NO300209B1 - Analogous Process for Preparation of Side-Chain Homologous Vitamin D Derivatives - Google Patents

Abstract

The description relates to novel side-chain-homologous vitamin D derivatives of formula (I), in which R<1>, R<2>, R<3>, R<4>, R<5> and R<6> have the significance given in the description, B and D are either a hydrogen atom or together a second bond (E-configuration double bond) and either A is a direct bond between the carbon atoms 20 and 22 and X is an alkyloxy radical -(CH2)nO- with n = 1 to 3 or A is a methylene bridge (-CH2-) between the carbon atoms 20 and 22 and X is an alkylene radical -(CH2)n- or an alkylenoxy radical -(CH2)nO- with n = 1 to 3, or if A is a direct bond and B and D together are a second bond, (a) is one of the radicals (b) , -(CH2)2-= or -(CH2)2-=, and a process for their production, pharmaceutical preparations containing this compound and its use in making medicaments. The new compounds have proliferation-inhibiting and cell-differentiating effects.

Description

The present invention relates to an analogue method for the production of side-chain homologous vitamin D derivatives of formula I

in which

R<1> denotes a hydrogen atom, a hydroxy or

acyloxy group with 1 to 9 carbon atoms,

R<2> denotes a hydrogen atom or an acyl group with 1

to 9 carbon atoms,

R<3> or R4 denotes a hydroxy or acyloxy group with 1

to 9 carbon atoms and the second substituent represents a hydrogen atom, or R3 and R4 together represent an oxygen atom,

R<5> and R6 independently denote a linear or branched alkyl residue with up to 5 carbon atoms, a trifluoromethyl group or form together with the tertiary carbon atom a saturated, unsaturated or aromatic carbocyclic, or by inclusion at 1 or 2 N- , 0 or S atoms, a heterocyclic 3-, 4-, 5- or 6-membered ring,

B and D denote either a hydrogen atom or together another

bond (E-configured double bond) and

A denotes either a direct bond between carbon atoms 20 and 22 and

X denotes an alkyleneoxy acid residue -(CH2)nO- where n = 1 to 3;

or

A denotes a methylene bridge (-CH2-) between the carbon atoms

20 and 22 and

X denotes an alkylene residue -(CH2)n- or an alkyleneoxy acid residue -(CH2)n0- where n = 1 to 3;

or;

when A denotes a direct bond and B and D together denote

draws a second bond, denotes

one of the residues characterized in that a compound of general formula IV

in which

R<1>' denotes a hydrogen atom or a protected hydroxy group and

R 2 denotes a hydroxy protecting group and

A, X, R<5> and R6 have the designations given for formula I,

if desired after selective hydration of the double bond in the side chain, is converted into a compound of general formula IVa

in which R<1>', R<2>', A, X, R<5> and R6 have the designation indicated in formula IV and, if desired, a reduction of the carbonyl function and optionally after separation of the mixture of the epimers formed by the reduction hydroxy compounds of general formula Illa and Illb

in which

R<1>', R<2>', A, X, R<5> and R6 have the designations indicated in formula IV, and B and D have the designations indicated in formula I, upon irradiation with ultraviolet light while inverting the stereoisomerism by The 5,6-double bond is converted into a compound of general formula II

in which

R<1>', R<2>', A, B, D, X, R<5> and R6 have the designations given in formula Illa/IIIb,

and this compound then, by splitting off the present hydroxy protecting groups and optionally by partial or complete esterification of the hydroxy groups, is transferred to a compound of general formula I.

Those for the residues R<1>, R2, and within the residues R<3> or R<4>, possible acyloxy groups, respectively acyl groups, are especially derived from saturated carboxylic acids or also benzoic acid. Other suitable acyl residues in R<1>, R2, R3 and R<4> include cyclic, acyclic, carbocyclic or heterocyclic residues, all of which may optionally also be unsaturated. The preferred residues are derived from Cx- to C9-, preferably C2- to C5-alkanecarboxylic acids, such as e.g. acetic acid, propionic acid and butyric acid.

When R<5> and R6 together with the tertiary carbon atom form a saturated carbocyclic ring, in particular a cyclopropyl ring or cyclohexyl ring comes into consideration. As alkyl groups for R<5> and R<6> in particular those with 1 to 5 carbon atoms come into consideration, which can be straight-chain or branched. Examples include methyl, ethyl, propyl, as well as

the t-butyl group.

Preferred compounds prepared according to the present invention are side chain homologous vitamin D derivatives of general formula I, wherein

R<1>, R<3> or R<4> denotes a hydroxy group or,

R<5> and R<6> denote a methyl group or together with the tertiary carbon atom denote a cyclopropyl ring,

R<2> denotes a hydrogen atom and n is 1 or 2.

Between carbon atoms 22 and 23 (when A denotes a direct bond) or between carbon atoms 23 and 24 (when A denotes a methylene group) there is preferably a double bond. Particularly preferred are the compounds

24-(1(R)-hydroxy-4-methylpentyl)-9,10-secochola-5Z,7E,10(19),23E-tetraene-1(S),3(R)-diol,

24-(1(S)-hydroxy-4-methylpentyl)-9,10-secochola-5Z,7E,10(19),23E-tetraene-1(S),3(R)-diol,

24-(1(R)-hydroxy-3-methylbutyl)-9,10-secochola-5Z,7E,10(19),23E-tetraene-1(S),3(R)-diol,

24-(1(S)-hydroxy-3-methylbutyl)-9,10-secochola-5Z,7E,10(19),23E-tetraene-1(S),3(R)-diol,

24-(1(R)-hydroxy-3-methylbutyl)-9,10-secochola-5Z,7E,10(19)triene-1(S),3(R)-diol,

24-(1(S)-hydroxy-3-methylbutyl)-9,10-secochola-5Z,7E,10(19)triene-1(S),3(R)-diol,

24-(1(R)-hydroxy-3-isopropoxypropyl)-9,10-secochola-5Z,7E,10(19),23E-tetraene-1(S),3(R)-diol,

24-(1(S)-hydroxy-3-isopropoxypropyl)-9,10-secochola-5Z,7E,10(19),23E-tetraene-1(S),3(R)-diol,

24-isopropoxymethyl-9,10-secochola-5Z,7E,10(19),22E-tetraene-1(S),3(R),24(R)-triol,

24-isopropoxymethyl-9,10-secochola-5Z,7E,10(19),22E-tetraene-1(S),3(R),24(S)-triol,

24-(2-isopropoxyethyl)-9,10-secochola-5Z,7E,10(19),22E-tetraene-1(S),3(R),24(R)-triol, 2 4-(2- i sopropoxyethyl 1)-9,10-secochola-5Z,7E,10(19),22E-tetraene-1(S),3(R),24(S)-triol,

26,27-cyclo-24a,24b-dihomo-9,10-secocholesta-5Z,7E,10(19),23E-tetraene-1(S),3(R),24a(R)-triol,

26,27-cyclo-24a,24b-dihomo-9,10-secocholesta-5Z,7E,10(19),23E-tetraene-1(S),3(R),24a(S)-triol,

The natural vitamins D2 and D3 (see general formula IV) are in themselves biologically inactive and are only converted after hydroxylation in the 25-position in the liver, respectively in the 1-position in the kidneys, into their biologically active metabolites. The effect of vitamins D2 and D3 consists in the stabilization of the plasma Ca<++->mirror and the plasma phosphate mirror: they counteract a lowering of the plasma Ca<++->mirror.

Ergocalciferol: R<a>=R<b>=H, R<C>=CH3, Vitamin D2

double bond C 22/23

Cholecalciferol: Ra=R<b>=R<c>=H Vitamin D3

25-hydroxycholecalciferol: R<a>=R<c>=H, R<b>=0H 1a-hydroxycholecalciferol: R<a>=0H, R<b>=Rc=H 1a,25-dihydroxycholecalciferol: R<a >=R<b>=0H, R<C>=H Calcitriol

In addition to their marked effect on calcium metabolism and phosphate metabolism, vitamins D2 and D3, and their synthetic derivatives, also possess proliferation-inhibiting and cell-differentiating effects (H.F. De Luca, The Metabolism and Function of Vitamin D in Biochemistry of Steroid Hormones, Ed. H.L.J. Makin, 2nd Edition, Blackwell Scientific Publications 1984, pp. 71-116).

When using vitamin D, however, overdose phenomena (hypercalcemia) can also occur.

la-cholecalciferol hydroxylated in the 24-position is already described in DE-AS-25 26 981; this compound has a lower toxicity than the corresponding non-hydroxylated la-cholecalciferol. The hydroxylated compounds show a selective activation of intestinal calcium absorption and a weaker bone absorption effect than la-cholecalciferol. The 24-hydroxy-vitamin D analogue described in the international patent application WO 87/00834 can be used to treat disorders in humans and animals that originate from abnormal cell proliferation and/or cell differentiation.

For various 1,25-dihydroxy-homo-vitamin D derivatives, a dissociation with regard to the properties of bone absorption action and HL-60 cell differentiation has already been briefly mentioned by De Luca. The bone absorption effect in vitro is thus a direct measure of calcium mobilization in vivo.

It has now been found that the side-chain homologous vitamin-D derivatives of general formula I produced according to the present invention surprisingly exhibit a more favorable spectrum of action compared to the vitamin-D derivative calcitriol (1α,25-dihydroxycholecalciferol). While the effect on calcium metabolism and phosphate metabolism is significantly weakened (reduction of side effects in case of overdose or necessary higher

dosage), the proliferation-inhibiting and cell-differentiating effects remain almost maintained (dissociation).

The vitamin D activity of the compounds prepared according to the present invention is determined by means of the calcitriol receptor test. The test is carried out using a

specific receptor protein from the intestine of rickety hens. Receptor-containing binding protein is incubated with <3>H-calcitriol (0.5 ng/ml) in a reaction volume of 0.575 ml, in the absence and presence of the sample material, for one hour in a small test tube. For the separation of free and receptor-bound calcitriol, a

adsorption with activated carbon-dextran. In addition, 200 ul of an activated charcoal-dextran suspension is added to each test tube and it is incubated at 22 "C for 30 min. The samples are then centrifuged at 1500 x g for 10 min at 4 °C. The supernatant is decanted and counted after approx. one hour of equilibration in the "Atom -Light" in a (3-

counting.

The competitive curves obtained with different concentrations of sample material, as well as reference material, (unlabeled calcitriol) at a constant concentration of the reference material (<3>H-calcitriol), were compared with each other and a competitive factor (KF) was determined.

This factor is defined as the ratio between the concentration of the respective sample material and the reference material required for 50% competitiveness:

After this possess

24-(1-hydroxy-3-methylbutyl)-9,10-secochola-5Z,7E,10(19),23E-tetraene-1(S),3(R)-diol (compound A) a KF value of 2.0 and

24-(1-hydroxy-3-methylbutyl)-9,10-secochola-5Z,7E,10(19),23E-tetraene-1(S),3(S)-diol (compound B) a KF value of 3.6.

In order to determine the antiproliferative potency of the compounds produced according to the present invention, the following described test is performed with compounds A and B as alternating test materials: Keratinocytes from newborn mice were prepared and cultured by modification of the method according to Yuspa, S. und Harris, CC, "Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro", Exp. Cell Res. 86:95-105, 1974.

Newborn NMRI mice of both sexes were sacrificed by decapitation, the skin was dissected from, washed in an antibiotic-antimycotic solution and incubated with the dermal side down in dispase II solution (1.2 U/ml in tissue culture medium M199 + 25 mmol/l HEPES + 15% fetal calf serum (FCS) + 50 U/ml penicillin/streptomycin (P/S) (preparation medium, PM) at 4 °C overnight Epidermis was removed and by treatment with trypsin a single cell suspension was prepared After centrifugation, the cell pellet was resuspended, the number of live, small, round cells was determined after trypan blue staining and the cells were inoculated at a thickness of 4 x 10<5> cells/cm<2> in primaria-24 perforated plates in tissue culture medium (M199 + 15% FCS + 50 U/ml P/S).After incubation for 24 h at 37 °C,

in the cells with phosphate-buffered saline (PBS) washed and incubated for an additional 24 h in serum-free tissue culture medium (Ml99 + 50 U/ml P/S + 0.5% ethanol) with and without test compounds. Then 0.4 µCi/50 µl <3>H-methylthymidine (40 Ci/mmol) was added. After 4 hours, the medium was aspirated and the reaction was stopped by adding 500 ul of ice-cold 10% trichloroacetic acid (TCA). The cells were washed with TCA and PBS, lysed by incubation in a proteinase K solution (10 mmol/l tris-HCl,

10 mmol/1 EDTA, 10 mmol/1 NaCl, 0.2% triton-X 100, pH 8.0,

50 ug/ml protein kinase K), and the lysate was cleared by centrifugation. The radioactivity was determined in the supernatant scintillation photometrically and, after specific staining of DNA with diamidinophenylindole (DAPI), the DNA concentration was determined fluorescence photometrically.

Depending on the dose, both calcitriol and compounds A and B thus inhibit the incorporation of <3>H-thymidine into DNA with the following IC50<->values:

The differentiation-stimulating effects of both calcitriol and the compounds prepared according to the present invention 26,27-cyclo-24a,24b-dihomo-9,10-secocholesta-5Z,7E,10(19),23E-tetraene-1(S),3( R),24a(R)-triol (compound C) and 26,27-cyclo-24a,24b-dihomo-9,10-secocholesta-5Z,7E,10(19),23E-tetraene-1(S), 3(R),24a(S)-triol (compound D) are practically not different.

It is known in the literature (Mangelsdorf, D.J. et al, in J. Cell. Biol. 98: 391-398 (1984)) that the treatment of human leukemia cells (promyelocyte cell line HL 60) in vitro with calcitriol induces the differentiation of the cells into macrophages.

For quantification of the differentiation-stimulating effect of calcitriol analogues, the test described below was carried out:

HL 60 cells were grown in tissue culture medium (RPMI -

10% fetal calf serum) at 37 °C in an atmosphere of 5% CO2 in air.

For testing the compounds, the cells were centrifuged and 2.8 x 10<5> cells/ml were transferred into a tissue culture medium without phenol red. The test compounds were dissolved in ethanol and diluted to the desired concentration with tissue culture medium without phenol red. The various diluted solutions were mixed with the cell suspension in a ratio of 1:10 and portions of 100 µl of the cell suspension spiked with compound were pipetted into wells of a 96-well plate. As a control, an analogous cell suspension with only solvent was added.

After incubation for 96 hours at 37 °C in 5% CO2 in air, 100 µl of an NBT-TPA solution (nitro-blue tetrazolium (NBT), final concentration in the preparation) was pipetted into each cell suspension in the wells of the 96-well plate 1 mg/ml, tetradecanoylphorbol myristate-13-acetate (TPA), final concentration in the preparation 2 x 10"<7> mol/l).

Upon incubation for 2 hours at 37 °C and 5% C02 in air, as a result of the intracellular oxygen radical release, stimulated through TPA in the cells differentiated into macrophages, NTB was reduced to insoluble formazan.

To terminate the reaction, the wells of the 96-well plate were aspirated and the adherent cells were fixed by adding methanol and dried after fixation.

To dissolve the formed intracellular formazan crystals, 100 ul of potassium hydroxide (2 val/l) and 100 ul of dimethylsulfoxide were pipetted into each well, followed by treatment with ultrasound for one minute. The concentration of formazan was measured spectrophotometrically at 650 nm.

As a measure for the differentiation induction of the HL 60 cells into macrophages, the concentration of formed formazan applies. The relative activity of the test compound is given from the quotient ED50 test compound/ED50 calcitriol.

Based on this, calcitriol, compound C and compound D have ED50 values of 1.8 x IO"<9> mol/l, 2.2 x IO'<9>, 2.5 x IO"<9> mol/l respectively .

Due to the reduced risk of hypercalcemia, the compounds produced according to the present invention are particularly suitable for the production of drugs for the treatment of diseases, which are characterized by a hyperproliferation, e.g. hyperproliferative diseases of the skin (psoriasis) and malignant tumors (leukaemia, colon cancer, breast cancer). In a particularly preferred embodiment, calcitriol receptors are detected in the target organ before the treatment.

The present invention also relates to the use of the compounds according to formula I for the production of medicinal products.

The production of the side-chain homologous vitamin D derivatives of formula I is carried out according to the present invention in that a compound of general formula IV

in which

R<1>' denotes a hydrogen atom or a protected hydroxy group and

R 2 denotes a hydroxy protecting group and

A, X, R<5> and R6 have the designations given for formula I,

if desired after selective hydration of the double bond in the side chain, is converted into a compound of general formula IVa

in which R<1>', R<2>', A, X, R<5> and R6 have the designation given in formula IV and, if desired, a reduction of the carbonyl function and optionally after separation of the mixture of the epimeric hydroxy compounds formed by the reduction with general formula Illa and Illb

in which

R<1>, R<2>, A, X, R<5> and R6 have the designations given in formula IV, and B and D have the designations given in formula I,

upon irradiation with ultraviolet light during inversion of the stereoisomerism at the 5,6-double bond, is converted into a compound of general formula II

in which

R<1>', R<2>', A, B, D, X, R<5> and R6 have the designations given in formula Illa/IIIb,

and this compound then, by splitting off the present hydroxy protecting groups and optionally by partial or complete esterification of the hydroxy groups, is transferred to a compound of general formula I.

Reduction of the side chain carbonyl functions in connection with general formula IV is preferably carried out with cerium-(III) chloride-sodium borohydride in a polar solvent. During the reduction, both the R-hydroxy isomer and the S-hydroxy isomer with general formula Illa and Illb respectively are formed. Both of these isomers can be separated chromatographically.

If desired, the double bond in the side chain can be selectively hydrogenated before reduction of the carbonyl function. Lithium-tri-tertiary-butoxy-aluminium hydride in a polar solvent is suitable as a hydrogenating agent.

The subsequent conversion of a compound of general formula IIa/IIIb into a compound of general formula II is carried out, e.g. by irradiation with ultraviolet light in the presence of a so-called "triplet sensitizer". For this, within the scope of the present invention, anthracene is used. By cleavage of the pi bond of the 5,6-double bond, rotation of the A-ring 180 <0> around the 5,6-single bond and re-establishment of the 5,6-double bond, the stereoisomerism of the 5,6-double bond is inverted.

In connection with this, the present hydroxy protecting groups are split off, preferably by using tetra-n-butyl-ammonium fluoride, and if desired, the free hydroxy groups are partially or completely esterified with the corresponding carboxylic acid halide (halide = chloride, bromide) or carboxylic acid anhydride, according to usual methods.

Production of output materials

1. 1(S),3(R)-bis-(tert-butylmethylsilyloxy)-20(S)-formyl-9,10-secopregna-5E,7E,10(19)-triene 1: The preparation of 1. performed according to M.J. Calverley, Tetrathron 43, 4609 (1987); see also international patent application WO 87/00834. In this publication, the preparation of the starting compound, in which R<1>' is a hydrogen atom, is also described. 2. 1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-20(R)-methyl-9,10-secopregna-5E,7E,10(19)-triene-21-carbaldehyde 2.

The aldehyde 2 is produced according to a new method. a. To a suspension of 1.8 g of sodium hydride (80% in oil) in 70 ml of absolute THF, a solution of 15.57 g of diethylphosphonoethoxyacetic acid ethyl ester (prepared according to W. Grell und H. Machleidt, Liebigs Ann . Chem. 699, 53 (1966)) in 200 ml of THF. After the addition, the mixture is stirred for a further 90 min at 60 °C. It is then cooled to 25 °C and a solution of 6.2 g of compound 1 in 70 ml of THF. It is stirred for 2 hours under reflux, the cooled reaction solution is then poured into water and extracted with ethyl acetate. After drying (Na2SO4) and evaporation, the crude product obtained is chromatographed on silica gel with hexane/ethyl acetate. The main fraction gives 5.2 g 1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-23-(ethoxy-9,10-secochola-5E,7E,10(19)-tetraene-24-acid ethyl ester in the form of a oily mixture of C-22 double bonds in the sommer.

b. Dissolve 5.2 g of the product obtained under point a. in 120 ml of toluene and slowly add 20 ml at 0 °C

of a 20% solution of diisobutylaluminum hydride in toluene. After 30 min at 0 °C, the reaction solution is carefully poured into NH4Cl solution and extracted with ethyl acetate. After usual work-up, 4.88 g of 1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-23-ethoxy-9,10-secochola-5E, 7E, 10(19), 22-tetraen- 24-ol in the form of a colourless, oily isomer mixture which is used in the subsequent steps without further purification. c. The compound prepared under point b. (4.88 g) is stirred in a mixture of 55 ml of dichloromethane and 55 ml of 70% aqueous acetic acid for 4 hours at room temperature.

The mixture is neutralized by adding NH3 solution and extracted with dichloromethane. The crude product is chromatographed on silica gel with hexane/ethyl acetate. In this way, 2.02 g of 1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-24-hydroxy-9,10-secochola-5E,7E,10(19)-trien-23-one are obtained 5 in the form of a colorless oil.

<1>H-NMR (CDC13): Y = 0.01 ppm (s, 12H, Si-CH3), 0.52 (s,3H,H-18), 0.81 and 0.84 (s,9H -Si-t-butyl), 0.90 (d,J=7Hz,3H,H-21), 3.09 (t,J=5Hz,1H,OH), 4.10 (dd,lH,H- 24), 4.16 (m,lH,H-3), 4.21 (dd,1H,H-24), 4.39 (m,lH,H-l), 4.88, 4.93 (s, ie 1H,H-19), 5.77, 6.39 (d,J=HHz, 1H,H-6,H-7). d. The product obtained under point c. (2.02 g) is dissolved in 25 ml of methanol and 25 ml of THF and 300 mg of sodium borohydride is added at 0 °C. It is stirred for 1.5 hours at 0 °C, the reaction mixture is then poured into NH4Cl solution and extracted with ethyl acetate. 1.75 g of 1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-9,10-secochola-5E,7E,10(19)-triene-23,24-diol 6 are obtained. 1 form of colorless, oily mixture of the 23-epimers, which is added as such in the subsequent reaction.

e. 1.75 g of the product obtained under point d. is dissolved in 40 ml of toluene and 1.23 g of lead tetraacetate is added in portions while cooling with ice water. It is stirred for 30 min, a further 1.0 g is added

Pb(0Ac)4 and stirred for a further 15 min at + 5 to + 10 °C.

NaHCO 3 solution is added to the mixture, the resulting suspension is filtered over celite and the filtrate is extracted with ethyl acetate. The crude product is chromatographed on silica gel with hexane/ethyl acetate. After crystallization of the main fraction from ethanol, 560 mg of 1(S),-3(R)-bis-(tert.-butyldimethylsilyloxy)-20(R)-methyl-9,10-secopregna-5E,7E, 10( 19) are obtained -triene-21-carbaldehyde with melting point 101-104 °C. The reaction of the aldehyde 1, respectively 2, with a phosphorane of formula

gives compounds of general formula IV (Wittig reaction).

Preparation of the phosphorylides used:

1. Isobutylcarbonylmethylenetriphenylphosphorane a. Bromomethylisobutylketone 50 ml of isobutylmethylketone in 240 ml of methanol is added to 20 ml of bromine at 0 °C and stirred for 1.5 hours at 10 °C.

360 ml of water are then added and the mixture is stirred for a further 16 hours at room temperature.

Saturated sodium chloride solution is added to the reaction mixture, the separated organic phase is separated and the aqueous phase is extracted with ethers. The combined organic phases are washed with 10% Na 2 CO 3 solution and dried over Na 2 SO 4 . After filtration, the solvent is sucked off in a water jet vacuum and the precipitate is distilled. The main fraction contains 53.7 g of bromomethyl isobutyl ketone with Kp<15>'<20> 67-69 <6>C.

b. Isobutylcarbonylmethyltriphenylphosphonium bromide Bronuethyl isobutyl ketone (53.6 g) and triphenyl phosphine (78.5 g) are thoroughly mixed in a 500 ml round bottom flask and after the initial strong reaction heat development has ceased, the mixture is allowed to stand for 12 hours under a nitrogen atmosphere at room temperature. The solid reaction mass is then extracted with 330 ml of methylene chloride and heated for 30 min under reflux cooling. After adding 500 ml of ether, cool to room temperature and the product is isolated by filtration. After drying, 111.7 g of the phosphonium salt with melting point 244-245 °C are obtained. c. Isobutylcarbonylmethylenetriphenylphosphorane 111.6 g of the phosphonium bromide obtained under point b. is successively added to 1500 ml of methylene chloride and 1500 ml of 2N-NaOH and stirred for 30 min at room temperature. The organic phase is separated, washed with water and dried over Na 2 SO 4 . The solid precipitate obtained after evaporation is recrystallized from tertiary butyl methyl ether and gives 72.2 g of the ylide with a melting point of 120-121 °C. 2. Isoamylcarbonylmethylenetriphenylphosphorane The preparation of the title compound is carried out analogously to the method described under point 1, by bromination of isoamylmethyl ketone, reaction of the bromide with triphenylphosphine to a phosphonium salt and formation of the ylide with 2N NaOH.

From 50.0 ml of isoamyl methyl ketone and 18.2 ml of bromine, 54.68 g of 1-bromo-5-methyl-hexan-one with Kp<15>"<20> 80-86 °C are obtained after purification by distillation.

From 54.58 g of the bromide and 74.14 g of diphenylphosphine, 91.6 g of a phosphonium salt with a melting point of 230-233 °C is obtained.

From 91.6 g of the phosphonium salt, after treatment with NaOH and recrystallization of the crude product from methylene chloride/ester, 69.8 g of the title compound with a melting point of 64-67 °C are obtained. 3. Isopropoxymethylcarbonylmethylenetriphenylphosphorane 2.43 g sodium dissolved in 150 ml isopropanol. After addition of 20.0 g of chloromethylcarbonylmethylenetriphenylphosphorane (R.F. Hudson et al., J. Org. Chem. 24 2446, 1963), dissolved in 200 ml isopropanol, heated for 8 hours under reflux.

The cooled reaction mixture is poured into sodium chloride solution and extracted with ethyl acetate. The oily residue obtained after evaporation is chromatographed on silica gel with ethyl acetate. 9.53 g of the title compound with a melting point of 134 °C are obtained.

4. (2-isopropoxyethyl)-carbonylmethylenetriphenylphosphorane a. 1-bromo-4-isopropoxy-butan-2-one

A solution of 68.2 g of 4-isopropoxy-2-butanone (F.B. Hassan et al., J. Biolog. Chem. 256. 7781, 1981) in 315 ml of methanol is added 26.9 ml of bromine dropwise at 0 °C and stirred then for 1.5 hours at + 10 °C. Then 470 ml of water is added to the reaction solution and it is stirred for 16 hours at room temperature. Saturated sodium chloride solution is added and extracted with ether. Distillation of the crude product gives 78.07 g of the bromine derivative with Kp<15>'<20> 95 °C.

b. 4-isopropoxy-2-oxo-butyl-triphenylphosphonium bromide From 78.0 g of the bromide obtained under point a. and 97.85 g of triphenylphosphine, 133.35 g of the phosphonium salt with a melting point of 183 °C is obtained according to the method described under point 1.

c. (2-isopropoxyethyl)-carbonylmethylenetriphenylphosphorane The phosphonium bromide (133.2 g) obtained under point b. is treated with 2N-NaOH in methylene chloride as described under point 1. After recrystallization of the crude product from ethyl acetate, 64.38 g of the title compound with melting point 97 is obtained °C. 5. (1-Ethylpropoxymethyl)-carbonylmethylenetriphenylphosphorane A solution of 3.04 g of sodium in 100 ml of 3-pentanol is reacted with 25.0 g of chloromethylcarbonylmethylenetriphenylphosphorane analogously to the preparation of isopropoxymethylcarbonylmethylenetriphenylphosphorane. The title compound is obtained as a crystallized oil with a melting point of 66-70 °C. 6. Cyclopropylmethoxymethylcarbonylmethylenetriphenylfuran

A solution of 5.58 g of sodium in 25.0 g of cyclopropyl methanol and 200 ml of toluene is reacted with 30.0 g of chloromethylcarbonylmethylenetriphenylphosphorane analogously to the preparation of isopropoxymethylcarbonylmethylenetriphenylphosphorane. The title compound is obtained as a solid with a melting point of 121 °C.

7. (3-Butynyl)-carbonylmethylenetriphenylphosphorane Dissolve 20.0 g of methylcarbonylmethylenetriphenylphosphorane in 628 ml of tetrahydrofuran and add dropwise 41.3 ml of butyl lithium (1.6 molar solution hexane) at -78 °C. After heating to room temperature, the reaction mixture is added to the ice/common salt solution and the mixture is extracted with vinegar. After drying the organic phase with sodium sulfate, 23.4 g of solid material is obtained. Column chromatographic purification (silica gel/ethyl acetate) gives 15.4 g of the title compound with melting point 135-136 °C.

8. (3-butenyl)-carbonylmethylenetriphenylphosphorane

By reaction of 15.0 g of methylcarbonylmethylenetriphenylphosphorane in 471 ml of tetrahydrofuran with 31.0 ml of butyllithium and 4.28 ml of allyl bromide, the title compound is obtained as a crystallized oil with a melting point of 92-93 °C.

By varying the keto components used for the preparation of the Wittig reagents, as described below, further phosphoranes can be produced, which can be converted with aldehyde 1 or 2 into further compounds of general formula IV.

Example 1

A solution of 1.6 g of 1(S),3(R)-bis-(tert-butyl-dimethylsilyloxy)-20(R)-methyl-9,10-secopregna-5E,7E,10(19)- triene-21-carbaldehyde in 50 ml of toluene is stirred after the addition of 3.02 g of isoamylcarbonylmethylenetriphenylphosphorane for 16 hours at 80 °C under argon. The solvent is then suctioned off under reduced pressure and the residue is chromatographed on silica gel with hexane/ethyl acetate. The main fraction gives 1.15 g of [1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-9,10-secochola-5E,7E,10(19),-23(E)-tetraene-24 -yl]-4-methyl-pentan-1-one as a colorless oil.

<1>H-NMR (CDCl3); 6 = 0.01 ppm (s, 12H, Si-CH3); 0.56 (s,3H,H-18); 0.87 (s,18H.Sit.-butyl); 0.88 (d,J=7Hz,6H,C-(CH3)2); 0.95 (d,J=7Hz,3H,H-21); 4.25 (m, 1H,H-3); 4.55 (m,1H,H-1); 4.94 and 5.00 (s,1H,H-19), 5.82 and 6.46 (d,J=11Hz, 1H,H-6,H-7); 6.10 (d,J=16Hz,1H,H-24); 6.80 (m,1H,H-23).

' Example 2

Dissolve 572 mg of cerium(III) chloride heptahydrate in

10 ml of methanol and the compound prepared according to example 1

(1.10 g) dissolved in 5 ml of methanol, is added. After addition

of 61 mg of sodium borohydride is stirred for 30 min at 0 °C. Water is added, extracted with dichloromethane, dried (Na 2 SO 4 ) and evaporated. The thus obtained mixture of diastereomeric alcohols is separated by chromatography on silica gel with hexane/ethyl acetate. 290 mg of 1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-24-(1-hydroxy-4-1 methylphenyl)-9,10-seco-5E,7E,10( 19),23(E)-cholatetetraene (epimer A) and 120 mg of epimer B. The epimers show identical NMR spectra.

<4>1-NMR (CDC13): 6 = 0.01 ppm (s, 12H, Si-CH3), 0.49 (s,3H,H-18), 0.86 (s,18H.Si-t .-butyl), 0.86 (d,J=7Hz,6H,C-(CH3)2), 0.88 (d,J=7Hz,3H,H-21); 4.16 (m, 1H,H-3); 4.48

(m,1H,H-1), 4.88 and 4.93 (s,1H,H-19), 5.40 (dd,J=15.5 and 7Hz, 1H,H-24); 5.55 (m, 1H, H-23); 5.77 and 6.40 (d,J=11Hz,1H,H-6,H-7).

Example 3

A solution of 290 mg of the product obtained according to example 2 (epimer A) in 80 ml of toluene is, after the addition of 44 mg of anthracene and 0.01 ml of triethylamine, irradiated in a pyrex immersion reactor by means of a mercury high-pressure lamp (Philips HPK 125 ). The irradiation time is 3.5 min, a thorough mixing of the solution is ensured by supplying a nitrogen stream. After evaporation and chromatography on silica gel with hexane/ethyl acetate, 241 mg of 1(S),3(R)-bis-(tert-butyl-dimethylsilyloxy)-24-(1-hydroxy-4-methylpentyl)-9,10- secochola-5Z,7E,10(19),23(E)-tetraene in the form of a colorless oil.

[o]?° + 49.6 0 (CHCl 3 , o = 0.425.

Analogous treatment of 120 mg of the polar isomer (epimer B), obtained according to Example 2, gives 113 mg in the form of a colorless oil.

[α]2° + 41.4 <0> (CHCl 3 , o = 0.285.

Example 4

A solution of 225 mg of the product obtained from epimer A according to example 3 in 5 ml of THF is stirred after the addition of 1.31 ml of a 1 M solution of tetrabutylammonium fluoride in THF for 60 min at 60 °C. After cooling, saturated sodium chloride solution is added and it is extracted with ethyl acetate. The crude product is chromatographed on silica gel with hexane/ethyl acetate and gives 85 mg of 24-(1-hydroxy-4-methylpentyl)-9,10-secochola-5Z,7E,10(19),23E-tetraene-1(S),3( R)-diol in the form of a white foam.

<1>H-NMR (CDCl3): δ = 0.57 ppm (s, 3H,H-18); 0.84 (d,J=7Hz,3H,H-21); 0.92 (d,J=7Hz 6H,C-(CH3)2; 4.03 (m,1H,H-25); 4.23 (m,1H,H-3); 4.43 (m, 1H,H-1); 5.00 and 5.33 (s,1H,H-19 ); 5.45 (dd,J=15.5 and 7Hz,1H,H-24); 5.60 (m,1H ,H-23); 6.02 and 6.38 (d,J=1H,11H,H-6,H-7).

An analogous treatment of the product obtained from epimer B according to example 3 (95 mg) gives 35 mg of the epimeric triols as a colorless oil. The NMR spectra of the epimers are identical.

Example 5

Analogous to the procedure described in example 1, 2.05 g of 1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-20-(R)-methyl-9,10-secopregna-5E, 7E, 10 (19)-triene-21-carbaldehyde in 53 ml of toluene with 3.4 g of isobutylcarbonylmethylenetriphenylphosphorane. After chromatographic purification, [1(S),3(R)-bis-(tert.-butyldimethylsilyloxy)-9,10-secochola-5E, 7E, 10(19 ), - 23(E)-tetraen-24-yl is obtained ]-3-methyl-butan-l-one with melting point 79-81 °C (from ethanol),

[α]2.<0>+ 52.6° (CHCl 3 , δ = 0.500).

Example 6

By reducing 1.75 g of the product obtained from example 5, under the conditions according to example 2, 1 (S),3(R)-bis-(tert-butyldimethylsilyloxy)-24-(1(R,S)- hydroxy-3-methylbutyl )-9,10-secopregna-5E, 7E, 10(19),23E-tetraene in the form of an oily mixture of the epimers. Chromatography on silica gel with hexane/ethyl acetate yields in order of elution 780 mg of epimer A and 600 mg of epimer B, in the form of colorless oils which cannot be rarefied by NMR spectroscopy.

Example 7

By triplet sensitized photoisomerization analogous to example 3, and subsequent silyl ether cleavage analogous to example 4, 240 mg of 24-(1-hydroxy-3-methylbutyl)9-10-secochola-5Z,7E are obtained from 700 mg of the epimer A prepared according to example 6 ,10(19),23E-tetraene-1(S),3(R)-diol (compound A) with a decomposition temperature in the range 119-125 °C,

[a]<2>,<0> + 38.8 0 (methanol, o = 0.505).

Analogous treatment of 330 mg of epimer B yields 129 mg of 24-(1-hydroxy-3-methylbutyl)9-10-secochola-5Z,7E,10(19),23E-tetraene-1(S),3(S)- diol (compound B) with decomposition temperature in the range 139-145 °C, [a]<2>,<0> + 54.8 (methanol,

o = 0.505).

Example 8

A solution of 170 mg of the product obtained according to

in example 5, 5 ml of THF is stirred after the addition of 200 mg of lithium-tri-tertiary-butoxy-aluminium hydride for 90 min at room temperature. 0.8 ml of saturated NH4C1 solution is added, filtered and the filtrate evaporated. Chromatography of the crude product on A1203 (Merck, neutral, stage III) gives 108 mg

1-[1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-9,10-secochola-5E,7E,10(19)-trien-24-yl]-3-methyl-butan- l-on in the form of colorless oil.

3-NMR (CDCl 3 ): δ = 0.53 ppm (s, 3H, H-18); 4.22 (m,1H,H-3); 4.54 (m,1H,H-1); 4.93 and 4.98 (m,1H,H-19); 5.82 and 6.46 (d,J=11Hz,1H,H-6,H-7).

Example 9

Photochemical double bond isomerization analogously to example 3 and silyl ether cleavage analogously to example 4 gives from 100 mg of the product from example 8 50 mg of 1-[1(S),3(R)-di-hydroxy-9,10-secochola-5Z,7E ,10(19)-trien-24-yl]-3-methyl-butan-1-one.

UV (methanol): = 212 nm (e = 14,300), 265 (15,860).

Example 10

Conversion of 1.6 g of 1(S),3(R)-bis-(tert-butyldimethyl-silyloxy)-20(R)-methyl-9, 10-secopregna-5E, 7E, 10(19)-triene -21-carbaldehyde with (2-isopropoxyethyl)-carbonylmethylenetriphenyl-phosphorane analogously to example 1, gives 1.15 g of l-[l(S),3(R)-bis-(tert.-butyldimethylsilyloxy)-9,10- secochola-5E,7E,10(19 )-23(E)-tetraen-24-yl]-3-isopropoxy-propan-1-one in the form of a colorless oil.

3-NMR (CDCl 3 ): δ = 0.01 ppm (s, 12 H, Si-CH 3 ); 0.55 (s,3H,H-18); 0.86 and 0.90 (s,9H,Si-t-butyl); 0.96 (d,J=7Hz, 3H,H-21); 1.15 (d,J=7Hz,6H,C-(CH3)2); 3.60 (m, 1H, CH-O); 3.73 (t, J=7 Hz, 2 H, CH 2 -O); 4.23 (m,1H,H-3); 4.55 (m,1H,H-1), 4.95 and 5.00 (m,1H,H-19); 5.83 and 6.46 (d,J=11Hz,1H,H-6,H-7); 6.11 (d,J=15.5Hz,1H,H-24); 6.87 (m,1H,H-23).

Example 11

By reduction analogous to example 2, photoisomerization analogous to example 3, and silyl ether cleavage analogous to example 4, 1.05 g of the product prepared according to example 10 yield 143 mg of 24-(1(R,S)-hydroxy-3-isopropoxy- propyl)-9,10-secochola-5Z,7E,10(19),23-tetraene-1(S),3(R)-diol in the form of a 1:1 mixture of diastereomers which can be separated by high-pressure liquid chromatography. The isomers exhibit identical NMR spectra.

3-NMR (CDCl 3 ): δ = 0.57 ppm (s,3H,H-18), 0.94 (d,J=7Hz,3H,H-21); 1.15 (d. J=7Hz, 6H,C(CH3)2), 4.17 (m,1H,H-3);

4.21 (m,1H,H-25); 4.38 (m,1H,H-1); 4.98 and 5.29 (m,1H,H-19);

5.45 (dd, J=15.5 and 7Hz,1H,H-24); 5.63 (m, 1H, H-23); 6.02 and

in 6.38 (d,J=11Hz,1H,H-6,H-7).

Example 12

Starting from aldehyde 1 and isopropoxymethylcarbonylmethylenetriphenylphosphorane is obtained analogously to series

> the sequence according to example 1-4 isomer B (5Z,7E,22E-1(S),3(R),-24(S)-9,10-seco-24a,24b-dihomo-24b-oxacholestra-5, 7,10(19)22-tetraene-1,3,24-triol) with melting point 131-132 "C.

Example 13

> Starting from aldehyde 1 and (2-isopropoxyethyl)-carbonylmethylenetriphenylphosphorane, isomer B (5Z,7E,22E-1(S),3(R),-24(S)- 9,10-seco-24a,24b,24c-trichomo-24c-oxacholestra-5,7,10-(19)22-tetraene-1,3,24-triol) with melting point 125-126 °C.

Example 14

Analogous to example 1, 0.85 g of 1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-20(R)-methyl-9,10-secopregna-5E,7E,10(19)-triene is reacted -21-carbaldehyde with 4.5 g of cyclopropylmethyl-5-carbonyltriphenylphosphorane. After chromatographic purification on silica gel with hexane/acetic ester, 500 mg of 1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-26,27-cyclo-24a, 24b-dihomo-9,10-secocholesta-5E is obtained ,7E,10(19)-23E-tetraen-24a-one in the form of a

colorless foam.

3<X>H-NMR (CDCl 3 ): δ = 0.01 ppm (s, 12 H, Si-CH 3 ); 0.09 and 0.50 (m,2H,H-26 and H-27); 0.50 (s,3H,H-18); 0.83 and 0.85 (s, 9H,Si-t-butyl); 0.91 (d,J=7.3Hz,3H,H-21; 0.96 (m,1H,H-25);

2.47 (d,J=6Hz,2H,H-24b); 4.16 (m,1H,H-3); 4.47 (m,1H,H-1);

4.89 and 4.93 (pp. 1H,H-19); 5.77 and 6.40 (d. J=11Hz,1H,H-6 and

5H-7); 6.08 (d. J=15.5Hz, H-24); 6.75 (ddd. J=15.5, 9, 6.5Hz, 1H,H-23).

Example 15

Reduction of the product obtained according to example 14, analogously to example 2, gives 200 mg of 1(S),3(R)-bis-(tert-butyldimethylsilyloxy)-26,27-cyclo-24a,24b-dihomo-9, 10-secocholesta-5E,7E,10(19),23E-tetraen-24a(R,S)-ol in the form of an oily mixture of epimers, which cannot be rarefied NMR spectroscopically.

<1>H-NMR (CDCl 3 ): δ = 0.01 ppm (s, 12H, Si-CH 3 ), 0.09 and 0.40 (m, 2H, H-26 and H-27); 0.50 (s,3H,H-18); 0.68 (m, 1H, H-25); 0.81 and 0.86 (s,9H,Si-t-butyl); 0.88 (d,J=7Hz,3H,H-21); 1.40 (t,J=7Hz,H-24b); 4.13 (m,1H,H-24a); 4.17 (m,1H,H-3); 4.49 (m, 1H,H-1); 4.88 and 4.93 (pp. 1H,H-19); 5.45 (dd. J=15.5, 6.5Hz, 1H,H-24); 5.59 (ddd. J=15.5, 7, 6.5Hz, 1H, H-23); 5.77 and 6.40 (d. J=11Hz,1H,H-6 and H-7).

Example 16

Analogous to example 3, by triplet sensitized photoisomerization and removal of the protective groups analogously to example 4, from 190 mg of the compound described under example 15, 85 mg of 26,27-cyclo-24a,24b-dihomo-9,10-secocholesta is obtained -5Z,7E,10(19),23(E)-tetraene-1(S),3(R),24a-(R,S)-triol in the form of a 1:1 mixture of diastereomers, which separate by high pressure liquid chromatography. The NMR spectra of both diastereomers are identical.

3-NMR (CDCl 3 ): δ = 0.09 and 0.49 (m, 2H, H-26 and H-27); 0.53 (s,3H,H-18); 0.70 (m,1H,H-25); 0.93 (d,J=7Hz,3H,H-21); 4.18 (m,1H,H-24a); 4.22 (m,1H,H-3); 4.43 (m,1H,H-1); 5.00 and 5.32 (s,1H,H-19); 5.50 (dd,J=15.5, 6.5Hz,H-24); 5.64 (ddd. J=15.5, 7, 6.5Hz, 1H, H-23); 6.02 and 6.38 (d. J=11Hz,1H, H-6 and H-7).

Example 17

Starting from aldehyde 1. and (1-ethylpropoxymethyl)-carbonylmethylenetriphenylphosphorane, isomer B is obtained analogously to the sequence according to examples 1-4 (5Z,7E,22E-1(S),3(R),24(S)-26,27 -dimethyl-24a,24b-dihomo-24b-oxa-9,10-secocholesta-5,7,10(19)22-tetraene-1,3,24-triol) with melting point 103-105 °C.

Example 18

Starting from aldehyde 1 and cyclopropylmethoxymethylcarbonylmethylenetriphenylphosphorane, isomer B (5Z,7E,22E-1(S),-3(R),24(S)-26,27-cyclo-24a) is obtained analogously to the sequence according to example 1-4 ,24b,24c-trihomo-24b-oxa-9,10-secocholesta-5,7,10(19)22-tetraene-1,3,24-triol).

<X>H-NMR (DMS0-d3): S=0.16 ppm (m,2H); 0.43 (m, 2H); 0.53 (S.3H); 1.00 (d,J=6Hz,3H); 3.21 (m, 4H); 4.00 (m, 2H); 4.19 (m, 1H); 4.51 (d,J=5Hz,1H); 4.70 (d,J=5Hz,1H); 4.75 (m,1H); 4.82 (d,J=5Hz,1H); 5.21(m,1H); 5.39 (m, 2H); 5.98 (d, J=11Hz, -1H); 6.18 (d,J=11Hz,1H).

Example 19

Starting from aldehyde 1 and (3-butynyl)-carbonyl-methylenetriphenylphosphorane, isomer B (5Z,7E,22E-1(S),3(R),24(S)-24- (3-butynyl)-9,10-secochola-5,7,10(19)22-tetraene-1,3,24-triol) with melting point 115-118 °C.

Example 20

Starting from aldehyde 1 and (3-butenyl)-carbonyl-methylenetriphenylphosphorane, isomer B (5Z,7E,22E-1(S),3(R),24(S)-24- (3-butenyl)-9,10-secochola-5,7,10(19)22-tetraene-1,3,24-triol) with melting point 146-147 °C.

Claims (1)

Analogy method for the preparation of therapeutic active side chain homologous vitamin D derivatives of formula I in which R<1> denotes a hydrogen atom, a hydroxy or acyloxy group with 1 to 9 carbon atoms, R<2> denotes a hydrogen atom or an acyl group with 1 to 9 carbon atoms, R3 or R4 denotes a hydroxy or acyloxy group with 1 to 9 carbon atoms and the second substituent denotes a hydrogen atom, or R3 and R<4> together denote an oxygen atom, R5 and R<6> independently denote a linear or pre- branched alkyl radical with up to 5 carbon atoms, a tri-fluoromethyl group or together with the tertiary carbon atom forms a saturated, unsaturated or aromatic carbocyclic, or by inclusion at 1 or 2 N-, 0- or S-atoms, a heterocyclic 3-, 4- , 5- or 6-membered ring, B and D denote either a hydrogen atom or together another bond (E-configured double bond) and A denotes either a direct bond between carbon the atoms 20 and 22 and X denotes an alkyleneoxy acid residue -(CH2)n0- where n = 1 to 3; or A denotes a methylene bridge (-CH2-) between carbon atoms 20 and 22 and X denotes an alkylene residue -(CH2)n- or an alkylene oxy radical -(CH2)n0- where n = 1 to 3; or; when A denotes a direct bond and B and D together be draws a second bond, denotes one of the residues -CH2-0-CH2-<J , -(CH2)2-h or -(CH2)2-<=,> characterized in that a compound of general formula IV in which R<1>' denotes a hydrogen atom or a protected hydroxy group and R<2>' denotes a hydroxy protecting group and A, X, R<5> and R<6> have the designations given for formula I, if desired after selective hydration of the double bond in the side chain, is converted into a compound of general formula IVa wherein R<1>', R<2>', A, X, R5 and R6 have the designation given in formula IV and if desired, a reduction of the carbonyl function and optionally after separation of the mixture of the epimeric hydroxy compounds formed by the reduction with general formulas Illa and Illb in which R<1>', R<2>', A, X, R<5> and R6 have the designations given in formula IV, and B and D have the designations given in formula I, by irradiation with ultraviolet light during inversion of the stereoisomerism at the 5,6-double bond is converted into a compound of general formula II in which R<1>', R<2>', A, B, D, X, R5 and R6 have the designations given in formula Illa/IIIb, and this compound then, by splitting off the present hydroxy protecting groups and optionally by partial or complete esterification of the hydroxy groups, is transferred to a compound of general formula I.