WO2006061714A2 - Anticoronaviral compounds and compositions, their pharmaceutical uses and materials for their synthesis - Google Patents

Abstract

The invention relates to methods of inhibiting SARS-related coronavirus viral replication activity comprising contacting a SARS-related coronavirus protease with a therapeutically effective amount of a SARS 3C like protease inhibitor, and compositions comprising the same.

Description

ANTICORONAVIRAL COMPOUNDS AND COMPOSITIONS, THEIR PHARMACEUTICAL USES AND MATERIALS FOR THEIR SYNTHESIS

Background

The invention relates to compounds and methods of inhibiting Severe Acute Respiratory Syndrome viral replication activity comprising contacting a SARS-related coronavirus 3C-like proteinase with a therapeutically effective amount of a SARS 3C-like protease inhibitor. The invention further relates to pharmaceutical compositions containing the SARS 3C-like proteinase inhibitor in a mammal by administering effective amounts of such coronavirus 3C-like proteinase inhibitor. A worldwide outbreak of Severe Acute Respiratory Syndrome-related coronavirus

("SARS") has been associated with exposures originating from a single ill health care worker from Guangdong Province, China. Recently, the causative agent has been identified as a novel coronavirus. There is an acute need in the art for an effective treatment for the SARS-related coronavirus. Recent evidence strongly implicates a new coronavirus as the causative agent of SARS

(Centers for Disease Control, CDC). Coronavirus replication and transcription function is encoded by the so-called "replicase" gene (Thiel, Herold et al. 2001), which consists of two overlapping polyproteins that are extensively processed by viral proteases. The C-proximal region is processed at eleven conserved interdomain junctions by the coronavirus main or "3C- like" protease (Ziebuhr, Snijder et al. 2000). The name "3C-like" protease derives from certain similarities between the coronavirus enzyme and the well-known picornavirus 3C proteases (Gorbalenya, Koonin et al. 1989). These include substrate preferences, use of cysteine as an active site nucleophile in catalysis, and similarities in their putative overall polypeptide folds. Very recently Hilgenfeld and colleagues published a high-resolution X-ray structure of the porcine transmissible gastroenteritis coronavirus main protease (Anand, Palm et al. 2002). Atomic coordinates are available through the Protein Data Bank under accession code 1LVO.

Summary

The present invention relates to compounds of formula (I):

(I) or a pharmaceutically acceptable salt, solvate or salt/solvate thereof; wherein: m is an integer selected from 0 and 1; Y is selected from the group consisting of H, -CH₃, and -CH₂CH₃;

R¹ is selected from C-i to C₇ alkyl, C₃ to C₁₀ cycloalkyl, and benzyl wherein said alkyl, benzyl and cycloalkyl ares unsubstituted or independently substituted with 1 to 3 R⁷ substituents; R² is selected from

R is independently selected from H and C₁ to C₃ alkyl; each R⁴ and R^4a is independently H₁ Ci to C₃ alkyl or C₃ to C₆ cycloalkyl, wherein each alkyl and cycloalkyl is unsubstituted or substituted with oxo, 1 to 3 halogens or 1 to 3 hydroxyls;

R⁵ is H or selected from R⁷ substituents; each R⁶ and R^6a is independently H, C₁ to C₃ alkyl, and -C(O)R³ or R⁶ and R^6a form a 5 to 7 membered heterocycle; each R⁷ is independently selected from halogen, oxo, C₁ to C₄ alkyl, C₂ to C₆ alkenyl, C₂ to C₆ alkynyl, C₃ to C₆ cycloalkyl, -OR⁴, -NR⁴C(O)R⁴, -NR⁴R^4a , SR⁴, -SOR⁴, -SO₂R⁴, - C(O)R⁴, -CO₂R⁴, -SO₂NR⁴R⁴³, -C(O)NR⁴R⁴³, -NR⁴SO₂NR⁴R⁴⁸, 4 to 10 member heterocycle and -OC(O)R⁴, wherein the foregoing alkyl, alkenyl, alkynyl, cycloalkyl and heterocycle groups are each optionally substituted with halogen, hydroxy, C₁ to C₆ alkoxy, and oxo;

Z is selected from the group consisting of

and

n is O to 3;

A is selected from a 4 to 10 member heterocycle, C₃ to C₁₀ cycloalkyl, C₆ to C-JQ aryl and Ci to C7 alkyl , wherein said heterocycle, cycloalkyl, alkyl and aryl are unsubstituted or independently substituted with 1 to 3 R⁷ substituents;

E is

then m is O; and

X is selected from -CH₂OH, -CH₂OR⁶, -CHO, -CH(OR⁶)(OR^6a) and -C(R³JO.

The compounds of the invention also include compounds with the following structure

(H):

(II) or a pharmaceutically acceptable salt, solvate or salt/solvate thereof; wherein:

Y is selected from the group consisting of H, -CH₃, and -CH₂CH₃; R¹ is C₁ to C₇ alky), C₃ to Ci₀ cycloalkyl, and benzyl wherein said alkyl, benzyl and cycloalkyl is unsubstituted or independently substituted with 1 to 3 R⁷ substituents;

R^z is selected from

R³ is selected from H and C-i to C₃ alkyl; each R⁴ and R^4a is independently H or Ci to C₃ alkyl;

R⁵ is H or selected from R⁷ substituents; each R⁶ and R^6a is independently H, C₁ to C₃ alkyl, and -C(O)R³ or R⁶ and R^6a form a 5 to 7 membered heterocycle; each R⁷ is independently selected from halogen, oxo, C₁ to C₄ alkyl, C₂ to C₆ alkenyl, C₂ to C₆ alkynyl, -OR⁴, -NC(O)R⁴, -NR⁴R^4' , SR⁴, -SOR⁴, -SO₂R⁴, -C(O)R⁴, -CO₂R⁴, -

C(O)NR⁴R^4', -SO₂NR⁴R^4', -NR⁴SO₂NR⁴R^4', 4 to 10 member heterocycle and

-OC(O)R⁴, wherein the foregoing R⁷ groups are each optionally substituted with halogen, hydroxy, C-i to C₆ alkoxy, and C₃ to C₆ cycloalkyl wherein said cycloalkyl is unsubstituted or independently substituted with 1 to 3 of substituents independently selected from halogen, hydroxy and C₁ to C₆ alkoxy;

Z is selected from the group consisting of

R and n wherein n is O to 3;

A is selected from a 4 to 10 member heterocycle, C₄ to C₁₀ cycloalkyl, C₆ to Ci₀ aryl and C₁ to C₇ alkyl , wherein said heterocycle, cycloalkyl, alkyl and aryl are unsubstituted or independently substituted with 1 to 3 R⁷ substituents; and

X is selected from -CH₂OH, -CH₂OR⁶, -CHO, -CH(OR^e)(OR^6a) and -C(R³)0.

Also included herein are compounds of formula (I) or (II), wherein R² is

Also included herein are compounds of formula (I) or (II), wherein R is

i

Also included herein are compounds of formula (I) or (II), wherein R² is

Also included herein are compounds of formula (I) or (II), wherein Z is

- A -

Also included herein are compounds of formula (!) or (II), wherein Z is

n

Also included herein are compounds of formula (I) or (II), wherein X is -CH₂OH.

Also included herein are compounds of formula (I) or (II), wherein X is -CH₂OR⁶.

Also included herein are compounds of formula (I) or (II), wherein X is -CHO. Also included herein are compounds of formula (I) or (II), wherein X is -CH(OR⁶)(OR^6a).

Also included herein are compounds of formula (I) or (II), wherein X is -C(R³)O.

The present invention provides methods of inhibiting the activity of a coronavirus 3C protease (also known as proteinase), comprising contacting the coronavirus 3C protease with an effective amount of a SARS 3C protease inhibitor compound or agent. In one embodiment of the present invention, the SARS coronavirus 3C-like protease inhibitor is administered orally or intravenously.

The present invention also provides a method of treating a condition that is mediated by coronavirus 3C-like protease activity in a patient by administering to said patient a pharmaceutically effective amount of a SARS protease inhibitor. The present invention also provides a method of targeting SARS inhibition as a means of treating indications caused by SARS-related viral infections.

The present invention also provides a method of identifying cellular or viral pathways interfering with the functioning of the members of which could be used for treating indications caused by SARS infections by administering a SARS protease inhibitor. The present invention also provides a method of using SARS protease inhibitors as tools for understanding mechanism of action of other SARS inhibitors.

The present invention also provides a method of using SARS 3C-like protease inhibitors for carrying out gene profiling experiments for monitoring the up or down regulation of genes for the purposed of identifying inhibitors for treating indications caused by SARS or SARS like infections.

The present invention further provides a pharmaceutical composition for the treatment of SARS in a mammal containing an amount of a SARS 3C-like protease inhibitor that is effective in treating SARS and a pharmaceutically acceptable carrier.

For purposes of the present invention, as described and claimed herein, the following terms are defined as follows:

As used herein, the terms "comprising" and "including" are used in their open, non- limiting sense. The term "treating", as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment", as used herein, unless otherwise indicated, refers to the act of treating as "treating" is defined immediately above. The term "halo", as used herein, unless otherwise indicated, means fluoro, chloro, bromo or iodo. Preferred halo groups are fluoro, chloro and bromo.

The term "alkyl", as used herein, unless otherwise indicated, includes saturated and unsaturated monovalent hydrocarbon radicals having straight or branched moieties.

The term "alkenyl", as used herein, unless otherwise indicated, includes alkyl moieties having at least one carbon-carbon double bond wherein alkyl is as defined above and including E and Z isomers of said alkenyl moiety.

The term "alkynyl", as used herein, unless otherwise indicated, includes alkyl moieties having at least one carbon-carbon triple bond wherein alkyl is as defined above.

The term "alkoxy", as used herein, unless otherwise indicated, includes O-alkyl groups wherein alkyl is as defined above.

The term "Me" means methyl, "Et" means ethyl, and "Ac" means acetyl.

The term "cycloalkyl", as used herein, unless otherwise indicated refers to a non- aromatic, saturated or partially saturated, monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbon referred to herein containing a total of from 3 to 10 carbon atoms, preferably 5-8 ring carbon atoms. Exemplary cycloalkyls include monocyclic rings having from 3-7, preferably 3-6, carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. Illustrative examples of cycloalkyl are derived from, but not limited to, the following:

The term "aryl", as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl.

The term "4 to 10 membered heterocyclic", as used herein, unless otherwise indicated, includes aromatic and non-aromatic heterocyclic groups containing one to four heteroatoms each selected from O, S and N, wherein each heterocyclic group has from 4 to 10 atoms in its ring system, and with the proviso that the ring of said group does not contain two adjacent O or S atoms. Non-aromatic heterocyclic groups include groups having only 4 atoms in their ring system, but aromatic heterocyclic groups must have at least 5 atoms in their ring system. The heterocyclic groups include benzo-fused ring systems. An example of a 4 membered heterocyclic group is azetidinyl (derived from azetidine). An example of a 5 membered heterocyclic group is thiazolyl and an example of a 10 membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups are pyrrol idinyl, tetrahydrofuranyl, ctihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6- tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3- dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3- azabicyclo[4.1.0]heptanyl, 3H-indolyl and quinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazoiyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The foregoing groups, as derived from the groups listed above, may be C-attached or N-attached where such is possible. For instance, a group derived from pyrrole may be pyrrol- 1-yl (N-attached) or pyrrol-3-yl (C-attached). Further, a group derived from imidazole may be imidazol-1-yl (N-attached) or imidazol-3-yl (C-attached). The 4 to 10 membered heterocyclic may be optionally substituted on any ring carbon, sulfur, or nitrogen atom(s) by one to two oxo, per ring. An example of a heterocyclic group wherein 2 ring carbon atoms are substituted with oxo moieties is 1,1-dioxo-thiomorpholinyl. Other illustrative examples of 4 to 10 membered heterocyclic are derived from, but not limited to, the following:

Unless otherwise indicated, the term "oxo" refers to =0.

The phrase "pharmaceutically acceptable salt(s)", as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the compounds of formula L The compounds of formula I that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of formula I are those that form non-toxic acid addition salts, La, salts containing pharmacologically acceptable anions, such as the acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edislyate, estolate, esylate, ethylsuccinate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, phospate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodode, and valerate salts.

Certain compounds of formula I may have asymmetric centers and therefore exist in different enantiomeric forms. All optical isomers and stereoisomers of the compounds of formula I₁ and mixtures thereof, are considered to be within the scope of the invention. With respect to the compounds of formula i, the invention includes the use of a racemate, one or more enantiomeric forms, one or more diastereomeric forms, or mixtures thereof. The compounds of formula I may also exist as tautomers. This invention relates to the use of all such tautomers and mixtures thereof. Certain functional groups contained within the compounds of the present invention can be substituted for bioisosteric groups, that is, groups which have similar spatial or electronic requirements to the parent group, but exhibit differing or improved physicochemical or other properties. Suitable examples are well known to those of skill in the art, and include, but are not limited to moieties described in Patini et al., Chem. Rev, 1996, 96, 3147-3176 and references cited therein. The subject invention also includes isotopically-labelled compounds, which are identical to those recited in Formula I₁ but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as ²H, ³H, ¹³C₁ ¹⁴C, ¹⁵N₁ ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶CI, respectively. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labelled compounds of the present invention, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C₁ isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., ²H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances, lsotopically labelled compounds of Formula I of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples and Preparations below, by substituting a readily available isotopically labelled reagent for a non- isotopically labelled reagent. This invention also encompasses pharmaceutical compositions containing and methods of treating SARS infections through administering prodrugs of compounds of the formula I. Compounds of formula I having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxy or carboxylic acid group of compounds of formula I. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by three letter symbols and also includes 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma- aminobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine sulfone. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters. Free hydroxy groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs, sulfonate esters and sulfate esters of hydroxy groups. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may be an alkyl ester, optionally substituted with groups including but not limited to ether, amine and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, are also encompassed. Prodrugs of this type are described in J. Med. Chem. 1996, 39, 10. Free amines can also be derivatized as amides, sulfonamides or phosphonamides. All of these prodrug moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities.

The compounds of the invention can also be used in combination with other drugs. For example, dosing a SARS coronavirus infected patient with the SARS coronavirus 3C-like protease inhibitor of the invention and an interferon, such as interferon alpha, or a pegylated interferon, such as PEG-intron or Pegasus, may provide a greater clinical benefit than dosing either the interferon, pegylated interferon or the SARS coronavirus inhibitor alone. Examples of greater clinical benefits could include a larger reduction in symptoms, a faster time to alleviation of symptoms, reduced lung pathology, a larger reduction in the amount of SARS coronavirus in the patient (viral load), and decreased mortality.

The SARS coronavirus infects cells which express p-glycoprotein. Some of the SARS coronavirus 3C-like protease inhibitors of the invention are p-glycoprotein substrates. Compounds which inhibit the SARS coronavirus which are also p-glycoprotein substrates may be dosed with p-glycoprotein inhibitor. Examples of p-glycoprotein inhibitors are verapamil, vinblastine, ketoconazole, nelfinavir, ritonavir or cyclosporine. The p-glycoprotein inhibitors act by inhibiting the efflux of the SARS coronavirus inhibitors of the invention out of the cell. The inhibition of the p-glycoprotein based efflux will prevent reduction of intracellular concentrations of the SARS coronavirus inhibitor due to p-glycoprotein efflux. Inhibition of the p-glycoprotein efflux will result in larger intracellular concentrations of the SARS coronavirus inhibitors. Dosing a SARS coronavirus infected patient with the SARS coronavirus 3C-like protease inhibitors of the invention and a p-glycoprotein inhibitor may lower the amount of SARS coronavirus 3CL protease inhibitor required to achieve an efficacious dose by increasing the intracellular concentration of the SARS coronavirus 3C-like protease inhibitor. Among the agents that may be used to increase the exposure of a mammal to a compound of the present invention are those that can as inhibitors of at least one isoform of the cytochrome P450 (CYP450)enzymes. The isoforms of CYP450 that may be beneficially inhibited include, but are not limited to, CYP1A2, CYP2D6, CYP2C9, CYP2C19 and CYP3A4. The compounds of the invention include compounds that are CYP3A4 substrates and are metabolized by CYP3A4. Dosing a SARS coronavirus infected patient with a SARS coronavirus inhibitor which is a CYP3A4 substrate, such as a SARS coronavirus 3CL protease inhibitor, and a CYP3A4 inhibitor, such as ritonavir, nelfinavir or delavirdine, will reduce the metabolism of the SARS coronavirus inhibitor by CYP3A4. This will result in reduced clearance of the SARS coronavirus inhibitor and increased SARS coronavirus plasma concentrations. The reduced clearance and higher plasma concentrations may result in a lower efficacious dose of the SARS coronavirus inhibitor.

The term "SARS-inhibiting agent" means any SARS related coronavirus 3C-like protease inhibitor compound represented by formula I or a pharmaceutically acceptable salt, hydrate, prodrug, active metabolite or solvate thereof.

The term "interfering with or preventing" SARS-related coronavirus ("SARS") viral replication in a cell means to reduce SARS replication or production of SARS components necessary for progeny virus in a cell as compared to a cell not being transiently or stably transduced with the ribozyme or a vector encoding the ribozyme. Simple and convenient assays to determine if SARS viral replication has been reduced include an ELISA assay for the presence, absence, or reduced presence of anti-SARS antibodies in the blood of the subject (Nasoff et al., PNAS 88:5462-5466, 1991), RT-PCR (Yu et al., in Viral Hepatitis and Liver Disease 574-477, Nishioka, Suzuki and Mishiro (Eds.); Springer-Verlag Tokyo, 1994). Such methods are well known to those of ordinary skill in the art. Alternatively, total RNA from transduced and infected "control" cells can be isolated and subjected to analysis by dot blot or northern blot and probed with SARS specific DNA to determine if SARS replication is reduced. Alternatively, reduction of SARS protein expression can also be used as an indicator of inhibition of SARS replication. A greater than fifty percent reduction in SARS replication as compared to control cells typically quantitates a prevention of SARS replication. If an inhibitor compound used in the method of the invention is a base, a desired salt may be prepared by any suitable method known to the art, including treatment of the free base with an inorganic acid (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like), or with an organic acid (such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid (such as glucuronic acid or galacturonic acid), alpha-hydroxy acid (such as citric acid or tartaric acid), amino acid (such as aspartic acid or glutamic acid), aromatic acid (such as benzoic acid or cinnamic acid), sulfonic acid (such as p-toluenesulfonic acid or ethanesulfonic acid), and the like.

If an inhibitor compound used in the method of the invention is an acid, a desired salt may be prepared by any suitable method known to the art, including treatment of the free acid with an inorganic or organic base (such as an amine (primary, secondary, or tertiary)), an alkali metal hydroxide, or alkaline earth metal hydroxide. Illustrative examples of suitable salts include organic salts derived from amino acids (such as glycine and arginine), ammonia, primary amines, secondary amines, tertiary amines, and cyclic amines (such as piperidine, morpholine, and piperazine), as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

In the case of inhibitor compounds, prodrugs, salts, or solvates that are solids, it is understood by those skilled in the art that the hydroxamate compound, prodrugs, salts, and solvates used in the method of the invention, may exist in different polymorph or crystal forms, all of which are intended to be within the scope of the present invention and specified formulas. In addition, the hydroxamate compound, salts, prodrugs and solvates used in the method of the invention may exist as tautomers, all of which are intended to be within the broad scope of the present invention. Solubilizing agents may also be used with the compounds of the invention to increase the compounds solubility in water or physiologically acceptable solutions. These solubilizing agents include cyclodextrans, propylene glycol, diethylacetamide, polyethylene glycol, Tween, ethanol and micelle forming agents. Oreffered solubilizing agents are cyclodextrans, particularly beta cyclodextrans and in particular hydroxypropyl betacyclodextran and sulfobutylether betacyclodextran.

In some cases, the inhibitor compounds, salts, prodrugs and solvates used in the method of the invention may have chiral centers. When chiral centers are present, the hydroxamate compound, salts, prodrugs and solvates may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates, and mixtures thereof are intended to be within the broad scope of the present invention.

As generally understood by those skilled in the art, an optically pure compound is one that is enantiomerically pure. As used herein, the term "optically pure" is intended to mean a compound comprising at least a sufficient optical activity against the target to be inhubited. Preferably, an optically pure amount of a single enantiomer to yield a compound having the desired pharmacological pure compound of the invention comprises at least 90% of a single isomer (80% enantiomeric excess), more preferably at least 95% (90% e.e.), even more preferably at least 97.5% (95% e.e.), and most preferably at least 99% (98% e.e.).

The term "treating", as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment", as used herein, unless otherwise indicated, refers to the act of treating as "treating" is defined immediately above. In a preferred embodiment of the present invention, "treating" or "treatment" means at least the mitigation of a disease condition in a human that is alleviated by the inhibition of the activity of one or more coronaviral 3C-like proteases, including, but not limited to the 3C-like protease of the causative agent for SARS. In the case of SARS, representative disease conditions include fever, dry cough, dyspnea, headache, hypoxemia, lymphopenia, elevated aminotransferase levels as well as viral titer. Methods of treatment for mitigation of a disease condition include the use of one or more of the compounds in the invention in any conventionally acceptable manner. According to certain preferred embodiments of the invention, the compound or compounds of the present invention are administered to a mammal, such as a human, in need thereof. Preferably, the mammal in need thereof is infected with a coronavirus such as the causative agent of SARS. The present invention also includes prophylactic methods, comprising administering an effective amount of a compound of the invention, or a pharmaceutically acceptable salt, prodrug, pharmaceutically active metabolite, or solvate thereof to a mammal, such as a human, at risk for infection by a coronavirus. According to certain preferred embodiments, an effective amount of one or more compounds of the invention, or a pharmaceutically acceptable salt, prodrug, pharmaceutically active metabolite, or solvate thereof is administered to a human at risk for infection by the causative agent for SARS. The prophylactic methods of the invention include the use of one or more of the compounds in the invention in any conventionally acceptable manner. The following are examples of specific embodiments of the invention.

Detailed Description The compounds of the invention can be made by the following general procedure:

A-1 A-2

Chart 1 depicts the general preparation of compounds of formula I wherein the definitions of R², Z, Y, X, A and B are given in the summary of the invention and PG is a suitable protecting group. Syntheses of compounds of general structure A-4 are possible by an individual skilled in the art, for example by the route shown in Scheme A. Compound A-1 (for its synthesis see Johnson, Theodore O. ef a/., J Med. Chem. 2002, 45, 2016 - 2023 or for a related compound see Dragovich, P. et al., J Med. Chem. 1999, 42, 1213) can be deprotected (i.e. protecting group (PG), such as t-butylcarbonyl (Boc), removed) and coupled, using a reagent such as 0-(7-azabenzotriazol-1-yl)-Λ/,Λ/,/V,Λ/^l-tetramethyluronium hexafluorophosphate (HATU), in the presence of an tertiary amine such as Λ/-methylmorpholine (NMM), to an amino acid analog capped with a protecting group, as an example Boc, generating compound A-2. Removal of the PG group, for example a Boc group can be removed with HCI₁ from A-2 and coupling, using a reagent such as O-(7-azabenzotriazol-1-yl)-Λ/,Λ/,Λf,Λ/'-tetramethyluronium hexafluorophosphate (HATU), in the presence of a tertiary amine, such as NMM, with R²CO₂H produces compound A-3. Oxidation of the primary alcohol in A-3, using appropriate conditions, for example Dess-Martin Periodinane or sulfur trioxide pyridine complex (SO₃Py) in dimethyl sulfoxide (DMSO) in the presence of a tertiary amine base such as diisopropylethylamine, will generate compound A-4. Recent evidence indicates that a new coronavirus is the causative agent of SARS. The nucleotide sequence of the SARS-associated coronavirus has also recently been determined and made publicly available.

The activity of the inhibitor compounds as inhibitors of SARS-related viral activity may be measured by any of the suitable methods available in the art, including in vivo and in vitro assays. The activity of the compounds of the present invention as inhibitors of coronavirus 3C- like protease activity (such as the 3C-like protease of the SARS coronavirus) may be measured by any of the suitable methods known to those skilled in the art, including in vivo and in vitro assays. Examples of suitable assays for activity measurements include the antiviral cell culture assays described herein as well as the antiprotease assays described herein, such as the assays described in the Example section.

Administration of the inhibitor compounds and their pharmaceutically acceptable prodrugs, salts, active metabolites, and solvates may be performed according to any of the accepted modes of administration available to those skilled in the art. Illustrative examples of suitable modes of administration include oral, nasal, pulmonary, parenteral, topical, intravenous, injected, transdermal, and rectal. Oral, intravenous, and nasal deliveries are preferred.

A SARS-inhibiting agent may be administered as a pharmaceutical composition in any suitable pharmaceutical form. Suitable pharmaceutical forms include solid, semisolid, liquid, or lyopholized formulations, such as tablets, powders, capsules, suppositories, suspensions, liposomes, and aerosols. The SARS-inhibiting agent may be prepared as a solution using any of a variety of methodologies. For example, the SARS-inhibiting agent can be dissolved with acid (e.g., 1 M HCI) and diluted with a sufficient volume of a solution of 5% dextrose in water (D5W) to yield the desired final concentration of SARS-inhibiting agent (e.g., about 15 mM). Alternatively, a solution of D5W containing about 15 mM HCI can be used to provide a solution of the SARS-inhibiting agent at the appropriate concentration. Further, the SARS-inhibiting agent can be prepared as a suspension using, for example, a 1% solution of carboxymethylcellulose (CMC).

Acceptable methods of preparing suitable pharmaceutical forms of the pharmaceutical compositions are known or may be routinely determined by those skilled in the art. For example, pharmaceutical preparations may be prepared following conventional techniques of the pharmaceutical chemist involving steps such as mixing, granulating, and compressing when necessary for tablet forms, or mixing, filling, and dissolving the ingredients as appropriate, to give the desired products for oral, parenteral, topical, intravaginal, intranasal, intrabronchial, intraocular, intraaural, and/or rectal administration.

Pharmaceutical compositions of the invention may also include suitable excipients, diluents, vehicles, and carriers, as well as other pharmaceutically active agents, depending upon the intended use. Solid or liquid pharmaceutically acceptable carriers, diluents, vehicles, or excipients may be employed in the pharmaceutical compositions. Illustrative solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, pectin, acacia, magnesium stearate, and stearic acid. Illustrative liquid carriers include syrup, peanut oil, olive oil, saline solution, and water. The carrier or diluent may include a suitable prolonged- release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., solution), or a nonaqueous or aqueous liquid suspension.

A dose of the pharmaceutical composition may contain at least a therapeutically effective amount of an SARS-inhibiting agent and preferably is made up of one or more pharmaceutical dosage units. The selected dose may be administered to a mammal, for example, a human patient, in need of treatment mediated by inhibition of SARS-related coronavirus activity, by any known or suitable method of administering the dose, including topically, for example, as an ointment or cream; orally; rectally, for example, as a suppository; parenterally by injection; intravenously; or continuously by intravaginal, intranasal, intrabronchial, intraaural, or intraocular infusion.

The phrases "therapeutically effective amount" and "effective amount" are intended to mean the amount of an inventive agent that, when administered to a mammal in need of treatment, is sufficient to effect treatment for injury or disease conditions alleviated by the inhibition of SARS viral replication. The amount of a given SARS-inihibiting agent used in the method of the invention that will be therapeutically effective will vary depending upon factors such as the particular SARS-inihibiting agent, the disease condition and the severity thereof, the identity and characteristics of the mammal in need thereof, which amount may be routinely determined by artisans.

It will be appreciated that the actual dosages of the SARS-inhibiting agents used in the pharmaceutical compositions of this invention will be selected according to the properties of the particular agent being used, the particular composition formulated, the mode of administration and the particular site, and the host and condition being treated. Optimal dosages for a given set of conditions can be ascertained by those skilled in the art using conventional dosage- determination tests. For oral administration, e.g., a dose that may be employed is from about 0.01 to about 1000 mg/kg body weight, preferably from about 0.1 to about 500 mg/kg body weight, and even more preferably from about 1 to about 500 mg/kg body weight, with courses of treatment repeated at appropriate intervals. For intervenous dosing a dose of up to 5 grams per day may be employed.

The terms "cytochrome P450-inhibiting amount" and "cytochrome P450 enzyme activity-inhibiting amount," as used herein, refer to an amount of a compound required to decrease the activity of cytochrome P450 enzymes or a particular cytochrome P450 enzyme isoform in the presence of such compound. Whether a particular compound of decreases cytochrome P450 enzyme activity, and the amount of such a compound required to do so, can be determined by methods known to those of ordinary skill in the art and the methods described herein.

Protein functions required for coronavirus replication and transcription are encoded by the so-called "replicase" gene. Two overlapping polyproteins are translated from this gene and extensively processed by viral proteases. The C-proximal region is processed at eleven conserved interdomain junctions by the coronavirus main or "3C-like" protease. The name "3C-like" protease derives from certain similarities between the coronavirus enzyme and the well-known picornavirus 3C proteases. These include substrate preferences, use of cysteine as an active site nucleophile in catalysis, and similarities in their putative overall polypeptide folds. A comparison of the amino acid sequence of the SARS-associated coronavirus 3C-)ike protease to that of other known coronaviruses shows the amino acid sequence to be highly conserved, particularly in the catalytically important regions of the protease.

Amino acids of the substrate in the protease cleavage site are numbered from the N to the C terminus as follows: -P3-P2-P1-P1'-P2'-P3', with cleavage occurring between the P1 and PT residues (Schechter & Berger, 1967). Substrate specificity is largely determined by the P2, P1 and P1 ' positions. Coronavirus main protease cleavage site specificities are highly conserved with a requirement for glutamine at P1 and a small amino acid at PT (Journal of General Virology 83, pp. 595-599 (2002)).

Recently, Hilgenfeld and colleagues published a high-resolution x-ray structure of the porcine transmissible gastroenteritis coronavirus main protease (The EMBO Journal, Vol. 21, pp. 3213-3224 (2002)). Atomic coordinates are available through the Protein Data Bank under accession code 1LVO. Our observations of the catalytic and structural similarities between rhinovirus 3C protease and coronavirus "3C-like" main protease, lead to the conclusion that selected inhibitors of rhinovirus 3C protease would be useful against the coronavirus main (3C- like) protease.

EXAMPLES

In the examples described below, unless otherwise indicated, all temperatures are set forth in degrees Celsius and all parts and percentages are by weight. Reagents may be purchased from commercial suppliers, such as Sigma-Aldrich Chemical Company, or Lancaster Synthesis Ltd. and may be used without further purification unless otherwise indicated. Tetrahydrofuran (THF) and N, N-dimethylformamide (DMF) may be purchased from Aldrich in Sure Seal bottles and used as received. All solvents may be purified using standard methods known to those skilled in the art, unless otherwise indicated.

The structures of the compounds of the following examples were confirmed by one or more of the following: proton magnetic resonance spectroscopy, elemental microanalysis and melting point. Proton magnetic resonance (^"Η NMR) spectra were determined using a Bruker spectrometers operating at a field strength of 300 to 400 megahertz (MHz). Chemical shifts are reported in parts per million (ppm, δ) downfield from an internal tetramethylsilane standard.

Alternatively, ^"^H NMR spectra were referenced to residual protic solvent signals as follows: CHCI3 = 7.26 ppm; DMSO = 2.49 ppnn, C6HD5 = 7.15 ppm. Peak multiplicities are designated as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; q, quartet; br, broad resonance; m, multiplet. Coupling constants are given in Hertz. Elemental microanalyses were performed by Atlantic Microlab Inc., Norcross, GA and gave results for the elements stated within ±0.4% of the theoretical values. Flash column chromatography was performed using Silica gel 60 (Merck Art 9385) or various MPLC systems. Analytical thin layer chromatography (TLC) was performed using precoated sheets of Silica 60 F254 (Merck Art 5719). All reactions were performed in septum-sealed flasks under a slight positive pressure of argon or dry nitrogen unless otherwise noted. Preferred compounds in accordance with the invention may be prepared in manners analogous to those specifically described below.

The examples and preparations provided below further illustrate and exemplify the compounds of the present invention and methods of preparing such compounds. It is to be understood that the scope of the present invention is not limited in any way by the scope of the following examples and preparations. In the following examples molecules with a single chiral center, unless otherwise noted, exist as a racemic mixture. Those molecules with two or more chiral centers, unless otherwise noted, exist as a racemic mixture of diastereomers. Single enantiomers/diastereomers may be obtained by methods known to those skilled in the art.

Where HPLC chromatography is referred to in the preparations and examples below, the general conditions used, unless otherwise indicated, are as follows. The column used is a ZORBAXμ RXC18 column (manufactured by Hewlett Packard) of 150 mm distance and 4.6 mm interior diameter. The samples are run on a Hewlett Packard- 1100 systemA gradient solvent method is used running 100 percent ammonium acetate / acetic acid buffer (0.2 M) to 100 percent acetonitrile over 10 minutes. The system then proceeds on a wash cycle with 100 percent acetonitrile for 1.5 minutes and then 100 percent buffer solution for 3 minutes. The flow rate over this period is a constant 3 ml / minute.

In the examples and specification, "Et" means ethyl, "Ac" means acetyl, "Me" means methyl, "ETOAC" or "ETOAc" means ethyl acetate, "THF" means tetrahydrofuran, and "Bu" means butyl Et₂O refers to diethyl ether, DMF refers to Λ/,Λ/-dimethylformamide. DMSO refers to dimethylsulfoxide. MTBE refers to fe/f-butyl methyl ether. Other abbreviations include: CH3OH (methanol), EtOH (ethanol), EtOAc (ethyl acetate), DME (ethylene glycol dimethyl ether), DCM refers to dichloromethane, 1,2 DCE referes to 1,2 dichloroethane, Ph (phenyl), Tr (triphenylmethyl), Cbz (benzyloxycarbonyl), Boc (ferf-butoxycarbonyl), TFA (trifluoroacetic acid), DIEA (Λ/,Λ/-diisopropylethylamine), TMEDA (Λ/,MΛ/',Λ/'-tetramethylethylenediamine), AcOH (acetic acid), Ac2θ (acetic anhydride), NMM (4-methyImorpholine), HOBt (1-hydroxybenzotriazole hydrate), HATU

[O-(7-azabenzotriazol-1-yl)-Λ/,Λ/,/v',Λ/4etramethyluronium hexafluorophosphate], EDC [1-(3-dimethylaminopropyl)-3-ethylcarbarbodiimide hydrochloride], TEA triethylamine, LDA lithium diisopropyl amide, DCC (dicyclohexyl-carbodiimide), DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone), DMAP (4-dimethylaminopyridine), GIn (glutamine), Leu (leucine), Phe (phenylalanine), Phe(4-F) (4-fluorophenylalanine), VaI (valine), amino-Ala (2,3-diaminopropionic acid), and (S)-Pyrrol-Ala

[(2S,3'S)-2-amino-3-(2'-oxopyrrolidin-3'-yl)-propionic acid]. Additionally, "L" represents the configuration of naturally occurring amino acids. General Scheme A

Syntheses of compounds of general structure A-4 are possible by an individual skilled in the art, for example by the route shown in Scheme A. Compound A-1 (for its synthesis see Johnson, Theodore O. ef a/., J Med. Chem. 2002, 45, 2016 - 2023 or for a related compound see Dragovich, P. et al., J Med. Chem. 1999, 42, 1213) can be deprotected {I.e. protecting group (PG), such as t-butylcarbonyl (Boc), removed) and coupled, using a reagent such as O- (7-azabenzotriazol-1-yl)-Λ/,Λ/,/V,Λ/'-tetramethyluronium hexafluorophosphate (HATU), in the presence of an tertiary amine such as Λ/-methylmorpholine (NMM), to an amino acid analog capped with a protecting group, as an example Boc, generating compound A-2. Removal of the PG group, for example a Boc group can be removed with HCI₁ from A-2 and coupling, using a reagent such as O-(7-azabenzotriazol-1-yl)-N,tø,N,W-tetramethyluronium hexafluorophosphate (HATU), in the presence of a tertiary amine, such as NMM, with R²CO₂H produces compound A-3. Oxidation of the primary alcohol in A-3, using appropriate conditions, for example Dess-Martin Periodinane or sulfur trioxide pyridine complex (SO₃Py) in dimethyl sulfoxide (DMSO) in the presence of a tertiary amine base such as diisopropylethylamine, will generate compound A-4. General Scheme B

As shown in Scheme B, the synthesis of analogs of A-4, such as B-1, B-2, B-3 and B-4 can be achieved directly from A-4 or from compound A-3. Compound B-1 can be generated from A-3 via reaction of the primary alcohol with an acid chloride or sulfonyl chloride, such as acetyl chloride or mesyl chloride, in the presence of a base, such as a tertiary amine like diisopropylethylamine. Alternatively, compound B-1 can be generated by subjecting A-4 to reductive amination conditions in the presence of an appropriate amine in contact with or latter subjected to a reducing agent, for example contacting A-4 with ethylamine in the presence of sodium cyanoborohydride. Conversion of A-4 to B-2 can be achieved by treatment with an appropriate organometallic reagent, for example lithium dimethylcuprate or diethylzinc (Reetz, Manfred T.; Griebenow, Nils, LJebig Annalen 1996, 335 - 348), in a suitable solvent, for example tetrahydrofuran, followed by oxidation of the resulting secondary alcohol, for example with SO₃Py in DMSO in the presence of diisopropylethylamine. Conversion of A-4 to B-3 can be accomplished by contact with R⁴OH, for example ethanol, to generate the R⁴ hemiacetal B- 3. Formation of B-4 is achieved by treatment of A-4 with the corresponding R^SOH, for example water or ethanol, in the presence of acid, such as pyridinium p-toluensulfonate, and possibly an agent like triethylorthoformate. General Scheme C

C-5 C-6

As shown in Scheme C one skilled in the art can readily generate compounds of general structure C-5. Coupling of readily available C-1, for example PG = t-butylcarbonyl and R² = t- butyl, with methoxymethylamine in the presence of a suitable coupling agent, such as O-(7- azabenzotriazol-1-yl)-Λ/,Λ/,Λf,Λ/^l-tetramethyluronium hexafluorophosphate (HATU), and in the presence of an tertiary amine such as Λ/-methylmorpholine (NMM) produces Weinreb amide C- 2. Removal of the protecting group (PG), such as Boc, with an appropriate reagent, such as TFA or HCI for Boc, followed by coupling to an activated N-protected aminoacid, such as the succimide ester, in the presence of a tertiary amine base, for example NMM. And then conversion of the R² ester or its corresponding acid to the R³,R⁴-substituted amide can be achieved by one skilled in the art. Removal of the PG, such as benzyloxycarbonyl (Cbz) or Boc, with the appropriate conditions, for example palladium on carbon in the presence of hydrogen (for Cbz) or trifluoroacetic acid (for Boc), followed by coupling to R⁶carboxylic acid with a coupling reagent, such as HATU, in the presence of a tertiary amine, such as NMM, produces C-4. Reduction of the Weinreb amide in C-4 with the appropriate reagent, for example diisobutylaluminum hydride or lithium aluminum hydride, directly or after subsequent oxidation, with for example sulfur trioxide pyridine complex in DMSO with a tertiary amine such as diisopropylethylamine, generates aldehyde C-5. Compound C-6 is readily generated from C-5 by reaction with an appropriate organometallic reagent, for example lithium dimethylcuprate or diethylzinc (Reetz, Manfred T.; Griebenow, Nils, Liebig Annalen 1996, 335 - 348), in a suitable solvent, for example tetrahydrofuran, followed by oxidation of the resulting secondary alcohol, for example with SO₃Py in DMSO in the presence of diisopropylethylamine. In addition C-6, can also be generated directly from Weinreb amide C-4 by treatment with the appropriate organometallic reagent, such as methyl lithium or methylmagnesium bromide. Example 1

Preparation of W²-(tert-butoxycarbonyl)-W¹ -((1 S)-2-hydroxy-1 -{[(3S)-2-oxopyrrolidin-3- yl]methyl}ethyl)-L-leucinamide

To solid tert-butyl (1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethylcarbamate (40.00 g, 0.1548 mol) in a 2 liter round-bottomed flask cooled to 0 ⁰C was added 5 portions of 100 mL of HCI in dioxane (4 N HCI in dioxane, 13 equiv.) resulting initially in vigorous bubbling. TLC analysis of the resultant white sticky solid showed that starting material had been consumed. The reaction mixture was concentrated In vacuo and 600 mL of 5:1 ethanol(abs):dioxane was added to dissolve all the solid. Upon concentration of the mixture In vacuo, a white foam formed. The crude ammonium salt was dissolved in DMF (600 mL) and the solution was cooled in an ice bath. Λ/-Boc-L-ieucine (35.81 g, 0.1548 mol, 1 equiv.) and O- (7-azabenzotriazol-1-yl)-Λ/,Λ/,Λf,Λ/'-tetramethyluronium hexafluorophosphate (HATU, 58.86 g, 0.1548 mol, 1 equiv.) were sequentially added followed by DMF (100 mL each) to was down the sides of the reaction vessel. After 15 minutes, Λ/-methylmorpholine (NMM, 35.7 mL, 0.325 mol, 2.1 equiv.) was added slowly to the solution. The solution warmed to room temperature overnight and then to the suspension was added NMM (11.9 mL, 1 equiv.), which dissolved most of the precipitate. After 1 h, to the solution was added 100 mL of ice and the material was concentrated in vacuo. The resulting residue was poured into a mixture of 200 mL ice and 600 mL saturated NaHCO₃. The aqueous layer was extracted with ethyl acetate (4x 1 L) and DCM (2x 1 L). The organic layers were combined, dried over magnesium sulfate, filtered and concentrated in vacuo. The material was purified by dissolving in 750 mL of 95(DCM):5(MeOH) and performing three silica gel column chromatographies (1 kg, SiO₂ loaded in 95:5, 3 L of 95:5 then 2 L 92.5:7.5 then 4 - 6 L 90:10) resulting in the isolation of Λ/²- (fert-butoxycarbonyl)-Λ/¹-((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)-L- leucinamide (45.75 g, 0.1232 mol) in 80% yield as a white solid. Mp 178.6 ⁰C. R_f = 0.34 (90:10 DCM-MeOH, ninhydrin stain); ¹H NMR (400 MHz, D₆-DMSO) δ 7.53 (d, J = 9 Hz, 1 H), 7.49 (s, 1H), 6.84 (d, J = 8 Hz, 1 H), 4.64 (t, J = 5 Hz, 1H), 3.89 - 3.82 (m, 1H), 3.82 - 3.72 (m, 1H), 3.34 - 3.29 (m, 1H), 3.24 - 3.18 (m, 1H)₁ 3.15 - 3.10 (m, 1H), 3.05 - 2.95 (m, 1H), 2.28 - 2.20 (m, 1H), 2.19 - 2.09 (m, 1H), 1.81 - 174 (m, 1H), 1.60 - 1.51 (m, 2H), 1.14 - 1.30 (m, 3H), 1.35 (s, 9H), 0.85 (d, J = 7 Hz, 3H), 0.83 (d, J = 7 Hz₁ 3H); MS (ESI+) for C₁₈H₃₃N₃O₅ mlz 372 (M+H); HRMS (ESI+) calcd for C₁₈H₃₃N₃O₅ +Na 394.2312, found 394.2307. Anal. Calcd for C₁₈H₃₃N₃O₅-0.5H₂O: C, 57.02; H, 8.98; N₁ 11.01. Found: C, 56.82; H, 9.01; N, 10.98. Example 2

Preparation of W-((1 S)-1 -{[((1 S)-2-hydroxy-1 -{[(3S)-2-oxopyrrolidin-3- yπmethy^ethylJaminolcarbonyl^S-methylbutylJ^-methoxy-IH-indole-Σ-carboxamide

Λ/²-(ferf-Butoxycarbonyl)-Λ/¹-((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)-

L-leucinamide (25.61 g, 68.9 mmol) was dissolved in 200 mL of warm dioxane in a 2 liter round-bottomed flask and briefly cooled in an ice bath (no freezing observed). To the chilled solution was added two 100 mL protions of HCI in dioxane (4 N HCI in dioxane, 12 equiv.). After a few minutes the ice bath was removed and the reaction was stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo and 600 mL of 5:1 ethanol(abs.):dioxane was added to dissolve all the solid. Upon concentration of the mixture in vacuo, a white powder formed. The crude ammonium salt was dissolved in DMF (220 mL) and the solution was cooled in an ice bath. To the chilled solution was added 4-methoxy-1H-indole- 2-carboxylic acid (13.18 g, 68.9 mmol, 1 equiv.) followed by DMF (40 mL) and O-(7- azabenzotriazol-1-yl)-Λ/,Λ/,Λ/',Λ/'-tetramethyluronium hexafluorophosphate (HATU, 26.20 g, 68.9 mmol, 1 equiv.) and again DMF (40 mL) to wash down the sides of the reaction vessel. After 15 minutes, Λ/-methylmorpholine (NMM, 15.9 mL, 0.145 mol, 2.1 equiv.) was added slowly to the solution. The solution warmed to room temperature overnight and then to the suspension was added NMM (7.6 mL, 1 equiv.). At this point the reaction was incomplete by LC/MS and more 4-methoxy-1H-indole-2-carboxylic acid (300 mg), HATU (200 mg) and NMM (11.4 mL) were added sequentially. After 1 h, to the solution was added 100 mL of ice and the material was concentrated in vacuo. The resulting residue was dissolved in 500 mL CH₂CI₂ and poured into a 500 mL mixture of ice and saturated NaHCO₃. A precipitate formed. The layers were separated and the precipitate filled aqueous layer was extracted with 500 mL CH₂CI₂ and again the layers were separated. The aqueous layer was filtered, washed with 100 mL water, 10O mL EtOAc and dried in a vacuum oven at 60 ⁰C overnight producing 18.00 g of pure title compound (40.5 mmol, 59% yield). The aqueous layer was extracted with ethyl acetate (2 x 0.5 L). The organic layers were combined, dried over magnesium sulfate, filtered and concentrated in vacuo. The crude material was purified by silica gel column chromatography ( 1 kg SiO₂ loaded in 95:5, mobile phase consisted of 95:5 then 93:7 then 90:10 to elute product, sample absorbed on 200 g of SiO₂ with 600 mL MeOH/ CH₂CI₂ and then 60OmL MeOH) resulting in the isolation of 5.96 g more of Λ/-((1S)-1-{[((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3- yl]methyl}ethyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide (13.4 mmol, 19 %) as a light yellow foam. A combined total yield of 78% (23.96 g) was realized. Mp 154.7⁰C (solid). ¹H NMR (400 MHz, D₆-DMSO) δ 11.56 (s, 1H), 8.32 (d, J = 8 Hz₁ 1H), 7.73 (d, J = 9 Hz₁ 1 H), 7.51 (s, 1H), 7.34 (s, 1H), 7.08 (t, J = 8 Hz, 1 H)₁ 6.99 (d, J = 8 Hz₁ 1H), 6.49 (d, J = 8 Hz, 1H), 4.66 (t, J = 6 Hz, 1H), 4.47 - 4.42 (m, 1H), 3.87 (s, 3H)₁ 3.84 - 3.73 (m, 1H)₁ 3.37 - 3.30 (m, 1H)₁ 3.26 - 3.20 (m, 1H)₁ 3.13 - 3.00 (m, 2H), 2.30 - 2.07 (m, 2H), 1.83 - 1.74 (m, 1H), 1.73 - 1.60 (m, 2H), 1.60 - 1.43 (m, 2H), 1.42 - 1.32 (m, 1H), 0.91 (d, J = 6 Hz₁ 3H)₁ 0.83 (d, J = 6 Hz, 3H); MS (ESI+) for C₂₃H₃₂N₄O₅ mlz 445 (M+H); HRMS (ESI+) calcd for C₂₃H₃₂N₄O₅ +H 445.2446, found 445.2432. Anal. Calcd for C₂₃H₃₂N₄O₅-ISH₂O: C₁ 58.58; H, 7.49; N, 11.88. Found: C, 58.36; H, 7.24; N, 11.62. Example 3

Preparation of /V-((1 S)-1 -{[((1 S)-2-hydroxy-1 -{[(3S)-2-oxopyrrolidin-3- yl]methyI}ethyl)amino]carbonyl}-3-methylbutyl)-1//-benzimidazole-2-carboxamide

To Λ/²-(tert-butoxycarbonyl)-Λ/¹-((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)-L- leucinamide (2.31 g, 6.22 mmol, 1 equiv.) was added dichloromethane (DCM, 50 ml_) followed by TFA (50 ml_). The resulting solution was stirred for 1 h at room temperature and then concentrated In vacuo. The resulting oil was dissolved in DCM (-20 ml.) and concentrated in vacuo twice. The residue was dissolved in DMF (20 ml_) with NMM (2.73 mL, 24.9 mmol, 4 equiv.) and the solution was added to a pre-cooled mixture of 1H-benzo[d]imidazole-2- carboxylic acid (1.01 g, 6.22 mmol), HATU (2.36 g, 6.22 mmol, 1 equiv.) and NMM (0.683 mL, 6.22 mmol, 1 equiv.) in DMF (20 mL) in an ice bath. The sides of the reaction vessel were rinsed with DMF (10 mL). After 15 min, the ice bath was removed. After overnight at room temperature the volatiles were removed in vacuo. To the residue was added DCM and saturated sodium carbonate and the layers were separated. The aqueous layer was extracted with DCM twice more and the organic layers were combined, dried over magnesium sulfate, filtered and concentrated in vacuo. The product was purified by silica gel column chromatography (~500 g SiO₂, 95:5 to 90:10 (DCM-MeOH), sample absorbed to -50 g SiO₂ with MeOH) resulting in the isolation of Λ/-((1S)-1-{[((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3- yl]methyl}ethyl)amino]carbonyl}-3-methylbutyl)-1H-benzimidazole-2-carboxamide (0.82 g, 1.97 mmol) in 32% yield as a white solid. Mp 246.5 ⁰C (dec). ¹H NMR (400 MHz, D₆-DMSO) δ 13.31 (s, 1H), 8.66 (d, J = 9 Hz, 1H), 7.86 (d, J = 9 Hz, 1 H), 7.74 (d, J = 8 Hz, 1H), 7.53 (s, 1H), 7.52 (d, J = 7 Hz, 1 H), 7.32 (t, J = 8 Hz, 1H), 7.27 (t, J = 8 Hz, 1H), 4.70 (t, J = 6 Hz₁ 1H)₁ 4.54 (dt, J = 5,9 Hz₁ 1H), 3.86 - 3.73 (m, 1H), 3.37 - 3.32 (m, 1H)₁ 3.27 - 3.20 (m, 1H), 3.14 - 3.02 (m, 2H), 2.28 - 2.20 (m, 1H)₁ 2.15 - 2.08 (m, 1H)₁ 1.85 - 1.78 (m, 1H)₁ 1.76 - 1.67 (m, 1H)₁ 1.67 - 1.50 (m, 3H), 1.40 - 1.33 (m, 1H), 0.90 (d, J = 6 Hz, 3H), 0.89 (d, J = 6 Hz, 3H); ¹³C NMR (100 MHz, D₆-DMSO) δ 178.8, 171.4, 158.4, 145.3, 142.4, 134.5, 124.2, 122.7, 119.9, 112.6, 63.9, 51.7, 48.7, 48.6, 41.3, 37.5, 32.4, 27.6, 24.5, 23.1, 21.6; MS (ESI+) for C₂₁H₂₉N₅O₄ mfz 416 (M+H); HRMS (ESI+) calcd for C_2IH₂₉N₅O₄ +H 416.2293, found 416.2281. Anal. Calcd for C₂₁H₂₉N₅O₄-1.5H₂O: C, 57.00; H, 7.29; N, 15.83. Found: C, 57.15; H, 7.14; N, 15.80. Example 4

Preparation of W-((1S)-1-{[((1S)-2-hydroxy-H[(3S)-2-oxopyrrolidin-3- yπmethytyethyOaminoJcarbonyty-S-methylbutyO-IH-indole^-carboxamide

To Λ/²-(te/t-butoxycarbonyl)-Λ/¹-((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)-L- leucinamide (0.720 g, 1.94 mmol, 1 equiv.) was added dichloromethane (DCM, 10 ml_) followed by TFA (10 ml_). The resulting solution was stirred for 1 h at room temperature and then concentrated in vacuo. The resulting oil was dissolved in DCM (-10 mL) and concentrated in vacuo twice. To the residue was added iH-indole-2-carboxylic acid (0.312 g, 1.94 mmol, 1 equiv.), HATU (0.737 g, 1.94 mmol, 1 equiv.) and DMF (15 mL). The vessel was cooled in an ice bath. To the chilled solution was added NMM (0.852 mL, 7.75 mmol, 4 equiv.) and after 15 min the ice bath was removed. After 56 h at ambient temperatures the volatiles were removed in vacuo. To the residue was added DCM and saturated potassium carbonate and the layers were separated. The aqueous layer was extracted with DCM twice more and the organic layers were combined, dried over magnesium sulfate, filtered and concentrated in vacuo. The crude product was purified by two radial chromatographies (1 mm plate, 95:5 to 90:10 (DCM- MeOH), sample loaded in DCM) generated 0.409 of Λ/-((1S)-1-{[((1S)-2-hydroxy-1-{[(3S)-2- oxopyrrolidin-S-yllmethylJethyOaminolcarbonylJ-S-methylbutylJ-IH-indole^-carboxamide (0.987 mmol, 51% yield). ¹H NMR (400 MHz, D₆-DMSO) δ 11.57 (s, 1H), 8.39 (d, J = 8 Hz, 1H), 7.79 (d, J = 9 Hz, 1 H)₁ 7.60 (d, J = 8 Hz, 1 H), 7.52 (s, 1H), 7.41 (d, J = 8 Hz, 1 H)₁ 7.24 (s, 1H), 7.16 (t, J = 8 Hz, 1 H), 7.02 (t, J = 8 Hz, 1 H), 4.67 (t, J = 6 Hz, 1H), 4.51 - 4.45 (m, 1H), 3.85 - 3.71 (s, 1H), 3.42 - 3.33 (m, 1H), 3.28 - 3.18 (m, 1H), 3.14 - 2.97 (m, 2H), 2.35 - 2.17 (m, 2H), 2.17 - 2.06 (m, 1H), 1.84 - 1.74 (m, 1H), 1.73 - 1.60 (m, 2H), 1.60 - 1.45 (m, 2H), 1.43 - 1.32 (m, 1H), 0.91 (d, J = 6 Hz, 3H), 0.88 (d, J = 6 Hz, 3H); MS (ESI+) for C₂₂H₃₀N₄O₄ m/z 415 (M+H). Example 5 Preparation of W-{(1 S)-1 -[({(1 S)-1 -formyl-2-[(3S)-2-oxopyrrolidin-3- yl]ethyl}amino)carbonyl]-3-methylbutyl}-4-methoxy-1H-indole-2-carboxamide

To Λ/-((1S)-1-{[((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)amino]carbonyl}-3- methylbutyl)-4-methoxy-1H-indole-2-carboxamide (0.654 g, 1.47 mmol, 1 equiv.) dissolved in dimethylsulfoxide (DMSO, 4 mL) was added DCM (4 mL) and Λ/,Λ/-diisopropylethylamine (DIEA, 0.769 mL, 4.41 mnnol, 3 equiv.) and the resulting solution was chilled in an ice bath. To this cooled solution was slowly added sulfur trioxide pyridine complex (SO₃Py, 0.703 g, 4.41 mmol, 3 equiv.) in DMSO (4 mL) and DCM (4 mL). The ice bath was removed after 15 minutes and after 2 h the reaction mixture was concentrated in vacuo (high vacuum roto-evaporator bath temperature was < 40 ⁰C). To the residue was added ethyl acetate and 10% potassium bisulfate. The layers were separated and the aqueous layer was extracted with ethyl acetate twice more. The organic layers were combined and extracted with brine, dried over magnesium sulfate, filtered and concentrated in vacuo resulted in 0.75 g of crude product. Purification was accomplished by recrystallization from acetonitrile or tetrahydrofuran (THF). Recrystallization from acetonitrile of 0.0750 g of the crude resulted in 55.4 mg of Λ/-{(1S)-1- t({(1S)-1-formyl-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)carbonyl]-3-methylbutyl}-4-methoxy- 1H-indole-2-carbbxamide (85% yield) as a white solid, which contained acetonitrile even after drying. Recrystallization from THF after a 2.05 mmol scale oxidation resulted in 0.482 g of W- {(1S)-1-[({(1S)-1-formyl-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)carbonyl]-3-methylbutyl}-4- methoxy-1H-indole-2-carboxamide (1.09 mmol, 53% yield) as a white fluffy powder (residual THF still present after drying). Material recrystallized from THF: mp - >130 ⁰C (morphology changes). ¹H NMR (400 MHz, D₆-DMSO) δ 11.58 (d, J = 2 Hz, 1H), 9.41 (s, 1H), 8.55 (d, J = 8 Hz, 1H), 8.44 (d, J = 8 Hz, 1 H), 7.63 (s, 1H), 7.36 (d, J = 2 Hz, 1H), 7.08 (t, J = 8 Hz, 1 H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 4.55 -4.50 (m, 1H), 4.23 - 4.18 (m, 1H), 3.87 (s, 3H), 3.15 - 3.05 (m, 2H), 2.16 - 2.09 (m, 1H), 1.95 - 1.87 (m, 1H), 1.76 - 1.54 (m, 5H), 0.93 (d, J = 6 Hz, 3H), 0.89 (d, J = 6 Hz, 3H); ¹³C NMR (100 MHz, D₆-DMSO) δ 200.9, 178.3, 173.0, 161.0, 153.6, 137.8, 129.9, 124.4, 118.0, 105.4, 101.2, 99.2, 56.3, 55.0, 51.4, 40.4, 39.4, 37.3, 29.3, 27.3, 24.4, 23.1, 21.4; MS (ESI+) for C₂₃H₃₀N₄O₅ m/z 443 (M+H); HRMS (ESI+) calcd for C₂₃H₃₀N₄O₅ +H 443.2289, found 443.2295. Example 6 Preparation of (2S)-2-({W-[(4-methoxy-1H-indol-2-yl)carbonyl]-L-leucyl}amino)-3-[(3S)-2- oxopyrrolidin-3-yl]propyl acetate

To /^-((ISJ-i-πcciS^-hydroxy-i-^JSS^-oxopyrrolidin-S-ylJmethylJethyOaminolcarbonylJ-S- methylbutyl)-4-methoxy-1H-indole-2-carboxamide (74.6 mg, 0,168 mmol) was added CH₂Cl₂ (3 mL) and cooled to 0 ⁰C. To the suspension was added sequentially NMM (27.7 μl_, 0.252 mmol, 1.5 equiv.), 4-(Λ/,Λ/-dimethylamino)pyridine(DMAP, catalytic) and acetyl chloride (12.5 μl_, 0.176 mmol, 1.05 equiv.). The suspension was warmed to rt overnight. The CH₂CI₂ was removed in vacuo and acetone (3 mL) and more acetyl chloride and NMM were added. After overnight the volatiles were removed in vacuo. The residue was treated with ethyl acetate and 10% KHSO₄, the layers were separated and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, dried over MgSO₄, filtered and concentrated in vacuo. The crude material was purified by silica gel column chromatography (mobile consisted of 95:5 (EtOAc-MeOH) and then 90:10, sample loaded in 95:5) resulting in the isolation of 39.1 mg of (2S)-2-({Λ/-[(4-methoxy-1H-indol-2-yl)carbonyl]-L-leucyl}amino)-3-[(3S)-2-oxopyrrolidin-3- yljpropyl acetate (0.0804 mmol, 48% yield) as a white, flaky glass. ¹H NMR (400 MHz, D_e- DMSO) δ 11.55 (bs, 1H), 8.32 (bs, 1H), 7.98 (bs, 1H), 7.54 (bs, 1H), 7.33 (bs, 1H), 7.05 (bs, 1H), 6.98 (bs, 1H), 6.47 (bs, 1H), 4.40 (bs, 1 H), 4.01 (bs, 2H), 3.85 (bs, 3H), 3.19 - 2.86 (m, 2H), 2.38 - 2.17 (m, 1H), 2.17 - 1.76 (m, 5H), 1.76 - 1.38 (m, 4H), 1.38 - 1.06 (m, 2H), 0.89 (bs, 6H); ¹³C NMR (100 MHz, D₆-DMSO) δ 178.6, 172.4, 170.2, 160.9, 153.6, 137.8, 129.9, 124.3, 118.1, 105.4, 101.1, 99.2, 65.9, 55.1, 51.7, 45.5, 40.6, 39.1, 37.4, 32.1, 27.4, 24.4, 22.9, 21.7, 20.6; MS (ESI+) for C₂₃H₃₀N₄O₅ m/z 443 (M+H); HRMS (ESI+) calcd for C₂₅H₃₄N₄O₆ +H 487.2551, found 487.2554. Example 7

Preparation of /V-((S)-1-((S)-1,1-dimethoxy-3-((S)-2-oxopyrroIidin-3-yl)propan-2- ylcarbamoyl)-3-methylbutyl)-4-methoxy-1W-indole-2-carboxamide

To Λ/-{(1S)-1-[({(1S)-1-formyl-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)carbonyl]-3-methylbutyl}-

4-methoxy-1/-/-indoIe-2-carboxamide (80.8 mg, 0.183 mmol) was dissolved in methanol (3 mL). To the solution was added trimethyl orthoformate (43.9 μL, 2.2 equiv.) and a catalytic quantity of pyridinium p-toluenesulfonate (PPTS). After overnight at ambient temperature, more trimethyl orthoformate (87.8 μL, 4.4 equiv.) and catalytic PPTS were added and the reaction was heated to reflux for 5 h. After cooling to room temperature the volatiles were removed in vacuo. The crude product was purified by silica gel column chromatography (SiO₂ and sample loaded in 95:5 (CH₂CI₂-MeOH) and run in 95:5 followed by 90:10) resulting in the isolation of 52.6 mg of Λ/-((S)-1-((S)-1,1-dimethoxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-ylcarbamoyl)-3- methylbutyl)-4-methoxy-1/-/-indole-2-carboxamide (0.108 mmol, 59%) as a white powder. Mp 213.4 ⁰C. ¹H NMR (400 MHz, D₄-MeOH) δ 7.28 (s, 1H), 7.15 (t, J = 8 Hz, 1H)₁ 7.03 (d, J = 8 Hz, 1H), 6.51 (d, J = 8 Hz, 1H), 4.62 (bs, 1H)₁ 4.27 (d, J = 4 Hz, 1 H), 4.13 (d, J = 8 Hz, 1H), 3.93 (s, 3 H), 3.39 (s, 6H), 3.30 - 3.14 (m, 2H), 2.59 - 2.43 (m, 1H), 2.39 - 2.23 (m, 1H), 2.01 (W = 11 Hz, 1 H), 1.90 - 1.64 (m, 4H), 1.55 (t, J = 12 Hz₁ 1H), 1.04 (d, J = 6 Hz, 3H), 1.01 (d, J = 6 Hz, 3H); ¹³C NMR (100 MHz, D₄-MeOH) δ 182.7, 175.4, 163.9, 155.6, 139.8, 130.3, 126.3, 120.1, 107.2, 106.2, 102.9, 101.4, 100.3, 56.2, 55.7, 55.2, 53.8, 42.0, 41.4, 39.3, 31.5, 28.9, 26.1, 23.3, 22.3; MS (ESI+) for C₂₅H₃₆N₄O₆ mlz 457 (M-OMe); HRMS (ESI+) calcd for C₂₅H₃₆N₄O₆ +H 489.2708, found 489.2695. Example 8 Preparation of W-((S)-1 -((S)-1 -formyl-Σ-US^-oxopyrrolidin-S-ylJethylcarbamoyO-S- methylbutyl)-1H-benzo[d]imidazole-2-carboxamide

To Λ/-((1S)-1-{[((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)amino]carbonyl}-3- methylbutyl)-1H-benzimidazole-2-carboxamide (0.600 g, 1.44 mmol, 1 equiv.) dissolved in dimethylsulfoxide (DMSO₁ 8 ml_) was added DCM (6 ml_) and Λ/,Λ/-diisopropylethylamine (DIEA₁ 0.754 ml_, 4.33 mmol, 3 equiv.) and the resulting solution was chilled in an ice bath. To this cooled solution was slowly added sulfur trioxide pyridine complex (SO₃Py, 0.690 g, 4.33 mmol, 3 equiv.) in DMSO (6 ml_) and DCM (6 ml_). The ice bath was removed after 15 minutes and after 3 h the reaction mixture was concentrated In vacuo (high vacuum roto-evaporator bath temperature was ≤ 40 ⁰C). To the residue was added ethyl acetate and 10% aqueous potassium bisulfate. The layers were separated and the aqueous layer was extracted with ethyl acetate twice more (later neutralization of the acidic aqueous layer with aqueous NaHCO₃ generated 0.310 g of what appears predominantly to be the aldehyde hydrate). The ethyl acetate layers were combined and extracted with a sat NaHCO₃/brine mixture (1:1), dried over magnesium sulfate, filtered and concentrated in vacuo resulted in 0.268 g of crude product. Purification was accomplished by recrystallization from THF, which resulted in 0.108 g of Λ/- ((S)-1-((S)-1-formyl-2-((S)-2-oxopyrrolidin-3-yl)ethylcarbamoyl)-3-methylbutyl)-1H- benzo[d]imidazole-2-carboxamide (0.262 mmol, 18% yield) as a white solid. Mp 196.5 ⁰C (dec). ¹H NMR (400 MHz, D₆-DMSO) δ 13.31 (s, 1H), 9.41 (s, 1H), 8.79 (d, J = 9 Hz₁ 1H)₁ 8.56 (d, J = 8 Hz, 1 H), 7.74 (d, J = 8 Hz, 1H), 7.63 (s, 1H), 7.52 (d, J = 8 Hz₁ 1 H)₁ 7.32 (t, J = 7 Hz, 1H), 7.27 (t, J = 8 Hz, 1H)₁ 4.63 - 4.54 (m, 2H)₁ 4.27 - 4.21 (m, 2H)₁ 3.16 - 2.98 (m, 2H), 2.35 - 2.27 (m, 1H), 2.16 - 2.09 (m, 1H)₁ 1.95 - 1.40 (m, 6H)₁ 0.91 (d, J = 6 Hz₁ 6H); MS (ES!+) for

C₂-_IH₂₇N₅O₄ m/z 414 (M+H); HRMS (ESI+) calcd for C₂₁H₂₇N₅O₄ +H 414.2136, found 414.2146.

Example 9

Preparation of W-((S)-1 -((S)-14ormyl-2-((S)-2-<>xopyrrolidin-3-yI)ethylcarbamoyl)-3- methylbutyl)-1H-indole-2-carboxamide

To Λ/-((1S)-1-{[((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)amino]carbonyl}-3- methylbutyl)-1H-indole-2-carboxamide (0.400 g, 0.955 mmol, 1 equiv.) dissolved in dimethylsulfoxide (DMSO₁ 3 ml_) was added DCM (3 mL) and Λ/,Λ/-diisopropylethylamine (DIEA, 0.500 mL, 2.86 mmol, 3 equiv.) and the resulting solution was chilled in an ice bath. To this cooled solution was slowly added sulfur trioxide pyridine complex (SO₃Py, 0.456 g, 2.86 mmol, 3 equiv.) in DMSO (3 mL) and DCM (3 mL). The ice bath was removed after 15 minutes and after 2 h the reaction mixture was incomplete by LC/MS and an additional 3 equiv. of DIEA was added and to this solution was slowly added sulfur trioxide pyridine complex (SO₃Py, 0.456 g, 2.86 mmol, 3 equiv.) in DMSO (3 mL) and DCM (3 mL). After 2 h more the reactions was concentrated in vacuo (high vacuum roto-evaporator bath temperature was ≤ 40 ⁰C). To the residue was added ethyl acetate and 5% potassium bisulfate. The layers were separated and the aqueous layer was extracted with ethyl acetate twice more. The organic layers were combined and extracted with a 1:1 brine/sat. NaHCO₃, dried over magnesium sulfate, filtered and concentrated in vacuo. Purification was accomplished by reverse-phase preparative HPLC (eluting with a gradient of MeCN containing 0.1% AcOH in H₂O containing 0.1% AcOH) to give a cream colored solid (0.020 g, 5% yield). Mp 122 ⁰C (dec). ¹H NMR (400 MHz, DMSO-D6) _ 11.58 (s, 1H)₁ 9.42 (s, 1H), 8.60 (d, J = 7Hz, 1H), 8.51 (d, J = 7Hz, 1H), 7.63 - 7.60 (m, 2H), 7.42 (d, J = 8Hz, 1H), 7.26 - 7.24 (m, 1H), 7.17 (t, J = 7Hz, 1H)₁ 7.04 (t, J = 7 Hz, 1H), 4.56 (m, 1H), 4.21 (m, 1H), 3.13 - 3.07 (m, 1H), 2.13 (m, 1H) 1.71 - 1.59 (m, 2H), 0.94 - 0.87 (m, 6H); MS (ESI+) for C₂₂H₂₈N₄O₄ m/z 413.21 (M+H); HRMS (ESI+) calcd for C₂₂H₂₈N₄O₄+H1 413.2184, found 413.2167. Anal. Calcd for C₂₂H₂₈N₄O₄ • 0.75 H₂O: C₁ 54.07; H, 6.21; N₁ 10.97. Found: C, 54.46; H, 6.01; N, 11.19.

Example 10 4-methoxy-/V-((1S)-3-methyl-1-{[(2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino] carbonyl}butyl)~1H-indole-2-carboxamide

To a. suspension of CuI (0.258 g, 1.36 mmol, 6 equiv.) in dry THF (5 ml_) at -78 ⁰C was added methyllithium (1.69 mL of 1.6 M in diethyl ether, 2.71 mmol, 12 equiv.). The mixture (Reetz, Manfred T.; Griebenow, Nils Liebig Annalen 1996, 335 - 348) was warmed to -35⁰C and went homogeneous and then was cooled back down to -78 ⁰C. A suspension of

Λ/-{(1S)-1-[({(1S)-1-formyl-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)carbonyl]-3-methylbutyl}-4- methoxy-1 H-indole-2-carboxamide (0.100 g, 0.226 mmol) in THF (5 mL) was added. The reaction was warmed to -35 ⁰C for 2 h, and then 0 ⁰C for 1h. The solution was cooled to -78 ⁰C and dimethyl zinc (0.300 mL, 1 M solution in heptane, 0.300 mmol, 1.3 equiv.) was added and the solution was allowed to warm to it The solution was poured into a mixture of saturated ammonium chloride (15 mL) and aqueous ammonia (1 N, 5 mL) and was stirred in air overnight. More aqueous ammonium (cone, 1 mL) was added and the mixture was extracted with ethyl acetate three times. The organic layers were combined, dried over magnesium sulfate, filtered and concentrated in vacuo. The 67.0 mg of crude material was dissolved in DMSO (2 mL) and DCM (2 mL) and treated with diisopropylethylamine (0.118 mL, 0.678 mmol, 3 equiv.) and the resulting solution was cooled in an ice bath. To the chilled solution was added sulfur trioxide pyridine complex (0.108 g, 6.78 mmol, 3 equiv.) in DMSO (1 mL) and DCM (1 mL). The ice bath was removed and the solution was stirred at room temperature for 1 h. The volatiles were removed in vacuo and DCM was added in addition to 10% potassium bisulfate. The separated aqueous layer was extracted with DCM three times and the combined organic layer was extracted with brine, dried over magnesium sulfate, filtered and concentrated in vacuo. Purification was accomplished by silica gel column chromatography (95:5 (DC- MeOH), DCM load) followed by reverse phase Dionex HPLC system (MeCN and water mobile phase with 0.1% acetic acid) resulting in 2.9 mg (0.00635 mmol, 3%) of 4-methoxy-Λ/-((1S)-3- methyl-1-{t(2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}butyl)-1H-indole-2- carboxamide as a white fluffy solid. ¹H NMR (400 MHz, D₆-DMSO₁ mixture of diastereomers) δ 11.67 (s, 2H), 8.56 - 8.51 (m, 2H)₁ 8.44 - 8.41 (m, 1H), 7.60 (s, 1H)₁ 7.42 (S₁ 1H), 7.34 (d, J = 3 Hz, 1H), 7.30 (d, J = 3 Hz, 1H), 7.11 - 7.06 (m, 2H), 7.01 - 6.98 (m, 3H), 6.51 - 6.48 (m, 2H)₁ 4.54 - 4.38 (m, 2H), 4.31 - 4.19 (m, 1H), 3.88 (s, 3 H), 3.87 (s, 3H), 3.17 - 2.98 (m, 2 H)₁ 2.34 -2.20 (m, 1H), 2.07 (d, J = 6 Hz, 3H), 2.12 -2.01 (m, 1H), 1.98 - 1.86 (m, 1H)₁ 1.79 - 1.70 (m, 2H), 1.68 (d, J = 5 Hz₁ 3H), 1.63 - 1.44 (m, 4H)₁ 0.93 - 0.87 (m, 12H); MS (ESI+) for C₂₄H₃₂N₄O₅ m/z 479 (M+Na), 457 (M+H); HRMS (ESI+) calcd for C₂₄H₃₂N₄O₅ + Na 479.2265, found 479.2245. Example 11

Preparation of W¹-((1S)-2-hydroxy-1 -{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)-L- leucinamide hydrochloride

A solution of ΛP-(fert-butoxycarbonyl)-Λ/¹-((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3- yl]methyl}ethyl)-L-leucinamide (2.07 g, 5.59 mmol) in anhydrous dioxane (6 ml_) at ambient temperature was treated with 4N HCl in dioxane (2.79 ml_, 11.2 mmol, 2 equiv.) After 14 h, the reaction mixture was first concentrated in vacuo and the resulting solid was dissolved in dichloromethane (DCM, 15 ml_) and concentration in vacuo three times resulting in Λ/¹-((1S)-2- hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)-L-leucinamide hydrochloride as an amorphous white solid that was used without further purification (1.67 g, 97%, 5.44 mmol). ¹H NMR (300 MHz, DMSO-D6) _ 8.33 (d, J = 9 Hz, 1H), 8.25 (br s, 3H), 7.56 (s, 1H), 4.68 (br. s, 1H), 3.82 - 3.75 (m, 1H), 3.71 - 3.67 (m, 1H), 3.37 - 3.25 (m, 2H), 3.18 - 3.04 (m, 2H), 2.38 - 2.24 (m, 1H), 2.23 - 2.18 (m, 1H), 1.86 - 1.75 (m, 1H), 1.69 - 1.52 (m, 4H)₁ 1.40 - 1.31 (m, 1H), 0.89 (d, J = 6 Hz₁ 3H), 0.87 (d, J = 6 Hz, 3H). MS (ESI+) for C₁₃H₂₅N₃O₃ m/z 272 (M+H)⁺.

Example 12

Preparation of W-[(benzyloxy)carbonyl]-L-valyl-/V¹-((1S)-2-hydroxy-1-{[(3S)-2- oxopyrrolidin-3-yl]methyl}ethyl)-L-leucinamide

Λ/¹-((1 S)-2-Hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)-L-leucinamide hydrochloride (1.14 g, 3.71 mmol) and (S)-2-benzyloxycarbonyl valine (0.932 g, 3.71 mmol, 1 equiv.) were added to anhydrous DMF (10 mL) and the resulting solution was cooled to 0 ⁰C. O-(7- Azabenzotriazol-1-yl)-Λ/, Λ/, Λ/', Λ/'-tetramethyluronium hexafluorophosphate (HATU, 1.62 g, 4.27 mmol, 1.15 equiv.) and diisopropylethylamine (DIEA, 1.45 mL, 8.35 mmol, 2.25 equiv.) were added. The mixture was stirred at 0 ⁰C for 2 h and the ice bath was removed. After 2 h at ambient temperature, the reaction mixture was concentrated in vacuo and the residue dissolved in ethyl acetate (EtOAc) (15 mL), washed with 2N HCI (1 x 5 mL), saturated NaHCO₃ (1 x 10 mL), H₂O (1 x 10 mL), saturated NaCI (1 x 10 mL), dried over MgSO₄, filtered and concentrated in vacuo resulting in an opaque viscous oil. Purification by silica gel column chromatography (Silica gel 100 g, gradient elutiong with dichloromethane (DCM) with 0 - 5% MeOH, substrate added in 20 mL DCM) resulted in the isolation of Λ/-[(benzyloxy)carbonyl]-L- valyl-Λ/¹-((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)-L-leucinamide as a white amorphous solid (0.760 g, 41%, 1.51 mmol). Mp 183 ⁰C. ¹H NMR (400 MHz, DMSO-D6) _ 7.93 (d, J = 8 Hz₁ 1H), 7.69 (d, J = 8 Hz, 1H), 7.49 (s, 1H), 7.35 - 7.25 (m, 6H), 5.02 (s, 2H), 4.64 (t, J = 6 Hz, 1H), 4.28 (q, J = 7 Hz, 1H), 3.88 (dd, J = 2, 1 Hz, 1H), 3.77 - 3.74 (m, 1H), 3.22 - 3.16 (m, 1H), 3.13 (t, J = 9 Hz, 1H), 3.05 - 2.97 (m, 1H), 2.24 - 2.07 (m, 2H), 1.97 - 1.88 (m, 1H)₁ 1.78 - 1.69 (m, 1H), 1.60 - 1.49 (m, 2H), 1.44 - 1.32 (m, 2H), 1.27 - 1.20 (m, 2H), 0.88 - 0.79 (m, 12H). Anal. Calcd for C₂₆H₄₀N₄O₆ ^»1.25 H₂O: C, 59.24; H₁ 8.13; N, 10.63. Found C₁ 58.99; H, 7.95; N, 10.61. MS (ESI+) for C₂₆H₄₀N₄O₆ m/z 505 (M+H)⁺. HRMS (ESI+) calcd for C₂₆H₄₀N₄O₆+H 1 505.3021 , found 505.3018.

Example 13

Preparation of W-[(benzyloxy)carbonyl]-L-valyl-/V¹-{-1 -formyl-2-[(3S)-2-oxopyrrolidin-3- yl]ethyl}-L-leucinamide

Λ/-[(Benzyloxy)carbonyl]-L-valyl-Λ/¹-((1S)-2-hydroxy-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)- L-leucinamide (0.200 g, 0.396 mmol) and diisopropylamine (DIEA, 0.256 g, 1.98 mmol, 3 equiv.) were added to anhydrous DMSO (6ml_) followed by dichloromethane (DCM, 6 mL) at ambient temperature. The reaction mixture was cooled to 0⁰C, followed by the addition of a 1:1 solution of DMSO and DCM containing sulfur trioxide pyridine complex (0.315 g, 1.98 mmol, 3 equiv.) dropwise over 15 min. After the addition, the ice bath was removed and the reaction mixture warmed to ambient temperature. After 5 h at ambient temperature, the reaction mixture was concentrated in vacuo and the residue was dissolved in DCM (15 mL) and washed with a 1:1 solution of 1% KHSO₄ and saturated NaCI (1 x 5 mL) followed by a 1:1 solution of saturated NaHCO₃ and saturated NaCI (1 x 5 mL), dried over MgSO₄, filtered and concentrated in vacuo producing a viscous light yellow oil. Purification by silica gel column chromatography (Silica gel 15 g, gradient elution with DCM containing 20 - 50% acetone, substrate added in 10 mL DCM) resulted in Λ/-[(benzyloxy)carbonyl]-L-valyl-Λ/¹-{(1S)-1-formyl-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}- L-leucinamide (2:1 mixture of diastereomers) as a white amorphous solid (25.0 mg, 13% 0.40 mmol). Mp 232 ⁰C (dec.) ¹H NMR (400 MHz, DMSO-D6) _ 9.39 (s, 0.67H), 9.36 (s, 0.33H), 8.55 (d, J = 7 Hz, 0.33H), 8.46 (d, J = 8 Hz, 0.67H), 8.01 (d, J = 8 Hz, 0.67H)₁ 7.95 (d, J = 8 Hz, 0.33H), 7.63 (s, 0.33H), 7.62 (s, 0.67H)₁ 7.37 - 7.25 (m, 6H), 5.01 (s, 2H)₁ 4.41 - 4.30 (m, 1H)₁ 4.24 - 4.17 (m, 1H), 3.89 (t, J = 7 Hz, 1H), 3.16 - 3.02 (m, 1H), 3.13 (t, J = 9 Hz, 1H), 2.32 - 2.19 (m, 1H), 2.16 - 2.05 (m, 1H), 1.98 - 1.83 (m, 1H), 1.65 - 1.57 (m, 1H), 1.50 - 1.42 (m, 3H), 1.27 - 1.23 (m, 2H), 0.91 - 0.80 (m, 12H). Anal. Calcd for C₂₆H₃₈N₄O₆ -1.20 H₂O: C, 59.77; H, 7.76; N, 10.72. Found C, 60.20; H, 7.77; N, 10.00. MS (ESI+) for C₂₆H₃₈N₄O₆ m/z 503 (M+H)⁺. HRMS (ESI+) calcd for C₂₆H₃₈N₄O₆+H1 503.2864, found 503.2877. Example 14

Preparation of tert-Butyl A^-methoxy-W'-methyl-L-α-glutaminate hydrochloride

A solution of terf-butyl Λ/²-(tert-butoxycarbonyl)-Λ/¹-methoxy-/V¹-methyl-L-_-glutaminate (1.14 g, 3.71 mmol) in anhydrous dioxane (6 ml_) at ambient temperature was treated with 4N HCI in dioxane (7.22 mL, 28.9 mmol, 2 equiv.) and was stirred at ambient temperature for 16 h. The reaction mixture was concentrated in vacuo and was then taken up in DCM (15 mL) and concentrated in vacuo three times, resulting in tert-butyl Λ/¹-methoxy-Λ/¹-methyl-L-_-glutaminate hydrochloride as a light yellow viscous oil that was used without further purification (3.82 g, 94% 14.4 mmol). ¹H NMR (300 MHz, DMSO-D6) _ 8.48 (br s, 3H), 4.22 (br s, 1H), 3.73 (s, 3H), 3.15 (s, 3H)₁ 2.38 - 2.31 (m, 2H), 1.02 - 2.87 (m, 2H), 1.38 (s, 9H). MS (ESI+) for C₁₁H₂₂N₂O₄ m/z 247(Mn-H)⁺. Example 15

Preparation of tert-Butyl N-[(benzyloxy)carbonyl]-L-leucyl-W¹-methoxy-Λ/¹-methyl-L-α- glutaminate

terf-Butyl Λ/¹-methoxy-Λ/¹-methyl-L-_-glutaminate hydrochloride (2.29 g, 8.12 mmol) and N- [(benzyloxy)carbonyl]-L-leucine (2.15 g, 8.12 mmol) were sequentially added to anhydrous DMF (10 mL). After cooling the solution to 0 ⁰C, 0-(7-azabenzotriazol-1-yl)-Λ/, N, N', /V- tetramethyluronium hexafluorophosphate (HATU, 3.55 g, 9.34 mmol, 1.15 equiv.) and diisopropylamine (DIEA, 3.20 mL, 18.3 mmol, 2.25 equiv.) were added. The reaction mixture was stirred at 0 ⁰C for 2 h, the ice bath was removed and the reaction mixture was warmed to ambient temperature for 30 min. The reaction mixture was concentrated in vacuo and the resulting residue was dissolved in ethyl acetate (EtOAc, 15 mL), washed with 2N HCI (1 x 5 mL), saturated NaHCO₃ (1 x 10 mL), H₂O (1 x 10 mL), saturated NaCI (1 x 10 mL), dried over MgSO₄ filtered and concentrated in vacuo resulted in an amber residue. Purification was accomplished via Biotage Horizon (65i silica gel cartridge, elutent gradient of MeOH 0-5% in DCM) resulted in ferf-butyl Λ/-[(benzyloxy)carbonyl]-L-leucyl-Λ/¹-methoxy-Λ/¹-methyl-L-_- T/IB2005/003766

- 32 -

glutaminate as a light yellow viscous oil (1.53 g, 38%, 3.10 mmol). ¹H NMR (400 MHz, DMSO- D6) _ 8.05 (d, J = 8 Hz, 1H), 7.37 - 7.28 (m, 6H), 5.01 (s, 2H), 4.74 (br s, 1H), 4.11- 4.03 (m, 1H), 3.71 (s, 3H), 3.10 (s, 3H), 2.28 - 2.22 (m, 2H), 1.86 - 1.81 (m, 1H)₁ 1.77 - 1.69 (m, 1H), 1.65 - 1.60 (m, 1H), 1.41-1.38 (m, 11H), 0.89 - 0.84 (m, 6H). MS (ESI +) for C₂₅H₃₉N₃O₇ m/z 494 (M+H)⁺.

Example 16

Preparation of W-[(benzyloxy)carbonyl]-L-leucyl-W¹-methoxy-W¹-methyl-L-n-glutamine

A solution of te/f-butyl Λ/-[(benzyloxy)carbonyl]-L-leucyl-Λ/¹-methoxy-Λ/¹-methyl-L-_-glutaminate (100 mg, 0.20 mmol) in anhydrous dichloromethane (DCM₁ 3 ml_) at 0 ⁰C was treated with TFA (3.0 ml.) and was stirred at 0 ⁰C for 3 h. The reaction mixture was concentrated in vacuo, and was dissolved in DCM (15 ml_) and concentrated in vacuo three times, resulting in N- [(benzyloxy)carbonyl]-L-leucyl-Λ/¹-methoxy-Λ/¹-methyl-L-_-glutamine as a light yellow viscous oil (84 mg, 92%, 0.87mmol) that was used without further purification. ¹H NMR (400 MHz, DMSO-D6) _ 8.04 (d, J = 8 Hz, 1H)₁ 7.38 - 7.28 (m, 6H), 5.01 (s, 2H), 4.72 (s, 1H), 4.09 - 4.03 (m, 1H), 3.70 (s, 3H), 3.09 (s, 3H), 2.28 - 2.18 (m, 2H), 1.89 - 1.82 (m, 1H), 1.77 - 1.67 (m, 1H), 1.64 - 1.58 (m, 1H), 1.45 - 1.37 (m, 2H), 0.87 - 0.83 (m, 6H). MS (ESI-) for C₂iH₃₁N₃O₆ m/z 436(M-H)-.

Example 17

Preparation of /V-[(benzyloxy)carbonyl]-L-leucyl-W¹-methoxy-W¹,W⁵,Λ/⁵-trimethyl-L- glutamamide

To a solution of Λ/-[(benzyloxy)carbonyl]-L-leucyl-Λ/¹-methoxy-Λ/¹-methyl-L-_-glutamine (91 mg, 0.21 mmol) in anhydrous DMF (10 ml.) was added 2.0M dimethylamine in THF (0.417 mL, 0.84 mmol, 4.0 equiv.). The reaction mixture was cooled to 0 ⁰C followed by the addition of O-(7- azabenzotriazol-1-yl)-Λ/, N₁ N\ Λf-tetramethyluronium hexafluorophosphate (HATU 91 mg, 0.24 mmol, 1.15 equiv.) and diisopropylamine (DIEA₁ 0.08 mL, 0.47 mmol, 2.25 equiv.). The mixture was stirred at 0 ⁰C for 2 h, the ice bath was removed and the reaction mixture warmed to ambient temperature for 30 min. The reaction mixture was concentrated in vacuo and the residue was dissolved in ethyl acetate (EtOAc, 15 mL), washed with 2N HCI (1 x 5 mL), saturated NaHCO₃ (1 x 5 mL), saturated NaCI (1 x 5 mL), dried over MgSO₄, filtered and concentrated in vacuo resulted in a light amber viscous oil. Purification was accomplished by silica gel column chromatography (Silica gel 1O g, eluted with DCM with increasing MeOH O- 2%, sample was loaded in 10 mL DCM) resulted in Λ/-[(benzyloxy)carbonyl]-L-leucyl-Λ/¹- metho><y-Λ/\Λ/^Λ/⁵4rimethyl-L-glutamamide as a clear viscous oil (0.052 g, 53%, 0.11 mmol). ¹H NMR (400 MHz, DMSO-D6) _ 8.07 (d, J = 8 Hz, 1H), 7.39 - 7.29 (m, 6H), 5.00 (s, 2H), 4.72 (br s, 1 H), 4.08 - 4.02 (m, 1H)₁ 3.70 (s, 3H), 3.10 (s, 3H)₁ 2.88 (s, 3H), 2.78 (s, 3H), 2.32 - 2.28 (m, 2H), 1.90 - 1.80 (m, 1H)₁ 1.75 - 1.68 (m, 1H)₁ 1.66 - 1.58 (m, 1H), 1.46 - 1.34 (m, 2H)₁ 127 - 1.20 (m, 1H), 0.87 - 0.83 (m, 6H). MS (ESI+) for C₂₃H₃₈N₄O₆ m/z 465 (M+H)⁺. HRMS (ESI+) calcd for C₂₃H₃₆N₄O₆+H1 465.2708, found 465.2711. Example 18 Preparation of

A solution of

(0.747 g, 1.61 mmol) in anhydrous MeOH (20 mL) was treated with 10% palladium on carbon (17.1 mg, 0.161 mmol). The reaction mixture was flushed with N₂ three times, charged with H₂ (50 psi) and was shaken at ambient temperature 16 h. The reaction mixture was filtered through celite and concentrated in vacuo resulting in L-leucyl-Λ/¹-methoxy-Λ/¹,Λ/⁵,Λ/⁵-trimethyl-L- glutamamide as a light yellow viscous oil (0.520 g , 98%, 1.60 mmol). The material was used without further purification. ¹H NMR (400 MHz, DMSO-D6) _ 8.06 (d, J = 8 Hz₁ 1H), 4.75 (br S₁ 1H), 3.71 (s, 3H), 3.14 - 3.10 (m, 4H), 2.90 (s, 3H), 2.79 (s, 3H), 2.28 (t, J = 7 Hz, 2H), 1.89 - 1.79 (m, 1H), 1.76 - 1.64 (m, 4H), 1.41 - 1.31 (m, 1 H), 1.25 - 1.15 (m, 1H), 0.87 - 0.83 (m, 6H). MS (ESI+) for C₁₅H₃₀N₄O₄ m/z 331(M+H)⁺. Example 19 Preparation of W-[(4-methoxy-1W-indol-2-yl)carbonyl]-L-leucyl-Λ/¹-methoxy-Λ/¹,Λ/⁵,W⁵- trimethyl-L-glutamamide

A solution of L-leucyl-Λ/^methoxy-Λ/^Λ^.Λ^-trimethyl-L-glutamamide (0.538 g, 1.63 mmol) and

4-methoxy-1H-indole-2-carboxylic acid (0.311 g, 1.63 mmol) in anhydrous DMF (8 mL) was cooled to 0 ⁰C and HATU (0.712 g, 1.87 mmol, 1.15 equiv.) and diisopropylamine (DIEA, 0.242 g, 1.87 mmol, 2.25 eq.) were added. The mixture was stirred at O ⁰C for 2 h, the ice bath was removed and the reaction mixture warmed to ambient temperature for 30 min. The reaction mixture was concentrated In vacuo and the residue was dissolved in ethyl acetate (EtOAc, i5 mL), washed with 2N HCI (1 x 5 ml_), saturated NaHCO₃ (1 x 10 ml_), H₂O (1 x 10 mL), saturated NaCI (1 x 10 mL), dried over MgSO₄, filtered and concentrated in vacuo resulted in an amber residue. Purifiication was accomplished by silica gel column chromatography (Silica gei 10Og, gradient elution with DCM containing 0 - 5% MeOH, sample loaded in 20 mL DCM) followed by dissolving the white amorphous solid in MeOH (25 mL) and filtering through basic Dowex monosphere (550A OH anion exchange) resin, concentrating in vacuo resulted in N- [(1 S)-1 -({[( 1 S)-4-(dimethylami no)-1 -f ormyl-4-oxobutyl]amino}carbonyl)-3-methylbutyl]-4- methoxy-1/-/-indole-2-carboxamide as a white amorphous solid (0.308 g, 38%, 0.61 mmol). Mp 158 ⁰C. ¹H NMR (400 MHz, DMSO-D6) _ 11.53 (s, 1H), 8.35 (d, J = 8 Hz, 1H), 8.16 (d, J = 8 Hz, 1H), 7.32 (s, 1H), 7.09 (t, J = 8 Hz, 1H), 7.00 (d, J = 8 Hz, 1H), 6.50 (d, J = 8 Hz, 1H), 4.73 (br s, 1H), 4.56 - 4.53 (m, 1H), 3.87 (s, 3H), 3.70 (s, 3H), 3.09 (s, 3H), 2.87 (s, 3H), 2.76 (s, 3H), 2.32 (t, J = 7 Hz, 2H), 1.89 - 1.82 (m, 1H), 1.77 - 1.62 (m, 3H), 1.54 - 1.47 (m, 1H), 0.91 (d, J = 6, Hz₁ 3H), 0.89 (d, J = 6, Hz, 3H). MS (ESI+) for C₂₅H₃₇N₅O₆ m/z 504 (M+H)⁺.HRMS (ESI+) calcd for C₂₅H₃₇N₅O₆+H 504.2817, found 504.2804. Example 20 Preparation of PF-02378867: W-[(1S)-1-({[(1S)-4-(dimethylamino)-1-formyl-4- oxobutyl]amino}carbonyl)-3-methylbutyl]-4-methoxy-1H-indole-2-carboxamide

A solution of Λ/-[(4-methoxy-1H-indol-2-yl)carbonyl]-L-leucyl-Λ/¹-methoxy-Λ/¹,Λ/⁵,Λ/⁵-trimethyl-L- glutamamide (0.15 g, 0.396 mmol) in anhydrous THF (1 mL) was cooled to -78 ⁰C. The reaction mixture was treated with 1.0M lithium aluminum hydride in THF (LAH, 0.044 mL, 1.05 mmol, 3.5 equiv.). The reaction mixture was stirred at -78 ⁰C for 3 h and was subsequentially quenched with EtOAc (1 mL) warmed to ambient temperature for 30 min and diluted with DCM (15 mL). The mixture was washed with 1% KHSO₄ (1 X 15 mL), saturated NaCI (1 x 15 mL), H₂O (1 x 10 mL), dried over MgSO₄, filtered and concentrated in vacuo resulting in a cream colored solid. Purification was accomplished via HPLC chromatography (Dionex preparative HPLC; Phenomex C₁₈ semi-prep column, elutent of acetilnitrile 10-100% in H₂O with 0.1% acetic acid) resulting in Λ/-[(1S)-1-({[(1S)-4-(dimethylamino)-1-formyl-4- oxobutyl]amino}carbonyl)-3-methylbutyl]-4-methoxy-1H-indole-2-carboxamide as a white amorphous solid (25.0 mg, 19%, 0.45 mmol). Mp 104 ⁰C. ¹H NMR (400 MHz, DMSO-D6) _ 11.55 (s, 1H), 9.40 (s, 1H), 8.47 - 8.44 (m, 2H), 7.34 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 7.00 (d, J = 8 Hz₁ 1H), 6.49 (d, J = 8 Hz, 1H), 4.54 - 4.50 (m, 1H), 4.14 - 4.06 (m, 1H), 3.87 (s, 3H), 2.86 (s, 3H), 2.76 (S, 3H), 2.32 (t, J = 7 Hz, 2H), 2.06 - 1.98 (m, 1H), 1.75 - 1.65 (m, 3H), 1.61 - 1.49 (m, 1H), 0.94 - 0.88 (m, 6H) . Anal. Calcd for C₂₃H₃₂N₄O₅ -2.1 H₂O: C₁ 57.27; H₁ 7.57; N, 11.62. Found C₁ 56.94; H, 7.14; N, 11.29. MS (ESI+) m/z 445 (M+H)⁺. HRMS (ESI+) calcd for C₂₃H₃₂N₄O₅+H 445.2446, found 445.2428.

Protection from SARS Infection: Neutral Red Endpoint

The ability of compounds to protect cells against infection by the SARS coronavims is measured by a cell viability assay similar to that described in Borenfreund, E., and Puerner, J. 1985. Toxicity determined in vitro by morphological alterations and neutral red absorption Toxicology Letters. 24:119-124, utilizing neutral red staining as an endpoint. Briefly, medium containing appropriate concentrations of compound or medium only is added to Vero cells. Cells are infected with SARS-associated virus or mock-infected with medium only. One to seven days later, the medium is removed and medium containing neutral red is added to the test plates. Following incubation at 37⁰C for two hours, cells are washed twice with PBS and a 50% EtOH, 1% acetic acid solution is added. The cells are shaken for 1 to 2 minutes and incubated at 37°C for 5 to 10 minutes. The amount of neutral red is quantified spectrophotometrically at 540nm. Data is expressed as the percent of neutral red in wells of compound-treated cells compared to neutral red in wells of uninfected, compound-free cells. The fifty percent effective concentration (EC50) is calculated as the concentration of compound that increases the percent of neutral red production in infected, compound-treated cells to 50% of that produced by uninfected, compound-free cells. The 50% cytotoxicity concentration (CC50) is calculated as the concentration of compound that decreases the percentage of neutral red produced in uninfected, compound-treated cells to 50% of that produced in uninfected, compound-free cells. The therapeutic index is calculated by dividing the cytotoxicity (CC50) by the antiviral activity (EC50).

Protection from SARS Infection: GIo endpoint

The ability of compounds to protect cells against infection by the SARS coronavims can also be measured by a cell viability assay utilizing luciferase to measure intracellular ATP as an endpoint. Briefly, medium containing appropriate concentrations of compound or medium only is added to Vero cells. Cells are infected with SARS-associated virus or mock-infected with medium only. One to seven days later, the medium is removed and the amount of intracellular ATP is measured as per Promega Technical Bulletin No. 288: CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, Wl). The CellTiter-Glo® reagent is added to the test plates and following incubation at 37⁰C for 1.25 hours, the amount of signal is quantified using a luminometer at 490nm. Data is expressed as the percent of luminescent signal from wells of compound-treated cells compared to the luminescent signal from wells of uninfected, compound-free cells. The fifty percent effective concentration (EC50) is calculated as the concentration of compound that increases the percent of the luminescent signal from infected, compound-treated cells to 50% of the luminescent signal from uninfected, compound-free cells. The 50% cytotoxicity concentration (CC50) is calculated as the concentration of compound that decreases the percentage of the luminescent signal from uninfected, compound-treated cells to 50% of the luminescent signal from uninfected, compound-free cells. The therapeutic index is calculated by dividing the cytotoxicity (CC50) by the antiviral activity (EC50). Cytotoxicity

The ability of compounds to cause cytotoxicity in cells is measured by a cell viability assay similar to that described in Weislow, O.S., Kiser, R., Fine, D.L, Bader, J., Shoemaker, R.H., and Boyd, M. R.1989. New Soluble-Formazan Assay for HIV-1 Cytopathic Effects: Application to High-Flux Screening of Synthetic and Natural Products for Al DS-Anti viral Activity. Journal of the National Cancer Institute 81(08): 577-586), utilizing formazan as an endpoint. Briefly, Vero cells are resuspended in medium containing appropriate concentrations of compound or medium only. One to seven days later, XTT and PMS are added to the test plates and following incubation at 37⁰C for two hours the amount of formazan produced is quantified spectrophotometrically at 540nm. Data is expressed as the percent of formazan produced in compound-treated cells compared to formazan produced in wells of compound-free cells. The 50% cytotoxicity concentration (CC50) is calculated as the concentration of compound that decreases the percentage of formazan produced in uninfected, compound-treated cells to 50% of that produced in uninfected, compound-free cells. Protection from Coronavirus 229E Infection The ability of compounds to protect cells against infection by human coronavirus 229E is measured by a cell viability assay similar to that described in Weislow, O.S., Kiser, R., Fine, D.L, Bader, J., Shoemaker, R.H., and Boyd, M.R.1989. New Soluble-Formazan Assay for HIV-1 Cytopathic Effects: Application to High-Flux Screening of Synthetic and Natural Products for AIDS-Antiviral Activity. Journal of the National Cancer Institute 81(08): 577-586), utilizing formazan as an endpoint. Briefly, medium containing appropriate concentrations of compound or medium only is added to MRC-5 cells. Cells are infected with human coronavirus 229E or mock-infected with medium only. One to seven days later, XTT and PMS are added to the test plates and following incubation at 37⁰C for two hours the amount of formazan produced is quantified spectrophotometrically at 540nm. Data is expressed as the percent of formazan in wells of compound-treated cells compared to formazan in wells of uninfected, compound-free cells. The fifty percent effective concentration (EC50) is calculated as the concentration of compound that increases the percent of formazan production in infected, compound-treated cells to 50% of that produced by uninfected, compound-free cells. The 50% cytotoxicity concentration (CC50) is calculated as the concentration of compound that decreases the percentage of formazan produced in uninfected, compound-treated cells to 50% of that produced in uninfected, compound-free cells. The therapeutic index is calculated by dividing the cytotoxicity (CC50) by the antiviral activity (EC50). Coronavirus 3C Protease FRET Assay and Analysis

Proteolytic activity of Coronavirus 3C protease is measured using a continuous fluorescence resonance energy transfer assay. The SARS 3CI_^pro FRET assay measures the protease catalyzed cleavage of TAMRA- SITSAVLQSGFRKMK-(DABCYL)-OH to TAMRA - SITSAVLQ and SGFRKMK- (DABCYL)-OH . The fluorescence of the cleaved TAMRA (ex. 558 nm / em. 581 nm) peptide was measured using a TECAN SAFIRE fluorescence plate reader over the course of 10 min. Typical reaction solutions contained 20 mM HEPES (pH 7.0), 1 mM EDTA, 4.0 uM FRET substrate, 4% DMSO and 0.005% Tween-20. Assays were initiated with the addition of 25 nM SARS 3CL^pro (nucleotide sequence 9985-10902 of the Urbani strain of SARS coronavirus complete genome sequence (NCBI accession number AY278741)). Percent inhibition was determined in duplicate at O.OOImM level of inhibitor. Data was analyzed with the non-linear regresssion analysis program Kalidagraph using the equation:

FU = offset + (limit)(1- _e-^(kobs)t) where offset equals the fluorescence signal of the uncleaved peptide substrate, and limit equals the fluorescence of fully cleaved peptide substrate. The kobs is the first order rate constant for this reaction, and in the absence of any inhibitor represents the utilization of substrate. In an enzyme start reaction which contains an irreversible inhibitors, and where the calculated limit is less than 20% of the theoretical maximum limit, the calculated kobs represents the rate of inactivation of coronavirus 3C protease. The slope (kobs/ I) of a plot of kobs vs. [I] is a measure of the avidity of the inhibitor for an enzyme. For very fast irreversible inhibitors, kobs/l is calculated from observations at only one or two [I] rather than as a slope.

The following table describes the mean antiviral EC50 and the inhibition of the C3 like protease in the FRET assay.

While the invention has been described in terms of various preferred embodiments and specific examples, the invention should be understood as not being limited by the foregoing detailed description, but as being defined by the appended claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A compound of formula (I),

wherein: m is an integer selected from 0 and 1;

Y is selected from the group consisting of H, -CH₃, and -CHzCH₃;

R¹ is C₁ to C₇ alkyl, C₃ to C₁₀ cycloalkyl, and benzyl wherein said alkyl, benzyl and cycloalkyl is unsubstituted or independently substituted with 1 to 3 R⁷ substituents;

R² is selected from

R³ is independently selected from H and Ci to C₃ alkyl; each R⁴ and R^4a is independently H, C₁ to C₃ alkyl or C₃ to C₆ cycloalkyl, wherein each alkyl and cycloalkyl is unsubstituted or substituted with oxo, 1 to 3 halogens or 1 to 3 hydroxyls;

R⁵ is H or selected from R⁷ substituents; each R⁶ and R^6a is independently H, C₁ to C₃ alkyl, and -C(O)R³ or R⁶ and R^6a form a 5 to 7 membered heterocycle; each R⁷ is independently selected from halogen, oxo, Ci to C₄ alkyl, C₂ to C₆ alkenyl, C₂ to C₆ alkynyl, C₃ to C₆ cycloalkyl, -OR⁴, -NR⁴C(O)R⁴, -NR⁴R^4a, SR⁴, -SOR⁴, -SO²R⁴, - C(O)R⁴, -CO₂R⁴, -C(O)NR⁴R⁴³, -SO₂NR⁴R⁴³, -NR⁴SO₂NR⁴R⁴⁹, 4 to 10 member heterocycle and -OC(O)R⁴, wherein the foregoing alkyl, alkenyl, alkynyl, cycloalkyl and heterocycle groups are each optionally substituted with halogen, hydroxy, C₁ to C₆ alkoxy, and oxo;

Z is selected from the group consisting of

and

n is O to 3; A is 4 to 10 member heterocycle, C₃ to Cio cycloalkyl, C₆ to Ci₀ aryl and Ci to C₇ alkyl , wherein said heterocycle, cycloalkyl, alkyl and aryl are unsubstituted or independently substituted with 1 to 3 R⁷ substituents;

E is

then m is 0; and

X is selected from -CH₂OH, -CH₂OR⁶, -CHO, -CH(OR⁶)(OR^6a) and -C(R³)O; or a pharmaceutically acceptable salt or solvate thereof.

2. A compound of claim 1, wherein R is

3. A compound of claim 1, wherein R is

4. A compound of claim 3, wherein A is a C₄ to Ci₀ heterocycle unsubstituted or substituted with 1 to 3 substituents independently selected from: halogen, C₁ to C₄ alkyl, -OR⁴, -NR⁴C(O)R⁴, -NR⁴R^4a, SR⁴, SOR⁴, SO²R⁴, -C(O)R⁴, -CO₂R⁴, -SO₂NR⁴R⁴³, -NR⁴SO₂NR⁴R⁴⁹ and -OC(O)R⁴

5. A compound of claim 3, wherein X is -CHO and -CH(OR⁶)(OR^6a).

6. A compound of claim 5, wherein Z is

R

7. A compound of claim 6, wherein R¹ is C-i to C₇ alkyl, said alkyl is unsubstituted or substituted with C₃ to C₆ cycloalkyl.

8. A method of interfering with or preventing SARS related coronavirus viral replication activity comprising contacting a SARS related coronavirus protease with a therapeutically effective amount of a compound of claim 1.

9. A pharmaceutical composition, comprising a therapeutically effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 10, further comprising at least one of an interferon, a p-glycoprotein inhibitor or a CYP3A4 inhibitor.

11. Use of a compound of claim 1 in the manufacture of a medicament for treatment of a SARS infection in a SARS infected human.