WO1992018499A1 - Novel ester derivatives of ryanodine and dehydroryanodine - Google Patents

Abstract

Novel 010eq-derivatives of ryanodine and dehydroryanodine characterized as binding strongly to ryanodine receptor, useful in affecting Ca++ efflux in tissue and also in isolating ryanodine receptor from sarcoplasmic reticulum.

Description

NOVEL ESTER DERIVATIVES OF RYANODINE AND DEHYDRORYANODINE

REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of our copending application Serial No. 07/687,712 filed April 18, 1991.

FIELD OF THE INVENTION

This invention relates to novel O_{10 e qu ato r ia l} (O_{10 eq} ) esters of the alkaloids ryanodine and dehydroryanodine.

BACKGROUND OF THE INVENTION Ryanodine and dehydroryanodine are represented by the following formula:

wherein, when R is H and Z is C(H)CH₃, ryanodine is

represented, and when R is H and Z is C=CH₂,

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

insecticide than the crude alkaloidal extract, and was first isolated by Rogers et al, J.Am Chem Soc. 70 3086 (1948). Its structure was determined by Wiesner et al, Tetrahedron

Letters 1967 221. The purification and structure of

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.

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

(irreversible contracture) and a negative inotropic effect on mammalian cardiac muscle. Jenden and Fairhurst conclude that ryanodine specifically interferes with vertebrate skeletal muscle relaxation, an activity believed to be effected by sequestration of Ca⁺⁺ ions by the sarcoplasmic reticulum.

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.

Specific information about the mode of action of

ryanodine on cardiac SR was first published by Sutko,

Willerson, Besch et al J.P.E.T. 209 37 and Jones, Besch,

Sutko et al id 40 (1979). More specific information is to be found in a paper by Inui et al, J.B.C. 262 15637 (1987). The authors find that ryanodine reacts with Ca⁺⁺ release

channels localized in the terminal cisternae of the SR. The ryanodine receptor from cardiac SR used by the authors was purified by selective chromatography.

SUMMARY OF THE INVENTION

This invention provides certain 10_eq-ester derivatives of ryanodine and dehydroryanodine having the following formula:

in which Z is C(H)CH₃ or C=CH₂ and R is

-CO-CH₂CH₂-COOH, -CO-CH₂-CH₂-CONHCH₃ or

R¹NH(CH₂)_n-CO-, wherein n is 1-3; and R¹ is H or a

lipophilic group. Lipophilic groups of particular interest which R¹ represents include adamantanecarbonyl,

adamantylmethylcarbonyl, adamantyl-1-oxycarbonyl,

benzyloxycarbonyl, benzoyl, substituted benzoyl, phenylacetyl and the like. Other groups of interest which R¹ can

represent include senecioyl, geranoyl, farnesoyl and the like lipophilic groups as well as N-biotinyl-β-alanyl,

β-alanyl, and the like groups of biological interest. The term "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

substituents; ie., 5-azido-2-nitro,2,3-dimethoxy, 2,4

dichloro, 2-methyl-4-chloro, etc. Of the above substituted benzoyl derivatives, the iodo derivative is of particular interest because by employing radio-iodine, a tracer compound of great interest is produced, as will be delineated

hereafter.

Compounds coming within the scope of the formula of

Figure 2 include: benzoyl-β-alanyl-dehydroryanodine,

1-adamantanecarbonyl-β-alanyl-dehydroryanodine,

1-adamantanecarbonyl-γ-aminobutyryl-ryanodine, benzoyl-β- alanyl-ryanodine, benzoyl-γ-aminobutyryl-ryanodine,

p-iodobenzoyl-β-alanyl-ryanodine, p-iodobenzoyl-β-alanyl-dehydroryanodine, p-ethoxybenzoyl-β-alanyl-ryanodine,

phenylacetyl-glycyl-ryanodine, phenylacetyl-γ-aminobutyryl- ryanodine, l-adamantylmethylcarbonyl-glycyl-ryanodine,

1-adamantylmethylcarbonyl-β-alanyl-dehydroryanodine,

1-adamantylmethylcarbonyl-γ-aminobutyryl-ryanodine,

adamantyl-1-oxycarbonylglycyl-ryanodine, adamantyl-1-oxycarbonyl-β-alanyl-ryanodine, adamantyl-1-oxycarbonyl-γ-aminobutyryl-ryanodine, glycyl-ryanodine, β-alanyl-ryanodine and γ-aminobutyryl-ryanodine and the like compounds.

The chief utility of the compounds of this invention wherein R is -CO-CH₂CH₂-CONHCH₃ or R¹ as defined with

the proviso that n is only 2 when Z is C=CH₂, is a

pharmacologic action╌the ability of the compounds to affect the function of the functional SR Ca⁺⁺-release channel of striated muscle. The effect of the parent compounds,

ryanodine and dehydroryanodine, is complex; at low

concentrations of drug (<μM), the Ca⁺⁺ release channel is opened thereby permitting an increased efflux of Ca⁺⁺, whereas at higher concentrations (>μM), 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.

The compounds of this invention, because of their

profound effects on Ca⁺⁺ ion movement within striated

muscle, are potentially useful in the treatment of heart disease, particularly as anti-fibrillatory agents.

Compounds represented by the above formula, wherein R¹ is benzyloxycarbonyl and n is 1-3, are prepared by direct acylation of the 10_eq-hydroxyl of ryanodine or of

dehydroryanodine. Hydrogenolysis of those derivatives in which Z is C(H)CH₃ yields the corresponding compounds

wherein R¹ is H (the unsubstituted amino acid esters).

Compounds in which R¹ is other than benzyloxycarbonyl and n is 2 (the β-alanyl derivatives) are also prepared by direct acylation using the appropriately substituted β-alanine acylating reagent. However, those compounds in which n is 1 or 3 and R¹ is other than CBZ, are not readily prepared by direct acylation but rather by acylation of the corresponding amino acid derivative, the glycyl or γ-butyryl ester

derivative, obtained by hydrogenolysis of the corresponding CBZ ryanodine derivative, with the appropriate acylating agent.

In addition, benzyloxycarbonyl-β-alanyl-ryanodine (n=2) and its hydrogenolysis product, β-alanyl-ryanodine, likewise are useful in the preparation of compounds represented by the above formula in which Z is CH(CH₃) and in which n = 2.

Although, as mentioned above, such derivatives in which n=2 can be made by direct acylation of ryanodine, the

hydrogenolysis route by way of β-alanyl-ryanodine is

preferred when the preparation of such derivatives involves the incorporation of a radioactive or photo-activatable label.

The hydrogenolysis of O_10eq-CBZ-β-alanyl-ryanodine

(Figure 3) is complex. Hydrogenolysis with a Pd catalyst under acidic conditions yields the anhydro derivative,

O_10eq-β-alanyl-anhydro-ryanodinehydrochloride (V) which converts over time to the desired

10_10eq-β-alanyl-ryanodine (V-II). Surprisingly, however, if the hydrogenolysis is carried out in the presence of base, triethyl amine for example, the hydrogenolysis proceeds smoothly in good yield to give O_10eq-β-alanyl-ryanodine

(V-II) directly.

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)

This invention is further illustrated by the following specific examples.

EXAMPLE 1

Preparation of O_10eq-[(N'Carbobenzyloxy)- Glycyl]-Ryanodine(CBZ-glycyl-Ry) (I)

Dicyclohexylcarbodiimide (DCC, 154 mg . , 0.75 mmol) was added at once to a 10 ml magnetically stirred solution of ryanodine (246.5 mg, 0.5 mmol), CBZ-glycine

(Carbobenzyloxyglycine) (136 mg., 0.6 mmol), and

dimethylaminopyridine (DMAP, 2.3 mg. 0.075 mmol, each dried in a vacuum desiccator over P₂O₅) in metnylenechloride

(dried 24 hours over Molecular Sieve).

After 30 min. the solution became turbid due to

crystallization of dicyclohexylurea. An additional amount (40 mg.) of DCC was added and stirring continued for 3 hours. The solids (132 mg.) were filtered by gravity, washe with methylenechloride and discarded. The combined methylen chloride solutions (50 ml.) were washed first with 5 ml of iced 2N HCl solution to remove DMAP, then with 5 ml of iced 5% NaHCO₃ solution, filtered by gravity, and finally dried with anhydrous sodium sulfate.

Thin layer chromatography (TLC) using Silica Gel-backed plates (Merck Silica GEL 60, F-254 with 254 nm fluorescent indicator) with a chloroform: methanol: 40% aqueous

methylamine solution (85:15:2, system A) revealed a major product (Rf = 0.52) with traces of ryanodine (Rf = 0.28) and of DMAP (Rf = 0.6).

The dried methylenechloride solution (containing

CBZ-glycyl-Ry, I) was concentrated under reduced pressure to a volume of 2 ml., applied to a chromatography column (inner diameter 9mm) containing 24 g. Silica Gel (100-120 mesh). Elution proceeded first with chloroform, then with

chloroform: methanol: 40%-methylamine (98:2:0.1) and, finall a 96:4:0.1 mixture of the same solvents. Fractions eluted with the latter system containing CBZ-gly-Ry were combined, the solvents removed under reduced pressure and the residue was taken up in 5 ml. of recently distilled dioxane. Lyophilization of the dioxane solution yielded the amorphous product (120 mg) which, upon digestion with n-pentane, yielded 90 mg. (26% of theory) of crystalline product (m.p. 182-184), Rf system A 0.52.

High Performance Liquid Chromatography (HPLC) was performed using a Waters C.g-microbondapak (4μ) Radial Pak liquid chromatography cartridge. The mobile phase used consisted of methanol-water mixtures in the following gradient protocol: 0-10 minutes 60% MeOH; 10-20 minutes 60-80% MeOH linear gradient; 20-30 minutes 80% MeOH.

(Gradient Protocol A) Retention time CBZ-gly-Ry; 17.9 min.

Melting Point, 182-184°C.

TLC Rf system A 0.52; HPLC retention time 17.9 min.

Mol. wt. found (HRMS by FAB, glycerol) :M⁺+Na⁺ =

707.3; M⁺+NH₄ ⁺ = 702.3. Calcd. for

C₃₅H₄₄N₂O₁₂:684.

U.V. λ=272, ε₂₇₂=15,000

I.R.(CHCl₃): 3700-3100 (C-OH groups, pyrrole-NE and

-OOCNH-), 1800-1600 shoulder at 1750 (esters- and acarbamate-C=0), 1520(pyrrole) cm

¹ H. NMR (CDCl₃); 7.25-7.35 (5 aromatic phenyl

hydrogens), 6.95, 6.90, and.6.25 (three doublets for pyrrole hydrogens), 5.40 (doublet, 10-H), 5.3 (s-3-H), 514 (s, phenyl-CH₂-O-), 2.50(d,-N-CH₂COO-), 2.33 (m, C₁₃-H),

0.84 (s,C₂₀-H₃).

**) Note: Most of the NMR bands in the spectrum of the ryanodine moiety of CBZ-Gly-Ry conform to those of ryanodine with one clear exception: the C₁₀-H band, which is present in ryanodine at 3.94 ppm, in the CBZ-Gly-Ry spectrum due to C₁₀-ester formation has shifted down-field and now appears in the CBZ-Gly-Ry Spectrum at 5.40 ppm. EXAMPLE 2

Preparation of O_10eq-(N-Benzoyl-β-alanyl)- Dehydroryanodine (II)

Following the above procedure, 100 mg. (0.2 mmol) of dehydroryanodine were reacted with 48 mg. (0.25 mmol) of

N-benzoyl-β-alanine in the presence of DMAP and DCC to yiel the compound of the title. The compound was isolated and purified also by the procedure of Example 1. 17 mg of the desired product were obtained having a TLC (system A) Rf 0.4 HPLC retention time (60% CH₃OH) 11.2 min. with shoulder at 12.4 min. Preparative HPLC purification using a Waters C₁₈microbondapak (10μ) Sep-Pak semipreparative liquid chromatography cartridge was done with 60% methanol.

Fractions containing II were combined, methanol was

evaporated at temperatures below 50°C under reduced pressure and the aqueous solution freeze-dried to give 6 mg. of amorphous product (II) having the following characteristics:

TLC (system A) Rf = 0.42 HPLC (gradient protocol A) retention time 9.9 min. ***(New guard column)***.

IR(CHCl₃): 3650-3150 (C-OH groups, pyrrole-NH, and

-CONH-), 1550-1650(esters- and-.amide-C=0),

1520(pyrrole ring)cm^-1.

EXAMPLE 3

Preparation of p-Iodobenzoyl-β-alanyl-dehydroryanodine (XII) A solution of p-iodo-benzoylchloride (2.4 g, 10 mmol) in anhydrous pyridine (5 ml) was added to a stirred solution of β-alanyl ethyl ester hydrochloride (2 g, 13.5 mmol) in 20 ml of anhydrous pyridine. The reaction mixture was kept at room temperature for 18 hrs. with stirring. Pyridine was

evaporated under reduced pressure and the residue was taken up in ethyl acetate. The ethyl acetate solution was washed first with 1N HCl, then with 1N NaOH solution and dried with anhydrous sodium sulfate. After evaporation of the solvent the residue was triturated with a mixture of pentane-ethyl ether (8:2), yielding 1.1 g. of N-(p-iodobenzoyl)-β-alanine ethyl ester, m.p. 94-96°C.

This ester was hydrolyzed for 18 hours in a stirred mixture of IN NaOH (12 ml) and ethanol (6 ml). Removal of the ethanol was followed by acidification with 6N HCl to pH=3-4 and extraction with ethyl acetate. Evaporation to small volume and cooling yielded 0.685 g. of the acid,

N-(p-iodobenzoyl)-β-alanine, m.p. 166-168°C.

Dehydroryanodine (100 mg, 0.2 mmol),

N-(p-iodobenzoyl)-β-alanine (50 mg, 0.22 mmol) and DMAP (2 mg, 0.02 mmol), all dried over P₂O₅, were dissolved in a solvent mixture of CH₂Cl₂ (10 ml) and tetrahydrofuran (0.1 ml) dried over Molecular Sieve. To the stirred

solution, 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 CH₂Cl₂.

The filtrate was evaporated under reduced pressure to small volume, the residue taken up in CHCl₃ and again

evaporated to small volume. Crystals of dicyclohexylurea were filtered off, the filtrate was concentrated to a volume of 0.5-1 ml., and applied to the top of a column (6 mm inner diameter) containing 12 g. of Silica Gel suspended in

CHCl₃. Elution of the product proceeded first with CHCl₃ (50 ml), then with mixtures (50 ml) of chloroform, methanol, and aq. 40% methyl amine (98:2:0.2, 96:4:0.4, and finally 94:6:0.6). Fractions eluted with the 96:4:0.4 mixture containing the product (XII) were combined and the solvents removed under reduced pressure. The semi-solid residue was triturated with pentane-ethyl ether (8:2) and allowed to stand at room temperature for 4 hours. The white,

crystalline product (XII), 12 mg, melts >220°C dec. TLC Rf (system A) = 0.47. HPLC (60-80% CH₃OH)

retention time = 20.15 minutes.

Calculated for C₃₅H₄₁IN₂O₁₁ Mol. Wt. 792. HRMS,

FAB, glycerol, LiI:799. IC_5Q(nM) = 16.0. K_D(nM) = 5.2.

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.

Conversion of the derivative XII - and other suitable iodinated Ryanodine-ligand derivatives - to the radio-active species is in progress using the radio-iodo-destannylation method [Blaszczak, L.C., Halligan, N.G., and Seitz, D.E.

J. Labelled Compds, Radiopharm., 27, 401 (1989); see also Mais, D.E., et al. ibid. 29, 75-79 (1991) and J. Med. Chem. 34, 1511 (1991)].

EXAMPLE 4

O_10eq-[N-(p-n-Butoxybenzoyl)-β-Alanyl]-Dehydroryanodine

(XIII)

A solution of p-n-butoxybenzoylchloride (4.24 g., 20 mmol) in 5 ml of anhydrous pyridine was added to a stirred solution of β-alanine ethyl ester hydrochloride (4.6 g., 30 mmol) in 25 ml of anhydrous pyridine. The reaction mixture was kept at room temperature for 18 hours with stirring.

Pyridine was evaporated under reduced pressure, and the residue was taken up in ethyl acetate. The ethyl acetate solution was washed first with 1N HCl and then with 1N NaOH solution and dried with anhydrous sodium sulfate. After evaporation of the solvent the residue was triturated with a mixture of pentane and ethyl ether (9:1) yielding 4 g., of the white, crystalline product

N-(p-n-butoxybenzoyl)-β-alanine ethyl ester, m.p. 86-88°C. This ester was hydrolyzed for 18 hours in a stirred mixture of 1N sodium hydroxide (15-ml.) and ethanol (10 ml.). Removal of the ethanol, was followed by acidification of the aqueous solution with 6N HCl to pH = 2-4 and

extraction with ethyl acetate yielded the acid

N-(p-n-butoxybenzoyl)-β-alanine,

1.8 g., mp. 144-146°C.

Dehydro-ryanodine (60 mg., 0.125 mmol),

N-(p-n-butoxybenzoyl)-β-alanine (40 mg. 0.15 mmol) and DMAP (2 mg., 0.02 mmol), dried over P₂O₅, were dissolved in a solvent mixture of CH₂Cl₂(10 ml.) and tetrahydofuran (0.1 ml) dried over Molecular Sieve. To the stirred solution dicyclohexylcarbodiimide (35 mg., 0.15 mmol) was added at once and the reaction mixture maintained at room temperature for 6 hours. Water (0.1 ml) was added and stirring continued for 30 minutes. The solids formed (dicyclohexylurea) were filtered and washed twice with CH₂Cl₂.

The combined filtrates were evaporated under reduced pressure to small volume, the residue taken up in CHCl₃ and again evaporated to small volume. Crystalls of

dicyclohexylurea were filtered off and the filtrate

concentrated to a volume of 1-2 ml.

This concentrated solution was applied to the top of a column (6 mm. inner diameter) containing 12 g. of silica gel suspended in chloroform. Elution proceeded first with chloroform (50 ml.), then with a mixture of

chloroform/methanol, and 4% aq. methylamine (98:2:0.1); then this mixture (96:4:0.2). Fractions eluted with the latter mixture containing the product (XIII) were combined and the solvents removed under reduced pressure. The semi-solid residue was triturated with a pentane-ethyl ether mixture (9:1) and allowed to stand at room temperature. A white crystalline product (XIII) was obtained, 4 mg ., mp.p.

210-220°C. (with dec). TLC Rf(system A) = 0.56. HPLC

(60-80% CH₃OH) retention time = 24.0 min. Calculated for C_3gH₅₀N₂O₁₂ Mol. Wt: 7?8, Mass spec.

HRMS, FAB, glycerol with Lil: 745; - IC₅₀(nM)=20.2;

KD(nM)=6.0

EXAMPLE 5

Preparation of

O_10eq- [N-(1-Adamantanecarbonyl)-β-Alanyl]

-Dehydroryanodine (III)

A. N-1-(Adamantanecarbonyl)β-alanine was prepared from

1-adamantanecarbonyl-chloride and β-alanine under modified Schotten-Bauman conditions as follows: A solution

1-adamantanecarbonylchloride (10 g., 0.05 mmol) in ether (5 ml.) was added to a vigorously stirred solution of β-alanine sodium salt (6.25 g., 0.055 mmol) and NaHCO₃ (5 g., 0.055 mmol) in 10 ml. of water. Stirring was continued for 18 hrs. The solution was acidified with iced concentrated hydrochloric acid, allowed to stand at room temperature for 1 hr. The precipitated solids - a mixture of

1-adamantanecarboxylic acid and the desired

1-adamantanecarbonyl-β-alanine - were filtered, dried in a vacuum desiccator over solid KOH, and then thoroughly

digested with ether to remove 1-Adamantanecarboxylic acid.

The remaining crystalline material was recrystallized from a chloroform-ether mixture (5:1). to yield 1.2 g. of

N-(1-Adamantanecarbonyl)-β-alanine, m.p. 180-182°C having the following characteristics:

TLC using Silica Gel plates without fluorescent indicator

Rf = 0.8, using a chloroforiτr.methanol:acetic acid mixture

(85:15:3) and chlorine vapors for detection.

I.R. (CHCl₃):3440(-CONH-), 1720 (-COOH), and

1605 (-CONH-) cm^-1.

H-NMR(dco-DMSO); 7.0(q. -CONH), 3.05-2.75 and

2.1-1.8(2 m, -CH₂CH₂-) 1.6(s), 1.4(d), and

1.3 (s)(adamanty1-9H). B. Dehydroryanodine (100 mg., 0.2 mmole),

1-adamantanecarbonyl-β-alanine (61-mg., 0.25 mmole) and 2 mg. of DMAP, each dried ov P₂O₅, ^were dissolved in 10 ml. of dried CH₂Cl₂. To the stirred solution was added 60 mg. (0.3 mmol) of DCC at once. Crystalls of dicyclohexylure appeared within ten minutes. After three hours the solids were filtered by gravity, washed with CH₂Cl₂; the

combined oganic layers were washed with IN HCl, then with 5% NaHCO₃, and dried with anhydrous Na₂SO₄. The solvent was removed under reduced pressure to small volume (0.5 ml.) and applied to the top of a chromatography column ( internal diameter 6 mm., 6 g. of Silica Gel, 100-200 Mesh). Elution was effected with first chloroform, then

chloroform:methanol:40 aq. methylamine (98:2:0.1). Fractions from the latter elution which contained the desired product (III) were combined and the solvents removed under reduced pressure. Lyophilization of the residue from recently distilled dioxane gave 32.5 mg. of amorphous product (III).

TLC Rf (system A) 0.49. HPLC (gradient protocol A) retention time 23.1 min.

O_{10 eq}-β-alanyl-ryanodine (V-II) as obtained by

hydrogenolysis of O_10eq-carbobenzyloxy-β-alanyl-ryanodine (IV), and its (V-II) subsequent acylation to

O_{10 eq}-5-azido-2-nitrobenzoyl-β-alanyl-ryanodine and its

(V-II) acylation to other molecular probes (XIV, XV, and XVI) is described below.

EXAMPLE 6

Preparation of O_10eq.-Carbobenzyloxy-β-alanyl-ryanodine. (IV)

Ryanodine (150 mg., 0.3 mmol), CBZ-β-alanine(82.5mg., 0.36 mmol), both dried by P₂O₅, and DMAP(2 m9.) were dissolved in CH₂Cl₂ (20 ml.) dried-with Molecular Sieve.

To the stirred solution dicyclohexylcarbodiimide(103 mg., 0.5 mmol) was added at once.

After 3 hrs. the product was obtained as described in Example 1, column chromatography (18 g. Silica Gel) using CHCl₃ and CHCl₃:CH₃OH:40% CH₃NH₂ mixture

(98:2:0.2). Fractions containing IV were combined and solvents removed under reduced pressure. The residue yielded a crystalline product, IV, (44 mg.):m.p. - 178-180°C. TLC (system A) Rf = 0.57. Analytical HPLC (gradient protocol A) retention time 19.2 min. U.V. λ max. = 272 nm., ε₂₇₂ =

14,450. Mol. Formula Calcd. C₃₆H₄₆N₂O₁₂; (mol. wt.

698).

EXAMPLE 7

Alternate Preparation of O_10eq-CBZ-β-Alanyl-Ryanodine (IV)

Ryanodine (300 mg., 0.6 mmol), CBZ-β-alanine (160 mg., 0.7 mmol) and DMAP (7 mg., 0.07 mmol), dried over P₂O₅, were dissolved in a magnetically stirred mixture of

CH₂Cl₂ (20 ml.) and tetrahydrofuran (0.2 ml.) dried with a Molecular Sieve. To the stirred solution

dicyclohexylcarbodiimide (180 mg., 0.9 mmol) was added at once and the reaction was maintained at room temperature for 18 hrs.

Water (0.25 ml.) was added and stirring continued for 30 minutes. The solids formed (dicyclohexylurea) were filtered and washed twice with CH₂Cl₂. The filtrate was

evaporated under reduced pressure to small volume, the residue taken up in CHCl₃ and again evaporated to small volume. Crystals of dicyclohexyl urea were filtered off, washed with CHCl₃, and the filtrate again concentrated to small volume (2 - 3 ml.).

This concentrated solution was applied to the top of a column (9 mm. inner diameter) containing 24 g. of Silica Gel in chloroform. Elution first proceeded with CHCl₃

(100 ml.), then with a mixture of CHCl₃, CH₃OH, and 40% aqu. methylamine solution (98:2:0.1), then this mixture

(96:4:0.2), and finally (94:6:0.3). Fractions eluted with the (96:4:0.2) mixture containing the product (IV) were combined and the solvents removed under reduced pressure.

The residue was redissolved in chloroform and the solvent removed under reduced pressure. The residue was triturated with a pentane:ethyl ether mixture (10:1, 10 ml.) and allowed to stand at 4°C. The white, crystalline product (IV),

150 mg. (36% of theoretical yield), melted at 178-180°C.

TLC (system A) Rf=0.57. Analytical HPLC (60-80% CH₃OH) retention time 19.2 min. U.V., λ_max = 272nm., ε₂₇₂ =

14,450.

Mol. Formula. Calcd. C₃₆H₄₆N₂O₁₂; (mol.wt. 698).

Mol.wt. (HRMS, FAB, Glycerol with Lil): M⁺+Li⁺ = 705.3.

1 H nmr(CD₃OD, δppm): 7.32(m, 5H,phenyl-aromatic

protons), 7.06, 6.87, 6.23(three double doublets for pyrrole hydrogens), 5.58(s, 1H, H-C10), 5.07(d, 2H, Ar-CH₂-O),

3.45(t, 2H, -NHCH₂-), 2.59(m, H-Cl3), 1.40(s, CH₃-Cl),

1.11(d, 3H, CH₃-Cl3), 0.90(s, 3H, CH₃-C5), 0.82(d, 3H,

CH₃-C9), and 0.74(d, 3H, CH₃-Cl3)

The above column chromatography fractions eluted with the (94:6:0.3) mixture containing unreacted ryanodine were collected. Solvent removal, followed by re-dissolving the residue in water, and freeze-drying, recovered 35 mg. (7%) of amorphous ryanodine.

EXAMPLE 8

O_10eq-β-Alanyl-Anhydroryanodine Hydrochloride (V),

O_10eq-β-Alanyl-Anhydroryanodine (V-1) and

O_10eq-β-Alanyl-Ryanodine (V-II)

Palladium on charcoal (10%, 15 mg.) was added to a

solution of O₁₀-CBZ-β-alanyl-ryanodine (IV, 285 mg., 0.42 mmol) in 50 ml. of ethanol containing 5 ml. (0.5 mmol) of aqueous 0.1 N HCl. The compound (IV) was hydrogenolysed under a hydrogen pressure of 50 lbs/m 2 with continuous shaking for 3 hrs. The catalyst was filtered, ethanol was removed under reduced pressure and the residue diluted with

5 ml. of water. The clear aqueous solution was freeze-drie to yield 190 mg. of the light-tan hydrochloride (V).

TLC of V showed a single spot (system A) Rf = 0.4. TLC

(CHCl₃:CH₃OH, 85:15, system B) Rf = 0.05. U.V.

λ_max 272 nm; ε ₂₇₂= 14,500.

Mol. Formula Calcd. C₂₈H₃₈N₂O₉.HCl

(Mol. wt. 546(+ 36.5).

Mass spectrum was run after three to four weeks of storage of the product at 4°C. At this time, a second product (V-II), Rf = 0.22 (Rf = 0.025 in system B) was present. The mass spectrum (FAB, Glycerol, with Lil) of this mixture (V, V-II) showed a peak M⁺+Li⁺ = 571.3(564 + 7) and a peak at 553.3(546 + 7). Ryanodine itself when assayed (Li-technique) shows no dehydration (M⁺ - 18) peak in the mass spectrum. A small peak is seen also at 705.3,

corresponding to a residue of the precursor

CZB-β-alanyl-ryanodine (Mol. Wt. 698).

The mixture of V and V-II hydrochlorides was separated by prior conversion to free-base (Na₂CO₃, CHCl₃) and

chromatography on SILICAR using CHCl₃, and CHCl₃:CH₃OH

(2, 4 and 6%) with 5, 10, and 15 drops of Et₃N,

respectively. Evaporation of fractions containing

O_10eq-β-alanyl-anhydroryanodine (V-I, free base) and those containing O_10eq-β-alanylryanodine (v-n, free base), and trituration of the respective residues after evaporation of solvents with a pentane-ether mixture (9:1) gave the

respective crystalline products V-I, Rf = 0.4 and V-II, Rf = 0.22, System A. Rf V-II 0.025 in System B. IC₅₀(nM) = 4.2

Mass spec, the anhydro-product (V-I free base): HRMS (FAB. Lil) gave a peak M⁺+Li⁺ at 553.2 (Calc. for

C₂₈H₃₈N₂O₉ = 546). V-I: ¹H nmr(CD₃OD, δ ppm); 7.06, 6.87, 6.26 (three double doublets for pyrrole hydrogens), 6.15(q, 1H, HC3), 5.63(d, 1H, HCl0), 3.42(d, H_fa) and 2.59(d, H_a) (AB

pattern, H₂Cl4), 2.71(m, 1H, HCl3), 3.07(t, 2H,

-CH₂NH₂), 2.61(t, 2H, -CH₂NH₂),

2.61(t, 2H, -CH₂CO-), 2.05(m, 1H, EC9), 1.82(d, CH₃-Cl), 1.1(d, 3H, CH₃-Cl3), 0.99(d, 3H, CH₃-C9),

0.96(s, 3H, CH₃-C5), 0.91(d, 3H, CH₃-Cl3).

Mass. spec (M⁺+Li⁺) of V-II, free base 571.3(564 +

7). Calculated for C₂₈H₄₀N₂O₁₀ = 564.

V-II: ¹H-nmr (CD₃OD, δ ppm); 7.03, 6.87, and 6.23

(three double doublets for pyrrole hydrogens),

5.58(s, 1H, HC3), 5.4(d, 1H, HCl0), 3.0(t, 2H, -CH₂-NH₂), 2.60(t, 2H, -CH₂CO-), 2.56(d, H_b) and 1.94(d, H_a)

(AB pattern, H₂Cl4), 2.26(m, 1H, HCl3), 2.10(m, 1H, HC9), 1.40(s, 3H, CH₃-Cl), 1.03(d, 3H, CH₃-Cl3),

0.89(S, 3H, CH₃-C5), 0.85(d, 3H, CH-C9), and

0.74(d, 3H, CH₃-Cl3).

EXAMPLE 9

Direct preparation of O_10eq-β-Alanyl-Ryanodine (V-II)

To a solution of CBZ-β-alanyl-ryanodine (IV, 112 mg.,

0.16 mmol) in ethanol (25 ml.) containing 25 mg (0.25 mmol) of triethylamine was added 35 mg of 10% Palladium-on-Carbon.

The mixture was shaken under 40 lbs/in² of hydrogen pressure for one hour. Filtration to remove the catalyst and evaporation of the solvent under reduced pressure, yielded a residue which crystallized by trituration with a

pentane-ethyl ether mixture (9:1); yield 37 mg. A second crop (7 mg.) was obtained by evaporation of the filtrate and freeze-drying the residue in dioxane. The product thus obtained consisted mainly of β-alanyl-Ry (V-II), some

ryanodine (2-3%), and a small amount (1-2%) of

β-alanyl-anhydro-ryanodine (V-I). Chromatography over SILICAR gel (20 g.) of 42 mg. of the above product using successively,. CHCl₃-CH₃OH(98-2), then CHCl₃-CH₃OH-Et₃N(98-2-0.2), (96-4-0.4), (94-6-0.6) and

(92-8-0.8) yielded 29 mg. of purified crystalline

β-alanyl-ryanodine (V-II), m.p. = 220-230°C. (dec).

TLC: Rf (system A) = 0.22 - 0.24; Rf (System B) = 0.025. HPLC (system B) retention time 8.8 min.

That the conversion of the anhydro-derivative (V) to the ryanodine derivative (V-II) would occur readily and

spontaneously upon storage at refrigerator temperatures was unexpected in view of the difficult experience encountered in the conversion of synthetic anhydro-ryanodol to ryanodol in the classic work of Deslongchamps, Ruest et al. on "The total synthesis of (+)-ryanodol" Parts I, II, III and IV

(Can. J.Chem., 68, 115, 127, 153 and 187, 1990).

On the basis of the reported difficulty and obstacles encountered in the conversion of anhydro-ryanodol to ryanodol and in the conversion of anhydro-ryanodine to ryanodine by Professor Deslongchamps and co-workers in the above quoted publication Part IV, the facile, spontaneous conversion of β-alanyl-anhydro-ryanodine HCl (V) to β-alanyl-ryanodine (V-II) is most surprising indeed. The common identity of V-II as obtained by

a) the spontaneous conversion of β-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

hydrogenolysis of CBZ-β-alanyl-Ry (IV) in the presence of triethylamine

is evidenced by their identical behavior (a) on TLC(Rf =0.22 system A), (b) on HPLC (retention time 8.8 min., (50%

CH₃ /H₂O, 1% Et₃N), (c) in the N.M.R. spectrum, and - most significantly - (d) in the Receptor Binding Assay

[IC₅₀(nM) = 4.2 and 4.3, respectively].

As stated above, compounds according to Figure 2 above in which n is 1 or 3 and R¹ is other than carbobenzyloxy or adamantyloxycarbonyl cannot be made in any satisfactory yield by a direct acylation of ryanodine or dehydroryanodine with the required esterifying moiety. These compounds, however, as well as the directly accessible derivatives in which n = 2 when such are desired, carrying a photo- or radio-label, are readily accessible by direct acylation of the hydrogenolysis products O_10eq-glycyl-, O_10eq-Υ-aminobutyryl-, and

β-alanyl-ryanodine, respectively.

To illustrate this procedure, utilization of

O_10eq-β-alanyl-ryanodine (V-II), as obtained by

hydrogenolysis of Q_{10 eq}-carbobenzyloxy-β-alanyl-ryanodine (IV), in acylation to provide

O_10eq-5-azido-2-nitrobenzoyl-β-alanyl-ryanodine (VI

Example 10) and three other molecular probes XIV (Example 16), XV (Example 11) and XIV (Example 12), is described below. EXAMPLE 10

Preparation of O_10eq-[N-(5-Azido-2-nitrobenzoyl)-β-Alanyl]

Ryanodine (VI)

a) From β-Alanyl-Ryanodine (V-II)

5-Azido-2-nitrobenzoyloxysuccinimide (ANB-NOS, Pierce Chem. Co., 27 mg., 0.09 mmol) was added to a stirred solution of β-alanyl-ryanodine (V-II, 25 mg., 0.045 mmol) in freshly distilled dioxane (10 ml.) containing triethylamine (16 mg., 0.16 mmol). This and all subsequent operations were

performed with exclusion of direct light. After 20 hrs. the clear, light-yellow solution was evaporated to dryness using a rotary evaporator. The residue was taken up in chloroform (1-2 ml.), and applied to the top of a chromatography column (internal diam. 15 mm.) containing SILICAR absorbent (20 g.) in chloroform. The product was eluted first with CHCl₃

(75 ml.) and followed by CHCl₃/CH₃OH/40% aqu. CH₃NH₂

mixtures: (98/2/0.2, 96/4/0.4, and 94/6/0.6). Fractions containing the desired product (VI) were combined and the solution evaporated to dryness under reduced pressure. The residue was dissolved in dioxane (5 ml.) and lyophilyzed to give the pale-yellow, photo-activatable product (VI), 10 mg.

TLC (System A) Rf = 0.44. On the TLC plate the single spot of V-I turns yellow on exposure to U.V. light.

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,

λmax=320 nm) in a 1:1.05 molar ratio.

I.R. (CHCl₃): 3650(C-OH, CONH-, pyrrole-NH),

2100(-NO₂), 2750(ester- and amide-C=O), 1518(aromatic pyrolle ring) cm- ¹.

Calcd. for C₃₅H₄₂N₆O₁₃ Mol. Wt.: 754. Found

HMRS(FAB, glycerol, Lil): 761. IC₅₀(nM) = 36.6 ± 2.8: K_D(nM) - 12.5 ± 1.0. b) From β-Alanyl-anhydro-ryanodine Hydrochloride (V)

N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS Pierce Chem. Co.) 6 mg., 0.02 mmol was added to a stirred solution of β-alanyl-ryanodine HCl (V) 10 mg., 0.18 mmol in 10 ml of freshly distilled dioxan and triethylamine (4 mg., 0.04 mmol). This operation and all following steps were done essentially in the dark. After 18 hrs., the clear,

light-brown solution was freeze-dried and the residue taken up in CHCl₃. The organic layer was washed with ice-cold 1N HCl, with 5% NaHCO₃ solution and dried over anhydrous

Na₂SO₄. The chloroform solution was reduced to a small volume (0.4 ml.) and 4-6 ml of n-pentane were added. The resulting precipiate was gravity-filtered to yield a pale yellow photo-activatable product (VI, 9 mg.).

TLC (System A) Rf = 0.46. The spot for VI turns yellow on the TLC plate upon exposure to UV light. HPLC (gradient protocol A) retention time 12.3 min. The UV spectrum of VI in MeOH solution in equimolar concentrations shows the respective maxima at 272 and 320 nm. of its two chromophoric moieties (ryanodine λ_max = 272; 5-azido-2-nitrobenzoic acid λ_max = 320 nm.)

I.R. (CHCL₃: 3650-3250 (C-OH groups; -CONH-,

pyrrole-NH), 2100 (N₃), 2750 (esters- and amide C=O), 1518 (pyrrole ring) cm ^-1

The product (VI) obtained (a) from β-alanyl-ryanodine

(V-II, free base), was in all respects identical to the product (VI) prepared (b) by the same, base-assisted

procedure from β-alanyl-anhydro-ryanodine HCl (V). TLC

(system A) Rf = 0.46 and 0.44, respectively. When run together on the same plate the two samples of VI in this system ran with identical Rf = 0.47.

The product (VI), prepared from V, in binding experiments to the ryanodine receptor revealed an affinity:

IC₅₀(nM) = 37.2 + 9.7; K_D = 14.0 ± 3.6

This result compares favorably with the analogous values determined above for this product (VI) prepared from

β-alanyl Ryanodine (V-II), namely:

IC₅₀(nM) = 36.6 ± 2.8; K_D(50) = 12.5 ± 1.0.

It is interesting to note that compound VI prepared from the anhydroryanodine-derivative (V) is the same as VI

prepared from the ryanodine derivative (V-II). This finding is a second example of the conversion of an anhydro-ryanodine species to the corresponding ryanodine-derivative under the influence of mild base. In the first example - the

conversion of β-alanyl-anhydro-ryanodine hydrochloride (V) to β-alanyl-ryanodine (V-II) - this mild base function presumably is the primary arnine of the C₁₀-β-alanyl side chain, while in the above preparation of the azido-nitro product (VI) from β-alanyl-anhydro-ryanodine HCl (V), triethylamine is present to act as a mild organic base.

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

determination is a satisfactory binding affinity or

comparable biological quality of the photo-generation label, here compound VI. The demonstrated binding affinity of the azido-derivative (VI) is of a similar magnitude as that of ryanodine which fact further accentuates the usefullness of this derivative. The use of such photo-affinity labels in a related field╌with the anticancer alkaloid, vinblastine╌is described in papers by Nasioulas et al, CAN. RES. 50 403 (1990) and by Gambitter et al,

CAN. RES. & CLIN. ONCOL. 109 Abs. Bio 06 (1985). See also, a chapter by Hagan Bayley "Photoregenerated Reagents in

Biochemistry and Molecular Biology" appearing in LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY. Ed. Work and Burdon (Elsevier Amsterdam, New York and Oxford, 1983) for other types of labels that have been used with molecules of biological interest.

EXAMPLE 11

Preparation of O_10eq-N-BODIPY(FL C3)-β-Alanyl- Ryanodine(XV) β-Alanyl-ryanodine (V-II, 5.6 mg, 0.01 mmol) dissolved in anhydrous dioxane (0.2 ml) was added to a magnetically stirred solution of

4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenepropionic acid N-hydroxysuccinimidyl ester (BODIPY FL-C3,

MOLECULAR PROBES INC., 5 mg. 0.013 mmol) in anhydrous dioxane (0.8 ml) containing triethylamine (2 mg, 0.02 mmol). The reaction was maintained at room temperature for 16 hrs. and the solvent was then removed by freeze-drying. The semi-solid residue was taken up in CHCl₃ and the highly fluorescent solution was filtered. The residue obtained on evaporation of the solvent was applied to the top of a chromatography column (0.6 mm inner diameter) containing 12 g. absorbent (SILICAR) in CHCl₃. The product was eluted with 50 ml portions of CHCl₃/CH₃-OH, then with successive CHCl₃/CH₃)OH/ 40% aq. CH₃NH₂ amine mixtures

98:2:0.2, 96:4:0.4, respectively. Fractions of the latter two mixtures containing the product (XV) were combined and evaporated under reduced pressure to give a brown-red, semi-solid residue. Trituration with pentane-ethyl ether mixture (8:2) gave a red-brown crystalline product (2 mg). TLC revealed a major, single, fluorescent product, Rf (system A) = 0.56. The ultraviolet/visible light absorption measured in methanol revealed maxima of the ryanodine-moiety (ε=8.000 at λ = 272 nM) and the BODIPY moiety (ε=72.000 at

λ=530nM), respectively, in a molar ratio of 1:1.2.

Calcd. for C H BF₂N₄O₁₁Mol Wt . 838. HRMS

(FAB, glycerol, LiI): 845.

IC₅₀(nM)=41.2 ± 2.7; K_D(nM) = 15.8 ± 1.0

The above BODIPY Ryanodine fluorescent agent (XV) is useful in localization by microscopy of tissue ryanodine binding sites.

EXAMPLE 12

Preparation of O_10eq-N-(7-Amino-4-methylcoumarin-3-acetyl)

-β-Alanyl-Ryanodine (XVI)

This fluorescent ryanodine derivative is prepared in a manner analogous to the preparation of the BODIPY-derivative (xv) - EXAMPLE 11 - from β-alanyl-ryanodine (V) and the

N-Hydroxy-succinimidyl 7-amino-4-methylcoumarin-3-acetate reagent (AMCA-NHS, Pierce Chemical Co.) in dioxane solution. The desired product was present in the reaction mixture, but on the small scale of the reaction employed the presence of several by-products rendered the isolation of pure XVI impractical. TLC system A: Rf = 0.71; system B Rf = 0.18.

Calcd. for C₄₀H₄₉N₃O₁₃Mol Wt 779.

EXAMPLE 13

Preparation of Dehydroryanodine-O_10eq-Hemi-Succinate (XVII)

To a stirred solution of dehydroryanodine (250 mg., 0.5 mmol) and dimethylaminopyridine (DMAP, 180 mg., 1.5 mmol) in 10 ml. of tetrahydrofuran (dried over Molecular Sieve) succinic anhydride (150 mg., 1.5 mmol) was added and stirring continued for 3 hrs. A further 50 mg. of succinic anhydride was added at this time. After 24 hrs. water (0.5 ml.) was added and the solution stirred for 1/2 hr. Tetrahydrofuran was removed by distillation under reduced pressure at a water bath temperature not exceeding 45°C.

The remaining residue was dissolved in water (5 ml.), redistilled triethylamine (0.5 ml.) was added and the aqueous layer then extracted with chloroform (three portions of 5 ml.) to remove DMAP.

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 effluent aqueous solution, pH 2.8, containing the product (XVII), 8,000 Opt. Density units), was lyophilyzed to give 300 mg . of (XVII) still containing succinic acid.

TLC analysis (system A) shows the presence of E (Rf 0.1) with a small amount of an un-identified by-product Rf = 0.15. Preparative HPLC using a Waters 10μ C₁₈-Bondaρak

Prep-Pak semi-preparative column and elution with 50% methanol provided fractions of purified succinate (XVII). Removal of methanol by distillation under red. pressure, freeze-drying of the remaining aqueous solution gave 30 mg . of amorphous product (XVII).

TLC (system A) Rf = 0.1. HPLC (CH₃OH:H₂O: acetic

acid; 50:50:0.5, system C) retention time = 11.4 min.

1H-NMR and mass. spec, data are available and confirm the structure of XVII as dehydoryanodine-O_10eq-hemisuccinate. As in CBZ-glycyl-ryanodine the HC₁₀-OH peak in the NMR spectrum in CD₃OD) has moved from its position in

dehydroryanodine at 5.04(m, 10-H_ax) downfield to 5.92.

U.V. and I.R. spectra compatible with structure XVII Mol. Formula: C₂₉H₃₇NO₁₂, Mol. Wt. 591; Hrms

(M⁺+Na⁺) = 614.

EXAMPLE 14

Preparation of N-Methyl-Dehydroryanodine-Succinamate (XVIII)

To a magnetically stirred solution of purified

dehydroryanodine hemisuccinate (XVII) from Example 11 (30 mg., 0.05mmol) in water was added methyl amine hydrochloride (6.7.mg., 0.2 mmol) in 2 ml of water,

1-(3-dimethylaminopropyl)- 3-ethyl-carbodiimide hydrochloride (40 mg., 0.2 mmol) and N-hydroxysulfosuccinimide, Na salt (Pierce Chemical Co.)

(11 mg., 0.05 mmol). The pH of the stirred solution (3-4) was adjusted dropwise to pH=5-6 with 0.1 N NaOH or 0.1 N HCl, as required. A slight turbidity initially present disappears gradually. After 2 hrs., a further sample of EDC (20 mg.) was added. The final ρH=5.5 (after 18 hrs.). The pH is then adjusted to about 8.5 with 5% NaHCO₃ and the resulting solution extracted with chloroform (3 × 10 ml). The solvent is evaporated; the residue taken up in water and the aqueous solution freeze dried to yield 12 mg. of a white amorphous, powdery product (XVIII).

TLC(system A) R_f = 0.43. HPLC (system C), retention time = 10.6 min.

I.R.(CHCl₃): 3650-3150 (C-OH groups, pyrrole-NH,

-CONH-), esters and amide-C=O) cm ^-1

Mol. Formula: C₃₀H₄₀N₂O₁₁; Mol, Wt . 604.

EXAMPLE 15

AFFINITY CHROMATOGRAPHY REAGENTS

Coupling of Dehydroryanodine O_10eq-Hemisuccinate (XVII) to AH-Sepharose 4B for Use in Affinity Chromatography of

Ryanodine Receptor

The resin (AH-Sepharose 4B) is suspended in a

phosphate-buffered aqueous solution of the hemisuccinate (XVII) containing catalytic amounts of

N-Hydroxysulfosuccinimide sodium salt (S-NHS). A two to three fold excess of water-soluble carbodiimide (EDC) is added to the stirred solution while the pH is maintained at 5-6.

After 18 hrs. the resin is washed extensively with distilled water and collected.

The substitution-rate percentage of the available amino groups covered through amide linkage by succinate (XVII) is determined by base hydrolysis and U.V. analysis at 272 nm. This substitution rate can be expressed as mmoles

dehydroryanodine/mg. of dried resin and was found to be satisfactory for affinity chromatography studies.

In the above example, the procedure of Inui et al, J. Biol. Chem. 262 15637 (1987) was modified by using the ryanodine-linked affinity chromatography described above en lieu of the resin types used by the authors. EXAMPLE 16

Preparation of O_10eq-N-Biotinyl-β-Alanyl-Ryanodine (XIV) β-Alanyl-ryanodine (V-II, 28 mg., 0.05 mmol) dissolved in dry FMF (0.2 ml) was added to a stirred solution of

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

temperature for 18 hrs. DMF was then evaporated under reduced pressure. The residue was treated with chloroform

(3 ml). To the resulting suspension ethyl acetate (1 ml) was added, the suspension centrifuged, and the supernatant was removed. The white solid residue was treated with anhydrous ether (1 ml), centrifuged and the supernatant decanted. The residual powdery white solid (9 mg) was collected.

TLC (CHCl₃:DMF, 75:25) revealed a single product (RF = 0.3). Calcd. for C₃₈H₅₄N₄O₁₂S Mol. Wt. 790.52; found

(HRMS, FAB, glycerol, Lil): 797.4

IC₅₀(nM) = 65.1 ± 3.5; K_D(nM) = 24.9 ± 1.4 The structures of the product of Examples 11, 12 and 16 are given in Figure 4 below.

While the affinity chromatography procedure for the isolation of Ryanodine receptor was illustrated by the use of dehydroryanodine-O_10eq-hemisuccinate (XVII), those skilled in the art will recognize that other compounds coming within the scope of this invention or their biotin-based equivalents would also be operable and even superior in procedures for the isolation of Ryanodine receptor.

In the use of the above Biotinyl-β-alanyl-Ryanodine

(XIV) for recognition, separation, and isolation of the ryanodine receptor by affinity chromatography advantage is taken of the high affinity of the biotin moiety for avidin, e.g. in the form of avidin-linked microspheres.

In case difficulties are encountered in liberating ryanodine-moiety from the Avidin-Biotin-β-alanyl- ryanodine -receptor complex, the use of a cleavable

di-sulfide biotinylating reagent, NHS-SS-Biotin (Pierce

Chemical Company) is envisaged.

PHARMACOLOGIC FINDINGS

The binding affinities of several compounds I_XVIII were determined in a traditional relative binding affinity assay

(Table I). Briefly, skeletal sarcoplasmic reticulum membrane vesicles were incubated in the presence of

6.7nM[³H] ryanodine and increasing concentrations of the various unlabelled derivatives to competitively displace the [³H] ryanodine. The IC₅₀ value for each derivative was

determined from the appropriate displacement curve and

compared to the IC₅₀ values for unlabelled ryanodine and dehydroryanodine.

Two of the compounds in Table 1 have been more

extensively characterized pharmacologically. Both

N'-methyl-dehydroryanodine-succinamate (NMDS, XVIII) and

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. At low concentrations (<μM) ryanodine opens the SR Ca⁺⁺ release channel/ryanodine receptor, permitting an increased efflux of

Ca⁺⁺ from the SR or from junctional SR Ca⁺⁺ vesicles. At higher concentrations of (>μM) ryanodine closes or

inactivates the SR Ca⁺⁺ release channels interdicting

Ca⁺⁺ accumulation by junctional SR vesicles. In contrast, neither XVII (NMDS) nor CBZ-glycyl-ryanodine (I) exhibits significant ability to close the channel at concentrations as high as 300μM. However, both are only slightly less active than ryanodine for opening the channel. These data suggest that the addition of a side chain at the 10_eq-OH confers selective properties on the ryanodine derivatives. In addition, 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).

The base-substituted ryanodine derivative,

β-alanyl-ryanodine (V-II) in addition to being a highly useful intermediate for the synthesis of ryanodine molecular probes (e.g. VI, XIV, XV and XVI), binds 3.5-4 times

stronger to the ryanodine receptor than does ryanodine itself. The product (V-II), O_10eq-β-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.

IC₅₀(nM) = 2.8 ± 0.8 ; K_D(nM) = 1.0 ± 0.27 Product (V-I), β-alanyl-anhydro-ryanodine, binds to the receptor with an affinity of 1/10 that of Ryanodine

IC₅₀ (nM) = 106.0 ; K_D(nM) = 37.0 Anhydro-Ryanodine itself binds. to the receptor with an affinity of about 1% of the affinity of Ryanodine.

IC₅₀(nM) >1,000; K_D(nM) = > 350

These observations suggest that addition of the β-alanyl side chain at C₁₀- is beneficial to ligand binding not only in the case of Ryanodine itself but also of anhydro-ryanodine,

This derivative (V-II) also displays pharmacological activity different from that of its parent, ryanodine.

Studies of Ca⁺⁺ flux across sarcoplasmic membrane vesicles of both cardiac and skeletal muscle reveal that ryanodine has a biphasic effect on the receptor, the sarcoplasmic reticulum (SR) Ca⁺⁺ release channel. At low concentrations (<uM) ryanodine enhances Ca⁺⁺ flux across the SR membrane by

opening the SR Ca⁺⁺ release channel but at higher

concentrations (>uM) ryanodine inhibits Ca⁺⁺ flux by

inactivating or closing the channel.

In contrast, β-alanyl-ryanodine (V-II), 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 β-alanyl-ryanodine (V-II), is exhibited also by the

O_10eq-aminoacyl derivatives CBZ-glycyl- (I) and

CBZ-β-alanyl-ryanodine (IV).

It is, therefore, anticipated that this novel derivative (V-II) may appreciably facilitate an understanding of the mechanism of ryanodine-receptor interaction.

The following table gives the relative binding affinities for ryanodine, dehydroryanodine and O_10eq-ester

derivatives thereof. TABLE I

Relative Binding Affinities

Name of O_10eq Esters IC₅₀(nM) K_D(nM) (I to XVIII)

Ryanodine 11.9±1.6 4.4±0.8

Dehydroryanodine 16.1±4.1 5.4±0.1

CBZ-glycyl-Ryanodine (I) 22.3±7.3 6.6±2.0

Benzoyl-β-Alanyl-dehydro- 25.1±8.2 8.2±3.2 Ryanodine (II)

Adamantoyl-β-Alanyl-dehydro- 72.8±6.2 26.9±3.6 Ryanodine (III)

CBZ-β-Alanyl-Ryanodine (IV) 12.3±1.0 4.0±0.2 β-Alanyl-anydro-Ryanodine HCl (V) 106.9±19 37

β-Alanyl-anhydro-Ryanodine (V-I) 37 NA β-Alanyl-Ryanodine (V-II) 2.8±o.8 1.0±0.3

N-(5-azido-2-nitrobenzoyl)-β-Alanyl- 36.6±2.8 12.5±1.0

Ryanodine (VI)

N-(p-Iodobenzoyl)-β-Alanyl 16.0 5.2

Dehydroryanodine (XII)

N-(p-n-Butoxybenzoyl)-β-Alanyl 28.0+6.6 9.7±2.5 Dehydroryanodine (XIII)

N-Biotinyl)-β-Alanyl-Ryanodine (XIV) 65.1±5.5 24.9±1.4

N-[BODIPY(FL C3 )]-β-Alanyl- 41.2±2.7 15.8±1.0 Ryanodine (XV)

Dehydroryanodine-hemisuccinate (XVII) >1.0μM

N-Methyl-Dehydroryanodine-succin49.4± 10.6 19.9±5.8 amidate (XVIII)

In the Table, CBZ = benzyloxycarbonyl

The above numbers are mean values of replicate determinations,± standard deviation. Compunds of this invention suitably modified are useful in assaying for ryanodine receptor in various tissue fluids.

Accordingly, one of the groups represented by R or R¹ in

Figure 2 can be modified by substitution therein of a

chromophore, of an isotopic atom ( ¹³C for example) or by a radioatom (Radio-iodine or ¹⁴C for example), as will be apparent to those skilled in the art. A preferred label would involve the use of tritium-labelled β-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.

In order to assay for 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

obtained for the presence of the chromophore or isotope.

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.

EXAMPLE 17

COUPLING OF DEHYDRORYANODINE-O_10eq-

HEMISUCCINATE (XVII) TO BSA TO SERVE AS AN

ANTIGEN TO GENERATE "ANTI-RYANODINE" ANTIBODIES

A.) Dehydro-ryanodine succinate - BSA Conjugate Antigen (XIX).

To a stirred solution O_10eq-dehydoryanodine

hemi-succinate (30 rag, 5.1 mmol), bovine serum albumin (BSA, 3.27 mmol), and N-hydroxy-sulfosuccinimide (NHS, 1.2 mg, 0.5 mmol) in phosphate buffer (10 ml 0.1 M, pH=7.4) was added

1-(3,3-dimethylaminopropyl)-3-ethylcarbodiimide HCl (EDCI, 192 mg, 1 mmol) at once. The stirred reaction mixture was allowed to remain at room temperature for 2 hours and then at 4°C for 16 hours. The solution was then transferred to dialysis tubing and dialyzed for 12 hour intervals first against 0.01 M phosphate buffer, then against 0.001 M

phosphate buffer.

A total volume of 14.5 ml. of solution containing

lysine-substituted BSA-dehydroryanodine-succinate conjugate (2.82 mg/ml) was obtained. Spectroscopic analysis of the solution - based on the molar absorption of dehydro-ryanodine (E=16,000) and the optical density (=0.694) of the (diluted) solution (47 ug/ml) relative to that of the corresponding unconjugated protein (BSA) control (=0.047) - showed that an average of 70% of the 56 lysine residues present per mole of BSA were substituted by dehydro-ryanodine-succinate. B.) Antibodies against BSA-dehydro-ryanodine succinate

antigen.

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.

C.) Dhydro-rvanodine succinate - Keyhole Limpet Hemocyanin Antigen

This antigen was prepared - analogous to the above

BSA-antigen - using Keyhole Limpet Hemocyanin in soluble from obtained from Pierce Chemical Co. Spectroscopic analysis of the resulting solution (22.0 ml) containing KLH-dehydroryanodine succinate conjugate (4.54 mg/ml) revealed an optical density (0.230, 0.15 mg/ml

relative to the control unconjugated KLH (0.140, 0.15 mg/ml). The above conjugates of ryanodine and dehydro-ryanodine with bovine serum albumin (BSA) and keyhole lympet hemocyanin (KLH) were prepared to serve as antigens for the generation of ryanodine antibodies. Ryanodine antibodies are of

interest for the following purposes:

a) 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

(serum) and/or in agricultural samples in areas where

ryanodine-containing insecticide preparation are being used. b) Ryanodine-antibodies would act as an antidote to treat animals or humans accidentally poisoned by an overdose of ryanodine.

c) Ryanodine-antibodies may serve as a tool to

recognize, locate, and isolate (sequester) putative native peptido-mimetic biological agents involved in the

physiological control of the mammalian ryanodine receptor. d) Anti-iodiotypic antibodies would constitute a

peptide model for and act as a peptido-mimetic of ryanodine.

The usefulness of these studies may be enhanced

appreciably by the use of Ryanodine antibody-gold complexes.

While the invention has been illustrated and described in detail in the figures and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred

embodiments have been shown and described and that all

changes and modifications that come within the spirit of the invention are desired to be protected.

Claims

What is claimed is

1. A compound of the formula

in which Z is C(H)-CH₃ or C=CH₂ and R is

-CO-CH₂CH₂-COOH or R¹HN(CH₂)_n-CO- wherein n is 1-3

and R¹ is H, methyl or a lipophilic group.

2. A compound according to Claim 1 in which R¹ is a lipophilic group of the class adamantanecarbonyl,

adamantylmethylcarbonyl, adamantyl-1-oxycarbonyl,

benzyloxycarbonyl, adamantoyl-glycyl, benzoyl, substituted benzoyl, phenylacetyl, biotinyl-β-alanyl, BODIPY (FL-C₃), and 7-amino-4-methyl coumarinyl-3-acetyl.

3. A compound according to Claim 2, said compound being O_10eq-adamantoyl-glycyl-dehydroryanodine

4. A compound according to Claim 2, said compound being O_10eq-[(N'-Carbobenzyloxy)-glycyl]-ryanodine

5. A compound according to Claim 2, said compound being O_10eq-(N-benzoyl-β-alanyl)-dehydroryanodine.

6. A compound according to Claim 2, said compound being O_10eq-[N-(1-adamantanecarbonyl)-β-alanyl]-dehydroryanodine

7. A compound according to Claim 2, said compound being O_10eq-carbobenzyloxy-β-alanyl-ryanodine

8. A compound according to Claim 1, said compound being O_10eq-β-alanyl-ryanodine.

9. A compound according to Claim 1, said compound being dehydroryanodine-O_10eq -hemisuccinate.

10. A compound according to Claim 1, said compound being N-methyl-dehydroryanodine-O_10eq-succinamate.

11. A compound according to Claim 2, said compound being O_10eq-N-BODIPY(FL-C )-β-alanyl-ryanodine.

12. A compound according to Claim 2, said compound being O_10eq-N-(Biotinyl)-β-alanyl-ryanodine.

13. A compound according to Claim 2, said compound being O_10eq-N-(7-amino-4-methylcoumarin-3-acetyl)-β-alanylryanodine.

14. A labelled O_10eq derivative of ryanodine or of dehydroryanodine in which the label is in the O_10eq

substituent.

15. A labelled O_10eq derivative according to Claim 14 in which the label is a photo label, an isotopic label or a radioactive label.

16. A photo-activatable derivative according to Claim 14, said derivative being O_10eq-[N-(5-azido-2-nitrobenzoyl)- β-alanyl]-ryanodine.

17. A method for isolating and/or purifying ryanodine receptor which comprises modifying a suitable carrier by attaching thereon a compound according to Claim 1, passing a fluid mixture containing ryanodine receptor over said

modified carrier, said mixture being derived from cardiac or skeletal muscle sarcoplasmic reticulum, whereby the ryanodine receptor is abstracted from the fluid mixture by the modified carrier, and then separating the ryanodine receptor thus isolated from the modified carrier.

18. A method for the preparation of a ryanodine

derivative according to Claim 1 wherein n is 1, 2 or 3, which comprises preparing a compound according to Claim 1 in which n is 1, 2 or 3 and R¹ is H, and then acylating said intermediate compound to prepare a compound according to Claim 1 in which n is 1, 2 or 3 and R¹ is other than H.

19. A compound according to Claim 1 in which the phenyl ring of a group represented by R¹ contains a

photo-activatable substituent.

20. A method for affecting the function of the junctional sarcoplasmic Ca ⁺⁺ release channel of striated muscle in a mammal which comprises administering thereto a dose effective to affect Ca ⁺⁺ efflux of a compound according to Claim 2, thereby opening said Ca ⁺⁺ release channel and permitting greater Ca ⁺⁺ efflux.

21. A method for affecting the function of the junctional sarcoplasmic Ca⁺⁺ release channel of striated muscle in a mammal which comprises administering thereto a dose effective to affect Ca⁺⁺ efflux of a compound according to Claim 10, thereby opening said Ca⁺⁺ release channel and permitting greater Ca⁺⁺ efflux.

22. A compound according to claim 2 in which substituted benzoyl includes halobenzoyl, C_1-4alkyl benzoyl and C_1-4 alkyloxy benzoyl.

23. Compounds according to Claim 1, said compounds being O_{10 eq}-(N-p-Iodobenzoyl-β-alanyl)dehydroryanodine and

O_10eq-(N-p-Iodobenzoyl-β-alanyl) ryanodine.

24. Compounds according to Claim 1, said compounds being O_{10 eq}-[N-p-(n-butyloxy)-β-alanyl] dehydroryanodine and

O_10eq-[N-p-(n-butyloxy)-β-alanyl] ryanodine.

25. In a process for the hydrogenolysis of

O_10eq-CBZ-β-alanyl-ryanodine in the presence of a noble metal catalyst to yield 0_10eq-β-alanyl-ryanodine, the

improvement which comprises carrying out the hydrogenolysis under basic conditions so as to avoid formation of inactive anhydroryanodine species.

26. The process for the preparation of

O_10eq-β-alanyl-ryanodine which comprises hydrogenolyzing

O_10eq-CBZ-β-alanyl-ryanodine to

O_10eq-β-alanyl-anhydroryanodine and then converting said

O_10eq-β-alanyl-anhydroryanodine to

O_10eq-β-alanyl-ryanodine.

27. A process according to Claim 26 in which said

conversion of O_10eq-β-alanyl-anhydroryanodine to

O_10eq-β-alanyl-ryanodine is catalyzed by a mild organic base.