# Patent

## Title
Cysteine protease inhibitors

## Abstract

Compounds of the formula I




wherein

    R1a is H; and R1b is C1-C6alkyl, Carbocyclyl or Het; or
    R1a and R1b together define a saturated cyclic amine with 3-6 ring atoms;
    R2a and R2b are independently H, halo, C1-C4alkyl, C1-C4haloalkyl or C1-C4alkoxy, or R2a and
    R2b together with the carbon atom to which they are attached form a C3-C6cycloalkyl;
    R3 is a branched C5-C10 alkyl chain, C2-C4haloalkyl or —CH2C3-C7 cycloalkyl;
    R4 is C1-C6alkyl, C1-C6haloalkyl, C1-C6alkylamino or C1-C6dialkylamino;

for use in the prophylaxis or treatment of a disorder characterised by inappropriate expression or activation of cathepsin S.

## Background
This application is the National Stage Under 35 U.S.C. §371 of PCT International Application No. PCT/IB2010/055738 filed on Dec. 10, 2010, which claims priority under 35 USC §119 of Application No. 0950951-4 filed in Sweden on Dec. 10, 2009 and of Application No. 1010084.0 filed in Great Britain on Jun. 16, 2010. The entire contents of each of which are incorporated by reference.
TECHNICAL FIELD
This invention relates to inhibitors of cathepsin S, and their use in methods of treatment for disorders involving cathepsin S such as autoimmune disorders, allergy and chronic pain conditions.
BACKGROUND TO THE INVENTION
The papain superfamily of cysteine proteases are widely distributed in diverse species including mammals, invertebrates, protozoa, plants and bacteria. A number of mammalian cathepsin enzymes, including cathepsins B, F, H, K, L, O, S, and W, have been ascribed to this superfamily, and inappropriate regulation of their activity has been implicated in a number of metabolic disorders including arthritis, muscular dystrophy, inflammation, glomerulonephritis and tumour invasion. Pathogenic cathepsin like enzymes include the bacterial gingipains, the malarial falcipains I, II, III et seq and cysteine proteases from Pneumocystis carinii, Trypanosoma cruzei and brucei, Crithidia fusiculata, Schistosoma spp.
In WO 97/40066, the use of inhibitors against Cathepsin S is described. The inhibition of this enzyme is suggested to prevent or treat disease caused by protease activity. Cathepsin S is a highly active cysteine protease belonging to the papain superfamily. Its primary structure is 57%, 41% and 45% homologous with human cathepsin L and H and the plant cysteine protease papain respectively, although only 31% homologous with cathepsin B. It is found mainly in B cells, dendritic cells and macrophages and this limited occurrence suggests the potential involvement of this enzyme in the pathogenesis of degenerative disease. Moreover, it has been found that destruction of Ii by proteolysis is required for MHC class II molecules to bind antigenic peptides, and for transport of the resulting complex to the cell surface. Furthermore, it has been found that Cathepsin S is essential in B cells for effective Ii proteolysis necessary to render class II molecules competent for binding peptides. Therefore, the inhibition of this enzyme may be useful in modulating class II-restricted immune response (WO 97/40066). Other disorders in which cathepsin S is implicated are asthma, chronic obstructive pulmonary disease, endometriosis and chronic pain.
BRIEF DESCRIPTION OF THE INVENTION
According to a first aspect of the invention there is provided a compound of the formula I:




wherein

    R1a is H; and
    R1b is C1-C6alkyl, optionally substituted with 1-3 substituents independently selected from:
    halo, hydroxy, cyano, azido, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy, C1-C4alkoxycarbonyl, C1-C4alkylcarbonyl, amine, C1-C4alkylamine, C1-C4dialkylamine, C1-C4alkylsulfonyl, C1-C4alkylsulfonylamino, aminocarbonyl, aminosulphonyl, Carbocyclyl and Het; or
    R1b is Carbocyclyl or Het; or
    R1a and R1b together with the N atom to which they are attached define a saturated cyclic amine with 3-6 ring atoms;
    
        wherein the Carbocyclyl, Het or cyclic amine is optionally substituted with 1-3 substituents independently selected from halo, hydroxy, cyano, azido, C1-C4alkyl, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy, C1-C4alkoxycarbonyl, C1-C4alkylcarbonyl, amine, C1-C4alkylamine, C1-C4dialkylamine, C1-C4alkylsulfonyl, C1-C4alkylsulfonylamino, aminocarbonyl, aminosulphonyl, RxOOC—C0-C2alkylene (where Rx is H, C1-C4alkyl or C1-C4haloalkyl), phenyl, benzyl or C3-C6cycloalkylC0-C2alkylene;
        
            wherein the phenyl, benzyl or cycloalkyl moiety is optionally substituted with 1-3 substituents independently selected from halo, C1-C4alkyl, C1-C4haloalkyl or C1-C4alkoxy);
        
        
    
    
    R2a and R2b are independently selected from H, halo, C1-C4alkyl, C1-C4haloalkyl, C1-C4alkoxy, or R2a and R2b together with the carbon atom to which they are attached form a C3-C6cycloalkyl;
    R3 is a C5-C10 alkyl, optionally substituted with 1-3 substituents independently selected from halo, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy; or
    R3 is a C2-C4alkyl chain with at least 2 chloro or 3 fluoro substituents; or
    R3 is C3-C7cycloalkylmethyl, optionally substituted with 1-3 substituents independently selected from C1-C4alkyl, halo, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy;
    R4 is C1-C6alkyl, C1-C6haloalkyl, C1-C6alkylamino, C1-C6dialkylamino;


Het is a stable, monocyclic or bicyclic, saturated, partially saturated or aromatic ring system containing 1-4 heteroatoms independently selected from O, S and N, each ring having 5 or 6 ring atoms;

    Carbocyclyl is C3-C6cycloalkyl, C5-C6cycloalkenyl or phenyl;

or a pharmaceutically acceptable salt, hydrate or N-oxide thereof.



In some embodiments, R1a is H and R1b is C1-C4alkyl, such as methyl, ethyl, isopropyl, t-butyl or preferably methyl, optionally substituted with one or more substituents as defined above, preferably 1-3 halo (e.g. F) or a C1-C4alkoxy (e.g. methoxy) group.
In other embodiments R1a is H and R1b is methyl, cyclopropyl, 1-phenylethyl, or a 5 or 6 membered heterocyclic ring containing 1-3 nitrogen atoms and 0 or 1 sulphur atoms, the cyclopropyl, phenyl or heterocyclic ring being optionally substituted with up to three substituents independently selected from:

    
    
        C1-C4alkyl, halo, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy, C1-C4alkoxycarbonyl, C1-C4alkylcarbonyl, amine, C1-C4alkylamine, C1-C4-dialkylamine, C1-C4alkylsulfonyl, C1-C4alkylsulfonylamino, aminocarbonyl, aminosulphonyl, RxOOC—C0-C2alkylene (where Rx is H or C1-C4alkyl) or C3-C6cycloalkylC0-C2alkylene or benzyl (the cycloalkyl, or the phenyl ring of the benzyl being optionally substituted with 1-3 substituents selected from C1-C4alkyl, halo, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy).
    
    


Examples of the 5 or 6 membered aromatic heterocyclyl for R1b include pyridyl or pyrimidyl and especially pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, thiadiazolyl, triazolyl or tetrazolyl, optionally substituted with any of which is optionally substituted with C1-C4alkyl (e.g. Me), halo (e.g. F), C1-C4haloalkyl (e.g. CF3), C1-C4alkoxy (e.g. MeO), C3-C6cycloalkylC0-C1alkylene (e.g. cyclopropyl or cyclopropylmethyl, benzyl or C0-C2alkyleneCOOH and its C1-C4alkyl esters. An exemplary species is 1-methyl-pyrazol-5-yl.
Typically according to this embodiment, the heterocyclic ring is pyrrolyl, pyrazolyl, imidazolyl, triazolyl, thiazolyl or thiadiazolyl, any of which is optionally substituted with C1-C4alkyl, halo, C1-C4haloalkyl, C1-C4alkoxy, C3-C6cycloalkyl or C3-C6cycloalkylmethyl.
Typically according to this embodiment, the heterocyclic ring is pyrazol-1-yl, which is optionally substituted with C1-C4alkyl, halo, C1-C4haloalkyl or cyclopropyl, preferably C1-C4alkyl, such as ethyl or preferably methyl.
A preferred value for R1b is pyrazol-1-yl which is N-substituted with C1-C4alkyl, such as ethyl or methyl.
A further typical value for R1b according to this embodiment, is methyl or cyclopropyl.
In other embodiments R1a is H and R1b is methyl or ethyl which is substituted in the 1-position with a cyclic group such as phenyl, or R1b is a monocyclic heterocyclyl such as pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, tetrazolyl, pyrazolyl, indolyl and the like. The phenyl or heterocyclyl is optionally substituted, for example with 1-3 substituents independently selected from hydroxy, amino, C1-C4alkyl, halo, C1-C4haloalkyl, C1-C4alkoxy, amino, C1-C4alkylamine, C1-C4dialkylamine and the like. An exemplary species is 1-phenylethyl.
In other embodiments R1a is H and R1b is C3-C6cycloalkyl, preferably cyclobutyl or cyclopropyl, optionally substituted as defined above. Preferably the cycloalkyl is unsubstituted or substituted with 1-3 substituents selected from halo (e.g. 1 or 2 fluoro), hydroxy, C1-C4alkyl (e.g. 1 or 2 methyl), C1-C4haloalkyl (e.g. a CF3 group) C1-C4alkoxy (e.g. an MeO group), C1-C4alkylamine (e.g. an MeNH— group), C1-C4dialkylamine (e.g. an (Me)2N— group) and the like. An exemplary species is cyclopropyl, or monofluoro- or gemdifluorocyclopropyl.
In some embodiments, R1a is H and R1b is a 6 or preferably 5 membered aromatic, heterocyclic ring containing 1-3 nitrogen atoms and 0 or 1 sulphur atoms, optionally substituted as defined above. Preferably the heterocyclic ring is linked to the adjacent nitrogen atom of the alpha keto amide group through a carbon atom of the heterocyclic ring. Exemplary substituents include C1-C4alkyl, halo, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy, C1-C4alkoxycarbonyl, C1-C4alkylcarbonyl, amine, C1-C4alkylamine, diC1-C4alkylamine, C1-C4alkylsulfonyl, C1-C4alkylsulfonylamino, aminocarbonyl, aminosulphonyl, RxOC(═O)C0-C2alkylene (where Rx is H or C1-C4alkyl) or C3-C6cycloalkylC0-C2alkylene or benzyl (the cycloalkyl or the phenyl ring of benzyl group) being optionally substituted with 1-3 substituents selected from C1-C4alkyl, halo, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy)
In some embodiments, R1a, R1b and the N-atom to which they are attached form a 3-6 membered cyclic amine, such as aziridine, azetidine, pyrrolidine, and preferably morpholine, piperazine or piperidine. These cyclic amines may be unsubstituted or substituted as described above, preferably with 1-3 substituents selected from halo (e.g. 1 or 2 fluoro), hydroxy, C1-C4alkyl (e.g. 1 or 2 methyl), C1-C4haloalkyl (e.g. a CF3 group) C1-C4alkoxy (e.g. an MeO— group), C1-C4alkylamine (e.g. an MeNH— group), C1-C4dialkylamine (e.g. an (Me)2N— group) and the like.
One embodiment of the invention includes compounds of formula I wherein R2a and R2b are both hydrogen.
In an alternative embodiment, at least one of R2a and R2b is halo, C1-C4alkyl, C1-C4haloalkyl or C1-C4alkoxy. Typically according to these embodiments, one of R2a and R2b is H, and the other is Cl, F, CF3 or MeO; especially F or MeO.
In a preferred embodiment, one of R2a and R2b is H, and the other is F. Specially preferred according to this embodiment are compounds having the stereochemistry shown in formula (Ia):




In some embodiments, R2a and R2b are both F, thus providing compounds of formula (Ib):




In other embodiments R2a and R2b together with the carbon atom to which they are attached form a C3-C6cycloalkyl.
In some embodiments R3 is cycloalkylalkyl, optionally substituted, for example with halo, (such as F) or alkoxy (such as MeO). Exemplary species include 1-methylcyclopentylmethyl, 1-methylcyclohexylmethyl, 1-methylcyclobutylmethyl, 1-methyl-3,3-difluorocyclobutylmethyl, 1-methyl-4,4-difluorocyclohexylmethyl, cyclopropylmethyl or 1-methyl-3,3-difluorocyclopentylmethyl.
Preferred R3 species include t-butylmethyl, cyclobutylmethyl, 1-methylcyclobutylmethyl and 1-methylcyclopentylmethyl, any of which is optionally substituted with one or two F or MeO. Representative species are 1-fluorocyclobutylmethyl and 1-fluorocyclopentylmethyl.
Further representative R3 species include 1-methylcyclopentylmethyl and 1-fluorocyclopentylmethyl,
Other embodiments have R3 as a straight or branched alkyl chain of 5-10 C-atoms, optionally substituted with 1-3 halo, (e.g. Cl or F), or a C1-C4alkoxy (e.g. MeO). Exemplary species include 2,2-dimethylpropyl, 3,3-dimethylpentyl, 2,2,3,3-tetramethylbutyl. Exemplary species of halogenated alkyl include 2,2-dichloroethyl, 3,3,3-trifluoropropyl, 2,2-trifluoromethylethyl, or 2,2,2-trifluoroethyl.
One embodiment of the invention include compounds wherein R4 is C1-C6alkyl, such as methyl or ethyl.
Another embodiment includes compounds wherein R4 is C1-C6haloalkyl, such as C1-C6chloroalkyl or C1-C6fluoroalkyl.
The compounds of formula I are characterised by various advantageous pharmaceutical properties and exhibit at least one improved property in view of the compounds of the prior art. In particular, the inhibitors of the present invention are superior in one or more of the following pharmacological related properties, i.e. potency, decreased cytotoxicity, improved pharmacokinetics, acceptable dosage and pill burden.
Without in any way wishing to be bound by theory, or the ascription of tentative binding modes for specific variables, P1, P2 and P3 as used herein are provided for convenience only and have their conventional meanings and denote those portions of the inhibitor believed to fill the S1, S2 and S3 subsites respectively of the enzyme, where S1 is adjacent the cleavage site and S3 remote from the cleavage site.
A further aspect of the invention comprises a method employing the compounds of formula I for the prophylaxis or treatment of diseases caused by aberrant expression or activation of cathepsin, i.e. diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.
A further aspect of the invention provides the use of the compounds of formula I prophylaxis or treatment of diseases caused by aberrant expression or activation of cathepsin, ie diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.
A further aspect of the invention provides the use of the compounds of formula I for the manufacture of a medicament for the prophylaxis or treatment of diseases caused by aberrant expression or activation of cathepsin S, i.e. diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.
Examples of such diseases or conditions defined in the immediately preceding three paragraphs include those enumerated in WO 97/40066, such as autoimmune diseases, allergies, such as asthma and hay fever, multiple sclerosis, rheumatoid arthritis and the like. A further example is the treatment of endometriasis, and especially chronic pain, as disclosed in WO03/20287. The invention further provides the use of the compounds of formula I or any subgroup of formula I in therapy and in the manufacture of a medicament for the treatment of diseases or conditions alleviated or moderated by inhibition of cathepsin S.
In one series of embodiments, the methods are employed to treat mammals, particularly humans at risk of, or afflicted with, autoimmune disease. By autoimmunity is meant the phenomenon in which the host's immune response is turned against its own constituent parts, resulting in pathology. Many human autoimmune diseases are associated with certain class II MHC-complexes. This association occurs because the structures recognized by T cells, the cells that cause autoimmunity, are complexes comprised of class II MHC molecules and antigenic peptides. Autoimmune disease can result when T cells react with the host's class II MHC molecules when complexed with peptides derived from the host's own gene products. If these class II MHC/antigenic peptide complexes are inhibited from being formed, the autoimmune response is reduced or suppressed. Any autoimmune disease in which class II MHC/antigenic complexes play a role may be treated according to the methods of the present invention.
Such autoimmune diseases include, e.g., juvenile onset diabetes (insulin dependent), multiple sclerosis, pemphigus vulgaris, Graves' disease, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis and Hashimoto's thyroiditis.
In another series of embodiments, the methods are employed to treat mammals, particularly humans, at risk of, or afflicted with, allergic responses. By “allergic response” is meant the phenomenon in which the host's immune response to a particular antigen is unnecessary or disproportionate, resulting in pathology. Allergies are well known in the art, and the term “allergic response” is used herein in accordance with standard usage in the medical field.
Examples of allergies include, but are not limited to, allergies to pollen, “ragweed,” shellfish, domestic animals (e.g., cats and dogs), bee venom, house dust mite allergens and the like. Another particularly contemplated allergic response is that which causes asthma. Allergic responses may occur, in man, because T cells recognize particular class II MHC/antigenic peptide complexes. If these class II MHC/antigenic peptide complexes are inhibited from being formed, the allergic response is reduced or suppressed. Any allergic response in which class II MHC/antigenic peptide complexes play a role may be treated according to the methods of the present invention. Immunosuppression by the methods of the present invention will typically be a prophylactic or therapeutic treatment for severe or life-threatening allergic responses, as may arise during asthmatic attacks or anaphylactic shock.
In another series of embodiments, the methods are employed to treat mammals, particularly humans, which have undergone, or are about to undergo, an organ transplant or tissue graft. In tissue transplantation (e.g., kidney, lung, liver, heart) or skin grafting, when there is a mismatch between the class II MHC genotypes (HLA types) of the donor and recipient, there may be a severe “allogeneic” immune response against the donor tissues which results from the presence of non-self or allogeneic class II MHC molecules presenting antigenic peptides on the surface of donor cells. To the extent that this response is dependent upon the formation of class II MHC/antigenic peptide complexes, inhibition of cathepsin S may suppress this response and mitigate the tissue rejection. An inhibitor of cathepsin S can be used alone or in conjunction with other therapeutic agents, e.g., as an adjunct to cyclosporin A and/or antilymphocyte gamma globulin, to achieve immunosuppression and promote graft survival. Preferably, administration is accomplished by systemic application to the host before and/or after surgery. Alternatively or in addition, perfusion of the donor organ or tissue, either prior or subsequent to transplantation or grafting, may be effective.
The above embodiments have been illustrated with an MHC class II mechanism but the invention is not limited to this mechanism of action. Suppression of cathepsin S as a treatment of COPD or chronic pain may not, for example, involve MHC class II at all.
A related aspect of the invention is directed to a method of treating a patient undergoing a therapy wherein the therapy causes an immune response, preferably a deleterious immune response, in the patient comprising administering to the patient a compound of Formula I or a pharmaceutically acceptable salt, n-oxide or hydrate thereof. Typically, the immune response is mediated by MHC class II molecules. The compound of this invention can be administered prior to, simultaneously, or after the therapy. Typically, the therapy involves treatment with a biologic, such as a protein, preferably an antibody, more preferably a monoclonal antibody. More preferrably, the biologic is Remicade®, Refacto®, ReferonA®, Factor VIII, Factor VII, Betaseron®, Epogen®, Enbrel®, Interferon beta, Botox®, Fabrazyme®, Elspar®, Cerezyme®, Myobloc®, Aldurazyrne®, Verluma®, Interferon alpha, Humira®, Aranesp®, Zevalin® or OKT3. Alternatively the treatment involves use of heparin, low molecular weight heparin, procainamide or hydralazine.
Assays for the assessment of cathepsin S inhibitors in the treatment of chronic pain, including neuropathic or inflammatory pain are as described in WO 03/20287.
Currently preferred indications treatable in accordance with the present invention include: Psoriasis;
Autoimmune indications, including idiopathic thrombocytopenic purpura (ITP), rheumatoid arthritis (RA), multiple schlerosis (MS), myasthenia gravis (MG), Sjögrens syndrome, Grave's disease and systemic lupus erythematosis (SLE);
Non-automimmune indications include allergic rhinitis, asthma, artherosclerosis, chronic obstructive pulmonary disease (COPD) and chronic pain.
The compounds of the invention can form salts which form an additional aspect of the invention. Appropriate pharmaceutically acceptable salts of the compounds of the invention include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate, benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids.
The compounds of the invention may in some cases be isolated as the hydrate. Hydrates are typically prepared by recrystallisation from an aqueous/organic solvent mixture using organic solvents such as dioxin, tetrahydrofuran or methanol. Hydrates can also be generated in situ by administration of the corresponding keton to a patient.
The N-oxides of compounds of the invention can be prepared by methods known to those of ordinary skill in the art. For example, N-oxides can be prepared by treating an unoxidized form of the compound of the invention with an oxidizing agent (e.g., trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta-chloroperoxybenzoic acid, or the like) in a suitable inert organic solvent (e.g., a halogenated hydrocarbon such as dichloromethane) at approximately 0° C. Alternatively, the N-oxides of the compounds of the invention can be prepared from the N-oxide of an appropriate starting material.
Compounds of the invention in unoxidized form can be prepared from N-oxides of the corresponding compounds of the invention by treating with a reducing agent (e.g., sulphur, sulphur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus dichloride, tribromide, or the like) in an suitable inert organic solvent (e.g., acetonitrile, ethanol, aqueous dioxane, or the like) at 0 to 80° C.
The present invention also includes isotope-labelled compounds of formula I or any subgroup of formula I, wherein one or more of the atoms is replaced by an isotope of that atom, i.e. an atom having the same atomic number as, but an atomic mass different from, the one(s) typically found in nature. Examples of isotopes that may be incorporated into the compounds of formula I or any subgroup of formula I, include but are not limited to isotopes of hydrogen, such as 2H and 3H (also denoted D for deuterium and T for tritium respectively), carbon, such as 11C, 13C and 14C, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O, phosphorus, such as 31P and 32P, sulphur, such as 35S, fluorine, such as 18F, chlorine, such as 36Cl, bromine such as 75Br, 76Br, 77Br and 82Br, and iodine, such as 123I, 124I, 125I and 131I.
The choice of isotope included in an isotope-labelled compound will depend on the specific application of that compound. For example, for drug or substrate tissue distribution assays, compounds wherein a radioactive isotope such as 3H or 14C is incorporated will generally be most useful. For radio-imaging applications, for example positron emission tomography (PET) a positron emitting isotope such as 11C, 18F, 13N or 15O will be useful. The incorporation of a heavier isotope, such as deuterium, i.e. 2H, may provide greater metabolic stability to a compound of formula I or any subgroup of formula I, which may result in, for example, an increased in vivo half life of the compound or reduced dosage requirements. For example, 2H isotope(s) are typically incorporated at position(s) disposed to metabolic liability. In the compounds of the present invention, suitable positions for incorporation of 2H isotopes are e.g. as substituents to the cyclobutylene group, i.e. one or both of R2a and R2b is 2H.
Isotopically labelled compounds of formula I or any subgroup of formula I can be prepared by processes analogous to those described in the Schemes and/or Examples herein below by using the appropriate isotopically labelled reagent or starting material instead of the corresponding non-isotopically labelled reagent or starting material, or by conventional techniques known to those skilled in the art.
It should be noted that the radical positions on any molecular moiety used in the definitions may be anywhere on such moiety as long as it is chemically stable.
As used herein, the following terms have the meanings as defined below:
Cm—Cnalkyl used on its own or in composite expressions such as Cm—Cnhaloalkyl, Cm—Cnalkylcarbonyl, Cm—Cnalkylamine, Cm—Cnalkylsulphonyl, Cm—Cnalkylsulfonylamino etc. represents a straight or branched alkyl radical having the number of carbon atoms indicated by m and n, e.g. C1-C4alkyl means an alkyl radical having from 1 to 4 carbon atoms. Preferred alkyl radicals for use in the present invention are C1-C4alkyl and includes methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl and isobutyl. Methyl and t-butyl are typically preferred. C1-C0alkyl has a corresponding meaning, including also all straight and branched chain isomers of pentyl and hexyl. Other recitals of Cm—Cnalkyl, such as C5-C10 alkyl have the corresponding meaning.
The term Me means methyl, MeO means methoxy, Et means ethyl and Ac means acetyl.
C0-C2alkylene used in composite expressions such as C3-C6cycloalkylC0-C2alkylene refers to a divalent radical derived from a methyl or ethyl group, or in the case of C0 the term C0-C2alkylene means a bond.
C1-C4haloalkyl refers to a C1-C4alkyl radical, wherein at least one C atom is substituted with a halogen, preferably chloro or fluoro. Trifluoromethyl is typically preferred C1-C4alkoxy represents a radical C1-C4alkyl-O wherein C1-C4alkyl is as defined above, and includes methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n-butoxy and isobutoxy. Methoxy and isopropoxy are typically preferred. C1-C6alkoxy has a corresponding meaning, expanded to include all straight and branched chain isomers of pentoxy and hexoxy. Other recitals of Cm-Cnalkoxy, such as C5-C10alkoxy have the corresponding meaning.
C1-C4haloalkoxy as used herein is meant to include C1-C4alkoxy wherein at least one C-atom is substituted with one or more halogen atom(s), typically chloro or fluoro. In many cases trifluoromethyl is preferred.
C1-C4alkoxycarbonyl means a radical C1-C4alkyl-O—C(═O).
Carbocyclyl includes cyclopentyl, cyclohexyl and especially cyclopropyl and cyclobutyl. Carbocyclyl further includes cyclopentenyl and cyclohexenyl, in each case with a single double bond. A frequently preferred value for Carbocyclyl is phenyl.
Cyclic amine includes aziridine, azetidine, pyrrolidine, piperidine, piperazine and morpholine.
Het is a stable, monocyclic or bicyclic, saturated, partially saturated or aromatic ring system, containing 1-4 hetero atoms independently selected from O, S and N, and each ring having 5 or 6 ring atoms; Exemplary aromatic Het include furan, thiophene, pyrrole, imidazole, pyrazole, triazole, tetrazole, thiazole, oxazole, isoxazole, oxadiazole, thiadiazole, isothiazole, pyridine, pyridazine, pyrazine, pyrimidine, quinoline, isoquinoline, benzofuran, benzothiophene, indole, indazole and the like. Exemplary unsaturated Het include tetrahydrofuran, pyran, dihydropyran, 1,4-dioxane, 1,3-dioxane, piperidine, pyrrolidine, morpholine, tetrahydrothiopyran, tetrahydrothiophene, 2-H-pyrrole, pyrroline, pyrazoline, imidazoline, thiazolidine, isoxazolidine and the like.
The compounds of the invention include a number of handles such as OH, NH or COOH groups to which conventional prodrug moieties can be applied. Prodrugs are typically hydrolysed in vivo to release the parent compound in the plasma, liver or intestinal wall. Favoured prodrugs are esters of hydroxyl groups such as a phenolic hydroxyl group at R4, or amine functions such as a sulphonamide amine function. Preferred pharmaceutically acceptable esters include those derived from C1-C6 carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubstituted or substituted with substituents broadly as described for R1a, typically 1-3 halo (e.g. F), C1-C4alkyl (e.g. Me), C1-C4haloalkyl (e.g. CF3) or C1-C4alkyloxy (e.g. MeO) groups. Favoured sulphonamide prodrugs include aminoacyls derived from C1-C6 carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubstituted or substituted with substituents broadly as described for variable R1a, typically 1-3 halo (e.g. F), C1-C4alkyl (e.g. Me), C1-C4haloalkyl (e.g. CF3) or C1-C4alkyloxy (e.g. MeO) groups.
Unless otherwise mentioned or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms, which said compound may possess. Said mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of said compound. All stereochemically isomeric forms of the compounds of the present invention both in pure form or mixed with each other are intended to be embraced within the scope of the present invention.
Pure stereoisomeric forms of the compounds and intermediates as mentioned herein are defined as isomers substantially free of other enantiomeric or diastereomeric forms of the same basic molecular structure of said compounds or intermediates. In particular, the term “stereoisomerically pure” concerns compounds or intermediates having a stereoisomeric excess of at least 80% (i.e. minimum 90% of one isomer and maximum 10% of the other possible isomers) up to a stereoisomeric excess of 100% (i.e. 100% of one isomer and none of the other), more in particular, compounds or intermediates having a stereoisomeric excess of 90% up to 100%, even more in particular having a stereoisomeric excess of 94% up to 100% and most in particular having a stereoisomeric excess of 97% up to 100%. The terms “enantiomerically pure” and “diastereomerically pure” should be understood in a similar way, but then having regard to the enantiomeric excess, and the diastereomeric excess, respectively, of the mixture in question.
Compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomer. While resolution of enantiomers can be carried out using covalent diasteromeric derivatives of compounds of Formula I, dissociable complexes are preferred (e.g., crystalline; diastereoisomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities. The diastereomers can be separated by chromatography, for example HPLC or, preferably, by separation/resolution techniques based upon differences in solubility. The optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques Andre Collet, Samuel H. Wilen, Enantiomers, Racemates and Resolutions, John Wiley & Sons, Inc. (1981).
While it is possible for the active agent to be administered alone, it is preferable to present it as part of a pharmaceutical formulation. Such a formulation will comprise the above defined active agent together with one or more acceptable carriers/excipients and optionally other therapeutic ingredients. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
The formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, but preferably the formulation is an orally administered formulation. The formulations may conveniently be presented in unit dosage form, e.g. tablets and sustained release capsules, and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the above defined active agent with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of Formula I or its pharmaceutically acceptable salt in conjunction or association with a pharmaceutically acceptable carrier or vehicle. If the manufacture of pharmaceutical formulations involves intimate mixing of pharmaceutical excipients and the active ingredient in salt form, then it is often preferred to use excipients which are non-basic in nature, i.e. either acidic or neutral.
Formulations for oral administration in the present invention may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion and as a bolus etc.
With regard to compositions for oral administration (e.g. tablets and capsules), the term suitable carrier includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring or the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.
Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatine and glycerine, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.
As with all pharmaceuticals, the appropriate dosage for the compounds or formulations of the invention will depend upon the indication, the severity of the disease, the size and metabolic vigour and the patient, the mode of administration and is readily determined by conventional animal trials. Dosages providing intracellular (for inhibition of physiological proteases of the papain superfamily) concentrations of the order 0.01-100 μM, more preferably 0.01-10 μM, such as 0.1-5 μM are typically desirable and achievable.
Compounds of the invention are prepared by a variety of solution and solid phase chemistries.
A typical first step is the preparation of a P1 building block of the formula V




where R2a and R2b are as defined above, PG is a conventional N protecting group such as Boc, CBz or Fmoc and PG* is H or a conventional carboxy protecting group, such as a C1-C4alkyl or benzyl. These building blocks are novel and constitute a further aspect of the invention.

Building blocks of formula V are typically prepared as described in scheme 1 below.




A suitable starting material is an N-protected cyclobutyl amino acid, of which several are available commercially or can be prepared as shown in the following Examples or as described by Allan et al. in J. Med. Chem., 1990 33(10) 2905-2915.
The carboxylic acid (1a) is transformed via a Weinreb synthesis to a N,O-dimethylhydroxamic acid (1b) which provides the corresponding aldehyde (1c). The aldehyde may also be accessed by reduction of the carboxylic function of a cyclobutyl amino acid and oxidation under Dess Martin conditions. The aldehyde (1c) can be subsequently reacted with the appropriate isocyanide in a Passerini reaction to afford the required α-hydroxy R1aR1b amide (1d). However, in the case where the appropriate isocyanide is not readily available, t-butylisocyanide can alternatively be used, thus affording the t-butyl amide, which after hydrolysis of the amide, provides the α-hydroxycarboxylic acid P1 building block (1e). Generally the strongly acidic conditions required to hydrolyse the amide also lead to loss of the NBoc protection, if used, hence, the amine can be used directly to couple to a P2 building block or else if it needs to be stored, the amine can be reprotected.
The P1 building block thus afforded is then extended at the C and N termini to provide compounds of formula I. In scheme 2, a general route to compounds wherein R4 is C1-C6alkyl or C1-C6haloalkyl.




Typically the C terminus is extended first by reaction of the building block of formula V (2a) with an R1a* R1b* amine, where R1a* and R1b* are R1a and R1b respectively or synthons therefor (selected in view of the sensitivity of the R1b function for the P3 elongation conditions outlined below). The reaction proceeds with conventional peptide chemistries as discussed below. The thus prepared P1-prime side unit (2b) is thereafter deprotected at the N terminus and elongated with the P2 building block, providing the N-protected intermediate amine 2c, and subsequently P3 building block, providing the amide 2d, using conventional peptide chemistries. For example a P2 residue can be introduced via BocP2-OH using standard coupling conditions such as HATU, DIPEA in DMF. The terminal Boc protection is again removed with acetyl chloride in methanol or equivalent and the P3 residue is introduced using standard peptide coupling techniques, such as by reaction with the appropriate P3-acid using conditions like HATU, DIPEA in DMF or by reaction with a P3-acid halide such as the acid chloride or the like. The final steps will generally comprise conversion of the R1a*/R1b* synthons (if present) to their final form and finally oxidation of the alpha hydroxy amide function using Dess Martin conditions to provide the desired alpha keto amide compound 2e.
An extensive range of appropriately protected L-amino acids suitable for P2 building blocks and carboxylic acids, carboxylic acid halides and carbamoyl halides suitable for P3 building blocks are commercially available or accessed by simple chemistries or as shown in WO06/064286. The P3 and P2 building blocks may alternatively be coupled first and then reacted with the P1-prime side unit.
Elongation is typically carried out in the presence of a suitable coupling agent e.g., benzotriazole-1-yloxytrispyrrolidinophosphonium hexafluorophosphate (PyBOP), O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate (HATU), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), or 1,3-dicyclohexyl carbodiimide (DCC), optionally in the presence of 1-hydroxybenzotriazole (HOBT), and a base such as N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, and the like. The reaction is typically carried out at 20 to 30° C., preferably at about 25° C., and requires 2 to 24 h to complete. Suitable reaction solvents are inert organic solvents such as halogenated organic solvents (e.g., methylene chloride, chloroform, and the like), acetonitrile, N,N-dimethylformamide, ethereal solvents such as tetrahydrofuran, dioxane, and the like.
Alternatively, the above elongation coupling step can be carried out by first converting the P3/P2 building block into an active acid derivative such as succinimide ester and then reacting it with the P1 amine. The reaction typically requires 2 to 3 h to complete. The conditions utilized in this reaction depend on the nature of the active acid derivative. For example, if it is an acid chloride derivative, the reaction is carried out in the presence of a suitable base (e.g. triethylamine, diisopropylethylamine, pyridine, and the like). Suitable reaction solvents are polar organic solvents such as acetonitrile, N,N-dimethylformamide, dichloromethane, or any suitable mixtures thereof.
The term “N-protecting group” or “N-protected” as used herein refers to those groups intended to protect the N-terminus of an amino acid or peptide or to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis” (John Wiley & Sons, New York, 1981), which is hereby incorporated by reference. N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoracetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl, and the like, carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Favoured N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl (bz), t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz).
Hydroxy and/or carboxy protecting groups are also extensively reviewed in Greene ibid and include ethers such as methyl, substituted methyl ethers such as methoxymethyl, methylthiomethyl, benzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl and the like, silyl ethers such as trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl, triphenylsilyl, t-butyldiphenylsilyl triisopropyl silyl and the like, substituted ethyl ethers such as 1-ethoxymethyl, 1-methyl-1-methoxyethyl, t-butyl, allyl, benzyl, p-methoxybenzyl, dipehenylmethyl, triphenylmethyl and the like, aralkyl groups such as trityl, and pixyl (9-hydroxy-9-phenylxanthene derivatives, especially the chloride). Ester hydroxy protecting groups include esters such as formate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate, pivaloate, adamantoate, mesitoate, benzoate and the like. Carbonate hydroxy protecting groups include methyl vinyl, allyl, cinnamyl, benzyl and the like.


DETAILED DESCRIPTION OF THE EMBODIMENTS
Various embodiments of the invention will now be described by way of illustration only with reference to the following Examples.
In the examples below, the following systems are typically employed:
Nuclear Magnetic Resonance (NMR) spectra were recorded on a Varian Gemini 7 Tesla 300 MHz instrument, or a Bruker Avance 400 MHz instrument in the solvent indicated. Chemical shifts are given in ppm down- and upfield from tetramethylsilane (TMS). Resonance multiplicities are denoted s, d, t, m, br and app for singlet, doublet, triplet, multiplet, broad and apparent, respectively. The Mass Spectrometry (MS) spectra were recorded on a Finnigan SSQ7000 TSP or a Finnigan SSQ710 DI/EI instrument. LC-MS was obtained with a Waters 2790 LC-system equipped with a Waters XterraTM MS C8 2.5 μm 2.1×30 mm column, a Waters 996 Photodiode Array Detector and a Micromass ZMD. High pressure liquid chromatography (HPLC) assays were performed using a Hewlett Packard 1100 Series HPLC system equipped with a Zorbax column SB-C8 4.6 mm×15 cm. Column chromatography was performed using silica gel 60 (230-400 mesh ASTM, Merck) and thin layer chromatography (TLC) was performed on TLC precoated plates, silica gel 60 F254 (Merck).
Preparation of Building Block 1, a P1 Building Block




Step a) [1-(Methoxy-methyl-carbamoyl)-cyclobutyl]-carbamic acid tert-butyl ester (BB1-a)
To a solution of 1-tert-butoxycarbonylamino-cyclobutanecarboxylic acid (3 g, 13.94 mmol) in dry DMF (50 mL) was added N,O-dimethylhydroxylamine x HCl (1.36 g, 13.94 mmol) and DIEA (9.21 mL, 55.75 mmol). The reaction flask was cooled to 0° C. and after 10 minutes HATU (5.30 g, 13.94 mmol) was added to the solution (which turned yellow on addition). After 2 hrs the DMF was removed by rotary evaporation at reduced pressure. The residue was dissolved in EtOAc (100 mL) and washed twice with 10% citric acid (aq) and saturated NaHCO3(aq) solution. The organic phase was dried with Na2SO4, filtered and evaporated on silica. The product was purified by flash chromatography (heptane: ethyl acetate (1:1) to give the product as a colourless oil that slowly crystallizes (3.13 g) in 87% yield.
Step b) (1-Formyl-cyclobutyl)-carbamic acid tert-butyl ester (BB1-b)
LiAlH4 (202 mg, 5.33 mmol) was added to a solution of the Weinreb amide BB1-a (1.10 g, 4.27 mmol) dissolved in dry diethyl ether (35 mL) at 0° C. The solution was stirred at 15 minutes before the reaction was quenched with slow addition of potassium hydrogen tartaric acid (sat, aq) and stirred for 10 minutes. The solution was poured into a separatory funnel and the water phase was extracted with ethyl acetate twice. The combined organic phases were washed with 0.5 M HCl (3 times), NaHCO3(aq) (2 times) and brine (1 time). The organic phase was dried with Na2SO4, filtered and evaporated on silica. The product was purified by flash chromatography (heptane: ethyl acetate (4:1→3:1) to give the product as white crystals (0.647 g) in 76% yield.
Step c) [1-(tert-Butylcarbamoyl-hydroxy-methyl)-cyclobutyl]-carbamic acid tert-butyl ester (BB1-c)
BB1-b, (1.75 g, 8.78 mmol) was dissolved in CH2Cl2 (18 mL) and cooled in an ice bath, under inert gas. Pyridine (2.85 mL) was added, followed by t-butyl isocyanide (1.50 mL, 13.3 mmol). Trifluoroacetic acid (1.35 mL, 17.5 mmol) was then added dropwise over 30 min. The yellow solution was stirred at RT overnight. The mixture was concentrated, diluted with EtOAc (100 mL) and washed successively with 1N HCl (50 mL), saturated NaHCO3 (50 mL) and saturated NaCl (2×50 mL). Drying (Na2SO4) and concentration under vacuum. The afforded crude product was treated with THF (2.5 mL) and 1M LiOH in 3/1 MeOH-water (2.5 mL) at RT. TLC (3/1 petroleum ether—EtOAc) showed complete ester hydrolysis after 15 min. After 45 min reaction time, 1N HCl (2.5 mL), water (10 mL) and EtOAc (20 mL) were added, and the layers were separated. The organic phase was washed with saturated NaHCO3 (20 mL) and then saturated NaCl (2×20 mL), dried (Na2SO4) and concentrated. Flash chromatography (75 g silica, 5/1 to 1/1 petroleum ether—EtOAc) gave a white solid (2.36 g, 89%).
Step d (1-Tert-butoxycarbonylamino-cyclobutyl)-hydroxyacetic acid (BB1)
BB1-c (1.30 g, 4.33 mmol) was refluxed with 6N HCl (40 mL) until amide hydrolysis was complete as monitored by LCMS. The mixture was evaporated, co-evaporating several times with water. 1M NaOH (15 mL) was added to the residue and the basic solution was stirred under vacuum for 15 min. Boc2O (1.92 g, 8.80 mmol) in dioxane (10 mL) was added, keeping pH at 10-11, and the mixture was stirred at RT overnight. The mixture was diluted with water (50 mL), acidified with 1N HCl to pH 3, in an ice bath, and then extracted with EtOAc (2×50 mL, then 30 mL). The organic phase was washed with saturated NaCl (50 mL), dried (Na2SO4) and evaporated to give crude P1 building block BB1 (0.649 g).
1HNMR (400 MHz, d6-DMSO) δ 6.88 (br s, 1H), 4.15 (s, 1H), 2.40 (br m, 2H), 1.98 (br m, 2H), 1.80 (br m, 2H), 1.35 (s, 9H); ms ES+ m/z 146 (100%), 190 (50%).
Preparation of Building Block 2, a P1 Building Block




Step a) ((1-Bromo-3-chloropropan-2-yloxy)methyl)benzene (BB2-a)
To a stirred mixture of benzyl bromide (185 g, 1.08 mol) and (1.5 g) of mercurous chloride was added epichlorohydrin (100 g, 1.08 mol). The reaction mixture was heated for 12 hr at 100° C. TLC analysis confirmed formation of product. The product was separated from the dark brown reaction mixture by column chromatography using petroleum ether as eluent. TLC system; Petroleum ether: ethyl acetate (9:1), RF=0.7. Yield; 148 g, 51%.
Step b) 3-Benzyloxy-cyclobutane-1,1-dicarboxylic acid diethyl ester (BB2-b)
To a stirred suspension of sodium hydride (22.5 g, 0.562 mol) in 800 mL of dry dioxane, was added diethyl malonate (90 g, 0.562 mol) drop-wise over 20 min. After this addition was complete, BB2-a (148 g, 0.56 mol) was added drop-wise over 20 min. The mixture was then heated at reflux for 24 hr. After cooling to room temperature, sodium hydride (22.5 g, 0.562 mol) in a little dioxane ( ̃20 mL) was added to the mixture and heating at reflux was continued for an additional 48 hr. The solvent was partially removed under reduced pressure and the mixture was treated with 800 mL of water. This mixture was then extracted with ethyl acetate (500 mL×3), extracts were dried (Na2SO4) and concentrated in vacuo and the residue was purified by column chromatography using petroleum ether: ethyl acetate (10%) which gave the title compound. TLC system; petroleum ether: ethyl acetate (9:1), RF=0.3. Yield: 100 g, 58%
Step c) Diethyl 3-hydroxycyclobutane-1,1-dicarboxylate (BB2-c)
To a solution of compound BB2-b (40 g) in EtOH (500 mL) was added 10% palladium on charcoal (4 g) and the mixture was hydrogenated for 3.5 hours at 50 psi at room temperature. The catalyst was removed by filtration, washed with ethyl acetate, EtOH and the solvent was then removed under reduced pressure. The residue was purified by silica gel chromatography with hexane/ethyl acetate as eluent to provide the title compound. TLC system; Petroleum ether: ethyl acetate (9:1), RF=0.3. Yield: 18 g, 64%.
Step d) Diethyl 3-oxocyclobutane-1,1-dicarboxylate (BB2-d)
To a solution of compound BB2-c (18 g, 0.0833 mol) in DCM (200 mL) was added PCC (37 g, 0.176 mol) and the mixture was stirred for four hours at room temperature. The solution was filtered through a silica gel column and the residue was washed with DCM/MeOH 98/2 and then filtered through a similar column. The combined fractions were evaporated under reduced pressure to provide the desired compound. TLC system; Petroleum ether: ethyl acetate (9:1), RF=0.3. Yield: 11 g, 62%.
Step e) Diethyl 3,3-difluorocyclobutane-1,1-dicarboxylate (BB2-e)
To a cooled solution of compound BB2-d (11 g, 0.0513 mol) in dry DCM (150 mL) was added drop-wise a solution of DAST (18.72 g, 0.116 mol) and the mixture was stirred at room temperature overnight. The mixture was added to ice water and was extracted three times with DCM. The solution was dried with sodium sulphate and evaporated under reduced pressure. The residue was purified by silica gel chromatography employing hexane/ethyl acetate as eluent to provide the title compound. TLC system; Petroleum ether: ethyl acetate (7:3), RF=0.5. Yield: 7.7 g, 64%.
Step f) 1-(Ethoxycarbonyl)-3,3-difluorocyclobutanecarboxylic acid (BB2-f)
Compound BB2-e (7.7 g, 0.0325 mol) was dissolved in ice cooled 0.5 M ethanolic potassium hydroxide solution (30 mL) and water (6 mL). The mixture was stirred at room temperature overnight. Water was added and most of the ethanol was removed under reduced pressure. The mixture was acidified with 2M HCl and extracted three times with ethyl acetate. The organic phase was dried with sodium sulphate and evaporated under reduced pressure to give the desired compound. TLC system: petroleum ether: ethyl acetate (1:1), RF=0.3. Yield: 5.8 g, 86%.
Step g) Ethyl 1-(tert-butoxycarbonylamino)-3,3-difluorocyclobutanecarboxylate (BB2-g)
To a solution of compound BB2-f (5.8 g, 0.0273 mol) in dry dioxane (100 mL) was added tert-butanol (24.4 mL), DPPA (7.87 g, 0.027 mol) and TEA (2.87 g, 0.0284 mol) and the mixture was refluxed for five hours. Ethyl acetate (about 200 mL) was added and the organic phase was washed twice with 5% citric acid and saturated sodium hydrogen carbonate. The solution was dried and evaporated under reduced pressure. The desired product was isolated by silica gel chromatography with hexane/ethyl acetate. TLC system; Petroleum ether: ethyl acetate (1:1), RF=0.5. Yield: 4 g, 51.4%.
Step h) tert-Butyl 3,3-difluoro-1-(hydroxymethyl)cyclobutylcarbamate (BB2-h)
To a ice cooled solution of compound BB2-g (4 g, 0.0143 mol) in dry THF (100 mL) was slowly added a solution of 2M lithium borohydride (30 mL) and the mixture was allowed to warm up to room temperature. The mixture was stirred for three hours at room temperature. Ice water and 5% citric acid were added and the mixture was extracted three times with DCM. The organic phase was dried (Na2SO4), filtered and evaporated under reduced pressure which gave the title compound. TLC system petroleum ether: ethyl acetate (1:1), RF=0.3. Yield: 3.1 g, 91%.
Step i) tert-Butyl 3,3-difluoro-1-formylcyclobutylcarbamate (BB2-i)
To a solution of compound BB2-h (3.1 g, 0.0130 mol) in dry DCM (100 mL) was added Dess Martin Period inane (19.9 g, 0.0470 mol) and the mixture was stirred for three hours at room temperature. Ethyl acetate (200 mL) was added and the organic phase was washed twice with 10% sodium thiosulphate solution, twice with 0.5 M NaOH and with brine. The organic phase was dried and evaporated under reduced pressure. The residue was purified by silica gel chromatography with hexane/ethyl acetate as eluent which gave the title compound. TLC system; petroleum ether: ethyl acetate (1:1), RF=0.4. Yield: 2.7 g, 87%.
Step j) tert-Butyl 1-(2-(tert-butylamino)-1-hydroxy-2-oxoethyl)-3,3-difluorocyclobutylcarbamate (BB2-j)
To a ice cooled solution of compound BB2-i (1.5 g, 0.0064 mol) in dry DCM (100 mL) was added tert-butylisocyanate (0.81 g, 0.009 mol) and pyridine (2.04 g, 0.027 mol). Trifluoroacetic acid (1.58 g, 0.015 mol) was added over a ten minutes period. The mixture was stirred for five hours at room temperature. Ethyl acetate was added and the organic phase was washed twice with 5% citric acid and brine. The organic phase was evaporated and dissolved in dioxane (50 mL). 1M LiOH solution (100 mL) was added and the mixture was stirred overnight at room temperature. 5% Citric acid was added and the mixture was extracted three times with ethyl acetate. The organic phase was washed with brine, dried (Na2SO4), filtered and evaporated under reduced pressure. The product was purified by silica gel chromatography with hexane/ethyl acetate as eluent. TLC system; Petroleum ether: ethyl acetate (1:1), RF=0.4. Yield: 1.0 g, 46%.
Step k) 2-(1-(Tert-Butoxycarbonylamino)-3,3-difluorocyclobutyl)-2-hydroxyacetic acid (BB2)
Compound BB2-j (1 g) was dissolved in 6N HCl (40 mL), and heated to reflux for 24 h after which TLC showed that the reaction had reached completion. The reaction mixture was concentrated in vacuo and residue was dissolved in THF; H2O (7; 3, 50 mL), and TEA (1.8 mL, 0.012 mol) and Boc anhydride (2.6 g, 0.012 mol) were both added. The mixture was stirred at RT for 8 h when TLC confirmed the reaction had reached completion. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography using 5% methanol in chloroform which gave the title compound. TLC system; MeOH: CHCl3 (1:9), RF=0.4. Yield: 0.6 g, 72%.
1H NMR (400 MHz, d6-DMSO) δ 7.30 (br s, 1H), 4.11 (s, 1H), 2.90 (br m, 2H), 2.61 (br m, 2H), 1.35 (s, 9H); ms ES+ m/z 281 (100%).
Preparation of Building Block 3—a P1 Building Block




Step a) ((1-Bromo-3-chloropropan-2-yloxy)methyl)benzene (BB3-a)
A mixture of benzyl bromide (46.0 g, 0.269 mol) and epichlorohydrin (24.9 g, 0.269 mol) and mercurous chloride (0.04 g, 0.085 mmol) was heated for 12 h at 150° C. The crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 1% EtOAc in pet ether) which afforded the title compound as a viscous liquid (50 g, yield 70%). TLC system: 10% EtOAc in pet ether, Rf=0.6.
Step b) Diethyl 3-(benzyloxy)cyclobutane-1,1-dicarboxylate (BB3-b)
In a three-neck flask equipped with stirrer, additional funnel and reflux condenser was place NaH (4.6 g, 0.192 mol) in dry dioxane (150 mL). To this stirred reaction mixture, diethyl malonate (30.75 g, 0.192 mol) was added drop-wise over 30 min. After the addition was complete, compound BB3-a (50 g, 0.19 mol) was added drop-wise over a period of 30 min. The reaction mixture was refluxed for 24 h. After cooling to room temperature, NaH (4.6 g, 0.192 mol) and dry dioxane (40 mL) was added to the reaction mixture and further heated to reflux for another 48 h. The solvent was partially removed under reduced pressure and the mixture was treated with water (150 mL). The product was extracted with diethyl ether (3×100 mL), the organic layer was washed with brine and dried over anhydrous Na2SO4. Solvent was concentrated in vacuum and the crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 2% EtOAc in pet ether) which afforded the title compound as a viscous liquid (33 g, yield 57%). TLC system: 15% EtOAc in pet ether, Rf=0.5.
Step c) Diethyl 3-hydroxycyclobutane-1,1-dicarboxylate (BB3-c)
To a solution of compound BB3-b (33 g, 0.108 mol) in EtOH (300 mL) was added 10% palladium on charcoal (10 g) and the mixture was hydrogenated for 48 h with 50 psi pressure at room temperature. The catalyst was removed by filtration through a celite bed and washed thoroughly with EtOAc. The solvent was removed under reduced pressure. The product was purified by silica gel chromatography (silica gel 60-120 mesh, eluent 20% EtOAc in pet ether) which afforded the title compound as a viscous liquid (12 g, yield 51%). TLC system: 30% EtOAc in pet ether, Rf=0.3.
Step d) Diethyl 3-fluorocyclobutane-1,1-dicarboxylate (BB3-d)
Compound BB3-c (0.8 g, 0.0037 mol) was dissolved in dry DCM (16 mL) and cooled to 0° C. DAST (1.8 g, 0.011 mol) was added drop-wise to the cold solution. The reaction mixture was warmed to room temperature stirred for 12 h. The reaction mixture was quenched with cold saturated NaHCO3 solution. The crude product was extracted with DCM (100 mL). The organic layer was washed with 10% NaHCO3 solution, water followed by brine and dried over anhydrous Na2SO4. Solvent was concentrated in vacuum and the crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 1-2% EtOAc in pet ether) which afforded the title compound as a pale yellow liquid (460 mg, yield 57%). TLC system: 10% EtOAc in pet ether, Rf=0.4.
Step e) 1-(Ethoxycarbonyl)-3-fluorocyclobutanecarboxylic acid (BB3-e)
Compound BB3-d (0.46 g, 0.0021 mol) was dissolved in ice cooled 0.5M potassium hydroxide solution in EtOH (4.2 mL) and water (1.4 mL). The mixture was stirred at room temperature overnight. Water was added and most of the ethanol was removed under reduced pressure. The mixture was acidified with 2N HCl and extracted with EtOAc (3×50 mL). The organic phase was dried over anhydrous Na2SO4. Solvent was concentrated in vacuum to afford the crude title compound (0.35 g, crude) which was used as such for the next step. TLC system: 50% EtOAc in pet ether, Rf=0.3.
Step f) Ethyl 1-(tert-butoxycarbonylamino)-3-fluorocyclobutanecarboxylate (BB3-f)
To a solution of compound BB3-e (0.35 g, 0.0018 mol) in dry dioxane (6 mL) was added tert-butanol (1.8 mL), diphenyl phosphoryl azide (0.56 g, 0.002 mol) and triethylamine (0.2 g, 0.002 mol) and the mixture was refluxed for 5 h. After completion of the reaction, EtOAc (60 mL) was added to the reaction mixture and the organic layer was washed with 5% citric acid (2×20 mL) followed by saturated NaHCO3 (50 mL). The organic solvent was evaporated under reduced pressure. To the residue EtOAc (100 mL) was added and the organic layer was washed with brine and dried over anhydrous Na2SO4. Solvent was concentrated in vacuum and the crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 5-10% EtOAc in pet ether) which afforded the title compound as white crystals (0.27 g, yield 56%). TLC system: 20% EtOAc in pet ether, Rf=0.4.
Step g) tert-Butyl 3-fluoro-1-(hydroxymethyl)cyclobutylcarbamate (BB3-g)
To a ice cooled solution of compound BB3-f (0.27 g, 0.001 mol) in dry THF (10 mL) was slowly added a solution of 2M lithium borohydride (2 mL, 0.004 mol) and the mixture was allowed to warm up to room temperature. The mixture was stirred for 3 h at room temperature. The reaction mixture was quenched with ice water (2 mL) and 5% citric acid (5 mL) and the crude product was extracted with DCM (2×50 mL). The organic layer was washed with brine and dried over anhydrous Na2SO4. Solvent was concentrated in vacuum and the crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 15-18% EtOAc in pet ether) which afforded the title compound as white solid (90 mg, yield 39%). TLC system: 50% EtOAc in pet ether, Rf=0.5.
Step h) tert-Butyl 3-fluoro-1-formylcyclobutylcarbamate (BB3-h)
To a degassed solution of compound BB3-g (90 mg, 0.0004 mol) in dry DCM (4.5 mL) was added Dess-Martin Periodinane (0.21 g, 0.0005 mol) and the mixture was stirred for 3 h at room temperature. EtOAc (30 mL) was added and the organic layer was washed with 10% sodium thiosulphate solution (2×10 mL), 0.5 M NaOH (20 mL) and with brine. The organic layer was dried over anhydrous Na2SO4. Solvent was concentrated in vacuum and the crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 10-15% EtOAc in pet ether) which afforded the title compound as a white crystalline solid (75 mg, yield 87%). TLC system: 20% EtOAc in pet ether, Rf=0.4.
Step i) tert-Butyl 1-(2-(tert-butylamino)-1-hydroxy-2-oxoethyl)-3-fluorocyclobutylcarbamate (BB3-i)
To an ice cooled solution of compound BB3-h (1.3 g, 0.0059 mol) in dry DCM (25 mL) was added tert-butyl isocyanide (0.75 g, 0.0089 mol) and dry pyridine (2.6 mL). Trifluoroacetic acid (0.9 mL, 0.0118 mol) was added over a period of ten minutes maintaining the temperature at 0° C. The reaction mixture was slowly warmed to room temperature and stirred for 16 h. EtOAc (50 mL) was added and the organic phase was washed twice with 5% citric acid and brine. The organic phase was evaporated and the crude product was dissolved in THF (25 mL). 1M LiOH solution in MeOH—H2O (3:2v/v) (2.6 mL) was added and the mixture was stirred for 2 h at room temperature. The reaction mixture was quenched with 5% citric acid and the mixture was extracted with ethyl acetate (2×25 mL). The organic layer was washed with brine and dried over anhydrous Na2SO4. Solvent was evaporated in vacuum and to afford the title compound which was pure enough to be used in the next step (1.6 g, yield 84%). TLC system: 20% EtOAc in pet ether, Rf=0.3.
Step j) 2-(1-(tert-Butoxycarbonylamino)-3-fluorocyclobutyl)-2-hydroxyacetic acid (BB3)
Compound BB3-i (1.6 g, 0.005 mol) was refluxed with 6N HCl (60 mL) for 16 h until the amide hydrolysis was complete. The solvent was evaporated under reduced pressure and co-evaporated several times with water. The product was dissolved in THF:H2O (7:3 v/v, 50 mL), cooled to 0° C. and Et3N (2.1 mL, 0.015 mol) was added followed by di-tert-butyl dicarbonate (2.18 g, 0.01 mol). The mixture was stirred at room temperature overnight (pH was monitored in a regular interval and kept  ̃11 throughout the reaction). The reaction mixture was neutralized with 1N HCl and the product was extracted with EtOAc (2×50 mL). The organic layer was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure followed by purification by column chromatography (silica gel 60-120 mesh, eluent 5% MeOH in CHCl3) which afforded the title P1 building block as a solid (0.65 g, yield 50%). TLC system: 30% MeOH in CHCl3, Rf=0.3.
1H NMR (400 MHz, d6-DMSO) δ 7.01 (br s, 1H), 5.16 (br m, 1H), 4.97 (br m, 1H), 2.49 (br m, 5H), 1.36 (s, 9H); ms ES+ m/z 262 (100%).
Building Block 4—a P1-Prime Side Building Block




Step a) tert-butyl 1-(hydroxymethyl)-3-methoxycyclobutylcarbamate (BB4-a)
500 mg (1.51 mmol) of tert-butyl 1-((tert-butyldimethylsilyloxy)methyl)-3-hydroxycyclobutylcarbamate (prepared by reduction of ethyl-1[[(tert-butyloxy)carbonyl]amino]-3-hydroxycyclobutane-1-carboxylate as described in J. Med. Chem., 1990 33(10) 2905-2915) and proton sponge (N,N,N′,N′ tetramethylnapthalene-1,8 diamine) (1.63 g, 6.04 mmol) were dissolved in DCM (18 mL), cooled down to 0° C., and 447 mg (3.02 mmol) of trimethyloxonium borontetrafluoride was added in one portion as a solid under vigorous stirring. The reaction mixture was stirred for 3 h and diluted with DCM (50 mL) and brine (20 mL), added under vigorous stirring. The organic phase was washed with sodium bicarbonate, brine, dried over sodium sulphate, evaporated and purified on short silica column (DCM as an eluent). The resulting product was dissolved in THF (5 mL), and a solution of tetrabutylammonium fluoride in THF (1M, 4.5 mL) was added, and the reaction was stirred at room temperature for 4.5 h. The reaction was monitored by TLC and once deemed to have reached completion, it was absorbed onto silica and purified on silica (EtOAc-hexane 1:1 to neat EtOAc) to give the title compound (251 mg, 72%). LC/MS 232 (M+1).
Step b) tert-Butyl 1-formyl-3-methoxycyclobutylcarbamate (BB4-b)
Alcohol BB4-a was dissolved in DCM (20 mL) and Dess-Martin reagent was added in one portion. The reaction was stirred for 2.5 hours. Once the reaction was deemed to have reached completion, it was diluted with 50 mL of DCM and 20 mL of 10% Na2S2O3 was added. The mixture was stirred, washed with sodium bicarbonate, brine, and the organic phase was dried over sodium sulphate. Purification on silica (EtOAc-hexane 1:1 to neat EtOAc) gave the title compound (500 mg, 59%).
Step c) tert-Butyl 1-(2-(cyclopropylamino)-1-hydroxy-2-oxoethyl)-3-methoxycyclobutylcarbamate (BB4)
Aldehyde BB4-b 498 mg (1.56 mmol) was dissolved in dry DCM (8 mL). Pyridine (0.52 mL) was added under stirring conditions, followed by adding cyclopropyl isonitrile. The reaction was placed in an ice-bath and TFA (0.25 mL) was added dropwise during 20 min. The reaction mixture was stirred overnight. The reaction was then deemed to have reached completion and was washed with 1 M HCl, sodium bicarbonate, brine, and the organic phase was dried over sodium sulphate and evaporated. The remaining residue was dissolved in dioxane and stirred with lithium hydroxide solution overnight and then neutralized with citric acid. The product was extracted with EtOAc from the resulting solution and purified on silica (EtOAc-hexane 1:3 to 1:1) which gave 263 mg of the title compound (54%) LC/MS 314 (M+1).
Building Block 5 (BB5)—a P2 Building Block




Step a) 2-tert-Butoxycarbonylamino-3-chloro-propionic acid methyl ester (BB5-a)
A solution of triphenylphosphine (65.8 g, 0.251 mol)) and hexachloroethane (59.4 g, 0.251 mol) in dichloromethane (850 mL) was added in one portion to a solution of N-Boc-serine methyl ester (50 g, 0.228 mol) in dichloromethane (170 mL) under argon atmosphere. The reaction mixture was stirred at room temperature for 2 h and then the reaction was quenched with a saturated solution of NaHCO3 (150 mL). The organic product was extracted with dichloromethane (2×300 mL) and the combined organic layers was washed with brine (300 mL) and dried over anhydrous sodium sulphate. The solution was concentrated under reduced pressure and then triturated with Et2O (500 mL). After filtration and evaporation of the solvent, the crude product was purified by chromatography on a silica column eluted with 6-8% EtOAc in pet ether) which gave the title compound (42 g, 78%).
Step b) (2-Chloro-1-hydroxymethyl-ethyl)-carbamic acid tert-butyl ester (BB5-b)
Lithium borohydride (4.3 g, 0.195 mol) was added in portions to a stirred solution of BB5-a (42 g, 0.177 mol) in EtOH-THF 9:1 at 0° C. under argon atmosphere. The reaction was stirred for 8 h at room temperature then quenched with a saturated solution of ammonium chloride (20 mL). The product was extracted with EtOAc (2×300 mL). The combined organic layers was washed with brine (300 mL) and dried over anhydrous sodium sulphate. The solvent was evaporated under reduced pressure and the afforded crude product was purified by chromatography on a silica column eluted with 15% EtOAc in pet ether, which gave the title compound (27.5 g, 74%).
Step c)[2-Chloro-1-(2-trimethylsilanyl-ethoxymethoxymethyl)-ethyl]-carbamic acid tert-butyl ester (BB5-c)
(2-Chloromethoxy-ethyl)-trimethyl-silane (26.18 g, 0.157 mol) was added drop-wise to a stirred solution of compound BB5-b (27.5 g, 0.131 mol) and N,N-diisopropylethylamine (27.4 mL, 0.157 mol) in dichloromethane (350 mL) at 0° C. under argon atmosphere. The reaction was allowed to attain room temperature and stirred for 18 h. The reaction mixture was concentrated under vacuum and then diluted with EtOAc (150 mL). The product was extracted with EtOAc (2×200 mL) and the organic layer was washed with brine (100 mL) and dried over anhydrous sodium sulphate. The solvent was evaporated under reduced pressure and the afforded crude product was purified by chromatography on silica a column eluted with 5% EtOAc in pet ether, which gave the title compound (25.5 g, 57%).
Step d)[2-(1-Hydroxy-cyclobutyl)-1-(2-trimethylsilanyl-ethoxymethoxymethyl)-ethyl]-carbamic acid tert-butyl ester (BB5-d)
n-BuLi (10 mL, 0.016 mol, 1.6 M solution in hexanes) was added drop-wise to a stirred solution of compound BB5-c (2 g, 5.88 mmol) in THF (170 mL) at −78° C. under argon atmosphere. The stirring was continued for 15 min, followed by drop-wise addition of LiNp (104 mL, 0.42 M solution in THF, 0.044 mol) over 5 min. The dark solution was stirred at −78° C. for 1 h and then cyclobutanone (0.88 mL, 11.77 mmol) was added drop-wise. The reaction mixture was stirred at −78° C. for 16 h then quenched with a saturated solution of NH4Cl (50 mL) and allowed to warm to room temperature. The reaction was diluted with ether (100 mL) and a saturated solution of NH4Cl (10 mL). The layers were separated and the aqueous layer was extracted with ether (2×100 mL). The combined organic layers was dried (Na2SO4) and the solvent was evaporated under reduced pressure. The crude product was purified by chromatography on a silica column eluted with heptane:ether 3:2, which gave the title compound (1.54 g, 70%,).
Step e) [2-(1-Fluoro-cyclobutyl)-1-(2-trimethylsilanyl-ethoxymethoxymethyl)-ethyl]-carbamic acid tert-butyl ester (BB5-e)
BB5-d (0.5 g, 1.33 mmol), 50% Deoxofluor in THF (excess) and pyridine (excess) were mixed in DCM (10 mL). The resulting mixture was stirred at rt over night. The reaction mixture was washed with 10% citric acid (aq) and sat. NaHCO3 (aq). The organic phase was dried (Na2SO4) and evaporated. The afforded crude product was purified by chromatography on a silica column using hexane:EtOAc (7:1 to 2:1) as eluent, which gave the title compound (192 mg, 38%).
Step f) [2-(1-Fluoro-cyclobutyl)-1-hydroxymethyl-ethyl]-carbamic acid tert-butyl ester (BB5-f)
A solution of BB5-e (192 mg, 0.51 mmol) in 0.1 M HCl in MeOH (20 mL) was stirred for 3 hours, then triethylamine (1 mL) was added and the solution was concentrated. The afforded crude product was purified by chromatography on a silica column using hexane:EtOAc (2:1) as eluent, which gave the title compound (69.3 mg, 55%) as a white solid.
Step g) 2-tert-Butoxycarbonylamino-3-(1-fluoro-cyclobutyl)-propionic acid (BB5)
BB5-f (69 mg, 0.279 mmol) and pyridine dichromate (1.15 g, 3.05 mmol) were dissolved in dry DMF (5 mL). After five hours H2O (15 mL) was added and the product was extracted with DCM (3×20 mL). The combined organic layers was dried (Na2SO4) and evaporated. The crude was purified by chromatography on a silica column using hexane:EtOAc (1:1) followed by EtOH (100%) as eluent. This afforded the title compound as a white solid (22.3 mg, 31%), 262.4 [M+H]+.
Building Block 6 (BB6)—a P1-Prime Side Building Block




Step a) Acetoxy-(1-tert-butoxycarbonylamino-cyclobutyl)-acetic acid BB6-a
(1-Tert-butoxycarbonylaminocyclobutyl)-hydroxyacetic acid (201.5 mg, 0.822 mmol) was stirred with acetic anhydride (95 mL) in pyridine (1.5 mL) for 24 h. The mixture was first concentrated under vacuum, and then diluted with 5 mL EtOAc. The organic solution was washed with 1N HCl (2 mL) followed by saturated aqueous NaCl (2 mL), dried (Na2SO4), and evaporated under vacuum which gave the title compound. LC-UV/MS API-ES− m/z 286 [M−H]−1H NMR (400 MHz, DMSO-d6) δ ppm 7.11 (s, 1H), 5.00 (s, 1H, CHOAc), 2.28 (m, 2H), 2.07 (s, 3H, Me), 2.04 (m, 2H), 1.81 (m, 1H), 1.67 (m, 1H), 1.35 (s, 9H, Boc).
Step b) Acetic acid (1-tert-butoxycarbonylamino-cyclobutyl)-(4-methyl-thiazol-2-ylcarbamoyl)-methyl ester (BB6-b)
A mixture of the α-acetoxy carboxylic acid BB6-a (0.23 mmol) and 1,1′-carbodiimidazole (87 mg, 0.54 mmol) in dry THF (5.6 mL) was stirred at rt. After 18 h, 4-methyl-2-aminothiazole hydrochloride (0.28 mmol), imidazole (20 mg, 0.29 mmol), and DMAP (0.5 mg) were added and stirring was continued at rt. After 26 h, very little amide was formed, so the mixture was heated at 65° C. for 4 h, and then concentrated under vacuum. The residue was partitioned between 10 mL EtOAc and 10 mL saturated aqueous NaCl. The aqueous phase was extracted further with EtOAc (2×10 mL). The organic phases were combined, dried (Na2SO4), and evaporated in vacuo to give 152 mg oil as crude material. Flash column chromatography (silica, 80/20 CH2Cl2—acetone) gave the title compound as white solids (62.8 mg, 71% yield).
TLC Rf (9/1 CH2Cl2—acetone) 0.70, LC-UV/MS 97% DAD, API-ES+ m/z 384 [M+H]+1H NMR (400 MHz, DMSO-d6) δ ppm 6.95 (s, 1H), 6.76 (s, 1H, thiazole), 5.16 (s, 1H, CHOAc), 2.26 (s, 3H, thiazole Me), 2.14 (s, 3H, Ac), 2.4-2.1 (m, 4H), 1.8-1.6 (m, 2H), 1.33 (m, 9H).
Step c) {1-[Hydroxy-(4-methyl-thiazol-2-ylcarbamoyl)-methyl]-cyclobutyl}-carbamic acid tert-butyl ester (BB6)
The acetyl group was hydrolyzed by stirring BB6-b (56 mg, 0.15 mmol) with aqueous LiOH (1N, 0.30 mL) in 1 mL methanol for 1 h at rt. The reaction mixture was concentrated, and then partitioned between 5 ml each EtOAc and saturated aqueous NaCl. The aqueous phase was extracted further with EtOAc (2×5 mL). The organic phases were combined, dried (Na2SO4), and evaporated in vacuo to give 39.6 mg solids as crude material. Flash column chromatography (silica, 95/5 CH2Cl2—acetone) gave the title compound as white solids (36.2 mg, 71% yield). LC-UV/MS 100% DAD, AP-ES+ m/z 342 [M+H]+
Building Block 7 (BB7)—a P1 Building Block




Step a) Tert-butyl 3-fluoro-1-(1-hydroxy-2-(methylamino)-2-oxoethyl)cyclobutylcarbamate (BB7)
Potassium carbonate (147.5 mg, 1.06 mmol) was added to a solution of the P1-building block BB3 (254 mg, 0.96 mmol) in DMF (5 mL) followed by addition of methyl iodide (72 μL, 1.15 mmol). The reaction mixture was stirred at room temperature for 2.5 h and then partitioned between DCM and aq. NaHCO3 (sat.). The phases were separated and the organic layer was washed with water, dried (Na2SO4) and concentrated. The crude residue of the formed methyl ester was dissolved in a solution of methylamine in ethanol (33% (w/w), 10 mL), heated at 60° C. for 2 days and then concentrated to give the α-hydroxy amide BB7 which was pure enough to be used in the next step without further purification. MS m/z 277.4 (M+H)+.
Building Block 8—a P1-Prime Side Building Block (BB8)




Step a) [1-(tert-Butylcarbamoyl-hydroxy-methyl)-3-methoxy-cyclobutyl]-carbamic acid tert-butyl ester (BB8-a)
Aldehyde BB4-b, 4.45 mmol, was reacted according to the procedure as described for the preparation of BB4-c, but using tert-butyl isocyanide (6.68 mmol) instead of cyclopropyl isonitrile, which gave the title compound (850 mg, 58%), (TLC: rf=0.61 (EtOAc:hexane 1:1).
Step d) (1-tert-Butoxycarbonylamino-3-methoxy-cyclobutyl)-hydroxy-acetic acid (BB8-b)
Compound DJ14 (850 mg, 2.57 mmol) was refluxed with 6N HCl (60 mL) for 16 h until the amide hydrolysis was complete. The solvent was evaporated under reduced pressure and co-evaporated with water. The product was dissolved in THF:H2O (7:3 v/v, 50 mL), cooled to 0° C. and Et3N (1.4 mL, 10.2 mmol) was added followed by di-tert-butyl dicarbonate (2.25 g, 10.2 mol). The mixture was stirred at room temperature overnight. The reaction mixture was washed with EtOAc followed by acidifying to pH3 with 1N HCl and extracted with EtOAc (2×50 mL). The organic layer was washed with brine and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure which afforded the title P1 building block as a solid (360 mg, yield 51%).
Step e) {1-[Hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-3-methoxy-cyclobutyl}-carbamic acid tert-butyl ester (BB8)
The hydroxy acid BB8-b was reacted with 1-methyl-1H-pyrazol-3-amine according to the procedure described in Example 1, step a, which gave the title compound (232 mg, yield 50%).
Building Block 9—a P1-Prime Side Building Block (BB9)




Tert-butyl (1R,3S)-3-fluoro-1-((S)-1-hydroxy-2-(1-D3-methyl-1H-pyrazol-3-ylamino)-2-oxoethyl)cyclobutylcarbamate (BB9)
D3-methyliodide (4.42 mmol, 0.281 mL) was added to a stirred solution of 3-nitropyrazole (4.42 mmol, 500 mg) and DBU (0.75 mL) in DMF. After 16 h, aq. NaHCO3 was added and the mixture was extracted with DCM. The organic phase was carefully concentrated and the afforded crude product was purified by column chromatography on silica gel eluted with DCM. The afforded residue was dissolved in MeOH, hydrogenated over Pd/C-10%, filtered through Celite and concentrated, which gave 1-(D3-methyl)-1H-pyrazol-3-ylamine. 1-(D3-methyl)-1H-pyrazol-3-ylamine was the reacted with BB3 according to the procedure described in Example 1, step a, which gave the title compound.
Example A1




Step a) {3-Fluoro-1-[hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutyl}-carbamic acid tert-butyl ester (A1-a)
1-Methyl-1H-pyrazol-3-amine (1 eq.) and DIEA (4 eq) was added to a solution of the P1-building block BB3 (1 eq) dissolved in DMF. The solution was cooled to 0° C. and after 10 minutes HATU (1 eq) was added. After approximately 2 hours at RT, LC-MS showed product and no starting material and the solvent was removed by rotary evaporation. The crude product was dissolved in 40 mL EtOAc and washed with 25 mL sat. NaHCO3 (aq). The organic phase was dried with Na2SO4, filtered and evaporated to dryness. The crude product was purified on a 25 g silica column on a Biotage Flashmaster, which gave the title product.
Step b) 2-(1-Amino-3-fluoro-cyclobutyl)-2-hydroxy-N-(1-methyl-1H-pyrazol-3-yl)-acetamide (A1-b)
Compound A1-a (363 mg, 1.06 mmol) was treated with a 7.5 mL of a mixture of TFA:DCM:TIS:water (20:80:1:1). After 2 h at room temperature, LC/MS analysis confirmed complete removal of the Boc group and the solution was concentrated in vacuo and azeotroped with dichloromethane (3×) to remove excess TFA. The crude product (339 mg, 0.9 mmol) was dissolved in DCM (10 mL) and added to a solution of 2-amino-3-(1-methyl-cyclopentyl)-propionamide (prepared as described in Ex. 1 of WO2006/064286) (1.2 eq), PyBOP (1.2 eq) and diisopropylethylamine (4 eq) in DCM (5 mL) that had been stirred at room temperature for 5 min. The mixture was stirred at room temperature until LC/MS analysis indicated complete amide formation ( ̃30 min) DCM was added to the reaction and the organic phase was washed with 0.1 M HCl (aq) (2×) and 10% NaHCO3 (aq) (2×). The organic phase was dried and concentrated in vacuo to afford the title compound which was used in subsequent step without further purification.
Step c) N-{3-Fluoro-1-[hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutyl}-3-(1-methyl-cyclopentyl)-2-propionylamino-propionamide (A1-c)
Compound A1-b (0.9 mmol) was treated with a 7.5 mL of a mixture of TFA:DCM:TIS:water (20:80:1:1). After 2 h at room temperature, LC/MS analysis confirmed complete removal of the BOC group and the solution was concentrated in vacuo and azeotroped with dichloromethane (3×) to remove excess TFA. The residue was dissolved in DCM and extracted with 0.1M HCl (aq) (3×). The pooled aqueous layers were basified to pH 10 with NaOH (aq) and the product extracted with DCM (3×). The pooled organic layers were dried (MgSO4) and concentrated in vacuo. The afforded crude compound (33 mg, 83 mmol) was dissolved in DCM (2 mL) and DIEA (3 eq.) and propionylchloride (91.3 mmol) was added. The reaction was stirred at room temperature for 1 h after which time LC/MS analysis indicated complete acylation. The reaction was quenched by addition of Methanol (0.5 mL) and the reaction was concentrated in vacuo. The residue was dissolved in DCM (5 mL) and the organic phase was washed with 0.1M HCl (aq) (2×) and 10% NaHOC3 (aq) (2×), eluted through a hydrophobic Phase separator and concentrated in vacuo, which gave the title compound.
Step d) N-[3-Fluoro-1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutyl]-3-(1-methyl-cyclopentyl)-2-propionylamino-propionamide (A1)
The residue afforded in step c was re-dissolved in DCM (1.5 mL) and Dess-Martin periodinane (91.3 μmol) was added in one portion at room temperature. The reaction was stirred at room temperature for 2 h after which time LC/MS analysis indicated complete oxidation. The reaction mixture was diluted with DCM and the solution washed with a 1:1 mixture of 10% Na2S2O3 (aq) and 10% NaHCO3 (aq). The organic layer was eluted through a hydrophobic Phase Separator and concentrated in vacuo. The residue was purified by preparative LC/MS to afford the target compound (Yield: 47%, LC/MS: tR=4.55 min, 450.2 [M+H]+)
Examples A2-A10
The compounds illustrated in the table below were prepared analogously to the procedure outlined in Example 1 using the appropriate R1aR1b amines, P1/P2 and P3 building blocks acid, followed by Dess Martin oxidation to the end product α-keto amide.







TABLE A1













 













Ex.
R4
R3
R2a
R2b
R1b
[M + H]+
















A2
methyl
1-methylcyclopentylmethyl
F
H
1-methylpyrazol-3-yl
436.3


A3
t.butyl
1-methylcyclopentylmethyl
F
H
1-methylpyrazol-3-yl
478.14


A41
ethyl
1-fluorocyclopentylmethyl
F
H
1-methylpyrazol-3-yl
453.96


A51
t.butyl
1-fluorocyclopentylmethyl
F
H
1-methylpyrazol-3-yl
481.98


A62,3
methyl
1-methylcyclopentylmethyl
H
H
1-methylpyrazol-3-yl
418.2


A7
t.butyl
1-fluorocyclopentylmethyl
F
F
1-methylpyrazol-3-yl
500.2


A84,5
methyl
1-methylcyclopentylmethyl
F
H
1-cyclopropylmethyl-
504







pyrazol-3-yl



A94,5
methyl
1-methylcyclopentylmethyl
F
H
1-(2,2,2-trifluoroethyl)-
476







pyrazol-3-yl



A102
methyl
1-fluorocyclobutylmethyl
F
F
1-methylpyrazol-3-yl
444.16





1Removal of the Boc group in step b was performed using 4M HCl in dioxane and/or MeOH.


2Removal of the Boc group in steps b and c was performed using 4M HCl in dioxane and/or MeOH.


3The P2 building block was coupled to the P1-P1′-building block using DMF as solvent and HATU as coupling reagent.


4The P1′ amine was coupled to the P1-building block using HATU as coupling agent.


5Removal of the Boc group in steps b and c was performed using MeOH:AcCl 9:1.






Example A11




Step a) {3,3-Difluoro-1-[hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutyl}-carbamic acid tert-butyl ester (A11-a)
1-Methyl-1H-pyrazol-3-amine (158 mg, 1.63 mmol) and DIEA (1.08 mL, 6.52 mmol) was added to a solution of (1-tert-Butoxycarbonylamino-3,3-difluoro-cyclobutyl)-hydroxy-acetic acid (1.63 mmol) dissolved in DMF (20 mL). The solution was cooled to 0° C. and after 10 minutes HATU (620 mg, 1.63 mmol) was added. After approximately 2 hours at RT, LC-MS showed product and no starting material and the solvent was removed by rotary evaporation. The crude product was dissolved in 40 mL EtOAc and washed with 25 mL sat. NaHCO3 (aq). The organic phase was dried with Na2SO4, filtered and evaporated to dryness. The crude product was purified on a 25 g silica column on a Biotage Flashmaster, which gave the title product as a white solid (92%). TLC rf: 0.07 in heptane:ethyl acetate 1:1.
Step b) [1-{3,3-Difluoro-1-[hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutylcarbamoyl}-2-(1-methyl-cyclopentyl)-ethyl]-carbamic acid tert-butyl ester (A11-b)
A11-a (0.629 mmol) was dissolved in MeOH (3 mL). A solution of HCl in dioxane (4M, 1.5 mL) was added. The solution was stirred at RT for 16 h, then concentrated and co-evaporated with toluene. The afforded residue (0.63 mmol) was dissolved in anhydrous DMF (1 mL) and then added at 0° C. to a cold solution of 2-amino-3-(1-methyl-cyclopentyl)-propionamide (prepared as described in Ex. 1 of WO2006/064286) (187.5 mg, 0.69 mmol) and HATU (263 mg, 0.69 mmol) in dry DMF (2 mL). DIEA (550 μL, 3.15 mmol) was added, and the reaction mixture was stirred at 0° C. for 30 minutes, then at RT for 2 h. The solution was concentrated to vacuum, the residue was dissolved in DCM (3 mL) and applied to a silica column (10 g). The compound was purified by flash chromatography (heptane: ethyl acetate 75:25-25:75) which gave the title compound (90%) as two chromatographic peaks. MS m/z 478.2 (M+H)+.
Step c) [1-[3,3-Difluoro-1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutylcarbamoyl]-2-(1-methyl-cyclopentyl)-ethyl]-carbamic acid tert-butyl ester (A11-c)
The α-hydroxy amide A11-b was oxidized according to the method described in Example A1 step d. Purification by prep LCMS (50-70% gradient, mobile phase: acetonitrile-water, 1% NH4OH) gave the title compound. MS m/z 476.2 (M+H)+. Purity assessed by analytical LCMS 99.4%.
Step d) 2-Acetylamino-N-[3,3-difluoro-1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutyl]-3-(1-methyl-cyclopentyl)-propionamide (A11)
Carbamate A11-c (196 mg, 0.383 mmol) was dissolved in a pre maid mixture of methanol and acetyl chloride 9:1 (10 mL). The mixture was stirred for 6 hrs whereafter the solvent was removed and the crude product dried over night under high vacuum. The crude product was dissolved in DMF (10 mL) and AcCl (27 μL, 0.383 mmol) was added followed by DIEA (253 μL, 1.53 mmol). After approximately 2 hours at RT, LC-MS showed product and no starting material and the solvent was removed by rotary evaporation. The crude product was dissolved in 40 mL of EtOAc and washed with 25 mL of sat. NaHCO3 (aq). The organic phase was dried with Na2SO4, filtered and evaporated on silica. The crude product was purified on a pre packed 10 g silica column using a gradient going from heptane: EtOAC 1:1 towards pure EtOAc, towards pure acetone, which gave the title compound (109 mg, 63%) yield. [M+H]+454.
The compounds illustrated in the table below were prepared according to the procedure outlined in Example A11, using the appropriate R1aR1bamine, P1-, P2-building blocks, and acid chloride.







TABLE A2













 













Ex.
R4
R3
R2a
R2b
R1b
[M + H]+
















A12
methyl
2-fluoro-2-methylbutyl
F
F
1-methylpyrazol-3-yl 
432


A13
methyl
1-methylcyclobutylmethyl
F
F
1-methylpyrazol-3-yl 
440


A14
methyl
cyclohexylmethyl
F
F
1-methylpyrazol-3-yl 
454


A15
methyl
homo-t-butyl
F
F
1-methylpyrazol-3-yl 
428


A162 
methyl
1-methylcyclopentylmethyl
OMe
H
cyclopropyl
408


A17
methyl
1-fluorocyclopentylmethyl
F
H
1-methylpyrazol-3-yl 
440


A18 
methyl
1-methylcyclobutylmethyl
F
H
1-methylpyrazol-3-yl 
422


A191
chloromethyl
1-methylcyclopentylmethyl
OMe
H
cyclopropyl
442


A202
methyl
1-methylcyclopentylmethyl
F
F
4-methylthiazol-2-yl
435


A212
methyl
1-fluorolcyclopentylmethyl
F
F
1-methylpyrazol-3-yl 
458


A22
methyl
1-methylcyclobutylmethyl
F
H
cyclopropyl
382


A23
methyl
1-fluorolcyclopentylmethyl
F
H
cyclopropyl
396


A24
methyl
1-methylcyclopentylmethyl
F
H
cyclopropyl
400


A252,3
isopropyl
1-methylcyclopentylmethyl
F
F
1-methylpyrazol-3-yl 
482


A264
ethyl
1-methylcyclopentylmethyl
F
F
1-methylpyrazol-3-yl
467.3


A274
chloroethyl
1-methylcyclopentylmethyl
F
F
1-methylpyrazol-3-yl
487.2





1The stereochemistry at the steric centre to which R2a and R2b is attached is not defined.


2The Boc group of the carbamate was removed using 2M HCl in dioxane.


3The P3 moiety was introduced using the appropriate acid anhydride.


4The P3 moiety was introduced in a HATU promoted coupling with the appropriate acid.






Example A28




Step a) Acetyl-cyclohexylglycine (A28-a)
L-Boc-cyclohexylglycine (0.500 g, 1.94 mmol) was dissolved in DCM (5 mL) and cooled to 0° C. TFA (5 mL) was added and the reaction was stirred for 30 min. The mixture was concentrated in vacuo and the residue was resuspended in a few mL of toluene and the mixture evaporated by rotary evaporation (process repeated thrice) followed by drying under vacuum. The crude TFA salt was dissolved in H2O (6 mL), cooled to 0° C. and NaOH (776 mg, 19.4 mmol) was added. Stirring was continued until a homogeneous solution was achieved. Acetic anhydride (3.88 mmol, 0.388 mL) was added and the reaction was stirred for 2 h. The mixture was diluted with 1 M NaOH (25 mL) and the resulting solution was washed with Et2O, then acidified with 3 M HCl to pH=1, and extracted with EtOAc (3×20 mL). The combined organic phases were dried (MgSO4), filtered and evaporated to give 120 mg of the title compound in high purity. LC-MS ES+200.2 [M+H]+.
Step b) 2-[1-(2-Acetylamino-2-cyclohexyl-acetylamino)-cyclobutyl]-2-hydroxy-N-(1-methyl-1H-pyrazol-3-yl)-acetamide (A28-b)
2-(1-Amino-cyclobutyl)-2-hydroxy-N-(1-methyl-1H-pyrazol-3-yl)-acetamide (prepared according to the method described in Example 1, step a (50 mg, 0.154 mmol) was treated with 2 mL 4 M HCl in dioxane to yield the dihydrochloric salt of the free amine after freeze-drying. This was reacted with compound A28-a according to the coupling procedure described in Example C9 step b and the product was purified by gradient column chromatography (1-10% MeOH in DCM) to give the title compound (30 mg, 48%) LC-MS ES+406.3 [M+H]+.
Step c) 2-[1-(2-Acetylamino-2-cyclohexyl-acetylamino)-cyclobutyl]-N-(1-methyl-1H-pyrazol-3-yl)-2-oxo-acetamide (A28)
Compound A28-b was oxidized according to Example 1 step d and the product was purified by preparative HPLC on a Phenomenex Gemini-NX column Eluents: A 0.01M NH3 in H2O; B 45% MeCN, 45% THF, 10% H2O, gradient 15-28% B. This gave 15 mg of the title compound (50% yield) after lyophilisation. Purity >99% (HPLC-DAD), LC-MS ES+403.1.
Example A29




2-Acetylamino-3-(1-fluoro-cyclopentyl)-N-[1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutyl]-propionamide (A29)
The title compound was prepared according to the method described in Example 1, using the appropriate building blocks. Removal of the Boc group in steps b and c was performed by treatment with 4M HCl in dioxane/MeOH. Yield: 16%, LC/MS: tR=4.0 min, 421.2 [M+H]+).
Example U1




Step a) 3-(1-Methyl-cyclopentyl)-2-[(pyrrolidine-1-carbonyl)-amino]-propionic acid lithium salt (U1-a)
(S)-2-(tert-Butoxycarbonylamino)-3-(1-methylcyclopentyl)propanoic acid, (prepared as described in Ex. 1 of WO2006/064286) (272 mg, 1 mmol) was dissolved in MeOH (1 mL). 4M
HCl in dioxane was added dropwise (3 mL) at room temperature. After approximately 3 hours, the solvent was removed by rotary evaporation and co-evaporated with MeOH (2×) to remove excess HCl. The afforded crude compound was used in the following step without further purification.
Step b) 2-(3,3-Diethyl-ureido)-3-(1-methyl-cyclopentyl)-propionic acid lithium salt (U1-b)
Compound U1-a (19 mg, 86 mmol) was dissolved in THF (1 mL) and triethylamine (3 eq) and diethylcarbamoyl chloride (1 eq) were added. The reaction was heated to 50° C. in a sealed tube for 16 h. LC/MS analysis showed 90% conversion. EtOAc was added to the reaction solution and the organic phase was washed with 01M HCl (aq) (3×). The organic layer was dried (MgSO4), filtered and the solvent removed in vacuo. The resulting crude methyl ester was dissolved in THF (mL), 1M LiOH in methanol (3 eq) was added, and the solution stirred at room temperature for 16 h. LC/MS analysis indicated complete ester hydrolysis and the solvent was removed in vacuo to afford the lithium salt which was used in subsequent step without further purification.
Step c) 2-(3,3-Diethyl-ureido)-N-{3-fluoro-1-[hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutyl}-3-(1-methyl-cyclopentyl)-propionamide (U1-c)
Compound 1a was treated with a 7.5 mL of a mixture of TFA:DCM:TIS:water (20:80:1:1). After 2 h at room temperature, LC/MS analysis confirmed complete removal of the BOC group and the solution was concentrated in vacuo and azeotroped with dichloromethane (3×) to remove excess TFA. The afforded crude TFA salt (64 μmol) was dissolved in DCM (2 mL) and added to a solution of U1-b (1.2 eq) PyBOP (1.2 eq) in DCM (2 mL) that had been pre-stirred at room temperature for 10 min. The mixture was stirred at room temperature for 16 h after which time the reaction was interrupted. DCM was added to the reaction and the organic phase was washed with 0.1 M HCl (aq) (2×) and 10% NaHCO3 (aq) (2×). The organic phase was dried and concentrated in vacuo and the residue purified by preparative LC/MS.
Step d) 2-(3,3-Diethyl-ureido)-N-[3-fluoro-1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutyl]-3-(1-methyl-cyclopentyl)-propionamide (U1)
The alcohol U1-c was dissolved in DCM (1.5 mL) and Dess-Martin periodinane (1.5 eq) was added in one portion at room temperature. The reaction was stirred at room temperature for 2 h after which time LC/MS analysis indicated complete oxidation. The reaction was diluted with DCM and the solution washed with a 1:1 mixture of 10% Na2S2O3 (aq) and 10% NaHCO3 (aq). The organic layer was eluted through a hydrophobic Phase Separator and concentrated in vacuo. The residue was purified by preparative LC/MS which gave the title compound. (Yield: 4.2 mg, LC/MS: tR=5.5 min, 493.16 [M+H]+).
Examples A30-A38
The compounds illustrated in the table below were prepared analogously to the procedure outlined in Example 1 using the appropriate R1aR1b, amine, P1- and P2-building blocks, and acid chlorides, followed by Dess Martin oxidation to the end product α-keto amide.







TABLE A3













 













Ex.
R4
R3
R2a
R2b
R1b
[M + H]+
















A302,3
isopropyl
1-fluorocyclopentylmethyl
F
H
1-methylpyrazol-3-yl
468.0


A312,3,6
tert.butyl
1-fluorocyclopentylmethyl
OMe
H
1-methylpyrazol-3-yl
494.4


A323,4
tert.butyl
1-fluorocyclopentylmethyl
H
H
1-methylpyrazol-3-yl
464.1


A332
methyl
cyclohexylethyl
H
H
1-methylpyrazol-3-yl
432.36


A342,3,7
2,2,2-tri-
1-fluorocyclopentylmethyl
F
F
1-methylpyrazol-3-yl
526.01



fluoro-








ethyl







A352,3,7
difluoro-
1-methylcyclopentyl-
F
F
1-methylpyrazol-3-yl
490.29



methyl
methyl






A362,3,7
difluoro- 
1-fluorocyclopentylmethyl
F
F
1-methylpyrazol-3-yl
494.29



methyl







A371,2,7
D9-t.butyl
1-fluorocyclopentylmethyl
F
F
1-methylpyrazol-3-yl
508.9


A385,7
sec. butyl
1-methylcyclopentyl-
H
H
1-methylpyrazol-3-yl
460




methyl





1Removal of the Boc group in step b was performed using 4M HCl in dioxane and/or MeOH.


2Removal of the Boc group in steps b and c was performed using 4M HCl in dioxane and/or MeOH.


3The P2 building block was coupled to the P1-P1′-building block using DMF as solvent and HATU as coupling reagent.


4The P1′ amine was coupled to the P1-building block using HATU as coupling agent.


5Removal of the Boc group in steps b and c was performed using MeOH:AcCl 9:1.


6The isomeric ratio of the OMe in R2a/R2b is 1:1.


7In step c, the P3 building block was introduced in a HATU-promoted coupling with the appropriate acid.






Example U2




Step a) 3-(1-Fluoro-cyclopentyl)-N-[3-fluoro-1-(1-methyl-1H-pyrazol-3-ylaminooxalyl)-cyclobutyl]-2-(3-isopropyl-ureido)-propionamide (U2)
The α-hydroxy amide precursor of carbamate C18 was treated with 4M HCl in dioxane for 0.5 h and then lyophilised. The thus afforded HCl-salt was treated with DIEA (2 eq.) and isopropyl isocyanate (1.1 eq.) in CH2Cl2. After 0.5 h, LC/MS analysis indicated complete turnover and the reaction mixture was diluted with CH2Cl2, and the organic layer was washed with 10% citric acid (2×) and 10% NaHCO3 (2×), dried through a hydrophobic frit and concentrated in vacuo. The afforded crude compound was oxidized according to the procedure described in Example 1 step d, which gave the title compound. The residue was purified by preparative LC/MS to afford the target compound, M/Z 483.4 [M+H]+.
Examples U3-U5
The compounds illustrated in the table below were prepared analogously to the procedure outlined in Example U2, using the appropriate α-hydroxy amide and ethyl isocyanate.







TABLE U1













 











Ex.
R3*
R2a
R2b
M/Z





U3
F
F
H
467.3 [M − H]−


U4
F
H
H
449.4 [M − H]−


U5
methyl
F
H
465.3 [M + H]+









Example A39







Step a) (9H-fluoren-9-yl)methyl 1-(1-hydroxy-2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoethyl)cyclobutylcarbamate (A39-a)
{1-[Hydroxy-(1-methyl-1H-pyrazol-3-ylcarbamoyl)-methyl]-cyclobutyl}-carbamic acid tert-butyl ester (209 mg, 0.64 mmol) was treated with HCl (4M in dioxane, 5 mL) overnight and then concentrated. The resulting hydrochloride salt was added to a solution of Fmoc-Cl (232 mg, 0.89 mmol) in dioxane:water 2:3, whereafter NaHCO3 (181 mg, 2.24 mmol) was added. The reaction mixture was stirred at room temperature and after for 40 min the solution was diluted with DCM and extracted. The organic phase was washed with brine, dried (NaSO4) and concentrated. Purification by flash column chromatography (EtOAc/iso-Hexane, 4:6-6:4) gave the title compound as a white solid (257 mg, 90%). MS m/z 446.9 (M+H)+.
Step b) (9H-fluoren-9-yl)methyl 1-(2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoacetyl)cyclobutylcarbamate (A39-b)
Dess-Martin periodinane (668 mg, 1.57 mmol) was added to a solution of alcohol A39-a (503 mg, 1.13 mmol) in anh. DCE (7 ml). The reaction mixture was stirred at room temperature for 30 min and then quenched with Na2S2O3 (10% aq) and NaHCO3 (aq, sat.). The phases were separated and the organic layer was dried (Na2SO4) and concentrated yielding the title compound which was used in the next step without further purification. MS m/z 444.9 (M+H)+.
Step c) (Z)-4-((2-(1-(1-(((9H-fluoren-9-yl)methoxy)carbonylamino)cyclobutyl)-2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoethylidene)hydrazinecarboxamido)methyl)cyclohexane-carboxylic acid (A39-c)
A trifluoroacetic salt of Webb's acid linker (593 mg, 1.78 mmol) was added to a solution of the ketone A39-b (1.12 mmol, crude) in methanol (25 mL). The reaction mixture was stirred at room temperature overnight and then concentrated. Purification by flash column chromatography (MeOH/DCM, 0:1-1:9) gave the title compound (0.61 g, 85%). MS m/z 642.9 (M+H)+.
Step d) Resin bound (Z)-4-((2-(1-(1-(((9H-fluoren-9-yl)methoxy)carbonylamino)cyclobutyl)-2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoethylidene)hydrazinecarboxamido)methyl)cyclohexane-carboxylic acid (A39-d)
Amine resin (TG resin NovaSyn, 01-64-0043, 0.25 mmol/g, 2 g, 0.5 mmol) was added to a solution of acid A39-c (546 mg, 0.85 mmol), HBTU (760 mg, 2 mmol), HOBt (306 mg, 2 mmol) and NMM (550 μL, 4 mmol) in DMF (13 mL). The slurry was agitated at room temperature overnight. The resin was filtered and then washed several times alternating with DMF, DCM and MeOH.
Step e) Resin bound 4-(((Z)-2-(1-(1-(3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(1-methylcyclopentyl)-2-oxobutyl)cyclobutyl)-2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoethylidene)hydrazinecarboxamido)methyl)cyclohexanecarboxylic acid (A39-e)
Resin A39-d (0.5 mmol) was treated with a solution of 20% piperidine in DMF (30 mL) for 1 h, then filtered and washed with DMF (3×20 mL), and several times alternating with DCM and MeOH. The resulting amine resin suspended in DMF was added to Fmoc-protected 2-amino-3-(1-methyl-cyclopentyl)-propionic acid (294 mg, 0.75 mmol), HBTU (380 mg, 1 mmol), HOBt (153 mg, 1 mmol) and NMM (350 μL, 2 mmol). The slurry was agitated at room temperature over night. The resin was filtered, washed as described above, and dried under vacuum.
Step f) Resin bound (S,Z)-4-((2-(2-(1-methyl-1H-pyrazol-3-ylamino)-1-(1-(4-(1-methylcyclopentyl)-2-oxo-3-propionamidobutyl)cyclobutyl)-2-oxoethylidene)hydrazinecarboxamido)methyl)cyclohexanecarboxylic acid (A39-f)
Resin bound compound A39-e (0.05 mmol) was treated with a solution of 20% piperidine in DMF (6 mL) for 1 h, filtered and washed with DMF (3×20 mL), and several times alternating with DCM and MeOH. To the resulting amine resin suspended in DMF was added, ethyl carboxylic acid (15 μL, 0.2 mmol), HBTU (76 mg, 0.2 mmol), HOBt (30 mg, 0.2 mmol) and NMM (55 μL, 0.4 mmol). The slurry was agitated at room temperature over night. The resin was filtered, washed as described above, and dried under vacuum.
Step g) (S)—N-(4-(1-(2-(1-methyl-1H-pyrazol-3-ylamino)-2-oxoacetyl)cyclobutyl)-1-(1-methylcyclopentyl)-3-oxobutan-2-yl)propionamide (A39)
Resin bound amide A39-e (0.05 mmol) was treated with a solution of TFA:H2O 95:5 (4 mL) for 4 h. The resin was filtered and washed with DCM (3×5 mL) and MeOH (3×5 mL). The filtrate and the washes were pooled and concentrated and the afforded crude product was purified by RP-LC-MS (0.1% NH4OH in acetonitrile-0.1% aq. NH4OH, 40-65% over 9 min). Overall yield 4%. MS m/z 431.9 (M+H)+.
BIOLOGICAL EXAMPLES
Determination of Cathepsin K Proteolytic Catalytic Activity
Convenient assays for cathepsin K are carried out using human recombinant enzyme, such as that described in PDB.

    ID BC016058 standard; mRNA; HUM; 1699 BP.
    DE Homo sapiens cathepsin K (pycnodysostosis), mRNA (cDNA clone MGC:23107
    RX MEDLINE; RX PUBMED; 12477932.
    DR RZPD; IRALp962G1234.
    DR SWISS-PROT; P43235;


The recombinant cathepsin K can be expressed in a variety of commercially available expression systems including E coli, Pichia and Baculovirus systems. The purified enzyme is activated by removal of the prosequence by conventional methods.
Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically H-D-Ala-Leu-Lys-AMC, and were determined in either 100 mM Mes/Tris, pH 7.0 containing 1 mM EDTA and 10 mM 2-mercaptoethanol or 100 mMNa phosphate, imM EDTA, 0.1% PEG4000 pH 6.5 or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and 20 mM cysteine, in each case optionally with 1M DTT as stabiliser. The enzyme concentration used was 5 nM. The stock substrate solution was prepared at 10 mM in DMSO. Screens were carried out at a fixed substrate concentration of 60 μM and detailed kinetic studies with doubling dilutions of substrate from 250 μM. The total DMSO concentration in the assay was kept below 3%. All assays were conducted at ambient temperature. Product fluorescence (excitation at 390 nm, emission at 460 nm) was monitored with a Labsystems Fluoroskan Ascent fluorescent plate reader. Product progress curves were generated over 15 minutes following generation of AMC product.
Cathepsin S Ki Determination
The assay uses baculovirus-expressed human cathepsin S and the boc-Val-Leu-Lys-AMC fluorescent substrate available from Bachem in a 384 well plate format, in which 7 test compounds can be tested in parallel with a positive control comprising a known cathepsin S inhibitor comparator.
Substrate Dilutions
280 μL/well of 12.5% DMSO are added to rows B-H of two columns of a 96 deep well polypropylene plate. 70 μL/well of substrate is added to row A. 2×250 μL/well of assay buffer (100 mM Na phosphate, 100 mM NaCl, pH 6.5) is added to row A, mixed, and double diluted down the plate to row H.
Inhibitor Dilutions
100 μL/well of assay buffer is added to columns 2-5 and 7-12 of 4 rows of a 96 well V bottom polypropylene plate. 200 μL/well of assay buffer is added to columns 1 and 6.
The first test compound prepared in DMSO is added to column 1 of the top row, typically at a volume to provide between 10 and 30 times the initially determined rough Ki. The rough Ki is calculated from a preliminary run in which 10 μL/well of 1 mM boc-VLK-AMC (1/10 dilution of 10 mM stock in DMSO diluted into assay buffer) is dispensed to rows B to H and 20 μl/well to row A of a 96 well MicrofluorTM plate. 2 μl of each 10 mM test compound is added to a separate well on row A, columns 1-10. Add 90 μl assay buffer containing 1 mM DTT and 2 nM cathepsin S to each well of rows B-H and 180 μl to row A. Mix row A using a multichannel pipette and double dilute to row G. Mix row H and read in the fluorescent spectrophotometer. The readings are Prism data fitted to the competitive inhibition equation, setting S=100 μM and KM=100 μM to obtain an estimate of the Ki, up to a maximum of 100 μM.
The second test compound is added to column 6 of the top row, the third to column 1 of the second row etc. Add 1 μL of comparator to column 6 of the bottom row. Mix column 1 and double dilute to column 5. Mix column 6 and double dilute to column 10. Using an 8-channel multistepping pipette set to 5×10 μL, distribute 10 μL/well of substrate to the 384 well assay plate. Distribute the first column of the substrate dilution plate to all columns of the assay plate starting at row A. The tip spacing of the multichannel pipette will correctly skip alternate rows. Distribute the second column to all columns starting at row B. Using a 12-channel multistepping pipette set to 4×10 μL, distribute 10 μL/well of inhibitor to the 384 well assay plate. Distribute the first row of the inhibitor dilution plate to alternate rows of the assay plate starting at A1. The tip spacing of the multichannel pipette will correctly skip alternate columns. Similarly, distribute the second, third and fourth rows to alternate rows and columns starting at A2, B1 and B2 respectively.
Mix 20 mL assay buffer and 20 μL 1M DTT. Add sufficient cathepsin S to give 2 nM final concentration.
Using the a distributor such as a Multidrop 384, add 30 μL/well to all wells of the assay plate and read in fluorescent spectrophotometer such as an Ascent.
Fluorescent readings, (excitation and emission wavelengths 390 nm and 460 nm respectively, set using bandpass filters) reflecting the extent of enzyme cleavage of the fluorescent substrate, notwithstanding the inhibitor, are linear rate fitted for each well.
Fitted rates for all wells for each inhibitor are fitted to the competitive inhibition equation using SigmaPlot 2000 to determine V, Km and Ki values.
Cathepsin L Ki
The procedure above with the following amendments is used for the determination of Ki for cathepsin L.
The enzyme is commercially available human cathepsin L (for example Calbiochem). The substrate is H-D-Val-Leu-Lys-AMC available from Bahcem. The assay buffer is 100 mM sodium acetate 1 mM EDTA, pH5.5) The DMSO stock (10 mM in 100% DMSO) is diluted to 10% in assay buffer. Enzyme is prepared at 5 nM concentration in assay buffer plus 1 mM dithiothreitol just before use. 2 μL of 10 mM inhibitor made up in 100% DMSO is dispensed into row A. 10 μL of 50 μM substrate (=1/200 dilution of 10 mM stock in DMSO, diluted in assay buffer).
Inhibition Studies
Potential inhibitors are screened using the above assay with variable concentrations of test compound. Reactions were initiated by addition of enzyme to buffered solutions of substrate and inhibitor. Ki values were calculated according to equation 1.




  
    
      
        
          v
          0
        
        =
        
          VS
          
            
              
                K
                M
              
              ⁡
              
                (
                
                  1
                  +
                  
                    I
                    
                      K
                      i
                    
                  
                
                )
              
            
            +
            S
          
        
      
    
    
      
        (
        1
        )
      
    
  




where v0 is the velocity of the reaction, V is the maximal velocity, S is the concentration of substrate with Michaelis constant of KM, and I is the concentration of inhibitor.

The inhibition of cathepsin S, cathepsin K and cathepsin L exhibited by a selection of the compounds of the invention represented as Ki values in nM is presented in Table 1.












TABLE 1







Example
Ki Cat. S
Ki Cat. K
Ki Cat. L





















A1
0.8
850
3900



A6
0.81
580
5200



A13
3.8
340
870



A15
20
1200
6500



A16
10
12000
33000



A19
5.6
7400
25000



A25
1.0
1100
2300



A30
1.0
720
3300



A31
1.8
340
1200



A36
3
1100
380



A37
3
640
1800



U1
1.2
510
860



U2
1.3
1300
2200



U4
0.63
620
2100










The compounds of formula I are thus potent inhibitors of cathepsin S and yet selective over the closely related cathepsin K and L.
Permeability
This experiment measures transport of inhibitors through the cells of the human gastroenteric canal. The assay uses the well known Caco-2 cells with a passage number between 40 and 60.
Apical to Basolateral Transport
Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1.5 mL and 0.4 mL transport buffer (TB), respectively, and the standard concentration of the tested substances is 10 μM. Furthermore all test solutions and buffers will contain 1% DMSO. Prior to the experiment the transport plates are pre-coated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material. After 21 to 28 days in culture on filter supports, the cells are ready for permeability experiments.
Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denoted Wash, row 2 “30 minutes” and row 3 “60 minutes”. Transport plate no 2 comprises 3 rows of 4 wells, one denoted row 4 “90 minutes”, row 5 “120 minutes and the remaining row unassigned.
The culture medium from the apical wells is removed and the inserts are transferred to a wash row (No. 1) in a transport plate (plate no. 1) out of 2 plates without inserts, which have already been prepared with 1.5 mL transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A→B screening the TB in basolateral well also contains 1% Bovine Serum Albumin
0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts and the cell monolayers equilibrated in the transport buffer system for 30 minutes at 37° C. in a polymix shaker. After being equilibrated to the buffer system the Transepithelial electrical resistance value (TEER) is measured in each well by an EVOM chop stick instrument. The TEER values are usually between 400 to 1000Ω per well (depends on passage number used).
The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to the 30 minutes row (No. 2) and fresh 425 μL TB (pH 6.5), including the test substance is added to the apical (donor) well. The plates are incubated in a polymix shaker at 37° C. with a low shaking velocity of approximately 150 to 300 rpm.
After 30 minutes incubation in row 2, the inserts are moved to new pre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60 minutes), 4 (90 minutes) and 5 (120 minutes).
25 μL samples are taken from the apical solution after  ̃2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.
300 μL will be taken from the basolateral (receiver) wells at each scheduled time point and the post value of TEER is measured at the end the experiment. To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at −20° C. until analysis by HPLC or LC-MS.
Basolateral to Apical Transport
Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1.55 mL and 0.4 mL TB, respectively, and the standard concentration of the tested substances is 10 μM. Furthermore all test solutions and buffers will contain 1% DMSO. Prior to the experiment the transport plates are precoated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material.
After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments. The culture medium from the apical wells are removed and the inserts are transferred to a wash row (No. 1) in a new plate without inserts (Transport plate).
The transport plate comprises 3 rows of 4 wells. Row 1 is denoted “wash” and row 3 is the “experimental row”. The transport plate has previously been prepared with 1.5 mL TB (pH 7.4) in wash row No. 1 and with 1.55 mL TB (pH 7.4), including the test substance, in experimental row No. 3 (donor side).
0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts in row No. 1 and the cell monolayers are equilibrated in the transport buffer system for 30 minutes, 37° C. in a polymix shaker. After being equilibrated to the buffer system the TEER value is measured in each well by an EVOM chop stick instrument.
The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to row 3 and 400 μL fresh TB, pH 6.5 is added to the inserts. After 30 minutes 250 μL is withdrawn from the apical (receiver) well and replaced by fresh transport buffer. Thereafter 250 μL samples will be withdrawn and replaced by fresh transport buffer every 30 minutes until the end of the experiment at 120 minutes, and finally a post value of TEER is measured at the end of the experiment. A 25 μL samples will be taken from the basolateral (donor) compartment after  ̃2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.
To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at −20° C. until analysis by HPLC or LC-MS.
Calculation
Determination of the cumulative fraction absorbed, FAcum, versus time. FAcum is calculated from:




  
    FA
    cum
  
  =
  
    ∑
    
      
        C
        RI
      
      
        C
        DI
      
    
  




Where CRi is the receiver concentration at the end of the interval i and CDi is the donor concentration at the beginning of interval i. A linear relationship should be obtained.
The determination of permeability coefficients (Papp, cm/s) are calculated from:




  
    P
    app
  
  =
  
    
      (
      
        k
        ·
        
          V
          R
        
      
      )
    
    
      (
      
        A
        ·
        60
      
      )
    
  




where k is the transport rate (min−1) defined as the slope obtained by linear regression of cumulative fraction absorbed (FAcum) as a function of time (min), VR is the volume in the receiver chamber (mL), and A is the area of the filter (cm2).

Reference Compounds













Category of absorption in man
Markers
absorption in man (%)















PASSIVE TRANSPORT









Low (0-20%)
Mannitol
16



Methotrexate
20


Moderate (21-75%)
Acyclovir
30


High (76-100%)
Propranolol
90



Caffeine
100







ACTIVE TRANSPORT









Amino acid transporter
L-Phenylalanine
100







ACTIVE EFFLUX









PGP-MDR1
Digoxin
30









Greater permeability through the gastrointestinal tissue is advantageous in that it allows for the use of a smaller dose to achieve similar levels of exposure to a less permeable compound administered in a higher dose. A low dose is advantageous in that it minimizes the cost of goods for a daily dose, which is a crucial parameter in a drug which is taken for protracted time periods.
All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.
Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.
The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims.

## Claims

1. A compound of the formula I:



wherein
R1a is H; and
R1b C1 -C6alkyl, optionally substituted with 1-3 substituents independently selected from: halo, hydroxy, cyano, azido, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy, C1-C4alkoxycarbonyl, C1-C4alkylcarbonyl, amine, C1-C4alkylamine, C1-C4 dialkylamine, C1-C4alkylsulfonyl, C1-C4alkylsulfonylamino, aminocarbonyl, aminosulphonyl, Carbocyclyl and Het; or

R1b is Carbocyclyl or Het; or
R1a and R1b together with the N atom to which they are attached define a saturated cyclic amine with 3-6 ring atoms;


wherein the Carbocyclyl, Het or cyclic amine is optionally substituted with 1-3 substituents independently selected from halo, hydroxy, cyano, azido, C1-C4alkyl, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy, C1-C4alkoxycarbonyl, C1-C4alkylcarbonyl, amine, C1-C4alkylamine, C1-C4dialkylamine, C1-C4alkylsulfonyl, C1-C4alkylsulfonylamino, aminocarbonyl, aminosulphonyl, RxOOC—C0-C2alkylene (where Rx is H, C1-C4alkyl or C1-C4haloalkyl), phenyl, benzyl or C3-C6cycloalkylC0-C2alkylene;

wherein the phenyl, benzyl or cycloalkyl moiety is optionally substituted with 1-3 substituents independently selected from halo, C1-C4alkyl, C1-C4haloalkyl or C1-C4alkoxy);
R2a and R2b are independently selected from H, halo, C1-C4alkyl, C1-C4haloalkyl, C1-C4alkoxy, or R2a and R2b together with the carbon atom to which they are attached form a C3-C6cycloalkyl;
R3 is a C5-C10alkyl, optionally substituted with 1-3 substituents independently selected from halo, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy; or

R3 is a C2-C4alkyl chain with at least 2 chloro or 3 fluoro substituents; or
R3 is C3-C7cycloalkylmethyl, optionally substituted with 1-3 substituents independently selected from C1-C4alkyl, halo, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy;
R4 is C1-C6alkyl, C1-C6dihaloalkyl, C1-C6alkylamino or C1-C6dialkylamino;
Het is a stable, monocyclic or bicyclic, saturated, partially saturated or aromatic ring system containing 1-4 heteroatoms independently selected from O, S and N, each ring having 5 or 6 ring atoms;
Carbocyclyl is C3-C6cycloalkyl, C5-C6cycloalkenyl or phenyl;


or a pharmaceutically acceptable salt, hydrate or N-oxide thereof.

2. A compound according to claim 1, wherein R1b is methyl, cyclopropyl, 1-phenylethyl, or a 5 or 6 membered heterocyclic ring containing 1-3 nitrogen atoms and 0 or 1 sulphur atoms, the cyclopropyl, phenyl or heterocyclic ring being optionally substituted with up to three substituents independently selected from C1-C4alkyl, halo, C1-C4haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy, C1-C4alkoxycarbonyl, C1-C 4alkylcarbonyl, amine, C1-C4alkylamine, C1-C4-dialkylamine, C1-C4alkylsulfonyl, C1-C4alkylsulfonylamino, aminocarbonyl, aminosulphonyl, RxOOC—C0-C2alkylene (where Rx is H or C1-C4alkyl) or C3-C6cycloalkylC0C2alkylene or benzyl (the cycloalkyl, or the phenyl ring of the benzyl being optionally substituted with 1-3 substituents selected from C1-C4alkyl, halo, C1-C4haloalkyl, C1 -C4alkoxy, C1 -C4haloalkoxy).

3. A compound according to claim 2, wherein the heterocyclic ring is pyrrolyl, pyrazolyl, imidazolyl, triazolyl, thiazolyl, or thiadiazolyl, any of which is optionally substituted with C1-C4alkyl, halo, C1-C4haloalkyl, C1-C4alkoxy, C3-C6cycloalkyl or C3-C6cycloalkylmethyl.

4. A compound according to claim 3, wherein the heterocyclic ring is pyrrazol-1-yl, optionally substituted with C1-C4alkyl, halo, C1-C4haloalkyl, C1-C4alkoxy or cyclopropyl.

5. A compound according to claim 4, wherein the pyrazolyl is N-substituted with C1-C4alkyl, such as ethyl or methyl.

6. A compound according to claim 1, wherein R2a and R2b are both F.

7. A compound according to claim 1, wherein one of R2a and R2b is H, and the other is Cl, F, F3C or MeO.

8. A compound according to claim 1, wherein R2a and R2b are both H.

9. A compound according to claim 1, wherein R3 is t-butylmethyl, cyclobutylmethyl, 1-methylcyclobutylmethyl or 1-methylcyclopentylmethyl, any of which is optionally substituted with one or two F or MeO.

10. A compound according to claim 1, wherein
R3 is 1- methylcyclopentylmethyl or 1-fluorocyclopentylmethyl.

11. A compound according to claim 1,wherein R4 is C1-C6alkyl, especially methyl or ethyl.

12. A compound according to claim 1,wherein R4 is C1-C6haloalkyl, especially chloroalkyl or fluoroalkyl.

13. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable vehicle therefor.

14. The compound according to claim 1, wherein
R1a is H;
R1b is methylpyrazolyl;
R 2a and R2b are each H;
R3 is 1-fluorocyclopentylmethyl; and
R 4 is methyl.

15. A pharmaceutical composition comprising a compound according to claim 14 and a pharmaceutically acceptable vehicle therefor.