IE51714B1 - 5,7-diene intermediates useful in preparing calcitroic acid and esters thereof - Google Patents

Description

This invention relates to novel intermediates useful in the production of' biologically active vitamin D compounds, in particular esters of calcitroic acid.

Calcitroic acid is an in vivo metabolite of 5 la,25-dihydroxyvitamin (la,25-(0H)jD^). This latter compound is the most potent known metabolite in the vitamin D series for the regulation of calcium and phosphate homeostasis (see De Luca and Schnoes, Ann.

Rev. Biochem. 45, 631 1976). Recently it was discovered that rats rapidly metabolize la,25-(OH)to a compound having an acid function in the steroid side chain.

This novel metabolite was isolated as the corresponding methyl ester and identified as methyl la,3(i-dihydroxy24-nor-9,10-secochola-5,7,10(19)-trien-23-oate depicted by structure 1 (R = CH^) for which the trivial name methyl calcitroate has been proposed: (Esvelt et al, Biochemistry 18, 3977, 1979).

Th® in vivo metabolite is therefore the corresponding free acid, namely calcitroic acid as shown by structure 1(R = H) below. (An alternative trivial name for this compound is Ια-hydroxycalcitroic acid).

I COOR HO^ 1: R = H calcitroic acid R « CHj methyl calcitroate A process for the chemical synthesis of calcitroic acid and of esters thereof (where R is hydrogen, alkyl or benzyl) has now been developed. The general process is outlined in Process Schematic 1.

Arabic numerals identifying specific intermediates and products refer to this Process Schematic.

In this specification the term alkyl denotes a lower alkyl radical of from 1 to 5 carbons which may be straight chain or branched (e.g. methyl, ethyl, butyl, isopropyl, isobutyl) and the term acyl denotes an aliphatic acyl group of from 1 to 5 carbons, such as formyl, acetyl, propionyl, butyryl, or an aromatic acyl group such as benzoyl or substituted benzoyl, e.g. toluoyl, nitrobenzoyl or halobenzoyl.

The process uses commercially available acid 2 (where R is a hydroxy-protecting group, e.g. acyl, tetrahydropyranyl, methoxymethyl, alkylsilyl) as starting material which, by an Arndt-Eistert homologation sequence using the general method of Ryer and Gebert (J.Am.Chem.Soc. 74. 43, 1959) gives the side chain desired in the final product. Use of methanol in the silver oxide-catalyzed Wolff rearrangement gives the methyl ester .3 (e.g. R. = acyl) in about 60% yield after recrystallization. Ester 3. is converted to diene 4a. by allylic bromination and dehydrobromination according to well-known procedures (Hunziker and Mullner, Helv. Chim. Acta 41. 70, 1958; Napoli et al Arch. Biochem. Biophys. 197. 119, 1979).

Removal of the hydroxy-protecting group (e.g. by mild acid or base hydrolysis, depending on the protecting group present, and according to well-known procedures) yields the hydroxy ester 4b (R = CHg). More vigorous basic hydrolysis (e.g. 10% NaOH/dil. methanol, 60-100°C, 1-3 hr.) also cleaves the ester function to give the acid (4b.

R = H) and from this acid other alkyl esters are readily prepared, if desired. For example, esterification of the acid or the acid halide with any desired low molecular weight alcohol according to procedures well known in the art, yields other alkyl or aryl esters suitable for subsequent interconversions (e.g. 4b where R is lower alkyl such as methyl, ethyl, propyl, butyl, or a benzyl group) . Irradiation of ester 4b (R = alkyl) with ultraviolet light yields the previtamin ester 5 (R = alkyl), which is isomerized by heating (60-80°C) in an alcohol or benzene solvent to the vitamin ester 6 (R » alkyl). Alternatively, ester 6 (R = alkyl) can, of course, also be obtained by subjecting the 3-0-protected derivatives of the 5,7-diene intermediate of general structure 4b to the irradiation/thermal isomerization sequence described above, to give the 3-0-protected derivative of ester 6. Subsequent removal of the protecting group yields hydroxy ester 6. The free acid (compound 6, R = H, can readily be obtained from the ester 6 (R - alkyl) by vigorous base hydrolysis (e.g. 1094 NaOH, e.g. alcohol, 1-3 hr, 60-90°C) and from the acid the corresponding 3-0protected derivatives are readily prepared, if desired, by standard procedures. The 3-0-acylates are preferred derivatives.

Introduction of the Ια-hydroxy function can be achieved by the method of Paaren et al (Proc. Nat. Acad. Sci. USA 75, 2080, 1978). Ester 6 (R = alkyl) is converted to the cyclovitamin ester 7, by a two-step process involving tosylation of £ to the 3-0-tosyl derivative followed by bicarbonate buffered methanolysis of the tosylate (Paaren et al, supra; sheves and Mazur, J. Am. Chem. Soc. 97. 6249, 1975). Subsequent allylic oxidation of 7 (R « alkyl) with selenium dioxide and t-butyl hydroperoxide in a halocarbon solvent yields the desired Ια-hydroxy cyclovitamin ester 8 (R = alkyl). Vigorous base hydrolysis of either 7 or 8 (where R = alkyl) using conditions as described for ester 6 provides the corresponding free acids. The 1-hydroxy group of ester 8 (R = alkyl) can be protected (e.g. by acyl, tetrahydropyranyl, methoxy-methyl, alkylsilyl groups) using standard derivatization conditions, the 1-0-acylates (e.g. acetyl, formyl, benzoyl) being preferred derivatives. The 1-0-acylates can be subjected to acid catalyzed solvolysis (e.g. using formic, acetic or paratoluene sulfonic acid) as described by Paaren et al (supra.) to yield either 1,3-di-0-acyl-, or l-0-acyl-3-hydroxyvitamin D esters, depending upon the solvolysis conditions chosen, as a mixture of the 5.6-cis and 5.6-trans-isomers.

For example, solvolysis in carboxylic acids (e.g. formic or acetic acid) leads to 1,3-di-0-acylates where the C-3-acyl group corresponds to the acyl moiety of the acid used, whereas solvolysis with sulfonic acids in aqueous media yields l-0-acyl-3-hydroxy-products, as fully described by Paaren et al. After separation of the 5.6-cis andtrans mixture, the 5.6-cis product is hydrolyzed in mild alkali to yield the desired calcitroic acid ester (compound 1, R = alkyl).

A preferred procedure consists of the direct solvolysis of unprotected hydroxy ester 8^ with a low molecular weight carboxylic acid (acetic acid being a preferred acid) to give Ια-hydroxyvitamin D ester 3-0-acyl (9, R · alkyl) and the corresponding 5.6-transisomer (la-hydroxy-5,6-transvitamin D ester 3-0-acyl) in a mixture ratio of ca. 3:1. These isomers are readily separated by chromatography (e.g. high pressure liquid chromatography, thin layer chromatography). Mild basic hydrolysis of 9 (R = alkyl) then yields the desired calcitroic acid ester 1 (R » alkyl). Vigorous alkaline hydrolysis of the ester (e.g. 10% NaOH, dilute alcohol, 60-100°C, 1-3 hr) provides the corresponding acid, calcitroic acid, compound 1 (R = H), in pure form.

Acylated derivatives of these calcitroic acid esters (e.g. compounds of structure 1, R « alkyl) with 1-0-acyl or l,3-di-0-acyl groups can be obtained by the use of alternative solvolysis conditions as described above or by acylation of intermediate 9 to the corresponding 1,3-di-0-acyl derivative (where the acyl groups may be the same or different) using well known acylation procedures. Similarly the l,3-di-0-acyl derivatives of calcitroic acid (i, R = H) can readily be obtained by direct acylation of this acid. If calcitroic acid or its esters are desired with oth.er 1,3-0-protecting groups, such groups may be conveniently selected from tetrahydropyranyl, methoxymethyl or alkylsilyl.

The 5.6-trans intermediates obtained after solvolysis of the cyclovitamin intermediate (e.g. compound with 5.6-trans double bond configuration) can be converted by hydrolysis of the acyl group to 5.6-trans calcitroic acid esters and further hydrolysis of the ester yields, 5.6-trans calcitroic acid. These hydrolysis steps can be conducted exactly as described for the 5,6-cis compounds. Any of these 5.6-trans intermediates or products are of course convertible to the corresponding natural 5.6-cis compound, by the wellknown photochemical isomerization process of Xnhoffen et al, Chem. Ber. 90, 2544 (1957). The novel la-substituted cis and trans products as well as the lot-substituted cis products and their previtamins are described and claimed in our. Patent Specification .No. 51713 The present invention provides the diene c intermediates of the formula: where R^ is hydrogen or a hydroxy-protecting group such as acyl and R^ is hydrogen, alkyl, for example methyl, or benzyl. It will, of course, be appreciated that the compounds in which R2 is hydrogen and R^ is a hvdroxy-protecting group can readily be obtained from the hydroxy-protected esters 4a For the following specific Exanples NMR were taken in CDC13 with a Bruker WH-270 FT spectrometer. Mass spectra were obtained at 110-112*0 above ambient at 70 eV with an AEI MS-9 spectrometer coupled to a DS-60 data system. Ultraviolet (UV) absorption spectra were recorded in methanol with a Beckman (Trade Mark) Model 24 recording spectrophotometer. HPI£ was performed on a Waters Associates Model ALC/GPC 204 using Zorbax-SIL (Dupont) 6.4 mm x 25 cm or 4.8 mm x 25 cm columns monitoring at 313 nm for preparative samples or 254 nm for analytical samples. Liquid scintillation counting of radioactivity was determined with a Packard (Trade Mark) model 3255 using a scintillation solution consisting of 0.4% 2,5diphenyl oxazole and 0.03% dimethyl-l,4-bis(2(520 phenyloxazolyl)) benzene in toluene. All reactions are preferably conducted under an inert atmosphere.

EXAMPLE 1 Methyl 33-acetoxy-24-nor-5-cholen-23-oate (3, R = acetyl) A solution of 5 g (12.3 nm) of 2 (R = acetyl) in 10 ml of freshly distilled thionyl chloride was stirred at 25 °C for 90 min. Excess thionyl chloride was removed by distillation following 5 additions of 20 ml benzene. The brown residue was suspended in 50 ml benzene and slowly added to a 130 ml ether solution containing approximately 2 g of diazomethane (2-fold excess) at 0°C. The reaction mixture was left at room temperature for 18 h resulting in the formation of pale yellow crystals . Solvents were evaporated and the crude diazoketone, dissolved in 50 ml benzene and 110 ml methanol, was heated to 60°C and a suspension of 6 mmoles silver oxide in 50 ml methanol was added slowly. After refluxing at 70°C for 20 h the solvents were evaporated and the residue (taken up in ether) was filtered through Celite (Trade Mark). The ether solution was adjusted to 100 ml and washed with 0.1 NHCl, dilute NaHCO^, water, and dried over sodium sulfate. The methyl ester product, 3 (R = acetyl), was recrystallized from 10½ acetone in methanol and 100% ethanol to give 3.2 g (60%) of white needles, mp 126.0127.2°C; mass spectrum m/e (rel.int.) 356 (100 M+ - HOAc), 341 (29), 325 (2.4), 282 (4.5), 255 (24); nmr (CDC13) 6.66 (s, 3H, 18-CH3), .93 (d, 3H. 21-CH3) .97 (s, 3H, 19-CHj), 2.01 (s, 3H, -OAc), 2.27 (d, 2H, 22-CH2), 3.60 (s, 3H, OACMe), 4.5(m,lH, 3a-H), 5.33 (m, IH, 6-H).

EXAMPLE 2 Methyl 3p-hydroxy-24-norchola-5,7-dien-23-oate (4b, R = CHj).

To a solution oi 3 (R « acetyl) (500 mg, 1.2 iranoles) in 22 ml benzene and 17 ml hexane was added 500 mg NaHCOj and 1.5 eg. l,3-dibromo-5,5-dimethyl-hydantoin. The reaction mixture was refluxed at 75°C for 20 minutes then rapidly cooled and filtered.. The residue obtained upon solvent evaporation was dissolved in 17 ml xylene and 4 ml s-collidine and refluxed for 90 minutes. Ether was added and the organic phase was thoroughly washed with 1 N HCl, dilute NaHCOj, water, saturated NaCl, and then dried over Na^SO^. The residue (containing 5,7- and 4,6-diene products) was heated in dry dioxane with 80 mg p-toluene sulfonic acid at 70°C for 35 minutes. The mixture was diluted with ether and washed with water, dilute bicarbonate, water and saturated NaCl. The dried residue was chromatographed on a silica gel column (2 x 15 cm) eluted with 15% EtOAc in hexane.

The product (4a) (R = apetyl) eluting between 51 and 102 ml obtained in 2S% yield from 2 was stirred in 10 ml ether and 10 ml 5% (w/v) KOH in 95% methanol for 30 minutes at room temperature. The reaction mixture was diluted with ether and the organic phase washed as above. The product was purified by tic (40% EtOAc in hexane, developed twice, Rf 0.33) to give 96 mq of 4b (R = CH.) (21% from 3). UV 262, 271, 282,292, nm.

High resolution mass spectrum, calc'd. for C^Hj^Oji 372.2664; found: 372.2652. nmr δθ.66 (s, 3H, 18-CH3). .94 (s, 3H, 19-CH3), 1.01 (d, 3H, 21-CH3), 3.67 (s, 3H. COOCHj). 2.76 (m. IH. 3a-H), 5.39 (d, IH, 7-H), 5.56 (d, IH, 6-H). EXAMPLE 3 Methyl 3S-hvdroxy-24-nor-9.10-seco-chola-5,7,10(19) trien-23-oate (6) (R = CHj). Ether solutions of approximately 20 mg of 4b (R = CH3) were irradiated on ice and under nitrogen for 10 minutes with a mercury arc lanp (Hanovia (Trade Mark) 9A-1) fitted with a Corex (Trade Mark) filter. The residues obtained after solvent evaporation were chromatographed on HPLC (6.4 mm x 25 cm Zorbax-SIL, 4 ml/min 1500 psi) eluted with 1.5% 2-propanol in hexane. Pure previtamin, , (R = CH,) was collected at 45 ml (UV: λ„.26Ο nm λ · 231 nm). The combined previtamin containing fractions min were heated in 10 ml ethanol at 80°C for 150 minutes to yield 10.S mg of 6 (R = CHj) (12% yield from 4).

UV, λ , 264 nm, λ . 228 nm. High resolution mass max · min spectrum calc'd. for Cj^HjgC^: 372.2664; found; 372.2661; m/e (rel.int.) 372 (44), 354 (3), 341 (6), 313 (4), 298 (1), 271 (4). 253 (7), 136 (97), 118 (100). nmr δ.58 (s, 3H, 18-CH3), .99 (d, 3H, 21-CH3). 3.67 (s. 3H. COOCHj) 3.95 (m, IH, 3a-H), 4.81 (s, IH, 19(Z)-H), 5.05 (s, IH, 19(E)-H) 6.03 (d, IH, 7-H), 6.23 (d, IH, 6-H).

EXAMPLE 4 Methyl la.33-dihydroxy-24-nor-9,10-seco-5.7,10(19)-cholatrien-23-oate (calcitroic acid methyl ester, 1, R = CH3).

A solution of 6 (R = CH3) (10.2 mg, 27 pmoles) in pyridine (0.2 ml) was treated with 30 mg p-toluene sulfonyl chloride at 4® for 22 hr. After addition of dilute bicarbonate (2 ml) the product was extracted with CHClg ether (10 ml); the combined organic phases were washed with IN HCl, dilute bicarbonate, water, and saturated NaCl and dried over MgSO^. The 3-tosyl derivative was then solvolyzed in 0.3 ml benzene, 2 ml methanol, and 50 mg NaHCOg, heated to 56°C for 18 hr. The resulting cyclovitamin product (2) (R = CHg) was extracted into ether, washed with water and saturated NaCl, dried, and purified on silica gel tic (30% EtOAc/hexane, Rf. 54). This product in 0.7 ml CHgCl^ was then added to an ice-cooled solution containing 0.5 eg. SeOg and 2 eq. t-BuOOH in 0.5 ml CHgClg. The reaction, followed by tic, was allowed to proceed at room temperature for a total of 40 minutes and was stopped with the addition of NaHCOg and ether. The organic phase was washed with dilute bicarbonate, water, and saturated NaCl, and dried over MgSO^. Evaporation of solvent gave la-hydroxy derivative 8, (R = CHg) which was dissolved in 0.5 ml glacial acetic acid and heated at 55° for 15 mins.Products (9, R = CHg) and the corresponding 5.6-trans isomer in ca 3:1 ratio) were extracted with ether, and the ether phase was washed as before. Compound 9 (R = CHg) was purified by tic (50% EtOAc in hexane rf. 0.32) followed by HPLC, (6.4 x 250 mM column, 2.5% of 2-propanol in hexane, at 2 ml/min and 900 psi). Product 9 (R = CHg) eluting t 4 at 63 ml, was recycled through the column and obtained in pure form in 7.156 yield from 6 (UV Xmax 264, 228 nm).

Mild hydrolysis of 9 (75 μΐ 0.1 M KOH/MeOH and 200 μΐ ether, 15®, 60 min) provided 1 (R =s CK3); UV Xmax 264 nm, Xm£n228 nm; high resolution mass spectrum: calc'd for C24H36°4388·26145 found 388.2645; m/e (rel.int., 388 (18), 370 (61), 357 (3), 352 (24). 314 (1). 287 (1), 269 (4), 251 (7). 152 (31), 134 (100); nmr δ 0.58 (s, 3H. 18 CH3), 0.99 (d, 3H, 21 CH3). 3.67 (s. 3H, COOCH3), 4.23 (m. Hi, 3α-Η), 4.43 (m, lH, 10-H). .00 (s, 1H, 19(Z)-H) 5.33 (s, lH, 19(E)-H), 6.02 (d, 1H, 7-H), 6.38 (d, 1H, 6-H).

Claims (4)

1. A compound having the formula where Rg is hydrogen, or a hydroxy-protecting group and 5 R 2 is hydrogen, alkyl or benzyl.

2. A compound according to Claim 1 where Rg is hydrogen or acyl and Rg is alkyl.

3. The compound according to Claim 1 where Rg is hydrogen and Rg is methyl. 10

4. A compound according to Claim 1 where Rg and Rg are as defined in Claim 1 with the proviso that if Rg is a hydroxyprotecting group, Rg is not hydrogen.