CA2097997C - Vitamin d compounds and method of preparing these compounds - Google Patents
Vitamin d compounds and method of preparing these compounds Download PDFInfo
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- CA2097997C CA2097997C CA002097997A CA2097997A CA2097997C CA 2097997 C CA2097997 C CA 2097997C CA 002097997 A CA002097997 A CA 002097997A CA 2097997 A CA2097997 A CA 2097997A CA 2097997 C CA2097997 C CA 2097997C
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/34—Alcohols
- A61K8/345—Alcohols containing more than one hydroxy group
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/67—Vitamins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/02—Nutrients, e.g. vitamins, minerals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q7/00—Preparations for affecting hair growth
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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
Abstract
The invention relates to a new vitamin D compound of the general formula wherein:
R1 is a hydrogen atom or a hydroxy group;
R2 is a (C1-C3)alkyl group, a hydroxy(C1-C3)alkyl group, a (C1-C2) alkoxymethyl group or a (C2-C3) alkenyl or alkynyl group;
R3 is a branched or non-branched, saturated or unsaturated aliphatic 3- to 5-membered hydrocarbon or oxahydrocarbon biradical, having at least 3 atoms in the main chain and being optionally substituted with one or more substituents selected from epoxy, fluoro and hydroxy;
R4 is a sec. or tert. (C3-C6)alkyl group or a (C3-C6)cycloalkyl group; and A and B are each individually hydrogen atoms or methyl groups, or A and B form together a methylene group.
The invention further relates to a method of preparing these compounds and to their use in pharmacotherapy and cosmetics.
R1 is a hydrogen atom or a hydroxy group;
R2 is a (C1-C3)alkyl group, a hydroxy(C1-C3)alkyl group, a (C1-C2) alkoxymethyl group or a (C2-C3) alkenyl or alkynyl group;
R3 is a branched or non-branched, saturated or unsaturated aliphatic 3- to 5-membered hydrocarbon or oxahydrocarbon biradical, having at least 3 atoms in the main chain and being optionally substituted with one or more substituents selected from epoxy, fluoro and hydroxy;
R4 is a sec. or tert. (C3-C6)alkyl group or a (C3-C6)cycloalkyl group; and A and B are each individually hydrogen atoms or methyl groups, or A and B form together a methylene group.
The invention further relates to a method of preparing these compounds and to their use in pharmacotherapy and cosmetics.
Description
209'99"7 Vitamin D comDOUnds and method of preparing these comuounds The invention relates t~o new vitamin D compounds, to a method of preparing these compounds and to their use in pharmacotherapy and cosmetics. The invention further relates to valuable new intermediates.
It is generally known, that vitamin-D compounds or vitamin-D related compounds ("vitamin-D compounds") have a strong biological activity and may be used in all thoe;e cases in which problems with the calcium metabolism play a part. A few years ago it was found that various active vitamin-D compounds also have other pharmacotherapeutic activities and may be used successfully, for example, for the treatment of certain skin and bone diseases, for cosmetic applications and for treating diseases which are related to cell differentiation, cell proliferation or imbalance in the immune system, including diabetes mellitus, hypertension and inflammatory diseases such as rheumatoid arthritis and asthma. In addition, these compounds may be used in various veterinary applications, and for diagnostic purposes.
Vitamin D compounds which are of interest for the above applications are hydroxylated vitamin D compounds, in particular vitamin D compounds hydroxylated in the la-, 24- and/or 25-positions. Recent developments in the field of active vitamin D compounds are 19-nor-vitamin D compounds (EP-A-0387077) and C,8-modified vitamin D compounds (EP-A-0521550), preferably also hydroxylacted in the la-position and optionally in the C,~-side chain. Other modifications of the C,~-side chain have been proposed, likewise to improve the intended activity and to suppress detrimental side-effects. Examples of modifications of the C,~-side chain are chain elongations (homo compounds), 22-oxa modifications, fluor substitutions, epo:Ky groups (e.g. WO 92/21695), etc. In addition certain 24-cyclopropyl-modified vitamin D compounds are disclosed in literature, e.g. in WO 87/00834 (for- treating abnormal cell differentiation and proliferation) anal in an article by Farach-Carson et al. in Endocrinology 1991, 129, 1876-84. Generally, however, the above C,.,-side chain modified vit~imin D compounds are still not completely satisfactory as regards their scalectiv~=_ activity, i.e. the intended activity without detrimental side-effects.
It is generally known, that vitamin-D compounds or vitamin-D related compounds ("vitamin-D compounds") have a strong biological activity and may be used in all thoe;e cases in which problems with the calcium metabolism play a part. A few years ago it was found that various active vitamin-D compounds also have other pharmacotherapeutic activities and may be used successfully, for example, for the treatment of certain skin and bone diseases, for cosmetic applications and for treating diseases which are related to cell differentiation, cell proliferation or imbalance in the immune system, including diabetes mellitus, hypertension and inflammatory diseases such as rheumatoid arthritis and asthma. In addition, these compounds may be used in various veterinary applications, and for diagnostic purposes.
Vitamin D compounds which are of interest for the above applications are hydroxylated vitamin D compounds, in particular vitamin D compounds hydroxylated in the la-, 24- and/or 25-positions. Recent developments in the field of active vitamin D compounds are 19-nor-vitamin D compounds (EP-A-0387077) and C,8-modified vitamin D compounds (EP-A-0521550), preferably also hydroxylacted in the la-position and optionally in the C,~-side chain. Other modifications of the C,~-side chain have been proposed, likewise to improve the intended activity and to suppress detrimental side-effects. Examples of modifications of the C,~-side chain are chain elongations (homo compounds), 22-oxa modifications, fluor substitutions, epo:Ky groups (e.g. WO 92/21695), etc. In addition certain 24-cyclopropyl-modified vitamin D compounds are disclosed in literature, e.g. in WO 87/00834 (for- treating abnormal cell differentiation and proliferation) anal in an article by Farach-Carson et al. in Endocrinology 1991, 129, 1876-84. Generally, however, the above C,.,-side chain modified vit~imin D compounds are still not completely satisfactory as regards their scalectiv~=_ activity, i.e. the intended activity without detrimental side-effects.
Further, the accessibility of the C,~ side chain modified vitamin D
compounds is often insufficient or unattractive. As an example, the preparation of the above vitamin D compound disclosed by Farach-Carson et al, seems very laborious, while the C,~ side chain build-up, described in the above WO 87/00834, also requires various laborious synthetic steps, using a not readily available ketone as a synthon. In this connection there is a need for better accessible C,~-side chain modified vitamin D compounds. As a matter of fact, both the starting compounds for the preparation of such vitamin-D compounds must be easily available or accessible, and the multistep preparation process must lead to the intended purpose with sufficient selectivity and efficiency.
The present invention provides a new class of vitamin D compounds, which is well accessible from readily available or accessible starting materials.
According to the present invention there is provided a new vitamin D compound of the general formula Hac~~/~a\ ~/Ry ~oy ~y (I) wherein:
R, is a hydrogen atom or a hydroxy group;
RZ is a (C,-C3)alkyl groug, a hydroxy(C,-C3)alkyl group, a (C,-Cz)alkoxymethyl group or a (CZ C3)alkenyl or alkynyl group;
R3 is a branched or non-branched, saturated or unsaturated aliphatic 3- to 5-membered hydrocarbon or oxahydrocarbon biradical, having at least 3 atoms in the main chain and being optionally substituted with one or more substituents selected from epoxy, 24'7997 fluoro and hydroxy;
R4 is a sec. or tert. (C3-C6)alkyl group or a (C3-C6)cycloalkyl group; and A and B are each individually hydrogen atoms or methyl groups, or A and B form together a methylene group.
The above new vitamin D compounds of the invention, presented by the general formula I, are valuable substances. The biological results, as illustrated in the Examples, indicate that these compounds are promising as biologically active substances and may be used in all above-mentioned pharmacotherapeuti.c indications, more in particular for the treatment of osteoporosis, renal osteodystrophy, osteomalacia, skin disorders such as psoriasis (and other hyperproliferative skin diseases), eczema and dermatitis, myopathy, leukemia, breast and colon cancer, osteosarcomas, squamous cell carcinomas, melanoma, certain immunological disorders, and transplant rejections.
Furthermore, the new vitamin D compounds of the invention may be used for wound healing .and may be incorporated in cosmetic compositions, such as creams, lotions, ointments and the like, in order to preserve, condition and/or protects the skin and to improve various skin conditions, such as wrinkles, dry skin, skin slackness and insufficient sebum secretion. The ne~a vitamin D compounds may also be used for diagnostic purposes.
Suitable example:; of the above substituent R4 are: isopropyl, cyclopropyl, tert.-butyl, thexyl (1,1,2-trimethylpropyl), 3-pentyl and cyclopentyl.
A vitamin D compound is preferred, having the above general formula I, wherein:
R, is a hydroxy group;
Rz has the meaning given above;
R3 is a biradi.cal of the formula - O - CHz - ( C:HZ ) o - ,, - CH~ - CHI - ( CH~ ) " - , 3 5 - CH = CH - ( CHz ) ~ - Or - CH, - CHz - CH ( CH3 ) - , wherein n is J. or 2;
R, is an isopropyl group, a cyclopropyl group or a tert.-butyl group; and A and B are hydrogen. atoms or form together a methylene group.
Examples of pre-eminently suitable vitamin D compounds according to the invention are vii:amin D compounds of the above general formula I, wherein the symbo7_s R" Rz, R3, A and B have the above-defined meanings, and R, is an isopropyl group;
because of their extreme7.y favourable biological properties.
It is a special merit of the present invention that the above new vitamin D compounds of the invention can easily be prepared from readily available startin<; materials. In particular, it has been found, that the desired Cu-configuration, i.e. the attachment of the appropriate substituents to C,S, can easily be achieved by starting from a readily accessible ester compound.
Consequently, the invention also relates to a method of preparing a vitamin D compound of then general formula I, as defined above, wherein R, is a hydroxy group, which method is characterized according to the present invention, in than an ester compound of the general formula NC-~C ~R3~ C/°
~O Rb . ,,.
nS
(IX) wherein:
RZ, R3, A and 13 have 'the above-defined meanings, RS is a protected hyclroxy group, and R6 is a ( C,-C6) alkyl group;
is reacted with an organometallic compound of the general formula RaM(X)P (III) ~os~ss~
wherein:
R, has the above meaning, X is C1, Br or I, M is a metal selected from Li and Mg, and 5 p is, dependent on the valence of M, 0 or 1;
followed by deprot:ection.
In an equally attractive manner the C,~ - side chain can first be finalized. Therefore the invention also relates to a method of preparing a vitamin D compound as defined above, which method is characterized according to the present invention, in that an ester compound of the general formula H3c~.n ~ ~3 ~ corn RZ i H
°R6 (II) wherein:
R2, R3 and R6 have the' above meanings, and Rs' is an optionally protected hydroxy group;
is reacted with are organometallic compound of the general formula R4M(X)P (III) wherein the symbols have the above meanings;
after which the hy<irindane compound obtained, having the general formula 3o H3c ~~~ / '~a ~ ~ i Ry R.s H I ~ o H
~y IRh_ (IV) is deprotected, if' RS' is a protected hydroxy group, and then oxidized to the corresponding hydrindane-4-one compound of the general formula 2(l9'799'~
Hjc;~C /~ R3 ' / Ry H C
Rz I \ OH
~y I
0 (v) which compound of formula V, if desired after protection of the hydroxy group, is then converted either (a) with a Wittig reagent of the general formula ( q ;~ """~~~~
S 1 (VI) wherein R,' is a hydrogen atom or a protected hydroxy group, and the other symbols have the above meanings;
or (b), after enolization and derivatization of the enolic hydroxy group, with an enyne compound of the general formula R ,,,, ~?
(VII) wherein the symbols have the above meanings, followed by hydrogenation and isomerization, to produce a compound of the general formula I, wherein A and B form together a methylene group;
followed by deprotection.
Aydroxy groups in 'the above intermediates or reactants may be protected by a reaction with a suitable esterification or etherification agent. A
suitable esterification agent is an alkylchlorocarbonate having 2 to 5 carbon atoms, or an aromatic carboxylic acid or saturated aliphatic carboxylic acid having 1 to 4 carbon atoms such as benzoic acid, or a derivative of such acids suitable for the esterification reaction. In order to protect hydroxy groups in the form of an ether, in principle any etherification agent: known for this purpose is suitable: for zo~~~~~
example, a trialkylsilylimidazole, a trialkylsilylhalide, a trialkyl-silyltriflate (-trifluoromethanesulfonate), a diphenylalkylsilylhalide, or a diphenylalkylsilylt.riflate, or a derivative thereof, the alkyl groups of which have 1 to 6 carbon atoms.
Particularly suitable for this purpose are trimethylsilylchloride, tert.-butyldimethylsilylc:hloride, dimethyl-(1,1,2-trimethylpropyl)-silylchloride, tert.-butyldimethylsilyl triflate, or trimethylsilyl-imidazole, because these etherification agents readily react with the hydroxy group to tie protected to form an ether function, which on the one hand is sufficiently stable under the conditions of the reaction or reactions in view, but on the other hand can easily be removed [deprotection] to recover the original hydroxy group; tert.-butyldime-thylsilylchloride or triflate is to be preferred, because the tert.-butyldimethylsilyl group has been found to be excellently suitable as a protective group.
The enolic hydroxy group is preferably derivatized by a reaction with N-phenyltriflimide to produce a triflate.
The starting compounds of formula II can conveniently be prepared from readily available substances, e.g. for the synthesis of vitamin D
compounds with the above-defined preferred C,~ side chains as follows:
r,,, vitamin DZ \d H 1 /. oion. ~ ~ ~oo(~xafr'on x. ted, _~C I I a ) chain extension: O N
r,, o G, ~OH
H
Z
-a .,, I
.\CN
nil off .,od..~
/,T sCC pyz~:.G~.e 1. DIBAL o ff (XIIb) z. NacN(Dl~lso~ z. N~,BHy 0 ~r 209"x'99"?
° DIR 0518 c/ O
CND CHCOVR~ ~~,yb XII
(2,, ~ ~'u r~ Svmc'Ca t c'o-h~
OH
wherein n is 1 or 2.
The introduction of a C,g modification (RZ) into the vitamin D compound of the invention can convE:niently be achieved as described in the above-mentioned EP-A-0521550.
Suitable examples of orc~anometallic compounds of the above general formula III are lithium compounds, such as isopropyllithium, cyclopropyllithium and tert.-butyllithium, and Grignard reagents, such as isopropylmagnesium chloride, cyclopropylmagnesium chloride and tert.-butylmagnesium chloride, as well as the corresponding bromides.
The intermediate eater compound of the general formula IX, presented above, is new. Therefore the present invention also relates to this intermediate, as wall as to a method of preparing this compound.
The ester compound of the general formula IX, wherein A and B form together a methylene group, can conveniently be prepared by reacting an ester of the general formula "t~ /H
Rz ,~ ~ ~R6 (VIII) wherein:
Rz, R3 and R6 have the above meanings, and R~ is a derivatized hydroxy group, is reacted with an enyne compound of the general formula 3 5 F~~ ~~ ~s ( V I I -A ) wherein the symbol:a have lthe above meanings;
followed by hydrogenation and isomerization.
~0~~9J'?
This reaction is preferably carried out in two reaction steps, viz. by first reacting the ingredients under the influence of an organic base such as triethylamine, arid in the presence of a palladium catalyst such as (PPh3)ZPdClz, send by then subjecting the product obtained to a hydrogenation with hydrogen under the influence of a suitable catalyst such as Lindlar catalyst (Pd on CaCO3, poisoned with lead), followed by an isomerization of the previtamin configuration obtained to the vitamin structure of the general formula IX.
Alternatively, said ester compound of the general formula IX can easily be synthetized by reacting a modified Windaus Grundmann ketone of the general formula X or XI with a Wittig reagent as follows:
H3~~ ~ R3~ , ~o I~z CH ~ ~'p R6 I4 ~'~~~ ~z.
~'-. $ ~ ( I X ) I ~ ',,,"
VI-A
p (X) S S' ( ) or:
,~~ ~ '~'I
usC'~.,aG ~~~
+ VI-A
de zoftcf.
~al~'~a.f~Qx ~ (XI) RS
,,, ~~ B
Rs Rs (XIII) CH1-CH~:OOR~
XIII ----~ IX, wherein R3 = (CHZ)3 or (CHz)4.
~Zr~ ~ Cu .z~ -~ohncc ~-i'° ~c~
The symbols in thsa above formulas are defined hereinbefore.
The intermediate hydrindane-4-one compound of the general formula V, presented above, is new. 'therefore the present invention also relates to this intermediate and to a method of preparing this compound, viz. by oxidizing a hydrindane compound of the general formula IV, as defined above, with an oxidizing agent, preferably selected from a chromium-to containing oxidant such as pyridinium chlorochromate or pyridinium dichromate, and ruthenium tetroxide.
The intermediate hydrindane compound of the general formula IV, presented above, is also new. Consequently, the present invention relates in addition to this intermediate and to a method of preparing this compound, viz. by reacting a compound of the general formula II, as defined above, with a metal-organic compound of the general formula III, as also defined above, in an inert organic solvent.
To improve the applicability of the new vitamin D compounds of the invention for the above-described pharmacotherapeutic indications, the compounds are usually processed to pharmaceutical compositions, comprising an effective amount of said vitamin D compound as the active ingredient in addition to a pharmaceutically acceptable carrier and/or at least one pharmaceutically acceptable auxiliary substance. Such a composition may be delivered in a dosage unit form for oral, topical (dermal) or parenteral administration, comprising approx. O.1 Ng to approx. 0.1 mg active ingredient per dosage unit.
A composition for diagnostic purposes may comprise, in addition to the vitamin D compound of the present invention, a compatible, non-toxic carrier and/or at least one auxiliary substance.
A cosmetical composition may comprise, in addition to an effective amount (in the range of approx. 0.1 pg to approx. 0.1 mg per dosage unit in.a dosage unit form) of the vitamin D compound of the present invention, a cosmetically acceptable, non-toxic carrier and/or at least one auxiliary substance.
The invention also relates to a method for the treatment and prophylaxis of a number of disease states including autoiummune diseases (including diabetes mellitus), acne, a,lopecia, skin aging (including photo-aging), imbalance in the immune system, inflammatory diseases such as rheumatoid arthritis and asthma, as well as diseases related to abnormal cell differentiation and/or proliferation, in a warm-blooded living being, comprising administering to said being or treating said being with a pharmaceutical composition as defined above in a quantity effective for the intended purpose. Examples of such diseases are psoriasis and other hyperproliferative skin diseases.
The present invention also relates to the use of the above pharmaceutical compositions for the treatment of solid, skin and blood cancers, in particular of blood cancers such as leukemia, of breast cancer, and of skin cancers such as melanoma and squamous cell carcinoma.
The above-defined cosmetical compositions, in particular selected from the group consisting of creams, lotions, ointments, liposomes and gels, can be used for the treatment and prevention of a number of skin disorders, such as inadequate skin firmness or texture, insufficient skin hydration, wrinkles and insufficient sebum secretion.
The invention also provides for use of the compounds and compositions of the invention in the treatment and prophylaxis of the noted diseases and disorders as well as use of the compounds and compositions for preparing medicaments.
The invention also provides commercial packages comprising the compounds or compositions of the invention and associated therewith instructions for the use thereof in the treatment and prophylaxis of the noted diseases and disorders.
The invention will now be described in greater detail with reference to the following specific Examples.
lla Examples Example I
Pre aration of vitamin ester reaction equation:
OH
(a) (b) ---;
------;
..-----;
1 0 (e)' (c) (d) -' TBSO''~~ (6~OTB5 ~09"?9~1'7 C o GH
7~ s '' to °''~ ,.~~ ~,~ TBSo~' m i,~,~
(a). Ph3P (6.5 g) and imidazole (4.8 g) are added to a solution of diol (1) (5.0 g) in THE' (100 ml). The suspension is cooled to -20°C, and IZ
(6.28 g) is added in portions. After being stirred for 15 min, the reaction mixture is warmed to room temp., further stirred for 15 min., cooled to 0°C, an~9 poured into saturated aqueous NaHC03 ( 50 ml ) .
The mixture is extracted with Et2o and the extract is washed with saturated aqueous NazS~o3 anc~ H~O, dried and filtered. Concentration affords a residue which is purified. by flash chromatography (8$ EtOAc/hexane) to give 7.32 g of iodide (2). After crystallization from EtOAc/hexane, the product has a melting point of 51°C; identification by NMR and elem.
analysis.
(b). Pyridinium dichromate (8.66 g) is added to a solution of compound ( 2 ) ( 3. 99 g ) in 50 ml CEf~Cl2. The mixture is stirred for 6 h at room temp. Etzo (60 ml) is added, and the resulting suspension is stirred for 15 min and filtered. The :filtrate is washed with brine, dried, filtered and concentrated. Purification by flash chromatography (10~
EtOAc/hexane) gives the desired iodo ketone (3) in a yield of 3.57 g;
crystallization from EtiO,/hexane: m.p. 65°C. Identification by NMR and elem. analysis.
(c). Lithium diisopropyleunine is prepared by addition of i-Pr~NH (3.9 mmol) to a cooled [-78°C) solution of n-BuLi in hexane (3.5 mmol in 1.43 ml). After stirring for 10 min, the mixture is diluted with THF (4 ml), stirred at 0°C for 30 min, and cooled to -78°C. A solution of ketone (3) (1.0 g) in 14 ml THF is slowly added, followed by a solution of N
phenyltriflimide (1.225 g) in 4 ml THF. The mixture is stirred for 2 h 2U~'~99'7 at -78°C. After being warmed to 0°C, the reaction is quenched by addition of a few drops of MeOH and water. Concentration gives a crude product which is diluted with EtOAc/hexane (30 ml), washed with brine, dried, filtered a.nd concentrated. The resulting residue is purified by flash chromatography (2%. EtOAc/hexane), affording 1.29 g of the iodo triflate (4) as a colourless oil. Identification by NMR and elem.
analysis.
(d). A suspension of CuI (201 mg) and Zn (161 mg) in EtOH/Hz0 (6 ml 7:3;
deoxygenated) is aonicated for 5 min. Methyl acrylate (637 girl, freshly distilled), and a solution of the iodide (4) (160 mg) in EtOH/H20 (1 ml 7:3) are successively added, and the resulting mixture is sonicated for 40 min. Dilution with Et20 (15 ml) and filtration gives a solution that is washed with brine. The aqueous phase is extracted with Et~O (30 ml) and the combined organic extracts are dried, filtered and concentrated.
Flash chromatography of 1=he residue (6$ EtOAc/hexane) affords 96 mg of the methyl ester t5) (co:Lourless oil). Identification by NMR and elem.
analysis.
(e). The palladium-catalyzed coupling between vinyl triflate (5) and the enyne (6) is performed as follows:
A mixture of enyne (6) (507 mg), triflate (5) (500 mg), Et3N (4.85 mmol) and (Ph3P)ZPdCl2 (1.6 mg) in 21 ml DMF is heated at 75°C for 1 h. The mixture is cooled to room temp., diluted with EtoAc/hexane (50 ml 1:3), and washed with brine. lJrying, filtration and concentration gives a residue which is p~urified~ by flash chromatography (2-4$ Et20/hexane) to afford 675 mg of dienyne (7) (viscous liquid). Identification by NMR.
(f). Product (7) is hydrogenated by Lindlar catalyst as follows:
A solution (0.2 m:L) of 50 pl quinoline in 10 ml hexane is added to a solution of dienyn,e (7) (305 mg) in 12 ml hexane. Lindlar catalyst (50 mg), previously dried, is added and the resulting solution is exposed to hydrogen gas at ~3tmosph~eric pressure. After stirring for 8 h, the reaction mixture is filtered and concentrated. The residue is purified by flash chromatography (1-3$ Et~o/hexane) to give 295 mg of the protected previtamin D compound.
Isomerization of previtamin D compound to vitamin D compound (8):
20979'7 The previtamin D compound obtained (295 mg) is dissolved in 15 ml isooctane and refluxed in the dark for 5 h. Concentration gives a residue which is purified by flash chromatography (2-4~ Etzo/hexane) to afford 290 mg of c~ompouncl (8). the product is identified by 'H-NMR, "C-NMR and elem. analysis.
'H-NMR ( b, CDC13) : 6.24 and 6.02 (d, 2H) , 5.18 (m, 1H) , 4.87 (m, 1H) , 4.37 (m, 1H), 4.1E~ (m, 1H), 3.67 (s, 3H), 0.93 (d, 3H), 0.88 (s, 18H), 0.53 (s, 3H), 0.07 ( s, 1.2 H).
'3C-NMR (b, CDC13): 173.1, 148.4, 141.0, 135.0, 132.2, 118.0, 111.2, 72.1, 67.5, 56.3, 51.3, 46.0, 45.7, 44.8, 40.6, 35.8, 35.3, 34.4, 31.5, 28.8, 27.6, 25.8, 25.7, 23.4, 22.6, 22.1, 21.5, 18.7, 18.1, 18.0, 14.0, 11.9, -4.8, -4.8, -4.9, -5.2.
Elem. anal.: Calcd.. for C:~H~04Siz: C, 70,75; H, 10.62. Found: C, 70.42;
H, 10.43.
In a corresponding manner the following ester compounds are prepared:
general formula 3---' C oz RC
25 iaso~~., vi r~J
compound no . R~ R, R6 ( 9 ) CHI ( CFi2 ) 4 CZHs ( 10 ) C~HS ( CH~ ) 3 C,HS
Compd. (9): This 24-homo compound is prepared by using in the above step (a) as the starting substance a homologue of compd. ( 1 ) , having a 1-methyl-3-hydroxypropyl side chain; in step (d) ethyl acrylate is used as the olefin.
'H-NMR (b, CDC13): 0.08 (s, 12H), 0.52 (s, 3H), 0.87 (s, 18H), 0.90 (d, 3H), 1.25 (t, 3H), 4.11 (q, 2H), 4.20 (m, 1H), 4.37 (m, 1H), 4.87 (d, 1H), 5.18 (d, 1H), 6.01 (d, 1H), 6.24 (d, 1H).
Compd. (10): This, 18-homa compound is prepared by using in the above ~o~~9s~r step (a) as the starting compound a homologue of compd. (1), prepared as described in published European patent application 521550 (compound no.
(64)].
~H-NMR (b, CDC13): 0.87 (s, 6H), 0.88 (t, 3H), 1.01 (d, 3H), 1.25 (t, 3H), 1.98 (t, 1H), 2.25 (m, 2H), 2.44 (dd, 1H), 4.13 (q, 2H), 4.18 (m, 1H), 4.37 (m, 1H),. 4.86 (s, 1H), 5.17 (s, 1H), 6.01 (d, 1H), 6.23 (d, 1H).
Example II
Preparation of vitamin D compound from vitamin ester reaction equation:
20 T~S~ viOJ ~Q~''~' vn Compound (11) is prepared as follows:
Cyclopropyl bromide (0.51 mmol) is slowly added to a cooled (-20°C) solution of t-BuLi in Et20 (0.51 mmol in 0.602 ml), to produce cyclopropyllithium. The resulting mixture is warmed to room temp. and diluted with 2.4 ;ml Et,O. 1 ml of this solution is slowly added to a cooled (-78°C) solution of compd. (8) (50 mg) in 3 ml Etzo. The reaction mixture is allowed to come to -40°C and quenched with a few drops of water. The resulting solution is diluted with Et,O, washed with brine, dried, filtered and concentrated. The concentrate is filtered through a flash chromatography column (2$ Etzo/hexane), affording a product (46 mg) which is dissolved in 7 ml THF and stirred in the dark at room temp.
with tetrabutyl ammoniumfluoride in THF (0.36 mmol in 0.36 ml) for 24 h.
Concentration gives a residue which is diluted with EtOAc (20 ml), dried, filtered, concentrated and flash chromatographed (60$
EtOAc/hexane) to give 23 mg of the desired compound (11) as a white solid.
~H-NMR (S, CDZC1=): 6.44 a.nd 5.99 (d, 2H), 5.27 (br-d, 1H), 4.95 (br-d, 209'99"?
1H), 4.35 (m, 1H), 4.15 (m, 1H), 0.92 (d, 3H), 0.81 (m, 2H), 0.53 (s, 3H), 0.34 (m, 8H).
'3C-NMR (S, CDZC12): 148.6, 143.5, 133.9, 125.1, 117.6, 111.8, 71.2, 71.0, 67.2, 57.2, 56.8, 45.8, 43.5, 43.4, 41.0, 37.1, 36.6, 29.4, 28.0, 24.0, 22.6, 20.8, 19.0, 12.1, C1.8, -0.5.
In a corresponding manner the following vitamin D compounds are prepared:
general foraula ~y off Ry compound no. RZ R3 R4 ( 12 ) CH3 ( CFiz C ( CHs ) 3 ) 3 (13) CH3 (CHz)3 CH(CH3)~
(14) CzHS (CH,)3 CH(CH3)=
(15) C.,HS (CH~)3 cyclopropyl ( 16 ) CH3 ( CH~ ) cyc lopropyl a ( 17 ) CH3 ( CH~ ) CH ( CH3 4 ) 2 Compd. (12) is prepared by using t-butyllithium instead of cyclopropyllithium.
Compounds (13), (7.4) and (17) are prepared by using isopropyllithium instead of cyclopropyllit:hium.
Compd. (12): 'H-NMR (5, CDC:Ij): 6.38 and 6.01 (d, 2H), 5.33 (m, 1H), 5.00 (m, 1H), 4.43 (m, 1H), 4.23 (m, 1H), 1.00 (s, 18H), 0.93 (d, 3H), 0.54 ( s, 3H) . "C-NMR ( ~~, CDC13) : 148.7, 143.4, 134.0, 125.0, 117.6, 111.8, 80.0, 71.1, 67.1, fi0.6, 5fi.9, 56.8, 46.3, 45.8, 43.4, 42.8, 40.9, 37.0, 209'99?' 36.5, 34.2, 29.4, :?8.0, 24.0, 23.3, 22.7, 19.2, 28.8, 12.1.
Compel. (13): (5, C:D30D): 0.55 (s, 3H), 0.90 2.22(dd, 'H-NM:R (m, 15H), 1H), 2.48 (dd, 2.83(dd, 1H), 4.09 (m, 1H), 4.31 4.86(b?, 1H), (t, 1H), 1H), 5.25 (b, 6.05(d, 1H), 6.29 (d, 1H).
1H), Compel. (14): (b, CDC13): 0.84 (t, 3H), 0.95 (m, 1.00(d, 'H-NNIR 12H), 3H), 2.32 (dd, 2.60(dd, 1H), 2.83 (dd, 1H), 4.24 4.44(m, 1H), (m, 1H), 1H), 5.01 (b, 5.33(b, 1H), 6.01 (d, 1H), 6.39 1H), (d, 1H).
Compel. (15): (5, 0.91(t, 'H-NMR CD3oD):
0.70-0.40 (m, 8H), 0.76 (m, 2H), 3H), 1.01 (d, 2.00(m, 1H), 2.22 (dd, 1H), 2.29 2.48(dd, 3H), (b, 1H), 1H), 2.83 (dd, 4.09 5.25(b, 1H), (m, 1H), 4.31 (t, 1H), 4.85 (b, 1H), 1H), 6.04 (d, 6.29(d, 1H).
1H), Compel . (16): 'H-NMR(b, 0.79(m, CCi30D):
0.20-0.40 (m, 8H), 0.57 (s, 3H), 2H), 0.95 (d, (dd, 1H), 2.51 (dd, 1H), 2.86 4.12(m, 3H), 2.25 (dd, 1H), 1H), 4.35 (t, 4.89(b, 1H), 5.28 (b, 1H), 6.08 6.32(d, 1H)" (d, 1H), 1H).
Compel. (17): 'H-NMR (S, C:D3oD): 0.53 (s, 3H), 0.90 (m, 12H), 2.22 (dd, 1H), 2.48 (dd, 1H), 2.83 (dd, 1H), 4.09 (m, 1H), 4.31 (t, 1H), 4.86 (b, 1H), 5.25 (b, 1H), 6.05 (d, 1H), 6.29 (d, 1H).
Example III
Preparation of 1-(1-methyl-5-hvdroxv-5 5-diisopropvl-pentvll-hvdrindanol-4 (19) Reaction equation:
2 5 ~. ~, ' ~z Cz.NS
a., - - c3H v J p~
35 (a). Starting compound (1) is converted to the corresponding iodide (2) as described in Example I(a).
(b). The compound (2) obtained is converted in a corresponding manner as described in Example I(d), using ethyl acrylate as the olefin, to produce ester compound (:18).
(c ) . Ester compound ( 18 ) is converted to compound ( 19 ) by a reaction with an excess of isopropyllithium, in a corresponding manner as described in Example II. The product is identified by 'H-NMR.
In a corresponding manner the following compounds are prepared:
general formula R
to ~ joy Ry is off compound no. R~ R3 R4 ( 20 ) CH == CH~ ( CHZ ) 3 cyc lopropyl ( 21 ) CH == CH, ( CHZ ) 3 isopropyl (22) CEi3 (CH2)4 isopropyl 20 (23) CEi3 (CHZ), cyclopropyl The products are i_dentifi_ed by 'H-NMR.
Example IV
Preparation of 1-(1-methyl-5-hydroxy-5 5-diisopropyl-oentyll-25 hvdrindanone-4 (2~1 Reaction equation Oxidation of compound (19) by using pyridinium dichromate as the oxidant, in a corresponding manner as described in Example I(b), affords the desired ketone~ (24) in a yield of 84~. The product is identified by ' H-NMR .
In a corresponding manner the following ketones are prepared:
general forrula ~3~
C
I woy ~y compound no. RZ R3 R4 (25) CH -= CHI (CHz)3 cyclopropyl ( 2 6 ) CH - CHI ( CHz ) 3 i sopropyl (27) CH3 (CH,)4 isopropyl (28) Cli3 (CHz)4 cyclopropyl The products are :identif.ied by ~H-NMR.
Example V
Prevaration of vii:amin D compound (131 from ketone (241 reaction equation:
- ''~~.
~'oTMS
,a ~z y~ --2 5 f~ ~Q ~ OT~
,;, ~
0 'z '~~ ~07 ~d~
i. zed.
z, r'so~ne r.
3. d~ptot~
H o ,,,...
~H
TBSO~''~~~ arss %$s0'"1 ~'oTr~s 2~~"~99'?
(a). The free hydroxy group is protected by a reaction with trimethylsilyltriflate (~TBS-triflate) in the presence of triethylamine and in methylene <:hloride as the solvent; temp. -78°C -- 0°C;
yield of compound (29) is .30%.
(b) The enolisation is carried out in a corresponding manner as described in Example I(c), producing compound (30) in a yield of 71%.
(c). In a corree3ponding manner as described in Example I(e), the coupling reaction with enyne (6) is performed, affording compound (31) in a yield of 94%.
(d). The final reaction step is carried out in a corresponding manner as described in Example I(f), followed by deprotection (desilylation), as described in Example II,, with tetrabutyl a.mmoniumfluoride. The final vitamin D compound (13) is obtained in an overall yield of 65%. The product is identical with the product obtained according to Example II.
In a corresponding manner the following vitamin D compounds are prepared.
general formula R3~C~~y ~ ~ off Ry ,, H 0 \h, v h.
compound no. R, R3 R4 ( 32 ) CH :- CH= ( CH,) 3 cyclopropyl (33) CH =- CHz (CHz)3 isopropyl ( 17 ) CH3 ( CH~ ) 4 isopropyl ( 16 ) Cfi3 ( CHz ) 4 cyc lopropyl The products are identified by 'H-NMR. The last two vitamin D compounds are identical with the corresponding vitamin D compounds prepared according to Example II.
_. ~09"T99'7 Example VI
Preparation of 19-nor-vitamin D compound (351 from ketone (291 Reaction equation:
r rMS
\ ~..
i s s o' o TB s ~~ ~3 ~J
x. dcpzGE.
,,,.
Hog, ~~ c~~~
A solution of 1.14 g (2 nvnol) of phosphine oxide (34) in 15 ml of dry THF is cooled to -78°C. n-Buthyllithium (BuLi), as a 2.5 M
solution in hexane, is added d~copwise until the red colour persists. Then 0.8 ml of a 2.5 M solution of BuLi is added. Stirring is continued for 15 min, followed by the dropwise addition of 0.73 g (1.8 mmol) of ketone (29) in 5 ml THF. After another hour of stirring, the reaction mixture is allowed to reach 0°C and then quenched by the addition of 50 ml of a saturated NH,C1-solution. lExtractive work-up and flash chromatography (2$
EtOAc in hexane) then affords the protected diene compound. Desilylation by reaction with 10 eq. of tetrabutylammonium fluoride (TBAF.3aq) in THF
(10 ml) during 48 hours gives compound (35), which is purified by flash chromatography using EtOAc as an eluent, followed by recrystallization from MeOH/EtOAc. The overall yield is 53$. Identification by 'H-NMR.
In a corresponding manner the following vitamin D compounds are prepared:
general formula ~3~. ~ ~ ay ~ ~ off Ry y°\ UH
209'7~9'~
compound RZ R3 R4 A B
no .
(36) CH := (CHZ)3 isopropyl H H
( 3'1 ) CH := ( cyc H H
CHz CHZ lopropyl ) ( 38 ) CFizOH ( isopropyl H H
CHI
) The products are i_dentif~Led by 'H-NMR.
Example VII
Affinity to intracellular vitamin D receptor Vitamin D compounds according to the invention are dissolved in ethanol in concentrations ranging from 10-'3 to 10-' M. The affinity towards the calf thymus intra<:ellular vitamin D receptor (VDR) is determined in a biological assay. In this: assay, 3H-1a,25-dihydroxycholecalciferol (3H
1a,25-DHCC), which is specifically bound to the VDR, is replaced by the tested compounds. Especially the tested compounds 11, 13 and 14 have a very high VDR-ai:finity.. A high VDR-affinity is indicative for biologically active substances.
Example VIII
Affinity to vitamin D binding protein Vitamin D binding protein (DBP) is the specific carrier for vitamin D
and its metabolites in :blood. The biological activity of vitamin D
compounds depends nn the it binding to DBP, because strong binding to DBP
will reduce the intracellular access to the VDR. Binding to the DBP may also influence t:he ha7Lf-life of the vitamin D derivatives in circulation. Weak binders are rapidly metabolized, which is a favourable aspect in topical application.
In the assay, DBP :s incubated with 3H-1a,25-DHCC and 1a,25-DHCC or with several vitamin D <:ompounds according to the invention. To this purpose, the vitamin compounds are dissolved in ethanol in concentrations ranging from 10'x' to 2. 5 x 10'6 M. The percentage bound/unbound 'H-la, 25-DHCC is then calculated. DBP is purified from total human serum. The results are shown in the appended Figures 1 and 2. Figures 1 and 2 show the binding of vitamin D compounds to human vitamin D binding protein.
['H]1a,25(OH)~D~ _ '13-1a,25-DHCC; in both Figures ~ - 1a,25-DHCC (known compound); in Fig. 1 ~ = compound 13 and D - compound 14; in Fig.
2 Q = compound 11.
_.. 20~'~00:
Compounds 14 and 11 bind rather weakly to the DBP, compared to the known 1x,25-DHCC. Compound 13 is a very weak binder.
Example IX
Cell differentiation Vitamin D compoundls according to the invention are dissolved in ethanol in concentrations ranging from 10-'2 to 10-6 M and tested for their capacity to induce cell differentiation in a HL-60 assay. In this assay, morphologic and biochemical examination of the human leukemic cell line HL-60 is done, in order to establish whether cell differentiation has taken place.
Differentiation is expressed as the maturation parameters nitroblue tetrazolium (NBT;~ reduction, non-specific esterase, and as the percentage of mature cells beyond the myelocyte stage which is visible after staining with May-Grunwald Giemsa. After culturing with the known 1a,25-DHCC or with vitamin D compounds of the invention, the percentage of cells containing black formazan deposits is determined. An increase in the percentage of NBT reducing cells indicates an increase in cell differentiation.
Proliferation and vitality of the cell cultures are established by counting the number of cells and by the trypan blue exclusion method.
The vitality and proliferation of the cells in the HL-60 cultures are good in all conditions tested. 1a,25-DHCC (known), compound 12, compound 11, compound 13 and compound 14 all induce differentiation and maturation of the HL-60 cells. In the cytological test (non-specific esterase and May-Grunwal~d Giemsa) especially compounds 13 and 11 are good differentiators. The optimum effect is found at concentrations in the range of 10-e to 10-' M.
The NBT-reduction inducing capacity of compounds 13 and 14 is about 10 x stronger than that of 'the known 1a,25-DHCC. Compounds 11 and 12 are about 5 x more potent in inducing NBT-reduction than 1a,25-DHCC (Figures 3 and 4).
The above implies that the tested new vitamin D compounds of the invention display a higher cell differentiating activity than the known 1a,25-DHCC.
zos~s9~
Figures 3 and 4 (appended) show the differentiating effect of the tested vitamin D compounds on human leukemia cells of the HL-60 line. In both Figures ~ = 1a,25-DHCC; in Fig. 3 O is compound 13 and O is com:..pound 14; in Fig. 4 d =- compound 11 and D = compound 12.
Example X
Calciotropic effects The most well-known efect of la, 25-DHCC is its action on the calcium metabolism, the ca.lciotropic effect. Calciotropic target organs are the intestine, the bone and t:he kidney.
The vitamin D compounds according to the invention are dissolved in ethanol and tested in the so-called Caco-2 assay for intestinal calcium (Ca) transport. In this ~3ssay, the vitamin D - induced influx of 'SCaZ+
is measured in monolayers of the intestinal cancer cell line Caco-2.
This influx is corrected for the concentration-driven Ca'-+ influx and is a measure for the Ca transport across the intestinal wall. The Caco-2 cells are known to have vitamin D receptors.
Increased intestinal calcium transport can be the first step leading to a rise in blood calcium levels (and eventually to hypercalcemia).
In Table A below 'the effects of vitamin D compounds of the invention, compared with the known 1a,25-DHCC, on the Ca'+ influx in intestinal Caco-2 cell cultures are presented. The values in the table represent the relative increase in Ca=+ influx tthe va7uP fnr ~n_~S-nu~r ;
arbitrarily fixed at 100).
The results in Table A demonstrate that compound 11, compound 13 and compound 14 are we.3ker at:Lmulators of intestinal calcium absorption than 1a,25-DHCC.
Table A
experiment 1:
compound 7.0-9 M
1a,25-DHCC 100 compound 11 79 compound 12 99 209"997 experiment 2:
compound 1.0-9 M
1a,25-DHCC 100 compound 13 65 compound 14 78 Example XI
Calciotropic effect Together with the :intestine and the bone, the kidney is one of the major target organs of 1a,25-D1HCC. The kidney plays an extremely important role in calcium hc~meosta:ois, since about 98~ of the calcium has to be reabsorbed in the kidneys in order to prevent calcium loss and hypocalcemia.
The vitamin D compounds according to the invention are dissolved in ethanol and tested in the rabbit kidney cell assay. In this assay, reabsorption of 'SCa'' is measured in monolayers of rabbit kidney cells.
The cells are isolated by immunodissection of connecting tubules with the aid of monoclonal antibodies.
In Table B below the effE~cts of vitamin D compounds of the invention, compared with the known 1.x,25-DHCC, on the Ca'+-reabsorption in rabbit renal cell cultures are presented. The values in the Table represent the increase in Ca=+-reabsorpt.ion in nmol/cm~/h.
The results in Table B demonstrate that compound 13 and compound 11 are weaker stimulators of renal Ca-reabsorption than compound 12 and 1a,25-DHCC itself.
Table B
compound ~D-9 M
1a,25-DHCC ~~----13.4 compound 11 2.4 compound 12 12.8 compound 13 ~-3,7 Example XII
Cell differentiation versus calciotropic effect 209'?99'7 One of the set-bac:ks of t:he highly active vitamin D compounds, such as the well-known 1a,25-DHCC, is its calciotropic effect, which may lead to toxic hypercalciuria, hypercalcemia and urolithiasis. Therefore, it should be very advantageous to develop compounds with a high selectivity of biological action. In other words, compounds in which the ratio between the induction of cell differentiation and calciotropic effects, e.g. the stimulation of intestinal Ca transport, is changed compared to 1a,25-DHCC.
The ratio between differentiation-inducing capacity and the stimulation of intestinal Ca transport is defined as the fraction between the concentration at which 50~ NBT reduction in HL-60 cells is obtained, and the concentration at which a half-maximal increase in intestinal Caz+
transport is reached. The smaller the ratio, the higher the relative cell differentiating capacity.
The results are presentecl in Table C.
Table C
compound fraction ~ ratio relative to DHCC
1a,25-DHCC 107 ~ 1.00 compound 12 29,7 0.2g compound 11 14.4 0.13 compound 14 2.6 0.02 compound 13 2.5 0.02 Table C shows, that the tested new vitamin D compounds of the invention have better rela;.ive cell differentiating properties than the known 1a,25-DHCC. Compound 14 and compound 13 have 50 x more selective actions than 1a,25-DHCC. C:ompouncl 11 has a 8 x better ratio. Compound 12 has a 4 x better ratio. This makes the new vitamin D compounds of the present invention extremely suitable for applications where the differentiation of cells (such as in hyperproliferative conditions) is desired.
compounds is often insufficient or unattractive. As an example, the preparation of the above vitamin D compound disclosed by Farach-Carson et al, seems very laborious, while the C,~ side chain build-up, described in the above WO 87/00834, also requires various laborious synthetic steps, using a not readily available ketone as a synthon. In this connection there is a need for better accessible C,~-side chain modified vitamin D compounds. As a matter of fact, both the starting compounds for the preparation of such vitamin-D compounds must be easily available or accessible, and the multistep preparation process must lead to the intended purpose with sufficient selectivity and efficiency.
The present invention provides a new class of vitamin D compounds, which is well accessible from readily available or accessible starting materials.
According to the present invention there is provided a new vitamin D compound of the general formula Hac~~/~a\ ~/Ry ~oy ~y (I) wherein:
R, is a hydrogen atom or a hydroxy group;
RZ is a (C,-C3)alkyl groug, a hydroxy(C,-C3)alkyl group, a (C,-Cz)alkoxymethyl group or a (CZ C3)alkenyl or alkynyl group;
R3 is a branched or non-branched, saturated or unsaturated aliphatic 3- to 5-membered hydrocarbon or oxahydrocarbon biradical, having at least 3 atoms in the main chain and being optionally substituted with one or more substituents selected from epoxy, 24'7997 fluoro and hydroxy;
R4 is a sec. or tert. (C3-C6)alkyl group or a (C3-C6)cycloalkyl group; and A and B are each individually hydrogen atoms or methyl groups, or A and B form together a methylene group.
The above new vitamin D compounds of the invention, presented by the general formula I, are valuable substances. The biological results, as illustrated in the Examples, indicate that these compounds are promising as biologically active substances and may be used in all above-mentioned pharmacotherapeuti.c indications, more in particular for the treatment of osteoporosis, renal osteodystrophy, osteomalacia, skin disorders such as psoriasis (and other hyperproliferative skin diseases), eczema and dermatitis, myopathy, leukemia, breast and colon cancer, osteosarcomas, squamous cell carcinomas, melanoma, certain immunological disorders, and transplant rejections.
Furthermore, the new vitamin D compounds of the invention may be used for wound healing .and may be incorporated in cosmetic compositions, such as creams, lotions, ointments and the like, in order to preserve, condition and/or protects the skin and to improve various skin conditions, such as wrinkles, dry skin, skin slackness and insufficient sebum secretion. The ne~a vitamin D compounds may also be used for diagnostic purposes.
Suitable example:; of the above substituent R4 are: isopropyl, cyclopropyl, tert.-butyl, thexyl (1,1,2-trimethylpropyl), 3-pentyl and cyclopentyl.
A vitamin D compound is preferred, having the above general formula I, wherein:
R, is a hydroxy group;
Rz has the meaning given above;
R3 is a biradi.cal of the formula - O - CHz - ( C:HZ ) o - ,, - CH~ - CHI - ( CH~ ) " - , 3 5 - CH = CH - ( CHz ) ~ - Or - CH, - CHz - CH ( CH3 ) - , wherein n is J. or 2;
R, is an isopropyl group, a cyclopropyl group or a tert.-butyl group; and A and B are hydrogen. atoms or form together a methylene group.
Examples of pre-eminently suitable vitamin D compounds according to the invention are vii:amin D compounds of the above general formula I, wherein the symbo7_s R" Rz, R3, A and B have the above-defined meanings, and R, is an isopropyl group;
because of their extreme7.y favourable biological properties.
It is a special merit of the present invention that the above new vitamin D compounds of the invention can easily be prepared from readily available startin<; materials. In particular, it has been found, that the desired Cu-configuration, i.e. the attachment of the appropriate substituents to C,S, can easily be achieved by starting from a readily accessible ester compound.
Consequently, the invention also relates to a method of preparing a vitamin D compound of then general formula I, as defined above, wherein R, is a hydroxy group, which method is characterized according to the present invention, in than an ester compound of the general formula NC-~C ~R3~ C/°
~O Rb . ,,.
nS
(IX) wherein:
RZ, R3, A and 13 have 'the above-defined meanings, RS is a protected hyclroxy group, and R6 is a ( C,-C6) alkyl group;
is reacted with an organometallic compound of the general formula RaM(X)P (III) ~os~ss~
wherein:
R, has the above meaning, X is C1, Br or I, M is a metal selected from Li and Mg, and 5 p is, dependent on the valence of M, 0 or 1;
followed by deprot:ection.
In an equally attractive manner the C,~ - side chain can first be finalized. Therefore the invention also relates to a method of preparing a vitamin D compound as defined above, which method is characterized according to the present invention, in that an ester compound of the general formula H3c~.n ~ ~3 ~ corn RZ i H
°R6 (II) wherein:
R2, R3 and R6 have the' above meanings, and Rs' is an optionally protected hydroxy group;
is reacted with are organometallic compound of the general formula R4M(X)P (III) wherein the symbols have the above meanings;
after which the hy<irindane compound obtained, having the general formula 3o H3c ~~~ / '~a ~ ~ i Ry R.s H I ~ o H
~y IRh_ (IV) is deprotected, if' RS' is a protected hydroxy group, and then oxidized to the corresponding hydrindane-4-one compound of the general formula 2(l9'799'~
Hjc;~C /~ R3 ' / Ry H C
Rz I \ OH
~y I
0 (v) which compound of formula V, if desired after protection of the hydroxy group, is then converted either (a) with a Wittig reagent of the general formula ( q ;~ """~~~~
S 1 (VI) wherein R,' is a hydrogen atom or a protected hydroxy group, and the other symbols have the above meanings;
or (b), after enolization and derivatization of the enolic hydroxy group, with an enyne compound of the general formula R ,,,, ~?
(VII) wherein the symbols have the above meanings, followed by hydrogenation and isomerization, to produce a compound of the general formula I, wherein A and B form together a methylene group;
followed by deprotection.
Aydroxy groups in 'the above intermediates or reactants may be protected by a reaction with a suitable esterification or etherification agent. A
suitable esterification agent is an alkylchlorocarbonate having 2 to 5 carbon atoms, or an aromatic carboxylic acid or saturated aliphatic carboxylic acid having 1 to 4 carbon atoms such as benzoic acid, or a derivative of such acids suitable for the esterification reaction. In order to protect hydroxy groups in the form of an ether, in principle any etherification agent: known for this purpose is suitable: for zo~~~~~
example, a trialkylsilylimidazole, a trialkylsilylhalide, a trialkyl-silyltriflate (-trifluoromethanesulfonate), a diphenylalkylsilylhalide, or a diphenylalkylsilylt.riflate, or a derivative thereof, the alkyl groups of which have 1 to 6 carbon atoms.
Particularly suitable for this purpose are trimethylsilylchloride, tert.-butyldimethylsilylc:hloride, dimethyl-(1,1,2-trimethylpropyl)-silylchloride, tert.-butyldimethylsilyl triflate, or trimethylsilyl-imidazole, because these etherification agents readily react with the hydroxy group to tie protected to form an ether function, which on the one hand is sufficiently stable under the conditions of the reaction or reactions in view, but on the other hand can easily be removed [deprotection] to recover the original hydroxy group; tert.-butyldime-thylsilylchloride or triflate is to be preferred, because the tert.-butyldimethylsilyl group has been found to be excellently suitable as a protective group.
The enolic hydroxy group is preferably derivatized by a reaction with N-phenyltriflimide to produce a triflate.
The starting compounds of formula II can conveniently be prepared from readily available substances, e.g. for the synthesis of vitamin D
compounds with the above-defined preferred C,~ side chains as follows:
r,,, vitamin DZ \d H 1 /. oion. ~ ~ ~oo(~xafr'on x. ted, _~C I I a ) chain extension: O N
r,, o G, ~OH
H
Z
-a .,, I
.\CN
nil off .,od..~
/,T sCC pyz~:.G~.e 1. DIBAL o ff (XIIb) z. NacN(Dl~lso~ z. N~,BHy 0 ~r 209"x'99"?
° DIR 0518 c/ O
CND CHCOVR~ ~~,yb XII
(2,, ~ ~'u r~ Svmc'Ca t c'o-h~
OH
wherein n is 1 or 2.
The introduction of a C,g modification (RZ) into the vitamin D compound of the invention can convE:niently be achieved as described in the above-mentioned EP-A-0521550.
Suitable examples of orc~anometallic compounds of the above general formula III are lithium compounds, such as isopropyllithium, cyclopropyllithium and tert.-butyllithium, and Grignard reagents, such as isopropylmagnesium chloride, cyclopropylmagnesium chloride and tert.-butylmagnesium chloride, as well as the corresponding bromides.
The intermediate eater compound of the general formula IX, presented above, is new. Therefore the present invention also relates to this intermediate, as wall as to a method of preparing this compound.
The ester compound of the general formula IX, wherein A and B form together a methylene group, can conveniently be prepared by reacting an ester of the general formula "t~ /H
Rz ,~ ~ ~R6 (VIII) wherein:
Rz, R3 and R6 have the above meanings, and R~ is a derivatized hydroxy group, is reacted with an enyne compound of the general formula 3 5 F~~ ~~ ~s ( V I I -A ) wherein the symbol:a have lthe above meanings;
followed by hydrogenation and isomerization.
~0~~9J'?
This reaction is preferably carried out in two reaction steps, viz. by first reacting the ingredients under the influence of an organic base such as triethylamine, arid in the presence of a palladium catalyst such as (PPh3)ZPdClz, send by then subjecting the product obtained to a hydrogenation with hydrogen under the influence of a suitable catalyst such as Lindlar catalyst (Pd on CaCO3, poisoned with lead), followed by an isomerization of the previtamin configuration obtained to the vitamin structure of the general formula IX.
Alternatively, said ester compound of the general formula IX can easily be synthetized by reacting a modified Windaus Grundmann ketone of the general formula X or XI with a Wittig reagent as follows:
H3~~ ~ R3~ , ~o I~z CH ~ ~'p R6 I4 ~'~~~ ~z.
~'-. $ ~ ( I X ) I ~ ',,,"
VI-A
p (X) S S' ( ) or:
,~~ ~ '~'I
usC'~.,aG ~~~
+ VI-A
de zoftcf.
~al~'~a.f~Qx ~ (XI) RS
,,, ~~ B
Rs Rs (XIII) CH1-CH~:OOR~
XIII ----~ IX, wherein R3 = (CHZ)3 or (CHz)4.
~Zr~ ~ Cu .z~ -~ohncc ~-i'° ~c~
The symbols in thsa above formulas are defined hereinbefore.
The intermediate hydrindane-4-one compound of the general formula V, presented above, is new. 'therefore the present invention also relates to this intermediate and to a method of preparing this compound, viz. by oxidizing a hydrindane compound of the general formula IV, as defined above, with an oxidizing agent, preferably selected from a chromium-to containing oxidant such as pyridinium chlorochromate or pyridinium dichromate, and ruthenium tetroxide.
The intermediate hydrindane compound of the general formula IV, presented above, is also new. Consequently, the present invention relates in addition to this intermediate and to a method of preparing this compound, viz. by reacting a compound of the general formula II, as defined above, with a metal-organic compound of the general formula III, as also defined above, in an inert organic solvent.
To improve the applicability of the new vitamin D compounds of the invention for the above-described pharmacotherapeutic indications, the compounds are usually processed to pharmaceutical compositions, comprising an effective amount of said vitamin D compound as the active ingredient in addition to a pharmaceutically acceptable carrier and/or at least one pharmaceutically acceptable auxiliary substance. Such a composition may be delivered in a dosage unit form for oral, topical (dermal) or parenteral administration, comprising approx. O.1 Ng to approx. 0.1 mg active ingredient per dosage unit.
A composition for diagnostic purposes may comprise, in addition to the vitamin D compound of the present invention, a compatible, non-toxic carrier and/or at least one auxiliary substance.
A cosmetical composition may comprise, in addition to an effective amount (in the range of approx. 0.1 pg to approx. 0.1 mg per dosage unit in.a dosage unit form) of the vitamin D compound of the present invention, a cosmetically acceptable, non-toxic carrier and/or at least one auxiliary substance.
The invention also relates to a method for the treatment and prophylaxis of a number of disease states including autoiummune diseases (including diabetes mellitus), acne, a,lopecia, skin aging (including photo-aging), imbalance in the immune system, inflammatory diseases such as rheumatoid arthritis and asthma, as well as diseases related to abnormal cell differentiation and/or proliferation, in a warm-blooded living being, comprising administering to said being or treating said being with a pharmaceutical composition as defined above in a quantity effective for the intended purpose. Examples of such diseases are psoriasis and other hyperproliferative skin diseases.
The present invention also relates to the use of the above pharmaceutical compositions for the treatment of solid, skin and blood cancers, in particular of blood cancers such as leukemia, of breast cancer, and of skin cancers such as melanoma and squamous cell carcinoma.
The above-defined cosmetical compositions, in particular selected from the group consisting of creams, lotions, ointments, liposomes and gels, can be used for the treatment and prevention of a number of skin disorders, such as inadequate skin firmness or texture, insufficient skin hydration, wrinkles and insufficient sebum secretion.
The invention also provides for use of the compounds and compositions of the invention in the treatment and prophylaxis of the noted diseases and disorders as well as use of the compounds and compositions for preparing medicaments.
The invention also provides commercial packages comprising the compounds or compositions of the invention and associated therewith instructions for the use thereof in the treatment and prophylaxis of the noted diseases and disorders.
The invention will now be described in greater detail with reference to the following specific Examples.
lla Examples Example I
Pre aration of vitamin ester reaction equation:
OH
(a) (b) ---;
------;
..-----;
1 0 (e)' (c) (d) -' TBSO''~~ (6~OTB5 ~09"?9~1'7 C o GH
7~ s '' to °''~ ,.~~ ~,~ TBSo~' m i,~,~
(a). Ph3P (6.5 g) and imidazole (4.8 g) are added to a solution of diol (1) (5.0 g) in THE' (100 ml). The suspension is cooled to -20°C, and IZ
(6.28 g) is added in portions. After being stirred for 15 min, the reaction mixture is warmed to room temp., further stirred for 15 min., cooled to 0°C, an~9 poured into saturated aqueous NaHC03 ( 50 ml ) .
The mixture is extracted with Et2o and the extract is washed with saturated aqueous NazS~o3 anc~ H~O, dried and filtered. Concentration affords a residue which is purified. by flash chromatography (8$ EtOAc/hexane) to give 7.32 g of iodide (2). After crystallization from EtOAc/hexane, the product has a melting point of 51°C; identification by NMR and elem.
analysis.
(b). Pyridinium dichromate (8.66 g) is added to a solution of compound ( 2 ) ( 3. 99 g ) in 50 ml CEf~Cl2. The mixture is stirred for 6 h at room temp. Etzo (60 ml) is added, and the resulting suspension is stirred for 15 min and filtered. The :filtrate is washed with brine, dried, filtered and concentrated. Purification by flash chromatography (10~
EtOAc/hexane) gives the desired iodo ketone (3) in a yield of 3.57 g;
crystallization from EtiO,/hexane: m.p. 65°C. Identification by NMR and elem. analysis.
(c). Lithium diisopropyleunine is prepared by addition of i-Pr~NH (3.9 mmol) to a cooled [-78°C) solution of n-BuLi in hexane (3.5 mmol in 1.43 ml). After stirring for 10 min, the mixture is diluted with THF (4 ml), stirred at 0°C for 30 min, and cooled to -78°C. A solution of ketone (3) (1.0 g) in 14 ml THF is slowly added, followed by a solution of N
phenyltriflimide (1.225 g) in 4 ml THF. The mixture is stirred for 2 h 2U~'~99'7 at -78°C. After being warmed to 0°C, the reaction is quenched by addition of a few drops of MeOH and water. Concentration gives a crude product which is diluted with EtOAc/hexane (30 ml), washed with brine, dried, filtered a.nd concentrated. The resulting residue is purified by flash chromatography (2%. EtOAc/hexane), affording 1.29 g of the iodo triflate (4) as a colourless oil. Identification by NMR and elem.
analysis.
(d). A suspension of CuI (201 mg) and Zn (161 mg) in EtOH/Hz0 (6 ml 7:3;
deoxygenated) is aonicated for 5 min. Methyl acrylate (637 girl, freshly distilled), and a solution of the iodide (4) (160 mg) in EtOH/H20 (1 ml 7:3) are successively added, and the resulting mixture is sonicated for 40 min. Dilution with Et20 (15 ml) and filtration gives a solution that is washed with brine. The aqueous phase is extracted with Et~O (30 ml) and the combined organic extracts are dried, filtered and concentrated.
Flash chromatography of 1=he residue (6$ EtOAc/hexane) affords 96 mg of the methyl ester t5) (co:Lourless oil). Identification by NMR and elem.
analysis.
(e). The palladium-catalyzed coupling between vinyl triflate (5) and the enyne (6) is performed as follows:
A mixture of enyne (6) (507 mg), triflate (5) (500 mg), Et3N (4.85 mmol) and (Ph3P)ZPdCl2 (1.6 mg) in 21 ml DMF is heated at 75°C for 1 h. The mixture is cooled to room temp., diluted with EtoAc/hexane (50 ml 1:3), and washed with brine. lJrying, filtration and concentration gives a residue which is p~urified~ by flash chromatography (2-4$ Et20/hexane) to afford 675 mg of dienyne (7) (viscous liquid). Identification by NMR.
(f). Product (7) is hydrogenated by Lindlar catalyst as follows:
A solution (0.2 m:L) of 50 pl quinoline in 10 ml hexane is added to a solution of dienyn,e (7) (305 mg) in 12 ml hexane. Lindlar catalyst (50 mg), previously dried, is added and the resulting solution is exposed to hydrogen gas at ~3tmosph~eric pressure. After stirring for 8 h, the reaction mixture is filtered and concentrated. The residue is purified by flash chromatography (1-3$ Et~o/hexane) to give 295 mg of the protected previtamin D compound.
Isomerization of previtamin D compound to vitamin D compound (8):
20979'7 The previtamin D compound obtained (295 mg) is dissolved in 15 ml isooctane and refluxed in the dark for 5 h. Concentration gives a residue which is purified by flash chromatography (2-4~ Etzo/hexane) to afford 290 mg of c~ompouncl (8). the product is identified by 'H-NMR, "C-NMR and elem. analysis.
'H-NMR ( b, CDC13) : 6.24 and 6.02 (d, 2H) , 5.18 (m, 1H) , 4.87 (m, 1H) , 4.37 (m, 1H), 4.1E~ (m, 1H), 3.67 (s, 3H), 0.93 (d, 3H), 0.88 (s, 18H), 0.53 (s, 3H), 0.07 ( s, 1.2 H).
'3C-NMR (b, CDC13): 173.1, 148.4, 141.0, 135.0, 132.2, 118.0, 111.2, 72.1, 67.5, 56.3, 51.3, 46.0, 45.7, 44.8, 40.6, 35.8, 35.3, 34.4, 31.5, 28.8, 27.6, 25.8, 25.7, 23.4, 22.6, 22.1, 21.5, 18.7, 18.1, 18.0, 14.0, 11.9, -4.8, -4.8, -4.9, -5.2.
Elem. anal.: Calcd.. for C:~H~04Siz: C, 70,75; H, 10.62. Found: C, 70.42;
H, 10.43.
In a corresponding manner the following ester compounds are prepared:
general formula 3---' C oz RC
25 iaso~~., vi r~J
compound no . R~ R, R6 ( 9 ) CHI ( CFi2 ) 4 CZHs ( 10 ) C~HS ( CH~ ) 3 C,HS
Compd. (9): This 24-homo compound is prepared by using in the above step (a) as the starting substance a homologue of compd. ( 1 ) , having a 1-methyl-3-hydroxypropyl side chain; in step (d) ethyl acrylate is used as the olefin.
'H-NMR (b, CDC13): 0.08 (s, 12H), 0.52 (s, 3H), 0.87 (s, 18H), 0.90 (d, 3H), 1.25 (t, 3H), 4.11 (q, 2H), 4.20 (m, 1H), 4.37 (m, 1H), 4.87 (d, 1H), 5.18 (d, 1H), 6.01 (d, 1H), 6.24 (d, 1H).
Compd. (10): This, 18-homa compound is prepared by using in the above ~o~~9s~r step (a) as the starting compound a homologue of compd. (1), prepared as described in published European patent application 521550 (compound no.
(64)].
~H-NMR (b, CDC13): 0.87 (s, 6H), 0.88 (t, 3H), 1.01 (d, 3H), 1.25 (t, 3H), 1.98 (t, 1H), 2.25 (m, 2H), 2.44 (dd, 1H), 4.13 (q, 2H), 4.18 (m, 1H), 4.37 (m, 1H),. 4.86 (s, 1H), 5.17 (s, 1H), 6.01 (d, 1H), 6.23 (d, 1H).
Example II
Preparation of vitamin D compound from vitamin ester reaction equation:
20 T~S~ viOJ ~Q~''~' vn Compound (11) is prepared as follows:
Cyclopropyl bromide (0.51 mmol) is slowly added to a cooled (-20°C) solution of t-BuLi in Et20 (0.51 mmol in 0.602 ml), to produce cyclopropyllithium. The resulting mixture is warmed to room temp. and diluted with 2.4 ;ml Et,O. 1 ml of this solution is slowly added to a cooled (-78°C) solution of compd. (8) (50 mg) in 3 ml Etzo. The reaction mixture is allowed to come to -40°C and quenched with a few drops of water. The resulting solution is diluted with Et,O, washed with brine, dried, filtered and concentrated. The concentrate is filtered through a flash chromatography column (2$ Etzo/hexane), affording a product (46 mg) which is dissolved in 7 ml THF and stirred in the dark at room temp.
with tetrabutyl ammoniumfluoride in THF (0.36 mmol in 0.36 ml) for 24 h.
Concentration gives a residue which is diluted with EtOAc (20 ml), dried, filtered, concentrated and flash chromatographed (60$
EtOAc/hexane) to give 23 mg of the desired compound (11) as a white solid.
~H-NMR (S, CDZC1=): 6.44 a.nd 5.99 (d, 2H), 5.27 (br-d, 1H), 4.95 (br-d, 209'99"?
1H), 4.35 (m, 1H), 4.15 (m, 1H), 0.92 (d, 3H), 0.81 (m, 2H), 0.53 (s, 3H), 0.34 (m, 8H).
'3C-NMR (S, CDZC12): 148.6, 143.5, 133.9, 125.1, 117.6, 111.8, 71.2, 71.0, 67.2, 57.2, 56.8, 45.8, 43.5, 43.4, 41.0, 37.1, 36.6, 29.4, 28.0, 24.0, 22.6, 20.8, 19.0, 12.1, C1.8, -0.5.
In a corresponding manner the following vitamin D compounds are prepared:
general foraula ~y off Ry compound no. RZ R3 R4 ( 12 ) CH3 ( CFiz C ( CHs ) 3 ) 3 (13) CH3 (CHz)3 CH(CH3)~
(14) CzHS (CH,)3 CH(CH3)=
(15) C.,HS (CH~)3 cyclopropyl ( 16 ) CH3 ( CH~ ) cyc lopropyl a ( 17 ) CH3 ( CH~ ) CH ( CH3 4 ) 2 Compd. (12) is prepared by using t-butyllithium instead of cyclopropyllithium.
Compounds (13), (7.4) and (17) are prepared by using isopropyllithium instead of cyclopropyllit:hium.
Compd. (12): 'H-NMR (5, CDC:Ij): 6.38 and 6.01 (d, 2H), 5.33 (m, 1H), 5.00 (m, 1H), 4.43 (m, 1H), 4.23 (m, 1H), 1.00 (s, 18H), 0.93 (d, 3H), 0.54 ( s, 3H) . "C-NMR ( ~~, CDC13) : 148.7, 143.4, 134.0, 125.0, 117.6, 111.8, 80.0, 71.1, 67.1, fi0.6, 5fi.9, 56.8, 46.3, 45.8, 43.4, 42.8, 40.9, 37.0, 209'99?' 36.5, 34.2, 29.4, :?8.0, 24.0, 23.3, 22.7, 19.2, 28.8, 12.1.
Compel. (13): (5, C:D30D): 0.55 (s, 3H), 0.90 2.22(dd, 'H-NM:R (m, 15H), 1H), 2.48 (dd, 2.83(dd, 1H), 4.09 (m, 1H), 4.31 4.86(b?, 1H), (t, 1H), 1H), 5.25 (b, 6.05(d, 1H), 6.29 (d, 1H).
1H), Compel. (14): (b, CDC13): 0.84 (t, 3H), 0.95 (m, 1.00(d, 'H-NNIR 12H), 3H), 2.32 (dd, 2.60(dd, 1H), 2.83 (dd, 1H), 4.24 4.44(m, 1H), (m, 1H), 1H), 5.01 (b, 5.33(b, 1H), 6.01 (d, 1H), 6.39 1H), (d, 1H).
Compel. (15): (5, 0.91(t, 'H-NMR CD3oD):
0.70-0.40 (m, 8H), 0.76 (m, 2H), 3H), 1.01 (d, 2.00(m, 1H), 2.22 (dd, 1H), 2.29 2.48(dd, 3H), (b, 1H), 1H), 2.83 (dd, 4.09 5.25(b, 1H), (m, 1H), 4.31 (t, 1H), 4.85 (b, 1H), 1H), 6.04 (d, 6.29(d, 1H).
1H), Compel . (16): 'H-NMR(b, 0.79(m, CCi30D):
0.20-0.40 (m, 8H), 0.57 (s, 3H), 2H), 0.95 (d, (dd, 1H), 2.51 (dd, 1H), 2.86 4.12(m, 3H), 2.25 (dd, 1H), 1H), 4.35 (t, 4.89(b, 1H), 5.28 (b, 1H), 6.08 6.32(d, 1H)" (d, 1H), 1H).
Compel. (17): 'H-NMR (S, C:D3oD): 0.53 (s, 3H), 0.90 (m, 12H), 2.22 (dd, 1H), 2.48 (dd, 1H), 2.83 (dd, 1H), 4.09 (m, 1H), 4.31 (t, 1H), 4.86 (b, 1H), 5.25 (b, 1H), 6.05 (d, 1H), 6.29 (d, 1H).
Example III
Preparation of 1-(1-methyl-5-hvdroxv-5 5-diisopropvl-pentvll-hvdrindanol-4 (19) Reaction equation:
2 5 ~. ~, ' ~z Cz.NS
a., - - c3H v J p~
35 (a). Starting compound (1) is converted to the corresponding iodide (2) as described in Example I(a).
(b). The compound (2) obtained is converted in a corresponding manner as described in Example I(d), using ethyl acrylate as the olefin, to produce ester compound (:18).
(c ) . Ester compound ( 18 ) is converted to compound ( 19 ) by a reaction with an excess of isopropyllithium, in a corresponding manner as described in Example II. The product is identified by 'H-NMR.
In a corresponding manner the following compounds are prepared:
general formula R
to ~ joy Ry is off compound no. R~ R3 R4 ( 20 ) CH == CH~ ( CHZ ) 3 cyc lopropyl ( 21 ) CH == CH, ( CHZ ) 3 isopropyl (22) CEi3 (CH2)4 isopropyl 20 (23) CEi3 (CHZ), cyclopropyl The products are i_dentifi_ed by 'H-NMR.
Example IV
Preparation of 1-(1-methyl-5-hydroxy-5 5-diisopropyl-oentyll-25 hvdrindanone-4 (2~1 Reaction equation Oxidation of compound (19) by using pyridinium dichromate as the oxidant, in a corresponding manner as described in Example I(b), affords the desired ketone~ (24) in a yield of 84~. The product is identified by ' H-NMR .
In a corresponding manner the following ketones are prepared:
general forrula ~3~
C
I woy ~y compound no. RZ R3 R4 (25) CH -= CHI (CHz)3 cyclopropyl ( 2 6 ) CH - CHI ( CHz ) 3 i sopropyl (27) CH3 (CH,)4 isopropyl (28) Cli3 (CHz)4 cyclopropyl The products are :identif.ied by ~H-NMR.
Example V
Prevaration of vii:amin D compound (131 from ketone (241 reaction equation:
- ''~~.
~'oTMS
,a ~z y~ --2 5 f~ ~Q ~ OT~
,;, ~
0 'z '~~ ~07 ~d~
i. zed.
z, r'so~ne r.
3. d~ptot~
H o ,,,...
~H
TBSO~''~~~ arss %$s0'"1 ~'oTr~s 2~~"~99'?
(a). The free hydroxy group is protected by a reaction with trimethylsilyltriflate (~TBS-triflate) in the presence of triethylamine and in methylene <:hloride as the solvent; temp. -78°C -- 0°C;
yield of compound (29) is .30%.
(b) The enolisation is carried out in a corresponding manner as described in Example I(c), producing compound (30) in a yield of 71%.
(c). In a corree3ponding manner as described in Example I(e), the coupling reaction with enyne (6) is performed, affording compound (31) in a yield of 94%.
(d). The final reaction step is carried out in a corresponding manner as described in Example I(f), followed by deprotection (desilylation), as described in Example II,, with tetrabutyl a.mmoniumfluoride. The final vitamin D compound (13) is obtained in an overall yield of 65%. The product is identical with the product obtained according to Example II.
In a corresponding manner the following vitamin D compounds are prepared.
general formula R3~C~~y ~ ~ off Ry ,, H 0 \h, v h.
compound no. R, R3 R4 ( 32 ) CH :- CH= ( CH,) 3 cyclopropyl (33) CH =- CHz (CHz)3 isopropyl ( 17 ) CH3 ( CH~ ) 4 isopropyl ( 16 ) Cfi3 ( CHz ) 4 cyc lopropyl The products are identified by 'H-NMR. The last two vitamin D compounds are identical with the corresponding vitamin D compounds prepared according to Example II.
_. ~09"T99'7 Example VI
Preparation of 19-nor-vitamin D compound (351 from ketone (291 Reaction equation:
r rMS
\ ~..
i s s o' o TB s ~~ ~3 ~J
x. dcpzGE.
,,,.
Hog, ~~ c~~~
A solution of 1.14 g (2 nvnol) of phosphine oxide (34) in 15 ml of dry THF is cooled to -78°C. n-Buthyllithium (BuLi), as a 2.5 M
solution in hexane, is added d~copwise until the red colour persists. Then 0.8 ml of a 2.5 M solution of BuLi is added. Stirring is continued for 15 min, followed by the dropwise addition of 0.73 g (1.8 mmol) of ketone (29) in 5 ml THF. After another hour of stirring, the reaction mixture is allowed to reach 0°C and then quenched by the addition of 50 ml of a saturated NH,C1-solution. lExtractive work-up and flash chromatography (2$
EtOAc in hexane) then affords the protected diene compound. Desilylation by reaction with 10 eq. of tetrabutylammonium fluoride (TBAF.3aq) in THF
(10 ml) during 48 hours gives compound (35), which is purified by flash chromatography using EtOAc as an eluent, followed by recrystallization from MeOH/EtOAc. The overall yield is 53$. Identification by 'H-NMR.
In a corresponding manner the following vitamin D compounds are prepared:
general formula ~3~. ~ ~ ay ~ ~ off Ry y°\ UH
209'7~9'~
compound RZ R3 R4 A B
no .
(36) CH := (CHZ)3 isopropyl H H
( 3'1 ) CH := ( cyc H H
CHz CHZ lopropyl ) ( 38 ) CFizOH ( isopropyl H H
CHI
) The products are i_dentif~Led by 'H-NMR.
Example VII
Affinity to intracellular vitamin D receptor Vitamin D compounds according to the invention are dissolved in ethanol in concentrations ranging from 10-'3 to 10-' M. The affinity towards the calf thymus intra<:ellular vitamin D receptor (VDR) is determined in a biological assay. In this: assay, 3H-1a,25-dihydroxycholecalciferol (3H
1a,25-DHCC), which is specifically bound to the VDR, is replaced by the tested compounds. Especially the tested compounds 11, 13 and 14 have a very high VDR-ai:finity.. A high VDR-affinity is indicative for biologically active substances.
Example VIII
Affinity to vitamin D binding protein Vitamin D binding protein (DBP) is the specific carrier for vitamin D
and its metabolites in :blood. The biological activity of vitamin D
compounds depends nn the it binding to DBP, because strong binding to DBP
will reduce the intracellular access to the VDR. Binding to the DBP may also influence t:he ha7Lf-life of the vitamin D derivatives in circulation. Weak binders are rapidly metabolized, which is a favourable aspect in topical application.
In the assay, DBP :s incubated with 3H-1a,25-DHCC and 1a,25-DHCC or with several vitamin D <:ompounds according to the invention. To this purpose, the vitamin compounds are dissolved in ethanol in concentrations ranging from 10'x' to 2. 5 x 10'6 M. The percentage bound/unbound 'H-la, 25-DHCC is then calculated. DBP is purified from total human serum. The results are shown in the appended Figures 1 and 2. Figures 1 and 2 show the binding of vitamin D compounds to human vitamin D binding protein.
['H]1a,25(OH)~D~ _ '13-1a,25-DHCC; in both Figures ~ - 1a,25-DHCC (known compound); in Fig. 1 ~ = compound 13 and D - compound 14; in Fig.
2 Q = compound 11.
_.. 20~'~00:
Compounds 14 and 11 bind rather weakly to the DBP, compared to the known 1x,25-DHCC. Compound 13 is a very weak binder.
Example IX
Cell differentiation Vitamin D compoundls according to the invention are dissolved in ethanol in concentrations ranging from 10-'2 to 10-6 M and tested for their capacity to induce cell differentiation in a HL-60 assay. In this assay, morphologic and biochemical examination of the human leukemic cell line HL-60 is done, in order to establish whether cell differentiation has taken place.
Differentiation is expressed as the maturation parameters nitroblue tetrazolium (NBT;~ reduction, non-specific esterase, and as the percentage of mature cells beyond the myelocyte stage which is visible after staining with May-Grunwald Giemsa. After culturing with the known 1a,25-DHCC or with vitamin D compounds of the invention, the percentage of cells containing black formazan deposits is determined. An increase in the percentage of NBT reducing cells indicates an increase in cell differentiation.
Proliferation and vitality of the cell cultures are established by counting the number of cells and by the trypan blue exclusion method.
The vitality and proliferation of the cells in the HL-60 cultures are good in all conditions tested. 1a,25-DHCC (known), compound 12, compound 11, compound 13 and compound 14 all induce differentiation and maturation of the HL-60 cells. In the cytological test (non-specific esterase and May-Grunwal~d Giemsa) especially compounds 13 and 11 are good differentiators. The optimum effect is found at concentrations in the range of 10-e to 10-' M.
The NBT-reduction inducing capacity of compounds 13 and 14 is about 10 x stronger than that of 'the known 1a,25-DHCC. Compounds 11 and 12 are about 5 x more potent in inducing NBT-reduction than 1a,25-DHCC (Figures 3 and 4).
The above implies that the tested new vitamin D compounds of the invention display a higher cell differentiating activity than the known 1a,25-DHCC.
zos~s9~
Figures 3 and 4 (appended) show the differentiating effect of the tested vitamin D compounds on human leukemia cells of the HL-60 line. In both Figures ~ = 1a,25-DHCC; in Fig. 3 O is compound 13 and O is com:..pound 14; in Fig. 4 d =- compound 11 and D = compound 12.
Example X
Calciotropic effects The most well-known efect of la, 25-DHCC is its action on the calcium metabolism, the ca.lciotropic effect. Calciotropic target organs are the intestine, the bone and t:he kidney.
The vitamin D compounds according to the invention are dissolved in ethanol and tested in the so-called Caco-2 assay for intestinal calcium (Ca) transport. In this ~3ssay, the vitamin D - induced influx of 'SCaZ+
is measured in monolayers of the intestinal cancer cell line Caco-2.
This influx is corrected for the concentration-driven Ca'-+ influx and is a measure for the Ca transport across the intestinal wall. The Caco-2 cells are known to have vitamin D receptors.
Increased intestinal calcium transport can be the first step leading to a rise in blood calcium levels (and eventually to hypercalcemia).
In Table A below 'the effects of vitamin D compounds of the invention, compared with the known 1a,25-DHCC, on the Ca'+ influx in intestinal Caco-2 cell cultures are presented. The values in the table represent the relative increase in Ca=+ influx tthe va7uP fnr ~n_~S-nu~r ;
arbitrarily fixed at 100).
The results in Table A demonstrate that compound 11, compound 13 and compound 14 are we.3ker at:Lmulators of intestinal calcium absorption than 1a,25-DHCC.
Table A
experiment 1:
compound 7.0-9 M
1a,25-DHCC 100 compound 11 79 compound 12 99 209"997 experiment 2:
compound 1.0-9 M
1a,25-DHCC 100 compound 13 65 compound 14 78 Example XI
Calciotropic effect Together with the :intestine and the bone, the kidney is one of the major target organs of 1a,25-D1HCC. The kidney plays an extremely important role in calcium hc~meosta:ois, since about 98~ of the calcium has to be reabsorbed in the kidneys in order to prevent calcium loss and hypocalcemia.
The vitamin D compounds according to the invention are dissolved in ethanol and tested in the rabbit kidney cell assay. In this assay, reabsorption of 'SCa'' is measured in monolayers of rabbit kidney cells.
The cells are isolated by immunodissection of connecting tubules with the aid of monoclonal antibodies.
In Table B below the effE~cts of vitamin D compounds of the invention, compared with the known 1.x,25-DHCC, on the Ca'+-reabsorption in rabbit renal cell cultures are presented. The values in the Table represent the increase in Ca=+-reabsorpt.ion in nmol/cm~/h.
The results in Table B demonstrate that compound 13 and compound 11 are weaker stimulators of renal Ca-reabsorption than compound 12 and 1a,25-DHCC itself.
Table B
compound ~D-9 M
1a,25-DHCC ~~----13.4 compound 11 2.4 compound 12 12.8 compound 13 ~-3,7 Example XII
Cell differentiation versus calciotropic effect 209'?99'7 One of the set-bac:ks of t:he highly active vitamin D compounds, such as the well-known 1a,25-DHCC, is its calciotropic effect, which may lead to toxic hypercalciuria, hypercalcemia and urolithiasis. Therefore, it should be very advantageous to develop compounds with a high selectivity of biological action. In other words, compounds in which the ratio between the induction of cell differentiation and calciotropic effects, e.g. the stimulation of intestinal Ca transport, is changed compared to 1a,25-DHCC.
The ratio between differentiation-inducing capacity and the stimulation of intestinal Ca transport is defined as the fraction between the concentration at which 50~ NBT reduction in HL-60 cells is obtained, and the concentration at which a half-maximal increase in intestinal Caz+
transport is reached. The smaller the ratio, the higher the relative cell differentiating capacity.
The results are presentecl in Table C.
Table C
compound fraction ~ ratio relative to DHCC
1a,25-DHCC 107 ~ 1.00 compound 12 29,7 0.2g compound 11 14.4 0.13 compound 14 2.6 0.02 compound 13 2.5 0.02 Table C shows, that the tested new vitamin D compounds of the invention have better rela;.ive cell differentiating properties than the known 1a,25-DHCC. Compound 14 and compound 13 have 50 x more selective actions than 1a,25-DHCC. C:ompouncl 11 has a 8 x better ratio. Compound 12 has a 4 x better ratio. This makes the new vitamin D compounds of the present invention extremely suitable for applications where the differentiation of cells (such as in hyperproliferative conditions) is desired.
Claims (30)
1. A vitamin D compound of the general formula (I):
wherein:
R2 is a (C1-C3) alkyl group, a hydroxy (C1-C3) alkyl group, a (C1-C2) alkoxymethyl group or a (C2-C3) alkenyl or alkynyl group;
R3 is a branched or non-branched, saturated or unsaturated aliphatic 3- to 5-membered hydrocarbon or oxahydrocarbon biradical having at least 3 atoms in the main chain and being optionally substituted with one or more substituents selected from epoxy, fluoro and hydroxy;
iPr is an isopropyl group: and A and B are each individually hydrogen atoms or methyl groups; or A and B form together a methylene group.
wherein:
R2 is a (C1-C3) alkyl group, a hydroxy (C1-C3) alkyl group, a (C1-C2) alkoxymethyl group or a (C2-C3) alkenyl or alkynyl group;
R3 is a branched or non-branched, saturated or unsaturated aliphatic 3- to 5-membered hydrocarbon or oxahydrocarbon biradical having at least 3 atoms in the main chain and being optionally substituted with one or more substituents selected from epoxy, fluoro and hydroxy;
iPr is an isopropyl group: and A and B are each individually hydrogen atoms or methyl groups; or A and B form together a methylene group.
2. A vitamin D compound as claimed in claim 1, wherein:
R2 is as defined in claim 1;
R3 is a biradical of the formula:
-0-CH2- (CH2)n-, r -CH2-CH2-(CH2)n-, -CH=CH-(CH2)n- or -CH2-CH2-CH(CH3)-, wherein n is 1 or 2; and A and B are hydrogen atoms or form together a methylene group.
R2 is as defined in claim 1;
R3 is a biradical of the formula:
-0-CH2- (CH2)n-, r -CH2-CH2-(CH2)n-, -CH=CH-(CH2)n- or -CH2-CH2-CH(CH3)-, wherein n is 1 or 2; and A and B are hydrogen atoms or form together a methylene group.
3. A vitamin D compound as claimed in claim 1, wherein:
R2 is a methyl, ethyl or vinyl group;
R3 is a trimethylene or tetramethylene group; and A and B are hydrogen atoms or form together a methylene group.
R2 is a methyl, ethyl or vinyl group;
R3 is a trimethylene or tetramethylene group; and A and B are hydrogen atoms or form together a methylene group.
4. A method of preparing a vitamin D compound as claimed in claim 1, comprising reacting an ester compound of the general formula (IX):
wherein:
R2, R3, A and B are as defined in claim 1, R1 is a protected hydroxy group, and R4 is a (C1-C6) alkyl group;
with an organometallic compound of the general formula (III):
iPrM(X)P (III) wherein:
iPr is an isopropyl group, X is Cl, Br or I, M is a metal selected from Li and Mg, and p is, dependent on the valence of M, 0 or 1;
followed by deprotection.
wherein:
R2, R3, A and B are as defined in claim 1, R1 is a protected hydroxy group, and R4 is a (C1-C6) alkyl group;
with an organometallic compound of the general formula (III):
iPrM(X)P (III) wherein:
iPr is an isopropyl group, X is Cl, Br or I, M is a metal selected from Li and Mg, and p is, dependent on the valence of M, 0 or 1;
followed by deprotection.
5. A method of preparing a vitamin D compound as claimed in claim 1, comprising reacting an ester compound of the general formula (II):
wherein:
R2 and R3 are as defined in claim 1, R5 is an optionally protected hydroxy group, and R4 is as defined in claim 4;
with an organometallic compound of the general formula (III) as defined in claim 4;
after which the hydrindane compound obtained, having the general formula (IV):
wherein R2, R3 and iPr are as defined in claim 1, and R5 is an optionally protected hydroxy group, is deprotected, if R5 is a protected hydroxy group, and then oxidized to the corresponding hydrindane-4-one compound of the general formula (V):
wherein R2, R3 and iPr are as defined in claim 1, which compound of general formula (V), optionally after protection of the hydroxy group, is then converted either (a) with a Wittig reagent of the general formula (VI):
wherein:
R1 is a protected hydroxy group; and A and B are as defined in claim 1; or (b) after enolization and derivatization of the enolic hydroxy group, with an enyne compound of the general formula (VII):
wherein R1 is as defined above, followed by hydrogenation and isomerization, to produce a compound of the general formula (I), wherein A and B form together a methylene group:
followed by deprotection.
wherein:
R2 and R3 are as defined in claim 1, R5 is an optionally protected hydroxy group, and R4 is as defined in claim 4;
with an organometallic compound of the general formula (III) as defined in claim 4;
after which the hydrindane compound obtained, having the general formula (IV):
wherein R2, R3 and iPr are as defined in claim 1, and R5 is an optionally protected hydroxy group, is deprotected, if R5 is a protected hydroxy group, and then oxidized to the corresponding hydrindane-4-one compound of the general formula (V):
wherein R2, R3 and iPr are as defined in claim 1, which compound of general formula (V), optionally after protection of the hydroxy group, is then converted either (a) with a Wittig reagent of the general formula (VI):
wherein:
R1 is a protected hydroxy group; and A and B are as defined in claim 1; or (b) after enolization and derivatization of the enolic hydroxy group, with an enyne compound of the general formula (VII):
wherein R1 is as defined above, followed by hydrogenation and isomerization, to produce a compound of the general formula (I), wherein A and B form together a methylene group:
followed by deprotection.
6. A hydrindane-4-one compound of the general formula (V), as defined in claim 5.
7. A method of preparing a hydrindane-4-one compound of the general formula (V), as defined in claim 6, wherein a hydrindane compound of the general formula (IV), as defined in claim 5, wherein R5 is a hydroxy group, is oxidized with an oxidizing agent.
8. A method as claimed in claim 7, wherein the oxidizing agent is a chromium-containing oxidant or ruthenium tetroxide.
9. A hydrindane compound of the general formula (IV), as defined in claim 5.
10. A method of preparing a hydrindane compound of the general formula (IV), as defined in claim 9, comprising reacting a compound of the general formula (II), as defined in claim 5, with an organometallic compound of the general formula (III), as defined in claim 5, in an inert organic solvent.
11. A pharmaceutical composition comprising, in addition to a pharmaceutically acceptable carrier and/or at least one pharmaceutically acceptable auxiliary substance, as the active ingredient at least one compound as defined in claim 1, 2 or 3.
12. A composition as claimed in claim 11, in a dosage unit form, for oral, topical or parenteral administration, comprising about 0.1 µg to about 0.1 mg active ingredient per dosage unit.
13. A composition as claimed in claim 12, for dermal administration.
14. A composition for diagnostic purposes comprising, in addition to a compatible, non-toxic carrier and/or at least one auxiliary substance, as the active ingredient at least one compound as defined in claim 1, 2 or 3 in a diagnostically effective amount.
15. A cosmetical composition comprising in addition to a cosmetically acceptable, non-toxic carrier and/or at least one auxiliary substance, as the active ingredient at least one compound as defined in claim 1, 2 or 3 in a cosmetically effective amount.
16. A cosmetical composition according to claim 15, selected from the group consisting of creams, lipocreams, lotions, ointments, liposomes and gels.
17. Use of a compound according to claim 1, 2 or 3, or a composition according to claim 11, 12 or 13 for preparing a medicament for the treatment or prophylaxis of a disease state selected from the group consisting of an autoimmune disease, acne, alopecia, skin aging, an imbalance in the immune system, an inflammatory disease and a disease related to abnormal cell differentiation and/or proliferation.
18. Use according to claim 17, wherein the inflammatory disease is rheumatoid arthritis or asthma.
19. Use according to claim 17, wherein the disease related to abnormal cell differentiation and/or proliferation is psoriasis or other hyperproliferative skin disease.
20. Use of a compound according to claim 1, 2 or 3, or a composition according to claim 11, 12 or 13 for preparing a medicament for the treatment or prophylaxis of a solid, skin or blood cancer.
21. Use according to claim 20, wherein the cancer is breast cancer, melanoma, squamous cell carcinoma or leukemia.
22. Use of a compound according to claim 1, 2 or 3, or a composition according to claim 15 or 16, for preparing a topical medicament for the treatment or prophylaxis of inadequate skin firmness or texture, insufficient skin hydration, wrinkles and/or insufficient sebum secretion.
23. Use of a compound according to claim 1, 2 or 3, or a composition according to claim 11, 12 or 13, for the treatment or prophylaxis of a disease state selected from the group consisting of an autoimmune disease, acne, alopecia, skin aging, an imbalance in the immune system, an inflammatory disease and a disease related to abnormal cell differentiation and/or proliferation in a warm-blooded animal.
24. Use according to claim 23, wherein the inflammatory disease is rheumatoid arthritis or asthma.
25. Use according to claim 23, wherein the disease related to abnormal cell differentiation and/or proliferation is psoriasis or other hyperproliferative skin disease.
26. Use of a compound according to claim 1, 2 or 3, or a composition according to claim 11, 12 or 13, for the treatment or prophylaxis of a solid, skin or blood cancer in a warm-blooded animal.
27. Use according to claim 26, wherein the cancer is breast cancer, melanoma, squamous cell carcinoma or leukemia.
28. Use of a compound according to claim 1, 2 or 3, or a composition according to claim 15 or 16, for the topical treatment or prophylaxis of inadequate skin firmness or texture, insufficient skin hydration, wrinkles and/or insufficient sebum secretion in a warm-blooded animal.
29. A commercial package comprising a compound according to claim 1, 2 or 3, or a composition according to claim 11, 12 or 13, and associated therewith instructions for use thereof in the treatment or prophylaxis of a disease as defined in any one of claims 23 to 27.
30. A commercial package comprising a compound according to claim 1, 2 or 3, or a composition according to claim 15 or 16, and associated therewith instructions for use thereof in the topical treatment or prophylaxis of inadequate skin firmness or texture, insufficient skin hydration, wrinkles and/or insufficient sebum secretion in a warm-blooded animal.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002097997A CA2097997C (en) | 1993-06-08 | 1993-06-08 | Vitamin d compounds and method of preparing these compounds |
| JP18547893A JP3633639B2 (en) | 1993-06-08 | 1993-06-29 | Vitamin D compounds and methods for producing them |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002097997A CA2097997C (en) | 1993-06-08 | 1993-06-08 | Vitamin d compounds and method of preparing these compounds |
| JP18547893A JP3633639B2 (en) | 1993-06-08 | 1993-06-29 | Vitamin D compounds and methods for producing them |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2097997A1 CA2097997A1 (en) | 1994-12-09 |
| CA2097997C true CA2097997C (en) | 2006-01-10 |
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ID=25676261
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002097997A Expired - Fee Related CA2097997C (en) | 1993-06-08 | 1993-06-08 | Vitamin d compounds and method of preparing these compounds |
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| Country | Link |
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| JP (1) | JP3633639B2 (en) |
| CA (1) | CA2097997C (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100596595B1 (en) | 2000-04-04 | 2006-07-06 | 칼라 액세스, 인크. | Composition for Improving Skin Lipid Barrier Function |
-
1993
- 1993-06-08 CA CA002097997A patent/CA2097997C/en not_active Expired - Fee Related
- 1993-06-29 JP JP18547893A patent/JP3633639B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP3633639B2 (en) | 2005-03-30 |
| JPH0717947A (en) | 1995-01-20 |
| CA2097997A1 (en) | 1994-12-09 |
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