WO2006120472A2 - Novel beta-steroid compounds - Google Patents

Abstract

The present invention relates to a compound of formula (I). The invention provides the use of the compound in the inhibition of the sodium pump, use of the compound in medicine and particularly in the treatment of heart disease, hypertension, impaired renal function, renal disease, diabetes, metabolic disorders, neurological disorders, pulmonary disease, chronic obstructive pulmonary disease and cancer.

Description

NOVEL BETA-STEROID COMPOUNDS

The present application relates to novel compounds, their use in the inhibition of the sodium pump, their use in medicine and particularly in the treatment of heart disease, hypertension, impaired renal function, renal disease, diabetes, metabolic disorders, neurological disorders, pulmonary disease, chronic obstructive pulmonary disease and cancer. The invention also provides processes for manufacture of said compounds, compositions containing them and processes for manufacturing such compositions.

The sodium pump is essential to the integrity and functioning of the animal cell. It maintains the high intracellular potassium and low intracellular sodium that characterises almost all our cells. Not only can this chemical disequilibrium be made to perform work such as the transporting of other substances by co- or counter- transport, it also is the basis for the functioning of excitable cells and for muscular contraction.

Evidence that the activity of the sodium pump may be altered in various clinical conditions was first produced in the 1960's when Welt and his colleagues (Welt et al, Trans. Ass. Am. Physicians 77: 169-181 (1964)) showed that in uraemia the sodium pump of erythrocytes was inhibited and that this inhibition could be transferred to normal erythrocytes by uraemic plasma (Cole et al, Trans. Assoc. Am. Physicians 81: 213-220 (1968)). Reductions in the activity of the sodium pump and the transferability of this effect have also been shown in volume expansion and essential hypertension (Edmondson et al, Lancet 1:1003-1005 (1975); Haddy et al, Clin. Exp. Hypertens, 1: 295-336 (1978); Poston et al, Br. Med. J. 282: 947-849 (1981); Haddy et al, Fed. Proc. 44: 2789-2794 (1985); Gray et al, Clin. ScL 70: 583-586 (1986)).

Cardiac glycosides, such as digoxin, are known to be sodium pump inhibitors and have been used in medicine for centuries in the treatment of congestive heart failure. Cellular calcium levels are increased as an indirect result of inhibition of the sodium pump by the cardiac glycosides, leading to an increased force of contraction of the heart and therefore improved cardiac output. The glycosidic aspect of the structure of the cardiac glycosides is however not essential for activity and the aglycones of the cardenolides (also referred to as genins) also have similar properties. The activity of chemicals derived from toads, now known to be the family of bufadienolides, is also similar. These compounds, when derived from amphibia, are typically all aglycones and have approximately ten times the potency of the cardenolides.

Cardiac glycosides such as digoxin have a very small therapeutic index. Even minor variations in bioavailability lead to serious toxicity problems, therefore careful administration of the drug is required to avoid such drug toxicity. Both the cardenolides and bufadienolides are efficient poisons of the sodium pump, being possessed of low dissociation constants from the ATP-ase and exhibiting no obvious specificity of action in humans.

There is therefore a need for a compound having a greater specificity for the sodium pump such that the positive therapeutic effects are achieved but with reduced toxicity. There has therefore been extensive research carried out in an attempt to discover the nature of the endogenous substance responsible for sodium pump inhibition.

Research directed towards identifying the circulating inhibitor of the sodium pump of humans has tended to focus on the fundamental structural similarities of the cardenolides and bufadienolides. Other groups have attempted to extract and characterise sodium pump inhibitors from human material. Mathews et al (Mathews et al, Hypertension 17: 930-935 (1991)) reported the isolation of ouabain on the basis of its mass-spectral properties from 750 litres of human plasma. Subsequent work by Haupert et al (Tymiak et al, Proc. Nat. Acad. ScL 90: 8189-8193 (1993)) argued that the material in question was not ouabain but an isomer, though the current position in that respect is not clear. Lichtstein et al (Lichtstein et al, Eur. J. Biochem. 216: 261- 268 (1993)) isolated a sodium pump inhibitor from human cataractous lenses which they considered to be a bufadienolide and Bagrov et al reported that a bufadienolide - marinobufagin (3β-OH,5β-OH,14:15β epoxy bufadienolide) could be identified by radioimmunoassay and later by mass spectrometry in human plasma (Bagrov et al, Cardiovasc. Res. 207: 296-305 (1996); Bagrov et al, Hypertension 31: 1097-1103 (1998)).

The investigation of the endogenous sodium pump inhibitor has not however produced conclusive results. The inventors of the present application have therefore focussed on synthetic compounds and have now found that compounds similar in structure to known cardiac glycosides, cardenolides and bufadienolides but lacking a 14-hydroxyl group result in a much higher dissociation constant and a degree of specificity of action on different sodium pumps. Their studies point to the possibility of inhibiting different subsets of the sodium pump by this type of compound over different concentration ranges. The differential inhibition of sodium pumps in various tissues forms the basis of a useful pharmacological effect. These compounds can therefore be used to inhibit the sodium pump in medical applications in the treatment of diseases where inhibition of the sodium pump is desirable.

According to the present invention there is provided a compound of formula (I):

(I)

wherein R¹ is a hydroxyl group, hydrogen, a sugar, an optionally substituted C_1-6 alkyl group, C_3-12 aryl group, a C_3-12 heterocyclyl group or =0 9

R is absent or is hydrogen, a hydroxyl group, a halogen, an optionally substituted C_{1- 6} alkyl group, C_3-I2 aryl group or a C_3-12 heterocyclyl group

R³ is hydrogen, a hydroxyl group, a halogen, an optionally substituted Ci_-6 alkyl group, C_3-12 aryl group or a C_3-12 heterocyclyl group

R⁴ is a group of formula (II), (III), (IV), (V) or (VI)

(II) (III) (IV) (V) (VI)

wherein R¹⁰, R¹¹, R¹²and R¹³ are independently oxygen, nitrogen, sulphur or CH₂

R is hydrogen, methyl or ethyl

R⁶ is absent or is hydrogen, methyl, ethyl, an aldehyde or CH₂OH

R⁷ is hydrogen or fluorine

R⁸ is hydrogen, fluorine or CH₃COO

R⁹ is hydrogen or fluorine

and wherein R is either in the alpha or beta position, R is in the alpha or beta position, R³ and R⁴ are in the beta position and R⁵ and R are in the alpha or beta position

with the proviso that R¹ and R³ are not both hydroxyl groups. The term "sugar" includes monosaccharides, disaccharides, polysaccharides and oligosaccharides. Preferably one to four sugars may be attached either directly at the 3-beta position or to a hydroxyl group at the 3-beta position. In a preferred aspect, the sugar is selected from the group consisting of rhamnose, glucose, digitoxose, digitalose, digginose, sarmentose, vallarose and fructose. These sugars may be in either the L or D configuration, however preferably the sugars are selected from the group consisting of L-rhamnose, D-glucose, D-digitoxose, D-digitalose, D-digginose, D-sarmentose, L-vallarose and D-fructose.

For the purposes of this invention, alkyl relates to both straight chain and branched alkyl radicals of 1 to 6 carbon atoms including but not limited to methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl n-pentyl, and n-hexyl. The term alkyl also encompasses cycloalkyl radicals of 3 to 6 carbon atoms, including but not limited to cyclopropyl, cyclobutyl, CH₂-cyclopropyl, CH₂-cyclobutyl, cyclopentyl or cyclohexyl. Cycloalkyl groups may be optionally substituted or fused to one or more carbocyclyl or heterocyclyl group. Haloalkyl relates to an alkyl radical preferably having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms substituted with one or more halide atoms for example one or more of fluorine, chlorine and/or bromine. Possible haloalkyl radicals include but are not limited to CH₂CH₂Br, CF₃ or CCl₃.

"Aryl" means an aromatic 3 to 12 membered hydrocarbon containing one ring or being fused to one or more saturated or unsaturated rings including but not limited to phenyl, napthyl, anthracenyl or phenanthracenyl.

"Heterocyclyl" means a 3 to 12 membered ring system containing one or more heteroatoms selected from nitrogen, oxygen or sulphur and includes heteroaryl. The heterocyclyl system can contain one ring or may be fused to one or more saturated or unsaturated rings; the heterocyclyl can be fully saturated, partially saturated or unsaturated and includes but is not limited to heteroaryl and heterocarbocyclyl. Examples of carbocyclyl or heterocyclyl groups include but are not limited to cyclohexyl, phenyl, acridine, benzimidazole, benzofuran, benzothiophene, benzoxazole, benzothiazole, carbazole, cinnoline, dioxin, dioxane, dioxolane, dithiane, dithiazine, dithiazole, dithiolane, furan, imidazole, imidazoline, imidazolidine, indole, indoline, indolizine, indazole, isoindole, isoquinoline, isoxazole, isothiazole, morpholine, napthyridine, oxazole, oxadiazole, oxathiazole, oxathiazolidine, oxazine, oxadiazine, phenazine, phenothiazine, phenoxazine, phthalazine, piperazine, piperidine, pteridine, purine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, pyrroline, quinoline, quinoxaline, quinazoline, quinolizine, tetrahydrofuran, tetrazine, tetrazole, thiophene, thiadiazine, thiadiazole, thiatriazole, thiazine, thiazole, thiomorpholine, thianaphthalene, thiopyran, triazine, triazole, and trithiane.

"Carbocyclyl" relates to a saturated, partly unsaturated or unsaturated 3 to 12 membered hydrocarbon ring, including cycloalkyl and aryl.

"Heteroaryl" means an aromatic 3 to 12 membered aryl containing one or more heteroatoms selected from nitrogen, oxygen or sulphur and containing one ring or being fused to one or more saturated or unsaturated rings.

"Halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine. When C5 is fluorinated then R¹ is not in the beta conformation and when C9 is fluorinated then R is not in the beta conformation.

Where R¹, R² or R³ are a C_1-6 alkyl group or cycloalkyl group, C_3-12 aryl group, or a C_3-12 heterocyclyl group they may be optionally substituted by one or more of halogen, C_1-6 alkyl, haloalkyl, C_3-12 aryl, C_3-I2 heteroaryl, hydroxyl, C_1-6 alkoxy, SH, NH₂, CO₂H, COH, NHCO₂H, SO₃H, NO₂ or CN and wherein the C_1-12 alkyl group optionally contains one or more insertions selected from -0-, -N(H)-, -S-, - S(O)- and -S(O₂)-.

For the avoidance of doubt, where the bonds between the ring structure and R , R , R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are drawn as straight lines, the groups may be in either the alpha conformation or the beta conformation, Where the bonds are shown as wedge shaped then the groups are in the beta conformation.

Where bonds are shown as dotted lines, these bonds are optional. Therefore, in the formula of group (II) there may be two double bonds present in the locations shown by the dotted line. Alternatively there may be only one double bond present in either of the positions shown by the dotted line in group (II), or may be no double bonds present.

In one embodiment of the first aspect of the invention one of the bonds between C4:C5, C5:C6, C15:C16 or C16:C17 may be a double bond, with the remaining bonds being single bonds or C1O:C1, C2:C3, C4:C5 may be double bonds and C5:C6 and at least one of C15:C16 and C16:C17 are single bonds

It will be appreciated that when a double bond is present at C4:C5 or C5:C6 then R is absent. When a double bond is present at C10:Cl then R must be absent.

A preferred compound of the invention comprises 3-beta-hydroxyl-5-beta-14-beta- bufa-20,22-dienolide.

Other preferred compounds may comprise 3-beta-hydroxyl-5-beta-14-beta bufatrienolide and 3-beta-hydroxyl-5-beta-14-beta bufenolide. For the avoidance of doubt, the following figure shows the numbering of carbon atoms referred to within this document.

In a second aspect of the invention, there is provided a pharmaceutical composition comprising the compound of the invention as described above and optionally a pharmaceutically acceptable carrier and/or diluent.

The compounds of the invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms.

Suitable carriers and/or diluents are well known in the art and include pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, (or other sugar), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile). The composition may be a mixed preparation of a composition or may be a combined preparation for simultaneous, separate or sequential use (including administration).

The composition according to the invention for use in the aforementioned indications may be administered by any convenient method, for example by oral (including by inhalation), parenteral, mucosal (e.g. buccal, sublingual, nasal), rectal or transdermal administration and the compositions adapted accordingly.

For oral administration, the composition can be formulated as liquids or solids, for example solutions, syrups, suspensions or emulsions, tablets, capsules and lozenges.

A liquid formulation will generally consist of a suspension or solution of the compound or physiologically acceptable salt in a suitable aqueous or non-aqueous liquid carrier(s) for example water, ethanol, glycerine, polyethylene glycol or oil. The formulation may also contain a suspending agent, preservative, flavouring or colouring agent.

A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and microcrystalline cellulose.

A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, powders, granules or pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatine capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatine capsule.

Compositions for oral administration may be designed to protect the active ingredient against degradation as it passes through the alimentary tract, for example by an outer coating of the formulation on a tablet or capsule.

Typical parenteral compositions consist of a solution or suspension of the compound or physiologically acceptable salt in a sterile aqueous or non-aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.

Compositions for nasal or oral administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve, which is intended for disposal once the contents of the container have been exhausted. Where the dosage form comprises an aerosol dispenser, it will contain a pharmaceutically acceptable propellant. The aerosol dosage forms can also take the form of a pump-atomiser.

Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.

Compositions for rectal or vaginal administration are conveniently in the form of suppositories (containing a conventional suppository base such as cocoa butter), pessaries, vaginal tabs, foams or enemas.

Compositions suitable for transdermal administration include ointments, gels, patches and injections including powder injections.

Conveniently the composition is in unit dose form such as a tablet, capsule or ampoule.

The composition can be manufactured using standard techniques well known in the art and involves combining a compound according to the first aspect of the invention and the pharmaceutically acceptable carrier or diluent. The composition may be in any form including a tablet, a liquid, a capsule, and a powder.

A compound of the present invention may be administered simultaneously, subsequently or sequentially with one or more other active agents, such as other cardioactive drugs (e.g. diuretics and nitrates) and other anti-cancer drugs.

The compounds of the invention will normally be administered in a daily dosage regimen (for an adult patient) of, for example, an oral dose of between 1 mg and 2000 mg, preferably between 30 mg and 1000 mg, e.g. between 0.5 and 20 mg or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 50 mg, e.g. between 1 and 25 mg of the compound of the formula (I) or a physiologically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day. Suitably the compounds will be administered for a period of continuous therapy, for example for a week or more.

In a third aspect of the invention there is provided the compounds or composition or the first or second aspect for use in medicine. The compounds may be used in the treatment of any disease where inhibition of sodium pump activity is beneficial.

Examples of such diseases include heart disease, hypertension, impaired renal function, renal disease, diabetes, metabolic disorders such as aldosteronism, neurological disorders such as bipolar disorder or Alzheimer's disease, pulmonary disease, chronic obstructive pulmonary disease and cancer. The compounds of the present invention may therefore be used in the treatment of any of these sodium pump mediated disorders.

The sodium pump, or sodium-potassium adenosine triphosphatase, is a plasma membrane-associated protein complex and is expressed in most eukaryotic cells. The pump controls directly or indirectly many essential cellular functions by coupling the energy released in the intracellular hydrolysis of ATP to the export of sodium ions and the import of potassium ions, thereby maintaining an electrochemical gradient, The pump comprises a membrane spanning protein consisting of two dimers composed of alpha and beta subunits. Four alpha and three beta isoforms of the subunits of the sodium pump have been identified.

In a fourth aspect of the invention there is provided the compound of the first aspect of the invention for use as a sodium pump inhibitor. The compounds of the invention are selective sodium pump inhibitors. The selective nature of these compounds may be achieved through binding to specific isoforms of the subunits of the pump. This selective binding may then lead to the provision of beneficial therapeutic effects in the absence of the toxic effects which usually result from treatment with cardiac glycosides.

In a fifth aspect of the invention the compound of the first aspect of the invention is used in the manufacture of a medicament for the treatment of heart disease, hypertension, impaired renal function, renal disease, diabetes, metabolic disorders, neurological disorders, pulmonary disease, chronic obstructive pulmonary disease and cancer.

In one embodiment of the fifth aspect, the invention further provides a method of treating or preventing heart disease, hypertension, impaired renal function, renal disease, diabetes, metabolic disorders, neurological disorders, pulmonary disease, chronic obstructive pulmonary disease and cancer, which method comprises administering an effective amount of a compound of the first aspect or a composition of the second aspect to a patient in need of treatment thereof.

One or more other active agents may be administered to the individual simultaneously, subsequently or sequentially to administering the compound. The other active agent may be a cardioactive drug (e.g. a diuretics or a nitrate) or an anticancer drug. Examples of diuretics include acetazolamide, chlorothiazide, cyclopenthiazide, chlorthialidone, indapamide, xipamide, metolazonehydro- chlorothiazide, bendrofluazide, frusemide, bumetanide, piretanide, torasemide, spironolactone, amiloride and triamterene. Examples of nitrates are isosorbide mononitrate and glyceryl trinitrate. Examples of anti-cancer drags are pentostatin, 6- mercaptopurine, 6-thioguanine, methotrexate, cytarabine, crisantapase, 5-fluorouracil, bleomycins, mitomycin, cisplatin, carboplatin, doxorubicin, etoposide, amsacrine, dactinomycin, taxoids, cyclophophamide, estramustine, chlorambicil, melphalan, lomustine, carmustine, bulsulphan, fludarabine, pentostatin, claribine, mercaptopurine, thioguanine, azathioprine, allopurinol, doxorubicin, idarubicin, epinibicin, aclarabian, mitozantrone, dactinomycin, mitomycin, vincristine, vinblastine, vindesine, vinorelbine, etopodise, paclitaxel, docelatel, irinatecan, topotecan, gluocorticoids, oestrogens, progestogens, goerelin, cyproterone, octreotide, flutamide, cyproterone, procarbazine, hydroxyurea, crisantaspase, milotane, amsacrine, γ-interferon, tamoxifen, aldesleukin and tretinoin.

In a sixth aspect the invention provides a method for the production of a compound of the first aspect of the invention comprising selectively reducing the 16:17 double bond on a compound of formula (VII):

(VII)

While any suitable catalyst could be used in this reaction, preferably selective reduction of the C16:17 double bond is catalysed by tris(triphenylphosphine) rhodium chloride. This catalyst is also known as Wilkinson's catalyst.

When the desired end product is a bufenolide compound, i.e. where the R group has one double bond present in its ring, the method may further comprise a subsequent step of performing the Diels Alder reaction (Diels, et al, Ann. 460, 98 (1928)) to produce bufenolides free of contaminating dienes and trienes.

The compound of formula (VII) may be produced by triflating a compound of formula (VIII)

(Vπi) and then coupling the triflated compound to a group of formula (II), (III), (IV), (V) or (VI)

(H) (in) (IV) (V) (VI)

In a preferred aspect, the compound of formula (VIII) is triflated to form a vinyl triflate and then coupled to a 2 pyrone-5-boronate ester in the presence of a suitable palladium catalyst using Suzuki cross coupling. Suitable palladium catalysts for the purpose of this invention include (PPh₃)₂PdCl₂, (PPh_S)₄Pd, Pd(OAc)₂, [PdCl(η³- C₃H₅]₂, Pd₂(dba)₃, Pd(dba)₂ (dba = dibenzylidenacetone), Pd/P(t-Bu)₃. The Suzuld cross coupling can be carried out according to Suzuki, Pure Appl. Chern. 63: 419 (1991) or Littke et al, Am. Chem. Soc. Ill: 4020 (2000) or Gravett, et al, Tetrahedron Letters 42: 9081-9084 (2001). In an alternative aspect, Stille coupling could be used to couple the compound of formula (VIII) to the group of formula (II), (III), (IV), (V) or (VI) as described in Stille et at, Chem., Int. ed, Engl. 25: 508 (1986); Mitchell, Synthesis, 803 (1992), or Littke et al, J. Am. Chem. Soc. 124: 6343 (2002).

The compound of formula (VIII) may suitably be produced by performing the following steps: a) epimerising and oxidising the compound of formula (IX) to introduce a double bond at 15:16

b) migrating the C15:C16 double bond to C 14 :C 15 followed by reduction to form a β epimer.

The 3-hydroxyl group may be temporarily protected e.g. with a tert-butyldimethylsilyl group prior to epimerisation. The epimerisation step of C 14 from the alpha to beta conformation may be performed via the formation of a trimethylsilyl-enol ether at C17. The compound may be subsequently oxidised to yield a 15-en-17-one moiety and the C15:16 double bond then migrated to C14:C15. The compound may then be reduced to form predominantly the beta epimer. The reduction may be catalysed by a palladium catalyst or a platinum catalyst.

Preferably, the compound of formula (XI) comprises 3-β hydroxyl-5 β-14α~ androstan-17-one. The use of this compound as a starting material results in the production of 3 beta hydroxyl 5-beta 14-beta-bufa-20,22-dienolide using the method of the sixth aspect of the invention.

Where a compound of the first aspect of the invention is fluorinated, fluorination may be carried out, where required, by the use of inorganic fluorine or by other known fluorinating agents, for example, l-Chloromethyl-4-fluoro-l,4-Diazoniabicyclo- [2.2.2]Octane Bis-(tetrafluoroborate) or l-Methyl-4-Fluoro-l,4-Diazoniabicyclo- [2.2.2]Octane Bis-(tetrafluoroborate).

In a seventh aspect of the invention, a compound of the first aspect of the invention can alternatively be produced by the steps of: a) addition of a phenylseleninyl group to C23 on a bufanolide of formula (XI)

b) subsequent elimination of the phenylseleniyl group. The phenylseleninyl group may be eliminated using a mild oxidising system such as hydrogen peroxide or bleach.

The bufanolide compound of formula (XI) may be produced by hydrogenation of a compound of formula (X)

(X) using sulphuric acid or phosphorous oxychloride to introduce a double bond at C14:C15 followed by reduction. The reaction can be catalysed using, for example, a platinum catalyst on charcoal.

Alternatively, the bufanolide may be produced by hydrogenation of a bufadienolide of formula (XII):

A bufadienolide produced by the method of the sixth aspect may therefore be converted to a bufenolide via the method of the seventh aspect by hydrogenation of the bufadienolide to form a bufanolide and subsequent addition and then elimination of a phenylseleninyl group to re-introduce one double bond. In an eighth aspect of the invention, the compound of the invention can be used to identify isoforms of subunits of the sodium potassium ATPase. The compound can therefore be used as a pharmacological tool to further elucidate the structure of the sodium pump.

In a preferred embodiment of the first aspect of the invention, the compounds comprise 3-beta-hydroxyl-5-beta- 14-beta-bufa-20,22-dienolide, 3-beta-hydroxyl-5- beta-14-beta bufatrienolide or 3-beta-hydroxyl-5-beta-14-beta bufenolide.

In a preferred embodiment of the sixth aspect of the invention, the compound of the first aspect may be produced by the following method.

The 3-OH group of an optionally substituted steroid of formula (IX) having a 14α configuration may be temporarily protected, such as for example, with a tert- butyldimethylsilyl (TBDMS) group. Epimerisation of C14 from the α to the β configuration may then be performed via the formation of the trimethylsilyl-enol ether at C17 and its subsequent oxidation to yield the 15-en-17-one moiety. The C15:16 double bond may then be migrated to C14:15 and then reduced to predominantly the β epimer. This compound may then be triflated on C 17, and coupled to 2-pyrine-5- boronate ester using a Suzuki cross coupling. The resulting compound is an optionally substituted bufatrienolide (16:17, 20:21, 22:23-ene) of the invention.

The bufatrienolide may however be converted to the corresponding bufadienolide by selective reduction of the 16:17 double bond, for example by using Wilkinson's catalyst (tris(triphenylphosphine) rhodium chloride). The bufadienolide is also a compound of the invention.

The bufadienolide compound may be converted to a bufenolide compound, also having activity against the sodium pump, by the steps of hydrogenation, addition of a phenylseleninyl group to C23 and subsequent elimination of the phenylseleninyl group. Preferred features of the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.

The invention will now be illustrated by the following non-limiting examples. In the Examples reference is made to a number of Figures in which:

Figure 1- shows the effect of 3-beta-hydroxyl-5-beta-14-beta bufadienolide on various sodium pumps.

Figure 2- shows the effect of bufalin and ouabain on various sodium pumps. Figure 3 -shows the effect of 3-beta-hydroxyl-5-beta-14-beta bufadienolide on the sodium pump of human leucocytes

Figure 4-shows the effect of bufalin and ouabain on the sodium pumps of human leucocytes.

EXAMPLES

Example 1: Synthesis of a sodium pump inhibitor

Step a: Bromination of 2-pyrone to 5,6-dibromo-5,6-dihydro-2H-pyran-2-one (a photodibromide) (Gravett, Edward C; Hilton, Philip J.; Jones, Keith; Romero, Fernando; Tetrahedron Letters; EN; 42; 51 ; 2001 ; 9081- 9084).

In a dry flask under N₂ was placed DCM (20OmL) and freshly distilled 2H-pyran-2- one (α-pyrone) (10.56 g, 110.0 mmol). The flask was cooled to -78 ⁰C (dry-ice - acetone). Bromine (17.56 g, 110.0 mmol) was placed in a dropping funnel, diluted in

DCM (50ml) and chilled using dry-ice pellet around the funnel. The cold solution was added to the α-pyrone solution over 1 hour whilst shining light on the flask (500 W floodlight). The irradiation was continued for about 5 hours (TLC: SiO₂, EtOAc/hexane 3:7, UV detection, Rf_(α-pyrone)=0.20, Rf(_Photodi_bromi_de)=0.54) with stirring, maintaining the temperature at -78 ⁰C. The light source was removed and the contents of the flask were allowed to warm to 20⁰C. The solvent was removed under reduced pressure to afford the title compound as a light-orange oil (27.42 g, 97%). By ¹H NMR traces of starting material remained. The photodibromide is known to readily lose HBr at room temperature and was hence used without further purification.

Step b: Dehvdrodebromination of photodibromide to 5-bromo-2H-pyran-2-one {Gravett, Edward C; Hilton, Philip J.; Jones, Keith; Romero, Fernando; Tetrahedron Letters; EN; 42; 51 ; 2001 ; 9081- 9084).

The title compound (18.35 g) was obtained as a crude light brown solid. Two column chromatographies (SiO₂, DCM; TLC: SiO₂, DCM, UV detection, Rf(_photodi_bromi_de)=0.82, Rf_(product)=0.34) afforded an off-white solid (13.69 g, 73 %) and sublimation of the residue from relevant fractions from the second column chromatography allowed isolation of colourless needles (1.45 g) mp 61-63 ⁰C 60- 61 ⁰C). An overall yield of 15.14 g (81 %) was achieved.

Step c: Preparation of 2-pyrone-5-boronate (Suzuki precursor). (Gravett, E. C;

Hilton. P. J.; Jones, K.: Peron, J. M. Synlett 2003, 253-255).

PdCl₂(PPh₃)₂, Et₃N_(anh)

Toluene, Reflux

A mixture of 5-bromo-2-pyrone (13 g, 74 mmol), pinacolborane (16.2 mL, 14.7 g,

111 mmol), triethylamine (31 mL, 22.1 g, 222 mmol) and PdCl₂(PPh₃)₂ (1.56 g, 2.2 mmol) in dry toluene (100 mL) was heated under reflux conditions under a N₂ atmosphere for 6 hours. After this reaction time, TLC analysis showed no remaining 5-bromo-2-pyrone. The dark solution was filtered through a short plug of silica and this was further eluted with dichloromethane (250 mL). The solvent was removed under reduced pressure to give a dark crystalline solid. Flash column chromatography using dichloromethane as eluent gave the 2-pyrone-5-boronate (13.6 g, 83%) as a pale yellow crystalline solid, mp 91-93 °C. This material was used in all coupling reactions. An analytical sample with the same mp could be obtained as white crystals by sublimation at 0.7 mm Hg pressure (Found: C, 59.66; H, 6.83. C₁₁H₁₅BO₄ requires C, 59.50; H, 6.81%); v_max (NaCiycm^"1: 2982 (s, CH-alkane), 1753 (s, C=O), 1633 (s, C=C-CO-), 1318 and 1225 (s, unsat. ester); δ_H (300MHz, CDCl₃) 1.29 (6H, s), 6.27 (IH, dd, J= 9.6 and 1.2Hz), 7.50 (IH, dd, J= 9.6 and 2.1Hz) and 7.84 (IH, dd, J= 2.1 and 1.2Hz); δ_c (75MHz; CDC13) 24.8, 84.5, 96.9, 115.6, 146.3 and 160.1; m/z (FAB) 222.1059 (M+. C₁₁H₁₅ ¹¹BO₄ requires 222.1063).

Step 1: Protection of 3-hydroxy function of 3β-etiocholanolone as a tert- butyldimethylsilyl ether.

3β-etiocholanolone (3β-hydroxy-5β-androstan-17-one), a steroid with the 14α configuration, was obtained from Sigma- Aldrich.

The 3-OH was temporarily protected with a te/t-butyldimethylsilyl (TBDMS) group using standard procedures (cf. Greene T.W and Wuts P.G.M: Protective Groups in Organic Synthesis, 3^rd Edition (1999). John Wiley & Sons Inc., Chichester). The TBDMS -protected etiocholan-3β-ol-17-one was obtained as white solid (2.698 g, 97 %), mp 129-130 ⁰C; m/z (EI) M^+' = 404. Step 2; Preparation of 17-silylenol ether of protected steroid (14-position epimerisation step 1).

The C 14 was epimerised from the α- to the β-configuration via the formation of the trimethylsilyl-enol ether at C17 using the following method. In a dry flask under a N₂ atmosphere were placed the steroid starting material (2.269 g, 5.61 mmol) and dry THF (35 mL). The flask was cooled to -78 ⁰C (dry-ice - acetone) and 2M LDA in THF (3.50 mL, 7.01 mmol) was introduced over a couple of minutes using a syringe. The enolate formation was allowed to proceed for a couple of hours. In a dropping funnel (non-pressure equalising) featuring a sinter at its base (above the tap), were premixed TMSCl (6.09 g, 7.09 mL, 56.1 mmol) and Et₃N (5.67 g, 7.8I mL, 56.1 mmol) in THF (5 mL), in order to trap any HCl impurity originating from TMSCl. After mixing for a few minutes the suspension was filtered slowly into the reaction flask by pressurising the funnel over a 2-3 minute period. After 15 min. the reaction mixture was allowed to warm to 20 ⁰C and stirred for 1 h. The reaction mixture was quenched in saturated aqueous NaHCO₃ (16OmL). The organic layer was separated from the aqueous one which was further extracted in THF. The THF layers were combined and dried (Na₂S O₄(_anh)) and the solvent was removed under reduced pressure affording a yellow oily residue. The residue was reconstituted in DCM and transferred to a separating funnel where it was washed with NaHCO₃(_sat) followed by back extraction of the aqueous layer in DCM. The combined DCM fractions were washed with brine, dried over Na₂SO₄(_anh) and evaporated to dryness to afford the title compound (2.554 g, 96 %) as an orange-yellow fluffy solid mp decomp. > 116 ⁰C; [α]f ^c + 72.0 (c 0.85 in CH₃Cl). Step 3: Tsuji-type oxidation of 17-silylenol ether to corresponding 15:16-enone (14- position epimerisation step 2)

The method used was derived from the work by Tsuji et al. The TMS enol ether (2.559 g, 5.366 mmol) was dissolved in a mixture of dry DCM (35 mL) and dry acetonitrile (12 mL) in a flask under a N₂ atmosphere. The solution was heated to 60 ⁰C for a few minutes and then palladium(II) acetate was introduced into the reaction flask as once portion and the orange solution stirred 40 ⁰C for at least 6 hours. The crude material was filtered through a plug of Celite to remove the solid byproduct and then chromatrographed twice (silica gel 60 using DCM as eluent (Rf = 0.5)). The still impure title compound (1.487 g, 73 %) was obtained as yellow- off white solid mp 113-121 ⁰C. Subsequently we discovered that the compound was more stable on silica 60 when the column was prepared and performed using the eluent DCM/Hexane 85:15 containing 196^"WV Et₃N.

Step 4: Acid catalysed C-C double bond isomerisation of 15:16-enone to 14:15-enone (14-position epimerisation step 3).

In. a dry flask under nitrogen were placed the steroid starting material (0.923 g, 2.292 mmol) dry THF (25 mL) and p-toluene sulphonic acid monohydrate, p-TSA, (0.098 g, 0.515 mmol, 10%W/W). The reaction mixture was heated under reflux overnight. Triethylamine, Et₃N, (0.5 mL, 3.587 mmol) was added whilst still under reflux; and after 10 minutes stirring the contents of the flask were allowed to cool to 20 ⁰C. The solvent was removed in vacuo to afford about 2 ml of oily material. After reconstitution in DCM (25 mL) the solution was transferred to a separating funnel and washed with water (2 x 25 mL). Then the aqueous layers were back extracted in DCM. The combined DCM fractions were washed once with brine and then dried (Na₂SO₄). The solvent was evaporated to yield a yellow solid (0.889 g, crude). The material was purified by column chromatography (silica gel 60, Hexane/DCM 1:1, Rf = 0.3) which furnished the title compound (0.469 g, 51 %) as an off-white crystalline material mp 102-104⁰C (from Hexane/DCM); MD^*0 + 102.0 (c 1.02 in CHCl₃). Subsequently we discovered that the compound was more stable on silica 60 when the column was prepared and performed using DCM/Hexane 1:1 containing 1%V/V Et₃N as eluent. Vm_ax (1 drop of material in CDCl₃ evaporated on a NaCl plate)/cm⁴: 2929 (vs), 2859 (s), 1745 (s, C=O), 1060 (s).

Step 5: Palladium catalysed hydrogenation of 14:15-enone to 14βH-17-keto steroid (14-position epimerisation step 4)

The starting steroid (0.469 g, 1.16 mmol) was placed in a flask with palladium on charcoal (20%W/W, 0.094 g) and ethanoi (50 mL) was introduced in the flask. The vessel was sealed using a septum and was successively evacuated with nitrogen and the hydrogen. A hydrogen balloon (ca IL) was connected to the flash by means of a needle, the extremity of which stood in the trough of the vortex resulting from a vigorous stirring. The hydrogenation was allowed to proceed overnight. Then the hydrogen gas was evacuated and replaced with nitrogen. The reaction mixture was filtered through a plug of Celite and the filtrate concentrated to produce a white amorphous solid (0.435 g). Column chromatography (silica gel 60 packed using DCM containing 0.1% Et₃N, DCM eluent, Rf = 0.48) afforded the TBDMS -protected 14- epi-3β-etiocholanolone as white solid (0.300 g, 64%) mp 124.0-126.0 ⁰C. MD ^C +66.0 (c 1.09 in CH₃Cl); v_max (1 drop of material in CDCl₃ evaporated on a NaCl plateycm^'1: 2936 (vs), 2885 (m), 2857 (m), 1740 (s, C=O), 1252 (m), 1076 (m), 1064 (S).

Deprotection of TBDMS -protected 14-epi-3β-etiocholanolone

TBDMS -protected 14-epi-3β-etiocholanolone (0.070 g, 0.17 mmol) was placed in a dry flask under nitrogen and dry DCM (5ml) was added to dissolve the material. The mixture was chilled to 0 ⁰C (ice-water bath) and an excess of pyridine-HF complex was added (0.25 ml, 70 %W/W HF in pyridine). The flask was allowed to warm to 20 °C with stirring for 1 h. The reaction mixture was quenched with saturated NaHCO₃, and the bilayer transferred to a separating funnel. The organic layer was collected and the aqueous layer further extracted in DCM. The combined organic phases were treated once with brine, dried over Na₂SO₄(_atlh) and concentrated to dryness. Further drying under high vacuum afforded 14-epi-3β-etiocholanolone

(0.046 g, 91 %) as a white solid mp 170-172 ⁰C. MD ^'C +77.0 (c 0.92 in CH₃Cl); v_max (1 drop of material in CDC13 evaporated on a NaCl plateycm^"1: 3500 (sharp, free OH) 2936 (s), 1724 (s, C=O); m/z (ES, QTOF) 291.2326 ([M+H]⁺, Ci₉H₃JO₂ requires 291.2324). Step 6: Preparation of vinyl triflate of TBDMS -protected M-epi-Sβ-etiocholanolone.

TBDMS-protected 14-epi-3β-etiocholanolone (0.200 g, 0.49 mmol) was placed in a dry two-necked round bottom flask and dissolved in dry THF (7 mL). The flask was cooled to - 78 ⁰C (acetone-dry ice bath) and 2M LDA in THF (0.42 mL, 0.84 mmol) was introduced over a couple of minutes using a syringe. The enolate formation was allowed to proceed for at least I h. In a separate dry flask was dissolved N- phenyltriflamide (0.278 g, 0.74 mmol) in dry THF (5 mL). The enolate was treated with this solution at -78 ⁰C and after about 15 minutes stirring the cooling bath was removed. Stirring was continued at 20⁰C for at least 1 h. The reaction mixture was transferred to another flask and the solvent removed under reduced pressure to yield a slowly-crystallising orange oil. This material was reconstituted in DCM and washed successively with water and brine (back-extracting the first aqueous layer with DCM). After drying and solvent removal (Na₂S0_4(anh)) the new residue (0.246 g) showed presence of starting material (¹H NMR) and hence the reaction was performed once more on this residue. The stirring of the reaction mixture was extended to one night. After an extractive work up (as above) the new residue was chromatographed (silica gel 60, DCM as eluent, Rf = 0.68). After purification the desired vinyl triflate was obtained (0.087 g, 33%) but was not analytically pure. It was used in the next synthetic step without further purification. The yields of this reaction proved to be variable. Step 7: Suzuki cross coupling of vinyl triflate of TBDMS -protected 14-epi-3β- etiocholanolone with 2-pyrone-5-boronate as prepared in steps a, b and c.

Steroid-vinyl triflate (0.746 g, 1.39 mmol) (only traces of 14α-epimer as impurity), 2- ρyrone-5-boronate (0.411 g, 1.81 mmol), and PdCl₂(dppf) (0.114 g, 0.14 mmol) were dissolved in dry DMF (5 mL) under a N₂ atmosphere. Finely crushed fresh K₃PO₄ (0.885 g, 4.17 mmol) was added to the DMF solution and this slurry was heated to 60 °C for 6.5 h. The DMF was removed under reduced pressure to give a dark solid that was purified two flash column chromatography (silica gel 60, DCM as eluent, Rf = 0.34). The desired product, 3β-TBDMS-oxy-5β-14β-bufatrienolide, was obtained as white crystalline material (0.566 g, 84 %) mp 155-158 ⁰C (decomp.);

MD ^C + 106.0 (c 1.02 in CH₃Cl); v_maχ (1 drop of material in CDCl₃ evaporated on a NaCl plateVcm^"1: 2928 (vs), 2883, 2859 (m), 1745 (vs, C=O).

Deprotection of TBDMS -protected bufatrienolide.

Following the same procedure as described earlier (from 66 mg protected material) 3β-hydroxy-5β-14β-bufatrienolide was obtained (50 mg, crude) and was purified by preparative HPLC. The bufatrienolide may be used as a sodium pump inhibitor or may be further converted to a bufadienolide or bufenolide.

Step 8; Hydrogenation of 16:17-double bond using Wilkinson's.

Following a method in the literature (Can. J. Chem. (1963) 46:377-383), 3β-TBDMS- oxy-5β-14β-bufatrienolide (0.46O g, 0.95 mmol) and Wilkinson's catalyst (0.174 g, 0.19 mmol) were dissolved in dry benzene (38.25 mL) in a nitrogen-purged flask. The flask was carefully evacuated and purged with hydrogen and a hydrogen balloon (ca IL) was connected to the flask by means of a needle (the red catalyst turned orange in the presence of H₂). The solution was vigorously stirred overnight. The solvent was removed under reduced pressure to afford a dark red residue. The crude material was chromatographed twice using the well known "dry flash" technique (silica gel 60 (4 x 1.8 cm φ and 6 x 2.0 cm φ), DCM as eluent, Rf = 0.24). Removal of the solvent under reduced pressure afforded an off-white crystalline material (0.434 g, 87 %) MP 205- 207 ⁰C (decomp.>207 ⁰C) (appeared contaminated with ca 9 % triene precursor under ¹H NMR). [On another instance a purer analytical sample (0.208 g) was prepared by recrystallisation m.p. 209-210 ⁰C (from hexane/EtOAc) (by ¹H NMR, it contained

4 % or less triene precursor)]. MD +84.0 (c 1.04 in CHCl₃); v_maχ (1 drop of material in CDCl₃ evaporated on a NaCl PIaIe)ZCm^"1: 2929 (vs), 2880 (m), 2857 (s), 1744 (vs), 1060 (s); m/z (ES, QTOF) 485.3436 ([M+H]⁺, C₃₀H₄₉O₃Si requires 485.3451), 471.2585 ([M-C₆H₁₅Si +H]⁺, C₂₄H₃₅O₃ requires 471.2586). eprotection of TBDMS -protected bufadienolide.

Following the same procedure as previously described (see before step 8), 3β- TBDMS-oxy-5β-14β-bufatrienolide (0.434 g, 0.89 mmol) was deprotected to 5β- hydroxy-5β-14β-bufatrienolide (0.380 g, crude). The material was recrystallised from CHC1₃/Hexane 1:4 at ca -4 ⁰C overnight to afford a first batch of white crystals (46mg) which was used for all the analyses and for pharmacological evaluation. From the mother liquor a second crop of crystals was obtained (0.273 g) and was used in subsequent hydrogenation step. First crop, MP 196-198 ⁰C (from CHC1₃/Hexane 1:4);

MD ^C + 98.0 (c 2.25 in CHCl₃); V_maχ (1 drop of material in CDCl₃ evaporated on a NaCl plateVcm^"1: 3436 (broad, w, OH), 2934 (s), 2863 (m), 1740 (s); m/z (ES, QTOF) 371.2585 ([M+H]⁺, C₂₄H₃₅O₃ requires 371.2586).

The bufadienolide may be used as a sodium pump inhibitor or may be further converted to a bufenolide using the methods of steps 9 to 12.

3-βTBDMS-Oxy-5β-14β-bufadienolide (0.334 g, 0.69 mmol) and 5 % w/w rhodium on alumina (10 % w/w, 0.036 g) were placed in a single neck flask with EtOAc (30 ml). The contents of the flask were successively purged with nitrogen and hydrogen and a hydrogen balloon was attached to the flash by means of a needle. The suspension was stirred vigorously overnight. The mixture was then filtered through a small plug of Celite and the solvent evaporated under reduced pressure. A bright white solid was obtained (0.334 g) showing incomplete hydrogenation. The hydrogenation procedure was applied once more to this product (as above) and yielded a mixture of two TBDMS-Oxy-5β-14β-bufanolides, (isomers at C-20 (0.310 g, 92 %)). This mixture showed a slight excess of one diastereoisomer over the other. It was not possible to resolve the components by chromatography and hence the mixture was used in the next step without further purification.

Step 10: Preparation of phenylseleninyl-steroid adduct at 3-position on tetrahydro-2- pyrone moiety of the 3-βTBDMS-oxy-5β-14β-bufanolides

The mixture of 3-βTBDMS-oxy-5β-14β-bufanolides (0.154 g, 0.315 mmol) was placed in a dry two-necked round bottom flask and dissolved in dry THF (7 mL). The flask was cooled to -78 C (acetone-dry ice bath) and 2M LDA in THF (0.20 mL, 0.41 mmol) was introduced over two minutes using a syringe. The enolate formation was allowed to proceed for at least 2 h. In a separate dry flask phenylseleninyl chloride (0.095 g, 0.47 mmol) was dissolved in dry THF (3 mL). The enolate was treated with this solution at -78 ⁰C and stirring was continued for at least 4 h at -78⁰C. The cooling bath was removed and the stirring continued overnight at 20⁰C. The reaction mixture was poured onto 0.5 N HCl (15 mL) and transferred to a separating funnel. The aqueous layer was treated with diethyl ether (2 x 20 mL) and the combined organic phases were successively treated with water (1 x 50 ml), NaHCO₃(_sat) and brine. The solvents were removed under reduced pressure and furnished a crude yellow solid (0.311 g). The steroid-phenyseleninyl mixture of adducts is known to be unstable and to readily oxidise and undergo ■S}>rc-elemination to furnish our next target molecule (in our hands). Hence no further purification was made before use in the next step.

Step 11: Preparation of TBDMS-oxy-5β-14β-buf-22:23-enolides bv oxidation of phenylseleninyl-steroid adduct and subsequent ffyn-erimination of phenylseleninic acid

The phenylseleninyl-bufanolide adduct mixture (0.311 g) was treated with an excess of H₂O_2(aq) (0.25 mL, 30 % w/w) in THF (25 mL) at O⁰C with stirring for 30 min. The mixture was allowed to warm to 20 ⁰C and stirred for about 4 hours (quenching of the yellow adduct accompanied by the formation of a white precipitate). The reaction mixture was treated with diethyl ether (50 mL) and was transferred to a separating funnel. The organic layer was washed with NaHCO₃(_Sat) (50 mL) and the resulting aqueous layer back extracted with some diethyl ether (2 x 50 mL). The combined ether fractions were washed once with brine (75 mL), dried (Na2SO4(_anh)) and evaporated to dryness. A crude (by ¹H NMR) 3β-TBDMS-oxy-5β-14β-buf-22:23- enolides mixture was obtained (0.157 g). Purification by flash column chromatography (silica gel 60 (4 x 1.5 cm φ), DCM as eluent, Rf(buf-22:23-enoiides) = 0.16) afforded a mixture of buf-22:23-enolides as expected (0.075 g, 49 %), contaminated with traces of dienolide and buf-20:21-enolide impurity (according to ¹H NMR). Step 12: Deprotection of 3-position of 3β-TBDMS-oχy-5β-14β-buf-22:23-enolides.

Using the previously described method, pyridine-HF deprotection method 3β- TBDMS-oxy-5β-14β-buf-22:23-enolide mixture (0.075 g, 0.154 mmol) was quantitatively deprotected to furnish 3β-hydroxy-5β-14β-buf-22:23-enolide as a 1:1.6 mixture of diastereoisomers (0.059 g). By proton NMR, 7% of dienolide impurity was present. The material for further purified by preparative HPLC.

Example 2: Sodium transport in intact cells

Sodium efflux in human erythrocytes was studied by the method of Walter & Distler (Walter U et al. Hypertension 4:205-210 (1982)). The erythrocytes were loaded with ²²Na by incubation in buffer containing ²²NaCl for 30 min. The cells were then washed three times in non-radioactive buffer and incubated at 37°C for 75 min with aliquots being removed every 15 min. These were immediately chilled to 0°C and spun at 120Og for 3 min. The supernatant was removed and counted in a gamma counter (Wallac 1282). The increase in radioactivity of the supernatant was related back to the radioactivity of a sample of cells removed at time 0.

Sodium efflux in human leucocytes was studied by the method described in Hilton PJ et al. Cell P/ι^røZ;109:323-332 (1981)) with minor modifications. The cells were again loaded in a ²²NaCl-containing buffer, washed once and incubated at 37° for 16 min, with aliquots of the cell suspension being removed every 3 min. The cell suspension was immediately chilled to 0°C before being spun for 1 min at 320Og. The supernatant was removed and the cell pellet counted in a gamma counter. The cell pellet was digested in NaOH (1 mol/1) and the protein content determined by the Folin method.

In both cases, the efflux was calculated as a pseudo-first order rate constant. The ouabain-sensitive (sodium pump) portion of the flux was derived by subtraction of the residual sodium efflux seen in the presence of 10^"4 mol/1 ouabain. The effects of the inhibitors were studied over a range of concentrations from 3xlO^"8 to 10^"4 mol/1.

An estimate of the dissociation of 3β-OH,5β,14β bufadienolide relative to ouabain from human erythrocytes was undertaken as follows. Human erythrocytes loaded with ²²Na were incubated for 5 min in ouabain 10^"5 mol/1 or 3β-OH,5β,14β bufadienolide 10^"4 mol/1 i.e. a concentration sufficient to give maximum achievable binding to the sodium pumps of these cells and the rate-constant for sodium efflux was determined in these media. A second batch of cells similarly loaded with the inhibitor was washed three times in approximately 10 volumes of non-radioactive buffer that contained no inhibitor (total time 16 minutes). Sodium efflux was then studied as previously described.

The ouabain pre-treated cells increased their rate-constant for sodium efflux by only 3%, demonstrating ouabain's low dissociation constant from their sodium pumps. In contrast, the rate constant for sodium efflux in the cells pre-treated with 3β- OH,5β,14β bufadienolide increased to 85% of control values. While this experiment does not provide a quantitative estimate of the compound's dissociation constant, this is clearly much greater than that of ouabain.

Example 3: ATP-ase studies

Dog kidney and pig brain ATP-ases were obtained from Sigma- Aldrich. Activity of the preparation was studied by the release of inorganic phosphate over a 28 minute period at 37⁰C in a medium containing Na⁺ 100 mmol/1, K⁺ 3mmol/l Mg²⁺ 4.5 mmol/1

ATP 0.3mmol/l, EDTA O.lmmol/1, EGTA 5 mmol/1, buffered to pH 7.40 with Tris (40mmol/l). The ouabain-sensitive portion of the flux was calculated by subtraction of the residual phosphate generation in the presence of ouabain. Inorganic phosphate was measured in samples taken every 7 min by the method of Kraml (Kraml M. Clin C/wm Acta;13:442-448 (1966))

The effect of 3β-OH,5β,14β bufadienolide on the activity of the four ATP-ase/sodium pump preparations is shown in Fig 1. By comparison, the typical pattern of inhibition by a plant poison or toad venom is a sigmoidal pattern of inhibition over approximately two orders of magnitude of concentration (Fig 2).

Comparison of Figure 1 with Figure 2 shows that the pattern of inhibition of the dog kidney and pig brain ATP-ases closely resembles that of ouabain or bufalin but requires a higher concentration to achieve these effects. The pattern of inhibition of erythrocytes in Figure 1 appears identical to that of the ATP-ases at the lower concentrations of the inhibitor though significantly, only 70% inhibition can be achieved at even the highest concentration that can be studied, this being limited by the compound's solubility in water. At this concentration, the inhibition of erythrocyte sodium efflux appears to be reaching a plateau.

The pattern of inhibition in the case of the leucocytes is obviously different. Here a clear maximum is reached at a concentration of the inhibitor of 10^"5 mol/1, at which point only 47% inhibition of the total sodium pump activity has been achieved. In the concentration range 3xlO^~8 to 10^"6 mol/1 there is further significant deviation of the pattern of inhibition from that seen in dog kidney, pig brain or erythrocytes, with zero inhibition not being observed until dilution to 3xlO^"8 mol/1. This discrepancy could be explained on the basis of inhibition of a further subset of the sodium pump that became saturated by a concentration of the inhibitor of approximately 10^"6 mol/1 (Fig 3). The results of Figure 3 can be compared with that of Figures 2 and 4 which show the effect of bufalin and ouabain on the sodium pumps of leucocytes.

These experiments demonstrate that a bufadienolide with the 14β configuration but lacking an oxygen function at this site is an effective inhibitor of four different preparations of the sodium pump (Na/K-ATPase). The lability of its effect on erythrocytes points to a relatively high dissociation constant from the pump as compared with ouabain. The more complex patterns of inhibition seen in erythrocytes and leucocytes as opposed to the ATP-ase preparations may not be simply a manifestation of the prevalence of different α isoforms of the pump on the cell surface but rather reflect the fact that these studies are performed in intact cells with the sodium pump situated in its native environment, the cell membrane.

The studies point to the possibility of inhibiting different subsets of the sodium pump by the compounds of the invention over different concentration ranges. The effect of

3β-OH,5β,14β bufadienolide on the differential inhibition of sodium pumps in various tissues might form the basis of a useful pharmacological effect. It has, for example, been argued that the usefulness of cardiac glycosides might be substantially improved and their toxicity reduced if their effects on the α₃ isoform were more pronounced than those on the (X₁.

Claims

1. A compound of formula (I):

wherein R¹ is a hydroxyl group, hydrogen, a sugar, an optionally substituted C_1-6 alkyl group, C_3-12 aryl group, a C_3-12 heterocyclyl group or =0

R² is absent or is hydrogen, a hydroxyl group, a halogen, an optionally substituted C_1- β alkyl group, C_3-12 aryl group or a C_3-12 heterocyclyl group

R³ is hydrogen, a hydroxyl group, a halogen, an optionally substituted C_1-6 alkyl group, C_3-12 aryl group or a C_3-12 heterocyclyl group

R⁴ is a group of formula (H), (in), (IV), (V) or (VI)

(II) (IH) (IV) (V) (VI)

wherein R , R , R and R are independently oxygen, nitrogen, sulphur or CH₂

R ^■, 5 : is hydrogen, methyl or ethyl R is absent or is hydrogen, methyl, ethyl, an aldehyde or CH₂OH

R⁷ is hydrogen or fluorine

R⁸ is hydrogen, fluorine or CH₃COO

R⁹ is hydrogen or fluorine

and wherein R¹ is either in the alpha or beta position, R² is in the alpha or beta ppoossiittiioonn, R³ and R⁴ are in the beta position and R⁵ and R⁶ are in the alpha or beta position

with the proviso that R¹ and R³ are not both hydroxy! groups.

2. The compound of claim 1 wherein the compound comprises 3-beta-hydroxyl- 5-beta-14-beta-bufa-20,22-dienolide.

3. A pharmaceutical composition comprising a compound of claim 1 or 2 and optionally a pharmaceutically acceptable carrier and/or diluent.

4. The compound of claims 1 or 2 for use in medicine.

5. The compound of claims 1 or 2 for use as a sodium pump inhibitor

6. The compound of claims 1 or 2 for use in the treatment of heart failure, cardiac arrhythmia, kidney disease or cancer.

7. Use of the compound of claims 1 or 2 in the manufacture of a medicament for the treatment of heart disease, hypertension, impaired renal function, renal disease, diabetes, metabolic disorders, neurological disorders, pulmonary disease, chronic obstructive pulmonary disease and cancer.

8. A method of treatment of heart disease, hypertension, impaired renal function, renal disease, diabetes, metabolic disorders, neurological disorders, pulmonary disease, chronic obstructive pulmonary disease and cancer comprising administering an effective amount of a compound of claim 1 or claim 2 to patient in need of treatment thereof.

9. A method for the production of a compound of claim 1 or claim 2 comprising selectively reducing the 16: 17 double bond on a compound of formula (VII):

(vπ)

10. The method of claim 9 wherein selective reduction of the C16: 17 double bond is catalysed by tris(triphenylphosphine) rhodium chloride.

11. The method of claims 9 or 10 further comprising a subsequent step of performing the Diels Alder reaction to produce bufenolides free of contaminating dienes and trienes.

12. The method of claims 9 or 10 wherein the compound of formula VII is produced by triflating a compound of formula (VTfl)

and then coupling the triflated compound to a group of formula (II), (III), (IV), (V) or (VI)

(H) (HI) (IV) (V) (VI)

13. The method of claim 12 wherein the coupling is performed using Suzuki cross coupling.

14. The method of claim 12 wherein the coupling is performed using Stille coupling.

15. The method of claims 7 to 14 comprising performing the following steps to form a compound of formula (VIII)

a) epimerising and oxidising the compound of formula (IX) to introduce a double bond at 15: 16.

b) migrating the C15:C16 double bond to C14:C15 followed by reduction to form a β epimer

16. The method of claims 7 to 9 wherein the compound of formula (IX) comprises 3-β hydroxyl-5β-14α-androstan-17-one.

17. A method for the production of a compound of claim 1 or claim 2 comprising the steps of

b) addition of a phenylseleninyl group to C23 on a bufanolide of formula (XI)

b) subsequent elimination of the phenylseleninyl group.

18. The method of claim 17 wherein the compound of formula (XI) is produced by hydrogenation of a compound of formula (X)

19. The method of claim 17 wherein the compound of formula (XI) is produced by hydrogenation of a compound of formula (XII):

20. A compound of claim 1 or claim 2 for use in identifying subunits of the sodium pump.