Patent Application
20080207683
The present invention relates to certain biaryl-substituted tetrahydro-pyrazolo-pyridine compounds, pharmaceutical compositions containing them, and methods of using them for the treatment of disease states, disorders, and conditions mediated by cathepsin S activity.
Cathepsin S is one of the major cysteine proteases expressed in the lysosome of antigen presenting cells, mainly dendritic cells, B cells and macrophages. Cathepsin S is best known for its critical function in the proteolytic digestion of the invariant chain chaperone molecules, thus controlling antigen presentation to CD4+ T cells by major histocompatibility complex class II molecules or to NK1.1+ T cells via CD1 molecules. Cathepsin S also appears to participate in direct processing of exogenous antigens for presentation by MHC class II to CD4+ T cells or crosspresentation by MHC class I molecules to CD8+ T cells. In addition, cathepsin S in secreted form is implicated in degradation of extracellular matrix, which may contribute to the pathology of a number of diseases, including arthritis, atherosclerosis, and chronic obstructive pulmonary disease. Therefore, inhibition of cathepsin S is a promising target for the development of novel therapeutics for a variety of indications. For a review, see: Thurmond, R. L. et al. Curr. Opin. Invest. Drugs 2005, 6(5), 473-482.
Pyrazole inhibitors of cathepsin S were disclosed in a series of applications from Ortho-McNeil, and publications on part of this work have appeared (See: Intl. Patent Appl. Publ. Nos. WO02/14314 (Feb. 21, 2002), WO02/14315 (Feb. 21, 2002), and WO02/14317 (Feb. 21, 2002). See also: Thurmond, R. L. et al. J. Pharm. Exp. Ther. 2004, 308, 268-276; and Thurmond, R. L. et al. J. Med. Chem. 2004, 47, 4799-4801). However, there remains a need for potent cathepsin S modulators with desirable pharmaceutical properties.
In one aspect the invention relates to compounds of the following Formula (I):
wherein:
In certain embodiments, the compound of Formula (I) is a compound selected from those species described or exemplified in the detailed description below.
In a further aspect, the invention relates to pharmaceutical compositions each comprising: (a) an effective amount of at least one chemical entity selected from compounds of Formula (I), and pharmaceutically acceptable salts, prodrugs, and metabolites thereof; and (b) a pharmaceutically acceptable excipient.
In another aspect, the invention is directed to a method of treating a subject suffering from or diagnosed with a disease, disorder, or medical condition mediated by cathepsin S activity, comprising administering to the subject in need of such treatment an effective amount of at least one chemical entity selected from compounds of Formula (I), and pharmaceutically acceptable salts, prodrugs, and metabolites thereof. Diseases, disorders and medical conditions that are mediated by cathepsin S activity include those referred to herein.
Additional embodiments, features, and advantages of the invention will be apparent from the following detailed description and through practice of the invention.
For the sake of brevity, the disclosures of the publications, including patents, cited in this specification are herein incorporated by reference.
As used herein, the terms “including”, “containing” and “comprising” are used herein in their open, non-limiting sense.
The term “alkyl” refers to a saturated, straight- or branched-chain alkyl group having from 1 to 12 carbon atoms in the chain. Examples of alkyl groups include methyl (Me, which also may be structurally depicted by a bond, “/”), ethyl (Et), n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl (tBu), pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and groups that in light of the ordinary skill in the art and the teachings provided herein would be considered equivalent to any one of the foregoing examples.
The term “cycloalkyl” refers to a saturated or partially saturated, monocyclic, fused polycyclic, or spiro polycyclic carbocycle having from 3 to 12 ring atoms per carbocycle. Illustrative examples of cycloalkyl groups include the following entities, in the form of properly bonded moieties:
A “heterocycloalkyl” refers to a monocyclic, or fused, bridged, or spiro polycyclic ring structure that is saturated or partially saturated and has from 3 to 12 ring atoms per ring structure selected from carbon atoms and up to three heteroatoms selected from nitrogen, oxygen, and sulfur. The ring structure may optionally contain up to two oxo groups on carbon or sulfur ring members. Illustrative entities, in the form of properly bonded moieties, include:
The term “heteroaryl” refers to a monocyclic, fused bicyclic, or fused polycyclic aromatic heterocycle (ring structure having ring atoms selected from carbon atoms and up to four heteroatoms selected from nitrogen, oxygen, and sulfur) having from 3 to 12 ring atoms per heterocycle. Illustrative examples of heteroaryl groups include the following entities, in the form of properly bonded moieties:
Those skilled in the art will recognize that the species of heteroaryl, cycloalkyl, and heterocycloalkyl groups listed or illustrated above are not exhaustive, and that additional species within the scope of these defined terms may also be selected.
The term “halogen” represents chlorine, fluorine, bromine, or iodine. The term “halo” represents chloro, fluoro, bromo, or iodo.
The term “substituted” means that the specified group or moiety bears one or more substituents. The term “unsubstituted” means that the specified group bears no substituents. The term “optionally substituted” means that the specified group is unsubstituted or substituted by one or more substituents. Where the term “substituted” is used to describe a structural system, the substitution is meant to occur at any valency-allowed position on the system that yields a stable chemical structure.
Any formula given herein is intended to represent compounds having structures depicted by the structural formula as well as certain variations or forms. In particular, compounds of any formula given herein may have asymmetric centers and therefore exist in different enantiomeric forms. All optical isomers and stereoisomers of the compounds of the general formula, and mixtures thereof, are considered within the scope of the formula. Thus, any formula given herein is intended to represent a racemate, one or more enantiomeric forms, one or more diastereomeric forms, one or more atropisomeric forms, and mixtures thereof. Furthermore, certain structures may exist as geometric isomers (i.e., cis and trans isomers), as tautomers, or as atropisomers. Additionally, any formula given herein is intended to represent hydrates, solvates, and polymorphs of such compounds, and mixtures thereof.
To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about”. It is understood that, whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. Whenever a yield is given as a percentage, such yield refers to a mass of the entity for which the yield is given with respect to the maximum amount of the same entity that could be obtained under the particular stoichiometric conditions. Concentrations that are given as percentages refer to mass ratios, unless indicated differently.
Reference to a chemical entity herein stands for a reference to any one of: (a) the actually recited form of such chemical entity, and (b) any of the forms of such chemical entity in the medium in which the compound is being considered when named. For example, reference herein to a compound such as R—COOH, encompasses reference to any one of, for example, R—COOH(s), R—COOH(sol), and R—COO−(sol). In this example, R—COOH(s) refers to the solid compound, as it could be for example in a tablet or some other solid pharmaceutical composition or preparation; R—COOH(sol) refers to the undissociated form of the compound in a solvent; and R—COO−(sol) refers to the dissociated form of the compound in a solvent, such as the dissociated form of the compound in an aqueous environment, whether such dissociated form derives from R—COOH, from a salt thereof, or from any other entity that yields R—COO− upon dissociation in the medium being considered. In another example, an expression such as “exposing an entity to compound of formula R—COOH” refers to the exposure of such entity to the form, or forms, of the compound R—COOH that exists, or exist, in the medium in which such exposure takes place. In this regard, if such entity is for example in an aqueous environment, it is understood that the compound R—COOH is in such same medium, and therefore the entity is being exposed to species such as R—COOH(aq) and/or R—COO−(aq), where the subscript “(aq)” stands for “aqueous” according to its conventional meaning in chemistry and biochemistry. A carboxylic acid functional group has been chosen in these nomenclature examples; this choice is not intended, however, as a limitation but it is merely an illustration. It is understood that analogous examples can be provided in terms of other functional groups, including but not limited to hydroxyl, basic nitrogen members, such as those in amines, and any other group that interacts or transforms according to known manners in the medium that contains the compound. Such interactions and transformations include, but are not limited to, dissociation, association, tautomerism, solvolysis, including hydrolysis, solvation, including hydration, protonation, and deprotonation. No further examples in this regard are provided herein because these interactions and transformations in a given medium are known by any one of ordinary skill in the art.
Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as 2H, 3H, 11C, 13C, 14C, C15N, 18O, 17O, 32P, 33P, 35S, 18F, 36Cl, 125I, respectively. Such isotopically labelled compounds are useful in metabolic studies (preferably with 14C), reaction kinetic studies (with, for example 2H or 3H), detection or imaging techniques [such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT)] including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an 18F or 11C labeled compound may be particularly preferred for PET or SPECT studies. Further, substitution with heavier isotopes such as deuterium (i.e., 2H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements. Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
When referring to any formula given herein, the selection of a particular moiety from a list of possible species for a specified variable is not intended to define the same choice of the species for the variable appearing elsewhere. In other words, where a variable appears more than once, the choice of the species from a specified list is independent of the choice of the species for the same variable elsewhere in the formula, unless stated otherwise.
By way of a first example on substituent terminology, if substituent S1example is one of S1 and S2, and substituent S2example is one of S3 and S4, then these assignments refer to embodiments of this invention given according to the choices S2example is S1 and S2example is S3; S1example is S1 and S2example is S4; S1example is S2 and S2example is S3; S1example is S2 and S2example is S4; and equivalents of each one of such choices. The shorter terminology “S1example is one of S1 and S2, and S2example is one of S3 and S4” is accordingly used herein for the sake of brevity, but not by way of limitation. The foregoing first example on substituent terminology, which is stated in generic terms, is meant to illustrate the various substituent assignments described herein. The foregoing convention given herein for substituents extends, when applicable, to any generic substituent symbol used herein.
Furthermore, when more than one assignment is given for any member or substituent, embodiments of this invention comprise the various groupings that can be made from the listed assignments, taken independently, and equivalents thereof. By way of a second example on substituent terminology, if it is herein described that substituent Sexample is one of S1, S2, and S3, this listing refers to embodiments of this invention for which Sexample is S1; Sexample is S2; Sexample is S3; Sexample is one of S1 and S2; Sexample is one of S1 and S3; Sexample is one of S2 and S3; Sexample is one of S1, S2 and S3; and Sexample is any equivalent of each one of these choices. The shorter terminology “Sexample is one of S1, S2, and S3” is accordingly used herein for the sake of brevity, but not by way of limitation. The foregoing second example on substituent terminology, which is stated in generic terms, is meant to illustrate the various substituent assignments described herein. The foregoing convention given herein for substituents extends, when applicable, to any generic substituent symbol used herein.
The nomenclature “Ci-j” with j>i, when applied herein to a class of substituents, is meant to refer to embodiments of this invention for which each and every one of the number of carbon members, from i to j including i and j, is independently realized. By way of example, the term C1-3 refers independently to embodiments that have one carbon member (C1), embodiments that have two carbon members (C2), and embodiments that have three carbon members (C3).
The term Cn-malkyl refers to an aliphatic chain, whether straight or branched, with a total number N of carbon members in the chain that satisfies n≦N≦m, with m>n.
Any disubstituent referred to herein is meant to encompass the various attachment possibilities when more than one of such possibilities are allowed. For example, reference to disubstituent -A-B-, where A≠B, refers herein to such disubstituent with A attached to a first substituted member and B attached to a second substituted member, and it also refers to such disubstituent with A attached to the second substituted member and B attached to the first substituted member.
According to the foregoing interpretive considerations on assignments and nomenclature, it is understood that explicit reference herein to a set implies, where chemically meaningful and unless indicated otherwise, independent reference to embodiments of such set, and reference to each and every one of the possible embodiments of subsets of the set referred to explicitly.
In some embodiments of Formula (I), —NR1R2 is a structure of Formula (II):
wherein:
In other embodiments, R1 and R2 taken together with the nitrogen to which they are attached form azetidine, pyrrolidine, piperidine, piperazine substituted with Ra, morpholine, or thiomorpholine, each unsubstituted or substituted with one, two, or three Rb substituents as described for Formula (I). In still other embodiments, R1 and R2 taken together with the nitrogen to which they are attached form pyrrolidine or piperidine, each unsubstituted or substituted with one, two, or three Rb substituents as described for Formula (I).
In some embodiments, Ra is H, methyl, isopropyl, acetyl, or tert-butoxycarbonyl.
In some embodiments, each Rb substituent is independently OH, methyl, propyl, CF3, dimethylamino, acetamido, tert-butoxycarbamoyl, fluoro, or methoxy. In other embodiments, Rb is pyrrolidinyl, 2-oxo-pyrrolidinyl, or piperidinyl. In still other embodiments, Rb is pyrrolidin-1-yl or 2-oxo-pyrrolidin-1-yl.
In some embodiments, R3 is H or OH.
In some embodiments, R4 is H, methyl, —SO2CH3, acetyl, or tert-butoxycarbonyl. In other embodiments, R4 is —SO2CH3.
In some embodiments, R5 is chloro or CF3. In other embodiments, R5 is chloro.
In some embodiments, R6 is H.
In some embodiments, R7 is H.
In some embodiments, R8 is Ar. In other embodiments, R3 is —CH(Ri)Ar. In still other embodiments, R3 is —(CH2)2N(Rh)Ar.
In some embodiments, each Rg is H or methyl. In other embodiments, two Rg groups together form a carbonyl.
In some embodiments, Rh is H or methyl.
In some embodiments, Ri is H, methyl, or ethyl.
In some embodiments, Ar is a phenyl, naphthyl, pyridinyl, pyrimidinyl, oxazolyl, thiophenyl, thiazolyl, indanyl, indolyl, benzimidazolyl, or benzothiazolyl group, unsubstituted or substituted with one, two, or three Rj substituents. In still other embodiments, Ar is 4-methoxyphenyl, 4-methylphenyl, indan-4-yl, 3-chloro-4-methoxyphenyl, 4-cyclopentylmethoxy-phenyl, 6-methoxy-pyridin-3-yl, pyridin-3-yl, oxazol-2-yl, 1H-indol-2-yl, thiophen-2-yl, 5-methyl-1H-benzoimidazol-2-yl, 1H-benzoimidazol-2-yl, thiazol-2-yl, 5-chloro-1H-benzoimidazol-2-yl, 4-tert-butyl-thiazol-2-yl, 4-phenyl-thiazol-2-yl, 5-fluoro-benzothiazol-2-yl, benzothiazol-2-yl, 5,6-difluoro-1H-benzoimidazol-2-yl, 3,4-dimethyl-phenyl, or 4-isopropyl-phenyl.
In some embodiments, —N(Rh)—Ar is 2,3-dihydro-indolyl (or “indanyl”), unsubstituted or substituted with one or two additional Rj substituents. In other embodiments, —N(Rh)—Ar is 5-fluoro-2,3-dihydro-indol-1-yl, 7-dimethylsulfamoyl-2,3-dihydro-indol-1-yl, 6-dimethylsulfamoyl-2,3-dihydro-indol-1-yl, 6-fluoro-2,3-dihydro-indol-1-yl, or 5-methyl-2,3-dihydro-indol-1-yl.
In some embodiments, each Rj substituent is independently methyl, isopropyl, tert-butyl, cyclopentyl, phenyl, methoxy, isopropoxy, cyclopentylmethoxy, cyclohexyloxy, chloro, fluoro, CF3, —NO2, —SO2N(CH3)2, or —SO3H, or two adjacent Rj substituents together form —(CH2)3—. In other embodiments, an Rj substituent taken together with Rh forms —CH2CH2—.
The invention includes also pharmaceutically acceptable salts of the compounds represented by Formula (I), preferably of those described above and of the specific compounds exemplified herein, and methods of treatment using such salts.
A “pharmaceutically acceptable salt” is intended to mean a salt of a free acid or base of a compound represented by Formula (I) that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to the subject. See, generally, S. M. Berge, et al., “Pharmaceutical Salts”, J. Pharm. Sci., 1977, 66:1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH and VHCA, Zurich, 2002. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of patients without undue toxicity, irritation, or allergic response. A compound of Formula (I) may possess a sufficiently acidic group, a sufficiently basic group, or both types of functional groups, and accordingly react with a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, methane-sulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.
If the compound of Formula (I) contains a basic nitrogen, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, and the like, or with an organic acid, such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxymaleic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as mandelic acid, citric acid, or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid, or cinnamic acid, a sulfonic acid, such as laurylsulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, any compatible mixture of acids such as those given as examples herein, and any other acid and mixture thereof that are regarded as equivalents or acceptable substitutes in light of the ordinary level of skill in this technology.
If the compound of Formula (I) is an acid, such as a carboxylic acid or sulfonic acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide, alkaline earth metal hydroxide, any compatible mixture of bases such as those given as examples herein, and any other base and mixture thereof that are regarded as equivalents or acceptable substitutes in light of the ordinary level of skill in this technology. Illustrative examples of suitable salts include organic salts derived from amino acids, such as glycine and arginine, ammonia, carbonates, bicarbonates, primary, secondary, and tertiary amines, and cyclic amines, such as benzylamines, pyrrolidines, piperidine, morpholine, and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.
The invention also relates to pharmaceutically acceptable prodrugs of the compounds of Formula (I), pharmaceutical compositions containing such pharmaceutically acceptable prodrugs, and treatment methods employing such pharmaceutically acceptable prodrugs. The term “prodrug” means a precursor of a designated compound that, following administration to a subject, yields the compound in vivo via a chemical or physiological process such as solvolysis or enzymatic cleavage, or under physiological conditions (e.g., a prodrug on being brought to physiological pH is converted to the compound of Formula (I)). A “pharmaceutically acceptable prodrug” is a prodrug that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to the subject. Illustrative procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.
Examples of prodrugs include compounds having an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues, covalently joined through an amide or ester bond to a free amino, hydroxy, or carboxylic acid group of a compound of Formula (I). Examples of amino acid residues include the twenty naturally occurring amino acids, commonly designated by three letter symbols, as well as 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine sulfone.
Additional types of prodrugs may be produced, for instance, by derivatizing free carboxyl groups of structures of Formula (I) as amides or alkyl esters. Examples of amides include those derived from ammonia, primary C1-6alkyl amines and secondary di(C1-6alkyl) amines. Secondary amines include 5- or 6-membered heterocycloalkyl or heteroaryl ring moieties. Examples of amides include those that are derived from ammonia, C1-3alkyl primary amines, and di(C1-2alkyl)amines. Examples of esters of the invention include C1-7alkyl, C5-7cycloalkyl, phenyl, and phenyl(C1-6alkyl) esters. Preferred esters include methyl esters. Prodrugs may also be prepared by derivatizing free hydroxy groups using groups including hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, following procedures such as those outlined in Adv. Drug Delivery Rev. 1996, 19, 115. Carbamate derivatives of hydroxy and amino groups may also yield prodrugs. Carbonate derivatives, sulfonate esters, and sulfate esters of hydroxy groups may also provide prodrugs. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers, wherein the acyl group may be an alkyl ester, optionally substituted with one or more ether, amine, or carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, is also useful to yield prodrugs. Prodrugs of this type may be prepared as described in J. Med. Chem. 1996, 39, 10. Free amines can also be derivatized as amides, sulfonamides or phosphonamides. All of these prodrug moieties may incorporate groups including ether, amine, and carboxylic acid functionalities.
The present invention also relates to pharmaceutically active metabolites of compounds of Formula (I), and uses of such metabolites in the methods of the invention. A “pharmaceutically active metabolite” means a pharmacologically active product of metabolism in the body of a compound of Formula (I) or salt thereof. Prodrugs and active metabolites of a compound may be determined using routine techniques known or available in the art. See, e.g., Bertolini, et al., J. Med. Chem. 1997, 40, 2011-2016; Shan, et al., J. Pharm. Sci. 1997, 86 (7), 765-767; Bagshawe, Drug Dev. Res. 1995, 34, 220-230; Bodor, Adv. Drug Res. 1984, 13, 224-331; Bundgaard, Design of Prodrugs (Elsevier Press, 1985); and Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen, et al., eds., Harwood Academic Publishers, 1991).
The compounds of Formula (I) and their pharmaceutically acceptable salts, pharmaceutically acceptable prodrugs, and pharmaceutically active metabolites (collectively, “active agents”) of the present invention are useful in the methods of the invention. The active agents may be used in the inventive methods for the treatment or prevention of medical conditions, diseases, or disorders mediated through modulation of cathepsin S, such as those described herein. Symptoms or disease states are intended to be included within the scope of “medical conditions, disorders, or diseases.”
Accordingly, the invention relates to methods of using the active agents described herein to treat subjects diagnosed with or suffering from a disease, disorder, or condition mediated through cathepsin S activity, such as an autoimmune disease, an allergic condition, inflammation, a bowel disorder, tissue transplant rejection, pain, or cancer. Active agents according to the invention may therefore be used as immunomodulating agents, immunosuppressants, anti-allergy agents, anti-inflammatory agents, analgesics, or anti-cancer agents.
In some embodiments, an active agent of the present invention is administered to treat lupus, asthma, allergic reaction, atopic allergy, hay fever, atopic dermatitis, food allergy, rhinitis (such as allergic rhinitis and the inflammation caused by non-allergic rhinitis), skin immune system disorders (such as psoriasis), uveitis, inflammation, upper airway inflammation, Sjögren's syndrome, arthritis, rheumatoid arthritis, osteoarthritis, type I diabetes, atherosclerosis, multiple sclerosis, coeliac disease, inflammatory bowel disease (IBD), chronic obstructive pulmonary disorder (COPD), tissue transplant rejection, pain, chronic pain (such as pain due to conditions such as cancer, neuropathic pain, rheumatoid arthritis, osteoarthritis and inflammatory conditions), or cancer (and cancer-related processes such as angiogenesis, tumor growth, cell proliferation, and metastasis). In certain embodiments, an active agent of the present invention is administered to treat psoriasis, pain, multiple sclerosis, atherosclerosis, or rheumatoid arthritis.
Thus, the active agents may be used to treat subjects diagnosed with or suffering from a disease, disorder, or condition mediated through cathepsin S activity. The term “treat” or “treating” as used herein is intended to refer to administration of an active agent or composition of the invention to a subject for the purpose of effecting a therapeutic or prophylactic benefit through modulation of cathepsin S activity. Treating includes reversing, ameliorating, alleviating, inhibiting the progress of, lessening the severity of, or preventing a disease, disorder, or condition, or one or more symptoms of such disease, disorder or condition mediated through modulation of cathepsin S activity. The term “subject” refers to a mammalian patient in need of such treatment, such as a human. “Modulators” include both inhibitors and activators, where “inhibitors” refer to compounds that decrease, prevent, inactivate, desensitize or down-regulate cathepsin S expression or activity, and “activators” are compounds that increase, activate, facilitate, sensitize, or up-regulate cathepsin S expression or activity.
In treatment methods according to the invention, an effective amount of at least one active agent according to the invention is administered to a subject suffering from or diagnosed as having such a disease, disorder, or condition. An “effective amount” means an amount or dose sufficient to generally bring about the desired therapeutic or prophylactic benefit in patients in need of such treatment for the designated disease, disorder, or condition. Effective amounts or doses of the active agents of the present invention may be ascertained by routine methods such as modeling, dose escalation studies or clinical trials, and by taking into consideration routine factors, e.g., the mode or route of administration or drug delivery, the pharmacokinetics of the agent, the severity and course of the disease, disorder, or condition, the subject's previous or ongoing therapy, the subject's health status and response to drugs, and the judgment of the treating physician. An exemplary dose is in the range of from about 0.001 to about 200 mg of active agent per kg of subject's body weight per day, preferably about 0.05 to 100 mg/kg/day, or about 1 to 35 mg/kg/day, or about 0.1 to 10 mg/kg daily in single or divided dosage units (e.g., BID, TID, QID). For a 70-kg human, an illustrative range for a suitable dosage amount is from about 0.05 to about 7 g/day, or about 0.2 to about 2.5 g/day.
Once improvement of the patient's disease, disorder, or condition has occurred, the dose may be adjusted for preventative or maintenance treatment. For example, the dosage or the frequency of administration, or both, may be reduced as a function of the symptoms, to a level at which the desired therapeutic or prophylactic effect is maintained. Of course, if symptoms have been alleviated to an appropriate level, treatment may cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms.
In addition, the active agents of the invention may be used in combination with additional active ingredients in the treatment of the above conditions. The additional active ingredients may be coadministered separately with an active agent of Formula (I) or included with such an agent in a pharmaceutical composition according to the invention. In an exemplary embodiment, additional active ingredients are those that are known or discovered to be effective in the treatment of conditions, disorders, or diseases mediated by cathepsin S activity, such as another cathepsin S modulator or a compound active against another target associated with the particular condition, disorder, or disease. The combination may serve to increase efficacy (e.g., by including in the combination a compound potentiating the potency or effectiveness of an agent according to the invention), decrease one or more side effects, or decrease the required dose of the active agent according to the invention.
The active agents of the invention are used, alone or in combination with one or more additional active ingredients, to formulate pharmaceutical compositions of the invention. A pharmaceutical composition of the invention comprises: (a) an effective amount of at least one active agent in accordance with the invention; and (b) a pharmaceutically acceptable excipient.
A “pharmaceutically acceptable excipient” refers to a substance that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological composition or otherwise used as a vehicle, carrier, or diluent to facilitate administration of a agent and that is compatible therewith. Examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
Delivery forms of the pharmaceutical compositions containing one or more dosage units of the active agents may be prepared using suitable pharmaceutical excipients and compounding techniques known or that become available to those skilled in the art. The compositions may be administered in the inventive methods by a suitable route of delivery, e.g., oral, parenteral, rectal, topical, or ocular routes, or by inhalation.
The preparation may be in the form of tablets, capsules, sachets, dragees, powders, granules, lozenges, powders for reconstitution, liquid preparations, or suppositories. Preferably, the compositions are formulated for intravenous infusion, topical administration, or oral administration.
For oral administration, the active agents of the invention can be provided in the form of tablets or capsules, or as a solution, emulsion, or suspension. To prepare the oral compositions, the active agents may be formulated to yield a dosage of, e.g., from about 0.05 to about 50 mg/kg daily, or from about 0.05 to about 20 mg/kg daily, or from about 0.1 to about 10 mg/kg daily.
Oral tablets may include the active ingredient(s) mixed with compatible pharmaceutically acceptable excipients such as diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservative agents. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquid oral excipients include ethanol, glycerol, water, and the like. Starch, polyvinyl-pyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid are exemplary disintegrating agents. Binding agents may include starch and gelatin. The lubricating agent, if present, may be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract, or may be coated with an enteric coating.
Capsules for oral administration include hard and soft gelatin capsules. To prepare hard gelatin capsules, active ingredient(s) may be mixed with a solid, semi-solid, or liquid diluent. Soft gelatin capsules may be prepared by mixing the active ingredient with water, an oil such as peanut oil or olive oil, liquid paraffin, a mixture of mono and di-glycerides of short chain fatty acids, polyethylene glycol 400, or propylene glycol.
Liquids for oral administration may be in the form of suspensions, solutions, emulsions or syrups or may be lyophilized or presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may optionally contain: pharmaceutically-acceptable excipients such as suspending agents (for example, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and the like); non-aqueous vehicles, e.g., oil (for example, almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol, or water; preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents such as lecithin; and, if desired, flavoring or coloring agents.
The active agents of this invention may also be administered by non-oral routes. For example, compositions may be formulated for rectal administration as a suppository. For parenteral use, including intravenous, intramuscular, intraperitoneal, or subcutaneous routes, the agents of the invention may be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity or in parenterally acceptable oil. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Such forms may be presented in unit-dose form such as ampules or disposable injection devices, in multi-dose forms such as vials from which the appropriate dose may be withdrawn, or in a solid form or pre-concentrate that can be used to prepare an injectable formulation. Illustrative infusion doses range from about 1 to 1000 μg/kg/minute of agent admixed with a pharmaceutical carrier over a period ranging from several minutes to several days.
For topical administration, the agents may be mixed with a pharmaceutical carrier at a concentration of about 0.1% to about 10% of drug to vehicle. Another mode of administering the agents of the invention may utilize a patch formulation to affect transdermal delivery.
Active agents may alternatively be administered in methods of this invention by inhalation, via the nasal or oral routes, e.g., in a spray formulation also containing a suitable carrier.
Exemplary chemical entities useful in methods of the invention will now be described by reference to illustrative synthetic schemes for their general preparation below and the specific examples that follow. Artisans will recognize that, to obtain the various compounds herein, starting materials may be suitably selected so that the ultimately desired substituents will be carried through the reaction scheme with or without protection as appropriate to yield the desired product. Alternatively, it may be necessary or desirable to employ, in the place of the ultimately desired substituent, a suitable group that may be carried through the reaction scheme and replaced as appropriate with the desired substituent. In addition, artisans will note that the various transformations described in the following Schemes may be performed in a different order than that depicted. Unless otherwise specified, the variables are as defined above in reference to Formula (I).
Referring to Scheme A, the tetrahydro-pyrazolo-pyridine core structure of Formula (I) may be prepared from commercially available piperidones (X). Installation of the R4 substituent is accomplished through, for example, alkylation, acylation, sulfonylation, amide formation, or other suitable methods known in the art to provide ketones (XI). Enamine formation according to general methods gives enamines (XII), which are then reacted with acyl chlorides, ArC(O)Cl, where Ar is a suitably substituted phenyl group, in the presence of a suitable tertiary amine base, to form enamines (XIII) (not isolated). In situ reaction of the enamines with hydrazine generates pyrazoles (XIV).
Where Ar is a suitably substituted group as in Formula (XV), where X is iodide or triflate, formation of biaryl acids (XVI) is accomplished palladium-mediated coupling with metallic reagents (XVa) or (XVb) such as boronic acids (where M is —B(OH)2) or tin reagents (where M is Sn(alkyl)3). Coupling with acids (XVa) yields biaryl acids (XVIa), which are converted into amides (XVII) by coupling with amines HNR7R8 using standard amide coupling methods known in the art. Amines HNR7R8 are commercially available or are prepared according to methods known in the art. In preferred embodiments, acids (XVI) are activated by coupling with HOSu, and the resulting succinamide analogs are reacted with amines HNR7R8. Alternatively, coupling of compounds (XV) with metal reagents (XVb) provide amides (XVII) directly.
Two variations for the installation of the propyl amino chain are shown in Scheme C. Pyrazoles (XXI) are alkylated with optionally protected aldehydes (XXII), where R3 is H, C1-4alkyl, or —OC1-4alkyl, and LG is a suitable leaving group, such as a chloride, bromide, iodide, mesylate or tosylate, to give compounds (XXIII). If the aldehyde group is protected, for example, as an acetal, deprotection of (XXIII) is accomplished under general conditions. The resulting aldehydes are reacted with amines (XXIV) under reductive amination conditions, to provide propyl amines (XXV) where R3 is H, C1-4alkyl, or —OC1-4alkyl. Alternatively, pyrazoles (XXI) are reacted with epichlorohydrin, in the presence of a suitable base, to give epoxides (XXVI). Epoxide opening with amines (XXIV), preferably at elevated temperatures, yields propyl amines (XXV) where R3 is OH.
In another embodiment, addition of pyrazoles (XXI) to α,β-unsaturated nitriles (XXVI), in the presence of a suitable base, such as aq. NaOH, generates nitriles (XXVII). Reduction of the nitriles to the corresponding aldehydes (XXIII, not shown) is accomplished with a reducing agent such as DIBAL-H. Reductive amination of aldehydes (XXIII) with amines (XXIV) gives amines (XXV) as described in Scheme C.
Compounds of Formula (I) may be converted to their corresponding salts using methods described in the art. For example, an amine of Formula (I) may be treated with trifluoroacetic acid, HCl, or citric acid in a solvent such as Et2O, CH2Cl2, THF, or MeOH to provide the corresponding salt form.
Compounds prepared according to the schemes described above may be obtained as single enantiomers, diastereomers, or regioisomers, by enantio-, diastero-, or regiospecific synthesis, or by resolution. Compounds prepared according to the schemes above may alternately be obtained as racemic (1:1) or non-racemic (not 1:1) mixtures or as mixtures of diastereomers or regioisomers. Where racemic and non-racemic mixtures of enantiomers are obtained, single enantiomers may be isolated using conventional separation methods known to one skilled in the art, such as chiral chromatography, recrystallization, diastereomeric salt formation, derivatization into diastereomeric adducts, biotransformation, or enzymatic transformation. Where regioisomeric or diastereomeric mixtures are obtained, single isomers may be separated using conventional methods such as chromatography or crystallization.
The following specific examples are provided to further illustrate the invention and various preferred embodiments.
In obtaining the compounds described in the examples below and the corresponding analytical data, the following experimental and analytical protocols were followed unless otherwise indicated.
Unless otherwise stated, reaction mixtures were magnetically stirred at room temperature (rt). Where solutions are “dried,” they are generally dried over a drying agent such as Na2SO4 or MgSO4. Where mixtures, solutions, and extracts were “concentrated”, they were typically concentrated on a rotary evaporator under reduced pressure.
Microwave reactions were performed on a Personal Chemistry Emrys Optimizer. Individual reactions were heated to the desired temperature and held at that temperature for the allotted time.
Analytical HPLC retention times are reported in minutes, and were obtained on an Agilent HP-1100 instrument with a Phenomenex Luna C-18 (5 uM, 4.6×150 mm) column, with a flow rate of 1 mL/min, detection at 230, 254, and 280 nM, and a gradient of 10 to 100% CH3CN (0.05% TFA)/H2O (0.05% TFA).
Preparatory HPLC purifications were typically performed on a Phenomenex Synergi column (4 μm, 21×150 mm), with a flow rate of 25 mL/min, and solvent conditions as described for Analytical HPLC.
Mass spectra (MS) were obtained on an Agilent series 1100 MSD using electrospray ionization (ESI) in positive mode unless otherwise indicated. Calculated (calcd.) mass corresponds to the exact mass. The MS data presented is the m/z found (typically [M+H]+) for the molecular ion.
Nuclear magnetic resonance (NMR) spectra were obtained on Bruker model DRX spectrometers (400, 500, or 600 MHz). NMR interpretation was performed using ACD Spec/Manager software to assign chemical shift and multiplicity. The format of the 1H NMR data below is: chemical shift in ppm downfield of the tetramethylsilane reference (multiplicity, coupling constant J in Hz, integration). All 1H NMR data was acquired in CD3OD solvent unless otherwise indicated.
Chemical names were generated using ChemDraw Version 6.0.2 (CambridgeSoft, Cambridge, Mass.).
A. 1-Methanesulfonyl-piperidin-4-one. To a solution of 4-piperidone monohydrate hydrochloride (90 g, 586 mmol) in CHCl3 (300 mL) and H2O (300 mL) was added K2CO3 (324 g, 2340 mmol). The slurry was cooled to 0° C. and treated with MsCl (136 mL, 1.76 mmol) by dropwise addition over a 1 h period (gas evolution was observed). The reaction mixture was allowed to stir for 72 h and was partitioned between CH2Cl2 (500 mL) and aq. NaHCO3 (500 mL). The aqueous layer was extracted with CH2Cl2 (3×200 mL). The organic layer was washed with 1% KHSO4 (250 mL), dried over MgSO4, and concentrated to give the desired product (90.5 g, 87%) as a white solid. HPLC: Rt=2.2. MS (ESI): mass calcd. for C6H11NO3S, 178.1; m/z found, 178.1 [M+H]+.
B. 3-(4-Chloro-3-iodo-phenyl)-5-methanesulfonyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine. To a solution of the above piperidone (10 g, 56 mmol) and p-toluenesulfonic acid (40 mg) in benzene (60 mL) was added morpholine (4.9 mL, 56 mmol). The reaction mixture was heated in a flask equipped with a condenser and a Dean-Stark trap at 90° C. for 16 h. The reaction mixture was cooled and concentrated to give the desired enamine as a beige solid, which was used without further purification. The enamine was dissolved in CH2Cl2 (40 mL), treated with TEA (9.4 mL, 67.2 mmol), and cooled to 0° C. To this solution was added 4-chloro-3-iodobenzoyl chloride* (16.9 g, 56 mmol). The reaction mixture was allowed to warm to rt, stirred for 14 h, and then concentrated. The resulting red oil was diluted with EtOH (56 mL) and treated with hydrazine (5.34 mL, 170 mmol) at 0° C. The resulting slurry was allowed to warm to rt and stirred for 16 h. EtOAc (120 mL) was added, and after 2 h the resulting precipitate was filtered and washed with additional EtOAc to afford the desired product as a white solid (8.80 g, 36%). HPLC: Rt=6.08. MS (ESI): mass calcd. for C13H13ClIN3O2S, 437.7; m/z found, 438.1 [M+H]+. 1H NMR (DMSO-d6): 8.05 (d, J=1.9, 1H), 7.51 (d, J=8.3, 1H), 7.43 (dd, J=8.4, 1.9, 2H), 4.30 (s, 2H), 3.36 (t, J=5.8, 2H), 3.30 (br s, 1H), 2.86 (s, 3H), 2.69 (t, J=5.6, 2H).
*Prepared by dissolving 4-chloro-3-iodobenzoic acid (15.8 g, 56 mmol) in CH2Cl2 (40 mL) and treating with oxalyl chloride (4.1 mL, 46.7 mmol) and a catalytic amount of DMF (0.40 mL; vigorous gas evolution). The mixture was stirred at rt for 3 h. The reaction mixture was concentrated to afford a white solid, which was used without further purification.
C. 3-(4-Chloro-3-iodo-phenyl)-1-(2-[1,3]dioxolan-2-yl-ethyl)-5-methanesulfonyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine. A slurry of the above pyrazole (10 g, 22.8 mmol) and Cs2CO3 (11.9 g, 45.6 mmol) in DMF (75 mL) was stirred at rt for 2 h. 2-(2-Bromoethyl)1,3-dioxolane (3.5 mL, 34.2 mmol) was added dropwise and stirring maintained for 12 h. Ice H2O was added slowly to form a precipitate. The white solid was collected by suction filtration and washed with H2O and Et2O to afford the desired product (10.4 g, 85%). HPLC: Rt=6.98. MS (ESI): mass calcd. for C18H21ClIN3O4S, 537.8; m/z found, 538.2 [M+H]+. 1H NMR (CDCl3): 8.15 (s, 1H), 7.46-7.45 (m, 2H), 4.83 (t, J=4.6, 1H), 4.49 (s, 2H), 4.17 (t, J=7.1, 2H), 4.01-3.97 (m, 2H), 3.89-3.86 (m, 2H), 3.65 (t, J=5.8, 2H), 2.89 (s, 3H), 2.87 (t, J=5.8, 2H), 2.28-2.26 (m, 2H).
D. 3-(4-Chloro-3-iodo-phenyl)-5-methanesulfonyl-1-(3-morpholin-4-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine. A mixture of the above acetal (4.65 g, 8.64 mmol) and 1 N HCl (19 mL) in acetone (75 mL) was heated at 55° C. for 5 h. The clear solution was diluted with CH2Cl2 and washed with saturated aqueous (satd. aq.) NaHCO3. The combined organic extracts were dried (Na2SO4), filtered, and concentrated to give a white solid, which was used directly in the next reaction. The crude aldehyde was dissolved in CH2Cl2 (80 mL) and pyrrolidine (2.5 mL, 17.3 mmol) and acetic acid (1.0 mL) were added sequentially. After 10 min, NaBH(OAc)3 (3.48 g, 13 mmol) was added and stirring was continued for 2 h. After the addition of satd. aq. NaHCO3, the layers were separated and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic extracts were washed with brine, dried (MgSO4), filtered, and concentrated to give an orange oil.
E. 2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid. To a solution of the above aryl iodide (6 g, 11.2 mmol), 3-carboxyphenyl boronic acid (2 g, 12.3 mmol), Pd-dppf (0.914 g, 1.1 mmol) in DMF (35 mL) was added 2 M aq. K2CO3 (11.2 mL). The reaction mixture was stirred under N2 at 90° C. for 3 h. Upon cooling to rt, the mixture was diluted with CH2Cl2 and a black precipitate resulted. The solution was decanted away and the desired product precipitate was collected. HPLC: Rt=1.494. MS (ESI): mass calcd. for C27H31ClN4O4S, 542.2; m/z found, 543.1 [M+H]+.
F. 2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid 2,5-dioxo-pyrrolidin-1-yl ester. A solution of the above carboxylic acid (1.6 g, 2.9 mmol) was dissolved in DMF (10 mL) and CH2Cl2 (20 mL) was treated with hydroxysuccinimide (0.366 g, 3.2 mmol), HATU (1.3 g, 3.5 mmol), and DIEA (1 mL, 5.8 mmol). After stirring for 2 h, the mixture was diluted with CH2Cl2 (50 mL) and washed with satd. aq. NaHCO3 (20 mL). The organic layer was dried over MgSO4 and concentrated to give the desired product. HPLC: Rt=1.657. MS (ESI): mass calcd. for C31H34ClN5O6S, 639.2; m/z found, 640.1 [M+H]+.
A. N1-(4-Methoxy-phenyl)-ethane-1,2-diamine. A solution of 2-oxazolidinone (1.5 g, 17.2 mmol) in 2-(2-ethoxyethoxy)ethanol (20 mL) was treated with p-anisidine hydrochloride (2.1 g, 17.2 mmol) and heated in the microwave at 150° C. at 300 W for 10 min. The solution was cooled to rt and Et2O (50 mL) was added. The resulting solid precipitate was filtered and washed with Et2O (3×20 mL). HPLC: Rt=0.23. MS (ESI): mass calcd. for C9H14N2O, 166.1; m/z found, 150 [M-16]+.
B. 2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid [2-(4-methoxy-phenylamino)-ethyl]-amide. To a solution of Intermediate 1 (0.100 g, 0.2 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (1 mL) was added N1-(4-methoxy-phenyl)-ethane-1,2-diamine (0.029 g, 0.2 mmol). After stirring for 2 h at rt, the mixture was concentrated to give a black oil. Purification via preparatory reverse phase HPLC followed by lyophilization afforded the title compound as an off-white solid. HPLC: Rt=1.425. MS (ESI): mass calcd. for C36H43ClN6O4S, 690.3; m/z found, 691.3 [M+H]+.
The compounds in Examples 2-4 were prepared according to the methods described for Example 1, substituting the appropriate amine for p-anisidine in Example 1, Step A.
HPLC: Rt=1.600. MS (ESI): mass calcd. for C36H43ClN6O3S, 674.3; m/z found, 675.3 [M+H]+.
HPLC: Rt=1.592. MS (ESI): mass calcd. for C38H45ClN6O3S, 700.3; m/z found, 701.3 [M+H]+.
HPLC: Rt=1.754. MS (ESI): mass calcd. for C36H42Cl2N6O4S, 724.2; m/z found, 725.3 [M+H]+.
A. 1-Bromo-4-cyclopentylmethoxy-benzene. To a 0° C. solution of 4-bromophenol (2.56 g, 14.8 mmol), Ph3P (4 g, 14.8 mmol), and cyclopentyl-methanol (1.6 mL, 14.8 mmol) in THF (50 mL) was added DEAD (2.5 mL, 14.8 mmol) dropwise. The reaction solution was allowed to warm to rt and stirred for 12 h. The mixture was concentrated and the resulting residue was diluted in hexanes until a white precipitate formed. The precipitate was removed by filtration and the filtrate was concentrated. Purification (SiO2; 25% EtOAc in hexanes) provided the desired product (2.84 g, 75.1%). HPLC: Rt=2.35.
B. N1-(4-Cyclopentylmethoxy-phenyl)-ethane-1,2-diamine. A solution of the above aryl bromide (1.4 g, 5.5 mmol) in 1,2-diaminoethane (1.5 mL, 22 mmol) was treated with CuSO4 (0.175 g, 1.1 mmol) and heated in the microwave at 150° C. at 300 W for 20 min. The solution was diluted with EtOAc (20 mL) and washed with H2O (3×20 mL). The organic layer was separated, dried over MgSO4, and concentrated. HPLC: Rt=2.32. MS (ESI): mass calcd. for C14H22N2O, 234.2; m/z found, 235.1 [M+H]+.
C. 2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid [2-(4-cyclopentylmethoxy-phenylamino)-ethyl]-amide. To a solution of Intermediate 1 (0.100 g, 0.2 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (1 mL) was added the above amine (0.051 g, 0.2 mmol). After stirring for 2 h at rt, the mixture was concentrated to give a black oil. Purification via preparatory reverse phase HPLC followed by lyophilization afforded the title compound as an off-white solid. HPLC: Rt=1.629. MS (ESI): mass calcd. for C41H51ClN6O4S, 758.3; m/z found, 759.3 [M+H]+.
A. N1-(6-Methoxy-pyridin-3-yl)-ethane-1,2-diamine. A solution of 5-bromo-2-methoxy-pyridine (0.2 g, 0.1 mmol) in 1,2-diaminoethane (0.5 mL) was treated with CuSO4 and heated in the microwave at 150° C. at 300 W for 10 min. The solution was diluted with EtOAc (10 mL) and washed with H2O (3×10 mL). The organic layer was separated, dried over MgSO4, and concentrated. HPLC: Rt=0.257. MS (ESI): mass calcd. for C8H13N3O, 167.1; m/z found, 168 [M+H]+.
B. 2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid [2-(6-methoxy-pyridin-3-ylamino)-ethyl]-amide. To a solution of Intermediate 1 (0.100 g, 0.2 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (1 mL) was added the above amine (0.051 g, 0.2 mmol). After stirring for 2 h at rt, the mixture was concentrated to give a black oil. Purification via preparatory reverse phase HPLC followed by lyophilization afforded the title compound as an off-white solid. MS (ESI): mass calcd. for C35H42ClN7O4S 691.27; m/z found, 692.3 [M+H]+.
A. N1-Pyridin-3-yl-ethane-1,2-diamine. A solution of 3-bromo-pyridine (0.2 g, 0.1 mmol) in 1,2-diaminoethane (0.5 mL) was treated with CuSO4 and heated in the microwave at 150° C. at 300 W for 10 min. The solution was diluted with EtOAc (10 mL) and washed with H2O (3×10 mL). The organic layer was separated, dried over MgSO4 and the solution was concentrated. HPLC: Rt=0.227. MS (ESI): mass calcd. for C7H11N3, 137.1; m/z found, 138 [M+H]+.
B. 2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid [2-(pyridin-3-ylamino)-ethyl]-amide. To a solution of Intermediate 1 (0.100 g, 0.2 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (1 mL) was added the above amine (0.051 g, 0.2 mmol). After stirring for 2 h at rt, the mixture was concentrated to give a black oil. Purification via preparatory reverse phase HPLC followed by lyophilization afforded the title compound as an off-white solid. HPLC: Rt=1.423. MS (ESI): mass calcd. for C34H40ClN7O3S, 661.3; m/z found, 662.3 [M+H]+.
A. 2-(5-Fluoro-2,3-dihydro-indol-1-yl)-ethylamine. To a solution of 5-fluoro-2,3-dihydro-(1H)-indole (0.196 g, 1.43 mmol) in MeOH (20 mL) was added N-Boc-glycine (0.250 mL, 1.57 mmol). After stirring for 10 min, a solution of NaBH3CN (1 M in THF, 0.650 mL, 3.14 mmol) and 3 drops of acetic acid were added. After stirring for 12 h, the solution was diluted with EtOAc (20 mL) and washed with satd. aq. NaHCO3 (3×20 mL). The organic layer was separated, dried over MgSO4, and concentrated. To a solution of the resulting residue in dioxane (2 mL) was added 2 N HCl in Et2O (4 mL). After stirring for 1 h, the mixture was concentrated to yield the crude product, which was used in the next step without further purification. HPLC: Rt=0.967. MS (ESI): mass calcd. for C10H13FN2, 180.1; m/z found, 181.2 [M+H]+.
B. 2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid [2-(5-fluoro-2,3-dihydro-indol-1-yl)-ethyl]-amide. To a solution of Intermediate 1 (0.100 g, 0.2 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (1 mL) was added 2-(5-fluoro-2,3-dihydro-indol-1-yl)-ethylamine (0.040 g, 0.2 mmol). After stirring for 2 h at rt, the mixture was concentrated to give a black oil. Purification via preparatory reverse phase HPLC followed by lyophilization afforded the title compound as an off-white solid. HPLC: Rt=1.571. MS (ESI): mass calcd. for C37H42ClFN6O3S, 704.3; m/z found, 705.3 [M+H]+.
The title compound was prepared according to the method described for Example 8, substituting N-Boc-alanine for N-Boc-glycine in Example 8, Step A. HPLC: Rt=1.726. MS (ESI): mass calcd. for C38H44ClFN6O3S, 718.3; m/z found, 719.3 [M+H]+.
The compounds in Examples 10-11 were prepared according to the method described for Example 8, substituting the appropriate indole for 5-fluoro-2,3-dihydro-(1H)-indole in Example 8, Step A.
HPLC: Rt=1.617. MS (ESI): mass calcd. for C39H48ClN7O5S2, 793.3; m/z found, 794.3 [M+H]+.
HPLC: Rt=1.640. MS (ESI): mass calcd. for C39H48ClN7O5S2, 793.3; m/z found, 794.3 [M+H]+.
The compounds in Examples 12-19 were prepared according to the method described for Example 1, omitting Example 1, Step A, and substituting the appropriate amine for N1-(4-methoxy-phenyl)-ethane-1,2-diamine in Example 1, Step B.
HPLC: Rt=1.431. MS (ESI): mass calcd. for C31H35ClN6O4S, 622.2; m/z found, 623.2 [M+H]+.
HPLC: Rt=1.710. MS (ESI): mass calcd. for C36H39ClN6O3S, 670.3; m/z found, 671.3 [M+H]+.
HPLC: Rt=1.621. MS (ESI): mass calcd. for C32H36ClN5O3S2, 637.2; m/z found, 638.2 [M+H]+.
HPLC: Rt=1.640. MS (ESI): mass calcd. for C35H40ClN5O4S, 661.3; m/z found, 662.3 [M+H]+.
HPLC: Rt=1.453. MS (ESI): mass calcd. for C37H42ClN7O3S, 699.3; m/z found, 700.3 [M+H]+.
HPLC: Rt=1.491. MS (ESI): mass calcd. for C38H44ClN7O3S, 713.3; m/z found, 714.4 [M+H]+.
HPLC: Rt=1.405. MS (ESI): mass calcd. for C36H40ClN7O3S, 685.3; m/z found, 686.3 [M+H]+.
HPLC: Rt=1.481. MS (ESI): mass calcd. for C31H35ClN6O3S2, 638.2; m/z found, 639.2 [M+H]+.
A. [1-(5-Methyl-1H-benzoimidazol-2-yl)-ethyl]-carbamic acid tert-butyl ester. To a solution of N-Boc-Ala-OH (1.9 g, 10 mmol) at 0° C. in DMF (50 mL) was added NMM (1.1 mL, 19 mmol). After stirring for 10 min, 4-methyl-benzene-1,2-diamine (1.2 g, 10 mmol) in DMF (5 mL) was added. After stirring at rt for 2 h, the mixture was concentrated and the resulting solid was dissolved in EtOAc (50 mL) and washed with satd. aq. NaHCO3 (3×50 mL). The organic layer was separated, dried over MgSO4, and concentrated. The crude mono-acylated amine was dissolved in acetic acid (30 mL) and heated to 70° C. for 1 h. The solution was concentrated, and the resulting cyclized intermediate was dissolved in EtOAc (50 mL) and washed with satd. aq. NaHCO3 (3×50 mL). The organic layer was dried over MgSO4 and concentrated to give the desired product.
B. 1-(5-Methyl-1H-benzoimidazol-2-yl)-ethylamine. The above intermediate was dissolved in TFA (30 mL) and stirred for 1 h. The mixture was concentrated to give the crude product, which was used in the next step without further purification.
C. (S)-2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid [1-(5-methyl-1H-benzoimidazol-2-yl)-ethyl]-amide. To a solution of Intermediate 1 (0.100 g, 0.2 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (1 mL) was added 1-(5-methyl-1H-benzoimidazol-2-yl)-ethylamine (0.039 g, 0.2 mmol). After stirring for 2 h at rt, the mixture was concentrated to give a black oil. Purification via preparatory reverse phase HPLC followed by lyophilization afforded the title compound as an off-white solid. HPLC: Rt=1.461. MS (ESI): mass calcd. for C37H42ClN7O3S, 699.3; m/z found, 700.3 [M+H]+.
The title compound was prepared according to the methods described for Example 20, substituting N-Boc-Gly-OH for N-Boc-Ala-OH in Example 20, Step A. HPLC: Rt=1.457. MS (ESI): mass calcd. for C35H37Cl2N7O3S, 705.2; m/z found, 706.2 [M+H]+.
A. Thiocarbamoylmethyl carbamic acid tert-butyl ester. To a solution of Boc-Gly-NH2 (11 g, 62.9 mmol) in CH2Cl2 (300 mL) was added Lawesson's Reagent (13.2 g, 32.7 mmol). After stirring at rt for 12 h, the mixture was concentrated. Purification of the residue (SiO2; 50% Et2O in hexanes) provided the desired product (5.0 g, 42%).
B. (4-tert-Butyl-thiazol-2-ylmethyl)-carbamic acid tert-butyl ester. To a solution of the above compound (0.2 g, 1.05 mmol) in EtOH (5 mL) was added 1-bromo-3,3-dimethyl-butan-2-one (0.188 g, 1.05 mmol). After stirring 12 h at rt, the mixture was concentrated. Purification of the residue (SiO2; 20% EtOAc in hexanes) provided the desired product. HPLC: Rt=1.79. MS (ESI): mass calcd. for C13H22N2O2S, 270.1; m/z found, 271.1 [M+H]+.
C. (4-tert-Butyl-thiazol-2-yl)-methylamine. The above intermediate was dissolved in TFA (30 mL) and stirred for 1 h. The mixture was concentrated to provide the crude product, which was used in the next step without further purification. HPLC: Rt=0.402. MS (ESI): mass calcd. for C8H14N2S, 170.1; m/z found, 171.1 [M+H]+.
D. 2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid (4-tert-butyl-thiazol-2-ylmethyl)-amide. To a solution of Intermediate 1 (0.100 g, 0.2 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (1 mL) was added (4-tert-butyl-thiazol-2-yl)-methylamine (0.037 g, 0.2 mmol). After stirring for 2 h at rt, the mixture was concentrated to give a black oil. Purification via preparatory reverse phase HPLC followed by lyophilization afforded the title compound as an off-white solid. HPLC: Rt=1.699. MS (ESI): mass calcd. for C35H43ClN6O3S2, 694.3; m/z found, 695.3 [M+H]+.
The title compound was prepared according to the methods described for Example 22, substituting 2-bromo-1-phenyl-ethanone for 1-bromo-3,3-dimethyl-butan-2-one in Example 22, Step B. HPLC: Rt=1.583. MS (ESI): mass calcd. for C37H39ClN6O3S2, 714.2; m/z found, 714.3 [M]+.
The title compound was prepared according to the methods described in Example 23, substituting N-Boc-D-Ala-NH2 for N-Boc-Gly-NH2 in Step A. HPLC: Rt=1.753. MS (ESI): mass calcd. for C38H41ClN6O3S2, 728.2; m/z found, 729.2 [M+H]+.
A. (5-Fluoro-benzothiazol-2-ylmethyl)-carbamic acid tert-butyl ester. A solution of thiocarbamoyl-acetic acid tert-butyl ester (0.2 g, 1.05 mmol), 4-fluoro-2-iodo-phenylamine (0.230 g, 1.05 mmol), Pd2 dba3 (0.096 g, 0.105 mmol), and dppf (0.117 g, 0.210 mmol) in DMF (10 mL) was heated at 60° C. for 12 h. The mixture was diluted with EtOAc (20 mL) and washed with stad. aq. NaHCO3 (3×20 mL). The organic layer was dried over MgSO4 and concentrated. Purification of the residue (SiO2; 20% EtOAc in hexanes) provided the desired product (0.216 g, 78%).
B. (5-Fluoro-benzothiazol-2-yl)-methylamine. A solution of the above intermediate in TFA (30 mL) was stirred for 1 h. The mixture was concentrated to give the crude product, which was used in the next step without further purification.
C. 2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid (5-fluoro-benzothiazol-2-ylmethyl)-amide. To a solution of Intermediate 1 (0.100 g, 0.2 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (1 mL) was added (5-fluoro-benzothiazol-2-yl)-methylamine (0.037 g, 0.2 mmol). After stirring for 2 h at rt, the mixture was concentrated to give a black oil. Purification via preparatory reverse phase HPLC followed by lyophilization afforded the desired product as an off-white solid. MS (ESI): mass calcd. for C35H36ClFN6O3S2, 706.2; m/z found, 707.2 [M+H]+.
A. 2-Amino-N-(4-methoxy-phenyl)-acetamide. A solution of p-anisidine (0.25 g, 2.0 mmol), N-Boc-Gly-OH (0.26 g, 2.2 mmol), and HATU (0.926 g, 2.4 mmol) in CH2Cl2 (10 mL) was treated with DIEA (0.653 mL, 4 mmol), and stirred for 2 h. The mixture was diluted with CH2Cl2 (20 mL) and washed with 1 M HCl (2×10 mL). The organic layer was dried over MgSO4 and concentrated. The resulting solid was dissolved in dioxane (10 mL) and treated with 4 N HCl (20 mL) and stirred for 1 h. Removal of solvent gave the desired product which was used in the next step without further purification. HPLC: Rt=0.293. MS (ESI): mass calcd. for C9H12N2O2, 180.1; m/z found, 181 [M+H]+.
B. 2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid [(4-methoxy-phenylcarbamoyl)-methyl]-amide. To a solution of Intermediate 1 (0.100 g, 0.2 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (1 mL) was added the above amine (0.039 g, 0.2 mmol). After stirring for 2 h at rt, the mixture was concentrated to give a black oil. Purification via preparatory reverse phase HPLC followed by lyophilization afforded the title compound as an off-white solid. HPLC: Rt=1.651. MS (ESI): mass calcd. for C36H41ClN6O5S, 704.3; m/z found, 705.3 [M+H]+.
A. N1-(3-Methoxy-benzyl)-ethane-1,2-diamine. A solution of N-Boc-glycinal (0.1 g, 0.5 mmol), 3-methoxy benzylamine (0.076 g, 0.5 mmol), and NaBH(OAc)3 (0.117 g, 0.6 mmol) in 1,2-dichloroethane (10 mL) was treated with AcOH (2 drops) and stirred for 1 h. The mixture was diluted with CH2Cl2 (10 mL) and washed with 1 M HCl (1×10 mL). The organic layer was dried over MgSO4 and concentrated. The resulting solid was dissolved in dioxane (10 mL), treated with 4 N HCl (10 mL), and stirred for 1 h. Removal of solvent resulted in the desired product which was used in the next step without further purification.
B. 2′-Chloro-5′-[5-methanesulfonyl-1-(3-pyrrolidin-1-yl-propyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-3-yl]-biphenyl-3-carboxylic acid [2-(3-methoxy-benzylamino)-ethyl]-amide. To a solution of Intermediate 1 (0.100 g, 0.2 mmol) and DIEA (0.1 mL, 0.6 mmol) in DMF (1 mL) was added the above amine (0.039 g, 0.2 mmol). After stirring for 2 h at rt, the mixture was concentrated to give a black oil. Purification via preparatory reverse phase HPLC followed by lyophilization afforded the title compound as an off-white solid. HPLC: Rt=1.554. MS (ESI): mass calcd. for C37H45ClN6O4S, 704.3; m/z found, 705.3 [M+H]+.
The title compound was prepared according to the methods described for Intermediate 1 and Example 1, substituting 2-pyrrolidinol for pyrrolidine in Intermediate 1, Step D. HPLC: Rt=1.375. MS (ESI): mass calcd. for C36H43ClN6O5S, 706.3; m/z found, 707.3 [M+H]+.
The compounds in Example 29-30 were prepared according to the methods described for Intermediate 1 and Example 1, substituting 2-pyrrolidinol for pyrrolidine in Intermediate 1, Step D, omitting Example 1, Step A, and substituting the appropriate amine for N1-(4-methoxy-phenyl)-ethane-1,2-diamine in Example 1, Step B.
HPLC: Rt=1.415. MS (ESI): mass calcd. for C31H35ClN6O5S, 638.2; m/z found, 639.2 [M+H]+.
HPLC: Rt=1.579. MS (ESI): mass calcd. for C35H37ClN6O4S2, 704.2; m/z found, 705.2 [M+H]+.
Intermediate 2; 1-Piperidin-4-yl-pyrrolidin-2-one.
A. 1-(1-Benzyl-piperidin-4-yl)-pyrrolidin-2-one. To a mechanically-stirring heterogeneous mixture of 1-benzyl-4-piperidone (5.0 g, 27.1 mmol) and ethyl-4-aminobutyrate hydrochloride (5 g, 32.5 mmol) in anhydrous dichloroethane (100 mL) was added NaBH(OAc)3 (7.5 g, 35.2 mmol) portion-wise over 15 min. The resultant solution was stirred for 20 min at rt and was then treated with TEA (13.4 mL, 135.6 mmol) dropwise over 5 min. The resulting mixture was heated at 60° C. for 4 h. The mixture was cooled to rt, quenched by the slow addition of H2O (100 mL), and extracted with CH2Cl2 (3×50 mL). The combined organic layers were dried over MgSO4 and concentrated. The residue was partitioned between 5% EtOAc/hexanes and 1 N HCl (3×50 mL). The combined aqueous layers were adjusted to pH˜11 with aq. NaOH and extracted with EtOAc (3×50 mL). The organic layers were combined, washed with brine, dried over MgSO4, and concentrated. Recrystallization using 5% EtOAc in hexanes gave the desired white solid (6.24 g, 89%).
B. 1-Piperidin-4-yl-pyrrolidin-2-one. A Parr bottle containing a solution of the above compound (4.8 g, 18.7 mmol) in absolute EtOH (35 ml) and 10 wt. % Pd/C (0.48 g) was stoppered and placed on a Parr shaker under H2 (45 psi). After shaking for 36 h, the mixture was filtered through a pad of diatomaceous earth, washing with EtOAc. The filtrate was concentrated. The residue was diluted with warm Et2O and filtered to remove insoluble particulates. The filtrate was concentrated to yield the desired product (2.8 g, 90%). HPLC: Rt=0.128. MS (ESI): mass calcd. for C9H16N2O, 168.1; m/z found, 169.1 [M+H]+.
The title compound was prepared according to the methods described for Intermediate 1 and Example 1, substituting Intermediate 2 for pyrrolidine in Intermediate 1, Step D. HPLC: Rt=1.361. MS (ESI): mass calcd. for C41H50ClN7O5S, 787.3; m/z found, 788.3 [M+H]+.
The title compound was prepared according to the methods described for Intermediate 1 and Example 1, substituting Intermediate 2 for pyrrolidine in Intermediate 1, Step D, omitting Example 1, Step A, and substituting oxazol-2-yl-methylamine for N1-(4-methoxy-phenyl)-ethane-1,2-diamine in Example 1, Step B. HPLC: Rt=1.376. MS (ESI): mass calcd. for C36H42ClN7O5S, 719.3; m/z found, 720.3 [M+H]+.
The title compound was prepared according to the methods described for Intermediate 1 and Example 8, substituting Intermediate 2 for pyrrolidine in Intermediate 1, Step D and the appropriate indole for 5-fluoro-2,3-dihydro-(1H)-indole in Example 8, Step A. HPLC: Rt=1.580. MS (ESI): mass calcd. for C42H49ClFN7O4S, 801.3; m/z found, 802.3 [M+H]+.
The compounds in Example 34-35 were prepared according to the methods described for Intermediate 1 and Example 1, substituting Intermediate 2 for pyrrolidine in Intermediate 1, Step D, omitting Example 1, Step A, and substituting the appropriate amine for N1-(4-methoxy-phenyl)-ethane-1,2-diamine in Example 1, Step B.
HPLC: Rt=1.582. MS (ESI): mass calcd. for C40H44ClN7O4S2, 785.3; m/z found, 786.2 [M+H]+.
HPLC: Rt=1.402. MS (ESI): mass calcd. for C42H49ClN8O4S, 796.3; m/z found, 797.3 [M+H]+.
The compounds in Example 36-41 were prepared according to the methods described for Intermediate 1 and Example 1, omitting Intermediate 1, Step A, substituting 1-acetyl-piperidin-4-one for piperidone in Intermediate 1, Step B, omitting Example 1, Step A, and substituting the appropriate amine for N1-(4-methoxy-phenyl)-ethane-1,2-diamine in Example 1, Step B.
HPLC: Rt=1.555. MS (ESI): mass calcd. for C39H44ClFN6O2, 682.3; m/z found, 682.0 [M]+.
HPLC: Rt=1.407. MS (ESI): mass calcd. for C37H38ClF2N7O2, 685.3; m/z found, 686.3 [M+H]+.
HPLC: Rt=1.688. MS (ESI): mass calcd. for C36H37ClN6O2S, 652.24; m/z found, 653.2 [M+H]+.
HPLC: Rt=1.403. MS (ESI): mass calcd. for C38H42ClN7O2, 663.3; m/z found, 664.3 [M+H]+.
HPLC: Rt=1.568. MS (ESI): mass calcd. for C36H40ClN5O3, 625.3; m/z found, 626.3 [M+H]+.
The compounds in Example 42-47 were prepared according to the methods described for the immediately preceding examples, with the appropriate substituent changes.
HPLC: Rt=1.387. MS (ESI): mass calcd. for C36H43ClN6O2, 626.3; m/z found, 627.3 [M+H]+.
HPLC: Rt=1.448. MS (ESI): mass calcd. for C37H45ClN6O2, 640.3; m/z found, 641.3 [M+H]+.
HPLC: Rt=1.325. MS (ESI): mass calcd. C35H41ClN6O3, 628.3; m/z found, 628.3 [M]+.
HPLC: Rt=1.487. MS (ESI): mass calcd. for C38H46ClN7O4S, 731.3; m/z found, 732.3 [M+H]+.
HPLC: Rt=1.561. MS (ESI): mass calcd. for C40H49ClN6O3, 696.36; m/z found, 697.3 [M+H]+.
Human CatS was cloned into pFB+HT (from Stratagene clone), expressed in Sf9 cells, and purified over a Ni column. Fractions were concentrated and activated at pH 4.0 for 6 hr and then purified over a thiopropylsepharose column and eluted with 1×TBS, 500 mM NaCl, 1 mM EDTA, 25 mM DTT pH 7.6. Glycerol (50%) was added in a 1:1 (vol/vol) ratio (25% glycerol final) and protein was stored at −80° C. in 5 μL aliquots. Compounds were tested for their ability to inhibit CatS hydrolysis of the fluorescent substrate Z-Val-Val-Arg-AFC (catalog #I-1540, Bachem). Inhibitor solutions in DMSO were serially diluted and mixed with a solution of substrate in 150 mM sodium acetate pH 5.0 containing 1.5 mM DTT and 150 mM NaCl (optionally also containing 0.005% Triton X-100), which yielded a final substrate concentration of 10 μM and DMSO concentration of 1%. Reactions were initiated by the addition of CatS (1.5 nM final concentration of active enzyme; determined by titration against a tight-binding known standard inhibitor [Ki=35 nM] using Equation 1 below) and brief mixing. The increase in fluorescence over time was monitored using λexcitation=400 nm and λemission=505 nm. Initial rates were fit to the Morrison equation (Williams, J. W.; Morrison, J. F. The Kinetics of Reversible Tight-Binding Inhibition. Methods in Enzymology 1979, 63, 437-467) and apparent Ki (Kiapp) determined using Graphpad Prism software.
In equation 1, v is the initial rate measured in the presence of [I]0, the inhibitor concentration, using an enzyme concentration [E]0. v0 is the initial rate measured in the absence of inhibitor. Kiapp values are given in Table 1.
While the invention has been illustrated by reference to examples, it is understood that the invention is intended not to be limited to the foregoing detailed description.
This application claims the benefit of U.S. provisional patent application Ser. No. 60/889,979, filed Feb. 15, 2007 which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60889979 | Feb 2007 | US |
