USOO8686.184B2

(12) United States Patent (10) Patent No.: US 8,686,184 B2

FleurV et al. 45) Date of Patent: Apr. 1,9 2014

(54) NEPRILYSIN INHIBITORS 6,660,756 B2 12/2003 Challenger et al. 7.956,054 B2 6, 2011 Blair et al. (75) Inventors: Mstis R& d E"SS CA 2005/00148168,212,052 B2A1 7/20121/2005 CompereChoi et al. et al. (US); Roland Gendron, San Francisco, 2008/0269305 A1 10/2008 Allegretti et al. CA (US); Adam D. Hughes, Belmont, 2010.0113801 A1 5/2010 Hook et al. CA (US); Jane Schmidt, San Francisco, 2010/028614.6 A1 11/2010 Macket al. CA (US) 2010/0305131 A1 12/2010 Coppola et al. 2010/0305145 A1 12/2010 Coppola et al. 2011/0021527 A1 1, 2011 Fath tal. (73) Assignee: Theravance, Inc., South San Francisco, 2011, 0046397 A1 2/2011 As CA (US) 2011/O124695 A1 5/2011 Iwaki et al. - 2012/O122844 A1 5, 2012 Foo (*) Notice: Subject to any disclaimer, the term of this 2012/O122977 A1 5/2012 Coppola et al. patent is extended or adjusted under 35 U.S.C. 154(b) by 113 days. FOREIGN PATENT DOCUMENTS (21) Appl. No.: 13/475,017 EP O 751. 145 A2 1/1997 JP 55OOO355 * 1 198O 1-1. JP 2004256435 9, 2004 (22) Filed: May 18, 2012 WO WO99.5O249 A2 10, 1999 O O WO WO 2004/104001 A2 12/2004 (65) Prior Publication Data WO WO 2011/0887.97 A1 T 2011 US 2012/0308588 A1 Dec. 6, 2012 WO WO 2011/092.187 A1 8, 2011 Related U.S. Application Data OTHER PUBLICATIONS (60) Provisional application No. 61/491.746, filed on May Ha et al. Tetrahedron: Asymmetry, 1999, (10), 2327-2336.* 31, 2011. U.S. Appl. No. 13/325.425. Unpublished, Gendron et al. U.S. Appl. No. 13/325.453, Unpublished, Smith et al. (51) Int. Cl. U.S. Appl. No. 13/398,015, Unpublished, Fluery et al. C07C 229/34 (2006.01) A6 IK3I/85 (2006.01) (Continued) A 6LX3L/95 (2006.01) (52) U.S.USPC Cl...... s62444 sco,39: 548,253: 548/371.1. Primary Examiner Shailendra Kumar 548/377.1546/322:544/168; 544/278; 514/171 (74) Attorney, Agent, or Firm — Jeffrey A. Hagenah; Shelley 514/231.2: 514/260.1; 514/356; 514/381: Eberle 514/404: 514/406; 514/539; 514/563 (58) Field of Classification Search USPC ...... 562/444; 560/39: 548/253,371.1, (57) ABSTRACT 548/377.1:546/322:544/168,278: In one aspect, the invention relates to compounds having the 514/171,2312,260.1356,381,404,514/.406, 539,563 E.OUIa spect, p 9. See application file for complete search history. (56) References Cited R2 (I) U.S. PATENT DOCUMENTS R NH ( )a sasyR4 4,189.604 A 2, 1980 Umezawa et al. r as -Rs 4,206,232 A 6/1980 Ondetti et al. O O al 4,374,829 A 2f1983 Harris et al. eX 6 4.513,009 A 4, 1985 Roques et al. R 4,722,810 A 2/1988 Delaney et al. 4,906,615 A 3/1990 Berger R3 4,929,641 A 5/1990 Haslanger et al. 4,939,261 A 7, 1990 KSander 4,975.444 A 12/1990 Danilewicz et al. 1 2 3 4 5 6 5,021,430 A 6, 1991 KSander where R', Ri, R., a, R, R, and R are as defined in the 5,030,654 A 7, 1991 Barnish et al. specification, or a pharmaceutically acceptable salt thereof. 6. g A 1918: 5A, al These compounds have neprilysin inhibition activity. In 5.27996 A 6, 1993 KSander another aspect, the invention relates to pharmaceutical com 5,294.632 A 3, 1994 Erion et al. positions comprising Such compounds; methods of using SE A g s s aquevent et al.1 Such compounds; and process and intermediates for prepar 5,677.297 A 10/1997 Waldecket al. ing Such compounds. 5,977.075 A 11/1999 KSander et al. 6,297.269 B1 10/2001 Hulin et al. 6,602,866 B2 8/2003 Flynn et al. 25 Claims, No Drawings US 8,686,184 B2 Page 2

(56) References Cited International Search Report for PCT/US2012/038517 dated Jul 23, 2012. Lassiani et al., “Anthranilic acid based CCK antagonists: OTHER PUBLICATIONS Blocking the receptor with the same words of the endogenous ligand”. Bioorganic & Medicinal Chemistry, 17: 2336-2350 (2009). U.S. Appl. No. 13/398,027, Unpublished, Fluery et al. Lee et al., “Synthesis and biological evaluation of chromone Ksander et al., “Dicarboxylic acid dipeptide neutral endopeptidase carboxamides as calpain inhibitors'. Bioorganic & Medicinal Chem inhibitors”, Journal of Medicinal Chemistry, 38(10): 1689-1700 istry Letters, 15:2857-2860 (2005). (1995). U.S. Appl. No. 13/474.987. Unpublished, Gendron et al. Misawa et al., “Structure-based design of dipeptide derivatives for U.S. Appl. No. 13/475,076, Unpublished, Fluery et al. the human neutral endopeptidase'. Bioorganic & Medicinal Chem istry, 19: 5935-5947 (2011). * cited by examiner US 8,686,184 B2 1. 2 NEPRLYSIN INHIBITORS R" is selected from —C-alkyl, -C-7cycloalkyl; - Calkylene-COOR: NR'R'': NHC(O)O CROSS-REFERENCE TO RELATED Calkyl; NHC(O)R: NHC(O)NH R: phenyl sub stituted with 1 or -2 groups independently selected from halo, APPLICATIONS —O C-alkyl, -Co-alkylene-COOR', -SO.NH2, —C(O)NR'R'', and tetrazole; and—Coalkylene-Chet This application claims the benefit of U.S. Provisional eroaryl or a partially unsaturated —Co-alkylene-C-shetero Application No. 61/491,746, filedon May 31, 2011; the entire cycle both optionally substituted with 1-3 groups indepen disclosure of which is incorporated herein by reference. dently selected from halo, —OH, =O, —CHO. —Calkyl, CF, and COOR: BACKGROUND OF THE INVENTION 10 R is selected from H, halo, -OH, -Calkyl, -O- Calkyl, phenyl, and morpholine; 1. Field of the Invention R is Horhalo: The present invention relates to novel compounds having R'' and R' are independently selected from H, neprilysin-inhibition activity. The invention also relates to —Calkyl, -Calkylene-Colaryl, -C-alkylene pharmaceutical compositions comprising Such compounds, 15 Coheteroaryl, -C-cycloalkyl, -(CH2)2OCH, processes and intermediates for preparing Such compounds —Calkylene-OC(O)R', - Calkylene-NR'R'', and methods of using Such compounds to treat diseases Such —Calkylene-C(O)R'', —Co-alkylenemorpholine, as hypertension, heart failure, pulmonary hypertension, and —Calkylene-SO, C-alkyl, renal disease. 2. State of the Art Neprilysin (neutral endopeptidase, EC 3.4.24.11) (NEP), is an endothelial membrane bound Zn" metallopeptidase found in many organs and tissues, including the brain, kid neys, lungs, gastrointestinal tract, heart, and the peripheral vasculature. NEP degrades and inactivates a number of 25 endogenous peptides, such as enkephalins, circulating brady kinin, angiotensin peptides, and natriuretic peptides, the latter of which have several effects including, for example, vasodi lation and natriuresis/diuresis, as well as inhibition of cardiac hypertrophy and ventricular fibrosis. Thus, NEP plays an 30 important role in blood pressure homeostasis and cardiovas cular health. NEP inhibitors, such as thiorphan, candoxatril, and can doXatrilat, have been studied as potential therapeutics. Com pounds that inhibit both NEP and angiotensin-I converting 35 enzyme (ACE) are also known, and include omapatrilat, gem patrilat, and Sampatrilat. Referred to as vasopeptidase inhibi tors, this latter class of compounds is described in Roblet al. R" is selected from —C-alkyl, -O-C-alkyl, -C-7cy (1999) Exp. Opin. Ther. Patents 9(12): 1665-1677. cloalkyl, —O—C-7cycloalkyl, phenyl, -O-phenyl, 40 NR'R'', and -CH(NH)CHCOOCH: R* and Rare SUMMARY OF THE INVENTION independently selected from H. —Calkyl, and benzyl, or R'' and R'' are taken together as —(CH) , —C(O)— The present invention provides novel compounds that have (CH2)4-, or -(CH2). O-(CH2): ; R' is —C-alkyl been found to possess neprilysin (NEP) enzyme inhibition or -Co-alkylene-Coaryl; and R' is selected from activity. Accordingly, compounds of the invention are 45 —O Calkyl, -O-benzyl, and —NR'R'': expected to be useful and advantageous as therapeutic agents R’ is Horistaken together with R' to form—CR'R' for treating conditions such as hypertension and heart failure. is taken together with R' to form - C(O) : R* and Rare One aspect of the invention relates to a compound of for independently selected from H. —Calkyl, and —O— mula I: Cacycloalkyl, or R'' and R are taken together to 50 form —O; R" is selected from Calkyl, —C-alkylene (I) COOR', phenyl, and pyridine: R2 R" is selected from —C-alkyl, -C-alkylene H R4 COOR', phenyl, and thiophene-COOR', where thiophene R N 55 is optionally substituted with CH: ( ) SS% R" is selected from H, -OH, - OC(O)R', HR: -CHCOOH, -O-benzyl, pyridyl, and —OC(S)NR'R'': O O 2a R' is selected from H. —C-alkyl, -Co-oaryl, —OCH2— R6 Coaryl, —CHO Coaryl, and —NR'R'': 60 R' is selected from H, Calkyl, and C(O)R'; R' is selected from H. —Calkyl, -C,cycloalkyl, -Caryl, and —Coheteroaryl; where: R', R, R, and Rare independently Hor-Calkyl: R is OR or NRR79; or a pharmaceutically acceptable salt thereof. R is Hor OR; 65 Another aspect of the invention relates to pharmaceutical R is selected from H. Cl, F, —CH, and CF; compositions comprising a pharmaceutically acceptable car a is 0 or an integer from 1 to 3: rier and a compound of the invention. Such compositions may US 8,686,184 B2 3 4 optionally contain other therapeutic agents. Accordingly, in process of preparing compounds of formula I, comprising the yet another aspect of the invention, a pharmaceutical compo steps of: sition comprises a compound of the invention as the first (a) coupling compound 1 with compound 2: therapeutic agent, one or more secondary therapeutic agent, and a pharmaceutically acceptable carrier. Another aspect of 5 the invention relates to a combination of active agents, com R2 prising a compound of the invention and a second therapeutic R4 agent. The compound of the invention can be formulated R NH2 HO ( )a sasy together or separately from the additional agent(s). When as formulated separately, a pharmaceutically acceptable carrier 10 O -- rO G-R. O may be included with the additional agent(s). Thus, yet 2. another aspect of the invention relates to a combination of pharmaceutical compositions, the combination comprising: a first pharmaceutical composition comprising a compound of 15 the invention and a first pharmaceutically acceptable carrier, (1) and a second pharmaceutical composition comprising a sec ond therapeutic agent and a second pharmaceutically accept able carrier. In another aspect, the invention relates to a kit (b) coupling compound 1 with compound 3 to form com containing Such pharmaceutical compositions, for example pound 4: where the first and second pharmaceutical compositions are separate pharmaceutical compositions. L Compounds of the invention possess NEP enzyme inhibi tion activity, and are therefore expected to be useful as thera 25 peutic agents for treating patients Suffering from a disease or lrts --R -e- disorder that is treated by inhibiting the NEP enzyme or by (1) + ". )a Nea increasing the levels of its peptide Substrates. Thus, one O aspect of the invention relates to a method of treating patients suffering from a disease or disorder that is treated by inhib (3) iting the NEP enzyme, comprising administering to a patient 30 L a therapeutically effective amount of a compound of the invention. Another aspect of the invention relates to a method orts --R of treating hypertension, heart failure, or renal disease, com )a N-al s prising administering to a patient a therapeutically effective 35 amount of a compound of the invention. Still another aspect O R6 of the invention relates to a method for inhibiting a NEP enzyme in a mammal comprising administering to the mam mal, a NEP enzyme-inhibiting amount of a compound of the invention. 40 (4) Since compounds of the invention possess NEP inhibition activity, they are also useful as research tools. Accordingly, one aspect of the invention relates to a method of using a compound of the invention as a research tool, the method 45 comprising conducting a biological assay using a compound where L is a leaving group (e.g., halo), and reacting com of the invention. Compounds of the invention can also be used pound 4 with compound 5: to evaluate new chemical compounds. Thus another aspect of the invention relates to a method of evaluating a test com pound in a biological assay, comprising: (a) conducting a 50 biological assay with a test compound to provide a first assay (5) value; (b) conducting the biological assay with a compound of the invention to provide a second assay value; wherein step (a) is conducted either before, after or concurrently with step (b); and (c) comparing the first assay value from step (a) with 55 the second assay value from Step (b). Exemplary biological assays include a NEP enzyme inhibition assay. Still another aspect of the invention relates to a method of studying a biological system or sample comprising a NEP enzyme, the method comprising: (a) contacting the biological system or 60 sample with a compound of the invention; and (b) determin in a palladium-catalyzed coupling reaction, where each R is ing the effects caused by the compound on the biological independently selected from halo. —O—Calkyl, system or sample. - Co-alkylene-COOR', -SONH, C(O)NR'R'', and Yet another aspect of the invention relates to processes and 65 tetrazole; or intermediates useful for preparing compounds of the inven (c) reacting compound 3 with compound 5 to form com tion. Accordingly, another aspect of the invention relates to a pound 6 in a palladium-catalyzed coupling reaction: US 8,686,184 B2 6 When a specific number of carbon atoms is intended for a (6) particular term used herein, the number of carbon atoms is shown preceding the term as Subscript. For example, the term "—Calkyl means an alkyl group having from 1 to 6 car HO () 17s. bon atoms, and the term “—C,cycloalkyl means a cycloalkyl group having from 3 to 7 carbon atoms, respec 1 Nee tively, where the carbon atoms are in any acceptable configu O / ration. The term “alkylene' means a divalent saturated hydrocar 10 bon group that may be linear or branched. Unless otherwise defined. Such alkylene groups typically contain from 0 to 10 and coupling compound 6 with compound 1; and carbon atoms and include, for example, —Co-alkylene-, (d) optionally deprotecting the product of step (a) or (b) or —Co-alkylene-, and —C-alkylene-. Representative alky (c), to produce a compound of formula I or a pharmaceuti lene groups include, by way of example, methylene, ethane cally acceptable salt thereof; where R', R. R., a, R. R. and 15 1,2-diyl (“ethylene'), propane-1,2-diyl, propane-1,3-diyl. Rare as defined for formula I. Another aspect of the inven butane-1,4-diyl, pentane-1,5-diyl and the like. It is under tion relates to a process of preparing a pharmaceutically stood that when the alkylene term include Zero carbons such acceptable salt of a compound of formula I, comprising con as —Co-alkylene-, such terms are intended to include the tacting a compound of formula I infree acid or base form with absence of carbon atoms, that is, the alkylene group is not a pharmaceutically acceptable base or acid. In other aspects, present except for a covalent bond attaching the groups sepa the invention relates to products prepared by any of the pro rated by the alkylene term. cesses described herein, as well as novel intermediates used The term “aryl means a monovalent aromatic hydrocar in Such process. In one aspect of the invention novel interme bon having a single ring (i.e., phenyl) or one or more fused diates have formula VI, as defined herein. rings. Fused ring systems include those that are fully unsat Yet another aspect of the invention relates to the use of a 25 urated (e.g., naphthalene) as well as those that are partially compound of formula I or a pharmaceutically acceptable salt unsaturated (e.g., 1.2.3,4-tetrahydronaphthalene). Unless thereof, for the manufacture of a medicament, especially for otherwise defined. Such aryl groups typically contain from 6 the manufacture of a medicament useful for treating hyper to 10 carbon ring atoms and include, for example, tension, heart failure, or renal disease. Another aspect of the —Coaryl. Representative aryl groups include, by way of invention relates to use of a compound of the invention for 30 example, phenyl and naphthalene-1-yl, naphthalene-2-yl, inhibiting a NEP enzyme in a mammal. Still another aspect of and the like. the invention relates to the use of a compound of the invention The term "cycloalkyl means a monovalent saturated car as a research tool. Other aspects and embodiments of the bocyclic hydrocarbon group. Unless otherwise defined. Such invention are disclosed herein. cycloalkyl groups typically contain from 3 to 10 carbonatoms 35 and include, for example, —C-scycloalkyl, —Clicy DETAILED DESCRIPTION OF THE INVENTION cloalkyl and —C-7cycloalkyl. Representative cycloalkyl groups include, by way of example, cyclopropyl, cyclobutyl, Definitions cyclopentyl, cyclohexyl and the like. The term "halo' means fluoro, chloro, bromo and iodo. When describing the compounds, compositions, methods 40 The term "heterocycle' is intended to include monovalent and processes of the invention, the following terms have the unsaturated (aromatic) heterocycles having a single ring or following meanings unless otherwise indicated. Additionally, two fused rings as well as monovalent Saturated and partially as used herein, the singular forms “a” “an and “the' include unsaturated groups having a single ring or multiple con the corresponding plural forms unless the context of use densed rings. The heterocycle ring can contain from 3 to 15 clearly dictates otherwise. The terms “comprising”, “includ 45 total ring atoms, of which 1 to 14 are ring carbon atoms, and ing.” and "having are intended to be inclusive and mean that 1 to 4 are ring heteroatoms selected from nitrogen, oxygen or there may be additional elements other than the listed ele Sulfur. Typically, however, the heterocycle ring contains from ments. All numbers expressing quantities of ingredients, 3 to 10 total ring atoms, of which 1 to 9 are ring carbonatoms, properties such as molecular weight, reaction conditions, and and 1 to 4 are ring heteroatoms. The point of attachment is at so forth used herein are to be understood as being modified in 50 any available carbon or nitrogen ring atom. Exemplary het all instances by the term “about unless otherwise indicated. erocycles include, for example, —Cheterocycle, Accordingly, the numbers set forth herein are approximations —C-sheterocycle. —Cheterocycle, —Cheterocycle, that may vary depending upon the desired properties sought —Cs-cheterocycle. —Coheterocycle, —Cheterocycle, to be obtained by the present invention. At least, and not as an and —Cheterocyle. attempt to limit the application of the doctrine of equivalents 55 Monovalent unsaturated heterocycles are also commonly to the scope of the claims, each number should at least be referred to as "heteroaryl groups. Unless otherwise defined, construed in light of the reported significant digits and by heteroaryl groups typically contain from 5 to 10 total ring applying ordinary rounding techniques. atoms, of which 1 to 9 are ring carbon atoms, and 1 to 4 are The term “alkyl means a monovalent saturated hydrocar ring heteroatoms, and include, for example, —Coheteroaryl bon group which may be linear or branched. Unless otherwise 60 and —Csoheteroaryl. Representative heteroaryl groups defined. Such alkyl groups typically contain from 1 to 10 include, by way of example, pyrrole (e.g., 3-pyrrolyl and carbon atoms and include, for example, —Calkyl, 2H-pyrrol-3-yl), imidazole (e.g., 2-imidazolyl), furan (e.g., —Csalkyl, —C-salkyl, -Calkyl, and —Coalkyl. 2-furyland 3-furyl), thiophene (e.g.,2-thienyl), triazole (e.g., Representative alkyl groups include, by way of example, 1,2,3-triazolyl and 1,2,4-triazolyl), pyrazole (e.g., 1H-pyra methyl, ethyl, n-propyl, isopropyl. n-butyl, S-butyl, isobutyl, 65 Zol-3-yl), oxazole (e.g., 2-oxazolyl), isoxazole (e.g., 3-isox t-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl azolyl), thiazole (e.g., 2-thiazolyl and 4-thiazolyl), and and the like. isothiazole (e.g., 3-isothiazolyl), pyridine (e.g., 2-pyridyl, US 8,686,184 B2 7 8 3-pyridyl, and 4-pyridyl), pyridylimidazole, pyridyltriazole, used in the invention. For example, the term “pharmaceuti pyrazine, pyridazine (e.g., 3-pyridazinyl), (e.g., cally acceptable carrier refers to a material that can be incor 2-pyrimidinyl), tetrazole, triazine (e.g., 1,3,5-triazinyl), porated into a composition and administered to a patient indolyle (e.g., 1H-indol-2-yl, 1H-indol-4-yl and 1H-indol-5- without causing unacceptable biological effects or interacting yl), benzofuran (e.g., benzofuran-5-yl), benzothiophene in an unacceptable manner with other components of the (e.g., benzobthien-2-yl and benzob thien-5-yl), benzimi composition. Such pharmaceutically acceptable materials dazole, benzoxazole, benzothiazole, benzotriazole, quinoline typically have met the required standards of toxicological and (e.g., 2-quinolyl), isoquinoline, quinazoline, quinoxaline and manufacturing testing, and include those materials identified the like. as suitable inactive ingredients by the U.S. Food and Drug 10 administration. Monovalent saturated heterocycles typically contain from The term “pharmaceutically acceptable salt' means a salt 3 to 10 total ring atoms, of which 2 to 9 are ring carbon atoms, prepared from a base or an acid which is acceptable for and 1 to 4 are ring heteroatoms, and include, for example administration to a patient, such as a mammal (for example, —C-sheterocycle. Representative monovalent saturated het salts having acceptable mammalian safety for a given dosage erocycles include, by way of example, monovalent species of 15 regime). However, it is understood that the salts covered by pyrrolidine, imidazolidine, pyrazolidine, piperidine, 1,4-di the invention are not required to be pharmaceutically accept oxane, morpholine, thiomorpholine, piperazine, 3-pyrroline able salts, such as salts of intermediate compounds that are and the like. In some instances, moieties may be described as not intended for administration to a patient. Pharmaceutically being taken together to form a saturated —Cheterocycle acceptable salts can be derived from pharmaceutically optionally containing an oxygen atom in the ring. Such acceptable inorganic or organic bases and from pharmaceu groups include: tically acceptable inorganic or organic acids. In addition, when a compound of formula I contains both a basic moiety, Such as an amine, pyridine or imidazole, and an acidic moiety Such as a carboxylic acid or tetrazole, Zwitterions may be 25 formed and are included within the term "salt as used herein. Salts derived from pharmaceutically acceptable inorganic bases include ammonium, calcium, copper, ferric, ferrous, lithium, , manganic, manganous, potassium, /o Sodium, and salts, and the like. Salts derived from phar 30 maceutically acceptable organic bases include salts of pri mary, secondary and tertiary amines, including Substituted amines, cyclic amines, naturally-occurring amines and the like, such as arginine, betaine, , choline, N,N'-diben // Zylethylenediamine, diethylamine, 2-diethylaminoethanol, 35 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glu cosamine, histidine, hydrabamine, isopropylamine, lysine, Representative monovalent partially unsaturated hetero methylglucamine, morpholine, piperazine, piperadine, cycles include, by way of example, pyran, benzopyran, ben polyamine resins, procaine, , , triethy 40 lamine, trimethylamine, tripropylamine, tromethamine and Zodioxole (e.g., benzo 1.3dioxol-5-yl), tetrahydropy the like. Salts derived from pharmaceutically acceptable inor ridazine, and 2,5-dihydro-1H-pyrrole. In some instances, ganic acids include salts of boric, carbonic, hydrohalic (hy moieties may be described as being taken together to form a drobromic, hydrochloric, hydrofluoric or hydroiodic), nitric, partially unsaturated —Csheterocycle. Such groups phosphoric, sulfamic and sulfuric acids. Salts derived from include: 45 pharmaceutically acceptable organic acids include salts of aliphatic hydroxyl acids (for example, citric, gluconic, gly colic, lactic, lactobionic, malic, and tartaric acids), aliphatic monocarboxylic acids (for example, acetic, butyric, formic, propionic and trifluoroacetic acids), amino acids (for 50 example, aspartic and glutamic acids), aromatic carboxylic acids (for example, benzoic, p-chlorobenzoic, diphenylace 7. tic, gentisic, hippuric, and triphenylacetic acids), aromatic hydroxyl acids (for example, o-hydroxybenzoic, p-hydroxy benzoic, 1-hydroxynaphthalene-2-carboxylic and 3-hydrox 55 ynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic The term “optionally substituted” means that group in acids (for example, fumaric, maleic, oxalic and Succinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, question may be unsubstituted or it may be substituted one or pamoic, pantothenic, Sulfonic acids (for example, benzene several times, such as 1 to 3 times or 1 to 5 times. For example, Sulfonic, camphoSulfonic, edisylic, ethanesulfonic, a phenyl group that is “optionally substituted with halo 60 isethionic, methanesulfonic, naphthalenesulfonic, naphtha atoms, may be unsubstituted, or it may contain 1, 2, 3, 4, or 5 lene-1,5-disulfonic, naphthalene-2,6-disulfonic and p-tolu halo atoms. enesulfonic acids), Xinafoic acid, and the like. As used herein, the phrase “having the formula' or “having As used herein, the term “prodrug is intended to mean an the structure' is not intended to be limiting and is used in the inactive (or significantly less active) precursor of a drug that same way that the term “comprising is commonly used. 65 is converted into its active form in the body under physiologi The term “pharmaceutically acceptable” refers to a mate cal conditions, for example, by normal metabolic processes. rial that is not biologically or otherwise unacceptable when The term is also intended to include certain protected deriva US 8,686,184 B2 10 tives of compounds of formula I that may be made prior to a final deprotection stage. Such compounds may not possess (I) pharmacological activity at NEP, but may be administered orally or parenterally and thereafter metabolized in the body R4 to form compounds of the invention which are pharmacologi S cally active at NEP. Thus, all protected derivatives and pro %. R5 drugs of compounds formula I are included within the scope 2a of the invention. Prodrugs of compounds of formula I having R6 a free carboxyl group can be readily synthesized by tech niques that are well known in the art. These prodrug deriva 10 tives are then converted by Solvolysis or under physiological conditions to be the free carboxyl compound. Exemplary or a pharmaceutically acceptable salt thereof. prodrugs include esters such as Calkylesters and aryl As used herein, the term “compound of the invention s Calkylesters. In one embodiment, the compounds of for 15 includes all compounds encompassed by formula I Such as mula I have a free carboxyl and the prodrug is an ester deriva the species embodied in formulas Ia-Ib, II, IIa-II, III, IV. V. tive thereof, i.e., the prodrug is an ester Such as —C(O) Va-V, as well as the compounds encompassed by formula VI. OCHCH In addition, the compounds of the invention may also contain The term “therapeutically effective amount’ means an several basic or acidic groups (for example, amino or car amount sufficient to effect treatment when administered to a boxyl groups) and therefore, Such compounds can exist as a patient in need thereof, that is, the amount of drug needed to free base, free acid, or in various salt forms. All such salt obtain the desired therapeutic effect. For example, a thera forms are included within the scope of the invention. Further peutically effective amount for treating hypertension is an more, the compounds of the invention may also exist as pro amount of compound needed to, for example, reduce, Sup drugs. Accordingly, those skilled in the art will recognize that press, eliminate, or prevent the symptoms of hypertension, or 25 reference to a compound herein, for example, reference to a to treat the underlying cause of hypertension. In one embodi “compound of the invention' or a “compound of formula I ment, a therapeutically effective amount is that amount of includes a compound of formula I as well as pharmaceutically drug needed to reduce blood pressure or the amount of drug acceptable salts and prodrugs of that compound unless oth needed to maintain normal blood pressure. On the other hand, 30 erwise indicated. Further, the term "or a pharmaceutically the term “effective amount’ means an amount sufficient to acceptable salt and/or prodrug thereof is intended to include obtain a desired result, which may not necessarily be a thera all permutations of salts and prodrugs, such as a pharmaceu peutic result. For example, when studying a system compris tically acceptable salt of a prodrug. Furthermore, solvates of ing a NEP enzyme, an “effective amount” may be the amount compounds of formula I are included within the scope of this needed to inhibit the enzyme. 35 invention. The term “treating or “treatment” as used herein means The compounds of formula I may contain one or more the treating or treatment of a disease or medical condition chiral centers and therefore, these compounds may be pre (such as hypertension) in a patient, such as a mammal (par pared and used in various stereoisomeric forms. Accordingly, ticularly a human) that includes one or more of the following: the invention also relates to racemic mixtures, pure stereoi 40 Somers (e.g., enantiomers and diastereoisomers), Stereoiso (a) preventing the disease or medical condition from occur mer-enriched mixtures, and the like unless otherwise indi ring, i.e., preventing the reoccurrence of the disease or medi cated. When a chemical structure is depicted herein without cal condition or prophylactic treatment of a patient that is any stereochemistry, it is understood that all possible stere pre-disposed to the disease or medical condition; (b) amelio oisomers are encompassed by Such structure. Thus, for rating the disease or medical condition, i.e., eliminating or 45 example, the terms “compound of formula I.” “compounds of causing regression of the disease or medical condition in a formula II, and so forth, are intended to include all possible patient; (c) Suppressing the disease or medical condition, i.e., Stereoisomers of the compound. Similarly, when a particular slowing or arresting the development of the disease or medi stereoisomer is shown or named herein, it will be understood cal condition in a patient; or (d) alleviating the symptoms of by those skilled in the art that minor amounts of other stere the disease or medical condition in a patient. For example, the 50 oisomers may be present in the compositions of the invention term “treating hypertension” would include preventing unless otherwise indicated, provided that the utility of the hypertension from occurring, ameliorating hypertension, composition as a whole is not eliminated by the presence of Suppressing hypertension, and alleviating the symptoms of such other isomers. Individual stereoisomers may be obtained hypertension (for example, lowering blood pressure). The by numerous methods that are well known in the art, includ term “patient' is intended to include those mammals, such as 55 ing chiral chromatography using a suitable chiral stationary humans, that are in need of treatment or disease prevention or phase or support, or by chemically converting them into dias that are presently being treated for disease prevention or tereoisomers, separating the diastereoisomers by conven treatment of a specific disease or medical condition, as well as tional means such as chromatography or recrystallization, test Subjects in which compounds of the invention are being then regenerating the original stereoisomer. evaluated or being used in an assay, for example an animal 60 Additionally, where applicable, all cis-trans or E/Z isomers model. (geometric isomers), tautomeric forms and topoisomeric forms of the compounds of the invention are included within All other terms used herein are intended to have their the scope of the invention unless otherwise specified. More ordinary meaning as understood by those of ordinary skill in specifically, compounds of formula I contain at least one the art to which they pertain. 65 chiral center when R is H, and contain at least two chiral In one embodiment, the invention relates to compounds of centers when R is —OR''. These chiral centers are indicated formula I: by the symbols * and * in the following formulas Ia and Ib: US 8,686,184 B2 11 12

(Ia) H OR20 (Ib-1) R :::::: N )a H. E H 4 and RS 2N-N-N-( a sé O O rá.al i 1 -R O O 2a R6 R3 (Ib) 10 R3 OR20 H N In this embodiment, compounds have the (R,R) configuration at the * and ** carbon atoms or are enriched in a stereoiso O O 15 meric form having the (R,R) configuration at these carbon atoms. In another stereoisomer of the compound of formula Ib, both carbonatoms identified by the * and ** symbols have the (S) configuration. This embodiment of the invention is shown in formula Ib-2:

(Ib-2) In one stereoisomer of the compounds of formula Ia, the carbon atom identified by the ** symbol has the (R) configu 25 ration. This embodiment of the invention is shown informula Ia-1:

(Ia-1) 30

R -N - is -s%SS O O 2a 35 In this embodiment, compounds have the (S,S) configuration at the * and ** carbon atoms or are enriched in a stereoiso meric form having the (S,S) configuration at these carbon atoms. In yet another Stereoisomer of the compound of for 40 mula Ib, the carbon atom identified by the symbol * has the (S) configuration and the carbon atom identified by the sym In this embodiment, compounds have the (R) configuration at bol ** has the (R) configuration. This embodiment of the the ** carbon atom or are enriched in a stereoisomeric form invention is shown in formula Ib-3: having the (R) configuration at this carbon atom. In another Stereoisomer of the compounds of formula Ia, the carbon 45 atom identified by the ** symbol has the (S) configuration. (Ib-3) This embodiments of the invention is shown in formula Ia-2: H )a 50 (Ia-2) i H R4 R! iss N ( )a sa 3.1 %. R5 O O 2a 55 R6

In this embodiment, compounds have the (S,R) configuration 60 In this embodiment, compounds have the (S) configuration at at the * and ** carbon atoms or are enriched in a stereoiso the ** carbon atom or are enriched in a stereoisomeric form meric form having the (S,R) configuration at these carbon having the (S) configuration at this carbon atom. atoms. In still another stereoisomer of the compound of for In one stereoisomer of the compound of formula Ib, both mula Ib, the carbon atom identified by the symbol * has the carbonatoms identified by the * and ** symbols have the (R) 65 (R) configuration and the carbon atom identified by the sym configuration. This embodiment of the invention is shown in bol ** has the (S) configuration. This embodiment of the formula Ib-1: invention is shown in formula Ib-4: US 8,686,184 B2 13 14 alkylene-NR'R''. Calkylene-C(O)R''. Co-alkylen

(Ib-4) emorpholine. —Calkylene-SO Calkyl,

R16, 2 and

O o O 10 Y O In this embodiment, compounds have the (R.S) configuration at the * and ** carbon atoms or are enriched in a stereoiso O meric form having the (R.S) configuration at these carbon 15 atOmS. The compounds of the invention, as well as those com O; pounds used in their synthesis, may also include isotopically labeled compounds, that is, where one or more atoms have been enriched with atoms having an atomic mass different from the atomic mass predominately found in nature. where R' is selected from —Coalkyl, -O-C-calkyl, Examples of isotopes that may be incorporated into the com —C-cycloalkyl, -O-C cycloalkyl, phenyl, -O-phe pounds of formula I, for example, include, but are not limited nyl, NR'R'', and -CH(NH)CHCOOCH: R'' and R' to, H, H, C, C, SN, O, 7.O., SS, C1, and F. Of are independently selected from H. —Calkyl, and benzyl, particular interest are compounds of formula I enriched in 25 or R'' and R'aretaken togetheras —(CH2). , —C(O)— tritium or carbon-14 which can be used, for example, in tissue distribution studies; compounds of formula I enriched in deu (CH) , or -(CH), O-(CH.) : R is —C-alkyl terium especially at a site of metabolism resulting, for or -Co-alkylene-Coaryl; and R'' is selected from example, in compounds having greater metabolic stability; - O Calkyl, -O-benzyl, and—NR'R''. The R' moi and compounds of formula I enriched in a positron emitting 30 ety is selected from H, -OH, - OC(O)R', -CHCOOH, isotope, such as 'C, F, 'O and 'N, which can be used, for -O-benzyl, pyridyl, and OC(S)NR'R'': where R is example, in Positron Emission Topography (PET) studies. Selected from H. —Calkyl, -Caryl, —OCH The nomenclature used herein to name the compounds of Coaryl, -CHO Coaryl, and—NR'R'; and Rand the invention is illustrated in the Examples herein. This Rare independently H or - Calkyl. The R' moiety is nomenclature has been derived using the commercially avail 35 selected from H. —Calkyl, and —C(O)R'; where R' is able AutoNom software (MDL, San Leandro, Calif.). selected from H. —Calkyl, —C,cycloalkyl, -Co-oaryl, and —Cheteroaryl. Representative Embodiments In one embodiment, R' is selected from —OR'' and - NR'R'': where R' is Hor-Calkyl (e.g., -CHCH The following substituents and values are intended to pro 40 —CH(CH), —(CH2)CH, -(CH2)CH, and vide representative examples of various aspects and embodi -(CH2)CH); R' is OH; and R' is H. ments of the invention. These representative values are In one embodiment, R' is selected from —OR" and intended to further define and illustrate such aspects and NRR79, where R is H, R is Hor OH, and R7 is H. embodiments and are not intended to exclude other embodi In another embodiment, R is OR', where R' is ments or to limit the scope of the invention. In this regard, the 45 selected from —C-alkyl (e.g., —CHCH, -(CH2)CH, representation that a particular value or Substituent is pre —CH(CH), —CH2CH(CH), —(CH2)CH, -(CH2) ferred is not intended in any way to exclude other values or CH, and —(CH2)2CHCCH)), —C-alkylene-Caryl Substituents from the invention unless specifically indicated. (e.g., benzyl), —C-alkylene-Coheteroaryl, —C,cy In one embodiment, this invention relates to compounds of cloalkyl, -(CH2)2OCH (e.g., -(CH2)2OCH and formula I: 50 -(CH2)2O2CH), —Coalkylene-OC(O)R' (e.g., —CH OC(O)CH, CH, OC(O)CHCH, and —CH, OC(O)CCHCH), —Calkylene-NR'R' (e.g., (I) —(CH2) N(CH), R2 R ( )a s/ s O O 2é--Rs 60 x-O and

The R' moiety is OR' or NR'R''. The R' moiety is selected from H. —Calkyl, —C-alkylene-Coaryl, 65 —C-alkylene-Cheteroaryl, —C-cycloalkyl, -(CH2)2O), CH, -C-calkylene-OC(O)R', -C, US 8,686,184 B2 15 16 —Calkylene-C(O)R' (e.g., —CHC(O)OCH, —CHC (O)O-benzyl, —CHC(O) N(CH), and

10 such as —O CH-5-methyl-1,3dioxol-2-one: —Co-alkylenemorpholine. —Calkylene-SO Calkyl (e.g., -(CH2)2SOCH), R1 = CH3, 15 or-( ty , and “Y”

“Y” making the compound a medoxomil ester. O The R moiety is H or -OR. The R moiety is H or is taken together with R' to form –CR'R' or is taken 25 together with R' to form - C(O) ; where R and Rare independently selected from H. —Calkyl, and —O— C-cycloalkyl, or R'' and R are taken together to form =O. In one embodiment, R is H. In another embodi ment, R is OR', where R is H. 30 When R is —OR'' and R' is taken together with R' to form—CR'R'' , this embodiment can be depicted as: In another embodiment, R' is NR'R'', where R' is selected from OC(O)R'', -CHCOOH, -O-benzyl, O pyridyl, and—OC(S)NR'R''; and R' is H. In still another 35 embodiment, R' is NR'R'', where R' is Hor-OH, and R" is Calkylor-C(O)R''. In yet another embodiment, R" is NR'R'', where R is selected from OC(O)R', - X -CHCOOH, -O-benzyl, pyridyl, and —OC(S)NR'R'': R"-- and R' is Calkyl or —C(O)R’. In one aspect of the 40 R19 invention, these compounds may find particular utility as prodrugs or as intermediates in the synthetic procedures described herein. For example, in one embodiment, R is and when R and R’ are taken together to form =O, this —OR'' and R' is Calkylene-OC(O)R', such as embodiment can be depicted as: —O CH(CH)OC(O) O-cyclohexyl: 45

R1 = Xrro 50 making the compound a cilexetil ester; or R' is —OR'' and When Ris-ORandR is taken together with R' to form R" is -Co-galkylenemorpholine such as-O-(CH2)2-mor 55 —NHC(O)—, this embodiment can be depicted as: pholine:

O

60 O X O

65 In one aspect of the invention, these compounds may find making the compound a 2-morpholinoethyl or mofetil ester; particular utility as prodrugs or as intermediates in the Syn or R' is OR'' and R' is thetic procedures described herein.

US 8,686,184 B2 19 20 urated —Co-alkylene-C-sheterocycle (e.g., dihydro -continued pyrazole and tetrahydrothienopyrimidine) both optionally substituted with 1-3 groups independently selected from halo, =O. —CHO. —Calkyl (e.g., —CH and -(CH),CH), —CF, and COOR' (e.g., -COOH); R' 5 * - N X lN 8. is Hor—C-alkyl (e.g., —CH2CH3); R' is Hor—C, alkyl (e.g., -CH3); R is —C-alkyl (e.g., —CHs and ul- J->S. d -(CH2)CH); R is selected from —Calkyl (e.g., C —(CH2)CH), - Calkylene-COOR' (e.g., —CHCOOH), phenyl, and pyridine; R is selected from 10 —Calkyl (e.g., -CHCH and —(CH2)CH), —C. alkylene-COOR' (e.g., -CH(COOH)|CHCH(CH)), 7tyS. phenyl, and thiophene-COOR" (e.g., -COOH), where OH: thiophene is optionally substituted with CH, such as: 15 tetrazoles Such as:

O OH, O OH, and A.2 S A,s ) O OH.

25 21 and pyridines such as:

S

30 O Exemplary optionally Substituted —Co-alkylene-Chet eroaryl groups include pyrroles Such as: N. N OH, and 35 4. 21

40 N OH. 2N

45 Exemplary optionally substituted partially unsaturated —Co-alkylene-C-sheterocycle groups include dihydropy razoles such as:

50

55 pyrazoles such as: 60

*-y 7ty ( 65 X U78, s US 8,686,184 B2 21 22 and tetrahydrothienopyrimidines such as: —C(O)NR'R'', and tetrazole; and Co-alkylene-Chet eroaryl or a partially unsaturated —Co-alkylene-C-shetero cycle both optionally substituted with 1-3 groups indepen dently selected from halo, =O, —CHO. —Calkyl, —CF, and COOR: R is H or Calkyl; R is H or —Calkyl; R is Calkyl; R is selected from A —Coalkyl, -Cisalkylene-COOR", phenyl, and pyridine: O 21 R is selected from —Calkyl, Calkylene-COOR', S phenyl, and thiophene-COOR", where thiophene is option sers ally substituted with CH; R is selected from H, fluoro, chloro, bromo, -OH, -CH, —CHCH, —CH(CH), —OCHs, phenyl, and morpholine; and R is selected from H, In one particular embodiment, R is -Co-alkylene fluoro, and chloro. COOR', where R' is H. In another particular embodiment, 15 In one embodiment, this invention relates to compounds of R" is - Co-alkylene-COOR', where R' is selected from formula II: —C-alkyl, -C-alkylene-Co-oaryl, -C-alkylene Coheteroaryl, -C-cycloalkyl, -(CH2)2OCH, —Calkylene-OC(O)R', - Calkylene-NR'R'', (II) C-alkylene-C(O)R''. -Co-alkylenemorpholine. —C. alkylene-SO Calkyl,

ty , and 25

“Y” 30 O In one exemplary embodiment, compounds of the invention have formula II, where R' is —OR' or - NR'R'': R' is H or Calkyl; R is OH: R' is H; R is Hor OR: R' is H; R is selected from H, Cl, and CF; R is selected from 35 —Calkyl, -C-cycloalkyl, -Co-alkylene-COOR': NR'R''. NHC(O)O Calkyl; NHC(O)R; NHC(O)NH-R: phenyl substituted with one or two groups independently selected from halo. —O—Calkyl, - Co-alkylene-COOR', -SONH, C(O)NR'R'', and In one aspect of the invention, these latter compounds may 40 tetrazole; and —Co-alkylene-Coheteroaryl or a partially find particular utility as prodrugs or as intermediates in the unsaturated —Co-alkylene-C-sheterocycle both optionally synthetic procedures described herein. substituted with 1-3 groups independently selected from The R moiety is selected from H, halo, -OH, halo, =O, —CHO, C-alkyl, -CF, and COOR': R' is —Calkyl, —O—Calkyl, phenyl, and morpholine. In Hor—Calkyl, R' is Hor—Calkyl; R is Calkyl; one embodiment, R is selected from H, fluoro, chloro, 45 R is selected from Calkyl, C-alkylene-COOR". bromo, —OH, -CH —CH2CH, —CH(CH), —OCH, phenyl, and pyridine; R is selected from Calkyl, phenyl, and morpholine: - Calkylene-COOR', phenyl, and thiophene-COOR', where thiophene is optionally substituted with CH; R is selected from H. halo. —OH, -Calkyl, —O—Calkyl, 50 phenyl, and morpholine; and R is selected from H, fluoro, and chloro; or a pharmaceutically acceptable salt thereof. Specific examples of compounds of formula II include compounds of formulas IIa to II, as described below: 55 The R moiety is selected from Handhalo. In one embodi (IIa) ment, R is selected from H, fluoro, and chloro. OH sa 5 In one exemplary embodiment, compounds of the inven H -HR tion have formula I, where R' is OR' or NR'R'': R' 60 R! N-N-x is Hor-Calkyl: Ris-OH: R7 is H; R is Hor–OR: R6 R’ is H; R is selected from H, C1, and CF; a is 0 or 3: R' is selected from —Calkyl; —C,cycloalkyl, -Co-alky O O lene-COOR': NR'R'': NHC(O)O Calkyl; NHC(O)R: NHC(O)NH R: phenyl substituted 65 with one or two groups independently selected from halo, C —O C-alkyl, -Co-alkylene-COOR', SO-NH2, US 8,686,184 B2 23 24 -continued -continued (IIb) (Ig)

OH 5 H HR R N 2 R6 O O

F 10

F F (IIh) 15 (IIc) R4 OH sé, H --R R! N eXal R6 O O (III) 25

(IId) 30 R4

35 (II)

40

(IIe)

O H 45 sé, R5 R! 2a In an exemplary embodiment of compounds of formula IIa, R" is OR or NR'R'': R is Hor Calkyl; R is - OH: R' is H; R is selected from phenyl substituted with C 50 - Co-alkylene-COOR, and a partially unsaturated —Co-alkylene-C-sheterocycle selected from —Co-alky Y as F lene-dihydropyrazole and tetrahydrothienopyrimidine, both of which are optionally substituted with 1-3 groups indepen dently selected from =O. —Calkyl, and CF; R' is H: 55 R is selected from H. halo. —Coalkyl, and —O— Calkyl; and R is selected from H. fluoro, and chloro. In an (IIf) exemplary embodiment of compounds of formula IIc, R' is —OR'; R' is H or - Calkyl; R is selected from Sé,--R —C-alkyl, -C-cycloalkyl, -Co-alkylene-COOR': R! 2 60 NR'R''. NHC(O)O Calkyl; NHC(O)R; OYa Y Ya Y R6 - NHC(O)NH-R: phenyl substituted with one or two groups independently selected from halo. —O—Calkyl, - Calkylene-COOR', SONH, C(O)NR'R'', and tetrazole; and —Co-alkylene-Coheteroaryl or a partially 65 unsaturated —Co-alkylene-C-sheterocycle selected from C —Co-alkylene-pyrrole, —Co-alkylene-imidazole, —Co alkylene-pyrazole, —Co-alkylene-tetrazole. —Co-alky US 8,686,184 B2 25 26 lene-pyridine. —Co-alkylene-dihydropyrazole and tetrahy -continued drothienopyrimidine, all of which are optionally substituted with 1-3 groups independently selected from halo. —O. CHO,-Calkyl, CF, and COOR': R' is H; R' is (Vc) H or Calkyl; R is Calkyl; R is selected from —Coalkyl, -Cisalkylene-COOR", phenyl, and pyridine: R" is selected from Calkyl, C-alkylene-COOR". phenyl, and thiophene-COOR', where thiophene is option ally substituted with CH3; R is selected from H, halo, -OH, —Coalkyl, phenyl, and morpholine; andR is Horfluoro. In 10 an exemplary embodiment of compounds of formula IIf R is —OR': R' is H or - Calkyl; R is selected from phenyl substituted with - Co-alkylene-COOR"; and a partially (Vd) unsaturated —Co-alkylene-C-sheterocycle selected from dihydropyrazole and tetrahydrothienopyrimidine, both all of 15 which are substituted with 1-3 groups independently selected RR! from =O and —Calkyl; R' is H; R is selected from H. halo, and —C-alkyl; and R is H. In an exemplary embodi OO R5R5 ment of compounds of formula Ilg, R is —OR'; R' is Hor Ye^SYaS —Coalkyl; R is phenyl substituted with -Co-alkylene COOR'; R is H; R is Calkyl; and R is H. In still another embodiment, this invention relates to com (Ve) pounds of formula V: (V) 25 aS R2 × H R4 R N Sé, O --Rs 30 O O 2 F

(Vf) H R! N 35 Sá. In one exemplary embodiment, compounds of the invention O O 2a have formula V, where R is —OR': R' is Hor-Calkyl: R6 R’ is H or -OR': R is H: R is selected from H, C1, and —CF; R is - Co-alkylene-COOR': R' is H; R is Hor C 40 halo; and R is H; or a pharmaceutically acceptable salt (Vg) thereof. Specific examples of compounds of formula V examples of as which include compounds of formulas Vato V, as described × below: O 45

(Va) OH F 50 (Vh)

55 (Vb) OH C (Vi) 60 as× 65 C US 8,686,184 B2 27 28 -continued thesis, Fourth Edition, Wiley, New York, 2006, and references (V) cited therein. More specifically, the following abbreviations R! R4 and reagents are used in the schemes presented below: S w P" represents a "carboxy-protecting group,” a term used is herein to mean a protecting group Suitable for preventing O O 2a undesired reactions at a carboxy group. Representative car R6 boxy-protecting groups include, but are not limited to, F methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), trimethylsilyl (TMS), t-butyldim 10 ethylsilyl (TBDMS), diphenylmethyl (benzhydryl, DPM) In an exemplary embodiment of compounds of formula Va, and the like. Standard deprotection techniques and reagents R" is —OR'; R' is Hor—Calkyl; R is Coalkylene are used to remove the P' group, and may vary depending COOR': R' is H; R is H or halo; and R is H. In an upon which group is used. For example, sodium or lithium exemplary embodiment of compounds of formula Vb, R' is 15 hydroxide is commonly used when P' is methyl, an acid such —OR': R' is H or - Calkyl; R is Coalkylene as TFA or HCl is commonly used when P' is ethyl ort-butyl, COOR'; R is H; and R is H. In an exemplary embodiment and H2/Pd/C may be used when P' is benzyl. of compounds of formula Vc, R' is OR'; R' is H or P represents an "amino-protecting group,” a term used —Calkyl; R is - Co-alkylene-COOR': R' is H; R is herein to mean a protecting group Suitable for preventing H; and R is H. In an exemplary embodiment of compounds of undesired reactions at an amino group. Representative formula Vf R is —OR'; R' is H or - Calkyl; R is amino-protecting groups include, but are not limited to, t-bu - Co-alkylene-COOR': R' is H: R is Horhalo; and R is toxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz), H. 9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl In addition, particular compounds of formula I that are of (TMS), t-butyldimethylsilyl (TBDMS), and the like. Stan interest include those set forth in the Examples below, as well 25 dard deprotection techniques are used to remove the P group. as pharmaceutically acceptable salts thereof. For example, a BOC group can be removed using an acidic reagent such as TFA in DCM or HCl in 1,4-dioxane, while a General Synthetic Procedures Cbz group can be removed by employing catalytic hydroge nation conditions such as H. (1 atm) and 10% Pd/C in an Compounds of the invention can be prepared from readily 30 alcoholic solvent (“H/Pd/C). available starting materials using the following general meth Prepresents a “hydroxyl-protecting group,” a term that is ods, the procedures set forth in the Examples, or by using used hereinto mean a protecting group suitable for preventing other methods, reagents, and starting materials that are known undesired reactions at a hydroxyl group. Representative to those of ordinary skill in the art. Although the following hydroxyl-protecting groups include, but are not limited to procedures may illustrate a particular embodiment of the 35 Calkyls, silyl groups including triC-alkylsilyl groups, invention, it is understood that other embodiments of the such as trimethylsilyl (TMS), triethylsilyl (TES), and tert invention can be similarly prepared using the same or similar butyldimethylsilyl (TBDMS): esters (acyl groups) including methods or by using other methods, reagents and starting Calkanoyl groups, such as formyl, acetyl, and pivaloyl, and materials known to those of ordinary skill in the art. It will aromatic acyl groups such as benzoyl: arylmethyl groups also be appreciated that where typical or preferred process 40 such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenyl conditions (for example, reaction temperatures, times, mole methyl (Fm), and diphenylmethyl (benzhydryl, DPM); and ratios of reactants, solvents, pressures, etc.) are given, other the like. Standard deprotection techniques and reagents are process conditions can also be used unless otherwise stated. used to remove the P group, and may vary depending upon In some instances, reactions were conducted at room tem which group is used. For example, H2/Pd/C is commonly perature and no actual temperature measurement was taken. It 45 used when P is benzyl, while NaOH is commonly used when is understood that room temperature can be taken to mean a P is an acyl group. temperature within the range commonly associated with the In addition, L is used to designate a “leaving group, a term ambient temperature in a laboratory environment, and will that is used herein to mean a functional group or atom which typically be in the range of about 18°C. to about 30°C. In can be displaced by another functional group or atom in a other instances, reactions were conducted at room tempera 50 Substitution reaction, Such as a nucleophilic Substitution reac ture and the temperature was actually measured and recorded. tion. By way of example, representative leaving groups While optimum reaction conditions will typically vary include halo groups such as chloro, bromo and iodo groups; depending on various reaction parameters such as the particu Sulfonic ester groups, such as mesylate, tosylate, brosylate, lar reactants, solvents and quantities used, those of ordinary nosylate and the like; and acyloxy groups, such as acetoxy, skill in the art can readily determine suitable reaction condi 55 trifluoroacetoxy and the like. tions using routine optimization procedures. Suitable bases for use in these schemes include, by way of Additionally, as will be apparent to those skilled in the art, illustration and not limitation, potassium carbonate, calcium conventional protecting groups may be necessary or desired carbonate, sodium carbonate, triethylamine, pyridine, 1.8- to prevent certain functional groups from undergoing undes diazabicyclo-5.4.0]undec-7-ene (DBU), N,N-diisopropyl ired reactions. The choice of a Suitable protecting group for a 60 ethylamine (DIPEA), 4-methylmorpholine, sodium hydrox particular functional group as well as Suitable conditions and ide, potassium hydroxide, potassium t-butoxide, and metal reagents for protection and deprotection of Such functional hydrides. groups are well-known in the art. Protecting groups other than Suitable inert diluents or solvents for use in these schemes those illustrated in the procedures described herein may be include, by way of illustration and not limitation, tetrahydro used, if desired. For example, numerous protecting groups, 65 furan (THF), acetonitrile (MeCN), N,N-dimethylformamide and their introduction and removal, are described in T. W. (DMF), N,N-dimethylacetamide (DMA), dimethylsulfoxide Greene and G. M. Wuts, Protecting Groups in Organic Syn (DMSO), toluene, dichloromethane (DCM), chloroform US 8,686,184 B2 29 30 (CHCl), carbon tetrachloride (CCI), 1,4-dioxane, metha step may be followed by a deprotection step to provide a nol, , water, and the like. compound of formula I where R' is a group such as —OH. Suitable carboxylic acid/amine coupling reagents include Thus, one method of preparing compounds of the invention benzotriazol-1-yloxytris(dimethylamino)phosphonium involves coupling compounds 1 and 2, with an optional hexafluorophosphate (BOP), benzotriazol-1-yloxytripyrroli dinophosphonium hexafluorophosphate (PyBOP), N.N.N', deprotection step to form a compound of formula I or a N-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium pharmaceutically acceptable salt thereof. hexafluorophosphate (HATU), 1,3-dicyclohexylcarbodiim Compounds of formula I and their salts, where R is sub ide (DCC), N-(3-dimethylaminopropyl)-N'-ethylcarbodiim stituted phenyl, can also be prepared as shown in Scheme II: ide (EDCI), carbonyldiimidazole (CDI), 1-hydroxybenzot 10 riazole (HOBt), and the like. Coupling reactions are conducted in an inert diluent in the presence of a base such as Scheme II DIPEA, and are performed under conventional amide bond L forming conditions. All reactions are typically conducted at a temperature 15 within the range of about -78 C to 100° C., for example at (1) + HO ( ) - H 1N--R —- room temperature. Reactions may be monitored by use of thin layer chromatography (TLC), high performance liquid chro O 1 SueR6 matography (HPLC), and/or LCMS until completion. Reac tions may be complete in minutes, or may take hours, typi (3) cally from 1-2 hours and up to 48 hours. Upon completion, the resulting mixture or reaction product may be further treated in order to obtain the desired product. For example, the resulting k mixture or reaction product may be subjected to one or more B-O of the following procedures: concentrating or partitioning 25 (for example, between EtOAc and water or between 5% THF L e in EtOAc and 1M phosphoric acid); extraction (for example, R2 l with EtOAc, CHCl, DCM. chloroform); washing (for H N N ITR example, with saturated aqueous NaCl, saturated NaHCO R! N ( ), it -R (5) NaCO (5%), CHCl or 1M NaOH); drying (for example, 30 r Sux (I) over MgSO4, over Na2SO, or in vacuo); filtering: crystalliz O O R ing (for example, from EtOAc and hexanes); being concen trated (for example, in vacuo); and/or purification (e.g., silica R3 gel chromatography, flash chromatography, preparative HPLC, reverse phase-HPLC, or crystallization). 35 (4) Compounds of formula I, as well as their salts, can be prepared as shown in Scheme I:

40 Scheme I Again, as with Scheme I, this is a standard coupling reac tion between compound 1 and compound 3 to yield the com pound 4. In compounds 3 and 4, L is a leaving group Such as halo (e.g., bromo and chloro) or a triflate (e.g., —OSOCF). 45 Compound 4 is then reacted with a substituted 4.4.5.5-tetram ethyl-2-phenyl-1,3,2dioxaborolane compound 5 in a palla dium-catalyzed coupling reaction. In compound 5, the phenyl ring is substituted with one or two groups (depicted as R.) independently selected from halo. —O—Calkyl, 50 - Co-alkylene-COOR', -SONH, C(O)NR'R'', and (1) R2 tetrazole. Examples of compound 5 include 2-t-butoxycarbo H R4 nylphenylboronic acid pinacol ester. The palladium-cata lyzed coupling reaction is typically conducted in an inert R N Jan 1s^ 55 diluent in the presence of a suitable base such as potassium O O 2aHR carbonate or sodium carbonate, and may be followed by a R6 deprotection step to provide a compound of formula I where R" is a group such as —OH. Thus, another method of prepar R3 ing compounds of the invention involves coupling compound 60 1 and compound 3 to form compound 4, coupling compound (I) 4 with a substituted 4.4.5.5-tetramethyl-2-phenyl-1,3,2di oxaborolane compound 5, and an optional deprotection step to form a compound of formula I or a pharmaceutically 65 acceptable salt thereof. Compound 1 is coupled with compound 2. In instances where Compounds of formula I and their salts, where R is sub R" is a group such as —OCH or —OCH2CHs, the coupling stituted phenyl, can also be prepared as shown in Scheme III: US 8,686,184 B2 31 32 enzyme-catalytic activity. In another embodiment, the com Scheme III pounds do not exhibit significant inhibitory activity of the angiotensin-converting enzyme. One measure of the ability (3) + (5) -- of a compound to inhibit NEP activity is the inhibition con stant (pK). The pK value is the negative logarithm to base 10 HO ( ) 17s. of the dissociation constant (K), which is typically reported (1) in molar units. Compounds of the invention of particular 1 Nue - (I) interest are those having a pK, at NEP greater than or equal to O / 6.0, particularly those having a pK, greater than or equal to 10 7.0, and even more particularly those having a pK, greater than or equal to 8.0. In one embodiment, compounds of interest have a pK, in the range of 6.0–6.9; in another embodi (6) ment, compounds of interest have a pK, in the range of 7.0- 7.9; in yet another embodiment, compounds of interest have As with Scheme II, the first step is a palladium-catalyzed 15 a pK, in the range of 8.0-8.9; and in still another embodiment, coupling reaction between compound 3 and a Substituted compounds of interest have a pK, in the range of greater than 4.4.5.5-tetramethyl-2-phenyl-1,3,2dioxaborolane CO or equal to 9.0. Such values can be determined by techniques pound 5 to form compound 6. Compound 6 is then coupled that are well known in the art, as well as in the assays with compound 1, followed by an optional deprotection step described herein. to provide a compound of formula I where R' is a group such Another measure of the ability of a compound to inhibit as —OH. Thus, another method of preparing compounds of NEP activity is the apparent inhibition constant (ICs), which the invention involves coupling compound 3 with a Substi is the molar concentration of compound that results in half tuted 4,4,5,5-tetramethyl-2-phenyl-1,3,2dioxaborolane maximal inhibition of substrate conversion by the NEP compound 5 to form compound 6, coupling compound 6 and enzyme. The plCso value is the negative logarithm to base 10 compound 1, and an optional deprotection step to form a compound of formula I or a pharmaceutically acceptable salt 25 of the ICso. Compounds of the invention that are of particular thereof. interest, include those that exhibit a plCso for NEP greater Compounds 1, 2, 3, and 5 are generally commercially than or equal to about 5.0. Compounds of interest also include available or can be prepared using procedures that are known those having a plCs for NEPaabout 6.0 or a plCs for in the art as well as those that are set forth in the Examples NEPaabout 7.0. In another embodiment, compounds of inter herein. 30 est have a plCs for NEP within the range of about 7.0-11.0: Certain intermediates described herein are believed to be and in another embodiment, within the range of about 8.0- novel and accordingly, such compounds are provided as fur 11.0, such as within the range of about 8.0-10.0. ther aspects of the invention including, for example, the com It is noted that in Some cases, compounds of the invention pounds of formula VI, or a salt thereof: may possess weak NEP inhibition activity. In such cases, 35 those of skill in the art will recognize that these compounds still have utility as research tools. (VI) Exemplary assays to determine properties of compounds R2 of the invention, such as the NEP inhibiting activity, are P R4 described in the Examples and include by way of illustration 40 and not limitation, assays that measure NEP inhibition (de scribed in Assay 1). Useful secondary assays include assays O O 1. -R. to measure ACE inhibition (also described in Assay 1) and r 2. aminopeptidase P (APP) inhibition (described in Sulpizio et al. (2005).JPET315:1306-1313). A pharmacodynamic assay 45 to assess the in vivo inhibitory potencies for ACE and NEP in anesthetized rats is described in Assay 2 (see also Seymour et al. (1985) Hypertension 7(Suppl 41-35-1-42 and Wigle et al. where P is selected from-O-P'. - NHP, and NH(O- P); P' is a carboxy-protecting group selected from methyl, (1992) Can. J. Physiol. Pharmacol. 70:1525-1528), where ethyl, t-butyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl, ACE inhibition is measured as the percent inhibition of the trimethylsilyl, t-butyldimethylsilyl, and diphenylmethyl; P 50 angiotensin I pressor response and NEP inhibition is mea is an amino-protecting group selected from t-butoxycarbonyl, Sured as increased urinary cyclic 3',5'-monophos trity1, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, phate (cGMP) output. formyl, trimethylsilyl, and t-butyldimethylsilyl; and P is a There are many in Vivo assays that can be used to ascertain hydroxyl-protecting group selected from Calkyl, silyl further utilities of the compounds of the invention. The con groups, esters, and arylmethyl groups; and a and R-R are as 55 scious spontaneously hypertensive rat (SHR) model is a renin defined for formula I. Thus, another method of preparing dependent hypertension model, and is described in Assay 3. compounds of the invention involves deprotecting a com See also Intengan et al. (1999) Circulation 100(22):2267 pound of formula VI. 2275 and Badyal et al. (2003) Indian Journal of Pharmacol Further details regarding specific reaction conditions and ogy 35:349-362. The conscious desoxycorticosterone other procedures for preparing representative compounds of 60 acetate-salt (DOCA-salt) rat model is a volume dependent the invention or intermediates thereof are described in the hypertension model that is useful for measuring NEP activity, Examples set forth below. and is described in Assay 4. See also Trapani et al. (1989).J. Cardiovasc. Pharmacol. 14:419–424, Intengan et al. (1999) Utility Hypertension 34(4):907-913, and Badyaletal. (2003) supra). 65 The DOCA-salt model is particularly useful for evaluating Compounds of the invention possess neprilysin (NEP) the ability of a test compound to reduce blood pressure as well inhibition activity, that is, the compounds are able to inhibit as to measure a test compounds ability to prevent or delay a US 8,686,184 B2 33 34 rise in blood pressure. The Dahl salt-sensive (DSS) hyperten sive rat model is a model of hypertension that is sensitive to dietary salt (NaCl), and is described in Assay 5. See also Rapp and (1982) Hypertension 4:753-763. The rat monocrotaline model of pulmonary arterial hypertension described, for example, in Kato et al. (2008) J. Cardiovasc. Pharmacol. 51(1):18-23, is a reliable predictor of clinical efficacy for the treatment of pulmonary arterial hypertension. Heart failure animal models include the DSS rat model for heart failure and the aorto-caval fistula model (AV shunt), the latter of which is 10 described, for example, in Norling et al. (1996).J. Amer: Soc. Nephrol. 7:1038-1044. Other animal models, such as the hot plate, tail-flick and formalin tests, can be used to measure the analgesic properties of compounds of the invention, as well as the spinal nerve ligation (SNL) model of neuropathic pain. 15 See, for example, Malmberg et al. (1999) Current Protocols in Neuroscience 8.9.1-8.9.15. Compounds of the invention are expected to inhibit the R" is NR'R'': R is selected from OC(O)R', NEP enzyme in any of the assays listed above, or assays of a -CHCOOH, -O-benzyl, pyridyl, and —OC(S)NR'R'': similar nature. Thus, the aforementioned assays are useful in and R' is H; or determining the therapeutic utility of compounds of the R" is NR'R'': R is selected from OC(O)R', invention, for example, their utility as antihypertensive agents -CHCOOH, -O-benzyl, pyridyl, and —OC(S)NR'R'': or antidiarrheal agents. Other properties and utilities of com and R" is Calkyl or C(O)R'; or pounds of the invention can be demonstrated using other in 25 R" is NR'R'': R is Hor OH; and R' is Calkyl vitro and in vivo assays well-known to those skilled in the art. or C(O)R7'; or Compounds of formula I may be active drugs as well as R" is OR'; R is —OR'; and R is taken together with prodrugs. Thus, when discussing the activity of compounds R9 to form CRR’ ; or of the invention, it is understood that any such prodrugs may R" is NR'R'': R is OR; and R is taken together not exhibit the expected activity in an assay, but are expected 30 with R' to form C(O) ; and R, a, R. R. R. R. R'', to exhibit the desired activity once metabolized. RS, R, R7, R, R2, R, R2, and R7 are as defined for Compounds of the invention are expected to be useful for formula I. the treatment and/or prevention of medical conditions respon Cardiovascular Diseases sive to NEP inhibition. Thus it is expected that patients suf 35 fering from a disease or disorder that is treated by inhibiting By potentiating the effects of vasoactive peptides like the the NEP enzyme or by increasing the levels of its peptide natriuretic peptides and bradykinin, compounds of the inven Substrates, can be treated by administering a therapeutically tion are expected to find utility in treating and/or preventing effective amount of a compound of the invention. For medical conditions such as cardiovascular diseases. See, for example, by inhibiting NEP, the compounds are expected to 40 example, Rogues et al. (1993) Pharmacol. Rev. 45:87-146 potentiate the biological effects of endogenous peptides that and Dempsey et al. (2009) Amer: J. of Pathology 174(3):782 are metabolized by NEP, such as the natriuretic peptides, 796. Cardiovascular diseases of particular interest include bombesin, bradykinins, calcitonin, endothelins, enkephalins, hypertension and heart failure. Hypertension includes, by neurotensin, Substance P and vasoactive intestinal peptide. way of illustration and not limitation: primary hypertension, Thus, these compounds are expected to have other physi 45 which is also referred to as essential hypertension or idio ological actions, for example, on the renal, central nervous, pathic hypertension; secondary hypertension; hypertension reproductive and gastrointestinal systems. with accompanying renal disease; severe hypertension with In one embodiment of the invention, patients suffering or without accompanying renal disease; pulmonary hyperten from a disease or disorder that is treated by inhibiting the NEP Sion, including pulmonary arterial hypertension; and resistant enzyme, are treated by administering a compound of the 50 hypertension. Heart failure includes, by way of illustration invention that is in its active form, i.e., a compound of formula and not limitation: congestive heart failure; acute heart fail I where R' is selected from —OR'' and - NR'R'', where ure; chronic heart failure, for example with reduced left ven R' is H, R is Hor OH, and R' is H; and R,R, a, R,R, tricular ejection fraction (also referred to as systolic heart and Rare as defined for formula I. failure) or with preserved left ventricular ejection fraction In another embodiment, patients are treated by administer 55 (also referred to as diastolic heart failure); and acute and ing a compound that is metabolized in vitro to form a com chronic decompensated heart failure, with or without accom pound of formula I, where R' is —OR' or - NR'R'': RD) panying renal disease. Thus, one embodiment of the invention is H, R is Hor OH, R' is H; and R. R., a, R,R, and R' relates to a method for treating hypertension, particularly are as defined for formula I. primary hypertension or pulmonary arterial hypertension, In another embodiment, patients are treated by administer 60 comprising administering to a patient a therapeutically effec ing a compound of the invention that is in its prodrug form at tive amount of a compound of the invention. the R' group, i.e., a compound of formula I where: R' is For treatment of primary hypertension, the therapeutically —OR'; and R' is selected from Calkyl, Calky effective amount is typically the amount that is sufficient to lene-Co-oaryl, -C-alkylene-Coheteroaryl, -C-7cy lower the patient’s blood pressure. This would include both cloalkyl, -(CH2)2OICH, Calkylene-OC(O)R', 65 mild-to-moderate hypertension and severe hypertension. —Calkylene-NR'R''. —Calkylene-C(O)R'7, Co. When used to treat hypertension, the compound may be alkylenemorpholine, SO Cealkyl, administered in combination with other therapeutic agents US 8,686,184 B2 35 36 Such as aldosterone antagonists, aldosterone synthase inhibi receptor antagonists, natriuretic peptides and their analogs, tors, angiotensin-converting enzyme inhibitors and dual-act natriuretic peptide clearance receptor antagonists, nitric ing angiotensin-converting enzyme? neprilysin inhibitors, oxide donors, prostaglandin analogs, PDE-V inhibitors, angiotensin-converting enzyme 2 (ACE2) activators and soluble guanylate cyclase activators and stimulators, and stimulators, angiotensin-II Vaccines, anti-diabetic agents, vasopressin receptor antagonists. In one particular embodi anti-lipid agents, anti-thrombotic agents, AT receptor ment of the invention, a compound of the invention is com antagonists and dual-acting AT /nepril bined with an aldosterone antagonist, a B-adrenergic recep ysin inhibitors, B-adrenergic receptor antagonists, dual-act tor antagonist, an AT receptor antagonist, or a , and ing f-adrenergic receptor antagonist/C.-receptor antagonists, used to treat congestive heart failure. calcium channel blockers, , endothelin receptor 10 antagonists, endothelin converting enzyme inhibitors, nepril Diarrhea ysin inhibitors, natriuretic peptides and their analogs, natri uretic peptide clearance receptor antagonists, nitric oxide As NEP inhibitors, compounds of the invention are donors, non-steroidal anti-inflammatory agents, phosphodi expected to inhibit the degradation of endogenous enkepha esterase inhibitors (specifically PDE-V inhibitors), prostag 15 lins and thus Such compounds may also find utility for the landin receptor agonists, renin inhibitors, soluble guanylate treatment of diarrhea, including infectious and secretory/wa cyclase stimulators and activators, and combinations thereof. tery diarrhea. See, for example, Baumer et al. (1992) Gut In one particular embodiment of the invention, a compound of 33:753-758: Farthing (2006) Digestive Diseases 24:47-58: the invention is combined with an AT receptor antagonist, a and Marçais-Collado (1987) Eur: J. Pharmacol. 144(2):125 calcium channel blocker, a diuretic, or a combination thereof, 132. When used to treat diarrhea, compounds of the invention and used to treat primary hypertension. In another particular may be combined with one or more additional antidiarrheal embodiment of the invention, a compound of the invention is agents. combined with an AT receptor antagonist, and used to treat hypertension with accompanying renal disease. When used to Renal Diseases treat resistant hypertension, the compound may be adminis 25 tered in combination with other therapeutic agents such as By potentiating the effects of vasoactive peptides like the aldosterone synthase inhibitors. natriuretic peptides and bradykinin, compounds of the inven For treatment of pulmonary arterial hypertension, the tion are expected to enhance renal function (see Chen et al. therapeutically effective amount is typically the amount that (1999) Circulation 100:2443-2448; Lipkin et al. (1997) Kid is sufficient to lower the pulmonary vascular resistance. Other 30 ney Int. 52:792-801; and Dussaule et al. (1993) Clin. Sci. goals of therapy are to improve a patient’s exercise capacity. 84:31-39) and find utility in treating and/or preventing renal For example, in a clinical setting, the therapeutically effective diseases. Renal diseases of particularinterest include diabetic amount can be the amount that improves a patient’s ability to nephropathy, chronic kidney disease, proteinuria, and par walk comfortably for a period of 6 minutes (covering a dis ticularly acute kidney injury or acute renal failure (see Shark tance of approximately 20-40 meters). When used to treat 35 ovska et al. (2011) Clin. Lab. 57:507–515 and Newaz et al. pulmonary arterial hypertension the compound may be (2010) Renal Failure 32:384-390). When used to treat renal administered in combination with other therapeutic agents disease, the compound may be administered in combination Such as C.-adrenergic receptor antagonists, B-adrenergic with other therapeutic agents such as angiotensin-converting receptor antagonists, f2-adrenergic receptor agonists, angio enzyme inhibitors, AT receptor antagonists, and diuretics. tensin-converting enzyme inhibitors, anticoagulants, calcium 40 channel blockers, diuretics, endothelin receptor antagonists, Preventative Therapy PDE-V inhibitors, prostaglandin analogs, selective serotonin reuptake inhibitors, and combinations thereof. In one particu By potentiating the effects of the natriuretic peptides, com lar embodiment of the invention, a compound of the invention pounds of the invention are also expected to be useful in is combined with a PDE-V inhibitor or a selective serotonin 45 preventative therapy, due to the antihypertrophic and antifi and used to treat pulmonary arterial hyper brotic effects of the natriuretic peptides (see Potter et al. tension. (2009) Handbook of Experimental Pharmacology 191:341 Another embodiment of the invention relates to a method 366), for example in preventing the progression of cardiac for treating heart failure, in particular congestive heart failure insufficiency after myocardial infarction, preventing arterial (including both systolic and diastolic congestive heart fail 50 restenosis after angioplasty, preventing thickening of blood ure), comprising administering to a patient a therapeutically vessel walls after vascular operations, preventing atheroscle effective amount of a compound of the invention. Typically, rosis, and preventing diabetic angiopathy. the therapeutically effective amount is the amount that is sufficient to lower blood pressure and/or improve renal func Glaucoma tions. In a clinical setting, the therapeutically effective 55 amount can be the amount that is sufficient to improve cardiac By potentiating the effects of the natriuretic peptides, com hemodynamics, like for instance reduction in wedge pres pounds of the invention are expected to be useful to treat Sure, right atrial pressure, filling pressure, and vascular resis glaucoma. See, for example, Diestelhorst et al. (1989) Inter tance. In one embodiment, the compound is administered as national Ophthalmology 12:99-101. When used to treat glau an intravenous dosage form. When used to treat heart failure, 60 coma, compounds of the invention may be combined with one the compound may be administered in combination with or more additional antiglaucoma agents. other therapeutic agents such as receptor antago nists, advanced glycation end product breakers, aldosterone Pain Relief antagonists, AT receptor antagonists, B-adrenergic receptor antagonists, dual-acting B-adrenergic receptor antagonist/C.- 65 As NEP inhibitors, compounds of the invention are receptor antagonists, chymase inhibitors, digoxin, diuretics, expected to inhibit the degradation of endogenous enkepha endothelin converting enzyme (ECE) inhibitors, endothelin lins and thus Such compounds may also find utility as anal US 8,686,184 B2 37 38 gesics. See, for example, Rogues et al. (1980) Nature 288: therapy. For example, in treating hypertension, blood pres 286-288 and Thanawala et al. (2008) Current Drug Targets sure measurements may be used to determine the effective 9:887-894. When used to treat pain, the compounds of the ness of treatment. Similar indicators for other diseases and invention may be combined with one or more additional conditions described herein, are well known and are readily antinociceptive drugs such as aminopeptidase Nor dipeptidyl available to the treating physician. Continuous monitoring by peptidase III inhibitors, non-steroidal anti-inflammatory the physician will insure that the optimal amount of the com agents, monoamine reuptake inhibitors, muscle relaxants, pound of the invention will be administered at any given time, NMDA receptor antagonists, opioid receptor agonists, as well as facilitating the determination of the duration of 5-HT serotonin receptoragonists, and tricyclic antidepres treatment. This is of particular value when secondary agents SantS. 10 are also being administered, as their selection, dosage, and duration of therapy may also require adjustment. In this way, Other Utilities the treatment regimen and dosing schedule can be adjusted over the course of therapy so that the lowest amount of active Due to their NEP inhibition properties, compounds of the agent that exhibits the desired effectiveness is administered invention are also expected to be useful as antitussive agents, 15 and, further, that administration is continued only so long as as well as find utility in the treatment of portal hypertension is necessary to successfully treat the disease or medical con associated with liver cirrhosis (see Sansoe et al. (2005) J. dition. Hepatol. 43:791-798), cancer (see Vesely (2005) J. Investi gative Med. 53:360-365), depression (see Noble et al. (2007) Research Tools Exp. Opin. Ther: Targets 11:145-159), menstrual disorders, preterm labor, pre-eclampsia, endometriosis, reproductive Since compounds of the invention possess NEP enzyme disorders (for example, male and female infertility, polycystic inhibition activity, Such compounds are also useful as ovarian syndrome, implantation failure), and male and female research tools for investigating or studying biological sys sexual dysfunction, including male erectile dysfunction and tems or samples having a NEP enzyme, for example to study female sexual arousal disorder. More specifically, the com 25 diseases where the NEP enzyme or its peptide substrates pounds of the invention are expected to be useful in treating plays a role. Any Suitable biological system or sample having female sexual dysfunction (see Pryde et al. (2006) J. Med. a NEP enzyme may be employed in such studies which may Chem. 49:4409-4424), which is often defined as a female be conducted either in vitro or in vivo. Representative bio patient’s difficulty or inability to find satisfaction in sexual logical systems or samples Suitable for Such studies include, expression. This covers a variety of diverse female sexual 30 but are not limited to, cells, cellular extracts, plasma mem disorders including, by way of illustration and not limitation, branes, tissue samples, isolated organs, mammals (such as hypoactive sexual desire disorder, sexual arousal disorder, mice, rats, guinea pigs, rabbits, dogs, pigs, humans, and so orgasmic disorder and sexual pain disorder. When used to forth), and the like, with mammals being of particular inter treat Such disorders, especially female sexual dysfunction, est. In one particular embodiment of the invention, NEP compounds of the invention may be combined with one or 35 enzyme activity in a mammal is inhibited by administering a more of the following secondary agents: PDE-V inhibitors, NEP-inhibiting amount of a compound of the invention. dopamine agonists, estrogen receptoragonists and/orantago Compounds of the invention can also be used as research tools nists, androgens, and estrogens. Due to their NEP inhibition by conducting biological assays using Such compounds. properties, compounds of the invention are also expected to When used as a research tool, a biological system or have anti-inflammatory properties, and are expected to have 40 sample comprising a NEP enzyme is typically contacted with utility as such, particularly when used in combination with a NEP enzyme-inhibiting amount of a compound of the statins. invention. After the biological system or sample is exposed to Recent studies suggest that NEP plays a role in regulating the compound, the effects of inhibiting the NEP enzyme are nerve function in insulin-deficient diabetes and diet induced determined using conventional procedures and equipment, obesity. Coppey et al. (2011) Neuropharmacology 60:259 45 Such as by measuring receptor binding in a binding assay or 266. Therefore, due to their NEP inhibition properties, com measuring ligand-mediated changes in a functional assay. pounds of the invention are also expected to be useful in Exposure encompasses contacting cells or tissue with the providing protection from nerve impairment caused by dia compound, administering the compound to a mammal, for betes or diet induced obesity. example by i.p. p.o. i.V., s.c., or inhaled administration, and The amount of the compound of the invention administered 50 So forth. This determining step can involve measuring a per dose or the total amount administered per day may be response (a quantitative analysis) or can involve making an predetermined or it may be determined on an individual observation (a qualitative analysis). Measuring a response patient basis by taking into consideration numerous factors, involves, for example, determining the effects of the com including the nature and severity of the patient’s condition, pound on the biological system or sample using conventional the condition being treated, the age, weight, and general 55 procedures and equipment, Such as enzyme activity assays health of the patient, the tolerance of the patient to the active and measuring enzyme substrate or product mediated agent, the route of administration, pharmacological consid changes in functional assays. The assay results can be used to erations such as the activity, efficacy, pharmacokinetics and determine the activity level as well as the amount of com toxicology profiles of the compound and any secondary pound necessary to achieve the desired result, that is, a NEP agents being administered, and the like. Treatment of a patient 60 enzyme-inhibiting amount. Typically, the determining step Suffering from a disease or medical condition (such as hyper will involve determining the effects of inhibiting the NEP tension) can begin with a predetermined dosage or a dosage enzyme. determined by the treating physician, and will continue for a Additionally, compounds of the invention can be used as period of time necessary to prevent, ameliorate, Suppress, or research tools for evaluating other chemical compounds, and alleviate the symptoms of the disease or medical condition. 65 thus are also useful in screening assays to discover, for Patients undergoing Such treatment will typically be moni example, new compounds having NEP-inhibiting activity. In tored on a routine basis to determine the effectiveness of this manner, a compound of the invention is used as a standard US 8,686,184 B2 39 40 in an assay to allow comparison of the results obtained with a Any conventional carrier or excipient may be used in the test compound and with compounds of the invention to iden pharmaceutical compositions of the invention. The choice of tify those test compounds that have about equal or Superior a particular carrier or excipient, or combinations of carriers or activity, if any. For example, pK data for a test compound or excipients, will depend on the mode of administration being a group of test compounds is compared to the pK, data for a 5 used to treat a particular patient or type of medical condition compound of the invention to identify those test compounds or disease state. In this regard, the preparation of a Suitable that have the desired properties, for example, test compounds composition for a particular mode of administration is well having a pK value about equal or Superior to a compound of within the scope of those skilled in the pharmaceutical arts. the invention, if any. This aspect of the invention includes, as Additionally, carriers or excipients used in Such compositions separate embodiments, both the generation of comparison 10 are commercially available. By way of further illustration, data (using the appropriate assays) and the analysis of test conventional formulation techniques are described in Rem data to identify test compounds of interest. Thus, a test com ington: The Science and Practice of Pharmacy, 20" Edition, pound can be evaluated in a biological assay, by a method Lippincott Williams & White, Baltimore, Md. (2000); and H. comprising the steps of: (a) conducting a biological assay 15 C. Ansel et al., Pharmaceutical Dosage Forms and Drug with a test compound to provide a first assay value; (b) con Delivery Systems, 7" Edition, Lippincott Williams & White, ducting the biological assay with a compound of the invention Baltimore, Md. (1999). to provide a second assay value; wherein step (a) is conducted Representative examples of materials which can serve as either before, after or concurrently with step (b); and (c) pharmaceutically acceptable carriers include, but are not lim comparing the first assay value from Step (a) with the second ited to, the following: Sugars, such as lactose, glucose and assay value from step (b). Exemplary biological assays Sucrose; starches, such as corn Starch and potato starch; cel include a NEP enzyme inhibition assay. lulose, such as microcrystalline cellulose, and its derivatives, Such as Sodium carboxymethyl cellulose, ethyl cellulose and Pharmaceutical Compositions and Formulations cellulose acetate; powdered tragacanth; malt, gelatin; talc; 25 excipients, such as cocoa butter and Suppository waxes; oils, Compounds of the invention are typically administered to Such as peanut oil, cottonseed oil, safflower oil, sesame oil, a patient in the form of a pharmaceutical composition or olive oil, corn oil and Soybean oil; glycols, such as propylene formulation. Such pharmaceutical compositions may be glycol; polyols. Such as glycerin, Sorbitol, and poly administered to the patient by any acceptable route of admin ethylene glycol; esters, such as ethyl oleate and ethyl laurate; istration including, but not limited to, oral, rectal, vaginal, 30 agar, buffering agents, such as magnesium hydroxide and nasal, inhaled, topical (including transdermal), ocular, and aluminum hydroxide; alginic acid; pyrogen-free water, iso parenteral modes of administration. Further, the compounds tonic saline: Ringer's solution; ethyl ; phosphate of the invention may be administered, for example orally, in buffer Solutions; compressed propellant gases, such as chlo multiple doses per day (for example, two, three, or four times rofluorocarbons and hydrofluorocarbons; and other non-toxic daily), in a single daily dose or a single weekly dose. It will be 35 compatible Substances employed in pharmaceutical compo understood that any form of the compounds of the invention, sitions. (that is, free base, free acid, pharmaceutically acceptable salt, Pharmaceutical compositions are typically prepared by solvate, etc.) that is suitable for the particular mode of admin thoroughly and intimately mixing or blending the active agent istration can be used in the pharmaceutical compositions with a pharmaceutically acceptable carrier and one or more discussed herein. 40 optional ingredients. The resulting uniformly blended mix Accordingly, in one embodiment, the invention relates to a ture may then be shaped or loaded into tablets, capsules, pills, pharmaceutical composition comprising a pharmaceutically canisters, cartridges, dispensers and the like using conven acceptable carrier and a compound of the invention. The tional procedures and equipment. compositions may contain other therapeutic and/or formulat In one embodiment, the pharmaceutical compositions are ing agents if desired. When discussing compositions, the 45 suitable for oral administration. Suitable compositions for “compound of the invention” may also be referred to hereinas oral administration may be in the form of capsules, tablets, the “active agent, to distinguish it from other components of pills, lozenges, cachets, dragees, powders, granules; Solu the formulation, such as the carrier. Thus, it is understood that tions or Suspensions in an aqueous or non-aqueous liquid; the term “active agent' includes compounds of formula I as oil-in-water or water-in-oil liquid emulsions; elixirs or Syr well as pharmaceutically acceptable salts, Solvates and pro 50 ups; and the like; each containing a predetermined amount of drugs of that compound. the active agent. The pharmaceutical compositions of the invention typi When intended for oral administration in a solid dosage cally contain a therapeutically effective amount of a com form (capsules, tablets, pills and the like), the composition pound of the invention. Those skilled in the art will recognize, will typically comprise the active agent and one or more however, that a pharmaceutical composition may contain 55 pharmaceutically acceptable carriers, such as Sodium citrate more than atherapeutically effective amount, such as in bulk or dicalcium phosphate. Solid dosage forms may also com compositions, or less thana therapeutically effective amount, prise: fillers or extenders, such as starches, microcrystalline that is, individual unit doses designed for multiple adminis cellulose, lactose. Sucrose, glucose, mannitol, and/or silicic tration to achieve a therapeutically effective amount. Typi acid; binders, such as carboxymethylcellulose, alginates, cally, the composition will contain from about 0.01-95 wt % 60 gelatin, polyvinyl pyrrolidone. Sucrose and/or acacia; humec of active agent, including, from about 0.01-30 wt %, such as tants, such as , disintegrating agents. Such as agar from about 0.01-10 wt %, with the actual amount depending agar, calcium carbonate, potato or tapioca Starch, alginic acid, upon the formulation itself, the route of administration, the certain silicates, and/or sodium carbonate; solution retarding frequency of dosing, and so forth. In one embodiment, a agents, such as paraffin, absorption accelerators, such as qua composition Suitable for an oral dosage form, for example, 65 ternary ammonium compounds; wetting agents, such as cetyl may contain about 5-70 wt %, or from about 10-60 wt % of alcohol and/or glycerol monostearate; absorbents, such as active agent. kaolin and/or bentonite clay; lubricants, such as talc, calcium US 8,686,184 B2 41 42 Stearate, magnesium Stearate, Solid polyethylene glycols, patient's respiratory tract. An exemplary nebulizer formula Sodium lauryl Sulfate, and/or mixtures thereof coloring tion comprises the active agent dissolved in a carrier to form agents; and buffering agents. a solution, or micronized and combined with a carrier to form Release agents, wetting agents, coating agents, Sweeten a suspension of micronized particles of respirable size. Dry ing, flavoring and perfuming agents, preservatives and anti powder inhalers administer the active agent as a free-flowing oxidants may also be present in the pharmaceutical compo powder that is dispersed in a patient’s air-stream during inspi sitions. Exemplary coating agents for tablets, capsules, pills ration. An exemplary dry powder formulation comprises the and like, include those used for enteric coatings, such as active agent dry-blended with an excipient Such as lactose, cellulose acetate phthalate, polyvinyl acetate phthalate, starch, mannitol, dextrose, polylactic acid, polylactide-co glycolide, and combinations thereof. Metered-dose inhalers hydroxypropyl methylcellulose phthalate, methacrylic acid 10 discharge a measured amount of the active agent using com methacrylic acid ester copolymers, cellulose acetate trimel pressed propellant gas. An exemplary metered-dose formu litate, carboxymethyl ethyl cellulose, hydroxypropyl methyl lation comprises a solution or Suspension of the active agent cellulose acetate Succinate, and the like. Examples of phar in a liquefied propellant. Such as a chlorofluorocarbon or maceutically acceptable antioxidants include: water-soluble hydrofluoroalkane. Optional components of Such formula antioxidants, such as ascorbic acid, cysteine hydrochloride, 15 tions include co-solvents, such as ethanol or pentane, and sodium bisulfate, sodium metabisulfate sodium sulfite and Surfactants, such as Sorbitan trioleate, oleic acid, lecithin, the like; oil-soluble antioxidants, such as ascorbyl palmitate, glycerin, and sodium lauryl Sulfate. Such compositions are butylated hydroxyanisole, butylated hydroxytoluene, leci typically prepared by adding chilled or pressurized hydrof thin, propyl gallate, alpha-tocopherol, and the like; and metal luoroalkane to a suitable container containing the active chelating agents, such as citric acid, ethylenediamine tet agent, ethanol (if present) and the Surfactant (if present). To raacetic acid, Sorbitol, tartaric acid, phosphoric acid, and the prepare a suspension, the active agent is micronized and then like. combined with the propellant. Alternatively, a Suspension Compositions may also be formulated to provide slow or formulation can be prepared by spray drying a coating of controlled release of the active agent using, by way of Surfactant on micronized particles of the active agent. The example, hydroxypropyl methyl cellulose in varying propor 25 formulation is then loaded into an aerosol canister, which tions or other polymer matrices, liposomes and/or micro forms a portion of the inhaler. spheres. In addition, the pharmaceutical compositions of the Compounds of the invention can also be administered parenterally (for example, by Subcutaneous, intravenous, invention may contain opacifying agents and may be formu intramuscular, or intraperitoneal injection). For such admin lated so that they release the active agent only, or preferen istration, the active agent is provided in a sterile solution, tially, in a certain portion of the gastrointestinal tract, option 30 Suspension, or emulsion. Exemplary solvents for preparing ally, in a delayed manner. Examples of embedding Such formulations include water, saline, low molecular compositions which can be used include polymeric sub weight alcohols such as propylene glycol, polyethylene gly stances and waxes. The active agent can also be in micro col, oils, gelatin, fatty acid esters such as ethyl oleate, and the encapsulated form, optionally with one or more of the above like. Parenteral formulations may also contain one or more described excipients. 35 anti-oxidants, solubilizers, stabilizers, preservatives, wetting Suitable liquid dosage forms for oral administration agents, emulsifiers, and dispersing agents. Surfactants, addi include, by way of illustration, pharmaceutically acceptable tional stabilizing agents or pH-adjusting agents (acids, bases emulsions, microemulsions, Solutions, Suspensions, syrups or buffers) and anti-oxidants are particularly useful to provide and elixirs. Liquid dosage forms typically comprise the active stability to the formulation, for example, to minimize or avoid agent and an inert diluent, Such as, for example, water or other 40 hydrolysis of ester and amide linkages that may be present in Solvents, Solubilizing agents and emulsifiers, such as ethyl the compound. These formulations may be rendered sterile by alcohol, , ethyl carbonate, ethyl acetate, use of a sterile injectable medium, a sterilizing agent, filtra benzyl alcohol, benzyl benzoate, propylene glycol. 1,3-buty tion, irradiation, or heat. In one particular embodiment, the lene glycol, oils (for example, cottonseed, groundnut, corn, parenteral formulation comprises an aqueous cyclodextrin germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl 45 Solution as the pharmaceutically acceptable carrier. Suitable cyclodextrins include cyclic molecules containing six or alcohol, polyethylene glycols andfatty acid esters of sorbitan, more C-D-glucopyranose units linked at the 1.4 positions by and mixtures thereof. Suspensions may contain Suspending a linkages as in amylase, B-cyclodextrin or cycloheptaamy agents such as, for example, ethoxylated isostearyl alcohols, lose. Exemplary cyclodextrins include cyclodextrin deriva polyoxyethylene Sorbitol and Sorbitan esters, microcrystal tives such as hydroxypropyl and sulfobutyl ether cyclodex line cellulose, aluminium metahydroxide, bentonite, agar 50 trins such as hydroxypropyl-3-cyclodextrin and Sulfobutyl agar and tragacanth, and mixtures thereof. ether B-cyclodextrin. Exemplary buffers for such formula When intended for oral administration, the pharmaceutical tions include carboxylic acid-based buffers such as citrate, compositions of the invention may be packaged in a unit lactate and maleate buffer solutions. dosage form. The term “unit dosage form' refers to a physi Compounds of the invention can also be administered cally discrete unit Suitable for dosing a patient, that is, each 55 transdermally using known transdermal delivery systems and unit containing a predetermined quantity of the active agent excipients. For example, the compound can be admixed with calculated to produce the desired therapeutic effect either permeation enhancers, such as propylene glycol, polyethyl alone or in combination with one or more additional units. For ene glycol monolaurate, azacycloalkan-2-ones and the like, example, such unit dosage forms may be capsules, tablets, and incorporated into a patch or similar delivery system. pills, and the like. 60 Additional excipients including gelling agents, emulsifiers In another embodiment, the compositions of the invention and buffers, may be used in Such transdermal compositions if are suitable for inhaled administration, and will typically be desired. in the form of an aerosol or a powder. Such compositions are generally administered using well-known delivery devices, Secondary Agents Such as a nebulizer, dry powder, or metered-dose inhaler. 65 Nebulizer devices produce a stream of high velocity air that The compounds of the invention may be useful as the sole causes the composition to spray as a mist that is carried into a treatment of a disease or may be combined with one or more US 8,686,184 B2 43 44 additional therapeutic agents in order to obtain the desired can be packaged separately or may be packaged together in a therapeutic effect. Thus, in one embodiment, pharmaceutical kit. When administered at separate times, the secondary agent compositions of the invention contain other drugs that are will typically be administered less than 24 hours after admin co-administered with a compound of the invention. For istration of the compound of the invention, ranging anywhere example, the composition may further comprise one or more from concurrent with administration of the compound of the drugs (also referred to as “secondary agents(s)). Such thera invention to about 24 hours post-dose. This is also referred to peutic agents are well known in the art, and include adenosine as sequential administration. Thus, a compound of the inven receptor antagonists, C.-adrenergic receptor antagonists, tion can be orally administered simultaneously or sequen f-adrenergic receptor antagonists, f-adrenergic receptor tially with another active agent using two tablets, with one agonists, dual-acting B-adrenergic receptor antagonist/ol-re 10 ceptor antagonists, advanced glycation end product breakers, tablet for each active agent, where sequential may mean being aldosterone antagonists, aldosterone synthase inhibitors, administered immediately after administration of the com aminopeptidase N inhibitors, androgens, angiotensin-con pound of the invention or at Some predetermined time later Verting enzyme inhibitors and dual-acting angiotensin-con (for example, one hour later or three hours later). It is also Verting enzyme/neprilysin inhibitors, angiotensin-converting 15 contemplated that the secondary agent may be administered enzyme 2 activators and stimulators, angiotensin-II Vaccines, more than 24 hours after administration of the compound of anticoagulants, anti-diabetic agents, antidiarrheal agents, the invention. Alternatively, the combination may be admin anti-glaucoma agents, anti-lipid agents, antinociceptive istered by different routes of administration, that is, one orally agents, anti-thrombotic agents, AT receptor antagonists and and the other by inhalation. dual-acting AT receptor antagonist/neprilysin inhibitors and In one embodiment, the kit comprises a first dosage form multifunctional angiotensin receptor blockers, bradykinin comprising a compound of the invention and at least one receptor antagonists, calcium channel blockers, chymase additional dosage form comprising one or more of the sec inhibitors, digoxin, diuretics, dopamine agonists, endothelin ondary agents set forth herein, in quantities sufficient to carry converting enzyme inhibitors, endothelin receptor antago out the methods of the invention. The first dosage form and nists, HMG-CoA reductase inhibitors, estrogens, estrogen 25 the second (or third, etc.) dosage form together comprise a receptor agonists and/or antagonists, monoamine reuptake therapeutically effective amount of active agents for the treat inhibitors, muscle relaxants, natriuretic peptides and their ment or prevention of a disease or medical condition in a analogs, natriuretic peptide clearance receptor antagonists, patient. neprilysin inhibitors, nitric oxide donors, non-steroidal anti Secondary agent(s), when included, are present in a thera inflammatory agents, N-methyl d-aspartate receptor antago 30 peutically effective amount such that they are typically nists, opioid receptor agonists, phosphodiesterase inhibitors, administered in an amount that produces a therapeutically prostaglandin analogs, prostaglandin receptoragonists, renin beneficial effect when co-administered with a compound of inhibitors, selective serotonin reuptake inhibitors, sodium the invention. The secondary agent can be in the form of a channel blocker, Soluble guanylate cyclase stimulators and pharmaceutically acceptable salt, Solvate, optically pure Ste activators, tricyclic antidepressants, vasopressin receptor 35 reoisomer, and so forth. The secondary agent may also be in antagonists, and combinations thereof. Specific examples of the form of a prodrug, for example, a compound having a these agents are detailed herein. carboxylic acid group that has been esterified. Thus, second Accordingly, in yet another aspect of the invention, a phar ary agents listed herein are intended to include all Such forms, maceutical composition comprises a compound of the inven and are commercially available or can be prepared using tion, a second active agent, and a pharmaceutically acceptable 40 conventional procedures and reagents. carrier. Third, fourth etc. active agents may also be included In one embodiment, compounds of the invention are in the composition. In combination therapy, the amount of administered in combination with an compound of the invention that is administered, as well as the antagonist, representative examples of which include, but are amount of secondary agents, may be less than the amount not limited to, naxifylline, rolofylline, SLV-320, theophyl typically administered in monotherapy. 45 line, and tonapofylline. Compounds of the invention may be physically mixed with In one embodiment, compounds of the invention are the second active agent to form a composition containing both administered in combination with an O-adrenergic receptor agents; or each agent may be present in separate and distinct antagonist, representative examples of which include, but are compositions which are administered to the patient simulta not limited to, doxazosin, prazosin, tamsulosin, and tera neously or at separate times. For example, a compound of the 50 Zosin. invention can be combined with a second active agent using Compounds of the invention may also be administered in conventional procedures and equipment to form a combina combination with a B-adrenergic receptor antagonist (“(B- tion of active agents comprising a compound of the invention blockers'). Representative B-blockers include, but are not and a second active agent. Additionally, the active agents may limited to, acebutolol, alprenolol, amoSulalol, arotinolol. be combined with a pharmaceutically acceptable carrier to 55 atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopin form a pharmaceutical composition comprising a compound dolol, bucindolol, bucumolol, bufetolol, bufuralol, bunitrolol, of the invention, a second active agent and a pharmaceutically bupranolol, bubridine, butofilolol, carazolol, carteolol, acceptable carrier. In this embodiment, the components of the carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, composition are typically mixed or blended to create a physi epanolol, esmolol, indenolol, labetolol, levobunolol, mepin cal mixture. The physical mixture is then administered in a 60 dolol, metipranolol, metoprolol such as metoprolol Succinate therapeutically effective amount using any of the routes and metoprolol tartrate, moprolol, nadolol, nadoxolol. described herein. nebivalol, nipradillol, oxprenolol, penbutolol, perbutolol, pin Alternatively, the active agents may remain separate and dolol, practolol, pronethalol, propranolol, Sotalol, Sufinalol, distinct before administration to the patient. In this embodi talindol, tertatolol, tilisolol, timolol, toliprolol. xibenolol, and ment, the agents are not physically mixed together before 65 combinations thereof. In one particular embodiment, the administration but are administered simultaneously or at B-antagonist is selected from atenolol, bisoprolol, meto separate times as separate compositions. Such compositions prolol, propranolol, Sotalol, and combinations thereof. Typi US 8,686,184 B2 45 46 cally, the B-blocker will be administered in an amount suf also be included. In one particular embodiment, the ACE/ ficient to provide from about 2-900 mg per dose. NEP inhibitor is selected from: AVE-7688, enalaprilat, fasi In one embodiment, compounds of the invention are dotril, fasidotrilate, omapatrilat, Sampatrilat, and combina administered in combination with a B-adrenergic receptor tions thereof. agonist, representative examples of which include, but are not 5 In one embodiment, compounds of the invention are limited to, albuterol, bitolterol, fenoterol, formoterol, inda administered in combination with an angiotensin-converting caterol, isoetharine, levalbuterol, metaproterenol, pirbuterol, enzyme 2 (ACE2) activator or stimulator. salbutamol, Salmefamol, salmeterol, terbutaline, Vilanterol, In one embodiment, compounds of the invention are and the like Typically, the f-adrenoreceptor agonist will be administered in combination with an angiotensin-II Vaccine, administered in an amount Sufficient to provide from about 10 examples of which include, but are not limited to ATR12181 0.05-500 g per dose. and CYT006-AngOb. In one embodiment, compounds of the invention are In one embodiment, compounds of the invention are administered in combination with an advanced glycation end administered in combination with an anticoagulant, represen product (AGE) breaker, examples of which include, by way tative examples of which include, but are not limited to: of illustration and not limitation, alagebrium (or ALT-711), 15 coumarins such as warfarin; heparin; and direct thrombin and TRC4149. inhibitors such as argatroban, bivalirudin, dabigatran, and In another embodiment, compounds of the invention are lepirudin. administered in combination with an aldosterone antagonist, In yet another embodiment, compounds of the invention representative examples of which include, but are not limited are administered in combination with an anti-diabetic agent. to, , , and combinations thereof. Representative anti-diabetic agents include injectable drugs Typically, the aldosterone antagonist will be administered in as well as orally effective drugs, and combinations thereof. an amount sufficient to provide from about 5-300 mg per day. Examples of injectable drugs include, but are not limited to, In one embodiment, compounds of the invention are insulin and insulin derivatives. Examples of orally effective administered in combination with an aminopeptidase N or drugs include, but are not limited to: biguanides such as dipeptidyl peptidase III inhibitor, examples of which include, 25 metformin; glucagon antagonists; C-glucosidase inhibitors by way of illustration and not limitation, bestatin and PC18 Such as acarbose and miglitol; dipeptidyl peptidase IV inhibi (2-amino-4-methylsulfonylbutane thiol, methionine thiol). tors (DPP-IV inhibitors) such as alogliptin, denagliptin, lina Compounds of the invention can also be administered in gliptin, saxagliptin, Sitagliptin, and Vildagliptin: meglitinides combination with an angiotensin-converting enzyme (ACE) Such as repaglinide; oxadiazolidinediones; Sulfonylureas inhibitor. Representative ACE inhibitors include, but are not 30 Such as chlorpropamide, glimepiride, glipizide, glyburide, limited to, accupril, alacepril, benazepril, benazeprilat, cap and tolaZamide; thiazolidinediones such as pioglitaZone and topril, ceranapril, cilazapril, delapril, enalapril, enalaprilat, rosiglitazone; and combinations thereof. fosinopril, fosinoprilat, imidapril, lisinopril, moexipril, In another embodiment, compounds of the invention are monopril, moveltopril, pentopril, perindopril, quinapril, administered in combination with antidiarrheal treatments. quinaprilat, ramipril, ramiprilat, Saralasin acetate, spirapril, 35 Representative treatment options include, but are not limited temocapril, trandolapril, Zofenopril, and combinations to, oral rehydration solutions (ORS), loperamide, diphenoxy thereof. In a particular embodiment, the ACE inhibitor is late, and bismuth Sabsalicylate. selected from: benazepril, captopril, enalapril, lisinopril, In yet another embodiment, a compound of the invention is ramipril, and combinations thereof. Typically, the ACE administered in combination with an anti-glaucoma agent. inhibitor will be administered in an amount sufficient to pro 40 Representative anti-glaucoma agents include, but are not lim vide from about 1-150 mg per day. ited to: C.-adrenergic agonists such as brimonidine; B-adren In another embodiment, compounds of the invention are ergic receptor antagonists; topical f-blockers such as betax administered in combination with a dual-acting angiotensin olol, levobunolol, and timolol; carbonic anhydrase inhibitors converting enzyme/neprilysin (ACE/NEP) inhibitor, Such as , brinzolamide, or dorzolamide; cho examples of which include, but are not limited to: AVE-0848 45 linergic agonists such as cevimeline and DMXB-anabaseine; ((4S,7S.12bR)-7-3-methyl-2 (S)-sulfanylbutyramido)-6- epinephrine compounds; miotics Such as pilocarpine; and oxo-1,2,3,4,6,7,8,12b-octahydropyrido 2,1-a2-benza prostaglandin analogs. Zepine-4-carboxylic acid); AVE-7688 (ilepatril) and its par In yet another embodiment, compounds of the invention ent compound; BMS-182657 (2-2-oxo-3(S)-3-phenyl-2 are administered in combination with an anti-lipid agent. (S)-sulfanylpropionamido)-2,3,4,5-tetrahydro-1H-1- 50 Representative anti-lipid agents include, but are not limited benzazepin-1-yl)acetic acid); CGS-35601 (N-1-4-methyl-2 to: cholesteryl ester transfer protein inhibitors (CETPs) such (S)-sulfanylpentanamidocyclopentylcarbonyl-L- as anacetrapib, dalcetrapib, and torcetrapib; statins such as tryptophan); fasidotril; fasidotrilate; enalaprilat; ER-32935 atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin ((3R,6S.9aR)-6-3(S)-methyl-2(S)-sulfanylpentanamido-5- and simvastatin; and combinations thereof. oXoperhydrothiazolo3.2-aaZepine-3-carboxylic acid); 55 In one embodiment, compounds of the invention are gempatrilat; MDL-101264 ((4S,7S.12bR)-7-2(S)-(2-mor administered in combination with an anti-thrombotic agent. pholinoacetylthio)-3-phenylpropionamido-6-oxo-1,2,3,4,6, Representative anti-thrombotic agents include, but are not 7,8,12b-octahydropyrido2,1-a2benzazepine-4-carboxy limited to: aspirin; anti-platelet agents such as , lic acid); MDL-101287 (4S-4C,7c.(R*),12b?3-7-2- , and ; heparin, and combinations (carboxymethyl)-3-phenylpropionamido-6-oxo-1,2,3,4,6,7, 60 thereof. 8,12b-octahydropyrido2,1-a2benzazepine-4-carboxylic In one embodiment, compounds of the invention are acid); omapatrilat; RB-105 (N-2(S)-(mercaptomethyl)-3 administered in combination with an AT receptor antagonist, (R)-phenylbutyl-L-alanine); sampatrilat; SA-898 ((2R, also known as angiotensin II type 1 receptor blockers (ARBs). 4R)—N-2-(2-hydroxyphenyl)-3-(3-mercaptopropionyl) Representative ARBs include, but are not limited to, abite thiazolidin-4-ylcarbonyl-L-phenylalanine); Sch-50690 (N- 65 Sartan, azilsartan (e.g., azilsartan medoxomil), benzyllosar 1(S)-carboxy-2-N2-(methanesulfonyl)-L-lysylamino tan, candesartan, candesartancilexetil, elisartan, embusartan, ethyl-L-Valyl-L-tyrosine); and combinations thereof, may enoltaSosartan, eprosartan, EXP3174, fonsartan, forasartan, US 8,686,184 B2 47 48 glycyllosartan, irbesartan, isoteoline, losartan, medoximil, acid and other phenoxyacetic acid compounds Such as tienilic milfasartan, olmesartan (e.g., olmesartan medoxomil), acid, and quincarbate; osmotic diuretics such as opomisartan, pratosartan, ripisartan, Saprisartan, Saralasin, mannitol; potassium-sparing diuretics, which include aldos sarmesin, TAK-591, tasosartan, telmisartan, Valsartan, Zola terone antagonists such as spironolactone, and Na channel Sartan, and combinations thereof. In a particular embodiment, 5 inhibitors such as and ; and the ARB is selected from azilsartan medoxomil, candesartan thiazide-like diuretics such as althiazide, bendroflumethiaz cilexetil, eprosartan, irbesartan, losartan, olmesartan medox ide, benzylhydrochlorothiazide, benzthiazide, buthiazide, omil, irbesartan, Saprisartan, tasosartan, telmisartan, Valsar chlorthalidone, , cyclopenthiazide, cyclothiaz tan, and combinations thereof. Exemplary salts and/or pro ide, epithiazide, ethiazide, fenduizone, flumethiazide, hydro drugs include candesartan cilexetil, eprosartan mesylate, 10 chlorothiazide, , , methyl losartan potassium salt, and olmesartan medoxomil. Typi clothiazide, , , paraflutizide, cally, the ARB will be administered in an amount sufficient to , , teclothiazide, and trichlorome provide from about 4-600 mg per dose, with exemplary daily thiazide; and combinations thereof. In a particular embodi dosages ranging from 20-320 mg per day. ment, the diuretic is selected from amiloride, , Compounds of the invention may also be administered in 15 chlorothiazide, chlorthalidone, dichlorphenamide, combination with a dual-acting agent, such as an AT receptor ethacrynic acid, , , hydroflu antagonist/neprilysin inhibitor (ARB/NEP) inhibitor, methiazide, indapamide, methylclothiazide, metolaZone, examples of which include, but are not limited to, compounds torsemide, triamterene, and combinations thereof. The described in U.S. Publication Nos. 2008/02693.05 and 2009/ diuretic will be administered in an amount sufficient to pro 0023228, both to Allegretti et al. filed on Apr. 23, 2008, such 20 vide from about 5-50 mg per day, more typically 6-25 mg per as the compound, 4-2-ethoxy-4-ethyl-5-(S)-2-mercapto-4- day, with common dosages being 6.25 mg, 12.5 mg or 25 mg methylpentanoylamino)-methyl)imidazol-1-ylmethyl-3- per day. fluorobiphenyl-2-carboxylic acid. Compounds of the invention may also be administered in Compounds of the invention may also be administered in combination with an endothelin converting enzyme (ECE) combination with multifunctional angiotensin receptor 25 inhibitor, examples of which include, but are not limited to, blockers as described in Kurtz & Klein (2009) Hypertension phosphoramidon, CGS 26303, and combinations thereof. Research 32.826-834. In a particular embodiment, compounds of the invention In one embodiment, compounds of the invention are are administered in combination with an endothelin receptor administered in combination with a bradykinin receptor antagonist. Representative endothelin receptor antagonists antagonist, for example, icatibant (HOE-140). It is expected 30 include, but are not limited to: selective endothelin receptor that this combination therapy may present the advantage of antagonists that affect endothelin A receptors, such as avosen preventing angioedema or other unwanted consequences of tan, ambrisentan, atrasentan, BQ-123, clazosentan, darusen elevated bradykinin levels. tan, sitaxentan, and Zibotentan; and dual endothelin receptor In one embodiment, compounds of the invention are antagonists that affect both endothelin A and B receptors, administered in combination with a calcium channel blocker. 35 Such as bosentan, macitentan, tezosentan). Representative calcium channel blockers include, but are not In yet another embodiment, a compound of the invention is limited to, amlodipine, anipamil, aranipine, barnidipine, ben administered in combination with one or more HMG-CoA cyclane, benidipine, bepridil, clentiazem, cilnidipine, cin reductase inhibitors, which are also known as statins. Repre narizine, diltiazem, efonidipine, elgodipine, etafenone, felo sentative statins include, but are not limited to, atorvastatin, dipine, fendiline, flunarizine, gallopamil. isradipine, 40 fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin lacidipine, lercanidipine, lidoflazine, lomerizine, manid and simvastatin. ipine, mibefradil, nicardipine, nifedipine, niguldipine, nilu In one embodiment, compounds of the invention are dipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, administered in combination with a monoamine reuptake nivaldipine, perhexyline, prenylamine, ryosidine, semotiadil, inhibitor, examples of which include, by way of illustration terodiline, tiapamil, Verapamil, and combinations thereof. In 45 and not limitation, norepinephrine reuptake inhibitors such as a particular embodiment, the calcium channel blocker is atomoxetine, buproprion and the buproprion metabolite selected from amlodipine, bepridil, diltiazem, felodipine, hydroxybuproprion, maprotiline, reboxetine, and Viloxazine; isradipine, lacidipine, nicardipine, nifedipine, niguldipine, selective serotonin reuptake inhibitors (SSRIs) such as citalo niludipine, nimodipine, nisoldipine, ryosidine, Verapamil, pram and the citalopram metabolite desmethylcitalopram, and combinations thereof. Typically, the calcium channel 50 dapoxetine, escitalopram (e.g., escitalopram oxalate), fluox blocker will be administered in an amount sufficient to pro etine and the fluoxetine desmethyl metabolite norfluoxetine, vide from about 2-500 mg per dose. fluvoxamine (e.g., fluvoxamine maleate), paroxetine, Sertra In one embodiment, compounds of the invention are line and the sertraline metabolite demethylsertraline; dual administered in combination with a chymase inhibitor, Such serotonin-norepinephrine reuptake inhibitors (SNRIs) such as TPC-806 and 2-(5-formylamino-6-oxo-2-phenyl-1,6-di- 55 as bicifadine, dulloxetine, milnacipran, nefazodone, and ven hydropyrimidine-1-yl)-N-3,4-dioxo-1-phenyl-7-(2-py lafaxine; and combinations thereof. ridyloxy)-2-heptyl)acetamide (NK3201). In another embodiment, compounds of the invention are In one embodiment, compounds of the invention are administered in combination with a muscle relaxant, administered in combination with a diuretic. Representative examples of which include, but are not limited to: carisopro diuretics include, but are not limited to: carbonic anhydrase 60 dol, chlorZoxaZone, cyclobenzaprine, diflunisal, metaxalone, inhibitors such as acetazolamide and dichlorphenamide; loop methocarbamol, and combinations thereof. diuretics, which include derivatives such as In one embodiment, compounds of the invention are acetazolamide, ambuside, azosernide, bumetanide, butazola administered in combination with a natriuretic peptide or mide, chloraminophenamide, , , clor analog, examples of which include but are not limited to: exolone, disulfamide, ethoxolamide, furosemide, , 65 carperitide, CD-NP (Nile Therapeutics), CU-NP. nesiritide, methazolamide, , torsemide, tripamide, and Xipa PL-3994 (Palatin Technologies, Inc.), ularitide, cenderitide, mide, as well as non-sulfonamide diuretics such as ethacrynic and compounds described in Ogawa et al (2004) J. Biol. US 8,686,184 B2 49 50 Chem. 279:28625-31. These compounds are also referred to organic nitrates such as pentaerythritol tetranitrate; and Syd as natriuretic peptide receptor-A (NPR-A) agonists. In nonimines such as linsidomine and molsidomine. another embodiment, compounds of the invention are admin In yet another embodiment, compounds of the invention istered in combination with a natriuretic peptide clearance are administered in combination with a non-steroidal anti receptor (NPR-C) antagonist such as SC-46542, cANF inflammatory agent (NSAID). Representative NSAIDs (4-23), and AP-811 (Veale (2000) Bioorg Med Chem Lett include, but are not limited to: acemetacin, acetyl salicylic 10:1949-52). For example, AP-811 has shown synergy when acid, alclofenac, alminoprofen, amfenac, amiprilose, amox combined with the NEP inhibitor, thiorphan (Wegner (1995) iprin, anirolac, apaZone, azapropaZone, benorilate, benox Clin. Exper. Hypert. 17:861-876). aprofen, bezpiperylon, broperamole, bucloxic acid, carpro 10 fen, clidanac, diclofenac, diflunisal, diftalone, enolicam, In another embodiment, compounds of the invention are etodolac, etoricoxib, fenbufen, fenclofenac, fenclozic acid, administered in combination with a neprilysin (NEP) inhibi fenoprofen, fentiazac, feprazone, flufenamic acid, flufenisal, tor. Representative NEP inhibitors include, but are not limited fluprofen, flurbiprofen, furofenac, ibufenac, ibuprofen, to: AHU-377; candoxatril; candoxatrilat; dexecadotril ((+)- N-2(R)-(acetylthiomethyl)-3-phenylpropionylglycine ben indomethacin, indoprofen, isoxepac, isoxicam, ketoprofen, 15 ketorolac, lofemizole, lornoxicam, meclofenamate, meclofe Zyl ester); CGS-24128 (3-3-(biphenyl-4-yl)-2-(phospho namic acid, mefenamic acid, meloxicam, mesalamine, miro nomethylamino)propionamidopropionic acid); CGS-24592 profen, mofebutaZone, nabumetone, naproxen, niflumic acid, ((S)-3-3-(biphenyl-4-yl)-2-(phosphonomethylamino)propi oxaprozin, oXpinac, oxyphenbutaZone, phenylbutaZone, onamidopropionic acid); CGS-25155 (N-9(R)-(acetylthi piroXicam, pirprofen, pranoprofen, Salsalate, Sudoxicam, Sul omethyl)-10-oxo-1-azacyclodecan-2(S)-ylcarbonyl-4(R)- fasalazine, Sulindac, Suprofen, tenoxicam, tiopinac, tiapro hydroxy-L-proline benzyl ester);3-(1-carbamoylcyclohexyl) fenic acid, tioxaprofen, tolfenamic acid, tolmetin, triflumi propionic acid derivatives described in WO 2006/027680 to date, Zidometacin, Zomepirac, and combinations thereof. In a Hepworth et al. (Pfizer Inc.); JMV-390-1 (20R)-benzyl-3-(N- particular embodiment, the NSAID is selected from etodolac, hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine); eca flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, dotril; phosphoramidon; retrothiorphan; RU-42827 (2-(mer 25 meloxicam, naproxen, oxaprozin, piroxicam, and combina captomethyl)-N-(4-pyridinyl)benzenepropionamide); tions thereof. RU-44004 (N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl) In one embodiment, compounds of the invention are propionamide); SCH-32615 ((S)- N—N-(1-carboxy-2- administered in combination with an N-methyl d-aspartate phenylethyl)-L-phenylalanyl-3-alanine) and its prodrug (NMDA) receptor antagonist, examples of which include, by SCH-34826 ((S) N—N-1-(2,2-dimethyl-1,3-dioxolan 30 way of illustration and not limitation, including amantadine, 4-yl)methoxycarbonyl-2-phenylethyl-L-phenylalanyl-B- dextromethorphan, dextropropoxyphene, ketamine, ketobe alanine); sialorphin; SCH-42495 (N-2(S)-(acetylsulfanylm midone, memantine, methadone, and so forth. ethyl)-3-(2-methylphenyl)propionyl-L-methionine ethyl In still another embodiment, compounds of the invention ester); spinorphin; SQ-28132 (N-2-(mercaptomethyl)-1- are administered in combination with an opioid receptorago oxo-3-phenylpropylleucine); SQ-28.603 (N-2-(mercaptom 35 nist (also referred to as opioid analgesics). Representative ethyl)-1-oxo-3-phenylpropyl-B-alanine); SQ-29072 (7-2- opioid receptor agonists include, but are not limited to: (mercaptomethyl)-1-oxo-3-phenylpropylaminoheptanoic buprenorphine, butorphanol, codeine, dihydrocodeine, fenta acid); thiorphan and its prodrug racecadotril:UK-69578 (cis nyl, hydrocodone, hydromorphone, levallorphan, levorpha 4-1-2-carboxy-3-(2-methoxyethoxy)propylcyclopentyl nol, meperidine, methadone, morphine, nalbuphine, carbonyl)aminocyclohexanecarboxylic acid); UK-447.841 40 nalmefene, nalorphine, naloxone, naltrexone, nalorphine, (2-1-3-(4-chlorophenyl)propylcarbamoyl-cyclopentylm oxycodone, oxymorphone, pentazocine, propoxyphene, tra ethyl-4-methoxybutyric acid); UK-505,749 ((R)-2-methyl madol, and combinations thereof. In certain embodiments, 3-(1-3-(2-methylbenzothiazol-6-yl)propylcarbamoyl the opioid receptoragonist is selected from codeine, dihydro cyclopentyl)propionic acid); 5-biphenyl-4-yl-4-(3- codeine, hydrocodone, hydromorphone, morphine, oxyc carboxypropionylamino)-2-methylpentanoic acid and 45 odone, oxymorphone, tramadol, and combinations thereof. 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methyl In a particular embodiment, compounds of the invention pentanoic acid ethyl ester (WO 2007/056546); daglutril (3S, are administered in combination with a phosphodiesterase 2R)-3-1-2'-(ethoxycarbonyl)-4'-phenylbutyl-cyclopen (PDE) inhibitor, particularly a PDE-V inhibitor. Representa tan-1-carbonylamino)-2,3,4,5-tetrahydro-2-oxo-1H-1- tive PDE-V inhibitors include, but are not limited to, avanafil. benzazepine-1-acetic acid described in WO 2007/106708 to 50 lodenafil, mirodenafil, sildenafil (Revatio(R), tadalafil (Ad Khder et al. (Novartis AG); and combinations thereof. In a circaR), Vardenafil (Levitra(R), and udenafil. particular embodiment, the NEP inhibitor is selected from In another embodiment, compounds of the invention are AHU-377, candoxatril, candoxatrilat, CGS-24128, phos administered in combination with a prostaglandin analog phoramidon, SCH-32615, SCH-34826, SQ-28603, thior (also referred to as prostanoids or prostacyclin analogs). Rep phan, and combinations thereof. In a particular embodiment, 55 resentative prostaglandin analogs include, but are not limited the NEP inhibitor is a compound such as daglutril or CGS to, beraprost sodium, bimatoprost, epoprostenol, iloprost, 26303 (N-2-(biphenyl-4-yl)-1 (S)-(1H-tetrazol-5-yl)ethyl latanoprost, tafluprost, travoprost, and treprostinil, with aminomethylphosphonic acid), which have activity both as bimatoprost, latanoprost, and tafluprost being of particular inhibitors of the endothelin converting enzyme (ECE) and of interest. NEP. Other dual acting ECE/NEP compounds can also be 60 In yet another embodiment, compounds of the invention used. The NEP inhibitor will be administered in an amount are administered in combination with a prostaglandin recep sufficient to provide from about 20-800 mg per day, with toragonist, examples of which include, but are not limited to, typical daily dosages ranging from 50-700 mg per day, more bimatoprost, latanoprost, travoprost, and so forth. commonly 100-600 or 100-300 mg per day. Compounds of the invention may also be administered in In one embodiment, compounds of the invention are 65 combination with a renin inhibitor, examples of which administered in combination with a nitric oxide donor, include, but are not limited to, alliskiren, enalkiren, remikiren, examples of which include, but are not limited to nicorandil; and combinations thereof. US 8,686,184 B2 51 52 In another embodiment, compounds of the invention are with starch (89 mg), microcrystalline cellulose (89 mg) and administered in combination with a selective serotonin magnesium Stearate (2 mg). The mixture is then passed reuptake inhibitor (SSRI). Representative SSRIs include, but through a No. 45 mesh U.S. sieve and loaded into a hard are not limited to: citalopram and the citalopram metabolite gelatin capsule (200 mg of composition per capsule). desmethylcitalopram, dapoxetine, escitalopram (e.g., escit Alternately, a compound of the invention (30 g), a second allopram oxalate), fluoxetine and the fluoxetine desmethyl ary agent (20g), 440 g spray-dried lactose and 10 g magne metabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine sium Stearate are thoroughly blended, and processed as maleate), paroxetine, Sertraline and the Sertraline metabolite described above. demethylsertraline, and combinations thereof. In one embodiment, compounds of the invention are 10 Exemplary Gelatin Capsule Formulation for Oral administered in combination with a 5-HT serotonin recep Administration toragonist, examples of which include, by way of illustration and not limitation, triptans Such as almotriptan, avitriptan, A compound of the invention (100 mg) is thoroughly eletriptan, froVatriptan, naratriptan rizatriptan, Sumatriptan, blended with polyoxyethylene sorbitan monooleate (50 mg) and Zolmitriptan. 15 and starch powder (250mg). The mixture is then loaded into In one embodiment, compounds of the invention are a gelatin capsule (400 mg of composition per capsule). Alter administered in combination with a sodium channel blocker, nately, a compound of the invention (70 mg) and a secondary examples of which include, by way of illustration and not agent (30 mg) are thoroughly blended with polyoxyethylene limitation, carbamazepine, fosphenyloin, lamotrignine, Sorbitan monooleate (50 mg) and Starch powder (250 mg), lidocaine, mexiletine, Oxcarbazepine, phenyloin, and combi and the resulting mixture loaded into a gelatin capsule (400 nations thereof. mg of composition per capsule). In one embodiment, compounds of the invention are Alternately, a compound of the invention (40 mg) is thor administered in combination with a soluble guanylate cyclase oughly blended with microcrystalline cellulose (Avicel PH stimulator or activator, examples of which include, but are not 103: 259.2 mg) and magnesium stearate (0.8 mg). The mix limited to ataciguat, riociguat, and combinations thereof. 25 ture is then loaded into a gelatin capsule (Size #1, White, In one embodiment, compounds of the invention are Opaque) (300 mg of composition per capsule). administered in combination with a tricyclic antidepressant (TCA), examples of which include, by way of illustration and Exemplary Tablet Formulation for Oral not limitation, amitriptyline, amitriptylinoxide, butriptyline, Administration clomipramine, demexiptiline, desipramine, dibenzepin, 30 dimetacrine, doSulepin, doxepin, imipramine, imipraminox A compound of the invention (10 mg), starch (45 mg) and ide, lofepramine, melitracen, metapramine, nitroxazepine, microcrystalline cellulose (35 mg) are passed through a No. nortriptyline, noxiptiline, pipofezine, propizepine, protrip 20 mesh U.S. sieve and mixed thoroughly. The granules so tyline, quinupramine, and combinations thereof. produced are dried at 50-60° C. and passed through a No. 16 In one embodiment, compounds of the invention are 35 mesh U.S. sieve. A Solution of polyvinylpyrrolidone (4 mg as administered in combination with a vasopressin receptor a 10% solution in sterile water) is mixed with sodium car antagonist, examples of which include, by way of illustration boxymethyl starch (4.5 mg), magnesium Stearate (0.5 mg), and not limitation, conivaptain and . and talc (1 mg), and this mixture is then passed through a No. Combined secondary therapeutic agents may also be help 16 mesh U.S. sieve. The sodium carboxymethyl starch, mag ful in further combination therapy with compounds of the 40 nesium Stearate and talc are then added to the granules. After invention. For example, compounds of the invention can be mixing, the mixture is compressed on a tablet machine to combined with a diuretic and an ARB, or a calcium channel afford a tablet weighing 100 mg. blocker and an ARB, or a diuretic and an ACE inhibitor, or a Alternately, a compound of the invention (250mg) is thor calcium channel blocker and a statin. Specific examples oughly blended with microcrystalline cellulose (400 mg), include, a combination of the ACE inhibitor enalapril (in the 45 silicon dioxide fumed (10 mg), and Stearic acid (5 mg). The maleate salt form) and the diuretic hydrochlorothiazide, mixture is then compressed to form tablets (665 mg of com which is sold under the mark Vaseretic(R), or a combination of position per tablet). the calcium channel blocker amlodipine (in the besylate salt Alternately, a compound of the invention (400 mg) is thor form) and the ARB olmesartan (in the medoxomil prodrug oughly blended with cornstarch (50 mg), croScarmellose form), or a combination of a calcium channel blocker and a 50 Sodium (25 mg), lactose (120mg), and magnesium Stearate (5 statin, all may also be used with the compounds of the inven mg). The mixture is then compressed to form a single-scored tion. Other therapeutic agents such as C.-adrenergic receptor tablet (600 mg of composition per tablet). agonists and vasopressin receptor antagonists may also be Alternately, a compound of the invention (100 mg) is thor helpful in combination therapy. Exemplary C.-adrenergic oughly blended with cornstarch (100 mg) with an aqueous receptor agonists include clonidine, dexmedetomidine, and 55 Solution of gelatin (20 mg). The mixture is dried and ground guanfacine. to a fine powder. Microcrystalline cellulose (50 mg) and The following formulations illustrate representative phar magnesium Stearate (5 mg) are then admixed with the gelatin maceutical compositions of the invention. formulation, granulated and the resulting mixture com pressed to form tablets (100 mg of the compound of the Exemplary Hard Gelatin Capsules for Oral 60 invention per tablet). Administration Exemplary Suspension Formulation for Oral A compound of the invention (50 g), 440 g spray-dried Administration lactose and 10g magnesium Stearate are thoroughly blended. The resulting composition is then loaded into hard gelatin 65 The following ingredients are mixed to form a Suspension capsules (500 mg of composition per capsule). Alternately, a containing 100 mg of the compound of the invention per 10 compound of the invention (20 mg) is thoroughly blended mL of Suspension: US 8,686,184 B2 53 54 Solution is administered using a nebulizer device that pro vides about 10 ug to about 500 lug of the compound of the Ingredients Amount invention per dose. Compound of the invention 1.0 g Fumaric acid 0.5 g. 5 EXAMPLES Sodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 g The following Preparations and Examples are provided to Granulated Sugar 25.5 g illustrate specific embodiments of the invention. These spe Sorbitol (70% solution) 12.85 g cific embodiments, however, are not intended to limit the Veegum (R) K (magnesium aluminum silicate) 1.0 g 10 Flavoring O.O35 mL. Scope of the invention in any way unless specifically indi Colorings 0.5 mg cated. The following abbreviations have the following mean Distilled water q.S. to 100 mL. ings unless otherwise indicated and any other abbreviations used herein and not defined have their standard, generally accepted meaning: 15 Exemplary Liquid Formulation for Oral AcOHacetic acid Administration Cbz carbobenzyloxy ( C(O)O-benzyl) DCM dichloromethane (i.e., methylene chloride) DCM dichloromethane or methylene chloride A suitable liquid formulation is one with a carboxylic DIPEAN,N-diisopropylethylamine acid-based buffer such as citrate, lactate and maleate buffer Solutions. For example, a compound of the invention (which DMF N,N-dimethylformamide may be pre-mixed with DMSO) is blended with a 100 mM Dnp 2,4-dinitrophenyl ammonium citrate buffer and the pH adjusted to pH 5, or is EDCI N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide blended with a 100 mM citric acid solution and the pH EtOAc ethyl acetate adjusted to pH 2. Such solutions may also include a solubi 25 EtOH ethanol lizing excipient such as a cyclodextrin, for example the solu HATU N.N.N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl) tion may include 10 wt % hydroxypropyl-P-cyclodextrin. uronium hexafluorophosphate Other suitable formulations include a 5% NaHCO solu HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic tion, with or without cyclodextrin. acid 30 HOBt 1-hydroxybenzotriazole hydrate Mca (7-methoxycoumarin-4-yl)acyl Exemplary Injectable Formulation for MeCN acetonitrile Administration by Injection MeOH methanol Pd(PPh) tetrakis(triphenylphosphine)palladium(0) A compound of the invention (0.2g) is blended with 0.4M 35 TFA trifluoroacetic acid sodium acetate buffer solution (2.0 mL). The pH of the result THF tetrahydrofuran ing Solution is adjusted to pH 4 using 0.5 Naqueous hydro Unless noted otherwise, all materials, such as reagents, chloric acid or 0.5 N aqueous Sodium hydroxide, as neces starting materials and solvents, were purchased from com sary, and then sufficient water for injection is added to provide mercial suppliers (such as Sigma-Aldrich, Fluka Riedel-de a total volume of 20 mL. The mixture is then filtered through 40 Haén, and the like) and were used without further purifica a sterile filter (0.22 micron) to provide a sterile solution suit tion. able for administration by injection. Reactions were run under nitrogen atmosphere, unless noted otherwise. The progress of reactions were monitored by Exemplary Compositions for Administration by thin layer chromatography (TLC), analytical high perfor Inhalation 45 mance liquid chromatography (anal. HPLC), and mass spec trometry, the details of which are given in specific examples. A compound of the invention (0.2 mg) is micronized and Solvents used in analytical HPLC were as follows: solvent A then blended with lactose (25 mg). This blended mixture is was 98% HO/2% MeCN/1.0 mL/LTFA: solvent B was 90% then loaded into a gelatin inhalation cartridge. The contents of MeCN/10% HO/1.0 mL/L TFA. the cartridge are administered using a dry powder inhaler, for 50 Reactions were worked up as described specifically in each example. preparation for example; commonly reaction mixtures were purified by extraction and other purification methods such as Alternately, a micronized compound of the invention (10g) temperature-, and solvent-dependent crystallization, and pre is dispersed in a solution prepared by dissolving lecithin (0.2 cipitation. In addition, reaction mixtures were routinely puri g) indemineralized water (200 mL). The resulting Suspension 55 fied by preparative HPLC, typically using Microsorb C18 and is spray dried and then micronized to form a micronized Microsorb BDS column packings and conventional eluents. composition comprising particles having a mean diameter Progress of reactions was typically measured by liquid chro less than about 1.5 Lum. The micronized composition is then matography mass spectrometry (LCMS). Characterization of loaded into metered-dose inhaler cartridges containing pres isomers were done by Nuclear Overhauser effect spectros Surized 1,1,1,2-tetrafluoroethane in an amount Sufficient to 60 copy (NOE). Characterization of reaction products was rou provide about 10 ug to about 500 g of the compound of the tinely carried out by mass and H-NMR spectrometry. For invention per dose when administered by the inhaler. NMR measurement, samples were dissolved in deuterated Alternately, a compound of the invention (25 mg) is dis solvent (CDOD, CDC1, or DMSO-d), and H-NMR spec solved in citrate buffered (pH 5) isotonic saline (125 mL). The tra were acquired with a Varian Gemini 2000 instrument (400 mixture is stirred and Sonicated until the compound is dis 65 MHz) under standard observation conditions. Mass spectro solved. The pH of the solution is checked and adjusted, if metric identification of compounds was typically conducted necessary, to pH 5 by slowly adding aqueous 1 NNaOH. The using an electrospray ionization method (ESMS) with an US 8,686,184 B2 55 56 Applied Biosystems (Foster City, Calif.) model API 150 EX To a solution of compound (1) (80.0g, 240 Limol) in DCM instrument or an Agilent (Palo Alto, Calif.) model 1200 (500 mL) was added EDCI (50.6 g.,264 umol), HOBt (35.6 g. LC/MSD instrument. 264 umol), N.O-dimethylhydroxylamine hydrochloride (51.5 g, 528 umol) and triethylamine (111 mL, 790 umol). The mixture was stirred at room temperature overnight. Then the mixture was washed with 2N HCl (3x500 mL) and satu Preparation 1 rated aqueous NaHCOs (3x500 mL), respectively. The organic layer was dried over anhydrous NaSO and concen (R)-1-(2-Chloro-benzyl)-2-cyano-2-hydroxy-ethyl trated to yield compound (2) as a yellow oil (71.0 g). carbamic Acid Benzyl Ester 10 To a suspension of LiAlH4 (7.2g, 188 umol) in THF (800 mL) was added dropwise a solution of compound (2) (71.0 g, 188 umol) in THF (200 mL) at -20° C. The mixture was stirred at -20°C. for 2 hours. Then the reaction was quenched C 15 carefully with 1N HC1. The mixture was extracted with EtOAc (2x600 mL) and the combined extracts were dried -e- over anhydrous Na2SO4 and concentrated to yield compound (3) (59.0 g), which was used directly without further purifi cation. HN1 NcooH To a solution of compound (3) (59.0g, 188 umol) in THF C (500 mL) was added aqueous NaHSO (19.5g in 500 mL of water) and the mixture was stirred at 0°C. overnight. NaCN Hs (9.2g, 188 umol) was added and the resulting mixture was stirred for 3 hours. The mixture was extracted with EtOAc 25 (2x500 mL) and the combined extracts were dried over anhy Cbz-N1)-COOH drous NaSO and concentrated to yield the title compound (64.0 g), which was used directly without further purification. (1) C 30

-er Preparation 2 (2R,3R)-3-Amino-4-(2-chloro-phenyl)-2-hydroxy 35 butyric acid Methyl Ester

C C C 40

HN -N-- 45 5 H

50 A mixture of (R)-1-(2-chloro-benzyl)-2-cyano-2-hy droxy-ethyl-carbamic acid benzyl ester (55.0g, 157 umol) in dioxane (300 mL) and 6N HCl (300 mL) was heated under reflux overnight. The solvent was removed under reduced To a suspension of (R)-2-amino-3-(2-chloro-phenyl)-pro 55 pressure and the residue was dissolved in a 3NHC1-MeOH pionic acid (100.0 g, 0.5 mol) in water (1 L) was added solution. The resulting mixture was refluxed for 4 hours and dropwise 4N aqueous NaOH (125 mL) at 0°C. Then a solu the solvent was removed under reduced pressure. The residue tion of N-(benzyloxycarbonyloxy)succinimide (125.0 g, 0.5 was taken up in EtOAc (500 mL) and aqueous NaHCO, (500 mol) in acetone (300 mL) was added in one portion. The pH mL). The organic layer was separated, dried over anhydrous of the mixture was maintained at 8-9 by addition of 3N 60 Na2SO and concentrated to give a mixture of the title com aqueous NaOH. After the mixture was stirred for 4 hours, the pound and its (R.S)-isomer, which was subjected to flash pH was adjusted to 1 with 6N HCl and the mixture was column chromatography (DCM:MeOH=100:1 to 50:1) to extracted with EtOAc (2x500 mL). The combined extracts yield the title compound (8.1 g). were washed with 1 NHCl (2x500 mL), dried over anhydrous 65 H NMR (CDC1): 8 7.37 (m, 1H), 7.22 (m, 3H), 4.30 (d. Na2SO and concentrated to yield compound (1) as a white J=3.3 Hz, 1H), 3.80 (s.3H), 3.51 (m, 1H), 2.96 (m. 1H), 2.71 solid (155.0 g). (m. 1H), 2.06 (brs, 2H). MS (m/z): 244 M+H". US 8,686,184 B2 57 58 Preparation 3 Example 1 (2R,3R)-3-Amino-4-(2-chloro-phenyl)-2-hydroxy butyric Acid Ethyl Ester H R C O O 10

C HN -Nulls, 1N 15 5 H 2-3-(1R,2R)-2-Carboxy-1-(2-chlorobenzyl)-2- hydroxy-ethylcarbamoyl-propyl-benzoic Acid (R-OH) A mixture of (R)-1-(2-chloro-benzyl)-2-cyano-2-hy 2-(3-Carboxy-propyl)benzoic acid (335 mg, 1.6 mmol. 1.0 eq.) was combined with DIPEA (897 uL, 3.2 eq.) and HATU droxy-ethyl-carbamic acid benzyl ester (64.0g, 188 umol) in (0.612g, 1.61 mmol. 1.0 eq.) in DCM (40 mL). The resulting dioxane (300 mL) and 6N HCl (300 mL) was heated under mixture was stirred for 10 minutes and (2R,3R)-3-amino-4- reflux overnight. The solvent was removed under reduced (2-chloro-phenyl)-2-hydroxy-butyric acid butyl ester (HCl pressure and the residue was dissolved in a 3N HC1-EtOH 25 salt, 519 mg, 1.6 mmol. 1.0 eq.) was added. The reaction was solution. The resulting mixture was refluxed for 4 hours and quenched after 1 hour by adding saturated NHCl and the the solvent was removed under reduced pressure. The residue product was extracted with DCM, dried and evaporated. The product was dissolved into AcOH (5 mL) and purified using was taken up in EtOAc (500 mL) and aqueous NaHCO, (500 preparative HPLC. The clean fractions were combined and mL). The organic layer was separated, dried over anhydrous lyophilized to yield 2-3-(1R,2R)-2-butoxycarbonyl-1-(2- Na2SO and concentrated to give a mixture of the title com 30 chloro-benzyl)-2-hydroxy-ethylcarbamoyl-propyl-ben pound and its (R.S)-isomer, which was subjected to flash Zoic acid (150 mg). column chromatography (DCM:MeOH=100:1 to 50:1) to 2-3-(1R,2R)-2-Butoxycarbonyl-1-(2-chloro-benzyl)-2- yield the title compound (9.7g). hydroxy-ethylcarbamoyl-propyl-benzoic acid (110 mg. 230 umol. 1.0 eq.) was combined withTHF (2.1 mL), and 1 M "H NMR (CDC1): 8 7.37 (m, 1H), 7.22 (m, 3H), 4.25 (m, 35 aqueous NaOH (480 uL 2.0 eq.). The mixture was stirred at 3H), 3.50 (s, 1H), 2.97 (d. J=10.8 Hz, 1H), 2.74 (t, J=11.4 Hz, room temperature for 1 hour. Upon completion of the reaction 1H), 2.06 (brs, 2H), 1.35 (t, J=7.1 Hz, 3H). MS (m/z): 258 was completed, AcOH (1 mL) was added and the product was M+H". purified by preparative HPLC. The clean fractions were com bined and lyophilized to yield the title compound (95 mg. 40 99% purity). MS m/z. M+H" calc'd for CHCINO, 420.11. found 420. Preparation 4 2-3-(1R,2R)-1-(2-Chlorobenzyl)-2-ethoxycarbo nyl-2-hydroxy-ethylcarbamoyl-propyl-benzoic (2R,3R)-3-amino-4-(2-chloro-phenyl)-2-hydroxy 45 Acid (R'—OCHCH.) butyric Acid Butyl Ester 2-(3-Carboxy-propyl)benzoic acid (50 mg, 240 umol 1.0 eq.) was combined with DIPEA (134 uL, 3.2 eq.) and HATU (91.3 mg, 240 umol. 1.0 eq.) in DCM (5 mL). (2R,3R)-3- Amino-4-(2-chloro-phenyl)-2-hydroxy-butyric acid ethyl H 50 ester (69.9 mg, 240 umol. 1.0 eq.) was added and the mixture was stirred. The reaction was quenched after 1 hour by adding N-1 nu saturated NHCl and the product was extracted with DCM, dried and evaporated. The product was purified using pre O parative HPLC to yield the title compound (30 mg, 100% 55 purity). MS m/z. M+H" calc’d for CHCINO. 448.14. found 448. 2-3-(1R,2R)-2-Butoxycarbonyl-1-(2-chloroben Zyl)-2-hydroxy-ethylcarbamoyl-propyl-benzoic 60 Acid (R'—O(CH2)CH) 2-(3-Carboxy-propyl)benzoic acid (300 mg, 1.4 mmol. 1.0 (2R,3R)-3-Amino-4-(2-chloro-phenyl)-2-hydroxy-butyric eq.) was combined with DIPEA (803 uL, 3.2 eq.) and HATU acid ethyl ester (100 mg, 0.4 mmol) was combined with (548 mg, 1.4 mmol. 1.0 eq.) in DCM (30 mL). The mixture 1-butanol (5 mL) and concentrated HCl (1 mL) and heated at 65 was stirred for 10 min and (2R,3R)-3-amino-4-(2-chloro 70° C. for 24 hours. Excess solvent was removed to yield the phenyl)-2-hydroxy-butyric acid butyl ester (412 mg, 1.4 title compound as an HCl salt. mmol. 1.0 eq.) was added. The reaction was quenched after 1 US 8,686,184 B2 59 60 hour by adding saturated NHCl and the product was 3-Bromo-4,5-difluoro-benzoic acid methyl ester (194 mg. extracted with DCM, dried and evaporated. AcOH (1 mL) 772 umol. 1.0 eq.) was mixed with 2-t-butoxycarbonylphe was added and the resulting product was purified by prepara nylboronic acid pinacol ester (282 mg. 927 umol. 1.2 eq.). tive HPLC to yield the title compound (260 mg, 99% purity). Toluene (1.1 mL) was added followed by MeOH (409 uL), MS m/z. M+H" calc’d for CHCINO. 476.18. found 5 then KCO (214 mg, 1.5 mmol. 2.0 eq.) predissolved in 476.2. water (202 LL). The mixture was stirred, and the reaction vessel was capped, placed under vacuum and purged with Example 2 nitrogen. Tetrakis(triphenylphosphine)palladium(0) (89.2 mg, 77.2 umol) was added. The vessel was recapped and 10 microwaved at 110° C. for 50 minutes. The organic layer was removed and the product was evaporated under vacuum. THF (Va) (3 mL) and 1 Maqueous NaOH (1.5 mL, 2.0 eq.) was added OH and the mixture was stirred for 2 hours. EtOAc was added and mixture was acidified with saturated NHC1. The organic 15 layers were extracted, dried and evaporated to yield 5'6'- difluoro-biphenyl-2,3'-dicarboxylic acid 2-t-butyl ester. 5",6'-Difluoro-biphenyl-2,3'-dicarboxylic acid 2-t-butyl ester (60 mg, 180 umol. 1.0 eq.), (2R,3R)-3-amino-4-(2- chloro-phenyl)-2-hydroxy-butyric acid ethyl ester (46.3 mg, 180 umol. 1.0 eq.), DIPEA (94 uL), and HATU (68.4 mg, 180 umol) were combined in DCM (2 mL) and was stirred for 45 Following the procedures described in the examples minutes at room temperature. The reaction was quenched herein, and Substituting the appropriate starting materials and with saturated NHCl and extracted with DCM, dried and reagents, compounds having formula Va were prepared: evaporated. 1:1 DCM:TFA (0.5 mL each) was added and the 25 mixture was stirred for 30 minutes. The solvent was evapo rated under vacuum. THF (2 mL) and 10N NaOH (0.3 mL) were added and the mixture was stirred for 2 hours. The R4 reaction was quenched with AcOH and the product was puri R4 fied by preparative HPLC to yield the title compound (5.8 mg, ^S 30 97% purity). MS m/z. M+H" calc’d for C.H.ClFNO ré, 490.08. found 491. 2 Example 4

35 (2R,3R)-4-(2-Chlorophenyl)-3-4-ethyl-3-(6-methyl 2,4-dioxo-1,4-dihydro-2H-thieno 2,3-dipyrimidin-3- yl)-benzoylamino-2-hydroxy-butyric Acid

MS miz: M + H 40 Ex. RI R4 R Formula calcd found 1 OH COOH F CHCIFNO 438.10 438.4 OH O 1.2-3-(1R,2R)-2-Carboxy-1-(2-chlorobenzyl)-2-hydroxy HO N ethylcarbamoyl-propyl-4-fluoro-benzoic acid 45 n N NH O E O Example 3 O 4N 5'-(1R,2R)-2-Carboxy-1-(2-chlorobenzyl)-2-hy droxy-ethylcarbamoyl-2',3'-difluoro-biphenyl-2- 50 C carboxylic Acid Methyl 2-isocyanato-5-methylthiophene-3-carboxylate (116 mg, 588 umol. 1.5 eq.) was dissolved in DMF (1000 uL) 55 then combined with 3-amino-4-ethyl-benzoic acid (64.8 mg, 392 umol. 1.0 eq.) and DIPEA (137 uL 2.0 eq.). The mixture was heated at 70° C. overnight. The solvent was removed and H the product was combined with DMF (500 uL) and HATU Y (298 mg, 784 umol. 2.0 eq.). The mixture was stirred at room 60 temperature for 15 minutes before adding (2R,3R)-3-Amino 4-(2-chloro-phenyl)-2-hydroxy-butyric acid ethyl ester (TFA O O O salt, 146 mg, 392 umol. 1.0 eq.) in DIPEA (137 uL 2.0 eq.) and DMF (500 uL). The mixture was stirred overnight at OH room temperature. The solvent was removed and the product C 65 was dissolved in THF (600 uL) and 2 Maqueous NaOH (600 uL). The mixture was heated at 80°C. overnight. The product was purified by preparative HPLC to yield the title compound US 8,686,184 B2 61 62 (12.3 mg, 100% purity). MS m/z. M+H" calc’d for MeOH (2 mL) and stirred. Hydroxylamine hydrochloride CHCINOS, 542.11. found 542.0. (118 mg, 1.69 mmol. 1.5 eq.) in MeCH (1 mL) was added to the stirred solution. While maintaining the temperature at 0° Example 5 C., 25 wt % NaOCH, in MeOH (25:75, NaOCH:MeOH, 1.29 mL, 5.64 mmol. 5.0 eq.) was slowly added. The solution was stirred for 1 hour until the reaction was complete. Excess Solvent was removed and the product was purified by prepara tive LC (1" column; gradient 2-50% MeCN/HO, 0.05% TFA, over 80 minutes; 15 mL/min flow; UV 280 nm). The ')N / 10 fractions were collected, frozen, and lyophilized to yield the OH NN title compound as a white solid (30 mg, 100% purity). MS m/z. M+H calcd for CHCINOs 445.12. found 445.6. Example 6 O E O 15

C R4 (IIa) OH Sé, (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-4-(3- R NR J exJ' methyl-5-oxo-4,5-dihydro-pyrazol-1-yl)-benzoy R6 lamino-butyric acid (R'—OH) O O 25 4-(3-Methyl-5-oxo-2-pyrazolin-1-yl)benzoic acid (42 mg, 0.2 mmol. 1.0 eq.) was combined with DMF (500 uL) and C HATU (88 mg 0.2 mmol. 1.2 eq.) and stirred at room tem perature for 15 minutes. (2R,3R)-3-Amino-4-(2-chloro-phe nyl)-2-hydroxy-butyric acid methyl ester (47 mg, 0.2 mmol. 30 Following the procedures described in the examples 1.0 eq.) in DIPEA (70 uL, 2.0 eq.) and DMF (500 uL) was herein, and Substituting the appropriate starting materials and then added and the resulting mixture was stirred overnight at reagents, compounds having formula IIa, were prepared: room temperature. THF (500 uL) and 2 Maqueous NaOH (600 uL. 1 mmol) was added and the mixture was heated at 50° C. overnight. The solvent was removed and the product 35 R6 was dissolved in AcOH/HO (50%) and purified by prepara tive HPLC to yield the title compound (58.3 mg, 56% purity). R4 R5 MS m/z. M+H" calc’d for CHCINOs, 430.11. found 43 O.O. Sé,H-R O 40 2a (2R,3R)-4-(2-Chlorophenyl)-2-hydroxy-3-4-(3- R6 O methyl-5-oxo-4,5-dihydro-pyrazol-1-yl)-benzoy lamino-butyric acid ethyl ester (R'—OCH2CH) OH 4-(3-Methyl-5-oxo-2-pyrazolin-1-yl)benzoic acid (100 45 mg, 0.6 mmol. 1.0 eq.) was combined with DMF (1000 uL) and HATU (250 mg, 0.7 mmol. 1.2 eq.) and stirred at room temperature for 15 minutes. (2R,3R)-3-Amino-4-(2-chloro phenyl)-2-hydroxy-butyric acid ethyl ester (TFA salt, 200 MS miz: M + HT mg, 0.6 mmol. 1.0 eq.) in DIPEA (200LL, 2.0 eq.) and DMF 50 Ex. RI R R6 Formula calcd found (1000 uL) was then added and the resulting mixture was 1 OH H F C2HgCIFNO 472.09 472.O stirred overnight at room temperature. Excess solvent was 2 OH H C C24H10Cl2NO6 488.06 488.O removed and the product was dissolved in AcOH/HO (50%) 3 OH C H C2H10Cl2NO6 488.06 488.O and purified by preparative LC (1" column; gradient 2-50% 4 OH F CI CHCl2FNO. 506.05 507.0 MeCN/HO, 0.05% TFA, over 80 minutes; 15 mL/min flow: 55 5 OH CH3 H C25H2CINO 468.11 468.2 UV 280 nm). The fractions were collected, frozen, and lyo 6 OH CH3 H C25H2CINO 468.11 468.2 philized to yield the title compound (100 mg, 98% purity). 7 OH OCH3 H C25H2CINO, 484.11 48S.O MS m/z. M+H calc’d for CHCINOs, 458.14. found 4582. 1. 5'-(1R,2R)-2-Carb oxy-1-(2-chlorobenzyl)-2-hydroxy 60 ethylcarbamoyl-3'-fluoro-biphenyl-2-carboxylic acid N-(1R,2R)-1-(2-Chlorobenzyl)-2-hydroxy-2-hy 2. 5'-(1R,2R)-2-Carb oxy-1-(2-chlorobenzyl)-2-hydroxy droxycarbamoyl-ethyl-4-(3-methyl-5-oxo-4,5-dihy ethylcarbamoyl-3'-chloro-biphenyl-2-carboxylic acid dro-pyrazol-1-yl)-benzamide (R'=-NH(OH) 3. 5'-(1R,2R)-2-Carb oxy-1-(2-chlorobenzyl)-2-hydroxy ethylcarbamoyl-2'-chloro-biphenyl-2-carboxylic acid (2R,3R)-4-(2-Chlorophenyl)-2-hydroxy-3-4-(3-methyl 65 4. 5'-(1R,2R)-2-Carb oxy-1-(2-chlorobenzyl)-2-hydroxy 5-oxo-4,5-dihydro-pyrazol-1-yl)-benzoylamino-butyric ethylcarbamoyl-3'-chloro-2'-fluoro-biphenyl-2-carboxy acid ethyl ester (517 mg, 1.13 mmol. 1.0 eq.) was dissolved in lic acid

US 8,686,184 B2 65 66 Preparation 5 (2x300 mL) and the combined extracts were dried over anhy drous NaSO and concentrated to yield compound (4) (32.1 (2R,3R)-3-Amino-2-hydroxy-4-(2-trifluoromethyl g, quantitative), which was used directly without further puri phenyl)-butyric Acid Ethyl Ester fication.

CF CF CF

Hs H HN 1Noooh CbzHN1 NCOOH can- No (1) | (2)

CF CF CF

a -e- HN -n-COOEt coin-ry: CN can : H 6H OH O (4) (3)

30 To a suspension of (R)-2-amino-3-(2-trifluoromethyl-phe A mixture of compound (4) (32.1 g, 85 mol) in dioxane nyl)-propionic acid (25.0 g, 107 umol) in water (230 mL) was (200 mL) and 6N HCl (200 mL) was heated under reflux added dropwise 4N aqueous NaOH (25 mL) at 0°C. Then a overnight. The solvent was removed under reduced pressure solution of N-(benzyloxycarbonyloxy)succinimide (26.8 g. 35 and the residue was dissolved in a 3N HC1-EtOH solution. 107 umol) in acetone (100 mL) was added in one portion. The The resulting mixture was refluxed overnight and the solvent pH of the mixture was maintained at 8-9 by addition of 3N aqueous NaOH. After the mixture was stirred for 4 hours, the was removed under reduced pressure. The residue was taken pH was adjusted to 1 with 6N HCl and the mixture was up in EtOAc (300 mL) and aqueous NaHCO (300 mL). The organic layer was separated, dried over anhydrous Na2SO extracted with EtOAc (2x200 mL). The combined extracts 40 were washed with 1 NHCl (2x200 mL), dried over anhydrous and concentrated to give a mixture of the title compound and NaSO and concentrated to yield compound (1) as a white its (R.S)-isomer, which was subjected to flash column chro solid (35.0 g). matography (DCM:MeOH=100:1 to 50:1) to yield the title To a solution of compound (1) (35.0 g, 95 umol) in DCM compound (6.0 g). (300 mL) was added EDCI (20.0g, 104 umol), HOBt (14.0 g, 45 104 umol), N.O-dimethylhydroxylamine hydrochloride "H NMR (CDC1): 8 7.68 (d. J=7.8 Hz, 1H), 7.52 (t, J–7.5 (20.0g, 208 Limol) and triethylamine (33.0g, 312 umol). The HZ, 1H), 7.37 (m, 2H), 4.31 (m, 3H), 3.42 (m, 1H), 3.00 (dd. mixture was stirred at room temperature overnight. Then the J=2.4, 14.4 Hz, 1H), 2.79 (m, 1H), 1.37 (t, J=7.1 Hz, 3H). MS mixture was washed with 2N HCl (3x250 mL) and saturated (m/z): 292 M+H". aqueous NaHCOs (3x200 mL), respectively. The organic 50 layer was dried over anhydrous NaSO and concentrated to Example 7 yield compound (2) (35.0 g). To a suspension of LiAlH4 (3.3 g, 85 umol) in THF (300 mL) was added dropwise a solution of compound (2) (35.0 g, 55 85umol) in THF (150 mL) at -20°C. The mixture was stirred at -20° C. for 2.5 hours. Then the reaction was quenched carefully with 1N HC1. The mixture was extracted with EtOAc (2x300 mL) and the combined extracts were dried over anhydrous NaSO and concentrated to yield compound 60 (3) (29.8 g), which was used directly without further purifi cation. To a solution of compound (3) (29.8 g. 85 umol) in THF (250 mL) was added aqueous NaHSO (8.84 g in 250 mL of water) and the mixture was stirred at 0°C. overnight. NaCN 65 (4.2 g, 85 umol) was added and the resulting mixture was stirred for 3 hours. The mixture was extracted with EtOAc US 8,686,184 B2 67 68 2-3-(1R,2R)-2-Ethoxycarbonyl-2-hydroxy-1-(2- and 10 Maqueous NaOH (134 uL, 4.0 eq.). The reaction was trifluoromethyl-benzyl)-ethylcarbamoyl-propyl stirred for 4 hours. Upon completion of the reaction was benzoic Acid (R'—OCHCH) completed, AcOH (1.5 mL) was added and the product was purified by preparative HPLC to yield the title compound (50 2-(3-Carboxy-propyl)benzoic acid (50 mg, 240 umol. 1.0 5 mg, 95% purity). MS m/z. M+H" calc'd for CHNO eq.) was combined with DIPEA (134 uL, 3.2 eq) and HATU 386.15. found 386.2. (91.3 mg, 240 umol. 1.0 eq.) in DCM (5 mL). (2R,3R)-3- Amino-2-hydroxy-4-(2-trifluoromethyl-phenyl)-butyric acid 2-3-((1R,2R)-1-Benzyl-2-butoxycarbonyl-2-hy ethyl ester (69.9 mg, 240 umol. 1.0 eq.) was added and the droxy-ethylcarbamoyl)-propyl-benzoic Acid resulting mixture was stirred for 1 hour. The reaction was 10 (R'= O(CH),CH) quenched by adding saturated NHCl and the product was extracted with DCM, dried, evaporated, and purified by pre (2R,3R)-3-t-Butoxycarbonylamino-2-hydroxy-4-phenyl parative HPLC to yield the title compound (40 mg, 100% butyric acid (244 mg. 827 umol. 1.0 eq.) and 1-butanol (4 mL) purity). MS m/z. M+H" calc'd for CHFNO. 482.17. 15 were combined in 4 MHCl in 1,4-dioxane (1 mL), and stirred found 482. for 2 hours at 60° C. The solvent was evaporated and the reaction mixture was azeotroped with toluene. 2-(3-Carboxy 2-3-(1R,2R)-2-Carboxy-2-hydroxy-1-(2-trifluo propyl)benzoic acid (160 mg, 790 umol. 1.0 eq.), DIPEA romethyl-benzyl)-ethylcarbamoyl-propyl-benzoic (439 uL, 3.2 eq.), and HATU (300 mg, 788 umol. 1.0 eq.) Acid (R'= OH) were combined in DCM (20 mL) and stirred for 5 minutes at room temperature. This mixture was then added to the azeo 2-3-(1R,2R)-2-Ethoxycarbonyl-2-hydroxy-1-(2-trifluo troped mixture and stirred for 1 hour. The solvent was evapo romethyl-benzyl)-ethylcarbamoyl-propyl-benzoic acid rated and the crude material was dissolved in AcOH (2 mL) was combined withTHF (5 mL) and 10 Maqueous NaOH (96 and purified using preparative HPLC to yield the title com LL, 4.0 eq.). The reaction was stirred for 4 hours. Upon 25 pound (80 mg, 99% purity). MS m/z. M+H" calc’d for completion of the reaction was completed, AcOH (1.5 mL) CHNO. 442.22. found 442.2. was added and the product was purified by preparative HPLC to yield the title compound (10 mg, 100% purity). MS m/z. Example 9 M+H calcd for CHFNO. 454.14. found 454. 30

Example 8

g H R

O O 40

5'-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcar bamoyl)-2'-fluoro-biphenyl-2-carboxylic Acid 45 (R'= OH) 2-3-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hy droxy-ethylcarbamoyl)-propyl-benzoic Acid 3-Bromo-4-fluorobenzoic acid (109 mg. 497 umol. 1.0 (R'= OCHCH.) eq.), DIPEA (273 uL, 3.2eq.), HATU (189 mg,497 umol. 1.0 eq.), and (2R,3R)-3-amino-2-hydroxy-4-phenyl-butyric acid 2-(3-Carboxy-propyl)benzoic acid (69.9 mg, 336 umol. 50 ethyl ester (129 mg. 497 umol. 1.0 eq.) were combined in 1.0 eq.) was combined with DIPEA (187 uL, 3.2 eq.) and DCM (2 mL) and stirred for 1 hour. The crude reaction was HATU (128 mg,336 umol. 1.0 eq.) in DCM (7 mL). (2R,3R)- then chromatographed (0-80% EtOAc/hexanes gradient) to 3-Amino-2-hydroxy-4-phenyl-butyric acid ethyl ester (75 yield (2R,3R)-3-(3-bromo-4-fluoro-benzoylamino)-2-hy mg, 340 umol. 1.0 eq.) was added and the resulting mixture droxy-4-phenyl-butyric acid ethyl ester. was stirred for 1 hour. The reaction was quenched by adding 55 2-t-Butoxycarbonylphenylboronic acid pinacol ester (173 saturated NHCl and the product was extracted with DCM, mg, 568 umol. 1.2 eq.) and toluene (661 LL) were added to dried, and evaporated. One third of the crude product (was (2R,3R)-3-(3-bromo-4-fluoro-benzoylamino)-2-hydroxy-4- dissolved in AcOH (1 mL) and purified by preparative HPLC phenyl-butyric acid ethyl ester (201 mg, 474 umol. 1.0 eq.), to yield the title compound (12 mg, 95% purity). MS m/z. followed by EtOH (361 LIL) then KCO (131 mg,947 umol. M+H calcd for CH, NO. 414.18. found 414.2. 60 2.0 eq) predissolved in water (124 ul). The mixture was stirred to aid in dissolution. The reaction vessel was purged 2-3-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethyl with nitrogen then Pd(PPh3)4 (54.7 mg, 47.4 umol) was added carbamoyl)-propyl-benzoic Acid (R'= OH) quickly. The reaction vessel was capped and heated to 100° C. for 30 minutes. The organics were removed and evaporated Two thirds of the crude 2-3-((1R,2R)-1-benzyl-2-ethoxy 65 under vacuum to yield 5'-((1R,2R)-1-benzyl-2-ethoxycarbo carbonyl-2-hydroxy-ethylcarbamoyl)-propyl-benzoic acid nyl-2-hydroxy-ethylcarbamoyl)-2'-fluoro-biphenyl-2-car for the previous example, was combined with THF (8 mL) boxylic acid t-butyl ester. US 8,686,184 B2 69 70 5'-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy-eth ylcarbamoyl)-2'-fluoro-biphenyl-2-carboxylic acid t-butyl ester (100 mg, 192 umol. 1.0 eq.) was combined with 1:1 MS miz: TFA/DCM (1 mL each) and stirred for 1 hour. The solvents M+ H were evaporated and THF (3 mL) and 1 M aqueous NaOH Ex. Ra R Formula calcd found (577 uL, 3.0 eq.) were added. The mixture was stirred for 2 OH CH CH 428.13 428O hours and EtOAc was added. The aqueous was extracted out OH H H 366.14 366.2 and the organic was washed with NaOH (0.2 mL), stirred and OH H CF 434.13 434.O the aqueous extracted. The aqueous was acidified to pH ~5 OH CH CH 394.17 394.2 with concentrated HCl yielding a gummy Solid, which was 10 dissolved in AcOH and purified by preparative HPLC to yield the title compound (12.7 mg, 99% purity). MS m/z. M+H" . (2R,3R)-3-4-(4-Chloro-3,5-dimethyl-pyrazol-1-yl)-ben calc’d for CHFNO. 438.13. found 438. Zoylamino-2-hydroxy-4-phenyl-butyric acid . (2R,3R)-2-Hydroxy-4-phenyl-3-(4-pyrazol-1-yl-benzoy 5'-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy 15 lamino)-butyric acid (TFA salt) ethylcarbamoyl)-2'-fluoro-biphenyl-2-carboxylic (2R,3R)-2-Hydroxy-4-phenyl-3-4-(3-trifluoromethyl Acid (R'—OCHCH.) pyrazol-1-yl)-benzoylamino-butyric acid (TFA salt) 5'-((1R,2R)-1-benzyl-2-ethoxycarbonyl-2-hydroxy-ethyl (2R,3R)-3-4-(3,5-Dimethyl-pyrazol-1-yl)-benzoy carbamoyl)-2'-fluoro-biphenyl-2-carboxylic acid t-butyl lamino-2-hydroxy-4-phenyl-butyric acid (TFA salt) ester (100 mg, 192 umol. 1.0 eq.) was combined with 1:1 TFA/DCM (1 mL each) and stirred for 1 hour. The solvent was evaporated and THF (3 mL) and 1 Maqueous NaOH (577 R4 uL, 3.0 eq.) were added. The mixture was stirred for 2 hours 25 as and EtOAc was added. The aqueous was extracted and the ’s organic was washed with NaOH (0.2 mL), stirred, and the 2a aqueous was extracted. The aqueous were acidified to pH~5 R6 with concentrated HCl to yield a gummy Solid. The aqueous was removed and the gummy solid was dissolved in AcOH 30 and purified by preparative HPLC to yield the title compound Ra (11.6 mg, 99% purity). MS m/z. M+H" calc'd for CHFNO. 466. 16. found 466.2. 35 Example 10 MS miz: M+H

Ex. R. R Re Re Formula calcd found (IIc) OH H CH CH 394.17 394.2 40 OH F CH CH 412.16 412.2 OH CH CH CH 408.18 4082 OH CH H COOH 424.14 424.2

45 5. (2R,3R)-3-3-(3,5-Dimethyl-pyrazol-1-yl)-benzoy lamino-2-hydroxy-4-phenyl-butyric acid (TFA salt) 6. (2R,3R)-3-3-(3,5-Dimethylpyrazol-1-yl)-4-fluoroben Zoylamino-2-hydroxy-4-phenyl-butyric acid (TFA salt) 7. (2R,3R)-3-3-(3,5-Dimethylpyrazol-1-yl)-4-methylben 50 Zoylamino-2-hydroxy-4-phenyl-butyric acid (TFA salt) Following the procedures described in the examples 8. 1-5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxyethylcar herein, and Substituting the appropriate starting materials and bamoyl)-2-methyl-phenyl)-1H-pyrazole-3-carboxylic reagents, compounds having formula IIc, were prepared as acid (TFA salt) the parent compound or as a TFA salt: 55

RC R4 sas Na ’s R4 60 N aS 2aR6 C Rb ’s Ra Ra 2aR6 65 OH

US 8,686,184 B2 75 76 -continued

Ex. RI Formula calcd found

42 OCHCH 424.18 424.2

43 OH C2 HoN2O 365.14 365.2

OH 379.16 379.2

45 OH C20H2 NSO 396.16 396.2

46 OH 380.15 380.2

47 OH 394.17 394.2

48 OH 434.15 434.O

OH

49 OH 434.15 434.4

OH

50 OH 434.15 434.4

OH

US 8,686,184 B2 85 86 Example 11 was added and the resulting mixture was stirred for 30 min utes. The solvents were evaporated and THF (3 mL) and 1 M HO aqueous NaOH (706 uL, 3.0 eq.) were added. The mixture O O was stirred for 2 hours and EtOAc was added. HO The aqueous was extracted out and the organic was washed with NaOH (0.2 mL), stirred and the aqueous extracted. The s aqueous was acidified to pH-5 with concentrated HCl yield ing a gummy Solid, which was dissolved in AcOH and puri 10 fied by preparative HPLC to yield the title compound (50 mg. 95% purity). MS m/z. M+H" calc’d for CHCINOs, C 452.12. found 452.0.

Example 13 2-3-((R)-1-Carboxymethyl-2-(2-chlorophenyl)- 15 ethylcarbamoyl-propyl-benzoic Acid (IIf) 2-(3-Carboxy-propyl)benzoic acid (160 mg, 790 umol. 1.0 eq.), DIPEA (439 uL, 3.2 eq) and HATU (300 mg, 788 umol. 1.0 eq.) were combined in DCM (20 mL) and stirred for 5 minutes at room temperature. (R)-3-amino-4-(2-chloro-phe nyl)-butyric acid ethyl ester (200 mg. 827 umol. 1.1 eq.) was added and the resulting mixture was stirred for 1 hour. The reaction was quenched with saturated NHCl, and the product was extracted with DCM, dried and evaporated. THF (20 mL) 25 and 10 Maqueous NaOH (315 uL) was added and the mixture was stirred for 1 hour. The crude material was dissolved in AcOH (1.5 mL) and purified using preparative HPLC to yield the title compound (25 mg, 95% purity). MS m/z. M+H" Following the procedures described in the examples calc’d for CHCINOs, 404.12. found 404.4. 30 herein, and Substituting the appropriate starting materials and reagents, compounds having formula IIf were prepared: Example 12

35 2. O 40

MS miz: M-H 5'-(R)-2-Carboxy-1-(2-chloro-benzyl)-ethylcarbam 45 oyl-2'-methyl-biphenyl-2-carboxylic Acid Ex. R R R Formula calcd found 1 OH H CHCOOH CHCINOs 452.12 453.2 3-Bromo-4-methylbenzoic acid (331 mg, 1.5 mmol. 1.0 2 OH H COOH C2H2CINOs 438.10 439.2 3 OH CH CHCOOH CHCINOs 466.13 466.2 eq.) and (R)-2-(2-chloro-phenyl)-1-ethoxycarbonylmethyl 50 ethyl-ammonium chloride (428 mg, 1.5 mmol. 1.0 eq.) were combined with DIPEA (844 uL, 3.2 eq) and HATU (585 mg. 1. (R)-3-(2-Carboxymethyl-biphenyl-3-carbonyl)-amino 1.5 mmol. 1.0 eq.) in DCM (8 mL). The resulting mixture was 4-(2-chloro-phenyl)-butyric acid stirred for 1 hour, then chromatographed (0-80% EtOAc/ hexanes gradient) to yield (R)-3-(3-bromo-4-methyl-benzoy 55 2. 3'-(R)-2-Carboxy-1-(2-chloro-benzyl)-ethylcarbamoyl lamino)-4-(2-chloro-phenyl)-butyric acid ethyl ester. biphenyl-2-carboxylic acid (R)-3-(3-Bromo-4-methyl-benzoylamino)-4-(2-chloro 3. (R)-3-(2-Carboxymethyl-6-methyl-biphenyl-3-carbo phenyl)-butyric acid ethyl ester (103 mg, 235 umol. 1.0 eq.) nyl)-amino-4-(2-chloro-phenyl)-butyric acid was combined with 2-t-butoxycarbonylphenylboronic acid R5 pinacol ester (85.9 mg, 282 umol. 1.2 eq.). Toluene (328 uL) 60 was added followed by EtOH (180 uL) then KCO (65.1 mg, 471 umol. 2.0 eq.) predissolved in water (61.5 LL). The mix ture was stirred to aid in dissolution. The reaction vessel was HR purged with nitrogen then Pd(PPh) (27.2 mg, 23.5 umol) eaR6 was added quickly. The reaction vessel was capped and 65 heated to 100° C. for 30 minutes. The organics were removed and evaporated under vacuum. 1:1 TFA/DCM (0.2 mL each)

US 8,686,184 B2 90 -continued

MS miz: M + HT

Ex. R. R4 R Formula calcd found 10 OH x - O CHCH CH2CINOS 526.11 526.2

O 21 S

ser

8. (R)-4-(2-Chlorophenyl)-3-3-(3-methyl-5-oxo-4,5-dihy Solvents were removed by evaporation and the resulting prod dro-pyrazol-1-yl)-benzoylamino-butyric acid uct was purified by preparative HPLC (using AcOH as sol 9. (R)-4-(2-Chlorophenyl)-3-3-(5-oxo-3-propyl-4,5-dihy vent) to yield the title compound (57 mg, 95% purity). MS dro-pyrazol-1-yl)-benzoylamino-butyric acid m/z. M+H calc’d for CHFNOs, 486.15. found 486.4. 10. (R)-4-(2-Chlorophenyl)-3-4-ethyl-3-(6-methyl-2,4-di oxo-1,4-dihydro-2H-thieno2,3-dipyrimidin-3-yl)-ben ASSAY1 Zoylamino-butyric acid In vitro Assays for the Quantitation of Inhibitor Example 14 25 Potencies at Human and Rat NEP, and Human ACE The inhibitory activities of compounds at human and rat neprilysin (EC 3.4.24.11: NEP) and human angiotensin con Verting enzyme (ACE) were determined using in vitro assays 30 as described below. HO n O Extraction of NEP Activity from Rat Kidneys O O E Rat NEP was prepared from the kidneys of adult Sprague 35 HO Dawley rats. Whole kidneys were washed in cold phosphate buffered saline (PBS) and brought up in ice-cold lysis buffer (1% Triton X-114, 150 mM. NaCl, 50 mM tris(hydroxym ethyl)aminomethane (Tris) pH 7.5; Bordier (1981) J. Biol. Chem. 256:1604-1607) in a ratio of 5 mL of buffer for every 40 gram of kidney. Samples were homogenized on ice using a 5'-(R)-2-Carboxy-1-(2-trifluoromethyl-benzyl)- polytron hand held tissue grinder. Homogenates were centri ethylcarbamoyl-2'-methyl-biphenyl-2-carboxylic fuged at 1000xg in a Swinging bucket rotor for 5 minutes at 3 Acid C. The pellet was resuspended in 20 mL of ice cold lysis 45 buffer and incubated on ice for 30 minutes. Samples (15-20 3-Bromo-4-methylbenzoic acid (100 mg. 465 umol. 1.0 mL) were then layered onto 25 mL of ice-cold cushion buffer eq.), DIPEA (434 LL), HATU (177 mg, 465 umol. 1.0 eq.), (6% w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, and DCM (4 mL) were combined and stirred for 5 minutes at Triton X-114), heated to 37° C. for 3-5 minutes and centri room temperature. Predissolved (R)-3-amino-4-(2-trifluo fuged at 1000xg in a Swinging bucket rotor at room tempera romethylphenyl)butanoic acid hydrochloride (132 mg, 465 50 ture for 3 minutes. The two upper layers were aspirated off, umol. 1.0 eq.) and DIPEA (0.2 mL) in DCM (10 mL) was then leaving a viscous oily precipitate containing the enriched added and the resulting mixture was stirred for 2 hours. The membrane fraction. Glycerol was added to a concentration of reaction was quenched with saturated NHCl and the mixture 50% and samples were stored at -20°C. Protein concentra was extracted with DCM, dried and evaporated to yield a tions were quantitated using a BCA detection system with crude material. 2-t-Butoxycarbonylphenylboronic acid pina 55 bovine serum albumin (BSA) as a standard. col ester (140 mg. 460 umol. 1.0 eq.) and toluene (540 uL) were added to the crude material, followed by EtOH (0.3 mL) Enzyme Inhibition Assays then KCO (128 mg, 930 umol. 2.0 eq.) predissolved in water (0.1 mL). The mixture was stirred to aid in dissolution. Recombinant human NEP and recombinant human ACE The reaction vessel was purged with nitrogen then Pd(PPh.) 60 were obtained commercially (R&D Systems, Minneapolis, (53.7 mg, 46.5umol) was added quickly. The reaction vessel Minn., catalog numbers 1182-ZN and 929-ZN, respectively). was capped and heated to 100° C. for 30 minutes. Upon The fluorogenic peptide Substrate Mica-D-Arg-Arg-Leu-Dap completion of the reaction, the mixture was acidified to pH~5 (Dmp)-OH (Medeiros et al. (1997) Braz. J. Med. Biol. Res. with 1N HC1. EtOAc was added, with stirring. The organic 30:1157-62; Anaspec, San Jose, Calif.) and Abz-Phe-Arg layers were removed and evaporated under vacuum to yield a 65 Lys(Dnp)-Pro-OH (Araujo et al. (2000) Biochemistry crude material. The crude material was then dissolved in 39:8519-8525; Bachem, Torrance, Calif.) were used in the DCM and TFA (0.3 mL each) and stirred for 2 hours. The NEP and ACE assays respectively. US 8,686,184 B2 91 92 The assays were performed in 384-well white opaque -continued plates at 37°C. using the fluorogenic peptide Substrates at a concentration of 10 uM in Assay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM. NaCl, 0.01% polyethylene glycol sorbitan monolaurate (Tween-20), 10 uM ZnSO; ACE: 50 5 mM HEPES, pH 7.5, 100 mM. NaCl, 0.01% Tween-20, 1 uM ZnSO). The respective enzymes were used at concentrations that resulted in quantitative proteolysis of 1 uM of substrate after 20 minutes at 37° C. Test compounds were assayed over the range of concen 10 trations from 10LM to 20 pM. Test compounds were added to the enzymes and incubated for 30 minute at 37° C. prior to initiating the reaction by the addition of substrate. Reactions were terminated after 20 minutes of incubation at 37° C. by the addition of glacial acetic acid to a final concentration of 15 3.6% (v/v). Plates were read on a fluorometer with excitation and emis sion wavelengths set to 320 nm and 405 nm, respectively Inhibition constants were obtained by nonlinear regression of the data using the equation (GraphPad Software, Inc., San Diego, Calif.): where v is the reaction rate, V is the uninhibited reaction rate, I is the inhibitor concentration and K" is the apparent inhibi 25 tion constant. Compounds of the invention were tested in this assay and found to have pK values at human NEP as follows. In general, either the prodrug compounds did not inhibit the enzyme in this in vitro assay, or the prodrugs were not tested (n.d.) since 30 activity would not be expected.

Ex pK, 35 1; R = -OH e9.0 1; R = -OCHCH n.d. 1; R = —O(CH2)3CH n.d. 2-1 e9.0 3 e9.0 40 4 e9.0 5; R = OH 7.O-7.9 5; R = -OCHCH n.d. 5; R = NH(OH 8.0-8.9 6-1 8.0-8.9 6-2 e9.0 6-3 e9.0 45 6-4 8.0-8.9 6-5 7.O-7.9 6-6 e9.0 6-7 7.O-7.9 6-8 8.0-8.9 6-9 8.0-8.9 50 6-10 n.d. 6-11 n.d. 6-12 n.d. 6-13 n.d. 6-14 7.O-7.9 6-15 8.0-8.9 55 6-16 7.O-7.9 6-17 8.0-8.9 6-18 7.O-7.9 6-19 7.O-7.9 7; R = -OCHCH n.d. 7; R = OH 8.0-8.9 8; R = -OCHCH n.d. 60 8; R = -OH 8.0-8.9 8; R = —O(CH2)3CH3 n.d. 9; R = -OH 8.0-8.9 9; R = -OCHCH n.d. 1O-1 7.O-7.9 10-2 7.O-7.9 65 US 8,686,184 B2 93 94 -continued mitter is kept intraperitoneally and secured to the abdominal wall while closing of the abdominal incision with a non Ex pK, absorbable suture. The outer skin is closed with suture and O-82 7.O-7.9 Staples. The animals are allowed to recover with appropriate 1 8.0-8.9 post operative care. On the day of the experiment, the animals 2 8.0-8.9 3-1 7.O-7.9 in their cages are placed on top of the telemetry receiver units 3-2 8.0-8.9 to acclimate to the testing environment and baseline record 3-3 8.0-8.9 ing. After at least of 2 hours baseline measurement is taken, 3-4 7.O-7.9 the animals are then dosed with vehicle or test compound and 3-5 7.O-7.9 10 3-6 7.O-7.9 followed out to 24 hours post-dose blood pressure measure 3-7 n.d. ment. Data is recorded continuously for the duration of the 3-8 7.O-7.9 3-9 7.O-7.9 study using Notocord Software (Kalamazoo, Mich.) and 3-10 7.O-7.9 stored as electronic digital signals. Parameters measured are 4 8.0-8.9 15 blood pressure (systolic, diastolic and mean arterial pressure) and heart rate.

ASSAY 2 ASSAY 4 Pharmacodynamic (PD) assay for ACE and NEP In Vivo Evaluation of Antihypertensive Effects in the Activity in Anesthetized Rats Conscious DOCA-Salt Rat Model of Hypertension Male, Sprague Dawley, normotensive rats are anesthetized CD rats (male, adult, 200-300 grams, Charles River Labo with 120 mg/kg (i.p.) of inactin. Once anesthetized, the jugu ratory, USA) are allowed a minimum of 48 hours acclimation lar vein, carotid artery (PE 50 tubing) and bladder (flared PE 25 upon arrival at the testing site before they are placed on a high 50 tubing) catheters are cannulated and a tracheotomy is salt diet. One week after the start of the high salt diet (8% in performed (Teflon Needle, size 14 gauge) to facilitate spon food or 1% NaCl in drinking water), a deoxycorticosterone taneous respiration. The animals are then allowed a 60 minute acetate (DOCA) pellet (100 mg, 90 days release time, Inno stabilization period and kept continuously infused with 5 vative Research of America, Sarasota, Fla.) is implanted sub mL/kg/h of saline (0.9%) throughout, to keep them hydrated 30 cutaneously and unilateral nephrectomy is performed. At this and ensure urine production. Body temperature is maintained time, the animals are also Surgically implanted with Small throughout the experiment by use of a heating pad. At the end rodent radiotransmitters for blood pressure measurement (see of the 60 minute stabilization period, the animals are dosed Assay 3 for details). The animals are allowed to recover with intravenously (i.v.) with two doses of Ang (1.0 ug/kg, for appropriate post operative care. Study design, data recording, ACE inhibitor activity) at 15 minutes apart. At 15 minutes 35 and parameters measured is similar to that described for post-second dose of Ang, the animals are treated with vehicle Assay 3. or test compound. Five minutes later, the animals are addi tionally treated with a bolus i.v. injection of atrial natriuretic peptide (ANP; 30 g/kg). Urine collection (into pre-weighted ASSAY 5 eppendorf tubes) is started immediately after the ANP treat 40 ment and continued for 60 minutes. At 30 and 60 minutes into In Vivo Evaluation of Antihypertensive Effects in the urine collection, the animals are re-challenged with Ang. Conscious Dahl/SS Rat Model of Hypertension Blood pressure measurements are done using the Notocord system (Kalamazoo, Mich.). Urine samples are frozen at -20° Male, Dahl salt sensitive rats (Dahl/SS, 6-7 weeks of age C. until used for the coiMPassay. Urine coMP concentrations 45 from Charles River Laboratory, USA) are allowed at least 48 are determined by Enzyme Immuno Assay using a commer hours of acclimation upon arrival at the testing site before cial kit (Assay Designs, AnnArbor, Mich. Cat. No. 901-013). they were placed on a 8% NaCl high salt diet (TD.92012, Urine volume is determined gravimetrically. Urinary c0MP Harlan, USA) then surgically implanted with small rodent output is calculated as the product of urine output and urine radiotransmitters for blood pressure measurement (see Assay cGMP concentration. ACE inhibition is assessed by quanti 50 3 for details). The animals are allowed to recover with appro fying the % inhibition of pressor response to Angl. NEP priate post operative care. At approximately 4 to 5 weeks from inhibition is assessed by quantifying the potentiation of ANP the start of high Salt diet, these animals are expected to induced elevation in urinary c0MP output. become hypertensive. Once the hypertension level is con firmed, these animals are used for the study while continued ASSAY 3 55 with the high salt diet to maintain their hypertension level. Study design, data recording, and parameters measured is In Vivo Evaluation of Antihypertensive Effects in the Con similar to that described in Assay 3. scious SHRModel of Hypertension While the present invention has been described with refer Spontaneously hypertensive rats (SHR, 14-20 weeks of ence to specific aspects or embodiments thereof, it will be age) are allowed a minimum of 48 hours acclimation upon 60 understood by those of ordinary skilled in the art that various arrival at the testing site with free access to food and water. changes can be made or equivalents can be substituted with For blood pressure recording, these animals are Surgically out departing from the true spirit and scope of the invention. implanted with small rodent radiotransmitters (telemetry Additionally, to the extent permitted by applicable patent unit; DSI Models TA11PA-C40 or C50-PXT, Data Science statutes and regulations, all publications, patents and patent Inc., USA). The tip of the catheter connected to the transmit 65 applications cited herein are hereby incorporated by refer ter is inserted into the descending aorta above the iliac bifur ence in their entirety to the same extent as if each document cation and secured in place with tissue adhesive. The trans had been individually incorporated by reference herein.

US 8,686,184 B2 97 98 7. The compound of claim 1, wherea is 0 or 3. 14. The compound of claim 8, where R' is —OR' or 8. The compound of claim 7, whereina is 0; R is OR' NRR79; R is Hor Calkyl; R is OH: R79 is H: or NR'R'': R is Hor Calkyl; R is OH: R7 is H: R’ is OR'; R is H: R is Cl; R is selected from phenyl R’ is Hor–OR': R is H; R is selected from Cland–CF; substituted with —Coalkylene-COOR, and a partially R" is selected from —C-alkyl, -C-cycloalkyl: unsaturated —Co-alkylene-C-sheterocycle selected from - Coalkylene-COOR': NR'R'': NHC(O)C C. -Co-alkylene-dihydropyrazole and tetrahydrothienopyrimi dine, both of which are optionally substituted with 1-3 groups alkyl; NHC(O)R: NHC(O)NH R: phenyl substi independently selected from =O. —Calkyl, and —CF: tuted with one or two groups independently selected from R" is H; R is selected from H, halo. —Calkyl, and halo, —O—Calkyl, - Co-alkylene-COOR', —O Calkyl; and R is selected from H. fluoro, and —SO.NH2, C(O)NR'R'', and tetrazole; and Co-alky 10 chloro. lene-Coheteroaryl or a partially unsaturated —Co-alky 15. The compound of claim 8, where R' is —OR': R' is lene-C-sheterocycle both optionally substituted with 1-3 Hor—C-alkyl; R is H; R is Cl; R is selected from phenyl groups independently selected from halo, =O, —CHO, substituted with - Co-alkylene-COOR'; and a partially —Calkyl, -CF, and COOR'; R' is Hor—Calkyl: unsaturated —Co-alkylene-C-sheterocycle selected from R' is H or - Calkyl; R is Calkyl; R is selected 15 dihydropyrazole and tetrahydrothienopyrimidine, both of from —Calkyl, -Calkylene-COOR', phenyl, and which are substituted with 1-3 groups independently selected pyridine: R is selected from —C-alkyl, -C-alkylene from =O and —Calkyl; R' is H; R is selected from H. COOR', phenyl, and thiophene-COOR', where thiophene halo, and —C-alkyl; and R is H. is optionally substituted with CH; R is selected from H. 16. The compound of claim 8, where R' is —OR': R' is halo. —OH, -Calkyl, —O—Calkyl, phenyl, and mor H or Calkyl; R is H; R is CF; R is phenyl substi pholine; and R is selected from H. fluoro, and chloro. tuted with -Co-alkylene-COOR'; R is H; R is 9. The compound of claim 7, whereina is 3: R is —OR': —Calkyl; and R is H. R" is H or Calkyl; R is Hor–OR: R is H; R is 17. The compound of claim 9, where R' is —OR': R' is selected from C1 and CF; R is - Co-alkylene-COOR': H or Calkyl; R is OR: R is H; R is Cl; R is R' is H; R is H or halo; and R is H. 25 - Co-alkylene-COOR': R' is H: R is Horhalo; and R is 10. The compound of claim 1, where R is selected from H. —Coalkyl, -C-cycloalkyl, -Co-alkylene-COOR". 18. The compound of claim 9, where R' is —OR': R' is NR'R'': NHC(O)C Calkyl; NHC(O)R: Hor Calkyl; R is OR; R is H; R is CF; R is -NHC(O)NH-R: phenyl substituted with one or two - Co-alkylene-COOR': R' is H; R is H; and R is H. groups independently selected from halo. —O—Calkyl, 30 19. The compound of claim 9, where R is H; R is Cl; R' - Co-alkylene-COOR', -SONH2, C(O)NR'R'', and is —OR'; R' is H or - Calkyl; R is - Co-alkylene tetrazole; and —Co-alkylene-Coheteroaryl or a partially COOR': R is H; R is Horhalo; and R is H. unsaturated —Co-alkylene-C-sheterocycle both optionally 20. A pharmaceutical composition comprising the com substituted with 1-3 groups independently selected from pound of claim 1 and a pharmaceutically acceptable carrier. halo, =O,-CHO,-Calkyl, -CF, and COOR': R' 35 21. The pharmaceutical composition of claim 20, further is H or - Calkyl; R' is H or - Calkyl; R is comprising a therapeutic agent selected from adenosine —Calkyl; R is selected from —Coalkyl, -C-alky receptor antagonists, C.-adrenergic receptor antagonists, lene-COOR", phenyl, and pyridine; and R is selected from B-adrenergic receptor antagonists, B-adrenergic receptor —Coalkyl, -C-alkylene-COOR", phenyl, and agonists, dual-acting B-adrenergic receptor antagonist/ol-re thiophene-COOR', where thiophene is optionally substi 40 ceptor antagonists, advanced glycation end product breakers, tuted with CH. aldosterone antagonists, aldosterone synthase inhibitors, 11. The compound of claim 1, where R is selected from H. aminopeptidase N inhibitors, androgens, angiotensin-con fluoro, chloro, bromo. —OH, -CH, —CHCH. —CH Verting enzyme inhibitors and dual-acting angiotensin-con (CH), —OCH phenyl, and morpholine. Verting enzyme/neprilysin inhibitors, angiotensin-converting 12. The compound of claim 1, where R is selected from H. 45 enzyme 2 activators and stimulators, angiotensin-II Vaccines, fluoro, and chloro. anticoagulants, anti-diabetic agents, antidiarrheal agents, 13. The compound of claim 1, where R' is —OR' or anti-glaucoma agents, anti-lipid agents, antinociceptive NR'R'': R is Hor Calkyl; R is OH: R7 is H: agents, anti-thrombotic agents, AT receptor antagonists and R’ is Hor–OR: R is H; R is selected from Cland–CF: dual-acting AT receptor antagonist/neprilysin inhibitors and a is 0 or 3: R is selected from —C-alkyl, -C-cycloalkyl; 50 multifunctional angiotensin receptor blockers, bradykinin - Coalkylene-COOR': NR'R'': NHC(O)O receptor antagonists, calcium channel blockers, chymase Calkyl; NHC(O)R: NHC(O)NH-R: phenyl sub inhibitors, digoxin, diuretics, dopamine agonists, endothelin stituted with one or two groups independently selected from converting enzyme inhibitors, endothelin receptor antago halo, —O—Calkyl, - Co-alkylene-COOR', nists, HMG-CoA reductase inhibitors, estrogens, estrogen —SO.NH2, C(O)NR'R'', and tetrazole; and Co-alky 55 receptor agonists and/or antagonists, monoamine reuptake lene-Coheteroaryl or a partially unsaturated —Co-alky inhibitors, muscle relaxants, natriuretic peptides and their lene-C-sheterocycle both optionally substituted with 1-3 analogs, natriuretic peptide clearance receptor antagonists, groups independently selected from halo, =O, —CHO, neprilysin inhibitors, nitric oxide donors, non-steroidal anti —Calkyl, -CF, and o: -COOR, R is H or inflammatory agents, N-methyl d-aspartate receptor antago —Calkyl; R' is Hor—Chalkyl; R is Calkyl; R' 60 nists, opioid receptor agonists, phosphodiesterase inhibitors, is selected from Calkyl, -Calkylene-COOR', phe prostaglandin analogs, prostaglandin receptoragonists, renin nyl, and pyridine; R is selected from Calkyl, inhibitors, selective serotonin reuptake inhibitors, sodium —Calkylene-COOR', phenyl, and thiophene-COOR', channel blocker, soluble guanylate cyclase stimulators and where thiophene is optionally substituted with CH3; R is activators, tricyclic antidepressants, vasopressin receptor selected from H. fluoro, chloro, bromo, OH, -CH 65 antagonists, and combinations thereof. —CH2CH, —CH(CH), —OCH phenyl, and morpho 22. The pharmaceutical composition of claim 21, wherein line; and R is selected from H, fluoro, and chloro. the therapeutic agent is an AT receptor antagonist. US 8,686,184 B2 99 100 23. A process for preparing the compound of claim 1, in a palladium-catalyzed coupling reaction, where each R is comprising the steps of independently selected from halo. —O—Calkyl, -Co (a) coupling compound 1 with compound 2: alkylene-COOR', -SONH = C(O)NR'R'', and tetra Zole; or 5 (c) reacting compound 3 with compound 5 to form com pound 6 in a palladium-catalyzed coupling reaction: HO ( )a Sé, O al" (6) (2) (1) 1 Nee 15 O / (b) coupling compound 1 with compound 3 to form com pound 4: L and coupling compound 6 with compound 1; and N (d) optionally deprotecting the product of step (a) or (b) or (l) Ho rs. -- (c), to produce a compound of formula I or a pharma ceutically acceptable salt thereof. 24. An intermediate useful in the synthesis of the com 25 pound of claim 1, having formula VI: L

(VI) IIrts --R, R2 4 ". )a N 30 P N - - -a× r R5 O O 2.1. 35 (4) where L is a leaving group, and reacting compound 4 with where P is selected from-O-P'. - NHP, and NH(O- compound 5: P); P is a carboxy-protecting group selected from methyl, 40 (5) ethyl, t-butyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl, t-butyldimethylsilyl, and diphenylmethyl; P is an amino-protecting group selected from t-butoxycarbonyl, trity1, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, formyl, trimethylsilyl, and t-butyldimethylsilyl; and P is a 45 hydroxyl-protecting group selected from Calkyl, silyl groups, esters, and arylmethyl groups, or a salt thereof. 25. A method for treating hypertension, heart failure, or renal disease, comprising administering to a patient a thera 50 peutically effective amount of the compound of claim 1. k k k k k