AND ANALOGS THEREOF

Technical Field This invention relates generally to synthetic methods involving steroids, and more particularly relates to a novel method for preparing cholesta-5,7-diene-3B,25-diol and analogs thereof. The invention additionally relates to novel steroids useful as intermediates in the aforementioned process.

Background of the Invention

The present invention derives from the development of a biotechnological fermentation process that produces a yeast sterol mixture enriched in cholesta-5,7,24-triene-3B-ol and accompanied by other di-olefinic yeast sterol metabolites. The aforementioned trienol, having the chemical structure (I)

is a valuable compound useful, inter alia, as an intermediate in the synthesis of a variety of compounds related to vitamin D3 derivatives. Commonly assigned U.S. patent application Serial

No. 07/869,574, filed April 15, 1992 on even date herewith and incorporated by reference herein, entitled "Isolation of Steroids Containing a 5,7 -Diene Functionality From a Sterol Mixture" (inventors J. Johansson et al.), relates to a method for isolating and purifying the trienol (I) from a fermentation mixture containing yeast sterol metabolites, including lanosterol, 4,4- dimethylzymosterol, 4-methylzymosterol, zymosterol and cholesta-7,24-diene-3B-ol. That method involves the preparation and isolation of certain Diels-AIder adducts of the 5,7-diene- containing steroid to be isolated.

The present invention derives from the aforementioned process, and involves the rnodificafion of the Diels-AIder adducts at the Δr^ double bond prior to adduct cleavage. The present process is a simple, straightforward synthesis which is especially useful in the preparation of cholesta-5,7-diene~3B,25-dioI, a pro-vitamin D3 metabolite which may be converted by sunlight or otherphotochemical methods to yield 25-hydroxy vitarniα D3. The invention also enables the preparation of analogs of cholesta-5,7-diene-3B,25-diols which contain structural modifications at the C-l, C-2, C-3 and or C-ll positions. The compounds which may be prepared using the techniques of the present invention are valuable as synthetic intermediates in the preparation of vitamin D analogs. They also constitute, in a general sense, high value-added chemical products and may be used in a variety of contexts, e.g., in topical pharmaceutical formulations for the treatment of skin diseases or the like, in oral vitamin compositions, and as livestock feed additives.

Overview of the Art

The following references relate to one or more aspects of the present invention: S.C. Eyley et aL, J.C.S. Perkins Trans. I. pp. 731-735 (1976), describe a method for synthesizing 25-hydroxyprovitamin D3 and 25ξ,26-dihy droxyprovitamin D3. The method involves initial reaction of the C-22 aldehyde derived by degradation of ergosterol with a Grignard reagent derived from 4-chloro-2-methylbut-l-ene, followed by reductive elimination of the mesylate of the resulting C-22 alcohol. J.P. Moreau et al., J. OTJΓ. Chem.22(14):2018-2023 (1974), is a background reference which describes the synthesis of 5α-cholesta-7,24-dien-3B-ol and cholesta-5,7,24-trien-3β-ol.

J.W. Blunt et al., Biochemistry 8(2):671-675 (February 1969), describe methods of synthesizing cholesta-5,7-diene-3B,25-diol, followed by conversion to 25-hydroxycholecaliferoL

S.S. Yang et al., Tetrahedron Letters 27:2315-2316 (1977), is a background reference describing a method for synthesizing 25-fluorovitamin D3.

D.R. Crump et aL. J.C.S. Perkins Trans. I. pp. 2731-2733 (1973), describes a method for synthesizing (22S)-hydroxyvitamin D using ergosterol acetate as a starting material. The synthesis involves selective epoxidation of the 22,23-double bond of ergosterol acetate, followed by a Grignard reaction on the hexanor-22-aldehyde, and irradiation. A.M. Moiseenkov et al., Bioorg. Khim..9(1):118-122 (1983) disclose a cyclopropylcarbinyl rearrangement for preparing 25-hydroxychloresterol and 25- hy droxyprovitamin D3. The synthesis involves an intermediate which is a Diels-AIder adduct of a

5,7-diene.

G.M. Segal et al.. Bioorg. Khim..7(3):429-435 (1981) set forth a synthesis of (Z)- 17(20)- dehydrochloesterol and 25-hydroxprovitamin D3 starting from 17-ketosteroids. The reference shows a protected 5,7-diene-containing steroid in the form of a Diels-AIder adduct.

Summary of the Invention

Accordingly, it is a primary object of the invention to address the above-mentioned need in the art, and to provide a simple, straightforward method for preparing cholesta-5,7-diene-3B,25- diol in isolated, purified form.

It is another object of the invention to provide a method for preparing analogs of cholesta- 5,7-diene-3B,25-diol which contain structural modifications at the C-l, C-2, C-3 and/or C-ll positions.

It is still another object of the invention to provide such a method which involves treating a Diels-AIder adduct of a steroid containing a 5,7-diene functionality and a Δ^ double bond with an oxidizing agent, followed by reduction of the resulting 24,25-oxido moiety and adduct cleavage.

It is still a further object of the invention to provide novel steroids which are analogs of cholesta-5,7-diene-3B,25-diol as will be described in detail below.

Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.

In one aspect of the invention, a synthetic method is provided which involves (a) reacting a Diels-AIder adduct having the structural formula

with an oxidizing agent effective to convert the C-24 olefin functionality to a 24,25-oxido moiety; and (b) treating the 24,25-oxido intermediate so provided with reducing agent selected to (i) reduce the 24,25-oxido moiety to a 25-hydroxyl group, (ii) cleave the Diels-AIder adduct to yield the corresponding 5,7-diene, and (iii) convert the C-3 -OR^ moiety to a C-3 hydroxyl group. In the above structural formula: the R's are both N or both C-Q wherein the Q's are H or together form a third bond; X and Y are electron-withdrawing groups and independently are -COOH, -CHO, -NO2,

-CN, -COORI or -COR* wherein R* is lower alkyl, or X and Y may be linked together to form a -(CO)-Z-(CO)- bridge in which Z is lower alkylene, lower alkenylene, monocyclic arylene of 5 to

7 carbon atoms with up to 4 ring substituents, -S-, or -NR ² - wherein R ² is H, lower alkyl or monocyclic aryl of 5 to 7 carbon atoms and containing up to 5 ring substituents, wherein the ring substituents are -(CO2) _n -NH2, -(CH ₂ )-COOH, -NO2, halogen or lower alkyl, wherein n is an integer in the range of 0 to 6 inclusive;

R3 is H or R'CO- wherein R' is lower alkyl or monocyclic aryl of 5 to 7 carbon atoms; and R , R5 and R" are independently H, hydroxyl or lower alkyl.

In a second aspect of the invention, novel compounds are provided having the structural formula

wherein the R's, X, Y, R^, R^, R^ and R > are as defined above.

In another aspect of the invention, novel compounds are provided having the structural formula

wherein R^, R and R-* are as defined above.

Detailed Description of the Invention

Before the present methods are disclosed and described, it is to be understood that this invention is not limited to the use of specific Diels-AIder adducts, specific 5,7- diene-containing steroids, or to the use of specific synthetic reagents, i.e., dienophiles, oxidizing agents, reducing agents, or the like, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a sterol" includes mixtures of sterols, reference to "a steroid" includes mixtures of two or more steroids, and the like. In this specification and in the claims which follow reference will be made to a number of terms which shall be defined to have the following meanings:

"Alkyl" refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Preferred "alkyl" groups herein contain 1 to 12 carbon atoms. "Lower alkyl" refers to an alkyl group of 1 to 6, more preferably 1 to 4, carbon atoms.

"Alkylene" refers to a difunctional saturated branched or unbranched hydrocarbon chain containing from 1 to 24 carbon atoms, and includes, for example, methylene (-CH2-), ethylene (-

CH ₂ -CH ₂ -), propylene (-CH ₂ -CH ₂ -CH ₂ -), 2-methylpropylene [-CH2-CH(CH ₃ )-CH ₂ -], hexylene [-(Q^g-] and the like. "Lower alkylene" refers to an alkylene group of 1 to 6, more preferably 1 to 4, carbon atoms.

"Alkenylene" refers to a difunctional branched or unbranched hydrocarbon chain containing from 2 to 24 carbon atoms and at least one double bond. "Lower alkenylene" refers to an alkenylene group of 2 to 6, more preferably 2 to 5, carbon atoms. "Alkynyl" refers to a branched or unbranched acetylenically unsaturated hydrocarbon group of 2 to 24 carbon atoms such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, octynyl, decynyl, tetradecenyl, hexadecynyl, and the like. "Lower alkynyl" refers to an alkynyl group of 2 to 6, more preferably 2 to 4, carbon atoms.

"Acyl" refers to a group of the structure -(C=O)-R\ where R' is as described herein. Acyl, therefore, includes such groups as, for example, acetyl, propanoyl (or propionyl), isopropanoyl, n-butanoyl (or n-butyryl), benzoyl, phenylacetyl, and the like. "Lower acyl" refers to an acyl group wherein R' is lower alkyl as defined above.

"Aryl" refers to a phenyl or 1- or 2-naphthyl group. "Monocyclic aryl" refers to a phenyl group. Optionally, these groups are substituted with up to five ring substituents selected from the group consisting of -(CH2) _n -NI_2, -(CH ₂ ) _n -COOH, -NO2, halogen and lower alkyl, where n is an integer in the range of 0 to 6 inclusive.

"Arylene" refers to a difunctional aromatic moiety; "monocyclic arylene" refers to a phenylene group. These groups may be substituted with up to four ring substituents selected from the group consisting of -(CH2) _n -NH2, -(CH2) _n -COOH, -NO2, halogen and lower alkyl, where n is an integer in the range of 0 to 6 inclusive.

"Halo" or "halogen" refers to fluoro, chloro, bromo or iodo, usually regarding halo substitution for a hydrogen atom in an organic compound. Of the halos, chloro and fluoro are generally preferred with fluoro generally being the more preferred.

"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, "optionally substituted phenyl" means that the phenyl may or may not be substituted and that the description includes both unsubstituted phenyl and phenyl wherein there is substitution.

In describing the location of groups and substituents, the following numbering system will be employed.

This system is intended to conform the numbering of the cyclopentanophenanthrene nucleus to the convention used by the IUPAC or Chemical Abstracts Service. The term "steroid" as used herein is intended to mean a chemical compound having the aforementioned cyclopentanophenanthrene nucleus.

The symbols "α" and "β" indicate the specific stereochemical configuration of a substituent at an asymmetric carbon atom in a chemical structure as drawn. Thus "α", denoted by a broken line, indicates that the group at the position in question is below the general plane of the molecule as drawn, and "β", denoted by a bold line, indicates that the group at the position in question is above the general plane of the molecule as drawn. In the present disclosure, if bonds are not explicitly indicated to be "α" or "β", it should be assumed that the structural formula encompasses both types of compounds, with the stereochemical configuration of the naturally occurring steroid molecule preferred.

In addition, the five- or sϊx-membered rings of the steroid molecule are often designated A, B, C and D as shown.

The term "sterol" as used herein is intended to mean a steroid molecule having the backbone structure illustrated above, and containing at least one hydroxyl group, typically a single hydroxyl group at the 3-position.

The term "purified compound" as used herein intends a composition which contains at least about 80 wt.% of that compound, preferably at least about 90 wt.%, and most preferably at least about 99 wt.%.

The Diels-AIder adducts which serve as the starting materials in the present methods have the general structural formula

wherein the R's, X, Y, R , R^ and R^ are as defined above. The R^ moiety, if other than H, is thus a hydroxyl-protecting group; typical R^ moieties are H, CH3CO-, and Cg^CO-. If R , R-> and R" are other than H and OH, they will generally be methyl or ethyl, more typically methyl.

These Diels-AIder adducts may be prepared using the method set forth in detail in commonly assigned, co-pending U.S. Patent Application Serial No. 07/869,574 incorporated by reference above. Briefly, that method involves treating a 5,7-diene-containing steroid with a dienophile having the structural formula X-R=R-Y wherein the R's are both N or both C-Q where the Q's are H or together form a third bond. Such dienophiles will thus have the structure X-N=N-Y, X- (CQ)=(CQ)-Y, or X-C≡C-Y. The substituents X and Y are electron-withdrawing groups which are as defined earlier herein. Examples of particular dienophiles within the aforementioned group include the following:

These dienophiles are available commercially from a number of sources, e.g., from the Aldrich Chemical Company, Milwaukee, WI. As will be appreciated by those skilled in the art, such dienophiles may also be readily synthesized using conventional techniques (see, e.g., S.W. Moje and P. Beak, J. Org. Chem. 39f20.:2951 (1974), and K. Rufenacht, Helv. Chim. Acta 51:518 (1968)).

Alternatively, a dienophiie precursor is used which may be converted to a dienophiie with a suitable oxidizing agent. In this case, the dienophiie precursor has the structural formula X-NH- NH-Y wherein X and Y are as defined above. Exemplary dienophiie precursors are wherein X and Y are linked together to form a -(CO)-Z-(CO)- bridge, with Z as defined above. Preferably, in this embodiment, Z is monocyclic arylene of 5 to 7 carbon atoms substituted with up to 2 substituents selected from the group consisting of -(Cf_2) _n -NI_2 and -(CH2) _n -COOH, wherein n is an integer in the range of 0 to 6 inclusive. Dienophiie precursors within the aforementioned group may be available commercially or may be readily synthesized using starting materials and techniques known to those skilled in the art of synthetic organic chemistry. Examples of particular dienophiie precursors useful herein (again, such compounds are available commercially, or may be readily synthesized) include the following:

(XIII) (XIV)

Any oxidizing agent capable of oxidizing the dienophiie precursor to an active dienophiie may be used, with the exception of oxidizing agents which could interfere with the formation of the Diels-Alder adduct or which could interact detrimentally in some other way with any of the sterols in the sterol mixture. Exemplary oxidizing agents include potassium peroxymonosulfate, lead tetraacetate, iodosobenzene diacetate, N-bromosuccinimide and t-butyl hypochlorite.

Either of the aforementioned reactions, i.e., treatment of the sterol mixture with a dienophiie having the structure X-R=R- Y, or with a dienophiie precursor of the structure X-NH-NH- Y and an oxidizing agent, results in the formation of a Diels-Alder adduct. These reactions are illustrated in the following schemes:

Reaction (1) (II)

(XVI)

Reaction (2)

As explained in co-pending U.S. Serial No. 07/869,574, incorporated by reference above, both types of reactions are carried out in an inert atmosphere, in a non-reactive, preferably polar organic solvent effective to dissolve the reactants. With the second type of reaction, it is preferred that the oxidizing agent be added gradually to a solution of the sterol and the dienophiie in the selected solvent, and that the procedure be carried out at a relatively low temperature, i.e., 10°C or lower (0°C to 5 ^* C, as may be obtained by an iceXwater bath, is optimal). At least about 15 minutes, preferably at least 1 hour, should be allowed for the reaction to occur.

At this point, the adduct is removed from the reaction mixture, preferably chromatographically. A silica gel column which will preferentially bind the adduct is particularly preferred. The adduct is then purified using conventional methods, e.g., using recrystallization, precipitation or chromatographic techniques, prior to use in the present synthetic method. The chemical and physical properties of the Diels-Alder adduct can be varied by manipulating the substituents present on the dienophiie as well as by varying R^. For example, basic properties can be imparted to the Diels-Alder adduct by the use of a dienophiie containing a basic substituent, e.g., - I_2, -(C__2) _n -N__2, or the like. The adduct is then a basic molecule and separable from the reaction mixture using acid extraction. Similarly, acidic properties can be imparted to the Diels-Alder adduct by the use of a dienophiie containing an acid substituent, e.g., -COOH,

-(C__2) _n -COOH, or the like. The adduct will then be an acidic molecule and separable from the reaction mixture using basic extraction.

Also, as alluded to above, after preparation of the Diels-Alder adduct, the moiety present at

R3 may be converted to a functionality which imparts desirable crystallization and/or precipitation parameters. For example, a hydroxyl group present at C-3 may be readily converted to a benzoate species, which in turn will make the adduct more crystalline and more readily separable from the reaction mixture.

As noted above, the present invention involves reaction of the Diels-Alder adduct with an oxidizing agent effective to convert the Δ ² double bond to a 24,25-oxido moiety, followed by treatment with a reducing agent selected to effect adduct cleavage, opening of the 24,25-epoxide, and conversion of the C-3 moiety to a hydroxyl group. The first reaction, oxidation, may be represented by the following scheme:

40

The reaction is preferably carried out on the adduct as isolated and purified as described in the preceding paragraph. Virtually any oxidizing agent may be used here, providing that the agent is effective to convert the 24,25-double bond to an epoxide functionality and does not cause any side reactions to occur in the remainder of the molecule. Suitable oxidizing agents include, for example, peracetic acid, __2θ /base, perphthahc acid, N-bromosuccinimide (e.g., in an acetone/water rαixture), and m-chloroperoxybenzoϊc acid. The reaction is carried out by gradually adding the oxidizing agent to a solution of the Diels-Alder adduct in a selected solvent, preferably a nonpolar organic solvent, at a relatively low temperature, i.e., 10 ^β C or lower (0 ^β C to 5°C, as may be obtained by an ice/water bath, is optimal). At least about 15 minutes, preferably 30 minutes or more, is allowed for the reaction to occur. The 24,25-oxido derivative may be isolated by evaporating the solvent from the reaction mixture.

The 24,25-oxido isolated in the preceding step is then treated with a reducing agent selected such that three separate molecular transformations will occur approximately simultaneously: (1) cleavage of the Diels-Alder adduct to regenerate the 5,7-diene moiety; (2) conversion of the 24,25- epoxide moiety to yield a 25-hydroxy functionality; and (3) if R^ is other than hydrogen in the Diels-Alder adduct, conversion of the C-3 moiety to a hydroxyl group. This reaction may be represented as follows:

40

Suitable reducing agents include, for example, lithium aluminum hydride ("LAH"), diisobutyl aluminum hydride ("DiBAL"), Red-Al® (a solution of sodium bis(2-methoxy-ethoxy) aluminum hydride in toluene, available from the Aldrich Chemical Company, Inc., Milwaukee WI.) or the like. Lithium aluminum hydride is particularly preferred. The reaction proceeds initially at a low temperature, i.e., 10 ^* C or lower (again, as may be obtained by an ice/water bath), followed by, after at least about 30 minutes, warming to at least about 50° C for at least several minutes. Excess reducing agent and any salts or derivatives thereof are then removed, e.g., by filtration through celite or the like. Evaporation of the reaction mixture will then give rise to the 5,7-diene- containing, 25-hydroxy steroid.

It should be pointed out that the 24,25-oxido Diels-Alder adduct described hereinabove is believed to be a novel compound, i.e., having the structural formula

wherein the R's, X, Y, R ³ , R ⁴ , R ⁵ and R ⁶ are defined above. Preferred compounds within this class include the following:

20

40

25

40

40

It is to be understood that while the invention has been described in conjunction with preferred specific embodiments thereof, the foregoing description, as well as the examples which follow, are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Example 1

To a stirred solution under argon of a crude sterol mixture (obtained from Amoco; 50 g) containing squalene, lanosterol, 4,4-dimethylzymosterol, 4-methyl-zymosterol, zymosterol, cholesta-7,24-diene-3β-ol and cholesta-5,7,24-triene-3B-ol (11.0 g, pure trienol, 28.8 mmol) dissolved in dichloromethane (600 ml) was added phthalhydrazide (obtained from Aldrich; 15.0 g, 92.5 mmol). This solution was cooled in an ice/water bath (0-5"C). To the cooled solution was added dropwise a solution of lead tetraacetate (15.0 g, 33.9 mmol) and acetic acid (1.95 ml) in dichloromethane (215 ml). Addition time 30 min. After stirring at 0-5°C for 1.5 hr, the reaction mixture was stirred at room temperature for a total of 4 hr. TLC on silica gel impregnated with silver nitrate showed no trienol present. The reaction mixture was filtered through celite and the combined dichloromethane solution was washed with water and sodium bicarbonate, and again with water. Evaporation of the solvent gave a yellow crude product that was purified in the following way. The crude product was dissolved in a mixture of ethyl acetate and 20% hexane, and filtered through silica gel (125 g). After all the non-reacted sterols had been washed off the column, the adduct was eluted with 50% ethyl acetate in hexane. The yield of pure adduct was

14.1 g, or 90.4%. NMR, IR and mass. spec, were in agreement with the proposed structure.

E ample 2

(XXVI) (xxvπ)

Procedure: To a solution of the Diels-Alder adduct (13.8 g, 25.4 mmol) in dichloromethane (250 ml) and cooled to (0-5 ^* C) was added portionwisem-chloroperoxybenzoic acid (50-60%, 10.4 g =33.1 mmol) under a period of 5 minutes. The reaction mixture was stirred for 30 minutes, when a TLC sample showed no starting material remaining. The reaction mixture was washed with sodium bisulfite (10%) sodium bicarbonate (5%) and water. The clear dichloromethane solution was dried over sodium sulfate. Evaporation of the solvent gave the epoxide 13.4 g (94.0%) mp 129-131 ^* C. Analytical for C ₃₅ H4 ₆ N ₂ O4 Calcd: C, 75.23; H, 8.30;

N, 5.01. Found: C, 74.83; H, 8.09; N, 4.89. NMR, IR, and Mass spec are in agreement with proposed structure.

Example 3

(XXVTJD

(xxvn)

(XXVH)

The following procedure enables recovery of compound (XXVIII) as well as compound

(XXVII):

A solution of epoxy Diels-Alder adduct (I) (0.50 g, 0.90 mmole) in THF (10 ml) was cooled by submersion in an ice/water bath at 5°C under an argon atmosphere was treated with 1 M LLAIH4 in THF (3.1 ml, 3.1 mmole) at a dropwise rate. The ice bath was removed and the reaction mixture was stirred at room temperature for 1.5 hours. Again cooled, the reaction mixture in the ice bath and added dropwise a saturated NH4CI solution (1 ml). The mixture was filtered, washed the solid with THF (10 ml), and evaporated to dryness leaving 0.45 g of tan solid.

Purification by flash column chromatography on Merck grade 60 silica gel (10 g) using EtOAc/CH Cl ₂ (10:90) gave 81 mg (23% yield) of epoxy diene (XXVIII) which was crystallized from acetone mp 124- 126 ^* C. Anal, for C ₂ 7H42θ ₂ -acetone: Calcd: C, 78.99; H, 10.59.

Found: C, 78.89; H, 10.67. Further elution gave 143 mg (40% yield) of diol (XXVII) which was crystallized from MeOH, mp 185-187°C.

Example 4

Procedure: To the Diels-Alder epoxide (12.9 g, 23.1 g) dissolved into THF (275 ml) and cooled to (0-5 ^* C) was added dropwise with stirring a solution of lithium aluminum hydride 1 M in THF (90 ml, 90 mmol). The reaction mixture was heated to 60 ^* C for 30 minutes. (The reaction proceeds in two steps: first the protective group is removed, and second the epoxide is opened). The reaction was cooled to 0-5 ^* C and a saturated ammonium chloride solution was added slowly (until excess LAH was destroyed =25 ml). The reaction mixture was stirred for 30 minutes at (0- 5 ^* C) and was then filtered through celite. The filterbed was washed with some THF. Evaporation of the solvent in vacuo gave the crude material. The crude material was recrystallized from methanol to give a total yield of 6.49 g (70.2%) mp 183-185° C. A second recrystallization raised the mp to 190-193 ^β C. NMR, IR, and Mass spec were all in agreement with the proposed structure (XXVE).

Example 5 The procedure of Examples 1 through 4 may be repeated, except that 4-phen l-l,2,4- triazoIine-3,5-dione (e.g., as may be obtained from Aldrich Chemical Co.) is substituted for phthalhydrazid in the procedure of Example 1 and no lead tetraacetate is used.