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WO1999063992A9 - Nouveaux medicaments pour traiter les etats dus aux canaux calcium et leurs utilisations - Google Patents

Nouveaux medicaments pour traiter les etats dus aux canaux calcium et leurs utilisations

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Publication number
WO1999063992A9
WO1999063992A9 PCT/US1999/012672 US9912672W WO9963992A9 WO 1999063992 A9 WO1999063992 A9 WO 1999063992A9 US 9912672 W US9912672 W US 9912672W WO 9963992 A9 WO9963992 A9 WO 9963992A9
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WIPO (PCT)
Prior art keywords
ligand
linker
ligands
linkers
compounds
Prior art date
Application number
PCT/US1999/012672
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English (en)
Other versions
WO1999063992A1 (fr
WO1999063992A8 (fr
Inventor
Yu-Hua Ji
Maya Natarajan
John H Griffin
Original Assignee
Advanced Medicine Inc
Yu-Hua Ji
Maya Natarajan
John H Griffin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Advanced Medicine Inc, Yu-Hua Ji, Maya Natarajan, John H Griffin filed Critical Advanced Medicine Inc
Priority to AU45493/99A priority Critical patent/AU4549399A/en
Priority to EP99928427A priority patent/EP1085863A4/fr
Priority to CA002318901A priority patent/CA2318901A1/fr
Priority to JP2000553061A priority patent/JP2002517440A/ja
Publication of WO1999063992A1 publication Critical patent/WO1999063992A1/fr
Publication of WO1999063992A8 publication Critical patent/WO1999063992A8/fr
Publication of WO1999063992A9 publication Critical patent/WO1999063992A9/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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • A61K47/545—Heterocyclic compounds
    • 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
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • This invention relates to novel multibinding compounds that bind to Ca * channels and modulate their activity.
  • the compounds of this invention comprise 2-10 Ca ⁇ channel ligands covalently connected by a linker or linkers, wherein the ligands in their monovalent (i.e. unlinked) state bind to and are capable of modulating the activity of one or more types of Ca ++ channel.
  • the manner of linking the ligands together is such that the multibinding agents thus formed demonstrate an increased biologic and/or therapeutic effect as compared to the same number of unlinked ligands made available for binding to the Ca ** channel.
  • the invention also relates to methods of using such compounds and to methods of preparing them.
  • the compounds of this invention are particularly useful for treating diseases and conditions of mammals that are mediated by Ca ++ channels. Accordingly, this invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a compound of this invention.
  • Voltage-gated Ca ++ channels mediate the influx of Ca ++ into cells in response to changes in membrane potential. Because of their central roles in ion homeostasis and in cell signalling events, these channels are involved in a wide variety of physiological activities, e.g., muscle contraction, cardiovascular function, hormone and neurotransmitter secretion, and tissue growth and remodeling processes.
  • the high-voltage activated Ca ++ channels are formed by the heteromeric association of membrane proteins comprising at least three subunits ⁇ ( ⁇ dress ⁇ 2 ), ⁇ , ⁇ (and ⁇ in skeletal muscle).
  • the ⁇ , subunit alone is sufficient to form a functional channel, although the functional properties of the channel are subject to modification , particularly by the ⁇ subunit.
  • the ⁇ , subunit is organized into four homologous domains (ITV), each domain including 6 transmembrane segments (S1-S6) ( Figure 1). It is thought that the channel pore is formed from S5, S6 and the region between them, and that the voltage sensor resides in S4. These channels exist in resting (closed), activated (open ) or inactivated
  • channels differ in their activation and inactivation properties.
  • Ca ++ channels are recognized as important targets for drug therapy. They are implicated in a variety of pathologic conditions, including, e.g., essential hypertension, angina, congestive heart failure, arrythmias, migraine and pain.
  • Calcium channel antagonists are potent vasodilators and are widely used in the treatment of hypertension and angina pectoris.
  • the compounds approved for clinical use in the U.S. fall into several chemical classes: the dihydropyridines (e.g., amlodipine, felodipine, nifedipine, nicardipine, isradipine, nimodipine); the benzothiazepines (e.g., diltiazem), phenylalkylamines (e.g., verapamil); and diarylaminopropylamine ether (e.g., bepridil).
  • dihydropyridines e.g., amlodipine, felodipine, nifedipine, nicardipine, isradipine, nimodipine
  • benzothiazepines e.g., diltiazem
  • phenylalkylamines e.g.
  • the dihydropyridines, benzothiazepines and phenylalkylamines bind to distinct, but functionally coupled, sites on the ⁇ , subunit of L-type channels at the interface of the IIIS6 and IVS6 transmembrane segments, such that the binding of any one class of drug can allosterically modulate the binding of drugs in the other two classes and the high affinity Ca ++ binding site in the channel (see GH Hockerman et al, Annu. Rev. Pharmacol. Toxicol. 37: 361-96 (1997)). It has been suggested that more than one high affinity binding site may exist for dihydropyridines in voltage- dependent calcium channels (Kokubun et al, Molec. Pharmacol. 30: 571-584 (1986)).
  • This invention is directed to novel multibinding compounds that bind to Ca ++ channels in mammalian tissues and can be used to treat diseases and conditions mediated by such channels.
  • this invention is directed to a multibinding 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 or different, each of said ligands comprising a ligand domain capable of binding to a Ca ++ channel.
  • the multibinding compounds of this invention are preferably represented by Formula I: (L) p (X) q I
  • each L is a ligand that may be the same or different at each occurrence;
  • X is a linker that may be the same or different at each occurrence;
  • p is an integer of from 2 to 10; and
  • q is an integer of from 1 to 20; wherein each of said ligands comprises a ligand domain capable of binding to a Ca ++ channel.
  • q is less than p.
  • linker is represented by the following formula:
  • m is an integer of from 0 to 20;
  • X' at each separate occurrence is -O-, -S-, -S(O)-, -S(O) 2 -, -NR-, -N + R R-, -C(O)-, -C(O)O-, -C(O)NH-, -C(S), -C(S)O-, -C(S)NH- or a covalent bond, where R and R at each separate occurrence are as defined below for R' and R";
  • Z is at each separate occurrence selected from alkylene, substituted alkylene, alkylalkoxy, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted alkenylene, arylene, substituted arylene, heteroarylene, heterocyclene, substituted heterocyclene, crown compounds, or a covalent bond;
  • Y' and Y' at each separate occurrence are selected from -S-S- or a covalent bond;
  • n 0, 1 or 2; and R' and R" at each separate occurrence are selected from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl or heterocyclic.
  • the ligands are selected from Ca ++ channel modulators such as verapamil, diltiazem, benziazem clentiazem, nicardipine, nifedipine, nilvadipine, nitredipine, nimodipine, isradipine, lacidipine, amlodipine, nisoldipine, isradipine, mibefrodil, amlodipine, felodipine, nimodipine, bepridil, SQ 32,910 and SQ 32,428.
  • Ca ++ channel modulators such as verapamil, diltiazem, benziazem clentiazem, nicardipine, nifedipine, nilvadipine, nitredipine, nimodipine, isradipine, lacidipine, amlodipine, nisoldipine, isradipine, mi
  • this invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of one or more multibinding compounds (or pharmaceutically acceptable 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 or different, each of said ligands comprising a ligand domain capable of binding to a Ca ++ channel of a cell mediating mammalian diseases or conditions, thereby modulating the diseases or conditions.
  • this invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of one or more multibinding compounds represented by Formula I,
  • each L is a ligand that may be the same or different at each occurrence;
  • X is a linker that may be the same or different at each occurrence; j? is an integer of from 2 to 10; and q is an integer of from 1 to 20; wherein each of said ligands comprises a ligand domain capable of binding to a Ca ++ channel of a cell mediating mammalian diseases or conditions, thereby modulating the diseases or conditions.
  • q is less than/?.
  • this invention is directed to a method for modulating the activity of a Ca ++ channel in a biologic tissue, which method comprises contacting a tissue having a Ca ++ channel with a multibinding compound (or pharmaceutically acceptable salts thereof) under conditions sufficient to produce a change in the activity of the channel in said tissue, wherein the multibinding compound comprises 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, each of said ligands comprising a ligand domain capable of binding to a Ca ++ channel.
  • this invention is directed to a method for treating a disease or condition in a mammal resulting from an activity of a Ca ⁇ channel, which method comprises administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more multibinding compounds (or pharmaceutically acceptable 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 or different, each of said ligands comprising a ligand domain capable of binding to a Ca ++ channel of a cell mediating mammalian diseases or conditions.
  • a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more multibinding compounds (or pharmaceutically acceptable 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 or different, each of said ligands comprising a ligand domain capable of binding to a
  • this invention is directed to a method for treating a disease or condition in a mammal resulting from an activity of a Ca ++ channel, which method comprises administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more multibinding compounds represented by Formula I,
  • each L is a ligand that may be the same or different at each occurrence;
  • X is a linker that may be the same or different at each occurrence;
  • p is an integer of from 2 to 10; and
  • q is an integer of from 1 to 20; wherein each of said ligands comprises a ligand domain capable of binding to a Ca ++ channel of a cell mediating mammalian diseases or conditions.
  • q is less than/?.
  • this invention relates to processes for preparing the multibinding agents of Formula I.
  • this invention is directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties for Ca ++ channels.
  • 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 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 for Ca ⁇ channels. 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 a receptor.
  • this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for Ca ++ channels which method comprises: (a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality;
  • each linker in said 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 for Ca ++ channels which method comprises: (a) identifying a library of ligands wherein each ligand contains at least one reactive functionality;
  • 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 heterodimeric 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 for Ca ++ channels which library is prepared by the method comprising:
  • each linker in said 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 may possess multivalent properties for Ca ++ channels which library is prepared by the method comprising: (a) identifying a library of ligands wherein each ligand contains at least one reactive functionality;
  • each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and (c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
  • 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 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 100 °A.
  • the ligand or mixture of ligands is selected to have reactive functionality at different sites on said ligands in order to provide for a range of orientations of said ligand on said multimeric ligand compounds.
  • Such reactive functionality includes, by way of example, carboxyhc acids, carboxyhc 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 heterodimeric (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 a receptor.
  • this method aspect is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for Ca ++ channels which method comprises:
  • steps (e) 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.
  • Figure 1 is a highly schematic illustration of the transmembrane organization of the ⁇ l subunit of the voltage-gated Ca ++ channel.
  • Figure 2 illustrates a method for optimizing the linker geometry for presentation of ligands (filled circles) in bivalent compounds:
  • FIG. 1 shows exemplary linker "core" structures.
  • Figures 4-20 illustrate convenient methods for preparing the multibinding compounds of this invention.
  • Biological systems in general are controlled by molecular interactions between bioactive ligands and their receptors, in which the receptor "recognizes" a molecule or a portion thereof (i.e., a ligand domain) to produce a biological effect.
  • the voltage- gated CsX channels are considered to be pharmacological receptors: they possess specific binding sites for ligands having agonist and antagonist activities; the binding of ligands to such sites allosterically modulates Ca ++ flux through the channel; the channel properties (i.e., gating and ion selectivity) are regulatable; and various channels are known to associate with G-proteins (D. Rampe and D.J. Triggle, Prog. Drug Res. 40: 191-238 (1993). Accordingly, diseases or conditions that involve, or are mediated by, Ca ++ channels can be treated with pharmacologically active ligands that interact with such channels to initiate, modulate or abrogate transporter activity .
  • the interaction of a Ca ++ channel and a Ca ++ channel-binding ligand may be described in terms of "affinity” and "specificity".
  • the "affinity” and “specificity” of any given ligand-Ca ++ channel interaction is dependent upon the complementarity of molecular binding surfaces and the energetic costs of complexation (i.e., the net difference in free energy G between bound and free states). Affinity may be quantified by the equilibrium constant of complex formation, the ratio of on/off rate constants, and/or by the free energy of complex formation. Specificity relates to the difference in binding affinity of a ligand for different receptors.
  • the net free energy of interaction of such ligands with a Ca ++ channel is the difference between energetic gains (enthalpy gained through molecular complementarity and entropy gained through the hydrophobic effect) and energetic costs (enthalpy lost through decreased solvation and entropy lost through reduced translational, rotational and conformational degrees of freedom).
  • the compounds of this invention comprise 2 to 10 Ca ++ channel-binding ligands covalently linked together and capable of acting as multibinding agents.
  • the enhanced activity of these compounds is believed to arise at least in part from their ability to bind in a multivalent manner with multiple ligand binding sites on a Ca ++ channel or channels, which gives rise to a more favorable net free energy of binding.
  • Multivalent interactions differ from collections of individual monovalent (univalent) interactions by being capable of providing enhanced biologic and/or therapeutic effect.
  • Multivalent binding cam amplify binding affinities and differences in binding affinities, resulting in enhanced binding specificity as well as affinity. Definitions As used herein:
  • alkyl refers to a monoradical branched or unbranched saturated hydrocarbon chain, preferably having from 1 to 40 carbon atoms, preferably 1-10 carbon atoms, more preferably 1-6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, secondary butyl, tert-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, 2- ethyldodecyl, tetradecyl, and the like, unless otherwise indicated.
  • substituted alkyl refers to an alkyl group as defined above having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, 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, -SO-alkyl, -SO-ary
  • alkylene refers to a diradical of a branched or unbranched saturated hydrocarbon chain, preferably having from 1 to 40 carbon atoms, preferably 1-10 carbon atoms, more preferably 1-6 carbon atoms. This term is exemplified by groups such as methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), the propylene isomers (e.g., - CH 2 CH 2 CH 2 - and -CH(CH 3 )CH 2 -) and the like.
  • substituted alkylene refers to an alkylene group as defined above having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyacylamino, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, thioaryloxy, heteroaryl, heteroaryloxy, thioheteroaryloxy, heterocyclic, heterocyclooxy, thioheterocyclooxy, nitro, and -NR a R b , wherein R a and R b may be the same or different and
  • 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.
  • substituted alkylene optionally includes an alkylene chain as defined above in which the carbon chain is interrupted by one or more atoms chosen from O, S or N (e.g., ethers, sulfides and amines).
  • alkaryl or "aralkyf'refers to the groups -alkylene-aryl and - substituted alkylene-aryl in which alkylene and aryl are as 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, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-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.
  • Alkenyl refers to a monoradical of a branched or unbranched unsaturated hydrocarbon preferably having from 2 to 40 carbon atoms, preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms, and preferably having 1-6 double bonds. This term is further exemplified by such radicals as vinyl, prop-2-enyl, pent-3-enyl, hex-5-enyl, 5-ethyldodec-3,6-dienyl, and the like.
  • substituted alkenyl refers to an alkenyl group as defined above having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, heteroaryl, heterocyclic, aryloxy, thioaryloxy, heteroaryloxy, thioheteroaryloxy, heterocyclooxy, thioheterocyclooxy, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substitute
  • R a and R b may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.
  • Alkenylene refers to a diradical of an unsaturated hydrocarbon, preferably having from 2 to 40 carbon atoms, preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms, and preferably having 1-6 double bonds. This term is further exemplified by such radicals as 1,2-ethenyl, 1 ,3-prop-2-enyl, l,5-pent-3-enyl, 1,4-hex- 5-enyl, 5-ethyl-l,12-dodec-3,6-dienyl, and the like.
  • substituted alkenylene refers to an alkenylene group as defined above having from 1 to 5 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyacylamino, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, thioaryloxy, heteroaryl, heteroaryloxy, thioheteroaryloxy, heterocyclic, heterocyclooxy, thioheterocyclooxy, nitro, and NR a R b , wherein R a and R b may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl
  • 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, preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms, and preferably having 1-6 triple bonds. This term is further exemplified by such radicals as acetylenyl, prop-2-ynyl, pent-3-ynyl, hex-5-ynyl, 5- ethyldodec-3,6-diynyl, and the like.
  • substituted alkynyl refers to an alkynyl group as defined above having from 1 to 5 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyacylamino, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, thioaryloxy, heteroaryl, heteroaryloxy, thioheteroaryloxy, heterocyclic, heterocyclooxy, thioheterocycloxy, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, SO 2 -alkyl, -SO 2 -substituted
  • Alkynylene refers to a diradical of an unsaturated hydrocarbon radical, preferably having from 2 to 40 carbon atoms, preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms, and preferably having 1-6 triple bonds. This term is further exemplified by such radicals as l,3-prop-2-ynyl, l,5-pent-3-ynyl, l,4-hex-5- ynyl, 5-ethyl-l,12-dodec-3,6-diynyl, and the like.
  • acyl refers to the groups -CHO, 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 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., morpholine) 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 refers to the group -NRC(O)OR 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.
  • acyloxy refers to the groups alkyl-C(O)O-, substituted alkyl-
  • 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).
  • such aryl groups can optionally be substituted with from 1 to 5 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, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioaryloxy, thioheteroaryloxy, -
  • 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 a diradical derived from aryl or substituted aryl as defined above, and is exemplified by 1 ,2-phenylene, 1,3-phenylene, 1 ,4-phenylene, 1 ,2-naphthylene and the like.
  • carboxyalkyl refers to the group “-C(O)Oalkyl” where alkyl is as defined above.
  • 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 selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, 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, -SO-alkyl, -SO- substituted alkyl, -
  • cycloalkenyl refers to cyclic alkenyl groups of from 4 to 20 carbon atoms having a single cyclic ring or fused rings 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 selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, 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, -SO-alkyl,
  • 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 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, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, - SO
  • heteroaryloxy refers to the group heteroaryl-O-.
  • heteroarylene refers to the diradical group derived from heteroaryl or 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-pyridinylene, 1,3-morpholinylene, 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, aminoacyl, aminoacyloxy, oxyaminoacyl, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO- alkyl
  • 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, mo ⁇ holino, piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen
  • 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-j where m is equal to or greater than 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-.
  • heterocyclooxy refers to the group heterocyclic-S-.
  • heterocyclene refers to the diradical group derived from a heterocycle as defined herein, and is exemplified by the groups 2,6-mo ⁇ holino, 2,5- mo ⁇ holino and the like.
  • oxyacylamino 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.
  • 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.
  • any of the above groups which contain one or more substituents it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
  • the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds.
  • Alkyl optionally interrupted by 1-5 atoms chosen from O, S, or N refers to alkyl as defined above in which the carbon chain is interrupted by O, S, or N.
  • ethers, sulfides, and amines for example 1 -methoxydecyl, 1 - pentyloxynonane, l-(2-isopropoxyethoxy)-4-methylnonane, l-(2- ethoxyethoxy)dodecyl, 2-(t-butoxy)heptyl, 1 -pentylsulfanylnonane, nonylpentylamine, and the like.
  • Heteroarylalkyl refers to heteroaryl as defined above linked to alkyl as defined above, for example pyrid-2-ylmethyl, 8-quinolinylpropyl, and the like.
  • Optional or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.
  • optionally substituted alkyl means that alkyl may or may not be substituted by those groups enumerated in the definition of substituted alkyl.
  • 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(wo-propyl) amine, tri( «-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, mo ⁇ holine, N-ethylpiperidine, and the like.
  • carboxyhc acid derivatives would be useful in the practice of this invention, for example, carboxyhc acid amides, including carboxamides, lower alkyl carboxamides, dialkyl carboxamides, and the like.
  • 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.
  • 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.
  • protecting group refers to any group which when bound to one or more hydroxyl, thiol, amino or carboxyl groups of the compounds 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. See, generally, T. W. Greene & P. G.M. Wuts "Protective Groups in Organic Synthesis, " 2 nd Ed, 1991 , John Wiley and Sons, N. Y.
  • 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 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 hydrolysis conditions compatible with the nature of the product.
  • inert organic solvent or “inert solvent” mean a solvent inert under the conditions of the reaction being described in conjunction therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”), chloroform (“CHCl ), methylene chloride (or dichloromethane or "CH 2 C1 2 "), diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol, ethanol, propanol, isopropanol, tert-butanol, dioxane, pyridine, and the like].
  • the solvents used in the reactions of the present invention are inert solvents.
  • Ca ++ channel refers to a structure comprised of integral membrane proteins that functions to allow Ca ++ to equilibrate across a membrane according to its electrochemical gradient and at rates that are diffusion-limited. Examples of various types of Ca ++ channels are given in Table 1.
  • Ligand denotes a compound that is a binding partner for a Ca ++ channel receptor, and is bound thereto, for example, by complementarity.
  • the specific region or regions of the ligand molecule that is recognized by the ligand binding site of a Ca ++ channel 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. ions, a lipid molecule, a solvent molecule, and the like).
  • Ligands useful in this invention comprise Ca ⁇ channel modulators such as verapamil (a phenylalkylamine), diltiazem (a benzothiazepine), nicardipine, nifedipine, isradipine, amlodipine, felodipine and nimodipine (dihydropyridines), and bepridil (a diarylaminopropylamine ether). See Tables 4 and 5, for structures of various dihydropyridine and benzothiazepine ligands.
  • Ca ⁇ channel modulators such as verapamil (a phenylalkylamine), diltiazem (a benzothiazepine), nicardipine, nifedipine, isradipine, amlodipine, felodipine and nimodipine (dihydropyridines), and bepridil (a diarylaminopropylamine ether).
  • ligand is not intended to be limited to compounds known to be useful as Ca ++ channel receptor - binding compounds (e.g., known drugs), in that ligands that exhibit marginal activity or lack useful activity as monomers can be highly active as multibinding compounds, because of the biological benefit conferred by multivalency.
  • the primary requirement for a ligand as defined herein is that it has a ligand domain, as defined above, which is available for binding to a recognition site on a Ca ++ channel.
  • the term "ligand” or "ligands” is intended to include the racemic ligands as well as the individual stereoisomers of the ligands, including pure enantiomers and non-racemic mixtures thereof.
  • a large number of known calcium channel ligands have at least one chiral center and show stereoselective pharmacokinetics and pharmacologic activity (reviewed in Tokuma, Y and Noguchi, H, J. Chromatography A, 694: 181-193 (1995)).
  • the scope of the invention as described and claimed encompasses the racemic forms of the ligands as well as the individual enantiomers and non-racemic mixtures thereof.
  • ligand binding site denotes a site on a Ca ++ channel receptor 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, modulation, or may maintain an ongoing biological event, and the like.
  • Ca ++ channel ligand binding sites of Ca ++ channel receptors that participate in biological multivalent binding interactions are constrained to varying degrees by their intra- and intermolecular associations.
  • Ca ++ channel ligand binding sites may be covalently joined in a single structure, noncovalently associated in one or more multimeric structures, embedded in a membrane or biopolymer matrix, and so on, and therefore have less translational and rotational freedom than if the same sites were present as monomers in solution.
  • agonist refers to a ligand that when bound to a Ca ++ channel stimulates its activity.
  • antagonist refers to a ligand that when bound to a Ca ++ channel inhibits its activity.
  • Channel block or activation may result from allosteric effects of ligand binding to the channel rather than occupancy of the channel pore. These allosteric effects may produce changes in protein conformation that affect Ca ++ binding sites, gating mechanisms and/or the pore region (i.e., ion permeation).
  • a channel can exist in three modes: mode 0 (where the channel has zero probability of opening); mode 1 (where the channel opens frequently but transiently) and mode 2, where the channel remains open for relatively long periods of time (Hess et al, Nature 311: 538-544 (1984)).
  • mode 0 where the channel has zero probability of opening
  • mode 1 where the channel opens frequently but transiently
  • mode 2 where the channel remains open for relatively long periods of time.
  • the probability that a channel will exist in one of these three states changes with voltage.
  • a given ligand may have different binding affinities for different states, and thereby be capable of producing agonist or antagonist activity.
  • Multibinding agent or “multibinding compound” refers herein to a compound that has from 2 to 10 Ca ++ channel ligands as defined herein (which may be the same or different) covalently bound to one or more linkers (which may be the same or different), and is capable of multivalency, as defined below.
  • a multibinding compound provides an improved biologic and/or therapeutic effect compared to that of the same number of unlinked ligands available for binding to the ligand binding sites on a Ca ++ channel or channels.
  • improved "biologic and/or therapeutic effect” include increased ligand-receptor binding interactions (e.g., increased affinity, increased ability to elicit a functional change in the target, improved kinetics), increased selectivity for the target, increased potency, increased efficacy, decreased toxicity, increased therapeutic index, improved duration of action, improved bioavailability, 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.
  • Univalency refers to a single binding interaction between one ligand with one ligand binding site as defined herein. It should be noted that a compound having multiple copies of a ligand (or ligands) exhibits univalency when only one ligand of that compound interacts 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 ligand binding sites, which may be the same or different.
  • An example of trivalent binding is depicted below for illustrative pu ⁇ oses.
  • linker refers to a group or groups that covalently links from 2 to 10 ligands (as defined above) in a manner that provides a compound capable of multivalency.
  • the linker is a ligand- orienting entity that permits attachment of multiple copies of a ligand (which may be the same or different) thereto.
  • linker includes everything that is not considered to be part of the ligand, e.g., ancillary groups such as solubilizing groups, lipophilic groups, groups that alter pharmacodynamics or pharmacokinetics, groups that modify the diffusability of the multibinding compound, spacers that attach the ligand to the linker, groups that aid the ligand-orienting function of the linker, for example, by imparting flexibility or rigidity to the linker as a whole, or to a portion thereof, and so on.
  • ancillary groups such as solubilizing groups, lipophilic groups, groups that alter pharmacodynamics or pharmacokinetics, groups that modify the diffusability of the multibinding compound, spacers that attach the ligand to the linker, groups that aid the ligand-orienting function of the linker, for example, by imparting flexibility or rigidity to the linker as a whole, or to a portion thereof, and so on.
  • linker does not, however, cover solid inert supports such as beads, glass particles, rods, and the like, but it is to be understood that the multibinding compounds of this invention can be attached to a solid support if desired, for example, for use in separation and purification processes and for similar applications.
  • linker or linkers that joins the ligands presents them to their array of ligand binding sites. Beyond presenting these ligands for multivalent interactions with ligand binding sites, the linker spatially constrains these interactions to occur within dimensions defined by the linker.
  • the linkers used in this invention are selected to allow multivalent binding of ligands to any desired ligand binding sites of a Ca ++ channel , whether such sites are located interiorly (e.g., within a channel/translocation pore), both interiorly and on the periphery of a channel, at the boundary region between the lipid bilayer and the channel, or at any intermediate position thereof.
  • the preferred linker length will vary depending on the distance between adjacent ligand binding sites, and the geometry, flexibility and composition of the linker.
  • the length of the linker will preferably be in the range of about 2A to about lOOA, more preferably from about 2A to about 5 ⁇ A and even more preferably from about 3 A to about lOA.
  • the ligands are covalently attached to the linker or linkers using conventional chemical techniques.
  • the reaction chemistries resulting in such linkage are well known in the art and involve the use of reactive functional groups present on the linker and ligand.
  • the reactive functional groups on the linker are selected relative to the functional groups available on the ligand for coupling, or which can be introduced onto the ligand for this pu ⁇ ose. Again, such reactive functional groups are well known in the art.
  • reaction between a carboxyhc 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 3 illustrates numerous reactive functional groups and the resulting bonds formed by reaction therebetween. Where functional groups are lacking, they can be created by suitable chemistries that are described in standard organic chemistry texts such as J. March, "Advanced Organic Chemistry", 4 th Edition, (Wiley-Interscience (New York), 1992.
  • the relative orientation in which the ligand domains are displayed depends both on 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 of the ligand and/or congeners and/or structural information about ligand-receptor complexes (e.g., X-ray crystallography, NMR, and the like).
  • linker-ligand conjugate may be tested for retention of activity in a relevant assay system (see Utility and Testing below for representative assays).
  • the multibinding compound is a bivalent compound in which two ligands are covalently linked, or a trivalent compound, in which three ligands are covalently linked.
  • Linker design is further discussed under Methods of Preparation.
  • “Potency” as used herein 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 receptor. 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 biologic or therapeutic effect at a lower concentration than the aggregate unlinked ligand (e.g. on a per weight, per mole or per ligand basis) is indicative of enhanced potency.
  • Selectivity is a measure of the binding preferences of a ligand for different receptors.
  • the selectivity of a ligand with respect to its target receptor relative to another receptor 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 or IC 50 s (i.e., the concentrations that produce 50% of the maximum response for the ligand interacting with the two distinct receptors).
  • treatment refers to any treatment of a disease or condition in a mammal, particularly a human, and includes: (i) preventing the disease or 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 pathologic condition;
  • disease or condition which is modulated by treatment with a multibinding Ca ++ channel ligand covers all disease states and/or conditions that are generally acknowledged in the art to be usefully treated with a ligand for a Ca ++ channel in general, and those disease states and/or conditions that have been found to be usefully treated by a specific multibinding compound of our invention, i.e., the compounds of Formula I.
  • Such disease states include, by way of example only, hypertension, angina pectoris (particularly vasospastic angina and unstable angina), cerebral ischemia, cardiac arrythmias (particularly, arrythmias resulting from calcium- related changes in membrane potential and conduction), cardiac hypertrophy due to systolic or diastolic overload, congestive heart failure, migraine, Raynaud's disease, acute renal failure due to prolonged renal ischemia, and the like.
  • therapeutically effective amount refers to that amount of multibinding compound that 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.
  • pharmaceutically acceptable excipient is intended to include vehicles and carriers capable of being coadministered with a multibinding compound to facilitate the performance of its intended function.
  • vehicles and carriers capable of being coadministered with a multibinding compound to facilitate the performance of its intended function.
  • the use of such media for pharmaceutically active substances is well known in the art.
  • vehicles and carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. Any other conventional carrier suitable for use with the multibinding compounds also falls within the scope of the present invention.
  • the linker or linkers when covalently attached to multiple copies of the ligands, provides a biocompatible, substantially non-immunogenic multibinding compound.
  • the biological activity of the multibinding Ca ++ channel compound is highly sensitive to the geometry, composition, size, length, flexibility or rigidity, the presence or absence of anionic or cationic charge, the relative hydrophobicity/ hydrophihcity, and similar properties of the linker. Accordingly, the linker is preferably chosen to maximize the biological activity of the compound.
  • the linker may be biologically "neutral,” i.e., not itself contribute any additional biological activity to the multibinding compound, or it may be chosen to further enhance the biological activity of the compound.
  • the linker may be chosen from any organic molecule construct that orients two or more ligands for binding to the receptors 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 of ligands can be achieved by varying the geometry of the framework (linker) by use of mono- or polycyclic groups, such as aryl and/or heteroaryl groups, or structures inco ⁇ orating one or more carbon-carbon multiple bonds (alkenyl, alkenylene, alkynyl or alkynylene groups).
  • mono- or polycyclic groups such as aryl and/or heteroaryl groups, or structures inco ⁇ orating one or more carbon-carbon multiple bonds (alkenyl, alkenylene, alkynyl or alkynylene groups).
  • the optimal geometry and composition of frameworks (linkers) used in the multibinding compounds of this invention are based upon the properties of their intended receptors.
  • rigid cyclic groups e.g., aryl, heteroaryl
  • non- rigid cyclic groups e.g., cycloalkyl or crown groups
  • hydrophobic/hydrophilic characteristics of the linker as well as the presence or absence of charged moieties can readily be controlled by the skilled artisan.
  • 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" (class of surfactants).
  • Different frameworks can be designed to provide preferred orientations of the ligands.
  • the identification of an appropriate framework geometry for ligand domain presentation is an important first step in the construction of a multi binding agent with enhanced activity.
  • Systematic spatial searching strategies can be used to aid in the identification of preferred frameworks through an iterative process.
  • Figure 2 illustrates a useful strategy for determining an optimal framework display orientation for ligand domains and can be used for preparing the bivalent compounds of this invention.
  • Various alternative strategies known to those skilled in the art of molecular design can be substituted for the one described here.
  • the ligands are attached to a central core structure such as phenyldiacetylene (Panel A) or cyclohexane dicarboxylic acid (Panel B).
  • the ligands are spaced apart from the core by an attaching moiety of variable lengths m and n. If the ligand possesses multiple attachment sites (see discussion below), the orientation of the ligand on the attaching moiety may be varied as well.
  • the positions of the display vectors around the central core structures are varied, thereby generating a collection of compounds.
  • the process may require the use of multiple copies of the same central core structure or combinations of different types of display cores. It is to be noted that core structures other than those shown here can be used for determining the optimal framework display orientation of the ligands.
  • core structures other than those shown here can be used for determining the optimal framework display orientation of the ligands.
  • the above-described technique can be extended to trivalent compounds and compounds of higher-order valency.
  • linkers A wide variety of linkers is commercially available (see, e.g., Chem Sources USA and Chem Sources International; the ACD electronic database; and Chemical Abstracts). Many of the linkers that are suitable for use in this invention fall into this category. Others can be readily synthesized by methods known in the art, and as described below. Examples of linkers include aliphatic moieties, aromatic moieties, steroidal moieties, peptides, and the like. Specific examples are peptides or polyamides, hydrocarbons, aromatics, heterocyclics, ethers, lipids, cationic or anionic groups, or a combination thereof.
  • linker can be modified by the addition or insertion of ancillary groups into the linker, for example, to change the solubility of the multibinding compound (in water, fats, lipids, biological fluids, etc.), hydrophobicity, hydrophihcity, linker flexibility, antigenicity, stability, and the like.
  • the introduction of one or more poly(ethylene glycol) (PEG) groups onto the linker enhances the hydrophihcity 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 that enhance the water solubility /hydrophihcity 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., tetraethylenepentamine), and the like to enhance the water solubility and/or hydrophihcity of the multibinding compounds of this invention.
  • the ancillary group used to improve water solubilify/hydrophilicity will be a polyether.
  • the ancillary group will contain a small number of repeating ethylene oxide (-CH 2 CH 2 O-) units.
  • Lipophilic groups useful with the linkers of this invention include, but are not limited to, lower alkyl, aromatic groups and polycyclic aromatic groups.
  • the aromatic groups 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.
  • aromatic groups inco ⁇ orates both aromatic hydrocarbons and heterocyclic aromatics.
  • Other lipophilic groups useful with the linkers of this invention include fatty acid derivatives which may or may not form micelles in aqueous medium and other specific lipophilic groups which modulate interactions between the multibinding compound and biological membranes.
  • lipid refers to any fatty acid derivative that is capable of forming a bilayer or 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, carboxyhc, 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 phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl- ethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoyl-phosphatidylcholine and dilinoleoylphosphatidylcholine.
  • 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 freedom is restrained by the presence of rings and/or ⁇ -bonds, for example, aryl, heteroaryl and heterocyclic groups.
  • Other groups which can impart rigidity include polypeptide groups such as oligo- or polyproline chains.
  • Rigidity can also be imparted electrostatically.
  • the ancillary groups are either positively or negatively charged, the similarly charged ancillary groups will force the 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, which is inversely related to the square of the distance between the groups, will tend to hold the linker in a configuration that maintains the separation between the like-charged ancillary groups.
  • ancillary groups bearing opposite charges will tend to be attracted to their oppositely charged counte ⁇ arts and potentially may enter into both inter- and intramolecular ionic bonds.
  • 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 inco ⁇ orating one or more carbon-carbon -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 alicyclic (e.g., cycloalkyl), aromatic and heterocyclic groups. In other preferred embodiments, this comprises one or more six-membered rings. In still further preferred embodiments, the ring is an aryl group such as, for example, phenyl or naphthyl, or a macrocyclic ring such as, for example, a crown compound.
  • Eliminating or reducing antigenicity of the multibinding compounds described herein is also within the scope of this invention.
  • the antigenicity of a multibinding compound may be eliminated or reduced by use of groups such as, for example, poly(ethylene glycol).
  • ligands attached covalently to a linker that links the ligands in a manner that allows their multivalent binding to ligand binding sites of Ca ++ channels.
  • the linker spatially constrains these interactions to occur within dimensions defined by the linker. This and other factors increases the biologic and/or therapeutic effect of the multibinding compound as compared to the same number of ligands used 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. This is intended to include the several ways in which the ligands can be linked together in order to achieve the objective of multivalency, and a more detailed explanation is provided below.
  • 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 thereof.
  • the simplest and most preferred multibinding compound is a bivalent compound which can be represented as L-X-L, where L is a ligand and is the same or different and X is the linker.
  • 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 is X.
  • a trivalent compound can also comprise three ligands attached to a central core, and thus be represented as (L) 3 X, where the linker X could include, for example, an aryl or cycloalkyl group.
  • Tetravalent compounds can be represented in a linear array , L-X- L-X-L-X-L , or a branched array, L-X-L-X-L , i.e., a branched construct analogous to the isomers of butane (w-butyl,
  • I L iso-butyl, sec-butyl, and t- butyl).
  • it could be represented as an aryl or cycloalkyl derivative as described above with four (4) ligands attached to the core linker.
  • the same considerations apply to higher multibinding compounds of this invention containing from 5-10 ligands.
  • a central linker such as an aryl , cycloalkyl or heterocyclyl group, or a crown compound
  • a central linker such as an aryl , cycloalkyl or heterocyclyl group, or a crown compound
  • the formula (L) p (X) q is also intended to represent a cyclic compound of formula (-L- X-) n , where n is 2-10.
  • a preferred linker may be represented by the following formula:
  • m is an integer of from 0 to 20;
  • X' at each separate occurrence is -O-, -S-, -S(O)-, -S(O) 2 -, -NR-, -N + R R -, -C(O)-, -C(O)O-, -C(O)NH-, -C(S), -C(S)O-, -C(S)NH- or a covalent bond, where R and R at each separate occurrence are as defined below for R' and R";
  • Z is at each separate occurrence selected from alkylene, substituted alkylene, alkylalkoxy, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted alkenylene, arylene, substituted arylene, heteroarylene, heterocyclene, substituted heterocyclene, crown compounds, or a covalent bond;
  • Y' and Y' at each separate occurrence are selected from -S-S- or a covalent bond;
  • n 0, 1 or 2;
  • R' and R" at each separate occurrence are selected from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl or 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, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic group.
  • the simplest (and preferred) construct is a bivalent compound which can be represented as L-X-L, where L is a Ca ++ channel ligand that is the same or different at each occurrence, and X is the linker. Accordingly, examples of the preparation of a bivalent ligand are given below as an illustration of the manner in which multibinding compounds of Formula I are obtained.
  • reaction schemes that follow illustrate preferred linking strategies for linking dihydropyridine, benzothiazepine and phenylalkylamine classes of calcium channel modulators. These strategies are intended to apply as well to any Ca ++ channel ligand that includes, or can be functionalized with groups compatible with the chosen linker (e.g., mibefradil).
  • the linker or linkers can be attached to different positions on the ligand molecule to achieve different orientations of the ligand domains and thereby facilitate multivalency.
  • the positions that are potentially available for linking a dihydropyridine of formula (1), a benzothiazepine of formula (2) verapamil, a phenylalkylamine (3) and mibefradil, a tetralol derivative and selective T-channel ligand (4) are indicated by arrows in the structures shown below.
  • the ligands are covalently attached to the linker using conventional chemical techniques.
  • the reaction chemistries resulting in such linkage are well known in the art and involve the coupling of reactive functional groups present on the linker and ligand. In some cases, it may be necessary to protect portions of the ligand that are not involved in linking reactions. Protecting groups for this pu ⁇ ose are well known in the art and are indicated generally in the reaction schemes by the symbols PG and PG'.
  • the reactive functional groups on the linker are selected relative to the functional groups on the ligand that are available for coupling, or can be introduced onto the ligand for this pu ⁇ ose.
  • the linker is coupled to ligand precursors, with the completion of ligand synthesis being carried out in a subsequent step (see, e.g., Figure 7).
  • functional groups are lacking, they can be created by suitable chemistries that are described in standard organic chemistry texts such as J. March, "Advanced Organic Chemistry", 4' h Edition (Wiley- Interscience, N. Y, 1992). Examples of the chemistry for connecting ligands by a linker are shown in Table 3 where R 1 and R 2 represent a ligand and/or the linking group.
  • R 1 and R 2 represent a ligand and/or the linking group.
  • the linker to which the ligands or ligand precursors are attached comprises a "core" molecule having two or more functional groups with reactivity that is complementary to that of the functional groups on the ligand.
  • Figure 3 illustrates the diversity of "cores” that are useful for varying the linker size, shape, length, orientation, rigidity, acidity/basicity, hydrophobicity/hydrophilicity, hydrogen bonding characteristics and number of ligands connected. This pictorial representation is intended only to illustrate the invention, and not to limit its scope to the structures shown.
  • a solid circle is used to generically represent a core molecule. The solid circle is equivalent to a linker as defined above after reaction.
  • Reactions performed under standard amide coupling conditions are carried out in an inert polar solvent (e.g., DMF, DMA) in the presence of a hindered base (e.g., TEA, DIPEA) and standard amide coupling reagents (e.g., DPPA, PyBOP, HATU, DCC).
  • an inert polar solvent e.g., DMF, DMA
  • a hindered base e.g., TEA, DIPEA
  • standard amide coupling reagents e.g., DPPA, PyBOP, HATU, DCC
  • Figure 4 illustrates a preferred method for linking molecules at positions R 2 (R 6 ) of the dihydropyridine ring.
  • a multistep Hantzsch DHP-cyclization is used to introduce the appropriate groups [see, e.g., Arrowsmith et al, J. Med. Chem. 29: 1696-1702 (1986); Bossert et al, Angew. Chem. Int. Ed. Engl. 20: 762-769 (1981)).
  • the starting materials are a compound of formula (4) having an azide- substituted -keto ester and a substituted benzaldehyde (5). These compounds are reacted in benzene in the presence of piperidine and acetic acid to yield an intermediate product, which, when reacted with a compound of formula (6) in benzene yields DHP (7), where R 2 is CH 2 OCH 2 CH 2 N 3 .
  • the ring nitrogen is protected to yield the compound of formula (8), following which the azide is reduced to an amine compound (9) using hydrogen and an appropriate catalyst, such as palladium/calcium carbonate.
  • Amine (9) can then be coupled to a core molecule using a variety of well known reactions, examples of which are shown in Figure 4 and described below.
  • the compound of formula (9) is reacted with a bifunctional alkylating core (e.g. a dibromide linker (10)) in an inert solvent (e.g. DMF) with a hindered base (e.g. DIPEA) to produce, after deprotection, the amine linked compound of Formula I (11).
  • Compound (11) can also be formed by reacting (9) with a dialdehyde core (12) under standard reductive amination conditions (e.g. sodium cyanoborohydride in ethanol with acid), followed by deprotection.
  • the compound of form (9) is reacted with an activated diacid core (13) in a polar inert solvent (e.g.
  • the diacid may be preactivated (e.g. by using the acid chloride), or activated in situ using conventional coupling conditions (e.g. DCC, DMAP, THF).
  • the compound of formula (9) can be reacted with a diisocyanate core (15) in an inert solvent (e.g. THF) to yield, after deprotection, the urea-linked compound of Formula I (16).
  • the compound of formula (9) can also be reacted with an activated sulfonate core (17) in an inert solvent (e.g. THF) with a hindered base (e.g. DIPEA) to yield, after deprotection, the sulfonamide linked compound of Formula I (18).
  • Figures 5 and 6 illustrate the preparation of dihydropyridine compounds of
  • Formula I having different functional linking groups at positions R 2 (R 6 ) of the dihydropyridine ring.
  • a dihydropyridine with an alcohol side chain at R 2 is prepared by reacting a compound of formula (19) with (5) and (6) (using the same conditions as above), to make a compound of formula (20).
  • the compound of formula (21) can be coupled to various cores, as exemplified in Figure 5.
  • a compound of formula (21) can be prepared as described in Alker, D and Denton, S. M., Tetrahedron, 46, 3693- 3702, (1990).
  • the compound of formula (25) is reacted with a bifunctional alkylating core
  • the compound of formula (21) is reacted with an activated diacid core (13) in a polar inert solvent (e.g. THF) with base to yield, after deprotection, an ester- linked compound of Formula I (23).
  • the compound of formula (21) is reacted with a diisocyanate core (15) in an inert solvent (e.g. THF) to yield, after deprotection, a carbamic ester-linked compound of Formula I (24).
  • Figure 6 illustrates reactions for converting a nucleophihc side chain into an electrophilic one.
  • the compound of formula (21) is reacted under standard chlorinating conditions (e.g. SO 2 Cl 2 in the presence of a suitable base such as imidazole in DMF) to yield chloride (25).
  • the compound of formula (21) is reacted with mesyl chloride in an inert solvent (e.g., THF) in the presence of a base to yield mesylate (26).
  • the compound of formula (21) is reacted with oxidating agents (e.g. CrO 3 )to yield acid (27).
  • the compound of form (21) is reacted under mild oxidating conditions (e.g. PCC/CH 2 C1 2 ) to yield aldehyde (28).
  • the electrophilic groups thus generated can then be used in standard coupling reactions such as those shown in Figures 4 and 5, for example, the reaction illustrated in Figure 6 (bottom).
  • a molar equivalent of a diol core (29) is reacted with 2 molar equivalents of a mesylated dihydropyridine (26) in an inert solvent with a base to yield, after deprotection, an ether-linked compound of Formula I (30).
  • Figure 7 illustrates a preferred method for forming bivalent DHP compounds, which involves coupling of the linker to ligand precursors, followed by synthesis of the ligand.
  • a molar equivalent of a diol core (29) is coupled with approximately two molar equivalents of a chloride-substituted diketone (31) in an inert solvent (e.g. DMF) in the presence of a strong base (e.g. NaH) to yield an ether- linked compound of formula (32).
  • Compound (32) is reacted with (5) and (6), as described above with reference to Figures 4 and 5, to form an ether-linked compound of Formula I (30).
  • Dihydropyridine dimers may be linked through an ester linkage at R 7 or R 8 , as is shown in Figure 8.
  • a t-butyl ester of formula (33) is reacted with compounds (5) and (6) as described previously with reference to Figures 4 and 5, to yield a dihydropyridine of formula (34).
  • Cleavage of the t-butyl group of (34) with dilute acid yields (35), which is then amine-protected to form (36).
  • Standard activation techniques e.g. DCC/DMAP/THF
  • DCC/DMAP/THF Standard activation techniques
  • Figure 9 illustrates another procedure for synthesizing an ester-linked dihydropyridine compound of Formula I.
  • a molar equivalent of a diol core (29) is coupled with two molar equivalents of a diketone compound of formula (38) in an acid-catalyzed transesterification reaction to form a compound of formula (39).
  • a compound of formula (41) is prepared as described in U.S. Patent No. 4,552,695.
  • Compound (41) is reacted with a dihalide core (10), in an inert solvent (e.g. THF) in the presence of a strong base (e.g. NaH) to form an amide N-linked compound of Formula I (42).
  • a strong base e.g. NaH
  • the R 12 side chain can be deprotected if necessary and acylated as described in the above-mentioned patent.
  • the starting material is a compound of formula (43) (where R 12 is an ether-protected hydroxyl group), which is prepared as described in the above- referenced patent. Deprotection of this compound yields the alcohol (44), which can be reacted with an activated diacid core (13) in an inert solvent (e.g. DMF) to yield ester-linked (45) as shown here. Alternative coupling reactions can be used, such as those shown in Figure 5.
  • a compound of formula (77) may be prepared as described in the above-referenced patent. Treatment of compound (77) with BBr 3 in CH 2 C1 2 affords cleavage of the aryl ether to phenol (78). Compound (78) can be selectively alkylated at the phenolic hydroxyl group by reaction with dihalide (10) in K 2 CO 3 /acetone solution to yield ether-linked (79).
  • Figure 1 1 illustrates methods for linking PAA molecules (as exemplified by verapamil).
  • Phenyl acetonitrile (80) is treated with a basic condensing agent (e.g. sodium amide) in an inert solvent (e.g. toluene).
  • N-protected (81) is slowly added to the amidine salt, as described in US Patent No. 3,261 ,859.
  • compound (82) is coupled to an electrophilic core, for example, with a dihalide core (10) in an inert solvent (e.g. DMF) in the presence of a hindered base (e.g. DIPEA) to form an amine-linked compound of Formula I (83).
  • a dihalide core e.g. DMF
  • DIPEA hindered base
  • Another strategy that can be used with all ligands, and for the preparation of both bivalent and higher order multibinding compounds, is to introduce a 'spacer' before coupling to a central core.
  • a spacer can itself be selected from the same set as the possible core compounds. Examples of this linking strategy are shown in Figures 12 and 13, where the spacer is represented by a gray circle. As defined herein, the linker comprises the spacer + core.
  • a dihydropyridine compound of formula (21), synthesized according to Figure 5 above, is coupled to a heterobifunctional spacer (46) having an electrophilic group (e.g., Br) and a masked nucleophihc group (e.g., protected alcohol).
  • the protecting groups, PG and PG' are different (e.g., PG is Boc and PG' is Cbz) and are capable of selective removal.
  • the reaction is carried out in an inert solvent (e.g. DMF) in the presence of a strong base (e.g. NaH).
  • a strong base e.g. NaH
  • a dihydropyridine compound of formula (36) is coupled to heterobifunctional spacer (49) under standard coupling conditions (e.g. DCC/ DMAP/ CH 2 C1 2 ).
  • the unmasked nucleophile (50) is coupled to a dialdehyde core (12) by reductive amination (or to another core with chemically compatible functional groups).
  • an ester-linked compound of formula I (51) is obtained.
  • Figure 13 illustrates the use of spacers to make bivalent benzothiazepine compounds.
  • a compound of formula (78) is coupled to a heterobifunctional spacer (46) in basic conditions (e.g.
  • the unmasked nucleophile (52) is coupled to an activated diacid core (13) using standard coupling conditions to yield an ether- linked compound of Formula I (53).
  • compound (41) is coupled to heterobifunctional spacer (54) in an inert solvent solvent (e.g. DMF) in the presence of strong base (e.g. NaH).
  • the resulting compound (55) is coupled to an activated diacid core (13) to yield an N-alkylated compound of Formula I (56).
  • suitable cores may be substituted if desired.
  • Compounds of Formula I of higher order valency can be prepared by simple extension of the above strategies.
  • compounds (58) and (61) are prepared by coupling ligands to a central core bearing multiple functional groups.
  • the reaction conditions are the same as described above for the preparation of bivalent compounds, with appropriate adjustments made in the molar quantities of ligand and reagents.
  • Compound (36), synthesized as described above with reference to Figure 5, is coupled to a polypeptide core with a sidechain spacer (62) to make (63).
  • Solid phase peptide synthesis can be used to produce a wide variety of peptidic core molecules.
  • Orthogonal protecting groups are used to selectively protect functional groups on the core molecule, thus allowing ancillary groups to be inserted into the linker of the multibinding compound and/or the preparation of "heterovalomers" (i.e., multibinding compounds with nonidentical ligands).
  • Figure 15 illustrates the preparation of bivalent dihydropyridine compounds of formulae (64) and (65), wherein R 2 of a first ligand is attached through a linker to R 7 of a second ligand, and the preparation of a bivalent benzothiazepine compound of formula (67), wherein the linker is attached between R" of a first ligand and R 13 of a second ligand.
  • the coupling steps are carried out as described previously.
  • a linear strategy can also be used to prepare heterovalomers, as shown in Figures 16 and 17.
  • Heterovalomers comprising different chemical classes of Ca ++ channel ligands (e.g., dihydropyridines and benzothiazepines), different ligands within the same chemical class (e.g. amlodipine and isradipine) and different enantiomers of a ligand (e.g., the (+) and (-) enantiomers of a dihydropyridine) are all encompassed by the present invention.
  • Figure 16 illustrates methods of preparing bivalent compounds comprising dihydropyridine and benzothiazepine ligands in which the orientation of the ligands is varied.
  • Figure 17 illustrates the preparation of a mixed agonist antagonist heterovalomer.
  • the compound of formula (66), a Ca ++ channel antagonist with an attached spacer is coupled to the compound of formula (75), a Ca ++ channel agonist, and deprotected to yield a compound of formula (76).
  • Figures 18-20 illustrate alternate methods of preparing bivalent compounds comprising dihydropyridine and benzothiazepine derivatives. Isolation and Purification of the Compounds
  • Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography, thick-layer chromatography, preparative low or high-pressure liquid chromatography or a combination of these procedures. Characterization is preferably by NMR and mass spectroscopy.
  • 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.
  • 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. Favorable properties include demonstrated safety and efficacy in human patients, appropriate PK/ADME profiles, synthetic accessibility, and desirable physical properties such as solubility, log P, etc.
  • 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, hydrophihcity
  • 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 receptor(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 protease inhibitor bound to its target allows one to identify one or more sites where linker attachment will not preclude the enzyme:inhibitor 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 receptor antagonist ligand bound to its target receptor 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 receptor molecule at sites proximal to the antagonist binding site, which include elements of the receptor that are not part of the formal antagonist binding site and/or elements of the matrix surrounding the receptor such as the membrane.
  • the most favorable orientation for interaction of the second ligand molecule with the receptor/matrix may be achieved by attaching it to the linker at a position which abrogates activity of the ligand at the formal antagonist 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.
  • bivalent interaction focused on bivalent interactions of dimeric compounds bearing two copies of the same ligand joined to a single linker through different attachment points, one of which may abrogate the binding/activity of the monomeric ligand. It should also be understood that bivalent advantage may also be attained with heterodimeric constructs bearing two different ligands that bind to common or different targets.
  • a 5HT 4 receptor antagonist and a bladder- selective muscarinic M 3 antagonist may be joined to a linker through attachment points which do not abrogate the binding affinity of the monomeric ligands for their respective receptor sites.
  • the dimeric compound may achieve enhanced affinity for both receptors due to favorable interactions between the 5HT 4 ligand and elements of the M 3 receptor proximal to the formal M 3 antagonist binding site and between the M 3 ligand and elements of the 5HT 4 receptor proximal to the formal 5HT 4 antagonist binding site.
  • the dimeric compound may be more potent and selective antagonist of overactive bladder and a superior therapy for urinary urge incontinence.
  • linkages are those that are compatible with the overall structure of the ligand (or protected forms of the ligand) readily and generally formed, stable and intrinsically inocuous under typical chemical and physiological conditions, and compatible with a large number of available linkers. Amide bonds, ethers, amines, carbamates, ureas, and sulfonamides are but a few examples of preferred linkages.
  • Linkers spanning relevant multibinding parameters through selection of valency, linker length, linker geometry, rigidity, physical properties, and chemical functional groups
  • linkers employed in this library of linkers takes into consideration the following factors: Valency: In most instances the library of linkers is initiated with divalent linkers. The choice of ligands and proper juxtaposition of two ligands relative to their binding sites permits such molecules to exhibit target binding affinities and specificities more than sufficient to confer biological advantage. Furthermore, divalent linkers or constructs are also typically of modest size such that they retain the desirable biodistribution properties of small molecules. Linker length:
  • 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, typically enzymes and soluble receptor targets.
  • high-resolution structural information is not available (such as 7TM G-protein coupled receptors)
  • preferred linker distances are 2-20 , with more preferred linker distances of 3-12 .
  • 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.
  • linker geometry is varied by attaching two ligands to the ortho, meta, and para positions of a benzene ring, or in cis- or tr ⁇ ns-arrangements at the 1,1- vs. 1 ,2- vs. 1,3- vs. 1,4- positions around a cyclohexane core or in cis- or trfl «s-arrangements at a point of ethylene unsaturation.
  • 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, hydrophihcity, amphiphilicity, polarization, acidity, and basicity) in the candidate multibinding compounds.
  • 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.
  • Combinatorial synthesis Having chosen a set of n ligands (n being determined by the sum of the number of different attachment points for each ligand chosen) and 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:
  • a library of 150 candidate multibinding compounds results.
  • common chemistries are preferably used to join the reactive functionalies on the ligands with complementary reactive functionalities on the linkers.
  • the library therefore lends itself to efficient parallel synthetic methods.
  • 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. Alternatively and preferably, 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). Analysis of array by biochemical, analytical, pharmacological, and computational methods:
  • 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). Second, ascertaining the structure of those compounds which exhibit multibinding properties can be accomplished via art recognized procedures. For example, 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. Alternatively, 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
  • 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. By 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 carboxyhc acids, sulfonylhalides, aldehydes, ketones, halides, isocyanates, amines and diols.
  • the carboxyhc 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 -amine hydroxyamine sulfonyl halide sulfonamide carboxyl acid amine amide hydroxyl alkyl/aryl halide ether aldehyde amine/NaCNBH 3 amine ketone amine NaCNBH 3 amine amine isocyanate urea
  • Exemplary linkers include the following linkers identified as X-1 through X- 418 as set forth below:
  • 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-21 above and the second ligand and linker is selected from the following:
  • the multibinding compounds of this invention can be used to modulate calcium channels in various tissues including heart, muscle, and neurons. They will typically be used for the treatment of diseases and conditions in mammals that involve or are mediated by Ca ⁇ channels, such as hypertension, cardiac arrythmias, angina pectoris, cerebral ischemia, congestive heart failure, migraine, Raynaud's disease, asthma and bronchospasm, renal impairment and acute renal failure due to prolonged renal ischemia, retinal ischemia, and pain.
  • the multibinding compounds of this invention are tested in well-known and reliable assays and their activities are compared with those of the corresponding unlinked (i.e., monovalent) ligands.
  • Binding affinity to calcium channels The binding affinity is determined by a radioligand competitive displacement assay, essentially as described in Eltze et al, Chirality 2: 233-240 (1990). The ability of the present compounds to displace (+)- [ 3 H] isradipine or a similar radioactive ligand from calcium binding sites of guinea pig skeletal muscle T-tubule membranes is measured in vitro. The binding affinity, calculated from competition curves, is compared with that of the monovalent ligand and/or monovalent linker-ligand conjugate.
  • Ca ++ channel activity The effects of compounds of this invention on channel activity are determined by measurement of whole-cell Ba ⁇ currents in voltage- clamped Xenopus oocytes that express various types of voltage-gated Ca ++ channels, as described in Bezprozvanny and Tsien, Molec. Pharmacol. 48: 540-549 (1995).
  • Antivasoconstrictor activity Antivasoconstrictor activity is determined as described in Brittain et al, Physiologist 28: 325 (1985) as the concentration of a compound required to produce 50% vasorelaxation in KCl-contracted rabbit thoracic aorta strips in the presence of calcium.
  • the concentration of a compound required to inhibit coronary vasoconstriction induced by a thromboxane mimetic (U-46619, i.e., 9, 11 -methanoepoxy-PGH 2 ) in guinea pig Langendorff heart preparation is measured as described in Eltze et al, Chirality 2: 233-240 (1990).
  • Antihypertensive activity is determined in male spontaneously hypertensive rats by measurement of mean arterial blood pressure (Rovnyak et al, J. Med. Chem. 35: 3254-3263 (1992)).
  • Tissue selectivity Selectivity for vascular smooth muscle as compared with cardiac muscle can be assessed by comparing the concentration of a multibinding compound that produces a 50% increase in coronary blood flow in an isolated guinea- pig heart with that required to inhibit myocardial contractility. See, e.g., Osterrieder, W. and Hoick, M., J. Cardiovasc. Pharmacol. 13: 754-9 (1989; and Cremers et al, J. Cardiovasc. Pharmacol. 29: 692-696 (1997).
  • compositions which contain, as the active ingredient, one or more of the compounds of Formula I above or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients, carriers, diluents, permeation enhancers, solubilizers and adjuvants.
  • the compounds may be administered alone or in combination with other therapeutic agents (e.g., other antihypertensive drugs, diuretics and the like).
  • Such compositions are prepared in a manner well known in the pharmaceutical art (see, e.g., Remington 's Pharmaceutical
  • the compounds of Formula I may be administered by any of the accepted modes of administration of agents having similar utilities, for example, by oral, parenteral, rectal, buccal, intranasal or transdermal routes. The most suitable route will depend on the nature and severity of the condition being treated. Oral administration is a preferred route for the compounds of this invention.
  • the active ingredient is usually 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 when it 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.
  • the 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.
  • compositions of the active agents may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, e.g., by J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4' h Ed. (New York: Wiley- Interscience, 1992).
  • 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.
  • 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.
  • Controlled release drug delivery systems for oral administration include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are given in U.S. Patent Nos. 3,845,770; 4,326,525; 4,902514; and 5,616,345.
  • Another preferred formulation for use in the methods of the present invention employs transdermal delivery devices ("patches"). 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 Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
  • compositions are preferably formulated in a unit dosage form.
  • 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 (e.g., a tablet, capsule, ampoule).
  • the active compound is effective over a wide dosage range and is generally administered in a pharmaceutically effective amount.
  • each dosage unit contains from 1-250 mg of a compound of Formula I, and for parenteral administration, preferably from 0.1 to 60 mg of a compound of Formula I or a pharmaceutically acceptable salt thereof.
  • 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 pharmaceutical excipient for preparing solid compositions such as tablets, 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.
  • these preformulation compositions as homogeneous, it is meant that 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.
  • 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.
  • the above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.
  • Quantity Ingredient (mg/tablet)
  • a dry powder inhaler formulation is prepared containing the following components:
  • the active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly.
  • the solution of polyvinylpyrrohdone 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 °C 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.
  • the active ingredient, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg quantities.
  • Suppositories each containing 25 mg of active ingredient are made as follows:
  • 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.
  • Suspensions each containing 50 mg of medicament per 5.0 mL dose are made as follows:
  • 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 subcutaneous formulation may be prepared as follows: Ingredient Quantity
  • Indirect techniques usually involve formulating the compositions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid-soluble drugs.
  • Latentiation is generally achieved through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups present on the drug to render the drug more lipid soluble and amenable to transportation across the blood-brain barrier.
  • the delivery of hydrophilic drugs may be enhanced by intra-arterial infusion of hypertonic solutions which can transiently open the blood-brain barrier.
  • Method B A solution of amlodipine [structure 9, where PG is H; R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-C1; and R 10 is H] (2 mmol), the linker molecule 1 ,6-dibromohexane (1 mmol), and diisopropylethylamine (0.2 mL) in DMF (3 mL) is stirred and warmed under an inert atmosphere. The progress of the reaction is followed by tic and when reaction is complete, the solution is poured into aqueous 5% NaHCO 3 and the aqueous mixture is extracted with methylene chloride. The organic extract solution is dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired compound of Formula I (structure 11, where R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-C1; and R 10 is H) is obtained by purification of the crude product by use of HPLC.
  • N-BOC-amlodipine [structure 9, where PG is t-butyloxycarbonyl (BOC); R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-C1; and R 10 is H] (2 mmol)
  • the linker molecule 3,6-dioxaoctanedioic acid (1 mmol) in CH 2 C1 2 (5 mL) is prepared under argon in a flask equipped with magnetic stirrer and a drying tube.
  • dicyclohexylcarbodiimide solid, 2.2 mmol).
  • reaction solution is quenched in water, aqueous sodium bicarbonate is added and the aqueous mixture is extracted with methylene chloride. The organic layer is washed with aqueous Na 2 CO 3 and with H 2 O, dried
  • the desired Formula I compound (structure 14, where R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-C1; and R 10 is H) is obtained by purification of the crude product with the use of HPLC.
  • the progress of the reaction is followed by tic and when reaction has occurred, the reaction solution is quenched in cold 5% aqueous Na 2 CO 3 .
  • the layers are separated and the organic layer is washed with aqueous Na 2 CO 3 , with water and is dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired Formula I compound (structure 16, where R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-Cl; and R 10 is H) is obtained by purification of the crude product with the use of HPLC.
  • the progress of the reaction is followed by tic and when reaction has occurred, the reaction solution is quenched in cold 5% aqueous Na 2 CO 3 .
  • the layers are separated and the organic layer is washed with aqueous Na 2 CO 3 , with water and is dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired Formula I compound (structure 18, where R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-Cl; and R 10 is H) is obtained by purification of the crude product with the use of HPLC.
  • the progress of the reaction is followed by tic and when reaction is complete, the solution is poured into aqueous 5% NaHCO 3 and the aqueous mixture is extracted with methylene chloride. The organic extract solution is dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired compound of Formula I (structure 22, where R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-Cl; and R 10 is H) is obtained by purification of the crude product by use of HPLC.
  • Step 1 A solution of N-BOC-l,4-dihydropyridine [structure 21, where PG is t- butyloxycarbonyl (BOC); R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-Cl; and R 10 is H] (2 mmol), the linker molecule benzene- 1 ,4-bisacetic acid (1 mmol), and 4- dimethylaminopyridine (10 mg) in CH 2 C1 2 (5 mL) is prepared under argon in a flask equipped with magnetic stirrer and a drying tube. To this solution is added dicyclohexylcarbodiimide (solid, 2.2 mmol).
  • reaction solution is quenched in water, aqueous sodium bicarbonate is added and the aqueous mixture is extracted with methylene chloride.
  • organic layer is washed with aqueous Na 2 CO 3 and with H 2 O, dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired compound is obtained by purification of the crude product with the use of
  • the progress of the reaction is followed by tic and after reaction occurs, the reaction solution is quenched in water, aqueous sodium bicarbonate is added and the aqueous mixture is extracted with methylene chloride. The organic layer is washed with aqueous Na 2 CO 3 and with H 2 O, dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired compound of Formula I (structure 23, where R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-Cl; and R 10 is H) is obtained by purification of the crude product with the use of HPLC.
  • the progress of the reaction is followed by tic and when reaction has occurred, the reaction solution is quenched in cold 5% aqueous Na 2 CO 3 .
  • the layers are separated and the organic layer is washed with aqueous Na 2 CO 3 , with water and is dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired compound of Formula I (structure 24, where R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-Cl; and R 10 is H) is obtained by purification of the crude product with the use of HPLC.
  • N-BOC-l,4-dihydropyridine structure 36, where PG is t- butyloxycarbonyl (BOC); R 2 , R 6 and R 8 are methyl; R 9 is 2-Cl; and R 10 is H] (2 mmol), the linker molecule 1 ,4-bis(hydroxymethyl)benzene (1 mmol), and 4- dimethylaminopyridine (10 mg) in CH 2 C1 2 (5 mL) is prepared under argon in a flask equipped with magnetic stirrer and a drying tube. To this solution is added dicyclohexylcarbodiimide (solid, 2.2 mmol).
  • Method B A solution of dihydropyridine (structure 36, where PG is H; R 2 , R 6 and R 8 are methyl; R 9 is 2-Cl; and R 10 is H] (2 mmol), the linker molecule 1,4- bis(hydroxymethyl)benzene (1 mmol), and 4-dimethylaminopyridine (10 mg) in CH 2 C1 2 (5 mL) is prepared under argon in a flask equipped with magnetic stirrer and a drying tube. To this solution is added dicyclohexylcarbodiimide (solid, 2.2 mmol).
  • the progress of the reaction is followed by tic and after reaction occurs, the reaction solution is quenched in water, aqueous sodium bicarbonate is added and the aqueous mixture is extracted with methylene chloride. The organic layer is washed with aqueous Na 2 CO 3 and with H 2 O, dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired compound of Formula I (structure 37, where R 2 , R 6 and R 8 are methyl; R 9 is 2-Cl; and R 10 is H) is obtained by purification of the crude product with the use of HPLC.
  • Step l A solution of N-BOC-l,4-dihydropyridine (structure 36, where PG is t- butyloxycarbonyl (BOC); R 2 , R 6 and R 8 are methyl; R 9 is 2-Cl; and R 10 is H] (2 mmol), the linker molecule 1,5-diaminopentane (1 mmol), and 4- dimethylaminopyridine (10 mg) in CH 2 C1 2 (5 mL) is prepared under argon in a flask equipped with magnetic stirrer and a drying tube. To this solution is added dicyclohexylcarbodiimide (sohd, 2.2 mmol).
  • reaction solution is quenched in water, aqueous sodium bicarbonate is added and the aqueous mixture is extracted with methylene chloride.
  • organic layer is washed with aqueous Na 2 CO 3 and with H 2 O, dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired compound is obtained by purification of the crude product with the use of
  • a mixture of NaH (2.1 mmol) and DMF (1 mL) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube.
  • the resulting mixture is stirred and the course of the reaction is followed by thin layer chromatography. After reaction occurs, the reaction is quenched with cold dilute aq. Na 2 CO 3 and extracted with methylene chloride.
  • the organic layer is dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired compound of Formula I (structure 42, where R 12 is OAc; R 13 is Me; and R 14 is H) is obtained by purification of the crude product by use of HPLC.
  • N-BOC-l,4-dihydropyridine structure 36, where PG is t- butyloxycarbonyl (BOC); R 2 is 2-(N-BOC-amino)ethoxymethyl; R 6 and R 8 are methyl; R 9 is 2-Cl; and R 10 is H] (1 mmol), the compound [structure 52, where R" is 2-(N,N-dimethylamino)ethyl; R 12 is Oac; and R' 4 is H] (1 mmol) obtained in the preceding reaction (1 mmol), and 4-dimethylaminopyridine (10 mg) in CH 2 C1 2 (5 mL) is prepared under argon in a flask equipped with magnetic stirrer and a drying tube.
  • the desired compound of Formula I [structure 68, where R 2 is (2-amino)ethoxymethyl; R 6 and R 8 are methyl; R 9 is 2-Cl; R 10 and R 14 are H; R 11 is 2-(N,N-dimethylamino)ethyl; and R 12 is OAc] is obtained by purification of the crude product with the use of HPLC.
  • a mixture of NaH (1.1 mmol) and DMF (1 mL) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube. To this is added a solution of the linker molecule l-hydroxymethyl-4-benzyloxybenzene (1 mmol) in dry DMF (5 mL) and the resulting mixture is stirred for 1 hour.
  • a mixture of NaH (2.1 mmol) and DMF (1 mL) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube. To this is added a solution of the linker molecule l-hydroxymethyl-4-benzyloxybenzene (1 mmol) in dry DMF (5 mL) and the resulting mixture is stirred for 1 hour. Then a solution of the 1 ,4-dihydropyridine [(see Figure 12) structure 21, where PG is H; R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-Cl; and R 10 is H] (1 mmol) in dry DMF (2 mL) is added.
  • Step 2 Ammonium formate (96 mg, 1.5 mmol) and 10% Pd-C (50 mg) are added to a solution of the compound obtained in the preceding reaction in methanol (3 mL) and THF (2 mL). The mixture is stirred at room temperature and the progress of the reaction is monitored by tic.
  • EXAMPLE 17 (see Figure 16) Preparation of a Formula I compound wherein p is 2, q is 1 , and one ligand, L bland is the 1 ,4-dihydropyridine moiety linked via a 2-aminomethyl group to the linker, X, and the second ligand, L 2 , is the benzothioazepine moiety linked to X via the amide nitrogen of the thioazepine ring
  • a mixture of NaH (1.1 mmol) and DMF (lmL) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube.
  • a solution of the benzothiazepine see Figure 13 structure 41, where R 12 is OAc; R 13 is methyl; and R 14 is H
  • (1 mmol) (1 mmol) in dry DMF (3 mL) followed by the linker molecule l-bromomethyl-4-(N-Cbz-N-methyl)aminobenzene (l mmol) in dry DMF (1 mL).
  • the resulting mixture is stirred and the course of the reaction is followed by thin layer chromatography.
  • EXAMPLE 18 (see Figure 16) Preparation of a Formula I compound wherein p is 2, q is 1, and one ligand, L,, is the 1 ,4-dihydropyridine moiety linked via a 2-hydroxymethyl group to the linker, X, and the second ligand, L 2 , is the benzothioazepine moiety linked to X via the amide nitrogen of the thioazepine ring
  • Step 1 A solution, cooled to the temperature of an ice-water bath, containing the N- BOC-l,4-dihydropyridine ⁇ structure 47 [see example 16, method A, step 2)] where PG is BOC; R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-Cl; and R 10 is H ⁇ (1 mmol), triphenylphosphine (1.5 mmol), and carbon tetrabromide (2 mmol) in CH 2 C1 2 (10 mL) is prepared and is stirred. The cooling bath is removed and the solution is stirred at room temperature.
  • a mixture of NaH (1.1 mmol) and DMF (lmL) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube.
  • a solution of the N-BOC-l,4-dihydropyridine [structure 59, where PG is t- butyloxycarbonyl (BOC); R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-Cl; and R'° is H] (1 mmol) in DMF (3 mL) followed by a solution of the benzothiazepine (structure 41, where R 12 is OAc; R 13 is methyl; and R 14 is H) (1 mmol) in dry DMF (3 mL).
  • a solution, cooled to the temperature of an ice- water bath, containing the 1 ,4- dihydropyridine ⁇ structure 47 [see example 16, method B, step 2] where PG is H; R 6 and R 8 are methyl; R 7 is ethyl; R 9 is 2-Cl; and R 10 is H ⁇ (1 mmol), triphenylphosphine (1.5 mmol), and carbon tetrabromide (2 mmol) in CH 2 C1 2 (10 mL) is prepared and is stirred. The cooling bath is removed and the solution is stirred at room temperature.
  • Step 2 A mixture of NaH (1.1 mmol) and DMF (lmL) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube.
  • the organic layer is dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired compound of Formula I [structure 71, where R 6 , R 8 and R 13 are methyl; R 7 is ethyl; R 9 is 2-Cl; R 10 and R 14 are H; R 12 is OAc] is obtained by purification of the crude product by use of HPLC.
  • N-BOC-l,4-dihydropyridine structure 36, where PG is t- butyloxycarbonyl (BOC); R 2 , R 6 and R 8 are methyl; R 9 is 2-Cl; and R 10 is H] (1 mmol), the linker molecule l-hydroxymethyl-4-bromomethylbenzene (1 mmol), and 4-dimethylaminopyridine (10 mg) in CH 2 C1 2 (5 mL) is prepared under argon in a flask equipped with magnetic stirrer and a drying tube. To this solution is added dicyclohexylcarbodiimide (solid, 2.2 mmol).
  • a mixture of NaH (1.1 mmol) and DMF (lmL) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube.
  • a mixture of NaH (1.1 mmol) and DMF (lmL) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube.
  • the resulting mixture is stirred and the course of the reaction is followed by thin layer chromatography.
  • EXAMPLE 20 (see Figure 17) Preparation of a Formula I compound wherein p is 2, q is 1, and one ligand, L bland is the 1 ,4-dihydropyridine moiety linked via a 6-amino group to the linker, X, and the second ligand, L 2 , is the benzthioazepine moiety linked to X via the hydroxyl function of the phenolic ring.
  • Method B Step l A solution, cooled to the temperature of an ice-water bath, containing the benzothioazepine ⁇ structure 52 [see example 9, method A, step 2], where R" is 2- (N,N-dimethylamino)ethyl; R 12 is OAc; and R 14 is H ⁇ (1 mmol), triphenylphosphine (1.5 mmol), and carbon tetrabromide (2 mmol) in CH 2 C1 2 (10 mL) is prepared and is stirred. The cooling bath is removed and the solution is stirred at room temperature.
  • Step 1 A mixture of NaH (1.1 mmol) and DMF (1 mL) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube. To this is added a solution of the benzothiazepine (structure 41 where R 12 is OAc; R 13 is methyl; and R 14 is H) (1 mmol) in dry DMF (3 mL) followed by 1 ,2-dibromoethane (10 mmol). The resulting mixture is stirred and the course of the reaction is followed by thin layer chromatography. After reaction occurs, the reaction is quenched with cold dilute aq. Na 2 CO 3 and extracted with methylene chloride. The organic layer is dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product. The desired compound (structure 88) is obtained by purification of the crude product by use of HPLC. Step 2
  • the desired compound (structure 91) is obtained by purification of the crude product by use of HPLC.
  • Step 2 A solution of the product (structure 91) from the preceding reaction and trifluoroacetic acid (3 mL) in CH 2 C1 2 (5 mL) is stirred at room temperature. The progress of the reaction is followed by tic. After reaction occurs, more CH 2 C1 2 is added and the solution is washed with aqueous Na 2 CO 3 and with H 2 O. The organic layer is dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired compound (structure 92) is obtained by purification of the crude product with the use of HPLC.
  • Step 2 t-Butyldimethylsilyl chloride(0.4 mol) is added to a solution of N-BOC- ethanolamine (0.1 mole) and imidazole (0.1 mol) in dry pyridine (75 mL) and the resulting solution is stirred at RT. The progress of the reaction is followed by tic. When reaction is complete, water (5 mL) is added to the solution which is then concentrated by evaporation under reduced pressure (>25 mm Hg, 30° C). The residue is dissolved in EtOAc and the solution is extracted with saturated aq. CuSO 4 to remove residual pyridine. The EtOAc solution is washed with water, dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product. The pure product, N-BOC-ethanolamine-O-TBDMS, is obtained by purification of the crude product by flash chromatography over silica gel. Step 3
  • a solution of N-BOC-ethanolamine-O-TBDMS (0.05 mol) in dry DMF (3 mL) is added dropwise to a stirred mixture of NaH (0.05 mol) and dry DMF (10 mL) under an inert atmosphere.
  • the resulting mixture is stirred for 1 hr and then is tranferred by cannulation to a stirred solution of 1 ,2-dibromoethane (0.3 mol) in dry DMF (10 mL).
  • the resulting solution is stirred and the progress of the reaction is followed by tic. After reaction occurs, the reaction solution is quenched with aqueous 5% NaHCO 3 (100 mL) and brine (100 mL).
  • a mixture of NaH (1.1 mmol) and dry DMF (1 mL) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube.
  • a solution of N-BOC-N-(2- bromoethyl)ethanolamine-0-TBDMS (1 mmol) in dry DMF (2 mL) is added and the resulting mixture is stirred and monitored for reaction by tic.
  • reaction solution is quenched with aqueous 5% NaHCO 3 (25 mL) and brine (25 mL).
  • the mixture is extracted with CH 2 C1 2 (4 x 20 mL) and the combined organic extracts are back-washed with water (3x).
  • the organic layer is dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure to give the crude product.
  • the desired compound (structure 93) is obtained by purification of the crude product with the use of HPLC. Step 5 A solution of the product (structure 93) (1 mmol) from the preceding reaction and Et 3 N-(HF) 3 in MeCN (5 mL) is stirred at room temperature.
  • DHP 1.4-dihy ⁇ dropyridines
  • Ctx conotoxin
  • Aga agatoxin
  • E- C excitation-contraction
  • SS stimulus-secretion.
  • FIG. 5 Benzothiazapines

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Abstract

L'invention concerne de nouveaux composés à liaisons multiples comprenant 2 à 10 ligands fixés par covalence, chacun de ces ligands étant capable de se lier à un site de liaison de ligand dans un canal Ca++ pour moduler les activités biologiques de ce canal.
PCT/US1999/012672 1998-06-08 1999-06-07 Nouveaux medicaments pour traiter les etats dus aux canaux calcium et leurs utilisations WO1999063992A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU45493/99A AU4549399A (en) 1998-06-08 1999-06-07 Novel calcium channel drugs and uses
EP99928427A EP1085863A4 (fr) 1998-06-08 1999-06-07 Nouveaux medicaments pour traiter les etats dus aux canaux calcium et leurs utilisations
CA002318901A CA2318901A1 (fr) 1998-06-08 1999-06-07 Nouveaux medicaments pour traiter les etats dus aux canaux calcium et leurs utilisations
JP2000553061A JP2002517440A (ja) 1998-06-08 1999-06-07 新規カルシウムチャンネル薬物および用途

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US7101909B2 (en) 1998-10-12 2006-09-05 Theravance, Inc. Calcium channel drugs and uses
ATE437641T1 (de) 2003-05-15 2009-08-15 Roskamp Res Llc Verfahren zur herstellung von medikamenten zur verringerung der amyloid-abscheidung, amyloid- neurotoxizität und mikrogliosis
EP2214666B1 (fr) 2007-10-05 2013-12-11 Alzheimer's Institute of America, Inc. Procédé de réduction du dépôt amyloïde, de la neurotoxicité amyloïde et de la microgliose avec l'énantiomère (-)-nilvadipine

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DE3042769A1 (de) * 1980-11-13 1982-06-09 Bayer Ag, 5090 Leverkusen C-3 verknuepfte 1,4-dihydropyridine, ihre verwendung in arzneimitteln und verfahren zu ihrer herstellung
NZ201395A (en) * 1981-07-30 1987-02-20 Bayer Ag Pharmaceutical compositions containing 1,4-dihydropyridines and certain of these dihydropyridines
US4771057A (en) * 1986-02-03 1988-09-13 University Of Alberta Reduced pyridyl derivatives with cardiovascular regulating properties
WO1992005802A1 (fr) * 1990-09-28 1992-04-16 Neorx Corporation Porteurs polymeres servant a la liberation d'agents a liaison covalente
KR100517210B1 (ko) * 1994-12-12 2006-06-07 오메로스 코포레이션 통증,염증및경련억제용관주용액
WO1996033972A1 (fr) * 1995-04-28 1996-10-31 Glaxo Group Limited Procedes de synthese de divers ensembles de pyridines, pyrimidines, derives 1,4-dihydro de ces composes et derives de piperidine
NZ280378A (en) * 1995-11-01 1998-04-27 Apotex Inc 4-phenyl-1,4-dihydropyridine-3,5-dicarboxylic acid compounds, preparation, intermediate compounds
US5965532A (en) * 1996-06-28 1999-10-12 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6403305B1 (en) * 1997-02-06 2002-06-11 Cornell Research Foundation, Inc. Methods of identifying peptide agonists or negative antagonists of a G protein coupled receptor

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EP1085863A1 (fr) 2001-03-28
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SG80038A1 (en) 2001-04-17
EP1085863A4 (fr) 2001-03-28
WO1999063992A8 (fr) 2001-02-22
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AU4549399A (en) 1999-12-30
CA2318901A1 (fr) 1999-12-16

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