JP2003513926A - Protease inhibitor - Google Patents

Abstract

SUMMARY OF THE INVENTION The present invention is an inhibitor of cysteine proteases, particularly cathepsin K, which is useful for treating diseases in which inhibiting bone loss or cartilage degradation is a factor, formula (I): Formula 1 Or a pharmaceutically acceptable salt thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION       [0001] (Field of the Invention)   The present invention relates to novel 4-amino-azepan-3-one protease inhibitors. This
Are especially inhibitors of cysteine and serine proteases,
Specifically, it is a cysteine protease inhibitor. The compound of the present invention further comprises
Specifically, it inhibits the cysteine protease of the papain superfamily,
More specifically, it inhibits the cysteine proteases of the cathepsin family.
Harm. Most particularly, the present invention relates to compounds that inhibit cathepsin K.
I do. Such compounds are useful in diseases involving cysteine proteases, especially in excessive bone.
Or for the treatment of cartilage loss disorders such as osteoporosis, periodontitis and arthritis.
Useful for       [0002] (Prior art)   Cathepsin K is part of the papain superfamily of cysteine proteases
Is one of a series of enzymes that Cathepsins B, H, L, N and S are described in the literature.
It is listed. Recently, cathepsin K polypeptides and such polypeptides have been
Encoding cDNA was disclosed in U.S. Pat. No. 5,501,969 (in that specification).
Is referred to as cathepsin O). Cathepsin K has recently been revealed
Purified, purified and characterized. Bossard, M.J. et al. (1996) J. Biol. Che.
m.271, 12517-12524; Drake, F.H. et al. (1996) J. Biol. Chem.
271, 12511-12516; Bromme, D. et al. (1996) J. Biol. Chem.
71, 2126-2132.   Cathepsin K is described in the literature as cathepsin O, cathepsin X or cathepsin O2.
And various names. Cathepsin K appears to be more appropriate
(Nomenclature Committee of the International Union of Biochemistry
and the name given by Molecular Biology).       [0003]   Cathepsins of the papain superfamily of cysteine proteases are human
In animals containing normal physiological processes of proteolysis, such as binding
Works in tissue degradation. However, high levels of these enzymes in the body
Some things can lead to pathological situations leading to disease. Thus, cathepsin
Pneumocystis carinii, trypanoma crew
Di (trypsanoma cruzi), trypsanoma brusei (trypsanoma b)
rucei brucei) and Crithidia fusiculata
Infections and schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dysfunction
Associated with, but limited to various conditions, such as trophies and muscular atrophy
Not something. International Publication Number WO94 / 04172 (released on March 3, 1994)
And the references cited therein. In addition, European Patent Application EP06
See 03873A1 and the references cited therein. Gingine pine
Two bacterial systems from P. gingivallis
In proteases have been implicated in the pathogenesis of gingivitis. Potempa, J. et al. (
1994) Perspectives in Drug Discovery and Design, 2, 445-458.
.       [0004]   Cathepsin K is thought to cause a disease of excessive bone or cartilage loss. Bone
Is a protein matrix that incorporates axial or plate-like crystals of hydroxyapatite.
It consists of tricks. Type I collagen is the main structural protein of bone
And accounts for about 90% of its structural proteins. The remaining 10% of the matrix
, Osteocalcin, proteoglycans, osteopontin, osteonecti
, Including thrombospondin, fibronectin and bone sialoprotein.
It is composed of many non-collagenous proteins. Skeletal bones are lifelong in individual lesions
Through the remodeling action. These lesions or remodeling units are used during the bone resorption phase,
Undergoes a cycle of bone replacement.   Bone resorption is performed by osteoclasts, which are multinucleated cells of the hematopoietic lineage. Osteoclasts attach to bone surface
A wide band that forms an airtight zone and then folds its tip (ie, the absorbent surface)
A film is formed over the surroundings. This forms a closed extracellular compartment on the bone surface.
The extracellular compartment is acidified by a wave-like proton pump in the membrane.
Osteoclasts secrete proteolytic enzymes. The low pH of the compartment will
Dissolves xiapatite crystals while proteolytic enzymes
Digest the cousin. In this way, absorption cavities or depressions are formed. Cycle
At the end of this phase, osteoblasts are transformed into a new protein matrix that is later mineralized.
Build a box. In some conditions, such as osteoporosis and Paget's disease, bone
The normal balance of resorption and formation is disrupted, and ultimately bone loss occurs in each cycle. After all,
This weakens the bone and consequently increases the risk of breaking with a small impact.       [0005]   The over-selective expression of cathepsin K in osteoclasts indicates that this enzyme
It strongly suggests that it is essential for yield. Thus, the selective inhibition of cathepsin K
Gingival diseases such as, but not limited to, porosis, gingivitis and periodontal disease
Disease, including Paget's disease, malignant hypercalcemia, and metabolic bone disease,
It may provide an effective treatment for diseases of excessive bone loss. Cathepsin
K levels were also measured to be high in cartilage resorbing cells of the synovium of osteoarthritis.
Was decided. Thus, selective inhibition of cathepsin K is also limited to this
But not excessive cartilage or malaria, including osteoarthritis and rheumatoid arthritis
It may be useful in the treatment of trix decomposition disorders. Metastatic tumor cells also
High levels of proteolytic enzymes that typically break down the surrounding matrix
Express. Thus, selective inhibition of cathepsin K may also cure certain tumor diseases.
May also be useful for treatment.       [0006]   Now, certain novel compounds are protease inhibitors, most specifically
It is an inhibitor of tepsin K.
Bone loss, such as gingival disease, or osteoarthritis and rheumatoid arthritis
And other diseases characterized by excessive cartilage or matrix degradation
Was found to be useful.       [0007] (Disclosure of the Invention)   It is an object of the present invention to provide 4-amino-azepan-3-one protease inhibitors, especially cis
It is to provide inhibitors of tein and serine protease. More specific
The present invention relates to such compounds that inhibit cysteine proteases,
More specifically, it relates to the cysteine protease of the papain superfamily.
. The present invention preferably inhibits cysteine proteases of the cathepsin family.
The offending compounds most preferably relate to compounds that inhibit cathepsin K. Book
The compounds of the invention may be therapeutically modified by altering the activity of such proteases.
It is useful for the treatment of diseases that can be treated.       [0008]   Thus, in one aspect, the present invention provides a compound of formula (I): Embedded image A 5-methoxybenzofuran-2-carboxylic acid {(S) -3-methyl
1- [3-oxo-1- (pyridin-2-sulfonyl) -azepan-4-ylcarbamoy
[-Butyl] amide.       [0009]   In another aspect, the invention provides compounds of formula (I) and pharmaceutically acceptable
A pharmaceutical composition comprising a carrier.   In yet another embodiment, the invention relates to a protease, such as cysteine.
And therapeutic modification of the disease state by inhibiting serine proteases
And a method for treating a disease. In particular, the method comprises a cysteine protease
Specifically inhibits cysteine proteases of the papain superfamily
And optionally treating the disease. More specifically, cathepsin K
Inhibiting cysteine proteases of the cathepsin family of the present invention.   In another embodiment, the compounds of the present invention are used for treating osteoporosis, gingival disease, e.g.
Bone loss, such as gingivitis and periodontal disease, or osteoarthritis and chronic joint disease
Cures for diseases characterized by excessive cartilage or matrix degradation, such as equine
It is especially useful for treatment.       [0010] (Best Mode for Carrying Out the Invention)   The present invention provides a compound of formula (I): Embedded image A 5-methoxybenzofuran-2-carboxylic acid {(S) -3-methyl
1- [3-oxo-1- (pyridin-2-sulfonyl) -azepan-4-ylcarbamoy
[-Butyl] amide.       [0011]   The present invention relates to all hydrates, solvates, complexes and polymorphs of the compounds of formula (I)
And prodrugs. Prodrugs are active parent compounds of formula (I) in vivo.
Any covalently bound compound that releases the drug. Prod of the Compound of the Invention
The lag comprises a ketone derivative, specifically a ketal or hemiketal. Henin
Chiral centers are present in compounds of the present invention, including thiomers and diastereomers.
All the isomers obtained by the above are included in the scope of the present invention.
It is intended. The compounds of the present invention are racemic mixtures, enantiomerically enriched
Or a racemic mixture using well-known methods.
And the individual enantiomers may be used alone.   The compounds of the present invention may exist in tautomeric forms, such as keto-enol tautomers.
When present, each tautomeric form is in equilibrium or one form predominates
Any of these cases are considered to be within the scope of the present invention.   When compared to the corresponding 5- and 6-membered ring compounds, the carbon center is α
The 7-membered ring compound of the invention in the -position is more stable.       [0012] Definition   Abbreviations and symbols commonly used in the field of peptides and chemistry
As used herein, the compounds of the present invention are described. Generally, abbreviations for amino acids are
Biochemical life described in Eur. J. Biochem., 158, 8 (1984).
According to IUPAC-IUB Joint Commission on Names
is there. In particular, throughout this application, m-CPBA means meta-chloroperoxybenzoic acid
EDC is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
Means DMSO means methylsulfoxide; TEA triethylamine
means.       [0013] Preparation method   Compounds of formula (I) are generally prepared according to Scheme 1. 5-methoxy
Benzofuran-2-carboxylic acid {(S) -3-methyl-1- [3-oxo-1- (pyridine-
2-sulfonyl) -azepan-4-ylcarbamoyl] -butyl} amide (10) and
Individual diastereomers of (11) can be prepared according to the scheme outlined in Scheme 1.
it can. Allyl-carbamic acid benzyl ester (1) converted to sodium hydride
Alkylation with 5-bromo-1-pentene in the presence of a base of
Get) Diene (2) is a bis (trichloride) developed by Grubbs.
Rohexylphosphine) benzylidene Ruthenium (IV) dichloride
2,3,4,7-tetrahydro-azepine-1-carboxylic acid benzyl ester (3
Get) Epoxidation of azepine (3) is common to the field such as m-CPBA
The epoxide (4) can be obtained by using the standard oxidizing agent. Natori
(4) is subjected to a ring-opening reaction with a nucleophilic epoxide using a reagent such as
This gives the did alcohol (not shown in the scheme). The intermediate azide alcohol
With 1,3-propanedithiol and triethylamine in methanol or
Common in the art, such as tetrahydrofuran and triphenylphosphine in water
It is reduced to the amino alcohol (5) under normal conditions. Coupling such as EDC
(5) is acylated with an acid such as N-Boc-leucine in the presence of an agent.
Can be. The benzyloxycarbonyl protecting group is hydrogenated in the presence of 10% Pd / C
Removal with gas gives amine (6). Amine (6) containing triethylamine
With 2-pyridinesulfonyl chloride in the presence of
Removal of the t-butoxycarbonyl protecting group gives (7). (7) is 5-methoxy
Coupling using nzofuran-2-carboxylic acid and a coupling agent such as EDC
To give the intermediate alcohol (8). The alcohol (8) is converted to an oxidizing agent such as D
Oxidized with pyridine sulfur trioxide complex in MSO and triethylamine to give dias
The ketone (9) is obtained as a mixture of teleomers. HP for diastereomer (9)
Separation by LC gives compounds (10) and (11).       [0014] Embedded image      [0015] Reagents and conditions: a) NaH, 5-bromo-1-pentene, DMF; b) bis (
Trichlorohexylphosphine) benzylidine ruthenium (IV) dichloride
, CH₂Cl₂C) m-CPBA, CH₂Cl₂D) NaN₃, CH₃OH
, H₂O, NH₄Cl; e) 1,3-propanedithiol, TEA, methanol
F) N-Boc-leucine, EDC, CH₂Cl₂G) 10% Pd / C, H ₂ H) 2-pyridinesulfonyl chloride, triethylamine, CH₂Cl₂
I) 4N HCl / dioxane, methanol; j) 5-methoxybenzofuran-
2-carboxylic acid, EDC, CH₂Cl₂K) pyridine-sulfur trioxide complex, D
MSO, TEA; l) HPLC separation       [0016]   Starting materials used in the present invention are either commercially available or are well known to those skilled in the art.
Manufactured by the conventional method, COMPENDIUM OF ORGANIC SYNTHETIC METHODS, Vol.
It can be found in standard reference books such as I-VI (published by Wiley-Interscience).
Wear.   The coupling method for forming an amide bond in the present invention is known in the art.
Generally well known. In general, Bodansky et al., THE PRACTICE OF PEPTIDE SY
NTHESIS, Springer-Verlag, Berlin (1984); E. Gross and J. Meienhofer
, THE PEPTIDES, Vol. 1, 1-284 (1979); and J.M.Stewart and J.D.You.
ng, SOLID PHASE PEPTIDE SYNTHESIS, 2nd edition, Pierce Chemical Co., Rockfield,
 The peptide synthesis method generally disclosed by III. (1984) is a typical method.
, Incorporated herein by reference.       [0017]   Synthetic methods useful in the preparation of the compounds of the present invention are useful for masking reactive functional groups.
Or use of protecting groups to minimize unwanted side reactions.
Many. Generally, such protecting groups are described in Green, T.W., PROTECTIVE GROUPS IN ORGAN
IC SYNTHESIS, John Wiley & Sons, New York (1981).
The term "amino protecting group" generally refers to Boc, A, as known in the art.
Refers to cetyl, benzoyl, Fmoc and Cbz groups. Protection and deprotection methods
In addition, methods for substituting an amino protecting group with another group are well known.   The acid addition salt of the compound of formula (I) may be prepared by adding the parent compound and hydrochloric acid in a suitable solvent,
Hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, maleic
Manufactured by standard methods from excess acid, such as acid, succinic acid or methanesulfonic acid.
It is.       [0018] New intermediate   The present invention also provides compounds of formula (II) useful for the synthesis of compounds of formula (I) according to Scheme 1.
): Embedded image A novel intermediate, 5-methoxybenzofuran-2-carboxylic acid {(S) -3
-Methyl-1- [3-hydroxy-1- (1-oxy-pyridine-2-sulfonyl) -azepane
-4-ylcarbamoyl] -butyl} amide (8-Scheme-1).       [0019] Method for synthesizing compounds of the present invention   Referring to Scheme 1 above, the present invention provides a method for converting a suitable compound of formula (II) into an oxidizing agent
To give the compound of formula (I) as a mixture of diastereomers.
And a method for synthesizing a compound of formula (I), comprising: Preferably, the oxidizing agent is a triacid
Complex of sulfur halide / pyridine / DMSO and triethylamine.   The process further comprises the step of separating the diastereomers of formula (I), preferably a high pressure liquid.
And a step of separating by chromatography (HPLC).       [0020] Utility of the present invention   The invention also relates to a compound of formula (I) and a pharmaceutically acceptable carrier, excipient or
A pharmaceutical composition comprising a diluent is provided. Therefore, the compounds of formula (I) can be
It may be used for construction. A pharmaceutical composition of a compound of formula (I) prepared as described above
May be formulated as a solution or lyophilized powder for parenteral use. Before use
The powder can be reconstituted by the addition of excipients or other pharmaceutically acceptable carriers.
Good. The liquid formulation may be a buffered isotonic aqueous solution. Examples of suitable diluents are
Standard isotonic saline solution, 5% dextrose in standard water or buffered sodium acetate
Or ammonium acetate solution. Such a formulation is particularly suitable for parenteral administration
May be used for oral administration or in a metered dose inhaler or nebulizer for inhalation.
No. Polyvinyl pyrrolidone, gelatin, hydroxycellulose, acacia, poly
Ethylene glycol, mannitol, sodium chloride or sodium citrate
It is desirable to add an excipient such as       [0021]   Alternatively, the compound may be encapsulated and tableted for oral administration.
Alternatively, they may be prepared as emulsions or syrups. Pharmaceutically acceptable solid
A body or liquid carrier may be added to enhance or stabilize the composition, or
Preparation of the composition may be facilitated. Solid carriers include starch, lactose,
Lucium dihydrate, clay, magnesium or stearic acid, tar
Includes grape, pectin, acacia, agar or gelatin. Liquid carrier is syrup,
Contains peanut oil, olive oil, saline and water. Carrier is monostearin
Glyceryl acid or glyceryl distearate (alone or with wax)
Contains any sustained release material. The amount of solid carrier varies but, preferably, is
Will be between 20 mg to about 1 g. If in tablet form, crush, mix, condyle
Pharmaceutical preparations are prepared according to conventional pharmaceutical methods, including granulation and, if necessary, tableting.
Prepared in the form of hard gelatin capsules, including crushing, mixing and filling.
The pharmaceutical preparation is manufactured according to conventional pharmaceutical methods. If a liquid carrier is used,
Products can be syrups, elixirs, emulsions or aqueous or non-aqueous suspensions.
Will be in the form. Such a liquid formulation may be administered directly orally, or soft gelatin
It may be filled in a capsule.       [0022]   For rectal administration, the compounds of the present invention can be incorporated with cocoa butter, glycerin, gelatin or
It may be mixed with excipients such as ethylene glycol and formed into a suppository.   Compounds of formula (I) are useful as protease inhibitors, especially cysteine and serine protease.
Inhibitors of theases, more specifically inhibitors of cysteine protease,
More specifically, inhibitors of the cysteine protease of the papain superfamily
And even more specifically, the inhibition of cathepsin family cysteine proteases.
It is useful as a harmful agent, most specifically as an inhibitor of cathepsin K. The invention also provides
Useful compositions of the compounds of the invention, including pharmaceutical compositions and formulations of the compounds of the invention
Articles and formulas are provided.   The compounds of the present invention can be used in the form of Pneumocystis carini, Trypanoma cruzi,
Infections caused by Lipsanoma bursei and Criticia fushiclata; and
Schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, muscle
Atrophy, especially diseases involving cathepsin K, most particularly osteoporosis, gingivitis
Diseases that cause excessive bone or cartilage loss, including periodontal disease, including and periodontitis, and arthritis
, More specifically osteoarthritis and rheumatoid arthritis, Paget's disease, malignant
Cysteine proteases, including hypercalcemia, and metabolic bone disease
It is useful for the treatment of diseases in which is involved.       [0023]   Typically, metastatic tumor cells also contain proteins that degrade the surrounding matrix.
It expresses high levels of proteolytic enzymes and certain tumors and metastatic tumors
It can be effectively treated with a substance.   The invention also relates to pathological levels of proteases, specifically cysteine and
And serine proteases, more specifically cysteine proteases, and even more
Specifically, cysteine proteases from the papain superfamily, and even more
More specifically, it is caused by the cysteine proteases of the cathepsin family.
A method for treating a disease to be treated, the animal requiring treatment, specifically a mammal,
Specifically, there is provided a method comprising administering to a human a compound of the invention. The present invention
Is, inter alia, the treatment of diseases caused by pathological levels of cathepsin K
A method of treating an animal, particularly a mammal, and most particularly a human, in need of treatment.
A method comprising administering an inhibitor of cathepsin K comprising a compound of the invention.
Offer. The invention relates, inter alia, to Pneumocystis carini,
Infections caused by Luzhi, Trypanoma brusei, and Criticia fusicata
; And schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy
Ee, muscular atrophy, especially diseases involving cathepsin K, most specifically osteoporosis
Excessive bone or cartilage loss, including periodontal disease, including gingivitis and periodontitis, arthritis
Diseases, more specifically osteoarthritis and rheumatoid arthritis, Paget's disease
Cysteine pro, including malignant hypercalcemia, and metabolic bone disease
A method for treating a disease in which thease is involved is provided.       [0024]   The invention further provides an effective amount of a compound of formula (I) alone or with a bisphosphonate.
Acid (ie alendronate), hormone replacement therapy, antiestrogen or
Intravenous administration to patients in combination with other bone resorption inhibitors such as calcitonin
A method for treating osteoporosis or inhibiting bone loss. In addition,
Treatment with light compounds and anabolic agents such as bone morphogenetic protein and iproflavone
May be used to prevent bone loss or increase bone mass
.       [0025]   According to the present invention, an effective amount of a compound of formula (I) is administered to produce a particular condition or disease.
Inhibits proteases involved in Of course, this dose will administer the compound
Can be modified according to type. For example, for the treatment of acute illness, the formula (I)
It is preferred to administer the compound parenterally. 5% dextrose in water or standard sey
Intravenous injection of a compound of the invention in the line, or a similar formulation with appropriate excipients
While infusion is most effective, intramuscular bolus injections are also useful. Typical
In some cases, parenteral doses maintain effective drug concentrations in plasma to inhibit cathepsin K.
About 0.01 to about 100 mg / kg, preferably less than 0.1
Would be 20 mg / kg. Total daily dose of about 0.4 to about 400 mg / kg
Per day, the compound of the present invention is administered one to four times a day. Therapeutic
The exact amount of the compound of the invention that is effective, as well as the
Is to compare the blood levels of the drug with the concentrations needed to achieve a therapeutic effect.
More easily determined by those skilled in the art.       [0026]   Prodrugs of the compounds of the present invention can be prepared by any suitable method.
. The prodrug moiety is a ketone functional moiety, specifically a ketal and / or
In the case of hemiacetal, its deformation can be carried out by conventional means.   If the drug concentration is sufficient to inhibit bone resorption, or
Orally administer a compound of the present invention to a patient in such a manner that the therapeutic effect of
It may be administered. Typically, a pharmaceutical composition containing a compound of the invention is administered to a patient.
Administer an oral dose between about 0.1 and about 50 mg / kg in a manner consistent with the condition.
Give. Preferably, the oral dose will be from about 0.5 to about 20 mg / kg.   When a compound of the present invention is administered in accordance with the present invention, unacceptable toxicological effects are
Unthinkable.       [0027] Biological assays   The compounds of the invention are tested in one of several biological assays and
The concentration of the compound required to exert the pharmacological effect of the compound may be determined.       [0028] Measurement of cathepsin K protein degradation catalytic activity   All assays for cathepsin K were performed using human recombinant enzymes.
Standard assay conditions for determination of rate constants include fluorogenic peptide substrates, typically
For Cbz-Phe-Arg-AMC, 20 mM cysteine and 5 m
Rate constant in 100 mM sodium acetate (pH 5.5) containing M EDTA
It was measured. 10 mM in DMSO, used at a final substrate concentration of 20 μM in the assay
Alternatively, a stock substrate solution was prepared at a concentration of 20 mM. All assays are
Contains 10% DMSO. Independent experiments have shown that this level of DMSO
It was found to have no effect on activity or rate constants. Ambient temperature for all assays
It went in. Product fluorescence (excitation at 360 nm; emission at 460 nm)
Monitoring was performed with a eptive Biosystems Cytofluor II fluorescent plate reader.
Product formation progress curves were obtained over 20 to 30 minutes after the formation of the AMC product.       [0029] Inhibition experiment   Potential inhibitors were evaluated using the reaction progress curve method. Testing of different concentrations
Assays were performed in the presence of compound. Add enzyme to buffered solution of inhibitor and substrate
The reaction was started by addition. Morphology of the reaction progress curve in the presence of inhibition
Data analysis was performed by one of two methods, depending on The reaction progress curve
For compounds that are linear, Equation 1 (Brandt et al., Biochemistry, 1989, 2).
8, 140):     ν = ν_mA / [K_a(1 + I / K_{i, app}) + A] (1) [Wherein v is the reaction rate, V_mIs the maximum reaction rate, A is the substrate concentration, K_aIs Michaelis
Constant, where I is the inhibitor concentration] by the apparent inhibition constant (K_{i, app})
did.       [0030]   For compounds whose reaction progress curves show a downward curvature that is characteristic of temporal inhibition,
Equation 2:     [AMC] = ν_sst + (ν₀−ν_ss) [1-exp (-k_obst)] / k _obs
(2) [Where [AMC] is the concentration of the product formed after the passage of time t, v₀Is the initial speed, v _ss Is the final steady-state velocity] according to the data from the individual sets
Then k_obsI got Then k_obsAs a linear function of inhibitor concentration.
Analysis to determine the apparent second order rate constant (k_obs/ Inhibitor concentration or k_{ob s} / [I]) to explain the inhibition over time. Complete about this kinetic process
A detailed description can be found in Morrison et al., Adv. Enzymol. Relat. Areas Mol. Biol., 1988, 61.
, 201.       [0031]   Those skilled in the art will appreciate that K less than 50 micromolar_iCompounds with
You will think it is possible. Preferably, the compound used in the method of the invention
Compound is K less than 1 micromolar_iHas a value. Most preferably, the compound
Is K less than 100 nanomolar_iHas a value.       [0032] Human osteoclast resorption assay   An aliquot of the suspension of osteoclastoma-derived cells was removed from the liquid nitrogen stock and 3
Warm immediately at 7 ° C and centrifuge (1000 rpm, 5 minutes, 4 ° C) to RPMI
Washed once in -1640 medium. The medium is aspirated and 1 in RPMI-1640 medium
: Substitute with 3 diluted murine anti-HLA-DR antibody and incubate on ice for 30 minutes
did. The cell suspension was mixed frequently.   Centrifugation (1000 rpm, 5 minutes, 4 ° C.) with cold RPMI-1640 medium
Cells were washed twice and transferred to a sterile 15 ml centrifuge tube. Improved Neubauer counting chip
The number of mononuclear cells was counted in the chamber.   Store enough magnetic beads (5 per mononuclear cell) coated with goat anti-mouse IgG
Removed from bottle and placed in 5 ml fresh medium (this preserves toxic azide
Wash away the ingredients). The medium is removed by fixing the beads on a magnet and fresh medium
Was replaced with       [0033]   The beads were mixed with the cells and the suspension was incubated on ice for 30 minutes. Suspension
Mix frequently. The bead-coated cells were fixed on a magnet and the remaining cells (rich in osteoclasts)
Fraction) was placed in a 50 ml centrifuge tube sterilized by decantation.
Fresh media was added to the bead-coated cells to remove trapped osteoclasts
. This washing operation was repeated 10 times. The bead-coated cells were discarded.   Change the sample using a large-hole disposable plastic Pasteur pipette.
Bars were placed and osteoclasts were counted in a counting chamber. Pellet cells by centrifugation
And osteoclast density was increased to 10% fetal bovine serum and 1.7 g / l charcoal.
1.5x10 in EMEM medium supplemented with sodium hydrogen oxy⁴Adjusted to pcs / ml
. Decant 3 ml aliquot (per treatment) of cell suspension
Into a 15 ml centrifuge tube. Pellet these cells by centrifugation.
Was. 3 ml of an appropriate processing solution (diluted to 50 μM in EMEM medium) in each centrifuge tube
) Was added. Appropriate vehicle controls, positive controls (8 diluted to 100 μg / ml)
7MEM1) and isotype control (IgG2a diluted to 100 μg / ml)
Was also incubated. The tubes were incubated at 37 ° C. for 30 minutes.       [0034]   Aliquot 0.5 ml aliquots onto sterile tooth slices in a 48 well plate.
The cells were seeded and incubated at 37 ° C. for 2 hours. Each processing is screened by quadruple repetition operation
Learning. Six times with warm PBS (10 ml / well in a 6-well plate)
Replace and wash slices, then place in fresh processing or control solution at 37 ° C
For 48 hours. The slice is then phosphate buffered saline
And 5 minutes with 2% glutaraldehyde (in 0.2 M sodium cacodylate)
And then rinse the slices with water and incubate in buffer at 37 ° C for 5 minutes.
I was vate. The slice is then washed with cold water and cold acetate buffer / fast
Incubation was performed at 4 ° C. for 5 min. Aspirate excess buffer
The slices were air-dried and then washed with water.   TRAP-positive osteoclasts were counted by brightfield microscopy and then sonicated.
Removed from the tooth surface. Pit using Nikon / Lasertec ILM21W confocal microscope
The volume was measured.       [0035] Example   In the following synthesis examples, all starting materials were obtained from commercial products unless otherwise specified.
Was. Without further elaboration, those skilled in the art will recognize, in accordance with the above description, the present invention.
Can be used to the fullest. These examples are provided to illustrate the invention.
And does not limit the scope of the invention. For the applicant
Matters to be retained are mentioned in the claims.       [0036] Example 1 5-methoxybenzofuran-2-carboxylic acid {(S) -3-methyl-1- [3-oxo-1- (
Preparation of pyridine-2-sulfonyl) -azepan-4-ylcarbamoyl] -butyl} amide
Made a) Allyl-pent-4-enyl-carbamic acid benzyl ester   To a suspension of NaH (1.83 g, 76.33 mmol of 90% NaH) in DMF
, Allyl-carbamic acid benzyl ester (7.3 g, 38.2 mmol) was added dropwise
Added by method. The mixture is stirred at room temperature for about 10 minutes, after which the 5-bromo-1-pe
Content (6.78 mL, 57.24 mmol) was added dropwise. Reactants
Heated to 40 ° C. for about 4 hours after which the reaction was partitioned between dichloromethane and water
. The organic layer was washed twice with water, brine and dried (MgSO 4).₄), Filter and concentrate
Shrunk. The residue was subjected to column chromatography (10% ethyl acetate: hexane).
This gave 10.3 g of the title compound as an oil. MS (EI) 260 (M + H ⁺ ).       [0037] b) 2,3,4,7-tetrahydro-azepine-1-carboxylic acid benzyl ester   Bis (tricyclohexane) was added to a solution of the compound of Example 1a (50 g) in dichloromethane.
Hexylphosphine) benzylidine ruthenium (IV) dichloride (5.0 g)
Was added. Heat it to reflux until the completion of the reaction is observed by TLC analysis.
Was. The reaction was concentrated under reduced pressure. The residue was subjected to column chromatography (50%
(Chloromethane: hexane) to give 35 g of the title compound. MS (EI) 23
2 (M + H⁺).       [0038] c) Benzyl 8-oxa-3-aza-bicyclo [5.1.0] octane-3-carboxylate
Steal   To a solution of the compound of Example 1b (35 g, 1.5 mol) in dichloromethane was added m
-CPBA (78 g, 0.45 mol) was added. Stir the mixture at room temperature overnight
The solid was then removed by filtration. The filtrate is washed with water and saturated NaHCO₃ And washed several times. Dry the organic layer (MgSO₄), Filtered and concentrated to 35 g
The title compound was obtained and its sufficiently pure compound was used for the next step: MS (EI
) 248 (M + H⁺), 270 (M + Na⁺).       [0039] d) 4-azido-3-hydroxy-azepan-1-carboxylic acid benzyl ester   The epoxide of Example 1c (2.0 g, 8.1 mmol) in methanol: water (8:
1 solution) in the solution₄Cl (1.29 g, 24.3 mmol) and sodium
Muazide (1.58 g, 24.30 mmol) was added. Starting by TLC analysis
Heat the reaction to 65-75 ° C until complete consumption of the material epoxide is observed
did. Most of the solvent is removed under reduced pressure and the remaining solution is diluted with ethyl acetate and pH 4 buffer.
Partitioned between liquids. The organic layer was washed with saturated NaHCO₃Wash with water, brine and dry
Dry (MgSO₄), Filtered and concentrated. The residue is subjected to column chromatography (
20% ethyl acetate: hexane to give 1.3 g of the title compound: MS (E
I) 291 (M + H⁺). An additional 0.14 g of trans-4-hydroxy-3-azi
De-hexahydro-1H-azepine was obtained.       [0040] e) Benzyl 4-amino-3-hydroxy-azepan-1-carboxylate   Example 1d azido alcohol (1.1 g, 3.79 mmol) in methanol
To the solution was added triethylamine (1.5 mL, 11.37 mmol) and 1,3-pro
Pandithiol (1.1 mL, 11.37 mmol) was added. By TLC analysis
The reaction is stirred until complete consumption of the starting material is observed, and then the reaction is
Concentrated under reduced pressure. The residue was subjected to column chromatography (20% methanol: dichloromethane).
Chloromethane) to give 0.72 g of the title compound: MS (EI) 265 (M
+ H⁺).       [0041] f) 4-((S) -2-tert-butoxycarbonylamino-4-methyl-pentanoylamino
G) -3-Hydroxy-azepan-1-carboxylic acid benzyl ester   CH of the amino alcohol of Example 1e (720 mg, 2.72 mmol)₂C
l₂EDC (521 mg), HOBt (368 mg) and N-Bo
c-Leucine (630 mg) was added. Complete disappearance of starting material by TLC analysis
The reaction was kept at room temperature until expense was observed. Dilute the reaction with ethyl acetate,
1N HCl, saturated K₂CO₃Washed with water, brine and dried (MgSO 4)₄
), Filtered and concentrated. The residue was subjected to column chromatography (3% methanol
: Dichloromethane) to give 1.0 g of the title compound: MS (EI) 478
(M + H⁺).       [0042] g) [(S) -1- (3-hydroxy-azepan-4-ylcarbamoyl) -3-methyl-butyi
Ru] -carbamic acid tert-butyl ester   Compound of Example 1f (1.0 g) and 10% Pd / C (catalytic amount) in ethyl acetate
A hydrogen balloon was attached to the solution in toluene: methanol (2: 1 solution). TLC minutes
The reaction was stirred until complete consumption of the starting material was observed by precipitation. Reactant
Was filtered to remove the catalyst and the filtrate was concentrated to give 0.82 g of the title compound: MS
(EI) 344 (M + H⁺).       [0043] h) {(S) -1- [3-hydroxy-1- (pyridin-2-sulfonyl) -azepan-4-yl
Carbamoyl] -3-methyl-butyl} -carbamic acid tert-butyl ester   Formation of 2-pyridinesulfonyl chloride: 2-mercaptopyridine (2.23
g, 9N HCl (33 mL) solution was cooled to 0 ° C. Keep the temperature at 0 ° C
Meanwhile, chlorine gas was bubbled through the solution for about 90 minutes. The pH of the aqueous layer is about
9 until ice-cold saturated NaHCO₃While slowly dropping ice-cooled acetic acid
Ethyl was added. Afterwards, the organic layer was washed with brine and dried over MgSO₄Dry with
Was. 3.5 g of crude 2-pyridine sulfo as yellow liquid from the ethyl acetate evaporate
Nyl chloride was obtained.   Example 1g of [(S) -1- (3-hydroxy-azepan-4-ylcarbamoyl) -3-
Methyl-butyl] -carbamic acid tert-butyl ester (12 g, 34.93 mm
ol) in dichloride was added to 2-pyridinesulfonyl chloride (7.45).
g, 41.92 mmol) in small portions.
(5.8 mL, 41.92 mmol) was added. Reaction completed by TLC analysis
The reaction was stirred until was observed. Thereafter, the mixture was washed with saturated NaHCO₃,water
, Brine, dried (Na₂SO₄), Filtered and concentrated. Chromatographic residue
Chromatography (75% ethyl acetate: hexane to 100% ethyl acetate).
g of the title compound were obtained: MS (EI) 484 (M⁺).       [0044] i) (S) -2-Amino-4-methyl-pentanoic acid- [3-hydroxy-1- (pyridine-2-
Sulfonyl) -azepan-4-yl] -amide   Example 1h {(S) -1- [3-hydroxy-1- (pyridine-2-sulfonyl) -azepa
4-N-ylcarbamoyl] -3-methyl-butyl} -carbamic acid tert-butyl ester
To a solution of toluene (14.3 g) in methanol was added 4M HCl in dioxane.
. The reaction was stirred until complete reaction was observed by TLC analysis and then
Was concentrated to give 14 g of the title compound: MS (EI) 385 (M + H⁺).       [0045] j) 5-methoxybenzofuran-2-carboxylic acid {(S) -3-methyl-1- [3-oxo
1- (pyridin-2-sulfonyl) -azepan-4-ylcarbamoyl] -butyl} amide   CH of the compound of Example 1i (0.35 g, 0.76 mmol)₂Cl₂Medium solution
In addition, triethylamine (0.26 mL, 1.9 mmol), EDC (146 mg,
0.76 mmol), HOBt (103 mg, 0.76 mmol) and 5-methoyl
Xybenzofuran-2-carboxylic acid (147 mg, 0.76 mmol) was added.
. The reaction was stirred until completion was observed by TLC analysis. Post treatment of residue
And subjected to column chromatography (5% methanol: dichloromethane).
To give 0.33 g of the title compound: MS (EI) 559 (M + H⁺).       [0046] k) 5- (Methoxybenzofuran-2-carboxylic acid {(S) -3-methyl-1- [3-oxo-
1- (pyridin-2-sulfonyl) -azepan-4-ylcarbamoyl] -butyl} amide   To a solution of the alcohol of Example 1j (0.32 g, 0.57 mmol) in DMSO
, TEA (0.48 mL) and pyridine / sulfur trioxide complex (274 mg)
Was added. The reaction was stirred at room temperature for about 2 hours, then the reaction was combined with ethyl acetate.
Partitioned between water. The organic layer was washed with brine, dried, filtered and concentrated.
The residue was subjected to column chromatography (10% CH₃OH: CH₂Cl₂)
300 mg of the title compound were obtained as a mixture of diastereomers:¹HNMR (
CDCl₃): Δ1.0 (m, 6H), 1.5-2.1 (m, 5H), 2.2 (
m, 2H), 2.7 (m, 1H), 3.7 (d, 1H), 4.0 (m, 1H),
4.7 (m, 2H), 5.0 (m, 1H), 7.0 (m, 4H), 7.6 (m, 2H)
3H), 8.0 (m, 2H), 8.7 (m, 1H); MS (EI): 557 (M
+ H⁺, 70%).       [0047]   The diastereomeric mixture is separated by HPLC and the faster eluting diastereomer
Ma;¹HNMR (CDCl₃): Δ1.0 (m, 6H), 1.5-2.1 (m
, 5H), 2.2 (m, 2H), 2.7 (t, 1H), 3.7 (m, 4H), 4
. 0 (d, 1H), 4.7 (m, 2H), 5.0 (d, 1H), 7.0 (m, 4
H), 7.6 (m, 3H), 8.0 (m, 2H), 8.7 (d, 1H); MS (
EI): 557 (M + H⁺, 100%) and the late-eluting diastereomer;
MS (EI): 557 (M + H⁺, 100%).   K_i= 0.4 nM.   Pit assay = 75 nM.       [0048]   The above specification and examples more fully describe the preparation and use of the compounds of the present invention.
Disclose. However, the invention is not limited to the specific embodiments described above.
And includes all modifications that fall within the scope of the following claims. Miscellaneous quoted in this specification
The various literature, including journals, patents and other publications, constitute the status quo and
Is a part of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 19/10 A61P 19/10 21/00 21/00 21/04 21/04 29/00 101 29/00 101 33/02 33/02 33/06 33/06 33/08 33/08 35/04 35/04 43/00 111 43/00 111 // C07M 7:00 C07M 7:00 (72) Inventor U.S.A. 19087 Hancock Lane 1509 (72), Wayne, PA Daniel Frank Baber United States 19002 Ambler, PA, United States 190F Beirson Road 290 F-term (reference) 4C063 AA03 BB08 BB09 CC19 DD12 EE01 4C086 AA01 AA02 AA03 AA04 BC31 GA02 GA07 GA08 MA01 MA04 MA05 MA17 MA23 MA35 MA37 MA44 MA52 MA55 NA14 ZA67 ZA94 ZA96 ZA97 ZB15 ZB26 ZB38 ZC20 ZC21

Claims (1)

[Claim 1] 5- (Methoxybenzofuran-2-carboxylate {(S) -3-methyl-carboxylate)
1- [3-oxo-1- (pyridin-2-sulfonyl) -azepan-4-ylcarbamoyl]-
Formula (I), also known as butyl} amide, Or a pharmaceutically acceptable salt thereof. 2. A pharmaceutical composition comprising the compound according to claim 1 and a pharmaceutically acceptable carrier, excipient or diluent. 3. A method for inhibiting a protease selected from the group consisting of a cysteine protease and a serine protease, comprising administering an effective amount of the compound of claim 1 to a patient in need of the inhibition of the protease. . 4. The method according to claim 3, wherein the protease is a cysteine protease. 5. The method according to claim 4, wherein the cysteine protease is cathepsin K. 7. A method of treating a disease characterized by bone loss comprising inhibiting a bone loss by administering an effective amount of a compound of claim 1 to a patient in need thereof. 8. The method according to claim 7, wherein the disease is osteoporosis. 9. The method according to claim 7, wherein the disease is periodontitis. 10. The method according to claim 7, wherein the disease is gingivitis. 11. Excessive cartilage comprising inhibiting an excessive cartilage or matrix degradation by administering an effective amount of a compound of claim 1 to a patient in need thereof. Or a method of treating a disease characterized by matrix degradation. 12. The method according to claim 11, wherein the disease is osteoarthritis. 13. The method of claim 11, wherein the disease is rheumatoid arthritis. 14. Methoxybenzofuran-2-carboxylate {(S) -3-methyl-1- [3-hydroxy-1- (pyridine-2-sulfonyl) -azepan-4-ylcarbamoyl] -butyl} Formula (II), known as an amide: Or a pharmaceutically acceptable salt, hydrate or solvate thereof. 15. A compound of formula (I): A method for synthesizing a compound represented by the formula (II): A method of oxidizing a compound represented by the formula (I) with an oxidizing agent to obtain a compound represented by the formula (I) as a mixture of diastereomers. 16. The method of claim 15, wherein the oxidizing agent is a sulfur trioxide-pyridine complex in DMSO and triethylamine. 17. The method according to claim 15, further comprising a step of separating diastereomers by a separation means. 18. The method according to claim 17, wherein the separation means is high pressure liquid chromatography (HPLC).