GB2321244A

GB2321244A - Vitamin D3 analogues, process for preparing them and their use as antiproliferative and antitumor agents

Info

Publication number: GB2321244A
Application number: GB9701116A
Authority: GB
Inventors: Achille Panzeri; Nicola Mongelli; Enzo Santaniello; Patrizia Ferraboschi
Original assignee: Pharmacia and Upjohn SpA
Current assignee: Pfizer Italia SRL
Priority date: 1997-01-20
Filing date: 1997-01-20
Publication date: 1998-07-22
Anticipated expiration: 2017-01-20
Also published as: GB2321244B; GB9701116D0

Abstract

Vitamin D 3 analogues of formula: wherein: W is a C 1 -C 3 alkyl group; A is selected from: a C 1 -C 3 alkylene group; an ethenylene group -CH=CH-; an ethynylene group -C#C-; and an oxygen atom -O-; B is selected from: a single bond; a C 1 -C 6 alkylene group optionally substituted by a hydroxy group or a halogen atom, preferably fluorine; a C 1 -C 4 alkenylene group; a C 1 -C 4 alkynylene group; a C 6 -C 10 arylene group; and a C 7 -C 9 arylalkylene group; T is selected from: a group, wherein R 1 and R 2 are, each independently, C 1 -C 4 alkyl groups, optionally substituted by one or more fluorine atoms, and X is hydrogen or hydroxy; and a cyclopropyl group. Such compounds are useful as antiproliferative and antitumor agents.

Description

VITAMIN 3 ANALOGUES, PROCESS FOR PREPARING THEM, AND THEIR USE AS ANTIPROLIFERATIVE AND ANTITUMOR AGENTS.

The present invention refers to vitamin D3 analogues, to a process for their preparation, and to their use as antiproliferative and antitumor agents.

1,25-dihydroxyvitamin D3, a biologically active form of vitamin D3, plays an important role as a major calciumregulating hormone. However, in recent years new studies have demonstrated that vitamin D3 plays a much wider role in biology than it was previously thought. In particular, evidences have been provided showing that 1,25dihydroxyvitamin D3 can regulate production and secretion of several cytokines (such as interleukin-2 from lymphocytes and tumor necrosis factor from monocytes), and can decrease proliferation and increase differentiation of lymphocytes, monocytes, fibroblast, keratinocytes, and bone cells. Similar effects on cell proliferation and/or differentiation have been observed in malignant cell lines, e.g. myeloid leukaemia and breast carcinoma cell (New England Journal of Medicine 320, 980-981, 1989; Journal of Steroid Biochemistry 37, 873876, 1990). Furthermore, the antiproliferative effect of 1,25-dihydroxyvitamin D2 has been confirmed also in vivo: prolonged survival of nude mice inoculated with murine myeloid leukaemia cells (MI-cells) (Proc. Nat. Acad. Sci. USA 80, 201-204 (1983)), and growth inhibition of human malignant melanoma, colonic cancer cells and breast cancer cell xenografts in immuno-suppressed mice (Cancer Res. 47, 21-25 (1987); Lancet i, 181-191, 1989) were observed.

Clinical studies, in patients with psoriases have already demonstrated the favorable effects deriving from topically administered 1,25-dihydroxyvitamin D3 (Endocrine Reviews 14 3-19 (1993)).

However, to obtain antiproliferative and antitumor effects, supraphysiological doses of systematically administered 1,25dihydroxyvitamin D3 are needed, thus causing side effects like hypercalcemia and hypercalciuria.

These severe complications have prompted researchers to find new vitamin D3 analogues in the attempt of separating calcemic activity from antiproliferative and cell differentiating effects.

We have found that this goal can be achieved with novel vitamin D3 analogues as defined hereinunder, which, differently from known vitamin D3 derivatives having a side chain at position 17ss, bear a side chain in position 16a or 16ss.

Therefore, the present invention provides compounds of formula:

wherein: W is a C1-C3 alkyl group; A is selected from: a Cl-C3 alkylene group; an ethenylene group -CH=CH-; an ethynylene group -C--C-; an oxygen atom -0-; B is selected from: a single bond; a C1-C6 alkylene group optionally substituted by a hydroxy group or a halogen atom, preferably fluorine; a C1-C4 alkenylene group; a C1-C4 alkynylene group; a C6-Cl0 arylene group; and a C7-Cg arylalkylene group; T is selected from: a

group, wherein Rl and R2 are, each independently, C1-C4 alkyl groups, optionally substituted by one or more fluorine atoms, and X is hydrogen or hydroxy; a cyclopropyl group.

A C1-C6 alkylene group may have a straight or branched chain, preferably a straight chain, and is preferably selected from: CH2 -CH2-CH2-, -CH2-CH2-CH2-, -CH2-(CH2)2 CH2 -(CH2 -CH2- (CH2)3 -CH2-, -CH2- (CH2)4 -CH2 -,

When a C1-C6 alkylene group is substituted by a hydroxy group it is preferably selected from:

When a C1-C6 alkylene group is substituted by a fluorine atom, it is preferably selected from:

A C1-C4 alkenylene group is preferably selected from: -CH=CH-,

-CH=CH-CH=CH-, -CH=CH-CH2-, wherein the double bond(s) is (are) preferably in E configuration.

A C1-C4 alkynylene group is preferably selected from: -CEC-, -C=C-CH2 , -CH2 -CC-, -CH2 -CH,-C#C-CH2-.

A C6-C10 arylene group is preferably phenylene.

A C7-C9 arylalkylene group is preferably selected from:

A C1-C3 alkyl group is preferably selected from: methyl, ethyl, n-propyl, and iso-propyl.

A C1-C3 alkylene group is preferably selected from: -CH2

-CH2-CH2-, -CH2-CH2 -CH2 -,

A group T equal to

wherein R1 and R2 are, each independently, C1-C4 alkyl groups and X is hydrogen or hydroxy, is preferably selected from:

A group T equal to

wherein Rl and R2 are, each independently, Cl-C4 alkyl groups substituted by one or more fluorine atoms and X is hydrogen or hydroxy is preferably selected from:

The group W is preferably methyl or ethyl, more preferably methyl.

The group A is preferably a Cl-C3 alkylene group, such as -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, or an ethenylene group.

When B is a C1-C6 alkylene group, it is preferably a C1-C3 straight alkylene group.

When B is a C1-C6 alkylene group substituted by a hydroxy group, it is preferably:

When B is a Cl-C6 alkylene group substituted by a fluorine atom, it is preferably,

When B is a Cl-C4 alkenylene group, it is preferably -CH=CHor -CH=CH-CH2-.

When B is a Cl-C4 alkynylene group, it is preferably -CC- or -C=-C-CH2-.

When B is an Cl-Cl0 arylene or a C-Cg arylalkylene group, it is preferably:

When T is a group

wherein Rl and R2 are, each independently, Cl-C4 alkyl groups and X is hydrogen or hydroxy, it is preferably:

When T is a group

wherein R1 and R2 are, each independently, C1-C4 alkyl groups substituted by fluorine atoms and X is hydrogen or hydroxy, it is preferably:

In formula (I), preferred side chains in position 16a or 16ss are the following:

(cont'd)

Among the compounds of formula (I), those bearing the side chain in position 16ss are preferred. Both the single (2 OR) and (20S) epimers and the (20RS) racemic mixture are within the scope of the present invention.

Particularly preferred compounds within the scope of formula (I) are reported in the following Table 1.

TABLE 1

Compound W A B T 1) ) CH, -CH3 -CH2- -CH2-CH2- H3 -C-OH CH3 2) -CH3 -CH2- -CH2-CH2-CH2- H3 -C-OH CH3 3) -CH3 -CH2- -CH2-CH2-CH2-CH2- 3 -C-OH CH3 4) -CH3 -CH2- -CH2-CH2-CH2-CH2- CH, -C-OH I 5) -CH3 -CH=CH- -CH2- -C-OH -C-OH CHO 6)-CH3-CH=CH- -CH=CH- -C-OH CH, (cont'd) TABLE 1 (cont'd)

Compound W A B T 7) -CH3 -CH2- -CH2-CH2- 3 -C-OH CF3 8) -CH3 -O- O - - CH2 - CH2 - /3 -C-OH CH3 9) -CH3 -O- -CH2-CH2-CH2- 3 -C-OH CH3 10) -CH3 -O- - CH2 - CH2 - CH2 - CH2 - AITCH 2 3 -C-OH cH2CH3 11) -CH3 -CH2- -C:::C- H3 -C-OH CH3 12) -CH3 -CH2- -C-C- F3 -C-OH CF3 13) -CH3 -CH2-CH2- -C-C- H3 -C-OH CH3 14) - CH3 -CH2-CH2- -CC- / 3 C-OH CF3 15) -CH3 -CH2 -CH2 - -C=C- -C-OH -C-OH CH2CH3 16) -CH3 -O- -CH2-C=C- / 3 -C-OH CH3 The compounds of formula (I) according to the present invention may be prepared starting from the corresponding ialkoxy-protected 16-substituted androstane of formula (IX) as reported hereinunder according to two alternative routes: 1) the la-OH group is introduced in the steroid template already containing in ring B the 5,7-diene system typical of vitamin D3 precursor, optionally protected with a suitable protecting group, e.g. 4-phenyl-1,2,4 triazoline-3,5-dione (PTAD) (see Scheme I reported below); or: 2) the la-OH group is introduced in the steroid template with simultaneous formation of a double bond at position 5,6 of the B ring, which is subsequently transformed into the 5,7-diene system typical of vitamin D3 precursor (see Scheme II reported below).

Both routes are based on a sequence of reactions which are well known to those skilled in the art for the synthesis of vitamin D3 derivatives having a side chain in position 17ss and not in positions 16a or 16ss as in the present invention.

For instance, a synthetic route analogous to Scheme I is described in: J. Chem. Soc. Perkin I, 820-822 (1977); Tetrahedron 30, 2071-2075 (1974); J. Chem. Soc. Perkin Trans I, 1330-1336 (1985); J. Chem. Soc. Perkin Trans I, 1809-1815 (1994); Chem. Pharm. Bull. 42 (11) , 2349-2351 (1994) . A synthetic approach analogous to Scheme II can be found for instance in: Chem. Pharm. Bull. 23, 695 (1975).

w SCHEME I A-B-T w HO g A- B- T 1) hv H OR OR > HO (11) (I) A-B-T LiAlH4 2J RO eki A-B-T Me ss 1) MCPBA I or tBu-SiO( < / t 2) nBuNF RO Ne Ph 4 R=H N0N\ Ph R=tBuMe2Sj R=tBuSe2Si (Iv) (III) 2) tBuMe2SiCl, imidazole 1) PTAD W A-B-T AcO A-B-T (V) (VI) }yrOAc 11 w w A-B-T DDQ -B-T HOCfI DDQ 0y̆A B- T (VIII) (VII) N E30+ W A-B-T I OR3 (IX)

w SCiEDSE II A-B-T w HO - A-B-T 1) hv HO OH 21 HO (1) o 1) Br O | Br O 2) 2) collidine, p-xylene w w A-B-T Li , OH A-B-T L A-B-T 0 (x) HO (xI) t B.202 MCPBA W W A-B-T A-B-T DDQ 0 HO (VII) (VIII) t 50+ W CA-B-T OR3 (IX) According to the above Scheme I, a compound of formula (I) can be prepared by a process which comprises: (a) reacting a compound of formula:

wherein: W is a C1-C3 alkyl group; A is selected from: a C1-C3 alkylene group; an ethenylene group -CH=CH-; an ethynylene group -CrC-; an oxygen atom -0-; B is selected from: a single bond; a C1-C6 alkylene group optionally substituted by a hydroxy group or a halogen atom, preferably fluorine; a C1-C4 alkenylene group; a C1-C9 alkynylene group; a C6-C10 arylene group; and a C7-Cg arylalkylene group; T is selected from: a

group, wherein Rl and R2 are, each independently, C1-C4 alkyl groups, optionally substituted by one or more fluorine atoms, and X is hydrogen or optionally protected hydroxy; a cyclopropyl group; R3 is a Cl-C4 alkyl group, preferably methyl or ethyl; with an inorganic or organic acid in the presence of water, in an organic solvent; so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (b) reacting the compound of formula (VIII) as defined above with 2, 3-dichloro-5, 6-dicyano-1,4-benzoquinone (DDQ) in an organic solvent, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (c) reacting the compound of formula (VII) as defined above with isopropenyl acetate, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (d) reducing the compound of formula (VI) as defined above, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (e) reacting the compound of formula (V) as defined above with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and then protecting the hydroxy group, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above and Q is a hydroxy protecting group; (f) reacting the compound of formula (IV) as defined above with an oxidizing agent, and then removing the protecting group Q, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (g) reducing the compound of formula (III) as defined above, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (h) treating the compound of formula (II) as defined above by photolysis, so obtaining a compound of formula (I) as defined above, and optionally removing any protecting group that could be present in A, B, or T.

As to step (a), the acid is preferably sulfuric acid or ptoluene sulfonic acid and the organic solvent is usually methanol, ethanol or tetrahydrofurane. The reaction is usually carried out for some .hours at a temperature varying from room temperature to the reflux temperature of the reaction mixture.

As to step (b), the organic solvent is preferably -selected from dioxane, benzene, toluene, chlorobenzene, xylene. The reaction is usually carried out at the reflux temperature of the reaction mixture for a time varying from 4 to 36 hours.

As to step (c), the reaction is preferably carried out in an organic solvent such as e.g. ethyl acetate, isopropyl acetate or, more preferably, butyl acetate, in the presence of a strong acid, preferably p-toluene sulfonic acid, at the reflux temperature of the reaction mixture, for a time varying from 4 to 8 hours.

As to step (d), the reduction is preferably carried out by adding a solution of compound (VI) in a solvent such as e.g.

diethylether, at a temperature from about -150C to about -100C for about 2 hours, to a mixture obtained by mixing an alcohol (preferably ethanol) solution of sodium borum hydride to an alcohol (preferably methanol) solution of calcium chloride, at a temperature from about OOC to about 50C, for about 1 hour.

As to step (e), the reaction with PTAD may be carried out in a solvent such as e.g. ethyl acetate, by adding PTAD until the red color persists. The hydroxy group is preferably protected by reaction with tertbutyldimethylsilylchloride in dimethylformamide in the presence of imidazole

As to step (f), the oxidizing agent is preferably mchloroperbenzoic acid (MCBA) dr alkaline hydrogen peroxide in an organic solvent, such as e.g. chloroform, methylene chloride, ethyl acetate or a mixture thereof. The reaction is usually carried out at a temperature varying from OOC to room temperature, for a time of from 8 to 24 hours. The protecting group Q can be removed e.g. by treatment with tetrahydrofurane, acetic acid, water, or with tetrabutylammonium fluoride in tetrahydrofurane.

As to step (g), the reduction is preferably carried out with lithium aluminum hydride in an organic solvent such as e.g.

tetrahydrofurane or diethylether, at the reflux temperature of the reaction mixture, for a time varying from 1 to 3 hours.

As to step (h), the reaction can be carried out by irradiating a solution of compound (II) in an organic solvent such as e.g. diethylether, tetrahydrofurane, ethanol, or a mixture thereof, with a high-energy (e.g. 450 W), highpressure mercury lamp, for a time varying from a few second to about 5 minutes, at a temperature of from -300C to 100C.

Then, after evaporating the organic solvent and adding ethanol, benzene or ethyl acetate, or mixture thereof, the solution is heated at the reflux temperature for a time varying from about 0.5 to 3 hours.

According to the above Scheme II, a compound of formula (I) can be prepared by a process which comprises: (a') reacting a compound of formula:

wherein: W is a C1-C3 alkyl group; A is selected from: a C1-C3 alkylene group; an ethenylene group -CH=CH-; an ethynylene group -C-C-; an oxygen atom -0-; B is selected from: a single bond; a C1-C6 alkylene group optionally substituted by a hydroxy group or a halogen atom, preferably fluorine; a C1-C4 alkenylene group; a C1-C4 alkynylene group; a C6-C10 arylene group; and a C7-Cg arylalkylene group; T is selected from: a

group, wherein R1 and R2 are, each independently, C1-C4 alkyl groups, optionally substituted by one or more fluorine atoms, and X is hydrogen or optionally protected hydroxy; a cyclopropyl group; R3 is a Cl-C4 alkyl group, preferably methyl or ethyl; with an inorganic or organic acid in the presence of water, in an organic solvent; so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (b') reacting the compound of formula (VIII) as defined above with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in an organic solvent, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (c') reacting the compound of formula (VII) as defined above with an oxidizing agent, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (d') reacting the compound of formula (X) as defined above with lithium in a mixture of liquid ammonia and tetrahydrofurane, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (e) reacting the compound of formula (XI) as defined above with dibromodimethylhydantoin or N-bromosuccinimide (NBS) in an organic solvent, and then dehydrobrominating the resulting bromo derivative, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (f') treating the compound of formula (II) as defined above by photolysis, so obtaining a compound of formula (I) as defined above, and optionally removing any protecting group that could be present in A, B, or T.

As to step (d'), lithium is used in a large excess and a proton donor (such as e.g. ammonium chloride at -330C) is usually added at the reaction end.

As to step (e'), the organic solvent is preferably carbontetrachloride or hexane. The reaction is usually carried out at a temperature from room temperature to the reflux temperature of the reaction mixture, for a time varying from a few minutes to 2 hours. The dehydrobromination can be carried out by ref fluxing the resulting bromo derivative in a high boiling solvent such as e.g. xylene, in the presence of an organic base such as e.g. collidine.

A compound of formula (IX) having as side chain A-B-T:

wherein Rl and R2 are as defined above, m is an integer from 0 to 2 and n is an integer from 0 to 5, may be obtained according to the following synthetic Scheme III.

SCHEME III

w CH20H < CHO 2 cHo Cr03 CH OH (XIII) (Collins) v 9 (Collins) 9 (XII) (XIII) OR3 OR3 p, w Ph3P=CH~(CH=CH)=-l (CH2)n C OP r > R -cl-Op (CH=CH) - (CH) R m 2 R2 (XIV) OR3 H2/P y ldeprotect w W R (CH ~~~R1 (CH=CH) (CH2) c-Op COH y [ (XVt (XV) OR3 OR3 deprotect w d: (CH2)n pl -C--OH R2 OR3 (XVI) wherein: W is a Cl-C3 alkyl group; R3 is a C1-C4 alkyl group, preferably methyl or ethyl; and P is a hydroxy protecting group, such as: tetrahydropyranyl (THP) methoxymethyl (MOM), trimethylsilyl (TMS), dimethyltertbutylsilyl (TBDMS), etc.

The reactions of Scheme III can be carried out as described, e.g., in: Zh. Obshch. Khim., 44, 2596 (1974), Zh. Obshch.

Khim., 45, 925 (1975).

A compound of formula (IX) having as side chain A-B-T:

wherein Rl and R2 are as defined above, m is an integer from 0 to 3, may be obtained according to the following Scheme IV: SCHEME IV

w w g TsC1 OTS pyridine J OR3 OR3 (XII) (XVIII) w CH2 CR,I I Nal nBuLi + PhS02R DXF OR 2 (XIX) (Xx) w PbS 02 RlOTHP < (CH ) AR 1) 5% Na/Hg 2 2=-1 2 2)pTsOH/H20/Dioxane OR3 (xxI) W R1 OH (CH2) (CH rC v OR3 (XXII) wherein: W is a Cl-C3 alkyl group; R3 is a Cl-C4 alkyl group, preferably methyl or ethyl; and P is a hydroxy protecting group, such as: tetrahydropyranyl (THP), methoxymethyl (MOM), trimethylsilyl (TMS), dimethyltertbutylsilyl (TBDMS), etc.

The reactions of Scheme IV can be carried out as described e.g. in Chem. Pharm. Bull., 42 (11), 2349-51 (1994).

Scheme IV can be carried out in a slightly modified manner as reported in the following Scheme IV: SCHEME IV'

w w CH2OH TSC1 < CH2OTS Tscl WAS (XII) pyridine OR3 (XVIII) OR3 (XII) OR3 (XVIII)

W O mO (CH2)2-.lR 1) 590 Na/H9 R, 2)pTsOH/H,O/Dioxane OR, i 2) (XXIV) OR

A compound of formula (IX) having as side chain A-B-T:

wherein R 1 and RF2 are R1 and R2 groups as defined above substituted by one or more fluorine atoms, and n is an integer from 0 to 5, may be obtained according to the following reaction Scheme V: SCHEME V

w w CHO Cr03 pyridine (XII) < (XII) (Collins) 7 < (XIII) OR3 OR3 W wP-CH2(CH)COOEt (CH2)n /Pd/EtoH Eto\ll -CII, (CB) coOEt H2 /Pd/EtOII (XXVI) OR3 w (CH,)=COOEt < W (CH,) w (cH2) COOEti-l (cOH LiAlE4 (XXVII) (XazII) OR3 OR3 w w 2n Tscl < LiBr < dine Ay7) (XXIX) < (XXX) OR3 OR3 so,Ph R1 + (xxxII) (XXXT) (XXXII) 0L w SO Ph W ,1rS" 2 of < /t flR2POH cmnrIv, 04 04 wherein: W and R3 are as defined above.

The reactions of Scheme V can be carried out as described e.g. in Chem. Pharm. Bull., 30 (12), 4297-303 (1982).

A compound of formula (IX) having as side chain A-B-T:

wherein R1 and R2 are as defined above, and n is an integer from 0 to 5, may be prepared according to the following Scheme VI.

SCHEME VI

w w 1) DABCO, t 1) CH2OH < ~ 1/ N C CHO '-bipyridyl > | CrO3 + | 2,2'-bipyridyl pyridine (Collins) 2) NaRH4 (XII) (XII) & (XIII) OR2 OR3

wherein W, R1 and R2 are as defined above, and TBDMS is a group tertbutylsilyl.

The reactions of Scheme VI can be carried out as described e.g. in Biorg. Med. Chem. Lett. 3 (2), 341-44 (1993) A compound of formula (IX) having as side chain A-B-T:

wherein R1 and R2 are as defined above, and n is an integer from 0 to 3, may be prepared according to the following Scheme VII: SCHEME VII

wherein W, R1, R2 and R3 are as defined above and TBDMS is a tertbutyldimethylsilyl group.

The reactions of Scheme VII can be carried out as described e.g. in J.O.C. 60, 767-71 (1995).

A compound of formula (IX) having as side chain A-B-T:

wherein RF1 and RF2 are R1 and R2 groups as defined above substituted by one or more fluorine atoms, and n is an integer from 1 to 5, may be prepared according to the following Scheme VIII.

SCHEME VIII

W w CH OH 3 < CHO 2 Cr03 pyridine pyridineOR, (Collins) 3 OR (XII) (XIII)

wherein W, RF1 and RF2 are as defined above and MOM is a methoxymethyl group.

The reactions of Scheme VIII can be carried out as described in Chem. Pharm. Bull., 40 (6), 1647-49 (1992).

A compound of formula (IX), wherein the side chain A-B-T is:

wherein R1 and R2 are as defined above, may be prepared according to the following Scheme IX.

SCHEME IX

wherein MOM is a methoxymethyl group.

The reaction of Scheme IX can be carried out as reported e.g.

in Biorg. Med. Chem. Lett., 3 (2), 341-344 (1993).

A compound of formula (IX) having as side chain A-B-T:

wherein R1 and R2 are as defined above, m and n are integers from 0 to 2, and the substituents on the aromatic ring are in meta or para position, may be prepared according to the following Scheme X: SCHEME X

n410H deprotect yr(cH2) i (H2) 2 (LIII) OR3 The reactions of Scheme X can be carried out analogously to what reported in, e.g., J. Med. Chem., 34, 2452-63 (1991).

A compound of formula (IX) having as side cha-in A-B-T:

wherein R1 and R2 are as defined above, m and n are integers from 0 to 2, and the substituents on the aromatic ring are in meta or para position, may be prepared according to the following Scheme XI: SCHEME XI

wherein Rl, R2, W, m, and n are as defined above, P is a hydroxy protecting group, such as: tetrahydropyranyl (THP), methoxymethyl (MOM), trimethylsilyl (TMS), dimethyltertbutylsilyl (TBDMS), etc., and X is a halogen atom such as fluorine, chlorine, bromine, or iodine.

The reactions of Scheme XI can be carried out analogously to what reported in, e.g., Biorg. Med. Chem. Lett., 3 (2), 341 A compound of formula (IX) having as side chain A-B-T:

wherein T is

or cyclopropyl, may be obtained according to the following synthetic Scheme XII as reported below.

SCHEME XII

w w CH2OH CHO cur03 pyridine (Collins) (XII) i (XIII) OR3 OR3 W Ph3P=CH-Co-T T < (LVII) 1 o NaB4 / EtOH H2/Pd/EtoH w W T T 1 OR3 (LVIII) (LIX) NaBHq /EtOH W > ' X r OOH WT (a) OH activation F ~~~~~~~~~~~ OH (b) F OR3 (LX) OR3 (LXI) OR3 ( LXI ) The reactions of Scheme XII can be carried out analogously to what reported, e.g., in Tetr., 43 (20), 4609-4619 (1987).

The compounds of formula (XII) and (XXXIX) -may be obtained according to the following reaction Scheme XIII.

SCHEME XIII

OH CrC3 0 Cornforth OR OR3 (LXII) (XXXIX) ( LX I I ) w w Ph3P COOEt > COOEt H2 EtONa/EtOH l | \ 10% Pd/C reflux 14b OR3 OR3 (LXIV) (LXIII) (LXIV)

The oxidation of compound (LXII) to compound (XXXIX) is preferably carried out with the Cornforth reagent, as reported e.g. in Tetr. 18, 1351 (1962).

To transform compound (XXXIX) into compound (LXIII), a Wittig-Horner reaction is carried out, according to the method reported, e.g., in J. Chem. Soc. Perkin Trans. I, 1282 (1978) for the 17-keto analogue. A mixture of 17E and 17Z stereoisomers is so obtained, which can be separated or maintained as a mixture.

The hydrogenation of compound (LXIII) to yield compound (LXIV) is preferably carried out in an alcoholic solvent such as, for example, methanol or ethanol, in the presence of a suitable hydrogenation catalyst, such as 10% Pd/C, in hydrogen atmosphere with a pressure of about 1 atm, for about 12 hours at about room temperature.

Compound (LXIV) can be reduced to compound (XII) by means of lithium aluminum hydride in an ethereal solvent such as, for example, diethylether or tetrahydrofurane.

The compound of formula (LXII) may be obtained from dehydroepiandrosterone (LXV) according to the following Scheme XIV: SCHEME XIV

After protection of the 3-hydroxy-A -moiety as i-alcohoxy (R3 is, as defined above, but preferably ethoxy), the 16,17 double bond is formed according to the method reported, e.g., in Arzneim Forsch./Drug Res. 30 (1), no. 2a, 342 (1980).

The selective hydration of the double bond at the 16 position is carried out according to what reported in J. Chem. Soc.

Perkin Trans. I, 1985 (1991).

PHARMACOLOGY The analogues of vitamin D3 related to this invention, with the side chain transposed from the 17ss position to the 16a/16ss position maintain a biological activity similar to vitamin D3, and constitute an entirely novel class of vitamin D2 analogues.

Such novel compounds have a selective activity on cell function, such as inhibition of cell proliferation (nonmalignant cells such as keratinocytes as well as malignant cell such as breast carcinoma, osteo-sarcoma and leukemia cells) and also have a high potency for induction of cell differentiation (e.g. cell types as just mentioned) but on the other hand have strikingly lower effect on calcium and bone homeostasis as evaluated in rachitic chicks (by measuring serum and bone calcium, and by measurement of two vitamin D3-dependent proteins, serum osteocalcin and duodenal calbindin D) as well as in vitamin D3 repleted normal mice (using similar end points). Thus, unlike the classical vitamin D3 compounds, the new drugs do not have the same toxic effect on calcium and bone homeostasis.

In light of prior art it was unexpected and surprising that the transfer of the side chain from the 17ss position to the 16a/16 position could cause such a modification of the spectrum of activities of vitamin D3.

Specifically the new drugs can be used for the therapy or prevention of the following diseases.

- Immune disorders, such as autoimmune diseases (such as, but not limited to diabetes mellitus type 1, multiple sclerosis, lupus like disorders, asthma, glomerulonephritis, etc.) selective dysfunctions of the immune system (e.g. AIDS) and prevention of immune rejection (such as rejections of grafts or prevention of graft versus host disease). The newly invented drugs can either be used alone or in combination with other drugs known to interfere with the immune system (e.g.

cyclosporin, FK 506, glucocorticoids, monoclonal antibodies, cytokines of growth factors, and the like). In analogy with the immune activity of the new compounds, similar effects can be expected in other inflammatory diseases (e.g. rheumatoid arthritis).

Skin disorders either characterized by hyperproliferation and/or inflammation and/or (auto) immune reaction (e.g.

psoriasis, dyskeratosis, acne). Moreover since these drugs can stimulate the differentiation of skin cells they can be used for the treatment or prevention of alaopecia of different origin including alopecia due to chemotherapy or irradiation.

Hyperproliferative disorders and cancer such as hyperproliferative skin diseases (e.g. psoriasis) and several types of cancers and their metastases (all types of cancer which have or can be induced to have vitamin D3 receptors such as but not limited to breast cancer, leukemia, myelo-dysplastic syndromes and lymphomas, squamous cell carcinomas and gastrointestinal cancers, melanomas, osteosarcoma, etc.). The newly invented drugs can, again as for the other indications, be used alone in the appropriate form and route of administration or used in combination with other drugs known to be of therapeutic value in such disorders. These new drugs may be particularly advantageous for such disease as they can, in contrast to classical chemo-therapeutic agents, also stimulate cell differentiation.

- Endocrine disorders since vitamin D3 analogues can modulate hormone secretion, such as increased insulin secret ion or selective suppression of parthyroid hormone secretion (e.g. in chronic renal failure and secondary hyperparathyroidism).

- Diseases characterized by abnormal intracellular calcium handling since the new drugs have favorable effects in cells whose functions depend largely on intracellular calcium movements (e.g. endocrine cells, muscle).

The use of the compounds can find application as well in human disorders as in veterinary medicine.

The amount of the new compounds necessary for their therapeutic effect can vary according to its indication, route of administration and species (animal/man) treated. The compounds can be administered by enteral, parenteral or local topical route. In the treatment of dermatological disorders a topical application as ointment, cream or lotion is to be preferred over systemic treatment, preferably in a dose of 0.1 to 500 pg/g. The systemic administration as tablets, capsules, liquid or as sterile preparation in an appropriate carrier, diluent and/or solvent for parenteral injection will use microgram quantities of the compounds per day depending on the indication and the clinical/veterinary situation.

The advantage of the new compounds over the natural or existing vitamin D3 metabolites or analogues is due to their intrinsic activity in induction of cell differentiation, inhibition of cell proliferation and modulation of the cellular activity in general, while nevertheless displaying reduced effects in vivo. Indeed such calcemic effects, present in other vitamin D3 metabolites or analogues are to be considered as undesired side effects since the doses required for to above mentioned indications are sometimes supraphysiologic and would results in serious calcemic abnormalities when other vitamin D3 metabolites or analogues would be used.

Biological evaluation of the novel vitamin D3 analogues 1. Bindings properties of the new novel vitamin D3 analogues The methods used to evaluate the binding properties of the new analogues are examples of the state of the art techniques used for steroid hormone (including vitamin D3) binding assays as described previously.

The affinity of the analogues of la,25-(OH2)D3 to vitamin D3 receptor was evaluated by their ability to compete with [3H]la,25-(OH2)D3 (specific activity 180 Ci/mmol Amersham, Buckinghamshire, UK) for binding to the high speed supernatant from intestinal mucosa homogenates obtained from normal pigs. The incubation was performed at 40C for 20 h and phase separation was obtained by addition of dextrane-coated charcoal. The affinity for la,25-(OH2)D3 was 1.06 + 0.38 x 1010 M (M + SD, n=10). The relative affinity of the analogues was calculated from their concentration needed to displace 50% of [3H]la,25-(OH2)D3 from its receptor compared with la,25-(OH2)D3 (assigned a 100% value). (Table 2).

The relative affinity for hDBP was measured by incubating [ H]1a,25-(OH2)D3 and increasing concentrations of la,25 (OH2)D3 or its analogues with purified hDBP (0.2 zM) in 1 ml (0.01 M Tris-HCl, 0.154 M NaCl, pH 7.4) for 3 h at 40C, followed by phase separation by addition of cold dextran coated charcoal.

The results obtained with some examples of the new analogues are given in Table 2. These data clearly show a binding to the vitamin D3 receptor, necessary for their biological activity, while their binding for the vitamin D3 binding protein, known as DBP, is decreased in comparison with la,25-(OH2)D3. We and others have previously demonstrated for other vitamin D3 analogues that such reduced binding to DBP enhances its ratio of cell differentiating over calcemic effects.

2. Effects of the novel vitamin D3 analogues on cell proliferation and cell differentiation The cell culture systems were used according to the state of the art: - to evaluate the effects on cell proliferation of non malignant cells and especially to evaluate their potential for use for dermatological disorders, the new compounds were tested in cultures of human normal keratinocytes.

Human skin keratinocytes were isolated and cultured using a modification of the method of Kitano and Okada.

Briefly, the skin from biopsies of patients with breast tumors, was cutted into pieces measuring 3-5 mm and soaked overnight at 40C ' in a solution of dispase (20 Boehringher units/ml). The epidermis was peeled from the dermis, washed with calcium- and magnesium-free phosphate buffered saline and incubated and shacked in a 0.25% trypsin solution for 10 min at room temperature.

The reaction was then stopped by addition of PBS containing 10% FCS. The cells were collected after centrifugation at 40C for 10 min at 800 rpm. After an additional washing with PBS, the pellet was suspended in culture medium into 25 cm2 primaria flaks from Becton Dickinson, The keratinocytes were cultivated at 370C in an atmosphere of 5% CO2 in air. A few hours later, the medium was replaced by new one. The medium [Keratinocytes from Gibco containing Epidermal Growth Factor (5 ng/ml), Bovine Pituitary Extract (35-50 Fg/ml) and antibiotics] was renewed every other day until confluency.

Keratinocytes were cultured in 96-well plate and, after 24 hours, were treated with various concentrations of vitamin D3 analogues, followed by pulse labeling with 1 zCi of [3H]thymidine for 3 hours. Cultures were washed 3 times with PBS and twice with 10% (v/v) ice cold trichloroacetic acid. cells were solubilized with 1M NaOH and radioactivity was counted in a scintillation counter.

to evaluate the effects on cell proliferation and induction of cell differentiation malignant cells were grown in vitro and their proliferation was evaluated by measuring cell number, protein content and the incorporation of radioactive thymidine. As examples of malignant cell human leukemia cells (HL 60), human osteosarcoma cells (MG 63 cells) and both murine and human breast cancer cells (MCF 7, MFM 223 and GR cells) were used. In addition the effect of the new drugs showed additive effects when tested in combination with other anticancer drugs (e.g. retinoic acids, antiestrogens...).

HL-60 cells were seeded at 1.2 x 105 cells/ml and la,25 (OH2)D3 or its analogues were added in ethanol (final concentration < 0.2%) in RPMI 1640 medium supplemented with 10W heat-inactivated fetal calf serum (FCS) for 4 d at 370C. Cells were then assayed for maturation by NBT reduction assay as described using a hemacytometer, or for proliferation by cell counting and [3H]thymidine incorporation. MG 63 cells, seeded at 5 x 10 cells/ml in 96 well flat bottomed culture plates (Falcon, Becton Dickinson, NJ) in a volume of 0.2 ml of DMEM and 2t FCS, were incubated with la,25-(OH2)D3 or its analogues for 72 h. Osteocalcin was then measured in the culture medium using a homologous human osteocalcin RIA. Breast carcinoma cells (MCF-7 or GR) were grown in DMEM/nut.mix F-12 (HAM) medium supplemented with 10% FCS. Cells (5000/well) were incubated during 24 h in 96 well tissue culture plates (Falcon 3072) followed by a 72 h incubation with/without la, 25- (OH2) D3 or analogues.

The cells were then incubated with [3H]thymidine (1 pCi/well) for 4 h and harvested thereafter in NaOH (0.1 M) and the radioactivity counted. The protein content of the cells was measured by the Pierce BCA protein assay (Rockford, IL).

to evaluate the immune potential of the new drugs their biological activity was tested in a mixed lymphocyte test in vitro according to state of the art procedures; in addition the effects of the analogues for induct ion of differentiation of HL 60 cells into mature monocytes was tested in vitro.

The following examples are given to better illustrate the present invention but do not limit the scope of the invention itself.

The key compound (XII) is synthesized as follows: Example 1 6 -Ethoxy-3a,5-cyclo-androstan-17-one (compound LXVI: R3 = Et) a.) For the preparation of the intermediate dehydroepiandrosterone (DHA) tosylate, to a solution of DRA [compound LXV] (20 g, 69.3 mmol) in pyridine (50 mL) at 0 "C tosyl chloride (20 g) is added portionwise. The reaction is kept for 24 h at room temperature (TLC benzene-ethyl acetate, 8:2) and then is poured into crushed ice (ca. 100 g). The precipitate is filtered off and the solid tosylate is recovered (30 g).

b.) To a solution of dried potassium acetate (37.6 g) in absolute ethanol (1.9 L) under nitrogen, the above tosylate (30 g) is added and the mixture refluxed (3 h; TLC benzeneethyl acetate, 8:2) .The reaction is cooled at room temperature and the solvent is gently removed at reduced pressure. The precipitate is taken with diethyl ether, filtered off and the ethereal solution dried at reduced pressure. The i-ethoxy derivative is obtained as an oil, that solidifies at 1 mm Hg (17.5 g, 80t).

NMR (60 MHz, 3 in ppm): 0.9 (s, 18 CH3), 1.05 (s, 19 CH3), 3.0 (m, 6a H), 3.7 (m, CH2 of i-ethoxy; the corresponding triplet for CH3 is centered at 1.15 ppm). Tetramethylsilane is used as internal standard and the resonances of the cyclopropyl ring of i-ethoxy group are not evidenced.

6 -Ethoxy-3a,5-cyclo-androstan-17-one hydrazone (compound LXVII: R3 = Et] To a solution of the above 17-ketone (17.6 g, 55.4 mmol) in ethanol (210 mL) under nitrogen triethylamine (27 mL) and monohydrated hydrazine (98%, 66.5 mL) are sequentially added and the solution refluxed (3 h; TLC benzene-ethyl acetate, 8:2). Water (210 mL) is added, the product is extracted with dichloromethane (3x200 mL) and the solvent is dried on sodium sulfate. Evaporation at reduced pressure leaves the oily hydrazone pure by TLC (18 g).

17-Bromo-6 -ethoxy-3a,5-cyclo-androst-16-ene compound LXVIII: R3 = Et) To a solution of the above hydrazone (18 g, 54.5 mmol) in anhydrous pyridine (120 mL) at -10 "C a solution of NBS (Nbromosuccinimide, 15.2 g) in dry pyridine (150 mL) is added dropwise, keeping the temperature around -10 "C. The reaction is complete at the end of the addition of NBS (TLC benzeneethyl acetate, 8:2) and a cold solution of 5% HCl (1.5 L) is added. The product is extracted with dichloromethane (3x800 mL) and the solvent is dried on sodium sulfate. Evaporation at reduced pressure leaves the crude bromoene (18.4 g) that is rapidly purified, pouring a hexane solution on neutral aluminum oxide (370 g). The title compound is recovered (10 is rapidly purified, pouring a hexane solution on neutral aluminum oxide (370 g). The title compound is recovered (10 g, 48%) pure by TLC.

NMR (60 MHz, 8 in ppm): 0.9 (s, 18 CH3), 1.05 (s, 19 CH3), 3.0 (m, 6a H), 3.7 (m, CH2 of i-ethoxy; the corresponding triplet for CH3 is centered at 1.15 ppm), 5.95 (m. 16-H).

6-Ethoxy-3(x,5-cyclo-androst-16-ene [compound LXIX: R3 = Et) To a solution of the previous 17-bromo derivative (10 g) in absolute ethanol (580 mL), under nitrogen and at room temperature, sodium (37 g) is added portionwise (12 h). The reaction is monitored by NMR following the integration and molteplicity of the signal at 5.95 ppm. At the end of the addition of sodium, the reaction is cooled at 0-5 C and is carefully poured into chilled water and the product is recovered by extraction with diethyl ether (3x300 mL). The organic solution is dried onto sodium sulfate, the solvent is removed at reduced pressure and the title compound is obtained, pure by TLC (7 g, 90%).

NMR (60 MHz, 8 in ppm): 0.8 (s, 18 CH3), 1.05 (s, 19 CH3), 3.0 (m, 6a H), 3.7 (m, CH2 of i-ethoxy; the corresponding triplet for CH3 is centered at 1.15 ppm), 5.95 (m. 16 and 17 -H).

l6-Hydroxy-6-ethoxy-3a, 5-cyclo-androstane (compound LXII: R3 = Et) Under nitrogen to a solution of 16,17-ene (7 g. 23.3 mmol) in anhydrous tetrahydrofuran (80 mL) a solution of 0.5 M 9-BBN (9-[3.3.1]borabicyclononane) in tetrahydrofuran (70 mL) is added and the solution is reflexed (8 h). After this time (TLC, benzene/ethyl acetate, 6:4) 9-BBN is still added (35 mL) and the solution is additionally refluxed (4 h). The reaction is cooled to 0-5"C and addition of the following reactants is performed: 3 N NaOH (290 mL), 30% H202 (290 mL), potassium carbonate (3.6 g). The solution is stirred for 1 h at 0 "C, then the product is extracted with diethyl ether (3x300 mL), washing the organic solution with a saturated solution of ferrous sulfate until complete elimination of peroxides. The organic solution is dried on sodium sulfate and the solvent removed at reduced pressure, leaving a residue (10 g) that is poured onto an aluminum oxide column (200 g). Elution with hexane affords the unreacted 16,17-ene (1 g) and with hexane/ethyl acetate (7:3) title 16-hydroxy is obtained (5.8 g, 79%).

NMR (500 MHz, 8 in ppm): 0.35 and 0.60 (two multiplets for CH2 of i-ethoxy), 0.75 [s, 18 CH3 of the 16a-isomer corresponding to 70% of the mixture; at 0.97 ppm 18 CH3 of the 16 -isomer (30t)], 0.95 (s, 19 CH3), 1.15 (s, CH3 of iethoxy), 2.85 (m, 6a H), 3.35 and 3.60 (two m, CH2 of i ethoxy) , 4.35 (q, 16a-H, 30%), 4.44 (q, 16 -H, 70%).

6 -Ethoxy-3α,5-cyclo-androstan-16-one (compound XXXIX: R3 = Et) To a solution of 16-hydroxy (5.8 g, 18.2 mmol) in pyridine (20 mL), 44 mL of a solution of the Cornforth's reagent [chromium trioxide (1 g) in water (1 mL) added to 10 mL of pyridine at 0 "C] is added. After 3 h at room temperature (TLC, benzene/ethyl acetate, 8:2) the chromium reagent is still added (22 mL). The reaction is then kept under stirring for additional 2 h and then poured onto Florisil (150 g) to remove the chromium reagent. Elution with hexane/ethyl acetate (1:1) affords the crude mixture that was purified by column chromatography (aluminum oxide, 150 g). Elution with hexane/ethyl acetate (9:1) affords the title 16-ketone (3.84 g, 67%), whereas the unreacted 16-hydroxy (0.6 g) is obtained with hexane/ethyl acetate (7:3) NMR (60 MHz, 6 in ppm): 0.8 (s, 18 CH3), 1.05 (s, 19 CH3), 3.0 (m, 6a H), 3.7 (q, CH2 of i-ethoxy; the corresponding triplet for CH3 is centered at 1.15 ppm).

Ethyl 2- (6-ethoxy-3a,5-cyclo-androstan-l6-y1)propenoate [compound LXIII: R3 = Et, W = Me] To a solution of 16-keto (3 g, 9.5 mmol) in absolute ethanol (48 mL) under nitrogen triethyl 2-phosphono propionate (Aldrich) is added (6.2 mL, 29 mmol) and the temperature is raised to 35-40 OC. A solution of sodium ethylate (from 0.761 g Na and 24.1 mL absolute ethanol) is added. The solution is then refluxed (16 h) (TLC, benzene/ethyl acetate, 8:2) and triethyl 2-phosphonopropionate (3 mL) and sodium ethylate are added and the solution is additionally refluxed (8 h). The reaction is cooled and the solvent is evaporated, then water is added and the product is extracted with diethyl ether (3x100 mL). The organic solution is diried on sodium sulfate and the solvent removed at reduced pressure, leaving a residue (4 g) that is poured onto an aluminum oxide column (80 g). Elution with hexane affords the title compound (1.92 g, 51%), with hexane/ethyl acetate (95:5) a mixture of the product with starting material (0.16 g) was obtained and the unreacted 16-keto (0.33 g) was eluted with hexane/ethyl acetate (9:1) NMR (60 MHz, 5 in ppm): 0.8 (s, 18 CH3), 1.05 (s, 19 CH3), 1.4 (t, for the CH3 of the ethyl ester), 3.0 (m, 6a H), 3.7 (q, CH2 of i-ethoxy; the corresponding triplet for CH3 is centered at 1.15 ppm), 4.4 (CH2 of the ethyl ester).

Ethyl 2-(6 -ethoxy-3a,5-cyclo-androstan-16-yl)propanoate compound LXIV: R3 = Et, W = Mew A solution of the above unsaturated ester (1.9 g, 4.75 mmol) in absolute ethanol (20 mL) is hydrogenated at ambient presure in the presence of 10W Pd-C (0.19 g). The reaction is monitored by GLC (HP-W5 column, T=280 OC). When the reation is complete, filtration of the catalyst and removal of the solvent affords the saturated 16-propanoate (1.8 g, 94%).

2- (6-Ethoxy-3a, 5-cyclo-androstan-l6-yl)propan-1-ol compound XII: R3 = Et, W = Me] To a solution of the above 16-ester (1.8 g, 4.48 mmol) in anhydrous tetrahydrofuran (10 mL) under nitrogen lithium aluminum hydride (0.536 g) is added at a the temperature of 0 OC. After 2 h (TLC, benzene/ethyl acetate, 8:2) addition of the following reactants is performed: water 0.536 mL, 15% NaOH (0.536 mL), H20 (0.536 mL). The reaction mixture is filtered and the organic solution is removed at reduced pressure, leaving a residue (1.34 g, 83a) of essentially pure alcohol. The most significant resonances are reported for the 500 MHz NMR spectrum (6 in ppm): 0.8 and 1.0 (s, 18 CH3), 0.9 (s, 19 CH3), 0.90-0.97 (two d, CH3 CH), 1.10- 1.15 (two t, CH3 CH2 0), 3.35 and 3.60 (two m, CH2 of i-ethoxy and CH2 OH).

Starting from this compound the different side chains are tailored as described in the general part (schemes III XII), following the bibliographic references herein reported.

For example, a cpompound of the scheme IV' is obtained as follows: Example 2 2-(6 -Ethoxy-3a,5-cyclo-androstan-16-yl)propan-1-iodide compound XIX: R3 = Et, W = Me] a) To a solution of the above alcohol (1.34 g, 3.72 mmol) in anhydrous pyridine (3 mL) p-tosyl chloride (1.1 g) is added at a the temperature of 0 OC. After 24 h at room temperature (TLC, benzene/ethyl acetate, 8:2) the solution is poured into chilly water and pure tosylate [compound XVIII: R3 = Et, W = Me] (1.83 g) is recovered by extraction with dichloromethane (3x5 mL).

b) To a solution of the above tosylate (1.84 g) in acetone (18 mL), lithium iodide (1.8 g) is added portionwise and the solution is refluxed for 8 h (TLC, benzene/ethyl acetate, 9:1). The solvent is evaporated then water is added and the product is extracted with diethyl ether (3x10 mL). The organic solution is washed with 10% sodium thiosulfate, dried on sodium sulfate and the solvent removed at reduced pressure, leaving a residue (1.46 g, 83.5% from 16-propanol).

The most significant resonances (60 MHz NMR, 5 in ppm):for the derivative XIX are: 3.0 (m, 6a H), 3.2-3.8 (m, CH2 I and CH2 O).

1-Phenylsulfonyl-3-butanone - -butanone A mixture of sodium benzenesulfinate (3.9 g, 23.7 mmol), potassium dihydrogenophosphate (3.4 g) and methyl vinylketone (3.1 mL) in water (37 mL) is kept under stirring at room temperature (60 h; TLC, benzene/ethyl acetate, 8:2). The product is filtered off and recrystallized from a concentrated solution in methylene chloride with diethyl ether (3.8 g); M.P. 88 OC NMR (60 MHz, 6 in ppm):2(s, CH3CO), 3.0 (t, CH2CO), 3.4 (t, CH2 SO2), 7.7-8.2 (m, aromatic).

Ketal of l-Phenylsulfonyl-3 -butanone [compound XXIII: m = 1, R1 = Me) To a solution of the previous ketone (3 g) in benzene (10 mL), ethylene glycol (0.8 mL) and concentrated sulfuric acid (4 drops) are added and the reaction is refluxed (5 h) separating the water formed by a Dean-Stark apparatus. The reaction is monitored by NMR and at the end it is poured into a 10% solution of sodium hydrogencarbonate. The solution is brought to neutrality and the organic solution is dried on sodium sulfate and the solvent removed at reduced pressure, leaving a residue (3.15 g, 66% from sodium benzenesulfi

16-(2,2-ethylendioxo-4-sulphonylphenylhept-6-yl)-6ss-ethOxy- 3a, 5-cyclo-androstane compound XXIV: m = 1, R1 = Me, R3 = Et, W = Me) A solution of the above ketal (0.56 g, 2.12 mmol) in tetrahydrofuran (3 mL) is added to a solution of lithium diisopropilamide [prepared from 1.6 M n-butyllithium in hexane (1.36 mL) and diisopropylamine (0.31 mL) at 0 OC] at 70 OC (10 min). A solution of 2-(6b-ethoxy-3a;5-cyclo- androstan-16-yl)propan-l-iodide (0.44 g1 0.93 mmol) in a mixture of tetrahydrofuran (3 mL) and HMPTA (1 mL) is added and the mixture is kept at 0 OC (4h; TLC, benzene/ethyl acetate, 8:2). At the end, ammonium chloride is added to neutrality and the solvent is evaporated, then the product is extracted with dichloromethane (3x10 mL). The organic solution is dried on sodium sulfate and the solvent removed at reduced pressure, leaving a residue (1.74 g) purified by aluminum oxide (40 g) chromatography. Elution with hexane/ethyl acetate (9:1) afforded the steroidal phenylsulfonyl derivative (0.416 g, 47%).

NMR (60 MHz, 8 in ppm): 0.8 -1.6 (complex signal for 18 and 19 CH3, two CH3 of the side chain, CH3 of the i-ethoxy group and methylenes), 2.4 (m, CH2 adjacent to the carbon bearing the ketal moiety), 2.9 (m, 6a H), 3.4 (CHSO2 superimposed with CH2 of the i-ethoxy), 3.9 (m, CH2CH2 of the ketal moiety), 7.7-8.2 (m, aromatic).

16-(2-ethylenedioxohept-6-yl)-6ss-ethoxy-3a,5-cyclo-androstane [compound XXIV: m = 1, R1 = Me, R3 = Et, W = Me, without the phenylsulphonyl group] To a solution of the above phenyl sulphonyl derivative (0.42 g, 0.7 mmol) in absolute ethanol (3 mL) freshly prepared sodium amalgam [Na/Hg (6%)] is added in four portions (4x1.14 g) during 4 days at room temperature under nitrogen (TLC; benzene/ethyl acetate, 8:2). At the end, a saturated solution of ammonium chloride is added to neutrality and the solvent is evaporated, then the product is extracted with dichloromethane (3x10 mL). The organic solution is dried on sodium sulfate and the solvent removed at reduced pressure, leaving a residue (0.31 g) purified by aluminum oxide (10 g) chromatography. Elution with hexane/ethyl acetate (95:5) afforded the title compound (0.18 g, 56W), whereas the unreacted phenylsulfonyl derivative (0.125 g) was obtained with hexane/ethyl acetate (8:2).

NMR (60 MHz, 8 in ppm): 0.8 -1.6 (complex signal for 18 and 19 CH3, two CH3 of the side chain, CH3 of the i-ethoxy group and methylenes), 2.4 (m, CH2 adjacent to the carbon bearing the ketal moiety), 2.9 (m, 6a H), 3.4 (CH2 of the iethoxy), 3.9 (m, CH2CH2 of the ketal moiety).

6- (6-Ethoxy-3a,5-cyclo-androstan-l6-yl)heptan-2-one [compound XXV: m = 1, R1 = Me, R3 =Et, W = Me) To a solution of the above steroidic ketal (0.165 g, 0.36 mmol) in acetone (6 mL), p-toluenesulfonic acid (6 mg) in water (0.6 mL) is added and the solution is kept at room temperature for 19 h (TLC; benzene/ethyl acetate, 8:2). At the end, water is added and .the pH is brought to 7 with a saturated solution of sodium hydrogen carbonate and the solvent is evaporated, then the product is extracted with dichloromethane (3x10 mL). The organic solution is dried on sodium sulfate and the solvent removed at reduced pressure, leaving a residue (0.31 g) purified by aluminum oxide (10 g) chromatography. Elution with hexane/ethyl acetate (95:5) afforded the title compound (0.1 g).

NMR (60 MHz, 6 in ppm): 0.8 -1.6 (complex signal for 18 and 19 CH3, CH3 of the side chain, CH3 of the i-ethoxy group and methylenes), 2.2 (s, CH3CO), 2.4 (m, CH2CO), 2.9 (m, 6a H), 3.4 (CH2 of the i-ethoxy). A signal at 3.9 ppm in the spectrum shows that ca. 20% of unreacted ketal is present in the sample.

2-Methyl-6-(6ss-ethoxy-3a,5-cyclo-androstan-16-yl)heptan-2-ol (compound XXII: m = 1, Rl = R2 = Me, R3 = Et, W = Met To a solution of methylmagnesium bromide in toluenetetrahydrofuran [(3:1)1.4 M, 0.18 mL}, a solution of the above ketone (0.1 g) in tetrahydrofuran (0.5 mL) is added at 0 OC under nitrogen (TLC; benzene/ethyl acetate, 8:2). After 10 min the reaction is worked-up by addition of crushed ice, and 6 N sulfuric acid to pH 5. The solvent is evaporated, water is added and the product is extracted with diethyl ether (3x5 mL), washing the organic solution with sodium hydrogen carbonate. The organic solution is dried on sodium sulfate and the solvent removed at reduced pressure, leaving a residue (84 mg) purified by aluminum oxide (3 g) chromatography. Elution with hexane/ethyl acetate (8:2) afforded the title compound (50 mg). The most significant resonances at 500 MHz NMR are.as follows: (6 in ppm): 0.78 (s, 18 CH3), 0.84 (two d, CH3CH), 0.99 (s, 19 CH3), 1.18 (s, (CH3)2COH) 2.9 (m, 6a H), 3.35 and 3.6 (two m, CH2 of the i-ethoxy).

Using an analogue procedure and the suitable reagents, the following compounds are prepared: compound XXII, wherein: m = 2, R1 = R2 = Me, R3 = Et, W = Me compound XXII, wherein: m = 2, R1 = R2 = Et, R3 = Et, W = Me Following the procedure of the scheme III, starting from the compound of formula (XII), the compounds of formula (XVI) listed below are prepared: compound XVI, wherein: m = 0, n = 2, R1 = R2 = Me, R3 = Et, W = Me compound XVI, wherein: m = 1, n = 1, R1 = R2 = Me, R3 = Et, W = Me compound XVI, wherein: m = 2, n = 0, Rl = R2 = Me, R3 = Et, W = Me Following the procedure of the scheme V, starting from the compound of formula (XII), the compound of formula (XXXIV), wherein: n = 1, R1,R2 = CF3, R3 = Et, W = Me is prepared.

Following the procedure of the scheme VI, starting from the compound of formula (XII), the compounds of formula (XXXVIII) listed below are prepared: compound XXXVIII, wherein: n = 1, Rl = R2 = Me, R3= Et, W = Me compound XXXVIII, wherein: n =' 3, R1 = R2 = Me, R3 = Et, W = Me compound XXXVIII, wherein: n = 3, R1 = R2 = Et, R3= Et, W = Me Following the procedure of the scheme VII, starting from the compound of formula (XII), the compounds of formula (XLIV) listed below are prepared: compound XLIV, wherein: n = 1, R1 = R2 = Me, R3 = Et, W = Me compound XLIV, wherein: n = 2, R1 = R2 = Me, R3 = Et, W = Me compound XLIV, wherein: n = 2, R1 = R2 = Et, R3 = Et, W = Me Following the procedure of the scheme VIII, starting from the compound of formula (XII), the compounds of formula (XLVII) are prepared: compound XLVII, wherein: n = 1, R1 ,R2 = = CF3, R3 = Et, W = Me compound XLVII, wherein: n = 2, R1 F, R2F = CF3, R3 = Et, W = Me Following the procedure of the scheme IX, starting from the compound of formula (XII), the compound of formula (L), wherein: R1 ,R2 = Me, R3 = Et, W = Me is obtained.

All these compounds so obtained, of general formula IX, can be transformed into the final compounds following either the procedure of the scheme I or the procedure of the scheme II.

For example: Example 3 2-Nethyl-6- (3-hydroxy-androst-5-en-16-yl) heptan-2-ol [compound VIII: W = Me, A = CH2, B = CH2CH2, T = C(CH3)2OH] To a solution of the above alcohol (1.075 g) in ethanol (40 mL) p-toluensulphonic acid (0.19 g) are added and the solution is heated to reflux for 1 h. The solvent is removed under vacuum, the crude residue is taken up with ethyl acetate, washed with water until neutral and anhydrified over sodium sulphate. The solvent is evaporated at reduced pressure and the crude solid thus obtained is purified by flash chromatography on silica gel (eluant hexane/ethyl acetate 6:4) to yield 0.86 g of the title diol.

The most significant resonances at 500 MHz NMR are as follows: (6 in ppm): 0.71 (s, 18 CH3), 0.84 (two d, CH3CH) , 0.99 (s, 19 CH3), 1.18 (s, (CH3)2COH) , 3.51 (m, 3a H), 5.36 (m, H 6).

16- (2-Hydroxy-2-methylhept-6-yl)androsta-1,4,6-trien-3-one [compound VII: W = Me, A = CH2, B = CH2CH2, T = C(CH3)2OH] To a solution of the above diol (0.80 g) in dioxane ( 40 mL) DDQ ( 2.1 g) is added and the mixture is refluxed for 15h.

The solution is poured onto a short column of neutral aluminum oxide and eluted with methylene chloride.The solvent was evaporated under vaccum and the reddish solid so obtained is purified again by chromatography on silica gel (eluant: chloroform/ethyl acetate 95:5) to afford 0.45 g of the title compound.

The most significant resonances at 500 MHz NMR are as follows: (6 in ppm): 0.62 (s, 18 CH3), 0.84 (two d, CH3CH), 0.99 (s, 19 CH3), 1.18 (s, (CH3)2COH), 6.12 (d, H 4), 6.5 (m, H 6 + H 7), 6.82 (dd, H 2), 7.08 (d, H 1).

16- (2-Hydroxy-2-methylhept-6-yl) -la,2a-epoxy-androsta-4,6- dien-3-one compound X: W = Me, A = CH2, B = CH2CH2, T = C(CH3)2OH) A solution of the 1,4,6-trien-3-one (0.4 g) in methanol (10 mL) is treated with 10% methanolic sodium hydroxide (0.1 mL) and 30% hydrogen peroxide ( 0.6 mL) and left overnight at room -temperature. The resulting crystalline epoxide was filtered off, washed with cold methanol and dried (0.28 g).

The most significant resonances at 500 MHz NMR are as follows: (8 in ppm)= 0.62 (s, 18 CH3), 0.84 (two d, CH3CH), 0.99 (s, 19 CH3), 1.18 (s, (CH3)2COH), 5.50 (s, H 4), 5.95 (m, H 6 + H 7).

16- (2-Hydroxy-2-methylhept-6-yl) -la,3-dihydroxy-androst-5- ene [compound XI: W = Me, A = CH2, B = CH2CH2, T = C(CH3)2OH) A solution of the above epoxyde (0.21 g) in THF (2 mL) is added to a solution of litium (0.26 g) in a mixture of THF/liquid ammonia (ca. 50:50) at -780C. After half an hour the temperature of the reaction is raised to -330C, and a further amount of litium (0.26 g) and ammonium chloride (0.4 g) is added. The stirring is continued fo 1 h, then the blue colour is discharged with ethanol. afterevaporating the solvents the crude oil is purified by flash chromatography on silica gel (eluant ethyl acetate/chloroform 20:80) to yield 0.13 g of the title compound.

The most significant resonances at 500 MHz NMR are as follows: (6 in ppm): 0.62 (s, 18 CH3), 0.84 (two d, CH3CH), 0.99 (s, 19 CH3), 1.18 (s, (CH3)2COH), 3.76 (s, H 1), 4.07 (m, H 3).

16- (2-Hydroxy-2-methylhept-6-yl) -la,3J3-dihydroxy-androsta- 5,7-diene compound II: W = Me, A = CH2, B = CH2CH2, T = C(CH3)20H] To a ref fluxing solution of the above diol (0.12 g) in carbon tetrachloride (l0mL), N-bromosuccinimide (0.071 g) is added.

The reaction mixture is refluxed for 20 minutes under an argon atmoshere and then filtered. The filtrate is evaporated under vacuum; the residue is dissolved in xylene (8 mL), added dropwise to a ref fluxing solution of s-collidine (1 mL) in xylene (6 mL) under argon and refluxed for 10 min. After cooling, the reaction mixture is extracted with ehyl acetate; the combined extracts are washed with 1N hydrochloric acid, with brine and anhydrified over sodium sulphate. The solvent is removed under vacuum and the crude is purified by flash chromatography on silica gel (eluant: hexane/ethyl acetate 65:35) to afford 52 mg of the title compound.

The most significant resonances at 500 MHz NMR are as follows: (6 in ppm) : 0.62 (s, 18 CH3) , 0.84 (two d, CH3CH), 0.99 (s, 19 CH3), 1.18 (s, (CH3)2COH), 3.76 (s, H 1), 4.07 (m, H 3), 5.37 and 5.71 (H 6 + H 7).

(lS,3R,5Z,7E) -16- (2-Hydroxy-2-methylhept-6-yl) -la,3J3- dihydro::y-9,10-secoandrosta-5,7,10(19) -triene compound (I): W = Me , A = CH2, B = CH2CH2, T = C(CH3)2OH] The 5,7-diene (49 mg) over obtained is dissolved in a mixture of diethylether and THF (500 mL; 1:3 , v/v) and irradiated with a 450 W high-pressure mercury lamp for 2 minutes using 1.2% potassium nitrate solution as filter. The solution is evaporated, the residue is dissolved in ethanol (5 ml) and refluxed for 1 h. After evaporation of the solvent the residue is purified by flash chromatography (eluant: ethyl acetate/chloroform 10:90) to yield 11 mg of the title compound.

MS (m/z): 416 M+, 398 M+ - H2O, 380 398 - H2O NMR (CDCl3) : (8 in ppm): 0.62 (s, 18 CH3), 0.84 (two d, CH3CH), 0.99 (s, 19 CH3), 1.18 (s, (CH3)2COH), 4.21(m, H 3), 4.43 (m, H 1), 5.00 (s, H 19Z), 5.34 (s, H 19E), 6.03 and 6.40 (ABq, H 6 + H 7).

Following an analogous procedure, all of the other preferred compounds 2 to 16 as reported in Table 1 were obtained.

Claims

1. A compound of formula:

wherein: W is a C;-C3 alkyl group; A is selected from: a C-- C3 alkylene group; an ethenylene group -CH=CH-; an ethynylene group -C#C-; and an oxygen atom -0-; B is selected from: a single bond; a C1-C6 alkylene group optionally substituted by a hydroxy group or a halogen atom; a C1-C4 alkenylene group; a C,-C, alkynylene group; a C6-Cl0 arylene group; and a C-Cg arylalkylene group; T is selected from: a

group, wherein Rl and R2 are, each independently, Ci-Cz alkyl groups, optionally substituted by one or more fluorine atoms, and X is hydrogen or hydroxy; and a cyclopropyl group.

2. A compound according to claim 1, wherein group T is selected from:

3. A compound according to claim 1, wherein group T is selected from:

4. A compound according to claim 1, wherein group W is methyl or ethyl.

5. A compound according to claim 1, wherein group A is a C1-C3 alkylene group or an ethenylene group.

6. A compound according to claim 1, wherein group B is selected from: a C1-C3 straight alkylene group;

-CH=CH-, -CH=CH-CH2-; -CEC-, -C=C-CH2-;

7. A compound according to claim 1, wherein: W is -CH3, A is -CH2-, B is -CH2-CH2-, T is

W is -CH3, A is -CH2-, B is -CH2-CH2-CH2-, T is

W is -CH3, A is -CH2-, B is -CH2-CH2-CH2-CH2-, T is

W is -CH3, A is -CH=CH-, B is -CH2-, T is

W is -CH3, A is -CH=CH-, B is -CH=CH-, T is

W is -CH3, A is -CH2-, B is -CH2-CH2-, T is

W is -CM3, A is -O-, B is -CH2-CH2-, T is

i W is -CM3, A is -O-, B is -CH2-CH2-CH2-, T is

W is -CM3, A is -O-, B is -CH2-CH2-CH2-CH2-, T is

W is -CH3, A is -CH2-, B is -C#C-, T is

W is -CM3, A is -CH2-, B is -C#C-, T is

W is -CM3, A is -CH2-CH2-, B is -C-C-, T is

W is -CH3, A is -CH2-CH2-, B is -CC-, T is

W is -CH3, A is -CH2-CH2-, B is -CrC-, T is

W is -CH3, A is -O-, B is -CH2-C-C-, T is

8. A process for preparing a compound of formula (I) according to claim 1, which comprises: (a) reacting a compound of formula:

wherein: W is a C1-C3 alkyl group; A is selected from: a C1-C3 alkylene group; an ethenylene group -CH=CH-; an ethynylene group -CEC-; an oxygen atom -0-; B is selected from: a single bond; a C1-C6 alkylene group optionally substituted by a hydroxy group or a halogen atom, preferably fluorine; a C1-C4 alkenylene group; a Cl-C4 alkynylene group; a C6-C10 arylene group; and a C,-C9 arylalkylene group; T is selected from: a

group, wherein Ri and R2 are, each independently, C1-C4 alkyl groups, optionally substituted by one or more fluorine atoms, and X is hydrogen or optionally protected hydroxy; a cyclopropyl group; R3 is a C1-C4 alkyl group, preferably methyl or ethyl; with an inorganic or organic acid in the presence of water, in an organic solvent; so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (b) reacting the compound of formula (VIII) as defined above with 2,3-dichloro-5, 6-dicyano-1,4-benzoquinone (DDQ) in an organic solvent, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (e) reacting the compound of formula (V) as defined above with 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD), and then protecting the hydroxy group, so obtaining a compound of formula:

9. A process for preparing a compound of formula (I) according to claim 1, which comprises: (a') reacting a compound of formula:

wherein: W is a C1-C3 alkyl group; A is selected from: a C1-C3 alkylene group; an ethenylene group -CH=CH-; an ethynylene group -CC-; an oxygen atom -0-; B is selected from: a single bond; a C1-C6 alkylene group optionally substituted by a hydroxy group or a halogen atom, preferably fluorine; a C1-C4 alkenylene group; a C1-C4 alkynylene group; a C6-C10 arylene group; and a C7-Cg arylalkylene group; T is selected from: a

group, wherein Rl and R2 are, each independently, C1 -C4 alkyl groups, optionally substituted by one or more fluorine atoms, and X is hydrogen or optionally protected hydroxy; a cyclopropyl group; R3 is a C1-C4 alkyl group, preferably methyl or ethyl; with an inorganic or organic acid in the presence of water, in an organic solvent; so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (b') reacting the compound of formula (VIII) as defined above with 2,3-dichloro-5, 6-dicyano-l,4-benzoquinone (DDQ) in an organic solvent, so obtaining a compound of formula:

wherein W, A, B, and T are defined as above; (e') reacting the compound of formula (XI) as defined above with dibromodimethylhydantoin or N-bromosuccinimide (NBS) in an organic solvent, and then dehydrobrominating the resulting bromo derivative, so obtaining a compound of formula:

10. A pharmaceutical compostition, which comprises a compound of formula (I) according to claim 1 as an active principle, in association with one or more pharmaceutically acceptable carrier and/or diluent.

11. A compound of formula (I) according to claim 1, for use in a method of treating the human or animal body by therapy.

12. A compound as claimed in claim 11, for use as an antiproliferative and antitumor agent.

13. Use of a compound of formula (I) according to claim 1, in the manufacture of a medicament for use in a method for treating cancer.