WO2014153667A1 - Cosmetic uses and methods for indoline granzyme b inhibitor compositions - Google Patents

Abstract

Cosmetic uses and methods for indoline granzyme B inhibitor compounds in compositions with a cosmetically acceptable carrier. Uses and methods for treating, reducing or inhibiting the appearance of ageing in the skin are provided. Also provided are compositions and formulation for cosmetic uses and methods of maintaining a youthful appearance, reducing an appearance of ageing, inhibiting an appearance of ageing, reducing a rate of an appearance of ageing, reducing a skin inelasticity, reducing a rate of increasing skin inelasticity, maintaining a skin elasticity, and increasing the density of hair follicles of a skin of a subjecl. The uses and methods comprise applying/administering an indoline granzyme B inhibitor to a skin, or a portion of a skin of the subject.

Description

COSMETIC USES AND METHODS FOR INDOLINE GRANZYME B

INHIBITOR COMPOSITIONS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 61/806,767, filed March 29, 2013, and U.S. Patent Application No. 61/941 ,358, filed February 18,

2014, each expressly incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This disclosure relates to the field of cosmetic compositions and formulations, and their use. More particularly, it relates to compositions for treating, reducing and inhibiting the appearance of ageing of skin comprising as at least one of the active ingredients an indoline granzyme B inhibitor compound and a cosmetically acceptable carrier.

BACKGROUND OF THE INVENTION

The skin, the primary defensive barrier of the human body, protects various organs in the body from changes in temperature, humidity, and stimuli from the external environment, such as ultraviolet rays and pollutants, and plays an important role in the regulation of biological homeostasis such as thermoregulation. Like other organs of the human body, the skin will age with time and, as a result, show skin-aging effects, including loss of skin elasticity, keratinization, wrinkle formation, skin atrophy and the like. The reasons for such skin-aging effects can be largely classified into two categories: internal factors such as cellular genetic mutations and tissue changes; and external factors such as UV light, heat and humidity.

The skin is one of the largest organs in the body and its condition and appearance are determined, in part, by the amount and the state of collagen that is contained in the skin. Collagen confers tensile strength to the skin and contributes to overall skin appearance. Loss of skin tone or collagenicity, is associated with collagen alteration in connective tissue structure.

Various compositions and methods for manipulating the quality of skin are available. For example, anti-ageing agents, including 3,3'-thiodipropionic acid or derivatives have been found to improve the aesthetic appearance of skin (WO2004/100889). One method for manipulating the quality of the skin is cosmetic surgery. Other methods involve application of caustic compositions alone or in combination with physical sloughing of the outer layers of the skin, such as 'dermabrasion' and 'chemical peel' processes. Still other methods comprise the use of various compositions that modulate the moisture, oil, or some other characteristic that can improve the appearance, suppleness, and/or condition of the skin.

Granzyme B is a pro-apoptotic serine protease found in the granules of cytotoxic lymphocytes (CTL) and natural killer (NK) cells. Granzyme B is released towards target cells, along with the pore-forming protein, perforin, resulting in its perforin-dependent internalization into the cytoplasm and subsequent induction of apoptosis (see, for e.g. , Medema et al , Eur. J. Immunol. 27:3492-3498, 1997). However, during aging, inflammation and chronic disease, granzyme B can also be expressed and secreted by other types of immune (e.g. , mast cell, macrophage, neutrophil, and dendritic cells) or non-immune (keratinocyte, chondrocyte) cells and has been shown to possess extracellular matrix remodeling activity [Choy et al , Arterioscler. Thromb. Vase. Biol. 24:2245-2250, 2004 and Buzza et al, J. Biol. Chem. 280:23549-23558, 2005).

Buzza et al, (J. Biol. Chem. 280:23549-23558, 2005) also describes that granzyme B possesses a potent extracellular matrix remodeling activity, that both native and recombinant granzyme B cause detachment of immortalized and transformed cell lines, primarily endothelial cells and chondrocytes, and that granzyme B cleaves three proteins involved in extracellular matrix structure and function, including vitronectin, fibronectin, and laminin.

Inhibitors of granzyme B in humans have been limited to (a) relatively weak, nonspecific inhibitors such as isocoumarins (Odake et al , Biochemistry 20:22 1-2221, 1991 ); (b) biological inhibitors such as serpinB9 (Sun et al , J. Biol. Chem. 271 :27802- 27809, 1996); (c) covalently coupled inhibitors such as aldehydes (Willoughby et al , Bioorg. Med. Chem. Lett. 12:2197, 2002), halomethyl ketones (Kam et al Biochim. Biophy. Acta 1477:307-323, 2000), phosphonates (Mahrus and Craik, Chem. Biol 12:567-577, 2005 and Kam et al , 2000, supra); and (d) tricyclic inhibitors (Willoughby et al , 2002, supra).

Nonspecific inhibitors (such as isocoumarins) are not sufficiently potent or specific to be effective treatments for granzyme-B-related diseases, disorders, and conditions. Likewise, the use of biological inhibitors such as serpins is limited by the ability to deliver the inhibitor to the target mammal, the cost of manufacturing the biological agents, and other, off-target activities, such as inhibition of other serine proteases such as human neutrophil elastase (Dahlen et al , Biochim. Biophys. Acta. 1451 :233-241 , 1999), Caspase-1 (Annaud et al. , Biochem. J. 342:655-665, 1999; Krieg et a!. , Mol Endocrinol. 15: 1971 -1982, 2001 ; and Young et al., J. Exp. Med. 191 : 1535- 1544, 2000); and Caspase-4 and Caspase-8 (Annaud et al , (1 99, supra).

The tricyclic inhibitors (Willoughby et al, 2001 , supra) also suffer from synthetic complexity/high manufacturing cost due to the complex core and accompanying low water solubility.

The cosmetic industry is continuously searching for novel ingredients to counter the adverse effects of premature skin aging as well as ways to reduce undesirable effects associated with skin inflammation where possible. Thus, there is a need for novel cosmetic and/or dermatological compositions to counter conditions associated with premature skin aging. The present disclosure provides compounds that inhibit granzyme B with selectivity, that are relatively simple to manufacture at low cost, that are highly water soluble, and that seek to fulfill this need and provides further related advantages.

SUMMARY OF THE INVENTION

This invention is based, in part, on the observation that granzyme B is induced in keratinocytes and immune cells, such as mast cells in the skin during aging. When released by these cells, it cleaves extracellular matrix proteins such as decorin which can result in collagen disorganization. This invention is also based in part on the observation that granzyme B cleaves decorin, in addition to other extracellular matrix proteins, in the interstitial space surrounding cells.

In one aspect of the disclosure, a cosmetic composition is provided, the composition comprises a cosmetically acceptable carrier and a compound having Formula (I):

Formula (I)

its stereoisomers and cosmetically acceptable salts thereof, wherein:

Ria is a heteroaryl group selected from

(a) 1,2,3-triazolyl optionally substituted with Ci-C₂ alkyl,

(b) 1 ,2,3,4-tetrazolyl, and

(c) 1 ,2,3-thiadiazolyl;

Rib and R]C are independently selected from hydrogen, methyl, and halogen; R₂a and R₂b are independently selected from hydrogen and methyl, or R₂a and R₂b taken together are oxo;

n is 1 or 2;

R₃ is selected from hydrogen and C1 -C4 alkyl optionally substituted with a carboxylic acid or a carboxylate group or an amine group;

Z is an acyl group selected from the group

H 0 (a) R4 .

(b) herein 0 is 1 or 2, and R¾a is C₄-acyl substituted with a carboxylic aci

(c)

wherein p is 1 or 2, and R & is C₄-acyl substituted with a carboxylic acid; and (d)

wherein Y is heteroaryl;

wherein

R4 is selected from

(i) C , -C,2 alkyl,

(ii) C1 -C6 heteroalkyl optionally substituted with C 1 -C6 alkyl,

(iii) C3-C6 cycloalkyl,

(iv) Ce-Ci o aryl,

(v) heterocyclyl,

(vi) C3-C10 heteroaryl,

(vii) aralkyl, and

(viii) heteroalkylaryl;

R5 is -C(=0)-Rio, wherein Rio is selected from

(i) C1 -C12 alkyl optionally substituted with C₆-Cio aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(ii) Ci-Cio heteroalkyl optionally substituted with Ci-C₆ alkyl or carboxylic acid,

(iii) C3-C6 cycloalkyl optionally substituted with Ci-Ce alkyl, optionally substituted C₆-Ci₀ aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(iv) C₆-Cio aryl optionally substituted with C_rC6 alkyl, optionally substituted C6-C10 aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(v) heterocyclyl,

(vi) C3-C10 heteroaryl,

(vii) aralkyl, and

(viii) heteroalkylaryl; and

Re, R₇, R₈, and R9 are independently selected from (a) hydrogen,

(b) halogen,

(c) C,-C₆ alkyl,

(d) -XRn, wherein X is selected from O, C(=0), S, S=0, or S(=0)₂,

(e) -C(=0)N(R₁₂)(R₁₃),

(f) -N(Ri i) (R₁₂)(R,₃),

(g) -N-C(=0)-R_n, and

(h) -N-C(=0)0-R,„

wherein Ru, R12, and R13 are independently selected from the group consisting of hydrogen, Ci-C₆ alkyl, C1-C6 heteroalkyl, C₂-C₆ alkenyl, C₆-Cio aryl, aralkyl, and C3-C 10 heteroaryl.

In another embodiment of the disclosure, the cosmetic composition comprises the cosmetically acceptable carrier and a compound having Formula (I):

Formula (I)

its stereoisomers and cosmetically acceptable salts thereof, wherein: Ria is a heteroaryl group selected from

(a) 1,2,3-triazolyl optionally substituted with Ci-C₂ alkyl, and

(b) 1 ,2,3,4-tetrazolyl;

Rib and R| C are independently selected from hydrogen and methyl; R₂a and R₂b are independently selected from hydrogen and methyl; n is 1 ;

R₃ is selected from hydrogen and C1-C4 alkyl optionally substituted with carboxylic acid or a carboxylate group or an amine group; Z is an acyl group selected from the group

(b) , wherein o is 1 or 2, and R.5a is C₄-acyl substituted with a carboxylic acid;

wherein p is 1 or 2, and Rs_a is C₄-acyl substituted with a

0

Y.

(d) ^R4 , wherein Y is heteroaryl;

wherein

R₄ is selected from

(i) C ,-C₁₂ alkyl,

(ii) C3-C cycloalkyl,

(iii) C6-C10 aryl, and

(iv) C3-C10 heteroaryl;

R5 is -C(=0)-Rio, wherein Rio is selected from

(i) C1 -C12 alkyl optionally substituted with C₆-Cio aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(ii) C1-C10 heteroalkyl optionally substituted with C!-C₆ alkyl or carboxylic acid, (iii) C₃-C₆ cycloalkyl optionally substituted with C)-C₆ alkyl, optionally substituted C6-C 10 aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(iv) C₆-C] o aryl optionally substituted with C] -C₆ alkyl, optionally substituted C₆-C io aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid, and

(v) C3-C10 heteroaryl; and

R R₇, R₈, and R9 are independently selected from

(a) hydrogen,

(b) halogen,

(c) C , -C₆ alkyl,

(d) -XRi wherein X is selected from O and C(=0),

(e) -N(R₁ ,)(R₁₂)(R ), and

(f) -N-C(=0)-Rn ,

wherein Ru , Ri₂, and R13 are independently selected from the group consisting of hydrogen, C\-Ce alkyl, C 1-C6 heteroalkyl, C₂-C₆ alkenyl, C₆-Cio aryl, aralkyl, and C3-C10 heteroaryl.

In yet another embodiment of the disclosure the composition comprises a compound wherein R_ja is tetrazole or triazole; R_| b and R_j c are H; n is 1 ; R₂a and R₂b are H; R₃ is H or C 1 -C4 alkyl substituted with a carboxylic acid or carboxylate group; Rg- R₉ are H; and Z is

In still yet another embodiment of the disclosure the compound of Formula (I) and Formula (II) comprises the compound wherein R_] a is tetrazole or triazole; R_jb and R_j c are H; n is 1 ; R₂a and R₂b are H; R₃ is H or C 1 -C4 alkyl substituted with a carboxylic acid or carboxylate group; R5-R9 are H; and Z is

In an further embodiment of the disclosure the composition comprises the compound of Formula I or Formula II, wherein R_j a is tetrazole or triazole; R_jb and ] C are H; n is 1 ; R₂a and R₂b are H; R₃ is H or CJ-C4 alkyl substituted with a carboxylic acid or carboxylate group; R₆-R₉ are H; and Z is

In yet a further embodiment of the disclosure the cosmetic composition comprises the compound of Formula I or the compound of Formula II wherein Rja is tetrazole or triazole; R_jb and R_jc are H; n is 1 ; R₂a and R₂b are H; R₃ is H or C_rC₄ alkyl substituted with a acid or carboxylate group; R₆-R₉ are H; and Z is

In still yet a further embodiment of the disclosure the cosmetic composition ses a cosmetically acceptable carrier and a compound having Formula (II):

Formula (II)

its stereoisomers and cosmetically acceptable salts thereof, wherein:

R_]a is tetrazole or triazole;

R_jb and R_jc are H;

n is 1 ;

R₂a and R₂b are H;

R₃ is H or C 1-C4 alkyl substituted with a carboxylic acid or carboxylate group; R₆-R₉ are H;

R₄ is C₃-C₆ cycloalkyl or C_j-C₆ alkyl optionally substituted with hydro xyl or C ₎ -C₆ alkoxy; and

R₅ is

alkyl substituted with a carboxylic acid or carboxylate group, or C3-C5 cycloalkyl substituted with a carboxylic acid or carboxylate group.

In yet still another further embodiment of the disclosure the cosmetic composition comprises a cosmetically acceptable carrier and a compound having Formula (III):

Formula (III)

its stereoisomers and cosmetically acceptable salts thereof, wherein:

R_ja is tetrazole or triazole;

R₃ is H or C C4 alkyl substituted with a carboxylic acid or carboxylate group; R4 is C3-C5 cycloalkyl or C Cg alkyl optionally substituted with hydroxyl or C_] -C₆ alkoxy; and

R_] 0 is -(CH₂)n-CC>2H, wherein n is 2, 3, 4, 5, or 6;

optionally wherein one or more single methylene carbons are substituted with a fluoro, hydroxy, amino, C C₃ alkyl, or C₆-C₁₀ aryl group;

optionally wherein one or more single methylene carbons are substituted with two fluoro or C_j -C₃ alkyl groups;

optionally wherein one or more single methylene carbons are substituted with two alkyl groups that taken together with the carbon to which they are attached form a 3, 4, 5, or 6-membered carbocyclic ring; or

optionally wherein adjacent carbon atoms from an unsaturated carbon-carbon bond or taken together form a benzene ring. The present disclosure also provides a cosmetic composition, comprising a cosmetically acceptable carrier and a compound having Formula (III):

Formula (III)

its stereoisomers and cosmetically acceptable salts thereof, wherein:

R_j a is tetrazole or triazole;

R₃ is H or C 1 -C4 alkyl substituted with a caiboxylic acid or carboxylate group; R4 is C3-C5 cycloalkyl or C C₆ alkyl optionally substituted with hydroxyi -Cg alkoxy; and

R₁₀ is

(CH₂)n

C0₂H

wherein n is 1 , 2, 3 , or 4; and

optionally, for n = 3 or 4, wherein adjacent carbon atoms from an unsaturated carbon-carbon bond,

The disclosure further provides a cosmetic composition, comprising a cosmetically acceptable carrier and a compound having Formula

its stereoisomers and cosmetically acceptable salts thereof, wherein:

R i a is tetrazole or triazole;

R3 is H or C 1 -C alkyl substituted with a carboxylic acid or carboxylate group; R4 is C₃-C₆ cycloalkyl or C_j-C₆ alkyl optionally substituted with hydroxyi or C _] -C₆ alkoxy; and R_{] [} is selected from:

(a) optionally substituted C₆-C₁₀ aryl; and

(b) optionally substituted C3-C ₁₀ heteroaryl.

The cosmetic compositions comprising a compound of any one of Formulae (I), (II), (III) or (IV) can further comprise a thickener, a diluent, a buffer, a preservative, a surface active agent, a neutral or cationic lipid, a lipid complex, a liposome, a penetration enhancer, a carrier compound and other cosmetically acceptable carrier or excipient.

The present disclosure also provides a method for treating, reducing and inhibiting (.he appearance of ageing in the skin, wherein the method comprises applying a cosmetic composition comprising a cosmetically acceptable carrier and any one of the indoline granzyme B inhibitor compounds of Formulae (I), (II), (III), or (IV) to a skin or a portion of a skin of a subject. The compositions can also further comprise a thickener, a diluent, a buffer, a preservative, a surface active agent, a neutral or cationic lipid, a lipid complex, a liposome, a penetration enhancer, a carrier compound and other cosmetically acceptable carrier or excipient. The treating, reducing and inhibiting the appearance of ageing in the skin can be by a reduction and inhibiting the appearance of ageing characterized by a reduction and inhibition of skin fragility, skin atrophy, skin wrinkles, fine lines, skin discoloration, skin sagging, skin fatigue, skin stress, skin inelasticity, skin fragility, skin softening, skin flakiness, skin dryness, enlarged pore size, skin thinning, reduced rate of skin cell turnover, or deep and deepening of skin wrinkles.

In another aspect of the present disclosure there is provided a method wherein cosmetic composition comprising the indoline granzyme B inhibitor compound of Formulae (I), (II), (III) or (IV) is applied topically to a skin of a subject or to a portion of a skin of the subject. BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a schematic illustration of a representative synthetic pathway for the preparation of indoline granzyme B inhibitor compounds P5-P4-P3-P2-P1 starting from PI .

FIGURE 2 is a schematic illustration of another representative synthetic pathway for the preparation of indoline granzyme B inhibitor compounds P5-P4-P3-P2-P 1 starting from P5.

FIGURE 3 is a schematic illustration of a further representative synthetic pathway for the preparation of indoline granzyme B inhibitor compounds P5-P4-P3-P2-P1 starting from a component other than PI or P .

DETAILED DESCRIPTION OF THE INVENTION

Skin is comprised of three main layers: the epidermis, the dermis and subcutaneous layers. Each of these three layers has individual compositions. The functions and structures of these layers are known to a person of skill in the art. The epidermis is the outermost layer of skin and includes both living and dead cell layers. The dermis is the middle layer of skin and is comprised of arrangements of collagen fibers, which surround many specialized cells and structures. Hair follicles are found within the dermis, and produce the hair shaft which grows out through layers of the dermis and epidermis to become visible as hair. The lowermost layer of the skin is the subcutaneous layer, often called the sub-dermis. The subcutaneous layer is comprised largely of fat and connective tissue and houses larger blood vessels and nerves. Collagen may be found in all layers of the skin, but is most prominently in the dermis layer.

A youthful appearance is achieved by not having at least one of the characteristic signs of age. This is often achieved by being young. Nevertheless, there are circumstances in which being young does not confer a youthful appearance as a disease or disorder or other non-time related event has conferred the characteristics associated with age. A youthful appearance is often characterized by the condition of the skin and the following skin qualities are typically associated with, but not limited to, a youthful appearance: small pore size, healthy skin tone, radiance, clarity, tautness, firmness, plumpness, suppleness, elasticity, softness, healthy skin texture, healthy skin contours, such as few or no wrinkles, shallow wrinkle depth, few or no fine lines, healthy skin luster and brightness, moisturized skin, healthy skin thickness and resilient skin. If a skin of a subject comprises any one or more of these characteristics then a youthful appearance is achieved.

The appearance of ageing can occur for a variety of reasons, but typically happens at a normal rate associated with the passage of time. A rate of appearance of ageing will be different for different subjects, depending on a variety of factors including age, gender, diet and lifestyle. An appearance of ageing is often characterized by the condition of the skin. Characteristics associated with an appearance of ageing in the skin include, but are not limited to, skin fragility, skin atrophy, skin wrinkles, fine lines, skin discoloration, skin sagging, skin fatigue, skin stress, skin inelasticity, skin fragility, skin softening, skin flakiness, skin dryness, enlarged pore size, skin thinning, reduced rate of skin cell turnover, deep and deepening of skin wrinkles. The rate of appearance of ageing can be measured by measuring the rate at which any one or more of the above characteristics appear. An appearance of ageing may be inhibited, reduced or treated by reducing or maintaining a state of any one or more of these skin characteristics.

In many circumstances a reduction in the appearance of ageing of skin occurs when the rate of collagen cleavage exceeds the rate of collagen formation. In many other circumstances, a youthful appearance of skin is maintained when the rate of collagen formation is equal to the rate of collagen cleavage. In many other circumstances, a reduction in a rate of appearance of ageing of skin is achieved when the rate of decorin cleavage and collagen disorganization and cleavage is slowed such that the rate of collagen fibrillogenesis exceeds the rate of collagen cleavage and the ratio of the rate of collagen fibrillogenesis to the rate of collagen cleavage is greater after application of an indoline granzyme B inhibitor compound compared to the ratio before application of the granzyme B inhibitor. In many other circumstances, an extracellular protein, other than decorin, is also cleaved by granzyme B, and the beneficial effects of inhibiting granzyme B can be enhanced beyond what is realized by inhibiting decorin cleavage alone.

Cosmetic and Topical Compositions and Formulations and their Use

Suitable cosmetic compositions can be formulated by means known in the art and their mode of administration and the amount of an indoline granzyme B inhibitor compound as described herein can be determined by a person of skill in the art. Compositions for use in the methods described herein can comprise one of more of the indoline Granzyme B inhibitor compound (e.g. , a compound of Formulae (I), (II), (III), or (IV)) as an active ingredient, or a cosmetically acceptable salt thereof, in combination with one or more suitable diluents, a cosmetically acceptable carrier, other relatively inert ingredients, and optionally other cosmetic ingredients. Such compositions can include any of the variety of preservatives, solvents, binding agents, emulsions stabilizers, film formers, anti-caking agents, moisturizers, and other ingredients commonly used in cosmetic creams, dermatologic products, and other topically-applied products.

The cosmetic uses and methods of the disclosure utilize an indoline granzyme B inhibitor compound and the compositions of the disclosure. As used herein "indoline granzyme B inhibitor compounds" refers to the compounds described below (i.e. , compounds of Formulae I, II, III, and IV). The terms "granzyme B inhibitors," "indoline compounds," and "indoline inhibitors" are used interchangeably with the term "indoline granzyme B inhibitor compounds."

The term "cosmetically acceptable salt" refers to a salt prepared from a cosmetically acceptable base including an inorganic base and an organic base. Representative salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, ammonium, potassium, sodium, and zinc salts. Representative salts derived from cosmetically acceptable organic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, Ν,Ν'- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, and trimethamine.

The cosmetic compositions and formulations of the present disclosure include cosmetically acceptable carriers, diluents, solubilizing or emulsifying agents and salts of the type that are available in the art. Examples of suitable agents that can be used include cosmetically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other cosmetically acceptable carriers or excipients and the like, in addition to the indoline granzyme B inhibitor compounds described herein.

Numerous types of penetration enhancers are known, such as fatty acids, bile salts, chelating agents, surfactants and non-surfactants (Lee et al , Critical Reviews in Therapeutic Drug Carrier Systems 8:91-192, 1991 ; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 7: 1-33, 1990. One or more penetration enhancers can be included in the compositions described herein.

Various fatty acids and their derivatives which act as penetration enhancers include, for example, cabrylic acid, oleic acid, lauric acid, capric acid, caprylic acid, hexanoic acid, myristic acid, palmitic acid, valeric acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, oleic acid, elaidic acid, erucic acid, nervonic acid, dicaprate, tricaprate, recinleate, monoolein (also known as 1-monooleoyl-rac-glycerol), dilaurin, arachidonic acid, glyceryll-monocaprate, l-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono- and di -glycerides and physiologically acceptable salts thereof (/^'. e. , oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, and the like). Lee et al , Critical Reviews in Therapeutic Drug Carrier Systems page 92, 1991 ; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 7: 1 , 1990; El-Hariri et al. , J. Pharm. Pharmacol. 44:651 -654, 1992).

The compositions described herein can be administered topically in any of a variety of forms. Such formulations are well known to those of skill in the art. Such formulations are suitable in the context of the use described herein for application to the skin or to the surface of the eye. The use of patches, corneal shields (see, U.S. Patent 5,185, 152), and ophthalmic solutions (see, for example, U.S. Patent No. 5,710,182) and ointments is within the skill in the art.

Formulations for topical administration include dermal patches, ointments, lotions, serums, creams, gels, hydrogels, pastes, foams, oils, semi-solids, shampoos, soaps, drops, sprays, films, liquids, powders, and the like. Conventional cosmetic carriers, aqueous, powder or oily bases, thickeners and the like can be used.

As used herein, a formulation of an indoline granzyme B inhibitor compound described herein for cosmetic, skincare, and/or dermatological applications includes, for example, known anti-wrinkle active ingredients, including for example, flavone glycosides (e.g., alpha-glycosylrutin), coenzyme Q10, vitamin E and derivatives and the like, as well as known sunblock ingredients, moisturizers, and perfume.

The indoline granzyme B inhibitor-containing compositions described herein can be administered for "cosmetic" or "skincare" (i.e., dermatologic) applications, either alone or as an "additive" in combination with other suitable agents or ingredients. As used herein, "cosmetic" and "skincare" applications includes, for example, preventive and/or restorative applications in connection with dermatological changes in the skin, such as, for example, during pre-mature skin aging; dryness; roughness; formation of dryness wrinkles; itching; reduced re-fatting (e.g. , after washing); visible vascular dilations (e.g. , telangiectases, cuperosis); flaccidity; formation of wrinkles and lines; local hyperpigmentation; hypopigmentation; incorrect pigmentation (e.g. , age spots); increased susceptibility to mechanical stress (e.g., cracking) and the like); skin-sagging (e.g. , lack of firmness) and the appearance of dry or rough skin surface features.

The indoline granzyme B inhibitors described herein can be formulated as dermalogical formulations for topical administration. Examples of such a formulation includes encapsulation of a cosmetically effective amount of an indoline granzyme B inhibiter in a vehicle selected from the group consisting of macro-capsules, micro-capsules, nano-capsules, liposomes, chylomicrons and microsponges. Another example of such a composition or formulation includes absorption of an indoline granzyme B inhibitor as described herein on a material selected from powdered organic polymers, talcs, bentonites, and other mineral supports. A third example of such a composition or formulation includes a mixture of a cosmetically effective amount of the indoline granzyme B inhibitor compound with other ingredients selected from a group comprising extracted lipids, vegetable extracts, liposoluble active principles, hydrosoluble active principles, anhydrous gels, emulsifying polymers, tensioactive polymers, synthetic lipids, gelifying polymers, tissue extracts, marine extracts, Vitamin A, Vitamin C, Vitamin D, Vitamin E, solar filter compositions, and antioxidants. Other examples of suitable compositions can be found, for example, in US2005/0249720.

The indoline granzyme B inhibitor described herein can be incorporated into any gelanic form, such as oil/water emulsions and water/oil emulsions, milks, lotions, gelifying and thickening, tensioactive and emulsifying polymers, pomades, lotions, capillaries, shampoos, soaps, powders, sticks and pencils, sprays, body oils. Regardless of the compound or formulation described herein, application/administration to a subject as a colloidal dispersion system can be used as a delivery vehicle to enhance the in vivo stability of the indoline granzyme B inhibitor compound and/or to target the indoline granzyme B inhibitor compound to a particular skin layer, tissue or cell type. Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres , beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:inhibitor complexes of uncharacterized structure. An example of a colloidal dispersion system is a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration (see, generally, Chonn et al , Current Op. Biotech. 6:698-708, 1995). Sustained-release dosage forms of the compounds described herein can also be used.

Dosing and Administration

The precise amount of the indoline granzyme B inhibitor compound administered or applied to a subject is not critical, except that it should be an amount sufficient to effect improvement of the condition for which the composition containing the indoline Granzyme B inhibitor compound is administered/applied. Application can be dependent on a number of factors, including severity and responsiveness of the condition to be treated, and with the course of treatment lasting from several days to several months, or until improvement of a condition is effected or a diminution of a symptom is achieved.

A "cosmetically effective amount" of an indoline granzyme B inhibitor compound includes a cosmetically effective amount or a prophylactically effective amount. A "cosmetically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired cosmetic result, such as improved skin elasticity, skin durability, skin firming, skin texture, decrease the appearance or decrease rate of appearance of aging, and the like. A cosmetically effective amount of a compound may vary according to factors such as the skin state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens can be adjusted to provide the optimum cosmetic response. A cosmetically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the cosmetically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as improved skin elasticity, skin durability, skin firming, skin texture, a decrease appearance or a decrease in the rate of appearance of aging, and the like. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of skin deterioration, so that a prophylactically effective amount may be less than a cosmetically effective amount.

It is to be noted that amount of an indoline granzyme B inhibitor applied may vary with the severity of the appearance, or rate of appearance, of age of the skin. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the judgment of the person applying or supervising the applying of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected. The amount of indoline granzyme B inhibitor compound(s) in the composition or formulation can vary according to factors such as the skin state, age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum response. For example, a single application can be applied/administered, several divided doses can be applied/administered over time or the amount of the indoline granzyme B inhibitor composition applied can be proportionally reduced or increased as indicated by the exigencies of the situation. It can be advantageous to formulate the indoline granzyme B inhibitor compounds in a compositions into a dosage unit form for ease of administration and uniformity of application.

By way of example, an indoline granzyme B inhibitor compound described herein can be applied/administered to achieve from about 0.01 micrograms per milliliter ^g/mL) to about 10 milligrams per milliliter, from about 0.1 μg/mL to about 500 μg mL, from about 0.1 μg/mL to about 1500 μ /mL, from about 1 μg/mL to about 2000 μg mL, and from about 0.1 μg/mL to about 5000 μg/mL, including any range within these ranges, final concentrations at a target site. Similarly, appropriate dosage values can be estimated based on the experimental data provided herein.

Appropriate dosage values can depend on the characteristics of the site to which the composition is to be applied/administered and on the form of the indoline granzyme B inhibitor compound used. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for different uses and the indoline granzyme B inhibitor compound used. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the indoline granzyme B inhibitor compound in, for example, a bodily fluid or a tissue. Following successful treatment, it can be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the condition, wherein a selected compound is applied/administered in maintenance doses applied, for example, once or more daily, to once every few days. In certain embodiments, indoline granzyme B inhibitor compounds provided herein, including indole granzyme B inhibitors, including indoline granzyme B inhibitors of Formulae (I), (II), (III), or (IV) as described herein are applied/administered in an amount to achieve ex vivo concentrations from about 1 micromolar to about 10 millimolar, from about 10 micromolar to about 5000 micromolar, or from about 30 micromolar to about 3000 micromolar, and from about 25 micromolar to about 3000 micromolar final concentration over a site of interest, and including, about 25 micromolar, or about 1600 micromolar, or about 3000 micromolar final concentration over the site, and still more typically between about 1 micromolar to about 1000 micromolar.

Compositions containing granzyme B inhibitors can also include penetrating agents. Penetrating agents can improve the ability of the granzyme B inhibitors to be delivered to deeper layers of the skin. Penetrating agents that can be used are known to a person of skill in the art and include, but are not limited to, hyaluronic acid, insulin, liposome, or the like, as well as L-arginine or the arginine-containing amino acids.

Compounds or compositions of granzyme B inhibitors can be administered by means of a device or appliance such as an implant, graft, prosthesis, garment of clothing, stent, and the like. Also, implants can be devised which are intended to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted to release the compound over a period of time. Such implants can be placed into a garment to be worn by a subject, for example a glove, shirt, mask or hat.

As used herein, a "subject" can be humans and non-human mammals, e.g. , nonhuman primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, transgenic non-human animals, and the like. The subject can be suspected of having or at risk for having an appearance of ageing of the skin. Diagnostic methods for various stages of the appearance of ageing of skin, including skin wrinkling and skin sagging, are known to those of ordinary skill in the art, see for example, Measuring the Skin by Agache et al., Springer, 2004.

Granzyme B inhibitors can be used to inhibit or reduce the appearance of ageing. Ageing is a natural phenomenon that cannot be reversed per se, but the appearance of ageing, such as skin deterioration including, but not limited to, skin inelasticity, skin fragility, skin softening, skin flakiness, skin dryness, enlarged pore size, skin thinning, reduced rate of skin cell turnover, skin wrinkling, deepening of skin wrinkles, skin sagging, fine lines, and skin discoloration may be inhibited or reduced.

Granzyme B inhibitors can be used to increase or decrease a rate of increasing or a rate of decreasing occurrences of a particular skin characteristic. In other words, a granzyme B inhibitor, when applied to the skin or a portion of the skin of a subject delays the onset of an appearance of aging. For example, in a population of subjects where half of the population applies a granzyme B inhibitor to their skin and another half of the population does not apply a granzyme B inhibitor to their skin, the half which applied a granzyme B inhibitor would not appear as aged as the half which did not apply the granzyme B inhibitor after a period of time had elapsed. The half of the population which applied a granzyme B inhibitor to the skin would also have maintained a youthful appearance.

The rate at which a particular subject experiences a change in the rate of appearance of a particular skin characteristic, i. e. , an increasing or decreasing rate of the appearance of a particular skin characteristic will depend on a variety of factors, including, but not limited to age, weight, sex and lifestyle of the subject. As such, rates are not necessarily constant, but a normal rate of increase or of decrease of an appearance of a characteristic, defined as being the new occurrence of a particular characteristic over a predetermined period of time under a set of conditions that do not include the presence of a granzyme B inhibitor applied by a method or use of this invention, is increased or decreased by applying a granzyme B inhibitor in accordance with a method or use of this invention. Methods of measuring skin characteristics, rates of increasing appearance of skin characteristics and rates of decreasing appearance of skin characteristics are known to a person of skill in the art, see for example, Measuring the Skin by Agache et al., Springer (2004). Surprisingly, granzyme B inhibitors can also be used to increase the density of hair follicles of a skin of a subject and may be used to reduce the occurrences of cutaneous xanthomas of a skin of a subject. Actively growing hair follicles contain melanocytes that transfer pigment to matrix keratinocytes, imparting color to hair. Additionally, sebum, produced in sebaceous glands, is often secreted via hair follicles. Increased density of hair follicles results in increased pigment production and increased sebum secretion resulting in improved hair appearance (e.g. , hair that is less grey in color or not grey at all) as well as healthier hair and skin. Granzyme B inhibitors also cause hair follicles to appear deeper in the skin which provide stronger hair that is less susceptible to mechanical damage. Additionally, a characteristic sign of ageing is the reduction in hair follicle density. It is known in the art that age and follicular miniaturization are weak predictors of total hair count (see Chapman et ah, Brit. J. Dermatol. 152:646-649, 2005). Consequently, the characteristic sign of age associated with hair follicle density is not predictive of hair density.

A granzyme B inhibitor or composition comprising a granzyme B inhibitor may be applied to a portion of the skin of a subject or to the whole of the skin of the subject. For example, granzyme B inhibitors and composition comprising granzyme B inhibitors may be applied to the skin, only on the face, only on the scalp, on the whole head or to each part of the body.

In one aspect, the indoline granzyme B inhibitor compounds have Formula (I):

Formula (I)

its stereoisomers and cosmetically acceptable salts thereof, wherein:

Ria is a heteroaryl group selected from

(a) 1 ,2,3-triazolyl optionally substituted with Ci-C₂ alkyl,

(b) 1 ,2,3,4-tetrazolyl, and

(c) 1 ,2,3-thiadiazolyl;

Rib and Ric are independently selected from hydrogen, methyl, and halogen; R₂a and R₂b are independently selected from hydrogen and methyl, or R₂a and R₂b taken together are oxo (i.e., =0, R₂a and R₂b together with the carbon to which they are attached form a carbonyl group);

n is 1 or 2;

R₃ is selected from hydrogen and C 1 -C4 alkyl optionally substituted with a carboxylic acid or a carboxylate group (-C0₂H or -C0₂ ^") or an amine group (-NH₂);

Z is an acyl group selected from the group

(b) , wherein o is 1 or 2, and R _a is C₄-acyl substituted with a carboxylic acid;

(c)

C4-acyl substituted with a carboxylic acid; and

O

(d) ^R4 , wherein Y is heteroaryl;

wherein

R is selected from

(i) C1 -C12 alkyl,

(ii) C1 -C6 heteroalkyl optionally substituted with C 1 -C6 alkyl,

(iii) C₃-C₆ cycloalkyl,

(iv) C₆-Cio aryl,

(v) heterocyclyl,

(vi) C₃-Cio heteroaryl,

(vii) aralkyl, and (viii) heteroalkylaryl;

Rs is -C(=0)-Rio, wherein Rio is selected from

(i) Ci-Cj₂ alkyl optionally substituted with C6-C10 aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(ii) C1-C10 heteroalkyl optionally substituted with C1-C6 alkyl or carboxylic acid,

(iii) C3-C6 cycloalkyl optionally substituted with C 1-C6 alkyl, optionally substituted C₆-Cio aryl, optionally substituted C₃-Cio heteroaryl, amino, or carboxylic acid,

(iv) C6-C10 aryl optionally substituted with Ci-C₆ alkyl, optionally substituted Ce-Ci o aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(v) heterocyclyl,

(vi) C3-C 10 heteroaryl,

(vii) aralkyl, and

(viii) heteroalkylaryl; and

R₆, R7, R_$, and R9 are independently selected from

(a) hydrogen,

(b) halogen,

(c) C,-C₆ alkyl,

(d) -XRi 1, wherein X is selected from O, C(=0), S, S=0, or S(=0)₂,

(e) -C(=0)N(R₁₂)(R₁₃),

(f) -N(R„) (Ri2)(Ri3),

(g) -N-C(=0)-R_u, and

(h) -N-C(=0)0-R„,

wherein Rn, Ri₂, and R13 are independently selected from the group consisting of hydrogen, C[-C₆ alkyl, Ci-Q heteroalkyl, C₂-C₆ alkenyl, C6-C10 aryl, aralkyl, and C3-C10 heteroaryl.

Representative compounds of the above embodiment include Examples A41 and

B2.

In certain embodiments, the compounds having Formula (I) have:

Ria is a heteroaryl group selected from

(a) 1 ,2,3-triazolyl optionally substituted with C1-C2 alkyl, and (b) 1 ,2,3,4-tetrazolyl;

Rib and Rjc are independently selected from hydrogen and methyl;

R₂a and R₂b are independently selected from hydrogen and methyl;

n is 1 ;

R₃ is selected from hydrogen and C 1 -C4 alkyl optionally substituted with a carboxylic acid or a carboxylate group (-C0₂H or -C0₂ ^") or an amine group (-NH₂);

Z is an acyl group selected from the group

, wherein o is 1 or 2, and R₅a is C4-acyl substituted with carboxylic acid;

(c)

wherein p is 1 or 2, and R₅a is C4-acyl substituted with a carboxylic acid; and

0

(d) ^R4 , wherein Y is heteroaryl;

R4 is selected from

(i) Ci-C₁₂ alkyl,

(ii) C₃-C₆ cycloalkyl,

(iii) C₆-Cio aryl, and

(iv) Q3-C10 heteroaryl;

R5 is -C(=0)-R)o, wherein Rio is selected from

(i) C1-C12 alkyl optionally substituted with C6-C10 aryl, optionally substituted C3-C 10 heteroaryl, amino, or carboxylic acid, (ii) Ci -Cio heteroalkyl optionally substituted with Ci -Ce alkyl or carboxylic acid,

(iii) C3-C6 cycloalkyl optionally substituted with C 1 -C6 alkyl, optionally substituted C6-C10 aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(iv) C6-C10 aryl optionally substituted with Ci-C₆ alkyl, optionally substituted C6-C 10 aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(v) C3-C 10 heteroaryl; and

R-6, R7, Rg, and R9 are independently selected from

(a) hydrogen,

(b) halogen,

(c) C,-C₆ alkyl,

(d) -XRi 1 , wherein X is selected from O and C(=0),

(g) -N-C(=0)-Ri i , and

wherein Ru, R12, and R_]3 are independently selected from the group consisting of hydrogen, C1-C6 alkyl, Ci-C₆ heteroalkyl, C2-C6 alkenyl, C₆-Cio aryl, aralkyl, and C3-C10 heteroaryl.

Representative compounds of the above embodiment include Examples C25, C26, C28, C29, C36, and C37.

Other representative compounds of the above embodiment include Examples C23, C24, and C38.

Other representative compounds of the above embodiment include Examples B3, B4, 1-5, A6, A7, A15, A29 and C47.

In further embodiments, the indoline granzyme B inhibitor compounds have:

R^ a is tetrazole or triazole; R_]b and R_j c are H; n is 1 ; R2a and R2b are H; R3 is H or C 1 -C4 alkyl substituted with a carboxylic acid or carboxylate group; R₆-R₉ are H; and

Z is

R_ja is tetrazole or triazole; R_jb and R_jc are H; n is 1 ; R2a and R2b are H; R3 is H or C1 -C4 alkyl substituted with a carboxylic acid or carboxylate group; R6-R9 are H; and Z is

R_ja is tetrazole or triazole; R_jb and R_jc are H; n is 1 ; R2a and R2b are H; R3 is H or C_]-C₄ alkyl substituted with a carboxylic acid or carboxylate group; R6-R9 are H; and Z is

R_ta is tetrazole or triazole; R_jb and R] C are H; n is 1 ; R₂a and R₂b are H; R₃ is H or C_] -C₄ alkyl substituted with a carboxylic acid or carboxylate group; R₆-R₉ are H; and Z is

wherein R4, R5a, o, p, and Y are as described above.

In another embodiment, the indoline granzyme B inhibitor compounds have Formula (II):

Formula (II) its stereoisomers and cosmetically acceptable salts thereof, wherein: R _j a is tetrazole or triazole; R_] b and R_j c are H; n is 1 ; R2a and R₂b are H; R3 is H or C 1 -C4 alkyl substituted with a carboxylic acid or carboxylate group; R6-R are H;

R₄ is C3-C6 cycloalkyl or C_j-Cg alkyl optionally substituted with hydroxyl or C C₆ alkoxy; and

R5 is

wherein Rio is selected from

(i) Ci-Ci₂ alkyl optionally substituted with C -C10 aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(ii) C1-C10 heteroalkyl optionally substituted with Ci -C₆ alkyl or carboxylic acid,

(iii) C3-C6 cycloalkyl optionally substituted with C1-C6 alkyl, optionally substituted C6-C10 aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(iv) C6-C10 aryl optionally substituted with C1 -C6 alkyl, optionally substituted C -C10 aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(v) C3-C 10 heteroaryl.

Representative compounds of the above embodiment include Examples B5, B6, B7, C 19, C20, C21 , C22, C27, C30, C3 1 , C32, C33, C34, and C35.

In a further embodiment, the indoline granzyme B inhibitor compounds have Formula (III):

Formula (III) its stereoisomers and cosmetically acceptable salts thereof, wherein:

R_ja, R₃, R₄, and R₁₀ are as above for Formula (II);

wherein R_{] Q}, when defined as C _]-C ₁₂ alkyl substituted with a carboxylic acid or carboxylate group, is:

-(CH₂)_n-C0₂H, where n is 2, 3, 4, 5, or 6;

optionally wherein one or more single methylene carbons are substituted with a fluoro, hydroxy, amino, C_j-C₃ alkyl (e.g. , methyl), or C₆-Ci ₀ aryl group; optionally wherein one or more single methylene carbons are substituted with two fluoro (e.g. , difluoro, perfluoro) or C1 -C3 alkyl (e.g. , gem-dimethyl) groups;

optionally wherein one or more single methylene carbons are substituted with two alkyl groups that taken together with the carbon to which they are attached form a 3, 4, 5, or 6-membered carbocyclic ring (e.g. , spiro groups such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl); and

optionally wherein adjacent carbon atoms from an unsaturated carbon-carbon bond (e.g. , alkenyl such as -CH=CH-) or taken form a benzene ring (e.g. , 1 ,2-, 1 ,3-, and 1 ,4-phenylene); or

wherein R_{] 0}, when defined as C3- 5 cycloalkyl substituted with a carboxylic acid or carboxylate group, is:

<CH₂),

wherein n is 1 , 2, 3, or 4; and optionally, for n = 3 or 4, wherein adjacent carbon atoms from an unsaturated carbon-carbon bond (e.g., cyclopentenyl cyclohexenyl).

Representative compounds of the above embodiment include Examples A5, A13, A14, A16, A17-1 , A17-2, A18, A19, A20- 1 , A20-2, A21 -1 , A21 -2, A22-1 , A22-2, A23-1 , A23-2, A24, A25, A26- 1 , A26-2, A30- 1 , A30-2, A31 , A32, A33, A36, A37, A38, A39, A40, A43, A44, A45, A46, A48, A51 , A52, A53, A54-1 , A54-2, A55, A56, A57- 1 , and A 57-2.

In another embodiment, the indoline granzyme B inhibitor compounds have Formula (IV):

Formula (IV) its stereoisomers and cosmetically acceptable salts thereof, wherein:

R_j a, R3, and R₄ arc as above for Formulae (II) and (III); and R\ \ is selected from: (a) optionally substituted C₆-C_jg aryl (e.g. , unsubstituted phenyl and substituted phenyl such as carboxyphenyl, aminophenyl, alkylaminophenyl, dialkylaminophenyl); and

(b) optionally substituted C3-C10 heteroaryl (e.g. , optionally substituted pyridyl, optionally substituted thiazolyl, optionally substituted benzothiophenyl, optionally substituted tetrazolyl, and optionally substituted triazolyl (e.g., -NH₂ substituted).

Representative compounds of the above embodiment include Examples Al , A2, A3, A4, A8, A9, AlO, Al 1 , A12, A27, A28, A34, A35, A42, A49, A50, B l, CI, C2, C3, C4, C5, C6, C7, C8, C9, CI O, Cl l , C12, C 13, C14, C 15, C 16, C17, C 18, and C39.

Each of the inhibitor compounds contain asymmetric carbon centers and give rise to stereoisomers (i. e. , optical isomers such as diastereomers and enantiomers). It will be appreciated that the present invention includes such diastereomers as well as their racemic and resolved enantiomerically pure forms. It will also be appreciated that in certain configurations, the relative stereochemistry of certain groups may be depicted as "cis" or "trans" when absolute stereochemistry is not shown.

Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.

When the compounds are basic, salts may be prepared from cosmetically acceptable non-toxic acids, including inorganic and organic acids. Examples of such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, and p-toluenesulfonic acids.

The compounds are described using the following definitions unless otherwise indicated.

As used herein, the term "alkyl" refers to a saturated or unsaturated, branched, straight-chain or cyclic monovalent hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene, or alkyne. Representative alkyl groups include methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-l -yl, propan-2-yl, cyclopropan-l-yl, prop-l -en-l -yl, prop-l -en-2- yl, prop-2-en-l -yl (allyl), cycloprop-l-en-l -yl; cycloprop-2-en-l-yl, prop-l-yn-l -yl, and prop-2-yn-l -yl; butyls such as butan-l -yl, butan-2-yl, 2-methyl-propan-l -yl, 2-methyl- propan-2-yl, cyclobutan-l -yl, but-l-en-l -yl, but- 1 -en-2-yl, 2-methyl-prop-l -en- l -yl, but- 2-en- l -yl, but-2-en-2-yl, buta-l ,3-dien-l-yl, buta-l,3-dien-2-yl, cyclobut-l-en-l-yl, cyclobut-l -en-3-yl, cyclobuta-l ,3-dien-l -yl, but-l -yn- l -yl, but- l -yn-3-yl, and but-3-yn-l - yl; and the like. Where a specific level of saturation is intended, the expressions "alkanyl," "alkenyl," and "alkynyl" are used. Alkyl groups include cycloalkyl groups. The term "cycloalkyl" refers to mono-, bi-, and tricyclic alkyl groups having the indicated number of carbon atoms. Representative cycloalkyl groups include cyclopropyl, cyclopentyl, cycloheptyl, adamantyl, cyclododecylmethyl, and 2-ethyl-l- bicyclo[4.4.0]decyl groups. The alkyl group may be unsubstituted or substituted as described below.

"Alkanyl" refers to a saturated branched, straight-chain, or cyclic alkyl group. Representative alkanyl groups include methanyl; ethanyl; propanyls such as propan- l -yl, propan-2-yl(isopropyl), and cyclopropan-l -yl; butanyls such as butan-l-yl, butan-2-yl (sec-butyl), 2-methyl-propan-l-yl(isobutyl), 2-methyl-propan-2-yl(t-butyl), and cyclobutan- l -yl; and the like. The alkanyl group may be substituted or unsubstituted. Representative alkanyl group substituents include

— R,4,— ORi4,— SR,4,— NR,₄(R,j),

—X,— CX₃,— CN,— N0₂,

— C(=0)R,4,— C(=0)OR₁₄,— C(=0)NRi₄(R,₅),— C(=0)SR,₄,

— C(=S)Ri4,— C(=S)ORi4,— C(=S)NR₁₄(R₁₅),— C(=S)SR₁₄,

— NR₁₄C(=0)NR₁₄(Ri₅),— NR₁₄(=NRi4)NRi4(R₁₅),— NR_l4C(=S)NR,4(Ri₅),

— S(=0)₂Ri4,— S(=0)₂OR₁₄,— S(=0)₂NR₁₄(R₁₅),

— OC(=0)R,4,— OC(=0)ORi4,— OC(=0)NR,₄(Ri₅),— OC(=0)SR_H,

— OS(=0)₂ORi4,— OS(=0)₂NRi₄(R₁₅), and

wherein each X is independently a halogen; and Ri₄ and R15 are independently hydrogen, C_j-C₆ alkyl, C5-C14 aryl, arylalkyl, C₃-C_{] 0} heteroaryl, and heteroarylalkyl, as defined herein.

In certain embodiments, two hydrogen atoms on a single carbon atom can be replaced with =0, =NR]₂, or =S. "Alkenyl" refers to an unsaturated branched, straight-chain, cyclic alkyl group, or combinations thereof having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The group may be in either the cis or trans conformation about the double bond(s). Representative alkenyl groups include ethenyl; propenyls such as prop-l-en-l -yl, prop-l-en-2-yl, prop-2- en- l-yl (allyl), prop-2-en-2-yl, and cycloprop-l -en-l -yl; cycloprop-2-en-l -yl; butenyls such as but-l -en-l-yl, but-l -en-2-yl, 2-methyl-prop-l -en-l -yl, but-2-en-l -yl, but-2-en- l- yl, but-2-en-2-yl, buta-l ,3-dien- l -yl, buta-l ,3-dien-2-yl, cyclobut-l -en-l-yl, cyclobut-1 - en-3-yl, and cyclobuta-l ,3-dien- l -yl; and the like. The alkenyl group may be substituted or unsubstituted. Representative alkenyl group substituents include

— |4,

—X,— CX₃,— CN,

— C(=NRi4)Ri4,— C(=NR|₄)OR,4,— C(=NR₁₄)NR₁₄(R,₅),— C(=NR,₄)SR₁₄, — C(=S)R₁₄,— C(=S)ORi4,— C(=S)NRi4(R|₅),— C(=S)SR₁₄,

wherein each X is independently a halogen; and RH and R15 are independently hydrogen, C_j-Cg alkyl, C6-C_j4 aryl, arylalkyl, C3-C 1 Q heteroaryl, and heteroarylalkyl, as defined herein.

"Alkynyl" refers to an unsaturated branched, straight-chain, or cyclic alkyl group having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne. Representative alkynyl groups include ethynyl; propynyls such as prop-l-yn-l -yl and prop-2-yn-l -yl; butynyls such as but-l -yn-l -yl, but-l-yn-3-yl, and but-3-yn-l-yl; and the like. The alkynyl group may be substituted or unsubstituted. Representative alkynyl group substituents include those as described above for alkenyl groups.

The term "haloalkyl" refers to an alkyl group as defined above having the one or more hydrogen atoms replaced by a halogen atom. Representative haloalkyl groups include halomethyl groups such as chloromethyl, fluoromefhyl, and trifluoromethyl groups; and haloethyl groups such as chloroethyl, fluoroethyl, and perfluoroethyl groups. The term "heteroalkyl" refers to an alkyl group having the indicated number of carbon atoms and where one or more of the carbon atoms is replaced with a heteroatom selected from O, N, or S. Where a specific level of saturation is intended, the expressions "heteroalkanyl," "heteroalkenyl," and "heteroalkynyl" are used. Representative heteroalkyl groups include ether, amine, and thioether groups. Heteroalkyl groups include heterocyclyl groups. The term "heterocyclyl" refers to a 5- to 10-membered non- aromatic mono- or bicyclic ring containing 1-4 heteroatoms selected from O, S, and N. Representative heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropuranyl, and morpholinyl groups. The heteroalkyl group may be substituted or unsubstituted. Representative heteroalkyl substituents include

— R,4,— ORi4,— SRi4,— NRi₄(Ris),

—X,— CX₃,— CN,— N0₂,

— C(=0)Ri4,— C(=0)OR₁₄,— C(=0)NR₁₄(R₁₅),— C(=0)SR₁₄,

— C(=NR|₄)Ri4,— C(=NR₁₄)ORi₄,— C(=NR₁₄)NR,₄(R₁₅),— C(=NR₁₄)SR|₄,

— C(=S)R,4,— C(=S)OR,4,— C(=S)NRi4(Ris),— C(=S)SR₁₄,

— NR₁₄C(=0)NR₁₄(Ri 5),— NR₁₄(=NR₁₄)NR,4(R₁₅),— NR₁₄C(=S)NR,₄(Ri ₅),

— S(=0)₂R,₄,— S(=0)₂OR, 4,— S(=0)₂NR₁₄(R₁₅),

— OC(=0)NR |₄(R, ₅),— OC(=0)SR| ₄,

— OS(=0)₂OR, 4,— OS(=0)₂NR,₄(R_I5), and

_ OP(=0)₂(OR, ₄),

wherein each X is independently a halogen; and R|₄ and R s are independently hydrogen, C _t -C₆ alkyl, C₆-C _] 4 aryl, arylalkyl, C3-C 10 heteroaryl, and heteroarylalkyl, as defined herein.

In certain embodiments, two hydrogen atoms on a single carbon atom can be replaced with =0, =NRi₂, or =S.

The term "alkoxy" refers to an alkyl group as described herein bonded to an oxygen atom. Representative C -_[-C_j alkoxy groups include methoxy, ethoxy, propoxy, and isopropoxy groups.

The term "alkylamino" refers an alkyl group as described herein bonded to a nitrogen atom. The term "alkylamino" includes monoalkyl- and dialkylaminos groups. Representative C Cg alkylamino groups include methylamino, dimethylamino, ethylamino, methylethylamino, diethylamino, propylamino, and isopropylamino groups.

The term "alkylthio" refers an alkyl group as described herein bonded to a sulfur atom. Representative C _t -C alkylthio groups include methylthio, propylthio, and isopropylthio groups. The term "aryl" refers to a monovalent aromatic hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Suitable aryl groups include groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and the like. In certain embodiments, the aryl group is a C5-C 14 aryl group. In other embodiments, the aryl group is a C5-C 10 aryl group. The number of carbon atoms specified refers to the number of carbon atoms in the aromatic ring system. Representative aryl groups are phenyl, naphthyl, and cyclopentadienyl. The aryl group can be substituted or unsubstituted. Representative aryl group substituents include

— R,4,— OR,4,— SR,4,— NR,₄(Ris),

— X,— CX₃,— CN,— N0₂,

— C(=0)R,4,— C(=0)OR_14!— C(=0)NR₁₄(R,₅),— C(=0)SR,₄,

— C(=NR₁₄)R,4,— C(=NR₁₄)OR,4,— C(=NR|₄)NR₁₄(R₁₅),— C(=NR,₄)SR₁₄,

— C(=S)R₁₄,— C(=S)OR,4,— C(=S)NRi4(Ris),— C(=S)SR,₄,

— NR,₄C(=0)NR₁₄(R, 5),

— S(=0)₂R,₄,— S(=0)₂OR_l4,— S(=0)₂NR₁₄(Ri₅),

— OS(=0)₂ORi4,— OS(=0)₂NR₁₄(Ri₅), and

— OP(=0)₂(OR₁₄),

wherein each X is independently a halogen; and Ri₄ and R15 are independently hydrogen, C_j-C₆ alkyl, C₆-C₁₄ aryl, arylalkyl, C₃-C₁₀ heteroaryl, and heteroarylalkyl, as defined herein.

The term "aralkyl" refers to an alkyl group as defined herein with an aryl group, optionally substituted, as defined herein substituted for one of the alkyl group hydrogen atoms. Suitable aralkyl groups include benzyl, 2-phenylethan-l-yl, 2-phenylethen- 1 -yl, naphthylmethyl, 2-naphthylethan-l-yl, 2-naphthylethen- 1 -yl, naphthobenzyl, 2-naphthophenylethan-l-yl, and the like. Where specific alkyl moieties are intended, the terms aralkanyl, aralkenyl, and aralkynyl are used. In certain embodiments, the aralkyl group is a C₆-C2o aralkyl group, (e.g. , the alkanyl, alkenyl, or alkynyl moiety of the aralkyl group is a C Cg group and the aryl moiety is a C5-C_] group). In other embodiments, the aralkyl group is a Cg-C^ aralkyl group (e.g. , the alkanyl, alkenyl, or alkynyl moiety of the aralkyl group is a C 1-C3 group and the aryl moiety is a C₅-C I Q aryl group. In certain embodiments, the aralkyl group is a benzyl group.

The term "heteroaryl" refers to a monovalent heteroaromatic group derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system, which may be monocyclic or fused ring (i. e. , rings that share an adjacent pair of atoms). A "heteroaromatic" group is a 5- to 14-membered aromatic mono- or bicyclic ring containing 1-4 heteroatoms selected from O, S, and N. Representative 5- or 6-membered aromatic monocyclic ring groups include pyridine, pyrimidine, pyridazine, furan, thiophene, thiazole, oxazole, and isooxazole. Representative 9- or 10-membered aromatic bicyclic ring groups include benzofuran, benzothiophene, indole, pyranopyrrole, benzopyran, quionoline, benzocyclohexyl, and naphthyridine. Suitable heteroaryl groups include groups derived from acridine, arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like. In certain embodiments, the heteroaryl group is a 5-14 membered heteroaryl group. In other embodiments, the heteroaryl group is a 5- 10 membered heteroaryl group. Preferred heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole, and pyrazine. The heteroaryl group may be substituted or unsubstituted. Representative heteroaryl group substituents include those described above for aryl groups.

The term "heteroarylalkyl" refers to an alkyl group as defined herein with a heteroaryl group, optionally substituted, as defined herein substituted for one of the alkyl group hydrogen atoms. Where specific alkyl moieties are intended, the terms heteroarylalkanyl, heteroarylalkenyl, or heteroarylalkynyl are used. In certain embodiments, the heteroarylalkyl group is a 6-20 membered heteroarylalkyl (e.g. , the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is a C C₆ group and the heteroaryl moiety is a 5-14-membered heteroaryl group. In other embodiments, the heteroarylalkyl group is a 6-13 membered heteroarylalkyl (e.g. , the alkanyl, alkenyl or alkynyl moiety is C1-C3 group and the heteroaryl moiety is a 5-10-membered heteroaryl group).

The term "acyl" group refers to the -C(=0)— R' group, where R' is selected from optionally substituted alkyl, optionally substituted aryl, and optionally substituted heteroaryl, as defined herein.

The term "halogen" or "halo" refers to fluoro, chloro, bromo, and iodo groups. The term "substituted" refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s).

Representative indoline compounds and related intermediates were prepared from commercially available starting materials or starting materials prepared by conventional synthetic methodologies. Representative indoline compounds were prepared according to Methods A to P as described below and illustrated in FIGURES 1-3. The preparations of certain intermediates (1-1 to 1-15) useful in the preparation of the indoline compounds are described in the Synthetic Intermediate section below.

FIGURES 1-3 present schematic illustrations of representative synthetic pathways for the preparation of representative compounds P5-P4-P3-P2-P1. As used herein, "P5-P4-P3-P2-P1 " refers to compounds prepared from five (5) components: P I, P2, P3, P4, and P5. Protected version of the components useful in the preparation of the compounds are designated as, for example, "PG-P2," "PG-P2-P 1," "PG-3," and "PG-P3- P2-P1 ," where "PG" is refers to a protecting group that allows for the coupling of, for example, PI to P2 or P3 to P1 -P2, and that is ultimately removed to provide, for example, Pl-P2 or Pl-P2-P3.

FIGURE 1 is a schematic illustration of a representative synthetic pathway for the preparation of representative indoline compounds P5-P4-P3-P2-P 1 starting from PI . In this pathway, compound P5-P4-P3-P2-P1 is prepared in a stepwise manner starting with PI by sequential coupling steps, separated as appropriate by deprotection steps and other chemical modifications. As shown in FIGURE 1 , PI is coupled with PG-P2 to provide PG-P2-P1 , which is then deprotected to provide P2-P1 and ready for coupling with the next component, PG-P3. The process is continued with subsequent couplings PG-P4 with P3-P2-P1 and PG-P5 with P4-P3-P2-P1 to ultimately provide P5-P4-P3-P2-P 1. Examples A1-A57 were prepared by this pathway.

FIGURE 2 is a schematic illustration of another representative synthetic pathway for the preparation of representative indoline compounds P5-P4-P3-P2-P1 starting from P5. In this pathway, compound P5-P4-P3-P2-P1 is prepared in a stepwise manner starting with P5 by sequential coupling steps, separated as appropriate by deprotection steps and other chemical modifications. As shown in FIGURE 2, P5 is coupled with PG-P4 to provide P5-P4-PG, which is then deprotected to provide P5-P4 and ready for coupling with the next component, P3-PG. The process is continued with subsequent couplings PG-P2 with P5-P4-P3 and PG-P l with P5-P4-P3-P2 to ultimately provide P5-P4-P3-P2-P1. Examples B1-B7 were prepared by this method.

FIGURE 3 is a schematic illustration of a further representative synthetic pathway for the preparation of representative indoline compounds P5-P4-P3-P2-P 1 starting from a component other than PI or P5. In this pathway, compound P5-P4-P3-P2-P1 is prepared in a stepwise manner starting with P2 by sequential coupling steps, separated as appropriate by deprotection steps and other chemical modifications. As shown in FIGURE 3, there are multiple pathways to P5-P4-P3-P2-P1. Pathways are shown involving representative Synthetic Intermediates (1- 1 , 1-2, 1-3, 1-4, 1-6, 1-7, 1-9, 1- 10, 1- 1 1 , and 1-12). Examples C1-C39 were prepared by this method.

The preparation of representative indoline compounds and their characterization are described in Examples Al to A57, Bl to B7, and CI to C39. The structures of representative compounds are set forth in Table 1.

Table 1. Representative Compounds.

-39-

-44-

A general kinetic enzyme assay useful for determining the inhibitory activity of the compounds is described in Example Dl .

Granzyme B enzymatic inhibition assays are described in Examples D2 and D6. The compounds identified in Table 1 exhibited Granzyme B inhibitory activity. In certain embodiments, select compounds exhibited IC50 < 50,000 nM. In other embodiments, select compounds exhibited IC50 < 10,000 nM. In further embodiments, select compounds exhibited IC50 < 1,000 nM. In still further embodiments, select compounds exhibited IC50 ^< 100 ⁿM. In certain embodiments, select compounds exhibited IC₅₀ from 10 nM to 100 nM, preferably from 1 nM to 10 nM, more preferably from 0.1 nM to 1 nM, and even more preferably from 0.01 nM to 0.1 nM.

Caspase enzymatic inhibition assays are described in Examples D3 and D7. None of the compounds tested demonstrated an ability to significantly inhibit any of the caspases evaluated at a concentration of 50 μΜ. In certain embodiments, the compounds exhibited less than 50% inhibition at 50 μΜ. In other embodiments, the compounds exhibited greater than 50% inhibition at 50 μΜ, but less than 10% inhibition at 25 μΜ. The results demonstrate that select compounds of the invention selectively inhibit Granzyme B without significantly inhibiting caspases. A method for determining kinetic solubility is described in Example D4. The results demonstrate that select compounds of the invention have significantly greater solubility than Willoughby 20, a representative Granzyme B inhibitor known in the art.

Each reference cited is incorporated herein by reference in its entirety.

Abbreviations

As used herein, the following abbreviations have the indicated meanings.

Ή NMR: proton nuclear magnetic resonance

¹⁹F NMR: fluorine- 19 nuclear magnetic resonance

%Inh: Percent inhibition

Ac-IEPD-AMC: acetyl-isoleucyl-glutamyl-prolyl-aspartyl-(7-amino-4- methylcoumarin) substrate

ACN: acetonitrile

BHET : bis-2 -hy droxyethyl-terephthal te

Boc: ferf-butoxycarbonyl

CHAPS : 3 - [(3 -cholamidopropyl)dimethylammonio] - 1 -propanesulfonate

DCM: dichloromethane

DIPEA: diisopropylethylamine

DMAP: 4-dimethylaminopyridine

DMF: dimethylformamide

DMSO: dimethylsulfoxide

DMSO-dtf: dimethylsulfoxide-e?(5

DTT: dithiothreitol

EDC: 1 -(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

EDTA: 2-({2-[5w(carboxymethyl)amino]ethyl}(carboxymethyl)amino)acetic acid

ESI: Electrospray ionization

EtOAc: ethyl acetate

eq.: equivalent(s)

GzmB: Granzyme B

HATU : 2-(7-aza- 1 H-benzotriazole- 1 -yl)- 1,1 , 1 ,1 -tetramethyluronium

hexafluorophosphate

HC1: hydrochloric acid HEPES: 4-(2-hydroxyethyl)- l -piperazineethanesulfonic acid hGzmB: human Granzyme B

HPLC: high performance liquid chromatography

HOBt: 1-hydroxy-benzotriazol

IC50: inhibitory concentration that provides 50% inhibition

LC/MS: liquid chromatography / mass spectrometry

MeOH: methanol

mGzmB: murine Granzyme B

MS: mass spectrometry

m/z: mass to charge ratio.

Oxyma: ethyl 2-cyano-2-(hydroxyimino)acetate

PBS: phosphate buffered saline (pH 7.4)

RPM: revolution per minute

RT: room temperature

THF: tetrahydrofuran

TFA: trifluoroacetic acid

General Methods A-P

Representative indoline compounds were prepared according to Methods A to P as described below and illustrated in FIGURES 1 -3.

The Willoughby 20 compound was synthesized by published procedures (see

Willoughby et al, Bioorg. Med. Chem. Lett. 12:2197-2200, 2002 and WO 03/065987). It will be appreciated that in the following general methods and preparation of synthetic intermediates, reagent levels and relative amounts or reagents/intermediates can be changed to suit particular compounds to be synthesized, up or down by up to 50% without significant change in expected results.

Method A: General method for deprotcction followed bv coupling reaction siim EDC / HOBt / DIPEA.

HCI Solution in dioxane (4 M, 5 ml) was added to respective carbamate compound (0.125 mmol) and stirred for 2 hrs at RT. The reaction mixture was concentrated to dryness under vacuum and swapped with MeOH (5 mL) three times. Resulting residue was dried well under vacuum and subjected to next reaction as it is. The residue obtained above, respective acid moiety (0.125 mmol), EDC (0.19 mmol), HOBt (0.16 mmol) and DIPEA (0.5 mmol) were stirred in anhydrous DCM (5 mL) for 16 hrs. The reaction mixture was concentrated under vacuum to give the crude product which was purified on a CI 8 column using 10-50% MeOH in water to yield product as an off-white solid (35-55%).

Method B; General method Tor deprotection followed by reaction with anhydride.

HC1 Solution in dioxane (4 M, 5 mL) was added to a representative Boc-protected compound (0.125 mmol) and stirred for 2 hrs at RT. The reaction mixture was concentrated to dryness under vacuum and washed with MeOH (5 mL) three times. Resulting residue was dried well under vacuum and subjected to next reaction as it is. The residue obtained above, the respective anhydride moiety (0.125 mmol), and triethylamine (0.5 mmol) were added to anhydrous DCM (5 mL) and stirred for 16 hrs. The mixture was concentrated under vacuum to give the crude product which was purified on a CI 8 column using 10-50% MeOH in water to yield product as an off-white solid (40-60%).

Method C: General method of coupling reaction usin^ HATU/DIPEA.

The respective acid moiety (0.125 mmol), HATU (0.17 mmol), DIPEA (0.5 mmol) and respective amine moiety (0.125 mmol) were stirred in anhydrous DCM (5 ml) for 16 hrs. The reaction mixture was concentrated under vacuum to give the crude product which was purified on a CI 8 column using 10-50% MeOH in water (or similar ratio as needed) to yield product as an off-white solid (35-55%).

To the stirring solution of the ester compound (0.08 mmol) in ethanol (1 mL) was added solution of lithium hydroxide monohydrate (0.4 mmol) in water (0.5 mL). After stirring the reaction mixture for 5 hrs at RT, the mixture was acidified using citric acid (saturated solution) and concentrated under vacuum to give the crude product which was purified on a C I 8 column using 10-40% MeOH in water to yield product as an off-white solid (50-65%).

Method E: General method for Boc deprotection.

HC1 Solution in dioxane (4M, 0.5 mL) was added to the respective carbamate compound (0.06 mmol) and stirred for 3 hrs at RT. The reaction mixture was concentrated under vacuum to give the crude product which was purified on a C I 8 column using 10-40% MeOH in water to yield product as an off-white solid (50-60%).

VI el hod F: General method for incthanesu lfonyl (mcsyl ) chloride mediated amino acid coupling.

A solution of an appropriate acid (1 .0 mmol) in THF (10 mL) was cooled to

-40 °C. To this solution was added DIPEA (1.7 mmol) followed by CH₃S0₂C1 (1.3 mmol). The reaction mixture was allowed to stir for 20 min before addition of an appropriate amine (such as (S)-methyl indoline-2-carboxylate) (1.1 mmol). After stirring for further 10 min at the same temperature the ice bath was removed and reaction mixture was allowed to warm up to room temperature. The reaction mixture was diluted with EtOAc and then washed with saturated aqueous solution of NaHC0₃. The aqueous layer was then extracted with EtOAc (2 x 20 mL). Combined organic layer was washed with brine and dried over anhydrous Na₂S0₄, filtered and concentrated in vacuo. Flash chromatography (MeOH: DCM 2-5%) afforded final compound.

Method G: EDC/Oxyma amino acid coupling.

To a mixture of an appropriate acid (0.05 mmol) in DMSO (0.5 mL) DIPEA (0.23 mmol), an appropriate acid (0.075 mmol), EDC (0.1 mmol), and oxyma (0.1 mmol) were added at RT. After stirring for 16 hrs the reaction mixture was purified by preparative HPLC (column: Ascentis CI 8, 25 cm x 21.2 mm, 10 μπι, gradient 0%→100% methanol/water with 0.1% TFA, 10 mL/min or similar) to furnish 1 1 mg of powder.

Method H: General method of acid chloride synthesis followed bv amide formation.

Acid compound (1.5 mmol) and thionyl chloride (90 mmol) were stirred together for 1 hrs. at room temperature. Thionyl chloride was removed by distillation under vacuum. The acid chloride was added to the stirring solution of L-isoleucine (1 .35 mmol) in NaOH (2 N, 1.8 mL) at 0°C. The resulting reaction mixture was warmed to RT and stirred overnight. The reaction mixture was diluted with water (2 mL) and washed with diethyl ether (3 mL). Separated aqueous layer was acidified to pH 2 by adding mixture of

1 : 1 HCl-water. The precipitated solid was filtered and washed with water to get the product as a white to off-white solid (45-75%).

Method I: General ineihod lbr dcprotcction followed by reaction with anhydride ).

3) Acidify with formic acid

This method is an improved procedure for the method B. HCI Solution in dioxane (4 M, 5 mL) was added to a representative Boc-protected compound (0.125 mmol) and stirred for 2 hrs at T. The reaction mixture was concentrated to dryness under vacuum and swapped with MeOH (5 mL) three times. The resulting residue was dried well under vacuum and subjected to next reaction as it was. The residue obtained above, the respective anhydride moiety (0.19 mmol, 1.5 eq.), and triethylamine (0.5 mmol, 4 eq.) were added to anhydrous DCM (5 mL) and stirred for 16 hrs. The mixture was acidified with formic acid and then concentrated under vacuum to give the crude product which was purified on a C18 column using 25-65% MeOH in water to yield product as an off- white solid (30-80%).

Method J : General method (br deprotcction followed by reaction with a i-acid .

OH O

O OH

3) Acidify with formic acid

HCI Solution in dioxane (4 M, 5 mL) was added to a representative Boc-protected compound (0.125 mmol) and stirred for 2 hrs at RT. The reaction mixture was concentrated to dryness under vacuum and swapped with MeOH (5 mL) three times. Resulting residue was dried well under vacuum and subjected to next reaction as it was. The residue obtained above, the respective di-acid moiety (0.62 mmol, 5 eq.), EDC (0.19 mmol), HOBt (0.15 mmol) and DIPEA (1.6 mmol, 13 eq.) were added to anhydrous DCM (5 mL) and stirred for 4 hrs. The mixture was acidified with formic acid and then concentrated under vacuum to give the crude product which was purified on a CI 8 column using 25-65% MeOH in water to yield product as an off-white solid (30-80%).

Method K: General method to Boc protection .

H₂NL

To respective amine compound (6.1 mmol) in dioxane (6 mL) and NaOH solution (0.5 M, 12 mL) was added slowly solution of di-tert-butyl dicarbonate (6.7 mmol) in dioxane (6 mL) at 0°C. The reaction mixture was warmed to RT and stirred overnight. The reaction mixture was then washed with hexanes (10 mL). The separated water layer was acidified using saturated solution of citric acid and extracted with ethyl acetate (3 x 15 mL). The organic layer was washed with brine, separated, dried over sodium sulfate and concentrated to give Boc protected amine compound as off-white solid (65-90%).

Method ,: General method of hydrolysis fol lowed by coupl ing!, reaction usint; EDC

HOBt / DIPEA.

O LiOH H₂0 , EDC . HOBt. DIPEA 0 R-^O PG

ErOH ί OH D F -^ N

n

To the stirring solution of the ester compound (0.1 1 mmol) in ethanol (4 mL) was added solution of lithium hydroxide monohydrate (0.23 mmol) in water (2 mL). After stirring the reaction mixture for 5 hrs at RT, the mixture was acidified using sat. citric acid solution and concentrated under vacuum to remove ethanol. The aqueous residue obtained was extracted with EtOAc (2 X 15 mL). Combined organic layer was dried over anhydrous ₂S04, filtered and concentrated in vacuo. Resulting residue was dried well under vacuum and subjected to next reaction as it was. The residue obtained above, the respective amine moiety (0.1 1 mmol), EDC (0.17 mmol), HOBT (0.15 mmol) and DIPEA (0.46 mmol) were stirred in DMF (4 mL) for 16 hrs. The mixture was concentrated under vacuum to give the crude product which was purified on a CI 8 column using 10-50% MeOH in water to yield product as an off-white solid (50-60%).

Method M: General method for EDC/1 lOBt/DIPEA coupling of an intermediate existing as an HC1 salt and a free carboxylic acid.

NH₂ 'HCI

R'

To an intermediate collected as an HC1 salt (0.125 mmol) were added the carboxylic acid (0.125 mmol), EDC (0.19 mmol), HOBt (0.16 mmol), and anhydrous DCM (5 mL). The flask was purged with N₂, sonicated for 20 s and DIPEA (0.5 mmol) was added. The reaction was stirred at room temperature for 6 hrs then concentrated under reduced pressure. The residue was purified on a CI 8 column using 10-80% MeOH in water to yield the product as an off-white solid (40-90%).

Method N : General method for anhydride ri tm openi ng by an inlennedi ate existi ng as an HC1 salt.

To an intermediate collected as an HC1 salt (0.11 mmol) were added the respective anhydride (0.13 mmol), and anhydrous DCM (6 mL). The flask was purged with N₂, sonicated for 20 s and triethylamine (0.44 mmol) was added. The reaction was stirred at room temperature for 5.5 hrs. The reaction was acidified to approximately pH 5 using citric acid (aqueous, saturated solution), and then concentrated under reduced pressure. The residue was purified on a CI 8 column using 10-75% MeOH in water to yield the product as an off-white solid (40-67%). Method 0: General method lor coupling (21 l-Teirazol-5 -yl }nicthylaminc and a free carboxvlic acid.

To the carboxylic acid (0.18 mmol), were added the (2H-tetrazol-5- yl)methylamine (0.22 mmol), EDC (0.275 mmol), HOBt (0.22 mmol), and anhydrous DMF (15 mL). The flask was purged with N₂, sonicated for 20 s and DIPEA (0.73 mmol) was added. The reaction was stirred at room temperature for 16 hrs. Analysis of the reaction by LC/MS showed approximately 75% conversion of the acid. An additional one half of the portion of the amine, EDC, HOBt, and DIPEA were added and the reaction was heated at 45°C for another 6 hrs then concentrated under reduced pressure. The residue was purified on a CI 8 column using 10-70% MeOH in water to yield the product as an off-white solid (40-95%).

Method P: General method for (a) preparation of unsymmetric anhydride ring opening products with proximal substitution {via anhydride ring openim; with MeOH ) followed by ( b ) coupling to an amine (ex. 1- 15 ) and c) subsequent hydrolysis.

R-NH₂ (ex. 1-15)

EDC, HOBt, DIPEA

DMF

To the respective anhydride (2.8 mmol) were added methanol (5.6 mmol), anhydrous DCM (28 mL) and triethylamine (1 1 mmol). The reaction was stirred at RT for 4 hrs then diluted with diethyl ether (30 mL) and HCl (1M, aqueous, 30 mL) and transferred to a separatory funnel. The organic layer was collected, washed with a NaCl solution (saturated, aqueous) and then dried over anhydrous Na₂SC>4. After filtration and concentration under reduced pressure, the crude ring-opened product was collected as a colourless oil and as a mixture of isomers. The ring-opened product mixture was used in the subsequent step without further purification. To the crude ring-opened product (0.1 1 mmol) were added the amine (ex. 1-15) (0.1 1 mmol) EDC (0.17 mmol), HOBt (0.13 mmol) and anhydrous DCM (6 mL). The flask was purged with N₂, sonicated for 20 s and triethylamine (0.44 mmol) was added. The reaction was stirred at RT for 3 hrs. The reaction mixture was concentrated under reduced pressure and the residue was purified on a CI 8 column using 10-70% MeOH in water to yield the methyl ester product as an off-white solid and as a mixture of isomers (49-65%). To the mixture of isomers (0.12 mmol) were added LiOH H₂0 (0.23 mmol), methanol (3 mL), and H₂0 (4.5 mL). The reaction was stirred at room temperature in air for 10 hrs. The reaction was acidified to approximately pH 5 using citric acid (aqueous, saturated), and then concentrated under reduced pressure. The residue was purified on a CI 8 column using 10-75%) MeOH in water to yield the product as an off-white solid ( 17-54%).

The following examples are provided for the purpose of illustrating, not limiting, the invention.

EXAMPLES

Synthetic Intermediates

The following is a description of synthetic intermediates (1-1 to 1-15) useful for making representative compounds of the invention.

Intermediate I- 1

1-1

tert- uty\ (25)-2-[(2H-l ,2,3,4-tetrazol-5-ylmethyl)carbamoyl]-2,3-dihydro-lH- indole-l-carboxylate (1-1):

To (2iS)-2,3-dihydro-lH-indole-2-carboxylic acid (1 g, 6.13 mmol) in dioxane

(6 mL) and NaOH solution (0.5 M, 12 mL) was added slowly di- ri-butyl dicarbonate (1.47 g, 6.74 mmol) in dioxane (6 mL) at 0°C. The reaction mixture was warmed to RT and stirred overnight. The reaction mixture was then washed with hexanes (10 mL). The separated water layer was acidified using saturated solution of citric acid and extracted with ethyl acetate (3 x 15 mL). The organic layer was washed with brine, separated, dried over sodium sulfate and concentrated to give (25)-l -[(iert-butoxy)carbonyl]-2,3-dihydro-lH-indole-2-carboxylic acid, as light orange colored solid (1.45 g, 90%), used further as described. 1H NMR (400 MHz, DMSO-< 6) δ 1.41 (9H, s), 2.99-3.04 (IH, d, J=20 Hz), 3.48-3.55 (IH, dd, J=12, 16 Hz), 4.74-4.78 (IH, dd, J=4, 12 Hz), 6.91 -6.95 (IH, t, J=8 Hz), 7.14-7.18 (2H, m), 7.72-7.74 (IH, d, J=8 Hz), 12.85 (I H, bs), MS (LC/MS) m/z observed 285.89, expected 286.1 1 [M+Na].

(25)-l -[(/'iir/-Butoxy)carbonyl]-2,3-dihydro-lH-indole-2-carboxylic acid (3 g, 1 1.40 mmol), 2H-l,2,3,4-tetrazol-5-ylmethanamine (1.16 g, 11.40 mmol), EDC (2.4 g, 12.53 mmol), HOBt (1.54 g, 1 1.40 mmol) and DIPEA (15.9 mL, 91.15 mmol) were stirred in anhydrous DMF (60 mL) for 16 hrs. The reaction mixture was concentrated under vacuum and re-dissolved in ethyl acetate (180 mL) and washed with citric acid (aqueous, saturated solution), water and brine. The organic layer was separated, dried over sodium sulfate and concentrated to give the crude product as an orange-red oil. The crude compound recrystallized in diethyl ether (80 mL) to yield feri-butyl (25)-2-[(2H-l,2,3,4-tetrazol-5-ylmethyl)carbamoyl]-2,3-dihydro-lH-indole- 1-carboxylate (1-1) as an off-white solid (2.8 g, 71%). 1H NMR (400 MHz, DMSO- d6) δ 1.32 (9H, s), 2.94-2.99 (IH, dd, J=4, 15 Hz), 3.41-3.48 (IH, dd, J=12, 20 Hz), 4.50-4.64 (2H, dq, J=4, 20 Hz), 4.73-4.77 (IH, dd, J=4, 12 Hz), 6.89-6.83 (IH, t, J=8 Hz ), 7.12-7.16 (2H, t, J=8 Hz), 7.71 (IH, bs), 7.86 (IH, bs), MS (LC/MS) m/z observed 344.73, expected 345.17 [M+H].

Intermediate 1-2

(25)-yV-(2H-l,2,3,4-Tetrazol-5-ylmethyl)-2,3-dihydro-lH-indole-2-carboxamide hydrochloride (1-2):

HC1 Solution in dioxane (4 M, 28 mL) was added to 1-1 (1.4 g, 4.07 mmol) and stirred for 2 hrs at RT. Precipitated solid was filtered under nitrogen and washed with diethyl ether (2 X 15 mL) to yield (2S N-(2H-l ,2,3,4-tetrazol-5-ylmethyl)-2,3- dihydro-lH-indole-2-carboxamide hydrochloride(I-2) as a light pink colored solid (1.1 g, 96%), 1H NMR (400 MHz, DMSO-tf6) δ 2.93-2.99 (1H, dd, J=8, 16 Hz), 3.26-3.33 (1 H, dd, .7=12, 16 Hz), 4.24-4.29 (1H, t, J=8 Hz), 5.56-5.58 (2H, dd, J=1.6, 8 Hz), 5.89 (1H, bs), 6.55-6.59 (2H, t, J=8 Hz), 6.91 -6.95 (1H, t, J=8 Hz), 6.99-7.01

(1H, d, J=8 Hz), 8.59-8.62 (1H, t, J=8 Hz), MS (LC/MS) m/z observed 244.97, expected 245.12[M+H].

Intermediate 1-3

tert-Butyl 7V-{2-oxo-2-[(25)-2-[(2H-l,2,3,4-tetrazol-5-ylmethyl)carbamoyl]-2,3- dihydro-lH-indol-l-yl]ethyl}carbamate (1-3):

1-2 (0.5 g, 1.78 mmol), N-Boc-glycine (0.37 g, 2.14 mmol), EDC (2.4 g, 12.5 mmol), HOBt (0.75 g, 3.92 mmol) and DIPEA (1.24 mL, 7.125 mmol) were stirred in anhydrous DCM (20 mL) for 16 hrs. The reaction mixture was diluted using DCM (15 mL) and washed with citric acid (aqueous, saturated solution), followed by water and then brine. The organic layer was separated, dried over sodium sulfate and concentrated to give the crude product as a yellow-orange colored oil. The crude compound was purified on a CI 8 column using 10-50% MeOH in water to yield tert-butyl N-{2-oxo-2-[(2S)-2-[(2H-l,2,3,4-tetrazol-5- ylmethyl)carbamoyl]-2,3-dihydro-lH-indol-l -yl]ethyl}carbamate (1-3) as an off- white solid (0.4 g, 56%), 1H NMR (400 MHz, DMSO-<½) 8 1.39 (9H, s), 3.08-3.12 (2H, d, J=16 Hz), 3.56-3.63(lH, dd, J=12, 20 Hz), 3.92-3.97 (1H, dd, J=4, 16 Hz), 4.56-4.62 (2H, m), 5.09-5.12 (IH, d, 12 Hz), 6.96-7.00 (2H, t, J=8 Hz), 7.13-7.17 (IH, t, J=8 Hz), 7.19-7.21 (IH, d, J=8 Hz), 8.00-8.02 (IH, d, J=8 Hz), 9.06 (IH, s), MS (LC MS) m/z observed 401.82, expected 402.19 [M+H], and observed 424.04, expected 424.17 [M+Na].

Intermediate 1-4

(25 -l-(2-Aminoacetyl)-/V-(2H-l,2,3,4-tetrazol-5-ylmethyl)-2,3-dihydro-lH- indole-2-carboxamide hydrochIoride(I-4):

HCI Solution in dioxane (4 M, 5 mL) was added to 1-3 (0.24 g, 0.6 mmol) and stirred for 2 hrs at RT. The precipitated solid was filtered under nitrogen and washed with diethyl ether (2 X 5 mL) and dried to yield (2S)-l -(2-aminoacetyl)-N-(2H- l,2,3,4-tetrazol-5-ylmethyl)-2,3-dihydro- lH-indole-2-carboxamide hydrochloride (I- 4) as an off-white solid (0.1 1 g, 54%), 1H NMR (400 MHz, DMSO-< 6) δ 2.20-2.24 (2H, d, J=16 Hz), 3.46-3.50(lH, d, J=16 Hz), 3.56-3.65 (IH, m), 4.05-4.09 (IH, d, J=16 Hz), 5.09-5.12 (IH, d, 12 Hz), 4.60-4.61 (2H, d, J=4 Hz), 5.19-5.22 (IK d, J=12 Hz), 7.04-7.08 (IH, t, J=8 Hz), 7.19-7.23 (IH, t, J=8 Hz), 7.25-7.27 (IH, d, J=8 Hz), 8.03-8.05 (I H, d, J=8 Hz), 8.30 (2H, bs), 9.30 (2H, s), MS (LC/MS) m/z observed 301 .98, expected 302.14 [M+H].

Intermediate 1-5

fe^-Butyl V-[(15',2₁y)-2-methyl-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-tetrazol-5- ylmethyl)carbamoylJ-2,3-dihydro-lH-indol-l- yl]ethyl}carbamoyl)butyl]carbamate (1-5): 1-4 (0.15 g, 0.44 mmol), N-Boc-Isoleucine (0.10 g, 0.44 mmol), EDC (0.13 g, 0.67 mmol), HOBt (0.078 g, 0.58 mmol) and DIPEA (0.31 mL, 1.78 mmol) were stirred in anhydrous DCM (15 mL) for 16 hrs. The reaction mixture was diluted using DCM (15 mL) and washed consecutively with citric acid (aqueous, saturated solution), water and brine. The organic layer was separated, dried over sodium sulfate and concentrated to give the crude product as a yellow-orange colored oil. The crude compound was purified on a C18 column using 10-50% MeOH in water to yield tert-butyl N-[(15,25)-2-methyl-l-({2-oxo-2-[(25 -2-[(2H-l ,2,3,4-tetrazol-5- ylmethyl)carbamoyl]-2,3-dihydro-lH-indol- l-yl]ethyl}carbamoyl)butyl]carbamate (1-5) as an off-white solid (0.14 g, 60%). Ή NMR (400 MHz, DMSO-</6) δ 0.80- 0.84 (3H, t, J=8 Hz), 0.85-0.87 (3H, d, J=8 Hz), 1.39 (9Η, s), 1.40-1.46 (IH, m), 1.69- 1.75 (IH, m), 3.02-3.12 (2H, m), 3.52-3.66 (3H, m), 3.89-3.93 (I H, t, J=8 Hz), 4.12-4.16 (IH, d, J=16 Hz), 4.33-4.38 (IH, dd, J=4, 16 Hz), 4.50-4.55 ( I H, dd, J=4, 16 Hz), 5.13-5.16 (IH, d, J=12 Hz), 6.73-6.75 (IH, d, J=8 Hz), 6.98-7.02 (I H, t, J=8 Hz), 7.15-7.22 (2H, m), 8.04-8.06 (IH, d, J=8 Hz), 8.10 )1H, s), 8.73 (IH, s), MS (LC/MS) m/z observed 514.86, expected 515.27[M+H].

Intermediate 1-6

1-6

2-Phenylacetic acid (1-6):

A solution of methyl 2-phenylacetate (10 g, 64 mmol) in methanol (60 mL) was treated with solution of sodium hydroxide (5.1 g, 127 mmol) in water (40 mL) at 70°C for 3 hrs. The resulting mixture was concentrated under vacuum to remove the methanol. The residue was diluted with water (40 mL) and washed with diethyl ether (40 mL). The separated water layer was acidified to pH 2 using a mixture of water and HC1 (1 : 1) and extracted with DCM (3 X 80 mL). Combined organic extracts were washed with brine, 80 mL, separated, dried over sodium sulfate and concentrated to give 2-phenylacetic acid (1-6) as a white solid (9 g, 96%) used without further characterization. Ή NMR (400 MHz, CDC1₃) δ 3.64 (2H, s), 7.27- 7.35 (5H, m), 1 1.5 (lH, bs). Intermediate 1-7

(25',35)-3-Methyl-2-(2-phenylacetamido)pentanoic acid (1-7):

1-6 (2.0 g, 14.7 mmol) and thionyl chloride (6.6 mL, 90.3 mmol) were stirred together for 1 hr at RT. Thionyl chloride was removed by distillation under vacuum. The acid chloride was added to the stirring solution of L-isoleucine (1 .75 g, 13.37 mmol) in NaOH (2 N, 17 mL) at 0°C. The resulting mixture was warmed to RT and stirred overnight. The mixture was washed with diethyl ether (20 mL) and acidified to pH 4-5 by adding citric acid (aqueous, saturated solution). The precipitated solid was filtered, washed with diethyl ether and dried to yield (2S S)-3- methyl-2-(2-phenylacetamido)pentanoic acid (1-7) as a white solid (1.6 g, 44%). Ή NMR (400 MHz, DMSO-rf6) δ 0.78-0.82 (6H, t, J=8 Hz), 1.12-1.18 (IH, m), 1.34- 1.40 (I H, m), 1.72-1 .78 (IH, m), 3.41 -3.53 (2H, q, J=16 Hz), 4.13-4.16 ( I H, dd, J=4,12 Hz), 7.15-7.19 (IH, m), 7.22-7.28 (IH, m), 8.19-8.21(1 H, d, J=8 Hz), 12.54 (IH, s), MS (LC/MS) m/z observed 250.02, expected 250.14 [M+H].

2- [(25,3»S)-3-Methyl-2-(2-phen lacetamido)pentanamido] acetic acid (1-8) :

To a suspension of 1-7 (24 g, 96.3 mmol) in DCM (400 mL) was added DIPEA (87 mL, 500 mmol) at 0°C. The reaction mixture was allowed to stir for

10 min, upon which a homogeneous mixture was obtained. To this was added glycine ethyl ester hydrochloride (15.1 g, 108 mmol). After stirring a further 5 min at the same temperature, EDC (24.5 g, 1 16 mmol) and oxyma (18.8 g 1 16 mmol) were added. The reaction mixture was then allowed to stir overnight (approximately 15 hrs) while allowed to slowly warm to RT. Another batch of glycine ethyl ester hydrochloride (2.1 g) was added and the reaction mixture was allowed to stir a further 4 hrs at ambient temperature. Reaction mixture was transferred to a separatory funnel and washed with NaHC0₃ (300 mL, aqueous, saturated), followed by HCl (3M, 50 mL), and brine (200 mL). The organic layer was then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Ethyl 2-[(25,35)-3-methyl-2-(2- phenylacetamido)pentanamido]acetate was obtained from flash column chromatography (MeOH: DCM 1 -5%) followed by recrystallization from acetonitrile ( 1 6 g, colourless crystals) and used further as described. Ή NMR (300 MHz, CDjOD) δ 7.32-7.21 (m, 5H), 4.34 (m, 1H), 4.17 (q, 2H), 4.03-3.96 (m, 1H), 3.89- 3.81 (m, lH), 3.68-3.52 (m, 2H), 1.98-1.89 (m, 1 H), 1.62-1.35 (m, 1H), 1 .26 (t, 3H), 1.21 -1.09 (m, 1H), 0.96-0.86 (m, 6H), MS (ESI) m/z observed 357.29, expected 357.18 [M+Na].

Intermediate 1-8 was obtained from hydrolysis of ethyl 2-[(25',3S)-3-methyl-2- (2-phenylacetamido)pentanamido]acetate (8.1 g, 24 mmol) using Method D (6 g, white powder). 1H NMR (300 MHz, CD₃OD) δ 12.40 (br s, 1H), 7.31-7.16 (m, 5H), 4.23-4.18 (m, 1H), 3.76-3.70 (m, 2H), 3.57-3.40 (m, 2H), 1.83-1.68 (m, 1H), 1.48- 1.35 (m, 1 H), 1.13-0.99 (m, 1H), 0.83-0.75 (m, 6H), MS (ESI) m/z observed 305.20, expected 305.15 [M-H],

Intermediate 1-9

1-9 (25)-l-{2-[(25,35)-3-Methyl-2-(2-phenylacetamido)pentanamido]acetyl}-2,3- dihydro-lH-indole-2-carboxylic acid (1-9):

Intermediate 1-9 was obtained from 1-8 (170 mg, 0.37 mmol) using Method F followed by Method D (120 mg, white powder) and used further as is. Ή NMR (300 MHz, DMSO-rftf) δ 8.37-8.18 (m, IH), 8.06 (d, IH), 7.28-7.08 (m, 7H), 6.95- 6.90 (m, IH), 4.57-4.53 (m, IH), 4.44-4.24 (m, IH), 4.13-3.89 (m, 2H), 3.59-3.18 (m, 4H), 1.92-1.71 (m, IH), 1.47-1.22 (m, I H), 1 .13-0.99 (m, IH), 0.84-0.75 (m, 6H).

Intermediate 1-10

1-10

Ethyl (25)-l-(2-{[(^r/-butoxy)carbonyl]ainino}aceryl)-2,3-dihydro-lH-indole-2- carboxylate (1-10)

(25)-2,3-dihydro-lH-indole-2-carboxylic acid (500 mg, 306 mmol) was suspended in EtOH (5 mL) at 0°C and thionyl chloride (0.45 mL, 6.13 mmol, 2 eq.) was added. The resulting clear mixture was allowed to come to RT and stirred for 16 hrs. The reaction mixture was then concentrated to dryness and swapped with EtOH (2 x 10 mL). The solid obtained was dried well under reduced pressure to give ethyl (2S -2,3-dihydro-lH-indole-2-carboxylate hydrochloride as a light brown solid (0.58 g, quantitative). 1H NMR (400 MHz, DMSO-rf6) δ 1.18 (3H, s), 3.10-3.18 (IH, m), 3.30-3.40 (IH, m), 4.05-4.17 (2H, m), 4.55 (I H, bs), 6.80 (2H, bs), 7.02-7.08 (2H, m), 7.7 (2H, bs). Compound was used further as described.

1-10 was prepared from ethyl (25)-2,3-dihydro-lH-indole-2-carboxylate hydrochloride and 2-((teri-butoxycarbonyi)amino)acetic acid using method C however the purification was performed on normal phase using 0% to 50% ethyl acetate in hexanes as the eluent. Ή NMR (400 MHz, DMSO-rfd) 81.18 (3H, t, J=6Hz), 1.38 (9H, s), 3.19 (IH, d, J=16Hz), 3.48-3.62 (2H, m), 3.95-4.20 (3H, m), 5.35 (I H, d, J=l l Hz), 7.00 (2H, t, J=8Hz), 7.15-7.25 (2H, m), 8.01 (IH, d, J=8Hz), MS (LC/MS) m/z 370.95 [M+Na]. Intermediate I-ll

Ethyl (2^-6-amino-2,3-dihydro-lH-indole-2-carboxylate (I-ll)

(25)-2,3-Dihydro-lH-indole-2-carboxylic acid (5 g, 30.640 mmol) was dissolved in sulfuric acid (concentrated, 40 mL) at 0°C. The resulting reaction mixture was chilled to -20°C and nitric acid (concentrated, 1.4 mL, 33.1 mmol) was then added dropwise. The reaction mixture was stirred at -20°C for 1 hr and then allowed to warm to RT and stirred for 2 hrs. The reaction mixture was diluted with cold water (100 mL), neutralized to pH 4 using NaOH solution (10 N) and extracted with EtOAc (3 X 200 mL). The combined organic layers were dried over sodium sulphate and concentrated to give an orange solid. This solid was dissolved in ethanol (200 mL) and treated with thionyl chloride (4.45 mL, 61.3 mmol) at 0°C and stirred overnight at RT. The reaction mixture was concentrated and purified by column chromatography on silica gel using 0% to 50% ethyl acetate in hexanes as the eluent to give ethyl (2S -6-nitro-2,3-dihydro- lH-indole-2-carboxylate, as a yellow solid (1.61 g, 22%). 1H NMR (400 MHz, CDC1₃) δ 1.30- 1.34 (3H, t, J=8 Hz), 3.30-3.57 (2H, m), 4.21-4.27 (2H, q, .7=8 Hz), 4.47-4.52 (1H, q, J=8 Hz), 4.70 (1H, bs), 7.16- 7.18 (1H, d, J=8 Hz), 7.47-7.48 (1H, d, J=4 Hz), 7.62-7.65 (1H, dd, J=8, 4 Hz), MS

(LC/MS) m/z observed 237.00, expected 237.08 [M+H].

Ethyl (25)-6-mtro-2,3-dihydro-lH-indole-2-carboxylate (2 g, 8.474 mmol) and 10% Pd on activated carbon (0.4 g, 20% wt./wt.) in ethanol (60 mL) was stirred under hydrogen (balloon) for 3 hrs. The reaction mixture was filtered through a bed of CELITE^® and washed with ethanol (4 X 30 mL). Collected filtrate was concentrated and dried well under vacuum to yield I-ll as pale yellow oil (1.4 g, 80%). 1H NMR (400 MHz, CDC1₃) δ 1.14-1.18 (3H, t, J=8 Hz), 2.94-3.01 (3H, m), 3.30 (1H, s), 4.57-4.61 (1H, t, J=8 Hz), 7.45-7.48 (1H, q, J=4 Hz), 8.03 (1H, s), 8.06- 8.09 (IH, m), 8.41-8.42 (IH, d, J=4 Hz), 8.96-8.97 (IH, d, J=4 Hz), MS (LC/MS) m/z observed 207.09, expected 207.1 1 [M+H].

Intermediate 1-12

(5)-Ethyl 6-((fc^-butoxycarbonyl)amino)-l-(2-((2,S',35)-3-methyl-2-(2- phenylacetamido)pentanamido)ace y])indoline-2-carboxylate (1-12)

1-11 (0.6 g, 2.91 mmol) and isoindoline-l ,3-dione (0.43 g, 2.91 mmol) in toluene was heated at 90°C for 4 hrs. Resulting reaction mixture was concentrated and purified by column chromatography on silica gel using 0% to 30% ethyl acetate in hexanes as the eluent to give (iS)-6-(l ,3-dioxo-l,3-dihydro-isoindolin-2-yl)-2,3- dihydro-lH-indole-2-carboxylic acid ethyl ester, as an off-white solid (0.7 g, 72%). Ή NMR (400 MHz, CDC1₃) δ 1.29-1.33 (3H, t, J=8 Hz), 3.38-3.44 (2H, m), 4.20- 4.26 (2H, m), 4.42-4.46 (IH, dd, J=12,8 Hz), 4.56 (I H, bs), 6.72 ( I H, s), 6.75-6.78

(IH, dd, J=12,4 Hz), 7.17-7.19 (I H, d, J=12 Hz), 7.76-7.78 (2H, m), 7.92-7.95 (2H, m), MS (LC/MS) m/z observed 337.07, expected 337.1 1 [M+H]. (5)-l -(2-/eri-Butoxycarbonylamino-acetyl)-6-(l ,3-dioxo-l ,3-dihydro-isoindol- 2-yl)-2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester was prepared from (S -6- (l ,3-dioxo-l ,3-dihydro-isoindolin-2-yl)-2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester and Boc-glycine using method C and purified by column chromatography on silica gel using 0% to 50% ethyl acetate in hexanes as the eluent to give (S)-l-(2-

/eri-Butoxycarbonylamino-acetyl)-6-(l ,3-dioxo-l ,3-dihydro-isoindol-2-yl)-2,3- dihydro- lH-indole-2-carboxylic acid ethyl ester, as an off-white solid (42%). MS (LC/MS) m/z observed 493.87, expected 494.19[M+H], Compound was confirmed using LC/MS and moved to next step as it was.

(S)- 1 -(2-fer Butoxycarbonylamino-acetyl)-6-( 1 ,3-dioxo-l ,3-dihydro-isoindol-

2-yl)-2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester and 1-7 were combined using method A to produce (5)-6-(l,3-dioxo-l ,3-dihydro-isoindol-2-yl)-l -[2-(3- methyl-2-phenylacetylamino-pentanoylamino)-acetyl]-2,3-dihydro- lH-indole-2- carboxylic acid ethyl ester. MS (LC/MS) m/z observed 625.09, expected 625.26 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

(₁S)-6-(l ,3-Dioxo-l ,3-dihydro-isoindol-2-yl)-l -[2-(3-methyl-2- phenylacetylamino-pentanoylamino)-acetyl]-2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester (0.3 g, 0.48 mmol) was dissolved in 1 :4 mixture of DMF:dioxane (10 mL) and hydrazine monohydrate (0.026 mL, 0.53 mmol) was added. The reaction mixture was heated at 50°C for 2 hrs, concentrated and dried well under vacuum to give a yellow solid. This solid was dissolved in 1 :5 mixture of water:dioxane (12 mL, v/v) and treated with DIPEA (0.67 mL, 3.85 mmol) and di-ier/-butyl dicarbonate (0.42 g, 1.92 mmol) and stirred overnight at RT. The reaction mixture was concentrated and purified by column chromatography on silica gel using 0% to 50% ethyl acetate in hexanes as the eluent to give 1-12, as a pale yellow solid (0.16 g, 56%). MS

(LC/MS) m/z observed 595.09, expected 595.31 [Μ+Η] used further as needed. Intermediates 1-13 and 1-14

(»S)-Ethyl 5-nitroindoline-2-carboxylate (1-12) and (5)-ethyl 7-nitroindoline-2- carboxylate (1-13)

(5 -2,3-Dihydro- lH-indole-2-carboxylic acid (20 g, 0.123 mol) was suspended in absolute ethanol (200 mL) and the suspension was cooled to 0°C. SOCl₂ (18 mL, 0.25 mol) was then added dropwise to the reaction mixture and it was allowed to warm to RT and was stirred for 16 hrs. The solvent was then evaporated and the residue was swapped twice with ethanol (100 mL) to get a brown solid. This solid was dissolved triethylamine (34.2 mL, 0.245 mol) and acetic anhydride (170 mL) and the mixture was stirred at RT for 2 hrs. Then it was diluted with EtOAc (500 mL) and washed with NaHC0₃ (2 x 200 mL, aqueous, saturated solution) and citric acid (2 x 200 mL, aqueous, saturated solution). The combined organic layers were dried over sodium sulphate and concentrated to give a brown solid. This solid was dissolved in acetic anhydride (200 mL) and a solution of nitric acid (11.1 mL) in acetic anhydride (161 mL) was added dropwise at 0°C. The reaction mixture was left at 0°C for 15 min then warm to RT and stirred for 3 hrs. Cold water (250 mL) was added to the reaction mixture and the product was extracted with EtOAC (3 x 450 mL). The combined organic layers were dried over sodium sulphate and concentrated. The two isomers obtained were separated by normal phase column chromatography using 10 % to 30% ethyl acetate in hexanes as the eluent to give first, (5^)-l -acetyl-5-nitro-2,3-dihydro-l H-indole-2-carboxylic acid ethyl ester as a yellow solid (18.49 g, 54%) and second, (5)-l -acetyl-7-nitro-2,3-dihydro- lH-indole-2- carboxylic acid ethyl ester as an orange oil (8.21 g, 24%).

(¾-l -Acetyl-5-nitro-2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester: 'Η NMR (400 MHz, CDC1₃) δ 1.30 (3H, t, J=7.0Hz), 2.22 (3H, bs), 3.34 (1H, m), 3.65 (1H, m), 4.19-4.34 (2H, m), 5.02 (1H, bs), 8.03 (1H, s), 8.17 (1H, dd, J=2, 9Hz), 8.34 (1H, bs), MS (LC/MS) m/z observed 279.01 , expected 279.10 [M+H].

(5)- l -Acetyl-7-nitro-2,3-dihydro- lH-indole-2-carboxylic acid ethyl ester: Ή

NMR (400 MHz, CDC1₃) δ 1.28 (3H, t, J=7.0Hz), 2.28 (3H, bs), 3.41 ( 1 H, d, J=16Hz), 3.62 (1H, dd, J=10, 17Hz), 4.19-4.30 (2H, m), 5.05 (1H, dd, J=2, 10Hz), 7.16 (1 H, t, J=8Hz), 7.42 (1H, dd, J=2, 9Hz), 7.67 (1H, d, J=8Hz), MS (LC/MS) m/z observed 279.01 , expected 279.10 [M+H].

1-13: (¾-l -Acetyl-5-nitro-2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester

(18.49 g) was refluxed in a aqueous HCl solution (1 N, 500 mL) for 2 hrs until the compound dissolved completely. Then, the reaction mixture was concentrated to dryness and the residue was dissolved in ethanol (500 mL). SOCl₂ (60 mL) was then added dropwise and the reaction was left at RT for 16 hrs. The solvent was then evaporated and the product was purified by normal phase column chromatography using 5% to 20% ethyl acetate in hexanes as the eluent to give intermediate 1-13 as a yellow solid (10.81 g, 69%). 1H NMR (400 MHz, CDC1₃) δ 1.32 (3H, t, =7.0Hz), 3.35-3.52 (2H, m), 4.22-4.30 (2H, q, J=7Hz), 4.57 (1H, dd, J=5, 10Hz), 5.01 (1 H, bs,), 6.63 (1H, d, J- 9Hz), 7.98 (1H, bs), 8.05 (1H, dd, J=2, 9Hz), MS (LC/MS) m z observed 237.03, expected 237.09 [M+H].

1-14: (5)-l-Acetyl-7-nitro-2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester (1.6 g) was refluxed in a HCl solution (1 N, aqueous, 80 mL) for 2 hrs until the compound dissolved completely. Then, the reaction mixture was concentrated to dryness and the residue was dissolved in ethanol (60 mL). SOCl₂ (10 mL) was then added dropwise and the reaction was left at RT for 16 hrs. The solvent was then evaporated and the product was purified by normal phase column chromatography using 5% to 20% ethyl acetate in hexanes as the eluent to give intermediate 1-14 as a yellow solid (0.97 g, 71 %). 1H NMR (400 MHz, CDC1₃) δ 1.31 (3H, t, J=7.0Hz), 3.37-3.53 (2H, m), 4.19-4.30 (2H, q, J=7Hz), 4.69 ( H, dd, J=4, 10Hz), 6.66 (I H, dd, J=7, 8Hz), 7.03 (IH, bs), 7.24 (IH, d, J= 7Hz), 7.83 (IH, d, J=8Hz), MS (LC/MS) m/z observed 237.03, expected 237.09 [M+H].

Intermediate 1-15

(l^Z^- -Methyl-l-iZ-oxo-l-ii ^- -K H-l^^^-tetrazol-S- lmethyl)- carbamoyl]-2,3-dihydro-indol-l-yl}-ethylcarbamoyl)-butyl-ammoniuin; chloride (1-15)

1-15 was collected as a faint pink solid from deprotection of 1-5 using method E. MS (LC/MS) m/z observed 415.09, expected 415.22 [M-Cl]. The compound was confirmed using LC/MS and moved to next step as it was.

Representative Granzyme B Inhibitor Compounds

The following is a description of representative Granzyme B inhibitor compounds.

Examples A1 -A57 were prepared by the representative synthetic pathway illustrated schematically in FIGURE 1.

EXAMPLE A 1

(25")-1-{2-[(25,35)-3-METHYL-2-(2-PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-7V- (2H-L,2,3,4-TETRAZOL-5-YLMETHYL)-2,3-DIHYDRO-LH-INDOLE-2-CARBOXALVIIDE

Title compound Al was prepared from 1-5 and 1-6 using method A: Ή NMR (400 MHz, DMSO-i 6) δ 0.76-0.80 (3H, t, J=8 Hz), 0.82-0.84 (3H, d, .7=8 Hz), 1.04-1.1 1 (IH, m), 1.39-1.45 (I H, m), 1.70- 1.76 (IH, m), 3.10-3.14 (2H, d, J=16 Hz), 3.42-3.45 (I H, d, j=8 Hz), 3.53-3.56 (IH, d, J=8 Hz), 3.62-3.65 (IH, m), 3.66-3.69 (I H, m), 4.14- 4.16 (IH, t, J=4 Hz), 4.24-4.28 (I H, t, J=8 Hz), 4.47-4.51 (I H, d, J=16 Hz), 4.59-4.63 (IH, d, J=16 Hz), 5.14-5.16 (I H, d, J=16 Hz), 6.97-7.01 (IH, t, J=8 Hz), 7.15-7.30 (7H, m), 8.08-8.1 1 (IH, d, J=12 Hz), 8.24(1H, bs), 8.99(1 H, bs), MS (LC/MS) m/z observed 533.05, expected 533.26 [M+H] and observed 555.17, expected 555.24 [M+Na].

EXAMPLE A2

(2S)-1-{2-[(2S,3£)-3-METHYL-2-[2-(PYRIDIN-4- YL)ACETAMIDOjPENTANAMIDO]ACETYL}-7V-(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)-2,3-

DIHYDRO-1H-INDOLE-2-CARBOXAMIDE

Title compound A2 was prepared from 1-5 and 4-pyridineacetic acid using method A: Ή NMR (400 MHz, DMSO-ί/ό) δ 0.76-0.80 (3H_: t, J=8 Hz), 0.82-0.84 (3H, d, J=8 Hz), 1.04-1.1 1 (IH, m), 1.38-1.46 (IH, m), 1.71-1.77 (IH, m), 3.07-3.1 1 (2H, t, J=8 IIz), 3.48-3.61 (211, m), 4.1 1-4.15 (IH, d, J=16 Hz), 4.25-4.29 (IH, t, J^~% Hz), 4.39- 4.43 (IH, d, J=16 Hz), 4.52-56 (IH, d, J=16 Hz), 5.12-5.15 (IH, d, J=12 Hz), 6.95-6.99 (IH, t, J=8 Hz), 7.13-7.22 (2H, m), 7.25-7.26 (2H, d, J=4 Hz), 7.25-7.28 (IH, d, J=8 Hz), 7.30 (IH, bs), 8.44-8.45(2H, d, J=4 Hz), 8.81 (1H, bs), MS (LC/MS) m/z observed 534.1 1 , expected 534.26 [M+H].

EXAMPLE A3

(25)-1-{2-[(25',35)-3-METHYL-2-[2-(PYRIDIN-3-

YL)ACETAMIDO] PENTANAMIDO]ACETYL}-/V-(2H-l,2,3,4-TETRAZOL-5-YLlMETHYL)-2,3-

DIHYDRO-1H-INDOLE-2-CARBOXAIYI1DE

Title compound A3 was prepared from 1-5 and 3-pyridineacetic acid using method A: Ή NMR (400 MHz, DMSO-</6) δ 0.76-0.80 (3H, t, J=8 Hz), 0.82-0.84 (3H, d, J=8 Hz), 1.04-1.12 (IH, m), 1.37-1.46 (IH, m), 1.72-1.77 (IH, m), 3.06-3.12 (2H, m), 3.47-3.62 (2H, m), 4.12-4.16 (IH, d, J=16 Hz), 4.24-4.28 (IH, t, J=8 Hz), 4.39-4.43 (IH, d, j=16 Hz), 4.53-4.57 (IH, d, J=\ 6 Hz), 5.13-5.15 (IH, d, J=8 Hz), 6.97-7.01 (IH, t, J=8 Hz), 7.14-7.18 (IH, t, J=8 Hz), 7.19-7.21 (IH, d, J=8 Hz), 7.28-7.31 (I H, q, J=8 Hz), 7.65-7.67 (IH, d, J=8 Hz), 8.02-8.04(lH, d, J=8 Hz), 8.23-8.26(lH, d, J=12 Hz), 8.29 (I H, bs), 8.40-8.41 (IH, d, J=4 Hz), 8.44 (IH, s), 8.82 (IH, bs), MS (LC MS) m/z observed 534.15, expected 534.26 [M+H]. EXAMPLE A4

(25)-1-{2-[(25,35)-3-METHYL-2-[2-(PYRIDIN-2-

YL)ACETAMIDO]PENTANAMIDO]ACETYL}- V-(2H-l,2,3,4-TETRAZOL-5-YL ETHYL)-2,3-

DIHYDRO-1H-INDOLE-2-CARBOXAMIDE

Title compound A4 was prepared from 1-5 and 2-pyridineacetic acid using method A: Ή NMR (400 MHz, DMSO-i/6) δ 0.77-0.81 (3H, t, J=8 Hz), 0.83-0.85 (3H, d, J=8 Hz), 1 .06- 1.13 (I H, m), 1.42- 1.47 (I H, m), 1.74-1.81 (I H, m), 3.06-3.12 (2H, m), 3.53-3.62 (2H, m), 4.15-4.19 (I H, d, J=16 Hz), 4.24-4.28 (IH, t, J=8 Hz), 4.25-4.29 (HI, q, J=12 Hz), 4.44-4.47 (IH, d, J=8 Hz), 4.59-4.63 (IH, d, J=16 Hz), 5.1 1 -5.14 (IH, d, J=12 Hz), 6.94-6.98 (III, t, J=8 Hz), 7.12-7.22 (3H, m), 7.68-7.72 (IH, td, J=4, 8 Hz), 8.01-8.03 (IH, d, J=8 Hz), 8.18-8.20(1H, d, J=8 Hz), 8.34 (IH, bs), 8.43-8.45 (IH, d, j=8 Hz), 8.87 (IH, bs), MS (LC/MS) m/z observed 534.12, expected 534.26 [M+H].

EXAMPLE A5

5- { [(1J?,25)-2-METH YL-1 -( {2-OXO-2- [{lS)-2- [(2H-1 ,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L-

YL]ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}PENTANOIC ACID

The starting compound (5-{[(15^,,2₁S)-2-methyl-l -({2-oxo-2-[(2,S)-2-[(2H-l ,2,3,4- tetrazol-5 -ylmethyl)carbamoyl] -2,3 -dihydro- 1 H-indol- 1 - yl]ethyl}carbamoyl)butyl]carbamoyl}pentanoic acid, ethyl ester) was prepared from 1-5 and adipic acid, monoethyl ester using method A: MS (LC/MS) m/z observed 571.02, expected 571.29 [Μ+Η], Compound was confirmed using LCMS and moved to next step as it was.

Title compound A5 was prepared from 5-{ [(15^,,2.S)-2-methyl-l -({2-oxo-2-[(25 -2- [(2H-l ,2,3,4-tetrazol-5-ylmethyl)carbamoyl]-2,3-dihydro-lH-indol-l- yl] ethyl }carbamoyl)butyl] carbamoyl }pentanoic acid, ethyl ester using method D: Ή NMR (400 MHz, DMSO-rftf) δ 0.78-0.82 (3H, t, J=8 Hz), 0.84-0.86 (3H, d, J=8 Hz), 1.06-1.13 (IH, m), 1.41 - 1.50 (5H, m), 1.68- 1.75 (IH, m), 2.08-2.20 (4H, m), 3.10-3.14 (IH, d, J=16 Hz), 3.50-3.62 (2H, m), 4.11 -4.13 (I H, d, J=8 Hz), 4.22-4.26 (IH, m), 4.52- 4.56 (IH, d, J=16 Hz), 4.62-4.66 (I H, d, J=16 Hz), 5.14-5.17 (IH, d, J=12 Hz), 6.97-7.01 (IH, t, J=8 Hz), 7.13-7.17 (IH, t, J=8 Hz), 7.20-7.22 (IH, d, J=8 Hz), 7.82-7.85 (IH, q, J=8 Hz), 8.00-8.02 (IH, d, J=8 Hz), 8.13 (IH, bs), 8.22-8.25 (IH, t, J=6 Hz) 9.09 (IH, bs), MS (LC/MS) m/z observed 542.99, expected 543.27 [M+H] and observed 565.09, expected 565.25 [M+Na].

EXAMPLE A6

(2iS)-l-{2-[(2iS',3iS)-2-(3-AMINOPROPANAMIDO)-3-METHYLPENTANAMIDO]ACETYL}-N- (2H-1,2,3,4-TETRAZOL-5-YLMETHYL)-2,3-DIHYDRO-lH-lNDOLE-2-CARBOXA IDE

(25)-l-{2-[(2LS',3_lS}-2-(3-(ieri-Butoxylcarbonyl)aminopropanamido)-3- methylpentanamido]acetyl}-N-(2H-l ,2,3,4-tetrazol-5-ylmethyl)-2,3-dihydro-lH-indole- 2-carboxamide was prepared from 1-5 and 3-((te^butoxycarbonyl)amino)propanoic acid using method A: 1H NMR (400 MHz, DMSO-i/6) δ 0.78-0.82 (3H, t, J=8 Hz), 0.84-0.86 (3H, d, J=8 IIz), 1.05-1.12 (III, m), 1.35 (9H, s), 1.39-1.46 (IH, m), 1.70-1.75 (IH, m), 2.23-2.34 (2H, m), 3.06-3.12 (3H, m), 3.53-3.59 (2H, m), 4.10-4.14 (IH, d, J=16 Hz), 4.22-4.26 (IH, t, J=8 Hz), 4.37-4.42 (IH, dd, J=4, 16 Hz), 4.52-4.57 (IH, dd, J=8, 16 Hz), 5.12-5.14 (IH, d, J=8 Hz), 6.96-7.00 (IH, t, J=8 Hz), 7.12-7.16 (IH, t, j=8 Hz), 7.18-7.20 (I H, d, J=8 Hz), 7.87-7.89 (IH, d, J=8 Hz), 8.01-8.03 (IH, d, J=8 Hz), 8.16 (IH, bs), 8.80 (IH, bs), MS (LC/MS) m/z observed 585.98, expected 586.31 [M+H] and observed 608.16, expected 608.29 [M+Na]. The compound was used further as described.

Title compound A6 was prepared from (2S)-l-{2-[(2S,3S)-2-(3-(tert- butoxylcarbonyl)aminopropanamido)-3-methylpentanamido] acetyl }-N-(2H- 1,2,3,4- tetrazol-5-ylmethyl)-2,3-dihydro-lH-indole-2-carboxamide using method E: 1H NMR (400 MHz, DMSC 6) δ 0.80-0.84 (3H, t, J=8 Hz), 0.87-0.89 (3H, d, J=8 Hz), 1.07-1.15 (I H, m), 1.43-1.52 (IH, m), 1.72-1.78 (IH, m), 2.52-2.60 (2H, m), 2.97-3.09 (3H, m), 3.51-3.64 (2H, m), 4.09-4.12 (IH, d, J=12 Hz), 4.19-4.23 (IH, t, J=8 Hz), 4.33-4.41 (2H, m), 5.10-5.13 (IH, d, j=12 Hz), 6.96-7.00 (IH, t, J=8 Hz), 7.12-7.16 (IH, t, J=8 Hz), 7.18-7.20 (IH, d, J=8 Hz), 7.72 (2H, bs), 8.01-8.03 (IH, d, J=8 Hz), 8.22 (IH, s), 8.24 (IH, bs), 8.59 (IH, bs), MS (LC/MS) m/z observed 486.14, expected 486.26 [M+H].

EXAMPLE A7

(25)-1-{2-[(25',35)-2-(4-A INOBUTANAMIDO)-3-IVIETHYLPENTANAMIDO]ACETYL}-/V- (2H-L,2,3,4-TETRAZOL-5-YLMETHYL)-2,3-DIHYDRO-LH-INDOLE-2-CARBOXAMIDE

HYDROCHLORIDE

(25)-l-{2-[(25,35)-2-(4-(/er/-Butoxycarbonyl)aminobutanamido)-3- methylpentanamido]acetyl}-N-(2H-l,2,3,4-tetrazol-5-ylmethyl)-2,3-dihydro-lH-indole- 2-carboxamide was prepared from 1-5 and 4-((iert-butoxycarbonyl)amino)butanoic acid using method A: MS (LC/MS) m/z observed 599.97, expected 600.32 [M+H], observed 622.15, expected 622.31 [M+Na]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound A7 was prepared from (25 -l -{2-[(25',3S)-2-(4-(ier/- butoxycarbonyl)aminobutanamido)-3-methylpentanamido]acetyl}-N-(2H- 1 ,2,3,4- tetrazol-5-ylmethyl)-2,3-dihydro- 1 H-indole-2-carboxamide using method E: 1H NMR (400 MHz, DMSO-i 6) δ 0.80-0.84 (3H, t, J=8 Hz), 0.88-0.90 (3H, d, J=8 Hz), 1.08-1.16 (IH, m), 1.47-1.53 (I H, m), 1.72-1.84 (3H, m), 1.18-1.34 (2H, m), 2.75-2.79 (2H, t, J=6 Hz), 3.03-3.07 (I H, d, J=8 Hz), 3.51-3.64 (2H, m), 4.07-4.1 1 (IH, m), 4.12-4.16 (IH, t, J=12 Hz), 4.30-4.35 (IH, dd, J=6,16 Hz), 4.41 -4.46 (IH, dd, J=6,16 Hz), 5.08-5.1 1 (IH, d, j=12 Hz), 6.96-7.00 (IH, t, J=8 Hz), 7.12-7.16 (IH, t, J=8 Hz), 7.18-7.20 (IH, d, j=8 Hz), 7.92 (3H, bs), 8.00-8.02 (IH, d, J=8 Hz), 8.13-8.16 (IH, t, J=6 Hz), 8.60 (IH, bs), MS (LC/MS) m/z observed 500.13, expected 500.27 [M+H].

EXAMPLE A8

(25 -1-{2-[(2S,35)-2-[2-(4-AMINOPHENYL)ACETAMIDO]-3-

ΜΕΤΗΥΕΡΕΝΤΑΙΝΑΐνΐΙΟθ]ΑΟΈΤΥΕ}-Λ^-(2^1,2,3,4-ΤΕΤΚΑΖΟΕ-5-ΥίΐνΐΕΤΗΥί)-2,3- DIHYDRO-lH-INDOLE-2-CARBOXAMIDE HYDROCHLORIDE

(25)-l-{2-[(25',35)-2-[2-(4-(/eri-Butoxycarbonyl)aminophenyl)acetamido]-3- methylpentanamido] acetyl }-N-(2H- 1 ,2,3 ,4-tetrazol-5-ylmethyl)-2,3-dihydro-lH-indole- 2-carboxamide was prepared from 1-5 and 2-(4-((tert- butoxycarbonyl)amino)phenyl)acetic acid using method A: MS (LC/MS) m/z observed 648.03, expected 648.31 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound A8 was prepared from (2S)-l-{2-[(2S,3S)-2-[2-(4-(tert- butoxycarbonyl)aminophenyl)acetamido]-3-methylpentanamido]acetyl} -N-(2H- 1 ,2,3,4- tetrazol-5-ylmethyl)-2,3-dihydro-lH-indole-2-carboxamide using method E: ^XH NMR (400 MHz, DMSO-rf6) δ 0.75-0.79 (3H, t, J=8 Hz), 0.81-0.83 (3H, d, J=8 Hz), 1.01 -1.06 (I H, m), 1 .32- 1.43 (IH, m), 1.62-1.74 (IH, m), 3.09-3.13 (2H, d, J=16 Hz), 3.19-3.26 (2H, m), 3.55-3.62 (2H, m), 4.10-4.15 (IH, dd, J=4, 16 Hz), 4.21-4.25 (IH, t, J=8 Hz), 4.51 -4.55 (IH, d, J=16 Hz), 4.62-4.66 (IH, d, J=16 Hz), 5.13-5.15 (IH, d, J=8 Hz), 6.44- 6.46 (2H, d, .7=8 Hz), 6.88-6.90 (2H, d, J=8 Hz), 6.96-7.00 (IH, t, J=8 Hz), 7.14-7.18 (1H, t, J=8 Hz), 7.19-7.21 (1H, d, J=8 Hz), 7.79-7.81 (1H, d, J=8 Hz), 8.01 -8.03 (1H, d, J=8 Hz), 8.20(1H, bs), 9.06(1H, bs), MS (LC/MS) m/z observed 548.06, expected 548.62 [M+H] and observed 570.17, expected 570.26 [M+Na] .

EXAMPLE A9

(2S l-{2-[(2S3S)-2-[2-(3-A INOPHENYL)ACETAMIDO]-3-

METHYLPENTANAMlDO]ACETYL}-iV-(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)-2,3- DIHYDRO-lH-rNDOLE-2-CARBOXAMIDE HYDROCHLORIDE

(25)- l-{2-[(25',3_JS)-2-[2-(3-(ier/-Butoxylcai-bonyl)aminophenyl)acetamido]-3- methylpentanamido]acetyl}-N-(2H- l ,2,3,4-tetrazol-5-ylmethyl)-2,3-dihydro-lH-indole- 2-carboxamide was prepared from 1-5 and 2-( -((tert- butoxycarbonyl)amino)phenyl)acetic acid using method A: MS (LC/MS) m/z observed 647.96, expected 648.33 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound A9 was prepared from (2S)-\ -{2-[(2S,3S)-2-[2-(3-(tert- butoxylcarbonyl)aminophenyl)acetamido]-3-methylpentanamido]acetyl}-N-(2H-l,2,3,4- tetrazol-5 -ylmethyl)-2,3 -dihydro- 1 H-indole-2-carboxamide using method E: Ή NMR (400 MHz, DMSO-./6) δ 0.76-0.80 (3H, t, J=8 Hz), 0.82-0.84 (3H, d, J=8 Hz), 1.03-1.1 1 (1H, m), 1.39-1.46 (1H, m), 1.68-1.74 (1H, m), 3.08-3.12 (2H, d, J=16 Hz), 3.17-3.25 (2H, m), 3.54-3.61 (2H, m), 4.10-4.15 (1H, dd, J=4, 16 Hz), 4.22-4.27 (1 H, m), 4.42-4.48 (1H, m), 4.51-4.56 (1 H, d, m), 5.12-5.14 (1H, d, J=8 Hz), 6.36-6.40 (2H, t, J=8 Hz), 6.43 (1H, s), 6.86-6.90 (1H, t, J=8 Hz), 6.96-7.00 (1H, t, J=8 Hz), 7.13-7.17 (1H, t, J=8 Hz), 7.19-7.21 (1 H, d, J=8 Hz), 7.92-7.96 (1H, t, J=8 Hz), 8.01 -8.03(1H, d, J=8 Hz), 8.23(1 H, bs), 8.36(1 H, bs), 8.88(1H, bs), MS (LC/MS) m/z observed, 548.03, expected 548.27 [M+H].

EXAMPLE A10

(25)-1-{2-[(25^,,35)-2-[2-(2-AMINO-1,3-THIAZOL-4-YL)ACETAIVIIDO]-3-

METHYLPENTANAlVlIDO]ACETYL}- V-(2H-l,2,3,4-TETRAZOL-5-YLlVIETHYL)-2,3- DIHYDRO-1H-INDOLE-2-CARBOXAMIDE HYDROCHLORIDE

Thionyl chloride (0.460 mL, 6.321 mmol, 2 eq) was added to a stirred mixture of 2-(2-aminothiazol-4-yl)acetic acid (500 mg, 3.2 mmol) and ethanol (5 mL) at 0 °C. The resulting clear reaction mixture was stirred at RT for 16 hr and then concentrated under vacuum to dryness and then swapped with ethanol twice. Ethyl 2-(2-aminothiazol-4- yl)acetate was obtained as a brown oil in a quantitative yield (588 mg). Ή NMR (400 MHz, DMSO-i/6) δ 1.17 (3H, bs), 3.72 (2H, bs), 4.08 (2H, bs), 6,68 (1H, bs), 9.43 (2H, bs). The compound was used further as described.

Ethyl 2-(2-aminothiazol-4-yl)acetate (250 mg, 1.123 mmol) was treated with triethylamine (0.630 mL, 4.494 mmol, 4 eq.), N,N-dimethylaminopyridine (30 mg, 0.225 mmol, 0.2 eq.) and di-toV-butyl dicarbonate (300 mg, 1.348 mmol, 1.2 eq.) in dichloromethane (10 mL) for 4 hr. The reaction mixture was diluted with dichloromcthane (10 mL) and sequentially washed with water (1 10 mL), saturated citric acid solution (1 x 10 mL) and brine (1 x 10 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to give a brown residue that was purified by column chromatography using a gradient hexanes/ethyl acetate (0% ethyl acetate to 50% ethyl acetate). Ethyl 2-(2-((?eri-butoxycarbonyl)amino)thiazol-4-yl)acetate was obtained as a yellow oil (130 mg, 34%). ¹H NMR (400 MHz, CDC1₃) δ 1.22 (3H, t, J=7Hz), 1.55 (9H, s), 3.72 (2H, s), 4.1 1 (2H, q, J=7Hz), 6.75 (1H, s), 9.55 (1H, bs).

Ethyl 2-(2-((rert-butoxycarbonyl)amino)thiazol-4-yl)acetate (100 mg,

0.350 mmol) was dissolved in ethanol (2 mL) and water (1 mL) and treated with lithium hydroxide (44 mg, 1.05 mmol, 3 eq.) at RT for 2 hrs. The reaction mixture was acidified by adding a saturated solution of citric acid to pH 5 and then concentrated to remove all ethanol. The residue was then extracted with ethyl acetate (2 x 15 mL) and the combined organic layers were dried over sodium sulfate, filtered and concentrated to give 2-(2- ((/eri-butoxycarbonyl)amino)thiazol-4-yl)acetic acid as a yellow oil (80 mg, 89%). MS (LC/MS) m/z observed 258.74, expected 259.08 [M+H]. Compound was confirmed using LC/MS and moved to next step as is.

(25)-l -{2-[(25',35)-2-[2-(2-(/eri-Butoxycarbonyl)amino-l,3-thiazol-4- yl)acetamido]-3-methylpentanamido]acetyl}-N-(2H-l ,2,3,4-tetrazol-5-ylmethyl)-2,3- dihydro- lH-indolc-2-carboxamide was prepared from 2-(2-((tert- butoxycarbonyl)amino)thiazol-4-yl)acetic acid and 1-5 using method A: MS (LC/MS) m/z observed 654.97, expected 655.28 [Μ+Η]. The compound was confirmed using LC/MS and moved to next step as it was.

Title compound A10 was prepared from (2₁S)-l -{2-[(2_tS',3₁S -2-[2-(2-(feri- butoxycarbonyl)amino-l ,3-thiazol-4-yl)acetamido]-3-methylpentanamido]acetyl}-N-(2H- l ,2,3,4-tetrazol-5-ylmethyl)-2,3-dihydro-lH-indole-2-carboxamide using method E: 1H NMR (400 MHz, DMSO-< 6) δ 0.77-0.92 (6H, m), 1.05-1.15 (IH, m), 1.40-1.52 (IH, m), 1.72-1.82 (IH, m), 3.00-3.65 (5H, m), 4.17 (IH, d, J=15Hz), 4.30 (I H, t, J=7Hz), 4.51 - 4.72 (2H, m), 5.20 (IH, d, J=10Hz), 6.27 (I H, s), 6.85 (2H, m), 7.02 (I H, t, J=7Hz), 7.10-7.30 (2H, m), 7.88 (IH, d, J=9Hz), 8.10 (IH, d, J=8Hz), 8.25-8.35 (IH, m), 9.10 (IH, bs), MS (LC MS) m/z observed 555.08, expected 555.23 [M+H].

EXAMPLE Al 1

(25)-l - {2- [(25)-2-C YCLOHEXYL-2-(2-PHEN YLACETAMIDO) ACETA IDO] ACETYL}-/V- (2H-l,2,3,4-TETRAZOL-5-YL ETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE feri-Butyl ((5)-2-((2-((5 -2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-2- oxoethyl)ainino)-l-cyclohexyl-2-oxoethyl)carbamate was prepared from 1-3 and (S)-2- ((ier -butoxycarbonyl)amino)-2-cyclohexylacetic acid using method A: MS (LC/MS) m/z observed 540.84, expected 541.29 [Μ+Η], Compound was confirmed using LC/MS and moved to next step as it was.

Title compound All was prepared from teri-butyl ((S)-2-((2-((S)-2-(((2H- tetrazol-5-yl)methyl)carbamoyl)indolin-l-yl)-2-oxoethyl)amino)-l -cyclohexyl-2- oxoethyl)carbamate and 1-6 using method A: Ή NMR (400 MHz, DMSO-rffi) δ 0.93- 1.00- (2H, m), 1.05-1.14 (3H, m), 1.55-1.67 (6H, m), 3.05-3.1 1 (2H, q, J=8 Hz), 3.52 (I H, s), 3.55-3.59 (2H, m), 4.1 1 -4.15 (IH, d, J=16 Hz), 4.23-4.27 (I H, t, J=8 Hz), 4.37- 4.41 (I H, d, J=16 Hz), 4.52-4.56 (IH, d, J=16 Hz), 5.12-5.14 (I H, d, J=8 Hz), 6.96-7.00 (IH, t, J=8 Hz), 7.14-7.21 (3H, m), 7.24-7.29 (4H, m), 8.02-8.04 (IH, d, J=8 Hz), 8.06- 8.08 (IH, d, J=8 Hz), 8.27 (IH, bs), 8.78 (IH, bs), MS (LC/MS) m/z observed 559.01 , expected 559.28 [M+H] and observed 581.13, expected 581.26 [M+Na].

EXAMPLE A12

(2S)-l-{2-[(25)-2-CYCLOPENTYL-2-(2-PHENYLACETAMIDO)ACETAMIDO]ACETYL}-N- (2H-l,2,3,4-TETRAZOL-5-YLMETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAlMlDE

/er -Butyl ((5)-2-((2-((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-2- oxoethyl)amino)-l -cyclopentyl-2-oxoethyl)carbamate was prepared from 1-3 and (S)-2- ((/eri-butoxycarbonyl)amino)-2-cyclopentylacetic acid using method A: MS (LC/MS) m/z observed 526.81 , expected 527.27 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound A12 was prepared from /er/-butyl ((S)-2-((2-((S)-2-(((2H- tetrazol-5-yl)methyl)carbamoyl)indolin- 1 -yl)-2-oxoethyl)amino)- 1 -cycIopentyl-2- oxoethyl)carbamate and 1-6 using method A: 1H NMR (400 MHz, DMSC 6) δ 1.36- 1.44 (4H, m), 1.47-1.64 (4H, m), 2.12-2.1 8 (IH, m), 3.03-3.09 (2H, q, J=8 Hz), 3.39-3.43 (IH, d, J=16 Hz), 3.48-3.58 (2H, m), 4.09-4.17 (IH, t, J=16 Hz), 4.25-4.29 (IH, t, J=8 Hz), 4.35-4.43 (IH, t, J=16 Hz), 4.47-4.55 (IH, t, J=16 Hz), 5.09-5.13 (IH, T, J=8 Hz), 6.96-7.00 (IH, t, J=8 Hz), 7.13-7.20 (3H, m), 7.23-7.28 (4H, m), 8.02-8.04 (IH, d, J=8 Hz), 8.18-8.21 (IH, d, J=12 Hz), 8.26 (I H, bs), 8.73 (IH, bs), MS (LC/MS) m z observed 544.99, expected 545.26 [M+H].

EXAMPLE Al 3

3- {[(15,25)-2-METHYL-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l-

YL] ETHYL}CARBAM0YL)BUTYL]CARBAM0YL} PROPANOIC ACID

Title compound A13 was prepared from 1-5 and succinic anhydride using method B: 1H NMR (400 MHz, DMSCM6) δ 0.79-0.83 (3H, t, J=8 Hz), 0.85-0.87 (3H, d, j=8 Hz), 1.10-1.16 (I H, m), 1.23-1.25 (4H, d, J=8 Hz), 1.43-1.52 (IH, m), 1.73-1.82 (IH, m), 3.05-3.09 (2H, d, J=16 Hz), 3.55-3.63 (2H, m), 4.13-4.22 (2H, m), 4.46-4.50 (2H, d, J=16 Hz), 5.11-5.13 (IH, d, J=8 Hz), 6.97-7.01 (IH, t, J=8 Hz), 7.15-7.19 (IH, d, J=16 Hz), 7.94-7.96 (IH, d, J=8 Hz), 8.02 (IH, s), 8.15 (IH, bs), 8.86 (IH, bs), MS (LC/MS) m/z observed 515.07, expected 515.24 [M+H] and observed 537.1 1 , expected 537.22 [M+Na],

EXAMPLE Al 4

4- {[(lS,25)-2-METHYL-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5-

YL ETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l- YL] ETHYL} CARBAMOYL)BUTYL] CARBAM0YL}BUTAN0IC ACID

Title compound A14 was prepared from 1-5 and glutaric anhydride using method B: Ή NMR (400 MHz, DMSCMtf) δ 0.78-0.82 (3H, t, J=8 Hz), 0.84-0.86 (3H, d, 7=8 Hz), 1.06-1.13 (IH, m), 1.40-1.47 (IH, m), 1.66- 1.75 (3H, m), 2.13-2.19 (4H, q, J=8 Hz), 3.03-3.07 (I H, d, J=16 Hz), 3.51-3.61 (2H, m), 4.09-4.1 1 (IH, d, j=8 Hz), 4.21- 4.25 (IH, t, J=8 Hz), 4.28-4.32 (I H, d, J=12 Hz), 4.42-4.45 (IH, d, J=12 Hz), 5.10-5.12 (IH, d, j=8 Hz), 6.95-6.99 (IH, t, J=8 Hz), 7.12-7.16 (IH, t, J=8 Hz), 7.18-7.20 (IH, d, J=8 Hz), 7.85-7.87 (IH, d, J=8 Hz), 8.02-8.04 (I H, d, .7=8 Hz), 8.19 (IH, bs), 8.58 (IH, bs), MS (LC MS) m/z observed 528.97, expected 529.24 [M+H] and observed 551.1 1, expected 551.23 [M+Na]. EXAMPLE Al 5

(25)- 1 - {2-[(2£,3iS3-2-ACETAMIDO-3-METHYLPENTANAMIDO] ACETYL} -N-(2H- 1 ,2,3,4- TETRAZOL-5-YLMETHYL)-2,3-DIIIYDRO-lH-INDOLE-2-CARBOXA IDE

To 1-5 (0.12 mmol) was added HC1 (4M) solution in dioxane (4 ml) and the reaction mixture was stirred for 2 hr at RT, then concentrated to dryness under vacuum and swapped with MeOH (5 ml) three times. The resulting residue was dried well under vacuum and treated with a mixture of acetic anhydride/pyridine (1 : 1 , 1.5 mL) for 15 minutes at RT. Then the reaction mixture was concentrated and the residue was submitted to reverse phase CI 8 column using 10-50% MeOH in water to yield the title compound A15 as an off-white solid (5 mg): Ή NMR (400 MHz, DMSO-rf6) δ 0.77- 0.92 (6H, m), 1.05-1.15 (IH, m), 1.40- 1.50 (IH, m), 1.68-1.77 (IH, m), 1.87 (3H, s), 3.08-3.15 (IH, m), 3.51 -3.65 (2H, m), 4.10-4.18 (IH, m), 4.24 (IH, t, J=9Hz), 4.48-4.68 (2H, m), 5.15 (IH, d, J=10Hz), 6.95 (IH, t, J=7Hz), 7.15-7.27 (2H, m), 7.90 (IH, d, J=7Hz), 8.10 (IH, d, J=8Hz), 8.18-8.33 (IH, m), 9.05 (I H, bs), MS (LC/MS) m/z observed 456.99, expected 457.23 [M+H].

EXAMPLE Al 6

3-{[(15,25)-2-METHYL-l-({2-Oxo-2-[(25)-2-[(lH-l,2,3-TRlAZOL-4-

YLMETHYL)CARBA OYL]-2,3-DIHYDRO-lH-INDOL-l- YL]ETHYL}CARBAM0YL)BUTYL]CARBAM0YL} PR0PAN0IC ACID

2-(((2H-l ,2,3-triazol-4-yl)methyl)carbamoyl)indoline-l -carboxylate was prepared from (5)-l -(feri-butoxycarbonyl)indoline-2-carboxylic acid, and (2H- 1 ,2,3- triazol-4-yl)methyl-amine following the same procedure for the preparation of 1-1. MS (LC/MS) m/z observed 365.99, expected 366.15 [M+Na]. Compound was confirmed using LC/MS and moved to next step as it was.

(SHerf-But l (2-(2-(((2H- 1 ,2,3 -triazol-4-yl)methyl)carbamoyl)indolin- 1 -yl)-2- oxoethyl)carbamate was prepared from (S)-tert-b ty\ 2-(((2H-l ,2,3-triazol-4- yl)methyl)carbamoyl)indoline-l -carboxylate following the same procedure for the preparation of 1-2 followed by the same procedure for the preparation of 1-3. MS (LC/MS) m/z observed 400.83, expected 401.19 [Μ+Η], Compound was confirmed using LC/MS and moved to next step as it was.

Title compound A16 was prepared from (S)-tert-butyl (2-(2-(((2H-l ,2,3-triazol-4- yl)methyl)carbamoyl)indolin-l-yl)-2-oxoethyl)carbamate and (2S,3S)-2-((tert- butoxycarbonyl)amino)-3-methylpentanoic acid using method A followed reaction with succinic anhydride using method B: 1H NMR (400 MHz, DMSC 6) δ 0.79-0.83 (3H, t, J=8 Hz), 0.85-0.87 (3H, d, J=8 Hz), 1.15-1.20 (IH, m), 1.40-1.50 (IH, m), 1.68-1.80 (IH, m), 2.31-2.45 (4H, m), 3.01-3.10 (IH, m), 3.50-3.63 (2H, m), 4.05-4.15 (I H, m), 4.25 (IH, t, J= 7Hz), 4.30-4.45 (2H, m), 5.11 (IH, d, J=9 Hz), 6.98 (IH, t, J=8 Hz), 7.10-7.25 (2H, m), 7.90 (IH, d, J=9 Hz), 8.02 (IH, d, J-8 Hz), 8.13 (IH, bs), 8.86 (IH, bs), 12.02 (IH, bs), MS (LC/MS) m/z observed 514.02, expected 514.24 [M+H].

EXAMPLE Al 7

3-METHYL-4-{[(15,25)-2-METHYL-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL] -2,3-DIHYDRO- LH-INDOL-1 -

YL]ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}BUTANOIC ACID Title compound A17 was prepared from 1-5 and 3-methylglutaric anhydride using method I and was separated by chromatography, into two diastereomers, A17-1 and A 17-2, each characterized as below:

A17-1:

Ή NMR (400 MHz, DMSO-rfd) δ 0.82 (3H, t, J=7.4Hz), 0.84-0.90 (6H, m), 1.12 (IH, m), 1.45 (IH, m), 1.75 (IH, m), 2.05 (IH, m), 2.15-2.30 (4H, m), 3.10 (IH, m), 3.55-3.65 (2H, m), 4.15 (IH, dd, J=4, 18Hz), 4.28 (IH, t, J=15Hz), 4.55 (IH, dd, J=4, 16Hz), 4,65 (IH, dd, J=6, 16Hz), 5.16 (IH, d, J=l lHz), 7.00 (IH, t, J=8Hz), 7.16 (IH, t, J=8Hz), 7.22 (IH, d, J=8Hz), 7.87 (IH, d, J=9Hz), 8.03 (IH, d, J=8Hz), 8.17 (IH, bs), 9.08 (IH, bs), MS (LC/MS) m/z observed 543.10, expected 543.27 [M+H].

A17-2

1H NMR (400 MHz, DMSO-rf6) δ 0.82 (3H, t, J=7.4Hz), 0.84-0.90 (6H, m), 1.12 (IH, m), 1.45 (IH, m), 1.75 (IH, m), 2.05 (IH, m), 2.08-2.15 (2H, m), 2.20-2.30 (2H, m), 3.10 (IH, m), 3.55-3.65 (2H, m), 4.13 (IH, d, J=17Hz), 4.28 (IH, t, J=19Hz), 4.53 (IH, dd, J=4, 16Hz), 4,65 (IH, dd, J=6, 16Hz), 5.15 (IH, d, J=l lHz), 7.00 (IH, t, J=8Hz), 7.16 (IH, t, J=8Hz), 7.22 (IH, d, J=8Hz), 7.87 (IH, d, J=9Hz), 8.03 (IH, d, J=8Hz), 8.17 (IH, bs), 9.08 (IH, bs), MS (LC/MS) m/z observed 543.07, expected 543.27 [M+H]. EXAMPLE Al 8

3,3-DlMETHYL-4-{[(15,25)-2-M ETHYL-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5-

YLMETHYL)CARBA OYL]-2,3-DIHYDRO-lH-INDOL-l-

YL] ETHYL}CARBAMOYL)BUTYL] CARBAMOYL}BUTANOIC ACID Title compound A18 was prepared from 1-5 and 3,3-dimethylglutaric anhydride using method I without acidification with formic acid. The product was obtained as a triethylammonium salt: Ή NMR (400 MHz, DMSO-i/6) δ 0.82 (3H, t, J=7.4Hz), 0.87 (3H, d, J=7Hz), 1.02 (6H, s), 1.05-1.10 (10H, m), 1.45 (I H, m), 1.75 (I H, m), 2-22-2-29 (4H, m), 2.77-2.90 (6H, m), 3.05 (IH, d, J=17Hz), 3.55-3.65 (2H, m), 4.13 (I H, d, J=16Hz), 4.26-4.33 (211, m), 4.53 (IH, m), 5.15 (IH, d, J-l l Hz), 6.98 (IH, t, J=8Hz), 7.12-7-24 (2H, m), 7.97 (IH, d, J=9Hz), 8.03 (IH, d, J=8Hz), 8.21 (IH, bs), 8.62 (IH, bs), MS (LC/MS) m/z observed 557.14, expected 557.28 [M+H] ,

EXAMPLE Al 9

2-[l-({[(15,25)-2-METHYL-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l-

YL] ETHYL}CARBAMOYL)BUTYL] CARBA OYL}METHYL)CYCLOPENTYL] ACETIC ACID Title compound A19 was prepared from 1-5 and 3,3-tetramethyleneglutaric anhydride using method I without acidification with formic acid. The product was obtained as a triethylammonium salt: 1H NMR (400 MHz, DMSO-rf6) δ 0.82 (3H, t, J=7.4Hz), 0.87 (3H, d, J=7Hz), 1.07-1.13 (10H, m), 1.40-1.62 (9H, m), 1.75 (I H, m), 2-31 -2-38 (3H, m), 2.45 (IH, d, J=15Hz), 2.86-2.93 (6H,m), 3.05 (III, d, J=17Hz), 3.55- 3.65 (2H, m), 4.13 (IH, d, J=16Hz), 4.26-4.33 (2H, m), 4.49 (IH, dd, J=6, 1 5Hz), 5.15 (I H, d, J=H Hz), 6.98 (IH, t, J=8Hz), 7.12-7-24 (2H, m), 7.97 (IH, d, J=9Hz), 8.03 (IH, d, J=8Hz), 8.23 (I H, bs), 8.65 (I H, bs), MS (LC/MS) m/z observed 583.1 1 , expected 583.30 [M+H]. EXAMPLE A20

2-{[(15,25)-2-ΜΕΤΗΥί-1-({2-θΧθ-2-[(25)-2-[(2^1,2,3,4-ΤΕΤΚΑΖθί-5-

YLMETHYL)CARBAMOYL]-2,3-DlHYDRO-lH-INDOL-l- YLl ETHYL}CARBAMOYL)BUTYL]CARBAIVIOYL}-r ? l/V5-CYCLOHEXANE-l-CARBOXYL[C

ACID

Title compound A20 was prepared from 1-5 and trans-1 ,2- cyclohexanedicarboxylic anhydride using method I without acidification with formic acid and was separated by chromatography, into two diastereomers, A20-1 and A20-2, each characterized below as triethylammonium salts:

A20-1 :

Ή NMR (400 MHz, DMSO-i 6) δ 0.79 (3H, t, J=7.4Hz), 0.85 (3H, d, J=7Hz), 1.06- 1.26 (14H, m), 1.46 (IH, m), 1.65-1.77 (3H, m), 1.85 (I H, m), 1.95 (I H, m), 2.40- 2.45 (2H, m), 2.85-2.99 (3H, m), 3.05 (IH, d, J=17Hz), 3.32-3.47 (3H, m), 3.52-3.65 (2H, m), 4.08-4.24 (2H, m), 4.34 (IH, m), 4.47 (IH, m), 5.13 (IH, d, J=l lHz), 6.98 (IH, t, J=8Hz), 7.12-7-24 (2H, m), 7.75 (IH, d, J=9Hz), 8.00-8-10 (2H, m), 8.65 (IH, bs), MS (LC/MS) m/z observed 569.1 1 , expected 569.28 [M+H].

A20-2:

Ή NMR (400 MHz, DMSO-rftf) δ δ 0.80 (3H, t, J=7.4Hz), 0.85 (3H, d, J=7Hz), 1.10-1-30 (14H, m), 1.50 (IH, m), 1.68-1.77 (3H, m), 1.91 (IH, m), 2.1 1 (I H, m), 2.43 (IH, m), 2.65 (IH, t, J=10.7Hz), 2.90-3.06 (4H, m), 3.32-3.50 (3H, m), 3.60 (IH, d, J=l l , 17Hz), 3.85 (I H, dd, J=5, 17Hz), 4.08 (IH, dd, J=5, 9Hz), 4.22 (IH, dd, J=6, 17Hz), 4.35 (IH, dd, J=6, 17Hz), 4.47 (I H, dd, J=6, 15Hz), 5.07 (IH, d, J=\ l Hz), 6.97 (IH, t, J=8Hz), 7.1 1 -7-21 (2H, m), 7.95-8-06 (3H, m), 8.85 (IH, bs), MS (LC/MS) m/z observed 569.12, expected 569.28 [M+H]. EXAMPLE A21

6-{[(15,25}-2-ΜΕΤΗΥΕ-1-({2-ΟΧΟ-2-[(25)-2-[(2^1,2,3,4-ΤΕΤ ΑΖΟΕ-5-

YLMETHYL)C ARBAMOYL] -2,3-DIH YDRO- lH-INDOL-1 - YL]ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}-C/5-CYCLOHEX-3-ENE-l-CARBOXYLIC

ACID

Title compound A21 was prepared from 1-5 and c/s-l ,2,3,6-tetrahydrophtalic anhydride using method I and was separated by chromatography, into two diastereomers, A21-1 and A21-2, each characterized as below:

A21-1 :

Ή NMR (400 MHz, DMSO-tfd) δ 0.81 (3H, t, J=7.4Hz), 0.86 (3H, d, J=7Hz),

1.10 (I H, m), 1.48 (IH, m), 1.75 (IH, m), 2.15-2.25 (2H, m), 2.33 (IH, m), 2.43 (IH, m), 2.82-2.95 (2H, m), 3.1 1 (IH, m), 3.54-3.66 (2H, m), 4.1 1 (I H, d, J=17Hz), 4.23 (IH, t, ,/=8Hz), 4.54 (IH, dd, J=6, 17Hz), 4.65 (I H, dd, J=6, 15Hz), 5.16 (IH, d, J=l l Hz), 5.60 (m, 2H), 7.00 (IH, t, J=8Hz), 7.1 1-7-26 (2H, m), 7.65 ( IH, d, J=9Hz), 8.02 (IH, d, J=9Hz), 8.16 (IH, m), 9.07 (IH, bs), MS (LC/MS) m/z observed 567.1 1 , expected 567.27 [M+H].

A21-2:

Ή NMR (400 MHz, DMSO-ί/ό) δ 0.81 (3H, t, J=7.4Hz), 0.86 (3H, d, J=7Hz),

1.1 1 (IH, m), 1.45 (IH, m), 1.81 (IH, m), 2.22 (IH, m), 2.30-2.36 (2H, m), 2.46 (IH, m), 2.82 (I H, m), 2.94 (IH, m), 3.1 1 (IH, d, J=17Hz), 3.54-3.66 (2H, m), 4.12-4.25 (2H, m),

4.50-4.65 (2H, m), 5.15 (IH, d, J=l lHz), 5.62 (m, 2H), 7.00 (IH, t, J=8Hz), 7.1 1 -7-26 (2H, m), 7.71 (IH, d, J=9Hz), 8.02 (IH, d, J=9Hz), 8.16 (IH, bs), 9.05 (IH, bs), MS (LC/MS) m/z observed 567.1 1, expected 567.27 [M+H].

EXAMPLE A22

2-{[(lS,25)-2-METHYL-l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YL ETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L- YL] ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}-C/5-CYCLOPENTANE-1-CARBOXYLIC

ACID

Title compound A22 was prepared from 1-5 and c s-l ,2-cyclopentanedicarboxylic anhydride using method I and was separated by chromatography, into two diastereomers, A22-1 and A22-2, each characterized as below:

A22-1 :

Ή NMR (400 MHz, DMSO-rftf) δ 0.81 (3H, t, J=7.4IIz), 0.86 (311, d, J=7Hz), 1.1 1 ( I H, m), 1.44-1.55 (2H, m), 1.66-1.85 (5H, m), 1.95 (IH, m), 2.82 (IH, m), 3.05 (IH, m), 3.11 (IH, m), 3.54-3.66 (2H, m), 4.10-4.23 (2H, m), 4.45-4.70 (2H, m), 5.16 (IH, d, J=l IHz), 7.00 (IH, t, J=8Hz), 7.14-7-26 (2H, m), 7.71 (IH, d, J=9Hz), 7.99-8.10 (2H, m), 9.08 (IH, bs), MS (LC/MS) m/z observed 555.10, expected 555.27 [M+H]. A22-2:

Ή NMR (400 MHz, DMSO-i/6) δ 0.81 (3H, t, J=7.4Hz), 0.85 (3H, d, J=7Hz), 1.14 (IH, m), 1.43 (I H, m), 1.52 (IH, m), 1.66-1.87 (5H, m), 1.96 (IH, m), 2.82 (IH, m), 3.08 ( IH, m), 3.14 (I H, m), 3.56-3.69 (2H, m), 4.15-4.27 (2H, m), 4.52-4.68 (2H, m), 5.16 (I H, d, J=l IHz), 7.00 (IH, t, J=8Hz), 7.14-7-26 (2H, m), 7.77 (IH, d, J=9Hz), 8.00- 8.08 (2H, m), 9.08 (IH, bs), MS (LC/MS) m/z observed 555.09, expected 555.27 [M+H].

EXAMPLE A23

2-{[(15,25)-2-METHYL-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAIMOYL]-2,3-DIHYDRO-LH-INDOL-L- YL]ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}-C/5-CYCLOHEXANE-1-CARBOXYLIC ACID

Title compound A23 was prepared from 1-5 and cis- 1 ,2-cyclohexanedicarboxylic anhydride using method I and was separated by chromatography, into two diastereomers, A23-1 and A23-2, each characterized as below:

A23-1:

Ή NMR (400 MHz, DMSO-</<5) δ 0.79 (3H, t, J=7.4Hz), 0.83 (3H, d, J=7Hz), 1.08 (IH, m), 1.20-1.32 (2H, m), 1.34-1.57 (4H, m), 1.63 (IH, m), 1.73 (IH, m), 1.87 (IH, m), 1.99 (IH, m), 2.55 (IH, m), 2.77 (IH, m), 3.10 (IH, m), 3.54-3.65 (2H, m), 4.10-4.23 (2H, m), 4.48-4.67 (2H, m), 5.14 (I H, d, J=HHz), 6.99 (I H, t, J=8Hz), 7.12-7- 24 (2H, m), 7.56 (IH, d, J=9Hz), 7.97-8.08 (2H, m), 9.07 (IH, bs), MS (LC/MS) m/z observed 569.08, expected 569.28 [M+H].

A23-2:

Ή NMR (400 MHz, DMSO-rf(i) δ 0.78 (3H, t, J=7.4Hz), 0.84 (3H, d, J=7Hz),

1.1 1 (IH, m), 1.20-1.48 (4H, m), 1.50- 1.69 (3H, m), 1.77 (IH, m), 1.90 (IH, ra), 2.03 (IH, m), 2.53 (IH, m), 2.82 (IH, m), 3.10 (IH, m), 3.54-3.65 (2H, m), 4.12-4.23 (2H, m), 4.46-4.64 (2H, ra), 5.13 (IH, d, J=l lHz), 6.97 (IH, t, J=8Hz), 7.12-7-24 (2H, m), 7.62 (IH, d, J=9Hz), 8.00 (IH, d, J=8Hz), 8.1 1 (IH, bs), 9.03 (IH, bs), MS (LC/MS) m/z observed 569.06, expected 569.28 [M+H].

EXAMPLE A24

(2 )-3-{[(lS,2₁S)-2-METHYL-l-({2-oxo-2-[(2-S)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l-

YL]ETHYL}CARBA OYL)BUTYL]CARBA OYL}PROP-2-ENOIC ACID Title compound A24 was prepared from 1-5 and maleic anhydride using method I without acidification with formic acid: 1H NMR (400 MHz, DMSO-rf6) δ 0.82 (3H, t, J=7.4Hz), 0.88 (3H, d, J=7Hz), 1.08 (I H, m), 1.12 (9H, t, J=7Hz), 1.47 (I H, m), 1.82 ( I H, m), 2.96 (6H, q, J=7Hz), 3.05 (IH, d, J=17Hz), 3.50-3.65 (2H, m), 4.13 (IH, d, J=16Hz), 4.26 (IH, t, J=7Hz), 4.38 (IH, m), 4.50 (IH, dd, J=6, 15Hz), 5.13 (IH, d, J=l lHz), 6.1 1-6.15 (2H, m), 6.98 (IH, t, J=8Hz), 7.12-7-24 (2H, m), 8.04 (IH, d, J=8Hz), 8.37 (IH, bs), 8.85 (IH, bs), MS (LC/MS) m/z observed 513.08, expected 513.22 [M+H] .

EXAMPLE A25

2-{[(15,2-S)-2-METHYL-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l-

YL] ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}BENZOIC ACID

Title compound A25 was prepared from 1-7 and phthalic anhydride using method I without acidification with formic acid: 1H NMR (400 MHz, DMSO-<f6) δ 0.82 (3H, t, J=7.4Hz), 0.93 (3H, d, J=7Hz), 1.10 (9H, t, ,/=7Hz), 1.25 (IH, m), 1.55 (IH, m), 1.97 (IH, m), 2.90 (6H, q, J=7Hz), 3.00 (IH, d, J=17Hz), 3.56 (I H, m), 3.73 (IH, m), 4.16 (IH, dd, J=6, 16Hz), 4.25 (IH, dd, J=6, 8Hz), 4.47-4.51 (2H, m), 5.18 (IH, d, J=l lHz), 6.98 (IH, t, J=8Hz), 7.12-7-20 (2H, m), 7.28 (IH, d, J=8Hz), 7.42-7-50 (2H, m), 7.88 (IH, d, J=9Hz), 8.03 (IH, d, J=8Hz), 8.62 (IH, d, J=8Hz), 8.71 (IH, bs), 9.21 (IH, bs), MS (LC/MS) m/z observed 563.10, expected 563.24 [M+H].

EXAMPLE A26

2-{[(lS,25)-2-METHYL-l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l-

YL] ETHYL} C ARBAMOYL)BUTYL]C ARBAMO YL} -C/S-CYCLOPROPANE-1 -CARBOXYLIC

ACID

Title compound A26 was prepared from 1-5 and c i-3-oxabicyclo[3.1.0]hexane- 2,4-dione using method I and was separated by chromatography, into two diastereomers, A26-1 and A26-2, each characterized as below:

A26-1 :

Ή NMR (400 MHz, DMSO-i 6) δ 0.81 (3H, t, J=7.4Hz), 0.87 (3H, d, J=7Hz), 1 .04- 1.14 (2H, m), 1 .31 (I H, m), 1.47 (IH, m), 1.75 (IH, m), 1.92 (l H, m), 2.13 (I H, m), 3.1 1 (IH, m), 3.55-3.66 (2H, m), 4.15 (IH, dd, J=6, 16Hz), 4.25 (IH, dd, J=7, 9Hz), 4.54 (I H, m), 4.65 (IH, m), 5.17 (IH, d, J=l lHz), 7.01 (IH, t, J=8Hz), 7.14-7-26 (2H, m), 8.03 (IH, d, J=8Hz), 8.18-8.30 (2H, m), 9.10 (IH, bs), MS (LC/MS) m/z observed 527.07, expected 527.24 [M+H].

A26-2:

Ή NMR (400 MHz, DMSCM6) δ δ 0.81 (3H, t, J=7.4Hz), 0.87 (3H, d, J=7Hz), 1.06- 1.17 (2H, m), 1.31 (IH, m), 1.47 (IH, m), 1.76 (IH, m), 1.91 (IH, m), 2.13 (IH, m), 3.1 1 (IH, m), 3.55-3.66 (2H, m), 4.16 (IH, dd, J=6, 16Hz), 4.29 (IH, dd, J=7, 9Hz), 4.54 (I H, m), 4.65 (IH, m), 5.17 (IH, d, J=l lHz), 7.01 (IH, t, J=8Hz), 7.14-7-26 (2H, m), 8.03 (IH, d, J=8Hz), 8.18-8.30 (2H, m), 9.10 (IH, bs), MS (LC/MS) m/z observed 527.06, expected 527.24 [M+H].

EXAMPLE A27

(25 -l-{2-[(2S,35)-2-[2-(l-BENZOTHIOPHEN-3-YL)ACETAiVIIDO]-3- IMETHYLPENTANAMIDO]ACETYL}-/V-(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)-2,3- DIHYDRO-1H-INDOLE-2-CARBOX AMIDE

Title compound A27 was prepared from 1-5 and 2-(benzo[ >]thiophen-3-yl)acetic acid using method A except a 2: 1 ratio DCM/DMF as solvent, was used for the coupling reaction: Ή NMR (400 MHz, DMSCM6) δ 0.80 (3H, t, J=7.4Hz), 0.86 (3H, d, J=7Hz), 1.10 (I H, m), 1.47 (IH, m), 1 .76 (I H, m), 3.1 1 (I H, m), 3.55-3.66 (2H, m), 3.73 (IH, d, J=5Hz), 3.83 (I H, d, J=5Hz), 4.15 (IH, dd, J=6, 16Hz), 4.30 (IH, dd, J=7, 9Hz), 4.54 (IH, m), 4.65 (IH, m), 5.17 (IH, d, J=l lHz), 7.01 (IH, t, J=8Hz), 7.14-7-26 (2H, m), 7.32-7.40 (2H, m), 7.52 (IH, s), 7.86 (IH, m), 7.95 (IH, m), 8.04 (IH, d, J=8Hz), 8.23- 8.32 (2H, m), 9.08 (IH, bs), MS (LC/MS) m/z observed 589.05, expected 589.23 [M+H].

EXAMPLE A28

(25)-l-{2-[(2^,35 -3-METHYL-2-[2-(2H-l,2,3,4-TETRAZOL-5-

YL)ACETA rDO]PENTAIN AMIDO]ACETYL}-7V-(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)-2,3-

DIHYDRO-1H-INDOLE-2-CARBOXAM1DE

Title compound A28 was prepared from 1-5 and 2-(2H-tetrazol-5-yl)acetic acid using method A bul with DMF as solvent for the coupling reaction: Ή NMR (400 MHz, DMSO-i/6) δ 0.82 (3H, t, J=7.4Hz), 0.88 (3H, d, J=7Hz), 1.12 (I H, m), 1.50 (IH, m), 1.77 (I H, m), 3.12 (IH, m), 3.56-3.67 (2H, m), 3.95-4.05 (2H, m), 4.16 (IH, dd, J=6, 16Hz), 4.31 (IH, t, J=8Hz), 4.56 (IH, m), 4.65 (IH, m), 5.17 (I H, d, J=l lHz), 7.01 (IH, t, J=8Hz), 7.14-7-26 (2H, m), 8.04 (IH, d, J=8Hz), 8.34 (IH, bs), 8.43 (I H, d, J=9Hz), 9.08 (IH, bs), MS (LC/MS) m/z observed 525.08, expected 525.24 [M+H].

EXAMPLE A29

METHYL (3^)-3-METHYL-3-{[(lS,2S)-2-METHYL-l-({2-oxo-2-[(2S)-2-[(2H-l,2,3,4-

TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l- YL] ETHYL} CARBAMOYL)BUTYL] CARBAMOYL} PROPANO ATE Title compound A29 was prepared from 1-5 and (i?)-4-methoxy-2-methyl-4- oxobutanoic acid using method E, followed by method C: Ή NMR (400 MHz, DMSO- d6) δ 0.83 (3H, t, J=7.4Hz), 0.89 (3H, d, J=7Hz), 1.05 (3H, d, J= 7Hz), 1.14 (IH, m), 1.47 (IH, m), 1.77 (I H, m), 2.33 (I H, dd, J=6, 16Hz), 2.57 (I H, dd, J=8, 16Hz), 2.87 ( I H, m), 3.12 (I H, m), 3.56 (3H, s), 3.58-3.67 (2H, m), 4.16 (IH, m), 4.25 (I H, t, J=8Hz), 4.56 (IH, m), 4.66 (I H, m), 5.17 (IH, d, J=l lHz), 7.01 (IH, t, J=8Hz), 7.14-7- 26 (2H, m), 7.91 (I H, d, J=9Hz), 8.02-8.12 (2H, m), 9.10 (IH, bs), MS (LC/MS) m/z observed 543.13, expected 543.27 [M+H]. EXAMPLE A30

3-METHYL-3-{[(15,25)-2-METHYL-l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYLj-2,3-DIHYDRO-lH-INDOL-l-

YL] ETHYL}CARBAMOYL) BUTYL] CARBAMOYLJPROPANOIC ACID Title compound A30 was prepared from A29 using method D with 3 eq. of

LiOH H₂0 and was separated by chromatography, into two diastereomers, A30-1 and A30-2, each characterized as below:

A30-1 :

Ή NMR (400 MHz, DMSCM6) δ 0.79 (3H, t, J=7.4Hz), 0.85 (3H, d, J=7Hz), 1.01 (3H, d, J= 7Hz), 1.10 (IH, m), 1.45 (IH, m), 1.74 (IH, m), 2.17 (IH, dd, J=6, 16Hz), 2.43 (IH, m), 2.79 (I H, m), 3.08 (IH, m), 3.54-3.64 (2H, m), 4.12 (IH, m), 4.22 (IH, t, J=8Hz), 4.50 (IH, m), 4.62 (IH, m), 5.14 (IH, d, J=l lHz), 6.98 (IH, t, J=8Hz), 7.1 1-7-23 (2H, m), 7.82 (IH, d, J=9Hz), 7.98-8.08 (2H, m), 9.01 (IH, bs), MS (LC/MS) m/z observed 529.08, expected 529.25 [M+H].

A310-2:

Ή NMR (400 MHz, DMSO-</6) 0.79 (3H, t, J=7.4Hz), 0.85 (3H, d, J=7Hz), 1.03 (3H, d, J= 7Hz), 1.10 (IH, m), 1.44 (IH, m), 1.72 (IH, m), 2.22 (IH, dd, J=7, 15Hz), 2.51 (IH, m), 2.68 (IH, m), 3.08 (IH, m), 3.54-3.64 (2H, m), 4.1 1 (IH, m), 4.22 (IH, t, J=8Hz), 4.48 (IH, m), 4.60 (IH, m), 5.14 (IH, d, J=l lHz), 6.98 (IH, t, J=8Hz), 7.1 1-7-23 (2H, m), 7.82 (IH, d, J=9Hz), 8.02 (IH, m), 8.15 (I H, bs), 8.96 (IH, bs), MS (LC/MS) m/z observed 529.10, expected 529.25 [M+H].

EXAMPLE A31

2-[3-({[(l₁S,2j -2-METHYL-l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l- YL] ETHYL}CARBAMOYL)BUTYL] CARBAMOYL}METHYL)PHENYL] ACETIC ACID

Title compound A31 was prepared from 1-5 and l -cyclohexene- l ,2-dicarboxylic anhydride using method I without acidification with formic acid: Ή NMR (400 MHz, DMSO-rf<5) δ 0.78 (3H, t, J=7.4Hz), 0.84 (3H, d, J=7Hz), 1.08 (IH, m), 1.13 (9H, t, J=7Hz), 1.17- 1.25 (2H, m), 1.43- 1.63 (3H, m), 1.99 (IH, m), 2.17-2.25 (3H, m), 2.28 (IH, m), 2.97-3.05 (7H, m), 3.54-3.65 (2H, m), 4.07-4.18 (2H, m), 4.38-4.50 (2H, m), 5.12 (IH, d, ^=1 1 Hz), 6.95 (IH, t, J=8Hz), 7.12-7-20 (2H, m), 7.97 (IH, d, J=8Hz), 8.07 (IH, d, J=8Hz), 8.45 (IH, bs), 9.05 (IH, bs), MS (LC/MS) m/z observed 566.96, expected 567.27 [M+H].

EXAMPLE A32

(2R,3R)-2,3-DlHYDROXY-3-{[(15,25)-2-METHYL-l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4- TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l-

YL] ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}PROPANOIC ACID Title compound A32 was prepared from 1-5 and L- +^-tartaric acid using method J in DMF: Ή NMR (400 MHz, DMSO-Λί) 6 0.83 (3H, t, J=7.4Hz), 0.87 (3H, d, J=7Hz), 1.08 (I H, m), 1.48 (IH, m), 1.80 (IH, m), 3.11 (IH, m), 3.56-3.68 (2H, m), 4.17 (I H, m), 4.26 (IH, s), 4.30-4.37 (2H, m), 4.55 (IH, m), 4.66 (IH, m), 5.16 (IH, d, J=l lHz), 7.00 (IH, t, J=8Hz), 7.15-7-24 (2H, m), 7.57 (IH, d, J=9Hz), 8.07 (IH, d, J=8Hz), 8.33 (IH, bs), 9.10 (IH, bs), MS (LC/MS) m/z observed 547.06, expected 547.23 [M+H].

EXAMPLE A33 (25,3₁S)-2,3-DiHYDROXY-3-{[(15,25)-2-METHYL-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-

TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l-

YL]ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}PROPANOIC ACID

Title compound A33 was prepared from 1-5 and D- -,)-tartaric acid using method J in DMF: 1H NMR (400 MHz, DMSO-t/6) δ 0.83 (3H, t, J=7.4Hz), 0.87 (3H, d, J=7Hz), 1.06 (IH, m), 1.51 (IH, m), 1.73 (IH, m), 3.1 1 (IH, m), 3.55-3.67 (2H, m), 4.17 (IH, m), 4.22 (IH, s), 4.31 (IH, s), 4.37 (IH, dd, J=7, 9Hz), 4.55 (IH, m), 4.63 (IH, m), 5.16 (IH, d, J=l lHz), 7.00 (IH, t, J=8Hz), 7.15-7-24 (2H, m), 7.44 (IH, d, J=9Hz), 8.02 (IH, d, J=8Hz), 8.47 (IH, bs), 9.10 (IH, bs), MS (LC/MS) m/z observed 546.92, expected 547.23 [M+H].

EXAMPLE A34

2-[4-({[(15,25)-2-METHYL-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5-

YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-lNDOL-l- YL]ETHYL}CARBA 0YL)BUTYL]CARBAM0YL} METHYL)PHENYL]ACETIC ACID

Title compound A34 was prepared from 1-7 and 1 ,4-phenylenediacetic acid using method J: Ή NMR (400 MHz, DMSO-tftf) 6 0.80 (3H, t, J=7.4Hz), 0.86 (3H, d, J=7Hz), 1.10 (I H, m), 1.44 (IH, m), 1.75 (IH, m), 3.1 1 (IH, m), 3.43 (2H, d, J=14Hz), 3.53 (2H, d, J=14Hz), 3.56-3.65 (2H, m), 4.15 (IH, m), 4.27 (IH, t, J=8Hz), 4.54 (IH, m), 4.65 (IH, m), 5.17 (IH, d, J=l lHz), 7.01 (IH, t, J=8Hz), 7.14-7-24 (6H, m), 8.04 (IH, d, J=8Hz), 8.10 (IH, d, J=9Hz), 8.25 (IH, bs), 9.08 (IH, bs), MS (LC/MS) m/z observed 591.15, expected 591.27 [M+H].

EXAMPLE A35

2-[3-({[(15,2-?)-2-METHYL-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5-

YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L- YL] ETHYL} CARBA OYL)BUTYL]CARBAMOYL} METHYL)PHENYL] ACETIC ACID

Title compound A35 was prepared from 1-7 and 1,3-phenylenediacetic acid using method J: Ή NMR (400 MHz, DMSC 6) δ 0.80 (3H, t, J=7.4Hz), 0.86 (311, d, J=7Hz), 1.10 (IH, m), 1.44 (IH, m), 1.75 (IH, m), 3.11 (IH, m), 3.43 (2H, d, J=14Hz), 3.55 (2H, d, J=14Hz), 3.57-3.66 (2H, m), 4.15 (IH, m), 4.27 (IH, t, J=8Hz), 4.55 (IH, m), 4.65 (IH, m), 5.17 (IH, d, J=l lHz), 7.01 (IH, t, J=8Hz), 7.08-7-24 (6H, m), 8.04 (IH, d, J=8Hz), 8.12 (IH, d, J=9Hz), 8.25 (IH, bs), 9.08 (IH, bs), MS (LC/MS) m/z observed 590.98, expected 591.27 [M+H].

EXAMPLE A36

(2£)-3-{[(l-?,25)-2-METHYL-l-({2-oxo-2-[(2S)-2-[(2H-l,2,3,4-TETRAZOL-5-

YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l-

YL]ETHYL}CARBAMOYL)BUTYL] CARBA OYL}PROP-2-ENOIC ACID Title compound A36 was prepared from 1-5 and fumaric acid using method J: Ή

NMR (400 MHz, DMSC 6) δ 0.83 (3H, t, J=7.4Hz), 0.90 (3H, d, J=7Hz), 1.13 (IH, m), 1.46 (IH, m), 1.78 (IH, m), 3.11 (IH, m), 3.57-3.66 (2H, m), 4.15 (IH, m), 4.37 (IH, t, J=8Hz), 4.55 (IH, m), 4.65 (IH, m), 5.17 (IH, d, J=l lHz), 6.52 (IH, d, J=15Hz), 7.01 (IH, t, J=8Hz), 7.12-7-24 (3H, m), 8.04 (IH, d, J=8Hz), 8.35 (IH, bs), 8.60 (IH, d, J=9Hz), 9.08 (IH, bs), MS (LC/MS) m/z observed 513.46, expected 513.22 [M+H].

EXAMPLE A37

(35^*)-3-AMiNO-3-{[(15,25)-2-METHYL-l-({2-oxo-2-[(2₁S)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L- YL] ETHYL} CARBAMOYL)BUTYL] CARBAMOYL} PROPANOIC ACID The intermediate compound ((S)-tert-Butyl 4-(((25,35)-l-((2-(2-(((2H-tetrazol-5- yl)methyl)carbamoyl)indolin-l-yl)-2-oxoethyl)amino)-3-methyl-l-oxopentan-2- yl)amino)-3-(S^)-((ier/^l-butoxycarbonyl)amino)-4-oxobutanoate) was prepared from 1-5 and Boc-Z-aspartic acid-P- e - -butyl ester using method A. MS (LC MS) m/z observed 685.99, expected 686.36 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

(S)-fert-Butyl 4-(((2S,35)-l-((2-(2-(((2H-tetrazol-5-yl)me l)carbamoyl)indolin-

1 -yl)-2-oxoethyl)amino)-3-methyl- 1 -oxopentan-2-yl)amino)-3-(5)-((ieri- butoxycarbonyl)amino)-4-oxobutanoate (30mg) was dissolved in a 1 : 1 mixture TFA/DCM (4 mL) and left under stirring at RT for 2 h. The solvents were evaporated and the product was purified on a CI 8 column using 10-30% MeOH in water to give title compound A37 as an off-white solid: Ή NMR (400 MHz, DMSC 6) δ 0.83 (3H, t, J=7.4Hz), 0.90 (3H, d, J=7Hz), 1.15 (IH, m), 1.55 (IH, m), 1.82 (I H, m), 2.68-2.84 (2H, m), 3.17 (IH, d, J=16Hz), 3.57-3.67 (2H, m), 4.07-4.18 (3H, m), 4.44 (IH, m), 4.56 (IH, m), 5.08 (IH, d, J=l lHz), 6.99 (IH, t, J=8Hz), 7.12-7-24 (2H, m), 8.04 (IH, d, J=8Hz), 8.32 (IH, bs), 8.45 (IH, m), 9.08 (IH, bs), MS (LC MS) m/z observed 530.17, expected 530.25 [M+H].

EXAMPLE A38

(S/iJ-S-AMINO-S-ililS^-l-METHYL-l-ii -OXO-Z-^^- -tilH-l^^^-TETRAZOL-S- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l- YL] ETHYL}CARBAM0YL)BUTYL] CARBAMOYL} PROPANOIC ACID

(S)-fer -Butyl 4-(((2₁S',35)-l-((2-(2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin- l -yl)-2-oxoethyl)amino)-3-methyl-l -oxopentan-2-yl)amino)-3-(i?)-(( er - butoxycarbonyl)amino)-4-oxobutanoate was prepared from 1-7 and Boc-Z aspartic acid- β-Zeri-butyl ester dicyclohexylammonium salt using method A. MS (LC/MS) m/z observed 686.07, expected 686.36 [Μ+Η], Compound was confirmed using LC/MS and moved to next step as it was.

(5)-feri-Butyl 4-(((25',3₁S -l-((2-(2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin- l-yl)-2-oxoethy])amino)-3-methyl-l -oxopentan-2-yl)amino)-3-(7?)-((½ri- butoxycarbonyl)amino)-4-oxobutanoate (30 mg) was dissolved in a 1 : 1 mixture trifluoroacetic acid/DCM (4 mL) and left under stirring at RT for 2 h. The solvents were evaporated and the product was purified on a CI 8 column using 10-30% MeOH in water to give title compound A38 as an off-white solid: *H NMR (400 MHz, DMSC 6) δ 0.83 (3H, t, J=7.4Hz), 0.90 (3H, d, J=7Hz), 1.13 (IH, m), 1.46 (IH, m), 1.78 (IH, m), 2.55-2.72 (2H, m), 3.08 (IH, d, J=16Hz), 3.52-3.65 (2H, m), 4.05 (IH, m), 4.18 (IH, m), 4.27 (IH, m), 4.44-4.49 (2H, m), 5.14 (IH, d, J=l lHz), 7.00 (IH, t, J=8Hz), 7.12-7-24 (2H, m), 8.03 (IH, d, J=8Hz), 8.35 (IH, bs), 8.52 (IH, m), 8.87 (IH, bs), MS (LC/MS) m/z observed 530.16, expected 530.25 [M+H].

EXAMPLE A39

(25)-2-AMiNO-3-{[(iS,25)-2-iviETHYL-l-({2-oxo-2-[(2S)-2-[(2H-l,2,3,4-TETRAZOL-5-

YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l- YL] ETHYL}CARBAM0YL)BUTYL]C ARB AMOYL} PROPANOIC ACID

(S -feri-Butyl 4-((l -((2-(2-(5)-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin- 1 - yl)-2-oxoethyl)ainiiio)-3-(₁S)-methyl-l -oxopentan-2-yl)amino)-2-(.S -((re; - butoxycarbonyl)amino)-4-oxobutanoate was prepared from 1-5 and Boc-L-aspartic acid- a-iert-butyl ester using method A. MS (LC/MS) m/z observed 686.09, expected 686.36 [Μ+Η], Compound was confirmed using LC/MS and moved to next step as it was.

(Syerf-Butyl 4-((l -((2-(2-(S)-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l - yl)-2-oxoethyl)amino)-3-(S)-methyl-l -oxopentan-2-yl)amino)-2-(₁5)-((½ /- butoxycarbonyl)amino)-4-oxobutanoate (30mg) was dissolved in a 1 : 1 mixture trifiuoroacetic acid/DCM (4 mL) and left under stirring at RT for 2 h. The solvents were evaporated and the product was purified on a CI 8 column using 10-30% MeOH in water to give title compound A39 as an off-white solid: 1H NMR (400 MHz, DMSO-r/6) δ 0.83 (3H, t, J=7.4Hz), 0.88 (3H, d, J=7Hz), 1.20 (IH, m), 1.51 (IH, m), 1.86 (IH, m), 2.37 (IH, d, J=15Hz), 2.88 (IH, dd, J=10, 14Hz), 3.01 (IH, d, J=16Hz), 3.52-3.63 (2H, m), 3.86 (I H, m), 4.10-4.20 (2H, m), 4.49 (IH, m), 4.61 (I H, m), 5.1 1 (I H, d, J=l lHz), 6.98 (IH, t, J=8Hz), 7.12-7-23 (2H, m), 8.03 (IH, d, J=8Hz), 8.35 (IH, d, J=8Hz), 8.52 (I H, m), 9.30 (IH, bs), MS (LC/MS) m/z observed 530.10, expected 530.25 [M+H].

EXAMPLE A40

(2/?)-2-AiviiNO-3-{[(i5^,,25)-2-lviETHYL-l-({2-oxo-2-[(2-S)-2-[(2H-l,2,3,4-TETRAZOL-5-

YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l- YL] ETHYL}CARBAMOYL)BUTYL] CARBAMOYL} PROPANOIC ACID

(S)-(ert-B ty\ 4-((l-((2-(2-(5)-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l- yl)-2-oxoethyl)amino)-3-(5)-methyl-l -oxopentan-2-yl)amino)-2-(i?)-((teri- butoxycarbonyl)amino)-4-oxobutanoate was prepared from 1-5 and Boc- -aspartic acid- oc-/er/-butyl ester using method A. MS (LC/MS) m/z observed 686.05, expected 686.36 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

4-((l-((2-(2-(5)-(((2H-tetrazol-5-yl)metliyl)carbamoyl)indolin-l- yl)-2-oxoethyl)amino)-3-(5)-methyl-l -oxopentan-2-yl)amino)-2-(R)-((/er/- butoxycarbonyl)amino)-4-oxobutanoate (30mg) was dissolved in a 1 : 1 mixture trifluoroacetic acid/DCM (4 mL) and left under stirring at RT for 2 h. The solvents were evaporated and the product was purified on a CI 8 column using 10-30% MeOH in water to give title compound A40 as an off-white solid: ^LU NMR (400 MHz, DMSCM6) δ 0.82 (3H, t, J=7.4Hz), 0.86 (3H, d, J=7Hz), 1.20 (IH, m), 1.50 (IH, m), 1.81 (IH, m), 2.50 (IH, m), 2.97-3.11 (2H, m), 3.55 (IH, m), 3.70 (IH, m), 3.82 (IH, m), 4.03 (IH, t, J= 8Hz), 4.18 (IH, dd, J=7, 16Hz), 4.49 (IH, dd, J=5, 6Hz), 4.66 (IH, dd, J=6, 17Hz), 5.08 (IH, d, J=l lHz), 6.98 (IH, t, J=8Hz), 7.12-7-23 (2H, m), 8.03 (IH, d, J=8Hz), 8.35 (IH, m), 8.48 (IH, d, J=8Hz), 9.34 (IH, bs), MS (LC MS) m/z observed 530.13, expected 530.25 [M+H].

EXAMPLE A41

(25)-1-{2-[3-FLUO O-3-METHYL-2-(2-PHENYLACETA IDO)BUTANAMIDO]ACETYL}-/V- (2H-L,2,3₅4-TETRAZOL-5-YLMETHYL)-2,3-DLHYDRO-LH-INDOLE-2-CARBOXAIVIIDE

3-Fluoro-3-methyl-2-(2-phenylacetamido)butanoic acid was prepared from 1-6 and 3-fluoro-DX-valine using method Η: 1H NMR (400 MHz, DMSO-i/6) δ 1.33 (3H, d, J=219Hz), 1.39 (3H, d, J=19Hz), 3.52 (IH, d, J=14Hz), 3.59 (IH, d, J=14Hz), 4.41, 4.46 (IH, 2 x d, J=19Hz), 7.20 (IH, m), 7.22-7.29 (3H, m), 8.50 (IH, d, J=9Hz), 12.90 (IH, bs), MS (LC/MS) m/z observed 254.02, expected 254.12 [M+H]. Compound was used further as described.

Title compound A41 was prepared from 1-4 and 3-fluoro-3-methyl-2-(2- phenylacetamido)butanoic acid using method C (as a mixture of diastereomers): MS (LC/MS) m/z observed 537.09, expected 537.24 [M+H].

EXAMPLE A42

(25)-l-{2-[(25',3 i)-3-METHYL-2-(2-PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-7A^r- (2H-l,2,3,4-TETRAZOL-5-YL ETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE (2S,3R)-3-Methyl-2-(2-phenylacetamido)pentanoic acid was prepared from 1-6 and a//o-isoleucine using method Η: 1H NMR (400 MHz, DMSO-</6) δ 0.75-0.90 (6H, m), 1.10 (IH, m), 1.25 (IH, m), 1.85 (IH, m), 3.47 (IH, d, J=14Hz), 3.56 (IH, d, J=14Hz), 4.35 (IH, m), 7.20 (I H, m), 7.24-7.30 (3H, m), 8.15 (IH, d, J=9Hz), 12.57 (IH, bs), MS (LC/MS) m/z observed 250.04, expected 250.14 [M+H]. Compound was used further as described.

Title compound A42 was prepared from 1-3 and (2>S',3/?)-3-methyl-2-(2- phenylacetamido)pentanoic acid using method A: Ή NMR (400 MHz, MeOH-rf4) δ 0.87-0.97 (6H, m), 1.18 (I H, m), 1.45 (I H, m), 1.99 (IH, m), 3.21 (IH, d, J=8Hz), 3.23 (I H, d, J=8Hz), 3.55-3.67 (2H, m), 3.69-3.76 (2H, m), 4.51 (IH, d, J=5Hz), 4.58-4.74 (2H, m), 5.16 (IH, d, J=l lHz), 7.05 (IH, t, J=8Hz), 7.15-7-35 (7H, m), 8.13 (IH, bs), MS (LC/MS) m/z observed 533.12, expected 533.26 [M+H].

EXAMPLE A43

^^^-TETRAFLUORO-S-llil^^-l- ETHYL-l-iil-OXO-Z-Iil^-l-KZH-l^^^- TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l- YL]ETHYL}CARBAMOYL)BUTYL]CARBAMOYL} PROPANOIC ACID

Title compound A43 was prepared from 1-5 and 2,2,3,3-tetrafluorosuccinic acid using method J in DMF: 1H NMR (400 MHz, DMSO-< 6) δ 0.80-0.84 (3H, t, J=8 Hz), 0.88-0.90 (3H, d, J=8 Hz), 1.08-1.15 (IH, m), 1.45-1.51 (IH, m), 1.83-1.91 (IH, m), 2.92-3.12 (2H, m), 3.56-3.64 (2H, m), 3.13-3.17 (IH, d, J=16 Hz), 4.27-4.31 (IH, t, J=8 Hz), 4.42-4.46 (IH, d, J=8 Hz), 4.58-4.62 (I H, d, J=16 Hz), 5.13-5.15 (IH, d, J=8 Hz), 6.98-7.02 (IH, t, J=8 Hz), 7.15-7.22 (2H, m), 8.03-8.05 (IH, d, J=8 Hz), 8.39(1H, s), 8.97(1H, bs), 9.90 (IH, bs) ¹⁹F NMR (376 MHz, DMSCM6) δ - 1 14.28 , -1 15.00 , -1 15.62, -1 16.35, -1 16.56, -1 17.28 , -1 18.35, -1 19.06., MS (LC/MS) m/z observed 587.01 , expected 587.19 [M+H].

EXAMPLE A44

2-[METHYL({[(15',25)-2-METHYL-l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL- 5-YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l-

YL] ETHYL}CARBAMOYL)BUTYL]CARBA OYL}METHYL)AMINO]ACETIC ACID

Title compound A44 was prepared from 1-5 and 4-methylmorpholine-2,6-dione using method I: ^lH NMR (400 MHz, DMSO-i 6) δ 0.82-0.86 (3H, t, J=8 Hz), 0.88-0.90 (3H, d, J=8 Hz), 1.04-1.1 1 (IH, m), 1.47-1.53 (IH, m), 1.77-1.84 (IH, m), 2.73 (lH,s), 2.89 (IH, s), 2.98-3.04 (4H, q, J=8 Hz), 3.53-3.67 (2H, m), 4.13-4.17 (IH, d, J=16 Hz), 4.30-4.34 (I H, t, J=8 Hz), 4.36-4.40 (IH, dd, J=12, 4 Hz), 4.48-4.52 (IH, dd, J=12, 4 Hz), 5.10-5.13 (IH, d, J=12 Hz), 6.97-7.01 (IH, t, 7=8 Hz), 7.14-7.18 (IH, t, J=8 Hz), 7.20-7.22 (IH, d, J=8 Hz), 7.95 (IH, s), 7.98-8.04 (IH, m), 8.42(1H, bs), 8.79(1 H, bs), MS (LC/MS) m/z observed 544.17, expected 544.26 [M+H].

EXAMPLE A45

3-{[( -CYCLOPENTYL({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TET AZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L-

YL]ETHYL}CARBAMOYL) ETHYL]CARBAMOYL}PROPANOIC ACID

((₁S)-2-((2-((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-2- oxoethyl)amino)-l-cyclopentyl-2-oxoethyl)carbamate was prepared from 1-3 and (S)-2- ((fer/-butoxycarbonyl)amino)-2-cyclopentylacetic acid using general method A. MS (LC/MS) m/z observed 527.03, expected 527.27 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as is.

Title compound A45 was prepared from teri-butyl ((S)-2-((2-((5)-2-(((2H- tetrazol-5-yl)methyl)carbamoyl)indolin-l-yl)-2-oxoethyl)amino)-l-cyclopentyl-2- oxoefhyl)carbamate and succinic anhydride using method I: Ή NMR (400 MHz, DMSC 6) δ 1.26-1.34 (2H, m), 1.41 -1.48 (2H, m), 1.51-1.67 (4H, m), 2.14-2.20 (IH, m), 2.37-2.44 (3H, m), 3.54-3.67 (3H, m), 4.16-4.26 (3H, m), 4.34-4.38 (IH, d, J=16 Hz), 4.44-4.48 ( IH, d, J=16 Hz), 5.09-5.11 (IH, d, J=8 Hz), 6.96-7.00 (IH, t, J=8 Hz), 7.13- 7.17 (IH, t, J=8 Hz), 7.19-7.21 (IH, d, J=8 Hz), 8.02-8.09 (2H, dd, J=20, 8 Hz), 8.16 (I H, bs), 8.72 (I H, bs), MS (LC MS) m/z observed 527.10, expected 527.23 [M+H].

EXAMPLE A46

3-{[(l.S)-2-METHYL-l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5-

YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l- YL]ETHYL}CARBAMOYL)PROPYL]CARBAMOYL}PROPANOIC ACID ter/-Butyl ((S)-\ -((2-((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-2- oxoethyl)amino)-3-methyl-l-oxobutan-2-yl)carbamate was prepared from 1-3 and (S)-2- ((/er^-butoxycarbonyl)amino)-3-methylbutanoic acid using general method A. MS (LC/MS) m/z observed 500.98, expected 501 .25 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as is.

Title compound A46 was prepared from /eri-butyl ((S)-\-((2-((S)-2-(((2H- tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-2-oxoethyl)amino)-3 -methyl- l -oxobutan-2- yl)carbamate and succinic anhydride using method I: 1H NMR (400 MHz, DMSO-rftf) δ 0.90-0.96 (6H, m), 2.01 -2.08 (IH, m), 2.44-2.50 (2H, m), 3.15-3.22 (2H, m), 3.57-3.66 (2H, m), 4.18-4.22 (IH, d, J=16 Hz), 4.26-4.30 (IH, t, J=8 Hz), 4.57-4.61 (IH, d, J=16 Hz), 4.64-4.68 (IH, d, J=16 Hz), 5.20-5.23 (IH, d, J=12 Hz), 7.03-7.07 (IH, t, J=8 Hz), 7.18-7.22 (IH, t, J=8 Hz), 7.26-7.28 (IH, d, J=8 Hz), 7.91 -7.95 (IH, dd, .7=16, 4 Hz), 8.06-8.08 (IH, d, 7=8 Hz), 8.21 (IH, bs), 8.27-8.31 (IH, t, J=8 Hz), 9.14 (IH, bs), MS (LC/MS) m/z observed 501.07, expected 501.21 [M+H] and observed 523.19, expected 523.21 [M+Na].

EXAMPLE A47

2-[(2,2-DIMETHYLPROPANOYL)OXY1ETHYL 3-{[(15,25)-2-METHYL-1-({2-OXO-2-[(25')- 2-[(2H-L,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L- YL]ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}PROPANOATE

Triethylamine (4.13 mL, 29.63 mmol) was added to ethylene glycol (22.5 mL). Pivaloyl chloride (3.1 mL, 25.19 mL) was then added slowly to this mixture and it was left at RT for 2 hrs. The reaction mixture was diluted with water (25 mL) and the product was extracted with DCM (4 x 20 mL). The combined organic layers were dried over sodium sulphate and concentrated. The product was purified on normal phase using 0% to 40% ethyl acetate in hexanes as the eluent to give a 2-hydroxyethyl pivalate as a colorless oil (3.99 g, 97%). 1H NMR (400 MHz, CDC1₃) δ 1.22-1.25 (9H, s), 2.02 (IH, bs), 3.80-3.85 (2H, m), 4.19-4.23 (2H, m).

Title compound A47 (75 mg, 0.146 mmol), 2-hydroxyethyl pivalate (32 mg, 0.219 mmol), EDC (42 mg, 0.219 mmol), DMAP (3.6 mg, 0.0292 mmol) were dissolved in DMF (5 mL). DIPEA (59 μΕ, 0.584 mmol) was then added and the reaction was heated to 50 for 6 hrs. The reaction mixture was concentrated and the product was purified on a CI 8 column using 10-65% MeOH in water to yield A47 as an off-white solid (35 mg, 37%). 1H NMR (400 MHz, DMSO-</6) δ 0.81 (3H, t, J=7.4Hz), 0.87 (3H, d, J-7Hz), 1.06-1.16 (10H, m), 1.45 (IH, m), 1.75 (IH, m), 2.35-2.48 (4H, m), 3.11 (IH, m), 3.55-3.65 (2H, m), 4.11-4.28 (6H, m), 4.52 (IH, m), 4.65 (IH, m), 5.13 (IH, d, J=l lHz), 7.01 (IH, t, J=8Hz), 7.14-7-26 (2H, m), 7.92 (IH, d, J=9Hz), 8.03 (IH, d, J=8Hz), 8.18 (IH, bs), 9.08 (IH, bs), MS (LC/MS) m/z observed 643.15, expected 643.32 [M+H]. EXAMPLE A48

(15,25)-2-{[(15,2S)-2-METHYL-l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBA OYL]-2,3-DIHYDRO-LH-INDOL-L- YL] ETHYL}CARBAMOYL)BUTYL] CARBAMOYL}CYCLOPROPANE-1-CARBOXYLIC CID To the stirring mixture of (lS^^-diethyl cyclopropane- 1,2-dicarboxylate (0.5 gm,

2.69 mmol) in 1 : 1 mixture of water:THF (2.7 ml) was added ammonium hydroxide (28%, 3.8 ml). The resulting reaction mixture was sealed in a flask and stirred at RT for 16 hrs. The reaction mixture was concentrated to dryness under vacuum. The resulting residue of (lS^S^cyclopropane-l^-dicarboxylic acid, was dried well under vacuum and subjected to next reaction as it is.

Title compound A48 was prepared from 1-5 and (lS^^-cyclopropane-l^- dicarboxylic acid using method J. MS (LC MS) m/z observed 525.99, expected 527.55 [M+H]. Compound was confirmed using LCMS.

EXAMPLE A49

(2S)-l-{2-[(2S',3S)-3-METHYi^2-l(2if)-2-

PHENYLPROPANAMrDO]PENTANAMIDO]ACETYL}-/V-(2H-1,2,3,4-TETRAZOL-5- YLMETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

Title compound A49 (7 mg, 0.01 mmol, 10%) was collected as an off white solid from intermediate 1-15 (60 mg, 0.13 mmol) using general method M via reaction with ( -2-phenyl-propionic acid. Ή NMR (400 MHz, DMSO-rf6) δ 9.00 (IH, bs), 8.23 (IH, bs), 8.04 (IH, d, J=7Hz), 7.96 (IH, d, J=9Hz), 7.35 (2H, d, J=7Hz), 7.28 (2H, t, J=7Hz), 7.25-7.15 (3H, m), 7.01 (IH, t, J=7Hz), 5.17 (IH, d, J=9Hz), 4.64 (IH, d, J=16Hz), 4.50 (IH, d, J=16Hz), 4.23 (IH, t, J=8Hz), 4.10 (IH, dd, J=10, 5Hz), 3.81 (IH, q, J=7Hz), 3.70-3.55 (2H, m), 3.16 (IH, d, J=5Hz), 1.68 (IH, m), 1.31 (3H, d, J=7Hz), 1.25 (IH, m), 0.97 (IH, m), 0.72 (3H, d, J=7Hz), 0.66 (3H, t, J=7Hz), MS (LC/MS) m/z observed 547.09, expected 547.28 [M+H]. EXAMPLE A50

(2S)-l-{2-[(2^,35)-3-METHYL-2-[(25)-2- PHENYLPROPANAMIDO]PENTANAMIDO]ACETYL}-7V-(2H-l,2,3,4-TET AZOL-5- YLMETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE Title compound A50 (10 mg, 0.01 mmol, 15%) was collected as an off white solid from intermediate 1-15 (60 mg, 0.13 mmol) using general method M via reaction with (5 -2-phenyl-propionic acid. Ή NMR (400 MHz, DMSO-</6) δ 9.06 (IH, bs), 8.15 (IH, bs), 8.00 (2H, d, J=9Hz), 7.32 (2H, d, J=7Hz), 7.27 (2H, t, J=7Hz), 7.23-7.13 (3H, m), 7.00 (IH, t, J=7Hz), 5.13 (IH, d, J=10Hz), 4.64 (IH, dd, J=16, 5Hz), 4.53 (IH, dd, J=16, 5Hz), 4.30 (IH, t, J=8Hz), 4.10 (IH, dd, J=16, 5IIz), 3.81 (III, q, J=7IIz), 3.67-3.50 (2H, m), 3.13 (IH, d, J=16Hz), 1.77 (IH, m), 1.47 (IH, m), 1.33 (3H, d, J=7Hz), 1.18 (IH, m), 0.88 (3H, d, J=7Hz), 0.84 (3H, t, J=7Hz), MS (LC/MS) m/z observed 547.05, expected 547.28 [M+H].

EXAMPLE A51

4-{[(15',25)-2-METHYL-l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBA OYL]-2,3-DIHYDRO-LH-INDOL-L- YL] ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}-2-PHENYLBUTANOIC ACID

Title compound A51 (20 mg, 0.035 mmol, 49%) was collected as an off white solid from intermediate 1-15 (30 mg, 0.07 mmol) using general method N via reaction with 3-phenyl-dihydro-pyran-2,6-dione, as an inseparable mixture of diastereomers. ^lH NMR (400 MHz, DMSO-i 6) δ 9.06 (IH, bs), 8.14 (IH, bs), 8.03 (IH, bs), 7.87 (IH, t, J=7Hz), 7.40-7.10 (7H, m), 7.01 (IH, t, J=7Hz), 5.16 (IH, m), 4.65 (IH, d, J=16Hz), 4.53 (IH, d, J=16Hz), 4.25 (IH, t, J=7Hz), 4.14 (IH, m), 3.60 (2H, m), 3.50 (IH, t, J=7Hz), 3.13 (IH, d, J=12Hz), 2.25-2.00 (3H, m), 1.86 (IH, m), 1.73 (IH, m), 1.43 (IH, m), 1.10 (IH, m), 0.86 (3H, d, J=6Hz), 0.82 (3H, t, J=6Hz), (MS (LC/MS) m/z observed 605.06, expected 605.28 [M+H]. EXAMPLE A52

^-Dl ETHYL^-iKl^^-METHYL-l-iil-OXO-Z-Ii ^^-fi H-l ^^^-TETRAZOL-S- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l- YL]ETHYL}CARBAIVtOYL)BUTYL]CARBAIV10YL}BUTANOIC ACID Title compound A52 (24 mg, 0.04 mmol, 40%) was collected as an off white solid from intermediate 1-15 (50 mg, 0.11 mmol) using general method N via reaction with 3,3-dimethyl-dihydro-pyran-2,6-dione. 1H NMR (400 MHz, DMSO-rf6) δ 9.05 (IH, bs), 8.15 (IH, bs), 8.03 (IH, d, J=8Hz), 7.89 (IH, d, J=9Hz), 7.22 (IH, d, J=7Hz), 7.17 (IH, t, J=7Hz), 7.01 (IH, t, J=8Hz), 5.17 (IH, d, J=10Hz), 4.65 (IH, dd, J=16, 6Hz), 4.53 (IH, dd, J=16, 4Hz), 4.24 (IH, t, J=8Hz), 4.15 (IH, dd, J=16, 4IIz), 3.60 (211, m), 3.14 (III, d, J=16Hz), 2.14 (2H, m), 1.80-1.62 (3H, m), 1.45 (IH, m), 1.20-1.10 (7H, m), 0.86 (3H, d, J=7Hz), 0.81 (3H, t, J=8Hz), MS (LC/MS) m/z observed 557.16, expected 557.28 [M+H].

EXAMPLE A53

2,2-DlMETHYL-3-{[(15',2S)-2-METHYL-l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIIIYDRO-LH-[NDOL-L- YL] ETHYL}CARBAMOYL)BLITYL] CARBAMOYL}PROPANOIC ACID

Title compound A53 (22 mg, 0.04 mmol, 62%) was collected as an off white solid from intermediate 1-15 (30 mg, 0.07 mmol) using general method N via reaction with 3,3-dimethyl-dihydro-furan-2,5-dione. Ή NMR (400 MHz, DMSO-i 6) δ 9.09 (IH, bs), 8.17 (I H, bs), 8.02 (IH, d, J=9Hz), 7.81 (IH, d, J=9Hz), 7.22 (IH, d, J=7Hz), 7.17 (I H, t, J=8Hz), 7.01 (IH, t, J=8Hz), 5.17 (IH, d, J=12Hz), 4.66 (IH, dd, J=16, 5Hz), 4.55 (IH, dd, J=16, 4Hz), 4.26 (IH, t, J=8Hz), 4.14 (IH, dd, J=16, 5Hz), 3.60 (2H, m), 3.14 (IH, d, J=16Hz), 2.46 (IH, d, J=l lHz), 2.41 (IH, d, J=l lHz), 1.74 (IH, m), 1.45 (IH, m) 1.19 (IH, m), 1.12 (3H, s), 1.11 (3H, s), 0.86 (3H, d, J=6Hz), 0.81 (3H, t, J=7Hz), MS (LC/MS) m/z observed 543.04, expected 543.27 [M+H]. EXAMPLE A54

4-{[(15,2.S)-2-METHYL-l-({2-oxo-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5-

YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L- YL] ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}-4-PHENYLBUTANOIC ACID

Title compound A54 (25mg, 0.08 mmol, 37%) was prepared from 1-15 (l OOmg,

0.22 mmol) and 3-phenyl-dihydro-pyran-2,6-dione using method P, where the hydrolysis step was stopped after 8h. A54 was separated by chromatography, into two diastereomers, A54-1 (eluted at 63% MeOH in water) and A54-2 (eluted at 65% MeOH in water), each characterized as below:

A54-1:

1H NMR (400 MHz, DMSO-rf6) 5 8.70 (IH, bs), 8.22-8.10 (2H, bs), 8.01 (IH, d, J=8Hz), 7.37-7.10 (7H, m), 6.99 (IH, t, J=8Hz), 5.08 (IH, d, J=9Hz), 4.51 (IH, d, J=14Hz), 4.40-4.20 (2H, m), 4.06 (IH, d, J=13Hz), 3.66 (IH, m), 3.51 (IH, m), 3.38 (IH, m), 3.06 (IH, d, J=18Hz), 2.20-2.05 (3H, m), 1.83 (IH, m), 1.77 (IH, m), 1.45 (IH, m), 1.12 (IH, m), 0.89 (3H, d, J=7Hz), 0.84 (3H, t, J=7Hz), MS (LC MS) m/z observed 605.09, expected 605.28 [M+H].

A54-2:

Ή NMR (400 MHz, DMSO-ifa) δ 9.07 (IH, bs), 8.20 (IH, bs), 8.10-8.00 (2H, m), 7.40-7.10 (7H, m), 7.01 (IH, t, J=7Hz), 5.17 (IH, d, J=10Hz), 4.65 (IH, dd, j=16, 5Hz), 4.54 (IH, dd, J=16, 4Hz), 4.28-4.12 (2H, m), 3.68 (IH, t, J=6Hz), 3.61 (IH, m), 3.39 (IH, m), 3.14 (IH, d, J=16Hz), 2.25-2.00 (3H, m), 1.84 (IH, m), 1.70 (IH, m), 1.23 (IH, m), 0.89 (IH, m), 0.70 (3H, d, J=6Hz), 0.82 (3H, t, J=7Hz), MS (LC/MS) m/z observed 605.13, expected 605.28 [M+H].

EXAMPLE A55

4,4-DlMETHYL-4-{[(lS,2S)-2-METHYL-l-({2-OXO-2-[(2S)-2-[(2H-l,2,3,4-TETRAZOL-5-

YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L- YL] ETHYL}CARBAMOYL)BUTYL]CARBAMOYL}BUTANOIC ACID

Title compound A55 (9 mg, 0.03 mmol, 30%) was prepared from 1-15 (51 mg, 0.11 mmol) and 3,3-dimethyl-dihydro-pyran-2,6-dione using method P, where the hydrolysis step was stopped after 20 h. 1H NMR (400 MHz, DMSO-i 6) δ 9.06 (1 H, bs), 8.15 (IH, bs), 8.05 (IH, d, J=8Hz), 7.30-7.15 (3H, m) 7.02 (IH, t, J=8Hz), 5.17 (IH, d, J=10Hz), 4.64 (IH, dd, J=16, 5Hz), 4.54 (IH, dd, J=16, 5Hz), 4.24 (IH, t, J=8Hz), 4.19 (IH, m), 3.61 (2H, m), 3.39 (IH, m), 3.14 (IH, d, J=16Hz), 2.12 (2H, m), 1.86 (IH, m), 1.75 (IH, m), 1.48 (IH, m), 1.20-1.05 (7H, m), 0.89 (3H, d, J=7Hz), 0.83 (3H, t, J=7Hz), MS (LC/MS) m/z observed 557.10, expected 557.28 [M+H].

EXAMPLE A56

3,3-DlMETHYL-3-{[(15,25)-2-METHYL-l-({2-OXO-2-[(2S)-2-[(2H-l,2,3,4-TETRAZOL-5-

YLMETHYL)CARBA OYL]-2,3-DIHYDRO-LH-INDOL-L- YL] ETHYL}CARBAMOYL)BUTYL] CARBAMOYL}PROPANOIC ACID

Title compound A56 (1 1 mg, 0.02 mmol, 11%) was prepared as an off-white solid, from 1-15 (80 mg, 0.18 mmol) and 3,3-dimethyl-dihydro-furan-2,5-dione using method P, where the hydrolysis step was stopped after 7 h. Ή NMR (400 MHz, DMSCW6) δ 9.04 (IH, bs), 8.17 (IH, bs), 8.02 (IH, d, J=8Hz), 7.82 (IH, d, J=9Hz), 7.22 (IH, d, J=7Hz), 7.17 (IH, t, J=8Hz), 7.01 (IH, t, J=8Hz), 5.17 (IH, d, J=10Hz), 4.63 (IH, dd, J=16, 5Hz), 4.53 (IH, dd, J=16, 4Hz), 4.25 (IH, t, J=8Hz), 4.14 (IH, dd, J=16, 4Hz), 3.62 (2H, m), 3.14 (IH, d, ,/=16Hz), 2.48 (IH, d, J=16Hz), 2.42 (IH, d, J=16Hz), 1.76 (IH, m), 1.45 (IH, m), 1.25-1.05 (7H, m), 0.86 (3H, d, J=6Hz), 0.82 (3H, t, J=7Hz), MS (LC/MS) m/z observed 543.10, expected 543.27 [M+H].

EXAMPLE A57

2-{[(lS,2S)-2-METHYL-l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L- YL] ETHYL}CARBAMOYL)BUTYL] CARBAMOYL}-TRANS-CYCLOPENTANE-1-CARBOXYLIC

ACID

Title compound A57 was prepared from 1-15 (71 mg, 0.16 mmol) and cis- cyclopentane- 1 ,2-dicarboxylic acid using method J without HC1 treatment and was further separated by chromatography, into two diastereomers, A57-1 (9.9 mg, 0.04 mmol, 23%) and A57-2 (6.7 mg, 0.02 mmol, 15%), each characterized as below.

A57-1:

¹H NMR (400 MHz, DMSO-< 6) δ 8.90 (IH, bs), 8.15 (IH, bs), 8.03 (IH, d, J=8Hz), 7.80 (IH, d, J=9Hz), 7.22 (IH, d, J=7Hz), 7.17 (IH, t, J=8Hz), 7.00 (IH, t, J=7Hz), 5.15 (IH, d, J=9Hz), 4.57 (IH, d, J=12Hz), 4.45 (IH, d, J=12Hz), 4.27 (IH, t, J=8Hz), 4.16 (IH, dd, J=9, 5Hz), 3.69 (I H, dd, J=8, 4Hz), 3.64 (IH, m), 3.1 1 (IH, d, J=14Hz), 3.06-2.85 (2H, m), 2.10-1.82 (2H, m), 1.80-1.66 (2H, m), 1.66-1.52 (3H, m), 1.46 (IH, m), 1.09 (IH, m), 0.86 (3H, d, J=6Hz), 0.81 (3H, t, J=7Hz), MS (LC/MS) m/z observed 554.94, expected 555.27 [M+H].

A57-2:

Ή NMR (400 MHz, DMSO-rffi) δ 8.81 (IH, bs), 8.13 (IH, bs), 8.10-7.95 (2H, m), 7.21 (I H, d, J=7Hz), 7.16 (IH, t, J=8Hz), 6.99 (IH, t, J=8Hz), 5.15 (IH, d, J=10Hz), 4.49 (IH, dd, J=17, 6Hz), 4.45 (IH, d, J=17, 6Hz), 4.25 (I H, t, J=8Hz), 4.17 (IH, dd, j=14, 5Hz), 3.69 (I H, dd, J=13, 4Hz), 3.58 (IH, dd, J=15, 8Hz), 3.05 (IH, d, J=16Hz), 2.97 (2H, m), 2.00-1.85 (2H, m), 1.85-153 (5H, m), 1.13 (IH, m), 0.87 (3H, d, J=6Hz), 0.82 (3H, t, J=7Hz), MS (LC/MS) m/z observed 554.98, expected 555.27 [M+H].

Examples B 1-B7 were prepared by the representative synthetic pathway illustrated schematically in FIGURE 2.

EXAMPLE Bl

(2-y)-l-{2-[(2-S',3-?)-3-METHYL-2-(2-PHENYLACETAMIDO)PENTANAIVIIDOjACETYL}-N- (lH-l,2,3-TRIAZOL-4-YL ETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE Title compound Bl was prepared from 1-9 and (lH-[l,2,3]triazol-4- yl)methanamine using method G followed by purification by preparative HPLC (Column: ASCENTIS™ CI 8, 25 cm x 21.2 mm, 10 μπι, gradient 0%→100% Methanol/water with 0.1% TFA, 10 mL/min) (11 mg, cream-coloured powder). Ή NMR, (300 MHz, DMSO-i/6) δ 8.91-8.83 (m, IH), 8.29-8.02 (m, 2H), 7.74-7.64 (m, IH), 7.30-7.15 (m, 7H), 7.03-6.97 (m, IH), 5.15-5.04 (m, IH), 4.44-4.07 (m, 4H), 3.63-3.02 (m, 5H), 1.90- 1.68 (m, I H), 1.50-1.21 (m, IH), 1.16-1.01 (m, IH), 0.85-0.76 (m, 6H). MS (ESI) m/z observed 532.33 observed, expected 532.27 [M+H].

EXAMPLE B2

(2S)-l-{2-[(25,3S)-3-METHYL-2-(2-PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-N- (l,2,3-THIADIAZOL-4-YLMETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

Title compound B2 was prepared from 1-9 and ([l ,2,3]thiadiazol-4- yl)methanamine using method G. 1H NMR, (300 MHz, DMSO-< 6) δ 9.15-8.96 (m, IH), 8.26-7.98 (m, 2H), 7.31-7.15 (m, 8H), 7.04-6.94 (m, IH), 5.19-5.10 (m, IH), 4.87-4.68 (m, 2H), 4.31-4.22 (m, IH), 4.18-4.01 (m, IH), 3.66-3.43 (m, 4H), 3.12-3.02 (m, I H), 1.82-1.66 (m, IH), 1.51-1.37 (m, IH), 1.18-1.03 (m, IH), 0.89-0.76 (m, 6H). MS (ESI) m/z observed 583.20, expected 583.19 [M+Cl] and observed 549.20, expected 549.23 [M+H] and observed 571.20, expected 571.21 [M+Na]. EXAMPLE B3

(25)-1-{2-[(25,3-S)-3-METHYL-2-(2-PHENYLACETA IDO)PENTANAIVIIDO]ACETYL}-_J/V-[1- (LH-L,2,3-TRIAZOL-4-YL)ETHYL]-2,3-DIHYDRO-LH-INDOLE-2-CARBOXAMIDE

Title compound B3 was prepared from 1-9 and l -(2H-tetrazol-5-yl)ethanamine using method G as a mixture of diastereomers. Ή NMR, (300 MHz, CD₃OD) δ 7.35- 7.21 (m, 8H), 7.09-7.03 (m, IH), 5.39-5.32 (m, I H), 5.17-5.09 (m, IH), 4.38-4.32 (m, 2H), 3.62-3.58 (m, 3H), 3.26-3.14 (m, 2H), 1.95- 1.84 (m, IH), 1.68-1.61 (m, 3H), 1.59- 1.47 (m, IH), 1.22-1.09 (m, IH), 0.98-0.86 (m, 6H). MS (ESI) m/z observed 545.48, expected 545.26 [M-H].

EXAMPLE D4

(25)-7V-[(4-METHYL-lH-l,2,3-TRIAZOL-5-YL)METHYL]-l-{2-[(25,35)-3-METHYL-2-(2- PHENYLACETA IDO)PENTANAlVIIDO]ACETYL}-2,3-DIHYDRO-lH-INDOLE-2-

CARBOXAMIDE

Title compound B4 was prepared from 1-9 and (4-methyl-lH-[l ,2,3]triazol-5- yl)methanamine using method C. 1H NMR, (300 MHz, DMSO-i/6) δ 8.76 (br s, IH), 8.20 (br s, IH), 8.1 1-8.00 (m, IH), 7.31 -7.15 (m, 8H), 7.03-6.97 (m, IH), 4.43-4.24 (m, 3H), 4.19-4.05 (m, IH), 3.61 -3.34 (m, 5H), 3.04-2.98 (m, IH), 2.17 (s, 3H), 1.89-1.69 (m, IH), 1.48-1.19 (m, 2H), 0.85-0.77 (m, 6H). MS (ESI) m/z observed 544.52, expected 544.27 [M-H].

EXAMPLE B5

(25)-l-{2-[(25^,,3S)-3-METHYL-2-(2-PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-A^?- (lH-l,2,3-TRIAZOL-4-YLMETHYL)-l,2,3,4-TETRAHYDROQUINOLINE-2-CARBOXAMIDE

Title compound B5 was prepared from 1-8 and l ,2,3,4-tetrahydroquinoline-2- carboxylic acid using method F followed by addition of l -(l H-[l ,2,3]triazol-4- yl)methanamine using method C, as a mixture of diastereomers. Ή NMR, (300 MHz, acetone-^6) δ 7.83 (br s, IH), 7.61-7.17 (m, 9H), 5.18 (t, IH), 4.45-4.33 (m, 4H), 3.85- 3.76 (m, IH), 3.67-3.55 (m, 2H), 2.79-2.69 (m, IH), 2.62-2.45 (m, 2H), 1.96-1.78 (m, 2H), 1.53-1.40 (m, IH), 1.15-1.04 (m, IH), 0.88-0.78 (m, 6H). MS (ESI) m/z observed 546.60, expected 546.28 [M-H]. EXAMPLE B6

6-METHOXY-l-{2-[(2S,3S)-3-METHYL-2-(2- PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-A'-(lH-l,2,3-TRIAZOL-5-YLMETHYL)- 2,3-DIHYDRO-lH-INDOLE-2-CARBOXA IDE

Title compound B6 was prepared from 1-8 and 5-methoxy-indoline-2-carboxylic acid methyl ester using method F followed by ester hydrolysis using method D and finally addition of l -(lH-[l ,2,3]triazol-4-yl)methanamine using method C. Ή NMR, (300 MHz, DMSO-< 6) δ 8.87-8.78 (m, IH), 8.26-7.98 (m, 2H), 7.74-7.64 (m, 1H), 7.30- 7.24 (m, 5H), 7.24-7.16 (m, IH), 7.12-7.06 (m, IH), 6.63-6.56 (m, IH), 5.15-5.06 (m, I H), 4.44-4.24 (m, 3H), 3.72 (s, 3H), 3.62-3.43 (m, 4H), 3.16 (s, IH), 3.01 -2.92 (m, I H), 1.90- 1.70 (m, I H), 1.49-1.26 (m, I H), 1.15-1.02 (m, IH), 0.85-0.77 (m, 6H). MS (ESI) m/z observed 560.59, expected 560.26 [M+H].

EXAMPLE B7

5-METH YL-1 - {2- [(25,35)-3-METHYL-2-(2- PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-N-(lH-l,2,3-TRIAZOL-4-YLMETHYL)-

2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

Title compound B7 was prepared from 1-8 and 6-methoxy-indoline-2-carboxylic acid ethyl ester using method C followed by ester hydrolysis using method D and finally addition of l -(lH-[l ,2,3]triazol-4-yl)methanamine using method C as a mixture of diastereomers. Ή NMR, (300 MHz, CD₃OD) δ 8.86-8.62 (m, IH), 8.32-7.93 (m, 2H), 7.68 (m, IH), 7.37-6.95 (m, 8H), 5.18-5.02 (m, 2H), 4.55-4.44 (m, 3H), 4.38-4.29 (m, IH), 3.70-3.54 (m, 3H), 3.22-3.05 (m, IH), 2.30 (s, 3H), 2.03-1.84 (m, I H), 1.60-1.49 (m, I H), 1.25-1.10 (m, I H), 0.96-0.86 (m, 6H). MS (ESI) m/z observed 544.53, expected 544.27 [M-H].

Examples C1 -C39 were prepared by the representative synthetic pathway illustrated schematically in FIGURE 3.

EXAMPLE C I

(25)-l-{2-[(25)-3-METHYL-2-(2-PHENYLACETAIVIIDO)BlJTANAMIDO]ACETYL}-7V-(2H- l,2,3,4-TETRAZOL-5-YLIVIETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE 1-6 (0.5 g, 3.67 mmol) and thionyl chloride (1.6 ml, 22 mmol) were stirred together for 1 hr at room temperature. Thionyl chloride was removed by distillation under vacuum. The acid chloride was added to the stirring solution of L-valine (0.39 g, 3.305 mmol) in NaOH (2 N, 4.2 ml) at 0 °C. The resulting reaction mixture was warmed to RT and stirred overnight. The mixture was washed with diethyl ether (5 ml) and acidified to pH 4-5 by adding citric acid (aqueous, saturated solution). The precipitated solid was filtered, washed with diethyl ether and dried to yield (S)-3-methyl-2-(2- phenylacetamido)butanoic acid as a white solid (0.64 g, 74%). Ή NMR (400 MHz, DMSO-i 6) 5 0.82-0.84 (3H, d, J=8 Hz), 0.85-0.87 (3H, d, J=8 Hz), 1.99-2.06 (1H, m), 3.44-3.55 (2H, q, J=12 Hz), 4.10-4.14 (1H, dd, J=8, 12 Hz), 7.16-7.21 (1H, m), 7.24-7.29 (4H, m), 7.19-7.21 (1H, d, J=8 Hz), 12.55 (1H, s), MS (LC/MS) m/z observed 236.04, expected 236.13 [M+H]. The compound was used further as described.

Title compound CI was prepared from 1-3 and (»S)-3-methyl-2-(2- phenylacetamido)butanoic acid using method A: Ή NMR (400 MHz, DMSC 6) δ 0.81-0.85 (6H, m), 1.94-2.01 (1H, m), 3.04-3.10 (2H, m), 3.42-3.45 (1H, d, J=12 Hz), 3.53-3.58 (3H, m), 4.1 1-4.15 (1H, d, J=16 Hz), 4.22-4.26 (1H, q, J=12 Hz), 4.37-4.43 (1H, t, J=12 Hz), 4.47-4.54 (1H, m), 5.12-5.14 (1H, d, J=8 Hz), 6.96-7.00 (1H, t, J=8 Hz), 7.13-7.21 (3H, m), 7.25-7.26 (4H, d, J=4 Hz), 8.02-8.03 (1H, d, J=4 Hz), 8.05- 8.13 (1H, m), 8.26(1 H, bs), 8.77(1H, bs), MS (LC/MS) m/z observed 519.01 , expected 519.25 [M+H] and observed 541.1 1 , expected 541.23 [M+Na].

EXAMPLE C2

(2I¾-1-{2-[(25^,,3_JK)-3-HYDROXY-2-(2-PHENYLACETAMIDO)BUTANAMIDO]ACETYL}-7V-

(2H-l ,2,3,4-TETRAZOL-5-YL ETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXA IDE

1-6 (0.5 g, 3.67 mmol) and thionyl chloride (1.6 ml, 22 mmol) were stirred together for 1 hr at room temperature. Thionyl chloride was removed by distillation under vacuum. The acid chloride was added to the stirring solution of L-threonine (0.39 g, 3.305 mmol) in NaOH (2 N, 4.2 ml) at 0 °C. The resulting reaction mixture was warmed to RT and stirred overnight. The reaction mixture was washed with diethyl ether (5 ml) and acidified to pH 4-5 by adding citric acid (aqueous, saturated solution). The precipitated solid was filtered, washed with diethyl ether and dried to yield (2S,3R)-3- hydroxy-2-(2-phenylacetamido)butanoic acid as a white solid (0.71 g, 82%). Ή NMR (400 MHz, DMSC 6) δ 0.99-1.01 (3H, d, J=8 Hz), 3.49-3.60 (2H, q, J=12 Hz), 4.08- 4.13 (1 H, m), 4.17-4.20 (1H, dd, J=4,8 Hz), 4.89 (lH,bs), 7.16-7.31 (5H, m), 7.97-7.99 (IH, d, J=8 Hz), 12.42 (IH, bs), MS (LC/MS) m/z observed 238.00, expected 238.1 1 [M+H]. The compound was used further as described.

Title compound C2 was prepared from 1-3 and (25',3^)-3-hydroxy-2-(2- phenylacetamido)butanoic acid using method A: Ή NMR (400 MHz, DMSO-rf6) δ 1.00-1.02 (3H, d, J=8 Hz), 1.66- 1.74 (I H, m), 3.06-3.12 (2H, m), 3.49-3.53 (IH, t, J=8 Hz), 3.55-3.62 (2H, m), 3.98-4.02 (IH, t, J=8 Hz), 4.14-4.18 (IH, d, J=16 Hz), 4.24-4.27 (IH, dd, J=4, 8 Hz), 4.44-4.49 (IH, m), 4.54-4.62 (IH, m), 5.12-5.18 (IH, m), 6.96-7.02 (IH, m), 7.16-7.22(3H, m), 7.26-7.28 (4H, q, J=4 Hz), 7.99-8.03 (I H, t, J=8 Hz), 8.08 (IH, bs), 8.99 (I H, bs), MS (LC/MS) m/z observed 521.04, expected 521.23 [M+H] and observed 543.1 1 , expected 543.21 [M+Na].

EXAMPLE C3

(2- )-l-{2-[(2S,3-S)-3-HYDROXY-2-(2-PHENYLACETAMIDO)BUTANAMIDO]ACETYL}-N- (2H-l,2,3,4-TETRAZOL-5-YL ETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

(25',35)-3-Hydroxy-2-(2-phenylacetamido)butanoic acid was prepared from 1-6 and (25,3S)-2-amino-4-hydroxybutanoic acid using method H: 1H NMR (400 MHz, DMSO-(/6) δ 1.05 (3H, d, J=6.4 Hz), 3.55 (2H, s), 3.85 (IH, m), 4.17 (IH, dd, J=4.0, 8.4 Hz), 4.89 (lH,bs), 7.13-7.30 (5H, m), 8.19 (IH, d, J=8.4 Hz), 12.35 (IH, bs), MS (LC/MS) m/z observed 237.86, expected 238.1 1 [M+H]. Compound was used further as described.

Title compound C3 was prepared from (25",35)-3 -hydro xy-2-(2- phenylacetamido)butanoic acid and 1-4 using method A: Ή NMR (400 MHz, DMSO- (16) δ 0.97-1.07 (3H, m), 2.93-3.15 (2H, m), 3.45-3.70 (3H, m), 3.82-3.88 (IH, m), 4.11 - 4.20 (I H, m), 4.22-4.30 (I H, m), 4.32-4.42 (IH, m), 4.45-4.55 (IH, m), 5.15 (IH, d, J=9.5Hz), 6.97 (IH, t, J=7.4Hz), 7.13-7.30 (7H, m), 7.92-8.28 (3H, m), 8.75 (IH, bs), MS (LC/MS) m/z observed 521.00, expected 521.23 [M+H].

EXAMPLE C4

(25)-l-{2-[(2S)-4-METHYL-2-(2-PHENYLACETAl IDO)PENTANAMIDO]ACETYL}-7V-(2H- l,2,3,4-TETRAZOL-5-YLMETHYL)-2,3-DIHYDRO-l H-INDOLE-2-CARBOXA IDE

(<S)-4-Methyl-2-(2-phenylacetamido)pentanoic acid was prepared from 1-6 and (5)-2-amino-4-methylpentanoic acid using method Η: ^JH NMR (400 MHz, DMSO-rf6) δ 0.85 (3H, d, J=6.4 Hz), 1.05 (3H, d, J=6.4 Hz), 1.45-1.53 (2H, m), 1.56-1.66 (IH, m), 3.40-3.50 (2H, m), 4.15-4.25 (IH, m), 7.13-7.30 (5H, m), 8.30 (I H, d, J=8.1 Hz), 12.50 (IH, bs), MS (LC/MS) m/z observed 249.98, expected 250.14 [M+H]. Compound was used further as described.

Title compound C4 was prepared from (S)-4-methyl-2-(2- phenylacetamido)pentanoic acid and 1-3 using method A: Ή NMR (400 MHz, DMSO- d6) δ 0.77 (3H, d, J=6.4 Hz), 0.88 (3H, d, J=6.4 Hz), 1.45- 1.53 (2H, m), 1.55-1.66 (I H, m), 2.95-3.12 (2H, m), 3.40-3.65 (3H, m), 4.07-4.15 (IH, m), 4.31 -4.43 (2H, m), 4.45- 4.55 (IH, m), 5.12 (IH, m), 7.01 (I H, t, J=7Hz), 7.15-7.33 (7H, m), 7.97-8.10 (IH, d, J=8Hz), 8.20-8.38 (2H, m), 8.72 (I H, bs), MS (LC/MS) m/z observed 533.00, expected 533.26 [M+H].

EXAMPLE C5

(25 -l-{2-[(2,S)-2-(2-PHENYLACETAIVlIDO)PENTANAlVlIDO]ACETYL}-/V-(2H-l,2,3,4- TETRAZOL-5-YLMETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

(5)-2-(2-Phenylacetamido)pentanoic acid was prepared from 1-6 and (S)-2- aminopentanoic acid using method Η: 1H NMR (400 MHz, DMSO-i/6) δ 0.85 (3H, d, J=7 Hz), 1.25-1.35 (2H, m), 1.52-1.70 (2H, m), 3.42-3.50 (2H, m), 4.13-4.20 (IH, m), 7.15-7.32 (5H, m), 8.30 (IH, d, J=8 Hz), 12.50 (IH, bs), MS (LC MS) m/z observed 235.97, expected 236.13 [M+H]. The compound was used further as described.

Title compound C5 was prepared from (5)-2-(2-phenylacetamido)pentanoic acid and 1-3 using method A: 1H NMR (400 MHz, DMSO-i/6) δ 0.83 (3H, d, J=6.4 Hz), 1.25-1.35 (2H, m), 1.45- 1.57 (IH, m), 1.61 -1.73 (IH, m), 2.95-3.12 (2H, m), 3.42-3.65 (3H, m), 4.07-4.18 (IH, m), 4.31-4.43 (2H, m), 4.45-4.55 (IH, m), 5.12 (IH, m), 7.01 (IH, t, J=7Hz), 7.15-7.33 (7H, m), 7.97-8.10 (I H, d, J=8Hz), 8.20-8.38 (2H, m), 8.75 (IH, bs), MS (LC/MS) m/z observed 518.99, expected 519.25 [M+H],

EXAMPLE C6

(25)-l-{2-[(25)-3,3-DlMETHYL-2-(2-PHENYLACETAMIDO)BUTANAMIDO]ACETYL}-/V- (2H-l,2,3,4-TETRAZOL-5-YLIMETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

(S -3,3-Dimethyl-2-(2-phenylacetamido)butanoic acid was prepared from 1-6 and (5)-2-amino-3,3-dimethylbutanoic acid using method Η: 1H NMR (400 MHz, DMSO- d6) δ 0.93 (9H, s), 3.47 (IH, d, J=14Hz), 3.57 (IH, d, J=14Hz), 4.10 (I H, d, J=9Hz), 7.10-7.22 (IH, m), 7.24-7.32 (4H, m), 8.15 (IH, d, J=9 Hz), 12.58 (IH, bs), MS (LC/MS) m/z observed 249.96, expected 250.14 [M+H]. The compound was used further as described. Title compound C6 was prepared from (S)-3,3-dimethyl-2-(2- phenylacetamido)butanoic acid and 1-3 using method A: 1H NMR (400 MHz, DMSO- (16) δ 0.91 (9H, s), 2.95-3.12 (2H, m), 3.40-3.70 (3H, m), 4.07-4.18 (IH, m), 4.28-4.40 (2H, m), 4.45-4.55 (IH, m), 5.12 (IH, m), 6.99 (IH, t, J=7Hz), 7.12-7.31 (7H, m), 7.95- 8.07 (IH, m), 8.30-8.50 (2H, m), 8.73 (IH, bs), MS (LC/MS) m/z observed 533.03, expected 533.26 [M+H].

EXAMPLE C7

(25)-l-{2-[(25,3J?)-3-METHOXY-2-(2-PHENYLACETAMIDO)BUTANAMIDO]ACETYL}-N- (2H-l,2,3,4-TETRAZOL-5-YLMETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAlVIIDE (2S^,,3i?)-3-Methoxy-2-(2-phenylacetamido)butanoic acid was prepared from 1-6 and (25,3i?)-2-amino-3-methoxybutanoic acid using method Η: 1H NMR (400 MHz, DMSO-i 6) δ 1.01 (3H, d, J=6Hz), 3.34 (3H, s), 3.51 (I H, d, J=14Hz), 3.61 (I H, d, J=14Hz), 3.81-3.85 (I H, m), 4.35 (IH, dd, J=3Hz, 9Hz), 7.16-7.22 (IH, m), 7.24-7.32 (4H, m), 8.17 (IH, d, J=9 Hz), 12.61 (IH, bs), MS (LC/MS) m/z observed 252.01 , expected 252.12 [M+H]. The compound was used further as described.

Title compound C7 was prepared from (2£,3.ft)-3-methoxy-2-(2- phenylacetamido)butanoic acid and 1-4 using method C: 1H NMR (400 MHz, DMSO- (16) δ 1.04 (3H, m), 3.10-3.16 (2H, m), 3.24 (3H, s), 3.45-3.75 (4H, m), 4.1 1 -4.25 (IH, m), 4.37-4.42 (I H, m), 4.50-4.70 (2H, m), 5.15 (IH, d, J=9.5Hz), 6.97 (IH, t, J=7.4Hz), 7.13-7.30 (7H, m), 7.97-8.18 (3H, m), 9.12 (IH, bs), MS (LC/MS) m/z observed 535.04, expected 535.24 [M+H].

EXAMPLE C8

(25)-l-{2-[(25)-3-(r£^,ffr-BUTOXY)-2-(2-PHENYLACETAMIDO)PROPANAMIDO]ACETYL}- A^r-(2H-l,2,3,4-TETRAZOL-5-YL ETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE (5)-3-(iert-Butoxy)-2-(2-phenylacetamido)propanoic acid was prepared from 1-6 and (iS)-2-amino-3-(ieri-butoxy)propanoic acid using method Η: 1H NMR (400 MHz, DMSCM6) δ 1.06 (9H, s), 3.48 (IH, m), 3.49-3.57 (2H, m), 3.63 (IH, dd, J=5Hz, 9Hz), 4.36 (IH, m), 7.16-7.22 (IH, m), 7.24-7.32 (4H, m), 8.15 (IH, d, J=9 Hz), 12.61 (IH, bs), MS (LC/MS) m/z observed 279.86, expected 280.16 [M+H]. The compound was used further as described.

Title compound C8 was prepared from (5)-3-(fer?'-butoxy)-2-(2- phenylacetamido)propanoic acid and 1-3 using method A: ^XH NMR (400 MHz, DMSO- d6) 8 1.08 (9H, ms), 2.93-3.15 (2H, m), 3.43-3.70 (5H, m), 4.1 1-4.20 (1H, m), 4.30-4.55 (3H, m), 5.15 (IH, m), 6.97 (IH, t, J=7.4Hz), 7.13-7.33 (7H, m), 7.95-8.25 (3H, m), 8.75 (IH, bs), MS (LC MS) m/z observed 563.00, expected 563.27 [M+H].

EXAMPLE C9 (2S)-l-{2-[(2S,3i?)-3-(r£'/?r-BuTOXY)-2-(2-

PHENYLACETAMlDO)BUTANAMIDO]ACETYL}-N-(2H-l,2,3,4-TETRAZOL-5-YLIViETHYL)- 2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMlDE

(25^,,3i?)-3-(ie i-Butoxy)-2-(2-phenylacetamido)butanoic acid was prepared from 1-6 and (25',3 ?)-2-amino-3-(/er -butoxy)butanoic acid using method Η: Ή NMR (400 MHz, DMSO-i/6) δ 1.06 (3H, d, J=6Hz), 1.09 (911, s), 3.48 (III, d, J=14Hz), 3.56 (IH, d, J=14Hz), 3.93 (IH, m), 4.33 (IH, dd, J=5Hz, 9Hz), 7.16-7.22 (IH, m), 7.24-7.32 (4H, m), 8.18 (IH, d, J=9 Hz), 12.61 (I H, bs), MS (LC/MS) m/z observed 293.85, expected 294.17 [M+H]. The compound was used further as described.

Title compound C9 was prepared from (2S,3R)-3-(tert-butoxy)-2-(2- phenylacetamido)butanoic acid and 1-4 using method C: 1H NMR (400 MHz, DMSO- d6) δ 1.02 (3H, m), 1.13 (9H, s), 3.10-3.16 (2H, m), 3.50-3.70 (3H, m), 3.88-3.95 (IH, m), 4.1 1-4.25 (IH, m), 4.30-4.37 (IH, m), 4.47-4.67 (2H, m), 5.15 (IH, d, J=9.5Hz), 6.97 (I H, t, J=7.4Hz), 7.13-7.30 (7H, m), 7.85 (IH, d, J=8Hz), 8.00-8.10 (2H, m), 9.12 (IH, bs), MS (LC/MS) m/z observed 577.11 , expected 577.29 [M+H].

EXAMPLE C I O

(25)-1-{2-[(25,35)-3-(Γ£ΛΓ-Βυτοχγ)-2-(2-

PHENYLACETAMIDO)BUTANAMIDO]ACETYL}-N-(2H-l,2,3,4-TETRAZOL-5-YLIVIETHYL)- 2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

(25',35)-3-( eri-Butoxy)-2-(2-phenylacetamido)butanoic acid was prepared from 1-6 and (2S,3£)-2-amino-3-(t£T/-butoxy)butanoic acid using method Η: 1H NMR (400 MHz, DMSO-rf6) δ 1.03 (3H, d, J=6Hz), 1.09 (9H, s), 3.52 (I H, d, J=14Hz), 3.62 (IH, d, J=14Hz), 4.10 (IH, m), 4.27 (IH, dd, J=3Hz, 9Hz), 7.16-7.22 (IH, m), 7.24-7.32 (4H, m), 7.83 ( I H, d, J=9 Hz), 12.53 (I H, bs), MS (LC/MS) m/z observed 293.92, expected 294.17 [M+H]. The compound was used further as described.

Title compound CIO was prepared from (25',35 -3-(feri-butoxy)-2-(2- phenylacetamido)butanoic acid and 1-4 using method C: MS (LC/MS) m/z observed 577.04, expected 577.29 [M+H]. EXAMPLE CI 1

(2S)- 1- [(2S)-2- [(25,35)-3-METHYL-2-(2- PHENYLACETAMIDO)PENTANAMIDO]PROPANOYL]-N-(2H-l,2,3,4-TETRAZOL-5- YL ETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAiVIIDE ri-Butyl ((S)-l -((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-l - oxopropan-2-yl)carbamate was prepared from 1-2 and (S)-2-((tert- butoxycarbonyl)amino)propanoic acid using method C: MS (LC/MS) m/z observed 415.76, expected 416.20 [Μ+Η], and observed 438.01 , expected 438.19 [M+Na], Compound was confirmed using LC/MS and moved to next step as it was.

Title compound Cll was prepared fruni r/-butyl ((.S)-l -((5)-2-(((2H-tetrazol-5- yl)methyl)carbamoyl)indolin-l-yl)-l-oxopropan-2-yl)carbamate and (25^,,35)-3-methyl-2- (2-phenylacetamido)pentanoic acid using method A: Ή NMR (400 MHz, CD₃OD) δ 0.80-0.88 (6H, m), 1.06-1.13 (IH, m), 1.26-1.28 (3H, d, J=8 Hz), 1.42-1.48 (IH, m), 1.75-1.83 (IH, m), 3.20-3.32 (2H, m), 3.48-3.64 (2H, m), 4.20-4.22 (I H, d, J=8 Hz), 4.37-4.41 (IH, d, j=16 Hz), 4.54-4.58 (IH, d, J=16 Hz), 4.66-4.70 (IH, d, J=16 Hz), 5.49-5.51 (IH, d, J=8 Hz), 7.02-7.05 (IH, t, J=6 Hz), 7.15-7.27 (7H, m), 8.10-8.12 (IH, d, J=8 Hz), MS (LC/MS) m/z observed 546.97, expected 547.28 [M+H].

EXAMPLE C12

(25)-l-[(2i?)-2-[(2₁S,35)-3-METHYL-2-(2- PHENYLACETAMIDO)PENTANAMIDO]PROPANOYL]-7V-(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)-2,3-DIHYDRO-lH-]NDOLE-2-CARBOXAMIDE te -Butyl ((i?)-l-((₁S -2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-l- oxopropan-2-yl)carbamate was prepared from 1-2 and (R)-2-((tert- butoxycarbonyl)amino)propanoic acid using method C: MS (LC/MS) m/z observed 415.79, expected 416.20 [Μ+Η], and observed 438.03, expected 438.19 [M+Na], Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C12 was prepared from tert-buty\ ((7?)-l -((S -2-(((2H-tetrazol-5- yl)methyl)carbamoyl)indolin- 1 -yl)- 1 -oxopropan-2-yl)carbamate and

(2-phenylacetamido)pentanoic acid using method A: Ή NMR (400 MHz, CD₃OD) δ 0.83-0.91 (6H, m), 1.10-1.13 (IH, m), 1.34-1.36 (3H, d, J=8 Hz), 1.48-1.56 (IH, m), 1.82-1.91 (IH, m), 3.18-3.28 (2H, m), 3.48-3.64 (2H, m), 4.20-4.22 (I H, d, J=8 Hz), 4.36-4.40 (IH, d, J=16 Hz), 4.54-4.58 (IH, d, J=16 Hz), 4.68-4.72 (IH, d, J=16 Hz), 5.50-5.52 (IH, d, J=8 Hz), 7.02-7.06 (IH, t, J=8 Hz), 7.20-7.34 (7H, m), 8.1 1-8.13 (IH, d, J=8 Hz), MS (LC/MS) m/z observed, 546.99, expected 547.28 [M+H].

EXAMPLE CI 3

(45^-4-[(25,3₁S -3-METHYL-2-(2-PHENYLACETAMIDO)PENTANA IDOJ-5-OXO-5-[(25)-2- [(2H-L,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L-

YLjPENTANOIC ACID

(S)-tert-Buty\ 5-((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin- 1 -yl)-4- ((teri-butoxycarbonyl)amino)-5-oxopentanoate was prepared from 1-2 and (S)-5-(tert- butoxy)-2-((teri-butoxycarbonyl)amino)-5-oxopentanoic acid using method C: MS (LC/MS) m/z observed 529.83, expected 530.27 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

(5)-Ethyl 5-((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-4-((25,3»S)- 3-methyl-2-(2-phenylacetamido)pentanamido)-5-oxopentanoate was prepared from (5)- tert-butyl 5-((5")-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-4-((^ri- butoxycarbonyl)amino)-5-oxopentanoate and 1-7 using method F: MS (LC/MS) m/z observed 633.02, expected 633.32 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C13 was prepared from (5)-ethyl 5-((S)-2-(((2H-tetrazol-5- yl)methyl)carbamoyl)indolin-l -yl)-4-((25',35)-3-methyl-2-(2- phenylacetamido)pentanamido)-5-oxopentanoate using method D: Ή NMR (400 MHz, DMSO-i/6) δ 0.74-0.81 (6H,m), 1.01 -1.05 (I H, m), 1.20-1.27 (IH, m), 1.70-1.81 (2H, m), 2.25-2.31 (IH, m), 3.1 1-3.19 (IH, t, J=16 Hz), 3.36-3.61 (4H, m), 4.19-4.25 (IH, m), 4.32-4.38 (IH, m), 4.42-4.48 (IH, dd, J=8, 16 Hz), 4.54-4.60 (I H, dd, J=8, 16 Hz), 4.62- 4.68 (IH, dd, J=8, 16 Hz), 5.17-5.20 (IH, d, J=12 Hz) 6.99-7.03 (IH, t, J=8 Hz), 7.15- 7.26 (6H, m), 7.99-8.07(2H, m), 8.81 (IH, bs), 8.98 (IH, bs), MS (LC/MS) m/z observed 604.97, expected 605.28 [M+H].

EXAMPLE C14

(35)-3-[(2S,35 -3-METHYL-2-(2-PHENYLACETAMIDO)PENTANAMIDO]-4-OXO-4-[(25)-2- [(2H-L,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L- YL]BUTANOIC ACID

(5 -/er -Butyl 4-((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-3- ((½ri-butoxycarbonyl)amino)-4-oxobutanoate was prepared from 1-2 and (S)-4-(tert- butoxy)-2-((/er -butoxycarbonyl)amino)-4-oxobutanoic acid using method C: MS (LC/MS) m/z observed 515.74, expected 516.26 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

(S)-Ethyl 4-((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-3-((25,35)- 3-mcthyl-2-(2-phenylacetamido)pentanamido)-4-oxobutanoate was prepared from (S)- terr-butyl 4-((5 -2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-3-((feri- butoxycarbonyl)amino)-4-oxobutanoate and 1-7 using method F: MS (LC/MS) m/z observed 618.93, expected 619.30 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C14 was prepared from (5)-ethyl 4-((5 -2-(((2H-tetrazol-5- yl)methyl)carbamoyl)indolin-l-yl)-3-((25,35)-3-methyl-2-(2- phenylacetamido)pentanamido)-4-oxobutanoate using method D: Ή NMR (400 MHz, DMSO-rf6) 5 0.68-0.78 (6H,m), 0.95-1.04 (IH, m), 1.22- 1.29 (IH, m), 1.66- 1.73 (IH, m), 2.55-2.64 (2H, m), 3.15-3.19 (IH, d, J=16 Hz), 3.38-3.42 (IH, d, J=16 Hz), 3.48-3.56 (2H, t, J=16 Hz), 4.29-4.31 (IH, t, J=16 Hz), 4.39-4.44 (IH, dd, J=4, 16 Hz), 4.57-4.66 (2H, dt, J=4, 16 Hz), 5.61 -5.63 (IH, d, J=8 Hz), 7.00-7.04 (IH, t, J=8 Hz), 7.14-7.25 (6H, m), 7.96-7.98 (I H, d, J=8 Hz), 8.04-8.06 (IH, d, J=8 Hz), 8.75 (IH, bs), 9.07 (I H, bs), MS (LC/MS) m/z observed 590.97, expected 591.27 [M+H] and observed 613.05, expected 613.25 [M+Na].

EXAMPLE C 15

(3^)-3-[(25',3₁S)-3-METHYL-2-(2-PHENYLACETAMIDO)PENTANAMIDO]-4-OXO-4-[(25 -2-

[(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3-DiHYDRO-lH-INDOL-l-

YL]BUTANOIC ACID

(R)-tert-Butyl 4-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin- l-yl)-3- ((/erf-butoxycarbonyl)amino)-4-oxobutanoate was prepared from 1-2 and (R)-4-(tert- butoxy)-2-((teA -butoxycarbonyl)amino)-4-oxobutanoic acid using method C: MS (LC/MS) m/z observed 515.77, expected 516.26 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

(7?)-Ethyl 4-((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin- 1 -yl)-3 -((25,35)- 3-methyl-2-(2-phenylacetamido)pentanamido)-4-oxobutanoate was prepared from (R)- ter/-butyl 4-((LV)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l-yl)-3-((ie /- butoxycarbonyl)amino)-4-oxobutanoate and 1-7 using method F: MS (LC/MS) m/z observed 618.94, expected 619.30 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C15 was prepared from (i?)-ethyl 4-((5^r)-2-(((2H-tetrazoI-5- yl)methyl)carbamoyl)indolin- l-yl)-3-((2S,3S)-3-methyl-2-(2- phenylacetamido)pentanamido)-4-oxobutanoate using method D: ^]H NMR (400 MHz, DMSO-i/6) δ 0.66-0.76 (6H,m), 0.96-1.05 (I H, m), 1.32-1.37 (IH, m), 1.60-1.66 (IH, m), 2.57-2.63 (2H, m), 3.13-3.17 (IH, t, J=8 Hz), 3.37-3.40 (I H, d, J=12 Hz), 3.46-3.52 (2H, t, J=12 Hz), 4.16-4.20 (IH, t, J=8 Hz), 4.39-4.42 (IH, d, J=12 Hz), 4.60-4.65 (2H, m), 5.60-5.63 (IH, d, J=12 Hz), 7.00-7.04 (I H, t, J=8 Hz), 7.14-7.23 (6H, m), 8.04-8.08 (IH, t, J=8 Hz), 8.04-8.06 (IH, d, J=8 Hz), 8.80 (IH, bs), 9.02 (IH, bs), MS (LC/MS) m/z observed 590.95, expected 591.27 [M+H] and observed 613.08, expected 613.25 [M+Na] .

EXAMPLE CI 6

(4i?)-4-[(25',35)-3-METHYL-2-(2-PHENYLACETAMIDO)PENTANA IDO]-5-OXO-5-[(25)-2- [(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-l-

YLjPENTANOlC ACID

(R)-tert-Buty\ 5-((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-4- (( r/-butoxycarbonyl)amino)-5-oxopentanoate was prepared from 1-2 and (R)-5-(tert- butoxy)-2-((/erf-butoxycarbonyl)amino)-5-oxopentanoic acid using method C: MS (LC/MS) m/z observed 529.86, expected 530.27 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

(_¾)-Ethyl 5-((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l-yl)-4-((25',35)- 3-methyl-2-(2-phenylacetamido)pentanamido)-5-oxopentanoate was prepared from (R)-tert-buty\ 5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-4-((ieri- butoxycarbonyl)amino)-5-oxopentanoate and 1-7 using method F: MS (LC/MS) m/z observed 633.01 , expected 633.32 [Μ+Η] . Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C16 was prepared from (R)-ethyl 5-((5^r)-2-(((2H-tetrazol-5- yl)methyl)carbamoyl)indolin-l-yl)-4-((25',3_lS)-3-methyl-2-(2- phenylacetamido)pentanamido)-5-oxopentanoate using method D: Ή NMR (400 MHz, DMSO-i/6) δ 0.75-0.82 (6H,m), 1 .00-1.05 (IH, m), 1.21-1.27 (IH, m), 1.73-1.83 (2H, m), 2.27-2.33 (IH, m), 3.12-3.20 (IH, t, J=16 Hz), 3.36-3.61 (4H, m), 4.20-4.24 (I H, t, J=8 Hz), 4.32-4.36 (I H, t, J=8 Hz), 4.42-4.48 (IH, dd, J=8, 16 Hz), 4.54-4.60 (IH, dd, J=8, 16 Hz), 4.62-4.68 (IH, dd, J=8, 16 Hz), 5.17-5.20 (IH, d, J=12 Hz) 6.99-7.03 (IH, t, J=8 Hz), 7.12-7.26 (6H, m), 7.99-8.07(2H, m), 8.80 (IH, bs), 9.02 (IH, bs), MS (LC/MS) m/z observed 604.95, expected 605.28 [M+H] and observed 627.08, expected 627.27 [M+Na].

EXAMPLE C 17

(S₁_T)-5-[(25,35)-3-METHYL-2-(2-PHENYLACETA IDO)PEI TANAMIDO]-6-OXO-6-[(2₁S)-2- [(2H-L,2,3,4-TET AZOL-5-YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L-

YL] HEX ANOIC ACID

(S)-tert-Butyl 6-((5)-2-(((2H-letrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-5-

((tert-butoxycarbonyl)amino)-6-oxohexanoate was prepared from 1-2 and (S)-6-(tert- butoxy)-2-((/er -butoxycarbonyl)amino)-6-oxohexanoic acid using method C: MS (LC/MS) m/z observed 543.87, expected 544.29 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

CS Ethyl 6-((5 -2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l-yl)-5-((2₁S',35)-

3-methyl-2-(2-phenylacetamido)pentanamido)-6-oxohexanoate was prepared from (S)- tert-butyl 6-((₁S -2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-5-((tert- butoxycarbonyl)amino)-6-oxohexanoate and 1-7 using method F: MS (LC/MS) m/z observed 647.01 , expected 647.33 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C17 was prepared from (5)-ethyl 6-((5 -2-(((2H-tetrazol-5- yl)methyl)carbamoyl)indolin-l-yl)-5-((25',35)-3-methyl-2-(2- phenylacetamido)pentanamido)-6-oxohexanoate using method D: Ή NMR (400 MHz, DMSC 6) δ 0.68-0.83 (6H, m), 0.95-1.08 (IH, m), 1.16-1.25 (IH, m), 1.30-1 .40 (IH, m), 1.45- 1.80 (4H, m), 2.00-2.25 (2H, m), 3.10-3.55 (4H, m), 4.18 (IH, bs), 4.35 (IH, dd, J= 6Hz, 9Hz), 4.50 (IH, m), 4.65 (IH, dd, J=6Hz, 16Hz), 5.56 (IH, dd, J=3Hz, 12 Hz), 6.93-7.02 (IH, m), 7.10-7.25 (7H, m), 7.90 (IH, d, J=9 Hz), 8.05 (IH, d, J=8 Hz), 8.52 (IH, d, J=7Hz), 9.10 (IH, bs), MS (LC/MS) m/z observed 618.98, expected 619.30 [M+H]. EXAMPLE CI 8

(2-S -1-[(25)-6-A INO-2-[(2-S',35)-3-METHYL-2-(2-

PHENYLACETAIMIDO)PENTANAMIDO] HEXANOYL]-yV-(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAIMIDE (5)-(5-Allyloxycarbonylamino-l -{(S)-2-[(2H-tetrazol-5-ylmethyl)-carbamoyl]- 2,3-dihydro-indole- l -carbonyl}-pentyl)-carbamic acid ieri-butyl ester was prepared from 1-2 and (^-6-(((allyloxy)carbonyl)amino)-2-((ier/-butoxycarbonyl)amino)hexanoate dicyclohexylammonium salt using method C: MS (LC/MS) m/z observed 556.86, expected 557.28 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

(_lS)-l-[6-(l-Allyloxy-vinylamino)-((53-2-((2_iS,3¾-3-methyl-2-(2- phenylacetamido)pentanamido)hexanoyl)]-indoline-2-carboxylic acid (2H-tetrazol-5- ylmethyl)-amide was prepared from (S)-(5-allyloxycarbonylamino-l-{(S)-2-[(2H- tetrazol-5-ylmethyl)-carbamoyl]-2,3-dihydro-indole-l -carbonyl}-pentyl)-carbamic acid ieri-butyl ester and 1-7 using method A: MS (LC MS) m/z observed 688.04, expected 688.36 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

To a stirred solution of (5 -l -[6-(l-allyloxy-vinylamino)-((₁S -2-((2S,35 -3- methyl-2-(2-phenylacetamido)pentanamido)hexanoyl)]-indoline-2-carboxylic acid (2H- tetrazol-5-ylmethyl)-amide (0.145 mmol) and palladium on charcoal (10%, 10 mg) in methanol (2 mL), was added triethylsilane (145 mmol, 10 equivalents) under nitrogen. The reaction mixture was stirred at RT for 1 hr then concentrated to give a residue that was purified by reverse phase column chromatography to give the title compound C18. (1.5 mg): MS (LC/MS) m/z observed 604.24, expected 604.34 [Μ+Η]. EXAMPLE C19

r£/f7--BUTYLN-[(25)-l-{2-[(2S,3,S -3-lVIETHYL-2-(2- PHENYLACETAMIDO)PENTANA IDO]ACETYL}-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-6-YL] CARBAMATE

Title compound C19 was prepared from 1-12 and (2H-tetrazol-5-yl)methyl-amine using general method L. Ή NMR (400 MHz, CD₃OD) δ 0.88-0.98 (6H, m), 1.14-1.22 (IH, m), 1.30-1.41 (IH, m), 1.52 (9H, s), 1.87-2.01 (lh, m), 3.12-3.21 (IH, m), 3.59-3.69 (2H, m), 3.84-3.92 (IH, t, J=16 Hz), 4.16-4.24 (IH, t, J=16 Hz), 4.34-4.36 (IH, d, J=8 Hz), 4.51-4.53 (IH, d, J=8 Hz), 4.63-4.72 (2H, m), 5.15-5.17 (IH, d, 7=8 Hz), 7.06- 7.15 (2H, m), 7.18-7.22 (IH, m), 7.20-7.27 (IH, m), 7.29-7.34 (4H, m), 7.55 (IH, bs), 8.21 (IH, bs), 8.91 (IH, bs) MS (LC/MS) m/z observed 648.13, expected 648.32 [M+H]

EXAMPLE C20

(2-y)-6-AMlNO-l-{2-[(2-S^,,35)-3-METHYL-2-(2-

PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-N-(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)- 2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

Title compound C20 was prepared from C19 using general method E. Ή NMR (400 MHz, CD₃OD) δ 0.76-0.85 (6H, m), 1.05-1.10 (IH, m), 1.30-1.42 (IH, m), 1.72- 1.86 (IH, m), 3.13-3.17 (IH, d, J=16 Hz), 3.39-3.47 (2H, m), 3.54-3.71 (3H, m), 4.10- 4.15 (IH, dd, J=16, 4 Hz), 4.19-4.26 (IH, m), 4.38-4.41 (IH, dd, J=8, 4 Hz), 4.53-4.69 (2H, m), 5.23-5.26 (IH, d, J=12 Hz), 6.96-6.98 (IH, d, J=8 Hz), 7.21-7.29 (5H, m), 8.01 - 8.12 (2H, m), 8.27-8.33 (IH, m), 9.17-9.22 (IH, m), 10.01 (IH, bs) MS (LC MS) m/z observed 548.12, expected 548.32 [M+H].

EXAMPLE C21

(25)-6-(BENZYLAMINO)-1-{2-[(25,35)-3-METHYL-2-(2- PHENYLACETAMIDO)PENTANAMIDO] ACETYL}-7 -(2H-L,2,3,4-TETRAZOL-5-YLMETHYL)-

2,3-DIHYDRO-LH-INDOLE-2-CARBOXAMIDE

A solution of compound C20 (0.025 g, 0.0385 mmol), DIPEA (0.032 ml, 0.23 mmol) and benzyl bromide (0.015 ml, 0.116 mol) in DCM (4 ml) was sealed in a microwave tube and heated to 70 °C for 10 hr. by microwave irradiation. The reaction mixture was concentrated to dryness and dried well under vacuum to give the crude product, which was purified on preparative HPLC using 0-80% MeOH in water to yield product as an off-white solid 0.0022 g (9%). MS (LC/MS) m/z observed 638.11 , expected 638.31 [M+H]. Compound was confirmed using LC/MS and used as directed.

EXAMPLE C22

3-{f(25)-l-{2-[(2S,3,S)-3-METHYL-2-(2-PHENYLACETAMIDO)PENTANA]VIIDO]ACETYL}- 2-[(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-6-

YL] CARBAMOYL} PROPANOIC ACID

Title compound C22 was prepared from CI 9 and succinic anhydride using method B: Ή NMR (400 MHz, DMSC 6) δ 0.78-0.87 (6H, m), 1.06-1.13 (IH, m), 1.30-1.42 (IH, m), 1.75-1.86 (IH, m), 3.03-3.07 (IH, d, J=16 Hz), 3.44-3.61 (511, m), 4.09-4.19 (IH, m), 4.23-4.27 (IH, t, J=8 Hz), 4.40-4.44 (IH, t, J=8 Hz), 4.48-4.61 (2H, m), 5.14-5.17 (IH, d, J=12 Hz), 7.09-7.1 1 (IH, d, J=8 Hz), 7.18-7.22 (IH, m), 7.25-7.31 (4H, m), 7.40-7.44 (IH, t, J=8 Hz), 8.04-8.06 (I H, d, .7=8 Hz), 8.12-8.14 (I H, t, J=8 Hz), 8.22 (IH, bs), 8.28-8.32 (IH, t, J=8 Hz), 8.95 (IH, bs), 9.96 (IH, s) 12.10 (IH, bs) MS (LC/MS) m/z observed 648.12, expected 648.28 [M+H].

EXAMPLE C23

3-{[(S)-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3- DIHYDRO-lH-INDOL-l-YL]ETHYL}CARBAMOYL)(THIOPHEN-3- YL)METHYL]CARBAMOYL}PROPANOIC ACID

(5)-2-((feri-Butoxycarbonyl)amino)-2-(thiophen-3-yl)acetic acid was prepared from (5")-2-amino-2-(thiophen-3-yl)acetic acid using general method K. MS (LC/MS) m/z observed 279.92, expected 280.07 [M+Na]. Compound was confirmed using LC/MS and moved to next step as it was.

(S)-Ethyl l-(2-((S)-2-((ierr-butoxycarbonyl)amino)-2-(thiophen-3- yl)acetamido)acetyl)indoline-2-carboxylate was prepared from 1-10 and (R)-2-((tert- butoxycarbonyl)amino)-2-(thiophen-3-yl)acetic acid using general method A. The purification was performed by column chromatography on silica gel using 0 % to 50 % ethyl acetate in hexanes as the eluent. MS (LC/MS) m/z observed 487.84, expected 488.18 [M+H], observed 510.06, expected 510.18 [M+Na]. Compound was confirmed using LC/MS and moved to next step as it was.

te -Butyl ((5)-2-((2-((5)-2-(((2H-tetrazol-5-yl)melhyl)carbamoyl)indolin- l-yI)-2- oxoethyl)amino)-2-oxo-l -(thiophen-3-yl)ethyl)carbamate was prepared from (5)-ethyl 1 - (2-(( ?)-2-((ierf-butoxycarbonyl)amino)-2-(thiophen-3-yl)acetamido)acetyl)indoline-2- carboxylate and (2H-tetrazol-5-yl)methyl-amine using general method L. MS (LC/MS) m/z observed 541.02, expected 541.19 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C23 was prepared from terf-butyl ((5)-2-((2-((5)-2-(((2H- tetrazol-5-yl)methyl)carbamoyl)indol in- 1 -yl)-2-oxoethyl)amino)-2-oxo- 1 -(thiophen-3 - yl)ethyl)carbamate and succinic anhydride using method I: Ή NMR (400 MHz, DMSO-tftf) δ 2.39-2.48 (2H, m), 3.12-3.16 (2H, d, J=16 Hz), 3.58-3.66 (3H, t, J=16 Hz), 4.20-4.24 (I H, d, J=16 Hz), 4.55-4.59 (IH, m), 4.62-4.69 (IH, m), 5.15-5.18 (IH, d, J=12 Hz), 5.64-5.68 (IH, t, J=8 Hz), 7.00-7.04 (I H, t, J=8 Hz), 7.18-7.22 (3H, m), 7.48-7.52 (2H, m), 8.04-8.06 (IH, d, J=8 Hz), 8.53-8.55 (IH, d, J=8 Hz), 8.61 -8.64 (IH, m), 9.10 (I H, bs), MS (LC/MS) m/z observed 541.03, expected 541.15 [M+H].

EXAMPLE C24

3-{[(/?)-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)CARBAIVIOYL]-2,3- DlHYDRO-lH-INDOL-l-YL]ETHYL}CARBAMOYL)(THIOPHEN-2-

YL)METHYL]CARBAM0YL} PROPANOIC ACID

(i?)-2-((ier/-Butoxycarbonyl)amino)-2-(thiophen-2-yl)acetic acid was prepared from (^)-2-amino-2-(thiophen-2-yl)acetic acid using general method K. MS (LC/MS) m/z observed 279.92, expected 280.07 [M+Na]. Compound was confirmed using LC/MS and moved to next step as it was.

(S -Ethyl l-(2-(( ?)-2-((ieri-butoxycarbonyl)amino)-2-(thiophen-2- yl)acetamido)acetyl)indoline-2-carboxylate was prepared from 1-10 and (R)-2-((tert- butoxycarbonyl)amino)-2-(thiophen-2-yl)acetic acid using general method A. The purification was performed on normal phase using 0 % to 50 % ethyl acetate in hexanes as the cluent. MS (LC/MS) m/z observed 487.89, expected 488.18 [M+H], observed 510.06, expected 510.18 [M+Na]. Compound was confirmed using LC/MS and moved to next step as it was.

teri-Butyl ((5 -2-((2-((.S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l -yl)-2- oxoethyl)amino)-2-oxo-l-(thiophen-2-yl)ethyl)carbamate was prepared from (S)-ethyl 1 - (2-((R)-2-(( eri-butoxycarbonyl)amino)-2-(thiophen-2-yl)acetamido)acetyl)indoline-2- carboxylate and (2H-tetrazol-5-yl)methyl-amine using general method L. MS (LC/MS) m/z observed 541.04, expected 541.19 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C24 was prepared from iert-butyl ((S)-2-((2-((5)-2-(((2H- tetrazol-5-yl)methyl)carbamoyl)indolin-l-yl)-2-oxoethyl)amino)-2-oxo-l -(thiophen-2- yl)ethyl)carbamate and succinic anhydride using method I: Ή NMR (400 MHz, DMSO-i/6) δ 2.41-2.45 (2H, m), 3.10-3.14 (2H, d, J=16 Hz), 3.56-3.64 (3H, t, J=16 Hz), 4.19-4.23 (1 H, d, J=16 Hz), 4.52-4.62 (2H, m), 5.14-5.17 (1H, d, J=\ 2 Hz), 5.80-5.84 (1 H, t, J=8 Hz), 6.95-6.98 (1 H, m), 7.18-7.22 (3H, m), 6.99-7.01 ( l H, d, J=8 Hz), 7.13- 7.18 (2H, m), 7.20-7.22 (1H, d, J=8 Hz), 7.40-7.42 (1H, d, J=8 Hz), 8.01-8.03 (1H, d, J=8 Hz), 8.62-8.64 (2H, d, J=8 Hz), 9.04 (1H, bs), MS (LC/MS) m/z observed 541 .01 , expected 541.15 [M+H].

EXAMPLE C25

4-Oxo-4-[(2^)-2-({2-oxo-2-[(2S)-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L- YL]ETHYL}CARBAMOYL)PIPERIDIN-1-YL]BUTANOIC ACID

(5)-l -(/eri-Butoxycarbonyl)piperidine-2-carboxylic acid was prepared from (S)- piperidine-2-carboxylic acid using general method K. MS (LC MS) m/z observed 251.99, expected 252.13 [M+Na]. Compound was confirmed using LC/MS and moved to next step as it was.

l -{2-[(l-ierf-Butoxycarbonyl-piperidine-(25)-2-carbonyl)-amino]-acetyl}-(25)- 2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester was prepared from 1-10 and (S)-\ - (/e^/-butoxycarbonyl)piperidine-2-carboxylic acid using general method A. The purification was performed by column chromatography on silica gel using 0% to 50% ethyl acetate in hexanes as the eluent. MS (LC/MS) m/z observed 459.96, expected 460.24 [Μ+Η] observed 482.16, expected 482.24 [M+Na]. Compound was confirmed using LC/MS and moved to next step as it was.

(25)-2-(2-Oxo-2- {(2₁S)-2-[(2H-tetrazol-5-ylmethyl)-carbamoyl]-2,3-dihydro- indol- l -yl}-ethylcarbamoyl)-piperidine-l-carboxylic acid tert-butyl ester was prepared from l -{2-[(l-/e?-i-butoxycarbonyl-piperidine-(25 -2-carbonyl)-amino]-acetyl}-(25)-2,3- dihydro- 1 H-indole-2-carboxylic acid ethyl ester and (2H-tetrazol-5-yl)methyl-amine using general method L. MS (LC/MS) m/z observed 513.08, expected 513.25 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was. Title compound C25 was prepared from (2S)-2-(2-oxo-2-{(2>S)-2-[(2H-tetrazol-5- ylmethyl)-carbamoyl]-2,3-dihydro-indol-l -yl}-ethylcarbamoyl)-piperidine-l-carboxylic acid tert-b tyl ester and succinic anhydride using method I: 1H NMR (400 MHz, DMSO-rf6) δ 1.32-1.40 (2H, m), 1.55-1.64 (2H, m), 3.56-3.64 (3H, t, J=16 Hz), 2.19- 2.22 (IH, d, J=12 Hz), 2.39-2.47 (2H, m), 2.60-2.64 (2H, t, J=8 Hz), 3.12-3.16 (IH, d, J=16 Hz), 3.79-3.83 (2H, d, J=16 Hz), 4.18-4.26 (IH, m), 4.32-4.35 (IH, d, J=12 Hz), 4.53-4.56 (2H, m), 5.10 (IH, s), 5.16-5.19 (IH, d, J=12 Hz), 6.99-7.03 (IH, t, J=8 Hz), 7.15-7.19 (IH, t, J=8 Hz), 7.22-7.24 (IH, d, J=8 Hz), 7.99-8.04 (2H, m), 9.09 (IH, bs), MS (LC/MS) m/z observed 512.99, expected 51 .21 [M+H].

EXAMPLE C26

( ^-l-l -K ^^-S-METHYL- -^PYRIMIDIN- -Y^AMI OlPENTA AMIDOlACETYLj-W- (2H-l,2,3,4-TETRAZOL-5-YLIMETHYL)-2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

(25)-Ethyl 1 -(2-((2 30>2-((te^butoxycarbonyl)amirio)-3- methylpentanamido)acetyl)indoline-2-carboxylate was prepared from 1-10 and Boc-L- isoleucine using method A. MS (LC/MS) m/z observed 461.98, expected 462.26 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

(25)- 1 - [2-((2S,3S)-2- Amino-3 -methyl-pentanoylamino)-acetyl] -2,3 -dihydro- 1 H- indole-2-carboxylic acid ethyl ester hydrochloride was prepared from (25)-ethyl l -(2- ((25',35 -2-((ie i-butoxycarbonyl)amino)-3-methylpentanamido)acetyl)indoline-2- carboxylate using method E. MS (LC/MS) m/z observed 362.08, expected 362.20 [Μ+Η], Compound was confirmed using LC/MS and moved to next step as it was.

A solution of (2_lS)-l -[2-((2₁S',35)-2-amino-3-methyl-pentanoylamino)-acetyl]-2,3- dihydro- 1 H-indole-2-carboxylic acid ethyl ester hydrochloride (0.3 g, 0.7537 mmol), DIPEA (0.46 ml, 2.638 mmol) and 2-chloropyrimidine (0.1 8 g, 1.131 mol) in ACN (9 ml) was sealed in a microwave tube and heated to 130°C for 16 hrs by microwave irradiation. The reaction mixture was concentrated to dryness and dried well under vacuum to give the crude product, which was purified by column chromatography on silica gel to get the pure product, (25)-l -{2-[(iS)-3-Methyl-( )-2-(pyrimidin-2-ylamino)-pentanoylamino]- acetyl}-2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester, as off-white solid, 0.07 g (21%). MS (LC/MS) m/z observed 440.10, expected 440.22 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as is. Title compound C26 was prepared from (2S)-l -{2-[(S)-3-Methyl-(S)-2- (pyrimidin-2-ylamino)-pentanoylamino]-acetyl}-2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester and (2H-tetrazol-5-yl)methyl-amine using general method L. Ή NMR (400 MHz, DMSO-rf6) δ 0.82-0.86 (3H, t, J=8 Hz), 0.92-0.94 (3H, d, J=8 Hz), 1.16- 1.25 (IH, m), 1.50-1.58 (I H, m), 1.84-1.92 (I H, m), 3.1 1-3.15 (2H, d, J=16 Hz), 3.52-3.60 (2H, m), 4.15-4.19 (IH, d, J=16 Hz), 4.37-4.41 (IH, t, J=8 Hz), 4.50-4.54 (I H, d, J=16 Hz), 4.62-4.66 (IH, d, J=16 Hz), 5.14-5.17 (IH, d, J=16 Hz), 6.60-6.62 (l h, t, J=4 Hz), 6.92-6.94 (IH, d, J=8 Hz), 6.98-7.02 (IH, t, J=8 Hz), 7.14-7.18 (IH, t, J=8 Hz), 7.21 - 7.23 (IH, d, J=8 Hz), 8.00-8.02 (IH, d, J=8 Hz), 8.14(1H, bs), 8.27-8.29(2H, d, J=8 Hz), 9.05 ( I H, bs), MS (LC/MS) m/z observed 493.14, expected 493.23 [M+H].

EXAMPLE C27

( ^^-ACETAIVIIDO-l-IZ-KZS^-S-METHYL-Z-i - PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-N-(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)- 2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

Acetic acid (0.3 g, 4.854 mmol), EDC (1.02 g, 5.339 mmol), HOBt (0.74 g,

4.854 mmol), DIPEA (2.5 ml, 14.560 mmol) and 1-11 (1 g, 4.845 mmol) were stirred in anhydrous DCM (100 ml) for 16 hrs. The reaction mixture was concentrated under vacuum to give the crude product, which was purified by column chromatography on silica gel using 0% to 50% ethyl acetate in hexanes as the eluent to give (5 -ethyl 6- acetamidoindoline-2-carboxylate as a pale yellow solid (72%). Ή NMR (400 MHz, CDC1₃) δ 1.27-1.31 (3H, t, J=8 Hz), 2.12 (3H, s), 3.32-3.37 (2H, m), 4.17-4.23 (2H, q, J=8 Hz), 4.35-4.38 (I H, q, J=4 Hz), 4.51 (IH, s), 6.71 -6.73 (IH, d, J=8 Hz), 6.97-6.99 (IH, d, J=8 Hz), 7.05 (IH, s), 7.47 (IH, s), MS (LC/MS) m/z observed 249.07, expected 249.1 2[M+H].

(.S)-Ethyl 6-acetamido-l-(2-((/e^butoxycarbonyl)amino)acetyl)indoline-2- carboxylate was prepared from (5)-ethyl 6-acetamidoindoline-2-carboxylate and Boc- glycine using method C and purified by column chromatography on silica gel using 0% to 80% ethyl acetate in hexanes as the eluent to give (5)-ethyl 6-acetamido-l-(2-((ter/- butoxycarbonyl)amino)acetyl)indoline-2-carboxylate, as an off- white solid (62%). MS (LC/MS) m/z observed 405.86, expected 406.19[M+H], observed 428.10, expected 428.19 [M+Na], Compound was confirmed using LC/MS and moved to next step as it was. (5)-Ethyl 6-acetamido-l-(2-((25,3S)-3-methyl-2-(2- phenylacetamido)pentanamido)acetyl)indoline-2-carboxylate was prepared from the intermediate (5)-ethyl 6-acetamido-l-(2-((ier/-butoxycarbonyl)amino)acetyl)indoline-2- carboxylate and 1-7 using method A. MS (LC/MS) m/z observed 537.03, expected 537.26 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C27 was prepared from (S)-ethyl 6-acetamido-l-(2-((25',3₁S)-3- methyl-2-(2-phenylacetamido)pentanamido)acetyl)indoline-2-carboxylate and (2H- tetrazol-5-yl)methyl-amine using general method L. Ή NMR (400 MHz, DMSO-rfd) δ 0.78-0.86 (6H, m), 1 .06-1 .13 (I H, m), 1.29-1.44 (IH, m), 1.75-1.86 (IH, m), 2.01 (3H, s), 3.05-3.09 (2H, d, J=16 Hz), 3.43-3.61 (4H, m), 4.09-4.13 (IH, d, J=16 Hz), 4.19-4.28 (IH, m), 4.38-4.44 (IH, m), 4.51-4.59 (2H, m), 5.15-5.18 (IH, d, J=8 Hz), 7.09-7.1 1 (IH, d, j=8 Hz), 7.18-7.22 (IH, m), 7.27-7.31 (3H, m), 7.40-7.44 (IH, t, J=8 Hz), 8.02-8.04 (IH, d, J=8 Hz), 8.15-8.23 (IH, m), 8.26-8.31 (IH, m), 9.04 (IH, bs), 9.93 (IH, bs), MS (LC/MS) m/z observed 590.15, expected 590.28 [M+H], m/z observed 612.28, expected 612.28 [M+Na].

EXAMPLE C28

4_Oxo-4-[(25)-2-({2-OXO-2-[(2S)-2-[(2H-l,23,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-LH-INDOL-L- YL]ETHYL}CARBAMOYL)PYRROLIDIN-1-YL]BUTANOIC ACID l -{2-[(l-^/-Butoxycarbonyl-pyrrolidine-(2iS)-2-carbonyl)-amino]-acetyl}-(25}- 2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester was prepared from 1-10 and Boc-L- proline using method A. MS (LC/MS) m/z observed 445.94, expected 446.23 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

1 - {2- [( 1 -/eri-Butoxycarbonyl-pyrrolidine-(2¾-2-carbonyl)-amino]-acetyl} -(25)-

2,3-dihydro-lH-indole-2-carboxylic acid was prepared from l-{2-[(l -½rt- butoxycarbonyl-pyrrolidine-(25 -2-carbonyl)-amino]-acetyl} -(25)-2,3-dihydro- 1 H- indole-2-carboxylic acid ethyl ester using method D with 2 eq. of LiOH Ή₂0. MS (LC/MS) m/z observed 417.90, expected 418.20 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

(5)-reri-Butyl 2-((2-((5)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)indolin-l-yl)-2- oxoethyl)carbamoyl)pyrrolidine-l-carboxylate was prepared from \-{2-[(\-tert- butoxycarbonyl-pyrrolidine-(25)-2-carbonyl)-amino]-acetyl}-(25)-2,3-dihydro-lH- indole-2-carboxylic acid and 2H-tetrazol-5-yl)methyl-amine using method A in DMF but without HC1 treatment. MS (LC/MS) m/z observed 498.94, expected 499.24 [M+H], Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C28 was prepared from (5)-terf-butyl 2-((2-((5)-2-(((2H-tetrazol-

5-yl)methyl)carbamoyl)indolin-l -yl)-2-oxoethyl)carbamoyl)pyrrolidine-l-carboxylate and succinic anhydride using method I. ^lH NMR (400 MHz, DMSO-rf6) δ 1.86- 1.91 (2H, m), 1.95-2.05 (2H, m), 2.40-2.65 (4H, m), 3.1 1 (IH, m), 3.36 (IH, m), 3.46 (IH, m), 3.63 (IH, m), 4.15 (IH, dd, J=6, 17 Hz), 4.32 (IH, d, J=7Hz), 4.48 (IH, dd, J=3, 9Hz), 4.56 (I H, m), 4.65 (I H, m), 5.17 (IH, d, J=l lHz), 7.01 (IH, t, J=8Hz), 7.12-7-25 (2H, m), 8.00-8.10 (2H, m), 9.08 (IH, bs), MS (LC/MS) m/z observed 499.1 1, expected 499.21 [M+H],

EXAMPLE C29

3-{[l-({2-OXO-2-[(25)-2-[(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3- DlHYDRO-lH-INDOL-1-

YL]ETHYL}CARBAMOYL)CYCLOPENTYL]CARBAMOYL}PROPANOIC ACI D

1 - { 2- [( 1 -ieri-Butoxycarbonylamino-cyclopentanecarbonyl)-amino] -acetyl } -(25)- 2,3-dihydro-lH-indole-2-carboxylic acid ethyl ester was prepared from 1-10 and Boc-I- cycloleucine using method A. MS (LC/MS) m/z observed 459.95, expected 460.24 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

1 - {2-[(l -/erf-Butoxycarbonylamino-cyclopentanecarbonyl)-amino]-acetyl} -(25)- 2,3-dihydro-lH-indole-2-carboxylic acid was prepared from l-{2-[(l-ieri- Butoxycarbonylamino-cyclopentanecarbonyl)-amino]-acetyl}-(25)-2,3-dihydro-lH- indole-2-carboxylic acid ethyl ester using method D with 2 eq. of LiOH'H₂0. MS (LC/MS) m/z observed 431.93, expected 432.21 [M+H] . Compound was confirmed using LC/MS and moved to next step as it was.

(25)-[l-(2-Oxo-2-{2-[(2H-tetrazol-5-ylmethyl)-carbamoyl]-2,3-dihydro-indol-l- yl }-ethylcarbamoyl)-cyclopentyl]-carbamic acid rt-butyl ester was prepared from l -{2- [(l -½ri-butoxycarbonylamino-cyclopentanecarbonyl)-amino]-acetyl}-(25)-2,3-dihydro- lH-indole-2-carboxylic acid and (2H-tetrazol-5-yl)methyl-amine using method A in DMF but without HC1 treatment. MS (LC/MS) m/z observed 512.90, expected 13.26 [M+H] . Compound was confirmed using LC/MS and moved to next step as it was. Title compound C29 was prepared from (2S)-[l -(2-Oxo-2-{2-[(2H-tetrazol-5- ylmethyl)-carbamoyl]-2,3-dihydro-indol-l-yl}-ethylcarbamoyl)-cyclopentyl]-carbamic acid /erf-butyl ester and succinic anhydride using method I. Ή NMR (400 MHz, DMSCM6) δ 1.55-1.67 (4H, m), 1.83-1.93 (2H, m), 1.98-2.14 (2H, m), 2.34-2.39 (2H, m), 2.41 -2.46 (2H, m), 3.1 1 (1H, m), 3.50-3.63 (2H, m), 4.05 (1H, m), 4.52 (1H, dd, J=7, 16IIz), 4.68 (1H, dd, J=7, 16Hz), 5.17 (1H, d, J=l lHz), 7.01 (1H, t, J=8Hz), 7.13-7-25 (2H, m), 7.65 (1H, t, J=5Hz), 8.02 (1H, d, J=8Hz), 8.12 (1H, bs), 9.10 (1 H, bs), MS (LC/MS) m/z observed 513.06, expected 513.22 [M+H].

EXAMPLE C30

(2S)-7-ACETAMIDO-l-{2-[(25',3-S -3-METHYL-2-(2-

PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-/V-(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)- 2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMrDE

Intermediate 1-14 (500 mg, 2.427 mmol) and palladium on charcoal (100 mg) were placed in a round bottom flask under nitrogen. Ethanol (10 mL) was then added and hydrogen was bubbled in the reaction mixture for 4 hrs. Then the reaction mixture was filtered over CELITE^® and the CELITE^® was washed with ethanol (3 x 15 mL). The filtrate and washings were concentrated to give a brown oil that was further dissolved in DCM (50 mL) and to this was added acetic acid (140 uL, 2.427 mmol), HOBt (372 mg, 2.427 mmol), EDC (512 mg, 2.670 mmol) and DIPEA (845 μί, 4.854 mmol). The reaction was left at RT for 16 hrs and the solvent was evaporated. The product was purified by normal phase column chromatography using 25% to 55% ethyl acetate in hexanes as the eluent to give (5)-ethyl 7-acetamidoindoline-2-carboxylate as an off white solid (103 mg, 17%). Ή NMR (400 MHz, CDC1₃) δ 1.32 (3H, t, J=7.0Hz), 2.19 (3H, s), 3.31 (1 H, dd, J=8, 16Hz), 3.45 (1H, dd, J=l l , 16Hz), 4.19-4.30 (2H, qd, J=2, 7Hz), 4.51 (1H, dd, J=7, 10Hz), 5.20 (1H, bs), 6.70 (1H, t, J=8Hz), 6.85 (1H, d, J=8Hz), 6.96 (1H, d, J= 8Hz), 7.40 (1H, bs), MS (LC/MS) m/z observed 249.07, expected 249.12 [M+H].

(S)-Ethyl 7-acetamido- 1 -(2-((i.;r -butoxycarbonyl)amino)acetyl)indoline-2- carboxylate was prepared from (5)-ethyl 7-acetamidoindoline-2-carboxylate and Boc- glycine using method C but purified by normal phase column chromatography using 25 % to 55% ethyl acetate in hexanes as the eluent. MS (LC MS) m/z observed 406.09, expected 406.20 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was. (S)-Ethyl 7-acetamido-l -(2-((25,3_S)-3-methyl-2-(2- phenylacetamido)pentanamido)acetyl)indoline-2-carboxylate was prepared from (S)-ethyl 7-acetamido- 1 -(2-((ie i-butoxycarbonyl)amino)acetyl)indoline-2-carboxylate and 1-7 using method A but the product was purified by normal phase column chromatography using 20% to 80% ethyl acetate in hexanes as the eluent. MS (LC/MS) m/z observed 537.08, expected 537.27 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

The intermediate ((S)-7-Acetamido-l-(2-((2S',35)-3-methyl-2-(2- phenylacetamido)pentanamido)acetyl)indoline-2-carboxylic acid) was prepared from (S)- ethyl 7-acetamido- l-(2-((2S,3S)-3-methyl-2-(2- phenylacetamido)pentanamido)acetyl)indoline-2-carboxylate using method D with 2 eq. of LiOH H₂0. MS (LC/MS) m/z observed 509.08, expected 509.24 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C30 was prepared from (<S)-7-acetamido-l-(2-((25',3¾-3-methyl- 2-(2-phenylacetamido)pentanamido)acetyl)indoline-2-carboxylic acid and (2H-tetrazol-5- yl)methyl-amine using method A in DMF but without HC1 treatment. Ή NMR (400 MHz, CD₃OD) δ 0.88-0.92 (3H, t, J= 7Hz), 0.99 (3H, d, J=7Hz), 1.15 (1H, m), 1.45 (1H, m), 1.92 (1H, m), 2.07 (3H, s), 3.25 (1H, d, J=16Hz), 3.53-3.70 (3H, m), 4.05 (1H, m), 4.21-4.35 (2H, m), 4.51 (1H, dd, J=6, 12Hz), 4.80 (1H, m), 5.28 (1H, t, J=9 Hz), 7.12-7.18 (3H, m), 7.23 (1H, m), 7.26-7.34 (4H, m), MS (LC/MS) m/z observed 590.17, expected 590.28 [M+H].

EXAMPLE C31

^/ir-BUTYL N-fiZ^-l-I -Ii ^^-S-iVIETHYL- -i - PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DlHYDRO-lH-INDOL-5-YL]CARBAIVIATE

Intermediate 1-13 ( 1 g, 4.850 mmol) and palladium on charcoal (200 mg) were placed in a round bottom flask under nitrogen. Ethanol (20 mL) was then added and hydrogen was bubbled in the reaction mixture for 4 hrs. Then the reaction mixture was filtered over CELITE^® and the CELITE^® was washed with ethanol (3 x 25 mL). The filtrate and washings were concentrated to give a brown oil. This brown oil was the dissolved in toluene (50 mL) and phtalic anhydride (718 mg, 4.85 mmol) was added. The reaction was heated to 80^CC for 7 hrs and the solvent was evaporated. The product was purified by normal phase column chromatography using 5% to 20% ethyl acetate in hexanes as the eluent to give (S)-Ethyl 5-(l ,3-dioxoisoindolin-2-yl)indoline-2-carboxylate as an off white solid (750 mg, 46%). Ή NMR (400 MHz, CDC1₃) δ 1.31 (3H, t, J=7.0Hz), 3.38 (IH, dd, J=6, 17Hz), 3.45 (IH, dd, J=10, 17Hz), 4.19-4.27 (2H, qd, J=2, 7Hz), 4.43 ( IH, dd, J=7, 10Hz), 4.58 (IH, bs), 6.78 (IH, d, J=8Hz), 7.05 (IH, dd, J=2, 8Hz), 7.08 (IH, bs), 7.76-7.78 (2H, m), 7.91 -7.95 (2H, m), MS (LC/MS) m/z observed 337.10, expected 337.12 [M+H],

(5 -Ethyl 1 -(2-((fe /-butoxycarbonyl)amino)acetyl)-5-(l ,3-dioxoisoindolin-2- yl)indoline-2-carboxylate was prepared from (5 -ethyl 5-(l ,3-dioxoisoindolin-2- yl)indoline-2-carboxylate and Boc-glycine using method C. MS (LC/MS) m/z observed 493.87, expected 494.19[M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

(5)-Ethyl 5-(l ,3-dioxoisoindolin-2-yl)-l -(2-((25,3S)-3-methyl-2-(2- phenylacetamido)pentanamido)acetyl)indoline-2-carboxylate was prepared from (5)-ethyl l-(2-((?er/-butoxycarbonyl)amino)acetyl)-5-(l ,3-dioxoisoindolin-2-yl)indoline-2- carboxylate and 1-7 using method A but the product was purified by filtration of the reaction mixture. The solid obtained was the pure compound. MS (LC/MS) m/z observed 625.07, expected 625.27 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

(S Ethyl 5-(l,3-dioxoisoindolin-2-yl)-l-(2-((2S,35)-3-methyl-2-(2- phenylacetamido)pentanamido)acetyl)indoline-2-carboxylate (420 mg, 0.672 mmol) was dissolved in a 2: 1 mixture DMF/ EtOH (90 mL) and hydrazine hydrate 60% solution in water was added (75uL 1.01 mmol). The reaction was heated to 40°C for 1 hr and then to 80°C for 5 hrs. LC/MS showed that the starting material was converted to the desired free amine. The solvents were evaporated and the residue was dissolved in EtOH (25 mL) and lithium hydroxide monohydrate (141 mg, 3.360 mmol) dissolved in water (25 mL) was added. The reaction was left at RT for 4 hrs and then acidified to pH 4 with a saturated solution of citric acid. The mixture was concentrated to dryness and the residue was suspended in water (25 mL) and the solid in suspension was filtered and washed with water (3 x 10 mL). (5)-5-amino-l -(2-((25,35)-3-methyl-2-(2- phenylacetamido)pentanamido)acetyl)indoline-2-carboxylic acid hydrochloride was obtained as a brown solid (325 mg, 96%). MS (LC/MS) m/z observed 466.99, expected 467.23 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

(5)-5-Amino-l-(2-((25,35)-3-methyl-2-(2- phenylacetamido)pentanamido)acetyl)indoline-2-carboxylic acid hydrochloride (325 mg, 0.646 mmol) was dissolved in dioxane (20 mL) and NaOH (1 M, 2 mL, 1.938 mmol) was added, followed by a solution of B0C2O (1.41 g, 6.461 mmol) in dioxane (5 mL). The reaction was left at RT for 16 hrs and then acidified to pH 4 with citric acid (aqueous, saturated solution). The mixture was concentrated and the product was purified on a C 18 column using 10-55% MeOH in water to yield (5)-5-((ier/-butoxycarbonyl)amino)-l -(2- ((2iS',3_>S)-3-methyl-2-(2-phenylacetamido)pentanamido)acetyl)indoline-2-carboxylic acid as an off-white solid (255 mg, 70%). MS (LC/MS) m/z observed 567.10, expected 567.28 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C31 was prepared from (5)-5-((ie -butoxycarbonyl)amino)-l -(2- ((2S,3S)-3 -methyl-2-(2-phenylacetamido)pentanamido)acetyl)indoline-2-carboxylic acid and (2H-tetrazol-5-yl)methyl-amine using method A in DMF but without HC1 treatment. Ή NMR (400 MHz, DMSO-rf6) δ 0.65-0.87 (6H, m), 1.10 (IH, m), 1.43 (IH, m), 1.75 (IH, m), 3.00 (IH, d, J=16Hz), 3.42-3.63 (4H, m), 4.10 (IH, m), 4.25 (IH, m), 4.45-4.67 (2H, m), 5.05 (IH, d, J=l l Hz), 6.35 (I H, d, J=8 Hz), 6.44 (IH, m), 7.23 (I H, m), 7.26- 7.32 (4H, m), 7.72 (I H, d, J=9 Hz), 8.00-8.25 (2H, m), 8.95 (I H, bs), MS (LC/MS) m/z observed 648.10, expected 648.33 [M+H].

EXAMPLE C32

(2-?)-5-AiviiNO-l-{2-[(25,3_lS)-3-iviETHYL-2-(2-

PHENYLACETAIMIDO)PENTANAIVIIDO]ACETYL}-N-(2H-l,2,3,4-TETRAZOL-5-YLIVlETHYL)- 2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE HYDROCHLORIDE

Title compound C32 was prepared from C31 using method E. Ή NMR (400 MHz, DMSCM6) δ 0.75-0.87 (6H, m), 1.08 (IH, m), 1.40-1.50 (10H, m), 1.74 (IH, m), 3.10 (I H, d, J=16Hz), 3.42-3.63 (4H, m), 4.10 (IH, m), 4.25 (IH, t, J=l l Hz), 4.40- 4.67 (2H, m), 5.13 (IH, d, J=l 1 Hz), 7.14-7.21 (2H, m), 7.24-7.30 (4H, m), 7.36 (IH, m), 7.88 (IH, d, .7=9 Hz), 8.1 1 (IH, d, J=9Hz), 8.24 (IH, m), 9.00 (IH, bs), 9.27 (IH, bs), MS (LC/MS) m/z observed 548.05, expected 548.27 [M+H]. EXAMPLE C33

(25)-5-(Γ£·ΛΓ-ΒυΤΥίΑΜΙΝθ)-1-{2-[(25,35)-3-ΜΕΤΗΥί-2-(2- PHENYLACETA IDO)PENTANAlVIIDO]ACETYL}-A⁷-(2H-l,2,3,4-TETRAZOL-5-YLIVIETHYL)- 2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

Title compound C32 (25 mg, 0.0428 mmol) was dissolved in DMF (5 mL) in a microwaved vial. Triethylamine (2 mL) and 2-bromo-2-methyl bromide (500 uL) were then added. The reaction was then microwaved at 100°C for 15 minutes. Additional triethylamine (3 mL) and 2-bromo-2-methyl bromide (1 mL) were added and the reaction was microwaved for an additional 50 minutes at 100°C. Additional triethylamine (2 mL) 2-bromo-2-methyl bromide (1 mL) were added and the reaction was microwaved for an additional 30 minutes at 100°C. The reaction mixture was then filtered to remove salts and the filtrate was concentrated. The product was purified on a CI 8 column using 10-55% MeOH in water to yield title compound C33 as a brown solid (1.2 mg, 5%). MS (LC/MS) m/z observed 604.20, expected 604.34 [M+H].

EXAMPLE C34

(2-?)-N^V^-TRIMETHYL-l-{2-[(2S,3₁?)-3-METHYL-2-(2- PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-2-[(2H-l,2,3,4-TETRAZOL-5- YLMETHYL)CARBAMOYL]-2,3-DIHYDRO-lH-INDOL-5-AMINIUM IODIDE

C32 (25 mg, 0.0428 mmol) was dissolved in DMF (8 mL) in a microwavable vial. Triethylamine (500 μΕ) and methyl iodide (150 μΕ) were then added. The reaction was then microwaved at 75°C for 35 minutes. The reaction mixture was then concentrated and the product was purified on a CI 8 column using 10-55% MeOH in water to yield title compound C34 as an off-white solid (4 mg, 13%). Ή NMR (400 MHz, DMSO-rf6) δ 0.75-0.87 (6H, m), 1.08 (1H, m), 1.33 (1H, m), 1.74 (1H, m), 2.80-2.86 (9H, m), 3.03 (1H, d, J=16Hz), 3.42-3.62 (4H, m), 4.10 (1H, m), 4.40 (1 H, m), 4.54 (1H, m), 4.63 (1H, m), 5.10 (1H, m), 6.54 (1H, m), 6.64 (1H, bs), 7.20 (1H, m), 7.25-7.32 (4H, m), 7.86 (1H, m), 8.03 (1H, m), 8.09-8.18 (2H, m), 9.07 (1H, bs), MS (LC/MS) m/z observed 590.1 1, expected 590.32[M]. EXAMPLE C35

(Z^-S-iBENZYLAMINOj-l-il-til^S^-S-METHYL-l-il- PHENYLACETAMIDO)PENTANAMIDO]ACETYL}-N-(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)- 2,3-DIHYDRO-lH-INDOLE-2-CARBOXAMIDE

C32 (36 mg, 0.0616 mmol) was dissolved in DMF (5 mL) in a microwaved vial.

Triethylamine (1 mL) and benzyl bromide (8 μΐ_^, 0.0678 mmol) were then added. The reaction was then microwaved at 50°C for 35 minutes. Additional benzyl bromide (15 μL, 0.126 mmol) was added and the reaction was then microwaved at 50°C for 30 additional minutes. The reaction mixture was then concentrated and the product was purified on a CI 8 column using 10-55% MeOH in water to yield title compound C35 as an off-white solid (5 mg, 13%). 1H NMR (400 MHz, DMSO-</6) δ 0.73-0.85 (6H, m), 1 .08 (1 H, m), 1.44 (1H, m), 1.75 (1H, m), 2.90 (1H, d, J=16Hz), 3.42-3.62 (6H, m), 4.08 (1 H, m), 4.41 (1H, m), 4.56 (1H, m), 4.68 (1H, m), 5.05 (1 H, d, J=l lHz), 6.35 (1 H, m), 6.45 (1H, m), 7.19 (1 H, m), 7.21-7.32 (6H, m), 7.33-7.39 (3H, m), 7.78 (1 H, m), 8.03 (1H, m), 8.09-8.18 (2H, m), 9.10 (1H, bs), MS (LC/MS) m/z observed 637.99, expected 638.32 [M+H],

EXAMPLE C36

(2_iS)-l-{2-[(2,S',35)-2-[(DlMETHOXY-l,3,5-TRIAZIN-2-YL)AMINO]-3- ETHYLPENTANAMIDO]ACETYL}-N-(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)-2,3- DIHYDRO-1H-INDOLE-2-CARBOXAMIDE

A solution of (25 -l-[2-((25',35)-2-amino-3-methyl-pentanoylamino)-acetyl]-2,3- dihydro-lH-indole-2-carboxylic acid ethyl ester hydrochloride (0.1 gm, 0.1941 mmol) that was prepared as in Example C26, DIPEA (0.085 ml, 0.291 mmol) and 2-chloro-4,6- dimethoxy-l ,3,5-triazine (0.051 gm, 0.291 mmol) in ACN (3 ml) was sealed in a microwave tube and heated to 100°C for 10 min by microwave irradiation. The reaction mixture was concentrated to dryness and dried well under vacuum to give the crude product, which was purified by column chromatography on silica gel to get the pure product (5}-l -(2-((25',35)-2-((4,6-dimethoxy-l,3,5-triazin-2-yl)amino)-3- methylpentanamido)acetyl)indoline-2-carboxylic acid ethyl ester as an off-white solid, 0.1 gm (80%). MS (LC/MS) m/z observed 501.15, expected 501.24 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C36 was prepared from (5)-l -(2-((25,35)-2-((4,6-dimethoxy- 1 ,3,5-triazin-2-yl)amino)-3-methylpentanamido)acetyl)indoline-2-carboxylic acid ethyl ester and (2H-tetrazol-5-yl)methyl-amine using general method L. Ή NMR (400 MHz, DMSCM6) δ 0.82-0.86 (3H, t, J=8 Hz), 0.90-0.92 (3H, d, J=8 Hz), 1.16-1 .24 (1 H, m), 1.46- 1.53 (1 H, m), 1.80- 1.87 (1H, m), 3.1 1-3.15 (2H, d, J=16 Hz), 3.52-3.60 (2H, m), 3.84 (6H, s), 4.16-4.20 (1H, t, J=8 Hz), 4.35-4.42 (1H, m), 4.56-4.62 (1H, d, J=\6 Hz), 5.16 (1 H, s), 5.14-5.17 (1H, d, J=16 Hz), 6.60-6.62 (lh, t, J=4 Hz), 6.99-7.03 (1H, t, j=8 Hz), 7.17-7.23 (2H, m), 7.81-7.85 (1 H, t, J=8 Hz), 8.02 (1H, s), 8.20 (1H, bs), 8.28 (1H, bs), 9.06 (1H, bs) MS LC/MS) m/z observed 554.18, expected 554.25 [M+H].

EXAMPLE C37

(25)-l-{2-[(2.ii)-2-[(DlMETHOXY-l,3,5-TRIAZIN-2-YL)AMINO]-3- ETHYLBUTANA IDO]ACETYL}- V-(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)-2,3- DIHYDRO-1H-INDOLE-2-CARBOXAMIDE

A solution of

2-amino-3-methylbutanoate hydrochloride (0.5 g, 2.8 mmol), DIPEA (1.16 ml, 6.7 mmol) and 2-chloro-4,6-dimethoxy-l ,3,5-triazine (0.4 g, 1.9 mmol) in ACN (10 ml) was sealed in a microwave tube and heated to 100°C for 10 min by microwave irradiation. The reaction mixture was concentrated to dryness and dried well under vacuum to give the crude product, which was purified by column chromatography on silica gel to get the pure product

2-((4,6-dimethoxy- l ,3,5-triazin-2-yl)amino)-3-methylbutanoate as an off-white solid, 0.45 g (76%). Ή NMR (400 MHz, CDC1₃) δ 0.96-1.00 (6H, t, , J=8 Hz), 1.45 (9H, s), 2.19-2.27 (1 H, m), 3.94 (3H, s), 3.95 (3H, s), 4.58-4.61 (1H, q, J=4H), 5.76-5.78 (1H, d, J=8 Hz). MS (LC/MS) m/z observed 313.06, expected 313.18 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

(S)-tert-B ty\ 2-((4,6-dimethoxy- 1 ,3,5-triazin-2-yl)amino)-3-methylbutanoate (0.1 g, 0.32 mmol) was stirred in 1 : 1 mixture of DCM:TFA (4 ml) for 2 hrs at RT. The reaction mixture was concentrated to dryness under vacuum and swapped with DCM (4 ml) three times. Resulting residue was dried well under vacuum and subjected to next reaction as it is. The residue obtained above, 1-4 (0.1 1 g, 0.32 mmol), EDC (0.092 g, 0.48 mmol), HOBt (0.064 g, 0.42 mmol) and DIPEA (0.22 ml, 1.28 mmol) were stirred in anhydrous DCM (5 ml) for 16 hrs. The reaction mixture was concentrated under vacuum to give the crude product which was purified on a C 18 column using 10-50%) MeOH in water to yield the title compound C37 as an off-white solid (45%). 1H NMR (400 MHz, DMSO-i 6) δ 0.92-0.98 (6H, m), 2.08-2.06 (I H, m), 1.80-1.87 (IH, m), 2.98-3.1 1 (2H, m), 3.52-3.58 (2H, m), 3.83 (6H, s), 4.13-4.17 (IH, t, J=8 Hz), 4.32-4.40 (2H, m), 4.47- 4.52 (I H, m), 5.13-5.16 (IH, d, J=12 Hz), 6.98-7.02 (IH, t, J=8 Hz), 7.14-7.22 (2H, m), 7.77-7.81 (I H, t, J=8 Hz), 8.03-8.05 (IH, d, J=8 Hz), 8.23 (IH, bs), 8.34 (IH, bs), 8.72 (I H, bs) MS (LC/MS) m/z observed 540.17, expected 540.24 [M+H].

EXAMPLE C38

3-{[(₁S)-({2-OXO-2-[(2₁S)-2-[(2H-l,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-2,3-

DIHYDRO-lH-INDOL-1-

YL]ETHYL}CARBAMOYL)(PHENYL)METHYL]CARBAMOYL}PROPANOIC ACID

(2iS)-2-{[(ier/-Butoxy)carbonyl]amino}-2-phenylacelie acid (3.0 g, 12.1 mmol,

92%) was collected as a colorless oil from (^-phenylglycine (2.0 g, 13.2 mmol) using general method . Ή NMR (400 MHz, CDC1₃) δ 7.98 (IH, bs), 7.44 (2H, d, J=7Hz), 7.34 (3H, m), 5.14 (I H, d, J=7Hz), 1.23 (9H, s), MS (LC/MS) m/z observed 273.96, expected 274.10 [M+Na].

(2S Ethyl l-{2-[(25)-2-{[(rerr-butoxy)carbonyl]amino}-2- phenylacetamido]acetyl}-2,3-dihydro-lH-indole-2-carboxyIate (131 mg, 0.27 mmol, 96%) was collected as an off white solid from the coupling of 1-10 (80 mg, 0.28 mmol) with (25)-2-{ [(ierf-butoxy)carbonyl]amino}-2-phenylacetic acid (71 mg 0.28 mmol) using general method M. MS (LC/MS) m/z observed 481.88, expected 482.23 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

(25)- 1 - {2-[(2iS)-2- { [(/er/-Butoxy)carbonyl]amino } -2-phenylacetamido]acetyl } - 2,3-dihydro-lH-indole-2-carboxylic acid (130 mg, 0.28 mmol, 98%) was collected as an off white solid from (2S)-ethyl l -{2-[(2S)-2-{ [(iert-butoxy)carbonyl]amino} -2- phenylacetamido]acetyl}-2,3-dihydro-lH-indole-2-carboxylate (137 mg, 0.285 mmol) using general method D. In this example, the reaction was stopped after 30 min and a gradient of 10-70% MeOH in H₂0 was used during purification. MS (LC/MS) m/z observed 453.89, expected 454.20 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

iert-Butyl N-[(5)-({2-oxo-2-[(2^-2-[(2H-l,2,3,4-tetrazol-5-ylmethyl)carbamoyl]- 2,3-dihydro-lH-indol-l-yl]ethyl}carbamoyl)(phenyl)methyl]carbamate (96 mg, 0.18 mmol, 63%) was collected as an off white solid from (2S)-\-{2-[(2S)-2-{[{tert- butoxy)carbonyl]amino}-2-phenylacetamido]acetyl}-2,3-dihydro-lH-indole-2-carboxylic acid (130 mg, 0.28 mmol) using general method O. MS (LC/MS) m/z observed 534.91 , expected 535.24 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C38 (41 mg, 0.08 mmol, 43%) was collected as an off white solid from tert-butyl N-[(>S)-({2-oxo-2-[(2₁S)-2-[(2H-l ,2,3,4-tetrazol-5-ylmethyl)carbamoyl]- 2,3-dihydro-lH-indol-l -yl]ethyl}carbamoyl)(phenyl)methyl]carbamate (96 mg, 0.18 mmol) using general method I. 1H NMR (400 MHz, DMSO-rf6) δ 9.08 (IH, bs), 8.65 (IH, bs), 8.56 (IH, d, J=8Hz), 8.05 (IH, d, J=8Hz), 7.47 (2H, d, J=7Hz), 7.34 (2H, t, J=7Hz), 7.28 (IH, d, J=7Hz), 7.26-7.15 (2H, m), 7.01 (IH, d, J=7Hz), 5.60 (IH, d, J=6Hz), 5.1 7 (I H, d, ,/=10Hz), 4.65 (IH, dd, J=16, 5Hz), 4.53 (IH, dd, J=16, 4Hz), 4.22 (IH, dd, J=16, 5Hz), 3.68-3.50 (2H, m), 3.14 (I H, d, J=16Hz), 2.48-2.39 (4H, m), MS (LC/MS) m/z observed 534.97, expected 535.20 [M+H].

EXAMPLE C39

(25)-l-{2-[(25^,,35)-3-METHYL-2-[2-(2H-l,2,3,4-TETRAZOL-5- YL)ACETAlVIIDO] PENTANAMIDO]ACETYL}-iV-(lH-l,2,3-TRIAZOL-4-YLIVIETHYL)-2,3-

DIHYDRO-1H-INDOLE-2-CARBOXAMIDE

(25 -l-[2-((2iS',35^,)-2-ieri-Butoxycarbonylamino-3-methyl-pentanoylamino)-acetyl]-2,3- dihydro-lH-indole-2-carboxylic acid ethyl ester was prepared from 1-10 and Boc-L- isoleucine using method A. MS (LC MS) m/z observed 461.98, expected 462.26 [Μ+Η]. Compound was confirmed using LC/MS and moved to next step as it was.

(2₁S¹)-l-[2-((25',35^,)-2-/er/-Butoxycarbonylamino-3-methyl-pentanoylamino)- acetyl]-2,3-dihydro-lH-indole-2-carboxylic acid was prepared from (25)-l-[2-((25',35)-2-^■eri-butoxycarbonylamino-3-methyl-pentanoylamino)-acetyl]-2,3-dihydro- lH-indole-2- carboxylic acid ethyl ester using method D with 2 eq. of LiOH Ή₂0. MS (LC/MS) m/z observed 433.96, expected 434.23 [M+H] , Compound was confirmed using LC/MS and moved to next step as it was.

N-[( 15,25)-2-Methyl- 1 -( {2-oxo-2-[(2S)-2-[( 1 H- 1 ,2,3 -triazol-4- ylmethyl)carbamoyl]-2,3-dihydro-lH-indol-l-yl]ethyl}carbamoyl)butyl]carbamic acid /eri-butyl ester was prepared from (25)-l-[2-((2>S',3S)-2-ier/-Butoxycarbonylamino-3- methyl-pentanoylamino)-acetyl]-2,3-dihydro- lH-indole-2-carboxylic acid and (2H- 1 ,2,3- triazol-4-yl)methyl-amine) using method A in DMF but without HC1 treatment. MS (LC/MS) m/z observed 513.97, expected 514.28 [M+H]. Compound was confirmed using LC/MS and moved to next step as it was.

Title compound C39 was prepared from N-[(15',2iS)-2-methyl-l -({2-oxo-2-[(25)- 2- [( 1 H- 1 ,2,3 -triazol-4-ylmethyl)carbamoyl] -2,3-dihydro- 1 H-indol- 1 - yl]ethyl}carbamoyl)butyl]carbamic acid tert-butyl ester and 2-(2H-tetrazol-5-yl)acetic acid using method A but with DMF as solvent for the coupling reaction: Ή NMR (400 MHz, DMSO-i 6) δ 0.82 (3H, t, J=7.4Hz), 0.88 (311, d, J=7Hz), 1.12 (1 H, m), 1.50 (1H, m), 1.78 (1H, m), 3.05 (1H, m), 3.56-3.67 (2H, m), 3.92-4.02 (2H, m), 4.13 (1H, m), 4.31 (1H, t, J=8Hz), 4.37-4.43 (2H, m), 5.13 (1H, d, J=8Hz), 7.01 (1H, t, J=8Hz), 7.14-7- 28 (2H, m), 7.70 (1H, s), 8.04 (1H, d, J=8Hz), 8.34 (1H, bs), 8.47 (1H, d, J=9Hz), 8.88 (1H, bs), MS (LC/MS) m/z observed 524.62, expected 524.25 [M+H],

EXAMPLE Dl

GENERAL KINETIC ENZYME ASSAY PROTOCOL

A specific 2X assay buffer was prepared for the enzyme to be tested (see Table 2 for final IX assay buffer compositions). If the assay buffer included DTT, it was added immediately prior to running the assay. A 2X enzyme mix was prepared (see Table 3 for enzyme assay conditions) at 80 \L per well. Compounds were screened at one or two appropriate concentrations (to determine the percent inhibition at those concentrations) and/or a full dose response curve (typically 8 points, to identify the IC50) in duplicate, triplicate, or higher replicates as needed. An appropriate control was also assessed in full dose response, in duplicate for each assay/plate. Background control wells consisted of IX assay buffer, DMSO (5% v/v) and substrate. Positive control wells consisted of enzyme, DMSO (5% v/v) and substrate. Test compounds and control compounds were diluted in DMSO to 40X the final desired concentration. For example, a test compound may be tested in dose response, in serial, tripling dilution condition starting at 20 μΜ and ending at 9.1 nM (or any appropriate concentration range and dilution scheme). Control compounds were prepared similarly. Diluted compounds were prepared in a dilution plate and transferred to the reaction plate (96-well medium binding plate (Greiner Bio- One FLUOTRAC™)) to allow for the desired final concentrations when added to the enzyme with AB. After mixing, the reaction plate was placed on a shaker (at 300 RPM) for 5 min, followed by incubation (covered) on the bench, for 20 min. Plates were warmed to reaction temperature (see Table 3) for a total incubation time of 30 min. Plates so prepared were ready for addition of substrate and the subsequent reaction.

An appropriate substrate for each assay was prepared in advance at 2X the final desired concentration (see Table 2) in DMSO. The appropriate substrate mix was added to each appropriate well on the reaction plate, and the plate was read immediately in the TEC AN plate reader (TEC AN INFINITE^® Ml 000 Pro), set to the correct wavelength as needed for each assay (see Table 3) using 25 cycles, kinetic interval of 1 min, number of reads per well of 20 with shaking set to 1 sec, double orbital, 2 mm amplitude. For fluorescent assays the gain was set to optimal (50%).

Table. 2. Assay Buffer Composition.

Enzyme Assay Buffer Composition

50 mM HEPES pH 7.2

50 mM NaCl

Caspase 1 , 3, 4, 5, 7, 8*, 9 & 10/a

0.1 % (w/v) CHAPS

(General caspase assay buffer)

l O mM EDTA

5% (v/v) Glycerol

l O mM DTT

50 mM HEPES pH 7.5

10% (w/v) sucrose

GzmB & Caspase 8

0.2% (w/v) CHAPS

5 mM DTT

*Can also use GzmB assay buffer for the Caspase-8 assay; Assay buffer components were sourced as follows: HEPES, DTT, Glycerol and sucrose: Sigma- Aldrich, St. Louis, MO, USA, NaCl and EDTA: Fisher Scientific, Pittsburgh, PA, USA, CHAPS: Calbiochem, Billerica, MA, USA. Table 3. Enzyme assay conditions.

* Ex/Em λ is the excitation and emission wavelengths at which to measure fluorescence. Enzyme and substrate concentrations are the final concentrations in the well. Note that most protocols require preparing 2X enzyme and substrate mixes, as they are diluted 2-fold in the well.

Enzymes were sourced as follows: hGzmB, Froelich Lab, Northshore University Health Systems Research Institute, Evanston, IL, USA; Caspases, Biovision Inc., Milpitas, CA, USA. Substrates were sourced as follows: Ac-IEPD-AMC, California Peptide Research Inc., Napa, CA, USA; YVAD-AFC, Biovision Inc., Milpitas, CA, USA; Ac-DEVD-AMC, LEHD-AFC, AC-WEHD-AFC and Ac-IETD-AMC, Enzo Life Sciences Inc, Farmingdale, NY, USA. Control inhibitors were sourced as follows: Ac-IEPD-CHO, Ac-WEHD-FMK and Q-LEHD-Oph, Biovision Inc., Milpitas, CA, USA; Z-VAD-FMK, R&D Systems, Minneapolis, MN, USA; and Ac-AEVD-CHO, Enzo Life Sciences Inc, Farmingdale, NY, USA. EXAMPLE D2

HUMAN GRANZYME B ENZYMATIC INHIBITION ASSAY

An in vitro fluorogenic detection assay for assessing the IC50 and/or percent inhibition at a given concentration of inhibitors against human Granzyme B (hGzmB) enzyme was performed as described in Example Dl . When appropriate, percent inhibition data was collected and fitted to generate IC50 data using GraphPad Prism 5 (GraphPad Software, La Jolla California USA) and its non-linear regression analysis tools or other equivalent tools.

Select compounds of Examples A1-A57, B1 -B7 and C1-C39 exhibited inhibitory activity against hGzmB. Each of the compounds of the invention identified in Table 1 exhibited Granzyme B inhibitory activity.

In certain embodiments, select compounds exhibited IC50 < 50,000 nM. In other embodiments, select compounds exhibited IC50 < 10,000 nM. In further embodiments, select compounds exhibited IC50 < 1 ,000 nM. In still further embodiments, select compounds exhibited IC50 < 100 nM. In certain embodiments, select compounds exhibited TC50 from 10 nM to 100 nM, preferably from 1 nM to 10 nM, more preferably from 0.1 nM to 1 nM, and even more preferably from 0.01 nM to 0.1 nM.

EXAMPLE D3

HUMAN CASPASE ENZYMATIC INHIBITION ASSAY

In vitro fluorogenic detection assays for assessing the IC50 and/or percent inhibition at a given concentration of inhibitors, against a set of human Caspase enzymes, was performed as described in Example Dl . Representative compounds of the invention do not significantly inhibit any caspase enzyme tested at a concentration of 50 μΜ.

In certain embodiments, the compounds exhibited less than 50% inhibition at 50 μΜ. In other embodiments, the compounds exhibited greater than 50% inhibition at 50 μΜ, but less than 10% inhibition at 25 μΜ.

EXAMPLE D4

KINETIC SOLUBILITY DETERMINATION

Kinetic solubility buffer (phosphate buffered saline (PBS, IX) at pH 7.4) was prepared from PBS (10X) solution, (Fisher Scientific, Pittsburgh, PA, USA, 10X), by adding PBS (50mL, 10X) to approximately water (450mL HPLC grade). The volume of the solution was then adjusted to 500 niL for a total dilution factor of 1 : 10 and a final PBS concentration of IX. The pH of the final solution (PBS (IX)) was measured and found to be 7.4.

A DMSO stock solution (typically lOmM or greater) was used for each compound to be tested. A final DMSO concentration of 2.0% and maximum theoretical compound concentration of typically 200 μΜ (or greater) was achieved by diluting an aliquot (6 μΐ) of each stock with kinetic solubility buffer (294 μΐ, PBS (l x) at pH 7.4) using a liquid handling system (Hamilton STARlet, Hamilton Robotics, Inc., Reno, NV, USA) and incubated directly in a solubility filter plate (Millipore, Billerica, MA, USA). Following 24 hour incubation at ambient temperature (20.5-21.7 °C), each sample was vacuum filtered. The filtrate was injected into the chemiluminescent nitrogen detector for quantification (Automated Discovery Workstation, Analiza, Inc., Cleveland, OH, USA). Each result was reported both in μΜ and μg/mL units. Three separate on-board performance indicating standards were assayed in triplicate with each test compound to ensure test results were within the acceptable range.

The equimolar nitrogen response of the detector was calibrated using standards which spanned the dynamic range of the instrument from 0.08 to 4500 μg/ml nitrogen. Each filtrate was quantified with respect to this calibration curve. Each calculated solubility value was corrected for background nitrogen present in the DMSO, and buffer used to prepare each sample. All reported values for samples containing adjacent nitrogen atoms in a ring structure were corrected for the expected non-equimolar response. Each calculated solubility result assumed that each sample was free of nitrogen containing impurities and was stable under the assay conditions. Analyses were performed at Analiza Inc. (Cleveland, OH, USA) by published methods (Bhattachar, S.N., et ah , J. Pharma. BioMedAnal. 41 :152-157, 2006).

Table 4. Kinetic solubility in PBS.

A25 696 462

A26-1 >750 >395

A26-2 685 361

A27 53 3 1

A28 >750 >393

A30-1 >750 >396

A30-2 >750 >396

A32 >750 >410

A34 >750 >443

A43 583 342

A44 724 467

A45 682 359

A46 >750 >375

A52 >750 >4 1 8

A53 >750 >407

A54-1 45 1 273

A54-2 >750 >454

A55 >750 >41 8

A56 >750 >407

Bl 97 62

B2 12.6 8.4

CI > 150 >78

Cll 125 68

C12 1 12 61

C13 >750 >454

C20 596 348

C22 >750 >486

C24 >750 >405

C27 >750 >442

1 C32 >750 >438 EXAMPLE D5

GENERAL KINETIC ENZYME ASSAY PROTOCOL (384 WELL) A specific 2X assay buffer was prepared for the enzyme to be tested (see Table 5 for final IX assay buffer compositions). If the assay buffer included DTT, it was added immediately prior to running the assay. A 2X enzyme mix was prepared (see Table 3 for enzyme assay conditions) at 26 per well. Compounds were screened at one or two appropriate concentrations (to determine the percent inhibition at those concentrations) and/or a full dose response curve (typically 12 points, to identify the IC50) in duplicate, triplicate, or higher replicates as needed. An appropriate control was also assessed in full dose response, in duplicate for each assay/plate. Background control wells consisted of IX assay buffer and substrate. Positive control wells consisted of enzyme (no DMSO) and substrate. Test compounds and control compounds were diluted in IX Assay Buffer to 15X the final desired concentration. For example, a test compound may be tested in dose response, in serial, tripling dilution condition starting at 20uM and ending at 0.1 nM (or any appropriate concentration range and dilution scheme). Control compounds were prepared similarly. Diluted compounds were prepared in a dilution plate and transferred to the reaction plate (384-well medium binding plate (Greiner Bio-One FLUOTRAC™)) to allow for the desired final concentrations when added to the enzyme with AB. After mixing, the reaction plate was placed on a shaker (at 300 RPM) for 5 min, followed by incubation (covered) on the bench, for 20 min. Plates were warmed to reaction temperature (see Table 6) for 5 mins for a total incubation time of 30 min. Plates so prepared were ready for addition of substrate and the subsequent reaction.

An appropriate substrate for each assay was prepared in advance at 2X the final desired concentration (see Table 5) in assay buffer. 30 μί, of the appropriate substrate mix was added to each appropriate well on the reaction plate, and the plate was read immediately in the TECAN plate reader (TEC AN INFINITE^® Ml 000 Pro), set to the correct wavelength as needed for each assay (see Table 6) using 15 cycles, kinetic interval of lmin, number of reads per well of 20 with shaking set to Is, double orbital, 2mm amplitude. For fluorescent assays the gain was set to optimal (100% with gain regulation) for all assays except human GzmB which was set to 85 (with the z set at 23000 μηι).

Table 5. Assay Buffer Composition.

Enzyme Assay Buffer Composition

50 mM HEPES pH 7.2

50 mM NaCl

Caspase 1 , 3, 4, 5, 7, 8*, 9 & 10/a

0.1 % (w/v) CHAPS

(General caspase assay buffer)

10 mM EDTA

5% (v/v) Glycerol

l O mM DTT

50 mM HEPES pH 7.5

GzmB & Caspase 8

0.2% (w/v) CHAPS

5 mM DTT

320mM Tris-HCL pH 7.4

Cathepsin G

3.2 M NaCl

*Can also use GzmB assay buffer for the Caspase-8 assay; Assay buffer components were sourced as follows: HEPES, DTT, Glycerol and sucrose: Sigma- Aldrich, St. Louis, MO, USA, NaCl and EDTA: Fisher Scientific, Pittsburgh, PA, USA, CHAPS: Calbiochem, Billerica, MA, USA.

Table 6. Enzyme assay conditions.

* Ex/Em λ is the excitation and emission wavelengths at which to measure fluorescence. Enzyme and substrate concentrations are the final concentrations in the well. Note that most protocols require preparing 2X enzyme and substrate mixes, as they are diluted 2-fold in the well.

Enzymes were sourced as follows: hGzmB, Froelich Lab, Northshore University Health Systems Research Institute, Evanston, IL, USA; Caspases and Elastase, Biovision Inc., Milpitas, CA, USA; Cathepsin G, Athens Research and Technologies, Athens, GA, USA. Substrates were sourced as follows: Ac-IEPD-AMC, California Peptide Research Inc., Napa, CA, USA; YVAD-AFC and MeOSuc-AAPF-AFC Biovision Inc., Milpilas, CA, USA; LEHD-AFC and Suc-AAPF-pNA Millipore, Billerica MA, USA. Ac-DEVD- AMC, AC-WEHD-AFC and Ac-IETD-AMC, Enzo Life Sciences Inc, Farmingdale, NY, USA. Control inhibitors were sourced as follows: Ac-IEPD-CHO, Ac-WEHD-FMK, Q- LEHD-Oph and CatG inhibito,r Biovision Inc., Milpitas, CA, USA; Z-VAD-FMK, R&D Systems, Minneapolis, MN, USA; and Ac-AEVD-CHO, Enzo Life Sciences Inc, Farmingdale, NY, USA. Sivelestat, Tocris Bioscience, Bristol, UK.

EXAMPLE D6

HUMAN GRANZYME B ENZYMATIC INHIBITION ASSAY

An in vitro fluorogenic detection assay for assessing the IC₅o and/or percent inhibition at a given concentration of inhibitors against human Granzyme B (hGzmB) enzyme was performed as described in Example D5. When appropriate, percent inhibition data was collected and fitted to generate IC50 data using GraphPad Prism 5 (GraphPad Software, La Jolla California USA) and its non-linear regression analysis tools or other equivalent tools.

Select compounds of Examples A1 -A57, B 1 -B7 and C1 -C39 exhibited inhibitory activity against hGzmB. Each of the compounds of the invention identified in Table 1 exhibited Granzyme B inhibitory activity.

In certain embodiments, select compounds exhibited IC50 < 50,000 nM. In other embodiments, select compounds exhibited IC50 < 10,000 nM. In further embodiments, select compounds exhibited IC50 < 1 ,000 nM. In still further embodiments, select compounds exhibited IC50 < 100 nM. In certain embodiments, select compounds exhibited IC5₀ from 10 nM to 100 nM, preferably from 1 nM to 10 nM, more preferably from 0.1 nM to 1 nM, and even more preferably from 0.01 nM to 0.1 nM.

EXAMPLE D7

HUMAN CASPASE ENZYMATIC INHIBITION ASSAY

In vitro fluorogenic detection assays for assessing the IC50 and/or percent inhibition at a given concentration of inhibitors, against a set of human Caspase enzymes, was performed as described in Example D5. Representative compounds of the invention do not significantly inhibit any caspase enzyme tested at a concentration of 50 μΜ.

In certain embodiments, the compounds exhibited less than 50% inhibition at 50 μΜ. In other embodiments, the compounds exhibited greater than 50% inhibition at 50 μΜ, but less than 10% inhibition at 25 μΜ.

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A cosmetic composition, comprising a cosmetically acceptable carrier and a compound having Formula (I):

its stereoisomers and cosmetically acceptable salts thereof, wherein:

Ria is a heteroaryl group selected from

(a) 1,2,3-triazolyl optionally substituted with Ci-C₂ alkyl,

(b) 1 ,2,3,4-tetrazolyl, and

(c) 1 ,2,3-thiadiazolyl;

R]b and Ric are independently selected from hydrogen, methyl, and halogen; R₂a and R₂b are independently selected from hydrogen and methyl, or R₂a and R₂b taken together are oxo;

n is 1 or 2;

R₃ is selected from hydrogen and C 1 -C4 alkyl optionally substituted with a carboxylic acid or a carboxylate group or an amine group;

Z is an acyl group selected from the group

herein o is 1 or 2, and Rs_a is C4-acyl substituted with

wherein p is 1 or 2, and Rs_a is C₄-acyI substituted with a carboxylic acid; and

(d)

, wherein Y is heteroaryl;

wherein

R4 is selected from

(i) C 1 -C12 alkyl,

00 C 1 -C6 heteroalkyl optionally substituted with Ci-C₆ alkyl,

(iii) C3-C6 cycloalkyl,

(iv) C₆-C,o aryl,

(v) heterocyclyl,

(vi) C3-C 10 heteroaryl,

(vii) aralkyl, and

(viii) heteroalkylaryl;

R5 is -C(=0)-Rio, wherein Rio is selected from

(i) Ci-Ci2 alkyl optionally substituted with C₆-Cio aryl, optionally substituted C3-C 10 heteroaryl, amino, or carboxylic acid,

(ii) C 1 -C10 heteroalkyl optionally substituted with C 1 -C6 alkyl or carboxylic acid,

(iii) C3-C6 cycloalkyl optionally substituted with C1 -C6 alkyl, optionally substituted C₆-Cio aryl, optionally substituted C3-C 10 heteroaryl, amino, or carboxylic acid, (iv) C_fi-Cio aryl optionally substituted with C1 -C6 alkyl, optionally substituted C6-C10 aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(v) heterocyclyl,

(vi) C₃-Cio heteroaryl,

(vii) aralkyl, and

(viii) heteroalkylaryl; and

R6, R₇, R₈, and R9 are independently selected from

(a) hydrogen,

(b) halogen,

(c) d-Ce alkyl,

(d) -XR11, wherein X is selected from O, C(=0), S, S=0, or S(=0)₂,

(e) -C(=0)N(R₁₂)(R₁₃),

(f) -N(Rn) (R₁₂)(R₁₃),

(g) -N-C(=0)-R_n, and

(h) -N-C(=0)0-R_n,

wherein Rn, Ri₂, and R13 are independently selected from the group consisting of hydrogen, Ci-C₆ alkyl, i-C^ heteroalkyl, C₂-C₆ alkenyl, C₆-Cio aralkyl, and C3-C10 heteroaryl.

2. The composition of Claim 1 , wherein the compound has Formula (I) :

Formula (I) its stereoisomers and cosmetically acceptable salts thereof, wherein:

Ria is a heteroaryl group selected from

(a) 1,2,3-triazolyl optionally substituted with C]-C₂ alkyl, and (b) 1 ,2,3,4-tetrazolyl;

Rib and Rjc are independently selected from hydrogen and methyl;

R₂a and R₂b are independently selected from hydrogen and methyl;

n is 1 ;

R₃ is selected from hydrogen and C 1 -C4 alkyl optionally substituted with a carboxylic acid or a carboxylate group or an amine group;

Z is an acyl group selected from the group

H 0

(a)

, wherein 0 is 1 or 2, and Rsa is C4-acyl substituted with a carboxylic acid;

0

H

(c) P , wherein p is 1 or 2, and Rsa is C₄-acyl substituted with a carboxylic acid; and

0

(d) R4 , wherein Y is heteroaryl;

wherein

R4 is selected from

(i) C,-C₁₂ alkyl,

(ii) C -C6 cycloalkyl,

(iii) C6-C 10 aryl, and

(iv) C₃-C 10 heteroaryl;

R5 is -C(=0)-Rio, wherein Rio is selected from (i) C1-C12 alkyl optionally substituted with C₆-Cio aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(ii) C1-C10 heteroalkyl optionally substituted with

alkyl or carboxylic acid,

(iii) C3-C6 cycloalkyl optionally substituted with C1-C6 alkyl, optionally substituted C₆-Cio aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid,

(iv) C₆-Cio aryl optionally substituted with C -Ce alkyl, optionally subsdluled C6-C10 aryl, optionally substituted C3-C10 heteroaryl, amino, or carboxylic acid, and

(v) C3-C10 heteroaryl; and

Re, R₇, Rs, and R are independently selected from

(a) hydrogen,

(b) halogen,

(c) C,-C₆ alkyl,

(d) -XRi 1 , wherein X is selected from O and C(=0),

(e) -N(Rn)(R.2)(Ri 3), and

(f) -N-C(=0)-R_n,

wherein Rn , R12, and R13 are independently selected from the group consisting of hydrogen, C]-C₆ alkyl, C1-C6 heteroalkyl, C₂-C6 alkenyl, C6-C10 aryl, aralkyl, and C3-Cio heteroaryl.

3. The composition of Claims 1 or 2, wherein R_ja is tetrazole or triazole; R_jb and R[ C are H; n is 1 ; R2a and R₂b are H; R₃ is H or C_j -C₄ alkyl substituted with a carboxylic acid or carboxylate group; R₆-R9 are H; and Z is

4. The composition of Claims 1 or 2, wherein The compound of Claims 1 or 2, wherein R_ja is tetrazole or triazole; R_jb and R_jc are H; n is 1 ; R₂a and R₂b are H; R-3 is H or C1 -C4 alkyl substituted with a carboxylic acid or carboxylate group; Rg H; and Z is

5. The composition of Claims 1 or 2, wherein R_ja is tetrazole or triazole; R _[b and R_jc are H; n is 1 ; R₂a and R₂b are H; R₃ is H or C 1-C4 alkyl substituted with a carboxylic acid or carboxylate group; R6- are H; and Z is

6. The composition of Claims 1 or 2, wherein R_ja is tetrazole or triazole; R_jb and R] C are H; n is 1 ; R₂a and R₂b are H; R₃ is H or C 1 -C4 alkyl substituted with a carboxylic acid or carboxylate group; Rg-Rg are H; and Z is

7. A cosmetic composition, comprising a cosmetically acceptable carrier and a compound having Formula (11):

Formula (II) its stereoisomers and cosmetically acceptable salts thereof, wherein:

R_ja is tetrazole or triazole;

R_jb and R_jc are H; n is 1 ;

R₂a and R₂b are H;

R3 is H or C_j-C₄ alkyl substituted with a carboxylic acid or carboxylate group; R₆-R₉ are H;

R₄ is C₃-C₆ cycloalkyl or C C₆ alkyl optionally substituted with hydroxyl or C_j- C₆ alkoxy; and

R₅ is

alkyl substituted with a carboxylic acid or carboxylate group, or C3-C5 cycloalkyl substituted with a carboxylic acid or carboxylate group.

8. A cosmetic composition, comprising a cosmetically acceptable carrier and a compound having Formula III):

Formula (III) its stereoisomers and cosmetically acceptable salts thereof, wherein:

R_ja is tetrazole or triazole;

R₃ is H or C 1 -C4 alkyl substituted with a carboxylic acid or carboxylate group; R4 is C3-C5 cycloalkyl or C C₆ alkyl optionally substituted with hydroxyl or Cj- C alkoxy; and

R_jO is -(ΟΗ₂)_η-00₂Η, wherein n is 2, 3, 4, 5, or 6;

optionally wherein one or more single methylene carbons are substituted with a fiuoro, hydroxy, amino, C_j-C₃ alkyl, or C₆-CJQ aryl group;

optionally wherein one or more single methylene carbons are substituted with two fluoro or C_] -C₃ alkyl groups;

optionally wherein one or more single methylene carbons are substituted with two alkyl groups that taken together with the carbon to which they are attached form a 3, 4, 5, or 6-membered carbocyclic ring; or optionally wherein adjacent carbon atoms from an unsaturated carbon-carbon bond or taken together form a benzene ring.

9. A cosmetic composition, comprising a cosmetically acceptable carrier and a compound havin Formula (III):

Formula (III) its stereoisomers and cosmetically acceptable salts thereof, wherein:

R_|a is tetrazole or triazole;

R₃ is H or C 1 -C4 alkyl substituted with a carboxylic acid or carboxylate group; R4 is C3-C5 cycloalkyl or C_] -C₆ alkyl optionally substituted with hydroxyl or C^ - C₆ alkoxy; and

R₁₀ is

(CH₂)n wherein n is 1 , 2, 3, or 4; and

optionally, for n = 3 or 4, wherein adjacent carbon atoms from an unsaturated carbon-carbon bond.

10. A cosmetic composition, comprising a cosmetically acceptable carrier and a compound having Formula IV):

Formula (IV) its stereoisomers and cosmetically acceptable salts thereof, wherein: R_] a is tetrazole or triazole;

R₃ is H or C_]-C₄ alkyl substituted with a carboxylic acid or carboxylate group; R4 is C3"C₆ cycloalkyl or C_j-C₆ alkyl optionally substituted with hydroxyl or C\ - C₆ alkoxy; and

R_] 1 is selected from:

(a) optionally substituted C₆-C J O aryl; and

(b) optionally substituted C3-C 10 heteroaryl.

1 1. The composition of any one of Claims 1-10, wherein the the composition further comprises a thickener, a diluent, a buffer, a preservative, a surface active agent, a neutral or cationic lipid, a lipid complex, a liposome, a penetration enhancer, a carrier compound and other cosmetically acceptable carrier or excipient.

12. A method for treating, reducing and inhibiting the appearance of ageing in the skin, comprising applying a composition of any one of Claims 1 to 1 1 to a skin or a portion of a skin of a subject.

13. The method of Claim 12, wherein the reduction and inhibiting the appearance of ageing is characterized by a reduction and inhibition of skin fragility, skin atrophy, skin wrinkles, fine lines, skin discoloration, skin sagging, skin fatigue, skin stress, skin inelasticity, skin fragility, skin softening, skin flakiness, skin dryness, enlarged pore size, skin thinning, reduced rate of skin cell turnover, or deep and deepening of skin wrinkles.