EP1438324A1

EP1438324A1 - Cohalogenations of selected double bonded compounds using n-halo-succinimide.

Info

Publication number: EP1438324A1
Application number: EP02799524A
Authority: EP
Inventors: Levan Kita Kavtaradze; Merilyn Manley-Harris
Original assignee: University of Waikato
Current assignee: University of Waikato
Priority date: 2001-09-26
Filing date: 2002-09-26
Publication date: 2004-07-21
Also published as: EP1438324A4; WO2003027133A1

Abstract

The present invention relates to a method of producing a cohalogenated compound by adding halogen(s) and another group(s) to a multiple bond in an initial compound, the method characterised by the step of reacting the initial compound with a primary reagent in the presence of a catalyst wherein the primary reagent reacts with the multiple bond to produce the cohalogenated compound. In preferred embodiments, the catalyst is hypophosphorous acid, the primary reagent consists of N-halo-succinimide and acetone and the initial compound is organic. This method is particularly applicable for the production of cohalogenated steroids, including lanosterol.

Description

Cohalogenations of selected double bonded compounds using N-halo-succinimide

TECHNICAL FIELD

This invention relates to a chemical synthesis.

More specifically, this invention relates to synthesising compounds by cohalogenation of double or triple bonds.

Cohalogenation is the electrophilic addition of a halogen in the presence of a nucleophilic solvent like water, dimethyl sulfoxide, dimethyl formamide, carboxylic acids, alcohols, nitriles and ethers. The nucleophilic solvent competes with halide ion and leads to incorporation of a group other than the second halogen atom. For example, cohalogenation may result in the addition of halogen plus hydroxyl (hydroxyhalogenation) or halogen plus methoxy or halogen plus nitrate.

The above process, for ease of reference, will be referred to as "cohalogenation" of a compound.

BACKGROUND ART

Cohalogenation of alkene functionalities in organic compounds is of great interest in synthetic organic chemistry.

This is because the cohalogenation reaction introduces two new chiral centers, which are differently substituted. In contrast, simple halogenation introduces two new chiral centers, but these have the same substituent. Thus, after cohalogenation, the two new stereocenters will have differential reactivity toward, for example, oxidation.

Hydroxyhalogenation (the addition of HOX) is a form of cohalogenation in which the electrophilic addition of a halogen atom is followed by nucleophilic addition of water, which with elimination of a proton, results effectively in addition of -OH (an hydroxyl group).

HOX can be generated in situ by the reaction of the halogen with water in the case of Br₂ and Cl₂ (HOI requires the presence of tetramethylenesulfone-CHCl₃ and HOF is difficult to prepare and can cause detonations). The reaction proceeds by addition of an electrophilic halogen atom, from the positive end of the HOX dipole, followed by nucleophilic attack by water to insert OH. A competing nucleophile can also attack instead of the water.

The handling and storage of Br₂ and Cl₂ requires special precautions: Br₂ is toxic and corrosive and concentrations of lOppm are immediately dangerous to life and health: The J.T. Baker MSDS carries the following warning about bromine:

DANGER! CORROSIVE. MAY BE FATAL IF SWALLOWED OR INHALED. CAUSES SEVERE BURNS TO EVERY AREA OF CONTACT. AFFECTS RESPIRATORY SYSTEM, EYES, CENTRAL NERVOUS SYSTEM AND SKIN. STRONG OXIDIZER. CONTACT WITH OTHER MATERIAL MAY CAUSE FIRE.

Similar warnings apply to chlorine, which also constitutes an environmental hazard in aquatic environments. In fact the use of chlorine is avoided in industrial situations, for example pulp mills, where possible because of the environmental risks.

Tetramethylene sulfone is hazardous by inhalation and skin contact and CHC1 (chloroform) is a chlorinated solvent that requires special disposal procedures (chlorinated hydrocarbons can produce dioxins upon incineration) and is a potential greenhouse gas. The use of chloroform is discouraged in 'Green Chemistry'.

HOX can also be safely and conveniently added by the use of N-halosuccinimides in the presence of a small amount of water in a solvent such as dioxane or dimethylsulf oxide. Typical yields from this reaction are 50-100% for smaller compounds and less for larger, lipophilic molecules such as steroids. Reaction times for this reaction can vary from 2 to 24 hours.

The cohalogenation of more complex organic compounds can prove more difficult as there may be other functionalities present that may be affected including other alkene groups, which one desires to remain unreacted.

Another synthetic method for the production of hydroxyhalides was undertaken by Masuda, H.; Takase, K.; Nishio, M.; Hasegawa, A.; Nishiyama, Y.; Ishii, Y. 'A New Synthetic Method of Preparing Iodohydrin and Bromohydrin Derivatives through in situ Generation of Hypohalous Acids from H₅IO₆ and NaBrO₃ in the presence of NaHSO₃\ J. Org. Chem. (1994) 59, 5550-5555.

This paper describes hydroxyhalogenation (adding either Br and ΟH or I and OH) to a number of different alkenes with good yields. The yields were determined by GC rather than isolated. Isolated yields are usually lower than GC yields, as material is lost during isolation. Total reaction times were 3 hours (1 hour for the addition of NaHSO₃ and 2 hours stirring thereafter).

It would be beneficial to have a method of producing cohalogenated compounds that is not toxic, does not have storage, and therefore commercial production limitations, and has increased yields and reduced reaction times, thereby reducing production costs.

The bulk of the discussion in this specification shall be directed towards the present invention in Lanosterol derivative synthesis.

One reason is that lanosterol is a complicated molecule with two double bonds plus a

(protected) hydroxyl functionality - only one double bond reacts. This makes it a perfect example to illustrate the effectiveness of the present invention. A second reason is that lanosterol and its side chain derivatives are important starting materials for organic syntheses. Examples of other applications of the present invention are discussed later on in the specification.

Lanosterol is the core steroid from which others are derived by biological modification. It can be sourced from wool fat in sheep (Merck Index, 10^th Edition, [1983]).

Lanosterol is included in a number of products, including cosmetics and de-inking materials. However, most of the interest in uses of stereochemically pure lanosterol derivatives seems to focus on two subjects: anti-fungal activity and steroid biosynthesis inhibition.

Fungal infections are a major clinical problem in infectious diseases, chemotherapy and immune-compromised individuals (e.g. AIDS sufferers). Current medications of choice are azole drugs, but resistance to these is now beginning to develop. The use of Polyene drugs for similar treatment has shown toxic side effects.

Both ergosterol and cholesterol, the main sterols in fungi and mammals respectively are synthesized via lanosterol. C-4 and C-14 demethylations are common to both ergosterol and cholesterol biosynthesis, but C-24 methylation only occurs in fungi. Therefore recent works have identified in particular amino and thio derivatives of the side chain of lanosterol as potent anti-fungals due to their inhibition of the enzyme that brings about C-24 methylation.

Similar activity, which is due to inhibition of Δ ²⁴'²⁵⁾ -sterol methyl transferase, has also been demonstrated by 24(R,S),25-epiminolanosterol against Trypanosoma cruzi, the protozoan cause of Chagas Disease, a disease which gives rise to much human misery and economic loss in South America. Commercially available lanosterol is a mixture of four closely related compounds, in which lanosterol (5α-lanosta-8,24-diene-3β-ol) and dihydrolanosterol (5α-lanosta-8- ene-3β-ol) predominate in the approximate ratio of 1:1.

Lanosterol is a highly desirable starting material for derivatisation to other steroids. Attempts have been made to separate lanosterol from dihydrolanosterol (and other impurities) by different methods. Unfortunately, common separation methods such as column chromatography or fractional crystallisation are almost impossible.

Earlier methods of isolating lanosterol from other sterols were based on the selective addition of bromine to the double bond in the side chain of lanosterol, isolation of the dibromo- derivative and debromination by sodium iodide in acetone or by zinc dust in acetic acid or benzene.

Low yields and impurity of the separated sterols have lead to the search for methods with improved yields.

The most successful results were obtained by Rodewald and Jagodzinski (Polish J Chem 1978, 52, 2473-2477). The reaction of acetylated commercial lanosterol with mercury acetate in aqueous tetrahydrofuran, followed by the in situ reduction of the mercurial intermediate with NaBH₄, provided a quantitative yield of 3β-acetoxy-5α- lanost-8-en-25-ol, which was separated from dihydrolanosterol by column chromatography, such as HPLC. Unfortunately, the use of HPLC is highly expensive which contributes significantly to the cost of the end product.

Also, mercury acetate is categorized as being poisonous and use of many mercury- based compounds is not preferred due to their detrimental environmental impact.

Alternatively, acetylated commercial lanosterol was selectively epoxidized at the

24,25- position, separated from dihydrolanosterol, and after reduction with LiAlH₄ and reacetylation, afforded 3β-acetoxy-5α-lanost-8-en-25-ol. Finally the 25-hydroxy derivatives were refluxed with 20% Ac₂O in acetic acid and 3β-acetoxylanosta-8,24- diene was obtained in 75% overall yield in relation to its content in commercial lanosterol.

LiAlH is highly flammable and corrosive and reacts violently with water releasing flammable hydrogen gas. For example, J.T. Baker Material Safety Data Sheet (MSDS) issues the following warnings about LiAlH₄:

"DANGER! CAUSES BURNS TO ANY AREA OF CONTACT. HARMFUL IF SWALLOWED OR INHALED. FLAMMABLE SOLID. WATER REACTIVE. MAY IGNITE IF HEATED OR CONTACTED WITH WATER OR ACIDS".

Separate storage is recommended by the MSDS. It is obvious that LiAlH₄ would constitute a severe health and safety problem in large-scale operations.

More recently a solvomercuration-demercuration procedure has been provided as a general method for separation of unsaturated steroids as well as lanosterol from wool grease. The steroid mixture containing unsaturated steroids was treated with organo- mercuric salts to give unsaturated steroid Hg compounds, which were converted to monoalkylmercury chlorides and then reduced to unsaturated steroids (JP 07258285 A2).

Again, mercury based compounds are not preferred due to their detrimental environmental impact.

Reports of successful separation of lanosterol from dihydrolanosterol are few, and none of them have solved the problem of the commercial production of pure lanosterol because the practical applicability of their approaches is limited by the hazardous and reactive nature of most mercury salts as well as reducing reagents.

As a result of this, pure lanosterol is prohibitively expensive. At the time of writing, Sigma-Aldrich has available for sale lanosterol with a purity grade of 50-60% for 35.30USD for 25g. Sigma-Aldrich also sells lanosterol with a purity grade of 97% for 46.60USD for lmg.

The discovery of a technically simple, environmentally acceptable separation technique for providing high purity lanosterol would have the advantage of providing a substantially pure source of product as a starting material for specific syntheses.

The discovery of a technically simple, environmentally acceptable separation technique for providing cohalogenated compounds, and in particular, those derived from lanosterol based compounds without recourse to HPLC would have the advantage of providing precursors to a number of other end products.

The ability to separate these cohalogenated compounds into epimerically pure amounts of both R and S would provide a number of advantages. Optically pure derivatives of bioactive materials treat problem areas directly, without introduction of other isomers that dilute the efficacy of the bioactivity and thus cause a requirement for higher dosages. (There is also a possibility of unknown activity of the unwanted diastereomer). This is a distinct advantage when treating, for example, AIDS patients with fungal infections, as the patient cannot afford to be weakened further by a treatment that not only does not heal the infection, but also brings about the possibility of damaging the surrounding tissue. Steroids also suffer from a problem of low solubility in aqueous media, so minimizing the dosage requirement is an advantage.

As mentioned earlier, the protozoa, Trypanosoma cruzi, causes Chagas Disease. Inhibition of the enzyme of the protozoa, Λ⁽²⁴'²⁵⁾ -sterol methyl transferase, has been demonstrated by 24(R,S),25-epiminolanosterol (an unresolved mixture of diastereomers). Since the enzyme has a preferred stereochemistry for addition to the C-24 position (S) pure diastereomers will have differing inhibitory power and the appropriate diastereomerically pure C-24 derivative will be a more potent inhibitor than the unresolved R,S mixture.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in New Zealand or in any other country.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description that is given by way of example only.

DISCLOSURE OF INVENTION

According to one aspect of the present invention there is provided a method of producing a cohalogenated compound by adding halogen(s) and another group(s) to a multiple bond in an initial compound,

the method characterised by the step of

a) reacting the initial compound with a primary reagent in the presence of a catalyst wherein the primary reagent reacts with the multiple bond to produce the cohalogenated compound.

According to another aspect of the present invention there is provided a method of producing a hydroxyhalogenated compound by adding halogen(s) and hydroxyl group(s) to a multiple bond in an initial compound,

the method characterised by the step of

a) reacting the initial compound with a primary reagent in the presence of a catalyst

wherein the primary reagent reacts with the multiple bond to produce the hydroxyhalogenated compound.

The inventors consider that a preferred feature of the catalyst to provide this effect is for the catalyst to have, as its conjugate base, a weak nucleophile. The inventors consider a strong, directly competing nucleophile (as opposed to a weak one) would compete with a component of the reagent, which adds the group in addition to the halogen group.

The present invention covers cohalogenation of compounds generally, which is the electrophilic addition of halogen in the presence of a nucleophilic solvent. The nucleophilic solvent competes with halide ion and leads to incorporation of a group other than the second halogen atom. Cohalogenation can occur on compounds containing double or triple bonds where cohalogenation of a triple bond leads to two halogens being added, and two non-halogen atoms or groups of atoms.

It is envisaged that in most instances of the present invention the initial compound will be organic. This could include alkenes, cyclohexenes or alkynes, but should not be seen as limiting. It should be appreciated that the term 'organic' should be understood to mean a carbon-based compound and is known to someone skilled in the art.

Halogens can include the addition of any member of the halogen series, those being fluorine, chlorine, bromine, or iodine.

The non-halogen group can include methoxy, ethoxy, carboxy, or hydroxy but these are listed by way of example only and should not be seen to be limiting. Addition comes through the oxygen or other atom such as sulfur or nitrogen possessing a non- bonding pair of electrons.

If the non-halogen group to be added by cohalogenation is a hydroxy group, the process is known as hydroxyhalogenation, which is a subset of cohalogenation.

The term 'multiple bond' in accordance with the present invention should be understood to mean any chemical bond that is greater than a single bond. This includes double and triple bonds, but these should not be seen to be limiting.

In some embodiments, the initial compound can be part of a solution containing a steroid or steroids where not all components of that solution are desired in the end product.

The initial compound may in some embodiments be a mixture of lanosterol, or lanosterol derivatives, and other impurities such as dihydrolanosterol. The initial compound can include the extract from sheep's wool, which is a naturally occurring mixture of Lanosterol and dihydrolanosterol in a 1 : 1 ratio and is the most common source of starting material to produce Lanosterol.

The initial compound can also include commercially available lanosterol (not available as 100% pure lanosterol, it generally contains dihydrolanosterol as the impurity), or the initial compound can also include lanosterol, or lanosterol derivatives, with a protecting group attached, but all the above examples are listed by way of example only and should not be seen to be limiting.

The term 'primary reagent' in relation to the present invention includes, in addition to a solvent(s), a halogenating compound(s). The primary reagent react(s) with the multiple bond on the initial compound in the presence of a catalyst, to provide a cohalogenated compound.

In preferred embodiments, the solvent in the primary reagent is an organic solvent, that facilitates the reaction of the chemical(s) in the primary reagent with the initial compound.

The organic solvent increases the solubility of most of the compounds to be reacted (alkenes/alkynes), as most are insoluble in water. If the reaction were carried out solely in water, it is possible that two phases would form and the more polar components of the reagent would migrate into the aqueous phase and not contact the non-polar compound to be reacted.

The organic solvent can include dimethylsulfoxide or dioxane, but in preferred embodiments the organic solvent making up part of the primary reagent is acetone.

The halogenating compound can be chloramine T in acetone-water, Br₂ or Cl₂ or I₂ in water, t-butylhypohalite or acetoxy chloride, but these are listed by way of example only and should not be seen to be limiting.

In preferred embodiments, the primary reagent can include an N-halo-succinimide, where the term 'halo' is a general term to describe the inclusion of any member of the halogen series, those being fluorine, chlorine, bromine, or iodine.

The term 'catalyst' in accordance with the present invention should be understood to mean any substance that increases the rate of a reaction without itself being consumed. In preferred embodiments, the catalyst is hypophosphorous acid. Preferably, this catalyst is in water (in addition to the primary reagent).

Hypophosphorous acid is a moderately strong acid that is strongly reducing, and has a conjugate base that is a weak nucleophile.

The term 'strongly reducing' in accordance with the present invention should be understood to mean a substance having a reduction potential of greater than +0.3V. This terminology is known to someone skilled in the art.

The term 'weak nucleophile' is a term known to someone skilled in the art and is generally understood to mean a nucleophile that reacts slowly with a given substrate under a certain reaction condition.

It has not been possible to define an absolute scale of nucleophilicity and therefore the terms weak and strong are relative only when measured against a specified substrate under specified conditions. In one case the rate of the S_N reaction with iodomethane for a given nucleophile is compared to the rate for the same reaction with methanol as a substrate. [Swain, C.G. and Scott, C. B. J.Am Chem. Soc. 15, 141 (1953)].

Some features which weaken the nucleophilicity of a reagent are strong solvation, weak bonding to the substrate, bulkiness, high electronegativity of the atom bearing the lone pair of electrons needed to form the bond to the substrate and low polarizability. Any or all of these may be operating in this or other instances.

The catalyst could also include oxalic acid or sulphurous acid however it is preferred that that catalyst has a combination of features, those being; a strong acid with very reducing properties and having a conjugate base, which is a weak nucleophile. Oxalic acid has the same pKa and the same reduction potential as hypophosphorous acid, but its conjugate base provides a slightly stronger nucleophile. Hypophosphorous acid (H₃PO ) is cheap and readily available, and its residues are environmentally benign, which makes its use preferable to the traditional production methods for producing co-halogenated compounds.

As far as the inventors are aware, hypophosphorous acid has never been used to catalyse the synthesis of cohalogenated compounds. In particular, the use of hypophosphorous acid in catalytic amounts in relation to the cohalogenation of lanosterol is understood never to have been undertaken before.

One advantage of using hypophosphorous acid is that it acts as a catalyst and reduces the reaction time from several hours to 15-16 minutes, as well as giving excellent yields.

According to another aspect of the present invention there is provided a cohalogenated compound

characterised in that

the cohalogenated compound is produced by the method as described earlier in the text, which includes the step of

wherein the primary reagent reacts with the multiple bond to produce the cohalogenated compound.

In other embodiments, it should be appreciated that the cohalogenated compounds could be used in pharmaceutical compositions.

The applicant has found that the present invention has particular application to the formation of cohalogenated lanosterol derivatives, which are then available for either further reaction to form lanosterol, or further reaction to form other products. In some cases the cohalogenated lanosterol derivatives can be reacted directly to form lanosterol.

The bulk of the discussion in this specification shall now be directed to the application of the present invention to lanosterol-based derivatives. Examples of other applications are discussed later on in the specification.

According to another aspect of the present invention there is provided a method of producing cohalogenated compounds from which lanosterol can be derived,

the method characterised by the step of

a) reacting an initial lanosterol solution with a primary reagent in the presence of a catalyst

wherein the primary reagent reacts with the Δ⁽²⁴'²⁵⁾ of the lanosterol to produce the cohalogenated compounds.

The term 'lanosterol' in relation to the present invention is defined as lanosta-8,24- diene-3β-ol, and is also known trivially as kryptosterol. Its molecular formula is ₀H₅₀O, and its molecular weight is 426.70.

The term 'initial lanosterol solution' in relation to the present invention is defined as a mixture of lanosterol, or lanosterol derivatives, and other impurities such as dihydrolanosterol. An initial lanosterol solution can include the extract from sheep's wool, which is a naturally occurring mixture of lanosterol and dihydrolanosterol in a 1:1 ratio and is the most common source of starting material to produce lanosterol. An initial lanosterol solution can also include commercially available lanosterol (not available as 100% pure lanosterol, it generally contains dihydrolanosterol as the impurity). An initial lanosterol solution can also include lanosterol or lanosterol derivatives with a protecting group attached, however these are listed by way of example only and should not be seen to be limiting.

The term 'protecting group' in accordance with the present invention should be understood to mean a chemical group that is used in synthesis to temporarily mask the characteristic chemistry of a functional group because it interferes with another reaction. A good protecting group should be easy to put on, easy to remove and be inert to the conditions of the reaction required and the term is known to someone skilled in the art.

In preferred embodiments, the protecting group is an acetyl group. The addition of an acetyl group to the oxygen on the C-3 position of crude lanosterol is preferred, however this is listed by way of example only and should not be seen to be limiting as other protecting groups such as propionate, methyl or trimethylsilyl could also be used. The term C-3 refers to the third carbon in a carbon-based molecule and is standard nomenclature for counting carbon atoms in carbon based molecules.

The primary reagent is a compound or mixture of compounds capable of adding X⁺ (where X = halogen, Fluorine, chlorine, bromine or iodine) to the multiple bond initially followed by a nucleophile that adds to the resultant carbocation. The primary reagent may also include an organic solvent to increase the solubility of lipophilic substrates such as lanosterol

In some embodiments that produce hydroxyhalogenated lanosterol, the primary reagent used is N-halo-succinimide in acetone and water and the catalyst is hypophosphorous acid. This has the advantage of greatly increasing the yield and decreasing the time of reaction relative to the uncatalyzed reaction. Another advantage of using hypophosphorous acid in a catalytic amount to produce cohalogenated compounds is that use of hypophosphorous acid permits the use of acetone as the solvent with N-halo-succinimide for the first time.

This is an advantage as acetone is a useful component of a primary reagent. Acetone (B.Pt 56.5° C) is more readily removed from the reaction mixture than dioxane (B.Pt 101.1° C) or dimethylsulfoxide (B.Pt 189° C), the other commonly used solvents for this reaction (as mentioned earlier). In fact, dimethylsulfoxide is noted as being a solvent, which is difficult to remove after use without the application of heat. Acetone is preferable to dioxane, which is a health hazard and is described by the Merck Index in the words "This substance may reasonably be anticipated to be a carcinogen".

Dichloromethane is sometimes used in reactions involving N-halo-succinimide, however dichloromethane is now less favoured due to its environmental impact.

If the primary reagent includes N-halo succinimide, then the outcome will be a diastereomeric mix of hydroxyhalogenated lanosterol derivatives, where the term 'halo or halogenated' is a general term to describe the inclusion of any member of the halogen series, those being fluorine, chlorine, bromine, or iodine.

The term 'Δ ²⁴'²⁵⁾' is the term used to describe the double bond between carbons 24 (C-24) and 25 in a molecule, in this case a steroid, and the nomenclature for counting carbon atoms in a steroid molecule is known to someone skilled in the art.

The term 'derivatives' in accordance with the present invention will, in preferred embodiments, be a diastereomeric mix of hydroxyhalogenated lanosterol derivatives. The choice of primary reagent will determine which derivative is formed.

It is envisaged that in a number of embodiments the derivative may be highly labile. Thus, it is likely that immediately following production of the derivatives, additional process steps will be taken to produce either lanosterol or possibly more useful lanosterol based derivatives.

Lanosterol and its major impurity, dihydrolanosterol have physical and chemical properties that are very similar. This similarity is what makes them very difficult to separate. By synthesising certain derivatives of lanosterol, the difference in properties between dihydrolanosterol and the derivatives is maximised, making it possible to separate them by standard, well known methods. This provides a distinct advantage over current methods, as not only is the process of producing lanosterol an environmentally 'green' one (especially in comparison with mercury), but it also utilises standard, easily applied separation techniques.

This method of producing lanosterol derivatives has the advantage of using chemicals that are considered to be 'green', especially when compared to lanosterol produced by using mercury dependent reactions.

In preferred embodiments, hydroxyhalogenation of lanosterol produces '24-Halo-24- hydroxy-lanosterol derivatives, where the term 'Halo' is a general term to describe the inclusion of any member of the halogen series.

In preferred embodiments, the halogens of choice are iodine, bromine and chlorine.

As discussed previously in preferred embodiments of the present invention a diastereomeric mix of hydroxyhalogenated lanosterol derivatives is created.

The term 'diastereomeric' in accordance with the present invention should be understood to mean stereoisomers that are not mirror images of each other, and in this case relates specifically to derivatives of lanosterol differing in the absolute configuration of C-24, and is a term known to someone skilled in the art. Diastereomers have similar chemical properties, but different physical properties, allowing them to be separated by physical means. The advantage of producing diastereomeric hydroxyhalogenated lanosterol derivatives is that they can be divided by a separating means to provide epimerically pure amounts of both 24(R) and 24(S) hydroxyhalogenated derivatives separately.

The terms 'R' and 'S' are used to describe the absolute configuration of the carbons that differ between the members of a pair of diastereomers, in this case only one carbon, carbon 24, is involved. The configuration is specified 'R' (Latin: rectus, right) if the eye travels in a clockwise direction from the highest priority ligand to the lowest priority ligand, when the carbon in question is viewed from opposite the lowest priority ligand. The configuration is specified 'S' (Latin: sinister, left) if the eye travels in an anti-clockwise direction. This naming regime is known to someone skilled in the art.

Pure R and S hydroxyhalides are then further available to be derivatised to other steroid based compounds, which are bioactive.

Diastereomerically pure lanosterol side chain derivatives have not been easily available to the medical profession/chemical industry. Up until now, there has been no process available for providing a 'green' and easily achievable means of dividing the precursor materials.

The advantage diastereomerically pure derivatives would have is that they would treat problem areas directly, without introduction of other isomers that dilute the efficacy of the bioactivity and thus cause a requirement for higher dosages. (There is also a possibility of unknown activity of the unwanted diastereomer). This is a distinct advantage when treating for example, AIDS patients with fungal infections, as the patient cannot afford to be weakened further by a treatment that not only does not heal the infection, but also brings about the possibility of damaging the surrounding tissue. Steroids also suffer from a problem of low solubility in aqueous media so minimizing the dosage requirement is an advantage. One embodiment of the present invention produces 24-bromo-25-hydroxylanosterol diastereomers. These may be separated by crystallization to give the individual 24(R) and 24(S) diastereomers. This provides a starting point for the production of stereochemically pure side chain derivatives of lanosterol and also provides an alternative route to the diastereomerically pure 24(R),25-epoxylanosterol and 24(S),25-epoxylanosterol, a convenient alternative route to these two compounds which have in the past been difficult to separate.

Side chain derivatives of lanosterol (3-β-hydroxy-5- -lanosta-8,24-diene) have been shown to act as inhibitors of Δ²⁴⁽²⁵⁾ sterol methyl transferase, which is an essential enzyme in the sterol biosynthesis pathway of protozoa, fungi and plants.

[Nes, W.D. 'Sterol methyl transferase: enzymology and inhibition', Biochim. Biophys. Acta, 2000, 1529 (1-3), 63-88.]

Because of this activity these compounds have potential therapeutic applications. Reaction of the Δ²⁴⁽²⁵⁾ bond in general leads to the production of a mixture of C-24 diastereomers.

It is probable that, where a mixture of diastereomers has been used in a bioactivity assay the activity resides principally in one diastereomer and it is thus not possible accurately to gauge this activity.

[Chung, S-K., Ryoo, C.H., Yang, H.W., Shim, J-Y., Myung, G.K., Lee, K.W., Kang, H.I., 'Synthesis and bioactivities of steroid derivative as anti-fungal agents'. Tetrahedron 1998, 54, 15899-15914. Urbina, J,.A., Vivas, J., Lazardi, K., Molina, J., Payares, G., Piras, M.M., Piras, R., Antiproliferative effects of A^24(25> sterol methyl transferase inhibitors on Trypanosoma (Schizotrypanum) cruzi: in vutro and in vivo studies'. Chemotherapy, 1996, 42, 294-307.]

Differential bioactivity of 24(R)- and 24(S)-epoxylanosterols has been demonstrated [Panini, S.R., Sexton, R.C., Gupta, A.K., Parish, E.J., Chitrakorn, S., Rudney, H., 'Regulation of 3-hydroxy-3-meihylglutaryl coenzvme A reductase activity and cholesterol biosynthesis by oxylanosterols' , J. Lipid Res. 1986, 27, 1190-1204.].

Early reports of the separation of the diastereomers of 24,25-epoxylanosterol acetate by crystallization have not been amenable to emulation more recently and instead separation of the isomers was achieved by repetitive HPLC. No other separations of C-24 diastereomers of lanosterol have been reported.

[Boar, R.B., Lewis, D.A., McGhie, J.F., 'Isolation and some reactions of lanosterol. A synthesis of agnosteroV . J. Chem. Soc. Perkin Trans I, 1973, 15, 1583-1588.]

[Emmons, G.T., Wilson, W.K., Schroepfer, G.J., '24.25-epoxysterols. Differentiation of 24R and 24S epimers by¹³C nuclear magnetic resonance spectroscopy' , J. Lipid Res. 1989, 30, 133-138. Panini, S.R., Sexton, R.C., Gupta, A.K., Parish, E.J., Chitrakorn, S., Rudney, H., 'Regulation of 3-hydroxy-3-methylglutaryl coenzvme A reductase activity and cholesterol biosynthesis by oxylanosterols' , J. Lipid Res. 1986, 27, 1190-1204]

The advantage of using hypophosphorous acid is that it acts as a catalyst and reduces the reaction time from several hours to 15-16 minutes as well as giving excellent yields.

Lanosterol occurs in a natural mixture with dihydrolanosterol. This occurrence has the disadvantage of providing researchers and industry alike with an impure starting material, thereby reducing yields _*

The provision of derivatives, which will subsequently allow for separation of lanosterol from dihydrolanosterol, has not been undertaken in a 'green' and easily achieved manner before. The advantages provided by the above-described method are that pure lanosterol (free from the dihydrolanosterol and other steroid impurities) can be made available as a starting material. In other embodiments the initial compound may be cyclohexene, which can be converted to cohalogenated cyclohexane via the present invention.

The term 'cyclohexene' in relation to the present invention is defined as 1,2,3, 4-tert- hydrobenzene. Its molecular formula is C₆Hio, and its molecular weight is 82.14

The term 'cyclohexane' in relation to the present invention is defined as a cyclic alkane containing 6 carbons. Its molecular formula is C₆H₁₂, and its molecular weight is 84.16

Cyclohexene can be reacted with the addition of water to the primary reagent plus catalyst to yield trøτw-2-bromocyclohexanol.

Cyclohexene can also be reacted with the addition of acetic acid to the primary reagent plus catalyst to yield l-acetoxy-2-iodocyclohexane or methanol can be used as the organic solvent to yield 2-iodo-l-methoxycyclohexane.

Advantages of producing cohalogenated cyclohexane in accordance with the present invention are mild reaction conditions, low toxicity, inexpensive chemical reagents and excellent yields.

In other embodiments, the initial compound could include, for example, trans- stilbene.

BRIEF DESCRIPTION OF DRAWINGS

Further aspects of the present invention will become apparent from the following description, which is given by way of example only and with reference to the accompanying drawings in which:

Figure 1 is an illustration of a preferred embodiment of the present invention showing a scheme for the synthesis of hydroxyhalogenated lanosterol derivatives (where R=H or Ac). Figure 2 is an illustration of a preferred embodiment of the present invention showing a scheme for the separation of diastereomers (where R=H or Ac).

Figure 3 is an illustration of a preferred embodiment of the present invention showing the hydroxyhalogenation of cyclohexene.

Figure 4 is an illustration of a preferred embodiment of the present invention showing the hydroxyhalogenation of trans-stilbene

BEST MODES FOR CARRYING OUT THE INVENTION

1H and ¹³C NMR spectra were recorded in chloroform- i using a Bruker ADV DRX 400 MHz Spectrometer. Mass spectra (MS) were determined on a HP5970B spectrometer at ionising voltage of 70 eV interfaced with an ultra HP5890 gas chromatograph fitted with an HP-1 column (25m x 0.22mm). Column chromatography was performed on Merck silica gel (70-230 mesh). Thin layer chromatography was carried out on Merck precoated silica gel 60 F ₅₄ plates (0.25mm thick). Gas chromatography was performed on a HP5890 Gas chromatograph fitted with an ultra HP Ultra 2 column (25m x 0.32 mm). All melting points were obtained on a micro-melting point determination apparatus and were uncorrected. Lanosterol (62% purity) was purchased from Sigma and used as such. All commercial reagents were used as such, without purification.

All numbers that appear in bold relate to the figures. A number 1 is molecule number one, and so forth.

A) Production of Lanosterol acetate

Commercial Lanosterol (mix of 1 and 2 where R=H) (80g), in pyridine (460mL) and acetic anhydride (250ml) was heated at 90°C for 3 hours and then left to cool overnight. The solution was poured into ice water and filtered. The precipitate was recrystallised from aqueous acetone to afford lanosterol acetate 3β-acetoxy-5α- lanosta-8,24-diene (1 where R=Ac) (69g, 61.5% purity), the rest being made up of dihydrolanosterol acetate (2 where R=Ac) and other small impurities.

B) Figure 1 (The Halo [X=I] hydroxy (3) Route)

To a solution of lanosterol acetates (1 and 2 where R=Ac) (lOg) in acetone (900mL), water (15 mL) and hypophosphorous acid [2.7 mL (50% in water)] was added N- iodosuccinimide (3.82 g, 0.017mol).

C) Figure 1 (The Halo [X=Br] hydroxy (3) Route)

To a solution of lanosterol acetates (1 and 2 where R=Ac) (lOg) in acetone (900mL), water (15 mL) and hypophosphorous acid [2.7 mL (50% in water)] was added N- bromosuccinimide (3.04g, 0.017 mol).

D) Figure 2 (The separation of bromo hydroxy-lanosterol (3) into 24(R)-3β- acetoxy-24-bromo-25-hydroxy-5α-lanost-8-ene (3R where X=Br, R=Ac) and 24(S)-3β-acetoxy-24-bromo-25-hydroxy-5α-lanost-8-ene (3S where X=Br, R=Ac)):

The reaction mixture was stirred at room temperature (15 rnin) and diluted with water. The product was extracted into ether, washed thoroughly with saturated aqueous sodium thiosulphate, dried (MgSO₄) and evaporated under reduced pressure to give a crude mixture of dihydrolanosterol acetate (2 where R=Ac) and 24(i?,S)-3β- acetoxy-24-bromo-25-hydroxy-5α-lanost-8-ene (3 where X=Br, R=Ac) (11.7g), which was fractionated by flash column chromatography. Elution with hexane/ether (5:1) gave dihydrolanosterol acetate (2 where R=Ac)(3.7 g) with a minor by-product. Further elution with hexane/ether gave 3 (5.12 g, 69%).

Threefold recrystallisation from hexane/ethyl acetate afforded 24(S)-3β-acetoxy-24- bromo-25-hydroxy-5 -lanost-8-ene (3S where X=Br, R=Ac) (2.3 g), m.pt 163- 165°C; [α]_D +23.1° (cl.6,CHCl₃); Anal. Calcd. For C₃₂H₅₃O₃Br: C, 68.00; H, 9.38; Br,14.14. Found: C, 68.14; H, 9.21; Br, 14.20.

The filtrates were evaporated under reduced pressure and crystallized twice from hexane/ethyl acetate to afford 24(R)-3β-acetoxy-24-bromo-25-hydroxy-5α-lanost-8- ene (3R where X=Br, R=Ac) (2.1 g), m.pt 157-159 °C; [α]_D +66.8 ° (cl.6,CHCl₃); Anal. Calcd. For C₃₂H₅₃O₃Br: C, 68.00; H, 9.38; Br,14.14. Found: C, 68.28; H, 9.64; Br, 14.01. Structures were assigned by NMR and the absolute configurations confirmed by X-ray crystallography.

E) Figure 2 (Conversion of 3S to 3-β-acetoxy-24(R),25-epoxy-5-α-lanost-8-ene (4R) and 3 to 3-β-acetoxy-24(S),25-epoxy-5-α-lanost-8-ene (4S)):

A solution of 3S (R=Ac) (2g, 3.5 mmol) in dry isopropanol (150 mL) and potassium acetate (0.6 g, 6.1 mmol) was refluxed for 4 hours. The reaction mixture was diluted with water, extracted into ether and evaporated under reduced pressure. Crystallization from acetone yielded pure 4J? (R=Ac) (1.48g, 87%). Similar treatment of 3R (R=Ac) afforded pure 4S (R=Ac) (1.52 g, 89%).

F) Figure 3 (Formation of trans -2-bromocyclohexanol)

To a solution of cyclohexene 5 (1 mL (0.807g, 0.0098 mol) in acetone (20 mL), water (lmL) and hypophosphorous acid (1.75 mL[50% in water]) was added N- bromosuccinimide (2.26g, 0.0127 mol). The reaction mixture was stirred at room temperature (15 mins), a saturated solution of NaCl (5 mL) was added and the acetone removed under reduced pressure. The resulting mixture was extracted with diethyl ether (80mL x 3) and the combined organic extracts washed with water until neutral and concentrated. The direct analysis of crude extracts by gas chromatography found quantitative conversion (>99%) of cyclohexene 5 to trans-2- bromocyclohexanol 6. G) Figure 3 (Formation of traras -2-iodocyclohexanol)

Trans-2-iodocyclohexanol 7 was synthesized according to the above procedures and led to 99% conversion.

H) Figure 4 (Formation of eryt/*rø-2-Bromo-l,2-Diphenylethanol (9)) To a solution of tr ns-stilbene 8 (0.5g, 0.00277 mol) in acetone (20ml), water (1ml) and hypophosphorous acid [0.250 ml (50% in water) was added N-bromosuccinimide (0.35g, 0.00126 mol). The reaction mixture was stirred at room temperature (30 min) and additional hypophosphorous acid [0.174 ml (50%in water] and N- bromosuccinimide (0.35g, 0.00126 mol) was added.

The reaction mixture was stirred at room temperature (90 min) then acetone was removed under reduced pressure and the resulting mixture was extracted with diethyl ether (30 ml x 2). The organic extracts were washed with water until neutral and concentrated.

The dry residue was dissolved with petroleum ether and purified by short column flash chromatography [elution with petroleum ether then with petroleum ether: diethyl ether (5:1)] to afford 9 as a colorless solid (0.61g, 80%), m.p. 82-83°C.[α]_D=0 (1.6 in CCI₃).

It is important to note that hydroxybromination of stilbene yielded two products, which were isolated by flash chromatography and proved to be unreacted 8 and reaction product 9. When unreacted 8 was re-exposed to the reaction conditions, a 95% yield of 9 was obtained. The yield of hydroxybromination reaction for stilbene according to the literature data is 50% (by GC, not isolated) (Masuda et al. J. Org. Chem. 1994, 59, 5550-5555).

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined by the appended claims.

Claims

WHAT WE CLAIM IS:

1. A method of producing a co-halogenated compound by adding halogen(s) and another group(s) to a multiple bond in an initial compound,

the method characterised by the step of

2. A method as claimed in claim 1, wherein the group(s) to be added to the co- halogenated compound is a hydroxyl group(s).

3. A method as claimed any one of claims 1 or 2, wherein the initial compound is a carbon based compound.

4. A method as claimed any one of claims 1 to 3, wherein the primary reagent includes a halogenating compound and a solvent.

5. A method as claimed in claim 4, wherein the halogenating compound is N- halo-succinimide.

6. A method as claimed in any one of claims 4 or 5 wherein the solvent is organic.

7. A method as claimed in claim 6 wherein the solvent is acetone.

8. A method as claimed in any one of claims 1 to 7 wherein the catalyst is:

a) a moderately strong acid with a pKa of less than or equal to 2.0,

b) with a reduction potential of greater than or equal to +3.0V, and c) a conjugate base which is a weak nucleophile.

9. A method as claimed in any one of claims 1 to 8 wherein the catalyst is hypophosphorous acid.

10. A method as claimed in any one of claims 1 to 8 wherein the catalyst is oxalic acid.

11. A method as claimed in any one of claims 1 to 8 wherein the catalyst is sulphurous acid.

12. A method as claimed in any one of claims 1 to 11 wherein the initial compound is in a solution containing steroid(s).

13. A method as claimed in 12 wherein the steroid solution is an initial lanosterol solution.

14. A method as claimed in any one of claims 12 to 13 wherein the initial compound includes a protecting group.

15. A method as claimed in claim 14 wherein the protecting group is an acetyl group.

16. A method as claimed any one of claims 12 to 15 wherein the protecting group is bonded to the oxygen on the C-3 position.

17. A method as claimed in any one of claims 13 to 16 wherein the co-halogenated compound is a diastereomeric mix of hydroxy-halogenated lanosterol derivatives.

18. A method as claimed in claim 17 wherein the hydroxy-halogenated lanosterol derivatives are 24(i?,S)-halo-25-hydroxylanosterol diastereomers.

19. A method as claimed in claim 18 wherein the 24(R,S)-halo-25- hydroxylanosterol diastereomers are separated into 24(R)-halo-25- hydroxylanosterol and 24(S)-halo-25-hydroxylanosterol.

20. A mixture of R and S diastereomers produced by the method as claimed in any one of claims 1 to 18.

21. A co-halogenated compound produced by the method as claimed in any one of claims 1 to 18.

22. A pharmaceutical composition including a co-halogenated compound as claimed in any one of claims 1 to 18.

23. An intermediate for the production of lanosterol and the lanosterol derivatives characterised in that the intermediate is formed by the method as claimed in any one of claims 1 to 18.

24. A method of producing a co-halogenated compound substantially as herein described and with reference to figures 1 to 4.

25. A method of producing a co-halogenated compound substantially as herein described and with reference to examples A to H.