WO1993016040A1 - Fluorine-containing vitamin d3 analogues - Google Patents

Fluorine-containing vitamin d3 analogues Download PDF

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Publication number
WO1993016040A1
WO1993016040A1 PCT/JP1993/000088 JP9300088W WO9316040A1 WO 1993016040 A1 WO1993016040 A1 WO 1993016040A1 JP 9300088 W JP9300088 W JP 9300088W WO 9316040 A1 WO9316040 A1 WO 9316040A1
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Prior art keywords
compound
reference example
protecting group
acetyl
vitamin
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PCT/JP1993/000088
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French (fr)
Inventor
Nobuo Ikekawa
Takeo Taguchi
Yoko Tanaka
Yoshiro Kobayashi
Yutaka Ohira
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Daikin Industries, Ltd.
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Priority to DE69300355T priority Critical patent/DE69300355T2/en
Priority to EP93902528A priority patent/EP0579840B1/en
Priority to JP5513939A priority patent/JP2658574B2/en
Priority to CA002107471A priority patent/CA2107471C/en
Publication of WO1993016040A1 publication Critical patent/WO1993016040A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C401/00—Irradiation products of cholesterol or its derivatives; Vitamin D derivatives, 9,10-seco cyclopenta[a]phenanthrene or analogues obtained by chemical preparation without irradiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C35/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring
    • C07C35/48—Halogenated derivatives
    • C07C35/52—Alcohols with a condensed ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
    • C07F7/1804—Compounds having Si-O-C linkages
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • This invention relates to novel fluorine-containing vitamin Do analogues which have excellent pharmacological activities, such as tumor cell differentiation-inducing activity and are expected to be used as a medicament.
  • active-type vitamin D3 a bio-metabolite of vitamin D 3 , l ⁇ ,25-dihydroxyvitamin D3
  • active-type vitamin D3 has an activity of promoting absorption of calcium via intestinal tract and thereby is useful as a medicament for the treatment of bone diseases.
  • active-type vitamin D and analogues thereof have a differentiation-inducing activity for recovering normal cells from cancerated cells (cf. Hirobu i Tanaka et al., "Seikagaku” (Biochemistry), Vol. 55, 1323, 1983) and further that some of these compounds have a remarked activity of inhibiting the progress of cancer (K. . Colton et al., Lancet, Jan. ⁇ 8, 188, 1989).
  • the present inventors have intensively studied as to novel fluorine-containing vitamin D 3 analogues which have excellent cell differentiation-inducing activity as well as high selectivity in calcium metabolism with less side effects, i.e. inhiDition of hepercalcaemia .
  • An object of the invention is to provide novel fluorine-containing vitamin D3 analogues having pharmacological activities, especially anti-tumor activity owing tc the cell differentiation-inducing activity.
  • a further object of the invention is to provide a novel intermediate suitable for the preparation of the active fluorine-containing vitamin D3 analogues.
  • the fluorine-containing vitamin D 3 analogues of this -invention have the following formula [I]:
  • R- ⁇ , R ⁇ and R J are independently hydrogen atom or a hydroxy-protecting group.
  • the hydroxy-protecting group includes a group being capable of forming acetal type protecting group (e.g. methoxymethyl, ethoxyethyl, methoxyethoxymethyl, tetrahydropyranyl, etc.), a silyl ether type protecting group (e.g. trimethylsilyl, t- butyldimethylsilyl, t-butyldiphenylsilyl, etc.), an acyl group (e.g. acetyl), and the like.
  • acetal type protecting group e.g. methoxymethyl, ethoxyethyl, methoxyethoxymethyl, tetrahydropyranyl, etc.
  • silyl ether type protecting group e.g. trimethylsilyl, t- butyldimethylsilyl, t-butyldiphenylsilyl
  • the compounds [I] of this invention can be prepared by various processes. One of the best processes is illustrated below.
  • R is a hydroxy-protecting group
  • R 7 and R3 are each a hydroxy-protecting group
  • Ph means phenyl, to give a condensed product of the formula [I] of this invention.
  • the above coupling reaction of the compound [II] and the compound [III] is usually carried out at a low temperature, for example -100°C to -50°C, preferably -78°C to -20°C, in an ether solvent (e.g. diethyl ether, tetra- hydrofuran (THF), etc.).
  • the conversion of the [ring A] fragment into the corresponding carbanion is carried out by treating the fragment with an appropriate base such as an alkyl-lithium (e.g. n-butyllithium, etc).
  • the reaction time is for 10 minutes to 24 hours, preferably for 30 minutes to 2 hours.
  • the obtained product [I] can be purified by a conventional method, for example, by silica gel column chromatograph .
  • the removal of the hydroxy-protecting group from the compound [I] can optionally be carried out by a conventional method.
  • R and R are each a hydroxy-protecting group, and MOM means methoxymethyl group.
  • the starting compound [II] ([II-l] and [II-2]) can be prepared by reacting the aldehyde compound (1) with the bromide compound
  • R4 is acetyl and compound [III] wherein R 7 and R ⁇ are t- butyldimethylsilyl:
  • Example 1-1 The silyl compound obtained in Example 1-1 (99 mg) is added to a suspension of ion-exchange resin (50 , 3 g) in methanol (30 ml) and stirred for 24 hours at room temperature. After filtering the solution and distilling off the solvent, the residue is purified by column chromatography to give the desired compound (A) (66 mg) .
  • Subculture cells derived from human colonic cancer were inoculated onto a 24-well plate for tissue culture and was cultured in RPMI-1640 medium (added with 10 % fetal calf serum). After culturing for about 24 hours, the supernatant was removed. To the residue was added a medium containing 2 x 10 — J 3M sodium butyrate and the test compound as mentioned below (exchange of the medium), and the mixture was subjected to station culture in a culture vessel containing carbon dioxide (5 I C0 2 - 95 % air) at
  • _ cells were treated by 2 x 10 ⁇ sodium butyrate and the compounds of this invention, the cells were differentiated into mucin-producing cells.
  • Vitamin D- defficient rats were prepared and the calcium concentration in serum was measured.
  • Wistar rats (5 rats per each group) was fed with vitamin D-free, low calcium (0.02 %) feed for about 3 weeks.
  • the test compound (650 pmole) solubilized in solvent (95 % propylene glycol + 5 % ethanol) was subcutaneously injected at the behind of the rats in a dose of 0.1 ml per day. In a control group, only the solvent was injected lilkewise. Three and four days after the first injection, the blood was taken and the calcium concentration in the serum was measured, and the obtained data were shown in the average thereof.
  • Test compounds The same as used in the above Experiment 1.
  • Test compouds Increase of serum calcium concentration* (mg/dl) l ,25-dihydroxyvitamin D3 3.1 ⁇ 0.6

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
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  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

Fluorine-containing vitamin D3? analogues of formula (I), wherein R?1, R?2 and R?3 are independently hydrogen atom, a hydroxy-protecting group, having excellent pharmacological activities, such as tumor cell differentiation-inducing activity, which are useful as a medicament.

Description

DESCRIPTION FLUORINE-CONTAINING VITAMIN D3 ANALOGUES
Technical Field
This invention relates to novel fluorine-containing vitamin Do analogues which have excellent pharmacological activities, such as tumor cell differentiation-inducing activity and are expected to be used as a medicament.
Background Art
It is known that a bio-metabolite of vitamin D3, lα ,25-dihydroxyvitamin D3 is called as "active-type vitamin D3" and has an activity of promoting absorption of calcium via intestinal tract and thereby is useful as a medicament for the treatment of bone diseases. Recently, it has been found that the active-type vitamin D and analogues thereof have a differentiation-inducing activity for recovering normal cells from cancerated cells (cf. Hirobu i Tanaka et al., "Seikagaku" (Biochemistry), Vol. 55, 1323, 1983) and further that some of these compounds have a remarked activity of inhibiting the progress of cancer (K. . Colton et al., Lancet, Jan. ^8, 188, 1989). It has, however, been known that these active-type vitamin D compounds have high antagonistic activity against calcium metabolism which induces hypercalcaemia and hence can not be used in a high dose. Accordingly, these compounds are not necessarily usable for the treatment of diseases which require continuous administration in a comparatively high dose, for example, for the treatment of leukemia. Disclosure of Invention
The present inventors have intensively studied as to novel fluorine-containing vitamin D3 analogues which have excellent cell differentiation-inducing activity as well as high selectivity in calcium metabolism with less side effects, i.e. inhiDition of hepercalcaemia .
An object of the invention is to provide novel fluorine-containing vitamin D3 analogues having pharmacological activities, especially anti-tumor activity owing tc the cell differentiation-inducing activity. A further object of the invention is to provide a novel intermediate suitable for the preparation of the active fluorine-containing vitamin D3 analogues. These and other objects and advantages of the invention will be apparent to the skilled persons in this field from the following description.
The fluorine-containing vitamin D3 analogues of this -invention have the following formula [I]:
Figure imgf000004_0001
wherein R-~ , R^ and RJ are independently hydrogen atom or a hydroxy-protecting group. -* In the present specification and claims, the hydroxy-protecting group includes a group being capable of forming acetal type protecting group (e.g. methoxymethyl, ethoxyethyl, methoxyethoxymethyl, tetrahydropyranyl, etc.), a silyl ether type protecting group (e.g. trimethylsilyl, t- butyldimethylsilyl, t-butyldiphenylsilyl, etc.), an acyl group (e.g. acetyl), and the like.
Suitable examples of the compounds [I] are as follows.
1) Compound A: 26, 26, 26,27, 27 , 27-Hexafluoro-24- homo-24-yne-lα ,22S,25-trihydroxyvitamin D3
Figure imgf000005_0001
2) Compound B: 26, 26, 26, 27, 27,27-Hexafluoro-24- homo-24-yne-lα ,22R,25-trihydroxyvitamin D3
Figure imgf000005_0002
3) lα , 3-Bis ( t-butyldimethylsilyl ) ether 22-acetate of Compound A
4) lα ,3-Bis( t-butyldimethylsilyl) ether 22-acetate of Compound B
The compounds [I] of this invention can be prepared by various processes. One of the best processes is illustrated below.
A [ring CD] fragment of the formula [II]:
Figure imgf000006_0001
wherein R is a hydroxy-protecting group, is subjected to coupling reaction with an anion derived from a protected [ring A] fragment of the formula [III]:
Figure imgf000006_0002
wherein R 7 and R3 are each a hydroxy-protecting group, and
Ph means phenyl, to give a condensed product of the formula [I] of this invention.
The above coupling reaction of the compound [II] and the compound [III] is usually carried out at a low temperature, for example -100°C to -50°C, preferably -78°C to -20°C, in an ether solvent (e.g. diethyl ether, tetra- hydrofuran (THF), etc.). The conversion of the [ring A] fragment into the corresponding carbanion is carried out by treating the fragment with an appropriate base such as an alkyl-lithium (e.g. n-butyllithium, etc). The reaction time is for 10 minutes to 24 hours, preferably for 30 minutes to 2 hours. The obtained product [I] can be purified by a conventional method, for example, by silica gel column chromatograph . The removal of the hydroxy-protecting group from the compound [I] can optionally be carried out by a conventional method.
Starting compound [II] can be prepared by the process as illustrated by the following reaction scheme:
Figure imgf000007_0001
Protecting >
Figure imgf000007_0002
Removal of Protecting Group
Figure imgf000008_0001
Oxidation
Figure imgf000008_0002
Figure imgf000008_0003
wherein R and R are each a hydroxy-protecting group, and MOM means methoxymethyl group.
According to the above process, the starting compound [II] ([II-l] and [II-2]) can be prepared by reacting the aldehyde compound (1) with the bromide compound
(2) to give the compounds (3) and (4), protecting the hydroxy group of the resultant compounds (3) and (4) with a hydroxy-protecting group in a usual manner, removing the hydroxy-protecting group R from the resultant compounds (5) and (6), and finally oxdizing the resultant compounds (7) and (8).
Best Mode for Carrying Out of the Invention
The compounds of this invention are illustrated by the following Examples and Reference Examples, but should not be constructed to be limited thereto.
Example 1
1-1) Preparation of lα,3-bis(t-butyldimethylsilyl) ether of compound (A) by Wittig reaction of compound [II-l] wherein
R4 is acetyl and compound [III] wherein R 7 and R ~ are t- butyldimethylsilyl:
To a solution of the compound [III] wherein R2 and R3 are t-butyldimethylsilyl (1.0 g) in anhydrous THF (10 ml) is added n-BuLi (2.5 M, 0.68 ml) at -78°C, and the mixture is stirred for 5 minutes. To the solution is added a solution of the compound [II-l] wherein R is acetyl (80 mg) in anhydrous THF (5 ml) is added and the mixture is stirred for 10 minutes after warming to room temperature. To the reaction mixture is poured a saturated ammonium chloride solution and the mixture is extracted with ethyl acetate. The ethyl acetate layer is washed with water and dried over anhydrous magnesium sulfate. After distilling off the solvent, the residue is purified by column chromatography to give the desired compound (106.9 g, 78 %) as a colorless solid.
1H-NMR (CDC13) 6: 0.05 (s, 6H), 0.06 (s, 6H), 0.54 (s, 3H), 0.866 (s, 9H), 0.875 (s, 9H), 0.94 (d, J=7.1Hz, 3H), 4.86 (d, J=2.8Hz, IH), 5.18 (d, J=2.8Hz, IH), 6.03 (d, J=12.2Hz, IH), 6.24 (d, J=12.2Hz, IH)
IR (KBr): 3431, 2954, 1221, 834 cm-1 1-2) Preparation of compound (A) by removal of the protecting silyl group:
The silyl compound obtained in Example 1-1 (99 mg) is added to a suspension of ion-exchange resin (50 , 3 g) in methanol (30 ml) and stirred for 24 hours at room temperature. After filtering the solution and distilling off the solvent, the residue is purified by column chromatography to give the desired compound (A) (66 mg) .
XH-NMR (CDCI3) δ: 0.58 (S, 3H), 0.91 (d, J=5.6Hz, 3H), 3.85-4.40 (m, 3H) , 4.91 (brs, IH) , 5.29 (brs, IH), 6.11 (d, J=12Hz, IH), 6.34 (d, J=12Hz, IH)
IR (KBr): 3383, 2948, 1221, 858 cm"1
Example 2
Preparation of compound (B) from compound [II-2] wherein R is acetyl:
Following the procedure of Example 1 except substituting compound [II-2] for compound [II-l], the desired compound (B) is obtained as a colorless solid. XH-NMR (CDCI3) δ: 0.56 (s, 3H) , 0.95 (d, J=6Hz, 3H), 2.59 (dd, J=llHz, 3Hz ) , 2.85 (dd, J=llHz, 3Hz ) , 3.94 (m, IH), 4.23 (m, IH), 4.43 ( , IH), 5.00 (brs, IH) , 5.33 (brs, IH), 6.02 (d, J=llHz, IH) , 6.37 (d, J=llHz, IH)
Reference Example 1
Preparation of compound (3) and (4) wherein R is t-butyldimethylsilyl by reacting compound (1) wherein R5 is t-butyldimethylsilyl with compound (2):
To a solution of the aldehyde compound (1) wherein R5 is t-butyldimethylsilyl group (1.14 g) and the bromide compound (2) (2.30 g) in DMF (8 ml) is added zinc powder (0.59 g) at 25°C and the mixture is stirred for 30 minutes. After adding a saturated ammonium chloride solution, the mixture is extracted with ether. The ether layer is washed with water and dried over anhydrous magnesium sulfate. After distilling off the solvent, the residue is purified by column chromatography to give the desired compound (3) (1.36 g) and the compound (4) wherein R5 is t-butyldimethylsilyl (0.54 g).
As to compound (3): 1H-NMR (CDCI3) δ: 0.00 (s, 3H) , 0.01 (s, 3H), 0.01 (s, 3H), 0.90 (s, 9H) , 0.91 (d, J=5Hz, 3H), 0.91 (s, 3H), 2.32 (dd, J=17Hz, 5Hz, IH) , 2.60 (dd, J=17Hz, 9Hz, IH), 3.47 (s, 3H) , 3.95 (m, IH) , 4.00 (brs, IH)", 5.07 (d, J=25Hz, IH), 5.09 (d, 25Hz, IH)
IR (KBr): 3470, 2250, 1230 cm-1
AS to compound (4): 1H-NMR (CDCI3) δ: 0.00 (s, 3H) , 0.01 (s, 3H), 0.89 (s, 9H), 0.93 (d, J=5Hz, 3H), 0.95 (s, 3H), 3.48 (s, 3H), 3.89 (m, IH), 4.00 (brs, IH), 5.08 (d, J=25Hz, IH), 5.10 (d, J=25Hz, IH)
IR (CHC13): 3520, 2260, 1230 cm"1
Reference Example 2
Preparation of compound (5) wherein R is acetyl and R is t-butyldimethylsilyl by protecting compound (3) obtained in Reference Example 1:
A solution of the alcohol compound (3) obtained in Reference Example 1 (149 mg), acetic anhydride (0.7 ml), pyridine (1.2 ml) and 4-dimethylaminopyridine (35 mg) in dichloromethane (2.5 ml) is stirred for 18 hours at room temperature. After completing the reaction, the mixture is extracted with ether, the ether extract is washed with 2% HC1, 5% sodium bicarbonate solution and brine. After distilling off the solvent, the residue is purified by column chromatography to give the desired compound (5) wherein R4 is acetyl and R5 is t-butyldimethylsilyl (140 mg) .
1H-NMR (CDCI3) δ: 0.00 (s, 6H) , 0.88 (s, 9H), 0.90 (s, 3H), 0.96 (d, J=6.8Hz, 3H), 2.05 (s, 3H) , 3.43 (s, 3H), 3.98 (brs, IH), 5.03 (s, 2H) , 5.08 (m, IH)
IR (neat): 2956, 2256, 1747, 1472, 1376 cm-1
Reference Example 3
Preparation of compound (6) wherein R4 is acetyl and R-1 is t-butyldimethylsilyl by protecting compound (4) obtained in Reference Example 1:
Following the procedure of Reference Example 2 except substituting the compound (4) obtained in Reference Example 1 for the compound (3), the desired compound (6) wherein R4 is acetyl and R5 is t-butyldimethylsilyl is obtained.
1H-NMR (CDC13) δ: 0.00 (s, 3H) , 0.01 (s, 3H), 0.88 (s, 9H), 0.92 (s, 3H), 0.93 (d, J=7Hz, 3H), 2.03 (s, 3H), 2.53 ( , 2H), 3.43 (ε, 3H) , 4.01 (brs, IH) , 5.02 (d, J=25Hz, IH), 5.04 (d, J=25Hz, IH) , 5.11 (m, IH)
Melting Point: 74.3°C to 75.5°C (ethanol)
Reference Example 4
Preparation of compound (7) wherein R4 is acetyl by removal of the protecting group of compound (5) obtained in Reference Example 2:
A mixture of the acetate compound (5) obtained in reference Example 2 (200 mg), dichloromethane (2.4 ml), acetic acid (2.4 ml) and 5% HC1 (0.4 ml) is refluxed for 5 hours. After completing the reaction, the mixture is extracted with ethyl acetate, and the extract is washed with 5% sodium bicarbonate solution and dried over magnesium sulfate. After distilling off the solvent, the residue is purified by column chromatography to give the desired compound (7) wherein R is acetyl (65 mg, 44%).
1H-NMR (CDCI3) δ: 0.94 (s, 3H), 0.98 (d, J=6.8Hz, 3H), 2.09 (s, 3H), 4.09 (brs, IH) , 4.77 (s, IH) , 5.23 (m, IH )
IR (KBr): 3545, 3219, 2937, 1719, 1250, 1200, 958 cm-1
Reference Example 5
Preparation of compound (8) wherein R4 is acetyl by removal of the protecting group of compound (6) obtained in Reference Example 3:
Following the procedure of Reference Example 4 except substituting the compound (6) obtained in Reference Example 3 for the compound (5), the desired compound (8) wherein R is acetyl is obtained.
^-H-NMR (CDC13) δ: 0.96 (s, 3H) , 0.97 (d, J=7Hz, 3H), 2.07 (s, 3H), 2.46 (m, 2H) , 4.09 (brs, IH), 5.20 (m, IH)
Melting point: 156°C to 157.5°C (ether/hexane)
Reference Example 6
Preparation of compound [II-l] wherein R4 is acetyl by oxidation of compound (7) obtained in Reference Example 4:
To a solution of pyridinium chlorochromate (PCC, 50 mg) in dichloromethane (2 ml) is added a solution of alcohol compound (7) obtained in Reference Example 4 (21 mg) in dichloromethane (2 ml), and the mixture is stirred for 4 hours at room temperature. After adding ether, the mixture is filtered. After distilling off the solvent of the filtrate, the residue is purified by column chromatography to give the desired compound [II-l] wherein R is acetyl (18.9 mg, 91%) .
1H-NMR (CDC13) δ: 0.64 (s, 3H), 1.04 (d, J=6.6Hz, 3H), 2.10 (s, 3H), 4.53 (brs, IH), 5.22 (m, IH)
IR (neat): 3262, 2964, 2252, 1738, 1713, 1698, 1240, 957 cm-1
Reference Example 7
Preparation of compound [II-2] wherein R4 is acetyl by oxidation of compound (8) obtained in Reference Example 5:
Following the procedure of Reference Example 6 except substituting the compound (8) obtained in Reference Example 5 for the compound (7), the desired compound [II-2] is obtained.
1H-NMR (CDCI3) δ: 0.65 (s, 3H) , 1.05 (d, J=7Hz, 3H), 3.26 (brs, IH) , 5.35 (dt, J=15Hz, IH), 5.45 (dd, J=15Hz, 5Hz, IH)
Experiment 1
Effects of the compound on differentiation-inducing activity:
Test method:
Subculture cells (HT-29) derived from human colonic cancer were inoculated onto a 24-well plate for tissue culture and was cultured in RPMI-1640 medium (added with 10 % fetal calf serum). After culturing for about 24 hours, the supernatant was removed. To the residue was added a medium containing 2 x 10 —J3M sodium butyrate and the test compound as mentioned below (exchange of the medium), and the mixture was subjected to station culture in a culture vessel containing carbon dioxide (5 I C02 - 95 % air) at
37°C. On every other day, the culture medium was exchanged with the same medium as mentioned above, and on 7th day, the number of the mucin-producing cells and shape of the cells were observed by the method of Augeron et al. [cf. Cancer
Res, Vol. 44, 3961, 1984].
It is known that the mucin-production is observed in normal cells of large intestine (the colon) but not in cancerated HT-29 cells. Accordingly, as a marker for measuring the fact that the cancer cells HT-29 was differentiated and could express characteristic of normal cells, the number of mucin-producing cells was measured.
Test compounds:
1. l , 25-dihydroxyvitamin D3
2. Compound A: 26,26,26 , 27,27 ,27-Hexafluoro-24- homo-24-yne-lα ,22S, 25-trihydroxyvitamin D3
3. Compound B: 26, 26, 26 , 27 , 27 , 27-HexafIuoro-24- homo-24-yne-lα ,22R, 25-trihydroxy itamin D
Results :
The data obtained above were shown in percentage based on whole cells (200 cells) measured. The results are shown in the following Table 1. Table 1
Test compound Concentration Number of mucin-producing
(M) cells (%)
Non 3 ± 3 lα,25-dihydroxy- vitamin D3 10 -7 100 lα ,25-dihydroxy- vitamin D3 10 -8 39
Compound A 10 -7 97 ± 3
Compound A 10' 79 ± 7
Compound A 10" 26 ± 9
Compound B 10" 90 ± 10
Compound B 10 -8 91 ± 9
Compound B 10 -9 49 ± 8
As is clear from the above results, when the HT-29
_ cells were treated by 2 x 10 ^ sodium butyrate and the compounds of this invention, the cells were differentiated into mucin-producing cells.
Experiment 2
Effects of the compounds on serum calcium concentration:
Test Method:
In accordance with the method of Mori et al [Vitamin-gaku Jikken-ho (Experiments in Vitamin Science), [I], Fat Soluble Vitamins, Ed. by Japan Vitamin Association, issued by Tokyo Kagaku Dojin, pages 120-135], Vitamin D- defficient rats were prepared and the calcium concentration in serum was measured.
That is, Wistar rats (5 rats per each group) was fed with vitamin D-free, low calcium (0.02 %) feed for about 3 weeks.
After confirming the low calcium concentration in serum (less than 6 mg/dl), the test compound (650 pmole) solubilized in solvent (95 % propylene glycol + 5 % ethanol) was subcutaneously injected at the behind of the rats in a dose of 0.1 ml per day. In a control group, only the solvent was injected lilkewise. Three and four days after the first injection, the blood was taken and the calcium concentration in the serum was measured, and the obtained data were shown in the average thereof.
Test compounds: The same as used in the above Experiment 1.
Results: The test results are shown in the following Table 2.
Table 2
Test compouds Increase of serum calcium concentration* (mg/dl) l ,25-dihydroxyvitamin D3 3.1 ± 0.6
Compound A 0.7 1 0.3
Compound B 0.6 ± 0.3 ;
*) It shows the data increased over the value (4.5 mg/dl) in the control group. As is clear from the above test results, the compounds of the present invention showed less increase of the serum calcium concentration in comparison with the known lα ,25-dihydroxyvitamin D3.

Claims

CLAIMS 1. A fluorine-containing vitamin D3 analogue of the formula [ I ] :
Figure imgf000020_0001
wherein R 1 ,
Figure imgf000020_0002
are independently hydrogen atom or a hydroxy-protecting group,
2. The compound according to claim 1, wherein the hydroxy-protecting group is selected from the group consisting of methoxymethyl, ethoxyethyl, methoxyethoxy¬ methyl, tetrahydropyranyl, trimethylsilyl, t-butyldimethyl¬ silyl, t-butyldiphenylsilyl, acetyl
3. 26,26,26,27,27, 27-Hexafluoro-24-homo-24-yne- lα ,22S, 25-trihydroxyvitamin D3, or lα , 3-bis( t-butyl¬ dimethylsilyl) ether 22S-acetate thereof.
4. 26, 26, 26, 27, 27, 27-Hexafluoro-24-homo-24-yne- l , 22R, 25-trihydroxyvitamin D3, or lα , 3-bis( t-butyl¬ dimethylsilyl) ether 22R-acetate thereof.
PCT/JP1993/000088 1992-02-10 1993-01-26 Fluorine-containing vitamin d3 analogues WO1993016040A1 (en)

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DE69300355T DE69300355T2 (en) 1992-02-10 1993-01-26 Fluorine-containing vitamin D3 analogs.
EP93902528A EP0579840B1 (en) 1992-02-10 1993-01-26 Fluorine-containing vitamin d3 analogues
JP5513939A JP2658574B2 (en) 1992-02-10 1993-01-26 Fluorine-containing vitamin D (lower 3) analogues
CA002107471A CA2107471C (en) 1992-02-10 1993-01-26 Fluorine-containing vitamin d3 analogues

Applications Claiming Priority (2)

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US07/832,888 US5200536A (en) 1992-02-10 1992-02-10 Fluorine-containing vitamin D3 analogues

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GB9017890D0 (en) * 1990-08-15 1990-09-26 Leo Pharm Prod Ltd Chemical compounds i
US20040009958A1 (en) * 1991-01-08 2004-01-15 Bone Care International, Inc. Methods for preparation and use of 1alpha,24(S)-dihydroxyvitamin D2
IL99368A (en) * 1991-09-02 1996-01-19 Teva Pharma Compositions for topical treatment of psoriasis and atopic dermatitis comprising a xanthine derivative
DE4220757A1 (en) * 1992-06-24 1994-01-05 Schering Ag Derivatives in the vitamin D series with modifications in the 20-position, process for their preparation, intermediates for this process, pharmaceutical preparations containing these derivatives and their use in the manufacture of medicaments
DE4221961A1 (en) * 1992-06-30 1994-01-05 Schering Ag 22-en-25-oxa derivatives in the vitamin D series, processes for their preparation, pharmaceutical preparations containing these derivatives and their use as medicines
CA2147264C (en) * 1992-10-16 2006-05-16 Tetsuhiro Mikami Vitamin d derivatives and a process for producing them
GB9223061D0 (en) * 1992-11-04 1992-12-16 Leo Pharm Prod Ltd Chemical compounds
US5401733A (en) * 1993-10-01 1995-03-28 Hoffmann-La Roche Inc. Stable and active metabolites of 1,25-dihydroxy-16-ene-cholecalciferol
NO971934L (en) * 1996-05-23 1997-11-24 Hoffmann La Roche Fluorinated vitamin D3 analogues
JP2005504279A (en) * 2001-09-27 2005-02-10 ザ・コカ−コーラ・カンパニー Vitamin fortification of food

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EP0326875A1 (en) * 1988-01-20 1989-08-09 F. Hoffmann-La Roche Ag Didehydro-vitamin D3 derivatives

Patent Citations (1)

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EP0326875A1 (en) * 1988-01-20 1989-08-09 F. Hoffmann-La Roche Ag Didehydro-vitamin D3 derivatives

Non-Patent Citations (1)

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Title
CHEMICAL AND PHARMACEUTICAL BULLETIN. vol. 40, no. 6, June 1992, TOKYO JP pages 1647 - 1649 Y. OHIRA ET AL 'Preparation of (22S)- and (22R)-24-Homo-26,26,26,27,27,27-Hexafluoro -1,22,25-Trihydroxyvitamin D3' *

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