HK1050197B - Dipeptide nitrile cathepsin k inhibitors - Google Patents

Description

Dipeptide nitrile cathepsin K inhibitors

The present invention relates to inhibitors of cysteine proteases, in particular to dipeptide nitrile cathepsin K inhibitors and their pharmaceutical use for the treatment or prevention of diseases or medical conditions associated with cathepsin K.

Cathepsin K is one of a family of lysosomal cysteine cathepsins, such as cathepsin B, K, L and S, which are associated with a variety of diseases including inflammation, rheumatoid arthritis, osteoarthritis, osteoporosis, tumors (particularly tumor invasion and tumor metastasis), coronary diseases, atherosclerosis (including atherosclerotic platelet rupture and destabilization), autoimmune diseases, respiratory diseases, infectious diseases and immunologically mediated diseases (including graft rejection).

Our co-pending international patent application WO 99/24460 describes dipeptide nitriles as cysteine cathepsin inhibitors and their use in the treatment of cysteine cathepsin dependent diseases or medical conditions. Novel dipeptide nitrile compounds have been prepared as inhibitors of cathepsin K, and said compounds also possess desirable properties for pharmaceutical applications.

According to the present invention there is provided a compound of formula I or a pharmaceutically acceptable salt or ester thereof

Wherein R is₁And R₂Independently of one another, H or C₁-C₇Lower alkyl, or R₁And R₂Together with the carbon atom to which they are attached form C₃-C₈A cycloalkyl ring, and Het is a substituted or unsubstituted nitrogen-containing heterocyclic substituent, with the proviso that Het is not 4-pyrrol-1-yl.

The Het substituent may be located at the 2-or 3-position of the phenyl ring, of course, preferably at the 4-position.

In the present invention, "nitrogen-containing heterocycle" refers to a heterocyclic ring system comprising at least 1 nitrogen atom, 2 to 10 carbon atoms, preferably 3 to 7 carbon atoms, most preferably 4 or 5 carbon atoms, and optionally comprising one or more other heteroatoms selected from O, S or preferably N.

Het may comprise an unsaturated heterocycle, such as an aromatic nitrogen-containing heterocycle; of course, saturated nitrogen-containing heterocycles are preferably included.

Particularly preferred saturated nitrogen-containing heterocycles are piperazinyl, preferably piperazin-1-yl, or piperidinyl, preferably piperidin-4-yl.

Het may be substituted with one or more substituents, for example, up to 5 substituents independently selected from halo, hydroxy, amino, nitro, substituted or unsubstituted C₁-C₄Alkyl (e.g., alkyl substituted with hydroxy, alkoxy, amino, substituted or unsubstituted alkyl)Amino, substituted or unsubstituted dialkylamino, aryl or heterocyclic), C₁-C₄An alkoxy group.

Preferably Het is substituted at the nitrogen atom, most preferably monosubstituted at the nitrogen atom.

Het preferably has the substituent C₁-C₇Lower alkyl, C₁-C₇Lower alkoxy-C₁-C₇Lower alkyl, C₅-C₁₀aryl-C₁-C₇Lower alkyl or C₃-C₈A cycloalkyl group.

R₁And R₂As C₁-C₇Lower alkyl is preferably the same, e.g. methyl, or R₁And R₂Together with the carbon atom to which they are attached form C₃-C₈Cycloalkyl rings, such as cyclopropyl rings. Most preferred is R₁And R₂Are all H.

Thus, in a particularly preferred embodiment of the present invention, there is provided a compound of formula II or a pharmaceutically acceptable salt or ester thereof

Wherein X is CH or N, and

r is H, C₁-C₇Lower alkyl, C₁-C₇Lower alkoxy-C₁-C₇Lower alkyl, C₅-C₁₀aryl-C₁-C₇Lower alkyl or C₃-C₈A cycloalkyl group.

Thus, R is C₁-C₇Specific examples of lower alkyl are methyl, ethyl, n-propyl or isopropyl.

R as C₁-C₇Lower alkoxy-C₁-C₇A specific example of lower alkyl is methoxyethyl.

R as C₅-C₁₀Aryl radical C₁-C₇A specific example of lower alkyl is benzyl.

R as C₃-C₈A specific example of a cycloalkyl group is cyclopentyl.

Examples of specific compounds of formula II are:

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (piperazin-1-yl) -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (4-methyl-piperazin-1-yl) benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (4-ethyl-piperazin-1-yl) -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- [4- (1-propyl) -piperazin-1-yl ] -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (4-isopropyl-piperazin-1-yl) -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (4-benzyl-piperazin-1-yl) -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- [4- (2-methoxy-ethyl) -piperazin-1-yl ] -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (1-propyl-piperidin-4-yl) -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- [1- (2-methoxy-ethyl) -piperidin-4-yl ] -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (1-isopropyl-piperidin-4-yl) -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (1-cyclopentyl-piperidin-4-yl) -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (1-methyl-piperidin-4-yl) -benzamide, and

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (piperidin-4-yl) -benzamide.

The compounds of formulae I and II and the specific compounds above are hereinafter referred to as the compounds of the invention.

The compounds of the invention can be prepared by coupling the corresponding Het-substituted benzoic acid derivative with 1-amino-cyclohexanecarboxylic acid cyanomethylamide. For example, a benzoic acid derivative, preferably in its hydrochloride form, is mixed with 1-amino-cyclohexanecarboxylic acid cyanomethylamide, for example, in the presence of HOBT (1-hydroxybenzotriazole), WSCD and triethylamine in solution, for example in DMF, and stirred, for example at room temperature overnight. The product can be recovered, for example, by evaporating the solvent and washing with aqueous sodium carbonate, preferably under mildly alkaline conditions, followed by solvent extraction, for example with ethyl acetate, drying of the extract, for example with sodium sulfate, evaporation of the solvent and filtration. Other processes and reagents may be employed; such as the procedures and reagents described in the examples below.

Thus, in a further aspect, the present invention provides a process for the preparation of a compound of formula I, which process comprises reacting a corresponding Het-substituted benzoic acid derivative of formula III

Coupling with 1-amino-cyclohexanecarboxylic acid cyanomethyl-amide

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide can be prepared by: 1-amino-cyclohexanecarboxylic acid, usually in a suitable amino-protected form, e.g. FMOC-1-oxy-cyclohexanecarboxylic acid, is subjected to a coupling reaction with 2-aminoacetonitrile. For example, FMOC-1-amino-cyclohexanecarboxylic acid, e.g. with HOBT and WSCD, is added to a solution of 2-aminoacetonitrile and triethylamine in DMF and the mixture is stirred overnight at 25 ℃. 1-amino-cyclohexanecarboxylic acid cyanomethyl-amide was recovered as described in the examples. FMOC-1-amino-cyclohexanecarboxylic acid can be prepared as described in the examples.

The compounds of the invention may be obtained in free form or in the form of their salts (when salt-forming groups are present).

The compounds of the invention having basic groups may be converted into acid addition salts, in particular pharmaceutically acceptable salts. These salts are formed, for example, with inorganic acids, such as mineral acids, for example sulfuric acid, phosphoric acid or hydrochloric acid, or with organic carboxylic acids, such as (C)₁-C₄) Alkanecarboxylic acids which are unsubstituted or substituted by halogen, for example acetic acid, such as saturated or unsaturated dicarboxylic acids, for example oxalic acid, succinic acid, maleic acid or fumaric acid, such as hydroxycarboxylic acids, for example glycolic acid, lactic acid, malic acid, tartaric acid or citric acid, such as amino acids, for example aspartic acid or glutamic acid, or with organic sulfonic acids, such as (C)₁-C₄) Alkanesulfonic acids (e.g. methanesulfonic acid) or unsubstituted or substituted (e.g. by halogen) arylsulfonic acids. Salts with hydrochloric acid, methanesulfonic acid and maleic acid are preferred.

In view of the close relationship between the free compound and the compound in its salt form, the compounds referred to herein also include their corresponding salts, as long as such salts are possible or suitable in particular circumstances.

The compounds (including salts thereof) may also be obtained in the form of their hydrates or include other solvents used in their crystallization processes.

The compounds of the present invention exhibit valuable pharmacological properties in mammals and are particularly useful as inhibitors of cathepsin K.

The cathepsin K inhibition of the compounds of the invention can be demonstrated by measuring the inhibition of recombinant human cathepsin K in vitro.

The in vitro test was performed as follows:

for cathepsin K:

the assay was performed in 96-well microtiter plates at room temperature using recombinant human cathepsin K. Cathepsin K inhibition was measured at constant enzyme (0.16nM) and substrate concentration (54mM Z-Phe-Arg-AMCA, Peptide Institute inc., osaka, japan), in 100mM sodium phosphate buffer, pH 7.0, containing 2mM dithiothreitol, 20mM tween 80 and 1mM edta. Cathepsin K was pre-incubated with the inhibitor for 30 min and the reaction was initiated by addition of substrate. After incubation for 30 minutes, the reaction was stopped by addition of E-64(2mM) and the fluorescence intensity was read on a multiwell plate reader at excitation and emission wavelengths of 360 and 460nm, respectively. The compounds of the invention generally have a K value for cathepsin K of less than about 50nM, preferably about 5nM or less, e.g. about 1 nM.

In view of their activity as cathepsin K inhibitors, the compounds of the invention are particularly useful as therapeutics for the treatment and prevention of diseases and medical conditions associated with elevated levels of cathepsin K. These diseases include diseases associated with infection by organisms such as pneumocystis carinii, trypanosoma cruzi, trypanosoma brucei, brachymyces molitorum (crithidia fusiculata) and parasitic diseases such as schistosomiasis and malaria, tumors (tumor invasion and tumor metastasis), and other diseases such as metachromatic leukodystrophy, muscular dystrophy, muscular atrophy and the like.

Cathepsin K is associated with diseases of excessive bone loss and, therefore, the compounds of the invention are useful in the treatment and prevention of such diseases, including osteoporosis, gum diseases such as gingivitis and periodontitis, paget's disease, hypercalcemia of malignancy, e.g. hypercalcemia due to tumors and metabolic bone disease. The compounds of the invention are also useful in the treatment or prevention of diseases of excessive cartilage or matrix degradation, including osteoarthritis and rheumatoid arthritis as well as certain neoplastic diseases associated with high levels of proteolytic enzymes and matrix degradation.

The compounds of the invention are also shown to be useful in the prevention or treatment of coronary disease, atherosclerosis (including atherosclerotic platelet rupture and destabilization), autoimmune disease, respiratory disease and immunologically-mediated disease (including transplant rejection).

The compounds of the invention are particularly useful in the prevention or treatment of osteoporosis of various genesis (e.g. juvenile, menopausal, postmenopausal, post-traumatic, geriatric or corticosteroid treatment or inactivity).

The effective effect was evaluated using in vitro and in vivo pharmacological tests generally known in the art, as described below.

The above properties may be demonstrated in vitro and in vivo experiments using advantageously mammals, such as mice, rats, dogs, rabbits, monkeys or isolated organs and tissues, and mammalian enzyme preparations, either native or prepared using recombinant techniques. The compounds of the invention may be used in vitro assays in the form of solutions, e.g. preferably in aqueous solution or suspension, and or in vivo assays in enteral or parenteral, e.g. oral, e.g. in suspension or aqueous solution, or in the form of solid capsules or tablets. In vitro reagent range of about 10^-5Molar sum of 10^-9Molar concentration. The in vivo dose ranges are about 0.1 and 100mg/kg depending on the mode of administration.

According to the present invention, it was found that the compounds of the present invention have excellent bioavailability, in particular excellent oral bioavailability. Thus, for example, selected compounds of the invention have an absolute oral bioavailability of 50% or greater, e.g., about 80% or greater.

The anti-arthritic efficacy of the compounds of the invention in the treatment of rheumatoid arthritis can be measured using the rat model or similar models described previously in the literature (r.e. esser et al, j.rhematology, 1993, 20, 1176.).

The efficacy of the compounds of the invention in the treatment of osteoarthritis can be measured using the rabbit partial meniscectomy model or similar model described previously: colombo et al, Arth. Rheum.199326, 875-886). The efficacy of the compounds of the invention in this model can be quantified using histological scoring, as described previously in O' Byrne et al, Inflamm Res 1995, 44, S117-S118).

The efficacy of a compound of the invention in treating osteoporosis can be measured using animal models such as ovariectomized rats or other similar species such as rabbits or monkeys, wherein the test compound is administered to the animal and the presence of a marker for bone resorption is measured in urine or serum (e.g., Osteporos Int (1997) 7: 539-.

Accordingly, another aspect of the present invention provides:

the use of a compound of the invention as a medicament;

a pharmaceutical composition comprising a compound of the present invention as an active ingredient;

a method of treating a patient suffering from or susceptible to a disease or medical condition associated with cathepsin K, comprising administering to the patient a therapeutically effective amount of a compound of the invention;

the use of a compound of the invention in the manufacture of a medicament for the treatment or prevention of a disease or medical condition associated with cathepsin K.

The invention also relates to methods of inhibiting cathepsin K in mammals using the compounds of the invention and pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof, for treating cathepsin K dependent diseases, such as inflammatory diseases, osteoporosis, rheumatoid arthritis and osteoarthritis.

In particular, the present invention relates to a method for selectively inhibiting cathepsin K in a mammal, the method comprising administering to a mammal in need thereof an effective cathepsin K inhibiting amount of a compound of the invention.

More particularly, this relates to the treatment of osteoporosis, rheumatoid arthritis, osteoarthritis and inflammation (and other conditions as previously identified) in a mammal, comprising administering to a mammal in need thereof an effective amount of a compound of the present invention.

The following examples are intended to illustrate the invention and are not intended to be limiting. The temperatures are given in degrees Celsius. All evaporation is carried out under reduced pressure, preferably about 15-100mmHg (20-133 mbar), unless otherwise indicated. The structures of the final products, intermediates and starting materials were determined using standard analytical methods, such as microanalysis and spectroscopic properties (e.g. MS, IR, NMR). The abbreviations used are those conventionally used in the art.

Examples

Synthesis of 1-amino-cyclohexanecarboxylic acid cyanomethyl-amides

FMOC-1-aminocyclohexanecarboxylic acid

The title compound was prepared according to conventional method from 1-aminocyclohexanecarboxylic acid (700mmol), FMOC-chloride (770mmol), diisopropyl-ethylamine (770mmol) and 770ml sodium hydroxide 1N in 950ml dioxane using conventional method. Mp.180-182 ℃; rf 0.21 (dichloromethane/methanol 95: 5).

Acetonitrile may be used as a solvent instead of dioxane.

FMOC-1-amino-cyclohexanecarboxylic acid cyanomethyl-amide

2-aminoacetonitrile hydrochloride (564mmol) and triethylamine (564mmol) were dissolved in DMF (1700 ml). FMOC-1-aminocyclohexanecarboxylic acid (564mmol), HOBt (564mmol) and WSCD (564mmol) were added and the mixture was stirred at 25 ℃ overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring slightly alkaline conditions) and extracted three times with ethyl acetate. The extracts were washed with water, 10% citric acid, brine, sodium bicarbonate, brine, dried over magnesium carbonate and evaporated. The residue was suspended in diethyl ether, the solid filtered off and dried (vacuum). A white powder was obtained, mp.167-169 ℃, Rf 0.27 (n-hexane: ethyl acetate 1: 1).

Alternatively, THF can be used as the solvent, 1-chloro-3, 5-dimethoxytriazine (CDMT) can be used as the activating agent, together with N-methylmorpholine (NMM) for the coupling reaction; in this case, the product can be recovered by the following process: isopropyl acetate and water were added, the organic phase was separated, followed by washing with brine, partial evaporation of the solvent, and recovery of the crystallized product by filtration and drying.

C.1-amino-cyclohexanecarboxylic acid cyanomethyl-amides

FMOC-1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (248mmol) was dissolved in DMF (200ml), piperidine (248mmol) was added and the mixture was stirred at room temperature for 2 h. The reaction mixture was poured into water (3000ml) and stirred for an additional 30 minutes. The suspension was filtered, and the filtrate was acidified with 4N hydrochloric acid and extracted with ethyl acetate. 1N sodium hydroxide was added to make the aqueous phase alkaline, and the mixture was extracted three times with ethyl acetate. The organic fraction was dried over sodium sulfate and the solvent was evaporated. The residue was dried (vacuum) to give a pale yellow oil. Rf 0.26 (dichloromethane/methanol 95: 5).

1H-NMR (d 6-DMSO): 1.05-1.80(m, 10H); 4.0(br.s, 2H); NH very wide signal.

Alternatively, THF can be used in place of DMF and diethylamine in place of piperidine in the FMOC deprotection step.

Example 1: synthesis of N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4-piperazin-1-yl-benzamide

A.4-piperazin-1-yl-benzoic acid methyl ester

1- (4-cyanophenyl) -piperazine (11mmol) was dissolved in a mixture of 15ml concentrated sulfonic acid and methanol (5N)To the mixture, and stirred in a sealed tube at 110 ℃ for 3 hours. After the solvent was distilled off, the residue was dissolved in water and extracted with ethyl acetate. Sodium carbonate was added to the aqueous phase until pH 9 to give a white solid precipitate which was filtered and dried (vacuum). A white powder was obtained, Rf ═ 0.59 (dichloromethane/methanol (+ NH)₃ 3N)＝9∶1)。

B.4-piperazin-1-yl-benzoic acid hydrochloride

4-piperazin-1-yl-benzoic acid methyl ester (17mmol) was dissolved in 6N hydrochloric acid (25ml) and heated under reflux for 3 hours. The mixture was cooled to 0-4 ℃ in an ice bath, and the solid formed was filtered off, washed with acetone and dried (vacuum). A white powder was obtained, mp. > 240 ℃.

C.4- (4-FMOC-piperazin-1-yl) -benzoic acid

4-piperazin-1-yl-benzoic acid hydrochloride (10.5mmol) was dissolved in 15ml dioxane and 11.6ml sodium hydroxide (2N) and cooled to 0 ℃. At the same time, FMOC-chloride (11.6mmol) in dioxane (5ml) and diisopropyl-ethylamine (11.6mmol) in dioxane (5ml) were added dropwise over 20 minutes at 0 ℃, the mixture was stirred for 15 minutes, then allowed to warm to room temperature and stirred overnight. The mixture was diluted with water (50ml) and extracted 2 times with ether. The aqueous phase is acidified with hydrochloric acid (4N) at 0-4 ℃ and the solid material formed is filtered off, washed with water and dried (vacuum). A white powder was obtained with Rf 0.2 (dichloromethane/methanol 95: 5).

D.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (4-FMOC-piperazin-1-yl) -benzamide

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (8.3mmol), 4- (4-FMOC-piperazin-1-yl) -benzoic acid (8.3mmol), HOBT (8.3mmol) and WSCD (8.3mmol) were dissolved in DMF (20ml) and stirred at room temperature overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring slightly alkaline conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel using (ethyl acetate/hexane 4: 1) as mobile phase. The product containing fractions were combined and evaporated. The residue was suspended in diethyl ether and the solid was filtered off and dried (vacuum). A white powder was obtained, mp.192-194 ℃, Rf 0.26 (dichloromethane/methanol 95: 5).

E.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (piperazin-1-yl) -benzamide

Reacting N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (4-FMOC-piperazin-1-yl) -benzamide (4.4mmol) was dissolved in DMF (15ml), piperidine (4.4mmol) was added and the mixture was stirred at room temperature for 4 h. 4 drops of piperidine were added and the mixture was stirred overnight. The reaction mixture was poured into water and ethyl acetate, the suspension was filtered, and the filtrate was acidified with 4N hydrochloric acid and then extracted with ethyl acetate. Saturated sodium carbonate solution was added to make the aqueous phase alkaline, and the mixture was extracted three times with ethyl acetate. The aqueous phase was saturated with sodium chloride and extracted three times with ethyl acetate. The organic fraction was dried over sodium sulfate and the solvent was evaporated. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol (3 NNH)₃) 95: 5 as mobile phase. The product containing fractions were combined and evaporated. The residue was suspended in diethyl ether, the solid filtered off and dried (vacuum). A white powder was obtained, mp.206-210 ℃, Rf ═ 0.28 (dichloromethane/methanol (3N NH)₃)＝9∶1)。

1H-NMR(d6-DMSO)：1.15-1.35(m，1H)；1.4-1.6(m，5H)；1.65-1.8(m，2H)；2.05-2.15(m，2H)；2.8(m，4H)；3.15(m，4H)；4.0(d，2H)，6.95(d，2H)；7.65(s，1H)；7.75(d，2H)，8.15(m，1H)。

Example 2: synthesis of N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (4-methyl-piperazin-1-yl) - Benzamide derivatives

A.4- (4-methyl-piperazin-1-yl) -benzoic acid methyl ester

Methyl 4-fluorobenzoate (34mmol), 1-methyl-piperazine (75mmol) and potassium carbonate (34mmol) were suspended in acetonitrile (30ml) and stirred at reflux for three days. After the solvent was distilled off, the residue was dissolved in water and extracted three times with ethyl acetate. The extract was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel using (dichloromethane/methanol 95: 5) as mobile phase. The product containing fractions were combined and evaporated. The residue was suspended in ether/pentane and the solid was filtered off and dried (vacuum). A pale yellow powder was obtained, mp.117-119 ℃, Rf 0.20 (dichloromethane/methanol 95: 5).

B.4- (4-methyl-piperazin-1-yl) -benzoic acid hydrochloride

4- (4-methyl-piperazin-1-yl) -benzoic acid methyl ester (8.5mmol) was dissolved in 4N hydrochloric acid (15ml) and heated under reflux for 8 hours. The mixture is cooled to 0-4 ℃ in an ice bath, diluted with 5ml of acetone, and the solid material formed is filtered off, washed with acetone and dried (vacuum). A white powder was obtained with mp. > 270 ℃ and Rf 0.11 (dichloromethane/methanol 9: 1).

C.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (4-methyl-piperazin-1-yl) benzamide

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (1.38mmol), 4- (4-methyl-piperazin-1-yl) -benzoic acid hydrochloride (1.38mmol), HOBT (1.38mmol), WSCD (1.38mmol) and triethylamine (1.38mmol) were dissolved in DMF (5ml) and stirred at room temperature overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring slightly alkaline conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was suspended in diethyl ether and the solid was filtered off and dried (vacuum). A light-colored powder was obtained, mp.218-220 ℃, Rf 0.19 (dichloromethane/methanol 9: 1).

1H-NMR(d6-DMSO)：1.15-1.35(m，1H)；1.4-1.6(m，5H)；1.65-1.8(m，2H)；2.05-2.15(m，2H)；2.2(s，3H)；2.4(m，4H)；3.2(m，4H)；4.0(d，2H)，6.95(d，2H)；7.65(s，1H)；7.75(d，2H)，8.15(m，1H)。

Examples3: synthesis of N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (4-ethyl-piperazin-1-yl) - Benzamide derivatives

A.4- (4-Ethyl-piperazin-1-yl) -benzoic acid methyl ester

Methyl 4-fluorobenzoate (53mmol), 1-ethyl-piperazine (44mmol) and potassium carbonate (44mmol) were suspended in dimethylacetamide (50ml) and stirred under reflux overnight. After the solvent was distilled off, the residue was dissolved in water and extracted three times with ethyl acetate. The extract was dried over sodium sulfate and evaporated. The residue was suspended in ether/pentane and the solid was filtered off and dried (vacuum). A brownish powder was obtained, mp.102-104 ℃, Rf 0.22 (dichloromethane/methanol 95: 5).

B.4- (4-Ethyl-piperazin-1-yl) -benzoic acid hydrochloride

4- (4-Ethyl-piperazin-1-yl) -benzoic acid methyl ester (15mmol) was dissolved in 4N hydrochloric acid (35ml) and heated under reflux for 8 hours. The mixture was cooled to 0-4 ℃ in an ice bath and the solid material formed was filtered off, washed with acetone and dried (vacuum). A grey powder was obtained, mp. > 270 ℃, Rf 0.08 (dichloromethane/methanol 9: 1).

C.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (4-ethyl-piperazin-1-yl) -benzamide

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (0.9mmol), 4- (4-ethyl-piperazin-1-yl) -benzoic acid hydrochloride (0.9mmol), HOBT (0.9mmol), WSCD (0.9mmol) and triethylamine (0.9mmol) were dissolved in DMF (5ml) and stirred at room temperature overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring slightly alkaline conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol (3N NH)₃) 93: 7 as the mobile phase. The product containing fractions were combined and evaporated. The residue was suspended in diethyl ether and the solid was filtered off and dried (vacuum). A white powder was obtained.

1H-NMR(d6-DMSO)：1.0(t，3H)，1.15-1.35(m，1H)；1.4-1.6(m，5H)；1.65-1.8(m，2H)；2.05-2.15(m，2H)；2.35(q，2H)；2.45(m，4H)；3.2(m，4H)；4.0(d，2H)，6.95(d，2H)；7.65(s，1H)；7.75(d，2H)，8.15(m，1H)。

Example 4: synthesis of N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- [4- (1-propyl-piperazine-1-) Phenyl) -benzamides

4- [4- (1-propyl) -piperazin-1-yl]-benzoic acid methyl ester

Methyl 4-fluorobenzoate (165mmol), 1- (1-propyl) -piperazine dihydrobromide (138mmol) and potassium carbonate (690mmol) were suspended in dimethylacetamide (320ml) and stirred under reflux overnight. After the solvent was distilled off, the residue was dissolved in water and extracted three times with ethyl acetate. The extract was dried over sodium sulfate and evaporated. The residue was suspended in ether/pentane and the solid was filtered off and dried (vacuum). A brownish powder was obtained, mp.99-101 ℃, Rf 0.23 (dichloromethane/methanol 95: 5).

In the above process, Cs₂CO₃Can replace K₂CO₃。

4- [4- (1-propyl) -piperazin-1-yl]-benzoic acid hydrochloride

4- [4- (1-propyl) -piperazin-1-yl ] -benzoic acid methyl ester (38mmol) was dissolved in 4N hydrochloric acid (60ml) and heated under reflux for 7 hours. The mixture was cooled to 0-4 ℃ in an ice bath and the solid material formed was filtered off, washed with cold water and dried (vacuum). A light-colored powder was obtained, mp. > 270 ℃, Rf 0.19 (dichloromethane/methanol 9: 1).

Alternatively, the 4- [4- (1-propyl) -piperazin-1-yl ] -benzoic acid product may be produced as an internal salt with acetic acid. For example, 4- [4- (1-propyl) piperazin-1-yl ] -benzoic acid methyl ester is suspended in water/methanol at 70 ℃ and hydrolyzed by addition of 1 equivalent of sodium hydroxide; the solution was clarified filtered, and the product precipitated by addition of 1 eq acetic acid, filtered and dried.

C.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- [4- (1-propyl) -piperazin-1-yl]-benzamides

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (22mmol), 4- [4- (1-propyl) -piperazin-1-yl ] -benzoic acid hydrochloride (22mmol), HOBT (22mmol), WSCD (22mmol) and triethylamine (22mmol) were dissolved in DMF (50ml) and stirred at room temperature overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring slightly alkaline conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel using (dichloromethane/methanol-9: 1) as mobile phase. The product containing fractions were combined and evaporated. The residue was suspended in diethyl ether and the solid was filtered off and dried (vacuum). A white powder was obtained, mp.216-218 ℃, Rf 0.34 (dichloromethane/methanol 9: 1).

1H-NMR(d6-DMSO)：0.85(t，3H)，1.2-1.3(m，1H)；1.4-1.6(m，7H)；1.65-1.8(m，2H)；2.05-2.15(m，2H)；2.25(t，2H)；2.45(m，4H)；3.2(m，4H)；4.0(d，2H)，6.95(d，2H)；7.65(s，1H)；7.75(d，2H)，8.15(m，1H)。

In another procedure, the acetic acid inner salt of 4- [4- (1-propyl) -piperazin-1-yl ] -benzoic acid was treated with HOBt, NMM and Diisopropylcarbodiimide (DICI) in acetonitrile, and after stirring for 1 hour at 40 ℃, a solution of 1-amino-cyclohexanecarboxylic acid cyanomethyl-amide in acetonitrile was added. After completion of the reaction, the product was precipitated by adding water to the reaction mixture, filtered, boiled with ethanol and dried to obtain the final product.

Example 5: synthesis of N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (4-isopropyl-piperazine-1-) Phenyl) -benzamides

A.4- [ 4-isopropyl-piperazin-1-yl]-benzoic acid methyl ester

In an oxygen-free atmosphere (N)₂) Tris- (dibenzylidene-acetone) -dipalladium (0.05mmol), (2' -dicyclohexylphosphinyl biphenyl-2-yl) -dimethyl-amine (0.1mmol) and potassium carbonate (4.6mmol) were suspended in 1, 2-dimethoxyethane (10 ml). 4-bromo-benzoic acid methyl ester (3.3mmol) and 1-isopropyl-piperazine (3.9mmol) were added and the stirred mixture was heated under reflux for 28 h. After cooling, the solvent was evaporated and water was added to the residue, which was then extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel using (dichloromethane/methanol 95: 5) as mobile phase. The product containing fractions were combined and evaporated. The residue was suspended in ether/pentane and the solid was filtered off and dried (vacuum). A light brown powder was obtained with Rf 0.23 (dichloromethane/methanol 95: 5).

B.4- (4-isopropyl-piperazin-1-yl) -benzoic acid hydrochloride

4- (4-isopropyl-piperazin-1-yl) -benzoic acid methyl ester (0.9mmol) was dissolved in 4N hydrochloric acid (2ml) and heated under reflux for 7 hours. The mixture was cooled to 0-4 ℃ in an ice bath and acetone was added. The solid material formed is filtered off, washed with cold acetone and dried (vacuum). A light brown powder was obtained, mp. > 270 ℃, Rf 0.08 (dichloromethane/methanol 9: 1).

C.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (4-isopropyl-piperazin-1-yl) -benzamide

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (0.6mmoL), 4- (4-isopropyl-piperazin-1-yl) -benzoic acid hydrochloride (0.6mmoL), HOBT (0.6mmoL), WSCD (0.6mmoL) and triethylamine (0.6mmoL) were dissolved in DMF (2ml) and stirred at room temperature overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring slightly alkaline conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was suspended in ethyl acetate/diethyl ether and the solid was filtered off and dried (vacuum). A white powder was obtained, mp.218-220 ℃, Rf 0.28 (dichloromethane/methanol 9: 1).

1H-NMR(d6-DMSO)：1.0(d，6H)，1.2-1.3(m，1H)；1.4-1.6(m，5H)；1.65-1.8(m，2H)；2.05-2.15(m，2H)；2.45(m，4H)；2.65(m，1H)；3.2(m，4H)；4.0(d，2H)，6.95(d，2H)；7.65(s，1H)；7.75(d，2H)，8.15(m，1H)。

Example 6: synthesis of N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (4-benzyl-piperazin-1-yl) - Benzamide derivatives

A.4- (4-benzyl-piperazin-1-yl) -benzoic acid methyl ester

In an oxygen-free atmosphere (N)₂) Tris- (dibenzylidene-acetone) -dipalladium (0.03mmol), (2' -dicyclohexylphosphinyl biphenyl-2-yl) -dimethyl-amine (0.9mmol) and NaOtBu (6.5mmol) were suspended in toluene (20 ml). 4-bromo-benzoic acid methyl ester (4.65mmol) and 1- (benzyl) -piperazine (5.6mmol) were added and the stirred mixture was heated under reflux for 4 h. After cooling, ethyl acetate and diethyl ether were added and the mixture was filtered. The solvent was then evaporated and the residue was suspended in diethyl ether, the solid was filtered off and dried (vacuum). A light-colored powder was obtained, mp.105-107 ℃, Rf 0.67 (dichloromethane/methanol 95: 5).

B.4- (4-benzyl-piperazin-1-yl) -benzoic acid hydrochloride

4- (4-benzyl-piperazin-1-yl) -benzoic acid methyl ester (0.84mmol) was dissolved in 4N hydrochloric acid (2ml) and heated under reflux for 8 h. The mixture was cooled to 0-4 ℃ in an ice bath and the solid material formed was filtered off, washed with cold acetone and dried (vacuum). A grey powder was obtained with mp. > 270 ℃ and Rf 0.18 (dichloromethane/methanol 95: 5).

C.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (4-benzyl-piperazin-1-yl) -benzamide

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (0.84mmol), 4- [4- (2-propyl) -piperazin-1-yl ] -benzoic acid hydrochloride (0.84mmol), HOBT (0.84mmol), WSCD (0.84mmol) and triethylamine (0.84mmol) were dissolved in DMF (2ml) and stirred at room temperature overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring slightly alkaline conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was suspended in methanol, the solid filtered off and dried (vacuum). A light-colored powder was obtained, mp.210-212 ℃, Rf 0.20 (dichloromethane/methanol 95: 5).

1H-NMR(d6-DMSO)：1.15-1.35(m，1H)；1.4-1.6(m，5H)；1.65-1.8(m，2H)；2.05-2.15(m，2H)；2.45(m，4H)；3.2(m，4H)；3.5(s，2H)；4.0(d，2H)，6.9(d，2H)；7.2-7.4(m，5H)，7.65(s，1H)；7.75(d，2H)，8.15(m，1H)。

Example 7: synthesis of N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- [4- (2-methoxy-ethyl-) Piperazin-1-yl]-benzamides

A.4- (4-benzyl-piperazin-1-yl) -benzoic acid methyl ester

Methyl 4-fluorobenzoate (200mmol), 1-benzyl-piperazine (300mmol) and potassium carbonate (300mmol) were suspended in acetonitrile (400ml) and stirred at reflux for 6 days. After the solvent was distilled off, the residue was dissolved in water and extracted three times with diethyl ether. The extract was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel using (dichloromethane first and then dichloromethane/methanol 15: 1) as mobile phase. The product containing fractions were combined and evaporated. The residue was suspended in ethanone/pentane and the solid was filtered off and dried (vacuum). A powder was obtained, mp.105-107 ℃.

B.4- (piperazin-1-yl) -benzoic acid methyl ester

4- (4-benzyl-piperazin-1-yl) -benzoic acid methyl ester (19.4mmol) was dissolved in methanol (150ml) and Pd on charcoal (0.6g) was added. The mixture was stirred under hydrogen atmosphere until consumption ceased. The catalyst was filtered off and the filtrate was evaporated. The residue was suspended in ether/pentane and the solid was filtered off and dried (vacuum). A powder was obtained, mp.95-97 ℃.

[4- (2-methoxy-ethyl) -piperazin-1-yl]-benzoic acid methyl ester

4- (piperazin-1-yl) -benzoic acid methyl ester (19mmol), 2-bromoethyl methyl ether (21mmol) and potassium carbonate (22.8mmol) were suspended in acetonitrile (50ml) and stirred at 80 ℃ for 8 h. After the solvent was distilled off, the residue was dissolved in water and extracted three times with dichloromethane. The extract was dried over sodium sulfate and evaporated. The residue was suspended in ether/pentane and the solid was filtered off and dried (vacuum). A powder was obtained, mp.103-105 ℃.

[4- (2-methoxy-ethyl) -piperazin-1-yl]-benzoic acid hydrochloride

[4- (2-methoxy-ethyl) -piperazin-1-yl ] -benzoic acid methyl ester (17mmol) was dissolved in 4N hydrochloric acid (70ml) and heated under reflux for 5 hours. After cooling, the solvent was evaporated, the residue was suspended in ethanol, the solid was filtered off, washed with diethyl ether and dried (vacuum). A powder was obtained with mp. > 270 ℃ and Rf 0.35 (dichloromethane/methanol 9: 1).

E.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- [4- (2-methoxy-ethyl) piperazin-1-yl]-benzene Carboxamides

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (1.0mmol), [4- (2-methoxyethyl) -piperazin-1-yl ] -benzoic acid hydrochloride (1.0mmol), HOBT (1.0mmol), WSCD (1.0mmol) and triethylamine (1.0mmol) were dissolved in DMF (4ml) and stirred at room temperature overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring slightly alkaline conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol 92.5: 7.5 as mobile phase. The product containing fractions were combined and evaporated. The residue was suspended in diethyl ether, the solid filtered off and dried (vacuum). A light-colored powder was obtained, mp.166-168 ℃, Rf 0.37 (dichloromethane/methanol 9: 1).

1H-NMR(d6-DMSO)：1.15-1.35(m，1H)；1.4-1.6(m，5H)；1.65-1.8(m，2H)；2.05-2.15(m，2H)；2.45(m，6H)；3.2(m，7H)；3.45(t，2H)；4.0(d，2H)，6.95(d，2H)；7.65(s，1H)；7.75(d，2H)，8.15(m，1H)。

Example 8: synthesis of N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (1-propyl-piperidin-4-yl) - Benzamide derivatives

A.1- (4-phenyl-piperidin-1-yl) -ethanones

4-phenylpiperidine (87mmol) and pyridine (96mmol) were dissolved in anhydrous dichloromethane (100ml) and a solution of acetyl chloride (96mmol) in dichloromethane (40ml) was added dropwise to the stirred solution at 10 ℃. The reaction was stirred at room temperature for 1 hour. The mixture was extracted three times with water and the aqueous phase was extracted with dichloromethane. The combined organic phases are dried over sodium sulfate and evaporated. A light brown oil was obtained with Rf 0.13 (ethyl acetate/hexane 1: 1).

B.4-piperidin-4-yl-benzoic acid

1- (4-phenyl-piperidin-1-yl) -ethanone (84mmol) was dissolved in dichloromethane (250rnl) and oxalyl chloride (336mmol) was added dropwise at-20 to-10 ℃. After complete addition of oxalyl chloride, aluminium trichloride (260mmol) was added in portions at-10 ℃. The mixture was stirred at-10 ℃ for 3 hours. The cooling bath was removed and the mixture was stirred at room temperature overnight. The mixture was poured into ice/water (600ml) and extracted three times with dichloromethane. The combined organic phases are washed with water, dried over sodium sulfate and evaporated. The residue was dissolved in aqueous sodium hydroxide (2N, 250ml) and 6N hydrochloric acid was added at 0 ℃ to acidify the solution. The precipitate formed is filtered off and washed with water. The solid material was suspended in 6N hydrochloric acid (300ml), and the mixture was heated under reflux for 18 hours. After cooling to room temperature, the solvent was removed and resuspended in ethanol. The solid material was filtered off and dried. A brown powder was obtained, mp. > 270 ℃.

C.4-piperidin-4-yl-benzoic acid methyl ester

4-piperidin-4-yl-benzoic acid (47mmol) was dissolved in methanol (300ml) and 1ml of concentrated sulfuric acid was added. The mixture was heated to reflux overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring basic conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. A brown powder was obtained with Rf 0.18 (dichloromethane/methanol 9: 1).

4- (1-propyl-piperidin-4-yl) -benzoic acid methyl ester

4-piperidin-4-yl-benzoic acid methyl ester (28mmol), ethyldiisopropylamine (31mol) and 1-iodopropane (42mmol) were dissolved in 1, 2-dimethoxyethane (100ml), and the mixture was heated at 70 ℃ overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring basic conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol 9: 1 as mobile phase. The product containing fractions were combined and evaporated. The residue was suspended in diethyl ether, the solid filtered off and dried (vacuum). A light brown powder was obtained with Rf 0.35 (dichloromethane/methanol 9: 1).

E.4- [ 1-propyl-piperidin-4-yl) -benzoic acid hydrochloride

4- (1-propyl-piperidin-4-yl) -benzoic acid methyl ester (32mmol) was dissolved in 4N hydrochloric acid (45ml) and heated under reflux for 7 hours. The mixture was cooled to 0-c in an ice bath and the solid material formed was filtered off, washed with cold acetone and dried (vacuum). A brown powder was obtained with mp. > 270 ℃ and Rf 0.08 (dichloromethane/methanol 9: 1).

F.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (1-propyl-piperidin-4-yl) -benzamide

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (23mmol), 4- (1-propyl-piperidin-4-yl) -benzoic acid hydrochloride (23mmol), HOBT (23mmol), WSCD (23mmol) and triethylamine (23mmol) were dissolved in DMF (50mol) and stirred at room temperature overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring basic conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was suspended in ether/pentane and the solid was filtered off and dried (vacuum). A light-colored powder was obtained, mp.198-200 ℃, Rf 0.34 (dichloromethane/methanol 9: 1).

1H-NMR(d6-DMSO)：0.85(t，3H)；1.2-1.3(m，1H)；1.4-1.6(m，7H)；1.6-1.8(m，6H)；1.9-2.0(m，2H)；2.05-2.15(m，2H)；2.25(t，2H)；2.55(m，1H)；2.95(d，2H)；4.0(d，2H)，7.35(d，2H)；7.8(d，2H)，7.9(s，1H)；8.15(m，1H)。

Example 9: synthesis of N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- [1- (2-methoxy-ethyl) - Piperidin-4-yl radical]-benzamides

A.4-Carboxyphenylboronic acid methyl ester

4-Carboxyphenylboronic acid (300mmol) was dissolved in methanol (400ml), and 1.5ml of concentrated hydrochloric acid was added to the stirred solution. The reaction was heated to reflux for 30 hours. The solvent was evaporated, the remaining residue was suspended in ether, the solid was filtered off and dried (vacuum). A light-colored powder was obtained, mp.201-205 ℃, Rf 0.28 (dichloromethane/methanol 95: 5). The powder is a mixture of 4-carboxyphenylboronic acid methyl ester and 4-carboxyphenylboronic acid methyl ester dimer anhydride, and can be used without purification.

B.4-pyridin-4-yl-benzoic acid methyl ester

Methyl 4-carboxyphenylborate from A (248mmol), 4-bromopyridine (248mmol), tetrakis- (triphenylphosphine) -palladium (2.5mmol) and potassium carbonate (744mmol) were suspended in 1, 2-dimethoxyethane (1100 ml). The stirring mixture was heated to reflux for 8 hours. After cooling, the solvent was evaporated and water was added to the residue, which was then extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was suspended in diethyl ether and the solid was filtered off and dried (vacuum). A light brown powder was obtained, mp.99-101 ℃, Rf 0.39 (dichloromethane/methanol 95: 5).

4- (4-methoxycarbonyl-phenyl) -1- (2-methoxy-ethyl) -pyridinium; bromide compound

4-pyridin-4-yl-benzoic acid methyl ester (70mmol) and 2-bromoethyl-methyl ether (28ml) were heated at 110 ℃ for 1 hour. After cooling, the reaction mixture was suspended in acetone, the solid was filtered off and dried (vacuum). A light brown powder was obtained, mp.170-171 ℃, Rf 0.22 (dichloromethane/methanol 9: 1).

4- [1- (2-methoxy-ethyl) -piperidin-4-yl]-benzoic acid methyl ester

4- (4-methoxycarbonyl-phenyl) -1- (2-methoxy-ethyl) -pyridinium bromide (67mmol) was suspended in methanol (250ml) and platinous oxide (1.2g) was added. The mixture was stirred under hydrogen atmosphere at normal pressure until consumption ceased. The catalyst was filtered off and the filtrate was evaporated. The residue was dissolved in dichloromethane and extracted with aqueous sodium carbonate. The organic phase was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol 9: 1 as mobile phase. The product containing fractions were combined and evaporated. A pale yellow oil was obtained with Rf 0.22 (dichloromethane/methanol 95: 5).

E.4-1- (2-methoxy-ethyl) -piperidin-4-yl]-benzoic acid hydrochloride

Reacting 4- [1- (2-methoxy-ethyl) -piperidin-4-yl]Methyl benzoate (47mmol) was dissolved in 4N hydrochloric acid (80ml) and heated under reflux for 12 hours. After cooling, the solvent was evaporated, the residue was suspended in acetone, the solid was filtered off, washed with acetone and dried (vacuum). A white powder was obtained, mp. > 270 ℃.F.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- [1- (2-methoxy-ethyl) -piperidin-4-yl]- Benzamide derivatives

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (107mmol), 4- [1- (2-methoxy-ethyl) -piperidin-4-yl]Benzoic acid hydrochloride (107mmol), HOBT (107mmol), WSCD (107mmol) and triethylamine (107mmol) were dissolved in DMF (250ml) and stirred at room temperature overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring slightly alkaline conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol (2N NH)₃) As mobile phase 9: 1. The product containing fractions were combined and evaporated. The residue was suspended in diethyl ether/ethyl acetate and the solid was filtered off and dried (vacuum). A light-colored powder was obtained, mp.160-162 ℃, Rf ═ 0.42 (dichloromethane/methanol (3N NH)₃)＝9∶1)。

1H-NMR(d6-DMSO)：1.2-1.3(m，1H)；1.4-1.6(m，5H)；1.6-1.8(m，6H)；2.0-2.2(m，4H)；2.45(m，2H)；2.55(m，1H)；2.95(br.d，2H)；3.2(s，3H)；3.4(dd，2H)；4.0(d，2H)；7.35(d，2H)；7.8(d，2H)；7.9(s，1H)；8.15(m，1H)。

Example 10: n- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (1-isopropylpiperidin-4-yl) -benzene Carboxamides

1-isopropyl-4- (4-methoxycarbonyl-phenyl) -pyridinium; bromide compound

4-pyridin-4-yl-benzoic acid methyl ester (2.3mmol) and 2-iodopropane (1.0ml) were heated at 90 ℃ for 24 hours. After cooling, the solvent was evaporated, the residue was suspended in acetone, the solid was filtered off and dried (vacuum). A pale yellow powder was obtained, mp.187-189 ℃, Rf 0.27 (dichloromethane/methanol 9: 1).

B.4- (1-isopropyl-piperidin-4-yl) -benzoic acid methyl ester hydroiodide

Reacting 1-isopropyl-4- (4-methoxycarbonyl-phenyl) -pyridinium; the bromide (1.9mmol) was suspended in methanol (10ml) and platinous oxide (80mg) was added. The mixture was stirred under a hydrogen atmosphere at normal pressure until consumption ceased. The catalyst was filtered off and the filtrate was evaporated. The residue was suspended in ether/pentane and the solid was filtered off and dried (vacuum). A light-colored powder was obtained, mp.219-224 ℃, Rf 0.41 (dichloromethane/methanol 9: 1).

C.4- (1-isopropyl-piperidin-4-yl) -benzoic acid hydrochloride

Methyl 4- (1-isopropyl-piperidin-4-yl) -benzoate hydroiodide (1.7mmol) was dissolved in 4N hydrochloric acid (5ml) and heated under reflux for 10 hours. After cooling, the solvent was evaporated, the residue was suspended in acetone, the solid was filtered off, washed with acetone and dried (vacuum). A grayish brown powder was obtained, mp. > 270 ℃.

D.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (1-isopropyl-piperidin-4-yl) -benzamide

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (0.95mmol), 4- (1-isopropyl-piperidin-4-yl) -benzoic acid hydrochloride (0.95mmol), HOBT (0.95mmol), WSCD (0.95mmol) and triethylamine (0.95mmol) were dissolved in DMF (5ml) and stirred at room temperature overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonate (ensuring basic conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was suspended in diethyl ether and the solid was filtered off and dried (vacuum). A white powder was obtained, mp.214-216 ℃, Rf ═ 0.38 (dichloromethane/methanol (3 NNH)₃)＝9∶1)。

1H-NMR(d6-DMSO)：0.95(d，6H)；1.2-1.3(m，1H)；1.4-1.8(m，11H)；2.05-2.25(m，4H)；2.55(m，1H)；2.7(m，1H)；2.85(d，2H)；4.0(d，2H)，7.35(d，2H)；7.8(d，2H)，7.9(s，1H)；8.15(m，1H)。

Example 11: n- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (1-cyclopentyl-piperidin-4-yl) -benzene Carboxamides

1-cyclopentyl-4- (4-methoxycarbonyl-phenyl) -pyridinium; bromide compound

4-pyridin-4-yl-benzoic acid methyl ester (2.35mmol) and 1-iodocyclopentane (1.0ml) were heated at 110 ℃ for 4 hours. 1-Iodocyclopentane (0.5ml) was added and the mixture was heated at 120 ℃ for a further 4 hours. After cooling, the solvent was evaporated, the residue was suspended in acetone, the solid was filtered off and dried (vacuum). The solid residue was purified by flash chromatography on silica gel using dichloromethane/methanol 9: 1 as mobile phase. The product containing fractions were combined and evaporated. The residue was suspended in diethyl ether and the solid was filtered off and dried (vacuum). A yellow powder was obtained, mp.183-185 ℃, Rf 0.35 (dichloromethane/methanol 9: 1).

B.4- (1-cyclopentyl-piperidin-4-yl) -benzoic acid methyl ester hydroiodide

Reacting 1-cyclopentyl-4- (4-methoxycarbonyl-phenyl) -pyridinium; the bromide (1.27mmol) was suspended in methanol (8ml) and platinous oxide (50mg) was added. The mixture was stirred under a hydrogen atmosphere at normal pressure until consumption ceased. The catalyst was filtered off and the filtrate was evaporated. The residue was suspended in ether/pentane and the solid was filtered off and dried (vacuum). A light-colored powder was obtained, mp.204-210 ℃, Rf 0.27 (dichloromethane/methanol 95: 5).

C.4- (1-cyclopentyl-piperidin-4-yl) -benzoic acid hydrochloride

Methyl 4- (1-cyclopentyl-piperidin-4-yl) -benzoate hydroiodide (1.06mmol) was dissolved in 4N hydrochloric acid (5ml) and heated under reflux for 10 hours. After cooling, the solvent was evaporated, the residue was suspended in acetone, the solid was filtered off, washed with acetone and dried (vacuum). A grayish brown powder was obtained, mp. > 270 ℃.

D.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (1-cyclopentyl-piperidin-4-yl) -benzamide

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (0.74mmol), 4- (1-cyclopentyl-piperidin-4-yl) -benzoic acid hydrochloride (0.74mmol), HOBT (0.74mmol), WSCD (0.74mmol) and triethylamine (0.74mmol) were dissolved in DMF (5ml) and stirred at room temperature overnight. After distilling off the solvent, the residue was dissolved in a mixture of water and sodium carbonateThe mixture (ensuring basic conditions) was extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was suspended in diethyl ether and the solid was filtered off and dried (vacuum). A white powder was obtained, mp.233-234 ℃, Rf ═ 0.34 (dichloromethane/methanol (3 NNH)₃)＝9∶1)。

1H-NMR(d6-DMSO)：1.2-1.85(m，20H)；1.9-2.15(m，4H)；2.4-2.6(m，2H)；3.05(d，2H)；4.0(d，2H)，7.35(d，2H)；7.8(d，2H)，7.9(s，1H)；8.15(m，1H)。

Example 12: n- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (1-methyl-piperidin-4-yl) -benzyl Amides of carboxylic acids

A.4-phenyl-1-methyl-piperidine

4-phenylpiperidine (12.4mmol), paraformaldehyde (24.8mmol) and titanium tetraisopropoxide (12.4mmol) were suspended in 1, 2-dimethoxyethane (20ml) and heated to 60 ℃ over 30 minutes and stirred at room temperature for a further 1 hour. Sodium borohydride (12.4mmol) was added portionwise and the mixture was stirred at room temperature for 2h and at 60 ℃ for 3 h. After cooling, the solvent was evaporated and the residue was dissolved in a mixture of aqueous ammonia (60ml) and ethyl acetate and carefully filtered. The mixture was extracted three times with ethyl acetate and the combined organic phases were dried over sodium sulfate and evaporated. A light brown oil was obtained.

B.4- (1-methyl-piperidin-4-yl) -benzoic acid methyl ester

4-phenyl-1-methyl-piperidine (9.9mmol) was dissolved in dichloromethane (60ml) and oxalyl chloride (39.6mmol) was added dropwise at-20 to-10 ℃. After complete addition of oxalyl chloride, aluminium trichloride (260mmol) was added in portions at-10 ℃. The mixture was stirred at-10 ℃ for 1.5 hours. The cooling bath was removed and the mixture was stirred at room temperature for an additional 2 hours. The mixture was cooled to 0 ℃ again, and methanol (30ml) was added dropwise. After the addition of methanol was complete, the cooling bath was removed and the mixture was stirred at room temperature overnight. The reaction mixture was poured into a mixture of aqueous sodium carbonate (ensuring basic conditions) and ethyl acetate and the suspension was carefully filtered. The filtrate was extracted three times with ethyl acetate and the combined raffinate was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol 9: 1 as mobile phase. The product containing fractions were combined and evaporated. A light yellow oil was obtained with Rf 0.29 (dichloromethane/methanol 9: 1).

C.4- (1-methyl-piperidin-4-yl) -benzoic acid hydrochloride

4- (1-methyl-piperidin-4-yl) -benzoic acid methyl ester (4.55mmol) was dissolved in 4N hydrochloric acid (10ml) and heated under reflux for 8 hours. After cooling, the solvent was evaporated, the residue was suspended in acetone, the solid was filtered off, washed with acetone and dried (vacuum). A light brown powder was obtained, mp. > 270 ℃.

D.N- [1- (cyanomethyl-carbamoyl) -cyclohexyl]-4- (1-methyl-piperidin-4-yl) -benzamide

1-amino-cyclohexanecarboxylic acid cyanomethyl-amide (0.98mmol), 4- (1-methylpiperidin-4-yl) -benzoic acid hydrochloride (0.98mmol), HOBT (0.98mmol), WSCD (0.98mmol) and triethylamine (0.98mmol) were dissolved in DMF (5ml) and stirred at room temperature overnight. After the solvent was distilled off, the residue was dissolved in a mixture of water and sodium carbonate (ensuring basic conditions) and extracted three times with ethyl acetate. The combined extract was dried over sodium sulfate and evaporated. The residue was suspended in ether/pentane and the solid was filtered off and dried (vacuum). A white powder was obtained, mp.215-217 ℃, Rf ═ 0.32 (dichloromethane/methanol (3 NNH)₃)＝9∶1)。

1H-NMR(d6-DMSO)：1.2-1.3(m，1H)；1.4-1.8(m，11H)；1.85-2.0(m，2H)；2.05-2.2(m，5H)；2.55(m，1H)；2.95(d，2H)；4.0(d，2H)，7.35(d，2H)；7.8(d，2H)，7.9(s，1H)；8.15(m，1H)。

Similarly, N- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (piperidin-4-yl) -benzamide was obtained according to substantially the same conditions as in example 12 above; for example, step A is omitted and the synthesis begins in step B, starting with 4-phenylpiperidine.

Claims (5)

1. A compound selected from the group consisting of:

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- [4- (1-propyl) -piperazin-1-yl ] -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (4-isopropyl-piperazin-1-yl) -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- (1-propyl-piperidin-4-yl) -benzamide;

n- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- [1- (2-methoxy-ethyl) -piperidin-4-yl ] -benzamide.

N- [1- (cyanomethyl-carbamoyl) -cyclohexyl ] -4- [4- (1-propyl) -piperazin-1-yl ] -benzamide or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition comprising the compound of claim 1 as an active ingredient.

4. Use of a compound of claim 1 in the manufacture of a medicament for the treatment or prevention of a disease or medical condition associated with cathepsin K.

5. The use of a compound according to claim 1 in the manufacture of a medicament for the treatment or prophylaxis of osteoporosis, rheumatoid arthritis, osteoarthritis, gingival disease, paget's disease or hypercalcemia of malignancy.