DK172567B1 - 3,5-cyclovitamin D2 derivative intermediates for use in the preparation of 1alpha, 25-dihydroxylated vitamin D2 derivatives - Google Patents

Description

in DK PR 172567 B1

The present invention relates to novel 3,5-cyclovitamin D 2 derivatives as intermediates at. the preparation of 24-epi-1α, 25-dihydroxylated vitamin D 2 derivatives.

5

The subject 3,5-cyclovitamin D 2 derivatives are peculiar to the characterizing part of the claim.

The importance of the hydroxylated forms of vitamin D as 10 regulators of calcium and phosphorus metabolism in animals and humans is well known and described in several patents and in the literature, and as a result, these hydroxyvitamin D derivatives are increasingly finding clinical and veterinary use as drugs. in the treatment of diseased states in calcium metabolism and associated bone disorders. Vitamin D 2 is known to be hydroxylated in vivo to 25-hydroxyvitamin D 1 and then to 1α, 25-dihydroxyvitamin D 2, the latter compound being generally accepted as the active hormonal form of vitamin D 2. Likewise, the extremely potent vitamin D2 metabolite le, 25-dihydroxyvitamin D2 (le, 25- (OH) 2D2) is formed from vitamin D2 via 25-hydroxyvitamin D2 (25-OH-D2). Both of these hydroxylated vitamin D 2 compounds have been isolated and identified (DeLuca 25 et al., U.S. Patent Nos. 3,585,221 and 3,880,894); derived from vitamin D 2, these metabolites are characterized by (S) stereochemistry at carbon 24.

Surprisingly, it has been found that the aforementioned 24-epi-1α, 25-30 dihydroxylated vitamin D Although highly effective in stimulating intestinal calcium transport and mineralization of new bones, they do have minimal effect on bone calcium mobilization. These properties therefore open up the possibility of controlling various vitamin D-dependent processes in a way that has not previously been possible with more common vitamin D derivatives.

2 DK PR 172567 B1

The 24-epi-1α, 25-dihydroxylated vitamin D3 derivatives have the general formula I

a 10 having R configuration at C-24, wherein each of R 1 - R 2 and R 9, which are the same or different, represents hydrogen, alkylcarbonyl of 1-6 carbon atoms, arylcarbonyl or an acyl group of formula: R00C (CH2) nC0 or R00CCH2-O-CH2 CO- where n is an integer from 0 to 4 and R represents an alkyl group of 1-6 carbon atoms and X represents alkyl of 1-4 carbon atoms.

Particular examples of compounds prepared with these intermediates include Ia, 25-dihydroxyvitamin D2, the corresponding (24R) epimer, Ia, 25-dihydroxy-24-epivitamin D2, the corresponding 5,6-trans isomer, ie. 5,6-trans-25a, 25-dihydroxyvitamin D2 and 5,6-trans-1a, 25-dihydroxy-24-epivitamin D2 as well as the C-25 alkyl or aryl homologues of these compounds i. compounds of the above formula wherein X represents ethyl, propyl, isopropyl, butyl or isobutyl.

30

An embodiment of the process for preparing vitamin D 2 derivatives is illustrated in the attached Scheme I. In the following depiction of this process, the numbers (1, 2, 3, etc.) denoting the specific compounds refer to the formulas, numbered in this way in Reaction Scheme I.

3 DK PR 172567 B1

A suitable starting material for the preparation is the vitamin D ketal derivative of formula (1). It may be convenient to use the compound (1) as a mixture of the 24R and S epimers, the separation of the individual 24R and 5 S epimers being carried out at a later stage during the process.

However, the pure 24R epimer of (1) is also well suited as a starting material to obtain the corresponding (24R) -la, 25-dihydroxylated final compound.

The starting material (1) is converted to the desired Ia, hydroxy cleaved form via the cyclovitamin D derivatives (DeLuca et al., U.S. Patents 4,195,027 and 4,260,549). Thus, treating the compound (1) with toluene sulfonyl chloride in the usual manner gives the corresponding C-3 tosylate 15 (2) which is solvolysed in an alcoholic substrate to obtain the 3,5-cyclovitamine D derivative (3).

Solvolysis in methanol gives the cyclovitamin of formula (3) where Z = methyl, while the use of other alcohols such as e.g. ethanol, 2-propanol or butanol at this reaction give the analogous cyclovitamin D compounds (3) wherein Z represents an alkyl group derived from the alcohol e.g. ethyl, isopropyl or butyl. Allyloxidation of the intermediate (3) with selenium dioxide and a hydroperoxide gives the Ια-hydroxy analog of formula (4).

Subsequent acetylation of compound (4) gives the 1-acetate of formula (5, R 2 = acetyl). Optionally, other 1-O acylates (formula 5 wherein R 1 = acyl, for example, the formate, propionate, butyrate or benzoate) are prepared by analogous ordinary acylations. The 1-O-acyl derivative 30 is then subjected to acid-catalyzed solvolysis. If this solvolysis is carried out in a solvent containing water, the 5,6-cis-vitamin D intermediate of formula (6, R R = acyl, R R *H) and the corresponding 5,6-trans compound (formula 7 = acyl, R2 = H) in a ratio of 3-4: 1. These 5,6-cis and 5,6-trans isomers can be separated at this stage e.g. using high capacity liquid chromatography. If desired, the C10-acyl group can be removed by base hydrolysis to give compounds (6) and (7) wherein R 1 and R 2 = H. Optionally, these 1-O manoacetyls can also be further acylated at C The 3-hydroxy groups using ordinary acylation conditions to give the corresponding 1,3-di-0-acylates of formulas (6) and (7) wherein R 1 and R 2, which may be the same or different, represent acyl groups. Alternatively, the hydroxy-cyclovitamin of formula (4) may be subjected to acid-catalyzed solvolysis in a substrate containing a low molecular weight organic acid to give 5,6-cis and trans compounds of formulas (6) and (7) wherein R and R 2 = acyl, wherein the acyl group is derived from the acid used in the solvolysis.

The next step of the process comprises removing the metal protecting group to prepare the corresponding 25 ketone. This step is critical as the conversion of the ketone to the ketone must be accomplished without simultaneous isomerization of the 22- (23) double bond to the conjugated 23 (24) position which may occur under the acidic conditions required. for ketal hydrolysis. Further, the conditions must be selected so as to avoid the removal of the sensitive allyl C-1 oxygen function. The conversion is successfully carried out by gentle hydrolysis at moderate temperatures using organic acid catalysis. Thus, treatment of the 5,6-cis compound (6) in aqueous alcohol with p-toluene-sulfonic acid gives the corresponding ketone (8).

In order to avoid unwanted removal of the C-1 oxygen function during the reaction, it is advantageous to protect the C-1-hydroxy group 30 in the compound (6) (eg = acyl, R 2 = acyl, R 2 = hydrogen or acyl).

Subsequent reaction of ketone (8) with a methyl Grig-nard reagent then gives the desired 1α, 25-dihydroxy-vitamin D 2 compound of formula (9). If the starting material, compound (1) used in the above process is a mixture of the two C-24 epimers, then compound (9) will be obtained as a mixture of the 24-S and R epimers. (9a and 9b, respectively). The separation of. this epimeric mixture can be obtained by chromatographic methods to give Ia, 25-dihydroxyvitamin D2 (Formal 9a, 524S stereochemistry) and its 24R epimer, Ia, 25-dihydroxy-24-epivitamin of formula 9b, both compounds in pure form. Of course, such separation of the epimers is not necessary if the compounds are to be used in the form of a mixture.

The 5,6-trans-25 ketal intermediate of formula (7) will similarly undergo ketal hydrolysis to give the 5,6-transketone intermediate of formula (10) which, via a Grignard reaction with methyl magnesium bromide or an analogous reagent, gives 5 The 6-trans-1α, 25-dihydroxyvitamin D2 compounds of formula (11), as the 24S or 24R epimer, or as a mixture of both epimers depending on the nature of the starting material (1) used in the process. If obtained as an epimeric mixture, the 20 epimers can be separated by chromatography to give 5,6-trans-1α, 25-dihydroxyvitamin D2 (Ha) and its 24R epimer 5,6-trans-1α, 25-dihydroxy -24-epivitamin D2 of formula (11b). These reactions using the 5,6-trans intermediate are carried out in a manner completely analogous to those used for the 5,6-cis compounds described above.

The side-chain ketones of formulas (8) or (10) are the most valuable and easily convertible intermediates because they can be used to prepare a whole series of 1α, 25-dihydroxyvitamin D2 side-chain analogues. Specifically, these keto intermediates may serve to prepare 5,6-cis or 5,6-trans-1α, 25-dihydroxyvitamin D2 analogs of the general side-chain formula set out below 35 *.

r *> X

6 DK PR 172567 B1 wherein X represents an alkyl or aryl group.

Eg. For example, treatment of the ketone (8) with ethylmagnesium bromide gives the corresponding hydroxyvitamin analog having the above-mentioned side chain formula, wherein X represents an ethyl group. Similarly, treatment of (8) with propyl, butyl or isopropylmagnesium bromide or phenylmagnesium bromide gives the side-chain analogs where X represents propyl, butyl, isopropyl or phenyl, respectively. Analogous treatment of the 5,6-trans-25-ketone intermediate of formula (10) with alkyl or aryl-Grignard reagents gives the 5,6-trans-vitamin D2 analog having the above side chain, wherein x represents alkyl or the aryl group introduced by the Grignard reagent used.

15

It is also understood that the reaction of the ketomel intermediates (8) or (10) with an isotope-labeled Grignard reagent (e.g., C CHgMgBr, 14CH 14MgBr or C ^H ^MgBr) provides a convenient way to prepare 1a, 25-dihydroxyvitamin D2 20 or its trans isomers, and the corresponding C-24 epimers in isotope labeled form e.g. compounds having the aforementioned side chain, wherein X represents C 1 H 2, C 4 H 4, C 2 H 2, C 1 H 2, or any other isotope-labeled alkyl or aryl group.

25

The above alkyl or aryl homologue of 5,6-cis or trans-1α, 25-dihydroxyvitamin D 2 is valuable substitutes for the parent compounds in situations where a higher fat solubility is desired, while the isotope-labeled compounds mentioned above are used as reagents for analytical purposes.

Although the free hydroxy compounds of formulas I above (wherein R 2, and = H) are further usually used for therapeutic purposes, it may in some cases be advantageous to use the corresponding hydroxy protective derivatives. These hydroxy protective derivatives are e.g. the acylated compounds of the general formula I above, wherein one or more R 1, R 2 and represents an acyl group.

These acyl derivatives are conveniently prepared from the free hydroxy compounds by ordinary acylation methods, ie. treating any of the hydroxyvitamin D2 final compounds with an acyl halide or acid anhydride in a suitable solvent such as e.g. pyridine or an alkyl pyridine. By appropriate selection of reaction time, acylating agent, temperature and solvent well known in the art, the partially or completely acylated derivatives of the above formulas are obtained. gives treatment of 1α, 25-dihydroxyvitamin D2 (9a) in pyridine solvent with acetic anhydride 15 at room temperature the 1,3-diacetate, while the same reaction carried out at elevated temperature gives the corresponding 1,3,25-triacetate. The 1,3-diacetate can be further acylated at C-25 with another acyl group e.g. by treatment with benzoyl chloride or succinic anhydride to give the 1,3-diacetyl-25-benzoyl or 1,3-diacetyl-25-succinoyl derivative, respectively. The 1,3,25-triacyl derivative can be seen to be selectively hydrolyzed in the mild base to give the 1,3-dihydroxy-25-O-acyl compound, the free hydroxy groups of which can be acylated again optionally with other acyl groups. Similarly, the 1,3-diacyl derivative may be partially subjected to acyl hydrolysis to give the 1-O-acyl and the 3-O-acyl compounds, which in turn can be acylated with other acyl groups. Similar treatment of any of the other hydroxyvitamin D2 end products (e.g.

(9b, 11a / b or their corresponding 25-alkyl or aryl analog) gives the corresponding acyl derivatives of formulas I, wherein any or all of R 1, R 2 and R 2 represent acyl.

35 8 DK PR 172567 B1

EXAMPLE I

1'-hydroxy-3,5-cyclovitamin D (4, Z = methyl)

A solution of compound (1) (50 mg) (as a mixture of 24 R and S epimers) in dry pyridine (300 ° 1) is treated with 50 mg of p-toluenesulfonyl chloride for 30 hours at 4 ° C.

The mixture is poured over ice / saturated NaHCO 3 with stirring and the compound is extracted with benzene. The combined organic phases are washed with aqueous NaHOC 3, H 2 O, aqueous CuSO 4 and water, dried over MgSO 4 and evaporated.

The crude 3-tosyl derivative (2) is directly solvated in anhydrous methanol (10 mL) and NaHOC® (150 mg) by heating 8.5 hours with stirring at 55 ° C. Then, the reaction mixture is cooled to room temperature and concentrated to about 2 ml in vacuo. Benzene (80 ml) is then added and the organic layer is washed with water, dried and evaporated. The produced cyclovitamine (3, Z * methyl) 20 can be used in the subsequent oxidation without further purification.

The crude compound (3) in CH2 Cl2 (4.5 ml) is added to an ice-cooled solution of SeO2 (5.05 mg) and t-BuOOH (16.5 25 µl) in CH2 Cl2 (8 ml) containing anhydrous pyridine (50 µl ). After stirring at 0 ° C for 15 minutes, the temperature of the reaction mixture is allowed to rise to room temperature.

After an additional 30 minutes, transfer the mixture to a separatory funnel and shake with 10% NaOH (30 ml). Ether 30 (150 ml) is added and the separated organic phase is washed with 10% NaOH, water, dried and evaporated. The oily residue is purified on silica gel thin-layer plates (20 x 20 cm plates, AcOEt / hexane 4: 6) to give 20 mg of the le-hydroxy derivative (4, Z = methyl): mass spectrum, m / e: 35 470 (M ;, 5), 438 (20), 87 (100); NMR (CDCl3) δ 0.53 (3H, s, 18-H3), 0.63 (1H, m, 3-H), 4.19 (1H, d, j = 9.5 Hz, 6-H) , 4.2 (1H, m, 1-H), 4.95 (1H, d, J = 9.5 Hz, 7-H), 5.17, and 5.25 (2H each m , 19-H2>, 5.35 (2H, m, 22-H and 23-H).

Example 2 Acetylation of Compound (4).

A solution of cyclovitamine (4, Z = methyl) (18 mg) in pyridine (1 ml) and acetic anhydride (0.33 ml) is heated at 55 ° C for 2 hours. The mixture is poured into ice-cold saturated NaHCO 3 and extracted with benzene and ether. The combined organic extracts are washed with water, saturated CuSO ^ and aqueous NaHCO ^ solutions, dried and evaporated to give the 1-acetoxy derivative (5, Z = methyl, acyl = acetyl) (19 mg): mass spectrum, m / e: 512 (M +, 5) 420 (5), 87 (100); NMR (CDCl3) δ 0.53 (3H, s, 18-H3) 4.18 (1H, d, J = 9.5 Hz, 6-H), 4.97 (2H, m, 7-H and 19 -H), 5.24 (2H, m, 1-H and 19-H), 5.35 (2H, m, 22-H and 23-H).

EXAMPLE 3 20

Solvolysis of 1 O-acetoxy-3,5-cyclovitamine (5) (R 2 = acetyl)

A solution of cyclovitamine (5) (4.5 mg) in 3: 1 mixture of dioxane / H 2 O (1.5 ml) is heated at 55 ° C. Then p-toluenesulfonic acid (1mg in 20 ml of water) is added and heating is continued for 15 minutes. The mixture is poured into saturated NaHCO 3 / ice and extracted with benzene and ether. The organic phase is washed with NaHCO 3 and water and dried over 30 MgSO 4. Evaporation of the solvents gives a residue containing compounds (6) (wherein R 2 = acetyl, R 2 = H) and (7) (wherein R x = acetyl and R 2 = H) which are separated by chromatography on HPLC (6.2 mm x 25 cm "Zor-bax-Sil") using 2% 2-propanol in hexane as eluant. Optionally, the final compounds can be further purified by repeated chromatography.

Example 10 PR 172567 B1 EXAMPLE 4

Ketal hydrolysis of compound (6) to give the ketone (8).

To the solution of the ketal (6, R 2 = acetyl, R 2 = H) (1.35 mg) in ethanol (1.5 ml) is added p-toluenesulphonic acid (0.34 mg in 45 μΐ water) and the mixture is heated. minutes of reflux. The reaction mixture is poured into dilute 10 NaHCO 3 and extracted with benzene and ether. The combined organic extracts are washed with water, dried over MgSO 4 and evaporated. High pressure liquid chromatography of the crude mixture (4% 2-propanol / hexane, 6.2 mm x 25 cm "Zorbax-Sil") gives unreacted ketone (6) (0.12 mg, collected 15 at 48 ml) and the desired ketone ( 8, R 2 = acetyl, R 2 = H) (0.36 mg, collected at 52 ml), characterized by the following results: mass spectrum m / e: 454 (M +, 9), 394 (17), 376 ( 10), 134 (23), 43 (100); NMR (CDCl3) δ 0.53 (3H, s, 18-H3), 1.03 (3H, d, J = 6.5 Hz, 21-H3), 1.13 (3H, d, J = 7.0 Hz, 28-H3), 2.03 (3H, s, OL 2 COO), 2.12 (3H, s, CH 2 CO), 4.19 (1H, m, 3-H), 5.03 ( IH, m, 19-H), 5.33 (3H, wide m, 19-H, 22-H and 23-H), 5.49 (1H, m, 1H), 5.93 (1H, d, J - 11 Hz, 7-H), 6.37 (1H, d, J = 11 Hz, 6- H); UV (EtOH λ 266 nm, 250 nm, λ. 225 nm.

EXAMPLE 5

Reaction of ketone (8) with methyl magnesium bromide to prepare compounds (9a) and (9b).

30

Ketone (8, R 2 = acetyl, R 2 = H) in anhydrous ether is treated with excess CH 3 MgBr (2.85 M solution in ether). The reaction mixture is stirred for 30 minutes at room temperature, then quenched with aqueous NH 2 Cl, extracted with benzene, ether and CH 2 Cl 2 · The organic phases are washed with dilute NaHCO 3, dried over MgSO 4 and evaporated. The mixture of (9a) and (9b) prepared in this way is separated by high pressure liquid chromatography (6% 2-propanol / hexane, 4.5 mm x 25 cm "Zorbaz-Sil") to obtain in sequence after elution the pure epimers (9a) and (9b). 1α, 25-dihydroxyvitamin D_ (9a); UV (EtOH) λ 265.5 nm, λ. 227.5 nm; in max min 5 mass spectrum, m / e 428 (M +, 6) 410 (4), 352 (4), 287 (6), 269 (10), 251 (10), 152 (42), 134 (100), 59 (99); NMR (CDCl3) δ 0.56 (3H, s, 18-H3), 1.01 (3H, d, J - 6.5 Hz, 28-H3), 1.03 (3H, d, J = 6, 5 Hz, 21-H3), 1.14 and 1.18 (6H, each s, 26-H3 and 27-H3), 4.24 (1H, m, 3-H), 4.43 (1H, m, 1-H), 5.01 (1H, m, 19-H), 5.34 (3H, broad -m, 19-H, 22-H and 23-H), 6.02 (1H, d, J = 11 Hz, 7-H), 6.39 (1H, d, J = 11 Hz, 6-H).

1α, 25-dihydroxy-25-epivitamin D- (9b): UV (EtOH) λ fnsx 265.5 nm, λ ^ 227.5 nm; mass spectrum, m / e 428 (M, 13), 410 (9), 352 (7), 287 (11), 269 (15), 251 (13), 152 (52), 134 (100), 59 (97) ).

EXAMPLE 6 20

Conversion of compound (7) to 5,6-trans-1α, 25-dihydroxyvitamin D2 compounds (11a) and (11b).

Hydrolysis of the ketal intermediate (7, = acetyl, R 2 = 25 H) under the conditions of Example 4 gives the corresponding 5,6-trans-25 ketone of the formula (10, = acetyl, r 2 = H) and subsequently Reaction of this ketone with methyl magnesium bromide using conditions analogous to that of Example 5 gives a mixture of the epimeric compounds (11a) and (11b) which are separated by high pressure liquid chromatography (HPLC) to give the pure 1α, 25-dihydroxy-5,6-trans-vitamin D2 (11a) and 1α, 25-dihydroxy-5,6-trans-24-epi-vitamin D2 (11b). If desired, the formula can be confined by isomerization to the respective 5,6-cis compounds (9a, 9b) by well known methods.

12 DK PR 172567 B1 5.6- trans-1α, 25-dihydroxyvitamin D2 (11a); UV (EtOH) AmQx 273.5 nm, 230 µm, mass spectrum, m / e 428 (ML +, 8), 410 (3), 287 (3), 269 (7), 251 (7), 152 (34), 134 (100), 59 (78).

5

5.6-trans-1α, 25-dihydroxy-24-epi-vitamin D2 (11b); UV

(EtOH) * max 273.5 nm, λ ^ 230 nm, mass spectrum, m / e 428 (M +, 10), 410 (4), 352 (4), 287 (5), 269 (9), 251 (8 ), 152 (37), 134 (100), 59 (82).

10

A suitable starting material for the vitamin D2 derivatives is the vitamin-D ketal derivative of formula (1), which can be obtained by working as described in Schemes II and III as described in English Patent No. 2 127 023 or U.S. Patent No. 5,407. It is usually convenient (for example, if both C-24 epimers are desired) to use the compound (1) as a mixture of 24R and 24S epimers, since the separation of the individual 24R and 24S epimers can be carried out. later. However, the pure 24S or pure 24R-20 epimer of (1) is suitable, the former providing the 24S, 1a, 25-dihydroxy final compound and the latter compound corresponding to the 24R final compound.

25 30 35 DK PR 172567 B1

REACTION SCHEME I

13 5 γΉι l_? r "* '"' i i_? ? _? YES so .. J ZO-. 10 RO ·· * ^^^ OR,

I: R = Η Λ 4: R, = H

R - Acyl

I ^ Sl L_? O 1 OH

j | -Ϊ * »- fj ^ || 20

R20 - '' ^ OR, R20 '' 'kx ^ OR, ΗΟ -' '^^ OH

6 8 9a: 24 p

b: £ 24

R, 0V'V ^ / V0R2 R, R 2 M O ^ '^' o1I

7 ro II a: 2 ^, S

35 h: 2 ^

SCHEME OF REACTION II

14 DK PR 172567 B1 5 YcH ° Yc "° ... γ Ύ oy ^ ° o ocH> ίο 'Ύ vj

J. Suli-o- Λ OH

ί ° i ^ '' '(¥%> is * »£ ζρ ic./j A ·“ ivct · Jh

* ° Υϊµ QtT

• Y- r 25 <· ^ Χ'Φ '5 v- ^ x' r. *; j] (*) rf '35 (<?)

SCHEME OF REACTION III

15 DK PR 172567 B1

-) Ph ~ S

o o o

T

10 II »o o σ o i — i i __ /

SUiro // A

15 20 25 30 35

Claims (2)

16 DK PR 172567 B1 Patent claim: 3,5-cyclovitamin D2 derivative, characterized in that it has the general formula III: 10 ZOy / f 111 KAy 15 wherein Y represents hydroxyl and Z represents an alkyl group. 20 25 30 35