USOO8871792B2

(12) United States Patent (10) Patent No.: US 8,871,792 B2 Hughes et al. (45) Date of Patent: Oct. 28, 2014

(54) NEPRILYSIN INHIBITORS C07D 231/14 (2013.01); C07F 9/65312 (2013.01); C07F 9/653 (2013.01) (71) Applicants: Adam D. Hughes, Belmont, CA (US); USPC ...... 514/369; 514/380: 514/381: 514/406; Melissa Fleury, San Francisco, CA (US) 514/378: 514/384: 514/383: 548/266.8; 548/263.2: 548/188: 548/248: 548/243: (72) Inventors: Adam D. Hughes, Belmont, CA (US); 548/374.1: 548/253 Melissa Fleury, San Francisco, CA (US) (ss) Field of classification search (73) Assignee: Theravance Biopharma R&D IP, LLC, None South San Francisco, CA (US) See application file for complete search history. (*) Notice: Subject to any disclaimer, the term of this (56) References Cited patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. U.S. PATENT DOCUMENTS (21) Appl. No.: 13/911,552 4,189.604 A 2/1980 Umezawa et al. 4,206,232 A 6, 1980 Ondetti et al. (22) Filed: Jun. 6, 2013 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2013/033O365A1 Dec. 12, 2013 WO WO 2011/O887.97 A1 T 2011

OTHER PUBLICATIONS Related U.S. Application Data The International Search Report for PCT/US2013/044485 dated (60) Provisional application No. 61/774,148, filed on Mar. Aug. 27, 2013. 7, 2013, provisional application No. 61/657,220, filed U.S. Appl. No. 13/905,338, Mammen et al. on Jun. 8, 2012. (Continued) (51) Int. Cl. CO7D 26L/2 (2006.01) Primary Examiner — Michael Barker CO7D 249/2249/04 (2006.01) (74)Eberle Attorney, ey, Agent, or Firm — Jeffreyy A. Hagenah;Flagenan, Shelley CO7D 26L/08 (2006.01) C07D 277/34 (2006.01) CO7D 23L/2 (2006.01) (57) ABSTRACT C07D 257/04 (2006.01) A6 IK3 L/45 (2006.01) In one aspect, the invention relates to compounds having the A6 IK3I/492 (2006.01) formula X: A6 IK3I/42 (2006.01) A6 IK3I/426 (2006.01) A6 IK3I/4I (2006.01) O X (X) CO7D 413/2 (2006.01) O N A6 IK3I/496 (2006.01) CO7D 26L/8 (2006.01) RN NH A 6LX3/5377 (2006.01) O N CO7D 405/2 (2006.01) O C07D 277/56 (2006.01) R21 A6 IK3I/675 (2006.01) O A6 IK 45/06 (2006.01) Rb CO7D 319/06 (2006.01) CO7D 231/4 (2006.01) C07F 9/653 (2006.01) Ra (52) U.S. Cl. CPC ...... C07D 249/12 (2013.01); C07D 413/12 where R,R,R,R, and Xareas defined in the specification, (2013.01); A61 K3I/4 196 (2013.01); C07D or a pharmaceutically acceptable salt thereof. The com 249/04 (2013.01); C07D 261/18 (2013.01); pounds described herein are prodrugs of compounds having A61 K3I/5377 (2013.01); C07D405/12 neprilysin inhibition activity. In another aspect, the invention (2013.01); C07D 277/56 (2013.01); A6IK relates to pharmaceutical compositions comprising these 31/426 (2013.01); A61 K3I/675 (2013.01); compounds; methods of using these compounds; and pro A61K 45/06 (2013.01); A61 K3I/42 (2013.01); cesses and intermediates for preparing these compounds. A6 IK3I/415 (2013.01); A61 K3I/41 (2013.01); C07D 319/06 (2013.01); A61 K 31/4192 (2013.01); C07D 257/04 (2013.01); 24 Claims, No Drawings US 8,871,792 B2 Page 2

(56) References Cited 2010/0305131 A1 12/2010 Coppola et al. 2010/0305145 A1 12/2010 Coppola et al. U.S. PATENT DOCUMENTS 2011, 0046397 A1 2/2011 Hook et al. 2011/O124695 A1 5, 2011 Iwaki et al. 4,374,829 2, 1983 Harris et al. 2012/O122844 A1 5, 2012 Foo 4.513,009 4, 1985 Roques et al. 2012/O122977 A1 5/2012 Coppola et al. 4,722,810 2, 1988 Delaney et al. 2012. O157383 A1 6/2012 Gendron et al. 4,906,615 3, 1990 Berger et al. 2012. O157386 A1 6, 2012 Smith et al. 4,929,641 5, 1990 Haslanger et al. 2012fO213806 A1 8/2012 Fleury et al. 4,939,261 7, 1990 Ksander 2012fO213807 A1 8/2012 Fleury et al. 4,975.444 12, 1990 Danilewicz et al. 2012/0308587 A1 12/2012 Gendron et al. 5,021,430 6, 1991 Ksander 2012/0308588 Al 12/2012 Fleury et al. 5,030,654 7, 1991 Barnish et al. 2012/0309724 A1 12/2012 Fleury et al. 5,155,100 10, 1992 Erion et al. 2013,0109639 A1 5/2013 Hughes et al. 5,208,255 5, 1993 Duhamel et al. 5,217,996 6, 1993 Ksander OTHER PUBLICATIONS 5,294,632 3, 1994 Erion et al. 5,508,272 4, 1996 Robl Ksander et al., “Dicarboxylic acid dipeptide neutral endopeptidase 5,599.951 2, 1997 Plaquevent et al. 5,677,297 10, 1997 Waldeck et al. inhibitors”, Journal of Medicinal Chemistry, 38(10): 1689-1700 5,977.075 11, 1999 Ksander et al. (1995). 6,602,866 8, 2003 Flynn et al. Misawa et al., “Structure-based design of dipeptide derivatives for 6,660,756 12, 2003 Challenger et al. the human neutral endopeptidase'. Bioorganic & Medicinal Chem 2010.0113801 A1 5, 2010 Hook et al. istry, 19: 5935-5947 (2011). US 8,871,792 B2 1. NEPRLYSIN INHIBITORS O X (X) CROSS-REFERENCE TO RELATED APPLICATIONS O n RN O 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. R21 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 where: 15 (i) R' is F: R is Cl; X is 1. Field of the Invention 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 N cesses and intermediates for preparing these compounds and methods of using these compounds to treat diseases such as hypertension, heart failure, pulmonary hypertension, and ( NO O 25 renal disease. 2. State of the Art Commonly-assigned U.S. Patent Publication No. 2012/ N O 0157386, filed on Dec. 14, 2011 to Smith et al., describes novel compounds that have activity as neprilysin inhibitors, 30 als the disclosure of which is incorporated herein by reference. In - particular, compounds of the genus:

35

40 N O

( NO 45 R’ is H and R7 is selected from CHCH, -CHCH (CH), —CHCF. —(CH2)CF, —CHCFCH. —CHCFCF, —C(CH) (CF), —CH(CH2CH)CF, are described. Depending upon the variables, compounds —CH(CH)CFCF, -(CH), OH, -CH-CH(NH) 50 COOCH, -(CH)OCH, -CHROC(O) Calkyl, within this genus can be referred to as being in the active form - CHROC(O)O Calkyl, -CHROC(O)O-cyclohexyl, or as being a prodrug, which is metabolized in Vivo to gener —Calkylene-N(CH), -CHOC(O)CHR NH, ate the active form of the compound. —CHOC(O)CHR NHC(O)C Calkyl, benzyl, and In spite of these compounds however, there remains a need for compounds and prodrugs within this genus that have 55 different metabolic and cleavage properties. For example, there remains a need for active compounds and/or prodrug compounds having improved oral absorption and for prodrug compounds that undergo rapid cleavage to form the active 60 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 8,871,792 B2 7 8 COOCH, -(CH)OCH, -CHROC(O) Calkyl, DETAILED DESCRIPTION OF THE INVENTION —CHROC(O)C) Calkyl, -CHROC(O)C)-cyclohexyl, —Calkylene-N(CH), CHOC(O)CHR NH, When describing the compounds, compositions, methods —CHOC(O)CHR NHC(O)C Calkyl, and and processes of the invention, the following terms have the following meanings unless otherwise indicated. Additionally, 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”, “includ ing.” and "having are intended to be inclusive and mean that 10 there may be additional elements other than the listed ele ments. All numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used herein are to be understood as being modified in all instances by the term “about unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations or R is selected from C(O) Calkyl, -C(O)CHR 15 that may vary depending upon the desired properties sought NH, -C(O)CHR NHC(O)C Calkyl, and -P(O) to be obtained by the present invention. At least, and not as an (OR), R is -OH, and R7 is H; or R is H, R is selected attempt to limit the application of the doctrine of equivalents from —OCHROC(O) Calkyl, OCHOC(O)CHCH to the scope of the claims, each number should at least be (CH), NH –OCHOC(O)CHCH(CH), NHC(O) construed in light of the reported significant digits and by OCH and applying ordinary rounding techniques. The term “alkyl means a monovalent saturated hydrocar bon group which may be linear or branched. Unless otherwise defined, such alkyl groups typically contain from 1 to 10 ? CH3, carbonatoms and include, for example, —Calkyl, meaning A Yo o 25 an alkyl group having from 1 to 6 carbon atoms where the carbon atoms are in any acceptable configuration. Represen tative alkyl groups include, by way of example, methyl, ethyl, “Y” n-propyl, isopropyl. n-butyl, S-butyl, isobutyl, t-butyl, n-pen O tyl, n-hexyl, and the like. 30 As used herein, the phrase “having the formula' or “having and R7 is H: the structure' is not intended to be limiting and is used in the where each R is independently Hor—C-alkyl; each R' same way that the term “comprising is commonly used. For is independently H. —CH, —CH(CH), phenyl, or benzyl; example, if one structure is depicted, it is understood that all and each R is independently H. —Calkyl, or phenyl: Stereoisomer and tautomer forms are encompassed, unless or a pharmaceutically acceptable salt thereof. 35 stated otherwise. The present invention provides compounds which are The term “pharmaceutically acceptable' refers to a mate metabolized in vivo to compounds that have been found to rial that is not biologically or otherwise unacceptable when possess neprilysin (NEP) enzyme inhibition activity. Accord used in the invention. For example, the term “pharmaceuti ingly, compounds of the invention are expected to be useful cally acceptable carrier refers to a material that can be incor and advantageous as therapeutic agents for treating patients porated into a composition and administered to a patient 40 without causing unacceptable biological effects or interacting suffering from a disease or disorder that is treated by inhib in an unacceptable manner with other components of the iting the NEP enzyme or by increasing the levels of its peptide composition. Such pharmaceutically acceptable materials Substrates. Thus, one aspect of the invention relates to a typically have met the required standards of toxicological and method of treating hypertension, heart failure, or renal dis manufacturing testing, and include those materials identified ease, comprising administering to a patient a therapeutically 45 as suitable inactive ingredients by the U.S. Food and Drug effective amount of a compound of the invention. administration. Another aspect of the invention relates to pharmaceutical The term “pharmaceutically acceptable salt' means a salt compositions comprising a pharmaceutically acceptable car prepared from a base or an acid which is acceptable for rier and a compound of the invention. administration to a patient, such as a mammal (for example, Yet another aspect of the invention relates to processes and 50 salts having acceptable mammalian safety for a given dosage intermediates useful for preparing compounds of the inven regime). However, it is understood that the salts covered by tion. Another aspect of the invention relates to a process of the invention are not required to be pharmaceutically accept preparing a pharmaceutically acceptable salt of a compound able salts, such as salts of intermediate compounds that are of formula I, comprising contacting a compound of formula I not intended for administration to a patient. Pharmaceutically in free acid or base form with a pharmaceutically acceptable acceptable salts can be derived from pharmaceutically base oracid. In other aspects, the invention relates to products 55 acceptable inorganic or organic bases and from pharmaceu prepared by any of the processes described herein, as well as tically acceptable inorganic or organic acids. In addition, novel intermediates used in Such process. when a compound of formula I contains both a basic moiety, Yet another aspect of the invention relates to the use of a Such as an amine, pyridine or imidazole, and an acidic moiety compound of formula I or a pharmaceutically acceptable salt Such as a carboxylic acid or tetrazole, Zwitterions may be thereof, for the manufacture of a medicament, especially for 60 formed and are included within the term "salt as used herein. the manufacture of a medicament useful for treating hyper Salts derived from pharmaceutically acceptable inorganic tension, heart failure, or renal disease. Another aspect of the bases include ammonium, calcium, copper, ferric, ferrous, invention relates to use of a compound of the invention for lithium, magnesium, manganic, manganous, potassium, inhibiting a NEP enzyme in a mammal Still another aspect of Sodium, and Zinc salts, and the like. Salts derived from phar the invention relates to the use of a compound of the invention 65 maceutically acceptable organic bases include salts of pri as a research tool. Other aspects and embodiments of the mary, secondary and tertiary amines, including Substituted invention are disclosed herein. amines, cyclic amines, naturally-occurring amines and the US 8,871,792 B2 10 like. Such as arginine, betaine, caffeine, choline, N,N-diben term “treating hypertension” would include preventing Zylethylenediamine, diethylamine, 2-diethylaminoethanol, hypertension from occurring, ameliorating hypertension, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, Suppressing hypertension, and alleviating the symptoms of N-ethylmorpholine, N-ethylpiperidine, glucamine, glu hypertension (for example, lowering blood pressure). The cosamine, histidine, hydrabamine, isopropylamine, lysine, term “patient' is intended to include those mammals, such as methylglucamine, morpholine, piperazine, piperadine, humans, that are in need of treatment or disease prevention or polyamine resins, procaine, purines, theobromine, triethy that are presently being treated for disease prevention or lamine, trimethylamine, tripropylamine, tromethamine and treatment of a specific disease or medical condition, as well as the like. Salts derived from pharmaceutically acceptable inor test Subjects in which the crystalline compound is being ganic acids include salts of boric, carbonic, hydrohalic (hy 10 drobromic, hydrochloric, hydrofluoric or hydroiodic), nitric, evaluated or being used in an assay, for example an animal phosphoric, sulfamic and sulfuric acids. Salts derived from model. pharmaceutically acceptable organic acids include salts of All other terms used herein are intended to have their aliphatic hydroxyl acids (for example, citric, gluconic, gly ordinary meaning as understood by those of ordinary skill in colic, lactic, lactobionic, malic, and tartaric acids), aliphatic 15 the art to which they pertain. monocarboxylic acids (for example, acetic, butyric, formic, The compound of the invention contain one or more chiral propionic and trifluoroacetic acids), amino acids (for centers and therefore, these compounds may be prepared and example, aspartic and glutamic acids), aromatic carboxylic used in various stereoisomeric forms. In some embodiments, acids (for example, benzoic, p-chlorobenzoic, diphenylace in order to optimize the therapeutic activity of the compounds tic, gentisic, hippuric, and triphenylacetic acids), aromatic hydroxyl acids (for example, o-hydroxybenzoic, p-hydroxy of the invention, e.g., to treat hypertension, it may be desir benzoic, 1-hydroxynaphthalene-2-carboxylic and 3-hydrox able that the carbon atoms have a particular (R,R), (S,S), ynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic (S,R), or (R.S) configuration or are enriched in a stereoiso acids (for example, fumaric, maleic, oxalic and Succinic meric form having such configuration. In other embodiments, acids), glucoronic, mandelic, mucic, nicotinic, orotic, the compounds of the invention are present as racemic mix pamoic, pantothenic, Sulfonic acids (for example, benzene 25 tures. Accordingly, the invention also relates to racemic mix Sulfonic, camphoSulfonic, edisylic, ethanesulfonic, tures, pure stereoisomers (e.g., enantiomers and diastereoiso isethionic, methanesulfonic, naphthalenesulfonic, naphtha mers), Stereoisomer-enriched mixtures, and the like unless lene-1,5-disulfonic, naphthalene-2,6-disulfonic and p-tolu otherwise indicated. When a chemical structure is depicted enesulfonic acids), Xinafoic acid, and the like. herein without any stereochemistry, it is understood that all As used herein, the term “prodrug is intended to mean an 30 possible stereoisomers are encompassed by Such structure. inactive (or significantly less active) precursor of a drug that Similarly, when a particular stereoisomer is shown or named is converted into its active form in the body under physiologi herein, it will be understood by those skilled in the art that cal conditions, for example, by normal metabolic processes. minor amounts of other stereoisomers may be present in the Such compounds may not necessarily possess pharmacologi compositions of the invention unless otherwise indicated, cal activity at NEP, but may be administered orally or 35 provided that the utility of the composition as a whole is not parenterally and thereafter metabolized in the body to form a eliminated by the presence of such other isomers. Individual compound that is pharmacologically active at NEP. Stereoisomers may be obtained by numerous methods that are The term “therapeutically effective amount’ means an well known in the art, including chiral chromatography using amount sufficient to effect treatment when administered to a a suitable chiral stationary phase or Support, or by chemically patient in need thereof, that is, the amount of drug needed to 40 converting them into diastereoisomers, separating the diaste obtain the desired therapeutic effect. For example, a thera reoisomers by conventional means Such as chromatography peutically effective amount for treating hypertension is an or recrystallization, then regenerating the original stereoiso amount of compound needed to, for example, reduce, Sup C. press, eliminate, or prevent the symptoms of hypertension, or Additionally, where applicable, all cis-trans or E/Z isomers to treat the underlying cause of hypertension. In one embodi 45 (geometric isomers), tautomeric forms and topoisomeric ment, a therapeutically effective amount is that amount of forms of the compounds of the invention are included within drug needed to reduce blood pressure or the amount of drug the scope of the invention unless otherwise specified. needed to maintain normal blood pressure. On the other hand, The compounds of the invention, as well as those com the term “effective amount’ means an amount sufficient to pounds used in their synthesis, may also include isotopically obtain a desired result, which may not necessarily be a thera 50 labeled compounds, that is, where one or more atoms have peutic result. For example, when studying a system compris been enriched with atoms having an atomic mass different ing a NEP enzyme, an “effective amount” may be the amount from the atomic mass predominately found in nature. needed to inhibit the enzyme. Examples of isotopes that may be incorporated into the com The term “treating or “treatment” as used herein means pounds of formula I, for example, include, but are not limited the treating or treatment of a disease or medical condition 55 to, H, H, C, C, SN, O, 7.O., SS, 3°C1, and F. Of (such as hypertension) in a patient, such as a mammal (par particular interest are compounds of formula I enriched in ticularly a human) that includes one or more of the following: tritium or carbon-14 which can be used, for example, in tissue (a) preventing the disease or medical condition from occur distribution studies; compounds of the invention enriched in ring, i.e., preventing the reoccurrence of the disease or medi deuterium especially at a site of metabolism resulting, for cal condition or prophylactic treatment of a patient that is 60 example, in compounds having greater metabolic stability; pre-disposed to the disease or medical condition; (b) amelio and compounds of formula I enriched in a positron emitting rating the disease or medical condition, i.e., eliminating or isotope, such as ''C, F, 'O and 'N, which can be used, for causing regression of the disease or medical condition in a example, in Positron Emission Topography (PET) studies. patient; (c) Suppressing the disease or medical condition, i.e., The nomenclature used herein to name the compounds of slowing or arresting the development of the disease or medi 65 the invention is illustrated in the Examples herein. This cal condition in a patient; or (d) alleviating the symptoms of nomenclature has been derived using the commercially avail the disease or medical condition in a patient. For example, the able AutoNom software (MDL, San Leandro, Calif.). US 8,871,792 B2 11 12 U.S. Patent Publication No. 2012/0157386 specifically -continued disclosed (R)-3-(N-(5'-chloro-2'-fluorobiphenyl-4-ylm (Ib) ethyl)-N'-1-(3-chlorophenyl)-5-oxo-4,5-dihydro-1H-1.2. 4triazole-3-carbonylhydrazino-2-hydroxypropionic acid, which is represented by formula I': O NN (I") N

NH) {N O N1 H 10 R2 -O

C HO N 15 OH F For compounds of formula Ia, R is Hand R is selected from —CHCH. —CHCH(CH), —CHCF. —(CH2)CF, —CHCFCH. —CHCFCF, —C(CH)(CF), —CH (CHCH)CF, —CH(CH)CFCF —(CH), OH, In one embodiment, this compound is referred to as the active CH-CH(NH)COOCH, -(CH),OCH, CHROC form and is administered as a prodrug, which is metabolized (O) Calkyl, -CHROC(O)O Calkyl, -CHROC in vivo to form the compound of formula I. This compound 25 can also exist in its tautomer form, (R)-3-N-(5'-chloro-2'- (O)O-cyclohexyl, —Calkylene-N(CH), —CHOC(O) fluorobiphenyl-4-ylmethyl)-N'-1-(3-chlorophenyl)-5-hy CHR NH, CHOC(O)CHR NHC(O)O Calkyl, droxy-1H-1.2.4 triazole-3-carbonylhydrazino-2-hydrox benzyl, and ypropionic acid. One aspect of the invention relates to other prodrugs of the 30 compound of formula I". These prodrugs are represented by formula X, where R is F. R is C1, and X is:

35 C O

( O 40 or R is selected from C(O) Coalkyl, -C(O)CHR NH, -C(O)CHR" NHC(O)O Calkyl, and -P(O) (OR), and R7 is H; where each R is independently H or C - Cisalkyl; each R" is independently H, CH, -CH (CH), phenyl, or benzyl; and each R is independently H, als 45 —Calkyl, or phenyl; or a pharmaceutically acceptable salt S thereof. For compounds of formula Ib, R is H, R is -OH, In one embodiment, these compounds are represented by and R7 is selected from CHOC(O)CH, and —CHOC(O) formula Ia or Ib: CHCH(CH), NH, or R is H, R is selected from –OCHOC(O)CH and OCHOC(O)CHCH(CH), 50 NH, and R7 is H; where each R is independently H or (Ia) —C-alkyl; each R" is independently H, —CH, -CH (CH), phenyl, or benzyl; and each R is independently H, —Calkyl, or phenyl: or a pharmaceutically acceptable salt thereof. O N C 55 In one particular embodiment of the compounds of For mula Ia, R is H and R7 is selected from —CHCH and 7 O ) { -l —CH-CH(CH). In one particular embodiment of the com RN NH N O pounds of Formula Ib, R is H. Ris-OH, and R is selected 60 from CHOC(O)CH, and —CHOC(O)CHCH(CH), NH; or R is H, R is selected from —OCHOC(O)CH and –OCHOC(O)CHICH(CH), NH, and R7 is H. C The compound (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- ylmethyl)-N'-(2-hydroxythiazole-5-carbonyl)hydrazino-2- 65 hydroxypropionic acid is also specifically disclosed in U.S. Patent Publication No. 2012/0157386, and is represented by formula II': US 8,871,792 B2 13 14 - Cisalkyl; each R" is independently H, CH, -CH (II) (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 Hand R7 is CHCH. HO The compound (R)-3-(N-(5'-chloro-2'-fluorobiphenyl-4- ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5-carbonyl-hy OH drazino-2-hydroxypropionic acid is also specifically dis 10 closed in U.S. Patent Publication No. 2012/0157386, and is represented by formula III':

(III) 15 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 II". U.S. Patent Publication No. 2012/0157386 also disclosed the isobutyl HO ester prodrug of the compound of formula II". OH Another aspect of the invention relates to other prodrugs of the compound of formula II". These prodrugs are represented by formula X, where R is F. R is C1, and X is: 25

30 In one embodiment, this compound is referred to as the active In one embodiment, these compounds are represented by form and is administered as a prodrug, which is metabolized formula II: in vivo to form the compound of formula III". U.S. Patent Publication No. 2012/0157386 also disclosed the ethyl ester (II) and mofetil ester prodrugs of the compound of formula III". Another aspect of the invention relates to other prodrugs of 35 the compound of formula III". These prodrugs are represented by formula X, where R is F. R is C1, and X is:

40

45 In one embodiment, these compounds are represented by formula III: where R is H and R is selected from —CH2CHs.

—CHCF. —(CH2)CF, —CHCFCH. —CHCFCF, (III) —C(CH) (CF), —CH(CHCH)CF, —CH(CH) 50 CFCF, -(CH), OH, -CH-CH(NH)COOCH, —(CH)OCH, -CHROC(O) Calkyl, -CHROC (O)O Calkyl, —CHROC(O)O-cyclohexyl, Calky lene-N(CH), —CHOC(O)CHR NH, -CHOC(O) CHR NHC(O)O Calkyl, benzyl, and 55

)." 60 OY O O or R is selected from C(O) Coalkyl, -C(O)CHR 65 where R is H and R is selected from CH-CH(CH), NH, -C(O)CHR NHC(O)O Calkyl, and -P(O) —CHCF. —(CH2)CF, —CHCFCH. —CHCFCF, (OR), and R is H; where each R is independently H or —C(CH) (CF), —CH(CHCH)CF, —CH(CH) US 8,871,792 B2 15 16 CFCF, -(CH), OH, -CH-CH(NH)COOCH, in vivo to form the compound of formula IV. U.S. Patent -(CH)OCH, -CHROC(O) Calkyl, -CHROC Publication No. 2012/0157386 also disclosed the ethyl ester, (O)O Calkyl, —CHROC(O)O-cyclohexyl, Calky isopropyl ester, and isobutyl ester prodrugs of the compound lene-N(CH), —CHOC(O)CHR NH, -CHOC(O) of formula IV". CHR NHC(O)O Calkyl, benzyl, and Another aspect of the invention relates to other prodrugs of the compound of formula IV". These prodrugs are represented by formula X, where R is F. R is C1, and X is:

10

15 where R is H or —CH. In one embodiment, these com or R is selected from C(O) Coalkyl, -C(O)CHR pounds are represented by IV: NH, -C(O)CHR NHC(O)C 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 thereof. In one particular embodiment of the compounds of For O Y.M mula III, R is Hand R is selected from —CH2CH(CH), 25 O O —CHCFCF —(CH)OCH, —CHOC(O)CH, RN NH —CHOC(O)(CH),CH, —CHOC(O)OCHCH O N —CHOC(O)OCH(CH), —CH(CH)OC(O)O-cyclo hexyl, -CHOC(O)CHCH(CH), NHC(O)OCH, and O 30 R21 C

F 35 where R is H and R7 is selected from —CHCF, —(CH2)CF, —CHCFCH. —CHCFCF, —C(CH) (CF), —CH(CHCH)CF, -CH(CH)CFCF, 40 —(CH), OH, -CH-CH(NH)COOCH,-(CH)OCH, or Ris-P(O)(OH), and R is H. - CHROC(O) Calkyl, -CHROC(O)O Calkyl, The compound (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- - CHROC(O)O-cyclohexyl, —Calkylene-N(CH), ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hydrazino CHOC(O)CHR NH, CHOC(O)CHR NHC(O) 2-hydroxypropionic acid is also specifically disclosed in U.S. O—C-alkyl, benzyl, Patent Publication No. 2012/0157386, and is represented by 45 formula IV':

(IV) 50 >~~~ Nuk and O Y. CH3: O O 55 NH HO N1

OH

60 or R is selected from C(O) Calkyl, -C(O)CHR NH, -C(O)CHR" NHC(O)O 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 65 alkyl; each Risindependently H, CH, CH(CH4), phe In one embodiment, this compound is referred to as the active nyl, or benzyl, and each R is independently H. —Calkyl, form and is administered as a prodrug, which is metabolized or phenyl; or a pharmaceutically acceptable salt thereof. US 8,871,792 B2 17 18 In one particular embodiment of the compounds of For In one embodiment, these compounds are represented by mula IV, R is —CH, R is H. and R7 is selected from formula V:

(V) hexyl, -CHOC(O)CHCH(CH), NH, -CHOC(O)CH O CH(CH),NHC(O)OCH, is N O N / N 10

15

In another particular embodiment of the compounds of For where R is Hand R is selected from CHCH-CHCH mula IV, R is —CH: R is selected from C(O)CHCH (CH), —CH2CF, -(CH2)CF, —CHCFCH C(O)CH-NH C(O)CH(CH)NH, C(O)CHCH —CHCFCF, —C(CH)(CF), —CH(CHCH)CF, (CH), NH, and C(O)CHCH(CH), NHC(O) 25 —CH(CH)CFCF, -(CH), OH, -CH-CH(NH) OCH; and R7 is H. In yet another particular embodiment of COOCH, -(CH)OCH, -CHROC(O) Calkyl, the compounds of Formula IV, R is —CH, R is C(O) - CHRO-C(O)O Calkyl, -CHROC(O)O-cyclo CH-NH and R7 is —CHCH. hexyl, C-alkylene-N(CH), —CHOC(O)CHR The compound (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- NH, -CHOC(O)CHR" NHC(O)O Calkyl, benzyl, ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triaz 30 and ole-3-carbonyl)hydrazino-2-hydroxypropionic acid is spe cifically disclosed in U.S. Patent Publication No. 2012/ 0157386, and is represented by formula V':

35 (V) O )." in “Y” O O s N 40 or R is selected from C(O) Calkyl, -C(O)CHR NH, -C(O)CHR" NHC(O)O Calkyl, and -P(O) HO (OR), and R is H; where each R is independently H or OH 45 - Calkyl; each R" is independently H, CH, CH (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 50 mula V. R is H and R is selected from —CH2CHs. —CHCH(CH), —CHCFCF, -CHOC(O)CH, —CHOC(O)—(CH2)CH, —CHOC(O)OCHCH In one embodiment, this compound is referred to as the active —CHOC(O)OCH(CH), —CH(CH)OC(O)O-cyclo form and is administered as a prodrug, which is metabolized hexyl, -CHOC(O)CHCH(CH), NHC(O)OCH, and in vivo to form the compound of formula V. 55 Another aspect of the invention relates to other prodrugs of the compound of formula V. These prodrugs are represented by formula X, where R is F. R is C1, and X is:

60 is O w N

w N 65 w w Another aspect of the invention relates to compounds of formula X, where R is F. R is C1, and X is: US 8,871,792 B2 19 20

—CH-CH(CH), where each R is independently H or —Calkyl; each R" is independently H, —CH, -CH (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 VIa and VIb, R is H, R is H, and R7 is selected from H, —CHCH. —CHCH(CH), —CHCFCH. —(CH) In one embodiment, these compounds are represented by O CH, —CHOC(O)OCHCH —(CH), N(CH), formula VIa or VIb: 10 —(CH2). N(CH), —(CH), N(CH), —CH2OC(O) CHCH(CH), NH, "Y-N4 (VIa) O S. 15 O O R No N1 NH R21 O

C

25 F 4 (VIb) R M N-N 30 o, l/ In yet another embodiment of the compounds of Formula VIa O O and VIb, R is H, R is R NH selected from —CHOC(O)CHCH(CH), NHC(O) No N1 OCH and —CHOC(O)CHCH(CH), NH, and R7 is H. 35 In still another particular embodiment of the compounds of Formula VIa and VIb, R is selected from C(O)CH, —C(O)CH(CH), and –C(O)CHCH(CH), R is H, and R7 is H. In yet another embodiment of the compounds of Formula 40 VIa and VIb, R is H, R is —CH OP(O)(OH), or —CHOC(O)CHCH(CH), NH, and R7 is CHCH or —CH-CH(CH). where RandR are H, and R7 is selected from H, CHCH In yet another embodiment of the compounds of Formula —CH-CH(CH), —CHCF, —(CH2)CF, VIa and VIb, R is –C(O)CHCH(CH), NH R is H, and —CHCFCH. —CHCFCF, —C(CH)(CF), —CH 45 R’ is CHCH or -CHCH(CH). (CHCH)CF —CH(CH)—CFCF —(CH), OH, The compound (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- CH-CH(NH)COOCH, -(CH)OCH, CHROC ylmethyl)-N'-(1H-tetrazole-5-carbonyl)hydrazino-2-hy (O) Calkyl, -CHROC(O)O C. alkyl, -CHROC droxypropionic acid is specifically disclosed in U.S. Patent (O)O-cyclohexyl, —Calkylene-N(CH), —CHOC Publication No. 2012/0157386, and is represented by formula (O) CHR NH, CHOC(O)CHR NHC(O)O VII': Calkyl, benzyl, 50 (VII)

CH3 - H(CH2)2 N N ~ o 55 N- O HO N OH or R is H, R is selected from CHOC(O)CHCH 60 (CH), NHC(O)OCH and CHOC(O)CHCH(CH), NH, and R7 is H; or R is selected from C(O) Calkyl, C(O)CHR NH, C(O)CHR NHC(O)C C. alkyl, and -P(O)(OR), R is H, and R7 is H; or R is H. R. is —CHOP(O)(OR), or -CHOC(O)CHCH(CH), 65 NH, and R7 is —CHCH or -CHCH(CH); or R is C —C(O)CHCH(CH)NHR' is H, and R7 is CHCH or US 8,871,792 B2 21 22 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 VII". 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. U.S. Patent Publication No. 2012/0157386 also disclosed the ethyl ester, isopropyl ester, and isobutyl ester prodrugs of the compound of formula VII". 10 Another aspect of the invention relates to other prodrugs of or R is H, R is selected from CHOC(O)CHCH the compound of formula VII". These prodrugs are repre (CH), NHC(O)OCH and —CHOC(O)CHCH(CH), sented by formula X, where R is F. R is C1, and X is: NH, and R is selected from H and —CH2CH; or R is selected from C(O) Calkyl, -C(O)CHR NH, 15 —C(O)CHR NHC(O)C Calkyl, and -P(O)(OR), R and R' and Rare H; where each R is independently H or M A w N N R4 —Calkyl; each R" is independently H, —CH, -CH , N/ O a N1 . (CH), phenyl, or benzyl; and each R is independently H, W / —Calkyl, or phenyl; or a pharmaceutically acceptable salt w NRN thereof. In one particular embodiment of the compounds of For mula VIIa and VIIb, Rand Rare H, and R is selected from In one embodiment, these compounds are represented by —CHCFCF, -CHOC(O)CH, CHOC(O)CHCH formula VIIa or VIIb: —CHOC(O)(CH),CH, —CHOC(O)OCH(CH), 25 CHOC(O)CHCH(CH), NH, CHOC(O)CHCH (CH), NHC(O)OCH, and (VIIa) R N-N N \ 30 O S. M O N )." O O RN NH O N Y O 35 R21 O In another embodiment of the compounds of Formula VIIa and VIIb, R is H, R is CHOC(O)CHCH(CH), NH, C and R is —CHCH. In still another embodiment of the compounds of Formula VIIa and VIIb, or R is selected from 40 - C(O)CH, -C(O)CHCH, C(O)CHCH(CH), NH, F and –C(O)CHICH (CH), NHC(O)OCH, and R and R' (VIIb) are H. The compound (R)-3-N-(3'-chlorobiphenyl-4-ylmethyl)- R4 N'-(3-hydroxy-isoxazole-5-carbonyl)hydrazino-2-hydrox

e \ 45 ypropionic acid is specifically disclosed in U.S. Patent Pub N Y lication No. 2012/0157386, and is represented by formula VIII: O s NM

RN O N NH 50 (VIII) R21 O

C 55 HO

F OH where R and Rare H, and R is selected from —CHCF, 60 —(CH),CF, —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, -CHROC (O)O-cyclohexyl, —Calkylene-N(CH), —CHOC(O) 65 In one embodiment, this compound is referred to as the active CHR NH, CHOC(O)CHR NHC(O)O Calkyl, form and is administered as a prodrug, which is metabolized benzyl, and in vivo to form the compound of formula VIII". U.S. Patent US 8,871,792 B2 23 24 Publication No. 2012/0157386 also disclosed the ethyl ester, OCH(CH), –OCHOC(O)CHCH(CH), NH, isopropyl ester, butyl ester, isobutyl ester, hexyl ester, heptyl –OCHOC(O). CHCH(CH), NHC(O)CCH, and ester, benzyl ester, medoxomil ester, 2-fluoro-1-fluorom ethyl-ethyl ester, and 2.2.3,3,3-pentafluoropropyl ester pro CH3, drugs of the compound of formula VIII". 5 A no o Another aspect of the invention relates to other prodrugs of the compound of formula VIII". These prodrugs are repre sented by formula X, where R is H. R. is C1, and X is: “Y” 10 O and R7 is H. R3 The compound (R)-3-N-(2,5'-dichlorobiphenyl-4-ylm ethyl)-N'-(1-hydroxy-1H-1.2.3 triazole-4-carbonyl)hy In one embodiment, these compounds are represented by 15 drazino-2-hydroxypropionic acid is specifically disclosed in formula VIII: U.S. Patent Publication No. 2012/0157386, and is repre sented by formula IX': (VIII) (IX) N 2O N2e Y r O S. OH O

NH HO N1 25 OH O C

C where R is H, R is -OH, and R is selected from —CHCF. —(CH2)CF, —C(CH) (CF), —CH In one embodiment, this compound is referred to as the (CHCH)CF, —CH(CH)CFCF —(CH), OH, 35 active form and is administered as a prodrug, which is CH-CH(NH)COOCH, -(CH)OCH, CHROC metabolized in vivo to form the compound of formula IX'. (O) Calkyl, -CHROC(O)O C, alkyl, -CHROC U.S. Patent Publication No. 2012/0157386 also disclosed the (O)O-cyclohexyl, —Calkylene-N(CH), —CHOC(O) isopropylester, isobutyl ester, and heptylesterprodrugs of the CHCH(CH), NH, and —CHOC(O)CHCH(CH)– compound of formula IX'. NHC(O)CCH; or R is selected from –C(O) Calkyl, 40 Another aspect of the invention relates to other prodrugs of -C(O)CHR NH, C(O)CHR NHC(O)O the compound of formula IX'. These prodrugs are represented Calkyl, and -P(O)(OR), R is -OH, and R7 is H; or R by formula X, where R is C1, R is C1, and X is: is H, R is selected from —OCHROC(O) Calkyl, OCHOC(O)CHCH(CH), NH, OCHOC(O)CH NN CH(CH), NHC(O)CCH, and ' \ 45 , S N O CH3, S&o1-( In one embodiment, these compounds are represented by “Y” 50 formula IXa or IXb: O (IXa) and R is H; where each Risindependently Hor—C-alkyl: N 2N each R" is independently H, CH, -CH(CH), phenyl, or 55 NN R4 benzyl; and each R is independently H. —Calkyl, or phe O S. nyl; or a pharmaceutically acceptable salt thereof. O In one particular embodiment of the compounds of For mula VIII, R is H, R is -OH, and R7 is selected from RN O N NH —CHOC(O)CH, CHOC(O)CHCH(CH), NH, and —CHOC(O)CHCH(CH), NHC(O)CCH. In another 60 -O particular embodiment of the compounds of Formula VIII, R R2 is selected from —C(O)CHCH. —C(O)CHCH(CH), —C(O)CHCH(CH), NH, and C(O)CHCH(CH)– O C NHC(O)CCH R is -OH, and R7 is H. In yet another particular embodiment of the compounds of Formula VIII, R is H, R is selected from —OCHOC(O)CH, OCHOC C (O)(CH),CH, OCHOC(O)OCHCH, OCHOC(O) US 8,871,792 B2 25 26 -continued 4 (IXb) R A N- N N O M O RN NH O N 10 In yet another embodiment of the compounds of Formula IXa R21 O and IXb, R is H, R is selected from —OCHOC(O)CH, –OCHOC(O)(CH2)CH, OCHOC(O)CHCH (CH), NH, OCHOC(O)CHCH(CH), NHC(O) 15 OCH and C CH3, where R is H, R is -OH, and R is selected from —CHCF. —(CH2)CF. —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, -CHROC (O)O-cyclohexyl, —Calkylene-N(CH), —CHOC(O) 25 CHR NH, CHOC(O)CHR NHC(O)O Calkyl, and R7 is H. and General Synthetic Procedures 30 Compounds of the invention can be prepared from readily available starting materials using the following general meth ods, the procedures set forth in the Examples, or by using other methods, reagents, and starting materials that are known 35 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 starting 40 materials known to those of ordinary skill in the art. It will or R is selected from C(O) Coalkyl, -C(O)CHR also be appreciated that where typical or preferred process NH, -C(O)CHR NHC(O)C Calkyl, and -P(O) conditions (for example, reaction temperatures, times, mole (OR), R is -OH, and R7 is H; or R is H, R is selected ratios of reactants, solvents, pressures, etc.) are given, other from —OCHROC(O) Calkyl, - OCHOC(O)CHCH process conditions can also be used unless otherwise stated. (CH), NH –OCHOC(O)CHCH(CH), NHC(O) 45 In some instances, reactions were conducted at room tem OCH and perature and no actual temperature measurement was taken. It is understood that room temperature can be taken to mean a temperature within the range commonly associated with the CH3, ambient temperature in a laboratory environment, and will 50 typically be in the range of about 18°C. to about 30°C. In ? O o other instances, reactions were conducted at room tempera ture and the temperature was actually measured and recorded. While optimum reaction conditions will typically vary “Y” depending on various reaction parameters such as the particu O 55 lar reactants, solvents and quantities used, those of ordinary skill in the art can readily determine suitable reaction condi and R7 is H; where each R is independently Hor—C-alkyl; tions using routine optimization procedures. each R" is independently H, CH, -CH(CH), phenyl, or Additionally, as will be apparent to those skilled in the art, benzyl; and each R is independently H. —Calkyl, or phe conventional protecting groups may be necessary or desired nyl; or a pharmaceutically acceptable salt thereof. 60 to prevent certain functional groups from undergoing undes ired reactions. The choice of a Suitable protecting group for a In one particular embodiment of the compounds of For particular functional group as well as Suitable conditions and mula IXa and IXb, R is H, Ris-OH and R7 is selected from reagents for protection and deprotection of Such functional —CHOC(O)CH, -CHOC(O)(CH),CH, -CHOC groups are well-known in the art. Protecting groups other than (O) OCHCH, -CHOC(O)OCH(CH), —CH(CH) 65 those illustrated in the procedures described herein may be OC(O)O-cyclohexyl, -CHOC(O). CHCH(CH), NHC used, if desired. For example, numerous protecting groups, (O)OCH, and and their introduction and removal, are described in T. W. US 8,871,792 B2 27 28 Greene and G. M. Wuts, Protecting Groups in Organic Syn resulting mixture or reaction product may be further treated in thesis, Fourth Edition, Wiley, New York, 2006, and references order to obtain the desired product. For example, the resulting cited therein. mixture or reaction product may be subjected to one or more Carboxy-protecting groups are Suitable for preventing of the following procedures: concentrating or partitioning undesired reactions at a carboxy group, and examples (for example, between EtOAc and water or between 5% THF include, but are not limited to, methyl, ethyl, t-butyl, benzyl in EtOAc and 1M phosphoric acid); extraction (for example, (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), tri with EtOAc, CHCl, DCM. chloroform); washing (for methylsilyl (TMS), t-butyldimethylsilyl (TBDMS), diphe example, with Saturated aqueous NaCl, saturated aqueous nylmethyl (benzhydryl, DPM) and the like. Amino-protect NaHCO, NaCO (5%), CHC1 or 1M NaOH); drying (for ing groups are suitable for preventing undesired reactions at 10 an amino group, and examples include, but are not limited to, example, over MgSO4, over NaSO4, or in vacuo); filtering; t-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl crystallizing (for example, from EtOAc and hexanes); being (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimeth concentrated (for example, in vacuo); and/or purification ylsilyl (TMS), t-butyldimethylsilyl (TBDMS), and the like. (e.g., silica gel chromatography, flash chromatography, pre Standard deprotection techniques and reagents are used to 15 parative HPLC, reverse phase-HPLC, or crystallization). remove the protecting groups, and may vary depending upon By way of illustration, compounds of the invention, as well which group is used. For example, Sodium or lithium hydrox as their salts, can be prepared as shown in Schemes I-IV. ide is commonly used when the carboxy-protecting group is methyl, an acid such as TFA or HCl (e.g., 4.0 M HCl in 1,4-dioxane) is commonly used when the carboxy-protecting group is ethyl or t-butyl, and H/Pd/C may be used when the Scheme I carboxy-protecting group is benzyl. A BOC amino-protect ing 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 catalytic hydrogenation 25 conditions such as H. (1 atm) and 10% Pd/C in an alcoholic solvent (“H/Pd/C). Leaving groups are functional groups or atoms that can be displaced by another functional group or atom in a Substitu tion reaction, such as a nucleophilic Substitution reaction. By 30 way of example, representative leaving groups include chloro, bromo and iodo groups; sulfonic ester groups, such as Ra mesylate, tosylate, brosylate, nosylate and the like; and acy loxy groups. Such as acetoxy, trifluoroacetoxy and the like. HO-R He Suitable bases for use in these schemes include, by way of 35 illustration and not limitation, potassium carbonate, calcium carbonate, sodium carbonate, triethylamine (Et-N), pyridine, 1,8-diazabicyclo-5.4.0]undec-7-ene (DBU), N,N-diisopro pylethylamine (DIPEA), 4-methylmorpholine, sodium hydroxide, potassium hydroxide, potassium t-butoxide, and 40 metal hydrides. Suitable inert diluents or solvents for use in these schemes include, by way of illustration and not limitation, tetrahydro furan (THF), acetonitrile (MeCN), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dimethylsulfoxide 45 (DMSO), toluene, dichloromethane (DCM), chloroform Ra (CHCl), carbon tetrachloride (CCI), 1,4-dioxane, metha nol, ethanol, water, diethyl ether, acetone, and the like. Scheme I is a transesterification reactions. Generally, this Suitable carboxylic acid/amine coupling reagents include reaction involves reacting the ester with heat, the desired benzotriazol-1-yloxytris(dimethylamino)phosphonium 50 hexafluorophosphate (BOP), benzotriazol-1-yloxytripyrroli alcohol (HO R7) and a suitable acid catalyst, for example dinophosphonium hexafluorophosphate (PyBOP), N.N.N', hydrochloric acid. The HO Ralcohols are either commer N-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium cially available or can be prepared by techniques that are hexafluorophosphate (HATU), 1,3-dicyclohexylcarbodiim known in the art or described herein. Exemplary HO R7 ide (DCC), N-(3-dimethylaminopropyl)-N'-ethylcarbodiim 55 compounds include HO CHCF HO—(CH2)CF, ide (EDC), carbonyldiimidazole (CDI), 1-hydroxybenzotria HO CHCFCH HO CHCFCF HO C(CH) Zole (HOBt), and the like. Coupling reactions are conducted (CF), HO CH(CHCH)CF, HO CH(CH)CFCF, in an inert diluent in the presence of a base such as DIPEA, benzyl alcohol, and and are performed under conventional amide bond-forming conditions. 60 All reactions are typically conducted at a temperature CH3. within the range of about -78 C to 100° C., for example at room temperature. Reactions may be monitored by use of thin 101-( layer chromatography (TLC), high performance liquid chro O O matography (HPLC), and/or LCMS until completion. Reac 65 tions may be complete in minutes, or may take hours, typi YO cally from 1-2 hours and up to 48 hours. Upon completion, the US 8,871,792 B2

-continued O X Scheme II O X O N NH O N HO N1 NH HO N1 R21 O R21 O 10 Rb + Ra

Ra 15 Scheme III is a nucleophilic substitution reaction, where L is a suitable leaving group. Generally, this reaction is conducted L-R - - in the presence of a suitable base such as N,N-diisopropyl ethylamine in a suitable inert diluent or solvent such as dichloromethane. The L-R compound is either commer O X cially available or can be prepared by techniques that are O N known in the art or described herein. Exemplary L-R com RN O N NH pounds include C1 C(O)—CH, C1 C(O)—CH(CH), and Cl—C(O)—CH2CH(CH). O R21 25 Rb Scheme IW

Ra 30

Scheme II is a nucleophilic substitution reaction, where L is a Suitable leaving group. Generally, this reaction is conducted in the presence of a Suitable base Such as triethylamine in a 35 suitable inert diluent or solvent such as acetone. The L-R compounds are either commercially available or can be pre pared by techniques that are known in the art or described herein. Exemplary L-R7 compounds include Br—(CH),OH, Br (CH),OH, Br (CH)OCH, Br CHOC(O)CH, 40 C1 CHOC(O)(CH) CH, C1 CHOC(O)OCHCH C1 CHOC(O)CCH(CH), C1 CHOC(O)O-cyclo hexyl, (S)-2-benzyloxycarbonylamino-3-methyl-butyric acid chloromethyl ester, and (S)-2-t-butoxycarbonylamino 3-methyl-butyric acid chloromethyl ester. 45 Alternately, in Scheme II, an alcohol have be used in place of the L-R7, for example HO Calkylene-N(CH) in a coupling reaction using HOBt and EDC. 50

Scheme III Ra O X O n Scheme IV is a coupling reaction, where P is H or a suitable NH 55 amino-protecting group. When P is an amino protecting HO N1 group, the process further comprises deprotecting the com pound before or in situ with the coupling step. Exemplary O coupling reagents include HATU and HOBt with EDC. Gen R21 erally, these reactions are conducted in the presence of a base Rb + 60 such as DIPEA or 4-methylmorpholine, and an inert diluent or solvents such as DMF or DMA. The carboxylic acid start ing materials are generally commercially available or can be prepared using procedures that are known in the art. Ra Further details regarding specific reaction conditions and 65 L-R2 -- other procedures for preparing representative compounds of the invention or intermediates thereof are described in the Examples set forth below. US 8,871,792 B2 31 32 Utility Thus, the compounds are expected to have other physiologi cal actions, for example, on the renal, central nervous, repro The compounds of formula I-V and VII'-IX" have activity ductive and gastrointestinal systems. as neprilysin inhibitors, and are expected to have therapeutic utility as a neprilysin inhibitor. Prodrugs of these compounds, Cardiovascular Diseases once metabolized in vivo, are expected to have the same utility. Thus, when discussing the activity of the compounds By potentiating the effects of vasoactive peptides like the of the invention, it is understood that these prodrugs have the natriuretic peptides and bradykinin, compounds of the inven expected activity once metabolized. tion are expected to find utility in treating and/or preventing Exemplary assays include by way of illustration and not 10 medical conditions such as cardiovascular diseases. See, for limitation, assays that measure NEP inhibition. Useful sec example, Rogues et al. (1993) Pharmacol. Rev. 45:87-146 ondary assays include assays to measure ACE inhibition and and Dempsey et al. (2009) Amer: J. of Pathology 174(3):782 aminopeptidase P (APP) inhibition (e.g., as described in 796. Cardiovascular diseases of particular interest include Sulpizio et al. (2005).JPET315:1306-1313). A pharmacody hypertension and heart failure. Hypertension includes, by namic assay to assess the in vivo inhibitory potencies for ACE 15 way of illustration and not limitation: primary hypertension, and NEP in anesthetized rats is described in Seymour et al. which is also referred to as essential hypertension or idio (1985) Hypertension 7(Suppl I):I-35-I-42 and Wigle et al. pathic hypertension; secondary hypertension; hypertension (1992) Can. J. Physiol. Pharmacol. 70:1525-1528), where with accompanying renal disease; severe hypertension with ACE inhibition is measured as the percent inhibition of the or without accompanying renal disease; pulmonary hyperten angiotensin I pressor response and NEP inhibition is mea Sion, including pulmonary arterial hypertension; and resistant Sured as increased urinary cyclic guanosine 3',5'-monophos hypertension. Heart failure includes, by way of illustration phate (cGMP) output. and not limitation: congestive heart failure; acute heart fail There are also many in vivo assays that can be used. The ure; chronic heart failure, for example with reduced left ven conscious spontaneously hypertensive rat (SHR) model is a tricular ejection fraction (also referred to as systolic heart refill dependent hypertension model. See for example, Inten 25 failure) or with preserved left ventricular ejection fraction gan et al. (1999) Circulation 100(22):2267-2275 and Badyal (also referred to as diastolic heart failure); and acute and et al. (2003) Indian Journal of Pharmacology 35:349-362. chronic decompensated heart failure, with or without accom The conscious desoxycorticosterone acetate-salt (DOCA panying renal disease. Thus, one embodiment of the invention salt) rat model is a Volume dependent hypertension model that relates to a method for treating hypertension, particularly is useful for measuring NEP activity. See for example, Tra 30 primary hypertension or pulmonary arterial hypertension, pani et al. (1989) J. Cardiovasc. Pharmacol. 14:419-424, comprising administering to a patient a therapeutically effec Intengan et al. (1999) Hypertension 34(4):907-913, and tive amount of a compound of the invention. Badyaletal. (2003) supra). The DOCA-salt model is particu For treatment of primary hypertension, the therapeutically larly useful for evaluating the ability of a test compound to effective amount is typically the amount that is sufficient to reduce blood pressure as well as to measure a test com 35 lower the patient’s blood pressure. This would include both pounds ability to prevent or delay a rise in blood pressure. mild-to-moderate hypertension and severe hypertension. The Dahl salt-sensive (DSS) hypertensive rat model is a When used to treat hypertension, the compound may be model of hypertension that is sensitive to dietary salt (NaCl), administered in combination with other therapeutic agents and is described, for example, in Rapp (1982) Hypertension Such as aldosterone antagonists, angiotensin-converting 4:753-763. The rat monocrotaline model of pulmonary arte 40 enzyme inhibitors and dual-acting angiotensin-converting rial hypertension described, for example, in Kato et al. (2008) enzyme? neprilysin inhibitors, angiotensin-converting J. Cardiovasc. Pharmacol. 51(1):18-23, is a reliable predictor enzyme 2 (ACE2) activators and stimulators, angiotensin-II of clinical efficacy for the treatment of pulmonary arterial vaccines, anti-diabetic agents, anti-lipid agents, anti-throm hypertension. Heart failure animal models include the DSS botic agents, AT receptor antagonists and dual-acting AT rat model for heart failure and the aorto-caval fistula model 45 receptor antagonist/neprilysin inhibitors, B-adrenergic (AV shunt), the latter of which is described, for example, in receptor antagonists, dual-acting B-adrenergic receptor Norling et al. (1996) J. Amer: Soc. Nephrol. 7:1038-1044. antagonist/ol-receptor antagonists, calcium channel block Other animal models, such as the hot plate, tail-flick and ers, diuretics, endothelin receptor antagonists, endothelin formalin tests, can be used to measure the analgesic proper converting enzyme inhibitors, neprilysin inhibitors, natri ties of a compound, as well as the spinal nerve ligation (SNL) 50 uretic peptides and their analogs, natriuretic peptide clear model of neuropathic pain. See, for example, Malmberg etal. ance receptor antagonists, nitric oxide donors, non-steroidal (1999) Current Protocols in Neuroscience 8.9.1-8.9.15. anti-inflammatory agents, phosphodiesterase inhibitors (spe Other properties and utilities of the compounds can be dem cifically PDE-V inhibitors), prostaglandin receptor agonists, onstrated using various invitro and in vivo assays well known renin inhibitors, soluble guanylate cyclase stimulators and to those skilled in the art. 55 activators, and combinations thereof. In one particular The compounds of the invention are expected to be useful embodiment of the invention, a compound of the invention is for the treatment and/or prevention of medical conditions combined with an AT receptor antagonist, a diuretic, a cal responsive to NEP inhibition. Thus it is expected that patients cium channel blocker, or a combination thereof, and used to suffering from a disease or disorder that is treated by inhib treat primary hypertension. In another particularembodiment iting the NEP enzyme or by increasing the levels of its peptide 60 of the invention, a compound of the invention is combined Substrates, can be treated by administering a therapeutically with an AT receptor antagonist, and used to treat hyperten effective amount of a compound of the invention. For sion with accompanying renal disease. example, by inhibiting NEP, the compound is expected to For treatment of pulmonary arterial hypertension, the potentiate the biological effects of endogenous peptides that therapeutically effective amount is typically the amount that are metabolized by NEP, such as the natriuretic peptides, 65 is sufficient to lower the pulmonary vascular resistance. Other bombesin, bradykinins, calcitonin, endothelins, enkephalins, goals of therapy are to improve a patient’s exercise capacity. neurotensin, Substance P and vasoactive intestinal peptide. For example, in a clinical setting, the therapeutically effective US 8,871,792 B2 33 34 amount can be the amount that improves a patient’s ability to nephropathy, chronic kidney disease, proteinuria, and par walk comfortably for a period of 6 minutes (covering a dis ticularly acute kidney injury or acute renal failure (see Shark tance of approximately 20-40 meters). When used to treat ovska et al. (2011) Clin. Lab. 57:507–515 and Newaz et al. pulmonary arterial hypertension the compound may be (2010) Renal Failure 32:384-390). When used to treat renal administered in combination with other therapeutic agents disease, the compound may be administered in combination Such as C.-adrenergic antagonists, B-adrenergic receptor with other therapeutic agents such as angiotensin-converting antagonists, f2-adrenergic receptor agonists, angiotensin enzyme inhibitors, AT receptor antagonists, and diuretics. converting enzyme inhibitors, anticoagulants, calcium chan nel blockers, diuretics, endothelin receptor antagonists, Preventative Therapy PDE-V inhibitors, prostaglandin analogs, selective serotonin 10 reuptake inhibitors, and combinations thereof. In one particu By potentiating the effects of the natriuretic peptides, com lar embodiment of the invention, a compound of the invention pounds of the invention are also expected to be useful in is combined with a PDE-V inhibitor or a selective serotonin preventative therapy, due to the antihypertrophic and antifi reuptake inhibitor and used to treat pulmonary arterial hyper brotic effects of the natriuretic peptides (see Potter et al. tension. 15 (2009) Handbook of Experimental Pharmacology 191:341 Another embodiment of the invention relates to a method 366), for example in preventing the progression of cardiac for treating heart failure, in particular congestive heart failure insufficiency after myocardial infarction, preventing arterial (including both systolic and diastolic congestive heart fail restenosis after angioplasty, preventing thickening of blood ure), comprising administering to a patient a therapeutically vessel walls after vascular operations, preventing atheroscle effective amount of a compound of the invention. Typically, rosis, and preventing diabetic angiopathy. the therapeutically effective amount is the amount that is sufficient to lower blood pressure and/or improve renal func Glaucoma tions. In a clinical setting, the therapeutically effective amount can be the amount that is sufficient to improve cardiac By potentiating the effects of the natriuretic peptides, com hemodynamics, like for instance reduction in wedge pres 25 pounds of the invention are expected to be useful to treat Sure, right atrial pressure, filling pressure, and vascular resis glaucoma. See, for example, Diestelhorst et al. (1989) Inter tance. In one embodiment, the compound is administered as national Ophthalmology 12:99-101. When used to treat glau an intravenous dosage form. When used to treat heart failure, coma, compounds of the invention may be combined with one the compound may be administered in combination with or more additional anti-glaucoma agents. other therapeutic agents such as adenosine receptor antago 30 nists, advanced glycation end product breakers, aldosterone Pain Relief antagonists, AT receptor antagonists, f-adrenergic receptor antagonists, dual-acting (3-adrenergic receptor antagonist/ As NEP inhibitors, compounds of the invention are C-receptor antagonists, chymase inhibitors, digoxin, diuret expected to inhibit the degradation of endogenous enkepha ics, endothelin converting enzyme (ECE) inhibitors, endot 35 lins and thus Such compounds may also find utility as anal helin receptor antagonists, natriuretic peptides and their gesics. See, for example, Rogues et al. (1980) Nature 288: analogs, natriuretic peptide clearance receptor antagonists, 286-288 and Thanawala et al. (2008) Current Drug Targets nitric oxide donors, prostaglandinanalogs, PDE-Vinhibitors, 9:887-894. When used to treat pain, compounds of the inven soluble guanylate cyclase activators and stimulators, and tion may be combined with one or more additional antinoci vasopressin receptor antagonists. In one particular embodi 40 ceptive drugs such as aminopeptidase N or dipeptidyl pepti ment of the invention, a compound of the invention is com dase III inhibitors, non-steroidal anti-inflammatory agents, bined with an aldosterone antagonist, a B-adrenergic recep monoamine reuptake inhibitors, muscle relaxants, NMDA tor antagonist, an AT receptor antagonist, or a diuretic, and receptor antagonists, opioid receptor agonists, 5-HT sero used to treat congestive heart failure. tonin receptor agonists, and tricyclic antidepressants. 45 Diarrhea Other Utilities As NEP inhibitors, compounds of the invention are Due to their NEP inhibition properties, compounds of the expected to inhibit the degradation of endogenous enkepha invention are also expected to be useful as antitussive agents, lins and thus such compounds may also find utility for the 50 as well as find utility in the treatment of portal hypertension treatment of diarrhea, including infectious and secretory/wa associated with liver cirrhosis (see Sansoe et al. (2005) J. tery diarrhea. See, for example, Baumer et al. (1992) Gut Hepatol. 43:791-798), cancer (see Vesely (2005) J. Investi 33:753-758: Farthing (2006) Digestive Diseases 24:47-58: gative Med. 53:360-365), depression (see Noble et al. (2007) and Marcais-Collado (1987) Eur: J. Pharmacol. 144(2):125 Exp. Opin. Ther: Targets 11:145-159), menstrual disorders, 132. When used to treat diarrhea, compounds of the invention 55 preterm labor, pre-eclampsia, endometriosis, reproductive may be combined with one or more additional antidiarrheal disorders (for example, male and female infertility, polycystic treatmentS. ovarian syndrome, implantation failure), and male and female sexual dysfunction, including male erectile dysfunction and Renal Diseases female sexual arousal disorder. More specifically, the com 60 pounds of the invention are expected to be useful in treating By potentiating the effects of vasoactive peptides like the female sexual dysfunction (see Pryde et al. (2006) J. Med. natriuretic peptides and bradykinin, compounds of the inven Chem. 49:4409-4424), which is often defined as a female tion are expected to enhance renal function (see Chen et al. patient’s difficulty or inability to find satisfaction in sexual (1999) Circulation 100:2443-2448; Lipkin et al. (1997) Kid expression. This covers a variety of diverse female sexual ney Int. 52:792-801; and Dussaule et al. (1993) Clin. Sci. 65 disorders including, by way of illustration and not limitation, 84:31-39) and find utility in treating and/or preventing renal hypoactive sexual desire disorder, sexual arousal disorder, diseases. Renal diseases of particularinterest include diabetic orgasmic disorder and sexual pain disorder. When used to US 8,871,792 B2 35 36 treat Such disorders, especially female sexual dysfunction, not limited to, cells, cellular extracts, plasma membranes, compounds of the invention may be combined with one or tissue samples, isolated organs, mammals (such as mice, rats, more of the following secondary agents: PDE-V inhibitors, guinea pigs, rabbits, dogs, pigs, humans, and so forth), and the dopamine agonists, estrogen receptoragonists and/orantago like, with mammals being of particular interest. In one par nists, androgens, and estrogens. Due to their NEP inhibition ticular embodiment of the invention, NEP enzyme activity in properties, compounds of the invention are also expected to a mammal is inhibited by administering a NEP-inhibiting have anti-inflammatory properties, and are expected to have amount of a compound of the invention. These compounds utility as such, particularly when used in combination with can also be used as research tools by conducting biological statins. assays using Such compounds. Recent studies suggest that NEP plays a role in regulating 10 When used as a research tool, a biological system or nerve function in insulin-deficient diabetes and diet induced sample comprising a NEP enzyme is typically contacted with obesity. Coppey et al. (2011) Neuropharmacology 60:259 a NEP enzyme-inhibiting amount of a compound of the 266. Therefore, due to their NEP inhibition properties, com pounds of the invention are also expected to be useful in invention. After the biological system or sample is exposed to providing protection from nerve impairment caused by dia 15 the compound, the effects of inhibiting the NEP enzyme are betes or diet induced obesity. determined using conventional procedures and equipment, The amount of the compound of the invention administered Such as by measuring receptor binding in a binding assay or per dose or the total amount administered per day may be measuring ligand-mediated changes in a functional assay. predetermined or it may be determined on an individual Exposure encompasses contacting cells or tissue with the patient basis by taking into consideration numerous factors, compound, administering the crystalline compound to a including the nature and severity of the patient’s condition, mammal, for example by i.p., p.o. i.V., s.c., or inhaled admin the condition being treated, the age, weight, and general istration, and so forth. This determining step can involve health of the patient, the tolerance of the patient to the active measuring a response (a quantitative analysis) or can involve agent, the route of administration, pharmacological consid making an observation (a qualitative analysis). Measuring a erations such as the activity, efficacy, pharmacokinetics and 25 response involves, for example, determining the effects of the toxicology profiles of the compound and any secondary compound on the biological system or sample using conven agents being administered, and the like. Treatment of a patient tional procedures and equipment, Such as enzyme activity Suffering from a disease or medical condition (such as hyper assays and measuring enzyme Substrate or product mediated tension) can begin with a predetermined dosage or a dosage changes in functional assays. The assay results can be used to determined by the treating physician, and will continue for a 30 determine the activity level as well as the amount of com period of time necessary to prevent, ameliorate, Suppress, or pound necessary to achieve the desired result, that is, a NEP alleviate the symptoms of the disease or medical condition. enzyme-inhibiting amount. Typically, the determining step Patients undergoing Such treatment will typically be moni will involve determining the effects of inhibiting the NEP tored on a routine basis to determine the effectiveness of enzyme. therapy. For example, in treating hypertension, blood pres 35 Additionally, the compounds of the invention can be used sure measurements may be used to determine the effective as research tools for evaluating other chemical compounds, ness of treatment. Similar indicators for other diseases and and thus are also useful in Screening assays to discover, for conditions described herein, are well known and are readily example, new compounds having NEP-inhibiting activity. available to the treating physician. Continuous monitoring by Thus another aspect of the invention relates to a method of the physician will insure that the optimal amount of the com 40 evaluating a test compound in a biological assay, comprising: pound of the invention will be administered at any given time, (a) conducting a biological assay with a test compound to as well as facilitating the determination of the duration of provide a first assay value; (b) conducting the biological assay treatment. This is of particular value when secondary agents with a compound of the invention to provide a second assay are also being administered, as their selection, dosage, and value; wherein step (a) is conducted either before, after or duration of therapy may also require adjustment. In this way, 45 concurrently with step (b); and (c) comparing the first assay the treatment regimen and dosing schedule can be adjusted value from step (a) with the second assay value from step (b). over the course of therapy so that the lowest amount of active Exemplary biological assays include a NEP enzyme inhibi agent that exhibits the desired effectiveness is administered tion assay. In this manner, the compounds of the invention are and, further, that administration is continued only so long as used as standards in an assay to allow comparison of the is necessary to successfully treat the disease or medical con 50 results obtained with a test compound and with the compound dition. of the invention to identify those test compounds that have about equal or Superior activity, if any. For example, pK data Research Tools foratest compound or a group of test compounds is compared to the pK, data for a compound of the invention to identify Since the compounds of the invention are metabolized in 55 those test compounds that have the desired properties, for Vivo to compounds having activity as neprilysin inhibitors, example, test compounds having a pK value about equal or they are also useful as a research tools for investigating or Superior to the compound of the invention, if any. This aspect studying biological systems or samples having a NEP of the invention includes, as separate embodiments, both the enzyme, for example to study diseases where the NEP generation of comparison data (using the appropriate assays) enzyme or its peptide Substrates plays a role. Accordingly, 60 and the analysis of test data to identify test compounds of one aspect of the invention relates to a method of using a interest. compound of the invention as a research tool, comprising Still another aspect of the invention relates to a method of conducting a biological assay using a compound of the inven studying a biological system or sample comprising a NEP tion. Any suitable biological system or sample having a NEP enzyme, the method comprising: (a) contacting the biological enzyme may be employed in Such studies which may be 65 system or sample with a compound of the invention; and (b) conducted either in vitro or in vivo. Representative biological determining the effects caused by the compound on the bio systems or samples Suitable for Such studies include, but are logical system or sample. US 8,871,792 B2 37 38 Pharmaceutical Compositions and Formulations excipients, such as cocoa butter and Suppository waxes; oils, Such as peanut oil, cottonseed oil, safflower oil, sesame oil, Compounds of the invention are typically administered to olive oil, corn oil and Soybean oil; glycols, such as propylene a patient in the form of a pharmaceutical composition or glycol; polyols. Such as glycerin, Sorbitol, mannitol and poly formulation. Such pharmaceutical compositions may be ethylene glycol; esters, such as ethyl oleate and ethyl laurate; administered to the patient by any acceptable route of admin agar, buffering agents, such as magnesium hydroxide and istration including, but not limited to, oral, rectal, vaginal, aluminum hydroxide; alginic acid; pyrogen-free water, iso nasal, inhaled, topical (including transdermal), ocular, and tonic saline; Ringer's Solution; ethyl alcohol; phosphate parenteral modes of administration. Further, the compounds buffer Solutions; compressed propellant gases, such as chlo of the invention may be administered, for example orally, in 10 rofluorocarbons and hydrofluorocarbons; and other non-toxic multiple doses per day (for example, two, three, or four times compatible Substances employed in pharmaceutical compo daily), in a single daily dose or a single weekly dose. It will be sitions. understood that any form of the compounds of the invention, Pharmaceutical compositions are typically prepared by (that is, free base, free acid, pharmaceutically acceptable salt, thoroughly and intimately mixing or blending the active agent solvate, etc.) that is suitable for the particular mode of admin 15 with a pharmaceutically acceptable carrier and one or more istration can be used in the pharmaceutical compositions optional ingredients. The resulting uniformly blended mix discussed herein. ture may then be shaped or loaded into tablets, capsules, pills, Accordingly, in one embodiment, the invention relates to a canisters, cartridges, dispensers and the like using conven pharmaceutical composition comprising a pharmaceutically tional procedures and equipment. acceptable carrier and a compound of the invention. The In one embodiment, the pharmaceutical compositions are compositions may contain other therapeutic and/or formulat suitable for oral administration. Suitable compositions for ing agents if desired. When discussing compositions, the oral administration may be in the form of capsules, tablets, “compound of the invention” may also be referred to hereinas pills, lozenges, cachets, dragees, powders, granules; Solu the “active agent, to distinguish it from other components of tions or Suspensions in an aqueous or non-aqueous liquid; the formulation, such as the carrier. Thus, it is understood that 25 oil-in-water or water-in-oil liquid emulsions; elixirs or Syr the term “active agent' includes compounds of formula I as ups; and the like; each containing a predetermined amount of well as pharmaceutically acceptable salts, Solvates and pro the active agent. drugs of that compound. 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 30 will typically comprise the active agent and one or more pound of the invention. Those skilled in the art will recognize, pharmaceutically acceptable carriers, such as Sodium citrate however, that a pharmaceutical composition may contain or dicalcium phosphate. Solid dosage forms may also com more than atherapeutically effective amount, such as in bulk prise: fillers or extenders, such as starches, microcrystalline compositions, or less thana therapeutically effective amount, cellulose, lactose. Sucrose, glucose, mannitol, and/or silicic that is, individual unit doses designed for multiple adminis 35 acid; binders, such as carboxymethylcellulose, alginates, tration to achieve a therapeutically effective amount. Typi gelatin, polyvinyl pyrrolidone. Sucrose and/or acacia; humec cally, the composition will contain from about 0.01-95 wt % tants, such as glycerol, disintegrating agents. Such as agar of active agent, including, from about 0.01-30 wt %, such as agar, calcium carbonate, potato or tapioca Starch, alginic acid, from about 0.01-10 wt %, with the actual amount depending certain silicates, and/or sodium carbonate; solution retarding upon the formulation itself, the route of administration, the 40 agents, such as paraffin, absorption accelerators, such as qua frequency of dosing, and so forth. In one embodiment, a ternary ammonium compounds; wetting agents, such as cetyl composition Suitable for an oral dosage form, for example, alcohol and/or glycerol monostearate; absorbents, such as may contain about 5-70 wt %, or from about 10-60 wt % of kaolin and/or bentonite clay; lubricants, such as talc, calcium active agent. Stearate, magnesium Stearate, Solid polyethylene glycols, Any conventional carrier or excipient may be used in the 45 Sodium lauryl Sulfate, and/or mixtures thereof coloring pharmaceutical compositions of the invention. The choice of agents; and buffering agents. a particular carrier or excipient, or combinations of carriers or Release agents, wetting agents, coating agents, Sweeten excipients, will depend on the mode of administration being ing, flavoring and perfuming agents, preservatives and anti used to treat a particular patient or type of medical condition oxidants may also be present in the pharmaceutical compo or disease state. In this regard, the preparation of a suitable 50 sitions. Exemplary coating agents for tablets, capsules, pills composition for a particular mode of administration is well and like, include those used for enteric coatings, such as within the scope of those skilled in the pharmaceutical arts. cellulose acetate phthalate, polyvinyl acetate phthalate, Additionally, carriers or excipients used in Such compositions hydroxypropyl methylcellulose phthalate, methacrylic acid are commercially available. By way of further illustration, methacrylic acid ester copolymers, cellulose acetate trimel conventional formulation techniques are described in Rem 55 litate, carboxymethyl ethyl cellulose, hydroxypropyl methyl ington. The Science and Practice of Pharmacy, 20" Edition, cellulose acetate Succinate, and the like. Examples of phar Lippincott Williams & White, Baltimore, Md. (2000); and H. maceutically acceptable antioxidants include: water-soluble C. Ansel et al., Pharmaceutical Dosage Forms and Drug antioxidants, such as ascorbic acid, cysteine hydrochloride, Delivery Systems, 7" Edition, Lippincott Williams & White, sodium bisulfate, sodium metabisulfate sodium sulfite and Baltimore, Md. (1999). 60 the like; oil-soluble antioxidants, such as ascorbyl palmitate, Representative examples of materials which can serve as butylated hydroxyanisole, butylated hydroxytoluene, leci pharmaceutically acceptable carriers include, but are not lim thin, propyl gallate, alpha-tocopherol, and the like; and metal ited to, the following: Sugars, such as lactose, glucose and chelating agents, such as citric acid, ethylenediamine tet Sucrose; starches, such as corn Starch and potato starch; cel raacetic acid, Sorbitol, tartaric acid, phosphoric acid, and the lulose. Such as microcrystalline cellulose, and its derivatives, 65 like. Such as Sodium carboxymethyl cellulose, ethyl cellulose and Compositions may also be formulated to provide slow or cellulose acetate; powdered tragacanth; malt, gelatin; talc; controlled release of the active agent using, by way of US 8,871,792 B2 39 40 example, hydroxypropyl methyl cellulose in varying propor Surfactant on micronized particles of the active agent. The tions or other polymer matrices, liposomes and/or micro formulation is then loaded into an aerosol canister, which spheres. In addition, the pharmaceutical compositions of the forms a portion of the inhaler. invention may contain opacifying agents and may be formu Compounds of the invention can also be administered lated so that they release the active agent only, or preferen parenterally (for example, by Subcutaneous, intravenous, tially, in a certain portion of the gastrointestinal tract, option intramuscular, or intraperitoneal injection). For such admin ally, in a delayed manner. Examples of embedding istration, the active agent is provided in a sterile solution, compositions which can be used include polymeric Sub Suspension, or emulsion. Exemplary solvents for preparing stances and waxes. The active agent can also be in micro Such formulations include water, saline, low molecular encapsulated form, optionally with one or more of the above 10 weight alcohols such as propylene glycol, polyethylene gly described excipients. col, oils, gelatin, fatty acid esters such as ethyl oleate, and the Suitable liquid dosage forms for oral administration like. Parenteral formulations may also contain one or more include, by way of illustration, pharmaceutically acceptable anti-oxidants, solubilizers, stabilizers, preservatives, wetting emulsions, microemulsions, Solutions, Suspensions, syrups agents, emulsifiers, and dispersing agents. Surfactants, addi and elixirs. Liquid dosage forms typically comprise the active 15 tional stabilizing agents or pH-adjusting agents (acids, bases agent and an inert diluent, Such as, for example, water or other or buffers) and anti-oxidants are particularly useful to provide Solvents, Solubilizing agents and emulsifiers, such as ethyl stability to the formulation, for example, to minimize or avoid alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, hydrolysis of ester and amide linkages, or dimerization of benzyl alcohol, benzyl benzoate, propylene glycol. 1,3-buty thiols that may be present in the compound. These formula lene glycol, oils (for example, cottonseed, groundnut, corn, tions may be rendered sterile by use of a sterile injectable germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl medium, a sterilizing agent, filtration, irradiation, or heat. In alcohol, polyethylene glycols andfatty acid esters of sorbitan, one particular embodiment, the parenteral formulation com and mixtures thereof. Suspensions may contain Suspending prises an aqueous cyclodextrin solution as the pharmaceuti agents such as, for example, ethoxylated isostearyl alcohols, cally acceptable carrier. Suitable cyclodextrins include cyclic polyoxyethylene Sorbitol and Sorbitan esters, microcrystal 25 molecules containing six or more C-D-glucopyranose units line cellulose, aluminium metahydroxide, bentonite, agar linked at the 1.4 positions by a linkages as in amylase, B-cy agar and tragacanth, and mixtures thereof. clodextrin or cycloheptaamylose. Exemplary cyclodextrins When intended for oral administration, the pharmaceutical include cyclodextrin derivatives Such as hydroxypropyl and compositions of the invention may be packaged in a unit sulfobutyl ether cyclodextrins such as hydroxypropyl-(3-cy dosage form. The term “unit dosage form' refers to a physi 30 clodextrin and sulfobutyl ether B-cyclodextrin. Exemplary cally discrete unit Suitable for dosing a patient, that is, each buffers for such formulations include carboxylic acid-based unit containing a predetermined quantity of the active agent buffers such as citrate, lactate and maleate buffer solutions. calculated to produce the desired therapeutic effect either Compounds of the invention can also be administered alone or in combination with one or more additional units. For transdermally using known transdermal delivery systems and example, such unit dosage forms may be capsules, tablets, 35 excipients. For example, the compound can be admixed with pills, and the like. permeation enhancers, such as propylene glycol, polyethyl In another embodiment, the compositions of the invention ene glycol monolaurate, azacycloalkan-2-ones and the like, are suitable for inhaled administration, and will typically be and incorporated into a patch or similar delivery system. in the form of an aerosol or a powder. Such compositions are Additional excipients including gelling agents, emulsifiers generally administered using well-known delivery devices, 40 and buffers, may be used in Such transdermal compositions if Such as a nebulizer, dry powder, or metered-dose inhaler. desired. Nebulizer devices produce a stream of high velocity air that causes the composition to spray as a mist that is carried into a Secondary Agents patient's respiratory tract. An exemplary nebulizer formula tion comprises the active agent dissolved in a carrier to form 45 The compounds of the invention may be useful as the sole a solution, or micronized and combined with a carrier to form treatment of a disease or may be combined with one or more a Suspension of micronized particles of respirable size. Dry additional therapeutic agents in order to obtain the desired powder inhalers administer the active agent as a free-flowing therapeutic effect. Thus, in one embodiment, pharmaceutical powder that is dispersed in a patient’s air-stream during inspi compositions of the invention contain other drugs that are ration. An exemplary dry powder formulation comprises the 50 co-administered with a compound of the invention. For active agent dry-blended with an excipient such as lactose, example, the composition may further comprise one or more starch, mannitol, dextrose, polylactic acid, polylactide-co drugs (also referred to as 'secondary agents(s)). Such thera glycolide, and combinations thereof. Metered-dose inhalers peutic agents are well known in the art, and include adenosine discharge a measured amount of the active agent using com receptor antagonists, C.-adrenergic receptor antagonists, pressed propellant gas. An exemplary metered-dose formu 55 B-adrenergic receptor antagonists, B-adrenergic receptor lation comprises a solution or Suspension of the active agent agonists, dual-acting f-adrenergic receptor antagonist/C-re in a liquefied propellant. Such as a chlorofluorocarbon or ceptor antagonists, advanced glycation end product breakers, hydrofluoroalkane. Optional components of Such formula aldosterone antagonists, aldosterone synthase inhibitors, tions include co-solvents, such as ethanol or pentane, and aminopeptidase N inhibitors, androgens, angiotensin-con Surfactants, such as Sorbitan trioleate, oleic acid, lecithin, 60 Verting enzyme inhibitors and dual-acting angiotensin-con glycerin, and Sodium lauryl Sulfate. Such compositions are Verting enzyme/neprilysin inhibitors, angiotensin-converting typically prepared by adding chilled or pressurized hydrof enzyme 2 activators and stimulators, angiotensin-II Vaccines, luoroalkane to a Suitable container containing the active anticoagulants, anti-diabetic agents, antidiarrheal agents, agent, ethanol (if present) and the Surfactant (if present). To anti-glaucoma agents, anti-lipid agents, antinociceptive prepare a suspension, the active agent is micronized and then 65 agents, anti-thrombotic agents, AT receptor antagonists and combined with the propellant. Alternatively, a suspension dual-acting AT receptor antagonist/neprilysin inhibitors and formulation can be prepared by spray drying a coating of multifunctional angiotensin receptor blockers, bradykinin US 8,871,792 B2 41 42 receptor antagonists, calcium channel blockers, chymase ondary agents set forth herein, in quantities sufficient to carry inhibitors, digoxin, diuretics, dopamine agonists, endothelin out the methods of the invention. The first dosage form and converting enzyme inhibitors, endothelin receptor antago the second (or third, etc.) dosage form together comprise a nists, HMG-CoA reductase inhibitors, estrogens, estrogen therapeutically effective amount of active agents for the treat receptor agonists and/or antagonists, monoamine reuptake ment or prevention of a disease or medical condition in a inhibitors, muscle relaxants, natriuretic peptides and their patient. analogs, natriuretic peptide clearance receptor antagonists, Secondary agent(s), when included, are present in a thera neprilysin inhibitors, nitric oxide donors, non-steroidal anti peutically effective amount such that they are typically inflammatory agents, N-methyl d-aspartate receptor antago administered in an amount that produces a therapeutically nists, opioid receptor agonists, phosphodiesterase inhibitors, 10 beneficial effect when co-administered with a compound of prostaglandin analogs, prostaglandin receptoragonists, renin the invention. The secondary agent can be in the form of a inhibitors, selective serotonin reuptake inhibitors, sodium pharmaceutically acceptable salt, Solvate, optically pure Ste channel blocker, Soluble guanylate cyclase stimulators and reoisomer, and so forth. The secondary agent may also be in activators, tricyclic antidepressants, vasopressin receptor the form of a prodrug, for example, a compound having a antagonists, and combinations thereof. Specific examples of 15 carboxylic acid group that has been esterified. Thus, second these agents are detailed herein. ary agents listed herein are intended to include all Such forms, Accordingly, in yet another aspect of the invention, a phar and are commercially available or can be prepared using maceutical composition comprises a compound of the inven conventional procedures and reagents. tion, a second active agent, and a pharmaceutically acceptable In one embodiment, compounds of the invention are carrier. Third, fourth etc. active agents may also be included administered in combination with an adenosine receptor in the composition. In combination therapy, the amount of antagonist, representative examples of which include, but are compound of the invention that is administered, as well as the not limited to, naxifylline, rolofylline, SLV-320, theophyl amount of secondary agents, may be less than the amount line, and tonapofylline. typically administered in monotherapy. In one embodiment, compounds of the invention are Compounds of the invention may be physically mixed with 25 administered in combination with an O-adrenergic receptor the second active agent to form a composition containing both antagonist, representative examples of which include, but are agents; or each agent may be present in separate and distinct not limited to, doxazosin, prazosin, tamsulosin, and tera compositions which are administered to the patient simulta Zosin. neously or at separate times. For example, a compound of the Compounds of the invention may also be administered in invention can be combined with a second active agent using 30 combination with a f-adrenergic receptor antagonist (“B- conventional procedures and equipment to form a combina blockers'). Representative B-blockers include, but are not tion of active agents comprising a compound of the invention limited to, acebutolol, alprenolol, amosulalol, arotinolol, and a second active agent. Additionally, the active agents may atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopin be combined with a pharmaceutically acceptable carrier to dolol, bucindolol, bucumolol, bufetolol, bufuralol, bunitrolol, form a pharmaceutical composition comprising a compound 35 bupranolol, bubridine, butofilolol, carazolol, carteolol, of the invention, a second active agent and a pharmaceutically carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, acceptable carrier. In this embodiment, the components of the epanolol, esmolol, indenolol, labetolol, levobunolol, mepin composition are typically mixed or blended to create a physi dolol, metipranolol, metoprolol such as metoprolol Succinate cal mixture. The physical mixture is then administered in a and metoprolol tartrate, moprolol, nadolol, nadoxolol. therapeutically effective amount using any of the routes 40 nebivalol, nipradillol, oxprenolol, penbutolol, perbutolol, pin described herein. dolol, practolol, pronethalol, propranolol, Sotalol, Sufinalol, Alternatively, the active agents may remain separate and talindol, tertatolol, tilisolol, timolol, toliprolol. xibenolol, and distinct before administration to the patient. In this embodi combinations thereof. In one particular embodiment, the ment, the agents are not physically mixed together before B-antagonist is selected from atenolol, bisoprolol, meto administration but are administered simultaneously or at 45 prolol, propranolol, Sotalol, and combinations thereof. Typi separate times as separate compositions. Such compositions cally, the B-blocker will be administered in an amount suf can be packaged separately or may be packaged together in a ficient to provide from about 2-900 mg per dose. kit. When administered at separate times, the secondary agent In one embodiment, compounds of the invention are will typically be administered less than 24 hours after admin administered in combination with a f-adrenergic receptor istration of the compound of the invention, ranging anywhere 50 agonist, representative examples of which include, but are not from concurrent with administration of the compound of the limited to, albuterol, bitolterol, fenoterol, formoterol, inda invention to about 24 hours post-dose. This is also referred to caterol, isoetharine, levalbuterol, metaproterenol, pirbuterol, as sequential administration. Thus, a compound of the inven salbutamol, Salmefamol, salmeterol, terbutaline, Vilanterol, tion can be orally administered simultaneously or sequen and the like. Typically, the f-adrenergic receptoragonist will tially with another active agent using two tablets, with one 55 be administered in an amount sufficient to provide from about tablet for each active agent, where sequential may mean being 0.05-500 ug per dose. administered immediately after administration of the com In one embodiment, compounds of the invention are pound of the invention or at Some predetermined time later administered in combination with an advanced glycation end (for example, one hour later or three hours later). It is also product (AGE) breaker, examples of which include, by way contemplated that the secondary agent may be administered 60 of illustration and not limitation, alagebrium (or ALT-711), more than 24 hours after administration of the compound of and TRC4149. the invention. Alternatively, the combination may be admin In another embodiment, compounds of the invention are istered by different routes of administration, that is, one orally administered in combination with an aldosterone antagonist, and the other by inhalation. representative examples of which include, but are not limited In one embodiment, the kit comprises a first dosage form 65 to, eplerenone, spironolactone, and combinations thereof. comprising a compound of the invention and at least one Typically, the aldosterone antagonist will be administered in additional dosage form comprising one or more of the sec an amount sufficient to provide from about 5-300 mg per day. US 8,871,792 B2 43 44 In one embodiment, compounds of the invention are insulin and insulin derivatives. Examples of orally effective administered in combination with an aminopeptidase N or drugs include, but are not limited to: biguanides such as dipeptidyl peptidase III inhibitor, examples of which include, metformin; glucagon antagonists; C-glucosidase inhibitors by way of illustration and not limitation, bestatin and PC18 Such as acarbose and miglitol; dipeptidyl peptidase IV inhibi (2-amino-4-methylsulfonylbutane thiol, methionine thiol). 5 tors (DPP-IV inhibitors) such as alogliptin, denagliptin, lina Compounds of the invention can also be administered in gliptin, saxagliptin, Sitagliptin, and Vildagliptin: meglitinides combination with an angiotensin-converting enzyme (ACE) Such as repaglinide; oxadiazolidinediones; Sulfonylureas inhibitor. Representative ACE inhibitors include, but are not Such as chlorpropamide, glimepiride, glipizide, glyburide, limited to, accupril, alacepril, benazepril, benazeprilat, cap and tolaZamide; thiazolidinediones such as pioglitaZone and topril, ceranapril, cilaZapril, delapril, enalapril, enalaprilat, 10 fosinopril, fosinoprilat, imidapril, lisinopril, moexipril, rosiglitaZone; and combinations thereof. monopril, moveltipril, pentopril, perindopril, quinapril, In another embodiment, compounds of the invention are quinaprilat, 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. 15 to, oral rehydration solutions (ORS), loperamide, diphenoxy In a particular embodiment, the ACE inhibitor is selected late, and bismuth Subsalicylate. from: benazepril, captopril, enalapril, lisinopril, ramipril, and In yet another embodiment, a compound of the invention is combinations thereof. Typically, the ACE inhibitor will be administered in combination with an anti-glaucoma agent. administered in an amount Sufficient to provide from about Representative anti-glaucoma agents include, but are not lim 1-150 mg per day. In another embodiment, compounds of the ited to: C.-adrenergic agonists such as brimonidine; B-adren invention are administered in combination with a dual-acting ergic receptor antagonists; topical f-blockers such as betax angiotensin-converting enzyme/neprilysin (ACE/NEP) olol, levobunolol, and timolol; carbonic anhydrase inhibitors inhibitor, examples of which include, but are not limited to: Such as acetazolamide, brinzolamide, or dorzolamide; cho AVE-0848 ((4S,7S,12bR)-7-3-methyl-2 (S)-sulfanylbutyra linergic agonists such as cevimeline and DMXB-anabaseine; mido-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido2,1-a2 25 epinephrine compounds; miotics Such as pilocarpine; and benzazepine-4-carboxylic acid); AVE-7688 (ilepatril) and its prostaglandin analogs. parent compound; BMS-182657 (2-2-oxo-3 (S)-3-phenyl-2 In yet another embodiment, compounds of the invention (S)-sulfanylpropionamido)-2,3,4,5-tetrahydro-1H-1-benza are administered in combination with an anti-lipid agent. Zepin-1-yl)acetic acid); CGS-35601 (N-1-4-methyl-2(S)- Representative anti-lipid agents include, but are not limited Sulfanylpentanamidocyclopentyl-carbonyl-L-tryptophan); 30 to: cholesteryl ester transfer protein inhibitors (CETPs) such fasidotril; fasidotrilate; enalaprilat; ER-32935 ((3R,6S,9aR)- as anacetrapib, dalcetrapib, and torcetrapib; statins such as 6-3 (S)-methyl-2(S)-sulfanylpentanamido-5-oxoperhy atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin drothiazolo 3.2-aaZepine-3-carboxylic acid); gempatrilat; and simvastatin; and combinations thereof. MDL-101.264 ((4S,7S.12bR)-7-2(S)-(2-morpholino In one embodiment, compounds of the invention are acetylthio)-3-phenylpropionamido-6-oxo-1,2,3,4,6,7,8, 35 administered in combination with an anti-thrombotic agent. 12b-octahydropyrido 2,1-a2benzazepine-4-carboxylic Representative anti-thrombotic agents include, but are not acid); MDL-101287 (4S-4C,7O.(R*),12b?3-7-2-(car limited to: aspirin; anti-platelet agents such as clopidogrel, boxymethyl)-3-phenylprop ionamido-6-oxo-1,2,3,4,6,7,8, prasugrel, and ticlopidine; heparin, and combinations 12b-octahydropyrido 2,1-a2benzazepine-4-carboxylic thereof. acid); omapatrilat; RB-105 (N-2(5)-(mercaptomethyl)-3 40 In one embodiment, compounds of the invention are (R)-phenylbutyl-L-alanine); sampatrilat; SA-898 ((2R, administered in combination with an AT receptor antagonist, 4R)—N-2-(2-hydroxyphenyl)-3-(3-mercaptopropionyl) also known as angiotensin II type 1 receptor blockers (ARBs). thiazolidin-4-ylcarbonyl-L-phenylalanine); Sch-50690 (N- Representative ARBs include, but are not limited to, abite 1(S)-carboxy-2-N2-(methanesulfonyl)-L-lysylamino Sartan, azilsartan (e.g., azilsartan medoxomil), benzyllosar ethyl-L-Valyl-L-tyrosine); and combinations thereof, may 45 tan, candesartan, candesartancilexetil, elisartan, embusartan, also be included. In one particular embodiment, the ACE/ enoltaSosartan, eprosartan, EXP3174, fonsartan, forasartan, NEP inhibitor is selected from: AVE-7688, enalaprilat, fasi glycyllosartan, irbesartan, isoteoline, losartan, medoxomil. dotril, fasidotrilate, omapatrilat, Sampatrilat, and combina milfasartan, olmesartan (e.g., olmesartan medoxomil), tions thereof. opomisartan, pratosartan, ripisartan, Saprisartan, Saralasin, In one embodiment, compounds of the invention are 50 sarmesin, TAK-591, tasosartan, telmisartan, Valsartan, Zola administered in combination with an angiotensin-converting Sartan, and combinations thereof. In a particular embodiment, enzyme 2 (ACE2) activator or stimulator. the ARB is selected from azilsartan medoxomil, candesartan In one embodiment, compounds of the invention are cilexetil, eprosartan, irbesartan, losartan, olmesartan medox administered in combination with an angiotensin-II Vaccine, omil, Saprisartan, tasosartan, telmisartan, Valsartan, and com examples of which include, but are not limited to ATR12181 55 binations thereof. Exemplary salts and/or prodrugs include and CYT006-AngOb. candesartan cilexetil, eprosartan mesylate, losartan potas In one embodiment, compounds of the invention are sium salt, and olmesartan medoxomil. Typically, the ARB administered in combination with an anticoagulant, represen will be administered in an amount sufficient to provide from tative examples of which include, but are not limited to: about 4-600mg perdose, with exemplary daily dosages rang coumarins such as warfarin; heparin; and direct thrombin 60 ing from 20-320 mg per day. inhibitors such as argatroban, bivalirudin, dabigatran, and Compounds of the invention may also be administered in lepirudin. combination with a dual-acting agent, Such as an AT receptor In yet another embodiment, compounds of the invention antagonist/neprilysin inhibitor (ARB/NEP) inhibitor, are administered in combination with an anti-diabetic agent. examples of which include, but are not limited to, compounds Representative anti-diabetic agents include injectable drugs 65 described in U.S. Publication Nos. 2008/02693.05 and 2009/ as well as orally effective drugs, and combinations thereof. 0023228, both to Allegretti et al. filed on Apr. 23, 2008, such Examples of injectable drugs include, but are not limited to, as the compound, 4-2-ethoxy-4-ethyl-5-(S)-2-mercapto US 8,871,792 B2 45 46 4-methylpentanoylamino)-methyl)imidazol-1-ylmethyl-3- vide from about 5-50 mg per day, more typically 6-25 mg per fluorobiphenyl-2-carboxylic acid. day, with common dosages being 6.25 mg, 12.5 mg or 25 mg Compounds of the invention may also be administered in per day. combination with multifunctional angiotensin receptor Compounds of the invention may also be administered in blockers as described in Kurtz & Klein (2009) Hypertension combination with an endothelin converting enzyme (ECE) Research 32.826-834. inhibitor, examples of which include, but are not limited to, In one embodiment, compounds of the invention are phosphoramidon, CGS 26303, and combinations thereof. administered in combination with a bradykinin receptor In a particular embodiment, compounds of the invention antagonist, for example, icatibant (HOE-140). It is expected are administered in combination with an endothelin receptor that this combination therapy may present the advantage of 10 antagonist. Representative endothelin receptor antagonists preventing angioedema or other unwanted consequences of include, but are not limited to: selective endothelin receptor elevated bradykinin levels. antagonists that affect endothelin A receptors, such as avosen In one embodiment, compounds of the invention are tan, ambrisentan, atrasentan, BQ-123, claZosentan, darusen administered in combination with a calcium channel blocker. tan, sitaxentan, and Zibotentan; and dual endothelin receptor 15 antagonists that affect both endothelin A and B receptors, Representative calcium channel blockers include, but are not Such as bosentan, macitentan, tezosentan). limited to, amlodipine, anipamil, aranipine, barnidipine, ben In yet another embodiment, a compound of the invention is cyclane, benidipine, bepridil, clentiazem, cilnidipine, cin administered in combination with one or more HMG-CoA narizine, diltiazem, efonidipine, elgodipine, etafenone, felo reductase inhibitors, which are also known as statins. Repre dipine, fendiline, flunarizine, gallopamil. isradipine, sentative statins include, but are not limited to, atorvastatin, lacidipine, lercanidipine, lidoflazine, lomerizine, manid fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin ipine, mibefradil, nicardipine, nifedipine, niguldipine, nilu and simvastatin. dipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, In one embodiment, compounds of the invention are nivaldipine, perhexyline, prenylamine, ryosidine, semotiadil, administered in combination with a monoamine reuptake terodiline, tiapamil, Verapamil, and combinations thereof. In 25 inhibitor, examples of which include, by way of illustration a particular embodiment, the calcium channel blocker is and not limitation, norepinephrine reuptake inhibitors such as selected from amlodipine, bepridil, diltiazem, felodipine, atomoxetine, buproprion and the buproprion metabolite isradipine, lacidipine, nicardipine, nifedipine, niguldipine, hydroxybuproprion, maprotiline, reboxetine, and Viloxazine; niludipine, nimodipine, nisoldipine, ryosidine, Verapamil, selective serotonin reuptake inhibitors (SSRIs) such as citalo 30 pram and the citalopram metabolite desmethylcitalopram, and combinations thereof. Typically, the calcium channel dapoxetine, escitalopram (e.g., escitalopram oxalate), fluox blocker will be administered in an amount sufficient to pro etine and the fluoxetine desmethyl metabolite norfluoxetine, vide from about 2-500 mg per dose. fluvoxamine (e.g., fluvoxamine maleate), paroxetine, Sertra In one embodiment, compounds of the invention are line and the sertraline metabolite demethylsertraline; dual administered in combination with a chymase inhibitor, Such 35 serotonin-norepinephrine reuptake inhibitors (SNRIs) such as TPC-806 and 2-(5-formylamino-6-oxo-2-phenyl-1,6-di as bicifadine, dulloxetine, milnacipran, nefazodone, and ven hydropyrimidine-1-yl)-N-3,4-di oxo-1-phenyl-7-(2-py lafaxine; and combinations thereof. ridyloxy)-2-heptyl)acetamide (NK3201). In another embodiment, compounds of the invention are In one embodiment, compounds of the invention are administered in combination with a muscle relaxant, administered in combination with a diuretic. Representative 40 examples of which include, but are not limited to: carisopro diuretics include, but are not limited to: carbonic anhydrase dol, chlorZoxaZone, cyclobenzaprine, diflunisal, metaxalone, inhibitors such as acetazolamide and dichlorphenamide; loop methocarbamol, and combinations thereof. diuretics, which include sulfonamide derivatives such as In one embodiment, compounds of the invention are acetazolamide, ambuside, azosemide, bumetanide, butazola administered in combination with a natriuretic peptide or mide, chloraminophenamide, clofenamide, clopamide, clor 45 analog, examples of which include but are not limited to: exolone, disulfamide, ethoXZolamide, furosemide, mefru carperitide, CD-NP (Nile Therapeutics), CU-NP. nesiritide, side, methazolamide, piretanide, torsemide, tripamide, and PL-3994 (Palatin Technologies, Inc.), ularitide, cenderitide, Xipamide, as well as non-sulfonamide diuretics such as and compounds described in Ogawa et al (2004) J. Biol. ethacrynic acid and other phenoxyacetic acid compounds Chem. 279:28625-31. These compounds are also referred to Such as tienilic acid, indacrinone and quincarbate; osmotic 50 as natriuretic peptide receptor-A (NPR-A) agonists. In diuretics Such as mannitol; potassium-sparing diuretics, another embodiment, compounds of the invention are admin which include aldosterone antagonists such as spironolac istered in combination with a natriuretic peptide clearance tone, and Na channel inhibitors such as amiloride and triam receptor (NPR—C) antagonist such as SC-46542, cANF terene; thiazide and thiazide-like diuretics such as althiazide, (4-23), and AP-811 (Veale (2000) Bioorg Med Chem Lett bendroflumethiazide, benzylhydrochlorothiazide, benzthiaz 55 10:1949-52). For example, AP-811 has shown synergy when ide, buthiazide, chlorthalidone, chlorothiazide, cyclopenthi combined with the NEP inhibitor, thiorphan (Wegner (1995) azide, cyclothiazide, epithiazide, ethiazide, fenguizone, flu Clin. Exper. Hypert. 17:861-876). methiazide, hydrochlorothiazide, hydroflumethiazide, In another embodiment, compounds of the invention are indapamide, methylclothiazide, meticrane, metolaZone, administered in combination with a neprilysin (NEP) inhibi paraflutizide, polythiazide, quinethaZone, teclothiazide, and 60 tor. Representative NEP inhibitors include, but are not limited trichloromethiazide; and combinations thereof. In a particu to: AHU-377; candoxatril; candoxatrilat; dexecadotril ((+)- lar embodiment, the diuretic is selected from amiloride, N-2(R)-(acetylthiomethyl)-3-phenylpropionylglycine ben bumetanide, chlorothiazide, chlorthalidone, dichlorphena Zyl ester); CGS-24128 (3-3-(biphenyl-4-yl)-2-(phospho mide, ethacrynic acid, furosemide, hydrochlorothiazide, nomethylamino)propionamidopropionic acid); CGS-24592 hydroflumethiazide, indapamide, methylclothiazide, metola 65 ((S)-3-3-(biphenyl-4-yl)-2-(phosphonomethylamino)propi Zone, torsemide, triamterene, and combinations thereof. The onamidopropionic acid); CGS-25155 (N-9 (R)-(acetylthi diuretic will be administered in an amount sufficient to pro omethyl)-10-oxo-1-azacyclodecan-2(S)-ylcarbonyl-4 (R)- US 8,871,792 B2 47 48 hydroxy-L-proline benzyl ester);3-(1-carbamoylcyclohexyl) fenic acid, tioxaprofen, tolfenamic acid, tolmetin, triflumi propionic acid derivatives described in WO 2006/027680 to date, Zidometacin, Zomepirac, and combinations thereof. In a Hepworth et al. (Pfizer Inc.); JMV-390-1 (20R)-benzyl-3-(N- particular embodiment, the NSAID is selected from etodolac, hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine); eca flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, dotril; phosphoramidon; retrothiorphan; RU-42827 (2-(mer meloxicam, naproxen, oxaprozin, piroxicam, and combina captomethyl)-N-(4-pyridinyl)benzenepropionamide); tions thereof. RU-44004 (N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl) In one embodiment, compounds of the invention are propionamide); SCH-32615 ((S)- N—N-(1-carboxy-2- administered in combination with an N-methyl d-aspartate phenylethyl)-L-phenylalanyl-3-alanine) and its prodrug (NMDA) receptor antagonist, examples of which include, by SCH-34826 ((S) N—N-1-(2,2-dimethyl-1,3-dioxolan 10 4-yl)methoxycarbonyl-2-phenylethyl-L-phenylalanyl-B- way of illustration and not limitation, including amantadine, alanine); sialorphin; SCH-42495 (N-2(S)-(acetylsulfanylm dextromethorphan, dextropropoxyphene, ketamine, ketobe ethyl)-3-(2-methylphenyl)propionyl-L-methionine ethyl midone, memantine, methadone, and so forth. ester); spinorphin; SQ-28132 (N-2-(mercaptomethyl)-1- In still another embodiment, compounds of the invention oxo-3-phenylpropylleucine); SQ-28.603 (N-2-(mercaptom 15 are administered in combination with an opioid receptorago ethyl)-1-oxo-3-phenylpropyl-(3-alanine); SQ-29072 (7-2- nist (also referred to as opioid analgesics). Representative (mercaptomethyl)-1-oxo-3-phenylpropylaminoheptanoic opioid receptor agonists include, but are not limited to: acid); thiorphan and its prodrug racecadotril:UK-69578 (cis buprenorphine, butorphanol, codeine, dihydrocodeine, fenta 4-1-2-carboxy-3-(2-methoxyethoxy)propylcyclopentyl nyl, hydrocodone, hydromorphone, levallorphan, levorpha carbonyl)aminocyclohexanecarboxylic acid); UK-447.841 nol, meperidine, methadone, morphine, nalbuphine, (2-1-3-(4-chlorophenyl)propylcarbamoyl-cyclopentylm nalmefene, nalorphine, naloxone, naltrexone, nalorphine, ethyl-4-methoxybutyric acid); UK-505,749 ((R)-2-methyl oxycodone, oxymorphone, pentazocine, propoxyphene, tra 3-(1-3-(2-methylbenzothiazol-6-yl)propylcarbamoyl madol, and combinations thereof. In certain embodiments, cyclopentyl)propionic acid); 5-biphenyl-4-yl-4-(3- the opioid receptoragonist is selected from codeine, dihydro carboxypropionylamino)-2-methylpentanoic acid and 25 codeine, hydrocodone, hydromorphone, morphine, oxyc 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methyl odone, oxymorphone, tramadol, and combinations thereof. pentanoic acid ethyl ester (WO 2007/056546); daglutril (3S, In a particular embodiment, compounds of the invention 2R)-3-1-2'-(ethoxycarbonyl)-4'-phenylbutyl-cyclopen are administered in combination with a phosphodiesterase tan-1-carbonylamino)-2,3,4,5-tetrahydro-2-oxo-1H-1- (PDE) inhibitor, particularly a PDE-V inhibitor. Representa benzazepine-1-acetic acid described in WO 2007/106708 to 30 tive PDE-V inhibitors include, but are not limited to, avanafil. Khder et al. (Novartis AG); and combinations thereof. In a particular embodiment, the NEP inhibitor is selected from lodenafil, mirodenafil, sildenafil (RevatioR), tadalafil (Ad AHU-377, candoxatril, candoxatrilat, CGS-24128, phos circaR), Vardenafil (Levitra(R), and udenafil. phoramidon, SCH-32615, SCH-34826, SQ-28603, thior In another embodiment, compounds of the invention are phan, and combinations thereof. In a particular embodiment, 35 administered in combination with a prostaglandin analog the NEP inhibitor is a compound such as daglutril or CGS (also referred to as prostanoids or prostacyclin analogs). Rep 26303 (N-2-(biphenyl-4-yl)-1 (S)-(1H-tetrazol-5-yl)ethyl resentative prostaglandin analogs include, but are not limited aminomethylphosphonic acid), which have activity both as to, beraprost sodium, bimatoprost, epoprostenol, iloprost, inhibitors of the endothelin converting enzyme (ECE) and of latanoprost, tafluprost, travoprost, and treprostinil, with NEP. Other dual acting ECE/NEP compounds can also be 40 bimatoprost, latanoprost, and tafluprost being of particular used. The NEP inhibitor will be administered in an amount interest. sufficient to provide from about 20-800 mg per day, with In yet another embodiment, compounds of the invention typical daily dosages ranging from 50-700 mg per day, more are administered in combination with a prostaglandin recep commonly 100-600 or 100-300 mg per day. toragonist, examples of which include, but are not limited to, In one embodiment, compounds of the invention are 45 bimatoprost, latanoprost, travoprost, and so forth. administered in combination with a nitric oxide donor, Compounds of the invention may also be administered in examples of which include, but are not limited to nicorandil; combination with a renin inhibitor, examples of which organic nitrates such as pentaerythritol tetranitrate; and Syd include, but are not limited to, alliskiren, enalkiren, remikiren, nonimines such as linsidomine and molsidomine. and combinations thereof. In yet another embodiment, compounds of the invention 50 In another embodiment, compounds of the invention are are administered in combination with a non-steroidal anti administered in combination with a selective serotonin inflammatory agent (NSAID). Representative NSAIDs reuptake inhibitor (SSRI). Representative SSRIs include, but include, but are not limited to: acemetacin, acetyl salicylic are not limited to: citalopram and the citalopram metabolite acid, alclofenac, alminoprofen, amfenac, amiprilose, aloX desmethylcitalopram, dapoxetine, escitalopram (e.g., escit iprin, anirolac, apaZone, azapropaZone, benorilate, benox 55 allopram oxalate), fluoxetine and the fluoxetine desmethyl aprofen, bezpiperylon, broperamole, bucloxic acid, carpro metabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine fen, clidanac, diclofenac, diflunisal, diftalone, enolicam, maleate), paroxetine, Sertraline and the Sertraline metabolite etodolac, etoricoxib, fenbufen, fenclofenac, fenclozic acid, demethylsertraline, and combinations thereof. fenoprofen, fentiazac, feprazone, flufenamic acid, flufenisal, In one embodiment, compounds of the invention are fluprofen, flurbiprofen, furofenac, ibufenac, ibuprofen, 60 administered in combination with a 5-HT serotonin recep indomethacin, indoprofen, isoxepac, isoxicam, ketoprofen, toragonist, examples of which include, by way of illustration ketorolac, lofemizole, lornoxicam, meclofenamate, meclofe and not limitation, triptans Such as almotriptan, avitriptan, namic acid, mefenamic acid, meloxicam, mesalamine, miro eletriptan, froVatriptan, naratriptan rizatriptan, Sumatriptan, profen, mofebutaZone, nabumetone, naproxen, niflumic acid, and Zolmitriptan. oxaprozin, oXpinac, oxyphenbutaZone, phenylbutaZone, 65 In one embodiment, compounds of the invention are piroXicam, pirprofen, pranoprofen, Salsalate, Sudoxicam, Sul administered in combination with a sodium channel blocker, fasalazine, Sulindac, Suprofen, tenoxicam, tiopinac, tiapro examples of which include, by way of illustration and not US 8,871,792 B2 49 50 limitation, carbamazepine, fosphenyloin, lamotrigine, Sorbitan monooleate (50 mg) and Starch powder (250 mg), lidocaine, mexiletine, Oxcarbazepine, phenyloin, and combi and the resulting mixture loaded into a gelatin capsule (400 nations thereof. mg of composition per capsule). Alternately, a compound of the invention (40 mg) is thor In one embodiment, compounds of the invention are oughly blended with microcrystalline cellulose (Avicel PH administered in combination with a soluble guanylate cyclase 103: 259.2 mg) and magnesium stearate (0.8 mg). The mix stimulator or activator, examples of which include, but are not ture is then loaded into a gelatin capsule (Size #1, White, limited to ataciguat, riociguat, and combinations thereof. Opaque) (300 mg of composition per capsule). In one embodiment, compounds of the invention are administered in combination with a tricyclic antidepressant Exemplary Tablet Formulation for Oral (TCA), examples of which include, by way of illustration and 10 Administration not limitation, amitriptyline, amitriptylinoxide, butriptyline, clomipramine, demexiptiline, desipramine, dibenzepin, A compound of the invention (10 mg), starch (45 mg) and dimetacrine, doSulepin, doxepin, imipramine, imipraminox microcrystalline cellulose (35 mg) are passed through a No. ide, lofepramine, melitracen, metapramine, nitroxazepine, 20 mesh U.S. sieve and mixed thoroughly. The granules so nortriptyline, noxiptiline, pipofezine, propizepine, protrip 15 produced are dried at 50-60° C. and passed through a No. 16 tyline, quinupramine, and combinations thereof. mesh U.S. sieve. A Solution of polyvinylpyrrolidone (4 mg as In one embodiment, compounds of the invention are a 10% solution in sterile water) is mixed with sodium car administered in combination with a vasopressin receptor boxymethyl starch (4.5 mg), magnesium Stearate (0.5 mg), antagonist, examples of which include, by way of illustration and talc (1 mg), and this mixture is then passed through a No. and not limitation, conivaptain and tolvaptan. 16 mesh U.S. sieve. The sodium carboxymethyl starch, mag Combined secondary therapeutic agents may also be help nesium Stearate and talc are then added to the granules. After ful in further combination therapy with compounds of the mixing, the mixture is compressed on a tablet machine to invention. For example, compounds of the invention can be afford a tablet weighing 100 mg. combined with a diuretic and an ARB, or a calcium channel Alternately, a compound of the invention (250mg) is thor 25 oughly blended with microcrystalline cellulose (400 mg), blocker and an ARB, or a diuretic and an ACE inhibitor, or a silicon dioxide fumed (10 mg), and Stearic acid (5 mg). The calcium channel blocker and a statin. Specific examples mixture is then compressed to form tablets (665 mg of com include, a combination of the ACE inhibitor enalapril (in the position per tablet). maleate salt form) and the diuretic hydrochlorothiazide, Alternately, a compound of the invention (400 mg) is thor which is sold under the mark Vaseretic(R), or a combination of oughly blended with cornstarch (50 mg), croScarmellose the calcium channel blocker amlodipine (in the besylate salt 30 Sodium (25 mg), lactose (120mg), and magnesium Stearate (5 form) and the ARB olmesartan (in the medoxomil prodrug mg). The mixture is then compressed to form a single-scored form), or a combination of a calcium channel blocker and a tablet (600 mg of composition per tablet). statin, all may also be used with the compounds of the inven Alternately, a compound of the invention (100 mg) is thor tion. Other therapeutic agents such as C2-adrenergic receptor oughly blended with cornstarch (100 mg) with an aqueous agonists and vasopressin receptor antagonists may also be 35 Solution of gelatin (20 mg). The mixture is dried and ground helpful in combination therapy. Exemplary a-adrenergic to a fine powder. Microcrystalline cellulose (50 mg) and receptor agonists include clonidine, dexmedetomidine, and magnesium Stearate (5 mg) are then admixed with the gelatin guanfacine. formulation, granulated and the resulting mixture com The following formulations illustrate representative phar pressed to form tablets (100 mg of the compound of the maceutical compositions of the invention. 40 invention per tablet). Exemplary Hard Gelatin Capsules for Oral Exemplary Suspension Formulation for Oral Administration Administration A compound of the invention (50 g), 440 g spray-dried 45 The following ingredients are mixed to form a Suspension lactose and 10g magnesium Stearate are thoroughly blended. containing 100 mg of the compound of the invention per 10 The resulting composition is then loaded into hard gelatin mL of Suspension: capsules (500 mg of composition per capsule). Alternately, a compound of the invention (20 mg) is thoroughly blended with starch (89 mg), microcrystalline cellulose (89 mg) and 50 Ingredients Amount magnesium Stearate (2 mg). The mixture is then passed Compound of the invention 1.0 g through a No. 45 mesh U.S. sieve and loaded into a hard Fumaric acid 0.5 g. gelatin capsule (200 mg of composition per capsule). Sodium chloride 2.0 g Alternately, a compound of the invention (30 g), a second Methyl paraben 0.15 g ary agent (20g), 440 g spray-dried lactose and 10 g magne 55 Propyl paraben 0.05 g Granulated Sugar 25.5 g sium Stearate are thoroughly blended, and processed as Sorbitol (70% solution) 12.85 g described above. Veegum (R) K (magnesium aluminum silicate) 1.0 g Flavoring O.O35 mL. Exemplary Gelatin Capsule Formulation for Oral Colorings 0.5 mg Administration 60 Distilled water q.S. to 100 mL. A compound of the invention (100 mg) is thoroughly blended with polyoxyethylene sorbitan monooleate (50 mg) Exemplary Liquid Formulation for Oral and starch powder (250mg). The mixture is then loaded into Administration a gelatin capsule (400 mg of composition per capsule). Alter 65 nately, a compound of the invention (70 mg) and a secondary A suitable liquid formulation is one with a carboxylic agent (30 mg) are thoroughly blended with polyoxyethylene acid-based buffer such as citrate, lactate and maleate buffer US 8,871,792 B2 51 52 Solutions. For example, a compound of the invention (which EtN triethylamine may be pre-mixed with DMSO) is blended with a 100 mM EtO diethyl ether ammonium citrate buffer and the pH adjusted to pH 5, or is blended with a 100 mM citric acid solution and the pH EtOAc ethyl acetate adjusted to pH 2. Such solutions may also include a solubi EtOH ethanol lizing excipient such as a cyclodextrin, for example the solu EtSiH triethylsilane tion may include 10 wt % hydroxypropyl-3-cyclodextrin. HATU N.N.N.N'-tetramethyl-O-(7-azabenzotriazol-1-yl) Other suitable formulations include a 5% NaHCO, solu tion, with or without cyclodextrin. uronium hexafluorophosphate 10 HCTU (2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tet Exemplary Injectable Formulation for ramethylaminium hexafluorophosphate) Administration by Injection HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid A compound of the invention (0.2g) is blended with 0.4M sodium acetate buffer solution (2.0 mL). The pH of the result 15 HOBt 1-hydroxybenzotriazole ing Solution is adjusted to pH 4 using 0.5 Naqueous hydro MeCN acetonitrile chloric acid or 0.5 N aqueous Sodium hydroxide, as neces MeOH methanol sary, and then sufficient water for injection is added to provide a total volume of 20 mL. The mixture is then filtered through MeTHF 2-methyltetrahydrofuran a sterile filter (0.22 micron) to provide a sterile solution suit Pd(dppf)Cl. 1,1-bis(diphenylphosphino) ferrocene palla able for administration by injection. dium chloride Pd(PPh) tetrakis(triphenylphosphine)palladium(0) Exemplary Compositions for Administration by Inhalation PE petroleum ether 25 PMB p-methoxybenzyl A compound of the invention (0.2 mg) is micronized and PyBOP benzotriazol-1-yloxytris(pyrrolidino)phospho then blended with lactose (25 mg). This blended mixture is nium hexafluorophosphate then loaded into a gelatin inhalation cartridge. The contents of SilicaCat RDPP-Pd silica based diphenylphosphine palla the cartridge are administered using a dry powder inhaler, for dium (II) catalyst example. 30 Alternately, a micronized compound of the invention (10g) TFA trifluoroacetic acid is dispersed in a solution prepared by dissolving lecithin (0.2 THF tetrahydrofuran g) indemineralized water (200 mL). The resulting Suspension Unless noted otherwise, all materials, such as reagents, is spray dried and then micronized to form a micronized starting materials and solvents, were purchased from com composition comprising particles having a mean diameter 35 mercial suppliers (such as Sigma-Aldrich, Fluka Riedel-de less than about 1.5 Lum. The micronized composition is then Haén, and the like) and were used without further purifica loaded into metered-dose inhaler cartridges containing pres tion. Surized 1,1,1,2-tetrafluoroethane in an amount Sufficient to provide about 10 ug to about 500 g of the compound of the Reactions were run under nitrogen atmosphere, unless invention per dose when administered by the inhaler. 40 noted otherwise. The progress of reactions were monitored by Alternately, a compound of the invention (25 mg) is dis thin layer chromatography (TLC), analytical high perfor solved in citrate buffered (pH 5) isotonic saline (125 mL). The mance liquid chromatography (anal. HPLC), and mass spec mixture is stirred and Sonicated until the compound is dis trometry, the details of which are given in specific examples. solved. The pH of the solution is checked and adjusted, if Solvents used in analytical HPLC were as follows: solvent A necessary, to pH 5 by slowly adding aqueous 1 NNaOH. The 45 was 98% HO/2% MeCN/1.0 mL/LTFA: solvent B was 90% Solution is administered using a nebulizer device that pro MeCN/10% HO/1.0 mL/L TFA. vides about 10 ug to about 500 lug of the compound of the Reactions were worked up as described specifically in each invention per dose. preparation for example; commonly reaction mixtures were purified by extraction and other purification methods such as EXAMPLES 50 temperature-, and solvent-dependent crystallization, and pre cipitation. In addition, reaction mixtures were routinely puri The following Preparations and Examples are provided to fied by preparative HPLC, typically using Microsorb C18 and illustrate specific embodiments of the invention. These spe Microsorb BDS column packings and conventional eluents. cific embodiments, however, are not intended to limit the Progress of reactions was typically measured by liquid chro Scope of the invention in any way unless specifically indi 55 matography mass spectrometry (LCMS). Characterization of cated. isomers were done by Nuclear Overhauser effect spectros The following abbreviations have the following meanings copy (NOE). Characterization of reaction products was rou unless otherwise indicated and any other abbreviations used tinely carried out by mass and H-NMR spectrometry. For herein and not defined have their standard, generally accepted NMR measurement, samples were dissolved in deuterated meaning: 60 solvent (CDOD, CDC1, or DMSO-d), and H-NMR spec AcOH acetic acid tra were acquired with a Varian Gemini 2000 instrument (400 BOC t-butoxycarbonyl ( C(O)OC(CH)) MHz) under standard observation conditions. Mass spectro DCM dichloromethane or methylene chloride metric identification of compounds was typically conducted DIPEAN,N-diisopropylethylamine using an electrospray ionization method (ESMS) with an DMAN,N-dimethylacetamide 65 Applied Biosystems (Foster City, Calif.) model API 150 EX DMF N,N-dimethylformamide instrument or an Agilent (Palo Alto, Calif.) model 1200 EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide LC/MSD instrument. US 8,871,792 B2 53 54 Preparation 1: (R)-3-N-(4-bromobenzyl)-N'-t-bu -continued BOC toxycarbonylhydrazino-2-hydroxypropionic Acid BOC Methyl Ester NH Br N1 NH HN

-o-

-- 1 HN n BOC Br Br 10 (1) Br BOC To a stirred solution of t-butyl carbazate (50 g., 0.4 mol) in dry THF (400 mL) was added dropwise a solution of 4-bro NH mobenzaldehyde (70 g., 0.4 mol) in dry THF (200 mL). The 15 mixture was stirred at room temperature for 2 hours, and then concentrated in vacuo to yield Compound 1 as a yellow solid (113.8 g). LC-MS: 243 M-tBu+H". To a solution of Compound 1 (113.8g, 0.4 mol) in dry THF Br (1L) was added NaCNBH (36 g. 0.6 mol) in portions at 0°C. AcOH (180 mL) was added dropwise and the resulting mix (1) ture was stirred at room temperature overnight. Water (2 L) 4-Bromobenzyl bromide (5.0g, 20 mmol) and DIPEA (3.5 and EtOAC (1.5 L) were added and the aqueous phase was mL. 20.0 mmol) were dissolved in DMF (20 mL). t-Butyl adjusted to pH-7 with a saturated aqueous NaCO, solution. carbazate (7.9 g, 60.0 mmol) was added and the mixture was 25 The organic layer was separated, washed with saturated aque stirred at room temperature until the reaction was complete. ous NaCl and water (200 mL), dried over anhydrous NaSO, The mixture was partially concentrated, then the residue was and concentrated in vacuo. The residue was treated with partitioned between EtOAc and a saturated aqueous NaHCO MeOH (2 L) and 1N NaOH (1.5 L), and then stirred at room solution. The EtOAc layer was then dried over NaSO and temperature for 2 hours. After the removal of the MeCH concentrated. The crude product was purified by flash chro 30 solvent, the precipitate was collected by filtration to yield the matography to yield Compound 1 (3.8 g). title compound as a white solid (112 g). LC-MS. 245 M-tBu+H". O Preparation 3: (R)-3-N'-t-Butoxycarbonyl-N-(5'- (1) -- 1. O 35 chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- hydroxypropionic Acid Methyl Ester O H O BOC

NH 40 BOC HO OH S-rOH NH N1 HN F Br 45 (2) CI Br Compound 1 (1.9 g, 6.3 mmol) was dissolved in isopropyl BOC alcohol (26.4 mL). Methyl (2R)-glycidate (1.1 mL, 12.6 mmol) was added and the mixture was heated at 90° C. until NH the reaction was complete (~4 days). The mixture was cooled 50 HN1 to room temperature and concentrated to yield the title com pound as a white solid (2.5 g). Preparation 2: N'-(4-Bromobenzyl)hydrazinecarboxylic Acid 55 t-Butyl Ester O C C F (1) 60 To a solution of N'-(4-bromobenzyl)hydrazinecarboxylic acid t-butyl ester (60g, 0.2 mol) in 1,4-dioxane (1.5 mL) was HN n BOC added 5-chloro-2-fluorophenylboronic acid (38 g., 0.2 mol) and Pd(dppf)Cl. (7.3 g). The mixture was stirred at room 65 temperature under nitrogen for 10 minutes, and then, KCO Br (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 US 8,871,792 B2 55 56 room temperature and concentrated in vacuo. The residue was followed by the addition of SilicaCat RDPP-Pd(0.28 mmol/g extracted with EtOAc (3x300 mL). The combined organic loading: 886 mg, 248 umol). The mixture was heated at 90° C. layers were dried over anhydrous NaSO and concentrated until the reaction was complete (2 hours). The precipitate was in vacuo. The product was purified by column chromatogra filtered off, and the filtrate was concentrated and purified by phy (hexanes/EtOAc=10:15:1) to yield Compound 1 as a pink 5 reverse phase chromatography (30-95% MeCN in water with solid (56 g). LC-MS: 701 2M--H". 0.5% TFA). The clean fractions were collected, lyophilized, and combined with 4M HCl in dioxane (8 mL, 30 mmol) and O EtOH (10 mL, 200 mmol). The resulting mixture was stirred at room temperature until the reaction was complete (7 (1) + 1 - 10 H hours). The mixture was concentrated to yield an oil, which O was stirred in ether with few drops of EtOH overnight. The O precipitate was filtered off and rinsed with ether to yield the title compound (140 mg). No N1 NBOC 15 Alternate Preparation of (R)-3-N-(5'-Chloro-2'-fluo OH robiphenyl-4-ylmethyl)hydrazino-2-hydroxypropi onic Acid Ethyl Ester C O No N1Y BOC (2) OH To a solution of Compound 1 (20g, 57 mmol) in isopropyl 25 alcohol (250 mL) was added methyl (2R)-glycidate (8.7g, 86 mmol) under nitrogen. The mixture was stirred at 85°C. for 3 days, then cooled to room temperature. The precipitated Solid was collected by filtration to yield the title compound as an 30 off-white solid (18.5 g). LC-MS. 397 M-tEu+H". Preparation 4: (R)-3-N-(5'-Chloro-2'-fluorobiphe nyl-4-ylmethyl)hydrazino-2-hydroxypropionic Acid OH Ethyl Ester 35

C O oc NH No N1 40

OH -- A solution of (R)-3-N'-t-butoxycarbonyl-N-(5'-chloro-2'- fluorobiphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic

Br 45 acid methyl ester (20 g, 16 mmol) in HC1/EtOH (1.1 M, 200 OH mL) was stirred overnight and then concentrated in vacuo. The residue was dispersed in EtOAc (2x40 mL), and the B C precipitate was collected by filtration to give the title com pound as an off-white solid HCl salt (8.8 g). LC-MS: 367 M+H". "H NMR (300 MHz, DMSO-d) & 1.05 (t, J=7.2 Hz, F 50 3H), 3.05-3.03 (q, J–7.2 Hz, 2H), 4.06-3.95 (m, 4H), 4.42 (br. O 1H), 6.46 (br. 1H), 7.62-7.40 (m, 7H), 9.42 (s.3H). 1N O N1 NH2 Preparation 5: 1-(3-Chlorophenyl)-5-oxo-4,5-dihy dro-1H-1,2,4-triazole-3-carboxylic Acid OH 55

60 -y (R)-3-N-(4-Bromobenzyl)-N'-t-butoxycarbonylhy drazino-2-hydroxypropionic acid methyl ester (1.0 g, 2.5 mmol), 5-chloro-2-fluorophenylboronic acid (865 mg, 5.0 65 C mmol), and KCO (857 mg, 6.2 mmol), were combined in EtOH (30 mL, 500 mmol) and water (8 mL, 400 mmol), US 8,871,792 B2 57 58 -continued -continued

HO \ HO N OH

C 10

A mixture of 1-(3-chlorophenyl)-5-oxo-4,5-dihydro-1H (1) 1,2,4-triazole-3-carboxylic acid ethyl ester (200.0 mg, 747 mmol), LiOH (71.6 mg, 1.5 mmol) in water (2 mL), and MeOH (10.0 mL, 247 mmol) was stirred at room temperature 15 LiOH hydrate (3 g, 73 mmol) in water (60 mL) was added overnight then concentrated. The residue was acidified with to (R)-3-N'-t-butoxycarbonyl-N-(5'-chloro-2'-fluorobiphe 1N HCl to pH 3-4, forming a precipitate, which was filtered, nyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid methyl washed with water (2x5 mL), and dried in vacuo to yield the ester (16.5g, 36.5 mmol) in MeOH (300 mL). The mixture title compound (100.6 mg). was stirred at room temperature for 2 hours, and the MeCH was evaporated in vacuo. The mixture was adjusted to pH=5 Preparation 6: (R)-3-N-(4-Bromobenzyl)hy with 1 M aqueous HCl, and the residue was extracted with EtOAc (2x300 mL). The combined organic layers were dried drazino-2-hydroxypropionic Acid Ethyl Ester over anhydrous NaSO, and concentrated in vacuo to yield 25 Compound 1 as a white solid (18 g). LC-MS: 383 M-tBu+ O BOC H". N1 NH (2) He O N->OH ON O f or NH2 O Br O OH O NH2 O C 1N --> N1 OH F

40 To a solution of Compound 1 (1.5 g., 3.42 mmol), KCO Br (0.95 g. 6.84 mmol) and potassium iodide (20 mg) in DMF (40 mL) was added 4-(bromomethyl)-5-methyl-1,3-dioxol A solution of (R)-3-N-(4-bromobenzyl)-N'-t-butoxycar 2-one (0.8 g. 4.1 mmol) in DMF (15 mL). The resulting bonylhydrazino-2-hydroxypropionic acid methyl ester (25 mixture was stirred for 4 hours at room temperature. Satu g, 62 mmol) in EtOH/HCl (1M,310 mL, 0.3 mol) was stirred 45 rated aqueous NaCl (30 mL) was added and the mixture was overnight until the reaction was complete. The mixture was extracted with EtOAc (2x50 mL). The combined organic then concentrated and the residue was washed with EtOAc layers were dried over anhydrous NaSO and concentrated (120 mL) and filtered. The solids were collected to yield the in vacuo. The residue was purified by column chromatogra title compound as a white solid HCl salt (15g). phy (hexanes/EtOAc=1:1) to yield Compound 2 as a yellow 50 solid (930 mg). LC-MS: 495 M-tBu+H". Preparation 7: (R)-3-N-(5'-Chloro-2'-fluorobiphe nyl-4-ylmethyl)hydrazino-2-hydroxypropionic Acid (2) -e- 5-methyl-2-oxo-1,3-dioxol-4-ylmethyl Ester O 55 O.N O or NH2 O OH N 1 Y BOC 60 OH O C

C F

65 Compound 2 (400 mg, 0.73 mmol) was dissolved in MeCN F (20 mL), and cooled to 0°C. N-trimethylsilylimidazole (290 mg, 1.46 mmol) was added dropwise and the resulting mix US 8,871,792 B2 59 60 ture was stirred for 2 hours. MeOH (50 mL) was added to mg, 21.1 mmol) dissolved in water (10 mL). The resulting quench the reaction. The mixture was washed with Saturated mixture was stirred at room temperature for 3 hours. Any aqueous NaCl (2x50 mL) and extracted with DCM (2x80 solids were filtered off and the filtrate was concentrated by mL). The combined organic layers were dried over anhydrous rotary evaporation. EtOAc (50 mL) was added and the mix NaSO and concentrated in vacuo. The product was col 5 ture was dried. This was repeated two more times. The prod lected to yield the title compound as a yellow solid (200 mg). uct was then dried under high vacuum at room temperature to LC-MS: 451 M+H". yield the title compound. Preparation 8: 5-Oxo-1-phenyl-4,5-dihydro-1H-1.2. Preparation 10: (R)-3-N-(5'-Chloro-2'-fluorobiphe 4triazole-3-carboxylic Acid 10 nyl-4-ylmethyl)-N'-(2-trityl-2H-tetrazole-5-carbonyl) hydrazino-2-hydroxypropionic Acid

15

O N \ Ho- N HO N-NH

O rOH 25 Methyl 2,5-dihydro-5-oxo-1-phenyl-1h-1,2,4-triazole-3- carboxylate (300.0 mg, 1.4 mmol) was mixed with MeOH (4.5 mL, 110 mmol) and water (0.5 mL, 30 mmol) at room temperature, then treated with LiOH monohydrate (0.1 g, 2.7 Tr mmol) at room temperature overnight. The mixture was con 30 \ centrated and the resulting residue was acidified to pH-1 with N1 \ N 1Naqueous HC1. The resulting solids were filtered and rinsed O M -3- with water, then dried in vacuo to give the title compound as N a yellowish solid (185 mg), which was used without further 35 purification. O Li" Preparation 9: 2-Trityl-2H-tetrazole-5-carboxylate Tr Lithium Salt N1 \v. 40 N O M O N

NH HO N1 45 OH

C

50 F

2-Trityl-2H-tetrazole-5-carboxylate lithium salt (385.2 55 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 min utes. (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)hy Ethyl-5-tetrazolecarboxylate sodium salt (2.3 g 14 mmol) drazino-2-hydroxypropionic acid ethyl ester (300 mg, 818 was dissolved in DMF (20 mL, 200 mmol). The mixture was umol) was added followed by DIPEA (285 uL, 1.6 mmol). cooled at 0°C. Triphenylmethyl chloride (3.9 g, 14.1 mmol) 60 The resulting mixture was stirred at room temperature over was added and the resulting mixture was stirred at room night. The mixture was partitioned between EtOAc (10.0 mL) temperature overnight, yielding a slurry. The slurry was and water (2.0 mL). The organic layer was washed with water slowly poured into cold stirred water (200 mL). The resulting (2.0 mL), dried over NaSO filtered and concentrated to slurry was stirred for 15 minutes (bicarbonate was added to give a yellowish oil. The oily residue was then purified by keep the pH basic), then filtered and dried to yield a white 65 flash chromatography (0-50% EtOAc/hexanes). The desired solid (5.1 g). The solid was suspended in MeOH (50 mL, 1.0 fractions were combined and concentrated to give a light mol), followed by the addition of LiOH monohydrate (886 yellowish oil (combined with other lots for a total of 147.2 US 8,871,792 B2 61 mg). This residue was dissolved in MeCH (5.0 mL, 120 -continued mmol) and water (0.5 mL, 30 mmol) at room temperature. H H LiOH monohydrate (17.5 mg, 417 umol) was added and N1YN BOC N1 N NBOC allowed to sit for 30 minutes. The mixture was concentrated, and the resulting residue was treated with EtOAc (10.0 mL) -- and acidified with 1N HCl till pH-3. The organic layer was washed with saturated aqueous NaCl (3x3.0 mL), dried over sodium NaSO filtered and concentrated to yield the title Br Br compound as a white foam (99.4 mg). (1) (2) 10 Preparation 11: Lithium 1-Allyl-1H-tetrazole-5-carboxylate To a stirred solution of t-butyl carbazate (50 g., 0.4 mol) in dry THF (400 mL) was added dropwise a solution of 4-bro mobenzaldehyde (70 g., 0.4 mol) in dry THF (200 mL). The 15 mixture was stirred at room temperature for 2 hours, and then concentrated in vacuo to yield Compound 1 as a yellow solid (113.8 g). LC-MS: 243 M-tEu+H". To a solution of Compound 1 (113.8 g., 0.4 mol) in dry THF (1L) was added NaCNBH (36 g. 0.6 mol) in portions at 0°C. AcOH (180 mL) was added dropwise and the resulting mix ture was stirred at room temperature overnight. Water (2 L) and EtOAc (1.5 L) were added and the aqueous phase was adjusted to pH 7 with a saturated aqueous NaCO solution. 25 The organic layer was separated, washed with Saturated aque ous NaCl and water (200 mL), dried over anhydrous NaSO, O and concentrated in vacuo. The residue was treated with LiO Z MeOH (2 L) and 1N NaOH (1.5 L), and then stirred at room -N4 30 temperature for 2 hours. After the removal of the MeCH solvent, the precipitate was collected by filtration to yield | \ Compound 2 as a white solid (112 g). LC-MS. 245 (M-tBu+ NéN HI.

35 To a stirred solution of 1H-tetrazole-5-carboxylic acid HO OH ethyl ester (2.0 g, 14.1 mmol) in DMF (20 mL) was added 1. KCO (2.3 g, 16.9 mmol) and 3-bromoprop-1-ene (1.9 g, 15.4 mmol) at 0°C. The mixture was warmed to room tem (2) + perature, stirred overnight, then poured into water (200 mL). The resulting solution was extracted with EtOAc (3x100 40 mL). The combined organic layers were washed with Satu C rated aqueous NaCl (100 mL), dried over anhydrous NaSO, filtered and concentrated in vacuo to yield Compound 1 as a yellow oil (2.3 g). LC-MS: 183 M+H". N n BOC To a solution of Compound 1 (2.3 g, 12.6 mmol) in EtOH 45 (20 mL) was added a solution of LiOH.H2O (636 mg, 15.2 mmol) in water (10 mL). The mixture was stirred at room temperature for 3 hours, the solids were filtered off, and the C filtrate was concentrated in vacuo to yield the title compound as a yellow solid (2.0 g), which was used without further 50 purification. F Preparation 12: (R)-3-N-(5'-Chloro-2'-fluorobiphe (3) nyl-4-ylmethyl)hydrazino-2-hydroxypropionic Acid Ethyl Ester 55 To a solution of Compound 2 (60g, 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 room temperature under nitrogen for 10 minutes, and 60 KCO (55.2g, 0.4 mol) in water (240 mL) was added. The resulting mixture was stirred at 60°C. for 3 hours, then cooled to room temperature and concentrated in vacuo. The residue HN1 N BOC was extracted with EtOAc (3x300 mL). The combined organic layers were dried over anhydrous NaSO and con 65 centrated in vacuo. The product was purified by column chro Br matography (PE:EtOAc=10:1-5:1) to yield Compound 3 as a pink solid (56 g). LC-MS: 701 2M--H". US 8,871,792 B2 64 -continued O - O O (3) + 1 —- H O HO N-NH

O 10 No N1Y BOC OH Hip 15 C

F (4)

O 25 1No N1 NH2 OH

30 O C (1)

To a solution of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- 35 ylmethyl)hydrazino-2-hydroxypropionic acid ethyl ester (3.4 g, 8.3 mmol) and lithium 1-allyl-1H-tetrazole-5-car To a solution of Compound 3 (20g, 57 mmol) in isopropyl boxylate (2.0 g, 12.50 mmol) in DMF (40 mL) were added alcohol (250 mL) was added methyl (2R)-glycidate (8.7g, 86 PyBOP (8.7 g. 16.7 mmol) and DIPEA (2.1 g, 16.7 mmol) mmol) under nitrogen. The mixture was stirred at 85°C. for 3 dropwise at 0°C. under nitrogen. The resulting mixture was days, then cooled to room temperature. The precipitated Solid 40 stirred for 2.5 hours, then poured into water (400 mL). The was collected by filtration to yield Compound 4 as an off resulting solution was extracted with EtOAc (2x200 mL). white solid (18.5 g). LC-MS. 397 M-tEu+H". The combined organic layers were washed with saturated aqueous NaCl (100 mL), dried over anhydrous NaSO, fil A solution of Compound 4 (20g, 16 mmol) in HCl/EtOH tered, and concentrated in vacuo. The residue was purified by (1.1 M, 200 mL) was stirred overnight and then concentrated 45 column chromatography (PE:EtOAc=5:1-4:1-3:1) to yield in vacuo. The residue was dispersed in EtOAc (2x40 mL), and Compound 1 as a yellow oil (2.5 g). LC-MS. 503 M+H". the precipitate was collected by filtration to give the title compound as an off-white solid HCl salt (8.8 g). LC-MS: 367 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. 50 1H), 6.46 (br. 1H), 7.62-7.40 (m, 7H), 9.42 (s.3H). LiO

Preparation 13: Lithium (R)-3-(2-(1-Allyl-1H-tetra Zole-5-carbonyl)-1-((5'-chloro-2'-fluorobiphenyl-4- 55 (1) -> yl)methyl)hydrazinyl)-2-hydroxypropanoate

60

To a solution of Compound 1 (2.5g, 4.97 mmol) in EtOH 65 (25 mL) was added a solution of LiOH.H2O (250 mg. 6.0 mmol) in water (10 mL). The mixture was stirred at room temperature for 3 hours, the solids were filtered off, and the US 8,871,792 B2 65 66 filtrate was concentrated in vacuo to yield the title compound (0.5 mL), and dried to yield a white powder (0.6 g; HCl salt). as a yellow solid (2.2 g), which was used without further The powder was then dissolved in IPA (15 mL) and heated to purification. reflux. The resulting slurry was allowed to cooled to room temperature and stirred for 1 hour. The solids were collected Preparation 14: (R)-3-(N-(3'-Chlorobiphenyl-4-ylm to yield Compound 2 as a white solid. ethyl)-N'-3-(4-methoxybenzyloxy)isoxazole-5-car bonylhydrazino-2-hydroxypropionic Acid Isopro pyl Ester

10

15 (R)

O H To a stirred solution of methyl 3-hydroxyisoxazole-5-car boxylate (5.0 g, 35 mmol) in DMF (20 mL, 300 mmol) at 0° C. was added KCO (5.4g, 39.4 mmol). After 10 minutes at room temperature p-methoxybenzyl chloride (5.5 mL, 40.2 mmol) was added in one portion. The resulting mixture was OH 25 heated at 60°C. for 2 hours and then cooled to room tempera ture and stirred overnight. 1.0 M HCl in water (150 mL) and EtOAc (150 mL) were added and the phases were separated. The organic layer was washed with Saturated aqueous NaCl (10 mL), dried over NaSO, and the solvent removed by 30 rotary evaporation to yield a thick oil. The oil was dissolved in THF (35 mL) and MeOH (35 mL), followed by addition of (1) LiOH monohydrate (2.9 g, 69.9 mmol) dissolved in water (35 mL). The resulting mixture was stirred at room temperature N'-(3'-Chlorobiphenyl-4-ylmethyl)hydrazinecarboxylic and the reaction monitored for completion (s3 hours). Sol acid t-butyl ester (400.0 g, 1.2 mol) was combined with IPA 35 vent was removed by rotary evaporation at 30° C. to yield a (7.0 L, 91 mol) and (R)-oxirane-2-carboxylic acid methyl pasty solid. Toluene (100 mL) was added and the volume was reduced (to ~50 ml). EtOAc (200 mL) was added and the ester (105.2 mL, 1.2 mol) under nitrogen. The mixture was volume was reduced (to ~50 ml). Filtration and drying heated at 83° C. for 51 hours. Additional (R)-oxirane-2-car yielded a solid (10g), which was dissolved in water (200 mL), boxylic acid methyl ester (52.61 mL, 600.9 mmol) was added 40 and the pH was adjusted slowly with concentrated HCl to s2. and the mixture was heated at 84° C. for 48 hours. Sodium EtOAc (200 mL) was added and the phases were separated. cyanoborohydride (1.0 g, 16 mmol) was added and the mix The aqueous layer was back extracted with EtOAc (200 mL). ture was heated at 80° C. and the reaction monitored (s48 The combined organic layers were dried over NaSO, fol hours). Additional Sodium cyanoborohydride (1 g) was added lowed by solvent removal. The product was reslurried in and the mixture was heated at reflux (s1 days). The mixture 45 EtOAc:hexanes (1:1) followed by filtration to yield Com was then cooled slowly at 15° C.; filtered, and dried to yield pound 3 (purity).99%). Compound 1 (470 g).

(2) + (3) -> (1) -> 50 N O-1 N 1. -->O N1 NH, HCI O S. OH 55 1. O NH

r OH

60 (2) O C C

Compound 1 (880 mg, 1.9 mmol) was combined with 3 M HCl in CPME (7 mL, 20 mmol). The mixture was then stirred 65 Compound 2 (18.0g, 26.8 mmol) in DMF (90 mL) was on an ice bath and the reaction monitored for completion combined with Compound 3 (7.3 g, 29 mmol) in DIPEA (12 (s2.5 hours). The solids were collected, washed with CPME mL, 67 mmol). The mixture was cooled at 0°C. followed by US 8,871,792 B2 67 68 the portion-wise addition of PyBOP (18 g., 35 mmol) and the -continued reaction monitored for completion (s30 minutes at 0°C.). O-1 N Water (540 mL) and EtOAc (540 mL) were added and the N OH phases were separated. The organic layer was washed with O S. saturated aqueous NaCl (500 mL) and dried over NaSO O followed by solvent removal. The crude product was purified NH (SiG chromatography: 300 g column, 10-30-50% EtOAc/ --> N1 hexanes) to yield the title compound (9 g, purity).98%). O OH

10 Preparation 15: (R)-3-N-(3'-Chlorobiphenyl-4-ylm ethyl)-N'-(3-hydroxyisoxazole-5-carbonyl)hy drazino-2-hydroxypropionic Acid 2-oxo-2-phenyl ethyl Ester 15 2-Bromoacetophenone (44.6 mg, 224 umol) was added to a mixture of Compound 1 (100.0 mg, 179.2 umol) and KCO (49.5 mg, 358.5 umol) in DMF (2.0 mL, 26 mmol). The N PMB O e resulting mixture was stirred at room temperature for 30 N O/ minutes, concentrated, and purified by flash chromatography O (EtOAc-hexanes=20-80%) to yield a solid (107.7 mg). The O solid was combined with TFA (82.86 uL. 1.075 mmol) and NH !C anisole (194.8LL, 1.8 mmol) in DCM (5.0 mL, 78 mmol), and 1. N1 -- stirred at room temperature for 24 hours. The mixture was concentrated and the residue was dissolved in AcOH (2 mL), OH 25 filtered, and purified by reverse phase preparative HPLC. The desired fractions were combined and lyophilized to yield the C title compound (79.4 mg). Preparation 16: 30 (S)-2-t-Butoxycarbonylamino-3-methylbutyric Acid N PMB O-1 N M Chloromethyl Ester O S. O 35

O N NH Li" H

40 O O C

(1) 45 To a mixture of (S)-2-(t-butoxycarbonylamino)-3-meth ylbutanoic acid (28.6 g. 130 mmol) and NaHCO, (44.g. 520 (R)-3-(N-(3'-Chlorobiphenyl-4-ylmethyl)-N'-3-(4-meth mmol) and BuNHSO (4.4g, 13 mmol) in DCM (200 mL) oxybenzyloxy)isoxazole-5-carbonylhydrazino-2-hydrox and water (200 mL) was added chloromethyl sulfochloridate ypropionic acid isopropyl ester (2.0 g, 3.4 mmol) was com 50 (26 g. 158 mmol) at 0°C. The mixture was stirred at room bined with MeOH(40 mL, 1.0 mol). LiOH monohydrate (170 temperature for 24 hours, and then was extracted with DCM mg, 4.0 mmol) dissolved in water (5 mL, 300 mmol) was (3x150 mL). The combined organic layers were washed with added, and the resulting mixture was stirred at room tempera water (2x300 mL), and the DCM layer was purified by flash ture overnight. The mixture was concentrated by rotary column (PE:EtOAc=15:1) to yield the title compound as a evaporation. The residue was mixed with MeOH and again 55 yellow solid (35 g). LC-MS. 266 M+H". concentrated by rotary evaporation. The residue was then Preparation 17: dried under high vacuum at room temperature to yield crude (S)-2-Methoxycarbonylamino-3-methylbutyric Acid Compound 1 (2 g). Chloromethyl Ester 60 O O N-s-s -- (1) + 65 Yr- O C US 8,871,792 B2 69 70 -continued Preparation 19: O 1-Allyloxy-1H-1.2.3 triazole-4-carboxylic Acid o1 No 5 N2N - OH -- (1) N-' S. O A solution of (S)-2-t-butoxycarbonylamino-3-methylbu 10 tyric acid chloromethyl ester (35 g, 132 mmol) in DCM (200 e N mL) was added dropwise a solution of TFA (50 mL) in DCM N Y-o1\1 - (100 mL) at 0°C. The resulting mixture was stirred at room N S. temperature overnight and then concentrated in vacuo to yield crude Compound 1 as a yellow oil (21.8 g). LC-MS. 166 15 M+H". (1)

O O N2V N-O (1) + ls -- O HO C o1 1. 1N O O To a solution of 1-hydroxy-1H-1.2.3 triazole-4-carboxy To a mixture of Compound 1 (21.8 g. 139 mmol) and 25 lic acid ethyl ester (5 g, 31.8 mmol) in DMF (20 mL) was methyl chloroformate (12 mL, 157 mmol) in THF (1 L) was added KCO (5.3.g., 38.2 mmol) at room temperature. After added TEA (38 mL. 278 mmol) at 0°C. The resulting mixture 10 minutes, allyl bromide (4.g., 33.4 mmol) was added. The was stirred at room temperature for 12 hours, then concen mixture was stirred at room temperature overnight. Water trated in vacuo. The residue was purified by flash column (150 mL) was added, and the mixture was extracted with (PE:EtOAc=6:1) to yield the title compound as a yellow solid 30 EtOAc (3x50 mL). The combined organic layers were (20.3 g). LC-MS: 224 M+H". "H NMR (400 MHz, DMSO washed with saturated aqueous NaCl (50 mL) and dried over d): 80.97-102 (m, 6H), 2.16-2.21 (m, 1H), 3.68 (s, 1H), 4.14 anhydrous NaSO. The solution was evaporated to and the (d. J–4 Hz, 1H), 5.76-5.91 (m, 2H). residue was purified by silica gel chromatography (silica gel: 35 200-300 mesh, eluted with PE:EA=10:1 to 5:1 to 1:1) to yield Preparation 18: (R)-3-N-(4-Bromobenzyl)hy Compound 1 as a yellow oil (4.3 g). LC-MS: 198 M+H". drazino-2-hydroxypropionic Acid Methyl Ester To a solution of Compound 1 (4.3 g, 22.0 mmol) in EtOH (30 mL) was added a solution of LiOH (1.2g, 28.5 mmol) in water (10 mL). The resulting mixture was stirred at room 40 temperature overnight. The mixture was concentrated, water (10 mL) was added, and the mixture was extracted with O EtOAc (2x20 mL). The aqueous layer was acidified by 1N HCl to pH 3, and extracted with EtOAc (3x30 mL). The O > O combined organic layers were washed with Saturated aqueous No N1 NH 45 NaCl (30 mL) and dried over anhydrous NaSO. The solu -e- tion was evaporated to yield the title compound as a white OH solid (3.5 g). LC-MS: 170M+H".

50 Br Preparation 20: (R)-3-N'-(1-Allyloxy-1H-1,2,3- O triazole-4-carbonyl)-N-(2,5'-dichlorobiphenyl-4- ylmethyl)-hydrazino-2-hydroxypropionic Acid N O N1 NH2 55 OH

Br (R)-3-N-(4-Bromobenzyl)-N'-t-butoxycarbonylhy 60 drazino-2-hydroxypropionic acid methyl ester (1.1 g, 2.8 mmol) was dissolved in MeCN (10 mL) and of 4N HCl in dioxane (6 mL, 20 mmol). The mixture was stirred at room temperature until deprotection was complete (1 hour). The 65 precipitate was filtered and dried to yield the title compound HO O (840 mg) as an HCl salt. US 8,871,792 B2 71 72 -continued 1N HCl to pH 3 and extracted with EtOAc (3x50 mL). The combined organic layers were washed with Saturated aqueous - O NaCl (50 mL) and dried over anhydrous NaSO. The solu O tion was evaporated to yield the title compound as a yellow 5 solid (3.5 g). LC-MS. 506 M+H", 508 (M+2)+H". HO N-NH Example 1A (R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-1-(3-chlorophenyl)-5-oxo-4,5-dihydro-1H-1.2. 10 4triazole-3-carbonylhydrazino-2-hydroxypropi ( ) onic Acid - O O 15 HO N-NH - O O /— O O M /Nl HO N-N N2 H ( ) --

25

Cl

C 30

(1) / N O --> To a solution of (R)-3-N-(2,5'-dichlorobiphenyl-4-ylm \ y N ethyl)hydrazino-2-hydroxy-propionic acid ethyl ester (HCl H salt, 4 g., 9.9 mmol) and 1-allyloxy-1H-1.2.3 triazole-4-car 35 O boxylic acid (1.7g, 9.9 mmol) in DMF (30 mL) was added PyBOP (5.2g, 9.9) and DIPEA (3.2g, 24.8 mL) at 0°C. The OH mixture was stirred at room temperature for 4 hours. Water (200 mL) was added, and the mixture was extracted with C EtOAc (3x100 mL), and the combined organic layers were 40 washed with saturated aqueous NaCl (100 mL) and dried over O NN N anhydrous NaSO. The mixture was concentrated and the residue was purified by silica gel chromatography (silica gel: O )-( -l 200-300 mesh; eluted with PE:EtOAc=10:1 to 5:1 to 1:1) to NH N O yield Compound 1 as a light yellow solid (4.2 g). LC-MS. 534 45 -r H M+H", 536 (M+2)+H". OH O / HO O O C 50 HO F 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 (1) --> mg, 316 umol) were combined in DMF (1.3 mL, 17.2 mmol). 55 and stirred at room temperature for 15 minutes. (R)-3-N-(5'- Chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hydrox ypropionic acid ethyl ester (105 mg, 287 umol) and DIPEA (150 mL. 862 umol) were added, and the resulting mixture was stirred at room temperature for 15 minutes. The mixture 60 was evaporated under reduced pressure. The residue was dissolved in EtOH (1.0 mL, 17.2 mmol) and a solution of 1 M To a solution of Compound 1 (4.2g, 7.9 mmol) in THF (20 LiOH in water (1.4 mL, 1.4 mmol) was added. The resulting mL) and water (5 mL) was added LiOH (0.5g, 11.8 mmol) at mixture was stirred at room temperature for 1 hour, then room temperature. The reaction mixture was stirred at room evaporated under reduced pressure. The residue was purified temperature overnight. The mixture was concentrated, water 65 by reverse phase preparative HPLC to yield the title com (50 mL) was added, and the resulting mixture was extracted pound (75 mg, purity 100%) as a TFA salt. MS m/z. M+H" with EtOAc (2x20 mL). The aqueous layer was acidified by calc’d for CHCIFN-Os, 560.08. found 559.6. US 8,871,792 B2 73 74 Example 1B and 1C (R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-1-(3-chlorophenyl)-5-oxo-4,5-dihydro-1H-1.2. 4triazole-3-carbonylhydrazino-2-hydroxypropi onic Acid Ethyl Ester (Compound 1) and (R)-3-(N- (5'-Chloro-2'-fluorobiphenyl-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)

C

O S.

s'sNH O N1

OH

--

Co.F (1) (2)

1-(3-Chlorophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triaz Example 1D ole-3-carboxylic acid (27.6 mg, 115 mol) and HATU (52.5 mg, 138 umol) were stirred in DMA (1.0 mL, 11 mmol) for 10 45 (S)-2-Amino-3-methylbutyric Acid (R)-3-N-(5'- minutes. (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl) Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-1-(3-chlo hydrazino-2-hydroxypropionic acid ethyl ester (42.2 mg, rophenyl)-5-hydroxy-1H-1,2,4-triazole-3-carbonyl 115umol) and DIPEA (60.1 uL., 345umol) were added, and hydrazino-2-hydroxypropionyloxymethyl Ester resulting mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure and the 50 residue was dissolved in EtOAc (20 mL). The organic layer was washed with water (2x5 mL), dried over MgSO, and concentrated. One half of the material was dissolved in 50% acetic acid-water (1.5 mL), filtered, and purified by reverse phase preparative HPLC to yield Compound 1 (1.3 mg, purity 55 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 1,4-dioxane (115uL. 460 Limol) and stirred at room tempera 60 ture 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 reverse phase preparative HPLC to yield the Compound 2 (1.8 mg, purity 65 100%) as a TFA salt. MS m/z. M+H" calc’d for Using the procedures described herein, the title compound CHCl2FNOs, 616.15. found 616.4. can also be prepared.

US 8,871,792 B2 77 78

-continued -continued

HO

OH OH

10

15

2-Hydroxy-5-thiazolecarboxylic acid (43.5 mg 0.3 mmol) 2-Hydroxy-5-thiazolecarboxylic acid (9.0 mg. 62 umol) and HCTU (124 mg. 0.3 mmol) were stirred in DMF (1.3 mL, and HATU (28.3 mg, 74 umol) were stirred in DMA (0.5 mL, 16.4 mmol) for 15 minutes at room temperature. (R)-3-N- 5 mmol) for 10 minutes. (R)-3-N-(5'-Chloro-2'-fluorobiphe (5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hy nyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid ethyl droxypropionic acid ethyl ester (100 mg. 0.3 mmol) and ester (22.7 mg, 62 umol) and DIPEA (32.4 uL, 186 umol) DIPEA (142 uL. 818 umol) were added, and resulting mix were added, and resulting mixture was stirred at room tem 25 ture was stirred at room temperature for 30 minutes. The perature for 2 hours. The mixture was then concentrated mixture was then evaporated under reduced pressure. The under reduced pressure and the residue was dissolved in 50% residue was dissolved in EtOH (955uL. 16.4 mmol). A solu AcOH-water (1.5 mL), filtered, purified by reverse phase tion of 1.0 M LiOH in water (1.4 mL, 1.4 mmol) was added preparative HPLC, and lyophilized to yield the title com and the resulting mixture was stirred at 40° C. for 3 hours. 30 LC/MS showed completion. The solvent was removed in pound (11.9 mg, purity 96%) as a TFA salt. MS m/z. M+H" vacuo and the residue was purified by preparative HPLC to calc'd for CHCIFNOS, 494.09. found 494.4. yield the title compound (22 mg) as a TFA salt. MS m/z. M+H" calc'd for C.H.ClFNOS, 466.06. found 466.0. 35 Example 3A Example 2B (R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- 40 N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy N'-(2-hydroxythiazole-5-carbonyl)hydrazino-2- drazino-2-hydroxypropionic Acid hydroxypropionic Acid Ethyl Ester

45 O

O -N O NH2 N-NH2 1No N1 50 HO -- OH O -- 55 Br C

F OH O -, 60 -e-

HO 65 O US 8,871,792 B2 79 80 -continued Example 3B O O ONN (R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- -N N N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy N-N 5 drazino-2-hydroxypropionic Acid Isobutyl Ester HO F

10

Br O (1)

15 HO (R)-3-N-(4-Bromobenzyl)hydrazino-2-hydroxypropi NNNH, 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-fluorophe nyl)isoxazole-5-carboxylic acid (417 mg, 2.0 mmol) were O added. The resulting mixture was stirred at room temperature for 15 minutes. The solvent was removed under pressure and F the crude residue was purified (reverse phase chromatogra 25 phy) to yield Compound 1 (5695 mg).

HO OH 1. 30

(1) +

35 C

40

45 HO

OH

3-(2-fluorophenyl)isoxazole-5-carboxylic acid (15.4 mg. 74 umol) and HATU (33.9 mg,89 umol) were stirred in DMA 55 (0.5 mL, 5 mmol) for 10 minutes. (R)-3-N-(5'-Chloro-2'- Compound 1 (700 mg, 1 mmol) and 5-chloro-2-fluorophe fluorobiphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic nylboronic acid (273 mg, 1.6 mmol) were combined with acid ethyl ester (27.3 mg, 74 umol) and DIPEA (38.9 uL., 223 KCO (541 mg, 3.9 mmol), EtOH (4.6 mL, 78.3 mmol), umol) were added, and the resulting mixture was stirred at 60 room temperature for 1 hour. The mixture was then concen toluene (13.9 mL, 130 mmol), and water (1.2 mL, 65.2 trated under reduced pressure, and the residue was dissolved mmol). Pd(PPh) (151 mg, 130 umol) was then added under EtOAc (20 mL) and washed with water (2x2 mL). The nitrogen, and the mixture was stirred at 90° C. for 3 hours. The organic layer was dried over MgSO4, filtered, and concen mixture was filtered and evaporated and purified by prepara trated. The product was then mixed with isobutyl alcohol (0.5 65 mL, 5 mmol) and 4.0 M HCl in 1,4-dioxane (93 uL., 372 tive HPLC to yield the title compound (40 mg). MS m/z. mmol), and stirred at room temperature overnight. The mix M+H" calc'd for CHCIFNO. 464.09. found 464.0. ture was concentrated under reduced pressure, and the residue US 8,871,792 B2 81 82 was dissolved in 50% AcOH-water (1.5 ml), filtered, and mL). The combined organic layers were dried over anhydrous purified by reverse phase preparative HPLC to yield the title NaSO and concentrated in vacuo. The residue was purified compound (1.7 mg, purity 100%) as a TFA salt. MS m/z. by column chromatography (hexanes/EtOAc=1:1) to yield M+H calcd for CHCIFNOs 584.17. found 584.4. the title compound as a white solid (58 mg). LC-MS: 640.2 M+H". H-NMR: (CDC1) 2.07 (s.3H), 3.43 (br, 2H), 4.4- Example 3C 4.2 (m, 2H), 4.3 (br. 1H), 4.94-4.86 (m, 2H), 7.24-6.85 (m, 5H), 7.97-7.27 (m, 8H). (R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy Example 3D drazino-2-hydroxypropionic Acid 5-Methyl-2-oxo 10 1,3-dioxol-4-ylmethyl Ester (R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy drazino-2-hydroxypropionic acid 2,2,3,3,3-pen 15 tafluoropropyl Ester

HO N-NH2

25 O -- HO

30 OH

35

F OH 40 F F F F F F F O 45 / F F O O 7 N HO N-N O H 50

55

EDCI (92 mg, 480 umol) and HOBT (65 mg, 480 umol) were added to a solution of (R)-3-N-(5'-chloro-2'-fluorobi 60 phenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid A mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl 5-methyl-2-oxo-1.3dioxol-4-ylmethyl ester (108 mg, 240 methyl)-N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy umol) and 3-(2-fluorophenyl)isoxazole-5-carboxylic acid drazino-2-hydroxypropionic acid (30.0 mg, 57 umol), EDC (50 mg, 240 umol) in DMF (20 mL). DIPEA (62 mg, 480 HCl (65.4 mg., 341 umol), and HOBt hydrate (52.2 mg, 341 umol) was added and the mixture was stirred for 5 hours at 65 umol) in DCM (0.5 mL, 8 mmol) was stirred at room tem room temperature. The mixture was washed with saturated perature for 10 minutes. 2.2.3,3,3-Pentafluoro-1-propanol aqueous NaCl (2x30 mL) and extracted with EtOAc (2x50 (45.3 uL, 455umol) was added and the resulting mixture was US 8,871,792 B2 83 84 stirred at room temperature for 1 hour then concentrated. The solved in AcOH (2 mL), filtered, and purified by reverse phase residue was dissolved in AcOH (2 mL), filtered, and purified prep HPLC. The desired fractions were frozen and lyo by reverse phase prep HPLC to yield the title compound (4.8 philized to yield the title compound (8.5 mg, purity 98.5%) as mg, purity 90.2%) as a TFA salt. MS m/z. M+H" calc’d for 5 a TFA salt. MS m/z. M+H" calc'd for CHCIFNO, CHCIF.N.O.s, 660.11. found 660.3. 600.13. found 600.1. Example 3E Example 3F 10 (R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- (R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-3-(2-fluorophenyl)-isoxazole-5-carbonylhy N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy drazino-2-hydroxypropionic Acid Acetoxymethyl drazino-2-hydroxypropionic Acid 2-methoxyethyl Ester 15 Ester

F F

25 O Y.M O Y.M O O O O NH -- NH -- HO N1 HO N1 30 OH OH

C

35 F

40

45

HO

50

Ethane, 1-bromo-2-methoxy (5.7 mL, 0.1 mmol) was 55 C added to a mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl 4-ylmethyl)-N'-[3-(2-fluorophenyl)isoxazole-5-carbonyl hydrazino-2-hydroxypropionic acid (20.0 mg. 38 umol) and Bromomethyl acetate (11.9 mL, 121 umol) was added to a EtN (13.2 uL, 0.1 mmol) in acetone (1.0 mL, 14 mmol), and mixture of (R)-3-(N-(5'-chloro-2'-fluorobiphenyl-4-ylm 60 resulting mixture was stirred at 50° C. overnight. The mixture ethyl)-N'-[3-(2-fluorophenyl)isoxazole-5-carbonylhy was concentrated, and the residue was dissolved in AcOH drazino-2-hydroxypropionic acid (40.0 mg, 76 umol) and (1.5 mL), filtered, and purified by reverse phase preparative HPLC to yield the title compound (10.7 mg, purity 95.7%) as EtN (21.1 mL, 152 umol) in acetone (1.0 mL, 14 mmol). The 65 resulting mixture was stirred at room temperature for 1 hour. a TFA salt. MS m/z. M+H calc’d for CHCIF.N.O. The mixture was then concentrated and the residue was dis 586.15. found 585.8. US 8,871,792 B2 85 86 Example 3G Example3H

Butyric acid (R)-3-N-(5'-chloro-2'-fluorobiphenyl (R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- 4-ylmethyl)-N'-[3-(2-fluorophenyl)isoxazole-5-car N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy bonylhydrazino-2-hydroxypropionyloxymethyl 5 drazino-2-hydroxypropionic Acid Isopropoxycarbo Ester nyloxymethyl Ester

10

15

O -- HO

OH

C 25

F O

1. O ls 1N -( O o- F o- O O 7 N HO N-N O 40 H

45

Chloromethylbutyrate (11.4 uL, 0.1 mmol) and NaI (13.6 mg, 0.1 mmol) were combined in acetone (0.7 mL, 10 mmol) Chloromethyl isopropyl carbonate (17.3 mg, 114 umol) and heated at 65° C. for 1 hour. The mixture was then cooled and NaI 17.0 mg, 114 umol) were combined in acetone (1.0 to room temperature. A mixture of (R)-3-N-(5'-chloro-2'- mL, 14 mmol) and heated at 60° C. for 1 hour. The mixture fluorobiphenyl-4-ylmethyl)-N'-3-(2-fluorophenyl)isox was then cooled to room temperature. A mixture of (R)-3-(N- azole-5-carbonylhydrazino-2-hydroxypropionic acid (16.0 55 mg, 30 umol) dissolved in acetone (0.2 mL) and treated with (5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-3-(2-fluo EtN (8.5 ul, 61 umol) was added and the resulting mixture rophenyl)isoxazole-5-carbonylhydrazino-2-hydroxypro was stirred at room temperature for 25 minutes. The mixture pionic acid (20.0 mg. 38 umol) dissolved in acetone (1.0 mL) was concentrated and the residue was dissolved in AcOH (2.0 and treated with Et N (10.6 uL, 76 umol) was added and the mL), filtered and purified by reverse phase preparative HPLC. 60 resulting mixture was stirred at room temperature for 5 hours. The desired fractions were combined and freeze dried to yield The mixture was concentrated and the residue was dissolved a yellowish solid. This solid was dissolved in AcOH (1.5 mL), filtered, and purified by reverse phase preparative HPLC. The in AcOH (2.0 mL), filtered and purified by reverse phase desired fractions were combined and freeze dried to yield the preparative HPLC. The product was freeze dried and purified title compound (5.7 mg, purity 100%) as a TFA salt white 65 by reverse phase preparative HPLC to yield the title com solid. MS m/z. M+H" calc’d for CHCIF.N.O., 628.16. pound (1.8 mg, purity 100%). MS m/z. M+H" calc’d for found 628. CHCIFN-Os, 644.15. found 644.1. US 8,871,792 B2 87 88 Example 3I Example 3J

(R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- (S)-2-Methoxycarbonylamino-3-methylbutyric acid N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy 5 (R)-3-(N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- drazino)-2-phosphonooxypropionic Acid N'-3-(2-fluorophenyl) is oxazole-5-carbonylhy drazino-2-hydroxypropionyloxymethyl Ester

O O HO F N1 NH OH -- OH O C

F O o, 1. o1 NC --

N O X ( F S O- O V O O / N

45 (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- droxy-propionic acid (12.0 mg, 22.7 umol) in EtOH (80 uL. ylmethyl)-N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy 1.4 mmol) was combined with a solution of 4.0 M HCl in 50 drazino-2-hydroxypropionic acid (200 mg. 380 umol) in 1,4-dioxane (227 uL. 909 mmol), and the resulting mixture DMF (10 mL) was added 2.6-lutidine (407 mg, 3.8 mmol), was stirred at room temperature for 1 hour. The solvent was (S)-2-methoxycarbonylamino-3-methylbutyric acid chlo removed in vacuo and the residue was dissolved in pyridine romethyl ester (170 mg, 760 umol) and NaI (114 mg, 760 55 mmol). The resulting mixture was stirred overnight at room (20 uL. 250 mmol). The resulting solution was added to a temperature. The Solution was washed with Saturated aque solution of phosphoryl chloride (19 ul, 0.2 mmol) in acetone ous NaCl (2x20 mL) and extracted with EtOAc (2x30 mL). (67 u , 0.9 mmol) and stirred at room temperature for 10 The combined organic layers were dried over anhydrous minutes. The solvent was removed in vacuo and the residue NaSO and concentrated in vacuo. The residue was purified was dissolved in EtOH (80 uL. 1.4 mmol). A solution of 1.0 M 60 by column chromatography (PE/EA=4/1-1/2) to yield the LiOH in water (1.4 mL, 1.4 mmol) was then added until the title compound as a white solid (90 mg). LC-MS: 714.8 M+H". H-NMR (CD3OD-d): 8 0.91 (d. J=9.6 Hz, 6H), pH reached-12. The mixture was stirred for 1 hour and the 2.05-2.13 (m. 1H), 3.39-3.45 (m, 2H), 3.66 (s.3H), 406-4.08 solvent was removed in vacuo. The residue was purified by 65 (m. 1H), 4.23-4.25 (m, 2H), 4.66-4.48 (m, 1H), 5.79-5.86 (m, preparative HPLC to yield the title compound (5 mg). MS m/z. 2H), 7.15-7.26 (m. 1H), 7.24-7.55 (m, 10H), 7.97-7.95 (m, M+H" calc'd for CHCIFNOP 608.07. found 608.0. 1H). US 8,871,792 B2 89 90 Example 3K Example 3L

(R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- (R)-3-(N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy 5 N'-3-(2-fluorophenyl)isoxazole-5-carbonylhy drazino-2-hydroxypropionic Acid Ethoxycarbony drazino-2-hydroxypropionic Acid 1-Cyclohexy loxymethyl Ester loxycarbonyloxyethyl Ester

10

15 HO N-NH --

O

HO

OH --

C 25

F O ---> Hs 30 - O o-( F O- O 35 O O / HO o-N

40

45

2.6-Lutidine (407 mg, 3.8 mmol) was added to a solution of 50 (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-3- (1) (2-fluorophenyl)isoxazole-5-carbonylhydrazino-2-hy droxypropionic acid (200 mg,380 mmol), carbonic acid chlo To a solution of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- ylmethyl)hydrazino-2-hydroxypropionic acid ethyl ester romethyl ester ethyl ester (105 mg, 760 umol) and NaI (114 55 mg, 760 umol) in DMF (10 mL). The mixture was stirred (1.5 g., 3.7 mmol), EDC (928 mg, 4.8 mmol), HOBt (653 mg, overnight at room temperature. Water (30 mL) was added and 4.8 mmol) and 3-(2-fluorophenyl)isoxazole-5-carboxylic the mixture was extracted with EtOAc (3x40 mL). The com acid (848 mg, 4.1 mmol) in DCM (20 mL) was added DIPEA bined organic layers were dried over anhydrous NaSO and (1.9 mL, 11.2 mmol) under nitrogen. The resulting mixture 60 was stirred at room temperature overnight, then concentrated concentrated in vacuo. The residue was purified by prepara to dryness. The residue was dissolved in EtOAc (20 mL), tive HPLC (MeCN HO (0.1% TFA); Gradient 60-70) to washed with 0.5N aqueous HCl (10 mL), saturated aqueous yield the title compound as a white solid (56 mg). LC-MS: NaHCO (10 mL) and saturated aqueous NaCl (10 mL), dried 630.1 M+H". H-NMR: (CDOD-d 400 MHz) & 1.24 (t, over anhydrous Na2SO4, filtered, and concentrated in vacuo. J=5.9 Hz, 3H), 3.30-3.33 (m, 2H), 4.17-4.32 (m, 4H), 4.45 (t, 65 The residue was purified by column chromatography (PE: J=4.2 Hz, 1H), 5.78 (br 2H), 7.20-7.28 (m. 1H), 7.57-7.29 EtOAc, 10:1-3:1) to yield Compound 1 as a solid (1.4 g). (m. 10H), 7.92-795 (m. 1H). LC-MS. 556 M+H". US 8,871,792 B2 91 92 -continued O O \ -e- O W -N O (1) + o-( He OH o-( O C 10 O Y O O

NH N HO N1 o, M O 15 OH ls 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'-fluoro To a solution of Compound 1 (1.4g, 2.5 mmol) in MeOH 25 biphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid (15 mL) was added a solution of LiOH.H2O (317 mg, 7.6 ethyl ester (300.0 mg, 1.0 mmol) and DIPEA (0.3 mL, 1.6 mmol) in water (3 mL). The mixture was stirred at room mmol). The resulting mixture was stirred at room temperature temperature for 1 hour, and the insoluble solid was filtered off overnight until the reaction was complete. The mixture was and the filtrate was concentrated in vacuo to yield a yellow partitioned between EtOAc (10.0 mL) and water (3.0 mL). solid (1.2g). LC-MS:528 M+H". The yellow solid (400 mg, 30 The organic layer was washed with water (2x30 mL), satu 760 umol) was dissolved in 2.6-lutidine (814 mg, 7.6 mmol) rated aqueous NaCl (3.0 mL), dried over NaSO filtered and and carbonic acid 1-chloro-ethyl ester cyclohexyl ester (1.6 g. concentrated to yield a yellowish oil. The oil was purified by flash chromatography (2x4 g stacker column, 0-100% 7.6 mmol) was added. The vial was sealed and the resulting EtOAc/hexanes). The desired fractions were combined and mixture was then irradiated for 30 minutes at 90° C. under concentrated to yield a light yellowish oil. The oily residue microwave irradiation. Water (10 mL) was added, and the 35 was then treated with a mixture of MeCH (5.0 mL, 120 mmol) mixture was extracted with EtOAc (3x10 mL). The combined and water (1.0 mL, 56 mmol). LiOH monohydrate (68.6 mg, organic layers were washed with 0.5Naqueous HCl (5x5 mL) 1.6 mmol) was added. After stirring at room temperature for and saturated aqueous NaCl (10 mL), dried over anhydrous 30 minutes, the mixture was concentrated. The residue was NaSO, filtered and concentrated in vacuo. The crude prod treated with EtOAc (10.0 mL) and acidified with 1N HCl until uct was purified by column chromatography (PE:EtOAc, pH-3. The organic layer was washed with Saturated aqueous 40 NaCl (2x3.0 mL), dried over NaSO filtered and concen 10:1-2: 1) to yield the title compound as a white solid (60 mg). trated to yield the title compound as a white foam (289.7 mg). LC-MS: 698 M+H". "H NMR: (CDC1,400 MHz) & 1.28 MS m/z. M+H" calc’d for CHCIFNO. 464.09. found 1.37 (m, 6H), 1.54 (d. 3H), 1.72-1.75 (m, 2H), 1.90-1.95 (m, 464.0. 2H), 3.33-3.38 (m, 2H), 4.24-4.30 (m, 2H), 4.38-440 (m, Example 4B 1H), 4.61-4.66 (m. 1H), 6.60 (t, 0.5H), 6.80 (t, 0.5H), 7.00 45 (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- 7.10 (m, 1H), 7.20-7.26 (m,3H) 7.36-7.41 (m, 2H), 7.48-7.52 N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- (m, 5H), 7.85 (s, 0.5H), 8.00-8.04 (m, 1H), 8.15 (s, 0.5H). hydroxypropionic Acid 5-Methyl-2-oxo-1,3-di Example 4A oxol-4-ylmethyl Ester O (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- 50 N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- Y- O hydroxypropionic Acid O 2 O

55

HO

OH 60

65 US 8,871,792 B2 93 94 -continued -continued F

/ F OH --> W F F O -- F

O o O OH 10 N N o, M t O F O O NH , ) or 15 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 mmol), were added to a solution of (R)-3-N-(5'-chloro-2'-fluorobi and HOBt hydrate (39.62 mg, 0.2587 mmol) were combined phenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid in DCM (0.5 mL, 8 mmol) and stirred at room temperature. 5-methyl-2-oxo-1.3dioxol-4-ylmethyl ester (200 mg. 440 30 After 10 minutes, 2.2.3,3,3-pentafluoro-1-propanol (51.8 mg, umol) and 3-methoxyisoxazole-5-carboxylic acid (63 mg. 345 umol) was added. The resulting mixture was stirred at 440 umol) in DMF (10 mL). DIPEA (114 mg. 880 umol) was room temperature overnight to yield the title compound (5.9 added and the mixture was stirred for 5 hours at room tem mg, purity 100%) as a TFA salt. MS m/z. M+H" calc’d for perature. The mixture was washed with Saturated aqueous CHCIFNO. 596.09. found 596. NaCl (2x30 mL) and extracted with EtOAc (2x50 mL). The 35 combined organic layers were dried over anhydrous Na2SO and concentrated in vacuo. The residue was purified by col Example 4D umn chromatography (hexanes/EtOAc=1:1) to yield the title compound as a white solid (60 mg). LC-MS: 576.1 M+H". 40 (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- H-NMR: (DMSO-d) 2.14 (s.3H), 3.21-3.19 (m, 2H), 3.91 N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- (s, 3H), 4.17-4.11 (m, 2H), 4.31 (br. 1H), 4.98 (s. 2H), 5.57 hydroxypropionic Acid Acetoxymethyl Ester (br. 1H), 6.73 (s, 1H), 7.57-7.34 (m, 7H), 10.07 (s, 1H).

Example 4C 45 O (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- Y. hydroxypropionic acid 2.2.3,3,3-pentafluoropropyl 50 O M Ester O O

O o NH HO N1 -- OH 55 O Y.M O O

NH HO N1 O C C 60 OH F

C

65 US 8,871,792 B2 95 96 -continued -continued O - O -N N 1. O O Y. O O O O NH 1. N-1No N 1. NH ---> O N1 OH OH 10

15 1-Bromo-2-methoxyethane (12.2 uL. 129 umol) was To a solution of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- added to a mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hydrazino 4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hy 2-hydroxypropionic acid (40.0 mg., 0.1 mmol) in acetone (1.0 drazino-2-hydroxypropionic acid (20.0 mg. 43 Lmol) and mL, 14 mmol) was added bromomethyl acetate (16.9L. 172 DIPEA (45.1 uI, 259 umol) in acetone (1.0 mL, 14 mmol). umol) followed by EtN (24.0 uL. 172 umol), and resulting The resulting mixture was heated at 60° C. for 4 hours. NaI mixture was stirred for 90 minutes. The reaction was 25 (19.4 mg, 129 umol) was added and the reaction was moni quenched with AcOH (19.6 uL. 345 umol) and the mixture tored for 2 hours. Additional 1-bromo-2-methoxyethane (3 was concentrated. The residue was dissolved in AcOH (3 eq.), DIEA (4 eq.), and NaI(3 eq.) were added and the heating continued overnight. Additional 1-bromo-2-methoxyethane mL), filtered, and purified by reverse phase preparative 30 HPLC. The desired fractions were combined and lyophilized. (3 eq.), NaI(3 eq.), and DIEA (3 eq.) were added and heating The solid was dissolved in AcOH (1.5 mL), filtered, and continued overnight. The mixture was then concentrated, and purified by reverse phase preparative HPLC to yield the title the residue 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 salt. MS m/z 35 M+H" calc'd for CHCIFN.O., 536.12. found 536.2. compound (2.6 mg, purity 99%). MS m/z. M+H" calc'd for CHCIFNO, 522.14. found 522.4. Example 4E Example 4F 40 (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- Butyric acid (R)-3-N-(5'-chloro-2'-fluorobiphenyl N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- 4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl) hydrazino-2-hydroxypropionyloxymethyl Ester hydroxypropionic Acid 2-Methoxy-Ethyl Ester 45

50

HO N 55 HO N OH OH

60

65 US 8,871,792 B2 97 98 -continued -continued

5

HO HO 10

15

A mixture of chloromethyl ethyl carbonate (17.9 mg, 129 A mixture of chloromethylbutyrate (16.2 LL, 129 umol) and NaI (19.4 mg, 129 umol) in acetone (0.7 mL, 10 mmol) umol) and NaI (19.4 mg, 129 umol) in acetone (0.7 mL, 10 was heated at 65° C. for 1 hour. The mixture was then cooled mmol) was heated at 65° C. for 1 hour. The mixture was then to room temperature and a mixture of (R)-3-N-(5'-Chloro 25 cooled to room temperature and a mixture of (R)-3-N-(5'- 2'-fluorobiphenyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5- chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3-methoxyisox carbonyl)hydrazino-2-hydroxypropionic acid (20.0 mg) and azole-5-carbonyl)hydrazino-2-hydroxypropionic acid (20.0 DIPEA (15.0 mL, 0.1 mmol) in acetone (0.3 mL) was added. The resulting mixture was stirred at room temperature for 1 mg) and DIPEA (15.0LL, 0.1 mmol) in acetone (0.3 mL) was hour then concentrated. The residue was dissolved in AcOH 30 added. The resulting mixture was stirred at room temperature (2.0 mL), filtered, and purified by reverse phase preparative for 1 hour then concentrated. The residue was dissolved in HPLC. The desired fractions were combined and freeze dried to yield a white solid. The solid was dissolved in acetic acid AcOH (2.0 mL), filtered, and purified by reverse phase pre (1.5 mL), filtered and purified by reverse phase preparative parative HPLC. The desired fractions were combined and HPLC to yield the title compound (6.5 mg, purity 100%). MS 35 freeze dried to yield a white solid. The solid was dissolved in m/z. M+H calcd for CH,ClFNOs 564.15. found. 564. acetic acid (1.5 mL), filtered and purified by reverse phase preparative HPLC to yield the title compound (5.8 mg, purity Example 4G 94%). MS m/z. M+H" calc'd for CHCIFNO. 566.13. 40 found 566. (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- Example 4H hydroxypropionic Acid Ethoxycarbonyloxymethyl Ester 45 (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- hydroxypropionic Acid 2-Morpholin-4-ylethyl Ester O 50

O Y. O O

NH HO N1 55 -- OH

HO N O C 60 OH

F C O 1No ls o1 No 65 US 8,871,792 B2 99 100 -continued -continued O OH o, 1. o1N -e- O 1NE

OO 10 OO ON NN-\ O O W \, O O 15 N -NH HO

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 mmol), (S)-2-methoxy carbonylamino-3-methylbutyric acid chloromethyl ester 25 (193 mg, 860 mmol), NaI (129 mg, 860 umol) and pyridine (136 mg, 1.7 mmol) in DMF (5.0 mL) was stirred at 25° C. A mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl overnight. The mixture was then poured into water (30 mL) methyl)-N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- and extracted with EtOAc (3x20 mL). The combined organic hydroxypropionic acid (20.0 mg. 43 mmol), EDC (45.8 LL. 30 layers were washed with saturated aqueous NaCl (30 mL), 259 umol) and HOBt hydrate (39.6 mg, 259 umol) in DCM dried over anhydrous Na2SO4, and concentrated in vacuo. (0.5 mL, 8 mmol) was stirred at room temperature for 10 The residue was purified by column chromatography (PE: minutes. 4-Morpholineethanol (41.8 LL 345 umol) was EtOAc=1:1) to yield the title compound as a colorless liquid added, and the resulting mixture was stirred at room tempera (7 mg). LC-MS: 651.1 M+H". "H NMR (CDOD, 400 ture overnight. The mixture was concentrated and the residue 35 MHz): 80.95-0.97 (m, 6H), 2.06-2.14 (m. 1H), 3.34-3.39 (m, was dissolved in AcOH (1.5 mL), filtered, and purified by 2H), 3.69 (s.3H), 3.98 (s.3H), 4.08-4.11 (m. 1H), 4.22-4.24 reverse phase preparative HPLC to yield the title compound (m. 2H), 4.38-440 (m, 1H), 5.78-5.91 (m, 2H), 6.54 (s, 1H), (14.1 mg) as a TFA salt. MS m/z. M+H" calc’d for 7.18-7.22 (m. 1H), 7.34-7.36 (m, 1H), 7.46-7.55 (m, 5H). CHCIFNO,577.18. found 577. 40 Example 4J Example 4I (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- (S)-2-Methoxycarbonylamino-3-methylbutyric Acid hydroxypropionic Acid Isopropoxycarbonyloxym (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- 45 ethyl Ester N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- hydroxypropionyloxymethyl Ester

50

O

O Y. 55 O O HO N OH NH HO N1 C OH 60

65 US 8,871,792 B2 101 102 -continued To a mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- - O OS ylmethyl)hydrazino-2-hydroxypropionic acid ethyl ester - O O W \ (300.0 mg, 818 mmol) and 3-methoxy-isoxazole-5-carboxy lic acid (140.4 mg., 981 umol) in DMF (4.0 mL, 52 mmol) at O -\, - room temperature was added HATU (373.2 mg, 981 umol) N-N HO and DIPEA (427 uL., 2.4 mmol). The resulting mixture was stirred at room temperature for 1 hour. The mixture was 10 partitioned between EtOAc (10.0 mL) and water (2.0 mL). The organic layer was washed with water (2x2.0 mL), dried over NaSO, filtered, and concentrated to yield a yellowish oil. The oil was purified by flash chromatography (2x4 g 15 stacker column, 0-100% EtOAc/hexanes). The desired frac tions were combined and concentrated to yield a colorless oil. A mixture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl methyl)-N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- The oil (28.8 mg, 58.5umol) was combined with DIPEA hydroxypropionic acid (200 mg. 430 mmol), chloromethyl (30.6 uL, 176 umol) and added to a mixture of N-O-(t-butoxy isopropyl carbonate (132 mg, 860 mmol), NaI (129 mg, 860 carbonyl)glycine (12.3 mg, 70.2 mol) and HATU (26.7 mg, umol) and pyridine (136 mg, 1.7 mmol) in DMF (5.0 mL) was 70.2 umol) in DMF (0.5 mL, 6 mmol) at room temperature. stirred at 25°C. overnight. The mixture was then poured into After stirred at room temperature overnight, the mixture was water (30 mL) and extracted with EtOAc (3x20 mL). The partitioned between EtOAc (10.0 mL) and water (2.0 mL). combined organic layers were washed with Saturated aqueous NaCl (30 mL), dried over anhydrous NaSO, and concen 25 The organic layer was dried over NaSO filtered and con trated in vacuo. The residue was purified by column chroma centrated to give a light yellowish oil. The oily residue was tography (PE:EtOAc=1:1) to yield the title compound as a then dissolved in DCM (0.2 mL) and treated with 4.0 M HCl colorless liquid (10mg). LC-MS. 580M+H". "HNMR (400 in 1,4-dioxane (0.2 mL, 0.8 mmol) at room temperature for 30 MHz, CDOD): & 1.28 (d. J=6 Hz, 6H), 3.37-3.39 (m. 2H), 3.98 (s.3H), 4.22-4.24 (m, 2H), 4.38-440 (m, 1H), 5.78-5.91 30 minutes. The mixture was concentrated, and the resulting (m. 2H), 6.54 (s, 1H), 7.22-7.18 (t, J=9 HZ, 1H), 7.34-7.36 (m, residue was co-evaporated with EtOAc (3x2.0 mL) to yield a 1H), 7.46-7.55 (m, 5H). white solid. The solid was dissolved in AcOH (1.5 mL), filtered, and purified by reverse phase preparative HPLC. The Example 4K 35 desired fractions were combined and freeze dried to yield the (R)-2-(2-Aminoacetoxy)-3-N-(5'-chloro-2'-fluorobi title compound as a white solid TFA salt (8 mg). MS m/z. phenyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5-car M+H" calc'd for CHCIFNO. 549.15. found 549. bonyl)hydrazinopropionic Acid Ethyl Ester O Example 4L 40 (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- HO N-NH N'-(3-methoxisoazole-5-carbonyl)hydrazino-2-pro ON pionyloxypropionic Acid O. M. 45 O Y OH -e- O O

Boc1 Nulls OH O e O Y. O O N N 55 NH HO N1 -- O O OH sN H 1n --> N1 O 60

O y O C F Co.O HN O 65 F Suls.C US 8,871,792 B2 103 104 -continued -continued O

O Y./ O

NH HO Or O 10

C

15 F

Propanoyl chloride (7.3 uL. 84 umol) was added to a mix ture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- A mixture of NaI (19.4 mg, 129 umol) and (S)-2-t-butoxy N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2-hydrox carbonylamino-3-methyl-butyric acid chloromethyl ester ypropionic acid (17.0 mg. 36.7 mol) and DIPEA (12.8 LL. (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 73 umol) in DCM (0.5 mL, 8 mmol). The mixture was stirred temperature and treated with a solution of (R)-3-N-(5'- at room temperature for 30 minutes, then concentrated. The 25 chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3-methoxyisox residue was dissolved in AcOH (1.5 mL), filtered, and puri azole-5-carbonyl)hydrazino-2-hydroxypropionic acid (20.0 mg, 43.1 umol) and DIPEA (15.0 uL. 86.2 umol) in acetone fied by reverse phase preparative HPLC. The desired frac (0.3 mL, 4 mmol). The mixture was stirred at room tempera tions were combined and freeze dried to yield the title com ture for 2 hours, then concentrated. The residue was parti pound as a white solid (1.2 mg). MS m/z. M+H" calc’d for 30 tioned between EtOAc (5.0 mL) and water (2.0 mL). the organic layer was washed with water (2.0 mL), Saturated CHCIFNO. 520.12. found 520.1. aqueous NaCl (2.0 mL), dried over NaSO, filtered, and concentrated to yield a colorless oil. The oil was further dried Example 4M in vacuo for 30 minutes and then stored in the freezer over 35 night. The oil was then treated with a 1:1 mixture of DCM/ TFA (0.3 mL) at room temperature for 30 minutes, and con (S)-2-Amino-3-methylbutyric Acid (R)-3-N-(5'- centrated. The residue was dissolved in AcOH (1.5 mL), chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3-meth filtered, and purified by reverse phase preparative HPLC. The desired fractions were combined and freeze dried to yield the oxyisoxazole-5-carbonyl)-hydrazino-2-hydroxypro 40 title compound as a white solid TFA salt (7.3 mg). MS m/z. pionyloxymethyl Ester M+H" calc'd for CHCIFNO. 593.17. found 593.1. Example 4N 45 (S)-2-Methoxycarbonylamino-3-methylbutyric Acid (R)-1-Carboxy-2-N-(5'-chloro-2'-fluorobiphenyl-4- ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hy drazinoethyl Ester 50

O HO N --

OH 55 O Y.M O O

NH HO N1 -- 60 OH

C

65 US 8,871,792 B2 105 106 -continued -continued O N-1 O HO : ls He 1sH o1 O Y.M O O O O NH HO N Y 10 O Y./ O O '8 C NH O N1 HN '', O O 15 O O F

C (S)-2-t-Butoxycarbonylaminopropionic acid (12.2 mg, NH O 64.7 umol) and HATU (24.6 mg, 64.7 umol) were stirred in DMA (1.0 mL, 11 mmol) for 10 minutes. (R)-3-N-(5'- es F Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3-methoxyisox azole-5-carbonyl)hydrazino-2-hydroxypropionic acid (20.0 (S)-2-Methoxycarbonylamino-3-methylbutyric acid (5.7 25 mg, 43.1 umol) and DIPEA (22.5 L, 129 umol) were added, mg, 32.3 Lumol) and HATU (12.3 mg, 32.3 Lumol) were stirred and the resulting mixture was stirred at room temperature in DMA (1.0 mL, 11 mmol) for 15 minutes. (R)-3-N-(5'- overnight then concentrated. 4.0 M HCl in 1,4-dioxane (0.2 Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3-methoxyisox mL, 0.8 mmol) was added and the resulting mixture was azole-5-carbonyl)hydrazino-2-hydroxypropionic acid (10.0 30 allowed to stand for 1 hour, then concentrated under reduced mg, 21.6 umol) and DIPEA (11.3 uL. 64.7 umol) were added, pressure. The residue was dissolved in AcOH (1.5 mL), fil and the resulting mixture was stirred at room temperature overnight. The mixture was concentrated, and the residue was tered, and purified by reverse phase preparative HPLC to dissolved in AcOH (1.5 mL), filtered, and purification by yield the title compound as a TFA salt (3.9 mg). MS m/z. reverse phase preparative HPLC to yield the title compound M+H" calc'd for CHCIFNO, 535.13. found 535.4. (2.7 mg). MS m/z. M+H" calc’d for CHCIFNO. 35 621.17. found 621.3. Example 4P Example 40 40 (S)-2-Amino-3-methylbutyric Acid (R)-1-Carboxy (S)-2-Aminopropionic Acid (R)-1-Carboxy-2-N-(5'- 2-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3- chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(3-meth methoxyisoxazole-5-carbonyl)hydrazinoethyl Ester oxyisoxazole-5-carbonyl)hydrazinoethyl Ester 45

O

50 O Y. O O

NH s1 HO N -- O N -- 55 OH OH O C 60 F O

1 N -e- BOC OH 65 US 8,871,792 B2 107 108 -continued -continued O O

O Y.M O / O O

NH 1. NH C HO N

--> N Y O HN 8.y O O C HN Co.F C F

N-(t-Butoxycarbonyl)-L-valine (14.0 mg, 64.7 umol) and N-O-(t-Butoxycarbonyl)glycine (11.3 mg, 64.7 umol) and HATU (24.6 mg, 64.7 umol) were stirred in DMA (1.0 mL, 11 HATU (24.6 mg, 64.7 umol) were stirred in DMA (1.0 mL, 11 mmol) for 10 minutes. (R)-3-N-(5'-Chloro-2'-fluorobiphe mmol) for 10 minutes. (R)-3-N-(5'-Chloro-2'-fluorobiphe nyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hy nyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5-carbonyl)hy drazino-2-hydroxypropionic acid (20.0 mg, 43.1 umol) and drazino-2-hydroxypropionic acid (20.0 mg, 43.1 umol) and DIPEA (22.5 LL, 129 umol) were added, and the resulting DIPEA (22.5 LL, 129 umol) were added, and the resulting 25 mixture was stirred at room temperature overnight then con centrated. 4.0 M HCl in 1,4-dioxane (0.2 mL, 0.8 mmol) was mixture was stirred at room temperature overnight then con added and the resulting mixture was allowed to stand for 1 centrated. 4.0 M HCl in 1,4-dioxane (0.2 mL, 0.8 mmol) was hour, then concentrated under reduced pressure. The residue added and the resulting mixture was allowed to stand for 1 was dissolved in AcOH (1.5 mL), filtered, and purified by hour, then concentrated under reduced pressure. The residue 30 reverse phase preparative HPLC to yield the title compound was dissolved in AcOH (1.5 mL), filtered, and purified by as a TFA salt (5.8 mg). MS m/z. M+H" calc'd for reverse phase preparative HPLC to yield the title compound CHCIFNO. 521.12. found 521.6. as a TFA salt (3.5 mg). MS m/z. M+H" calc'd for Example 4R CHCIFNO. 563.16. found 563.4. 35 (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- Example 4Q N'-(3-methoxyisoxazole-5-carbonyl)hydrazino-2- hydroxypropionic Acid 1-Cyclohexyloxycarbony 40 loxyethyl Ester (R)-2-(2-Aminoacetoxy)-3-N-(5'-chloro-2'-fluorobi phenyl-4-ylmethyl)-N'-(3-methoxyisoxazole-5-car bonyl)hydrazinopropionic Acid 45 VO

O HO N-NH

50

O Y. O O

NH sY HO N -- 55

OH

O C 60

F O

N 65 Y Nulls -- BOC OH OH US 8,871,792 B2 109 110 -continued C. under microwave irradiation. Water (10 mL) was added, O and the mixture was extracted with EtOAc (3x10 mL). The combined organic layers were washed with 0.5N aqueous HCl (4x5 mL) and saturated aqueous NaCl (10 mL), dried O Y. over anhydrous NaSO filtered and concentrated in vacuo. O O The residue was purified by preparative HPLC (Gemini-C18, 150x21.2 mm, 5u, MeCN HO (0.1% TFA); from 43% to 1no N NH 43%) to yield the title compound as a white solid (7 mg). LC-MS: 634 M+H". "H NMR: (CDC1,400 MHz) & 1.28 OH 10 1.37 (m, 6H), 1.54 (d. 3H), 1.70-1.73 (m, 2H), 1.84-1.87 (n, 2H), 3.26-3.29 (m, 2H), 3.38-3.43 (m, 1H), 3.97 (s, 3H), 4.19-4.20 (m, 2H), 4.38-4.50 (m, 1H), 4.50-4.57 (m, 1H), 6.50 (m, 1H), 6.75 (t, 1H), 7.18-7.20 (m, 1H), 7.35-7.37 (m, 1H), 7.48-7.52 (m, 4H). 15 Example 5A (1) (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole To a solution of compound (R)-3-N-(5'-chloro-2'-fluoro 3-carbonyl)hydrazino-2-hydroxypropionic Acid biphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid ethyl ester (1.2g, 3.0 mmol), EDC (742 mg, 3.9 mmol), HOBt (523 mg, 3.9 mmol) and 3-methoxyisoxazole-5-carboxylic acid (468 mg, 3.3 mmol) in DCM (20 mL) was added DIPEA (1.48 mL, 8.9 mmol) under nitrogen. The resulting mixture 25 O was stirred at room temperature overnight, then concentrated to dryness. The residue was dissolved in EtOAc (20 mL), washed with 0.5N aqueous HCl (10 mL), saturated aqueous HO N-NH NaHCO (10 mL) and saturated aqueous NaCl (10 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. 30 The residue was purified by column chromatography (PE: EtOAc, 10:1-3:1) to yield Compound 1 as a solid (970 mg). ( ) -- LC-MS. M+H": 492. 35 F ( ) C

(1) -- 40 i-K -( O )-NV O HN N 45

M o, O OD OH O Ol lsO 1. O NH O O or 50 in OH O S. N O N

O C NH HO N1 55 F OH

To a solution of Compound 1 (970 mg, 2.0 mmol) in MeOH C (15 mL) was added a solution of LiOH.HO (248 mg, 5.9 mmol) in water (3 mL). The mixture was stirred at room 60 temperature for 1 hour, and the insoluble solid was filtered off and the filtrate was concentrated in vacuo to yield a yellow F solid (780 mg). LC-MS: 464 M+H". The yellow solid (200 mg, 430 umol) was dissolved in 2.6-lutidine (460 mg, 4.3 5-Oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole-3-car mmol) and carbonic acid 1-chloro-ethyl ester cyclohexyl 65 boxylic acid (56.4 mg, 275umol) was combined with HCTU ester (888 mg, 4.3 mmol) was added. The vial was sealed and (154 mg. 371 umol) in DMF (852 uL, 11 mmol) and stirred the resulting mixture was then irradiated for 30 minutes at 90° for 15 minutes at room temperature. DIPEA (72 uL., 413 US 8,871,792 B2 111 112 umol) and (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylm drazino-2-hydroxypropionic acid 5-methyl-2-oxo-1.3di ethyl)hydrazino-2-hydroxypropionic acid ethyl ester (50 oxol-4-ylmethyl ester (68 mg, 330 umol) in DMF (10 mL). mg, 0.1 mmol) were added, and the resulting mixture was DIPEA (86 mg. 660 umol) was added, and the resulting stirred overnight at room temperature. EtOH (402 uL, 6.9 mixture was stirred for 5 hours at room temperature. The mmol) and 1 M LiOH in water (1.1 mL, 1.1 mmol) were mixture was then washed with saturated aqueous NaCl (2x30 added and the resulting mixture was stirred at room tempera mL) and extracted with EtOAc (2x50 mL). The combined ture for 1 hour. The mixture was evaporated under reduced organic layers were dried over anhydrous NaSO and con pressure and the residue was purified by preparative HPLC to centrated in vacuo. The residue was purified by column chro yield the title compound (39.8 mg, purity 100%). MS m/z. matography (PE:EtOAc=1:1) to yield the title compound as a 10 white solid (67 mg). LC-MS. 638.2 M+H". H-NMR (400 M+H" calc'd for CHCIFNOs 526.12. found 526.0. MHz, DMSO-d): 8 2.13 (s, 3H), 3.31-3.16 (m, 2H), 4.18 Example 5B 4.21 (q, 2H), 4.35 (br. 1H), 4.98-5.01 (m, 2H), 5.54 (br. 1H), 7.26-7.90 (m, 12H), 9.98 (s, 1H), 12.74 (s, 1H). (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole 15 Example 5C 3-carbonyl)hydrazino-2-hydroxypropionic Acid (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- 5-methyl-2-oxo-1,3-dioxol-4-ylmethyl Ester N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole 3-carbonyl)-hydrazino-2-hydroxypropionic Acid Ethyl Ester

O O - O 25 O

HO N-NH HO N-NH -- 30 ( ) --

35

O 40 N \ HN N N1 N YO es 45 \ N I H O O O OH O OH

50 - O O O NN N HO N-N N HO N- N) {N O 55

60 F ( ) C 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 65 1.2.4 triazole-3-carboxylic acid (150 mg, 330 umol) and 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 US 8,871,792 B2 113 114 (244 mg, 589 umol) in DMF (1.0 mL, 13 mmol) and stirred 5-Oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole-3-car for 10 minutes. (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl boxylic acid (89.5 mg, 436 umol) was combined with HCTU methyl)hydrazino-2-hydroxypropionic acid ethyl ester (80.0 (244 mg, 589 umol) in DMF (1.0 mL, 13 mmol) and stirred mg, 0.2 mmol) and DIPEA (0.1 mL, 0.7 mmol) were added, for 10 minutes. (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-yl and the resulting mixture was stirred overnight. The mixture methyl)hydrazino-2-hydroxypropionic acid ethyl ester (80.0 was diluted with EtOAc (10.0 mL) and washed with water mg, 0.2 mmol) and DIPEA (0.1 mL, 0.7 mmol) were added, (3.0 mL), saturated aqueous NaHCO, (2x30 mL), and satu and the resulting mixture was stirred overnight. The mixture rated aqueous NaCl (3.0 mL), then dried over NaSO fil was diluted with EtOAc (10.0 mL) and washed with water tered and concentrated to give a yellowish solid. A portion (20 (3.0 mL), saturated aqueous NaHCO (2x30 mL), and satu mg) of the solid was dissolved in AcOH (1.5 mL), filtered and 10 rated aqueous NaCl (3.0 mL), then dried over NaSO fil purified by reverse phase preparative HPLC to yield the title tered and concentrated to give a yellowish solid. A portion (20 compound (1.6 mg, purity 100%). MS m/z. M+H" calc’d for mg) of the solid was treated with isobutyl alcohol (170LL, 1.8 CHCIFNO. 554.15. found 554.4. mmol) and 4.0 M HCl in 1,4-dioxane (36.0LL, 144 umol) at room temperature overnight. The mixture was concentrated, Example 5D 15 and the residue was dissolved in AcOH (1.5 mL), filtered, and purified by reverse phase preparative HPLC to yield the title compound (1.6 mg, purity 100%). MS m/z. M+H" calc’d for (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole CHCIFNO. 582. 18. found 582.4. 3-carbonyl)hydrazino-2-hydroxypropionic Acid Example 5E Isobutyl Ester (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-hydroxypropionic Acid -v O 25 2.2.3,3,3-Pentafluoropropyl Ester O

HO N-NH2 30 O ( ) -- HO Oy C 35 HO N-N N O

--

40

HO N O -- F sy- D 45 \ N F H -e- O F OH F F OH F 50 F F O N-N N- > 55 F F O O C Y-, N O HO N- N N O N-N HO

60

65

US 8,871,792 B2 119 120 mg, 335umol) in dry DMF (6 mL) was added chloromethyl ole-3-carbonyl)hydrazino-2-hydroxypropionic acid (200 isopropyl carbonate (76 mg, 503 umol), NaI (101 mg, 670 mg, 335 umol) in dry DMF (6 mL) was added (S)-2-meth umol) and 2,6-dimethylpyridine (143 mg, 1.3 mmol) in por oxycarbonylamino-3-methylbutyric acid chloromethyl ester tions at room temperature. The resulting mixture was stirred (112 mg, 503 umol), NaI (101 mg, 670 umol), 2,6-dimeth at room temperature for 8 hours. Water (12 mL) was added 5 ylpyridine (143 mg, 1.3 mmol) in portions at room tempera and the mixture was extracted with EtOAc (3x10 mL). The ture. The resulting mixture was stirred at room temperature combined organic layers were dried over anhydrous NaSO for 8 hours. Water (12 mL) was added and the mixture was and concentrated in vacuo. The residue was purified by col extracted with EtOAc (3x20 mL). The combined organic umn chromatography (PE:EtOAc=1:1) to yield the title layers were dried over anhydrous NaSO and concentrated compound as a white solid (12.8 mg). LC-MS: 641.9 10 in vacuo. The residue was purified by column chromatogra M+H". "H NMR (DMSO-d400MHz) & 1.21 (d.J=6.2 Hz, phy (PE:EtOAc=3:1) to yield the title compound as a white 6H), 3.21-3.28 (m, 2H), 4.12-4.30 (m, 2H), 4.40-4.52 (m, solid (8.4 mg). LC-MS: 712.9 M+H". "H NMR: (MeOD, 1H), 4.77-4.79 (m. 1H), 5.68-5.76 (m, 2H), 7.41-7.45 (m, 400MHz) & 0.85 (dd, J=14.1, 6.8 Hz, 6H), 2.01-2.17 (m, 1H), 1H), 7.50-7.87(m, 9H), 7.90 (d. J–7.7 Hz, 2H), 9.98 (s, 1H), 3.42-3.51 (m, 2H), 3.69 (s.3H), 4.00 (d. J=5.9 HZ, 1H), 4.23 12.76 (s, 1H). 15 (br, 2H), 4.42 (t, J=4.7 Hz, 1H), 5.83 (dd, J-31.5, 5.6 Hz, 2H), 7.19 (t, J=9.4 Hz, 1H), 7.32-7.39 (m,3H), 7.47-7.51 (m, 4H), Example 5J 7.56-7.58 (m, 2H), 7.91 (d. J=8.0 Hz, 2H). (S)-2-Methoxycarbonylamino-3-methylbutyric Acid (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- Example 5K N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole 3-carbonyl)hydrazino-2-hydroxypropionyloxym (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- ethyl Ester N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole 3-carbonyl)hydrazino-2-hydroxypropionic Acid 25 1-cyclohexyloxycarbonyloxyethyl Ester

N-N X=O O N O H -- NH 35 HO N1

OH

40

Using the procedures described herein, the title compound can also be prepared. 45 Example 6A

50 chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- hydroxypropionic acid

O 55 HO -N O

HO

60 O -- C 65 To a solution of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4- F ylmethyl)-N'-(5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triaz US 8,871,792 B2 121 122

-continued -continued

3-Acetyl-1H-pyrazole-5-carboxylic acid (69.3 mg, 450 3-Acetyl-1H-pyrazole-5-carboxylic acid (168 mg, 1.1 umol) and HCTU (186 mg. 450 umol) were combined in 25 mmol) and HCTU (451 mg, 1.1 mmol) were combined in DMF (6.8 mL, 87.2 mmol), and stirred at room temperature. DMF (1.9 mL. 24.5 mmol), and stirred at room temperature. After 15 minutes, DIPEA (570 uL, 3.3 mmol) and (R)-3-N- After 15 minutes, DIPEA (214 uL. 1.2 mmol) and (R)-3-N- (5'-chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hy (5'-chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hy 30 droxypropionic acid ethyl ester (400 mg, 1.1 mmol) were droxypropionic acid ethyl ester (150 mg, 0.4 mmol) were added. The mixture was stirred for 20 minutes at room tem added. The mixture was stirred for 30 minutes at room tem perature, then evaporated under reduced pressure to yield the perature, then evaporated under reduced pressure. The resi title compound. MS m/z. M+H" calc’d for CHCIFNOs, due was dissolved in EtOH (1.4 mL. 24.5 mmol). A solution 503.14. found 503.2. of 1.0 M LiOH in water (2.0 mL, 2.0 mmol) was added, and 35 the resulting mixture was stirred at 40° C. for 3 hours. The Example 6C Solvent was removed under pressure and the residue was purified by preparative HPLC to yield the title compound (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- (110 mg, purity 100%) as a TFA salt. MS m/z. M+H" calc’d 40 chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- for CHCIFN.O.s, 475.11. found 475.1. hydroxypropionic Acid 2-Morpholin-4-ylethyl Ester

Example 6B 45 HN1 NN (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- O S. chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- O O hydroxypropionic Acid Ethyl Ester 50 NH HO N1 -- OH

55 O C N -NH2 HO F OH 60 N He

65 CO D US 8,871,792 B2 123 124 -continued ypropionic acid (15.0 mg, 32 umol) was combined with N HN N isobutyl alcohol (876 uL. 9.5 mmol). A solution of 4 M HCl

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 prod uct lyophilized to yield the title compound (19 mg, purity 99%) as a white powder TFA salt. MS m/z. M+H" calc’d for CHCIFNO. 531.17. found 531.1. 10 Example 6E

(R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hydr 15 hydroxypropionic Acid 5-Methyl-2-oxo-1,3-di oxy-propionic acid (41.0 mg, 86 umol), HOBt (70.0 mg, 518 umol) and EDC (92 uL. 520 mol) were combined in DCM oxol-4-ylmethyl Ester (0.7 mL, 10 mmol). The resulting solution was stirred for 10 minutes. 4-Morpholineethanol (84 uL 691 umol) was added, and the mixture was stirred at room temperature until the reaction was complete (s2 hours). The mixture was concen HN1 N trated by rotary evaporation and purified (reverse phase col umn) to yield the title compound (7.5 mg, purity 98%) as a O S. TFA salt. MS m/z. M+H" calc’d for CHCIFNO, O O 588.19. found 588.1. NH HO N1 Example 6D -- OH (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- hydroxypropionic Acid Isobutyl Ester O C

F

HN-N

40

45

50

(R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- 55 chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hydrox ypropionic acid (30.0 mg, 63 mmol), HOBt (26 mg, 190 umol) and EDC (34 ul, 190 umol) were combined in DCM (243 uL, 3.8 mmol) and stirred for 10 minutes. 4-Hydroxym 60 ethyl-5-methyl-1,3dioxol-2-one (66 mg, 0.5 mmol) and 4-methylmorpholine (28 uL. 250 umol) were added and the 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 65 the title compound (4.6 mg, purity 97%) as a TFA salt. MS (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- m/z. M+H" calc’d for CHCIFNOs, 587.13. found chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hydrox 587.1. US 8,871,792 B2 125 126 Example 6F -continued (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- 1'N-1 —- hydroxypropionic Acid 2.2-Difluoropropyl Ester HN1 \ N HN1 O S. N O O O S. 1'N-1- N Y NH 10 OH

15

chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hydrox ypropionic acid (10.0 mg, 21 Jumol) was combined with 2-methoxyethanol (0.1 mL, 1.3 mmol). A solution of 4 MHCl in dioxane (53 uL, 0.2 mmol) was added, and the resulting 25 mixture was stirred for 1 hour at room temperature. LC/MS showed the mass of the desired product. The mixture was concentrated by rotary evaporation and purified (reverse phase column) to yield the title compound (2.7 mg, purity 30 95%) as a white solid TFA salt. MS m/z. M+H" calc'd for C CHCIFNO. 533.15. found 533.1.

Example 6H F 35 chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hydrox (S)-2-Amino-3-methylbutyric Acid 3-Acetyl-5-N'- ypropionic acid (15.0 mg, 32 umol), HOBt (12.8 mg, 95 ((R)-2-carboxy-2-hydroxyethyl)-N'-(5'-chloro-2'- umol) and EDC (16.8 uL. 95 umol) were combined in DCM fluorobiphenyl-4-ylmethyl)hydrazinocarbonylpyra (121 uL, 1.9 mmol) and stirred for 10 minutes. 2.2-Difluoro 40 Zol-1-ylmethyl Ester propanol (24.3 mg, 253 Limol) 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 phase column) to yield the title compound (13.8 mg, purity 96%) as 45 HN1 N a TFA salt. MS m/z. M+H calc’d for CHCIFNOs, 553.14. found 553.1. O S. O O

Example 6G NH 50 HO N1 (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- -- chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- OH hydroxypropionic Acid 2-Methoxyethyl Ester HN-N 55 O C C F

HO N BOC 60 M OH iN H O Her

65 (C US 8,871,792 B2 127 128 -continued -continued

HO O O O '. HO 21NH

O O ?- o HO N f N N 10 H N

15

(S)-2-Amino-3-methylbutyric acid 3-acetyl-5-N'-((R)-2- carboxy-2-hydroxyethyl)-N'-(5'-chloro-2'-fluorobiphenyl-4- chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2-hydrox ylmethyl)hydrazinocarbonylpyrazol-1-ylmethyl ester (21.0 ypropionic acid (95.5 mg, 0.2 mmol) was dissolved in mg, 35umol) was combined with DCM (134 uL., 2.1 mmol) acetone (886 uL, 12.1 mmol). EtN (70 uL, 503 umol) and and Et-N (15 uL, 0.1 mmol). Methyl chloroformate (2.7 uL. (S)-2-t-butoxycarbonylamino-3-methyl-butyric acid chlo 25 35 umol) was added and the mixture was stirred at room temperature for 20 minutes. The solvent was removed in romethyl ester (56.1 mg, 211 umol) were added, and the vacuo and the residue was purified by preparative HPLC to resulting mixture was stirred at 60°C. for 6 hours. The solvent yield the title compound (0.7 mg). MS m/z. M+H calc’d for was removed in vacuo and the residue was purified using CHCIFNO, 662.20. found 662.1. flashy chromatography (normal phase; MeOH:EtOAc=0:50). 30 The pure fractions were collected, concentrated, then dis Example 6.J solved in MeCN (630 uD, 12.1 mmol). A solution of 4.0 M Isobutyric Acid (R)-2-N'-(5-Acetyl-2H-pyrazole-3- HCl in 1,4-dioxane (503 uL 2.0 mmol) was added, and the carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylm resulting mixture was stirred at room temperature for 2 hours. ethyl)hydrazino-1-carboxyethyl Ester The solvent was removed in vacuo to yield the title compound 35 (90 mg). N HN1 N Example 6I O S. 40 O O

(S)-2-Methoxycarbonylamino-3-methylbutyric acid 3-acetyl-5-N'-((R)-2-carboxy-2-hydroxyethyl)-N'- HO (5'-chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazi nocarbonylpyrazol-1-ylmethyl Ester 45

50

55

60

65 US 8,871,792 B2 129 130 Isobutyryl chloride (23.4 uL. 221.1 umol) and (R)-3-N'- Example 6L (5-acetyl-2H-pyrazole-3-carbonyl)-N-(5'-chloro-2'-fluoro biphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid (R)-2-Acetoxy-3-N'-(5-acetyl-2H-pyrazole-3-carbo 5 nyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)hy (10.5 mg, 22.1 umol) were combined in THF (108 uL. 1.3 drazinopropionic Acid mmol), and stirred overnight at room temperature. The Sol vent was evaporated and the residue was purified by prepara tive HPLC to yield the title compound (4.9 mg) as a TFA salt. O \ MS m/z. M+H" calc’d for CHCIFNO. 545.15. found 10 O S--(NH O 545.1 HO N -- OH Example 6K 15 O C 3-Methylbutyric Acid (R)-2-N'-(5-acetyl-2H-pyra Zole-3-carbonyl)-N-(5'-chloro-2'-fluorobiphenyl-4- F ylmethyl)hydrazino-1-carboxyethyl Ester O

C ls HN-N

O \ 25 HN-N O NH O HO N

HO 30 N. C

35 F Acetyl chloride (30 ul, 421.2 Lumol) and (R)-3-N'-(5- acetyl-2H-pyrazole-3-carbonyl)-N-(5'-chloro-2'-fluorobi phenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid 40 (20.0 mg, 42.1 umol) were combined in THF (205 uL., 2.5 mmol), and stirred overnight at room temperature. The Sol vent 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 CHCIFN.O. 45 517.12. found 517.2.

Example 6M

50 (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2- hydroxypropionic acid 3-dimethylaminopropyl Ester HN-N 55 O \ Isovaleryl chloride (51.4 uL, 421.2 umol) and (R)-3-N'- O N O (5-acetyl-2H-pyrazole-3-carbonyl)-N-(5'-chloro-2'-fluoro NH biphenyl-4-ylmethyl)hydrazino-2-hydroxypropionic acid HO N1 60 -- (20.0 mg, 42.1 umol) were combined in THF (205 uL., 2.5 OH mmol), and stirred overnight at room temperature. The Sol vent was evaporated and the residue was purified (reverse phase HPLC column) to yield the title compound (11.8 mg) as O c C 65 a TFA salt. MS m/z. M+H calc’d for C.H.ClFNO. F 559.17. found 559.1. US 8,871,792 B2 131 132 -continued -continued OH

10

15 chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2-hydr oxypropionic acid (10.0 mg, 21 Jumol) 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. 4-Dim ethylamino-1-butanol (22.4 uL. 168 umol) was added and the resulting mixture was stirred at room temperature and moni 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 (4.2 mg). MS m/z. M+H" calc’d for CHCIFNSOs, chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2-hydr 574.22. found 574.1. oxypropionic acid (10.0 mg, 21 Jumol) was combined with 25 HOBt (17.1 mg, 126 umol) and EDC (22 uL, 130 umol) in Example 6O DCM (0.2 mL, 3 mmol) and stirred for 10 minutes. 3-Dim ethylamino-1-propanol (19.9 uL. 168 umol) was added and (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- the resulting mixture was stirred at room temperature and chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2- monitored for completion (s4 hours). The mixture was con 30 hydroxypropionic Acid 3-Morpholin-4-yl-propyl centrated by rotary evaporation and the residue was purified Ester by preparative HPLC to yield the title compound as a TFA salt (6.4 mg). MS m/z. M+H" calc’d for C.H.ClFNSOs, 560.20. found 560.1. 35 HN-N

O Example 6.N O S O NH (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- 40 HO N1 chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2- -- hydroxypropionic Acid 4-dimethylaminobutyl Ester OH

45 HN-N O N \ coF ?/ OH 50 HO N OH (),O )

55 - O OS-S--( 1n 1a NH O r N O N hu OH

F 65 1N (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2-hydrox US 8,871,792 B2 133 134 ypropionic acid (10.0 mg, 21 umol) was combined with HOBt Example 6O (17.1 mg, 126 umol) and EDC (22 uL, 130 umol) in DCM (0.2 mL, 3 mmol) and stirred for 10 minutes. 3-Morpholinopro panol (24.5 mg, 168 umol) was added and the resulting mix (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- ture was stirred at room temperature and monitored for chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2- completion (s4 hours). The mixture was concentrated by hydroxypropionic Acid 4-Morpholin-4-yl-yutyl Ester 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 CHCIFNO, 602.21. found 6O2.1. 10 HN-N O O N Example 6P NH O 15 HO N (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- -- chloro-2'-fluorobiphenyl-4-ylmethyl)-hydrazino-2- OH hydroxypropionic Acid 2-Dimethylaminoethyl Ester O C

HN-N F O \ O N O 25 / " HO N NH -- OH ( HN-N 30 C O OS-S--( \ NH O F O H 35 hur~~~~. OH Her O C

N 40 F HN-N O \ Using the procedures described herein, the title compound O can also be prepared. N NH O 45 1N-1S N Y Example 6R OH (S)-2-Amino-3-methylbutyric Acid (R)-2-N'-(5- 50 Acetyl-2H-pyrazole-3-carbonyl)-N-biphenyl-4-ylm ethylhydrazino-1-isobutoxycarbonylethyl Ester

F co N HN1 55 N The title compound can be prepared as follows: (R)-3-N'- (5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'-chloro-2'-fluoro biphenyl-4-ylmethyl)-hydrazino-2-hydroxypropionic acid (10.0 mg, 21 umol) is combined with HOBt (17.1 mg, 126 umol) and EDC (22 uL, 130 umol) in DCM (0.2 mL, 3 mmol) 60 and stirred for 10 minutes. N,N-Dimethylaminoethanol (16.9 LL, 168 umol) is added and the resulting mixture is stirred at room temperature and monitored for completion. The mix ture is concentrated by rotary evaporation and the residue is 65 purified by preparative HPLC to yield the title compound as a TFA salt. US 8,871,792 B2 135 136 Using the procedures described herein, the title compound Example 6U can also be prepared. (S)-2-Amino-3-methylbutyric Acid 3-Acetyl-5-N- Example 6S 5 biphenyl-4-ylmethyl-N'-((R)-2-ethoxycarbonyl-2- hydroxyethyl)hydrazinocarbonylpyrazol-1-ylmethyl (S)-2-Amino-3-methylbutyric Acid 3-Acetyl-5-N'- Ester biphenyl-4-ylmethyl-N'-((R)-2-hydroxy-2-isobu 10 toxycarbonylethyl)hydrazinocarbonylpyrazol-1- ylmethyl Ester

O Using the procedures described herein, the title compound can also be prepared. 35 Using the procedures described herein, the title compound can also be prepared. Example 6V Example 6T 40

(S)-2-Amino-3-methylbutyric Acid (R)-2-N'-(5- (S)-2-Amino-3-methylbutyric acid (R)-3-N'-(5- Acetyl-2H-pyrazole-3-carbonyl)-N-biphenyl-4-ylm- 45 Acetyl-2H-pyrazole-3-carbonyl)-N-(5'-chloro-2'- ethylhydrazino-1-ethoxycarbonylethyl Ester fluorobiphenyl-4-ylmethyl)-hydrazino-2-hydrox ypropionyloxymethyl Ester

N 50 HN e N HN-N N O O O S. O O

1N O N NH 55 H2 Nulls NH 1n O N1 O O OH HN r 60 O O C F

Using the procedures described herein, the title compound 65 Using the procedures described herein, the title compound can also be prepared. can also be prepared. US 8,871,792 B2 137 138 Example 6W Example 7A

(R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- (R)-3-N'-(5-Acetyl-2H-pyrazole-3-carbonyl)-N-(5'- 5 N'-(2H-tetrazole-5-carbonyl)hydrazino-2-hydrox chloro-2'-fluorobiphenyl-4-ylmethyl)hydrazino-2- ypropionic Acid 5-methyl-2-oxo-1,3-dioxol-4-ylm hydroxypropionic Acid Ethoxycarbonyloxymethyl ethyl Ester Ester 10 Tr M N1 N N 15 O / O O O O Y NH ls N HO N 1No 1a--> N1 OH -- OH 2O O C O C

F 25 F

Using the procedures described herein, the title compound OX-ra -e- can also be prepared. O 30 O H N Example 6X N Y O 35 O N (R)-3-N'-(5-Acetyl-2-phosphonooxymethyl-2H- ---NH S O N1 pyrazole-3-carbonyl)-N-(5'-chloro-2'-fluorobiphenyl- O 4-ylmethyl)-hydrazino-2-hydroxypropionic Acid 40 y- O H

Isobutyl Ester O O C

45 F

(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 (0.2 55 mL) and 2M HCl in a mixture of dioxane and DCM (0.2 mL) at room temperature for 1 hour. The mixture was concen trated, 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 solid TFA salt (3.3 mg). MS m/z. M+H calc'd for C.H.CIFNO, 547.11. found 547. Note that as explained herein, compounds such as this can existina tautomerform, for example, as (R)-3-N-(5'-chloro s 2'-fluorobiphenyl-4-ylmethyl)-N'-(1H-tetrazole-5-carbonyl) Using the procedures described herein, the title compound 6 hydrazino-2-hydroxypropionic acid 5-methyl-2-oxo-1.3 can also be prepared. dioxol-4-ylmethyl ester. US 8,871,792 B2 139 140 Example 7B Example 7C

(R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- (S)-2-Amino-3-methylbutyric Acid 5-N'-(5'-chloro N'-(2H-tetrazole-5-carbonyl)hydrazino-2-hydrox 2'-fluorobiphenyl-4-ylmethyl)-N'-((R)-2-ethoxycar ypropionic Acid 2.2.3,3,3-Pentafluoropropyl Ester bonyl-2-hydroxyethyl)-hydrazinocarbonyltetrazol 2-ylmethyl Ester

10 Tr N1 \v. N \ O M N1 V N O N O M 15 O N NH HO N1 NH -- HO N1 -- OH OH C O C F F F 25 F He O OH F F F c1 No YBOC —- N1 v N 30 O W NH2 F O N F NH - O N1 N1 Nv (; F 35 N F F OH O W O N C 1No N1 NH 40 OH

(R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- (2-trityl-2H-tetrazole-5-carbonyl)hydrazino-2-hydroxypro- 45 pionic acid (42.4 mg, 63 umol) was combined with EDC (66.5 uL, 376 umol) and HOBt hydrate (57.5 mg, 376 umol) in DCM (0.7 mL, 10 mmol), and stirred at room temperature (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- for 10 minutes. 2.2.3,3,3-Pentafluoro-1-propanol (75.2 mg, 50 (2-trityl-2H-tetrazole-5-carbonyl)hydrazino-2-hydroxypro 501 umol) was added and the resulting mixture was stirred at pionic acid ethyl ester (15.1 mg, 21.4 umol) was stirred in a room temperature overnight. The mixture was concentrated mixture of DCM (0.2 mL, 3 mmol) and 4.0 M HCl in 1,4- and the residue was dissolved in DCM (0.4 mL, 6 mmol) at dioxane (0.1 mL, 0.4 mmol) at room temperature for 1 hour, room temperature and treated with 4.0 MHCl in 1,4-dioxane then concentrated. To this was added CsCO (14.0 mg, 42.8 55 umol) in acetone (0.5 mL) and a mixture of (S)-2-t-butoxy (0.2 mL, 0.8 mmol) for 2 hours. The mixture was concen carbonylamino-3-methylbutyric acid chloromethyl ester trated, and the residue was dissolved in AcOH (1.5 mL), (17.1 mg, 64.2 umol) and NaI (9.6 mg, 64.2 Lumol) that had filtered, and purified by reverse phase preparative HPLC. The been stirred in acetone (0.5uL, 7 umol) at 60° C. for 1 hour. desired fractions were combined and freeze dried to yield the 60 The resulting mixture was stirred at 60° C. for 4 hours, then title compound as a white solid TFA salt (8.3 mg). MS m/z. concentrated. TFA (0.1 mL, 1 mmol) and DCM (0.1 mL, 2 M+H" calc'd for C.H.ClFNO. 567.09. found 567. mmol) were added to the residue and stirred at room tempera Note that as explained herein, compounds such as this can ture for 1 hour. The mixture was then concentrated, and the exist in a tautomer form, for example, as (R)-3-N-(5'-chloro residue was dissolved in AcOH (1.5 mL), filtered, and puri 2'-fluorobiphenyl-4-ylmethyl)-N'-(1H-tetrazole-5-carbonyl) 65 fied by reverse phase preparative HPLC to yield the title hydrazino-2-hydroxypropionic acid 2.2.3,3,3-pentafluoro compound as a white solid TFA salt (4.3 mg). MS m/z. propyl ester M+H" calc’d for CHCIFN.O. 592.20. found 592.4. US 8,871,792 B2 141 142 In addition, it was found that the regioisomer of the title stirred at 60° C. for 1 hour, then it was added to a mixture of compound was also produced, (S)-2-amino-3-methylbutyric (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(2- acid 5-N'-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'- trityl-2H-tetrazole-5-carbonyl)hydrazino-2-hydroxypropi ((R)-2-ethoxycarbonyl-2-hydroxyethyl)hydrazinocarbonyl onic acid (12.3 mg, 18.2 Lumol) and DIPEA (4.8 uL. 27.2 tetrazol-1-ylmethyl ester, as a white solid TFA salt (2.7 mg): umol) in acetone (0.5 mL). The resulting mixture was stirred at 40° C. for 2 hours, concentrated and partitioned between HN EtOAc (10 mL) and water (2 mL). The organic layer was dried over MgSO, filtered, and concentrated. 4.0 M HCl in 1,4-dioxane (40.0 uL. 160 umol) in MeCN (0.2 mL, 4 mmol) O o-r -N was added and the resulting mixture was stirred at room N \ temperature for 20 minutes. The mixture was concentrated N O S. M and the residue was dissolved in AcOH (1.5 mL), filtered, and O N purified by reverse phase preparative HPLC to yield the title 15 compound (1.3 mg). MS m/z. M+H" calc’d for 1No N1 NH CHCIFNO. 535. 14. found 535.1. Note that as explained herein, compounds such as this can OH exist in a tautomer form, for example, as butyric acid (R)-3- N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(1H-tetra Zole-5-carbonyl)hydrazino-2-hydroxypropionyloxymethyl ester.

Example 7E Both regioisomers were isolated and characterized by NMR, 25 HPLC, and LCMS. (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- Example 7D N'-(1H-tetrazole-5-carbonyl)hydrazino-2-hydrox ypropionic Acid Acetoxymethyl Ester Butyric Acid (R)-3-N-(5'-chloro-2'-fluorobiphenyl 30 4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl)hydrazino 2-hydroxypropionyloxymethyl Ester

LiO

35 Tr

e

O MN O N 40

NH HO N1 --

OH 45

50

NaN O S. NH 55 O O N ~~~~ NH 60

65 A mixture of chloromethylbutyrate (6.8 uL, 54.5 umol) and NaI(8.2 mg, 54.5umol) in acetone (0.5 mL, 7 mmol) was US 8,871,792 B2 143 144 -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 HO 10 (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- N'-(1H-tetrazole-5-carbonyl)hydrazino-2-hydrox ypropionic Acid Isopropoxycarbonyloxymethyl 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 Limol) in DMF (3 mL) was added bromomethyl acetate (144 mg,936 umol), NaI (140 mg,936 umol) and 2.6-lutidine (134 mg, 1.2 mmol) dropwise at 0°C. under nitrogen. The resulting mixture was 25 stirred for 3.5 hours, then poured into water (30 mL). The resulting solution was extracted with EtOAc (2x15 mL). The combined organic layers were washed with Saturated aqueous NaCl (15 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by prepara 30 tive 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 temperature 35 under nitrogen for 30 hours. The solids were filtered off and the filtrate was concentrated in vacuo. The residue was puri fied 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 40 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, 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 45 exist in a tautomer form, for example, as (R)-3-N-(5'-chloro 2'-fluorobiphenyl-4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl) (1) hydrazino-2-hydroxypropionic acid acetoxymethyl ester.

Example 7F 50 (S)-2-Amino-3-methylbutyric Acid (R)-3-N-(5'- Chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(1H-tetra Zole-5-carbonyl)hydrazino-2-hydroxypropiony

loxymethyl Ester 55 HN1 N HO N O O O sNs NH HNulls 1. or 60 OH co 65 US 8,871,792 B2 145 146 To a solution of lithium (R)-3-(2-(1-allyl-1H-tetrazole-5- Example 7I carbonyl)-1-(5'-chloro-2'-fluorobiphenyl-4-yl)methyl)hy drazinyl)-2-hydroxypropanoate (250 mg, 526 umol) in chlo (R)-2-Acetoxy-3-N-(5'-chloro-2'-fluorobiphenyl-4- romethyl isopropyl carbonate (2 mL) was added NaI (113 mg, ylmethyl)-N'-(1H-tetrazole-5-carbonyl)hydrazino 1.1 mmol) and 2.6-lutidine (158 mg, 1.1 mmol). The resulting propionic Acid 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, 10 and concentrated in vacuo. The residue was purified by col umn chromatography (PE:EtOAc=5: 1-4:1-3:1) to yield Compound 1 as a colorless oil (165 mg). LC-MS. 591 M+H". 15 To a solution of Compound 1 (150 mg, 254 umol) in dry DCM (5 mL) was added Pd(PPh) (88 mg, 76 mmol), trieth ylsilane (148 mg, 1.3 mmol) and AcOH (76 mg, 1.3 mmol). The resulting mixture was stirred at room temperature under nitrogen for 2 days then concentrated in vacuo. The residue was purified by preparative HPLC Gemini-C18, 150x21.2 Using the procedures described herein, the title compound can also be prepared. Note that as explained herein, com mm, 5u: MeCN HO (0.1% TFA) from 50% to 80% to pounds such as this can existina tautomerform, for example, yield the title compound as a white solid (15 mg). LC-MS: as (R)-2-acetoxy-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylm 551 M+H". "H NMR (CDC1) & 1.25-129 (d. 6H), 3.44 25 ethyl)-N'-(2H-tetrazole-5-carbonyl)hydrazinopropionic 3.56 (m,2H), 4.23-4.31 (dd, 2H), 4.54-4.56 (t, 1H), 4.85-4.91 acid. (m. 1H), 5.74 (s. 2H), 7.04-7.08 (t, 1H), 7.23-7.25 (m. 1H), 7.35-7.36 (m. 1H), 7.46 (s, 4H), 9.06 (s, 1H). Example 7J Note that as explained herein, compounds such as this can 30 (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- exist in a tautomer form, for example, as (R)-3-N-(5'-chloro N'-(1H-tetrazole-5-carbonyl)-hydrazino-2-hydrox 2'-fluorobiphenyl-4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl) ypropionic acid ethoxycarbonyloxymethyl ester hydrazino-2-hydroxypropionic acid isopropoxycarbony loxymethyl ester. 35 Example 7H LiO

(S)-2-Amino-3-methylbutyric acid (R)-1-carboxy-2- 40 N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(1H tetrazole-5-carbonyl)hydrazinoethyl Ester

45 N HN1 N\ O S. M O N arry NH O O

55 HO

60 Using the procedures described herein, the title compound can also be prepared. Note that as explained herein, com pounds such as this can existina tautomer form, for example, as (S)-2-amino-3-methylbutyric acid (R)-1-carboxy-2-N- 65 (5'-chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(2H-tetrazole (1) 5-carbonyl)hydrazinoethyl ester. US 8,871,792 B2 147 148

-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) (1) hydrazinyl)-2-hydroxypropanoate (250 mg, 526 umol) in No chloromethylethylcarbonate (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 O NH temperature, then poured into water (10 mL). The resulting 25 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 col y umn chromatography (PE:EtOAc=4:1-3:1-2: 1) to yield Compound 1 as a yellow solid (170 mg). LC-MS. 577 30 N M+H". O O To a solution of Compound 1 (160 mg, 277 umol) in dry DCM (5 mL) was added Pd(PPh) (96 mg,83 mmol), trieth ylsilane (161 mg, 1.4 mmol) and AcOH (83 mg, 1.4 mmol). The resulting mixture was stirred at room temperature under HO O nitrogen for 2 days then concentrated in vacuo. The residue 35 was purified by preparative HPLC Gemini-C18, 150x21.2 Nn NH mm, 5u: MeCN HO (0.1% TFA) from 50% to 60% to N N yield the title compound as a white solid (7 mg). LC-MS. 537 H N M+MI", "H NMR (CDC1) & 1.28-133 (t, 3H), 3.47-3.50 (t, N M 2H), 4.21-4.24 (m, 4H), 4.50-4.52 (t, 1H), 5.72-5.77 (dd, 2H), 7.06-7.11 (t, 1H), 7.25-7.27 (m, 1H), 728-7.38 (m, 1H), 7.49 40 O NN (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'-chloro 2'-fluorobiphenyl-4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl) hydrazino-2-hydroxypropionic acid ethoxycarbonyloxym 45 F O ethyl ester. C Example 7K (S)-2-Methoxycarbonylamino-3-methylbutyric Acid To a suspension of (R)-3-N'-(1-allyl-1H-tetrazole-5-car (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- bonyl)-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)hy N'-(1H-tetrazole-5-carbonyl)-hydrazino-2-hydrox 50 drazino-2-hydroxypropionic acid and methane (200 mg. 421 ypropionyloxymethyl Ester umol) in THF (5 mL), was added (S)-2-methoxycarbony O lamino-3-methylbutyric acid chloromethyl ester (1.88 g, 8.42 HO mmol), NaI(126 mg, 842 umol) and 2.6-lutidine (90 mg. 842 55 mmol). The mixture was refluxed under nitrogen for 30 hours, then cooled to room temperature and poured into water (20 HO mL). The mixture was extracted with EtOAc (2x10 mL). The combined organic layers were dried over anhydrous NaSO filtered, and concentrated in vacuo. The residue was then 60 purified by column chromatography (PE:EtOAc, 5:1-4:1-3: 1) to yield Compound 1 as a yellow oil (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 mmol), EtSiH 65 (97 mg, 831 umol) and AcOH (50 mg. 831 mmol). The mixture was stirred at room temperature under nitrogen for 2 days, then concentrated in vacuo. The residue was purified by US 8,871,792 B2 149 150 preparative HPLC (Gemini-C18, 150x21.2 mm, 5u, Note that as explained herein, compounds such as this can MeCN HO (0.1% TFA); from 50% to 60%) to yield the existina tautomerform, for example, as (R)-3-N-(5'-chloro title compound as a white solid (20 mg). LC-MS. 622M--H. 2'-fluorobiphenyl-4-ylmethyl)-N'-(1H-tetrazole-5-carbonyl) H NMR (CDC1,400 MHz): & 1.02-1.05 (d. 6H), 2.06-2.09 hydrazino-2-propionyloxypropionic acid. (m. 1H), 3.47 (s.2H), 3.85 (s.3H), 4.23-4.37 (dd, 2H), 4.42 (s, 5 1H), 4.70 (t, 1H), 5.27-5.28 (d. 1H), 5.69-5.74 (dd, 2H), Example 7M 7.06-7.08 (m, 1H). 7.24-7.26 (m, 1H), 7.34-7.34 (d. 1H), 742-744 (dd, 2H), 7.50-7.52 (dd, 2H). (S)-2-Methoxycarbonylamino-3-methylbutyric Acid Note that as explained herein, compounds such as this can 10 exist in a tautomer form, for example, as (S)-2-Methoxycar (R)-1-carboxy-2-N-(5'-chloro-2'-fluorobiphenyl-4- bonylamino-3-methylbutyric acid (R)-3-N-(5'-chloro-2'- ylmethyl)-N'-(2H-tetrazole-5-carbonyl)hydrazino fluorobiphenyl-4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl) ethyl Ester hydrazino-2-hydroxypropionyloxymethyl ester. 15 Example 7L N 2N (R)-3-N-(5'-Chloro-2'-fluorobiphenyl-4-ylmethyl)- O O s NH N'-(2H-tetrazole-5-carbonyl)hydrazino-2-propiony loxypropionic acid NH HO N1 --

OH

25 N1 v N O M O C C O N F NH 30 O OH HO N1 -- O

OH ul -e- No HN1",r O C 35 N 2N NH F O O s O C NH He 40 HO N1

O O O N1 \ N l '' C O M 45 O N No NH r O NH F HO N1

O O 50 (S)-2-((methoxycarbonyl)amino)-3-methylbutanoic acid (12.9 mg, 73 umol) was combined with HOBt (12.4 mg. 92 O C umol) and EDC (11.4 mg, 73 umol) in DCM (2 mL) and stirred for 15 minutes. (R)-3-N-(5'-chloro-2'-fluorobiphe nyl-4-ylmethyl)-N'-(2H-tetrazole-5-carbonyl)hydrazino-2- F 55 hydroxypropionic acid (26.6 mg, 61 umol) and 4-methylmor pholine (7.4 mg, 73 umol) were added and the resulting Propionyl chloride (24 mg, 55umol) was added to a mix mixture was stirred at room temperature overnight. The Sol ture of (R)-3-N-(5'-chloro-2'-fluorobiphenyl-4-ylmethyl)- vent was evaporated and the residue was purified by prepara N'-(2H-tetrazole-5-carbonyl)hydrazino-2-hydroxypropi 60 tive HPLC to yield the title compound as a TFA salt (6 mg). onic acid (6.1 mg. 66 umol) and DCM (2 mL), and the MS m/z. M+H" calc’d for CH-CIFN.O. 592.16. found resulting mixture was stirred at room temperature for 1 hour. 5922. The mixture was then heated to 60°C. for 1 hour. The reaction Note that as explained herein, compounds such as this can was then stopped and the mixture was concentrated and puri exist in a tautomer form, for example, as (S)-2-methoxycar fied by preparative HPLC to yield the title compound as a 65 bonylamino-3-methylbutyric acid (R)-1-carboxy-2-N-(5'- TFA salt (1 mg). MS m/z. M+H calc’d for CHCIFNOs, chloro-2'-fluorobiphenyl-4-ylmethyl)-N'-(1H-tetrazole-5- 491.12. found 491.2. carbonyl)hydrazinoethyl ester. US 8,871,792 B2 151 152 Example 8A -continued C (R)-3-N-(3'-Chlorobiphenyl-4-ylmethyl)-N'-(3-hy droxyisoxazole-5-carbonyl)hydrazino-2-propiony Or loxypropionic Acid o-N O N N OH O S. O S. O ! OH

10 NH O N O OH ir O C 15 "N.S. -- O Isovalery chloride (15.5 uL. 127 umol) was added to a O N mixture of (R)-3-N-(3'-Chlorobiphenyl-4-ylmethyl)-N'-(3- hydroxyisoxazole-5-carbonyl)hydrazino-2-hydroxypropi O S. onic acid (25.0 mg, 58 umol) and EtN (40.3 uL, 289 umol) in O DCM (0.5 mL, 8 mmol). The resulting mixture was stirred at N H IC room temperature for 1 hour and concentrated. The residue N 25 was combined with saturated aqueous NaHCO (10:90, --> Y NaHCO: water, 0.1 mL, 0.1 mmol) in MeOH (1.0 mL), O 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). 30 MS m/z. M+H" calc’d for CHCINO, 516.15. found O co 516.5. Propanoyl chloride (3.5 uL, 40 umol) was added to a mix Example 8C ture of (R)-3-N-(3'-chlorobiphenyl-4-ylmethyl)-N'-(3-hy droxyisoxazole-5-carbonyl)hydrazino-2-hydroxypropionic 35 acid 2-oxo-2-phenylethyl ester (20.0 mg. 36.4 umol) and (R)-3-N'-(3-Acetoxymethoxyisoxazole-5-carbonyl)- EtN (12.7 L. 90.9 umol) in DCM (0.5 mL, 8 mmol). The N-(3'-chlorobiphenyl-4-ylmethyl)hydrazino-2-hy resulting mixture was stirred at room temperature for 1 hour, droxypropionic Acid concentrated, and purified by flash chromatography (EtOAc N hexanes=0-100%) to give a solid (15.5 mg). The solid was 40 O dissolved in AcOH (1.5 mL, 26 mmol). Zinc (119 mg, 1.8 O S. mmol) was added and the resulting mixture was stirred at O ! OH room temperature for 2 hours. The mixture was filtered and NH the zinc powder was washed with AcOH (0.5 mL). The com sor -- bined washes were purified by reverse phase preparative 45 O OH HPLC to yield the title compound (3.9 mg). MS m/z. M+H" calc'd for CHCINO, 488.11. found 488.3. O C Example 8B 50 3-Methylbutyric Acid (R)-1-carboxy-2-N-(3'-chlo O robiphenyl-4-ylmethyl)-N'-(3-hydroxyisoxazole-5- carbonyl)hydrazinoethyl Ester o1 NB Her H O O-N N OH 55 O S. HO -()- O O ls HO N O 60 OH ( ) es

65 US 8,871,792 B2 153 154 A mixture of bromomethylacetate (16.0LL, 164 umol) and A mixture of chloromethylbutyrate (20.5uL. 164 Lumol) NaI (24.5 mg, 164 Lumol) in acetone (2.0 mL. 27 mmol) was and NaI (24.5 mg, 164 Lumol) in acetone (2.0 mL. 27 mmol) stirred at 60° C. for 1 hour, then cooled to room temperature. was stirred at 60° C. for 1 hour, then cooled to room tempera A mixture of (R)-3-N-(3'-Chlorobiphenyl-4-ylmethyl)-N'- ture and added to a mixture of (R)-3-N-(3'-chlorobiphenyl (3-hydroxyisoxazole-5-carbonyl)hydrazino-2-hydroxypro 4-ylmethyl)-N'-(3-hydroxyisoxazole-5-carbonyl)hy pionic acid 2-oxo-2-phenylethyl ester (30.0 mg, 54.5umol) drazino-2-hydroxypropionic acid 2-oxo-2-phenylethyl ester and EtN (38.0 uL. 273 umol) in acetone (1 mL) was then added. The resulting mixture was stirred at room temperature (30.0 mg, 54.5 umol) and Et-N (38.0 uL. 273 umol) in for 2 hours, concentrated, dissolved in AcOH (2 mL), filtered, acetone (1 mL). The resulting mixture was stirred at room and purified by reverse phase preparative HPLC. The desired temperature for 2 hours, concentrated, dissolved in AcOH (2 fractions were combined and lyophilized to yield a solid (19.2 10 mL), filtered, and purified by reverse phase preparative mg). The solid was combined with zinc (178 mg, 2.7 mmol) HPLC. The desired fractions were combined and lyophilized. in AcOH (1.0 mL, 18 mmol) and stirred at room temperature Zinc (178 mg, 2.7 mmol) and AcOH (1.0 mL, 18 mmol) was for 2 hours. The mixture was filtered and purified by reverse added and the resulting mixture was stirred at room tempera phase preparative HPLC. The desired fractions were com bined, lyophilized, dissolved in AcOH (1.5 mL), and purified 15 ture for 2 hours. The mixture was filtered and purified by by reverse phase preparative HPLC to yield the title com reverse phase preparative HPLC to yield the title compound pound (3.8 mg) as a TFA salt. MS m/z. M+H" calc’d for (2.5 mg). MS m/z. M+H" calc’d for CHCINOs 532.14. CHCINOs 504.11. found 504.0. found 532.2. Example 8D Example 8E Butyric acid 5-N'-((R)-2-carboxy-2-hydroxyethyl)- (R)-3-N-(3'-Chlorobiphenyl-4-ylmethyl)-N'-(3- N'-(3'-chlorobiphenyl-4-ylmethyl)hydrazinocarbo ethoxycarbonyloxymethoxyisoxazole-5-carbonyl) nylisoxazol-3-yloxymethyl Ester hydrazino-2-hydroxypropionic acid 25

30

HO HO

35

40

45

50

55

60

65 US 8,871,792 B2 155 156 A mixture of chloromethyl ethyl carbonate (22.7 mg, 164 A mixture of chloromethyl isopropyl carbonate (15.6 mg. umol) and NaI (24.5 mg, 164 umol) in acetone (2.0 mL. 27 102 umol) and NaI (15.3 mg, 102 umol) in acetone (0.7 mL, mmol) was stirred at 60° C. for 1 hour, then cooled to room 10 mmol) was stirred at 65° C. for 1 hour, then added (R)-3- temperature and added to a mixture of (R)-3-N-(3'-chloro N-(3'-chlorobiphenyl-4-ylmethyl)-N'-(3-hydroxyisoxazole biphenyl-4-ylmethyl)-N'-(3-hydroxyisoxazole-5-carbonyl) 5-carbonyl)hydrazino-2-hydroxypropionic acid 2-oxo-2- hydrazino-2-hydroxypropionic acid 2-oxo-2-phenylethyl phenylethyl ester (20.0 mg, 36.4 umol) that had been ester (30.0 mg, 54.5 umol) and cesium carbonate (17.8 mg, dissolved in acetone (0.4 mL, 5 mmol) at room temperature and treated with cesium carbonate (13.0 mg. 40 umol). The 0.054.5umol) in acetone (1 mL). The resulting mixture was resulting mixture was then heated at 40°C. for 1 hour. DIPEA stirred at room temperature for 2 hours, concentrated, dis (0.2 mL, 1 mmol) was added and heating continued for 1 hour. solved in AcOH (2 mL), filtered, and purified by reverse phase 10 The mixture was cooled to room temperature and concen preparative HPLC. The desired fractions were combined and trated. The residue was partitioned between EtOAc (10.0 mL) concentrated. Zinc (178 mg, 2.7 mmol) and AcOH (1.0 mL. and water (2.0 mL). The organic layer was washed with 18 mmol) were added and the resulting mixture was stirred at saturated aqueous NaHCOs (3.0 mL), Saturated aqueous room temperature for 2 hours. The mixture was filtered and NaCl (3.0 mL), dried over NaSO, filtered, and concen purified by reverse phase preparative HPLC to yield the title 15 trated. Zinc 90.8 mg, 1.4 mmol) and AcOH (0.5 mL, 9 mmol) compound (2.2 mg). MS m/z. M+H" calc’d for were added and the resulting mixture was stirred at room CHCINO. 534.12. found 534.3. temperature for 1 hour. The solids were washed with AcOH (1.0 mL) then filtered. The filtrates were combined and puri Example 8F fied by reverse phase preparative HPLC to yield the title compound (3.8 mg). MS m/z. M+H calc’d for (R)-3-N-(3'-Chlorobiphenyl-4-ylmethyl)-N'-(3-iso CHCINO. 548.14. found 548.5. propoxycarbonyloxymethoxyisoxazole-5-carbonyl) hydrazino-2-hydroxypropionic Acid Example 8G (R)-3-N-(3'-Chlorobiphenyl-4-ylmethyl)-N'-3-(5- methyl-2-oxo-1,3-dioxol-4-ylmethoxy)-isoxazole 5-carbonylhydrazino-2-hydroxypropionic Acid

30

O

HO O O O 9NN 35 HO N-N N H OH -

40

45 KX C

50 a O

55 " ) O ONN HO N-N Vl H O

60 US 8,871,792 B2 157 158 A mixture of 4-chloromethyl-5-methyl-1,3-dioxol-2-one A mixture of (S)-2-t-butoxycarbonylamino-3-methylbu (11.9 uL. 109 umol) and NaI (16.4 mg, 109 umol) in acetone tyric acid chloromethyl ester (52.8 mg, 198 umol) and NaI (29.8 mg, 198 umol) in acetone (1.0 mL, 14 mmol) was (2.0 mL. 27 mmol) was stirred at 60° C. for 1 hour, then heated at 65° C. for 1 hour, then (R)-3-N-(3'-chlorobiphenyl cooled to room temperature and added to a mixture of (R)-3- 4-ylmethyl)-N'-(3-hydroxyisoxazole-5-carbonyl)hy N-(3'-chlorobiphenyl-4-ylmethyl)-N'-(3-hydroxyisoxazole drazino-2-hydroxypropionic acid 2-oxo-2-phenylethyl ester 5-carbonyl)hydrazino-2-hydroxypropionic acid 2-oxo-2- (36.4 mg. 66.2 mol) and cesium carbonate (25.9 mg, 79.4 phenylethyl ester (20.0 mg. 36.4 umol) and cesium carbonate umol) were added. The resulting mixture was stirred at 65° C. (14.2 mg, 43.6 umol) in acetone (1 mL). The resulting mix for 3 hours, concentrated, purified by flash chromatography ture was stirred at 60° C. for 2 hours, concentrated, purified by (EtOAc/hexanes=0-100%). Zinc (216 mg, 3.3 mmol) and 10 AcOH (1.0 mL, 18 mmol) were added and the resulting flash chromatography (EtOAc/hexanes=0-100%). The mixture was stirred at room temperature for 1 hour, then desired fractions were combined and concentrated. Zinc (178 concentrated by rotary evaporation. TFA (0.1 mL, 1 mmol) mg, 2.7 mmol) and AcOH (1.0 mL, 18 mmol) were added and and DCM (0.1 mL, 2 mmol) were added and the mixture was the resulting mixture was stirred at room temperature for 2 stirred for 30 minutes then concentrated. The residue was dissolved in AcOH (1.5 mL), filtered, and purified by reverse hours. The solids were washed with AcOH (0.5 mL) then 15 phase preparative HPLC to yield the title compound as a TFA filtered. The filtrates were combined and purified by reverse salt (3.0 mg). MS m/z. M+H calcd for CHCINOs, phase preparative HPLC to yield the title compound (2.8 mg). 561.17. found 561.2. MS m/z. M+H calc’d for CHCINO. 544.10. found 544.5. Example 8I (S)-2-Methoxycarbonylamino-3-methylbutyric Acid Example 8H (R)-1-carboxy-2-N-(3'-chloro-biphenyl-4-ylm ethyl)-N'-(3-hydroxyisoxazole-5-carbonyl)hy (S)-2-Amino-3-methylbutyric Acid 5-N'-((R)-2- drazinoethyl Ester carboxy-2-hydroxyethyl)-N'-(3'-chloro-biphenyl-4- ylmethyl)hydrazinocarbonylisoxazol-3-yloxymethyl Ester 25

30 O HO O O O 9NN

HO N-NX-Cl OH -- 35

40

C

45

O Her

H N V 50 BOC O HO O 55 HO N-NH y O O 60

65 US 8,871,792 B2 159 160 (S)-2-Methoxycarbonylamino-3-methylbutyric acid (9.6 mg, 54.5umol) and HATU (23.5 mg. 62 mol) were stirred in (1) -> DCM (1.0 mL, 16 mmol) for 10 minutes. (R)-3-N-(3'-Chlo O robiphenyl-4-ylmethyl)-N'-(3-hydroxyisoxazole-5-carbo nyl)hydrazino-2-hydroxypropionic acid 2-oxo-2-phenyl r NH2 ethyl ester (20.0 mg. 36.4 umol) and DIPEA (31.7 L, 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/hex anes=0-100%). Zinc (119 mg, 1.8 mmol) and AcOH (0.5 mL, 9 mmol) were added. The mixture was stirred for 2 hours and (2) filtered. The solids were washed with AcOH (1 mL) and 15 filtered. The combined filtrates were purified by reverse phase 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 preparative HPLC to yield the title compound (0.8 mg). MS mmol) was added and the resulting mixture was stirred at m/z. M+H" calc’d for CHCINO, 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 step Example 8J without any further purification.

(S)-2-t-Butoxycarbonylamino-3-methylbutyric Acid 25 O biphenyl-4-ylmethyl)hydrazinocarbonylisoxazol-3- in-Q yloxymethyl Ester O O

30 (2) + - --

O O N O / N O N O NH 35 le HO N1 -es OH O OH

40

O O O 45 O O HO N-N1 N O H O-n H O N 50 f a O C (1) 55

(R)-3-N'-t-Butoxycarbonyl-N-(3'-chlorobiphenyl-4-ylm 3-((S)-2-t-Butoxycarbonylamino-3-methylbutyry ethyl)hydrazino-2-hydroxy-propionic acid (406 mg, 965 loxymethoxy)isoxazole-5-carboxylic acid (44.1 mg, 123 umol) was dissolved in DMF (5 mL). Potassium carbonate 60 umol) and HATU (78 mg, 205umol) were combined in DMF (333 mg, 2.4 mmol) was added followed by 2-bromo-1-phe (1 mL) and stirred at room temperature for 15 minutes. Com nylethanone (230 mg, 1.2 mmol). The resulting mixture was pound 2 (45 mg, 103 umol) and DIPEA (54 uL, 307 umol) were added, and the resulting mixture was stirred at room stirred at room temperature overnight. The solvent was temperature for 15 minutes. The solvent was removed in removed in vacuo and the residue was purified (CombiFlash 65 vacuo and the residue was purified (CombiFlash normal normal phase column). The clean fractions were collected phase column). The clean fractions were collected and com and combined to yield Compound 1 (280 mg). bined to yield Compound 3 (41 mg). US 8,871,792 B2 161 162 Example 8M (3) -> (S)-2-Methoxycarbonylamino-3-methylbutyric Acid O sk (R)-3-N-(3'-Chlorobiphenyl-4-ylmethyl)-N'-(3-hy N Sk 5 droxyisoxazole-5-carbonyl)hydrazino-2-hydrox O- Y-O /No O ypropionyloxymethyl Ester O O S. arry NH 10 Ny-oh O s OH O O C 1.O Nulls o1 No N1 NH O 15 1N OH O C Compound 3 (41 mg, 53 Limol) was dissolved in AcOH (1 mL) and Zinc (172 mg, 2.6 mmol) was added. The mixture was stirred at room temperature for 45 minutes to 1 hour until the reaction was complete. The zinc was filter off and the 20 Using the procedures described herein, the title compound solution was purified (reverse phase column) to yield the title can also be prepared. compound (7 mg). MS m/z. M+H calc’d for CHCINO 661.22. found 661.2. Example 8N Example 8K 25 (S)-2-Amino-3-methylbutyric Acid (R)-1-carboxy-2- (R)-3-(N-(3'-Chlorobiphenyl-4-ylmethyl)-N'-(3 -hy- N-(3'-chlorobiphenyl-4-ylmethyl)-N'-(3-hydroxy downsylasilylox isoxazole-5-carbonyl)hydrazinoethyl Ester

N 30 f\-oh NH2 O S. ''' O ul-o1 No N1 NH 35 %. o, N'NN -N OH OH Nii HO Cl 40 O O

Using the procedures described herein, the title compound C can also be prepared. 45 Example 8L (S)-2-Amino-3-methylbutyric Acid (R)-3-N-(3'- Using the procedures described herein, the title compound Chlorobiphenyl-4-ylmethyl)-N'-(3-hydroxyisox- can also be prepared. aZole-5-carbonyl)-hydrazino-2-hydroxypropiony- 50 loxymethyl Ester Example 9A (R)-3-N-(2,5'-Dichlorobiphenyl-4-ylmethyl)-N'-(1- O- NY-OH hydroxy-1H-1.2.3 triazole-4-carbonyl)hydrazino O S. 55 2-hydroxypropionic Acid O O

H2 Nulls 1n O Y NH 1. OH O HO 60 NH V No N1 2 N-N C \ O O H -- N -- 65 Using the procedures described herein, the title compound Br O OH can also be prepared. US 8,871,792 B2 164 -continued -continued HON -OH Br O B N1 1 Cl -- O N 2N N-O O O S. N1 C O N 2N O N-OH NH O S. 10 HO N1 O OH NH HO N1

OH 15 O C

C Bromomethyl acetate (15.3 mg, 100 umol) was added to a solution of (R)-3-N-(2,5'-dichlorobiphenyl-4-ylmethyl)-N'- (1-hydroxy-1H-1,2,3-triazole-4-carbonyl)hydrazino-2-hy droxypropionic acid (31.2 mg, 66.8 umol) in acetone (0.5 mL, 6.8 mmol) followed by EtsN (18.6 uL, 134 umol). The result 1-Hydroxy-1H-1,2,3-triazole-4-carboxylic acid (42.6 ing mixture was stirred at 55°C. for 1 hour. The mixture was mg, 330 Lumol) and HATU (125 mg. 330 umol) were com 25 then concentrated in vacuo to yield a yellow liquid. The crude bined in DMF (2 mL) and stirred for 5 minutes at room liquid was purified (preparative scale C18 column chroma temperature. DIPEA (86 ul, 495 umol) and (R)-3-N-(4- tography, small column, using 30-90% MeCN in water with bromobenzyl)hydrazino-2-hydroxypropionic acid methyl 0.05%TFA) to yield the title compound (5.2 mg, purity 96%) as a white solid. MS m/z. M+H" calc'd for CHC1.N.O. ester (50 mg, 0.2 mmol) were added, and the resulting mix 538.08. found 538.1. ture was stirred for 30 minutes. The mixture was evaporated 30 under reduced pressure and the product dissolved in EtOH Example 9C (0.8 mL, 10 mmol) and water (0.2 mL, 10 mmol). 2,5-Dichlo Butyric Acid 4-N4(R)-2-carboxy-2-hydroxyethyl)- rophenylboronic acid (57 mg, 297 umol), KCO (68 mg,495 N'-(2,5'-dichlorobiphenyl-4-ylmethyl)hydrazinocar umol), and SilicaCat RDPP-Pd (0.28 mmol/g loading; 58.9 bonyl-1,2,3triazol-1-yloxymethyl Ester mg, 16.5 umol) were added and the resulting mixture was 35 heated at 120° C. for 10 minutes. The mixture was filtered, and 1 Maqueous LiOH (1.2 mL, 1.2 mmol) was added to the N 2N filtered. The mixture was stirred until the reaction was com N-OH plete (1 hour), then vacuumed to dryness and purified by O S. preparative HPLC to yield the title compound as a TFA salt 40 O (14 mg. purity 95%). MS m/z. M+H" calc’d for NH CH.ClNOs, 466.06. found 466.2. HO N1 -- OH Example 9B 45

(R)-3-N'-(1-Acetoxymethoxy-1H-1,2,3-triazole-4- carbonyl)-N-(2,5'-dichlorobiphenyl-4-ylmethyl) O c C hydrazino-2-hydroxypropionic Acid 50 C C n-is-n- -e- O N2N N 2N N-OH 55 N-O O O S. O S. N1 O O O NH NH HO N1 -- HO N1 60 OH OH O c C 65 US 8,871,792 B2 165 166 Chloromethylbutyrate (13.7 mg, 100 umol) was added to scale C18 column chromatography, Small column, using a solution of (R)-3-N-(2,5'-dichlorobiphenyl-4-ylmethyl)- 30-90% MeCN in water with 0.05% TFA) to yield the title N'-(1-hydroxy-1H-1,2,3-triazole-4-carbonyl)hydrazino-2- compound (10.0 mg, purity 99%) as a white solid. MS m/z. hydroxypropionic acid (31.2 mg, 66.8 umol) in acetone (0.5 5 M+H" calc'd for CHC1.N.O.s, 578.08. found 578.1. mL, 6.8 mmol) followed by Et N (18.6 uL, 134 umol). The resulting mixture was stirred at 65° C. for 2 hours. The mix Example 9E ture was then concentrated in vacuo to yield a yellow liquid. The crude liquid was purified (preparative scale C18 column 10 (S)-2-Methoxycarbonylamino-3-methylbutyric Acid chromatography, small column, using 30-90% MeCN in (R)-3-N-(2,5'-dichlorobiphenyl-4-ylmethyl)-N'-(1- water with 0.05% TFA) to yield to yield the title compound hydroxy-1H-1,2,3-triazole-4-carbonyl)hydrazino (6.1 mg, purity 99%) as a white solid. MS m/z. M+H" calc’d 2-hydroxypropionyloxymethyl Ester for CHCINO, 566.11. found 566.1. 15 Example 9D NaN N-OH (R)-3-(N-(2',5'-Dichlorobiphenyl-4-ylmethyl)-N'-1- O s (5-methyl-2-oxo-1,3-dioxol-4-ylmethoxy)-1H-1,2, O O 3 triazole-4-carbonylhydrazino-2-hydroxypropi 1. O Nulls o1 No N1 NH onic Acid O OH

25 1N O C

C

30 Using the procedures described herein, the title compound NH can also be prepared. HO N1 -- OH Example 9F 35 O C (R)-3-N-(2,5'-Dichlorobiphenyl-4-ylmethyl)-N'-(1- hydroxy-1H-1.2.3 triazole-4-carbonyl)hydrazino C 2-hydroxypropionic acid isopropoxycarbonyloxym O 40 C ethyl Ester O -e- O N N2V O O o N-O 45 O S. O HO O -O /— O O NH ) { HO N-N N2 N HO N1 H 50 ( ) -- 55 C ( ) C 4-Chloromethyl-5-methyl-1,3-dioxol-2-one (14.9 mg, 100 umol) was added to a solution of (R)-3-N-(2,5'-dichlorobi 60 phenyl-4-ylmethyl)-N'-(1-hydroxy-1H-1,2,3-triazole-4-car r" O O bonyl)hydrazino-2-hydroxypropionic acid (31.2 mg, 66.8 N -e- umol) in acetone (0.5 mL, 6.8 mmol) followed by EtsN (18.6 uL, 134 umol). The resulting mixture was stirred at 65°C. for 65 2 hours. The mixture was then concentrated in vacuo to yield N. a yellow liquid. The crude liquid was purified (preparative US 8,871,792 B2 167 168 -continued Example 9G (R)-3-N-(2,5'-Dichlorobiphenyl-4-ylmethyl)-N'-(1- -( hydroxy-1H-1.2.3 triazole-4-carbonyl)hydrazino o-( 5 O O 2-hydroxypropionic acid acetoxymethyl Ester O O NS N 2N -OH HO ) { N O S. H ON-2 O O --~. N1 NH

15

Using the procedures described herein, the title compound To a mixture of (R)-3-N'-(1-allyloxy-1H-1.2.3 triazole can also be prepared. 4-carbonyl)-N-(2,5'-dichlorobiphenyl-4-ylmethyl)-hy drazino-2-hydroxypropionic acid (400 mg, 790 umol) and Example 9H carbonic acid chloromethyl ester isopropyl ester (1.5 mL) 25 (R)-3-N-(2,5'-Dichlorobiphenyl-4-ylmethyl)-N'-(1- were added NaI(237 mg, 1.6 mmol) and lutidine (166 mg, 1.6 hydroxy-1H-1.2.3 triazole-4-carbonyl)hydrazino mmol). The mixture was stirred at 50° C. for 3 hours. After 2-hydroxypropionic Acid 5-methyl-2-oxo-1,3-di cooling to room temperature, the mixture was diluted with oxol-4-ylmethyl Ester water (15 mL) and extracted with EtOAc (2x15 mL). The 30 NaN combined organic layers were washed with Saturated aqueous -OH O S. NaCl (20 mL), dried over anhydrous NaSO, concentrated, O and purified by column chromatography (PE:EtOAc=5:1 to NH 2:1) to yield Compound 1 as a light yellow solid (230 mg). 35 O O N1

LC-MS. 622 M+H". O={ | OH O

(1) He O C 40

C O S. 1. O O Using the procedures described herein, the title compound O ls ^^ - NH 45 can also be prepared. OH Example 9I

C (R)-3-N-(2,5'-Dichlorobiphenyl-4-ylmethyl)-N'-(1- 50 hydroxy-1H-1.2.3 triazole-4-carbonyl)hydrazino 2-hydroxypropionic acid Ethoxycarbonyloxymethyl C Ester NaN To a solution of Compound 1 (230 mg, 370 umol) in THF N-OH 55 O S. (5 mL) was added Pd(PPh.) (64 mg, 56 mmol) and 1,3- O O dimethylbarbituric acid (577 mg, 3.7 mmol). The mixture l NH was stirred at room temperature for 2 hours and then concen 1No ^ - OH trated. The residue was purified by preparative HPLC Dai 60 sogel-C18, 250x50 mm, 10u: MeCN HO (0.1% TFA) from 50% to 90% to yield the title compound as a white solid C (120 mg). LC-MS: 582 M+H". H-NMR (MeOD, 400 Hz): C & 1.13-1.24 (d. 6H), 3.33-3.36 (m, 2H), 408-4.11 (m, 2H), 65 4.34-4.35 (m. 1H), 5.66 (s. 2H), 7.15-7.38 (m, 4H), 7.39-7.44 Using the procedures described herein, the title compound (m,3H), 8.11 (s, 1H). can also be prepared. US 8,871,792 B2 169 170 Example 9J To a solution of Compound 1 (220 mg, 360 umol) in THF (5 mL) was added Pd(PPh) (22 mg, 20 umol) and 1,3- Butyric Acid (R)-3-N-(2,5'-Dichlorobiphenyl-4- dimethylbarbituric acid (525 mg, 3.6 mmol). The mixture ylmethyl)-N'-(1-hydroxy-1H-1.2.3 triazole-4-carbo was stirred at room temperature for 2 hours and then concen nyl)hydrazino-2-hydroxypropionyloxymethyl Ester trated. The residue was purified by preparative HPLC Dai sogel-C18, 250x50 mm, 10u: MeCN HO (0.1% TFA) from 60% to 90% to yield the title compound as a white solid O /— (80 mg). LC-MS: 566 M+H, 568 (M+2)+H". H-NMR HO O O (CDOD, 400 Hz): 8 0.93 (t, 3H), 1.62 (q, 2H), 2.31 (t, 2H), 3.32-3.37 (m, 2H), 4.20 (m, 2H), 4.41 (m. 1H), 5.75 (dd, 2H), 10 7.35 (m, 4H), 7.47-7.54 (m, 3H), 8.20 (s, 1H). - ) ( e N. HO N-N N2 H Example 9K (R)-3-N-(2,5'-Dichlorobiphenyl-4-ylmethyl)-N'-(1- 15 hydroxy-1H-1,2,3-triazole-4-carbonyl)hydrazino 2-hydroxypropionic acid 1-cyclohexyloxycarbony loxyethyl Ester

C

O o HO O / / 25 HO N-N 2N o- O H O NN HO ) { N-N Yo N-2 30

35 C

(1) To a mixture of (R)-3-N'-(1-allyloxy-1H-1.2.3 triazole 4-carbonyl)-N-(2,5'-dichlorobiphenyl-4-ylmethyl)-hy 40 drazino-2-hydroxypropionic acid (300 mg, 590 umol) and chloromethylbutyrate (1.5 mL) were added NaI (178 mg, 1.2 mmol) and lutidine (124 mg, 1.2 mmol). The mixture was stirred at 50° C. for 5 hours. After cooling to room tempera ture, the mixture was diluted with water (15 mL) and 45 extracted with EtOAc (2x15 mL). The combined organic layers were washed with saturated aqueous NaCl (20 mL), dried over anhydrous NaSO 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 50 yellow solid (220 mg). LC-MS: 606 M+H", 608 (M+2)+ H*.

(1) -- 55 NaN N-OH O S. O O l NH N-1 no ro 60 OH C

C (1)

65 C To a mixture of (R)-3-N'-(1-allyloxy-1H-1.2.3 triazole 4-carbonyl)-N-(2,5'-dichlorobiphenyl-4-ylmethyl)-hy US 8,871,792 B2 171 172 drazino-2-hydroxypropionic acid (300 mg, 590 umol) and Example 9M carbonic acid 1-chloro-ethyl ester cyclohexyl ester (1.5 mL) were added NaI(178 mg, 1.2 mmol) and lutidine (124 mg, 1.2 (S)-2-Methoxycarbonylamino-3-methylbutyric Acid mmol). The mixture was stirred at 50° C. for 5 hours. After 4- N'-((R)-2-carboxy-2-hydroxy-ethyl)-N'-(2,5'- cooling to room temperature, the mixture was diluted with 5 dichlorobiphenyl-4-ylmethyl)hydrazinocarbonyl-1, water (15 mL) and extracted with EtOAc (2x15 mL). The 2.3 triazol-1-yloxymethyl Ester combined organic layers were washed with Saturated aqueous NaCl (20 mL), dried over anhydrous NaSO concentrated, and purified by silica gel chromatography (PE:EtOAc=5:1 to 2:1) to yield Compound 1 as a light yellow solid (200 mg). 10 O LC-MS. 676 M+H". NaNN-o1 No ... O n O O (1) -e- --> 15 ors N1 NH N-OH OH O S. Ol lO 1. O NH O C O O ors N Y OH C

O C Using the procedures described herein, the title compound 25 can also be prepared. C ASSAY To a solution of Compound 1 (200 mg, 296 umol) in THF (5 mL) was added Pd(PPh), (46 mg, 40 umol) and 1,3- dimethylbarbituric acid (461 mg, 3.0 mmol). The mixture 30 In Vitro Assays for the Quantitation of Inhibitor was stirred at room temperature for 2 hours and then concen Potencies (ICs) at Human and Rat NEP, and Human trated. The residue was purified by preparative HPLC Dai ACE sogel-C18, 250x50 mm, 10u: MeCN HO (0.1% TFA) from 60% to 90% to yield the title compound as a white solid The inhibitory activities of compounds at human and rat (60 mg). LC-MS. 636M--H". H-NMR (MeOD,400 Hz): 8 35 neprilysin (EC 3.4.24.11: NEP) and human angiotensin con 1.29-1.55 (m, 9H), 1.70-1.74 (m, 2H), 1.85-1.89 (m, 2H), Verting enzyme (ACE) were determined using in vitro assays 3.33-3.40 (m, 2H), 4.19-4.22 (m, 2H), 4.37-4.40 (m, 1H), as described below. 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). Extraction of NEP Activity from Rat Kidneys 40 Example 9L Rat NEP was prepared from the kidneys of adult Sprague Dawley rats. Whole kidneys were washed in cold phosphate (S)-2-Amino-3-methylbutyric acid 4-N'-((R)-2-car buffered saline (PBS) and brought up in ice-cold lysis buffer boxy-2-hydroxyethyl)-N'-(2,5'-dichlorobiphenyl-4- (1% Triton X-114, 150 mM. NaCl, 50 mM tris(hydroxym ylmethyl)hydrazinocarbonyl-1,2,3triazol-1- 45 ethyl)aminomethane (Tris) pH 7.5; Bordier (1981) J. Biol. yloxymethyl Ester Chem. 256:1604-1607) in a ratio of 5 mL of buffer for every gram of kidney. Samples were homogenized on ice using a polytron hand held tissue grinder. Homogenates were centri fuged at 1000xg in a Swinging bucket rotor for 5 minutes at 3 O 50 C. The pellet was resuspended in 20 mL of ice cold lysis N 2N NH2 buffer and incubated on ice for 30 minutes. Samples (15-20 N-o1 No mL) were then layered onto 25 mL of ice-cold cushion buffer O S. O (6% w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, 55 Triton X-114), heated to 37° C. for 3-5 minutes and centri NH fuged at 1000xg in a Swinging bucket rotor at room tempera HO N1 ture for 3 minutes. The two upper layers were aspirated off, leaving a viscous oily precipitate containing the enriched OH membrane fraction. Glycerol was added to a concentration of 50% and samples were stored at -20°C. Protein concentra C tions were quantitated using a BCA detection system with bovine serum albumin (BSA) as a standard. C Enzyme Inhibition Assays 65 Using the procedures described herein, the title compound Recombinant human NEP and recombinant human ACE can also be prepared. were obtained commercially (R&D Systems, Minneapolis, US 8,871,792 B2 173 174 Minn., catalog numbers 1182-ZN and 929-ZN, respectively). based upon the activity of the active form, this prodrug is are The fluorogenic peptide Substrate Mca-D-Arg-Arg-Leu-Dap expected to have in vivo NEP activity. (Dnp)-OH (Medeiros et al. (1997) Braz. J. Med. Biol. Res. The compound of Example 6A was tested in this assay and 30:1157-62; Anaspec, San Jose, Calif.) and Abz-Phe-Arg found to have a pK value at human NEP of 9.0. The prodrug Lys(Dnp)-Pro-OH (Araujo et al. (2000) Biochemistry compounds of Examples 6B-P either did not inhibit the 39:8519-8525; Bachem, Torrance, Calif.) were used in the enzyme in this in vitro assay, or were not tested since activity NEP and ACE assays respectively. would not be expected in this assay; however, based upon the The assays were performed in 384-well white opaque activity of the active form, these prodrugs are expected to plates at 37°C. using the fluorogenic peptide Substrates at a have in vivo NEP activity. 10 The compound of formula VII' was tested in this assay and concentration of 10 uM in Assay Buffer (NEP: 50 mM found to have a pK value at human NEP of 9.0. The prodrug HEPES, pH 7.5, 100 mM. NaCl, 0.01% polyethylene glycol compounds of Examples 7A-E and 7J either did not inhibit sorbitan monolaurate (Tween-20), 10 uM ZnSO; ACE: 50 the enzyme in this in vitro assay, or was not tested since mM HEPES, pH 7.5, 100 mM. NaCl, 0.01% Tween-20, 1 uM activity would not be expected in this assay; however, based ZnSO). The respective enzymes were used at concentrations 15 upon the activity of the active form, this prodrug is are that resulted in quantitative proteolysis of 1 uM of substrate expected to have in vivo NEP activity. after 20 minutes at 37° C. The compound of formula VIII' was tested in this assay and Test compounds were assayed over the range of concen found to have a pK value at human NEP of 9.0. The prodrug trations from 10LM to 20 pM. Test compounds were added to compounds of Examples 8A-I either did not inhibit the the enzymes and incubated for 30 minute at 37° C. prior to enzyme in this in vitro assay, or was not tested since activity initiating the reaction by the addition of substrate. Reactions would not be expected in this assay; however, based upon the were terminated after 20 minutes of incubation at 37° C. by activity of the active form, this prodrug is are expected to have the addition of glacial acetic acid to a final concentration of in vivo NEP activity. 3.6% (v/v). Plates were read on a fluorometer with excitation and emis The compound of Example 9A was tested in this assay and 25 found to have a pK value at human NEP of 9.0. The prodrug sion wavelengths set to 320 nm and 405 nm, respectively compounds of Examples 9B-K either did not inhibit the Inhibition constants were obtained by nonlinear regression of enzyme in this in vitro assay, or was not tested since activity the data using the equation (GraphPad Software, Inc., San would not be expected in this assay; however, based upon the Diego, Calif.): activity of the active form, this prodrug is are expected to have 30 in vivo NEP activity. While the present invention has been described with refer where v is the reaction rate, vo is the uninhibited reaction rate, ence to specific aspects or embodiments thereof, it will be I is the inhibitor concentration and K" is the apparent inhibi understood by those of ordinary skilled in the art that various tion constant. changes can be made or equivalents can be substituted with The compound of formula I (Example 1A) was tested in 35 out departing from the true spirit and scope of the invention. this assay and found to have a pKi value at human NEP of Additionally, to the extent permitted by applicable patent >9.0. The prodrug compounds of Examples 1B and 1C either statutes and regulations, all publications, patents, and patent did not inhibit the enzyme in this in vitro assay, or were not applications cited herein are hereby incorporated by refer tested since activity would not be expected in this assay; ence in their entirety to the same extent as if each document however, based upon the activity of the active form, these 40 had been individually incorporated by reference herein. prodrugs are expected to have in vivo NEP activity. The compound of formula II" (Example 2A) was tested in What is claimed is: this assay and found to have a pK value at human NEP of 1. A compound of formula X: >9.0. The prodrug compound of Example 2B either did not inhibit the enzyme in this in vitro assay, or was not tested 45 (X) since activity would not be expected in this assay; however, based upon the activity of the active form, this prodrug is expected to have in vivo NEP activity. The compound of formula III (Example 3A) was tested in this assay and found to have a pK value at human NEP of 50 >9.0. The prodrug compounds of Examples 3B-L either did not inhibit the enzyme in this in vitro assay, or were not tested since activity would not be expected in this assay; however, based upon the activity of the active form, this prodrug is expected to have in vivo NEP activity. 55 The compound of formula IV" (Example 4A) was tested in Ra this assay and found to have a pK value at human NEP of >9.0. The prodrug compounds of Examples 4B-Q either did (i) R' is F: R is Cl; X is not inhibit the enzyme in this in vitro assay, or were not tested since activity would not be expected in this assay; however, 60 based upon the activity of the active form, these prodrugs are expected to have in vivo NEP activity. C The compound of formula V" (Example 5A) was tested in O this assay and found to have a pK value at human NEP of >9.0. The prodrug compound of Examples 5B-K either did 65 not inhibit the enzyme in this in vitro assay, or was not tested d O since activity would not be expected in this assay; however,

US 8,871,792 B2 181 182 3. The compound of claim 2, where: 5. The compound of claim 4, where X is: R’ is H, R is H, and R7 is selected from H, -CHCH —CH-CH(CH), —CHCFCH —(CH)OCH, —CHOC(O)OCHCH —(CH2) N(CH), —(CH), N(CH), —(CH), N(CH), —CHOC (O)CHCH(CH), NH, Cl,

( O >~~ un-rr 10 Nuk V Nu. O R’ is H and R7 is selected from CHCH and —CHCH (CH), or X is: 15

Cl, W R’ is H, R is selected from CHOC(O)CHCH (CH), NHC(O)OCH and —CHOC(O)CHCH als R3 (CH4), NH, and R is H; or R is selected from C(O)CH, C(O)CH(CH), and R’ is H, R is -OH, and R7 is selected from —CHOC(O) —C(O)CHCH(CH), R is H, and R7 is H; or 25 CH, and —CHOC(O)CHCH(CH)NH; or R is H, R is R’ is H, R is CH, OP(O)(OH), or CHOC(O)CH selected from OCHOC(O)CH, and —OCHOC(O)CH CH(CH), NH, and R7 is CHCH or -CHCH CH(CH), NH, and R7 is H. (CH); or 6. The compound of claim 1, of the formula II: R’ is –C(O)CHICH(CH)NH R is H, and R7 is 30 —CHCH or —CH2CH(CH).

4. The compound of claim 1, of the formula Ia or Ib: (II)

(Ia) 35

C O N 40 7 O ) { -N n -NH H O

O R21 45

C 7. The compound of claim 6, where R is H and R is —CHCH. F 50 8. The compound of claim 1, of the formula III: (Ib)

(III) O-1 N C. N O N 55 O S. 7 O ) { -N n -NH H O 60

65

US 8,871,792 B2 187 188 20. A process for preparing the compound of claim 1, (d) reacting a compound of formula comprising the step of (a) reacting a compound of formula

1NO N1 NH 10 R21 O

Rb Ra

15 with a compound of formula HOOC X in a coupling Ra reaction, where P is H or an amino-protecting group; to produce a compound of formula X. 21. A pharmaceutical composition comprising a pharma with a compound of formula HO R7 in a transesterifi ceutically acceptable carrier and the compound of claim 1. cation reaction; or 2O 22. The pharmaceutical composition of claim 21, further (b) reacting a compound of formula comprising a therapeutic agent selected from adenosine receptor antagonists, C.-adrenergic receptor antagonists, B-adrenergic receptor antagonists, B-adrenergic receptor agonists, dual-acting B-adrenergic receptor antagonist/ol-re ceptor antagonists, advanced glycation end product breakers, NH aldosterone antagonists, aldosterone synthase inhibitors, HO N1 aminopeptidase N inhibitors, androgens, angiotensin-con Verting enzyme inhibitors and dual-acting angiotensin-con O 2 Y Verting enzyme/neprilysin inhibitors, angiotensin-converting R 30 enzyme 2 activators and stimulators, angiotensin-II Vaccines, anticoagulants, anti-diabetic agents, antidiarrheal agents, Rb anti-glaucoma agents, anti-lipid agents, antinociceptive agents, anti-thrombotic agents, AT receptor antagonists and dual-acting AT receptor antagonist/neprilysin inhibitors and Ra multifunctional angiotensin receptor blockers, bradykinin 35 receptor antagonists, calcium channel blockers, chymase inhibitors, digoxin, diuretics, dopamine agonists, endothelin with a compound of formula L-R in a nucleophilic converting enzyme inhibitors, endothelin receptor antago Substitution reaction, where L is a leaving group; or nists, HMG-CoA reductase inhibitors, estrogens, estrogen (c) reacting a compound of formula receptor agonists and/or antagonists, monoamine reuptake 40 inhibitors, muscle relaxants, natriuretic peptides and their analogs, natriuretic peptide clearance receptor antagonists, O X neprilysin inhibitors, nitric oxide donors, non-steroidal anti O N inflammatory agents, N-methyl d-aspartate receptor antago NH nists, opioid receptor agonists, phosphodiesterase inhibitors, HO N1 45 prostaglandin analogs, prostaglandin receptoragonists, renin O inhibitors, selective serotonin reuptake inhibitors, sodium R21 channel blocker, soluble guanylate cyclase stimulators and activators, tricyclic antidepressants, vasopressin receptor Rb antagonists, and combinations thereof. 50 23. The pharmaceutical composition of claim 22, wherein the therapeutic agent is an AT receptor antagonist. Ra 24. A method for treating hypertension, heart failure, or renal disease, comprising administering to a patient a thera with a compound of formula L-R in a nucleophilic 55 peutically effective amount of the compound of claim 1. Substitution reaction, where L is a leaving group; or k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 8,871,792 B2 Page 1 of 1 APPLICATIONNO. : 13/91.1552 DATED : October 28, 2014 INVENTOR(S) : Adam D. Hughes et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

In the Claims At Column 183, line 1, the second occurrence of “R” should be “R”. At Column 183, line 20, the second occurrence of “R” should be “R”. At Column 183, line 44, “R” should be “R”.

Signed and Sealed this Twenty-eighth Day of April, 2015 74-4-04- 2% 4 Michelle K. Lee Director of the United States Patent and Trademark Office