USOO9683 002B2

(12) United States Patent (10) Patent No.: US 9,683,002 B2 Hughes et al. (45) Date of Patent: *Jun. 20, 2017

(54) NEPRILYSIN INHIBITORS 3 1/675 (2013.01); A61K 45/06 (2013.01); C07D 231/14 (2013.01); C07D 249/04 (71) Applicant: THERAVANCE BIOPHARMA R&D (2013.01); C07D 249/12 (2013.01); C07D IP, LLC, South San Francisco, CA 257/04 (2013.01); C07D 261/18 (2013.01); (US) C07D 277/56 (2013.01); C07D 319/06 (72) Inventors: Adam D. Hughes, Half Moon Bay, CA (2013.01); C07D405/12 (2013.01); C07D (US); Melissa Fleury, Brisbane, CA 413/12 (2013.01); C07F 9/65312 (2013.01) (58) Field of Classification Search (US) None (73) Assignee: Theravance Biopharma R&D IP, See application file for complete search history. LLC, South San Francisco, CA (US) (56) References Cited (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S. PATENT DOCUMENTS U.S.C. 154(b) by 0 days. 4,189.604 A 2, 1980 Umezawa et al. This patent is Subject to a terminal dis 4,206,232 A 6, 1980 Ondetti et al. claimer. (Continued) (21) Appl. No.: 14/816,409 FOREIGN PATENT DOCUMENTS WO WO 2011/O887.97 A1 T/2011 (22) Filed: Aug. 3, 2015 WO WO2012O82857 A1 * 6/2012 ...... CO7D 231/14 (65) Prior Publication Data OTHER PUBLICATIONS US 2016/0090393 A1 Mar. 31, 2016 Ksander et al., “Dicarboxylic acid dipeptide neutral endopeptidase Related U.S. Application Data inhibitors”, Journal of Medicinal Chemistry, 38(10): 1689-1700 (63) Continuation of application No. 14/477,030, filed on (1995). Sep. 4, 2014, now abandoned, which is a continuation (Continued) of application No. 13/911,552, filed on Jun. 6, 2013, now Pat. No. 8,871,792. Primary Examiner — Michael Barker (74) Attorney, Agent, or Firm — Jeffrey A. Hagenah; (60) Provisional application No. 61/774,148, filed on Mar. Wendy Petka 7, 2013, provisional application No. 61/657,220, filed on Jun. 8, 2012. (57) ABSTRACT (51) Int. Cl. In one aspect, the invention relates to compounds having the CO7D 249/10 (2006.01) formula X: C07D 277/56 (2006.01) CO7D 26L/8 (2006.01) CO7D 413/12 (2006.01) (X) C07F 9/653 (2006.01) CO7D 319/06 (2006.01) CO7D 231/4 (2006.01) CO7D 249/04 (2006.01) A6 IK 3/4I (2006.01) A6 IK 3L/45 (2006.01) A6 IK 3/492 (2006.01) A6 IK 3/496 (2006.01) A6 IK 3/42 (2006.01) A6 IK 3/426 (2006.01) A 6LX 3/5.377 (2006.01) A6 IK3I/675 (2006.01) A6 IK 45/06 (2006.01) where R", R. R. R', and X are as defined in the specifi CO7D 249/12 (2006.01) cation, or a pharmaceutically acceptable salt thereof. The C07D 257/04 (2006.01) compounds described herein are prodrugs of compounds CO7D 405/2 (2006.01) having neprilysin inhibition activity. In another aspect, the (52) U.S. Cl. invention relates to pharmaceutical compositions compris CPC ...... C07F 9/653 (2013.01); A61K 31/41 ing these compounds; methods of using these compounds; (2013.01); A61K 31/415 (2013.01); A61 K and processes and intermediates for preparing these com 31/4192 (2013.01); A61K 31/4196 (2013.01); pounds. A61K 31/42 (2013.01); A61K 31/426 (2013.01); A61 K3I/5377 (2013.01); A61 K 3 Claims, No Drawings US 9,683,002 B2 Page 2

(56) References Cited 8,686,184 B2 4/2014 Fleury et al. 8,691,868 B2 4/2014 Hughes et al. U.S. PATENT DOCUMENTS 8,871,792 B2 * 10/2014 Hughes ...... CO7D 319,06 514,369 4,374,829 2, 1983 Harris et al. 8,901,169 B2 12/2014 Fenster et al. 4.513,009 4, 1985 Roques et al. 9,045,443 B2 6/2015 Mammen et al. 4,722,810 2, 1988 Delaney et al. 9,108,934 B2 8/2015 Hughes et al. 4,906,615 3, 1990 Berger et al. 9,126,956 B2 9/2015 Fleury et al. 4,929,641 5, 1990 Haslanger et al. 2010/0113801 A1 5, 2010 Hook et al. 4,939,261 7, 1990 Ksander 2010/0305131 A1 12/2010 Coppola et al. 4,975.444 12, 1990 Danilewicz et al. 2010/0305145 A1 12/2010 Coppola et al. 5,021,430 6, 1991 Ksander 2011 0046397 A1 2/2011 Hook et al. 5,030,654 7, 1991 Barnish et al. 2011 0124695 A1 5, 2011 Iwaki et al. 5,155,100 10, 1992 Erion et al. 2012/O122844 A1 5, 2012 Foo 5,208,255 5, 1993 Duhamel et al. 2012/O122977 A1 5/2012 Coppola et al. 5,217,996 6, 1993 Ksander 2012/0309724 A1 12/2012 Fleury et al. 5,294,632 3, 1994 Erion et al. 5,508,272 4, 1996 Robl 5,599.951 2, 1997 Plaquevent et al. OTHER PUBLICATIONS 5,677,297 10, 1997 Waldeck et al. 5,977.075 11, 1999 Ksander et al. Misawa et al., “Structure-based design of dipeptide derivatives for 6,602,866 8, 2003 Flynn et al. the human neutral endopeptidase'. Bioorganic & Medicinal Chem 6,660,756 12, 2003 Challenger et al. istry, 19: 5935-5947 (2011). 8.449,890 5, 2013 Fleury et al. 8,481,044 T/2013 Fleury et al. The International Search Report for PCT/US2013/044485 dated 8,513,244 8, 2013 Gendron et al. Aug. 27, 2013. 8,563,512 10, 2013 Smith et al. 8,586,536 11, 2013 Gendron et al. * cited by examiner US 9,683,002 B2 1. NEPRLYSIN INHIBITORS (X) CROSS-REFERENCE TO RELATED APPLICATIONS N NH

This application claims the benefit of U.S. Provisional O Application No. 61/657,220, filed on Jun. 8, 2012 and U.S. R2 Y Provisional Application No. 61/774,148, filed on Mar. 7, 2013; the entire disclosures of which are incorporated herein 10 Rb by reference. Ra BACKGROUND OF THE INVENTION 15 where: Field of the Invention (i) R' is F: R is Cl; X is The present invention relates to novel compounds which are metabolized in vivo to compounds having activity as neprilysin inhibitors. The invention also relates to pharma ceutical compositions comprising these compounds, pro cesses and intermediates for preparing these compounds and methods of using these compounds to treat diseases such as hypertension, heart failure, pulmonary hypertension, and 25 Ny O renal disease. State of the Art Commonly-assigned U.S. Patent Publication No. 2012/ 0157386, filed on Dec. 14, 2011 to Smith et al., describes novel compounds that have activity as neprilysin inhibitors, 30 the disclosure of which is incorporated herein by reference. d In particular, compounds of the genus:

35

40

( NO 45 R’ is H and R7 is selected from CHCH, -CHCH (CH), —CH2CF, -(CH2)CF, —CHCFCH are described. Depending upon the variables, compounds —CHCFCF, —C(CH)(CF), —CH(CHCH)CF, 50 —CH(CH)CFCF, -(CH), OH, -CH-CH(NH) within this genus can be referred to as being in the active COOCH, -(CH)OCH, -CHROC(O) Calkyl, form or as being a prodrug, which is metabolized in vivo to - CHROC(O)O Calkyl, -CHROC(O)O-cyclohexyl, generate the active form of the compound. —Calkylene-N(CH), -CHOC(O)CHR NH, In spite of these compounds however, there remains a —CHOC(O)CHR NHC(O)C Calkyl, benzyl, and need for compounds and prodrugs within this genus that 55 have different metabolic and cleavage properties. For example, there remains a need for active compounds and/or prodrug compounds having improved oral absorption and H3 for prodrug compounds that undergo rapid cleavage to form 60 the active compound. This invention is directed to that need. ) (

SUMMARY OF THE INVENTION 65 or R is selected from C(O) Coalkyl, -C(O)CHR One aspect of the invention relates to a compound of the NH = C(O)CHR NHC(O)C Calkyl, and -P(O) formula X: (OR), and R is H; and

US 9,683,002 B2 8 DETAILED DESCRIPTION OF THE INVENTION When describing the compounds, compositions, methods and processes of the invention, the following terms have the following meanings unless otherwise indicated. Addition ally, as used herein, the singular forms “a,” “an,” and “the include the corresponding plural forms unless the context of use clearly dictates otherwise. The terms “comprising”. 10 “including, and “having are intended to be inclusive and mean that there may be additional elements other than the or R is selected from —C(O) Calkyl, -C(O)CHR listed elements. All numbers expressing quantities of ingre NH, -C(O)CHR" NHC(O)O Calkyl, and -P(O) dients, properties such as molecular weight, reaction condi (OR), R is -OH, and R7 is H; or R is H, R is selected tions, and so forth used herein are to be understood as being from OCHROC(O) OCHOC(O)CHCH(CH), NH, 15 modified in all instances by the term “about, unless other –OCHOC(O)CHCH(CH), NHC(O)OCH, and wise indicated. Accordingly, the numbers set forth herein are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At CH3, least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each number should at least be construed in light of the reported significant digits and by applying ordinary rounding tech niques. The term “alkyl means a monovalent saturated hydro 25 carbon group which may be linear or branched. Unless otherwise defined, such alkyl groups typically contain from and R7 is H: 1 to 10 carbon atoms and include, for example, —Calkyl, where each R is independently Hor—C-alkyl; each R' meaning an alkyl group having from 1 to 6 carbon atoms is independently H. —CH, —CH(CH), phenyl, or benzyl; where the carbon atoms are in any acceptable configuration. 30 Representative alkyl groups include, by way of example, and each R is independently H. —Calkyl, or phenyl: methyl, ethyl, n-propyl, isopropyl. n-butyl, S-butyl, isobutyl, or a pharmaceutically acceptable salt thereof. t-butyl, n-pentyl, n-hexyl, and the like. The present invention provides compounds which are As used herein, the phrase “having the formula” or metabolized in vivo to compounds that have been found to “having the structure' is not intended to be limiting and is possess neprilysin (NEP) enzyme inhibition activity. 35 used in the same way that the term "comprising is com Accordingly, compounds of the invention are expected to be monly used. For example, if one structure is depicted, it is useful and advantageous as therapeutic agents for treating understood that all stereoisomer and tautomer forms are patients suffering from a disease or disorder that is treated by encompassed, unless stated otherwise. inhibiting the NEP enzyme or by increasing the levels of its The term “pharmaceutically acceptable' refers to a mate peptide Substrates. Thus, one aspect of the invention relates 40 rial that is not biologically or otherwise unacceptable when to a method of treating hypertension, heart failure, or renal used in the invention. For example, the term “pharmaceu disease, comprising administering to a patient a therapeuti tically acceptable carrier refers to a material that can be cally effective amount of a compound of the invention. incorporated into a composition and administered to a Another aspect of the invention relates to pharmaceutical patient without causing unacceptable biological effects or compositions comprising a pharmaceutically acceptable car 45 interacting in an unacceptable manner with other compo nents of the composition. Such pharmaceutically acceptable rier and a compound of the invention. materials typically have met the required standards of toxi Yet another aspect of the invention relates to processes cological and manufacturing testing, and include those and intermediates useful for preparing compounds of the materials identified as suitable inactive ingredients by the invention. Another aspect of the invention relates to a 50 U.S. Food and Drug administration. process of preparing a pharmaceutically acceptable salt of a The term “pharmaceutically acceptable salt' means a salt compound of formula I, comprising contacting a compound prepared from a base or an acid which is acceptable for of formula I in free acid or base form with a pharmaceuti administration to a patient, such as a mammal (for example, cally acceptable base or acid. In other aspects, the invention salts having acceptable mammalian safety for a given dos relates to products prepared by any of the processes 55 age regime). However, it is understood that the salts covered described herein, as well as novel intermediates used in such by the invention are not required to be pharmaceutically process. acceptable salts, such as salts of intermediate compounds Yet another aspect of the invention relates to the use of a that are not intended for administration to a patient. Phar compound of formula I or a pharmaceutically acceptable salt maceutically acceptable salts can be derived from pharma thereof, for the manufacture of a medicament, especially for 60 ceutically acceptable inorganic or organic bases and from the manufacture of a medicament useful for treating hyper pharmaceutically acceptable inorganic or organic acids. In tension, heart failure, or renal disease. Another aspect of the addition, when a compound of formula I contains both a invention relates to use of a compound of the invention for basic moiety, Such as an amine, pyridine or imidazole, and inhibiting a NEP enzyme in a mammal. Still another aspect an acidic moiety such as a carboxylic acid or tetrazole, of the invention relates to the use of a compound of the 65 Zwitterions may be formed and are included within the term invention as a research tool. Other aspects and embodiments “salt as used herein. Salts derived from pharmaceutically of the invention are disclosed herein. acceptable inorganic bases include ammonium, calcium, US 9,683,002 B2 10 copper, ferric, ferrous, lithium, magnesium, manganic, man patient that is pre-disposed to the disease or medical con ganous, potassium, Sodium, and Zinc salts, and the like. Salts dition; (b) ameliorating the disease or medical condition, derived from pharmaceutically acceptable organic bases i.e., eliminating or causing regression of the disease or include Salts of primary, secondary and tertiary amines, medical condition in a patient; (c) Suppressing the disease or including Substituted amines, cyclic amines, naturally-oc medical condition, i.e., slowing or arresting the development curring amines and the like. Such as arginine, betaine, of the disease or medical condition in a patient; or (d) caffeine, choline, N,N'-dibenzylethylenediamine, diethyl alleviating the symptoms of the disease or medical condition amine, 2-diethylaminoethanol, 2-dimethylaminoethanol, in a patient. For example, the term “treating hypertension” ethanolamine, ethylenediamine, N-ethylmorpholine, N-eth would include preventing hypertension from occurring, ylpiperidine, glucamine, glucosamine, histidine, hydrabam 10 ameliorating hypertension, Suppressing hypertension, and ine, isopropylamine, lysine, methylglucamine, morpholine, alleviating the symptoms of hypertension (for example, piperazine, piperadine, polyamine resins, procaine, purines, lowering blood pressure). The term “patient' is intended to theobromine, triethylamine, trimethylamine, tripropylam include those mammals. Such as humans, that are in need of ine, tromethamine and the like. Salts derived from pharma treatment or disease prevention or that are presently being ceutically acceptable inorganic acids include Salts of boric, 15 carbonic, hydrohalic (hydrobromic, hydrochloric, hydroflu treated for disease prevention or treatment of a specific oric or hydroiodic), nitric, phosphoric, Sulfamic and Sulfuric disease or medical condition, as well as test Subjects in acids. Salts derived from pharmaceutically acceptable which the crystalline compound is being evaluated or being organic acids include salts of aliphatic hydroxyl acids (for used in an assay, for example an animal model. example, citric, gluconic, glycolic, lactic, lactobionic, malic, All other terms used herein are intended to have their and tartaric acids), aliphatic monocarboxylic acids (for ordinary meaning as understood by those of ordinary skill in example, acetic, butyric, formic, propionic and trifluoro the art to which they pertain. acetic acids), amino acids (for example, aspartic and gluta The compound of the invention contain one or more chiral mic acids), aromatic carboxylic acids (for example, benzoic, centers and therefore, these compounds may be prepared and p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and tri 25 used in various stereoisomeric forms. In some embodiments, phenylacetic acids), aromatic hydroxyl acids (for example, in order to optimize the therapeutic activity of the com o-hydroxybenzoic, p-hydroxybenzoic, 1-hydroxynaphtha pounds of the invention, e.g., to treat hypertension, it may be lene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylic desirable that the carbon atoms have a particular (R,R), (S,S), (S,R), or (R.S) configuration or are enriched in a acids), ascorbic, dicarboxylic acids (for example, fumaric, Stereoisomeric form having Such configuration. In other maleic, oxalic and Succinic acids), glucoronic, mandelic, 30 embodiments, the compounds of the invention are present as mucic, nicotinic, orotic, pamoic, pantothenic, Sulfonic acids racemic mixtures. Accordingly, the invention also relates to (for example, benzenesulfonic, camphosulfonic, edisylic, racemic mixtures, pure Stereoisomers (e.g., enantiomers and ethanesulfonic, isethionic, methanesulfonic, naphthalene diastereoisomers), Stereoisomer-enriched mixtures, and the Sulfonic, naphthalene-1,5-disulfonic, naphthalene-2,6-disul like unless otherwise indicated. When a chemical structure fonic and p-toluenesulfonic acids), Xinafoic acid, and the 35 is depicted herein without any stereochemistry, it is under like. stood that all possible stereoisomers are encompassed by As used herein, the term “prodrug is intended to mean an Such structure. Similarly, when a particular Stereoisomer is inactive (or significantly less active) precursor of a drug that shown or named herein, it will be understood by those is converted into its active form in the body under physi skilled in the art that minor amounts of other stereoisomers ological conditions, for example, by normal metabolic pro 40 may be present in the compositions of the invention unless cesses. Such compounds may not necessarily possess phar otherwise indicated, provided that the utility of the compo macological activity at NEP, but may be administered orally sition as a whole is not eliminated by the presence of Such or parenterally and thereafter metabolized in the body to other isomers. Individual stereoisomers may be obtained by form a compound that is pharmacologically active at NEP. numerous methods that are well known in the art, including The term “therapeutically effective amount’ means an 45 chiral chromatography using a suitable chiral stationary amount sufficient to effect treatment when administered to a phase or Support, or by chemically converting them into patient in need thereof, that is, the amount of drug needed to diastereoisomers, separating the diastereoisomers by con obtain the desired therapeutic effect. For example, a thera ventional means such as chromatography or recrystalliza peutically effective amount for treating hypertension is an tion, then regenerating the original stereoisomer. amount of compound needed to, for example, reduce, Sup 50 press, eliminate, or prevent the symptoms of hypertension, Additionally, where applicable, all cis-trans or E/Z iso or to treat the underlying cause of hypertension. In one mers (geometric isomers), tautomeric forms and topoiso embodiment, a therapeutically effective amount is that meric forms of the compounds of the invention are included amount of drug needed to reduce blood pressure or the within the scope of the invention unless otherwise specified. amount of drug needed to maintain normal blood pressure. 55 The compounds of the invention, as well as those com On the other hand, the term “effective amount’ means an pounds used in their synthesis, may also include isotopi amount Sufficient to obtain a desired result, which may not cally-labeled compounds, that is, where one or more atoms necessarily be a therapeutic result. For example, when have been enriched with atoms having an atomic mass studying a system comprising a NEP enzyme, an “effective different from the atomic mass predominately found in amount may be the amount needed to inhibit the enzyme. 60 nature. Examples of isotopes that may be incorporated into The term “treating or “treatment” as used herein means the treating or treatment of a disease or medical condition the compounds of formula I, for example, include, but are (such as hypertension) in a patient, such as a mammal not limited to, H, H, C, C, N, O, ''O, S, C1, and (particularly a human) that includes one or more of the F. Of particular interest are compounds of formula I following: (a) preventing the disease or medical condition 65 enriched in tritium or carbon-14 which can be used, for from occurring, i.e., preventing the reoccurrence of the example, in tissue distribution studies; compounds of the disease or medical condition or prophylactic treatment of a invention enriched in deuterium especially at a site of US 9,683,002 B2 11 12 metabolism resulting, for example, in compounds having greater metabolic stability; and compounds of formula I (Ia) enriched in a positron emitting isotope, such as 'C, F, 'O and 'N, which can be used, for example, in Positron Emission Topography (PET) studies. C The nomenclature used herein to name the compounds of O N the invention is illustrated in the Examples herein. This O ) { nomenclature has been derived using the commercially R NH -s, available AutoNom software (MDL, San Leandro, Calif.). 10 No N1 H U.S. Patent Publication No. 2012/0157386 specifically O disclosed (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylm R21 ethyl)-N'-1-(3-chlorophenyl)-5-oxo-4,5-dihydro-1H-1.2. C 4triazole-3-carbonylhydrazino-2-hydroxypropionic acid, 15 which is represented by formula I': F (Ib) (I)

C C O N O NN N O ) { 25 R No N1 NH H-s, NH ClN O HO N1 H O OH R21 30 C C

F 35 For compounds of formula Ia, R is H and R is selected In one embodiment, this compound is referred to as the from —CHCH, —CHCH(CH), —CHCF. —(CH), active form and is administered as a prodrug, which is CF, —CHCFCH. —CHCFCF, —C(CH) (CF). metabolized in vivo to form the compound of formula I. —CH(CHCH)CF, —CH(CH)CFCF —(CH), OH, This compound can also exist in its tautomer form, (R)-3- 40 CH-CH(NH)COOCH, -(CH)OCH, CHROC {N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-1-(3-chlo (O) Calkyl, -CHROC(O)O Calkyl, -CHROC rophenyl)-5-hydroxy-1H-1.2.4 triazole-3-carbonylhy (O)O-cyclohexyl, —Calkylene-N(CH), —CHOC(O) CHR NH, CHOC(O)CHR NHC(O)O Calkyl, drazino-2-hydroxypropionic acid. benzyl, and One aspect of the invention relates to other prodrugs of 45 the compound of formula I". These prodrugs are represented by formula X, where IV is F. R is C1, and X is:

50

C

NN N O 7 -l. 55 N O or R is selected from —C(O) Calkyl, -C(O)CHR NH, -C(O)CHR" NHC(O)O Calkyl, and -P(O) (OR), and R is H; where each R is independently H or C —C-alkyl; each R" is independently H, —CH, -CH NN 60 (CH), phenyl, or benzyl; and each R is independently H, / N —Calkyl, or phenyl: or a pharmaceutically acceptable salt thereof. For compounds of formula Ib, R is H, R is -OH, als and R7 is selected from —CHOC(O)CH and —CHOC (O)CHICH(CH)NH; or R is H, R is selected from 65 –OCHOC(O)CH and OCHOC(O)CHCH(CH), In one embodiment, these compounds are represented by NH, and R7 is H; where each R is independently H or formula Ia or Ib: - Cisalkyl; each R" is independently H, —CH, -CH US 9,683,002 B2 13 14 (CH), phenyl, or benzyl; and each R is independently H, where R is H and R is selected from —CH2CHs. —Calkyl, or phenyl; or a pharmaceutically acceptable salt —CHCF —(CH)CF, —CHCFCH. —CHCFCF, thereof. —C(CH) (CF), —CH(CHCH)CF, —CH(CH) In one particular embodiment of the compounds of For CFCF, -(CH), OH, -CH-CH(NH)COOCH, mula Ia, R is H and R7 is selected from —CHCH and —(CH)OCH, -CHROC(O) Calkyl, -CHROC —CH-CH(CH). In one particular embodiment of the (O)O Calkyl, —CHROC(O)O-cyclohexyl, —Ca compounds of Formula Ib, R is H, R is -OH, and R is alkylene-N(CH), —CHOC(O)CHR NH, —CHOC selected from —CHOC(O)CH and —CHOC(O)CHCH (O)CHR NHC(O)O Calkyl, benzyl, and (CH), NH, or R is H, R is selected from —OCHOC (O)CH and —OCHOC(O)CHCH(CH), NH, and R7 is 10 H. The compound (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- ylmethyl)-N'-(2-hydroxythiazole-5-carbonyl)hydrazino-2- hydroxypropionic acid is also specifically disclosed in U.S. 15 Patent Publication No. 2012/0157386, and is represented by formula II':

OH (II)II or R is selected from —C(O) Calkyl, -C(O)CHR O -( NH, -C(O)CHR" NHC(O)O Calkyl, and -P(O) R. N/ (OR), and R is H; where each R is independently H or HO N1 —Calkyl; each R" is independently H, —CH, -CH OH O 25 (CH), phenyl, or benzyl; and each R is independently H,

—Calkyl, or phenyl; or a pharmaceutically acceptable salt thereof. In one particular embodiment of the compounds of For mula II, R is H and R7 is CHCH. 30 The compound (R)-3-(N-(5'-chloro-2'-fluorobiphenyl-4- ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5-carbonyl-hy drazino-2-hydroxypropionic acid is also specifically dis closed in U.S. Patent Publication No. 2012/0157386, and is In one embodiment, this compound is referred to as the represented by formula III': active form and is administered as a prodrug, which is 35 metabolized in vivo to form the compound of formula II".

U.S. Patent Publication No. 2012/0157386 also disclosed (III) the isobutyl ester prodrug of the compound of formula II". Another aspect of the invention relates to other prodrugs of the compound of formula II". These prodrugs are repre 40 sented by formula X, where R is F. R is C1, and X is: HO

OH 45

In one embodiment, these compounds are represented by 50 formula II: (II) In one embodiment, this compound is referred to as the active form and is administered as a prodrug, which is metabolized in vivo to form the compound of formula III". 55 U.S. Patent Publication No. 2012/0157386 also disclosed the ethyl ester and mofetil ester prodrugs of the compound

of formula III". Another aspect of the invention relates to other prodrugs of the compound of formula III". These prodrugs are repre 60 sented by formula X, where R is F. R is C1, and X is:

65 US 9,683,002 B2 15 16 In one embodiment, these compounds are represented by 2-hydroxypropionic acid is also specifically disclosed in formula III: U.S. Patent Publication No. 2012/0157386, and is repre sented by formula IV':

(III) (IV)

10

HO N

15 OH

C where R is H and R is selected from —CH2CH(CH), F —CHCF. —(CH2)CF, —CHCFCH. —CHCFCF, —C(CH) (CF), —CH(CHCH)CF, —CH(CH) In one embodiment, this compound is referred to as the CFCF, -(CH), OH, -CH-CH(NH)COOCH, active form and is administered as a prodrug, which is -(CH)OCH, -CHROC(O) Calkyl, -CHROC 25 metabolized in vivo to form the compound of formula IV". (O)O Calkyl, —CHROC(O)O-cyclohexyl, —Ca U.S. Patent Publication No. 2012/0157386 also disclosed alkylene-N(CH), —CHOC(O)CHR NH, -CHOC the ethyl ester, isopropyl ester, and isobutyl ester prodrugs of (O)CHR" NHC(O)O Calkyl, benzyl, and the compound of formula IV". Another aspect of the invention relates to other prodrugs 30 of the compound of formula IV". These prodrugs are repre sented by formula X, where R is F. R is C1, and X is:

O YO 35 O where R is H or —CH. In one embodiment, these com or R is selected from —C(O) Calkyl, -C(O)CHR 40 pounds are represented by IV: NH, -C(O)CHR" NHC(O)O Calkyl, and -P(O) (OR), and R is H; where each R is independently H or —C-alkyl; each R" is independently H, —CH, -CH (IV) (CH), phenyl, or benzyl; and each R is independently H, O-R —Calkyl, or phenyl; or a pharmaceutically acceptable salt 45 thereof. In one particular embodiment of the compounds of For O Y.M mula III, R is H and R7 is selected from —CH-CH(CH), O O —CHCFCF —(CH)OCH, —CHOC(O)CH, —CHOC(O)(CH),CH, —CHOC(O)OCHCH 50 RN NH —CHOC(O)OCH(CH), —CH(CH)OC(O)O-cyclo O N hexyl, -CHOC(O)CHCH(CH), NHC(O)OCH, and

55

60 where R is H and R7 is selected from —CHCF, —(CH2)CF, —CHCFCH. —CHCFCF, —C(CH) (CF), —CH(CHCH)CF, -CH(CH)CFCF, —(CH), OH, -CH-CH(NH)COOCH,-(CH)OCH, - CHROC(O) Calkyl, -CHROC(O)O C. alkyl, or R is -P(O)(OH), and R7 is H. 65 - CHROC(O)O-cyclohexyl, —C-alkylene-N(CH), The compound (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- CHOC(O)CHR NH, CHOC(O)CHR NHC(O) ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hydrazino O—C-alkyl, benzyl, US 9,683,002 B2 17 18 In one embodiment, this compound is referred to as the active form and is administered as a prodrug, which is metabolized in vivo to form the compound of formula V. Another aspect of the invention relates to other prodrugs of the compound of formula V. These prodrugs are repre sented by formula X, where R is F. R is C1, and X is:

10 or R is selected from C(O) Coalkyl, -C(O)CHR NH, -C(O)CHR NHC(O)C Calkyl, and -P(O) (OR), and R7 is H; or R is –C(O)CH-NH and R7 is —CHCH; where each R is independently H or - C alkyl; each R" is independently H, —CH, -CH(CH), 15 phenyl, or benzyl; and each R is independently H. —C. alkyl, or phenyl; or a pharmaceutically acceptable salt In one embodiment, these compounds are represented by thereof. formula V: In one particular embodiment of the compounds of For mula IV, R is —CH, R is H. and R7 is selected from (V) —CHCFCF —(CH)OCH, —CHOC(O)CH, O —CHOC(O)—(CH2)CH, —CHOC(O)OCHCH —CHOC(O)OCH(CH), —CH(CH)OC(O)O-cyclo in hexyl, -CHOC(O)CHCH(CH), NH, -CHOC(O)CH N 25 O N / CH(CH),NHC(O)OCH, O N

35

In another particular embodiment of the compounds of Formula IV. R is —CH; R is selected from C(O) where R is H and R7 is selected from —CHCH CHCH –C(O)CH-NH = C(O)CH(CH)NH2, C(O) —CH-CH(CH), —CHCF, —(CH2)CF, CHCH(CH), NH, and C(O)CHCH(CH), NHC 40 —CHCFCH. —CHCFCF, —C(CH)(CF), —CH (O)OCH; and R7 is H. In yet another particular embodiment (CHCH)CF, —CH(CH)CFCF —(CH), OH, of the compounds of Formula IV. R is —CH, R is C(O) CH-CH(NH)COOCH, -(CH)OCH, CHROC CH-NH and R7 is CHCH (O) C alkyl, -CHRO C(O)O Calkyl, -CHROC The compound (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- (O)O-cyclohexyl, —Calkylene-N(CH), —CHOC(O) ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triaz 45 CHR NH, CHOC(O)CHR NHC(O)O Calkyl, ole-3-carbonyl)hydrazino-2-hydroxypropionic acid is spe benzyl, and cifically disclosed in U.S. Patent Publication No. 2012/ 0157386, and is represented by formula V':

50 (V) in O N 55 O S. M O N NH or R is selected from C(O) Coalkyl, -C(O)CHR HO N1 NH = C(O)CHR NHC(O)C Calkyl, and -P(O) 60 (OR), and R is H; where each R is independently H or OH —Calkyl; each R" is independently H, —CH, -CH (CH), phenyl, or benzyl; and each R is independently H, O C —Calkyl, or phenyl: or a pharmaceutically acceptable salt thereof. 65 In one particular embodiment of the compounds of For F mula V. R is H and R7 is selected from —CH2CH, - CH-CH(CH), —CHCFCF, -CHOC(O)CH,

US 9,683,002 B2 21 22 In yet another embodiment of the compounds of Formula -continued VIa and VIb, R is H, R is —CH OP(O)(OH), or (VIIb) —CHOC(O)CHCH(CH), NH, and R7 is —CHCH or R4 —CHCH(CH). / In yet another embodiment of the compounds of Formula 5 VIa and VIb, R is C(O)CHCH(CH), NH, R is H, and N1 N R7 is CHCH or -CHCH(CH). O / The compound (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- O ylmethyl)-N'-(1H-tetrazole-5-carbonyl)hydrazino-2-hy droxypropionic acid is specifically disclosed in U.S. Patent 10 RN NH Publication No. 2012/0157386, and is represented by for O N mula VII': (VII) N N1R 15 O N^Ys O H NH HO N1 where R and Rare H, and R7 is selected from —CHCF, —(CH2)CF, —CHCFCF —C(CH) (CF), —CH OH (CHCH)CF, —CH(CH)CFCF —(CH), OH, F CH-CH(NH)COOCH, -(CH),OCH, CHROC (O) Calkyl, -CHROC(O)O Calkyl, -CHROC (O)O-cyclohexyl, —Calkylene-N(CH), —CHOC(O) 25 CHR NH, CHOC(O)CHR NHC(O)O Calkyl, benzyl, and

C In one embodiment, this compound is referred to as the 30 active form and is administered as a prodrug, which is metabolized in vivo to form the compound of formula VII". Compounds Such as this can exist in a tautomer form, for example, as (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylm ethyl)-N'-(2H-tetrazole-5-carbonyl)hydrazino-2-hydroxy 35 propionic acid. U.S. Patent Publication No. 2012/0157386 also disclosed the ethyl ester, isopropyl ester, and isobutyl ester prodrugs of the compound of formula VII". or R is H, R is selected from CHOC(O)CHCH Another aspect of the invention relates to other prodrugs (CH), NHC(O)OCH and —CHOC(O)CHCH(CH), of the compound of formula VII". These prodrugs are 40 NH, and R7 is selected from H and —CHCH, or R is represented by formula X, where R is F. R is C1, and X is: selected from C(O) Coalkyl, -C(O)CHR NH2, —C(O)CHR NHC(O)O Calkyl, and -P(O)(OR), R N NN e p and R and R7 are H; where each R is independently H or w \ A. N 4 - Cisalkyl; each R" is independently H, —CH, -CH , M p N-R 45 (CH), phenyl, or benzyl; and each R is independently H, as N a YN17 —Calkyl, or phenyl; or a pharmaceutically acceptable salt w w O NRN thereof. In one embodiment, these compounds are represented by In one particular embodiment of the compounds of For formula VIIa or VIIb: mula VIIa and VIIb, RandR are H, and R7 is selected from 50 —CHCFCF, -CHOC(O)CH, CHOC(O)CHCH —CHOC(O)(CH),CH, —CHOC(O)OCH(CH), (VIIa) CHOC(O)CHICH(CH), NH, CHOC(O)CHCH R (CH), NHC(O)OCH, and N-NN. W. 55 O S. ) O N

R NH n 1

60

65 In another embodiment of the compounds of Formula VIIa and VIIb, R is H, R is CHOC(O)CHCH(CH), NH, and R is —CHCH. In still another embodiment of the US 9,683,002 B2 23 24 compounds of Formula VIIa and VIIb, or R is selected from (O)O-cyclohexyl, —Calkylene-N(CH), —CHOC(O) —C(O)CH –C(O)CHCH –C(O)CHCH(CH), NH, CHCH(CH), NH, and —CHOC(O)CHCH(CH)– and –C(O)CHCH(CH)NHC(O)CCH, and R and R' NHC(O)OCH; or R is selected from –C(O) Calkyl, are H. C(O)CHR NH, C(O)CHR NHC(O)O C. The compound (R)-3-N-(3'-chlorobiphenyl-4-ylmethyl)- alkyl, and -P(O)(OR), R is -OH, and R7 is H; or R is N'-(3-hydroxy-isoxazole-5-carbonyl)hydrazino-2-hydroxy H, R is selected from —OCHROC(O) Calkyl, propionic acid is specifically disclosed in U.S. Patent Pub OCHOC(O)CHCH(CH), NH, OCHOC(O)CH lication No. 2012/0157386, and is represented by formula VIII: CH(CH), NHC(O)CCH, and 10 Y& CH3, (VIII) o-N o1 N OH O S. 15 “Y” O and R is H; where each R is independently H or - C HO N alkyl; each R" is independently H, CH, -CH(CH), OH phenyl, or benzyl; and each R is independently H, —Calkyl, or phenyl; or a pharmaceutically acceptable salt C thereof. In one particular embodiment of the compounds of For mula VIII, R is H, R is -OH, and R7 is selected from 25 —CHOC(O)CH, -CHOC(O)CHCH(CH), NH, and —CHOC(O)CHICH(CH), NHC(O)OCH. In another In one embodiment, this compound is referred to as the particular embodiment of the compounds of Formula VIII, active form and is administered as a prodrug, which is metabolized in vivo to form the compound of formula VIII". R’ is selected from –C(O)CHCH –C(O)CHCH U.S. Patent Publication No. 2012/0157386 also disclosed (CH), C(O)CHCH(CH), NH, and –C(O)CHCH the ethyl ester, isopropyl ester, butyl ester, isobutyl ester, 30 (CH), NHC(O)OCH R is -OH, and R7 is H. In yet hexyl ester, heptyl ester, benzyl ester, medoxomil ester, another particular embodiment of the compounds of For 2-fluoro-1-fluoromethyl-ethyl ester, and 2.2.3,3,3-pentafluo mula VIII, R is H, R is selected from —OCHOC(O)CH, ropropyl ester prodrugs of the compound of formula VIII". –OCHOC(O)(CH2)CH, OCHOC(O)OCHCH Another aspect of the invention relates to other prodrugs –OCHOC(O)OCH(CH), OCHOC(O)CHCH of the compound of formula VIII". These prodrugs are 35 (CH), NH, OCHOC(O). CHCH(CH), INHC(O) represented by formula X, where R is H, R is C1, and X is: OCH and Y{ CH3, 40 or “Y” O In one embodiment, these compounds are represented by formula VIII: 45 and R7 is H. The compound (R)-3-N-(2,5'-dichlorobiphenyl-4-ylm ethyl)-N'-(1-hydroxy-1H-1.2.3 triazole-4-carbonyl)hy (VIII) drazino-2-hydroxypropionic acid is specifically disclosed in U.S. Patent Publication No. 2012/0157386, and is repre 50 sented by formula IX': (IX) NN

O O N \ s OH 55 NH HO N1

OH 60 C where R is H, R is -OH, and R7 is selected from —CHCF. —(CH2)CF, —C(CH) (CF), —CH C (CHCH)CF, —CH(CH)CFCF —(CH), OH, 65 CH-CH(NH)COOCH, -(CH)OCH, CHROC In one embodiment, this compound is referred to as the (O) Calkyl, -CHROC(O)O C. alkyl, -CHROC active form and is administered as a prodrug, which is US 9,683,002 B2 25 26 metabolized in vivo to form the compound of formula IX'. (OR), R is -OH, and R7 is H; or R is H, R is selected U.S. Patent Publication No. 2012/0157386 also disclosed from –OCHROC(O) C, OCHOC(O)CHCH the isopropyl ester, isobutyl ester, and heptyl ester prodrugs (CH), NH, OCHOC(O)CHCH(CH), NHC(O) of the compound of formula IX'. Another aspect of the invention relates to other prodrugs OCH and of the compound of formula IX'. These prodrugs are repre sented by formula X, where R is C1, R is C1, and X is: Y& CH3,

NN 10 or w \ O

w “Y” 3-S-sew O In one embodiment, these compounds are represented by 15 and R7 is H; where each R is independently H or - C formula IXa or IXb: alkyl; each R" is independently H, —CH, -CH(CH), phenyl, or benzyl; and each R is independently H. —C. alkyl, or phenyl; or a pharmaceutically acceptable salt (IXa) thereof. N=N In one particular embodiment of the compounds of For O O N NND4R mula IXa and IXb, R is H, R is –OH and R7 is selected from —CHOC(O)CH, —CHOC(O)(CH2)CH, —CHOC(O) OCHCH —CHOC(O)OCH(CH), RN O N NH —CH(CH)OC(O)O-cyclohexyl, -CHOC(O). CHCH 25 (CH), INHC(O)CCH, and R21 O

C 30

C 4 (IXb R M -N 35 ( \ In yet another embodiment of the compounds of Formula O 's-- IXa and IXb, R is H, R is selected from —OCHOC(O) RN NH CH, OCHOC(O)(CH),CH, OCHOC(O)CHCH O N 40 (CH), NH, OCHOC(O)CHCH(CH), NHC(O) OCH and R21 O

C 45 Y& CH3, C or where R is H, R is -OH, and R7 is selected from “Y” —CHCF. —(CH2)CF, —C(CH) (CF), —CH O (CHCH)CF, —CH(CH)CFCF —(CH), OH, 50 —CH-CH(NH)COOCH, -(CH), OCH, -CHROC (O) Calkyl, -CHROC(O)O C. alkyl, -CHROC and R7 is H. (O)O-cyclohexyl, —Calkylene-N(CH), —CHOC(O) General Synthetic Procedures CHR NH, CHOC(O)CHR NHC(O)O Calkyl, 55 and Compounds of the invention can be prepared from readily available starting materials using the following general methods, the procedures set forth in the Examples, or by using other methods, reagents, and starting materials that are 60 known to those of ordinary skill in the art. Although the following procedures may illustrate a particular embodiment of the invention, it is understood that other embodiments of the invention can be similarly prepared using the same or similar methods or by using other methods, reagents and 65 starting materials known to those of ordinary skill in the art. or R is selected from —C(O) Calkyl, -C(O)CHR It will also be appreciated that where typical or preferred NH, -C(O)CHR" NHC(O)O Calkyl, and -P(O) process conditions (for example, reaction temperatures, US 9,683,002 B2 27 28 times, mole ratios of reactants, solvents, pressures, etc.) are diisopropylethylamine (DIPEA), 4-methylmorpholine, given, other process conditions can also be used unless Sodium hydroxide, potassium hydroxide, potassium t-butox otherwise stated. In some instances, reactions were con ide, and metal hydrides. ducted at room temperature and no actual temperature Suitable inert diluents or solvents for use in these schemes measurement was taken. It is understood that room tempera include, by way of illustration and not limitation, tetrahy ture can be taken to mean a temperature within the range drofuran (THF), acetonitrile (MeCN), N,N-dimethylforma commonly associated with the ambient temperature in a mide (DMF), N,N-dimethylacetamide (DMA), dimethyl laboratory environment, and will typically be in the range of sulfoxide (DMSO), toluene, dichloromethane (DCM), chlo about 18° C. to about 30° C. In other instances, reactions roform (CHCl), carbon tetrachloride (CC1), 1,4-dioxane, were conducted at room temperature and the temperature 10 methanol, ethanol, water, diethyl ether, acetone, and the like. was actually measured and recorded. While optimum reac Suitable carboxylic acid/amine coupling reagents include tion conditions will typically vary depending on various benzotriazol-1-yloxytris(dimethylamino)phosphonium reaction parameters such as the particular reactants, solvents hexafluorophosphate (BOP), benzotriazol-1-yloxytripyrroli dinophosphonium hexafluorophosphate (PyBOP), N.N.N', and quantities used, those of ordinary skill in the art can 15 readily determine Suitable reaction conditions using routine N-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium optimization procedures. hexafluorophosphate (HATU), 1,3-dicyclohexylcarbodiim Additionally, as will be apparent to those skilled in the art, ide (DCC), N-(3-dimethylaminopropyl)-N'-ethylcarbodiim conventional protecting groups may be necessary or desired ide (EDC), carbonyldiimidazole (CDI), 1-hydroxybenzotri to prevent certain functional groups from undergoing unde azole (HOBt), and the like. Coupling reactions are sired reactions. The choice of a Suitable protecting group for conducted in an inert diluent in the presence of a base Such a particular functional group as well as Suitable conditions as DIPEA, and are performed under conventional amide and reagents for protection and deprotection of Such func bond-forming conditions. tional groups are well-known in the art. Protecting groups All reactions are typically conducted at a temperature other than those illustrated in the procedures described 25 within the range of about -78 C. to 100° C., for example at herein may be used, if desired. For example, numerous room temperature. Reactions may be monitored by use of protecting groups, and their introduction and removal, are thin layer chromatography (TLC), high performance liquid described in T. W. Greene and G. M. Wuts, Protecting chromatography (HPLC), and/or LCMS until completion. Groups in Organic Synthesis, Fourth Edition, Wiley, New Reactions may be complete in minutes, or may take hours, York, 2006, and references cited therein. 30 typically from 1-2 hours and up to 48 hours. Upon comple Carboxy-protecting groups are suitable for preventing tion, the resulting mixture or reaction product may be further undesired reactions at a carboxy group, and examples include, but are not limited to, methyl, ethyl, t-butyl, benzyl treated in order to obtain the desired product. For example, (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), the resulting mixture or reaction product may be subjected trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), 35 to one or more of the following procedures: concentrating or diphenylmethyl (benzhydryl, DPM) and the like. Amino partitioning (for example, between EtOAc and water or protecting groups are Suitable for preventing undesired between 5% THF in EtOAc and 1M phosphoric acid); reactions at an amino group, and examples include, but are extraction (for example, with EtOAc, CHCl, DCM. chlo not limited to, t-butoxycarbonyl (BOC), trityl (Tr), benzy roform); washing (for example, with Saturated aqueous loxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), 40 NaCl, saturated aqueous NaHCO, NaCO (5%), CHCl or formyl, trimethylsilyl (TMS), t-butyldimethylsilyl (TB 1M NaOH); drying (for example, over MgSO, over DMS), and the like. Na2SO, or in vacuo); filtering; crystallizing (for example, Standard deprotection techniques and reagents are used to from EtOAc and hexanes); being concentrated (for example, remove the protecting groups, and may vary depending upon in vacuo); and/or purification (e.g., silica gel chromatogra which group is used. For example, sodium or lithium 45 hydroxide is commonly used when the carboxy-protecting phy, flash chromatography, preparative HPLC, reverse group is methyl, an acid such as TFA or HCl (e.g., 4.0 MHCl phase-HPLC, or crystallization). in 1,4-dioxane) is commonly used when the carboxy-pro By way of illustration, compounds of the invention, as tecting group is ethyl or t-butyl, and H/Pd/C may be used well as their salts, can be prepared as shown in Schemes when the carboxy-protecting group is benzyl. A BOC 50 I-IV. amino-protecting group can be removed using an acidic reagent such as TFA in DCM or HCl in 1,4-dioxane, while a Cbzamino-protecting group can be removed by employing Scheme I catalytic hydrogenation conditions such as H. (1 atm) and O X 10% Pd/C in an alcoholic solvent (“H/Pd/C). 55 O N Leaving groups are functional groups or atoms that can be NH displaced by another functional group or atom in a Substi 1n O N Y tution reaction, Such as a nucleophilic Substitution reaction. By way of example, representative leaving groups include O R21 -- chloro, bromo and iodo groups; Sulfonic ester groups, such 60 as mesylate, tosylate, brosylate, nosylate and the like; and acyloxy groups, such as acetoxy, trifluoroacetoxy and the Rb like. Suitable bases for use in these schemes include, by way Ra of illustration and not limitation, potassium carbonate, cal 65 cium carbonate, Sodium carbonate, triethylamine (Et-N), HO-R -> pyridine, 1,8-diazabicyclo-5.4.0]undec-7-ene (DBU), N.N- US 9,683,002 B2 30 -continued can be prepared by techniques that are known in the art or O X described herein. Exemplary L-R7 compounds include Br— O N (CH),OH, Br (CH),OH, Br—(CH2)2OCH, RN NH Br CHOC(O)CH, C1 CHOC(O)(CH)CH, O N C1 CHOC(O)CCHCH, C1 CHOC(O)OCH(CH), C1 CHOC(O)O-cyclohexyl, (S)-2-benzyloxycarbo R21 O nylamino-3-methyl-butyric acid chloromethyl ester, and (S)- 2-t-butoxycarbonylamino-3-methyl-butyric acid chlorom ethyl ester. 10 Alternately, in Scheme II, an alcohol have be used in place of the L-R", for example HO C-alkylene-N(CH), in a Ra coupling reaction using HOBt and EDC.

Scheme I is a transesterification reactions. Generally, this 15 reaction involves reacting the ester with heat, the desired Scheme III alcohol (HO-R) and a suitable acid catalyst, for example hydrochloric acid. The HO Ralcohols are either commer O X cially available or can be prepared by techniques that are O N known in the art or described herein. Exemplary HO R' NH compounds include HO CHCF. HO-(CH2)CF, HO N1 HO CHCFCH HO CHCFCF HO C(CH) O (CF), HO CH(CHCH)CF, HO CH(CH)CFCF, R21 -- benzyl alcohol, and Rb 25

Ra L-R2 -> 30 O X “Y”O O N NH HO N1

35 O Scheme II R21 O X Rb O n NH 40 HO N1 Ra

O -- R21 Scheme III is a nucleophilic substitution reaction, where L Rb 45 is a Suitable leaving group. Generally, this reaction is con ducted in the presence of a suitable base such as N.N- diisopropylethylamine in a suitable inert diluent or solvent Ra such as dichloromethane. The L-R compound is either L-R -> commercially available or can be prepared by techniques 50 that are known in the art or described herein. Exemplary O X L-R compounds include C1-C(O) CH, C1-C(O)—CH O N (CH), and Cl—C(O)—CH-CH(CH). RN O N NH

O 55 Scheme IW R2 Y O P Rb RN O N NH

60 O Ra R21 -- Scheme II is a nucleophilic substitution reaction, where L is Rb a Suitable leaving group. Generally, this reaction is con ducted in the presence of a suitable base such as triethyl 65 amine in a suitable inert diluent or solvent such as acetone. Ra The L-R compounds are either commercially available or US 9,683,002 B2 32 -continued compound to reduce blood pressure as well as to measure a O test compounds ability to prevent or delay a rise in blood pressure. The Dahl salt-sensitive (DSS) hypertensive rat HO J X - model is a model of hypertension that is sensitive to dietary O X salt (NaCl), and is described, for example, in Rapp (1982) O N Hypertension 4:753-763. The rat monocrotaline model of RN NH pulmonary arterial hypertension described, for example, in O N Kato et al. (2008).J. Cardiovasc. Pharmacol. 51(1):18-23, is a reliable predictor of clinical efficacy for the treatment of O 10 R2 Y pulmonary arterial hypertension. Heart failure animal mod els include the DSS rat model for heart failure and the aorto-caval fistula model (AV shunt), the latter of which is described, for example, in Norling et al. (1996).J. Amer: Soc. 15 Nephrol. 7:1038-1044. Other animal models, such as the hot Ra plate, tail-flick and formalin tests, can be used to measure the analgesic properties of a compound, as well as the spinal Scheme IV is a coupling reaction, where P is H or a suitable nerve ligation (SNL) model of neuropathic pain. See, for amino-protecting group. When P is an amino protecting example, Malmberg et al. (1999) Current Protocols in group, the process further comprises deprotecting the com Neuroscience 8.9.1-8.9.15. Other properties and utilities of pound before or in situ with the coupling step. Exemplary the compounds can be demonstrated using various in vitro coupling reagents include HATU and HOBt with EDC. and in vivo assays well known to those skilled in the art. Generally, these reactions are conducted in the presence of The compounds of the invention are expected to be useful a base such as DIPEA or 4-methylmorpholine, and an inert for the treatment and/or prevention of medical conditions diluent or solvents such as DMF or DMA. The carboxylic 25 responsive to NEP inhibition. Thus it is expected that acid starting materials are generally commercially available patients suffering from a disease or disorder that is treated by or can be prepared using procedures that are known in the inhibiting the NEP enzyme or by increasing the levels of its art. peptide Substrates, can be treated by administering a thera Further details regarding specific reaction conditions and peutically effective amount of a compound of the invention. other procedures for preparing representative compounds of 30 For example, by inhibiting NEP, the compound is expected the invention or intermediates thereof are described in the to potentiate the biological effects of endogenous peptides Examples set forth below. that are metabolized by NEP, such as the natriuretic pep tides, bombesin, bradykinins, calcitonin, endothelins, Utility enkephalins, neurotensin, Substance P and vasoactive intes 35 tinal peptide. Thus, the compounds are expected to have The compounds of formula I-V and VIP-IX' have activity other physiological actions, for example, on the renal, cen as neprilysin inhibitors, and are expected to have therapeutic tral nervous, reproductive and gastrointestinal systems. utility as a neprilysin inhibitor. Prodrugs of these com pounds, once metabolized in vivo, are expected to have the Cardiovascular Diseases same utility. Thus, when discussing the activity of the 40 compounds of the invention, it is understood that these By potentiating the effects of vasoactive peptides like the prodrugs have the expected activity once metabolized. natriuretic peptides and bradykinin, compounds of the Exemplary assays include by way of illustration and not invention are expected to find utility in treating and/or limitation, assays that measure NEP inhibition. Useful sec preventing medical conditions such as cardiovascular dis ondary assays include assays to measure ACE inhibition and 45 eases. See, for example, Rogues et al. (1993) Pharmacol. aminopeptidase P (APP) inhibition (e.g., as described in Rev. 45:87-146 and Dempsey et al. (2009) Amer: J. of Sulpizio et al. (2005) JPET 315:1306-1313). A pharmaco Pathology 174(3):782-796. Cardiovascular diseases of par dynamic assay to assess the in vivo inhibitory potencies for ticular interest include hypertension and heart failure. ACE and NEP in anesthetized rats is described in Seymour Hypertension includes, by way of illustration and not limi et al. (1985) Hypertension 7 (Suppl I): I-35-I-42 and Wigle 50 tation: primary hypertension, which is also referred to as et al. (1992) Can. J. Physiol. Pharmacol. 70:1525-1528), essential hypertension or idiopathic hypertension; secondary where ACE inhibition is measured as the percent inhibition hypertension; hypertension with accompanying renal dis of the angiotensin I pressor response and NEP inhibition is ease; severe hypertension with or without accompanying measured as increased urinary cyclic guanosine 3',5'-mono renal disease; pulmonary hypertension, including pulmo phosphate (cGMP) output. 55 nary arterial hypertension; and resistant hypertension. Heart There are also many in vivo assays that can be used. The failure includes, by way of illustration and not limitation: conscious spontaneously hypertensive rat (SHR) model is a congestive heart failure; acute heart failure; chronic heart renin dependent hypertension model. See for example, failure, for example with reduced left ventricular ejection Intengan et al. (1999) Circulation 100(22):2267-2275 and fraction (also referred to as systolic heart failure) or with Badyal et al. (2003) Indian Journal of Pharmacology 60 preserved left ventricular ejection fraction (also referred to 35:349-362. The conscious desoxycorticosterone acetate as diastolic heart failure); and acute and chronic decompen salt (DOCA-salt) rat model is a volume dependent hyper sated heart failure, with or without accompanying renal tension model that is useful for measuring NEP activity. See disease. Thus, one embodiment of the invention relates to a for example, Trapani et al. (1989) J. Cardiovasc. Pharma method for treating hypertension, particularly primary col. 14:419–424, Intengan et al. (1999) Hypertension 34(4): 65 hypertension or pulmonary arterial hypertension, compris 907-913, and Badyal et al. (2003) supra). The DOCA-salt ing administering to a patient a therapeutically effective model is particularly useful for evaluating the ability of a test amount of a compound of the invention. US 9,683,002 B2 33 34 For treatment of primary hypertension, the therapeutically B-adrenergic receptor antagonists, dual-acting B-adrenergic effective amount is typically the amount that is sufficient to receptor antagonist/C.-receptor antagonists, chymase lower the patient’s blood pressure. This would include both inhibitors, digoxin, diuretics, endothelin converting enzyme mild-to-moderate hypertension and severe hypertension. (ECE) inhibitors, endothelin receptor antagonists, natriuretic When used to treat hypertension, the compound may be peptides and their analogs, natriuretic peptide clearance administered in combination with other therapeutic agents receptor antagonists, nitric oxide donors, prostaglandinana Such as aldosterone antagonists, angiotensin-converting logs, PDE-V inhibitors, soluble guanylate cyclase activators enzyme inhibitors and dual-acting angiotensin-converting and stimulators, and vasopressin receptor antagonists. In one enzyme? neprilysin inhibitors, angiotensin-converting particular embodiment of the invention, a compound of the enzyme 2 (ACE2) activators and stimulators, angiotensin-II 10 invention is combined with an aldosterone antagonist, a vaccines, anti-diabetic agents, anti-lipid agents, anti-throm B-adrenergic receptor antagonist, an AT receptor antago botic agents, AT receptor antagonists and dual-acting AT nist, or a diuretic, and used to treat congestive heart failure. receptor antagonist/neprilysin inhibitors, B-adrenergic receptor antagonists, dual-acting B-adrenergic receptor Diarrhea antagonist/C.-receptor antagonists, calcium channel block 15 As NEP inhibitors, compounds of the invention are ers, diuretics, endothelin receptor antagonists, endothelin expected to inhibit the degradation of endogenous converting enzyme inhibitors, neprilysin inhibitors, natri enkephalins and thus Such compounds may also find utility uretic peptides and their analogs, natriuretic peptide clear for the treatment of diarrhea, including infectious and secre ance receptor antagonists, nitric oxide donors, non-steroidal tory/watery diarrhea. See, for example, Baumer et al. (1992) anti-inflammatory agents, phosphodiesterase inhibitors (spe Gut 33:753-758: Farthing (2006) Digestive Diseases 24:47 cifically PDE-V inhibitors), prostaglandin receptor agonists, 58; and Marçais-Collado (1987) Eur: J. Pharmacol. 144(2): renin inhibitors, soluble guanylate cyclase stimulators and 125-132. When used to treat diarrhea, compounds of the activators, and combinations thereof. In one particular invention may be combined with one or more additional embodiment of the invention, a compound of the invention antidiarrheal treatments. is combined with an AT receptor antagonist, a diuretic, a 25 calcium channel blocker, or a combination thereof, and used Renal Diseases to treat primary hypertension. In another particular embodi By potentiating the effects of vasoactive peptides like the ment of the invention, a compound of the invention is natriuretic peptides and bradykinin, compounds of the combined with an AT receptor antagonist, and used to treat invention are expected to enhance renal function (see Chen hypertension with accompanying renal disease. 30 et al. (1999) Circulation 100:2443-2448: Lipkin et al. (1997) For treatment of pulmonary arterial hypertension, the Kidney Int. 52:792-801; and Dussaule et al. (1993) Clin. Sci. therapeutically effective amount is typically the amount that 84:31-39) and find utility in treating and/or preventing renal is sufficient to lower the pulmonary vascular resistance. diseases. Renal diseases of particular interest include dia Other goals of therapy are to improve a patient’s exercise betic nephropathy, chronic kidney disease, proteinuria, and capacity. For example, in a clinical setting, the therapeuti 35 particularly acute kidney injury or acute renal failure (see cally effective amount can be the amount that improves a Sharkovska et al. (2011) Clin. Lab. 57:507–515 and Newaz patient’s ability to walk comfortably for a period of 6 et al. (2010) Renal Failure 32:384-390). When used to treat minutes (covering a distance of approximately 20-40 renal disease, the compound may be administered in com meters). When used to treat pulmonary arterial hypertension bination with other therapeutic agents such as angiotensin the compound may be administered in combination with 40 converting enzyme inhibitors, AT receptor antagonists, and other therapeutic agents such as C.-adrenergic antagonists, diuretics. f-adrenergic receptor antagonists, f-adrenergic receptor agonists, angiotensin-converting enzyme inhibitors, antico Preventative Therapy agulants, calcium channel blockers, diuretics, endothelin By potentiating the effects of the natriuretic peptides, receptor antagonists, PDE-V inhibitors, prostaglandin ana 45 compounds of the invention are also expected to be useful in logs, selective serotonin reuptake inhibitors, and combina preventative therapy, due to the antihypertrophic and anti tions thereof. In one particular embodiment of the invention, fibrotic effects of the natriuretic peptides (see Potter et al. a compound of the invention is combined with a PDE-V (2009) Handbook of Experimental Pharmacology 191:341 inhibitor or a selective serotonin reuptake inhibitor and used 366), for example in preventing the progression of cardiac to treat pulmonary arterial hypertension. 50 insufficiency after myocardial infarction, preventing arterial Another embodiment of the invention relates to a method restenosis after angioplasty, preventing thickening of blood for treating heart failure, in particular congestive heart vessel walls after vascular operations, preventing athero failure (including both systolic and diastolic congestive Sclerosis, and preventing diabetic angiopathy. heart failure), comprising administering to a patient a thera peutically effective amount of a compound of the invention. 55 Glaucoma Typically, the therapeutically effective amount is the amount that is sufficient to lower blood pressure and/or improve By potentiating the effects of the natriuretic peptides, renal functions. In a clinical setting, the therapeutically compounds of the invention are expected to be useful to treat effective amount can be the amount that is sufficient to glaucoma. See, for example, Diestelhorst et al. (1989) improve cardiac hemodynamics, like for instance reduction 60 International Ophthalmology 12:99-101. When used to treat in wedge pressure, right atrial pressure, filling pressure, and glaucoma, compounds of the invention may be combined vascular resistance. In one embodiment, the compound is with one or more additional anti-glaucoma agents. administered as an intravenous dosage form. When used to treat heart failure, the compound may be administered in Pain Relief combination with other therapeutic agents such as adenosine 65 receptor antagonists, advanced glycation end product break As NEP inhibitors, compounds of the invention are ers, aldosterone antagonists, AT receptor antagonists, expected to inhibit the degradation of endogenous US 9,683,002 B2 35 36 enkephalins and thus Such compounds may also find utility medical condition. Patients undergoing Such treatment will as analgesics. See, for example, Rogues et al. (1980) Nature typically be monitored on a routine basis to determine the 288:286-288 and Thanawala et al. (2008) Current Drug effectiveness of therapy. For example, in treating hyperten Targets 9:887-894. When used to treat pain, compounds of Sion, blood pressure measurements may be used to deter the invention may be combined with one or more additional 5 mine the effectiveness of treatment. Similar indicators for antinociceptive drugs such as aminopeptidase N or dipepti other diseases and conditions described herein, are well dyl peptidase III inhibitors, non-steroidal anti-inflammatory known and are readily available to the treating physician. agents, monoamine reuptake inhibitors, muscle relaxants, Continuous monitoring by the physician will insure that the NMDA receptor antagonists, opioid receptor agonists, optimal amount of the compound of the invention will be 5-HT serotonin receptoragonists, and tricyclic antidepres 10 administered at any given time, as well as facilitating the SantS. determination of the duration of treatment. This is of par Other Utilities ticular value when secondary agents are also being admin istered, as their selection, dosage, and duration of therapy Due to their NEP inhibition properties, compounds of the 15 may also require adjustment. In this way, the treatment invention are also expected to be useful as antitussive regimen and dosing schedule can be adjusted over the course agents, as well as find utility in the treatment of portal of therapy so that the lowest amount of active agent that hypertension associated with liver cirrhosis (see Sansoe et exhibits the desired effectiveness is administered and, fur al. (2005) J. Hepatol. 43:791-798), cancer (see Vesely ther, that administration is continued only so long as is (2005) J. Investigative Med. 53:360-365), depression (see necessary to Successfully treat the disease or medical con Noble et al. (2007) Exp. Opin. Ther. Targets 11:145-159), dition. menstrual disorders, preterm labor, pre-eclampsia, endo metriosis, reproductive disorders (for example, male and Research Tools female infertility, polycystic ovarian syndrome, implanta tion failure), and male and female sexual dysfunction, 25 Since the compounds of the invention are metabolized in including male erectile dysfunction and female sexual Vivo to compounds having activity as neprilysin inhibitors, arousal disorder. More specifically, the compounds of the they are also useful as a research tools for investigating or invention are expected to be useful in treating female sexual studying biological systems or samples having a NEP dysfunction (see Pryde et al. (2006).J. Med. Chem. 49:4409 enzyme, for example to study diseases where the NEP 4424), which is often defined as a female patients difficulty 30 enzyme or its peptide Substrates plays a role. Accordingly, or inability to find satisfaction in sexual expression. This one aspect of the invention relates to a method of using a covers a variety of diverse female sexual disorders includ compound of the invention as a research tool, comprising ing, by way of illustration and not limitation, hypoactive conducting a biological assay using a compound of the sexual desire disorder, sexual arousal disorder, orgasmic invention. Any Suitable biological system or sample having disorder and sexual pain disorder. When used to treat such 35 a NEP enzyme may be employed in such studies which may disorders, especially female sexual dysfunction, compounds be conducted either in vitro or in vivo. Representative of the invention may be combined with one or more of the biological systems or samples Suitable for Such studies following secondary agents: PDE-V inhibitors, dopamine include, but are not limited to, cells, cellular extracts, plasma agonists, estrogen receptor agonists and/or antagonists, membranes, tissue samples, isolated organs, mammals (such androgens, and estrogens. Due to their NEP inhibition 40 as mice, rats, guinea pigs, rabbits, dogs, pigs, humans, and properties, compounds of the invention are also expected to So forth), and the like, with mammals being of particular have anti-inflammatory properties, and are expected to have interest. In one particular embodiment of the invention, NEP utility as such, particularly when used in combination with enzyme activity in a mammal is inhibited by administering statins. a NEP-inhibiting amount of a compound of the invention. Recent studies suggest that NEP plays a role in regulating 45 These compounds can also be used as research tools by nerve function in insulin-deficient diabetes and diet induced conducting biological assays using Such compounds. obesity. Coppey et al. (2011) Neuropharmacology 60:259 When used as a research tool, a biological system or 266. Therefore, due to their NEP inhibition properties, sample comprising a NEP enzyme is typically contacted compounds of the invention are also expected to be useful in with a NEP enzyme-inhibiting amount of a compound of the providing protection from nerve impairment caused by 50 invention. After the biological system or sample is exposed diabetes or diet induced obesity. to the compound, the effects of inhibiting the NEP enzyme The amount of the compound of the invention adminis are determined using conventional procedures and equip tered per dose or the total amount administered per day may ment, Such as by measuring receptor binding in a binding be predetermined or it may be determined on an individual assay or measuring ligand-mediated changes in a functional patient basis by taking into consideration numerous factors, 55 assay. Exposure encompasses contacting cells or tissue with including the nature and severity of the patient’s condition, the compound, administering the crystalline compound to a the condition being treated, the age, weight, and general mammal, for example by i.p. p.o. i.V., s.c., or inhaled health of the patient, the tolerance of the patient to the active administration, and so forth. This determining step can agent, the route of administration, pharmacological consid involve measuring a response (a quantitative analysis) or can erations such as the activity, efficacy, pharmacokinetics and 60 involve making an observation (a qualitative analysis). toxicology profiles of the compound and any secondary Measuring a response involves, for example, determining agents being administered, and the like. Treatment of a the effects of the compound on the biological system or patient Suffering from a disease or medical condition (Such sample using conventional procedures and equipment, Such as hypertension) can begin with a predetermined dosage or as enzyme activity assays and measuring enzyme substrate a dosage determined by the treating physician, and will 65 or product mediated changes in functional assays. The assay continue for a period of time necessary to prevent, amelio results can be used to determine the activity level as well as rate, Suppress, or alleviate the symptoms of the disease or the amount of compound necessary to achieve the desired US 9,683,002 B2 37 38 result, that is, a NEP enzyme-inhibiting amount. Typically, pound of the invention. Those skilled in the art will recog the determining step will involve determining the effects of nize, however, that a pharmaceutical composition may inhibiting the NEP enzyme. contain more than a therapeutically effective amount, Such Additionally, the compounds of the invention can be used as in bulk compositions, or less than a therapeutically as research tools for evaluating other chemical compounds, 5 effective amount, that is, individual unit doses designed for and thus are also useful in screening assays to discover, for multiple administration to achieve a therapeutically effective example, new compounds having NEP-inhibiting activity. amount. Typically, the composition will contain from about Thus another aspect of the invention relates to a method of 0.01-95 wt % of active agent, including, from about 0.01-30 evaluating a test compound in a biological assay, compris wt %, such as from about 0.01-10 wt %, with the actual ing: (a) conducting a biological assay with a test compound 10 amount depending upon the formulation itself, the route of to provide a first assay value; (b) conducting the biological administration, the frequency of dosing, and so forth. In one assay with a compound of the invention to provide a second embodiment, a composition Suitable for an oral dosage form, assay value; wherein step (a) is conducted either before, for example, may contain about 5-70 wt %, or from about after or concurrently with step (b); and (c) comparing the 10-60 wt % of active agent. first assay value from step (a) with the second assay value 15 Any conventional carrier or excipient may be used in the from step (b). Exemplary biological assays include a NEP pharmaceutical compositions of the invention. The choice of enzyme inhibition assay. In this manner, the compounds of a particular carrier or excipient, or combinations of carriers the invention are used as standards in an assay to allow or excipients, will depend on the mode of administration comparison of the results obtained with a test compound and being used to treat a particular patient or type of medical with the compound of the invention to identify those test condition or disease state. In this regard, the preparation of compounds that have about equal or Superior activity, if any. a suitable composition for a particular mode of administra For example, pK data for a test compound or a group of test tion is well within the scope of those skilled in the pharma compounds is compared to the pK, data for a compound of ceutical arts. Additionally, carriers or excipients used in Such the invention to identify those test compounds that have the compositions are commercially available. By way of further desired properties, for example, test compounds having a 25 illustration, conventional formulation techniques are pK value about equal or superior to the compound of the described in Remington. The Science and Practice of Phar invention, if any. This aspect of the invention includes, as macy, 20" Edition, Lippincott Williams & White, Baltimore, separate embodiments, both the generation of comparison Md. (2000); and H. C. Ansel et al., Pharmaceutical Dosage data (using the appropriate assays) and the analysis of test Forms and Drug Delivery Systems, 7" Edition, Lippincott data to identify test compounds of interest. 30 Williams & White, Baltimore, Md. (1999). Still another aspect of the invention relates to a method of Representative examples of materials which can serve as studying a biological system or sample comprising a NEP pharmaceutically acceptable carriers include, but are not enzyme, the method comprising: (a) contacting the biologi limited to, the following: Sugars, such as lactose, glucose cal system or sample with a compound of the invention; and and Sucrose; starches, such as corn starch and potato starch; (b) determining the effects caused by the compound on the 35 cellulose, such as microcrystalline cellulose, and its deriva biological system or sample. tives, such as Sodium carboxymethyl cellulose, ethyl cellu lose and cellulose acetate; powdered tragacanth; malt, gela Pharmaceutical Compositions and Formulations tin, talc, excipients, such as cocoa butter and Suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, Compounds of the invention are typically administered to 40 sesame oil, olive oil, corn oil and Soybean oil; glycols, such a patient in the form of a pharmaceutical composition or as propylene glycol, polyols, such as glycerin, Sorbitol, formulation. Such pharmaceutical compositions may be mannitol and polyethylene glycol; esters, such as ethyl administered to the patient by any acceptable route of oleate and ethyl laurate; agar; buffering agents, such as administration including, but not limited to, oral, rectal, magnesium hydroxide and aluminum hydroxide; alginic vaginal, nasal, inhaled, topical (including transdermal), ocu 45 acid; pyrogen-free water, isotonic Saline; Ringer's solution; lar, and parenteral modes of administration. Further, the ethyl alcohol; phosphate buffer Solutions; compressed pro compounds of the invention may be administered, for pellant gases, such as chlorofluorocarbons and hydrofluoro example orally, in multiple doses per day (for example, two, carbons; and other non-toxic compatible Substances three, or four times daily), in a single daily dose or a single employed in pharmaceutical compositions. weekly dose. It will be understood that any form of the 50 Pharmaceutical compositions are typically prepared by compounds of the invention, (that is, free base, free acid, thoroughly and intimately mixing or blending the active pharmaceutically acceptable salt, Solvate, etc.) that is Suit agent with a pharmaceutically acceptable carrier and one or able for the particular mode of administration can be used in more optional ingredients. The resulting uniformly blended the pharmaceutical compositions discussed herein. mixture may then be shaped or loaded into tablets, capsules, Accordingly, in one embodiment, the invention relates to 55 pills, canisters, cartridges, dispensers and the like using a pharmaceutical composition comprising a pharmaceuti conventional procedures and equipment. cally acceptable carrier and a compound of the invention. In one embodiment, the pharmaceutical compositions are The compositions may contain other therapeutic and/or suitable for oral administration. Suitable compositions for formulating agents if desired. When discussing composi oral administration may be in the form of capsules, tablets, tions, the “compound of the invention' may also be referred 60 pills, lozenges, cachets, dragees, powders, granules; Solu to herein as the “active agent,” to distinguish it from other tions or Suspensions in an aqueous or non-aqueous liquid; components of the formulation, such as the carrier. Thus, it oil-in-water or water-in-oil liquid emulsions; elixirs or Syr is understood that the term “active agent” includes com ups; and the like; each containing a predetermined amount pounds of formula I as well as pharmaceutically acceptable of the active agent. salts, Solvates and prodrugs of that compound. 65 When intended for oral administration in a solid dosage The pharmaceutical compositions of the invention typi form (capsules, tablets, pills and the like), the composition cally contain a therapeutically effective amount of a com will typically comprise the active agent and one or more US 9,683,002 B2 39 40 pharmaceutically acceptable carriers, such as Sodium citrate dosage form. The term “unit dosage form” refers to a or dicalcium phosphate. Solid dosage forms may also com physically discrete unit Suitable for dosing a patient, that is, prise: fillers or extenders, such as starches, microcrystalline each unit containing a predetermined quantity of the active cellulose, lactose. Sucrose, glucose, mannitol, and/or silicic agent calculated to produce the desired therapeutic effect acid; binders, such as carboxymethylcellulose, alginates, either alone or in combination with one or more additional gelatin, polyvinyl pyrrolidone. Sucrose and/or acacia: units. For example, Such unit dosage forms may be capsules, humectants, such as glycerol; disintegrating agents. Such as tablets, pills, and the like. agar-agar, calcium carbonate, potato or tapioca starch, alg In another embodiment, the compositions of the invention inic acid, certain silicates, and/or sodium carbonate; Solution are suitable for inhaled administration, and will typically be retarding agents. Such as paraffin; absorption accelerators, 10 in the form of an aerosol or a powder. Such compositions are Such as quaternary ammonium compounds; wetting agents, generally administered using well-known delivery devices, Such as cetyl alcohol and/or glycerol monostearate; absor Such as a nebulizer, dry powder, or metered-dose inhaler. bents, such as kaolin and/or bentonite clay; lubricants. Such Nebulizer devices produce a stream of high velocity air that as talc, calcium Stearate, magnesium Stearate, Solid polyeth causes the composition to spray as a mist that is carried into ylene glycols, Sodium lauryl Sulfate, and/or mixtures thereof 15 a patient’s respiratory tract. An exemplary nebulizer formu coloring agents; and buffering agents. lation comprises the active agent dissolved in a carrier to Release agents, wetting agents, coating agents, Sweeten form a solution, or micronized and combined with a carrier ing, flavoring and perfuming agents, preservatives and anti to form a Suspension of micronized particles of respirable oxidants may also be present in the pharmaceutical compo size. Dry powder inhalers administer the active agent as a sitions. Exemplary coating agents for tablets, capsules, pills free-flowing powder that is dispersed in a patient's air and like, include those used for enteric coatings, such as stream during inspiration. An exemplary dry powder formu cellulose acetate phthalate, polyvinyl acetate phthalate, lation comprises the active agent dry-blended with an hydroxypropyl methylcellulose phthalate, methacrylic acid excipient such as lactose, starch, mannitol, dextrose, poly methacrylic acid ester copolymers, cellulose acetate trimel lactic acid, polylactide-co-glycolide, and combinations litate, carboxymethyl ethyl cellulose, hydroxypropyl methyl 25 thereof. Metered-dose inhalers discharge a measured cellulose acetate Succinate, and the like. Examples of phar amount of the active agent using compressed propellant gas. maceutically acceptable antioxidants include: water-soluble An exemplary metered-dose formulation comprises a solu antioxidants, such as ascorbic acid, cysteine hydrochloride, tion or Suspension of the active agent in a liquefied propel sodium bisulfate, sodium metabisulfate sodium sulfite and lant, Such as a chlorofluorocarbon or hydrofluoroalkane. the like; oil-soluble antioxidants, such as ascorbyl palmitate, 30 Optional components of Such formulations include co-sol butylated hydroxyanisole, butylated hydroxytoluene, leci vents, such as ethanol or pentane, and Surfactants, such as thin, propyl gallate, alpha-tocopherol, and the like; and sorbitan trioleate, oleic acid, lecithin, glycerin, and sodium metal-chelating agents, such as citric acid, ethylenediamine lauryl Sulfate. Such compositions are typically prepared by tetraacetic acid, Sorbitol, tartaric acid, phosphoric acid, and adding chilled or pressurized hydrofluoroalkane to a suitable the like. 35 container containing the active agent, ethanol (if present) Compositions may also be formulated to provide slow or and the Surfactant (if present). To prepare a Suspension, the controlled release of the active agent using, by way of active agent is micronized and then combined with the example, hydroxypropyl methyl cellulose in varying pro propellant. Alternatively, a Suspension formulation can be portions or other polymer matrices, liposomes and/or micro prepared by spray drying a coating of Surfactant on micron spheres. In addition, the pharmaceutical compositions of the 40 ized particles of the active agent. The formulation is then invention may contain opacifying agents and may be for loaded into an aerosol canister, which forms a portion of the mulated so that they release the active agent only, or inhaler. preferentially, in a certain portion of the gastrointestinal Compounds of the invention can also be administered tract, optionally, in a delayed manner. Examples of embed parenterally (for example, by Subcutaneous, intravenous, ding compositions which can be used include polymeric 45 intramuscular, or intraperitoneal injection). For such admin Substances and waxes. The active agent can also be in istration, the active agent is provided in a sterile solution, micro-encapsulated form, optionally with one or more of the Suspension, or emulsion. Exemplary solvents for preparing above-described excipients. Such formulations include water, Saline, low molecular Suitable liquid dosage forms for oral administration weight alcohols such as propylene glycol, polyethylene include, by way of illustration, pharmaceutically acceptable 50 glycol, oils, gelatin, fatty acid esters such as ethyl oleate, and emulsions, microemulsions, Solutions, Suspensions, syrups the like. Parenteral formulations may also contain one or and elixirs. Liquid dosage forms typically comprise the more anti-oxidants, solubilizers, stabilizers, preservatives, active agent and an inert diluent. Such as, for example, water wetting agents, emulsifiers, and dispersing agents. Surfac or other solvents, Solubilizing agents and emulsifiers. Such tants, additional stabilizing agents or pH-adjusting agents as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl 55 (acids, bases or buffers) and anti-oxidants are particularly acetate, benzyl alcohol, benzyl benzoate, propylene glycol, useful to provide stability to the formulation, for example, to 1,3-butylene glycol, oils (for example, cottonseed, ground minimize or avoid hydrolysis of ester and amide linkages, or nut, corn, germ, olive, castor and sesame oils), glycerol, dimerization of thiols that may be present in the compound. tetrahydrofuryl alcohol, polyethylene glycols and fatty acid These formulations may be rendered sterile by use of a esters of Sorbitan, and mixtures thereof. Suspensions may 60 sterile injectable medium, a sterilizing agent, filtration, irra contain Suspending agents such as, for example, ethoxylated diation, or heat. In one particular embodiment, the parenteral isostearyl alcohols, polyoxyethylene Sorbitol and Sorbitan formulation comprises an aqueous cyclodextrin solution as esters, microcrystalline cellulose, aluminium metahydrox the pharmaceutically acceptable carrier. Suitable cyclodex ide, bentonite, agar-agar and tragacanth, and mixtures trins include cyclic molecules containing six or more C-D- thereof. 65 glucopyranose units linked at the 1.4 positions by a linkages When intended for oral administration, the pharmaceuti as in amylase, B-cyclodextrin or cycloheptaamylose. Exem cal compositions of the invention may be packaged in a unit plary cyclodextrins include cyclodextrin derivatives Such as US 9,683,002 B2 41 42 hydroxypropyl and sulfobutyl ether cyclodextrins such as ing both agents; or each agent may be present in separate and hydroxypropyl-3-cyclodextrin and sulfobutyl ether B-cyclo distinct compositions which are administered to the patient dextrin. Exemplary buffers for such formulations include simultaneously or at separate times. For example, a com carboxylic acid-based buffers such as citrate, lactate and pound of the invention can be combined with a second active maleate buffer solutions. 5 agent using conventional procedures and equipment to form Compounds of the invention can also be administered a combination of active agents comprising a compound of transdermally using known transdermal delivery systems the invention and a second active agent. Additionally, the and excipients. For example, the compound can be admixed active agents may be combined with a pharmaceutically with permeation enhancers, such as propylene glycol, poly acceptable carrier to form a pharmaceutical composition ethylene glycol monolaurate, azacycloalkan-2-ones and the 10 comprising a compound of the invention, a second active like, and incorporated into a patch or similar delivery agent and a pharmaceutically acceptable carrier. In this system. Additional excipients including gelling agents, embodiment, the components of the composition are typi emulsifiers and buffers, may be used in such transdermal compositions if desired. cally mixed or blended to create a physical mixture. The 15 physical mixture is then administered in a therapeutically Secondary Agents effective amount using any of the routes described herein. Alternatively, the active agents may remain separate and The compounds of the invention may be useful as the sole distinct before administration to the patient. In this embodi treatment of a disease or may be combined with one or more ment, the agents are not physically mixed together before additional therapeutic agents in order to obtain the desired administration but are administered simultaneously or at therapeutic effect. Thus, in one embodiment, pharmaceutical separate times as separate compositions. Such compositions compositions of the invention contain other drugs that are can be packaged separately or may be packaged together in co-administered with a compound of the invention. For a kit. When administered at separate times, the secondary example, the composition may further comprise one or more agent will typically be administered less than 24 hours after drugs (also referred to as 'secondary agents(s)). Such 25 administration of the compound of the invention, ranging therapeutic agents are well known in the art, and include anywhere from concurrent with administration of the com adenosine receptor antagonists, C.-adrenergic receptor pound of the invention to about 24 hours post-dose. This is antagonists, f-adrenergic receptor antagonists, f-adrener also referred to as sequential administration. Thus, a com gic receptor agonists, dual-acting B-adrenergic receptor pound of the invention can be orally administered simulta antagonist/ol-receptor antagonists, advanced glycation end 30 neously or sequentially with another active agent using two product breakers, aldosterone antagonists, aldosterone syn tablets, with one tablet for each active agent, where sequen thase inhibitors, aminopeptidase N inhibitors, androgens, tial may mean being administered immediately after admin angiotensin-converting enzyme inhibitors and dual-acting istration of the compound of the invention or at Some angiotensin-converting enzyme/neprilysin inhibitors, angio predetermined time later (for example, one hour later or tensin-converting enzyme 2 activators and stimulators, 35 three hours later). It is also contemplated that the secondary angiotensin-II Vaccines, anticoagulants, anti-diabetic agents, agent may be administered more than 24 hours after admin antidiarrheal agents, anti-glaucoma agents, anti-lipid agents, istration of the compound of the invention. Alternatively, the antinociceptive agents, anti-thrombotic agents, AT receptor combination may be administered by different routes of antagonists and dual-acting AT receptor antagonist/nepri administration, that is, one orally and the other by inhalation. lysin inhibitors and multifunctional angiotensin receptor 40 In one embodiment, the kit comprises a first dosage form blockers, bradykinin receptor antagonists, calcium channel comprising a compound of the invention and at least one blockers, chymase inhibitors, digoxin, diuretics, dopamine additional dosage form comprising one or more of the agonists, endothelin converting enzyme inhibitors, endothe secondary agents set forth herein, in quantities Sufficient to lin receptor antagonists, HMG-CoA reductase inhibitors, carry out the methods of the invention. The first dosage form estrogens, estrogen receptor agonists and/or antagonists, 45 and the second (or third, etc.) dosage form together comprise monoamine reuptake inhibitors, muscle relaxants, natri a therapeutically effective amount of active agents for the uretic peptides and their analogs, natriuretic peptide clear treatment or prevention of a disease or medical condition in ance receptor antagonists, neprilysin inhibitors, nitric oxide a patient. donors, non-steroidal anti-inflammatory agents, N-methyl Secondary agent(s), when included, are present in a d-aspartate receptor antagonists, opioid receptor agonists, 50 therapeutically effective amount such that they are typically phosphodiesterase inhibitors, prostaglandin analogs, prosta administered in an amount that produces a therapeutically glandin receptor agonists, renin inhibitors, selective sero beneficial effect when co-administered with a compound of tonin reuptake inhibitors, sodium channel blocker, soluble the invention. The secondary agent can be in the form of a guanylate cyclase stimulators and activators, tricyclic anti pharmaceutically acceptable salt, Solvate, optically pure depressants, vasopressin receptor antagonists, and combina 55 Stereoisomer, and so forth. The secondary agent may also be tions thereof. Specific examples of these agents are detailed in the form of a prodrug, for example, a compound having herein. a carboxylic acid group that has been esterified. Thus, Accordingly, in yet another aspect of the invention, a secondary agents listed herein are intended to include all pharmaceutical composition comprises a compound of the Such forms, and are commercially available or can be invention, a second active agent, and a pharmaceutically 60 prepared using conventional procedures and reagents. acceptable carrier. Third, fourth etc. active agents may also In one embodiment, compounds of the invention are be included in the composition. In combination therapy, the administered in combination with an adenosine receptor amount of compound of the invention that is administered, antagonist, representative examples of which include, but as well as the amount of secondary agents, may be less than are not limited to, naxifylline, rolofylline, SLV-320, theoph the amount typically administered in monotherapy. 65 ylline, and tonapofylline. Compounds of the invention may be physically mixed In one embodiment, compounds of the invention are with the second active agent to form a composition contain administered in combination with an O-adrenergic receptor US 9,683,002 B2 43 44 antagonist, representative examples of which include, but sin (ACE/NEP) inhibitor, examples of which include, but are are not limited to, doxazosin, prazosin, tamsulosin, and not limited to: AVE-0848 ((4S,7S,12bR)-7-3-methyl-2(S)- teraZosin. sulfanylbutyramido)-6-oxo-1,2,3,4,6,7,8,12b-octahydro Compounds of the invention may also be administered in pyrido2,1-a2-benzazepine-4-carboxylic acid); AVE combination with a B-adrenergic receptor antagonist ("B- 5 7688 (ilepatril) and its parent compound; BMS-182657 blockers'). Representative B-blockers include, but are not (2-2-oxo-3(S)-3-phenyl-2(S)-sulfanylpropionamido)-2,3, limited to, acebutolol, alprenolol, amoSulalol, arotinolol. 4,5-tetrahydro-1H-1-benzazepin-1-yl)acetic acid); CGS atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopin 35601 (N-1-4-methyl-2(S)-sulfanylpentanamidocyclo dolol, bucindolol, bucumolol, bufetolol, bufuralol, buni pentyl-carbonyl-L-tryptophan); fasidotril; fasidotrilate; trolol, bupranolol, bubridine, butofilolol, carazolol, car 10 enalaprilat; ER-32935 ((3R,6S,9aR)-6-3(S)-methyl-2(S)- teolol, carvedilol, celiprolol, cetamolol, cloranolol. Sulfanylpentanamido-5-oxoperhydrothiazolo3.2-a dilevalol, epanolol, esmolol, indenolol, labetolol, azepine-3-carboxylic acid); gempatrilat; MDL-101264 ((4S, levobunolol, mepindolol, metipranolol, metoprolol Such as 7S.12bR)-7-2(S)-(2-morpholino-acetylthio)-3- metoprolol Succinate and metoprolol tartrate, moprolol. phenylpropionamido-6-oxo-1,2,3,4,6,7,8,12b nadolol, nadoxolol, nebivalol, nipradillol, oXprenolol, pen 15 octahydropyrido 2,1-a2benzazepine-4-carboxylic acid); butolol, perbutolol, pindolol, practolol, pronethalol, propra MDL-101.287 (4S-4a,7a(R*), 12bf8-7-2-(carboxym nolol, Sotalol, Sufinalol, talindol, tertatolol, tilisolol, timolol, ethyl)-3-phenylpropionamido-6-oxo-1,2,3,4,6,7,8,12b-oc toliprolol, Xibenolol, and combinations thereof. In one par tahydropyrido2,1-a2benzazepine-4-carboxylic acid); ticular embodiment, the B-antagonist is selected from omapatrilat; RB-105 (N-2(S)-(mercaptomethyl)-3(R)-phe atenolol, bisoprolol, metoprolol, propranolol, Sotalol, and nylbutyl-L-alanine); sampatrilat; SA-898 ((2R,4R)—N-2- combinations thereof. Typically, the B-blocker will be (2-hydroxyphenyl)-3-(3-mercaptopropionyl)thiazolidin-4- administered in an amount Sufficient to provide from about ylcarbonyl-L-phenylalanine); Sch-50690 (N-1 (S)- 2-900 mg per dose. carboxy-2-N2-(methanesulfonyl)-L-lysylaminoethyl-L- In one embodiment, compounds of the invention are valyl-L-tyrosine); and combinations thereof, may also be administered in combination with a B-adrenergic receptor 25 included. In one particular embodiment, the ACE/NEP agonist, representative examples of which include, but are inhibitor is selected from: AVE-7688, enalaprilat, fasidotril, not limited to, albuterol, bitolterol, fenoterol, formoterol, fasidotrilate, omapatrilat, Sampatrilat, and combinations indacaterol, isoetharine, levalbuterol, metaproterenol, pir thereof. buterol, salbutamol, salmefamol, salmeterol, terbutaline, In one embodiment, compounds of the invention are Vilanterol, and the like. Typically, the f-adrenergic receptor 30 administered in combination with an angiotensin-converting agonist will be administered in an amount Sufficient to enzyme 2 (ACE2) activator or stimulator. provide from about 0.05-500 ug per dose. In one embodiment, compounds of the invention are In one embodiment, compounds of the invention are administered in combination with an angiotensin-II Vaccine, administered in combination with an advanced glycation end examples of which include, but are not limited to ATR12181 product (AGE) breaker, examples of which include, by way 35 and CYT006-AngOb. of illustration and not limitation, alagebrium (or ALT-711), In one embodiment, compounds of the invention are and TRC4149. administered in combination with an anticoagulant, repre In another embodiment, compounds of the invention are sentative examples of which include, but are not limited to: administered in combination with an aldosterone antagonist, coumarins such as warfarin; heparin; and direct thrombin representative examples of which include, but are not lim 40 inhibitors such as argatroban, bivalirudin, dabigatran, and ited to, eplerenone, spironolactone, and combinations lepirudin. thereof. Typically, the aldosterone antagonist will be admin In yet another embodiment, compounds of the invention istered in an amount sufficient to provide from about 5-300 are administered in combination with an anti-diabetic agent. mg per day. Representative anti-diabetic agents include injectable drugs In one embodiment, compounds of the invention are 45 as well as orally effective drugs, and combinations thereof. administered in combination with an aminopeptidase N or Examples of injectable drugs include, but are not limited to, dipeptidyl peptidase III inhibitor, examples of which insulin and insulin derivatives. Examples of orally effective include, by way of illustration and not limitation, bestatin drugs include, but are not limited to: biguanides such as and PC18 (2-amino-4-methylsulfonyl butane thiol, methio metformin; glucagon antagonists; C-glucosidase inhibitors nine thiol). 50 Such as acarbose and miglitol; dipeptidyl peptidase IV Compounds of the invention can also be administered in inhibitors (DPP-IV inhibitors) such as alogliptin, denaglip combination with an angiotensin-converting enzyme (ACE) tin, linagliptin, saxagliptin, Sitagliptin, and Vildagliptin; inhibitor. Representative ACE inhibitors include, but are not meglitinides such as repaglinide; oxadiazolidinediones; Sul limited to, accupril, alacepril, benazepril, benazeprilat, cap fonylureas such as chlorpropamide, glimepiride, glipizide, topril, ceranapril, cilaZapril, delapril, enalapril, enalaprilat, 55 glyburide, and tolaZamide; thiazolidinediones such as pio fosinopril, fosinoprilat, imidapril, lisinopril, moexipril, glitaZone and rosiglitaZone; and combinations thereof. monopril, moveltipril, pentopril, perindopril, quinapril, qui In another embodiment, compounds of the invention are naprilat, ramipril, ramiprilat, Saralasin acetate, spirapril, administered in combination with antidiarrheal treatments. temocapril, trandolapril, Zofenopril, and combinations Representative treatment options include, but are not limited thereof. 60 to, oral rehydration solutions (ORS), loperamide, diphe In a particular embodiment, the ACE inhibitor is selected noxylate, and bismuth Subsalicylate. from: benazepril, captopril, enalapril, lisinopril, ramipril, In yet another embodiment, a compound of the invention and combinations thereof. Typically, the ACE inhibitor will is administered in combination with an anti-glaucoma agent. be administered in an amount sufficient to provide from Representative anti-glaucoma agents include, but are not about 1-150 mg per day. In another embodiment, com 65 limited to: C.-adrenergic agonists such as brimonidine; pounds of the invention are administered in combination B-adrenergic receptor antagonists; topical B-blockers such with a dual-acting angiotensin-converting enzyme? neprily as betaxolol, levobunolol, and timolol; carbonic anhydrase US 9,683,002 B2 45 46 inhibitors such as acetazolamide, brinzolamide, or dorzol niludipine, nilvadipine, nimodipine, nisoldipine, nitrendip amide; cholinergic agonists such as cevimeline and DMXB ine, nivaldipine, perhexiline, prenylamine, ryosidine, Semo anabaseine; epinephrine compounds; miotics such as pilo tiadil, terodiline, tiapamil, Verapamil, and combinations carpine; and prostaglandin analogs. thereof. In a particular embodiment, the calcium channel In yet another embodiment, compounds of the invention blocker is selected from amlodipine, bepridil, diltiazem, are administered in combination with an anti-lipid agent. felodipine, isradipine, lacidipine, nicardipine, nifedipine, Representative anti-lipid agents include, but are not limited niguldipine, niludipine, nimodipine, nisoldipine, ryosidine, to: cholesteryl ester transfer protein inhibitors (CETPs) such Verapamil, and combinations thereof. Typically, the calcium as anacetrapib, dalcetrapib, and torcetrapib; statins such as channel blocker will be administered in an amount sufficient atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin 10 and simvastatin; and combinations thereof. to provide from about 2-500 mg per dose. In one embodiment, compounds of the invention are In one embodiment, compounds of the invention are administered in combination with an anti-thrombotic agent. administered in combination with a chymase inhibitor, Such Representative anti-thrombotic agents include, but are not as TPC-806 and 2-(5-formylamino-6-oxo-2-phenyl-1,6-di limited to: aspirin; anti-platelet agents such as clopidogrel, 15 hydropyrimidine-1-yl)-N-3,4-dioxo-1-phenyl-7-(2- prasugrel, and ticlopidine; heparin, and combinations pyridyloxy)-2-heptylacetamide (NK3201). thereof. In one embodiment, compounds of the invention are In one embodiment, compounds of the invention are administered in combination with a diuretic. Representative administered in combination with an AT receptor antago diuretics include, but are not limited to: carbonic anhydrase nist, also known as angiotensin II type 1 receptor blockers inhibitors such as acetazolamide and dichlorphenamide; (ARBs). Representative ARBs include, but are not limited loop diuretics, which include sulfonamide derivatives such to, abitesartan, azilsartan (e.g., azilsartan medoxomil), ben as acetazolamide, ambuside, azosemide, bumetanide, buta Zyllosartan, candesartan, candesartan cilexetil, elisartan, Zolamide, chloraminophenamide, clofenamide, clopamide, embusartan, enoltaSosartan, eprosartan, EXP3174, fonsar clorexolone, disulfamide, ethoXZolamide, furosemide, tan, forasartan, glycylosartan, irbesartan, isoteoline, losar 25 mefruside, methazolamide, piretanide, torsemide, tripamide, tan, medoxomil, milfasartan, olmesartan (e.g., olmesartan and Xipamide, as well as non-sulfonamide diuretics Such as medoxomil), opomisartan, pratosartan, ripisartan, Saprisar ethacrynic acid and other phenoxyacetic acid compounds tan, Saralasin, Sarmesin, TAK-591, tasosartan, telmisartan, Such as tienilic acid, indacrinone and quincarbate; osmotic Valsartan, Zolasartan, and combinations thereof. In a par diuretics Such as mannitol; potassium-sparing diuretics, ticular embodiment, the ARB is selected from azilsartan 30 which include aldosterone antagonists such as spironolac medoxomil, candesartan cilexetil, eprosartan, irbesartan, tone, and Na channel inhibitors such as amiloride and losartan, olmesartan medoxomil, Saprisartan, tasosartan, triamterene; thiazide and thiazide-like diuretics such as telmisartan, Valsartan, and combinations thereof. Exemplary althiazide, bendroflumethiazide, benzylhydrochlorothiazide, salts and/or prodrugs include candesartan cilexetil, eprosar benzthiazide, buthiazide, chlorthalidone, chlorothiazide, tan mesylate, losartan potassium salt, and olmesartan 35 cyclopenthiazide, cyclothiazide, epithiazide, ethiazide, fen medoxomil. Typically, the ARB will be administered in an quizone, flumethiazide, hydrochlorothiazide, hydroflume amount sufficient to provide from about 4-600 mg per dose, thiazide, indapamide, methylclothiazide, meticrane, metola with exemplary daily dosages ranging from 20-320 mg per ZOne, paraflutizide, polythiazide, quinethaZone, day. teclothiazide, and trichloromethiazide; and combinations Compounds of the invention may also be administered in 40 thereof. In a particular embodiment, the diuretic is selected combination with a dual-acting agent, such as an AT from amiloride, bumetanide, chlorothiazide, chlorthalidone, receptor antagonist/neprilysin inhibitor (ARB/NEP) inhibi dichlorphenamide, ethacrynic acid, furosemide, hydrochlo tor, examples of which include, but are not limited to, rothiazide, hydroflumethiazide, indapamide, methylcloth compounds described in U.S. Publication Nos. 2008/ iazide, metolaZone, torsemide, triamterene, and combina 0269305 and 2009/0023228, both to Allegretti et al. filed on 45 tions thereof. The diuretic will be administered in an amount Apr. 23, 2008, such as the compound, 4-2-ethoxy-4-ethyl sufficient to provide from about 5-50 mg per day, more 5-((S)-2-mercapto-4-methylpentanoylamino)-methylimi typically 6-25 mg per day, with common dosages being 6.25 dazol-1-ylmethyl-3'-fluorobiphenyl-2-carboxylic acid. mg, 12.5 mg or 25 mg per day. Compounds of the invention may also be administered in Compounds of the invention may also be administered in combination with multifunctional angiotensin receptor 50 combination with an endothelin converting enzyme (ECE) blockers as described in Kurtz & Klein (2009) Hypertension inhibitor, examples of which include, but are not limited to, Research 32:826-834. phosphoramidon, CGS 26303, and combinations thereof. In one embodiment, compounds of the invention are In a particular embodiment, compounds of the invention administered in combination with a bradykinin receptor are administered in combination with an endothelin receptor antagonist, for example, icatibant (HOE-140). It is expected 55 antagonist. Representative endothelin receptor antagonists that this combination therapy may present the advantage of include, but are not limited to: selective endothelin receptor preventing angioedema or other unwanted consequences of antagonists that affect endothelin A receptors, such as elevated bradykinin levels. avosentan, ambrisentan, atrasentan, BQ-123, claZosentan, In one embodiment, compounds of the invention are darusentan, sitaxentan, and Zibotentan; and dual endothelin administered in combination with a calcium channel 60 receptor antagonists that affect both endothelin A and B blocker. Representative calcium channel blockers include, receptors. Such as bosentan, macitentan, tezosentan). but are not limited to, amlodipine, anipamil, aranipine, In yet another embodiment, a compound of the invention barnidipine, bencyclane, benidipine, bepridil, clentiazem, is administered in combination with one or more HMG-CoA cilnidipine, cinnarizine, diltiazem, efonidipine, elgodipine, reductase inhibitors, which are also known as Statins. Rep etafenone, felodipine, fendiline, flunarizine, gallopamil, 65 resentative statins include, but are not limited to, atorvasta isradipine, lacidipine, lercanidipine, lidoflazine, lomerizine, tin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuv manidipine, mibefradil, nicardipine, nifedipine, niguldipine, astatin and simvastatin. US 9,683,002 B2 47 48 In one embodiment, compounds of the invention are methoxybutyric acid); UK-505,749 ((R)-2-methyl-3-(1-3- administered in combination with a monoamine reuptake (2-methylbenzothiazol-6-yl)propylcarbamoyl inhibitor, examples of which include, by way of illustration cyclopentyl)propionic acid); 5-biphenyl-4-yl-4-(3- and not limitation, norepinephrine reuptake inhibitors such carboxypropionylamino)-2-methylpentanoic acid and as atomoxetine, buproprion and the buproprion metabolite 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methyl hydroxybuproprion, maprotiline, reboxetine, and vilox pentanoic acid ethyl ester (WO 2007/056546); daglutril(3S, azine; selective serotonin reuptake inhibitors (SSRIs) such 2R)-3-1-2'-(ethoxycarbonyl)-4'-phenylbutyl-cyclopen as citalopram and the citalopram metabolite desmethylcit tan-1-carbonylamino)-2,3,4,5-tetrahydro-2-oxo-1H-1- allopram, dapoxetine, escitalopram (e.g., escitalopram benzazepine-1-acetic acid described in WO 2007/106708 to oxalate), fluoxetine and the fluoxetine desmethyl metabolite 10 Khder et al. (Novartis AG); and combinations thereof. In a norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate), par particular embodiment, the NEP inhibitor is selected from oxetine, sertraline and the sertraline metabolite demethyl AHU-377, candoxatril, candoxatrilat, CGS-24128, phos Sertraline; dual serotonin-norepinephrine reuptake inhibitors phoramidon, SCH-32615, SCH-34826, SQ-28603, thior (SNRIs) such as bicifadine, duloxetine, milnacipran, nefa phan, and combinations thereof. In a particular embodiment, Zodone, and Venlafaxine; and combinations thereof. 15 the NEP inhibitor is a compound such as daglutril or In another embodiment, compounds of the invention are CGS-26303 (N-2-(biphenyl-4-yl)-1 (S)-(1H-tetrazol-5-yl) administered in combination with a muscle relaxant, ethylaminomethylphosphonic acid), which have activity examples of which include, but are not limited to: carisop both as inhibitors of the endothelin converting enzyme rodol, chlorZoxazone, cyclobenzaprine, diflunisal, metax (ECE) and of NEP. Other dual acting ECE/NEP compounds alone, methocarbamol, and combinations thereof. can also be used. The NEP inhibitor will be administered in In one embodiment, compounds of the invention are an amount sufficient to provide from about 20-800 mg per administered in combination with a natriuretic peptide or day, with typical daily dosages ranging from 50-700 mg per analog, examples of which include but are not limited to: day, more commonly 100-600 or 100-300 mg per day. carperitide, CD-NP (Nile Therapeutics), CU-NP. nesiritide, In one embodiment, compounds of the invention are PL-3994 (Palatin Technologies, Inc.), ularitide, cenderitide, 25 administered in combination with a nitric oxide donor, and compounds described in Ogawa et al (2004) J. Biol. examples of which include, but are not limited to nicorandil; Chem. 279:28625-31. These compounds are also referred to organic nitrates such as pentaerythritol tetranitrate; and as natriuretic peptide receptor-A (NPR-A) agonists. In Sydnonimines such as linsidomine and molsidomine. another embodiment, compounds of the invention are In yet another embodiment, compounds of the invention administered in combination with a natriuretic peptide clear 30 are administered in combination with a non-steroidal anti ance 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 10: include, but are not limited to: acemetacin, acetyl salicylic 1949-52). For example, AP-811 has shown synergy when acid, alclofenac, alminoprofen, amfenac, amiprilose, alox combined with the NEP inhibitor, thiorphan (Wegner (1995) iprin, anirolac, apaZone, azapropaZone, benorilate, benox Clin. Exper. Hypert. 17:861-876). 35 aprofen, bezpiperylon, broperamole, bucloxic acid, carpro In another embodiment, compounds of the invention are fen, clidanac, diclofenac, diflunisal, diftalone, enolicam, administered in combination with a neprilysin (NEP) inhibi etodolac, etoricoxib, fenbufen, fenclofenac, fenclozic acid, tor. Representative NEP inhibitors include, but are not fenoprofen, fentiazac, feprazone, flufenamic acid, flufenisal, limited to: AHU-377; candoxatril; candoxatrilat; dexecado fluprofen, flurbiprofen, furofenac, ibufenac, ibuprofen, indo tril ((+)-N-2(R)-(acetylthiomethyl)-3-phenylpropionylgly 40 methacin, indoprofen, isoxepac, isoxicam, ketoprofen, cine benzyl ester); CGS-24128 (3-3-(biphenyl-4-yl)-2- ketorolac, lofemizole, lornoxicam, meclofenamate, (phosphonomethylamino)propionamidopropionic acid); meclofenamic acid, mefenamic acid, meloxicam, mesala CGS-24592 ((S)-3-3-(biphenyl-4-yl)-2-(phosphonomethyl mine, miroprofen, mofebutaZone, nabumetone, naproxen, amino)propionamidopropionic acid); CGS-25155 (N-9 niflumic acid, oxaprozin, oXpinac, oxyphenbutaZone, phe (R)-(acetylthiomethyl)-10-oxo-1-azacyclodecan-2(S)-ylcar 45 nylbutaZone, piroxicam, pirprofen, pranoprofen, Salsalate, bonyl-4 (R)-hydroxy-L-proline benzyl ester); 3-(1-carbam Sudoxicam, SulfaSalazine, Sulindac, Suprofen, tenoxicam, oylcyclohexyl)propionic acid derivatives described in WO tiopinac, tiaprofenic acid, tioxaprofen, tolfenamic acid, tol 2006/027680 to Hepworth et al. (Pfizer Inc.); JMV-390-1 metin, triflumidate, Zidometacin, Zomepirac, and combina (2OR)-benzyl-3-(N-hydroxycarbamoyl)propionyl-L-iso tions thereof. In a particular embodiment, the NSAID is leucyl-L-leucine); ecadotril; phosphoramidon; retrothior 50 selected from etodolac, flurbiprofen, ibuprofen, indometha phan; RU-42827 (2-(mercaptomethyl)-N-(4-pyridinyl)ben cin, ketoprofen, ketorolac, meloxicam, naproxen, oxaprozin, Zenepropionamide); RU-44004 (N-(4-morpholinyl)-3- piroXicam, and combinations thereof. phenyl-2-(sulfanylmethyl)propionamide); SCH-32615 In one embodiment, compounds of the invention are ((S) N—N-(1-carboxy-2-phenylethyl)-L-phenylalanyl administered in combination with an N-methyl d-aspartate (3-alanine) and its prodrug SCH-34826 ((S)- N—N-1- 55 (NMDA) receptor antagonist, examples of which include, by (2,2-dimethyl-1,3-dioxolan-4-yl)methoxycarbonyl-2- way of illustration and not limitation, including amantadine, phenylethyl-L-phenylalanyl-B-alanine); sialorphin; SCH dextromethorphan, dextropropoxyphene, ketamine, ketobe 42495 (N-2(S)-(acetylsulfanylmethyl)-3-(2-methylphenyl) midone, memantine, methadone, and so forth. propionyl-L-methionine ethyl ester); spinorphin; SQ-28132 In still another embodiment, compounds of the invention (N-2-(mercaptomethyl)-1-oxo-3-phenylpropylleucine); 60 are administered in combination with an opioid receptor SQ-28.603 (N-2-(mercaptomethyl)-1-oxo-3-phenylpropyl agonist (also referred to as opioid analgesics). Representa B-alanine); SQ-29072 (7-2-(mercaptomethyl)-1-oxo-3- tive opioid receptor agonists include, but are not limited to: phenylpropylaminoheptanoic acid); thiorphan and its pro buprenorphine, butorphanol, codeine, dihydrocodeine, fen drug racecadotril: UK-69578 (cis-4-1-2-carboxy-3-(2- tanyl, hydrocodone, hydromorphone, levallorphan, levor methoxyethoxy)propylcyclopentylcarbonylamino 65 phanol, meperidine, methadone, morphine, nalbuphine, cyclohexanecarboxylic acid); UK-447.841 (2-1-3-(4- nalmefene, nalorphine, naloxone, naltrexone, nalorphine, chlorophenyl)propylcarbamoyl-cyclopentylmethyl-4- oxycodone, oxymorphone, pentazocine, propoxyphene, tra US 9,683,002 B2 49 50 madol, and combinations thereof. In certain embodiments, Combined secondary therapeutic agents may also be the opioid receptor agonist is selected from codeine, dihy helpful in further combination therapy with compounds of drocodeine, hydrocodone, hydromorphone, morphine, oxy the invention. For example, compounds of the invention can codone, oxymorphone, tramadol, and combinations thereof. be combined with a diuretic and an ARB, or a calcium In a particular embodiment, compounds of the invention channel blocker and an ARB, or a diuretic and an ACE are administered in combination with a phosphodiesterase inhibitor, or a calcium channel blocker and a statin. Specific (PDE) inhibitor, particularly a PDE-V inhibitor. Represen examples include, a combination of the ACE inhibitor tative PDE-V inhibitors include, but are not limited to, enalapril (in the maleate salt form) and the diuretic hydro avanafil, lodenafil, mirodenafil, sildenafil (Revatio(R), tada chlorothiazide, which is sold under the mark Vaseretic(R), or lafil (AdcircaR), Vardenafil (Levitra(R), and udenafil. 10 a combination of the calcium channel blocker amlodipine (in In another embodiment, compounds of the invention are the besylate salt form) and the ARB olmesartan (in the administered in combination with a prostaglandin analog medoxomil prodrug form), or a combination of a calcium (also referred to as prostanoids or prostacyclin analogs). channel blocker and a statin, all may also be used with the Representative prostaglandin analogs include, but are not compounds of the invention. Other therapeutic agents such 15 as O-adrenergic receptor agonists and vasopressin receptor limited to, beraprost sodium, bimatoprost, epoprostenol, antagonists may also be helpful in combination therapy. iloprost, latanoprost, tafluprost, travoprost, and treprostinil, Exemplary C-adrenergic receptor agonists include cloni with bimatoprost, latanoprost, and tafluprost being of par dine, dexmedetomidine, and guanfacine. ticular interest. The following formulations illustrate representative phar In yet another embodiment, compounds of the invention maceutical compositions of the invention. are administered in combination with a prostaglandin recep tor agonist, examples of which include, but are not limited Exemplary Hard Gelatin Capsules for Oral to, bimatoprost, latanoprost, travoprost, and so forth. Administration Compounds of the invention may also be administered in combination with a renin inhibitor, examples of which 25 A compound of the invention (50 g), 440 g spray-dried include, but are not limited to, alliskiren, enalkiren, lactose and 10 g magnesium Stearate are thoroughly remikiren, and combinations thereof. blended. The resulting composition is then loaded into hard In another embodiment, compounds of the invention are gelatin capsules (500 mg of composition per capsule). administered in combination with a selective serotonin Alternately, a compound of the invention (20 mg) is thor reuptake inhibitor (SSRI). Representative SSRIs include, 30 oughly blended with starch (89 mg), microcrystalline cel but are not limited to: citalopram and the citalopram metabo lulose (89 mg) and magnesium Stearate (2 mg). The mixture lite desmethylcitalopram, dapoxetine, escitalopram (e.g., is then passed through a No. 45 mesh U.S. sieve and loaded escitalopram oxalate), fluoxetine and the fluoxetine desm into a hard gelatin capsule (200 mg of composition per ethyl metabolite norfluoxetine, fluvoxamine (e.g., fluvox capsule). amine maleate), paroxetine, Sertraline and the Sertraline 35 Alternately, a compound of the invention (30 g), a sec metabolite demethylsertraline, and combinations thereof. ondary agent (20g), 440 g spray-dried lactose and 10 g In one embodiment, compounds of the invention are magnesium Stearate are thoroughly blended, and processed administered in combination with a 5-HT serotonin recep as described above. tor agonist, examples of which include, by way of illustra tion and not limitation, triptans Such as almotriptan, avitrip 40 Exemplary Gelatin Capsule Formulation for Oral tan, eletriptan, froVatriptan, naratriptan rizatriptan, Administration Sumatriptan, and Zolmitriptan. In one embodiment, compounds of the invention are A compound of the invention (100 mg) is thoroughly administered in combination with a sodium channel blocker, blended with polyoxyethylene sorbitan monooleate (50 mg) examples of which include, by way of illustration and not 45 and starch powder (250mg). The mixture is then loaded into limitation, carbamazepine, fosphenytoin, lamotrigine, lido a gelatin capsule (400 mg of composition per capsule). caine, mexiletine, oXcarbazepine, phenytoin, and combina Alternately, a compound of the invention (70 mg) and a tions thereof. secondary agent (30 mg) are thoroughly blended with poly In one embodiment, compounds of the invention are oxyethylene Sorbitan monooleate (50mg) and starch powder administered in combination with a soluble guanylate 50 (250 mg), and the resulting mixture loaded into a gelatin cyclase stimulator or activator, examples of which include, capsule (400 mg of composition per capsule). but are not limited to ataciguat, riociguat, and combinations Alternately, a compound of the invention (40 mg) is thereof. thoroughly blended with microcrystalline cellulose (Avicel In one embodiment, compounds of the invention are PH 103: 259.2 mg) and magnesium stearate (0.8 mg). The administered in combination with a tricyclic antidepressant 55 mixture is then loaded into a gelatin capsule (Size #1, White, (TCA), examples of which include, by way of illustration Opaque) (300 mg of composition per capsule). and not limitation, amitriptyline, amitriptylinoxide, butrip tyline, clomipramine, demexiptiline, desipramine, dibenze Exemplary Tablet Formulation for Oral pin, dimetacrine, do Sulepin, doxepin, imipramine, imip Administration raminoxide, lofepramine, melitracen, metapramine, 60 nitroxazepine, nortriptyline, noxiptiline, pipofezine, A compound of the invention (10 mg), starch (45 mg) and propizepine, protriptyline, quinupramine, and combinations microcrystalline cellulose (35 mg) are passed through a No. thereof. 20 mesh U.S. sieve and mixed thoroughly. The granules so In one embodiment, compounds of the invention are produced are dried at 50-60° C. and passed through a No. 16 administered in combination with a vasopressin receptor 65 mesh U.S. sieve. A solution of polyvinylpyrrolidone (4 mg antagonist, examples of which include, by way of illustra as a 10% solution in sterile water) is mixed with sodium tion and not limitation, conivaptain and tolvaptan. carboxymethyl starch (4.5 mg), magnesium Stearate (0.5 US 9,683,002 B2 51 52 mg), and talc (1 mg), and this mixture is then passed through hydrochloric acid or 0.5 N aqueous sodium hydroxide, as a No. 16 mesh U.S. sieve. The sodium carboxymethyl necessary, and then Sufficient water for injection is added to starch, magnesium Stearate and talc are then added to the provide a total volume of 20 mL. The mixture is then filtered granules. After mixing, the mixture is compressed on a tablet through a sterile filter (0.22 micron) to provide a sterile machine to afford a tablet weighing 100 mg. Solution Suitable for administration by injection. Alternately, a compound of the invention (250 mg) is thoroughly blended with microcrystalline cellulose (400 Exemplary Compositions for Administration by mg), silicon dioxide fumed (10 mg), and Stearic acid (5 mg). Inhalation The mixture is then compressed to form tablets (665 mg of composition per tablet). 10 A compound of the invention (0.2 mg) is micronized and Alternately, a compound of the invention (400 mg) is then blended with lactose (25 mg). This blended mixture is thoroughly blended with cornstarch (50 mg), croscarmellose then loaded into a gelatin inhalation cartridge. The contents Sodium (25 mg), lactose (120 mg), and magnesium Stearate of the cartridge are administered using a dry powder inhaler, (5 mg). The mixture is then compressed to form a single for example. scored tablet (600 mg of composition per tablet). 15 Alternately, a micronized compound of the invention (10 Alternately, a compound of the invention (100 mg) is g) is dispersed in a solution prepared by dissolving lecithin thoroughly blended with cornstarch (100 mg) with an aque (0.2 g) in demineralized water (200 mL). The resulting ous solution of gelatin (20 mg). The mixture is dried and Suspension is spray dried and then micronized to form a ground to a fine powder. Microcrystalline cellulose (50 mg) micronized composition comprising particles having a mean and magnesium Stearate (5 mg) are then admixed with the diameter less than about 1.5 Lum. The micronized composi gelatin formulation, granulated and the resulting mixture tion is then loaded into metered-dose inhaler cartridges compressed to form tablets (100 mg of the compound of the containing pressurized 1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 ug to about 500 ug of invention per tablet). the compound of the invention per dose when administered Exemplary Suspension Formulation for Oral 25 by the inhaler. Administration Alternately, a compound of the invention (25 mg) is dissolved in citrate buffered (pH 5) isotonic saline (125 mL). The following ingredients are mixed to form a suspension The mixture is stirred and Sonicated until the compound is containing 100 mg of the compound of the invention per 10 dissolved. The pH of the solution is checked and adjusted, mL of Suspension: 30 if necessary, to pH 5 by slowly adding aqueous 1 N NaOH. The solution is administered using a nebulizer device that provides about 10 ug to about 500 ug of the compound of the Ingredients Amount invention per dose.

Compound of the invention 1.0 g 35 EXAMPLES Fumaric acid 0.5 g. Sodium chloride 2.0 g Methyl paraben 0.15 g The following Preparations and Examples are provided to Propyl paraben 0.05 g illustrate specific embodiments of the invention. These spe Granulated Sugar 25.5 g cific embodiments, however, are not intended to limit the Sorbitol (70% solution) 12.85 g Veegum (R) K (magnesium aluminum silicate) 1.0 g 40 Scope of the invention in any way unless specifically indi Flavoring O.O35 mL. cated. Colorings 0.5 mg The following abbreviations have the following meanings Distilled water q.S. to 100 mL. unless otherwise indicated and any other abbreviations used herein and not defined have their standard, generally 45 accepted meaning: Exemplary Liquid Formulation for Oral AcOH acetic acid Administration BOC t-butoxycarbonyl ( C(O)OC(CH)) 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 50 DMA N,N-dimethylacetamide 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 EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide ammonium citrate buffer and the pH adjusted to pH 5, or is EtN triethylamine blended with a 100 mM citric acid solution and the pH EtO diethyl ether adjusted to pH 2. Such solutions may also include a solu 55 EtOAc ethyl acetate bilizing excipient such as a cyclodextrin, for example the EtOH ethanol solution may include 10 wt % hydroxypropyl-f-cyclodex EtSiH triethylsilane trin. HATU N.N.N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl) Other suitable formulations include a 5% NaHCO, solu uronium hexafluorophosphate tion, with or without cyclodextrin. 60 HCTU (2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-te tramethylaminium hexafluorophosphate) Exemplary Injectable Formulation for HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic Administration by Injection acid HOBt 1-hydroxybenzotriazole A compound of the invention (0.2 g) is blended with 0.4 65 MeCN acetonitrile M sodium acetate buffer solution (2.0 mL). The pH of the MeOH methanol resulting Solution is adjusted to pH 4 using 0.5 N aqueous MeTHF 2-methyltetrahydrofuran US 9,683,002 B2 53 54 Pd(dppf)Cl. 1,1-bis(diphenylphosphino) ferrocene palla -continued dium chloride BOC Pd(PPh) tetrakis(triphenylphosphine)palladium(O) NH PE petroleum ether PMB p-methoxybenzyl PyBOP benzotriazol-1-yloxytris(pyrrolidino)phospho nium hexafluorophosphate Br SilicaCat R. DPP-Pd silica based diphenylphosphine pal 10 ladium (II) catalyst (1) TFA trifluoroacetic acid 4-Bromobenzyl bromide (5.0 g, 20 mmol) and DIPEA (3.5 mL, 20.0 mmol) were dissolved in DMF (20 mL). THF tetrahydrofuran t-Butyl carbazate (7.9 g, 60.0 mmol) was added and the Unless noted otherwise, all materials, such as reagents, 15 mixture was stirred at room temperature until the reaction starting materials and solvents, were purchased from com was complete. The mixture was partially concentrated, then mercial suppliers (such as Sigma-Aldrich, Fluka Riedel-de the residue was partitioned between EtOAc and a saturated aqueous NaHCO, solution. The EtOAc layer was then dried Haén, and the like) and were used without further purifica over NaSO and concentrated. The crude product was tion. purified by flash chromatography to yield Compound 1 (3.8 Reactions were run under nitrogen atmosphere, unless g). noted otherwise. The progress of reactions were monitored by thin layer chromatography (TLC), analytical high per formance liquid chromatography (anal. HPLC), and mass spectrometry, the details of which are given in specific 25 (1) -- O examples. Solvents used in analytical HPLC were as fol lows: solvent A was 98% HO/2% MeCN/1.0 mL/L TFA: O BOC solvent B was 90% MeCN/10% HO/1.0 mL/L TFA. NH Reactions were worked up as described specifically in each preparation for example; commonly reaction mixtures 30 were purified by extraction and other purification methods OH Such as temperature-, and solvent-dependent crystallization, and precipitation. In addition, reaction mixtures were rou tinely purified by preparative HPLC, typically using Br Microsorb C18 and Microsorb BDS column packings and 35 (2) conventional eluents. Progress of reactions was typically Compound 1 (1.9 g, 6.3 mmol) was dissolved in isopropyl measured by liquid chromatography mass spectrometry alcohol (26.4 mL). Methyl (2R)-glycidate (1.1 mL, 12.6 (LCMS). Characterization of isomers were done by Nuclear mmol) was added and the mixture was heated at 90°C. until Overhauser effect spectroscopy (NOE). Characterization of 40 the reaction was complete (~4 days). The mixture was reaction products was routinely carried out by mass and cooled to room temperature and concentrated to yield the 'H-NMR spectrometry. For NMR measurement, samples title compound as a white solid (2.5 g). were dissolved in deuterated solvent (CDOD, CDC1, or DMSO-d), and H-NMR spectra were acquired with a Preparation 2: Varian Gemini 2000 instrument (400 MHz) under standard 45 N'-(4-Bromobenzyl)hydrazinecarboxylic Acid observation conditions. Mass spectrometric identification of t-Butyl Ester compounds was typically conducted using an electrospray ionization method (ESMS) with an Applied Biosystems (Foster City, Calif.) model API 150 EX instrument or an O Agilent (Palo Alto, Calif.) model 1200 LC/MSD instrument. 50 21 H Preparation 1: (R)-3-N-(4-bromobenzyl)-N'-t-bu Y Na toxycarbonylhydrazino-2-hydroxypropionic Acid HN BOC - > Methyl Ester 55

Br oc BOC NH NH HN 1 60

Her HN n BOC --

Br Br 65 Br (1) US 9,683,002 B2 55 To a stirred solution of t-butyl carbazate (50 g., 0.4 mol) -continued in dry THF (400 mL) was added dropwise a solution of O 4-bromobenzaldehyde (70 g., 0.4 mol) in dry THF (200 mL). The mixture was stirred at room temperature for 2 hours, and then concentrated in vacuo to yield Compound 1 as a yellow No N1N BOC solid (113.8 g). LC-MS: 243 M-tEu+H". OH To a solution of Compound 1 (113.8 g. 0.4 mol) in dry THF (1 L) was added NaCNBH (36 g., 0.6 mol) in portions at 0° C. AcOH (180 mL) was added dropwise and the C resulting mixture was stirred at room temperature overnight. Water (2 L) and EtOAc (1.5 L) were added and the aqueous 10 phase was adjusted to pH-7 with a saturated aqueous F Na2CO Solution. The organic layer was separated, washed with saturated aqueous NaCl and water (200 mL), dried over (2) anhydrous NaSO4, and concentrated in vacuo. The residue was treated with MeOH (2 L) and 1N NaOH (1.5 L), and 15 To a solution of Compound 1 (20g, 57 mmol) in isopropyl then stirred at room temperature for 2 hours. After the alcohol (250 mL) was added methyl (2R)-glycidate (8.7 g. removal of the MeOH solvent, the precipitate was collected 86 mmol) under nitrogen. The mixture was stirred at 85°C. by filtration to yield the title compound as a white solid (112 for 3 days, then cooled to room temperature. The precipi g). LC-MS. 245 (M-tEu+H". tated solid was collected by filtration to yield the title Preparation 3: (R)-3-N-t-Butoxycarbonyl-N-(5'- compound as an off-white solid (18.5 g). LC-MS. 397 chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- M-tBu+H". hydroxypropionic Acid Methyl Ester Preparation 4: (R)-3-N-(5'-Chloro-2'-fluorobiphe nyl-4-ylmethyl)hydrazino-2-hydroxypropionic Acid 25 Ethyl Ester BOC HN1 NH HO N1OH O BOC F NH -- -e- No N1

Br C OH -- BOC

NH HN1 Br OH

B C 40 HO1 -e-

F O

45 1N O N1 NH2

OH To a solution of N'-(4-bromobenzyl)hydrazinecarboxylic acid t-butyl ester (60 g, 0.2 mol) in 1,4-dioxane (1.5 mL) was added 5-chloro-2-fluorophenylboronic acid (38 g., 0.2 mol) and Pd(dppf)Cl. (7.3 g). The mixture was stirred at 50 room temperature under nitrogen for 10 minutes, and then, KCO (55.2g, 0.4 mol) in water (240 mL) was added. The resulting mixture was stirred at 60° C. for 3 hours, and then cooled to room temperature and concentrated in vacuo. The residue was extracted with EtOAc (3x300 mL). The com 55 (R)-3-N-(4-Bromobenzyl)-N'-t-butoxycarbonylhy bined organic layers were dried over anhydrous Na2SO and drazino-2-hydroxypropionic acid methyl ester (1.0 g, 2.5 concentrated in vacuo. The product was purified by column mmol), 5-chloro-2-fluorophenylboronic acid (865 mg, 5.0 chromatography (hexanes/EtOAc=10:1-5:1) to yield Com mmol), and KCO (857 mg, 6.2 mmol), were combined in pound 1 as a pink solid (56 g). LC-MS: 701 2M--H". EtOH (30 mL, 500 mmol) and water (8 mL, 400 mmol), 60 followed by the addition of SilicaCat R. DPP-Pd (0.28 mmol/g loading; 886 mg, 248 umol). The mixture was heated at 90° C. until the reaction was complete (2 hours). The precipitate was filtered off, and the filtrate was concen trated and purified by reverse phase chromatography (30 (1) + O 65 95% MeCN in water with 0.5% TFA). The clean fractions were collected, lyophilized, and combined with 4M HCl in dioxane (8 mL, 30 mmol) and EtOH (10 mL, 200 mmol). US 9,683,002 B2 57 58 The resulting mixture was stirred at room temperature until A mixture of 1-(3-chlorophenyl)-5-oxo-4,5-dihydro-1H the reaction was complete (7 hours). The mixture was 1,2,4-triazole-3-carboxylic acid ethyl ester (200.0 mg, 747 concentrated to yield an oil, which was stirred in ether with umol), LiOH (71.6 mg, 1.5 mmol) in water (2 mL), and few drops of EtOH overnight. The precipitate was filtered MeOH (10.0 mL, 247 mmol) was stirred at room tempera off and rinsed with ether to yield the title compound (140 5 ture overnight then concentrated. The residue was acidified mg). with 1N HCl to pH 3-4, forming a precipitate, which was filtered, washed with water (2x5 mL), and dried in vacuo to Alternate Preparation of (R)-3-N-(5'-Chloro-2'- yield the title compound (100.6 mg). fluorobiphenyl-4-ylmethyl)hydrazino-2-hydroxy Preparation 6: (R)-3-N-(4-Bromobenzyl)hy propionic Acid Ethyl Ester 10 drazino-2-hydroxypropionic Acid Ethyl Ester

O O BOC No N1Y BOC 15 NO N1 NH OH -e- OH

Br O NH2 1YO N Y 25 OH OH

Br C A solution of (R)-3-N-(4-bromobenzyl)-N'-t-butoxycar bonylhydrazino-2-hydroxypropionic acid methyl ester (25 F g, 62 mmol) in EtOH/HCl (1M, 310 mL, 0.3 mol) was A solution of (R)-3-N-t-butoxycarbonyl-N-(5'-chloro-2'- stirred overnight until the reaction was complete. The mix fluorobiphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic 35 ture was then concentrated and the residue was washed with acid methyl ester (20 g, 16 mmol) in HC1/EtOH (1.1 M, 200 EtOAc (120 mL) and filtered. The solids were collected to mL) was stirred overnight and then concentrated in vacuo. yield the title compound as a white solid HCl salt (15 g). The residue was dispersed in EtOAc (2x40 mL), and the Preparation 7: (R)-3-N-(5'-Chloro-2'-fluorobiphe precipitate was collected by filtration to give the title com 40 nyl-4-ylmethyl)hydrazino-2-hydroxypropionic Acid pound as an off-white solid HCl salt (8.8 g). LC-MS: 367 5-methyl-2-oxo-1.3dioxol-4-ylmethyl Ester M+H". "H NMR (300 MHz, DMSO-d) & 1.05 (t, J=7.2 Hz, 3H), 3.05-3.03 (q, J–7.2 Hz, 2H), 4.06-3.95 (m, 4H), 4.42 (br. 1H), 6.46 (br. 1H), 7.62-7.40 (m, 7H), 9.42 (s, 3H). 45 Preparation 5: 1-(3-Chlorophenyl)-5-oxo-4,5-di hydro-1H-1.2.4 triazole-3-carboxylic Acid

OH 50

55

HO N

OH 60 HO \

65 (1) US 9,683,002 B2 59 60 LiOH hydrate (3 g, 73 mmol) in water (60 mL) was added Preparation 8: 5-Oxo-1-phenyl-4,5-dihydro-1H-1.2. to (R)-3-N'-t-butoxycarbonyl-N-(5'-chloro-2'-fluorobiphe 4triazole-3-carboxylic Acid nyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid methyl ester (16.5g, 36.5 mmol) in MeOH (300 mL). The mixture was stirred at room temperature for 2 hours, and the MeOH was evaporated in vacuo. The mixture was adjusted to pH=5 with 1 M aqueous HCl, and the residue was extracted with O O EtOAc (2x300 mL). The combined organic layers were dried over anhydrous NaSO, and concentrated in vacuo to 10 Y V Y\ yield Compound 1 as a white solid (18 g). LC-MS: 383 HNNeN - HNNeN M-tBu+H".

15 D.\ D. (2) He O Methyl 2,5-dihydro-5-oxo-1-phenyl-1 h-1,2,4-triazole-3- NH2 carboxylate (300.0 mg, 1.4 mmol) was mixed with MeOH O O N1 (4.5 mL, 110 mmol) and water (0.5 mL, 30 mmol) at room O={ O OH temperature, then treated with LiOH monohydrate (0.1 g, 2.7 mmol) at room temperature overnight. The mixture was concentrated and the resulting residue was acidified to pH-1 O C with 1N aqueous HC1. The resulting solids were filtered and 25 rinsed with water, then dried in vacuo to give the title F compound as a yellowish Solid (185 mg), which was used without further purification. To a solution of Compound 1 (1.5g, 3.42 mmol), KCO Preparation 9: 2-Trityl-2H-tetrazole-5-carboxylate (0.95 g. 6.84 mmol) and potassium iodide (20 mg) in DMF 30 Lithium Salt (40 mL) was added 4-(bromomethyl)-5-methyl-1,3-dioxol 2-one (0.8 g. 4.1 mmol) in DMF (15 mL). The resulting mixture was stirred for 4 hours at room temperature. Satu O rated aqueous NaCl (30 mL) was added and the mixture was 35 N extracted with EtOAc (2x50 mL). The combined organic layers were dried over anhydrous NaSO and concentrated /No 2 y// - in vacuo. The residue was purified by column chromatog N-N raphy (hexanes/EtOAc=1:1) to yield Compound 2 as a Na" yellow solid (930 mg). LC-MS: 495 M-tBu+14". 40

(2) Ho O O O 45 O -Sf NH2 O O N1 Li" NRN ( ) O={ O OH 50 Ethyl-5-tetrazolecarboxylate sodium salt (2.3 g 14 mmol) O C was dissolved in DMF (20 mL, 200 mmol). The mixture was cooled at 0°C. Triphenylmethyl chloride (3.9 g, 14.1 mmol) F was added and the resulting mixture was stirred at room 55 temperature overnight, yielding a slurry. The slurry was slowly poured into cold stirred water (200 mL). The result Compound 2 (400 mg 0.73 mmol) was dissolved in ing slurry was stirred for 15 minutes (bicarbonate was added MeCN (20 mL), and cooled to 0°C. N-trimethylsilylimida to keep the pH basic), then filtered and dried to yield a white Zole (290 mg, 1.46 mmol) was added dropwise and the solid (5.1 g). The solid was suspended in MeOH (50 mL, 1.0 resulting mixture was stirred for 2 hours. MeOH (50 mL) 60 mol), followed by the addition of LiOH monohydrate (886 was added to quench the reaction. The mixture was washed mg, 21.1 mmol) dissolved in water (10 mL). The resulting mixture was stirred at room temperature for 3 hours. Any with saturated aqueous NaCl (2x50 mL) and extracted with solids were filtered off and the filtrate was concentrated by DCM (2x80 mL). The combined organic layers were dried rotary evaporation. EtOAc (50 mL) was added and the over anhydrous Na2SO and concentrated in vacuo. The 65 mixture was dried. This was repeated two more times. The product was collected to yield the title compound as a yellow product was then dried under high vacuum at room tem solid (200 mg). LC-MS: 451 M+H". perature to yield the title compound. US 9,683,002 B2 61 62 Preparation 10: (R)-3-N-(5'-Chloro-2'-fluorobiphe Preparation 11: Lithium nyl-4-ylmethyl)-N'-(2-trityl-2H-tetrazole-5-carbonyl) 1-Allyl-1H-tetrazole-5-carboxylate hydrazino-2-hydroxypropionic Acid

O \ y O /or -,\, tN -e- 10 O HO N-NH Y-,| \ -/ -e- 15 NéN ( ) -- (1) O LiO ~ W \ Tr NS 2N \ N N -3- O M To a stirred solution of 1H-tetrazole-5-carboxylic acid N 25 ethyl ester (2.0 g, 14.1 mmol) in DMF (20 mL) was added O KCO (2.3 g, 16.9 mmol) and 3-bromoprop-1-ene (1.9 g, Li" 15.4 mmol) at 0° C. The mixture was warmed to room Tr temperature, stirred overnight, then poured into water (200 mL). The resulting solution was extracted with EtOAc N1 \v. 30 (3x100 mL). The combined organic layers were washed N with saturated aqueous NaCl (100 mL), dried over anhy O A drous NaSO, filtered and concentrated in vacuo to yield O N Compound 1 as a yellow oil (2.3 g). LC-MS: 183 M+H". NH To a solution of Compound 1 (2.3 g, 12.6 mmol) in EtOH HO N1 35 (20 mL) was added a solution of LiOH.H2O (636 mg, 15.2 mmol) in water (10 mL). The mixture was stirred at room OH temperature for 3 hours, the solids were filtered off, and the filtrate was concentrated in vacuo to yield the title com C pound as a yellow solid (2.0 g), which was used without 40 further purification. Preparation 12: (R)-3-N-(5'-Chloro-2'-fluorobiphe F nyl-4-ylmethyl)hydrazino-2-hydroxypropionic Acid Ethyl Ester 2-Trityl-2H-tetrazole-5-carboxylate lithium salt (385.2 45 mg, 1.1 mmol) and HATU (404.3 mg, 1.1 mmol) in DMF (5.9 mL, 76 mmol) was stirred at room temperature for 10 minutes. (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl) hydrazino-2-hydroxypropionic acid ethyl ester (300 mg. 818 umol) was added followed by DIPEA (285 uL, 1.6 50 mmol). The resulting mixture was stirred at room tempera n ture overnight. The mixture was partitioned between EtOAc HN BOC (10.0 mL) and water (2.0 mL). The organic layer was washed with water (2.0 mL), dried over NaSO filtered and Br concentrated to give a yellowish oil. The oily residue was 55 then purified by flash chromatography (0-50% EtOAc/ n hexanes). The desired fractions were combined and concen N BOC N BOC trated to give a light yellowish oil (combined with other lots for a total of 147.2 mg). This residue was dissolved in MeOH (5.0 mL, 120 mmol) and water (0.5 mL, 30 mmol) at room temperature. LiOH monohydrate (17.5 mg, 417 60 umol) was added and allowed to sit for 30 minutes. The Br Br mixture was concentrated, and the resulting residue was (1) (2) treated with EtOAc (10.0 mL) and acidified with 1N HCl till pH-3. The organic layer was washed with Saturated aqueous To a stirred solution of t-butyl carbazate (50 g, 0.4 mol) NaCl (3x3.0 mL), dried over sodium NaSO filtered and 65 in dry THF (400 mL) was added dropwise a solution of concentrated to yield the title compound as a white foam 4-bromobenzaldehyde (70 g., 0.4 mol) in dry THF (200 mL). (99.4 mg). The mixture was stirred at room temperature for 2 hours, and US 9,683,002 B2 63 64 then concentrated in vacuo to yield Compound 1 as a yellow -continued solid (113.8 g). LC-MS: 243 M-tEu+H". O To a solution of Compound 1 (113.8 g. 0.4 mol) in dry THF (1 L) was added NaCNBH (36 g., 0.6 mol) in portions 1No N1 at 0° C. AcOH (180 mL) was added dropwise and the resulting mixture was stirred at room temperature overnight. OH Water (2 L) and EtOAc (1.5 L) were added and the aqueous phase was adjusted to pH 7 with a saturated aqueous Na2CO Solution. The organic layer was separated, washed with saturated aqueous NaCl and water (200 mL), dried over anhydrous NaSO4, and concentrated in vacuo. The residue 10 was treated with MeOH (2 L) and 1N NaOH (1.5 L), and F then stirred at room temperature for 2 hours. After the To a solution of Compound 3 (20g, 57 mmol) in isopropyl removal of the MeOH solvent, the precipitate was collected alcohol (250 mL) was added methyl (2R)-glycidate (8.7 g. by filtration to yield Compound 2 as a white solid (112 g). 86 mmol) under nitrogen. The mixture was stirred at 85°C. LC-MS. 245 M-tEu+H". 15 for 3 days, then cooled to room temperature. The precipi tated solid was collected by filtration to yield Compound 4 HO OH as an off-white solid (18.5 g). LC-MS. 397 M-tBu+H". 1 A solution of Compound 4 (20g, 16 mmol) in HCl/EtOH (1.1 M, 200 mL) was stirred overnight and then concentrated in vacuo. The residue was dispersed in EtOAc (2x40 mL), and the precipitate was collected by filtration to give the title compound as an off-white solid HCl salt (8.8 g). LC-MS: (2) + 367 IM+H". "H NMR (300 MHz, DMSO-d) & 1.05 (t, C J=7.2 Hz, 3H), 3.05-3.03 (q, J=7.2 Hz, 2H), 4.06-3.95 (m, 4H), 4.42 (br. 1H), 6.46 (br. 1H), 7.62-7.40 (m, 7H), 9.42 (s, N1N BOC 25 3H). Preparation 13: Lithium (R)-3-(2-(1-Allyl-1H-tetra Zole-5-carbonyl)-1-((5'-chloro-2'-fluorobiphenyl-4- yl)methyl)hydrazinyl)-2-hydroxypropanoate C 30 O LO Z F (3) ,W \ -/ -- 35 NeN To a solution of Compound 2 (60 g, 0.2 mol) in 1,4- \ y dioxane (1.5 mL) was added 5-chloro-2-fluorophenylbo O ronic acid (38 g., 0.2 mol) and Pd(dppf)C1 (7.3 g). The mixture was stirred at room temperature under nitrogen for 40 10 minutes, and KCO (55.2g, 0.4 mol) in water (240 mL) HO N-NH was added. The resulting mixture was stirred at 60° C. for 3 hours, then cooled to room temperature and concentrated in vacuo. The residue was extracted with EtOAc (3x300 mL). The combined organic layers were dried over anhydrous NaSO and concentrated in vacuo. The product was purified 45 by column chromatography (PE:EtOAc=10:1-5:1) to yield

Compound 3 as a pink solid (56 g). LC-MS: 701 2M--H".

O 50 (3) + o1 -e- O H O 55 No N1N BOC OH

C

65 (4) US 9,683,002 B2 65 66 To a solution of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- N'-(3'-Chlorobiphenyl-4-ylmethyl)hydrazinecarboxylic ylmethyl)hydrazino-2-hydroxypropionic acid ethyl ester acid t-butyl ester (400.0 g, 1.2 mol) was combined with IPA (3.4 g, 8.3 mmol) and lithium 1-allyl-1H-tetrazole-5-car (7.0 L, 91 mol) and (R)-oxirane-2-carboxylic acid methyl boxylate (2.0 g, 12.50 mmol) in DMF (40 mL) were added ester (105.2 mL, 1.2 mol) under nitrogen. The mixture was PyBOP (8.7g, 16.7 mmol) and DIPEA (2.1 g, 16.7 mmol) dropwise at 0°C. under nitrogen. The resulting mixture was heated at 83° C. for 51 hours. Additional (R)-oxirane-2- stirred for 2.5 hours, then poured into water (400 mL). The carboxylic acid methyl ester (52.61 mL, 600.9 mmol) was resulting solution was extracted with EtOAc (2x200 mL). added and the mixture was heated at 84° C. for 48 hours. The combined organic layers were washed with saturated Sodium cyanoborohydride (1.0 g, 16 mmol) was added and the mixture was heated at 80° C. and the reaction monitored aqueous NaCl (100 mL), dried over anhydrous NaSO, 10 filtered, and concentrated in vacuo. The residue was purified (s48 hours). Additional Sodium cyanoborohydride (1 g) was by column chromatography (PE:EtOAc=5: 1-4:1-3:1) to added and the mixture was heated at reflux (s3 days). The yield Compound 1 as a yellow oil (2.5 g). LC-MS. 503 mixture was then cooled slowly at 15° C.; filtered, and dried M+H", to yield Compound 1 (470 g). 15

Y N (1) -- OH

C

25

(2)

Compound 1 (880 mg, 1.9 mmol) was combined with 3 M 30 HCl in CPME (7 mL, 20 mmol). The mixture was then To a solution of Compound 1 (2.5g, 4.97 mmol) in EtOH stirred on an ice bath and the reaction monitored for comple (25 mL) was added a solution of LiOH.HO (250 mg. 6.0 tion (s2.5 hours). The solids were collected, washed with mmol) in water (10 mL). The mixture was stirred at room CPME (0.5 mL), and dried to yield a white powder (0.6 g; temperature for 3 hours, the solids were filtered off, and the HCl salt). The powder was then dissolved in IPA (15 mL) filtrate was concentrated in vacuo to yield the title com 35 and heated to reflux. The resulting slurry was allowed to pound as a yellow solid (2.2 g), which was used without cooled to room temperature and stirred for 1 hour. The solids further purification. were collected to yield Compound 2 as a white solid. Preparation 14: (R)-3-(N-(3'-Chlorobiphenyl-4- ylmethyl)-N'-3-(4-methoxybenzyloxy)isoxazole-5- 40 O-N carbonylhydrazino-2-hydroxypropionic Acid Iso O propyl Ester Y-Qu-o He --O O-N 45 OY-Q-9-y \ PMB

50 To a stirred solution of methyl 3-hydroxyisoxazole-5- carboxylate (5.0 g, 35 mmol) in DMF (20 mL, 300 mmol) at 0°C. was added KCO (5.4 g. 39.4 mmol). After 10 55 minutes at room temperature p-methoxybenzyl chloride (5.5 mL, 40.2 mmol) was added in one portion. The resulting mixture was heated at 60° C. for 2 hours and then cooled to room temperature and stirred overnight. 1.0 M HCl in water OH (150 mL) and EtOAc (150 mL) were added and the phases 60 were separated. The organic layer was washed with Satu rated aqueous NaCl (10 mL), dried over NaSO and the solvent removed by rotary evaporation to yield a thick oil. The oil was dissolved in THF (35 mL) and MeOH (35 mL), followed by addition of LiOH monohydrate (2.9 g, 69.9 65 mmol) dissolved in water (35 mL). The resulting mixture (1) was stirred at room temperature and the reaction monitored for completion (s3 hours). Solvent was removed by rotary US 9,683,002 B2 67 68 evaporation at 30° C. to yield a pasty solid. Toluene (100 -continued mL) was added and the volume was reduced (to ~50 ml). N PMB EtOAc (200 mL) was added and the volume was reduced (to O-1 Y-0M ~50 ml). Filtration and drying yielded a solid (10 g), which O S. was dissolved in water (200 mL), and the pH was adjusted 5 O slowly with concentrated HCl to s2. EtOAc (200 mL) was NH added and the phases were separated. The aqueous layer was O N1 back extracted with EtOAc (200 mL). The combined organic Li layers were dried over NaSO, followed by solvent OH removal. The product was reslurried in EtOAc:hexanes (1:1) 10 followed by filtration to yield Compound 3 (purity >99%). C

(2) + (3) 15 (1)

(R)-3-N-(3'-Chlorobiphenyl-4-ylmethyl)-N'-3-(4- methoxybenzyloxy)isoxazole-5-carbonylhydrazino-2-hy droxypropionic acid isopropyl ester (2.0 g, 3.4 mmol) was combined with MeOH (40 mL, 1.0 mol). LiOH monohy OH drate (170 mg, 4.0 mmol) dissolved in water (5 mL, 300 mmol) was added, and the resulting mixture was stirred at 25 room temperature overnight. The mixture was concentrated by rotary evaporation. The residue was mixed with MeOH and again concentrated by rotary evaporation. The residue was then dried under high vacuum at room temperature to 30 yield crude Compound 1 (2 g). Compound 2 (18.0 g, 26.8 mmol) in DMF (90 mL) was combined with Compound 3 (7.3g, 29 mmol) in DIPEA (12 O mL, 67 mmol). The mixture was cooled at 0°C. followed by Br the portion-wise addition of PyBOP (18 g. 35 mmol) and the 35 reaction monitored for completion (s30 minutes at 0°C.). Water (540 mL) and EtOAc (540 mL) were added and the (1) + -e- phases were separated. The organic layer was washed with saturated aqueous NaCl (500 mL) and dried over NaSO, o-N followed by solvent removal. The crude product was puri 40 N OH fied (SiG chromatography: 300 g column, 10-30-50% O S. EtOAc/hexanes) to yield the title compound (9 g, purity O >98%). NH

45 Preparation 15: (R)-3-N-(3'-Chlorobiphenyl-4-ylm OO OH ethyl)-N'-(3-hydroxyisoxazole-5-carbonyl)hy drazino-2-hydroxypropionic Acid 2-oxo-2-phenyl ethyl Ester 50

O-1 N PMB 2-Bromoacetophenone (44.6 mg, 224 umol) was added to a mixture of Compound 1 (100.0 mg, 179.2 umol) and O S. 55 KCO (49.5 mg, 358.5 umol) in DMF (2.0 mL, 26 mmol). The resulting mixture was stirred at room temperature for 30 minutes, concentrated, and purified by flash chromatography (EtOAc-hexanes 20-80%) to yield a solid (107.7 mg). The OH 60 solid was combined with TFA (82.86 uL. 1.075 mmol) and anisole (194.8 uL. 1.8 mmol) in DCM (5.0 mL, 78 mmol), and stirred at room temperature for 24 hours. The mixture was concentrated and the residue was dissolved in AcOH (2 65 mL), filtered, and purified by reverse phase preparative HPLC. The desired fractions were combined and lyophilized to yield the title compound (79.4 mg). US 9,683,002 B2 69 70 Preparation 16: -continued (S)-2-t-Butoxycarbonylamino-3-methylbutyric Acid O Chloromethyl Ester 1. O r Nulls o1 No O O

10 To a mixture of Compound 1 (21.8 g. 139 mmol) and methyl chloroformate (12 mL, 157 mmol) in THF (1 L) was Yr- -- added TEA (38 mL. 278 mmol) at 0° C. The resulting O mixture was stirred at room temperature for 12 hours, then CN-ON O Her concentrated in vacuo. The residue was purified by flash I C 15 column (PE:EtOAc=6:1) to yield the title compound as a O yellow solid (20.3 g). LC-MS: 224 M+H". "H NMR (400 MHz, DMSO-d): 8 0.97-102 (m, 6H), 2.16-2.21 (m, 1H), N-N- o1 No 3.68 (s, 1H), 4.14 (d. J–4 Hz, 1H), 5.76-5.91 (m, 2H). O Preparation 18: (R)-3-N-(4-Bromobenzyl)hy drazino-2-hydroxypropionic Acid Methyl Ester To a mixture of (S)-2-(t-butoxycarbonylamino)-3-meth ylbutanoic acid (28.6 g. 130 mmol) and NaHCO, (44g, 520 mmol) and BuNHSO (4.4g, 13 mmol) in DCM (200 mL) 25 and water (200 mL) was added chloromethyl sulfochloridate (26 g. 158 mmol) at 0°C. The mixture was stirred at room temperature for 24 hours, and then was extracted with DCM O (3x150 mL). The combined organic layers were washed O with water (2x300 mL), and the DCM layer was purified by 30 O -- flash column (PE:EtOAc=15:1) to yield the title compound No N1 NH as a yellow solid (35 g). LC-MS. 266 M+H". OH Preparation 17: (S)-2-Methoxycarbonylamino-3-methylbutyric Acid 35 Chloromethyl Ester Br O NH2 40 O No N1 y O r Nulls o1 Ne. He OH O 45 Br O 2 o1 No (R)-3-N-(4-Bromobenzyl)-N'-t-butoxycarbonylhy IN drazino-2-hydroxypropionic acid methyl ester (1.1 g, 2.8 50 mmol) was dissolved in MeCN (10 mL) and of 4N HCl in dioxane (6 mL, 20 mmol). The mixture was stirred at room (1) temperature until deprotection was complete (1 hour). The precipitate was filtered and dried to yield the title compound A solution of (S)-2-t-butoxycarbonylamino-3-methylbu (840 mg) as an HCl salt. tyric acid chloromethyl ester (35 g, 132 mmol) in DCM (200 55 mL) was added dropwise a solution of TFA (50 mL) in DCM Preparation 19: (100 mL) at 0°C. The resulting mixture was stirred at room 1-Allyloxy-1H-1.2.3 triazole-4-carboxylic Acid temperature overnight and then concentrated in vacuo to yield crude Compound 1 as a yellow oil (21.8 g). LC-MS: 60 166 M+H".

O (1) + --- -e- 65 US 9,683,002 B2 71 72 -continued -continued

N2N O N- S. HO N-NH O (1)

10 N2NN 1-4 HO S. 15 O

To a solution of 1-hydroxy-1H-1.2.3 triazole-4-carbox ylic acid ethyl ester (5 g, 31.8 mmol) in DMF (20 mL) was HO added KCO (5.3.g., 38.2 mmol) at room temperature. After 10 minutes, allyl bromide (4.g., 33.4 mmol) was added. The mixture was stirred at room temperature overnight. Water (150 mL) was added, and the mixture was extracted with 25 EtOAc (3x50 mL). The combined organic layers were washed with saturated aqueous NaCl (50 mL) and dried over anhydrous NaSO. The solution was evaporated to and the C residue was purified by silica gel chromatography (silica gel: 30 200-300 mesh, eluted with PE:EA=10:1 to 5:1 to 1:1) to (1) yield Compound 1 as a yellow oil (4.3 g). LC-MS: 198 M+H". To a solution of (R)-3-N-(2,5'-dichlorobiphenyl-4-ylm ethyl)hydrazino-2-hydroxy-propionic acid ethyl ester (HCl To a solution of Compound 1 (4.3 g, 22.0 mmol) in EtOH 35 salt, 4 g., 9.9 mmol) and 1-allyloxy-1H-1.2.3 triazole-4- (30 mL) was added a solution of LiOH (1.2g, 28.5 mmol) carboxylic acid (1.7 g, 9.9 mmol) in DMF (30 mL) was in water (10 mL). The resulting mixture was stirred at room added PyBOP (5.2g, 9.9) and DIPEA (3.2g, 24.8 mL) at 0° temperature overnight. The mixture was concentrated, water C. The mixture was stirred at room temperature for 4 hours. Water (200 mL) was added, and the mixture was extracted (10 mL) was added, and the mixture was extracted with with EtOAc (3x100 mL), and the combined organic layers EtOAc (2x20 mL). The aqueous layer was acidified by 1N 40 were washed with saturated aqueous NaCl (100 mL) and HCl to pH 3, and extracted with EtOAc (3x30 mL). The dried over anhydrous NaSO. The mixture was concen combined organic layers were washed with Saturated aque trated and the residue was purified by silica gel chromatog ous NaCl (30 mL) and dried over anhydrous NaSO. The raphy (silica gel: 200-300 mesh; eluted with Solution was evaporated to yield the title compound as a 45 PE:EtOAc=10:1 to 5:1 to 1:1) to yield Compound 1 as a white solid (3.5 g). LC-MS: 170 M+H". light yellow solid (4.2 g). LC-MS. 534 M+H", 536 (M+ 2)+H". O /— Preparation 20: (R)-3-N-(1-Allyloxy-1H-1.2.3 HO O / 1' triazole-4-carbonyl)-N-(2,5'-dichlorobiphenyl-4- 50 (1) -> ) ( e . ylmethyl)-hydrazino-2-hydroxypropionic Acid HO N-N N2

55

M 60 N-N / Na To a solution of Compound 1 (4.2 g, 7.9 mmol) in THF (20 mL) and water (5 mL) was added LiOH (0.5 g, 11.8 65 mmol) at room temperature. The reaction mixture was HO O stirred at room temperature overnight. The mixture was concentrated, water (50 mL) was added, and the resulting US 9,683,002 B2 73 74 mixture was extracted with EtOAc (2x20 mL). The aqueous -continued layer was acidified by 1N HCl to pH 3 and extracted with EtOAc (3x50 mL). The combined organic layers were washed with saturated aqueous NaCl (50 mL) and dried over anhydrous NaSO. The solution was evaporated to yield the C title compound as a yellow solid (3.5 g). LC-MS. 506 M+H", 508 (M+2)+H". Example 1A: (R)-3-(N-(5'-Chloro-2'-fluorobiphe HO N nyl-4-ylmethyl)-N'-1-(3-chlorophenyl)-5-oxo-4,5- OH dihydro-1H-1.2.4 triazole-3-carbonylhydrazino-2- 10 hydroxypropionic Acid

15 O 1-(3-Chlorophenyl)-5-oxo-4,5-dihydro-1H-1.2.4 triaz ole-3-carboxylic acid (75.8 mg, 316 umol) and HCTU (131 HO N-NH2 -- mg, 316 umol) were combined in DMF (1.3 mL, 17.2 mmol) and stirred at room temperature for 15 minutes. (R)-3-N- (5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hy droxypropionic acid ethyl ester (105 mg, 287 umol) and DIPEA (150 uL. 862 umol) were added, and the resulting mixture was stirred at room temperature for 15 minutes. The 25 mixture was evaporated under reduced pressure. The residue was dissolved in EtOH (1.0 mL, 17.2 mmol) and a solution of 1 M LiOH in water (1.4 mL, 1.4 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, then evaporated under reduced pressure. The residue was purified by reverse phase preparative HPLC to yield the title 30 compound (75 mg, purity 100%) as a TFA salt. MS m/z. M+h" calc’d for CHC1FNOs 560.08; found 559.6. Example 1B and 1C: (R)-3-(N-(5'-Chloro-2'-fluoro biphenyl-4-ylmethyl)-N'-1-(3-chlorophenyl)-5-oxo 35 4,5-dihydro-1H-1,2,4-triazole-3-carbonylhy drazino-2-hydroxypropionic Acid Ethyl Ester (Compound 1) and (R)-3-N-(5'-Chloro-2'-fluorobi phenyl-4-ylmethyl)-N'-1-(3-chlorophenyl)-5-oxo-4, 5-dihydro-1H-1.2.4 triazole-3-carbonylhydrazino 2-hydroxypropionic Acid Isobutyl Ester (Compound 2)

US 9,683,002 B2 75 76 1-(3-Chlorophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triaz Using the procedures described herein, the title compound ole-3-carboxylic acid (27.6 mg, 115umol) and HATU (52.5 mg, 138 umol) were stirred in DMA (1.0 mL, 11 mmol) for can also be prepared. 10 minutes. (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylm ethyl)hydrazino-2-hydroxypropionic acid ethyl ester (42.2 mg, 115umol) and DIPEA (60.1 uL, 345 umol) were added, and resulting mixture was stirred at room temperature for 2 Example 1F: (S)-2-Amino-3-methylbutyric Acid hours. The mixture was concentrated under reduced pressure 5-N' ((R)-2-Carboxy-2-hydroxyethyl)-N'-(5'- and the residue was dissolved in EtOAc (20 mL). The organic layer was washed with water (2x5 mL), dried over chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazinocar MgSO, and concentrated. One half of the material was 10 bonyl-2-(3-chlorophenyl)-2H-1,2,4-triazol-3- dissolved in 50% acetic acid-water (1.5 mL), filtered, and purified by reverse phase preparative HPLC to yield Com yloxymethyl Ester pound 1 (1.3 mg, purity 96%) as a TFA salt. MS m/z. M+H" calc’d for C.H.ClFNOs, 588.11: found 588.4. The remaining half of the material was combined with isobutyl alcohol (0.5 mL, 6 mmol), and 4.0 M of HCl in 15 1,4-dioxane (115 uL. 460 Limol) and stirred at room tem perature overnight. The mixture was then concentrated under reduced pressure and the residue was dissolved in 50% acetic acid-water (1.5 ml), filtered, and purified by C reverse phase preparative HPLC to yield the Compound 2 N-N O (1.8 mg, purity 100%) as a TFA salt. MS m/z. M+H" calc’d O for CHCl2FNOs, 616.15; found 616.4. N 2s o1 No s ls Example 1D: (S)-2-Amino-3-methylbutyric Acid (R)-3- E.R., -2'-fluorobiphenyl-4-ylmethyl)- NH NH2 N'-1-(3-chlorophenyl)-5-hydroxy-1H-1,2,4-triaz HO N1 ole-3-carbonylhydrazino-2-hydroxypropiony 25 loxymethyl Ester OH

C

30

N-N C os- 2n O O N OH Using the procedures described herein, the title compound HNullsan ^-r NH 35 can also be prepared. OH

C Example 1G: (R)-3-N'-(5-Acetoxymethoxy-1-(3- 40 chlorophenyl)-1H-1.2.4 triazole-3-carbonyl-N-(5'- chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- Using the procedures described herein, the title compound hydroxypropionic Acid can also be prepared. Example 1E: (R)-3-(N-(5'-Chloro-2'-fluorobiphe 45 nyl-4-ylmethyl)-N'-1-(3-chlorophenyl)-5-hydroxy 1H-1,2,4-triazole-3-carbonylhydrazino-2-hy droxypropionic Acid Acetoxymethyl Ester C 50 N-N O s-s-s N-N C 55 HO N NH O O os- N > OH OH NH --> O N1 60 OH O C

F

65 Co.F C Using the procedures described herein, the title compound can also be prepared. US 9,683,002 B2 77 78 Example 2A: (R)-3-N-(5'-Chloro-2'-fluorobiphe Example 2B: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl 4-ylmethyl)-N'-(2-hydroxythiazole-5-carbonyl)hy nyl-4-ylmethyl)-N'-(2-hydroxythiazole-5-carbonyl) drazino-2-hydroxypropionic Acid Ethyl Ester hydrazino-2-hydroxypropionic Acid O 1No N1 NH2 OH 10 O -- C

F -,OH

o

HO O

O 1No OH

2-Hydroxy-5-thiazolecarboxylic acid (9.0 mg. 62 umol) and HATU (28.3 mg, 74 umol) were stirred in DMA (0.5 mL, 5 mmol) for 10 minutes. (R)-3-N-(5'-Chloro-2'-fluo robiphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid ethyl ester (22.7 mg, 62 umol) and DIPEA (32.4 L, 186 umol) were added, and resulting mixture was stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure and the residue was dissolved in 50% AcOH-water (1.5 mL), filtered, purified by reverse phase preparative HPLC, and lyophilized to yield the title 2-Hydroxy-5-thiazolecarboxylic acid (43.5 mg, 0.3 compound (11.9 mg, purity 96%) as a TFA salt. MS m/z. M+H" calc’d for CHCIFNOS, 494.09: found 4944. mmol) and HCTU (124 mg. 0.3 mmol) were stirred in DMF 50 (1.3 mL, 16.4 mmol) for 15 minutes at room temperature. Example 3A: (R)-3-(N-(5'-Chloro-2'-fluorobiphe (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)hy nyl-4-ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5- drazino-2-hydroxypropionic acid ethyl ester (100 mg, 0.3 carbonylhydrazino-2-hydroxypropionic Acid mmol) and DIPEA (142 uL. 818 umol) were added, and 55 resulting mixture was stirred at room temperature for 30 minutes. The mixture was then evaporated under reduced pressure. The residue was dissolved in EtOH (955 uL. 16.4 mmol). A solution of 1.0 M LiOH in water (1.4 mL, 1.4 60 N-NH2 mmol) was added and the resulting mixture was stirred at HO 40°C. for 3 hours. LC/MS showed completion. The solvent was removed in vacuo and the residue was purified by preparative HPLC to yield the title compound (22 mg) as a 65 TFA salt. MS m/z. M+H" calc’d for CHCIFNOS, Br 466.06; found 466.0. US 9,683,002 B2 79 80 -continued Example 3B: (R)-3-(N-(5'-Chloro-2'-fluorobiphe nyl-4-ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5- carbonylhydrazino-2-hydroxypropionic Acid Isobutyl Ester

--

O O 9NN

-N \l HO N-N HO HO 15 F

Br (1)

(R)-3-N-(4-Bromobenzyl)hydrazino-2-hydroxypropi 25 onic acid ethyl ester (580 mg, 1.8 mmol), HCTU (756 mg, 1.8 mmol) and DMF (850 mL, 110 mmol) were combined. After 15 minutes, DIPEA (956 uL 5.5 mmol) and 3-(2- fluorophenyl)isoxazole-5-carboxylic acid (417 mg, 2.0 mmol) were added. The resulting mixture was stirred at 30 room temperature for 15 minutes. The solvent was removed under pressure and the crude residue was purified (reverse phase chromatography) to yield Compound 1 (56.95 mg).

35

HON B -OH

F

40 (1)1 -- C -e- 9N O S. 3-(2-fluorophenyl)isoxazole-5-carboxylic acid (15.4 mg. 45 O 74 umol) and HATU (33.9 mg. 89 umol) were stirred in NH F DMA (0.5 mL, 5 mmol) for 10 minutes. (R)-3-N-(5'- HO N1 Chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hy OH 50 droxypropionic acid ethyl ester (27.3 mg, 74 umol) and DIPEA (38.9 uL., 223 umol) were added, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was then concentrated under reduced pressure, and

55 the residue was dissolved EtOAc (20 mL) and washed with water (2x2 mL). The organic layer was dried over MgSO, Compound 1 (700 mg, 1 mmol) and 5-chloro-2-fluoro filtered, and concentrated. The product was then mixed with phenylboronic acid (273 mg, 1.6 mmol) were combined with isobutyl alcohol (0.5 mL, 5 mmol) and 4.0 M HCl in KCO (541 mg, 3.9 mmol), EtOH (4.6 mL, 78.3 mmol), 60 1,4-dioxane (93 uL, 372 umol), and stirred at room tem toluene (13.9 mL, 130 mmol), and water (1.2 mL, 65.2 perature overnight. The mixture was concentrated under mmol). Pd(PPh) (151 mg, 130 umol) was then added under reduced pressure, and the residue was dissolved in 50% nitrogen, and the mixture was stirred at 90° C. for 3 hours. AcOH-water (1.5 ml), filtered, and purified by reverse phase The mixture was filtered and evaporated and purified by preparative HPLC to yield the title compound (1.7 mg, preparative HPLC to yield the title compound (40 mg). MS 65 m/z. M+H" calc’d for CHCIFNO. 464.09: found purity 100%) as a TFA salt. MS m/z. M+H" calc’d for 464.0. CHCIFN-Os, 584.17; found 584.4. US 9,683,002 B2 81 82 Example 3C: (R)-3-(N-(5'-Chloro-2'-fluorobiphe 2H), 4.4–4.2 (m. 2H), 4.3 (br. 1H), 4.94-4.86 (m. 2H), nyl-4-ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5- 7.24-6.85 (m, 5H), 7.97-7.27 (m, 8H). carbonylhydrazino-2-hydroxypropionic Acid 5-Methyl-2-oxo-1.3dioxol-4-ylmethyl Ester Example 3D: (R)-3-(N-(S-Chloro-2'-fluorobiphe nyl-4-ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5- carbonylhydrazino-2-hydroxypropionic acid 2.2.3, 3.3-pentafluoropropyl Ester

10

HO N-NH 15

O -- HO

OH 25 C

F 30 F

F OH F F F 35 F

F F F O F F O O / / 40 N HO N-N O H

45

50

EDCI (92 mg, 480 umol) and HOBT (65 mg, 480 umol) were added to a solution of (R)-3-N-(5'-chloro-2'-fluorobi A mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl phenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid 55 methyl)-N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy 5-methyl-2-oxo-1.3dioxol-4-ylmethyl ester (108 mg, 240 drazino-2-hydroxypropionic acid (30.0 mg, 57 EDC HCl umol) and 3-(2-fluorophenyl)isoxazole-5-carboxylic acid (65.4 mg., 341 umol), and HOBt hydrate (52.2 mg, 341 (50 mg, 240 umol) in DMF (20 mL). DIPEA (62 mg, 480 umol) in DCM (0.5 mL, 8 mmol) was stirred at room umol) was added and the mixture was stirred for 5 hours at 60 temperature for 10 minutes. 2.2.3,3,3-Pentafluoro-1-propa room temperature. The mixture was washed with saturated aqueous NaCl (2x30 mL) and extracted with EtOAc (2x50 nol (45.3 uL. 455umol) was added and the resulting mixture mL). The combined organic layers were dried over anhy was stirred at room temperature for 1 hour then concen drous Na2SO and concentrated in vacuo. The residue was trated. The residue was dissolved in AcOH (2 mL), filtered, purified by column chromatography (hexanes/EtOAc=1:1) 65 and purified by reverse phase prep HPLC to yield the title to yield the title compound as a white solid (58 mg). LC-MS: compound (4.8 mg, purity 90.2%) as a TFA salt. MS m/z. 640.2 M+H". H-NMR: (CDC13) 2.07 (s, 3H), 3.43 (br. M+H" calc’d for CHCIF.N.O.s, 660.11: found 660.3. US 9,683,002 B2 83 84 Example 3E: (R)-3-(N-(5'-Chloro-2'-fluorobiphe Example 3F: (R)-3-(N-(5'-Chloro-2'-fluorobiphenyl nyl-4-ylmethyl)-N'-3-(2-fluorophenyl)-isoxazole-5- 4-ylmethyl)-N'-[3-(2-fluorophenyl)isoxazole-5-car carbonylhydrazino-2-hydroxypropionic Acid bonylhydrazino-2-hydroxypropionic Acid Acetoxymethyl Ester 2-methoxyethyl Ester

10

F F

15 O Y. O Y.M O O O O

NH -- NH HO N1 HO N1 --

OH OH O C 25 F

O --~. -e- 30

-( 35 o-w O O O A F N HO N-N O H 40

45

C

50 Bromomethyl acetate (11.9 uL. 121 umol) was added to a mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylm Ethane, 1-bromo-2-methoxy (5.7 uL, 0.1 mmol) was ethyl)-N'-[3-(2-fluorophenyl)isoxazole-5-carbonylhy 55 added to a mixture of (R)-3-N-(5'-chloro-2'-fluorobiphe drazino-2-hydroxypropionic acid (40.0 mg, 76 umol) and nyl-4-ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5-carbo EtN (21.1 uL. 152 umol) in acetone (1.0 mL, 14 mmol). nylhydrazino-2-hydroxypropionic acid (20.0 mg. 38 The resulting mixture was stirred at room temperature for 1 umol) and EtN (13.2 uL, 0.1 mmol) in acetone (1.0 mL, 14 hour. The mixture was then concentrated and the residue was 60 mmol), and resulting mixture was stirred at 50° C. overnight. dissolved in AcOH (2 mL), filtered, and purified by reverse The mixture was concentrated, and the residue was dis phase prep HPLC. The desired fractions were frozen and solved in AcOH (1.5 mL), filtered, and purified by reverse lyophilized to yield the title compound (8.5 mg, purity phase preparative HPLC to yield the title compound (10.7 65 98.5%) as a TFA salt. MS m/z. M+H" calc’d for mg, purity 95.7%) as a TFA salt. MS m/z. M+H" calc’d for CHCIFNO, 600.13: found 600.1. CHCIFNO. 586.15; found 585.8. US 9,683,002 B2 85 86 Example 3G: Butyric acid (R)-3-N-(5'-chloro-2'- Example 3H: (R)-3-(N-(5'-Chloro-2'-fluorobiphe fluorobiphenyl-4-ylmethyl)-N'-3-(2-fluorophenyl) nyl-4-ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5- isoxazole-5-carbonylhydrazino-2-hydroxypropio carbonylhydrazino-2-hydroxypropionic Acid Iso nyloxymethyl Ester propoxy carbonyloxymethyl Ester

35 HO

40

45

Chloromethylbutyrate (11.4 LL, 0.1 mmol) and Nat (13.6 mg, 0.1 mmol) were combined in acetone (0.7 mL, 10 mmol) and heated at 65° C. for 1 hour. The mixture was then cooled to room temperature. A mixture of (R)-3-N-(5'- Chloromethyl isopropyl carbonate (17.3 mg, 114 umol) chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-3-(2-fluorophe and Nat 17.0 mg, 114 umol) were combined in acetone (1.0 nyl)isoxazole-5-carbonylhydrazino-2-hydroxypropionic mL, 14 mmol) and heated at 60° C. for 1 hour. The mixture acid (16.0 mg. 30 mol) dissolved in acetone (0.2 mL) and was then cooled to room temperature. A mixture of (R)-3- treated with EtN (8.5 L, 61 umol) was added and the 55 {N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-[3-(2-fluo resulting mixture was stirred at room temperature for 25 rophenyl)isoxazole-5-carbonylhydrazino-2-hydroxypro minutes. The mixture was concentrated and the residue was pionic acid (20.0 mg, 38 umol) dissolved in acetone (1.0 dissolved in AcOH (2.0 mL), filtered and purified by reverse mL) and treated with EtN (10.6 uL, 76 umol) was added phase preparative HPLC. The desired fractions were com 60 and the resulting mixture was stirred at room temperature for bined and freeze dried to yield a yellowish solid. This solid 5 hours. The mixture was concentrated and the residue was was dissolved in AcOH (1.5 mL), filtered, and purified by dissolved in AcOH (2.0 mL), filtered and purified by reverse reverse phase preparative HPLC. The desired fractions were phase preparative HPLC. The product was freeze dried and combined and freeze dried to yield the title compound (5.7 65 purified by reverse phase preparative HPLC to yield the title mg, purity 100%) as a TFA salt white solid. MS m/z. M+H" compound (1.8 mg, purity 100%). MS m/z. M+H" calc’d calc’d for CHCIFNO7, 628.16; found 628. for CHCIFNOs, 644.15; found 644.1. US 9,683,002 B2 87 88 Example 3I: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl Example 3.J: (S)-2-Methoxycarbonylamino-3-meth 4-ylmethyl)-N'-[3-(2-fluorophenyl)isoxazole-5-car ylbutyric acid (R)-3-N-(5'-chloro-2'-fluorobiphe bonylhydrazino-2-phosphonooxypropionic Acid nyl-4-ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5- carbonylhydrazino-2-hydroxypropionyloxymethyl Ester

N O- N O S. 10 O 9N NH F HO N1 O S. -- O OH 15 NH F HO N1 -- O C OH

F O C

F O Cl V / He O P 25 o, N C1 No 1. o1 NC -- O -n. O-1 NN - O O S. O

NH F HO N1 35 HO

40

(R)-3-(N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- 3-(2-fluorophenyl)isoxazole-5-carbonylhydrazino-2-hy 45 droxy-propionic acid (12.0 mg, 22.7 umol) in EtOH (80 uL. To a solution of (R)-3-(N-(5'-chloro-2'-fluorobiphenyl-4- 1.4 mmol) was combined with a solution of 4.0 M HCl in ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy 1,4-dioxane (227 uL. 909 umol), and the resulting mixture 50 drazino-2-hydroxypropionic acid (200 mg. 380 umol) in was stirred at room temperature for 1 hour. The solvent was DMF (10 mL) was added 2.6-lutidine (407 mg, 3.8 mmol), removed in vacuo and the residue was dissolved in pyridine (S)-2-methoxycarbonylamino-3-methylbutyric acid chlo (20 uL. 250 umol). The resulting solution was added to a romethyl ester (170 mg, 760 umol) and Nat (114 mg, 760 umol). The resulting mixture was stirred overnight at room solution of phosphoryl chloride (19 uL, 0.2 mmol) in 55 temperature. The Solution was washed with Saturated aque acetone (67 uL, 0.9 mmol) and stirred at room temperature ous NaCl (2x20 mL) and extracted with EtOAc (2x30 mL). for 10 minutes. The solvent was removed in vacuo and the The combined organic layers were dried over anhydrous residue was dissolved in EtOH (80 uL, 1.4 mmol). A NaSO and concentrated in vacuo. The residue was purified solution of 1.0 M LiOH in water (1.4 mL, 1.4 mmol) was 60 by column chromatography (PE/EA=4/1-1/2) to yield the title compound as a white solid (90 mg). LC-MS: 714.8 then added until the pH reached ~12. The mixture was M+H". H-NMR (CD3OD-d): 8 0.91 (d. J=9.6 Hz, 6H), stirred for 1 hour and the solvent was removed in vacuo. The 2.05-2.13 (m. 1H), 3.39-3.45 (m, 2H), 3.66 (s, 3H), 4.06 residue was purified by preparative HPLC to yield the title 65 4.08 (m, 1H), 4.23-4.25 (m, 2H), 4.66-4.48 (m, 1H), 5.79 compound (5 mg). MS m/z. M+H" calc’d for 5.86 (m, 2H), 7.15-7.26 (m, 1H), 7.24-7.55 (m. 10H), CHCIFNOP 608.07; found 608.0. 7.97-7.95 (m, 1H). US 9,683,002 B2 89 90 Example 3K: (R)-3-(N-(5'-Chloro-2'-fluorobiphe Example 3Li: (R)-3-N-(5'-Chloro-2'-fluorobiphe nyl-4-ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5- nyl-4-ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5- carbonylhydrazino-2-hydroxypropionic Acid carbonylhydrazino-2-hydroxypropionic Acid Ethoxycarbonyloxymethyl Ester 1-Cyclohexyloxycarbonyloxyethyl Ester

O -n O N-NH2 HO --

15

25

30

35 N O-1 N O S. O 40 1No N1 NH F OH

45

(1) 2.6-Lutidine (407 mg, 3.8 mmol) was added to a solution 50 of (R)-3-(N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- 3-(2-fluorophenyl)isoxazole-5-carbonylhydrazino-2-hy To a solution of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- droxypropionic acid (200 mg, 380 umol), carbonic acid ylmethyl)hydrazino-2-hydroxypropionic acid ethyl ester chloromethyl ester ethyl ester (105 mg, 760 umol) and NaI (1.5 g., 3.7 mmol), EDC (928 mg, 4.8 mmol), HOBt (653 mg, (114 mg, 760 umol) in DMF (10 mL). The mixture was 55 4.8 mmol) and 3-(2-fluorophenyl)isoxazole-5-carboxylic stirred overnight at room temperature. Water (30 mL) was acid (848 mg, 4.1 mmol) in DCM (20 mL) was added added and the mixture was extracted with EtOAc (3x40 DIPEA (1.9 mL, 11.2 mmol) under nitrogen. The resulting mL). The combined organic layers were dried over anhy mixture was stirred at room temperature overnight, then drous Na2SO and concentrated in vacuo. The residue was 60 concentrated to dryness. The residue was dissolved in purified by preparative HPLC (MeCN HO (0.1% TFA): EtOAc (20 mL), washed with 0.5N aqueous HCl (10 mL), Gradient 60-70) to yield the title compound as a white solid saturated aqueous NaHCO (10 mL) and Saturated aqueous (56 mg). LC-MS: 630.1 M+H". H-NMR: (CDOD-d NaCl (10 mL), dried over anhydrous NaSO filtered, and 400 MHz) & 1.24 (t, J=5.9 Hz, 3H), 3.30-3.33 (m, 2H), 65 concentrated in vacuo. The residue was purified by column 4.17-4.32 (m, 4H), 4.45 (t, J=4.2 Hz, 1H), 5.78 (br, 2H), chromatography (PE:EtOAc, 10:1-3:1) to yield Compound 7.20-7.28 (m, 1H), 7.57-729 (m, 10H), 7.92-7.95 (m. 1H). 1 as a solid (1.4 g). LC-MS. 556 M+H". US 9,683,002 B2 91 92 -continued O-N- O W \ Hip (1) + O -3- o1 o-( OH -( O C 10 O Y O O

NH HO N1 M o, O 15 OH l NH O O or OH O C To a mixture of 3-methoxy-isoxazole-5-carboxylic acid F (140 mg, 1.0 mmol) and HATU (373 mg, 1.0 mmol) in DMF (5.0 mL. 64 mmol) was added (R)-3-N-(5'-chloro-2'-fluo 25 robiphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid To a solution of Compound 1 (1.4g, 2.5 mmol) in MeOH ethyl ester (300.0 mg, 1.0 mmol) and DIPEA (0.3 mL, 1.6 (15 mL) was added a solution of LiOH.HO (317 mg, 7.6 mmol). The resulting mixture was stirred at room tempera mmol) in water (3 mL). The mixture was stirred at room ture overnight until the reaction was complete. The mixture temperature for 1 hour, and the insoluble solid was filtered was partitioned between EtOAc (10.0 mL) and water (3.0 off and the filtrate was concentrated in vacuo to yield a 30 mL). The organic layer was washed with water (2x3.0 mL), yellow solid (1.2 g). LC-MS: 528 M+H". The yellow solid saturated aqueous NaCl (3.0 mL), dried over NaSO, (400 mg, 760 umol) was dissolved in 2.6-lutidine (814 mg. filtered and concentrated to yield a yellowish oil. The oil was 7.6 mmol) and carbonic acid 1-chloro-ethyl ester cyclohexyl purified by flash chromatography (2x4 g Stacker column, ester (1.6 g., 7.6 mmol) was added. The vial was sealed and 0-100% EtOAc/hexanes). The desired fractions were com the resulting mixture was then irradiated for 30 minutes at 35 bined and concentrated to yield a light yellowish oil. The 90° C. under microwave irradiation. Water (10 mL) was oily residue was then treated with a mixture of MeCH (5.0 added, and the mixture was extracted with EtOAc (3x10 mL, 120 mmol) and water (1.0 mL, 56 mmol). LiOH mL). The combined organic layers were washed with 0.5N monohydrate (68.6 mg, 1.6 mmol) was added. After stirring aqueous HCl (5x5 mL) and saturated aqueous NaCl (10 at room temperature for 30 minutes, the mixture was con centrated. The residue was treated with EtOAc (10.0 mL) mL), dried over anhydrous Na2SO4, filtered and concen 40 and acidified with 1 NHCl until pH-3. The organic layer was trated in vacuo. The crude product was purified by column washed with saturated aqueous NaCl (2x30 mL), dried over chromatography (PE:EtOAc, 10:1-2: 1) to yield the title NaSO filtered and concentrated to yield the title com compound as a white solid (60 mg). LC-MS: 698 M+H", pound as a white foam (289.7 mg). MS m/z. M+H calc’d NMR: (CDC1,400 MHz) & 1.28-1.37 (m, 6H), 1.54 (d. 3H), for CHCIFNO. 464.09: found 464.0. 1.72-1.75 (m, 2H), 1.90-1.95 (m, 2H), 3.33-3.38 (m, 2H), 45 4.24-4.30 (m. 2H), 4.38-440 (m. 1H), 4.61-4.66 (m. 1H), Example 4B: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl 6.60 (t, 0.5H), 6.80 (t, 0.5H), 7.00-7.10 (m, 1H), 7.20-7.26 4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl) (m, 3H) 7.36-7.41 (m, 2H), 7.48-7.52 (m, 5H), 7.85 (s, hydrazino-2-hydroxypropionic Acid 5-Methyl-2- 0.5H), 8.00-8.04 (m. 1H), 8.15 (s, 0.5H). oxo-1.3dioxol-4-ylmethyl Ester Example 4A: (R)-3-N-(5'-Chloro-2'-fluorobiphe nyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbo nyl)hydrazino-2-hydroxypropionic Acid

55

HO

60 OH

65 US 9,683,002 B2 93 94 -continued -continued F

He F OH F F F

O O 10 N N O M t O F O O 15 NH , ) or F O N1 F F F OH O C

F

25 (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- (3-methoxyisoxazole-5-carbonyl)hydrazino-2-hydroxypro EDCI (169 mg,880 umol) and HOBT (119 mg,880 umol) pionic acid (20 mg. 43 umol), EDC (40.16 mg, 0.2587 were added to a solution of (R)-3-N-(5'-chloro-2'-fluorobi phenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid mmol), and HOBt hydrate (39.62 mg, 0.2587 mmol) were 5-methyl-2-oxo-1.3dioxol-4-ylmethyl ester (200 mg. 440 30 combined in DCM (0.5 mL, 8 mmol) and stirred at room umol) and 3-methoxyisoxazole-5-carboxylic acid (63 mg. temperature. After 10 minutes, 2,2,3,3,3-pentafluoro-1-pro 440 umol) in DMF (10 mL). DIPEA (114 mg. 880 umol) was panol (51.8 mg, 345 umol) was added. The resulting mixture added and the mixture was stirred for 5 hours at room was stirred at room temperature overnight to yield the title temperature. The mixture was washed with Saturated aque ous NaCl (2x30 mL) and extracted with EtOAc (2x50 mL). 35 compound (5.9 mg, purity 100%) as a TFA salt. MS m/z. The combined organic layers were dried over anhydrous M+H" calc’d for CHCIFNO. 596.09: found 596. NaSO and concentrated in vacuo. The residue was purified by column chromatography (hexanes/EtOAc=1:1) to yield the title compound as a white solid (60 mg). LC-MS: 576.1 Example 4D: (R)-3-N-(5'-Chloro-2'-fluorobiphe M+H". H-NMR: (DMSO-d) 2.14 (s, 3H), 3.21-3.19 (m, 40 nyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbo 2H), 3.91 (s, 3H), 4.17-4.11 (m, 2H), 4.31 (br. 1H), 4.98 (s, nyl)hydrazino-2-hydroxypropionic Acid Acetoxym 2H), 5.57 (br. 1H), 6.73 (s, 1H), 7.57-7.34 (m, 7H), 10.07 (s, ethyl Ester 1H).

Example 4C: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl 45 4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl) hydrazino-2-hydroxypropionic acid 2.2.3,3,3-penta fluoropropyl Ester

50 O HO N

55 OH O Y O O

NH HO N1 -- 60 OH

65 US 9,683,002 B2 95 96 -continued 1-Bromo-2-methoxyethane (12.2 LL, 129 umol) was O added to a mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl 4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hy drazino-2-hydroxypropionic acid (20.0 mg, 43 umol) and O Y.M DIPEA (45.1 uL. 259 umol) in acetone (1.0 mL, 14 mmol). O O O The resulting mixture was heated at 60° C. for 4 hours. NaI (19.4 mg, 129 umol) was added and the reaction was NH monitored for 2 hours. Additional 1-bromo-2-methoxy ---, N1 ethane (3 eq.), DIEA (4 eq.), and NaI(3 eq.) were added and 10 OH the heating continued overnight. Additional 1-bromo-2- methoxyethane (3 eq.), NaI (3 eq.), and DIEA (3 eq.) were added and heating continued overnight. The mixture was then concentrated, and the residue was dissolved in AcOH (1.5 mL), filtered and purified by reverse phase preparative 15 HPLC to yield the title compound (2.6 mg, purity 99%). MS m/z. M+H" calc’d for CHCIFNO7, 522.14; found To a solution of (R)-3-(N-(5'-chloro-2'-fluorobiphenyl-4- 5224. ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hydrazino 2-hydroxypropionic acid (40.0 mg, 0.1 mmol) in acetone Example 4F: Butyric acid (R)-3-N-(5'-chloro-2'- (1.0 mL, 14 mmol) was added bromomethyl acetate (16.9 fluorobiphenyl-4-ylmethyl)-N'-(3-methoxyisox uL, 172 umol) followed by EtN (24.0 uL, 172 umol), and azole-5-carbonyl)hydrazino-2-hydroxypropiony resulting mixture was stirred for 90 minutes. The reaction loxymethyl Ester was quenched with AcOH (19.6 uL., 345 umol) and the mixture was concentrated. The residue was dissolved in AcOH (3 mL), filtered, and purified by reverse phase 25 preparative HPLC. The desired fractions were combined and O lyophilized. The solid was dissolved in AcOH (1.5 mL), filtered, and purified by reverse phase preparative HPLC to yield the title compound (14.3 mg, purity 97.8%) as a TFA N salt. MS m/z. M+H" calc’d for CHCIFNOs, 536.12: 30 O M found 536.2. O O NH Example 4E: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl HO N1 -- 4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl) hydrazino-2-hydroxypropionic Acid 2-Methoxy 35 OH Ethyl Ester O C (N 40 F O S. o1 O O

NH HO N1 -- 45 OH O C

50 F HO 1 O N-1N N O- N 55 O S. O 1. O 1. ON-1\o N1 NH

OH 60

A mixture of chloromethylbutyrate (16.2 LL, 129 pawl) and NaI (19.4 mg, 129 umol) in acetone (0.7 mL, 10 mmol) 65 was heated at 65° C. for 1 hour. The mixture was then cooled to room temperature and a mixture of (R)-3-N-(5'-Chloro 2'-fluorobiphenyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5- US 9,683,002 B2 97 98 carbonyl)hydrazino-2-hydroxypropionic acid (20.0 mg) bined and freeze dried to yield a white solid. The solid was and DIPEA (15.0 uL, 0.1 mmol) in acetone (0.3 mL) was dissolved in acetic acid (1.5 mL), filtered and purified by added. The resulting mixture was stirred at room tempera reverse phase preparative HPLC to yield the title compound ture for 1 hour then concentrated. The residue was dissolved (5.8 mg, purity 94%). MS m/z. M+H" calc'd for in AcOH (2.0 mL), filtered, and purified by reverse phase 5 CHCIFNO. 566.13: found 566. preparative HPLC. The desired fractions were combined and freeze dried to yield a white solid. The solid was dissolved Example 4H: (R)-3-N-(5'-Chloro-2'-fluorobiphe in acetic acid (1.5 mL), filtered and purified by reverse phase nyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbo preparative HPLC to yield the title compound (6.5 mg. nyl)hydrazino-2-hydroxypropionic Acid 2-Morpho purity 100%). MS m/z. M+H" calc’d for CHCIFNOs, 10 564.15; found. 564. lin-4-ylethyl Ester Example 4G: (R)-3-N-(5'-Chloro-2'-fluorobiphe nyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbo nyl)hydrazino-2-hydroxypropionic Acid Ethoxycar 15 O bonyloxymethyl Ester

O Y.M O O O

HO N NH --

O Y. OH O O 25 NH HO N1 -- OH co C 30 C

He F s O 35 ---> C O Y ( 40 o O O O O N

HO N-N) (?o-N 45 H - C

50 C

F F ( ) C 55 A mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl A mixture of chloromethyl ethyl carbonate (17.9 mg, 129 methyl)-N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- umol) and NaI (19.4 mg, 129 umol) in acetone (0.7 mL, 10 hydroxypropionic acid (20.0 mg. 43 pimp, EDC (45.8 LL. mmol) was heated at 65° C. for 1 hour. The mixture was then 259 umol) and HOBt hydrate (39.6 mg, 259 umol) in DCM cooled to room temperature and a mixture of (R)-3-N-(5'- 60 (0.5 mL, 8 mmol) was stirred at room temperature for 10 chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3-methoxyisox minutes. 4-Morpholineethanol (41.8 L., 345 umol) was azole-5-carbonyl)hydrazino-2-hydroxypropionic acid (20.0 added, and the resulting mixture was stirred at room tem mg) and DIPEA (15.0 L, 0.1 mmol) in acetone (0.3 mL) perature overnight. The mixture was concentrated and the was added. The resulting mixture was stirred at room residue was dissolved in AcOH (1.5 mL), filtered, and temperature for 1 hour then concentrated. The residue was 65 purified by reverse phase preparative HPLC to yield the title dissolved in AcOH (2.0 mL), filtered, and purified by reverse compound (14.1 mg) as a TFA salt. MS m/z. M+H" calc’d phase preparative HPLC. The desired fractions were com for CHCIFNO. 577.18; found 577. US 9,683,002 B2 99 100 Example 4I: (S)-2-Methoxycarbonylamino-3-meth Example 4.J. (R)-3-N-(5'-Chloro-2'-fluorobiphenyl ylbutyric Acid (R)-3-N-(5'-chloro-2'-fluorobiphe 4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl) nyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbo hydrazino-2-hydroxypropionic Acid Isopropoxycar nyl)hydrazino-2-hydroxypropionyloxymethyl Ester bonyloxymethyl Ester

O O 10

O Y O Y.M O O O O

NH 15 NH HO N1 -- HO N1 --

OH OH

O C C

F F

25 O H -e- 1. O N o1N O -N 30

H O O

35

40

45

A mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl methyl)-N-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- A mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl methyl)-N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- hydroxypropionic acid (200 mg, 430 umol), (S)-2-methoxy 50 carbonylamino-3-methylbutyric acid chloromethyl ester hydroxypropionic acid (200 mg, 430 umol), chloromethyl (193 mg, 860 umol), NaI (129 mg, 860 umol) and pyridine isopropyl carbonate (132 mg, 860 umol), NaI (129 mg, 860 (136 mg, 1.7 mmol) in DMF (5.0 mL) was stirred at 25° C. umol) and pyridine (136 mg, 1.7 mmol) in DMF (5.0 mL) overnight. The mixture was then poured into water (30 mL) 55 was stirred at 25°C. overnight. The mixture was then poured and extracted with EtOAc (3x20 mL). The combined into water (30 mL) and extracted with EtOAc (3x20 mL). organic layers were washed with Saturated aqueous NaCl The combined organic layers were washed with saturated (30 mL), dried over anhydrous NaSO, and concentrated in aqueous NaCl (30 mL), dried over anhydrous NaSO, and vacuo. The residue was purified by column chromatography concentrated in vacuo. The residue was purified by column (PE:EtOAc=1:1) to yield the title compound as a colorless 60 liquid (7 mg). LC-MS: 651.1 M+H". "H NMR (CDOD, chromatography (PE:EtOAc=1:1) to yield the title com 400 MHz): 8 0.95-0.97 (m, 6H), 2.06-2.14 (m, 1H), 3.34 pound as a colorless liquid (10 mg). LC-MS. 580M+H". 3.39 (m, 2H), 3.69 (s.3H), 3.98 (s, 3H), 4.08-4.11 (m. 1H), "H NMR (400 MHz, CDOD): & 1.28 (d. J=6 Hz, 6H), 4.22-4.24 (m, 2H), 4.38-440 (m, 1H), 5.78-5.91 (m, 2H), 65 3.37-3.39 (m, 2H), 3.98 (s, 3H), 4.22-4.24 (m, 2H), 4.38 6.54 (s, 1H), 7.18-7.22 (m. 1H), 7.34-7.36 (m. 1H), 7.46 4.40 (m, 1H), 5.78-5.91 (m, 2H), 6.54 (s, 1H), 7.22-7.18 (t, 7.55 (m, 5H). J=9 Hz, 1H), 7.34-7.36 (m, 1H), 7.46-7.55 (m, 5H). US 9,683,002 B2 101 102 Example 4K: (R)-2-(2-Aminoacetoxy)-3-N-(5'- concentrated, and the resulting residue was co-evaporated chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3- with EtOAc (3x2.0 mL) to yield a white solid. The solid was methoxyisoxazole-5-carbonyl)hydrazinopropionic dissolved in AcOH (1.5 mL), filtered, and purified by reverse Acid Ethyl Ester phase preparative HPLC. The desired fractions were com bined and freeze dried to yield the title compound as a white solid TFA salt (8 mg). MS m/z. M+H" calc’d for CHCIFNO. 549.15; found 549.

10 Example 4L: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl O 4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl) HO N-NH2 O W \ hydrazino-2-propionyloxypropionic Acid O1 OH 15 O

Boc1 Nulls OH

HO N O 25 OH N N o, / O O

NH 30 1n --> N Y O O

C 35 HN O F

To a mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- 40 ylmethyl)hydrazino-2-hydroxypropionic acid ethyl ester (300.0 mg, 818 mmol) and 3-methoxy-isoxazole-5-carbox ylic acid (140.4 mg., 981 umol) in DMF (4.0 mL, 52 mmol) at room temperature was added HATU (373.2 mg, 981 45 umol) and DIPEA (427 uL., 2.4 mmol). The resulting mix HO N ture was stirred at room temperature for 1 hour. The mixture was partitioned between EtOAc (10.0 mL) and water (2.0 O O mL). The organic layer was washed with water (2x2.0 mL), dried over NaSO, filtered, and concentrated to yield a 50 O C yellowish oil. The oil was purified by flash chromatography (2x4 g stacker column, 0-100% EtOAc/hexanes). The desired fractions were combined and concentrated to yield a F colorless oil. 55 The oil (28.8 mg, 58.5 umol) was combined with DIPEA Propanoyl chloride (7.3 uL. 84 umol) was added to a (30.6 LL., 176 umol) and added to a mixture of N-O-(t- mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylm butoxycarbonyl)glycine (12.3 mg, 70.2 mol) and HATU ethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- (26.7 mg, 70.2 umol) in DMF (0.5 mL, 6 mmol) at room hydroxypropionic acid (17.0 mg, 36.7 umol) and DIPEA temperature. After stirred at room temperature overnight, the 60 (12.8 LL, 73 umol) in DCM (0.5 mL, 8 mmol). The mixture mixture was partitioned between EtOAc (10.0 mL) and was stirred at room temperature for 30 minutes, then con water (2.0 mL). The organic layer was dried over NaSO, centrated. The residue was dissolved in AcOH (1.5 mL), filtered and concentrated to give a light yellowish oil. The filtered, and purified by reverse phase preparative HPLC. oily residue was then dissolved in DCM (0.2 mL) and 65 The desired fractions were combined and freeze dried to treated with 4.0 M HCl in 1,4-dioxane (0.2 mL, 0.8 mmol) yield the title compound as a white solid (1.2 mg). MS m/z. at room temperature for 30 minutes. The mixture was M+H" calc’d for CHCIFNO, 520.12; found 520.1. US 9,683,002 B2 103 104 Example 4M: (S)-2-Amino-3-methylbutyric Acid and concentrated. The residue was dissolved in AcOH (1.5 (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- mL), filtered, and purified by reverse phase preparative N'-(3-methoxyisoxazole-5-carbonyl)-hydrazino-2- HPLC. The desired fractions were combined and freeze hydroxypropionyloxymethyl Ester dried to yield the title compound as a white solid TFA salt (7.3 mg). MS m/z. M+H" calc’d for C.H.ClFNOs, 593.17; found 593.1. O Example 4N: (S)-2-Methoxycarbonylamino-3-meth 10 ylbutyric Acid (R)-1-Carboxy-2-N-(5'-chloro-2'- fluorobiphenyl-4-ylmethyl)-N'-(3-methoxyisox O Y. azole-5-carbonyl)hydrazinoethyl Ester O O

NH HO N1 15

OH

O

coF -- BOC HO

V 25

HO

40

45

A mixture of Nat (19.4 mg, 129 umol) and (S)-2-t- butoxycarbonylamino-3-methyl-butyric acid chloromethyl 50 ester (34.4 mg, 129 umol) in acetone (0.5 mL, 7 mmol) was heated at 65° C. for 1 hour. The mixture was then cooled to room temperature and treated with a solution of (R)-3-N- (5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3-methoxy 55 (S)-2-Methoxycarbonylamino-3-methylbutyric acid (5.7 isoxazole-5-carbonyl)hydrazino-2-hydroxypropionic acid mg, 32.3 umol) and HATU (12.3 mg, 32.3 umol) were (20.0 mg. 43.1 umol) and DIPEA (15.0 uL. 86.2 umol) in stirred in DMA (1.0 mL, 11 mmol) for 15 minutes. (R)-3- acetone (0.3 mL, 4 mmol). The mixture was stirred at room N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3- temperature for 2 hours, then concentrated. The residue was methoxyisoxazole-5-carbonyl)hydrazino-2-hydroxypropi partitioned between EtOAc (5.0 mL) and water (2.0 mL). the 60 onic acid (10.0 mg, 21.6 umol) and DIPEA (11.3 uL. 64.7 organic layer was washed with water (2.0 mL), Saturated umol) were added, and the resulting mixture was stirred at aqueous NaCl (2.0 mL), dried over NaSO filtered, and room temperature overnight. The mixture was concentrated, concentrated to yield a colorless oil. The oil was further and the residue was dissolved in AcOH (1.5 mL), filtered, dried in vacuo for 30 minutes and then stored in the freezer 65 and purification by reverse phase preparative HPLC to yield overnight. The oil was then treated with a 1:1 mixture of the title compound (2.7 mg). MS m/z. M+H calc’d for DCM/TFA (0.3 mL) at room temperature for 30 minutes, CHCIFN.O., 621.17; found 621.3. US 9,683,002 B2 105 106 Example 4O: (S)-2-Aminopropionic Acid (R)-1- Example 4P: (S)-2-Amino-3-methylbutyric Acid Carboxy-2-N-(5'-chloro-2'-fluorobiphenyl-4-ylm (R)-1-Carboxy-2-N-(5'-chloro-2'-fluorobiphenyl-4- ethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hy ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hy drazinoethyl Ester drazinoethyl Ester

O O 10

N O M O Y. O O O O

NH 15 NH HO N1 HO N1

OH OH O C +

F

25 O

Boc1 N. OH 30

O O

N O M O Y.M O O O O

NH 40 NH HO N1 --> N Y O O O

C C HN O 45 ''', F F

50 (S)-2-t-Butoxycarbonylaminopropionic acid (12.2 mg, N-(t-Butoxycarbonyl)-L-valine (14.0 mg, 64.7 umol) and 64.7 umol) and HATU (24.6 mg, 64.7 umol) were stirred in HATU (24.6 mg, 64.7 umol) were stirred in DMA (1.0 mL. DMA (1.0 mL, 11 mmol) for 10 minutes. (R)-3-N-(5'- 11 mmol) for 10 minutes. (R)-3-N-(5'-Chloro-2'-fluorobi Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3-methoxyisox phenyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl) azole-5-carbonyl)hydrazino-2-hydroxypropionic acid (20.0 55 hydrazino-2-hydroxypropionic acid (20.0 mg. 43.1 umol) mg, 43.1 umol) and DIPEA (22.5 LL, 129 umol) were added, and DIPEA (22.5 L, 129 umol) were added, and the and the resulting mixture was stirred at room temperature resulting mixture was stirred at room temperature overnight overnight then concentrated. 4.0 M HCl in 1,4-dioxane (0.2 60 then concentrated. 4.0 M HCl in 1,4-dioxane (0.2 mL, 0.8 mL, 0.8 mmol) was added and the resulting mixture was mmol) was added and the resulting mixture was allowed to allowed to stand for 1 hour, then concentrated under reduced stand for 1 hour, then concentrated under reduced pressure. pressure. The residue was dissolved in AcOH (1.5 mL), The residue was dissolved in AcOH (1.5 mL), filtered, and filtered, and purified by reverse phase preparative HPLC to 65 purified by reverse phase preparative HPLC to yield the title yield the title compound as a TFA salt (3.9 mg). MS m/z. compound as a TFA salt (3.5 mg). MS m/z. M+H" calc’d M+H" calc'd for CHCIFNO, 535.13: found 535.4. for CHCIFNO. 563.16; found 563.4. US 9,683,002 B2 107 108 Example 4Q: (R)-2-(2-Aminoacetoxy)-3-N-(5'- Example 4R. (R)-3-N-(5'-Chloro-2'-fluorobiphenyl chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3- 4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl) methoxyisoxazole-5-carbonyl)hydrazinopropionic hydrazino-2-hydroxypropionic Acid 1-Cyclohexy Acid loxycarbonyloxyethyl Ester

O \ y 10 O

O Y. HO N-NH O O

NH 15 HO N1

CH O C +

F 25 o1 O

BOC1 OH K. -- Nulls O 30 O O OH O 35 O Y. O O O Y NH O O HO N1 40 NH O O 1no N1 O C OH HN O 45 O C F

F N-O-(t-Butoxycarbonyl)glycine (11.3 mg, 64.7 umol) and 50 HATU (24.6 mg, 64.7 mol) were stirred in DMA (1.0 mL. To a solution of compound (R)-3-N-(5'-chloro-2'-fluoro 11 mmol) for 10 minutes. (R)-3-N-(5'-Chloro-2'-fluorobi biphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid phenyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl) ethyl ester (1.2 g 3.0 mmol), EDC (742 mg, 3.9 mmol), hydrazino-2-hydroxypropionic acid (20.0 mg, 43.1 umol) 55 HOBt (523 mg, 3.9 mmol) and 3-methoxyisoxazole-5-car and DIPEA (22.5 L, 129 umol) were added, and the boxylic acid (468 mg, 3.3 mmol) in DCM (20 mL) was resulting mixture was stirred at room temperature overnight added DIPEA (1.48 mL, 8.9 mmol) under nitrogen. The then concentrated. 4.0 M HCl in 1,4-dioxane (0.2 mL, 0.8 resulting mixture was stirred at room temperature overnight, mmol) was added and the resulting mixture was allowed to 60 then concentrated to dryness. The residue was dissolved in EtOAc (20 mL), washed with 0.5N aqueous HCl (10 mL), stand for 1 hour, then concentrated under reduced pressure. saturated aqueous NaHCO (10 mL) and saturated aqueous The residue was dissolved in AcOH (1.5 mL), filtered, and NaCl (10 mL), dried over anhydrous NaSO filtered, and purified by reverse phase preparative HPLC to yield the title 65 concentrated in vacuo. The residue was purified by column compound as a TFA salt (5.8 mg). MS m/z. M+H" calc’d chromatography (PE:EtOAc, 10:1-3:1) to yield Compound for CHCIFNO. 521.12; found 521.6. 1 as a solid (970 mg). LC-MS. M+H": 492. US 9,683,002 B2 109 110 -continued

O

(1) -- b-(b-( -e- C O 10 N M o, O ls NH O. O. or 15 OH O C HO N OH F To a solution of Compound 1 (970 mg, 2.0 mmol) in MeOH (15 mL) was added a solution of LiOH.HO (248 mg, 5.9 mmol) in water (3 mL). The mixture was stirred at 25 room temperature for 1 hour, and the insoluble solid was filtered off and the filtrate was concentrated in vacuo to yield a yellow solid (780 mg). LC-MS: 464 M+H". The yellow 5-Oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole-3-car solid (200 mg, 430 umol) was dissolved in 2.6-lutidine (460 boxylic acid (56.4 mg, 275umol) was combined with HCTU mg, 4.3 mmol) and carbonic acid 1-chloro-ethyl ester cyclo 30 (154 mg., 371 umol) in DMF (852 uL, 11 mmol) and stirred hexyl ester (888 mg, 4.3 mmol) was added. The vial was for 15 minutes at room temperature. DIPEA (72 uL., 413 sealed and the resulting mixture was then irradiated for 30 umol) and (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylm minutes at 90° C. under microwave irradiation. Water (10 ethyl)hydrazino-2-hydroxypropionic acid ethyl ester (50 mL) was added, and the mixture was extracted with EtOAc mg, 0.1 mmol) were added, and the resulting mixture was (3x10 mL). The combined organic layers were washed with stirred overnight at room temperature. EtOH (402 uL, 6.9 0.5N aqueous HCl (4x5 mL) and saturated aqueous NaCl 35 mmol) and 1 M LiOH in water (1.1 mL, 1.1 mmol) were (10 mL), dried over anhydrous NaSO, filtered and con added and the resulting mixture was stirred at room tem centrated in vacuo. The residue was purified by preparative perature for 1 hour. The mixture was evaporated under HPLC (Gemini-C18, 150x21.2 mm, 5u, MeCN HO reduced pressure and the residue was purified by preparative (0.1% TFA); from 43% to 43%) to yield the title compound HPLC to yield the title compound (39.8 mg, purity 100%). as a white solid (7 mg). LC-MS: 634 M-HI", "H NMR: 40 MS m/z. M+H" calc’d for CHCIFN-Os, 526.12; found (CDC1, 400 MHz) & 1.28-1.37 (m, 6H), 1.54 (d. 3H), 526.O. 1.70-1.73 (m, 2H), 1.84-1.87 (im, 2H), 3.26-3.29 (m, 2H), 3.38-3.43 (m. 1H), 3.97 (s, 3H), 4.19-4.20 (m, 2H), 4.38 Example 5B; (R)-3-N-(5'-Chloro-2'-fluorobiphenyl 4.50 (m, 1H), 4.50-4.57 (m, 1H), 6.50 (m. 1H), 6.75 (t, 1H), 4-ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1, 7.18-7.20 (m, 1H), 7.35-7.37 (m. 1H), 7.48-7.52 (m, 4H). 45 2.4 triazole-3-carbonyl)hydrazino-2-hydroxypropi onic Acid 5-methyl-2-oxo-1.3dioxol-4-ylmethyl Example 5A: (R)-3-N-(5'-Chloro-2'-fluorobiphe Ester nyl-4-ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro 1H-1,2,4-triazole-3-carbonyl)hydrazino-2-hy droxypropionic Acid 50 \ y O 55 HO N-NH HO N-NH

60

65 US 9,683,002 B2 111 112 -continued -continued

O YV N O HN N N1 Y N H 10 O O OD OH O OH t O O O - O 15 O O N

HO N-N N HO N-N NH O

25

EDC (127 mg, 660 umol) and HOBt (89 mg, 660 umol) were added to a solution of 5-oxo-1-phenyl-4,5-dihydro-1H 1.2.4 triazole-3-carboxylic acid (150 mg, 330 umol) and 30 5-Oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole-3-car (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)hy boxylic acid (89.5 mg, 436 umol) was combined with HCTU drazino-2-hydroxypropionic acid 5-methyl-2-oxo-1.3di (244 mg, 589 Lumol) in DMF (1.0 mL, 13 mmol) and stirred oxol-4-ylmethyl ester (68 mg, 330 Limol) in DMF (10 mL). for 10 minutes. (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl DIPEA (86 mg, 660 umol) was added, and the resulting methyl)hydrazino-2-hydroxypropionic acid ethyl ester mixture was stirred for 5 hours at room temperature. The 35 (80.0 mg, 0.2 mmol) and DIPEA (0.1 mL, 0.7 mmol) were mixture was then washed with saturated aqueous NaCl added, and the resulting mixture was stirred overnight. The (2x30 mL) and extracted with EtOAc (2x50 mL). The mixture was diluted with EtOAc (10.0 mL) and washed with combined organic layers were dried over anhydrous Na2SO water (3.0 mL), saturated aqueous NaHCO (2x30 mL), and and concentrated in vacuo. The residue was purified by saturated aqueous NaCl (3.0 mL), then dried over NaSO, column chromatography (PE:EtOAc=1:1) to yield the title 40 filtered and concentrated to give a yellowish solid. A portion compound as a white solid (67 mg). LC-MS: 638.2 M+H". (20 mg) of the solid was dissolved in AcOH (1.5 mL), 'H-NMR (400 MHz, DMSO-d): 8 2.13 (s, 3H), 3.31-3.16 filtered and purified by reverse phase preparative HPLC to (m. 2H), 4.18-4.21 (q, 2H), 4.35 (br. 1H), 4.98-5.01 (m, 2H), yield the title compound (1.6 mg, purity 100%). MS m/z. 5.54 (br. 1H), 7.26-7.90 (m, 12H), 9.98 (s, 1H), 12.74 (s, M+H" calc’d for CHCIFNOs 554.15; found 554.4. 1H). 45 Example 5D: (R)-3-N-(5'-Chloro-2'-fluorobiphe Example 5C: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl nyl-4-ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro 1H-1.2.4 triazole-3-carbonyl)hydrazino-2-hy 2.4 triazole-3-carbonyl)-hydrazino-2-hydroxypropi droxypropionic Acid Isobutyl Ester onic Acid Ethyl Ester 50 \ y O 55

HO N-NH HO N-NH --

60

65 US 9,683,002 B2 113 114 -continued -continued F F Her F 5 - HO He F F OH F N Y F ) -H F O O 10 F F O O N OH ( NN HO N N H-N O

N-N Y-, t \->N O

HO

25 A mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl methyl)-N-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole 3-carbonyl)hydrazino-2-hydroxypropionic acid (200 mg. 380 umol), 2.2.3,3,3-pentafluoropropan-1-ol (114 mg, 760 5-Oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole-3-car 30 umol), HOBT (103 mg, 760 umol), EDC (145 mg, 760 boxylic acid (89.5 mg, 436 umol) was combined with HCTU umol) and DIPEA (200 mg, 1.5 mmol) in DMF (5.0 mL) was (244 mg, 589 umol) in DMF (1.0 mL, 13 mmol) and stirred stirred at room temperature overnight. The mixture was then for 10 minutes. (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl poured into water (30 mL) and extracted with EtOAc (3x20 methyl)hydrazino-2-hydroxypropionic acid ethyl ester mL). The combined organic layers were washed with Satu (80.0 mg, 0.2 mmol) and DIPEA (0.1 mL, 0.7 mmol) were added, and the resulting mixture was stirred overnight. The 35 rated aqueous NaCl (30 mL), dried over anhydrous NaSO, mixture was diluted with EtOAc (10.0 mL) and washed with and concentrated in vacuo. The residue was purified by water (3.0 mL), saturated aqueous NaHCO, (2x3.0 mL), and column chromatography (PE:EtOAc=2:1) to yield the title saturated aqueous NaCl (3.0 mL), then dried over NaSO, compound as a white solid (20 mg). LC-MS: 658 M+H". filtered and concentrated to give a yellowish solid. A portion "H NMR (400 MHz, CDC1,) & 3.44-3.40 (m, 2H), 4.25 (br. (20 mg) of the solid was treated with isobutyl alcohol (170 40 2H), 4.58-4.61 (m, 3H), 7.14-7.08 (m, 1H), 7.41-7.55 (m, uL, 1.8 mmol) and 4.0 M HCl in 1,4-dioxane (36.0 uL, 144 umol) at room temperature overnight. The mixture was 8H), 7.84-7.92 (m, 3H). concentrated, and the residue was dissolved in AcOH (1.5 mL), filtered, and purified by reverse phase preparative Example 5F: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl HPLC to yield the title compound (1.6 mg, purity 100%). MS m/z. M+H" calc’d for CHCIFN-Os, 582.18; found 45 2.4 triazole-3-carbonyl)hydrazino-2-hydroxypropi 582.4. onic Acid Acetoxymethyl Ester Example 5E: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl 4-ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1, 2.4 triazole-3-carbonyl)hydrazino-2-hydroxypropi onic Acid 2,2,3,3,3-Pentafluoropropyl Ester 50

O

HO y & N 55 HO N-N N 1s, --

60 OH

65

US 9,683,002 B2 117 118 -continued -continued O 1n --~. Hs -( –0 O 5 O O O -\ O O N-- o- O N O O N )-( -l HO N-N N / N 10 H H O HO N-N) {H O

15

To a solution of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- To a solution of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triaz ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triaz ole-3-carbonyl)hydrazino-2-hydroxypropionic acid (200 ole-3-carbonyl)hydrazino-2-hydroxypropionic acid (200 mg, 335umol) in dry DMF (6 mL) was added chloromethyl mg, 335 mol) in dry DMF (10 mL) was added chlorom 25 isopropyl carbonate (76 mg, 503 umol), NaI (101 mg, 670 ethyl ethyl carbonate (69 mg, 503 umol), NaI (101 mg. 670 umol) and 2,6-dimethylpyridine (143 mg, 1.3 mmol) in umol) and 2,6-dimethylpyridine (143 mg, 1.3 mmol) in portions at room temperature. The resulting mixture was portions at room temperature. The resulting mixture was stirred at room temperature for 8 hours. Water (15 mL) was stirred at room temperature for 8 hours. Water (12 mL) was added and the mixture was extracted with EtOAc (3x20 30 added and the mixture was extracted with EtOAc (3x10 mL). The combined organic layers were dried over anhy mL). The combined organic layers were dried over anhy drous NaSO and concentrated in vacuo. The residue was drous Na2SO and concentrated in vacuo. The residue was purified by column chromatography (PE:EtOAc=3: 1-1: purified by column chromatography ((PE:EtOAc=1:1) to 100) to yield the title compound as a white solid (9.5 mg). yield the title compound as a white solid (12.8 mg). LC-MS: LC-MS. 627.9M+H". "H NMR: (CDC1,400 MHz) & 1.25 35 641.9 M+H". "H NMR (DMSO-de, 400 MHz) & 1.21 (d. (t, J–7.1 Hz, 3H), 3.44 (br, 2H), 4.17-4.35 (m, 4H), 4.50 (s, J=6.2 Hz, 6H), 3.21-3.28 (m, 2H), 4.12-4.30 (m, 2H), 1H), 5.75-5.78 (m, 2H), 7.10 (t, J=9.3 Hz, 1H), 7.28-7.40 4.40-4.52 (m. 1H), 4.77-4.79 (m, 1H), 5.68-5.76 (m, 2H), (m. 1H), 7.42-7.52 (m, 7H), 7.87 (s. 2H), 8.37 (s, 1H). 741-7.45 (m, 1H), 7.50-7.87 (n, 9H), 7.90 (d. J–7.7 Hz, Example 5I: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl 2H), 9.98 (s, 1H), 12.76 (s, 1H). 40 2.4 triazole-3-carbonyl)hydrazino-2-hydroxypropi Example 5.J: (S)-2-Methoxycarbonylamino-3-meth onic Acid Isopropoxycarbonyloxymethyl Ester ylbutyric Acid (R)-3-N-(5'-chloro-2'-fluorobiphe nyl-4-ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro 1H-1.2.4 triazole-3-carbonyl)hydrazino-2- 45 hydroxypropionyloxymethyl Ester

50

55 HO N

OH

HO N 60 OH

65 US 9,683,002 B2 119 120 -continued Using the procedures described herein, the title compound O can also be prepared. 1. O Nulls o1 Ne. He Example 6A: (R)-3-N'-(5-Acetyl-2H-pyrazole-3- O carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm - O ethyl)hydrazino-2-hydroxypropionic acid X-H O O \ { 10 - 0-y

HO N-NH n-soH 15 N-NH2 HO

To a solution of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triaz 25 ole-3-carbonyl)hydrazino-2-hydroxypropionic acid (200 mg, 335 umol) in dry DMF (6 mL) was added (S)-2- methoxycarbonylamino-3-methylbutyric acid chloromethyl ester (112 mg, 503 umol), NaI (101 mg, 670 umol). 2.6- dimethylpyridine (143 mg, 1.3 mmol) in portions at room 30 temperature. The resulting mixture was stirred at room temperature for 8 hours. Water (12 mL) was added and the mixture was extracted with EtOAc (3x20 mL). The com bined organic layers were dried over anhydrous Na2SO and concentrated in vacuo. The residue was purified by column 35 chromatography (PE:EtOAc=3:1) to yield the title com pound as a white solid (8.4 mg). LC-MS: 712.9 M+H". "H NMR: (MeOD, 400 MHz) & 0.85 (dd, J=14.1, 6.8 Hz, 6H), 2.01-2.17 (m, 1H), 3.42-3.51 (m, 2H), 3.69 (s.3H), 4.00 (d. J=5.9 Hz, 1H), 4.23 (br, 2H), 4.42 (t, J=4.7 Hz, 1H), 5.83 HO N (dd, J=31.5, 5.6 Hz, 2H), 7.19 (t, J=9.4 Hz, 1H), 7.32-7.39 40 (m, 3H), 747-7.51 (m, 4H), 7.56-7.58 (m, 2H), 7.91 (d. OH J=8.0 Hz, 2H). Example 5K: (R)-3-N-(5'-Chloro-2'-fluorobiphe nyl-4-ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro 45 1H-1,2,4-triazole-3-carbonyl)hydrazino-2-hy droxypropionic Acid 1-cyclohexyloxycarbonyloxyethyl Ester 3-Acetyl-1H-pyrazole-5-carboxylic acid (69.3 mg, 450 50 umol) and HCTU (186 mg. 450 umol) were combined in DMF (1.9 mL. 24.5 mmol), and stirred at room temperature. After 15 minutes, DIPEA (214 uL. 1.2 mmol) and (R)-3- N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- 55 O hydroxypropionic acid ethyl ester (150 mg, 0.4 mmol) were *-O : \-3 added. The mixture was stirred for 30 minutes at room - N-NY--> temperature, then evaporated under reduced pressure. The HO 60 residue was dissolved in EtOH (1.4 mL. 24.5 mmol). A solution of 1.0 M LiOH in water (2.0 mL, 2.0 mmol) was added, and the resulting mixture was stirred at 40° C. for 3 hours. The solvent was removed under pressure and the C 65 residue was purified by preparative HPLC to yield the title F compound (110 mg, purity 100%) as a TFA salt. MS m/z. M+H" calc’d for CHCIFNOs, 475.11; found 475.1. US 9,683,002 B2 121 122 Example 6B: (R)-3-N-(5-Acetyl-2H-pyrazole-3- -continued carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm OH ethyl)hydrazino-2-hydroxypropionic Acid Ethyl Ester

N -e- O -N N -NH2 O HO 10 -- -

O 15 N--~~~ C OH

F

N HN1/ N O (R)-3-N-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- -e- chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hy O droxy-propionic acid (41.0 mg, 86 umol), HOBt (70.0 mg. OH 25 518 umol) and EDC (92 uL. 520 umol) were combined in HN-N DCM (0.7 mL, 10 mmol). The resulting solution was stirred for 10 minutes. 4-Morpholineethanol (84 uL 691 umol) was O \ added, and the mixture was stirred at room temperature until the reaction was complete (s2 hours). The mixture was O s' - (O NH concentrated by rotary evaporation and purified (reverse 1n O N 1. 30 phase column) to yield the title compound (7.5 mg, purity 98%) as a TFA salt. MS m/z. M+H" calc’d for OH CHCIFNO. 588.19; found 588.1. Example 6D: (R)-3-N-(5-Acetyl-2H-pyrazole-3- carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm ethyl)hydrazino-2-hydroxypropionic Acid Isobutyl Ester coF C HN-N 3-Acetyl-1H-pyrazole-5-carboxylic acid (168 mg, 1.1 O \ mmol) and HCTU (451 mg, 1.1 mmol) were combined in 40 O S DMF (6.8 mL, 87.2 mmol), and stirred at room temperature. After 15 minutes, DIPEA (570 uL, 3.3 mmol) and (R)-3- NH O N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- HO N -- hydroxypropionic acid ethyl ester (400 mg, 1.1 mmol) were added. The mixture was stirred for 20 minutes at room OH temperature, then evaporated under reduced pressure to 45 yield the title compound. MS m/z. M+H calc’d for CHCIFNO. 503.14; found 503.2. O C Example 6C: (R)-3-N-(5-Acetyl-2H-pyrazole-3- carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm 50 F ethyl)hydrazino-2-hydroxypropionic Acid 2-Mor OH pholin-4-ylethyl Ester 1. HN-N HIN 55 O N \ \ O ---,O Y NH HO N 60 OH Sr. OH N O C

65 F US 9,683,002 B2 123 124 (R)-3-N-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- Example 6F: (R)-3-N'-(5-Acetyl-2H-pyrazole-3- chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hy carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm droxypropionic acid (15.0 mg, 32 umol) was combined with ethyl)hydrazino-2-hydroxypropionic Acid 2,2-Dif isobutyl alcohol (876 uL. 9.5 mmol). A solution of 4 M HCl luoropropyl Ester in dioxane (282 uL. 1.1 mmol) was added, and the resulting mixture was stirred for 15 minutes at room temperature. The mixture was concentrated by rotary evaporation and the O - product lyophilized to yield the title compound (19 mg, purity 99%) as a white powder TFA salt. MS m/z. M+H" 10 S--(O calc’d for CHCIFNOs, 531.17; found 531.1. HO N NH -- OH Example 6E: (R)-3-N-(5-Acetyl-2H-pyrazole-3- 15 carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm C ethyl)hydrazino-2-hydroxypropionic Acid 5-Methyl-2-oxo-1.3dioxol-4-ylmethyl Ester XnotF HN-N F F He HN -N O O N O 25 NH O O HO N -- NH---, N1 OH Xno F F OH

C 30

F O F 35 co O (R)-3-N-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- Hoss chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hy O 21 droxypropionic acid (15.0 mg, 32 umol), HOBt (12.8 mg, 95 OH umol) and EDC (16.8 uL. 95 umol) were combined in DCM HN-N 40 (121 uL. 1.9 mmol) and stirred for 10 minutes. 2,2-Difluo ropropanol (24.3 mg, 253 umol) was added and the resulting mixture was stirred at room temperature until the reaction was complete (s48 hours). The mixture was evaporated under reduced pressure and the product was purified (reverse 45 phase column) to yield the title compound (13.8 mg, purity 96%) as a TFA salt. MS m/z. M+H" calc’d for CHCIFNOs, 553.14; found 553.1. Example 6G: (R)-3-N-(5-Acetyl-2H-pyrazole-3- carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm 50 ethyl)hydrazino-2-hydroxypropionic Acid 2-Methoxyethyl Ester (R)-3-N-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- HN-N chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hy 55 droxypropionic acid (30.0 mg, 63 umol), HOBt (26 mg, 190 O N \ umol) and EDC (34 uL, 190 umol) were combined in DCM (243 uL, 3.8 mmol) and stirred for 10 minutes. 4-Hydroxym ethyl-5-methyl-1,3dioxol-2-one (66 mg, 0.5 mmol) and HO N 60 4-methylmorpholine (28 uL. 250 umol) were added and the OH resulting mixture was stirred at room temperature for 3 hours. The mixture was evaporated under reduced pressure, yielding a brown oil, which was purified by preparative HPLC to yield the title compound (4.6 mg, purity 97%) as 65 a TFA salt. MS m/z. M+H calc’d for C.H.ClFNOs, 587.13: found 587.1. US 9,683,002 B2 125 126 O -continued -continued

1. N1,No. -es O HO O HN-N

O \ *NH, O N HO N 1N1\oO N1 NH O 10 OH O C 15

F

(R)-3-N-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hy (R)-3-N-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- droxypropionic acid (10.0 mg, 21 umol) was combined with chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hy 2-methoxyethanol (0.1 mL, 1.3 mmol). A solution of 4 M droxypropionic acid (95.5 mg, 0.2 mmol) was dissolved in HCl in dioxane (53 uL, 0.2 mmol) was added, and the 25 acetone (886 uL, 12.1 mmol). EtN (70 uL, 503 umol) and resulting mixture was stirred for 1 hour at room temperature. (S)-2-t-butoxycarbonylamino-3-methyl-butyric acid chlo LC/MS showed the mass of the desired product. The mixture romethyl ester (56.1 mg, 211 umol) were added, and the resulting mixture was stirred at 60° C. for 6 hours. The was concentrated by rotary evaporation and purified (reverse Solvent was removed in vacuo and the residue was purified phase column) to yield the title compound (2.7 mg, purity 30 using flashy chromatography (normal phase; MeOH: 95%) as a white solid TFA salt. MS m/z. M+H" calc'd for EtOAc=0:50). The pure fractions were collected, concen CHCIFNO. 533.15; found 533.1. trated, then dissolved in Mecn (630 uL, 12.1 mmol). A solution of 4.0 M HCl in 1,4-dioxane (503 LL, 2.0 mmol) Example 6H: (S)-2-Amino-3-methylbutyric Acid 35 was added, and the resulting mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo 3-Acetyl-5-N'-((R)-2-carboxy-2-hydroxyethyl)- to yield the title compound (90 mg). N'-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)hy

drazinocarbonylpyrazol-1-ylmethyl Ester 40 Example 6I: (S)-2-Methoxycarbonylamino-3-meth ylbutyric acid 3-acetyl-5-N-((R)-2-carboxy-2- hydroxyethyl)-N'-(5'-chloro-2'-fluorobiphenyl-4- ylmethyl)hydrazinocarbonylpyrazol-1-ylmethyl Ester 45

HO N 50 O O % HO NH2 O

N 55 HO N YN -- s N \ i?

60

65 C US 9,683,002 B2 127 128 -continued Isobutyryl chloride (23.4 uL. 221.1 umol) and (R)-3-N'- (5-acetyl-2H-pyrazole-3-carbonyl)-N-(5'-chloro-2'-fluoro O biphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid O % (10.5 mg, 22.1 umol) were combined in THF (108 uL. 1.3 HO NH O mmol), and stirred overnight at room temperature. The a ?o solvent was evaporated and the residue was purified by HO N N /O preparative HPLC to yield the title compound (4.9 mg) as a 10 TFA salt. MS m/z. M+H" calc'd for CHCIFN.O. N- H W 545.15; found 545.1.

Example 6K: 3-Methylbutyric Acid (R)-2-N'-(5- O O 15 acetyl-2H-pyrazole-3-carbonyl)-N-(5'-chloro-2'- fluorobiphenyl-4-ylmethyl)hydrazino-1-carboxy F ethyl Ester

C (S)-2-Amino-3-methylbutyric acid 3-acetyl-5-N'-((R)- 2-carboxy-2-hydroxyethyl)-N'-(5'-chloro-2'-fluorobiphenyl 4-ylmethyl)hydrazinocarbonylpyrazol-1-ylmethyl ester (21.0 mg, 35 umol) was combined with DCM (134 uL., 2.1 mmol) and Et-N (15 uL, 0.1 mmol). Methyl chloroformate 25 (2.7 uL. 35umol) was added and the mixture was stirred at HO room temperature for 20 minutes. The solvent was removed in vacuo and the residue was purified by preparative HPLC to yield the title compound (0.7 mg). MS m/z. M+H" calc’d for CHCIFNO, 662.20; found 662.1. 30 Example 6.J: Isobutyric Acid (R)-2-N-(5-Acetyl 2H-pyrazole-3-carbonyl)-N-(5'-chloro-2'-fluorobi phenyl-4-ylmethyl)hydrazino-1-carboxyethyl Ester 35

40

HO O 45 r N1 NH 50

55 co Isovaleryl chloride (51.4 uL, 421.2 mol) and (R)-3-N'- (5-acetyl-2H-pyrazole-3-carbonyl)-N-(5'-chloro-2'-fluoro biphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid 60 (20.0 mg, 42.1 umol) were combined in THF (205 uL., 2.5 mmol), and stirred overnight at room temperature. The solvent was evaporated and the residue was purified (reverse

65 phase HPLC column) to yield the title compound (11.8 mg) as a TFA salt. MS m/z. M+H calc’d for C.H.ClFNO. 559.17; found 559.1. US 9,683,002 B2 129 130 Example 6L: (R)-2-Acetoxy-3-N'-(5-acetyl-2H -continued pyrazole-3-carbonyl)-N-(5'-chloro-2'-fluorobiphe nyl-4-ylmethyl)hydrazinopropionic Acid OH

1N -N 10 HNW O S. HO N --

15

(R)-3-N-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2-hy 25 droxypropionic acid (10.0 mg, 21 umol) was combined with HOBt (17.1 mg, 126 umol) and EDC (22 uL, 130 umol) in DCM (0.2 mL, 3 mmol) and stirred for 10 minutes. 3-Di methylamino-1-propanol (19.9 uL. 168 umol) was added and the resulting mixture was stirred at room temperature 30 and monitored for completion (s4 hours). The mixture was concentrated by rotary evaporation and the residue was purified by preparative HPLC to yield the title compound as a TFA salt (6.4 mg). MS m/z. M+H" calc’d for 35 CHCIFNOs 560.20; found 560.1. Example 6.N: (R)-3-N-(5-Acetyl-2H-pyrazole-3- Acetyl chloride (30 ul, 421.2 umol) and (R)-3-N'-(5- acetyl-2H-pyrazole-3-carbonyl)-N-(5'-chloro-2'-fluorobi carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm ethyl)-hydrazino-2-hydroxypropionic Acid 4-dim phenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid 40 (20.0 mg, 42.1 umol) were combined in THF (205 uL., 2.5 ethylaminobutyl Ester mmol), and stirred overnight at room temperature. The solvent was evaporated and the residue was purified (reverse phase HPLC column) to yield the title compound (12.2 mg) as a TFA salt. MS m/z. M+H" calc'd for CHCIFNO N 517.12; found 517.2. 45 HN1 N Example 6M; (R)-3-N-(5-Acetyl-2H-pyrazole-3- O S. carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm O O ethyl)-hydrazino-2-hydroxypropionic acid 3-dim NH ethylaminopropyl Ester 50 HO N1 --

OH

N HN1 N O C 55 O S.

F OH HO N --

OH 60

65 N r 1N US 9,683,002 B2 131 132 -continued N (R)-3-N-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2-hy HN1 v droxypropionic acid (10.0 mg, 21 umol) was combined with O S. O O HOBt (17.1 mg, 126 umol) and EDC (22 uL, 130 umol) in DCM (0.2 mL, 3 mmol) and stirred for 10 minutes. 3-Mor pholinopropanol (24.5 mg, 168 umol) was added and the --~~~~ resulting mixture was stirred at room temperature and moni OH tored for completion (s4 hours). The mixture was concen trated by rotary evaporation and the residue was purified by preparative HPLC to yield the title compound as a TFA salt (4.9 mg). MS m/z. M+H" calc'd for CHCIFN.O. 602.21; found 602.1.

(R)-3-N-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- 15 Example 6P: (R)-3-N'-(5-Acetyl-2H-pyrazole-3- chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2-hy carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm droxypropionic acid (10.0 mg, 21 umol) was combined with ethyl)-hydrazino-2-hydroxypropionic Acid 2-Dim HOBt (17.1 mg, 126 umol) and EDC (22 uL, 130 umol) in ethylaminoethyl Ester DCM (0.2 mL, 3 mmol) and stirred for 10 minutes. 4-Di methylamino-1-butanol (22.4 uL. 168 umol) was added and the resulting mixture was stirred at room temperature and monitored for completion (s4 hours). The mixture was concentrated by rotary evaporation and the residue was purified by preparative HPLC to yield the title compound as 25 a TFA salt (4.2 mg). MS m/z. M+H" calc’d for CHCIFNO. 574.22; found 574.1. HO N -- Example 6O: (R)-3-N-(5-Acetyl-2H-pyrazole-3- carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm OH ethyl)-hydrazino-2-hydroxypropionic Acid 3-Mor 30 pholin-4-yl-propyl Ester

35

40 HO N --

45

50

55 The title compound can be prepared as follows: (R)-3- N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'-chloro-2'- fluorobiphenyl-4-ylmethyl)-hydrazino-2-hydroxypropionic 60 acid (10.0 mg, 21 umol) is combined with HOBt (17.1 mg, 126 umol) and EDC (22 u, 130 umol) in DCM (0.2 mL, 3 mmol) and stirred for 10 minutes. N,N-Dimethylaminoetha nol (16.9 uL, 168 umol) is added and the resulting mixture is stirred at room temperature and monitored for completion. 65 The mixture is concentrated by rotary evaporation and the residue is purified by preparative HPLC to yield the title compound as a TFA salt. US 9,683,002 B2 133 134 Example 6O: (R)-3-N'-(5-Acetyl-2H-pyrazole-3- Example 6S: (S)-2-Amino-3-methylbutyric Acid carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm 3-Acetyl-5-N'-biphenyl-4-ylmethyl-N'-((R)-2- ethyl)-hydrazino-2-hydroxypropionic Acid 4-Mor hydroxy-2-isobutoxycarbonylethyl)hydrazinocarbo pholin-4-yl-yutyl Ester nylpyrazol-1-ylmethyl Ester HN O \ \ly 10 O S. O O

HO N -- O N OH 15 r -->OH NH

Using the procedures described herein, the title compound can also be prepared. Example 6T: (S)-2-Amino-3-methylbutyric Acid / He- 25 (R)-2-N-(5-Acetyl-2H-pyrazole-3-carbonyl)-N- biphenyl-4-ylmethylhydrazino-1-ethoxycarbonyl ethyl Ester O N HN1 N R 30 N O S. O S. O O ?r. NH O O CN-N--> NH OH 35 ~~~ C F C 40 HN y O Using the procedures described herein, the title compound can also be prepared. Using the procedures described herein, the title compound can also be prepared. Example 6R: (S)-2-Amino-3-methylbutyric Acid Example 6U: (S)-2-Amino-3-methylbutyric Acid (R)-2-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N- 45 3-Acetyl-5-N-biphenyl-4-ylmethyl-N'-((R)-2- biphenyl-4-ylmethylhydrazino-1-isobutoxycarbony ethoxycarbonyl-2-hydroxyethyl)hydrazinocarbonyl lethyl Ester pyrazol-1-ylmethyl Ester

50 N HN e N O S. O O 55 NH O N1

O O

DC''', Clus -

65 Using the procedures described herein, the title compound can also be prepared. US 9,683,002 B2 135 136 Using the procedures described herein, the title compound Using the procedures described herein, the title compound can also be prepared. can also be prepared. Example 6V: (S)-2-Amino-3-methylbutyric acid (R)-3-N-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- Example 7A: (R)-3-N-(5'-Chloro-2'-fluorobiphe chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2- nyl-4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl)hy hydroxypropionyloxymethyl Ester drazino-2-hydroxypropionic Acid 5-methyl-2-oxo 1.3dioxol-4-ylmethyl Ester

N HN N 10 O Tr O O \ HNulls NH N r O M OH 15 sNH O C HO N1

F

Using the procedures described herein, the title compound C can also be prepared. Example 6W: (R)-3-N'-(5-Acetyl-2H-pyrazole-3- OH co carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm F O ethyl)hydrazino-2-hydroxypropionic Acid Ethoxy 25 carbonyloxymethyl Ester s C O -e-

30 y O S. O O O O H N ls NH N e M N 1No ^-r M OH 35 O s N O C s O N NH O OH F 40 y Using the procedures described herein, the title compound O can also be prepared. O C Example 6X: (R)-3-N'-(5-Acetyl-2-phospho nooxymethyl-2H-pyrazole-3-carbonyl)-N-(5'-chloro 2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2-hy 45 F droxypropionic Acid Isobutyl Ester (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- (2-trityl-2H-tetrazole-5-carbonyl)hydrazino-2-hydroxypro 50 pionic acid (30.0 mg. 44 umol) was combined with 4-chlo romethyl-5-methyl-1,3-dioxol-2-one (9.9 mg, 66 umol) and DIPEA (15.4 uL. 89 umol) in acetone (0.4 mL, 5 mmol). The mixture was maintained at 56° C. overnight. The mixture was concentrated, and the residue was combined with DCM 55 (0.2 mL) and 2M HCl in a mixture of dioxane and DCM (0.2 mL) at room temperature for 1 hour. The mixture was concentrated, and the residue was dissolved in 50% water/ AcOH (1.5 mL), filtered, and purified by reverse phase preparative HPLC to yield the title compound as a white 60 solid TFA salt (3.3 mg). MS m/z. M+H" calc'd for CHCIFNO. 547.11: found 547. Note that as explained herein, compounds such as this can exist in a tautomer form, for example, as (R)-3-N-(5'- 65 chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(1H-tetrazole-5- carbonyl)hydrazino-2-hydroxypropionic acid 5-methyl-2- oxo-1.3dioxol-4-ylmethyl ester. US 9,683,002 B2 137 138 Example 7B: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl Example 7C: (S)-2-Amino-3-methylbutyric Acid 4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl)hydrazino 5-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- 2-hydroxypropionic Acid 2,2,3,3,3-Pentafluoropro ((R)-2-ethoxycarbonyl-2-hydroxyethyl)-hy pyl Ester drazinocarbonyltetrazol-2-ylmethyl Ester

Tr Tr \ \ 10 N1 \ N N O M O M O N O N NH NH HO N1 -- HO N1 -- 15 OH OH O C C

F F O F F

H -as 25 c1 No Y BOC

F F H N NH2 N e Y M. 30 - o, N F F O Y, G NH O M F --> N 1 O N 35 1No N1 NH O C OH

40 C F

(R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- F (2-trityl-2H-tetrazole-5-carbonyl)hydrazino-2-hydroxypro pionic acid (42.4 mg, 63 umol) was combined with EDC 45 (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- (66.5 uL, 376 umol) and HOBt hydrate (57.5 mg, 376 umol) (2-trityl-2H-tetrazole-5-carbonyl)hydrazino-2-hydroxypro in DCM (0.7 mL, 10 mmol), and stirred at room temperature pionic acid ethyl ester (15.1 mg, 21.4 Lmol) was stirred in for 10 minutes. 2.2.3,3,3-Pentafluoro-1-propanol (75.2 mg, a mixture of DCM (0.2 mL, 3 mmol) and 4.0 M HCl in 501 umol) was added and the resulting mixture was stirred 50 1,4-dioxane (0.1 mL, 0.4 mmol) at room temperature for 1 at room temperature overnight. The mixture was concen hour, then concentrated. To this was added CsCO, (14.0 mg, 42.8 Lmol) in acetone (0.5 mL) and a mixture of trated and the residue was dissolved in DCM (0.4 mL, 6 (S)-2-t-butoxycarbonylamino-3-methylbutyric acid chlo mmol) at room temperature and treated with 4.0 M HCl in romethyl ester (17.1 mg, 64.2 Lumol) and NaI (9.6 mg, 64.2 1,4-dioxane (0.2 mL, 0.8 mmol) for 2 hours. The mixture 55 umol) that had been stirred in acetone (0.5 LL, 7 umol) at 60° was concentrated, and the residue was dissolved in AcOH C. for 1 hour. The resulting mixture was stirred at 60° C. for (1.5 mL), filtered, and purified by reverse phase preparative 4 hours, then concentrated. TFA (0.1 mL, 1 mmol) and DCM HPLC. The desired fractions were combined and freeze (0.1 mL, 2 mmol) were added to the residue and stirred at dried to yield the title compound as a white solid TFA salt room temperature for 1 hour. The mixture was then concen 60 trated, and the residue was dissolved in AcOH (1.5 mL), (8.3 mg). MS m/z. M+H" calc’d for C.H.CIFNO filtered, and purified by reverse phase preparative HPLC to 567.09: found 567. yield the title compound as a white solid TFA salt (4.3 mg). Note that as explained herein, compounds such as this can MS m/z. M+H" calc’d for CHCIFN.O. 592.20; found exist in a tautomer form, for example, as (R)-3-N-(5'- 5924. chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(1H-tetrazole-5- 65 In addition, it was found that the regioisomer of the title carbonyl)hydrazino-2-hydroxypropionic acid 2.2.3,3,3- compound was also produced, (S)-2-amino-3-methylbutyric pentafluoropropyl ester acid 5-N'-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- US 9,683,002 B2 139 140 ((R)-2-ethoxycarbonyl-2-hydroxyethyl)hydrazinocarbonyl was stirred at 60° C. for 1 hour, then it was added to a tetrazol-1-ylmethyl ester, as a white solid TFA salt (2.7 mg): mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylm ethyl)-N'-(2-trityl-2H-tetrazole-5-carbonyl)hydrazino-2- hydroxypropionic acid (12.3 mg, 18.2 umol) and DIPEA HN (4.8 uL. 27.2 mol) in acetone (0.5 mL). The resulting mixture was stirred at 40° C. for 2 hours, concentrated and

O partitioned between EtOAc (10 mL) and water (2 mL). The o-n -N organic layer was dried over MgSO, filtered, and concen N1 W 10 trated. 4.0 M HCl in 1,4-dioxane (40.0 uL. 160 umol) in O S. N MeCN (0.2 mL, 4 mmol) was added and the resulting O N mixture was stirred at room temperature for 20 minutes. The NH mixture was concentrated and the residue was dissolved in 1No N1 15 AcOH (1.5 mL), filtered, and purified by reverse phase OH preparative HPLC to yield the title compound (1.3 mg). MS m/z. M+H" calc’d for CHCIFNO. 535.14; found 535.1. Note that as explained herein, compounds such as this can exist in a tautomer form, for example, as butyric acid (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(1H tetrazole-5-carbonyl)hydrazino-2-hydroxypropionyloxym Both regioisomers were isolated and characterized by ethyl ester. NMR, HPLC, and LCMS. 25 Example 7E: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl Example 7D: Butyric Acid (R)-3-N-(5'-chloro-2'- fluorobiphenyl-4-ylmethyl)-N'-(2H-tetrazole-5-car 4-ylmethyl)-N'-(1H-tetrazole-5-carbonyl)hydrazino 2-hydroxypropionic Acid Acetoxymethyl Ester bonyl)hydrazino-2-hydroxypropionyloxymethyl 30 Ester

Tr 35 LiO

N1 \v. N O A. O N 40 NH HO N1 --

OH O C 45

F O 50 1N -- N 2N O S. NH O O N 55 ~~ --> N1 NH OH 60

65 A mixture of chloromethylbutyrate (6.8 uL, 54.5 umol) and NaI (8.2 mg, 54.5 umol) in acetone (0.5 mL, 7 mmol) US 9,683,002 B2 141 142 -continued Using the procedures described herein, the title compound can also be prepared. Note that as explained herein, compounds such as this can exist in a tautomer form, for example, as (S)-2-amino-3- methylbutyric acid (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- ylmethyl)-N'-(2H-tetrazole-5-carbonyl)hydrazino-2-hy

droxypropionyloxymethyl ester. Example 7G: (R)-3-N-(5'-Chloro-2'-fluorobiphe HO nyl-4-ylmethyl)-N'-(1H-tetrazole-5-carbonyl)hy 10 drazino-2-hydroxypropionic Acid Isopropoxycarbo nyloxymethyl Ester

15

LiO

To a solution of lithium (R)-3-(2-(1-allyl-1H-tetrazole-5- carbonyl)-1-((5'-chloro-2'-fluorobiphenyl-4-yl)methyl)hy drazinyl)-2-hydroxypropanoate (300 mg. 624 umol) in DMF (3 mL) was added bromomethyl acetate (144 mg. 936 mmol), NaI (140 mg, 936 umol) and 2.6-lutidine (134 mg. 1.2 mmol) dropwise at 0°C. under nitrogen. The resulting mixture was stirred for 3.5 hours, then poured into water (30 25 mL). The resulting solution was extracted with EtOAc (2x15 mL). The combined organic layers were washed with Satu rated aqueous NaCl (15 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by preparative TLC (PE:EtOAc-1:2) to yield Compound 1 as a yellow oil (170 mg). LC-MS. 547 M+H". To a solution of Compound 1 (80 mg, 146 umol) in dry DCM (3 mL) was added Pd(PPh.) (50 mg, 43.9 umol), triethylsilane (51 mg, 439 umol) and AcOH (26 mg, 439 umol). The resulting mixture was stirred at room tempera ture under nitrogen for 30 hours. The solids were filtered off 35 and the filtrate was concentrated in vacuo. The residue was purified by preparative HPLC Gemini-C18, 150x21.2 mm, 5u: MeCN HO (0.1% TFA) from 43% to 43% to yield the title compound as a white solid (10 mg). LC-MS. 507 M+H", "H NMR (CDC1): 8 1.27 (s, 3H), 3.41-3.53 (m, 2H), 4.21-4.24 (dd, 2H), 4.51-4.53 (t, 1H), 5.68-5.75 (dd. 40 2H), 7.05-7.09 (t, 1H), 7.25-7.26 (m, 1H), 7.36-7.38 (dd, 1H), 7.48 (s, 4H), 8.84 (s, 1H). Note that as explained herein, compounds such as this can exist in a tautomer form, for example, as (R)-3-N-(5'- chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(2H-tetrazole-5- carbonyl)hydrazino-2-hydroxypropionic acid acetoxym 45 ethyl ester. Example 7F: (S)-2-Amino-3-methylbutyric Acid (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-(1H-tetrazole-5-carbonyl)hydrazino-2-hydroxy 50 propionyloxymethyl Ester

55 HO

60

C

65 To a solution of lithium (R)-3-(2-(1-allyl-1H-tetrazole-5- carbonyl)-1-((5'-chloro-2'-fluorobiphenyl-4-yl)methyl)hy US 9,683,002 B2 143 144 drazinyl)-2-hydroxypropanoate (250 mg, 526 umol) in chlo Example 7I: (R)-2-Acetoxy-3-N-(5'-chloro-2'-fluo romethyl isopropyl carbonate (2 mL) was added Nat (113 robiphenyl-4-ylmethyl)-N'-(1H-tetrazole-5-carbonyl) mg, 1.1 mmol) and 2.6-lutidine (158 mg, 1.1 mmol). The hydrazinopropionic Acid resulting mixture was stirred at 80°C. for 3 hours, cooled to room temperature, then poured into water (10 mL). The resulting solution was extracted with EtOAc (2x10 mL). The combined organic layers were dried over anhydrous NaSO filtered, and concentrated in vacuo. The residue was purified by column chromatography (PE:EtOAc=5:1-4: 10 1-3:1) to yield Compound 1 as a colorless oil (165 mg). LC-MS: 591 M+H". To a solution of Compound 1 (150 mg, 254 umol) in dry DCM (5 mL) was added Pd(PPh) (88 mg, 76 winmp, 15 triethylsilane (148 mg, 1.3 mmol) and AcOH (76 mg, 1.3 mmol). The resulting mixture was stirred at room tempera ture under nitrogen for 2 days then concentrated in vacuo. The residue was purified by preparative HPLC Gemini C18, 150x21.2 mm, 5u: MeCN HO (0.1% TFA) from Using the procedures described herein, the title compound 50% to 80% to yield the title compound as a white solid (15 can also be prepared. Note that as explained herein, com mg). LC-MS. 551 M+H". "H NMR (CDC1) & 1.25-129 pounds such as this can exist in a tautomer form, for 25 example, as (R)-2-acetoxy-3-N-(5'-chloro-2'-fluorobiphe (d. 6H), 3.44-3.56 (m, 2H), 4.23-4.31 (dd, 2H), 4.54-4.56 (t, nyl-4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl)hydrazino 1H), 4.85-4.91 (m. 1H), 5.74 (s. 2H), 7.04-7.08 (t, 1H), propionic acid. 723-7.25 (m, 1H), 7.35-7.36 (m. 1H), 7.46 (s, 4H), 9.06 (s, 1H). Example 7J: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl Note that as explained herein, compounds such as this can 30 4-ylmethyl)-N'-(1H-tetrazole-5-carbonyl)-hy exist in a tautomer form, for example, as (R)-3-N-(5'- drazino-2-hydroxypropionic acid ethoxycarbony chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(2H-tetrazole-5- loxymethyl ester carbonyl)hydrazino-2-hydroxypropionic acid isopropoxy carbonyloxymethyl ester. 35

Example 7H: (S)-2-Amino-3-methylbutyric acid LiO (R)-1-carboxy-2-N-(5'-chloro-2'-fluorobiphenyl-4- ylmethyl)-N'-(1H-tetrazole-5-carbonyl)hydrazino 40 ethyl Ester

45 HN1 NN O S. ) O N

NH 50 HO N1

O O

55 HN r O C F

60 Using the procedures described herein, the title compound can also be prepared. Note that as explained herein, com pounds such as this can exist in a tautomer form, for example, as (S)-2-amino-3-methylbutyric acid (R)-1-car boxy-2-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- 65 (2H-tetrazole-5-carbonyl)hydrazinoethyl ester. US 9,683,002 B2 145 146

-continued -continued

HO -e- 10

15

To a suspension of lithium (R)-3-(2-(1-allyl-1H-tetrazole 5-carbonyl)-1-((5'-chloro-2'-fluorobiphenyl-4-yl)methyl) hydrazinyl)-2-hydroxypropanoate (250 mg, 526 umol) in chloromethyl ethyl carbonate (2 mL) was added NaI (158 mg, 1.1 mmol) and 2.6-lutidine (113 mg, 1.1 mmol). The resulting mixture was stirred at 50° C. for 4 hours, cooled to room temperature, then poured into water (10 mL). The 25 resulting solution was extracted with EtOAc (2x10 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by column chromatography (PE:EtOAc=4:1-3: 1-2: 1) to yield Compound 1 as a yellow solid (170 mg). 30 LC-MS: 577 M+H". To a solution of Compound 1 (160 mg, 277 umol) in dry DCM (5 mL) was added Pd(PPh) (96 mg, 83 umol. HO O triethylsilane (161 mg, 1.4 mmol) and AcOH (83 mg, 1.4 N H mmol). The resulting mixture was stirred at room tempera N 35 NN N ture under nitrogen for 2 days then concentrated in vacuo. H N The residue was purified by preparative HPLC Gemini N M C18, 150x21.2 mm, 5u: MeCN HO (0.1% TFA) from 50% to 60% to yield the title compound as a white solid (7 mg). LC-MS. 537 M+H", "H NMR (CDC1) & 1.28-1.33 (t, O NN 3H), 3.47-3.50 (t, 2H), 4.21-4.24 (m, 4H), 4.50-4.52 (t, 1H), 40 5.72-5.77 (dd, 2H), 7.06-7.11 (t, 1H), 7.25-7.27 (m. 1H), 7.28-7.38 (m, 1H), 7.49 (s, 4H), 8.69 (s, 1H). Note that as explained herein, compounds such as this can exist in a tautomer form, for example, as (R)-3-N-(5'- F O chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(2H-tetrazole-5- C carbonyl)hydrazino-2-hydroxypropionic acid ethoxycarbo 45 nyloxymethyl ester. To a suspension of (R)-3-N'-(1-allyl-1H-tetrazole-5-car Example 7K: (S)-2-Methoxycarbonylamino-3-meth bonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)hy ylbutyric Acid (R)-3-N-(5'-chloro-2'-fluorobiphe drazino-2-hydroxypropionic acid and methane (200 mg. nyl-4-ylmethyl)-N'-(1H-tetrazole-5-carbonyl)-hy 50 421 umol) in THF (5 mL), was added (S)-2-methoxycarbo drazino-2-hydroxypropionyloxymethyl Ester nylamino-3-methylbutyric acid chloromethyl ester (1.88 g. 8.42 mmol), NaI (126 mg. 842 umol) and 2.6-lutidine (90 O mg, 842 Lumol). The mixture was refluxed under nitrogen for HO 30 hours, then cooled to room temperature and poured into 55 water (20 mL). The mixture was extracted with EtOAc (2x10 mL). The combined organic layers were dried over HO anhydrous Na2SO, filtered, and concentrated in vacuo. The residue was then purified by column chromatography (PE: EtOAc. 5: 1-4:1-3:1) to yield Compound 1 as a yellow oil 60 (110 mg). LC-MS: 662 M+H". To a solution of Compound 1 (110 mg, 166 umol) in dry DCM (3 mL) was added Pd(PPh.) (57 mg, 50 umol), EtSiH (97 mg. 831 umol) and AcOH (50 mg. 831 umol). The mixture was stirred at room temperature under nitrogen 65 for 2 days, then concentrated in vacuo. The residue was purified by preparative HPLC (Gemini-C18, 150x21.2 mm, MeCN HO (0.1% TFA); from 50% to 60%) to yield the US 9,683,002 B2 147 148 title compound as a white solid (20 mg). LC-MS. 622 chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(1H-tetrazole-5- M+H. 'H NMR (CDC1, 400 MHz): & 1.02-105 (d. 6H), carbonyl)hydrazino-2-propionyloxypropionic acid. 2.06-2.09 (m, 1H), 3.47 (s. 2H), 3.85 (s, 3H), 4.23-4.37 (dd. 2H), 4.42 (s, 1H), 4.70 (t, 1H), 5.27-5.28 (d. 1H), 5.69-5.74 (dd, 2H), 7.06-7.08 (m. 1H). 7.24-7.26 (m, 1H), 7.34-7.34 Example 7M: (S)-2-Methoxycarbonylamino-3- (d. 1H), 7.42-744 (dd, 2H), 7.50-7.52 (dd, 2H). methylbutyric Acid (R)-1-carboxy-2-N-(5'-chloro Note that as explained herein, compounds such as this can 2'-fluorobiphenyl-4-ylmethyl)-N'-(2H-tetrazole-5- exist in a tautomer form, for example, as (S)-2-Methoxy carbonyl)hydrazinoethyl Ester carbonylamino-3-methylbutyric acid (R)-3-N-(5'-chloro-2'- fluorobiphenyl-4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl) 10 hydrazino-2-hydroxypropionyloxymethyl ester. Example 7L: (R)-3-N-(5'-Chloro-2'-fluorobiphenyl 4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl)hydrazino 2-propionyloxypropionic acid 15

HO N

OH N1 v. O W O N

NH HO N1 -- 25

OH O OH O --

O C 30 N- N1 ''','o, H

F N 2N 35 O S. NH Her O N

NH HO N1

40 O O N1 v O N O W O N C H NH 45 F irrO O (S)-2-((methoxycarbonyl)amino)-3-methylbutanoic acid

50 (12.9 mg, 73 umol) was combined with HOBt (12.4 mg. 92 umol) and EDC (11.4 mg, 73 umol) in DCM (2 mL) and stirred for 15 minutes. (R)-3-N-(5'-chloro-2'-fluorobiphe F nyl-4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl)hydrazino-2- hydroxypropionic acid (26.6 mg, 61 umol) and 4-methyl Propionyl chloride (24 mg, 55 umol) was added to a 55 morpholine (7.4 mg, 73 umol) were added and the resulting mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylm mixture was stirred at room temperature overnight. The ethyl)-N'-(2H-tetrazole-5-carbonyl)hydrazino-2-hydroxy solvent was evaporated and the residue was purified by propionic acid (6.1 mg. 66 umol) and DCM (2 mL), and the preparative HPLC to yield the title compound as a TFA salt resulting mixture was stirred at room temperature for 1 hour. The mixture was then heated to 60° C. for 1 hour. The 60 (6 mg). MS m/z. M+H" calc'd for CHCIFN.O. reaction was then stopped and the mixture was concentrated 592.16; found 592.2. and purified by preparative HPLC to yield the title com Note that as explained herein, compounds such as this can pound as a TFA salt (1 mg). MS m/z. M+H calc’d for exist in a tautomer form, for example, as (S)-2-methoxycar CHCIFNOs, 491.12; found 491.2. 65 bonylamino-3-methylbutyric acid (R)-1-carboxy-2-N-(5'- Note that as explained herein, compounds such as this can chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(1H-tetrazole-5- exist in a tautomer form, for example, as (R)-3-N-(5'- carbonyl)hydrazinoethyl ester. US 9,683,002 B2 149 150 Example 8A: (R)-3-N-(3'-Chlorobiphenyl-4-ylm -continued ethyl)-N'-(3-hydroxyisoxazole-5-carbonyl)hy o-N drazino-2-propionyloxypropionic Acid N OH O N O S. N OH O NH HO N1 O 10

C

15 Isovalery chloride (15.5 uL. 127 umol) was added to a mixture of (R)-3-N-(3'-Chlorobiphenyl-4-ylmethyl)-N'-(3- hydroxyisoxazole-5-carbonyl)hydrazino-2-hydroxypropi onic acid (25.0 mg, 58 umol) and EtN (40.3 uL, 289 umol) in DCM (0.5 mL, 8 mmol). The resulting mixture was stirred at room temperature for 1 hour and concentrated. The residue was combined with saturated aqueous NaHCO HO N (10:90, NaHCO: water, 0.1 mL, 0.1 mmol) in MeOH (1.0 25 mL), stirred for 15 minutes, and then concentrated. The residue was dissolved in AcOH (1.5 mL), filtered, and purified by reverse phase preparative HPLC to yield the title compound (2.4 mg). MS m/z. M+H calc’d for CHCINO, 516.15; found 516.5. 30 Example 8C. (R)-3-N'-(3-Acetoxymethoxyisox azole-5-carbonyl)-N-(3'-chlorobiphenyl-4-ylmethyl) Propanoyl chloride (3.5 uL, 40 umol) was added to a hydrazino-2-hydroxypropionic Acid mixture of (R)-3-N-(3'-chlorobiphenyl-4-ylmethyl)-N'-(3- hydroxyisoxazole-5-carbonyl)hydrazino-2-hydroxypropi onic acid 2-oxo-2-phenylethyl ester (20.0 mg. 36.4 umol) 35 and EtN (12.7 uL. 90.9 umol) in DCM (0.5 mL, 8 mmol). O- N The resulting mixture was stirred at room temperature for 1 N OH hour, concentrated, and purified by flash chromatography O S. (EtOAc-hexanes=0-100%) to give a solid (15.5 mg). The O solid was dissolved in AcOH (1.5 mL, 26 mmol). Zinc (119 NH mg, 1.8 mmol) was added and the resulting mixture was Y stirred at room temperature for 2 hours. The mixture was --> N -- filtered and the zinc powder was washed with AcOH (0.5 O OH mL). The combined washes were purified by reverse phase preparative HPLC to yield the title compound (3.9 mg). MS 45 O C m/z. M+H calc’d for CHCINOz, 488.11: found 488.3. Example 8B: 3-Methylbutyric Acid (R)-1-carboxy 2-N-(3'-chlorobiphenyl-4-ylmethyl)-N'-(3-hydroxy O isoxazole-5-carbonyl)hydrazinoethyl Ester 50 o1 NB o-N HO O O N OH 2. NN O S. O

55 O N NH --

OH co C 60

65 Or e A mixture of bromomethyl acetate (16.0 uL, 164 Lumol) and NaI (24.5 mg, 164 Lumol) in acetone (2.0 mL. 27 mmol) US 9,683,002 B2 151 152 was stirred at 60° C. for 1 hour, then cooled to room A mixture of chloromethylbutyrate (20.5 L, 164 Lumol) temperature. A mixture of (R)-3-N-(3'-Chlorobiphenyl-4- and NaI (24.5 mg, 164 Lumol) in acetone (2.0 mL. 27 mmol) ylmethyl)-N'-(3-hydroxyisoxazole-5-carbonyl)hydrazino was stirred at 60° C. for 1 hour, then cooled to room 2-hydroxypropionic acid 2-oxo-2-phenylethyl ester (30.0 temperature and added to a mixture of (R)-3-N-(3'-chloro mg, 54.5umol) and EtN (38.0 uL. 273 umol) in acetone (1 biphenyl-4-ylmethyl)-N'-(3-hydroxyisoxazole-5-carbonyl) mL) was then added. The resulting mixture was stirred at hydrazino-2-hydroxypropionic acid 2-oxo-2-phenylethyl room temperature for 2 hours, concentrated, dissolved in ester (30.0 mg, 54.5umol) and Et-N (38.0 uL. 273 umol) in AcOH (2 mL), filtered, and purified by reverse phase acetone (1 mL). The resulting mixture was stirred at room preparative HPLC. The desired fractions were combined and temperature for 2 hours, concentrated, dissolved in AcOH (2 10 mL), filtered, and purified by reverse phase preparative lyophilized to yield a solid (19.2 mg). The solid was HPLC. The desired fractions were combined and combined with zinc (178 mg, 2.7 mmol) in AcOH (1.0 mL. lyophilized. Zinc (178 mg, 2.7 mmol) and AcOH (1.0 mL. 18 mmol) and stirred at room temperature for 2 hours. The 18 mmol) was added and the resulting mixture was stirred at mixture was filtered and purified by reverse phase prepara room temperature for 2 hours. The mixture was filtered and tive HPLC. The desired fractions were combined, 15 purified by reverse phase preparative HPLC to yield the title lyophilized, dissolved in AcOH (1.5 mL), and purified by reverse phase preparative HPLC to yield the title compound compound (2.5 mg). MS m/z. M+H" calc’d for (3.8 mg) as a TFA salt. MS m/z. M+H calc’d for CHCINOs 532.14; found 532.2. CHCINOs 504.11: found 504.0. Example 8E: (R)-3-N-(3'-Chlorobiphenyl-4-ylm ethyl)-N'-(3-ethoxycarbonyloxymethoxyisoxazole-5- Example 8D: Butyric acid 5-N-((R)-2-carboxy-2- hydroxyethyl)-N'-(3'-chlorobiphenyl-4-ylmethyl) carbonyl)hydrazino-2-hydroxypropionic acid hydrazinocarbonylisoxazol-3-yloxymethyl Ester

25

O

O 30 O O O O O O O 9NN \l HO N-NH OH HO N-N OH 35 ( ) -- ( ) -- 40

KX C C 45 s s -as Her O O l 50 O

O HO O O HO O 9NN ? \l 55 HO N-N HO N-N O H l O

60

C

65 A mixture of chloromethyl ethyl carbonate (22.7 mg, 164 umol) and NaI (24.5 mg, 164 umol) in acetone (2.0 mL. 27 US 9,683,002 B2 153 154 mmol) was stirred at 60° C. for 1 hour, then cooled to room isoxazole-5-carbonyl)hydrazino-2-hydroxypropionic acid temperature and added to a mixture of (R)-3-N-(3'-chloro 2-oxo-2-phenylethyl ester (20.0 mg. 36.4 umol) that had biphenyl-4-ylmethyl)-N'-(3-hydroxyisoxazole-5-carbonyl) been dissolved in acetone (0.4 mL, 5 mmol) at room hydrazino-2-hydroxypropionic acid 2-oxo-2-phenylethyl temperature and treated with cesium carbonate (13.0 mg. 40 ester (30.0 mg, 54.5 umol) and cesium carbonate (17.8 mg, umol). The resulting mixture was then heated at 40° C. for 0.054.5umol) in acetone (1 mL). The resulting mixture was 1 hour. DIPEA (0.2 mL, 1 mmol) was added and heating stirred at room temperature for 2 hours, concentrated, dis continued for 1 hour. The mixture was cooled to room solved in AcOH (2 mL), filtered, and purified by reverse temperature and concentrated. The residue was partitioned phase preparative HPLC. The desired fractions were com between EtOAc (10.0 mL) and water (2.0 mL). The organic bined and concentrated. Zinc (178 mg, 2.7 mmol) and AcOH 10 layer was washed with saturated aqueous NaHCOs (3.0 mL), (1.0 mL, 18 mmol) were added and the resulting mixture was stirred at room temperature for 2 hours. The mixture saturated aqueous NaCl (3.0 mL), dried over NaSO was filtered and purified by reverse phase preparative HPLC filtered, and concentrated. Zinc 90.8 mg, 1.4 mmol) and to yield the title compound (2.2 mg). MS m/z. M+H" calc’d AcOH (0.5 mL, 9 mmol) were added and the resulting 15 mixture was stirred at room temperature for 1 hour. The for CHCINO. 534.12; found 534.3. solids were washed with AcOH (1.0 mL) then filtered. The Example 8F: (R)-3-N-(3'-Chlorobiphenyl-4-ylm filtrates were combined and purified by reverse phase pre ethyl)-N'-(3-isopropoxycarbonyloxymethoxyisox parative HPLC to yield the title compound (3.8 mg). MS m/z. azole-5-carbonyl)hydrazino-2-hydroxypropionic M+H" calc’d for CHCINO. 548.14; found 548.5. Acid Example 8G: (R)-3-(N-(3'-Chlorobiphenyl-4-ylm ethyl)-N'-[3-(5-methyl-2-oxo-1.3dioxol-4-yl methoxy)-isoxazole-5-carbonylhydrazino-2-hy droxypropionic Acid 25

O O O O O

30 HO N-NH OH

35 ( ) -- N-N H OH

40 ( ) -- l

45 C

H / O HO O 50 o-s, O HO HO O O 55 HO - N-N H O

A mixture of chloromethyl isopropyl carbonate (15.6 mg, 102 umol) and NaI (15.3 mg, 102 umol) in acetone (0.7 mL, 65 10 mmol) was stirred at 65° C. for 1 hour, then added (R)-3-N-(3'-chlorobiphenyl-4-ylmethyl)-N'-(3-hydroxy US 9,683,002 B2 155 156 A mixture of 4-chloromethyl-5-methyl-1,3-dioxol-2-one (29.8 mg, 198 umol) in acetone (1.0 mL, 14 mmol) was (11.9 uL. 109 umol) and NaI (16.4 mg, 109 umol) in acetone heated at 65° C. for 1 hour, then (R)-3-N-(3'-chlorobiphe (2.0 mL. 27 mmol) was stirred at 60° C. for 1 hour, then nyl-4-ylmethyl)-N'-(3-hydroxyisoxazole-5-carbonyl)hy cooled to room temperature and added to a mixture of drazino-2-hydroxypropionic acid 2-oxo-2-phenylethyl (R)-3-N-(3'-chlorobiphenyl-4-ylmethyl)-N'-(3-hydroxy 5 ester (36.4 mg. 66.2 Lumol) and cesium carbonate (25.9 mg, isoxazole-5-carbonyl)hydrazino-2-hydroxypropionic acid 79.4 umol) were added. The resulting mixture was stirred at 2-oxo-2-phenylethyl ester (20.0 mg, 36.4 umol) and cesium 65° C. for 3 hours, concentrated, purified by flash chroma carbonate (14.2 mg, 43.6 Limol) in acetone (1 mL). The tography (EtOAc/hexanes=0-100%). Zinc (216 mg, 3.3 resulting mixture was stirred at 60° C. for 2 hours, concen mmol) and AcOH (1.0 mL, 18 mmol) were added and the trated, purified by flash chromatography (EtOAc/ 10 resulting mixture was stirred at room temperature for 1 hour, hexanes=0-100%). The desired fractions were combined and then concentrated by rotary evaporation. TFA (0.1 mL, 1 mmol) and DCM (0.1 mL, 2 mmol) were added and the concentrated. Zinc (178 mg, 2.7 mmol) and AcOH (1.0 mL. mixture was stirred for 30 minutes then concentrated. The 18 mmol) were added and the resulting mixture was stirred residue was dissolved in AcOH (1.5 mL), filtered, and at room temperature for 2 hours. The solids were washed purified by reverse phase preparative HPLC to yield the title with AcOH (0.5 mL) then filtered. The filtrates were com 15 bined and purified by reverse phase preparative HPLC to compound as a TFA salt (3.0 mg). MS m/z. M+H" calc’d yield the title compound (2.8 mg). MS m/z. M+H calc’d for CHCINOs 561.17; found 561.2. for CHCINO. 544.10; found 544.5. Example 8I: (S)-2-Methoxycarbonylamino-3-meth ylbutyric Acid (R)-1-carboxy-2-N-(3'-chloro-biphe Example 8H: (S)-2-Amino-3-methylbutyric Acid nyl-4-ylmethyl)-N'-(3-hydroxyisoxazole-5-carbonyl) 5-N'-((R)-2-carboxy-2-hydroxyethyl)-N'-(3'- hydrazinoethyl Ester chloro-biphenyl-4-ylmethyl)hydrazinocarbonylisox azol-3-yloxymethyl Ester

25

O O O O O 30 HO N-NX-Cl H OH

HO 35 ( ) --

40

O HO

45 O He H N V BOC 50 O HO O ONN \l H y 55 O O 60

65 A mixture of (S)-2-t-butoxycarbonylamino-3-methylbu tyric acid chloromethyl ester (52.8 mg, 198 umol) and NaI US 9,683,002 B2 157 158 (S)-2-Methoxycarbonylamino-3-methylbutyric acid (9.6 mg, 54.5 umol) and HATU (23.5 mg. 62 umol) were stirred (1) in DCM (1.0 mL, 16 mmol) for 10 minutes. (R)-3-N-(3'- Chlorobiphenyl-4-ylmethyl)-N'-(3-hydroxyisoxazole-5-car bonyl)hydrazino-2-hydroxypropionic acid 2-oxo-2-pheny NH2 lethyl ester (20.0 mg. 36.4 umol) and DIPEA (31.7 uL, 182 --> O OH umol) were added, and the resulting mixture was stirred at room temperature overnight. The mixture was then concen 10 trated and purified by flash chromatography (EtOAc/ hexanes=0-100%). Zinc (119 mg, 1.8 mmol) and AcOH (0.5 mL, 9 mmol) were added. The mixture was stirred for 2 (2) hours and filtered. The solids were washed with AcOH (1 15 mL) and filtered. The combined filtrates were purified by reverse phase preparative HPLC to yield the title compound Compound 1 (200 mg. 371 umol) was dissolved in MeCN (3 mL). A solution of 4.0 M HCl in dioxane (928 uL, 3.7 (0.8 mg). MS m/z. M+H" calc'd for C.H.CINO. mmol) was added and the resulting mixture was stirred at 589.16; found 589.3. room temperature for 1 hour. The solvent was removed in vacuo and the residue, Compound 2, was used in the next Example 8J: (S)-2-t-Butoxycarbonylamino-3-meth step without any further purification. ylbutyric Acid 5-N'-((R)-2-carboxy-2-hydroxy ethyl)-N'-(3'-chlorobiphenyl-4-ylmethyl)hy 25 drazinocarbonylisoxazol-3-yloxymethyl Ester in-Q O O 30 (2) + He O O O N NH HO r" N1 N O 35 1N OH

O OH 40

O O O HO --KllN 50 O H r r")O

(1) 55 C) (R)-3-N'-t-Butoxy carbonyl-N-(3'-chlorobiphenyl-4-yl (3) methyl)hydrazino-2-hydroxy-propionic acid (406 mg, 965 umol) was dissolved in DMF (5 mL). Potassium carbonate 60 3-((S)-2-t-Butoxycarbonylamino-3-methylbutyry (333 mg, 2.4 mmol) was added followed by 2-bromo-1- loxymethoxy)isoxazole-5-carboxylic acid (44.1 mg, 123 phenylethanone (230 mg, 1.2 mmol). The resulting mixture umol) and HATU (78 mg, 205 umol) were combined in DMF (1 mL) and stirred at room temperature for 15 minutes. was stirred at room temperature overnight. The solvent was Compound 2 (45 mg, 103 umol) and DIPEA (54 uL, 307 removed in vacuo and the residue was purified (CombiFlash 65 umol) were added, and the resulting mixture was stirred at normal phase column). The clean fractions were collected room temperature for 15 minutes. The solvent was removed and combined to yield Compound 1 (280 mg). in vacuo and the residue was purified (CombiFlash normal US 9,683,002 B2 159 160 phase column). The clean fractions were collected and Example 8M: (S)-2-Methoxycarbonylamino-3- combined to yield Compound 3 (41 mg). methylbutyric Acid (R)-3-N-(3'-Chlorobiphenyl-4- ylmethyl)-N'-(3-hydroxyisoxazole-5-carbonyl)hy (3) -> drazino-2-hydroxypropionyloxymethyl Ester 5 O

O-NN /NO SkO O S/O O 10 or NH OH O . C 15

Compound 3 (41 mg, 53 umol) was dissolved in AcOH (1 mL) and Zinc (172 mg, 2.6 mmol) was added. The mixture 2O was stirred at room temperature for 45 minutes to 1 hour Using the procedures described herein, the title compound until the reaction was complete. The Zinc was filter off and can also be prepared. the solution was purified (reverse phase column) to yield the title compound (7 mg). MS m/z. M+H" calc’d for Example 8N: (S)-2-Amino-3-methylbutyric Acid CHCINO 661.22; found 661.2. 25 (R)-1-carboxy-2-N-(3'-chlorobiphenyl-4-ylmethyl)- Example 8K: (R)-3-N-(3'-Chlorobiphenyl-4-ylm N'-(3-hydroxyisoxazole-5-carbonyl)hydrazinoethyl ethyl)-N'-(3-hydroxyisoxazole-5-carbonyl)hy Ester drazino-2-hydroxypropionic Acid Acetoxymethyl Ester 30 NH o-N N OH OS O S. 35 y- O O O O O. N ---, N1 NH 1. N-N OH HO OH 40 O

C

45 Using the procedures described herein, the title compound can also be prepared. Example 8L: (S)-2-Amino-3-methylbutyric Acid Using the procedures described herein, the title compound (R)-3-N-(3'-Chlorobiphenyl-4-ylmethyl)-N'-(3-hy 50 can also be prepared. droxyisoxazole-5-carbonyl)-hydrazino-2-hydroxy propionyloxymethyl Ester Example 9A: (R)-3-N-(2,5'-Dichlorobiphenyl-4- ylmethyl)-N'-(1-hydroxy-1H-2.3 triazole-4-carbo N OH nyl)hydrazino-2-hydroxypropionic Acid O S. 55 O O HN sus NH ~~~ N1 O OH 60 HO No N1 NH2 VN-N C W OH N-N

65 Using the procedures described herein, the title compound Br O OH can also be prepared. US 9,683,002 B2 161 162 -continued -continued HON B-OH N 2N N - O O C O S. N1 O O NH C HO N1 OH 10

HO N

OH 15 Bromomethyl acetate (15.3 mg, 100 umol) was added to C a solution of (R)-3-N-(2,5'-dichlorobiphenyl-4-ylmethyl)- N'-(1-hydroxy-1H-1,2,3-triazole-4-carbonyl)hydrazino-2- hydroxypropionic acid (31.2 mg, 66.8 umol) in acetone (0.5 mL, 6.8 mmol) followed by EtN (18.6 uL, 134 umol). The resulting mixture was stirred at 55° C. for 1 hour. The mixture was then concentrated in vacuo to yield a yellow 1-Hydroxy-1H-1,2,3-triazole-4-carboxylic acid (42.6 mg, liquid. The crude liquid was purified (preparative scale C18 330 umol) and HATU (125 mg. 330 mol) were combined 25 column chromatography, Small column, using 30-90% in DMF (2 mL) and stirred for 5 minutes at room tempera MeCN in water with 0.05%TFA) to yield the title compound ture. DIPEA (86 uL. 495 umol) and (R)-3-N-(4-bromoben (5.2 mg, purity 96%) as a white solid. MS m/z. M+H" Zyl)hydrazino-2-hydroxypropionic acid methyl ester (50 calc’d for CHC1.N.O., 538.08; found 538.1. mg, 0.2 mmol) were added, and the resulting mixture was stirred for 30 minutes. The mixture was evaporated under 30 Example 9C: Butyric Acid 4-N-((R)-2-carboxy reduced pressure and the product dissolved in EtOH (0.8 2-hydroxyethyl)-N'-(2,5'-dichlorobiphenyl-4-ylm mL, 10 mmol) and water (0.2 mL, 10 mmol). 2,5-Dichlo ethyl)hydrazinocarbonyl-1,2,3triazol-1-yloxym rophenylboronic acid (57 mg, 297 umol), KCO (68 mg, ethyl Ester 495 umol), and SilicaCat(R) DPP-Pd (0.28 mmol/g loading: 58.9 mg, 16.5 umol) were added and the resulting mixture 35 was heated at 120° C. for 10 minutes. The mixture was N2N filtered, and 1 M aqueous LiOH (1.2 mL, 1.2 mmol) was N-OH added to the filtered. The mixture was stirred until the O S. reaction was complete (1 hour), then vacuumed to dryness O 40 NH and purified by preparative HPLC to yield the title com 1 pound as a TFA salt (14 mg. purity 95%). MS m/z. M+H" HO N -- calc'd for CH,ClNOs, 466.06; found 466.2. OH Example 9B; (R)-3-N'-(1-Acetoxymethoxy-1H-1, 2.3 triazole-4-carbonyl)-N-(2,5'-dichlorobiphenyl-4- 45 O C ylmethyl)hydrazino-2-hydroxypropionic Acid C C N1 O 50 -e- O N2N N-O O O S. N1 55 O HO N O NH OH HO N1

OH 60 C

C 65 Chloromethylbutyrate (13.7 mg, 100 umol) was added to a solution of (R)-3-N-(2,5'-dichlorobiphenyl-4-ylmethyl)- US 9,683,002 B2 163 164 N'-(1-hydroxy-1H-1,2,3-triazole-4-carbonyl)hydrazino-2- Example 9E: (S)-2-Methoxycarbonylamino-3-meth hydroxypropionic acid (31.2 mg, 66.8 umol) in acetone (0.5 ylbutyric Acid (R)-3-N-(2,5'-dichlorobiphenyl-4- mL, 6.8 mmol) followed by EtN (18.6 uL, 134 umol). The ylmethyl)-N'-(1-hydroxy-1H-1,2,3-triazole-4-car resulting mixture was stirred at 65° C. for 2 hours. The bonyl)hydrazino-2-hydroxypropionyloxymethyl mixture was then concentrated in vacuo to yield a yellow Ester liquid. The crude liquid was purified (preparative scale C18 column chromatography, Small column, using 30-90% NaN. MeCN in water with 0.05% TFA) to yield to yield the title N-OH O s compound (6.1 mg, purity 99%) as a white solid. MS m/z. O O M+H calc’d for CHC1NO2, 566.11; found 566.1. 10 1. O NNulls roy NH Example 9D: (R)-3-(N-(2',5'-Dichlorobiphenyl-4- O 1N OH ylmethyl)-N'-1-(5-methyl-2-oxo-1.3dioxol-4-yl methoxy)-1H-1.2.3 triazole-4-carbonylhydrazino 15 C 2-hydroxypropionic Acid C Using the procedures described herein, the title compound can also be prepared. Example 9F: (R)-3-N-(2',5'-Dichlorobiphenyl-4- ylmethyl)-N'-(1-hydroxy-1H-1.2.3 triazole-4-carbo nyl)hydrazino-2-hydroxypropionic acid iso propoxycarbonyloxymethyl Ester 25 HO N

OH HO O O /- C 30 ) ( . HO N-N 2N H

C 35

40

HO N

OH 45

50

4-Chloromethyl-5-methyl-1,3-dioxol-2-one (14.9 mg, O 100 umol) was added to a solution of (R)-3-N-(2,5'- O NSN dichlorobiphenyl-4-ylmethyl)-N'-(1-hydroxy-1H-1,2,3-tri 55 HO azole-4-carbonyl)hydrazino-2-hydroxypropionic acid (31.2 H) (C. ON-2 mg, 66.8 mol) in acetone (0.5 mL, 6.8 mmol) followed by EtN (18.6 uL, 134 umol). The resulting mixture was stirred at 65° C. for 2 hours. The mixture was then concentrated in 60 vacuo to yield a yellow liquid. The crude liquid was purified (preparative scale C18 column chromatography, Small col umn, using 30-90% MeCN in water with 0.05% TFA) to yield the title compound (10.0 mg, purity 99%) as a white 65 solid. MS m/z. M+H" calc'd for CHCINOs 578.08: (1) found 578.1. US 9,683,002 B2 165 166 To a mixture of (R)-3-N'-(1-allyloxy-1H-1.2.3 triazole Example 9H: (R)-3-N-(2',5'-Dichlorobiphenyl-4- 4-carbonyl)-N-(2',5'-dichlorobiphenyl-4-ylmethyl)-hy ylmethyl)-N'-(1-hydroxy-1H-1.2.3 triazole-4-carbo drazino-2-hydroxypropionic acid (400 mg, 790 umol) and nyl)hydrazino-2-hydroxypropionic Acid 5-methyl carbonic acid chloromethyl ester isopropyl ester (1.5 mL) 2-oxo-1.3dioxol-4-ylmethyl Ester were added NaI (237 mg, 1.6 mmol) and lutidine (166 mg, 1.6 mmol). The mixture was stirred at 50° C. for 3 hours. After cooling to room temperature, the mixture was diluted N2N with water (15 mL) and extracted with EtOAc (2x15 mL). - OH The combined organic layers were washed with saturated O S. aqueous NaCl (20 mL), dried over anhydrous NaSO, 10 O concentrated, and purified by column chromatography (PE: NH EtOAc=5:1 to 2:1) to yield Compound 1 as a light yellow solid (230 mg). LC-MS. 622 M+H". O ={O OH 15 C (1) He

C O S. l O O Using the procedures described herein, the title compound ls NH can also be prepared. O ^^ - Example 9I: (R)-3-N-(2,5'-Dichlorobiphenyl-4- OH ylmethyl)-N'-(1-hydroxy-1H-1.2.3 triazole-4-carbo 25 nyl)hydrazino-2-hydroxypropionic acid Ethoxycar C bonyloxymethyl Ester

C 30 NaN N-OH To a solution of Compound 1 (230 mg, 370 umol) in THF O S. (5 mL) was added Pd(PPh.) (64 mg, 56 umol) and 1,3- O O dimethylbarbituric acid (577 mg, 3.7 mmol). The mixture was stirred at room temperature for 2 hours and then ls NH concentrated. The residue was purified by preparative HPLC 35 1no ^^ - Daisogel-C18, 250x50 mm, 10u: MeCN HO (0.1% OH TFA) from 50% to 90% to yield the title compound as a white solid (120 mg). LC-MS. 582 M+H". H-NMR C (MeOD, 400 Hz): & 1.13-1.24 (d. 6H), 3.33-3.36 (m, 2H), 4.08-4.11 (m, 2H), 4.34-4.35 (m, 1H), 5.66 (s. 2H), 7.15 40 7.38 (m, 4H), 7.39-7.44 (m, 3H), 8.11 (s, 1H). C Using the procedures described herein, the title compound Example 9G: (R)-3-N-(2,5'-Dichlorobiphenyl-4- can also be prepared. ylmethyl)-N'-(1-hydroxy-1H-1.2.3 triazole-4-carbo 45 Example 9J: Butyric Acid (R)-3-N-(2',5'-Dichloro nyl)hydrazino-2-hydroxypropionic acid acetoxym biphenyl-4-ylmethyl)-N'-(1-hydroxy-1H-1.2.3 triaz ethyl Ester ole-4-carbonyl)hydrazino-2-hydroxypropiony loxymethyl Ester 50

HO 55 HO OH

60

65 Using the procedures described herein, the title compound can also be prepared. US 9,683,002 B2 167 168 -continued Example 9K: (R)-3-N-(2,5'-Dichlorobiphenyl-4- ylmethyl)-N'-(1-hydroxy-1H-1.2.3 triazole-4-carbo nyl)hydrazino-2-hydroxypropionic acid 1-cyclo hexyloxycarbonyloxyethyl Ester

O /— HO O O 10

HO N-N) (N2 .N H

15

C C To a mixture of (R)-3-N-(1-allyloxy-1H-1.2.3 triazole 4-carbonyl)-N-(2',5'-dichlorobiphenyl-4-ylmethyl)-hy drazino-2-hydroxypropionic acid (300 mg, 590 umol) and N chloromethylbutyrate (1.5 mL) were added NaI (178 mg, 25 1.2 mmol) and lutidine (124 mg, 1.2 mmol). The mixture s" - was stirred at 50° C. for 5 hours. After cooling to room O temperature, the mixture was diluted with water (15 mL) and extracted with EtOAc (2x15 mL). The combined organic layers were washed with saturated aqueous NaCl (20 mL), 30 dried over anhydrous Na2SO, concentrated, and purified by silica gel chromatography (silica gel: 200-300 mesh; elute with PE:EtOAc=5:1 to 2:1) to yield Compound 1 as a light yellow solid (220 mg). LC-MS: 606 M+H", 608 (M+2)+ 35 O H*. O O O NSN (1) -e- 40 N HO N-N)-K NaN H ON-2 N-OH O S. O O 45 N-1 no l ^^ - NH OH C C C 50 CO (1) To a mixture of (R)-3-N'-(1-allyloxy-1H-1.2.3 triazole To a solution of Compound 1 (220 mg, 360 umol) in THF 55 4-carbonyl)-N-(2',5'-dichlorobiphenyl-4-ylmethyl)-hy (5 mL) was added Pd(PPh) (22 mg, 20 umol) and 1,3- drazino-2-hydroxypropionic acid (300 mg, 590 umol) and dimethylbarbituric acid (525 mg, 3.6 mmol). The mixture carbonic acid 1-chloro-ethyl ester cyclohexyl ester (1.5 mL) was stirred at room temperature for 2 hours and then were added NaI (178 mg, 1.2 mmol) and lutidine (124 mg. concentrated. The residue was purified by preparative HPLC 60 1.2 mmol). The mixture was stirred at 50° C. for 5 hours. After cooling to room temperature, the mixture was diluted Daisogel-C18, 250x50 mm, 10u: MeCN HO (0.1% with water (15 mL) and extracted with EtOAc (2x15 mL). TFA) from 60% to 90% to yield the title compound as a The combined organic layers were washed with saturated white solid (80 mg). LC-MS. 566 M+H", 568 (M+2)+Ht aqueous NaCl (20 mL), dried over anhydrous NaSO, 'H-NMR (CDOD, 400 Hz): 8 0.93 (t, 3H), 1.62 (q, 2H), 65 concentrated, and purified by silica gel chromatography 2.31 (t, 2H), 3.32-3.37 (m, 2H), 4.20 (m, 2H), 4.41 (m. 1H), (PE:EtOAc=5:1 to 2:1) to yield Compound 1 as a light 5.75 (dd, 2H), 7.35 (m, 4H), 7.47-7.54 (m, 3H), 8.20 (s, 1H). yellow solid (200 mg). LC-MS: 676 M+H". US 9,683,002 B2 169 170 Using the procedures described herein, the title compound (1) -e- can also be prepared. NaN Assay N-OH O s 5 In Vitro Assays for the Quantitation of Inhibitor ls NH Potencies (ICs) at Human and Rat NEP, and O O or Human ACE OH 10 The inhibitory activities of compounds at human and rat O C neprilysin (EC 3.4.24.11: NEP) and human angiotensin converting enzyme (ACE) were determined using in vitro C assays as described below. To a solution of Compound 1 (200 mg, 296 umol) in THF 15 Extraction of NEP Activity from Rat Kidneys (5 mL) was added Pd(PPh) (46 mg, 40 umol) and 1,3- dimethylbarbituric acid (461 mg, 3.0 mmol). The mixture Rat NEP was prepared from the kidneys of adult Sprague was stirred at room temperature for 2 hours and then concentrated. The residue was purified by preparative HPLC Dawley rats. Whole kidneys were washed in cold phosphate Daisogel-C18, 250x50 mm, 10u: MeCN HO (0.1% buffered saline (PBS) and brought up in ice-cold lysis buffer TFA) from 60% to 90% to yield the title compound as a (1% Triton X-114, 150 mM. NaCl, 50 mM tris(hydroxym white solid (60 mg). LC-MS. 636 M+H". H-NMR ethyl)aminomethane (Tris) pH 7.5; Bordier (1981).J. Biol. (MeOD, 400 Hz): & 1.29-1.55 (m, 9H), 1.70-1.74 (m, 2H), Chem. 256: 1604-1607) in a ratio of 5 mL of buffer for every 1.85-1.89 (m, 2H), 3.33-3.40 (m, 2H), 4.19-422 (m, 2H), gram of kidney. Samples were homogenized on ice using a 4.37-4.40 (m. 1H), 4.57-4.60 (m, 1H), 6.67-6.74 (q, 1H), 7.33-7.36 (m, 4H), 7.46-7.53 (m, 3H), 8.19 (s, 1H). 25 polytron hand held tissue grinder. Homogenates were cen Example 9L: (S)-2-Amino-3-methylbutyric acid trifuged at 1000xg in a swinging bucket rotor for 5 minutes 4-N'-((R)-2-carboxy-2-hydroxyethyl)-N'-(2,5'- at 3° C. The pellet was resuspended in 20 mL of ice cold dichlorobiphenyl-4-ylmethyl)hydrazinocarbonyl-1, lysis buffer and incubated on ice for 30 minutes. Samples 2.3 triazol-1-yloxymethyl Ester (15-20 mL) were then layered onto 25 mL of ice-cold O 30 cushion buffer (6% w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, Triton X-114), heated to 37° C. for 3-5 minutes and centrifuged at 1000xg in a Swinging bucket N1\,.N-O -a- O NH O S. rotor at room temperature for 3 minutes. The two upper O layers were aspirated off, leaving a viscous oily precipitate 35 containing the enriched membrane fraction. Glycerol was NH added to a concentration of 50% and samples were stored at HO N -20° C. Protein concentrations were quantitated using a OH BCA detection system with bovine serum albumin (BSA) as a standard. 40 C Enzyme Inhibition Assays Recombinant human NEP and recombinant human ACE C were obtained commercially (R&D Systems, Minneapolis, 45 Minn., catalog numbers 1182-ZN and 929-ZN, respec Using the procedures described herein, the title compound tively). The fluorogenic peptide substrate Mca-D-Arg-Arg can also be prepared. Leu-Dap-(Dnp)-OH (Medeiros et al. (1997) Braz. J. Med. Example 9M: (S)-2-Methoxycarbonylamino-3- Biol. Res. 30:1157-62; Anaspec, San Jose, Calif.) and Abz methylbutyric Acid 4-N'-((R)-2-carboxy-2-hy Phe-Arg-Lys(Dnp)-Pro-OH (Araujo et al. (2000) Biochem droxy-ethyl)-N'-(2,5'-dichlorobiphenyl-4-ylmethyl) istry 39:8519-8525; Bachem, Torrance, Calif.) were used in hydrazinocarbonyl-1,2,3triazol-1-yloxymethyl the NEP and ACE assays respectively. Ester The assays were performed in 384-well white opaque plates at 37°C. using the fluorogenic peptide Substrates at a O concentration of 10 uM in Assay Buffer (NEP: 50 mM NaN. O 55 HEPES, pH 7.5, 100 mM. NaCl, 0.01% polyethylene glycol 2 v N-o1 No n sorbitan monolaurate (Tween-20), 10 uM ZnSO; ACE: 50 O S. mM HEPES, pH 7.5, 100 mM. NaCl, 0.01% Tween-20, 1 uM O O ZnSO). The respective enzymes were used at concentra NH tions that resulted in quantitative proteolysis of 1 uM of or 60 substrate after 20 minutes at 37° C. OH Test compounds were assayed over the range of concen trations from 10 uM to 20 uM. Test compounds were added C to the enzymes and incubated for 30 minute at 37° C. prior to initiating the reaction by the addition of substrate. Reac 65 tions were terminated after 20 minutes of incubation at 37° C C. by the addition of glacial acetic acid to a final concen tration of 3.6% (v/v). US 9,683,002 B2 171 172 Plates were read on a fluorometer with excitation and The compound of Example 9A was tested in this assay emission wavelengths set to 320 nm and 405 nm, respec and found to have a pK value at human NEP of >9.0. The tively. Inhibition constants were obtained by nonlinear prodrug compounds of Examples 9B-K either did not inhibit regression of the data using the equation (GraphPad Soft the enzyme in this in vitro assay, or was not tested since ware, Inc., San Diego, Calif.): activity would not be expected in this assay; however, based upon the activity of the active form, this prodrug is are expected to have in vivo NEP activity. where v is the reaction rate, V is the uninhibited reaction While the present invention has been described with reference to specific aspects or embodiments thereof, it will rate, I is the inhibitor concentration and K is the apparent 10 inhibition constant. be understood by those of ordinary skilled in the art that The compound of formula I (Example 1A) was tested in various changes can be made or equivalents can be substi this assay and found to have a pKi value at human NEP of tuted without departing from the true spirit and scope of the >9.0. The prodrug compounds of Examples 1B and 1C either invention. Additionally, to the extent permitted by applicable did not inhibit the enzyme in this in vitro assay, or were not 15 patent statutes and regulations, all publications, patents, and tested since activity would not be expected in this assay; patent applications cited herein are hereby incorporated by however, based upon the activity of the active form, these reference in their entirety to the same extent as if each prodrugs are expected to have in vivo NEP activity. document had been individually incorporated by reference The compound of formula II" (Example 2A) was tested in herein. this assay and found to have a pK value at human NEP of >9.0. The prodrug compound of Example 2B either did not What is claimed is: inhibit the enzyme in this in vitro assay, or was not tested 1. A compound of: since activity would not be expected in this assay; however, (a) (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- based upon the activity of the active form, this prodrug is N'-1-(3-chlorophenyl)-5-oxo-4,5-dihydro-1H-1.2.4 expected to have in vivo NEP activity. 25 triazole-3-carbonylhydrazino-2-hydroxypropionic The compound of formula III" (Example 3A) was tested in acid ethyl ester; this assay and found to have a pK value at human NEP of (b) (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- >9.0. The prodrug compounds of Examples 3B-L either did N'-1-(3-chlorophenyl)-5-oxo-4,5-dihydro-1H-1.2.4 not inhibit the enzyme in this in vitro assay, or were not triazole-3-carbonylhydrazino-2-hydroxypropionic tested since activity would not be expected in this assay; 30 acid isobutyl ester; however, based upon the activity of the active form, this (c) (S)-2-amino-3-methylbutyric Acid (R)-3-(N-(5'- prodrug is expected to have in vivo NEP activity. Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-1-(3-chloro The compound of formula IV" (Example 4A) was tested in phenyl)-5-hydroxy-1H-1,2,4-triazole-3-carbonylhy this assay and found to have a pK value at human NEP of drazino-2-hydroxypropionyloxymethyl ester; >9.0. The prodrug compounds of Examples 4B-Q either did 35 (d) (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- not inhibit the enzyme in this in vitro assay, or were not N'-1-(3-chlorophenyl)-5-hydroxy-1H-1,2,4-triazole tested since activity would not be expected in this assay; 3-carbonylhydrazino-2-hydroxypropionic acid however, based upon the activity of the active form, these acetoxymethyl ester, prodrugs are expected to have in vivo NEP activity. (e) (S)-2-amino-3-methylbutyric Acid 5-N'-((R)-2-Car The compound of formula V" (Example 5A) was tested in 40 boxy-2-hydroxyethyl)-N'-(5'-chloro-2'-fluorobiphenyl this assay and found to have a pK value at human NEP of 4-ylmethyl)hydrazinocarbonyl-2-(3-chlorophenyl)- >9.0. The prodrug compound of Examples 5B-K either did 2H-1,2,4-triazol-3-yloxymethyl ester; not inhibit the enzyme in this in vitro assay, or was not tested (f) (R)-3-N'-5-acetoxymethoxy-1-(3-chlorophenyl)-1H since activity would not be expected in this assay; however, 1.2.4 triazole-3-carbonyl-N-(5'-chloro-2'-fluorobi based upon the activity of the active form, this prodrug is are 45 phenyl-4-ylmethyl)hydrazino-2-hydroxypropionic expected to have in vivo NEP activity. acid; or The compound of Example 6A was tested in this assay a pharmaceutically acceptable salt thereof. and found to have a pK value at human NEP of >9.0. The 2. A compound of: prodrug compounds of Examples 6B-P either did not inhibit (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(2- the enzyme in this in vitro assay, or were not tested since 50 hydroxythiazole-5-carbonyl)hydrazino-2-hydroxypro activity would not be expected in this assay; however, based pionic acid ethyl ester or a pharmaceutically acceptable upon the activity of the active form, these prodrugs are salt thereof. expected to have in vivo NEP activity. 3. A compound of: The compound of formula VII was tested in this assay (a) (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- and found to have a pK value at human NEP of >9.0. The 55 N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy prodrug compounds of Examples 7A-E and 7J either did not drazino-2-hydroxypropionic acid isobutyl ester; inhibit the enzyme in this in vitro assay, or was not tested (b) (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- since activity would not be expected in this assay; however, N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy based upon the activity of the active form, this prodrug is are drazino-2-hydroxypropionic acid 5-methyl-2-oxo-1, expected to have in vivo NEP activity. 60 3dioxol-4-ylmethyl ester; The compound of formula VIII' was tested in this assay (c) (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- and found to have a pK value at human NEP of >9.0. The N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy prodrug compounds of Examples 8A-I either did not inhibit drazino-2-hydroxypropionic acid 2.2.3,3,3-pentafluo the enzyme in this in vitro assay, or was not tested since ropropyl ester; activity would not be expected in this assay; however, based 65 (d) (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- upon the activity of the active form, this prodrug is are N'-3-(2-fluorophenyl)-isoxazole-5-carbonylhy expected to have in vivo NEP activity. drazino-2-hydroxypropionic acid acetoxymethyl ester