WO2009117831A1 - Molécules hybrides associant aux propriétés d'agonisme du récepteur de la vitamine d, des propriétés d'inhibition de l'histone désacétylase - Google Patents

Molécules hybrides associant aux propriétés d'agonisme du récepteur de la vitamine d, des propriétés d'inhibition de l'histone désacétylase Download PDF

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WO2009117831A1
WO2009117831A1 PCT/CA2009/000403 CA2009000403W WO2009117831A1 WO 2009117831 A1 WO2009117831 A1 WO 2009117831A1 CA 2009000403 W CA2009000403 W CA 2009000403W WO 2009117831 A1 WO2009117831 A1 WO 2009117831A1
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mmol
equiv
hybrid
group
solution
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WO2009117831A9 (fr
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John White
Jim Gleason
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The Royal Institution For The Advancement Of Learning/Mcgill University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C401/00—Irradiation products of cholesterol or its derivatives; Vitamin D derivatives, 9,10-seco cyclopenta[a]phenanthrene or analogues obtained by chemical preparation without irradiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to a se ⁇ es of new chemical agents that demonstrate antiproliferative and cytotoxic activity against cancer cells More particularly, but not exclusively, the present invention relates to hybrid molecules capable of mixed vitamin D receptor agonism and histone deacetylase inhibition The present invention also relates to methods of their synthesis
  • VDR vitamin D receptor
  • HDAC histone deacetylase
  • HDAC inhibitors (a further example of which is provided by SAHA (3)) have emerged as a new and promising class of anticancer agents capable of regulating transcription and inhibiting cancer cell proliferation by induction of cell cycle arrest in either G0/G1 or G2/M, cell differentiation and/or apoptosis. 8 ' 9
  • Trapoxin (11) a cytotoxic cyclic tetrapeptide, is a natural product with irreversible HDAC inhibitory activity that shows an unusual epoxyketone zinc binding group. Fujisawa pursues FK228, also called depsipeptide or romidepsin, which is a cyclic peptide that inihibits class I HDAC. FK288 is again a prodrug which releases a thiol upon reduction of its disulfide group.
  • the present invention relates to hybrid molecules capable of mixed vitamin
  • the present invention relates to hybrid molecules comprising a vitamin D receptor agonist moiety and an HDAC inhibitor moiety.
  • the present invention relates to hybrid molecules comprising a vitamin D receptor agonist moiety and an HDAC inhibitor moiety, wherein the vitamin D receptor agonist moiety is derived from vitamin D and wherein the HDAC inhibitor moiety is modelled after an HDAC inhibitor comprising a functionality including an N- hydroxyformate, a thioglycolate amide, a glycinamide, a bromoacetamide, a sulphonamide, a sulfamide, an ⁇ -keto ester or amide, or an ortho-aminoanilide.
  • the present invention relates to hybrid molecules or pharmaceutically acceptable salts thereof selected from the group consisting of:
  • Ri is sel H, OH and lower alkyl
  • R 2 is selected from the group consisting of H, lower alkyl and alkylene
  • R 3 is selected from the group consisting of H and OH;
  • R 4 and R 5 are independently selected from the group consisting of H, OH and lower alkyl;
  • R 6 is selected from the group consisting of H and lower alkyl
  • Y is selected from the group consisting of CO, SO 2 and NRj; [0021] n is an integer ranging from 0 to 3; and
  • m is an integer ranging from 0 to 3.
  • the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:
  • Ri and R 6 are independently selected from the group consisting of H and Me; and n is an integer ranging from 1 to 2.
  • the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:
  • n is an integer ranging from 1 to 2.
  • the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:
  • n is an integer ranging from 1 to 3.
  • the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:
  • n is an integer ranging from 1 to 3.
  • the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:
  • X is selected from the group consisting of CF 3 , Et, Bu, Ph, 4-CN-Ph and OH; n is 2; and m is either 0 or 2.
  • the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:
  • X is selected from the group consisting of SH, NMe 2 , NH 2 and Br; and n is either 1 or 2.
  • the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:
  • n is either 1 or 2.
  • the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:
  • X is selected from the group consisting of OMe and NHMe; and n is either 1 or
  • the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:
  • n is either 1 or 2.
  • the present invention relates to a hybrid molecule, or a pharmaceutically acceptable salt or prodrug thereof, comprising the formula:
  • n is either 1 or 2.
  • the present invention relates to a method for the treatment of disorders or diseases wherein inhibition of HDAC and/or vitamin D agonism is beneficial, the method comprising administering to a subject in need thereof an affective amount of one or more hybrid molecules as disclosed herein.
  • the present invention relates to a method of treating a patient afflicted with a condition selected from the group consisting of cancer, inflammation and auto-immune diseases, the method comprising administering to the patient a therapeutically effective amount of one or more of the hybrid molecules as disclosed herein.
  • the present invention relates to a method of wound healing, the method comprising administering to a patient in need thereof a therapeutically effective amount of one or more of the hybrid molecules as disclosed herein.
  • the present invention relates to a method of treating bacterial infections in a patient, the method comprising administering to the patient a therapeutically effective amount of one or more of the hybrid molecules as disclosed herein.
  • the present invention relates to a method of reducing proliferation of/or inducing i method comprising contacting the neoplastic cells with one or more of the hybrid molecules as disclosed herein.
  • the present invention relates to the use of one or more of the hybrid molecules as disclosed herein in the manufacture of a medicament for the treatment of a condition selected from the group consisting of cancer, inflammation and auto-immune diseases.
  • the present invention relates to the use of one or more of the hybrid molecules as disclosed herein in the manufacture of a medicament for inducing wound healing.
  • the present invention relates to the use of one or more of the hybrid molecules as disclosed herein in the manufacture of a medicament for treating bacterial infections.
  • the present invention relates to a pharmaceutical composition comprising an effective amount of one or more of the hybrid molecules as disclosed herein in association with one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the present invention relates to an admixture comprising an effective amount of one or more of the hybrid molecules as disclosed herein in association with one or more pharmaceutically acceptable carriers, excipients or diluents.
  • FIG. 1 is an illustration of the VDR agonist activity of hybrid molecules 4, and 16-40.
  • the capacity of the hybrid molecules to induce the expression of the 1,25D target gene encoding CYP24 was analyzed by RT/PCR using 1,25D (1) and hybrid molecule 4 as positive controls. Expression of the gene encoding GAPDH was used a negative control.
  • FIG. 2 is an illustration of the direct binding of 1 and hybrid molecules 5
  • FIG. 3 is an illustration of the capacity of selected hybrid molecules of the present invention to inhibit HDAC activity.
  • FIG. 3A illustrates the HDAC inhibitory activity of hybrid molecules 5 and 32 (using 2 as a positive control) as analyzed by colorimetric assay using HeLa cell nuclear extracts as a source of HDAC activity.
  • 10 Hybrid molecules were also tested using Western blotting for their capacity to induce hyperacetylation of tubulin (tub) or histones H3 and H4 (FIGs. 3B-E). As indicated, 2 or 4 were used as positive controls for induction of hyperacetylation. ⁇ -actin was used as control for protein loading.
  • FIG. 4 provides data from dose-response analyses of the capacity of hybrids to inhibit the activity of purified HDAC3 using the same colourimetric assay presented in FIG. 3A. IC50 values are presented. 3 was used as a positive control for HDAC inhibition.
  • Table. 1 provides a summary of the functional studies performed on compounds with 3-5 used as positive controls for analysis of HDAC inhibition and 1, 4 and 5 used as positive controls for VDR agonism.
  • Data for CYP24 induction (CYP24) are taken in part from FIG. 1, and data for fluorescence polarization assays for VDR binding are taken from FIG. 2.
  • Function of hybrids as HDAC inhibitors was tested using either a colorimetric assay or Western blottii ier tubulin (Tubulin) or histone (Histone) hyperacetylation using 2 and/or 4 as positive controls.
  • HDAC inhibitory activity of hybrids 4, 5, 17-19, 21, 22, 25, 26, 28, 30, 32 and 38 was measured using an HDAC colorimetric assay with purified fractions of HDAC2, HDAC3 or HDAC6, as indicated.
  • the IC 50 values ( ⁇ M) are presented for compounds tested. Mice fed a normal calcium diet were infused using minipumps with 1 (12 or 24 pmol/day) or hybrids 4, 17, 18, 21, 25, 26, and 32 (240 or 1,200 pmol/day). Unlike the animals treated with 1, which appeared unhealthy and were hypercalcemic (+++), all animals infused with hybrid compounds appeared healthy over the entire course of the experiment, and none developed hypercalcemia (-). Thus, all hybrids tested lacked the dose- limiting toxicity associated with treatment with 1.
  • FIG. 5 is an illustration of the capacity of hybrid molecules of the present invention to inhibit CYP24 enzymatic activity.
  • the CYP24 enzyme whose induction by 1 represents a physiological negative feedback loop, modifies 1 and inhibits its capacity to activate gene expression.
  • A illustrates the comparison between the capacities of 31 and 39 to boost expression of the gene encoding CYP24 in the presence of limiting (1OnM) concentrations of 1.
  • Cytochrome P450 inhibitor ketoconazole is used as a positive control for the effects of inhibition of CYP24 enzymatic activity.
  • B demonstrates that the effects of 31 and 39 are not linked to HDACi activity as 2 failed to boost CYP24 gene expression in the presence of limiting concentrations of 1.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.
  • NMR Nuclear Magnetic Resonance
  • MS Mass Spectrometry
  • m.p. melting point
  • HRMS High Resolution Mass Spectrometry
  • EtOAc Ethyl Acetate
  • CH 2 Cl 2 Dichloromethane
  • CDCl 3 Chloroform-d
  • DMAP 4-(N,N-dimethylamino)pyridine
  • TFA Trifluoroacetic acid
  • TCDI 1,1-thiocarbonyldiimidazole
  • AcOH Acetic acid
  • TLC Thin Layer Chromatography
  • FAB Fast Atom Bombardment
  • FCC Flash Column Chromatography.
  • alkyl can be straight-chain or branched. This also applies if they carry substituents or occur as substituents on other residues, for example in alkoxy residues, alkoxycarbonyl residues or arylalkyl residues. Substituted alkyl residues can be substituted in any suitable position.
  • alkyl residues containing from 1 to 18 carbon atoms are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl and octadecyl, the ⁇ -isomers of all these residues, isopropyl, isobutyl, isopentyl, neopentyl, isohexyl, isodecyl, 3-methylpentyl, 2,3,4-trimethylhexyl, sec-butyl, tert- butyl, or /erZ-pentyl.
  • a specific group of alkyl residues is formed by the residues methyl, ethyl, ⁇ -propyl, isopro ' terr-butyl.
  • lower alkyl can be straight-chain or branched. This also applies if they carry substituents or occur as substituents on other residues, for example in alkoxy residues, alkoxycarbonyl residues or arylalkyl residues. Substituted alkyl residues can be substituted in any suitable position. Examples of lower alkyl residues containing from 1 to 6 carbon atoms are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-buty ⁇ , pentyl, isopentyl, neopentyl, and hexyl.
  • alkylene can be a linear saturated divalent hydrocarbon radical of one to six carbon atoms or a branched saturated divalent hydrocarbon radical of three to six carbon atoms.
  • alkylene residues are methylene, ethylene, 2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, and pentylene.
  • alkenyl can be straight-chain or branched unsaturated alkyl residues that contain one or more, for example one, two or three double bonds which can be in any suitable position.
  • an unsaturated alkyl residue has to contain at least two carbon atoms.
  • alkenyl residues such as vinyl, 1-propenyl, allyl, butenyl or 3-methyl-2-butenyl.
  • alkynyl can be straight-chain or branched unsaturated alkyl residues that contain one or more, for example one, two or three, triple bonds which can be in any suitable position.
  • an unsaturated alkyl residue has to contain at least two carbon atoms.
  • alkynyl residues such as ethynyl, 1-propynyl or propargyl.
  • cycloalkyl can be monocyclic or polycyclic, for example monocyclic, bicyclic or tricyclic, i.e., they can for example be monocycloalkyl residues, bicycloalkyl residues and tricycloalkyl residues, provided they have a suitable number of carbon atoms and the parent hydrocarbon systems are stable.
  • a bicyclic or tricyclic cycloalkyl residue has to contain at least 4 carbon atoms.
  • a bicyclic or tricyclic cycloalkyl residue contain further embodiment, a bicyclic or tricyclic cycloalkyl residue contains at least 6 carbon atoms and up to the number of carbon atoms specified in the respective definition.
  • Cycloalkyl residues can be saturated or contain one or more double bonds within the ring system. In particular they can be saturated or contain one double bond within the ring system. In unsaturated cycloalkyl residues the double bonds can be present in any suitable positions.
  • Monocycloalkyl residues are, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl or cyclotetradecyl, which can also be substituted, for example by Ci-C 4 alkyl.
  • Examples of substituted cycloalkyl residues are 4-methylcyclohexyl and 2,3-dimethylcyclopentyl.
  • Examples of parent structures of bicyclic ring systems are norbornane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.1]octane.
  • aryl means an aromatic substituent which is a single ring or multiple rings fused together. When formed of multiple rings, at least one of the constituent rings is aromatic.
  • aryl substituents include phenyl and naphthyl groups.
  • substituted phenyl is understood as being phenyl having a substituent selected from the group consisting of amino, -NH(lower alkyl), and -N(lower alkyl) 2 , as well as being mono-, di- and tri-substituted phenyl comprising substituents selected from the group consisting of lower alkyl, methoxy, methylthio, halo, cyanao, hydroxy, amino, NH(lower alkyl), and -N(lower alkyl) 2 .
  • stereocenter or axis of chirality for which the stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and S forms, including racemic and non-racemic mixtures.
  • a novel class of chemical agents i.e. novel hybrid molecules having mixed vitamin D receptor agonism and histone deacetylase inhibitory properties are described herein.
  • Hybrid molecules have had considerable success in pharmacotherapy and offer several advantages over the use of the individual compounds (i.e. the compounds making-up the hybrid molecule) in combination therapy. 12'14 Moreover, analyses of dose/toxicity relationships of hybrid molecules are simpler than those of combination therapies, and problems associated with differing pharmacokinetic profiles of individual components are eliminated.
  • the design of the hybrid molecules of the present invention is based on structure-activity relationship (SAR) and X-ray studies.
  • SAR structure-activity relationship
  • a first generation of hybrid molecules, based on the structures of 1 and 2, has been disclosed in International Application PCT/CA2007/000885 published on November 22, 2007 under WO 2007/131364 Al .
  • the crystal structure of calcitriol bound to the VDR-LBD reveals hydrogen bonding to all three hydroxyl functionalities (Ser237 and Arg274 for 1-OH; Ser278 and Tyrl43 for 3-OH; and His305 and His397 for 25-OH).
  • the remainder of the binding pocket is filled with hydrophobic residues which contact the triene and C/D-ring sections, as well as a portion of the side chain.
  • the 25-OH group hydrogen bonds with His305 and His397, and this region is more tolerant of changes; with altered chain length and oxidation state, replacement of C26 and C27 with larger or cyclic structures, inversion of the C20 stereocenter and replacement of the 25-OH group with other hydrogen bonding groups all possible.
  • the x-ray crystal structure of 2 bound to archaebacterial HDAC homolog HDLP revealed 2 bound through bidentate coordination of the hydroxamic acid to the catalytic zinc ion, with the diene chain spanning the substrate-binding tube.
  • the top of the tube ends at a surface groove which has several hydrophobic residues which contact the dimethylaniline group of 2.
  • Most competitive HDACi consist of this general motif, a zinc-binding group (ZBG) connected by a saturated or unsaturated linker to a highly variable 'cap' group which binds at the protein surface.
  • ZBG zinc-binding group
  • 1 Hydrogen bonding to the hydroxyl functionalities of the A-ring of 1 is critical for binding, as deletion or alteration of the stereochemistry of the 1- or 3-OH group significantly decreases affinitv for the VDT?
  • 16 Mn «t nntpnt analogs of calcitriol have hydroxyl moieties in the vicinity of C-2: , . __r exact location (e.g. in EB 1089) is tolerated.
  • the central C/D-ring is less critical, as it may be partially or fully excised in favor of a single 5- or 6-membered ring or a linear chain. 17 ' 18 19-Nor and C-20 epi analogs are also well tolerated by the VDR. 16 ' 19
  • the crystal structure of 2 bound to an HDAC revealed a tube-like binding pocket possessing a zinc ion coordinated to two Asp residues and one His residue at a bottom portion of the tube-like binding pocket.
  • the hydroxamic acid function of 2 forms a bidentate chelate with the zinc ion.
  • the polyene chain of 2 spans the remainder of the tube-like binding pocket, consisting of hydrophobic residues.
  • the top portion of the tube-like binding pocket terminates at a surface groove comprising several hydrophobic residues which come into contact with the dimethylamino group of 2.
  • the ⁇ -methyl dienylhydroxamic acid unit is required.
  • the ketone and adjacent methyl substituted methyne may be excised, provided that the dimethylamino group is replaced with a larger unit such as an arylsulfonamide. 21
  • the dienyl chain in 2 seems to function as a tether, linking the zinc binding unit with a "cap” group which binds on the HDAC surface. Hydrogenation of the dienyl chain in analogs of 2 renders them inactive.
  • straight chain analogs lacking the ⁇ -methyl group e.g. SAHA, 3 have been found to be potent HDAC inhibitors.
  • a first hybrid molecule (36) was designed in which the 25-hydroxyl moiety required for binding to the VDR was replaced by an N-hydroxyformate. It was hypothesized that the N- hydroxyformamide terminus of this polar side chain would allow hydrogen bond formation in the active site of the VDR, as well as permitting chelation to the zinc ion in the HDAC binding site.
  • the backbone of the vitamin D core, including the A and C/D-ring systems were maintained along with the stereochemical relationships of the various substituents.
  • the present invention relates to pharmaceutical compositions comprising a pharmaceutically effective amount of one or more hybrid molecules as defined herein or pharmaceutically acceptable salts thereof, in association with one or more pharmaceutically acceptable carriers, excipients and/or diluents.
  • pharmaceutically effective amount is understood as being an amount of hybrid molecule required upon administration to a mammal in order to induce vitamin D receptor agonism and HDAC inhibition.
  • Therapeutic methods comprise the step of treating patients in a pharmaceutically acceptable manner with one or more hybrid molecules or compositions comprising one or more hybrid molecules as disclosed herein.
  • compositions may be in the form of tablets, capsules, caplets, powders, granules, lozenges, suppositories, reconstitutable powders, creams, lotions, or liquid preparations, such as oral or sterile parenteral solutions or suspensions.
  • the therapeutic agents of the present invention may be administered alone or in combination with pharmaceutically acceptable carriers.
  • the proportion of each carrier is determined by the solubility and chemical nature of the agent(s), the route of administration, and standard pharmaceutical practice.
  • the pharmaceutical composition is in the form of a unit dose.
  • the unit dose presentation forms for oral administration may be tablets and capsules and may contain conventional excipients.
  • Non-limiting examples of conventional excipients include binding agents such as acacia, gelatin, sorbitol, or polyvinylpyrolidone; fillers such as lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants such as magnesium stearate; disintegrants such as starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulphate.
  • binding agents such as acacia, gelatin, sorbitol, or polyvinylpyrolidone
  • fillers such as lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine
  • tabletting lubricants such as magnesium stearate
  • disintegrants such as starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose
  • pharmaceutically acceptable wetting agents such as sodium lau
  • the hybrid molecules of the present invention may be injected parenterally; this being intramuscularly, intravenously, or subcutaneously.
  • the hybrid molecules may be used in the form of sterile solutions containing solutes, for example sufficient saline or glucose to make the solution isotonic.
  • the hyi d orally in the form of tablets, capsules, or granules, containing suitable excipients such as starch, lactose, white sugar and the like.
  • the hybrid molecules may be administered orally in the form of solutions which may contain coloring and/or flavoring agents.
  • the hybrid molecules may also be administered sublingually in the form of tracheas or lozenges in which the active ingredient(s) is/are mixed with sugar or com syrups, flavoring agents and dyes, and then dehydrated sufficiently to make the mixture suitable for pressing into solid form.
  • the solid oral compositions may be prepared by conventional methods of blending, filling, tabletting, or the like. Repeated blending operations may be used to distribute the active agent(s) (i.e. hybrid molecules) throughout the compositions, employing large quantities of fillers. Such operations are, of course, conventional in the art.
  • the tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.
  • Oral liquid preparations may be in the form of emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or any other suitable vehicle before use. Such liquid preparations may or may not contain conventional additives.
  • Non limiting examples of conventional additives include suspending agents such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fats; emulsifying agents such as sorbitan monooleate or acaci; non-aqueous vehicles (which may include edible oils), such as almond oil, fractionated coconut oil, oily esters selected from the group consisting of glycerine, propylene glycol, ethylene glycol, and ethyl alcohol; preservatives such as for instance methyl parahydroxybenzoate, ethyl para-hydroxybenzoate, n- propyl para-hydroxybenzoate, «-butyl para-hydroxybenzoate and sorbic acid; and, if desired, conventional flavoring or coloring agents.
  • suspending agents such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogen
  • fluid unit dosage forms may be prepared by utilizing one or more hybrid molecules and a sterile vehicle, and, depending on the concentration employed, the hybrid molecule(s) may be either suspended or dissolved in the vehicle. Once in solution, the hybrid molecule(s) may be injected and filter sterilized before filling a suitable vial or ampoule followed by si " " " - ⁇ - • storage package. Adjuvants, such as a local anesthetic, a preservative or a tmrienng agent, may be dissolved in the vehicle prior to use. Stability of the pharmaceutical composition may be enhanced by freezing the composition after filling the vial and removing the water under vacuum, (e.g., freeze drying).
  • Parenteral suspensions may be prepared in substantially the same manner, except that the hybrid molecule(s) should be suspended in the vehicle rather than being dissolved and, further, sterilization is not achievable by filtration.
  • the hybrid molecule(s) may be sterilized, however, by exposing it to ethylene oxide before suspending it in the sterile vehicle.
  • a surfactant or wetting solution may be advantageously included in the composition to facilitate uniform distribution of the hybrid molecule(s).
  • Topical administration can be used as the route of administration when local delivery of one or more hybrid molecules is desired at, or immediately adjacent to the point of application of the composition or formulation comprising one or more hybrid molecules.
  • the pharmaceutical compositions of the present invention comprise a pharmaceutically effective amount of one or more hybrid molecules as described herein and one or more pharmaceutically acceptable carriers, excipients and/or diluents.
  • the pharmaceutical compositions contain from about 0.1% to about 99% by weight of a hybrid molecule as disclosed herein.
  • the pharmaceutical compositions contain from about 10% to about 60% by weight of a hybrid molecule as disclosed herein, depending on which method of administration is employed. Physicians will determine the most-suitable dosage of the present therapeutic agents (i.e. hybrid molecules). Dosages may vary with the mode of administration and the particular hybrid molecule chosen. In addition, the dosage may vary with the particular patient under treatment. The dosage of the hybrid molecule used in the treatment may vary, depending on the condition, the weight of the patient, the relative efficacy of the compound and the judgment of the treating physician.
  • ester 48 could be reduced to aldehyde 49 (Scheme 1) for subsequent chain extension or hydrolyzed to its corresponding acid (Scheme 2). Curtius rearrangement of 50 afforded amine 51.
  • Extended carboxylic acid 53 could be prepared by a methoxymethylene Wittig reaction on 49 followed by hydrolysis and oxidation of the olef ⁇ nation product.
  • One and two carbon homologues of amine 51 could be prepared by reductive amination from aldehydes 49 and 52.
  • Acids 50 and 53 and amines 51, 54 and 55 are the main precursors for almost all hybrids described below. imidazole r,, rreef ⁇ lu to rt 4 1 Ar uxx 3 o0 ⁇ m ⁇ nm
  • Hydroxamic acids 5 and 13 were synthesized by coupling acid chlorides derived from 53 and 50, respectively, with TBS protected hydroxylamine followed by deprotection (Scheme 3). Hydroxamic acid derivatives with methyl groups on either/both the nitrogen or/and oxygen could be prepared by a similar route using substituted hydroxylamines.
  • Orthoaminoanilides 17 and 18 were prepared in a simple sequence from acids 50 and 53 by formation of an acid chloride and then coupling to with 1,2-phenylene diamine to form anilides. Deprotection with tetrabutylamonnium fluoride formed the final hybrids (Scheme 4) o-Aminobenzamides
  • Hybrid molecules 19 to 30 were prepared from amines 51, 54 and 55 through straightforward coupling with either sulfonyl chlorides, to form sulfonamides, or with N-(tert- Butoxycarbonyl)-N-[4-(dimethylazaniumylidene)- 1 ,4-dihydropyridin- 1 -ylsulfonyl]azanide to form sulfamides (Scheme 5).
  • Hybrids 31 to 35 were formed by coupling amines 51 and 54 with S-acetyl thioglycolic acid, glycine derivatives or bromoacetic acid under standard peptide coupling conditions (Scheme 6).
  • N-Hydroxyformate hybrid 36 was prepared from aldehyde 59 by partial reduction of the corresponding oxime followed by formylation and deprotection (Scheme 7).
  • ⁇ -Ketoester and amide hybrids 37 and 38 were prepared by Horner-Emmons olefination using a silyloxy substituted phosphonate ester.
  • the resulting product, 69 could be deprotected directly to form 37 or partially desilylated and then aminated to form 38.
  • N-hydroxyurea 39 was achieved by treating amine 54 with carbonyl diimidizole to form an intermediate which was directly treated with TBS protected hydroxylamine and then deprotected with HF.
  • Trithiocarbonate 40 was prepared by addition of ethyl trithiocarbonate, formed from ethane thiol and carbon disulfide, to hybrid bromide 36.
  • VDR agonist activity of the hybrid molecules of the present invention was tested by screening of induction of expression of the 1,25D target gene encoding CYP24 (FIG. 1), the enzyme responsible for initiation catabolic degradation of 1,25D, thus representing a physiological negative feedback loop.
  • SCC4 cells were treated with hybrid molecules at either 10OnM or l ⁇ M, as indicated for 8h prior to harvesting RNA for reverse transcription/PCR analysis of CYP24 mRNA expression. 1 and/or 4 were used as positive controls for CYP24 mRNA induction. The results show that all hybrids, with the exceptions of compounds 33-35 and 39, showed substantial VDR agonist activity at 1 ⁇ M.
  • VDR ligand binding domain (LBD) was tested using a fluorescence polarization (FP) competition assay. 1,25D was used a positive control.
  • the assay was performed using a VDR competitor assay kit (Polarscreen, Invitrogen, Carlsbad, CA) set up using 0.5nM fix ires the decrease in FP accompanying loss of binding to the relatively high molecular weight VDR ligand binding domain of the fluorescent tracer due to the presence of a competitor.
  • FP was measured using an Analyst HT fluorimeter (Molecular Devices) configured with absorption and emissions filters as recommended by the kit manufacturers.
  • HDACi activity of hybrids 5, 17- 19, 21, 22, 25, 26, 28, 30, 32 and 38 was measured using the HDAC colorimetric assay kit following the supplier's protocol (Bio Vision, Mountain View, CA) using purified fractions of HDAC2, HDAC3 or HDAC6.
  • the IC 50 values ( ⁇ M) for compounds tested are presented in Table 1.
  • One notable compound was the thioglycolate (mercaptoamide) analogue 32, which was a potent VDR agonist, thus indicating that the thioglycolate moiety is an excellent substitute for the 25-OH group of 1.
  • 32 functioned with low micromolar potency as an HDAC6 inhibitor; its HDAC ⁇ i activity was within a factor of 5 of that of 3 (Table 1).
  • HDAC6 18.5- and >50-fold more potently than HDAC3 and HDAC2, respectively.
  • thioglycolates or mercaptoamides
  • thioglycolate analogues of 3 could be developed with low micromolar HDACi activity when measured against HDACs present in HeLa cell extracts.
  • Hybrid molecules were tested for their capacity to induce acetyl ation of tubulin or histones H3 and H4 using specific acetyl-protein antibodies and Western blotting. 5 and 32 induced ' " " . .. . tcetylation (FIG. 3B). Hybrid molecule 5, but not 32, also induced modest nistone nyperacetylation at 1 ⁇ M (FIGs. 3B, C). In contrast, while hybrid molecule 36 induced modest histone hyperacetylation, it had no effect on acetylation of tubulin (FIG. 3C).
  • hybrid molecules 21, 34 and 35 strongly induced hyperacetylation of histone H4, whereas hybrid molecule 20 induced moderate H4 hyperacetylation (FIG. 3D).
  • hybrid molecules 20, 21, 34 and 35 had no effect on tubulin acetylation (FIG. 3D).
  • the capacity of hybrid molecule 18 to induce protein acetylation was also tested by Western blotting (FIG. 3E). Remarkably, hybrid molecule 18 induced marked hyperacetylation of both tubulin and histone H3.
  • hypercalcemia is the dose-limiting toxicity associated with 1,25D treatment
  • 4 and hybrids 5, 17, 18, 21, 25, 26, and 32 were compared with 1 for their capacity to induce hypercalcemia in mice.
  • Compounds chosen had a range of affinities for the VDR as judged by FP assay (FIG. 2) and a variety of terminal zinc binding groups.
  • Mice fed a normal calcium diet were infused using minipumps with 1 (12 or 24 pmol/day) or hybrids (240 or 1,200 pmol/day) over 6-7 days. Elevated concentrations of hybrids were chosen based on literature data that suggested induction of hypercalcemia with 1, and also to account for the lower affinity for the VDR of some hybrids.
  • THF and Et 2 O were distilled from sodium metal/benzophenone ketyl under argon. All other commercial solvents and reagents were used as received from the Aldrich Chemical Company, Fischer Scientific Ltd., EMD Chemicals Inc., Strem or BDH. All glassware was flame dried and allowed to cool under a stream of dry argon.
  • Silica gel (6 ⁇ A, 230-400 mesh) used in flash column chromatography was obtained from Silicycle and was used as received.
  • Analytical thin-layer chromatography (TLC) was performed on pre-coated silica gel plates (Ultra Pure Silica Gel Plates purchased from Silicycle), visualized with a Spectroline UV2 5 4 lamp, and stained with a 20% phosphomolybdic acid in ethanol solution, or a basic solution of KMnO 4 . Solvent systems associated with R f values and flash column chromatography are reported as percent by volume values.
  • a flame-dried 100 mL three-necked flask was charged sequentially with 28 mg (0.33 mmol, 0.06 equiv) of NaHCO 3 , 20 mL of anhydrous MeOH, 60 mL of anhydrous CH 2 Cl 2 , and 2.0 g (5.12 mmol, 1 equiv) of ergocalciferol.
  • the solution was cooled to -78 0 C and treated with O 3 until a blue color appears.
  • the solution was subsequently flushed with Ar for 10- 15 min until the blue color faded.
  • PCC (546 mg, 2.52 mmol, 2 eq ⁇ iv) was added to a stirred solution of 46 (500 mg, 1.26 mmol) and celite (600 mg) in CH 2 Cl 2 (20 mL). The solution was stirred for 3 h, then the mixture was filtered on a silica pad. The precipitate was washed with CH 2 Cl 2 (3 x 20 mL) then the combined organic fractions were concentrated in vacuo to provide the crude ester as a colorless oil. The oil was diluted in CH 2 Cl 2 (10 mL) and CH 3 CN (10 mL) and a 48% solution of HF (1 mL) was added. The solution was stirred for 24 h at room temperature.
  • the reaction mixture was c on of a sat. solution of NaHCO 3 until no effervescence was observed.
  • the solution was extracted with CH 2 Cl 2 (3 x 20 mL), the combined organic layers were then washed with H 2 O (20 mL) and brine (20 mL), then dried (MgSO 4 ).
  • the solution was concentrated in vacuo, then diluted in CH 2 Cl 2 (20 mL), celite was added (500 mg) followed by PCC (546 mg, 2.52 mmol, 2 equiv). After stirring for 3 h at room temperature, the reaction mixture was filtered on a silica pad, the precipitate was washed with CH 2 Cl 2 (3 x 20 mL) and the solution was concentrated in vacuo.
  • Enol ether (40 mg, 0.063 mmol) was dissolved in a solution Of CHCl 3 (1 mL), distilled H 2 O (0.5 mL) and TFA (0.15 mL), and cooled to 0 0 C. The reaction was stirred at 0 0 C for approx. 30 min and monitored by TLC until complete consumption of the starting material, the reaction was quenched with sat. 44
  • nub wab ⁇ ic ⁇ cucu Hum ⁇ u J3 by following the same procedure described for 13.
  • the reagent used were as follows: acid 53 (6.5 mg, 0.010 mmol, 1 equiv), oxalylchloride (1.2 ⁇ L, 0.014 mmol, 1.5 equiv), DIPEA (1.6 ⁇ L, 0.027, 3 equiv) and 0-tert-butyldimethylsilyl- yV-methyl hydroxylamine (2.26 ⁇ L, 0.018 mmol, 2 equiv).
  • the reagents used were as follows: 62 (18.8 mg, 0.026 mmol, 1 equiv), TBAF (78 ⁇ L of a 1 M solution in THF ⁇ 078 mmnl 4 emiiv ⁇ and Et 3 N (15 ⁇ L, 0.078 mmol, 3 equiv). 18 was purified by oct; _, ⁇ ilumn chromatography (100% H 2 O to 100%
  • EDCHCl (1.6 mg, 0.011 mmol, 1.1 equiv) was added to a stirred solution of amine 54 (6.3 mg, 0.010 mmol, 1 equiv) and dimethyl glycine (1.2 mg, 0.011 mmol, 1.1 equiv) in CH 2 Cl 2 (0.2 m ⁇ ⁇ "* A 0 ⁇ ⁇ 1 " —' ⁇ *—- — ⁇ 4 at room temperature for 4 h then diluted with CH 2 Cl 2 (10 mL,, vvdoiivu vvxtij ou... 1 ,UiX ⁇ Wj v- - ,.
  • the product was then directly submited to deprotection: the oil was dissolved in CH 2 Cl 2 (100 ⁇ L) and CH 3 CN (100 ⁇ L) then a 48% solution of HF was added (2 drops) and the solution was stirred overnight.

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Abstract

La présente invention concerne des molécules hybrides comprenant un groupe fonctionnel agoniste du récepteur de la vitamine D et un groupe fonctionnel inhibiteur de l'histone désacétylase (HDAC). Ce groupe fonctionnel inhibiteur de HDAC peut être dérivé d'un inhibiteur de HDAC comprenant une fonctionnalité choisie dans un groupe comprenant N-hydroxyformate, thioglycolate amide, glycinamide, bromoacétamide, sulphonamide, sulfamide, alpha-céto ester ou amide, et ortho-aminoanilide. Ces molécules hybrides conviennent au traitement d'états tels qu'infections bactériennes, inflammations, et maladies auto-immunes, ou à l'induction de la guérison de blessures. Les molécules hybrides spécifiques selon la présente invention présentent des structures représentées par les formules (I) et (II).
PCT/CA2009/000403 2008-03-27 2009-03-27 Molécules hybrides associant aux propriétés d'agonisme du récepteur de la vitamine d, des propriétés d'inhibition de l'histone désacétylase WO2009117831A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN103058902A (zh) * 2012-12-21 2013-04-24 江西科技师范大学 光活性可逆加成-断裂链转移试剂及其制备与应用
WO2014153030A2 (fr) 2013-03-14 2014-09-25 Genentech, Inc. Méthodes de traitement du cancer et de prévention d'une résistance à un médicament anticancéreux

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US20240327427A1 (en) * 2020-10-23 2024-10-03 Icahn School Of Medicine At Mount Sinai New compounds for the treatment of alzheimer's disease

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CA2162272A1 (fr) * 1993-05-07 1994-11-24 Robert Henry Hesse Derives amides de la vitamine d
US5494905A (en) * 1991-11-07 1996-02-27 Research Institute For Medicine And Chemistry Vitamin D amide derivatives
WO2007131364A1 (fr) * 2006-05-16 2007-11-22 Mcgill University Molécules hybrides ayant des propriétés mixtes d'agonisme du récepteur de la vitamine d et inhibitrice de l'histone désacétylase

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US5494905A (en) * 1991-11-07 1996-02-27 Research Institute For Medicine And Chemistry Vitamin D amide derivatives
CA2162272A1 (fr) * 1993-05-07 1994-11-24 Robert Henry Hesse Derives amides de la vitamine d
WO2007131364A1 (fr) * 2006-05-16 2007-11-22 Mcgill University Molécules hybrides ayant des propriétés mixtes d'agonisme du récepteur de la vitamine d et inhibitrice de l'histone désacétylase

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TAVERNA-MENDOZA, L. E. ET AL.: "Incorporation of histone deacetylase inhibition into the structure of a nuclear receptor agonist", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 105, no. 24, 17 June 2008 (2008-06-17), pages 8250 - 8255 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103058902A (zh) * 2012-12-21 2013-04-24 江西科技师范大学 光活性可逆加成-断裂链转移试剂及其制备与应用
CN103058902B (zh) * 2012-12-21 2014-08-13 江西科技师范大学 光活性可逆加成-断裂链转移试剂及其制备与应用
WO2014153030A2 (fr) 2013-03-14 2014-09-25 Genentech, Inc. Méthodes de traitement du cancer et de prévention d'une résistance à un médicament anticancéreux

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