POSITION 16,17 WITH PYRROLIDINE RING

FIELD OF THE INVENTION

The present invention relates to novel anti-inflammatory and antiallergic compounds of the glucocorticosteroid series, methods of preparing such compounds, pharmaceutical compositions comprising them, combinations and therapeutic uses thereof. More particularly, the invention relates to glucocorticosteroids that are derivatives of pyrrolidine.

BACKGROUND OF THE INVENTION

Corticosteroids are potent anti-inflammatory agents, able to decrease the number, activity and movement of inflammatory cells.

Corticosteroids are commonly used to treat a wide range of chronic and acute inflammatory conditions including asthma, chronic obstructive pulmonary disease (COPD), allergic rhinitis, rheumatoid arthritis, inflammatory bowel disease and autoimmune diseases.

Corticosteroids mediate their effects through the glucocorticoid receptor (G ). The binding of corticosteroids to GR induces its nuclear translocation which, in turn, affects a number of downstream pathways via DNA-binding- dependent (e.g. transactivation) and DNA-binding -independent (e.g. transrepression) mechanisms.

Corticosteroid for treating chronic inflammatory conditions in the lung such as asthma and COPD are currently administered through inhalation. One of the advantages of employing inhaled corticosteroids (ICS) is the possibility of delivering the drug directly at the site of action, limiting systemic side-effects, thus resulting in a more rapid clinical response and a higher therapeutic ratio.

Although ICS treatment can yield important benefits, expecially in asthma, it is important to minimise ICS systemic exposure which leads to the occurrence and severity of unwanted side effects that may be associated with chronic administration. Moreover, the limited duration of action of ICS currently available in the clinical practice contributes to suboptimal management of the disease. While the inhaler technology is the key point to target the lung, the modulation of the substituents on the corticosteroids molecular scaffold is important for the optimization of pharmacokinetic and pharmacodynamic properties in order to decrease oral bioavailability, confine pharmacological activity only in the lung (prodrugs and soft drugs) and increase systemic clearance. Morever, long lasting ICS activity in the lung is highly desirable as once daily administration of ICS would allow the reduction of the frequency of administration and, thus, substantially improve patient compliance and, as a result, disease management and control. In sum, there is a pressing medical need for developing ICS with improved pharmacokinetic and pharmacodynamic characteristics.

Fluticasone furoate is an example of enhanced affinity glucocorticoid that has been developed as topical therapy for allergic rhinitis with a unique combination of pharmacodynamic and physicochemical properties which render this compound long acting in the lung and rapidly inactivated by hepatic metabolism to reduce systemic side effects (Salter et al., 2007).

To the extent of our knowledge, glucocorticoids pyrrolidine derivatives have never been described, except for the co-pending patent application PCT/EP201 1/051537, where some pyrrolidine derivatives were described.

Surprisingly, it has been found that the compounds of the invention show a particular good potency.

SUMMARY OF THE INVENTION

The invention is directed to compounds of general formula (I), and to the corresponding compounds of general formula (Γ) wherein, the configuration of some stereogenic centers is fixed. The invention is also directed to pharmaceutically acceptable acid addition salts of compounds of formula (I) and (Γ), to methods of preparing such compounds, to combinations with one or more active ingredients selected from the classes of 2-agonist, antimuscarinic agents, PDE4 inhibitors, P38 MAP kinase inhibitors and IKK2 inhibitors, to pharmaceutical compositions comprising them and to therapeutic uses thereof.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect, the invention is directed to compounds of general formula (I)

(I)

wherein

R is (CH ₂ ) _n -V-(CH ₂ ) _n '-R ₄ wherein n and n' are independently 0 or 1 ;

V is absent or is selected from -O-, -S- and -OC(O)-;

R ₄ is selected from the group consisting of:

- -H, -OH, -CN or halogen;

(Ci-C ₆ )alkyl optionally substituted by one or more groups selected from oxo, -CN, -SH or halogen;

R ₂ is (CH ₂ ) _m -R ₆ wherein m = 0 or an integer from 1 to 4;

R ₆ is selected from the group consisting of aryl and heteroaryl, optionally substituted by one or more groups selected from oxo, -OH, -CN, -COOH, (C C ₆ )alkoxy, (C C ₆ )alkyl, (C C ₆ )haloalkyl and halogen;

and pharmaceutically acceptable salts thereof.

The term "halogen atoms" includes fluorine, chlorine, bromine and iodine.

The expression "(Ci-C ₆ )alkyl" refers to linear or branched alkyl groups wherein the number of constituent carbon atoms is in the range 1 to 6. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, t-butyl, n- pentyl and n-hexyl.

The term "(Ci-C ₆ )alkoxy" refers to alkyl-oxy (e.g. alkoxy) groups, being the alkyl portion as above defined. Examples of said groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy and the like.

The expression "(Ci-C ₆ )haloalkyl" refers to (Ci-C ₆ )alkyl wherein one or more hydrogen atoms are replaced by halogen atoms, which can be the same or different from each other.

Examples of said (Ci-C ₆ )haloalkyl include halogenated, poly- halogenated and fully halogenated alkyl groups wherein all of the hydrogen atoms are replaced by halogen atoms, e.g. trifluoromethyl group.

The expression "aryl" refers to mono, bi- or tricyclic ring systems which have 5 to 20 ring atoms, preferably from 5 to 15 and wherein at least one ring is aromatic.

Examples of suitable aryl monocyclic systems include benzene and the like.

Examples of suitable aryl bicyclic systems include biphenyl and the like.

Examples of suitable aryl tricyclic systems include fluorene radicals and the like.

The expression "heteroaryl" refers to mono, bi- or tricyclic ring systems which have 5 to 20 ring atoms, preferably from 5 to 15, in which at least one ring is aromatic and in which at least one ring atom is a heteroatom or a heteroatomic group selected from N, NH, S and O. Examples of suitable heteroaryl monocyclic systems include thiophene, pyrrole, pyrazole, imidazole, isoxazole, oxazole, isothiazole, thiazole, pyridine, imidazolidine, pyrimidine, furan and the like.

Examples of suitable heteroaryl bicyclic systems include naphthalene, purine, pteridine, benzotriazole, benzoimidazole, quinoline, isoquinoline, indole, isoindole, benzofuran, benzodioxane, benzothiophene and the like.

Optionally, in any of the said rings including aryl, heteroaryl, one or more hydrogen atoms can be replaced by a group selected from halogen atoms, -OH, -CN, (Ci-C ₆ )alkyl, (Ci-C ₆ )haloalkyl, (Ci-C ₆ )alkoxy and oxo.

The compounds of formula (I) contain asymmetric centers at the positions 4a, 4b, 5, 6a, 6b, 9a, 10a, 10b and 12: the invention is directed to all the possible stereoisomers and to mixtures thereof.

Preferably, the configuration of stereogenic centers is fixed: the configuration of the carbon atom in position 4a is S, 4b is R, 5 is S, in position 6a is S, in position 6b is S, in position 9a is R, in position 10a is S, in position 10b is S and in position 12 is S, as represented by the formula (Γ) below

F

( )

Compounds of general formula (I) and (Γ) may form acid addition salts with inorganic or organic acids or with bases such as amines or alkaline or alkali earth metal salts.

Suitable inorganic acids include hydrohalogen acids such as hydrofluoric acid, hydrochloric acid, hydrobromic acid or hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid; suitable organic acids include aliphatic monocarboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid; aliphatic hydroxy acids such as lactic, citric, tartaric or malic acids; dicarboxylic acids such as maleic or succinic acid; aromatic carboxylic acids such as benzoic acid; aromatic hydroxy acids and sulfonic acids.

A preferred group of compounds of general formula (Γ) is that wherein R is (CH ₂ ) _n -V-(CH ₂ ) _n '-R ₄ wherein n and n' are independently 0 or 1 ; V is absent or is selected from -O-, -S- and -OC(O)-; R ₄ is selected from the group consisting of -H, -OH, -CN, halogen and (Ci-C ₆ )alkyl wherein one or more of the hydrogen atoms of the alkyl group may be optionally substituted by one or more groups selected from oxo, -CN, -SH or halogen.

Another preferred group of compounds of general formula (Γ) is that wherein Rx is (CH ₂ ) _n -V-(CH ₂ ) _n >-R ₄ wherein n is 0 or 1 and n' is 0; V is -O- and R ₄ is -H.

Another preferred group of compounds of general formula (Γ) is that wherein Rx is (CH ₂ ) _n -V-(CH ₂ ) _n >-R ₄ wherein n is 0, V is -S- or -O-, n' is 1 and R ₄ is fluorine or -CN.

Another preferred group of compounds of general formula (Γ) is that wherein Rx is (CH ₂ ) _n -V-(CH ₂ ) _n >-R ₄ wherein n is 1 , V is absent, n' is 0 and R ₄ is fluorine.

Another preferred group of compounds of general formula (Γ) is that wherein Rx is (CH ₂ ) _n -V-(CH ₂ ) _n >-R ₄ wherein n is 1 , V is -OC(O)-, n' is 1 and R ₄ is -H.

An even more preferred group of compounds of general formula (Γ) is that wherein R ₂ is (CH ₂ ) _m -R ₆ wherein m = 0 or 1 to 4; R ₆ is selected from the group consisting of aryl and heteroaryl, optionally substituted by one or more groups selected from oxo, -OH, -CN, -C(O)OH, (C C ₆ )alkoxy, (C C ₆ )alkyl, (Ci-C ₆ )haloalkyl and halogen.

Still more preferred are the compounds of general formula (Γ) wherein R ₂ is (CH ₂ ) _m -R ₆ wherein m = 0, R ₆ is selected from phenyl, benzoimidazolyl and quinolinyl, optionally substituted by one or more chlorine atoms.

An even more preferred group of compounds of general formula (Γ) is that wherein R ₂ is (CH ₂ ) _m -R ₆ wherein m = 1 , R ₆ is selected from phenyl, quinolinyl and benzothiophenyl, optionally substituted by one or more chlorine atoms or methyl groups.

An even more preferred group of compounds of general formula (Γ) is that wherein R ₂ is (CH ₂ ) _m -R ₆ wherein m = 3 and R ₆ is phenyl.

Whenever possible, it is to be understood that all of the preferred groups of compounds may be combined among each other.

The invention also provides pharmaceutical compositions comprising a compound of formula (I) or (Γ) and one or more carriers and/or excipients.

The invention also provides combinations of a compound of formula (I) or (Γ) with one or more active ingredients selected from p2-agonists, antimuscarinic agents, PDE4 inhibitors, P38 MAP kinase inhibitors, IKK2 inhibitors.

The invention also provides combinations of a compound of formula (I) or (Γ) with a 2-agonist selected from the group of carmoterol, GSK-642444, indacaterol, milveterol, arformoterol, formoterol, salbutamol, levalbuterol, terbutaline, AZD-3199, BI- 1744-CL, LAS- 100977, bambuterol, isoproterenol, procaterol, clenbuterol, reproterol, fenoterol and ASF- 1020.

The invention also provides combinations of a compound of formula (I) or (Γ) with an antimuscarinic agent selected from aclidinium, tiotropium, ipratropium, trospium, glycopyrronium and oxitropium.

The invention also provides combinations of a compound of formula (I) or (Γ) with a PDE4 inhibitor selected from cilomilast, roflumilast, BAY 19- 8004, SCH-351591.

The invention also provides combinations of a compound of formula (I) or (Γ) with a P38 inhibitor selected from semapimod, talmapimod, pirfenidone, PH-797804, GSK-725, minokine and losmapimod.

In a preferred embodiment, the invention provides combinations of a compound of formula (I) or (Γ) with a IKK2 inhibitor.

The invention also provides compounds of formula (I) or (Γ) for use as a medicament as well as the use of compounds of formula (I) or (Γ), alone or combined with one or more active ingredients, for the preparation of a medicament for the prevention or treatment of any disease wherein the decrease in the number, activity and movement of inflammatory cells is implicated, particularly of a disease of the respiratory tract characterized by airway obstruction such as asthma and COPD.

The invention provides a method for prevention and/or treatment of any disease wherein the decrease in the number, activity and movement of inflammatory cells is implicated, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of general formula (I) or (Γ), alone or combined with one or more active ingredients.

Examples of said diseases, comprise diseases of the respiratory tract characterized by airway obstruction such as asthma and COPD.

The pharmaceutical preparations of the invention are suitable for administration by inhalation, by injection, orally or intranasally.

Inhalable preparations include inhalable powders, propellant-containing metering aerosols or propellant-free inhalable formulations.

The invention is also directed to a device which may be a single- or multi-dose dry powder inhaler, a metered dose inhaler or a soft mist nebulizer comprising a compound of formula (I) or (Γ). The invention is also directed to a kit comprising the pharmaceutical compositions of compounds of formula (I) or (Γ) alone or in combination with or in admixture with one or more pharmaceutically acceptable carriers and/or excipients, and a device which may be a single- or multi-dose dry powder inhaler, a metered dose inhaler or a soft mist nebulizer.

Advantageously, the compounds of formula (I) or (Γ) are administered at a dosage comprised between 0.001 and 1000 mg/day, preferably between 0.1 and 500 mg/day.

When they are administered by inhalation route, the dosage is advantageously comprised between 0.01 and 20 mg/day, preferably between 0.1 and 10 mg/day.

The compounds of formula (I) or (Γ), alone or combined with other active ingredients, are administered for the prevention and/or treatment of any obstructive respiratory disease such as asthma, chronic bronchitis and chronic obstructive pulmonary disease (COPD).

The compounds of formula (I) or (Γ) may be administered for the prevention and/or treatment of any disease wherein the decrease in the number, activity and movement of inflammatory cells is implicated.

Examples of such diseases include: diseases involving inflammation such as asthma and other allergic disorders, COPD, acute rhinitis; reverse acute transplant rejection and acute exacerbations of selected autoimmune disorders, graft-versus-host disease in bone-marrow transplantation; autoimmune disorders such as rheumatoid and other arthritis; skin conditions such as systemic lupus erythematosus, systemic dermatomyositis, psoriasis; inflammatory bowel disease, inflammatory ophthalmic diseases, autoimmune hematologic disorders, and acute exacerbations of multiple sclerosis; kidney, liver, heart, and other organ transplantation; Behcet's acute ocular syndrome, endogenous uveitis, atopic dermatitis, and nephrotic syndrome; Hodgkin's disease and non-Hodgkin's lymphoma, multiple myeloma and chronic lymphocytic leukemia (CLL); autoimmune hemolytic anemia and thrombocytopenia associated with CLL; leukemia and malignant lymphoma.

Preferably the compounds of formula (I) or (Γ) are administered for the prevention and/or treatment of respiratory diseases such as from mild to acute severe conditions of asthma and COPD.

Preferred compounds of the invention are reported below:

(continued) (4aS,4bR,5S,6aS,6bS,9aR, 1 OaS, 1 ObS, 12S)-4b, 12- Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-4a,6a-

Compound 12 dimethyl-8-quinolin-2-ylmethyl-

4b,5 ,6,6a,6b,7,8,9,9a, 10, 10a, 1 Ob, 1 1 , 12-tetradecahydro- 4aH-8-aza-pentaleno[2, 1 -a]phenanthren-2-one

Acetic acid 2-((4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S) - 8-B enzo [b] thiophen-2-ylmethyl-4b , 12-dif uoro-5 -hy droxy- 4a,6a-dimethyl-2-oxo-

Compound 13

2,4b,5 ,6,6a,7,8,9,9a, 10, 10a, 1 Ob, 1 1 , 12-tetradecahydro-4aH- 8-aza-pentaleno[2, 1 -a]phenanthren-6b-yl)-2-oxo-ethyl ester

Acetic acid 2- [(4aS,4bR,5S,6aS,6bS,9aR, 1 OaS, 1 ObS, 12S)-4b, 12- difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo-8-(3-phenyl-

Compound 14

propyl)-2,4b,5,6,6a,7,8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12- tetradecahydro-4aH-8-aza-pentaleno[2, l-a]phenanthren- 6b-yl]-2-oxo-ethyl ester

(4aS,4bR,5S,6aS,6bS,9aR, 10aS, 10bS, 12S)-8-

Benzo[b]thiophen-2-ylmethyl-4b, 12-difluoro-5-hydroxy-

Compound 15 6b-(2-hydroxy-acetyl)-4a,6a-dimethyl-

4b,5 ,6,6a,6b,7,8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12-tetradecahydro-

4aH-8-aza-pentaleno[2, 1 -a ]phenanthren-2-one

Acetic acid 2-((4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)- 4b,12-difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo-8-

Compound 16 quinolin-2-yl-2,4b,5 ,6,6a,7,8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12- tetradecahydro-4aH-8-aza-pentaleno[2, 1 -ajphenanthren- 6b-yl)-2-oxo-ethyl ester

(4aS,4bR,5S,6aS,6bS,9aR, 1 OaS, 1 ObS, 12S)-4b, 12- Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-4a,6a-

Compound 17 dimethyl-8-quinolin-2-yl-

4b,5 ,6,6a,6b,7,8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12-tetradecahydro- 4aH-8-aza-pentaleno[2, 1 -a]phenanthren-2-one

(4aS,4bR,5S,6aS,6bS,9aR, 10aS, 10bS, 12S)-8-(lH-

Benzoimidazol-2-yl)-4b, 12-difluoro-5-hydroxy-6b-(2-

Compound 18 hydroxy-acetyl)-4a,6a-dimethyl-

4b,5 ,6,6a,6b,7,8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12-tetradecahydro- 4aH-8-aza-pentaleno[2, 1 -a]phenanthren-2-one

Acetic acid 2-[(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-

8-(lH-benzoimidazol-2-yl)-4b,12-difluoro-5-hydroxy-

4a,6a-dimethyl-2-oxo-

Compound 19

2,4b,5,6,6a,7,8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12-tetradecahydro-4aH- 8-aza-pentaleno[2, 1 -a]phenanthren-6b-yl]-2-oxo-ethyl ester

(continued) (4aS,4b ,5S,6aS,6bS,9a , 1 OaS, 1 ObS, 12S)-4b, 12- Difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo-8-(3-phenyl-

Compound 20 propyl)-2,4b,5,6,6a,7,8,9,9a, 10, 10a, 1 Ob, 1 1 , 12- tetradecahydro-4aH-8-aza-pentaleno[2, l- a]phenanthrene-6b-carboxylic acid f uoromethyl ester

(4aS,4bR,5S,6aS,6bS,9aR, 1 OaS, 1 ObS, 12S)-4b, 12- Difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo-8-(3-phenyl-

Compound 21 propyl)-2,4b,5,6,6a,7,8,9,9a, 10,1 Oa, 1 Ob, 1 1 , 12- tetradecahydro-4aH-8-aza-pentaleno[2, l- a]phenanthrene-6b-carboxylic acid cyanomethylester

Acetic acid 2-

[(4aS,4bR,5S,6aS,6bS,9aR, 10aS, 10bS, 12S)-8-(4-chloro- phenyl)-4b, 12-difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo-

Compound 22

2,4b,5 ,6,6a,7,8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12-tetradecahydro- 4aH-8-aza-pentaleno[2, 1 -a]phenanthren-6b-yl]-2-oxo- ethyl ester

(4aS,4bR,5S,6aS,6bS,9aR, 10aS, 10bS, 12S)-8-(4-Chloro- phenyl)-4b, 12-difluoro-5-hydroxy-6b-(2-hydroxy-

Compound 23 acetyl)-4a,6a-dimethyl-

4b,5 ,6,6a,6b,7,8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12-tetradecahydro- 4aH-8-aza-pentaleno[2, 1 -a]phenanthren-2-one

(4aS,4bR,5S,6aS,6bS,9aR, 10aS, 10bS, 12S)-8-(3-Chloro- phenyl)-4b, 12-difluoro-5-hydroxy-6b-(2-hydroxy-

Compound 24 acetyl)-4a,6a-dimethyl-

4b,5 ,6,6a,6b,7,8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12-tetradecahydro- 4aH-8-aza-pentaleno[2, 1 -a]phenanthren-2-one

(4aS,4bR,5S,6aS,6bS,9aR, 1 OaS, 1 ObS, 12S)-4b, 12- Difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo-8-phenyl-

Compound 26 2,4b,5 ,6,6a,7,8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12-tetradecahydro- 4aH-8-aza-pentaleno[2, 1 -a]phenanthrene-6b-carboxylic acid

(4aS,4bR,5S,6aS,6bS,9aR, 1 OaS, 1 ObS, 12S)-4b, 12- Difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo-8-phenyl-

Compound 27 2,4b,5 ,6,6a,7,8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12-tetradecahydro- 4aH-8-aza-pentaleno[2, 1 -a]phenanthrene-6b-carbothioic acid S-f uoromethyl ester

(4aS,4bR,5S,6aS,6bS,9aR, 1 OaS, 1 ObS, 12S)-4b, 12- Difluoro-6b-(2-fluoro-acetyl)-5-hydroxy-4a,6a-dimethyl-

Compound 32 8-phenyl-4b,5,6,6a,6b,7, 8,9,9a, 10, 1 Oa, 1 Ob, 1 1 , 12- tetradecahydro-4aH-8-aza-pentaleno[2, l-a]phenanthren- 2-one Scheme

m = 1

The compounds of formula (Γ) may be prepared according to different routes, described in Scheme, depending on the nature of the substituents i and R ₂ .

Compounds of formula (Γ) wherein Rx is (CH ₂ ) _n -V-(CH ₂ ) _n >-R ₄ , n=l , n'=0, V is -O- and R ₄ is -H, can be prepared by a four steps procedure following Route A, starting from the reaction of a compound of formula (III) with N-benzyl-N-(trimethylsilylmethyl)aminomethyl ether (compound (IV), which is an azomethine ylide precursor, wherein m=l and R ₆ =phenyl) under the 1 ,3-dipolar cycloaddition (1 ,3-DC) conditions of unsaturated compounds and azomethine ylides. The ylide is generated in situ from the suitable precursor, e.g. compound (IV). The reaction involves the use of 1 to 7 equivalents of azomethine ylide precursor and it is usually performed in a high boiling point solvent such as THF, dioxane, toluene or xylene. The reaction usually proceeds in a range of temperature from 50 to 150°C over a period of 1-5 hours and may be promoted by an acid such as trifluoroacetic acid, trimethylsilyl iodide or trimethylsilyl trifluoromethanesulfonate. Sodium, potassium or cesium fluoride could also be effective in catalyzing the reaction. Azomethine ylide precursor (IV) is commercially available or can be prepared as described in the literature, for example following the procedure described in J. Chem.- Soc, Perkin trans. 1 , 1998, p 3867-3872. Compounds of general formula (III) may be conveniently prepared according to standard procedures reported in the literature. For instance they may be prepared by treatment of compounds of general formula (II) with a base such as potassium acetate. This reaction is usually performed in a suitable polar solvent (e.g. dimethylformamide (DMF)) and typically proceeds at a temperature range from 80 to 1 10°C, over a period of 0.5 to 4 hours. Compounds of formula (II) are commercially available or may be readily prepared from known compounds by known methods (J. Med. Chem. 1982, 25, 1492-1495). The benzyl group can be removed by chemical dealkylation of tertiary amines with acyl chlorides, phosgene analogues or preferably chloroformates such as vinylchloroformate. The reaction is usually performed in a suitable solvent such as dichloromethane (DCM), tetrahydrofuran (THF) or acetonitrile at temperature range from room temperature (RT) to 60°C. The reaction requires the presence of a base such as alkali carbonates or bicarbonates or an organic base such as pyridine and it usually completes over a time range from 0.5 to 2 hours. The carbamate obtained from the first step of the reaction is deprotected under known conditions (Wiley-VCH; Wuts, Peter G. M./Greene, Theodora W. Greene's Protective Groups in Organic Synthesis p 503 and following) affording compound (VI). Compound (VI) can be easily further functionalized to obtain compounds of general formula (Γ).

In fact, aryl or heteroaryl groups can be introduced at N atom of pyrrolidine ring of compound (VI) by following described procedures for the N arylation of pyrrolidines. Compound of formula (VI) can be reacted with aryl boronic acids under metal catalyzed arylation conditions. The reaction is promoted by copper (I or II) salts, nickel(II) phosphine complexes and palladium complexes and often a base such as sodium or potassium terbutylate or triethylamine (TEA) is required. The reaction occurs in a solvent such as DMF, dimethylacetamide (DMA), acetonitrile, dioxane, THF, toluene, dichloromethane (DCM), N-methyl pyrrolidone (NMP) at a temperature range from 20 to 200°C by conventional thermal heating or by microwaves. In some cases the reaction can occur by simple heating, from 20 to 150°C, of a solution of compound (VI) and a suitable aryl or heteroaryl derivative, such as halides or trifluoromethane sulfonates (triflate), in a solvent such as ethanol, THF, acetonitrile, DMF, DMA, dioxane or NMP for a period of time from 0.5 to 24 hours. Alternatively this compound can be prepared by reacting compound (VI) with a benzyne. This very reactive species can be generated in situ from a suitable precursor following methods described in the literature. A valid protocol involves the treatment of 2-trimethylsilyl-phenyl trifluoromethane sulfonates with cesium fluoride. The reaction occurs at T in a polar solvent such as acetonitrile and it completes over a period from 1 to 72 hours. In the case the benzyne precursor features substituents on the benzene ring, the reaction can lead to a mixture of two regioisomers.

The alkylation of compound (VI) can be performed applying the conditions for the alkylation of pyrrolidines. The reaction involves the use of alkyl halides, methane sulfonates, tosylates or other alkyl derivatives suitable for amine alkylation and requires the presence of a base such as TEA, DIPEA or pyridine to complete. It proceeds at a temperature from RT to 100°C over 1 to 48 hours in a suitable solvent such as DCM, THF, acetonitrile or DMF. The presence of sodium or potassium iodide can in some cases accelerate the reaction rate. The same class of derivatives can be obtained reacting compound (VI) under reductive amination reaction conditions with a suitable aldehyde in a solvent such as acetonitrile. The imine intermediate is usually in situ reduced by treatment with reducing agents, such as formic acid and its salts at a temperature range from 70 to 150°C, over 10-30 minutes.

In all cases obtained products feature an acetoxy moiety on the side chain position at 6b of the steroid scaffold. This moiety can be easily hydrolyzed by treatment with a base such as LiOH, NaOH, KOH or K ₂ CO ₃ solid or dissolved in water, in organic solvents such as methanol, ethanol or THF or alternatively with an aqueous acid solution (for example HC1) in a suitable organic solvent such as THF or dioxane at 40 to 80°C over a period of 1 to 8 hours.

Route B - reaction of compounds of general formula (Γ) wherein Rx is

(CH ₂ ) _n -V-(CH ₂ ) _n -R ₄ , n=n'=l , V is -OC(O)- and R ₄ is -H, under well known oxidation conditions to afford the compounds of general formula (Γ) wherein Rx is (CH ₂ ) _n -V-(CH ₂ ) _n >-R ₄ , n=n'=0, V is -O- and R ₄ is -H. This reaction is usually performed in open air at RT over a period of 12 to 48 hours, in a suitable solvent such as THF or dioxane in the presence of an aqueous solution of an inorganic base, such as for example sodium or potassium hydroxide.

Route Bl - Conversion of the compounds of general formula (Γ) wherein ¾ is (CH ₂ )„-V-(CH ₂ )„'-R ₄ , n=n'=0, V is -O- and R ₄ is -H, into compounds of general formula (Γ) wherein Rx is (CH ₂ ) _n -V-(CH ₂ ) _n >-R ₄ , wherein n=0 and n'=l , V is -O- and R ₄ is fluorine or -CN, can be obtained by treating the acid of general formula (Γ) wherein Rx is (CH ₂ ) _n -V-(CH ₂ ) _n >-R ₄ , n=n'=0, V is -O- and R ₄ is -H, with an alkylating reagent such as bromofluoromethane or bromoacetonitrile in a suitable solvent such as DMF, at RT, in the presence of an inorganic base such as sodium carbonate over a period of 1-48 hours. These reactions are carried out as described in the literature for the synthesis of similar compounds and are well known.

Route B2 - Conversion of acids of formula (Γ) wherein Rx is (CH ₂ ) _n -V- (CH ₂ ) _n >-R ₄ , n=n'=0, V is -O- and R ₄ is -H, into compounds of general formula (Γ) wherein Rx is (CH ₂ ) _n -V-(CH ₂ ) _n -R ₄ , wherein n=0, n'=l, V is -S-, R ₄ is fluorine, derived from reaction of acid (Γ) with for example carbonyldiimidazole or N-[(dimethylamino)-lH-l,2,3-triazolo[4,5-b]pyridine-l-ylmeth ylene]-N- methylmethanaminium hexafluorophosphate N-oxide (HATU), followed by reaction with the sodium salt of thioacetic acid or with sodium hydrogen sulfide. The reaction is usually performed adding the solution of the preformed salt in the reaction solvent to the solution of the activated acid, at a temperature ranging from 0 to 20°C. The thioacid compounds of formula (VII) readily formed is in situ reacted with an alkylating reagent, such as bromofloromethane, leading to thioesters of general formula (Γ).

Route C - In another embodiment of the present invention, compounds of formula (Γ) wherein Rx is (CH ₂ ) _n -V-(CH ₂ ) _n >-R ₄ , n=0, V is absent, n'=l , R ₄ is halide can be prepared by a five steps procedure, starting from the conversion of the hydroxyl group of 2-hydroxy acetyl moiety at position 6b of compound (VIII) into a leaving group (LG) using methanesulfonyl chloride or p-toluenesulfonyl chloride (March's, "Advanced Organic Chemistry", Wiley-Interscience), in a suitable solvent, for example acetonitrile in the presence of an organic base such as DIPEA. This reaction is usually performed at T over a period of 1 to 24 hours and the obtained activate intermediates are submitted to the displacement of the LG by using a nucleophile such as a halide salt to obtain compounds of general formula (IX). This displacement is usually performed in situ for example by the addition of TBAF and/or potassium fluoride and refluxing the reaction mixture over a period of 24-48 hours. The compounds of formula (VIII) may be prepared hydrolyzing the compounds of formula (III). This reaction is preferably carried out by subjecting compounds (III) to the action of an enzyme, such as for example immobilized Lipase from Candida Antarctica (Sigma Aldrich) (Tetrahedron 1994, Vol. 50, N° 46, 13165- 13172).

The third step is the 1-3 dipolar cycloaddition reaction of compounds of general formula (IX) with compound (IV) performed as described above (Route A) to yield compounds of general formula (X). In particular, a compound of formula (IX) can react with N-benzyl-N- (trimethylsilylmethyl)aminomethyl ether (i.e. compound (IV), which is an azomethine ylide precursor, wherein m=l and R ₆ =phenyl) under the 1 ,3-dipolar cycloaddition (1 ,3-DC) conditions of unsaturated compounds and azomethine ylides. The ylide is generated in situ from the suitable precursor, e.g. compound (IV). The reaction involves the use of 1 to 7 equivalents of azomethine ylide precursor and it is usually performed in a high boiling point solvent such as dioxane. The reaction usually proceeds in a range of temperature from 50 to 150°C over a period of 1 -5 hours and may be promoted by an acid such as trifluoroacetic acid. The fourth step is the benzyl group removal of intermediate (X) by chemical dealkylation of tertiary amines with chloroformates such as vinylchloroformate as described above (Route A) to obtain compound of formula (XI). The reaction is usually performed in a suitable solvent such as dichloromethane (DCM), tetrahydrofuran (THF) or acetonitrile at temperature range from T to 60°C. The reaction requires the presence of a base such as alkali carbonates or bicarbonates or an organic base such as pyridine and it usually completes over a time range from 0.5 to 2 hours. The carbamate obtained from the first step of the reaction is deprotected under known conditions (Wiley- VCH; Wuts, Peter G. M ./Greene, Theodora W. Greene's Protective Groups in Organic Synthesis p 503 and following) affording intermediate (XI). The last step is the fictionalization of intermediate (XI) by arylation, alkylation or reductive amination reactions as previously described for compound (VI) (Route A) yielding compounds of general formula (Γ). In particular, aryl or heteroaryl groups can be introduced at N atom of pyrrolidine ring of compound (XI) by following described procedures for the N arylation of pyrrolidines. Compound of formula (XI) can be reacted with aryl boronic acids under metal catalyzed arylation conditions. The reaction is promoted by copper (I or II) salts, nickel(II) phosphine complexes and palladium complexes and often a base such as sodium or potassium terbutylate or triethylamine (TEA) is required. The reaction occurs in a solvent such as DMF, dimethylacetamide (DMA), acetonitrile, dioxane, THF, toluene, dichloromethane (DCM), N-methyl pyrrolidone (NMP) at a temperature range from 20 to 200°C by conventional thermal heating or by microwaves. In some cases the reaction can occur by simple heating, from 20 to 150°C, of a solution of compound (XI) and a suitable aryl or heteroaryl derivative, such as halides or trifluoromethane sulfonates (triflate), in a solvent such as ethanol, THF, acetonitrile, DMF, DMA, dioxane or NMP for a period of time from 0.5 to 24 hours. Alternatively this compound can be prepared by reacting compound (XI) with a benzyne. This very reactive species can be generated in situ by the treatment of 2-trimethylsilyl-phenyl trifluoromethane sulfonates with cesium fluoride. The reaction occurs at T in a polar solvent such as acetonitrile and it completes over a period from 1 to 72 hours. In the case the benzyne precursor features substituents on the benzene ring, the reaction can lead to a mixture of two regioisomers.

The alkylation of compound (XI) can be performed applying the conditions for the alkylation of pyrrolidines. The reaction involves the use of alkyl halides, methane sulfonates, tosylates or other alkyl derivatives suitable for amine alkylation and requires the presence of a base such as TEA, DIPEA or pyridine to complete. It proceeds at a temperature from RT to 100°C over 1 to 48 hours in a suitable solvent such as DCM, THF, acetonitrile or DMF. The presence of sodium or potassium iodide can in some cases accelerate the reaction rate. The same class of derivatives can be obtained reacting compound (XI) under reductive amination reaction conditions with a suitable aldehyde in a solvent such as acetonitrile. The imine intermediate is usually in situ reduced by treatment with reducing agents, such as formic acid and its salts at a temperature range from 70 to 150°C, over 10-30 minutes.

The present invention will now be further described by way of the following examples.

Example 1

Preparation of Acetic acid 2-((6S,8S,9R,10S,HS,13S,14S)-6,9- difluoro-ll-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,ll,12,13 ,14,15- decahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxo-ethyl ester (intermediate 2)

To a solution of butyric acid (6S,8S,9R, 10S, 1 l S,13S, 14S, 17R)-17-(2- acetoxyacetyl)-6,9-difluoro- 1 1 -hydroxy- 10, 13-dimethyl-3-oxo-

7,8,9, 10, 1 1 , 12, 13, 14, 15, 16, 17-dodecahydro-3H-cyclopenta[a]phenanthren- 17- yl ester (intermediate 1) (2.48 g, 4.88 mmol) in anhydrous DMF (60 ml), under nitrogen atmosphere, potassium acetate (3.83 g, 39.0 mmol) was added and the reaction mixture was stirred at 100°C for 1.5 hours. The reaction mixture was cooled to RT and then poured into ice and brine (200 ml) and the aqueous layer was extracted with AcOEt (3 x 150 ml). The combined organic extracts were washed with water and brine, dried over Na2SO4 and concentrated to afford 2.55 g of crude title compound which was used in the next step without further purification.

1H NMR (300 MHz, DMSO- ₆ ): δ ppm 7.29 (dd, 1 H), 6.99 (dd, 1 H),

6.29 (dd, 1 H), 5.98 - 6.15 (m, 1 H), 5.68 (dddd, 1 H), 5.56 (dd, 1 H), 5.10 (d,

1 H), 4.92 (d, 1 H), 3.98 - 4.23 (m, 1 H), 2.56 - 2.83 (m, 1 H), 2.26 - 2.44 (m, 3 H), 2.14 - 2.26 (m, 1 H), 2.09 (s, 3 H), 1.71 - 1.87 (m, 1 H), 1.55 - 1.65 (m,

2 H), 1.53 (s, 3 H), 1.15 (s, 3 H)

LC-MS (ESI POS): 421.2 (MH+)

Preparation of (6S,8S,9R,10S,HS,13S,14S)-6,9-Difluoro-ll- hydroxy-17-(2-hydroxy-acetyl)-10,13-dimethyl-6,7,8,9,10,ll,1 2,13,14,15- decahydro cyclopenta[a]phenanthren-3-one (intermediate 3)

To a solution of intermediate 2 (2.55 g, 6.06 mmol) in ethanol (100 ml), Candida Antarctica Lipase (2 U/mg) (510 mg, 6.06 mmol) was added and the reaction mixture was stirred at 37°C overnight. The reaction mixture was filtered, washing with methanol, and the residue was purified by flash chromatography on silica gel, in gradient elution from DCM/AcOEt 90: 10 to DCM/AcOEt 50: 50, to afford the title compound (1.62 g, 70.6% yield).

1H NMR (300 MHz, DMSO- ₆ ): ppm 7.29 (dd, 1 H), 6.87 (dd, 1 H), 6.29 (dd, 1 H), 6.09 - 6.17 (m, 1 H), 5.67 (dddd, 1 H), 5.53 (dd, 1 H), 4.77 (t, 1 H), 4.44 (dd, 1 H), 4.26 (dd, 1 H), 4.04 - 4.15 (m, 1 H), 2.56 - 2.79 (m, 1 H), 2.39 (dd, 1 H), 2.25 - 2.35 (m, 2 H), 2.09 - 2.25 (m, 1 H), 1.76 (td, 1 H), 1.55 - 1.66 (m, 2 H), 1.53 (s, 3 H), 1.17 (s, 3 H)

LC-MS (ESI POS): 379.2 (MH+)

Example 2

Preparation of Acetic acid 2-

((4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-benzyl-4b,12-di fluoro-5- hydroxy-4a,6a-dimethyl-2-oxo-2,4b,5,6,6a,7,8,9,9a,10,10a,10b ,ll,12- tetradecahydro-4aH-8-aza-pentaleno[2,l-a]phenanthren-6b-yl)- 2-oxo- ethyl ester (intermediate 4)

In a nitrogen atmosphere, to a mixture of intermediate 2 (3.63 g, 8.63 mmol) and N-benzyl-N-(trimethylsilylmethyl)aminomethyl ether (10.25 g, 43.2 mmol) in dioxane (50 ml) was stirred under nitrogen in a preheated bath at 100°C for 2hours. The solvent was evaporated and the residue was triturated several times with petroleum ether to give a pale yellow solid. The solid was purified by silica gel chromatography (AcOEt/petroleum ether 1 : 1) to yield the title intermediate (4.47 g, 94% yield).

1H NMR (300 MHz, DMSO-d ₆ ) δ ppm 6.79 - 7.68 (m, 6 H), 6.29 (dd, 1 H), 6.13 (s, 1 H), 5.48 - 5.82 (m, 1 H), 5.36 - 5.46 (m, 1 H), 4.95 (d, 1 H), 4.79 (d, 1 H), 4.01 - 4.24 (m, 1 H), 3.47 (s, 1 H), 3.03 - 3.21 (m, 1 H), 2.83 - 2.98 (m, 1 H), 2.54 - 2.61 (m, 1 H), 2.24 - 2.46 (m, 4 H), 2.1 1 (s, 3 H), 1.94 - 2.10 (m, 2 H), 1.85 (d, 1 H), 1 .52 - 1.74 (m, 3 H), 1.49 (s, 3 H), 1 .28 - 1 .43 (m, 1 H), 0.92 (s, 3 H)

LC-MS (ESI POS): 554.2 (MH+)

Example 3

Preparation of (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-6b-(2- Acetoxy-acetyl)-4b,12-difluoro-5-hydroxy-4a,6a-dimethyl-2-ox o- 2,4b,5,6,6a,6b,7,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8- aza- pentaleno[2,l-a]phenanthrene-8-carboxylic acid vinyl ester (intermediate 5)

Intermediate 4 (1.95 g, 3.52 mmol) and NaHCO ₃ (0.592 g, 7.04 mmol) were dissolved in acetonitrile (30 ml) and then vinyl chloroformate (0.599 ml, 7.04 mmol) was added. The reaction mixture was warmed at 50°C for 2 hours. The solution was partitioned between AcOEt and brine. The organic phase was separated while the aqueous solution was extracted with AcOEt. The combined organic phases were dried over Na ₂ SO ₄ and then evaporated to give a residue that was purified by silica gel column chromatography, eluting with Petroleum Ether/AcOEt 6:4 to 4:6, leading to pure title intermediate (1.1 g, 58.5% yield).

LC-MS (ESI POS): 534.1 (MH+)

Preparation of Acetic acid 2-

((4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-difluoro-5- hydroxy- 4a,6a-dimethyl-2-oxo-2,4b,5,6,6a,7,8,9,9a,10,10a,10b,ll,12- tetradecahydro-4aH-8-aza-pentaleno [2,l-a]phenanthren-6b-yl)-2-oxo- ethyl ester hydrochloride (intermediate 6)

Intermediate 5 (1.0 g, 1.874 mmol) was dissolved in dioxane (3 ml) and then HCI 4.0 M in Dioxane (5 ml, 1.874 mmol) was added. The solution was stirred at T for 2 hours and then the solvent was evaporated and the residue was dried under vacuum for 1 hour. The solid was then dissolved in methanol and warmed at 40°C for 1 hour. Methanol was evaporated and the residue was triturated with diethyl ether to give the title intermediate (0.93 g, 99% yield).

LC-MS (ESI POS): 464.0 (MH+)

Example 4

Preparation of Acetic acid 2-

[(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-difluoro-5- hydroxy- 4a,6a-dimethyl-8-(3-methyl-benzyl)-2-oxo- 2,4b,5,6,6a,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8-a za- pentaleno[2,l-a]phenanthren-6b-yl]-2-oxo-ethyl ester (compound 7)

In a nitrogen atmosphere, intermediate 6 (200 mg, 0.400 mmol) was dissolved in DCM (5 ml), triethylamine (166 \L, 1.20 mmol) and l-(chloromethyl)-3-methylbenzene (79 μL·, 0.60 mmol) were added and the

o

mixture was heated at 40 C for 2 hours and at RT for 16 hours. Then triethylamine (138 \L, 1.00 mmol) and 3-methylbenzyl bromide (54 \L, 0.40 mmol) were added to the mixture and stirring was continued for 2 hours. The reaction mixture was partitioned between water and DCM and the aqueous layer was extracted with DCM (3x15 mL), the organic phase was collected and concentrated. The crude was purified by silica gel flash chromatography (eluent DCM/MeOH 99: 1) to yield the title compound (130 mg, 57.2% yield). LC-MS (ESI-POS): 568.3 MH+

Compound 8 in Table 1 was prepared as described in Example 4 for compound 7, by reacting intermediate 6 with the commercially available 4-methylbenzylbromide.

Table 1

Example 5

Preparation of (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-

Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-4a,6a-dimethyl-8 -(3-methyl- benzyl)-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,ll,12-tetradecahydr o-4aH-8-aza- pentaleno[2,l-a]phenanthren-2-one (compound 9)

Compound 7 (130 mg, 0.229 mmol) was dissolved in MeOH (8 ml) and the mixture was degassed with nitrogen for 15 minutes. After cooling to 0°C, K ₂ CO ₃ (15.8 mg, 0.1 15 mmol) was added and the mixture was stirred for lhour. The reaction mixture was partitioned between a 5% NaHCO ₃ solution and AcOEt. The organic layer was separated, dried over Na ₂ SO ₄ and concentrated. The crude was purified by silica gel flash chromatography (eluent DCM/MeOH 99: 1 to 98:2) to give the title compound (91 mg, 76% yield).

1H NMR (300 MHz, DMSO- ₆ ) δ ppm 7.25 (d, 1 H), 7.09 - 7.21 (m, 1 H), 6.92 - 7.06 (m, 3 H), 6.29 (d, 1 H), 6.13 (s, 1 H), 5.45 - 5.81 (m, 1 H), 5.40 (br. s., 1 H), 4.83 (t, 1 H), 4.06 - 4.28 (m, 3 H), 3.33 - 3.53 (m, 3 H), 3.15 (d, 1 H), 2.84 (t, 1 H), 2.34 - 2.48 (m, 2 H), 2.19 - 2.34 (m, 1 H), 2.24 (s, 3 H), 1.94 - 2.14 (m, 2 H), 1.77 - 1.89 (m, 1 H), 1.42 - 1.77 (m, 3 H), 1.49 (s, 3 H), 1.35 (dd, 1 H), 0.87 (s, 3 H)

LC-MS (ESI POS): 526.30 MH+

[cc] _D ²⁵ + 81.0 (C 0.33, MeOH)

Compound 10 in Table 2 was prepared as described in Example 5 for compound 9, starting from compound 8.

Table 2

Compound Structure Yield Analytical

1H NMR (300 MHz,

HO 4.11 (dd, 1 H), 3.39 (s,

2 H), 3.05 - 3.21 (m, 1 H), 2.85 (t, 1 H), 2.55 -

10 47% 2.69 (m, 2 H), 2.36 (d,

1 H), 2.26 (s, 3 H), 2.21 - 2.32 (m, 1 H),

F 1.92 - 2.12 (m, 2 H),

1.77 - 1.92 (m, 1 H), 1.51 - 1.76 (m, 3 H), 1.49 (s, 3 H), 1.34 (dd, 1 H), 0.86 (s, 3 H)

LC-MS (ESI POS): 526.32 MH+

[cc] _D ²⁵ + 89.3 (c= 0.33, MeOH) Example 6

Preparation of Acetic acid 2-((4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)- 4b,12-difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo-8-quinolin-2-y lmethyl- 2,4b,5,6,6a,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8-a za- pentaleno[2,l-a]phenanthren-6b-yl)-2-oxo-ethyl ester (compound 11)

A solution of intermediate 6 (200 mg, 0.400 mmol), quinoline-2- carbaldehyde (251 mg, 1.600 mmol), triethylamine (61 mL, 0.440 mmol) and formic acid (263 iL, 3.20 mmol), in acetonitrile (4 ml) is irradiated with microwaves (130°C, 20 minutes).

The reaction mixture was partitioned between AcOEt and 5% NaHCO ₃ solution. The organic layers were then washed with brine, dried over Na ₂ SO ₄ and filtered. The solvent was evaporated and the crude was purified by silica gel chromatography (DCM/MeOH 99: 1) to give compound 1 1 (132 mg, 54.6% yield).

LC-MS (ESI POS): 605.3 MH+

Preparation of 4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12- Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-4a,6a-dimethyl-8-qu inolin-2- ylmethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,ll,12-tetradecahyd ro-4aH-8- aza-pentaleno[2,l-a]phenanthren-2-one (compound 12)

The title compound was prepared from compound 1 1 (130 mg, 0.215 mmol) following the procedure described in Example 5 for the synthesis of compound 9. Compound 12 (68 mg, 56% yield) was obtained. 1H NMR (300 MHz, DMSO-d ₆ ) δ ppm 8.29 (d, 1 H), 7.89 - 8.00 (m, 2 H), 7.72 (ddd, 1 H), 7.56 (ddd, 1 H), 7.46 (d, 1 H), 7.26 (dd, 1 H), 6.30 (dd, 1 H), 6.14 (s, 1 H), 5.50 - 5.87 (m, 1 H), 5.27 - 5.44 (m, 1 H), 4.85 (t, 1 H), 4.28 (dd, 1 H), 4.14 (dd, 1 H), 4.01 - 4.15 (m, 1 H), 3.76 (s, 2 H), 3.12 - 3.24 (m, 1 H), 2.94 (t, 1 H), 2.62 (d, 1 H), 2.54 - 2.62 (m, 2 H), 2.22 - 2.37 (m, 1 H), 2.17 (dd, 1 H), 2.01 - 2.13 (m, 1 H), 1.78 - 1.93 (m, 1 H), 1.52 - 1.78 (m, 3 H), 1.49 (s, 3 H), 1.39 (dd, 1 H), 0.88 (s, 3 H)

LC-MS (ESI POS): 563.35 MH+

[cc] _D ²⁵ + 76.1 (c 0.32, MeOH)

The compounds listed in Table 3 were prepared as described in Example 6 for compound 1 1 , starting from intermediate 6 and the suitable commercially available aldehyde:

Table 3

Compound 15 in Table 4 was prepared from compound 13 as described in Example 5 for the preparation of compound 9. Table 4

Example 7

Preparation of Acetic acid 2- ((4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-difluoro-5-hyd roxy- 4a,6a-dimethyl-2-oxo-8-quinolin-2-yl-

2,4b,5,6,6a,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH- 8-aza- pentaleno[2,l-a]phenanthren-6b-yl)-2-oxo-ethyl ester (compound 16)

Intermediate 6 (200 mg, 0.400 mmol) and 2-chloroquinoline (131 mg,

0.800 mmol) were placed in microwave vessel with EtOH (5 ml) and the reaction mixture was heated to 130°C for 2 hours by microwaves. The mixture was partitioned between AcOEt and NaHCO ₃ (5% solution). The organic phase was separated, dried over Na ₂ SO ₄ and evaporated to give a residue that was purified by a silica gel cartridge (DCM /MeOH 99: 1 to 95 :5) to give the title compound (171 mg, 72% yield).

LC-MS (ESI POS): 591.3 (MH+)

Preparation of (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12- Difluoro-5-hydroxy-6b-(2-hydroxy-acetyl)-4a,6a-dimethyl-8-qu inolin-2-yl- 4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8- aza- pentaleno[2,l-a]phenanthren-2-one (compound 17)

A solution of compound 16 (171 mg, 0.290 mmol) in MeOH (4 ml) was degassed bubbling nitrogen at T for 20 minutes. After cooling to 0°C K ₂ CO ₃ (12 mg, 0.0.87 mmol) was added and the mixture was stirred for 1 hour. The mixture was partitioned between AcOEt and brine. The organic phase was separated, dried over Na ₂ SO ₄ and concentrated. The crude was purified by preparative HPLC (CH ₃ CN/H ₂ O without CF ₃ COOH) to give the title compound (45 mg, 28% yield).

1H NMR (300 MHz, DMSO-d ₆ ) δ ppm 8.00 (d, 1 H), 7.68 (d, 1 H), 7.43 - 7.60 (m, 2 H), 7.27 (dd, 1 H), 7.19 (ddd, 1 H), 6.94 (d, 1 H), 6.29 (dd, 1 H), 6.07 (s, 1 H), 5.52 - 5.81 (m, 1 H), 5.49 (dd, 1 H), 4.94 (t, 1 H), 4.50 (dd, 1 H), 4.20 - 4.28 (m, 1 H), 4.16 (dd, 1 H), 3.78 (dd, 1 H), 3.72 (d, 1 H), 3.57 (d, 1 H), 3.34 - 3.54 (m, 2 H), 2.54 - 2.70 (m, 2 H), 2.12 - 2.31 (m, 1 H), 1.76 - 2.10 (m, 4 H), 1.52 - 1.66 (m, 1 H), 1.50 (s, 3 H), 1.03 (s, 3 H)

LC-MS (ESI POS): 549.32 (MH+)

[cc] _D ²⁵ + 37.3 (c 0.45, MeOH)

Compound 18 in Table 5 was prepared by a two steps procedure as described in Example 7 for compound 17, starting from intermediate 6 and 2- chloro- 1 H-benzo[d]imidazole.

Table 5

Example 8

Preparation of Acetic acid 2-

[(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-(lH-benzoimidaz ol-2-yl)- 4b,12-difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo- 2,4b,5,6,6a,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8-a za- pentaleno[2,l-a]phenanthren-6b-yl]-2-oxo-ethyl ester (intermediate 19)

A solution of compound 14 (800 mg, 1.375 mmol) and 6 N aqueous NaOH (1031 μΐ, 6.19 mmol) in dioxane (45 ml) and water (15 ml) was stirred at T in open air for 18 hours. HC1 6N (1 ml) was added to the reaction mixture until pH 4-5, then the organic solvent was evaporated and a solid precipitated, which was recovered by filtration. Mother liquors were extracted with AcOEt and the combined organics were washed with brine, dried over Na ₂ SO ₄ and concentrated to dryness affording a solid that was combined to the previously obtained one. The solid was triturated with Et ₂ O, to give the title intermediate (667 mg, 92% yield).

LC-MS (ESI POS): 526.0 MH+

Example 9

Preparation of (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12- Difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo-8-(3-phenyl-propyl)- 2,4b,5,6,6a,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8-a za- pentaleno[2,l-a]phenanthrene-6b-carboxylic acid fluoromethyl ester (compound 20)

To a solution of intermediate 19 (150 mg, 0.285 mmol) in dry DMF (4 ml), Na ₂ CO ₃ (60.5 mg, 0.571 mmol) was added and the suspension stirred at RT for 15 minutes, then the reaction was cooled to 0°C and bromofluoromethane (285 μΐ, 0.571 mmol) was added. After stirring at 0°C overnight, Na ₂ CO ₃ (181 mg, 1.707 mmol) and bromofluoromethane (570 μΐ, 1.140 mmol) were added and the mixture was stirred at T overnight. The reaction was diluted with AcOEt and poured onto brine, the aqueous layer was extracted with AcOEt twice and the combined organic extracts were washed with brine. After solvent removal, the crude was purified by silica gel chromatography (DCM/AcOEt 85 : 15) to give the title compound (60 mg, 38% yield).

1H NMR (300 MHz, DMSO-d ₆ ) δ ppm 7.26 (dd, 1 H), 7.06 - 7.27 (m, 5 H), 6.29 (dd, 1 H), 6.10 (s, 1 H), 5.80 (dd, 1 H), 5.79 (dd, 3 H), 5.51 - 5.73 (m, 1 H), 5.50 (dd, 1 H), 4.06 - 4.28 (m, 1 H), 2.99 - 3.18 (m, 1 H), 2.84 (t, 1 H), 2.63 (d, 1 H), 2.54 (d, 1 H), 2.40 - 2.58 (m, 3 H), 2.13 - 2.34 (m, 2 H), 2.06 (dd, 1 H), 1.95 - 2.03 (m, 1 H), 1.53 - 1.91 (m, 5 H), 1.49 (s, 3 H), 1.36 (dd, 1 H), 0.98 (s, 3 H))

LC-MS (ESI POS): 558.28 MH+

[cc] _D ²⁰ + 56.52 (c 0.27, MeOH)

Example 10

Preparation of (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12- Difluoro-5-hydroxy-4a,6a-dimethyl-8-(5-methyl-furan-2-ylmeth yl)-2-oxo- 2,4b,5,6,6a,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8-a za- pentaleno[2,l-a]phenanthrene-6b-carboxylic acid cyanomethyl ester (compound 21)

To a solution of intermediate 19 (151 mg, 0.287 mmol) in dry DMF (4ml), Na ₂ CO ₃ (54.8 mg, 0.517 mmol) was added and the suspension stirred at RT for 20 minutes. After cooling to 0°C 2-bromoacetonitrile (0.018 ml, 0.259 mmol) was added and the mixture was stirred for 3 hours. Then, Na ₂ CO ₃ (24 mg, 0.230 mmol) and 2-bromoacetonitrile (8.00 μΐ, 0.1 14 mmol) were added and the mixture was stirred 48 hours. The reaction was diluted with AcOEt and poured onto brine, the aqueous layer was extracted with AcOEt twice and the combined organic extracts were washed with brine. After solvent removal, the crude was purified by silica gel chromatography (DCM/AcOEt 3: 1 to 1.1) to title compound (75 mg, 46% yield).

1H NMR (300 MHz, DMSO-d6) δ ppm 7.05 - 7.34 (m, 6 H), 6.29 (dd, 1 H), 6.10 (s, 1 H), 5.54 - 5.76 (m, 1 H), 5.53 (dd, 1 H), 5.02 (s, 2 H), 4.03 - 4.28 (m, 1 H), 3.01 - 3.17 (m, 1 H), 2.83 (t, 1 H), 2.55 - 2.68 (m, 4 H), 2.40 - 2.47 (m, 2 H), 2.14 - 2.35 (m, 3 H), 1.92 - 2.12 (m, 2 H), 1.50 (s, 3 H), 1.43 - 1.88 (m, 5 H), 1.29 - 1.42 (m, 1 H), 0.97 (s, 3 H)

LC-MS (ESI POS): 565.29 MH+

[cc] _D ²⁵ +71.3 (c 0.29, MeOH)

Example 11

Preparation of Acetic acid 2-

[(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-(4-chloro-pheny l)-4b,12- difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo- 2,4b,5,6,6a,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8-a za- pentaleno[2,l-a]phenanthren-6b-yl]-2-oxo-ethyl ester (compound 22)

Intermediate 6 (250 mg, 0.500 mmol) and 4-chlorophenylboronic acid (235 mg, 1.500 mmol) were dissolved in DCM (10 ml), then triethylamine (0.485 ml, 3.50 mmol), and copper(II) acetate (91 mg, 0.500 mmol) were added and the reaction mixture was stirred at T for 15 days. The resulting suspension was filtered off and the solvent was evaporated. The crude was purified by silica gel flash chromatography (eluent DCM/MeOH 99: 1 to 98:2) to yield the title compound (35 mg, 12%).

1H NMR (300 MHz, DMSO-d6) d ppm 7.27 (d, 1 H), 7.03 - 7.23 (m, 2 H), 6.48 - 6.77 (m, 2 H), 6.29 (dd, 1 H), 6.09 (s, 1 H), 5.50 - 5.77 (m, 1 H), 5.47 (dd, 1 H), 5.12 (d, 1 H), 4.78 (d, 1 H), 4.04 - 4.27 (m, 1 H), 3.30 - 3.55 (m, 4 H), 3.1 1 (dd, 1 H), 2.57 - 2.69 (m, 1 H), 2.14 - 2.34 (m, 1 H), 2.08 (s, 3 H), 1.69 - 2.02 (m, 4 H), 1.51 - 1.63 (m, 1 H), 1.50 (s, 3 H), 1.37 - 1.49 (m, 1 H), 1.05 (s, 3 H)

LC-MS (ESI POS): 574.3 MH+

Preparation of (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-(4- Chloro-phenyl)-4b,12-difluoro-5-hydroxy-6b-(2-hydroxy-acetyl )-4a,6a- dimethyl-4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,ll,12-tetradecahyd ro-4aH-8- aza-pentaleno[2,l-a]phenanthren-2-one (compound 23)

Compound 22 (33 mg, 0.057 mmol) was hydrolyzed as described in

Example 5 for the synthesis of compound 9. Title compound 23 (24 mg, 78%) was isolated by silica gel flash chromatography (eluent DCM/MeOH 99: 1 to 98:2).

1H NMR (300 MHz, DMSO- ₆ ) δ ppm 7.26 (dd, 1 H), 7.10 - 7.22 (m, 2 H), 6.52 - 6.72 (m, 2 H), 6.29 (dd, 1 H), 6.09 (s, 1 H), 5.49 - 5.74 (m, 1 H), 5.45 (dd, 1 H), 4.92 (t, 1 H), 4.45 (dd, 1 H), 4.13 - 4.27 (m, 1 H), 4.12 (dd, 1 H), 3.43 - 3.55 (m, 1 H), 3.34 - 3.43 (m, 3 H), 3.06 (dd, 1 H), 2.59 - 2.67 (m, 1 H), 2.18 - 2.34 (m, 1 H), 1.71 - 2.00 (m, 4 H), 1.52 - 1.59 (m, 1 H), 1.50 (s, 3 H), 1.37 - 1.48 (m, 1 H), 0.99 (s, 3 H)

LC-MS (ESI POS): 532.28 MH+

[cc] _D ²⁵ + 19.3 (c 0.26, MeOH)

Compound 24 reported in Table 6 was prepared by a two steps procedure as described in Example 1 1 for compound 23, starting from intermediate 6 and 3-chlorophenylboronic acid.

Table 6

Example 12

Preparation of Acetic acid 2-((4aS,4bR,5S,6aS,6bS,9aR,10aS, lObS, 12S)-4b,12-difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo-8-phenyl- 2,4b,5,6,6a,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8-a za- pentaleno [2,l-a]phenanthren-6b-yl)-2-oxo-ethyl ester (intermediate 25)

In a nitrogen atmosphere compound 6 (150 mg, 0.300 mmol) was suspended in Acetonitrile (4 ml). 2-(trimethylsilyl)phenyl trifluoromethane sulfonate (0.146 ml, 0.600 mmol) and CsF (182 mg, 1.200 mmol) were added and the mixture was stirred for 45 minutes at T. Additional CsF (137 mg, 0.900 mmol) (not dried) was added and the mixture was stirred for 5 hours then it is was poured in water. Acetonitrile was evaporated and the reaction mixture was partitioned between water and AcOEt. The organic layer was separated dried over Na ₂ SO ₄ and concentrated. The crude was purified by silica gel flash chromatography (eluent DCM/MeOH 98:2) affording the title intermediate (49 mg, 30.3% yield).

LC-MS (ESI POS): 540.2 (MH+)

Example 13

Preparation of (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12- Difluoro-5-hydroxy-4a,6a-dimethyl-2-oxo-8-phenyl- 2,4b,5,6,6a,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8-a za- pentaleno[2,l-a]phenanthrene-6b-carboxylic acid (compound 26)

Intermediate 25 (550 mg, 1.019 mmol) was dissolved in a THF /water 2/1 mixture (15ml), 2M aqueous NaOH (2.039 ml, 4.08 mmol) was added and the mixture was stirred at open air for 1.5 hours. The reaction mixture was neutralised by adding 3N HC1 solution. THF was evaporated and the mixture was partitioned between AcOEt and water. The organic phase was dried over Na ₂ SO ₄ and concentrated to dryness affording the title compound (493 mg, quant, yield).

LC-MS (ESI POS): 484.3 MH+

(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12-Difluoro-5- hydroxy-4a,6a-dimethyl-2-oxo-8-phenyl-

2,4b,5,6,6a,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH- 8-aza- pentaleno[2,l-a]phenanthrene-6b-carbothioic acid S-fluoromethyl ester (compound 27)

A mixture of compound 26 (493 mg, 1.019 mmol), HATU (387 mg,

1.1019 mmol) and N-methylmorpholine (0.123 ml, 1.121 mmol) in dry DMF (7 ml) was stirred at RT under nitrogen for 40 minutes. Then, anhydrous sodium hydrogen sulfide (171 mg, 3.06 mmol) was added and the solution was stirred at RT for 1 hour. Bromofluoromethane (2.038 ml, 4.08 mmol, 2 M solution in DMF) was added and the mixture was stirred at RT for 2 hours. The reaction mixture was concentrated to dryness and the crude was purified by preparative HPLC (CH ₃ CN/H ₂ O, without CF ₃ COOH) to afford the title compound (158 mg, 29% yield).

1H NMR (300 MHz, DMSO-d ₆ ) δ ppm 7.26 (dd, 1 H), 7.04 - 7.21 (m, 2 H), 6.60 - 6.79 (m, 3 H), 6.30 (dd, 1 H), 6.09 (s, 1 H), 5.95 (dd, 1 H), 5.91 (dd, 1 H), 5.58 (dd, 1 H), 5.47 - 5.76 (m, 1 H), 4.04 - 4.36 (m, 1 H), 3.61 (dd, 1 H), 3.48 (d, 1 H), 3.34 (d, 1 H), 3.05 (dd, 1 H), 2.56 - 2.70 (m, 2 H), 2.14 - 2.32 (m, 1 H), 2.02 - 2.14 (m, 1 H), 1.74 - 2.02 (m, 3 H), 1.52 - 1.63 (m, 1 H), 1.50 (s, 3 H), 1.37 - 1.48 (m, 1 H), 1.08 (s, 3 H)

LC-MS (ESI POS): 532.30 MH+

[cc] _D ²⁵ + 12.4 (c 0.34, MeOH)

Example 14

Preparation of (6S,8S,9R,10S,HS,13S,14S)-6,9-difluoro-17-(2- fluoroacetyl)-ll-hydroxy-10,13-dimethyl-6,7,8,9,10,ll, 12,13,14,15- decahydro-3H-cyclopenta[a]phenanthren-3-one (intermediate 28)

In a nitrogen atmosphere, intermediate 3 (100 mg, 0.264 mmol) was dissolved in Acetonitrile (4 ml), Mesyl-Cl (32 μΐ, 0.41 1 mmol) and DIPEA (78 μΐ, 0.447 mmol) were added. The mixture was stirred at T for 30 minutes. Potassium fluoride (154 mg, 2.64 mmol) was added and the mixture was stirred for 1 h at 60°C. TBAF 1M in THF (529 μΐ, 0.529 mmol) was added and the mixture is stirred at 60°C for 4 hours. The reaction mixture was partitioned between water and AcOEt, the organic layer was separated, dried over Na ₂ SO ₄ and concentrated. The crude was purified by silica gel flash chromatography (eluent DCM/AcOEt = 1 : 1) to yield intermediate 28 (62 mg, 61.7% yield).

LC-MS (ESI POS): 381.0 (MH+)

Preparation of (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-8-Benzyl- 4b,12-difluoro-6b-(2-fluoro-acetyl)-5-hydroxy-4a,6a-dimethyl - 4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8- aza- pentaleno[2,l-a]phenanthren-2-one (intermediate 29)

In a closed vessel, to a mixture of intermediate 28 (125 mg, 0.329 mmol) and N-benzyl-l -methoxy-N- ((trimethylsilyl)methyl)methanamine (0.318 ml, 1.314 mmol) in 1 ,4-Dioxane (5 ml), 1 drop of TFA (cat) was added and the mixture was stirred at 100°C for 1 hour. The reaction mixture was concentrated and purified by silica gel flash chromatography (eluent DCM/AcOEt 7:3) and then by preparative HPLC to afford the title intermediate (45 mg, 26.7% yield).

1H NMR (300 MHz, DMSO- ₆ ) δ ppm 7.1 1 - 7.47 (m, 6 H), 6.29 (dd, 1

H), 6.12 (s, 1 H), 5.49 - 5.77 (m, 1 H), 5.40 (dd, 1 H), 5.26 (dd, 1 H), 5.1 1 (dd, 1 H), 4.05 - 4.22 (m, 1 H), 3.49 (d, 1 H), 3.44 (d, 1 H), 3.03 - 3.19 (m, 1 H), 2.92 (t, 1 H), 2.53 - 2.61 (m, 2 H), 2.38 (d, 1 H), 2.20 - 2.33 (m, 1 H), 1.93 - 2.10 (m, 2 H), 1.84 (d, 1 H), 1.51 - 1.77 (m, 3 H), 1.49 (s, 3 H), 1.37 (dd, 1 H), 0.91 (s, 3 H)

LC-MS (ESI POS): 514.39 MH+

[cc] _D ²⁵ + 84.2 (c 0.36 CHC13)

Example 15

Preparation of (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12- Difluoro-6b-(2-fluoro-acetyl)-5-hydroxy-4a,6a-dimethyl-2-oxo - 2,4b,5,6,6a,6b,7,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8- aza- pentaleno[2,l-a]phenanthrene-8-carboxylic acid vinyl ester (intermediate 30)

Intermediate 29 (614 mg, 1.196 mmol) and NaHCO ₃ (100 mg, 1.196 mmol) were dissolved in acetonitrile (12 ml) and then vinyl chloroformate (0.204 ml, 2.391 mmol) was added. The reaction mixture was warmed at 50°C for 1 hour. The solution was partitioned between AcOEt and water. The organic phase was separated and the aqueous solution was extracted with AcOEt. The combined organic phases were dried over Na ₂ SO ₄ and then evaporated to give a residue that was purified by silica gel column chromatography (eluent AcOEt/petroleum ether 2:8 to 8:2) leading to the pure title intermediate (41 1 mg, 69.7% yield).

LC-MS (ESI POS): 494.0 MH+

Preparation of (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12- Difluoro-6b-(2-fluoro-acetyl)-5-hydroxy-4a,6a-dimethyl-4b,5, 6,6a,6b,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8-aza- pentaleno[2,l-a]phenanthren-2-one hydrochloride (intermediate 31)

Intermediate 30 (294 mg, 0.596 mmol) was dissolved in DCM (20 ml) and then HCI 4.0 M in Dioxane (2.98 ml, 1 1.91 mmol) was added. The solution was stirred at RT for 5 hours and then the solvent was evaporated and the residue was dried under vacuum for 16 hours. The solid was then dissolved in methanol (30 ml) and warmed at 45°C for 20 minutes. Methanol was evaporated to give the title intermediate (274 mg, 100% yield).

LC-MS (ESI POS): 424.0 (MH+)

Example 16

Preparation of (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS,12S)-4b,12- Difluoro-6b-(2-fluoro-acetyl)-5-hydroxy-4a,6a-dimethyl-8-phe nyl- 4b,5,6,6a,6b,7,8,9,9a,10,10a,10b,ll,12-tetradecahydro-4aH-8- aza- pentaleno[2,l-a]phenanthren-2-one (compound 32)

Intermediate 31 (307 mg, 0.667 mmol) was suspended in acetonitrile (12 ml). CsF (406 mg, 2.67 mmol), 2-(trimethylsilyl)phenyl trifluoromethanesulfonate (239 mg, 0.194 mmol) and water (1 drop) were added and the mixture was stirred at RT for 24 hours. The reaction mixture was partitioned between water and AcOEt, the organic layer was separated, dried and concentrated. The crude was purified by silica gel flash chromatography (eluent DCM/MeOH = 99: 1) to yield the title compound (94 mg, 28%).

1H NMR (300 MHz, DMSO-d ₆ ) δ ppm 7.27 (dd, 1 H), 7.05 - 7.22 (m, 2 H), 6.55 - 6.81 (m, 3 H), 6.29 (dd, 1 H), 6.08 (s, 1 H), 5.50 - 5.74 (m, 1 H), 5.46 (dd, 0 H), 5.46 (dd, 1 H), 5.14 (dd, 1 H), 4.08 - 4.29 (m, 1 H), 3.44 - 3.60 (m, 1 H), 3.32 - 3.41 (m, 3 H), 3.25 (d, 1 H), 3.05 (dd, 1 H), 2.55 - 2.69 (m, 1 H), 2.15 - 2.34 (m, 1 H), 1.70 - 2.00 (m, 4 H), 1.51 - 1.59 (m, 1 H), 1.50 (s, 3 H), 1.39 - 1.48 (m, 1 H), 1.03 (s, 3 H)

LC-MS (ESI POS): 500.31 MH+

[cc] _D ²⁵ + 24, 1 (c= 0.48, MeOH)

s = singlet

d = doublet

t = triplet

q = quartet

dd = doublet of doublets

m = multiplet

br = broad

PHARMACOLOGICAL ACTIVITY OF THE COMPOUNDS OF THE INVENTION

In vitro studies

Example 17

Glucocorticoid Receptor (GR) translocation assay protocol

A quantitative measurement of GR nuclear translocation of the compounds of the present invention was performed according to ASSAY Drug Devel.Technol., 4(3), 263-272, 2006, through a novel cell-based GR-translocation assay in Enzyme Fragment Complementation (EFC) format developed by DiscoveRx (Fremont, CA).

The DiscoveRx assay uses EFC of b-galactosidase (b-gal) as an indicator of GR-translocation in engineered CHO-K1 biosensor cells. The enzyme acceptor (EA) fragment of b-gal resides in the nucleus, as designed through the use of a proprietary set of sequence additions and modifications. The small peptide enzyme donor (ED) fragment of b-gal is fused directly to the C-terminus of GR, and is localized in the cytoplasm is the absence of receptor signaling. Upon binding to a GR ligand, the complex translocates to the nucleus, where intact enzyme activity is restored by complementation and b-gal activity is detected.

CHO-K1 cells stably expressing NLS-enzyme acceptor fragment (EA) of b-gal and GR-enzyme donor (ED) fragment of b-gal were maintained in F12 medium (Invitrogen, Carlsbad, CA) at 37 °C under a humidified atmosphere containing 5% CO ₂ and 95% air. The medium contained 10% FBS, 2 mM L-glutamine, 50 U/ml penicillin 50 g/ml streptomycin, and 250 g/ml hygromycin and 500 g/ml G418 (Invitrogen).

GR-translocation was measured using the PathHunter Detection Kit containing cell membrane permeabilizing reagent and beta-gal substrate (DiscoveRx, Fremont, CA). All compounds were screened using varying concentrations ranging from 10 ^{"1 1} to 10 ^"6 M. The assay was performed in 48-wells (105 cells/well). Incubation with screened compounds was performed at 37°C for two hours. Detection was made by adding the detection buffer from the kit supplied by DiscoveRx and incubating at RT for one hour. Luminescence was detected by using a CENTRO LB 960 microplate reader (Berthold Technologies).

Statistical analysis and determinations of EC50s were performed by using Prism-version 3.0 Graphpad Software (San Diego, CA).

The compounds assayed with the GR translocation assay displayed a EC50 comprised between 1 nM and 10 nM.

Example 18

Inhibition of LPS-induced nitric oxide production in RAW 264.7 macrophages

An in vitro model based on macrophagic murine cell line RAW 264.7 was used for testing the anti-inflammatory effects of the corticosteroids of the present invention.

During the inflammatory process, large amounts of nitric oxide (NO) are generated by the inducible isoforms of NO synthase (iNOS). Bacterial lipopolysaccharide (LPS) is commonly used in experimental settings to stimulate inflammatory responses in macrophages.

Cells were grown in a culture medium ( PMI supplemented with heat- inactivated 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin) without phenol red. Cell stimulation was elicited by incubating cells for 24 hours with LPS to final concentrations ranging from 100 ng/ml. Treatments with the compounds of the invention were carried out by adding such compounds vehicled in DMSO (0.1% final concentration) to the final desired concentrations 15 minutes before LPS exposure. As an index of nitric oxide production, the concentration of nitrite was measured in the conditioned media by using the Griess colorimetric reaction (J. Neuroimmunol., 150, 29-36, 2004).

Statistical analysis and determinations of IC50s were performed by using Prism-version 3.0 Graphpad Software (San Diego, CA). The IC50 values tested on the compounds of the invention are comprised between 0.16 and 1 nM.