BRPI0712572A2 - compounds and compositions as channel activating protease inhibitors - Google Patents

Abstract

COMPOUNDS AND COMPOSITIONS AS CHANNEL ACTIVATING PROTEASE INHIBITORS. The present invention relates to compounds and pharmaceutical compositions thereof, which are useful for modulating channel activating protease, and methods for using such compounds to treat, ameliorate or prevent an associated condition as a channel activating protease, including but not limited to prostasin , PRSS22, TNPRSS11 (e.g., TMPRSS11B, TMPRSS11E), TMPRSS2, TMPRSS3, TMPRS4 (MTPS-2), matriptase (MTSP-1), CAP2, CAP3, trypsin, cathepsin A, or neutrophil elastase.

Description

Report of the Invention Patent for "COMPOUNDS AND COMPOSITIONS AS CHANNEL ACTIVATE PROTEASE INHIBITORS".

Cross Reference to Related Requests

This claim claims the benefit of provisional applications from

United States Serial Number 60 / 808,014, filed May 23, 2006; and serial number 60 / 860,622, filed November 22, 2006. Each of these applications is incorporated herein by reference in its entirety.

Technical Field

The present invention generally relates to channel activating protease (CAP) inhibitors.

Background Technique

Prostasin is a trypsin-like serine protease that is present in a variety of mammalian tissues. It is a membrane-anchored protease that is expressed on the extracellular membrane of cells, but can also be secreted into body fluids such as semen, urine and airway surface fluid. Prostasin (PRSS8), together with proteases such as matriptase, CAP2, CAP3, trypsin, PRSS22, TMPRSS11, cathepsin A, and neutrophil elastase, can stimulate activity to amiloride-sensitive epithelial sodium channel (ENaC) activity. Inhibition of these enzymes may induce changes in epithelial ion transport and therefore fluid homeostasis through epithelial mebranes. For example, inhibition of CAP in the kidney is supposed to promote diuresis, although inhibition of CAP in the airway promotes mucus and sputum clearance in the lung. CAP inhibition in the kidney can therefore be used therapeutically to treat hypertension. Inhibition of CAP in the airways prevents the stagnation of respiratory secretions that otherwise tends to make sufferers vulnerable to secondary bacterial infections.

Description of the Invention

The present invention provides compounds, pharmaceutical compositions and methods of using such compounds to modulate channel activating protease (CAP). For example, the compounds and compositions of the invention may be used to modulate prostasin, PRSS22, TMPRSS11 (e.g. TMPRSS11B, TMPRSS11E), TMPRSS2, TMPRSS3, TM-PRSS4 (MTSP-2), Matrixase (MTSP-1), CAP2, CAP3, trypsin, cathepsin A, and neutrophil elastase.

In one aspect, the present invention provides compounds of formula (1):

<formula> formula see original document page 3 </formula>

and pharmaceutically acceptable salts, hydrates, solvates and stereoisomers thereof,

on what

J is a 5- to 12-membered monocyclic or fused carbocyclic ring, aryl,

heteroaryl or heterocyclic ring containing Ν, O and / or S;

R1 is - (CR2) I-NR2, - (CR2), - NRC (= NR) -NR2, - (CR2) rC (= NR) -NR2 or a 5-7 membered non-aromatic nitrogen-containing heterocyclic ring;

W-R2 is a substituent at any position on ring A;

W is either -O (CR2) k-, -S (CR2) k-, -S (O) (CR2) k-, -SO2 (CR2) k- or -OC (O) (CR2) k-;

R2 is C1-6 alkyl1 C2-6alkenyl1 C2-6alkynyl, R6, -CR9 = CR9-R61 or

wherein ring E is an optionally substituted 5- to 7-membered monocyclic or fused carbocyclic or heterocyclic ring; or W-R2 together form a 5-7 membered C1-6alkyl or -OC (O) NR7R8;

R3 is NR7R8 or R6;

R4 and R5 are independently H, C1-6 alkyl, OH, or C1-6 alkoxy; R7 and R8 are independently H1 C1-6 alkyl, C2- and alkenyl, C2-6 alkynyl or - (CR2) I-R6; or R 7 and R 8 together with N may form an optionally substituted 5 to 7 membered monocyclic or fused heterocyclic ring;

R9 is H or C1-6 alkyl;

R10 is halo, C1-6alkyl, C1-6alkoxy, OR11 or - (CR2) 1-R11;

R 6, R 11 and X are independently an optionally substituted 5 to 7 membered carbocyclic ring, heterocyclic ring, aryl or heteroaryl; or R11 is H or C1-6 alkyl;

each R is H, or C 1-6 alkyl, C 2-6 alkenyl, or C 2-6 alkynyl, wherein a carbon may optionally be substituted or exchanged with NR, O or S;

i is 0 to 1; k and I are independently 0 to 6; m and n are independently 1 to 6; and ρ is 0 to 3.

In the above formula (1), R 1 is - (CH 2) 1 -NH 2, - (CH 2), -NHC (= NH) -NH 2 or - (CH 2) 1 -C (= NH) -NH 2 NH 2, where each I is 0 to 1; or R1 is piperidinyl. In particular examples, R1 is - (CH2) rNH2, - (CH2) 1-NHC (= NH) -NH2 or - (CH2) 1- C (= NH) -NH2NH2.

In the above formula (1), W is -O (CR2) k-, -S (CR2) k-, -S (O) (CR2) k-, -SO2 (CR2) k- or -OC (O) ( CR2) k-; and k is 1. In particular examples, W is -O (CR2) k-.

In some embodiments, R 2 is an optionally substituted phenyl, C 5-7 cycloalkyl, thienyl, furanyl, piperidinyl, methylene cyclohexyl,

<formula> formula see original document page 4 </formula>

In particular examples, R 2 is phenyl or C 5-7 cycloalkyl.

In other embodiments, R 3 is an optionally substituted phenyl, pyridyl, thiazolyl, piperidinyl, or NR 7 R 8; wherein R 7 and R 8 are both H, or R 7 and R 8 together with N form an optionally substituted piperidinyl. In some examples, R, R4, R5, R7 and R8 are each H.

In other embodiments, however, R 6 is an optionally substituted phenyl, C 3-7 cycloalkyl, pyridyl, thiazolyl, piperidinyl, cyclohexanol, imidazolyl, thienyl, furanyl,

<formula> formula see original document page 5 </formula>

In particular examples, R 6 is an optionally substituted phenyl, C 3-7 cycloalkyl, pyridyl, thiazolyl or piperidinyl.

However in other embodiments, X is cyclohexyl, phenyl or piperidinyl, each of which may be optionally substituted with C1-6 alkyl, C1.6 alkoxy, halo, or a combination thereof. In some examples, X is cyclohexyl or phenyl.

In formula (1) above, -J- (R10) p at the same time may be

<formula> formula see original document page 5 </formula>

Z is O or S;

Z1, Z2, Z3 or Z4 are independently N, CH1 or C when bonded to R10;

Z5, Z6 or Z7 are independently N, O, S, CH1 or C when bonded to R10;

R10 is C1-6 alkyl or - (CR2) i-R11;

R11 is phenyl or C5.7 cycloalkyl; and

p is 0-1.

In some examples, J is benzothiazolyl, benzoxazolyl, thiazolyl, or oxadiazolyl.

In one embodiment, the invention provides compounds of formula (2A) or (2B):

<formula> formula see original document page 5 </formula>

(2A) or <formula> formula see original document page 6 </formula>

wherein Z is O or S;

R 1 is NH 2, -NHC (= NH) -NH 2 or -C (= NH) -NH 2; W is -O (CH 2) k- or -S (O) (CH 2) k-;

R 2 is an optionally substituted phenyl, or W-R 2 together form C 1-6 alkyl or an optionally substituted phenyl;

R, R 4 and R 5 are independently H;

Y is a 5- to 7-membered aryl, heteroaryl or heterocyclic ring containing N,

O or S;

R 12 is halo, C 1-6 alkyl or -L- (CH 2) 1 -R 13; L is a bond, O, SO 2, NHCO, NHSO 2 or SO 2 NH;

R 13 is optionally halogenated C 1-6 alkyl, or an optionally substituted C 3-7 cycloalkyl, or a 5- to 7-membered aryl, heteroaryl or heterocyclic ring; i is 0;

k is 1;

1 is 0 to 1;

m and η are independently 1 to 4; and

q is 0 to 3.

In Formula (2A) or (2B) above, Y may be phenyl, pyridyl, thiazoyl or piperidinyl. In some examples, R 12 is -L- (CH 2) r R 13; and R13 is an optionally halogenated C1-6 alkyl, C3-7 cycloalkyl, morpholinyl, phenyl or piperidinyl.

In another aspect, the present invention provides pharmaceutical compositions comprising a compound having Formula (1), (2A) or (2B), and a pharmaceutically acceptable excipient. The present invention also provides methods for modulating a channel activating protease comprising administering to a system or an individual in need thereof a therapeutically effective amount of a compound having Formula (1), (2A) or (2B), or salts pharmaceutically acceptable drugs or pharmaceutical compositions thereof, thereby modulating said channel activating protease.

In yet another aspect, the invention provides methods for ameliorating a channel activating protease-mediated condition, comprising administering to a system or individual in need of such treatment an effective amount of a compound having Formula (1), (2A). ) or (2B), or pharmaceutically acceptable salts or pharmaceutical compositions thereof, and optionally in combination with a second therapeutic agent, thereby treating said condition. Examples of a second therapeutic agent that may be used with the compounds of the invention include, but are not limited to, an anti-inflammatory, bronchodilator, antihistamine, antitussive, antibiotic or DNase.

Examples of channel activating protease that can be modulated using the compounds of the invention include, but are not limited to, prostasin, PRSS22, TMPRSS11 (e.g., TMPRSS11B, TM-PRSS11E), TMPRSS2, TMPRSS3, TMPRSS4 (MTSP-2) , matriptase (MTSP-1), CAP2, CAP3, trypsin, cathepsin A, or neutrophil elastase. In particular examples, the invention provides methods for modulating prostasin, or methods for treating a prostasin-mediated condition.

In the above methods for using the compounds of the invention, a compound having Formula (1), (2A) or (2B) may be administered to a system comprising cells or tissues. For example, a compound having Formula (1), (2A) or (2B) may be contacted with bronchial epithelial cells, which may be human cells. In other embodiments, a compound having Formula (1), (2A) or (2B) may be administered to a human or animal subject.

In one embodiment, the invention provides methods for ameliorating a condition associated with fluid movement through ion transport epithelia or mucus or sputum accumulation in respiratory tissues, or a combination thereof. For example, the condition may be cystic fibrosis, primary ciliary dyskinesia, lung carcinoma, chronic bronchitis, chronic obstructive pulmonary disease, asthma, or a respiratory tract infection.

In addition, the present invention provides the use of a compound of formula (1), (2A) or (2B), or pharmaceutically acceptable salts or pharmaceutical compositions thereof, and optionally in combination with a second therapeutic agent, to modulate a channel activating protease (eg to inhibit prostasin). The present invention also provides the use of a compound having Formula (1), (2A) or (2B), or pharmaceutically acceptable salts or pharmaceutical compositions thereof, and optionally in combination with a second therapeutic agent, in the manufacture of a medicament for treating a channel activating protease-mediated condition (for example, a prostasin-mediated condition). Definitions

"Alkyl" refers to a moiety, and as a structural element of other groups, for example halosubstituted alkyl and alkoxy, and may be straight chain or branched. An optionally substituted alkyl, alkenyl, or alkenyl as used herein may be optionally halogenated (e.g. CF3), or may have one or more carbons which are substituted or exchanged with a heteroatom such as NR, O or S (e.g. -OCH 2 CH 2 O-, alkylthiols, thioalkoxy, alkylamines, etc.).

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

"Heteroaryl" as used herein is as defined above, where one or more of the ring members is a heteroatom. Examples of heteroaryl include, but are not limited to, pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl, benzothiopyranyl, benzo [1,3] dioxol, imidazolyl, benzoimidazole, pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, thienyl etc.

A "carbocyclic ring" as used herein refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring containing carbon atoms, which may optionally be substituted, for example, with = O. Examples of carbocyclic rings include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylene, cyclohexanone, and the like.

A "heterocyclic ring" as used herein is as defined for a carbocyclic ring above, wherein one or more ring carbons is a heteroatom. For example, a heterocyclic ring may contain N1 O, S, -N =, -S-, -S (O) 1 -S (O) 2-, or -NR- wherein R may be hydrogen, C1-4 alkyl or a protection group. Examples of heterocyclic rings include, but are not limited to, morpholine, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone, 1,4-dioxa-8-aza-spiro [4.5] dec-8-yl, etc.

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

The term "pharmaceutical combination" as used herein refers to a product obtained by mixing or combining active ingredients, and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, for example a compound of formula (1), (2A) or (2B) and a coagent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, for example a compound of formula (1), (2A) or (2B) and a coagent, are both administered to a patient as separate entities or simultaneously. concomitantly or sequentially without any specific time limit, wherein such administration provides therapeutically effective levels of the active ingredients in the patient's body. The latter also applies to cocktail therapy, for example, the administration of three or more active ingredients.

The term "therapeutically effective amount" means the amount of the object compound that will elicit a biological or medical response in a cell, tissue, organ, system, animal, or human being being researched by the researcher, veterinarian, physician, or other clinician.

The term "administering" and / or "administering" the subject compound should be understood as the method of providing a compound of the invention and prodrugs thereof to the individual in need of treatment.

As used herein, the terms "treating", "treating" and "treatment" refer to a method of alleviating or mitigating a disease and / or its accompanying symptoms.

The term "prostasin" may also be referred to as: human channel activating protease (hCAP); channel activator protease-1; and PRSS8, MERPOPS ID S01.159.

Ways to Carry Out the Invention

The present invention provides compounds, pharmaceutical compositions and methods of using such compounds to modulate channel activating protease (CAP).

In one aspect, the present invention provides compounds of formula (1):

<formula> formula see original document page 10 </formula>

and pharmaceutically acceptable salts, hydrates, solvates and stereoisomers thereof, wherein

J is a 5- to 12-membered monocyclic or fused carbocyclic ring, aryl, heteroaryl or heterocyclic ring containing Δ, O and / or S; R1 is - (CR2) rNR2, - (CR2), - NRC (= NR) -NR2, - (CR2), - C (= NR) -NR2 or a 5-7 membered non-aromatic nitrogen-containing heterocyclic ring ; W-R2 is a substituent at any position on ring A; W is OR -O (CR2) k-, -S (CR2) k-, -S (O) (CR2) k-, -SO2 (CR2) k- or -OC (O) (CR2) k-; R2 is C1-6alkyl, C2-6alkenyl, C2-6alkynyl, R61 -CR9 = CR9-R6, or <formula> formula see original document page 11 </formula>

wherein ring E is an optionally substituted 5- to 7-membered monocyclic or fused carbocyclic or heterocyclic ring; or W-R2 together form C1-6 alkyl, a 5-7 membered aryl or -OC (O) NR7R8; R3 is NR7R8 or R6;

R4 and R5 are independently H, C1-6 alkyl, OH, or C1-6 alkoxy; R 7 and R 8 are independently H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl or - (CR 2) i -R 6; or R 7 and R 8 together with N may form an optionally substituted 5 to 7 membered monocyclic or fused heterocyclic ring; R9 is H or C1-6 alkyl;

R10 is halo, C1-6alkyl, C1-6alkoxy, OR11 or - (CR2) 1-R11; R 6, R 11 and X are independently an optionally substituted 5 to 7 membered carbocyclic ring, heterocyclic ring, aryl or heteroaryl; or R11 is H or C1-6 alkyl;

each R is H1 or C1-6 alkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein a carbon may optionally be substituted or exchanged with NR, O or S; i is 0 to 1;

k and I are independently 0 to 6; m and n are independently 1 to 6; and p is 0 to 3.

In one embodiment, the invention provides compounds of formula (2A) or (2B): wherein Z is O or S;

R1 is NH2, -NHC (= NH) -NH2 or -C (= NH) -NH2; W is -O (CH 2) k- or -S (O) (CH 2) k-;

R2 is an optionally substituted phenyl, or W-R2 together form C1-6 alkyl or an optionally substituted phenyl; R, R4 and R5 are independently H;

Y is a 5- to 7-membered aryl, heteroaryl or heterocyclic ring containing N,

0 or S;

R 12 is halo, C 1-6 alkyl or -L- (CH 2) R 13; L is a bond, O, SO 2, NHCO, NHSO 2 or SO 2 NH; R 13 is optionally halogenated C 1-6 alkyl, or an optionally substituted C 3-7 cycloalkyl, or a 5- to 7-membered aryl, heteroaryl or heterocyclic ring; ion; k is 1;

l is 0 to 1;

m and n are independently 1 to 4; and q is 0 to 3.

In each of the above formulas, X, R 6 and R 11 may alternatively be an optionally substituted C 3-7 cycloalkyl.

In each of the above formulas, R1 may be NR'R ", NH-C (NR'R") = NH, NH-C (NHR1) = NR ", NH-C (R1) = NR", SC (NR1R11) -NH, S-C (NHR 1) -NR 11, C (NR 1 R 11) = NH, C (NHR 1) = NR "or CR = NR"; where R1R11 are equal or different and are H, C1-6 alkyl, C1.3 arylalkyl, aryl or where R1R11 forms a cyclic ring containing (CH2) p where ρ is an integer from 2 to 5. In each of the formulas above each optionally substituted moiety may be substituted with halo, = O1 amino, guanidinyl, amidino, C1-6 alkoxy; C1-6 alkyl, C2-6 alkenyl or C2-6 alchemy, each of which may optionally be halogenated or may optionally have a carbon that may be substituted or exchanged with Ν, O or S; CO2 R111 O- (CR2) m -C (O) -R11; - (CR2) m-R11, - (CR2) m-C (O) -R111 or - (CR2) m-SO2-R11; or a combination thereof, wherein each R 11 is H, C 1-6 alkyl, or an optionally substituted carbocyclic ring, heterocyclic ring, aryl or heteroaryl.

The compounds and compositions of the invention may be useful for modulating a channel activating protease. Examples of channel activating protease that can be modulated using the compounds and compositions of the invention include, but are not limited to, prostasin, PRSS22, TMPRSS11 (e.g., TMPRSS11B, TMPRSS11E), TMPRSS2, TM-PRSS3, TMPRSS4 (MTSP- 2), matriptase (MTSP-1), CAP2, CAP3, trypsin, cathepsin A, or neutrophil elastase. The novel compounds of this invention may also inhibit protease activity that stimulates ion channel activity, such as the epithelial sodium channel, and may be useful in treating CAP-associated diseases.

Pharmacology and Utility

Compounds of the invention modulate channel activating protease activity, for example tripin-like serine proteases such as prostasin, and as such, are useful for treating diseases or disorders in which prostasin contributes to the pathology and / or symptom of the disease.

Diseases mediated by inhibition of a channel activating protease, for example by a trypsin-like serine protease such as prostasin, include diseases associated with the regulation of fluid volumes across epithelial membranes. For example, airway surface fluid volume is a key regulator of mucociliary clearance and maintenance of lung health. Inhibition of a channel activating protease will promote fluid accumulation on the mucosal side of the airway epithelium, thereby promoting mucus clearance and preventing mucus and sputum accumulation in respiratory tissues (including lung airways). Such diseases include respiratory diseases such as cystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronic obstructive pulmonary disease (COPD), asthma, respiratory tract infections (acute and chronic; viral and bacterial) and lung carcinoma. Channel-activating protease inhibition-mediated diseases also include diseases other than respiratory diseases that are associated with abnormal fluid regulation through an epithelium, perhaps involving abnormal physiology of protective surface fluids on its surface, for example, dry mouth (dry mouth). ) or dry kerato conjunctivitis (dry eye). In addition, regulation of ENaC CAP in the kidney can be used to promote diuresis and thereby induce a hypotensive effect.

Chronic obstructive pulmonary disease includes chronic bronchitis or dyspnea associated with it, emphysema, as well as exacerbation of airway hyperactivity due to other drug therapy, in particular to other inhaled drug therapy. The present invention is also applicable to the treatment of bronchitis of any kind or gender including, for example, acute, arachid, catarrhal, croupal, chronic or phytoid bronchitis.

The compounds of the invention may be used for treating asthma, including but not limited to intrinsic (non-allergic) and extrinsic (allergic) asthma, mild asthma, moderate asthma, severe asthma, bronchitic asthma, exercise-induced asthma, occupational asthma. and induced asthma following bacterial infection. Asthma treatment is also understood to encompass treatment of individuals, for example, under 4 or 5 years of age, exhibiting symptoms of asthmatic breathing and diagnosed or diagnosed as "asthmatic children", an established patient category. of major medical concern and currently often identified as incipient, or early-stage asthmatics, or as "childhood asthmatic syndrome". The suitability of a channel inhibitor activating protease such as a prostasin inhibitor for the treatment of a disease mediated by inhibition of a channel activating protease can be tested by determining the inhibitory effect of the inhibitor activating protease. according to the assays described below and following methods known in the art.

Accordingly, the present invention also provides a method for preventing or treating any of the diseases or disorders described above in an individual in need of such treatment, which method comprises administering to said individual a therapeutically effective amount of a compound of formula (1), (2A) or (2B), or a pharmaceutically acceptable salt thereof. For any of the above uses, the dosage required will vary depending upon the mode of administration, the particular condition to be treated and the desired effect.

Pharmaceutical Administration and Compositions

In general, compounds of the invention will be administered in therapeutically effective amounts by any of the usual and acceptable methods known in the art, singly or in combination with one or more therapeutic agents.

Channel activating protease inhibitors of the invention are also useful as co-therapeutic agents for use in combination with other drug substances such as anti-inflammatory, boncodilatory, antihistamine or antitussive drug substances, particularly in the treatment of cystic fibrosis or obstructive or inflammatory airway diseases such as those mentioned above, for example, as enhancers of therapeutic activities of such drugs or as a method of reducing the required dosage or potential side effects of such drugs.

The channel inhibitor activating protease may be mixed with the other drug substance in a fixed pharmaceutical composition or may be administered separately before, concurrently with, or after the other drug substance.

Accordingly, the invention may include a combination of channel inhibitor activating protease with an anti-inflammatory, bronchodilator, antihistamine, antitussive, antibiotic or DNase drug substance, said channel inhibitor activating protease and said subset. drug substance being in the same or different pharmaceutical composition.

Suitable antiinflammatory drugs include steroids, in particular glucocorticosteroids such as budesonide, beclametasone dipropionate, fluticasone propionate, ciclesonide or metastone furoate, or steroids described in international patent application WO 02/88167, WO 02/12266, WO 02/100879, WO 02/00679 (e.g., Examples 3, 11, 14, 17, 19, 26, 34, 37, 39, 51, 60, 67, 72, 73, 90, 99 and 101), WO 03/35668, WO 03/48181, WO 03/62259, WO 03/64445, WO 03/72592, WO 04/39827 and WO 04/66920; non-steroidal glucocorticoid receptor agonites such as those described in DE 10261874, WO 00/00531, WO 02/10143, WO 03/82280, WO 03/82787, WO 03/86294, WO 03/104195, WO 03/101932 WO 04/05229, WO 04/18429, WO 04/19935 and WO 04/26248; LTD4 antagonists such as montelukast and zafirlukast; PDE4 inhibitors such as cilomilast (ARIFLO® GIaxoSmithKIine), ROFLUMILAST® (Byk Gulden), V-11294A (Napp), BAY19-8004 (Bayer), SCH-351591 (Schering-Plow), AROFILLINE® (Almirall Prodesfarma), PD189659 / PD168787 (Parke-Davis), AWD-12-281 (Asta Medica), CDC-801 (Celgene), SelCID (TM) CC-10004 (Celgene), VM554 / UM565 (Vernalis), T-440 ( Tanabe), KW-4490 (Kyowa Hakko Kogyo), and those described in WO 92/19594, WO 93/19749, WO 93/19750, WO 93/19751, WO 98/18796, WO 99/16766, WO 01/13953 WO 03/104204, WO 03/104205, WO 03/39544, WO 04/000814, WO 04/000839, WO 04/005258, WO 04/018450, WO 04/018451, WO 04/018457, WO 04/018465 WO 04/018431, WO 04/018449, WO 04/018450, WO 04/018451, WO 04/018457, WO 04/018465, WO 04/019944, WO 04/019945, WO 04/045607 and WO 04/037805 ; and adenosine A2B receptor antagonists such as those described in WO 02/42298, each of which is incorporated herein in its entirety.

Suitable bronchodilatory drugs include beta-2 adrenoceptor agonists such as albuterol (salbutamol), metaproterenol, terbutoline, salmeterol, fenoterol, procaterol, formoterol, and pharmaceutically acceptable salts thereof, and compounds of formula (1) as described. in WO 00/75114 (in free or salt or solvate form), which is incorporated herein by reference in its entirety, such as a compound of formula:

<formula> formula see original document page 17 </formula>

and pharmaceutically acceptable salts thereof; compounds of formula (1) of WO 04/16601; as well as compounds of EP 1440966, JP 05025045, WO 93/18007, WO 99/64035, US 2002/0055651, WO 01/42193, WO 01/83462, WO 02/66422, WO 02/70490, WO 02/76933, WO 03/24439, WO 03/42160, WO 03/42164, WO 03/72539, WO 03/91204, WO 03/99764, WO 04/16578, WO 04/22547, WO 04/32921, WO 04/33412, WO 04/37768, WO 04/37773, WO 04/37807, WO 04/39762, WO 04/39766, WO 04/45618 WO 04/46083 and WO 04/80964, each in free or salt or solvate form. Each of these publications is incorporated herein in its entirety.

Suitable bronchodilatory drugs also include anticholinergic or antimuscarinic agents, in particular ipratropium bromide, oxitropium bromide, tiotropium salts and CHF 4226 (Chiesi), glycopyrrolate, and also those described in EP 424021, US 3714357, US 5171744, WO 01 / 04118, WO 02/00652, WO 02/51841, WO 02/53564, WO 03/00840, WO 03/33495, WO 03/53966, WO 03/87094, WO 04/018422 and WO 04/05285 each of which is incorporated herein in its entirety.

Suitable dual anti-inflammatory and bronchodilatory drugs include beta-2 adrenoceptor agonists / dual muscarinic antagonists such as those described in US 2004/0167167, WO 04/74246 and WO 04/74812, each of which is incorporated herein in its entirety. .

Suitable antihistamine drug substances include cetirizine hydrochloride, acetaminophen, clemastine fumarate, promethazine, loratidine, desloratidine, diphenhydramine and fexofenadine hydrochloride, activastin, astemizole, azelastine, ebastine, mephine tephine, JP 2004107299, WO 03/099807 and WO 04/026841, each of which is incorporated herein in its entirety.

Suitable antibiotics include macrolide antibiotics, for example tobramycin (TOBI®).

Suitable DNase drug substances include dornase alfa (PULMOZYME®), a highly purified recombinant human deoxyribonuclease I (rhDNase) solution that selectively cleaves DNA. Alpha Dorase is used to treat cystic fibrosis.

Other useful combinations of channel activating protease inhibitors with anti-inflammatory drugs are those with chemosin receptor antagonists, for example, CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CCR -6, CCR-7, CCR-8, CCR-9 and CCR10, CXCR1, CXCR2, CXCR3, CX-CR4, CXCR5, particularly CCR-5 antagonists such as Schering-Plow antagonists SC-351125, SCH-55700 and SCH-D, Takeda antagonists such as N - [[4 - [[[6,7-dihydro-2- (4-methyl-phenyl) -5H-benzo-cycloepten-8-yl] carbonyl] chloride amino] phenyl] methyl] tetrahydro-N, N-dimethyl-2H-pyran-4-one

(TAK-770), and CCR-5 antagonists described in US 6166037, WO 00/66558, WO 00/66559, WO 04/018425 and WO 04/026873, each of which is incorporated herein in its entirety.

In the treatment of a Prostasin inhibition mediated disease according to the invention, a channel inhibitor activating protease of the invention in free or pharmaceutically acceptable salt form may be administered by any appropriate routine, for example orally. te, for example, in tablet, capsule or liquid form; parenterally, for example in the form of a solution or suspension for injection; intranasally, for example in the form of an aerosol another sprayable formulation using a suitable intranasal delivery device, for example a nasal spray such as those known in the art; or by inhalation, such as use with a nebuliser.

The channel inhibitor activating protease may be administered in a pharmaceutical composition together with a pharmaceutically acceptable diluent or carrier. Such compositions may be, for example, dry powders, tablets, capsules and liquids, but also injection solutions, infusion solutions or inhalation suspensions, which may be prepared using other formulation ingredients and techniques known in the art.

The dosage of the free or salt pharmaceutically acceptable channel inhibitor activating protease may depend on a number of factors, such as the activity and duration of action of the active ingredient, the severity of the condition to be treated, the mode of treatment. administration, the species, gender, ethnic origin, age and weight of the individual and / or individual condition. In a normal case, the daily dose for administration, for example oral administration to a warm-blooded animal, particularly a human weighing about 75 kg, is estimated to be approximately 0.7 mg to approximately 1400 mg. ; or in some examples from about 5 mg to about 200 mg. That dose may be administered in a single dose or in multi-part doses, for example from 5 to 200 mg.

Where the composition comprises an aerosol formulation, it may contain a hydrofluoroalkane (HFA) propellant such as HFA134a or HFA227 or a mixture thereof; one or more co-solvents known in the art such as ethanol (up to 20% by weight); one or more surfactants such as oleic acid or sorbitan trioleate; and / or one or more volume agents such as lactose. When the composition comprises a dry powder formulation, it may contain, for example, the channel inhibitor activating protease having a particular diameter of up to 10 microns, optionally together with a diluent or carrier, such as lactose, of the distribution. desired particular size blend and a compound that helps protect against deterioration of product performance due to moisture (eg magnesium stearate). When the composition comprises a nebulized formulation, it may contain, for example, the channel inhibitor activating protease dissolved or suspended in a vehicle containing water, a co-solvent such as ethanol or propylene glycol and a stabilizer, which can be a surfactant.

The present invention includes (A) a compound of the invention in inhalable form, for example in an aerosol or other atomizable composition or in an inhalable particulate, for example micronised form; (B) an inhalable medicament comprising a compound of the invention in inhalable form; (C) a pharmaceutical product comprising a compound of the invention in inhalable form in combination with an inhalation device; and (D) an inhalation device containing a compound of the invention in inhalable form.

Processes for Preparing Compounds of the Invention

The present invention also includes processes for preparing compounds of the invention. In a described reaction, reactive functional groups, where desired in the final product (e.g., hydroxy, amino, imino, thio or carboxy groups), may be protected using protecting groups known in the art to prevent their unwanted participation in the reactions. . Conventional protecting groups may be used according to standard practice, for example, see T.W. Greene and P. G. M. Wuts in "Protective Groups in Organic Chemistry", John Wiley and Sons, 1991.

The compounds of the invention may be prepared following reaction scheme I below: <formula> formula see original document page 21 </formula>

Scheme I

wherein R1 'is (CR2) n-X1-R1; R2 'is (CR2) kR2; R 1, R 2, R 3, X, Z, i, k and n are as defined in Formula (1); R7 is an alkyl carbamate protecting group (for example methyl, ethyl, t-butyl or benzyl and the like).

Intermediate II can be synthesized by reacting intermediate 1aa with an R2'-Y type alkyl reagent where Y is a leaving group in the presence of a suitable base and a suitable solvent. Examples of leaving groups in R2'-Y alkyl reagents include, but are not limited to halides such as chlorides and bromides, or a tosylate, mesylate, or besylate leaving group, and the like. These reactions may proceed in a temperature range of about 0 ° C to about 60 ° C and may take about 24 hours to complete.

Intermediate IV may be synthesized by reacting intermediate II with reagent III with a suitable peptide coupling reagent and a suitable base in the presence of a suitable solvent. Suitable bases for this reaction include but are not limited to triethylamine, DIEA1 pyridine, 2,4,6-collidine, and other suitable bases within the skill of the art. The reaction may proceed in a temperature range of about 0 ° C to about 40 ° C and may take up to about 24 hours to complete.

In the above reaction scheme I, intermediate V may be synthesized by removing the carbamate protecting group (for example, where R 7 is t-butyl) of intermediate IV with an appropriate acid, and optionally in the presence of a suitable solvent. Suitable acids include, but are not limited to, TFA1 p-TsOH, TfOH, HCl1 HBr1 HF, HBF4, and other suitable acids to the knowledge of those skilled in the art. The reaction may proceed within a temperature range of about -20 ° C to about 40 ° C and may take up to about 24 hours to complete.

Alternatively, intermediate V may be synthesized by removing intermediate group IV carbamate protecting group (for example, where R 7 is benzyl or any benzyl derivative) with hydrogen gas in the presence of a suitable catalyst and a suitable solvent or water. Examples of suitable catalysts include, but are not limited to, Pd / C, R 1 RO 2, Pt / C, Rh / C, and other suitable catalysts within the skill of the art. The reaction may proceed in a temperature range from about 0 ° C to about 80 ° C, with hydrogen pressures from about 1.05 kg / cm2 (15 psi) to about 5.62 kg / cm2 (80 psi) , and it may take up to 48 hours to complete.

Alternatively, a compound of formula V may be synthesized by removing the carbamate protecting group from a compound of formula IV (for example, where R 7 is derived from allyl or any allyl) by reacting a compound of formula IV with a suitable allylic scavenger (e.g. Et2NH, morpholine, piperidine, pyrrolidine, NaBH4, and the like) in the presence of a suitable catalyst (e.g. Pd2 (dba) 3, PdCI2, Pd (PPh3) 4 and the like) in the presence of a suitable solvent (e.g. water, methanol, ethanol, isopropanol, t-butanol, n-propanol, n-butanol, cyclohexanol, and mixtures thereof and the like).

Intermediate V11 can be synthesized by reacting intermediate V with reagent V1 in the presence of a suitable peptide coupling reagent and a suitable base (Et3N, DIEA, pyridine, 2,4,6-collidine, and similar) in the presence of a suitable solvent. The reaction may proceed in a temperature range of about 0 ° C to about 40 ° C, and may take up to about 24 hours to complete.

The final compound VIII may be synthesized by reacting a compound of formula VIII with the appropriate oxidant in the presence of a suitable solvent or water. Suitable oxidizers include, but are not limited to, periodinane Dess-Martinl 2-iodobenzoic acid with oxone, TIME with trichlorisocyanuric acid, TIME with NaOCI, DMSO with oxalyl chloride, pyridinium chlorochromate, Mn02, Cr02, and other suitable oxidants to the knowledge. of those skilled in the art. The reaction may proceed within a temperature range of about 0 ° C to about 40 ° C, and may take up to about 24 hours to complete.

Suitable peptide coupling reagents for use in a reaction described in Reaction Scheme I include, but are not limited to, DCC1 DIC, HATU, BOP1 PyBOP, EDC and other coupling reagents within the skill of the art.

Suitable bases for use in a reaction described in Reaction Scheme I include, but are not limited to hydroxides such as NaOH, KOH, or LiOH; carbonates such as K 2 CO 3 or CsCO 3; hydrides such as NaH or KH, and the like. Other suitable bases are amines, DIEA, pyridine, 2,4,6-collidine, and other suitable bases within the skill of the art.

Suitable organic solvents for use in a reaction described in Reaction Scheme I include, but are not limited to DMSO, THF, DMF, DMAc1 acetonitrile, acetone, 2-propanone, butanone, HMPA1 NMP, dichloromethane, chloroform, 1,2 - dichloroethane, diethyl ether, methanol, ethanol, t-butanol, isopropanol, propanol, n-butanol, cyclohexanol, acetonitrile, dioxane, MTBE1 benzene, toluene, and mixtures thereof, and other solvents suitable for those skilled in the art. . Additional Processes for Preparing Compounds of the Invention

A compound of the invention may be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt of a compound of the invention may be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base.

Alternatively, salt forms of the compounds of the invention may be prepared using salts of the starting materials or intermediates.

The free acid or free base forms of the inventive compounds may be prepared from the corresponding base addition salt or acid addition salt form, respectively, for example, a compound of the invention in an addition salt form. Acid may be converted to the corresponding free base by treatment with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of the invention in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid (eg hydrochloric acid etc.).

Compounds of the invention in non-oxidized form may be prepared from N-oxides of compounds of the invention by treatment with a reducing agent (eg sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride). , phosphorus trichloride, tribromide, or the like) in a suitable inert organic solvent (for example, acetonitrile, ethanol, aqueous dioxane, or the like) at 0 to 80 ° C.

Prodrug derivatives of the compounds of the invention may be prepared by methods known to those skilled in the art (e.g., for details, see Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985). For example, suitable prodrugs may be prepared by reacting a non-derivatized compound of the invention with a suitable carbamylating agent (e.g. 1,1-acyloxyalkylcarbonochloride, para-nitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the invention may be prepared by methods known to those skilled in the art. A detailed description of applicable techniques for creating and removing protecting groups can be found in TW Greene, "Protecting Groups in Organic Chemistry", 3rd edition, John Wiley and Sons, Inc., 1999. Compounds of the present invention they may conveniently be prepared or formed during the process of the invention as solvates (e.g. hydrates). Hydrates of compounds of the present invention may conveniently be prepared by recrystallization from an aqueous / organic soluble mixture using organic solvents such as dioxin, tetrahydrofuran or methanol.

Compounds of the invention may 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 enantiomers. Resolution of enantiomers may be performed using covalent diastereomeric derivatives of the inventive compounds, or using dissociable complexes (eg crystalline diastereomeric salts). Diastereomers have distinct physical properties (eg, melting points, boiling points, solubilities, reactivity etc.), and can be easily separated by taking advantage of these dissimilarities. Diastereomers may be separated by chromatography, or by separation / resolution techniques based on differences in solubility. The optically pure enantiomer is then recovered, together with the resolving agent, by any practical method that would not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds of their racemic mixture can be found in Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc. ., 1981.

In summary, the compounds of formula (1) may be prepared by a process involving:

(a) that of reaction Scheme I;

(b) optionally converting a compound of the invention into a pharmaceutically acceptable salt;

(c) optionally converting a salt form of a compound of the invention into a non-salt form;

(d) optionally converting an unoxidized form of a compound of the invention to a pharmaceutically acceptable N-oxide;

(e) optionally converting an N-oxide form of a compound of the invention to its non-oxidized form;

(f) optionally resolving an individual isomer of a compound of the invention from a mixture of isomers;

(g) optionally converting a non-derivatized compound of the invention into a pharmaceutically acceptable prodrug derivative; and

(h) optionally converting a prodrug derivative of a compound of the invention to its non-derivatized form.

As the production of starting materials is not particularly described, the compounds are known or may be prepared analogously to methods known in the art or as described in the following examples. One skilled in the art will appreciate that the above transformations are only representative of methods for preparing the compounds of the present invention, and that other well known methods may similarly be used. The present invention is also exemplified, but not limited to, by the following intermediates (reference compounds) and examples illustrating the preparation of the compounds of the invention.

In the synthetic methodologies below, the following common abbreviations known in the art are used: DCM (dichloromethane); THF (tetrahydrofuran); and DIEA (diisopropylethylamine). Reference Compound 1 <formula> formula see original document page 27 </formula>

Scheme 1

In the above reaction scheme, the reagents and conditions are: (a) iso-BuOCOCl, Et3N, THF; NaBH4, H2O. (b) Dess-Martin periodinane, CH 2 Cl 2; (c) / so-PrMgCl, benzoxazole, THF, -20 ° C, 30 minutes, then 1- C1 -20 ° C to room temperature, (d) H2 2.81 kg / cm2, (40 psi), EtOH, 10% Pd / C, rt, 18 hours.

1-B: The crude starting material, Z-Lys (Boc) OH 1-A (320 g, 842 mmols) is dissolved in THF (2.5 L), and the solution is cooled to -10 ° C followed by the addition of triethylamine. (115.2 mL, 1.0 eq) and dropwise addition of isobutylchloroformate (118.7 mL, 1.1 eq). The resulting suspension is stirred for 2 hours at 0 ° C. The reaction mixture is filtered and cooled to -10 ° C. NaBH4 (64.6 g, 2.1 eq) is dissolved in water (500 mL) at 0 ° C, and the solution is added portion by portion to the THF (heavy CO 2 evolution) solution. The reaction mixture is allowed to warm to room temperature and stirred for one hour. The reaction mixture is acidified with 1N HCl solution, and the aqueous phase is extracted several times with EtOAc. The combined organic layers are washed with water, saturated aqueous NaHCO 3 solution, and brine; dried over MgSO4, and the solvent is removed in vacuo. The product is purified by flash column chromatography (hexanes / ethyl acetate) to provide the desired product as a white foam. 1-C: Alcohol 1-B (200 g, 545.8 mmols) is dissolved in DCM (2.0 L) and resinated at 0 ° C. A solution of Dess-Martin reagent (231g, 1.0 eq) in DCM (2.0 L) is added portion by portion. The suspension is allowed to warm to room temperature and stirred until complete conversion (1 to 4 hours). A 1: 1 mixture of saturated aqueous NaHCO 3 solution and 1 M Na 2 S 2 O 3 solution is added, and the resulting biphasic system is stirred vigorously for 20 minutes. The organic layer is separated and the aqueous layer is extracted once with DCM. The combined organic layers are vacuum distilled, and the resulting oil is taken up in EtOAc and washed six times with the mixture of NaHCO3 / Na2S203, water, and brine; dried over MgSO4, and the solvent is removed in vacuo to afford the crude aldehyde as a yellowish oil. The material is directly used in the next step without further purification.

1-D: To a solution of isopropyl magnesium chloride (1.67 eq. Vs aldehyde, 15 390 ml of a solution of 2M THF from Sigma-Aldrich) in THF (1.5 L) is added benzoxazole (92 1.8 g, 1.67 eq) in THF (1.0 L) at -20 ° C. The reaction mixture is stirred at -20 ° C for 30 min (color change to deep red), and a solution of 1-C aldehyde (170 g, 466 mmol) in THF (1.5 L) is slowly added under controlled temperature. at -20 ° C to -15 ° C. The reaction mixture is allowed to warm to room temperature and stirred until completion. The reaction mixture is quenched with saturated aqueous NH 4 Cl solution and the solvent is removed in vacuo. The aqueous phase is extracted three times with EtOAc, and the combined organic layers are excessively washed with 1N HCl solution, water, and brine; dried over MgSO4, and the solvent is removed in vacuo to afford crude benzoxazole as an intense red oil. Purification on silica with EtOAc / hexanes (1: 5 to 1: 1) provided benzoxazole as a yellow solid. 1-E: A solution of intermediate 1-D (25.0 g, 51.7 mmol) is dissolved in ethanol (150 mL. Pd / C (10%, wet, Degussa type) is added, and the vial is placed on a Parr shaker overnight and subjected to 40 psi (2.81 kg / cm2) hydrogen gas The catalyst is filtered through Celite and the solvent removed in vacuo The crude material is purified by chromatography first using a hexanes / EtOAc gradient to remove colored and less polar impurities, then followed by a DCM / MeOH gradient to elute the desired compound The solvent is removed in vacuo and the compound triturated several times. in ether to provide the desired reference compound 1 as a white powder 1 H-NMR (DMSO-d 6,400 MHz) δ 7.73 - 7.70 (2H, m), 7.40 - 7, 34 (2H, m), 6.78 - 6.73 (1H, m), 4.55 -4.51 (1H, m), 3.05-3.01 (1H, m), 2.92- 2.83 (2H, m), 1.48-1.18 (14H, m) LCMS: 350.5 (M + H) + Reference Compound 2 <formula> formula see original document page 29 </formula> Scheme 2

In Scheme 2 above, the reagents and conditions are: a) Cbz-OSu, Et 3 N, THF, H 2 O, rt, 18h, (b) i. iso-BuOCOCI, Et 3 N, THF; ii. NaBH4, H2O; (c) periodinane Dess-Martin1 CH 2 Cl 2; (d) iso-PrMgCl, benzoxazole, THF, -20 ° C, 30 minutes, then 2-D, -20 ° C to room temperature; (e) indium, NH 4 Cl, EtOH, reflux, 5h; (f) β, β-Bis (tert-butoxycarbonyl) -1H-pyrazoM-carboxamidine, DIEA, MeOH; (g) H2, (2.81 kg / cm2), 10% Pd / C, EtOH. 2-B: L-Nitrophenylalanine Hydrochloride (4.45 g, 18.0 mmols) and N- (Benzyloxycarbonyloxy) sucinimide (Cbz-OSu) (4.49 g, 18.0 mmols) are added to a vial round base containing THF (60 mL) and water (20 mL). The mixture is stirred at room temperature and Et 3 N (10.1 mL, 72.0 mmol) is added, and the reaction is stirred overnight at room temperature. The clear solution is diluted with EtOAc (200 mL) and washed with 1N HCl (3 x 100 mL) and brine (1 x 100 mL), and dried with MgSO4. The solvent is evaporated in vacuo to afford 2-B as a white solid, which is used without further purification.

2-C to 2-E: These intermediates are prepared following methods analogous to those described for preparing intermediates 1-B to 1-D of reference compound 1, respectively.

2-F: The 2-E nitrophenyl analog (1.85 g, 4.15 mmol) is dissolved in EtOH (50 mL) and heated to reflux. Saturated aqueous NH 4 Cl (5 mL) is added, followed by indium powder (3.2 g, 27.9 mmol). The reaction mixture is stirred at reflux temperature for 5 h, cooled to room temperature, and the solvent removed in vacuo. The crude material is suspended in EtOAc (100 mL) and washed with saturated NaHCO 3 (3 x 100 mL), dried with MgSO 4, and filtered through Celite. The solvent is removed in vacuo to afford aniline 2-F as a non-all white waxy solid which is used without further purification.

2-G: Aniline 2-F (1.52 g, 3.67 mmol) is dissolved in MeOH (10 mL), and DIEA (0.7 mL, 4.4 mmol) and Δ, Ν-Bis (tert-methanol). butoxycarbonyl) -1H-pyrazol-1-carboxamidine (1.37 g, 4.4 mmol) is added, and the reaction mixture is stirred at room temperature. After 4 hours, an additional 0.5 equivalent of Ν, Ν-Bis (tert-butoxycarbonyl) -1H-pyrazol-1-carboxamidine (0.685 g, 2.2 mmol) is added, and the reaction is then stirred overnight. at room temperature. EtOAc (100 mL) is added, and the organic layer is washed with water, and brine; and dried over MgSO4. The solvent is removed in vacuo and the crude material is purified by chromatography on silica gel with EtOAc / hexanes (0 to 100% gradient) to afford the desired product 2- G as an oil.

2-H: This compound is prepared from 2-G using methods analogous to those described for the preparation of intermediate 1-E for reference compound 1.

Reference Compound 3

<formula> formula see original document page 31 </formula>

Scheme 3

In the above scheme 3, the reagents and conditions are: (a) HN (OMe) Me HCl1 BOP1 Et3 N, DMF1 O0C at rt; (b) n -BuLi (2.5 M in hexanes), benzothiazole, THF, -78 ° C, then 3-B, THF, -70 ° C at rt; (c) NaBH 4, Me-OH; (d) p-TsOH, CH 2 Cl 2 6h.

3-B: BOP (50 g, 112 mmols) is added in one portion to a stirring solution of 3-A (49.92 g, 102.6 mmols), α-β-dimethylhydroxylamine hydrochloride (30.4 g , 224 mmol), and triethylamine (88 mL, 616 mmol) in dry DMF (200 mL) under argon at 0 ° C. The reaction mixture is slowly allowed to warm to room temperature over 2 hours, filtered through diatomaceous earth, and concentrated in vacuo. The residue is dissolved in ethyl acetate, washed with 1 M H 2 O 1 of aqueous KHSO 4, saturated aqueous NaHCO 3, and brine; dried and concentrated in vacuo. The residue is column purified to provide compound 3-B.

3-C: n-Butyl lithium (2.5 M in hexanes, 272.2 mL, 681.4 mmol) is added dropwise at -78 ° C under argon to a stirring solution of benzothiazole (115.72 g , 850.7 mmols) in dry THF (1660 mL) at a rate that kept the reaction temperature below -64 ° C. Upon completion of the addition, the reaction mixture is stirred for 30 minutes at -70 ° C, and a solution of compound 3-B (45 g, 85.7 mmols) in dry THF (300 mL) is added at a rate. which kept the reaction temperature below -70 ° C. The reaction is stirred for 15 minutes, quenched with saturated aqueous NH 4 Cl, and stirred for 16 hours at room temperature. The resulting organic layer is separated, diluted with ethyl acetate, washed with water and brine; dried and concentrated in vacuo, and purified by silica gel chromatography to provide compound 3-C.

To a solution of 3-C (33.7 g, 55.82 mmol) in MeOH (407 mL) at 0 ° C is added NaBH4 (9.98 g). The reaction mixture is slowly warmed to room temperature over 1 hour, then warmed to 45 ° C over 1 hour, and then cooled back to room temperature. The reaction is quenched with acetone (60 mL), and concentrated in vacuo. The residue is dissolved in ethyl acetate, washed with brine, and dried over MgSO4. The crude material is purified by silica gel chromatography to provide the 3-D product.

.3-E: p-TsOH is added to a stirring solution of compound 3-D (28.2 g) in CH 2 Cl 2 (300 mL) at room temperature until the solution is saturated. The reaction is stirred at room temperature for 6 hours. Water is added, and the organic layer is extracted with EtOAc, washed with 1: 1 (V / V) mixture of brine and 10% aqueous Na 2 CO 3, dried over Na 2 SO 4, and purified by silica gel chromatography to provide the 3-E product. Reference Compound 4 <formula> formula see original document page 33 </formula>

Scheme 4

To prepare intermediate 4-Β, n-Butyl lithium (1.6 M in hexanes, 11.3 mL, 17.9 mmols) is added dropwise to a solution of i-Pr2NH (2.53 mL, 17.9 mmol) in THF (25 mL) cooled to -78 ° C and stirred for 10 minutes. Ketone 4-A (3.25 g, 16.3 mmol) is added dropwise and the continued solution stirred at -78 ° C for a further 25 minutes. N-phenyl-bis- (trifluoromethanesulfonamide) (6.26 g, 17.5 mmol) dissolved in THF (25 mL) is added dropwise, and the reaction mixture is placed in a water / ice bath at 0 ° C. ° C and stirred for 4 hours. The solvent is evaporated, and the crude residue taken up in a minimum amount of hexanes and then filtered through an alumina buffer using hexanes: EtOAc / 9: 1 as the eluent. The solvent is removed in vacuo to afford the desired triflate 4-B.

Intermediates 4-B (715 mg, 2.16 mmol) and 4-C (514 mg, 1.96 mmol) are dissolved in dioxane (10 mL) in a microwave reaction vial. K3PO4 (874 mg, 4.12 mmols) dissolved in water (2.5 mL) is added, and the solution is degassed by three freeze-pump-thawing liquid nitrogen cycles. Catalyst PdCl2 (dppf) (143 mg, 0.196 mmol) is added, and the nitrogen purged vial sealed. The reaction is heated in an Emrys Personal Chemistry microwave reactor to 150 ° C for 25 minutes. The dioxane is evaporated in vacuo, and the residue taken up and partitioned between EtOAc (50 mL) and 10% aqueous citric acid (50 mL). The layers are separated, and the organic phase is washed with 10% aqueous citric acid (2 x 50 mL) and brine (50 mL). The organic layer is dried with Na 2 SO 4 and evaporated, and the crude residue purified by silica gel chromatography (hexanes / EtOAc gradient) to afford intermediate 4-D.

Intermediate 4-D (45 mg, 0.142 mmol) is dissolved in EtOH (10 mL) in a 25 mL vial fitted with a rubber septum. A catalytic amount of Pd / C (10%) is added, and hydrogen gas is introduced from a balloon equipped with a needle inserted through the septum. The reaction is stirred at room temperature until the starting material is consumed (as indicated by LCMS). The catalyst is then filtered, and the solvent evaporated to dryness to afford Reference Compound 4.

Reference Compound 5

<formula> formula see original document page 34 </formula>

Scheme 5

In the above scheme 5, the reagents and conditions are: a) Cbz-OSu1 Et3 N, THF, H2 O, rt, 18h; (b) i. iso-BuOCOCl, Et 3 N, THF; ii. NaBH4, H2O; (c) Dess-Martin Periodinane, CH 2 Cl 2; (d) iso-PrMgCl, benzoxazole, THF, -20 ° C, 30 minutes, then 5-D, -20 ° C to rt; (e) NaBH 4, NiCl 2, Boc 2 O, MeOH, 0 ° C, (f) H 2 (2.81 kg / cm 2), 10% Pd / C, EtOH.

5-B to 5-E: This compound is prepared from L-4-cyanophenylalanine (5-A) using methods analogous to those described for the preparation of intermediate 2-B for reference compound 2. Intermediates 5 -C to 5-E are prepared following methods analogous to those described for preparing intermediates 1-B to 1-D of reference compound 1, respectively. 5-F: Nitrile 5-E (3.68 g, 8.64 mmol) is dissolved in methanol (60 mL), and cooled to 0 ° C. BCO 2 O (3.77 g, 2 eq) is added, followed by NiCb (210 mg, 0.1 eq). The mixture is stirred and NaBH 4 (2.29 g, 7 eq) is slowly added in small portions. The mixture is allowed to warm to room temperature and stirred for 1 hour. Diethylene triamine (0.94 mL, 1 eq) is added, and the mixture stirred for a further 30 minutes, and then the solvent is evaporated in vacuo. The residue is dissolved in EtOAc, and washed with saturated NaH-CO 3 (2 x 100 mL). The solution is dried with MgSO4 and the solvent is evaporated in vacuo. The crude material is purified by flash chromatography on 0 to 100% EtOAc and hexanes gradient silica gel to afford the title compound as an oil.

5-G: This compound is prepared from 5-F using methods analogous to those described for the preparation of intermediate 1-E for reference compound 1.

Reference Compound 6

<formula> formula see original document page 35 </formula>

Scheme 6

6J5: (R) -Boc-nipecotic acid (2.50 g, 10.9 mmol) and HOBt (1.77 g, 13.1 mmol) are dissolved in DMF (50 mL), and EDC (2.30 g , 12.0 mmols) is added. The reaction mixture is stirred for 1h, and 30% aqueous ammonia (2.8 mL) is added, and the reaction is stirred for a further 2 hours. The reaction is then diluted with EtOAc (200 mL) and partitioned with water (100 mL). The organic layer is washed with saturated aqueous NaHCO 3 (3 x 200 mL), and then dried with MgSO 4. The solvent is removed in vacuo to afford amide 6-B as a waxy solid.

6-C: Amide 6-B (2.40, 10.5 mmol) is dissolved in THF (50 mL). Lawesson's reagent (2.64 g, 6.54 mmol) is added, and the reaction mixture is stirred at room temperature for 4 hours. The solvent is evaporated, and the crude material is purified by silica gel chromatography using a 0 to 5% MeOH in CH 2 Cl 2 gradient as eluent. The solvent is evaporated to afford the desired thioamide.

6-D: 6-C thioamide (1.27 g, 5.20 mmol) is dissolved in CH 2 Cl 2 (50 mL). Ethyl 4-chloroacetoacetate (0.8 mL, 5.72 mmol) is added, and the solution is stirred at room temperature for 5 hours. The solvent is removed in vacuo, and the crude residue is then dissolved in EtOH (50 mL). Activated powdered molecular sieves (1.2 g) are added and the reaction mixture is heated at reflux for 18 hours. The mixture is then cooled to room temperature and filtered through celite. Saturated aqueous NaHCO 3 (50 mL) is added to the filtered solution, and then extracted with EtOAc (3 x 50 mL). The combined organic layer is washed with brine (100 mL) and dried (MgSO4). The solvent is evaporated in vacuo and the crude material is purified by silica gel flash chromatography using a 0 to 100% gradient of EtOAc and hexanes to provide thiazole 6-D.

Reference compound 6: LiOH-H 2 O (65 mg, 1.55 mmol) is dissolved in water (5 mL) and added dropwise to a stirring solution of ethyl ester 6-D dissolved in dioxane (20 mL) . The reaction is stirred for 2 hours, and dioxane is evaporated. The crude residue is partitioned between EtOAc and 1M NaHSO4; The combined organic layer is dried (MgSO4) and evaporated in vacuo; and crude material is purified by reverse phase HPLC (H20 / MeCN mobile phase).

Reference Compound 7 <formula> formula see original document page 37 </formula> Scheme 7

In the above scheme 7, the reagents and conditions are: a) i. iso- BuOCOCI, Et3 N1 THF; ii. NaBH41 H2O; (b) trichloroisocyanuric acid, TIME, 0 ° C to room temperature, CH 2 Cl 2, (c) Cbz-α-phosphonoglycine trimethyl ester, DBU, DCM; (d) H2 4.21 kg / cm2 (60 psi), (SiS) -Me-BPE-Rh (COD) + OTf1 MeOH, 96h; (e) LiOH 1 Dioxane 1 water; (f) i. iso-BuOCOCI, Et 3 N, THF; ii. NaBH4, H2O; (g) Periodinane Dess-Martin, CH 2 Cl 2; (h) iso- PrMgCl1 benzoxazole, THF1 -20 ° C, 30 minutes, then 7 -G, -20 ° C to room temperature; (i) H2l (2.81 kg / cm2 (40 psi), 10% Pd / C, EtOH. 7-A: This compound is prepared from trans-4- (tert-butoxycarbonylaminomethyl) cyclohexanecarboxylic acid (7- A) using methods analogous to those described for the preparation of intermediate 1-B for reference compound 1 in Scheme 1.

7-B: Alcohol 7-A (4.14 g, 17.0 mmol) is dissolved in CH 2 Cl 2 (35 mL) and the solution is cooled to 0 ° C. Trichloroisocyanuric acid (4.15 g, 17.8 mmol) is added, followed by TIME (28 mg, 0.17 mol). The reaction is then warmed to room temperature and stirred for a further 15 minutes at room temperature. A precipitate formed, and the reaction mixture was filtered through Celite and washed with CH 2 Cl 2. The combined CH 2 Cl 2 solution (-100 mL) is washed with saturated aqueous NaHCO 3 (2 x 50 mL), 1 M HCl (2 x 50 mL), and brine (50 mL), dried (MgSO 4) and evaporated solvent. used directly in the next step without further purification. 7-C: N-Benzyloxycarbonyl-α-phosphonoglycine trimethyl ester (5.63 g, 17 mmol) is dissolved in CH 2 Cl 2 (35 mL); DBU (5.1 ml, 34 mmol) is added and the solution is stirred for 20 minutes. Aldehyde 7-B (4.09 g, 17 mmol) is added dropwise as a solution in CH 2 Cl 2 (10 mL). The reaction is stirred overnight, then the solvent is evaporated and the residue dissolved in EtOAc (100 mL) and washed with 1 M NaHSO 4 (2 x 50 mL) and brine, then dried (MgSO 4) and evaporated in vacuum. The crude material is purified by flash chromatography using a gradient of 100-100% EtOAc / hexanes to provide the desired product as a white solid. 7-D: 7-C olefin (2.04 g, 4.56 mmol) is dissolved in MeOH (100 mL), and the solution is degassed before addition of catalyst, (-) - 1,2-sulfonate. Bis - ((2S, 3S) -2,5-dimethylphospholane) ethane (cyclooctadiene) rhodium (1) trifluoromethane (28 mg, 1 mol%). The reaction mixture is placed on a Parr shaker and stirred under 4.21 kg / cm2 (60 psi) H2 for 4 days. The solvent is then evaporated in vacuo, and the crude material is purified by flash chromatography using a 0 to 100% EtOAc / hexanes gradient to provide the desired product as a white solid.

7-E: 7-D methyl ester (1.81 g, 4.04 mmol) is dissolved in dioxane (50 mL) and stirred at 0 ° C. LiOH (203 mg, 4.84 mmol) dissolved in water (10 mL) is added dropwise, and the solution is then warmed to room temperature. After the starting material has disappeared (by LCMS), the solvent is evaporated, and the crude material is dissolved in EtOAc (100 mL), washed with 1 N NaHSO4 (2 x 50 mL) and brine (50 mL), and dried (MgSO4). The solvent is removed in vacuo and the product is used directly in the next step without further purification.

7-F to 7-H: Intermediates 7-F to 7-H are prepared following methods analogous to those described for preparing reference compound 1 intermediates.

Z1I: This compound is prepared from 7-H using methods analogous to those described for the preparation of intermediate 1-E for reference compound 1. MS m / z 404.2 (M + 1); 1H-NMR (CDCl3, 400 MHz)? 7.66 - 7.64 (1, m), 7.49 - 7.47 (1, m), 7.31 - 7.26 (2, m), 4.85 - 4.64 (1Ηm), 3.44 - 3.16 (1Ηm), 2.96 - 2.88 (2Ηm), 1.80 - 1.55 (4Ηm) ), 1.39 - 1.14 (13Η, m), 0.89 - 0.72 (4Η, m).

Reference Compound 8

<formula> formula see original document page 39 </formula>

Reference compound 8 is prepared from (S) -Bocnipecotic acid using methods analogous to those described for the preparation of reference compound 6.

Reference Compound 9

<formula> formula see original document page 39 </formula>

Reference compound 9 is prepared starting from 3-cyanophenylalanine using methods analogous to those described for preparing reference compound 5.

Reference Compound 10

<formula> formula see original document page 39 </formula>

Reference compound 10 is prepared starting from Boc-isonipecotic acid using methods analogous to those described for preparing reference compound 6. Reference Compound 11

<formula> formula see original document page 40 </formula>

Boc-isonipecotic acid methyl ester hydrochloride (3.24 g, 14.1 mmol), DL-4-Amino-3-hydroxybutyric acid (2.40 g, 14.1 mmol), and DIEA (7, 0 mL, 42.3 mL) are dissolved in CH 2 Cl 2 (50 mL). HATU (5.9 g, 15.5 mmol) is added, and the reaction mixture is stirred at room temperature for 2 hours. The solvent is evaporated, and the crude residue is taken up in EtOAc (100 mL). The organic phase is washed with 0.1 M HCl (2 x 100 mL) and brine (100 mL), dried with MgSO 4, and evaporated in vacuo. The crude material is purified by silica gel 10 chromatography (EtOAc / Hexanes Gradient) to afford intermediate 11-B.

11-C: Alcohol 11-B is oxidized to ketone using conditions analogous to those used for the preparation of Example 1-F.

11-D; Ketone 11-C (1.10 g, 3.21 mmol) is dissolved in THF (100 mL). Lawesson's reagent (650 mg, 1.61 mmol) is added, and the reaction mixture is stirred at room temperature for 48 hours. The solvent is removed in vacuo, and the crude material is purified by silica gel intantane chromatography (EtOAc / Hexanes gradient 0: 100).

11: Methyl ester 11-D is saponified using methods analogous to those described for the preparation of reference compound 6.

Reference Compound 12

<formula> formula see original document page 40 </formula>

The reagents and conditions in the above reaction for reference compound 12 are: a) (cyclohexylcarbonyloxy) succinimide, pyridine, DMAP, rt, THF; (b) 1N NaOH in water. (4-Amino-phenyl) -acetic acid ethyl ester (0.895 g, 5.0 mmols) and N- (cyclohexylcarbonyloxy) succinimide (1.051 g, 5.0 mmols) are added to a round base flask containing THF (20 mL), pyridine (0.60 mL) and DMAP (10 mg). The mixture is stirred at room temperature overnight. The clear solution is diluted with EtOAc (200 mL), washed with 1N HCl (3 x 100 mL) and brine (100 mL), and dried with MgSO4. The solvent is evaporated in vacuo to afford intermediate 12-B as a white solid which is used without further purification.

Ethyl ester 12-B (1.46 g, 5 mmol) is dissolved in 1N NaOH (15 mL, 15 mmol). After the starting material has disappeared (by LCMS), the reaction is acidified with 1 N HCl to afford reference compound 12 as a white precipitate.

Reference Compound 13

<formula> formula see original document page 41 </formula>

The reagents and conditions in the above reaction for reference compound 13 are: a) MeSO 2 Cl 1 Pyridine, rt, THF; (b) 1N NaOH in water.

(3-Amino-phenyl) -acetic acid ethyl ester (0.895 g, 5.0 mmols) and (0.281 mL, 5.0 mmols) are added to a round base flask containing THF (20 mL) and pyridine. (0.60 mL). The mixture is stirred at room temperature overnight. The clear solution is diluted with EtOAc (200 mL), washed with 1N HCl (3 x 100 mL) and brine (100 mL), and dried with MgSO4. The solvent is evaporated in vacuo to afford intermediate 13-B as a white solid. Ethyl ester 13-B is saponified following methods analogous to those described for the preparation of reference compound 12 to provide reference compound 13.

Reference Compound 14

<formula> formula see original document page 41 </formula>

The reagents and conditions in the above reaction for reference compound 14 are: a) CH 3 ClOCl, Pyridine, DMAP, rt, THF; (b) 1N NaOH in water.

(3-Amino-phenyl) -acetic acid ethyl ester (0.895 g, 5.0 mmols) and acetyl chloride (0.26 mL, 5.0 mmols) are added to a round base flask containing THF ( 20 mL), pyridine (0.60 mL) and DMAP (10 mg). The mixture is stirred at room temperature overnight. The clear solution is diluted with EtOAc (200 mL), washed with 1N HCl (3 x 100 mL) and brine (100 mL), and dried with MgSO4. The solvent is evaporated in vacuo to afford intermediate 14-B as a white solid which is used without further purification. Ethyl ester 14-B is saponified following analogous methods to those described above to provide the reference compound 14.

Reference Compound 15

<formula> formula see original document page 42 </formula>

The reagents and conditions in the above reaction for reference compound 15 are: a) EtNH 2, CH 2 Cl 2, DMAP; (b) 1N NaOH in water.

(4-Chlorosulfonyl-phenyl) -acetic acid methyl ester (0.895 g, 5.0 mmols) and diethylamine (1 mL) are added to a round base flask containing CH 2 Cl 2 (20 mL) and DMAP (10 mg). The mixture is stirred at room temperature overnight. The clear solution is diluted with EtOAc (200 mL), washed with 1N HCl (3 x 100 mL) and brine (1 x 100 mL), and dried with MgSO4. The solvent is evaporated in vacuo to afford intermediate 15-B, which is purified from a reaction mixture using a silica column. Ethyl ester 15-B is saponified following analogous methods to those described above to provide the reference compound 15.

Reference Compound 16

<formula> formula see original document page 42 </formula>

The reagents and conditions in the above reaction for reference compound 16 are: a) 4-CI-PhB (OH) 2, C 2 H 4 Cl 2, Cu (OAc) 2, pyridine, molecular sieves 4A; (b) 1N NaOH in water.

mmol), 4-chlorophenyl boronic acid (755 mg, 4.84 mmol), pyridine (0.388 mL, 4.8 mmol) and copper (11) acetate (477 mg, 2.4 mmol) are added to a base vial. containing 1,2-dichloroethane (10 mL) and 4Å molecular sieves (300 mg). The mixture is stirred at room temperature overnight. The clear solution is diluted with EtOAc (200 mL), washed with 1N HCl (3 x 100 mL) and brine (100 mL), and dried with MgSO4. The solvent is evaporated in vacuo to afford intermediate 16-B, which is purified from a reaction mixture using a silica column. Methyl ester 16-B is saponified following analogous methods to those described above to provide reference compound 16. Example 1 <formula> formula see original document page 43 </formula> (3-Hydroxy-phenyl) -acetic acid methyl ester (0.268 g, 1.6

JT> ^ O 600

NH

/

^ 0 ^ O ^

Q \\} Q-

NH

1-G NH. 1-B: Fine powder KOH (35 g, 0.622 mol) is dissolved in DMSO and stirred at room temperature for 20 min and then cooled to 0 ° C. N-Cbz-trans-4-hydroxy-L-proline (Cbz-Hyp-OH, 1-A) (10 g, 43.3 mmol) is dissolved in DMSO (10 mL) and added, and the reaction mixture is stirred. for a further 10 minutes at 0 ° C. Then bromomethylcycloexane (33 g, 0.204 mol) is added, and the reaction mixture is stirred at 0 ° C for a further 15 minutes, after which point the ice bath is removed and the reaction mixture is allowed to warm to room temperature. at room temperature and stirred for 4 hours. The reaction mixture is poured into water (300 mL), and a reaction vessel is filled with an additional aliquot of water (300 mL). The combined aqueous layer is extracted with ether (2 x 300 mL) and discarded. The aqueous layer is acidified with 87% H3PO4 to pH 2.3, and then extracted with ether (3 x 300 mL). The combined ether extracts are washed with water (2 x 400 mL) and brine (2 x 400 mL), dried over MgSO4, filtered, and concentrated in vacuo. The residue is purified by silica gel chromatography to afford compound 1-B as a clear oil.

1-C: A 40 mL vial is charged with 1-B (250 mg, 0.695 mmol), reference compound 1-E (243 mg, 0.695 mmol, 1. eq), HATU (291 mg, 0.765 mmol, 1 0.1 eq), IPr2EtN (0.15 mL, 0.834 mmol, 1.2 eq) and CH2 Cl2 (4mL).

The reaction is stirred at room temperature for 1 hour. Volatile reagents are removed under reduced pressure, and the reaction mixture is dissolved in EtOAc. The organic layers are washed with NaHSO 4, water, saturated NaHCOs, and brine. The organic layers are then dried with MgSO 4, and the product purified from a reaction mixture by silica gel chromatography to provide compound 1-C which is used in the preparation of 1-D.

1-D: A Parr reaction vessel is loaded with 1-C (2.5 g, 3.6 mmol), Pd / C (3.6 g, 0.36 mmol, 1 eq.), T-BuOH ( 20 mL) and water (5 mL). The vessel is placed in a Parr apparatus and shaken for 18 hours under a pressure of 3.51 kg / cm2 (50 psi) of H2 gas. The reaction mixture is filtered through a pad of Celite to provide compound 1-D, which is used directly in the preparation of 1-E. 1-Ε: A 40 mL vial is charged with 1-D (100 mg, 0.18 mmol), 4-phenylbutyric acid (30 mg, 0.18 mmol, 1. eq), HATU (75 mg, 0, 20 mmol, 1.1 eq), IPr2EtN (0.04 mL, 0.22 mmol, 1.2 eq) and CH2 Cl2 (2mL). The reaction is stirred at room temperature for 1 hour. Volatile reagents are removed under reduced pressure and the reaction mixture is dissolved in EtOAc. The organic layers are washed with NaHS04, water, saturated NaHCO3, and brine. The organic layers are then dried with MgSO4, and the product is purified from a reaction mixture by silica gel 1-E chromatography, which is used in the preparation of 1-F.

1-F: Alcohol 1-E (127 mg, 0.18 mmol) is dissolved in DCM (2 mL) and Dess-Martin periodin (85 mg, 0.2 mmol) is added. The reaction mixture is stirred overnight at room temperature. The solvent is removed in vacuo, and the crude is purified by flash chromatography (4 g silica gel column) using an EtOAc: hexanes gradient to afford ketone 1-F as a white foam which is used in the preparation of 1 -G.

1-G: Ketone 1-F (128 mg, 0.18 mmol) is dissolved in DCM (1 mL) and 50% TFA in DCM (5 mL) is added. The reaction is stirred at room temperature for 2 hours and the solvent removed in vacuo. The crude material is purified by reverse phase HPLC, and the solvent is lyophilized to afford 1-G as a white powder.

Examples 2-7

Example 2 is prepared following methods analogous to those described for example 1, using 3-tert-butoxycarbonylamino-propionic acid to provide intermediate 2-E.

Example 3 is prepared following methods analogous to those described for example 1, using Reference Compound 13 to provide intermediate 3-E.

Example 4 is prepared following methods analogous to those described for example 1, using Reference Compound 14 to provide intermediate 4-E.

Example 5 is prepared following methods analogous to those described for example 1 using (4-ethanesulfonylamino-phenyl) -acetic acid (prepared in a manner analogous to that described for reference compound 13) to provide intermediate 5-E .

Example 6 is prepared following methods analogous to those described for example 1, using Reference Compound 12 to provide intermediate 6-E.

Example 7 is prepared following methods analogous to those described for example 1 using 4- (methanesulfonylamino) phenylacetic acid to provide intermediate 7-E.

Example 8

<formula> formula see original document page 46 </formula>

.8B: frans-4-hydroxyproline (Hyp-OH) (7.90 g, 60.1 mmol) and N- (allyloxycarbonyloxy) succinimide (12.0 g, 60.1 mmol) are added to a flask. round base containing THF (220 mL), triethylamine (21 mL, 150.3 mmol) and water (55 mL). The mixture is stirred at room temperature overnight. The clear solution is acidified with 1N HCl (100 mL) and extracted with EtOAc (4 x 150 mL). The combined organic layer is washed with brine (100 mL) and dried with MgSO4. The solvent is evaporated in vacuo to afford the desired product as a white solid, which is used without further purification.

8-C: This compound is prepared by alkylation of 8-B and 1-chloro-4-chloromethylbenzene using methods analogous to those described for the preparation of intermediate 1-B in Example 1.

8-D: This compound is prepared by Coupling HATUde 8-C and reference compound 1-E using methods analogous to those described for the preparation of intermediate 1-C in Example 1.

8-E: A 50 ml round base flask is charged with 8-D alcohol (780 mg, 1.16 mmol), Et 2 NH (1.8 mL, 17.43 mmol, 15 eq), Pd 2 (dba) 3. (55 mg, 0.06 mmol 0.05 eq), phosphine dppe ligand (26 mg, 0.06 mmol, 0.05 eq) and THF (5 mL). The reagents are stirred at room temperature for 15 minutes and then concentrated to dryness. The product (as a mixture of diastereomers) is purified from a reaction mixture by silica gel chromatography using 2-9% MeOH / CH 2 Cl 2 gradient. The 8-E amine is then used in the preparation of 8-F. 8-F: Following methods analogous to those described for the preparation of intermediate 1-E in Example 1, compound 8-F is prepared by coupling 8-E HATU and (4-ethanesulfonylamino-phenyl) -acetic acid (prepared from a analogous to that described for the preparation of 4- (methanesulfonylamino) phenylacetic acid, which was prepared using methods analogous to those described for the preparation of reference compound 13).

8-G: This compound is prepared by oxidation of 8-F using methods analogous to those described for the preparation of intermediate 1-F in Example 1.

8-H: This compound is prepared by deprotecting 8-G using methods analogous to those described for the preparation Example 1. Examples 9-30

Example 9 is prepared following methods analogous to those described for example 8, using Reference Compound 15 to provide intermediate 9-F. Example 10 is prepared following methods analogous to those described for example 8, using (4-methoxy-phenyl) -acetic acid to provide intermediate 10-F.

Example 11 is prepared following methods analogous to those described for example 8, using pyridin-4-yl acetic acid to provide intermediate 11-F.

Example 12 is prepared following methods analogous to those described for example 8, using (3-chloro-phenyl) -acetic acid to provide intermediate 12-F.

Example 13 is prepared following methods analogous to those described for example 8 using [4- (morpholine-4-sulfonyl) -phenyl] -acetic acid to provide intermediate 13-F.

Example 14 is prepared following methods analogous to those described for example 8 using (3,5-dichloro-phenyl) -acetic acid to provide intermediate 14-F.

Example 15 is prepared following methods analogous to those described for example 8, using (4-phenyl-phenyl) -acetic acid to provide intermediate 15-F.

Example 16 is prepared following methods analogous to those described for example 8 using (2-chloro-phenyl) -acetic acid to provide intermediate 16-F.

Example 17 is prepared following methods analogous to those described for example 8, using (3-phenoxy-phenyl) -acetic acid to provide intermediate 17-F.

Example 18 is prepared following methods analogous to those described for example 8, using (2-phenyl-thiazol-4-yl) -acetic acid to provide intermediate 18-F.

Example 19 is prepared following methods analogous to those described for example 8 using (4-phenylmethanesulfonylamino-phenyl) -acetic acid (prepared in a manner analogous to that described for the preparation of reference compound 13) to provide intermediate 19-F Example 20 is prepared by following methods analogous to those described for example 8 using [4- (piperidine-1-sulfonyl) phenyl] acetic acid to provide intermediate 20-F.

Example 21 is prepared following methods analogous to those described for example 8 using [3- (4-chloro-phenoxy) -phenyl] -acetic acid reference compound 16 to provide intermediate 21-F.

Example 22 is prepared following methods analogous to those described for example 8 using [4- (trifluoroethanesulfonylamino) phenyl] acetic acid (following methods analogous to those described for reference compound 13) to provide intermediate 22 -F.

Example 23: This compound is prepared from 1-Boc-piperidin-4-yl-propionic acid following methods analogous to those described for the preparation of Example 8.

Example 24: This compound is prepared from N-Boc-4-piperidin-4-yl-butyric acid following methods analogous to those described for the preparation of Example 8.

Example 25: This compound is prepared from 1-Boc-piperidin-4-yl-acetic acid following methods analogous to those described for the preparation of Example 8.

Example 26: This compound is prepared from 1-Boc-piperidin-3-yl-acetic acid following methods analogous to those described for the preparation of Example 8.

Example 27: This compound is prepared from 1-Boc-piperidin-3-yl-propionic acid following methods analogous to those described for the preparation of Example 8.

Examples 28-30 are prepared following methods analogous to those described for the preparation of Example 8. Example 31

<formula> formula see original document page 50 </formula>

.31-B: Fine powder KOH (19.4 g, 0.346 mol) is dissolved in DMSO and stirred at room temperature for 20 minutes and then cooled to 0 ° C. N-Boc-trans-4-hydroxy-L-proline (Boc-Hyp-OH, 1-A) (10g, 43.3 mmols) is dissolved in DMSO (10 mL) and added, and the reaction mixture is stirred for 2 hours. another 10 minutes at 0 ° C. Then 4-chlorobenzyl chloride (33 g, 0.204 mol) is added, and the reaction mixture is stirred at 0 ° C for an additional 15 minutes, after which point the ice bath is removed and the reaction mixture is allowed to warm to at room temperature and stirred for 4 hours. The reaction mixture is poured into water (300 mL), and a reaction vessel is rinsed with an additional aliquot of water (300 mL). The combined aqueous layer is extracted with ether (2 x 300 mL) and discarded. The aqueous layer is acidified with 87% H3PO4 to pH 2.3, and then extracted with ether (3 x 300 mL). The combined ether extracts are washed with water (2 x 400 mL) and brine (2 x 400 mL), dried over MgSO4, filtered and concentrated in vacuo. The residue is purified by chromatography on silica gel with EtOAc / Hexanes (0 to 100% gradient) to afford compound 1-B as a clear oil. MS m / z 256.1 (M + 1 - Boc); 1H NMR (DMSO-D6, 400 MHz) δ 7.39 - 7.31 (4H, m), 4.52 - 4.40 (2H, m), 4.16 - 4.10 (2H, m), 3.48 - 3.41 (2H, m), 2.40 - 2.30 (1H, m), 2.03 -1.94 (1H, m), 1.39 -1.34 (9H, m ). 31-C: A solution of (trimethylsilyl) diazomethane (2M in diethyl ether) (4.7 mL, 9.45 mmol) is added to 1-B carboxylic acid (2.4 g, 8.6 mmol) dissolved in DCM / MeOH (5: 1, 25 mL). When starting material is consumed as determined by LCMS, the reaction mixture is concentrated in vacuo, and the crude residue is purified by flash chromatography (EtOAc: Hexanes gradient) to afford the methyl ester as a clear oil. 31-D: A round base flask is charged with a stir bar and Ι- Ο (510 mg, 1.38 mmol). TFA (50%) in DCM (6 mL) is added and the solution is stirred for 1 hour at room temperature. The solvent is removed in vacuo, hexanes is added and then evaporated again in vacuo to dryness, and repeated if necessary to azeotrop the remaining TFA. The raw material is used directly in the next step without further purification.

31-E: This compound is prepared from 31-D and commercially available {2- [1- (tert-Butoxycarbonyl) piperidin-4-yl] -5-methyl-1,3-thiazol-4-yl} acetic acid using methods analogous to those described for the preparation of Example 1-E.

31-F: This compound is prepared from 31-E by saponification according to the methods described for the preparation of reference compound 6. 31-G: This compound is prepared from 31-F and reference compound 3 using methods analogous to those described for the preparation of Example 1-C.

31-H: This compound is prepared from 31-G using methods analogous to those described for the preparation of Example 1-F. Reference compound 31 is prepared from 31-H using methods analogous to those described for the preparation of Example 1-G. Examples 32 - 43

Example 32 is prepared from reference compound 10, following methods analogous to those described for the preparation of Example 31.

Example 33 is prepared following methods analogous to those described for the preparation of Example 31.

Example 34 is prepared from Reference Compound 11, following methods analogous to those described for the preparation of Example 8.

Example 35 is prepared from Reference Compound 8, following methods analogous to those described for the preparation of Example 8.

Example 36 is prepared from Reference Compound 6, following methods analogous to those described for the preparation of Example 8.

Example 37 is prepared from Reference Compound 2, following methods analogous to those described for the preparation of Example 8.

Example 38 is prepared from Reference Compound 2, following methods analogous to those described for the preparation of Example 8.

Example 39 is prepared from Reference Compound 2, following methods analogous to those described for the preparation of Example 8.

Example 40 is prepared from Reference Compound 5 and {2- [1- (tert-Butoxycarbonyl) piperidin-4-yl] -5-methyl-1,3-thiazol-4-yl} acetic acid following methods analogous to those described for Example 8.

Example 41 is prepared from Reference Compound 9 and {2- [1- (tert-Butoxycarbonyl) piperidin-4-yl] -5-methyl-1,3-thiazol-4-yl} acetic acid following methods analogous to those described for Example 8.

Example 42 is prepared from Reference Compound 7 and {2- [1- (tert-Butoxycarbonyl) piperidin-4-yl] -5-methyl-1,3-thiazol-4-yl} acetic acid following methods analogous to those described for Example 8.

Example 43 is prepared from Reference Compound 2 and {2- [1- (tert-Butoxycarbonyl) piperidin-4-yl] -5-methyl-1,3-thiazol-4-yl} acetic acid following methods analogous to those described for Example 8.

Table 1 shows the compounds of formula (1) as described in examples 1-42. Table 1

<table> table see original document page 53 </column> </row> <table> Example Structure <table> table see original document page 54 </column> </row> <table> <table> table see original document page 55 </column> </row> <table> <table> table see original document page 56 </column> </row> <table> <table> table see original document page 57 </column> </row> < table> <table> table see original document page 58 </column> </row> <table> <table> table see original document page 59 </column> </row> <table> <table> table see original document page 60 </column> </row> <table> <table> table see original document page 61 </column> </row> <table> <table> table see original document page 62 </column> </row> < table> <table> table see original document page 63 </column> </row> <table> <table> table see original document page 64 </column> </row> <table> <table> table see original document page 65 </column> </row> <table>

Examples 51 - 53

Examples 51-53 in Table 2 are exemplary compounds of the invention having Formula (1) comprising 3-alkyl or 3-aryl substituted prolines, which may be prepared by repeating the procedures described in the above examples using appropriate starting materials evident. for those skilled in the art.

Table 2

<table> table see original document page 65 </column> </row> <table> <table> table see original document page 66 </column> </row> <table> Assays

The suitability of a channel inhibitor activating protease such as a prostasin inhibitor for the treatment of a disease mediated by inhibition of a channel activating protease can be tested by determining the inhibitory effect of the inhibitor activating protease. (1) the isolated, purified, or recombinant native channel activator protease using a suitable biochemical assay format using the method described in Shipway et al; Biochem. Biophys. Res. Commun.2004; 324 (2): 953-63); and / or (2) the ion / ion channel transport function in suitable isolated cells or confluent epithelia, using the methods described in Bridges et al; Am. J. Physiol. Lung Cell Mol. Physiol. 2001; 281 (1): L16-23; and Donaldson et al. J. Biol. Chem. 2002; 277 (10): 8338-45. Biochemical Assays

Recombinant human prostasin and guinea pig prostasin and matriptase are generated according to the methods described in Shipway et al., Biochem. Biophys. Res. Commun. 2004; 324 (2): 953-63). Recombinant enzymes are incubated in an electrolyte buffer containing test compounds or vehicle in a suitable multi-well assay plate such as a 96- or 384-well plate. At a defined time after mixing the enzyme with the compound or vehicle, a suitable fluorescent peptide substrate is added to the assay mixture. When the substrate becomes cleaved by the active enzyme, fluorescence (measured using a suitable fluorescence plate reader) increases and the rate of substrate regeneration (ie enzyme activity) can be quantified, and thereby the inhibitory effect of any test compound. The efficacy of the test compounds is expressed as the concentration that induces a 50% attenuation in enzyme activity (Ki).

In general, compounds of the invention may have Ki values from 0.1 nM to 5 μΜ. In some examples, compounds of the invention may have Ki values from 0.1 nM to 500 nM; from 0.1 nM to 50 nM; from 0.1 nM to 5 nM; or from 0.1 nM to 0.5 nM. In particular examples, the compounds of the invention may have 0.1 nM to 0.5 nM Kide values; from 0.5 nM to 5 nM; from 5 nM to 50 nM; from 50 nM to 500 nM; or from 500 nM to 5 μΜ. However in other examples, compounds may have Ki values less than 0.1 nM or more than 5 μΜ.

Epithelial Ion Transport

Human bronchial epithelial cells are cultured according to the methods described in Danahay et al., Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 282 (2): L226-36). When properly differentiated epithelial cells (days 14-21 after stabilization of an apical interface) are treated with either vehicle, aprotinin (200 pg / ml) or test compound for 90 minutes. Epithelia are then placed inserted using chambers as described in Danahay et al., Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 282 (2): L226-36) maintaining the concentration of vehicle, aprotin or test compound on the apical side of the epithelia. Short circuit current (ISC) is then measured by voltage by clipping the epithelia to zero millivolts. Amiloride-sensitive SSI is then measured by the addition of amiloride (10 μΜ) to the apical surface of the epithelia. The potency of the test compound is expressed as the concentration that induces 50% inhibition of the total aprotinin-sensitive component of amiloride-sensitive ISC.

In general, the compounds of the invention may have IC 50 values from 1 nM to 10 μΜ. In some examples, compounds of the invention may have IC 50 values from 1 nM to 1 μΜ; or more particularly from 1 nM to 100 nM. However in other examples, the compounds of the invention may have IC 50 values from 100 nM to 1 μΜ, or from 1 μΜ to 10 μΜ. In still other examples, the compounds may have IC50 values less than 1 nM or more than 10 μΜ. Tracheal potential difference (in vivo)

Guinea pigs are anesthetized using short-acting inhalation anesthesia such as halothane and N2O. At the same time as under short-acting anesthesia, an oral gavage needle is inserted into the trachea through the oropharangeal routine. Once inside the trachea, a small volume (50-200 μΙ) of vehicle or test compound in a suitable aqueous based diluent is instilled into the airway. The animals then recover and become fully ambulatory. Alternatively, test compounds may be administered to animals using aerosol or dry powder dosage. At a set time after dosing, animals are surgically anesthetized using appropriate anesthesia such as ketamine and xylazine. The trachea is then exposed and a plastic electrode agar bridge is inserted into the tracheal lumen. A reference electrode is also inserted into the muscle layers in the animal's neck. The tracheal potential difference is then measured using a suitable high impedance voltmeter as described in Takahashi et al., Toxicol Appl Pharmacol. 1995; 131 (1): 31-6. The potency of the test compound is expressed as the dose that induces a 50% reduction in the sensitive component of the tracheal potential difference.

It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in view of this will be suggested to persons skilled in the art and should be included in the spirit and competence of this application and scope. of the attached claims. All publications, patents, and patent applications cited herein are hereby incorporated by Reference for all purposes.

Claims (27)

1. Compound of formula (1): <formula> formula see original document page 69 </formula> and pharmaceutically acceptable salts, hydrates, solvates and stereoisomers thereof, wherein J is a 5 to 12 membered monocyclic or fused carbocyclic ring, aryl, heteroaryl or heterocyclic ring containing Ν, O and / or S; R1 is - (CR2) i-NR2, - (CR2) i-NRC (= NR) -NR2, - (CR2), - C (= NR) -NR2 or a 5-7 membered non-aromatic nitrogen-containing heterocyclic ring ; W-R2 is a substituent at any position on ring A; W is either -O (CR2) k-, -S (CR2) k-, -S (O) (CR2) k-, -SO2 (CR2) k- or -OC (O) (CR2) k-; R2 is C1-6 alkyl, C2 alkenyl, C2 alkynyl R6, -CR4 9 = CR9-R6, or <Rule> where ring E is an optionally substituted monocyclic or fused 5- to 7-membered carbocyclic or heterocyclic ring; or W-R 2 together form C 1-6 alkyl, a 5- to 7-membered aryl or -OC (O) NR 7 R 8; R 3 is NR 7 R 8 or R 6; R4 and R5 are independently H, C1-6 alkyl, OH, or C1-4 alkoxy; R 7 and R 8 are independently H, C 1-6 alkyl, C 2-4 alkenyl, C 2-6 alkynyl or - (CR 2) 1 -R 6; or R 17 and R 18 together with N may form an optionally substituted 5 to 7 membered monocyclic or fused heterocyclic ring; R19 is H or C1-6 alkyl; R 10 is halo, C 1-6 alkyl, C 1-6 alkoxy, OR 11 or - (CR 2) 1 -R 11; R 6, R 11 and X are independently an optionally substituted 5 to 7 membered carbocyclic ring, heterocyclic ring, aryl or heteroaryl; or R11 is H or C1-6 alkyl; each R is H, or C 1-6 alkyl, C 2-6 alkenyl, or C 2-6 alkynyl, wherein a carbon may optionally be substituted or exchanged with NR, O or S; i is 0 to 1; k and 1 are independently 0 to 6; m and η are independently 1 to 6; and p is 0 to 3. A compound according to claim 1, wherein R 1 is - (CH 2) i -NH 2, - (CH 2) i -NHC (= NH) -NH 2 or - (CH 2) i C (= NH) -NH 2 NH 2 each I is 0 to 1; or R1 is piperidinyl. A compound according to claim 1 wherein W is -O (CR2) k-, -S (CR2) k-, -S (O) (CR2) k-, -SO2 (CR2) k- or - OC (O) (CR 2) 4 -; and k is 1. A compound according to claim 1, wherein R2 is an optionally substituted phenyl, C5-7 cycloalkyl, thienyl, furanyl, piperidinyl, methylene cyclohexyl, <formula> formula see original document page 70 </formula> A compound according to claim 1, wherein R 3 is an optionally substituted phenyl, pyridyl, thiazolyl, piperidinyl, or NR 7 R 8; wherein R 7 and R 8 are both H, or R 7 and R 8 together with N form an optionally substituted piperidinyl. A compound according to claim 1 wherein R, R 4, R 5, R 7 and R 8 are each H. A compound according to claim 1, wherein R 6 is an optionally substituted phenyl, C 3-7 cycloalkyl, pyridyl, thiazolyl, piperidinyl, cyclohexanol, imidazolyl, thienyl, furanyl, <formula> see original document page 70 > A compound according to claim 1, wherein X is cyclohexyl, phenyl or piperidinyl. A compound according to claim 1, wherein -J- (R 10) p at the same time is <formula> formula see original document page 71 </formula> Z is O or S; Z11 Z21 Z3 or Z4 are independently N, CH1 or C when bonded to R10; Z5, Z6 or Z7 are independently N, O, S1 CH1 or C when bonded to R10; R10 is C1-6 alkyl or - (CR2) 1-R11; R11 is phenyl or C5-7 cycloalkyl; and foot 0-1. A compound according to claim 1, wherein J is benzothiazolyl, benzoxazolyl, thiazolyl, or oxadiazolyl. A compound according to claim 1, wherein said compound is of formula (2A) or (2B): wherein Z is O or S; R1 is NH2, -NHC (= NH) -NH2 or -C (= NH) -NH2; W is -O (CH 2) k- or -S (O) (CH 2) k-; R 2 is an optionally substituted phenyl, or W-R 2 together form C 1-6 alkyl or an optionally substituted phenyl; R, R4 and R5 are independently H; Y is one of a 5- to 7-membered aryl, heteroaryl or heterocyclic ring containing Δ, O or S; R 12 is halo, C 1-6 alkyl or -L- (CH 2) 1 -R 13; L is a bond, O, SO 2, NHCO 1 NHSO 2 or SO 2 NH; R 13 is optionally halogenated C 1-6 alkyl, or an optionally substituted C 3-7 cycloalkyl, or a 5- to 7-membered aryl, heteroaryl or heterocyclic ring; i is 0; k is 1; l is 0 to 1; m and n are independently 1 to 4; and q is 0 to 3. A compound according to claim 11 wherein Y is phenyl, pyridyl, thiazolyl or piperidinyl. The compound of claim 11, wherein R 12 is -L- (CH 2) 1 -R 13; and R 13 is an optionally halogenated C 1-6 alkyl, C 3-7 cycloalkyl, morpholinyl, phenyl or piperidinyl. A pharmaceutical composition comprising a therapeutically effective amount of a compound as defined in claim 1, and a pharmaceutically acceptable excipient. A method of modulating a channel activating protease comprising administering to a system or an individual in need thereof a therapeutically effective amount of the compound as defined in claim 1, or pharmaceutically acceptable salts or pharmaceutical compositions thereof, thereby modulating said protease. channel activator. The method of claim 15, wherein said channel activating protease is prostasin, PRSS22, TMPRSS11 (e.g., TMPRSS11B, TMPRSS11E), TMPRSS2, TMPRSS3, TMPRSS4 (MTSP-2), matriptase (MTSP- 1), CAP2, CAP3, trypsin, cathepsin A, or neutrophil elastase. The method of claim 16, wherein said channel activating protease is prostasin. The method of claim 15, comprising administering the compound of claim 1, or pharmaceutically acceptable salts or pharmaceutical compositions thereof, to the cells or tissue system, or to a human or animal subject. The method of claim 18, wherein said cells are bronchial epithelial cells. A method for ameliorating a channel activating protease-mediated condition comprising administering to a system or individual in need of such treatment an effective amount of a compound as defined in claim 1, or pharmaceutically acceptable salts thereof. or pharmaceutical compositions thereof, and optionally in combination with a second therapeutic agent, thereby treating said condition. The method of claim 20, wherein said channel activating protease is prostasin, PRSS22, TMPRSS11 (e.g. TMPRSS11B, TMPRSS11E), TMPRSS2, TMPRSS3, TMPRSS4 (MTSP-2), matriptase ( MTSP-1), CAP2, CAP3, trypsin, cathepsin A, or neutrophil elastase. The method of claim 21, wherein said channel activating protease is prostasin. The method of claim 20, wherein said condition is associated with the movement of fluid through the ion transport epithelia or the accumulation of mucus and sputum in respiratory tissues, or a combination thereof. The method of claim 20, wherein said condition is cystic fibrosis, primary ciliary dyskinesia, lung carcinoma, chronic bronchitis, chronic obstructive pulmonary disease, asthma, or a respiratory tract infection. The method of claim 20, wherein said second therapeutic agent is an anti-inflammatory, bronchodilator, antihistamine, antitussive, antibiotic or DNase. Use of a compound as defined in claim 1, or pharmaceutically acceptable salts or pharmaceutical compositions thereof, and optionally in combination with a second therapeutic agent, to modulate a channel activating protease. Use of a compound as defined in claim 1, or pharmaceutically acceptable salts or pharmaceutical compositions thereof, and optionally in combination with a second therapeutic agent, in the manufacture of a medicament for treating a channel activating protease mediated condition.