
  • Compounds having such functionality are either known in the art or can be prepared by routine modification of known compounds using conventional reagents and procedures.
  • the ligand can be covalently attached to the linker through any available position on the ligand, provided that when the ligand is attached to the linker, the ligand retains its ability to inhibit 5-HT 3 .
  • a first group of preferred bivalent ligands for use in this invention are prepared via attachment through the basic nitrogen atom of the indole ring to provide for N-N linked compounds.
  • the preparation of such N-N bivalent ligands is illustrated in FIG. 1. Specifically, in FIG. 1
  • a p ⁇ r ⁇ -substituted phenyl hydrazine, compound I is combined with at least a stoichiometric equivalent and preferably a slight excess of 1,3-cyclohexanedione wherein, in compound I, R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, acyl, aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, aryloxy, heteroaryloxy and the like.
  • this reaction is conducted in an inert diluent, such as methanol, ethanol, isopropanol and mixtures thereof, at a temperature of about 25 °C to about 100°C until reaction completion as evidenced by, for example, tic.
  • an inert diluent such as methanol, ethanol, isopropanol and mixtures thereof
  • the resulting N-phenyl, N'-cyclohex-2-en-l-on-3-yl hydrazine, compound II is readily recovered by stripping the solvent. Purification, if desired, can be achieved by conventional methods such as chromatography, crystallization, filtration and the like.
  • N-phenyl, N'-cyclohex-2-en-l-on-3-yl hydrazine, compound II is then cyclized under conventional conditions to provide for tricyclic compound III.
  • this reaction is conducted in a diluent comprising acetic acid and concentrated HCI and the reaction is preferably conducted at reflux until reaction completion as evidenced by, for example, tic.
  • the resulting tricyclic compound III is readily recovered by stripping the solvent. Purification, if desired, can be achieved by conventional methods such as chromatography, crystallization, filtration and the like.
  • Tricyclic compound III is then coupled via a linking arm utilizing the basic amine group of the indole portion of the molecule to form compound IV.
  • the linking arm selected for linkage is illustrated by an ⁇ , ⁇ dibromoalkylene compound wherein n is preferably an integer from 2 to 12. It is understood, however, that other linking groups can be employed in this reaction.
  • this reaction is conducted by contacting compound III with about one half of an equivalent of a ⁇ , ⁇ -dibromoalkylene in an inert diluent such as toluene, benzene, tetrahydrofuran, and the like.
  • a stoichiometric excess of a base such as potassium carbonate, sodium bicarbonate, sodium hydroxide, etc. is employed to scavenge the acid generated by the reaction.
  • the reaction is preferably conducted at a temperature of from about 0°C to about 50 °C and is continued until reaction completion as evidenced by, for example, tic.
  • the resulting compound IV is readily recovered by extraction, filtration, stripping, etc. Purification, if desired, can be achieved by conventional methods such as chromatography, crystallization, filtration and the like to provide for dimeric compound IV.
  • Compound IV is then alkylated at each of the methylene groups located to the carbonyl of the cyclohexenone group to provide for compound V.
  • alkylation provides for substitution at this position with an N,N- dialkylaminomethylene group (illustrated by N,N-dimethylaminomethylene).
  • this reaction is conducted by contacting compound IV in acetic acid in the presence of at least 2 equivalents of paraformaldehyde, and dimethylamine hydrochloride (or other dialkylamine hydrochloride).
  • the reaction is preferably conducted at a temperature of from about 60 °C to about 120°C and is continued until reaction completion as evidenced by, for example, tic.
  • the resulting compound V is readily recovered by extraction, filtration, stripping, etc. Purification, if desired, can be achieved by conventional methods such as chromatography, crystallization, filtration and the like to provide for dimeric compound V.
  • each of R 2 , R 3 and R 4 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, aryloxy, heteroaryloxy and the like.
  • this reaction is conducted by contacting at least two equivalents of the imidazole compound with compound V in an inert diluent.
  • the reaction is preferably conducted at a temperature of from about 60 °C to about 120°C and is continued until reaction completion as evidenced by, for example, tic.
  • the resulting compound VI is readily recovered by extraction, filtration, stripping, etc. Purification, if desired, can be achieved by conventional methods such as chromatography, crystallization, filtration and the like to provide for dimeric compound VI.
  • a second group of preferred bivalent ligands for use in this invention are prepared via attachment through the saturated methylene group to the carbonyl of the cyclohexenone group to provide for C-C linked compounds.
  • FIG. 2 illustrates a reaction scheme resulting in bivalent 5-HT 3 ligands coupled via a suitable linking group (illustrated as the -NH(CH 2 ) n NH- group wherein n is preferably from 2 to 40, more preferably, from 2 to 12, although longer alkyl chains are contemplated).
  • a suitable linking group illustrated as the -NH(CH 2 ) n NH- group wherein n is preferably from 2 to 40, more preferably, from 2 to 12, although longer alkyl chains are contemplated.
  • compound VII is contacted with at least a stoichiometric amount of a diaminoalkylene compound to provide for compound VIII wherein R 1 and n are as defined above and R 2 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, and the like. It being understood that when R 2 is hydrogen, then a suitable protecting group is employed to protect the basic aromatic nitrogen during the reaction.
  • this reaction is conducted by contacting compound VII with a half of an equivalent of the alkylene diamine linking agent in an inert diluent.
  • the reaction is preferably conducted at a temperature of from about 60 °C to about 120°C and is continued until reaction completion as evidenced by, for example, tic.
  • the resulting compound VI is readily recovered by extraction, filtration, stripping, etc. Purification, if desired, can be achieved by conventional methods such as chromatography, crystallization, filtration and the like to provide for dimeric compound VI.
  • FIG. 3 illustrates the preparation of compounds similar to those depicted in FIG. 2 having a different linking agent showing versatility in the linker arm.
  • the displacement reaction employs an amino acid which, if necessary, is optionally protected at the carboxyl group to provide for an N- substituted amino acid identified as compound IX.
  • Subsequent reaction of the amino acid group with the alkylene diamine provides for bivalent compound X.
  • additional amino acids can be inco ⁇ orated into the linker group by conventional peptide chemistry prior to reaction with the alkylene diamine and typically 1 to 10 amino acid groups are inco ⁇ orated into compound IX.
  • this reaction is conducted by contacting compound VII with an at least an equivalent of an amino acid (R 3 is hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic and the like) in an inert diluent.
  • the reaction is preferably conducted at a temperature of from about 60 °C to about 120°C and is continued until reaction completion as evidenced by, for example, tic.
  • the resulting compound IX is readily recovered by extraction, filtration, stripping, etc. Purification, if desired, can be achieved by conventional methods such as chromatography, crystallization, filtration and the like to provide for compound IX.
  • the reaction coupling two copies of compound IX via the alkylene diamine is achieved through conventional acylation reactions well known in the art.
  • Such acylation reactions are typically conducted using conventional coupling reagents and procedures optionally using well known coupling reagents such as carbodiimides with or without the use of well known additives such as N- hydroxysuccinimide, 1-hydroxybenzotriazole, etc. can be used to facilitate coupling.
  • the reaction is conventionally conducted in an inert aprotic polar diluent such as dimethylformamide, dichloromethane, chloroform, acetonitrile, tetrahydrofuran and the like.
  • a third group of preferred multiligand compounds per this invention are prepared by attachment of two or more different ligands to the linkers wherein at least one of the ligands comprises a ligand domain capable of binding to one or more 5-HT 3 receptors.
  • at least one of the other ligands is a ligand which binds 5-HT 3 receptors.
  • at least one of the other ligands is does not bind 5-HT 3 receptors and includes, for example, corticosteriods, ligands binding D 2 receptors, and the like.
  • Examples of corticosteroid are cortisone, desoximetasone, dexamethazone, hydrocortisone, betamethasone, fluorinated derivatives, and the like.
  • Examples of ligands which bind to D 2 receptors include, for instance, metopimazine, e.g. ,
  • FIG. 4 illustrates a synthetic method for preparing such heterologous bivalent multibinding compounds. Specifically, in FIG. 4, compound VII, described as above, is coupled with compound XI under standard coupling conditions, also described above, to provide for a heterologous bivalent multibinding compound of this invention, i.e. , compound XII.
  • factors such as the proper juxtaposition of the individual ligands of a multibinding compound with respect to the relevant array of binding sites on a target or targets is important in optimizing the interaction of the multibinding compound with its target(s) and to maximize the biological advantage through multivalency.
  • One approach is to identify a library of candidate multibinding compounds with properties spanning the multibinding parameters that are relevant for a particular target. These parameters include: (1) the identity of ligand(s), (2) the orientation of ligands, (3) the valency of the construct, (4) linker length, (5) linker geometry, (6) linker physical properties, and (7) linker chemical functional groups.
  • a single ligand or set of ligands is (are) selected for inco ⁇ oration into the libraries of candidate multibinding compounds which library is directed against a particular biological target or targets, i.e. , binding of 5HT 3 receptors.
  • the only requirement for the ligands chosen is that they are capable of interacting with the selected target(s).
  • ligands may be known drugs, modified forms of known drugs, substructures of known drugs or substrates of modified forms of known drugs (which are competent to interact with the target), or other compounds.
  • Ligands are preferably chosen based on known favorable properties that may be projected to be carried over to or amplified in multibinding forms.
  • ligands which display an unfavorable property from among the previous list may obtain a more favorable property through the process of multibinding compound formation; i.e. , ligands should not necessarily be excluded on such a basis.
  • a ligand that is not sufficiently potent at a particular target so as to be efficacious in a human patient may become highly potent and efficacious when presented in multibinding form.
  • a ligand that is potent and efficacious but not of utility because of a non- mechanism-related toxic side effect may have increased therapeutic index (increased potency relative to toxicity) as a multibinding compound.
  • Compounds that exhibit short in vivo half-lives may have extended half-lives as multibinding compounds.
  • Physical properties of ligands that limit their usefulness e.g. poor bioavailability due to low solubility, hydrophobicity, hydrophilicity
  • each ligand at which to attach the ligand to the linker.
  • the selected points on the ligand/linker for attachment are functionalized to contain complementary reactive functional groups. This permits probing the effects of presenting the ligands to their target binding site(s) in multiple relative orientations, an important multibinding design parameter.
  • the only requirement for choosing attachment points is that attaching to at least one of these points does not abrogate activity of the ligand.
  • Such points for attachment can be identified by structural information when available. For example, inspection of a co-crystal structure of a ligand bound to its target allows one to identify one or more sites where linker attachment will not preclude the ligand/target interaction.
  • positions of attachment that do abrogate the activity of the monomeric ligand may also be advantageously included in candidate multibinding compounds in the library provided that such compounds bear at least one ligand attached in a manner which does not abrogate intrinsic activity. This selection derives from, for example, heterobivalent interactions within the context of a single target molecule.
  • a ligand bound to its target and then consider modifying this ligand by attaching to it a second copy of the same ligand with a linker which allows the second ligand to interact with the same target at sites proximal to the first binding site, which include elements of the target that are not part of the formal ligand binding site and/or elements of the matrix surrounding the formal binding site, such as the membrane.
  • the most favorable orientation for interaction of the second ligand molecule may be achieved by attaching it to the linker at a position which abrogates activity of the ligand at the first binding site.
  • Another way to consider this is that the SAR of individual ligands within the context of a multibinding structure is often different from the SAR of those same ligands in momomeric form.
  • Linkers are chosen in a range of lengths to allow the spanning of a range of inter-ligand distances that encompass the distance preferable for a given divalent interaction.
  • the preferred distance can be estimated rather precisely from high-resolution structural information of targets.
  • high-resolution structural information is not available, one can make use of simple models to estimate the maximum distance between binding sites either on adjacent receptors or at different locations on the same receptor.
  • preferred linker distances are 2-20 A, with more preferred linker distances of 3-12 A.
  • preferred linker distances are 20-100 A, with more preferred distances of 30-70 A.
  • Linker Geometry and Rigidity The combination of ligand attachment site, linker length, linker geometry, and linker rigidity determine the possible ways in which the ligands of candidate multibinding compounds may be displayed in three dimensions and thereby presented to their binding sites.
  • Linker geometry and rigidity are nominally determined by chemical composition and bonding pattern, which may be controlled and are systematically varied as another spanning function in a multibinding array. For example, linker geometry is varied by attaching two ligands to the ortho, meta, and para positions of a benzene ring, or in cis- or trans-arrangements at the 1,1- vs. 1 ,2- vs. 1 ,3- vs.
  • Linker rigidity is varied by controlling the number and relative energies of different conformational states possible for the linker.
  • a divalent compound bearing two ligands joined by 1,8-octyl linker has many more degrees of freedom, and is therefore less rigid than a compound in which the two ligands are attached to the 4,4' positions of a biphenyl linker.
  • Linker Physical Properties The physical properties of linkers are nominally determined by the chemical constitution and bonding patterns of the linker, and linker physical properties impact the overall physical properties of the candidate multibinding compounds in which they are included.
  • a range of linker compositions is typically selected to provide a range of physical properties (hydrophobicity, hydrophilicity, amphiphilicity, polarization, acidity, and basicity) in the candidate multibinding compounds.
  • the particular choice of linker physical properties is made within the context of the physical properties of the ligands they join and preferably the goal is to generate molecules with favorable PK/ADME properties.
  • linkers can be selected to avoid those that are too hydrophilic or too hydrophobic to be readily absorbed and/or distributed in vivo.
  • Linker Chemical Functional Groups are selected to be compatible with the chemistry chosen to connect linkers to the ligands and to impart the range of physical properties sufficient to span initial examination of this parameter.
  • n being determined by the sum of the number of different attachment points for each ligand chosen
  • m linkers by the process outlined above
  • a library of (n ⁇ )m candidate divalent multibinding compounds is prepared which spans the relevant multibinding design parameters for a particular target. For example, an array generated from two ligands, one which has two attachment points (Al , A2) and one which has three attachment points (Bl , B2. B3) joined in all possible combinations provide for at least 15 possible combinations of multibinding compounds:
  • the combinatorial library can employ solid phase chemistries well known in the art wherein the ligand and/or linker is attached to a solid support.
  • the combinatorial libary is prepared in the solution phase.
  • candidate multibinding compounds are optionally purified before assaying for activity by, for example, chromatographic methods (e.g., HPLC).
  • Various methods are used to characterize the properties and activities of the candidate multibinding compounds in the library to determine which compounds possess multibinding properties. Physical constants such as solubility under various solvent conditions and logD/clogD values can be determined. A combination of NMR spectroscopy and computational methods is used to determine low-energy conformations of the candidate multibinding compounds in fluid media. The ability of the members of the library to bind to the desired target and other targets is determined by various standard methods, which include radioligand displacement assays for receptor and ion channel targets, and kinetic inhibition analysis for many enzyme targets. In vitro efficacy, such as for receptor agonists and antagonists, ion channel blockers, and antimicrobial activity, can also be determined. Pharmacological data, including oral abso ⁇ tion, everted gut penetration, other pharmacokinetic parameters and efficacy data can be determined in appropriate models. In this way, key structure-activity relationships are obtained for multibinding design parameters which are then used to direct future work.
  • the members of the library which exhibit multibinding properties can be readily determined by conventional methods. First those members which exhibit multibinding properties are identified by conventional methods as described above including conventional assays (both in vitro and in vivo).
  • each member of the library can be encrypted or tagged with appropriate information allowing determination of the structure of relevant members at a later time.
  • each member of the library can be encrypted or tagged with appropriate information allowing determination of the structure of relevant members at a later time. See, for example, Dower, et al., International Patent Application Publication No. WO 93/06121 ; Brenner, et al. , Proc. Natl. Acad. Sci.. USA. 89:5181 (1992); Gallop, et al., U.S. Patent No. 5,846,839; each of which are inco ⁇ orated herein by reference in its entirety.
  • the structure of relevant multivalent compounds can also be determined from soluble and untagged libaries of candidate multivalent compounds by methods known in the art such as those described by Hindsgaul, et al. , Canadian Patent Application No. 2,240,325 which was published on July 11 , 1998. Such methods couple frontal affinity chromatography with mass spectroscopy to determine both the structure and relative binding affinities of candidate multibinding compounds to receptors.
  • an optional component of the process is to ascertain one or more promising multibinding "lead” compounds as defined by particular relative ligand orientations, linker lengths, linker geometries, etc. Additional libraries can then be generated around these leads to provide for further information regarding structure to activity relationships. These arrays typically bear more focused variations in linker structure in an effort to further optimize target affinity and/or activity at the target (antagonism, partial agonism, etc.), and/or alter physical properties.
  • iterative redesign/ analysis using the novel principles of multibinding design along with classical medicinal chemistry, biochemistry, and pharmacology approaches one is able to prepare and identify optimal multibinding compounds that exhibit biological advantage towards their targets and as therapeutic agents.
  • suitable divalent linkers include, by way of example only, those derived from dicarboxylic acids, disulfonylhalides, dialdehydes, diketones, dihalides, diisocyanates,diamines, diols, mixtures of carboxylic acids, sulfonylhalides, aldehydes, ketones, halides, isocyanates, amines and diols.
  • carboxylic acid, sulfonylhalide, aldehyde, ketone, halide, isocyanate, amine and diol functional group is reacted with a complementary functionality on the ligand to form a covalent linkage.
  • complementary functionality is well known in the art as illustrated in the following table:
  • First Reactive Group Second Reactive Group Linkage hydroxyl isocyanate urethane amine epoxide ⁇ -hydroxyamine sulfonyl halide amine sulfonamide carboxyl acid amine amide hydroxyl alkyl/aryl halide ether aldehyde amine(+ reducing agent) amine ketone amine(+ reducing agent) amine amine isocyanate urea
  • Exemplary linkers include the following linkers identified as X-l through
  • Representative ligands for use in this invention include, by way of example, L-1 through L-6, where ondansetron (L-1), granisetron (L-2), tropisetron (L-3), dolasetron (L-4), mirtazapine (L-5) and itasetron (L-6).
  • Combinations of ligands (L) and linkers (X) per this invention include, by way example only, homo- and hetero-dimers wherein a first ligand is selected from L-1 through L-6 above and the second ligand and linker is selected from the following:
  • the compounds of this invention are usually administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These compounds are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
  • compositions which contain, as the active ingredient, one or more of the compounds described herein associated with pharmaceutically acceptable carriers.
  • the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container.
  • the excipient serves as a diluent, it can be a solid, semi- solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.
  • compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose.
  • the formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.
  • the compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • compositions are preferably formulated in a unit dosage form, each dosage containing from about 0.001 to about 1 g, more usually about 1 to about 30 mg, of the active ingredient.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • the compound of formula I above is employed at no more than about 20 weight percent of the pharmaceutical composition, more preferably no more than about 15 weight percent, with the balance being pharmaceutically inert carrier(s).
  • the active compound is effective over a wide dosage range and is generally administered in a pharmaceutically effective amount.
  • the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.
  • the tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • liquid forms in which the novel compositions of the present invention may be inco ⁇ orated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
  • Hard gelatin capsules containing the following ingredients are prepared:
  • Quantity Ingredient (mg/capsule)
  • Magnesium stearate 5.0 The above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.
  • Quantity Ingredient (mg/tablet)
  • the components are blended and compressed to form tablets, each weighing 240 mg.
  • Formulation Example 3 A dry powder inhaler formulation is prepared containing the following components: Ingredient Weight %
  • the active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.
  • the active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly.
  • the solution of poly vinylpyrrolidone is mixed with the resultant powders, which are then passed through a 16 mesh U.S. sieve.
  • the granules so produced are dried at 50° to 60 °C and passed through a 16 mesh U.S. sieve.
  • the sodium carboxymethyl starch, magnesium stearate, and talc previously passed through a No. 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 120 mg.
  • Suppositories each containing 25 mg of active ingredient are made as follows:
  • Active Ingredient 25 mg Saturated fatty acid glycerides to 2,000 mg The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.
  • the active ingredient, sucrose and xanthan gum are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water.
  • the sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.
  • a formulation may be prepared as follows:
  • Quantity Ingredient (mg/capsulel Active Ingredient 15.0 mg
  • Formulation Example 9 A formulation may be prepared as follows:
  • a topical formulation may be prepared as follows:
  • the white soft paraffin is heated until molten.
  • the liquid paraffin and emulsifying wax are inco ⁇ orated and stirred until dissolved.
  • the active ingredient is added and stirring is continued until dispersed .
  • the mixture is then cooled until solid.
  • transdermal delivery devices Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts.
  • the construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g. , U.S. Patent 5,023,252, issued June 11 , 1991 , herein inco ⁇ orated by reference in its entirety. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
  • Other suitable formulations for use in the present invention can be found in Remington 's Pharmaceutical Sciences, Mace Publishing Company. Philadelphia, PA, 17th ed. (1985).
  • the multibinding compounds of this invention are agonists, partial agonists, inverse agonists or antagonists of the 5HT 3 receptors, which are known to mediate numerous disorders. Accordingly, the multibinding compounds and pharmaceutical compositions of this invention are useful in the treatment and prevention of various disorders, in particular, chemotherapy induced and radiation induced emesis, anxiety, schizophrenia, drug withdrawal and cognitive disorders.
  • the compounds of this invention are typically delivered to a patient in need of such treatment by a pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of at least one compound of this invention.
  • a pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of at least one compound of this invention.
  • the amount of compound administered to the patient will vary depending upon what compound and/or composition is being administered, the pu ⁇ ose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like.
  • compositions are administered to a patient already suffering from, for example, emesis, in an amount sufficient to at least partially reduce the symptoms. Amounts effective for this use will depend on the judgment of the attending clinician depending upon factors such as the degree or severity of the disorder in the patient, the age, weight and general condition of the patient, and the like.
  • the pharmaceutical compositions of this invention may contain more than one compound of the present invention.
  • the compounds administered to a patient are in the form of pharmaceutical compositions described above which can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, etc. These compounds are effective as both injectable and oral deliverable pharmaceutical compositions.
  • Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
  • the multibinding compounds of this invention can also be administered in the form of pro-drugs, i.e. , as derivatives which are converted into a biologically active compound in vivo.
  • pro-drugs will typically include compounds in which, for example, a carboxylic acid group, a hydroxyl group or a thiol group is converted to a biologically liable group, such as an ester, lactone or thioester group which will hydrolyze in vivo to reinstate the respective group.
  • the compounds of the invention may be bound to affinity resins for affinity chromatography .
  • the compounds of the invention may be used as a tool in immunoprecipitation.
  • the compounds may be used to identify a receptor in vitro for example in microscopy, electrophoresis and chromatography.
  • the compounds can be assayed to identify which of the multimeric ligand compounds possess multibinding properties.
  • a ligand or mixture of ligands which each contain at least one reactive functionality and a library of linkers which each include at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand.
  • a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands with the library of linkers under conditions wherein the complementary functional groups react to form a covalent linkage between the linker and at least two of the ligands.
  • the multimeric ligand compounds produced in the library can be assayed to identify multimeric ligand compounds which possess multibinding properties.
  • the method can also be performed using a library of ligands and a linker or mixture of linkers.
  • the preparation of the multimeric ligand compound library can be achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands with the linkers.
  • the multimeric ligand compounds can be dimeric, for example, homomeric or heteromeric.
  • a heteromeric ligand compound library can be prepared by sequentially adding a first and second ligand.
  • Each member of the multimeric ligand compound library can be isolated from the library, for example, by preparative liquid chromatography mass spectrometry (LCMS).
  • the linker or linkers can be flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarization and/or polarizabihty or amphiphilic linkers.
  • the linkers can include linkers of different chain lengths and/or which have different complementary reactive groups. In one embodiment, the linkers are selected to have different linker lengths ranging from about 2 to 100A.
  • the ligand or mixture of ligands can have reactive functionality at different sites on the ligands.
  • the reactive functionality can be, for example, carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates, and precursors thereof, as long as the reactive functionality on the ligand is complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.
  • a library of multimeric ligand compounds can thus be formed which possesses multivalent properties.
  • Multimeric ligand compounds possessing multibinding properties can be identified in an iterative method by preparing a first collection or iteration of multimeric compounds by contacting at least two stoichiometric equivalents of the ligand or mixture of ligands which target the 5HT 3 receptors with a linker or mixture of linkers, where the ligand or mixture of ligands includes at least one reactive functionality and the linker or mixture of linkers includes at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand.
  • the ligand(s) and linker(s) are reacted under conditions which form a covalent linkage between the linker and at least two of the ligands.
  • the first collection or iteration of multimeric compounds can be assayed to assess which if any of the compounds possess multibinding properties.
  • the process can be repeated until at least one multimeric compound is found to possess multibinding properties.
  • a second collection or iteration of multimeric compounds which elaborates upon the particular molecular constraints can be assayed, and the steps optionally repeated to further elaborate upon the molecular constraints.
  • the steps can be repeated from between 2 and 50 times, more preferably, between 5 and 50 times.
  • Example 1 In vitro Radioligand binding studies.
  • Receptor binding assays for the 5HT 3 receptors are performed on rat membrane preparations.
  • crude membrane pellets are prepared by homogenization and centrifugation. Aliquots of membrane homogenate are added to test tubes containing buffer, radioligand, and various concentrations of inhibitor. Nonspecific binding is determined using an excess of the specified inhibitor for the receptor of interest.
  • the reactions are terminated by separating the membrane bound from free radioligand by rapid filtration over Whatman GF/C filters. Bound radioligand is quantified by scintillation spectroscopy. The percent inhibition of radioligand binding at each test concentration is calculated, and an IC 50 is determined by log-probit analysis.
  • the pKi for 5-HT 3 receptor binding is determined for the multi-binding compounds of this invention.
  • the hydrazone II from last step is mixed with acetic acid (20 mL) and concentrated hydrochloric acid (3.2 mL), refluxed for 1 h, and evaporated to dryness. The residue is taken up in CH 2 C1 2 and washed with water. The organic layer is concentrated, and the residue is purified by silica gel chromatography using ethyl acetate as eluant to afford the tetrahydrocarbazolone III.
  • the compound V (1.2 g, 2.0 mmol, 1.0 equiv) is mixed with 2- methylimidazole (0.5 g, 6.0 mmol, 3.0 equiv) and water (6 mL). The mixmre is stirred for 20 h at 100 °C. After cooling at 0 °C, the product is isolated by filtration, dried, and purified by silica gel chromatography with CH 2 C1 2 containing 5% methanol as eluant to afford the compound VI.
  • reaction mixture is diluted with 200 mL of diethyl ether to give a white precipitate which is collected by vacuum filtration.
  • the solid is purified by reverse-phase HPLC using a mixed acetonitrile/water solvent system to afford the final compound IX.

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Abstract

La présente invention concerne des composés ou agents à plusieurs liaisons et qui se lient aux récepteurs 5-HT3. Ces composés comportent une pluralité de ligands dont chacun peut se lier à de tels récepteurs, modulant ainsi les fonctions ou processus biologiques de ces récepteurs. Chacun des ligands est attaché par covalence à un lieur ou à des lieurs qui peuvent être identiques ou différents, et ce, de façon à constituer un composé capable de liaisons multiples. Le choix du lieur est fait pour que le composé à liaisons multiples fasse preuve d'une modulation accrue des processus biologiques à médiation du récepteur 5-HT3.
PCT/US1999/012768 1998-06-08 1999-06-08 Agonistes polyvalents, agonistes partiels, agonistes inverses et antagonistes des recepteurs 5-ht¿3? WO1999064046A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP99928450A EP1085888A1 (fr) 1998-06-08 1999-06-08 Agonistes polyvalents, agonistes partiels, agonistes inverses et antagonistes des recepteurs 5-ht 3?
CA002321191A CA2321191A1 (fr) 1998-06-08 1999-06-08 Agonistes polyvalents, agonistes partiels, agonistes inverses et antagonistes des recepteurs 5-ht3
AU45514/99A AU4551499A (en) 1998-06-08 1999-06-08 Multivalent agonists, partial agonists, inverse agonists and antagonists of the 5-HT3 receptors

Applications Claiming Priority (6)

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US8846698P 1998-06-08 1998-06-08
US60/088,466 1998-06-08
US9293898P 1998-07-15 1998-07-15
US60/092,938 1998-07-15
US12028299P 1999-02-16 1999-02-16
US60/120,282 1999-02-16

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WO2003100091A1 (fr) * 2002-05-24 2003-12-04 Epidauros Biotechnologie Ag Moyens et methodes de traitement ameliores utilisant des 'setrones'
FR2850381A1 (fr) * 2003-01-24 2004-07-30 Synthon Bv Procede pour preparer de l'ondansetron et ses intermediaires
EA013836B1 (ru) * 2003-02-18 2010-08-30 Хелсинн Хелткэр С.А. Способ лечения послеоперационной тошноты и рвоты

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003100091A1 (fr) * 2002-05-24 2003-12-04 Epidauros Biotechnologie Ag Moyens et methodes de traitement ameliores utilisant des 'setrones'
FR2850381A1 (fr) * 2003-01-24 2004-07-30 Synthon Bv Procede pour preparer de l'ondansetron et ses intermediaires
EA013836B1 (ru) * 2003-02-18 2010-08-30 Хелсинн Хелткэр С.А. Способ лечения послеоперационной тошноты и рвоты

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WO1999064046A9 (fr) 2000-04-20
EP1085888A1 (fr) 2001-03-28
WO1999064046A8 (fr) 2001-08-16
AU4551499A (en) 1999-12-30
CA2321191A1 (fr) 1999-12-16

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