NL8120067A - 25-Hydroxy-26,26,26,27,27,27,27-hexa:fluoro-cholecalciferol - useful as vitamin D3, like agent for calcium deficiency diseases - Google Patents

Abstract

(A)25-Hydroxy-26,26,26,27,27,27-hexafluoro cholecalciferol of formula (I) is new as are 25- Hydroxycholestene derivs. of formula (II) (R is H, tetrahydropyranyl, 1-4C alkyl, or PhCO opt. substd. and the broken bond is an optional double bond.) Cpd. (I) has excellent vitamin D3-like activity as measured by ability to stimulate Ca transport in the intestine and to mobilise Ca from bone and in its antirachitic activity, and so it can be used to treat diseases resulting from Ca metabolism disorders. Cpds. (II) are intermediates in the prepn. of (I).

Description

8 1 2 0 0 6 7

• pi j ·. I

25-Hydroxy-26,26,26,27,27,27-hexafluorocholecalciferol.

O · I

Technical Field: FY Y * I This invention relates to a compound characterized by a vitamin D-like action.

In particular, this invention relates to a derivative of vitamin D_.

5 3

Vitamin D ^ is a well-known means of controlling the calcium and phosphorus homeostasis. It is known that in normal animals or humans this compound stimulates calcium transport in the gut and mobilization of calcium in bones IQ and is effective in preventing rickets.

It is also now known that in order to be effective, vitamin D must be converted in vivo to its hydroxylated forms. For example, the vitamin is first hydroxylated in the liver to 25-hydroxy-vitamin D ^ and further hydroxylated in the kidneys to 1a, 25-dihydroxy vitamin or 2 ^, 25-dihydroxy vitamin D ^. The 1a-hydroxylated form of the vitamin is generally regarded as the physiologically active or hormonal form of the vitamin and is thought to be responsible for what is termed the vitamin D-like actions, such as an increased absorption of calcium and phosphate in the intestines, mobilizing minerals in the bones and retaining calcium in the kidneys.

Background of the prior art.

Since the discovery of biologically active metabolites of vitamin D, there has been much interest in the preparation of structural analogues of these metabolites, because such compounds may be useful therapeutic agents for treating diseases resulting from disorders of calcium metabolism . A 2Q number of vitamin D-like compounds has been synthesized. See, for example, U.S. Pat. Nos. 3,7 ^ 1,996 directed to 1a-hydroxycholecalciferol, 3,907.8H3 directed to 1a-hydroxy-8120067-2 - ergocalciferol, 3,786,062 directed to 22-dehydro-25-hydroxychole-calciferol, 3,906.01H directed to 3 -deoxy-1α-hydroxycholecalciferol and 1,069,321 directed to the preparation of various side chain fluorinated vitamin derivatives and side chain fluorinated dihydrotachysterol analogs.

Description of the Invention A novel derivative of vitamin D has been prepared to exert excellent vitamin D-like activity as measured by its ability to promote calcium transport in the gut, its ability to mobilize calcium from bones (to increase serum calcium and its antirachitic activity, as measured by a rat behavior test. Therefore, this compound can serve as a substitute for vitamin D in its many known applications and will be useful for treating various metabolic bone diseases.

This derivative has been identified as 25-hydroxy-26.26.26.27.27.27-hexafluorocholecalciferol (25-hydroxy-26,26,26, 27.27.27-hexafluoro vitamin D ^ or 25-OH-26,27-Fg-D ^).

The best way to practice the invention.

The compound of the invention was synthesized according to the following description and condensed scheme:

The starting material in the process, namely 3-hydroxyhydro-5-en-2L + ol tetrahydropyranyl ether (1), is readily available by treatment of commercially available cholic acid or esters of cholic acid. For example, by conversion of cholic acid or an ester of cholic acid into the 3-tetrahydropyranyl derivative, followed by reduction of the acid or ester function with a metal hydride, such as lithium aluminum hydride, all of which are known. See the diagram on the formula sheet.

Synthesis of 25-hydroxy-26,26,26,27,27,27-hexafluoro-vitamin D ^.

3B-hydroxychol-5-enyl bromide tetrahydropyranyl ether (2) _

To a solution of 36-hydroxychol-5-en-2i + -ol 8120067-3-tetrahydropyranyl ether (1) (1.1 g, 3 * 15 m.inol) in tetrahydrofuran (THF) (15 ml) was added at -78 ° C added a solution of n-butyl lithium (3.5 mmol) in hexane. After stirring for 5 min, p-toluene sulfonyl chloride (670 mg, 3.5 mole) in THF (5 ml) was added and the mixture was stirred for 1 h. The reaction mixture was poured onto ice water and extracted with methylene chloride. After washing with water and drying over magnesium sulfate, the solvent was removed by evaporation. The residue is dissolved in a mixture of THF (15 ml) and acetone (15 ml) and then added to lithium bromide (3.0 g). After heating under reflux for 2 hours, the precipitate was removed by filtration and the filtrate was chromatographed on a column of silica gel. Eluting with methylene chloride gave 2 (1.1 µg, 88%), mp: 117-119 ° C (from a mixture of methanol and acetone).

Analysis: calculated for C 16 H 20 Br: c 68.62%, H 9.33%.

Found: C 68.8% and H 9.3%.

33-acetoxy-26,26,26,27,27,27-hexafluoro-25-hydroxychole st-5-ene (1 *) _ 20 A suspension of potassium (150 mg) and magnesium c KLoride (200 mg) in THF (5 ml) was refluxed in an argon atmosphere for 2 hours and then cooled to room temperature. The bromide 2 (25¾mg, 0.5mmol) in THF (5ml) was added and the mixture was stirred at room temperature for 2 hours. Under cooling with a mixture of dry ice and acetone, an excess of hexafluoroacetone gas was introduced and the mixture was stirred for 15 min, methanol (5 ml was added and the mixture was stirred at room temperature for 10 min.) After addition of dilute HCl, the reaction mixture extracted with ether. The extract was chromatographed on a column of silica gel. From the eluate with a mixture of benzene and ether (30: 1), the hexafluoride was 3 ^ 8 mg, 16%), MS, m / e 510 ( M + ~ CnI) ** 92, 77, 255; NMR (CDC13), δ, 0.68 (s, C-18), 0.9¾ (d, J = 6 M, C-21), 1.00 (s, C-19), 3.88 (m , C-3), 3, U8 (πι, ^ νζΧ), 3> 88 <m> c “3)> h> J2 35 - 81 2 0 0 6 7 - It - (πι, ζ ^ Ο- '5 , 32 (m 'C'6)'

The hexafluoride 3 was dissolved in methanol (3ml) and methylene chloride (3ml) to which p-toluene sulfonic acid (10mg) was added to give the corresponding 3-5 hydroxy compound. The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was dissolved in methylene chloride (2ml) and stirred with acetic anhydride (1ml) and pyridine (1ml) for 16 hours.

The product was purified with a silica gel column to give 28 mg of the 3-acetate 1+, melting point: 165-166 ° C, MS, m / e 1 + 92 (M + -AcOH), 1 + 77, 381+, 371, 255; MR (CDC13), 6, 0.68 (s, C-18), 0.93 (d, J = 6 Bs, C-21), 1.01 (s, C-19), 2.02 (s , acetyl), 1 +, 56 (m, C-3), 5.3 * + (m, C-6).

If desired, other 0-acyl protecting groups than the 3-acetyl group can be used. For example, the tetrahydropyranyl group can be used, or an acyl group with 1 to about 1+ carbon atoms, for example, acetyl, propionyl, butyryl or an aromatic acyl group, such as benzoyl or substituted benzoyl (nitro-benzoyl, chlorobenzoyl) can be used and easily are obtained by reacting the 3-hydroxy compound with the appropriate anhydride or acyl halide, for example the chloride, as is known in the art. Thus, the substituent in the 3 position in compounds 1+ and 5 can be referred to as an RO group, wherein R is hydrogen, tetrahydropyranyl, an acyl group having 1 to about 1+ carbon atoms, benzoyl or substituted benzoyl group.

33-Acetoxy-26,26,26,27,27,27-Hexafluorocholesta- 5,7-dien-25-ol (5) ._

N-bromosuccinimide (9 mg) was added to a refluxing solution of the acetate 30 1+ (19 mg) in CCl 2 (2 ml) and the mixture was refluxed for 20 min in an argon atmosphere. After cooling, the resulting precipitate was filtered off and the filtrate evaporated under reduced pressure. The residue was dissolved in xylene (1.5ml) and dropped into a refluxed solution of s-collidine (0.5ml) in xylene (1.5ml).

fi 1 2 0 0 6 7 - 5 -

After heating under reflux for 10 minutes, the mixture was extracted with ethyl acetate. The crude product was dissolved in acetone (5ml) and treated with p-toluenesulfonic acid (10mg) by stirring at room temperature for 14 hours. After adding water, the mixture was extracted with ethyl acetate. The reaction product was purified by preparative thin layer chromatography (benzene ethyl acetate, 50: 1, twice) to give the 5,7-diene 5 (* +, 9 mg) UV, λ (EtOH) 262 (sch), 271, 282, 293 nm was obtained, max 25-hydroxy-26,26,26,27 <27,27-hxxafluorovitamin D ^ (6) 10 The 5 »T-8ien 5 was dissolved in an ethanol (40 ml) - benzene (90 ml ) solution 2.5 min. under argon and cooling with ice through a Vycor filter irradiated with a medium pressure mercury lamp. The reaction mixture was then refluxed for 1 hour. The solvent was evaporated and the residue was chromatographed on a silica gel column and then chromatographed by preparative thin layer chromatography (benzene-ethyl acetate, 50: 1, twice) to obtain 1.2 mg of the crude vitamin D acetate. The solution of the acetate in TH1 (¾ ml)

Treated with 5% KOH for 13 hours at room temperature and under argon

20 in methanol (5 ml). The product was extracted with ethyl acetate

and was purified by HPLC using a Zorbax-SIL

column (15 cm X * t, 6 mm. i.d.) and a solvent of methylene-cKLoride-hexane (2: 1), to give 0.75 mg of 6, UV, λ. (EtOH) 227.5, λ 264 nm; MS, m / e 508 (M +), 493, 490, mm max 25 475, 271, 253, 136, 118.

The 25-OH-26,27-Fg-D4 product can, if desired, be obtained in crystalline form by dissolving in a suitable solvent or solvent system, for example ether, ether-hexane, methanol-ether, ethyl acetate-alkane and then the solution. agent or the solvents by evaporation or other known manner.

If desired, the above-described process may also hydrolyze the -5,7-diene (5) by the foregoing process or by other mild, basic hydrolysis methods known in the art prior to irradiation. converting the acetoxy substituent in the 3-position to hydroxyl.

The biological effect.

The biological activity of 25-OH-26,27-Fg-D4 is confirmed by suitable in vivo test rats. Newly weaned 5 male rats were purchased from Holtzman Co. Erase. and ad libitum with water and either with a low calcium suitable phosphorus, vitamin D deficient diet, as described by Suda and med. (J. Nutrition 100, 1θ! +9, 1970), or a high calcium-low phosphorus, vitamin D deficient diet as described by Tanaka and DeLuca (PNAS 71,10 10 ^ 0, 197U) fed for 3 weeks.

The calcium transport in the intestines

The rats fed the low calcium, vitamin D deficient diet for 3 weeks were divided into 3 groups of 5 rats each and sacrificed 22 hours before being sacrificed, respectively. 650 pmoles of either 25-OH-26.27-O-O-1, or 25-hydroxy D 1 O (25-OHD-O) dissolved in 0.1 ml of 95 # ethanol in the jugular vein. The rats in the control group were given the ethanol carrier in the same manner. They were killed by decapitation and the blood was collected. Then their 12-fingered intestine was immediately removed to measure the visceral calcium transport activity according to the method described by Martin and DeLuca (Am. J. Physiology 216, 1351, 1969). The results are shown in Table 1, first column.

The concentration of serum calcium. The blood collected from the rats as described above was centrifuged to obtain the serum. 0.1 ml of the serum was mixed with 1.9 ml of a 0.1% lanthanum chloride solution and the calcium concentration was measured with an atomic absorption spectrophotometer (Perkin-Elmer 30 Model HO-21U). The results are shown in the second column of Table 1.

Because of the significantly greater ability of 25-OH-26.27-FgD ^ over that of 25-OHD ^ to increase the serum calcium concentration, as shown in Table 1, a study of the increase in the calcium content of the serum 8120067-7 - performed over time in response to the administration of 25-OHD ^ or 25-OH-26,27-F ^ D ^.

Rats fed the low calcium, vitamin D deficient diet for 3 weeks were divided into groups of 5 rats. The rats were administered 325 pmol 25-0H-26,27-Fg-D 4 or 25-OHD 4 dissolved in 0.1 ml 95% ethanol through the jugular vein. The materials were given 6.17.27 or U8 hours before sacrifice.

Table 1

The intestinal calcium transport and the increase in serum calcium concentration as a result of a single dose of 25-OH-26,27-F £ -D or 25-0HD_.

(65Ο mol) administered the Ca transport in serum calcium compound the intestines: (mg / 100 ml) _ ^ Ca inside / ^ Ca outside_ control 2.1 + 0.6 * 3.6+ 0.1 25-OH-26, 27-Fg-D ^ 5.6 + 0.8b ^ 5, it + 0.1 25-OHD3 lt, 9 + 0.8c) Π, 9 + '0.3 standard deviation from the mean b) and c) from a) p <0.001 b) from c) NS

e) and f) from d) p <0.001 e) from f) p <0.005

Control group rats similarly received only the ethanol carrier. The rats were killed by decapitation at the indicated times, the blood was collected and centrifuged to obtain the serum.

The serum calcium concentration was determined as described above. The results are shown in the following table.

8120067 t - 8 -

Table 2 the calcium content of the serum in mg / 100 ml the number of hours after the dose of compound 6 17 27 48 5 control 4.1 4.3 4.1 4.0 25-OH-D3 4.4 5.8 5, 3b) 5.3 25-OH-26.27-F6-D3 4.7 5.7 6.2a) 5.8 a) from b) p ^ 0.001

It is clear that the hexafluoro compound 10 of the invention not only induces a rapid increase in serum calcium (almost equivalent to that induced by 25-OH-D3), but it increases serum calcium at a higher level for the rest of the time. ^ level than 25-OH-D3.

The anti-rickets effect

15 Rats fed the low phosphorus, vitamin D.

deficient diet, as described above, were divided into 3 groups of 5 rats. 1 week before sacrifice, a single dose of 325 pmol of 25-0H-26.27-Fg-D3 or 25-OHD3 dissolved in 0.1 ml of 95% ethanol was delivered through the jugular vein. Rats from the control group similarly received the ethanol carrier.

A week later, the rats were killed by decapitation and their 12-finger intestines were used to measure the intestinal calcium transport activity as described above. The results are shown in Table 3, first column. Their radii and 25 ulnae were removed and assessed according to the following rat experiment (U.S. Pharmacopoeia, 15th Ev., Mack Publishing Co.,

Easton, Pa. 1955, biz. 889). The results obtained are included in the second column of Table 3.

8120067 * - 9 -

Table 3

Gut calcium transport and antirachitic activity due to a single dose (385 pmol) of 25-OH-

26,27-Fg-D or 25-OHD_ given a week before sacrifice 5. J

the administered Ca transport in the antirachitic compound viscera action (unit) 1 * 5. .1 * 5 _Ca in / Ca out_ control 2.0 + 0.3 * 0 10 25-OH-26,27-F6-d3 7,1 + 1, Hb) ^ 6 25-OHD3 6,9 + 0, 6 ^ 6 standard deviation from the mean b) and c) from a) p ώ 0.001 15 b) from c) NS

From the previous data it appears that 25-0H-26,27-Fg-D3 exerts a pronounced vitamin D-like effect and appears to exert as effective an effect in this respect as 25-OHD3> 20 The 25-OH- 26,27-F 1 -D 3 according to the invention can be easily administered in sterile parenteral solutions by injection or intravenously or via the digestive system in the form of oral dosages or suppositories. Doses of about 0.1 µg to about 2.5 µg per day are effective in obtaining the aforementioned physiological calcium balance responses characteristic of the vitamin D-like activity, with a maintenance dose of about 0.25 µg suitable .

A dosage form of the compound can be obtained by combining it with a non-toxic, pharmaceutically acceptable carrier, as is known in the art. Such carriers can be both solid and liquid, such as, for example, corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and water. When a solid carrier is used, the dosage forms of the compounds of the invention may be tablets, capsules, powders, Atsenij tablets or cough tablets. When using a liquid 8120067 ♦ - 10 carrier, soft gelatin capsales or syrupy or liquid suspensions, emulsions or solutions may be the dosage form. The dosage forms may also be additives, such as preservatives, stabilizers, wetting agents or emulsifying agents, solubilizers, etc. They can also contain other therapeutically valuable ingredients.

It will be appreciated that although dosage ranges have been given, the particular dose to be administered to a host will depend on the specific disease condition to be treated, in particular case end results desired as well as other factors known to those in the field of disease. therapeutic uses of such drugs are known.

15 8120067

Claims (3)

2. The compound of claim 1 in crystalline form. 3. Compounds of formula 7, wherein R is selected from the group consisting of hydrogen, tetrahydropyranyl, an alkyl group of 1 to about k carbon atoms, benzoyl or substituted benzoyl. 10 1+. 36-Acetoxy-26,26,26,27,27,27-Hexafluorocholesta-5,7-dien-25-ol. 5. 3B-Hydroxy-26,26,26,27,27,27-hexafluorchole sta- 5,7-dien-25-ol. 6. Compounds of formula 8, wherein R is selected from the group consisting of hydrogen, tetrahydropyranyl, an alkyl group having 1 to about H carbon atoms, benzoyl or substituted benzoyl. 7. 3B-Acetoxy-26,26,26,27 * 27,27-hexafluoro-25-hydroxychole st-5-ene. 8. 3B-hydroxy-26,26,26,27,27,27-hexafluoro-25-hydroxycholest-5-ene. ér ....... - / -? Improvement of errata in the description associated with patent application no .: 8120067 Ned. proposed by Applicant <w. 15 0Ki. 193! In the definition of R, "alkyl" should be "acyl". This is evidenced by the fact that the pre-hydrolyzed product can be an acetoxy derivative, i.e. R is acetyl. jchut: RR 8120067