HK1098470A

HK1098470A - Piperidinylcarbonyl-pyrrolidines and their use as melanocortin agonists

Info

Publication number: HK1098470A
Application number: HK07104781.8A
Authority: HK
Inventors: A．A．卡拉布雷斯; D．S．弗拉代; D.赫普沃思; M．兰斯代尔
Original assignee: 辉瑞大药厂
Priority date: 2004-02-04
Filing date: 2005-01-26
Publication date: 2007-07-20

Description

Piperidinylcarbonyl-pyrrolidines and their use as melanocortin agonists

no marking

The present invention relates to a novel class of melanocortin MCR4 agonist compounds, in particular to selective MCR4 agonist compounds, their use in medicine, compositions containing them, processes for their preparation, and intermediates used in such processes. Specifically, the present invention relates to a class of MCR4 agonist compounds useful for the treatment of sexual dysfunction and/or obesity.

The compounds of the invention are useful in the treatment of male and female sexual dysfunction, including female hypoactive sexual desire disorder, sexual arousal disorder, orgasm disorder and/or dyspareunia, male erectile dysfunction, as well as obesity (by reducing appetite, increasing metabolic rate, reducing fat uptake or reducing carbohydrate craving) and diabetes (by increasing glucose tolerance and/or reducing insulin resistance). The compounds of the invention are useful in the treatment of other diseases, disorders, or conditions, including but not limited to, hypertension, hyperlipidemia, osteoarthritis, cancer, gallbladder disease, sleep apnea, depression, anxiety, obsessive-compulsive, neurological disorders, insomnia/sleep disorders, substance abuse, pain, fever, inflammation, immune modulation, rheumatoid arthritis, tanning of the skin, acne and other skin disorders, neuroprotective and cognition and memory-enhancing effects, including the treatment of alzheimer's disease.

The compounds of the invention are particularly suitable for the treatment of female sexual dysfunction, male erectile dysfunction, obesity and diabetes.

Desirable properties of the MCR4 agonist compounds of the invention include: desirable MCR4 efficacy as detailed below; selectivity to MCR4 relative to MCR1 and/or MCR5 and/or MCR3 as detailed below; desirable MCR4 efficacy and selectivity for MCR4 over MCR1 and/or MCR5 and/or MCR 3; good biopharmaceutical properties, such as physical stability; solubility; appropriate metabolic stability.

General formula (VII)

The present invention provides compounds of formula (I)

Or a pharmaceutically acceptable salt, hydrate, solvate, isomer or prodrug thereof,

wherein R is¹Selected from: - (C)₁-C₆) Alkyl, - (C)₂-C₆) Alkenyl, - (C)₂-C₆) Alkynyl, - (C)₃-C₈) Cycloalkyl, - (C)₅-C₈) Cycloalkenyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, aryl, - (C)₁-C₂) Alkylaryl, heterocyclyl or- (C)₁-C₂) An alkyl heterocyclic group, a heterocyclic group having a heterocyclic group,

wherein each of the above R¹The groups are optionally substituted with one or more groups selected from: - (C)₁-C₄) Alkyl, - (CH)₂)_m(C₃-C₅) Cycloalkyl, halogen, - (CH)₂)_mOR⁶、-CN、-C(O)OR⁶、-(CH₂)_mNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃Wherein m is 0, 1 or 2;

R²is H, OH or OCH₃；

R³Selected from: H. - (C)₁-C₆) Alkyl, - (C)₂-C₆) Alkenyl, - (C)₂-C₆) Alkynyl, - (C)₃-C₈) Cycloalkyl, - (C)₅-C₈) Cycloalkenyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, aryl, - (C)₁-C₂) Alkylaryl, heterocyclyl or- (C)₁-C₂) An alkyl heterocyclic group, a heterocyclic group having a heterocyclic group,

wherein each of the latter ten R³The groups are optionally substituted with one or more groups selected from: -OH, - (C)₁-C₄) Alkyl, - (CH)₂)_n(C₃-C₅) Cycloalkyl, halogen, -CN, - (CH)₂)_nOR⁶Or- (CH)₂)_nNR⁷R⁸Wherein n is 0, 1 or 2;

R⁴selected from: H. - (C)₁-C₄) Alkyl, - (C)₂-C₄) Alkenyl, - (C)₂-C₄) Alkynyl, - (CH)₂)_p(C₃-C₅) Cycloalkyl, - (CH)₂)_p(C₅) Cycloalkenyl, halogen, - (CH)₂)_pOR⁶、-(CH₂)_pNR⁷R⁸、-CN、-C(O)R⁶、-C(O)OR⁶、-C(O)NR⁷R⁸、-(CH₂)_pNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃A group wherein p ═ 0, 1 or 2;

R⁵selected from: - (C)₁-C₄) Alkyl, - (C) ₂-C₄) Alkenyl, - (C)₂-C₄) Alkynyl, - (CH)₂)_p(C₃-C₅) Cycloalkyl, - (CH)₂)_p(C₅) Cycloalkenyl, halogen, - (CH)₂)_pOR⁶、-(CH₂)_pNR⁷R⁸、CN、-C(O)R⁶、-C(O)OR⁶、-C(O)NR⁷R⁸、-(CH₂)_pNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃A group wherein p ═ 0, 1 or 2;

or R⁴And R⁵May together form a fused 5-to 7-membered saturated or unsaturated ring;

R⁶、R⁷and R⁸Each independently selected from H, CH₃Or CH₂CH₃；

And wherein R¹And R³The heterocyclyl group of (a) is independently selected from a 4-to 10-membered ring system containing up to 4 heteroatoms independently selected from O, N or S.

Suitable heterocyclyl groups for use herein are 4-to 10-membered monocyclic or bicyclic heteroaryl rings containing one to three heteroatoms derived from N, S and O, and combinations thereof, wherein the bicyclic heteroaryl ring is a 9-or 10-membered ring system, which may be two heteroaryl rings fused together or a heteroaryl ring fused to an aryl ring.

Bicyclic heteroaryls suitable for use herein include: benzimidazolyl, benzotriazolyl, benzothiazolyl, indazolyl, indolyl, imidazopyridinyl, imidazopyrimidinyl, pyrrolopyridinyl, quinolinyl, isoquinolinyl, quinazolinyl, naphthyridinyl, and pyridopyrimidinyl.

Preferred for use herein are monocyclic 5-to 6-membered heteroaryl rings containing one or three heteroatoms from the group consisting of N and O, and combinations thereof.

Suitable 5-membered monocyclic heteroaryls for use herein include: triazinyl, oxadiazinyl, oxazolyl, thiazolyl, thiadiazolyl, furanyl, thienyl, pyrrolyl and imidazolyl.

Suitable 6-membered monocyclic heteroaryls for use herein include: pyridyl, pyrimidinyl, pyridazinyl and pyrazinyl.

Preferred R¹Heterocycles are monocyclic 5-to 6-membered heteroaryl rings containing one or two heteroatoms from N and O, and combinations thereof. More preferred R¹Heterocycles are monocyclic 5-to 6-membered heteroaryl rings containing one or two N heteroatoms. Very preferred R here¹Heterocyclic is a monocyclic 6-membered heteroaryl ring containing one or two N heteroatoms, e.g.Pyridyl and pyrimidyl.

R is particularly preferred here¹Heteroaryl is pyridyl.

Preferred R³Heterocycles are monocyclic 5-to 6-membered heteroaryl rings containing one or two heteroatoms from N and O, and combinations thereof, such as tetrahydropyranyl, pyridinyl, pyridazinyl, pyrazinyl, and pyrimidinyl. More preferred R³Heterocycles are monocyclic 5-to 6-membered heteroaryl rings containing one or two N heteroatoms. Even more preferred R³Heterocycles are monocyclic 6-membered heteroaryl rings containing one or two N heteroatoms, such as pyridyl, pyridazinyl, pyrazinyl and pyrimidinyl.

R is particularly preferably used herein³6-membered monocyclic heteroaryl is pyridin-2-yl, pyridin-3-yl, pyridazin-3-yl, pyrazinyl, pyrimidin-5-yl and pyrimidin-2-yl. R is particularly preferably used herein ³6-membered ring monocyclic heteroaryl includes pyridin-2-yl, pyridin-3-yl and pyridazin-3-yl. Of these groups, pyridazin-3-yl is most preferred.

Suitably represented by R⁴And R⁵The fused ring systems together may be carbocyclic or heterocyclic ring systems containing up to two heteroatoms selected from O, N or S. Including the phenyl ring to which they are attached, R⁴And R⁵Preferred ring systems which may be constituted are: 1, 2-indane, 1, 2, 3, 4-tetrahydronaphthalene, indolyl, indazolyl, naphthyl, quinolyl, benzothiazolyl, benzimidazolyl, benzo [1, 3 ] yl]Dioxolanes, 2, 3-dihydrobenzo [1, 4 ]]Dioxins, 2, 3-dihydrobenzofuran, 2, 3-dihydrobenzothiophene and 1, 3-dihydroisobenzofuran.

In the above definitions, unless otherwise indicated, alkyl, alkenyl and alkynyl groups having three or more carbon atoms, alkanoyl groups having four or more carbon atoms may be straight or branched. E.g. C₄The alkyl substituents may be in the form of n-butyl (n-butyl), isobutyl (i-butyl), sec-butyl (sec-butyl) or tert-butyl (t-butyl). For the avoidance of doubt, if R¹And/or R³Is optionalSubstituted alkyl, which alkyl may not be further substituted by further (unsubstituted) alkyl groups. Furthermore, if R ³The carbon atom (of the unsaturated group) directly bonded to the N atom, substituted by an alkenyl or alkynyl group, may not be itself unsaturated.

The term halogen includes Cl, Br, F and I.

The term "aryl" as used herein includes six-to ten-membered carbocyclic aromatic groups such as phenyl and naphthyl.

The present invention further provides compounds of the general formulae (IA) to (IF) and mixtures thereof as detailed below. For the avoidance of doubt, all references hereinafter to compounds of formula (I), such as their salts, polymorphs, prodrugs or optical, geometric and tautomeric forms are intended to encompass compounds of formulae (IA) to (IF) unless otherwise specifically stated.

Pharmaceutically acceptable salts of the compounds of formula (I) include their acid addition and base salts.

Pharmaceutically acceptable salts of the compounds of formula (I) can be readily prepared by mixing together a solution of the compound of formula (I) with the desired acid or base, as the case may be. The salt may precipitate out of solution, be collected by filtration or recovered by evaporation of the solvent.

Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate/carbonate, bisulfate/sulfate, borate, camphorsulfonate, citrate, cyclamate, edisylate, ethanesulfonate, formate, fumarate, glucoheptanoate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/biphosphate/dihydrogenphosphate, pyroglutamate, saccharate, stearate, dihydrogencarbonate, dihydrogenphosphate, dihydrogensulfate, dihydrogensulfonate, salicylate, and camphorate, Succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate.

Suitable base salts are formed from bases which form non-toxic salts. Examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, alkanolamine, potassium, sodium, tromethamine and zinc salts.

Hemisalts of acids and bases, such as hemisulfate and hemicalcium salts, may also be formed.

For a review of suitable Salts, see Handbook of Pharmaceutical Salts: properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable salts of the compounds of formula (I) may be prepared by one or more of the following three methods:

(i) reacting a compound of formula (I) with a desired acid or base;

(ii) removing acid or base labile protecting groups from suitable precursors of compounds of formula (I) or opening suitable cyclic precursors (e.g. lactones or lactams) using a desired acid or base; or

(iii) One salt of the compound of formula (I) is converted to another salt by reaction with an appropriate acid or base or by means of a suitable ion exchange column.

All three reactions are usually carried out in solution. The resulting salt may precipitate out and may be collected by filtration or recovered by evaporation of the solvent. The degree of ionization of the resulting salt may vary from completely ionized to almost unionized.

The compounds of the present invention may exist as solid continuous bodies ranging from completely amorphous to completely crystalline. The term "amorphous" denotes a state in which the substance lacks a long range order on the molecular level and can exhibit physical properties of a solid or liquid depending on temperature. In general, such materials do not produce a characteristic X-ray diffraction pattern and are more formally described as liquids while exhibiting solid properties. Upon heating, a change in properties occurs from solid to liquid, which is characterized by a change in state, usually secondary ("glass transition"). The term "crystalline" denotes a solid phase in which the substance has a regular ordered internal structure at the molecular level, giving rise to a characteristic X-ray diffraction pattern with defined peaks. Such materials will also behave as liquids when heated sufficiently, but the change from solid to liquid is characterized by a phase change, usually first order ("melting point").

The compounds of the present invention may also exist in unsolvated and solvated forms. The term "solvate" is used herein to describe a molecular complex comprising a compound of the invention and one or more pharmaceutically acceptable solvent molecules, such as ethanol. When the solvent is water, the term "hydrate" is used.

The currently accepted classification system for organic hydrates defines isolated sites, channels or metal ion coordinated hydrates-seePolymorphism in Pharmaceutical Solidsby k.r.morris (ed.h.g.brittain, Marcel Dekker, 1995). An isolated site hydrate is one in which water molecules are isolated from direct contact with each other by intermediate organic molecules. In channel hydrates, water molecules are located in lattice channels where they are adjacent to other water molecules. In the metal ion complex hydrate, water molecules are bonded to the metal ions.

When the solvent or water is intimately bound, the complex will have a well-defined stoichiometry, independent of humidity. However, when the solvent or water is weakly bound, as with channel solvates and hygroscopic compounds, the water/solvent content will depend on the humidity and drying conditions. In such cases, the non-stoichiometry would be the benchmark.

Multicomponent complexes (other than salts and solvates) in which the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts are also included within the scope of the present invention. Complexes of this type include clathrates (drugs)Host inclusion complex) and co-crystals. The latter is generally defined as a crystalline complex of neutral molecular components that are held together by non-covalent interactions, but may also be a complex of a neutral molecule and a salt. The co-crystals may be prepared by melt crystallization, recrystallization from a solvent or physical milling of the components together-see Chem Commun, 171889-. For a general review of multicomponent complexes, see J Pharm Sci,64(8)，1269-1288，by Haleblian(August 1975)。

the compounds of the invention may also be present in a mesomorphic state (mesophase or liquid crystal) when subjected to suitable conditions. A mesomorphic state is an intermediate state between a true crystalline state and a true liquid state (molten or dissolved). The mesogenicity that occurs as a result of a change in temperature is described as "thermotropic" and the mesogenicity caused by the addition of a second component, such as water or another solvent, is described as "lyophilic". Compounds having the potential to form lyophilic mesophases are described as "amphiphilic" and consist of a compound possessing an ionic (e.g., -COO)^-Na⁺、-COO^-K⁺or-SO₃ ^-Na⁺) Or of the non-ionic type (e.g. -N)^-N⁺(CH₃)₃) Molecular composition of the polar head group. For more information seeCrystals and the Polarizing Microscope by N.H.Hartshorne and A.Stuart，4th Edition(Edward Arnold，1970)。

All references hereinafter to compounds of formula (I) include their salts, solvates, multicomponent complexes and liquid crystals, and solvates of the salts, multicomponent complexes and liquid crystals.

The compounds of the invention include compounds of formula (I) as defined above, their polymorphs and crystal forms as defined above, their prodrugs and isomers (including optical, geometric and tautomeric isomers) and isotopically labeled compounds of formula (I).

As indicated above, so-called "prodrugs" of the compounds of formula (I) are also disclosed hereinWithin the scope of the invention. Thus, certain derivatives of the compounds of formula (I) which may themselves have little or no pharmacological activity, when administered into or onto the body, can be converted into compounds of formula (I) having the desired activity, for example by hydrolytic cleavage. Such derivatives are referred to as "prodrugs". For further information on the use of prodrugs can be found inPro-drugs as Novel Delivery SystemsVol.14, ACS Symposium Series (T Higuchi and W Stella) andBioreversible Carriers in Drug Design，Pergamon Press，1987(Ed.E B Roche，American Pharmaceutical Association)。

prodrugs according to the invention may be generated, for example, by replacing appropriate functional groups present in a compound of formula (I) with moieties known to those skilled in the art as "precursor moieties", for example inDesign of Prodrugsby H Bundgaard (Elsevier, 1985).

Some examples of prodrugs according to the invention include

(i) If the compound of the formula I contains a carboxylic acid function (-COOH) or an ester thereof, for example where the hydrogen of the carboxylic acid function of the compound of the formula (I) is- (C)₁-C₈) Alkyl substituted compounds;

(ii) if the compounds of the formula I contain an alcohol function (-OH) or an ether thereof, for example, where the hydrogen of the alcohol function of the compounds of the formula (I) is- (C)₁-C₆) Alkanoyloxymethyl substituted compounds, e.g. when R is ²OH or R³Preferred prodrugs herein when the group is substituted with an-OH group are ethers; and

(iii) if the compounds of the formula I contain primary or secondary amino functions (-NH)₂or-NHR, where R ≠ H), for example if R³Preferred prodrugs thereof are amides thereof, for example wherein one or both hydrogens of the amino function of the compound of formula (I) are, as the case may be, - (C)₁-C₁₀) Alkanoyl, preferably- (C)₁-C₆) Alkanoyl, more preferably methyl, ethyl or propyl alkanoylThe compound of (1).

Particularly preferred prodrugs here are ethers of the compounds of the formula (I) < CHEM > - (C)₁-C₄) Alkyl ethers and- (C)₁-C₄) Alkyl esters, esters are particularly preferred. Ester prodrugs are described in detail in the following references: "Design of ester precursors to enhance organic requirements of porous compositions: challenges to the discovery scientist ", Current Drug Metabolism, (2003), 4(6), 461-.

Further examples of substituent groups according to the above examples and other examples of prodrug types can be found in the above references.

Finally, certain compounds of formula (I) may themselves act as prodrugs of other compounds of formula (I).

Also included within the scope of the present invention are metabolites of the compounds of formula (I), i.e. compounds which are produced in vivo after administration of the drug. Some examples of metabolites according to the invention include

(i) If the compound of formula (I) contains a methyl group, the hydroxymethyl derivative (-CH) thereof₃→-CH₂OH)；

(ii) If the compound of formula (I) contains an alkoxy group, its hydroxy derivative (-OR → -OH);

(iii) if the compound of formula (I) contains a tertiary amino group, its secondary amino derivative (-NR)⁷R⁸→-NHR⁷or-NHR⁸Wherein R is⁷And R⁸Are different groups);

(iv) if the compound of formula (I) contains a secondary amino group, its primary amino derivative (-NHR)⁷→-NH₂)；

(v) If the compound of formula (I) contains a phenyl moiety, its phenolic derivative (-Ph → -PhOH); and

(vi) if the compound of formula (I) contains an amide group, its carboxylic acid derivative (-CONH)₂→-COOH)。

The compounds of formula (I) containing one or more asymmetric carbon atoms may exist as two or more stereoisomers. If the compounds of formula (I) contain alkenyl or alkenylene groups, geometric cis/trans (or Z/E) isomers are possible. If the structural isomers are interconvertible via a low energy barrier, tautomerism ("tautomerism") may occur. This may be in the form of proton tautomerism in compounds of formula (I) containing, for example, imino, keto, or oxime groups, or so-called valence tautomerism in compounds containing aromatic moieties. It follows that a single compound may exhibit more than one type of isomerism.

All stereoisomers, geometric isomers and tautomeric forms of the compounds of formula (I) are included within the scope of the invention, including compounds exhibiting more than one type of isomerism, and one or more mixtures thereof. Also included are acid addition or base salts in which the counterion is optically active, such as d-lactate or l-lysine, or racemic, such as dl-tartrate or dl-arginine.

Specifically included within the scope of the present invention are mixtures of stereoisomers of the compounds of formula (I), or diastereoisomerically enriched or diastereoisomerically pure isomers of the compounds of formula (I), or enantiomerically enriched or enantiomerically pure isomers of the compounds of formula (I).

The cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, such as chromatography and fractional crystallization.

Conventional preparation/separation techniques for individual enantiomers include chiral synthesis starting from precursors suitable for optical purity or resolution of the racemate (or the racemate of a salt or derivative) for example using chiral High Performance Liquid Chromatography (HPLC).

Alternatively, the racemate (or racemic precursor) may be reacted with a suitable optically active compound, for example an alcohol, or, in the case of compounds of formula (I) containing an acidic or basic moiety, an acid or base, for example tartaric acid or 1-phenylethylamine. The resulting diastereomeric mixtures can be separated by chromatography and/or fractional crystallization, and one or both diastereomers converted to the corresponding pure enantiomers by means well known to the skilled artisan.

Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically enriched form by chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2 to 20%, and possibly from 0 to 5% by volume of an alkylamine. The eluate is concentrated to give an enriched mixture. The absolute composition of the mobile phase will depend on the chiral stationary phase (asymmetric resin) chosen. The following preparative example 2 provides an example of such a separation technique.

When any racemate crystallizes, two different types of crystals are possible. The first type is the racemic compound (true racemate) mentioned above, in which a crystal of homogeneous form is formed, containing equimolar amounts of the two enantiomers. The second type is a racemic mixture or aggregate, in which equimolar amounts of the two forms of the crystal, each containing a single enantiomer, are produced.

Although both crystal forms present in the racemic mixture have the same physical properties, they may have physical properties different from the true racemate. The racemic mixture can be separated by conventional techniques known to those skilled in the art, see, e.g. Stereochemistry of Organic Compounds by E.L.Eliel and S.H.Wilen(Wiley，1994)。

Thus, the present invention additionally provides compounds of formula (IA), (IB), (IC), (ID), (IE), (IF), (IG) and (IH).

Wherein R is¹、R²、R³、R⁴And R⁵Is as defined above. Also included are compounds of the general formulae (IB) and (ID) in which the radicals 3 and 4 of the pyrrolidine ringThe stereochemistry of the clusters is cis with respect to each other.

Preferably, the present invention provides compounds of formula (IA), more preferably compounds of formula (IC), even more preferably compounds of formula (IE), especially compounds of formula (IF).

The present invention includes all pharmaceutically acceptable isotopically-labelled compounds of formula (I) wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.

Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of the following elements: hydrogen, e.g.²H and³h, carbon, e.g.¹¹C、¹³C and¹⁴c, chlorine, e.g.³⁶Cl, fluorine, e.g.¹⁸F, iodine, e.g.¹²³I and¹²⁵i, nitrogen, e.g.¹³N and¹⁵n, oxygen, e.g.¹⁵O、¹⁷O and¹⁸o, phosphorus, e.g.³²P, and sulfur, e.g.³⁵S。

Certain isotope-labeled compounds of formula (I), such as those incorporating a radioisotope, are useful in drug and/or substrate tissue distribution studies. Radioisotope tritium, i.e. ³H and carbon-14, i.e.¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ease of detection.

By heavier isotopes, e.g. deuterium, i.e.²H substitution may be preferred in some cases because greater metabolic stability may provide certain therapeutic benefits, such as increased in vivo half-life or reduced dosage requirements.

By positron-emitting isotopes, e.g.¹¹C、¹⁸F、¹⁵O and¹³n substitution, can be used in Positron Emission Tomography (PET) studies for examining the occupancy of substrate receptors.

Isotopically-labelled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the examples and preparations using appropriate isotopically-labelled reagents in place of the non-labelled reagents previously employed.

Pharmaceutically acceptable solvates according to the invention include those in which the crystallization solvent may be isotopically substituted, e.g. D₂O、d₆-acetone, d₆-DMSO。

According to a preferred embodiment, the present invention provides a group of compounds of formula (I), preferably of formula (IA), more preferably of formula (IC), even more preferably of formula (IE), especially of formula (IF), wherein R is¹Selected from: - (C)₁-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C) ₃-C₈) Cycloalkyl, phenyl, - (C)₁-C₂) Alkylaryl, heterocyclyl or- (C)₁-C₂) Alkyl heterocyclic radical, wherein R¹Optionally substituted with one or more groups selected from: - (C)₁-C₄) Alkyl, - (CH)₂)_mOR⁶、-(CH₂)_m(C₃-C₅) Cycloalkyl, halogen, OCH₃、OCH₂CH₃、CN、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃Wherein m is 1 or 2, and wherein when R is¹Is heterocyclyl or- (C)₁-C₂) Alkyl heterocyclyl when said heterocyclyl is independently selected from a monocyclic 5-to 6-membered ring system containing up to 3 heteroatoms independently selected from O, N or S and combinations thereof.

According to a more preferred embodiment, the present invention provides a group of compounds of formula (I), preferably of formula (IA), more preferably of formula (IC), even more preferably of formula (IE), especially of formula (IF), wherein R is¹Selected from: - (C)₁-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, phenyl, - (C)₁-C₂) Alkylaryl, heterocyclyl or- (C)₁-C₂) Alkyl heterocyclic radical, wherein each of the above R¹The groups are optionally substituted with one or more groups selected from: - (C)₁-C₄) Alkyl, halogen, - (CH)₂)_mOR⁶、CN、CF₃、OCF₃Wherein m is 1 or 2;

R²is OH;

R³selected from: H. - (C)₁-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, aryl, - (C)₁-C₂) Alkylaryl, heterocyclyl or- (C)₁-C₂) Alkylheterocyclyl, wherein each of the latter seven R' s³The groups are optionally substituted with one or more groups selected from: OH, - (C) ₁-C₄) Alkyl, - (CH)₂)_n(C₃-C₅) Cycloalkyl, halogen, CN, - (CH)₂)_nOR⁶Or- (CH)₂)_nNR⁷R⁸Wherein n is 0, 1 or 2;

R⁴selected from: H. - (C)₁-C₄) Alkyl, - (CH)₂)_p(C₃-C₅) Cycloalkyl, halogen, - (CH)₂)_pOR⁶、-(CH₂)_pNR⁷R⁸、CN、-C(O)R⁶、-C(O)OR⁶、-C(O)NR⁷R⁸、-(CH₂)_pNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃A group wherein p ═ 0, 1 or 2;

R⁵selected from: - (C)₁-C₄) Alkyl, - (CH)₂)_p(C₃-C₅) Cycloalkyl, halogen, - (CH)₂)_pOR⁶、-(CH₂)_pNR⁷R⁸、CN、-C(O)R⁶、-C(O)OR⁶、-C(O)NR⁷R⁸、-(CH₂)_pNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃Wherein p is 0, 1 or 2;

R⁶、R⁷and R⁸Each independently selected from H, CH₃Or CH₂CH₃；

Wherein R is³The heterocyclic group of (a) is selected from monocyclic 5-to 6-membered ring systems containing up to 2 heteroatoms independently selected from O or N and combinations thereof;

wherein R is¹The heterocyclic group of (a) is selected from monocyclic 5-to 6-membered ring systems containing 1N heteroatom.

Preference is given here to the group of compounds of the general formula (I), preferably of the formula (IA), more preferably of the formula (IC), and even more preferably of the formula (IE), in particular of the formula (IF), as defined above, in which R is³Is H, - (C)₁-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkylaryl or heterocyclyl, wherein each of the latter five R' s³The groups are optionally substituted with one or more groups selected from: -OH, - (C)₁-C₄) Alkyl, - (CH)₂)_n(C₃-C₅) Cycloalkyl, halogen, -CN or- (CH)₂)_nOR⁶Wherein n is 0 or 1, wherein R⁶Is H, CH₃Or CH₂CH₃And wherein when R is³When a heterocyclyl group, the heterocyclyl group is selected from monocyclic 5-to 6-membered ring systems containing up to 2 heteroatoms independently selected from O or N, and combinations thereof.

According to a further preferred embodiment, the present invention provides a group of compounds of general formula (I), preferably of formula (IA), more preferably of formula (IC), even more preferably of formula (IE), especially of formula (IF), wherein R is¹Selected from: - (C)₁-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, phenyl or heterocyclyl, wherein each of the above R¹The groups are optionally substituted with one or more groups selected from: - (C)₁-C₄) Alkyl, halogen, -OR⁶or-CN;

R²is OH;

R³selected from: H. - (C)₂-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl or heterocyclyl, wherein each of the latter four R' s³The groups are optionally substituted with one or more groups selected from: -OH, - (C)₁-C₄) Alkyl, - (CH)₂)_n(C₃-C₅) Cycloalkyl, halogen, -CN, -OR⁶Or- (CH)₂)_nNR⁷R⁸Wherein n is 0, 1 or 2;

R⁴selected from: H. f or Cl;

R⁵selected from: f or Cl;

R⁶、R⁷and R⁸Each independently selected from H, CH₃Or CH₂CH₃；

Preference is given here to the group of compounds of the general formula (I), preferably of the formula (IA), more preferably of the formula (IC), and even more preferably of the formula (IE), in particular of the formula (IF), as defined above, in which R is ¹The heterocyclic group of (a) if present is a monocyclic 6-membered ring system containing up to 1N heteroatom.

Preference is given here to the group of compounds of the general formula (I), preferably of the formula (IA), more preferably of the formula (IC), and even more preferably of the formula (IE), in particular of the formula (IF), as defined above, in which R is³The heterocyclic group of (a) if present is a monocyclic 6-membered ring system containing up to 2N heteroatoms.

R is preferably used herein¹The group is selected from (A) to (B)C₁-C₄) Alkyl, - (C)₃-C₆) Cycloalkyl, phenyl, pyridyl or pyrimidinyl, wherein R¹Optionally substituted with one or more groups selected from: CH (CH)₃、CH₂CH₃Halogen, OCH₃、OCH₂CH₃、CN、CF₃Or OCF₃。

More preferably R used herein¹The group is selected from n-propyl, isopropyl, n-butyl, methoxymethyl, cyclopropyl, cyclohexyl, phenyl, 3-fluorophenyl, 4-chlorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2, 6-difluorophenyl, 2, 4-difluorophenyl, 3, 4-difluorophenyl, pyridin-2-yl or pyridin-3-yl.

Very preferred for use herein R¹The group is selected from pyridin-2-yl, phenyl, 3-fluorophenyl, 4-chlorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2, 6-difluorophenyl, 2, 4-difluorophenyl or 3, 4-difluorophenyl.

R is preferably used herein ³The radicals being selected from-H, - (C)₂-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl or heterocyclyl, wherein each of the latter four R' s³The groups are optionally substituted with one or more groups selected from: -OH, - (C)₁-C₄) Alkyl OR-OR⁶Wherein R is⁶is-H, CH₃Or CH₂CH₃Wherein when R is³When a heterocyclyl group, the heterocyclyl group is a monocyclic 6-membered ring system containing up to 2N heteroatoms.

More preferably R used herein³The group is selected from: hydrogen, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, isobutyl, 2-methoxyethyl, cyclopentyl, cyclobutyl, cyclopentylmethyl, pyridin-2-yl, pyridin-3-yl, pyridazin-3-yl, pyrazinyl, pyrimidin-5-yl, pyrimidin-2-yl, pyrimidin-4-yl or tetrahydropyran-4-yl.

R is preferably used herein⁴Radical (I)Selected from H, F or Cl, preferably R for use herein⁵The group is selected from F or Cl.

Preferably used herein having R⁴And R⁵The phenyl groups of the substituents are: 2, 4-disubstituted phenyl, wherein R⁴And R⁵Each group is independently selected from F or Cl; or 4-monosubstituted phenyl, wherein R⁴Is H, R⁵Is F or Cl.

More preferably used herein are linked with R⁴And R⁵The phenyl group of (a) is 4-chlorophenyl or 2, 4-difluorophenyl.

When R is³When is H, in a preferred group of compounds of formula (IC), more preferably (IE), especially (IF) herein, R is ¹Is phenyl, 3-fluorophenyl, 4-fluorophenyl, 2, 6-difluorophenyl, 2, 4-difluorophenyl, 3, 4-difluorophenyl or pyridin-2-yl; r²Is OH; and R is⁴Selected from H or F; and R is⁵Selected from F or Cl.

Preferred herein are those wherein R³A compound that is H is a compound of examples 12, 16, 24, and 48 or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

When R is³Is a heterocyclic group as defined below, R in a preferred group of compounds of general formula (IC), more preferably (IE), especially (IF)¹Is phenyl or pyridin-2-yl; r²is-OH; r³Is a heterocyclic group selected from pyridin-2-yl, pyridin-3-yl, pyridazin-3-yl, pyrazinyl, pyrimidin-5-yl, pyrimidin-4-yl, pyrimidin-2-yl or tetrahydropyran-4-yl; and R is⁴And R⁵Are both F.

Preferred herein are those wherein R³Compounds which are heterocyclic groups selected from pyridin-2-yl, pyridin-3-yl, pyridazin-3-yl, pyrazinyl, pyrimidin-5-yl, pyrimidin-4-yl, pyrimidin-2-yl or tetrahydropyran-4-yl are the compounds of examples 31, 34, 35, 42 and 47 and pharmaceutically acceptable salts, solvates and hydrates thereof.

When R is³Is Et, i-Pr or t-Bu, a preferred group of formulae (IC) here) More preferably (IE), especially (IF) compounds, R ¹Is phenyl, 4-fluorophenyl, 4-chlorophenyl, 3-fluorophenyl, 2, 4-difluorophenyl, 3, 4-difluorophenyl, pyridin-2-yl; r²Is OH; and R is⁴And R⁵Is F.

Preferred herein are those wherein R³Compounds which are Et, i-Pr or t-Bu are the compounds of examples 1, 5, 6, 8, 9, 10, 13, 15, 22, 40, 50, 51, 52 and 53 and pharmaceutically acceptable salts, solvates and hydrates thereof.

Preferred compounds according to the invention include:

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol;

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (3, 4-difluorophenyl) -3, 5-dimethylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidin-3-yl ] carbonyl } -4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (4-chlorophenyl) -3, 5-dimethylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (2, 4-difluorophenyl) -3, 5-dimethylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -4- (2, 4-difluorophenyl) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-pyridin-2-ylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyridin-2-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyridin-3-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyridazin-3-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-propylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyrimidin-4-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyridazin-3-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-pyridin-2-ylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (4-chlorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -4- (4-chlorophenyl) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -4- (3, 4-difluorophenyl) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -4- (2, 4-difluorophenyl) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidin-4-ol hydrochloride;

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-ethylpyrrolidin-3-yl ] carbonyl } -4- (3-fluorophenyl) -3, 5-dimethylpiperidine-4-ol hydrochloride

And pharmaceutically acceptable acid salts, solvates and hydrates thereof.

Preferred compounds according to the invention are independently selected from the group consisting of:

(3R, 4R, 5S) -4- (4-chlorophenyl) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidine-4-ol hydrochloride

And pharmaceutically acceptable acid salts, solvates and hydrates thereof.

Very much preferred here are: (3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol, also known as [ 1-tert-butyl-4- (2, 4-difluoro-phenyl) -pyrrolidin-3-yl ] - (4-hydroxy-3, 5-dimethyl-4-phenyl-piperidin-1-yl) -methanone (compound of example 1) and/or a pharmaceutically acceptable acid salt thereof, for example (3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride, also known as [ 1-tert-butyl-4- (2, 4-difluoro-phenyl) -pyrrolidin-3-yl ] - (4-hydroxy-3, 5-dimethyl-4-phenyl-piperidin-1-yl) -methanone HCl salt (example 5 compound).

According to a further embodiment, the present invention provides a compound of formula (I)

Or a stereoisomeric mixture thereof, or a diastereoisomerically enriched or diastereomerically pure isomer thereof, or an enantiomerically enriched or enantiomerically pure isomer thereof, or a prodrug of such a compound, a mixture or isomer thereof, or a pharmaceutically acceptable salt of the compound, mixture, isomer or prodrug,

wherein R is¹Selected from: (C)₁-C₆) Alkyl, (C)₂-C₆) An alkenyl group,(C₂-C₆) Alkynyl, (C)₃-C₈) Cycloalkyl group, (C)₅-C₈) Cycloalkenyl group, (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, aryl, (C)₁-C₂) Alkylaryl, heterocyclyl or (C)₁-C₂) An alkyl heterocyclic group, a heterocyclic group having a heterocyclic group,

wherein each of the above R¹The groups are optionally substituted with one or more groups selected from: (C)₁-C₄) Alkyl group, (CH)₂)_m(C₃-C₅) Cycloalkyl, halogen, (CH)₂)_mOR⁶、(CH₂)_mNR⁷R⁸、CN、C(O)R⁶、C(O)OR⁶、CON(R⁷)₂、(CH₂)_mNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃、OCH₂CF₃SMe or sat, wherein m ═ 0, 1 or 2;

R²is H, OH or OMe;

R³selected from: H. (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl, (C)₃-C₈) Cycloalkyl group, (C)₅-C₈) Cycloalkenyl group, (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, aryl, (C)₁-C₂) Alkylaryl, heterocyclyl or (C)₁-C₂) An alkyl heterocyclic group, a heterocyclic group having a heterocyclic group,

wherein each of the latter ten R³The groups are optionally substituted with one or more groups selected from: OH, (C)₁-C₄) Alkyl group, (CH)₂)_n(C₃-C₅) Cycloalkyl, halogen, CN, (CH)₂)_nOR⁶、(CH₂)_nN(R⁷)₂SMe or SEt, wherein n is 0, 1 or 2

R⁴And R⁵Each independently selected from: (C)₁-C₄) Alkyl, (C)₂-C₄) Alkenyl, (C)₂-C₄) Alkynyl, (CH)₂)_p(C₃-C₅) Cycloalkyl group, (CH)₂)_p(C₅) Cycloalkenyl, halogen, (CH)₂)_pOR⁶、(CH₂)_pNR⁷R⁸、CN、C(O)R⁶、C(O)OR⁶、CON(R⁷)₂、(CH₂)_pNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃、OCH₂CF₃SMe or sat, wherein p ═ 0, 1 or 2;

R⁶、R⁷and R⁸Each independently selected from H, Me or Et;

wherein R is¹And R³Heterocyclyl, if present, is an optionally fused 4-to 10-membered ring system containing up to 4 heteroatoms selected from O, N or S.

A preferred group of compounds according to this further embodiment are those wherein R is¹Selected from: (C)₁-C₆) Alkyl, (C)₃-C₈) Cycloalkyl group, (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, aryl, (C)₁-C₂) Alkylaryl, heterocyclyl or (C)₁-C₂) Alkyl heterocyclic radical, wherein R¹Optionally substituted with one or more groups selected from: (C)₁-C₄) Alkyl group, (CH)₂)_m(C₃-C₅) Cycloalkyl, halogen, OMe, OEt, CN, halogen, CF₃、CH₂CF₃、OCF₃、OCH₂CF₃SMe or sat, wherein m ═ 0, 1 or 2; wherein said heterocyclyl is selected from: pyridyl, pyrimidinyl, triazinyl, oxadiazinyl, oxazolyl, thiazolyl, thiadiazolyl, imidazolyl, benzimidazolyl, benzeneThiazolyl, indazolyl, quinolinyl, or isoquinolinyl;

R²is H or OH;

R³is H, (C) ₁-C₆) Alkyl, (C)₃-C₈) Cycloalkyl group, (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl group, (C)₁-C₂) Alkylaryl, wherein each of the latter four R' s³The groups are optionally substituted with one or more groups selected from: OH, (C)₁-C₄) Alkyl group, (CH)₂)_n(C₃-C₅) Cycloalkyl, halogen, CN, (CH)₂)_nOR⁶SMe or sat, wherein n ═ 0 or 1;

R⁴and R⁵Independently selected from (C)₁-C₄) Alkyl, cyclopropyl, halogen, OR⁶、CN、CF₃、CH₂CF₃、OCF₃、OCH₂CF₃SMe, SEt or R⁴And R⁵Together form a fused 5-to 6-membered saturated or unsaturated ring; wherein R is⁶Is as defined above.

In a more preferred group of compounds according to this further embodiment, R¹Is (C)₁-C₄) Alkyl, (C)₃-C₆) Cycloalkyl group, (C)₁-C₂) Alkyl radical (C)₃-C₅) Cycloalkyl, phenyl, pyridyl or pyrimidinyl, wherein R¹Optionally substituted with one or more groups selected from: me, Et, halogen, OMe, OEt, CN, CF₃、OCF₃And SMe;

R²is OH;

R³is (C)₁-C₆) Alkyl, optionally substituted with one of the following groups: OH, OR⁶、CF₃；

R⁴And R⁵Each independently is (C)₁-C₄) Alkyl, halogen, OR⁶、CN、CF₃、CH₂CF₃、OCF₃、OCH₂CF₃；R⁶Is H or Me.

In a particularly preferred group of compounds according to this further embodiment, R¹Is n-butyl, cyclohexyl, phenyl or 4-methylphenyl;

R²is OH;

R³is ethyl or tert-butyl;

R⁴and R⁵Each independently is F.

Very preferred compounds according to this further embodiment are compounds of formula (IG), especially preferred compounds according to this further embodiment are compounds of formula (IH),

wherein R is¹、R²、R³、R⁴And R⁵Is as defined above.

More preferred compounds according to this further embodiment are compounds of formula (IG) or (IH), more preferably (IH) wherein the stereochemistry of the groups at the 3-and 4-positions of the pyrrolidine ring is trans relative to each other.

The following route illustrates the method of synthesizing the compound of formula (I).

Scheme 1 illustrates the preparation of compounds of formula (I) via peptide coupling of intermediates (II) and (III), with the addition of a suitable base and/or additive (e.g., 1-hydroxybenzotriazole hydrate or 4-dimethylaminopyridine), if necessary. Scheme 1a illustrates the preparation of compounds of formula (IA) via peptide coupling of intermediates (II) and (IIIA). Similarly, schemes 1b through 1e illustrate the preparation of compounds of formula (IC), (ID), (IE) and (IF) via peptide coupling of intermediates (IIA) and (IIIA), (IIB) and (III), (IIB) and (IIIA), (IIB) and (IIIB), respectively. The compounds of formulae (IB), (IG) and (IH) may be prepared in a similar manner from the relevant intermediates.

Scheme 1

Scheme 1a

Scheme 1b

Scheme 1c

Scheme 1d

Scheme 1e

With respect to the compounds (I), (II), (III) in schemes 1 and 1a to 1e, R¹、R²、R³、R⁴And R⁵Is as defined above for compounds of formula (I), unless otherwise specified.

Alternative conditions employed involve stirring solutions of piperidine (amine) of formula (II) with pyrrolidine (acid) of formula (III) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), triethylamine or N-methylmorpholine with 1-hydroxybenzotriazole hydrate (HOBt) in Dimethylformamide (DMF) or Tetrahydrofuran (THF) or ethyl acetate at room temperature. A suitable alternative process is the stirring of the intermediate compounds of the general formulae (II) and (III) and the CH of O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU) or 1-propylphosphonic acid cyclic anhydride₂Cl₂Or EtOAc solution. Any suitable inert solvent may be used in place of those mentioned above, wherein inert solvent means a solvent which does not contain a carboxylic acid or a primary or secondary amine. At least one equivalent of each coupling reagent should be used, and one or both may be used in excess if desired.

Thus, according to a further embodiment, the present invention provides a process for the preparation of a compound of formula (I) comprising coupling a piperidine (amine) of formula (II) with a peptide of formula (III) a pyrrolidine (acid). According to a preferred embodiment, the present invention provides a process for the preparation of a compound of formula (IA) comprising coupling a piperidine (amine) of formula (II) with a peptide of formula (IIIA) pyrrolidine (acid). According to a more preferred embodiment, the present invention provides a process for the preparation of a compound of formula (ID) comprising coupling a piperidine (amine) of formula (IIB) with a peptide of formula (III) a pyrrolidine (acid). According to a more preferred embodiment, the present invention provides a process for the preparation of a compound of formula (IC) comprising coupling a piperidine (amine) of formula (IIA) with a peptide of formula (IIIA) pyrrolidine (acid). According to a more preferred embodiment, the present invention provides a process for the preparation of a compound of formula (IE) comprising coupling a piperidine (amine) of formula (IIB) with a peptide of formula (IIIA) pyrrolidine (acid). According to a particularly preferred embodiment, the present invention provides a process for the preparation of a compound of formula (IF) comprising coupling a piperidine (amine) of formula (IIB) with a peptide of formula (IIIB) pyrrolidine (acid).

According to a further embodiment, the present invention provides intermediate compounds of the general formulae (II), (IIA) and (IIB),

Wherein R is¹And R²Is as defined above. Preference is given here to intermediates of the formula (II), more preferably of the formula (IIA), especially of the formula (IIB), in which R is²OH, and R¹Mono-substituted phenyl, or 2, 6-or 3, 4-or 2, 4-disubstituted phenyl, or pyridinyl, wherein the phenyl substituents are selected from F, Cl and OCH₃。

A highly preferred group of intermediates herein are compounds of formula (II), more preferably of formula (IIA), especially of formula (IIB), wherein R is²＝-OH，R¹Is phenyl, 4-fluorophenyl, 3, 4-difluorophenyl, 4-chlorophenyl, 3-fluorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2, 6-difluorophenyl, 2, 4-difluorophenyl, 3, 4-difluorophenyl or pyridin-2-yl.

Thus, according to a preferred embodiment, the present invention provides intermediates of formula (IIB) wherein R is²is-OH, and R¹Is phenyl, 4-fluorophenyl, 3, 4-difluorophenyl, 3-fluorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2, 6-difluorophenyl, 2, 4-difluorophenyl, 3, 4-difluorophenyl or pyridin-2-yl.

According to a further embodiment, the present invention provides intermediate compounds of general formulae (III), (IIIA) and (IIIB),

wherein R is³、R⁴And R⁵Is as defined above. Preferred herein are intermediates of formula (II), more preferably of formula (IIA), most preferably of formula (IIB), wherein R is ⁴Is H or F or Cl, wherein R⁵Is F or Cl, wherein R³Is H or- (C)₂-C₄) Alkyl, - (C)₃-C₈) Cycloalkyl group, (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl or heterocyclyl. Here a group ofPreferred intermediates are compounds of formula (III), more preferably of formula (IIIA), especially of formula (IIIB), wherein R³is-H, i-Pr, Et, or a heterocyclyl selected from: pyridin-2-yl, pyridin-3-yl, pyridazin-3-yl, pyrazinyl, pyrimidin-5-yl, pyrimidin-4-yl, pyrimidin-2-yl or tetrahydropyran-4-yl.

Thus, according to another embodiment, the present invention provides a process for the preparation of compounds of formula (I), more preferably of formula (IC), more preferably of formula (IE), especially of formula (IF), which process is coupled via peptides of intermediates (II) and (III), preferably of (IIA) and (IIIA), more preferably of (IIB) and (IIIA), especially of (IIB) and (IIIB), wherein: r²is-OH; r¹Is phenyl, 4-fluorophenyl, 3, 4-difluorophenyl, 3-fluorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2, 6-difluorophenyl, 2, 4-difluorophenyl, 3, 4-difluorophenyl or pyridin-2-yl; r³is-H, t-Bu, i-Pr, Et or a heterocyclic group selected from: pyridin-2-yl, pyridin-3-yl, pyridazin-3-yl, pyrazinyl, pyrimidin-5-yl, pyrimidin-4-yl, pyrimidin-2-yl or tetrahydropyran-4-yl; r ⁴Is H, Cl or F; and R is⁵Is Cl or F.

According to the preferred process herein, the compounds of general formula (IF) are prepared via peptide coupling of intermediates (IIA) and (IIIA), wherein: r²is-OH; r¹Is phenyl, 4-fluorophenyl, 3, 4-difluorophenyl, 3-fluorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2, 6-difluorophenyl, 2, 4-difluorophenyl, 3, 4-difluorophenyl or pyridin-2-yl; r³is-H, t-Bu, i-Pr, Et or a heterocyclic group selected from: pyridin-2-yl, pyridin-3-yl, pyridazin-3-yl, pyrazinyl, pyrimidin-5-yl, pyrimidin-4-yl, pyrimidin-2-yl or tetrahydropyran-4-yl; r⁴Is H, Cl or F; r⁵Is Cl or F.

According to a more preferred process herein, the compounds of general formula (IF) are prepared via peptide coupling of intermediates (IIA) and (IIIA), wherein: r²is-OH; r¹Is phenyl, 4-fluorophenyl, 3, 4-difluorophenyl, 3-fluorophenyl, 2, 4-difluorophenyl or 3, 4-difluorophenyl or pyridin-2-yl; r³Is t-Bu,i-Pr or Et; and R is⁴And R⁵Are both F.

Scheme 2 illustrates the preparation of a peptide having a series of R groups using a protecting group strategy³Alternative routes to the compounds of general formula (I) of the radicals. The compounds of general formulae (IA) to (IF) can also be prepared according to the route described in scheme 2, using the appropriate intermediates (II), (IIA) or (IIB) and the appropriately protected amines of formula (IV), (IVA) or (IVB) as required.

Scheme 2

With respect to compounds (I), (II), (IV) and (V) in scheme 2 or (IVA) or (IVB), R as described below¹、R²、R³、R⁴And R⁵Is as defined above for compounds of formula (I), unless otherwise specified. PG is a nitrogen protecting group.

In scheme 2, the amine intermediate of formula (II) and the protected pyrrolidinic acid intermediate of formula (IV) are coupled using standard peptide coupling procedures as described in scheme 1 above to provide a coupled and protected intermediate of formula (V) from which the nitrogen protecting group can be removed using standard deprotection strategies to provide a compound of formula (I), wherein R is R³H. Any suitable nitrogen Protecting group (e.g., "Protecting Groups in Organic Synthesis" 3) may be used^rdEdition t.w.greene and p.g.wuts, Wiley-Interscience, 1999). A common nitrogen Protecting Group (PG) suitable for use herein is t-butyloxycarbonyl, which can be readily removed by treatment with an acid such as trifluoroacetic acid or hydrogen chloride in an organic solvent such as dichloromethane or 1, 4-dioxane.

Can be carried out at R by conventional alkylation techniques³Where a replacement group (instead of H) is introduced. Suitable processes for secondary amine alkylation include:

(i) with an aldehyde and a hydride reducing agent, such as sodium triacetoxyborohydride, optionally in the presence of acetic acid, in an inert solvent, such as dichloromethane or acetonitrile;

(ii) With an alkyl halide or a suitably activated alcohol derivative (e.g. a sulfonate ester) in the presence of a base, such as triethylamine, in an inert solvent.

By replacing suitable leaving groups from the heteroaromatic precursor, aryl and heteroaryl groups can be introduced as R3. Suitable leaving groups include halogen. In some cases, a transition metal catalyst (e.g., palladium, copper) or optionally in combination with a phosphine ligand (e.g., 1 '-binaphthyl-2, 2' -diylbiphenylphosphine) may be required to obtain the desired coupled product.

Scheme 3a illustrates a route for the preparation of a pyrrolidinic acid intermediate of formula (III) from an unsaturated ester intermediate of formula (VI).

Scheme 3a

With respect to compounds (III), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII) in scheme 3a, R¹、R²、R³、R⁴And R⁵Is as defined above for compounds of formula (I), unless otherwise specified. PG (Picture experts group)²Are suitable carboxylic acid protecting groups.

Compounds of the general formula (VI) can be prepared by Wittig or a similar olefination of an aldehyde intermediate of the general formula (X) with a suitable ylium salt, for example methyl (triphenylphosphino) acetate, or a phosphonic acid anion, for example derived from deprotonation of trimethyl phosphonoacetate, predominantly the trans isomer.

With respect to the production of unsaturated ester intermediates of formula (VI), there are many alternatives in the literature, including esterification of the precursor cinnamic acid derivative (VII) using standard esterification methods, or Heck reaction of an aromatic halide (VIII) with a suitable acrylic acid ester (IX), such as t-butyl acrylate, in the presence of a palladium catalyst and a suitable base, such as triethylamine.

The resulting E-olefin intermediates of formula (VI) will react with compounds of formula (XI) to undergo a [3+2] -azomethine salt cycloaddition to give pyrrolidines of almost trans-stereochemistry only. This reaction requires an inert solvent, such as dichloromethane or toluene or tetrahydrofuran, and one or more of the following activation measures: (1) acid catalysts, such as TFA; (2) desilylating agents such as silver fluoride; (3) and (4) heating.

Alternatively, the compounds of formula (XI) are reacted with unsaturated esters or acids of Z-olefin configuration to give pyrrolidines of almost cis-stereochemistry only. Such Z-alkenes can be prepared by Lindlar reduction of alkynes or via Still-Gennari olefination.

The compound of formula (XII) obtained from the cycloaddition reaction is a racemate and may need to be resolved into its enantiomeric components, which can be achieved by preparative HPLC using a chiral stationary phase. Alternatively, the acid intermediates of formula (III) may be resolved by standard methods, for example by reaction with an enantiomerically pure reagent to give the diastereomeric derivative, separation of the resulting diastereomers by physical means and subsequent cleavage to the acid (III).

The intermediate compounds of the general formula (XII) can be converted into the compounds of the general formula (III) by hydrolysis of the ester. A number of methods are available for effecting this transformation (see Advanced organic chemistry: Reactions, mechanics, and Structure, Fouth edition. March, Jerry, 1992, pp 378-383 public by Wiley, New York, N.Y.USA). Specifically, treatment of a compound of formula (XII) with an aqueous alkali metal hydroxide solution, such as lithium hydroxide, sodium hydroxide or potassium hydroxide, in a suitable organic solvent will yield the corresponding compound of formula (III). Preferably, water-miscible organic cosolvents, such as 1, 4-dioxane or tetrahydrofuran, are also used in such reactions. A preferred method of ester hydrolysis of this type here involves treating the ester with potassium trimethylsilanolate in an inert solvent, such as diethyl ether, at room temperature. If desired, the reaction may be heated to assist in hydrolysis. Ester hydrolysis may also be effected using acidic conditions, for example by heating the ester in an aqueous acid, for example hydrochloric acid. Certain esters are preferably hydrolyzed under acidic conditions, such as t-butyl or benzhydryl esters. Such esters can be cleaved by treatment with anhydrous acids, such as trifluoroacetic acid or hydrogen chloride, in an inert organic solvent, such as methylene chloride.

Scheme 3b illustrates an alternative route to prepare a single enantiomer of a pyrrolidinoic acid intermediate of formula (III) from an unsaturated ester intermediate of formula (VI) using oxazolidinone as a chiral auxiliary. Hydrolysis of the unsaturated ester (VI) gives the acid of formula (XVIII), and oxazolidinone may be used as a chiral auxiliary (wherein R is preferably phenyl, tert-butyl or isopropyl) to give the intermediate of formula (XVIII). Alternatively, reaction of a compound of formula (VI) (when R ═ COt-Bu) with lithium oxazolidinone salt in a suitable solvent (e.g. THF) may also give a compound of formula (XVIII).

The compound of formula (XVIII) will undergo a [3+2] -azomethine ylide cycloaddition reaction with the compound of general formula (XI) to give diastereomers (XX) and (XVIX), which can be separated by chromatography or crystallization and hydrolyzed to give pyrrolidine of formula (III).

Scheme 3b

Scheme 4 illustrates that the synthesis of protected pyrrolidinic acid intermediates of formula (IV) can be achieved using methods similar to those described above for intermediates of formula (III), except that intermediates of formula (XIIA) contain a nitrogen protecting group, which can be subsequently removed in the synthetic scheme. Once the protecting group is removed, any suitable conventional technique is utilizedOther R can be introduced by the method described in scheme 2 ³A group.

Pyrrolidines of the general formula (IV) bearing nitrogen protecting groups can also be obtained enantioselectively in a similar manner as described in scheme 3b, using oxazolidinone chiral auxiliary.

Scheme 4

With respect to compounds (VI), (XIA), (XIIA), (XII) and (IV) in scheme 4, R¹、R²、R³、R⁴And R⁵Is as defined above for compounds of formula (I), unless otherwise specified. In formulas (XIA), (XIIA) and (IV), PG is selected from suitable nitrogen protecting groups. In formulae (VI), (XIIA) and (VII), PG²Selected from suitable carboxylic acid protecting groups.

The synthesis of the azomethine salt precursor compounds of general formula (XI) can be achieved as depicted in scheme 5. Thus, the primary amine of formula (XIII) can be alkylated by treatment with chloromethyl trimethylsilane, optionally neat or by heating the reaction in an inert solvent if desired. The resulting intermediate (XIV) can then be reacted with formaldehyde in methanol in the presence of a suitable base, such as potassium carbonate or tert-butylamine, to give intermediate (XI). To generate a similar intermediate (XIA) containing a nitrogen protecting group, a similar reaction sequence may be followed.

Scheme 5

With respect to compounds (XIII), (XIIIA), (XIV), (XIVA), (XIA) and (XI) in scheme 5, R ³Is as defined above for compounds of formula (I), unless otherwise specified. In formulas (XIIIA), (XIVA) and (XIA), PG is selected from suitable nitrogen protecting groups.

As depicted in scheme 6, wherein R²Piperidine intermediates of general formula (II) ═ OH can be prepared by addition of organometallic nucleophiles to ketones of general formula (XV) containing suitable nitrogen protecting groups to give intermediates of general formula (XVI). Such nucleophilic addition is generally carried out using Grignard, organolithium or other suitable organometallic reagents in anhydrous etheric or non-polar solvents at low temperatures. These organometallic reagents can be prepared by halogen-metal exchange using the appropriate halide precursors Y-Br or Y-I and n-butyllithium or tert-butyllithium. Suitable protecting groups include Bn, which CAN be removed by hydrogenation, or Boc, which CAN be removed by treatment with an acid (e.g., TFA), or PMB, which CAN be removed by treatment with DDQ, CAN or chloroethyl chloroformate to give the desired piperidine intermediate of formula (II). With certain protecting groups and under certain conditions, the protecting group may be unstable to treatment with organometallic reagents and thus both transformations may be accomplished in one step, for example when PG ═ Boc, the protecting group may sometimes be cleaved when the intermediate of formula (VII) is treated with organometallic reagents.

Scheme 6

With respect to the compounds (XV), (XVI) and (II) in scheme 6, R¹Is as defined above for compounds of formula (I), unless otherwise specified. In formulas (XV) and (XVI), PG is selected from suitable nitrogen protecting groups.

As depicted in scheme 7, when (3R, 5S) -1-benzyl-3, 5-dimethyl-piperidin-4-one is used, the stereochemistry of the addition favors the cis-form of the hydroxyl group in the product for the two methyl groups. The addition of control to carbonyl systems is described, for example, in the literature (Journal of medicinal chemistry (1964), 7(6), pp 726-8).

Scheme 7

With respect to compounds (XV), (XXI) and (II) in scheme 7, R¹Is as defined above for compounds of formula (I), unless otherwise specified. In formulas (XV) and (XXI), PG is selected from suitable nitrogen protecting groups.

In addition, scheme 8 illustrates hydrogenation under forced reduction conditions, e.g., high pressure and/or high temperature, or strong acid plus triethylsilane, where R²Intermediate compounds of the general formula (II) can be converted to where R²Other intermediate compounds of general formula (II) ═ H. In some cases, it may be desirable to protect the piperidine nitrogen atom to facilitate this transformation. Thus, intermediates of general formula (XVI) may be converted to compounds wherein R ²Other intermediate compounds of general formula (XXII) where H, followed by deprotection, give compounds wherein R²Compounds of general formula (II) ═ H.

Scheme 8

With respect to compounds (XVI) and (II) in scheme 8, R¹Is as defined above for compounds of formula (I), unless otherwise specified. In formulas (II) and (XXIII), PG is selected from suitable nitrogen protecting groups.

Additionally, scheme 9 illustrates wherein R²Intermediate compounds of the general formula (II) can be converted to where R²Other intermediate compounds of general formula (II) of OMe. This conversion can be achieved by means of a standard Williamson ether synthesis. That is, in an anhydrous solvent, such as tetrahydrofuran or dimethylformamide, wherein R²The alcohol group in the compounds of general formula (II) which are OH can be deprotonated by a strong base, for example sodium hydride, and the resulting anion is reacted with methyl iodide, if necessary with heating. Protection of the piperidine nitrogen atom may be required to facilitate this transformation. Thus, wherein R²Intermediates of general formula (XVI) OH can be converted into those wherein R²Other than OMeIntermediate compounds of formula (XXV) followed by deprotection to give compounds wherein R²Compounds of general formula (II) ═ OMe, as depicted in scheme 9.

Scheme 9

With respect to the compounds (XVI) and (II), R in scheme 9 ¹Is as defined above for compounds of formula (I), unless otherwise specified. In formulas (II) and (XVI), PG is selected from suitable nitrogen protecting groups.

The skilled person will appreciate that in addition to protecting the nitrogen group, as discussed above, at various stages during the synthesis of the compound of formula (I), it may be necessary to protect other groups, such as a hydroxy group, with a suitable protecting group, and then remove the protecting group. The method of deprotection of any particular group will depend on the protecting group. For examples of protection/deprotection methods, see "Protective groups in Organic synthesis", TW Greene and P G MW utz. For example, if the hydroxyl group is protected as a methyl ether, the deprotection conditions include refluxing in 48% aqueous HBr or stirring with borane tribromide in dichloromethane. Alternatively, if the hydroxyl group is protected as a benzyl ether, the deprotection conditions include hydrogenation with a palladium catalyst under a hydrogen atmosphere.

According to a preferred embodiment, the present invention provides a process for the preparation of compounds of general formula (I) in analogy to the preparation of the compound of example 1 via preparations 1 to 5 and 12 to 16, more preferably the preparation of the compound of example 5 via preparations 1, 21, 22b, 4, 5 and 12 to 16, having the stereochemistry defined herein.

According to a further embodiment, the present invention independently provides: the intermediate compound of preparation 1; and/or intermediate compounds of preparation 2; and/or the intermediate compound of preparation 3; and/or the intermediate compound of preparation 4; and/or the intermediate compound of preparation 5; and/or an intermediate compound of preparation 21; and/or the intermediate compound of preparation 22 b; and/or an intermediate compound of preparation 12; and/or an intermediate compound of preparation 13; and/or the intermediate compound of preparation 14; and/or an intermediate compound of preparation 15; and/or an intermediate compound of preparation 16.

The general reaction mechanisms described above in connection with the preparation of the novel starting materials used in the prior processes are reagents and reaction conditions which are conventional and suitable for their performance or preparation, and the methods for isolating the desired products will be well known to those skilled in the art with reference to the previous literature and the examples and preparations herein.

MCR4 Activity

The compounds of the invention are useful as MCR4 agonists for the treatment of various disease states.

Preferably, the MCR4 agonist exhibits functional potency at the MC4 receptor as EC₅₀EC representing less than about 1000nM, more preferably less than 150nM, even more preferably less than about 100nM, even more preferably less than about 50nM, especially less than about 10nM, wherein the functional potency of MCR4 ₅₀The assay can be performed using protocol C or E described below. Compounds according to the present invention, including the compounds of examples 12, 20, 16, 48, 1, 5, 6, 22, 13, 9, 10, 50, 14, 17, 19, 53, 40, 15, 52, 51, 8, 33, 31, 34, 35, 36, 42, 44 and 47, have been tested and demonstrated functional efficacy at the MC4 receptor of less than about 150 nM. Thus according to a further embodiment, the present invention provides a compound of formula (I) having a functional potency at the MC4 receptor of less than about 150 nM. A preferred group of compounds according to the invention, including the compounds of examples 1, 5, 6, 22, 13, 9, 17, 19, 53, 15, 52, 51, 8, 31, 34, 35, 42, 44 and 47, have been tested and have been found to demonstrate a functional potency at the MC4 receptor of less than about 50 nM. A further preferred group of compounds according to the invention has been tested, including the compounds of examples 1, 5, 9, 19, 8, 31, 34, 35, 42 and 47In particular, functional potency of less than about 10nM was found to be demonstrated for the MC4 receptor.

Preferred compounds herein exhibit functional potency as defined above for the MCR4 receptor and are selective for MCR4 relative to MCR 1. Preferably, the MCR4 agonist is selective for MCR4 over MCR1, wherein the MCR4 receptor agonist is at least about 10-fold, preferably at least about 20-fold, more preferably at least about 30-fold, even more preferably at least about 100-fold, even more preferably at least about 300-fold, even more preferably at least about 500-fold, especially at least about 1000-fold functionally selective for the MCR4 receptor as compared to the MCR1 receptor, wherein the relative selectivity assessment is based on an assay for the functional potency of MCR1 and MCR4, as may be performed using protocols a and C or E described below. The compounds according to the present invention, including the compounds of examples 1, 5, 6, 13, 10, 50, 14, 17, 33, 31 and 35, exhibited functional potency at the MCR4 receptor and were tested to exhibit greater than about 10-fold selectivity for MCR4 over MCR 1. Thus according to a further embodiment, the present invention provides compounds of formula (I) which exhibit functional potency at the MCR4 receptor and exhibit greater than about 10-fold selectivity for MCR4 over MCR 1. A preferred group of compounds according to the present invention, including the compounds of examples 1, 5, 13, 14, 17, 31 and 35, exhibit functional potency at the MCR4 receptor and have been tested to exhibit greater than about 30-fold selectivity for MCR4 over MCR 1. A further preferred group of compounds according to the invention, including the compounds of examples 13, 14, 31 and 35, exhibit functional potency at the MCR4 receptor and were tested to show greater than about 100-fold selectivity for MCR4 over MCR 1.

Preferably, the MCR4 agonist is selective for MCR4 over MCR3, wherein the MCR4 receptor agonist is at least about 10-fold, preferably at least about 30-fold, more preferably at least about 100-fold, even more preferably at least about 300-fold, even more preferably at least about 500-fold, and especially at least about 1000-fold more functionally selective for the MCR4 receptor as compared to the MCR3 receptor, wherein the relative selectivity assessment is based on an assay for the functional potency of MCR3 and MCR4, as may be performed using protocols a and B or E described below. The compounds of examples 1, 2 and 3 exhibited functional potency at the MCR4 receptor and were tested to show greater than about 30-fold selectivity for MCR4 over MCR 3.

Preferred compounds herein exhibit functional potency as defined above for the MCR4 receptor and are selective for MCR4 relative to MCR 5. Preferably, the MCR4 agonist is selective for MCR4 over MCR5, wherein the MCR4 receptor agonist is at least about 10-fold, preferably at least about 30-fold, more preferably at least about 100-fold, even more preferably at least about 300-fold, even more preferably at least about 500-fold, and especially at least about 1000-fold functionally selective for the MCR4 receptor as compared to the MCR5 receptor, wherein the relative selectivity assessment is based on an assay for the functional potency of MCR5 and MCR4, as may be performed using schemes D and E described below. Compounds according to the invention, including the compounds of examples 1, 5, 6, 22, 13, 9, 10, 50, 14, 17, 19, 53, 15, 52, 51, 33, 31, 35, 42, and 44, exhibited functional potency at the MCR4 receptor and were tested to exhibit greater than about 10-fold selectivity for MCR4 over MCR 5. Thus according to a further embodiment, the present invention provides compounds of formula (I) which exhibit functional potency at the MCR4 receptor and exhibit greater than about 10-fold selectivity for MCR4 over MCR 5. A group of preferred compounds according to the present invention, including the compounds of examples 1, 5, 22, 13, 9, 50, 17, 19, 53, 15, 52, 31, 33, 35, 42 and 44, exhibit functional potency at the MCR4 receptor and tested to exhibit greater than about 100-fold selectivity for MCR4 over MCR 5. A further preferred group of compounds according to the invention, including the compounds of examples 22, 13, 19, 15, 35, 42 and 44, exhibit functional potency at the MCR4 receptor and were tested to show greater than about 300-fold selectivity for MCR4 over MCR 5.

Preferably, the MCR4 agonist is selective for MCR4 over MCR1 and MCR3, wherein the MCR4 receptor agonist is at least about 10-fold, preferably at least about 30-fold, more preferably at least about 100-fold, even more preferably at least about 300-fold, even more preferably at least about 1000-fold functionally selective for the MCR4 receptor as compared to the MCR1 and MCR3 receptors.

Preferred compounds herein exhibit functional potency as defined above for the MCR4 receptor and are selective for MCR4 relative to MCR1 and MCR 5. Preferably, the MCR4 agonist is selective for MCR4 over MCR1 and MCR5, wherein the MCR4 receptor agonist is at least about 10-fold, preferably at least about 30-fold, more preferably at least about 100-fold, even more preferably at least about 300-fold, even more preferably at least about 500-fold, especially at least about 1000-fold functionally selective for the MCR4 receptor as compared to the MCR1 and MCR5 receptors.

Compounds according to the present invention, including the compounds of examples 1, 5, 6, 13, 10, 50, 14, 17, 33, 31 and 35, exhibited functional potency at the MCR4 receptor and were tested to exhibit greater than about 10-fold selectivity for MCR4 over MCR1 and MCR 5. Thus according to a further embodiment, the present invention provides compounds of formula (I) which exhibit functional potency at the MCR4 receptor and exhibit greater than about 10-fold selectivity for MCR4 over MCR1 and MCR 5. A preferred group of compounds according to the present invention, including the compounds of examples 1, 5, 13, 31 and 35, exhibited functional potency at the MCR4 receptor and were tested to exhibit greater than about 100-fold selectivity for MCR4 over MCR1 and MCR 5.

Preferably, the MCR4 agonist is selective for MCR4 over MCR3 and MCR5, wherein the MCR4 receptor agonist is at least about 10-fold, preferably at least about 30-fold, more preferably at least about 100-fold, even more preferably at least about 300-fold, and most preferably at least about 1000-fold functionally selective for the MCR4 receptor as compared to the MCR3 and MCR5 receptors.

In addition to their role in the treatment of sexual dysfunction, the compounds of the present invention are likely to be effective for a number of other indications as described below. The verb "treat" or the noun "treatment" as used herein is intended to encompass the prevention and control of the indicated condition, i.e. prophylactic and palliative treatment.

The compounds of the present invention are useful for treating diseases, disorders or conditions, including, but not limited to, male and female sexual dysfunction, including female hypoactive sexual desire disorder, sexual arousal disorder, orgasmic disorder and/or dyspareunia, male erectile dysfunction, as well as obesity (by reducing appetite, increasing metabolic rate, reducing fat uptake or reducing carbohydrate craving) and diabetes (by increasing glucose tolerance and/or reducing insulin resistance). The compounds of the invention are useful in the treatment of other diseases, disorders, or conditions, including but not limited to, hypertension, hyperlipidemia, osteoarthritis, cancer, gallbladder disease, sleep apnea, depression, anxiety, obsessive-compulsive, neurological disorders, insomnia/sleep disorders, substance abuse, pain, fever, inflammation, immune modulation, rheumatoid arthritis, tanning of the skin, acne and other skin disorders, neuroprotective and cognition and memory-enhancing effects, including the treatment of alzheimer's disease.

Some of the compounds of formula (I) exhibit highly specific activity at the melanocortin-4 receptor, making them particularly useful in the treatment of male and female sexual dysfunction and obesity.

The compounds of the invention are useful in the treatment of male and female sexual dysfunction, particularly male erectile dysfunction.

Female Sexual Dysfunction (FSD) includes Female Sexual Arousal Disorder (FSAD), sexual desire disorders, such as hypoactive sexual desire disorder (lack of interest in sex), and orgasmic disorders, such as loss of sexual arousal (inability to reach orgasm).

Male sexual dysfunction includes Male Erectile Dysfunction (MED) and ejaculatory disorders such as loss of libido (inability to achieve orgasm) or sexual desire disorders such as hypoactive sexual desire (lack of interest in sex).

The compounds of the invention are particularly useful in the treatment of female sexual dysfunction, including hypoactive sexual desire disorder, sexual arousal disorder, orgasm disorder, dyspareunia, and male erectile dysfunction.

Male Erectile Dysfunction (MED)

The compounds of the invention are useful in the treatment of male sexual dysfunction, in particular male erectile dysfunction. Male Erectile Dysfunction (MED), also known as male erectile dysfunction, is defined as:

"penile erection failure to achieve and/or maintain satisfactory sexual performance" (NIHConsenssus Development Panel on Impentance, 1993)

According to estimates, the prevalence of Erectile Dysfunction (ED) to all degrees (mild, moderate and complete impotence) is 52% in men between 40 and 70 years, with higher rates above 70 years (Melman et al 1999, j. urology, 161, p 5-11). This condition has a significant negative impact on the quality of life of individuals and their spouses, often resulting in increased anxiety and stress, causing depression and reduced self-esteem. Whereas MED was primarily considered a psychological disorder two decades ago (Benet al 1994 comp. ther., 20: 669-. As a result, great progress has been made in identifying the mechanism of normal penile erection and the pathophysiology of MED.

Penile erection is a hemodynamic event that depends on the balance of contraction and relaxation of the cavernous smooth muscle of the penis and the vasculature of the penis (Lerner et al 1993, J.Urology, 149, 1256-. The corpus cavernosum smooth muscle is also referred to herein as the corpus cavernosum smooth muscle or simply the corpus cavernosum of the penis. Relaxation of the smooth muscle of the corpora cavernosa causes increased blood flow into the spaces of the corpus cavernosa trabeculae, causing them to expand towards the surrounding capsule, compressing the drainage veins. This causes a large rise in blood pressure, resulting in an erection (Naylor, 1998, j. urology, 81, 424-.

The changes that occur during the erectile process are complex and require a high degree of coordinated control involving the peripheral and central nervous and endocrine systems (Naylor, 1998, j. urology, 81, 424-. Cavernous smooth muscle contraction is regulated by sympathetic norepinephrine innervation, via activation of the postsynaptic alpha 1 adrenoreceptor. MED may be associated with an increase in the intrinsic smooth muscle tone of the corpora cavernosa. However, the process of cavernous smooth muscle relaxation is partially affected by non-adrenergic energyMediation of non-cholinergic (NANC) neurotransmission. A number of other NANC neurotransmitters than NO are found in the penis, such as calcitonin gene-related peptide (CGRP) and Vasoactive Intestinal Peptide (VIP). The major relaxant responsible for mediating this relaxation is Nitric Oxide (NO), which is synthesized from L-arginine by Nitric Oxide Synthase (NOS) (Taub et al 1993 Urology, 42, 698-704). It is thought that reducing the smooth muscle tone of the corpus cavernosum may contribute to NO-induced relaxation of the corpus cavernosum. During male sexual arousal, NO is released from neurons and endothelium, binds to and activates soluble guanylate cyclase (sGC) located in smooth muscle cells and endothelium, causing an increase in intracellular cyclic guanosine 3 ', 5' -monophosphate (cGMP) levels. This cGMP increase is due to intracellular calcium concentration ([ Ca ] ²⁺]_i) The mechanism of the decrease leading to penile cavernous relaxation is unknown, but is thought to involve activation of protein kinase G (probably due to Ca)²⁺Activation of pumps and Ca²⁺Activated K⁺A channel).

Multiple sites of potential regulatory behavior have been identified within the central nervous system. Key neurotransmitters are believed to be serotonin, norepinephrine, oxytocin, nitric oxide, dopamine and melanocortins, such as alpha-melanocyte stimulating hormone. By mimicking the action of one of these key neurotransmitters, sexual function can be modulated.

Melanocortins are peptides derived from Proopiomelanocortin (POMC) that bind to and activate G-protein coupled receptors (GPCRs) of the melanocortin receptor family. Melanocortins regulate a variety of different physiological processes, including sexual function and sexual behavior, food intake and metabolism.

Five melanocortin receptors have been cloned, namely MCR1, MCR2, MCR3, MCR4, MCR5, and are expressed in various tissues. MCR1 is specifically expressed in melanocytes and melanoma cells, MCR2 is the ACTH receptor and is expressed in adrenal tissue, MCR3 is mainly expressed in the brain and limbic system, MCR4 is commonly expressed in the brain and spinal cord, and MCR5 is expressed in the brain and many peripheral tissues, including skin, adipose tissue, skeletal muscle, and lymphoid tissue. MCR3 may be involved in the control of sexual function, food intake and thermogenesis. Activation of MCR4 has been shown to induce penile erection in rodents, inactivation of MCR4 has been shown to cause obesity (Hadley, 1999, AnnN Y Acad Sci., 885: 1-21, Wikberg et al, 2000, Pharmacol Res., 42(5), 393-420).

Synthetic melanocortin receptor agonists have been found to elicit erection in men with psychogenic erectile dysfunction (Wessells et al, Int J Impot Res.2000 Oct; 12 Suppl 4: S74-9). Wessels et al describe the effect of melantotan II (MT II) on Erectile Dysfunction (ED) human subjects, a non-selective melanocortin receptor agonist. MTII was administered to 20 psychogenic and organic ED men using a double-blind, placebo-controlled crossover design. Penis firmness was monitored for 6 hours using RigiScan. The level of libido and side effects was reported with a questionnaire. Melantotan II elicits 17 penile erections in 20 men in the absence of sexual stimulation. Subjects experienced an average Rigiscan apical stiffness of > 80% for 41 minutes. Increased libido was reported after the 13/19 (68%) dose of MTII, with 4/21 (19%) placebo (P < 0.01). Nausea and yawning are frequently reported side effects caused by MTII; at a dose of 0.025mg/kg, 12.9% of subjects had severe nausea. The observed adverse effects of MT-II may be the result of activation of MC-1R, MC-2R, MC-3R and/or MC-5R.

It is suggested that selective MCR4 agonists may be administered orally (including buccally or sublingually) and will be effective in treating female sexual dysfunction or male erectile dysfunction, but will not have significant adverse side effects such as those observed by Wessels et al, that is, the selective drug will be better tolerated.

PT-141 of Palatin is another synthetic peptide analog of α -MSH. It is a melanocortin receptor agonist and includes MC3R and MC 4R. Molinoff et al (Ann N.Y.Acad.Sci. (2003), 994, 96-102) describe how administration of "PT-141 to rats and non-human primates results in penile erection. Systemic administration of PT-141 to rats activated hypothalamic neurons, as indicated by an increase in c-Fos immunoreactivity. Neurons in the same central nervous system region harbor pseudorabies virus injected into the corpus cavernosum of rat penis. Administration of PT-141 to normal males and patients with erectile dysfunction (intranasal or subcutaneous) resulted in a rapid, dose-dependent increase in erectile activity. "

U.S.5,576,290, U.S.6,579,968 and U.S.2002/0107,182a1 describe the use of PT-141 for sexual dysfunction. In addition, peptides such as MT-II or PT-141 are widely metabolized in the intestine, and thus parenteral administration is most effective, for example, by subcutaneous, intravenous, intranasal, or intramuscular routes, since when administered by the oral route, it is not absorbed into the systemic circulation.

Thus, there would be a need to develop MCR4 agonist compounds for the treatment of male and female sexual dysfunction, suitable for oral delivery (including buccal or sublingual administration), that reduce or overcome adverse side effects, such as nausea.

It is proposed herein that selective MCR4 agonists according to the invention will exhibit oral bioavailability and will therefore be capable of being otherwise administered orally (including buccally or sublingually).

Numerous reports have been made describing selective MCR4 agonists increasing erectile activity in rats (Martinet al, 2002, Eur J Pharmacol., 454(1), 71-79; Van Der Ployeget al, 2002, Proc. Natl. Acad. Sci. USA., 99(17), 11381-11386). Examples of MCR4 agonists that have been used in these studies are N- [ (3R) -1, 2, 3, 4-tetrahydroisoquinolinium-3-ylcarbonyl ] - (1R) -1- (4-chlorobenzyl) -2- [ 4-cyclohexyl-4- (1H-1, 2, 4-triazol-1-ylmethyl) piperidin-1-yl ] -2-oxoethylamine (1), a potent selective melanocortin subtype-4 receptor agonist (Sebhat et al, 2002, J.Med.chem., 45(21), 4589-4593).

Cragnolin et al (neuropeps, 34(3-4), 211-5) have indicated that α -MSH significantly increases lordotic behavior in female rats after injection into the ventral medial nucleus of the brain. Furthermore, they indicated that HS014 (a recognized MCR4 antagonist, Vergoni 1998, eur.j. pharmacol.362(2-3), 95-101) dose-dependently blocked the pro-active effect of α -MSH on lordosis in female rats. U.S.6,051,555 has disclosed the use of various methods of stimulating sexual responses in females to melanopeptides (similar to MTII).

Essentially, MCR4 is the initiator of male and female sexual behavior. The present invention therefore provides the use of a compound of formula (I) for the preparation of a medicament for the treatment of male and female sexual dysfunction, in particular male erectile dysfunction.

Mild to severe MED patients should benefit from treatment with a compound according to the invention. However, early studies suggest that the response rates of mild, moderate and severe MED patients may be greater in the combination of selective MCR4 agonist/PDE 5 inhibitor. Mild, moderate and severe MED will be terms known to those skilled in The art, although guidance can be found in The Journal of Urology, vol.151, 54-61(Jan 1994).

Early studies suggested that the group of MED patients mentioned below should benefit from treatment with a selective MCR4 agonist and/or a PDE5 inhibitor (or other combination as described below). These patient groups are described in more detail in Clinical android vol.23, No.4, p773-782 and the book "Erectile dye-Current Inyestion and Management" of I.Eardley and K.Sethia, chapter 3 of published by Mosby-Wolfe, as follows: psychogenic, organic, vascular, endocrine, neurological, arterial, drug-induced sexual dysfunction (lactogenic) and sexual dysfunction associated with cavernous factors, particularly venous causes.

The present invention therefore provides the use of a compound of formula (I) in combination with a PDE5 inhibitor for the manufacture of a medicament for the treatment of male erectile dysfunction.

Suitable PDE5 inhibitors are described below.

Female Sexual Dysfunction (FSD)

The compounds of the invention are useful in the treatment of Female Sexual Dysfunction (FSD), in particular FSAD.

According to the present invention, FSD can be defined as a satisfaction in sexual expression that is difficult or undetectable for women. FSD is a collective term for several different Female sexual disorders (Leiblum, S.R. (1998) -Definition and Classification of Female sexal disorders. Int. J.Impower Res., 10, S104-S106; Berman, J.R., Berman, L. & Goldstein, I. (1999) -Female sexal dynamics: index, pathology, evaluation and treatment options. Urology, 54, 385- & 391). Women may have hypoactive libido, arousal or orgasm difficulties, dyspareunia, or a combination of these problems. Several types of disease, medications, injuries or psychological problems can lead to FSD. Treatments in progress are targeted at treating specific FSD subtypes, primarily sexual desire and arousal disorders.

The FSD is preferably classified by the stage of sexual response with normal women: sexual desire, arousal, and orgasm contrast (leiplus, s.r. (1998) -Definition and classification of functional sextual disorders. int.j. inpotenceres, 10, S104-S106). Sexual desire or sexual impulse is the driving force for sexual expression. Its manifestation often includes sexual thoughts when a companion of interest accompanies or when exposed to other sexual stimuli. Arousal is a vascular response to sexual stimulation, an important component of which is genital engorgement, including increased vaginal lubricity, vaginal elongation, and increased genital sensation/sensitivity. Orgasm is the release of sexual tension that has reached orgasm during arousal.

Thus, FSD occurs in women at any of these stages, usually with inadequate or unsatisfactory response in sexual desire, arousal, or orgasm. The FSD classification includes hypoactive sexual desire disorder, sexual arousal disorder, orgasmic disorder and dyspareunia. While the compounds of the invention will improve the genital response to sexual stimulation (as in female sexual arousal disorder), at the same time it may also improve the pain, tension and discomfort associated with sexual intercourse, as well as treat other female sexual disorders.

Hyposexuality exists if the woman has no or little desire for sex, and no or little thoughts or fantasy for sex. This type of FSD may result from low testosterone levels, either due to natural menopause or surgical menopause. Other causes include disease, medication, fatigue, depression, and anxiety.

Female Sexual Arousal Disorder (FSAD) is characterized by an inadequate response of the genitals to sexual stimuli. The genitals do not experience congestion typical of normal sexual arousal. The lubricity of the vaginal wall is so poor that sexual intercourse is painful. Orgasm may also be hindered. Arousal disorders may result from estrogen deprivation during menopause or post partum and lactation, as well as from diseases with vascular components, such as diabetes and atherosclerosis. Other causes include treatment with diuretics, antihistamines, antidepressants (e.g., Selective Serotonin Reuptake Inhibitors (SSRIs)), or antihypertensive agents.

Sexual pain disorders (including dyspareunia and vaginismus) are characterized by pain from insertion, which may result from medications that reduce lubricity, endometriosis, pelvic inflammatory disease, inflammatory bowel disease or urinary tract problems.

As discussed previously, MCR4 is considered to be an initiator of sexual behavior. The clitoris are considered to be homologous to the penis (Levin, R.J (1991), exp. clin. endocrinol., 98, 61-69); the same mechanism provides an erectile response in men and an increase in genital blood flow in women with a related effect on FSD. In addition, there is a change in perception and sensitivity (lordosis).

Thus, according to a preferred aspect of the present invention there is provided the use of a compound of formula (I) in the manufacture of a medicament for the treatment or prevention of female sexual dysfunction, more particularly hypoactive sexual desire disorder, sexual arousal disorder, orgasmic disorder and dyspareunia.

Preferably, the compounds of formula (I) are useful for the treatment or prevention of sexual arousal disorders, orgasmic disorders and hypoactive sexual desire disorders, most preferably for the treatment or prevention of sexual arousal disorders.

In a preferred embodiment, the compounds of formula (I) are useful for treating female sexual arousal disorder with hypoactive sexual desire disorder.

The American psychiatric Association's Manual of Diagnostics and Statistics (DSM) IV defines Female Sexual Arousal Disorder (FSAD) as:

"… … continued or repeated failed to achieve or maintain a sufficient lubrication-swelling response to sexual arousal until sexual activity was complete. This disorder must result in significant pain or dyspareunia … … ".

Evoked responses consist of vascular engorgement of the pelvis, vaginal lubrication and distension, and swelling of the external genitalia. This disorder results in significant pain and/or dyspareunia.

FSAD is a very common sexual disorder affecting women during premenopausal, menopausal, and postmenopausal (+ Hormone Replacement Therapy (HRT)). It is associated with concomitant disorders such as depression, cardiovascular disease, diabetes and genitourinary (UG) disorders.

The primary consequences of FSAD are a lack of congestion/swelling, lack of lubrication, and lack of genital pleasure. Secondary consequences of FSAD are decreased libido, pain during intercourse, and difficulty reaching orgasm.

Recent hypothesis suggests that at least a portion of patients with FSAD symptoms present a vascular basis (Goldstein et al, int.j. impot. res., 10, S84-S90, 1998), and animal data support this view (Park et al, int.j. impot. res., 9, 27-37, 1997).

Levin teaches us that since "… … male and female genitalia develop embryologically from a common tissue primordium, homology of male and female genital structures to each other is controversial. Thus, the clitoris is a homolog of the penis, and the labia are homologous to the scrotum … … "(Levin, R.J. (1991), exp.

Drug candidates for FSAD that are under study are mainly erectile dysfunction therapy, promoting blood circulation in the male genitalia.

The compounds of the present invention are useful in providing a means to restore normal sexual arousal response-i.e., increased genital blood flow, resulting in vaginal, clitoral and labial engorgement. This will result in increased vaginal lubricity via protoplasmic exudation, increased vaginal compliance and increased genital sensitivity. Thus, the present invention provides a means to restore or enhance normal sexual arousal responses.

Thus, according to a preferred aspect of the present invention there is provided the use of a compound of formula (I) in the manufacture of a medicament for the treatment or prevention of female sexual arousal disorder.

Female genitalia herein means: the "reproductive organs consist of an inner and an outer part. Internal organs are located within the pelvis, consisting of the ovaries, uterine tube, uterus and vagina. The external organs are located outside the urogenital diaphragm and below the pelvic arch. They comprise the mons pubis, labia majora and labia minora, the clitoris, the vestibule, the bulbar vestibule and the large vestibular gland "(Gray's Anatomy, c.d. clemente, 13) ^th American Edition)。

The compounds of the invention were applied to the following subgroups of FSD patients: young, elderly, pre-menopausal, post-menopausal women, may or may not receive hormone replacement therapy.

The compounds of the invention are useful in FSD patients for the following reasons:

i) vascular etiologies such as cardiovascular or atherosclerotic diseases, hypercholesterolemia, smoking, diabetes, hypertension, radiation, perineal trauma, traumatic injury to the hypoiliac, ventral, pudendal vasculature;

ii) neurological etiologies such as spinal cord injury or central nervous system disease including multiple sclerosis, diabetes, parkinsonism, cerebrovascular accident, peripheral neuropathy, trauma or radical pelvic surgery;

iii) hormonal/endocrine etiologies such as dysfunction of the hypothalamic/pituitary/gonadal axis, dysfunction of the ovary, dysfunction of the pancreas, surgical or pharmaceutical castration, androgen deficiency, high circulating levels of prolactin (e.g., hyperprolactinemia), natural menopause, premature ovarian failure, hyperthyroidism and hypothyroidism;

iv) psychiatric etiology such as depression, obsessive-compulsive disorder, anxiety, postpartum depression/"baby depression", emotional and relational diarrhoea, behavioral anxiety, marital discordance, dysfunctional mood, sexual phobia, religious contraindications or traumatic past experience; and/or

v) drug-induced sexual dysfunction, caused by Selective Serotonin Reuptake Inhibitors (SSRIs) and other antidepressant therapies (tricyclic and primary sedative tranquilizers), antihypertensive therapies, sympatholytic drugs, long-term oral contraceptive pill therapies.

The compounds of the invention may be delivered in combination with auxiliary active ingredients to treat sexual dysfunction, obesity or diabetes. Auxiliary active ingredients suitable for use in the combinations of the invention include:

1) compounds which modulate the action of natriuretic factors, especially atrial natriuretic factor (also known as atrial natriuretic peptide), type B and type C natriuretic factors, such as neutral endopeptidase inhibitors, especially the compounds described and claimed in WO 02/02513, WO 02/03995, WO 02/079143 and EP- ｃA-1258474, especially the compound (2S) -2{ [1- {3-4 (-chlorophenyl) propyl ] amino } carbonyl) -cyclopentyl ] methyl } -4-methoxybutanoic acid of example 22 of WO 02/079143;

2) angiotensin converting enzyme inhibiting compounds, such as enalapril, and angiotensin converting enzyme in combination with neutral endopeptidase inhibitors, such as omapatrilat;

3) NO-synthetase substrates, such as L-arginine;

4) cholesterol lowering agents, such as statins (e.g., atorvastatin/Lipitor-trademark) and fibrates;

5) Estrogen receptor modulators and/or estrogen agonists and/or estrogen antagonists, preferably raloxifene or lasofoxifene, (-) -cis-6-phenyl-5- [4- (2-pyrrolidin-1-yl-ethoxy) -phenyl ] -5, 6, 7, 8-tetrahydronaphthalen-2-ol and pharmaceutically acceptable salts thereof, the preparation of which is described in detail in WO 96/21656;

6) PDE inhibitors, more precisely PDE2, 3, 4, 5, 7 or 8 inhibitors, preferably PDE2 or PDE5 inhibitors, most preferably PDE5 inhibitors (see below), said inhibitors preferably having an IC of less than 100nM for each enzyme₅₀(provided that the PDE3 and 4 inhibitors are administered only topically or by injection into the penis);

7) vasoactive Intestinal Protein (VIP), VIP mimetics, VIP analogues, are more specifically mediated by: one or more VIP receptor subtypes VPAC1, VPAC or PACAP (pituitary adenylate cyclase activating peptide), one or more VIP receptor agonists or VIP analogs (e.g., Ro-125-1553) or VIP fragments, one or more alpha-adrenoreceptor antagonists in combination with VIP (e.g., Invicorp, Aviptadil);

8) serotonin receptor agonists, antagonists or modulators, more specifically agonists, antagonists or modulators of the 5HT1A (including VML 670[ WO 02/074288] and flibanserin [ US2003/0104980]), 5HT2A, 5HT2C, 5HT3 and/or 5HT6 receptors, including those described in WO-09902159, WO-00002550 and/or WO-00028993;

9) Testosterone substitutes (including dehydroandrostenedione), testosterone (e.g., tosrelle, LibiGel), dihydrotestosterone, or testosterone implants;

10) selective androgen receptor modulators, such as LGD-2226;

11) estrogens, estrogens with medroxyprogesterone or medroxyprogesterone acetate (MPA) (i.e., as a combination) or estrogens with methyltestosterone hormone replacement therapies (e.g., HRT, especially Premarin, Cenestin, oestrofemal, Equin, Estrace, Estrofem, eleste Solo, Estring, eastaderm TTS, eastaderm Matrix, Dermestril, primrose, Preempro, Prempak, premix, Estratest HS, Tibolone);

12) modulators of norepinephrine, dopamine, and/or serotonin transporters, such as bupropion, GW-320659;

13) an oxytocin/vasopressin receptor agonist or modulator, preferably a selective oxytocin agonist or modulator; and

14) dopamine receptor agonists or modulators, preferably D3 or D4 selective agonists or modulators, such as apomorphine.

Preferred herein are combinations of a compound of the invention with one or more other therapeutic agents selected from the group consisting of: a PDE5 inhibitor; an NEP inhibitor; a D3 or D4 selective agonist or modulator; an estrogen receptor modulator and/or an estrogen agonist and/or an estrogen antagonist; a testosterone replacement agent, testosterone or a testosterone implant; estrogen, estrogen and medroxyprogesterone or medroxyprogesterone acetate (MPA) or estrogen and methyltestosterone hormone replacement therapy.

A preferred therapeutic combination of MED is a combination of a compound of the invention and one or more PDE5 inhibitors and/or NEP inhibitors.

A preferred therapeutic combination of FSD is a combination of a compound of the invention with: PDE5 inhibitors, and/or NEP inhibitors, and/or D3 or D4 selective agonists or modulators, and/or estrogen receptor modulators, estrogen agonists, estrogen antagonists, and/or testosterone substitutes, testosterone implants, and/or estrogen, estrogen and medroxyprogesterone or medroxyprogesterone acetate (MPA), estrogen and methyltestosterone hormone replacement therapeutics.

Particularly preferred PDE5 inhibitors for use in such combination MED or FSD treatment products are sildenafil, tadalafil, vardenafil and 5- [ 2-ethoxy-5- (4-ethylpiperazin-1-ylsulfonyl) pyridin-3-yl ] -3-ethyl-2- [ 2-methoxyethyl ] -2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one.

NEP inhibitors which are particularly preferred for use in such combination MED or FSD therapeutic products are exemplified by WO 02/079143.

Preferred combination MED or FSD therapeutic products herein are: sildenafil, tadalafil, vardenafil or 5- [ 2-ethoxy-5- (4-ethylpiperazin-1-ylsulfonyl) pyridin-3-yl ] -3-ethyl-2- [ 2-methoxyethyl ] -2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one in combination with the compound of example 1 herein; and/or combinations of any of the compounds exemplified in WO02/079143 with the compounds of example 1 herein.

By cross-reference herein to compounds contained in patents and patent applications which can be used in accordance with the present invention, therapeutically active compounds are defined as set forth in the claims (especially claim 1) and the specific examples (both incorporated herein by reference).

If a combination of active ingredients is administered, they may be administered simultaneously, separately or sequentially.

Auxiliary ingredient PDE5 inhibitor

Particularly preferred as co-active ingredients herein are PDE5 inhibitors.

The suitability of any particular cGMP PDE5 inhibitor can be readily determined by evaluating its potency and selectivity using literature procedures, followed by evaluating its toxicity, absorption, metabolism, pharmacokinetics, and the like in accordance with standard pharmaceutical practice.

IC of cGMP PDE5 inhibitors can be determined using the PDE5 assay₅₀Values (see below).

Preferably, the cGMP PDE5 inhibitor used in the pharmaceutical combination according to the invention is PDE5 enzyme selective. Preferably (when used orally) they are more selective relative to PDE3, more preferably relative to PDE3 and PDE 4. Preferably (when administered orally) the cGMP PDE5 inhibitor of the invention has a selectivity ratio over PDE3, more preferably over PDE3 and PDE4 of more than 100, more preferably more than 300.

The selectivity ratio can be easily determined by the skilled person. IC of PDE3 and PDE4 enzymes can be determined using established literature methods₅₀See, for example, S A Ballard et al, Journal of Urology, 1998, vol.159, pages 2164-.

cGMP PDE 5 inhibitors suitable for use in the present invention include:

(i)5- [ 2-ethoxy-5- (4-methyl-1-piperazinylsulfonyl) phenyl ] -1-methyl-3-n-propyl-1, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (sildenafil), also known as 1- [ [3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4, 3-d ] pyrimidin-5-yl) -4-ethoxyphenyl ] sulfonyl ] -4-methylpiperazine (see EP-A-0463756);

(ii)5- (2-ethoxy-5-morpholinoacetylphenyl) -1-methyl-3-n-propyl-1, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (see EP-A-0526004);

(iii) 3-ethyl-5- [5- (4-ethylpiperazin-1-ylsulfonyl) -2-n-propoxyphenyl ] -2- (pyridin-2-yl) methyl-2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (see WO 98/49166);

(iv) 3-ethyl-5- [5- (4-ethylpiperazin-1-ylsulfonyl) -2- (2-methoxyethoxy) pyridin-3-yl ] -2- (pyridin-2-yl) methyl-2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (see WO 99/54333);

(v) (+) -3-ethyl-5- [5- (4-ethylpiperazin-1-ylsulfonyl) -2- (2-methoxy-1 (R) -methylethoxy) pyridin-3-yl ] -2-methyl-2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one, also known as 3-ethyl-5- {5- [ 4-ethylpiperazin-1-ylsulfonyl ] -2- ([ (1R) -2-methoxy-1-methylethyl ] oxy) pyridin-3-yl } -2-methyl-2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (see WO 99/54333);

(vi)5- [ 2-ethoxy-5- (4-ethylpiperazin-1-ylsulfonyl) pyridin-3-yl ] -3-ethyl-2- [ 2-methoxyethyl ] -2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one, also known as 1- { 6-ethoxy-5- [ 3-ethyl-6, 7-dihydro-2- (2-methoxyethyl) -7-oxo-2H-pyrazolo [4, 3-d ] pyrimidin-5-yl ] -3-pyridylsulfonyl } -4-ethylpiperazine (see WO01/27113, example 8);

(vii)5- [ 2-isobutoxy-5- (4-ethylpiperazin-1-ylsulfonyl) pyridin-3-yl ] -3-ethyl-2- (1-methylpiperidin-4-yl) -2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (see WO01/27113, example 15);

(viii)5- [ 2-ethoxy-5- (4-ethylpiperazin-1-ylsulfonyl) pyridin-3-yl ] -3-ethyl-2-phenyl-2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (see WO01/27113, example 66);

(ix)5- (5-acetyl-2-propoxy-3-pyridyl) -3-ethyl-2- (1-isopropyl-3-azetidinyl) -2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (see WO01/27112, example 124);

(x)5- (5-acetyl-2-butoxy-3-pyridyl) -3-ethyl-2- (1-ethyl-3-azetidinyl) -2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one (see WO01/27112, example 132);

(xi) (6R, 12aR) -2, 3, 6, 7, 12, 12 a-hexahydro-2-methyl-6- (3, 4-methylenedioxyphenyl) -pyrazino [2 ', 1': 6,1]Pyrido [3, 4-b]Indole-1, 4-dione (tadalafil IC-351, Cialis)^®) Namely the compounds of examples 78 and 95 of published international application WO 95/19978, and the compounds of examples 1, 3, 7 and 8;

(xii)2- [ 2-ethoxy-5- (4-ethyl-piperazin-1-yl-1-sulfonyl) -phenyl ] -5-methyl-7-propyl-3H-imidazo [5, 1-f ] [1, 2, 4] triazin-4-one (vardenafil), also known as 1- [ [3- (3, 4-dihydro-5-methyl-4-oxo-7-propylimidazo [5, 1-f ] -as-triazin-2-yl) -4-ethoxyphenyl ] sulfonyl ] -4-ethylpiperazine, is the published international application WO 99/24433, examples 20, 19, 337 and 336 compound;

(xiii) Pyrazolo [4, 3-d ] pyrimidin-4-ones as disclosed in WO 00/27848, in particular N- [ [3- (4, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4, 3-d ] pyrimidin-5-yl) -4-propoxyphenyl ] sulfonyl ] -1-methyl-2-pyrrolidinopropionamide [ DA-8159 (example 68 of WO 00/27848) ];

(xiv) The compound of example 11 of published international application WO 93/07124;

(xv)4- (4-chlorobenzyl) amino-6, 7, 8-trimethoxy quinazoline; and

(xvi)7, 8-dihydro-8-oxo-6- [ 2-propoxyphenyl ] -1H-imidazo [4, 5-g ] quinazoline;

(xvii)1- [3- [1- [ (4-fluorophenyl) methyl ] -7, 8-dihydro-8-oxo-1H-imidazo [4, 5-g ] quinazolin-6-yl ] -4-propoxyphenyl ] carboxamide;

(xviii)5- (5-acetyl-2-butoxy-3-pyridinyl) -3-ethyl-2- (1-ethyl-3-azetidinyl) -2, 6-dihydro-7H-pyrazolo [4, 3-d ] pyrimidin-7-one; and

(xix)1- { 6-ethoxy-5- [ 3-ethyl-6, 7-dihydro-2- (2-methoxyethyl) -7-oxo-2H-pyrazolo [4, 3-d ] pyrimidin-5-yl ] -3-pyridylsulfonyl } -4-ethylpiperazine; and pharmaceutically acceptable salts and solvates thereof.

The suitability of any particular PDE5 inhibitor can be readily determined by evaluating its potency and selectivity using literature procedures, followed by evaluating its toxicity, absorption, metabolism, pharmacokinetics, and the like in accordance with standard pharmaceutical practice.

Preferably, the PDE5 inhibitor has an IC of less than 100 nanomolar, more preferably less than 50 nanomolar, and even more preferably less than 10 nanomolar₅₀。

Preferably, the PDE5 inhibitor used in the pharmaceutical combination according to the invention is PDE5 enzyme selective. Preferably, they have a selectivity for PDE5 of greater than 100, more preferably greater than 300, relative to PDE 3. More preferably, the PDE5 inhibitor has a selectivity over PDE3 and PDE4 of greater than 100, more preferably greater than 300. The skilled person can easily derive the relevant IC from ₅₀The value determines the selectivity ratio. IC of PDE3 and PDE4 enzymes can be determined using established literature methods₅₀Values such as those described in SA Ballard et al, Journal of Urology, 1998, vol.159, pages 2164-. The IC of the PDE5 enzyme can be determined using established literature methods and as described in WO01/27113₅₀The value is obtained.

In vivo prokinetic data

The MCR4 in vivo data for the compound of example 1 was evaluated by selective activation of the melanocortin MCR4 receptor using a method to assess spontaneous penile erection in conscious rats.

Intracavernosal pressure was measured using a surgically implanted telemetry device (TA11PA-C40, 8mm catheter, modified 3mm tip, Data Sciences International Inc.) and erectile responses recorded. Intracavernosal pressure increase is an indicator of penile erection, as intracavernosal pressure increase is a necessary hemodynamic event during initiation and maintenance of penile erection. Specific details of the procedures, data acquisition and analysis used herein to determine the increase in intracavernosal pressure can be found in Bernabe j, Rampin o, Sachs b.d., girliano f, "Intracavernous pressure reduction in rates: integral adaptive base on telematics recording ", am.j. physical.1999, Feb; 276(2 Pt 2): r441-9.

The test animals (rats) (during the dark cycle) were habituated for 18 hours prior to baseline assessment of erectile function. Baseline erectile activity (B) in terms of vehicle was assessed for 10 minutes prior to administration of the agent using telemetry recording of intracavernosal pressure. Following subcutaneous administration of the compound of example 1 (in the same vehicle), penile erection was assessed (using telemetry recording of intracavernosal pressure) for 10 minutes, with intervals of 30, 60 and 90 minutes following administration.

The compound of example 1 dose-dependently increased penile erection times when administered subcutaneously (s.c.) at levels of 1-100 μ g/kg (see figures 1 and 2). Baseline/vehicle treated animals showed minimal erectile activity (see figure 1).

Figure 1 illustrates the results of a preliminary study comparing the number of erections observed within 10 minutes after administration with 1, 10 and 100 μ g/kg of the compound of example 1 s.c. animals dosed with the compound starting from 60 minutes after dosing with baseline erectile activity (B). The data in figure 1 show that the compound of example 1 increased erectile activity relative to baseline erectile activity (B) at all doses tested. Figure 1 further shows that the compound of example 1 dose-dependently increases the number of spontaneous erection in conscious rats. The maximum effective dose observed in this preliminary study was 1 μ g/kg s.c.

Figure 2 illustrates the results of another more detailed study comparing the number of erections observed within 10 minutes after administration to animals dosed with 1, 10 and 100 μ g/kg of the compound of example 1 s.c. to baseline erectile activity (vehicle treatment) starting at 30 minutes after administration. Figure 2 data shows that the compound of example 1 increased erectile activity relative to baseline erectile activity (vehicle treatment) at all doses tested. Figure 2 further shows that the compound of example 1 dose-dependently increases the number of spontaneous erection in conscious rats. The maximum effective dose observed in this other, more detailed study was 10 μ g/kg s.c.

For the compound of example 1, the maximum effective dose observed in this preliminary study was 1 μ g/kg s.c., and the maximum effective dose observed in another, more detailed study was 10 μ g/kg s.c. The number of erections observed did not differ significantly between the two studies and at these dose levels. From both studies the same conclusion can be drawn independently, that the compound of example 1 dose-dependently increases the number of spontaneous erection in conscious rats. It is proposed here that the observed differences between preliminary and more detailed studies with respect to the maximum effective dose observed are a reflection of the expected biological differences associated with this type of animal model.

The data presented in figures 1 and 2 strongly suggest that MCR4 receptors are involved in the induction and maintenance of penile erection, where it is proposed that selective MCR4 agonists according to the invention may provide opportunities for the treatment of male erectile dysfunction.

Assay method

Stimulation of adenylate cyclase following receptor activation is a commonly used assay for the functional activity of a number of receptor systems. Functional assays for the determination of cyclic amp (camp) employ Human Embryonic Kidney (HEK) cells that stably express the human melanocortin MCR1, MCR3, or MCR4 receptors. Activation of MCR1, MCR3, or MCR4 receptors stimulates adenylate cyclase to produce cAMP using AlphaScreen^TM(PerkinElmer) assay kit.

AlphaScreen^TMThe cAMP assay kit consists of a "donor bead"),"acceptor beads" and biotinylated cAMP linking different beads together. In Fusion^TMExcitation of this attached complex at 680nM in an α microplate analyzer results in light emission between 520 and 620 nM.

cAMP generated in the assay competes with biotinylated cAMP for binding sites on the acceptor beads, preventing the attachment of the "donor" to the "acceptor" beads, thereby reducing light emission.

(i) MCR1, MCR3 and MCR4 Standard functional assays (protocols A, B and C, respectively)

Concept of determination

The activity of the compounds according to the invention on the human MCR1, MCR3 and MCR4 receptor subtypes was determined using three immortalized Human Embryonic Kidney (HEK) cell lines that were biologically processed to express the human melanocortin MCR1, MCR3 or MCR4 receptor subtypes. These cell lines were processed using a similar protocol as described by Gouarders et al (Gouarders, C., (2002) Neuroscience, 115 (2); 349-361).

Activation of these MCR1, MCR3, or MCR4 receptors induced by such compounds leads to stimulation of cellular enzymes adenylate cyclase, which in turn leads to cellular production and intracellular accumulation of cyclic adenosine monophosphate (cAMP). The magnitude of these intracellular cAMP increases was found to be proportional to the extent to which the test compound activates the MCR1, MCR3, or MCR4 receptors present in these cell lines. Using AlphaScreen commercially available from Perkinelmer^TMThe assay kit quantifies intracellular cAMP levels. Detailed protocol and an explanation of the concept of such a kit are available from the PerkinElmer website: (www.perkinelmer.com). The following scheme summarizes this information.

Using Fusion^TMThe α microplate analyzer measures the amount of intracellular cAMP produced by compound-induced activation of MCR1, MCR3 and MCR4 receptors in these three cell lines, the instrument being set to stimulate at 680nM wavelength and measure the emitted energy at 520 nM wavelength. The compound-induced increase in activation of the MCR1, MCR3, or MCR4 receptors was subsequently quantified as Reduction of the amount of light emitted at the wavelengths 520 nM and 620 nM. Data analysis was then performed using a curve-fitting program to extrapolate the apparent potency (in EC) of the test compound from the fitted curve₅₀Expressed as the concentration of effective compound that causes 50% of the maximum compound-induced response).

Material

From PerkinElmer: AlphaScreen^TMcAMP assay kit, Cat N ° 6760600M, Fusion^TMα microplate analyzer (stimulated at 680nM wavelength, recording light emitted at 520-620nM wavelength).

From Invitrogen: phosphate Buffered Saline (PBS) (w/o Ca²⁺And Mg²⁺) CatN ° 14190-; dulbecco's Modified Eagle Medium (DMEM) high glucose, Cat N.cndot.21969-; hank's Balanced Salt Solution (HBSS), Cat N14065-; geneticin, Cat N ° 10131-.

From Sigma: bovine Serum Albumin (BSA), Cat N ° a 7030; l-glutamine, Cat N ° G7513; n- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), Cat N ° H0887; cell dissociation solution, Cat N ° C5914; dimethyl sulfoxide (DMSO), Cat N ° D8418; cyclic adenosine monophosphate (cAMP), Cat N ° a 9501; 3-isobutyl-1-methylxanthine (IBMX), Cat N ° I5879; magnesium chloride (MgCl)₂)1M solution, Cat N ° M1028; trypan blue, Cat N ° T-8154, cell counting chamber (Bright-line 35, 962-9).

From PAA laboratories GmbH: fetal Calf Serum (FCS), Cat N ° A15-043.

From Gilson: 10 μ l to 1000 μ l pipettes.

From Hereaus: hera Cell CO₂A cell incubator.

From Medical Air Technology: BioMat²A class II microbial safety cabinet.

From Bachem: alpha-melanocyte stimulating hormone alpha-MSH, Cat N ° H1075, was used as a positive control.

Buffer solution

Stimulation buffer (according to AlphaScreen^TMScheme): HBSS supplemented with 0.5mM IBMX, 5mM HEPES, 0.1% (w/v) BSA and 10mM MgCl₂。

Lysis buffer (according to AlphaScreen^TMScheme): 5mM HEPES solution, supplemented with 0.1% (w/v) BSA and 0.3% (v/v) Tween-20.

Detection mixture (according to AlphaScreen^TMScheme): lysis buffer supplemented with biotinylated cAMP (10nM) and donor beads (10. mu.g/ml), as in AlphaScreen^TMSupplied in cAMP assay kit.

Consumable product

From Fisher: non-binding surface 384-well assay plate, Cat N ° DPS-172-.

From Costar: a sterile pipette of 2 to 50ml capacity, P10 up to the sterile tip of P1000; a sterile reservoir, Cat No. 4878; t225 vented cap, Cat No. 3001.

Compound preparation

For the standard functional assays of MCR1, MCR3, and MCR4, compounds were first dissolved in DMSO to give a compound concentration of 4mM, and then further diluted in stimulation buffer for assay to give an actual concentration 2-fold greater than the desired final assay concentration.

Routine cell culture

Three HEK cell lines expressing the human MCR1, MCR3 or MCR4 receptor subtypes as described above were grown in T225 vented cap flasks containing 50ml of growth medium (DMEM supplemented with 10% (v/v) FCS, 2mM L-glutamine, 25mM HEPES and 1.0mg/ml Geneticin), maintained at 37 ℃ and containing 5% CO₂In a cell incubator in the environment of (a). When 80-90% confluence was reached, the cells were harvested by first removing the existing growth medium and then washing with PBS which had been previously warmed to 37 ℃. The PBS was then removed and 5ml of cell dissociation solution was added to the flask. The cells were incubated at 37 ℃ and incubatedWith 5% CO₂The cells were detached by incubating in the cell incubator for 5 minutes. The flask was tapped sharply to dislodge the cells from the bottom of the flask. When the cells detached, growth medium previously warmed to 37 ℃ was added, and the cells were resuspended and gently mixed by pipetting to achieve a single cell suspension. The cell suspension is then counted using a cell counting chamber for experimentation or transferred to a new T225 flask to preserve the cell culture.

Determination process

The assay procedure used was essentially as AlphaScreen^TMKit methods of (A)www.perkinelmer.com) However, to facilitate liquid handling, all assay volumes were doubled.

First, 10 μ l of test compound solution was transferred to a non-binding surface 384 well assay plate.

Next, the assay cells were harvested as described above.

(i) For the MCR1 standard functional assay, cells were prepared in stimulation buffer (supplemented with 10. mu.l/ml anti-cAMP receptor bead solution in AlphaScreen^TMAvailable in cAMP assay kit) 3 × 10⁵Cells/ml suspension;

(ii) for the MCR3 standard functional assay, cells were prepared in stimulation buffer (supplemented with 10. mu.l/ml anti-cAMP receptor bead solution in AlphaScreen^TMAvailable in cAMP assay kit) 5 × 10⁴Cells/ml suspension;

(iii) for the MCR4 standard functional assay, cells were prepared in stimulation buffer (supplemented with 10. mu.l/ml anti-cAMP receptor bead solution in AlphaScreen^TMAvailable in cAMP assay kit) 1X 10⁵Cells/ml suspension.

Subsequently, 10 μ l of the cell suspension was transferred to each well of a non-binding surface 384-well assay plate. The assay plates were then incubated for 30 minutes at room temperature in the dark.

Third, addThe assay reaction was terminated by adding 30. mu.l/well of assay mixture. Plates were incubated overnight at room temperature in the dark and then transferred to Fusion^TMα microplate analyzer for quantification.

(ii) MCR5 Standard functional assay and MCR4 modified functional assay (protocols D and E, respectively)

Concept of determination

The activity of the compounds on the human MCR5 receptor subtype was determined using an immortalized Chinese hamster ovary cell line (CHO-K1) that was processed to stably express the recombinant human MCR5 receptor and a beta-lactamase reporter gene (CHO-K1-MC 5R-CRE-beta-lactamase). Similarly, the activity of the compounds on the human MCR4 receptor subtype was determined using an improved assay using an immortalized CHO-K1 cell line engineered to stably express the recombinant human MCR4 receptor and a beta-lactamase reporter gene (CHO-K1-MC 4R-CRE-beta-lactamase). These cell lines were processed using a similar protocol as described by Zaccolo et al (Zaccolo, M., (2000) Nature, 2 (1): 25-29).

Compound-induced activation of MCR5 or MCR4 receptors in both cell lines stimulates β -lactamase production and intracellular accumulation. The amount of beta-lactamase produced was directly proportional to the extent to which the test compound activated the MCR5 or MCR4 receptor present on these cells, quantified using a beta-lactamase reporter gene assay kit commercially available from Invitrogen Life Technologies. A deep description of this technique and protocol is available from the Invitrogen website ((R)) www.invitrogen.com). The following scheme summarizes this assay.

Using Lj1 Biosystems analysis^TMHT 96.384 plate reader quantified the amount of β -lactamase produced by compound-induced activation of MCR5 or MCR4 receptors expressed in these cell lines, the instrument being set to excite at 405nm and measure the emitted energy at 450nm and 530 nm. The measured energy emitted at 450nm wavelength was divided by the measured energy emitted at 530nm wavelength to quantify the cellular response. Subsequently using curve-fittingData analysis was performed in a closed procedure, and the apparent potency (in EC) of the test compound was extrapolated from the fitted curve₅₀Expressed as the concentration of effective compound that causes 50% of the maximum compound-induced response).

Material

From Invitrogen: dulbecco's Modified Eagle Medium (DMEM) containing Glutamax-1, Cat N.cndot.32430-; non-essential amino acids, Cat N ° 1140-0.35; geneticin (G418), Cat N ° 10131-; cell dissociation buffers (enzyme-free PBS), Cat N ° 13151-014; phosphate Buffered Saline (PBS) (w/o Ca²⁺And Mg²⁺) Cat N ° 14190-; CCF4-AM, Cat N ° K1028; pluronic F127 s solution (solution B), Cat N ° K1026N; 24% PEG with 18% TR40 solution (solution C), Cat N ° K1026N; zeocin, Cat N ° R250-05.

From Sigma: fetal Calf Serum (FCS), Cat N ° F7524; sodium pyruvate, Cat N ° S8636; n- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), Cat N ° H0887; dimethyl sulfoxide (DMSO), Cat N ° D-8418; cyclohexanamides, Cat N ℃ C-7698; trypan blue solution, Cat N ° T-4424; probenecid, Cat N ° P8761; bovine Serum Albumin (BSA), Cat N ° a 2153; pluronic F-127, Cat N9003-11-6.

From Gilson: 10 μ l to 1000 μ l pipettes.

From Hereaus: hera Cell CO₂A cell incubator.

From Medical Air Technology: BioMat²A class II microbial safety cabinet.

From Ljl Biosystems: analyst^TMHT 96.384 plate reader, set to excite at 405nm, and measure the emitted energy at wavelengths of 450nm and 530 nm.

Buffer solution

CCF4-AM was dissolved in 100% DMSO to give a final solution concentration of 1 mM. This solution is referred to as solution a.

Probenecid was dissolved in 200mM NaOH to give a final solution concentration of 200 mM. This solution is referred to as solution D.

Beta-lactamase assay the composition of the stain solution: for 1072. mu.L of assay stain solution, 12. mu.L of solution A, 60. mu.L of solution B, 925. mu.L of solution C and 75. mu.L of solution D were combined.

Consumable product

From Greiner: 384 well black clear bottom microtiter plates, Cat No. 781091.

Compound preparation

For the standard functional assay of MCR5, all test compounds were first dissolved in DMSO to give a compound concentration of 4mM, and then further diluted in PBS for assay, containing 1.25% v/v DMSO and 0.1% w/v BSA, to give an actual concentration 5-fold greater than the desired final assay concentration.

For the MCR4 modified functional assay method, all test compounds were first dissolved in DMSO to give a compound concentration of 4mM, and then further diluted in PBS for assay, containing 2.5% v/v DMSO and 0.05% w/v pluronic F-127, to give an actual concentration 5-fold greater than the desired final assay concentration.

Routine cell culture

Cells were grown in T225 vented cap flasks containing 50ml of growth medium, maintained at 37 ℃ and containing 5% CO₂Ambient cell incubator. The growth medium for CHO-K1-MC 5R-CRE-beta-lactamase consisted of 90% v/v DMEM supplemented with Glutamax-1, 25mM HEPES, 10% v/v Fetal Calf Serum (FCS), 1mM sodium pyruvate, 0.1mM non-essential amino acids and 800. mu.g/ml Geneticin. In the case of CHO-K1-MC 4R-CRE-beta-lactamase In particular, this growth medium was further supplemented with 200. mu.g/ml Zeocin. When 80-90% confluence was reached, the cells were harvested by first removing the existing growth medium and then washing with PBS which had been previously warmed to 37 ℃. The PBS was then removed and 5ml of cell dissociation solution was added to the flask. Cells were incubated at 37 ℃ and 5% CO₂The cells were detached by incubation in an ambient cell incubator for 5 minutes. When the cells detached, pre-warmed growth medium was added, the cells were resuspended by pipetting and gently mixed to achieve a single cell suspension. This cell suspension was then used for experiments or transferred to new T225 flasks to preserve the cell culture.

Determination process

On the first day of assay, cells were harvested as described above. For the standard functional assay of MCR5, 3.33X 10 cells were prepared in modified growth medium containing 1% (instead of 10%) FCS⁵Cells/ml suspension, 30. mu.l of this cell suspension was added to each well of a Greiner 384-well black clear-bottomed microassay plate.

For the MCR4 improved function assay, cells were prepared 2X 10 in modified growth medium containing 5% (instead of 10%) FCS⁵Cells/ml suspension, 40. mu.l of this cell suspension was added to each well of a Greiner 384-well black clear-bottomed microassay plate.

For each assay, the cell plates were then returned to 37 ℃ and 5% CO maintenance₂The cell incubator of (1) was left overnight, and then the measurement was performed the next day of measurement.

On the next day of the assay, for the standard functional assay of MCR5, the cell plate was removed from the cell incubator and 10. mu.L of a 5. mu.M solution of cyclohexylamine (in PBS containing 5% v/v DMSO) was added to each well of the assay plate. For the MCR4 modified function assay method, no cyclohexylamide solution was added. Subsequently, 10. mu.L of the test compound solution was transferred to an assay plate. The assay plates were then transferred to 37 ℃ and contained 5% CO₂Environmental cell incubators, left for 4 hours in MCR4 modified assay method, or in MCR5The standard assay was allowed to stand for 5 hours. After this incubation period, the plates were removed from the incubator, 10 μ L of beta-lactamase assay stain solution was added to each well, and the plates were then returned to the cell incubator. After further incubation, at 60 minutes in the MCR4 modified assay or 90 minutes in the MCR5 standard assay, the plates were removed from the incubator and transferred to an Lj1 biosystems analysis^TMHT 96.384 plate reader for quantification.

Obesity

The compounds of the present invention may also be used in combination with pharmaceutical ingredients to treat diseases, conditions and/or disorders related to obesity. Accordingly, there is also provided a composition (or a pharmaceutical product) for the treatment of obesity comprising a combination of a compound of the present invention and an anti-obesity agent. Suitable anti-obesity agents include cannabinoid 1(CB-1) receptor antagonists (e.g., rimonabant), apolipoprotein-B secretion/microsomal triglyceride transfer protein (apo-B/MTP) inhibitors (particularly gut-selective MTP inhibitors such as edipatamide or dirlotamide), 11 beta-hydroxysteroid dehydrogenase-1 (11 beta-HSD type 1) inhibitors, peptide YY_3-36And analogs thereof, cholecystokinin-A (CCK-A) agonists, monoamine reuptake inhibitors (e.g. sibutramine), sympathomimetics, beta₃Adrenergic receptor agonists, dopamine receptor agonists (e.g., bromocriptine), melanocyte-stimulating hormone receptor analogs, 5HT2c receptor agonists, melanin-concentrating hormone antagonists, leptin (OB protein), leptin analogs, leptin receptor agonists, galanin antagonists, lipase inhibitors (e.g., tetrahydropancreatin, orlistat), anorectic agents (e.g., bombesin agonists), neuropeptide-Y receptor antagonists (particularly NPY-5 receptor antagonists), thyromimetics, dehydroepiandrosterone or analogs thereof, glucocorticoid receptor agonists or antagonists, orexin receptor antagonists, glucagon-like peptide-1 receptor agonists, ciliary neurotrophic factors (e.g., okaxine) ^TMAvailable from Regeneron Pharmaceuticals, Inc., Tarrytown, NY and Procter&Gamble Company, Cincinnati, OH), human agouti-related protein (AGRP) inhibitors, ghrelin receptor antagonists, histamine 3 receptor antagonistsAnti-agents or inverse agonists, neuregulin U receptor agonists, and the like. Other anti-obesity agents, including the preferred agents described below, are well known or will be apparent to those of ordinary skill in the art in view of this disclosure. The compounds of the present invention may also be administered in combination with naturally occurring compounds that lower plasma cholesterol levels. Such naturally occurring compounds are commonly referred to as nutraceuticals and include, for example, garlic extract, Hoodia plant extract, and niacin.

Particularly preferred anti-obesity agents are selected from the group consisting of CB-1 antagonists, gut-selective MTP inhibitors, orlistat, sibutramine, bromocriptine, ephedrine, leptin, peptide YY_3-36And analogs thereof, and pseudoephedrine. Preferably, the compounds of the present invention and combination therapies for the treatment of obesity and related disorders are administered in combination with exercise and a sensible diet.

Preferred CB-1 antagonists include Rimonabant (SR141716A, also known under the trademark Acomplia) as described in U.S. Pat. No.5,624,941 ^TMAvailable from Sanofi-Synthelabo); and compounds described in the following documents: U.S. Pat. Nos. 5,747,524, 6,432,984 and 6,518,264, U.S. patent publication Nos. US2004/0092520, US2004/0157839, US2004/0214855 and US2004/0214838, U.S. patent application No.10/971599 filed on 10/22.2004, and PCT patent publications No. WO 02/076949, WO03/075660, WO 04/048317, WO 04/013120 and WO 04/012671.

Preferred gut-selective MTP inhibitors include dirlotapede as described in U.S. patent No.6,720,351; 4- (4- (4- (4- ((2- ((4-methyl-4H-1, 2, 4-triazol-3-ylthio) methyl) -2- (4-chlorophenyl) -1, 3-dioxolan-4-yl) methoxy) phenyl) piperazin-1-yl) phenyl) -2-sec-butyl-2H-1, 2, 4-triazol-3 (4H) -one described in U.S. patent nos. 5,521,186 and 5,929,075 (R103757); and impritapide (BAY 13-9952) as described in U.S. Pat. No.6,265,431.

Other representative anti-obesity agents for use in the combinations, pharmaceutical compositions and methods of the invention may be prepared using methods known to those of ordinary skill in the art, for example sibutramine may be as described in U.S. patent No.4,929,629; bromocriptine can be prepared as described in U.S. patent nos. 3,752,814 and 3,752,888; orlistat can be prepared as described in U.S. patent nos. 5,274,143, 5,420,305, 5,540,917, and 5,643,874; PYY _3-36(including analogs) may be prepared as described in U.S. patent publication No.2002/0141985 and WO 03/027637.

Food intake

The following screen was used to evaluate the efficacy of test compounds to inhibit food intake in Sprague-Dawley rats after an overnight fast.

Male Sprague-Dawley rats are available from Charles River Laboratories, Inc. (Wilmington, Mass.). Rats were individually housed and fed with crushed feed. They were maintained on a 12 hour day-night cycle and were free to receive food and water. Animals were allowed to acclimate for one week prior to testing. The test was completed during the diurnal portion of the diurnal cycle.

For food efficacy screening, rats were transferred to individual test cages without food the afternoon of the day before the test and were fasted overnight. After overnight fasting, rats were given vehicle or test compound the following morning. Known antagonists (3mg/kg) were administered as positive controls, the control group receiving vehicle alone (no compound). Between 0.1 and 100mg/kg of test compound is administered, depending on the compound. The standard carrier is a 0.5% (w/v) aqueous solution of methylcellulose and the standard route of administration is oral. However, different carriers and routes of administration may be used to accommodate the various compounds as desired. The rats were fed food 30 minutes after dosing and the Oxymax automatic food intake system (Columbus Instruments, Columbus, Ohio) was started. Food intake was recorded continuously for a single rat over two hours, 10 minutes apart. Manually recording food intake with an electronic balance when needed; food was weighed every 30 minutes after food was provided until four hours after food was provided. The efficacy of the compounds was determined by comparing the food intake pattern of compound-treated rats with vehicle and standard positive controls.

Administration and dosage range

The compounds of formula (I) should be evaluated for biopharmaceutical properties such as solubility, solution stability (over a range of pH), appropriate dosage levels and permeability in order to select the dosage form and route of administration that is most suitable for the treatment of the desired indication. Preliminary biopharmaceutical evaluation has shown that some compounds according to the invention may be particularly suitable for administration via the oral route (including buccal and sublingual) or the intranasal route. For example, the sublingual or intranasal route may be suitable for the compounds of examples 1 and 5, with the sublingual route being preferred. Other compounds may be more suitable for any oral administration, such as the compound of example 9.

Thus, according to a further embodiment, the present invention provides a pharmaceutical composition formulated for sublingual delivery comprising a compound of general formula (I) as defined above, preferably the compounds of examples 1 and 5.

The compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products. They can be obtained, for example, as solid plugs, powders or films, by methods such as precipitation, crystallization, freeze drying, spray drying or evaporation drying. Microwave or radio frequency drying may be used for this purpose.

They may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs (or as any combination thereof). Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any ingredient other than a compound of the present invention. The choice of excipient will depend to a large extent on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

Pharmaceutical compositions suitable for delivery of the compounds of the invention and methods for their preparation will be apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in "Remington's Pharmaceutical Sciences", 19th Edition (MackPublishing Company, 1995).

Any suitable route of administration may be employed to provide an effective dose of a compound of the invention to a mammal, especially a human. For example, oral (including buccal and sublingual administration), rectal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. Preferably, the compound of formula (I) is administered orally or intranasally.

The effective dosage of the active ingredient employed may vary with the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosages are readily determined by one skilled in the art.

For the treatment of sexual dysfunction, the compounds of the invention are administered in a dosage range of from about 0.001 milligram (mg) to about 1000mg, preferably from about 0.001mg to about 500mg, more preferably from about 0.001mg to about 100mg, even more preferably from about 0.001mg to about 50mg, especially from about 0.002mg to about 25mg per kilogram of body weight, preferably as a single dose orally or as a nasal spray. For example, oral administration may require a total daily dose of about 0.1mg to about 1000mg, while intravenous doses may require only about 0.001mg to about 100 mg. The total daily dose may be administered in single or multiple administrations and, at the discretion of the physician, may fall outside the typical ranges given herein.

In the treatment of obesity with diabetes and/or hyperglycemia or obesity alone, satisfactory results are generally obtained when the compounds of the present invention are administered at the following daily doses: from about 0.0001mg to about 1000mg, preferably from about 0.001mg to about 500mg, more preferably from about 0.005mg to about 100mg, in particular from about 0.005mg to about 50mg per kg of animal body weight, preferably in single or multiple administrations, two to six times per day, or in sustained release. In the case of a 70kg adult, the total daily dose will generally be from about 0.7mg to about 3500 mg. Adjustment of this dosage regimen provides the best therapeutic response.

In the treatment of diabetes and/or hyperglycemia, as well as other diseases or disorders for which compounds of formula (I) are indicated, satisfactory results are generally obtained when the compounds of the invention are administered at the following daily doses: from about 0.001mg to about 100mg per kilogram of animal body weight, preferably administered one or more times, two to six times per day, or in a sustained release manner. In the case of a 70kg adult, the total daily dose will generally be from about 0.07mg to about 350 mg. Adjustment of this dosage regimen provides the best therapeutic response.

These doses are based on normal human subjects weighing about 65kg to 70 kg. A physician will be readily able to determine dosages for subjects having a body weight outside this range, such as infants and elderly people.

Oral administration

The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual or sublingual administration, whereby the compound enters the blood stream directly from the oral cavity.

Formulations suitable for oral administration include solid, semi-solid and liquid systems, such as tablets; soft or hard capsules containing multiparticulates or nanoparticles, liquids or powders; lozenges (including liquid filled); a masticatory; gelling; a fast dispersing dosage form; a film; an ovoid; a spray; and oral/mucosal adhesive patches.

Liquid preparations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard gelatin capsules (e.g. made from gelatin or hydroxypropylmethyl cellulose), typically comprising a carrier, for example water, ethanol, polyethylene glycol, propylene glycol, methyl cellulose or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by reconstitution of a solid, for example from a sachet.

The compounds of the present invention may also be used in fast dissolving, fast disintegrating dosage forms, such as the Expert Opinion in Therapeutic Patents,11(6) 981-986 by Lianggard Chen (2001).

For tablet dosage forms, depending on the dosage, the drug may comprise from 1 wt% to 80 wt% of the dosage form, more typically from 5 wt% to 60 wt% of the dosage form. In addition to the drug, tablets typically contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropylcellulose, starch, pregelatinized starch, and sodium alginate. Generally, the disintegrant will comprise from 1 wt% to 25 wt%, preferably from 5 wt% to 20 wt% of the dosage form.

Binders are generally used to impart cohesive properties to the tablet. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycols, natural and synthetic gums, polyvinyl pyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methyl cellulose. Tablets may also contain diluents such as lactose (monohydrate, spray-dried monohydrate, anhydrous, etc.), mannitol, xylitol, glucose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.

The tablets may also optionally contain surfactants such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, the surfactant may comprise from 0.2 wt% to 5 wt% of the tablet and the glidant may comprise from 0.2 wt% to 1 wt% of the tablet.

Tablets typically also contain lubricating agents such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate and mixtures of magnesium stearate and sodium lauryl sulfate. The lubricant generally comprises from 0.25 wt% to 10 wt%, preferably from 0.5 wt% to 3 wt% of the tablet.

Other possible ingredients include antioxidants, colorants, flavorants, preservatives, and taste masking agents.

Exemplary tablets contain up to about 80% drug, about 10 wt% to about 90 wt% binder, about 0 wt% to about 85 wt% diluent, about 2 wt% to about 10 wt% disintegrant, and about 0.25 wt% to about 10 wt% lubricant.

The tablet blend may be compressed into tablets either directly or with rollers. The tablet blend or blend portion can either be wet-, dry-, or soft-granulated, soft frozen, or extruded and then tableted. The final formulation may comprise one or more layers, and may be coated or uncoated; it may even be encapsulated.

See "formulation of tabletsPharmaceutical Dosage Forms：Tablets，Vol.1″，by H.Lieberman and L.Lachman(Marcel Dekker，New York，1980)。

Human or veterinary consumable oral films are typically flexible water-soluble or water-swellable film dosage forms which dissolve rapidly or adhere to mucous membranes and typically comprise a compound of formula (I), a film-forming polymer, a binder, a solvent, a humectant, a plasticizer, a stabilizer or emulsifier, a viscosity modifier and a solvent. Some components of the formulation may perform more than one function.

The compounds of formula (I) may be water soluble or insoluble. The water soluble compound typically comprises from 1 wt% to 80 wt%, more typically from 20 wt% to 50 wt% of the solute. Compounds with low solubility may comprise a greater proportion of the composition, typically up to 88% by weight of the solute. Alternatively, the compound of formula (I) may be in the form of multiparticulate beads.

The film-forming polymer may be selected from natural polysaccharides, proteins or synthetic hydrocolloids, typically in an amount of from 0.01 to 99 wt%, more typically from 30 to 80 wt%.

Other possible ingredients include antioxidants, colorants, flavors and taste modifiers, preservatives, saliva stimulating agents, cooling agents, solubilizing agents (including oils), emollients, fillers, antifoams, surfactants and taste masking agents.

Films according to the present invention are typically prepared by evaporation drying an aqueous film coated on a releasable backing support or paper. This can be done in a drying oven or in a tunnel, usually a combination coating dryer, or by freeze drying or vacuum.

Solid formulations for oral administration may be formulated for immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-targeted-, and programmed-release.

Modified release formulations suitable for the purposes of the present invention are described in U.S. Pat. No.6,106,864. Other suitable release techniques, such as high energy dispersions and penetration and coating of particles for details seePharmaceutical Technology On-line25(2), 1-14 by Vermaet al (2001). Controlled release is achieved by chewing gum as described in WO 00/35298.

Parenteral administration

The compounds of the invention may also be administered directly into the bloodstream, muscle or internal organs. Means suitable for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial and subcutaneous. Devices suitable for parenteral administration include needle (including microneedle) syringes, needle-free syringes, and infusion techniques.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH of 3 to 9), but for some applications they may be more suitably formulated as sterile non-aqueous solutions or in dry form in association with a suitable carrier, for example sterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions is readily accomplished using standard pharmaceutical techniques well known to those skilled in the art, such as lyophilization.

The solubility of the compounds of formula (I) for use in the preparation of parenteral solutions can be increased by appropriate formulation techniques, for example, the incorporation of solubility enhancers.

Parenteral formulations may be formulated for immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release. Thus, the compounds of the present invention may be formulated as suspensions or as solid, semi-solid or thixotropic liquids for administration as an implantable depot (drug depot) providing modified release of the active compound. Examples of such formulations include drug-coated stents and semi-solids and suspensions containing drug-coated poly (dl-lactic-co-glycolic acid) (PGLA) microspheres.

Topical administration of drugs

The compounds of the invention may also be administered topically to the skin or mucous membranes, i.e., transdermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, sachets, implants, sponges, fibers, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohols, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Penetration enhancers may be incorporated; see, for example, J Pharm Sci,88(10)，955-958 by Finninand Morgan(October 1999)。

other topical administration means include the following delivery modes: electroporation, iontophoresis, phonophoresis (sonophoresis), sonophoresis (sonophoresis) and microneedles or needles-free (e.g. Powderject)^TM、Bioject^TMEtc.) for injection.

Topical formulations may be formulated for immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

Inhalation/intranasal administration

The compounds of the invention may also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone or, for example, as a dry blend with lactose or as admixed component particles, for example, with phospholipids such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, nebulizer, atomizer (preferably one that generates a fine mist using electro-hydraulics) or nebulizer, with or without the use of a suitable propellant, for example 1, 1, 1, 2-tetrafluoroethane or 1, 1, 1, 2, 3, 3, 3-heptafluoropropane, or as nasal drops. For intranasal use, the powder may contain a bioadhesive, such as chitosan or cyclodextrin.

A pressure vessel, pump, nebulizer, atomizer or nebulizer containing a solution or suspension of a compound of the invention, e.g., comprising ethanol, aqueous ethanol or a suitable substitute, for dispersion, solubilization or extended release of the active ingredient; a propellant as a solvent; and optionally a surfactant such as sorbitan trioleate, oleic acid or oligolactic acid.

Prior to use in dry powder or suspension formulations, the drug product is micronized to a size suitable for inhalation delivery (typically less than 5 microns). This can be achieved by any suitable comminution method, such as spiral jet milling, fluidized bed jet milling, supercritical fluid processing into nanoparticles, high pressure homogenization or spray drying.

Capsules (made, for example, from gelatin or hydroxypropylmethyl cellulose), blisters and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention, a suitable powder base such as lactose or starch and a performance-improving agent such as 1-leucine, mannitol or magnesium stearate. Lactose may be in the anhydrous or monohydrate form, the latter being preferred. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

Solution formulations suitable for use in an atomizer for generating fine mist using electro-hydraulics may contain 1. mu.g to 20mg of the compound of the present invention per actuation, and the actuation volume may vary from 1. mu.l to 100. mu.l. A typical formulation may comprise a compound of formula (I), propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents that may be used in place of propylene glycol include glycerol and polyethylene glycol.

Suitable flavouring agents, for example menthol or levomenthol, or sweetening agents, for example saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhalation/intranasal administration.

Formulations for inhalation/intranasal administration may be formulated for immediate and/or modified release, for example using PGLA. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

In the case of dry powder inhalers and aerosols, the dosage unit depends on the valve to which the dose is to be delivered. The units according to the invention are generally arranged to administer a metered dose or "spray" containing from 0.001mg to 10mg of a compound of formula (I). The total daily dose will typically range from 0.001mg to 40mg, which may be administered in a single dose or more typically in divided doses throughout the day.

Rectal/intravaginal administration

The compounds of the invention may be administered rectally or vaginally, for example in the form of suppositories, pessaries or enemas. Cocoa butter is a traditional suppository base, but various substitutes may be used as appropriate.

Rectal/vaginal formulations may be formulated for immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

Ophthalmic/otic administration

The compounds of the invention may also be administered directly to the eye or ear, usually in the form of drops of micronized suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ophthalmic and otic administration include ointments, gels, biodegradable (e.g., absorbable gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, paper sachets, lenses, and particulate or vesicular systems, such as niosomes or liposomes. Polymers such as crosslinked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, cellulosic polymers (e.g., hydroxypropylmethylcellulose, hydroxyethylcellulose, or methylcellulose), or heteropolysaccharide polymers (e.g., agarose gel), and preservatives such as benzalkonium chloride may be incorporated. Such formulations may also be delivered iontophoretically.

Ophthalmic/otic formulations may be formulated for immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, or programmed-release.

Other techniques

The compounds of the invention may be combined with soluble macromolecular entities such as cyclodextrins and suitable derivatives thereof or polyethylene glycol containing polymers in order to enhance their solubility, dissolution rate, taste masking, bioavailability and/or stability for use in any of the above modes of administration.

For example, drug-cyclodextrin complexes are found to be generally useful in most dosage forms and routes of administration. Inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, cyclodextrin may be used as an auxiliary additive, i.e. a carrier, diluent or solubiliser. The most commonly used for these purposes are alpha-, beta-and gamma-cyclodextrins, examples of which can be found in international patent applications No. WO 91/11172, WO 94/02518 and WO 98/55148.

Medicine box

Since it may be necessary to administer a combination of active compounds, for example for the purpose of treating a particular disease or condition, it is also within the scope of the present invention that two or more pharmaceutical compositions may suitably be combined in a kit form suitable for co-administration of the compositions, at least one composition containing a compound according to the invention.

Thus, the kit of the invention comprises two or more separate pharmaceutical compositions, at least one composition comprising a compound of formula (I) according to the invention, and means for separately containing said compositions, such as a container, a separate bottle or a separate foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like.

The kit of the invention is particularly suitable for administering different dosage forms, for example oral and parenteral dosage forms, for administering separate compositions at different dosage intervals, or for titrating separate compositions against one another. To aid compliance, the kit typically contains directions for administration, and may be provided with a so-called memo.

For the avoidance of doubt, references herein to "treatment" include references to curative, palliative and prophylactic treatment.

The following non-limiting examples further illustrate the invention, wherein the following abbreviations and definitions are used.

Abbreviations

APCI atmospheric pressure chemical ionization mass spectrum

[α]_DSpecific rotation at 587nm

Arbocel ® filtering agent

Chemical shift of delta

d double peak

dd double doublet

GC-MS gas chromatography mass spectrometry

HPLC high performance liquid chromatography

HRMS high resolution mass spectrometry

LC-MS liquid chromatography mass spectrometry

LRMS low resolution mass spectrometry

m multiplet

min for

Peak of mass m/z

NMR nuclear magnetic resonance

psi pounds per square inch

q quartet peak

s single peak

t triplet peak

For ease of synthesis, although in many cases the free base forms of the compounds are isolated first, they are often converted to their corresponding hydrochloride salts for analytical identification purposes. For the avoidance of doubt, both the free base and the HCl salt forms are considered to be provided herein.

X-ray crystal data

Crystalline materials of four compounds were obtained as follows: (1) the compound of example 5 was dissolved in refluxing 90: 5 i-PrOH/MeCN/AcOH and the solution was allowed to cool to room temperature to give a crystalline material which could be isolated for further analysis; (2) the compound of preparation 16 was dissolved in refluxing 95: 5MeCN/THF and the solution was allowed to cool to room temperature to give a crystalline material which could be isolated for further analysis; (3) the compound of preparation 22b was dissolved in hot EtOAc, then pentane was added to the cloud point, and the solution was allowed to cool to room temperature to give a crystalline material which could be isolated for further analysis; and (4) as (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl group]Pyrrolidine-3-carboxamide hydrochloride from EtOH/i-Pr via a vapor diffusion process₂O gives a crystalline material.

The stereochemistry of the resulting crystalline materials of these four compounds was determined using X-ray crystallography. The 3D structures of these compounds are shown below in figures 3, 4, 5 and 6.

Figure 3 illustrates an ORTEP plot of the asymmetric units of the crystal structure of the compound of example 5 plotted at a 50% confidence level with thermal ellipsoids.

Figure 4 illustrates an ORTEP plot of the asymmetric units of the crystal structure of the compound of preparation 16 plotted at a 50% confidence level with thermal ellipsoids.

Figure 5 illustrates an ORTEP plot of asymmetric units of the crystal structure of the compound of preparation 22b plotted at a 50% confidence level with thermal ellipsoids.

Figure 6 illustrates an ORTEP plot of asymmetric units of the (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl ] pyrrolidine-3-carboxamide hydrochloride crystal structure plotted at 50% confidence level with thermal ellipsoids. For clarity, the difluorophenyl ring and the disorder of the phenyl ring have been omitted.

Example 5 Compounds (wherein R¹Is phenyl, R²＝OH，R³＝Bu^t，R⁴And R⁵X-ray crystal data for ═ F) elucidate: the relative cis relationship of the methyl substituents on the piperidine ring; r on the piperidine ring²To the cis-arrangement of the methyl substituents; the relative trans arrangement of the groups at the C3 and C4 positions on the pyrrolidine ring; and the absolute configuration of pyrrolidine rings C3 and C4.

Preparation 16 intermediate Compound (wherein R¹Is phenyl, R²OH) of the X-ray crystal data elucidate: relative cis relationship of methyl substituents on the piperidine ring and R on the piperidine ring²To the cis-arrangement of the methyl substituents. The compound of preparation 16 is a direct precursor to the compound of example 5. X-ray data confirm that there is no stereochemical interconversion at the piperidine ring in the subsequent reaction of the compound of preparation 16 with the compound of preparation 1 to give the compound of example 5.

Preparation 22b intermediate Compounds (wherein R³＝Bu^t，R⁴And R⁵X-ray crystal data for ═ F) elucidate: the relative trans arrangement of the groups at the C3 and C4 positions on the pyrrolidine ring; and (in view of the known absolute configuration of the benzyl oxazolidinone moiety) the absolute configurations of C3 and C4. Preparation 22b hydrolysis of the intermediate compound gives the intermediate compound of preparation 1, which is a direct precursor of the compound of final example 5. X-ray data certificateIndeed, there was no stereochemical interconversion on the pyrrolidine ring during the synthesis of the intermediate of preparation 22b to the intermediate of preparation 16 and its subsequent reaction with the intermediate of preparation 1 to give the final compound of example 5.

The relative and absolute configuration of the compound of preparation 53 was determined by conversion to (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl ] pyrrolidine-3-carboxamide hydrochloride. This transformation is achieved by:

(i) reaction of the compound of preparation 53 with (R) - (+) - α -methylbenzylamine in the presence of 1- [3- (dimethylamino) propyl ] -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole in tetrahydrofuran at room temperature to give tert-butyl (3R, 4S) -3- (2, 4-difluorophenyl) -4- ({ [ (1R) -1-phenylethyl ] amino } carbonyl) pyrrolidine-1-carboxylate;

(ii) Boc-deprotection of a solution of tert-butyl (3R, 4S) -3- (2, 4-difluorophenyl) -4- ({ [ (1R) -1-phenylethyl ] amino } carbonyl) pyrrolidine-1-carboxylate in dichloromethane was treated with 4M hydrogen chloride in dioxane at room temperature to give (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl ] pyrrolidine-3-carboxamide hydrochloride. X-ray crystallography data of (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl ] pyrrolidine-3-carboxamide hydrochloride demonstrate the relative trans relationship of the C3 and C4 substituents and the absolute configurations of the C3 and C4 substituents of the pyrrolidine ring. This 3D structure behaves as described below with respect to fig. 6.

The stereochemistry of the compounds of the remaining examples and preparations was given on the basis of the stereochemistry established in the synthesis of the compounds of example 5, preparation 22b, preparation 16 and preparation 53 as described above. With the exception of the compound of example 7, it has a cis-arrangement at the 3-and 4-positions of the pyrrolidine ring.

X-ray diffraction data of single crystals of the compounds of example 5 and preparation examples 16 and 22b were recorded at room temperature using a Bruker AXS SMART-APEX CCD area detector diffractometer (Mo K.alpha.radiation). Intensities from several exposure series were integrated using the method described by SMART v5.622 (control) and SAINT v6.02 (integration) software Bruker AXS inc, Madison, WI 1994. Each exposure covered 0.3 ° ω, exposure times 60s (example 5), 10s (preparation 16) or 120s (preparation 22b), and the total data set was: substantially spherical (example 5); hemispherical (preparations 16 and 22 b). The multi-scan method described by programs SADABS, g.m. sheldrag, University of g ö ttingen, 1997, which utilized scaling and correcting the area detector data, corrected the data set according to absorption (methods based on r.h. blanking, Acta cryst.1995, a51, 33-38).

X-ray diffraction data were recorded for the crystalline (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl ] pyrrolidine-3-carboxamide hydrochloride at 100K using a Bruker AXS SMART-APEX CCD area detector diffractometer (Mo K.alpha.radiation) equipped with an Oxford Cryosystem Series 700 Liquid Nitrogen Cryogyostream. The intensities from several exposure series were integrated (as described above). Each exposure covered 0.3 ω, exposure time 60s, and the total data set was approximately spherical. The data set was corrected according to absorption using a multi-scan method (as described above).

The crystal structure was successfully resolved by direct methods using SHELXS-97 (as described in SHELXS-97, Program for crystal structure solution. G.M.Sheldrick, University of G ö ttingen, Germany, 1997, release 97-2): space group P2₁(example 5); space group Pna2₁(preparation example 16); space group P2₁2₁2₁Preparation 22b and (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl]Pyrrolidine-3-carboxamide hydrochloride) to locate all non-hydrogen atoms in the asymmetric unit from the resulting electron density map. From these figures and subsequent structure refinement it was found that: one cation and one chloride ion are present in the asymmetric unit of the compound of example 5 (as depicted in figure 3); the presence of one molecule of the compound of preparation 16 in an asymmetric unit (as depicted in FIG. 4); the presence of one molecule of the compound of preparation 22b in an asymmetric unit (as depicted in FIG. 5); in the asymmetric unit there is one (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl group ]Pyrrolidine-3-carboxamide hydrochloride cation and one chloride anion (as depicted in figure 6).

For all four crystals evaluated, the diffraction data were refined (refined) in terms of coordinates of non-hydrogen atoms by means of the least squares method using SHELXL-97 with anisotropic substitution parameters (as described in SHELXL-97, Program for crystal structure refinement. G.M. Sheldrick, University of G ö ttingen, Germany, 1997, release 97-2).

For the crystal of example 5, the positions of the hydroxyl and N-H + hydrogen atoms were located from the Fourier difference plot, and their coordinates were refined using constraints placed on the respective O-H and N-H bond distances and angles, so that the groups retained the idealized geometry. The remaining hydrogen atoms were placed in the calculated positions, refined using a ride model, and all hydrogen atoms were refined using anisotropic displacement parameters. The absolute configuration of the cation stereochemistry of the compound of example 5 was determined directly from the X-ray diffraction data by the method of Flack (as described in H.D. Flack, Acta Crystal.1983, A39, 876-one 881). The final refined enantiomer Flack parameter was 0.00(5), as depicted in FIG. 3.

For the crystal of preparative example 16, the positions of the amine and hydroxyl hydrogen atoms were located from the Fourier difference plot, and their coordinates were refined using constraints placed on the respective N-H and O-H bond distances and angles so that the groups retained the idealized geometry. The remaining hydrogen atoms were placed in the calculated positions, refined using a ride model, and all hydrogen atoms were refined using anisotropic displacement parameters.

For the crystal of preparation 22b, the hydrogen atoms were placed in the calculated positions, refined using a riding model, and all the hydrogen atoms were refined using anisotropic substitution parameters. The absolute stereochemistry of the compound of preparation 22b could not be determined directly from the diffraction data. However, this crystal structure establishes that the configuration may be only one of a pair of enantiomers (shown in FIG. 5 or a mirror image of all its chiral centers inverted). If the configuration of the oxazolidinone ring center C4 is assumed to be the same as the starting material, i.e., "S", it can be concluded that the other two centers are as depicted in FIG. 5.

For the crystals of (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl ] pyrrolidine-3-carboxamide hydrochloride, the large thermal ellipsoid associated with the two phenyl rings strongly suggests that these two groups are disordered. With two rotations about the C17 axis, a difluorophenyl ring model was established according to the two orientations involved, with a relative occupancy ratio of 80: 20. Finally, the unsubstituted phenyl ring was modeled for two overlapping orientations, with equal occupancy ratios. The hydrogen atoms are placed at the calculated positions, refined using a ride model, and all hydrogen atoms are refined using anisotropic displacement parameters. The positions of the amide and amine N-H hydrogen atoms were located from the Fourier difference plot and refined using anisotropic displacement parameters. The remaining hydrogen atoms were placed in the calculated positions, refined using a ride model, and all hydrogen atoms were refined using anisotropic displacement parameters. The absolute configuration of the stereochemistry of the (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl ] pyrrolidine-3-carboxamide hydrochloride cation was determined directly from the X-ray diffraction data by the method of Flack (as described above). The final refined diastereomer, Flack parameter, was-0.01 (7), as depicted in FIG. 6.

The final refined R-factor% (data 1 > 2. sigma.1) was as follows: example 5 was 4.15%; preparation 16 was 4.07%; preparation 22b was 4.62% and (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl ] pyrrolidine-3-carboxamide hydrochloride was 5.00%.

Excited powder X-ray diffraction data

(i) Example 5 Compounds

Using Accelrys Materials Studio^TMThe "Reflex powder diffraction" module (version 3.0), 2-theta angles, d-spacings and relative intensities were calculated from the single crystal structure of the compound of example 5. The relevant excitation parameters were in each case: wavelength 1.540562 Å (Cu K α); polarization factor is 0.5; pseudo-Voigt curve (U ═ 0.01, V ═ 0.001, W ═ 0.002).

The single crystal structure data derived via the above method was used in these calculations. Table 1 lists the most intense peaks of the powder pattern excited by example 5, from a summary of single crystal data (as depicted in fig. 7).

TABLE 1

Angle (° 2-theta)	Strength (%)	Angle (° 2-theta)	Strength (%)
Angle (° 2-theta)	Strength (%)	Angle (° 2-theta)	Strength (%)	8.6	17.4	24.7	12
10.9	42.3	25.3	29.5	8.6	17.4	24.7	12
10.9	42.3	25.3	29.5	11.3	86	25.8	11.8
11.7	53.6	28.1	10.3	11.3	86	25.8	11.8
11.7	53.6	28.1	10.3	13.9	100
16.1	61.5			13.9	100
16.1	61.5			18.2	13.6
18.9	51.4			18.2	13.6
18.9	51.4			19.7	72.5
20.3	32.2			19.7	72.5
20.3	32.2			23.5	33.5
24.1	22.2			23.5	33.5

Thus, according to a further aspect, the present invention provides the compound of example 5 having the excited PXRD pattern as described in fig. 7, the strongest peaks being as described in table 1, wherein the excited PXRD pattern was generated via the method described above.

(ii) Preparation of the Compound of example 16

Using Accelrys Materials Studio^TMThe "Reflex powder diffraction" module (version 3.0), the 2-theta angle, the d-spacing and the d-spacing are calculated from the single crystal structure of the compound of preparation 16Relative strength. The relevant excitation parameters were in each case: wavelength 1.540562 Å (Cu K α); polarization factor is 0.5; pseudo-Voigt curve (U ═ 0.01, V ═ 0.001, W ═ 0.002).

The single crystal structure data derived via the above method was used in these calculations. Table 2 lists the most intense peaks of the powder pattern excited by preparative example 16, from a summary of single crystal data (as depicted in fig. 8).

TABLE 2

Angle (° 2-theta)	Strength (%)
Angle (° 2-theta)	Strength (%)	9.2	65.5
11.6	14.4	9.2	65.5
11.6	14.4	14.3	100
15.5	41.6	14.3	100
15.5	41.6	16.6	78.9
18.1	85.7	16.6	78.9
18.1	85.7	20.8	13.5
22.6	16	20.8	13.5
22.6	16	24.6	17.1
25.7	12.5	24.6	17.1
25.7	12.5	26.4	14.9
27.2	15.5	26.4	14.9

Thus, according to a further aspect, the present invention provides the compound of preparative example 16 having an excited PXRD pattern as depicted in fig. 8, the strongest peaks being depicted in table 2, wherein the excited PXRD pattern was generated via the method described above.

(iii) Preparation of the Compound of example 22b

Using Accelrys Materials Studio^TMThe "Reflex powder diffraction" module (version 3.0), 2-theta angles, d-spacings and relative intensities were calculated from the single crystal structure of the compound of preparation 22 b. The relevant excitation parameters were in each case: wavelength 1.540562 Å (Cu K α); polarization factor is 0.5; pseudo-Voigt curve (U ═ 0.01, V ═ 0.001, W ═ 0.002).

The single crystal structure data derived via the above method was used in these calculations. Table 3 lists the most intense peaks of the powder pattern excited by preparative example 22b, from a summary of single crystal data (as depicted in fig. 9).

TABLE 3

Angle (° 2-theta)	Strength (%)	Angle (° 2-theta)	Strength (%)
Angle (° 2-theta)	Strength (%)	Angle (° 2-theta)	Strength (%)	6.5	20.7	20.1	23.7
9.1	34.3	20.3	17.3	6.5	20.7	20.1	23.7
9.1	34.3	20.3	17.3	9.4	60.1	20.6	28.2
10.5	17.4	21.9	16.2	9.4	60.1	20.6	28.2
10.5	17.4	21.9	16.2	14.9	35.2	23.4	27.3
16.9	100	23.7	11.3	14.9	35.2	23.4	27.3
16.9	100	23.7	11.3	17.1	19.8	24.1	24.1
17.5	43.6	25.4	12.5	17.1	19.8	24.1	24.1
17.5	43.6	25.4	12.5	18.3	24.5	27.4	19.2
18.9	73.1	29.2	16.2	18.3	24.5	27.4	19.2
18.9	73.1	29.2	16.2	19.4	28.3
19.7	13.3			19.4	28.3

Thus, according to a further aspect, the present invention provides the compound of preparation 22b having the excited PXRD pattern as depicted in fig. 9, the strongest peaks being depicted in table 2, wherein the excited PXRD pattern was generated via the method described above.

(iv) (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl ] pyrrolidine-3-carboxamide hydrochloride compound

Using Accelrys Materials Studio^TMThe "Reflex powder diffraction" module (version 3.0) from (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl]The single crystal structure of the pyrrolidine-3-carboxamide hydrochloride compound calculated the 2-theta angle, d-spacing, and relative intensity. The relevant excitation parameters were in each case: wavelength 1.540562 Å (Cu K α); polarization factor is 0.5; pseudo-Voigt curve (U ═ 0.01, V ═ 0.001, W ═ 0.002).

The single crystal structure data derived via the above method was used in these calculations. Table 4 lists the most intense peaks of the powder pattern excited by (3S, 4R) -4- (2, 4-difluorophenyl) -N- [ (1R) -1-phenylethyl ] pyrrolidine-3-carboxamide hydrochloride, from a summary of single crystal data (as depicted in figure 10).

TABLE 4

Angle (° 2-theta)	Strength (%)
Angle (° 2-theta)	Strength (%)	4.4	100
8.8	17.1	4.4	100
8.8	17.1	11.4	20.1
18.1	17.4	11.4	20.1
18.1	17.4	18.7	12.7
19.0	14.6	18.7	12.7
19.0	14.6	20.0	11.7
20.6	11.2	20.0	11.7
20.6	11.2	22.8	11.8
23.9	24	22.8	11.8
23.9	24	25.2	10.8
		25.2	10.8

Example 1

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol

To a stirred suspension of (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid hydrochloride (57mg, 0.2mmol) from preparation 1 in dichloromethane (1mL) was added O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium hexafluorophosphate (76mg, 0.2mmol) followed by N-methylmorpholine (132. mu.L, 0.4mmol) and then (3R, 4S, 5S) -3, 5-dimethyl-4-phenylpiperidin-4-ol (45mg, 0.2mmol), all in one portion, at room temperature under dry nitrogen. The resulting mixture was stirred at room temperature under dry nitrogen for 18 hours, quenched by addition of water (10mL), and extracted with dichloromethane (2X 10 mL). The organic layers were combined, dried (magnesium sulfate), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography on silica eluting with 10% methanol in dichloromethane to give the title compound as a white foam (72mg, 77%).

LRMS(APCI)471(100％)[MH⁺]，298(40％)，220(20％)；HRMS C₂₈H₃₇F₂O₂[MH⁺]Theoretical 471.2818 found 471.2815.

Example 2

(3R, 4R, 5S) -4-cyclohexyl-1- { [ (3S)^*，4R^*) -4- (2, 4-difluorophenyl) -1-ethylpyrrolidin-3-yl ]Carbonyl } -3, 5-dimethylpiperidin-4-ol

Stirred (3S) from preparation 17 under dry nitrogen at room temperature^*，4R^*) Suspension of-4- (2, 4-difluorophenyl) -1-ethylpyrrolidine-3-carboxylic acid hydrochloride (161mg, 0.6mmol) in N, N-dimethylformamide (10mL) was added triethylamine (0.2mL, 1.4mmol) followed by 1-hydroxybenzotriazole hydrate (77mg, 0.6mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (109mg, 0.6mmol) and (3R, 4S, 5S) -4-cyclohexyl-3, 5-dimethylpiperidin-4-ol from preparation 7 (100mg, 0.5mmol), all in one portion. The resulting mixture was stirred at 30 ℃ under dry nitrogen for 25 hours. The reaction was quenched by the addition of 2M sodium hydroxide solution (75mL) and then extracted with diethyl ether (80 mL). The organic layer was washed with brine (50mL), separated, dried over magnesium sulfate, filtered, and concentrated in vacuo to give the title compound as a clear oil (209mg, 99%). LRMS (APCI)449 (100%) [ MH⁺]，298(40％)，220(20％)；HRMS C₂₆H₃₉F₂O₂[MH⁺]Theoretical 449.2974 found 449.2970.

Example 3

(3R, 4R, 5S) -4-butyl-1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidin-4-ol

To a stirred suspension of (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid hydrochloride (71mg, 0.3mmol) from preparation 1 in N, N-dimethylformamide (10mL) at room temperature under dry nitrogen was added O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium hexafluorophosphate (95mg, 0.3mmol), N-methylmorpholine (83. mu.L, 0.8mmol) followed by (3R, 4S, 5S) -4-butyl-3, 5-dimethylpiperidin-4-ol (50mg, 0.3mmol), all in one portion, from preparation 9. The resulting mixture was stirred at room temperature under dry nitrogen for 2.5 days, then quenched by addition of water (5mL) and extracted with diethyl ether (10 mL). The organic layer was separated, dried (magnesium sulfate), filtered, and concentrated in vacuo. The residue was purified by HPLC using a Phenomenex Luna C18(2) column 150 x 15mm (10 micron particle size, 100 Å porosity) using 2 solvent eluents: acetonitrile, water trifluoroacetic acid (5: 95: 0.1) (solvent A) and acetonitrile (solvent B). The solvent gradient was as follows, flow rate 20 mL/min: 0 min-5% of B; 0.6 min-5% B; 9.5 min-95% B; 10.5 min-95% B. The title compound was obtained as an oil (18mg, 13%) with a retention time of 6.05 min.

LRMS(APCI)451(100％)[MH⁺]；HRMSC₂₆H₄₀F₂O₂Theoretical 451.3131 found 451.3114.

Example 4

(3R, 4R, 5S) -1- ([ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4- (4-methylphenyl) piperidin-4-ol

To a stirred suspension of (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid hydrochloride (172mg, 0.6mmol) from preparation 1 in N, N-dimethylformamide (10mL) at room temperature under dry nitrogen was added O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium hexafluorophosphate (231mg, 0.6mmol), N-methylmorpholine (201. mu.L, 1.8mmol) and then (3R, 4S, 5S) -3, 5-dimethyl-4- (4-methylphenyl) piperidin-4-ol (136mg, 0.6mmol), all at once, from preparation 10. The resulting mixture was stirred at room temperature under dry nitrogen for 2.5 days, quenched by addition of water (5mL), and then extracted with diethyl ether (10 mL). The organic layer was separated, then dried (magnesium sulfate), filtered, and concentrated in vacuo. The residue was purified by HPLC using a Phenomenex Luna C18(1) column 150 x 15mm (10 micron particle size, 100 Å porosity) using 2 solvent eluents: acetonitrile, water trifluoroacetic acid (5: 95: 0.1) (solvent A) and acetonitrile (solvent B). The solvent gradient was as follows, flow rate 20 mL/min: 0 min-5% of B; 0.6 min-5% B; 9.5 min-95% B; 10.5 min-95% B. The title compound was obtained as an oil (24mg, 9%) with a retention time of 6.15 min.

LRMS(APCI)485(100％)[MH⁺]；HRMS C₂₉H₃₉F₂O₂Theoretical 485.2974 found 485.2959.

Example 5

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

(3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid from preparation 1 (26.0g, 92mmoL) and (3R, 4S, 5S) -3, 5-dimethyl-4-phenylpiperidin-4-ol from preparation 16 (17.4g, 85mmoL) were suspended in dichloromethane (1000 mL). Triethylamine (14.2mL, 102mmol) was added and the mixture was cooled to 0 ℃ while stirring under nitrogen. 1-Propylphosphonic acid cyclic anhydride (50% in ethyl acetate) (54.5mL, 92mmol) was added dropwise, maintaining the temperature below 5 ℃. The mixture was then allowed to warm to room temperature while stirring continuously. After stirring at room temperature for 1 hour, acetic acid (5mL) was added to remove the last traces of (3R, 4S, 5S) -3, 5-dimethyl-4-phenylpiperidin-4-ol. The reaction mixture was stirred at room temperature for another 1 hour. 10% potassium carbonate solution (500mL) was added and the mixture was stirred vigorously at room temperature for 2 hours. The organic layer was separated and then stirred with 10% potassium carbonate solution (500mL) for 1 hour. The dichloromethane layer was then separated, washed with water (3 × 300mL), dried over sodium sulfate, and filtered. A4M solution of hydrogen chloride in dioxane (50mL) was then added to the dichloromethane solution. The solvent was then evaporated to give the crude hydrochloride salt as a white powder. Acetone (500mL) was added to the crude hydrochloride salt and the mixture was boiled for 30 minutes and then cooled to room temperature. The hydrochloride salt was filtered off and washed with acetone (5X 100 mL). The product was recrystallized from isopropanol to give an analytically pure hydrochloride salt (39.5 g).

MS(APCI⁺)471(M+H)

¹H NMR(400MHz，CD₃OD) δ (rotamer), 0.35(d, 2H), 0.50(m, 3.60H), 0.95(m, 0.6H), 1.22(s, 9H), 1.65(m, 0.75H), 1.97(m, 0.48H), 2.70(m, 1.02H), 2.87(m, 0.54H), 3.2(m, 0.66H), 3.70(m, 0.8H), 3.20-3.40(m, H), 3.57(m, 0.66H), 3.65(m, 0.24H), 3.80(m, 1.5H), 4.30(m, 1H), 7.05(m, 0.5H), 7.20(m, 1.5H), 7.25(m, 3.5H), 7.45(m, 0.5H), 7.60(m, 1H).

[□]²⁵ _D＝-51.9(c＝0.3，MeOH).

Example 6

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

To a stirred solution of (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidine-3-carboxylic acid (160mg, 0.58mmol) from preparation 33 and (3R, 4S, 5S) -3, 5-dimethyl-4-phenylpiperidin-4-ol (100mg, 0.48mmol) from preparation 16 in ethyl acetate (2mL) at 0 deg.C was added triethylamine (140. mu.L, 0.97mmol) and 1-propylphosphonic acid cyclic anhydride (50% in ethyl acetate) (290. mu.L, 0.48 mmol). The reaction mixture was stirred for 30 minutes, then warmed to room temperature and the solvent was removed in vacuo. The residue was partitioned between dichloromethane (20mL) and saturated potassium carbonate solution (2X 20 mL). The phases were separated and the organic phase was washed with brine (10mL), dried over magnesium sulfate, filtered and concentrated in vacuo. The residue was purified by column chromatography using dichloromethane: methanol: 0.88 ammonia (99: 1: 0.1-98: 2: 0.2-97: 3: 0.3) as eluent to give 170mg of the product as a colourless oil. This oil was dissolved in 1, 4-dioxane (3mL) and a 4M solution of hydrogen chloride in dioxane (6mL) was added slowly. The solvent was then removed in vacuo. Recrystallization from acetone gave 110.8mg of the desired product.

¹H NMR(400MHz，CD₃OD) (rotamers) □ 0.27-0.56(m, 6H), 1.45(m, 7H), 1.69-2.02(m, 1H), 2.75(m, 2H), 3.14(m, 2H), 3.40(m, 1H), 3.61(m, 1H), 3.77(m, 1H), 3.92(m, 2H), 4.01-4.17(m, 1H), 4.32(dd, 1H), 7.05-7.24(m, 4H), 7.34(m, 3H), 7.62-7.72(m, 1H).

LRMS(APCI)457[MH⁺].

[□]²⁵ _D＝-53.5(c＝0.26，MeOH).

Example 7

(3R，4S，5S)-1-{[(3R^*，4R^*) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl]Carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol

To the cooled (R) from preparation 36^*，R^*) A solution of-1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid (500mg, 1.76mmol) in dichloromethane (20mL) was added with a catalytic amount of N, N-dimethylformamide followed by oxalyl chloride (309. mu.L, 3.53 mmol). Will be reversedThe mixture was stirred for 2 hours, then the solvent was removed in vacuo. The resulting residual white powder was azeotroped with dichloromethane (2X 10 mL). The white powder was redissolved in dichloromethane (10mL) and added dropwise over 10 min at room temperature to a solution of (3R, 4S, 5S) -3, 5-dimethyl-4-phenylpiperidin-4-ol (prepared as in preparation 16) (362mg, 1.76mmol) and triethylamine (246. mu.L, 1.76mmol) in dichloromethane (10 mL). The resulting mixture was stirred for 24 h, diluted with dichloromethane (10mL) and partitioned with saturated sodium bicarbonate solution (2X 30 mL). The phases were separated and the organic phase was washed with brine (30mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give a crude residue. Purification by column chromatography on silica gel eluting with methylene chloride: methanol: 0.88 ammonia (99: 1: 0.1-98: 2: 0.2) gave the desired product as a white foam of 497 mg.

¹H NMR(400MHz，CD₃OD) (rotamer) □ 0.43-0.55(m, 6H), 0.75-0.79(m, 1H), 1.25(s, 9H), 1.87-1.97(m, 1H), 2.16-2.66(m, 2H), 3.09(t, 2H), 3.18-3.30(m, 2H), 3.41-3.61(m, 2H), 3.80-4.17(m, 3H), 6.91-7.09(m, 3H), 7.28-7.35(m, 3H), 7.47(q, 1H).

LCMS(APCI)＝471[MH⁺].

Example 8

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (3, 4-difluorophenyl) -3, 5-dimethylpiperidine-4-ol hydrochloride

A solution of (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid from preparation 1 (159mg, 0.49mmol), (3R, 4S, 5S) -4- (3, 4-difluorophenyl) -3, 5-dimethylpiperidin-4-ol from preparation 39 (100mg, 0.41mmol), 1-propylphosphonic acid cyclic anhydride (50% in ethyl acetate) (244. mu.L, 0.41mmol), and triethylamine (120. mu.L, 0.41mmol) in dichloromethane (2.5mL) was stirred at room temperature for 3 days. The reaction was then diluted with dichloromethane (20mL) and partitioned with 10% potassium carbonate solution (20 mL). The phases were separated and the organic phase was washed with brine, dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel using dichloromethane: methanol: 0.88 ammonia (99: 1: 0.1-96: 4: 0.4) as eluent gave the free base as 117mg of colorless oil. The oil was dissolved in dichloromethane (1mL) and treated with 2M hydrogen chloride in diethyl ether (3 mL). The solvent was then removed in vacuo and the residue azeotroped with diethyl ether to give the title compound as a white solid, 108 mg.

¹H NMR(400MHz，CD₃OD) (rotamer) □ 0.30-0.56(m, 6H), 1.48(s, 9H), 1.62-1.94(m, 1H), 2.64-2.75(m, 1H), 3.12(t, 2H), 3.40-3.54(m, 2H), 3.70-3.96(m, 2H), 4.00(m, 1H), 4.2(dd, 1H), 7.02-7.21(m, 4H), 7.52(m, 1H), 7.65(m, 1H).

LRMS(APCI)507[MH⁺].

[□]²⁵ _D＝-34.89(c＝0.23，MeOH).

Example 9

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol hydrochloride

1-Propylphosphonic acid cyclic anhydride (50% wt ethyl acetate solution) (0.67mL, 2.0mmol) was added dropwise to a mixture of (3R, 4S, 5S) -4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol (267mg, 1.2mmol) from preparation 41, (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid (450mg, 1.4mmol) from preparation 1 and triethylamine (0.48mL, 3.6mmol) in dichloromethane (25mL) at 0 deg.C under nitrogen. After the addition was complete, the resulting homogeneous solution was stirred at room temperature for 6 hours. The solution was washed with 10% aqueous potassium carbonate (3X 20mL), then dried over sodium sulfate,and (5) filtering. The solvent was removed in vacuo and the crude product was purified by column chromatography (reverse phase C-18, 40g Redisep ® cartridge) using an ISCO company ® automated purification system. Mobile phase gradient over 20 min: MeCN/H ₂O/TFA (5%/95%/0.1%) 95%: MeCN (100%) 5% elution to MeCN/H₂O/TFA (5%/95%/0.1%) 5%: MeCN (100%) 95%. The purified product was then dissolved in 1, 4-dioxane (100mL) and 4M hydrogen chloride in dioxane (20mL) was added. The solution was then evaporated to dryness, redissolved in 4M hydrogen chloride in dioxane (100mL), and evaporated again to dryness. The residue was then dried at 50 ℃ in vacuo to give the product hydrochloride salt (391mg) as a white amorphous solid.

¹H NMR(CD₃OD 400 MHz): (rotamers), 0.31-0.57(3xd, 6H), 0.83-2.08(3xm, 2H), 1.55(s, 9H), 1.60-2.07(3xm, 2H), 2.68-3.20(2xm, 2H), 3.20-4.12(m, 5H), 4.29(m, 1.H), 6.95-7.19(m, 5H), 7.38-7.85(m, 2H)

LRMS(APCI)＝489[MH⁺]

[□]²⁵ _D＝-42.7(c＝0.31，MeOH)

Example 10

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidin-3-yl ] carbonyl } -4- (4-fluorophenyl) -3, 5-dimethylpiperidine-4-ol hydrochloride

The title compound was prepared from the compounds of preparation 33 and 41 by a similar method to that described in example 9.

¹H NMR(400MHz，CD₃OD) delta (rotamer), 0.31-0.57(m, 6H), 0.83-2.08(m, 2H), 1.42(m, 6H), 1.64-2.35(m, 2H), 2.65(m, 1H), 3.11-4.18(m, 7H), 4.35(m, 1H), 6.95-7.19(m, 5H), 7.38-7.85(m, 2H) LRMS (APCI) MH [ 475[ MH, 1H ] ⁺]

[α]²⁵ _D＝-39.6(c＝0.3，MeOH)

Example 11

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-methylpyrrolidin-3-yl ] carbonyl } -4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol hydrochloride

The title compound was prepared from the compounds of preparation 62 and 41 by a similar method to that described in example 9.

¹H NMR(400MHz，CD₃OD) δ (rotamer), 0.23-0.60(m, 6H)1.03-1.98(m, 2H), 2.65(m, 1H), 3.11-4.18(m, 11H), 4.35(m, 1H), 6.95-7.19(m, 5H), 7.38-7.85(m, 2H)

LRMS(APCI)448[MH⁺]

[α]²⁵ _D＝+49.7(c＝0.3，MeOH)

Example 12

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol hydrochloride

1-Propylphosphonic acid cyclic anhydride (50% wt ethyl acetate solution) (0.67mL, 2.0mmol) was added dropwise to a mixture of (3R, 4S, 5S) -4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol (265mg, 1.2mmol) from preparation 41, (3S, 4R) -1- (tert-butoxycarbonyl) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid (0.75g, 1.4mol) from preparation 53 and triethylamine (0.48mL, 3.6mmol) in dichloromethane (25mL) at 0 deg.C under nitrogen. After the addition was complete, the resulting homogeneous solution was stirred at room temperature for 6 hours. The solution was washed with 10% aqueous potassium carbonate (3X 20mL), 3% aqueous citric acid (3X 50mL), then dried over sodium sulfate, and filtered. The solvent was then removed in vacuo and the residue was purified by column chromatography on silica eluting with ethyl acetate: pentane (gradient 1: 9 to 4: 6) to give the Boc-protected product as a white solid (529 mg). A portion of this product (300mg, 5.6mmol) was dissolved in 1, 4-dioxane (20mL) and 4M hydrogen chloride in dioxane (80mL) was added. The solution was stirred for another 8 hours, then the solvent was removed in vacuo. The residue was then dried under vacuum at 50 ℃ to give the title compound (311mg) as a white solid.

¹H NMR(400MHz，CD₃OD) delta (rotamer), 0.36-0.66(m, 6H), 0.81-1.97(m, 2H), 2.70(m, 1H), 3.19-4.05(m, 8H), 4.31(m, 1H), 6.85-7.31(m, 6H), 7.28-7.80(m, 2H)

LRMS(APCI)433[MH⁺]

[α]²⁵ _D＝-62.2(c＝0.3，MeOH)

Example 13

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl-4- (4-chlorophenyl) -3, 5-dimethylpiperidine-4-ol hydrochloride

The title compound was prepared from the compounds of preparation examples 1 and 43 according to a similar method to that described in example 9.

¹H NMR(400MHz，CD₃OD) delta (rotamer), 0.31-0.57(m, 6H), 0.79-1.99(m, 2H), 1.45(s, 9H), 1.60-2.07(m 2H), 2.68-3.20(m, 2H), 3.20-4.12(m, 5H), 4.29(m, 1H), 7.05-7.29(m, 5H), 7.40-7.75(m, 2H)

LRMS(APCI)505[MH⁺]

[α]²⁵ _D＝-37.6(c＝0.3，MeOH)

Example 14

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-ethylpyrrolidin-3-yl ] carbonyl } -4- (4-methoxyphenyl) -3, 5-dimethylpiperidine-4-ol hydrochloride

The title compound was prepared from the compounds of preparation 48 and 75 according to a similar method to that described in example 9.

¹H NMR(400MHz，CD₃OD) delta (rotamer), 0.20-0.57(m, 6H)1.05(t, 3H), 1.81(q, 2H), 0.79-1.99(m, 4H), 1.60-2.07(m, 3H), 2.68-3.20(m, 2H), 3.20-4.12(m, 5H), 4.29(m, 1H), 6.81-7.29(m, 5H), 7.60-7.74(m, 2H)

LRMS(APCI)472[MH⁺]

[α]²⁵ _D＝-42.7(c＝0.3，MeOH)

Example 15

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (2, 4-difluorophenyl) -3, 5-dimethylpiperidine-4-ol hydrochloride

The title compound was prepared from the compounds of preparation examples 1 and 49 according to a similar method to that described in example 9.

¹H NMR(400MHz，CD₃OD) delta (rotamer), 0.31-0.55(m, 6H), 0.83-1.95(m, 2H), 1.50(s, 9H), 1.57-2.01(m, 2H), 2.68-3.20(m, 2H), 3.12-4.17(m, 5H), 4.29(m, 1H), 7.04-7.28(m, 4H), 7.55-7.72(m, 2H)

LRMS(APCI)507[MH⁺]

[α]²⁵ _D＝-79.7(c＝0.3，MeOH)

Example 16

(3R, 4R, 5S) -4- (2, 4-difluorophenyl) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidine-4-ol hydrochloride

The title compound was prepared from the compounds of preparation 49 and 53 by a similar method to that described in example 12.

¹H NMR(400MHz，CD₃OD) delta (rotamer), 0.36-0.66(m, 6H), 0.81-1.97(m, 2H), 2.70(m, 1H), 3.19-4.05(m, 8H), 4.31(m, 1H), 7.05-7.35(m, 4H), 7.45-7.65(m, 2H)

LRMS(APCI)451[MH⁺]

[α]²⁵ _D＝-42.7(c＝0.3，MeOH)

Example 17

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (2, 6-difluorophenyl) -3, 5-dimethylpiperidine-4-ol hydrochloride

The title compound was prepared from the compounds of preparation examples 1 and 44 according to a similar method to that described in example 9.

¹H NMR(400MHz，CD₃OD) delta (rotamer), 0.31-0.59(m, 6H), 0.83-2.08(m, 2H), 1.55(s, 9H), 1.60-2.07(m, 2H), 2.68-3.20(m, 2H), 3.20-4.12(m, 5H), 4.29(m, 1H), 7.05-7.15(m, 4H), 7.45-7.65(m，2H)

LRMS(APCI)507[MH⁺]

[α]²⁵ _D＝-77.7(c＝0.3，MeOH)

Example 18

(3R, 4R, 5S) -4- (2, 6-difluorophenyl) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidine-4-ol hydrochloride

The title compound was prepared from the compounds of preparation 44 and 53 by a similar method to that described in example 12.

¹H NMR(400MHz，CD₃OD) delta (rotamer), 0.36-0.57(m, 6H), 0.81-1.97(m, 2H), 2.70(m, 1H), 3.15-4.05(m, 8H), 4.31(m, 1H), 7.05-7.35(m, 4H), 7.50-7.63(m, 2H).

LRMS(APCI)451[MH⁺]

[α]²⁵ _D＝-22.7(c＝0.3，MeOH)

Example 19

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (3-fluorophenyl) -3, 5-dimethylpiperidin-4-ol hydrochloride

The title compound was prepared from the compounds of preparation 1 and 46 by a similar method to that described in example 9.

¹H NMR(400MHz，CD₃OD) delta (rotamer), 0.31-0.54(m, 6H), 0.83-2.08(m, 2H), 1.55(s, 9H), 1.58-2.09(m, 2H), 2.68-3.20(m, 2H), 3.25-4.15(m，5H)，4.29(m，1.H)，6.95-7.19(m，4H)，7.33(m，1H)，7.38-7.85(m，2H))

LRMS(APCI)489[MH⁺]

[α]²⁵ _D＝-81.3(c＝0.3，MeOH)

Example 20

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4- (3-fluorophenyl) -3, 5-dimethylpiperidin-4-ol hydrochloride

The title compound was prepared from the compounds of preparation 46 and 53 by a similar method to that described in example 12.

¹H NMR(CD₃OD, 400MHz) (rotamer), 0.36-0.60, (m, 6H)0.81-2.01(m, 2H), 2.70(m, 1H), 3.19-4.05(m, 8H), 4.31(m, 1H), 6.95-7.15(m, 4H), 7.32(m, 1H), 7.28-7.80(m, 2H)

LRMS(APCI)433[MH⁺]

[α]²⁵ _D＝-72.7(c＝0.3，MeOH)

Example 21

(3R, 4R, 5S) -4- (4-chlorophenyl) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-methylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidin-4-ol hydrochloride

The title compound was prepared from the compounds of preparation 43 and 62 according to a similar method to that described in example 9.

¹H NMR(CD₃OD, 400MHz) (rotamer), 0.20-0.62(m, 6H)1.03-1.98(m, 2H))，2.67(m，1H)，3.09-4.16(m，10H)，4.31(m，1H)，6.95-7.19(m，5H)，7.38-7.85(m，2H)

LRMS(APCI)463[MH⁺]

[α]²⁵ _D＝-39.3(c＝0.3，MeOH)

Example 22

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-pyridin-2-ylpiperidin-4-ol hydrochloride

A solution of (3R, 4S, 5S) -3, 5-dimethyl-4-pyridin-2-ylpiperidin-4-ol (260mg, 1.26mmol) from preparation 74, (3R, 4S) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid (267mg, 0.94mmol) from preparation 1, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (181mg, 0.95mmol) and 1-hydroxybenzotriazole hydrate (9mg, 0.07mmol) in tetrahydrofuran (5ml) was stirred at room temperature for 18 hours. The solvent was removed in vacuo and the residue partitioned between water (5ml) and ethyl acetate (5 ml). The phases were separated and the aqueous phase was extracted with ethyl acetate (2X 5 ml). The combined organic extracts were dried over magnesium sulfate, filtered, and concentrated in vacuo to give a crude residue. Purification by column chromatography on silica gel eluting with dichloromethane: methanol (100: 0-99: 1-97: 3-92: 8) followed by dichloromethane: methanol: 0.88 ammonia (95: 5: 0.5) gave the desired product as a colourless oil, 11 mg. It is converted to the hydrochloride salt by treatment with a 4M solution of hydrogen chloride in dioxane, followed by evaporation of the solvent.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.32-0.64(m, 6H), 0.69-2.42(m, 5H), 2.60-3.22(m, 5H), 3.47-4.17(m, 10H), 4.46(m, 1H), 7.00-7.26(m, 2H), 7.97(m, 1H), 7.53-8.66(m, 3H), 8.71(d, 1H)

LRMS(APCI)472[MH⁺]

[α]_D ²⁵＝-42.46(c＝0.35，MeOH)

Example 23

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-pyridin-2-ylpiperidin-4-ol hydrochloride

To a stirred solution of tert-butyl (3R, 4R, 5S) -3- (2, 4-difluorophenyl) -4- ([ (3R, 4R, 5S) -4-hydroxy-3, 5-dimethyl-4-pyridin-2-ylpiperidin-1-yl ] carbonyl } pyrrolidine-1-carboxylate (250mg, 0.48mmol) from preparation 54 in dichloromethane (2mL) was added a 4M solution of hydrochloric acid in dioxane (3.9mL) at room temperature, the reaction mixture was stirred for 27 h, the solvent was removed in vacuo to give a white solid which was triturated with diethyl ether to give the desired product in quantitative yield.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.36-0.66(m, 6H), 1.08-2.41(m, 2H), 2.66-3.23(m, 2H), 3.45-4.10(m, 7H), 4.47(m, 1H), 7.01-7.24(m, 2H), 7.53-7.72(2xq, 1H), 7.74-8.22(m, 2H), 8.63(m, 1H), 8.72(d, 1H)

LRMS(APCI)472[MH⁺]

[α]_D ²⁵＝-44.00(c＝0.37，MeOH)

Example 24

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] -carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

The title compound was prepared from the compound of preparation 55 according to a similar method to that described for example 23.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.27-0.56(m, 6H), 0.77-2.06(m, 2H), 2.68-3.19(m, 2H), 3.40-4.08(m, 7H), 4.30(m, 1H), 6.98-7.39(m, 7H), 7.46-7.64(2xq, 1H)

LRMS(APCI)415[MH⁺]

[α]_D ²⁵＝-51.81(c＝0.47，MeOH)

Example 25

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-butyl-4- (2, 4-difluorophenyl) pyrazin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

To a stirred solution of (3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride (135mg, 0.3mmol) from example 24 in dichloromethane (2mL) was added triethylamine (85. mu.L, 0.61mmol) at room temperature. The reaction mixture was stirred for 10 min, butyraldehyde (54 μ L, 0.61mmol) was added and the solution was stirred for an additional 20 min. Sodium triacetoxyborohydride (95mg, 0.45mmol) was then added and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with dichloromethane (3X 2mL) and saturated sodium bicarbonate solution (6mL) was added. The phases were separated and the aqueous phase was extracted with dichloromethane (3X 2 mL). The organic fractions were combined, dried over magnesium sulfate and concentrated in vacuo to give a crude residue. Purification by column chromatography on silica gel using dichloromethane: methanol (100: 0-97: 3) as eluent gave 45mg of pure product. It is converted to the hydrochloride salt by dissolving in dichloromethane, treating with 2M hydrogen chloride in diethyl ether, and then evaporating the solvent.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.20-0.58(m, 6H), 0.65-2.06(m, 2H), 1.02(t, 3H), 1.47 (gm，2H)，1.77(m，2H)，2.67-3.20(m，2H)，3.32-4.22(m，9H)，4.32(m，1H)，7.00-7.40(m，7H)，7.54-7.74(m，1H)

LRMS(APCI+)＝471[MH⁺]

[α]_D ²⁵＝-60.39(c＝0.32，MeOH)

Example 26

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-isobutylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

The title compound was prepared from the compound of example 24 and isobutyraldehyde according to a similar method to that described for example 25.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.19-0.79(m, 6H), 1.10(t, 6H), 0.93-2.07(m, 2H), 2.16(m, 1H), 2.68-4.22(m, 11H), 4.32(m, 1H), 6.60-7.43(m, 7H), 7.56-7.76(m, 1H)

LRMS(APCI)471[MH⁺]

[α]_D ²⁵＝-71.94(c＝0.31，MeOH)

Example 27

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-propylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

To a solution of (3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride (250mg, 0.55mmol) from example 24 in acetonitrile (3mL) was added triethylamine (115. mu.L, 0.83mmol), potassium carbonate (151mg, 1.11mmol) and 1-bromopropane (55. mu.L, 0.61mmol) at room temperature. The reaction mixture was heated to 40 ℃ for 90 minutes. The mixture was cooled and the solvent was removed in vacuo. The residue was partitioned between water (40mL) and ethyl acetate (40 mL). The phases were separated and the organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo to give a crude residue. Purification by column chromatography on silica eluting with dichloromethane: methanol (100: 0-99: 1-98: 2-97: 3-96: 4) gave the desired product as a colourless oil, 193mg (70%). It was converted to the hydrochloride salt as follows: treatment with 4M dioxane solution of hydrochloric acid, followed by evaporation of the solvent, gave a white solid.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.20-0.57(m, 6H), 1.05(t, 3H), 1.81(q, 2H), 0.83-2.04(m, 2H), 2.69-3.18(2xm, 2H), 3.20-4.18(m, 9H), 4.31(m, 1H), 6.94-7.38(m, 7H), 7.51-7.70(m, 1H)

LRMS(APCI)457[MH⁺]

[α]_D ²⁵＝-62.57(c＝0.33，MeOH)

Example 28

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-methylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-pyridin-3-ylpiperidin-4-ol trifluoroacetate

To a solution of (3R, 4R) -4- (2, 4-difluorophenyl) -1-methylpyrrolidine-3-carboxylic acid from preparation 62 (323mg, 1.16mol) and (3R, 4S, 5S) -3, 5-dimethyl-4-pyridin-3-ylpiperidin-4-ol from preparation 51 (200mg, 0.96mmol) in ethyl acetate (5mL) at room temperature was added triethylamine (400. mu.L, 2.88mmol), followed by 1-propylphosphonic acid cyclic anhydride (50% in ethyl acetate) (570. mu.L, 0.96 mmol). The reaction mixture was stirred for 24 h, treated with saturated potassium carbonate solution (10mL), and diluted with ethyl acetate (5 mL). The phases were separated and the organic layer was washed with saturated potassium carbonate solution (2X 30mL), brine (1X 30mL) and dried over magnesium sulfate. The solvent was removed in vacuo to give a crude residue. Purification by reverse phase silica gel chromatography using acetonitrile/water/trifluoroacetic acid (5: 95: 0.1-100: 0) as eluent gave a colourless oil, azeotroped with toluene, triturated with diethyl ether and evaporated to dryness to give 44mg of a white solid.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.23-0.60(m, 6H), 1.03-2.13(m, 2H), 3.06(s, 3H), 2.67-3.18(m, 2H), 3.30-4.15(m, 7H), 4.38(m, 1H), 7.10(m, 2H), 7.48-7.67(m, 1H), 7.89(m, 1H), 8.05-8.81(m, 3H)

LRMS(APCI)430[MH⁺]

Example 29

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyrimidin-2-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

A solution of (3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride (250mg, 0.55mmol), 2-bromopyrimidine (123mg, 0.79mmol) and triethylamine (230. mu.L, 1.65mmol) in ethanol (5mL) from example 24 was heated at reflux for 24 hours. The solvent was removed in vacuo and the residue partitioned between ethyl acetate (5mL) and water (5 mL). The phases were separated and the organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo to give a crude residue. Purification by column chromatography on silica eluting with dichloromethane: methanol (100: 0-99: 1-98: 2-97: 3) gave the desired product as a white foam, 220mg (74%). It was converted to the hydrochloride salt as follows: treatment with 4M hydrogen chloride in dioxane, followed by evaporation of the solvent.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.36-0.60(m, 6H), 0.76-2.10(m, 2H), 2.70-3.25(m, 2H), 3.63-4.37(m, 9H), 6.98-7.44(m, 8H), 7.46-7.68(m, 1H), 8.64(d, 2H)

LRMS(ESI+)＝493[MH⁺]

[α]_D ²⁵＝-52.10(c＝0.44，MeOH)

Example 30

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-cyclobutyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

The title compound was prepared from the compound of example 24 and cyclobutanone according to a similar method to that described in example 25, except that ethanol was used as the solvent.

¹H NMR(40OMHz，CD₃OD) (rotamer) delta 0.15-0.51(3Xm, 6H), 0.58-2.01(m, 4H), 2.15-3.13(m, 6H), 3.25-4.17(m, 8H), 4.25(m, 1H), 6.93-7.35(m, 7H), 7.47-7.66(m, 1H)

LRMS(APCI)469[MH⁺]

[α]_D ²⁵＝-61.50(c＝0.45，MeOH)

Example 31

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyridin-2-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

To a solution of (3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride (200mg, 0.44mmol) from example 24 in toluene (4mL) was added sodium 2-methylpropan-2-ol (97mg, 1.31mmol), and the reaction was stirred at room temperature for 10 minutes. 2-bromopyridine (63. mu.L, 0.66mmol) was added followed by tris (dibenzylideneacetone) dipalladium (O) (40mg, 0.04mmol), (+/-)1, 1 '-binaphthyl-2, 2' -diylbis (diphenylphosphine) (55mg, 0.09mmol) and the reaction mixture was heated at reflux for 24 h. The solvent was removed in vacuo and the residue partitioned between ethyl acetate (4mL) and water (4 mL). The phases were separated and the organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo to give a crude residue. Purification by column chromatography on silica gel using dichloromethane: methanol (100: 0-96: 4) as eluent gave the desired product as a foam, 160mg (67%). It was converted to the hydrochloride salt as follows: treatment with 4M hydrogen chloride in dioxane, followed by evaporation of the solvent.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.38-0.62(m, 6H), 0.87-2.14(m, 2H), 2.70-3.24(m, 2H), 3.51-4.20(m, 7H), 4.33(m, 1H), 6.58-6.68(m, 2H), 6.93-7.62(m, 9H), 8.02(m, 1H)

LRMS(APCI)492[MH⁺]

[α]_D ²⁵＝-44.46(c＝0.37，MeOH)

Example 32

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1- (2-methoxyethyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

The title compound was prepared in a similar manner to that used in example 27 from the compound of example 24 and 1-bromo-2-methoxyethane.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.20-0.58(m, 6H), 0.77-2.05(m, 2H), 2.68-3.19(m, 2H), 3.43(s, 3)H)，3.30-4.20(m，11H)，4.31(m，1H)，6.98-7.38(m，7H)，7.50-7.71(m，1H)

LRMS(APCI)473[MH⁺]

[α]_D ²⁵＝-62.03(c＝0.32，MeOH)

Example 33

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyrazin-2-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

A solution of (3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride (200mg, 0.44mmol), 2-chloropyrazine (75mg, 0.84mmol) and triethylamine (122. mu.L, 0.88mmol) from example 24 in N, N-dimethylformamide (4mL) was heated at 100 ℃ for 24 hours. The solvent was removed in vacuo and the crude residue partitioned between water (4mL) and ethyl acetate (4 mL). The phases were separated and the organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo to give a crude residue. Purification by column chromatography on silica gel using ethyl acetate pentane (10: 90-100: 0) as eluent gave the desired product as a foam. 121mg (55%). It was converted to the hydrochloride salt as follows: treatment with 4M hydrogen chloride in dioxane, followed by evaporation of the solvent.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.37-0.60(m, 6H), 0.82-2.10(m, 2H), 2.70-3.26(m, 2H), 3.64-4.21(m, 7H), 4.34(m, 1H), 6.94-7.43(m, 7H), 7.43-7.64(m, 1H), 7.90(d, 1H), 8.26(m, 2H)

LRMS(APCI)493[MH⁺]

[α]_D ²⁵＝-46.98(c＝0.31，MeOH)

Example 34

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyridin-3-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

The title compound was prepared from the compound of example 24 and 3-bromopyridine according to a similar method to that described in example 31.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.36-0.57(m, 6H), 0.83-2.08(m, 2H), 2.69-3.24(m, 2H), 3.56-4.23(m, 7H), 4.33(m, 1H), 6.94-7.62(m, 8H), 7.70-7.83(m, 2H), 7.98-8.10(m, 2H)

LRMS(APCI)492[MH⁺]

[α]_D ²⁵＝-33.26(c＝0.36，MeOH).

Example 35

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyridazin-3-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

The title compound was prepared according to a similar method to that described in example 31 from the compound of example 24 and 3-chloropyridazine (j.med.chem.30(2), 239, 1987).

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.35-0.58(m, 6H), 0.73-2.08(m, 2H), 2.68-3.24(m, 2H), 3.60-4.27(m, 7H), 4.30(m, 1H), 6.97-8.11(m, 10H), 8.50-9.30(2xd, 1H)

LRMS(APCI)493[MH⁺]

[α]_D ²⁵＝-36.61(c＝0.31，MeOH)

Example 36

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyrimidin-5-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

The title compound was prepared from the compound of example 24 and 5-bromopyrimidine according to a similar method to that described in example 31.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.37-0.60(m, 6H), 0.85-2.12(m, 2H), 2.70-3.26(m, 2H), 3.60-4.22(m, 7H), 4.35(m, 1H), 6.93-7.43(m, 7H), 7.43-7.64(m, 1H), 8.52(m, 2H), 8.71-9.26(m, 1H)

LRMS(APCI)493[MH⁺]

[α]_D ²⁵＝-37.45(c＝0.25，MeOH)

Example 37

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4-isopropyl-3, 5-dimethylpiperidine-4-ol hydrochloride

The title compound was prepared from the compounds of preparation 1 and 57 according to a similar method to that described in example 8.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.37-0.97(m, 12H), 1.46(s, 9H), 1.33-2.07(m, 3H), 2.55-3.05(m, 2H), 3.07-4.06(m, 8H), 7.05(m, 2H), 7.60(m, 1H)

LRMS(APCI)437[MH⁺]

[α]_D ²⁵＝-26.07(c＝0.60，MeOH)

Example 38

(3R, 4R, 5S) -1- { [ (3S, 4R) -1- (cyclopropylmethyl) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

The title compound was prepared according to a similar method to that described for example 27 from the compound of example 24 and (bromomethyl) cyclopropane.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.20-0.80(m, 10H), 1.19(m, 1H), 0.83-2.02(m, 2H), 2.68-3.18(m, 2H), 3.19-4.18(m, 9H), 4.31(m, 1H), 7.00-7.38(m, 7H), 7.52-7.71(m, 1H)

LRMS(APCI+)＝469[MH⁺]

[α]_D ²⁵＝-66.40(c＝0.28，MeOH)

Example 39

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1- (tetrahydro-2H-pyran-4-yl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidine-4-ol hydrochloride

The title compound was prepared in a similar manner to that described in example 25 from the compound of example 24 and tetrahydro-4H-pyran-4-one, except that ethanol was used as the solvent.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.18-0.55(m, 6H), 0.782.20(m, 6H), 2.67-3.18(m, 2H), 3.30-4.18(m, 12H), 4.30(m, 1H), 7.00-7.35(m, 7H), 7.53-7.71(m, 1H)

LRMS(APCI)499[MH⁺]

[α]_D ²⁵＝-51.40(c＝0.37，MeOH)

Example 40

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-propylpiperidin-4-ol hydrochloride

The title compound was prepared from the compounds of preparation examples 1 and 59 according to a similar method to that described in example 8.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 1.45(s, 9H), 0.19-1.66(m, 15H), 2.49-304(m, 2H), 3.17-4.05(m, 7H), 4.11(m, 1H), 7.05(m, 2H), 7.59(m, 1H).

LRMS(APCI)437[MH⁺]

[α]_D ²⁵＝-28.03(c＝0.38，MeOH)

EXAMPLE 41

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-cyclopropyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

1M sodium hydroxide solution (15mL) was added to (3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride from example 24 (200mg, 0.48 mmol). The suspension was stirred and extracted with ethyl acetate (2X 30 mL). The organic layers were combined, dried over magnesium sulfate, filtered, and concentrated in vacuo. The residual oil was dissolved in methanol (5mL) and acetic acid (275. mu.L, 4.8mmol) and [ (1-ethoxycyclopropyl) oxy ] (trimethyl) silane (580. mu.L, 2.88mmol) and sodium triacetoxyborohydride (90mg, 0.96mmol) were added at room temperature. The reaction mixture was heated at reflux for 2 hours, cooled to room temperature, and methanol (5mL) was added. The mixture was filtered and the filtrate was concentrated in vacuo. The recovered solid was partitioned between 1M sodium hydroxide solution (10mL) and ethyl acetate (10 mL). The phases were separated and the aqueous phase was extracted with ethyl acetate (2X 5 mL). The organic extracts were combined, dried over magnesium sulfate, filtered, and concentrated in vacuo to give a crude residue. Purification by column chromatography on silica gel using dichloromethane: methanol (100: 0-97: 3) as eluent gave the desired product as a colourless oil 131 mg. It was converted to the hydrochloride salt as follows: treatment with 4M hydrogen chloride in dioxane, followed by evaporation of the solvent, gave a white solid.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.20-0.55(m, 6H), 1.03(m, 4H), 0.76-2.05(m, 2H), 2.67-3.18(m, 2H), 3.29-4.24(m, 8H), 4.30(m, 1H), 6.99-7.37(m, 7H), 7.54-7.71(m, 1H)

LRMS(ESI+)＝455[MH⁺]

[α]_D ²⁵＝-64.04(c＝0.26，MeOH)

Example 42

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyrimidin-4-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride

A solution of (3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride (200mg, 0.44mmol), 4-chloropyrimidine (Biorg. chem.30(3), 188, 2002) (140mg, 0.88mmol) and triethylamine (250. mu.L, 1.80mmol) in N, N-dimethylformamide (3mL) from example 24 was heated at 80 ℃ for 3 hours. The reaction mixture was cooled to room temperature and the solvent was removed in vacuo. The crude residue was partitioned between ethyl acetate (5mL) and water (5 mL). The phases were separated and the organic phase was washed with 1M sodium hydroxide solution (2 × 20mL) and brine (1 × 20mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give the desired compound as a yellow foam 192mg (81%). It was converted to the hydrochloride salt as follows: treatment with 4M hydrogen chloride in dioxane, followed by evaporation of the solvent, gave a yellow oil. The oil was triturated with diethyl ether to give a solidified product which was then isolated by filtration.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.32-0.58(m, 6H), 0.75-2.08(m, 2H), 2.67-3.23(m, 2H), 3.60-4.47(m, 9H), 6.84-7.40(m, 8H), 8.19(t, 1H), 8.71(m, 1H)

LRMS(ESI+)＝493[MH⁺]

[α]_D ²⁵＝-54.71(c＝0.35，MeOH)

Example 43

(3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4-cyclopropyl-3, 5-dimethylpiperidine-4-ol hydrochloride

The title compound was prepared from the compounds of preparation examples 1 and 61 according to a similar method to that described in example 8.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.19-0.53(m, 4H), 0.65-0.99(m, 6H), 1.27-1.74(m, 2H), 1.47(s, 9H), 2.50-3.01(m, 2H), 3.15-4.04(m, 8H), 4.12(m, 1H), 7.08(m, 2H), 7.49-7.64(m, 1H)

LRMS(ESI+)＝435[MH⁺]

[α]_D ²⁵＝-21.74(c＝0.33，MeOH)

Example 44

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyrimidin-4-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-pyridin-2-ylpiperidine-4-ol hydrochloride

The title compound was prepared according to a similar method to that described in example 42 from the compound of example 23 and 4-chloropyrimidine (biorg. chem.30(3), 188, 2002).

¹H NMR(400MHz，CD₃OD) rotamers delta 0.45-0.65(m, 6H), 1.07-2.50(m, 2H), 2.66-3.28(m, 2H), 3.79-4.52(m, 8H), 6.90-8.26(m, 7H), 7.96(m, 1H), 8.56(m, 1H), 8.73(m, 2H)

LRMS(APCI+)＝494[MH⁺]

[α]_D ²⁵＝-30.35(c＝0.30，MeOH)

Example 45

(3R, 4R, 5S) -4- (3, 4-difluorophenyl) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidine-4-ol hydrochloride

To a solution of tert-butyl (3R, 4S) -3- (2, 4-difluorophenyl) -4- { [ (3R, 4R, 5S) -4- (3, 4-difluorophenyl) -4-hydroxy-3, 5-dimethylpiperidin-1-yl ] carbonyl } pyrrolidine-1-carboxylate (176mg, 0.32mmol) from preparation 63 in dichloromethane (2mL) was added a 4M solution of hydrogen chloride in dioxane (2mL) at room temperature. The reaction mixture was stirred at room temperature overnight. The solvent was then evaporated and the residual solid triturated with diethyl ether (10mL), filtered and dried in a vacuum oven to give the title compound as a white solid, 116 mg.

¹H NMR(400MHz，CD₃OD) (rotamer) □ 0.31-0.76(m, 7H), 1.81-2.00(m, 1H), 2.65-2.81(m, 1.5H), 3.14(t, 0.5H), 3.48(m, 2H), 3.66-3.81(m, 3H), 3.89(m, 1H), 4.03(m, 1H), 4.34(d, 1H), 7.04-7.27(m, 4H), 7.46-7.60(m, 2H).

LRMS(APCI)451[MH⁺]

[□]²⁵ _D＝-65.29(c＝0.17，MeOH).

Example 46

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4- [4- (trifluoromethyl) phenyl ] piperidin-4-ol hydrochloride

The title compound was prepared from the compound of preparation 66 by a similar method to that described for example 45.

¹H-NMR(400MHz，CD₃OD): (rotamers) □ □ 0.31-0.56(4xd, 7H), 0.83-2.06(4xbm, 2H), 2.69-2.90(m, 1.5H), 3.16(t, 0.5H), 3.50(m, 2H), 3.56-3.82(m, 3H), 3.91(m, 1H), 4.05(m, 1H), 4.34(d, 1H), 7.02-7.23(m, 3H), 7.51-7.69(m, 3H).

LRMS(APCI)＝483[MH⁺]

[□]²⁵ _D＝-55.67(c＝0.26，MeOH).

Example 47

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-pyridazin-3-ylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-pyridin-2-ylpiperidine-4-ol hydrochloride

The title compound was prepared according to a similar method to that described in example 31 from the compound of example 23 and 3-chloropyridazine (j.med.chem.30(2), 239, 1987).

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.46-0.66(m, 6H), 1.02-2.49(m, 2H), 2.65-3.28(3xm, 2H), 3.78-4.27(m, 7H), 4.48(m, 1H), 6.98-7.22(m, 2H), 7.49-8.29(m, 5H), 8.55(d, 1H), 8.65(t, 1H), 8.73(d, 1H)

LRMS(APCI)494[MH⁺]

[α]_D ²⁵＝-21.45(c＝0.27，MeOH)

Example 48

(3R, 4R, 5S) -1- { [ (3S, 4R) -4- (4-chlorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidine-4-ol hydrochloride

The title compound was prepared from the compound of preparation 69 according to a similar method to that described in example 45.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.14-0.46(m, 6H), 0.42-1.93(m, 2H), 2.57-3.03(m, 2H), 3.25-3.72(m, 6H), 3.76-3.91(m, 1H), 4.20(m, 1H), 7.07-7.42(m, 9H).

LRMS(APCI)413[MH⁺]

[α]_D ²⁵＝-122.15(c＝0.36，MeOH)

Example 49

4- ((3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -4-hydroxy-3, 5-dimethylpiperidin-4-yl) benzonitrile

The title compound was prepared from the compounds of preparation 1 and 72 by a similar method to that described in example 9.

¹H NMR(400MHz，CD₃OD) delta (rotamer), 0.31-0.59(m, 6H), 0.83-2.08(m, 2H), 1.55(s, 9H), 1.60-2.07(m, 2H), 2.68-3.20(m, 2H), 3.20-4.12(m, 5H), 4.29(m, 1.H), 7.05-7.15(m, 3H), 7.62-7.73(m, 2H), 8.10-8.20(m, 2H)

LRMS(APCI)＝496[MH⁺]

[α]²⁵ _D＝-97.7(c＝0.30，MeOH)

Example 50

The title compound was prepared from the compounds of preparation 33 and 43 according to a similar method to that described in example 9.

¹H NMR(CD₃OD, 400 MHz): (rotamers), 0.30-0.54(3xd, 6H)0.81-2.18(3xm, 2H), 1.37(m, 6H), 1.55-2.28(3xm, 2H), 2.65(m, 1H), 3.11-4.18(m, 7H), 4.31(m, 1.H), 6.80-7.05(m, 5H), 7.30-7.65(m, 2H)

LRMS(APCI)＝492[MH⁺]

[□]²⁵ _D＝-43.8(c＝0.35，MeOH)

Example 51

(3R, 4R, 5S) -4- (3, 4-difluorophenyl) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidine-4-ol hydrochloride

The title compound was prepared from the compounds of preparation 39 and 33 according to a similar method to that described in example 8.

¹H-NMR(400MHz，CD₃OD): (rotamer) □ ═ 0.30-0.59(4xd, 6H), 1.36-1.41(m, 6H), 1.66-1.98(m, 1H), 2.66-2.81(m, 2H), 3.12(t, 1H), 3.38-3.49(m, 3H), 3.61-3.70(m, 1H), 3.71-3.83(m, 2H), 3.90-4.05(m, 2H), 4.35(dd, 1H), 7.02-7.31(m, 5H), 7.57-7.68(m, 1H).

LRMS(APCI)＝493[MH⁺].

[□]²⁵ _D＝-49.77(c＝0.21，MeOH).

Example 52

(3R, 4R, 5S) -4- (2, 4-difluorophenyl) -1- { [ (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidin-3-yl ] carbonyl } -3, 5-dimethylpiperidine-4-ol hydrochloride

The title compound was prepared from the compounds of preparation 33 and 49 according to a similar method to that described in example 9.

¹H NMR(CD₃OD, 400 MHz): (rotamers), 0.34-0.52(3xd, 6H)0.79-1.20(3xm, 2H), 1.39(m, 6H), 1.49-2.30(3xm, 2H), 2.65(m, 1H), 3.19-4.21(m, 7H), 4.35(m, 1H), 6.80-7.18(m, 4H), 7.28-7.75(m, 2H)

LRMS(APCI)＝493[MH⁺]

[□]²⁵ _D＝-49.8(c＝0.33，MeOH)

Example 53

The title compound was prepared from the compounds of preparation 46 and 75 according to a similar method to that described in example 9.

¹H NMR(CD₃OD, 400 MHz): (rotamers), 0.28-0.56(3xd, 6H)0.85-1.98(3xm, 2H), 1.47(m, 4H), 1.55-2.05(3xm, 2H), 2.55(m, 1H), 3.11-4.20(m, 7H), 4.31(m, 1H), 6.85-7.20(m, 5H), 7.35(m, 1H), 7.55-7.95(m, 1H)

LRMS(APCI)＝461[MH⁺]

[□]²⁵ _D＝-57.3(c＝0.35，MeOH)

Preparation example 1

(3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid hydrochloride

To a stirred solution of methyl (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylate (6.1g, 20.5mmol) from preparation 2 in diethyl ether (60mL) was added potassium trimethylsilanolate (3.5g, 24.6mmol) in one portion at room temperature under dry nitrogen. The resulting mixture was stirred at room temperature under dry nitrogen for 24 hours. 4M hydrogen chloride in dioxane (60mL) was then added and the resulting mixture was stirred at room temperature under dry nitrogen for 30 minutes and then concentrated in vacuo to give the hydrochloride salt of the title compound as a white solid containing potassium chloride (8.4g, ca. 100%).

¹H NMR(400MHz，CDCl₃)δ_H 1.40(9H，s)，3.45(2H，m)，3.90(4H，m)，7.00(2H，m)，7.60(1H，m)；LRMS(APCI)284(100％)[MH⁺].

Alternative methods (separation of zwitterions)

A solution of lithium hydroxide (0.93g, 39mmol) in water (15mL) was added dropwise to a stirred suspension of (4S) -4-benzyl-3- { [ (3S, 4R) -1-tert-butyl-4- (2, 4 difluorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one (8.63g, 19.5mmol) from preparation 22b in tetrahydrofuran (50 mL). The resulting reaction mixture was then stirred at room temperature for 1.5 h, diluted with water (50mL) and extracted with ethyl acetate (4X 150 mL). The aqueous layer was separated, treated with 2M aqueous hydrogen chloride (19.5mL), concentrated to dryness, and azeotroped with toluene (5X 50 mL). The residual white solid was triturated in dichloromethane (40mL) and filtered to remove insoluble lithium chloride. The filtrate was then evaporated to give the product as a white foam, 5.05 g.

MS m/z(APCI⁺)：284[MH⁺]；¹H NMR(CD₃OD，400MHz)δ1.44(s，9H)，3.36(m，2H)，3.64(t，1H)，3.25(dd，1H)，3.88(m，3H)，6.98(t，2H)，7.55(q，1H).

Preparation example 2

(3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid methyl ester

To a stirred mixture of methyl (2E) -3- (2, 4-difluorophenyl) acrylate from preparation 3 (10g, 50.5mmol) and N- (methoxymethyl) -2-methyl-N- [ (trimethylsilyl) methyl ester from preparation 4 under dry nitrogen at room temperature]A solution of propan-2-amine (10.3g, 50.5mmol) in dichloromethane (200mL) was added trifluoroacetic acid (0.39mL, 5.1 mmol). The resulting mixture was stirred at room temperature under dry nitrogen for 17 hours. Then another portion of N- (methoxymethyl) -2-methyl-N- [ (trimethylsilyl) methyl group from preparation 4 was added to the reaction mixture]Propan-2-amine (3.9g, 19.2mmol) and trifluoroacetic acid (0.39mL, 5.1 mmol). The resulting mixture was stirred at room temperature under dry nitrogen for 18 hours, then quenched by addition of saturated sodium bicarbonate solution (200mL) and extracted with ethyl acetate (3X 75 mL). The organic layers were combined, dried (magnesium sulfate), filtered, and concentrated in vacuo. The residue was purified by an automatic flash column chromatography System (CombiFlash ® Separation System Sg 100c by Isco) using a prepackaged column (Redisp)^TMDisposable Columns for Flash Chromatography by Isco, 40g column), eluted with a linear gradient of 5% ethyl acetate in pentane increasing the polarity to 100% ethyl acetate over 1 hour. The residue was then subjected to chiral HPLC (elution with 95: 5 hexane: isopropanol, flow rate 80mL/min, at ambient temperature, chiralpak AD 500 x 80mm column) to give the desired enantiomer of the title compound, here we call enantiomer 1, which is the faster eluting enantiomer (retention time 8 min), as a clear oil (6.1g, 80%), in enantiomeric excess > 99% according to the racemic standard as determined by chiral HPLC. The undesired enantiomer was the slower eluting component (enantiomer 2, retention time 8.7 min).

¹H NMR(400MHz，CDCl₃)δ_H 1.10(9H，s)，2.80(1H，m)，3.00(1H，m)，3.15(3H，m)，3.60(3H，s)，3.80(1H，m)，6.80(2H，m)，7.40(1H，m)；LRMS(APCI)298(100％)[MH⁺].

Preparation example 3

(2E) -methyl 3- (2, 4-difluorophenyl) acrylate

To a solution of 2, 4-difluorocinnamic acid (20g, 135mmol) in N, N-dimethylformamide (500mL) was added potassium carbonate (90g, 675mmol) followed by iodomethane (21mL, 337.5mmol) at room temperature under dry nitrogen. The resulting mixture was stirred at room temperature under dry nitrogen for 7 hours, then quenched by addition of water (1L) and extracted with diethyl ether (3X 200 mL). The organic extracts are combined and washed with magnesium sulfateDried, filtered and concentrated in vacuo. The residue was purified by an automatic flash column chromatography System (CombiFlash ® Separation System Sg 100c by Isco) using a prepackaged column (Redisp)^TMDisposable Columns for Flash chromatography by Isco, 120g column), linear gradient elution with 2% ethyl acetate in pentane to 10% ethyl acetate in pentane with increasing polarity over 1 hour to give the title compound as a white solid (20.5g, 77%).

¹H NMR(400MHz，CDCl₃)δ_H 3.80(3H，s)，6.50(1H，d)，6.85(1H，m)，7.50(1H，m)，7.75(1H，d)；LRMS(APCI)216[MNH₄ ⁺]，199[MH⁺].

Preparation example 4

N- (methoxymethyl) -2-methyl-N- [ (trimethylsilyl) methyl ] propan-2-amine

2-methyl-N- [ (trimethylsilyl) methyl ] propan-2-amine (4.31g, 27mmol) from preparation 5 was added over 45 minutes to an ice-cold mixture of methanol (1.29mL, 31.8mmol) and aqueous formaldehyde (37% w/v 2.49mL, 33 mmol). The heterogeneous mixture was stirred at 0 ℃ for 2 hours, then solid potassium carbonate (325 mesh) (1.08g, 13mmol) was added and the mixture was stirred at 0 ℃ for 30 minutes. The layers were separated and the aqueous phase was extracted with ethyl acetate (3X 20 mL). The combined organic layers were dried over sodium sulfate, filtered and evaporated under reduced pressure to give an 80: 20 mixture of the title compound and unreacted tert-butyl [ (trimethylsilyl) methyl ] amine as a colorless oil (5.09 g). The mixture was used directly in preparation 2 without further purification.

¹H NMR(400MHz，CD₃OD)δ_H 0.04(s，9H)，1.11(s，9H)，2.27(s，2H)，3.34(s，3H)，4.17(s，2H).

Preparation example 5

2-methyl-N- [ (trimethylsilyl) methyl ] propan-2-amine

The preparation of this intermediate is given in J.org.chem.53(1), 194, 1988. The alternative method is as follows: a solution of chloromethyltrimethylsilane (50g, 408mmol) and tert-butylamine (130mL) was heated in a sealed tube at 200 ℃ for 18 hours under dry nitrogen and quenched by addition of 2M sodium hydroxide solution (700 mL). The resulting mixture was extracted with diethyl ether (3X 100mL), the organic layers combined and distilled under 1 atmosphere of dry nitrogen to give the title compound as a clear oil (62g, 96%).

¹H NMR(400MHz，CDCl₃)δ_H 0.05(9H，s)，1.05(9H，s)，1.95(2H，s).

Alternative preparation example:

chloromethyl trimethylsilane (100mL, 730mmol) and tert-butylamine (250mL, 2400mmol) were placed in a sealed reaction vessel and heated for 18 hours while stirring vigorously. After cooling to room temperature, a slurry of the hydrochloride salt formed and residual excess tert-butylamine was poured into 4M sodium hydroxide solution (500mL) and stirred vigorously for 1 hour. The aqueous layer was separated and the organic layer was stirred vigorously with water (3X 500mL) (excess tert-butylamine was readily soluble in water and the product tert-butyl-trimethylsilylmethyl-amine was only slightly soluble). The residual organic layer was dried over sodium sulfate to give essentially pure tert-butyl-trimethylsilylmethyl amine (105.4g), which was used without further purification in preparation 23.

¹H NMR(400MHz，CD₃OD)0.05(s，9H)，1.05(s，9H)，1.95(s，2H).

Preparation example 6

(3R, 4S, 5S) -1-benzyl-4-cyclohexyl-3, 5-dimethylpiperidin-4-ol

(3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (prepared according to preparation 14; also as described in J.Med.chem.7, 726, 1964) (36g, 166mmol) is dissolved in anhydrous tetrahydrofuran (331mL) in a flame-dried flask under a dry nitrogen atmosphere. The solution was cooled to-78 deg.C and cyclohexylmagnesium chloride (2M in tetrahydrofuran) (2.42mL, 4.84mmol) was added dropwise over 2 hours. The reaction mixture was allowed to slowly reach room temperature over 18 hours. The reaction was quenched by careful addition of water (1L) and diluted with ethyl acetate (1L). The organic layer was separated, washed with water (2X 1L) and then brine. After drying over anhydrous sodium sulfate and filtration, the solution was evaporated to a yellow oily residue (about 50 g). The product was purified by flash chromatography on silica gel eluting with 10% acetone in hexane in two batches. To give the desired N-benzylpiperidinol intermediate¹H NMR estimates contained about 8% residual 1-benzyl-3, 5-dimethyl-piperidin-4-one.

Preparation example 7

(3R, 4S, 5S) -4-cyclohexyl-3, 5-dimethylpiperidin-4-ol

To a solution of (3R, 4S, 5S) -1-benzyl-4-cyclohexyl-3, 5-dimethylpiperidin-4-ol (23g, 76mmol) from preparation 6 in methanol (762mL) were added ammonium formate (24.07g, 381mmol) and palladium hydroxide (35%, 40.25g), followed by a 5M solution of hydrogen chloride in methanol (20 mL). The reaction flask was equipped with a condenser and heated in a 60 ℃ oil bath for 2 hours. The mixture was then filtered through Celite ®, washing the filter cake with ethyl acetate. The filtrate was concentrated in vacuo, then basified with 5M sodium hydroxide solution and extracted with ethyl acetate (approximately 600 mL). The organic layer was washed four times with 5M sodium hydroxide solution, and the resulting organic layer was dried over anhydrous sodium sulfate. After filtration, the aqueous layer was evaporated to give the title compound as a white powder (11g, 68%).

LC-MS 212[MH⁺]；¹H NMR(400MHz，CDCl₃) Delta 0.84(6H, d), 0.83-0.85[1H, m (mask)]，1.16(6H，m)，1.63-1.83(6H，m)，2.62-2.29(4H，m).

The relative stereochemistry of the product was established using X-ray crystallography, consistent with the stereochemistry reported in the literature for (3R, 4S, 5S) -3, 5-dimethyl-4-phenylpiperidin-4-ol [ j.med.chem.1964, 7, 726 ].

Preparation example 8

(3R, 4S, 5S) -1-benzyl-4-butyl-3, 5-dimethylpiperidin-4-ol

(3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (prepared according to preparation 14; also as described in J.Med.chem.7, 726, 1964) (500mg, 2.3mmol) is dissolved in anhydrous tetrahydrofuran (15mL) under a nitrogen atmosphere and placed in a flame-dried round-bottom flask. The solution was cooled to-78 deg.C, n-butylmagnesium chloride (2M in tetrahydrofuran) (2.42mL, 4.84mmol) was added dropwise via syringe, and the solution was allowed to reach room temperature. The reaction mixture was cooled again to 0 ℃, quenched by addition of water and diluted with ethyl acetate. The organic layer was separated, washed twice with water, then dried over anhydrous sodium sulfate and evaporated. The resulting residue was purified by flash chromatography on silica gel eluting with a gradient of 15% acetone in dichloromethane with increasing solvent polarity to 30% acetone in dichloromethane to give the title compound (300mg, 47%).¹H NMR(400MHz，CD₂Cl₂)δ0.78(6H，d)，0.91(3H，t)，1.00-1.20(2H，m)，1.26-1.33(2H，m)，1.47-1.52(2H，m)，1.80-1.85(2H，m)，1.98-2.03(2H，m)，2.48-2.51(2H，m)，3.43(2H，s)，7.21-7.30(5H，m).

Preparation example 9

(3R, 4S, 5S) -4-butyl-3, 5-dimethylpiperidin-4-ol

(3R, 4S, 5S) -1-benzyl-4-butyl-3, 5-dimethylpiperidin-4-ol from preparation 8 (300mg, 1.1mmol) is dissolved in methanol (10 mL). Palladium hydroxide on carbon (525mg) was added, followed by ammonium formate (237mg, 5.5mmol) and 2M hydrochloric acid solution (1.1mL, 2.2 mmol). The reaction mixture was heated to 60 ℃ overnight and then cooled to room temperature. The mixture was then filtered through Celite ® and the filter cake was washed with methanol (500 mL). The filtrate was evaporated, the residue diluted with water, adjusted to pH about 12 by addition of saturated sodium carbonate solution and extracted with ethyl acetate. The organic layer was washed with water, then dried over sodium sulfate and evaporated to give the title compound (75mg, 37%).

¹H NMR(400MHz，CD₂Cl₂)δ0.77(6H，d)，0.79-0.81(2H，m)，0.91(3H，t)，1.11-1.96(8H，m)，2.57-2.64(2H，m).

Preparation example 10

(3R, 4S, 5S) -3, 5-dimethyl-4- (4-methylphenyl) piperidin-4-ol

To a solution of 4-bromotoluene (0.80mL, 6.5mmol) in cyclohexane (2mL) at 0 deg.C under dry nitrogen was added n-butyllithium (2.5M in hexane) (2.5mL, 6.25 mmol). The resulting mixture was stirred at 0 ℃ under dry nitrogen for 2 hours. A solution of tert-butyl (3R, 5S) -3, 5-dimethyl-4-oxopiperidine-1-carboxylate (300mg, 1.3mmol) from preparation 11 in toluene (4.5mL) was then added, and the resulting mixture was stirred at 0 ℃ for 2.5 hours under dry nitrogen and then quenched with water (10mL) at 0 ℃. A2M hydrochloric acid solution (10mL) was added, the mixture was extracted with ethyl acetate (20mL), and the organic layer was discarded. The aqueous layer was basified to pH11 with 2M sodium hydroxide solution and extracted with ethyl acetate (2X 15 mL). The organic layers (from extraction of the basic aqueous phase only) were combined, dried over magnesium sulfate, filtered, and concentrated in vacuo to give the title compound as a white solid (136mg, 47%).

¹H NMR(400MHz，CDCl₃)δ_H 0.55(6H，m)，2.00(3H，m)，2.35(3H，s)，5.80(5H，m)，7.10(4H，m)；LRMS(APCI)220(100％)[MH⁺]；HRMS C₁₄H₂₂O[MH⁺]Theoretical 220.1695 found 220.1693.

Preparation example 11

(3R, 5S) -3, 5-dimethyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester

(3R, 5S) -1-benzyl-3, 5-dimethyl-piperidin-4-one from preparation 14 was dissolved in ethanol (200mL), di-tert-butyl dicarbonate (5.08g, 23mmol) was added, followed by palladium hydroxide on carbon (20%/carbon, 200mg), and the reaction mixture was placed under 40 atm of hydrogen and stirred at room temperature overnight. The reaction mixture was then filtered through a pad of Celite ® and Arbocel ® and concentrated in vacuo to give a yellow oil which crystallized upon standing to give the title compound (5.2g, 90%) pure enough to be used directly in the next stage (preparation 10).

¹H NMR(400MHz，CDCl₃) Δ 1.03(6H, d), 1.49 and 1.52[9H, 2xs (rotamers)]，2.48-2.76(4H，m)，4.24-4.53(2H，m).

Preparation example 12

2, 4-dimethyl-3-oxoglutarate dimethyl ester

Potassium carbonate (325 mesh) (298.8g, 2160mmol) was added to a solution of dimethyl 3-oxoglutarate (150.62g, 865mmol) in tetrahydrofuran (1.33L). Heating the suspension to 45 deg.C. Methyl iodide (107.7mL, 1.73mol) was added slowly at such a rate to maintain the temperature below 60 ℃. The slurry was stirred between 50-60 ℃ for 1 hour, then cooled to 20 ℃ and then filtered. The filter cake was washed with tetrahydrofuran (500mL), and the filtrates combined and concentrated to dryness in vacuo. Crude dimethyl 2, 4-dimethyl-3-oxoglutarate (179g) was quantitatively obtained as a pale yellow viscous oil. ¹H NMR indicated that the product was a tautomeric mixture of enol and ketone and was used without further purification in preparation 13.

MS(APCI^-)：201(M+H)；¹H NMR(400MHz，CDOD)1.25(s，6H)，3.65(s，6H).

Preparation example 13

1-benzyl-3, 5-dimethyl-4-oxopiperidine-3, 5-dicarboxylic acid dimethyl ester

A1M hydrochloric acid solution (69mL, 68.8mmol) was added to a cooled (9 ℃ C.) solution of dimethyl 2, 4-dimethyl-3-oxoglutarate (69.6g, 344mmol) from preparation 12 and benzylamine (37.6mL, 344mmol) in methanol (1.8L). 37% aqueous formaldehyde (56.8mL, 760mmol) was added and the solution was stirred at room temperature for 3 days and then concentrated to dryness. Crude dimethyl 1-benzyl-3, 5-dimethyl-4-oxopiperidine-3, 5-dicarboxylate (125.7g) was obtained as a light brown oil. GC-MS indicated that the product was 91% pure and was used in preparation 14 without further purification.

GC-MS：333(M+)；¹H NMR(400MHz，CDCl₃)δ7.27-7.38(m，5H)，3.64(s，6H)，3.62(s，2H)，3.48(d，2H)，2.21(d，2H)，1.26(s，6H).

Preparation example 14

(3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one

A mixture of crude dimethyl 1-benzyl-3, 5-dimethyl-4-oxopiperidine-3, 5-dicarboxylate (786.0g, ca. 2.3mol) and 1M hydrochloric acid solution (11.5L) was refluxed for 24 hours. The reaction mixture was cooled to 10 ℃ and 25% wt. aqueous sodium hydroxide solution (1.92kg) was slowly added. The mixture was extracted with dichloromethane (4X 4L) and the combined organic extracts were concentrated to dryness to give crude (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (475g) as a light brown oil. ¹H NMR indicated it to be a 6: 1 desired: undesired diastereomeric mixture. 205g of the crude product are purified over silica gel (4.7kg), eluting with hexane/ethyl acetate (20: 1 to 7: 1), to give 94.8g of pure (3R, 5S) -1-benzyl-3, 5-dimethyl-piperidin-4-one (> 19: 1 diastereomer ratio) and 44.8g of less pure product (. about.8: 1 diastereomer ratio). Both products were colorless oils.

Analytical data for (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one:

GC-MS：217(M+)；¹HNMR(400MHz，CDCl₃)δ7.27-7.38(m，5H)，3.60(s，2H)，3.15(m，2H)，2.70(m，2H)，2.04(t，J＝11.6Hz，2H)0.93(d，J＝6.6Hz，6H).

the alternative method comprises the following steps:

a mixture of crude methyl 1-benzyl-3, 5-dimethyl-4-oxo-piperidinecarboxylate (786.0g, ca. 2300mmol) from preparation 13 and 1M aqueous hydrochloric acid (11.5L) was refluxed for 24 hours. The reaction mixture was cooled to 10 ℃ and 25% wt. aqueous sodium hydroxide solution (1.92kg) was slowly added. The mixture was extracted with dichloromethane (4X 4L). The combined organic extracts were concentrated to dryness to give 1-benzyl-3, 5-dimethyl-piperidin-4-one (475g) as a light brown oil.¹H NMR indicated a 6: 1 cis: trans diastereomeric mixture.

A portion of the crude diastereomeric mixture (15g) was purified by an automated chromatographic purification system using a normal phase Redispe silica cartridge column (330g) with a solvent flow rate of 100mL/min, a cyclohexane/ethyl acetate 2-3% ethyl acetate linear gradient for 25 minutes, a 3-14% ethyl acetate linear gradient for 10 minutes, and elution was completed with 14% ethyl acetate. Pure (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (10.2g, 99% + by means of LC-MS) is obtained.

LC-MS(ESI⁺)：218(M+H)。

Alternatively, the crude cis/trans mixture may be enriched in the desired cis-component before purification by the following method:

the crude cis: trans mixture of 1-benzyl-3, 5-dimethylpiperidin-4-one (usually 6: 1 cis: trans) (45g) was added to a 5% solution of sodium methoxide in methanol (500mL) and stirred at room temperature for 6 hours. Saturated aqueous ammonium chloride (30mL) was added and the mixture was stirred at room temperature for another 30 minutes. The mixture was evaporated to dryness and then redissolved in dichloromethane (500 mL). Filtering off the insoluble solid and then evaporating the solvent to obtain an enriched mixture according to¹H NMR was 96: 4 cis: trans (quantitative mass recovery). Longer reaction times did not produce any further enrichment. If desired, purification by chromatography as described above can give the pure cis-product.

Preparation example 15

(3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4-phenylpiperidin-4-ol

(3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (prepared as preparation 14, also reported in J.Med.chem.7, 726, 1964) (5g, 23mmol) was dissolved in anhydrous tetrahydrofuran (77mL) in a flame-dried flask under a dry nitrogen atmosphere. The solution was cooled to-78 deg.C and phenyllithium (2M cyclohexane-ether solution) (34.6mL, 69mmol) was added dropwise. The reaction mixture was allowed to slowly reach room temperature overnight, then quenched by careful addition of water (50 mL). The resulting mixture was diluted with ethyl acetate and the organic layer was separated and then washed three times with water and once with brine. The organic layer was then dried (sodium sulfate), filtered and evaporated. The resulting residue was purified by flash chromatography on silica eluting with 10% to 50% ethyl acetate in hexane (1L) to give (3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4-phenylpiperidin-4-ol in > 90% purity.

LRMS：296(MH⁺)；¹H NMR(400MHz，CD₂Cl₂)δ0.52(6H，d)，2.09-2.24(4H，m)，2.67-2.71(2H，m)，3.54(2H，s)，7.22-7.38.

The alternative method comprises the following steps:

a solution of phenyllithium in diisopropyl ether (2M, 34.5mL, 690mmol) was added dropwise to a stirred solution of (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (10.0g, 46mmol) from preparation 14 in anhydrous diethyl ether (150mL) at-78 ℃. The mixture was stirred at-78 ℃ for another 30 minutes, then saturated aqueous ammonium chloride (10mL) was added and the mixture was allowed to warm to room temperature. The organic layer was separated, washed with water (3X 200mL), dried over sodium sulfate, and filtered. The solvent was then evaporated to give crude (3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4-phenylpiperidin-4-ol (12.8g) as a white solid. According to¹H NMR, crude compound > 95% pure, used directly in preparative example 21.

LC-MS(ESI⁺)：296(M+H)；¹¹H NMR(400MHz，CD₃OD)δ0.51(d，6H)，2.18(m，2H)，2.30(m，2H)，2.42(m，2H)，3.6(s，2H)，7.15(m，1H)，7.35(m，9H).

Preparation example 16

(3R, 4S, 5S) -3, 5-dimethyl-4-phenylpiperidin-4-ol

(3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4-phenylpiperidine-4-ol from preparation 15 was dissolved in methanol (156mL), ammonium formate (4.9g, 78mmol) was added followed by palladium on carbon hydroxide (8g) followed by 2M hydrochloric acid in diethyl ether (11mL, 22 mmol). The flask was fitted with a water condenser and the reaction was heated to 60 ℃ overnight. After cooling to room temperature, the reaction mixture was filtered through Celite ®, washing the filter cake with an additional 1L of methanol. The filtrates were combined, evaporated, the residue dissolved in a small amount of water and saturated sodium carbonate solution was added to make alkaline (pH 11). The resulting mixture was extracted twice with ethyl acetate, and the organic layers were combined, washed with water, dried over sodium sulfate, filtered, and concentrated to give the title compound (3.23g, 68%).

¹H NMR(400MHz，CD₂Cl₂)δ0.52(6H，d)，2.03-2.10(2H，m)，2.71-2.77(2H，m)，2.83-2.88(2H，dd)，7.22-7.38(5H，m).

The alternative method comprises the following steps:

(3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4-phenylpiperidin-4-ol (15g, 51mmol) from preparation 15 was dissolved in methanol, Pd (OH) was added₂/(C) 20% wt water (1.5 g). The mixture was hydrogenated at 50 deg.C/50 psi for 18 hours. The mixture was then filtered through an Arbocel filter and methanol was evaporated to give crude (3R, 4S, 5S) -3, 5-dimethyl-4-phenylpiperidin-4-ol as a white solid. The crude product was recrystallized from acetonitrile to give analytically pure (3R, 4S, 5S) -3, 5-dimethyl-4-phenylpiperidin-4-ol as white needles (9.6 g).

Preparation example 17

(3S^*，4R^*) -4- (2, 4-difluorophenyl) -1-ethylpyrrolidine-3-carboxylic acid hydrochloride

To a stirred solution of methyl (3S, 4R) -4- (2, 4-difluorophenyl) -1-ethylpyrrolidine-3-carboxylate (5.9g, 22mmol) from preparation 18 in diethyl ether (59mL) was added potassium trimethylsilanolate (2.36g, 26mmol) in one portion at room temperature under dry nitrogen. The resulting mixture was brought to room temperature N₂Stirred for 3 hours. Then add4M hydrogen chloride in dioxane (20mL) was added and the resulting mixture stirred at room temperature under dry nitrogen for 18 h, then concentrated in vacuo to give the title compound as a white solid containing a potassium chloride residue (7.0 g).

¹H NMR(400MHz，CDCl₃)δ_H 1.25(3H，m)，3.25(5H，m)，3.8(2H，m)，4.10(1H，m)，7.20(2H，m)，7.80(1H，m)；LRMS(APCI)256(100％)[MH⁺]；HRMS C₁₃H₁₅F₂O₂[MH⁺]Theoretical 256.1144 found 256.1142.

Preparation example 18

(3S^*，4R^*) -4- (2, 4-difluorophenyl) -1-ethylpyrrolidine-3-carboxylic acid methyl ester

To a stirred solution of methyl (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylate (10.5g, 43mmol) from preparation 19 in tetrahydrofuran (215mL) at room temperature under dry nitrogen was added iodomethane (3.8mL, 48mmol) and N, N-diisopropylethylamine (8.3mL, 48mmol) in one portion. The resulting mixture was stirred at room temperature under dry nitrogen for 72 hours, then quenched by addition of water (200mL) and extracted with ethyl acetate (2X 250 mL). The organic layers were combined, dried (magnesium sulfate), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography eluting with a 2: 1 mixture of pentane and ethyl acetate to increase the polarity to 1: 1. The title compound was obtained as a clear oil (7.9g, 68%).

¹H NMR(400MHz，CDCl₃)δ_H 1.15(3H，m)，2.45(1H，m)，2.55(1H，m)，2.65(1H，m)，2.95(3H，m)，3.15(1H，m)，3.65(3H，s)，3.85(1H，m)，6.80(2H，m)，7.40(1H，m)；LRMS(APCI)270(100％)[MH⁺].

Preparation example 19

(3S^*，4R^*) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid methyl ester

To a suspension of methyl (3S, 4R) -1-benzyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylate (15g, 45mmol) from preparation 20 in ethanol (225mL) under dry nitrogen at room temperature was added 10% palladium on carbon (Degussa type) (1.5g) and the reaction mixture was placed under hydrogen at 50psi pressure and heated to 40 ℃ overnight. After cooling to room temperature, the reaction mixture was filtered through Celite ® and concentrated in vacuo to give the title compound as an orange oil (10.8g, 98%).

¹H NMR(400MHz，CDCl₃)δ_H 2.85(1H，m)，3.15(1H，m)，3.30(2H，m)，3.45(1H，m)，3.65(4H，m)，6.80(2H，m)，7.20(1H，m)；LRMS(APCI)242(100％)[MH⁺]；HRMSC₁₂H₁₄F₂O₂[MH⁺]Theoretical 242.0987 found 242.0986.

Preparation example 20

(3S^*，4R^*) -1-benzyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid methyl ester

A solution of trifluoroacetic acid (2.42mL, 31.5mmol) in dichloromethane (5mL) was added to a stirred solution of N-benzyl-N- (methoxymethyl) trimethylsilylamine (45.1g, 190mmol) and methyl (2E) -3- (2, 4-difluorophenyl) acrylate from preparation 3 (25.1g, 126mmol) in dichloromethane (100mL) at 0-5 deg.C. After stirring at room temperature overnight, the organic solution was washed with saturated sodium bicarbonate solution and then brine. The resulting organic solution was dried over anhydrous sodium sulfate, filtered, and then evaporated. The residue was purified by flash chromatography on silica eluting with a toluene: tetrahydrofuran mixture (11: 1) to give the title compound (31.6mL, 71%) as a colorless oil.

¹H NMR(400MHz，CDCl₃)δ_H 2.80(1H，m)，3.05(3H，m)，(3.25(1H，m)，3.62(3H，s)，3.85(1H，m)，4.20(2H，s)，6.55(5H，m)，6.80(2H，m)，7.40(1H，m)；LRMS(APCI)332(100％)[MH⁺].

Preparation example 21

(4S) -4-benzyl-3- [ (2E) -3- (2, 4-difluorophenyl) prop-2-enoyl ] -1, 3-oxazolidin-2-one

A solution of oxalyl chloride (19mL, 216mmol) in dichloromethane (50mL) was added dropwise over a period of 0.5 h to an ice-cold stirred suspension of 2, 4-difluorocinnamic acid (20.0g, 108mmol) in dichloromethane (400mL) and N, N-dimethylformamide (0.4mL) (the off-gas from the reaction was purged with concentrated sodium hydroxide solution). Once the addition was complete, the reaction mixture was allowed to warm to room temperature and stirred at room temperature under nitrogen for 18 hours. The reaction mixture was then concentrated and azeotroped with dichloromethane (2X 50 mL). The resulting acid chloride was redissolved in dichloromethane (50mL) and the solution was added dropwise under nitrogen to a vigorously stirred suspension of lithium chloride (23.0g, 540mmol), triethylamine (76mL, 540mmol) and (S) - (-) -4-benzyl-2-oxazolidinone (18.3g, 103mmol) in dichloromethane (400mL) over 30 minutes. Once the addition was complete, the reaction mixture was stirred at room temperature under nitrogen for 2.5 hours. The reaction mixture was diluted with dichloromethane (200mL) and treated with 5% citric acid solution (500 mL). The organic layer was then separated and dried over magnesium sulfate. The dichloromethane was filtered and evaporated to give the crude product as an orange oil. The crude product was redissolved in dichloromethane (100mL) and the resulting solution was passed through a plug of silica eluting with dichloromethane. The filtrate (1L) was finally concentrated to give 30.8g of product as a white solid.

MS m/z(APCI⁺)：344[MH⁺]；¹H NMR(CDCl₃，400MHz)δ2.85(dd，1H)，3.36(dd，1H)，4.22(m，2H)，4.80(m，1H)，6.90(m，2H)，7.68(m，5H)，7.68(dd，1H)，7.91(d，1H)，8.01(dd，1H).

Preparation 22a

(4S) -4-benzyl-3- { [ (3R, 4S) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one

And

preparation 22b

(4S) -4-benzyl-3- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one

A stirred solution of (S) -4-benzyl-3- [3- (2, 4-difluoro-phenyl) -acryloyl ] -oxazolidin-2-one from preparation 21 (1.70g, 4.95mmol) and N- (methoxymethyl) -2-methyl-N- [ (trimethylsilyl) methyl ] propan-2-amine from preparation 4 (1.60g, 5.94mmol) in dichloromethane (15mL) was treated with trifluoroacetic acid (0.075mL, 1 mmol). The resulting mixture was stirred at room temperature under nitrogen for 4.5 hours. The reaction mixture was diluted with dichloromethane (50mL) and treated with saturated aqueous sodium bicarbonate (50 mL). The organic layer was separated and the aqueous layer was extracted with dichloromethane (50 mL). The organic fractions were combined and dried over magnesium sulfate. The dichloromethane was filtered and evaporated to give a crude mixture of diastereomers.

Chromatography on silica gel eluting with a gradient of pentane: ethyl acetate 80/20 to 10/90v/v gave first 0.74g (1.67mmol) of (4S) -4-benzyl-3- { [ (3R, 4S) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one as a colorless oil, 0.82g (1.85mmol) of (4S) -4-benzyl-3- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one was then obtained as a white solid.

(4S) -4-benzyl-3- { [ (3R, 4S) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one:

-MS m/z(APCI⁺)：443[MH⁺]；¹H NMR(CDCl₃，400MHz)δ1.12(s，9H)，2.77(dd，1H)，2.85(m，1H)，3.25(dd，1H)，3.17-3.47(m，1H)，4.15(m，3H)，4.65(m，1H)，6.74(t，1H)，6.82(t，1H)，7.17-7.42(m，6H).

(4S) -4-benzyl-3- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one:

-MS m/z(APCI⁺)：443[MH⁺]；¹H NMR(CDCl₃，400MHz)1.12(s，9H)，2.72(dd，1H)，2.83(m，2H)，3.20(m，2H)，3.36(t，1H)，4.14(m，3H)，4.29(m，1H)，4.67(m，1H)，6.77(t，1H)，6.85(t，1H)，7.08(m，2H)，7.24(m，3H)，7.43(m，1H).

the complete relative and absolute stereochemistry of (4S) -4-benzyl-3- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one was determined by X-ray analysis of the crystals obtained from ethyl acetate/pentane.

Preparation example 23

(45) -4-benzyl-3- [ (2E) -3- (4-chlorophenyl) prop-2-enoyl ] -1, 3-oxazolidin-2-one

A solution of oxalyl chloride (10.82mL, 124mmol) in dichloromethane (50mL) was added dropwise to a cooled solution of (2E) -3- (4-chlorophenyl) acrylic acid (11.33g, 62.0mmol) in dichloromethane (110mL) and N, N-dimethylformamide (0.4. mu.L, 0.01 mmol). After the reaction mixture was stirred for 24 h, the solution was added dropwise to a cooled solution of (4S) -4-benzyl-1, 3-oxazolidin-2-one (9.49g, 53.6mmol), triethylamine (39.2mL, 282mmol) and lithium chloride (11.95g, 282mmol) in dichloromethane (110 mL). The reaction mixture was slowly warmed to room temperature, stirred for 2 hours, and then water (50mL) was added. The mixture was diluted with dichloromethane (100mL) and 5% citric acid solution (2X 150mL) was added. The phases were separated and the organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo. Purification by column chromatography on silica gel using dichloromethane as eluent gave the desired product as a white solid, 14.6g (74%).

¹H NMR(400MHz，CDCl₃)δ2.86(dd，1H)，3.37(dd，1H)，4.23(m，2H)，4.81(m，1H)，7.21-7.41(m，7H)，7.57(d，2H)，7.87(2xd，2H)

LRMS(APCI)342[MH⁺]

Preparation example 24

(4S) -4-benzyl-3- { [ (3S, 4R) -1-benzyl-4- (4-chlorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one

To a cooled solution of (4S) -4-benzyl-3- [ (2E) -3- (4-chlorophenyl) prop-2-enoyl ] -1, 3-oxazolidin-2-one from preparation 23 (5g, 14.62mmol) and N-benzyl-1-methoxy-N- [ (trimethylsilyl) methyl ] methylamine (5.24mL, 20.47mmol) in dichloromethane (50mL) was added trifluoroacetic acid (60. mu.L, 0.73 mmol). The reaction mixture was stirred at 0 ℃ for 20 minutes, then warmed to room temperature and stirred for 24 hours. Sodium bicarbonate solution (80mL) was added and the reaction mixture was stirred for 10 minutes. The phases were separated, the organic phase was dried over magnesium sulfate and the solvent was removed in vacuo to give a yellow oil. Purification by column chromatography on silica gel using ethyl acetate: pentane (10: 50-50: 50) as eluent gave the desired product, which was the diastereomer from the second elution, as a white crystalline solid, 733mg (11%).

¹H NMR(400MHz，CDCl₃)δ2.63-2.82(m，3H)，3.09-3.25(m，3H)，3.67(dd，2H)，3.98-4.28(m，4H)，4.65(m，1H)，7.03(m，2H)，7.17-7.39(m，12H)

LRMS(APCI)475[MH⁺]

Preparation example 25

(3S, 4R) -1-benzyl-4- (4-chlorophenyl) pyrrolidine-3-carboxylic acid methyl ester

To a stirred solution of (4S) -4-benzyl-3- { [ (3S, 4R) -1-benzyl-4- (4-chlorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one (2.51g, 5.28mmol) from preparation 24 and dimethyl carbonate (2.22mL, 26.4mmol) in dichloromethane (40mL) was added sodium methoxide (1.42g, 26.4mmol) at room temperature. The reaction mixture was stirred for 24 hours and diluted with dichloromethane (50 mL). The phases were separated and the organic phase was washed with water (2 × 40mL), dried over magnesium sulfate and concentrated in vacuo. The crude residue was purified by column chromatography on silica gel using ethyl acetate: pentane (5: 95-20: 80) as eluent to give the desired product as a colorless oil 1.61g (79%).

¹H NMR(400MHz，CDCl₃)δ2.67-3.17(m，5H)，3.65(s，3H)，3.53-3.75(m，3H)，7.20-7.40(m，9H)

LRMS(APCI)330[MH⁺]

Preparation example 26

(3S, 4R) -4- (4-chlorophenyl) pyrrolidine-3-carboxylic acid methyl ester hydrochloride

To a solution of methyl (3S, 4R) -1-benzyl-4- (4-chlorophenyl) pyrrolidine-3-carboxylate (0.93g, 2.8mmol) from preparation 25 in dichloromethane (9mL) cooled with an ice bath was added 1-chloroethyl chloroformate (0.46 mL). The reaction mixture was allowed to warm to room temperature and stirred for 48 hours. The reaction mixture was then cooled to 0 deg.C and triethylamine (0.43mL, 3.1mmol) was added followed by additional 1-chloroethyl chloroformate (0.31mL, 2.8 mmol). The ice bath was removed and the reaction mixture was stirred at room temperature for 1.5 hours, then diluted with dichloromethane, washed with water (20mL), 5% aqueous citric acid (20mL), then dried over magnesium sulfate and filtered. The solvent was removed in vacuo and the residual oil refluxed in methanol (20mL) for 1 hour. The solvent was then removed in vacuo and the residue triturated with diethyl ether and filtered to give the desired product as a white solid, 0.874 g.

¹H NMR(400MHz，CD₃OD)δ3.64(s，3H)，3.31-3.83(m，6H)，7.36(s，4H)

LRMS(APCI)240[MH⁺]

Preparation example 27

(2E) -3- (2, 4-difluorophenyl) prop-2-enoylcarbonic acid tert-butyl ester

To a stirred solution of (2E) -3- (2, 4-difluorophenyl) acrylic acid (42.0g, 230mmol) in anhydrous tetrahydrofuran (400mL) was added triethylamine (37.5mL, 270mmol) and the reaction mixture was cooled to-70 ℃. Trimethylacetyl chloride (30mL, 250mmol) was added dropwise over 20 minutes and the solution was allowed to warm to room temperature over 1 hour. Thin layer chromatography analysis indicated that the desired product had formed and was used directly in the next step.

Preparation example 28

N-butyllithium (2.5M in hexane) (100mL, 250mmol) was added dropwise to a stirred solution of (S) - (-) -4-benzyl-2-oxazolidinone (43.55g, 250mmol) in anhydrous tetrahydrofuran (350mL) at 0 ℃. The resulting solution was cooled to-78 ℃ for 30 minutes and added dropwise via a cannula to a stirred solution of tert-butyl (2E) -3- (2, 4-difluorophenyl) prop-2-enoylcarbonate from preparation 27 at-78 ℃. The resulting suspension was warmed to 0 ℃ and saturated ammonium chloride solution (75mL) was added, followed by water (50 mL). The phases were separated and the aqueous layer was extracted with ethyl acetate (2X 300 mL). The combined organic extracts were dried over magnesium sulfate, filtered, and concentrated in vacuo to give a slurry. Cyclohexane (178.5mL) and tert-butyl methyl ether (126mL) were added to the slurry, and the mixture was stirred at room temperature for 2 hours. The resulting white solid was collected via filtration and dried in a vacuum oven at 40 ℃ to give the desired product 45.48g (61%).

¹H NMR(400MHz，CDCl₃)□2.82(dd，1H)，3.34(dd，1H)，4.20(m，2H)，4.77(m，1H)，6.84(m，1H)，6.91(t，1H)，7.20-7.33(m，3H)，7.65(m，2H)，7.96(m，3H).

Preparation example 29

(4S) -4-benzyl-3- { [ (3S, 4R) -1-benzyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one

To a stirred solution of (4S) -4-benzyl-3- [ (2E) -3- (2, 4-difluorophenyl) prop-2-enoyl ] -1, 3-oxazolidin-2-one from preparation 28 (46.83g, 140mmol) in dichloromethane (300mL) at room temperature was added N-methoxymethyl-N- (trimethylsilylmethyl) benzylamine (50.2mL, 210 mmol). The solution was cooled to-12 ℃ and a solution of trifluoroacetic acid (1.05mL) in dichloromethane (10mL) was added dropwise. The reaction mixture was warmed to room temperature, stirred for 24 hours, and saturated sodium bicarbonate solution (180mL) was added. The phases were separated and the aqueous phase was extracted with dichloromethane (180 mL). The organic extracts were combined, dried over magnesium sulfate, filtered, and concentrated in vacuo to give a crude residue. The residue was purified by column chromatography using toluene: methyl tert-butyl ether (12: 1) followed by dichloromethane: methyl tert-butyl ether (19: 1) as eluent to give the title compound, which was the diastereomer of the second elution, 63.0g (49%).

¹H NMR(400MHz，CDCl₃)□2.75(m，3H)，3.12(t，1H)，3.24(m，2H)，3.70(q，2H)4.13(m，2H)，4.27(q，1H)，4.33(m，1H)，4.67(m，1H)，6.57(m，1H)，6.84(t，1H)，7.13(m，2H)，7.16(m，1H)，7.24-7.41(m，8H).

Preparation example 30

(3S, 4R) -1-benzyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid methyl ester

To a stirred solution of (4R) -4-benzyl-3- { [ (3S, 4R) -1-benzyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -1, 3-oxazolidin-2-one (63g, 130mmol) from preparation 29 in methanol (350mL) was added samarium triflate (6.32g, 10mmol) at room temperature. The reaction mixture was stirred for 24 hours and the solvent was removed in vacuo. Dichloromethane (290mL) was added followed by saturated sodium bicarbonate solution (140mL) and the mixture was stirred for 15 minutes. The resulting precipitate was filtered and washed with dichloromethane (250mL) and water (25 mL). The phases were separated and the aqueous layer was extracted with dichloromethane (2X 40 mL). The organic extracts were combined, dried over magnesium sulfate, filtered, and concentrated in vacuo to give a crude residue. The residue was suspended in warm cyclohexane (300mL) and shaken until a solid formed. The mixture was left at room temperature for 24 hours. The solid was filtered and washed with cold cyclohexane (150 mL). The filtrate was concentrated in vacuo to give 38g (87%) of the desired compound.

¹H NMR(400MHz，CDCl₃)□2.67(t，1H)，2.86(m，1H)，2.93(t，1H)，3.04(m，2H)，3.64(s，3H)，3.65(t，1H)，3.84(m，1H)，6.72(m，1H)，6.80(t，1H)，7.23(m，2H)，7.29-7.38(m，5H).

[□]²⁵ _D＝-38(c＝0.5，MeOH)

Preparation example 31

(3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid methyl ester

Palladium hydroxide (20% Palladium on carbon, 1g) was added to a solution of methyl (3S, 4R) -1-benzyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylate (10g, 30mmol) from preparation 30 in ethanol (50mL) at room temperature. The reaction mixture was hydrogenated at 50psi for 24 hours, then filtered through Arbocel ®, washed with ethanol (50 mL). The solvent was removed in vacuo to give the desired compound as a colorless oil 7.19g (98%).

¹H NMR(400MHz，CD₃OD)□2.60(s，1H)，2.91(t，1H)，3.08(q，1H)，3.31-3.44(m，1H)，3.50(t，1H)，3.63(m，1H)，3.66(s，3H)，6.76(m，1H)，6.84(m，1H)，7.20(m，1H).LRMS(EI)242[MH⁺].

Preparation example 32

(3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidine-3-carboxylic acid methyl ester

Sodium triacetoxyborohydride (1.32g, 6.22mmol) and acetic acid (235. mu.L, 4.14mmol) were added to a solution of acetone (304. mu.L, 4.14mmol) and methyl (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylate (1g, 4.14mmol) from preparation 31 in dichloromethane (20mL) at room temperature. The resulting mixture was stirred for 2 hours and diluted with dichloromethane (10 mL). Aqueous sodium bicarbonate (2X 20mL) was added followed by brine (20 mL). The phases were separated and the organic phase was dried over magnesium sulfate, filtered and the solvent removed in vacuo to give a crude residue. The residue was purified by column chromatography using dichloromethane: methanol (99: 1-98: 2) as eluent to give 1.01g (86%) of the desired product.

¹H NMR(400MHz，CDCl₃)□1.10-1.13(m，6H)，2.48(m，1H)，2.72(t，1H)，3.00(q，1H)，3.05-3.12(m，3H)，3.65(s，3H)，3.83(q，1H)，6.73(m，1H)，6.82(t，1H)，7.37(q，1H).

LRMS(APCI)284[MH⁺].

Preparation example 33

(3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidine-3-carboxylic acid

Lithium hydroxide (171mg, 7.14mmol) was added to a solution of methyl (3S, 4R) -4- (2, 4-difluorophenyl) -1-isopropylpyrrolidine-3-carboxylate (1.01g, 3.59mmol) from preparation 32 in tetrahydrofuran (10mL) at room temperature. The reaction mixture was stirred for 3 hours and the solvent was removed in vacuo. The residue was dissolved in water (20mL) and washed with ethyl acetate (2X 20 mL). The phases were separated and the aqueous phase was acidified with 2M aqueous hydrochloric acid (3.59mL) and extracted with ethyl acetate (20 mL). The combined organic extracts were dried over magnesium sulfate and concentrated in vacuo to give the desired product as foam 686mg (71%).

¹H NMR(400MHz，CD₃OD)□1.42(m，6H)，3.31(m，3H)，3.32(m，1H)，3.57(m，2H)，3.91(m，1H)，7.03(t，2H)，7.55(m，1H).

LRMS(EI)270[MH⁺].

Preparation example 34

(2Z) -3- (2, 4-difluorophenyl) acrylic acid methyl ester

To a solution of 18-crown-6 (30g, 110mmol), bis (2, 2, 2-trifluoroethyl) (methoxycarbonylmethyl) phosphonate (6mL, 28mmol) in tetrahydrofuran was added potassium hexamethyldisilazide (0.5M in toluene) (50mL, 25mmol) followed by 2, 4-difluorobenzaldehyde (4g, 28mmol) at-78 ℃. The reaction mixture was stirred at this temperature for 8 hours and slowly warmed to room temperature over 24 hours. The reaction mixture was then poured into a saturated ammonium chloride solution (200 mL). The phases were separated and the organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo to give a crude residue. The residue was purified by column chromatography using pentane: ethyl acetate (99: 1-98: 2) as eluent to give the desired product as a colourless oil, 5.1g (91%).

¹H NMR(400MHz，CDCl₃)□3.70(s，3H)，6.05(d，1H)，6.80(m，1H)，6.86(m，1H)，6.97(d，1H)，7.69(q，1H).

LRMS(APCI)199[MH⁺]

Preparation example 35

(2Z) -3- (2, 4-difluorophenyl) acrylic acid

Methyl (2Z) -3- (2, 4-difluorophenyl) acrylate from preparation 34 (1.3g, 6.56mmol) was stirred at room temperature with a solution of 1M lithium hydroxide (314mg, 13.1mmol) in tetrahydrofuran (51mL) for 24 h. The solvent was removed in vacuo, the residue dissolved in water (10mL), and ethyl acetate (20mL) added. The phases were separated and the aqueous phase was acidified to pH2 with 2M hydrochloric acid solution (3 mL). The aqueous phase was extracted with diethyl ether (2X 30 mL). The organic extracts were combined, dried over magnesium sulfate, filtered, and concentrated in vacuo to give the desired product as a white solid, 1.03g (86%).

¹H NMR(400MHz，CD₃OD)：□6.09(d，1H)，6.93(m，2H)，6.97(d，1H)，7.66(q，1H).

Preparation example 36

(3R^*，4R^*) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid

To a stirred solution of (2Z) -3- (2, 4-difluorophenyl) acrylic acid (400mg, 2.17mmol) from preparation 35 and trifluoroacetic acid (17. mu.L, 0.2mmol) in dichloromethane (1mL) at 0 ℃ was added N- (methoxymethyl) -2-methyl-N- [ (trimethylsilyl) methyl ] propan-2-amine (882mg, 4.35mmol) from preparation 23 over 30 minutes. The reaction mixture was warmed to room temperature and stirred for 24 hours. The solvent was removed in vacuo and the resulting white residue triturated with diethyl ether (5mL) and the solid filtered off to give the desired product 400mg (65%).

¹H NMR(400MHz，CD₃OD)□1.46(s，9H)，3.31(s，1H)，3.59(m，1H)，3.69(m，1H)，3.78(d，1H)，3.89(t，1H)，3.97(m，1H)，6.93(m，2H)，7.41(m，1H).

LCMS(APCI)＝284[MH⁺].

Preparation example 37

(3S, 4R) -4- (2, 4-difluorophenyl) -1-methylpyrrolidine-3-carboxylic acid methyl ester

To a solution of methyl (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylate (500mg, 2.07mmol) from preparation 31 and formaldehyde (155. mu.L, 2.07mmol) in dichloromethane (20mL) at room temperature was added acetic acid (188. mu.L, 2.07mmol) followed by sodium triacetoxyborohydride (659mg, 3.11 mmol). The reaction mixture was stirred for 2 hours, diluted with dichloromethane (10mL) and partitioned with saturated sodium bicarbonate solution (40 mL). The phases were separated and the organic phase was washed with brine (20mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give the desired product as a colorless oil, 288mg (54%).

¹H NMR(400MHz，CDCl₃)δ2.41(s，3H)，2.68(t，1H)，3.00(m，2H)，3.01(q，1H)，3.11(m，1H)，3.65(s，3H)，3.88(m，1H)，6.78(m，1H)，6.82(t，1H)，7.37(m，1H).

LRMS：m/z APCI⁺256[MH⁺].

Preparation example 38

Tert-butyl (3R, 4S) -3- (2, 4-difluorophenyl) -4- { [ (3R, 4R, 5S) -4- (4-fluorophenyl) -4-hydroxy-3, 5-dimethylpiperidin-1-yl ] carbonyl } pyrrolidine-1-carboxylate

(3R, 4S, 5S) -4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol (265mg, 1.2mmol) from preparation 41, (3S, 4R) -1- (tert-butoxycarbonyl) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid (0.75mg, 1.4mmol) from preparation 53 and triethylamine (0.48mL, 3.6mmol) were added to dichloromethane (25 mL). The stirred suspension was cooled under nitrogen and 1-propylphosphonic acid cyclic anhydride (50% ethyl acetate solution) (0.67mL, 2mmol) was added dropwise. After the addition was complete, the resulting homogeneous solution was stirred at room temperature for another 6 hours. The solution was washed with 10% aqueous potassium carbonate (3X 20mL), 3% citric acid (3X 50mL), then dried over sodium sulfate and filtered. The dichloromethane was then removed in vacuo and the crude compound was purified by column chromatography on silica eluting with a gradient of ethyl acetate: pentane (10: 90) to ethyl acetate: pentane (40: 80) to give the desired product as a white solid (529 mg).

¹H NMR(CD₃OD, 10mg/mL, 400MHz) (rotamer), 0.21-0.58(m, 6H), 1.46(s, 9H), 0.81-1.97(m, 2H), 2.68(m, 1H), 4.35(m, 1H), 2.93-3.91(m, 7H), 4.31(m，1H)，6.90-7.29(m，5H)，7.38-7.85(m，2H)

[α]²⁵ _D＝-82.7(c＝0.3，MeOH)

Preparation example 39

(3R, 4S, 5S) -4- (3, 4-difluorophenyl) -3, 5-dimethylpiperidin-4-ol

A solution of 3, 4-difluorobromobenzene (4.45g, 21mmol) in diethyl ether (25mL) was cooled to-78 deg.C under nitrogen. N-butyllithium (2.5M hexane solution) (8.10mL, 20mmol) was added dropwise with stirring, maintaining the temperature below-65 ℃. The mixture was stirred at-78 ℃ for 4 hours. A solution of (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (5.90g, 20mmol) from preparation 14 in diethyl ether (25mL) is then added dropwise, maintaining the temperature below-65 ℃. The mixture was stirred at-78 ℃ for 1 hour and then warmed to room temperature. Saturated ammonium chloride solution (40mL) was added and the mixture was stirred for 30 minutes. The ether layer was separated, washed with water (3 × 50mL), dried over sodium sulfate, filtered and evaporated to dryness. The crude product was dissolved in methanol (100mL) and the solution was subjected to 20% palladium on carbon at 50psi and 50 ℃ for 18 hours. The mixture was filtered through Celite ® and the filtrate was evaporated to dryness. The crude product was recrystallized from acetonitrile to give the desired product as a solid, 1.58g (24%).

¹H NMR(400MHz，CD₃OD)δ0.60(d，6H)，2.21(m，2H)，3.10(m，4H)，7.38(d，2H)，7.05-7.20(m，1H)，7.25(m，1H)，7.30-7.50(m，1H)

LRMS：m/z APCI⁺242[MH⁺]

Preparation example 40

(3R, 4S, 5S) -1-benzyl-4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol

A solution of 4-fluorobromobenzene (4.51g, 0.024mol) in diethyl ether (20mL) was cooled to-78 deg.C under nitrogen. N-butyllithium (2.5M hexane solution) (8.40mL, 21mmol) was added dropwise with stirring, maintaining the temperature below-65 ℃. The mixture was stirred at-78 ℃ for 1 hour and then warmed to room temperature. The resulting 4-fluorophenyllithium solution was then added dropwise to a solution of (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (6g, 19mmol) from preparation 14 in diethyl ether (20mL) at-78 ℃ maintaining the temperature below-65 ℃. The mixture was stirred at-78 ℃ for 1 hour and then warmed to room temperature. Saturated ammonium chloride solution (40mL) was added and the mixture was stirred for 30 minutes. The organic phase was separated, washed with water (3 × 50mL), dried over sodium sulfate, filtered and evaporated to dryness. The crude product was used without further purification.

¹H NMR(400MHz，CD₃OD)δ0.51(d，6H)，2.18(m，2H)，2.39(m，2H)，2.71(m，1H)，3.58(s，1H)，3.65(s，2H)，7.12(m，2H)，7.35(m，7H)

LRMS(APCI)314[MH⁺]

Preparation example 41

(3R, 4S, 5S) -4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol

A solution of (3R, 4S, 5S) -1-benzyl-4- (4-fluorophenyl) -3, 5-dimethylpiperidin-4-ol (5.0g, 16mmol) from preparation 40 in methanol (100mL) was hydrogenated over 20% palladium on carbon (1.1g) at 50psi and 50 ℃ for 18 h. The mixture was then filtered through Celite ® and the filtrate was evaporated to dryness. The crude product was recrystallized from acetonitrile to give the desired product as a solid (3.81 g).

¹H NMR(400MHz，CD₃OD)δ0.54(d，6H)，2.18(m，2H)，2.85(m，4H)，7.05(m，2H)，7.20-7.45(m，2H)，

LRMS(APCI)224[MH⁺]

Preparation example 42

(3R, 5S) -1- (4-methoxybenzyl) -3, 5-dimethylpiperidin-4-one

A1M hydrochloric acid solution (69mL) was added to a solution of dimethyl 2, 4-dimethyl-3-oxoglutarate (69.6g, 344mmol) from preparation 12 in methanol (1.8L) with 4-methoxybenzylamine (44.81mL, 344 mmol). 37% aqueous formaldehyde (56.8mL, 760mmol) was added. The solution was stirred at room temperature for 72 hours and then evaporated to dryness. Crude dimethyl 1- (4-methoxybenzyl) -3, 5-dimethyl-4-oxo-piperidinedicarboxylate (126.2g) was added to a 1M hydrochloric acid solution (1735mL), and the mixture was heated at reflux for 24 hours. The reaction mixture was cooled to 10 ℃ and 20 wt% aqueous sodium hydroxide (400mL, 2.0mol) was slowly added. The mixture was extracted with dichloromethane (4X 400 mL). The combined organic extracts were evaporated to dryness to give the crude product as a light brown oil ¹H NMR indicated a 6: 1 cis: trans mixture. A portion of the crude diastereomeric mixture (15g) was purified by an automated chromatographic purification system using a normal phase Redispe ® silica cartridge column (330g), solvent flow 100mL/min, cyclohexane/ethyl acetate 2-3% ethyl acetate linear gradient for 35 minutes, 3-14% ethyl acetate linear gradient for 10 minutes, elution being completed with 14% ethyl acetate. The pure cis-isomer was obtained as a pale yellow oil which solidified upon standing (10.2g, 99% + by means of LCMS).

¹H NMR(400MHz，CD₃OD)δ0.91(d，6H)，2.02(m，2H)，2.75(m，2H)，3.18(m，2H)，3.58(s，2H)，3.95(s，3H)，6.85(d，2H)，7.25(d，2H)

LRMS(APCI)248[MH⁺]

Preparation example 43

(3R, 4S, 5S) -4- (4-chlorophenyl) -3, 5-dimethylpiperidin-4-ol

A solution of 4-chloroiodobenzene (4.6g, 25mmol) in anhydrous diethyl ether (200mL) was cooled to-78 ℃ under nitrogen. N-butyllithium (2.5M in hexane) (15.2mL, 20mmol) was added dropwise, maintaining the temperature below-65 ℃. The mixture was stirred at-78 ℃ for 2 hours and warmed to room temperature. Then 4-chlorophenyl lithium solution was added dropwise to a solution of (3R, 5S) -1- (4-methoxybenzyl) -3, 5-dimethylpiperidin-4-one (5.0g, 20mmol) from preparation 42 in diethyl ether (25mL) at-78 ℃. The mixture was stirred at-78 ℃ for an additional 2 hours, then warmed to room temperature. The mixture was quenched with saturated ammonium chloride (50 mL). The organic phase was separated, washed with water (3 × 50mL), dried over sodium sulfate, filtered and then evaporated to dryness to give the crude intermediate. The crude product was dissolved in anhydrous dichloromethane (150mL), triethylamine (4.0mL, 29mmol) was added and the solution cooled to 0 ℃ under nitrogen. To the stirred solution was added dropwise 1-chloroethyl chloroformate (3.21mL, 30mmol), and after the addition was complete the mixture was stirred at room temperature for an additional 3 hours.

The mixture was then washed with 10% aqueous potassium carbonate (3 × 25mL), dried over sodium sulfate and evaporated to dryness. The crude residue was heated under reflux in methanol (150mL) for 3 hours and the solvent was removed in vacuo. The residue was dissolved in dichloromethane (100mL), solid potassium carbonate (5g) was added and the heterogeneous mixture was stirred for 1 hour. The solid potassium carbonate was filtered off and the filtrate was evaporated to dryness. The crude product was then recrystallized from acetonitrile to give the desired product as fine white needles (3.90 g).

¹H NMR(400MHz，CD₃OD)δ0.60(m，6H)，2.25(m，2H)，3.10(m，4H)7.38(d，2H)，7.55(m，4H)

LRMS(APCI)240[MH⁺]

Preparation example 44

(3R, 4S, 5S) -4- (2, 6-difluorophenyl) -3, 5-dimethylpiperidin-4-ol

Tert-butyllithium (1.7M pentane solution) (9.62mL, 16.4mmol) was added dropwise to a stirred solution of 2, 6-difluorobromobenzene (3.0g, 15.5mmol) at-78 ℃. The solution was stirred at-78 ℃ for another 3 hours. A solution of (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (2.16g, 12mmol) from preparation 14 in diethyl ether (30mL) is then added dropwise, maintaining the temperature below-65 ℃. The mixture was stirred at-78 ℃ for 1 hour and then warmed to room temperature overnight. Saturated ammonium chloride solution (20mL) was added and the mixture was stirred for 30 min. The organic phase was washed with water (3X 50mL) and dried over sodium sulfate. The solvent was removed in vacuo, the residue dissolved in methanol (100mL), and the solution hydrogenated (50psi and 50 deg.C, 20% palladium on carbon) for 18 h. The mixture was filtered through Celite ® and the filtrate was evaporated to dryness. The crude product was recrystallized from acetonitrile to give the desired product (1.78g) as fine white needles.

¹H NMR(400MHz，CD₃OD) (rotamer) delta 0.60(d, 6H), 2.21(m, 2H), 3.10(m, 4H), 7.38(d, 2H), 7.05-7.20(m, 1H), 7.25(m, 1H), 7.31-7.50(m, 1.20H)

LRMS(APCI)242[MH⁺]

Preparation example 45

(3R, 4S, 5S) -4- (3-fluorophenyl) -1- (4-methoxybenzyl) -3, 5-dimethylpiperidin-4-ol

n-BuLi (2.5M in hexane) (7.2mL, 18mmol) was added dropwise to a stirred solution of 3-fluoroiodobenzene (1.91g, 8.0mmol) in anhydrous diethyl ether (10mL) at-78 ℃. The mixture was stirred at-78 ℃ for 3 h, then a solution of (3R, 5S) -1- (4-methoxybenzyl) -3, 5-dimethylpiperidin-4-one (1.85g, 7.5mmol) from preparation 42 in diethyl ether (10mL) was added dropwise, maintaining the temperature below-60 ℃. The mixture was then allowed to warm to room temperature. Saturated ammonium chloride solution (25mL) was added, the mixture was stirred for 30 min, and the organic phase was separated. The organic phase was washed with water (3 × 50mL), dried over sodium sulfate, filtered and the solvent removed in vacuo. Recrystallization from ethyl acetate: cyclohexane gave the desired product (2.88 g).

¹H NMR(400MHz，CD₃OD)δ0.51(d，6H)，2.18(m，2H)，2.35(m，2H)，2.71(m，2H)，3.58(s，2H)，3.65(s，3H)，7.12(m，3H)，7.35(m，5H)

LRMS(APCI)344[MH⁺]

Preparation example 46

(3R, 4S, 5S) -4- (3-fluorophenyl) -3, 5-dimethylpiperidin-4-ol

A solution of (3R, 4S, 5S) -4- (3-fluorophenyl) -1- (4-methoxybenzyl) -3, 5-dimethylpiperidin-4-ol (2.5g, 7.3mmol) from preparation 45 in methanol (25mL) was hydrogenated (50psi and 50 ℃, 20% palladium on carbon) for 18 h. The mixture was filtered through Celite ® and the filtrate was evaporated to dryness. The crude product was recrystallized from acetonitrile to give the title compound (1.52 g).

¹H NMR(400MHz，CD₃OD)δ0.59(d，6H)，2.10(m，2H)，2.85(m，5H)，6.95(m，1H)，7.35(m，2H)

LRMS(APCI)224[MH⁺]

Preparation example 47

(3R, 4S, 5S) -1-benzyl-4- (4-methoxyphenyl) -3, 5-dimethylpiperidin-4-ol

Tert-butyllithium (1.7M pentane solution) (33.0mL, 56mmol) was added to anhydrous diethyl ether (20mL) under nitrogen and cooled to-78 ℃. A solution of 4-methoxy-iodobenzene (6.89g, 29mmol) in anhydrous diethyl ether (25mL) was added dropwise to the tert-butyllithium solution, maintaining the temperature between-78 deg.C and-60 deg.C. After the addition was complete, the mixture was stirred at-78 ℃ for another 30 minutes and then warmed to room temperature. The resulting 4-methoxyphenyl lithium solution was then added dropwise to a solution of (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (4.0g, 18mmol) from preparation 14 in anhydrous diethyl ether (70mL) at-78 ℃. The mixture was stirred at-78 ℃ for 2 hours and then warmed to room temperature. Saturated ammonium chloride (20mL) was added dropwise and the mixture was stirred for 30 min. The organic layer was separated, washed with water (3 × 100mL), dried over sodium sulfate and evaporated to dryness to give the crude product which was recrystallized from cyclohexane/ethyl acetate to give the pure product (7.1 g).

¹H NMR(400MHz，CD₃OD)δ0.51(d，6H)，2.12(m，2H)，2.25(m，2H)，2.61(m，2H)，3.58(s，2H)，3.78(s，3H)，6.85(m，3H)，7.25(m，6H)

LRMS(APCI)326[MH⁺]

Preparation example 48

(3R, 4S, 5S) -4- (4-methoxyphenyl) -3, 5-dimethylpiperidin-4-ol

A solution of (3R, 4S, 5S) -1-benzyl-4- (4-methoxyphenyl) -3, 5-dimethylpiperidin-4-ol (7.1g, 21mmol) from preparation 47 in methanol (100mL) was hydrogenated over palladium on carbon (1.0g) (50psi and 50 ℃ C.) for 18 h. The mixture was filtered through Celite ® and the filtrate was evaporated to dryness to give the crude product. Recrystallization from acetonitrile gave the desired compound (3.1g) which was used without further purification.

¹H NMR(400MHz，CD₃OD)δ0.52(d，6H)，2.00(m，2H)，2.68(m，4H)，3.78(s，3H)，6.82(d，2H)，7.20-7.60(m，2H).

LRMS(APCI)235[MH⁺]

Preparation example 49

(3R, 4S, 5S) -4- (2, 4-difluorophenyl) -3, 5-dimethylpiperidin-4-ol

A solution of 2, 4-difluorobromobenzene (4.51g, 22mmol) in diethyl ether (20mL) was cooled to-78 deg.C under nitrogen. N-butyllithium (2.5M hexane solution) (8.40mL, 21mmol) was added dropwise with stirring, maintaining the temperature below-65 ℃. The mixture was stirred at-78 ℃ for 4 hours. A solution of (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (6.00g, 19mmol) from preparation 14 in diethyl ether (25mL) is then added dropwise, maintaining the temperature below-65 ℃. The mixture was stirred at-78 ℃ for 1 hour and then warmed to room temperature. Saturated ammonium chloride solution (40mL) was added and the mixture was stirred for 30 minutes. The ether layer was separated, washed with water (3 × 50mL), dried over sodium sulfate, filtered and evaporated to dryness. The product was dissolved in methanol (100mL) and the solution was hydrogenated (50psi and 50 deg.C, 20% palladium on carbon) for 18 hours. The mixture was filtered through Celite ® and the filtrate was evaporated to dryness. The crude product was recrystallized from acetonitrile to give the desired product (1.78g) as fine white needles.

¹H NMR(400MHz，CD₃OD)δ0.60(d，6H)，2.21(m，2H)，3.10(m，4H)，7.38(d，2H)，7.05-7.20(br，1.00H)，7.25(m，1H)，7.31-7.50(m，1.20H)

LRMS(APCI)242[MH⁺]

Preparation example 50

(3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4-pyridin-3-ylpiperidin-4-ol

A cooled solution of 3-bromopyridine (2.4mL, 25mmol) in anhydrous diethyl ether (2mL) was added to a solution of n-butyllithium (2.5M in hexane) (10mL, 25mmol) at-78 deg.C. The reaction mixture was stirred for 1 hour. A solution of (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (5.42mg, 25mmol) in tetrahydrofuran (2mL) was added at-78 deg.C and the reaction mixture was stirred for 1 hour. The reaction was allowed to warm to-20 ℃, saturated ammonium chloride solution (10mL) was added, and the resulting mixture was stirred at room temperature for 24 hours. The suspension was filtered and the solid was washed with diethyl ether (4X 50 mL). The solid was redissolved in dichloromethane: methanol (90: 10) and the solution was washed with brine. The phases were separated and the organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo to give 4.35g (61%) of the desired product.

¹H-NMR(400MHz，CDCl₃)：δ＝0.56(d，6H)，2.07-2.43(br，4H)，2.79(br，2H)，3.63(br，2H)，7.25-7.46(m，5H)，7.65(br，1H)，7.84(br，1H)，8.47(d，1H)，8.68(br，1H)

LRMS(APCI+)＝297[MH⁺]

Preparation example 51

(3R, 4S, 5S) -3, 5-dimethyl-4-pyridin-3-ylpiperidin-4-ol

A mixture of (3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4-pyridin-3-ylpiperidin-4-ol (3.0g, 10.12mmol) from preparation 50 and 20% by weight palladium hydroxide on carbon (0.45g) in ethanol (50mL) was hydrogenated at 40 ℃ and 40psi for 14 hours. The reaction mixture was then filtered through Arbocel ® and the filtrate was concentrated in vacuo to give the desired product as an off-white foam, 2.05 g.

¹H-NMR(400MHz，CDCl₃)：δ＝0.57(d，6H)，2.12(m，2H)，2.81(t，2H)，2.94(m，2H)，7.27(m，1H)，7.51-7.99(br，1H)，8.48(d，1H)，8.67(br，1H)

LRMS(APCI+)＝207[MH⁺]

Preparation example 52

(3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidine-1, 3-dicarboxylic acid 1-tert-butyl 3-methyl ester

To a solution of methyl (3S, 4R) -1-benzyl-4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylate (1.0g, 3.01mmol), 1-methylcyclohexa-1, 4-diene (1.25mL, 11.12mmol) from preparation 30 in di-tert-butyl dicarbonate (0.72g, 3.31mmol) in ethanol (10mL) was added palladium hydroxide on carbon (0.1g) at room temperature. The resulting mixture was heated at reflux for 4 hours, cooled to room temperature, and filtered through Arbocel ®. The filtrate was concentrated in vacuo to give a crude residue, which was partitioned between ethyl acetate (80mL) and 10% citric acid solution (5 mL). The phases were separated and the organic layer was washed with brine (60mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give the desired product as a colourless oil 940 mg.

¹H NMR(400MHz，CDCl₃)δ1.40(s，9H)，3.14-3.25(m，1H)，3.25-3.40(m，1H)，3.48-3.59(m，4H)，3.68-3.89(m，3H)，6.71-6.82(m，2H)，7.15(m，1H)

LRMS(APCI)242[MH⁺-BOC+1]

Preparation example 53

(3S, 4R) -1- (tert-Butoxycarbonyl) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid

Lithium hydroxide (130mg, 23.5mmol) was added dropwise to a stirred solution of (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidine-1, 3-dicarboxylic acid 1-tert-butyl 3-methyl ester (930mg, 2.72mmol) from preparation 52 in tetrahydrofuran (10mL) at room temperature. The reaction mixture was stirred for 48 hours, concentrated in vacuo, and diluted with water (15 mL). The phases were separated and the aqueous phase was extracted with ethyl acetate (1X 25 mL). The aqueous layer was acidified with 2M hydrochloric acid solution (2.7mL) and further extracted with ethyl acetate (2X 40 mL). The combined organic extracts were dried over magnesium sulfate, filtered, concentrated in vacuo, and azeotroped with dichloromethane to give 775mg (87%) of the desired product.

¹H NMR(400MHz，CDCl₃)δ1.45(s，9H)，3.23-3.46(m，2H)，3.56-3.65(m，1H)，3.74-3.93(m，3H)，6.75-6.87(m，2H)，7.20(m，1H)

LRMS(APCI)228[MH⁺-BOC+1]

LRMS(APCI-)＝326[M-1]

Preparation example 54

(3R, 4S) -3- (2, 4-difluorophenyl) -4- { [ (3R, 4R, 5S) -4-hydroxy-3, 5-dimethyl-4-pyridin-2-ylpiperidin-1-yl ] carbonyl } pyrrolidine-1-carboxylic acid tert-butyl ester

A solution of (3R, 4S, 5S) -3, 5-dimethyl-4-pyridin-2-ylpiperidin-4-ol (835mg, 4mmol) from preparation 74, (3S, 4R) -1- (tert-butoxycarbonyl) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid (1.32g, 4mmol) from preparation 53, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (776mg, 4mmol) and 1-hydroxybenzotriazole hydrate (62mg, 0.4mmol) in tetrahydrofuran (20mL) was stirred at room temperature for 20 hours. The solvent was removed in vacuo and the crude residue partitioned between water (15mL) and ethyl acetate (15 mL). The phases were separated and the organic phase was washed with saturated sodium bicarbonate solution (15mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give a crude residue. The residue was purified by column chromatography using ethyl acetate: pentane (10: 90-40: 60) as eluent to give the desired product as a white foam 380mg (43%).

¹H NMR(400MHz，CDCl₃) (rotamers) δ 0.27-0.52(m, 6H), 1.46(s, 9H), 0.81-1.97(m, 2H), 2.68(m, 1H), 2.93-3.24(m, 2H), 3.38-4.14(m, 7H), 4.41(m, 1H), 5.50(m, 1H), 6.82(m, 1H), 6.87-7.36(m, 3H), 7.71(m, 1H), 8.47(m, 1H).

LRMS(APCI)516[MH⁺].

Preparation example 55

(3R, 4S) -3- (2, 4-difluorophenyl) -4- { [ (3R, 4R, 5S) -4-hydroxy-3, 5-dimethyl-4-phenylpiperidin-1-yl ] carbonyl } pyrrolidine-1-carboxylic acid tert-butyl ester

To a solution of (3S, 4R) -1- (tert-butoxycarbonyl) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid (1000mg, 3mmol) from preparation 53, and (3R, 4S, 5S) -3, 5-dimethyl-4-phenylpiperidin-4-ol (522mg, 2.54mmol) from preparation 16 and triethylamine (706. mu.L, 0.73mmol) in ethyl acetate (10mL) at 0 ℃ was added 1-propylphosphonic acid cyclic anhydride (50% ethyl acetate solution) (1.5mL, 2.54mmol), and the resulting solution was stirred at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate (70mL), and a saturated potassium carbonate solution (2X 50mL) was added, followed by a 10% citric acid solution (1X 50 mL). The phases were separated and the organic phase was washed with brine (1 × 50mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give a crude residue. The residue was purified by column chromatography using ethyl acetate: pentane (10: 90-40: 60) as eluent to give the desired product as a white foam 560mg (43%).

¹H NMR(400MHz，CDCl₃) (rotamers). delta.0.41-0.62 (m, 6H), 0.94-1.24(m, 1H), 1.47(s, 9H),1.65-2.07(m，1H)，2.59-3.02(m，1H)，3.15(m，1H)，3.40-4.15(m，7H)，4.42(d，1H)，6.76-6.85(m，2H)，7.16-7.41(m，6H)

LRMS(APCI)515[MH⁺].

preparation example 56

(3R, 4S, 5S) -1-benzyl-4-isopropyl-3, 5-dimethylpiperidin-4-ol

To a solution of (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (500mg, 2.3mmol) from preparation 14 was added lithium isopropyl (0.7M pentane solution) (3.6mL, 2.53 mmol). The reaction mixture was stirred at-78 ℃ for 1 hour, then slowly warmed to 0 ℃ and stirred at this temperature for another 30 minutes. Saturated ammonium chloride solution (6mL) was then added at-10 ℃. The reaction mixture was partitioned between ethyl acetate (6mL) and water (6 mL). The phases were separated and the aqueous phase was extracted with ethyl acetate (6 mL). The organic extracts were combined, dried over magnesium sulfate, filtered, and the solvent was removed in vacuo to give a crude residue. The residue was purified by column chromatography using dichloromethane: methanol: 0.88 ammonia (100: 0-99: 1-96: 4: 0.4) as eluent to give 244mg (41%) of the desired product.

¹H NMR(400MHz，CDCl₃)δ0.82(d，6H)，0.99(d，6H)，1.88-2.03(m，4H)，2.08(m，1H)，2.48(m，2H)，3.45(m，2H)，7.19-7.34(m，5H)

LRMS(APCI)262[MH⁺]，244[MH⁺-H₂O]

Preparation example 57

(3R, 4S, 5S) -4-isopropyl-3, 5-dimethylpiperidin-4-ol

(3R, 4S, 5S) -1-benzyl-4-isopropyl-3, 5-dimethylpiperidin-4-ol from preparation 56 (1.42g, 5.44mmol) and a solution of palladium hydroxide on carbon (210mg) in ethanol (25mL) were hydrogenated at 40 ℃ and 40psi for 24 h. The reaction mixture was filtered through Arbocel ®, washed with ethanol (25 mL). The filtrate was concentrated in vacuo to give a crude residue which was recrystallized from acetonitrile to give the desired product as brown needles, 390mg (42%).

¹H NMR(400MHz，CDCl₃)δ0.83(d，6H)，0.99(d，6H)，1.74(m，2H)，2.08(m，1H)，2.64(m，4H)

LRMS(APCI)172[MH⁺].

Preparation example 58

(3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4-propylpiperidin-4-ol

To a stirred solution of (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (1.0g, 4.6mmol) from preparation 19 in tetrahydrofuran (7mL) at-78 deg.C was added propylmagnesium chloride (2M in diethyl ether) (7.5mL, 15 mmol). The reaction mixture was stirred for 1 hour, saturated ammonium chloride solution (20mL) was added, and the mixture was allowed to warm slowly to room temperature. The reaction mixture was diluted with ethyl acetate (40mL) and the phases were separated. The aqueous phase was extracted with ethyl acetate (1 × 40mL), and the organic extracts were combined, dried over magnesium sulfate, filtered, and concentrated in vacuo to give a crude residue. The residue was purified by column chromatography using dichloromethane: methanol: 0.88 ammonia (98: 2: 0-95: 5: 0.5) as eluent to give 790mg (66%) of the desired product.

¹H NMR(400MHz，CDCl₃)δ0.80(d，6H)，0.90(t，3H)，1.20(m，2H)，1.51(m，2H)，1.87(m，2H)，2.04(m，2H)，2.54(m，2H)，3.47(m，2H)，7.19-7.36(m，5H).

LRMS(APCI)262[MH⁺]，244[MH⁺-H₂O].

Preparation example 59

(3R, 4S, 5S) -3, 5-dimethyl-4-propylpiperidin-4-ol

(3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4-propylpiperidin-4-ol from preparation 58 (780mg, 3mmol) was hydrogenated with a solution of palladium hydroxide (20% palladium on carbon, 130mg) in ethanol (10mL) at 40 ℃ and 40psi for 24 h. The reaction mixture was filtered through Arbocel ®, washed with ethanol (10 mL). The filtrate was concentrated in vacuo to give 504mg (98%) of the desired product.

¹H NMR(400MHz，CDCl₃)：δ0.80(d，6H)，0.91(t，3H)，1.21(m，2H)，1.50(m，2H)，1.70(m，2H)，2.70(m，5H)

LRMS(APCI)172[MH⁺].

Preparation example 60

(3R, 4S, 5S) -1-benzyl-4-cyclopropyl-3, 5-dimethylpiperidin-4-ol

To a stirred solution of (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one (1.0g, 4.6mmol) from preparation 19 in tetrahydrofuran (8mL) at-78 deg.C was added bromine (cyclopropyl) magnesium (0.5M in tetrahydrofuran) (28mL, 14 mmol). The reaction mixture was stirred for 2 hours, saturated ammonium chloride solution (40mL) was added, and the mixture was allowed to warm slowly to room temperature. The reaction mixture was diluted with water (40mL) and the phases were separated. The aqueous phase was extracted with ethyl acetate (2 × 60mL), and the organic extracts were combined, dried over magnesium sulfate, filtered, and concentrated in vacuo to give a crude residue. The residue was purified by column chromatography using dichloromethane: methanol (100: 0-96: 4) as eluent to give the desired product as a colorless liquid 780mg (65%).

¹H NMR(400MHz，CDCl₃)δ0.35(m，4H)，0.52(m，1H)，0.90(d，6H)，1.95(m，4H)，2.56(d，2H)，3.50(s，2H)，7.20-7.37(m，5H)

LRMS(APCI+)＝260[MH⁺]，242[MH⁺-H₂O]

Preparation example 61

(3R, 4S, 5S) -4-cyclopropyl-3, 5-dimethylpiperidin-4-ol

(3R, 4S, 5S) -1-benzyl-4-cyclopropyl-3, 5-dimethylpiperidin-4-ol from preparation 60 (780mg, 3mmol) was hydrogenated with a solution of palladium hydroxide (20% palladium on carbon, 140mg) in ethanol (10mL) at 40 ℃ and 40psi for 24 h. The reaction mixture was filtered through Arbocel ®, washed with ethanol (10 mL). The filtrate was concentrated in vacuo to give 480mg (94%) of the desired product.

¹H NMR(400MHz，CDCl₃)δ0.35(m，4H)，0.55(m，1H)，0.92(d，6H)，1.72(m，2H)，1.84(m，1H)，2.65(m，4H)

LRMS(APCI+)＝170[MH⁺]，152[MH⁺-H₂O]

Preparation example 62

(3S, 4R) -4- (2, 4-difluorophenyl) -1-methylpyrrolidine-3-carboxylic acid

To a solution of methyl (3S, 4R) -4- (2, 4-difluorophenyl) -1-methylpyrrolidine-3-carboxylate (800mg, 3.13mmol) from preparation 37 in tetrahydrofuran (10mL) at room temperature was added lithium hydroxide (150mg, 6.27 mmol). The reaction mixture was stirred for 2 hours and the solvent was concentrated in vacuo. The crude residue was dissolved in water (20mL) and partitioned with ethyl acetate (2X 20 mL). The phases were separated and the aqueous phase was acidified with 2M hydrochloric acid solution (3.13 mL). The aqueous phase was evaporated and the residue azeotroped with toluene (6X 20mL) to give an oily residue (1000mg) which was used in the next step without further purification.

¹H NMR(400MHz，CD₃OD)δ3.05(s，3H)，3.41(m，1H)，3.50-3.81(m，3H)，3.91(m，3H)，7.06(m，2H)，7.62(m，1H).

LRMS(APCI+)：242[MH⁺]

LRMS(APCI-)：240(M-1)

Preparation example 63

Tert-butyl (3R, 4S) -3- (2, 4-difluorophenyl) -4- { [ (3R, 4R, 5S) -4- (3, 4-difluorophenyl) -4-hydroxy-3, 5-dimethylpiperidin-1-yl ] carbonyl } pyrrolidine-1-carboxylate

A solution of (3S, 4R) -1- (tert-butoxycarbonyl) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid from preparation 53 (160mg, 0.49mmol), (3R, 4S, 5S) -4- (3, 4-difluorophenyl) -3, 5-dimethylpiperidin-4-ol from preparation 39 (100mg, 0.42mmol), 1-propylphosphonic acid cyclic anhydride (50% in ethyl acetate) (244. mu.L, 0.41mmol), and triethylamine (120. mu.L, 0.82mmol) in dichloromethane (2mL) was stirred at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane (20mL) and saturated potassium carbonate solution (2X 20mL) was added. The phases were separated and the organic phase was washed with brine (20mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give the desired product as a white foam 257mg (77%).

¹H NMR(400MHz，CDCl₃) (rotamers) □ 0.44-0.61(4xd, 6H), 1.47(s, 9H), 2.59(t, 2H), 3.10(m, 2H), 3.51-3.91(m, 6H), 4.44(d, 2H),6.82(m，2H)，6.90(m，1H)，7.07-7.15(m，3H).

LRMS(APCI+)：551(MH⁺)

preparation 64

(3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4- [4- (trifluoromethyl) phenyl ] piperidin-4-ol

The title compound was prepared in 33% yield from (3R, 5S) -1-benzyl-3, 5-dimethylpiperidin-4-one and 4-bromo-trifluoromethylbenzene from preparation 14 in a similar manner to preparation 40 except that the compound was additionally purified by column chromatography using pentane: ethyl acetate (4: 1) as eluent.

¹H NMR(400MHz，CDCl₃)□0.54(d，6H)，1.58(s，1H)，2.12(t，2H)，2.24(m，2H)，2.71(dd，2H)，3.55(s，2H)，7.27-7.36(m，7H)，7.59(d，2H).

LRMS(APCI)364[MH⁺]

Preparation example 65

(3R, 4S, 5S) -3, 5-dimethyl-4- [4- (trifluoromethyl) phenyl ] piperidin-4-ol

A mixture of (3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4- [4- (trifluoromethyl) phenyl ] piperidin-4-ol (527mg, 1.45mmol), 20% palladium on carbon (65mg) and dihydrotoluene (570. mu.L, 5.4mmol) from preparation 64 in ethanol (10mL) was heated at reflux for 3 hours. The reaction mixture was filtered through Arbocel ®, washed with ethanol (100 mL). The solvent was removed in vacuo to give the desired compound as a brown foam 501mg (87%).

¹H NMR(400MHz，CDCl₃)□0.53(d，6H)，1.74(s，2H)，2.07(m，2H)，2.73(t，2H)，2.91(dd，2H)，7.26-7.70(m，4H).

LRMS(APCI)274[MH⁺]

Preparation example 66

(3R, 4S) -3- (2, 4-difluorophenyl) -4- ({ [ (3R, 4R, 5S) -4-hydroxy-3, 5-dimethyl-4- [4- (trifluoromethyl) phenyl ] piperidin-1-yl } carbonyl) pyrrolidine-1-carboxylic acid tert-butyl ester

The title compound was prepared in 94% yield from (3S, 4R) -1- (tert-butoxycarbonyl) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylic acid from preparation 53 and (3R, 4S, 5S) -3, 5-dimethyl-4- [4- (trifluoromethyl) phenyl ] piperidin-4-ol from preparation 65 according to a similar method to that described in preparation 38.

¹H NMR(400MHz，CDCl₃) (rotamers) □ 0.43.43-0.60 (m, 6H), 1.46(s, 9H), 2.63(m, 2H), 3.14(m, 2H), 3.45-3.90(m, 6H), 4.44(d, 2H), 6.82(m, 2H), 6.86(m, 1H), 7.16-7.32(m, 2H), 7.58(m, 2H).

LRMS(EI)583[MH⁺]

Preparation example 67

(3S, 4R) -4- (4-chlorophenyl) pyrrolidine-1, 3-dicarboxylic acid 1-tert-butyl 3-methyl ester

To a solution of methyl (3S, 4R) -4- (4-chlorophenyl) pyrrolidine-3-carboxylate hydrochloride (870mg, 2.8mmol) from preparation 26 and triethylamine (780. mu.L, 5.6mmol) in dichloromethane (5mL) was added a solution of di-tert-butyl dicarbonate (610mg, 2.8mmol) in dichloromethane (5 mL). The reaction mixture was stirred for 20 hours and diluted with ethyl acetate (50 mL). The phases were separated and the organic phase was washed with 5% citric acid solution (3X 20mL) and brine (1X 20 mL). The organic phase was dried over magnesium sulfate, filtered, and concentrated in vacuo to give the desired product in quantitative yield as a colorless oil.

¹H NMR(400MHz，CDCl₃)δ1.45(s，9H)，3.07-3.25(m，2H)，3.36(m，1H)，3.58(m，1H)，3.63(s，3H)，3.85(m，2H)，7.17(d，2H)，7.29(d，1H)

LRMS(APCI)340[MH⁺]，240[MH⁺-BOC+1]

Preparation example 68

(3S, 4R) -1- (tert-Butoxycarbonyl) -4- (4-chlorophenyl) pyrrolidine-3-carboxylic acid

To a solution of (3S, 4R) -4- (4-chlorophenyl) pyrrolidine-1, 3-dicarboxylic acid 1-tert-butyl 3-methyl ester (0.98g, 2.88mmol) from preparation 67 in tetrahydrofuran (8mL) was added lithium hydroxide (0.21g, 8.64mmol) at room temperature. The reaction mixture was stirred for 24 hours and the solvent was removed in vacuo. The crude residue was dissolved in water (8mL) and 1M hydrochloric acid solution (8.65mL) was added. The suspension was extracted with dichloromethane (2 × 40mL), the organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo to give the desired product as a white solid, 705mg (75%).

¹H NMR(400MHz，CDCl₃)δ1.45(s，9H)，3.17(m，1H)，3.36(m，1H)，3.61(m，2H)，3.88(m，2H)，7.18(d，2H)，7.29(d，2H)

LRMS(APCI)226[MH⁺-BOC+1]

LRMS(APCI-)＝324[M-1]

Preparation example 69

(3R, 4S) -3- (4-chlorophenyl) -4- { [ (3R, 4R, 5S) -4-hydroxy-3, 5-dimethyl-4-phenylpiperidin-1-yl ] carbonyl } pyrrolidine-1-carboxylic acid tert-butyl ester

To a solution of (3S, 4R) -1- (tert-butoxycarbonyl) -4- (4-chlorophenyl) pyrrolidine-3-carboxylic acid (250mg, 0.76mmol) from preparation 68, and (3R, 4S, 5S) -3, 5-dimethyl-4-phenylpiperidin-4-ol (190mg, 0.91mmol) from preparation 16 and triethylamine (320. mu.L, 2.28mmol) in ethyl acetate (5mL) was added 1-propylphosphonic acid cyclic anhydride (50% in ethyl acetate) (540. mu.L, 1.10mmol) at room temperature. The reaction mixture was stirred for 24 hours, 1M hydrochloric acid solution (20mL) was added, and the solution was stirred for 10 minutes. The phases were separated and the organic phase was diluted with ethyl acetate (3mL) and 1M sodium hydroxide solution (6mL) was added. The organic phase was separated, dried over magnesium sulfate, filtered and the solvent removed in vacuo. The crude residue was purified by column chromatography using pentane: ethyl acetate (90: 10-50: 50) as eluent to give the desired product as a white foam, 370mg (95%).

¹H NMR(400MHz，CDCl₃) (rotamers) delta 0.32-0.59(m, 6H), 1.46(s, 9H), 0.64-2.05(m, 2H), 2.63(m, 1H), 2.79-3.15(2xq, 1H), 3.30-4.01(m, 7H), 4.42(m, 1H), 7.16-7.40(m, 9H)

LRMS(APCI)513[MH⁺]，457[MH⁺-t-Bu+1]，413[MH⁺-BOC+1]

Preparation example 70

(3R, 4S, 5S) -4- (4-bromophenyl) -1- (4-methoxybenzyl) -3, 5-dimethylpiperidin-4-ol

n-BuLi (2.5M in hexane) (7.89mL, 195mmol) was added dropwise to a solution of 1, 4-dibromobenzene (4.9g, 20mmol) in diethyl ether (150mL) at-78C. The mixture was stirred for 3 hours and warmed to room temperature. A solution of 1- (4-methoxy-benzyl) -trans-3, 5-dimethyl-piperidin-4-one (5.0g, 20mmol) in diethyl ether (25mL) was added dropwise and the reaction mixture was stirred for an additional 2 hours. The mixture was quenched with saturated ammonium chloride (50mL) and the phases separated. The organic phase was washed with water (3 × 50mL), dried over sodium sulfate, filtered and the solvent removed in vacuo to give crude (3R, 4S, 5S) -4- (4-bromophenyl) -1- (4-methoxybenzyl) -3, 5-dimethylpiperidin-4-ol (7.9g) which was used without further purification.

LRMS(APCI)＝404[MH⁺]

Preparation 71

4- [ (3R, 4S, 5S) -4-hydroxy-1- (4-methoxybenzyl) -3, 5-dimethylpiperidin-4-yl ] benzonitrile

A solution of (3R, 4S, 5S) -4- (4-bromophenyl) -1- (4-methoxybenzyl) -3, 5-dimethylpiperidin-4-ol (3.50g, 8mmol), potassium cyanide (1.05g, 16mmol), tris (triphenylphosphino) palladate (1-) (0.462g, 0.4mmol) and copper iodide (1.52g, 8mmol) in acetonitrile (30mL) from preparation 70 was heated at reflux for 1 hour. The mixture was cooled to room temperature, diluted with ethyl acetate (30mL) and filtered through Celite ®. The filtrate was washed with water, brine, dried over sodium sulfate and filtered. Concentration in vacuo gave a crude residue which was purified by column chromatography on silica gel using ethyl acetate: hexane (3: 97-15: 85) as eluent to give the title compound as a yellow solid (2.51 g).

¹H NMR(400MHz，CD₃OD)δ0.51(d，6H)，2.25(m，2H)，2.42(m，2H)，2.79(m，2H)，3.58(s，2H)，3.65(s，3H)，7.12(m，4H)，7.52(d，2H)，8.10(m，2H).

LRMS(APCI)351[MH⁺]

Preparation example 72

4- [ (3R, 4S, 5S) -4-hydroxy-3, 5-dimethylpiperidin-4-yl ] benzonitrile

To a solution of (3R, 4S, 5S) -4- (4-isocyanophenyl) -1- (4-methoxybenzyl) -3, 5-dimethylpiperidin-4-ol (2.50g, 7.1mmol) from preparation 71 in dichloromethane (50mL) at-15 deg.C was added triethylamine (2.0mL, 14 mmol). To the stirred solution was added dropwise 1-chloroethyl chloroformate (1.50mL, 14mmol), and the mixture was stirred for 30 minutes while maintaining the temperature at-15 ℃. The solvent was removed in vacuo to give a crude residue, which was refluxed in methanol (150mL) for 3 hours. After cooling the reaction mixture to room temperature, the solvent was removed in vacuo and the residue was dissolved in dichloromethane (100 mL). Potassium carbonate (5g) was added, the mixture was stirred for 1 hour, then filtered and the solvent was removed in vacuo. The crude residue was recrystallized from acetonitrile to give the pure compound as fine white needles (1.23 g).

¹H NMR(400MHz，CD₃OD)δ0.60(d，6H)，2.25(m，2H)，3.1(m，4H)，7.62(d，2H)，8.15(d，2H)

LRMS(APCI)＝232[MH⁺]

Preparation example 73

(3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4-pyridin-2-ylpiperidin-4-ol

A solution of 2-bromopyridine (4.10mL, 0.024mol) in diethyl ether (50mL) was cooled to-78 ℃ under nitrogen. n-BuLi (2.5M/hexane) (10.10mL, 25.3mmol) was added dropwise while stirring, maintaining the temperature below-65 ℃. The mixture was stirred at-78 ℃ for 3 hours. A solution of (3R, 5S) -1-benzyl-3, 5-dimethylpiperidinone (6.10g, 28.0mmol) from preparation 14 in diethyl ether (50mL) is then added dropwise, maintaining the temperature below-65 ℃. The mixture was stirred at-78 ℃ for 1 hour and then warmed to room temperature. Saturated ammonium chloride solution (40mL) was added and the mixture was stirred for 30 minutes. The ether layer was separated, washed with water (3 × 50mL), dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography to give the desired product as an orange oil 7.31 g.

¹H-NMR(400MHz，CDCl₃)：δ＝0.43(d，6H)，2.09-2.30(m，4H)，2.71(d，2H)，3.59(s，2H)，5.48(s，1H)，7.19(m，1H)，7.22-7.42(m，6H)，7.71(t，1H)，8.48(d，1H)

LRMS(APCI+)＝297[MH⁺]

Preparation example 74

(3R, 4S, 5S) -3, 5-dimethyl-4-pyridin-2-ylpiperidin-4-ol

A mixture of (3R, 4S, 5S) -1-benzyl-3, 5-dimethyl-4-pyridin-2-ylpiperidin-4-ol (3.0g, 10.12mmol) from preparation 73 and palladium hydroxide (20% palladium on carbon) (0.45g) in ethanol (50mL) was hydrogenated at 40 ℃ and 40psi for 14 h. The reaction mixture was allowed to cool to room temperature and stirred at 40psi for 5 hours. The reaction mixture was filtered through Arbocel ® and the filtrate was concentrated in vacuo to give a crude residue. Purification by column chromatography on silica gel using dichloromethane: methanol: 0.88 ammonia (97.5: 2.5: 0.25-90: 10: 1) as eluent gave 2.05g (99%) of the desired product.

¹H NMR(400MHz，CDCl₃)δ0.43(d，6H)，2.00(m，2H)，2.84(m，4H)，5.50(br，1H)，7.20(m，1H)，7.33(d，1H)，7.72(t，1H)，8.49(d，1H)

LRMS(ESI+)＝207[MH⁺]，413[2MH⁺]

Preparation example 75

(3S, 4R) -4- (2, 4-difluorophenyl) -1-ethylpyrrolidine-3-carboxylic acid hydrochloride

To methyl (3S, 4R) -4- (2, 4-difluorophenyl) -1-ethylpyrrolidine-3-carboxylate (500mg, 1.85mmol) from preparation 76 was added concentrated aqueous hydrochloric acid (10mL), and the resulting solution was stirred at room temperature for 16 hours. The reaction mixture was then evaporated to dryness in vacuo and the resulting residue azeotroped with toluene (2X 50 mL). The title compound was obtained as an off-white foam 500 mg.

¹H-NMR(400MHz，CD₃OD)：□□1.10(t，3H)，2.51-2.62(m，2H)，2.69(t，1H)，2.86(t，1H)，3.05(t，1H)，3.10-3.13(m，2H)，3.92(q，1H)，6.80-6.86(m，2H)，7.45(q，1H).

LRMS(APCI)＝256[MH⁺]

Preparation example 76

(3S, 4R) -4- (2, 4-difluorophenyl) -1-ethylpyrrolidine-3-carboxylic acid methyl ester

A mixture of methyl (3S, 4R) -4- (2, 4-difluorophenyl) pyrrolidine-3-carboxylate from preparation 31 (500mg, 2.07mmol), ethyl tosylate (519mg, 2.59mmol) and potassium carbonate (573mg, 4.15mmol) in acetonitrile was heated at 70 ℃ under nitrogen for 16 hours. The reaction mixture was then cooled to room temperature and concentrated in vacuo. The residue was dissolved in dichloromethane (30mL) and partitioned with saturated aqueous sodium bicarbonate (30 mL). The organic layer was then washed with brine (20mL), dried over magnesium sulfate, filtered and the solvent removed in vacuo. The above procedure was performed in duplicate. The crude products from the two reactions were then combined and purified by column chromatography on silica eluting with methylene chloride: methanol (99: 1) to give the title compound as a pale yellow oil, 968 mg.

¹H-NMR(400MHz，CDCl₃)：□□1.14(t，3H)，2.55-2.62(m，1H)，2.63-2.68(m，1H)，2.70-2.73(m，1H)，2.95-3.05(m，2H)，3.11(t，1H)，3.69(s，3H)，3.89(q，1H)，6.76(t，1H)，6.83(t，1H)，7.37(q，1H).

LRMS(EI)＝270[MH⁺]

According to a preferred embodiment, the compounds according to the invention and the intermediate compounds, in particular the compounds and intermediates exemplified above, are obtained in pure isolated form. As defined herein, pure isolated form means that such compounds and/or intermediates are substantially free of compounds having alternative stereospecific characteristics. As defined herein, substantially free means that at least 90%, preferably at least 92%, more preferably at least 95%, even more preferably at least 98%, especially at least 99% of the compound is present in the desired stereospecific form.

Data of

Compounds according to the present invention, including the compounds of examples 12, 20, 16, 48, 1, 5, 6, 22, 13, 9, 10, 50, 14, 17, 19, 53, 40, 15, 52, 51, 8, 33, 31, 34, 35, 36, 42, 44, and 47, have been tested and found to demonstrate less than about 150nM functional potency at the MC4 receptor when tested using the assay described in scheme E.

MCR1, MCR3, MCR4 and MCR5 EC for the compounds of the invention produced using the assay methods described in schemes A, B, C and D₅₀The data and their relative selectivities to MCR4 relative to MCR3, MCR1, and MCR5 are set forth in table 5.

TABLE 5

Example No. 2	MCR4EC₅₀(nM)	MCR3EC₅₀(nM)	MCR1EC₅₀(nM)	MCR5EC₅₀(nM)	Selective MCR3/MCR4	Selective MCR1/MCR4	Selective MCR5/MCR4
Example No. 2	MCR4EC₅₀(nM)	MCR3EC₅₀(nM)	MCR1EC₅₀(nM)	MCR5EC₅₀(nM)	Selective MCR3/MCR4	Selective MCR1/MCR4	Selective MCR5/MCR4	1	25	1389	8100	6710	56	324	268
2	68	8380	-	-	123	-	-	1	25	1389	8100	6710	56	324	268
2	68	8380	-	-	123	-	-	3	44	2620	-	-	60	-	-
4	45	-	-	-	-	-	-	3	44	2620	-	-	60	-	-

MCR1, MCR3, MCR4 and MCR5 EC of the Compounds of the invention produced Using the assay methods described in schemes A, B, D and E₅₀The data and their relative selectivities to MCR4 relative to MCR3, MCR1, and MCR5 are set forth in table 6.

TABLE 6

Example No. 2	MCR4EC₅₀(nM)	MCR1EC₅₀(nM)	MCR5EC₅₀(nM)	Selective MCR1/MCR4	Selective MCR5/MCR4
Example No. 2	MCR4EC₅₀(nM)	MCR1EC₅₀(nM)	MCR5EC₅₀(nM)	Selective MCR1/MCR4	Selective MCR5/MCR4	1	9.6	1197	2738	125	285
6	19	541	1586	28	83	1	9.6	1197	2738	125	285
6	19	541	1586	28	83	22	23	-	17754	-	772
13	27	8403	16861	311	624	22	23	-	17754	-	772
13	27	8403	16861	311	624	31	3.6	10687	956	2969	266
34	4	-	-	-	-	31	3.6	10687	956	2969	266
34	4	-	-	-	-	12	75	667	20000	9	267
35	1.5	1270	20000	847	13,333	12	75	667	20000	9	267
35	1.5	1270	20000	847	13,333	15	6.3	-	3505	-	556
16	54	-	-	-	-	15	6.3	-	3505	-	556
16	54	-	-	-	-	5	9.6	1197	2738	125	285

Claims

1. A compound of the general formula (I)

wherein each of the above R¹The groups are optionally substituted with one or more groups selected from:

-(C₁-C₄) Alkyl, - (CH)₂)_m(C₃-C₅) Cycloalkyl, halogen, - (CH)₂)_mOR⁶、-CN、-C(O)OR⁶、-(CH₂)_mNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃Wherein m is 0, 1 or 2;

R²is H, OH or OCH₃；

R⁴selected from: -H, - (C)₁-C₄) Alkyl, - (C)₂-C₄) Alkenyl, - (C)₂-C₄) Alkynyl, - (CH)₂)_p(C₃-C₅) Cycloalkyl, - (CH)₂)_p(C₅) Cycloalkenyl, halogen, - (CH)₂)_pOR⁶、-(CH₂)_pNR⁷R⁸、-CN、-C(O)R⁶、-C(O)OR⁶、-C(O)NR⁷R⁸、-(CH₂)_pNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃A group wherein p ═ 0, 1 or 2;

R⁵selected from: - (C)₁-C₄) Alkyl, - (C)₂-C₄) Alkenyl, - (C)₂-C₄) Alkynyl, - (CH) ₂)_p(C₃-C₅) Cycloalkyl, - (CH)₂)_p(C₅) Cycloalkenyl, halogen, - (CH)₂)_pOR⁶、-(CH₂)_pNR⁷R⁸、-CN、-C(O)R⁶、-C(O)OR⁶、-C(O)NR⁷R⁸、-(CH₂)_pNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃A group wherein p ═ 0, 1 or 2;

R⁶、R⁷and R⁸Each independently selected from H, CH₃Or CH₂CH₃；

2. A compound according to claim 1, wherein R¹Selected from: - (C)₁-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, phenyl, - (C)₁-C₂) Alkylaryl, heterocyclyl or- (C)₁-C₂) An alkyl heterocyclic group, and wherein R¹Optionally substituted with one or more groups selected from: - (C)₁-C₄) Alkyl, - (CH)₂)_mOR⁶、-(CH₂)_m(C₃-C₅) Cycloalkyl, halogen, OCH₃、OCH₂CH₃、CN、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃Wherein m is 1 or 2,

wherein when R is¹Is heterocyclyl or- (C)₁-C₂) Alkyl heterocyclyl when said heterocyclyl is independently selected from a monocyclic 5-to 6-membered ring system containing up to 3 heteroatoms independently selected from O, N or S, and combinations thereof.

3. A compound according to claim 1 or 2, wherein R¹Selected from: - (C)₁-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, phenyl, - (C)₁-C₂) Alkylaryl, heterocyclyl or- (C)₁-C₂) An alkyl heterocyclic group, a heterocyclic group having a heterocyclic group,

wherein each of the above R¹The groups are optionally substituted with one or more groups selected from: - (C) ₁-C₄) Alkyl, halogen, - (CH)₂)_mOR⁶、CN、CF₃Or OCF₃Wherein m is 1 or 2;

R²is OH;

R³selected from: -H, - (C)₁-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, aryl, - (C)₁-C₂) Alkylaryl, heterocyclyl or- (C)₁-C₂) An alkyl heterocyclic group, a heterocyclic group having a heterocyclic group,

wherein each of the latter seven kinds of R³The groups are optionally substituted with one or more groups selected from: -OH, - (C)₁-C₄) Alkyl, - (CH)₂)_n(C₃-C₅) Cycloalkyl, halogen, CN, - (CH)₂)_nOR⁶Or- (CH)₂)_nNR⁷R⁸Wherein n is 0, 1 or 2;

R⁴selected from: -H, - (C)₁-C₄) Alkyl, - (CH)₂)_p(C₃-C₅) Cycloalkyl, halogen, - (CH)₂)_pOR⁶、-(CH₂)_pNR⁷R⁸、-CN、-C(O)R⁶、-C(O)OR⁶、-C(O)NR⁷R⁸、-(CH₂)_pNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃A group wherein p ═ 0, 1 or 2;

R⁵selected from: - (C)₁-C₄) Alkyl, - (CH)₂)_p(C₃-C₅) Cycloalkyl, halogen, - (CH)₂)_pOR⁶、(CH₂)_pNR⁷R⁸、CN、C(O)R⁶、C(O)OR⁶、C(O)NR⁷R⁸、(CH₂)_pNR⁷SO₂R⁸、CF₃、CH₂CF₃、OCF₃Or OCH₂CF₃A group wherein p ═ 0, 1 or 2;

R⁶、R⁷and R⁸Each independently selected from H, CH₃Or CH₂CH₃；

and wherein R¹The heterocyclic group of (a) is selected from monocyclic 5-to 6-membered ring systems containing 1 heteroatom independently selected from O or N.

4. A compound according to any preceding claim, wherein R is³is-H, - (C)₁-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) An alkylaryl or heterocyclyl group, and wherein each of the latter five R' s³The groups are optionally substituted with one or more groups selected from: -OH、-(C₁-C₄) Alkyl, - (CH)₂)_n(C₃-C₅) Cycloalkyl, halogen, -CN or- (CH)₂)_nOR⁶Wherein n is 0 or 1, wherein R⁶Is H, CH₃Or CH₂CH₃，

Wherein when R is³When a heterocyclyl group, the heterocyclyl group is selected from monocyclic 5-to 6-membered ring systems containing up to 2 heteroatoms independently selected from O or N, and combinations thereof.

5. A compound according to any preceding claim, wherein R is¹Selected from: - (C)₁-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, phenyl or heterocyclyl, wherein each of the above R¹The groups are optionally substituted with one or more groups selected from: - (C)₁-C₄) Alkyl, halogen, -OR⁶or-CN;

R²is-OH;

R³selected from: -H, - (C)₂-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl or heterocyclyl, wherein each of the latter four R' s³The groups are optionally substituted with one or more groups selected from: -OH, - (C)₁-C₄) Alkyl, - (CH)₂)_n(C₃-C₅) Cycloalkyl, halogen, -CN, -OR⁶Or- (CH)₂)_nNR⁷R⁸Wherein n is 0, 1 or 2;

R⁴selected from: H. f or Cl;

R⁵selected from: f or Cl;

R⁶、R⁷and R⁸Each independently selected from H, CH₃Or CH₂CH₃；

Wherein R is³The heterocyclic group of (a) is selected from monocyclic 6-membered ring systems containing up to 2 heteroatoms independently selected from O or N and combinations thereof;

and wherein R¹The heterocyclic group of (A) is selected from monocyclic 6-membered ring systems containing 1And each heteroatom is independently selected from O or N.

6. A compound according to any preceding claim, wherein R is¹The heterocyclic group of (a) if present is a monocyclic 6-membered ring system containing 1N heteroatom.

7. A compound according to any preceding claim, wherein R is³The heterocyclic group of (a) if present is a monocyclic 6-membered ring system containing up to 2N heteroatoms.

8. A compound according to any preceding claim having the general formula (IA)

Wherein R is¹、R²、R³、R⁴And R⁵Is as defined above, wherein the stereochemistry of the groups at the 3 and 4 positions of the pyrrolidine rings are trans relative to each other.

9. A compound according to any preceding claim having the general formula (IB)

Wherein R is¹、R²、R³、R⁴And R⁵Is as defined above, wherein the stereochemistry of the methyl groups at the 3 and 5 positions of the piperidine ring are cis with respect to each other.

10. A compound according to any preceding claim having the general formula (IB) wherein the stereochemistry of the groups at the 3 and 4 positions of the pyrrolidine ring are trans relative to each other.

11. A compound according to any preceding claim having the general formula (IC)

Wherein R is¹、R²、R³、R⁴And R⁵Is as defined above, wherein the stereochemistry of the groups at the 3 and 4 positions of the pyrrolidine rings is trans with respect to each other, and wherein the stereochemistry of the methyl groups at the 3 and 5 positions of the piperidine rings is cis with respect to each other.

12. A compound according to any preceding claim having the general formula (ID)

Wherein R is¹、R²、R³、R⁴And R⁵Is as defined above, wherein the stereochemistry of the methyl groups at the 3 and 5 positions of the piperidine ring are cis with respect to each other, wherein R at the 4 position is¹The radicals being trans with respect to the methyl groups in the 3 and 5 positions of the piperidine ring, and R²The group is cis with respect to the methyl group.

13. A compound according to any preceding claim having the general formula (ID) wherein the stereochemistry of the groups at the 3 and 4 positions of the pyrrolidine ring are trans relative to each other.

14. A compound according to any preceding claim having the general formula (IE)

Wherein R is¹、R²、R³、R⁴And R⁵Is as defined above, wherein the stereochemistry of the groups at the 3-and 4-positions of the pyrrolidine rings is trans with respect to each other, wherein the stereochemistry of the methyl groups at the 3-and 5-positions of the piperidine rings is cis with respect to each other, wherein the R at the 4-position is¹The radicals being trans with respect to the methyl groups in the 3 and 5 positions of the piperidine ring, and R²The group is cis with respect to the methyl group.

15. A compound according to any preceding claim, having the general formula (IF)

Wherein R is¹、R²、R³、R⁴And R⁵Is as defined above, wherein the stereochemistry of the groups at the 3-and 4-positions of the pyrrolidine rings is trans with respect to each other, wherein the stereochemistry of the methyl groups at the 3-and 5-positions of the piperidine rings is cis with respect to each other, wherein the R at the 4-position is ¹The radicals being trans with respect to the methyl groups in the 3 and 5 positions of the piperidine ring, and R²The radicals being cis relative to the methyl radical, wherein R⁴And R⁵At the 2 and 4 positions of the phenyl ring.

16. A compound according to any preceding claim, wherein R is¹Is selected from- (C)₁-C₄) Alkyl, - (C)₃-C₆) Cycloalkyl, phenyl or pyridyl, wherein R¹Optionally substituted with one or more groups selected from: CH (CH)₃、CH₂CH₃Halogen, OCH₃、OCH₂CH₃、CN、CF₃Or OCF₃。

17. According to any preceding claimA compound of formula (I) wherein R¹Selected from n-propyl, isopropyl, n-butyl, methoxymethyl, cyclopropyl, cyclohexyl, phenyl, 3-fluorophenyl, 4-chlorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2, 6-difluorophenyl, 2, 4-difluorophenyl, 3, 4-difluorophenyl, pyridin-2-yl or pyridin-3-yl.

18. A compound according to any preceding claim, wherein R is¹Selected from pyridin-2-yl, phenyl, 3-fluorophenyl, 4-chlorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2, 6-difluorophenyl, 2, 4-difluorophenyl or 3, 4-difluorophenyl.

19. A compound according to any preceding claim, wherein R is³is-H, - (C)₂-C₆) Alkyl, - (C)₃-C₈) Cycloalkyl, - (C)₁-C₂) Alkyl radical (C)₃-C₈) Cycloalkyl or heterocyclyl, wherein each of the latter four R' s ³The groups are optionally substituted with one or more groups selected from: -OH, - (C)₁-C₄) Alkyl OR-OR⁶Wherein R is⁶is-H, CH₃Or CH₂CH₃Wherein when R is³When a heterocyclyl group, the heterocyclyl group is a monocyclic 6-membered ring system containing up to 2N heteroatoms.

20. A compound according to any preceding claim, wherein R is³Selected from: hydrogen, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, isobutyl, 2-methoxyethyl, cyclopentyl, cyclobutyl, cyclopentylmethyl, pyridin-2-yl, pyridin-3-yl, pyridazin-3-yl, pyrazinyl, pyrimidin-5-yl, pyrimidin-2-yl, pyrimidin-4-yl or tetrahydropyran-4-yl.

21. A compound according to any preceding claim, wherein R is⁴Selected from H, F or Cl, and R⁵Selected from F or Cl.

22. A compound according to any preceding claim, wherein R is⁴And R⁵The phenyl groups of the substituents are: 2, 4-disubstituted phenyl, wherein R⁴And R⁵Each group is independently selected from F or Cl; or 4-monosubstituted phenyl, wherein R⁴Is H, and R⁵Is F or Cl.

23. A compound according to any preceding claim, wherein R is⁴And R⁵The substituted phenyl is selected from 4-chlorophenyl or 2, 4-difluorophenyl.

24. A compound according to any of claims 16 to 23 having the general formula (IF).

25. A compound of formula (IC) according to any preceding claim, wherein:

R¹is phenyl, 3-fluorophenyl, 4-fluorophenyl, 2, 6-difluorophenyl, 2, 4-difluorophenyl, 3, 4-difluorophenyl or pyridin-2-yl; r²Is OH; r³Is H; r⁴Selected from H or F; and R is⁵Selected from F or Cl.

26. A compound according to claim 25 selected from the compounds of examples 12, 16, 24 and 48 or a pharmaceutically acceptable salt, solvate or hydrate thereof.

27. A compound of general formula (IC) according to any of claims 1 to 24, wherein:

R¹is phenyl or pyridin-2-yl; r²Is OH; r³Is a heterocyclic group selected from pyridin-2-yl, pyridin-3-yl, pyridazin-3-yl, pyrazinyl, pyrimidin-5-yl, pyrimidin-4-yl, pyrimidin-2-yl or tetrahydropyran-4-yl; r⁴And R⁵Are both F.

28. A compound according to claim 27, selected from the compounds of examples 31, 34, 35, 42 and 47 and pharmaceutically acceptable salts, solvates or hydrates thereof.

29. A compound of general formula (IC) according to any of claims 1 to 24, wherein:

R¹is phenyl, 4-fluorophenyl, 4-chlorophenyl, 3-fluorophenyl, 2, 4-difluorophenyl, 3, 4-difluorophenyl, pyridin-2-yl; r²Is OH; r³Is t-Bu, i-Pr, Et; r ⁴And R⁵Is F.

30. A compound according to claim 29 selected from the compounds of examples 1, 5, 6, 8, 9, 10, 13, 15, 22, 40, 50, 51, 52 and 53 and pharmaceutically acceptable salts, solvates or hydrates thereof.

31. A compound according to claim 29 selected from the compounds of examples 1, 5, 6, 8, 9, 10, 12, 13, 15, 16, 22, 24, 31, 34, 35, 40, 42, 47, 48, 50, 51, 52 and 53 and pharmaceutically acceptable salts, solvates or hydrates thereof.

32. A compound according to any preceding claim selected from the compounds of examples 1, 5, 9, 12, 13 and pharmaceutically acceptable salts, solvates or hydrates thereof.

33. A compound according to claim 1 selected from (3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol, also known as [ 1-tert-butyl-4- (2, 4-difluoro-phenyl) -pyrrolidin-3-yl ] - (4-hydroxy-3, 5-dimethyl-4-phenyl-piperidin-1-yl) -methanone and pharmaceutically acceptable acid salts, hydrates or solvates thereof.

34. A compound according to claim 1 selected from (3R, 4R, 5S) -1- { [ (3S, 4R) -1-tert-butyl-4- (2, 4-difluorophenyl) pyrrolidin-3-yl ] carbonyl } -3, 5-dimethyl-4-phenylpiperidin-4-ol hydrochloride and [ 1-tert-butyl-4- (2, 4-difluoro-phenyl) -pyrrolidin-3-yl ] - (4-hydroxy-3, 5-dimethyl-4-phenyl-piperidin-1-yl) -methanone HCl salt.

35. A process for the preparation of a compound of formula (I) as defined in any of claims 1 to 34, via a coupling reaction of compounds (II) and (III)

Wherein R is¹、R²、R³、R⁴And R⁵Is as defined above.

36. A pharmaceutical composition comprising a compound of formula (I), (IA), (B), (IC), (ID), (IE) or (IF), or a pharmaceutically acceptable salt, hydrate, solvate or derivative thereof, together with one or more pharmaceutically acceptable excipients, diluents or carriers.

37. The pharmaceutical composition according to claim 36, comprising one or more additional therapeutic agents.

38. The pharmaceutical composition according to claim 36, wherein the additional therapeutic agent is one or more agents selected from the group consisting of: a PDE5 inhibitor; an NEP inhibitor; a D3 or D4 selective agonist or modulator; an estrogen receptor modulator and/or an estrogen agonist and/or an estrogen antagonist; a testosterone replacement agent, testosterone or a testosterone implant; estrogen, estrogen and medroxyprogesterone or medroxyprogesterone acetate (MPA) or estrogen and methyltestosterone hormone replacement therapy.

39. The pharmaceutical composition according to claim 36, wherein the additional therapeutic agent is a PDE5 inhibitor or a NEP inhibitor.

40. A compound of formula (I), (IA), (B), (IC), (ID), (IE) or (IF) or a pharmaceutically acceptable salt, solvate or derivative thereof or a pharmaceutical composition according to any of claims 37 to 39 for use as a medicament.

41. A compound of formula (I), (IA), (B), (IC), (ID), (IE) or (IF) or a pharmaceutically acceptable salt, hydrate, solvate or derivative thereof or a pharmaceutical composition according to any of claims 37 to 39 for use in the treatment of female sexual dysfunction, male erectile dysfunction, obesity or diabetes.

42. Use of a compound of formula (I), (IA), (B), (IC), (ID), (IE) or (IF), or a pharmaceutically acceptable salt, solvate or derivative thereof, or a pharmaceutical composition according to any of claims 37 to 39, for the manufacture of a medicament for the treatment of female sexual dysfunction, male erectile dysfunction, obesity or diabetes.

43. A method of treating female sexual dysfunction, male erectile dysfunction, obesity or diabetes comprising administering an effective amount of a compound of formula (I), (IA), (B), (IC), (ID), (IE) or (IF) or a pharmaceutically acceptable salt, solvate or derivative thereof or a pharmaceutical composition according to any of claims 37 to 39.