WO2004099185A1

WO2004099185A1 - 2-cyanopyrrolidine derivatives and their use as dpp-iv inhibitors

Info

Publication number: WO2004099185A1
Application number: PCT/JP2004/006568
Authority: WO
Inventors: Ichiro Shima; Toshio Kurosaki; Aiko Wada
Original assignee: Fujisawa Pharmaceutical Co. Ltd.
Priority date: 2003-05-09
Filing date: 2004-05-10
Publication date: 2004-11-18
Also published as: AU2003902260A0

Abstract

A compound of the formula (I) or a pharmaceutically acceptable salt thereof: [wherein X is CFH, or CF?2&num;191, R1 is the moiety represented by the formula: [wherein R2 is (lower)alkyl, R3 is phenyl-(lower)alkyl, and the like.], and the like.] Compounds of formula (I) inhibit DPP-IV activity. They are therefore useful in the treatment of conditions mediated by DPP-IV, such as NIDDM.

Description

DESCRIPTION

2-CYANOPYRROLIDINE DERIVATIVES AND THEIR USE AS DPP-IV INHIBITORS

TECHNICAL FIELD

This invention relates to the compounds and pharmaceutically acceptable salts thereof which inhibit dipeptidyl peptidase-IV (DPP-IV).

Moreover, this invention relates to medicament or pharmaceutical composition comprising the above-mentioned compounds or pharmaceutically acceptable salts thereof as an active ingredient , a method for treatment and/or prevention of NIDDM, or the like, and use of the above compound.

BACKGROUND ART

It is known that DPP-IV has various physiological functions in living body, especially has the action which inactivates Glucagon- like peptide-1 (GLP-1) by cleaving the terminal dipeptide (His-Ala) . That is, the resultant peptide is the receptor antagonist of GLP-1 and totally reduces the activity of GLP-1.

This GLP-1 has very important role in sugar metabolism. For example, (1) GLP-1 intensifies the secretion of insulin, (2) express genes which are indispensable for the secretion of insulin, (3) stimulate proliferation of β-cell, (4) suppresses secretion of glucagon, (5) suppresses the function about secretion and motility of digestive organs (especially, peristalsis), and (6) suppresses appetite. That is, GLP-1 restricts food ingestion, postpones the process of digestion and absorption, and raised the use of the sugar in blood.

Therefore, the inhibitor of DPP-IV can maintain the activity of GLP-1 , so it is expected as a medicine to treat and prevent various diseases, especially non-insulin dependent diabetes mellitus (NIDDM).

Hitherto, such inhibitors of DPP-IV are known so far. For example, in US 6,011,155, 2 -cyanopyrrolidine compounds like following are disclosed.

Pyrrolidine, l-[[2-[(5-cyano-2- pyridinyl)amino]ethyl]amino]actyl-2-cyano, (S)-, monohydrochloride

In US 6,110,949, 4 - cyanothiazolidine compounds like following are disclosed.

3-[(Cyclohexyl)amino]acetyl-4-cyano-(R)- thiazolid ne monohydrochloride

In US 6,124,305, 2 -cyanopyrrolidine compounds like following are disclosed.

Pyrrolidine, l-[(2,6,6-trimethylbicyclo[3.1.1]hept-3- yl)amino]acetyl-2-cyano, (S)[lS[lα,2β,3α(S),5α]] monohydrochloride

In WO 00/34241, 2 -cyanopyrrolidine compounds like following are disclosed.

"LAF-237"

Pyrrolidine, 1- [(3-hydroxy-l-adamantyl)amino]acetyl- 2-cyano, (S)

WO 02/02556 discloses following compounds as α4 integrin receptor antagonists for treating integrin mediated disorder such as asthma, rheumatoid arthritis, or the like.

In the above formula, R₃ and R₅ may be bonded to form a pyrrolidine ring. However, such compound or compounds, which has hydrophilic group on azabicyclo moiety, are not described specifically.

WO 03/002553 discloses ( 2S , 4S )- 4 - fluoro - 1 -({[ 1 - (isopropylsulfonyl) -4 - piperidinyl ] amino Jacetyl) -2-pyr rolidinecarbonitrile hydrochloride .

However, the other pyrrolidinecarbonitrile compounds substituted by [( lower ) alkyl ] sulfonyl group are not described .

WO 03/074500 discloses ( 2S , 4S ) - 4 -fluoro- 1 - ( 2 - { [ 1- (2-pyrazinyl)piperidin-4-yl] amino }acetyl) -2-pyrro lidinecarbonitrile.

DISCLOSURE OF INVENTION

Under the above situation, the inventors of this invention found that the introduction of the oxime derivative group at appropriate position of the compound result in remarkable improvement of the activity to inhibit DPP-IV, and completed this invention.

Accordingly, this invention relates to DPP-IV inhibitor. More particularly, this invention relates to DPP-IV inhibitor useful for treating or preventing conditions mediated by DPP-IV, more particularly useful for treating or preventing altered glucose tolerance, glucosuria, hyperlipidemia , metabolic acidosis, diabetes mellitus (IDDM and NIDDM) , diabetic neuropathy, nephropathy, and secondary diseases in mammals caused by diabetes mellitus.

That is, one object of this invention is to provide new compounds and pharmaceutically acceptable salts thereof, of which activity to inhibit DPP-IV is remarkably improved against known compounds.

Another object of this invention is to provide a medicament and pharmaceutical composition containing the compound(s) and/or pharmaceu ically acceptable salts thereof as an active ingredient. A further object of this invention is to provide a method for inhibiting DPP-IV comprising administering an effective amount of the compounds and/or pharmaceutically acceptable salts thereof.

A further object of this invention is to provide a use of the compounds and pharmaceutically acceptable salts thereof as medicaments.

A further object of this invention is to provide the compounds and pharmaceutically acceptable salts thereof which are useful for the manufacture of medicaments for treating or preventing conditions mediated by DPP-IV inhibition, more particularly useful for treating or preventing altered glucose tolerance, glucosuria, hyperlipidemia, metabolic acidosis, diabetes mellitus (IDDM and NIDDM), diabetic neuropathy, nephropathy, and secondary diseases in mammals caused by diabetes mellitus , especially NIDDM.

A further object of this invention is to provide the commercial package comprising the pharmaceutical composition containing the new compound.

The compounds of this invention can be represented by the following formula (I):

[wherein

X is CFH, or CF₂,

R¹ is the moiety represented by the formula

[wherein R is ( lower ) alkyl ,

R³ is cycloalkyl, aryl- ( lower ) alkyl (which may have 1 to 3 substituent ( s ) selected from the group described later on the aryl group), or heteroaryl -( lower ) alkyl (which may have 1 to 3 subs t ituent ( s ) selected from the group described later on the heteroaryl group)], or

the moiety represented by the formula:

[wherein R⁴ is ( lower ) alkyl ,

R^s is hydrogen, or ( lower ) alkyl , R⁶ is ( lower ) alkyl , cycloalkyl, aryl (which may have 1 to 3 substituen ( s ) selected from the group described later), or heteroaryl (which may have 1 to 3 substituent ( s ) selected from the group described later) , and the partial structure:

may form cycloalkylidene ] , the " substituent(s)" is(are) selected from the group consisting of ( lower ) alkyl , halogenated- ( lower ) alkyl , ( lower ) alkoxy , aryloxy, halogen, cyano, nitro, amino and hydroxy ]

In the above and subsequent description of the present specification, suitable examples of the various definitions to be included within the scope of the invention are explained in detail in the following.

The term "lower" is intended to mean a group having 1 to 6 carbon atom(s), unless otherwise provided.

Therefore, the "( lower ) alkyl " means a straight or branched chain aliphatic hydrocarbon, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like, and it is preferably (C1-C4 ) alkyl , more preferably ( CI -C2 ) alkyl , most preferably methyl. The "cycloalkyl" means ( C3 -CIO ) cycloalkyl group, such as cyclopropyl , cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, adamantyl, and the like, and it is preferably cyclopentyl or cyclohexyl.

The "cycloalkylidene" is divalent cycloalkyl group.

Therefore, the partial structure:

may be exemplified

and the like.

The "aryl" means an aromatic hydrocarbon group, such as phenyl, naphtyl, indenyl, and the like, and it is preferably ( C6 -CIO ) aryl , more preferably phenyl.

The " aryl- ( lower ) alkyl" means the "( lower ) alkyl " group mentioned above substituted by aryl group, and include benzyl, 1 -phenylethyl , 2 -phenylethyl , 3-phenylpropyl , 4 -phenylbutyl , naphtylmethyl , 2-naphtylethyl , and the like, and it is preferably phenyl- ( lower ) alkyl , phenyl- ( CI -C4 ) alkyl , more preferably phenyl- (Cl-C2)alkyl, most preferably benzyl.

Especially, the "phenyl- ( lower ) alkyl " means the "( lower ) alkyl " group mentioned above substituted by phenyl group, and include benzyl, 1-phenethyl, 2-phenethyl, 3-phenylpropyl, phenylisopropyl , 4 -phenylbutyl , 6 -phenylhexyl and the like, and it is preferably phenyl- ( CI -C4 ) alkyl , more preferably phenyl- ( CI -C2 ) alkyl , most preferably benzyl.

The "heteroaryl" means 5-, 6-membered or condensed polycyclic aromatic heterocyclic group which contains at least one hetero atom such as nitrogen, oxygen, sulfur atom. The "heteroaryl" may include 5-membered heteroaryl group such as pyrrolyl, imidazolyl, pyrazolyl, thienyl, furyl , oxazolyl, isoxazolyl, thiazolyl.

the heteroaryl group, more preferably b-memoere o 6 -membered heteroaryl group containing nitrogen atom(s) , more preferably pyridinyl .

Especially, the "pyridinyl" may be 2-pyridinyl, 3-pyridinyl and 4-pyridinyl, preferably 2-pyridinyl or 3 -pyridinyl . The "pyridinyl -( lower ) alkyl" includes pyridinylmethyl , 1 -pyridinylethyl , 2 -pyridinylethyl , 3 -pyridinylpropyl , pyridinylisopropyl , 4 -pyridinylbutyl , 6 -pyridinylhexyl and the like, and it is preferably pyridinyl- ( CI -C4 ) alkyl , more preferably pyridinyl- (C1-C2 ) alkyl , most preferably pyridinylmethyl . The "aryl- ( lower ) alkyl" , " heteroaryl- ( lower ) alkyl " , "aryl" and "heteroaryl" groups may respectively have 1 to 3 substituent ( s ) on the aryl or heteroaryl group, the number of substituent ( s ) is preferably 1 or 2, more preferably 1, in case that these groups has plural substituents, they may be the same or different each other , but, needless to say, these groups may not have substituent .

The "halogenated- ( lower ) alkyl" means the above (lower)alkyl substituted by halogen atom(s), such as fluoromethyl, chloromethyl , dif luoro ethyl , dichloromethyl , dibromomethyl , trifluoromethyl, trichloromethyl , fluoroethyl, chloroethyl, 2,2,2 - trifluoroethyl , 2,2,2 - trichloroethyl ,

2,2,3,3 , 3-pentafluoroethyl , fluoropropyl , fluorobutyl, fluorohexyl, and the like, and it is preferably halogen-substituted ( CI -C4 ) alkyl , more preferably halogen- substituted ( CI -C2 ) alkyl , more preferably fluorine- substituted ( CI -C2 ) alkyl , more preferably fluorine- substituted methyl, most preferably trifluoromethyl.

The "( lower ) lkoxy" means a straight or branched chain aliphatic hydrocarbon oxy group, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, and the like, and it is preferably ( CI -C4 ) alkoxy , more preferably ( CI -C2 ) alkoxy , most preferably methoxy.

The "aryloxy" means oxy group subsutituted with the above aryl, and includes phenyloxy, naphtyloxy, indenyloxy, and the like, and it is preferably phenyloxy. The "halogen" may include a fluorine atom, a chlorine atom, a bromine atom or a iodine atom, more preferably a chlorine atom.

The compounds of formula (I) may contain one or more asymmetric centers and thus they can exist as enantiomers or diastereoisomers . This invention includes both mixtures and separate individual isomers. However, in the portion of 2- cyanopyrrolidine , (2S) and/or (4S) isomer is more preferable. Oxime derivatives may have two kind of geometrical isomers, that is, syn form and anti form, this invention includes both isomers.

The compounds of the formula (I) may also exist in tautomeric forms and this invention includes both mixtures and separate individual tautomers.

The compounds of the formula (I) and its salts may be in a form of a solvate such as hydrate, which is included within the scope of the present invention.

Also included in the scope of invention are radiolabelled derivatives of compounds of formula (I) which are suitable for biological studies.

The compounds of this invention may be converted to salt according to a conventional method. Suitable salts of the compounds (I) are pharmaceutically acceptable conventional non-toxic salts such as an organic acid salt (e.g., acetate, trifluoroacetate , maleate, tartrate, methanesulfonate , benzenesulfonate , formate, toluenesulfonate , trifluoroacetate , or the like.), an inorganic acid salt (e.g., hydrochloride , hydrobromide , sulfate, phosphate, or the like.), a salt with an amino acid (e.g. , arginate, aspartate, glu amate , or t elike. ) , or the like, and the preferable salt is hydrochloride or trifluoroacetate salt.

The compound (I) may preferably include; a compound of the formula (II)

[wherein

X is CFH or CF₂,

R² is ( lower ) alkyl ,

R³ is cycloalkyl, aryl- ( lower ) alkyl (which may have 1 to 3 subs tituent ( s ) selected from the group described later on the aryl group), or heteroaryl- ( lower ) alkyl (which may have 1 to 3 substituent ( s ) selected from the group described later on the heteroaryl group), the "substituent(s) " is(are) selected from the group consisting of ( lower ) alkyl , halogenated- ( lower ) alkyl , ( lower ) alkoxy , aryloxy, halogen, cyano, nitro, amino and hydroxy ] , and

a compound of the formula (III)

[wherein

X is CFH, or CF₂,

R⁴ is ( lower ) alkyl ,

R⁵ is hydrogen, or ( lower ) alkyl ,

R⁶ is ( lower ) alkyl , cycloalkyl, aryl (which may have 1 to 3 substituent ( s ) selected from the group described later) , or heteroaryl (which may have 1 to 3 substituent ( s ) selected from the group described later), and

the partial structure:

may form cycloalkylidene , the " substituent (s)" is(are) selected from the group consisting of ( lower ) alkyl , halogenated- ( lower ) alkyl , ( lower ) alkoxy , aryloxy, halogen, cyano, nitro, amino and hydroxy] .

In the each definition of the compound formula (I) to ( III), preferably,

(1) X is CFH, (2) X is CF₂, (3) R² is (C1-C4 )alkyl, (4) R² is (C1-C2 )alkyl , (5) R² is methyl, (6) R³ is cycloalkyl, (7) R³ is cyclopentyl or cyclohexyl, (8) R³ is benzyl (which may have 1 to 3 substituent ( s ) on the phenyl group).

(9) R is benzyl (which may have 1 substituent on the phenyl group),

(10 R³ is pyridinylmethyl (which may have 1 to 3 subs tituent ( s ) on the pyridinyl group),

(11 R³ is pyridinylmethyl (which may have 1 substituent on the pyridinyl group),

(12 R⁴ is (Cl-C4)alkyl, (13 R⁴ is (Cl-C2)alkyl, (14 R⁴ is methyl. (15 R- is hydrogen, (16 R- is (Cl-C4)alkyl, (17 R- is (C1-C2 )alkyl. (18 R is methyl. (19 R is (Cl-C4)alkyl, (20 R⁶ is (Cl-C2)alkyl, (21 R⁶ is methyl, (22 R⁶ is phenyl (which may have 1 to 3 substituent ( s ) ) , (23 R⁶ is phenyl (which may have 1 substituent), (24 R⁶ is pyridinyl (which may have 1 to 3 substituent(s) ) , ( 25 R⁶ is pyridinyl (which may have 1 substituent). (26) R⁶ is thiazolyl (which may have 1 substituent),

(27) R⁶ is thienyl (which may have 1 substituent),

(28) R⁶ is pyrimidinyl (which may have 1 to 3 substituent(s) ) , (29) R⁶ is pyrimidinyl (which may have 1 substituent), (30) the "substituent(s)" is(are) selected from the group consisting of ( lower ) alkyl , halogenated- (lower) alkyl , (lower ) alkoxy , aryloxy and halogen, (31) the "substituent (s) " is(are) ( lower ) alkyl ,

(32) the " substituent ( s ) " is(are) halogenated- (lower) alkyl ,

(33) the " substituent ( s ) " is(are) aryloxy,

(34) the " substituent ( s ) " is(are) halogen,

The compound of the formula (I) of the present invention can be prepared according to the following Process A. [Process A]

(IV) (V) (I)

In the above formula, R¹ and X represent the same meanings as defined above, and "Hal" represents halogen atom, especially, chlorine or bromine atom. Process A is the process for preparing the Compound (I) from amine compound (IV) and halogenated compound (V) in solvent, preferably in the presence of base.

Compound (IV) and (V) may be purchased if it is commercial, or synthesized according to Processes C and B, respectively, mentioned after or other general methods obvious to the person skilled in the organic chemistry from commercial compounds.

The solvent employable in this . process is not particularly limited so long as it is inactive in this reaction and may include ethers such as diisopropyl ether, tetrahydrofuran , dioxane, preferably tetrahydrofuran .

The base employable in this process for making basic condition is not particularly limited so long as it accelerates this reaction and may include organic amine such as triethylamine, tributylamine , diisopropylethylamine ; alkali metal hydrogencarbonates such as litium hydrogencarbonate , sodium hydrogencarbonate and potassium hydrogencarbonate; alkali metal carbonates such as lithium carbonate, sodium carbonate and potassium carbonate; alkaline earth metal carbonates such as magnesium carbonate and calcium carbonate; alkali metal hydroxides such as lithium hydroxide, sodium hydroxide and potassium hydroxide, preferably organic amine, especially triethylamine.

To accelerate the reaction, sodium iodide may be added . This process is generally carried out by adding the compound (V) to the solution of compound (IV). The temperature at that time varies depending on the starting material, the solvent, etc. , but it is usually from -10°C to 40°C, preferably room temperature. The reaction time after the adding compound (V) varies depending on the starting material, the solvent, etc., but it is usually from Ihr to 24hrs, preferably from Ihr to lOhrs.

After the reaction, the mixture is quenched by aqueous solvent such as water, saturated aqueous solution of NH₄C1, hydrochloric acid , etc, and extracted with organic solvent insoluble with water such as ethyl acetate, CHC1₃, etc. Preferably, the organic layer is washed with water or the like, dried over anhydrous MgS0₄ or Na₂S0₄, evaporated in vacuo, and the target compound (I) is purified by the conventional method such as thin layer chromatography, silica gel column chromatography, etc.

Compound (V), which is the starting compound of Process A, can be synthesized by following Process B. [Process B]

(VI) (VII) (V)

In the above formula, X and "Hal" represent the same meanings as defined above. Process B is the process for preparing the Compound

(V) by forming amide bond in solvent in the presence of base . Compound (VI) and (VII) may be purchased if it is commercial, or synthesized according to general methods obvious to the person skilled in the organic chemistry from commercial compounds . The solvent employable in this process is not particularly limited so long as it is inactive in this reaction and may include ethers such as diisopropyl ether, tetrahydrofuran , dioxane, preferably tetrahydrof ran .

The base employable in this process is not particularly limited so long as it accelerates this process and may include organic amines such as triethylamine, tributylamine , diisopropylethylamine

(DIEA), preferably DIEA.

The additive such as sodium 2 -ethylhexanoate may be added to the mixture to improve the yield of this Process.

This process is generally carried out by adding the Compound (VI) to the solution of Compound (VII) and base and/or catalyst. The temperature at that time varies depending on the starting material, the solvent, etc., but it is usually from -10°C to 30°C , preferably from 0°C to 10 °C . After the addition, the. temperature is preferably raised to room temperature.

The reaction time after the adding varies depending on the starting material, the solvent, etc., but it is usually from lhrs to 24hrs.

After the reaction, the solvent is removed in vacuo, the target compound may be purified by the conventional method such as silica gel column chromatography, etc.

Compound (IV¹) and (IV²), bothof which is the starting Compound (VI) of Process A, canbe synthesized by following Process C. [Process C]

In the above formulae, R² to R⁶ represent the same meanings as defined above, and "Pro" means the protective group of amino grou . Process C is the process for preparing the Compound (IV¹) and (IV²) by forming oxyimino group from amino compound and carbonyl compound in solvent.

Starting Compound (VIII), (IX), (XI) and (XII) may be purchased if they are commercial, or synthesized according to general methods from commercial compounds .

For example, aminooxy group of Compound (XII) can be synthesized by application of Process D described later.

The solvent employable in this process is not particularly limited so long as it is inactive in this reaction and may include alcohols such as methanol, ethanol, isopropanol, preferably methanol . However, in case that Compound (XI) may be used as solvent, other solvent is not necessarily needed.

This process is generally carried out by respectively adding the solution of Compound (VIII) or (XI) to the solution of Compound (IX) or (XII). The temperature at that time varies depending on the starting material, the solvent, etc., but it is usually from 10°C to 50°C , preferably room temperature. The reaction time after the adding varies depending on the starting material, the solvent, etc., but it is usually from Ihr to 24hrs, preferably from 4hrs to 20hrs. After the reaction, the solvent is removed in vacuo, and the residue is triturated with n-hexane, diisopropylether , etc. Compound (X) and (XIII) can be obtained by filtration.

In the second step ( deprotecting ) , concerning the kind of protective group ("Pro") and cleavage reaction of the protective group, TpROTECTIVE GROUPS IN ORGANIC SYNTHESIS Second Edit ionJ T.W. Green and P .G.M. Wuts , John Wiley & Sons, INC. may be referred.

For example, when "Pro" group is carbamate group such as tert -butoxycarbonyl group, the cleavage reaction is carried out in the acidic condition in solvent by acid such as hydrochloric acid, trifluoroacet ic acid, or the like. After the deprotection , excess acid and solvent are removed in vacuo, the solution of residue is washed with aqueous solvent, dried over MgS0 or Na₂S0₄, and the target compound (IV¹) and (IV²) can be obtained by concentrating in vacuo.

The compound having aminooxy group such as Compound (XII) (the starting compound of Process C) can be synthesized by following Process D. [Process D]

In the above formula, "Pro" represents the same meanings as defined above, and R represents R or R' ( ( lower ) alkyl ) .

Process D is the process for preparing the compound having aminooxy group by functional group trans formation from hydroxy group. Inthisprocess, first. Compound (IX) is reduced with ordinary method, for example reduction by H₂ gas and catalyst such as platinum oxide.

The solvent employable in this process is not particularly limited so long as it is inactive in this reaction and may include alcohols such as methanol, ethanol, isopropanol; acetic acid. The temperature at that time varies depending on the starting material, the solvent, etc., but it is usually from 10 °C to 50°C , preferably room temperature. After the reaction, the target compound (XIV) can be obtained by ordinary treating.

Next, to the solution of Compound (XIV) having hydroxy group, hydroxyphthalimide and triphenylphosphine was added diisopropyl azodicarboxylate (DIAD). The solvent employable in this process is not particularly limited so long as it is. inactive in this reaction and may include ethers such as diisopropyl ether, tetrahydrofura , dioxane, preferably tetrahydrofuran .

The temperature at that time varies depending on the starting material, the solvent, etc., but it is usually from -10°C to 50°C, preferably from 0°C to 30°C.

The reaction time after the adding varies depending on the starting material, the solvent, etc., but it is usually from lOmin to 12hrs. After the reaction, to the solution water is added, then extracted with organic solvent insoluble with water such as ethyl acetate, CHC1₃, etc. The organic layer is washed with aqueous solvent such as saturated aqueous solution of NaCI, dried over anhydrous MgS0₄ or Na₂S0₄, and evaporated in vacuo . The phthalimide derivative (XV) can be obtained by triturating and further purification such as silica gel chromatography.

Then, phthalimide derivative (XV) was decomposed by hydrazine in solvent. That is, to the solution of phthalimide derivative, hydrazine is added.

The solvent employable in this process is not particularly limited so long as it is inactive in this reaction and may include alcohols such as methanol, ethanol, isopropanol, preferably ethanol. The temperature at that time varies depending on the starting material, the solvent, etc., but it is usually from 50°C to 150°C , preferably from 70°C to 130°C .

The reaction time after the adding varies depending on the starting material, the solvent, etc., but it is usually from lOmin to 6hrs .

After the reaction, the resulting precipitate is filtered off and washed with solvent, and filtrate is concentrated. Then, to the residue, aqueous solvent and organic solvent insoluble with water such as ethyl acetate are added, aqueous layer is basified and extracted. The organic layer is dried over anhydrous MgS0₄ or Na₂S0₄, evaporated in vacuo to obtain the target compound (XII). If necessary, further purification may be carried out.

In the above processes, functional group trans formation may timely be carried out so long as the other sites of the compounds are not affected. In the following reaction formula, the functional group trans formation of the pyrrolidine ring is shown as representative example.

[Process E]

Proces s E shows the repres entat ive example of functional group trans formation. Accordingly, other reactions of functional group trans formation may be carried out .

Above processes, all starting materials and product compounds may be salts. The compounds of above processes can be converted to salt according to a conventional method. For example of making hydrochloride or trif luoroacetate , to the compound, the solution of acid such as 4N hydrochloride/dioxane or trifluoroacetic acid is added, then the solvent and excess acid is removed and the reside is triturated appropriate solvent such as diethlether.

In the above compounds, which have reactive group, may be protected at the group on cue and be deprotected on cue. In these reactions (protecting or deprotecting steps), concerning the kind of protective group and the condition of the reaction, TPROTECTIVE GROUPS IN ORGANIC SYNTHESIS Second Edition] T . W . Green and P . G . . Wuts , John Wiley & Sons, INC. may be referred.

For therapeutic purpose, the compound (I) and a pharmaceutically acceptable salt thereof of the present invention can be used in a form of pharmaceutical preparation containing one of said compounds as an active ingredient, in admixture with a pharmaceutically acceptable carrier such as an organic or inorganic solid or liquid excipient suitable for oral, parenteral or external administration. The pharmaceutical preparations may be capsules , tablets, dragees, granules, inhalant, suppositories, solution, lotion, suspension, emulsion, ointment, gel, cream, or the like. If desired, there may be included in these preparations, auxiliary substances, stabilizing agents, wetting or emulsifying agents, buffers and other commonly used additives. While the dosage of therapeutically effective amount of the compound (I) will vary depending upon the age and condition of each individual patient, an average single dose of about 0.01 mg, 0.1 mg, 1 mg, 10 mg, 50 mg, 100 mg, 250 mg, 500 mg and 1000 mg of the compound (I) may be effective for treating the above-mentioned diseases. In general, amounts between 0.01 mg/body and about 1,000 mg/body may be administered per day.

BEST MODE FOR CARRYING OUT THE INVENTION

The following Examples are given only for the purpose of illustrating the present invention in more detail.

Although the present invention has been fully described by way of example, it is to be understood that various changes and modifications will be apparent to those skilled in the art. Therefore, unless otherwise such changes and modifications depart from the scope of the present ' invention hereinafter defined, they should be construed as being included therein.

Preparation 1-1

Methyl ( 2S , 4R ) - 4 -hydroxy- 2 -pyrrolidinecarboxylate hydrochloride

Hydroxyproline (155g) was dissolved in Hydrogen

Chlororide, Methanol Reagent 10 (TCI, 900mL), and this mixture was heated at reflux for 2hrs .

The resulting mixture was cooled to room temperature, and the solvent was removed in vacuo to give the target compound as a white powder (215g).

^-N R (in DMSO-d6) : δ 2.30 - 1.99 ( 2H , m), 3.14 - 2.97 ( 1H , m) , 3.45-3.25( 1H, m), 3.76(3H, s), 4.57 - 4.35 ( 2H , m), 9.23(1H, br-s), 10.32(1H, br-s).

Preparation 1-2

1-tert-Butyl 2-methyl

( 2S , 4R ) -4 -hydroxy- 1 , 2 -pyrrolidinedicarboxylate

To a solution of methyl ( 2S , 4R ) -4 -hydroxy- 2 -pyrrolidinecarboxylate hydrochloride obtained in Preparation 1-1 (215g) in water/dioxane (800/500mL) with cooling on an ice bath, was added a solution of di-tert -butyl dicarbonate (271g) in dioxane (150mL) and 6N NaOH (400mL) dropwise.

The reaction mixture was stirred at room temperature for 3hrs and quenched by adding with IN HC1. The aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with saturated aqueous NaCI solution, dried over MgS0₄ , and concentrated in vacuo. The resulting residue was triturated with n-hexane to give the target compound as a white powder (200g).

^XH-NMR (in CDC1₃) : <5 1.51-1.32(9H, m), 2.39-1.82(2H, m), 3.79-3.38 ( 5H, m), 4.58 - 4.31 ( 2H , m).

Preparation 1-3 1-tert-Butyl 2-methyl (2S.4S) -4-f luoro-1,2 -pyrrolidinedicarboxylate

1-tert-Butyl 2-methyl ( 2S , 4R ) - 4 -hydroxy- 1 , 2 - pyrrolidinedicarboxylate obtained in Preparation 1-2 ( 130g ) and cesium fluoride (105g) was dissolved in dioxane (600mL) , this mixture was cooled on an ice bath, and then a solution of diethylaminosulfur trifluoride (lOOg) in dioxane (20mL) was added dropwise for 30min. The reaction mixture was warmed to room temperature and stirred for 5hrs . To the resulting mixture was added NaHC0₃ (400g) , and the reaction mixture was quenched with saturated aqueous NaHC0₃ solution, then water (lOOOmL) and CaCl₂ (382g) in water (300mL) were added. The resulting suspension was filtered and the filtrate was extracted with ethyl acetate . The combined organic layer was washed with saturated aqueous NaCI solution, dried over MgS0₄ , and concentrated in vacuo to give the target compound as a yellow oil (127.5g). Further purification was not attempted.

¹H-NMR (in CDC1₃) : δ 1.55-1.35(9H, m), 2.62-2.16(2H, m), 3 . 94 - 3 . 4 9 ( 5 H , m ) , 4 . 6 0 - 4 . 36 ( 1H , m ) , 5 . 20 ( 1H , br - d , J = 52 . 8 H z ) .

Preparation 1-4 (2S,4S)-l-(tert-Butoxycarbonyl)-4-fluoro-2-pyrrolidin ecarboxylic acid

The crude product of 1-tert-butyl 2-methyl (2S,4S)-4-fluoro-l,2 -pyrrolidinedicarboxylate obtained in Preparation 1-3 (127.5g) was dissolved in methanol (400mL) and then IN NaOH (800mL) was added at room temperature.

After stirring for 1.5hrs , the resulting mixture was washed with diethyl ether , acidified with IN HC1 (lOOOmL), and then was extracted with ethyl acetate. The combined organic layer was washed with saturated aqueous NaCI solution, dried over MgS0₄, and concentrated in vacuo. The resulting residue was triturated with ethyl acetate to give the target compound as a white powder (64g).

^XH-NMR (inCDCl₃) : δ 1.62-1.31(9H, m), 2.94-2.09(2H, m),

4.01-3.44(2H, m), 4.66 - 4.37 ( 1H , m), 5.22(1H, br-d, J=51.9Hz) .

Preparation 1-5 tert-Butyl (2S,4S)-2 - aminocarbonyl- 4- fluoro -1-pyrroli dinecarboxylate

To a mixture of (2S,4S)-l-(tert -butoxycarbonyl ) -4 -fluoro- 2- yrrolidin ecarboxylic acid obtained in Preparation 1-4 (66g), 1 -hydroxybenzotriazole hydrate (45g) in acetonitrile (1500mL) with cooling on an ice bath, was added WSC ^• HC1 (water soluble carbodiimide hydrochloride, 82g). After the mixture was stirred for 45min, 28% aqueous NH₃ (43mL) was added at the same temperature. The resulting mixture was warmed to room temperature and stirred for 15min .

The reaction mixture was filtered and the filtration was evaporated in vacuo. After dilution with ethyl acetate, the reaction mixture was washed with IN HCl, saturated aqueous NaHC0₃ and saturated aqueous NaCI, dried over MgS0₄ , and filtered. After removal the solvent, the target compound was obtained as a white powder (46g).

^-N (inCDCl₃) : δ 1.58 - 1.36 ( 9H , m), 2.99 - 2.02 ( 2H , m), 3.99-3.43( 2H, m), 4.57 - 4.23 ( 1H , m), 5.23(1H, br-d, J=51.6Hz), 5.69-5.40 ( 1H, m), 6.79 - 6.05 ( 1H , m). MS : 233.10 (ES+) .

Preparation 1-6

(2S,4S)-4-Fluoro-2 -pyrrolidinecarboxamide hydrochloride

tert-Butyl ( 2S , 4S ) - 2 -aminocarbonyl- 4 -fluoro- 1 - pyrrolidinecarboxylate obtained in Preparation 1-5 (46g) was dissolved in 4N HCl in dioxane ( 200mL ) and the resulting mixture was stirred for lOmin at room temperature.

After removal of the solvent, the resulting residue was triturated with ethyl acetate to give the target compound as a white powder (34g).

^-NMR (in DMSO-d6) : δ 2.84 - 2.00 ( 2H , m), 4.10 - 3.09 ( 2H , m) , 4.44-4.15( 1H, m), 5.39(1H, br-d, J= 52.5Hz), 7.73(1H, br-s), 8.09(1H, br-s), 8.76(1H, br-s), 10.62(1H, br-s). MS : 132.94 (ES+) .

Preparation 1-7

(2S, 4S) -l-Chloroacetyl-4-fluoro-2 -pyrrolidinecarboxam ide To a mixture of

(2S, 4S) -4-fluoro-2 -pyrrolidinecarboxamide hydrochloride obtained in Preparation 1 - 6 (33g) and sodium 2-ethylhexanoate (70g) in tetrahydrofuran (500mL) with cooling on an ice bath, was added chloroacethyl chloride (24.3g) .

After stirring for 2hrs , the resulting residue was then poured onto buchner funnel/filter paper and washed with ethyl acetate. The solvent was removed in vacuo and the resulting residue was triturated with diethylether to give the target compound (34g) as a white powder.

^-NMR (inCDCl₃) : δ 2.58 - 2.03 ( 1H , m), 3.05 - 2.58 ( 1H , m), 4.17-3.68( 4H, m), 4.85 - 4.54 ( 1H , m), 5.36(1H, br-d, J= 52.5Hz), 5.88-5.49( 1H, m), 6.63 - 6.19 ( 1H , m).

Preparation 1-8

(2S, 4S) -l-Chloroacetyl-4-fluoro-2-pyrrolidinecarbonit rile

To a solution of

(2S,4S)-l-chloroacetyl-4-fluoro-2 -pyrrolidinecarboxam ide obtained in Preparation 1-7 (34g) in tetrahydrofuran (800m) , was added trifluoroacetic anhydride (28mL) at room temperature .

After stirring for 15min, the resulting mixture was concentrated in vacuo. The resulting residue was triturated with ethyl acetate to give the target compound (22g) as a white powder.

^-NM (in CDC1₃) : δ 2.52-2.22(lH, m), 2.87-2.59(lH, m), 4.33-3.75(4H, m), 5.12 - 4.87 ( 1H , m), 5.41(1H, br-d, J=50.7Hz) . Preparation 2-1

Ethyl 1 , 4-dioxaspiro[4.5]decane-8 -carboxylate

To a solution of ethyl 4 -oxocyclohexanecarboxylate (lOOg) in toluene (500mL), were added ethylene glycol

(36mL) and a catalytic amount of p- toluenesulfonic acid, and the resulting mixture was refluxed azeotropically for

2hrs .

Then the solution was cooled to room temperature, washed with saturated aqueous NaHC0₃ solution, saturated aqueous NaCI solution, and dried over MgS0₄. After removal of the solvent, the target compound was obtained

(130g) as a pale yellow oil.

^-NMR (inCDCl₃) : δ 1.18(3H, t, J= 6.9Hz), 1.68 - 1.48 ( 2H , m) , 2.01-1.70 ( 6H, m), 2.40 - 2.25 ( 1H , m), 3.94(4H, s), 4.13(2H, q, J=6.9Hz) . MS : 250.09 (ES+) .

Preparation 2-2

Ethyl 8 -methyl- 1 , 4 -dioxaspiro [ 4.5 ] dec.ane- 8 -carboxylat e

To a solution of diisopropylamine (70mL) in tetrahydrofuran ( 500mL ) , was added n-butyllithium ( 1.6M ,

300mL ) at -40°C dropwise for 25min and the reaction mixture was warmed to -10°C , then cooledto -65°C . To this solution was added a solution of ethyl

1 , 4 -dioxaspiro [ 4.5 ] decane-8 -carboxylate obtained in Preparation 2-1 in tetrahydrofuran (lOOmL) dropwise at the same temperature. The reaction mixture was stirred at -50°C for Ihr, and then methyl iodide (31mL) was added.

The reaction mixture was stirred at between -40 and -30°C for 0.5hr, then warmed to -10°C. The solution was poured into saturated aqueous NaCI solution and ethyl acetate. The combined organic layer was washed with saturated aqueous NaCI solution, and dried over MgS0₄. After removal of the solvent, the target compound was obtained as a yellow oil (70g).

Preparation 2-3

8 -Methyl- l,4-dioxaspiro[4.5]decane-8 -carboxylic acid

Ethyl 8 -methyl- 1 , 4 -dioxaspiro [4.5] decane-8 - carboxylate obtained in Preparation 2-2 (70g) was dissolved in 3N NaOH ( 5 OOmL ) -methanol (400mL), and the reaction mixture was heated at reflux for 0.5hr, and then was cooled to room temperature.

After removal of the organic solvent in vacuo, the aqueous solution was neutralized with 3N HCl and 10% aqueous citric acid to pH5. The solution was saponificated with NaCI, and extracted with ethyl acetate . The combined organic layer was dried over MgS0₄ , and filtered. After removal of the solvent in vacuo, the resulting residue was triturated with diisopropylether to give the target compound (46g) as a white powder.

¹H-NMR (in CDC1₃) : <5 1.26(3H, s), 1.62 - 1.45 ( 2H , m), 1.72-1.62 ( 4H, m), 2.19 - 2.08 ( 2H , m), 3.94(4H, s). MS : 199.78 (ES- ) .

Preparation 2-4 tert-Butyl 8 -methyl- 1 , 4 -dioxaspiro [4.5] dec-8 -ylcarbam ate

To a solution of

8 -methyl- l,4-dioxaspiro[4.5]decane-8 -carboxylic acid obtained in Preparation 2-3 (45g) in toluene (450mL) , were added diphenylphosphoryl azide (53mL) and triethylamine (35mL), and the reaction mixture was heated at 100°C for Ihr. The resulting mixture was cooled on an ice bath, washed with saturated aqueous NaHC0₃ solution, water and saturated aqueous NaCI solution, and dried over MgS0₄.

After removal of the solvent in vacuo, the isocyanate intermediate was obtained as a pale yellow oil. This oil was dissolved in dimethylacetamide (270mL), and to this solution was added potassium tert -butoxide (26g) as portions with cooling on an ice bath. After stirring for

Ihr, the reaction mixture was poured into ice-water (300mL). The resulting precipitate was collected, washed with water ( lOOmL ) to give the target compound ( 53g ) as a white powder.

^XH-NMR (in CDC1₃) : δ 1.33(3H, s) , 1.43(9H, s) , 1.76-1.53(6H, m) , 2.11-1.95(2H, m) , 3.94(4H, s) , 4.39(1H, br-s ) .

Preparation 2-5 tert-Butyl 1 -methyl- 4 -oxocyclohexylcarbamate

tert-Butyl 8 -methyl- 1 , 4-dioxaspiro[ 4.5 ] dec - 8 - ylcarbamate obtained in Preaparation 2-4 (53g) was dissolved in tetrahydrofuran (300mL) and p- toluenesulfonic acid (74g) in water (300mL) at room temperature, and the reaction mixture was stirred at the same temperature for 16hrs.

The resulting solution was neutralized with NaHC0₃, and extracted with ethyl acetate. The combined organic layer was washed with saturated aqueous NaCI solution, dried over MgS0₄, and filtered. After removal of the solvent, the residue was triturated with n-hexane to give the target compound (26g) as a white powder.

^XH-NMR (in CDC1₃) : <5 1.43(3H, s), 1.46(9H, s), 1.87-1.69(2H, m), 2.54 - 2.21 ( 6H , m), 4.50(1H, br-s). Example 1-1 tert-Butyl 4 -hydroxy- 1 -methylcyclohexylcarbamate

To a solution of tert-butyl 1-methyl- 4 -oxocyclohexylcarbamate obtained in Preparation 2-5 (128g) dissolved in acetic acid (lOOOmL), was added platinum oxide (6.4g). The reaction mixture was hydrogenated under H₂ 2.0atm at room temperature for 8hrs.

The resulting mixture was filtered, and the solvent was removed in vacuo. The residue was dissolved in saturated aqueous NaHC0₃ solution, and the aqueous layer was extracted with ethyl acetate. The combined organic layer was dried over MgS0₄, and filtered. After removal of the solvent in vacuo, the residue was triturated with ethyl acetate to give the target compound (128g, cis/trans=12 : 1 ) as a colorless oil.

^XH-NMR (in CDC1₃) (cis) : δ 1.89 - 1.18 ( 18H , m),

2.17-2.05(2H, m), 3.70 - 3.55 ( 1H , m), 4.36(1H, br-s).

Example 1-2 tert-Butyl 4-[ (l,3-dioxo-l,3-dihydro-2H-isoindol-2-y l)oxy] - 1 -methylcyclohexylcarbamate

To a solution of tert-butyl 4 -hydroxy- 1 -methylcyclohexylcarbamate obtained in Example 1-1 (75g), hydroxyphthalimide (54g) and triphenylphosphine (86g) in tetrahydrofuran (750mL) with cooling on an ice bath, was added diisopropyl azodicarboxylat e (64mL) dropwise for 20min, and the reaction mixture was stirred at the same temperature for 20min. The reaction mixture was quenched by pouring water. The organic layer was washed with saturated aqueous NaCI solution, dried over MgS0₄, and filtered. After removal of the solvent in vacuo, the residue was triturated with diethylether , and this precipitate was filtered off. After removal of the solvent, the resulting residue was purified with silica gel chromatography ( n-hexane : ethyl acetate= 4:l) to give the target compound as a white powder (84g). .

^XH-NMR (in CDC1₃ ) : δ 2.03 - 1.17 ( 2 OH , m), 4.41 - 4.30 ( 1H , m), 7.77-7.72(2H, m), 7.86-7.80(2H, m).

Example 1-3 tert-Butyl 4 -aminooxy- 1 -methylcyclohexylcarbamate

tert-Butyl 4- [ (l,3-dioxo-l,3-dihydro-2H-isoindol - 2 -yl ) oxy ]- 1 -methylcyclohexylcarbamate obtained in Example 1-2 (84g) was dissolved in ethanol (840mL) . This solution was warmed to 80°C and NH₂NH₂Η₂0 was added at the same temperature. The resulting mixture was heated at reflux for 30min, and then was cooled on an ice bath. The resulting precipitate was filtered off, washed with ethanol, and the filtrate was concentrated in vacuo.

The resulting mixture was diluted with water and acidified with concentrated HCl. The aqueous layer was washed with ethyl acetate, basified with NaOH, saturated with NaCI, and was extracted with ethyl acetate. The combined organic layer was washed with saturated aqueous NaCI solution, dried over MgS0 , and filtered. After removal of the solvent in vacuo, the target compound was given as a yellow oil (16g).

^-NMR (in CDC1₃) : δ 1.31(3H, s), 1.43(9H, s),

1.84-1.53(8H, m), 3.69 - 3.55 ( 1H , m), 4.38(1H, br-s), 5.24( 1H, br-s) . Example 1-4 tert-Butyl l-methyl-4-({[(lE) - 3 -pyridinylmethylidene ] amino}oxy ) cyclohexylcarbamate

To a cold stirred solut ion ( - 78 °C ) of tert-butyl

4 -( aminooxy )- 1 -methylcyclohexylcarbamate obtained in

Example 1-3 (16. Og) in methanol (160mL), was added nicotinaldehyde (6.18mL). The mixture was stirred at room temperature for 16hrs.

The solution was concentrated in vacuo. The residue was triturated with n-hexane, then filtered to provide the target compound (17. lg) as a white solid.

^-N (300MHz, CDC1₃ ) : δ 1.35(3H, s), 1.45(9H, s), 1.53-1.95(8H,m), 4.33(lH,br-s), 4.40 (1H, br-s), 7.31(1H, dd, J= 4.9, 8.0Hz), 7.96(1H, dt , J= 1.8, 8.0Hz), 8.11(1H, s), 8.59(1H, dd, J=1.8, 4.9Hz), 8.74(1H, d, J=1.8Hz).

Example 1-5

Nicotinaldehyde 0- ( 4 -amino - 4 -methylcyclohexyl ) oxime bis(trifluoroacetate)

Trifluoroacet ic acid (lOOmL) was added to a solution of 1 -methyl - 4 -({[( IE )- 3 -pyridinylmethylidene ] amino } - oxy ) cyclohexylcarbamate obtained in Example 1-4 (11. Og) in tetrahydrofuran (50mL) with cooling on an ice bath.

The reaction mixture was stirred at room temperature for

Ihr. The mixture was concentrated in vacuo. The residue was used in the next step without purification (28.4g).

^XH-NMR (300MHz, DMSO) : δ 1.30(3H, s), 1.56 - 1.83 ( 6H , m), 1.90-2.09(2H,m), 4.20(lH,m), 7.52(lH,dd, J=4.8, 7.9Hz), 7.90(2H, br-s), 8.09( 1H, d, J=7.9Hz), 8.33(1H, s), 8.65(1H, br - s ) , 8 . 8 2 ( 1 H , b r - s ) .

Examp l e 1 - 6

( 2S, 4S) -4 -Fluoro- 1- ( { [ l-methyl-4- ( { [ ( IE) - 3 -pyridinylm ethylidene ] amino >oxy ) cyclohexyl ] amino }acetyl) -2-pyrro lidinecarbonitrile

To a stirred solution of nicotinaldehyde 0- ( 4 -amino- 4 -methylcyclohexyl ) oxime bis ( trifluoroacetate ) obtained in Example 1-5 (28.4g) in dimethylformamide (130mL), were added K₂C0₃ (24.2g), (2S, 4S) -l-chloroacetyl-4-fluoro-2-pyrrolidinecarbonit rile obtained in Preparation 1-8 (5.73g), and sodium iodide ( 5 Omg ) . The mixture was stirred for 2hrs at 50°C and quenched by adding water (200mL). The aqueous layer was neutralized with IN HCl and extracted with ethyl acetate. The combined organic layers were extracted with 0.5N HCl . The aqueous layer was separated and basified with NaHC0₃ , and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, dried and concentrated in vacuo. The residue was purified by silica gel column chromatography ( chloroform/methanol= 5 : 1 ) . The residue was crystallized from ethanol to provide the target compound (7.2g) as a white solid.

^^•H-NMR (300MHz, CDC1₃) : <5 1.12(3X4/5H, s), 1.15(3Xl/5H, s), 1.38-1.53(2H, m), 1.57 - 1.79 ( 4H , m), 1.97(2H, br-s), 2.20-2.58(1H, m), 2.70(lX4/5H, t, J=15.7Hz), 2.77(lX 1/5H, t, J= 15.7Hz), 3.30-4.10(4H, m), 4.23 - 4.35 ( 1H , m), 4.97(1X4/5H, d, J=8.9 Hz), 5.11(lXl/5H, d, J=8.9Hz), 5.36(1 X 1/5H, br-d, J=51.2Hz), 5.45(1 X 4/5H, br-d, J= 51.2Hz), 7.30(1H, dd, J= 4.9, 7.8Hz), 7.97 ( 1H, dt, J= 2.0, 7.8Hz), 8.10(1H, s), 8.59(1H, dd, J=2.0, 4.9Hz), 8.73(1H, d, J=2.0Hz) .

MS (ES+) : m/e 388.18.

Example 2-1 2-(2-Pyridinylmethoxy)-lH-isoindole-l,3(2H)-dione

To a solution of 2 -pyridinylmethanol (50g),

2-hydroxy-lH-isoindole-l , 3 ( 2H) -dione (75g) and triphenylphosphine (144g) in tetrahydrofuran (500mL) at room temperature, was added diisopropyl. azodicarboxylate

(lOOmL) and stirred for 15min at the same temperature. The reaction precipitate was filtered off and washed with tetrahydrof ran to give the target compound (60g) as a white powder.

^XH-NMR (in CDC1₃) : δ 5.34(s, 2H), 7.30-7.26(m, 1H),

7.83-7.72(m, 6H), 8.56-8.55(m, 1H).

Example 2-2 0- ( 2 -Pyridinylmethyl ) hydroxylamine

2 - ( 2-Pyridinylmethoxy ) -lH-isoindole-l,3(2H)- dione obtained in Example 2-1 (60g) was dissolved in ethanol (lOOOmL). This solution was warmed to 80°C and NH₂NH₂ ^• H₂0 was added at the same temperature. The resulting mixture was heated at reflux for 30min and then was cooled to room temperature.

The resulting precipitate was filtered off, washed with ethyl acetate, and the filtrate was concentrated in vacuo. The resulting mixture was diluted with water and acidified with concentrated HCl. The aqueous layer was washed with ethyl acetate, basified with NaOH, saturated with NaCI, and was extracted three times with chloroform. The combined organic layer was washed with brine, dried over MgS0₄ , and filtered. After removal of the solvent in vacuo, the target compound was given as a yellow oil (20g) .

¹H-NMR (in CDC1₃) : δ 4.84(s, 2H), 5.63(br-s, 2H), 7.26-7.16(m, 1H), 7.43-7.34(m, 1H), 7.77-7.65(m, 1H), 8.66-8.52 (m, 1H) .

Example 2-3 tert-Butyl 1 -methyl - 4 - [ ( 2 -pyridinylmethoxy )imino]cycl ohexylcarbamate

The title compound (1.24g) was prepared from tert-butyl 1 -methyl- 4 -oxocyclohexylcarbamate obtained in Preparation 2-5 and O- ( 2 -pyridinylmethyl ) hydroxylamine obtained in Example 2-2 in a similar manner to that of Example 3-3 described later .

¹H-NMR (300MHz, CDC1₃ ) : δ 1.36(3H, s), 1.44(9H, s), 1.46-1.65(2H, m), 2.06 - 2.32 ( 5H , m), 2.98(1H, dt , J= 4.8,

15.0Hz), 4.40(1H, br-s), 5.19(2H, s), 7.18(1H, br-dd,

J=4.8, 7.6Hz), 7.35 1H, d, J=7.6Hz), 7.68(1H, dt , J=1.8,

7.6Hz), 8.57(1H, br-d, J=4.8Hz).

MS (ES+) : m/e 334.24.

Example 2-4

4 -Amino- 4 -methylcyclohexanone O- ( 2 -pyridinylmethyl ) ox ime

The title compound (918mg) was prepared from tert-butyl 1 -methyl- 4 - [ ( 2 -pyridinylmethoxy ) imino ] - cyclohexylcarbamate obtained in Example 2-3 in a similar manner to that of Example 3-4 described later.

^XH-NMR (300MHz, CDC1₃) : δ 1.20(3H, s), 1.47(2H, br-s), 1.54-1.70 ( 4H, m), 2.24(1H, dt , J = 6.0, 15.2Hz), 2.36-2.46 ( 1H, m), 2.54 - 2.64 ( 1H , m), 2.74(1H, dt , J = 6.0, 15.4Hz), 5.20(2H, s), 7.18(1H, br-dt, J = 4.8, 7.6Hz ) , 7.39(1H, d, J=7.6Hz), 7.68(1H, dt, J=1.8, 7.6Hz), 8.57(1H, ddd, J= 0.9 , 1.8, 4.8Hz) MS (ES+) : m/e 234.20.

Example 2-5

4-Fluoro-l-[({l -methyl- 4 - [ ( 2 -pyridinylmethoxy ) imino ] c yclohexyl } amino )acetyl]-2-pyrrolidinecarbonitrile dihydrochloride

The title compound (69.49g) was prepared from ( 2S, 4S) -l-chloroacetyl-4-fluoro-2-pyrrolidinecarbonit rile obtained in Preparation 1-8 and 4 -amino- 4 -methylcyclohexanone O- ( 2 -pyridinylmethyl ) - oxime obtained in Example 2-4 in a similar manner to that of Example 3-5 described later.

^-NMR (300MHz, CDC1₃, free-form) : δ 1.14(3X4/5H, s), 1.17(3Xl/5H, s), 1.45-2.03( 5H, m), 2.17 - 2.88 ( 5H , m), 3.30-4.20(4H, m), 4.97 (1H, d, J = 9.5Hz), 5.19(2H, s), 5.37(1 X 1/5H, br-d, J=51.5Hz), 5.45(1 X 4/5H, br-d, J=51.5Hz), 7.19(1H, br-dd, J=5.0, 7.7Hz ) , 7.35(1H, d, J = 7.7Hz), 7.69(1H, dt , J= 1.8, 7.7Hz ) , 8.57(1H, br-d, J = 5.0Hz ) . MS (ES+) : m/e 388.12 (free-form).

Example 3-1 2- ( 3 -Pyridinylmethoxy) -lH-isoindole-l,3(2H)-dione

The title compound (5.77g) was prepared from 3 -pyridinylmethanol in a similar manner to that of Example 2-1. Examp l e 3 - 2

3- [ (Aminooxy) methyl] yridine

The title compound (1.50g) was prepared from 2- ( 3 -pyridinylmethox ) -lH-isoindole-l,3(2H) -dione obtained in Example 3-1 in a similar manner to that of Example 2 - 2.

^XH-NMR (300MHz, CDC1₃ ) : δ 4.71(2H, s), 5.47(2H, br-s), 7.30(1H, dd, J=4.9, 7.7Hz), 7.70(1H, dt , J=1.7, 7.7Hz ) , 8.57(1H, dd, J=1.7, 4.9Hz), 8.62(1H, d, J=1.7Hz) MS (ES+) : m/e 124.93.

Example 3-3 tert-Butyl 1 -methyl- 4 -[( 3 -pyridinylmethoxy ) imino ] cycl ohexylcarbamate

To a stirred solution of tert-butyl

1 -methyl- 4 -oxocyclohexylcarbamate obtained in Preparation 2-5 (l.OOg) in methanol (0.5mL), was added

3 -[( aminooxy )methyl ] pyridine obtained in Example 3-2

(552mg). The mixture was stirred for 5hrs .

The solution was concentrated in vacuo. The residue was triturated with diisopropylether, then filtrated to provide the target compound (892mg) as a white solid.

^XH-NMR (300MHz, CDC1₃) : <5 1.35(3H, s), 1.44(9H, s), 1.45-1.60(2H, m), 2.05 - 2.29 ( 5H , m), 2.81 - 2.93 ( 1H , m), 4.40(1H, br-s), 5.06(2H, s), 7.27(1H, dd, J=4.8, 7.9Hz), 7.67(1H, d, J=1.7, 7.9Hz), 8.54(1H, dd, J=1.7, 5.0Hz), 8.60(1H, br-d, J=1.7Hz). MS (ES+) : m/e 334.20.

Example 3-4 4 -Amino- 4 -methylcyclohexanone 0- ( 3 -pyridinylmethyl ) ox ιme

To a cold stirred solution of tert-butyl 1 -methyl- 4- [ ( 3 -pyridinylmethoxy ) imino ] cyclohexylcarba mate obtained in Example 3-3 (250mg) in methanol (0.5mL), was added 4N HCl solution in dioxane (1.5mL).

The mixture was stirred for 2hrs , and concentrated in vacuo. The residue was acidified with IN HCl, rinsed with ethyl acetate. The aqueous layer was alkalized with saturated aqueous NaHC0₃ solution, then extracted with chloroform. The combined organic layer was dried and filtrated. The filtrate was concentrated in vacuo to provide the target compound (117mg) as an oil.

^XH-NMR (300 MHz, CDC1₃) : δ 1.19(3H, s), 1.50 - 1.63 ( 3H , m) , 1.77-1.83 ( 1H, m), 2.16 - 2.71 ( 4H , m), 5.07(2H, s), 7.27(1H, ddd, J=0.6, 4.8, 7.7Hz), 7.67(1H, dddd, J=0.6, 1.7, 2.0, 7.7Hz), 8.54(1H, dd, J=1.7, 4.8Hz ) , 8.61(1H, dd, J= 0.6 , 2.0Hz) . MS (ES+) : m/e 234.13.

Example 3-5

(2S,4S)-4-Fluoro-l-[ ( {l-methyl-4- [ ( 3 -pyridinylmethoxy ) imino ] cyclohexyl } amino )acetyl] -2 -pyrrolidinecarbonit rile dihydrochloride

To a stirred solution of 4 -amino- 4 -methylcyclohexanone 0- ( 3 -pyridinylmethyl ) - oxime obtained in Example 3-4 (llOmg) in tetrahydrofuran ( 2mL ) , were added triethylamine (0.066mL), (2S, 4S) -1-chloroacetyl- 4 -fluoro- 2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 (75.0mg), and sodium iodide ( 7mg ) .

The mixture was stirred for 6hrs and diluted with water. The aqueous layer was extracted with ethyl acetate . The combined organic layers were washed with saturated aqueous NaCI solution , dried and filtered . After removal of the solvent in vacuo, the residue was purified by preparative thin layer chromatography ( chloroform/methanol=5 : 1 ) . The provided oil was dissolved in ethyl acetate and 4N HCl-dioxane was added, then the precipitated was collected by filtration under N₂ to provide the target compound ( 66.5mg) as a white solid .

^XH-NMR (300MHz, DMSO) : δ 1.42(3H, s), 1.73 - 2.00 ( 3H , m), 2.03-2.57(6H, m), 3.10 (1H, br-d, J=11.5Hz), 3.70-4.30(4H, m) , 5.08(1H, d, J=7.9Hz), 5.19(2H, s), 5.57(1H, br-d, J=52.6Hz), 7.87(1H, br-s), 8.28(1H, br-s), 8.77(1H, s), 8.79(1H, s), 9.05(1H, br-s), 9.17(1H, br-s). MS (ES+) : m/e 388.16.

Example 4-1

2- [ ( 6 -Methy1-2 -pyridinyl) methoxy] -lH-isoindole-1, 3(2H ) -dione

The title compound (4.52g) was prepared from ( 6 -methyl- 2 -pyridinyl ) methanol in a similar manner to that of Example 2-1.

Example 4-2

2 - [ (Aminooxy) methyl ] - 6 -methylpyridine

The title compound (l.lδg) was prepared from 2- [ ( 6 -methyl -2 -pyridinyl) methoxy ] -lH-isoindole-1, 3(2H ) -dione obtained in Example 4-1 in a similar manner to that of Example 2-2.

^XH-NMR (300MHz, CDC1₃) : δ 2.57(3H, s), 4.80(2H, s), 5.61(2H, br-s), 7.08 ( 1H, d, J= 7.7Hz), 7.18(1H, d, J=7.7Hz) , 7.59(1H, t , J=7.7Hz) . MS (ES+) : m/e 139.01.

Example 4-3 tert-Butyl 1 -methyl - 4 - { [ ( 6 -methyl- 2- pyridinyl ) methox y ] imino}cyclohexylcarbamate

The title compound (260mg) was prepared from tert-butyl 1 -methyl- 4 -oxocyclohexylcarbamate obtained in Preparation 2-5 and 2 -[( aminooxy )methyl ]- 6 -methylpyridine obtained in

Example 4-2 in a similar manner to that of Example 3-3.

^XH-NMR (300MHz, CDC1₃ ) : δ 1.36(3H, s), 1.44(9H, s), 1.5-1.6(2H, m), 2.1-2.3(5H, m), 2.55(3H, s), 2.97(1H, dt, J=4.7, 15.2Hz), 4.41(1H, br-s), 5.15(2H, s), 7.04(1H, d, J= 7.8Hz ) , 7.14 ( 1H , d, J= 7.8Hz), 7.56 ( 1H , t, J= 7.8Hz ) MS (ES+) : m/e 384.24.

Example 4-4 4 -Amino - 4 -methylcyclohexanone O- [( 6 -methyl- 2 -pyridiny 1 ) methyl ] oxime

The title compound (143mg) was prepared from tert-butyl 1 -methyl - 4 - { [ (6 -methyl- 2-pyridinyl) - methoxy ] imino }cyclohexylcarbamate obtained in Example 4-3 in a similar manner to that of Example 3-4.

^-NM (300 MHz, CDC1₃) : δ 1.22(3H, s), 1.55 - 1.70 ( 3H , m), 2.24(1H, dt, J= 5.8, 14.5Hz), 2.41(1H, ddd, J= 6.2, 8.1, 14.7Hz), 2.55(3H, s), 2.59 ( 1H, dd, J=6.6, 14.7Hz), 2.61(1H, dd, J= 6.2, 11.5Hz), 2.74(1H, dt , J= 6.2, 15.1Hz), 5.16(2H, s), 7.04(1H, d, J=7.7Hz), 7.15(1H, d, J=7.7Hz), 7.57(1H, t , J=7.7Hz) . MS (ES+) : m/e 248.18. Examp l e 4 - 5

(2S, 4S) -4-Fluoro-l-{ [(l-methyl-4-{[ ( 6 -methyl- 2 -pyridi nyl ) methoxy ] imino } cyclohexyl ) amino ]acetyl}-2-pyrrolid inecarbonitrile dihydrochloride

The title compound (95.2mg) was prepared from (2S,4S)-l-chloroacetyl-4-fluoro-2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and 4 -amino- 4 -methylcyclohexanone O- [ ( 6 -methyl- 2 - pyridinyl ) methyl ] oxime obtained in Example 4-4 in a similar manner to that of Example 3-5.

^XH-NMR (300MHz, DMSO) : δ 1.43(3H, s), 1.80 - 2.10 ( 3H , m),

2.17-2.55(6H, m), 2.74(3H, s), 3.15 ( 1H, br-d, J= 14.7Hz), 3.70-4.27(4H, m), 5.08(1H, br-d, J= 8.1Hz), 5.36(2H, s),

5.57(1H, br-d, J=52.0Hz), 7.68(1H, d, J=8.0Hz), 7.75(1H, d, J= 8.0Hz), 8.35 ( 1H, t, J=8.0Hz), 9.12(1H, br-s), 9.36 ( 1H, br-s ) .

MS (ES+) : m/e 402.18.

Example 5-1

2- [ ( 2 -Methyl- 3 -pyridinyl) methoxy] -lH-isoindole-l,3(2H

) -dione

The title compound (1.8g) was prepared from ( 2 -methyl- 3 -pyridinyl ) methanol in a similar manner to that of Example 2-1.

Example 5-2 3- [ (Aminooxy )methyl ] - 2 -methylpyridine

The title compound (676mg) was prepared from 2- [ ( 2-met yl-3-pyridinyl)methoxy ] -lH-isoindole-l,3(2H ) -dione obtained in Example 5-1 in a similar manner to that of Example 2-2. ^XH-NMR (300MHz, CDC1₃) : δ 2.58(3H, s), 4.72(2H, s), 5.48(2H, br-s), 7.13(1H, dd , J= 4.9, 7.7Hz), 7.61(1H, dd, J=1.8, 7.7Hz), 8.45(1H, d , J=1.8, 4.9Hz). MS (ES+) : m/e 123.96.

Example 5-3 tert-Butyl 1 -methyl - 4 - { [ ( 2 -methyl -3 -pyridinyl )methox y ] imino } cyclohexylcarbamate

The title compound (314mg) was prepared from tert-butyl 1 -methyl- 4 -oxocyclohexylcarbamate obtained in Preparation 2-5 and

3 - [ ( aminooxy ) methyl ] - 2 -methylpyri ine by Example 5-2 in a similar manner to that of Example 3-3.

^XH-NMR (300MHz, CDC1₃) : <5 1.35(3H, s), 1.44(9H, s), 1.45-1.62 ( 2H, m), 2.06 - 2.30 ( 5H , m), 2.56(3H, s), 2.82-94(lH, m), 4.34(1H, br-s), 5.06(2H, s), 7.12(1H, dd, J= 4.8, 7.7Hz), 7.59(1H, d , J= 1.8, 7.7Hz), 8.43(1H, dd , J= l .8, 4.9Hz) . MS (ES+) : m/e 348.24.

Example 5-4 4 -Amino- 4 -methylcyclohexanone O- [( 2 -methyl- 3 -pyridiny 1 ) methyl ] oxime

The title compound (155.1mg) was prepared from tert-butyl 1 -methyl - 4 - { [ ( 2 -methyl- 3 -pyridinyl ) - methoxy ] imino }cyclohexylcarbamate obtained in Example 5-3 in a similar manner to that of Example 3-4.

^Ε-NMR ( 300MHz , CDC1₃) : δ 1.21 ( 3H, s), 1.54-1.65(4H, m), 1.80(2H, br-s), 2.23(1H, dt, J= 5.9, 14.7Hz), 2.34-2.71(3H, m), 2.56(3H, s), 5.07(2H, s), 7.12(1H, dd, J=5.0, 7.5Hz), 7.59(1H, dd, J=1.5, 7.5Hz ) , 8.43(1H, dd , J=1.5, 5.0Hz) . MS (ES+) : m/e 248.19.

Example 5-5 ( 2S , 4S) -4-Fluoro-l-{ [ ( l-methyl-4-{ [ ( 2 -methyl- 3 -pyridi nyl ) methoxy] imino }cyclohexyl ) amino ]acetyl}-2-pyrrolid inecarbonitrile dihydrochloride

The title compound (179mg) was prepared from (2S,4S)-l-chloroacetyl-4-fluoro-2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and 4 -amino - 4 -methylcyclohexanone O- [ ( 2 -methyl - 3 - pyridinyl ) methyl ] oxime obtained in Example 5-4 in a similar manner to that of Example 3-5.

^XH-NMR (300MHz, DMSO) : δ 1.42(3H, s), 1.70 - 1.99 ( 3H , m), 2.05-2.50 ( 6H, m), 2.71(3H, s), 3.10(1H, br-d, J=16.1Hz), 3.65-4.30( 4H, m), 5.08(1H, d, J=8.1Hz ) , 5.20(2H, s), 5.61(1H, br-d, J= 52.6Hz), 7.80(1H, br-s), 8.23 - 8.32 ( 1H , ), 8.67(1H, br-d, J=5.1Hz), 9.07(1H, br-s), 9.18(1H, br- s ) . MS (ES+) : m/e 402.17.

Example 6-1 2- [ ( 6 -Chloro -3 -pyridinyl )methoxy]-lH-isoindole-l,3(2H ) -dione

The title compound (1.65g) was prepared from ( 6 -chloro- 3 -pyridinyl ) methanol in a similar manner to that of Example 2-1.

Example 6-2

5- [ (Aminooxy ) methyl ] - 2 -chloropyridine

The title compound (40mg) was prepared from 2 - [ ( 6 -chloro -3 -pyridinyl )methoxy]-lH-isoindole-l,3(2H ) -dione obtained in Example 6-1 in a similar manner to that of Example 2-2.

^XH-NMR (300MHz, DMSO) : <5 5.11(2H, s), 7.61(1H, d, J=8.0Hz ) , 7.95(1H, dd, J=2.4 , 8.0Hz), 8.40 ( 1H, d, J=2.4Hz), 10.9(2H, br-s) . MS (ES- ) : m/e 158.92.

Example 6-3 tert-Butyl 4-{ [ ( 6-chloro-3 -pyridinyl ) methoxy ] imino}-l -methylcyclohexylcarbamate

The title compound (252mg) was prepared from tert-butyl 1 -methyl- 4 -oxocyclohexylcarbamate obtained in Preparation 2-5 and

5- [ ( aminooxy )methyl ] - 2 -chloropyridine by Example 6-2 in a similar manner to that of Example 3-3.

^Η-NMR (300MHz, CDC1₃) : <5 1.35(3H, s), 1.44(9H, s), 1.40-1.65 (2H, m), 2.08 - 2.50 ( 5H , m), 2.80 - 2.90 ( 1H , m), 4.38(1H, br-s), 5.02(2H, s), 7.32 ( 1H, d, J= 8.1Hz), 7.64(1H, dd, J=2.4, 8.1Hz), 8.36(1H, d, J=2.4Hz). MS (ES+) : m/e 368.12.

Example 6-4

4 -Amino- 4 -methylcyclohexanone 0-[(6-chloro-3 -pyridiny

1 ) methyl ] oxime

The title compound (174mg) was prepared from tert-butyl 4-{ [ ( 6 -chloro- 3 -pyridinyl )methoxy] imino} - 1 -methylcyclohexylcarbamate obtained in Example 6-3 in a similar manner to that of Example 3-4.

^-NMR (300MHz, CDC1₃) : δ 1.18(3H, s), 1.49 - 1.62 ( 4H , m), 2.20(1H, dt, J= 5.9, 14.8Hz), 2.33 - 2.54 ( 2H , m), 2.65(1H, dt, J=5.9, 14.8Hz), 5.03(2H, s), 7.31(1H, d, J=8.1Hz), 7.64(1H, dd, J=2.4, 8.1Hz), 8.36(1H, d, J=2.4Hz). MS (ES+) : m/e 268.15.

Example 6-5

(2S,4S)-l-{[(4-{[( 6 -Chloro -3 -pyridinyl) methoxy] imino}

- 1 -methylcyclohexyl ) amino ] acetyl}-4-fluoro-2-pyrrolid inecarbonitrile

The title compound (151mg) was prepared from (2S,4S)-l-chloroacetyl-4-fluoro-2-pyrrolidinecarbonit rile obtained in Preparation 1-8 and 4 -amino- 4 -methylcyclohexanone 0- [ (6-chloro-3- pyridinyl ) methyl ] oxime obtained in Example 6-4 in a similar manner to that of Example 3-5.

¹H-NMR (300MHz, CDC1₃) : δ 1.11(3X3/4H, s), 1.14(3Xl/4H, s), 1.40-1.69(4H,m), 2.10-2.47(4H,m), 2.62-2.80(2H,m), 3.28-4.00(4H, m), 4.96(1H, d, J=9.5Hz), 5.03(2H, s),

5.36(1 X 1/4H, br-d, J=51.3Hz), 5.44(1 X 3/4H, br-d,

J=51.3Hz), 7.31(1H, d, J= 8.2Hz), 7.64(1H, dd , J= 2.2.

8.2Hz), 8.36(1H, d, J=2.2Hz).

MS (ES+) : m/e 422.11.

Example 7-1

2 -Pyridinecarbaldehyde O- ( 4 -amino- 4 -methyl - cyclohexyl ) oxime bis(trifluoroacetate)

To a solution of tert-butyl trans - 1 -methyl- 4-({[(lE)-2 -pyridinylmethylene ] amino }o xy ) cyclohexyl ] carbamate (436mg) in tetrahydrofuran (1.5mL), was added trifluoroacetic acid ( 3mL ) in an ice-water bath. The reaction mixture was stirred at room temperature for 1.5hrs . The mixture was concentrated in vacuo. The residue was used in the next step without purification (1.09g).

Example 7-2 (2S,4S)-4-Fluoro-l-({[ 1 -methyl- 4- ( { [ ( IE ) - 2 -pyridinylm ethylidene ] amino }ox ) cyclohexyl ] amino Jacetyl) -2-pyrro lidinecarbonitrile

To a stirred solution of 2 -pyridinecarbaldehyde 0-(trans-4 -amino - 4 -methylcyclohexyl ) oxime bis ( trifluoroacetate ) in dimethylformamide (2mL), were added K₂C0₃ (935mg), ( 2S , 4S ) - 1 -chloroacetyl- 4 - fluoro- 2 -pyrrolidinecarbonitrile obtained in Preparation 1-8 (237mg) and sodium iodide (9mg). The mixture was stirred for 4hrs at 50°C and diluted with water. The aqueous layer was extracted with ethyl acetate. The organic layer was washed with saturated aqueous NaCI, dried and concentrated in vacuo. The residue was crystallized from ethanol to provide the target compound as a white solid (150mg).

^XH-NMR (300MHz, CDC1₃) : <5 1.12(3X4/5H, s), 1.15(3Xl/5H, s), 1.39-1.52(2H,m), 1.55-1.80(4H,m), 1.90-2.05(2H,m), 2.20-2.56(lH, m), 2.67(lX4/5H, t, J=15.0Hz), 2.77(lX 1/5H, t, J= 14.9Hz), 3.30 - 4.10 ( 4H , m ) , 4.32(1H, br-s), 4.71(1X4/5H, d, J=9.2Hz), 5.11(lXl/5H, d, J=8.8Hz), 5.36(lXl/5H, br-d, J= 52.2Hz), 5.43(1X4/ 5H, br-t, J= 3.5, 50.9Hz), 7.25(lH,m), 7.69(1H, dt, J=1.8, 7.9Hz), 7.80(1H, dt, J=0.9, 7.9Hz), 8.18(1H, s), 8.61(1H, ddd, J=0.9, 1.8, 5.0Hz) .

MS (ES+) : m/e 388.14.

Example 8-1

1-tert-Butyl 2-methyl ( 2S ) - 4 -oxo- 1 , 2 - pyrrolidinedicarboxylate To a solution of oxalyl chloride in dichloromethane

(50mL) at -70°C, was added dimethylsulfoxide dropwise.

After 15min, a solution of 1-tert-butyl 2-methyl ( 2S , 4R ) -4 -hydroxy- 1 , 2 -pyrrolidinedicarboxylate in

CH₂C1₂ (20mL) was added at the same temperature.

The reaction mixture was warmed to 0°C , and then cooled to -70°C . Triethylamine (23mL) was added to the reaction mixture, and warmed to room temperature. The reaction mixture was quenched by adding water. The aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with IN HCl and brine, dried over MgS0₄, and filtered. After removal of the solvent in vacuo, the residue was purified with silica gel chromatography (n-hexane/ethyl acetate = 4/1 to 2/1) to give the target compound as a yellow oil (2.61g).

^XH-NMR (inCDCl₃) : δ 1.55-1.38(9H, m), 2.66-2.51(lH, m), 3.05-2.84 ( 1H, m), 3.77(3H, s), 3.97 - 3.84 ( 2H , m), 4.88-4.65( 1H, m) .

Example 8-2

1-tert-Butyl 2-methyl ( 2S ) - 4 , 4 -difluoro- 1 , 2 - pyrrolidinedicarboxylate

To a solution of 1-tert-butyl 2-methyl ( 2S )- 4 -oxo- 1 , 2 -pyrrolidinedicarboxylate obtained in Example 8-1 (3.6g) in dichloromethane, was added diethylaminosulfur trifluoride ( 4mL ) dropwise with cooling on an ice bath. The reaction mixture was warmed to room temperature and stirred for llhrs.

To the resulting mixture was added NaHC0₃ , and the reaction mixture was quenched by adding saturated aqueous NaHC0₃ , and then water (lOOOmL) and CaCl₂ (11.5g) in water (50mL) were added. The resulting suspension was extracted with ethyl acetate, the combined organic layer was washed with saturated aqueous NaCI, dried over MgS0₄, and concentrated in vacuo. The residue was purified with silica gel chromatography ( n-hexane/ethyl acetate = 5/1) to give the target compound as a yellow oil (3.6g).

^XH-NMR (inCDCl₃) : δ 1.53-1.36(9H, m), 2.55-2.35(lH, m), 2.83-2.58( 1H, m), 3.94 - 3.71 ( 5H , m), 4.62 - 4.37 ( 1H , m). MS (ESI+) : m/z 266.18 (M+H).

Example 8-3

(2S)-l-(tert -Butoxycarbonyl ) -4 , 4-difluoro-2-pyrrolidi necarboxylic acid

The crude product of 1-tert-butyl 2-methyl ( 2S ) -4 , 4-difluoro-l , 2 -pyrrolidinedicarboxylate obtained in Example 8-2 (3.9g) was dissolved in methanol (llmL) and then IN NaOH (22mL) was added at room temperature. After stirring for 1.5hrs , the resulting mixture was washed with diethyl ether, acidified with IN HCl (30mL) , and then was extracted with ethyl acetate. The combined organic layer was washed with saturated aqueous NaCI, dried over MgS0₄ , and concentrated in vacuo. The residue was triturated with hexane to give the target compound as a white powder (3.1g).

^XH-NMR (in CDC1₃) : δ 1.62-1.30(9H, m), 2.92-2.39(2H, m), 4.02-3.60(2H, m), 4.69 - 4.41 ( 1H , m), 7.50(1H. br-s). MS (ESI-) : m/z 250.19 (M-H).

Example 8-4 tert-Butyl (2S)-2 -aminocarbonyl- 4 , 4-difluoro-l- pyrrolidinecarboxylate To a solution of ( 2S ) - 1 - ( tert -butoxycarbonyl ) - 4 , 4 - difluoro-2 -pyrrolidinecarboxylic acid obtained in Example 8-3 in acetonitrile (30mL), were added 1 -hydroxybenzotriazole ydrate (1.98g) and water soluble carbodiimide (3.11g) with cooling on an ice bath. After stirred for 20min, 28% aqueous NH₃ ( 2mL ) was added at the same temperature and the resulting mixture was stirred for Ihr.

The reaction mixture was filtered and the filtration was evaporated in vacuo. After dilution with ethyl acetate, the resulting mixture was washed with water and saturated aqueous NaCI, dried over MgS0 , and filtered. After removal of the solvent, the target compound was obtained as a yellow oil (3.2g).

^XH-NMR (in CDC1₃) : δ 1.48(9H, s), 3.10 - 2.44 ( 2H , m), 4.02-3.54 ( 2H, m), 4.65 - 4.41 ( 1H , m), 5.71 - 5.40 ( 1H , m), 6.97-6.64 ( 1H, m) . MS (ESI+) : m/z 251.22 (M+H).

Example 8-5

( 2S ) -4 , 4-Difluoro-2 -pyrrolidinecarboxamide hydrochloride

tert-Butyl ( 2S ) - 2 -aminocarbonyl- 4 , 4 -difluoro- 1 - pyrrolidinecarboxylate obtained in Example 8-4 (3.2g) was dissolved in 4N HCl in dioxane (13mL) and the resulting mixture was stirred for lOmin at room temperature.

After removal of the solvent, the resulting residue was triturated with ethyl acetate to give the target compound as a white powder (2.1g).

MS (ESI+) m/z 150.95 (M+H)

Example 8-6 (2S) -l-Chloroacetyl-4, 4 -difluoro- 2 -pyrrolidinecarboxa mide

To a mixture of ( 2S ) - 4 , 4 -difluoro- 2 - pyrrolidinecarboxamide hydrochloride obtained in Example 8-5 and sodium 2 -ethylhexanoate (4.35g) in tetrahydrof ran (30mL), was added chloroacetyl chloride with cooling on an ice bath. The reaction mixture was stirred for 30min at the same temperature. The resulting mixture was then poured onto buchner funnel/filter paper and washed with ethyl acetate. The solvent was removed in vacuo and the residue was purified with silica gel chromatography ( chloroform/methanol = 10/1 to 5/1) to give the target compound as a colorless oil (2.27 g) .

^XH-NMR (inCDCl₃) : δ 2.70 - 2.48 ( 1H , m), 3.13 - 2.89 ( 1H , m), 4.16-3.86 ( 4H, m), 4.86 - 4.75 ( 1H , m), 5.69(1H, br-s), 6.64( 1H, br-s) . MS (ESI-) : m/z 225.15 (M-H).

Example 8-7

(2S) -l-Chloroacetyl-4, 4-difluoro-2-pyrrolidinecarboni trile

To a solution of ( 2S )- 1 -chloroacetyl- 4 , 4 - difluoro- 2 -pyrrolidinecarboxamide obtained in Example 8-6 (14.5g) in tetrahydrofuran (25mL), was added tetrafluoroacetic anhydride ( 2.3mL ) at room temperature . After stirring for 30min, the resulting mixture was concentrated in vacuo. The residue was purified with silica gel chromatography ( n-hexane/ethyl acetate = 4/1 to 1/1) to give the target compound as a colorless solid (1.9g) . ^XH-NMR (in CDCl₃) : δ 2.95-2.67(2H, m) , 4.21-3.88(4H, m) , 5.06-4.87 ( 1H, m) .

Example 8-8 (2S)-4,4-Difluoro-l-({[l-methyl-4-({[ (IE) -3 -pyridinyl methylidene ] amino }oxy ) cyclohexyl ] amino Jacetyl) -2-pyrr olidinecarbonitrile

The title compound (83mg) was prepared from nicotinaldehyde 0- ( 4 -amino - 4 -methylcyclohexyl ) oxime bis ( trifluoroacetate ) obtained in Example 1-5 and ( 2S) -1- chloroacetyl -4 , 4-difluoro-2-pyrrolidinecarboni trile obtained in Example 8-7 in a similar manner to that of Example 7 - 2.

^XH-NMR (in DMSO) : <5 1.38(3H, s), 2.20 - 1.43 ( 8H , m), 3.06-2.77(2H, m), 4.48 - 3.90 ( 5H , m), 5.30 - 5.14 ( 1H , m), 7.99-7.69 ( 1H, m), 8.58 - 8.32 ( 2H , m), 8.90 - 8.68 ( 1H , ), 9.39-8.90 (3H, m) . MS (ESI+) : m/z 406.21 (M+H).

Example 9-1

6 -Trifluoromethylnicotinaldehyde 0- ( 4 -amino- 4 -methylcyclohexyl ) oxime

The title compound (65mg) was prepared from tert-butyl l-methyl-4-({[(lE) - 6 - trifluoromethyl- 3 - pyridinyl -methylidene ] amino} oxy ) cyclohexylcarbamate in a similar manner to that of Example 17-2 described later.

MS (ESI+) m/z 302.14 (M+H)

Example 9-2

(2S) -4 , 4 - difluoro- 1- [({l-methyl-4-[({(lE)-[6- (trifluo romethyl ) - 3 -pyridinyl ] methylidene} amino ) oxy] cyclohexy l}amino)acetyl] - 2 -pyrrolidinecarbonitrile dihydrochloride

The title compound (5.5mg) was prepared from (2S)-l-chloroacetyl-4,4-difluoro-2-pyrrolidinecarboni trile obtained in Example 8-7 and 6 - trif luoromethylnicot inaldehyde 0- ( 4 - amino - 4 -methyl - cyclohexyl ) oxime obtained in Example 9-1 in a similar manner to that of Example 7-2.

^XH-NMR (in DMSO) : ό^* 1.36(3H, s), 1.69 - 1.43 ( 2H , m),

1.99-1.69 ( 4H, m), 2.20 - 1.99 ( 2H , m), 3.05 - 2.78 ( 2H , m),

4.64-3.82 (5H, m), 5.31 - 5.12 ( 1H , m), 8.06 - 7.90 ( 1H , m),

8.37-8.19 ( 1H, m), 8.50 - 8.37 ( 1H , m). 9.22 - 8.90 ( 3H , m). MS (ESI+) : m/z 474.11 (M+H).

Example 10

(2S)-4,4-Difluoro-l-({[l-methyl-4-({[(lE)-l-( 3-pyridi nyl ) ethylidene ] amino } oxy ) cyclohexyl ] amino Jacetyl) -2-p yrrolidinecarbonitrile dihydrochloride

The title compound (42.1mg) was prepared from

1 -methyl -4 -({ [ (lE)-l-(3-pyridinyl) ethylidene ] amino }ox y ) cyclohexylamino and ( 2S )- 1 -chloroacetyl- 4 , 4 - dif luoro - 2 -pyrrolidinecarbonitrile obtained in Example

8-7 in a similar manner to that of Example 7-2.

¹H-NMR (in DMSO) : <5 1.38(3H, s), 2.20 - 1.44 ( 8H , m), 2.27(3H, s), 3.03-2.78(2H, m), 4.49 - 3.91 ( 5H , m), 5.29-5.13(1H, m), 8.00 - 7.82 ( 1H , m), 8.66 - 8.50 ( 1H , m), 8.90-8.78( 1H, m), 9.32 - 9.00 ( 3H , m). MS (ESI+) : m/z 420.21 (M+H).

Example 11 (2S) -4 , 4 -Dif luoro- 1- [ ( {1 -methyl- 4- [ ( 2 -pyridinylmethox y ) imino ] cyclohexyl } amino )acetyl] -2-pyrrolidinecarboni trile dihydrochloride

The title compound (249mg) was prepared from l-methyl-4-[(2 -pyridinylmethoxy) imino ] cyclohexyl }amin o and ( 2S ) - 1 -chloroacetyl- 4 , 4 -difluoro-2 - pyrrolidinecarbonitrile obtained in Example 8-7 in a similar manner to that of Example 7-2.

^XH-NMR (in DMSO) : δ 1.44(3H, s), 2.36 - 1.74 ( 7H , m),

3.02-2.74 (3H, m), 3.27 - 3.09 ( 1H , m), 4.46 - 4.04 ( 4H , m),

5.27-5.15 ( 1H, m), 5.37(2H, s), 8.01 - 7.80 ( 2H , m),

8.52-8.38 ( 1H, m), 8.90 - 8.76 ( 1H , m), 9.32 - 9.11 ( 1H , m), 9.54-9.32( 1H, m) . MS (ESI+) : m/z 406.23 (M+H).

Example 12-1 tert-Butyl l-methyl-4-({[(lE) - 4 -pyridinyl - methylidene ] amino }oxy ) cyclohexylcarbamate

To a cold (-78°C ) stirred solution of tert-butyl [ trans - 4 -aminooxy- 1 -methylcyclohexyl ] carbamate . (147mg) in methanol ( lmL ) , was added isonicot inaldehyde (0.057mL). The mixture was stirred to room temperature for 16hrs. The solution was concentrated in vacuo. The residue was triturated with isopropylether , then filtrated to provide the target compound as a white solid ( 128mg) .

^XH-NMR (300MHz, CDC1₃) : <5 1.34(3H, s), 1.45(9H, s), 1.57-1.94(8H, m), 4.35 ( 1H, br-s), 4.39(1H, br-s), 7.45(2H, dd, J=1.5, 4.4Hz), 8.04(1H, s), 8.62(2H, dd, J=1.5, 4.4Hz ) . MS (ES+) : m/e 334.22. Example 12-2

Isonicotinaldehyde 0-(4-amino-4 -methylcyclohexyl ) - oxime bis ( trifluoroacetate )

The title compound (165mg) was prepared from tert-butyl l-methyl-4-({[(lE) - 4 -pyridinylmethylidene ] amino }oxy ) cyclohexylcarbamate obtained in Example 12-1 in a similar manner to that of Example 7-1.

Example 12-3

(2S,4S)-4-Fluoro-l-({[ 1 -methyl- 4- ( { [ ( IE) - 4 -pyridinylm ethylidene ] amino} oxy ) cyclohexyl ] amino }acetyl) -2-pyrro lidinecarbonitrile

The title compound (89.0mg) was prepared from isonicotinaldehyde 0-(4- amino- 4 -methylcyclohexyl ) - oxime bis ( trif luoroacetate ) obtained in Example 12-2 and (2S)-l-chloroacetyl-4,4-difluoro-2-pyrrolidinecarboni trile obtained in Example 8-7 in a similar manner to that of Example 7-2.

^XH-NMR ( 300MHz , CDC1₃) : <5 1.12(3X4/5H, s), 1.15(3Xl/5H, s), 1.39-1.80(6H, m), 1.97(2H, br-s), 2.20 - 2.57 ( 1H , m), 2.70(1X4/5H, t), 2.77(lXl/5H, t), 3.30 - 4.40 ( 4H , m) , 4.31(1H, br-s), 4.97(lX4/5H, d, J= 9.3Hz), 5.09 ( 1 X 1/5H , d, J=9.0Hz), 5.37(lXl/5H, br-d, J=53.2Hz), 5.44(lX4/5H, br-t, J=3.6, 50.8Hz), 7.45(2H, dd, J=1.7, 4.6Hz), 8.03(1H, s), 8.62(2H, dd, J=1.7, 4.6Hz). MS (ES+) : m/e 388.15.

Example 13-1 tert-Butyl 4-({[(lE)-( 6 -chloro- 3 -pyridinyl ) - methylidene ] amino }oxy ) - 1 -methylcyclohexylcarbama e

The title compound (138mg) was prepared from tert-butyl (trans -4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and 6 - chloronicot inaldehyde in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : δ 1.34(3H, s), 1.44(9H, s), 1.63-1.95(8H, m), 4.32(1H, br-s), 4.38(1H, br-s), 7.33(1H, d, J=8.3Hz), 7.95(1H, dd , J=2.3, 8.3Hz), 8.07(1H, s), 8.47( 1H, d, J=2.3Hz) . MS (ES+) : m/e 368.13.

Example 13-2

6 -Chloronicot inaldehyde O- ( 4 -amino- 4 -methyl - cyclohexyl ) oxime bis(trifluoroacetate)

The title compound (146mg) was prepared from tert-butyl 4-({[(lE)-( 6 -chloro- 3 -pyridinyl ) - methylidene ] amino} oxy ) - 1 -methylcyclohexylcarbamate obtained in Example 13-1 in a similar manner to that of Example 7-1.

Example 13-3

(2S,4S)-l-({[4-({[(lE)- (6 -Chloro- 3 -pyridinyl) methylid ene ] amino }oxy ) - 1 -methylcyclohexyl ] amino }acetyl) -4-flu oro- 2 -pyrrolidinecarbonitrile

The title compound (13.3mg) was prepared from (2S, 4S) -l-chloroacetyl-4-fluoro-2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and 6 -chloronicotinaldehyde O- ( 4 -amino- 4 -methyl - cyclohexyl ) oxime bis ( trifluoroacetate ) obtained in Example 13-2 in a similar manner to that of Example 7-2.

^XH-NMR (300MHz, CDC1₃) : δ l.ll(3X4/5H, s), 1.15(3Xl/5H, s), 1.38-1.76 ( 6H, m), 1.96(2H, br-s), 2.20 - 2.58 ( 1H , m), 2.70(lX4/5H, t), 2.77(lXl/5H, t), 3.30 - 4.10 ( 4H , m). 4.28(1H, br-s), 4.97(lX4/5H, d, J=9.2Hz), 5.09(lXl/5H, d, J=9.0Hz), 5.37(lXl/5H, br-d, J=51.0Hz), 5.44(lX4/5H, br-d, J=51.0Hz), 7.33(1H, d, J=8.4Hz), 7.96(1H, dd, J=2.4, 8.4Hz), 8.07(1H, s), 8.47(1H, d, J=2.4Hz). MS (ES+) : m/e 422.09.

Example 14-1 tert-Butyl 4-({[(lE)-(6-methoxy -3-pyridiny1)- methylidene ] amino }oxy ) - 1 -methylcyclohexylcarbama e

The title compound (141mg) was prepared from tert-butyl (trans-4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and 6 -methoxynicot inaldehyde in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : <? 1.34(3H, s), 1.44(9H, s), 1.60-1.90 (8H, m), 3.96(3H, s), 4.27(1H, br-s), 4.39(1H, br-s), 6.75(1H, d, J=8.6Hz), 7.94(1H, dd, J=2.2, 8.6Hz), 8.05(1H, s), 8.18(1H, d, J=2.2Hz). MS (ES+) : m/e 364.19.

Example 14-2

6 -Methoxynicotinaldehyde 0-(4-amino- 4 -methyl - cyclohexyl ) oxime bis (trifluoroacetate)

The title compound (163mg) was prepared from tert-butyl 4-({[(lE)-(6 -methoxy- 3 -pyridinyl ) - methylidene ] amino } oxy ) - 1 -methylcyclohexylcarbamate obtained in Example 14-1 in a similar manner to that of Example 7-1.

Example 14-3

(2S, 4S) -4 -Fluoro- 1- ({[4-({[(lE)- ( 6 -methoxy- 3 -pyridiny 1 ) methylidene ] amino } oxy ) - 1 -methylcyclohexyl ] amino }ace tyl) - 2 -pyrrolidinecarbonitrile The title compound (74.4mg) was prepared from (2S, 4S) -1-chloroacetyl- 4 -fluoro-2- pyrrolidinecarbonit rile obtained in Preparation 1-8 and 6 -methoxynicot inaldehyde 0- ( 4 -amino - 4 -methyl- cyclohexyl ) oxime bis ( trif luoroacetate ) obtained in Example 14-2 in a similar manner to that of Example 7-2.

^XH-NMR (300MHz , CDC1₃) : <5 1.11(3X4/5H, s), 1.15(3Xl/5H, s), 1.30-1.78 ( 6H, m), 1.95(2H, br-s), 2.30 - 2.56 ( 1H , m), 2.69(lX4/5H, t, J=15.8Hz), 2.77(lXl/5H, t, J=15.5Hz), 3.30-4.02 (4H, m), 3.96(3H, s), 4.23(1H, br-s), 4.97(1 X4/5H, d, J=9.6Hz), 5.12(lXl/5H, d, J=9.5Hz), 5.36(1 X 1/5H, br-d, J=51.1Hz), 5.44(1 X 4/5H, br-d, J=3.4, 51.1Hz), 6.75(1H, d, J=8.6Hz), 7.95(1H, dd, J=2.4, 8.6Hz), 8.05(1H, s), 8.18(1H, d, J= 2.4Hz ) . MS (ES+) : m/e 418.12.

Example 15-1 tert-Butyl 1 -methyl- 4 - ( { [ ( IE ) - ( 6 -phenoxy- 3 - pyridinyl ) methylidene ] amino } oxy ) cyclohexylcarbamate

The title compound (203mg) was prepared from tert-butyl (trans-4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and 6 -phenoxynicot inaldehyde in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃ ) : δ 1.34(3H, s), 1.44(9H, s), 1.62-1.90(8H, m), 4.28(1H, br-s), 4.39 ( 1H, br-s), 6.91(1H, d, J=8.7 Hz), 7.15(2H, d, J=7.5Hz), 7.23(1H, t, J=7.5Hz), 7.42(2H, t, J=7.5Hz), 8.04( 1H, dd, J=2.1, 8.7Hz), 8.06(1H, s ) , 8.22 ( 1H, d, J = 2.1Hz) . MS (ES+) : m/e 426.16.

Example 15-2 6 -Phenoxynicotinaldehyde 0- ( 4-amino-4 -methyl - cyclohexyl ) oxime bis(trifluoroacetate)

The title compound (233mg) was prepared from tert-butyl 1 -methyl- 4 - ( { [ ( IE ) - ( 6 -phenoxy- 3 - pyridinyl ) methylidene ] amino }oxy ) cyclohexylcarbamate obtained in Example 15-1 in a similar manner to that of Example 7 - 1.

Example 15-3

(2S,4S)-4-Fluoro-l-({[ 1 -methyl_^- 4- ( { [ ( IE) - ( 6 -phenoxy- 3 -pyridinyl ) methylidene ] amino }oxy ) cyclohexyl ] amino }ace tyl) - 2 -pyrrolidinecarbonitrile

The title compound (35.9mg) was prepared from (2S,4S)-l-chloroacetyl-4-fluoro-2-pyrrolidinecarbonit rile obtained in Preparation 1-8 and 6 -phenoxynicot inaldehyde O- ( 4 -amino- 4 -methyl - cyclohexyl ) oxime bis ( trifluoroacetate ) obtained in Example 15-2 in a similar manner to that of Example 7-2.

^XH-NMR (300MHz, CDC1₃) : δ l.ll(3Xl/5H, s), 1.15(3X4/5H, s), 1.37-1.78(6H, m), 1.95(2H, br-s), 2.20 - 2.55 ( 1H , ), 2.70(lX4/5H, t, J=15.3Hz), 2.77(lXl/5H, t, J=15.2Hz), 3.30-4.10 (4H, m), 4.25(1H, br-s), 4.97(1 X 4/5H, d, J= 9.0Hz), 5.10(1X1/5H, d, J= 9.0Hz), 5.36 ( 1 X 1/5H , br-d, J=51.0Hz), 5.44(lX4/5H, br-d, J=50.4Hz), 6.92(1H, d, J=8.4Hz), 7.14(2H, d, J=7.9Hz), 7.23(1H, t, J=7.9Hz), 7.42(2H, t, J=7.9Hz), 8.04(1H, br-d, J=8.4Hz), 8.06(1H, s) , 8.22(1H, s) .

MS (ES+) : m/e 480.06.

Example 16-1 tert-Butyl 1 -methyl- 4- [ ( { ( IE) - [ 6- (trifluoromethyl) -3- pyridinyl ] methylidene } amino ) oxy] cyclohexylcarbamate The title compound (206mg) was prepared from tert-butyl ( trans - 4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and 6 -trif luoromethylnicot inaldehyde in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : δ 1.34(3H, s), 1.45(9H, s), 1.60-1.95(8H, m) , 4.38(2H, br-s), 8.68(1H, d, J= 8.4Hz), 8.12(1H, br-d, J=8.4Hz), 8.15(1H, s), 8.84(1H, br-s). MS (ES+) : m/e 346.21 ( M- tBu ) .

Example 16-2

6 - ( Trifluoromethyl ) nicotinaldehyde 0- ( 4 -amino- 4 - methylcyclohexyl ) oxime bis(trifluoroacetate)

The title compound (133mg) was prepared from tert-butyl l-methyl-4-({[(lE)-( 6 -Trifluoromethyl- 3 - pyridinyl ) methylidene ] amino }oxy ) cyclohexylcarbamate obtained in Example 16-1 in a similar manner to that of Example 7-1.

Example 16-3

(2S, 4S) -4 -Fluoro- 1- [ ( {l-methyl-4- [ ({ (IE) - [6- (trifluor omethyl ) - 3 -pyridinyl ] methylidene} amino ) oxy] cyclohexyl } amino )acetyl] -2 -pyrrolidinecarbonitrile

The title compound (23mg) was prepared from (2S, 4S) -l-chloroacetyl-4-fluoro-2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and 6 -( trifluoromethyl ) nicotinaldehyde O- ( 4 -amino- 4 - methylcyclohexyl ) oxime bis ( trifluoroacetate ) obtained in Example 16-2 in a similar manner to that of Example 7-2.

¹H-NMR (300MHz , CDC1₃) : <5 1.12(3X4/5H, s), 1.15(3Xl/5H, s), 1.37-1 ,80(6H, m), 1.97(2H, br-s), 2.20 - 2.55 ( 1H , m), 2.70(lX4/5H, t, J=15.2Hz), 2.77(lXl/5H, t, J=15.5Hz), 3.30-4.10(4H, m), 4.33(1H, br-s), 4.97(1 X 4/5H, d, J=9.3Hz), 5.08(lXl/5H, d, J=9.5Hz), 5.36(lXl/5H, br-d, J=51.0Hz), 5.44(lX4/5H, br-t, J=3.6, 51.5Hz), 7.88(1H, d, J=8.2Hz), 8.12(1H, d, J=8.2Hz), 8.15(1H, s), 8.84(1H, br- s ) . MS (ES+) : m/e 456.08.

Example 17-1 tert-Butyl 1 -methyl - 4 - { [ ( IE ) - 1 - ( 3 -pyridinyl ) - ethylidene ] amino }oxy } cyclohexylcarbamate

The title compound was prepared from tert-butyl ( trans - 4 -aminooxy- 1 -methylcyclohexyl ) carbamate and 3 - acetylpyridine in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : <5 1.36(3H, s), 1.45(9H, s), 1.66-1.90(8H, m), 2.26(3H, s), 4.35(1H, br-s), 4.40(1H, br-s), 7.29(1H, dd, J= 4.7, 8.1Hz), 7.9.6(1H, ddd, J= 1.7, 2.4, 8.1Hz), 8.58(1H, dd, J=1.7, 4.7Hz), 8.87 ( 1H, d, J= 2.4 Hz ) .

MS (ES+) : m/e 348.31.

Example 17-2

( IE ) - 1 - ( 3 -pyridinyl )ethanone 0-(4-amino-4 -methylcyclohexyl ) oxime

To a cold stirred solution of tert-Butyl 1 -methyl- 4-{[ (lE)-l-(3-pyridinyl) -ethylidene ] amino }ox y }cyclohexylcarbamate (50mg) in methanol (0.5mL), was added 4N HCl solution in dioxane ( 1. OmL ) . The mixture was stirred for 2hrs, and alkalized with saturated aqueous NaHC0₃ , then extracted with chloroform. The combined organic layer was dried and filtrated. The filtrate was concentrated in vacuo to provide the target compound as an oil ( 30mg ) .

¹H-NMR (300MHz, CDC1₃ ) : δ 1.17(3H, s), 1.30 - 1.80 ( 6H , m), 1.90-2.03(2H, m), 2.25(3H, s), 4.22-4.33(lH, m), 7.28 ( 1H, dd, J=4.9, 8.2Hz), 7.97(1H, dd , J=2.0, 8.2Hz), 8.58(1H, dd, J=2.0, 4.9Hz), 8.87(1H, d, J=2.0Hz ) . MS (ES+) : m/e 248.23.

Example 17-3

(2S,4S)-4-Fluoro-l-({[l-methyl-4-({[(lE)-l-( 3-pyridin yl ) ethylidene ] amino }oxy ) cyclohexyl ] amino} acetyl ) - 2 -py rrolidinecarbonitrile

The title compound (40mg) was prepared from ( 2S, 4S) -1 -chloroacetyl -4 -fluoro -2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and ( IE )- 1 -( 3 -pyridinyl ) ethanone 0- ( 4 - amino- 4 -methyl - cyclohexyl ) oxime obtained in Example 17-2 in a similar manner to that of Example 7-2.

^Η-NMR (300MHz, CDC1₃) : δ 1.12(3X4/5H, s), 1.16(3Xl/5H, s), 1.40-1.80 (6H, m), 1.96(2H, br-s), 2.19 - 2.56 ( 1H , m), 2.26(3H, s) , 2.70(lX4/5H, t, J=15.3Hz) , 2.76(lXl/5H, t, J=15.2Hz) , 3.30-4.12 (4H, mj , 4.30(1H, br-s) , 4.97(1 X4/5H, d, J=9.2Hz) , 5.12(1X1/5H, d, J=9.2Hz) , 5.36(1 X 1/5H, br-d, J=51.1Hz) , 5.44(1 X 4/5H, br-t, J=3.3, 51.1Hz) , 7.29(1H, ddd, J=0.9, 4.8, 8.1Hz) , 7.97(1H, ddd, J=1.7, 2.2 8.1Hz) , 8.58(1H, dd , J=1.7, 4.8Hz) , 8.87(1H, dd, J = 0.9 , 2.2Hz ) . MS (ES-) m/e 402.23.

Example 18-1 tert-Butyl 1 -methyl- 4 -({[( IE )-( 6 -methyl- 2 -pyridinyl ) - methylidene] amino }oxy ) cyclohexylcarbamate

The title compound was prepared from tert-butyl (trans -4 -aminooxy- 1 -methylcyclohexyl) carbamate and 6 -mehtyl- 2 -pyridinecarboxaldehyde in a similar manner to that of Example 12-1.

^-NMR (300MHz, CDC1₃) : δ 1.34(3H, s), 1.44(9H, s), 1.62-1.90 ( 8H, m), 2.56(3H, s), 4.36(2H, br-s), 7.11(1H, br-d, J=7.5Hz), 7.57(1H, t, J=7.5Hz), 7.62(1H, br-d, J= 7.5Hz ) , 8.16 ( 1H, s ) . MS (ES+) : m/e 348.32.

Example 18-2 6 -Methyl- 2 -pyridinecarbaldehyde 0- ( 4 -amino - 4 -methylcyclohexyl ) oxime bis(trifluoroacetate)

The title compound (158mg) was prepared from tert-butyl 1 -methyl- 4-({[(lE)-(6 -methyl -2-pyridinyl) - methylidene ] amino }oxy ) cyclohexylcarbamate obtained in

Example 18-1 in a similar manner to that of Example 7-1.

Example 18-3

(2S,4S)-4-Fluoro-l-({[l-methyl-4-({[(lE)- ( 6 -methyl- 2- pyridinyl ) methylidene ] amino} oxy ) cyclohexyl ] amino} acet yl) -2 -pyrrolidinecarbonitrile

The title compound (85.5mg) was prepared from (2S,4S)-l-chloroacetyl-4-fluoro-2-pyrrolidinecarbonit rile obtained in Preparation 1-8 and 6 -methyl- 2 -pyridinecarbaldehyde O- ( 4 -amino - 4 -methylcyclohexyl ) oxime bis ( trifluoroacetate ) obtained in Example 18-2 in a similar manner to that of Example 7-2.

^XH-NMR (300MHz, CDC1₃ ) : δ l.ll(3X4/5H, s), 1.15(3Xl/5H, s), 1.46(2H, br-s), 1.55 - 1.80 ( 4H , m), 1.96(2H, br-s), 2.17-2.50(1H, m), 2.57(3H, s), 2.70(lX4/5H, t, J=15.8Hz), 2.76(1 X 1/5H, t, J= 14.7Hz), 3.30 - 4.15 ( 4H , m), 4.23-4.37(1H, m), 4.97(lX4/5H, d, J= 9.3Hz), 5.12(lX 1/5H, d, J=9.0Hz), 5.36(1X1/5H, br-d, J=51.0Hz), 5.44(1 X 4/5H, br-t, J=3.5, 51.0Hz), 7.11(1H, d, J=7.7Hz), 7.57(1H, t, J=7.7Hz), 7.63(1H, t, J=7.7Hz), 8.16(1H, s). MS (ES+) : m/e 402.24.

Example 19-1 tert-Butyl 1 -methyl- 4- ( { [ ( IE) - ( 3 -methyl - 4 -pyridinyl ) - methylidene ] amino }oxy ) cyclohexylcarbamate

The title compound (194mg) was prepared from tert-butyl ( trans - 4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and 3 -mehtyl- 4 -pyridinecarboxaldehyde in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : δ 1.35(3H, s), 1.45(9H, s), 1.60-1.92 (8H, m), 2.41(3H, s), 4.36(1H, br-s), 4.40(1H, br-s), 7.54(1H, d, J=5.1Hz), 8.27(1H, s), 8.44(1H, d, J=5.1Hz) , 8.46 ( 1H, s) . MS (ES+) : m/e 348.32.

Example 19-2

3 -Methylisonicot inaldehyde 0- ( 4 -amino- 4 -methylcyclohexyl ) oxime bis ( trifluoroacetate )

The title compound (121mg) was prepared from tert-butyl 1 -methyl- 4 -({[( IE )-( 3 -methyl - 4 -pyridinyl ) - methylidene ] amino }oxy ) cyclohexylcarbamate obtained in

Example 19-1 in a similar manner to that of Example 7-1.

Example 19-3 (2S, 4S) -4- Fluoro- 1- ( { [ 1 -methyl- 4- ( { [ ( IE) - ( 3 -methyl- 4 - pyridinyl ) methylidene ] amino }oxy ) cyclohexyl ] amino }acet yl ) - 2 -pyrrolidinecarbonitrile

The title compound (48.2mg) was prepared from (2S,4S)-l-chloroacetyl-4-fluoro-2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and 3 -methylisonicotinaldehyde O- ( 4 -amino- 4 -methylcyclohexyl ) oxime bis ( trifluoroacetate ) obtained in Example 19-2 in a similar manner to that of Example 7-2.

^-NMR (300MHz , CDC1₃) : δ 1.12 ( 3X4/5H, s), 1.16(3Xl/5H, s), 1.36-1.80(6H, m), 1.98(2H, br-s), 2.20-2.56(lH, m), 2.40(3H, s), 2.70(lX4/5H, t, J=16.4Hz), 2.77(lXl/5H, t, J=14.9Hz), 3.30-4.10(4H, m), 4.31(1H, br-s), 4.97(1 X4/5H, d, J=9.2Hz), 5.09(lXl/5H, d, J=8.6Hz), 5.36(1 X 1/5H, br-d, J=51.7Hz), 5.44(1 X 4/5H, br-d, J=3.5, 51.1Hz), 7.54(1H, d, J=5.0Hz), 8.28(1H, s), 8.43(1H, d, J=5.0Hz) , 8.45 ( 1H, s) . MS (ES+) : m/e 402.25.

Example 20-1 tert-Butyl 1 -methyl- 4 - ( { [ ( IE ) - ( 1 , 3 - thiazol- 2 -yl ) - methylidene ] amino }oxy ) cyclohexylcarbamate

The title compound (208mg) was prepared from tert-butyl [trans-4-( aminooxy ) - 1 -methylcyclohexyl ] - carbamate and 2 -thiazolecarboxaldehyde in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : δ 1.34(3H, s), 1.45(9H, s), 1.60-1.90 (8H, m), 4.36(2H, br-s), 7.32(1H, dd , J= 0.9, 3.1Hz), 7.86(1H, d, J=3.1Hz), 8.32(1H, d, J=0.9Hz). MS (ES+) : m/e 340.21.

Example 20-2 1 , 3 -Thiazole- 2 -carbaldehyde O- ( 4 -amino- 4 -methylcyclohexyl ) oxime bis(trifluoroacetate)

The title compound (138mg) was prepared from tert-butyl 1 -methyl - 4 - ( { [ ( IE ) - ( 1 , 3 - thiazol-2 -yl ) - methylidene ] amino }oxy ) cyclohexylcarbamate obtained in

Example 20-1 in a similar manner to that of Example 7-1.

Example 20-3 ( 2S, 4S) -4 -Fluoro- 1- ( { [ l-methyl-4- ({[(lE)-(l,3-thiazol - 2 -yl ) methylidene ] amino }ox ) cyclohexyl ] amino }acetyl) - 2 -pyrrolidinecarbonitrile

The title compound (12.4mg) was prepared from (2S,4S)-l-chloroacetyl-4-fluoro- 2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and 1 , 3-thiazole-2 -carbaldehyde O- ( 4 -amino- 4 -methylcyclohexyl ) oxime bis ( trifluoroacetate ) obtained in Example 20-2 in a similar manner to that of Example 7-2.

^XH-NMR ( 300MHz , CDC1₃) : 1.12(3X4/5H,. s), 1.16(3Xl/5H, s), 1.38-1.80( 6H, m), 1.97(2H, br-s), 2.20 - 2.57 ( 1H , m), 2.70(lX4/5H, t, J=15.5Hz), 2.77(lXl/5H, t, J=15.2Hz), 3.30-4.10(4H, m), 4.31(1H, br-s), 4.97(1 X 4/5H, d, J= 9.0Hz), 5.09(lXl/5H, d, J=9.0Hz), 5.36 ( 1 X 1/ 5H , br-d, J=51.5Hz), 5.44(1X4/5H, br-d, J=51.5Hz), 7.32(1H, d, J=3.2Hz), 7.86(1H, d, J=3.2Hz), 8.32(1H, s). MS (ES+) : m/e 394.10.

Example 21-1 tert-Butyl trans-4-[( cyclohexylideneamino ) oxy] - 1 - methylcyclohexylcarbamate

The title compound (81.5mg) was prepared from tert-butyl ( trans - 4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and cyclohexanone in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : δ 1.33(3H, s), 1.44(9H, s), 1.55-1.85(14H, m), 2.20(2H, br-t, J=6.3Hz), 2.48(2H, br-s), 4.14(1H, br-s), 4.38(1H, br-s). MS (ES+) : m/e 325.33.

Example 21-2 Cyclohexanone 0- ( 4 -amino- 4 -methylcyclohexyl ) oxime

To tert-butyl { trans - 4 -[( cyclohexylideneamino ) - oxy ]- 1 -methylcyclohexyl }carbamate obtained in Example 21-1 (81.5mg), was added trifluoroacetic acid (0.5mL) at room temperature for lOmin. The mixture was alkalized with saturated aqueous NaHC0₃ , then extracted with chloroform. The combined organic layer was dried and filtrated. The filtrate was concentrated in vacuo to provide the target compound as an oil (70.7mg).

^XH-NMR (300MHz, CDC1₃) : δ 1.31(3H, s), 1.45 - 2.05 ( 14H , m), 2.19(2H, t, J=6.4Hz), 2.45(2H, br-s), 4.05(1H, br-s).

Example 21-3 (2S,4S)-l-[({4-[ (Cyclohexylideneamino ) oxy] -1-methyley clohexyl} amino )acetyl] -4 -fluoro - 2 -pyrrolidinecarbonit rile

The title compound (26.3mg) was prepared from (2S, 4S) -l-chloroacetyl-4-fluoro-2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and cyclohexanone

0- ( 4 -amino -4 -methylcyclohexyl ) oxime obtained in Example

21-2 in a similar manner to that of Example 7-2.

¹H-NMR (300MHz, CDCI₃) : β 1.10 ( 3 X 4 / 5H , s ) , 1.13 ( 3 X 1 / 5H , s), 1.33-1.48(2H, m), 1.50 - 1.72 ( 1 OH , m), 1.82 - 1.98 ( 2H , m), 2.18-2.58 ( 1H, m), 2.20(2H, br-t, J=6.0Hz), 2.48(2H, br-s), 2.69(1 X 4/5H, t, J=15.6Hz), 2.75 ( 1 X 1/5H , t, J=15.6Hz), 3.28-4.05(4H, m), 4.07(1H, br-s), 4.96(lX 4/5H, d, J=9.4Hz), 5.16(lXl/5H, d, J=9.4Hz), 5.35(lX 1/5H, br-d, J=51.5Hz), 5.43(lX4/5H, br-d, J=51.5Hz). MS (ES+) : m/e 379.24.

Example 22-1 tert-Butyl 1 -methyl - 4 -{[( 1 -methylethylidene ) amino ] - oxy } cyclohexylcarbamate

The title compound (72mg) was prepared from tert-butyl (trans -4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and acetone in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : δ 1.33(3H, s), 1.44(9H, s), 1.60-1.85(8H, m), 1.87(3H, s), 1.88(3H, s), 4.14(1H, br-s), 4.37 ( 1H, br-s) .

MS (ES+) : m/e 285.32.

Example 22-2

Acetone 0-(4 -amino- 4 -methylcyclohexyl ) oxime

The title compound (33.2mg) was prepared from tert-butyl 1 -methyl- 4 - { [ ( 1 -methylethylidene ) amino ] - oxy }cyclohexylcarbamate obtained in Example 22-1 in a similar manner to that of Example 17-2.

^XH-NMR (300MHz, CDC1₃) : <5 1.14(3H, s), 1.15 - 1.48 ( 4H , m), 1.50-1.76 (4H, m), 1.86(3H, s), 1.87(3H, s), 4.07(1H, br- s ) . MS (ES+) : m/e 185.13. Example 22-3

(2S,4S)-4-Fluoro-l-{[( l-methyl-4-{ [ ( 1 -methylethyliden e ) amino ] oxy } cyclohexyl ) amino ]acetyl}-2-pyrrolidinecar bonitrile

The title compound (30.6mg) was prepared from

(2S,4S)-l-chloroacetyl-4-fluoro-2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and acetone

0- ( 4 -amino- 4 -methylcyclohexyl ) oxime obtained in Example 22-2 in a similar manner to that of Example 7-2.

¹H-NMR ( 300MHz , CDC1₃) : δ 1.10(3X4/5H, s), 1.15(3Xl/5H, s), 1.32-1.48(2H,m), 1.50-1.70(4H,m), 1.72-2.00(2H,m),

1.86(3H, s), 1.87 ( 3H, s), 2.18-2.56(lH, m), 2.69(lX4/5H, t, J=15.0Hz) , 2.75(lXl/5H, t, J=15.3Hz), 3.30 - 4.10 ( 4H , m), 4.08(1H, br-s), 4.97(lX4/5H, d, J=9.5Hz), 5.16(1

X1/5H, d, J=9.5Hz), 5.35(lXl/5H, br-d, J=50.9Hz), 5.43(1

X4/5H, br-d, J=51.5Hz).

MS (ES+) m/e 339.24.

Example 23-1 tert-Butyl l-methyl-4- ({[(IE) -2-thienylmethylidene] - amino }oxy ) cyclohexylcarbamate

The title compound (64.8mg) was prepared from tert-butyl ( trans - 4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and 2 -thiophenecarboxaldehyde in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : δ 1.34(3H, s), 1.44(9H, s), 1.60-2.00(8H, m), 4.28(1H, br-s), 4.38(1H, br-s), 7.03(1H, dd, J=3.7, 5.1Hz), 7.16(1H, d, J=3.7Hz), 7.30(1H, d, J= 5.1Hz) , 8.25( 1H, s ) . MS (ES+) : m/e 339.20. Examp l e 2 3 - 2

2 -Thiophenecarbaldehyde O- (4- amino- 4 -methyl - cyclohexyl ) oxime

The title compound (34.5mg) was prepared from tert-butyl l-methyl-4-({[(lE) - 2 - thienylmethylidene ] - amino }oxy) cyclohexylcarbamate obtained in Example 23-1 in a similar manner to that of Example 17-2.

Example 23-3

(2S,4S)-4-Fluoro-l-({[ l-methyl-4- ( { [ ( IE) - 2 - thienylmet hy11dene ] amino }oxy ) cyclohexyl ] amino }acetyl) -2-pyrroli dinecarbonitrile hydrochloride

To a stirred solution of 2 - thiophenec'arbaldehyde 0- ( trans - 4 -amino-4 -methylcyclohexyl ) oxime obtained in Example 23-2 ( 34. lmg ) in dimethylformamide ( 1.5mL ) , were added K₂C0₃ (37.7mg), ( 2S , 4S )- 1 -chloroacetyl - 4 -fluoro- 2 -pyrrolidinecarbonitrile obtained in Preparation 1-8 (26.0mg) and sodium iodide (lmg).

The mixture was stirred for 5hrs at 50°C and diluted with water. The aqueous layer was extracted with ethyl acetate. The organic layer was washed with saturated aqueous NaCI, dried, and concentrated in vacuo. The residue was dissolved to ethyl acetate, then added 4M HCl in dioxane (30£6 L) . The precipitate was filtrated, then washed with ethyl acetate to provide the target compound as a white solid (22.5mg).

^XH-NMR (300MHz, CDC1₃ ) : δ 1.36(3H, s), 1.40 - 1.65 ( 2H , m), 1.70-2.17(6H, m), 2.35 - 2.60 ( 2H , m), 3.36(2H, s), 3.68-4.29 (4H, m), 5.09 (1H, d, J= 8.3Hz), 5.58(1H, br-d. J=52.2Hz), 7.17(1H, dd, J=3.7, 5.0Hz), 7.55(1H, d, J=3.7Hz), 7.82(1H, d, J=5.0Hz), 8.96(2H, br-s). MS (ES+) : m/e 393.17. Example 24-1 tert-Butyl l-methyl-4- ( { [ ( IE ) - phenylmethylidene ] - amino }oxy ) cyclohexylcarbamate

The title compound (314mg) was prepared from tert-butyl (trans-4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and benzaldehyde in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : <? 1.35(3H, s), 1.45(9H, s), 1.50-2.20(8H, m), 4.31(1H, br-s), 4.39(1H, br-s), 7.33-7.40( 3H, m), 7.62 - 7.55 ( 2H , m), 8.10(1H, s). MS (ES+) : m/e 333.26.

Example 24-2

Benzaldehyde O- ( 4 -amino-4 -methylcyclohexyl ) oxime

The title compound (206mg) was prepared from tert-butyl 1 -methyl- 4 - ( { [ ( IE ) -phenylmethylidene ] - amino }oxy) cyclohexylcarbamate obtained in Example 24-1 in a similar manner to that of Example 17-2.

^-NMR (300MHz, CDC1₃) : δ 1.16(3H, s), 1.30 - 1.43 ( 2H , m), 1.58-1.80(4H, m), 1.86 - 2.04 ( 2H , m), 4.18 - 4.30 ( 1H , m), 7.30-7.42 ( 3H, m), 7.52 - 7.64 ( 2H , m), 8.09 (1H, s). MS (ES+) : m/e 233.32.

Example 24-3 (2S,4S) -4 -Fluoro- 1- ({ [ 1 -methyl - 4 -( { [ (IE) -phenylmethyl idene ] amino }oxy ) cyclohexyl ] amino }acetyl) -2-pyrrolidin ecarbonitrile

The title compound (92.3mg) was prepared from (2S,4S)-1 -chloroacetyl- 4 -fluoro- 2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and benzaldehyde 0-( 4 -amino-4 -methylcyclohexyl) oxime obtained in Example 24-2 in a similar manner to that of Example 7-2.

^XH-NMR (300MHz, CDC1₃ ) : δ 1.12(3X5/4H, s), 1.15(3Xl/5H, s), 1.39-1.52 (2H, m), 1.52 - 1.49 ( 4H , m), 1.97(2H, br-s), 2.19-2.56(1H, m), 2.69(lX4/5H, t, J=15.7Hz), 2.76(lX 1/5H, t, J=15.7Hz), 3.28-4.12 (4H, m), 4.26(1H, br-s), 4.97(lX4/5H, d, J=9.1Hz), 5.13(lXl/5H, d, J=9.0Hz), 5.36(1X1/5H, br-dt, J=3.4, 51.3Hz), 5.44(lX4/5H, br-dt, J= 3.4, 51.1Hz), 7.33-7.43 ( 3H, m), 7.54 - 7.63 ( 2H , m), 8.09 ( 1H, s ) . MS (ES+) : m/e 387.18.

Example 25-1 tert-Butyl 1 -methyl- 4 - ( { [ ( IE ) - 1 -phenylethylidene ] - amino }oxy ) cyclohexylcarbamate

The title compound (222mg) was prepared from tert-butyl ( trans - 4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and acetophenone in a similar manner to that of Example 12-1.

^-N R (300MHz, CDC1₃) : δ 1.36(3H, s), 1.45(9H, s), 1.62-1.90(8H, m), 2.25(3H, s), 4.34(1H, br-t, J=4.5Hz), 4.40(1H, br-s), 7.32 - 7.41 ( 3H , ), 7.60 - 7.69 ( 2H , m. MS (ES+) m/e 347.2.7.

Example 25-2 ( IE ) - 1 -Phenylethanone O- ( 4 -amino- 4 -methylcyclohexyl ) - oxime

The title compound (68.5mg) was prepared from tert-butyl l-methyl-4- ({[(IE) -1-phenylethylidene]- amino}oxy) cyclohexylcarbamate obtained in Example 25-1 in a similar manner to that of Example 17-2.

H-NMR (300MHz, CDC1₃) : δ 1.17(3H, s), 1.32 - 1.45 ( 2H , m), 1.56-1.80(4H, m), 1.90 - 2.04 ( 2H , m), 2.24(3H, s), 4.20-4.32(1H, m), 7.30 - 7.40 ( 3H , m), 7.60 - 7.70 ( 2H , m). MS (ES+) : m/e 247.24.

Example 25-3

(2S,4S)-4-Fluoro-l-({[ l-methyl-4- ( { [ ( IE) - 1 -phenylethy lidene] amino} ox ) cyclohexyl ] amino }acetyl) -2 -pyrrolidi necarbonitrile

The title compound (58.7mg) was prepared from (2S, 4S) -1 -chloroacetyl -4 -fluoro- 2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and ( IE ) - 1 -phenylethanone O- ( 4 -amino- 4 -methylcyclohexyl ) - oxime obtained in Example 25-2 in a similar manner to that of Example 7 - 2.

^XH-NMR (300MHz , CDC1₃) : δ 1.12(3X4/5H, s), 1.16(3Xl/5H, s), 1.30-1.80 (6H, m) , 1.96(2H, br-s), 2.15 - 2.55 ( 1H , m), 2.25(3H, s), 2.69(lX4/5H, t, J=15.7Hz), 2.76(lXl/5H, t, J=15.7Hz), 3.31-4.40(4H, m), 4.28(1H, br-s), 4.97(1 X4/5H, d, J=9.0Hz), 5.15(lXl/5H, d, J=9.0Hz), 5.35(1 X 1/5H, br-d, J=51.1Hz), 5.44(1 X 4/5H, br-t, J=3.7, 51.5Hz), 7.32-7.40(3H, m), 7.60 - 7.70 ( 2H , m). MS (ES+) : m/e 401.28.

Example 26-1 tert-Butyl 1 -methyl- 4 - ( { [ ( IE ) - 5 -pyrimidinyl- methylidene ] amino }oxy ) cyclohexylcarbamate

The title compound (84.8mg) was prepared from tert-butyl (trans- 4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and^' 5 -pyrimidinecarbaldehyde in a similar manner to that of Example 12-1.

^-NMR (300MHz, CDC1₃ ) : δ 1.35(3H, s), 1.45(9H, s), 1.61-1.92(8H, m), 4.36(2H, br-s), 8.07(1H, s), 8.92(2H, s ) , 9.18 ( 1H, s) .

MS (ES+) m/e 335.23.

Example 26-2

5 -Pyrimidinecarbaldehyde O- ( 4 -amino- 4 -methyl - cyclohexyl ) oxime bis ( trifluoroacetate )

The title compound (147mg) was prepared from tert-butyl 1 -methyl -4-({[(lE)-5 -pyrimidinyl - methylidene ] amino }oxy ) cyclohexylcarbamate obtained in Example 26_^1 in a similar manner to that of Example 7-1.

Example 26-3

(2S,4S)-4-Fluoro-l-({ [l-methyl-4-({ [ (IE) - 5 -pyrimidiny lmethylidene ] amino }oxy ) cyclohexyl ] amino} acetyl ) - 2 -pyr rolidinecarbonitrile

The title compound (22.4mg) was prepared from (2S,4S)-l-chloroacetyl-4-fluoro-2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and 5 -pyrimidinecarbaldehyde 0- ( 4 -amino - 4 -methylcyclohexyl ) oxime bis ( trif luoroacetate ) obtained in Example 26-2 in a similar manner to that of Example 7-2.

^XH-NMR (300MHz, CDC1₃) : δ 1.12(3X4/5H, s), 1.15(3Xl/5H, s), 1.40-1.80 (6H, m), 1.97(2H, br-s), 2.18 - 2.60 ( 1H , m), 2.70(lX4/5H, t, J=15.6Hz), 2.77(lXl/5H, t, J=15.6Hz), 3.30-4.09(4H, m), 4.32(1H, br-s), 4.96(1 X 4/5H, d, J=9.4Hz), 5.08(lXl/5H, d, J=9.4Hz), 5.36(lXl/5H, br-d, J=51.5Hz), 5.45(lX4/5H, br-d, J=50.5Hz), 8.06(1H, s), 8.92(2H, s), 9.18(1H, s). MS (ES+) : m/e 389.18.

Example 27-1 tert-Butyl 4- [ ( cyclopentylideneamino ) oxy ] -1-methyl- cyclohexylcarbamate

The title compound (304mg) was prepared from tert-butyl (trans-4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and cyclopentanone in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : δ 1.33(3H, s), 1.43(9H, s), 1.60-1.83 (12H, m), 2.32 - 2.44 ( 4H , m), 4.14(1H, br-s), 4.37( 1H, br-s ) . MS (ES+) : m/e 311.28.

Example 27-2

Cyclopentanone 0-(4 -amino- 4 -methylcyclohexyl ) oxime

The title compound (137mg) was prepared from tert-butyl 4- [ ( cyclopentylideneamino ) oxy ] -1-methyl- cyclohexylcarbamate obtained in Example 27-1 in a similar manner to that of Example 17-2.

^XH-NMR (300MHz, CDC1₃ ) : <5 1.15(3H, s), 1.24 - 1.40 ( 2H , m),

1.44-1.68(4H, m), 1.68 - 1.80 ( 4H , m), 1.82 - 1.96 ( 2H , m),

2.30-2.44 (4H, m), 4.01 - 4.12 ( 1H , m). MS (ES+) : m/e 211.21.

Example 27-3

(2S,4S)-l-[({4-[( Cyclopentylideneamino ) oxy] -1 -methylc yclohexyl } amino )acetyl]-4-fluoro-2-pyrrolidinecarboni trile

The title compound (15mg) was prepared from (2S,4S)-l-chloroacetyl-4-fluoro-2-pyrrolidinecarbonit rile obtained in Preparation 1-8 and cyclopentanone O- ( 4 -amino- 4 -methylcyclohexyl ) oxime obtained in Example 27-2 in a similar manner to that of Example 7-2.

^-NMR (300MHz, CDC1₃) : δ l.ll(3X4/5H, s), 1.14(3Xl/5H, s), 1.35-1.50(2H,m), 1.50-1.69(4H, m), 1.69-1.80(4H, m), 1.83-1.97 (2H, m), 2.19 - 2.42 ( 5H , m), 2.69(lX4/5H, t, J=16.0Hz), 2.75(lXl/5H, t, J=16.1Hz), 3.30-4.02(4H, m), 4.08(1H, br-s), 4.95(lX4/5H, d, J=9.7Hz), 5.15(lXl/5H, d, J=9.5Hz), 5.35(lXl/5H, br-d, J=52.1Hz), 5.43(lX4/5H, br-dt, J= 3.5 , 51.5Hz ) . MS (ES+) : m/e 365.24.

Example 28-1 tert-Butyl 4 - { [ ( 1 -ethylpropylidene ) amino ] oxy} - 1 - methylcyclohexylcarbamate

The title compound (121mg) was prepared from tert-butyl ( trans - 4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and 3-pentanone in a similar manner to that of Example 12-1.

^XH-NMR (300MHz, CDC1₃) : δ 1.07(3H, t, J=7.7Hz), 1.08(3H, t, J = 7.5Hz), 1.33(3H, s), 1.44(9H, s), 1.58 - 1.99 ( 8H , m), 2.19(2H, q, J= 7.5Hz), 2.31(2H, q, J = 7.7Hz), 4.13 ( 1H, br-s), 4.37( 1H, br-s) . MS (ES+) : m/e 313.28.

Example 28-2

3-Pentanone 0-(4 -amino- 4 -methylcyclohexyl ) oxime

The title compound (74.4mg) was prepared from tert-butyl 4 - { [ ( 1 -ethylpropylidene ) amino ] oxy} - 1 - methylcyclohexylcarbamate obtained in Example 28-1 in a similar manner to that of Example 17-2.

^XH-NMR (300MHz, CDC1₃ ) : δ 1.06(3H, t, J=7.7Hz), 1.08(3H, t, J=7.5Hz), 1.13(3H, s), 1.20-1.70(8H,m), 1.80-1.93(2H, m), 2.19(2H, q, J=7.5Hz), 2.32(2H, q, J=7.7Hz), 4.00-4.12 ( 1H, m) . MS (ES+) : m/e 213.19.

Example 28-3 (2S,4S)-l-{[(4-{[( 1 -Ethylpropylidene) amino] oxy} -1-met hylcyclohexyl ) amino ]acetyl} -4 -fluoro -2-pyrrolidinecar bonitrile

The title compound (60.5mg) was prepared from (2S, 4S) -l-chloroacetyl-4-fluoro-2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and 3-pentanone

O- ( 4 -amino-4 -methylcyclohexyl ) oxime obtained in Example

28-2 in a similar manner to that of Example 7-2.

^-NMR (300MHz, CDC1₃) : δ 1.07(3H, t, J=7.7Hz), 1.08(3H, t, J=7.5Hz), 1.09(3 X 4/5H, s), 1.13(3 X 1/5H, s), 1.33-1.48( 2H, m), 1.50 - 1.68 ( 4H , m), 1.78 - 1.96 ( 2H , m), 2.13-2.57 ( 1H, m), 2.19(2H, q, J=7.5Hz), 2.32(2H, q, J=7.7Hz), 2.69(1X4/5H, t, J=15.6Hz), 2.75(lXl/5H, t, J=15.9Hz), 3.27-4.13 ( 5H, m), 4.96(lX4/5H, d, J=9.4Hz), 5.15(1X1/ 5H, d, J=9.4Hz), 5.35(lXl/5H, br-d, J= 51.1Hz ) , 5.43(1X4/5H, br-t, J=3.3, 51.1Hz). MS (ES+) : m/e 367.32.

Example 29-1 tert-Butyl l-methyl-4-({[(lE) - 2 -pyridinylmethylidene ] amino }oxy ) cyclohexylcarbamate

The title compound (436mg) was prepared from tert-butyl ( trans - 4 -aminooxy- 1 -methylcyclohexyl ) - carbamate and 2 -pyridinecarboxaldehyde in a similar manner to that of Example 12-1.

Example 29-2 2 -Pyridinecarboxaldehyde O- ( 4 -amino- 4 -methylcyclohexyl ) oxime

The title compound is prepared from tert-butyl 1 -methyl -4-({[(lE)-2 -pyridinyl -methylidene ] amino }oxy ) cyclohexylcarbamate obtained in Example 29-1 in a similar manner to that of Example 17-2.

Example 29-3

(2S,4S)-l-{[(4-{[( 2 -pyridinylmethylidene) amino] oxy}-l -methylcyclohexyl ) amino ]acetyl}-4-fluoro -2-pyrrolidin ecarbonitrile

The title compound is prepared from (2S, 4S) -l-chloroacetyl-4-fluoro-2 -pyrrolidinecarbonit rile obtained in Preparation 1-8 and 2 -pyridinecarboxaldehyde O- ( 4 -amino- 4 -methylcyclohexyl ) oxime obtained in Example 29-2 in a similar manner to that of Example 7-2.

Example 30-1

Nicotinaldehyde O- ( 4 -amino- 4 -methylcyclohexyl ) oxime

The title compound (171mg) was prepared from tert-butyl l-methyl-4- ( { [ ( IE) - 3 -pyridinyl - methylidene ] amino }oxy) cyclohexylcarbamate in a similar manner to that of Example 17-2.

MS (ESI+) : m/z 234.19 (M+H).

Example 30-2 (2S,4S)-l-{[(4-{[( 3 -pyridinylmethylidene) amino] oxy}-l -methylcyclohexyl )amino]acetyl}-4-fluoro-2 -pyrrolidin ecarbonitrile

The title compound is prepared from (2S,4S)-l-chloroacetyl-4-fluoro-2-pyrrolidinecarbonit rile obtained in Preparation 1-8 and nicotinaldehyde O- ( 4 -amino- 4 -methyl-cyclohexyl ) oxime obtained in Example 30-1 in a similar manner to that of Example 7-2.

In order to illustrate the usefulness of the object Compound (I), the pharmacological test is carried out as shown in the following.

[A] Inhibition test of human plasma DPP-IV : (i) Material and Method :

The effect of test compounds on DPP-IV activity in human plasma was evaluated with a modified version of the assay described by Hughes et al (Biochemistry, 38, ppll597-11603(1999) ) .

Briefly, 20£tL of human plasma were mixed with 20 μ L of 80mM MgCl₂ in assay buffer ( 25mM HEPES , 140mM NaCI , 1% RIA-grade BSA, pH7.8), and were incubated in a room temperature for 60min. Then the reaction was initiated by the addition of both 20 . L of test compounds and 20 β L of 0.2mM substrate ( H-glycine-proline-AMC ; AMC is 7 -amino- 4 -methylcoumarine ) , they were dissolved in the assay buffer.

After 20min incubation in a room temperature (kept in the dark ) , fluorescence was measured (Excitation 380nm , Emission 460nm) . A fluorescence-concentration curve of free AMC was obtained using AMC solution in the assay buffer with appropriate concentration. Plasma DPP-IV activities, with or without the test compounds, were expressed as the amount of product per minute per mL . The potency of the test compounds as DPP-IV inhibitor was expressed as IC₅₀.

(ii) Results : The following IC₅₀ values were obtained. T ab l e 1

It appeared, from the above-mentioned Inhibition test, that the compound (I) and (1) or pharmaceutically acceptable salts thereof of the present invention have an inhibiting activity against DPP-IV.

Therefore, the compound (I) and (1) or pharmaceutically acceptable salts thereof are useful for treating or preventing disease mediated by DPP-IV, more particularly useful for treating or preventing altered glucose tolerance, glucosuria, hyperlipidemia, metabolic acidosis, diabetes mellitus (IDDM and NIDDM), diabetic neuropathy , nephropathy, and secondary diseases in mammals caused by diabetes mellitus .

Further, the compound ( I ) and ( 1 ) or pharmaceutically acceptable salts thereof are useful for treating or preventing autoimmune disease, arthritis, rejection of transplanted organs , systemic lupus erythematosus (SLE), acquired immunodeficiency syndrome (AIDS), hypertension, atherosclerosis, gallbladder disease, cancer, intestinal disease and dwarfism.

The patents, patent applications and publications cited herein are incorporated by reference.

This application is based on Australian Provisional Application No .2003902260 filed on May 9, 2003, the contents of which are hereby incorporated by references.

Claims

C L A I M S

1. A compound of the formula (I) or pharmaceutically acceptable salt thereof.

[wherein

X is CFH, or CF₂,

R^J is the moiety represented by the formula:

[wherein R² is ( lower ) alkyl ,

R³ is cycloalkyl, aryl -( lower ) alkyl (which may have 1 to 3 substituent ( s ) selected from the group described later on the aryl group), or heteroaryl- ( lower ) alkyl (which may have 1 to 3 subs ituent ( s ) selected from the group described later on the heteroaryl group)], or

the moiety represented by the formula:

[wherein R⁴ is ( lower ) alkyl ,

R⁵ is hydrogen, or ( lower ) alkyl ,

R⁶ is ( lower ) alkyl , cycloalkyl, aryl (which may have 1 to 3 substituent ( s ) selected from the group described later), or heteroaryl (which may have 1 to 3 substituent ( s ) selected from the group described later) , and

R⁵

R^b "; the partial structure: ' may form cycloalkylidene] , the "substituent(s)" is(are) selected from the group consisting of ( lower ) alkyl , halogenated- ( lower ) alkyl , ( lower ) alkoxy , aryloxy, halogen, cyano, nitro, amino and hydroxy ]

2. A compound of the formula (II) or pharmaceutically acceptable salt thereof.

[wherein

X is CFH, or CF₂,

R^? is ( lower ) alkyl ,

R³ is cycloalkyl, aryl- ( lower ) alkyl (which may have 1 to 3 substituent ( s ) selected from the group described later on the aryl group), or heteroaryl- ( lower ) alkyl (which may have 1 to 3 subs tituent ( s ) selected from the group described later on the heteroaryl group), the "substituent(s)" is(are) selected from the group consisting of ( lower ) alkyl , halogenated- ( lower ) alkyl , ( lower ) alkoxy , aryloxy, halogen, cyano, nitro, amino and hydroxy]

3. The compound of Claim 2, wherein X is CFH.

4. The compound of Claim 2 or 3, wherein R is methyl

5. The compound of any one of Claims 2 to 4, wherein R^; is pyridinylmethyl (which may have 1 to 3 substituent(s) ) .

6. A compound of the formula (III) or pharmaceutically acceptable salt thereof. R⁵

^H o ^CN (iii) [wherein

X is CFH, or CF₂,

R⁴ is ( lower ) alkyl ,

R⁵ is hydrogen, or ( lower ) alkyl ,

R⁶ is ( lower ) alkyl , cycloalkyl, aryl (which may have 1 to 3 substituent ( s ) selected from the group described later), or heteroaryl (which may have 1 to 3 substituent(s) selected from the group described later), and

R⁵

3 ^ the partial structure: >' may form cycloalkylidene, the "substituent(s)" is(are) selected from the group consisting of ( lower ) alkyl , halogenated- ( lower ) alkyl , ( lower ) alkoxy , aryloxy, halogen, cyano, nitro, amino and hydroxy]

7. The compound of Claim 6, wherein X is CFH.

8. The compound of Claim 6 or 7, wherein R⁴ is methyl.

9. The compound of any one of Claims 6 to 8, wherein R⁵ is hydrogen.

10. The compound of any one of Claims 6 to 8, wherein

R .5° is methyl.

11. The compound of any one of Claims 6 to 10, wherein R⁶ is methyl.

12. The compound of any one of Claims 6 to 10, wherein R⁶ is pyridinyl (which may have 1 to 3 substituent ( s )) .

13. The compound of any one of Claims 6 to 10, wherein R⁶ is pyrimidinyl (which may have 1 to 3 substituent ( s ) ) .

14. A medicament comprising a compound of any one of Claims 1 to 13 as an active ingredient.

15. A pharmaceutical composition comprising a compoun of any one of Claims 1 to 13 as an active ingredient, in association with a pharmaceutically acceptable carrier or excipient .

16. An inhibitor of DPP-IV consisting of a compound of any one of Claims 1 to 13.

17. A method for treatment and/or prevention of NIDDM which comprises administering an effective amount of the compound of any one of Claims 1 to 13 to human beings or animals .

18. The compound of any one of Claims 1 to 13 for use in the treatment and/or prevention of NIDDM in human beings or animals .

19. Use of the compound of any one of Claims 1 to 13 for the manufacture of a medicament for treatment and/or prevention of NIDDM in human beings or animals.

20. A commercial package comprising the pharmaceutical composition containing the compound (I) identified in any one of Claims 1 to 13 and a written matter associated therewith, wherein the written matter states that the compound (I) can or should be used for preventing or treating NIDDM.