JP2003513927A - Protease inhibitor - Google Patents

Abstract

SUMMARY OF THE INVENTION The present invention relates to a compound of the formula (I), which is an inhibitor of cysteine proteases, in particular cathepsin K, and is useful in the treatment of diseases in which inhibiting bone loss or cartilage degradation is a factor. ): It relates to the compound represented by (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION       [0001] (Field of the Invention)   The present invention relates to novel deuterated 4-amino-azepan-3-one protease inhibitors
About. This compound is particularly an inhibitor of cysteine and serine proteases.
And more specifically inhibitors of cysteine protease. Compound of the present invention
The product more specifically inhibits the cysteine protease of the papain superfamily,
Furthermore, they specifically inhibit cysteine proteases of the cathepsin family. most
In a preferred embodiment, the invention relates to compounds that inhibit cathepsin K. Or
Such compounds may be useful in diseases involving cysteine proteases, especially excessive bone or softness.
Particularly useful in the treatment of bone loss diseases such as osteoporosis, periodontitis and arthritis
is there.       [0002] (Prior art)   Cathepsin K is a series of enzymes that are part of the papain superfamily of cysteine proteases.
One of the prime. Cathepsins B, H, L, N and S have been described in the literature.
Recently, cathepsin K polypeptides and cDN encoding such polypeptides
A was disclosed in U.S. Pat. No. 5,501,969, in which cathepsin O
Is called). Cathepsin K has recently been identified and purified,
And characterized. Bossard, M.J. et al. (1996) J. Biol. Chem. 271, 12
517-12524; Drake, F.H., et al. (1996) J. Biol. Chem. 271, 125.
11-12516; Bromme, D. et al. (1996) J. Biol. Chem. 271, 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]   Catapsin, a cysteine protease papain superfamily, is found in animals, including humans.
In the normal physiological processes of proteolysis, such as the breakdown of connective tissue.
Works. However, high levels of these enzymes in the body can lead to disease.
Pathological situation. Thus, cathepsins are pneumocystis
Lini (pneumocystis carinii), trypsanoma cruzi
), Trypsanoma brucei brucei and
Infections caused by Crithidia fusiculata, and schisitism
Fluke, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, muscular atrophy
Are associated with, but not limited to, various disease states. International public
WO 94/04172 (published March 3, 1994) and references therein
See published literature. In addition, European Patent Application EP0603873A1 and
See the references cited therein. Pee Gin called Gingipine
Two bacterial cysteine proteases from P. gingivallis
It has been linked to the pathogenesis of sarcoma. Potempa, J. et al. (1994) Perspective
s 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 hydroxyapatite axial or plate-like crystals.
It is composed of boxes. Type I collagen is the main structural protein of bone and its structure
It accounts for about 90% of the protein. The remaining 10% of the matrix is osteocalcin
, Proteoglycans, osteopontin, osteonectin, thrombospon
Many non-collagen proteins, including gin, fibronectin and bone sialoprotein
It is configured. Skeletal bone undergoes life-long remodeling at individual lesions. this
These lesions or remodeled units undergo a cycle consisting of a bone resorption phase followed by a bone replacement phase.
I can.   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 is
The patite crystals dissolve, while proteolytic enzymes digest the protein matrix. This
Thus, a cavity or depression is formed. At the end of this phase of the cycle,
Osteoblasts build a new protein matrix that is later mineralized. osteoporosis
In some conditions, such as and Paget's disease, there is a normal balance between bone resorption and formation.
Collapse and ultimately bone loss in each cycle. In the end, this weakens the bones,
As a result, the risk of fracture with a small impact is increased.       [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 osteoporosis, gingivitis and periodontal disease, Paget's disease and malignant disease
Excessive, including, but not limited to, calcemia, and metabolic bone disease
May provide an effective treatment for diseases of bone loss. Cathepsin K
Levels were also measured to be high in cartilage resorbing cells of the synovium of osteoarthritis.
Was. Thus, selective inhibition of cathepsin K is also associated with osteoarthritis and chronic involvement.
Excessive cartilage or matrix, including but not limited to rheumatoid arthritis
It may be useful in treating degradation disorders. Metastatic tumor cells also typically
Express high levels of proteolytic enzymes that degrade the surrounding matrix. Hide
Thus, selective inhibition of cathepsin K is also useful for treating certain tumor diseases
there is a possibility.       [0006]   Now, certain novel deuterated compounds are protease inhibitors, most specifically
Is an inhibitor of cathepsin K, which is useful in osteoporosis, gingivitis and tooth
Bone loss such as gum disease such as periodontal disease, or osteoarthritis and chronic joints
For diseases characterized by excessive cartilage or matrix degradation, such as rheumatism
It has been found useful in therapy.       [0007] (Disclosure of the Invention)   An object of the present invention is to provide a novel deuterated 4-amino-azepan-3-one proteinase.
To provide inhibitors, particularly inhibitors of cysteine and serine proteases.
You. More particularly, the present invention relates to such a method for inhibiting cysteine protease.
The invention furthermore relates to cysteine proteases of the papain superfamily.
The present invention preferably provides compounds that inhibit cysteine proteases of the cathepsin family.
And most preferably to compounds that inhibit cathepsin K. Compound of the present invention
Can be therapeutically modified by altering the activity of such proteases.
It is useful for the treatment of sick diseases.       [0008]   Thus, in one aspect, the present invention provides a compound of formula I: Embedded image               (I) Thieno [3,2-b] thiophene-2-carboxylic acid {(S)-
3-methyl-1-[(2,2 ', 4-trideuterio) -3-oxo-1- (pi
Lysine-2-sulfonyl) -azepan-4-ylcarbamoyl] -butyl} amide
I will provide a.       [0009]   In another embodiment, the present invention relates to compounds of formula I and a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising the body is provided.   In yet another embodiment, the invention relates to a protease, such as cysteine.
And therapeutic modification of disease states by inhibiting serine proteases
And a method for treating a disease. In particular, the method comprises a cysteine protease.
Specifically, by inhibiting cysteine proteases of the papain superfamily,
Treating the condition. More specifically, cathepsins such as cathepsin K
Inhibiting cysteine proteases of the family.   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               (I) Thieno [3,2-b] thiophene-2-carboxylic acid {(S)-
3-methyl-1-[(2,2 ', 4-trideuterio) -3-oxo-1- (pi
Lysine-2-sulfonyl) -azepan-4-ylcarbamoyl] -butyl} amide
Alternatively, a pharmaceutically acceptable salt, hydrate or solvate thereof is provided.       [0011]   The present invention relates to all hydrates, solvates, complexes and polymorphs of the compounds of formula (I)
And prodrugs. Prodrugs have the activity of formula (I) in vivo
Any covalently bonded compound that releases the parent drug. Compounds of the invention
Lodrugs include ketone derivatives, specifically ketal or hemi-ketal.
. Chiral compounds in the compounds of the present invention, including enantiomers and diastereomers,
All forms of the isomer obtained by having a center are included within the scope of the present invention.
It is intended to be The compounds of the present invention are racemic mixtures, enantiomerically enriched.
It can be used as an enriched mixture or a racemic mixture
Separation may be achieved using methods or individual enantiomers may be used alone.   The compounds of the present invention may exist in the form of tautomers 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
The 7-membered ring compounds of the present invention in the α-position are 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 salt
DMSO means methylsulfoxide; TEA triethylamido
Means       [0013] Preparation method   Compounds of formula (I) are generally prepared according to Scheme I. Chieno [3,
2-b] thiophen-2-carboxylic acid {(S) -3-methyl-1-[(2,2 ',
4-trideuterio) -3-oxo-1- (pyridine-2-sulfonyl) -aze
Pan-4-ylcarbamoyl] -butyl} amide (10) and (11)
Diastereomers can be prepared according to the scheme outlined in Scheme 1. Allyl-
Carbamate benzyl ester (1) in the presence of a base such as sodium hydride
Alkylation with 5-bromo-1-pentene gives the diene (2). Diene
(2) Bis (trichlorohexylphosph) developed by Grubbs
2,3,4,7-tein by reaction with benzylidine ruthenium (IV) dichloride
This gives trahydro-azepine-1-carboxylic acid benzyl ester (3). Azepi
A standard oxidizing agent common to the art such as m-CPBA
To obtain epoxide (4). Such as sodium azide
(4) is subjected to a ring-opening reaction of a nucleophilic epoxide using a reagent to give an azido alcohol (
(Not shown in the scheme). The intermediate azide alcohol is converted into methanol
1,3-propanedithiol and triethylamine or tetrahydrofuran
Under conditions common in the art, such as triphenylphosphine in water and water,
Reduction to mino alcohol (5). Acid, in the presence of a coupling agent such as EDC,
For example, (5) can be acylated using N-Boc-leucine. Ben
Removal of the diyloxycarbonyl protecting group using hydrogen gas in the presence of 10% Pd / C
To give the amine (6). Amine (6) with saturated sodium bicarbonate and dichloromethane
Reaction with 2-pyridinesulfonyl chloride in the presence of
The tert-butoxycarbonyl protecting group is removed under conditions to give (7). (7)
Coupling agents such as eno [3,2-b] thiophene-2-carboxylic acid and EDC
To give alcohol (8) as an intermediate. Al
Cole (8) is converted to an oxidizing agent such as pyridine in DMSO and triethylamine.
Oxidation with sulfur trioxide complex to obtain ketone (9) as a mixture of diastereomers.
Can be The ketone (9) and triethylamine are converted to CD by reflux.₃OD: D ₂ Treatment in O affords the deuterated analog as a mixture of diastereomers, which is
Separation by HPLC affords deuterated compounds (10) and (11).
Wear.       [0014] Embedded image Scheme 1      [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, H₂ H) 2-pyridinesulfonyl chloride, TEA, CH₂Cl₂I) 4N H
Cl / dioxane, methanol; j) thieno [3,2-b] thiophene-2-cal
Boric acid, EDC, CH₂Cl₂K) a pyridine-sulfur trioxide complex, DMSO,
TEA; l) CD₃OD; D₂O (10: 1), TEA; m) 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).
You.   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.Yo.
ung, SOLID PHASE PEPTIDE SYNTHESIS, 2nd edition, Pierce Chemical Co., Rockfield
, III. (1984) is a typical method for peptide synthesis.
, 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             (II) Thieno [3,2-b] thiophene-2-carboxylic acid}
(S) -3-methyl-1-[(2,2 ', 4-trideuterio) -3-hydro
Xy-1- (pyridine-2-sulfonyl) -azepan-4-ylcarbamoyl]-
Butyl} amide (8-scheme-1).       [0019] Method for synthesizing compounds of the present invention   With respect to scheme 1 described above, the present invention provides a method for preparing a suitable compound of formula (II)
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.   Referring to scheme 1, the present invention also provides a deuterated compound of formula (I)
Is provided. In particular, if the deuterated isomer is desired, the
Providing the deuterated compound of formula (I) as a mixture of astereomers;
Synthesizing an Additional Step of Deuteration of Protonated Isomers with Deuterating Agents
Add to Preferably, the deuterating agent is a CD in triethylamine.₃OD
: D₂O (10: 1).   The process may further comprise separating the diastereomer of formula (I), preferably a high pressure liquid.
And a step of separating by chromatography (HPLC).       [0020] Utility of the present invention   The compounds of formula (I) have superior chiral stability compared to the protonated isomers.
Show qualitative.   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. Polyvinylpyrrolidone, 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
And pectin, acacia, agar or gelatin. The liquid carrier is
Oil, olive oil, saline and water. Carrier is monos
Glyceryl theate or glyceryl distearate (alone or with wax
Or both). The amount of solid carrier varies, but is preferably
Would be between about 20 mg to about 1 g per dosage form. For tablet form, crushed
According to conventional pharmaceutical methods, including mixing, granulating and, if necessary, tableting.
Pharmaceutical preparations are manufactured and, if in the form of hard gelatin capsules, ground, mixed and filled
The pharmaceutical preparation is prepared according to conventional pharmaceutical methods, including filling. Liquid carrier
When used, preparations may be syrups, elixirs, emulsions or aqueous or
It may be in the form of a non-aqueous suspension. Such liquid formulations may be administered orally directly,
Or it may be filled in a soft gelatin 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.   The compounds of the formula (I) are protease inhibitors, in particular cysteine and serine.
Inhibitors of proteases, more particularly inhibitors of cysteine proteases,
More particularly, inhibitors of cysteine proteases of the papain superfamily, and even more
Specifically, inhibitors of the cysteine proteases of the cathepsin family, most particularly
Useful as an inhibitor of Psin K. The present invention also provides a pharmaceutical composition of a compound of the present invention.
Provided are useful compositions and formulations of the compounds of the present invention, including articles and formulations.   The compounds of the present invention can be used in the form of Pneumocystis carini, Trypsanoma cruzi,
Infection by Lipsano-ma bursei and Crisidian fushiclata; and
Schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, muscle
Atrophy, especially diseases involving cathepsin K, most particularly osteoporosis, gingivitis and
Diseases that cause excessive bone or cartilage loss, including periodontal disease, including periodontitis, and arthritis.
Disease, more specifically osteoarthritis and rheumatoid arthritis, Paget's disease, malignancy
Cysteine proteases, including hypercalcemia and metabolic bone disease
It is useful for the treatment of diseases involving zeolites.       [0023]   Typically, metastatic tumor cells also have proteolysis, which breaks down the surrounding matrix.
Expresses enzymes at high levels, and certain tumors and metastatic tumors use the compounds of the invention.
And can be treated effectively.   The present invention also relates to proteases at the pathological level, in particular cysteine and
Serine proteases, more particularly cysteine proteases, even more details
Cysteine proteases from the papain superfamily, and even more particularly, cathepsin
A method for treating a disease caused by a cysteine protease of the family
The compounds of the invention may be administered to animals in need of treatment, particularly mammals, most particularly humans.
Providing a method comprising providing The present invention relates, inter alia, to pathological level
A method for treating a disease caused by Psin K, wherein the animal requires treatment,
Specifically, in mammals, and most particularly in humans, cathepsin K containing a compound of the present invention.
There is provided a method comprising administering an inhibitor. The present invention is, inter alia,
Stis Carini, Trypsano-Ma Cluj, Trypsano-Ma Brusei, and
Infections caused by Criticia fusicata; schistosomiasis, malaria, tumor turnover
Transfer, metachromatic leukodystrophy, muscular dystrophy, muscular atrophy, especially cathepsin K involved
Diseases, most particularly periodontal diseases, including osteoporosis, gingivitis and periodontitis;
Diseases that cause excessive bone or cartilage loss, including arthritis, and more specifically osteoarthritis
And rheumatoid arthritis, Paget's disease, malignant hypercalcemia, and
Methods for treating diseases involving cysteine proteases, including boring bone diseases
I will provide a.       [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,
Using treatment with light-emitting compounds and anabolic agents such as bone morphogenetic protein and iproflavone
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 dye
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 ketal and / or
In the case of hemiacetal, its deformation can be effected 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 proteolytic 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)
The cells were monitored with an eptive Biosystems Cytofluor II fluorescent pre-reader. A
Product formation progress curves were obtained over 20 to 30 minutes after MC product formation.       [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):     v = V_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] = v_sst + (v₀-V_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] and analyze 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]   One skilled in the art will appreciate that K less than 50 micromoles_iIs a lead compound
I think that there is. Preferably, the compound used in the method of the invention
The material has a K of less than 1 micromol_iHas a value. Most preferably, the compound is 10
K less than 0 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 mouse 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 was removed by fixing the beads on a magnet,
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 medium 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 pastel pipette.
The osteoclasts were counted in a counting chamber. Pellet cells by centrifugation
Osteoclast density of 10% fetal calf serum and 1.7 g / l carbonated water
1.5x10 in EMEM medium supplemented with sodium hydrogen⁴It adjusted to the number / ml. Fine
A 3 ml aliquot (per treatment) of the cell suspension was decanted.
And placed in a 15 ml centrifuge tube. These cells were pelleted by centrifugation.
Dispense 3 ml of the appropriate treatment solution (diluted to 50 μM in EMEM medium) into each centrifuge tube.
Was added. Appropriate vehicle control, positive control (87M diluted to 100 μg / ml)
EM1) and isotype control (IgG2a diluted to 100 μg / ml).
Incubated. The centrifuge tube was incubated at 37 ° C for 30 minutes.       [0034]   Aliquot 0.5 ml aliquots on sterile tooth slices on a 48 well plate.
The cells were seeded and incubated at 37 ° C. for 2 hours. Each processing is screened by quadruple repetition operation
-I did it. 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)
The slices are then washed with water and incubated in buffer at 37 ° C for 5 minutes.
I got a bait. The slices are then washed with cold water and cold acetate buffer / fast
Incubation was performed at 4 ° C for 5 minutes in a dog garnet. 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 Thieno [3,2-b] thiophene-2-carboxylic acid {(S) -3-methyl-1- [
(2,2,4-trideuterio) -3-oxo-1- (1-oxy-pyridine
Preparation of -2-sulfonyl) -azepan-4-ylcarbamoyl] -butyl} amide
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 10 minutes and then 5-bromo-1
-Pentene (6.78 mL, 57.24 mmol) was added dropwise. reaction
Heat the material to 40 ° C. for about 4 hours, then partition the reaction between dichloromethane and water
did. The organic layer was washed twice with water, brine and dried (MgSO 4).₄) And filter
And concentrated. Column chromatography of the residue (10% ethyl acetate: hexane)
To give the title compound (10.3 g) 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 back until the reaction is complete, as determined by TLC analysis
Shed. The reaction was concentrated under reduced pressure. The residue was subjected to column chromatography (50%
(Dichloromethane: hexane) to give the title compound (35 g). MS (EI
) 232 (M + H⁺).       [0038] c) Benzene 8-oxa-3-aza-bicyclo [5.1.0] octane-3-carboxylate
Jill ester   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. The mixture is stirred at room temperature overnight,
Then, the solid was removed by filtration. The filtrate is washed with water and saturated NaHCO₃In number
Washed twice. Dry the organic layer (MgSO₄), Filter and concentrate to 35 g of title
The compound was obtained and its sufficiently pure compound was used for the next step: MS (EI) 2
48 (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 reaction to 65-75 ° C. until complete consumption of epoxide of material 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. Wash the organic layer with saturated NaHCO3, water and brine, dry
Dry (MgSO₄), Filtered and concentrated. The residue was 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-
Azide-hexahydro-1H-azepine was obtained.       [0040] e) 4-amino-3-hydroxy-azepan-1-carboxylic acid benzyl ester   Azide alcohol of Example 1d (1.1 g, 3.79 mmol) in methanol
To the solution was added triethylamine (1.5 mL, 11.37 mmol) and 1,3-butane.
Lopandithiol (1.1 mL, 11.37 mmol) was added. By TLC analysis
The reaction is stirred until complete consumption of the starting material is observed
Was concentrated under reduced pressure. The residue was purified by column chromatography (20% methanol:
Chloromethane) to give 0.72 g of the title compound: MS (EI) 265 (
M + H⁺).       [0041] f) 4-((S) -2-tert-butoxycarbonylamino-4-methyl-pentano
(Ilamino) -3-hydroxy-azepan-1-carboxylic acid benzyl ester   Amino alcohol of Example 1e (720 mg, 2.72 mmol) in CH₂C
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 urate K₂CO₃Washed with water, brine and dried (Mg
SO₄), Filtered and concentrated. The residue was subjected to column chromatography (3%
(Nol: 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-me
Tert-butyl] -carbamic acid tert-butyl ester   Compound of Example 1f (1.0 g) and 10% Pd / C (catalytic amount) in ethyl acetate
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- (1-oxy-pyridine-2-sulfoni)
L) -azepan-4-ylcarbamoyl] -3-methyl-butyl} -carbamic acid
ert-butyl ester   Formation of 2-pyridinesulfonyl chloride: 2-mercaptopyridine (33 mL
(2.23 g) in 9 M HCl was cooled to 0 ° C. About 90 minutes of chlorine gas
And maintained at 0 ° C. Add ice-cold ethyl acetate, then add ice-cold saturated N
aHCO₃Was slowly added until the pH of the aqueous layer was about 9. Then the organic layer
Washed with line, MgSO₄And dried. Ethyl acetate was evaporated and 3.5 g of crude
2-Pyridinesulfonyl chloride was obtained as a pale yellow liquid.   Example 1g of [(S) -1- (3-hydroxy-azepane-4) in dichloromethane.
-Ylcarbamoyl) -3-methyl-butyl] -carbamic acid tert-butyl ester
(12 g, 34.93 mmol) in a solution of triethylamine (5.8 mL,
41.92 mmol), followed by 2-pyridinesulfonyl chloride (7.
45 g, 41.92 mmol) were added dropwise. Measure reaction by TLC assay
Stir until possible. The mixture was then washed with saturated NaHCO₃Washed with water, brine
, Dried (Na₂SO₄), Filtered and concentrated. Column chromatography of the residue
(75% ethyl acetate: hexane to 100% ethyl acetate) and 15 g of target
The title compound was obtained: MS (EI) 484 (M⁺).       [0044] i) (S) -2-Amino-4-methyl-pentanoic acid- [3-hydroxy-1- (pyri)
Zin-2-sulfonyl) -azepan-4-yl] -amide   {(S) -1- [3-hydroxy-1- (pyridine-2-sulfonyl) of Example 1h
-Azepan-4-ylcarbamoyl] -3-methyl-butyl} -carbamic acid tert
-Butyl ester (14.3 g) in methanol in 4M H 2 in dioxane
Cl was added. The reaction was stirred at room temperature until measured by TLC analysis.
It was concentrated to give 14 g of the title compound: MS (EI) 385 (M + H⁺)
.       [0045] j) Thieno [3,2-b] thiophene-2-carboxylic acid {(S) -3-methyl-1
-[3-hydroxy-1- (pyridine-2-sulfonyl) -azepan-4-dolphin
Rubamoyl] -butyl} amide   CH of the amine salt of Example 1i (0.25 g, 0.55 mmol)₂Cl₂Medium solution
To the solution were added triethylamine (0.15 mL), EDC (105 mg), HOBt (
74 mg) and thieno [3,2-b] thiophene-2-carboxylic acid (101 mg)
) Was added. The reaction was stirred until the reaction was complete by TLC analysis. Remaining
After the residue was subjected to post-treatment, column chromatography (5% methanol: dichloromethane)
To give 0.23 g of the title compound: MS (EI) 550 (M + H)⁺ ).       [0046] k) Thieno [3,2-b] thiophene-2-carboxylic acid {(S) -3-methyl-1
-[3-oxo-1- (pyridine-2-sulfonyl) -azepan-4-ylcarba
Moyl] -butyl} amide   To a solution of the alcohol of Example 1j (0.23 g, 0.42 mmol) in DMSO
, TEA (0.35 mL) and pyridine-sulfur trioxide complex (199 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 (2.5% CH₃OH: CH₂Cl₂Attached to)
To give the title compound as a mixture of diastereomers:¹HNMR (CDCl₃ ): 1.0 (m, 6H), 1.5-2.2 (m, 6H), 2.2 (m, 2H),
2.7 (m, 1H), 3.8 (m, 1H); 4.1 (m, 1H), 4.7 (m, 1H)
2H), 5.2 (m, 1H), 7.4-8.0 (m, 8H); 8.7 (m, 1H)
); MS (EI): 548 (M⁺, 100%).       [0047] l) Thieno [3,2-b] thiophene-2-carboxylic acid {(S) -3-methyl-1
-[(2,2,4-trideuterio) -3-oxo-1- (pyridine-2-s
Ruphonyl) -azepan-4-ylcarbamoyl] butyl diamide   D₂O: CD₃Thieno [3,2-b] thiophene- in OD (0.4: 4 mL)
2-carboxylic acid {(S) -3-methyl-1- [3-oxo-1- (pyridine-2)
-Sulfonyl) -azepan-4-ylcarbamoyl] -butyl} amide (0.0
To the solution of 3 g) was added triethylamine (0.04 mL). Heat the reaction for the second time
At reflux, it was concentrated, concentrated and dried under reduced pressure. The residue is then mixed in a similar
And heated to reflux overnight. Concentrate the reaction, and column chromatograph the residue.
-To give the title compound. Diastereomers were separated by HPLC.       [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 19/08 A61P 19/08 19/10 19/10 21/04 21/04 29/00 101 29/00 101 33/02 33/02 33/06 33/06 33/08 33/08 33/12 33/12 35/04 35/04 43/00 111 43/00 111 // C07B 57/00 380 C07B 57/00 380 C07M 7:00 C07M 7:00 (72) Inventor Daniel Frank Baber United States 19002 Ambler, PA, Ambler, PA 290F F-term (reference) 4C071 AA01 BB01 CC22 DD04 EE13 FF23 HH28 JJ05 KK14 KK16 LL01 4C086 AA01 AA02 AA03 BC31 GA16 MA01 MA04 MA17 MA22 MA23 MA52 MA55 MA66 NA14 ZA67 ZA94 ZA96 ZA97 ZB15 ZB26 ZB38 ZC20 ZC21 4H006 AA02 AC83 AD17

Claims (1)

Claims: 1. Thieno [3,2-b] thiophene-2-carboxylic acid {(S)-
Also known as 3-methyl-1-[(2,2 ', 4-trideuterio) -3-oxo-1- (pyridine-2-sulfonyl) -azepan-4-ylcarbamoyl] -butyl} amide. , Formula (I): A compound of the formula (I) 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 protein selected from the group consisting of cysteine and serine proteases, which comprises administering an effective amount of a compound according to claim 1 to a patient in need of protease inhibition. -A method for inhibiting zeolites. 4. The method of 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. A compound of formula (I): A method for synthesizing a compound represented by the formula (I): a) Formula (II): Oxidizing the compound of formula (II) with an oxidizing agent to obtain the compound of formula (I) as a mixture of diastereomers: b) deuterating the protonated isomer with a deuterating material; Obtained as a mixture of diastereomers of the formula (I): 15. The method of claim 14, wherein the oxidizing agent is a sulfur trioxide-pyridine complex in DMSO and triethylamine. 16. The method according to claim 16, wherein the deuterating agent is CD ₃ OD: D ₂ O (
The method according to claim 14, wherein the ratio is 10: 1). 17. The method according to claim 14, further comprising a step of separating diastereomers by separation means. 18. The method according to claim 17, wherein the separation means is high pressure liquid chromatography (HPLC).