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WO1992018499A1 - Nouveaux derives esters de ryanodine et de dehydroryanodine - Google Patents

Nouveaux derives esters de ryanodine et de dehydroryanodine Download PDF

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Publication number
WO1992018499A1
WO1992018499A1 PCT/US1992/003193 US9203193W WO9218499A1 WO 1992018499 A1 WO1992018499 A1 WO 1992018499A1 US 9203193 W US9203193 W US 9203193W WO 9218499 A1 WO9218499 A1 WO 9218499A1
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WO
WIPO (PCT)
Prior art keywords
ryanodine
alanyl
compound
compound according
dehydroryanodine
Prior art date
Application number
PCT/US1992/003193
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English (en)
Inventor
Koert Gerzon
Rod Humerickhouse
Henry R. Besch, Jr.
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Indiana University Foundation
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Publication date
Application filed by Indiana University Foundation filed Critical Indiana University Foundation
Publication of WO1992018499A1 publication Critical patent/WO1992018499A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/022—Boron compounds without C-boron linkages

Definitions

  • This invention relates to novel O 10 e qu ato r ia l (O 10 eq ) esters of the alkaloids ryanodine and dehydroryanodine.
  • dehydroryanodine is represented.
  • Ryanodine (Merck Index number 8065-9th Edition) and dehydroryanodine are insecticidal alkaloids derived from the stem and roots of the plant Ryania speciosa Vahl, native to Trinidad. Crude extracts of the plant contain upwards of 25 alkaloids. Ryanodine is 700 times more potent as an
  • dehydroryanodine are disclosed in publications by Waterhouse et al, J. Chem Soc. Chem. Commun., 1984 1265 and J.Chem Soc., Perkin Trans 2. 1985 1011. A later paper by the same group published in J. Med. Chem., 30 710 (1987), discloses a number of derivatives of ryanodine as well as three new alkaloids. Ruest et al, Can. J. Chem., 63 2840 (1985) disclose a number of other Ryania speciosa alkaloids. The above publications disclose one derivatizate of the 10 eq -hydroxyl, the acetate.
  • ryanodine The pharmacology of ryanodine is summarized in an article by Jenden and Fairhurst, Pharmacological Reviews 21 1 (1969). In addition to its insecticidal properties, ryanodine also has a profound effect on mammalian skeletal muscle
  • Ryanodine has been shown to obstruct active uptake of Ca ++ by skeletal muscle sarcoplasmic reticulum (SR). It therefore follows that ryanodine interferes with intracellular Ca ++ transport mechanisms and inhibits the normal lowering of the sarcoplasmic Ca ++ concentration that effects relaxation. In cardiac muscle, ryanodine's inhibition of SR Ca ++ uptake results in a depletion of SR Ca ++ stores with a subsequent loss of contractility. Ryanodine is also postulated to have other pharmacologic actions in smooth muscle and in systems free of functional remnants of the SR such as nervous and hepatic tissue. Here again, these effects are also Ca ++ dependent.
  • the ryanodine receptor from cardiac SR used by the authors was purified by selective chromatography.
  • This invention provides certain 10 eq -ester derivatives of ryanodine and dehydroryanodine having the following formula:
  • R 1 NH(CH 2 ) n -CO-, wherein n is 1-3; and R 1 is H or a
  • Lipophilic groups of particular interest which R 1 represents include adamantanecarbonyl,
  • substituted benzoyl includes halo (chloro, bromo, and iodo), alkoxy (C 1-4 alkyloxy) including methoxy, ethoxy, n-butoxy, n-propoxy, isobutoxy and the like groups, and C 1-4 alkyl including methyl, ethyl; isopropyl, n-butyl and the like groups.
  • the substituents may be ortho, meta or para to the benzoyl carbonyl and there may be multiple
  • Figure 2 include: benzoyl- ⁇ -alanyl-dehydroryanodine,
  • adamantyl-1-oxycarbonylglycyl-ryanodine adamantyl-1-oxycarbonyl- ⁇ -alanyl-ryanodine, adamantyl-1-oxycarbonyl- ⁇ -aminobutyryl-ryanodine, glycyl-ryanodine, ⁇ -alanyl-ryanodine and ⁇ -aminobutyryl-ryanodine and the like compounds.
  • ryanodine and dehydroryanodine is complex; at low
  • the Ca ++ release channel is opened thereby permitting an increased efflux of Ca ++ , whereas at higher concentrations (> ⁇ M), the channel is closed, thereby interdicting Ca ++ efflux.
  • Addition of the side chain at the 10 eq -hydroxyl confers selectivity for the opening action of ryanodine.
  • the compounds of this invention are also useful in affinity chromatography for isolating and purifying the Ryanodine receptor and in photo-affinity labeling of the same receptor and in preparing anti-ryanodine anti-bodies using Ryanodine protein-conjugates.
  • muscle are potentially useful in the treatment of heart disease, particularly as anti-fibrillatory agents.
  • R 1 is H (the unsubstituted amino acid esters).
  • the preferred procedure for preparing the carbobenzyloxy derivatives comprehended by the above formula involves the use of a mixture of dicyclohexyl carbodiimide (DCC) and dimethylaminopyridine (DMAP) and is based on the procedure of Neises and Steglich, Angew. Chem., Int. Ed. Eng. 17 522 (1978)
  • HPLC High Performance Liquid Chromatography
  • N-(p-iodobenzoyl)- ⁇ -alanine 50 mg, 0.22 mmol
  • DMAP 2 mg, 0.02 mmol
  • dicyclohexylcarbodiimide (DCC, 52 mg., 0.25 mmol) was added at once and the stirred reaction maintained at room temperature for 6 hours. Water (0.1 ml) was added to inactivate excess DCC and stirring was continued for 30 minutes. The crystals of dicyclohexylurea thus formed were filtered off and washed twice with CH 2 Cl 2 .
  • the above compound (XII) is of interest in connection with the need for probes for the ryanodine binding site: since XII binds effectively to the ryanodine receptor, it serves as a model for radio-iodinated ligands. Such, more readily detectable I 125 -ligands are effective probes for the detection of further ryanodine receptor sites in diverse tissues not readily detected with ryanodine itself.
  • N-(p-n-butoxybenzoyl)- ⁇ -alanine 40 mg. 0.15 mmol
  • DMAP 2 mg., 0.02 mmol
  • a solvent mixture of CH 2 Cl 2 (10 ml.) and tetrahydofuran 0.1 ml
  • dicyclohexylcarbodiimide 35 mg., 0.15 mmol
  • Water 0.1 ml
  • the solids formed (dicyclohexylurea) were filtered and washed twice with CH 2 Cl 2 .
  • N-1-(Adamantanecarbonyl) ⁇ -alanine was prepared from
  • N-(1-Adamantanecarbonyl)- ⁇ -alanine m.p. 180-182°C having the following characteristics:
  • V-II 1 H-nmr (CD 3 OD, ⁇ ppm); 7.03, 6.87, and 6.23
  • V-II spontaneous conversion of ⁇ -alanyl-anhydro-ryanodine HCl (V) upon storage at 4°C. for four weeks to V-II, and (b) the direct preparation of V-II by
  • Example 10 and three other molecular probes XIV (Example 16), XV (Example 11) and XIV (Example 12), is described below.
  • EXAMPLE 10 is described below.
  • HPLC (Gradient system A) revealed a retention time of 12.3 min.
  • the ultraviolet absorption spectrum of VI in methanolic solution shows the respective maxima at 272 and 320 nm. of its two chromophoric moieties (ryanodine,
  • V-II ⁇ -alanyl Ryanodine
  • the above azido compound (VI) is photoactivatable and therefore can be used in photo-generation labelling studies to effect the covalent attachment of this ryanodine derivative (VI) to loci in, or adjacent to, the ryanodine receptor site.
  • This photo-generated labelling procedure permits localization of the ryanodine binding site within the receptor molecule and determination of the detailed molecular architecture of the ryanodine binding site and its environs. A prerequisite for successful receptor structure
  • BODIPY Ryanodine fluorescent agent (XV) is useful in localization by microscopy of tissue ryanodine binding sites.
  • This fluorescent ryanodine derivative is prepared in a manner analogous to the preparation of the BODIPY-derivative (xv) - EXAMPLE 11 - from ⁇ -alanyl-ryanodine (V) and the
  • the aqueous layer was held under reduced pressure (hi. vac.) to remove excess triethylamine and then acidified by stirring with DOWEX-50 H ion exchange resin which lowers the pH to pH ⁇ 7.
  • the filtrate from this resin suspension was passed through a 9 mm diam. column containing additional (4 g.) DOWEX-50 H + resin, followed by an additional 50 ml. of distilled water.
  • the resin (AH-Sepharose 4B) is suspended in a
  • N-Hydroxysulfosuccinimide sodium salt S-NHS
  • EDC water-soluble carbodiimide
  • substitution-rate percentage of the available amino groups covered through amide linkage by succinate is determined by base hydrolysis and U.V. analysis at 272 nm. This substitution rate can be expressed as mmoles
  • N-hydrosuccinimido-biotin (ImmunoPure* NHS-Biotin, Pierce Chemical Co., 16 mg, 0.05 mmol) in DMF (dried over Molecular Sieve, 1.5 ml) containing triethylamine (5 mg., 0.05 mmol). The reaction mixture was allowed to remain at room
  • CBZ-glycyl-ryanodine (I) exhibit pharmacology quite different from that of ryanodine and dehydroryanodine.
  • Ryanodine (and dehydroryanodine, its pharmacologically equivalent natural congener) exhibits a complex pharmacologic profile.
  • ryanodine opens the SR Ca ++ release channel/ryanodine receptor, permitting an increased efflux of
  • CBZ-glycyl-ryanodine (I) is more potent and more selective than XVIII (NMDS) suggesting that the electronic configuration of the carbamyl-function of the carbobenzyloxy functionof (I) (and of IV) is more favorable for binding to the specific polar receptor binding site than the amide function of NMDS (XVIII).
  • V-II ⁇ -alanyl-ryanodine
  • V-II O 10eq - ⁇ -alanyl-ryanodine
  • the product (V-II), O 10eq - ⁇ -alanyl-ryanodine is of great interest. It binds to the receptor with an affinity which is 4 times greater than that of ryanodine and is the f i rst der ivative with a receptor affinity higher than that of ryanodine itself.
  • V-II This derivative (V-II) also displays pharmacological activity different from that of its parent, ryanodine.
  • ⁇ -alanyl-ryanodine which binds with approximately four-fold higher affinity to the receptor, exhibits only the ability to enhance Ca ++ flux by opening the SR Ca ++ channel.
  • the same selective activity of only opening this channel albeit at higher dose levels than those of ⁇ -alanyl-ryanodine (V-II), is exhibited also by the
  • V-II novel derivative
  • V-I ⁇ -Alanyl-anhydro-Ryanodine
  • V-II NA ⁇ -Alanyl-Ryanodine
  • Figure 2 can be modified by substitution therein of a
  • chromophore of an isotopic atom ( 13 C for example) or by a radioatom (Radio-iodine or 14 C for example), as will be apparent to those skilled in the art.
  • a preferred label would involve the use of tritium-labelled ⁇ -alanine in one of the above synthetic procedures in which an alanyl derivative is prepared. All such labelled 10 derivatives of ryanodine or of dehydroryanodine are part of this invention since all such would be useful in the affinity labelling of ryanodine receptor.
  • a labelled ryanodine or dehydroryanodine derivatives is coupled with ryanodine receptor by adding the label-carrying derivative to a solution thought to contain ryanodine receptor, separating the coupled receptor and then assaying the material so
  • Dehydro-ryanodine succinate and ryanodine succinate can be coupled with various proteins to provide antigens which can in turn be used to provide ryanodine antibodies.
  • the preparation of such conjugates is illustrated below.
  • Serum samples (0.5 ml) were obtained from eight week old rabbits from an ear vein to serve as baseline. The rabbits were then injected intraperitoneally with
  • BSA-dehydro-ryanodine succinate solution (0.5 ml). Two booster injections three weeks apart were given thereafter. Controls using corresponding concentrations of BSA were prepared concurrently.
  • Antibodies generated in the above immunization process against the BSA-dehydro-ryanodine succinate antigen were determined by the Enzyme Linked Immuno Sorbent Assay (ELISA) using 6% Fetal calf serum and anti-rabbit IgG peroxidase conjugate.
  • ELISA Enzyme Linked Immuno Sorbent Assay
  • This antigen was prepared - analogous to the above
  • ryanodine antibodies An immediate use for ryanodine antibodies is the development of a RADIOIMMUNO ASSAY(RIA) or ENZYME IMMUNO ASSAY (EIA) which would allow the detection of ryanodine at micro- and even nano-molar levels in biological fluids
  • ryanodine-containing insecticide preparation are being used.
  • Ryanodine-antibodies would act as an antidote to treat animals or humans accidentally poisoned by an overdose of ryanodine.
  • Anti-iodiotypic antibodies would constitute a

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Nouveaux dérivés 0eq10 de ryanodine et de déhydroryanodine se caractérisant par le fait qu'ils se lient fortement au récepteur de la ryanodine, utiles pour influencer l'afflux de Ca++ dans les tissus et également pour isoler le récepteur de la ryanodine du réticulum sarcoplasmique.
PCT/US1992/003193 1991-04-18 1992-04-17 Nouveaux derives esters de ryanodine et de dehydroryanodine WO1992018499A1 (fr)

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US68771291A 1991-04-18 1991-04-18
US687,712 1991-04-18

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5492839A (en) * 1994-01-25 1996-02-20 University Of Iowa Research Foundation Immunogenic ryanodine derivative and related uses
WO2002022122A1 (fr) * 2000-09-15 2002-03-21 Novo Nordisk A/S Utilisation de composes pour le traitement de l'obesite
EP1392344A1 (fr) * 2001-05-17 2004-03-03 The Australian National University Procede de modulation de l'activite des canaux calciques dans des cellules cardiaques et reactifs pour la mise en oeuvre de ce procede

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL MEDICINAL CHEMISTRY, Volume 30, No. 4, issued 1987, ANDREW L. WATERHOUSE et al., "Structural aspects of ryanodine action and selectivity", see pages 710-716. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5492839A (en) * 1994-01-25 1996-02-20 University Of Iowa Research Foundation Immunogenic ryanodine derivative and related uses
WO2002022122A1 (fr) * 2000-09-15 2002-03-21 Novo Nordisk A/S Utilisation de composes pour le traitement de l'obesite
EP1392344A1 (fr) * 2001-05-17 2004-03-03 The Australian National University Procede de modulation de l'activite des canaux calciques dans des cellules cardiaques et reactifs pour la mise en oeuvre de ce procede
EP1392344A4 (fr) * 2001-05-17 2005-09-21 Univ Australian Procede de modulation de l'activite des canaux calciques dans des cellules cardiaques et reactifs pour la mise en oeuvre de ce procede

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