HK1166085B

HK1166085B - Isoxazolidine derivatives

Info

Publication number: HK1166085B
Application number: HK12106794.1A
Authority: HK
Inventors: E．阿曼尼; E．吉迪尼; I．佩雷托; A．沃蒂斯
Original assignee: 奇斯药制品公司
Priority date: 2009-09-11
Filing date: 2010-09-01
Publication date: 2017-01-06

Description

Isoxazolidine derivatives

Technical Field

The present invention relates to novel anti-inflammatory and antiallergic compounds of the glucocorticosteroid series, processes for preparing said compounds, pharmaceutical compositions comprising them, combinations and therapeutic uses thereof. More particularly, the present invention relates to glucocorticosteroids which are isoxazolidine derivatives.

Background

Corticosteroids are potent anti-inflammatory agents that are capable of reducing inflammatory cell number, activity and motility.

They 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 (GR). Binding of corticosteroids to GR induces their nuclear translocation, which in turn affects many downstream pathways via DNA-binding dependent (e.g., transactivation) and DNA-binding independent (e.g., trans expression) mechanisms.

Corticosteroids for the treatment of chronic inflammatory conditions of the lung, such as asthma and COPD, are currently administered by inhalation. One of the advantages of using Inhaled Corticosteroids (ICS) is the ability to deliver the drug directly at the site of action, limiting the systemic negative effects, resulting in a faster clinical response and higher therapeutic rates.

While ICS therapy can provide important benefits, it is important, especially in asthma, to minimize ICS systemic exposure that leads to the appearance and exacerbation of undesirable side effects that can be associated with chronic dosing. Furthermore, the limited duration of action of ICS currently available in clinical practice leads to suboptimal disease management. Since inhaler technology is a key point for targeting the lung, modulation of the substituents on the corticosteroid molecular skeleton is important for optimizing pharmacokinetic and pharmacodynamic properties, which reduces oral bioavailability, limits pharmacological activity only in the lung (prodrugs and soft drugs) and increases systemic clearance. In addition, long-lasting ICS activity in the lung is highly desirable, as once daily administration of ICS would allow for a reduction in dosing frequency, thereby substantially improving patient compliance and, as a result, disease management and control. In conclusion, there is an urgent medical need to develop ICS with improved pharmacokinetic and pharmacodynamic characteristics.

Glucocorticoid isoxazolidine derivatives are described in WO2006/005611, GB1578446 and "Synthesis and picacalandi-antiflammator derivatives of somesteroides [16 α, 17 α -d ] isoxazolidines" (J.Med.chem., 25, 1492-1495, 1982).

The isoxazolidine glucocorticoids of the present invention were characterized for in vitro potency and efficacy by: a) binding to GR, b) induction of GR nuclear translocation, and c) inhibition of inflammatory responses in macrophages. Furthermore, the objective of optimizing the pharmacokinetic/pharmacodynamic properties is to improve the anti-inflammatory efficacy, efficacy and duration of action in the lung as well as to reduce systemic side effects. In the case of local administration in the lungs of experimental animal models, isoxazolidine glucocorticoids of the present invention are characterized by good anti-inflammatory potency and efficacy, which is associated with long duration of action and limited systemic exposure.

Summary of The Invention

The present invention relates to compounds of general formula (I), to a process for preparing said compounds, to compositions comprising them and to their therapeutic use

(I)

Wherein

R1 is (CH)₂)_n-Z-(CH₂)_n-R4, wherein n and n' are each independently 0, 1 or 2;

z is a single bond or is selected from S, O, CO and NR3, wherein R3 is selected from H, linear or branched (C)₁-C₆) Alkyl radical (C)₁-C₆) Haloalkyl (C)₃-C₈) Cycloalkyl, aryl (C)₁-C₆) Alkyl and heteroaryl, optionally substituted with CN;

r4 is selected from:

-H, halogen, OH, SH, CN, NH₂；

Aryl (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkylsulfonyl group (C)₁-C₆) Alkylcarbonyl group, (C)₁-C₆) Alkylcarboxyl, O (C)₁-C₆) Alkylcarboxy (C)₁-C₆) Alkyl amides and (C)₁-C₆) Alkoxy, optionally substituted with oxo;

-(C₁-C₆) Alkyl, which may be optionally substituted with one or more substituents selected from: halogen atom, CN, OH, NH₂，NO₂，CF₃And SH;

-(C₂-C₆) An alkynyl group;

-a mono-, di-or tricyclic saturated or partially saturated or unsaturated ring, such as (C)₃-C₈) Cycloalkyl, aryl, (C)₅-C₁₀) Heterocycloalkyl or heteroaryl, optionally substituted with one or more halogen atoms or oxo groups;

r2 is selected from

-H；

-linear or branched (C)₁-C₆) An alkyl group;

-(CH₂)_mr5, wherein R5 is heteroaryl, optionally substituted with a substituent selected from: oxo, OH, halogen, CN, NH₂，NO₂Aryl, (C)₁-C₆) Alkylsulfonyl group (C)₁-C₆) Alkoxy group, (C)₁-C₆) Alkylthio (C)₁-C₆) Alkylcarboxy (C)₁-C₆) Alkylamides, aryl radicals (C)₁-C₆) Alkoxy and (C)₁-C₆) Alkyl, each of which is optionally substituted with one or more halogen atoms or COOH;

-(CH₂)_pNR6R7；

-(CH₂)_pNR6COR7；

-(CH₂)_pNR6SO₂R7；

-(CH₂)_mCONR6R7；

-(CH₂)_mSO₂NR6R7；

-(CH₂)_mCOR7；

-(CH₂)_pOR7；

-(CH₂)_mSO_qR7；

wherein R6 and R7 are independently H or selected from linear or branched (C)₁-C₆) Alkyl radical (C)₃-C₈) Cycloalkyl, aryl (C)₁-C₆) Alkyl and saturated, partially saturated or unsaturated, optionally fused rings, e.g. aryl, (C)₅-C₁₀) Heterocycloalkyl or heteroaryl, which groups may be optionally substituted with one or more substituents selected from: halogen, CN, oxo, NH₂，NO₂And (C)₁-C₆) An alkyl group;

-(CH₂)_pr8, wherein R8 is selected from halogen, oxo, CN, OH, NH₂，NO₂；(C₃-C₈) Cycloalkyl, aryl and saturated, partially saturated or unsaturated, optionally fused, rings such as (C)₅-C₁₀) Heterocycloalkyl, optionally substituted with one or more substituents selected from: halogen, CO, CN, (C)₁-C₆) Alkyl radical (C)₁-C₆) Haloalkyl (C)₁-C₆) Carboxyalkyl (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkoxy and (C)₁-C₆) An alkylsulfonyl group;

wherein m and p are each independently 0 or an integer of 1 to 6 and q is 0, 1 or 2 and

x and Y are independently selected from H and halogen atoms

And a pharmaceutically acceptable salt thereof,

provided that at R2 is (C)₁-C₆) In the case of alkyl, X and Y are not both H.

It is obvious to the person skilled in the art that the compounds of the general formula (I) contain asymmetric centers at least in positions 4a, 4b, 5, 6a, 6b, 9a, 10a, 10b, 12 and can therefore exist as a number of optical stereoisomers and mixtures thereof.

The invention therefore also relates to all of these forms.

Preferred compounds are those of formula (I) wherein the configuration of the chiral carbon atom is fixed, and in particular those wherein 4a is (R), 4b is (R), 5 is (S), 6a is (S), 6b is (R), 9a is (S), 10b is (S) and 12 is (S), which are represented by the following formula (I '), (I') wherein

(I’)

Wherein the values of R1, R2, X and Y are as defined hereinbefore.

The compounds of general formula (I) are capable of forming acid addition salts, in particular pharmaceutically acceptable acid addition salts.

Thus, pharmaceutically acceptable acid addition salts of the compounds of formula (I) also encompass those of formula (I'), including inorganic acids, for example hydrohalic acids such as hydrofluoric, hydrochloric, hydrobromic or hydroiodic acid; those of nitric acid, sulfuric acid, phosphoric acid; and organic acids, for example, aliphatic monocarboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, and propionic acid; aliphatic hydroxy acids such as lactic acid, citric acid, tartaric acid or malic acid; dicarboxylic acids such as maleic acid, fumaric acid, oxalic acid or succinic acid; aromatic carboxylic acids such as benzoic acid; aromatic hydroxy acids and sulfonic acids.

These salts may be prepared from the compounds of formula (I) by known salt-forming procedures.

The compounds of formula (I) object of the present invention can be prepared according to various synthetic procedures carried out according to conventional methods and techniques.

The invention also relates to a process for the preparation of compounds of general formula (I') wherein R1 ═ (CH)₂)_n-Z-(CH₂)_n-R4, wherein n ═ 1 and R4 are as defined hereinbefore, which comprises:

a compound of formula (VI)

(VI)

With methanesulfonyl chloride or p-toluenesulfonyl chloride to give a compound of the general formula (XI)

Wherein the Leaving Group (LG) can be replaced by a nucleophile.

The invention also relates to a process for the preparation of compounds of general formula (I') wherein R1 ═ (CH)₂)_n-Z-(CH₂)_n-R4, wherein n ═ 0, Z and R4 are as defined hereinbefore, which includes:

-reacting the compound of formula (VI) under oxidizing conditions to obtain an intermediate of general formula (XII)

-treating the compound of formula (XII) with one or more equivalents of an acid activator, followed by treatment with a nucleophile.

The invention also relates to a process for the preparation of compounds of general formula (I') wherein R1 ═ (CH)₂)_n-Z-(CH₂)_n-R4, wherein n is 0, Z is S and R4 are as defined hereinbefore, comprising:

-converting it into a compound of formula (XIII)

(XIII)

-alkylating the compound of formula (XIII).

The invention also relates to a process for the preparation of a compound of general formula (VI) comprising:

-reacting a compound of formula (IV)

With N-tetrahydropyranyl hydroxylamine (HO-NH-THP) to produce a compound of formula (V)

-optionally further functionalizing a compound of formula (V) and

deprotection.

-reacting a compound of formula (VII)

With compounds of the formula (X)

(X)

-reacting the compound of formula (VII) with N-tetrahydropyranyl hydroxylamine (HO-NH-THP) to obtain the compound of formula (VIII)

Protecting the compound of formula (VIII) to obtain a compound of formula (IX)

-optionally further functionalizing the compound of formula (IX) and

deprotection.

The invention also relates to a process for the preparation of compounds of general formula (I') wherein R1 ═ (CH)₂)_n-Z-(CH₂)_n-R4 wherein n ═ 1, Z ═ O and R4 ═ Ac, comprising the reaction of the intermediate of general formula (IV) with hydroxylamine of formula (X).

The invention also relates to a process for the preparation of compounds of general formula (I') wherein R1 ═ (CH)₂)_n-Z-(CH₂)_n-R4, wherein n ═ 1, Z ═ O, R4 ═ H and X ═ Cl, the process comprising:

-reacting a compound of formula (I') wherein n ═ 1, Z ═ O, R4 ═ Ac and X ═ H with methanesulfonyl chloride to obtain a compound of formula (XIV)

-reacting compound (XIV) with a chlorinating agent and

-carrying out the hydrolysis.

From all the above, it is clear to the skilled person that any of the possible stereoisomers of formula (I) can be obtained by selecting a starting material with a suitable stereochemical configuration.

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

The compounds of the invention may be administered as the sole active agent or in combination with other pharmaceutically active ingredients including those currently used in the treatment of respiratory disorders, such as β 2-agonists, antimuscarinics, corticosteroids, mitogen-activated protein kinase (P38MAP kinase) inhibitors, nuclear factor kappa-B kinase subunit beta (IKK2) inhibitors, Human Neutrophil Elastase (HNE) inhibitors, phosphodiesterase 4(PDE4) inhibitors, leukotriene modulators, non-steroidal anti-inflammatory agents (NSAIDs) and mucus regulators.

The invention also provides a combination of a compound of formula (I) or (I') with a β 2-agonist selected from 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 and salts thereof.

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

The invention also provides a combination of a compound of formula (I) or (I') with a PDE4 inhibitor selected from the group consisting of AN-2728, AN-2898, CBS-3595, apremilast, ELB-353, KF-66490, K-34, LAS-37779, IBFB-211913, AWD-12-281, simmeridine, cilomilast, roflumilast, BAY19-8004 and SCH-351591, AN-6415, indus-82010, TPI-PD3, ELB-353, CC-11050, GSK-256066, Omilast, OX-914, tetomilast, MEM-1414 and RPL-554.

The present invention also provides a combination of a compound of formula (I) or (I') with an inhibitor of P38MAP kinase selected from the group consisting of samimod, talmapimod, pirfenidone, PH-797804, GSK-725, a minute and a loshapimod and salts thereof.

In a preferred embodiment, the present invention provides a combination of a compound of formula (I) or (Γ) and an IKK2 inhibitor.

The present invention also provides a combination of a compound of formula (I) with an HNE inhibitor selected from the group consisting of AAT, ADC-7828, Aeriva, TAPI, AE-3763, KRP-109, AX-9657, POL-6014, AER-002, AGTC-0106, respriva, AZD-9668, zemaira, AATIV, PGX-100, elafin, SPHD-400, alpha 1-proteinase inhibitor C and alpha 1-proteinase inhibitor inhaled.

The invention also provides a combination of a compound of formula (I) with a leukotriene modulator selected from montelukast, zafirlukast and pranlukast.

The present invention also provides a combination of a compound of formula (I) and an NSAID selected from ibuprofen and ketoprofen.

The invention also provides a combination of a compound of formula (I) and a mucus modulator selected from INS-37217, diquafosol, siberian, CS-003, talnetant, DNK-333, MSI-1956 and gefitinib.

The invention also provides compounds of general formula (I) or (I') for use as medicaments.

The invention also relates to the use of a compound of general formula (I) or (I') for reducing the number, activity and movement of inflammatory cells in vitro and/or in vivo.

The invention also relates to the use of compounds of general formula (I) or (I') in the prevention or treatment of any disease in which a reduction in the number, activity and movement of inflammatory cells is implicated.

In other aspects of the invention, there is provided the use of a compound of formula (I) or (I') for the prevention and/or treatment of any disease in which a reduction in the number, activity and movement of inflammatory cells is implicated.

In particular, the compounds of general formula (I) or (I') may be administered alone or in combination with one or more active ingredients for the prevention and/or treatment of respiratory diseases characterized by airway obstruction such as asthma and COPD.

In a further aspect, the present invention provides the use of a compound of general formula (I) or (I') for the preparation of a medicament for the prevention and/or treatment of any disease in which a reduction in the number, activity and movement of inflammatory cells is implicated.

Furthermore, the present invention provides a method for the prevention and/or treatment of any disease in which a reduction in the number, activity and movement of inflammatory cells is implicated, which comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound of formula (I) or (Γ).

The invention also provides pharmaceutical formulations of compounds of formula (I) or (I') suitable for administration by inhalation, injection, orally or intranasally.

Inhalable formulations include inhalable powders, propellant-containing metered dose aerosols or propellant-free inhalable formulations.

The invention also relates to a device comprising a compound of formula (I) or (Γ), which may be a single-or multi-dose anhydrous powder inhaler, a metered dose inhaler or a nebulizer, in particular a nebulizer of the soft mist (softmist) type.

The invention also relates to kits comprising a pharmaceutical composition of a compound of general formula (I) or (Γ) alone or in combination with or admixed with one or more pharmaceutically acceptable carriers and/or excipients, and a device which may be a single-or multi-dose anhydrous powder inhaler, a metered dose inhaler or a nebulizer.

Definition of

The term "halogen" or "halogen atom" as used herein includes fluorine, chlorine, bromine and iodine.

As used herein, the expression "(C)₁-C₆) Alkyl "refers to straight and branched chain alkyl groups in which the number of constituent carbon atoms is from 1 to 6. Examples of alkyl are methyl, ethyl, n-propyl, isopropyl, tert-butyl, pentyl and hexyl.

Expression "(C)₂-C₆) Alkynyl "is explained in a similar manner. Examples of alkynyl groups include ethynyl, propynyl, butynyl, pentynyl and hexynyl.

The term "(C)₁-C₆) Alkoxy "refers to an alkyl-oxy (e.g., alkoxy) group. Thus, examples of the group may include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.

The term "(C)₁-C₆) Haloalkyl "and" (C)₁-C₆) Haloalkoxy "means" above "(C)₁-C₆) Alkyl groups "and" ("C)₁-C₆) Alkoxy "groups, in which one or more hydrogen atoms are replaced by one or more halogen atoms, can be identical or different from each other.

Similarly, the term "(C)₁-C₆) Alkylcarbonyl, "O (C)₁-C₆) Alkylcarbonyl "," (C)₁-C₆) Alkylcarboxy "and" (C)₁-C₆) Alkylamides "are alkyl-CO, O-alkyl-CO, alkyl-CO respectively₂And alkyl-NH₂alkyl-NH-alkyl or alkyl-N- (alkyl)₂A group.

Similarly, the term "(C)₁-C₆) Alkylsulfonyl "means alkyl-SO₂-a group.

The term "(C)₃-C₈) Cycloalkyl "refers to a cyclic non-aromatic hydrocarbon group having 3 to 8 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.

The term "(C)₅-C₁₀) Heterocycloalkyl "refers to a cyclic, non-aromatic hydrocarbon group in which at least one ring atom is a heteroatom (e.g., N, S or O). Examples include dihydropyridine and dihydrobenzodioxadiene residues.

The term "aryl" refers to a mono-, bi-or tricyclic ring system having from 5 to 20, preferably from 5 to 15 ring atoms, and wherein at least one ring is aromatic.

TermAryl radical (C)₁-C₆) Alkyl "and" aryl (C)₁-C₆) Alkoxy "means (C)₁-C₆) Alkyl, which is further substituted with an aryl, alkoxy, heteroaryl or cycloalkyl ring, respectively.

Suitable aryl radicals (C)₁-C₆) Examples of alkyl groups include benzyl, biphenylmethyl, and thienylmethyl.

The term "heteroaryl" as used herein refers to a mono-, bi-or tricyclic ring system having 5 to 20, preferably 5 to 15 ring atoms, wherein at least one ring is aromatic and wherein at least one ring atom is a heteroatom (e.g., N, S or O).

Examples of suitable monocyclic systems include thiophene, cyclopentadiene, benzene, pyrrole, pyrazole, imidazole, isoxazole, oxazole, isothiazole, thiazole, pyridine, imidazolidine, piperidine and furan residues such as tetrahydrofuran.

Examples of suitable bicyclic ring systems include naphthalene, biphenyl, purine, pteridine, benzotriazole, quinoline, isoquinoline, indole, isoindole, benzofuran, benzodioxan and benzothiophene residues.

Examples of suitable tricyclic systems include fluorene residues.

Detailed Description

The present invention relates to compounds that act as glucocorticosteroids, which are potent anti-inflammatory agents.

The compounds reduce the number, activity and movement to the bronchial submucosa of inflammatory cells, resulting in reduced airway responsiveness.

In particular, the present invention relates to compounds of general formula (I) as defined hereinbefore

(I)

Preferred compounds are those of formula (I) wherein the configuration of the chiral carbon atom is fixed and in particular wherein 4a is (R), 4b is (R), 5 is (S), 6a is (S), 6b is (R), 9a is (S), 10b is (S) and 12 is (S), which are represented by the following formula (I '), (I') wherein

(I’)

Wherein the values of R1, R2, X and Y are as defined hereinbefore, in free or salt form.

A first preferred group of compounds of the general formula (I') are those in which R is₁Is (CH)₂)_n-Z-(CH₂)_n-R4, wherein n is 0 or 1; z is a single bond or selected from S, O and NR3, wherein R3 is H or (C)₁-C₆) An alkyl group; n' is 0, 1 or 2; r4 is selected from H, halogen, CN, OH; (C)₁-C₆) Alkyl, optionally substituted with one or more substituents selected from: halogen atom, CN, OH, NH₂，NO₂，CF₃And SH; aryl radical (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkynyl (C)₁-C₆) Alkylsulfonyl group (C)₁-C₆) Alkylcarbonyl, O (C)₁-C₆) Alkylcarbonyl group, (C)₁-C₆) Alkyl amides, (C)₁-C₆) Alkylcarboxy (C)₅-C₁₀) Heterocycloalkyl and heteroaryl, optionally substituted with oxo; r2, X and Y have the meanings reported above.

Also more preferred in this class are compounds of formula (I') wherein R4 is selected from methyl, ethyl, benzothiazole, benzoxazole, tetrahydrofuran, tetrahydropyran, methanesulfonyl, methylcarbonyl, chloro, fluoro, trifluoromethyl, methylcarboxyl, ethylcarboxy, methoxy, ethoxy, trifluoromethyl and butynyl.

Even more preferred in this category are compounds of formula (I') wherein R3 is H or methyl.

Even more preferred in this category are compounds of formula (I') wherein R2 is linear or branched (C)₁-C₆) An alkyl group.

A second preferred group of compounds of formula (I') are those wherein R1, X and Y are as previously defined; r2 is H or selected from (CH)₂)_mR5, wherein m is 1 or 2, R5 is heteroaryl, optionally substituted with a group selected from: halogen, CN, OH, CF3, (C)₁-C₆) Alkyl radical (C)₁-C₆) Haloalkyl (C)₁-C₆) Alkoxy group, (C)₁-C₆) Alkylsulfonyl and aryl; (CH)₂)_mCONR6R7 wherein m is 0 and R6 is (C)₁-C₆) Alkyl and R7 is aryl; (CH)₂)_mCOR7 wherein m is 1, R7 is aryl, heteroaryl or (C)₅-C₁₀) A heterocycloalkyl group; (CH)₂)_pOR7 where p is 2 and R7 is aryl OR aryl (C)₁-C₆) An alkyl group; (CH)₂)_mSO_qR7 wherein m is 0 or 2, q is 0 or 2 and R7 is (C)₁-C₆) An alkyl or aryl group; and (CH)₂)_pR8 wherein p is 1, 2 or 3 and R8 is selected from aryl and (C)₅-C₁₀) Heterocycloalkyl, optionally substituted with one or more substituents selected from: halogen, oxo, CN, OH, CF₃，(C₁-C₆) Alkyl radical (C)₁-C₆) Haloalkyl (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Alkylcarboxy and (C)₁-C₆) An alkylsulfonyl group.

Still more preferred within this class are compounds of formula (I') wherein R2 is selected from the group consisting of phenyl, thienyl, pyridyl, imidazolyl, thiazolyl, benzyloxyethyl, thiophenyl, phenylpropyl, phenoxyethyl, isoxazolyl, benzoyl, furancarbonyl, methanesulfonyl, dihydropyridinylmethyl and methylphenylamide, cyclopentene copper, benzofuran, furan, dihydrobenzodioxadiene residues.

A third preferred group of compounds of formula (I') are those wherein R1 and R2 are as defined hereinbefore and X and Y are both H or a fluorine atom or X is chlorine and Y is H.

It has been found that the majority of the compounds of the formulae (I) and (I') is in the range of 10^-8To 10^-10M showed in vitro activity in all cell-free and cell-based assays used, and some of them had long-term duration of anti-inflammatory action in vivo in rodent experimental model lungs.

Examples of preferred compounds of the invention are reported below:

(continuation)

The compounds of formula (I) and (I') may be prepared conventionally according to methods disclosed in the art. As shown in the following schemes, certain processes that can be used to prepare compounds of formula (I') can also be applied to compounds of formula (I).

Process for preparing the Compounds of the invention

According to a particular embodiment of the invention, depending on the substituents R1, R2, X and Y, the compounds of the invention can be prepared according to different routes described in the schemes.

Scheme A1-reaction of a compound of formula (IV) with N-tetrahydropyranyl hydroxylamine (HO-NH-THP), preparation of a compound of formula (V) can conveniently be carried out in a protic solvent such as EtOH at a temperature of 80 to 100 ℃. The THP protecting group is directly cleaved under the reaction conditions.

These compounds may be further functionalized with alkyl halides, acid halides, isocyanates, carbamoyl chlorides or sulfonyl chlorides using methods readily available to those skilled in the art (J.Med.chem., 379-one 388, 1995; J.C.S.chem.Comm., 256-one 257, 1985) to provide compounds of formula (VI). These reactions are typically carried out in a solvent such as Dichloromethane (DCM) or Tetrahydrofuran (THF) and at temperatures from Room Temperature (RT) to reflux. A base such as triethylamine or diisopropylethylamine may be required to facilitate the reaction. The reaction with aryl halides can be carried out as known copper catalyzed N-arylation of isoxazolidines (bioorg.med.chem.lett., 2834, 2005). Acetyl esters can be readily hydrolyzed using standard conditions for alcohol deacetylation, for example by treating the compound with a base such as sodium or potassium hydroxide or potassium carbonate in a suitable solvent (e.g. methanol or ethanol). The reaction is generally carried out at room temperature over a period of 1 to 5 hours to give the compound of formula (VI).

The compounds of formula (IV) can be conveniently prepared according to standard procedures reported in the literature. For example they may be prepared by treating a compound of formula (III) with a base such as potassium acetate. The reaction is usually carried out in a suitable polar solvent such as Dimethylformamide (DMF) and is generally carried out at a temperature of 80 to 110 ℃ for 0.5 to 4 hours.

The compounds of formula (III) can be readily prepared from known compounds starting from compounds of formula (II) by methods well known to those skilled in the art (J.Med.chem.1982, 25, 1492-1495).

Route a 2-alternatively, the compounds of general formula (VI) may be prepared by: the compound of formula (VII) is first reacted with the compound of formula (X) in the presence of paraformaldehyde using the known procedure for nitrone cycloaddition for isoxazolidine formation (j.med.chem., 25, 1492-1495, 1982). The reaction is conveniently carried out in a protic solvent such as ethanol at a temperature of from 80 to 100 ℃. Hydroxylamines of the formula (X) are commercially available or can be readily prepared by procedures well known to those skilled in the art, for example, reduction of oximes with reducing agents such as borane pyridine complexes (J.Med.chem., 40, 1955-.

The compound of formula (VII) may be prepared by hydrolysis of the compound of formula (IV). The reaction is preferably carried out by treating compound (IV) with an enzyme such as immobilized lipase (Sigma Aldrich) from Candida antarctica (Candidaantarctica) (Tetrahedron, 50, 13165-13172, 1994).

Route A3-Compounds of general formula (VIII) can be prepared starting from the reaction of a compound of formula (VII) with HO-NH-THP. The reaction may conveniently be carried out in dioxane or in a protic solvent such as EtOH at a temperature of 80 to 100 ℃. The THP protecting group is directly cleaved under the reaction conditions. The obtained (VIII) can be conveniently and selectively protected by treatment with dihydropyran in a suitable solvent such as DCM or THF at a temperature from 0 ℃ to room temperature to obtain the compound of formula (IX). The reaction is completed in a time of 0.5 to 3 hours. The compounds of formula (IX) may be further functionalized with alkyl halides, acid halides, isocyanates, carbamoyl chlorides or sulfonyl chlorides as described in scheme A1. The THP protecting group can be easily removed by treating the protected intermediate with HCl in a suitable solvent such as THF or dioxane. The reaction is generally carried out at room temperature for 1 to 15 hours to give the compound of the formula (VI).

Scheme a-the conversion of the hydroxyl group of the 2-hydroxyacetyl moiety at position 6b of the compound of general formula (VI) into a Leaving Group (LG) of the compound of general formula (XI) can be carried out: the compound of formula (VI) is treated with methanesulfonyl chloride or p-toluenesulfonyl chloride in a suitable solvent such as pyridine (March, "advanced organic chemistry", Wiley-Interscience). The reaction is usually carried out at room temperature for 1 to 5 hours.

General formula (VII)LG of the compound (XI) can be easily replaced by nucleophiles such as halide anions, alcohols, thiols, thioacids, amines, amides and carbanions (J.Org.chem., 1042, 1999; J.Steroid.biochem.13, 311-322, 1980) to obtain compounds of the general formulae (I) and (I'), wherein R1 ═ (CH)₂)_n-Z-(CH₂)_n-R4, wherein n ═ 0, Z and R4 are as defined hereinbefore. The reaction is usually carried out in a suitable solvent such as DCM, THF or Dimethylformamide (DMF) at 0 to 80 ℃ over 1-5 hours and may be promoted with a base such as sodium or potassium carbonate or sodium hydride. The resulting product may be further functionalized by modifying the moieties introduced by the nucleophilic substitution reaction described.

Route B-reaction of the compound of formula (VI) under well-known oxidation conditions gives intermediates of general formula (XII). The reaction is typically carried out in a suitable solvent such as THF in the presence of an aqueous solution of an inorganic base such as sodium hydroxide or potassium hydroxide in open air at room temperature for 12 to 48 hours.

Scheme B1-conversion of intermediates of formula (XII) to compounds of general formulae (I) and (I'), wherein R1 ═ CH (CH)₂)_n-Z-(CH₂)_n-R4, wherein n ═ 0, Z and R4 are as defined hereinbefore: acid (XII) is treated with one or more equivalents of an acid active agent such as carbonyldiimidazole. The reaction is usually carried out in a suitable polar solvent such as DMF at a temperature of from 0 to 80 ℃ for 1 to 2 hours. The activated acid can be reacted with nucleophiles such as alcohols, thiols, thioacids, and amines. The reaction may be promoted with a base such as sodium or potassium carbonate, sodium hydride and carried out at a temperature of 0 to 20 ℃ for 1 to 24 hours.

Alternatively, intermediates of formula (XII) may be converted to the corresponding acid chlorides using oxalyl chloride in a suitable solvent such as DCM under well known conditions. The activated intermediate can be reacted with nucleophiles such as alcohols, thiols, thioacids, amines and carbanions such as alkyl, aryl and heteroaryl copper or other metal organic compounds reported in the literature for the conversion of acid chlorides to the corresponding ketones.

Route B2-conversion of an intermediate of formula (XII)Is a compound of the general formula (XIII): the acid (XII) is reacted with carbonyldiimidazole and subsequently with sodium thioacetate and/or anhydrous hydrogen sulfide. The reaction is generally carried out as follows: the solution of the preformed salt in the reaction solvent is added to the solution of the activating acid at 0 to 20 ℃. The readily formed thioacid intermediate (XIII) is reacted in situ with an alkylating agent such as bromoalkane to give thioacid esters of general formula (I) and (I') wherein R1 ═ (CH)₂)_n-Z-(CH₂)_n-R4, wherein n-0, Z-S and R4 are as defined hereinbefore. The selection of suitable bromoalkanes, such as bromochloromethane, allows the preparation of compounds of formulae (I) and (I') wherein R1 ═ (CH)₂)_n-Z-(CH₂)_n-R4, wherein n-0, Z-S and R4 are as defined hereinbefore, which may be further modified. For example, the reaction of these compounds wherein R4 is chloromethyl with potassium iodide followed by treatment with silver fluoride makes it possible to prepare compounds of formulae (I) and (I') wherein R4 ═ fluoromethyl. These reactions are well known to those skilled in the art (j.med.chem., 37, 3717-.

Scheme C-known procedure for the cycloaddition of nitrones to isoxazolidines, intermediates of general formula (IV) are reacted with hydroxylamines of formula (X) in the presence of paraformaldehyde. The reaction is conveniently carried out in a protic solvent such as ethanol. The reaction is conveniently carried out at elevated temperature, for example 60 to 85 ℃, to give compounds of general formulae (I) and (I') wherein R1 ═ CH (CH)₂)_n-Z-(CH₂)_n-R4, wherein n ═ 1, Z ═ O and R4 ═ Ac.

Intermediates of general formula (XIV) can be prepared by: compounds of formula (I) and (I') wherein R1 ═ CH (CH) in a suitable solvent such as DMF, in the presence of a base such as pyridine₂)_n-Z-(CH₂)_n-R4, where n ═ 1, Z ═ O, R4 ═ Ac and X ═ H, treated with methanesulfonyl chloride. The reaction is carried out at a temperature of 80 to 100 ℃ for 1 to 5 hours.

The compounds of the general formulae (I) and (I') can be obtained by reacting compounds of the formula (XIV) under the well-known conditions for preparing chlorohydrins starting from the corresponding olefins, wherein R1 ═ (CH)₂)_n-Z-(CH₂)_n-R4, wherein n ═ 1, Z ═ O, R4 ═ H, and X ═ Cl. The reaction involves the use of a chlorinating agent such as N-chlorosuccinimide or dichloro-5, 5-dimethylhydantoin, and is promoted by an acid such as perchloric acid. The reaction is usually carried out in a polar solvent such as THF at 0 to 20 ℃ for 1 to 4 hours. The acetyl ester of the compound of formula (XIV) can be readily hydrolyzed using standard conditions for alcohol deacetylation: for example, the compound is treated with a base such as sodium or potassium carbonate in a solvent such as methanol or ethanol. The reaction is usually carried out at a low temperature of 0 to 20 ℃ for 0.5 to 2 hours.

Route D-intermediates of general formula (VI) are reacted with acid chlorides using procedures well known to those skilled in the art. The reaction is conveniently carried out in DCM as solvent in the presence of a base such as triethylamine at room temperature for 20 to 50 hours. This procedure makes it possible to prepare compounds of formula (I') in which R1 ═ CH (CH)₂)_n-Z-(CH₂)_n-R4, wherein n ═ 1, Z ═ O, and R4 is as defined above.

Route E-one-pot procedure for the synthesis of compounds of general formula (XV) followed by cycloaddition reaction provides compounds of general formula (I'). The first step of the procedure: the corresponding mesylate was formed at C21 starting from intermediate (VII) by well-known conditions with methanesulfonyl chloride and N, N-Diisopropylethylamine (DIPEA) in anhydrous acetonitrile. The introduction of fluorine atoms can then be conveniently carried out: tetra-n-butylammonium fluoride (TBAF) and KI are added in situ and heated for 8 to 20 hours. The obtained intermediate (XV) is subjected to a cycloaddition reaction with a hydroxylamine of the formula (X) in the presence of paraformaldehyde under known conditions as described in scheme C, leading to the formation of a compound of the general formula (I'), wherein R1 ═ CH₂-F and R2 are as defined above.

Advantageously, the compounds of the invention may be administered, for example, in doses comprised between 0.001 and 1000 mg/day, preferably between 0.1 and 500 mg/day.

In the case of administration thereof by the inhalation route, the dose of the compounds of general formulae (I) and (I') advantageously comprises from 0.01 to 20 mg/day, preferably from 0.1 to 10 mg/day.

Preferably, the compounds of the present invention may be administered for the prevention and/or treatment of any obstructive respiratory disease such as asthma, chronic bronchitis and Chronic Obstructive Pulmonary Disease (COPD), either alone or in combination with other active ingredients.

However, the compounds of the present invention may be administered for the prevention and/or treatment of any disease in which a reduction 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; acute exacerbation of adverse acute transplant rejection and selected autoimmune disorders, graft versus host disease in bone marrow transplantation; autoimmune disorders such as rheumatoid arthritis and other arthritis; skin disorders such as systemic lupus erythematosus, systemic dermatomyositis, psoriasis; inflammatory bowel disease, inflammatory eye disease, autoimmune hematological disorders, and acute exacerbation of multiple sclerosis; kidney, liver, heart and other organ transplants; behcet acute ocular syndrome, endogenous uveitis, atopic dermatitis, inflammatory bowel disease, and nephrotic syndrome; hodgkin's disease and non-hodgkin's lymphoma, multiple myeloma and Chronic Lymphocytic Leukemia (CLL); CLL-associated autoimmune hemolytic anemia and thrombocytopenia; leukemia and malignant lymphoma.

Preferably, the compounds of the present invention may be administered for the prevention and/or treatment of the progression of respiratory diseases such as asthma and COPD from mild to acute severe conditions.

The invention is further described by the following non-limiting examples.

Example 1

Preparation of acetic acid 2- ((10R, 13S, 17R) -17-acetoxy-10, 13-dimethyl-3, 11-dioxo 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-dodecahydro-3H-cyclopenta [ a ] phenanthren-17-yl) -2-oxo-ethyl ester (intermediate 2)

To a suspension of acetic acid 2- ((10R, 13S, 17R) -17-hydroxy-10, 13-dimethyl-3, 11-dioxo-6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-dodecahydro-3H-cyclopenta [ a ] phenanthren-17-yl) -2-oxo-ethyl ester (intermediate 1) (2g, 4.99mmol) and p-toluenesulfonic acid (PTSA) (200mg, 1.051mmol) in acetic acid (5ml) at 0 ℃ was slowly added trifluoroacetic anhydride (5ml, 35.4mmol) over 10 min. After stirring at 0 ℃ for 20 minutes, the reaction mixture was stirred at room temperature for 3 hours.

The reaction mixture was poured into ice/water (130ml) and the resulting mixture was extracted with DCM (2x100ml) and AcOEt (2x100 ml). The combined organic extracts were dried over anhydrous Na₂SO₄Drying and concentrating. The crude product was purified by flash chromatography on silica gel eluting with a gradient of DCM to DCMAcOEt 50: 50 to provide the title compound (2.64g, quantitative yield).

LC-MS(ESIPOS)：445.2(MH+)

Preparation of acetic acid (10R, 11S, 13S, 17R) -17- (2-acetoxy-acetyl) -11-hydroxy-10, 13-dimethyl-3-oxo-6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-dodecahydro-3H-cyclopenta [ a ] phenanthren-17-yl ester (intermediate 3)

To an ice-cold solution of (intermediate 2) (2.64g, 5.97mmol) in THF (15ml) and MeOH (15.00ml) was added sodium borohydride (221mg, 5.84mmol) portionwise over a period of 2.5 h. The reaction mixture was poured into 1n hcl and ice (150 ml). The precipitate formed was extracted with AcOEt (3 × 100ml), and the combined organic layers were washed with anhydrous Na₂SO₄Drying and concentrating. The crude material was purified by flash chromatography on silica gel eluting with a gradient of DCM to DCM/AcOEt 40: 60 to provide the title compound (1.21g, 45.6% yield).

¹HNMR (300MHz, chloroform-d) ppm7.28(d, 1H), 6.30(dd, 1H), 6.05(t, 1H), 4.92(d, 1H), 4.69(d, 1H), 4.48-4.58(m, 1H), 2.75-2.91(m, 1H),2.61(m，1H)，2.37(ddd，1H)，2.18-2.21(m，3H)，2.09-2.28(m，3H)，2.07(s，3H)，1.74-1.98(m，3H)，1.51-1.70(m，1H)，1.48(s，3H)，1.26-1.39(m，2H)，1.11-1.19(m，1H)，1.05(s，3H)

LC-MS(ESIPOS)：445.2(MH+)

example 2

Preparation of acetic acid 2- ((6S, 9R, 10S, 11S, 13S) -6, 9-difluoro-11-hydroxy-10, 13-dimethyl-3-oxo-6, 7, 8, 9, 10, 11, 12, 13, 14, 15-decahydro-3H-cyclopenta [ a ] phenanthren-17-yl) -2-oxo-ethyl ester (intermediate 5)

To butyric acid (9R, 10S, 11S, 13S, 17R) -17- (2-acetoxy-acetyl) -9-chloro-11-hydroxy-10, 13-dimethyl-3-oxo-6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-dodecahydro-3H-cyclopenta [ a ] under a nitrogen atmosphere]A solution of phenanthren-17-yl ester (intermediate 4) (2.48g, 4.88mmol) in anhydrous DMF (60ml) was added potassium acetate (3.83g, 39.0mmol) and the reaction mixture stirred at 100 ℃ for 1.5 h. The cooled reaction mixture was poured into ice and brine (200ml) and the aqueous layer extracted with AcOEt (3 × 150 ml). The combined organic extracts were washed with water and brine, washed over Na₂SO₄Drying above, concentration afforded 2.55g of the crude title compound, which was used in the next step without further purification.

¹HNMR(300MHz，DMSO-d₆)：ppm7.29(dd，1H)，6.99(dd，1H)，6.29(dd，1H)，5.98-6.15(m，1H)，5.68(dddd，1H)，5.56(dd，1H)，5.10(d，1H)，4.92(d，1H)，3.98-4.23(m，1H)，2.56-2.83(m，1H)，2.26-2.44(m，3H)，2.14-2.26(m，1H)，2.09(s，3H)，1.71-1.87(m，1H)，1.55-1.65(m，2H)，1.53(s，3H)，1.15(s，3H)。

LC-MS(ESIPOS)：421.97(MH+)

Preparation of (6S, 9R, 10S, 11S, 13S) -6, 9-difluoro-11-hydroxy-17- (2-hydroxy-acetyl) -10, 13-dimethyl-6, 7, 8, 9, 10, 11, 12, 13, 14, 15-decahydro-cyclopenta [ a ] phenanthren-3-one (intermediate 6)

To a solution of (intermediate 5) (2.55g, 6.06mmol) in ethanol (100ml) was added Candida antarctica (Candidaantarctica) lipase (2U/mg) (510mg, 6.06mmol) and the reaction mixture was stirred at 37 ℃ overnight. The reaction mixture was filtered, washed with methanol and the residue was purified by flash chromatography on silica gel eluting with a gradient of DCM/AcOEt 90: 10 to DCM/AcOEt 50: 50 to afford 1.62g of the title compound (70.6% yield).

¹HNMR(300MHz，DMSO-d₆)：ppm7.29(dd，1H)，6.87(dd，1H)，6.29(dd，1H)，6.09-6.17(m，1H)，5.67(dddd，1H)，5.53(dd，1H)，4.77(t，1H)，4.44(dd，1H)，4.26(dd，1H)，4.04-4.15(m，1H)，2.56-2.79(m，1H)，2.39(dd，1H)，2.25-2.35(m，2H)，2.09-2.25(m，1H)，1.76(td，1H)，1.55-1.66(m，2H)，1.53(s，3H)，1.17(s，3H)。

LC-MS(ESIPOS)：379.99(MH+)

Intermediates 7 and 8 listed in table 1 were prepared starting from intermediate 3 as described previously for intermediates 5 and 6.

TABLE 1

Example 3

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -8-furan-3-ylmethyl-5-hydroxy-6 b- (2-hydroxy-acetyl) -4a, 6 a-dimethyl-4 a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthren-2-one (Compound 9)

A mixture of (intermediate 8) (100mg, 0.292mmol), N- (furan-3-ylmethyl) hydroxylamine (33mg, 0.292mmol) and paraformaldehyde (50mg, 0.999mmol) in ethanol (5ml) was stirred at 105 ℃ overnight. The solvent was evaporated and the residue was purified by preparative HPLC to provide 58mg of the pure compound (42% yield).

¹HNMR (300MHz, chloroform-d): ppm7.38(t, 1H), 7.32-7.36(m, 1H), 7.25(d, 1H), 6.36(dd, 1H), 6.30(dd, 1H), 6.05(t, 1H), 4.45-4.63(m, 1H), 4.45(d, 1H), 4.10(d, 1H), 3.76(d, 1H), 3.70(d, 1H), 3.38-3.61(m, 2H), 2.90(br.s., 1H), 2.48-2.69(m, 1H), 2.36(ddd, 1H), 2.14-2.29(m, 1H), 2.03-2.14(m, 2H), 1.99(dd, 1H), 1.65-1.85(m, 2H), 1.49-1.67(m, 4H), 1.04-1H, 1.47 (m, 1H), 1.47-0.47 (m, 1H).

LC-MS(ESIPOS)：468.18(MH+)

The compounds listed in table 2 were prepared as described previously for compound 9 by cycloaddition of intermediate 6 or 8 with the appropriate hydroxylamine.

The final compound was purified by silica gel column chromatography or preparative HPLC.

TABLE 2

(continuation)

Example 4

Preparation of methanesulfonic acid 2- [ (4aS, 4bR, 5S, 6aS, 6bR, 9aS, 12S) -4b, 12-difluoro-5-hydroxy-4 a, 6 a-dimethyl-2-oxo-8- (3-phenylpropyl) 2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-azapenta-penta-no [2, 1-a ] phenanthren-6 b-yl ] -2-oxo-ethyl ester (Compound 74)

After addition of methanesulfonyl chloride (0.030ml, 0.385 mmod. stirring at room temperature for 5 hours) under nitrogen to a solution of compound 30(100mg, 0.185mmol) in pyridine (5ml), the reaction mixture was poured into 2n hcl and ice (75ml), the aqueous layer was extracted with AcOEt (3 × 50 ml.) the combined organic extracts were dried over anhydrous sodium sulfate and concentrated to afford 115mg of crude compound 48.

LC-MS(ESIPOS)：620.4(MH+)

The compounds listed in table 3 were prepared as described previously for compound 74:

TABLE 3

Example 5

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -6b- (2-ethylsulfanyl-acetyl) -8-furan-3-ylmethyl-5-hydroxy-4 a, 6 a-dimethyl-4 a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthren-2-one (Compound 81)

Under nitrogen atmosphere, to K₂CO₃(41mg, 0.297mmol) in dry DMF (1ml) was added ethanethiol (15ml, 0.196 mmol). After stirring for 10 minutes, compound 76(107mg, 0.196mmol) dissolved in anhydrous DMF (2ml) was added dropwise thereto. The reaction mixture was stirred at room temperature for 1.5 h, then poured into 2N HCl (20ml) and ice. The aqueous layer was extracted with AcOEt (3 × 30ml), and the combined organic extracts were dried over anhydrous sodium sulfate and concentrated. The crude was purified by column chromatography on silica gel eluting with a gradient of AcOEt/petroleum ether 10: 90 to AcOEt/petroleum ether 40: 60 to provide 68mg of the title compound (68% yield).

¹HNMR (300MHz, chloroform-d) ppm7.38(t, 1H), 7.33-7.36(m, 1H), 7.26(d, 1H), 6.37(dd, 1H), 6.30(dd, 1H), 6.05(t, 1H), 4.50(quin, 1H), 3.73(s, 2H), 3.44(d, 1H), 3.35-3.61(m, 2H), 3.40(d, 1H), 2.43-2.73(m, 3H), 2.36(ddd, 1H), 1.92-2.28(m, 4H), 1.60-1.91(m, 3H), 1.47(s, 3H), 1.41-1.54(m, 1H))，1.23(t，3H)，1.08-1.38(m，3H)，0.99(s，3H)。

LC-MS(ESIPOS)：512.09(MH+)

The compounds listed in table 4 were prepared as described previously for compound 81.

TABLE 4

(continuation)

Example 6

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -8-furan-3-ylmethyl-5-hydroxy-6 b- (2-mercapto-acetyl) -4a, 6 a-dimethyl-4 a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthren-2-one (Compound 88)

To a solution of compound 82(63mg, 0.120mmol) in degassed ethanol (7ml) and water (3ml) was added 1N NaOH (120. mu.l, 0.120mmol) at 0 ℃ under nitrogen. After stirring at 0 ℃ for 3.5 h, the reaction mixture was poured into 2N HCl (1ml) and brine (10 ml). The aqueous layer was extracted with AcOEt (3 × 20ml) and the combined organic extracts were dried (Na)₂SO₄)，And (5) concentrating. The crude was purified by column chromatography on silica gel eluting with a gradient of DCM to DCM/AcOEt 80: 20 to afford 20mg of the pure compound (34% yield).

¹HNMR(300MHz，DMSO-d6)ppm7.58(t，1H)，7.52-7.57(m，1H)，7.31(d，1H)，6.41(dd，1H)，6.17(dd，1H)，5.93(t，1H)，4.70(d，1H)，4.14-4.44(m，1H)，3.76(d，1H)，3.66(d，1H)，3.62(dd，1H)，3.34(dd，1H)，3.29-3.53(m，2H)，2.52-2.62(m，1H)，2.34-2.44(m，1H)，2.20-2.35(m，1H)，1.87-2.22(m，3H)，1.73(d，2H)，1.50-1.71(m，2H)，1.38(s，3H)，1.30-1.51(m，1H)，0.99-1.13(m，1H)，0.95(dd，1H)，0.81(s，3H)。

LC-MS(ESIPOS)：484.09(MH+)

Example 7

Preparation of (4aS, 4bR, 5S, 6aS, 6bR, 9aS, 10aS, 10bS, 12S) -6b- (2-chloro-acetyl) -4b, 12-difluoro-5-hydroxy-4 a, 6 a-dimethyl-8- (3-phenyl-propyl) -4a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthren-2-one (Compound 89)

To a solution of lithium chloride (97mg, 2.285mmol) in anhydrous DMF (1ml) under a nitrogen atmosphere was added compound 74(118mg, 0.190mmol) dissolved in anhydrous DMF (2 ml). The reaction mixture was stirred at 65 ℃ for 3.5 hours. The reaction mixture was poured into 2n hcl and ice (20ml) and the aqueous layer was extracted with AcOEt (3 × 50 ml). The combined organic extracts were washed with water (2 × 40ml), brine, dried over anhydrous sodium sulfate, and concentrated. The crude was purified by column chromatography on silica gel eluting with a gradient of DCM to DCM/AcOEt 85: 15 to provide 78mg of the title compound (73.1% yield).

¹HNMR(300MHz，DMSO-d₆)ppm7.03-7.34(m，6H)，6.29(dd，J＝10.12，1.91Hz，1H)，6.11(s，1H)，5.49-5.78(m，1H)，5.45(d，J＝2.93Hz，1H)，4.69(d，J＝17.31Hz，1H)，4.54(d，J＝17.31Hz，1H)，3.98-4.24(m，1H)，3.45-3.62(m，1H)，3.33-3.45(m，1H)，2.70(t，J＝7.19Hz，2H)，2.59(t，J＝7.63Hz，2H)，2.05-2.35(m，2H)，1.97(br.s.，2H)，1.54-1.85(m，6H)，1.49(s，3H)，1.39-1.46(m，1H)，0.84(s，3H)。

LC-MS(ESIPOS)560.0(MH+)

The compounds listed in table 5 were prepared as described previously for compound 89.

TABLE 5

(continuation)

Example 8

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -8-furan-3-ylmethyl-5-hydroxy-4 a, 6 a-dimethyl-6 b- [2- (2-oxo-tetrahydro-furan-3-sulfanyl) -acetyl ] -4a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthren-2-one (Compound 93)

At 0 deg.CA solution of 3-mercapto-dihydro-furan-2-one (37mg, 0.312mmol) in dry THF (1ml) was added dropwise to a stirred suspension of NaH (13mg, 0.325mmol, 60% suspension in mineral oil) in dry THF (1.5 ml). The resulting solution was stirred at 0 ℃ for 30 minutes. A solution of compound 76(170mg, 0.312mmol) in dry THF (2ml) was slowly added dropwise, and the mixture was stirred at 0 ℃ for 2 hours and at room temperature for 3 hours. The reaction mixture was poured into water (20ml) and ice, the aqueous layer was extracted with AcOEt (3 × 20ml), the combined organic extracts were washed with brine and dried (Na)₂SO₄) And (4) concentrating. The crude was purified by column chromatography on silica gel eluting with a gradient of DCM to DCM/AcOEt 25: 75 to afford 97mg of the pure compound (55% yield).

¹HNMR (300MHz, chloroform-d) ppm7.32-7.43(m, 2H), 7.18-7.32(m, 2H), 6.33-6.43(m, 1H), 6.26-6.34(m, 1H), 5.82-6.13(m, 1H), 4.46-4.58(m, 1H), 4.24-4.69(m, 2H), 3.31-4.21(m, 7H), 2.48-2.87(m, 2H), 2.30-2.49(m, 1H), 1.86-2.29(m, 5H), 1.60-1.86(m, 2H), 1.47(s, 3H), 1.38-1.63(m, 1H), 1.25-1.37(m, 1H), 1.06-1.20(m, 2H), 0.98(s, 3H).

LC-MS(ESIPOS)：568.11(MH+)

The compounds listed in table 6 were prepared as described previously for compound 93:

TABLE 6

(continuation)

Example 9

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -5-hydroxy-4 a, 6 a-dimethyl-6 b- [2- (tetrahydro-pyran-2-yloxy) -acetyl ] -4a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthren-2-one (intermediate 97)

To a solution of (4aR, 5S, 6aS, 6bR, 9aS) -5-hydroxy-6 b- (2-hydroxy-acetyl) -4a, 6 a-dimethyl 4a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-and-cyclopenta [2, 1-a ] phenanthren-2-one (intermediate 21) (250mg, 0.64mmol) in DCM (10ml) at 0 deg.C under nitrogen was added DHP (161. mu.l, 1.9mmol) and the reaction mixture was stirred at room temperature for 1 h. The solvent was evaporated and the residue was purified by flash chromatography on silica gel, eluting with DCM/AcOEt 80: 20, to provide 260mg of the title compound (86% yield).

LC-MS(ESIPOS)：444.2(MH+)

Example 10

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -8-benzoyl-5-hydroxy-4 a, 6 a-dimethyl-6 b- [2- (tetrahydro-pyran-2-yloxy) -acetyl ] -4a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-pentaleno [2, 1-a ] phenanthren-2-one (intermediate 98)

To a solution of intermediate 97(130mg, 0.27mmol) in anhydrous THF (2.7ml) was added triethylamine (75. mu.l, 0.54mmol) under nitrogen at 0 ℃ and the mixture was stirred at 0 ℃ for 15 min. Benzoyl chloride (63. mu.l, 0.54mmol) was added and the mixture was stirred for a further 15 minutes. The mixture was diluted with AcOEt (20ml), the organic layer washed with water and brine, dried (Na)₂SO₄) And (4) concentrating. The crude was purified by flash chromatography on silica gel eluting with DCM/AcOEt 80: 20 to provide 120mg of the title compound as a colorless oil (77% yield).

LC-MS(ESIPOS)：576.15(MH+)

The intermediates listed in table 7 were prepared as described previously for intermediate 98 by reacting intermediate 97 with the appropriate acid chloride.

TABLE 7

Example 11

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -8-benzoyl-5-hydroxy-6 b- (2-hydroxy-acetyl) -4a, 6 a-dimethyl-4 a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-eno [2, 1-a ] phenanthren-2-one (Compound 100)

To a solution of intermediate 98(120mg, 0.20mmol) in THF (6ml) was added 1N HCl (3ml), and the mixture was stirred at room temperature for 14 hours. The mixture was diluted with AcOEt (30ml), the organic layer washed with water and brine, dried (Na)₂SO₄) And (4) concentrating. The crude was purified by preparative HPLC to provide 41.3mg of the title compound (42% yield).

¹HNMR (300MHz, chloroform-d) ppm7.57-7.71(m, 2H), 7.47-7.56(m, 1H), 7.37-7.46(m, 2H), 7.30(d, 1H), 6.39(dd, 1H), 6.15(t, 1H), 4.44-4.56(m, 1H), 4.42(d, 1H), 4.21(dd, 1H), 4.16(d, 1H), 3.73(td, 1H), 3.53(dd, 1H), 2.53-2.72(m, 1H), 2.42(ddd, 1H), 2.17-2.30(m, 1H), 2.06-2.17(m, 1H), 1.96-2.06 (m, 1H), (1.96-2.06: (m, 1H)m，1H)，1.85(ddd，1H)，1.67-1.79(m，2H)，1.62(dd，1H)，1.47(s，3H)，1.09-1.33(m，1H)，1.04(dd，1H)，1.01(s，3H)。

LC-MS(ESIPOS)：492.10(MH+)

Compound 101 was prepared as described previously for compound 100:

TABLE 8

Example 12

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -8- (furan-2-carbonyl) -5-hydroxy-6 b- (2-hydroxy-acetyl) -4a, 6 a-dimethyl-4 a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthren-2-one (Compound 102)

To a solution of intermediate 97 and triethylamine (117ml, 0.84mmol) in dry THF under nitrogen at 0 deg.C was added 2-furoyl chloride (76. mu.l, 0.77mmol) and the reaction mixture was stirred for 5 minutes. The solvent was removed under reduced pressure and the residue dissolved in THF (2ml), acetonitrile (2ml) and water (10 ml). 1N HCl was added to the mixture, which was stirred at room temperature for 30 minutes. DCM (15ml) was added, the phases separated and the aqueous layer extracted with DCM (2 × 15 ml). The combined organic layers were dried (Na)₂SO₄) And (4) concentrating. The crude was purified by preparative HPLC to provide 40mg of the title compound (11% yield).

¹HNMR1HNMR(300MHz，DMSO-d6)ppm7.90(dd，1H)，7.31(d，1H)，7.20(dd，1H)，6.67(dd，1H)，6.16(dd，1H)，5.91(s，1H)，4.80(br.s.，1H)，4.45(d，1H)，4.27-4.38(m，1H)，4.20(dd，1H)，4.19(d，1H)，3.53-3.67(m，1H)，3.46(dd，1H)，2.53-2.61(m，1H)，2.22-2.39(m，2H)，1.94-2.17(m，2H)，1.82-1.92(m，2H)，1.50-1.82(m，3H)，1.39(s，3H)，0.99-1.15(m，1H)，0.92-1.00(m，1H)，0.92(s，3H)。

LC-MS(ESIPOS)：482.00(MH+)

The compounds listed in table 9 were prepared as described previously for compound 102:

TABLE 9

(continuation)

Example 13

Preparation of acetic acid 2- ((4aR, 5S, 6aS, 6bR, 9aS) -5-hydroxy-4 a, 6 a-dimethyl-2-oxo-2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthren-6 b-yl) -2-oxo-ethyl ester (Compound 106)

The title compound was obtained in 55% yield starting from intermediate 7 as described previously for compound 9 (example 3).

LC-MS(ESIPOS)：430.3(MH+)

Preparation of acetic acid 2- ((4aR, 5S, 6aS, 6bR, 9aS) -5-hydroxy-8-methanesulfonyl-4 a, 6 a-dimethyl-2-oxo-2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthren-6 b-yl) -2-oxo-ethyl ester (Compound 107)

Compound 106(120mg, 0.28mmol) was dissolved in anhydrous DCM (5ml) under a nitrogen atmosphere. Triethylamine (75. mu.l, 0.54mmol) and methanesulfonyl chloride (75. mu.l, 0.54mmol) were added, and the reaction mixture was stirred at room temperature for 3 days. The mixture was diluted with DCM (20ml), the organic phase was washed with water and dried (Na)₂SO₄) And (4) concentrating. The crude was purified by flash chromatography on silica gel eluting with a gradient of DCM/AcOEt 90: 10 to DCM/AcOEt 50: 50 to provide 74mg of the pure compound (52% yield).

LC-MS(ESIPOS)：508.3(MH+)

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -5-hydroxy-6 b- (2-hydroxy-acetyl) -8-methanesulfonyl-4 a, 6 a-dimethyl-4 a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthren-2-one (Compound 108)

To a solution of acetic acid 2- ((4aR, 5S, 6aS, 6bR, 9aS) -5-hydroxy-8-methanesulfonyl-4 a, 6 a-dimethyl-2-oxo-2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-and-cyclopenta [2, 1-a ] phenanthren-6 b-yl) -2-oxo-ethyl ester (compound 107) (74mg, 0.16mmol) in degassed methanol (5ml) and water (5ml) at 0 ℃ under nitrogen atmosphere was added 1NNaOH (180 μ l) and the reaction mixture was stirred at 0 ℃ for 10 min. The reaction mixture was acidified to pH2, concentrated and the residue was purified by preparative LC-MS to provide 11.5mg of the title compound (17% yield).

¹HNMR (300MHz, chloroform-d): ppm7.26(d, 1H), 6.33(dd, 1H), 6.08(t, 1H), 4.72(d, 1H), 4.54(q, 1H), 4.23(d, 1H), 4.03(dd, 1H), 3.78(td, 1H), 3.06(s, 3H), 3.00(dd, 1H), 2.51-2.71(m, 1H), 2.39(ddd, 1H), 2.17-2.31(m, 1H)，2.01-2.17(m，3H)，1.77-1.95(m，1H)，1.58-1.76(m，2H)，1.47(s，3H)，1.09-1.32(m，2H)，1.01(s，3H)

LC_MS(ESIPOS)：466.09(MH+)

Example 14

Preparation of acetic acid 2- [ (4aR, 5S, 6aS, 6bR, 9aS) -5-hydroxy-4 a, 6 a-dimethyl-8- (methyl-phenyl-carbamoyl) -2-oxo-2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthren-6 b-yl ] -2-oxo-ethyl ester (Compound 109)

Compound 106(200mg, 0.50mmol) was dissolved in anhydrous DCM (7ml) under a nitrogen atmosphere. Triethylamine (71. mu.l, 0.51mmol) and N-methyl-N-phenylcarbamoyl chloride (87mg, 0.51mmol) were added, and the reaction mixture was stirred at 0 ℃ for 3 hours and at room temperature for 3 days. The mixture was diluted with water (15ml) and 1N HCl was added. The aqueous layer was extracted with DCM (3 × 20 ml). The combined organic layers were dried (Na)₂SO₄) And (4) concentrating. The crude was purified by preparative HPLC to provide 66mg of the pure compound (23% yield).

LC-MS(ESIPOS)：563.7(MH+)

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -5-hydroxy-6 b- (2-hydroxy-acetyl) -4a, 6 a-dimethyl-2-oxo-2, 4a, 4b, 5, 6, 6a, 6b, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthrene-8-carboxylic acid methyl-phenyl-amide (Compound 110)

To a solution of compound 109(66mg, 0.12mmol) in degassed methanol (1.5ml) and water (1.5ml) under nitrogen at 0 ℃ was added 1N NaOH (117. mu.l) and the reaction mixture was stirred at 0 ℃ for 10 min. The reaction mixture was acidified to pH2, concentrated and the residue was purified by preparative LC-MS to provide 21mg of the title compound (33% yield).

¹HNMR(300MHz，DMSO-d₆)ppm7.25-7.38(m，3H)，7.17-7.25(m，2H)，7.06-7.17(m，1H)，6.20(dd，1H)，5.94(s，1H)，4.64(br.s.，1H)，4.36(d，1H)，4.04-4.25(m，1H)，3.89(d，1H)，3.65(dd，1H)，3.40-3.46(m，1H)，3.14(s，3H)，2.98(dd，1H)，2.53-2.65(m，1H)，2.20-2.35(m，1H)，1.84-2.13(m，2H)，1.47-1.69(m，2H)，1.37-1.47(m，1H)，1.35(s，3H)，1.13-1.37(m，3H)，0.92-1.12(m，1H)，0.84(dd，1H)，0.78(s，3H)。

LC_MS(ESIPOS)：521.24(MH+)

Example 15

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -5-hydroxy-4 a, 6 a-dimethyl-2-oxo-8-thiophen-3-ylmethyl-2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthrene-6 b-carboxylic acid (Compound 111)

To a solution of 11(202mg, 0.418mmol) in THF (3ml) and water (1ml) was added 1N NaOH (836ul, 0.836mmol) and the reaction mixture was stirred at room temperature in an open vessel for 24 h. The reaction mixture was poured into 2n hcl and ice (30ml) and the aqueous layer was extracted with AcOEt (3 × 30 ml). The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, and concentrated to give 202mg of crude material (quantitative yield), which was used as such without additional purification.

1HNMR(300MHz，DMSO-d6)ppm7.42(dd，1H)，7.31-7.35(m，1H)，7.31(d，1H)，7.04(dd，1H)，6.16(dd，1H)，5.92(s，1H)，4.20-4.35(m，1H)，3.89(br.s.，2H)，3.31-3.58(m，2H)，2.53-2.60(m，1H)，2.22-2.37(m，1H)，1.93-2.20(m，3H)，1.81(d，1H)，1.49-1.71(m，3H)，1.35-1.48(m，1H)，1.39(s，3H)，0.97-1.15(m，1H)，0.96(s，3H)，0.91(dd，1H)。

LC-MS(ESIPOS)：470.13(MH+)

The compounds listed in table 10 were prepared as described previously for compound 111:

watch 10

Example 16

Preparation of (4aR, 5S, 6aS, 6bR, 9aS) -5-hydroxy-4 a, 6 a-dimethyl-2-oxo-8-thiophen-3-ylmethyl-2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthrene-6 b-carboxylic acid methyl ester (Compound 116)

Compound 111(110mg, 0.23mmol) was suspended in anhydrous THF (25ml) under a nitrogen atmosphere, CDI (65mg, 0.39mmol) was added, and the mixture was stirred at 60 ℃ for 3 hours. Methanol (5ml) was added and the reaction mixture was stirred at room temperature overnight. The solvent was evaporated and the crude was purified by preparative HPLC to afford 41mg of the pure compound (37% yield).

¹HNMR(300MHz，DMSO-d6)ppm7.42(dd，1H)，7.31(d，1H)，7.27-7.30(m，1H)，7.01(dd，1H)，6.16(dd，1H)，5.92(s，1H)，4.18-4.36(m，1H)，3.88(s，2H)，3.66(s，3H)，3.38-3.51(m，2H)，2.53-2.61(m，1H)，2.21-2.37(m，1H)，1.92-2.18(m，3H)，1.69-1.84(m，1H)，1.46-1.68(m，3H)，1.39(s，3H)，1.32-1.46(m，1H)，0.97-1.14(m，1H)，0.90(s，3H)，0.86-0.96(m，1H)。

LC-MS(ESIPOS)：484.28(MH⁺)

Example 17

Preparation of (4aS, 4bR, 5S, 6aS, 6bR, 9aS, 10aS, 10bS, 12S) -4b, 12-difluoro-5-hydroxy-4 a, 6 a-dimethyl-2-oxo-8- (3-phenyl-propyl) -2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthrene-6 b-thiocarboxylic acid S-butyl ester (Compound 117)

To a solution of compound 114(100mg, 0.190mmol) in anhydrous DMF (2ml) under nitrogen was added Carbonyldiimidazole (CDI) (61.5mg, 0.379mmol D, the reaction mixture was stirred at room temperature for 1.5 hours, stirred at 60 ℃ for 30 minutes, then, a solution of butylmercaptan (0.041ml, 0.379mmol) and sodium hydride (16.68mg, 0.417mmol) in anhydrous DMF (3ml) was slowly added dropwise, the reaction mixture was stirred at room temperature for 1.5 hours, the reaction mixture was poured into water and ice (50ml), the aqueous layer was extracted with AcOEt (3X50ml), the combined organic extracts were washed with water, brine, dried over anhydrous sodium sulfate, concentrated₂Column purification with a gradient of DCM to DCM/AcOEt 88: 12 afforded 66mg of the title compound (58.1% yield).

¹HNMR(300MHz，DMSO-d₆)ppm7.08-7.33(m，6H)，6.29(dd，1H)，6.11(s，1H)，5.50-5.78(m，1H)，5.47(d，1H)，4.06-4.30(m，1H)，3.44-3.64(m，1H)，3.32-3.44(m，1H)，2.79(td，2H)，2.54-2.74(m，6H)，2.18-2.32(m，1H)，1.94-2.18(m，3H)，1.70-1.94(m，4H)，1.53-1.68(m，1H)，1.49(s，3H)，1.40-1.48(m，2H)，1.25-1.39(m，2H)，0.87(s，3H)，0.86(t，3H)。

LC-MS(ESIPOS)：600.4(MH+)

[α]_D ²⁵＝+150.2°(c＝0.50，CHCl₃)

Example 18

Preparation of (4aS, 4bR, 5S, 6aS, 6bR, 9aS, 10aS, 10bS, 12S) -8- (4-chloro-benzyl) -4b, 12-difluoro-5-hydroxy-4 a, 6 a-dimethyl-2-oxo-2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthrene-6 b-carboxylic acid methylamide (Compound 118)

To a solution of compound 113(200mg, 0.375mmol) in anhydrous DMF (5ml) under nitrogen was added CDI (66.8mg, 0.412mmol) and the mixture was stirred at 50 ℃ for 1 h. The reaction was cooled to room temperature and 2M methylamine (0.749ml, 1.498mmol) in THF was added followed by DMAP (45.8mg, 0.375 mmol). The reaction was stirred at 50 ℃ for 4 hours: as some carboxylic acid was still present, a further 2M solution of methylamine (1.5ml, 3.00mmol) was added and the reaction heated at 50 ℃ overnight. The reaction mixture was cooled, poured into 100ml water and extracted with AcOEt (3 × 70 ml). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated. The crude (240mg) was dissolved in MeOH and residual carboxylic acid was removed on a PS-HCO 3-cartridge to yield 219mg of crude. Purification via chromatography on silica gel (DCM/AcOEt1/1) afforded 150mg of the title compound (73% yield).

¹HNMR(300MHz，DMSO-d₆)ppm7.27-7.46(m，4H)，7.24(dd，1H)，7.10(q，1H)，6.29(dd，1H)，6.11(s，1H)，5.50-5.79(m，1H)，5.38(dd，1H)，4.05-4.17(m，1H)，3.91(d，1H)，3.83(d，1H)，3.46-3.62(m，1H)，3.34-3.46(m，1H)，2.61(d，3H)，2.54-2.59(m，1H)，2.18-2.33(m，1H)，1.97-2.17(m，1H)，1.71-1.86(m，1H)，1.51-1.65(m，3H)，1.49(s，3H)，1.46-1.52(m，1H)，1.39(dd，1H)，0.88(s，3H)

LC-MS(ESIPOS)：547.19MH+[α]_D ²⁵＝+283.3，(c0.21，CHCl₃)

The compounds listed in table 11 were prepared as described previously for compound 118 using the appropriate alcohol, thiol or amine (or the corresponding hydrochloride salt of an amine):

TABLE 11

(continuation)

Example 19

Preparation of (4aS, 4bR, 5S, 6aS, 6bR, 9aS, 10aS, 10bS, 12S) -4b, 12-difluoro-5-hydroxy-4 a, 6 a-dimethyl-2-oxo-8- (3-phenyl-propyl) -2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthrene-6 b-carboxylic acid chloromethyl ester (Compound 135)

To a solution of compound 114(120mg, 0.227mmol) in N, N-dimethylacetamide (4ml) under a nitrogen atmosphere were added potassium carbonate (62.9mg, 0.455mmol) and bromochloromethane (0.075ml, 1.137mmol), and the reaction mixture was stirred at room temperature for 25 hours. The reaction mixture was diluted with AcOEt (40ml) and the organic layer was diluted with 5% NaHCO₃Washed with water and brine, dried (Na)₂SO₄) And (4) concentrating. The crude was purified by flash chromatography on silica gel eluting with a gradient of DCM to DCM/AcOEt 85: 15 to provide 31.5mg of the title compound (24% yield).

¹HNMR(300MHz，DMSO-d₆)ppm7.08-7.34(m，6H)，6.29(dd，1H)，6.11(s，1H)，5.95(d，1H)，5.92(d，1H)，5.53-5.80(m，1H)，5.53(dd，1H)，4.10-4.24(m，1H)，3.43-3.72(m，2H)，2.54-2.70(m，4H)，2.18-2.34(m，2H)，2.04-2.19(m，1H)，1.80-1.95(m，1H)，1.60-1.80(m，5H)，1.50-1.60(m，1H)，1.49(s，3H)，1.38-1.47(m，1H)，0.94(s，3H)。

LC-MS(ESIPOS)：576.07(MH+)

The compounds listed in table 12 were prepared as described previously for compound 135:

TABLE 12

(continuation)

Example 20

Preparation of (4aS, 4bR, 5S, 6aS, 6bR, 9aS, 10aS, 10bS, 12S) -4b, 12-difluoro-5-hydroxy-4 a, 6 a-dimethyl-2-oxo-8- (3-phenyl-propyl) -2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthrene-6 b-thiocarboxylic acid S-cyanomethyl ester (139)

A mixture of compound 114(220mg, 0.417mmol) and CDI (135mg, 0.834mmol) in dry DMF (4ml) was stirred at 65 ℃ under nitrogen for 1h30 min. The mixture was cooled at room temperature then at 0 ℃; a solution of freshly prepared thioacetic acid (0.096ml, 1.334mmol) and sodium hydride (50.0mg, 1.251mmol) in anhydrous DMF (2ml) was added to the mixture and the resulting solution was stirred at 0 ℃ for 10 min. 2-bromoacetonitrile (0.119ml, 1.710mmol) was then added and the resulting mixture was stirred at room temperature for 3 hours. The mixture was stirred in AcOEt (40ml) withWater (30ml) was partitioned and the organic phase was separated; the aqueous layer was extracted with AcOEt (2 × 40ml) and the combined organic layers were washed with brine over Na₂SO₄Drying and concentrating. The crude was purified by flash chromatography on silica eluting with a gradient of DCM to DCM/AcOEt 90: 10 to provide 100mg of the title compound (41.2% yield).

¹HNMR(300MHz，DMSO-d₆)ppm7.05-7.34(m，6H)，6.29(dd，1H)，6.11(s，1H)，5.55(dd，1H)，5.49-5.82(m，1H)，4.12-4.24(m，1H)，3.96(d，1H)，3.89(d，1H)，3.46-3.67(m，1H)，3.33-3.45(m，1H)，2.54-2.84(m，5H)，1.99-2.33(m，3H)，1.49(s，3H)，1.42-1.93(m，7H)，0.92(s，3H)。

LC-MS(ESIPOS)：583.98(MH+)

[α]_D ²⁵＝+118.2°(c＝0.49，CHCl3)

The compounds listed in table 13 were prepared as described previously for compound 139 using the appropriate bromoethane derivatives:

watch 13

(continuation)

Example 21

Preparation of (4aS, 4bR, 5S, 6aS, 6bR, 9aS, 12S) -4b, 12-difluoro-5-hydroxy-4 a, 6 a-dimethyl-2-oxo-8- (3-phenyl-propyl) -2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthrene-6 b-thiocarboxylic acid S-iodomethyl ester (Compound 157)

To an acetone (4ml) solution of compound 143 was added sodium iodide (122mg, 0.811mmol), and the reaction mixture was stirred at 65 ℃ for 9 hours. The reaction mixture was diluted with AcOEt (50ml), and the aqueous layer was washed with water, 10% sodium thiosulfate, 5% sodium bicarbonate and brine, dried over anhydrous sodium sulfate, and concentrated. The crude was purified by flash chromatography on silica gel eluting with a gradient of AcOEt/petroleum ether 5: 95 to AcOEt/petroleum ether 30: 70 to provide 16mg of the title compound (34.6% yield).

LC-MS(ESIPOS)：684.2(MH+)

Preparation of (4aS, 4bR, 5S, 6aS, 6bR, 9aS, 10aS, 10bS, 12S) -4b, 12-difluoro-5-hydroxy-4 a, 6 a-dimethyl-2-oxo-8- (3-phenyl-propyl) -2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthrene-6 b-thiocarboxylic acid S-fluoromethyl ester (Compound 158)

To a solution of compound 157(16mg, 0.023mmol) in acetonitrile (7ml) was added silver fluoride (41.6mg, 0.328mmol) and the reaction mixture was stirred at room temperature overnight, protected from light. The reaction mixture was filtered through a cotton pad, concentrated to give 28mg of crude material, which was purified through an RP18 cartridge in acetonitrile to give 13mg of a mixture containing the desired product. The mixture is passed through SiO₂The cartridge was further purified, eluting with a gradient of AcOEt/DCM 15: 85 to AcOEt/DCM 30: 70, to provide 2.1mg of the title compound (16% yield).

¹HNMR (300MHz, chloroform-d) ppm7.16-7.33(m, 5H), 7.12(dd, 1H), 6.46(s, 1H), 6.40(dd, 1H), 6.01(dd, 1H), 5.64(dd, 1H), 5.41(dddd, 1H), 4.25-4.50(m, 1H), 3.52-3.74(m, 2H), 2.76-2.94(m, 2H), 2.64-2.76(m, 2H), 2.41-2.64(m, 1H), 2.23-2.40(m, 2H), 1.65-2.19(m, 6H), 1.48(br.s., 1H), 1.28(s, 3H), 1.22-1.36(m, 2H), 1.03(s, 3H).

LC-MS(ESIPOS)：575.90(MH+)

The compounds listed in table 14 were prepared starting from the appropriate acid derivatives as described previously for compound 158:

TABLE 14

Example 22

Intermediate 7 was reacted with 3-phenyl-propyl-hydroxylamine aS described previously for compound 9 (example 3) to give 2- [ (4aR, 5S, 6aS, 6bR, 9aS) -5-hydroxy-4 a, 6 a-dimethyl-2-oxo-8- (3-phenyl-propyl) -2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-and-cyclopenta [2, 1-a ] phenanthren-6 b-yl ] -2-oxo-ethyl acetate (compound 160) in 79% yield.

Preparation of acetic acid 2- [ (4aS, 6aS, 6bR, 9aS) -4a, 6 a-dimethyl-2-oxo-8- (3-phenyl-propyl) -2, 4a, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-dodecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthren-6 b-yl ] -2-oxo-ethyl ester (Compound 161)

To a solution of compound 160(403mg, 0.736mmol) in DMF (8ml) under nitrogen was added pyridine (0.268ml, 3.31mmol) and methanesulfonyl chloride (0.172ml, 2.207 mmol). The reaction mixture was stirred at 90 ℃ for 3.5 hours and at room temperature overnight.

The reaction mixture was poured into ice and brine (40ml) and the aqueous layer was extracted with AcOEt (3 × 40 ml). The combined organic extracts were washed with water and brine and dried (Na)₂SO₄) And (4) concentrating. The crude was purified by flash chromatography on silica gel eluting with a gradient of DCM to DCM/AcOEt 85: 15,to provide 211mg of pure compound (54.1% yield).

LC-MS(ESIPOS)：530.4(MH+)

Preparation of acetic acid 2- [ (4aS, 4bR, 5S, 6aS, 6bR, 9aS) -4 b-chloro-5-hydroxy-4 a, 6 a-dimethyl-2-oxo-8- (3-phenyl-propyl) -2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthren-6 b-yl ] -2-oxo-ethyl ester (Compound 162)

To a solution of compound 161(211mg, 0.398mmol) in THF (12ml) was added a solution of perchloric acid (0.073ml, 1.214mmol) in water (164. mu.l) followed by 1, 3-dichloro-5, 5-dimethylhydantoin (57mg, 0.289mmol) at 0 ℃ under a nitrogen atmosphere. After 5 minutes, the cooling bath was removed and the mixture was stirred at room temperature for 3 hours. 1, 3-dichloro-5, 5-dimethylhydantoin (57.0mg, 0.289mmol) and perchloric acid (0.073ml, 1.215mmol) were then added and the reaction mixture was stirred at room temperature for a further 1 hour. The reaction mixture was poured to 0.64% NaHSO₃(100ml) solution, followed by addition of solid NaCl. The aqueous layer was extracted with AcOEt (3 × 75ml), and the combined organic extracts were washed with brine and dried (anhydrous Na)₂SO₄) And (4) concentrating. The crude was purified by flash chromatography on silica gel eluting with a gradient of DCM to DCM/AcOEt 70: 30 to provide 112mg of the title compound (48.3% yield).

LC-MS(ESIPOS)：582.3(MH+)

Preparation of (4aS, 4bR, 5S, 6aS, 6bR, 9aS) -4 b-chloro-5-hydroxy-6 b- (2-hydroxy-acetyl) -4a, 6 a-dimethyl-8- (3-phenyl-propyl) -4a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthren-2-one (Compound 163)

To a solution of compound 162(112mg, 0.192mmol) in MeOH (8ml) and THF (5.00ml) (degassed solvent) at 0 deg.C under a nitrogen atmosphere was added K₂CO₃(39.9mg, 0.289mmol), the reaction mixture was stirred at 0 ℃ for 1.5 h. The reaction mixture was poured into 2n hcl and ice (50ml) and the aqueous layer was extracted with AcOEt (3 × 60 ml). Warp beamThe combined organic extracts were washed with water and brine, dried over anhydrous sodium sulfate, and concentrated. The crude material was then purified by flash chromatography on silica gel eluting with a gradient of AcOEt/DCM 5: 95 to AcOEt/DCM 40: 60 to provide 73mg of the title compound (70.3% yield).

¹HNMR (300MHz, chloroform-d) ppm7.29-7.34(m, 1H), 7.11-7.25(m, 5H), 6.37(dd, 1H), 6.11(t, 1H), 4.87(d, 1H), 4.60(br.s., 1H), 4.22(d, 1H), 3.75-3.88(m, 1H), 3.59-3.75(m, 1H), 2.88-3.16(m, 2H), 2.68-2.79(m, 2H), 2.54-2.70(m, 3H), 2.36-2.53(m, 2H), 2.19-2.33(m, 1H), 1.99-2.17(m, 2H), 1.71-1.93(m, 3H), 1.67(s, 3H), 1.60 (dd), 1.40H), 1.00 (m, 1H), 1.54-3H)

LC-MS(ESIPOS)：540.01(MH+)

Compound 164 was prepared by reacting intermediate 7 with N-p-chlorobenzylhydroxylamine according to the previously described preparation of compound 163.

Watch 15

Example 23

Preparation of 2- ((4aS, 4bR, 5S, 6aS, 6bR, 9aS, 10aS, 10bS, 12S) -4b, 12-difluoro-5-hydroxy-4 a, 6 a-dimethyl-2-oxo-8-thiophen-2-ylmethyl-2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthren-6 b-yl) -2-oxo-ethyl propionate (Compound 165)

Triethylamine (0) was added to a solution of 37 deg.C in anhydrous dichloromethane (15ml) under a nitrogen atmosphere at 0 deg.C.161ml, 1.155mmol) and propionyl chloride (0.100ml, 1.155 mmol). The reaction mixture was stirred at 0 ℃ for 15 minutes, at room temperature for 24 hours, then additional triethylamine (0.080ml, 0.577mmol) and propionyl chloride (0.050ml, 0.577mmol) were added. The mixture was stirred at room temperature for a further 2 hours. The mixture was diluted with DCM (60ml), the organic phase was washed with 1N HCl, water and brine, dried (Na)₂SO₄) And (4) concentrating. The residue was purified by flash column on silica eluting with a gradient of DCM to DCM/AcOEt 85: 15 affording 248mg of 104 (75% yield).

¹HNMR(300MHz，DMSO-d₆)ppm7.44(dd，1H)，7.27(dd，1H)，7.00(dd，1H)，6.96(dd，1H)，6.31(dd，1H)，6.12(s，1H)，5.58(d，1H)，5.37-5.81(m，1H)，4.76(d，1H)，4.64(d，1H)，4.20-4.26(m，1H)，4.21(d，1H)，4.01(d，1H)，3.45-3.63(m，1H)，3.32-3.38(m，1H)，2.59-2.71(m，1H)，2.38(q，2H)，2.08-2.31(m，3H)，1.94-2.05(m，1H)，1.73(d，1H)，1.52-1.68(m，2H)，1.49(s，3H)，1.43(dd，1H)，1.05(t，3H)，0.85(s，3H)

LC-MS(ESIPOS)：576.21MH+

[α]_D ²⁵＝+162.0(c＝0.151，MeOH)

The compounds listed in table 16 were prepared starting from the appropriate isoxazolidine derivatives as described previously for compound 165:

TABLE 16

Example 24

Preparation of acetic acid 2- ((4aS, 4bR, 5S, 6aS, 6bR, 9aS, 10aS, 10bS, 12S) -4b, 12-difluoro-5-hydroxy-4 a, 6 a-dimethyl-2-oxo-2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-penta-cyclopenta [2, 1-a ] phenanthren-6 b-yl) -2-oxo-ethyl ester (Compound 168)

The title compound was obtained in 33% yield starting from intermediate 7 as described previously for compound 9 (example 3).

LC-MS(ESIPOS)：466.0(MH+)

Preparation of acetic acid 2- ((4aS, 4bR, 5S, 6aS, 6bR, 9aS, 10aS, 10bS, 12S) -4b, 12-difluoro-5-hydroxy-4 a, 6 a-dimethyl-2-oxo-2, 4a, 4b, 5, 6, 6a, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-N- (1-methyl-2-oxo-1, 2-dihydro-pyridin-3-ylmethyl) -aza-penta-cyclopenta [2, 1-a ] phenanthren-6 b-yl) -2-oxo-ethyl ester (Compound 169)

To a solution of compound 168(200mg, 0.430mmol), potassium iodide (164mg, 0.988mmol) and ethyldiisopropylamine (0.169ml, 0.988mmol) in anhydrous DMF (3ml) was added methyl (1-methyl-2-oxo-1, 2-dihydropyridin-3-yl) methanesulfonate (215mg, 0.988mmol) dissolved in anhydrous DMF (5 ml). The reaction mixture was stirred for 3 hours under microwave heating at 100 ℃. The reaction mixture was diluted with AcOEt (50ml), the organic phase washed with water and brine and dried (Na)₂SO₄) And (4) concentrating. The residue was purified by flash chromatography on silica gel eluting with a gradient of DCM/MeOH/AcOEt 49: 1: 50 to DCM/MeOH/AcOEt 47: 3: 50 to provide 115mg of the title compound (46% yield).

LC-MS(ESIPOS)：587.1(MH+)

Preparation of (4aS, 4bR, 5S, 6aS, 6bR, 9aS, 10aS, 10bS, 12S) -4b, 12-difluoro-5-hydroxy-6 b- (2-hydroxy-acetyl) -4a, 6 a-dimethyl-4 a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-N- (1-methyl-2-oxo-1, 2-dihydro-pyridin-3-ylmethyl) -aza-pentaleno [2, 1-a ] phenanthren-2-one (Compound 170)

To a solution of compound 169(115mg, 0.196mmol) in dioxane (12ml) and water (6.00ml) was added 12NHCl (1.5ml) and the reaction mixture was stirred at 60 ℃ for 3 hours. The reaction mixture was cooled to 0 ℃ and the pH was adjusted to 9. Brine was added and the aqueous phase was extracted with AcOEt (2 × 50 ml). The combined organic extracts were dried (Na)₂SO₄) And (4) concentrating. The crude was purified by flash column on silica gel, eluting with a gradient of AcOEt to AcOEt/MeOH 97: 3 to provide 73mg of the title compound (68% yield).

¹HNMR(300MHz，DMSO-d₆)ppm7.62(dd，1H)，7.37(dd，1H)，7.26(d，1H)，6.30(dd，1H)，6.17(t，1H)，6.12(s，1H)，5.49-5.83(m，1H)，5.43(dd，1H)，4.77(t，1H)，4.32(dd，1H)，4.10-4.22(m，1H)，4.01(dd，1H)，3.71(br.s.，2H)，3.44-3.56(m，2H)，3.42(s，3H)，2.58-2.71(m，1H)，2.07-2.35(m，3H)，1.83-1.99(m，1H)，1.52-1.68(m，3H)，1.49(s，3H)，1.38-1.47(m，1H)，0.81(s，3H)

LC-MS(ESIPOS)：545.25MH+[α]_D ²⁵＝+190.6(c＝0.136，MeOH)

The compounds listed in Table 16 were prepared as described previously for compound 170 with the appropriate alkylating agent

TABLE 17

Example 25

Preparation of (6S, 8S, 9R, 10S, 11S, 13S, 14S) -6, 9-difluoro-17- (2-fluoro-acetyl) -11-hydroxy-10, 13-dimethyl-6, 7, 8, 9, 10, 11, 12, 13, 14, 15-decahydro-cyclopenta [ a ] phenanthren-3-one (Compound 173)

To a solution of compound 6(0.5g, 1.321mmol) in dry acetonitrile (20ml) under nitrogen was added DIPEA (0.396ml, 2.246mmol) and Ms-Cl (0.155ml, 1.982mmol) and the reaction mixture was stirred at room temperature for 1 hour. Then, a solution of TBAF (2.64ml, 2.64mmol) in 1M THF and potassium fluoride (0.077g, 1.321mmol) were added and the mixture was heated at reflux overnight. The mixture was diluted with AcOEt, the organic phase washed with water, brine and dried (Na)₂SO₄) And (4) concentrating. The crude was purified by flash chromatography on silica gel eluting with a gradient of petroleum ether/AcOEt 8: 2 to AcOEt to provide the title compound (98% yield).

LC-MS(ESIPOS)：381.3MH+

Preparation of (4aS, 4bR, 5S, 6aS, 6bR, 9aS, 10aS, 10bS, 12S) -8- (4-chloro-benzyl) -4b, 12-difluoro-6 b- (2-fluoro-acetyl) -5-hydroxy-4 a, 6 a-dimethyl-4 a, 4b, 5, 6, 6a, 6b, 8, 9, 9a, 10, 10a, 10b, 11, 12-tetradecahydro-7-oxa-8-aza-cyclopenta [2, 1-a ] phenanthren-2-one (Compound 174)

Intermediate 111 was reacted with N- (4-chloro-benzyl) -hydroxylamine as described previously for compound 9 (example 3) to afford compound 173 in 21% yield.

¹HNMR(300MHz，DMSO-d6)ppm7.36-7.44(m，2H)，7.29-7.36(m，2H)，7.27(dd，1H)，6.30(dd，1H)，6.13(s，1H)，5.49-5.79(m，1H)，5.46(dd，1H)，4.92(dd，1H)，4.67-4.94(m，1H)，4.09-4.27(m，1H)，3.95(d，1H)，3.78(d，1H)，3.45-3.60(m，1H)，3.34-3.44(m，1H)，2.59-2.69(m，1H)，1.99-2.39(m，3H)，1.72-1.96(m，1H)，1.51-1.70(m，3H)，1.49(s，3H)，1.38-1.48(m，1H)，0.84(s，3H)

LC-MS(ESIPOS)：550.18MH+

[α]_D ²⁵＝+221.2(c＝0.2，MeOH)

Illustration of the drawings

＊NMR

s ═ singlet

d is doublet

t is triplet

q is quartet

ddd-doublet

m is multiplet

br is wide

ESI-POS (electrospray ionization-positive ionization)

LC-MS (liquid chromatography-mass spectrometry) combined use

Pharmacological Activity of the Compounds of the invention

In vivo studies

Example 26

Lipopolysaccharide (LPS) -induced pulmonary neutrophilia

The potency and duration of action of the compounds described in the present invention were evaluated in vivo in the acute model of pneumonia according to a simple modified procedure described in am.j.respir.crit.careed.voll 162.pp1455-1461, 2000.

The tests were carried out on Sprague-Dawley male rats (200 g).

Intratracheal instillation of LPS caused a statistically significant increase in neutrophil concentration in BALF, a hallmark of acute ongoing pneumonia.

For the test of evaluation of glucocorticoid dose producing 75% inhibition (ED75 dose), compound (0.01-3 micromoles/Kg body weight) was administered intratracheally as a suspension (0.2% tween 80 in nacl0.9%) 1 hour prior to LPS challenge.

Dose-response curves were drawn for the inhibitory effect of test compounds on LPS-induced pulmonary neutrophilia, taking the ED75 dose of glucocorticoid as a measure of potency in this bioassay. The dose value of ED75 for the compound of the invention is 0.62 to 1.62 micromoles per Kg body weight.

In a second series of experiments aimed at evaluating duration of action, the compound was administered intratracheally as a suspension at different time points (16h, 24h) prior to LPS challenge at the ED75 dose. At 16 hours, some compounds showed a percent inhibition of greater than 50%. The most interesting compounds were active given 24 hours before LPS challenge (percentage inhibition higher than 50%).

In vitro study

Example 27

Glucocorticoid Receptor (GR) translocation testing protocol

Quantitative measurements of GR nuclear translocation of the compounds of the invention were performed by a novel cell-based GR-translocation test in the Enzyme Fragment Complementation (EFC) method developed by discoverrx (Fremont, CA) according to assayregu devel.

In the absence of glucocorticoids, the Glucocorticoid Receptor (GR) is localized in the cytoplasm and complexes with a variety of proteins, including heat shock proteins.

In the case of glucocorticoid diffusion through the cell membrane into the cytoplasm and binding to the Glucocorticoid Receptor (GR), it causes the release and translocation of heat shock proteins into the nucleus where it regulates gene transcription.

DiscovexX test EFC of b-galactosidase (b-gal) was used as an indicator of GR-translocation in engineered CHO-K1 biosensor cells. As designed, by using a proprietary set of sequence additions and modifications, the enzyme receptor (EA) fragment of b-gal is localized in the nucleus. The small peptidase donor (ED) fragment of b-gal is directly fused to the C-terminus of GR and localized in the cytoplasm in the absence of receptor signaling. After binding to the GR ligand, the complex translocates to the nucleus where the intact enzyme activity is restored by complementation and b-gal activity is detected.

Humidified at 37 ℃ with 5% CO₂And 95% air, CHO-K1 cells stably expressing b-gal NLS-enzyme acceptor fragment (EA) and b-gal GR-Enzyme Donor (ED) fragment were maintained in F12 medium (Invitrogen, Carlsbad, Calif.). The above medium contained 10% FBS, 2 mML-glutamine, 50U/ml penicillin 50. mu.g/ml streptomycin, and 250. mu.g/ml hygromycin and 500. mu.g/ml G418 (Invitrogen).

GR-translocation was measured using the PathHunter assay kit containing a cell membrane permeabilizing agent and a β -gal substrate (DiscovexX, Fremont, CA). All compounds used 10^-11To 10^-6Varying concentrations of M were screened. Test at 48-well (10)⁵Individual cells/well). Incubate with the screening compound for 2 hours at 37 ℃. Detection was performed by adding detection buffer from the kit provided by discoverrx and incubating for one hour at room temperature. Luminescence was detected by using a CENTROLB960 microplate reader (Bertholdtechnologies).

Statistical analysis and determination of EC50s was performed using Prism-version 3.0GraphpadSoftware (SanDiego, Calif.).

Compounds tested with the GR translocation assay showed EC50 between 1nM and 10 nM.

Example 28

Inhibition of nitric oxide production in LPS-induced RAW264.7 macrophages

In an in vitro model based on macrophage murine, the cell line RAW264.7 was used to test the anti-inflammatory effect of the corticosteroids of the invention.

During inflammatory processes, large amounts of Nitric Oxide (NO) are produced by inducible NO synthase Isoforms (iNOS). Bacterial Lipopolysaccharides (LPS) are commonly used in experimental settings to stimulate inflammatory responses in macrophages.

Cells were grown in phenolsulfonphthalein-free medium (RPMI supplemented with heat-inactivated 10% fetal bovine serum, 2mM glutamine, 100U/ml penicillin and 0.1mg/ml streptomycin). Cell stimulation was induced by incubating the cells with LPS for 24 hours to a final concentration of 100 ng/ml. The treatment with the compounds of the invention is carried out in such a way that: 15 minutes before LPS exposure, the compound loaded in DMSO (0.1% final concentration) was added to the final desired concentration. Nitrous acid concentration was measured by Griess colorimetric reaction in conditioned media as an indicator of nitric oxide production (j.neurosimulunol, 150, 29-36, 2004).

Statistical analysis and determination of IC50s was performed using Prism version 3.0GraphpadSoftware (SanDiego, Calif.). The IC50 values of the compounds of the invention were measured to be between 0.06 and 5.3 nM.

Example 29

[³H]Dexamethasone binding assay

The glucocorticoid receptor affinity of the compounds described in this patent application is evaluated using radioligand binding assays as described in IRLPress, Oxford university Press, 247-.

The human lymphoblast cell line IM-9(ACC 117; DSMZ) has been used as a source of a soluble glucocorticoid receptor³H]Dexamethasone (GEHealthcare; SA 34. mu. Ci/nmol) was used as a source of radioligand (invest. Ophtalmol. Vis. Sci.37, 805-813, 1996). Nonspecific binding was assessed in the presence of excess unlabeled beclomethasone (10 μ M).

In the near [2 ]³H]Dexamethasone K_dBinding experiments were repeated with radioligand concentrations at values (1.5-2 nM). The samples were incubated at 0 ℃ for 16-18 hours in the dark. The reaction was stopped by adding a mixture of 10mM pH7.4Tris, 2% activated carbon in 1mM EDTA and 0.5% dextran. After incubation at 0 ℃ for 10 minutes, the samples were centrifuged at 4500rpm for 10 minutes. Aliquots of the sample supernatants were placed in vials containing 3ml scintillation cocktail filtercount (PerkinElmer) and counted using a PerkinElmer2500TRbeta counter.

The compounds of the invention were tested to have IC50 values of 0.5 to 2 nM.

Example 30

And (3) kinetic characterization: lung retention

Lung retention is measured by two parameters: MRT_L(mean residence time in lung), i.e. the residence of compound in lung, which is the time of the last measurable concentration of compound in rat lung measured after lung homogenization after intratracheal administration of 1 μmol/kg; and C₄₈L/C_0.5L (%), the percentage of the amount of compound in the lung 48 hours after intratracheal administration compared to the amount of the same compound in the lung 0.5 hours after administration.

MRT_LAnd C₄₈L/C_0.5L (%) is two meaningful and predictive parameters of the duration of drug effect after a single pulmonary administration.

The compounds of the invention show very slow pulmonary elimination, of which MRT_LGreater than 20 hours C₄₈L/C_0.5L is higher than 20%.

Claims

1. A compound of the general formula (I)

Wherein

R1 is (CH)₂)_n-Z-(CH₂)_n’-R4, wherein n and n' are each independently 0, 1 or 2;

z is a single bond or O;

r4 is selected from:

-H；

-(C₁-C₆) An alkylcarbonyl group;

r2 is selected from

-(CH₂)_mR5, wherein R5 is thienyl optionally substituted with halogen;

-(CH₂)_pr8 wherein R8 is phenyl optionally substituted with one or more substituents selected from halogen and CN;

wherein m and p are each independently 0 or an integer from 1 to 6; and

x and Y are fluorine;

and pharmaceutically acceptable salts thereof.

2. The compound according to claim 1, which is represented by the general formula (I

Wherein 4a is (R), 4b is (R), 5 is (S), 6a is (S), 6b is (R), 9a is (S), 10b is (S) and 12 is (S).

3. The compound according to claim 1 or 2, wherein R1 is (CH)₂)_n-Z-(CH₂)_n’-R4, wherein n is 0 or 1.

4. A compound according to claim 3, wherein R4 is methylcarbonyl.

5. A compound according to claim 1 or 2, wherein R2 is selected from (CH)₂)_mR5, wherein m is 1 or 2; and (CH)₂)_pR8 wherein p is 1, 2 or 3.

6. A compound according to claim 1 or 2, wherein R2 is selected from phenyl and thienyl.

7. Preparation of the catalyst as claimed in the preceding claimA method of treating a compound as defined in any one of the claims, wherein R1 ═ (CH)₂)_n-Z-(CH₂)_n’-R4, wherein n ═ 1, the method comprising:

a compound of formula (VI)

Wherein the Leaving Group (LG) can be replaced by a nucleophile.

8. A process for the preparation of a compound as defined in any one of the preceding claims, wherein R1 ═ (CH)₂)_n-Z-(CH₂)_n’-R4, wherein n is 0, the method comprising:

9. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 6, together with one or more pharmaceutically acceptable carriers and/or excipients.

10. A combination of a compound as defined in any one of claims 1 to 6 and one or more active ingredients selected from the following classes: beta 2-agonists, antimuscarinics, corticosteroids, mitogen-activated protein kinase inhibitors, nuclear factor kappa-B kinase subunit beta inhibitors, human neutrophil elastase inhibitors, phosphodiesterase 4 inhibitors, leukotriene modulators, non-steroidal anti-inflammatory agents, and mucus regulators.

11. Use of a compound as defined in any one of claims 1 to 6 for the preparation of a medicament for the prevention and/or treatment of any disease in which a reduction in inflammatory cell number, activity and motility is implicated.