CA2110158A1 - Vitamin d derivatives comprising a lateral chain expoxide group - Google Patents

Abstract

(57) ?tract Compounds derived from vitamin D or one of its analogs having, in particular, a lateral chain grafted in position 17 on the D ring of vitamin D. Said compounds are characterized in that they include an epoxide group grafted on said lateral chain, the epoxide group being separated in position 17 by a linear chain of at least two atoms. The invention also concerns a process for the preparation of said compounds and their use as drugs.

Description

e ~
FlLE, ~i~ THIS A~ED 21~ 91 d 7~T TRANS~ATION

VITAMIN D DERIVATIVES COMPRISING A
LATERAL CHAIN EPOXIDE GROUP

The present invention relates to novel derivatives of vitamin D, a process for the preparation of these derivatives, and tlleir therapeutic applications.
Vitamin D represents one of the most important 5 biological regulators for the metabolism of calcium, mineral and bone phosphorus. In association with two peptidal hormones, calcitonin and parathyroid hormone (PDH), vitamin D
is responsible for the control and maintenance of mineral and bone homeostasis.
Today vitamin D iS considered as a hormone, partly steroidal in nature and, more precisely, as a prohormone that after successive metabolisation between, amongst others, the liver and the kidneys, gives birth to a biologically active compound, 1-alpha,25-dihyd~oxy-vitamin D. This latter hormone 15 is the major element of a complex endocrinal system. It plays a role, not only in phospho-calcium metabolism, by acting on the classic target tissues (the intestines, bones, kidneys and parathyroid), but also in the control of important cellular processes (growth, differentiation and divers 20 biological functions) in a large number of other normal or cancerous tissues (1-3).
The general term vitamin D applies at the same time, to both vitamin D2 (ergocalciferol) oE vegetable origin and to vitamin D3 (cholecalciferol) that occurs naturally in animals.
25 In man, these two forms of vitamin D have a very similar biological role. It is therefore of little significance to ~, consider them separately. The D vitamins are defined as seco-steroids, that is molecules, chemically characterized by a steroid nucleus of 4 rings, of which the s ring is open at 30 the C-9 and C-10 carbons.
In the present case we therefore understand by vitamin D, vitamin D3 as well as vitamin D2.
In man the main source of vitamin D is endogenous and in the form of vitamin D3. Vitamin D3 is produced starting 35 from 7-dehydrocholesterol, a precursor synthesized in the

2~ 3 ~ 2 epidermis, under the influence oE ultra-violet rays o-E sun light. Insufficient solar exposure can be compensated by an exogenous supply of the vitamins D2 and D3.

5 STRUCTURE OF VITAMIN D:

~7~ 6 65~17 6~5 ~ o~
~a~

20Vitamin D3 Vitamin D2 cholecalciferol Vitamin D is subjected to an initial hydroxylation at position 25, its first metabolic transformation, generating 25 25-hydroxy-vitamin D ~hereafter called 25-(OH)D), one of the main forms of circulating vitamin D. This metabolite has a high affinity ~or a plasma protein carrier, designated by DBP
("vitamin D binding protein"), but a weak affinity for the intracellular receptor of vitamin D. After hydroxylation, in 30 the kidneys, at the 1-alpha position, 25-hydroxy-vitamin D is transformed into 1-alpha,25-dihydroxy-vitamin D, abbriviated ~ ;
to 1-alpha,25-(OH)2D3, which is the biologically active form of vitamin D. This metabolite has a weak affinity for DBP and a strong affinity for the intracellular receptor. Its level 35 in the blood is regulated within very narrow limits.
The metabolism of vitamin D produces a series of other metabolites of which the physiological role, if it exists, is still unclear. 1-alpha-hydroxylase, the enzyme responsible for activation of the 25-(O~i)D, is also present in the 2'~ 8 placenta, the intes-tines, the breasts, the epidermis (keratinocytes), the bone cells (embryonic and normal), certain haematopoie-tic-cells (macrophages and monocy-tes) as well as granulomatic tissue, such as sarcomas, during 5 granulomatic illnesses.
The main pharmacological role of vitamin D, is the ~ regulation of calcium and phosphorus metabolism in classic targeted tissues. It stimulates intestinal and renal absorption of calcium and phosphorus, mobilization in the 10 bones of these same minerals (resorption or mineralisation), as well, as regulating the secretion of parathyroid hormone, which controls the renal 1-alpha-hydroxylase. Thus an excessive production of vi-tamin D (accompanied by hypercalcemia, hypercalciuri and their consequences) is 15 observed in -the case of hyperparathyroid patients.
The vitamin D receptor has been identified in many normal tissues, such as skin, muscles, the pancreas, the brain, the thyroid, the placenta, the colon, bone (osteoblasts), the thymus, and immune system cells 20 (monocytes, macrophages, and activated B and T lymphocytes).
A large number of cancér cells from all origins possess vitamin D receptors; for instance leukocites, of monocytic (myeloid) or lymphocytic (lymphome) origin, melanomas and carcinomas of the colon, bone and breast. In these tissues, 25 vitamin D plays an important role in the control of cell proliferati.on and differentiation and in the regulation and modulation of the immune system, in the same way as oestrogens and glucocorticoids and, more recently, vitamin A.
It regulates the growth and differentiation of normal cells -30 (embryogenesis) and cancerous cells (stopping proliferation and/or induction cellular differentiation). It also acts as an immuno-modulatery substance (for example stimulation of non-specific immunity by monocytes, inhibition of specifically lymphocitic immunity).
-Vitamin D plays a fundamental role in many natural biological processes. Every anomaly in the supply or the regulation of vitamin D metabolism expresses itself in the more or less important pathologies. Nowadays the pathologies 21~ a~s associated with vitamin D, are classed into those deficient or excessive in vitamin D.
Deficiences o~ shortages of vitamin D are due to insufficient exposure to the sun associated with a deficient 5 intake in food or, during malabsorptions syndrome, to anomalies in the metabolism of vitamin D, or the tissues being resistant to the action of vitamin D. Anomalies in metabolism are associated with the connected problems (a) with the production of 25-(OH)D (liver diseases, prolonged usage of anticonvulsants or corticoids);
(b) with the production of 1-alpha,25-(OH)2D3, by the absence or deficiency of hydroxylation at the 1-alpha position (as in the case of vitamin resistant rickets type I), or 15 (c) to its biological action by the absence of, or a deficiency in, the cellular receptors of vitamin D (in the case of vitamin resistant rickets of type II).
Vitamin resistant rickets is hereditary. The metabolism of vitamin D can also be disturbed in the course of different 20 pathologies, in particular family hypophosphatemic rickets (tubulary renal deficiency for the transport of phosphate), a chronic renal insufficiency (renal osteodystrophie), sugar diabetis (increase in the incidence of osteopeny, osteoporosis and fractures), hypoparathyroidism and certain 25 cancers (multiple myelomas), or by pharmacalogical treatments, especially with anti-epileptic medicaments and corticoids.
The clinical manifestations of vitamin D deficiences appear most clearly in the bones: rickets in children and 30 osteomalacia in adults, and perhaps participation in osteoporosis phenomena. Other not so obvious problems also exist, for example immune deficiency amongst children with rickets, a higher incidence of certain cancers, especially of the colon, associated with the deficiency in calcium and 35 vitamin D or vascular and endocrine problems.
Excesses in vitamin D occur essentially with vitamin D
intoxication or with ectopic production of the active metabolites, for example during the course of granulomatic problems (sarcoidose). The same problem occurs with S S ~ SS ~

~ 5 2 .
hyperparathyroidism, stimulating the excessive production of 1-alpha,25-(OH)zD3.
The usual vitamin D deEiciences are easily corrected by an intake of vitamin D. The active derivatives of vitamin D
5 and in particular the 1-alpha,25-(OH)2D3 should be administered, if the metabolism is abnormal, especially with ~ renal insufficiency. These derivatives give very little indication of their therapeutic effects, such that vitamin poisoning often occurs at the time of their administration.
10 The 1-alpha,25(OH)2D3 has on the other hand a short half life, which often justifies two daily doses. In addition, prolonged treatments are accompanied by hypercalcemia, hypercalciurie and their consequences, especially in liver and kidney.
Research of derivatives having a different pharmocokinetic 15 profile, and a more favourable therapeutic indication is therefore very topical.
The particular regulatory role of vitamin D, especially in cell proliferation and differentiation, can be usefully employed in order to control certain pathologies that do not 20 show up as a direct result of a problem concerning the vitamin D metabolism.

- 2 ~ 8 .

The development of novel vitamin D analogues opens up possibilities in the area of already known applications, such as secondary hyperparathyroidism (1-4), skin pathologies, amongst others psoriasis (7-12), dyskeratosis and acne, 5 osteoporosis, including postmenopausal osteoporosis, senile osteoporosis or osteoporosis due to an excess of corticoids (13, 14), or potential endocrinological applications, such as the regulation of insulin secretion of the pancreas, immunological applications, such as autoimmune diseases or 10 immunoregulation for example with transplanted organs (15, lS) or with cancerology, such as leukaemia, lymphomes, and myelodysplasic syndromes (17-19).
The biomedical potential of a vitamin D analogue will essentially depend on the possibility of distinguishing or 15 dissociating between the different biologically active forms, especially between the calcemic and cellular effect. -~
An object of the present invention is to provide certain modifications -to the lateral chain of vitamin D in such a way as to obtain such a distinction, notably 20 conserving a similar or even superior biological activity to that of the natural hormone, whilst reducing the calcemic effect particularly in bone tissue.
l-alpha,25-(OH)2D3 and its precursor, l-alpha-OH-D3 are utilized in the circumstances of renal insufficiency and 25 osteoporosis (13,14). Calcipotriol, or 22-ene-24-cyclopropyl-l-alpha,24-(OH)2D3 of Leo Pharmaceutica (11, 12), has also been proposed Eor the treatment of psoriasis. Research and development is also being carried out on other derivatives.
Their therapeutic use underlines the growing irlterest for 30 vitamin D analogues in human medicin.
If, at the moment, the obtained results with these Eirst synthetic analogues of vitamin D, mentioned above, are interesting and encouraging their therapeutic efficiency is at the same time still limited.
An object of the present invention is to provide new analogues of vitamin D, allowing the improvement of biological selectivity and the therapeutic potential of vitamin D analogues.

~` 7 211~1~8 More particularly, an object of the present invention is -to provide new analogues, permitting the different biological vitamin D activities in the target cells to be modula~ed or dissociated, and for example providing active 5 compounds to the regions of cellular differentiation, but less active compounds to the areas of phospho-calcium metabolism, allowing an increase in administratable doses and a broadening of the therapeutic index for the therapeutic directory concerned.
The present invention has therefore as object derivatives of vitamin D, or of one of its analogues, having a lateral chain grafted on to position 17 of the D cycle of vitamin D, said lateral chain comprising an epoxide group separated from position 17 by a linear chain of at least two 15 atoms, preferably two carbon atoms.
In particular, the compound corresponds to the formula:

~ X ~o 20 (I) ~ R2

3 :

25 In which ~.
- V represents a part oE vitamin D, or one of its :
analogues wherein position 17 is vacant; : .
- X, grafted onto postition 17 of V represents a hydrocarbon chain, in particular an alkyl chain, having 2 to 15 carbon atoms, possibly substituted or modified, may in particular , comprise at one or several positions, one or more of the -~ :~
group(s) o hydroxyl, methyl, ethyl, halogen, preferably fluorine, o ester, notably an R1COO group where Rl is Cl to C7 alkyl or alkoxy, linear or branched, saturated or unsaturated, possibly substituted notably by one of the group(s) hydroxy or halogen, preferably fluorine, 21101r8 :- 8 ether, notably an Rl-0 group, wherein Rl represents Cl to C7 alkyl or alkoxy, linear or branched, saturated or unsaturated, possibly substituted by one or more of the group(s) hydroxy or halogen, o oxo in ketone form, a ring or heteroring, notably cycloalkyl, saturated or ~ unsaturated, aromatic or heteroaromatic, which may carry all the groups and modifications mentioned hereabove and/or o said chain X may comprise in the place of one or more of the carbon atoms, an cxygen, nitrogen or sulphur atom, said chain X may be unsaturated, with one or more double or triple carbon carbon bonds, this unsaturated chain may carry all the groups and modifications mentioned hereabove and -~ : :
o said chain and the different groups situated on the chain may adopt all the isomeric forms, o the epoxide group R

k~fl2 may be grafted onto anyone of the 13 carbon atoms of X, :~
in particular at its end, the epoxide group, wherein e Rl, R2 and R3 may be identical or different, represent a hydrogen, a C1 to ClO alkyl group, linear or branched, saturated or unsaturated, possibly substituted with one or more of the group(s) halogen, hydroxy, or cycloalkyl, also possibly substituted with halogen or ~
hydroxy groups; . ~:
R1 and R2 may be able to make up part of a same ring, such as a carboring or a heteroring, and in the case of R3 ~:
having the same meaning as hereabove;
~ R2 and R3 may form part of the same ring, such as a carboring or heteroring, and in the case of R1 having the same meaning as hereabove;
o different isomeric forms of the epoxide being possible.

2~101~8 It is understood that the rest of vitamin D or the analogues of vitamin D (v) comprises a compound of which the base skeleton structure is, ll Ç 3 L~10 3 ~ I

and of which the complete formula may comprise substitutions at all positions other than position 17 of ring D. . ~
In particular, V may represent a part :.
~ of analogues possessing a derivation at Cll, that has been described by the applicant in EP341158, `~
- derivatives possessing a double bond between C,6-C17 described in U.S. 199167 and EP 050325, .
20 - compounds with a heteroring between C6 and ClO that has been described in EP 81793, EP 78704, - derivatives possessing divers derivations at C3 and C~ that have been described in W0 90/10620.
The compounds according to the invention correspond 25 most often to the formula R~ :
X~R2 (Ib) y~ ~y 1 wherein X, R1 to R3 have the same meaning as given above and ~ represent a single bond or a double bond;

2~ 10~
..... :-` 1 o - A represents a hydrogen, a methyl or a methylene group - CH2;
- Y identical of different represents H, OH, an ester group, notably R1O, or ether, notably Rlo, wherein Rl has the same meaning as given hereabove.
Among these, the compounds in which X represents an alkyl chain having 2 to 6 carbon atoms, wherein a carbon atom is eventually replaced by an oxygen atom are preferred, possibly substituted, no-tably by one or more of the groups 10 methyl, hydroxyl, ether, notably an R1O group and/or unsa- ~.
turated with a dsuble or triple carbon-carbon bond, the epoxy group being grafted to the end of the alkyl chain X, R1, R2 and R3, may be identical or different, representing hydrogen, a hydroxy, an alkyl group, notably a 15 methyl, ethyl or hydroxyalkyl.
In particular, the compounds that correspond to the : :
formula II

y~, R2 ,, Jl H

(II) qo OR' ~ .

may be cited and e~pecially four isomaric forms pure or as a 30 mixture :

l ~ l ~ ~
35 IIa H H IIb .,,.,~,...,.. .;.,. . ~. .. ",. . .. .. ;

-~ 11 2~10~

, ~Z~I R2 { ~ Z~ 2 IIc IId 10 in which Z represents an alkyl chain, saturated or unsaturated having 0 to 3 atoms of carbon, possibly substituted or modified as menti.oned for X hereinbefore, in particular substituted by an hydroxy, an alkyl, such as methyl or ethyl, at one or more carbon atoms, and R1 to R3 and 15 R1 having the same meanings as given hereabove, the epoxide group being grafted to the end of 7. ~-Among these latter figures the compounds according to the formula II, in particular IIa or IIb with ~ : :
F~ I R2 R3 CH3 H 2 2 ~ :
CH3 H H -CH2-cHOH-H H H -C(CH3)2- :

The compounds according to the invention may be prepared by procedures known by a skilled person.
The present invention has also as object a process for :~
the preparation of a compound according to the invention, characterised in that the anion obtained from a composition, 30 whose base skeletal structure has formula IV, . .

PPh2 6~

4~ l10 and n-butyllithium, ~ ~ 12 21101~

is made to react with a compound whose base skeletal structure has the formula III :~

~4 ~

( I II ) . . : ~
1 0 , . . .
wherein the reactive groups grafted onto the skeletons of III ~ ::
and IV are possibly protected and X, R1, R2, R3 of which reactive groups are possibly protected, ~: :
have the same meanings as given hereinbefore, subsequently 15 protection of the said reactive groups, notably X, Rl, R2 and R3, and positions 1 and 3 hydroxylated is removed.
This type of honding is described in the literature (2iB).
At the same time the synthesis of derivati.ves of :: : :
20 formula IV has been described (29).
In particular, the derivatives of formula lb, in which 21~ 01~8 -~ 13 .
Y represen-ts OH or ORI, may be obt~ined Erom Cf'Ph2 ;~ ~

S ` `^-- t ``~ ~

(IV)~ (llt ~ ~

: The compound of structure III may be obtained in several ways, notably those described in the scheme la hereafter.
- A first sequence comprises the following stages:
15 a) a compound of base structural skeleton of formula IV in which Tos is the tosylate group, R2 is hydrogen or an alcohol protecting group and X has the same meaning as given hereinbeore, with possibly the reactive groups notably hydroxy in a protected form, and W and Z' represent hydrocarbon chains which make up part of X, such as X represents .
W~Z', the chain X being made up of chains W and Z' assemblied at one of their ends, W and Z' having the same meanings as X, notably Z' may represent Z, of which if needed, the reactive groups, such as hydroxy are protected, is reacted with an organometallic derivative of formula VII in which Met is lithium or halogenomagnesium.
30 b) subsequently the olefine thus obtained is epoxidized, and c) after possible removal of protecting groups, from the derivative of which the base structural skeleton has as formula VIII, the hydroxy group is oxidized to a ketone.

A second se~uence comprises the following stages:
a) the compound of skeleton VI is reacted with an organometallic derivative of formula IX, followed by hydrolysis that liberates the ketone group, b) it is then reacted with sulphurylides, and 21101~
- ~` 14 c) after possible removal of protection from the derivative VIII, oxidation is carried out as hereinabove.
It is also possible to use one or more of the following sequences, wherein an organometallic derivative of formula X

5 is reacted with the tosylate XI or XII and to then proceed with the respective epoxidations, such as have been described - hereinbefore. This last way will be advantageous, since W
(and therefore X) is a hydrocarbonated chain, wherein one carbon atom is replaced by ox~gen, sulphur or nitrogen. In 10 this case the organometallic derivative X will be prepared, preferably from Xa, wherein the hydrogen marked H+ is bonded to the oxygen, sulphur or nitrogen, by deprotonation. If no-t X may be prepared from a halogen derivative Xb by transmetallation.
It is noted, that in place of a tosylate in the derivative (VI, XI and XII) described hereinbefore another leaving group, :Eor example iodide, may be used.
~ ' - 2llal~

Scheme la.

3~_ y_ 10 ~r ~13~_ ¢ ~ 0~~
15 Y~ `o l \ ~ O
t ~ ~
1 `3~
-~0 ~0 ¢ . ~ ~
Y~ _ ~ ~
~ ~$ o~

~oS

~~ 16 2 ~ .L a 1 i33 .
In VII, IX, XI and XII, the groups t and `
may b~ grafted onto any position of the Z' chain, notably at its end.
Alternative ways of preparation of the compounds will appear in the light of the following examples.
The present invention has also for its object, the use of compounds according to the invention, as medicaments.
The analogues of vitamin D according to the invention, are characterized by the presence of an epoxide group in the lateral chain, constituting the first group of modifications 20 of this type, of vitamin D for therapeutic objectives. The epoxide group favourably substitutes the hydroxyl group, characteristic of the active form of vitamin D and situated at position 25. In effect, up until the present invention, all the biologically active analogues of vitamin D possessed 25 at least one hydroxyl group in the lateral chain, notably at position 25. It has been discovered, according to the present invention, that the epoxide function, situated on the lateral chain of vitamin D, allows a biologically active analogue, without the obligatory presence of an hydroxyl group in the 30 lateral chain to be obtained. Moreover the epoxide group ~, confers particularly interesting biological properties onto the corresponding products, not possessing this group. It appears, in effect, that the epoxide group confers on itself `
a biological activity and that this activity is different to 35 that conferred by the hydroxy group.
For one part, the epoxide group confers on the 1-alpha-hydroxy-vitamin D, an inactive metabolite of vitamin D, whose biological activities are characteristics of the active form, 1-alpha,25-(OH)2D3. For example, the derivative " 17 21ia158 1-alpha hydro~y,25,26-epoxy-1-vi~amin D3 (compound 24) increases by more than 100 times the capacity of the 1-alpha-OHD3 to differentiate human leukaemia cells (HL-60).
For another part, the epoxide group confers a biolo-5 gical property not connected with the usual analogues ofvitamin D that possesses a hydroxyl group at position 25 of ~ the lateral chain.
Concerning cellular differentiation properties, the epoxide analogues act in the same general way as the active 10 metabolite of vitamin D. They appear just as, if not more, active than the 1-alpha,25-(OH)2D3.
Notably may he cited the differentiation of human leukaemia cells and epidermis cells (human keratinocytes) as well as the anti-proliferation effect on tumerous cells, for 15 example MCF-7.
As to that concerning the phospho-calcium metabolism, the calcemic effect of the analogues is considerably reduced (marginal activity) and is not existing in the model of chicken rickets. They are also much less apt to stimulate the 20 secretion of osteocalcines by cultured human, osteoblastic cells.
This diminuation of secondary effects of the hypercalcemic type, observed in case of the epoxide analogues, does not prevent the efficiency of the compounds 25 where osteoporosis is concerned. In effect, the therapeutic effect under these circumstances, independent of the calcemic effect, results in the eEfect of differentiation in bone cells.
Thus, the epoxide derivation allows the calcemic effect 30 to be well disassociated from other forms of biological ~, - activity. For example the epoxide analogues and notably the derivative 26a : (245, 25R)-1-alpha,24 dihydroxy-25,26-epoxy, vitamin D3, are capable of stimulating leukaemic or epidermic cell differentiation, without, however, inducing a calcemic 35 effect. This notable reduction in the calcemic effect confers these analogues certain advantages with respect to vitamin D
or its active metabolite, especially that of increasing the concentration of compound, which may be administered, without ~

- ' :

18 2~101S8 influencing the phosho-calcic me-tabolism and thus increasing the therapeutic index in numerous applications.
The present invention has therefore, al~o for its object, the application of compounds according to the 5 invention as medicaments.
More precisely, the present invention has for its ~ object the use of this medicament notably for treatment and/or prophylaxis, of:
- skin diseases, amongst others psoriasis, dyskeratosis, acne, alopecia and skin aging including the effects of solar rays and lights;
- secondary hyperparathyroidism;
- cancers including leukaemia, lymphomes, syndromes, myelodysplasic syndromes, melanomas, cancers of the breast, lung or colon and their metastases;
- autoimmune diseases, including endoscinopathies, such as diabetes "mellitus"; neurological pathologies such as mutiple sclerosis, and systemic diseases, such as erythematic lupus, glomerulon-phrites;
20 - inflammatory processes, such as articulatory rheumatism, arthritis and asthma;
- osteoporosis, comprising senile osteoporosis, which accompanies sugar diabetes or corticoid treatment;
- arterial hypertension; ~;
25 - the modulation of the immune system (suppression of the proliferation of lymphocytes, differentiation of monocytes etc.) used for treatment and prophylaxis in case of, for example, autoimmune diseases and inflammatory processes, such as those in case of organ transplantation.
In effect it is possible to use the compounds, according - to the invention, to prevent the rejection of grafts by using the compounds independently, or in association with other compounds, such as cyclosporine, FK 506 etc.;
- renal insufficiency.
Other characteristics and advantages o the invention will be apparent from the following description and examples.

: ::

21~0~

In the following examples, the derivatives of formula V, have been obtained notably following one of the general methods mentioned in the following scheme lb:
s O ',' o~
~: ~

~ \

D ~

~ O ~. " ~, ' r (~
~, ~ ~" ~ ~ ~ .
7~o ~ o ~ "~ t : ~

~O ~
., . ~ ( ~ :
_`"~,"~ ,_ ~ ,~, ~' ::;
~

:.: ~;: - , `` 20 2 ~ 8 Three stages may be distinguished:
(a) the introduction of a fragment of a lateral chain, containing a group, possibly protected, which allows introduction of an epoxide; introduction of the fragment takes place advantageously by substitution of a tosylate group in lc, which method is described in the literature (30);
(b) introduction of an epoxide, which may originate from an olefin of type A', by one of the conventional epoxidation methods, or from a ketone of type A'' by using, for example, y-lures in which case the introduction of a portion of the chain jointly gives the epoxide;
(c) after possibly deprotection of the hydroxyl group in the ring, the hydroxyl is oxidized to a ketone. It is to be noted, that, in general, the epoxide formed will be present in two isomeric forms, which may suitably be separated.
The isomeric forms at C-20 (see structure IIc, IId) are prepared in the same way, but from a tosylate, which is 20 obtained from a primary isomeric alcohol corresponding to lb.
The latter may be obtained from la by the following sequence:
(a) oxidation of the primary alcohol to la, for example by pyridinium hydrobromide perbromide;
(b) isomerisation of the aldehyde 2a with a base such as diazabicyclo-undecene;
(c) reduction of a mixture of the aldehydes 2a and 2b by sodium borohydrate and separation of the alcohols la and lb.
Aldehyde 2a can also be obtained by direct oxidation of 30 the tosylate lc in dimethylsulphoxide. ~ ;~ In the examples, and in the Scheme 2, that follows, the synthesis of V precursors of the derivatives of vitamin D3, according to the invention, is further explicated.
The epoxide 5, obtained in the form of a mixture of ~ ;35 diastereoisomeres 5a and 5b, is obtained from the tosylate ~`
lc. The latter is prepared from the alcohol la with tosyle chloride in pyridine. The alcohol la results from ozonolysis ~-~ followed by reduction, by sodium borohydrate, of vitamin D2, 21iOlj3 or the acetate of vitamin D2, according to a process described in the literature (31). The tosylate lc is then bonded.
The isomeric forms at C-20 (see structure IIc, IId) are obtained in the same way, but from the tosylate, obtained 5 from the primary isomeric alcohol, corresponding to lb. The latter may be obtained from la by the following sequence:
~ (a) oxidation of the primary alcohol to la, for example by pyridinium hydrobromide perbromide;
(b) isomerisation of the aldehyde 2a with a base, such as diazadicyclo-undecene;
(c) reduction of a mixture of the aldehydes 2a and 2b by sodium borohydryde and separation of the alcohols la and lb.
The aldehyde 2a may also be obtained by direct 15 oxidation of the tosylate lc in dimethylsulphoxyde.
In the examples and Scheme 2 hereafter, the synthesis of the precursors V from the derivatives of vitamin D3 according to the invention, is further explicated.
The epoxide 5, obtained in the form of a mixture of 20 diastereoisomers 5a and 5b, is obtained from the tosylate lc.
The latter is prepared from the alcohol la with tosyle chloride in pyridine. The alcohol la results from ozonolysis, followed by reduction by sodium borohydrate of vitamin D2 or the vitamin D2 acetate, according to a process described in 25 the literature (31). The tosylate lc is then coupled with the organomagnesium derivative of 4-chloro-2-methyl-1-butene in the presence o Li2CuC14 following the process described for this derivative in the literature ~32). The epoxidation of the olefin 3, thus obtained, is carried out in the 30 conventional manner by m-chloroperbenzoic acid in dichloromethane at ambient temperature. The mixture of the two epoxides 4a and 4b, thus obtained, is advantageously ;
oxidized by pyridine dichromate in dichloromethane.
The epoxide lla, possessing a supplementary function in 35 the lateral chain, may be prepared in a selective manner Erom the olefin 3. After protection of the secondary alcohol in the form of trimethylsilysilic ether, the position 24 in the lateral chain is oxidized by selenium dioxide in the presence of tertiary butyl hydroperoxide in dichloromethane. The -- 21~ 01 ~8 .,, mixture of the resulting alcohol diastereoisomers 7a and 7b is not separated at this stage.

"

1015~

,~1 ., .~. _ ~0 ~_o ~0 \~ o Z , ' o~

r~ D <~ L l ~ ~ 8, c ~ O ~ _ \ / j~ S ~
~ 0 ~

O ~ ~ T T S

~~ L ~ a ~ ~ W
~ D ~ o 0 1~ : ' .

24 2l l ~15~

L L ~ L ~
~ ~\y~ I I

~ T ~
~1 3 '~
o~D~

15 (~ r X~

--~ ~I ~ d 1 y ~ ~ ~ 8 Y, ~` ~ o ~ ~ ~ ~' X` ~ ~

~.~ S ~Q' S ~ æ

~ ''- 0 :
O ~
O ~ ¦ ~ C ~ O . t~ Z ' ~
3 0 ~ ~ ~) ~

o ~_0 ~ O

--~ 25 21101~
In effect, epoxidation according to the Sharpless method (33) allows in the present case by using, in the presence of ~ diisopropyl-L-tartrate from tertiary butylhydroperoxide and Ti(i-PrO)4, the selective 5 transformation of alcohol 7a to the epoxide 8a, leaving the alcohol 7b unchanged. The latter may eventually be used in an epoxidation, such as described for olefin 3. After deprotection of the secondary alcohol, the diol 9a is selectively oxidized to a hydroxyl group, attached to the 10 ring. Finally the hydroxyl group in the lateral chain of ketone 10a is protected due to its coupling possibilities in the form of trimethylsilysilic ether (lla).
In an analogous manner, the protected epoxy-ketone 15b, a stereoisomer of lla is prepared from the olefin 6. After 15 allylic oxidation, the mixture of alcohols 7a and 7b is deprotected. This time, Sharpless oxidation is carried out on the mixture of the diols 12a and 12b. The epoxidation is carried out selectively on 12b, in the presence of (-)-diisopropyl-D-tartrate, tertiary butylhydroperoxide and 20 Ti(IV)isopropoxide, leaving 12a unchanged. The epoxide 13b is then oxidized and the free hydroxy group on the ketone 14b is protected in the form of trimethylsilicilic ether, due to the coupling possibilities o 15b. The synthesis of the epoxides 18a, 18b, just as 18'a and 18'b, is also carried out starting ~ -2S from the tosylate lc. When the latter is treated with the Grignard derivative, obtained from isopentenylchloride in ~-~
tetrahydrofuran in the presence of Li2Cu(II)Cl4, a mixture of the expected olefin 16 and an isomeric olefin 16' is obtained.
The latter is not an important product, since in the described stage iodide is used as the leaving group in place of the tosylate. After classical epoxidation the mixture of epoxides is obtained, that may be separated. In this way the epoxidation of the secondary alcohol may be carried out for 35 one part on the derivatives 17a, 17b, and for the other part on the derivatives 17'a, 17'b. The ketones 18a, 18b and 18'a~
18'b may be used immediately in the bonding stage.
Finally, the acetylene 22a, 22b is obtained from the tosylate by substitution with the lithiated derivative 2 ~

obtained from the acetal of 3-butyn-2-one. After deprotection of the acetal at 19, the ketone is treated with a ylure, obtained from trimethylsulphonium iodite. The epoxide in the diostereomeric forms 21a, 21b is oxidized and the 5 corresponding ketones 22a, 22b may be bonded immediately.
The synthesis of the vitamin D3 derivatives II by bonding of the derivatives V with the anion of the derivative IV is carried out according to scheme 3:

2 7 2 1 1 0 1 ~ 8 . .

C~ ~c~

; ~ 10 ~ , ~ ' ~ @ ~:
::
/ . .

'¦ ~1 ., o~
. .
~ r 2 5 ~<> D ~, . ~; . , 7~;~r \~_0 ~ ~
N ~:
~C~ , ' , ' .

~T

28 21101r~

~ o n 1~ r~ o ~
C~ r ~
,, V~

~ ~ I ~ 1 ~ T E ~ X : . .
u~ ;~

~ 0~ o~

2 0 ¢ . T ~ ~ T I ~ T T r I r ~;

¦ t~ ~ T I T T j~ ~ ~ S T ~:

25~ ~ T = i T T ~

I / I

~ ~a .0 ~' ~

, 211~1~8 The bonding of ketones possessing the epoxide group in the lateral chain, such as 5a/5b and lla, with the anion of derivative IV leads to the pro-tected derivatives, such as 23a/23b and 25a,respectively. The elimination of protective 5 groups from the bonding derivatives leads finally to -the obtaining of the alcohols derived from formula II.
An example is the condensation of the lithium salt, obtained from phosphonate IV with n-butyllithium in THF at -78C with the mixture of diastereoisomeres 5a and 5b, 10 yielding a mixture of trienes 23a and 23b. Deprotection of the silicilic ether groups is advantageously carried out with tetrabutylammonium fluoride. In the case of the epoxide lla, the same bonding leads to 25a and, after deprotection, to the triol 26a.
In exactly the same way, the epoxide 15b leads via 27b to the triol 28b. The reaction of the diastereoisomers mixtures 18a and 18b, 18'a and 18'b, as well as 22a and 22b, leads, after bonding and deprotection, to mixtures of diols, 30a and 30b, 30'a and 30'b and 32a and 32b, respectively.
EXAMPLE 1: Synthesis of the epoxy-alcohols 4a, 4b To a solution of 3 (0.1.6 g, 0.61 mmoles) in anhydrous dichloromethane (7 ml) m-chloroperbenzoic acid (mCPBA, 0.235 25 g, 1.36 mmole, 2.2 equivalents) was added, and the resulting solution was stirred for an hour at ambient temperature in an argon atmosphere. Potassium carbonate was added, and stirring continued for 10 minute~, the reaction mixture was then filtered over a bed of Merck Kieselgel 60 and celite, using a 30 mixture of n-hexane/ethyl acetate (7:3). The filtrate was concentrated under reduced pressure and the oily residue was purified by HPLC over a silica column, 10 ~m, with n-hexane/ethyl acetate (8:2) as eluent. This yielded the mixture of diastereoisomers 4a and 4b (0.146 g, 0.52 mmole, 35 86%) in the form of a white solid.
Characteristics: Rf (n~hexane/ethyl acetate, 7:3) : 0.31.
Spectroscopic results of 4a, 4b : IR(Ksr-film) : 3424 (s), 2943 (s), 2872 (s), 2285, 1461, 1444, 1390, 1373, 1266, 1170, 1066, 995, 943, 898, 887, 858, 835, 802, 737 and 702 cm~1;

, 21~0~
lH NMR (CDC13,360 MHz) : 4,07 (lH, q, 2.6 Hz), 2.61 (lH, dd, 4.9, 2.9 Hz), 2.57 (lH, d, 4.9 Hz), 1.99 (lH, m), 1.82 (3H, m), 1.30 (3H, s), 0.92 (3~, s) and 0.90 (3H, d, 6.5 Hz) ppm ;
MS : m/z to 262 (1), 247 (2), 234 (1), 180 (1), 179 (4), 162 5 (4), 151 (9), 136 (14), 125 (16), 111 (100), 96 (27), 81 (40), 67 (36) and 55 (75).

EXAMPLE 2: Synthesis of the epoxy-ketones 5a, 5b Pyridinium dichromate (PDC, 0.494 g, 1.31 mmole, 2.5 equivalents) was added to a solution of 4a, 4b (0.146 g, 0.52 mmole) in anhydrous dichloromethane (9 ml) together with a catalytic quantity of pyridinium para-toluenesulphonate ;
(PPTS) and the suspension was stirred for 4 hours at ambient 15 temperature under argon atmosphere. Then to 10 ml, ether was added and stirring continued for 5 minutes, the reaction mixture was filtered over a bed of Merck Kieselgel 60 and `
celite, using ether, and the chrome salt precipitates were washed several times with ether and then filtered. The 20 combined filtrates were concentrated under reduced pressure and the residue purified by HPLC, over a silica column, 10 ~m, with n-hexane/ethyl acetate (85:15) as eluent. In this way a mixture of diastereoisomers 5a and 5b (0.139 g, 0.5 mmole, 96 ~) in the form of an oil was obtained.
25 Characteristics : Rf (n-hexane/ethyl acetate, 7:3) : 0.35.
Spectroscopic results of Sa, 5b: I~ (KBr-film) : 3034, 2956 (s~, 2873 (s), 1714 (s), 1456, 13~8, 1307, ~255, 1234, 1187, 1152, 1107, 1056, 943, 902, 838, 795, 737 and 703 cm~1 ;
lH NMR (CDC13, 360 MHz) : 2.61 (lH, dd, 4.9, 2.8 Hz), 2.58 30 (lH, d, 4.9 Hz), 2.44 (lH, dd, 11.6, 7.5 Hz), 2.26 (2H, m), 2.12 (lH, ddd, 12, 4.1, 2.5 Hz), 2 (lH, m), 1.90 (ZH, m), 1.78 (lH, m), 1,31 (3H, s), 0.96 (3H, d, 6.1 Hz)t and 0.63 (3H, s) ppm; MS : m/z at 278 (M -, 1), 263 (2), 250 (2), 235 (2), 204 (2), 177 (4), 159 (4), 125 (2.4), 111 (36), 85 (~4), 35 81 (52), 67 (39) and 55 (100).

- 2 ~ 8 EXAMPLE 3: Synthesis of 25(S)-1-alpha-hydroxy-25,26-epoxy-vitamin D3 (24a) and 25R-l-alpha-hydroxy-25,26-epoxy-vitamin D3 (24 b)-A solution of IV (0.053 g, 0.091 mmole, 1.3 equivalents) in anhydrous tetrahydrofuran (0.5 ml) was placed in an argon atmosphere (1.1 bar), cooled to -70C, and n-butyllithium (2.41 M solution in n-hexane, 0.037 ml, 0.087 mmoleJ was added. The resulting dark solution was stiried f~r 10 30 minutes. Then, the ketones 5a and 5b dissolved in anhydrous tetrahydrofuran (0.9 ml) were added drop by drop (0.02 g, 0.072 mmole). The mixture was stirred at -70C for an hour and a half and then allowed to return to ambient temperature in a period of two and a half hours. Two or three 15 drops of water were added to the red solution and most of the solvents were eliminated under an argon flux. A solution of saturated sodium bicarbonate (2 ml~ was added and extra~ted three times with diethyl ether (2 ml), the combined extracts were filtered over a bed of Merck Kieselgel 100 (35-70 mesh) 20 and the solvent was eliminated under reduced pressure. The residue was subjected to rapid chromatograhpy over a HPLC
column, silica 10 ~m, with n-hexane/ethyl acetate (85:15) as eluent. The oil obtained (mixture of 23a and 23b), was immediately taken up in anhydrous tetrahydrofuran (0.3 ml), 25 tetrabutylammonium fluoride (TBAF, 1 M solution in tetrahydrofuran 0.255 ml) was added, and the solution was stirred for 15 hours at ambient temperature in an argon atmosphere, protected from light. The solvent was eliminated under an argon flux and the residue subjected to 30 chromatography over Merck Kieselgel 60 with dichloromethane/methanol (9:1) as eluent. An additional purification by HPLC (10 ~m silica, dichloromethane/methanol 92.5 : 7.5) provided the mixture of diastereoisomers 24a and `
24b (0.019 g, 0.046 mmole, 64 %).
35 Characteristics Rf (dichloromethane/methanol 9:1) :0.41.
Spectroscopic results of 24a, 24b: UV (MeOH)~2 max = 261 nm;
IR (KBr-film) : 3380 (s), 3048, 2946 (s), 2870 (s), 1644, 1446, 1377, 1266, 1218, 1148, 1055, 957, 898, 799, 737 and 703 cm~l ;

: ~

~ 21~01~8 IH NMR (CDC13, 360 MHz) : 6.38 (lH, d, 11.2 Hz), 6.01 (lH, d, 11.2 Hz), 5.32 (lH, m), 5 (lH, m), 4.44 (lH, dd, 7, 4.3 Hz), 4.22 (lH, ddd, 10, 7, 4 Hz), 2.82 (lH, dd, 11.5, 3.5 Hz), 2.61 (lH, dd, 4.8, 2.3 Hz), 2.57 (lH, d, 4.8 Hz), 2.31 (lH, 5 dd, 13.4, 6.6 ~z), 1.81-2.05 (4H, m), 1.31 (3H, s), 0.93 (3H, d, 6.4 Hz) and 0.54 (3H,s) ppm : MS : m/z at 415 (M +, 1, 2), 397 (2), 378 (2), 287 (1), 267 (2), 251 (3), 197 (3), 171 (5), 152 (24), 134 (60), 105 (31), 91 (42), 81 (40), 79 (40), 67 (35) and 55 (100).
' ' EXAMPLE 4: Synthesis of the alcohols 7a and 7b A solution of 3 (600 mg, 2.27 mmole) and N-(trimethylsilyl)-imidazol (953 mg, 6.81 mmole, 997 ~l) in 15 anhydrous CH2C12 (8 ml) was stirred at ambient temperature under nitrogen atmosphere for 3 hours. After concentration a residue was obtained, that was immediately subjected to chromatography (hexane/EtOAc, 16:1) to give 6 in the form of an oil (672 mg, 88 %).
20 Characteristics: Rf (hexane/EtOAc 8:2) : 0.95.
Spectroscopic results of 6 : lH NMR (CDCl3, 360 MHz) : 0.05 (9H, s), 0.88 (3H, s), 0.89 (3H, d, 6.64 Hz), 1.71 (3H, s), 3.99 (lH, m), 4.66 (lH, bs), 4.68 (lH, bs) ppm.
A mixture of selenium dioxide (33 mg, 0.30 mmole), t-butylhy-25 droperoxide (90 %, 90 mg, 0.90 mmole) and anhydrous CH2C12 (4ml) was stirred at 25C for half an hour.
A solution of 6 (200 mg : 0.60 mmole) in anhydrous CH2Cl2 (4 ml) was added, during the course of several minutes. The resulting mixture was stirred at 25C under nitrogen for half 30 an hour. Stirring was carried out at about -20C for 22 hours. The reaction continued until ambient temperature was attained, methyl sulphide (1 ml) was added, and the resulting mixture stirred at 25-30C for 5 hours. The reaction mixture was filtered over a small quantity of silica gel, 35 concentration of the filtrate provided a residue that was immediately subjected to chromatography (hexane/EtOAc 16:1) to give 6 (9 mg, 4.5 %) and a mixture o diastereoisomers 7a and 7b in an oily form (63 mg, 30 %).

2~10~5~

Characteristics: Rf (hexane/EtOAc, 9:1) : 0.36. [a]26 D
45.83 (c 1.857, CHCl3).
Spectroscopic results of 7a and 7b: lH NMR (CDCl3, 360 MHz) :
0.04 (9H, s~, 0.87 (3H, d, 6.51 Hz), 1.72 (SH, s), 4 (2H, m), 5 4.83 (lH, d), 4.92 (lH, m) ppm.
: .
~ EXAMPLE 5: Synthesis of alcohol 7b and -the epoxide 8a A 4~ moleculaire sieve of reactivated powder (27 mg) 10 was added to a solution of 7a, 7b (90 mg, 0.256 mmole) and (+)-diisopropyltartrate (9 mg, 0.038 mmole) in anhydrous CH2Cl2 (1.5 ml). The stirred mixture kept under a nitrogen atmosphere, was recooled to -10 to -20C, treated with titanium isoproproxide (IV) (7.4 mg, 0.026 mmole) and stirred 15 for 30 minutes at -20C. Then the reaction was treated with a solution of tertiary butyl hydroperoxide in isooctane (7 M, 0.178 mmole, 26 ~l, dried with the 3 ~ pellets, freshly activated for 30 minutes before addition) and added using a syringe. The reaction was stirred, at about 20 -20C for 27 hours. An aqueous solution of iron sulphate and tartric acid (2 ml) was cooled to 0C and treated dropwise with the reaction solution. The biphase mixture was stirred for 10 minutes and the aqueous solution extracted with two portions of ether (4 ml). The organic phase was treated with 25 2 ml of a 30 ~ NaOH solution precooled to 0C in saturated brine. The biphase mixture was vigorously stirred for 1 hour at 0C and after dilution with water (3 ml) the phases were separated and the a~ueous layer extracted with ether (2 x 10 ml). The organic mixture phase was washed with brine and then 30 dried over MgSO4. After filtration and concentration, a residue was obtained, which was separated by HPLC (hexane/E-tOAc, 9:1) providing an oil of pure 7b (27 mg) and a crude product of 8a. The latter was purified by HPLC (hexane/EtOAc, 9.5:1) to yield a product of pure 8a in the form of white ~ ' 35 chrystals (38 mg).
Compound 7b:
Characteristics: Rf (hexane/EtOAc, 8:3) = 0.72 ; [a]2~D +
56,05 (c 1.140, CHCl3).

- 21101~8 Spectroscopic results: IR (KBr-Film): 3360 (s, br), 3072 (w), 1650, 908 (s) cm~l;
lH NMR (360 MHz, CDCl3) : 0.05 (9H, s), 0.88 (3H, s), 0.90 (3H, d, 6.53 Hz), 1.72 (3H, s), 3.99 (2H, m), 4.83 (lH, t), 5 4.93 (lH, t) ppm; MS : m/z 334 (2), 309 (3), 262 (1), 223 (8), 197 (12), 183 (75), 73 (100).
Compound 8a:
Characteristics: Rf (hexane/EtOAc, 8:3) = 0.55 ; [a]20D +
46.81 (c 1.177, CHC13) ; mp : 65-66C.
10 Spectroscopic Results: IR (KBr~Film) : 3479 (s, br), 1250 (s), 839 (s), 745 cml ; lH NMR (360 MHz, CDCl3) : 0.04 (9H, s), 0.87 (3H, s), 0.91 (3H, d, 6.57 Hz), 1.34 (3H, s), 2.61 (lH, d, 4.77 Hz), 2.89 (lH, d, 4.77 Hz), 3.56 (lH, m), 3.99 ~lH, m) ppm ; MS : m/z 368 (M~., 1), 295 (2), 223 (3), 197 15 (11), 183 (66), 73 (100).

EXAMPLE 6: Synthesis of the diol 9a A solution of 8a (34 mg, 0.02 mmole) and of 20 tetrabutylammonium fluoride (solution lM in THF, 0.46 mmole, 460 ~l) in THF (2 ml) was stirred at ambient temperature for 2.5 hours. After elimination of THF under reduced pressure, the residue was treated with ether and the mixture washed with brine and then dried over MgSO4. After filtration and 25 concentration, a residue was obtained, which was subjected to chromatography (hexane/EtOAc, 6:4) to yield an oily and viscous compound 9a (26 mg, 96 %).
Characteristics: Rf (hexane/EtOAc, 6:4) = 0.38.
Spectroscopic results of 9a: lH NMR (360 MHz, CDCl3) : 0.92 30 (3H, d, 6.26 Hz), 0.92 (3H, s), 1.34 (3H, s) 2.60 (lH, d, 4.78 Hz), 2.88 (lH, d, 4.78 Hz), 3.55 (lH, m), 4.07 (lH, m) ppm.

EXAMPLE 7: Synthesis of the epoxyketone 10a A mixture of 9a (25 mg, 0.084 mmole), of pyridinium dichromate (47 mg, 0.125 mmole) and anhydrous CH2Cl2 (2 ml) was stirred at ambient temperature under nitrogen for 2 hours. The mixture was subjected to chromatography 211~
"~ 35 (hexane:EtOAc/CH3COCH3, 6:4:0.5) to yield the crude product 10a, which was purified by HPLC (hexane/EtOAc, 6:4) to yield the pure oil 10a (20 mg, 81 %).
Characteristics: Rf (hexane/EtOAc, 6:4) = 0.36.
5 Spectroscopic results of 10a: IR (KBr-Film) : 3444 (s, br), 1709 (s), 1236, 906, 812 cm~l ; lH NMR (500 MHz, CDC13) : 0.63 (3H, s), 0.97 (3H, d, 6.22 Hz), 1.34 (3H, s), 2.45 (lH, dd, 7.45 Hz, 11.70 Hz), 2.60 (lH, d, 4.81 Hæ), 2.87 (lH, d, 4.81 Hz), 3.55 (lH, d, 8.36 Hz) ppm.
EXAMPLE 8: Synthesis of the protected epoxyketone lla A solution of 10a (18 mg, 0.061 mmole) and N-(trimethyl-silyl)-imidazol (26 mg, 0.186 mmole, 27 ~l) in ;
15 anhydrous CH2C12 (1.5 ml) was stirred at ambient temperature in an atmosphere of nitrogen for 2 hours. The mixture was subjected to chromatography (hexane/EtOAc, 9:1) to yield compound lla in the form of an oil (20 mg, 89 %).
Characteristics: Rf (hexane/EtOAc, 8:2) = 0.48.
20 Spectroscopic results of lla: lH NMR ~360 MHz, CDCl3) : 0.09 (9H, s), 0.63 (3H, s), 0.96 (3H, d, 6.06 Hz), 1.28 (3H, s), 2.44 (lH, dd, 7.48 Hz, 11.64 Hz), 2.56 (lH, d, 5.12 Hz), 2.65 (lH, d, 5.12 Hz), 3.17 (lH, dd, 3.29 Hz., 8.88 Hz) ppm.

25 EXA~P~E ~: Synthesis of (24S,25R)-1-alpha,24-dihydroxy-25, ~ ;
26-epoxy-vitamin D3 (26a) To a stirred solution of phosphine oxide IV (57 mg, 0.098 mmole) in anhydrous THF (0.8 ml) at -80C in an argon 30 atmosphere, n-BuLi (2.46 M solution in hexane, 0.098 mmole, 40 ~l) was added to give a dark orange solution. The mixture was re-stirred for 1 hour. Compound lla (18 mg, 0.049 mmole) in anhydrous THF (0.8 ml) was then added dropwise to the solution. The mixture was stirred at -80C for 2 hours. The 35 solution was slowly re-heated to ambient temperature, while the colour turned into pale yellow and eventually became practically colourless. The mixture was subjected to chromotography (hexane/EtOAc, 12:1) to yield the crude . ~ ~

- 2llo3~3 product, which was purified by HPLC (hexane/EtOAc, 14:1) to yield the pure product 25a in the form of an oil (32 mg, 89 %).
Characteristics: Rf (hexane/EtOAc, 9:1) = 0.78.
5 A solution of 25a (32 mg, 0.044 mmole) and tetrabutylammonium fluoride (0.44 mmole, 440 ul, lM solution in THF), in THF (2 ml) was stirred at ambient temperature under nitrogen in the dark for 3.5 hours. Tetrabutylammonium fluoride (440 111 of a lM solution, 0.44 mmole) was added and the resulting mixture 10 stirred for 6 hours. Ether was added, the mixture was washed with brine and the aqueous phase extracted twice with ether.
The combined organic phases were dried over MgSO4. After filtration and concentration, a residue was obtained, which was filtered over a small column of silica gel by elution 15 with 100 ul of 5 % CH30H in CH~C12. Concentration of the filtrate provided a residue, which was purified by HPLC
(CH2Cl2/CH3OH 95:5) to yield pure product 26a (16 mg, 85 %).
Characteristics: Rf (CH2Cl2/CH30H 9.5:0.5) = 0.30.
Spectroscopic results for 26a: UV (CH30H): max = 263 nm; IR
20 tKBr-Film): 3379 (s, br), 1643 (br), 1059 (s) 896, 800, 736 cm~1; 1H NMR (500 MHz, CDCl3) : 0.54 (3H, s), 0.95 (3H, d,

6.52 Hz), 1.34 (3H, s), 2.60 (lH, d, 4.78 Hz), 2.88 (lH, bs), 5.32 (lH, bs), 6.01 t1H, d, 11.26 Hz), 6.37 (lH, d, 11.26 Hz) ppm ; MS : m/z 431 (M; -1, 2), 394 (1), 382 (2), 355 (2), 337 25 (2), 285 (1), 251 (a~), 227 (3), 197 (5), 134 (100).

EXAMPLE 10: Synthesis of the diols 12a, 12b A mixture of 31 mg (0.28 mmole) selenium dioxide, 83 mg 30 (0.93 mmole) tertiary butyl hydroperoxide (90 %) and 5 ml dichloromethane, was stirred together at about 254C under nitrogen for 0.5 hour. A solution of 1.85 mg (0.55 mmole) of 6 in 5 ml dichloromethane was then added over several ~;~
minutes. The obtained mixture was stirred at about 25C for 35 0.5 hour. Stirring was continued at -20C for 22 hours. 1 ml of methylsulphide was added, and the mixture stirred at 25-30C for 4 hours. Dichloromethane was removed using a rotatable evaporator. 4 ml of acetone are added, 10 % of hydrochloric acid is added dropwise, until a pH 3 is obtained. The mix-ture is stirred at 25C for 1 hour, then neutralized with aqueous satura-ted NaHCO3, extracted with ether and dried ~ver MgSO4. Concentration provides a residue, that is submitted to flash chroma-tography (hexane/EtOAc :
5 10/3) to obtain 63 mg (41 %) of 12a, 12b in the form of white chrystals.
Rf (hexane/EtOAc : 7/3) : 0.38 ; [a]D18 ~ 35.82 (c = 1.329 ;
CHC 13 ) ; pf 94-108C.
Spectral values of 12a, 12bi I~ (KT3r-film) : 3 408 (F, 10 large), 3 072 (f), 1 650, 899 cm~l.
lH-RMN (360 MHz, CDC13) : 0.91 (d, 6.56 Hz, 3H), 0.93 (s, 3H), 1.72 (s, 3H), 4.01 (t, lH), 4.07 (s large, lH), 4.83 (d, lH), 4.92 (m, lE~) ppm.

15 EXAMiPLE 11: Synthesis of the alcohol 12a and epoxide 13b To a solution at ambient temperature of 90 mg (0.32 mmole) of 12a, 12b and 11.23 mg (0.048 mmole) of (-)-diisopropyl tartrate in 2 ml of anhydrous 20 dichloromethane, 27 mg of 4A pulverized and activated pellets are added. The stirred mixture maint:ained under nitrogen is cooled to between -10 and -20C, treated with 9.09 mg (0.032 mmole) of Ti(o-i-Pr)4 and stirred continuously or 30 minutes at - 20C. The reaction mixture is then treated with a 25 solution of tertiary butyl hydroperoxide in isooctane (7.00 M
; 0.224 mmole ; 32 ~ul, dried with 3A pellets, newly activated, for 30 minutes, before addition), added with the aid of a gastight syringe. The reaction mixture is stirred at about -20C in a bath maintained at constant temperature for 30 23 hours. The reaction mixtu e is then cooled to about 0C
using a bath of iced water, 3 ml of an aqueous suspension of iron sulphate and tartaric acid. The epoxidation reaction mixture is left to warm up to about 0C and then poured slowly into a beaker, containing the solution of cooled 35 stirred iron sulphate (external recooling is not indispensable, during or after this additlon).-The mixture is stirred into two phases during about 10 minutes, and then transferred into a decanting beaker. The phases are separated, and the aqueous phase extracted with two portions 21101~

of 6 ml ether. The combined organic layers are treated with 3 ml of a preferably cooled (0C) solution of 30 % NaOH in saturated salt water. The two phase mixture is vigourously stirred for 1 hour. After transfer -to a decanting beaker and 5 dilution with 3 ml water, the phases are separated and the aqueous layer extracted with two portions of 15 ml ether. The ~ combined organic layers are washed with saturated aqueous NaCl and dried over MgSO4. It is then filtered and concentrated to obtain a residue that is separated by HPLC
10 (hexane/EtOAc : 7/4) to yield 32 mg of optically pure 13b in the form of white chrystals, 4 mg of a stereoisomer ~f 13b (characterized by 1H-RMN) and 55 mg of crude 12a in the form of white chrystals, also containing the isomer 24-(R) (according to 1H-RMN at 500 MHz). The crude product 12a is 15 submitted to a second kinetic Sharpless splitting, to obtain 33 mg of optically pure 12a.
For 12a: Rf (hexane/EtOAc : 1/1) : 0.61 ; [a]D18 + 32.58 (c =
1.016 ; CHCl3) ; pf 121.5-123,5.
Spectral values: IR (RBr-film) : 3 374 (F, large), 3 073 (f), 20 1 651 (f), 899 cm-1.
H-RMN (500 MHz, CDCl3) : 0.91 (d, 6.56 Hz, 3H), 0.93 (s, 3H), 1.72 (s, 3H), 4.02 (t, lH), 4.07 (s large, lH), 4.83 (t, lH), 4.92 (t, lH~ ppm.
SM : m/z 280 (Mt : 0.04), 262 (10.8), 247 (5.8), 229 (5.7), 25 190 (6.3), 163 (15.9), 135 (43,2).
For 13b: Rf (hexane/EtOAc : 1/1) : 0-44 ; [~]D20 + 40.89 (c =
1.010 ; CHCl3) ; pf 97-98C.
Theoretical analysis of C18H32O3 : C = 72,97 ; H - 10.81 ;
found: c = 72.83 ; H = 10.85.
30 Spectral values: IR (KRr-film) : 3 440 (F, large), 1 267, 942, 756 cm~1.
H-RMN (500 MHz, CDCl3) : 0.93 (s, 3H), 1,34 (s, 3H), 2.60 (d, 4.78 Hz, lH), 2.89 (d, 4.78 Hz, lH), 3.61 (m, lH), 4.07 (s large, lH) ppm.
35 SM : m/z 278 (0.7), 263 (1,3), 260 (0.7), 245 (0.7), 193 (2), 177 (4.7), 161 (7.3), 135 (17.4), 111 (100).

~`~` 39 2~ i 01~8 EXAMPLE 12: Synthesis of the epoxyketone 14b A mixture oE 25 mg (0.084 mmole) of 13b, 63 mg (0.168 mmole) of pyridinium dichromate (DCP) and 2.5 ml of 5 dichloromethane are stirred together at 0C, under nitrogen for 4 hours. 16 mg (0.043 mmole) of DCP is added, and the reaction mixture allowed to return ~o normal temperature and then stirred for 2 hours. The mixture is then immediately submitted to flash chromatography (hexane/EtOAc : 6/4) to 10 yield 20 mg of 14b in the form of white chrystals (80 %).
Rf (hexane/EtOAc : 6/4) : 0.33 ; pf 84-85C.
Spectral values of 14b : IR (KBr-film) : 3 446 (F, large), 1 711 (F), 1 266, 942, 814 cm~l.
1H-RMN (360 MHz, CDC13) : 0.64 (s, 3H), 0.97 (d, 5.53 Hz, 3H), 15 1.34 (s, 3H), 2.45 (dd, 11.64 Hz, 7.48 Hz, lH~, 2.60 (d, 4.75 Hz, lH), 2.89 (d, 4.75 Hz, lH) 3.61 (d, lH).

EXAMPLE 13: Synthesis of epoxyketone 15b A solution o 20 mg (0.068 mmole) of 14b and 19 mg (0.136 mmole ; 20 ,ul) of N-(trimethylsilisilic)imidazol in 2 ml CH2Cl2 are stirred together at normal temperature in an atmosphere of nitrogen for 1 hour. The mixture is then immediately subjected to flash chromatography (hexane/EtOAc :
25 9/1) to yield 22 mg of 15b in the form of an oil (88 Rf (hexane/EtOAc : 8/2 ) : 0.49.
Spectral values of 15b : 1H-RMN (500 MHz, CDCl3) : 0.09 (s, 9H), 0.63 (s, 3H), 0.96 (d, 5.98 Hz, 3H), 1.28 (s, 3H), 2.45 (dd, 11.61 Hz, 7.49 Hz, lH), 2.56 (d, 5.12 Hz, lH), 2.67 (d, 30 5.12 Hz, lH), 3.22 (dd, 8.56 Hz, 3.62 Hz, lH) ppm.

EXAMPLE 14: Synthesis of 28b (24R,25S)-la,24-dihydroxy-25, 26-epoxy-vitamin D3.

To a stirred solution of 54 mg (0.093 mmole) of phosphine IV oxide in 0.6 ml anhydrous THF under nitrogen at -80C, n-BuLi (solution 2.5 M in hexane ; 0.093 mmole ; 37 ~l) is added to obtain a dark orange solution. The mixture is stirred under these conditions for 1 hour. 17 mg (0.046 : . .

21101~3 , mmole) of 15b in 0.8 ml anhydrous THF is added dropwise to the solution. The obtained mixture is stirred at -80~C for 2 hours. The solution is allowed to slowly return to normal temperature, while it turns into pale yellow and finally 5 becomes practically colourless. The mixture is immediately submitted to flash chromatography (hexane/EtOAc: 12/1) to yield crude 27b, that is then purified by HPLC (hexane/EtOAc : 18/1) to yield 28 mg (82 %) of pure 27b in the form of an oil.
10 Rf (hexane/EtOAc: 9/1): 0.74.
A solution of 28 mg (0.038 mmole) of the latter compound and (n-Bu),~NF (solution lM in THF; O.75 mmole; 760 ul) in 1 to 5 ml of THF is shielded from light and stirred at normal temperature under nitrogen for 9 hours. The mixture is 15 immediately filtered over a short column of silica gel with 100 ml of 10 % CH30H in CH2CL2 acting as eluent. On concen-tration, a residue is obtained that is separated by HPLC
(CH2Cl2/CH3OH : 95/5) to yield 12 mg of pure 28b in the form of a white solid (73 %).
20 Rf (CH2Cl2/CH3OH: 9.5/0.5): 0.29; [a]Dl3 = 32.37C (c =
1.035, CHCl3).
Spectral values of 28b: UV (CH30H): lambdamaI = 262 nm.
H-RMN (500 MHz, CDCl3): 0.55 (s, 3H), 0.94 (d, 6.50 Hz, 3H), 1.34 (s, 3H), 2.31 (dd, 13.43 Hz, 6.63 Hz, lH), 2.60 (d, 4.77 25 Hz, lH), 2.89 (d, 4.77 Hz, lH), 3.61 (m, lH), 4.22 (s large, lH), 4.43 (s large, lH), 5.00 (s large, lH), 5.32 (t, lH), 6.02 (d, 11.27 Hz, lH), 6.38 (d, 11.27 Hz, lH) ppm.
SM: m/z 394 (2), 336 (2). 251 (3).

30 EXAMPLE 15: Synthesis of the alcenols 16 and 16' To a solution of 0.47 g (1.28 mmole) of 16 in 5.0 ml of THF at -78C under argon, is added dropwise 6 ml of a solution 1.25 M of isopentenylmagnesium chloride (7.5 mmole 35 6 equivalents), followed by 6.4 ml of a solution of 0.1 M of dilithiumcopper (II) tetrachloride (0.64 mmol ; 0.5 equiva- ~ ;
lents) in THF. The reaction mixture is allowed to return to normal temperature and then stirred for 16 hours, and then poured into a saturated solution of ammonium chloride 50 ml.
.~

, ,, ,- ,, .. ~ .. ,.,., , , .. ,, ,, , ",,, .. - . , . ,-2 ~101~ 8 The aqueous layer is extracted several times with diethyl ~ther, then the combined ethyl layers are washed with a saturated solution of sodium chloride, dried over anhydrous magnesium sulphate and concentrated under reduced pressure.
5 The residue is subjected to chromatography on silica gel and an HPLC silica 10 ~m column with a mixture of isooctane/ethyl acetate (9/1) as eluent, in both cases, to obtain the isomeric alcenols 16 and 16' (0.31 g; 92 %) in the form of a mixture (3/7 according to the 1H-RMN spectral values).
10 Rf (isooctane/ethyl acetate : 9/1) : 0.20.
Spectral values of 16: IR (KBr-film) : 3 403 (F), 2 936 (F), 2 867 (F), 1 641, 1 445, 1 376, 1 268, 1 234, 1 165, 1 118, 1 065, 1 035, 9gO, 942, 912, 886, 858, 822, 787, 725, 698 and 668 cm~l.
15 1H-RMN (CDC13 : 360 MHz) : 5.09 (lH, t, 7.1 Hz, LR 1.4 Hz), 4.07 (lH, s large), 2.01 (2H, m), 1.83 (4H, m), 1.68 (3H, s large), 1.60 (3H, s large), 0.93 (3H, s) and 0.91 (3H, d, 6.5 Hz) ;
SM : m/z at 264 (M+ : 2), 249 (3), 246 (3), 231 (4), 179 (7), 20 161 (12), 151 (26), 133 (22), 125 (11), 111 (28), 95 (21), 84 (43), 69 (46), 55 (44) and 49 (100).
Spectral values of 16' (for a mixture 3/7 of 16/16') :
IR (KBr-film) : 3 420 (F), 3 081 (F), 2 868 (F), 1 656, 1 638, 1 546, 1 453, 1 413, 1 378, 1 361, 1 268, 1 234, 25 1 165, 1 066, 1 032, 991, 959, 941, 908, 885, 858, 820, 785, 725 and 692 cm~l.
H-RMN (CDC13, 360 MHz) : 5.79 (lH, dd, 17.8, 10.4 Hz), 4.89 (lH, m), 4.85 (lH, s large), 0.98 (3H, s), 0.97 (3H, s), 0.93 (3H, s) and 0.91 (3H, d, 6.5 Hz).
30 SM : m/z at 264 (M+ : 2), 249 (4), 246 (4), 231 (5), 179 (9), 161 (15), 151 (34), 133 (27), 125 (18), 111 (60), 95 (34), 81 (39), 69 (74), 55 (82) and 43 (100).

EXAMPLE 16: Synthesis of the epoxyalcohols 17a, 17b and 17'a, 17'b To a solution of 0.29 g (1.1 mmole) of a 3/7 mixture of 16 and 16' in 13 ml of anhydrous dichloromethane, 0.43 mg (2.1 mmole) of m-chloroperbenzoic acid (AmCPB) is added and ;i --~
~ ~:

42 21101~
the solution stirred for 3 hours at normal temperature. 0.5 g oE potassium carbonate is added, stirring continued for 15 minutes, the reaction mixture filtered and the resulting solid matter washed with dichloromethane and filtered. The 5 combined filtrates are concentrated under reduced pressure and the residue purified on silica gel and in an HPLC silica ~ 10 ~g column with a mixture 3/2 isooctane/ethyl acetate, as eluent, in both cases, to obtain 0.21 g (68 %) of 17a, 17b and 17la, 17'b in the form of a mixture.
10 Rf (isooctane/ethyl acetate : 8/2) : 0.18.
Reversed phase chromatography of this mi~ture on an HPLC column, of RSilC18HL of 10 ~m with a mixture 8/2 of methanol/water as eluent, yields 0.131 g of diastereoisomers 17'a and 17'b. An additional purification of r0sulting matter 15 over an HPLC column silica 10 ~l, with a mixture 8/2 isooctane/ethyl acetate as eluent, yields 0.025 g of the diastereoisomers 17a and 17b.
Rf (isooctane/acetone : 9/1) : 17a, 17b : 0.19 ; 17'a, 17'b :
0.23.
20 Spectral values of 17a, 17b : IR (KBr-film) : 3 465 (F), 2 933 (F), 1 455, 1 378, 1 327, 1 266, 1 251, 1 180, 1 166, 1 118, 1 066, 1 035, 992, 943, 899, 859, 792, 737, 701 and 680 cm~l.
1H-RMN (CDC13, 360 MHz) : 4.07 (lH, s large), 2.68 (lH, t, 6.0 25 Hz), 1.99 (lH, dm, 11.9 Hz), 1.93-1.75 (3H, m), 1.30 (3H, s), 1.26 (3H, s), 0.93 (3H, s) and 0.91 (3H, d, 6.5 Hz).
SM: m/z at 280 (M+ : 1), 265 (3), 262 (5), 247 (3), 229 (4), 21g (4), 179 (5), 161 (10), 151 (20), 133 (20), 125 (16), 111 (60), 95 (32), 81 (41), 67 (38), 55 (72) and 43 (100!.
30 Spectral values of 17'a, 17'b : IR (KBr-film) : 3 454 (F), 2 930 (F), 2 870 (F), 1 472, 1 403, 1 375, 1 362, 1 267, ;~
1 167, 1 067, 1 033, 1 018, 992, 942, 913, ?386, 859, 835, 784, 722, 688, 668 and 658 cm~~.
lH-RMN (CDC13, 360 MHz) : 4.06 (lH, s large), 2.74 (lH, dd, 35 4.0, 3.0 Hz), 2.62 (2H, m), 2.00 (lH, dm, 13.2 Hz), 1.92 (lH, m), 1.81 (2H, m), 0.99 + 0.97 (3H, 2 x d, 6.2 Hz), 0.96 (3H, s), (0.89 + 0.87) (3H, 2 x s) and 0.83 + 0.81 (3H, 2 x s).

2~10~

SM: m/z at 262 (1), 247 (2), 219 (1), 203 (1), 191 (2), 179 (4), 162 (6), 151 (8), 135 (15), 125 (19), 111 (66), 95 (34), 81 (37), 69 (45) and 55 (100).

5 EXAMPLE 17: Synthesis of the epoxyketones 18a; 18b To a solution of 0.021 g (0.075 mmole) of 17a, 17b in 2.0 ml of anhydrous dichloromethane, is added 0.079 g (0.21 mmole ; 2.8 equivalents) pyridinium dichromate (PDC) and a 10 catalytic quantity of pyridinium p-toluenesulphonate (PTSP) and the suspension is stirred for 4.5 hours at normal temperature under argon. 3 ml diethyl ether is added, followed by continuous stirring for 5 minutes, the reaction mixture is filtered over a bed of Celite/silica gel using the 15 ether and the precipitated salts are washed several times with ether and then filtered. The combined filtrates are concentrated under reduced pressure and the residue purified by HPLC over a silica 10 ~m column, using a mixture 9/1 of isooctane/acetone as eluent. In this way 0.017 g (80 % of a 20 mixture of diastereoisomers 18a and 18b in the form of an oil) is obtained.
Rf (isooctane/acetone : 9/1) : 0.25.
Spectral values of 18a, 18b : IR (KBr-film) : 2 958 (F), 2 875 (F), 1 714 (F), 1 462, 1 378, 1 324, 1 308, 1 242, ; ~-~
25 1 223, 1 191, 1 172, 1 120, 1 056, 943, 900, 867, 837, 7 and 680 cm~l.
1H-RMN (CDC13, 360 MHz) : 2.67 (lH, t, 6.0 Hz), 2.45 (lH, ddd, 11.5, 7.2, 2.9 Hz), 2.32-2.16 (2H, m), 2.11 (lH, dm, 13.2 Hz), 2.05-1.82 (3H, m), 1.73 (lH, m), 1.30 (3H, s), 1.26 (3H, 30 s), 0.97 (3H, d, 6.0 Hz) and 0.64 (3H, s).
SM: m/z at 278 (M+ : 2), 260 (3), 235 (3), 205 (6), 192 (6), 177 (8), 149 (24), 135 (10) 124 (20), 111 (40), 36 (36), 81 (48), 67 (36), 55 (72) and 41 (100). ;
5 EXAMPLE 18: Synthesis of 24(S)-l~alpha-hydroxy-24,25-epoxy-vitamin D3 (30a) and of 24(R3-24,25-epoxy-1-alpha-OH-vitamin D3 (30b) ::

~- ~ 2llolrj8 A solution of IV 0.06 g (0.103 mmole ; 2.05 equiva-lents) cooled to -70C in 1.0 ml of anhydrous THF, is placed under an atmosphere of 1.1 bars of argon and 0.039 ml of a solution 2.5 M of n-butyllithium, in n-he~ane (0.096 mmole), 5 is added. The dark red solution obtained is stirred for 30 minutes. The ketones 18a and 18b 0.014 g (0.C5 mmole) dissolved in 0.5 ml of anhydrous THF are added dropwise. The mixture is stirred at - 70C for 1.5 hour and allowed to return to normal temperature, dur ng a period of 2.5 hours. 2 10 to 3 drops of water are added to the still red solution, and the major portion oE solvents is removed under an argon current. 2.0 ml of a saturated solution of sodium bicarbonate is added, that is extracted several times with 2 ml diethyl ether. The combined extracts are fil~ered over a Merck 15 Kieselg~l 60 bed and the solvent is removed under reduced pressure. The residue is rapidly subjected to chromatography over an HPLC silica 10 ~m column, using a mixture 85/15 of n-hexane/ethyl acetate as eluent. The obtained oil (a mixture of 29a and 29b) is immediately taken up in 0.4 ml anhydrous 20 THF, 0.2 ml of a solution 1 M of tetrabutylammonium fluoride (FTBA) 1 M in THF is added and the solution is stirred for 19 hours at normal temperature, under argon, in the absence of light. The solvent is removed under an argon current and the residue is subjected to chromatography over Merck Kieselgel 25 60, usina a mixture 9/1 of dichloromethane/methanol as ;
eluent. An additional HPLC purification (silica 10 ~m ;
mixture 95/5 of dichloromethane/methanol) yields 0.018 g (86 %) of a mixture of diastereoisomers 30a and 30b in the form of a vitreous material.
30 Rf (dichloromethane/methanol : 9/1) : 0.36. ~-Spectral value of 30a, 30b:
UV (MeOH) lambdamax = 263 nm.
IR (KBr-film) : 3 409 (F), 2 951 (F), 2 298 (F), 1 647, 1 447, 1 379, 1 266, 1 056, 958, 896, 800, 738 and 703 cm~1.
35 lH-RMN (CDC13, 360 MHz) : 6.37 (lH, d, 11.2 Hz), 6.01 (lH, d, 11.2 Hz), 5.32 (lH, m), 5.00 (lH, m), 4.43 (lH, m), 4.22 (lH, m), 2.82 (lH, dd, 11.8, 3.7 Hz), 2.68 (lH, t, 6.0 Hz), 2,59 (lH, dd, 13.2, 3.5 Hz), 2.31 (lH, dd, 13.2, 6.5 Hz), 2.05-. 2llal~s 1.85 (5H, m), 1.31 (3H, s), 1.26 (3H, s), 0.94 (3H, d, 6.3 Hz) and 0.55 (3H, s).
SM : m/z at 414 (M+ : 7), 396 (45), 378 (34), 269 (5), 251 (9), 195 (6), 181 (5), 171 (7), 169 (6), 161 (8), 159 (9), 5 157 (12), 155 (16), 152 (19), 134 (61), 105 (28), 91 (27), 81 (24), 71 (22), 55 (41) and 43 (100).
-EXAMPLE 19: Synthesis oE the epoxyketones 18'a, 18'b To a solution of 0.096 g (0.34 mmole) of 17'a, 17'b in 8.0 ml of anhydrous dichloromethane, is added 0.36 g (0.95 mmole ; 2.8 equivalents) o~ pyridinium dichromate (PDC) and a catalytic quantity o~ pyridinium p-toluenesulphonate (PTSP) and the suspension is stirred for 4.5 hours at normal 15 temperature under argon. 10 ml of diethyl ether is added, stirring is continued for 5 minutes, the reaction mixture is filtered over a Celite/silica gel bed, using ether and the salts of the chrome precipitates are washed several times with ether and then filtered. Tha combined filtrates are 20 concentrated under reduced pressure and the residue purified by HPLC on a silica 10 ~m column, using a mixture 95/5 isooctane/acetone. 0.070 g (74 ~) of the mixture of diastereoisomers 18'a and 18'b in the form of oil, are obtained.
25 Rf (isooctane/acetone : 95/5 ) : 0.16.
Spectral values of 18'a, 18'b~
Ir (KBr-film) : 2 960 (F), 2 348 (F), 2 282 (F), 1 714 (F), 1 470, 1 379, 1 308, 1 246, 1 225, 1 057, 943, 913, 840 and 733 cm~
30 1H-RMN (CDC13, 360 MHz) : 2.73 (lH, t, 3.5 Hz), 2.61 (2H, m~
2.44 (lH, dd, 11.7, 7.5 Hz), 2.31-2.16 (2H, m), 2.13 (lH, dm, 13.2 Hz), 2.04-1.81 (3H, m), 1.71 (lH, m), 1.30 (2H, m), 1.09 (lH, ddd, 14.1, 10.8, 8.3 Hz), 1.04 + 1.02 (3H, 2 x d, 6.7 -~
Hz), 0.87 + 0.85 (3H, 2 x s), 0.84 -t 0.82 (3H, 2 x s) and 35 0.66 + 0.65 (3H, 2 x s).
SM : m/z at 278 (M+ : 1), 248 (1), 193 (12), 178 (8), 163 (7), 149 (1~), 135 (14), 124 (22), 111 (36), 95 (30), 81 (43), 69 (36) and 55 (100).

~ 21101 ) 3 EXAMPLE 20: Synthesis of 24(S)-lalpha-hydroxy-24,25-epoxy-25,25-dinor-23,23-dimethyl-vitamin D3 (30'a) and 24(R)-24,25-epoxy-25,25-dinor-23,23-dimethyl-lalpha-OH-vitamin D3 (30'b).
A solution 0.085 g (0.146 mmole ; 2.03 equivalents) of IV in 1.4 ml of anhydrous THF is placed under an atmosphere of 1.1 bar of argon and 0.056 ml of a solution of n-butyllithium 2.5 M in n-hexane (0.14 mmole) is added. The 10 dark red solution obtained is stirred for 30 minutes. The ketones 18'a, 18'b 0.02 g (0.072 mmole) dissolved in 0.8 ml of anhydrous THF are added drop-~ise. The mixture is stirred at -70C for 1.5 hours and then allowed to return to normal temperature, during a period of 2.5 hours. Two to three drops 15 of water are added to the still red solution and the major portion of solvents are removed under an argon current. 2.0 ml of solution saturated with sodium bicarbonate, is added -and then extracted several times with 2.0 ml of diethyl ether. The combined extracts are filtered over a Merck 20 Kieselgel 60 bed and the solvent removed under reduced pressure. The residue is quickly subjected to chromatography over an HPLC column of 10 ~m, using a mixture 85/15 of n-he~ane/ethyl acetate as eluent. The obtained oil, a mixture of 29'a and 29'b3 is immediately taken up in 0.5 ml of 25 anhydrous THF, 0.25 ml of a solution of 1 M
tetrabuthylammonium fluoride (FTBA) in tetrahydrofuran is added and the solution is stirred for 30 hours at normal temperature under argon and in the absence of light. The solvent is removed under an argon flow and the residue is 30 subjected to chromatography on Merck ~ieselgel 60, using a mixture 9/1 of dichloromethane/methanol as eluent. An additional purification by HPLC (silica 10 ~m, mixture 95/5 of dichloromethane/methanol) yields 0.023 g (77 %) of a mixture of the diastereoisomers 30'a and 30'b in the form of 35 a vitreous substance.
Rf (dichloromethane/methanol : 9/1) : 0.41.
Spectral values of 30'a, 30'b :
UV (MeOH) lambdamax = 263 nm.
IR (KBr-film) : 3 404 (F), 2 950 (F), 2 250, 1 642, 1 444, 2~1Ql~
-~ 47 1 378, 1 054, 958, 910, 833, 800 and 735 cm~1.
lH-RMN (CDC13, 360 MHz) : 6.37 (lH, d, 11.2 Hz), 6.02 (lH, d, 11.2 Hz), 5.32 (lH, s large), 4.99 (lH, s large), 4.42 (lH, m), 4.22 (lH, m), 2.81 (lH, dd, 11.8, 3.8Hz), 2.75 llH, dd, 5 4.0, 3.0 Hz), 2.61 (3H, m), 2.31 (lH, dd, 13.4, 6.4 Hz), 2.05-1.87 (5H, m), 1.07 (lH, ddd, 14.1, 11.9, 8.3 Hz), 1.02 +
1.00 (3H, 2 x d, 6.5 Hz), 0.89 -~ 0.87 (3H, 2 x s), 0.83 +
0.81 (3H, 2 x s) and 0.58 + 0.57 (3H, 2 x s).
SM : m/z at 414 (M : 7), 396 (64), 378 (36), 367 (6), 352 10 (5), 285 (6), 26g (7), 251 (1~), 209 (10), 195 (9), 152 (27), 134 (83), 105 (38), 95 (27), 91 (34), 81 (31), 69 (51) and 55 (100) .

EXAMPLE 21: Synthesis of the ketone 20 Starting from tosylate lc, the reaction with the lithium derivative, obtained from the acetal of 3-butyne-2-one, yields the acetal 19, according to the Uskokovic operational mode, described for an analogous 20 product in ref. 32.
IR (KBr) : 3 475, 2 935, 2 867, 2 207, 1 675 cm~~
The hydrolysis is carried out in aqueous dioxane in the presence of sulphuric acid. The ketone 20 has the following spectroscopic values~
25 IR (KBr-film) : 3 475, 2 935, 2 867, 2 207, 1 675 cm~l.
1H-RMN (360 MHz, CDCl3) : 4.09 (m, lH), 2.41 (lH, dd, 17.2, 3.6 Hz), 2.33 (3H, s), 2.21 (lH, dd, 17.2, 7.6 Hz), 1.05 (3H, d, 6.6 Hz), 0.93 (3H, s) ppm.

30 EXAMPLE 22: Synthesis of the epoxides 21a, 21b A mixture of 131 mg (0.624 mmole) of trimethyl-sulphonium iodide, 54 mg (0.964 mmole) of powdered potassium hydroxide and 1 ml of acetonitrile is stirred at 60C under 35 nitrogen. 50 mg of a solution of 20 (0.160 mmole) in 1 ml of acetonitrile is added, producing an orange colour. The -obtained mixture is stirred for 1 hour and 40 minutes, the colour changes to yellow. The mixture is immediately filtered over a short silica gel column, using 220 ml of EtOAc, 50 ~

` 48 2~101~8 ~, in hexane as eluent. By concentration, a residue is obtained, that is separated by HPLC (hexane/EtOAc :8/2) to yield 13 mg (30 %) of 21a, 21b in the form of an oil.
Rf (hexane/EtOAc : 8/2) : 0.31.
5 Spectral values for 21a, 21b:
IR (KBr-film) : 3 456 (F, large), 3 047 (f), 2 240 (f), 1 271 (F), 943 (F), 760 cm~1.
1H-RMN (360 MHz, CDC13) : 0.93 (s, 3H), 1.04 (d, 6.58 Hz, 3H), 1.54 (s, 3H), 2.02 (m, lH), 2.25 (m, lH), 2.72 (d, 5.55 Hz, 10 lH), 2.97 (d, 5.55 Hz, lH), 4.08 (s large, lH) ppm.
SM : m/z 261 (1.7), 243 (2.1), 163 (10.3), 95 (40.2), 81 (60.3).

EXAMPLE 23: Synthesis of the epoxyketones 22a, 22b A mixture of 25 mg (0.091 mmole) of 21a, 21b, 103 mg (0.274 mmole) of PDC and 2 ml of dichloromethane is stirred under nitrogen for 4 hours. The mixture is immediately filtPred over a short column of silica gel, using 200 ml 20 EtOAc, 25 ~ in hexane as eluent. By concentation, a residue is obtained that is purified by HPLC (hexane/EtOAc : 8/2) to `~
obtain 19 mg (76 ~) of 22a, 22b in the form of an oil.
Rf (hexane/EtOAc : 8/2) : 0.33.
Spectral values of 22a, 22b: IR (KBr-film) : 2 239 (f), 1 712 25 (F), 1 271, 857, 763 cm~1.
lH-RMN (360 MHz, CDC13) : 0.63 (s, 3H), 1.09 (d, 6.20 Hz, 3H), 1.54 (s, 3H), 2.47 (dd, 11.61 Hz, 7.51 Hz, lH), 2.73 (d, 5.55 Hz, lH), 2.97 (d, 5.55 Hz, lH) ppm.

30 EXAMPLE 24: Synthesis of the vitamin D3 derivatives 32a, 32b To a stirred solution of 77 mg (0.132 mmole) of phosphine IV oxide in 1 ml of anhydrous THF under nitrogen at 35 -80C, is added 53 ml of a solution of 2.5 M n-BuLi in a mixture of hexanes, which yields a dark orange solution. The solution is stirred under these conditions for 1 hour. 18 mg (0.066 mmole) of 22a, 22b in 0.8 ml of anhydrous THF is slowly added dropwise to the solution. The mixture obtained, . 2~lalris ~ ~9 is stirred at -80C for 2 hours. The solution is allowed to slowly return to normal temperature, during which time the colour changes to pale yellow and finally becomes almost colourless. The mixture is immediately submitted to flash 5 chromatography (hexane/EtOAc : 9/1) to obtain a crude product, that is purified by HPLC (hexane/EtOAc : 18/1) to ~ yield 37 mg (88 %) of 31a, 31b, completely pure in the form of an oil.
Rf (hexane/EtOAc : 9/1) : 0.64.
A solution 37 mg (0.058 mmole) of 31a, 31b and 700 ~l of a solution 1 M of (n-Bu)4NF 1 M in (0.70 mmole) THF in 1.5 ml of THF, is protected from light and stirred under nitrogen for 8 hours. The mixture is immediately filtered over a short - column of silica, using 100 ml of CH30H, 8 % in CH2Cl2 as 15 eluent. By concentration, a residue is obtained, that is separated by HPLC (CH2C12/CH3OH : 96/4) to ohtain 19 mg (80 %) ~-of pure 20 in the form of a white solid.
Rf (CH2C12/CH3OH : 9.5/0.5) : 0-38 Spectral values for 32a, 32b :
- 20 UV (CH30H) : lambdamax = 262 nm-IR (KBr-film) : 3 304 (F, large), 2 239 (f), 1 647 (large), 1 271, 1 054 (F), 911, 863, 800, 737 cm~~
lH-RMN t360 MHz, CDCl3) : 0.54 (s, 3H), 1.06 (d, 6.51 Hz, 3H), 1.54 (s, 3H), 2.28 (m, 2H), 2.59 (m, lH), 2.73 (d, 5.56 Hz, 25 lH), 2.83 (m, lH), 2.97 (d, 5.56 Hz, lH), 4.22 (m, lH), 4.42 (m, lH), 4.99 (s large, lH), 5.32 (s large, lH), 6.01 (d, 11~25 ~Iz, lH), 6.37 (d, 11.25 Hz, lH) ppm.
SM : m/z 410 (M~ : 16), 392 (28), 374 (25), 359 (3), 315 (3), 297 (4), 279 (3), 134 (100).
- _XAMPLE 25: Synthesis of the diol lb .:
Diol la is oxidiæed by pyridinium dichromate (PDC) in the presence of pyridinium p-toluenesulphonate in 35 dichloromethane. The oxidation is not selective and a mixture oE hydroxyketone, ketoaldehyde and hydroxyaldehyde 2a is - obtained. After purification of the hydroxyaldehyde 2a (Rf:
0.22 ; ethyle acetate/hexane : 1/1) on silica gel, it is treated with a catalytic quantity of diazabicyclo-undecene in ~,,. 211015~

dichloromethane. After 3 hours it is treated with ether. The etherized solution is washed with 2 ml of 5 % hydrochloric acid. After drying over Na2SO4, filtration and concentration, a mixture of the aldehydes 2a and 2b is obtained (respecti-5 vely 36/64, according to lH-RMN).
RE (ether/hexane : 1/1) : 0.28.
101 mg (0.48 mmole) of this mixture is dissolved in 10 ml of methanol and treated at 0C with 90 mg (2.38 mmole) of sodium borohydride. After 30 minutes, it is concentrated under 10 vacuum ice and 20 ml ether is added. After washing of the organic phase with 1 ml of 5 % hydrochloric acid and water, the residue obtained by concentration under vacuum is dried (Na2SO4) and purified by chromatography on a silica gel (benzene/hexane/ether : 3/2/5). 62 mg (0.293 mmole) of isomer 15 lb, having an Rf of 0.18, compared to isomer la (Rf : 0.16), is thus obtained.
Spectral values of 2a :
IH-RMN (500 MHz, CDC13) : 9.57 (lH, d, 3.18 Hz), 4.11 (lH, m), 1.10 (3H, d, 6.83 Hz) and 0.98 (3H, s) ppm.
20 Spectral values of 2b :
9.53 (lH, d, 4.96 Hæ), 4.09 (lH, m), 1.02 (3H, d, 6.82 Hz) and 0.95 (3H, s) ppm.
Spectral values of lb :
4.08 (lH, m), 3.72 (lH, dd, 3.5, 10.7 Hz), 3.45 (lH, dd, 6.9, 25 10.6 Hz), 0.955 (3H, 5), 0.954 (3H, d, 6.6 Hz) ppm.

EXAMPLE 26: Synthesis of the hydroxyaldehyde 2a starting from tosylate lc.

A solution of tosylate lc (100 mg, 0.27 mmole) and collidine (66 mg, 0.55 mmole) in dimethylsulphoxide ~3 ml) is stirred at 150C for 30 minutes under argon. After cooling, water is added. After extraction by CH2C12, the organic phase is washed (NaCl) and dried. After concentration, the next 35 stage is immediately proceeded to. After RMN analysis, a mixture of 2a and 2b in the proportions 3:1 are obtained.

2 1 i ~ i ) 3 . 51 EXAMPLE 26: Experimental models for hiological evalua-tion 1) Study of the differentiation of leukaemia cells Human leukaemia cells HL-60 (origin promyelocytic) are 5 cultivated in T-25 flasks to a density of 1.2 x 105 cells per ml of RPMI 1640 medium (containing 10 % foetal bovine serum, ~ inactivated by heat and antibiotics) and in the presence of increasing concentrations of 1 alpha,25-(OH)2D3 or analogues.
The cells are cultivated in this way for 4 days at 37C in an 10 atmosphere of 5 % CO2 in air. To determine the differentiation of the promyelocytic cells into macrophagas, maturation of the cells is evaluated by the NBT reduction tPSt (salt of Tetrazolium Nitro Blue), according to the method of Ostrem et ~ -~
al [23]. The percentage of cells, containing formazan (black) 15 granules is determined by counting the cells using a hematocymeter.

2) Study of differentiation in human epidermic cells The pharmacological property of cellular 20 differentiation, is equally studied in human epidermic cells or keratinocytes in primary culture. The keratinocytes are isolated from skin fragments (biopsis, surgical resection), according to the method described by Matsumoto et al [24].
These skin fragments are incubated a night at 4C in the 25 presence of a buffered saline solution (150 mM PBS, pH 7.4), containing 100 U dispase per ml. The epidermis is then s~parated from the dermis, subsequently treated for 10 minutes in the presence of a 0.25 % trypsine solution, 0.02 %
EDTA, at ambient temperature. The reaction is stopped by 30 adding PBS, containing 10 ~ bovine serum. The cells are then washed twice by centrifugation, resuspended in the complete culture medium, and cultivated at 37C in 5 % of CO2 in air in the T-25 flasks. The complete culture medium is the modified MCDB 153 medium (Gibco) without serum, containing bovine 35 pituitary extract and the epidermic growth factor [24]. After 24 hours, the epidermis cells stick to the support and are multiplying progressively.
To study the biological activity of the compounds, the cells obtained after the second passage, are cultivated in 52 2~01~3 multi cup cultivation trays (24 or 96 cups) at a cell density of 5 to 8 x 104 cells/cm2. After 24 hours, concentrations increasing in 1-alpha,25-(OH)2D3 and the analogues are added to the culture medium. After different incubation times, the 5 cellular growth and differentiation are determined by the following evaluation parameters:
(a) cellular viability [MTT or tetrasolium salt test, according to Bio-Rad protocol ; measure of LDH]
~b) total proteins [BCA method or Pierce bicinchonic acid]
10 (c) cell growth [incorporation of tritiated thymidine] ;
(d) epidermic differentiation [differential colouration of cytokeratines to Rhodanile Bleu, cytochemical detection of keratin 67 k, from the filaggrin and involucrin by means of specific monoclonal antibodies (Biosoft ;
Biomedical Technologies)].

3) Effect on the pxoliferation of lymphocyte cells This test determines the capacity of a compound or an analogue of vitamin D, to inhibit the multiplication 20 (proliferation) of lymphocyte cells, previously stimulated by phytohemagglutinin.
The cells of the white line are isolated by centrifuging human blood, heparized on the Ficoll-Paque (buffy coat technique). The buffy coat cells are treated with 25 phytohemagglutinine and then cultivated in the presence of increasing compound concentrations and tritiated thimidine, according to the previously described protocol [25]. At the end of incubation, the radioactivity associated with the cells is measured. The cell multiplication is deEined as the 30 relationship between the radioactivity associated with the cells, in the presence of the compounds, and that associated with the control cells, expressed as a percentage.

(4) Study of the antiproliferation effect in human tumour cells Different tumour cell lines are used in order to determine the anti-tumour properties of the vitamin D
analogues. This concerns mainly the human cell lines, resulting from an adenocarcinoma oE the breast (MCF-7, T-~` 53 2~101~8 47D), -the colon (CX1), the lungs (LX1) from the melanomas (MM-96).
During the test, -the cells are cultivated in multi hollow trays of 96 wells. The initial cell density is of 2 to 5 5000 cells per cup, according to the envisaged line, in order to maintain the cells in a logarithmic growth phase during the duration of the experiment. After 24 hours the cells are incubated in the presence of increasing concentrations of vitamin D and its analogues, for different periods of time.
10 At the end of each incubation, the protein concentration is determined (BCA or Pierce method), along with the incorporation of tritiated thymidine and the cellular viability test (MTT and LDH). These different parameters allow the evaluation of cell growth and the cytotoxic effect 15 of the compounds. The anti-tumour activity is expressed as a percentage of growth inhibition, with respec-t to the control cells.

5) Calcemic ~ffect in chicken with rickets The chicken ricket model allows evaluation of the pharmacological effect of vitamin D on the phosphocalcium metabolism, in particular in the intestine (calcium absorption) and bone tissue (mineralisation). This model is concerned with studying the calcemic effect of vitamin D
25 [26].
Chickens with rickets are obtained by raising the chicks from the time of their birth in the absence of light and with a diet poor in calcium. After three weeks, the animals are treated for 10 days with vitamin D or its 30 analogues, by means of a daily intramuscular injection. At the end of the treatment, the animals are killed; the serum, the intestines and the bone tissuesare removed. The level of calcium in the serum and the bones is measured by atomic absorption; serum osteocalcin and the protein intestinale 35 calbindin-28k, two vitamin D-dependent proteins, are determined by a radioimmunological RIA test, using specific antibodies.
The measured parameters show the phosphocalcium metaholism well. Serum calcium and the pres~nce of intestinal ` 54 2~101~
cabindin witness the effect of vitamin D on the intestinal absorption of calcium. The content of calcium in bone and in serum osteocalcin is representative of mineralisation and bone formation.
6) Study of in vitro differentiation in human ~ osteosarcomas The cell line MG-63 derived from a human osteosarcoma presents osteoblast characteristics (cells of the bone 10 matrix). Cells of this line are used to evaluate the hypercalcemic or osteocalcic effect oE vitamin D in bone tissues.
In this test, the cells are incubated in the presence of increasing concentrations of the compounds. hfter 48 hours 15 of incubation, the concentration of intracellular calcium (flame spectrometry) and osteocalcin (RIA) is determined.
Osteocalcin is a specific protein, produced by the osteoblasts, during bone formation ; this parameter reflects the stimulating effect on bone cells well.

7~ Study of the formation of plurinuclear cells starting from bone marrow in culture.
In bone, vitamin D influences bone resorption and increases the number of osteoclastic cells. Different systems 25 have been described in order to evaluate the vitamin D
activity on bone cells in vitro. A particularly effective system consists of determining the formation of multinuclear cells from bone marrow culture, under the inductive effect of vitamin D and its analogues. The multinuclear cells formed 30 under these conditions present all the functional and morphological characteristics of osteoclasts.
The method used is that described by Ishizuka et al.
[27]. Briefly, bone marrow is removed from mouse femurs and resuspended in a culture medium MEM (Minimal Essential 35 Medium) containing 10 % of foetal bovine serum, previously inactivated using heat. Ths cellular suspension is then placed in a multi hollow dish of 24 cups, giving 106 cells per cup (1 ml). The cells are incubated at 37C in an atmosphere of 5 % C2 in air. After a day of culture, the culture medium --` 213 0~8 is replaced by completely fresh medium (culture control) or medium containing different concentrations of vitamin D and its analogues (culture -test). The medium is renewed every three days. In the course of the culture, the number of 5 multinuclear cells (>3 nuclei/cell) is determined by counting using an inversed phase contrast microscope. On day 8, the ~ cells are fixed with methanol and coloured by Giemsa to allow the morphological study of the cells.
., 10 EXAMPLE 27: Biological properties of vitamin D analogues, according to the invention.

A) The epoxide group confers the analogue, 1-alpha~
hydroxy-vitamin D the characteristic properties of an active metabolite of vitamin D.

By way of example, the biological activity of the epoxide derivatives of vitamin D and of 1-alpha-hydroxy-vitamin D, have been compared in the induction test for 20 differentiation for leukaemia cells HL-60.
In this test, cell multiplication is only weakly inhibited by 1-alpha-OHD3. A 50 % inhibition effect (ED50) of 1-alpha-OHD3 is obtained for an extra-cellular concentration increased to 3 x 10-6 M. The addition of an epoxide group, to 25 the 1-alpha-OHD3, for example at position 25 as in the case of compound 24 (1-alpa-hydroxy 25,26-epoxy, vitamin D3) allows inhibition and complete differentiation of the leukaemia cells clearly for the lower concentrations. So, the biological effect is obtained at a concentration, being 100 30 times less, 3 x 10~a M. ~ ~ ;
Thus the presence of an epoxide group in the lateral chain of 1-alpha-hydroxy-vitamin D confers biological proper-ties, namely the occurrence of cellular differentiation, due ;
to the hydroxylated active metabolite of vitamin D in the 35 lateral chain (1-alpha,25-(OH32D3).

~ 56 2~ 10 l ~
B) _itamin D epoxy an~logues clearly give dissociated bioloaical activities with respect to the active natural m abolite of vitamin D. hydroxylated at position 25.

sy way of example, the biological activity of the epoxide analogues is compared to that of 1-alpha25-(OH)2D3 in ~ the in vitro tests on human epidermic cells (keratinocytes), osteoblastic cells and leukaemia cells, and in the in vivo test in the chicken ricket model.
1. Differentiation of leukaemia cells In the induction of differentiation test in leukaemia cells HL-60, cell multiplication is remarkably inhibited by 1-alpha,25-dihydroxy-vitamin D3 and by the epoxide analogues 15 of vitamin D. A 50 % inhibiting effect of 1-alpha,25-(OH)2D3 is obtained for an extra cellular concentration of 1.24 x 10-8 M. The epoxide derivatives also inhibit remarkably the multiplication of HL-60 cells. For example, the inhibiting concentration of compound 32 (1-alpha-hydroxy-23,24-20 didehydro-25, 26-epoxy vitamin D3) is 1.34 x 10-3 M.
Thus, vitamin D's property of cell differentiation is conserved by the epoxide analogues characteriæed by the presence of an epoxide group in the lateral chain of the 1-alpha-hydroxy-vitamin D, and gives this even in the case 25 where no hydroxyl group is present at position 25.

2. Differentiation of human epidermic cells The comparative effect of 1-alpha-OHD3 epoxide analogues on the growth of epidermic cells was studied in 30 primary cultures of human keratinocytes.
At an extracellular concentration of 10-7 M, the ~
epoxide analogues, tested to this level, inhibit the growth ~ ;
of keratinocytes in culture by at least 45 %. A 50 % inhibi~
ting effect, on the growth of keratinocytes, is obtained, for 35 example in extracellular concentrations of 6.2 x 10-7 M and 2.8 x 10-7 M for compounds 30 (1-alpha hydroxy-24,25-epoxy-vitamin D3) and 32 respectively. In the same test the active metabolite of vitamin D, 1-alpha,25-(OH)2D3 has clearly less effect on the growth of epidermic cells, with an ED50 of 3.0 2llolrj~
`~ 57 x 10-6 M. A signlficant inhibiting effect of the epoxide analogues is already observed with a concentration of 10-8 M, where the 1-alpha,25-(OH)2D3 only gives a marginal eEfect. For example the compound 26a ((24S, 251)-1-alpha,24-dihydroxy-25, 5 26-epoxy-vitamin D3) gives an inhibiting effect to the order of 35 % at 10-8 M.
The obtained results show the excellent inhibiting power of the epoxide analogues on human keratinocytes in culture, which is clearly superior to that of 1-alpha,25-10 (OH)2D3, for example by a factor of 5 and 10 for the compounds30 (1-alpha hydroxy-24,25-epoxy vitamin D3) and 32 (1-alpha hydroxy-23,2~-didehydro,25,26-epoxy vitamin D3) respectively.

3. In vitro differentiation effect on human osteosarcomas The human osteoblasic cells MG-63, originating from an osteosarcoma, are incubated in the presence of different concentrations of 1-alpha,25-(OH)2D3 and epoxide analogues.
After 48 hours the osteocalcin secretion is measured in the culture medium. Under normal culture conditions, the MG-63 20 cells produce little or no osteocalcine.
The 1-alpha,25-(OH~2D3 rapidly induces the secretion of osteocalcin in the cells (ED50 = 1.6 x 10-lO M); the optimum effect is obtained at a concentration of 10-9 M. The epoxide analogues are clearly less active than the 1-alpha,25-(OH)2D3.
25 A biological activity of 50 % in the epoxide analogues is thus observed at very high extracellular concentrations. By way of example, the compound 28b (24R,25S)-1-alpha,24-dihydroxy-25,26-epoxy-vitamin D3~ induces only 50 % of the maximum osteocalcin secretion, by the MG-63 cells, at a 30 concentration of 4.2 x 10-9 M.
Thus, the property of osteoblastic stimulation in vitamin D is affected by the epoxide derivation of the lateral chain of 1-alpha-OHD3. The biological effect of the ~ -epoxide analogues is obtained at distinctly higher 35 concentrations; at the increased concentrations, the analogues act as 1-alpha,25-(OH)2D3. There is, however, a distinction between the effects on the cells of bone origin, ;~
where the effect is diminished, and the other cells, such as . 21101~g keratinocytes or leukaemia cells, where the effect is conserved or more oEten considerably increased.

4. Calcemic effect in chicken with rickets The in vivo biological activity of vitamin D and its derivatives were studied in the model of chicken rickets. The serum concentrations of calcium and osteocalcin were determined after 10 days of treatment, of the chickens with rickets, with 1-alpha,25-(OH)2D3 or the epoxide analogues of 10 1-alpha-OHD3.
In the case of 1-alpha,25-(OH)2D3, the calcium concentration in the blood increased proportionally with the administered dose, reaching normal values of those obtained in a chicken without rickets at the daily dose of 80 ng/kg.
15 Serum calcium reflects the absorption of calcium in the intestine induced by vitamin D. In the case of the epoxide analogues, the pharmacological effect is particularly reduced. For example, no calcium resorption in the intestine was observed for the derivative 26a (24S,25R)-1-alpha,24-20 dihydroxy 25,26-epoxy-vitamin D3) at a daily dose of 160 ng/kg.
Osteocalcin is specifically produced by the bone cells and represents an excellent parameter of bone metabolism.
1-alpha,25-(OH)2D3 stimulates, in a notable way, bone 25 metabolism; the maximum effect on serum osteocalcin is obtained with a daily dose of ~0 ng/kg. The epoxy derivatives show little or no pharmacological effects on bone tissue. sy way of example, compound 32 (1-alpha-hydroxy-23,24 didehydro-2.5,26-epoxy-vitamin D3) is totally inactive (tested until 160 30 ng/kg) whilst compound 24 ((24S,25R)-1-alpha,24-dihydroxy-25, 26-epoxy-vitamin D3) posesses only a marginal activity at a concentration of 160 ng/kg. Thus the supplied modifications to the lateral chain of 1-alpha-hydroxy-vitamin D, considerably affect the principal pharmacological property of 35 1-alpha,25-dihydroxy-vitamin D, regarding the phosphocalcium metabolism. The calcemic effect of the analogues is marginal to nonexistant.

~ 59 21101~8 C) Biological properties of epoxy analogues of vitamin D
can be modulated by their respective isomers.

In the case of the epoxy analogues of 1-alpha-hydroxy-5 vitamin D, different isomeric forms are possible in both the epoxy group and the lateral chain. Also, the effect of analogous epoxy isomers on the biological properties has been studied. For example the compounds 26a and 28b were compared with respect to their epidermic cell differentiation 10 properties and their antiproliferative effects on human tumour cells.
Considering human epidermic cells, the compound 26a inhibits by 50 % the growth of keratinocytes at an extra-cellular concentration (ED50) of 3.1 x 10-7 M. As for compound 15 28b, this is twice as inactive having an ED50 of 6.4 x 10-7 M.
At a concentration of 10-8 M, a significant inhibitive effect is obtained with compound 26a (35 %) whilst compound 28b does not even show a marginal effect.
Concerning the antiproliferative effect in human tumour 20 cells, compound 26a, inhibits the growth of MCF-7 cells, after 7 days incubation at a concentration of 10-7 N, by nearly 50 %. At this same concentration the antiproliferative effect of compound 28b is reduced by at least a factor of three.
Thus lateral chain isomers of the epoxy analogues of vitamin D and particularly those in the epoxy group, significantly influence the epoxides' cellular biological activities. These results also show the interest of considering the pure isomer forms of the epoxide analogues, 30 rather than their forms in a mixture.
-: :
D) The biological activity of epoxy analogues Qf 1-alpha-hydroxy-vitamin D can be superior to that of 1-alpha, 25-dihy~Lroxy-vltamin D.
Amongst the interesting biological properties of vitamin D and the epoxide analogues, the effects on human epidermic cell differentiation on the one hand and the antiproliferative effects on tumour cells MCF-7 on the other - 211 Q~rj8 hand, have been considered. For example compound 32 shows to be an excellent cell growth inhibitor with respect to the active metabolit~ of vitamin D,1-alpha,25-(OH)zD3.
In human epidermic cells, compound 32 is found to be at 5 least ten times as effective in differentiating the keratinocytes than 1-alpha,25-(OH)2D3 with ED50's of respectively 2.8 x 10-7 M and 3.0 x 10-~ M. Furthermore in the MCF-7 cells in culture, the antiproliferative effect is also in favour of the epoxy analogue. At a concentration of 10-a M, 10 1-alpha,25-(OH)2D3 is inactive, whilst at the same time roughly 20 % cell inhibition is o~tained for compound 32. 50 % biological activity of this analogue is observed at an extracellular concentration of 5.9 x 10-8 M.
As shown with this epoxide analogue, the addition of an 15 epoxide group in the lateral chain of 1-alpha-hydroxy-vitamin D, not only-conserves the cellular biological of 1-alpha,25-dihydroxy-vitamin D (cf. B), but also increases certain cellular properties characteristic of vitamin D.

20 E) General biological properties of the epoxide analogues of 1-alpha-hydroxy-vitamin D.

The vitamin D analogues, characterized by the presence of an epoxide group in the la~.eral chain, possess 25 particularly interesting in vitro and in vivo biological ;
properties. The epoxide group favourably substitutes the hydroxyl group characteristic of the active metabolite and the analogues of vitamin D. It confers particular biological properties allowing notably to distinguish between the 30 epoxide derivatives of 1-alpha,25-(OH)2D3 and its principal analogues. Thus, the epoxide group makes possible the dissociation between the calcemic effect and the other forms of biological activity.
The epoxide analogues of 1-alpha-hydroxy-vitamin D
35 possess in a general manner the characteristic cellular properties of vitamin D. Notably the differentiation of leukaemic and epidermic cells, and the antiproliferative effect on cancer and leukocyte cells. The epoxide analogues are particularly effective in the epidermic and cancerous 2~31~8 ~` 61 cells. At the same -time they only weakly stimulate the secretion of osteocalcin by cells osteoblastic in origin.
Concerning the phosphocalcium metabolism, the calcemic effect of the analogues is considerably reduced (marginal activity) 5 being nonexistan-t in the model of chicken rickets.
So, the notable reduction of the calcemic effect confers advantages on the epoxide analogues with respect to vitamin D or its active metabolite, notably to increase the concentration of the compound able to be administered without 10 influencing the phosphocalcium metabolism. This dissociation of activity makes it possible for example, to stimulate the differentiation of leukaemic or epidermic cells without at the same time inducing a harmful toxic calcemic effect.
The obtained results permit envisaging the therapeutic 15 application of epoxide analogues in the treatment of skin affections, such as psoriasis, secondary hyperparathyroidism (chronic renal disease), and certain cancers (leukaemia;
lymphomas, myelodisplasic syndrome, melanomas, breast, etc.), or as immunomodulators (suppression of the proliferation of T
20 lymphocytes, differentiation of monocytes, etc.), notably for autoimmune diseases and inflammatory processes during skin marrow or organ transplants.
`: ::
*****

` ~

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Claims (14)

1. Compounds derived from vitamin D or one of its analogues having in particular a lateral chain grafted in position 17 on the D ring of vitamin D, characterized in that they comprise an epoxide group grafted on said lateral chain, the epoxide group being separated in position 17 by a linear chain of at least two atoms.

2. Compounds derived from vitamin D or one of its analogues, possessing a lateral chain grafted in position 17 on the D ring, said lateral chain comprising an epoxide group, of formula:

I in which - V represents a part of vitamin D or one of its analogues wherein position 17 is vacant;
- X, grafted onto position 17 of V, represents a hydrocarbon chain, in particular an alkyl chain, having 2 to 15 carbon atoms, possibly substituted or modified, may in particular comprise at one or more positions, one or more of the group(s) ? hydroxyl, methyl, ethyl, halogen, preferably fluorine, ? ester, notably an R1COO group wherein R1 is C1 to C7 alkyl or alkoxy, linear or branched, saturated or unsaturated, possibly substituted notably by one or more of the group(s) hydroxy or halogen preferably fluorine, ? ether, in particular a R1-O group wherein R1 represents C1 to C7 alkyl or alkoxy, linear or branched, saturated or unsaturated, possibly substituted by one or more of the group(s) hydroxy or halogen, ? oxo in ketone form, ? a ring or heteroring, notably cycloalkyl, saturated or unsaturated, aromatic or heteroaromatic which may carry all the groups and modifications mentioned hereabove and/or ? said chain X may comprise in the place of one or more of its carbon atoms, an oxygen, nitrogen or sulphur atom, ? said chain X may be unsaturated, with one or more double or triple carbon-carbon bonds, this unsaturated chain may carry all the groups and modifications mentioned hereabove and ? said chain and the different groups situated on the chain may adopt all the isomeric forms, ? the epoxide group may be grafted onto any one of the 13 carbon atoms of X, in particular at its end, the epoxide group wherein ? R1, R2 and R3 may be identical or different, represent an hydrogen, an C1 to C10 alkyl group, linear or branched, saturated or unsaturated, possibly substituted with one or more of the group(s) halogen, hydroxy, cycloalkyl, also possibly substituted with halogen or hydroxy groups;
? R1 and R2 may make up part of the same ring, such as a carboring or a heteroring, and in the case of R3 having the same meaning as hereabove;
? R2 and R3 may form part of the same ring, such as a carboring or a heteroring, and in the case of R1 having the same meaning as hereabove;
? different isomeric forms of the epoxide being possible.

3. Compounds according to claim 2, characterized in that they correspond to the formula Ib wherein X, R1 to R3 have the meanings as given in claim 1 and A represents a hydrogen, a methyl or a methylene group = CH2, --- represents a single or double bond, Y represents H or OH or an ester group, in particular R1COO, or ether, in particular R1O, wherein R1 has the same meaning as given in claim 1.

4. Compound according to claim 2 or 3, characterized in that X represents an alkyl chain having 2 to 6 carbon atoms, wherein one carbon atom may be replaced by an oxygen atom and possibly substituted, in particular by one or more methyl, hydroxyl groups and/or - ether, in particular by a R1-O group, and X may be unsaturated with a double or triple carbon-carbon bond, - the epoxide group being grafted on one end of X, and R1, R2 and R3 represent a hydrogen, a hydroxyl, an alkyl group, such as methyl, ethyl or hydroxyalkyl.

5. Compounds according to one of the claims 2 to 4 characterized in that they correspond to the formula (II) wherein Z represents a saturated or unsaturated alkyl chain having 0 to 3 carbon atoms, possibly substituted by a hydroxy, an alkyl, such as methyl or ethyl, on one or more of the said carbon atoms, the epoxide group being grafted to one end of Z.

6. Compounds according to claim 5, characterised in that they correspond to the isomeric formulas IIa or IIb IIa IIb with

7. Process for the preparation of a compound according to any of the proceeding claims characterized in that the anion obtained from a compound having the skeletal base structure formula IV

and n-butyllithium is reacted with a compound which skeletal structure has formula III

wherein the grafted reactive groups on said skeletons of III
and IV are possibly protected and X, R1, R2, R3 of which the reactive groups are possibly protected, have the meanings as given in the claims 1 to 5, subsequently the said reactive groups are possibly deprotected notably X, R1, R2, R3 and hydroxyl at positions 1 and 3.

8. Compound according to one of the claims 1 to 6, for use as a medicament.

9. Compound according to one of the claims 1 to 6 for use in a method of treatment and/or prophylaxis of cancer, including leukaemia, lymphoma, myelodisplasic syndromes, melanomas, cancer of the breast, lungs and colon, as well as their metastases.

10. Compound according to one of the claims 1 to 6, for use in a method for the treatment and/or prophylaxis of psoriasis and dyskeratoses.

11. Compound according to one of the claims 1 to 6, for use as an immunomodulator to prevent rejection of grafts to be used alone or in association with other compounds such as cyclosporine or FK 506.

12. Compound according to one of the claims 1 to 6, for use in a method for the treatment and/or prophylaxis of autoimmune diseases.

13. Compound according to one of the claims 1 to 6, for use in a method for the treatment and/or prophylaxis of osteoporosis.

14. Compound according to one of the claims 1 to 6, for use in a method for the treatment and/or prophylaxis of hyperparthyroidism notably secondary and renal insufficiency.