EP2078027A1 - Cathepsin proteases inhibitors - Google Patents

Abstract

The invention provides compounds (I) and pharmaceutical compositions thereof, and more particularly, bicyclic carbamates, which are useful as cathepsin inhibitors, and methods for using such compounds or pharmaceutically acceptable salts thereof, wherein X is 0 or S; R1 is OR2, halo, (CR2)nR3, nitro, cyano, amino, aniido, sulfonamide, or an optionally substituted C1-6 alkyl, C2-6 alkenyl, or C3-6 alkynyl;R2 is H, (CR2)nR3, or an optionally substituted C1-6alkyl, C2-6 alkenyl or C3-6 alkynyl; R3 is an optionally substituted aryl, heteroaryl, carbocyciic ring or heterocyclic ring; m is 1-3; and n is 0-4.

Description

COMPOSITIONS AND METHODS FOR INHIBITING CATHEPSIN PROTEASES

Cross Reference to Related Applications

[0001] This application claims the benefit of U.S. provisional application serial no. 60/862,500, filed October 23, 2006, which is incorporated herein by reference in its entirety.

Background Art

[0002] Most cathepsins are endopeptidases belonging to the family of papain-like cysteine proteases. Cathepsins are generally highly concentrated in lysosomal and endosomal compartments and play a role in a broad array of physiological processes. Among them are nonspecific functions such as degradation of both internalized and cellular proteins, as well as more specialized functions in the processing of enzymes and hormones. Various diseases are linked to the overexpression of certain proteases. For example, inhibition of specific cathepsin proteases is believed to have therapeutic implications on pathological conditions associated with cellular homeostasis, apoptosis, tumor invasion and metastasis, bone resorption and antigen presentation.

[0003] Most small molecule inhibitors of proteases, including those for cathepsins, consist of a peptidomimetic that recognizes specific pockets in the enzyme and defines substrate selectivity, and an electrophilic "warhead" that makes important contacts to the catalytic domain. The nature of the electrophilic group determines the classification of these small molecules into reversible and irreversible inhibitors. Several electrophilic groups have been routinely employed in the inhibition of proteases by covalent interaction of enzyme with inhibitors. For example, aldehydes, semicarbazones, nitriles and ketones usually lead to reversible inhibition, whereas halomethylketones, epoxides and Michael acceptors are among the functionalities used for irreversible inhibition.

[0004] To date, relatively few protease inhibitors have successfully progressed through clinical trials. Major issues involve non-druglike properties of the peptidic part of the inhibitor and/or the lack of selectivity, often attributed to non-specific interactions of the electrophilic functionality with endogenous nucleophiles in vivo. In order to circumvent these drawbacks commonly seen with the classical approach of substrate-based drug design, novel molecular concepts need to be explored. Disclosure of the Invention

[0005] The invention provides compounds and pharmaceutical compositions thereof, which may be useful as inhibitors for cathepsin proteases.

[0006] In one aspect, the present invention provides compounds of Formula (1):

or pharmaceutically acceptable salts thereof, wherein:

X is O or S;

R¹ is OR², halo, (CR₂)_nR³, nitro, cyano, amino, amido, sulfonamide, or an optionally substituted Ci_6alkyl, C₂-6 alkenyl, or C3-6 alkynyl;

R² is H, (CR₂)_nR³, or an optionally substituted Ci_6alkyl, C₂-6 alkenyl or C3_6 alkynyl;

R³ is an optionally substituted aryl, heteroaryl, carbocyclic ring or heterocyclic ring; m is 1-3; and n is 0-4.

[0007] In one embodiment, m is 1. For example, R¹ is halo or OR², wherein R² may be an optionally substituted phenyl, benzyl or Ci_₆ alkyl. In some examples, compounds having Formula (1) have a cis stereoconformation. In other examples, compounds having Formula (1) have a trans stereoconformation.

[0008] In another aspect, the invention provides pharmaceutical compositions comprising compounds having Formula (1) and a pharmaceutically acceptable excipient.

[0009] In yet another aspect, the invention provides methods for inhibiting a cathepsin protease, comprising administering to a system or a subject in need thereof, a therapeutically effective amount of a compound of Formula (1), or pharmaceutically acceptable salts or pharmaceutical compositions thereof, thereby inhibiting said cathepsin protease.

[0010] The present invention also provides methods for treating a condition or disease mediated by cathepsin protease activity, comprising administering to a system or a subject in need thereof, a therapeutically effective amount of a compound of Formula (1), or pharmaceutically acceptable salts or pharmaceutical compositions thereof, thereby treating said cathepsin protease-mediated condition or disease. In one embodiment, the invention provides methods for treating a condition or disease mediated by papain-like cathepsin protease, including but not limited to cellular homeostasis, apoptosis, tumor invasion and metastasis, bone resorption and antigen presentation. For example, the compounds of the invention may be used to treat osteoporosis, arthritis, asthma, auto-immune disease and tumors.

[0011] In yet another aspect, the invention provides the use of compounds having Formula (1) for the manufacture of a medicament for treating a disease mediated by cathepsin protease, more particularly papain-like cathepsin protease.

[0012] In the above methods for using the compounds of the invention, a compound having Formula (1) may be administered to a system comprising cells or tissues. In other embodiments, a compound having Formula (1) may be administered to a human or animal subject.

Definitions

[0013] "Alkyl" refers to a moiety and as a structural element of other groups, for example halo-substituted-alkyl and alkoxy, and may be straight-chained or branched. An optionally substituted alkyl, alkenyl or alkynyl as used herein may be optionally halogenated (e.g., CF₃), or may have one or more carbons that is substituted or replaced with a heteroatom, such as NR, O or S (e.g., -OCH₂CH₂O-, alkylthiols, thioalkoxy, alkylamines, etc).

[0014] "Aryl" refers to a monocyclic or fused bicyclic aromatic ring containing carbon atoms. For example, aryl may be phenyl or naphthyl. "Arylene" means a divalent radical derived from an aryl group.

[0015] "Heteroaryl" as used herein is as defined for aryl above, where one or more of the ring members are a heteroatom. Examples of heteroaryls include but are not limited to pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl, benzothiopyranyl, benzo[l,3]dioxole, imidazolyl, benzo-imidazolyl, pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, thienyl, etc.

[0016] A "carbocyclic ring" as used herein refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring containing carbon atoms, which may optionally be substituted, for example, with =0. Examples of carbocyclic rings include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylene, cyclohexanone, etc. [0017] A "heterocyclic ring" as used herein is as defined for a carbocyclic ring above, wherein one or more ring carbons is a heteroatom. For example, a heterocyclic ring may contain N, O, S, -N=, -S-, -S(O), -S(O)₂-, or -NR- wherein R may be hydrogen, Ci^alkyl or a protecting group. Examples of heterocyclic rings include but are not limited to morpholino, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone, l,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc.

[0018] Unless otherwise indicated, when a substituent is deemed to be "optionally substituted," it is meant that the substituent is a group that may be substituted with one or more group(s) individually and independently selected from, for example, an optionally halogenated alkyl, alkenyl, alkynyl, alkoxy, alkylamine, alkylthio, alkynyl, amide, amino, including mono- and di-substituted amino groups, aryl, aryloxy, arylthio, carbonyl, carbocyclic, cyano, cycloalkyl, halogen, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, heterocyclic, hydroxy, isocyanato, isothiocyanato, mercapto, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, perhaloalkyl, perfluoroalkyl, silyl, sulfonyl, thiocarbonyl, thiocyanato, trihalomethanesulfonyl, and the protected compounds thereof. The protecting groups that may form the protected compounds of the above substituents are known to those of skill in the art and may be found in references such as Greene and Wuts, Protective Groups in Organic Synthesis, 3^rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, which are incorporated herein by reference in their entirety.

[0019] The terms "co-administration" or "combined administration" or the like as used herein are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

[0020] The term "pharmaceutical combination" as used herein refers to a product obtained from mixing or combining active ingredients, and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g. a compound of Formula (1) and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g. a compound of Formula (1) and a co-agent, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the active ingredients in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more active ingredients.

[0021] The term "therapeutically effective amount" means the amount of the subject compound that will elicit a biological or medical response in a cell, tissue, organ, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

[0022] The term "administration" and or "administering" of the subject compound should be understood to mean as providing a compound of the invention including a pro-drug of a compound of the invention to the individual in need of treatment.

Details Description of the Invention

[0023] The invention provides compounds and compositions for inhibiting cathepsin proteases, more particularly papain-like cathepsin proteases. The present invention also provides methods for treating a condition or disease mediated by cathepsin protease activity, particularly a condition or disease mediated by papain-like cathepsin protease, comprising administering to a system or a subject in need thereof, a therapeutically effective amount of a compound of Formula (1), or pharmaceutically acceptable salts or pharmaceutical compositions thereof, thereby treating said papain-like cathepsin protease-mediated condition or disease.

[0024] Papain-like cysteine proteases have been identified as key proteolytic activities in degenerative, invasive, and immune system related disorders. (Lecaille et al., Chem. Rev. 2002, 102:4459-4488; Bromine et al., Curr. Pharm. Des. 2002, 8:1639-1658). For example, cathepsin K is the major bone-degrading activity in osteoclasts, and its selective inhibition may be beneficial for treating osteoporosis and certain forms of arthritis. Cathepsin S plays an important role in MHC class II dependent antigen presentation; inhibition of cathepsin S significantly decreases the response to antigens, rendering cathepsin S as a drug target for asthma and certain auto-immune diseases. (Riese et al., J. Clin. Invest. 1998, 101:2351-2363). Cathepsins also have been implicated in tumor invasion and metastasis, and in tumors of the central nervous systems, such as astrocytoma, glioblastoma and meningioma. (Berquin et al., Perspect. Drug Discovery Design 1995, 2:371-388; Levicar et al., J. Neurooncol. 2002, 58:21- 32).

[0025] In one aspect, the compounds of the invention have Formula (1):

or pharmaceutically acceptable salts thereof, wherein:

X is O or S;

R¹ is OR², halo, (CR₂^R³, nitro, cyano, amino, amido, sulfonamide, or an optionally substituted Ci_6alkyl, C₂-6 alkenyl, or C3_6 alkynyl;

R² is H, (CR₂)_nR³, or an optionally substituted Ci_6alkyl, C₂-6 alkenyl or C3_6 alkynyl;

R³ is an optionally substituted aryl, heteroaryl, carbocyclic ring or heterocyclic ring; m is 1-3; and n is 0-4.

[0026] In one embodiment, the compounds of the invention may be used to inhibit papain- like cathepsin proteases. Table 1 shows the apparent inhibition constants (K^app), μM) of various compounds of the invention for inhibition of various cathepsins St_rel = relative stereoconformation.

entry X St₁ rel R CatB CatS CatC CatL CatK CatH CatX CatF CatV

8 O trans 0.046 0.473 1.727 1.270 2.438 >30 0.553 5.908 3.317

12 O cis 0.001 0.016 0.018 0.022 0.038 2.653 0.197 0.331 0.131

13 S cis 0.519 0.696 0.606 0.994 1.833 >100 7.149 >10 5.166

14 O cis 0.005 0.017 0.029 0.026 0.104 6.591 0.326 0.487 0.158

15 O cis 0.002 0.174 0.403 0.263 0.511 >10 0.547 3.760 1.289

16 O cis 0.008 0.298 0.191 0.230 1.267 >10 1.102 1.826 0.894

19 O trans OR = Br 0.158 4.553 >10 5.816 >30 >100 >10 >10 >10

6 >100 >100 >100 >100 >100 >100 >100 >100 >100 11 >100 >100 >100 >100 >100 >100 >100 >100 >100 21 >100 >100 >100 >100 >100 >100 >100 >100 >100

Table 1

[0027] Cyclic carbamates 8 and 12-16 showed inhibitory activity across the board of papain- like cathepsin proteases. Within this protease family, the apparent K₁ values are lowest for cathepsin B, followed by cathepsins S, C, L, and K. Moderate activities are seen against cathepsins V, X, F and H. The compounds are shown to be inactive against other families of cysteine proteases such as caspases and against members of the serine protease family such as hepsin, thrombin, MT-SPl or trypsin.

[0028] Generally, there is a drop in K₁ values when comparing the trans to the cis configured stereoisomers (cf. 8 vs 12). Analog 12, which contains the more hydrophobic dimethylphenyl group, seemed to be consistently more active in the cathepsin panel when compared to the phenyl substituted analog 14. The thiocarbamate 13 has substantially less inhibitory effects when compared to the oxo-carbamates. The alkyl-substituted analogs 15 (R = benzyl) and 16 (R = isopropyl) generally displayed about 10-fold higher K₁- values than 12 or 14. An exception is the low nanomolar activity of the benzyl substituted analog 15 against Cathepsin B, representing the most selective inhibitor for Cathepsin B in the series (100-fold).

[0029] Although the mechanism is not necessary to practice the invention, the mechanism of inhibition by the bicyclic carbamates of the invention is examined in a dialysis study. Briefly, cathepsin B is first incubated at 37⁰C with 0.2 μM of compound 12 (20Ox K₁) for complete suppression of catalytic activity (2.9 rfu/sec for inhibitor treated versus 121 rfu/sec for vehicle control). Then the mixtures are dialyzed extensively at 4⁰C to remove unbound inhibitor before the cathepsin activities are re-measured at 37⁰C (51 rfu/sec for inhibitor treated versus 71 rfu/sec for vehicle control). The apparent recovery of cathepsin B activity in compound 12 treated sample after dialysis suggests that compound 12 is a partially reversible inhibitor. However, a careful examination of the progress curves revealed that the substrate conversion is accelerated over the course of the assay (indicated by the dotted arrows). This rate increase may likely caused by a temperature sensitive release of covalently bound compound 12 from the active site, since the substrate conversion of the control is not affected by the incubation temperature.

[0030] The potential adduct formation between cathepsin B and inhibitor (compound 12) is analyzed by mass spectrometry following tryptic/chymotryptic digestion. Two active site fragments of the test enzyme (cathepsin B) are identified as ⁷⁹EIRDQGSCGSC^*W³⁰ and ²²DQGSCGSC W³⁰ from the untreated cathepsin B sample. For inhibitor treated cathepsin B, both fragments gained a mass of 247, corresponding to a single covalent conjugation of 12 to the catalytic cysteine residues (C ). The stability of this adduct is sensitive to temperature increase. Compared to the control that is kept on ice, a 2-hour incubation at 37⁰C promoted the dissociation of this enzyme-inhibitor adduct, resulting in a 4-fold increase in abundance of a metabolite. The multiple reaction monitoring (MRM) characteristics of the metabolite are identical to that of the suggested dissociation product 11.

[0031] In summary, the invention provides compounds of Formula (1) that are selective for the cathepsin family, with subtype selectivities following the order B > S > C,L,K > V,X,F,H. A preference for hydrophobic R-groups in cis configuration relative to the carbamate oxygen is observed, with apparent K₁ values for Cathepsin B in the single digit nanomolar range. The carbamate functionality in the scaffold is substantially destabilized, and offers a weak point for nucleophilic attack by the active site cysteine thiol. The bicycle subsequently undergoes ring- opening and covalently binds to the catalytic cysteine, leading to inhibition of the enzyme. This hypothesis is in accordance with the mass spectrometric analysis of digested enzyme. After incubation with compound 12, the fragments which include the catalytic cysteine covalently carry the inhibitor. The non-linear rate recovery seen in dialysis studies and characterization of metabolites via MRM suggest that the inhibitor slowly gets hydrolyzed off the enzyme as its ring-opened synthetic precursor 11 at ambient temperature.

Administration and Pharmaceutical Compositions

[0032] In general, compounds of the invention will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.03 to 2.5 mg/kg per body weight. An indicated daily dosage in the larger mammal, e.g. humans, is in the range from about 0.5 mg to about 100 mg, conveniently administered, e.g. in divided doses up to four times a day or in retard form. Suitable unit dosage forms for oral administration comprise from ca. 1 to 50 mg active ingredient.

[0033] Compounds of the invention may be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form.

[0034] Pharmaceutical compositions comprising a compound of the present invention in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent may be manufactured in a conventional manner by mixing, granulating or coating methods. For example, oral compositions may be tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets, together with c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; and if desired, d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions may be aqueous isotonic solutions or suspensions, and suppositories may be prepared from fatty emulsions or suspensions. [0035] The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Suitable formulations for transdermal applications include an effective amount of a compound of the present invention with a carrier. A carrier may include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used. Suitable formulations for topical application, e.g., to the skin and eyes, may be aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0036] Compounds of the invention may be administered in therapeutically effective amounts in combination with one or more therapeutic agents (pharmaceutical combinations). Where the compounds of the invention are administered in conjunction with other therapies, dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth.

[0037] For example, the compounds of the invention may be used in combination with a chemotherapeutic agent to treat a cell proliferative disorder and tumors. Examples of chemotherapeutic agents which may be used in the compositions and methods of the invention include but are not limited to anthracyclines, alkylating agents (e.g., mitomycin C), alkyl sulfonates, aziridines, ethylenimines, methylmelamines, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, folic acid analogs (e.g., dihydrofolate reductase inhibitors such as methotrexate), purine analogs, pyrimidine analogs, enzymes, podophy Ho toxins, platinum- containing agents, interferons, and interleukins. Particular examples of known chemotherapeutic agents which may be used in the compositions and methods of the invention include, but are not limited to, busulfan, improsulfan, piposulfan, benzodepa, carboquone, meturedepa, uredepa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, aclacinomycins, actinomycin F(I), anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carzinophilin, chromomycin, dactinomycin, daunorubicin, daunomycin, 6-diazo-5-oxo-l-norleucine, doxorubicin, epirubicin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, fluorouracil, tegafur, L-asparaginase, pulmozyme, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, carboplatin, cisplatin, defofamide, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, etoposide, flutamide, gallium nitrate, hydroxyurea, interferon- alpha, interferon-beta, interferon-gamma, interleukin-2, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2- ethylhydrazide, procarbazine, razoxane, sizofiran, spirogermanium, paclitaxel, tamoxifen, teniposide, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, urethane, vinblastine, vincristine, and vindesine.

[0038] Synergistic effects may also occur with other immunomodulatory or antiinflammatory substances, for example when used in combination with cyclosporin, rapamycin, or ascomycin, or immunosuppressant analogues thereof, for example cyclosporin A (CsA), cyclosporin G, FK-506, rapamycin, or comparable compounds, corticosteroids, cyclophosphamide, azathioprine, methotrexate, brequinar, leflunomide, mizoribine, mycophenolic acid, mycophenolate mofetil, 15-deoxyspergualin, immunosuppressant antibodies, especially monoclonal antibodies for leukocyte receptors, for example MHC, CD2, CD3, CD4, CD7, CD25, CD28, B7, CD45, CD58 or their ligands, or other immunomodulatory compounds, such as CTLA41g.

[0039] The invention also provides for a pharmaceutical combinations, e.g. a kit, comprising a) a first agent which is a compound of the invention as disclosed herein, in free form or in pharmaceutically acceptable salt form, and b) at least one co-agent. The kit may comprise instructions for its administration. Processes for Making Compounds of the Invention [0040] The compounds of the present invention may be prepared according to Scheme 1.

[0041] In Scheme 1, the reagents and conditions are: (a) CbzCl, 0.5 M Na₂Cθ₃/dioxane 5:2, rt, 5h; (b) mCPBA, DCM, O⁰C-It, 7h; (c) ROH, 2 M NaOH/MeCN 1:4, reflux, 48h; (d) 1 atm H₂, Pd/C (cat), EtOH, rt, 3h; (e) triphosgene, NEt₃, DCM, O⁰C, Ih; (f) NaH, rt, 18h; (g) PPh₃, DEAD, p-nitrobenzoic acid, THF, 5O⁰C, 48h; (h) NaOH, MeOH, rt, Ih; (i) 1 atm H₂, Pd/C (cat), EtOH, rt, 3h; Q) OsO₄, NMO, citric acid, tBuOH/H₂O 1:1, 5O⁰C, 12h; (k) HBr, HOAc, O°C-rt, 3h; (1) 2,3-dimethylphenol, K₂CO₃, MeCN, 6O⁰C, 12h; (m) MeNH₂, MeOH, 5O⁰C, 2h; (n) CDI, DMAP, benzene, reflux, 4h. Compounds 3-21 are racemic mixtures. [0042] According to synthetic procedures described by Paioni (U.S. patent no. 4,160,837, incorporated herein by reference in its entirety), the tetrahydropyridine 2 is converted to the racemic Cbz-protected epoxide 3 in two steps. Opening of the epoxide 3 (ROH = 2,3- dimethylphenol) under basic aqueous conditions gives a mixture of regioisomers. As expected for a favored trans-diaxial ring-opening, the ratio of 4-aryloxy piperidines 4 versus 3- aryloxypiperidines 5 is about 5:1. The regioisomers are chromatographic ally separated and hydrogenolytically deprotected to give the open forms 6 and 7. The cyclization of 6 requires a two step procedure. First the piperidine-nitrogen is selectively carbonylated to the corresponding chloroformate using triphosgene and triethylamine, then the 3 -hydroxy group is deprotonated using sodium hydride, which leads to spontaneous intramolecular cyclization to give racemic ?r«n5-4-(2,3-dimethyl-phenoxy)-6-oxa-l-aza-bicyclo[3.2.1]octan-7-one 8. Attempts to cyclize the chloroformate intermediate of 7 to the bicycle 9 are unsuccessful, probably due to the unfavorable boat conformation of the piperidine ring required for intramolecular ring closure.

[0043] The Mitsunobu reaction using p-nitrobenzoic acid as the nucleophile is employed to invert the stereocenter at position 3 of compound 4. Alternatively, the inversion may be accomplished by an oxidation/reduction sequence using Pyr-Sθ₃ and Red- Al. Saponification of the intermediate 10 in methanolic base followed by deprotection gives piperidine 11. Intramolecular cyclization analogous to the above described procedure gives ds-4-(2,3- dimethyl-phenoxy)-6-oxa-l-aza-bicyclo[3.2.1]octan-7-one 12.

[0044] Both stereoisomers 8 and 12 are isolated as white, stable solids. The compounds are also stable in aqueous basic or neutral solutions, while spontaneous ring opening is observed under fairly acidic conditions (pH<2). Analogs 13-16 (Table 1, supra) are synthesized in a similar fashion to the synthesis of 12. In the case of analog 13, thiophosgene is used instead of phosgene in the cyclization step; for analogs 15 and 16, the corresponding alcohol (benzylalcohol for 15, isopropanol for 16) is used as solvent together with NaH in the opening of epoxide 3. An alternative synthetic route via dihydroxylation leads to the protected tetrahydropyridine 17. Selective cyclization and alkylation of the deprotected diol failed to give the desired bicyclic carbamate analogs. Treatment of the epoxide 3 with HBr selectively gives bromide 18 which could be cyclized to the carbamate 19, but any attempts to displace the bromide were unsuccessful. The control compound 21 is synthesized in three steps starting from epibromohydrin 20. [0045] Furthermore, compounds of the present invention may be obtained in the free form, or as a salt thereof if salt forming groups are present, or as esters if ester forming groups are present. Compounds of the present invention that have acidic groups may be converted into salts with pharmaceutically acceptable bases, e.g., an aqueous alkali metal hydroxide, advantageously in the presence of an ethereal or alcoholic solvent, such as a lower alkanol. Resulting salts may be converted into the free compounds, e.g., by treatment with acids. These or other salts may also be used for purification of the compounds obtained. Ammonium salts may be obtained by reaction with the appropriate amine, e.g., diethylamine, and the like.

[0046] In certain aspects, compounds of the present invention having basic groups may be converted into acid addition salts, especially pharmaceutically acceptable salts. These may be formed, for example, with inorganic acids such as mineral acids (e.g., sulfuric acid, a phosphoric or hydrohalic acid); organic carboxylic acids (e.g., Ci-C₄ alkyl carboxylic acids such as acetic acid, which may be unsubstituted or substituted by halogen; saturated or unsaturated dicarboxylic acids, such as oxalic, succinic, maleic or fumaric acid; hydroxycarboxylic acids, such as glycolic, lactic, malic, tartaric or citric acid); amino acids, such as aspartic or glutamic acid); organic sulfonic acids (e.g., Ci-C₄ alkylsulfonic acids such as methanesulfonic acid); or arylsulfonic acids which may be unsubstituted or substituted (for example, by halogen). Preferred may be salts formed with hydrochloric acid, methanesulfonic acid and maleic acid.

[0047] In view of the close relationship between the free compounds and the compounds in the form of their salts or esters, whenever a compound is referred to in this context, a corresponding salt or ester is also intended, provided such is possible or appropriate under the circumstances. The compounds, including their salts, may also be obtained in the form of their hydrates, or include other solvents used for their crystallization.

[0048] The compounds of the present invention that comprise free hydroxyl groups may also exist in the form of pharmaceutically acceptable, physiologically cleavable esters, and as such may be included within the scope of the invention. Such pharmaceutically acceptable esters may be preferably prodrug ester derivatives, such being convertible by solvolysis or cleavage under physiological conditions to the corresponding compounds of the present invention which comprise free hydroxyl groups. Suitable pharmaceutically acceptable prodrug esters may be those derived from a carboxylic acid, a carbonic acid monoester or a carbamic acid, preferably esters derived from an optionally substituted lower alkanoic acid or an arylcarboxylic acid. [0049] As will be apparent to one of skill in the art, certain compounds of the present invention may possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, enantiomers, geometric isomers and individual isomers may be all intended to be encompassed within the scope of the present invention.

[0050] Detailed examples of the synthesis of a compound of Formula (1) can be found in the Examples, infra, to illustrate, but not limit the invention.

Example 1 trans-4-(2,3-Dimethyl-phenoxy)-6-oxa-l-aza-bicyclor3.2.11octan-7-one (8)

3,6-dihydro-2H-pyridine-l-carboxylic acid benzyl ester (2)

[0051] 1,2,3,6-Tetrahydropyridine (1.1 mL, 12 mmol) is dissolved in dioxane (10 mL). An aqueous solution of 0.5 M sodium carbonate (25 mL, 12.5 mmol) is added followed by benzyl chloroformate (1.8 mL, 12 mmol). The mixture is stirred for 3 h at rt, then diluted with ethyl acetate (50 mL). The organic layer is separated, washed with water and dried over magnesium sulfate. The solvents are removed in vacuo to give 3,6-dihydro-2H-pyridine-l-carboxylic acid benzyl ester as a colorless liquid. ¹H-NMR (400 MHz, CDCl₃) δ = 7.40-7.33 (m, 5H), 5.85 (m, IH), 5.68 (m, IH), 5.19 (s, 2H), 4.00 (t, J = 2 Hz, 2H), 3.60 (t, J = 5.7 Hz, 2H), 1.72 (m, 2H). MS calcd. for Ci₃H₁₆NO₂ (M+H⁺) 218.1, found 218.3.

7-oxa-3-aza-bicvclor4.1.01heptane-3-carboxylic acid benzyl ester (3) [0052] The 3,6-Dihydro-2H-pyridine-l-carboxylic acid benzyl ester 2 (2.6 g, 12 mmol) is dissolved in dichloromethane (100 mL). m-Chloroperbenzoic acid (4.5 g, 18 mmol) dissolved in dichloomethane (40 mL) is added dropwise over 20 min. The mixture is stirred at rt for 7 h, then the solution is washed with 1 M aqueous sodium carbonate three times and once with brine. The organic phase is separated, dried (MgSO₄) and concentrated in vacuo to give the racemic epoxide 7-oxa-3-aza-bicyclo[4.1.0]heptane-3-carboxylic acid benzyl ester (3) as a colorless oil. ¹H-NMR (400 MHz, CDCl₃) δ = 7.40-7.32 (m, 5H), 5.14 (s, 2H), 3.99 (m, IH), 3.80 (m, IH), 3.53 (m, IH), 3.31 (m, IH), 3.24 (m, IH), 2.10 (m, IH), 1.96 (m, IH). MS calcd. for Ci₃H₁₆NO₃ (M+H⁺) 234.1, found 234.3. trans-4-(2,3-Dimethyl-phenoxy)-3-hvdroxy-piperidine-l-carboxylic acid benzyl ester (4) and trans-3-(2,3-dimethyl-phenoxy)-4-hvdroxy-piperidine-l-carboxyric acid benzyl ester (5)

[0053] 2,3-Dimethylphenol and 7-oxa-3-aza-bicyclo[4.1.0]heptane-3-carboxylic acid benzyl ester (3) (1.0 g, 4.3 mmol) are dissolved in acetonitrile (20 mL). 2 N aqueous sodium hydroxide (5 mL) is added and the mixture is heated to reflux for 48 h. The acetonitrile is removed in vacuo, and the aqueous remainder is diluted with water (20 mL) and extracted twice with dichloromethane (50 mL). The combined organic layers are washed with water and brine, dried over MgSO₄ and concentrated. The crude products are separated by flash chromatography (hexanes/ethyl acetate gradient) to give trans-4-(2,3-dimethyl-phenoxy)-3-hydroxy-piperidine-l- carboxylic acid benzyl ester 4 and the regioisomer trans-3-(2,3-dimethyl-phenoxy)-4-hydroxy- piperidine-1-carboxylic acid benzyl ester 5 as white solids. 4: ¹H-NMR (400 MHz, CDCl₃) δ = 7.58-7.50 (m, 5H), 7.24 (t, J = 7.9 Hz, IH), 7.01 (d, J = 7.5 Hz, IH), 6.97 (d, J = 8.2 Hz, IH), 5.35 (s, 2H), 4.44 (m, IH), 4.26 (m, IH), 4.08 (m, IH), 4.01 (m, IH), 3.50 (m, 2H), 2.47 (s, 3H), 2.35 (s, 3H), 2.28 (m, IH), 1.85 (m, IH). MS calcd. for C₂]H₂₆NO₄ (M+H⁺) 356.2, found 356.3. 5: ¹H-NMR (400 MHz, CDCl₃) δ = 7.53-7.46 (m, 5H), 7.15 (t, br, IH), 7.01 (d, J = 7.5 Hz, IH), 6.95 (d, br, IH), 5.35 (br, 2H), 4.28-4.15 (m, 4H), 3.28-3.22 (m, 2H), 2.46 (s, 3H), 2.34 (m, IH), 2.33 (s, 3H), 1.84 (m, IH). MS calcd. for C₂]H₂₆NO₄ (M+H⁺) 356.2, found 356.3.

trans-4-(2,3-Dimethyl-phenoxy)-piperidin-3-ol (6)

[0054] trans-4-(2,3-Dimethyl-phenoxy)-3-hydroxy-piperidine-l-carboxylic acid benzyl ester 4 (700 mg, 2.0 mmol) is dissolved in EtOH (10 mL) and a catalytic amount of palladium (10% on charcoal) is added. After stirring for 3 h at rt under 1 atm hydrogen, the mixture is filtered over CELITE® and washed with EtOH. The filtrate is concentrated in vacuo to give the piperidinol 6 as a white solid: ¹H-NMR (400 MHz, MeOD) δ = 7.04 (t, J = 7.9 Hz, IH), 6.85 (d, J = 6.0 Hz, IH), 6.80 (d, J = 7.7 Hz, IH), 4.38 (m, IH), 3.96 (m, IH), 3.34 (m, IH), 3.16 (m, IH), 2.97 (m, 2H), 2.28 (s, 3H), 2.27 (m, IH), 2.21 (s, 3H), 1.83 (m, IH). MS calcd. for C]₃H₂₀NO₂ (M+H⁺) 222.1, found 222.3.

trans-4-(2,3-Dimethyl-phenoxy)-6-oxa-l-aza-bicyclor3.2.11octan-7-one (8) [0055] The piperidinol 6 (50 mg, 0.23 mmol) is dissolved in DCM (10 mL). Triphosgene (45 mg, 0.15 mmol) is added and the mixture is cooled to O ⁰C. Then triethylamine (96 μL, 0.69 mmol) is added slowly in increments over 50 min. Sodium hydride (30 mg, 0.75 mmol) is added, and the mixture is warmed to rt and stirred at rt for 18h. The mixture is washed with 0.25 M phosphate buffer (pH 6.2), the organic layer is separated, dried (MgSO₄) and concentrated. The crude product is purified on reverse phase HPLC (MeCN/H₂0 gradient, no acidic additives) to give 8 as a white solid: ¹H-NMR (400 MHz, CDCl₃) δ = 7.06 (t, J = 7.9 Hz, IH), 6.85 (d, J = 7.5 Hz, IH), 6.66 (d, J = 8.2 Hz, IH), 4.80 (t, J = 4.3 Hz, IH), 4.73 (t, J = 4.3 Hz, IH), 3.70 (d, J = 11.9 Hz, IH), 3.47-3.38 (m, 2H), 3.30 (m, IH), 2.31 (m, IH), 2.29 (s, 3H), 2.19 (s, 3H), 2.00 (m, IH). MS calcd. for C₁₄H₁₈NO₃ (M+H⁺) 248.1, found 248.3.

Example 2 trans-3-(2,3-Dimethyl-phenoxy)-piperidin-4-ol (7)

[0056] Trans-3-(2,3-Dimethyl-phenoxy)-piperidin-4-ol (7) is obtained as a white solid following the procedure for the piperidinol 6 in Example 1, replacing 4 with 5 as the reagent. ¹H-NMR (400 MHz, CDCl₃) δ = 7.03 (t, J = 7.9 Hz, IH), 6.82 (d, J = 6.2 Hz, IH), 6.81 (d, J = 7.5 Hz, IH), 4.08 (m, IH), 3.88 (m, IH), 3.34 (m, IH), 3.12 (m, IH), 2.73 (m, IH), 2.59 (m, IH), 2.27 (s, 3H), 2.16 (s, 3H), 2.12 (m, IH), 1.64 (m, IH). MS calcd. for C₁₃H₂₀NO₂ (M+H⁺) 222.1, found 222.3.

Example 3 cis-4-(2,3-Dimethyl-phenoxy)-6-oxa-l-aza-bicyclor3.2.11octan-7-one (12)

[0057] The benzyl carbamate 4 (0.93 g, 2.6 mmol) is dissolved in THF (50 mL) together with triphenylphosphine (2.07 g, 7.9 mmol) and p-nitrobenzoic acid (1.32 g, 7.9 mmol). The mixture is cooled to O ⁰C, then DEAD (1.24 mL, 7.9 mmol) is added slowly. The mixture is stirred at 50°C for 48h, concentrated and purified by flash chromatography (hexanes/ethyl acetate gradient) to yield cis-4-(2,3-Dimethyl-phenoxy)-3-(4-nitro-benzoyloxy)-piperidine-l- carboxylic acid benzyl ester as a white solid: ¹H-NMR (400 MHz, CDCl₃) δ = 8.21 (d, J = 8.8 Hz, 2H), 8.03 (d, J = 8.0 Hz, 2H), 7.37-7.24 (m, 5H), 6.99 (t, J = 7.8 Hz, IH), 6.79 (d, J = 7.4 Hz, IH), 6.73 (d, J = 8.2 Hz, IH), 5.36 (m, IH), 5.14 (m, IH), 5.03 (m, IH), 4.69 (m, IH), 4.16 (m, IH), 3.96-3.46 (m, 3H), 2.24 (s, 3H), 2.19 (m, IH), 2.05 (s, 3H), 2.02 (m, IH). MS calcd. for C₂₈H₂₉N₂O₇ (M+H⁺) 505.2, found 505.3. cis-4-(2,3-Dimethyl-phenoxy)-3-hvdroxy-piperidine-l-carboxylic acid benzyl ester (10) [0058] Powdered NaOH (0.4 g, 10 mmol) is dissolved in MeOH (20 mL). cis-4-(2,3- Dimethyl-phenoxy)-3-(4-nitro-benzoyloxy)-piperidine-l-carboxylic acid benzyl ester (0.5 g, 1 mmol) is added and the mixture is stirred for Ih at rt. The mixture is concentrated, DCM is added and the organic layer is washed with water twice. The organic layer is dried over MgSO₄ and concentrated to give cis-4-(2,3-dimethyl-phenoxy)-3-hydroxy-piperidine-l-carboxylic acid benzyl ester as a white solid: ¹H-NMR (400 MHz, CDCl₃) δ = 7.39-7.28 (m, 5H), 7.05 (t, J = 7.9 Hz, IH), 6.84 (d, J = 7.5 Hz, IH), 6.77 (d, J = 8.2 Hz, IH), 5.17 (s, 2H), 4.55 (m, IH), 3.94 (m, IH), 3.80 (m, IH), 3.65 (m, IH), 3.52 (m, 2H), 2.29 (s, 3H), 2.19 (s, 3H), 2.09 (m, IH), 1.74 (m, IH). MS calcd. for C₂]H₂₆NO₄ (M+H⁺) 356.2, found 356.3.

cis-4-(2,3-Dimethyl-phenoxy)-piperidin-3-ol (11)

[0059] cis-4-(2,3-Dimethyl-phenoxy)-3-hydroxy-piperidine-l-carboxylic acid benzyl ester (300 mg, 0.85 mmol) is dissolved in EtOH (20 mL) and a catalytic amount of palladium (10% on charcoal) is added. After stirring for 3 h at rt under 1 atm hydrogen, the mixture is filtered over CELITE® and washed with EtOH. The filtrate is concentrated in vacuo to give cis-4-(2,3- Dimethyl-phenoxy)-piperidin-3-ol (11) as a white solid: ¹H-NMR (400 MHz, MeOD) δ = 6.98 (t, J = 7.9 Hz, IH), 6.82 (d, J = 8.2 Hz, IH), 6.74 (d, J = 7.5 Hz, IH), 4.48 (m, IH), 3.90 (m, IH), 3.04 (m, IH), 2.91 (m, IH), 2.83 (m, IH), 2.64 (m, IH), 2.24 (s, 3H), 2.20 (s, 3H), 1.98 (m, IH), 1.70 (m, IH). MS calcd. for C₁₃H₂₀NO₂ (M+H⁺) 222.1, found 222.3.

cis-4-(2,3-Dimethyl-phenoxy)-6-oxa-l-aza-bicyclor3.2.11octan-7-one (12) [0060] cis-4-(2,3-Dimethyl-phenoxy)-piperidin-3-ol 11 (50 mg, 0.23 mmol) is dissolved in DCM (10 mL). Triphosgene (45 mg, 0.15 mmol) is added and the mixture is cooled to O ⁰C. Then triethylamine (96 μL, 0.69 mmol) is added slowly in increments over 50 min. After warming to rt, sodium hydride (30 mg, 0.75 mmol) is added, and the mixture is stirred at rt overnight. The mixture is diluted with DCM, washed with water, and the organic layer is separated, dried (MgSO₄) and concentrated. The crude product is purified on reverse phase HPLC (MeCN/H₂O gradient, no acidic additives) to give 12 as a white solid: ¹H-NMR (400 MHz, CDCl₃) δ = 7.03 (t, J = 7.9 Hz, IH), 6.85 (d, J = 7.5 Hz, IH), 6.72 (d, J = 8.2 Hz, IH), 4.87 (d, J = 5.1 Hz, IH), 4.43 (dd, J = 6.7 Hz, J = 9.5 Hz, IH), 3.56 (dd, J = 5.1 Hz, J = 12.2 Hz, IH), 3.47 (dd, J = 7.3 Hz, J = 14.1 Hz, IH), 3.47 (dt, J = 4.9 Hz, J = 13.7 Hz, IH), 2.89 (d, J = 12.1 Hz, IH), 2.35 (m, IH), 2.28 (s, 3H), 2.22 (m, IH), 2.18 (s, 3H). MS calcd. for C₁₄H₁₈NO₃ (M+H⁺) 248.1, found 248.3.

Example 4 cis-4-(2,3-Dimethyl-phenoxy)-6-oxa-l-aza-bicyclor3.2.11octane-7-thione (13)

[0061] Following the procedure for the bicyclic carbamate 12, replacing triphosgene with thiophosgene, the title compound 13 is prepared as a white solid: ¹H-NMR (400 MHz, CDCl₃) δ = 7.04 (t, J = 7.9 Hz, IH), 6.86 (d, J = 7.5 Hz, IH), 6.72 (d, J = 8.2 Hz, IH), 5.06 (d, J = 5.3 Hz, IH), 4.39 (t, J = 8.3 Hz, IH), 3.61 (m, 2H), 3.13 (m, IH), 3.05 (d, J = 12.1 Hz, IH), 2.31 (m, IH), 2.28 (s, 3H), 2.20 (s, 3H), 2.15 (m, IH). MS calcd. for C₁₄H₁₈NO₂S (M+H⁺) 264.1, found 264.3.

Example 5 cis-4-Phenoxy-6-oxa-l-aza-bicyclor3.2.11octan-7-one (14)

[0062] Following the procedure for the bicyclic carbamate 12, replacing 2,3-dimethylphenol with phenol, the title compound 14 is prepared as a white solid: ¹H-NMR (400 MHz, CDCl₃) δ = 7.34-7.28 (m, 2H), 7.05-6.94 (m, 3H), 4.93 (d, J = 4.6 Hz, IH), 4.50 (m, IH), 3.63 (m, IH), 3.51 (m, IH), 2.99 (m, IH), 2.94 (d, J = 11.9 Hz, IH), 2.39 (m, IH), 2.20 (m, IH). MS calcd. for C₁₂H₁₄NO₃ (M+H⁺) 220.1, found 220.3.

Example 6 cis-4-Benzyloxy-6-oxa-l-aza-bicyclor3.2.11octan-7-one (15)

[0063] The epoxide 3 (1.0 g, 4.3 mmol) is dissolved in benzyl alcohol (5 mL). Sodium hydride (0.86 g, 21.5 mmol) is added and the mixture is stirred at 50 °C overnight. Then the solvent is removed in vacuo and the remainder is purified by flash chromatography (hexanes/ethyl acetate gradient) to yield 3-hydroxy-4-isobutoxy-piperidine-l-carboxylic acid benzyl ester. Following the procedure for the bicyclic carbamate 12, the title compound 15 is prepared as a white solid: ¹H-NMR (400 MHz, CDCl₃) δ = 7.38-7.32 (m, 5H), 4.82 (d, J = 6.1 Hz, IH), 4.61 (s, 2H), 3.67 (m, IH), 3.52 (m, IH), 3.41 (m, IH), 2.86 (m, IH), 2.78 (d, J = 12.1 Hz, IH), 2.21 (m, IH), 2.04 (m, IH). MS calcd. for C₁₃H₁₆NO₃ (M+H⁺) 234.1, found 234.3. Example 7 cis-4-Isobutoxy-6-oxa-l-aza-bicyclor3.2.11octan-7-one (16)

[0064] Following the procedure for the bicyclic carbamate 15, benzyl alcohol with isobutanol, the title compound 16 is prepared as a white solid: ¹H-NMR (400 MHz, CDCI₃) δ = 4.83 (d, J = 5.1 Hz, IH), 3.56 (m, 2H), 3.43 (m, IH), 3.26 (m, 2H), 2.89 (m, IH), 2.81 (d, J = 12.0 Hz, IH), 2.21 (m, IH), 2.00 (m, IH), 1.84 (m, IH), 0.92 (d, J = 6.7 Hz, 6H). MS calcd. for Ci₀H₁₈NO₃ (M+H⁺) 200.1, found 200.3.

Example 8 trans-4-Bromo-6-oxa-l-aza-bicyclor3.2.11octan-7-one (19)

[0065] 7-oxa-3-aza-bicyclo[4.1.0]heptane-3-carboxylic acid benzyl ester (3) (1.0 g, 4.3 mmol) is dissolved in DCM (32 mL). The solution is cooled to 0⁰C, then hydrobromic acid (30% in acetic acid, 8 mL, 30 mmol) is added dropwise and the mixture is stirred for 3h at rt. The product precipitated and is filtered to give 4-bromo-piperidin-3-ol hydrobromide (18) as a white solid: ¹H-NMR (400 MHz, MeOD) δ = 4.46 (m, IH), 4.25 (m, IH), 3.77 (m, IH), 3.51 (m, IH), 3.36 (m, 2H), 2.88 (m, IH), 2.28 (m, IH). MS calcd. for C₅H₁₁BrNO (M+H⁺) 180.0, found 180.3.

[0066] 4-bromo-piperidin-3-ol hydrobromide (18) (100 mg, 0.4 mmol) and triphosgene (80 mg, 0.27 mmol) are suspended in DCM (20 mL) and cooled to O ⁰C. Then triethylamine (332 μL, 2.4 mmol) is added slowly in increments over 60 min. After warming to rt, the mixture is stirred at rt overnight. Purification by flash chromatography (hexanes/ethyl acetate gradient) to yield 19 as a white solid: ¹H-NMR (400 MHz, CDCl₃) δ = 4.71 (t, J = 4.2 Hz, IH), 4.42 (m, IH), 3.76 (d, J = 12.3 Hz, IH), 3.46-3.24 (m, 3H), 2.75 (m, IH), 2.07 (m, IH). MS calcd. for C₆H₉BrNO₂ (M+H⁺) 206.0, found 206.3.

Example 9 5-(2,3-Dimethyl-phenoxymethyl)-3-methyl-oxazolidin-2-one (21)

[0067] Epibromohydrin 20 (0.7 mL, 8.2 mmol), 2,3-dimethylphenol (1.0 g, 8.2 mmol) and K₂CO₃ (1.13 g, 8.2 mmol) are suspended in MeCN (25 mL) and stirred at 6O ⁰C overnight. The mixture is concentrated, the remainder diluted with DCM and washed with water twice. The organic layer is dried, concentrated and purified by flash chromatography (hexanes/ethyl acetate gradient) to yield 2-(2,3-dimethyl-phenoxymethyl)-oxirane as a colorless oil: ¹H-NMR (400 MHz, CDCl₃) δ = 7.05 (t, J = 7.9 Hz, IH), 6.81 (d, J = 7.5 Hz, IH), 6.70 (d, J = 8.2 Hz, IH), 4.21 (dd, J = 3.1 Hz, J = 11.0 Hz, IH), 3.97 (dd, J = 5.4 Hz, J = 11.0 Hz, IH), 3.38 (m, IH), 2.91 (t, J = 4.5 Hz, IH), 2.79 (dd, J = 2.7 Hz, J = 5.0 Hz, IH), 2.28 (s, 3H), 2.18 (s, 3H). MS calcd. for C_nH₁₅O₂ (M+H⁺) 179.1, found 179.1.

l-(2,3-dimethyl-phenoxy)-3-methylamino-propan-2-ol

[0068] 2-(2,3-Dimethyl-phenoxymethyl)-oxirane (0.3 g, 1.7 mmol) is dissolved in MeOH (2 mL). Methylamine (40% in H₂O, 0.3 mL, 3.4 mmol) is added and the mixture is stirred at 50 °C for 2 h. The solvents are removed in vacuo and the remainder is purified on reverse phase HPLC (MeCN/H₂O gradient) to give l-(2,3-dimethyl-phenoxy)-3-methylamino-propan-2-ol as a colorless oil: ¹H-NMR (400 MHz, CDCl₃) δ = 7.02 (t, J = 7.9 Hz, IH), 6.80 (d, J = 7.5 Hz, IH), 6.61 (d, J = 8.1 Hz, IH), 4.42 (m, IH), 3.98 (m, 2H), 3.26 (m, 2H), 2.80 (br, 3H), 2.24 (s, 3H), 2.10 (s, 3H). MS calcd. for Ci₂H₂₀NO₂ (M+H⁺) 210.1, found 210.1.

5-(2,3-Dimethyl-phenoxymethyl)-3-methyl-oxazolidin-2-one (21) [0069] l-(2,3-Dimethyl-phenoxy)-3-methylamino-propan-2-ol (180 mg, 0.86 mmol) is dissolved in benzene (10 mL). Carbonyl diimidazole (167 mg, 1.03 mmol) is added followed by a catalytic amount of DMAP. The mixture is heated to reflux for 4 h. The solvent is removed in vacuo, and the remainder is purified on reverse phase HPLC (MeCN/H₂O gradient) to give 21 as a colorless oil: ¹H-NMR (400 MHz, CDCl₃) δ = 7.05 (t, J = 7.9 Hz, IH), 6.82 (d, J = 7.5 Hz, IH), 6.67 (d, J = 8.2 Hz, IH), 4.84 (m, IH), 4.11 (d, J = 4.5 Hz, 2H), 3.74 (t, J = 8.7 Hz, IH), 3.58 (t, J = 5.7 Hz, J = 8.6 Hz, IH), 2.94 (s, 3H), 2.27 (s, 3H), 2.12 (s, 3H). MS calcd. for Ci₃H₁₈NO₃ (M+H⁺) 236.1, found 236.1.

Example 10 Mass Spectrometric Analysis of Cathepsin B Digests

[0070] LC-MS analysis of samples is performed using an LCQ Deca XP Plus mass spectrometer modified with a home-built nanospray source configured for online desalting as described in Licklider et al., Anal. Chem. 74:3076-3083 (2002). The peptide digests are loaded onto a 100 μm i.d. precolumn packed with 2 cm of Monitor, 5 μm, C18 (Column Engineering, Ontario, Canada), and desalted for 5 min at 5 μL/min with 0.1 M HOAc. After desalting, the precolumn is placed in line with a 75 μm i.d. pulled tip (5 μm opening) packed with 8 cm of the same packing material, and the ACN concentration is increased from 0% to 50% over 90 min. The column is then washed with 95% ACN before returning to 0%. The flow from the HPLC pump is passively split prior to the precolumn to achieve 250 nL/min. MS-MS are acquired in a data-dependent scanning mode with one full scan followed by three MS-MS scans on the three most intense precursor ions. The dynamic exclusion of previously selected precursors is set to 1 min. Tandem MS data are analyzed with TurboSequest (Thermo Electron). A custom database containing 8 proteins is searched using the following parameters: variable methionine oxidation, carboxamidomethyl adduct on cysteine, and carbamate adduct on cysteine (+247) or histidine (+247).

Example 11 Peptides identified by LC-MS/MS from inhibitor treated cathepsin B

[0071] Cathepsin B is digested with trypsin and chymotrypsin. Peptides are identified by nano-LC-MS/MS as described in the methods section. "^Λ" denotes carboxymethylated cysteines and "#" indicates modification by the strained carbamate compound. When controlled for the amount of injected material, the unmodified peptide is present at an amount 118-fold less than the modified peptide. As determined by Q-Tof analysis of the intact protein, there is an additional cleavage of the pro-enzyme upon auto-activation to yield the active enzyme with alternative N-terminal sequence starting points. The cleavage at position -4 relative to the expected major cleavage product is noted in Table 2.

Sequence range Peptide Identified

-4-5 EDLK LPASF

1 -5 LPASF

9-21 EQWPQC^ΛPTIKEI R

19-30 EIRDQGSC^ΛGSC#W

22-30 DQGSC^ΛGSC#W

22-30 DQGSC^ΛGSC^ΛW

31 -41 AFGAVEAISDR

33-41 GAVEAISDR

131 -140 IC^ΛEPGYSPTY

166-174 KNGPVEGAF

189-202 QHVTGEMMGGHAIR

203-214 ILGWGVENGTPY

215-221 WLVANSW

222-232 NTDWGDNGFFK

233-252 ILRGQDHC^ΛGIESEVVAGIPR

235-252 RGQDHC^ΛGIESEVVAGIPR

236-252 GQDHC^ΛGIESEVVAGIPR

Table 2

Example 12 Temperature Effects on Bicyclic Carbamate Adduct

[0072] Samples of cathepsin B are inhibited with compound 12 followed by overnight dialysis in order to remove any non-cathepsin bound compound. Dialyzed samples are then analyzed by LC-MS for the presence of the dissociation product 11. Prior to LC-MS analysis, the samples are incubated for 2 h on ice or at 37°C. The temperature effect on carbamate inhibited cathepsin B samples is evaluated using LC-MS with multiple reaction monitoring (MRM) on an Applied Biosystems/MDS SCIEX 4000 Q TRAP. MRM transitions for the open form of the carbamate are determined by infusion of a synthetic standard. Collision energy and exit cell potential are individually optimized for each of the selected transitions. Cathepsin B samples are loaded on a Phenomenex Luna C5 column (30 x 2mm) in 2% ACN, 0.1M HOAc and eluted using a 5 min linear gradient to 70% ACN, 0.1M HOAc and flow rate of 300 μL/min. Effluent from the column is introduced into the 4000 QTRAP using a TurboV ion source. Source parameters are: Cur, 10; IS, 4500; TEM, 450; GSl, 30; GS2, 15.

[0073] Cathepsin B is inhibited with compound 12 and dialyzed overnight. Compound 11 is detected using LC-MS with MRM post dialysis (panel A) and after incubation for 2 hours on ice (panel B) or at 37C (panel C). MRM transitions are chosen from collisionally activated dissociation of a synthetic standard of compound 11 (inset). Five MRM transitions are monitored for the detection of compound 11 (222.1 to 100.0, 71.1, 69.0, 81.9, and 55.0) and only the most intense transition is graphed for clarity (222.1 to 100.0).

Example 13 Effect of Temperature on the Stability of the Strained Carbamate-Cathepsin B Adduct

[0074] The presence of a metabolite of compound 12 is monitored by MRM. MRM transitions are selected and optimized using a synthetic analog of a metabolite (compound 11). The abundance of the compound 11 increased four-fold after incubation at 37°C for 2 hrs.

Temperature Time Metabolite Signal Intensity

0 0 900 0 2hrs 1000

37 2hrs 4000

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[0075] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

Claims

Claims

1. A compound having Formula (1)

or pharmaceutically acceptable salts thereof, wherein: X is O or S;

R¹ is OR², halo, (CR₂^R³ , nitro, cyano, amino, amido, sulfonamide, or an optionally substituted Ci_6alkyl, C₂-6 alkenyl, or C3-6 alkynyl;

R² is H, (CR₂)_nR³, or an optionally substituted Ci_6alkyl, C₂-6 alkenyl or C3_6 alkynyl; R³ is an optionally substituted aryl, heteroaryl, carbocyclic ring or heterocyclic ring; m is 1-3; and n is 0-4.

2. The compound of claim 1, wherein m is 1.

3. The compound of claim 2, wherein R¹ is halo.

4. The compound of claim 2, wherein R¹ is OR²; and R² is an optionally substituted Ci_₆alkyl, C₂-₆ alkenyl, or C₃_₆ alkynyl; or an optionally substituted aryl, heteroaryl, carbocyclic ring or heterocyclic ring.

5. The compound of claim 4, wherein R² is an optionally substituted phenyl, benzyl, or Ci-6 alkyl.

6. The compound of any one of claims 1-5, wherein said compound has a cis or trans stereoconformation.

7. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1-6 and a pharmaceutically acceptable carrier.

8. The use of a compound of any of claims 1-6, or pharmaceutically acceptable salts or pharmaceutical compositions thereof, in the manufacture of a medicament for treatment of a condition mediated by cathepsin protease activity, wherein said condition or disease is cellular homeostasis, apoptosis, tumor invasion and metastasis, bone resorption or antigen presentation, thereby treating said cathepsin protease-mediated condition or disease.

9. The use of claim 8, wherein said cathepsin protease is papain-like cathepsin protease.

10. The use of a compound of any of claims 1-6, or pharmaceutically acceptable salts or pharmaceutical compositions thereof, in the manufacture of a medicament for inhibiting a cathepsin protease activity.