NZ207413A - Steroid intermediates in the preparation of 24,24-difluoro-25-hydroxy-vitamin d3 - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £07413 Iff! il1 207413 Priority Date':. t>.' P. .17 Corrects S>ccj;':c-at;-on F;!ed:^."? 'JP.'ll >1 ^ D D ] g ] l i ■ • l 'Q'^' (&'bc+ - - - - ••<<iii jj v- ' - P.O. 984 Under the provisions of Regit** jjtion 23 (I) the , :n " 3-chted .... tZSGA Spscifi" to v . s£3 Qc&Staif!..„. I <=UL Initials NEW ZEALAND PATENTS ACT, 1953 J)ivided from: 191899 D>te: 23 October 1979 COMPLETE SPECIFICATION "NOVEL STEROID INTERMEDIATES USEFUL FOR PREPARATION OF 24, 24-DIFLUORO-25-HYDROXYCHOLECALCIFEROL" +VWe, WISCONSIN ALUMNI RESEARCH FOUNDATION, a corporation organised and existing under the laws of the State of Wisconsin, United States of America, of: 614 North Walnut Street, Madison, Wisconsin 53707, United States of America, hereby declare the invention for which jfc / we pray that a patent may be granted to oos/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 _ (followed by page la) 2074 1 3 - 1 «- Our New Zealand Patent Specification No. 191899 ! relates to a derivative of vitamin D^, specifically 24,24-difluoro-25-hydroxy chole-calciferol.

Vitamin is a well-known agent for the 5 control of calcium and phosphorus homeostasis. In the normal animal or human it is known to stimulate intestinal calcium transport and bone-calcium mobilization and is effective in preventing rickets.

It is also now well known that to be effective 10 vitamin must be converted in vivo to its hydroxylated forms. For example, the vitamin is first hydroxylated in the liver to form 25-hydroxy vitamin and is further hydroxylated in the kidney to produce lcc, 25-dihydroxy vitamin or 24,25-dihydroxy vitamin D^. The la-hydroxy- lated vitamin is generally considered to be the physiologically active or hormonal form of the vitamin and to be responsible for what are termed the "vitamin D-like activities"t such as increasing intestinal absorption of calcium and phosphate, mobilizing bone 20 mineral, and retaining calcium in the kidneys.

Various derivatives of vitamin have been disclosed prior to the invention of N.Z. Patent Specification No. 191899. That invention provides a new derivative of vitamin D3 which expresses excellent vitamin D-like activity and which, therefore, can serve as a substitute for vitamin in its various known applications, including the treatment of various diseases such as osteomalacia, osteodystrophy and hypoparathyroidism and such conditions as milk fever in dairy cows and leg weakness in turkeys. This derivative has been indentified as 24, 24-difluoro-25-hydroxychole calciferol (24,24-difluoro-25-hydroxy vitamin or 24 F2,25-OH D3).

The compound of that invention can be synthesized in accordance with the following abbreviated schematic: 2074 1 3 vT' HO' AcO Oftc, AcO F oAc AcO The present invention includes the novel intermediates (3) and (/) shown in the above schematic. In one asDect J] Ibl the invention consists in - X compound having the formula where R is hydrogen or acetyl.

In another aspect the invention consists in The compound having the formula Qftc AcO where Ac is acetyl. 207413 Example Cholenic acid 1 was treated with dihydropyran in a suitable organic solvent (CHjC^) at 0°C in the presence of p-toluene sulphonic acid and then with 5 IN Na OH in ethanol at 20°C to form the cholenic acid tetrahydropyranyl ether (protection of the hydroxyl group in the A ring). This compound was then treated with an excess of CH^Li in tetrahydrofuran (THF) - ethyl ether at 0°C for four hours after which the protective tetra-10 hydropyranyl group was removed by treatment with p -TsOH in CH2CI2 - methanol for 24 hours at 20°C. Subsequent acetylation (Ac20-pyridine-CH2Cl2, 20°C, 24 hours) gave the methylketone 2 (mp 148-151°C,6 2.12 (3H,s#C-25), m/e 354 (M-60)) (Yield = 6% overall from 1_). 15 The methylketone 2 was refluxed for seven hours in acetic anhydride in the presence of p-TsOH (enolacetyl-ation) to give the diacetate 3 (mp 109-110°C,6 5.02 (1H, m, C-23), 1.90 (3H,s,C-25) m/e 396 (M-60)). The diacetate was then converted to the difluorocyclopropane 4 by 20 heating with sodium chlorodifluoro-acetate in diglyme at 170°C for 0.5 hours. Yield, 34%: mp 112-115°C, 5.38 (lH,m,C-6), 4.60 (lH,m,C-3), 2.05 (3H,s,24-OAc), 2.02 (3H,s,3-OAc), 1.60 (3H,m,C-26), m/e 446 (M-60)).

Treatment of 4 with LiOH in THF-methanol-25 water at 20°C for two hours followed by acetylation j (Ac20-pyridine-CH2Cl2, 20°C, 24 hours) gave, after ^ 4 ~"\ i' ; V -6- • chromatography on silica cel., the difluoroketone 5 ' o (9.3% yield, mp 135-136.5UC, 6 2.26 (SH.t.J.- = 1Hz, i rir C-26), m/e 404 (M-60)). The difluoroketone was obtained in a mixture with the 23(E)- and the 23(Z)- conjugated 5 ketone, with the difluoroketone being separated by chromatography on silica gel.

The difluoroketone 5 was reacted with an excess of CH^Mgl in ethyl ether at 0°C for 15 minutes and was then acetylated (Ac^O-pyridine-Cf^C^, 20°C, 10 20 hours) to furnish the 25-carbinol, 6, in 85% yield (mp 163-164.5°C, 6 1.28 (6H,s,C-26,27), m/e 420 (M-60)). The carbinol, 6, was allylically brominated by reacting it with N-bromo-succinimide in refluxing CCl^ for 25 minutes. The brominated compound was then dehydro-15 brominated by treatment with s-collidine in refluxing xylene for 15 minutes to give a mixture of the 4,6-diene and the 5,7-diene, 1_. The 5,7-diene (Xmax 263, 272, 282 and 292 nm, m/e 419 (M-59)) was isolated in 2Q% yield, by treatment with p-TsOH in acetone at 20°C for 15 hours 20 followed by preparative thin-layer chromatography (benzene-ethyl acetate (15:1 volume/volume), 3 times). The recovered 5, 7-diene was saponified by treatment with 5% L >eight/va=ight KOH-methanol at 20°C for 15 hours and then irradiated (Hanovia high pressure quartz mercury vapour lamp, model 654A36: 200 W) 25 in a mixture of ethanol and benzene for 2.5 minutes at 0°C to give the previtamin 8 in solution. The irradiated 2074 1 3 -7- • solution was refluxed for one hour and then fractionated with thin-layer chromatography (silica gel, benzene- ethyl acetate, (5:1 volume/volume), 3 times) and high pressure liquid chromatography [(Zorbax SIL, 25 cm x 2.1 mm i.d., 5 available through the DuPont Co., Willmington, Delaware) CI^Clj ] to yield 24,24-difluoro-25-hydroxy vitamin , 9, (Xmax 264 nm, Xmin 228 nm, m/e 436 (M+), 421, 418, 403, 377, 271, 253, 136, 118).

If desired the acetylated 5»7—diene 7_ after 10 recovery as described above can be saponified by well known means (5% wsight/vaeight KQH in MeQH, 20°C, 15 hours) to convert the acetoxy group at the 3-position to hydroxyl.

Also, if desired, the previtamin 8 can be recovered by evaporation of solvent at 5°C and subsequent 15 chromatographing on silica gel, and subsequently converted to the vitamin by heating or by standing at room tenperature. Biological Activity Male weanling rats were housed in hanging wire cages and fed ad libitum the low calcium, vitamin D 20 deficient diet described by Suda et al (J. Nutr. 100, 1049 (1970)) for three weeks prior to their use in the following assays.

Intestinal Calcium Transport Groups of five or six rats fed as above were 25 given respectively a single dose (650p mole) of either 24,24-difluoro-25-hydroxy vitamin (24 F2»25-OH D^) <y 3> • -8- • or 25-hydroxy.vitamin D, (25-OHD^) dissolved in 0.05 ml of 95% by weight ethanol intrajugularly 8, 23 or 30 hours prior \ to sacrifice. The rats in the control group were given the ethanol vehicle only. They were then killed by 5 decapitation after the respective times prescribed and their duodena were used to measure the intestinal calcium transport activity in accordance with the techniques of Martin and DeLuca (Am. J. Physiology 216, 1351 (1969)). Results are shown in the table below. lO Table 1 45 45 _ , Ca serosal/ Ca mucosal Compound Given 8 h 23 h 30 h Control 2.7+0. 2*a) 2. +0.4a) 2. 6+0.2a) 24F2.25-OHD3 6.6+1. 2b) . 9+0.6b) 8. 2+2.lb) -OHD3 .0+0. 7C) . +0.8c) . 7+1.4C) Significance b) & c) from b) & c) from b) & c) from of difference a) a) a) p< 0.001 p <0.001 p <0.001 b) from c) b) from c) b) from c) p <0.025 N.S. p <0.05 * Standard deviation from the mean To show the effect of small doses of 24F2» -OHD3 on intestinal calcium transport rats fed the low 25 calcium diet as indicated above, in groups of 5 or 6 2074 1 3 -9- | t were given a single dose of 24F2<25-OHD3 or 25-OHD3 dissolved in 0.05 ml of 95% by wsiqht ethanol intrajugularly Rats in the control group received the vehicle alone. Either 20 hours or 168 hours after receiving the dose 5 the rats were killed and their duodena were used to measure the intestinal calcium transport activity in accordance with the Martin and DeLuca procedure referenced above. Results are shown in Table 2 below.

Table 2 - Compound Dosage 45 ,45 Ca serosol/ Ca mucosal Given (p mole/rat) h 168 h Control 1.5+0.5* a) 2.0+0.4a) 24F2,25-OHD3 6.5 1.9+0.6 b) 2.1+0.1b) 32.5 1.9+0.3b) 3.7+0.9c) 2 5-OHD'3 6.5 1.8+0.4b) 2.1+0.2b) 32.5 2.2+0.6b) 3.8+0.7d) Significance b) from a) b) from a) of difference N.S.

N.S. c) from a) p < 0.05 d) from a) p <0.001 * Standard deviation from the mean Serum Calcium Concentration Rats fed as indicated above were divided into groups of six rats each. The rats in one group were given a single dose of 650p mole of 24F^,25-OHD3, in the second ! 0 7 c -, s group a dose of 650 p mole of 25-OHE>2 (in each case the vitamin derivative was dissolved in 0.05 ml of 95% by weight ethanol) while the third group (control) was given the vehicle alone. The materials were administered i 1 « intrajugularly either 8 or 24 hours prior to sacrifice. i The rats were killed by decapitation after the indicated times, the blood collected and centrifuged i to obtain the .serum- The serum (0-1 ml) was mixed with ! 1.9 ml of 0.1% by weight lanthanum chloride solution and the calcium concentration was measured with an atcmic absorption spectraphotoneter (Perkin-Elmer Model HO-214). Results are shown in the table below.

Table 3 ' Serum calcium (mg/100 ml) 15 Compound Given 8 h 24 h Control 7.7+0.2*a) 3.9+0.1a) 24F2,25-OHD3 4.9+0.2b* 5.2+0.2b* -OHD3 4.7+0.3b) 5.3+0.2b) Significance b) from a) b) from a) of difference p<O.OOl p<O.OOl * Standard deviation from the mean I Antirachitic Acitivity Male weanling rats (Holtzman Co., Madison, Wisconsin) maintained in hanging wire cages were fed, in 25 groups of six, the low phosphorus diet described in 2074 J 3 -11- .

Am. J. Physiol 204, 833 (1963) (Guroff, DeLuca and Steenbock), and were simultaneously given either 24?or 25-CHD3 dissolved in 0.1 ml ethanol/propylene glycol (5/95, v/v) subcutaneously every day for two weeks. Rats in the control group were fed in like manner but received only the vehicle subcutaneously.

Twenty-four hours after receiving the last subcutaneous dose the rats were killed by decapitation and their duodena were used for measurement of intestinal calcium transport as described above. Their radii and ulnae were removed for measurement of widened epiphyseal plates, and femurs for determination of ash content (femurs were dried to constant weight and then ashed in a muffle furnace at 650°C for 8 hours).

Results obtained are shown in the table below.

Table 4 Compound Dosage . Intestinal Calcium ' Width of Epiphyseal Femur Ash Given (p mole) Transport (I/O) Plate (mm) Total (mg) Percent Control 2. 1+0. 3*a) 3.2+0. 3a) 29. 71+3. 06a) 27.3+2 a)" 24F7,25-OHDO 6.5 . 7+1.2b) 1. 5+0. 2b) 37. 53+3. 82b) . 9+1. lb) O 6.5 11.2+2. 3C) 0. 5HH0.1C) 52.95+3. 55C) 38.5+1. lc> -OHD, 6.5 - 4+0. 3d * 1. 5+0. 3d). 39. 83+6. 31d) 31. 7+2 d) o 6.5 .8+2.0e) 0. 6+0. 2e) 53. 66+6. 72e) 38.4+2.7 9 •Significance b), c),d) & e) b) from a) b) from a) b) from ^a) of difference from a) e < 0. 005 f <0.005 f<0. 01 f <0.001 d) from a) d) from a) d)from a) P<0. 025 p<0.025 ?< 0.025 c) & e) from a) c) & e) from a) c) & e) ?<0. 001 ? <0. 001 from a) p <0.001 ■"Standard . deviation from the mean 2074 96-. -13- - Male weanling rats were fed the low phosphorus diet referenced above and then divided into groups of five or six rats each. The rats in each group were given respectively a single dose (as shown in the table below) of either 24F2,25-OHD3 °r 25-OHD3 dissolved in 0.05 ml of 95% by vreight ethanol Rats in the control group received the ethanol vehicle alone. 168 hours after receiving the indicated dosage the rats were killed by decapitation, the blood of each group was collected and the radii and ulnae were removed to determine antirachitic acitivity in accordance with the rat line test (U. S. Pharmacopoeia, 15th Rev., Mack Publishing Co., Easton, Pa., 1955, p. 889). The blood was centrifuged immediately after collection to yield the serum. The inorganic phosphorus in the serum was determined by the method of Chen et al (Anal. Chem., 28, 1756, (1956)).

Results obtained are shown in the table below.

Table 5 Compound Given Dosage (p mole) Serum Inorganic Phosphorus (mg/100 ml) Line Test Score (Unit) Control 1.6+0.2*a) 0 24F2< 25-0HD3 130 3.0+0.2b* 4.4+1.4a) 325 3.5+p.4b) -OHD3 130 325 3.3+0.1b) 3.6+0.4b) 2. 6+0.6b ^ 3.5+0.6 Significance of difference b) f rom a) p < O.OOl b) from a) -pCO.025 * Standard deviation from the mean -14- • To determine the antirachitic activity in response to a daily dose of 24F2,25-OHD3 rats were fed the low phosphorus diet referenced above for three weeks. They were then given either.24F2,25-OHD3 or 25-OHD^ (in each case, 65 p mole dissolved in 0.1 ml ethanol/propylene glycol (5/95, v/v)) subcutaneously every day for 8 days while being maintained on the same diet (9 rats in each group). The rats in the control group (4 rats) were given only the ethanol/propylene glycol vehicle in the same IO manner.

Twenty-four hours after receiving the last dose they were killed and their radii and ulnae were removed and used for measuring antirachitic activity (rat line test, supra) while the femurs were removed and ashed as 15 described above.

Results obtained are shown in the table below.

Table 6 Compound Line Test Total Femur Ash Percent Ash Given Score (Unit) (mg) (%) Control 0 23.80+3.98*a* 19.5+3.4a) 24F2,25-OHD3 >> 5 37.03+4.94b) 26.2+1.8b5 -OHD3 » 5 38.56+5.79b) 27.4+2.4*^ Significance b) from a) b) from a) of difference p <0.001 p <0.005 * Standard deviation from the mean 207 -15- .

It is evident from the foregoing data that 24,24-difluoro-25-hydroxy vitamin exhibits pronounced vitamin D-like activity and appears to be wholly as effective in this regard as 25-hydroxy vitamin D3 (see U.S. Letters Patent No. 3,565,924).

The 24,24-difluoro-25-hydroxycholecal6iferol compound of Ekat invention may be readily administered as sterile parenteral solutions by injection or intravenously or by alimentary canal in the form of oral dosages, or by suppository. Doses of from about 0.1 |ag to about 2.5 ng per day are generally effective for obtaining the physiological calcium balance responses described which are characteristic of vitamin D-like activity; maintenance doses of about 0.25 |ig are generally suitable.

Dosage forms of the compound can be prepared by combining it with a conventional non-toxic pharmaceutically acceptable carrier. Such carriers may be either solid or liquid such as corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and water. If a solid carrier is used the dosage forms include tablets, capsules, powders, troches or lozenges. If a liquid carrier is used, soft gelatin capsules, or syrup or liquid suspensions, emulsions or solutions are typical dosage forms. The dosage forms may also contain adjuvants, such as preserving, stabilizing, -16- - wetting or emulsifying agents, and solution promoters.

They may also contain other therapeutically valuable substances.

It should be understood that although dosage ranges are given the particular dose to be administered to a host will depend upon the specific disease state being treated, the end results being sought in a particular case, as well as other factors known to those skilled in the art in the therapeutic use of such medicinal agents.

This new derivative may represent a preferred agent for many therapeutic applications, because it is blocked to further metabolism at the carbon 24. It is well known that la,25-dihydroxy vitamin , can undergo further metabolism in vivo to yield la,24R,25-trihydroxy vitamin D^. This 24-hydroxylated form is however less active than la,25-dihydroxy vitamin itself, and 24-hydroxylation may indeed represent the first step towards degradation and elimination of this compound from the animal system. In the derivative of thatr invention the presence of two fluorine atoms at carbon-24 will, of course, prevent hydroxylation of this carbon atom and the compound is not, therefore, subject to the activity-attenuating metabolism affecting 1«<, —Z5-dihydroxycholecalciferol. Prevention of this side chain metabolism should permit the maintenance of higher

Claims (3)

207 4 1 3 -17- * tissue levels of the analog for a longer period of time, a factor of obvious advantage in many therapeutic applications. -18- 207413 * WHAT WE "CLAIM IS:

1- A compound having the formula where P is hydrogen or acetyl.

2. The compound having the formula OAc AcO where Ac is acetyl.

3. A compound as claimed in claim 1 or 2 substantially as hereinbefore described. DATED THIS % day of 1*^ A. J. PARK & SON PER AGENTS FOR THf APPLICANTS