WO2009058856A1 - Macrocyclic inhibitors of hepatitis c virus ns3 serine protease - Google Patents

Abstract

The present invention discloses novel compounds which have HCV protease inhibitory activity as well as methods for preparing such compounds. In another embodiment, the invention discloses pharmaceutical compositions comprising such compounds as well as methods of using them to treat disorders associated with the HCV protease.

Description

MACROCYCLIC INHIBITORS OF HEPATITIS C VIRUS NS3 SERINE PROTEASE

Field of Invention

The present invention relates to novel hepatitis C virus ("HCV") protease inhibitors, pharmaceutical compositions containing one or more such inhibitors, methods of preparing such inhibitors and methods of using such inhibitors to treat hepatitis C and related disorders. This invention additionally discloses novel macrocyclic compounds as inhibitors of the HCV NS3/NS4a serine protease.

Background of the Invention

Hepatitis C virus (HCV) is a (+)-sense single-stranded RNA virus that has been implicated as the major causative agent in non-A, non-B hepatitis (NANBH), particularly in blood-associated NANBH (BB-NANBH) (see, international Patent

Application Publication No. WO 89/04669, equal to US 2003162167). NANBH is to be distinguished from other types of viral-induced liver disease, such as hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV), as well as from other forms of liver disease such as alcoholism and primary biliar cirrhosis.

Recently, an HCV protease necessary for polypeptide processing and viral replication has been identified, cloned and expressed; (see, e_.g., U.S. Patent No. 5,712,145). This approximately 3000 amino acid polyprotein contains, from the amino terminus to the carboxy terminus, a nucleocapsid protein (C), envelope proteins (E1 and E2) and several non-structurai proteins (NS1, 2, 3, 4a, 5a and 5b). NS3 is an approximately 68 kda protein, encoded by approximately 1893 nucleotides of the HCV genome, and has two distinct domains: (a) a serine protease domain consisting of approximately 200 of the N-terminaJ amino acids; and (b) an RNA-dependent ATPase domain at the C-terminus of the protein. The NS3 protease is considered a member of the chymotrypsin family because of similarities in protein sequence, overall three- dimensional structure and mechanism of catalysis. Other chymotrypsin-like enzymes are eiastase, factor Xa, thrombin, trypsin, plasmin, urokinase, tPA and PSA. The HCV NS3 serine protease is responsible for proteolysis of the polypeptide (polyprotein) at the NS3/NS4a, NS4a/NS4b, NS4b/NS5a and NS5a/NS5b junctions and is thus responsible for generating four viral proteins during virai replication. This has made the HCV NS3 serine protease an attractive target for antiviral chemotherapy. The inventive compounds can inhibit such protease. They also can modulate the processing of hepatitis C virus (HCV) polypeptide.

It has been determined that the NS4a protein, an approximately 6 kda polypeptide, is a co-factor for the serine protease activity of NS3. Autocleavage of the NS3/NS4a junction by the NS3/NS4a serine protease occurs intramolecularly (Le., cis) while the other cleavage sites are processed intermolecularly (i.e_;, trans). Analysis of the natural cleavage sites for HCV protease revealed the presence of cysteine at P1 and serine at P1 ' and that these residues are strictly conserved in the NS4a/NS4b, NS4b/NS5a and NS5a/NS5b junctions. The NS3/NS4a junction contains a threonine at P1 and a serine at P1 '. The Cys→Thr substitution at NS3/NS4a is postulated to account for the requirement of cis rather than trans processing at this junction. See, e^g., Pizzi et al. (1994) Proc. Natl. Acad. Sci (USA) 91:888-892, Faiila et al. (1996) Folding & Design 1 :35-42. The NS3/NS4a cleavage site is also more toierant of mutagenesis than the other sites. See, e.g.. Kollykhalov et ai. (1994) J. Virol. 68:7525-7533. It has also been found that acidic residues in the region upstream of the cleavage site are required for efficient cleavage. See, e.g., Komoda et al. (1994) J. Viroi. 68:7351-7357.

Inhibitors of HCV protease that have been reported include antioxidants (see, International Patent Application Publication No. WO 98/14181), certain peptides and peptide analogs (see, International Patent Application Publication No. WO 98/17679 (equal to US2002032175), Landro et al. (1997) Biochem. 36:9340-9348, lngaliinella et a|. (1998) Biochem. 37:8906-8914, Llinas-Brunet et al. (1998) Bioorg. Med. Chem. Lett. 8:1713-1718). inhibitors based on the 70~amino acid polypeptide eglin c (Martin et al. (1998) Biochem. 37:11459-11468. inhibitors affinity selected from human pancreatic secretory trypsin inhibitor (hPSTi-C3) and minibody repertoires (MBip) (Dimasi etal. (1997) J. Viroi. 71 : 7461 -7469). cV_HE2 (a "cameiized" variable domain antibody fragment) (Martin et al.f 1997) Protein Enα. 10:607-614), and α1 - antichymotrypsin (ACT) (Elzouki et al.) (1997) J. Hepat. 27:42-28). A ribozyme designed to selectively destroy hepatitis C virus RNA has recently been disclosed (see, BioWorld Today 9(217): 4 (November 10, 1998)). Reference is also made to the PCT Publications, No. WO 98/17679, published April 30, 1998 (Vertex Pharmaceuticals Incorporated); WO 98/22496, published May 28, 1998 (equal to U.S. 6,018,020 and U.S. 5,866,684; F. Hoffmann-La Roche AG); and WO 99/07734, published February 18, 1999 (equal to U.S. 6,143,715; Boehringer lngelheim Canada Ltd.).

HCV has been implicated in cirrhosis of the liver and in induction of hepatocellular carcinoma. The prognosis for patients suffering from HCV infection is currently poor. HCV infection is more difficult to treat than other forms of hepatitis due to the lack of immunity or remission associated with HCV infection. Current data indicates a less than 50% survival rate at four years post cirrhosis diagnosis. Patients diagnosed with localized resectable hepatocellular carcinoma have a five-year survival rate of 10-30%, whereas those with localized unresectable hepatocellular carcinoma have a five-year survival rate of less than 1%.

Reference is made to WO 00/59929 (equal to US2004002448 and U.S. 6,608,027; Assignee: Boehringer lngelheim (Canada) Ltd.; Published October 12, 2000) which discloses peptide derivatives of the formula:

Reference is made to A. Marchetti etal, Synlett, SJ_,, 1000-1002 (1999) describing the synthesis of bicylic analogs of an inhibitor of HCV NS3 protease. A compound disclosed therein has the formula:

Reference is also made to W. Han et al, Bioorganic & Medicinal Chem. Lett, (2000) 1_0, 711 -713, which describes the preparation of certain α-ketoamides, α- ketoesters and α-diketones containing allyl and ethyl functionalities.

Reference is also made to WO 00/09558 (Assignee: Boehringer lngelheim Limited; Published February 24, 2000) which discloses peptide derivatives of the formula:

where the various elements are defined therein. An illustrative compound of that series is:

Reference is also made to WO 00/09543 (equal to US2002016442 and US 2002037998; Assignee: Boehringer lngelheim Limited; Published February 24, 2000) which discloses peptide derivatives of the formula:

where the various elements are defined therein. An illustrative compound of that series is:

Current therapies for hepatitis C include interferon-α (INF₃) and combination therapy with ribavirin and interferon. See, e.g., Beremguer et ai. (1998) Proc. Assoc. Am. Physicians 110(2):98-112. These therapies suffer from a Sow sustained response rate and frequent side effects. See, e.g., Hoofnagle et al. (1997) N. Engl. J. Med. 336:347. Currently, no vaccine is available for HCV infection.

Reference is further made to WO 01/74768 (equal to US 2003236242; Assignee: Vertex Pharmaceuticals !nc) published October 11 , 2001 , which discloses certain compounds of the following general formula (R is defined therein) as NS3- serine protease inhibitors of Hepatitis C virus:

A specific compound disclosed in the afore-mentioned WO 01/74768 has the following formula:

PCT Publications WO 01/77113; WO 01/081325; WO 02/08198; WO 02/08256;

WO 02/08187; WO 02/08244; WO 02/48172; WO 02/08251 ; and pending U.S. patent application, Serial No. 10/052,386, filed January 18, 2002, disclose various types of peptides and/or other compounds as NS-3 serine protease inhibitors of hepatitis C virus. The disclosures of those applications are incorporated herein by reference thereto.

There is a need for new treatments and therapies for HCV infection. There is a need for compounds useful in the treatment or prevention or amelioration of one or more symptoms of hepatitis C. There is a need for methods of treatment or prevention or amelioration of one or more symptoms of hepatitis C. There is a need for methods for modulating the activity of serine proteases, particularly the HCV NS3/NS4a serine protease, using the compounds provided herein.

There is a need for methods of modulating the processing of the HCV polypeptide using the compounds provided herein.

Summary of the Invention

U.S. patent application, Serial No. 10/948367, filed February 24, 2005 (which published as 2005/0119168 on June 2, 2005), the entire dislosure of which, is incorporated herein, by reference.

In its many embodiments, the present invention provides novel compounds as inhibitors of the HCV protease, pharmaceutical compositions containing one or more of the compounds, methods of preparing pharmaceutical formulations comprising one or more of such compounds, methods of treatment or prevention of HCV or amelioration of one or more of the symptoms of hepatitis C using one or more of such compounds or one or more of such formulations, and methods of modulating the interaction of an HCV polypeptide with HCV protease using one or more of such compounds or one or more of such formulations. The present invention discloses compounds, as well as pharmaceutically acceptable salts, solvates or esters of said compounds, said compound being selected from the compounds of structures listed below:

I



A further feature of the invention is pharmaceutical compositions containing as active ingredient at least one compound of the present invention (or its salts, esters, solvate or isomers) together with a pharmaceutically acceptable carrier or excipient. The invention also provides methods for preparing compounds of the present invention as well as methods for treating diseases such as, for example, HCV, AIDS (Acquired Immune Deficiency Syndrome), and related disorders. The methods for such treatment comprise administering to a patient suffering from one or more of the above diseases or one or more related diseases a therapeutically effective amount of at least one compound of the present invention or a pharmaceutical composition comprising at least one compound of the present invention.

Also disclosed is the use of at least one compound of The present invention for the manufacture of a medicament for treating HCV, AIDS, and related disorders.

Further disclosed is a method of treatment of a hepatitis C virus associated disorder, comprising administering an effective amount of one or more of the inventive compounds.

In still yet further embodiments there is provided methods of modulating the activity of hepatitis C virus (HCV) protease, comprising contacting HCV protease with one or more inventive compounds as well as methods of treating or preventing HCV, or ameliorating one or more symptoms of hepatitis C, comprising administering an effective amount of one or more of the inventive compounds. Such modulation, treatment, prevention or amelioration can also be done with the inventive pharmaceutical compositions or formulations. Without being limited to theory, it is believed that the HCV protease may be the NS3 or NS4a protease. The inventive compounds can inhibit such protease. They can also modulate the processing of hepatitis C virus (HCV) polypeptide.

Description of the invention

As used above, and throughout this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: "Patient" includes both human and animals.

"Mammal" means humans and other mammalian animals.

"Alky!" means an aliphatic hydrocarbon group which may be straight or branched and comprising about 1 to about 20 carbon atoms in the chain. Preferred a!kyl groups contain about 1 to about 12 carbon atoms in the chain. More preferred alky] groups contain about 1 to about 6 carbon atoms in the chain. Branched means that one or more lower alky! groups such as methyl, ethyl or propyl, are attached to a linear alkyl chain. "Lower alkyi" means a group having about 1 to about 6 carbon atoms in the chain which may be straight or branched. The alkyl group may be optionally substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alky!, aryl, cycloalkyl, cyano, hydroxy, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), - N(alkyl)₂, carboxy and -C(O)O-aikyi. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl and t-butyl.

"Alkynyl" means an aliphatic hydrocarbon group containing at least one carbon- carbon triple bond and which may be straight or branched and comprising about 2 to about 15 carbon atoms in the chain. Preferred alkyny! groups have about 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a iinear alkynyl chain. "Lower alkynyl" means about 2 to about 6 carbon atoms in the chain which may be straight or branched. Non-limiting examples of suitable alkynyl groups include ethynyl, propynyl, 2-butynyl and 3- methylbutynyl. The term "substituted alkynyl" means that the alkynyi group may be substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of alkyl, ary! and cycloalkyl.

"Aliphatic" means and includes straight or branched chains of paraffinic, olefinic or acetylenic carbon atoms. The aliphatic group can be optionally substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of H, halo, halogen, alkyl, aryi, cycloalkyl, cycloalkylamino, alkenyf, heterocyclic, alkynyl, cycloalkylaminocarbonyi, hydroxyl, thio, cyano, hydroxy, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), - N(a!kyl)₂₎ carboxyl, -C(O)O-alkyl, heteroaryl, aralkyl, alkylaryl, aralkenyl, heteroaraikyl, alkylheteroaryl, heteroaralkenyl, heteroalkyl, carbonyl, hydroxyalkyl, aryloxy, aralkoxy, acyl, aroyl, nitro, amino, amido, ester, carboxylic acid aryloxycarbonyl, aralkoxycarbonyl, alkylsuifonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkenyl, heterocyclyi, heterocyclenyl, carbamate, urea, ketone, aldehyde, cyano, sulfonamide, sulfoxide, sulfone, suifony! urea, sulfonyl, hydrazide, hydroxamate, S(alkyl)YiY₂N-alkyl-, Y₁Y₂N^yI-, YiY₂NC(O)- and Y₁Y₂NSO₂-, wherein Y₁ and Y₂ can be the same or different and are independently selected from the group consisting of hydrogen, alkyl, aryl, and aralkyl.

"Heteroaliphatic" means an otherwise aliphatic group that contains at least one heteroatom (such as oxygen, nitrogen or sulfur). The term heteroaliphatic includes substituted heteroaliphatic.

"Aryl" means an aromatic monocyclic or multicyclic ring system comprising about 6 to about 14 carbon atoms, preferably about 6 to about 10 carbon atoms. The aryl group can be optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined herein. Non-limiting examples of suitable aryl groups include phenyl and naphthyl.

"Heteroalkyl" means an alkyl as defined above, wherein one or more hydrogen atoms are substituted by a heteroatom selected from N, S, or O.

"Heteroaryl" means an aromatic monocyclic or multicyclic ring system comprising about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the ring atoms is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. Preferred heteroaryls contain about 5 to about 6 ring atoms. The "heteroaryl" can be optionally substituted by one or more "ring system substituents" which may be the same or different, and are as defined herein. The prefix aza, oxa or thia before the heteroaryl root name means that at least a nitrogen, oxygen or sulfur atom respectively, is present as a ring atom. A nitrogen atom of a heteroaryl can be optionally oxidized to the corresponding N-oxide. Non-limiting examples of suitable heteroaryls include pyridyi, pyrazinyl, furanyl, thienyl, pyrimidinyl, pyridone (including N-substituted pyridones), isoxazoiyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazoiyJ, 1,2,4- thiadiazoiyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, oxindolyl, imidazo[1 ,2- a]pyridinyi, imidazo[2,1-b]thiazo!yl, benzofurazanyl, indolyS, azaindolyi, benzimidazolyl, benzothienyl, quinolinyl, imidazolyi, thienopyridyl, quinazolinyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, 1 ,2,4-triazinyl, benzothiazolyl and the like. The term "heteroaryl" also refers to partially saturated heteroaryl moieties such as, for example, tetrahydrσisoquinolyl, tetrahydroquinoly! and the like.

"Aralkyl" or "arylalkyl" means an aryi-alkyl- group in which the aryl and alkyl are as previously described. Preferred araikyls comprise a lower alkyl group. Non-limiting examples of suitable aralkyl groups include benzyl, 2-phenethyi and naphthalenylmethyl. The bond to the parent moiety is through the aikyl.

"Alkylaryl" means an alkyl-aryl- group in which the alkyl and aryl are as previously described. Preferred alkylaryls comprise a lower alkyl group. Non-limiting example of a suitable alkylaryl group is toiyl. The bond to the parent moiety is through the aryl.

"Cycloalkyl" means a non-aromatic mono- or multicycϋc ring system comprising about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms. Preferred cycioalkyl rings contain about 5 to about 7 ring atoms. The cycloalkyl can be optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined above. Non-limiting examples of suitable monocyclic cycloalkyls include cyclopropyi, cyciopentyl, cyciohexyl, cycloheptyl and the like. Non-limiting examples of suitable multicyclic cycloalkyls include 1-decalinyl, norbornyl, adamantyl and the like, as well as partially saturated species such as, for example, indanyl, tetrahydronaphthyl and the like. "Halogen" means fluorine, chlorine, bromine, or iodine. Preferred are fluorine, chlorine and bromine.

"Ring system substituent" means a substituent attached to an aromatic or non- aromatic ring system which, for example, replaces an available hydrogen on the ring system. Ring system substituents may be the same or different, each being independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heteroaryl, araikyl, alkylaryl, heteroaralkyl, heteroarylalkenyt, heteroarylalkynyl, alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl, aroyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaraikylthio, cycloalkyl, heterocyclyi, -C(=N-CN)-NH₂, -Cf=NH)-NH₂, -Cf=NH)- NH(alkyl), YiY₂N-, YiY₂N-alkyl-, Y₁Y₂NC(O)-, Y₁Y₂NSO₂- and -SO₂NYiY₂, wherein Y₁ and Y₂ can be the same or different and are independently selected from the group consisting of hydrogen, alkyl, aryl, cycloaikyl, and aralkyl. "Ring system substituent" may also mean a single moiety which simultaneously replaces two available hydrogens on two adjacent carbon atoms (one H on each carbon) on a ring system. Examples of such moiety are methylene dioxy, ethylenedioxy, -C(CH₃J₂- and the like which form moieties such as, for example:

"Heterocycly!" or "heterocycloalkyl" or "heterocyclic" means a non-aromatic saturated monocyclic or multicyciic ring system comprising about 3 to about 10 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the atoms in the ring system is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. There are no adjacent oxygen and/or sulfur atoms present in the ring system. Preferred heterocyciyls contain about 5 to about 6 ring atoms. The prefix aza, oxa or thia before the heterocyclyl root name means that at least a nitrogen, oxygen or sulfur atom respectively is present as a ring atom. Any - NH in a heterocyclyl ring may exist protected such as, for example, as an -N(Boc), - N(CBz), -N(Tos) group and the like; such protections are also considered part of this invention. The heterocyclyl can be optionally substituted by one or more "ring system substituents" which may be the same or different, and are as defined herein. The nitrogen or sulfur atom of the heterocyclyl can be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide. Non-limiting examples of suitable monocyclic heterocyciyl rings include piperidyl, pyrrolidinyl, piperaztnyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1 ,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, lactam, lactone, and the like.

It should be noted that in hetero-atom containing ring systems of this invention, there are no hydroxyl groups on carbon atoms adjacent to a N, O or S₁ as well as there are no N or S groups on carbon adjacent to another heteroatom. Thus, for example, in the ring:

there is no -OH attached directly to carbons marked 2 and 5. It should also be noted that tautomeric forms such as, for example, the moieties:

are considered equivalent in certain embodiments of this invention. "Alkynyialkyl" means an aikynyl-alkyl- group in which the aikynyl and alkyl are as previously described. Preferred alkynylalkyls contain a lower aikynyl and a lower alkyl group. The bond to the parent moiety is through the alkyl. Non-limiting examples of suitable aikynylalkyl groups include propargylmethyl.

"Heteroaralkyl" means a heteroaryl-aSkyi- group in which the heteroaryl and alkyl are as previously described. Preferred heteroaralkyls contain a lower aikyl group.

Non-limiting examples of suitable aralkyl groups include pyridylmethyl, and quinolin-3- ylmethyl. The bond to the parent moiety is through the alkyl.

"Hydroxyalkyl" means a HO-alkyl- group in which alkyl is as previously defined.

Preferred hydroxyaikyls contain lower alkyl. Non-limiting examples of suitable hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.

"Acyl" means an H-C(O)-, alkyl-C(O)- or cycloalkyl-C(O)-, group in which the various groups are as previously described. The bond to the parent moiety is through the carbonyl. Preferred acyls contain a lower alkyl. Non-limiting examples of suitable acyl groups include formyl, acetyl and propanoyl. "Aroyl" means an aryi-C(O)- group in which the aryl group is as previously described. The bond to the parent moiety is through the carbonyl. Non-limiting examples of suitable groups include benzoyl and 1- naphthoyl.

"Aikoxy" means an aikyi-O- group in which the alkyl group is as previously described. Non-limiting examples of suitable aikoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy and n-butoxy. The bond to the parent moiety is through the ether oxygen.

"Aryloxy" means an aryl-O- group in which the aryl group is as previously described. Non-limiting examples of suitable aryloxy groups include phenoxy and naphthoxy. The bond to the parent moiety is through the ether oxygen. "Aralkyloxy" means an aralkyl-O- group in which the aralkyl group is as previously described. Non-ϋmiting examples of suitable aralkyloxy groups include benzyloxy and 1- or 2-naphthalenemethoxy. The bond to the parent moiety is through the ether oxygen.

"Alkylthio" means an alkyl-S- group in which the alky! group is as previously described. Non-limiting examples of suitable alkylthio groups include methyithio and ethylthio. The bond to the parent moiety is through the sulfur.

"Aryithio" means an aryl-S- group in which the aryl group is as previously described. Non-limiting examples of suitable aryithio groups include phenylthio and naphthylthio. The bond to the parent moiety is through the sulfur.

"Aralkylthio" means an aralkyl-S- group in which the araikyl group is as previously described. Non-limiting example of a suitable araikylthio group is benzyithio. The bond to the parent moiety is through the sulfur.

"Alkoxycarbonyl" means an alkyl-O-CO- group. Non-limiting examples of suitable aikoxycarbonyl groups include methoxycarbonyl and ethoxycarbonyl. The bond to the parent moiety is through the carbonyl. "Aryloxycarbonyl" means an aryl-O-C(O)- group. Non-limiting examples of suitable aryloxycarbonyl groups include phenoxycarbonyl and naphthoxycarbonyl. The bond to the parent moiety is through the carbonyl.

"Araikoxycarbonyl" means an aralkyl-O-C(O)- group. Non-limiting example of a suitable araikoxycarbonyl group is benzyloxycarbonyl. The bond to the parent moiety is through the carbonyl.

"Alkylsulfonyl" means an alkyl-S(O₂)- group. Preferred groups are those in which the alkyl group is lower alky!. The bond to the parent moiety is through the sulfonyl.

"Arylsulfonyl" means an aryl-S(O₂)- group. The bond to the parent moiety is through the sulfonyi.

The term "substituted" means that one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. By "stable compound¹ or "stable structure" is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. The term "optionally substituted" means optional substitution with the specified groups, radicals or moieties.

The term "isolated" or "in isolated form" for a compound refers to the physical state of said compound after being isolated from a synthetic process or natural source or combination thereof. The term "purified" or "in purified form" for a compound refers to the physical state of said compound after being obtained from a purification process or processes described herein or well known to the skilled artisan, in sufficient purity to be characterizable by standard analytical techniques described herein or well known to the skilled artisan. It should also be noted that any carbon as well as heteroatom with unsatisfied valences in the text, schemes, examples and Tables herein is assumed to have the sufficient number of hydrogen atom(s) to satisfy the valences.

When a functional group in a compound is termed "protected", this means that the group is in modified form to preclude undesired side reactions at the protected site when the compound is subjected to a reaction. Suitable protecting groups will be recognized by those with ordinary skill in the art as well as by reference to standard textbooks such as, for example, T. W. Greene et al, Protective Groups in organic

Synthesis (1991), Wiley, New York.

When any variable (e.g., aryl, heterocycie, R², etc.) occurs more than one time in any constituent or in the present invention, its definition on each occurrence is independent of its definition at every other occurrence.

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

Prodrugs and solvates of the compounds of the invention are also contemplated herein. The term "prodrug", as employed herein, denotes a compound that is a drug precursor which, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield a compound of the present invention or a salt and/or solvate thereof. A discussion of prodrugs is provided in T.

Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems (1987) 14 of the ACS.

Symposium Series, and in Bioreversibfe Carriers in Drug Design, (1987) Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press, both of which are incorporated herein by reference thereto.

"Solvate" means a physical association of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like. "Hydrate" is a solvate wherein the solvent molecule is H₂O.

"Effective amount" or "therapeutically effective amount" is meant to describe an amount of compound or a composition of the present invention effective in inhibiting the desired diseases and thus producing the desired therapeutic, ameliorative, inhibitory or preventative effect. The compounds of the present invention can form salts which are also within the scope of this invention. Reference to a compound of the present invention herein is understood to include reference to salts thereof, unless otherwise indicated. The term "salt(s)", as employed herein, denotes acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. In addition, when a compound of the present invention contains both a basic moiety, such as, but not limited to a pyridine or imidazole, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful. Salts of the compounds of the the present invention may be formed, for example, by reacting a compound of the present invention with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by iyophilization.

Exemplary acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisuifates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartarates, thiocyanates, toiuenesulfonates (also known as tosyiates,) and the like. Additionally, acids which are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G, (eds.) Handbook of Pharmaceutical Salts. Properties, Sefection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66m 1-19; P. Gould, International J. of Pharmaceutics (1986) 33201-217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D.C. on their website). These disclosures are incorporated herein by reference thereto. Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamines, t-buty! amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quarternized with agents such as lower alkyi halides (e.g. methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, and dibutyl sulfates), long chain haiides (e.g. decyl, lauryl, and stearyl chlorides, bromides and iodides), aralkyl halides (e.g. benzyl and phenethyl bromides), and others.

All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the invention and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the invention.

One or more compounds of the invention may also exist as, or optionally converted to, a solvate. Preparation of solvates is generally known. Thus, for example, M. Caira et al, J. Pharmaceutical Sci., 93(3), 601-611 (2004) describe the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar preparations of solvates, hemisolvate, hydrates and the like are described by E. C. van Tonder et al, AAPS PharmSciTech., 50}, article 12 (2004); and A. L. Bingham et al, Chem. Commun., 603-604 (2001). A typical, non-limiting, process involves dissolving the inventive compound in desired amounts of the desired soivent (organic or water or mixtures thereof) at a higher than ambient temperature, and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example 1. R. spectroscopy, show the presence of the solvent (or water) in the crystals as a solvate (or hydrate). Compounds of the present invention, and salts, solvates, esters and prodrugs thereof, may exist in their tautomeric form (for example, as an amide or imϊno ether). All such tautomeric forms are contemplated herein as part of the present invention.

All stereoisomers (for example, geometric isomers, optical isomers and the like) of the present compounds (including those of the salts, solvates and prodrugs of the compounds as well as the salts and solvates of the prodrugs), such as those which may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may exist even in the absence of asymmetric carbons), rotameric forms, atropisomers, and diastereomeric forms, are contemplated within the scope of this invention, as are positional isomers (such as, for example, 4-pyridyl and 3-pyridyl). Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers, or may be admixed, for example, as racemates or with ail other, or other selected, stereoisomers. The chiral centers of the present invention can have the S or R configuration as defined by the IUPAC 1974 Recommendations. The use of the terms "salt", "solvate" "prodrug" and the like, is intended to equally apply to the salt, solvate and prodrug of enantiomers, stereoisomers, rotamers, tautomers, positional isomers, racemates or prodrugs of the inventive compounds.

Polymorphic forms of the compounds of the present invention and of the salts, solvates and prodrugs of the compounds of the present invention, are intended to be included in the present invention.

In one embodiment, the present invention discloses compounds of the present invention as inhibitors of HCV protease, especially the HCV NS3/NS4a serine protease, or a pharmaceutically acceptable derivative thereof, where the various definitions are given above. in another embodiment, R¹ is ketoamide, acid, ketoacid, ketoester, ketoaidehyde, diketone, boronic acid or trifluoroketone. In still yet another aspect of the invention there is provided a pharmaceutical composition comprising as an active ingredient a compound of the present invention which is for use in treating disorders associated with HCV. The composition would generally include a pharmaceutically acceptable carrier. The composition may contain one or more additional agents such as, for exampfe, an antiviral agent, an interferon or pegylated interferon and the like. A preferred antiviral agent is ribavirin and a preferred interferon is α-interferon.

A method of treating disorders associated with the HCV protease comprises administering to a patient in need of such treatment therapeutically effective amounts of a compound of the present invention, or a pharmaceutical composition which comprises therapeutically effective amounts of a compound of the present invention. The administration may be oral or subcutaneous.

The compounds of the present invention may be used for the manufacture of a medicament to treat disorders associated with the HCV protease, for example, the method comprising bringing into intimate contact a compound of the present invention a pharmaceutically acceptable carrier. These and other aspects of the invention are described in further detail below.

In embodiments described above, the present invention discloses compounds of the present invention as inhibitors of HCV protease, especially the HCV NS3/NS4a serine protease, or a pharmaceutically acceptable derivative thereof, where the various definitions are given above.

In another embodiment, this invention provides pharmaceutical compositions comprising the inventive peptides as an active ingredient. The pharmaceutical compositions generally additionally comprise a pharmaceutically acceptable carrier diluent, excipient or carrier (collectively referred to herein as carrier materials). Because of their HCV inhibitory activity, such pharmaceutical compositions possess utility in treating hepatitis C and related disorders. The HCV inhibitory activity can also lead to use of the inventive compounds and/or compositions for treating diseases (e.g., AIDS, etc) that are associated or connected with HCV.

In yet another embodiment, the present invention discloses methods for preparing pharmaceutical compositions comprising the inventive compounds as an active ingredient, in the pharmaceutical compositions and methods of the present invention, the active ingredients will typically be administered in admixture with suitable carrier materials suitably selected with respect to the intended form of administration, i.e. oral tablets, capsules (either solid-filled, semi-solid filled or liquid filled), powders for constitution, oral gels, elixirs, dtspersibie granules, syrups, suspensions, and the like, and consistent with conventional pharmaceutical practices. For example, for oral administration in the form of tablets or capsules, the active drug component may be combined with any oral non-toxic pharmaceutically acceptable inert carrier, such as lactose, starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, taic, mannitol, ethyl alcohol (liquid forms) and the like. Moreover, when desired or needed, suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated in the mixture. Powders and tablets may be comprised of from about 5 to about 95 percent inventive composition.

Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethylcelluiose, polyethylene glycol and waxes. Among the lubricants there may be mentioned for use in these dosage forms, boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include starch, methyicellulose, guar gum and the like. Sweetening and flavoring agents and preservatives may also be included where appropriate. Some of the terms noted above, namely disintegrants, diluents, lubricants, binders and the like, are discussed in more detail below.

Additionally, the compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimize the therapeutic effects, i.e. HCV inhibitory activity and the like. Suitable dosage forms for sustained release include layered tablets containing layers of varying disintegration rates or controlled release polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices. Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injections or addition of sweeteners and pacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.

For preparing suppositories, a low melting wax such as a mixture of fatty acid glycerides such as cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein by stirring or similar mixing. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.

The compounds of the invention may also be deliverable transdermally. The transdermal compositions may take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose. The compounds of the invention may also be administered orally, intravenously, intranasally or subcutaneously.

The compounds of the invention may also comprise preparations which are in a unit dosage form. In such form, the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose.

The quantity of the inventive active composition in a unit dose of preparation may be generally varied or adjusted from about 1.0 milligram to about 1 ,000 milligrams, preferably from about 1.0 to about 950 milligrams, more preferably from about 1.0 to about 500 milligrams, and typically from about 1 to about 250 milligrams, according to the particular application- The actual dosage employed may be varied depending upon the patient's age, sex, weight and severity of the condition being treated. Such techniques are well known to those skilled in the art.

Generally, the human oral dosage form containing the active ingredients can be administered 1 or 2 times per day. The amount and frequency of the administration will be regulated according to the judgment of the attending clinician. A generally recommended daily dosage regimen for oral administration may range from about 1.0 milligram to about 1,000 milligrams per day, in single or divided doses.

Some useful terms are described below:

Capsule - refers to a special container or enclosure made of methyl cellulose, polyvinyl alcohols, or denatured gelatins or starch for holding or containing compositions comprising the active ingredients. Hard shell capsules are typically made of blends of relatively high gel strength bone and pork skin gelatins. The capsule itself may contain small amounts of dyes, opaquing agents, plasticizers and preservatives.

Tablet- refers to a compressed or molded solid dosage form containing the active ingredients with suitable diluents. The tablet can be prepared by compression of mixtures or granulations obtained by wet granulation, dry granulation or by compaction.

Oral gel- refers to the active ingredients dispersed or soiubiiized in a hydrophillic semi-solid matrix.

Powder for constitution refers to powder blends containing the active ingredients and suitable diluents which can be suspended in water or juices.

Diluent - refers to substances that usually make up the major portion of the composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol and sorbitol; starches derived from wheat, corn, rice and potato; and celluloses such as microcrystalline cellulose. The amount of diluent in the composition can range from about 10 to about 90% by weight of the total composition, preferably from about 25 to about 75%, more preferably from about 30 to about 60% by weight, even more preferably from about 12 to about 60%.

Disintegrant - refers to materials added to the composition to help it break apart

(disintegrate) and release the medicaments. Suitable disintegrants include starches; "cold water soluble" modified starches such as sodium carboxymethyi starch; natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar; cellulose derivatives such as methylcellulose and sodium carboxymethylcelluiose; microcrystalline celluloses and cross-linked microcrystalline celluloses such as sodium croscarmellose; alginates such as alginic acid and sodium alginate; clays such as bentonites; and effervescent mixtures. The amount of disintegrant in the composition can range from about 2 to about 15% by weight of the composition, more preferably from about 4 to about 10% by weight.

Binder - refers to substances that bind or "glue" powders together and make them cohesive by forming granules, thus serving as the "adhesive" in the formulation. Binders add cohesive strength already available in the diluent or bulking agent.

Suitable binders include sugars such as sucrose; starches derived from wheat, corn rice and potato; natural gums such as acacia, gelatin and tragacanth; derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate; cellulosic materials such as methylcelfulose and sodium carboxymethylcellulose and hydroxypropyimethylcelSuIose; polyvinylpyrrolidone; and inorganics such as magnesium aluminum silicate. The amount of binder in the composition can range from about 2 to about 20% by weight of the composition, more preferably from about 3 to about 10% by weight, even more preferably from about 3 to about 6% by weight.

Lubricant - refers to a substance added to the dosage form to enable the tablet, granules, etc. after it has been compressed, to release from the mold or die by reducing friction or wear. Suitable lubricants include metallic stearates such as magnesium stearate, calcium stearate or potassium stearate; stearic acid; high melting point waxes; and water soluble lubricants such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols and d'l-leucine. Lubricants are usually added at the very last step before compression, since they must be present on the surfaces of the granules and in between them and the parts of the tablet press. The amount of lubricant in the composition can range from about 0.2 to about 5% by weight of the composition, preferably from about 0.5 to about 2%, more preferably from about 0.3 to about 1.5% by weight.

Glident - material that prevents caking and improve the flow characteristics of granulations, so that flow is smooth and uniform. Suitable glidents include silicon dioxide and talc. The amount of glident in the composition can range from about 0.1 % to about 5% by weight of the total composition, preferably from about 0.5 to about 2% by weight.

Coloring agents - excipients that provide coloration to the composition or the dosage form. Such excipients can include food grade dyes and food grade dyes adsorbed onto a suitable adsorbent such as clay or aluminum oxide. The amount of the coloring agent can vary from about 0.1 to about 5% by weight of the composition, preferably from about 0.1 to about 1 %.

Bioavailability - refers to the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed into the systemic circulation from an administered dosage form as compared to a standard or control. Conventional methods for preparing tablets are known. Such methods include dry methods such as direct compression and compression of granulation produced by compaction, or wet methods or other special procedures. Conventional methods for making other forms for administration such as, for example, capsules, suppositories and the like are also well known.

Another embodiment of the invention discloses the use of the pharmaceutical compositions disclosed above for treatment of diseases such as, for example, hepatitis C and the like. The method comprises administering a therapeutically effective amount of the inventive pharmaceutical composition to a patient having such a disease or diseases and in need of such a treatment.

In yet another embodiment, the compounds of the invention may be used for the treatment of HCV in humans in monotherapy mode or in a combination therapy (e.g., dual combination, triple combination etc.) mode such as, for example, in combination with antiviral and/or immunomodulatory agents. Examples of such antiviral and/or immunomodulatory agents include Ribavirin (from Schering-Plough Corporation, Madison, New Jersey) and Levovirin™ (from ICN Pharmaceuticals, Costa Mesa, California), VP 50406™ (from Viropharma, Incorporated, Exton, Pennsylvania), ISIS 14803™ (from ISIS Pharmaceuticals, Carlsbad, California),

Heptazyme™ (from Ribozyme Pharmaceuticals, Boulder, Colorado), VX 497™ (from Vertex Pharmaceuticals, Cambridge, Massachusetts), Thymosin™ (from SciClone Pharmaceuticals, San Mateo, California), Maxamine™ (Maxim Pharmaceuticals, San Diego, California), mycophenolate mofetil (from Hoffman-LaRoche, Nutley, New Jersey), interferon (such as, for example, interferon-alpha, PEG-interferon alpha conjugates) and the like. "PEG-interferon alpha conjugates" are interferon alpha molecules covaiently attached to a PEG molecule. Illustrative PEG-interferon alpha conjugates include interferon alpha-2a (Roferon™, from Hoffman La-Roche, Nutley, New Jersey) in the form of pegylated interferon alpha-2a (e.g., as sold under the trade name Pegasys™), interferon a!pha-2b (Intron™, from Schering-Plough Corporation) in the form of pegylated interferon a!pha-2b (e.g., as sold under the trade name PEG- Intron™), interferon alpha-2c (Berofor Alpha™, from Boehringer Ingelheim, tngelheim, Germany) or consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (Infergen™, from Amgen, Thousand Oaks, California).

As stated earlier, the invention includes tautomers, rotamers, enantiomers and other stereoisomers of the inventive compounds also. Thus, as one skilled in the art appreciates, some of the inventive compounds may exist in suitable isomeric forms. Such variations are contemplated to be within the scope of the invention.

Another embodiment of the invention discloses a method of making the compounds disclosed herein. The compounds may be prepared by several techniques known in the art. Representative illustrative procedures are outlined In the following reaction schemes. The invention disclosed herein is then further exemplified by preparative examples and example compounds which should not be construed to limit the scope of the invention which is defined in the appended claims. Alternative mechanistic pathways and analogous structures will be apparent to those skilled in the art.

It is to be understood that while the following illustrative schemes describe the preparation of a few representative inventive compounds, suitable substitution of any of both the natural and unnatural amino acids will result in the formation of the desired compounds based on such substitution. Such variations are contemplated to be within the scope of the invention.

For the procedures described below, the following abbreviations are used:

AcOH: Acetic acid

ADDP: 1 ,1'-(Azodicarbobyl)dipiperidine

Boc means t-butyloxy or tert-Butyloxycarbonyl 'Bu, TBu or Bu¹: terf-Butyl

Cbz: Benzyloxycarbonyl

Bop: Benzotriazol-1-yl-oxy-tris(dimethylamino)hexafiuorophosphate

Bn or BzI: Benzyl

Bz: Benzoyl Chg: Cyciohexylglycine

Cp: Cylcopentyidienyl

DCM means dicihioromethane;

DCC: 1 ^-Dicyclohexylcarbodiimide

DEAD: Diethylazodicarboxylate DMAP: 4-N,N-Dimethyiaminopyridine

DMF means /VjN-dimethylformamide;

DMSO means dimethyl sulfoxide;

EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Et: Ethyl;

EtOAc means ethyl acetate;

Et2θ: Diethyl ether;

HATU means O-(7-azabenzotriazo]-1-yl)-1 ,1 ,3,3-tetramethyluronium; HOOBt: 3-Hydroxy-1,2,3-benzotriazin-4(3/-/)-one;

HOBt: N-Hydroxybenzotriazo!e; iBoc: isobutoxycarbonyl; iPr: isopropyl;

KHMDS means Potassium hexamethyl disilylamide; LiHMDS means hexamethyldisiiazide;

Me: Methyl;

MS means mass spectrum; nBuLi means n-buty] lithium;

NMM means N-methyi morpholine; NMR means nuclear magnetic resonance;

Phg: Phenylglycine;

Ph: Phenyl;

Pd/C means palladium on charcoal catalyst;

PyBrOP: Bromo-fπs-pyrrolidinophosphonium hexafluorophosphate; TBuNCO means t-butyl isocyanate;

TEMPO: 2,2,6,6-Tetramethyl-i-piperidinyloxy;

THF means tetrahydrofuran;

THP means tetrahydrofuran;

TMSI means trimethyl silyf iodide; T₃N means triethyiamine;

Ts: p-toluenesulfonyl.

Several of the intermediates and/or preparative examples used in the following synthetic procedures have been disclosed in WO 01/77113; WO 01/081325; WO 02/08198; WO 02/08256; WO 02/08187; WO 02/08244; WO 02/48172; WO 02/08251 ; and pending U.S. patent application, Serial No. 10/052,386, filed January 18, 2002. The disclosures of those applications are incorporated herein by reference thereto.

The compounds of the present invention can be synthesized using the schemes and procedures for preparative examples disclosed in U.S. patent application, Serial No. 10/948367, filed February 24, 2005 (which published as 2005/0119168 on June 2, 2005), the entire dislosure of which, is incorporated herein, by reference.

General Preparative Schemes and Procedures for Preparative Examples

SCHEME 1

SCHEME 2

10

11 12

SCHEME 3

14 Procedures For Preparative Examples Preparative Example 1

Step A

1a 1b

The synthesis of 1b can be accomplished using the procedure of (1) Myers, A. G.; Gleason, J. L.; Yoon, T.; Kung, D. W.; J. Am, Chem. Soc 1997, 119 , 656; (2) Myers, A. G.; Schnider, P.; Kwon, S.; Kung, D. W.; J. Org. Chem., 1999, 64, 3322.; or (3) Myers, A. G.; Gleason, J. L.; Org. Synth. 1998, 76, 57.

A solution of amine 1a ( 24 g, 120 mmol) in THF (300 ml_) was treated with anhydrous LiCI ( 16.80 g, 400 mmol) over 0.5 h and stirred till the reaction mixture turns homogeneous. The reaction mixture was cooled to 0° C and treated with a THF solution of LiHMDS ( 66.80 g , 400 mmol in 300 ml of THF) over 20 min. The reaction mixture was stirred at 0° C for 0.5 h and treated with 6-bromohexene (19.44 g, 120 mmol) and stirred at rt. for 24 n. The reaction mixture was dissolved in aq. 1 M HC! and concentrated in vacuo to remove THF. The mostly aq. layer was further diluted with 3M aq HCI (300 mL) and extracted with ether (2x200 mL). The aqueous layer was basified to pH 14 using aq. NaOH (50%) and extracted with CH₂Cl₂ (3x 300 mL). The combined organic layers were dried with MgSO₄ filtered concentrated in vacuo to yield crude 1b (15.1 g ) that was used in next step without further purification. Step B

1b 1c A solution of 1b (12.5 g, 41.2 mmol) was dissolved in aq. NaOH (1 M, 88.0 mL,

88 mmol) and heated at reflux for 3 h. The reaction mixture was cooled to rt. and extracted with CH2C12 (3x100 mL). The aq. layer was treated with 100 mL of dioxane followed by NaHCO₃ (8.00 g, 95.2 mmol) and di-tertbutyl dicarbonate ( 8.95 g, 41 mmol) and stirred at rt. for 5 h. The reaction mixture was extracted with ether (2x250 mL) and the aqueous layer was acidified to pH~2 with aq. HCI and extracted with CH₂CI₂ (2x200 mL). The combined organic layers were dried with MgSO4, filtered concentrated in vacuo to yield acid 1c (10.8 g) as a colorless oil. Step C

A solution of acid 1c (5g, 19.44 mmol) and amine 1d (3.98 g, 19.44 mmol) in

CH₂Cl₂ (30 mL), DMF (30 mL) at 0° C was treated with HATU (8.87 g, 23.31 mmol) and NMM (4.91 g, 5.33 mL) and stirred overnight at 0° C. The reaction mixture was concentrated in vacuo and diluted with 650 mL of CH₂CI₂. The aqueous layer was washed with aq. HCI (1 M, 2x300 mL), aq. NaHCO₃ (1M₁ 2x300 mL). The organic layers were dried with MgSO₄, filtered concentrated in vacuo and purified by chromatography (SiO₂, Acetone/Hexanes 5:1 ) to yield 1e as a colorless oil (5.5 g). ¹H NfVIR: (CD₃OD, 300 MHz) 55.87-5.76 (m, 1 H), 4.97-4.92 (dd, 2 H), 4.26 (bt, 1 H, J=7.8 Hz), 3.98 (d, 1 H₁ J= 10.2 Hz), 3.61 (dd, 2 H, J=5.1 , 5.1 Hz), 3.73 (s, 3 H), 2.14- 2.07 (m, 2 H)₁ 1.74-1.42 (m, 9 H), 1.41 (s, 9 H)₁ 1.12 (s, 3 H)₁ 0.92 (s, 3 H). 1³C NMR: (CD₃OD, 75 MHz), d 173.8, 173.2, 158.0, 139.8, 115.0, 80.4, 60.91 , 53.42, 52.80, 34.7, 33.5, 32.3, 31.4, 29.8, 28.7, 26.4, 26.1 , 20.6, 12.9. Step D

1e I g

A solution of ester 1e (4g, 9.79 mmo!) in THF (20 ml), H₂O (20 m!_) and MeOH (10 mL) was treated with LiOH-H₂O (575 mg, 14 mmo!) and stirred at rt. for 4h. The reaction mixture was concentrated in vacuo to remove THF and MeOH. The mostly aqueous layer was acidified with aq. HCI and extracted into CH₂CI₂ (3x100 mL). The combined organic layers were dried with MgSO₄, filtered, concentrated in vacuo and used as it is.

A solution of acid obtained from hydrolysis of 1e, amine segment 1f (2.02 g, 9.79 mmol) in DMF (40 mL), CH₂CI₂ (40 mL) at 0 ⁰C was treated with HATU (4.46 g, 11.84 mmol) and NMM (3.5 g, 35 mmol) and stirred at 0° C for 24 h. The reaction mixture was concentrated in vacuo and diluted with aq. HCl (100 mL). The aqueous layer was extracted with CH₂Ci₂ (3x75 mL). The combined organic layers were washed with aq saturated NaHCO₃ (3x100 mL), brine dried with MgSO₄, filtered concentrated in vacuo and purified by silica gei chromatography ( EtOAc/Hex 1 :3) to yield 1g (4.5 g ) as a colorless foam. Step E

¹9 1h

A solution of diene 1g (1.1 g, 2.0 mmol) in dry CH₂CI₂ (20 mL) was treated with Grubbs catalyst [(Cy)₃RuCI₂=CHC₆H₅, 83.8 mg, 0.1 mmol) and stirred at rt. for 24 h. The reaction mixture was concentrated in vacuo and purified by chromatography (SiO₂, EtOAc/Hex 1 :3) to yield 1h (501 mg) as a colorless solid and mixture of E/Z isomers.

¹H NMR (CDCI₃, 300 MHz) δ, 7.38 (d, 1 H, J=8.1 Hz), 5.30-5.18 (m, 2 H)₁ 4.55 (dt, 1 H, J= 2.4, 9.6 Hz), 3.92 (bs, 1 H), 3.77 (s, 3 H), 3.79-3.77 (bm, 1 H), 2.06-2.1 (bm, 3 H). 1.95-1.81 (m, 2 H), 1.79-1.77 (m, 13 H), 1.31 ( s, 9 H), 1.05 (s, 3 H), 0.85 (s, 3H). IWS (ESI), m/z, relative intensity 542 [(M+Na)⁺ 45], 464 (20), 448 (25) 420 (100) . Step F

1 h ^{1 i}

A solution of ester 1h (100 mg, 0.19 mmol) in dry THF (1 mL) was treated with LiBH₄ (2M soln. in THF, 0.2 mL) and stirred at rt. for 16 h. The reaction mixture was quenched with aqueous HCI (1 M₁ 30 mL) and extracted with CH₂CI₂ (3x30 mL). The combined organic layers were washed with aq. NaHCO₃ (IOO ml) brine, dried with MgSO₄ filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 1 :3) to yield 1i (70 mg) as an amorphous solid. 1H NMR (CDCI₃, 300 MHz) δ 6.96 (d, 1 H, J=8.1 Hz), 5.32-5.21 (m, 2 H), 4.43-4.37 (m, 2 H) 4.01-3.93 (m, 1 H), 3.77 (dd_r 1 H, J=5.7, 4.8 Hz), 3.65 (dd, 1 H, J= 3.9, 6.6 Hz), 3.53 (dd, 1 H, J= 6.0, 10.8 Hz), 2.11-1.77 (m, 6 H), 1.55-1.31 (m, 12 H), 1.45 (s, 9 H), 1.05 (S₁ 3 H), 0.87 (s, 3 H).

MS (ESI), m/z, relative intensity 530 [(M+K)⁺, 10], 514 [(M+Na)⁺, 70], 492 [(M+1)\ 20], 392 (100).

Step H

1i 1j

A solution of alcohol 1i (70 mg, 0.15 mmol), in CHbCI₂ (3 mL) was treated with Dess Martin reagent (85 mg, 0.2 mmol) and stirred at rt. for 2 h. The reaction mixture was quenched with Na₂S₂O₃ solution (10%, 10 mL) and saturated NaHCO₃ solution (10 mL) and stirred at rt. for 0.5 h. The reaction mixture was extracted with CH₂CI₂ (50 mL). The organic layer was dried with MgSO₄, filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 4:1 ) to yield 1j (50 mg) as a colorless fluffy solid. 1H NMR (CDCI₃, 300 MHz) δ9.54 (s, 1 H), 7.43 (d, 1 H, J = 7.8 Hz), 5.30-5.19 (m, 2 H), 4.55-4.40 (m, 2 H), 3.93 (d, 1 H, J=10.2 Hz), 3.77 (dd, 1 H, J==5.4, 5.1 Hz), 2.04- 1.78 (m, 4 H), 1.55-1.27 (m, 14), 1.31 (s, 9 H), 1.02 (s, 3 H), 0.95 (s, 3 H). MS (ESI), m/z, relative intensity 512 [(M+Naf, 80], 490 [(M+1)\ 10], 434 (20), 390 (100). Step !

U ^{1 k}

A solution of aldehyde 1j (50 mg, 0.11 mmol) in dry CH₂CI₂ (2 mL) was treated with CH₃COOH (19 mg, 0.31 mmol) and methylisocyanoacetate (31 mg, 0.31 mmol). The reaction mixture was stirred at rt. for 48 h and concentrated in vacuo. The residue was purified by chromatography (SiO₂, acetone/hexanes 1 :2) to yield 1k (50 mg) as a mixture of diastereomers.

MS (ESI), m/z, relative intensity 671 [(M+Na)⁺, 45], 649 [(M-H)⁺, 30], 549 (100). Step J

1k 11

A solution of methyl ester 1k (50 mg, 0.078 mmol) in THF (2 ml_), H₂O (2 ml_) and CH₃OH (2 mL) was treated with LiOH-H₂O (20 mg, 0.5 mmol) and stirred at rt. for 2 h. After the completion of the reaction it was acidified with aq. HCi (2 mL) and concentrated in vacuo. The residue was dried in vacuo and used as it with out further purification.

The acid was dissolved in CH₂CI₂ (2 mL), DMF (2 mL) and treated with H-Phg- N(CH)₂ -HCl (26 mg, 0.12 mmol), NMM ( 32 mg, 0.32 mmol) HATU ( 45 mg, 0.12 mmol) and stirred at 0 ⁰C for 24 h. The yellow colored solution was concentrated in vacuo and diluted with CH₂CI₂ (70 mL). The organic layers were washed with saturated aq. NaHCO₃, aq. HCI and brine. The reaction mixture was dried (MgSO₄) filtered concentrated in vacuo and used as it is in next step (47 mg). Step K

A solution of alcohol 11 (50 mg, 0.066 mmol) in CH₂CI₂ (2 mL) was treated with Dess-Martin reagent (60 mg, 0.14 mmol) and stirred at rt. for 2 h. The reaction was diluted with aq Na2S2O3 solution and aq. NaHCO₃ solution (20 mL each) and extracted with CH₂Cf₂ (50 mL). The organic layer was washed with satd. NaHCO₃, brine, dried with MgSO₄ filtered concentrated in vacuo and purified by chromatography ( acetone/hexanes 2:3) to yield 1 (22 mg) as a colorless solid. MS (ESI), m/z, relative intensity 773 [(M+Na}⁺, 80], 751 [(M+1)⁺, 60], 651 (100). Preparative Example 2

Step A

M 2a

A solution of alcohol 1i (1.1g, 2.25 mmol) in methanol (30 ml_) was treated with Pd/C (10% w/w, 100 mg) and hydrogenated at 60 psi for 3 h. The reaction mixture was filtered through a plug of celite, concentrated in vacuo to yield 2a which was used in the next step without further purification. Step B

2a 2b

Crude 2a from step A was oxidized using Dess-Martin reagent (1.14 g, 2.68 mmol) following the procedure similar to step H (preparative example 1) to yield 2b (760 mg) as a colorless foam.

MS (ESI), m/z, relative intensity 1005 [(2M+Na)⁺, 10], 530 [(M+K)⁺, 20], 514 [(M+Na)⁺, 90], 492 [(M+1 )⁺, 30], 436 (40), 392 (100). Step C

2b ^2C

Compound 2b (200 mg, 0.41 mmol) from step B was converted to 2c (250 mg) using CH₃COOH (60 mg) and methylisocyanoacetate (99 mg, 1 mmol) following the procedure similar to step I (preparative example 1) as a mixture of diastereomers. ¹H NMR (CDCI₃, 300 MHz, mixture of diastereomers) 8.05, 7.93 (d, 1 H), 6.60 (d, 1 H, J=7.8 Hz), 5.20, 5.09 (d, 1 H), 4.58-4.49 (bt, 1 H)₁ 4.34 (s, 1 H), 4.34-4.31 (bt, 1 H), 4.11-4.06 (m, 1H)₁ 3.95-3.86 (m, 3 H), 3.73, 3.71 (s, 3 H), 2.21 , 2.19 (s, 3H)₁ 1.99-1.06 (m, 31 H), 0.99-0.94 (6 H). MS (ESl), m/z, relative intensity 689 [(M+K)⁺, 5], 673 [(M+Na)⁺, 30], 651 [(M+1)⁺, 35], 551 (100). Step D

2c 2d

Methyl ester 2c (250 mg, 0.39 mmol) was hydrolyzed to acid using LiOH-H₂O (42 mg, 1 mmol) and coupled to H-Phg-N(CH)₂ -HCI (90 mg, 0.42 mmol) using NMM ( 126 mg, 1.26 mmol) and HATU (160 mg, 0.42 mmol) as outlined in preparative example 1, step J to yield crude 2d directly used for oxidation. Step E

2d

Hydroxy amide 2d was oxidized using Dess-Martin reagent (200 mg, 0.48 mmol) which was purified by chromatography (Siθ₂, acetone/ChkCb 1 :4) to yield 2 (110 mg) as colorless solid.

MS (ESI), m/z, relative intensity 775 [(M+Naf, 60], 753 [(M+1)⁺, 50], 653 (100), 277 (80), 232 (60), 162 (30), 162 (40), 148 (80), 217 (95). Preparative Example 3

3

Step A

A solution of 2 (40 mg, 0.0053 mmol) in HCOOH (2 mL) was stirred at rt. for 2 h and concentrated in vacuo. The residue was repeatedly dissolved in toluene and dried in vacuo to remove residual formic acid. The residue was dissolved in CH₂CI₂/DMF (1 mL each) and treated with ¹BuNCO (10 μL) and NMM (15 μl_) at 0° C and left in the refrigerator for 12 h. The reaction mixture was concentrated in vacuo and purified by chromatography (Siθ₂, acetone/hexanes 1 :2) to yield 3 (21 mg) as a colorless solid. MS (ESl), m/z, relative intensity 774 [(M+Naf, 50], 752 [(M+1)⁺, 70], 653 (90), 420 (30), 297 (30), 148 (100), 134 (40). Preparative Example 4

Step A

2b 4a

A solution of aldehyde 2b (100 mg, 0.2 mrnol) in CH₂Ci₂ (2 ml_) was treated with Et₃N (50 mg, 0.5 mmo!) and acetone cyanohydrin (43 mg, 0.5 mmol). The reaction mixture was stirred at rt for 2 h and concentrated in vacuo. The residue was purified by chromatography (SiO2, acetone/hexanes 1:4) to yieid 4a (100 mg) as a colorless solid.

MS (ESI), m/z, relative intensity 541 [(IvH-Na)⁺, 60], 519 [(IVH-I)⁺, 10], 463 (30), 419 (100).

Step B

4a 4b

A solution of cyanohydrin 4a (100 mg, 0.2 mmo!) in DMSO (3 mL) was treated with H₂O₂ (35%, 0.3 mL) and K₂CO₃ (43 mg, 0.3 mL) and stirred at rt. for 4 h. The reaction mixture was diluted with CH₂CI₂ (150 mL) and washed with aq. Na₂S₂O₃ solution (10%, 30 mL) and brine (30 mL). The reaction mixture was dried (MgSO4) filtered concentrated in vacuo and directly used in step C without further purification. Step C

4b 4

A solution of hydroxy amide 4b (100 mg, 0.18 mmol) in toluene/DMSO (1 :1, 5 mL) at 0 ⁰C was treated with EDCI (356 mg, 1.86 mmol) and CI₂CHCOOH (120 mg, 0.93 mmo!) and stirred at 0 ⁰C for 3 h. The reaction mixture was diluted with EtOAc (150 mL) and washed with satd. aq. NaHCO₃ (100 mL) and brine (100 mL). The ethyl acetate layer was dried (MgSO₄), concentrated and purified by chromatography (SiO₂, acetone/hexanes 2:3) to yield 4 (20 mg) as colorless solid MS (ESI), m/z, relative intensity 435 [(M+1)⁺, 85], 390 (100). Preparative Example 5

Step A

Carbamate 4 (40 mg, 0.1 mmol) was converted to urea 5 (7.5 mg) following the procedure similar to preparative example 3, Step A. Preparative Example 6

Step A

The synthesis of 6 was achieved using the similar procedure so synthesis of 5. A solution of 4 (180 mg 0.34 mmol) in HCOOH (3.0 mL) was stirred at rt. for 3 h an concentrated in vacuo. The residue was dried in vacuo and taken in CH₂CI₂ (4 mL) and treated with methyl cyclohexylisocyanate (72 mg, 0.52 mmol) and Et₃N (52 mg, 0.52 mmol). The reaction mixture was stirred at 0 ⁰C for 16 h and concentrated in vacuo. The residue was purified by chromatography (SiO₂, acetone/hexanes 1:3) to yield 6 (10 mg) as colorless solid

MS (ESI), m/z, relative intensity 574 [(M+1)⁺, 20], 435 (100), 390 (50). Preparative Example 7

Step A

The synthesis of 7 was achieved using the similar procedure so synthesis of 5. A solution of 4 (180 mg 0.34 mmol) in HCOOH (3.0 ml_) was stirred at rt for 3 h and concentrated in vacuo. 50 mg (0.12 mmol) of this residue was dried in vacuo and taken in CH₂Ci₂ (4 mL) and treated with isocyanate of ferf-butyl glycine terfbuty! ester (74mg, 0.0.35 mmol) and Et₃N (35 mg, 0.0.35 mmol). The reaction mixture was stirred at 0 ⁰C for 16 h and concentrated in vacuo. The residue was diluted with CH₂CI₂ and washed with aq HCI, aq satd. NaHCO₃ and brine. The organic layers were dried (IVIgSO₄) and purified by chromatography (SiO₂, acetone/hexanes 1:3) to yield 7 (15 mg) as colorless solid.

MS (ESI), m/z, relative intensity 648 [(M+1 )⁺, 45], 592 (25), 435 (100). Preparative Example 8

Step A

2c 8a

A solution of methyl ester 2c (100 mg, 0.15 mmol) in THF (2 mL), H₂O (2 mL) and CH₃OH (2 mL) was treated with LiOH-H₂O (41 mg, 1.0 mmol) and stirred at rt. for 2 h. After the completion of the reaction it was acidified with aq. HCI (2 mL) and concentrated in vacuo. The residue was dried in vacuo and used as it with out further purification.

The acid was dissolved in CH₂CI₂ (2 mi_), DMF (2 ml_) and treated with benzyi amine (107 mg, 0.22 mmol), NMM (42 mg, 0.42 mmol) HATU (53 mg, 0.14 mmol) and stirred at 0 ⁰C for 24 h. The yellow colored solution was concentrated in vacuo and diluted with CH₂CI₂ (100 ml_). The organic layers were washed with saturated aq. NaHCO₃, aq. HCI and brine. The reaction mixture was dried (MgSO₄) filtered concentrated in vacuo and used as it is in next step (63 mg). Step B

"

8a 8

Hydroxyamide 8a (62 mg) in CH₂CI₂ (3 tτiL) was treated with Dess-Martin reagent (62 mg, 0.15 mmol) and stirred at it for 1.5 h. The reaction mixture was diluted with CH₂CI₂ (20 ml_) and treated with aq. soln of Na₂S₂O₃ (10%, 25 mL) and satd. NaHCO₃ (25 mL) and stirred for 20 min. The aqueous layer was separated and extracted once again with CH2CI2. The combined organic layers were dried (MgSO₄), filtered, concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 1 :2) to yield 8 as a colorless solid (21 mg). MS (ESI), m/z, relative intensity 704 [(M+Na)⁺, 40], 682 [(M-M)⁺, 20], 582 (100), 150 (70), 117 (30). Preparative Example 9

Step A

A solution of acid 9a (3.6 g, 18.1 mmol), amine 9b (5.53 g, 18.1 mmol) HATU (8.59 mmol, 22.62 mmol) and NMM in CH₂CI₂ (50 mL), DMF (50 ml_) was stirred at O⁰C overnight. The reaction mixture was concentrated in vacuo and diluted with aq. HCI (1 M, 500 mL) and extracted with CH₂CI₂ (3x250 mL). The combined organic layers were washed with aq. HCI 500 ml), aqueous saturated NaHCθ₃ (500 mL) brine (300 mL) and purified by chromatography (SiO₂, acetone/hexanes 1 :4) to yield 9c (6.7 g) as colorless solid. MS (ESI), m/z, relative intensity 495 (M+Na)⁺, 90], 473 [(M+1)⁺, 60], 429 (70), 391 (40), 200 (100), 140 (30). Step B

A solution of methyl ester 9c (5.5 g, 11.59 mmol) in CH₃OH/THF/H₂O (300 mL) was treated with LiOH-H₂O (700 mg, 16.7 mmol) and stirred at rt. for 1.5 h. The reaction mixture was diluted with aq. HCI and extracted into CH₂CI₂ (700 mL). The organic layer was dried with MgSO₄ filtered concentrated in vacuo and used as it is in subsequent steps.

A solution of crude acid in CH₂CI₂ (50 mL), DMF (50 mL) was treated with

HATU (5.5 g, 17.35 mmol), NMM (4,07 g, 40.32 mmol) and stirred at 0 °C for 24 h. The reaction mixture was concentrated in vacuo and taken in aq. HCI (30OmL). The acidic layers was extracted into CH₂CI₂ (2x200 mL) and the combined organic layers were washed with saturated NaHCO3, brine and purified by chromatography (SiO₂, acetone/hexanes 4:1 ) to yield 9d (7.1 g ) as a colorless solid.

Step C

9d 9e

A solution of diene 9d (2.0 g, 3.2 mmol) in CH2CI2 (64 m!_) was treated with Grubbs catalyst ( [(Cy)₃RuCI₂=CHC₆H₅, 404 mg, 0.48 mmol) and stirred at it for 24 h. The reaction mixture was concentrated in vacuo and purified by chromatography (SiO₂, EtOAc/Hex 1 :3) to yield 9e (1.1 g) as a brown solid and mixture of EfZ isomers. ¹H NMR (CDCi₃, 300 MHz) D, 7.36 (bm, 5 H), 7.13 (d, 1 H, 4.5 Hz), 5.73 (d, 1 H, J=8.1Hz), 5.28 (m, 2 H), 5.10 (s, 2 H), 4.75 (m, 1 H), 4.65 (m, 2 H), 4.52- 4.46 (m, 1 H), 3.90 (bd, 1 H), 3.74 (s, 3 H)₁ 3.61 (dd, 1 H₁ J= 15.6, 11.1 Hz)₁ 3.44 (dd, 1 H, J=6.9, 7.2 Hz), 2.12-2.01 (m, 5 H)₁ 1.79-1.67 (m, 3 H), 1.49-1.43 (m, 3 H), 1.36-1.34 (m, 4 H), 1.26 (bs, 5 H), 1.16 (bs, 3 H).

MS (ESI), m/z, relative intensity 606 [(M+Na)⁺ 70], 584 (100), 540 (30). Step D

9e Sf

A solution of ester 9e (200 mg, 0.32 mmo!) in dry THF (5 mL) was treated with LiBH₄ (2M soln. in THF, 0.32 mL) and stirred at rt. for 3 h. The reaction mixture was quenched with aqueous HCI (1M, 100 mL) and extracted with CH₂CI₂ (3x50 mL). The combined organic layers were washed with aq. NaHCO₃ (IOO ml) brine, dried with

MgSO₄ filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 1:3) to yield 9f (2.1 g). 1H NMR (CDCI₃, 300 MHz) δ.

MS (ESI), m/z, relative intensity 578 [(M+Na)^÷, 40], 556 [(M+1)⁺, 80], 512, (30), 295

(100).

Step E

9f 99

A solution of alcohol 9f (100 mg, 0.19 mmol), in CH₂CI₂ (3 ml_) was treated with Dess Martin reagent (106 mg, 0.25 mmol) and stirred at rt. for 2 h. The reaction mixture was quenched with Na₂S₂O₃ solution (10%, 10 mL) and saturated NaHCO₃ solution (10 mL) and stirred at rt. for 0.2 h. The reaction mixture was extracted with CH₂CI₂. The organic layer was dried with MgSO₄, filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 3:1) to yield 9g (80 mg). 1H NMR (CDCI₃, 300 MHz) δ 9.52 (s, 1 H), 7.36 (bs, 5 H), 7.11 (d, 1 H, J=7.2 Hz), 5.67 (d, 1 H, J=7.8 Hz), 5.24-5.11 (m, 2 H), 5.11 (s, 2 H), 4.77-4.45 (m, 5 H)₁ 3.92 (d, 1 H, J=12 Hz), 3.58 (dd, 1 H, J=6.6, 5.5 Hz), 3.51-3.46 (m, 1 H)₁ 2.17-1.00 (m, 25 H). MS (ESI)₁ m/z, relative intensity 576 [(M+Na)⁺, 15], 554 [(M+1)⁺, 100], 510 (40). Step F

_9g ^9h

A solution of aldehyde 9g (80 mg, 0.15 mmol) in dry CH₂Ci₂ (2 mL) was treated with CH₃COOH (30 mg, 0.50 mmol) and methylisocyanoacetate (50 mg, 0.50 mmol).

The reaction mixture was stirred at rt. for 24 h and concentrated in vacuo. The residue was purified by chromatography (SiO₂, acetone/hexanes 1:3) to yield 9h as a mixture of diastereomers.

MS (ESI)₁ m/z, relative intensity 735 [(M+Na)⁺, 70], 713 [(M+1 )⁺, 100]. Step F

Methyl ester 9h (600 mg, 0.92 mmoi) was hydrolyzed to acid using LiOH-H₂O and coupled to H-Phg-N(CH)₂ ΗC1 (235 mg, 1.09 mmol) using NMM (303 mg, 3.0 mmoi) and HATU (437 mg, 1.15 mmol) as outlined in preparative example 1 , step J to yield 9ϊ that was directly used for oxidation. Step G

Crude 9j (470 mg, 0.58 mmol) from step F was oxidized using Dess-Martin reagent (424 mg, 1.00 mmoi) following the procedure similar to step H (preparative example 1) to yield 9j (310 mg) as a colorless solid.

MS (ESI), m/z, relative intensity 869 [(M+CH₃OH+Na)⁺, 100], 815 [(M+1)⁺, 40], 770

(30).

Preparative Example 10

Step A

9h 10a

A solution of 9h (200 mg, 0.3 mmol) in methanol (5 m!) was treated with Pd(OH)2/C (wet, 10%) and hydrogenated for 3h. The reaction mixture was filtered through a plug of celite and the filtrate was concentrated in vacuo. The residue was dissolved in methylene chloride and treated with ditertbutyldicarbonate (200 mg, 0.92 mmol). The reaction mixture was stirred at rt. for 24 h and purified by chromatography (SiO₂, acetone/Hexanes 1 :2) to yield 10a (85 mg) as a colorless solid. Step B

10a 10b Methyl ester 10a (80 mg, 0.15 mmol) was hydrolyzed to acid using LiOH-H₂O

(41 mg, 1 mmol) and coupied to H-Phg-N(CH)₂ -HCI (32 mg, 0.15 mmo!) using NMM (40 mg, 0.40 mmol) and HATU (64.6 mg, 0.17 mmol) as outlined in preparative example 1 , step J to yield 10b directly used for oxidation. Step C

10b ^{1 Oc}

Hydroxy amide 10b (60 mg, 0.08 mmol) was oxidized using Dess-Martin reagent (60 mg, 0.14 mmol) which was purified by chromatography (SiO₂, acetone/CH₂CI₂ 1 :2) to yield 10c (21 mg) as colorless solid. MS (ESl), m/z, relative intensity 805 [(M+Naf, 20], 783 [(W+1)⁺, 20], 683 (30), 369 (40), 210 (70), 116 (100). Preparative Example 11

Step A

9g 11a

A solution of aldehyde 9g (400 mg, 0.73 mmol) in CH₂CI₂ was treated with Et₃N (150 mg, 1.5 mmoi) and acetone cyanohydrin (170 mg, 1.5 mmol). The reaction mixture was stirred at rt for 3 h and concentrated in vacuo. The residue was purified by chromatography (SiO₂, acetone/hexanes 1:4) to yield 4a (286 mg) as a colorless solid.

MS (ESI), m/z, relative intensity 603 [(M+Na)⁺, 60], 581 [(M+1)^*, 70], 464 (50), 420 (100). Step B

11a 11b

A solution of cyanohydrin 11a (600 mg, 1.1 mmo!) in DMSO (12 mL) was treated with H₂O₂ (35%, 1.0 mL) and K₂CO₃ (43 mg, 0.3 mL) and stirred at rt. for 8 h. The reaction mixture was diluted with CH₂CI₂ (150 mL) and washed with aq. Na₂S₂Oe solution (10%) and brine (30 mL). The reaction mixture was dried (MgSO₄) filtered concentrated in vacuo and directly used in step C without further purification. MS (ESI)₁ m/z, relative intensity 621 [(M+Na)⁺, 70], 599 [(M+1 )⁺, 100], 554 (40). Step C

11b 11

A solution of hydroxy amide 11b (320 mg, 0.54 mmol) in toluene/DMSO (1 :1 , 10 mt_) at 0 ⁰C was treated with EDCI (1.1 g, 5.40 mmol) and Ci₂CHCOOH (350 mg, 2.7 mmol) and stirred at rt. for 4 h. The reaction mixture was diluted with CH₂CI₂ (150 ml_) and washed with satd. aq. NaHCO₃ and brine. The organic layer was dried (MgSO₄), concentrated and purified by chromatography (SiO₂, acetone/hexanes 1 :2) to yield 11 (173 mg) as colorless solid. MS (ESI), m/z, relative intensity 619 [(M+1 )⁺, 20], 597 (100). Preparative Example 12

Step A

11a 11b

A solution of 11a was hydrogenated using Pd/C and the amine obtained was dissolved in CH₂CI₂ and treated with tert-butyϋsocyanide at 0 ⁰C. The reaction mixture was stirred at rt. for 12 h and diluted with water. The reaction mixture was extracted with CH₂CI₂ (30 ml_) and combined organic layers were dried (IvIgSO₄) filtered concentrated in vacuo to obtain 11b that was used in oxidation without further purification. Step B

11b 12

A solution of hydroxy amide 11b (320 mg, 0.54 mmol) in toluene/DMSO (1 :1 , 10 mL) at 0 ⁰C was treated with EDCl (1.1 g, 5.40 mmol) and CI₂CHCOOH (350 mg, 2.7 mmol) and stirred at rt. for 4 h. The reaction mixture was diluted with CH₂CI₂ (150 mL) and washed with satd. aq. NaHCO₃ and brine. The organic layer was dried (MgSO₄), concentrated and purified by chromatography (SiO₂, acetone/hexanes 1 :2) to yield 11 (173 mg) as colorless solid. MS (ESI), m/z, relative intensity 619 [(M+1 )⁺, 20], 597 (100). Preparative Example 13

13

Step A

2b 13a

A solution of aldehyde 2b (50 mg, 0.1 mmo!) in dry CH₂CI₂ (5 mL) was treated with CH₃COOH (21 mg, 0.3 mmol) and TOSMiC (59 mg, 0.3 mmol, 3.0 eq.). The reaction mixture was stirred at rt. for 40 h and concentrated in vacuo. The residue was purified by chromatography (SiO₂, EtOAc/hexanes 2:3) to yield 1k (60 mg) as a mixture of diastereomers. MS (ESI), m/z, relative intensity 769 [(M+Na)⁺, 30], 747 [(EvHI)⁺, 20], 647 (100). Step B

13

13a

A solution of 13a (βOmg, 0.08 mmol) in methanol was treated with a 8 drops of concentrated HCI and stirred at rt for 12 h. The acetate ester was hydroiyzed with partially deprotection of Boc group which was reprotected with ditertbutyldicarbonate (16 mg, 0.073 mmot).

The hydroxyamide (46 mg, 0.07 mmol) in CH₂CI₂ was treated with Dess-Martin reagent (55 mg, 0.13 mmol) and stirred at rt for 10 min. Satd aq. Na₂S₂O₃ was added and reaction mixture was extracted into CH₂Ci₂ . The reaction mixture was dried (MgSO₄) filtered concentrated in vacuo and purified by chromatography to yield 13 (61 mg).

MS (ESi), m/z, relative intensity 703 [(M+1)⁺, 11], 603 (100). Preparative Example 14

Step A

14a

A solution of methylrsobutyrate (2.Og, 19.5 mmof) in THF was added dropwise to a solution of KHMDS in THF (4.65 g, 23.5 mmol) at -78 ⁰C and stirred for 0.5 h. The reaction mixture was treated with 5-bromo-1-pentene (3.5 g, 23.5 mmol) and shirred at rt. for 1 h. The reaction mixture was quenched with aq. HCI and extracted into ether (150 mL). The organic layer was dried (MgSO₄) fiitered concentrated in vacuo and purified by chromatography (EtOAc/Hexane 1:19) to yield 2.1 g of 14b as colorless liquid.

¹H NMR: (CDCi₃, 300 MHz) δ, 5.83-5.70 (m, 1 H), 5.00-4.91 (dd, 2 H), 3.65 (s, 3 H), 2.01 (dt, 2 H), 1.53-1.48 (m, 2 H), 1.35-1.30 (m, 2 H), 1.1 (s, 9 H).

Step B

A solution of ester (2. 6 g, 16 mmol) in ether (30 mL) was treated with LiAIH₄ (1M soln in THF, 20 mL) at -78 ⁰C and warmed to it The reaction mixture was quenched with a solution of KHSO₄ and filtered through a plug of celite and MgSO₄. The filtrate was concentrated in vacuo and used as it is in the next step.

Step C o

^CH3 CH₃ CH₃" XH3

14c _14d

A solution of oxalyl chloride (1.48 g, 11.7 mmol) in dry CH₂CI₂ was treated with DMSO (1.53 g, 19.5 mmol) at -78 ⁰C and stirred for 15 min. To this mixture was added alcohol 14c {1.1 g, 7.8 mmoi) and stirred at -78 ⁰C for 15 min. Triethyi amine (5.0 mL, 35.5 mmol) was added and the reaction mixture was warmed to rt. The reaction mixture was acidified and extracted with EtOAc (200 mL). The combined organic layers were washed with aq. HCl₁ dried (MgSO4) filtered, concentrated in vacuo and used in next reaction.

¹H NIVIR (CDCl₃, 300 MHz) δ 9.42 (s, 1 H), 5.82-5.68 (m, 1 H), 5.00-4.91 (m, 2 H)₁ 2.03 (dt, 2 H)₁ 1.48-1.23 (m, 4 H)₁ 1.03 (s, 3 H). Step D

A solution of aldehyde 14d (18g, 129 mmoi) in CH₂CI₂ (150 mL) was treated with (R)-phenyglycinol (20.33 g, 148.3 mmoi ) and stirred at 0 ⁰C for 1 h. The reaction mixture was treated with TMS-CN (25.6g, 258 mmol) and stirred at rt. for 12 h. The reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with EtOAc (3x150 ml_). The combined organic layers were dried (MgSO₄) filtered concentrated in vacuo and the residue was dissolved in THF (100 mL) and treated with aq HCI (100 mL). The aqueous layer was basified with aq. NaOH (1 M) and extracted with (EtOAc, 450 mL). The combined organic layers were dried, filtered concentrated in vacuo and purified with chromatography (SiO₂, EtOAc/Hexanes 6:1 ) to yield 14e 21 g as a colorless oil. Step E

A solution of 14e (20 g) in CH₃OH (200 mL) was treated with H₂O₂ (60 mL) and

LiOH-H₂O (5.88 g, 209.6 mmol) at 0 ⁰C. The reaction mixture was stirred at rt. for 12 h and cooled to 0°C and carefully quenched with aq. Na₂S₂O₃ solution (10%). The reaction mixture was concentrated in vacuo and the aq. layer was extracted with EtOAc (600 mL). The combined organic layers were washed extensively with aq. Na₂S₂O₃, dried (MgSO,*) concentrated in vacuo and purified by crystallization (EtOAc/Hexanes) to yield pure diastereomer directly used in the next reaction. 1H NMR (CDCI₃, 300 MHz) δ 7.30 (bs, 5 H), 6.25 (s, 1 H), 6.17 (s, 1 H)₁ 5.79-5.66 (m, 2 H), 4.98-4.89 (m, 2 H), 3.71-3.60 (m, 3 H)₁ 2.68 (bs, 1 H), 1.98-1.90 (3 H), 1.03 (s, 3 H), 0.99 (s, 3 H), 1.03-0.99 (m, 1 H). Step F

A solution of amide 14f (8.0Og, 26.3 mmo!) in CH₂CI₂ (160 mL), CH3OH (80 mL) at 0⁰C was treated with Pb(OAc)₄ (13.45 mmol, 30.3 mmol), at 0° C for 1 h. the yellow solution was treated with aq. NaHCO₃ (250 mL, and stirred for 15 min. The reaction mixture was filtered and concentrated in vacuo. The mostly aqueous layer was extracted in CH₂CI₂ (3x300 m!_) concentrated in vacuo and directly used in further reaction.

A solution of the crude imine was taken in THF (200 mL) and treated with aq

HCL (1 M, 200 mL) and stirred at rt. for 1 h. The reaction mixture was concentrated in vacuo and extracted with Ether (2x250 mL). The aqueous layer was basified with aq.

NaOH (50%) at 0 ⁰C and extracted with CH₂CI₂ (600 mL). The combined organic layers were extracted with brine, dried (MgSO4) filtered concentrated in vacuo and directly used in the next reaction.

The residue was dissolved in CH₂CI₂ (200 mL) and cooled to -78⁰C and treated with NMM (4.2 g, 40 mmol) and Cbz-CI (5-4g, 31.58 mmol). The reaction mixture was stirred at rt. for 12 h and washed with aq. HCI . The organic layer was separated and the aq. layer was extracted with CH₂CI₂ (200 mL) The combined organic layers were extracted with brine, dried and purified by chromatography (SiO₂, EtOAc/Hexanes 2:3) to yield 14g (6.8 g) as a colorless solid. 1H NMR (CDCI₃, 300 MHz) δ 7.37-7.30 (m, 5 H), 6.23 (bs, 1 H), 5.86 (bs, 1 H), 5.82-

5.64 (m, 1 H)₁ 5.63 (d, 1 H, J= 9.3 Hz)₁ 5.12-4.93 (m, 4 H), 4.07 (d, 1 H, J=9 Hz), 2.0-

1.9(m, 2 H)₁ 1.42- 1.30 (m, 4 H)₁ 0.96 (s, 6 H).

IVIS (ESI), m/z, relative intensity 341[M+Na)⁺, 100], 319 [(M+1 )+, 30], 274 (50), 230

(70), 213 (30), 140 (30). Step G

A solution of amide 14 g (6.8 g, 21.4 mmo!) in CH₂CI₂ (200 mL) was treated with Me₃OBF₄ (10.36 g, 69.9 mmol) and K₃PO₄ (12.11 g, 69.52 mmol) and stirred at rt. for 12 h. The reaction mixture was concentrated in vacuo and dissolved in CH₃OH (280 mL) and aq. HCI (140 mL, 1 M) and heated at reflux for 1 h. The reaction mixture was concentrated and the aqueous layer was further extracted with CH₂CI₂ (3x150 mL). The combined organic layers were dried (MgSO₄), filtered concentrated in vacuo and purified by chromatography (SiO₂, EtOAc/hexanes 1 :19) to yield 14h (5.6g) as colorless oil ¹H NMR (CDCI₃, 300 MHz) δ 7.36 (bs, 5 H), 5.85-5.71 (m, 1 H), 5.32 (d, 1 H, J=9.9

Hz), 5.10 (dd, 2 H, J= 12, 3.9 Hz), 5.03-4.93 (m, 2 H)₁ 4.27 (d, 1 H, J=9.9 Hz), 3.72 (s,

3 H), 2.05—1.98 (m, 2 H), 1.47-1.24 (m, 4 H), 0.93 (s, 9 H).

MS (ESI), m/z, relative intensity 356 [M+Na)⁺, 95], 334 [(M+1)+, 10], 290 (100), 230

(60), 213 (20).

Step H

A solution of acid 14i (4.5g, 17.64 mmol) and amine 1f (3.66 g, 17.64 mmol) in CH₂Cl₂ (SO ml_), DMF (50 ml_) at 0° C was treated with HATU (8.39 g, 22.05 mmol) and NMM (5.35 g, 52.92 mmol) and stirred overnight at 0° C. The reaction mixture was concentrated in vacuo and diluted with 450 ml_ of CH₂CI₂. The aqueous layer was washed with aq. HCl (1 M, 2x300 mL), aq. NaHCO₃ (1 M, 2x300 mL). The organic layers were dried with MgSO₄, filtered concentrated in vacuo and purified by chromatography (SiO₂, Acetone/Hexanes 5:1) to yield 14j as a colorless oil (5.8 g). ¹H NMR (CDCI₃, 300 MHz) δ 7.03, 6.39 (d, 1 H₁ J = 7.5 Hz)₁ 5.8-5.7 (m, 1 H), 4.99- 4.90 (m, 2 H), 4.66 -4.54 (m, 1 H), 3.72 (s, 3 H), 3.62-3.42 (m, 2 H), 2.01 (bs, 2 H), 1.88-1.63 (m, 4 H), 1.61 , 1.43 (s, 9 H)₁ 1.6-1.3 (m, 4 H), 1.02 (s, 3 H), 0.90 (s, 3H). MS (ESl), m/z, relative intensity 431 [(M+Na)⁺, 60], 409 [(M+1)⁺, 40], 353 (40), 309 (100), 110 (80). Step 1

A solution of ester 14h (5.4g, 16.2 mmol) in H₂O (30 mL), THF (30 mL) and CH₃OH (30 mL) was stirred with LiOH-H₂O (1.36 g, 32.42 mmol) for 24 h and concentrated in vacuo. The aqueous layer was acidified with aq. HCI (1 M) and extracted into CH₂CI₂ (400 mL). The combined organic layers were dried (MgSO₄), filtered concentrated in vacuo and used as it is in further reactions. A solution of acid (4.0 g, 12.5 mmol) and deprotected amine^* in CH₂CI₂ (30 ml_), DMF (30 mL) at 0° C was treated with HATU (7.15 g, 18.79 mmol) and NMM (4.5 g, 45.0 mmol) and stirred at 0° C for 48 h, and 25°C for 24 h. The reaction mixture was concentrated in vacuo and diluted with 300 mL of CH₂CI₂. The aqueous layer was washed with aq. HCI (1M₁ 3x100 mL), aq. NaHCO₃ (satd, 3x100 mL). The organic layers were dried with MgSO₄, filtered concentrated in vacuo and purified by chromatography (SiO₂, EtOAc/Hexanes 3:1) to yield 14k as a colorless oil (4 g of pure 14k and 2 g of partially impure 14k). 1H NMR (CDCI₃, 300 MHz) δ 7.34-7.32 (bs, 5 H), 6.92 (d, 1H, J=7.5Hz), 5.48-5.69 (m, 2 H), 5.37 (d, 1 H, J=9.9 Hz), 5.08-4.92 (m, 6 H), 4.56-4.33 (M, 1 h), 3.97-3.93 (m, 2 H), 3.84-3.80 (m, 2 H), 3.74 (s, 3 H), 2.03-1.97 (m, 4 H), 1.86-1.87-1.39 (m, 12 H), 1.12 (s, 3 H), 0.98 (s, 6 H), 084 (s, 3 H)

MS (ESI), m/z, relative intensity 632 [(M+Na)⁺, 20], 610 [(M+1)⁺, 100], 309 (60). * Amine was obtained by the deportation of 14j with 4 M HCi in dioxane. Step J

A solution of diene 14k (4.00 g, 6.57 mmol) in CH₂CI₂ (65.0 mL) at it was saturated with N₂ and treated with Grubbs catalyst (551 mg, 0.657 mmol) and stirred for 24 h. The reaction mixture was concentrated in vacuo and purified by chromatography (SiO₂, EtOAc/hexanes 1 :3) to yield 141 (1.7 g) as a tan colored solid. ¹H NMR (CDCI₃, 300 MHz) δ 7.34-7.31 (bs, 5 H), 7.08 (d, 1 H, J = 7.8 Hz), 5.43 (d, 1 H, J = 10.2 Hz), 5.28 (m, 2 H), 5.13-5.02 (m, 2 H), 4.56-4.32 (m, 1 H), 4.49-4.28 (m, 2 H), 3.96-3.79 (m, 2 H), 3.74 (s, 9 H), 2.05-1.29 (m, 16 H), 1.0 (s, 3 H), 0.96 (s, 3 H), 0.94 (s, 3 H), 0.86 (s, 3 H). MS (ESI), m/z, relative intensity 550 [(M+1 )⁺, 50], 450 (100). Step K

A solution of alkene 141 (200 mg, 0.35 mmol) in CH3OH (2 0 ml_) was treated with Pd/C (5%, 200 mg), ditertbutyldicarbonate (200 mg, 0.92 mmol) and hydrogenated at rt. for 12 h. The reaction mixture was filtered through a plug of celite and concentrated in vacuo. The reaction mixture was purified by chromatography

(SiO₂, acetone/hexanes 1 :5) to yield 14m (81 mg).

¹H NMR (CDCi₃, 300 MHz) δ 6.84 (d, 1 H, J-7.8 Hz), 5.14 (d, 1 H)₁ 4.61-4.55 (m, 1 H),

4.31 (s, 1 H), 4.22 (d, 1 H, J=10 Hz), 4.03 (d, 1 H, J=10.5 Hz), 3.88-3.85 (m, 1 H), 3.75(s, 3 H), 1.89-1.76 (m, 1 H)₁ 1.59-1.76 (m, 28 H), 1.02 (s, 3 H), 0.97 (s, 3 H)₁ 0.94

(s, 3 H), 0.86 (s, 3 H).

MS (ESI), m/z, relative intensity 610 [(M+AcOH+1)⁺, 40], 550 [(M+1 )⁺, 50], 450 (100),

309 (20).

Step L

A solution of ester 14m (80 mg, 0.15 mmol) in dry THF (2 mL) was treated with LiBH₄ (2 M soln. in THF, 0.1 mL) and stirred at rt. for 4 h. The reaction mixture was quenched with aqueous HCI (1 M, drops) and extracted with CH₂CI₂ (3x30 mL). The combined organic layers were washed with aq. NaHCO₃(IOO ml) brine, dried with MgSO₄ filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 1 :3) to yield 14n (70 mg) as an amorphous solid. MS (ESI)₁ m/z, relative intensity 544 [(M+Na)⁺, 30], 522 [(M+1)⁺, 40], 422 (100). Step M

A solution of alcohol 14n (30 mg, 0.05 mmol), in CH₂CI₂ (2 ml_) was treated with Dess Martin reagent (30 mg, 0.07 mmol) and stirred at rt. for 2 h. The reaction mixture was quenched with Na₂S₂O₃ solution (10%, 10 ml_) and saturated NaHCO₃ solution (10 mL) and stirred at rt. for 0.5 h. The reaction mixture was extracted with CH₂CI₂ (3x10 mL). The organic layer was dried with MgSO₄, filtered concentrated in vacuo and used as it is in further reaction. MS (ESI)₁ m/z, relative intensity 552 [(M+1)⁺, 10O]₁ 248 (40). Step N

Compound 14o from step M was converted to 14p (40 mg) using CH₃COOH (20 DL) and methyϋsocyanoacetate (20 DL) following the procedure similar to step I (preparative example 1) as a mixture of diastereomers. MS (ESI), m/z, relative intensity 711 [(M+1)⁺, 100], 240 (20). Step O

A solution of methyl ester 14p (80 mg, 0.12 mmol) in THF (3 mL), H₂O (3 mL) and CH₃OH (3 mL) was treated with LiOH-H₂O (41 mg, 1 mmol) and stirred at rt. for 2 h. After the completion of the reaction it was acidified with aq. HCl (15 mL) and extracted with CH2CI2 (3x30 mL). The combined organic layers were dried (MgSO4), filtered and concentrated in vacuo. The residue was dried in vacuo and used as it with out further purification.

The acid was dissolved in CH₂CI₂ (2 mL), DMF (2 mL) and treated with H-Phg- N(CH)₂ ΗCi (40 mg, 0.2 mmol), NMM ( 40 mg, 0.4 mmoi) HATU ( 68 mg, 0.16 mrnol) and stirred at 0 ⁰C for 24 h. The yellow colored solution was concentrated in vacuo and diluted with CH₂Ci₂ (75 mL). The organic layers were washed with saturated aq. NaHCO₃, aq. HCI and brine. The reaction mixture was dried (MgSO-O filtered concentrated in vacuo and used as it is in next step (90 mg). Step P

A solution of alcohol 14q (90 mg, 0.11 mmof) in CH₂CI₂ (2 mL) was treated with Dess-Martin reagent (100 mg, 0.24 mmol) and stirred at rt. for 2 h. The reaction was diluted with aq Na₂S₂Os solution ( 30 mL) and aq. NaHCO₃ solution (30 mL each) and extracted with CH₂CI₂ (50 mL). The organic layer was washed with satd. NaHCO₃, brine, dried with MgSO₄ filtered concentrated in vacuo and purified by chromatography ( acetone/hexanes 2:3) to yield 14 (22 mg) as a colorless soiid. WIS (ESI), m/z, relative intensity 813 [(M+1f, 100], 768 (20).

Preparative Example 15

Step A

Ia 15a To 45 ml. THF, diisopropylamine {4.70 mL, 33.51 mmol, 2 eq.) and LiCI (4.26 g, 6 eq) at -78 ⁰C was added nBuLi (20.4mL, 1.95 eq) under nitrogen atmosphere. 10 min later, the solution of 1a/30 mL THF was transferred to the above solution over 10 min, After 20 min, the brownish yellow mixture was warmed up to 0 ⁰C. Another 20 min later, the solution became opaque bright yellow and 4-iodo-1-butene (3.35 g, 1.1 eq) was added in dropwise. The solution became even brighter and 60 min later 115 mL 1 M HCI was added to quench the reaction. The THF was removed and 150 mL EtOAc was added in for extraction. The organic layer was further washed with 115 mL 1M HCI. The aqueous layers were combined and adjusted to pH 14 by 6M NaOH at O⁰C. Extraction was done with dichforomethane 110 mL x 4. The organic layer was dried over sodium carbonate. Filtration through celite and removal of the solvent afforded 4 g of the oil which upon standing, became solid. Flash chromatography with

5:5:90 Et₃N/MeOH/DCM provided 2.63 g pure 15a in 57 % yield. (R_f = 0.64, 5:5:90

Et₃N/MeOH/DCM)

¹H NMR (4: 1 rotamer ratio. * denotes minor rotamer peaks. CDCI₃): δ 0.96* <d, 3H, J = 6.7 Hz) 1.15 (d, 3H₁ J = 6.9 Hz) 1.45-1.55 (m, 2H) 2.05-2.20 (m, 2H) 2.80 (s, 3H) 2.92^* (s, 3H) 3.55-3.60 (m, 2H) 4.00* (m, 1H) 4.35-4.45* (m, 1H) 4.60-4.65 (m, 2H) 4.92-5.02 (m, 2H) 5.68-5.80 (m, 1 H) 7.20-7.40 (m, 5H). 1³C NMR(CDCI₃): ^ 11.26 15.68 31.11 35.67 47.17 52.22 76.92 116.46 127.50 128.67 129.34 138.60 143.19 178.08. MS: C₁₆H₂₄N₂O₂: 277 (M+H)⁺ ; HRMS: calcd: 277.1916; found: 277.1917.

Step B

¹⁵³ 15b

1.9 g of 15a (6.88 mmol, 1 eq) was treated with 2N NaOH (7.0 mL, 2 eq), 7 mL of water and refluxed at 100 ⁰C for 3 h. The mixture was cooled to room temperature. 20 mL of DCM, 10 mL of water was added and the organic layer was separated. The aqueous layer was washed with 20 mL of DCM. The combined organic layers were further washed with 10 mL of water. The combined aqueous layer was treated with 1.3 mL 12 N HCI. 20 mL of dioxane was added and the solution was adjusted to pH 8-9 by adding saturated NaHCO₃. 1.48 g of iBOC-OSU (1 eq) was added and the mixture was stirred for overnight. After decreasing the solvent volume to one half, 10 mL of water and 10 mL DCM was added for extraction. The aqueous layer was then treated with 12 N HCI dropwise until it precipitated (pH 2). Extraction with EtOAc 40 mL x 2 followed by MgSO₄ drying and celite filtration afforded 1.52 g colorless oil 15b in 90 % yield.

¹H NWiR(CDCI₃): 6 0.88 (d, 6 H, J = 6.6 Hz) 1.78-2.00 (m, 3 H) 2.10-2.20 (m, 2 H) 3.80-3.82 (m, 2 H) 4.40 (m, 1 H) 5.00-5.06 (m, 2 H) 5.10 (m, 1 H) 5,80 (m, 1 H). ¹³C NIVIR(CDCI₃): δ 20.0 26.2 29.0 32.8 54.2 72.8 117.0 138.0 157.8 177.6. MS for CnH₁₉NO₄: 230 (M+H)⁺.

Step C

lmine 15c (9.42 g, 31.88 mmol, 1 eq) was mixed with the Corey's catalyst (J. Am. Chem. Soc, 1997, 119, 12414) (1.93 g, 0.1 eq), cesium hydroxide monohydrate (53.55 g, 10 eq) in 150 ml_ DCM. The solution was cooled down to -60⁰C followed by addition of 5-iodo-1-pentene (25 g, 4 eq) under nitrogen. The crude was stirred for 60 h when 100 mL ethyl ether was added in. After washing with water 100 ml_ x 2 and brine 70 mL x 1 , the organic layer was dried over MgSO₄. Celite filtration and removal of the solvent afforded the crude 28.56 g. 5.1 g of the crude was chromatographed with pure hexane first and then 1 : 40 to 1 :20 EtOAc/hexane. A 2.56 g of a mixture of 15d, 5-iodo-1-pentene and benzophenone (1 : 2.5 : 0.8) was obtained. (15d: R_f = 0.39, 1 : 20 EtOAc/hexane.) Step D

N. .COOtBu

15d _15β 0.5 g of the above crude 15 d (2.56 g) was treated with 4 mL HOAc/THF/water

1 : 1 : 1 for 90 min when TLC shows disappearance of the starting materia]. Two pipetful of saturated NaHCO₃ was added. 10 mL water and 20 mL hexane was added for extraction. The aqueous layer was then further basified to pH 9-10. (Boc)₂O (0.15 g) and dioxane 4 mL were added and after 2.5 h, the solvent was removed and the pH of the solution was adjusted to 3-4. Extraction with ether followed by chromatography with 1 : 10 EtOAc/hexane afforded 0.16 g of 15e in 48 % overall yield from 15c. (R_f = 0.44, 1 : 10 EtOAc/hexane.) Step E

H Boc-NL _^CHO

BoC-N_x^COOtBu

15e 15f

4.88 g of 15e (13.87 mmol) was dissolved in 20 mL of toluene at -78 ⁰C and was treated with 21 mL LiAIH₄ (1 M in Et₂θ, 1.6 eq) for 40 min. The mixture was warmed up to O⁰C and was quenched by EtOAc and 20 mL 5 % NaHSO₄. Extraction with ether, filtration through celite and removal of solvent afforded the residue which was chromatographed with 1/5 EtOAc/hexane. 2.8 g of the desired aldehyde 15f (R_f = 0.4) along with the alcohol (1.43 g, R_f = 0.04) were obtained. The latter could be converted to the aldehyde by Dess-Martin reaction. Step F

15f 15g

1.26 g of 15f (5.55 mmol, 1 eq), methyl isocyanoacetate (0.50 mL, 1 eq), acetic acid (0.32 mL, 1 eq) were mixed in 20 mL DCM and stirred for 80 h. Removal of the solvent and flash chromatography provided 1.10 g of 15g in 51 % yield. (R_f= 0.29, 1:1 EtOAc/hexane).

¹H NMR(CDCl₃): δ 1.42 (s, 9 H) 1.50-1.60 (m, 2 H) 1.99-2.20 (m, 4 H) 2.18 (s, 3 H) 3.76 and 3.78 (two singlets, 3 H, 1 : 1 diastereomers) 3.90-4.20 (m, 4 H) 4.90-5.00 (m, 2 H) 5.20 (br s, 1 H) 5.70 (m, 1 H) 6.62 (br s, 1 H). 1³C NIVIR(CDCI₃): δ 21.93 26.26 29.46 31.25 34.41 41.99 52.53 53.50 75.57 80.41 115.74 139.14 156.28 168.91 169.38 170.79.

HRMS fOr C₁₈H₃₀N₂O₇: calcd: 387.2131 (M+H)⁺; found 387.2133. Step G

Compound 15g (1.08 g, 2.8 mmol, 1 eq), 60 mg K₂CO₃ (0.15 eq) in 6 mL MeOH were stirred at room temperature for 1 h and then another 2 h at 40⁰C. Removal of solid followed by flash chromatography afforded the desired product 15h as white solid (0.65 g, 68 % yield).

¹H NMR(CDCI₃): δ 1.40 (s, 9 H) 1.40-1.70 (m, 4 H) 1.99-2.10 (m, 2 H) 3.70(s, 3 H)

3.80 (br, 1 H) 4.00-4.25 (m, 4 H) 4.90-5.00 (m, 2 H) 5.10 (br s, 1 H) 5.30 (m, 1 H) 5.78 (m, 1 H) 7.40 (br s, 1 H).

¹³C NIVIR(CDCI₃): δ 26.83 29.48 30.76 34.53 42.03 53.51 54.95 75.05 81.07

115.76 139.30 157.92 170.84 174.16.

C₁₆H₂₈N₂O₆: 345 (M+H)⁺.

HRMS: calcd: 345.2026; found: 345.2033. Step H

Compound 15h (0.39 g, 1.13 mmol) was stirred with 4 IVI HCI in dioxane (4 ml_) at room temperature for 2 h when solid precipitates formed. The solvent was removed and 20 mL DCM was added. The pH was adjusted to 7 by using Hunig's base. The solvent was then removed and the residue was treated with 10 mL THF, Boc-Pro-OH (0.73 g, 3 eq), HATU (1.29 g, 3 eq), Hunig's base (1.18 mL, 6 eq) and 1 mL DMF. After stirring at room temperature for 7 h, the solvent was removed in vacuo. The residue was dissolved in 20 mL EtOAc and washed with 10 mL saturated NaHCO₃, 10 mL 0.5 M HCl twice, water 20 mL and brine 5 mL. Chromatography provided 0.68 g 15i (Rf = 0.31 , 5 % MeOH in DCM). Step I

15j

15i was treated with 2 mL DCM, 3 mL 4 M HC! in dioxane for 1 h. 3OmL DCM was added followed by neutralization with Hunig's base at 0 ⁰C. The solvent was removed and the crude was dissolved in 5 mL DCM, 10 mL THF. After addition of 15b (0.26 g, 1 eq), HATU (0.43 g, 1 eq) and Hunig's base (0.41 mL, 2.1 eq) and stirred for 4 h, the solvent was removed and 30 mL EtOAc was added. The solution was then washed with 10 mL saturated NaHCO₃, 10 mL 1 M HCi, 10 mL 0.5 M HCi, water 20 mL, brine 5 mL. Chromatography gave the desired product 15j (0.3 g, 48 % from 15h). 1³C NWIR(CDCI₃): δ 20.20 26.26 26.72 29.18 29.55 30.58 33.25 34.60 41.95

48.57 52.90 53.00 53.40 54.68 61.56 72.34 75.68 115.64 116.73 138.07 139.33

157.47 171.04 171.15 173.06 174.23

C₂₇H₄₄N₄O₈: 553 (M+H)⁺.

HRWIS: calcd: 553.3237; found: 553.3259. Step J

15j 15k

Compound 15j (0.37 g, 0.67 mmol) was treated with 0.138 g Grubbs' catalyst

(0.25 eq) in 223 mL DCM under argon. After stirring at room temperature for 65 h,

NMR shows the mixture contained the S. M. 15j, the desired product 15k (about 20 % yield) and PO(C₆Hn)₃. The R_f for these three are 0.34, 0.24, 0.74, respectively in 5

% HOAc/EtOAc. Repeated flash chromatography could provide the pure sample of

15k.

¹H NMR(CDCI₃): δ 0.90 (d, 6 H, J = 6.6 Hz) 1.40-2.00 (m, 14 H) 2.05-2.50 (m, 3 H)

3.60(m, 1 H) 3.70 (s, 3 H) 3.75-4.00 (m, 3 H) 4.00-4.20 (m, 2 H) 4.50 (m, 1 H) 4.70 (d, 1 H, J = 7.5 Hz, diastereomer) 4.81 (d, 1 H₁ J = 7.9 Hz, another diastereomer)

5.38 (m, 1 H) 5.58 (m, 1 H) 5.65 (br s, 1 H) 7.20 (d, 1 H J = 7.0 Hz) 7.38 (d, 1 H, J

= 7.1 Hz).

¹³C NMR(CDCi₃): δ 20.26 23.05 26.54 27.02 27.67 27.73 29.21 31.06 34.03

41.97 48.71 52.40 52.80 53..53 60.54 72.43 75.08 130.44 130.56 157.02 171.13 172.01 173.13 173.38.

LC/MS: Tr = 5.11 min (gradient A (acetonitrϊle)/B (water with 0.1 % TFA): from 5% A/B to 95 % A/B in 10 min.) C₂₅H₄₀N₄O₈: 525 (M+1)⁺

HRMS: calcd: 525.2924; found: 525.2908. Step K

15k ₁₅|

Compound 15k (92 mg, 0.18 mmol, 1 eq), 60 mg K₂CO₃ (2.5 eq) in 5 ml_ MeOH were stirred at 40⁰C for 2 h when TLC shows complete disappearance of S. M. After removal of the solvent, 44 ml_ 0.01 M HCf in DCM (2.5 eq) was added to neutralize the solution. The solvent was removed followed by addition of 10 ml_ THF, 1 ml_ DMF, PhG-O-tBu (HCi salt, 51 mg, 1.2 eq), 80 mg of HATU (1.2 eq), 0.11 ml_ of Hunig's base (3.5 eq). The mixture was stirred for 12 h. After removal of solvent, direct chromatography provided the product 151 (97 mg, 79 % yield from 15j. Rf = 0.32, 5 % MeOH/DCM).

¹H NMR(CDCi₃): 6 0.90 (d, 6 H, J = 6.6 Hz) 1.30 (s, 9 H) 1.40-2.00 (m, 14 H) 2.15- 2.20 (m, 1 H) 3.60(m, 1 H) 3.75-3.90 (m, 3 H) 4.00-4.09 (m, 1 H) 4.10-4.35 (m, 2 H) 4.50 (m, 1 H) 4.62 (d, 1 H₁ J = 7.5 Hz, diastereomer) 4.72 (d, 1 H, J = 7.9 Hz, another diastereomer) 5.20-5.38 (m, 1 H) 5.44 (d, 1 H, J = 6.6 Hz) 5.50 (m, 1 H) 5.98 (m, 1 H) 7.30 (m, 5 H) 7.45 (d, 1H₁ J = 7.0 Hz) 7.55 (d, 1 H₁ J = 7.1 Hz) 7.70 (br s, 1 H). ¹³C NMR(CDCl₃): 6 20.30 23.35 26.38 26.78 27.29 28.02 29.18 31.42 34.89 43.97 48.70 51.90 52.93 58.22 60.40 72.44 74.96 75.93 83.80 120.88 128.10 128.12 129.63 129.70 130.33 137.74 157.20 169.32 170.69 173.70 174.47. LC/MS: Tr = 6.61 min (gradient A (acetonitrile)/B (water with 0.1 % TFA): from 5% A/B to 95 % A/B in 10 min.) MS: C₃₆H₅₃N₅O₉: 700 (M+H)⁺. Step L

Compound 151 (90 mg, 0.13 mmol) was treated with 109 mg of Dess-Martin reagent (2 eq) in 10 mL DCM at room temperature for 12 h. After removal of the solvent, direct chromatography with 7:3 EtOAc/hexane provided 15m (40 %) as white solid. ¹H NIVIR(CDCI₃): δ 0.95 (d, 6 H₁ J = 6.6 Hz) 1.40 (s, 9 H) 1.50-2.10 (m, 14 H) 2.20- 2.30 (m, 1 H) 3.60(m, 1 H) 3.75-3.90 (m, 3 H) 3.93 (dd, 1 H, J = 5.9, 16.8 Hz) 4.10 (m, 1 H) 4.50 (dd, 1 H₁ J = 8.0, 13.9 Hz) 4.80 (d, 1 H, J = 6.6 Hz) 5.20-5.40 (m, 3 H) 5.41 (d, 1 H₁ J = 6.6 Hz) 5.60 (dd, 1 H, J = 7.3, 10 Hz) 6.82 (d, 1 H, J = 7.3 Hz) 7.30 (m, 5 H) 7.50 (m, 1 H) 7.80 (d, 1 H₁ J - 6.7 Hz).

¹³C NMR(CDCI₃): ό 20.29 23.65 26.34 26.75 29.02 29.20 30.37 30.95 31.56 35.07 43.71 48.83 52.95 54.20 58.14 60.23 72.54 84.15 128.03 129.41 129.68 129.87 130.62 137.60 156.99 160.33 167.41 171.37 173.84 187.26 196.36. LC/MS: Tr = 6.81 min (gradient A (acetonϊtrile)/B (water with 0.1 % TFA): from 5% A/B to 95 % A/B in 10 min.) MS: C₃₆H_5I N₅O₉: 698 (M+H)⁺ HRMS: calcd 698.3765 found 698.3762. Step M

Compound 15m (4 mg) was treated with 5 mL MeOH₁ 2 mg of Pd-C under hydrogen balloon for 1.5 h. The solution was filtered through celite. The filtrate was dried in vacuo and the NMR shows exclusive formation of 15.

¹H NMR(CDCI₃): 6 0.95 (d, 6 H, J = 6.6 Hz) 1.40 (s, 9 H) 1.50-2.10 (m, 16 H) 2.20-

2.30 (m, 1 H) 3.60(m, 1 H) 3.75-3.90 (m, 3 H) 3.93 (dd, 1 H, J = 5.9, 16.8 Hz) 4.10

(m, 1 H) 4.50 (dd, 1 H, J = 8.0, 13.9 Hz) 4.80 (d, 1 H, J = 6.6 Hz) 5.30 (m, 1 H) 5.41 (d, 1 H₁ J - 6.6 Hz) 5.55 (d, 1 H, J = 7.0 Hz) 6.82 (d, 1 H, J = 7.3 Hz) 7.30 (m, 5 H)

7.50 (m, 1 H) 7.80 (d, 1 H, J = 6.7 Hz).

LC/MS: Tr = 5.26 min (gradient A (acetonitrile)/B (water with 0.1 % TFA): from 5%

A/B to 95 % A/B in 10 min.) MS: C₃₆H₅₃N₅O₉: 700 (M+H)⁺.

HRMS: caicd: 700.3922; found: 700.3925. Preparative Example 16

Step A

A solution of aldehyde 14o (590 mg, 1.15 mmol) in CH₂CI₂ (10 mL) was treated with EtβN (240 mg, 2.4 mmol) and acetone cyanohydrin (240 mg, 2.82 mmol). The reaction mixture was stirred at rt for 2 h and concentrated in vacuo. The residue was purified by chromatography (SiO₂, acetone/hexanes 1 :4) to yield 16a (600 mg) as a colorless solid.

MS (ESI), m/z, relative intensity 569 [(M+Na)⁺, 20], 547 [(M+1)⁺, 40], 447 (100). Step B

A solution of cyanohydrin 16a (600 mg, 1.1 mmol) in DMSO (10 mL) was treated with H₂O₂ (35%, 1.5 mL) and K₂CO₃ (252 mg, 1.83 mmol) and stirred at rt. for 15 h. The reaction mixture was diluted with CH₂Ci₂ (200 mL) and washed with aq. Na₂S₂Oa solution (10%, 50 mL) and brine (30 mL). The reaction mixture was dried (MgSO₄) filtered concentrated in vacuo and directly used in oxidation without further purification.

A solution of hydroxy amide in toluene/DMSO (2:1, 15 mL) was treated with EDCI (1.9 g, 10.00 mmol) and CI₂CHCOOH (317 mg, 2.49 mmol) and stirred at 0 ⁰C for 3 h. The reaction mixture was diluted with CH2CI2 (300 mL) and washed with satd. aq. NaHCO₃ (2x100 mL) and brine (100 mL). The organic layer was dried (MgSO₄), concentrated and purified by chromatography (SiO₂, acetone/hexanes 1 :5) to yield 16 as colorless solid.

MS (ESl), m/z, relative intensity 617 [(M+CH₃OH+Na)⁺, 20], 595 [(M+CH₃OH+1)⁺, 40],

507 [(M+1)\ 20], 463 (100).

Preparative Example 17

Step A

A solution of 16 (300 mg 0.54 mmo!) in HCOOH (10.0 mL) was stirred at rt for 2 h and concentrated in vacuo. The residue was dried in vacuo and used in further reactions without further purification. Step B

A solution of 17a (100 mg) in DMF/CH₂CI₂ (1 :1 , 3 mL) was treated with *^" BuNCO (50 DL and NMM (52 mg, 0.52 mmol). The reaction mixture was stirred at rt for 16 h and concentrated in vacuo, and diluted with CH₂Cb (60 mL) and washed with aq. HCi (1 M, 2x30 ml_), dried, concentrated in vacuo. The residue was purified by chromatography (SiO₂, acetone/hexanes 1:2) to yield 17 (34 mg) as colorless solid. MS (ESI), m/z, relative intensity 584 [(M+1)⁺, 30], 463 (100). Preparative Example 18

Step A

A solution of 17a (100 mg) in DMF/CH₂CI₂ (1 :1 , 3 ml_) was treated with isocyanate of tertbutylester of tert-butylglycine (100 mg, 0.46 mmol) and NMM (52 mg, 0.52 mmol). The reaction mixture was stirred at rt for 16 h and concentrated in vacuo, and diluted with CH₂CI₂ (60 mL) and washed with aq. HCI (1 M, 2x30 mL), dried, concentrated in vacuo. The residue was purified by chromatography (StO₂, acetone/hexanes 1:2) to yield 18 (42 mg) as colorless solid. IVIS (ESI), m/z, relative intensity 698 [(M+Na)⁺, 40], 676 [(M+1 )⁺, 100], 463 (20). Preparative Example 19

Step A

A solution of 17a (100 mg) in DMF/CH₂CI₂ (1 :1 , 3 ml_) was treated with isocyanate of α-methyl-cyclohexyiamine (100 DL) and NMM (52 mg, 0.52 mmol). The reaction mixture was stirred at rt for 16 h and concentrated in vacuo, and diluted with CH₂CI₂ (60 mL) and washed with aq. HCI (1M, 2x30 mL), dried, concentrated in vacuo. The residue was purified by chromatography (SiO₂, acetone/hexanes 1 :2) to yield 20 (21 mg) as coiorless solid.

MS (ESI), m/z, relative intensity 624 [(M+Na)⁺, 30], 602 [(M+1 )⁺, 15], 463 (100), 449 (20), 129 (30). Preparative Example 20

20

Step A

A solution of acyclic diene 20a (6.00 g, 10.954 mmol) in dry toluene (500 mL), degassed with Argon for 0.5 h, was treated with Grubbs catalyst (1.35 g, 1.643 mmot) and heated at 60° C for 12 h. The reaction mixture was concentrated in vacuo and purified by chromatography (S1O2, EtOAc/hexanes 1:3) to yield 20b as a brown foam. Step B

20b 20c

A solution of alkene 20b (5.0Og mg, 0.865 mmol) in methanol (100 ml_) was treated with Pd/C (1.2g, 5% w/w) and hydrogenated at 50 psi for 3 h. The reaction was filtered through a plug of celite and concentrated in vacuo. The residue was purified by chromatography using THF/hexanes gradient from 10-40% to isolated 20c (3.00 g) as a colorless solid.

Step C

20c 2Od

A solution of ester 20c (3.00 g, 5.75 mmol) in dry THF (50 mL) was treated with IJBH4 (2M soln in THF, 3.5 mL, 6.90 mmol) and stirred at rt for 3 h. The reaction was followed by TLC ( EtOAc/Hexanes 1 :2). The reaction was quenched with methanol (2 mL) and diluted with aq. HCI (1 M, 30 mL) and extracted into CH2CI2 (3x100 mL). The combined organic layers were washed with aq. saturated NaHCθ3 (30 mL), brine, dried (MgSθ4), filtered concentrated in vacuo and purified by chromatography (Siθ2, Acetone/Hexanes 1 :2) to yield 2Od (2.21 g) as colorless soiid. MS (m/z, relative intensity) 518 [(M+K)⁺, 15], 480 [(M+H)⁺, 75], 380(100). Step D

2Od 2Oe A solution of alcohol 2Od (2.2 g, 4.58 mmol) in dry CH2CI2 (50 mL) was treated with Dess-Martin reagent (2.91 g, 6.880 mmol) and stirred at rt for 2 h. The reaction mixture was diluted with aq. Na2δ2θ3 (5%, 50 mL) and aq. saturated NaHCO3 (50 mL) and stirred at rt. for 15 min. The reaction mixture was extracted with CH2CI2 (500 mL) and the combined organic layers were dried (MgSO4), filtered, concentrated in vacuo to yield crude 2Oe (1.9 g) that was used in the next reaction without further purification.

Step E

2Oe 2Of

A solution of crude 20e_(1.00 g, 2.094 mmo!) in CH2CI2 (15 ml) was cooled to 0° C and treated with acetone cyanohydriπ (356 mg, 4.187 mmol) and triethylamine (424 mg, 4.187 mmoi). The reaction mixture was stirred at 0° C for 12 h and concentrated in vacuo. The residue was purified by chromatography (Siθ2, EtOAc/Hexanes 1 :5— >1:1) to yield 2Of (500 mg) as a colorless oil. Step F

2Of 2Og

A solution of cyanohydrin 20f_(500 mg, ~1.00 mmoi) in DMSO (5 mL) was treated with H2O2 (5 mL), K2CO3 (276 mg, 2.00 mmoi) and stirred at rt. for 12 h. The reaction mixture was diluted with aq. Na2S2θ3 (5%, 100 mL) and extracted with CH2CI2 (2x100 mL). The combined organic layers were dried (MgSO4), filtered, concentrated in vacuo to yield 20g_ that was used as it is for further oxidation without purification. Step G:

2Og 2Oh

A solution of hydroxylamine 20g_(850 mg, 1.626 mmol) in toluene (5 mL) and DMSO (5 mL) was treated with EDCI (3.117 g, 16.26 mmol), and dichloroacetic acid ( 1.048 g, 8.13HTImOl₁ 698μL) and stirred at rt. for 3 h. The reaction mixture was diluted with CH2CI2 {200 mL) and washed with aq. saturated NaHCO3 (200 mL), aq. HCI (1 M.200 mL), brine (30 mL), dried (MgSθ4) filtered, concentrated in vacuo and purified by chromatography (Siθ2, acetone/Hexanes 1 :2) to yield 2Oh (300 mg) as a colorless solid. Step H:

2Oh 2Oi

A solution of Boc protected ketoamide 2Oh in formic acid (5 mL) was stirred at rt for 3 h and concentrated in vacuo and used as it is in the next step without further purification.

2Oi 20

A solution of amine 2Oi (40 mg, 0.1 mmol) in methylene chloride (3.0 mL) was treated with NMM (30 mg, 0.3 mmoi) and cooled to 0 ⁰C. A solution of isocyanate in CH₂Cb was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (60 mL) and washed with aq. HCI (1 M, 30 ml_). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 20->50%) to yield 20_as a colorless solid. MS {m/z, relative intensity) 588 [(M+H)⁺, 100], 421 (40). HRMS (ESl)

Caicd. for C31 H50N5O6: 588.3761 (M+H)⁺; Found: 588.3751. Preparative Example 21 :

2Oi 21

A solution of amine 2Oi (40 mg, 0.1 mmoi) in methylene chloride (3.0 mL) was treated with NMM (30 mg, 0.3 mmol) and cooled to 0 ⁰C. A solution of 2-cyclohexyl-1 - cyciopropyl-2-isocyanato ethanone (0.15 mmol) in CH2CI₂ was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (60 mL) and washed with aq. HCI (1 M, 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 20 -> 50%) to yield 21 as colorless solid. Preparative Example 22

22 Step A:

2Oe 22a

A solution of aldehyde 2Oe (100 mg, 0.210 mmol) in methylene chloride (4 ml_) was treated with allyl isocyanide (28.01 mg, 0.411 mmol) and acetic acid and stirred at rt. for 12 h. The reaction was concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 1 :4-^1 :1 ) to obtain 22a (75 mg) as colorless solid. MS (m/z, relative intensity) 605 [(M+H)⁺, 100], 505 (98). Step B:

22a 22b

A solution of 22b (275 mg, 0.454 mmol) in methanol (4 mL), THF(4.0 ml_) and water (4.0 mL) was treated with LiOH-H₂O (22 mg, 0.55 mmol) and stirred at rt. for 2 h. The reaction mixture was diluted with aq. HC! (1 M, 30 mL) and extracted in CH₂CI₂ (2x40 mL). The combined organic layer were dried (MgSO₄), filtered, concentrated in vacuo, and used as it is in next step without further purification. Step C:

22b 22

A solution of alcohol 22b (300 mg, 0.534 mmol) in dry CH2CI2 (15 mL) was treated with Dess-Martin reagent (453 mg, 1.06 mmol) and stirred at rt. for 2 h. The reaction mixture was diluted with aq. Na2S2θ3 (5%, 30 mL) and aq. saturated NaHCO3 (30 mL) and stirred at rt. for 15 min. The reaction mixture was extracted with CH2CI2 (3x50 ml_) and the combined organic layers were dried (MgSθ4), filtered, concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 0:1->1:1) to yield 22 as a colorless solid. MS (m/z, relative intensity) 561 [(M+H)⁺, 100], 461 (99). HRMS

(ESf) Calcd. for C3-[H5θN5θ6: 588.3761 (M+H)⁺; Found: 588.3751. Preparative Example 23

23

Step A:

23a 23b

A solution of amine 23a (900 mg, 3.40 mmol) in CH₂Ci₂ at 0 ⁰C was treated with NMM (511 mg, 5.10 mmol) and methanesuffonyl chioride (585 mg, 5.10 mmol) and stirred at 0 ⁰C for 12 h. The reaction mixture was diluted with CH2CI2 (300 ml_) and washed with excess aq. HCi (1 M, 500 mL). The organic layer was dried (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, Hex/EtOAc 1 :9-^1:1) to yield methylsulfonamide 23b (1.00 g). Step B:

23b 23c A solution methanesulfonamide 23b (1.0 g, 2.9 mmol) in methanol (30 mL) was treated with palladium (200 mg, 10% wt/C) and hydrogenated at 60 psi for 3 h. The reaction mixture was filtered through a plug of celite and the filtrate was concentrated in vacuo. The residue was directly used in further reaction without further purification. A solution of deprotected amine in CH₂CI₂ (10 mL) aq. saturated NaHCO₃ (10 mL) at 0⁰C was treated with phosgene (5 mL, 15% soln. in toluene) and stirred at 0 ⁰C for 2 h. The reaction mixture was diluted with CH₂CI₂ (50 mL) and the organic layer was washed with cold aq NaHCO₃. The organic iayer was dried (MgSO₄) filtered and further diluted with 10 mL toluene, concentrated the methylene chloride iayer and used as a solution of 23c. Step C:

2Oi 23

A soiution of amine 2Oi (40 mg, 0.1 mmol) in methylene chloride (3.0 mL) was treated with NMM (30 mg, 0.3 mmol) and cooled to 0 ⁰C. A soiution of isocyanate 23 in CH₂Cb was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (60 mL) and washed with aq. HC! (1 M, 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 20->50%) to yield 23. MS (m/z, relative intensity) 693 [(M+K)\ 10], 677 [(M+Na)⁺, 20], 655 [(M+H)⁺, 100], 449

(3O)₁ 421 (30); HRMS (ESI) Calcd. for C31 H54Neθ7SNa 677.3672 (M+Na)⁺; Found:

677.3685.

Preparative Example 24

24

Step A:

22 24a A solution of Boc protected ketoamide 22 (220 mg, 0.39 mmoi) in formic acid (5 ml.) was stirred at rt. for 3 h and concentrated in vacuo and used as it is in the next step without further purification. Step B:

24a 24

A solution of amine 24a (40 mg, 0.1 mmol) in methylene chloride (3.0 ml_) was treated with NMM (30 mg, 0.3 mmol) and cooled to 0 ⁰C. A solution of isocyanate in CH₂CI₂ was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (60 mL) and washed with aq. HCI (1 M, 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 20-^50%) to yield 24 (27_mg) MS (m/z, relative intensity) 734 [(M+K)⁺, 10], 695 [(M+H)⁺, 100], 461 (20), 443 (20);

HRMS (FAB) Calcd. for C34H59N6O7S 695.4166 (M+H)⁺; Found: 695.4161. Preparative Example 25

25

Step A:

23a 25a

A solution of amine 23a (900 mg, 3.40 mmol) in CH₂Cl₂ at 0 ⁰C was treated with NMM (511 mg, 5.10 mmol) and thiophene sulfony! chloride (928 mg, 5.10 mmol) and stirred at 0 ⁰C for 12 h. The reaction mixture was diluted with CH2CI2 (300 mL) and washed with excess aq. HCI (1M, 500 ml_). The organic layer was dried (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, Hex/EtOAc 1:9->1 :1) to yield sulfonamide 25a ( 1.00 g) of colorless solid. Step B:

25a 25b

A solution of Cbz-protected compound 25a (1.00 g, 2.118 mmo!) was treated with TFA (30 mL) and dimethylsulfide (7.78 ml_) at 0° C and stirred at rt. for 3 h. The reaction mixture was concentrated in vacuo and diluted with aq. NaOH (100 mL). The amine was extracted with methylene chloride (2x100 mL) and the combined organic layers were dried with (MgSO-t) filtered concentrated in vacuo and to yield 25b (800 mg) that was used in further reaction without purification. MS (m/z, relative intensity) 277 [(M+H)⁺, 100], 190 (50). Step C:

25b 25c

A solution of deprotected amine 25b (800 mg, 2.9 mmol) in CH₂Ci₂ (10 mL) aq. saturated NaHCO₃ (10 mL) at 0° C was treated with phosgene (5 mL, 15% soin. in toluene) and stirred at 0 ⁰C for 2 h. The reaction mixture was diluted with CH₂CI₂ (50 mL) and the organic layer was washed with co!d aq NaHCO₃. The organic layer was dried (IvIgSO₄) filtered and further diluted with 10 mL toluene, concentrated the methylene chloride layer and used as a solution of 25c. Step D:

24a 25

A solution of amine 24a (40 mg, 0.1 mmol) in methylene chloride (3.0 mL) was treated with NMM (30 mg, 0.3 mmol) and cooled to 0 ⁰C. A solution of isocyanate in CH₂Cb was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (60 ml_) and washed with aq. HCI (1 M, 30 ml_). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexaπes 20-> 50%) to yield 25 (39 mq) as a colorless solid. MS (m/z_t relative intensity) 801 [(M+K)⁺, 10], 763 [(M+H)\ 100], 461

(15), 277 (20); HRMS (ESl) Calcd. for C-37H58N6θ7S₂Na 785.3706 (M+Na)⁺; Found:

785.3706.

Preparative Example 26

26

Step A:

2OJ 26

A solution of amine 2Oi (40 mg, 0.1 mmol) in methylene chloride (3.0 ml_) was treated with NMM (30 mg, 0.3 mmol) and cooled to 0 °C. A solution of isocyanate in CH₂Cb was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (60 ml_) and washed with aq. HCI (1 M, 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 20-» 50%) to yield 26 as coloriess solid (31 mg). MS (m/z, relative intensity) 761 [(M+K)*_f 10], 720 [(M+H)⁺,

100], 421 (20); HRMS (ESI) Calcd. for 034^4NeOzS₂Na 745.3393 (M+Na)⁺; Found:

745.3396.

Preparative Example 27

27

Step A:

27a 27b

A solution of acid 27a (100 mg, 0.385 mmol) in toluene (5 mL) was treated with DPPA (116.5 mg, 0.425 mmol) and Et₃N (42.5 mg, 0.425 mmol) and stirred at reflux for 1.5 h. The reaction mixture was diluted with saturated NaHCO₃ (30 mL) and extracted into CH₂CI₂ (2x20 mL). The combined organic layers were washed with aq. NaHCO₃ (30 mL), brine (30 mL), dried (MgSO₄), filtered, concentrated in vacuo, and used as a solution of isocyanate in toiuene. Step B:

24a 27

A solution of amine 24a (40 mg, 0.1 mmol) in methylene chloride (3.0 mL) was treated with NMM (30 mg, 0.3 mmol) and cooled to 0 ⁰C. A solution of isocyanate 27b (3 equiv) in CH₂CI₂ was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (60 mL) and washed with aq. HCI (1 M, 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 20->50%) to yield 27 as a colorless solid. MS (m/z, relative intensity) 720 [(M+H)⁺, 85], 461(100); HRMS

(ESI) Calcd. for C37H6I NsOySNa 742.4189 (M+Na)⁺; Found: 742.4200. Preparative Example 28

28

Step A:

2Oi 28

A solution of amine 2Oi (40 mg, 0.1 mmol) in methylene chloride (3.0 mL) was treated with NMM (30 mg, 0.3 mmol) and cooled to 0 °C. A solution of isocyanate 27b (3.00 equiv) in CH₂CI₂ was added and the reaction mixture was stirred at it for 1.5 h. The reaction mixture was diluted with methylene chloride (60 mL) and washed with aq. HCI (1 M, 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 20-> 60%) to yield 28 (29 mg) as a colorless solid. MS (m/z, relative intensity) 718 [(M+K)⁺, 10], 702 [(M+Na)⁺, 20], 680 [(M+H)⁺, 80], 421 (100); HRMS (ESI) Calcd. for

C34H57N5θ7SNa 702.3876 (M+Na)⁺; Found: 702.3889. Preparative Example 29

29

Step A:

24a 29

A solution of amine 24a (50 mg, 0.1 mmol) in methylene chloride (3.0 ml_) was treated with NMM (30 mg, 0.3 mmol) and cooled to 0 ⁰C. A solution of isocyanate in CH₂Cl₂ was added and the reaction mixture was stirred at rt for 1.5 h. The reaction mixture was diluted with methylene chloride (60 ml_) and washed with aq. HCI (1 M, 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 20 -> 50%) to yield 29 as a colorless solid (41 mg). MS {m/z, relative intensity) 628 [(M+H)⁺, 100], 129 (35). Preparative Example 30

30

Step A:

24a 30

A solution of amine 24a (50 mg, 0.1 mmol) in methylene chloride (3.0 mL) was treated with NMM (30 mg, 0.3 mmol) and cooled to 0 °C. A solution of isocyanate (3.0 equiv.) in CH₂Ci₂ was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (60 mL) and washed with aq. HCi (1 M, 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 20— > 50%) to yield 30 as a colorless solid. MS (m/z, relative intensity) 668 [(M+H)⁺, 100], 169 (50), 128 (80). Preparative Example 31 : Preparation of:

Step A:

BOCHN_N X

31a 31b

A solution of BooGlu-OBn 31a (1.8 g, 5.36 mmol) and amine 1d (1 g, 4.87 mmol) was reacted as in preparative example 1 , step C and purified by siϋca gel chromatography (10% to 25% EtOAc/hexanes) to give 31b (1.28 g).

Step B:

A solution of benzyl ester 31b (1.25 g, 2.56 mmol) was treated with 10% Pd/C in EtOH and hydrogenated (1 atm., rt.) for 12 hours. The reaction mixture was filtered through a piug of ceWte and concentrated under vacuum to give 31c (997 mg) which was used in the next reaction without further purification. Step C

A solution of acid 31c (20.4 g, 48.7 mmol) in THF (300 ml) was cooled to O⁰C and treated with Et₃N (7.47 ml, 53.6 mmol) and ethyl chloroformate (4.89 ml, 51.2 mmol) and stirred for 2 hours. The white precipitate formed was filtered and washed with cold THF. The filtrate was cooled to O⁰C and NaBH₄ (2.39 g, 63.4 mmol) was added. MeOH (20ml) was added dropwise over 1 hour and stirred for an additional 2.5 hours. Solvent was removed under vacuum, CH₂CI₂ added and washed with water, brine and dried over Na₂SO₄. Na₂SO₄ was filtered and solvent removed to dryness. The residue was purified by silica gel chromatography (50% to 90% EtOAc/hexanes) to give 31 d (8.15 g). Step D:

A solution of ester 31 d (8 g, 20.8 mmol) in MeOH (120 ml) and H₂O (24 ml) was treated with LiOH-H₂O (2.62 g, 62.5 mmol) at room temperature for 12 hours. Solvent was removed under vacuum to dryness. CH₂CI₂ was added and stirred for 5 minutes with 1N. HCl (72.9 mmol). CH₂CI₂ layer was separated, washed with brine and dried over Na₂SO₄. Na₂SO₄WaS filtered and solvent was removed to dryness to give white solid 31 e (7.65 g). Step E:

A solution of acid 31 e in anhydrous DMF (75 mi) and anhydrous CH₂CI₂ (75 ml) was cooled to 0 ⁰C and stirred with HOOBt (3.68 g, 22.5 mmol), NMM (6.77 ml, 61.6 mmol) and EDCI (5.11 g, 26.7 mmol) for 5 minutes. H-Lys(Z)-OMe^«HC! (7.13 g, 21.5 mmol) was added and stirred for 3.5 hours at 0 ⁰C. Reaction was held 12 hours at 5 ⁰C after which CH₂Cl₂ was removed, EtOAc added and washed with sat. NaHCO₃, 5% H₃PO₄, Brine and filtered through Na₂SO₄- Solvent was removed under vacuum to dryness to give 31f (12.7g). Step F

A solution of 31f (5.5 g, 8.51 mmo!) was treated with 10% Pd/C in EtOH (100 ml) and hydrogenated (1atm., rt.) for 12 hours. The reaction mixture was filtered through a plug of celite and concentrated under vacuum to give 31g (4.25 g). Step G:

A solution of amine 31 g (4.25 g, 8.3 mmol) in anhydrous CHaCi₂ (750 ml) was stirred with triethylamine (1.5 ml, 10.7 mmol) and 4-nitropheny! chloroformate (2.0 g, 9.96 mmol) at room temperature for 5 hours. Solvent was removed under vacuum to -200 ml, then washed with sat. NaHCO₃, water, 5% H₃PO₄, brine and filtered through Na₂SO₄. Na₂SO₄ was filtered and solvent was removed to give 31 h (5.82 g). Step H:

A solution of 31 h (5.8 g, 8.3 mmoi) in anhydrous THF (600 ml) was treated with 60% NaH (996 mg, 24.9 mmol) at room temperature for 22 hours. Reaction was quenched by adding H₂O (5 ml) then 1N. HCI (50 ml) over 3 minutes. Solvent was removed under vacuum, CH₂Ci₂WaS added and washed with 5% H₃PO₄, Brine and filtered through NajSOφ Na₂SO₄ was filtered, solvent was removed and the residue was chromatographed on silica gel column with 0.25% to 3% MeOH/CH₂Cl₂ to give 31 i (2.86 g, 64 % yield). Step I:

A solution of 31i (613 mg, 1.13 mmol) was reacted as in preparative example 1 , step F and purified by silica gei chromatography (3% to 6% MeOH/CH₂CI₂) to give alcohol 31j (500 mg). Step J:

A solution of alcohol 31 j (480 mg, 0.94 mmol) was reacted as in preparative example 1 , step H and purified by silica gel chromatography (30% to 60% acetone/hexanes) to give aldehyde 31k (383 mg). Step K:

A solution of aldehyde 31j (365 mg, 0.71 mmol) was reacted as in preparative example 22, step A and purified by silica gei chromatography (30% to 50% acetone/hexanes) to give 31k (426 mg). Step L:

A solution of 311 (357 mg, 0.56 mmol) was reacted as in preparative example 22, step B to give 31m (426 mg). Step M:

A solution of 31m (350 mg, 0.59 mmol) was reacted as in preparative example 22, step C and purified by silica gel chromatography (30% to 50% acetone/hexanes) to give 31 (335 mg). MS (ES) m/z relative intensity 492 [(M-BOC+1)⁺, 80]; 592 [(M+1 )⁺, 100]. Calcd. for C₂₉H₄₆N₅O₈ [M+1 f: 592.3346; Found 592.3359. Preparative Example 32: Preparation of:

Step A:

20 e 32a

A solution of aldehyde 2Oe (200 mg, 0.42 mmol) in methylene chloride (10 mL) was treated with cyclopropylmethylisocyanide (66.5 mg, 4.11 mmol) and acetic acid (50 mg, 0.82 mmol) and stirred at rt. for 12 h. The reaction was concentrated in vacuo and residue was purified by chromatography (SiO₂, acetone/hexanes 1 :901 :1) to obtain 32a (230 mg).

MS (ES) m/z relative intensity 641 [(M+Na)⁺, 70]; 619 [(M-M)⁺, 100], 519 (50). Step B:

32a

32

A solution of acetate 32a (230 mg, 0.371 mmol) in methanol (5.0 mL), THF (5.0 mL) and water (5.0 mL) was treated with LiOH-H₂O (25 mg, 0.55 mmol) and stirred at rt. for 1 h. The reaction mixture was diluted with aq. HCI (1 M, 30 mL) and extracted in CH₂Ci₂ (2 x 50 mL). The combined organic layer were dried (MgSO₄), filtered, concentrated in vacuo, and used as it is in next step without further purification. A solution of alcohol in dry CH2CI2 (15 mL) was treated with Dess-Martin reagent (237 mg, 0.558 mmol) and stirred at rt. for 2 h. The reaction mixture was diluted with aq. Na2S2θ3 (5%, 30 mL) and aq. saturated NaHCO3 (30 mL) and stirred at rt. for 15 min. The reaction mixture was extracted with CH2O2 (3x50 mL) and the combined organic layers were dried (MgSO4), filtered, concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 0:1 ->1 :1) to yield 32_as a colorless solid (275 mg) MS (ES) m/z relative intensity 629 [(M+isobutene)⁺, 40], 575 [(M+1)⁺, 100], 475 (90). Similar procedures were used to synthesize compounds: 33 and 34 using cyciopropyS and ethyl isocyanide for Step A: preparative example 32: Preparative Example 35: Preparation of:

Step A

32 ^35a

32 (200 mg, 0.39 mmol) was deprotected by dissolving in formic acid 20 mL and standing for 2 h. The reaction mixture was concentrated in vacuo to yield 35a and used in further reactions without purification. Step B:

35a 35

A solution of amine 35a (70 mg, 0.13 mmol) in methylene chloride (3.0 mL) was treated with NMM (50 mg, 0.5 mmol) and cooled to 0 ⁰C. A solution of isocyanate 25c (1 ml, 0.25 mmoi) in CH₂CI₂ was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (150 mL) and washed with aq. HCI (1 M, 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, EtOAc/CH₂CI₂ 5OD 100%) to yield 35 as a colorless solid. MS (ES) m/z relative intensity 799 [(M+Na)⁺, 60]; 777 [(M+1)⁺, 100], Preparative Example 36: Preparation of: Step A:

³³ 36a

33 (200 mg, 0.39 mmol) was deprotected by dissolving in formic acid 20 mL and standing for 2 h. The reaction mixture was concentrated in vacuo to yield 36a and used in further reactions without purification. Step B:

A solution of amine 36a (70 mg, 0.13 mmol) in methylene chloride (3.0 mL) was treated with NMM (50 mg, 0.5 mmol) and cooled to 0 ⁰C. A solution of isocyanate 25c (1 ml, 0.25 mmol) in CH₂CI₂ was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (150 mL) and washed with aq. HCl (1 M₁ 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, EtOAc/CH₂Cj₂ 0^100%) to yield 36 as a colorless solid. MS (ES) m/z relative intensity 785 [(M+Na)⁺, 50]; 763 [(M+1)⁺, 100]; 593 (60). Preparative Example 37: Preparation of: Step A:

34 37a

34 (200 mg, 0.39 mmol) was deprotected by dissolving in formic acid 20 mL and standing for 2 h. The reaction mixture was concentrated in vacuo to yield 37a and used in further reactions without purification. Step B:

37a 37

A solution of deprotected amine 37a (70 mg, 0.13 mmol) in methylene chloride (3.0 mL) was treated with NMM (50 mg, 0.5 mmol) and cooled to 0 ⁰C. A solution of isocyanate 25c (1 ml, 0.25 mmol) in CH₂CI₂ was added and the reaction mixture was stirred at it for 1.5 h. The reaction mixture was diluted with methylene chloride (150 mL) and washed with aq. HCI (1 M, 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, EtOAc/CH₂Ci₂ 5OD 100%) to yield 37.

MS (ES) m/z relative intensity 773 [(M+Naf, 100]; 751 [(M+1 )⁺, 70]. Preparative Example 38: Preparation of: Step

A solution of deprotected amine 37a (70 mg, 0.13 mmol) in methylene chloride (3.0 ml_) was treated with NMM (50 mg, 0.5 mmol) and cooled to 0 ⁰C. A solution of isocyanate 27b (1.5 ml, 0.25 mmol) in CFhCI₂ was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (150 ml_) and washed with aq. HCI (1 M, 30 ml_). The organic layers were dried with (MgSO4 filtered concentrated in vacuo and purified by chromatography (SiO₂, EtOAc/CH₂C!₂ 500 100%) to yield 35 as colorless solid. MS (ES) m/z relative intensity 730 [(M+Na)⁺, 30]; 708 [(M+1)⁺, 100]; 409 (30). Preparative Example 39: Preparation of:

Step A:

A solution of amine 36a (70 mg, 0.13 mmol) in methylene chloride (3.0 mL) was treated with NMM (50 mg, 0.5 mmol) and cooled to 0 ⁰C. A solution of isocyanate 27b (1 mL, 0.25 mmol) in CH₂CI₂ was added and the reaction mixture was stirred at rt for 1.5 h. The reaction mixture was diluted with methylene chloride (150 mL) and washed with aq. HCI (1 M, 30 mL). The organic layers were dried with (MgSO₄) filtered concentrated In vacuo and purified by chromatography (SiO₂, EtOAcZCH₂CI₂ 5OD 100%) to yield 39. MS (ES) m/z relative intensity 742 [(M+Na)⁺, 70]; 720 [(M+1)⁺, 100]; 461 (40). HRMS Calcd. for C37H62N5O7S [M+1]⁺: 720.4370; Found 720.4350. Preparative Example 40: Preparation of:

Step A:

A solution of amine 32a (70 mg, 0.13 mmol) in methylene chloride (3.0 mL) was treated with NMM (50 mg, 0.5 mmol) and cooled to 0 ⁰C. A solution of isocyanate 27b (1 mL, 0.25 mmol) in CHaCI₂ was added and the reaction mixture was stirred at rt. for 1.5 h. The reaction mixture was diluted with methylene chloride (150 mL) and washed with aq. HCI (1 M, 30 mL). The organic layers were dried with (IvIgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, EtOAc/CH₂CI₂ 5OD

100%) to yield 40.

¹H NMR(dmso, 500 MHz), δ, 8.80 (t, 1 H₁ J=6.0 Hz)₁ 8.37 (d, 1 H, J=9.5 Hz), 6.22 (d, 1

H, J=8.8 Hz), 5.88 (s, 1 H), 5.31 (dt, 1 H, J=2.8 & 9.5 Hz), 4.35 (s, 1 H), 4.28-4.22 (m,

1 H), 3.85 (d, 1 H, J=10 Hz), 3.76 (q, 1 H, J=5.4 Hz), 3.59 (t, 1 H, J=13.5 Hz), 3.41 (d,

1 H, J-13.9 Hz), 3.07-2.95 (m, 2 H), 2.22-2.15 (m, 2 H)₁ 1.69-1.00 (b, 23 H), 1.25 (s, 9

H), 0.99 (s, 3 H), 0.99-0.70 (m, 1 H), 0.88 (s, 3 H)₁ 0.42-0.38 (m, 2 H)₁ 0.21-0.18 (m, 2

H).

¹³C NMR (dmso, 125 MHz) δ, 198.5, 172.1 , 171.3, 162.0, 157.3, 60.5, 60.1 , 54.4,

52.8, 51.5, 47.6, 43.8, 35.4, 35.1 , 34.8, 32.3, 31.6, 31.4, 28.3, 28.0, 27.9, 27.3, 26.9,

26.6, 25.8, 25.6, 24.6, 23.4, 22.4, 21.5, 19.5, 13.7, 11.5. MS (ES) m/z relative intensity

756 [(M+Na)⁺, 45]; 734 [(M+1)⁺, 100]; 475 (20). HRMS cacld. for C38H64N5O7S

[M+1]⁺: 734.4526; Found 734.4535.

Preparative Exampie 41 : Preparation of:

Step A:

22b 41

A solution of intermediate 22b (300 mg, 0.54 mmol) was taken in methanol (25 mL) and treated with 10% Pearlman's catalyst and hydrogenated at 50 psi for 4 h. The reaction mixture was filtered through a plug of ceiite^® and concentrated in vacuo to yield reduced product that was used in further reaction without purification. A solution of reduced alcohol in dry CH2CI2 (5 mL) was treated with Dess-Martin reagent (350 mg, 0.82 mmoi) and stirred at rt. for 2 h. The reaction mixture was diluted with aq. Na2S2θ3 (5%, 30 mL) and aq. saturated NaHCO3 (30 mL) and stirred at rt for 15 min. The reaction mixture was extracted with CH2CI2 (3x75 mL) and the combined organic layers were dried (MgSθ4), filtered, concentrated in vacuo and purified by chromatography (SiO₂, acetone/hexanes 0:1 ->1:1 ) to yield 41 (270 mg) as a colorless solid. Preparative Example 42: Preparation of:

Step

41 42a

41 was deprotected by dissolving in formic acid 20 mL and standing for 2 h. The reaction mixture was concentrated in vacuo to yield 42a and used in further reactions without purification. Step B:

42a 42

A solution of amine 42a (100 mg, 0.196 mmol) in methylene chloride (3.0 mL) was treated with NMM (60 mg, 0.6 mmol) and cooled to 0 ⁰C. A solution of isocyanate 25c (1.5 mL, 0.25 mmol, 0.38 mmoi) in toluene was added and the reaction mixture was stirred at rt. for 2 h. The reaction mixture was diluted with methylene chloride (100 mL) and washed with aq. HCI (1 M, 50 mL). The organic layers were dried with (MgSO₄) filtered, concentrated in vacuo and purified by chromatography (SiO₂, Ethyl acetate/hexanes 1 :1 D 1 :0) yield 42 (65 mg) as a, colorless solid. ¹H NMR (dmso, 500 MHz), δ, 8.71 (t, 1 H, J=6.3 Hz), 8.36 (d, 1 H, J=9 Hz), 8.00 (dd, 1 H, Jt=1.3 & 5.0 Hz), 7.65 (dd, 1 H, J=1.3 & 2.5 Hz), 7.25 (dd, 1 H₁ J= 3.8 &1.3 Hz), 6.15 (d, 1 H, J=9.0 Hz), 5.88 (d, 1 H, J=10 Hz), 5.31 (m, 1 H), 4.34 (s, 1 H), 4.30 (m, 1 H), 3.93 (d, 1 H, J=10.5 Hz), 3.79-3.75 (q, 1 H, J=5.0 Hz), 3.67-3.62 (dt, 1 H, J= 4.1 & 5.6 Hz), 3.12-3.05 (m, 2 H), 2.95-2.91 (m, 2 H), 2.67 (s, 3 H), 1.70-1.61 (m, 2 H) 1.40-1.00 (b, 20 H), 0.99 (s, 3 H), 0.85 (s, 3 H), 0.83 (s, 9 H), 0.83 (t, 3 H)-¹³C NMR (dmso, 125 MHz) D, 198.5, 172.0, 171.7, 162.2, 158.3, 137.7, 133.9, 133.1 , 129.0, 60.5, 55.8, 55.7, 52.7, 51.6, 51.5, 47.6, 36.0, 35.0, 32.2, 31.6, 31.3, 28.5, 27.9, 27.4, 27.1, 26.9, 26.7, 26.3, 24.4, 22.8, 22.3, 19.5, 13.7, 12.1. MS (ES) m/z relative intensity 788 [(M+Na)⁺, 50]; 765 [(M+1 )⁺, 100]. Preparative Example 43: Preparation of:

Step

42a

A solution of amine 42a (100 mg, 0.196 mmol) in methylene chloride (3.0 mL) was treated with NMM (60 mg, 0.6 mmol) and cooled to 0 °C. A solution of isocyanate 27b (3 mL, 0.1 M soln., 0.3 mmol) in toluene was added and the reaction mixture was stirred at rt. for 2 h. The reaction mixture was diluted with methylene chloride (100 mL) and washed with aq. HCI (1 M, 50 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, EtOAc/Hexanes 1:1-» 1 :0) yield 43 (42 mg) as a colorless solid.¹H NMR (dmso, 500 MHz) δ, 8.71 (t, 1 H, J=6.0 Hz), 8.36 (d, 1 H, J=9.0 Hz), 6.22 (d, 1 H, J=8.5 Hz), 5.88 (s, 1 H), 5.29 (dt, 1 H, J=9.5 & 2.5 Hz), 4.34 (s, 1 H), 4.23 (t, 1 H₁ J=9.0 Hz)₁ 3.86 (d, 1 H, J=10.5 Hz), 3.76 (dd, 1 H, J=5.0 & 5.5 Hz), 3.60 (d, 1 H, J=13.5 Hz), 3.41 (d, 1 H₁ ./=13.5 Hz), 3.13-3.04 (m, 2 H), 2.23-2.15 (m, 2 H), 1.67-0.9 (bm, 30 H), 1.25 (s, 9 H), 0.99 (s, 3 H), 0.88 (s, 3 H), 0.83 (t, 3 H, J=7.0 Hz). ¹³C NMR (dmso, 125 MHz) δ, 198.5, 172.1 , 171.3, 162.1 , 157.3, 60.5, 60.1 , 55.8, 54.3, 52.8, 51.0, 47.6, 35.4, 35.1, 32.3, 31.7, 31.3, 28.3, 28.0, 27.9, 27.3, 26.9, 26.6, 26.2, 25.8, 24.6, 23.3, 22.8, 21.5, 19.5, 13.7, 12.2. MS (ES) m/z relative intensity 744 [(M+Na)⁺, 40]; 722 [(M+1)⁺, 100]. Preparative Example 44: Preparation of:

Step A:

A solution of deprotected amine 44a (Busacca, C. A.; Grossbach, D.; Spinelli, E. Tetrahedron: Asymmetry; 2000, 9, 1907) in CH₂Cb (10 ml_) aq. saturated NaHCO₃ (10 rriL) at 0° C was treated with phosgene (5 mL, 15% soln. in toluene) and stirred at 0 ⁰C for 2 h. The reaction mixture was diluted with CH2CI₂ (50 mL) and the organic layer was washed with cold aq NaHCO₃. The organic layer was dried (MgSO₄) filtered and further diluted with 10 mL toluene, concentrated the methylene chloride layer and used as a solution Step B:

A solution of amine 24a (100 mg, 0.196 mmol) in methylene chloride (3.0 ml_) was treated with NMM (60 mg, 0.6 mmol) and cooled to 0 ⁰C. A solution of isocyanate 44b (2.5 ml_, 0.25 mmol,) in toluene was added and the reaction mixture was stirred at rt. for 2 h. The reaction mixture was diluted with methylene chloride (100 ml_) and washed with aq. HCI (1 M₁ 50 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, ethyl acetate/hexanes 1 :1 D 1 :0) yield 44 (31 mg) as a colorless solid. MS (ES) m/z relative intensity 755 [(M+Na)⁺, 40]; 733 [(M+1)⁺, 100]. Preparative Example 45: Preparation of:

Step A:

A solution of amine 45a* (2.00 g, 9.20 mmo!) in CH₂CI₂ at 0 ⁰C was treated with (C₂H₅)₃N (3.7 g, 37 mmol) and 2-pyridtnesulfonyl chloride (2.4 g, 11.2 and stirred at rt. for 12 h. The reaction mixture was diluted with CH2CI2 (300 mL) and washed with excess aq. NaHCO3 (1 M, 500 mL). The organic layer was dried (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, Acetone/Hexanes 0:1 -H :1) to yield sulfonamide (2.3 g). A solution of Boc-protected amine was deprotected by dissolving (2.1 g, 5.7 mmol) in 4M soln. of HCI in dioxane and stirred at rt. for 2 h. The reaction mixture was concentrated in vacuo and used as it is in next step without further purification.

* obtained by the protection of terf-leucine-N-methylamide (TCI-Jpn) with ditertbutyldicarbonate and subsequent reduction with BH₃OMS in THF (reflux, 2 h). Step B:

A solution of amine 45b (300 mg, 1 mmol) in CH₂Cb (3 ml_) aq. saturated NaHCθ₃ (3 ml_) at 0⁰C was treated with phosgene (2.5 mL, 15% soln. in toluene) and stirred at 0 ⁰C for 2 h. The reaction mixture was diluted with CH₂Ck (30 mL) and the organic layer was washed with cold aq NaHCO₃. The organic layer was dried (MgSO₄) filtered and further diluted with 3 mL toluene, concentrated the methylene chloride layer and used as a solution. Step C.

A solution of amine 24a (100 mg, 0.197 mmol) in methylene chloride (3.0 mL) was treated with NMM (60 mg, 0.6 mmol) and cooled to 0 ⁰C. A solution of isocyanate 45c (2.5 mL, 0.25 mmol,) in toluene was added and the reaction mixture was stirred at rt. for 2 h. The reaction mixture was diluted with methylene chloride (100 mL) and washed with aq. HCl (1 M, 50 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, ethyl acetate/hexanes 1 :1 -> 1 :0) yield product 45 as a colorless solid. The crude mixture was further purified using HPLC to yield pure product 45 (27 mg). ¹H NMR (dmso, 500 MHz) δ 8.89 (t, 1 H, J=7.0 Hz)₁ 8.72 (d, 1 H, J=6.0 Hz), 8.37 (d, 1 H, J=10.5 Hz), 8.07 (t, 1 H₁ J=9.0 Hz), 7.88 (d, 1 H, J=9.0 Hz)₁ 7.66 (dd, 1 H₁ J=6.5 &3.5 Hz), 6.12 (d, 1 H₁ J=11 Hz), 5.84-5.75 (m, 2 H), 4.27 (s, 1 H), 4.22 (bt, 1 H₁ J=11.5 Hz), 3.92 (d, 1 H, J=13 Hz), 3.77-3.60 (m, 4 H), 3.33 (bd, 1 H), 3.06 (bt, 1 H, J=12.5 Hz), 2.75 (s, 3 H), 1.68-1.59 (m, 2 H), 1.44-1.12 (m, 18 H), 0.98 (s, 3 H), 0.83 (s, 3 H), 0.78 (s, 9 H). ¹³C NMR (dmso, 125 MHz) δ, 198.3, 172.1 , 171.7, 162.1 , 158.3, 157.1 , 151.0, 139.6, 135.0, 127.9, 123.3, 116.4, 60.5, 55.8, 52.8, 52.2, 51.5, 36.4, 35,0, 28.0, 27.1 , 26.9, 26.3, 19.5, 13.7.MS (ES) m/z relative intensity 780 [(M+Naf, 50]; 758 [(M+1 )⁺, 100]. Preparative Example 46: Preparation of:

Step A:

46a 46b

A solution of (S) -te/t-leucinol (5.0 g, 42.7 mmol, Aldrich) 46a at 0 ⁰C in CH₂CI₂ (100.0 mL) was treated with benzyl chloroformate (6.7 mL, 47.0 mmol), followed by Hunig's base (9.3 mL, 53.3 mmol). The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (500 mL), washed with 10 % KH₂PO₄, followed by saturated NaHCO₃ and brine. The organic layer was dried over MgSO₄ and concentrated to yield protected leucinol (10.7 g, 100%) that was used in further reaction without any purification.

To a sofution of protected leucinol (crude) (10.7 g, 42.7 mmo!) in CH₂CI₂ (10O.O mL) at 0 ⁰C was added pyridine (20.0 mL) and methanesulfonyl chloride (3.63 mL, 47.0 mmo!). The reaction mixture was allowed to warm to room temperature and stirred overnight, concentrated, redissolved in ethyl acetate (500 mL), washed with saturated NaHCO₃ and brine. The organic layer was dried (MgSO₄), concentrated and purified by flash chromatography over SiO₂ using ethyl acetate/hexane (1 :4) to yield 46b (14.O g, 100 %). Step B:

A solution of 46b (3.1g, 9.9 mmo!) in toluene (72 ml_) containing water (400 μL) was treated with (C₄H₉J₄NBr (582 mg, 1.8 mmo!), K₂CO₃( 2.72 g, 1.97 mmol) and 2- hydroxypyridine (937 mg, 9.85 mmol). The reaction mixture was refluxed overnight with stirring, filtered and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography over SiO₂ using ethyl acetate/CH₂CI₂ (1 :9 to 1 :1 ) to yield 46c (1.15 g, 35 %) as a colorless oil. Step C:

A solution of pyridone 46c (1.15 g) in MeOH (50 ml_) was treated with Pd/C ( 10% w/w, 450 mg) and placed in a Parr^® shaker and hydrogenated at 40 psi for 4 h. The reaction mixture was filtered through a plug of celite® and concentrated in vacuo to yield 46d that was used in the next step without further purification. Step D:

A solution of amine 46d (600 mg, 3.03 mmoi) in CH₂CI₂ (10 mL) aq. saturated

NaHCO₃ (10 mL) at 0° C was treated with phosgene (5 mL, 15% soln. in toluene) and stirred at 0° C for 2 h. The reaction mixture was diluted with CH₂Ci₂ (50 mL) and the organic layer was washed with cold aq NaHCO₃. The organic layer was dried (MgSO^ filtered and further diluted with 3 mL toluene, concentrated the methylene chloride layer and used as a solution in toluene. Step E:

A solution of amine 24a (100 mg, 0.197 mmol) in methylene chloride {3.0 mL) was treated with NMM (60 mg, 0.6 mmol) and cooled to 0 °C. A solution of isocyanate 46e (1.5 mL, 0.25 mmol) in toluene was added and the reaction mixture was stirred at rt. for 2 h. The reaction mixture was diluted with methylene chloride (100 mL) and washed with aq. HCl (1 M, 50 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, ethyl acetate/hexanes 1 :10 1 :0) and 100% ethyl acetate to yield 46 (30 mg) as a colorless solid. ¹H NMR (dmso, 500 MHz) δ, 8.92 (t, 1 H, J=6.5 Hz)₁ 8.39 (d, 1 H, J=9.0 Hz), 6.17 (d, 1 H, J=9.0 Hz), 5.81 (m, 1 H), 5.69 (d, 1 H, J=10.5 Hz), 5.29 (bt, 1 H, J=10.0 Hz), 5.13-5.10 (m, 2 H)₁ 4.33 (s, 1H)₁ 4.30-4.26 (m, 1 H)₁ 3.86-3.65 (m, 6 H), 3.50 (bt, 1 H, J=12 Hz), 3.15-3.08 (m, 2 H), 2.21-2.05 (m, 2 H), 1.74-1.54 (bm, 6 H)₁ 1.46-1.11 (bm, 18 H), 0.99 (s, 3 H), 0.84 (s, 3 H), 0.82 (s, 9 H). ¹³C NMR (dmso, 125 MHz) δ, 198.2, 172.1 , 171.3, 169.3, 162.1 , 158.2, 135.0, 116.4, 60.5, 55.8, 55.1 , 52.8, 51.5, 48.3, 47.6, 47.0, 41.7. 34.6, 33.O₁ 32.4, 31.5, 28.3, 28.0, 27.8, 27.2, 26.9, 26.2, 24.5, 23.7, 22.4, 21.9, 19.5, 13.7.

Preparative Example 47: Preparation of:

Step A:

The amine, 47a, (C. A. Busacca etal, Tetrahedron: Asymmetry, (2000) 9 1907)

(1.5 g, 6.9 mmol, 1 equiv.) was dissolved in dry dϊchloromethane (20 ml) and cooled to -78⁰C. Added 3 ml (3 equiv.) of EtgN followed by the slow addition of dimethylsulfamyl chloride (1.5 eq., Sigma-Aldrich) dissolved in DCM. The temperature was kept at -78 ⁰C until the addition is complete and then stirred overnight allowing it to rise to room temperature. Diluted with methylene chloride and washed with water, aq. 1 N HCl and finally brine. The organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. Crude product isolated was purified via flash column (10^30 % EtOAc-Hexane) to afford 1.27g (58%) of 47b. ¹H NMR (CDCI₃, 300 MHz)

6, 4.6 (d, 1 H), 3.45 (m, 1 H₁), 3.25 (d, 1 H), 2.89 (s, 6 H)₁ 1.89 (bs, NH)₁ 1.22 (s, 9H),

0.98 (s, 9 H).

MS (ESI), m/z, relative intensity 324 [(M+1) 85], 268 (100), 224 (50).

Step B:

To the Boc protected sulfonyl urea 47b (440 mg, 1.25 mmol, 1 equiv.) in DMF (10 ml_) at 0° C was added Cs₂CO₃ (613 mg, 1.5 equiv, 1.88 mmol) and MeI (6.36 mmoi, 5 equiv., 0.601 ml_) under inert atmosphere. The reaction mixture was stirred at room temperature for 90 min and quenched with water. The aqueous layers were extracted with EtOAc₁ washed 4 times with water and brine. The organic layers were dried over anhydrous sodium sulfate, filtered and evaporated off the solvent to afford 420 mg (91%) of 47c that was used in the next reaction without further purification. ¹H NMR (CDCI₃, 300 MHz) D.4.59 (d, 1 H)₁ 3.62-3.58 (m, 1 H₁), 3.29-3.22 (m, 1 H)₁ 2.80 (s, 3 H), 2.79 (s, 6H), 1.89 (bs, NH), 1.22 (s, 9 H), 0.98 (s, 9 H). MS (ESI), m/z, relative intensity 338 [(M+1 ) 60], 282 (100), 238 (90). Step C:

To the Boc-protected sulfonyl urea 47c (890 mg, 1 equiv.) was added 4 M solution of HCI in dioxane (25 ml_) at room temperature and stirred for 1 hr. After the disappearance of starting material (TLC), the reaction mixture was concentrated and azeotroped with hexanes and ether. The residue was triturated with ether and the solid separating out was filtered and dried in vacuum to afford a pale yellow solid (720 mg, -iOO%). It was used in further reaction without purification. Step D:

To the amine hydrochloride salt 47d (720 mg, 2.63 mmol) in dichloromethane (1 5 ml) was added 15 ml of aq. saturated NaHCO₃ and stirred vigorously at 0 ⁰C for 5 min. A solution of phosgene (2 equiv. 20% in toluene) was syringed out to the lower layer and restored the vigorous stirring immediately. Checked the TLC at times and after 2 hrs, it showed complete consumption of starting material. The methylene chloride layer was separated and the aqueous layer was extracted with dichloromethane (30 ml). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated using rotary evaporator under reduced pressure at it to half the volume and then flushed N₂ for 15 minutes. Diluted the soiution to 130 ml_ with dichSoromethane and used as 0.02 M solution in further reactions. Step E:

24a 47

A solution of amine 24a (100 mg, 0.197 mmol) in methylene chloride (3.0 ml_) was treated with NMM (60 mg, 0.6 mmol) and cooled to 0 ⁰C. A solution of isocyanate 47e (1.5 mL, 0.25 mmol,) in toluene was added and the reaction mixture was stirred at rt for 2 h. The reaction mixture was diluted with methylene chloride (100 mL) and washed with aq. HCI (1 M, 50 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, ethy! acetate/hexanes 1 :10 1 :0) and 100% ethy! acetate to yield 47 (49 mg) as a colorless solid.

¹H NMR (dmso, 500 MHz) δ, 8.89 (t, 1 H, J=6 Hz), 8.37 (d, 1 H, J-9.0 Hz), 6.15 (d, 1 H, J=9.0 Hz) 5.83-5.76 (m, 2 H), 5.31-5.27 (m, 2 H), 4.33 (s, 1 H), 4.30-4.28 (m, 1 H), 3.91 (d, 1 H, J=10.5 Hz), 3.80-3.70 (m, 4 H), 3.63-3.59 (m, 1 H)₁ 2.93 (dd, 1 H), 2.7 (s, 3 H), 2.69 (s, 6 H), 1.73-1.65 (m, 2 H), 1.51-1.02 (m, 18 H), 0.99 (s, 3 H), 0.84 (s, 3 H)₁ 0.81 (m, 9 H) ¹³C NMR (dmso, 125 MHz) δ, 198.3, 172.1 , 171.7, 162.1 , 158.2, 135.0, 116.5, 60.5, 55.8, 52.8, 51.7, 1.3, 47.6, 41.1 , 38.5, 36.0, 34.9, 32.3, 31.6, 31.3, 28.5, 28.4, 27.9, 27.4, 27.4, 27.1.MS (ES) m/z refative intensity 746 [(M+Na)⁺, 4Oj;

724 [(M+1 )^*, 100].

Preparative Example 48: Preparation of:

Step A:

48a 43b

Compound 48b was prepared from 48a and 2-carbomethoxy-3- thiophenesulfonyl chloride according to the procedures described for the preparation of compound 45b. Step B:

4Sb 48c

To the solution of ester 48b (4.65 g, 11.1 mmol) in anhydrous toluene (40 mL) at -78 ⁰C was added a solution of DIBAL-H in toluene (23.0 mL, 34.5 mmol). The mixture was stirred at -78 ⁰C for 20 min and at rt. for 2 h. Methanol (20 mL) was added followed by 10% aqueous citric acid solution (100 mL). After stirred for 5 min, EtOAc (200 mL) was added and layers were separated. The aqueous solution was extracted with EtOAc (2 x100mL). The organic solutions were combined, dried (MgSO4), filtered and concentrated. The residue was purified by flash column chromatography using 10-50% acetone/hexanes to give 4.6 g (quant.) of 48c. Step C:

To a solution of 48c (1.04 g, 2.65 mmol) in CH₂CI₂ 1 (50 mL) at 0 ⁰C was added methanesulfonyl chloride (0.23 mL, 2.97 mmol) and triethylamine (0.80 mL, 5.74 mmol). The mixture was warmed to rt along with ice bath and stirred for 18 h. EtOAc (200 mL) and 5% H₃PO4 solution (100 mL) was added and the layers were separated. The organic solutions were washed with 1 N sodium carbonate solution (100 mL) before it was dried (MgSO4), filtered and concentrated- The residue was purified by fiash column chromatography using 10-50% acetone/hexanes to give 0.80 g (73%) of 48d. Step D:

A suspension of 48d (1.17 g, 2.85 mmol) and cesium carbonate (1.40 g, 4.30 mmoi) in anhydrous DMF (100 mL) was stirred at rt. for 18 h. Water (50 mL), brine (50 mL) and EtOAc (300 mL) were added and the layers were separated. The organic solution was washed water (3 x 150 mL) before it was dried, filtered and concentrated to give 0.99 g of the desired product 48e (93%). Step E:

48e 48f

Compound 48f was prepared from 48e according to the procedures described for the preparation of compounds 45b and 45c. Step F:

A solution of amine 24a (100 mg, 0.197 mmol) in methylene chioride (3.0 mL) was treated with NMM (60 mg, 0.6 mmol) and cooled to 0 ⁰C. A solution of isocyanate 48f (2 mL, 0.25 mmol,) in toluene was added and the reaction mixture was stirred at rt. for 2 h. The reaction mixture was diluted with methylene chloride (100 mL) and washed with aq. HCl (1 M, 50 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, ethyl acetate/hexanes 1:1->1:0) and 100% ethyl acetate to yield 48 as a colorless solid. Preparative Example 49: Preparation of:

Step A:

COOCH₃

49a 49b

A solution of 2 M LDA / THF-heptane (Acros Chemical Co.) in 50 mL of THF was cooled to -70° C, methyl cyclohexanecarboxylate 49a was added drop wise at < - 60° C. After an additional 0.5 hr stirring at -70 ⁰C, 2-picoiyl chloride in 40 mL ether was added drop wise at < -60° C. The temperature was then allowed to rise slowly to room temperature over 2 hr, and stirred an additional 2 hr. The reaction was quenched in a cold mixture of 200 mL 20% aqueous KH2PO4 and 5 mL of 12 N HCI, the mixture was extracted with EtOAc, the extract was washed with brine, and then dried with MgSO4. The mixture was filtered, the filtrate was evaporated, the residue was evaporated twice from xylene, and the final residue was chromatographed on silica gel (1 :3 Et2O-CH2Cl2 to 1 :1 acetone-CH2Cl2) to obtain 49b. Step B:

49b 49c

A solution of ester 49b in 20 mL of dioxane was treated with 30 mL of 1 N aqueous LiOH, and the mixture was stirred at 100° C for 6 hr. The mixture was quenched in ice-water, extracted with ether, and the cold aqueous was slowly acidified to pH ~4 with 3 N HCI. The precipitate was filtered, washed with water, and dried to leave product acid that was used in the following step without further purification. The conversion of the acid to the isocyanate 49c was identical to the synthesis of 27b in preparative example 27. Step C:

A solution of amine 24a (100 mg, 0.197 mmol) in methylene chloride (3.0 mL) was treated with NMM (60 mg, 0.6 mmol) and cooled to 0 ⁰C. A solution of isocyanate 49c (2.9 mL, 0.25 mmol,) in toluene was added and the reaction mixture was stirred at rt. for 2 h. The reaction mixture was diluted with methylene chloride (100 mL) and washed with aq. HCI (1 M, 50 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂, ethyl acetate/hexanes 1:10 1:0) and 100% ethyl acetate to yield 49 as a colorless solid. Preparative Example 50: Preparation of:

Step A:

50b

A solution of pyrazinecarboxyiic acid 50a (Aldrich, 3 g) in 150 ml_ of dry dichloromethane and 150 ml_ of dry DMF was stirred at 0 ⁰C and treated with HATU (1.4 eq, 6.03 g). L-cyclohexyiglycine-methyl ester hydrochloride (1.2 eq, 6.03 g) was added in small portions. Then, N-methylmorpholine (4 eq, 10 mL, d 0.920) was added dropwise. The reaction mixture was gradually warmed to room temperature and stirred for 20 h. All the volatiles were removed under vacuum and the residue was dissolved in 500 mL of ethyl acetate. The organic layer was washed with water (100 mL), aq. 1 N HCI (100 mL), aq. saturated sodium bicarbonate solution (100 mL), and brine (100 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 5:95 to 3:7) to afford the product 50b (6.5 g, 95%) as a white solid. Step B:

A solution of methyl ester 50b (6.5 g) in 270 mL of a 1 :1 :1 mixture of THF/MeOH/H₂O was cooled to 0 ⁰C and treated with lithium hydroxide monohydrate (2.5 eq, 2.45 g). The mixture was stirred and monitored by TLC (acetone/hexanes; 2:8). When ail the starting material had been consumed, the reaction mixture was treated with 100 mL of aq 1N HCI and the mixture was concentrated in vacuo. Dichforomethane (250 mL) was added and layers separated. The aqueous layer was extracted with dichloromethane (3 x 80 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated to afford acid 50c. Step C:

A solution of acid 24a (100 mg, 0.197 mmol) in dry CH2CI2 (2 mL) and DMF (2 mL) was cooled to 0° C and treated with acid 50c (51.8 mg, 0.197 mmol), HATU (94 mg, 0.25 mmol) and NMM (45 mg, 0.45 mmol). The reaction was stirred at 0° C for 12 h and concentrated in vacuo. The residue was dissolved in CH2CJ2 (100 mL) and washed with aq. HCI (1 M, 2x 30 mL), aq. saturated NaHCO3 (2x30mL), brine (30 mL), dried (MgSO4), filtered, concentrated in vacuo. The crude dipeptide was purified by chromatography (Siθ2, acetone/Hexanes 0:1 ->1 :1 ) to yield 50. ¹H NMR (dmso, 400 MHz) δ, 9.16 (δ, 1 H, J=12 Hz), 8.89 (d, 1 H, J=2.4 Hz), 8.74 (s, 1 H), 8.59 (d, 1 H, J=7.4 Hz), 8.43-8.38 (m, 2 H), 5.81-5.75 (m, 1 H), 5.28 (t, 1 H,

Hz), 5.11-5.03 (m, 2 H), 4.45-4.31 (m, 3 H), 3.88-3.70 (m, 5 H), 1.65-1.22 (m, 31 H), 0.97 (s, 3 H)₁ 0.83 (s, 3 H). MS (ES) m/z relative intensity 728 [(M+Na)⁺, 4]; 706 [(M-H)⁺, 80]. Preparative Example 51 : Preparation of:

Step A:

51a 51b

A solution of the alcohol 51a (1.00 g, 4.6 mmol) in anhydrous CH2CI2 (30 mL) in an inert atmosphere was treated with triphenylphosphine (1.52 g, 5.75 mmol) and dimethylglutarlmide (780 mg, 5.52 mmol). The reaction mixture was cooled to 0 ⁰C and treated with DIAD (930 mg, 4.60 rnmol, in 4 mL CH₂CI₂) dropwise and warmed to rt. It was stirred at rt. for 5 h and concentrated in vacuo. The residue was purified by chromatography (SiO₂, Hexanes/acetone 1:0-^1:1) to obtained 51b (600 mg) as a colorless solid.

Step B:

A solution of 51b (500 mg, 1.5 mmol) in HCI (15 mL, 4M soln. in dioxane) was stirred at rt. for 1 h and concentrated in vacuo. The residue was used in further reaction without purification. A solution of the deprotected amine in CH₂CI₂ (10 mL) aq. saturated NaHCO₃ (10 mL) at 0° C was treated with phosgene (5 mL, 15% soln. in toluene) and stirred at 0° C for 2 h. The reaction mixture was diluted with CH₂CI₂ (50 mL) and the organic layer was washed with cold aq. NaHCO₃. The organic layer was dried (MgSO₄) filtered and further diluted with 3 mL toluene, concentrated the methylene chloride layer and used as a solution. Step C:

A solution of amine 24a (100 mg, 0.196 mmol) in methylene chloride (3.0 mL) was treated with NMM (60 mg, 0.6 mmol) and cooled to 0° C. A solution of isocyanate 51c (2 mL, 0.5 mmol,) in toluene was added and the reaction mixture was stirred at rt. for 2 h. The reaction mixture was diluted with methylene chloride (100 mL) and washed with aq. HCI (1 M, 50 mL). The organic layers were dried with (MgSO₄) filtered concentrated in vacuo and purified by chromatography (SiO₂ Acetone/hexanes 0:1 ->1 :1) yield 51 as a colorless solid.¹H NMR (dmso, 500 MHz) δ, 8.91 (d, 1 H)₁ 6.19 (d, 1 H₁ J=8.5 Hz), 5.84-5.57 (m, 1 H), 5.58 (d, 1 H, J=10.5 Hz), 5.28 (t, 1 H, J=7.0 Hz), 5.10-5.05 (m, 2 H), 4.31 (s, 1 H), 4.18 (t, 1 H, J=8.5 Hz)₁ 3.83-3.57 (m, 7 H), 2.44- 2.38 (AB, 4 H), 1.66-1.62 (m, 2 H), 1.44-1.03 (m, 18 H), 0.98 & 0.96 (2s, 9 H), 0.84 & 0.81 (2s, 12 H). ¹³C NMR (dmso, 125 MHz) δ.198.2, 172.7, 172.1 , 171.3, 162.1 , 158.1 , 135.0, 116.4, 60.5, 55.5, 52.9, 51.3, 47.5, 46.4, 41.7, 39.6, 35.0, 32.4, 31.5, 31.3, 29.3, 28.3, 27.9, 27.0, 26.9, 26.6, 26.1, 24.5, 22.4, 19.5, 13.7.MS (ES) m/z relative intensity 749 [(M+Na)^*, 20]; 727 [(M+1 )⁺,.100]. Preparative Example 52: Preparation of:

Step A:

A solution of N-Boc-L-Ser-OMe (3.6 g, Aldrich) in 150 mL of dry THF was degassed (vacuum/N2-flush) and treated with allylmethyl carbonate (1.4 eq, 2.6 mL, d 1.022). A catalytic amount of tetrakis(triphenylphosphine)paliadium (0.02 mol%, 379 mg) was added. The slightly yellow mixture was degassed again and heated at 60⁰C for about 3 h until TLC analysis (acetone/hexanes; 2:8) showed no more starting material left (reaction mixture became brown). The THF was removed under reduced pressure and the residue was diluted with 300 mL of ethyl acetate and washed with 80 mL of aqueous saturated sodium bicarbonate solution and 80 mL of brine. The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 5:95 to 2:8) to afford the product 52b as a clear oil (2.7 g, 64%). Step B:

A solution of methyl ester 52b (1.5 g) in 90 ml_ of a mixture of THF/MeOH/H₂O (1 :1 :1) was treated with lithium hydroxide monohydrate (2.5 eq, 630 mg). Reaction was stirred at room temperature and monitored by TLC (acetone/hexanes; 1 :9). After 45 min, all the volatiles were removed under reduced pressure. The residue was partitioned between 80 mL of aqueous 1 N HCI and 200 m!_ of dichloromethane. The aqueous layer was back extracted with dichloromethane (2 x 80 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure to afford the product 52c as a clear oil (1.4 g, 95%). Step C:

A solution of acid 52c (6 mmoi) in 40 mL of dry dichloromethane and 40 mL of dry DMF was stirred at 0 ⁰C and treated with HATU (1.4 eq, 3.2 g). The amine hydrochloride 1d (1.3 eq, 1.6 g) and N-methy!morpholine (4 eq, 2.6 mL, d 0.92O) were successively added. The reaction mixture was gradually warmed to room temperature and stirred overnight. AH the volatiles were removed under vacuum and the residue was taken into 300 mL of ethyl acetate. The organic layer was washed with aqueous 1 N HCl (50 mL), aqueous saturated sodium bicarbonate (50 mL), and brine (50 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 5:95 to 2:8) to afford the desired product 52d (2.23 g, 93%) as a clear oil. Step D:

A solution of methyl ester 52d (2.23 g) in 45 ml_ of a mixture of THF/MeOH/H₂O (1 :1 :1) was treated with lithium hydroxide monohydrate (2.5 eq, 300 mg) at 0 ⁰C. The cooling bath was removed and the reaction mixture was stirred at room temperature and monitored by TLC (acetone/hexanes; 2:8). After 1 h, 10 mL of aq 1 N HCl were added and all the volatiles were removed under reduced pressure. The residue was partitioned between 30 mL of aqueous 1N HCI and 100 mL of dichSoromethane. The aqueous layer was back extracted with dichloromethane (2 x 50 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure to afford the product 52e (1.88 g, 88%) as a clear oiS. Step E:

A solution of acid 52e (830 mg) in 20 mL of dry dichloromethane and 20 mL of dry DMF was stirred at 0 ⁰C and treated with HATU (1.4 eq, 1.15 g). The amine hydrochloride 1f (1.1 eq, 227 mg) was added in 10 mL of dichloromethane followed by N-methylmorphoiine (4 eq, 0.95 mL, d 0.920). The reaction mixture was kept in the freezer (-20 ⁰C) for 48 h. AK the voiatiles were removed under vacuum and the residue was dissolved in 200 mL of ethyl acetate. The organic layer was washed with water (50 mL), aqueous 1 N HCI (50 mL), aqueous saturated sodium bicarbonate solution (50 mL), and brine (50 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 5:95 to 3:7) to afford the product 52f (680 mg) as a white solid along with a minor diastereomeric product (130 mg) for a combined yield of 70%. Step F:

A 0.01 M solution of diene 52f (670 mg) in toluene was degassed for 30 min

(argon bubbling) and treated with Grubb's catalyst (0.2 eq, 205 mg). The pink solution was heated to 60⁰C for 16 h (the solution became dark after heating 10 min). The solvent was removed under reduced pressure and the residue was chromatographed on silica gel (gradient; ethyl acetate/hexanes; 2:8 to 1:1) to afford the alkene product 52g (570 mg, 90%) as a mixture of E- and Z-isomers (approx 4:1). Step G:

A solution of alkene 52g (570 mg) in 20 mL of methanol was treated with palladium dihydroxide on carbon (0.1 mol%, 78 mg of 20% Pd(OH)2/C). The mixture was hydrogenated at 50 psi until all the starting material had been consumed. The reaction mixture was diluted with 100 mL of dichloromethane and filtered thru a short path of celite. The filtrate was concentrated and the residue was chromatographed on silica gel to afford the product 52h (590 mg, 70%) as a clear oil. Step H:

A solution of methyl ester 52h (580 mg) in 20 mL of dry THF was treated with lithium borohydride (2.1 eq, 1.2 mL of a 2M soln in THF). The reaction mixture was stirred at room temperature and monitored by TLC (acetone/hexanes; 3:7) for disappearance of the starting material. After 5 h, the excess lithium borohydride was quenched by addition of aqueous saturated ammonium chloride solution (3 mL). The mixture was partitioned between ethyl acetate (100 mL) and aqueous saturated sodium bicarbonate solution (50 mL). The aqueous layer was back extracted with ethyl acetate (2 x 30 mL) and dichloromethane (2 x 30 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 1 :9 to 5:5) to afford the product 52i (360 mg, 68%) as a white solid. Step I:

A solution of alcohol 52ϊ (350 mg) in 20 mL of dry dichioromethane was treated with Dess-Martin periodinane (3 eq, 925 mg). The reaction mixture was stirred at room temperature for 45 min. The mixture was treated with aqueous 1 M sodium thiosulfate solution (15 mL) and aqueous saturated sodium bicarbonate solution (15 mL) and stirred for 15 min. The mixture was extracted with dichloromethane (3 x 50 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 1:9 to 4:6) to afford the product 52j (285 mg, 83%) as a colorless solid. Step J:

A solution of aldehyde 52j (270 mg) in 10 ml_ of dry dichloromethane was treated with aliylisocyanide (2 eq, 77 mg) and acetic acid (2 eq, 0.064 ml_, d 1.049). The mixture was stirred for about 5 h. All the volatiles were removed under vacuum and the residue was chromatographed on silica gel (gradient: acetone/hexanes; 1 :9 to 1:1) to afford the product 52k (303 mg, 90%) as a white solid. Step K:

The acetate 52k (300 mg) was dissolved in 15 ml of a 1 : 1 :1 mixture of

THF/MeOH/H₂O and treated with lithium hydroxide monohydrate (2.5 eq, 51 mg). The flow of the reaction was followed by TLC (acetone/hexanes; 4:6). After 15 min the reaction mixture was concentrated in the rotavap and the residue was partitioned between dichloromethane (80 imL) and aqueous saturated sodium bicarbonate solution (20 mL). The aqueous layer was back extracted with dichloromethane (3 x 50 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated. The crude product 521 (276 mg, 98%) was used without further purification. Step L:

A solution of hydroxyamide 52I (276 mg) in 20 mL of dry dichloromethane was treated with Dess-Martin periodinane (3 eq, 424 mg). The reaction mixture was stirred at room temperature for 30 min. The mixture was treated with aqueous 1 M sodium thiosulfate solution (20 mL) and aqueous saturated sodium bicarbonate solution (10 mL) and stirred for 10 min. The mixture was extracted with dichloromethane (3 x 20 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 1:9 to 4:6) to afford the product 52 (236 mg, 86%) as a white solid. ¹H NMR (500 MHz, CDCI₃): J₁ 7.47 (d, 1H₁ J = 7.56 Hz), 7.03 (dd, 1 H, J = 5.68, 5.99 Hz), 5.88 (ddt, 1 H, J - 5.6, 10.0, 17.0 Hz), 5.50 (d, 1 H₁ J - 8.83 Hz)₁ 5.46 (m, 1 H), 5.28 (dd, 1H, J = 0.9, 17.0 Hz), 5.25 (dd, 1 H, J = 0.9, 10.0 Hz), 4.61 (m, 1 H), 4.51 (s, 1 H), 3.99 (dt, 2H, J = 1.2, 5.6 Hz), 3.88 (dd, 1 H, J = 5.0, 10.8 Hz)₁ 3.83 (d, 1 H, J = 11 -0 Hz)₁ 3.66 (m, 2H), 3.48 (dd, 2H, J = 4.7, 5.6 Hz), 1.95 (m, 1 H), 1.81 (d, 1 H, J = 7.56 Hz)₁ 1.47 (s, 9H), 1.27-1.63 (m, 12H), 1.09 (s, 3H), 0.93 (s, 3H); ¹³C NMR (CDCI₃, 125 MHz): ό 196.7, 170.9, 170.7, 159.3, 155.6, 133.2, 117.9, 80.4, 71.0, 70.8, 61.2, 54.5, 52.9, 48.2, 42.1 , 31.4, 29.3, 28.7, 27.8, 26.8, 26.6, 26.4, 23.6, 23.4, 19.2, 13.2 ppm; HRMS calcd for C₂₉H₄₇N₄O₇ [M+H]⁺: 563.3445, found 563.3457. Preparative Example 53: Preparation of:

Step A:

The N-Boc protected amine 52 (60 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes and stirred at room temperature for 1 h. Ail the volatiles were removed under reduced pressure and the product was placed under high vacuum for 3 h. No further purification was done for the product 53a (99%). Step B:

The amine salt 53a (31 mg) was dissolved in 5 ml_ of dry dichioromethane and cooled to 0 ⁰C. Then, 10 drops of aqueous saturated sodium bicarbonate solution were added. After 10 min, a solution of the isocyanate 27b (2.5 eq_» 0.8 rnl_ of a 0.2M solution in toluene) was added and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 3 h. The residue was chromatographed on silica gel (gradient: acetone/hexanes,- 1:9 to 4:6) to yield the product 53 (25 mg_τ 58%) as a white solid. ¹H NMR (500 MHz, CDCI3): δ, 7.90 (d, 1 H, J = 8.5 Hz), 7.38 (br s, 1 H), 5.9 (ddt, 1H₁ J = 5.6, 10.4, 17.0 Hz), 5.61 (ddd, 1H₁ J = 1.6, 8.8, 10.4 Hz)₁ 5.27 (dd, 1 H₁ J = 1.26, 17.3 Hz)₁ 5.24 (dd, 1H, J = 1.26, 10.0 Hz), 4.9 (dd, 1 H, J = 3.4, 8.8 Hz), 4.53 (s, 1 H), 3.94-4.08 (m, 4H)₁ 3.62 (dd, 1 H, J = 8.5, 8.8 Hz)₁ 3.56 (m, 1 H), 3.47 (dd. 1 H, J = 4.0, 7.9 Hz), 3.37 (ddd, 1 H₁ J = 2.2, 7.2, 9.4), 3.15 (d, 1 H, J = 13.5 Hz), 2.4 (m, 1 H), 2.24 (m, 1 H)₁ 1.95 (m, 1 H), 1.85 (m, 1 H), 1.27-1.70 (m, 20H), 1 A (s, 9H), 1.2 (m, 1 H), 1.07 (s, 3H), 0.94 (s, 3H), 0.92 (m, 1 H); ¹³C NMR (CDCI₃, 125 MHz): δ 198.0, 172.1 , 171.0, 159.5, 157.0, 133.3, 117.7, 70.8, 70.5, 61.0, 60.8, 54.9, 53.8, 51.0, 48.4, 42.2, 36.2, 32.0, 30.5, 28.7, 27.9, 27.2, 27.0, 26.8, 25.9, 24.1 , 23.9,23.5, 21.9, 21.8, 19.3, 13.4 ppm; HRMS calcd for C₃₆H₆₀N₅O₈S [M+Hf : 722.4163, found 722.4193. Preparative Example 54: Preparation of:

Step A:

A solution of amine salt 53a (17 mg) in 2 mL of dry dichloromethane was treated with solid sodium bicarbonate (3 eq, 8 mg) followed by the addition of isocyanate 54a (2.5 eq, 0.26 mL of a 0.307M solution in toiuene). The resulting heterogeneous mixture was stirred at room temperature for approximately 3 h. The mixture was diluted with 50 mL of ethyl acetate and washed with aqueous 1M HCI (10 mL), and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 1 :1) to yield the product 54 (8 mg, 34%) as a white solid. ¹H NMR {500 MHz_r CDCI₃): S, 7.91 (br s, 1H)₁ 7.40 (m, 1H), 6.37 (br s, 1 H), 5.91 (ddt, 1 H, J = 5.6, 10.4, 17.3 Hz), 5.88 (br s, 1 H), 5.62 (dt, 1 H, J = 1.26, 9.45 Hz), 5.28 (dd, 1 H, J = 1.26, 17.3 Hz), 5.23 (dd, 1 H₁ J = 1.26, 10.4 Hz), 4.92 (ddd, 1 H, J = 3.46, 8.5, 8.5 Hz), 4.77 (dd, 1H, J = 4.7, 8.8 Hz), 4.55 (s, 1H), 3.94-4.06 (m, 4H)₁ 3.63 (t, 1 H, J = 8.2 Hz), 3.54 (ddd, 1 H₁ J = 3.4, 6.6, 9.7 Hz), 3.47 (m, 1 H), 3.38 (m, 1 H), 2.09 (ddd, 1 H, J = 4.4, 7.8, 12.3 Hz), 1.91 (m, 2H), 0.91-1.83 (m, 27H), 1.07 (s, 3H), 0.94 (s, 3H); HRMS calcd for C₃₆H₅₆N₅O₇ [M+H]⁺: 670.4180, found 670.4177. Preparative Example 55: Preparation of:

Step A:

A solution of amine salt 53a (17 mg) in 2 mL of dry dichioromethane was treated with solid sodium bicarbonate (3 eq, 8 mg) followed by the addition of isocyanate 25c (2.5 eq, 0.45 mL of a 0.18M solution in toluene). The resulting heterogeneous mixture was stirred at room temperature for approximately 3 h. The mixture was diluted with 50 mL of ethyl acetate and washed with aq 1 M HCI (10 mL), and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 1 :1) to yield the product 55 (8 mg, 30%) as a white solid. HRMS caicd for C₃₆H₅₇N₆O₃S₂ [M+H]⁺: 765.3679, found 765.3687. Preparative Example 56: Preparation of:

Step A:

A solution of 4,4-dimethylglutarimide 56a (ASdrich, 1.5 eq, 4.86 g) in 200 mL of dry THF was cooled to 0 ⁰C and treated with triphenylphosphine (3 eq, 18.07 g) and S-Boc-tert-butytglycinol 56b (Aldrich, 5 g).

Diisopropylazodicarboxylate (2.5 eq, 11.3 mL, d 1.027) was added dropwise and the resulting solution was stirred at 0 ⁰C. After 10 min, the mixture became a slurry and stirring was continued overnight (0 to 25 ⁰C). The mixture was concentrated under reduced pressure and the residue was dissolved in 80 mL of ether. Hexanes (100 mL) was added and the precipitated solids were filtered off. The filtrate was concentrated to half its volume and hexanes (100 mL) was added again. The solids were filtered off. The filtrate was concentrated under reduced pressure. The residue was chromatographed on silica gel (ethyl acetate/hexanes; 2:8) to afford the product 56c (4.0 g, 51 %) as a white solid. Step B:

The N-Boc protected amine 56c (4.0 g) was dissolved in 200 ml_ of 4M HCI solution in dioxanes. The mixture was stirred at room temperature and a white solid precipitated after 10 min. The mixture was further stirred for 2 h. All the voiatϋes were removed under reduced pressure to afford the product 56d (3.24 g, 98%) as a white solid.

Step C:

A solution of amine hydrochloride 56d (1.5 g) in 60 mL of dichioromethane was treated with 50 mL of aqueous saturated sodium bicarbonate solution and stirred vigorously for 10 min at 0 ⁰C. Stirring was stopped and layers were allowed to separate. Phosgene (15 mL of 20% soin in toluene) was added through a needle to the organic layer {lower layer) in one portion. The mixture was vigorously stirred immediately after addition for 10 min at 0 ⁰C and further stirred at room temp for 2.5 h. The mixture was diluted with 100 mL of dichioromethane and layers were separated. The organic layer was washed with 40 mL of cold aqueous saturated sodium bicarbonate solution and dried over magnesium sulfate. The organic layer was filtered and diluted with 50 mL of toluene. The product 56e (1.44 g, 98%) was kept as a 0.216M solution in toluene- Step C:

The N-Boc amine 52 (200 mg) was dissolved in 20 mL of 4M HC! solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichioromethaπe and cooled to 0 ⁰C. Then, 10 drops of aqueous saturated sodium bicarbonate solution were added. After 10 min, a solution of the isocyanate 56e was added dropwise (1.2 eq, 1.97 mL of a 0.216M solution in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with dichloromethane (70 mL) and washed with aqueous saturated sodium bicarbonate solution (20 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 15:85 to 55:45) to afford the product 56 (172 mg, 66%) as a white solid. ¹H NMR (400 MHz₁ CDCI₃): 6, 7.95 (d, 1 H, J = 8.8 Hz), 7.59 (br s, 1 H), 5.91 (br s, 1 H), 5.84 (ddt, 1 H₁ J = 5.8, 10.2, 16.8 Hz), 5.61 (ddd, 1 H₁ J = 1.5, 8.7, 10.2 Hz)₁ 5.21 (dd, 1H₁ J = 1.4, 17.5 Hz), 5.17 (dd, 1 H, J = 1.4, 10.2 Hz), 5.13 (br s, 1 H)₁ 4.86 (br s, 1 H), 4.52 (s, 1 H), 4.05 (d, 1 H, J = 10.2 Hz), 3.80-3.99 (m, 6H), 3.50 (m, 2H)₁ 3.27 (m, 2H), 2.51 (d, 2H₁ J = 16.8 Hz), 2.43 (d, 2H₁ J = 16.8 Hz), 1.88 (m, 1 H), 1.77 (m, 1 H)₁ 0.84-1.58 (m, 12H), 1.05 (s, 6H)₁ 0.97 (s, 3H), 0.92 (s, 9H), 0.81 (s, 3H); ¹³C NMR (CDCI3, 125 MHz): 6 198.6, 172.8, 172.0, 171.2, 159.3, 157.8, 133.4, 117.7, 71.4, 70.8, 60.7, 57.0, 53.5, 48.5, 46.8, 42.2, 40.0, 34.9, 32.1 , 30.9, 29.4, 28.7, 28.1 , 27.7, 27.4, 26.9, 24.3, 19.3, 13.5 ppm; HRMS caicd for C₃₈H_6IN₆O₈ [M+H]⁺: 729.4551 , found 729.4529. Preparative Example 57: Preparation of:

Step A:

The N-Boc protected amine 52 (101 mg) was dissolved in 10 mL of formic acid and stirred at room temperature for 1 h. Ail the volatiles were removed in rotovap and the residue was chromatographed on siiica gel (gradient: acetone/hexanes; 2:8 to 6:4) to give the formylated product 57 (35 mg, 40%) as a white solid. ¹H NMR (500 MHz, CDCI₃) δ, 8.24 (s, 1 H), 7.35 (d, 1 H, J = 7.8 Hz), 7.07 (br s, 1 H), 6.83 (d, 1 H, J = 6.9 Hz), 5.89 (ddt, 1 H, J = 5.6, 10.0, 17.0 Hz), 5.47 (m, 1 H), 5.28 (dd, 1 H₁ J = 1.2, 17.3 Hz), 5.25 (dd, 1H₁ J = 1.2, 10.4 Hz), 4.95 (ddd, 1 H, J = 3.1, 5.9, 8.5 Hz), 4.51 (s, 1 H), 3.99 (m, 2H), 3.92 (dd, 1 H, J = 5.3, 11.0 Hz), 3.75 (d, 1 H, J = 11.0 Hz), 3.74 (m, 1 H), 3.70 (dd, 1 H₁ J = 5.6, 9.1 Hz), 3.48 (m, 2H), 1.96 (m, 1 H), 1.77 (d, 1 H, J = 7.8 Hz)₁ 1.76 (m, 1 H), 1.27-1.63 (m, 11 H), 1.10 (s, 3H)₁ 0.92 (s, 3H); ¹³C NMR (CDCi₃, 125 MHz) δ 196.8, 170.5, 169.4, 160.8, 159.3, 133.1, 117.9, 71.3, 70.0, 61.5, 54.4, 50.8, 48.2, 42.1 , 32.0, 31.5, 29.4, 28.6, 27.8, 26.7, 26.6, 23.6, 23.5, 19.3, 14.5, 13.2 ppm; HRMS calcd for C₂₅H₃₉N₄O₆ [M+H]⁺: 491.2870, found 491.2882. Preparative Example 58: Preparation of:

Ste

The N-Boc protected amine 52 (80 mg) was dissolved in 5 mL of 4M HCI soln in dioxanes and stirred at room temperature for 45 min. Ail the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 3 mL of dry dichloromethane and treated with N- methylmorpholine (3 eq, 0.05 mL, d 0.920). The isocyanate 58a was added in solution (2 eq, 3.8 mL of a 0.075M solution in toluene). The reaction mixture was stirred at room temperature for about 3 h. The mixture was diluted with ethyl acetate (50 mL) and washed with aqueous 1 M HCI (10 mL), aqueous saturated sodium bicarbonate (10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 3:7 to 7:3) to afford the product 58 (16 mg, 16%) as a white solid. ¹H NMR (500 MHz, CDCi₃) δ, 8.19-8.39 (br s, 1 H)₁ 8.05 (d, 1 H, J = 8.5 Hz), 6.22 (br s, 1 H), 5.91 (ddt, 1 H₁ J = 5.6, 10.0, 17.0 Hz), 5.71 (dd, 1 H₁ J = 9.4, 10.0 Hz)₁ 5.33 (d, 1H, J = 9.4 Hz), 5.28 (m, 1H), 5.26 (dd, 1 H, J = 1.2, 17.0 Hz)₁ 5.20 (dd, 1 H, J = 1.2, 10.4 Hz), 4.96 (ddd, 1 H, J = 4.0, 9.4, 9.4 Hz), 4.60 (s, 1H)₁ 4.32 (t, 1 H, J = 12.6 Hz), 4.12 (d, 1 H, J = 10.7 Hz)₁ 3.86-4.07 (m, 4H), 3.49-3.63 (m, 3H), 3.38 (dd, 1 H, J = 4.1, 7.9 Hz), 3.31 (m, 1 H), 3.16 (m, 1 H), 2.66 (dd, 1H₁ J = 2.8, 13.8 Hz), 2.39 (dt, 1H₁ J = 5.6, 17.3 Hz)₁ 2.27 (dt, 1H₁ J = 6.6, 17.3 Hz)₁ 1.89-2.04 (m, 2H), 1.71 -1.87 (m, 4H), 0.88-1.64 (m, 11 H), 1.03 (s, 3H), 0.93 (s, 9H)₁ 0.90 (s, 3H); ¹³C NMR (CDCI₃, 125 MHz) δ 196.2, 171.8, 171.3, 159.5, 158.2, 133.7, 117.3, 71.4, 70.8, 60.6, 55.7, 53.5, 51.O₁ 48.3, 48.1 , 46.5, 42.2, 34.3, 32.7, 31.8, 31.0, 28.7, 27.8, 27.6, 27.0, 26.9, 24.6, 24.4, 23.5, 21.7, 19.2, 13.5 ppm; HRMS calcd for C₃₆H₅₉N₆O₇ [M+H]⁺: 687.4445, found 687.4434. Preparative Example 59: Preparation of:

Step A:

The N-Boc amine 52 (56 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 °C. Then, 15 drops of aqueous saturated sodium bicarbonate solution were added. After 10 min, a soin of the isocyanate 59a was added dropwise (1.0 eq) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with aqueous 1 M HCI (10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 6:4) to afford the product 59 (35 mg, 50%) as a white solid. HRMS calcd for C₃SH₆₃N₆O₇ [M+Hf: 715.4758, found 715.4739. Preparative Example 60: Preparation of:

Step

A solution of aldehyde 52j (405 mg) in 15 mL of dry dichloromethane was treated with cyciopropylisocyanide (Oakwood Prod., 2 eq, 117 mg) and acetic acid (2 eq, 0.1 mL, d 1.049). The mixture was stirred at room temperature overnight. All the volatiles were removed under reduced pressure and the residue was chromatographed on silica gel (gradient: acetone/hexanes; 1:9 to 1 :1) to afford the product 60a (500 mg, 98%) as a white solid. Step B:

A solution of acetate 60a (500 mg) in 15 ml_ of a 1 :1 :1 mixture of THF/MeOH/water was treated with lithium hydroxide monohydrate (2.5 eq, 86 mg) and stirred for approx. 30 min until all the starting materia! had been consumed as determined by TLC analysis (ethyl acetate/hexanes; 6:4). The reaction mixture was diluted with 30 ml_ of aqueous saturated sodium bicarbonate solution and extracted with dichloromethane (3 x 50 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated to afford the crude product 60b (464 mg, 98%) as a colorless semi-solid which was used without further purification. Step C:

A solution of hydroxyamϊde 60b (0.824 mmol) in 20 mL of dry dichloromethane was treated with Dess-Martin periodinane (2.0 eq, 698 mg). The reaction mixture was stirred at room temperature for 30 min. The mixture was treated with aqueous 1 M sodium thiosuifate solution (15 mL) and stirred for 5 min. Aqueous saturated sodium bicarbonate solution (20 mL) was aiso added and stirring was continued for further 10 min. The mixture was extracted with dichloromethane (3 x 50 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 5:95 to 35:65) to afford the product 60c (333 mg, 72%) as white solid. Step D:

The N-Boc amine 60c (70 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum overnight. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 ⁰C. Then, 20 drops of aqueous saturated sodium bicarbonate solution were added followed by a solution of the isocyanate 56e (1.3 eq, 0.7 mL of a 0.241 M solution in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with dichloromethane (50 mL) and washed with aqueous saturated sodium bicarbonate solution (10 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 55:45) to afford the product 60 (70 mg, 77%) as a white solid. ¹H NMR (500 MHz₁ CDCI₃): δ 7.95 (br s, 1 H), 7.56 (br s, 1 H), 5.86 (br s, 1 H), 5.65 (t, 1H, J = 8.8 Hz)₁ 5.09 (br s, 1 H), 4.91 (br s, 1 H), 4.56 (s, 1 H), 4.07 (d, 1 H₁ J = 10.4 Hz), 3.98 (dd, 1 H, J = 5.0, 10.7 Hz), 3.91 (m, 3H)₁ 3.54 (m, 2H)₁ 3.34 (m, 2H)₁ 2.88 (ddd, 1H₁ J = 3.7, 7.5, 15.1 Hz), 2.56 (d, 2H, J = 16.7 Hz), 2.50 (d, 2H, J = 16.7 Hz), 1.94 (m, 1 H)₁ 0.87-1.76 (m, 15H)₁ 1.11 (s, 6H), 1.03 (S₁ 3H), 0.97 (s, 9H), 0.86 (s, 3H)_T 0.70 (m, 2H); ¹³C NMR (CDCI₃, 125 MHz) δ 198.0, 172.9, 172.0, 171.2, 160.8, 157.7, 71.5, 70.8, 60.7, 56.9, 53.4, 51.1 , 48.4, 46.8, 39.9, 34.9, 32.1 , 30.8, 29.4, 28.7, 28.1 , 27.7, 27.5, 26.9, 26.8, 24.4, 23.0, 19.2, 13.5, 6.8, 6.7 ppm. HRMS calcd for C₃₈H₆₁N₆O₈ [M+H]⁺: 729.4551, found 729.4558. Preparative Example 61: Preparation of:

Step A:

The N-Boc amine 60c (56 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichioromethane and cooled to 0 ⁰C. Then, 20 drops of aqueous saturated sodium bicarbonate solution were added followed by a soln of the isocyanate 59a in toluene (1.3 eq) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with dichioromethane (60 mL) and washed with aqueous saturated sodium bicarbonate solution (20 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica ge! (gradient: acetone/hexanes; 2:8 to 6:4) to afford the product 61 (52 mg, 73%) as a white solid. ¹H NIvIR (500 MHz, CDCI₃): O 8.30-8.53 (br s, 1 H)₁ 8.15 (d, 1 H, J = 8.8 Hz), 6.13 (br s, 1H), 5.74 (dd, 1 H₁ J = 8.8, 9.7 Hz)₁ 5.38 (d, 1H₁ J = 9.1 Hz), 4.96 (br s, 1 H), 4.59 (S₁ 1 H), 4.35 (dd, 1 H, J = 12.9, 12.9 Hz), 4.10 (d, 1H, J = 10.4 Hz), 4.01 (dd, 1 H, J = 5.0, 10.4 Hz), 3.94 (m, 1 H), 3.56 (m, 2H), 3.50 (dd, 1 H₁ J = 8.5, 8.8 Hz)₁ 3.31 (m, 2H)_T 3.17 (ddd, 1 H, J = 5.6, 6.0, 12.3 Hz)₁ 2.91 (ddd, 1 H, J = 4.0, 7.8, 15.4 Hz)₁ 2.67 (dd, 1 H, J = 3.4, 13.5 Hz)₁ 2.17 (d, 1 H, J = 17.0 Hz), 2.10 (d, 1H, J = 17.0 Hz), 1.94 (m, 3H), 1.24-1.70 (m, 12H), 1.14 (m, 1H)₁ 1.04 (s, 3H)₁ 1.02 (s, 3H), 1.00 (s, 3H), 0.91 (S₁ 9H), 0.89 (s, 3H)₁ 0.84 (m, 2H)₁ 0.73 (m, 2H); ¹³C NMR (CDCI₃, 125 MHz) δ 199.9, 171.9, 171.3, 171.0, 160.9, 158.0, 71.4, 70.9, 60.4, 55.4, 53.2, 48.2, 46.3, 45.0, 35.8, 34.6, 31.7, 30.3, 28.8, 28.7, 27.8, 27.7, 27.6, 27.1 , 26.9, 26.8, 24.8, 24.7, 23.2, 19.1 , 13.4, 6.4 ppm. HRMS calcd for C₃₃H₆₃N₆O₇ [M+H]⁺: 715.4758, found 715.4768. Preparative Example 62: Preparation of: Ste

The N-Boc amine 60c (60 mg) was dissoived in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. Ail the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 °C. Then, 20 drops of aqueous saturated sodium bicarbonate soiution were added followed by a soln of the isocyanate 27b in toluene (1.2 eq) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with dichloromethane (60 mL) and washed with aqueous saturated sodium bicarbonate solution (20 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 6:4) to afford the product 62 (65 mg, 85%) as a white solid. ¹H NMR (500 MHz, CDCI₃): δ 7.84 (d, 1 H, J = 8.2 Hz)₁ 7.28 (br s, 1 H), 5.73-6.02 (br s, 1H), 5.57 (ddd, 1 H, J = 1.9, 8.2, 8.5 Hz), 5.22 (br s, 1H), 4.88 (dd, 1 H₁ J = 3.4, 8.5 Hz), 4.51 (s, 1H), 4.01 (m, 3H), 3.62 (dd, 1 H, J = 8.5, 8.5 Hz), 3.55 (ddd, 1 H, J = 3.7, 6.3, 9.7 Hz), 3.48 (dd, 1 H₁ J = 4.0, 8.1 Hz), 3.38 (m, 1 H)₁ 3.18 (d, 1 H₁ J = 13.5 Hz)₁ 2.86 (ddd, 1H, J - 3.8, 7.2, 14.8 Hz)₁ 2.41 (d, 1 H, J = 11.6 Hz)₁ 2.24 (d, 1H, J = 11.6 Hz)₁ 1.93 (m, 1 H), 1.72-1.89 (m, 4H), 1.40 (s, 9H), 1.28-1.70 (m, 16H), 1.21 (m, 1 H)₁ 1.06 (s, 3H), 0.93 (s, 3H)₁ 0.91 (m, 2H)₁ 0.70 (m, 2H); ¹³C NMR (CDCI₃, 125 MHz) δ 197.9, 172.0, 171.0, 160.9, 157.0, 70.8, 70.6, 61.0, 60.8, 54.9, 53.7, 51.1 , 48.4, 36.2, 32.0, 30.5, 28.7, 27.9, 27.2, 26.9, 26.8, 25.9, 24.1, 23.8, 23.5, 23.0, 21.9, 21.8, 19.3, 13.4, 6.9, 6.8 ppm. Preparative Example 63: Preparation of:

Step A:

A solution of amine 63a (2.0 g) in 100 ml_ of dry dichloromethane was cooled to 0 °C and treated with pyridine (3.0 eq, 2.24 mL, d 0.978) and ethanesulfonyl chioride (1.2 eq, 1.05 mL, d 1.357). The resulting yellow homogeneous solution was stirred overnight (temp 0 to 25 ⁰C). The mixture was diluted with 200 mL of ether and washed with aqueous 1 M HCI (100 mL) and brine (100 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated. The residue was chromatographed on silica gel (gradient: dichloromethane to ethyl acetate/dichioromethane 3:7) to afford the product 63b (850 mg, 30%) as a white solid. Step B:

A solution of ethylsuifonamide 63b (850 mg) in dry DMF (30 mL) was treated with cesium carbonate (3.0 eq, 2.74 g) and iodomethane (3.0 eq, 0.51 mL, d 2.280). The reaction mixture was stirred for approximately 4 h. TLC analysis (acetone/hexanes; 2:8) showed that all the starting material had been consumed. The mixture was diluted with ethyl acetate (300 mL) and washed with water (3 x 50 mL) and brine (1 x 50 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure to afford the product 63c (860 mg, 97%) as a white solid. No further purification was carried out for the product Step C:

The N-Boc protected amine 63c (850 mg) was dissolved in 100 mL of 4M HCi solution in dioxanes. The resulting solution was stirred at room temperature until all the starting materia! had been consumed as determined by TLC (acetone/hexanes; 2:8). All the volatiles were removed under reduced pressure and the residue was placed under high vacuum to afford the product 63d (680 mg, 98%). Step D:

A solution of amine hydrochloride 63d (2.636 mmol) in 40 mL of dichloromethane was treated with 40 mL of aqueous saturated sodium bicarbonate solution and stirred vigorously for 10 min at 0 ⁰C. Stirring was stopped and layers were allowed to separate. Phosgene (10 mL of 20% soln in toluene) was added through a needle to the organic layer (lower layer) in one portion. The mixture was vigorously stirred immediately after addition for 10 min at 0 °C and further stirred at room temp for 2.5 h. The mixture was diluted with 100 mL of dichloromethane and layers were separated. The organic layer was washed with 30 mL of cold aqueous saturated sodium bicarbonate solution and dried over magnesium sulfate. The organic layer was filtered and the filtrate was diluted with 50 mL of toluene. The product 63e (654 mg, 98%) was concentrated and kept as a 0.131 M solution in toluene (the solution contains about 2 mL of dichioromethane). Step E:

The N-Boc amine 52 (60 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the vofatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 ⁰C. Then, 10 drops of aqueous saturated sodium bicarbonate solution were added. After 10 min, a soln of the isocyanate 63e was added dropwise (1.2 eq, 0.97 mL of a 0.131 M solution in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with dichloromethane (70 mL) and washed with aqueous saturated sodium bicarbonate solution (20 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 6:4) to afford the product 63 (49 mg, 65%) as a white solid. ¹H NMR (500 MHz, CDCI₃): 68.04 (d, 1 H₁ J = 8.5 Hz), 7.52 (br s, 1 H), 6.04 (br s, 1 H), 5.89 (ddt, 1 H, J = 5.6, 10.4, 17.0 Hz), 5.65 (dd, 1 H, J = 8.8, 10.4 Hz), 5.26 (dd, 1H₁ J = 1.2, 17.0 Hz), 5.22 (dd, 1 H, J = 1.2, 10.0 Hz), 5.17 (d, 1 H, J = 10.0 Hz), 4.99 (br s, 1 H), 4.61 (s, 1 H), 4.19 (d, 1 H, J = 10.7 Hz), 4.02 (m, 2H), 3.95 (m, 2H), 3.60 (dd, 1 H, J = 8.1 , 9.1 Hz), 3.54 (m, 1 H), 3.49 (d, 1 H, 1.9 Hz)₁ 3.44 (m, 1 H), 3.32 (m, 1 H), 3.07 (m, 3H)₁ 2.94 (s, 3H)₁ 1.93 (m, 1 H), 1.35 (t, 3H, J = 7.5 Hz), 1.27-1.62 (m, 15H)₁ 1.16 (m, 1 H), 1.03 (s, 3H), 0.92 (s, 9H), 0.91 (s, 3H); ¹³C NMR (CDCI₃, 125 MHz) 6, 198.6, 172.3, 171.3, 159.4, 158.0, 133.4, 117.6, 71.1 , 70.6, 60.7, 54.7, 53.5, 51.0, 50.6, 48.4, 45.8, 42.2, 34.8, 34.5, 32.0, 30.9, 28.7, 27.8, 27.3, 27.0, 26.9, 24.3, 24.2, 19.2, 13.5, 8.6 ppm; HRMS calcd for C₃₄H₅₉N₆O₈S [M+H]⁺: 711.4115, found 711.4133. Preparative Example 64: Preparation of:

Step A:

The N-Boc amine 60c (60 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 ⁰C. Then, 10 drops of aqueous saturated sodium bicarbonate solution were added. After 10 min, a soln of the isocyanate 63e was added dropwise (1.2 eq, 0.97 mL of a 0.131 M solution in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with dichloromethane (70 mL) and washed with aqueous saturated sodium bicarbonate solution (20 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 6:4) to afford the product 64 (62 mg, 82%) as a white solid. ¹H NMR (500 MHz₁ CDCI₃): δ 8.10 (br s, 1H), 7.47 (br s, 1H), 5.94-6.19 (br S₁ 1H), 5.65 (dd, 1 H, J = 8.8, 10.7 Hz), 5.21 (d, 1 H, J = 7.8 Hz), 5.00 (dd, 1 H, J = 3.7, 9.4 Hz), 4.59 (s, 1 H), 4.21 (d, 1 H, J = 10.7 Hz), 4.02 (dd, 1 H, J = 5.0, 10.7 Hz), 3.93 (dd, 1 H, J = 9.1, 9.7 Hz), 3.55 (m, 2H), 3.48 (d, 1 H, J = 12.6 Hz), 3.40 (m, 1 H), 3.29 (m, 1H), 3.07 (q, 2H, J = 7.2 Hz), 3.06 (m, 1 H), 2.93 (s, 3H), 2.85 (dddd, 1 H, J = 1.8, 4.0, 7.5, 15.1 Hz), 1.91 (m, 1 H), 1.34 (t, 3H, J = 7.2 Hz)₁ 1.25-1.61 (m, 12H), 1.13 (m, 1H)₁ 1.01 (s, 3H), 0.90 (s, 9H), 0.89 (s, 3H), 0.87 (m, 2H), 0.69 (m, 2H); ¹³C NMR (CDCi₃, 125 MHz) δ 198.9, 172.3, 171.4, 160.9, 158.0, 71.0, 70.6, 60.6, 53.2, 50.9, 50.6, 48.4, 45.9, 34.8, 34.5, 32.1 , 31.1 , 28.7, 27.8, 27.5, 27.0, 26.9, 24.4, 23.0, 19.2, 13.6, 8.6, 6.7 ppm; HRMS caicd for C₃₄H₅₉N₆O₃S [M+if : 711.4115, found 711.4133. Preparative Example 65: Preparation of: Step A:

65a 65b

A solution of (S)-allo-threoπine-OMe hydrochloride 65a (Chem-lmpex, 5 g) in dry dichioromethane (150 mL) was cooled to 0 ⁰C and treated with di-tert- butyldicarbonate (1.1 eq, 7.0 g) in 50 mL of dry dichioromethane. N-methylmorpholine (2.5 eq, 8.1 mL, d 0.920) was added dropwise and the mixture was stirred for 30 min. The cooling bath was removed and the mixture was stirred for further 3 h. The mixture was concentrated to one third of its volume and then diluted with ethyl acetate (300 mL) and washed with aqueous 1 M HCI (100 mL), aqueous saturated sodium bicarbonate (80 mL) and brine (80 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure to afford the product 65b (6.78 g, 98%) as a colorless oil. Step B:

A solution of Boc-L-allo-Thr-OMe 65b (6.8 g) in 250 mL of dry THF was degassed (vacuum/N2-flush) and treated with allylmethyl carbonate (1.3 eq, 4.3 mL, d 1.022). A catalytic amount of tetrakis(triphenylphosphine)pailadium (0.02 mol%, 673 mg) was added. The slightly yellow mixture was degassed again and heated at 60 ⁰C for about 3 h until TLC analysis (acetone/hexanes; 2:8) showed no more starting material left (reaction mixture became brown). The mixture was concentrated under reduced pressure and the residue was chromatographed on silica gel (ethyl acetate/hexanes; 1:9) to afford the product 65c (5.72 g, 72%) as a colorless oif. Step C:

A solution of methyl ester 65c (1.45 g) in 250 ml_ of a 4:2:1 mixture of THF/water/MeOH was cooled to 0 ⁰C and treated with lithium hydroxide monohydrate (2.5 eq, 2.19 mg). The cooling bath was removed after 30 min and the mixture was stirred at room temp for further 4 h until all the starting material had been consumed as determined by TLC analysis (acetone/hexanes; 15:85). The reaction mixture was treated with 200 ml_ of aqueous 11Vl HCI (pH of mixture = 1 ) and the product was taken into dichloromethane (4 x 100 mL). The combined organic extracts were dried over magnesium sulfate, filtered and concentrated under reduced pressure to afford the product. No further purification was carried out for the product 65d (5.42 g, 98%). Step D:

A solution of acid 65d (20.92 mmol) in 200 mL of dry dichloromethane and 100 mL of dry DMF was stirred at 0⁰C and treated with HATU (1.4 eq, 11.16 g). The amine salt 1d (1.2 eq, 5.16 g) was added followed by N-methylmorpholine (4 eq, 9.19 mL, d 0.920). The reaction mixture was stirred overnight. All the volatϊles were removed under vacuum and the residue was dissolved in 500 mL of ethyl acetate. The organic layer was washed with water (200 mL), aqueous 1 M HC! (100 mL), aqueous saturated sodium bicarbonate solution (100 mL), and brine (100 mL). The organic iayer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (ethyl acetate/hexanes; 2:8) to give the product 65e (7.6 g, 88%) as a colorless oil along with a small amount of its corresponding diastereomeric product. Step E:

A solution of methy^jester 65e (7.6 g) in 300 mL of a 2:1 mixture of THF/water was cooled to 0 ⁰C and treated with lithium hydroxide monohydrate (2.5 eq, 1.93 mg). The cooling bath was removed after 30 min and the mixture was stirred at room temp for further 4 h until all the starting material had been consumed as determined by TLC analysis (ethyl acetate/hexanes; 25:75). The reaction mixture was treated with 200 mL of aqueous 1 M HCI (pH of mixture = 1) and the product was taken into dichloromethane (4 x 100 mL). The combined organic extracts were dried over magnesium sulfate, filtered and concentrated under reduced pressure to afford the product 65f (6.86 g, 93%) as a colorless solid- Step F:

A solution of acid 65f (6.86 g) in 100 mL of dry dichloromethane and 100 mL of dry DMF was stirred at 0⁰C and treated with HATU (1.4 eq, 9.23 g). The amine salt 1f (1.1 eq, 4.21 g) was added in 100 mL of dichloromethane followed by addition of N- methylmorpholine (4 eq, 7.6 mL, d 0.920). The reaction mixture was stirred at 0 ⁰C overnight. AN the volatiles were removed under vacuum and the residue was dissolved in 500 mL of ethyl acetate. The organic layer was washed with water (2 x 100 mL), aqueous 1M HCI (100 mL), aqueous saturated sodium bicarbonate solution (100 mL), and brine (100 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (ethyl acetate/hexanes; 3:7) to afford the product 65g (8.17 g, 84%) as a colorless oil. Step G:

A solution of diene 65g (8.17 g) in 1.5 L of toluene was degassed for 30 min (argon bubbling) and treated with Grubb's catalyst (0.2 eq, 2.38 g). The pink solution was heated to 60⁰C for 18 h (the solution became dark after 10 min of heating). The solvent was removed under reduced pressure and the residue was chromatographed on silica gel (ethyl acetate/hexanes; 3:7) to give the alkene product 65h (7.0 g, 90%) as a mixture of E- and 2-isomers (approx 4:1). Step H:

A solution of aikene 65h (7.0 g) in 300 mL of methanol was treated with palladium on carbon (0.1 mol%, 1.37 g of 10% Pd/C). The mixture was hydrogenated at 35 psi until all the starting material had been consumed (approx 3 h). The reaction mixture was diluted with 300 mL of dichloromethane and filtered thru a short path of ceϋte. The filtrate was concentrated and the residue was chromatographed on silica gel (ethyl acetate/hexanes; 3:7) to afford the product 65i (5.33 g, 76%) as a white solid. Step I:

A solution of ethyl ester 65i (5.33 g) in 100 mL of dry THF was treated with lithium borohydride (2.1 eq, 10.4 mL of a 2M soln in THF). The reaction mixture was stirred at room temperature and monitored by TLC (acetone/hexanes; 3:7) for disappearance of the starting material. After 2 h, more lithium borohydride solution was added (1 eq) and stirring was continued for 1h. The excess lithium borohydride was quenched by addition of aqueous saturated ammonium chloride solution. The mixture was partitioned between ethyl acetate (300 mL) and aqueous saturated sodium bicarbonate solution (100 mL). The aqueous layer was back extracted with ethyl acetate (2 x 100 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (acetone/hexanes; 3:7) to afford the product 65j (3.93 g, 80%) as a white solid. Step J:

A solution of alcohol 65j (1.0 g) in 40 mL of dry dichloromethane was treated with Dess-Martin periodinane (1.5 eq, 1.28 g). The reaction mixture was stirred at room temperature for 3 h. The mixture was treated with aqueous 1M sodium thiosulfate solution (10 mL) and stirred for 5 min. Aqueous saturated sodium bicarbonate solution (30 mL) was also added and stirring was continued for further 10 min. The mixture was extracted with dichloromethane (3 x 80 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated. The residue was chromatographed on silica gel (gradient: ethyl acetate/hexanes; 4:6 to 8:2) to afford the product 65k (750 mg, 75%) as a colorless solid. Step K:

A solution of aldehyde 65k (750 mg) in 20 mL of dry dichioromethane was treated with allylisocyanide (2 eq, 0.26 mL, d 0.8) and acetic acid (2 eq, 0.17 mL, d 1.049). The mixture was stirred at room temperature for about 5 h. All the volatiles were removed under vacuum and the residue was chromatographed on silica gel (gradient: acetone/hexanes; 1 :9 to 45:55) to afford the product 65I (700 mg, 74%) as a white solid. Step L:

A solution of acetate 651 (700 mg) in 20 mL of a 2:1 mixture of THF/water was treated with lithium hydroxide monohydrate (2.5 eq, 118 mg) and stirred for approx 30 min unti! all the starting material had been consumed as determined by TLC analysis (ethyl acetate/hexanes; 8:2). The reaction mixture was diluted with 50 mL of aqueous saturated sodium bicarbonate solution and extracted with dichioromethane (3 x 80 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated to afford the product 65m (651 mg, 98%) as a colorless semi-solid which was used without further purification. Step M:

A solution of hydroxyamide 65m (1.127 mmol) in 25 mL of dry dichloromethane was treated with Dess-Martin periodinane (2.0 eq, 956 mg). The reaction mixture was stirred at room temperature for 30 min. The mixture was treated with aqueous 1 M sodium thϊosulfate solution (20 mL) and stirred for 5 min. Aqueous saturated sodium bicarbonate solution (30 mL) was also added and stirring was continued for further 10 min. The mixture was extracted with dichloromethane (3 x 80 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 1:9 to 45:55) to afford the product 65n (585 mg, 90%) as white solid. Step N:

The N-Boc amine 65n (60 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 ⁰C. Then, 10 drops of aqueous saturated sodium bicarbonate solution were added. After 10 min, a soln of the isocyanate 56e was added dropwise (1.2 eq, 0.57 mL of a 0.216M solution in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with dichioromethane (70 mL) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 15:85 to 5:5) to afford the product 65 (50 mg, 65%) as a white solid. ¹H NMR (500 MHz, CDCi₃): δ 8.13 (br s, 1H)₁ 7.42-7.82 (br s, 1 H), 6.30 (br s, 1 H)₁ 5.90 (ddt, 1 H, J = 5.6, 10.4, 17.0 Hz), 5.71 (br s, 1 H), 5.38 (br s, 1 H)₁ 5.27 (dd, 1 H, J = 1.2, 17.0 Hz), 5.23 (dd, 1 H, J = 1.2, 10.4 Hz), 4.63 (dd, 1H, J = 7.8, 8.1 Hz), 4.50 (br s, 1H), 4.23 (d, 1 H, J = 10.4 Hz), 4.05 (m, 2H), 3.98 (dd, 1 H, J = 5.6, 5.9 Hz), 3.95 (d, 1 H, J = 11.0 Hz), 3.88 (dd, 1 H, J = 10.7, 10.8 Hz), 3.82 (q, 1 H, J = 11.6 Hz), 3.71 (m, 1 H), 3.62 (ddd, 1 H, J = 5.0, 5.3, 9.4 Hz), 3.20 (m, 1 H)₁ 2.55 (d, 2H, J = 16.7 Hz), 2.47 (d, 2H, J = 16.7 Hz), 1.73-1.97 (m, 4H), 1.14 (d, 3H, J = 6.0 Hz), 1.10 (s, 6H), 1.00 (s, 3H), 0.99 (s, 9H)₁ 0.83 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz) δ 198.0, 172.8, 171.5, 159.4, 157.8, 117.7, 75.5, 68.1 , 60.8, 57.2, 55.9, 48.7, 46.8, 42.3, 35.2, 29.3, 28.7, 28.3, 27.8, 27.6, 26.9, 26.8, 24.7, 24.4, 19.4, 16.3, 13.6 ppm; HRMS calcd for C₃₉H₆₃N₆O₈ [M+Hf: 743.4707, found 743.4717. Preparative Example 66: Preparation of:

Ste

The N-Boc amine 65n (60 mg) was dissolved in 10 ml_ of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. Al! the voiatiles were removed under reduced pressure and the residue was placed under high vacuum overnight. The resulting amine salt was dissolved in 5 ml_ of dichloromethane and cooled to 0 ⁰C. Then, 10 drops of aqueous saturated sodium bicarbonate solution were added. After 10 min, a soln of the isocyanate 63e was added dropwise (1.2 eq, 0.95 mL of a 0.131M solution in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 3 h. The reaction mixture was diluted with dichioromethane (70 mL) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 6:4) to afford the product 66 (55 mg, 73%) as a white solid. ¹H NMR (500 MHz, CDCI₃): 68.20 (d, 1 H, J = 6.6 Hz), 7.58-7.77 (br s, 1H), 6.13 (br s, 1H), 5.90 (ddt, 1H, J = 5.6, 10.0, 17.0 Hz), 5.76 (br s, 1 H), 5.27 (dd, 1 H, J = 1.2, 17.0 Hz), 5.22 (dd, 1 H, J = 1.2, 10.0 Hz), 5.15 (d, 1 H, J = 9.1 Hz), 4.69 (dd, 1 H, J = 8.8, 8.8 Hz), 4.57 <s, 1 H), 4.29 (d_f 1H, J = 10.7 Hz)₁ 3.91-4.09 (m, 4H), 3.61 (m, 2H), 3.47 (dd, 1 H, J = 11.9, 13.5 Hz), 3.19 (m, 1H), 3.07 (m, 3H), 2.94 (s, 3H), 1.95 <m, 1H), 1.35 (t, 3H, J= 7.5 Hz), 1.27-1.69 (m, 12H)₁ 1.22 (d, 3H, J = 6.3 Hz), 1.14 (m, 1 H), 1.02 (s, 3H), 0.93 (s, 9H), 0.89 (s, 3H); ¹³C NMR (CDCi₃, 125 MHz) δ 199.0, 172.8, 171.5, 159.3, 158.1, 133.5, 117.5, 75.8, 68.4, 60.7, 56.2, 50.4, 48.6, 45.6, 42.2, 34.7, 34.5, 32.0, 31.6, 28.6, 27.7, 27.0, 26.9, 26.7, 24.8, 24.6, 19.3, 16.2, 14.5, 13.5, 8.5 ppm; HRMS calcd for C₃₅H₆₁N₆O₈S [M+H]⁺: 725.4272, found 725.4285. Preparative Example 67: Preparation of:

Ste

The N-Boc amine 33 (60 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the voiatiles were removed under reduced pressure and the residue was placed under high vacuum overnight. The resulting amine salt was dissolved in 5 mL of dry dichloromeihane and cooled to 0 ⁰C. Then, N-methylmorphofine (2 eq, 0.03 mL, d 0.920) was added. After 10 min, a soln of the isocyanate 56e was added dropwise (1.5 eq, 0.8 mL of a 0.2M solution in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with aq 1 M HCl (10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 1 :1) to afford the product 67 (50 mg, 64%) as a white solid. ¹H NMR (500 MHz, CDCI₃): 68.14 (d, 1 H, J - 6.9 Hz), 7.66- 7.82 (br s, 1 H), 6.11 (br s, 1 H), 5.70 <br s, 1 H), 5.32 (br s, 1 H)₁ 4.63 (br s, 1 H), 4.60 (s, 1H)₁ 4.19 (d, 1H, J = 10.0 Hz), 3.96 (dd, 1H₁ J 5.0, 10.0 Hz), 3.91 (m, 3H), 2.91 (ddd, 1 H, J = 3.7, 7.8, 15.1 Hz), 2.57 (d, 2H, J = 16.7 Hz), 2.50 (d, 2H, J = 16.7 Hz), 1.86 (m, 3H), 1.69 (m, 1H), 1.18-1.61 (m, 16H), 1.10 (s, 6H), 1.01 (s, 3H), 0.95 (s, 9H), 0.89 (m, 2H), 0.87 (s, 3H), 0.71 (m, 2H); ¹³C NMR (CDCI₃, 125 MHz) δ 199.2, 173.5, 172.9, 171.9, 160.7, 158.0, 60.5, 56.6, 51.5, 48.5, 46.8, 39.9, 35.0, 34.2, 31.4, 29.4, 28.1 , 27.8, 27.6, 27.4, 27.3, 27.0, 26.9, 26.5, 26.1 , 23.4, 23.1, 19.4, 13.6, 6.8, 6.7 ppm. Preparative Example 68: Preparation of:

Ste

The N-Boc amine 34 (60 mg) was dissolved in 10 ml_ of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 ml_ of dry dichloromethane and cooled to 0 ⁰C. Then, N-methylmorpholine (2 eq, 0.02 mL, d 0.920) was added. After 10 min, a soln of the isocyanate 56e was added dropwise (1.4 eq, 0.6 mL of a 0.241 M solution in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with aq 1 M HCI (10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 1 :9 to 45:55) to afford the product 68 (44 mg, 56%) as a white solid. ¹H NMR (500 MHz, CDCI₃): δ 8.08 (brs, 1H), 7.52-7,77 (br s, 1 H), 6.06 (br s, 1 H), 5.70 (br s, 1 H), 5.26 (br s, 1 H), 4.63 (m, 2H), 4.20 (d, 1 H, J = 10.0 Hz)₁ 3.97 (dd, 1 H, J = 5.0, 10.0 Hz), 3.92 (m, 3H), 3.43 (m, 2H), 2.57 (d, 2H, J = 16.7 Hz), 2.50 (d, 2H, J = 16.7 Hz), 1.90 (m, 1H), 1.74 (m, 2H), 1.27 (t, 3H, J = 7.2 Hz), 1.20-1.62 (m, 17H)₁ 1.11 (s, 6H), 1.02 (s, 3H), 0.96 (s, 9H), 0.88 (s, 3H); ¹³C NMR (CDCI₃, 125 MHz) 6 199.1 , 173.5, 172.9, 171.8, 159.3, 157.9, 60.6, 56.6, 51.5, 48.5, 46.8, 40.0, 34.9, 34.8, 34.1, 32.8, 29.4, 28.1 , 27.8, 27.5, 27.4, 27.3, 27.0, 26.9, 26.5, 26.0, 25.1 , 23.4, 19.4, 14.8, 13.6 ppm; HRMS calcd for C₃₈H₆₃N₆O₇ [M+H]⁺: 715.4758, found 715.4751. Preparative Example 69: Preparation of:

Ste

The N-Boc amine XX (93 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. AIi the volatiies were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 ⁰C. Then, N-methylmorpholine (2 eq, 0.04 mL, d 0.920) was added. After 10 min, a soln of the isocyanate 59a in toluene was added dropwise (1.2 eq) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with ethy! acetate (50 mL) and washed with aq 1 M HCI (10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetonθ/hexanes; 1:9 to 45:55) to afford the product 69 (45 mg, 38%) as a white solid. ¹H NMR (500 MHz, CDCl₃): 68.26-8.67 (br s, 1 H), 8.17 (br s, 1 H)₁ 6.19 (br s, 1 H), 5.92 (ddt, 1H₁ J = 5.6, 10.4, 17.3 Hz)₁ 5.74 (dd, 1 H, J = 8.8, 9.1 Hz), 5.41 (br s, 1 H)₁ 5.26 (dd, 1 H, J = 1.2, 17.3 Hz), 5.20 (d, 1 H, J = 10.0 Hz), 4.67 (br s, 1 H), 4.62 (s, 1 H), 4.35 (dd, 1 H₁ J = 1.9, 12.9 Hz), 4.20 (d, 1 H, J = 9.8 Hz), 3.99 (m, 4H), 3.58 (ddd, 1 H₁ J = 5.9, 6.9, 12.6 Hz), 3.18 (ddd, 1 H, J = 5.9, 5.9, 11.9 Hz)₁ 2.69 (d, 1 H, J = 10.7 Hz), 2.18 (d, 1 H, J = 17.0 Hz), 2.12 (d, 1H, J = 17.0 Hz), 1.96 (m, 1H)₁ 1.18-1.89 (m, 20H), 1.12 (m, 1 H), 1.04 (s, 3H), 1.03 (s, 3H), 1.00 (s, 3H)₁ 0.92 (s, 9H), 0.91 (s, 3H); ¹³C NMR (CDCI₃, 125 MHz) δ 199.3, 173.3, 171.8, 171.1 , 159.4, 158.2, 133.7, 117.3, 60.4, 55.3, 51.5, 48.3, 46.3, 45.0, 42.3, 35.8, 34.6, 34.0, 31.2, 30.3, 28.6, 27.8, 27.7, 27.6, 27.3, 27.2, 27.0, 26.3, 25.9, 25.4, 23.2, 19.3, 13.5 ppm. Preparative Example 70: Preparation of:

Ste

The N-Boc amine 34 (73 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiies were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichioromethane and cooled to 0 ⁰C. Then, N-methylmorpholine (2 eq, 0.03 mL, d 0.920) was added. After 10 min, a soln of the isocyanate 59a in toluene was added dropwise (1.2 eq) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with aqueous 1 M HCI (10 mL) and brine {10 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 1 :9 to 45:55) to afford the product 70 (63 mg, 69%) as a white solid. ¹H NMR (500 MHz, CDCI₃): δ 8.20-8.43 (br s, 1H)₁ 8.17 (br s, 1H), 6.20 (br s, 1 H), 5.75 (dd, 1 H₁ J - 8.2, 9.4 Hz)₁ 5.41 (br s, 1 H), 4.66 (d, 1 H, J = 9.1 Hz)₁ 4.63 (s, 1 H)₁ 4.36 (dd, 1 H₁ J = 12.6, 13.2 Hz), 4.18 (d, 1 H, J = 10.4 Hz), 3.96 (m, 2H), 3.57 (m, 1H), 3.41 (m, 2H)₁ 3.18 (ddd, 1H₁ J = 5.9, 11.9 Hz)₁ 2.69 (d, 1H, J - 13.2 Hz), 2.19 (d, 1 H, J = 17.0 Hz)₁ 2.14 (d, 1 H, 17.0 HZ)₁ 1.76-1.99 (m, 4H), 1.25 (t, 3H, J = 7.2 Hz), 1.18-1.75 (m, 17H), 1.12 (m, 1H)₁ 1.04 (s, 3H)₁ 1.02 (s, 3H), 1.01 (s, 3H), 0.92 (s, 9H), 0.91 (S₁ 3H); ¹³C NMR (CDCl₃, 125 MHz) δ 199.9, 173.3, 171.8, 171.1 , 159.4, 158.2, 60.4, 55.2, 53.5, 51.5, 48.3, 46.3, 35.8, 34.8, 34.6, 31.3, 30.3, 28.7, 27.8, 27.7, 27.6, 27.3, 27.0, 26.4, 26.0, 23.2, 19.3, 14.8, 13.5 ppm; HRMS calcd for C₃₈H₆₅N₆O₆ [M+H]⁺: 701.4966, found 701.4960. Preparative Example 71 : Preparation of:

Step

A solution of 31 (100 mg, 0.169 mmol) in 4 N. HCI in dioxane (5 ml) was stirred at room temperature for 1 hour. Solvent was removed to dryness to give 71a (120 mg) which was used without further purification Step B:

A solution of 71a (89 mg, 0.169 mmol) in CH₂CI₂ (IO ml) was treated with isocyanate 27b (3 equiv), sat. NaHCO₃ (3 ml) and stirred vigorously for 2 hours. The solution was allowed to stand at 5⁰C for 12 hours. The CH₂CI₂ layer was separated, washed with water, brine and filtered through Na₂SO₄. Solvent was removed to dryness and the residue was purified on silica gel column (40% to 60% acetone/hexanes) to give 71 (73 mg). MS (ES) m/z relative intensity 773 [(M+Na)⁺, 20]; 751 [(M+1 )⁺, 100]. Calcd. for C₃₆H₅₉N₆O₉S [M+1]⁺: 751.4064; Found 751.4075. Preparative Example 72: Preparation of:

Step A:

A solution of 71a (89 mg, 0.169 mmol) in CH₂CI₂(IO ml) was treated with isocyanate 51c (1.5 equiv), sat. NaHCO₃(4 ml) and stirred vigorously for 30 minutes. The solution was allowed to stand at 5 ⁰C for 12 hours. The CH₂C)₂ layer was separated, washed with water, brine and filtered through Na₂SO_4. Solvent was removed to dryness and the residue was purified on silica gel column (40% to 50% acetone/hexanes) to give 72 (95 mg). MS (ES) m/z relative intensity 790 t(M+CH₃OH+1 )⁺, 40]; 758 [(M+1)⁺, 100]. Calcd. for C₃₈H₅₉N₇O₉ [M+1]^÷: 758.4453;

Found 758.4449.

Preparative Example 73: Preparation of:

Z3

Step A:

73a

The N-(tert-Butoxycarbonyl)-L-serine-beta-lactone 73a will be prepared according to the procedure described by Vederas and co-workers (Arnold, L. D.; Kalantar, T. H.; Vederas, J. C. J. Am. Chem. Soc. 1985, 107, 7105-7109) starting from commercially available N-Boc-L-Ser-OH. Step B:

A solution of N-ftert-ButoxylcarbonyO-L-serine-beta-lactone 73a (1 mmol) in 20 ml_ of dry acetonitrile will be added dropwise at ambient temperature over 1 h to a stirred solution of allylamine (25 mmol) in 30 mL dry acetonitrile. After 2 h, the solution will be concentrated under reduced pressure. The residue will be slurried with acetonitriie and the acid product 73b will be recovered by filtration. Step C:

A solution of acid 2 (1 mmol) in aqueous saturated sodium bicarbonate solution (4 mL) and water (1 mL) at room temperature will be treated with benzyl chforoformate (1.12 mmol) in acetone (1 mL). The reaction mixture will be stirred for 2 h. The mixture will be partitioned between ether (20 mL) and water (20 mL). The aqueous layer will be cooled in an ice-water bath, brought to pH 2 using 5% aqueous HCI and extracted with dichloromethane (3 x 30 mL). The combined organic layers will be dried over magnesium sulfate, filtered and concentrated to afford the acid product 73c. Step D:

A solution of acid 73c (1 mmol) in 10 mL of dry dichloromethane and 10 mL of dry DMF will be stirred at 0 ⁰C and treated with HATU (1.4 mmol). The amine hydrochloride (1.3 mmol) and N-methylmorpholine (4 mmoi) will be successively added. The reaction mixture will be gradually warmed to room temperature and stirred overnight. All the volatiles will be removed under vacuum and the residue will be taken into 100 mL of ethyl acetate. The organic layer will be washed with water (20 mL), aqueous 1 N HCI (20 mL), aqueous saturated sodium bicarbonate solution (20 mL), and brine (20 mL). The organic layer will be dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The product 73d will be purified by column chromatography on silica gel. Step E:

A solution of methyl ester 73d (1 mmol) in 15 mL of a mixture of THF/MeOH/H2O (1:1:1) will be treated with lithium hydroxide monohydrate (2.5 mmol) at 0 ⁰C. The cooling bath will be removed and the reaction mixture stirred at room temperature and monitored by TLC (acetone/hexanes; 2:8). After 1 h, 10 mL of aqueous 1 N HCI will be added and all the volatiles will be removed under reduced pressure. The residue will be partitioned between 30 mL of aqueous 1N HCI and 100 mL of dichloromethane. The aqueous layer will be back extracted with dichloromethane (2 x 50 mL). The combined organic layers will be dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the acid product 73e. Step F:

A solution of acid 73e (1 mmol) in 10 mL of dry dichloromethane and 10 mL of dry DMF will be stirred at 0⁰C and treated with HATU (1.4 eq, 1.15 g). The amine hydrochloride 7 (1.2 mmol) will be added in 10 mL of dichloromethane followed by N- methylmorpholine (4 mmol). The reaction mixture will be stirred overnight (temp from 0 to 25 ⁰C). All the volatiles will be removed under vacuum and the residue will be dissolved in 100 mL of ethyl acetate. The organic layer will be washed with water (20 mL), aqueous 1 N HCl (20 mL), aqueous saturated sodium bicarbonate solution (20 mL), and brine (20 mL). The organic layer will be dried over magnesium sulfate, filtered and concentrated under reduced pressure. The product 73f will be purified by column chromatography on siiica gel. Step G:

A 0.01 M solution of diene 73f (1 mmol) in toluene will be degassed for 30 min

(argon bubbling) and treated with Grubb's catalyst (0.2 mmol). The pink solution will be heated to 60⁰C for 16 h. The solvent will be removed under reduced pressure and the residue wiil chromatographed on silica gel to afford the alkene product 73g as a mixture of E- and Z-isomers. Step H:

A solution of alkene 73g (1 mmol) in 20 mL of methanol will be treated with 5% palladium on carbon (0.1 mol%). The mixture will be hydrogenated at 50 psi until all the starting material is consumed. The reaction mixture wiil be diluted with 100 mL of dichloromethane and filtered thru a short path of celite. The filtrate will be concentrated and the product 73h will be purified by column chromatography on silica gel.

Step I:

A solution of macrocyclic amine 73h (1 mmol) in 10 ml_ of dichloromethane will be treated with potassium carbonate (2 mmol) and (trimethylsϊlyl)ethanesuifonyl chloride (1 mmol). The mixture is stirred for 1 day and solvent will be evaporated. The product 73i will be purified by column chromatography on silica gel. Step J:

A solution of methyl ester 73i (1 mmol) in 10 mL of dry THF will be treated with lithium borohydride (2.1 mmol). The reaction mixture will be stirred at room temperature. After 5 h, the excess lithium borohydride will be quenched by addition of aqueous saturated ammonium chloride solution (3 mL). The mixture will be partitioned between ethyl acetate (50 mL) and aqueous saturated sodium bicarbonate solution (30 mL). The aqueous layer will be back extracted with ethyl acetate (2 x 30 mL) and dichloromethane (2 x 30 mL). The combined organic layers will be dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue will be chromatographed on silica gel to afford the product 73j. Step K:

A solution of alcohol 73j (1 mmol) in 20 ml_ of dry dichioromethane wiii be treated with Dess-Martin periodinane (1.5 mmol). The reaction mixture will be stirred at room temperature for 45 min. The mixture will be treated with aqueous 1 M sodium thiosuifate solution (10 ml_) and aqueous saturated sodium bicarbonate solution (20 ml_) and stirred for 15 min. The mixture will be extracted with dichioromethane (3 x 40 ml_). The combined organic layers will be dried over magnesium sulfate, filtered, and concentrated. The residue will be chromatographed on silica gel to afford the aldehyde product 73k. Step L:

73k °>^s^^< ° r

A solution of aldehyde 73k (1 mmol) in 10 mL of dry dichioromethane will be treated with ailylisocyanide (2 mmol) and acetic acid (2 mmol). The mixture will be stirred for about 5 h. All the volatiles wiii be removed under vacuum and the residue wil) be chromatographed on silica gel to afford the acetate product 731. Step M:

The acetate 731 (1 mmol) will be dissolved in 16 mL of a 1 :1 mixture of THF/water and treated with lithium hydroxide monohydrate (2.5 mmol). After 30 min the mixture will be partitioned between dichioromethane (50 mL) and aqueous saturated sodium bicarbonate solution (20 mL). The aqueous layer will be back extracted with dichioromethane (3 x 30 mL). The combined organic layers will be dried over magnesium sulfate, filtered, and concentrated. The hydroxyamide product 73m will be used without further purification. Step N:

A solution of hydroxyamide 73m (1 mmol) in 20 mL of dry dichloromethane will be treated with Dess-Martin periodinane (2.5 mmol). The reaction mixture will be stirred at room temperature for 30 min. The mixture will be treated with aqueous 1 M sodium thiosulfate solution (20 mL) and aqueous saturated sodium bicarbonate solution (10 mL) and stirred for 15 min. The mixture will be extracted with dichloromethane (3 x 30 mL). The combined organic layers will be dried over magnesium sulfate, filtered, and concentrated. The ketoamide product 73n will be purified by column chromatography. Step O:

The N-Boc protected amine 73n (0.1 mmol) will be dissolved in 5 mL of 4M HCl solution in dioxanes. The resulting solution will be stirred for 30 min and then evaporated under reduced pressure to give the amine hydrochloride product 73o.

Step P:

The amine hydrochloride 73o (0.1 mmol) will be dissolved in 5 mL of dichloromethane and treated with 20 drops of aqueous saturated sodium bicarbonate solution followed by a solution of isocyanate 51c (0.12 mmol) in toluene. The mixture will be stirred for 5 h and then diluted with 50 mL of dichloromethane and dried over magnesium sulfate. The mixture wiil be filtered, and concentrated under reduced pressure. The product 73p wiil be purified by column chromatography on silica gel. Step Q:

The SES-protected amine 73p (0.1 mmol) will be dissolved in 2 mL of DMF and treated with cesium fluoride (0.4 mmol). The reaction mixture wiil be stirred at room temperature for 4 h and poured onto water (10 mL). The mixture wiil be extracted with ethyl acetate (3 x 20 mL). The combined organic layers will be dried over magnesium sulfate, filtered and concentrated under reduced pressure. The macrocyclic amine 73 will be purified by column chromatography on silica gel. Preparative Example 74: Preparation of:

Step A:

The N-Boc amine 65n (60 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 ⁰C. Then, 20 drops of aqueous saturated sodium bicarbonate solution were added followed by a soin. of the isocyanate 27b in toluene (1.2 eq, 0.6 mL of a 0.2M soln. in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with dichloromethane (60 mL) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 1:9 to 1 :1) to afford the product 74 (45 mg, 59%) as a white solid. ¹H NMR (500 MHz, CDCI₃): 68.11 (br s, 1H)₁ 7.40-7.69 (br s, 1H), 6.08-6.43 (br s, 1 H), 5.91 (ddt, 1 H₁ J = 5.6, 10.4, 17.3 Hz), 5.70 (br s, 1H)₁ 5.29 (dd, 1 H, J = 1.2, 17.3 Hz), 5.24 (dd, 1 H, J = 1.2, 10.4 Hz)₁ 4.66 (d, 1H, J = 9.4 Hz), 4.46 (br s, 1H), 4.38 (m, 1H), 4.25 (d, 1 H, J = 10.7 Hz), 4.06 (m, 2H)₁ 3.98 (m, 1 H), 3.71 (dq, 5.6, 11.6 Hz), 3.64 (ddd, 1 H, J = 5.0, 5.3, 9.7 Hz), 3.25 (m, 1H), 2.88 (d, 1H, J = 13.5 Hz), 2.47 (br s, 1 H), 2.19 (d, 1H, J = 11.6 Hz), 1.39 (s, 9H), 1.28-1.99 (m, 22H), 1.21 (d, 3H₁ J = 5.6 Hz), 1.13 (m, 1H), 1.04 (s, 3H), 0.91 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz) δ 198.7, 173.0, 171.3, 159.4, 157.0, 133.4, 117.7, 75.2, 67.7, 61.2, 60.8, 55.6, 54.6, 48.7, 42.2, 36.3, 31.6, 28.9, 27.9, 27.8, 27.1 , 26.8, 25.9, 24.8, 24.0, 23.6, 21.9, 21.5, 19.4, 15.8, 13.4 ppm; HRMS calcd for C₃₇H₆₂N₅O₈S [M+H]⁺: 736.4319, found 736.4325. Preparative Example 75: Preparation of: Step

A solution of aidehyde 65k (710 mg) in 30 mL of dry dichloromethane was treated with cyclopropylisocyanide (Oakwood Prod., 2.0 eq, 0.25 mL, d 0.8) and acetic acid (2 eq, 0.16 mL, d 1.049). The mixture was stirred at room temp for 5 h. All the volatiles were removed under reduced pressure and the residue was chromatographed on silica gel (gradient: acetone/hexanes; 15:85 to 55:45) to afford the product 75a (740 mg, 83%) as a white solid. Step B:

A solution of acetate 75a (740 mg) in 20 mL of a 2:1 mixture of THF/water was treated with lithium hydroxide monohydrate (2.5 eq, 125 mg) and stirred for approx 30 min until ail the starting material had been consumed as determined by TLC analysis (ethyl acetate/hexanes; 8:2). The reaction mixture was diluted with 50 mL of aqueous saturated sodium bicarbonate solution and extracted with dichloromethane (3 x 80 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated to afford the product 75b (688 mg, 98%) as a colorless semi-solid which was used without further purification. Step C:

A solution of hydroxyamide 75b (1.192 mmol) in 25 ml_ of dry dichioromethane was treated with Dess-Martin periodinane (2.0 eq, 1.01 g). The reaction mixture was stirred at room temperature for 30 min. The mixture was treated with aqueous 1 M sodium thiosulfate solution (30 ml_) and stirred for 5 min. Aqueous saturated sodium bicarbonate solution (30 ml_) was also added and stirring was continued for further 10 min. The mixture was extracted with dichioromethane (3 x 80 nriL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 5:95 to 4:6) to afford the product 75c (476 mg, 69%) as white solid. Step D:

The N-Boc amine 75c (60 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichioromethane and cooled to 0 °C. Then, 10 drops of aqueous saturated sodium bicarbonate solution were added. After 10 min, a soln of the isocyanate 56e was added dropwise (1.2 eq, 0.59 mL of a 0.216M solution in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with dichioromethane (70 mL) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 1 :9 to 1 :1) to afford the product 75 (41 mg, 53%) as a white solid. ¹H NMR (500 MHz, CDCI₃): δ 8.12 (br s, 1 H), 7.40-7.70 (br s, 1 H), 6.28 (br s, 1 H), 5.68 (br s, 1 H), 5.37 (br s, 1H), 4.62 (s, 1H)₁ 4.49 (br s, 1H), 4.22 (d, 1H₁ J = 10.7 Hz), 4.05 <dd, 1 H, J = 5.0, 10.4 Hz), 3.94 (d, 1 H₁ J = 1.6 Hz), 3.88 (dd, 1 H, J = 10.4, 10.7 Hz)₁ 3.82 (q, 1 H, J = 11.0 Hz), 3.69 (m, 1 H), 3.62 (ddd, 1 H, J = 5.0, 5.6, 9.4 Hz), 3.20 (m, 1 H)₁ 2.89 (ddd, 1H₁ J= 3.4, 7.2, 14.8 Hz)₁ 2.55 (d, 2H₁ J= 17.0 Hz), 2.48 (d, 2H, J = 17.0 Hz), 1.79- 1.99 (m, 4H), 1.28-1.69 (m, 10H), 1.14 (d, 3H, J = 6.0 Hz), 1.10 (s, 6H), 1.00 (s, 3H), 0.99 (s, 9H), 0.90 (m, 2H), 0.83 (s, 3H), 0.71 (m, 2H); ¹³C NMR (CDCI₃, 125 MHz) 6 198.8, 172.8, 171.5, 160.9, 157.8, 75.5, 68.1, 60.8, 57.2, 55.9, 48.7, 46.8, 35.2, 29.3, 28.6, 28.3, 27.7, 26.9, 26.8, 24.8, 24.4, 23.1, 19.3, 16.3, 13.6, 6.8 ppm; HRMS calcd for C₃₉H₆₃N₆O₈ [M+Hf: 743.4707, found 743.4686. Preparative Example 76: Preparation of:

Ste

The N-Boc amine 75c (60 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum overnight. The resulting amine salt was dissolved in 5 mL of dichloromethane and cooled to 0 ⁰C. Then, 10 drops of aqueous saturated sodium bicarbonate solution were added. After 10 min, a soln. of the isocyanate 63e was added dropwise (1.2 eq, 0.95 mL of a 0.131 M solution in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 3 h. The reaction mixture was diluted with dichloromethane (70 mL) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 6:4) to afford the product 76 (54 mg, 72%) as a white solid. ¹H NMR (500 MHz, CDCI₃): δ 8.22 (br s, 1H), 7.58 (br s, 1H), 6.13 (br s, 1H), 5.75 (br s, 1 H), 5.15 (d, 1H, J = 8.5 Hz), 4.68 (br s, 1H)₁ 4.56 (s, 1H), 4.28 (d, 1H₁ J = 10.7 Hz), 4.06 (dd, 1H, J = 4.7, 10.4 Hz)₁ 3.99 (dd, 1 H, J = 9.1, 9.7 Hz), 3.60 (m, 2H), 3.47 (dd, 1H₁ J 12.2, 13.2 Hz), 3.19 (m, 1 H), 3.07 (m, 3H), 2.94 (s, 3H), 2.87 (ddd, 1 H, J = 4.0, 7.8, 15.1 Hz), 1.72-1.99 (m, 4H), 1.37 (t, 3H, J = 7.5 Hz), 1.27-1.68 (m, 9H), 1.21 (d, 3H, J = 6.0 Hz), 1.13 (m, 1H), 1.01 (s, 3H), 0.92 (s, 9H), 0.89 (s, 3H), 0.87 (m, 2H), 0.71 (m, 2H); ¹³C NMR (CDCI₃, 125 MHz) δ 199.5, 172.8, 171.6, 160.8, 158.1, 75.8, 68.4, 60.6, 56.2, 54.4, 50.4, 48.5, 45.7, 34.7, 34.5, 32.1 , 31.6, 28.6, 27.8, 27.7, 27.0, 26.9, 26.7, 24.9, 24.6, 23.0, 19.2, 16.2, 13.5, 8.5, 6.7 ppm; HRMS calcd for C₃₅H_6IN₆O₈S [M+Hf: 725.4272, found 725.4292. Preparative Example 77: Preparation of:

Ste

The N-Boc amine 75c (60 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dich Io ro methane and cooled to 0 ⁰C. Then, 20 drops of aqueous saturated sodium bicarbonate solution were added followed by a soln of the isocyanate 27b in toluene (1.2 eq, 0.6 mL of a 0.2M soln in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 2 h. The reaction mixture was diluted with dichioromethane (60 mL) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on siiica gel (gradient: acetone/hexanes; 2:8 to 6:4) to afford the product 77 (50 mg, 65%) as a white solid. ¹H NMR (500 MHz₁ CDCi₃): δ 8.12 (br s, 1 H), 7.33-7.63 (br s, 1 H), 6.07-6.47 (br s, 1 H), 5.67 (br s, 1 H), 4.65 (d, 1 H, J = 9.7 Hz), 4.45 (br s, 1H)₁ 4.37 (m, 1H), 4.24 (d, 1 H, J = 10.7 Hz), 4.07 (dd, 1 H₁ J =5.0, 10.7 Hz), 3.70 (clq, 1 H, 5.9, 9.7 Hz), 3.64 (ddd, 1 H, J - 5.0, 5.6, 9.7 Hz), 3.24 (m, 1 H), 2.89 (ddd, 1 H, J - 3.7, 73, 14.5 Hz), 2.88 (m, 1H)₁ 2.47 (br s_r 1H), 2.18 (d, 1 H, J= 12.6 Hz)₁ 1.74-1.97 (m, 5H), 1.39 (s, 9H), 1.27-1.73 (m, 17H), 1.20 (d, 3H, J = 6.3 Hz), 1.11 (m, 1H), 1.04 (s, 3H), 0.91 (s, 3H), 0.90 (m, 2H), 0.73 (m, 2H); ¹³C NMR (CDCI₃, 125 MHz) δ 198.7, 173.0, 171.3, 161.0, 157.0, 75.2, 67.8, 61.1 , 60.8, 55.5, 54.6, 50.1 , 48.6, 36.3, 31.6, 28.8, 27.9, 27.0, 26.9, 25.9, 24.8, 24.0, 23.6, 23.1, 21.9, 21.5, 19.4, 15.8, 13.4, 6.9, 6.8 ppm. HRMS calcd for C₃₇H₆₂N₅O₈S [M+1f: 736.4319, found 736.4329. Preparative Example 78

Ste

The N-Boc amine 65n (60 mg) was dissolved in 10 ml_ of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. Ail the voiatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 ⁰C. Then, 20 drops of aqueous saturated sodium bicarbonate solution were added followed by a solution of the isocyanate 59a in toluene (1.2 eq, 0.6 mL of a 0.2M soln in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 3 h. The reaction mixture was diluted with dichloromethane (60 mi_) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/(hexanes-dichloromethane; 1:1); 1:9 to 1:1) to afford the product 78 (51 mg, 67%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) δ 8.17 (br s, 1 H), 6.42-6.79 (br s, 1 H), 5.90 (ddt, 1 H, J = 5.6, 10.7, 17.0 Hz), 5.73 (br s, 1H), 5.57 (br s, 1 H), 5.27 (d, 1 H, J = 17.0 Hz), 5.22 (d, 1 H, J = 10.0 Hz), 4.62 (dd, 1 H, J = 9.1 , 9.7 Hz), 4.52 (br s, 1H), 4.29 (m, 2H), 3.86-4.11 (m, 4H)₁ 3.64 (m, 3H), 3.17 (m, 2H), 2.74 (d, 1 H, J = 11.9 Hz), 2.24 (d, 1 H, J = 17.3 Hz), 2.10 (d, 1H, J = 17.0 Hz), 1.95 (m, 4H), 1.24-1.68 (m, 11 H), 1.16 (d, 3H, J = 5.9 Hz), 1.11 (m, 1 H), 1.02 (s, 3H), 1.01 (s, 6H)₁ 0.95 (s, 9H), 0.87 (s, 3H); ¹³C NMR (CDCI₃, 125 MHz) δ 196.8, 172.9, 171.6, 170.7, 159.4, 158.2, 133.6, 117.5, 75.7, 68.2, 60.8, 56.0, 55.3, 48.5, 46.8, 46.2, 44.9, 42.3, 35.7, 34.8, 32.3, 31.6, 30.2, 28.6, 28.4, 27.8, 27.7, 27.1 , 27.0, 26.8, 24.8, 24.5, 19.3, 16.5, 13.6 ppm. HRMS caicd for C₃₉H₆₅N₆O₇ [M+1]^*: 729.4915, found 729.4917. Preparative Example 79

Ste

The N-Boc amine 75c (60 mg) was dissolved in 10 ml_ of 4M HCl solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiies were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 rnL of dry dichloromethane and cooled to 0 °C. Then, 20 drops of aqueous saturated sodium bicarbonate solution were added followed by a solution of the isocyanate 59a in toluene (1.2 eq, 0.6 ml_ of a 0.2M soln in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 3 h. The reaction mixture was diluted with dichloromethane (60 mL) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/(hexanes-dichloromethane, 1 :1); 1:9 to 1 :1) to afford the product 79 (36 mg, 48%) as a white solid. ¹H NMR

(CDCI₃, 500 MHz) δ 8.19 (br s, 1 H), 6.38-6.70 (br s, 1 H), 5.73 (br S₁ 1 H)₁ 5.55 (d, 1 H, J = 7.8 Hz), 4.61 (t, 1 H, J - 9.7 Hz), 4.51 (br s, 1 H), 4.27 (m, 2H), 4.05 (dd, 1 H, J = 5.0, 10.4 Hz), 3.95 (dd, 1 H, J - 9.4, 9.7 Hz); 3.62 (m, 3H)₁ 3.18 (m, 2H), 2.90 (ddd, 1 H, J = 3.7, 7.2, 14.8 Hz), 2.73 (d, 1 H, J = 12.6 Hz), 2.21 (d, 1 H₁ J = 17.0 Hz)₁ 2.09 (d, 1 H, J = 17.3 Hz), 1.93 (br s, 4H), 1.27-1.68 (m, 11H), 1.15 (d, 3H, J = 5.9 Hz)₁ 1.11 (m, 1H)₁ 1.02 (s, 3H), 1.00 (s, 6H), 0.94 (s, 9H), 0.87 (m, 2H), 0.86 (s, 3H), 0.73 (m, 2H); ¹³C NMR (CDCi₃, 125 MHz) δ 197.1 , 172.9, 171.6, 170.6, 160.9, 158.2, 75.6, 68.3, 60.7, 56.0, 55.3, 48.5, 46.7, 46.3, 44.9, 35.7, 34.8, 32.4, 31.6, 30.2, 29.7, 28.6, 28.5, 27.8, 27.7, 27.0, 26.7, 24.8, 24.7, 23.1 , 19.3, 16.5, 13.6, 6.7, 6.6 ppm. HRMS caicd for C₃₉H₆₅N₆O₇ [M+1]⁺: 729.4915, found 729.4926. Preparative Example 80

Ste

The N-Boc amine 52 (60 mg) was dissolved in 10 mL of 4M HCi solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 ⁰C. Then, 20 drops of aqueous saturated sodium bicarbonate solution were added followed by a solution of the isocyanate 80a in toluene (1.2 eq, 0.8 ml_ of a 0.155M soln in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 3 h. The reaction mixture was diluted with dichloromethane {60 mL) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 6:4) to afford the product 80 (41 mg, 61 %) as a white solid. ¹H NMR (CDCI₃, 500 MHz) 6, 8.01 (d, 1H₁ J = 8.2 Hz)₁ 7.65 (br s, 1 H), 6.01 (br s, 1 H), 5.91 (ddt, 1 H, J = 5.6, 10.0, 17.0 Hz), 5.68 (dd, 1 H, J = 9.1 , 9.4 Hz), 5.27 (dd, 1 H, J = 1.2, 17.0 Hz), 5.23 (dd, 1 H, J = 1.2, 10.0 Hz), 5.20 (m, 1 H), 4.98 (br s, 1 H); 4.59 (s, 1 H), 4.13 (d, 1 H₁ J = 10.7 Hz), 4.01 (m, 3H), 3.89 (ddd, 1 H, J = 2.2, 10.4, 10.7 Hz)₁ 3.79 (dd, 1 H, J = 3.4, 12.9 Hz), 3.76 (m, 1 H), 3.56 (m, 2H), 3.36 (dd, 1 H, J = 4.1 , 7.5 Hz), 3.31 (m, 1H), 3.18 (brs, 1 H), 3.14 (br s, 1H), 2.22 (d, 1 H, J = 10.7 Hz), 2.07 (br s, 2H), 1.73-2.00 (m, 5H), 1.25-1.70 (m, 11 H), 1.16 (m, 1 H)₁ 1.02 (s, 3H)₁ 0.96 (s, 9H), 0.86 (s, 3H); ¹³C NMR (CDCI₃, 125 MHz) δ 198.8, 177.4, 172.1 , 171.3, 159.3, 157.9, 133.4, 117.7, 71.4, 70.7, 60.7, 56.7, 53.4, 50.8, 48.6, 45.4, 45.2, 42.2, 39.4, 34.7, 33.1 , 32.2, 31.0, 28.7, 27.7, 27.5, 27.3, 26.9, 24.3, 19.3, 13.5 ppm. HRMS calcd for C₃₈H₅₉N₆O₈ [M+1]⁺: 727.4394, found 727.4387. Preparative Example 81

Ste

The N-Boc amine 65n (60 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 ⁰C. Then, 20 drops of aqueous saturated sodium bicarbonate solution were added followed by a solution of the isocyanate 80a in toluene (1.2 eq, 0.8 mL of a 0.155M soln in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 3 h. The reaction mixture was diluted with dichioromethane (60 mL) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica ge! (gradient: acetone/hexanes; 2:8 to 6:4) to afford the product 81 (54 mg, 70%) as a white solid. ¹H NMR (CDCI₃, 500 MHz) δ, 8.12 (br s, 1H)₁ 7.39-7.79 (br s, 1 H), 6.29 (br s, 1 H), 5.91 (ddt, 1H, J = 5.9, 10.4, 17.0 Hz), 5.71 (br s, 1H), 5.40 (br s, 1H), 5.27 (dd, 1H₁ J = 1.2, 17.0 Hz), 5.23 (dd, 1H₁ J = 1.2, 10.4 Hz), 4.67 (dd, 1 H₁ J = 7.8, 8.1 Hz); 4.50 (br s, 1 H), 4.24 (d, 1 H, J = 10.7 Hz), 4.07 (dd, 1 H, J = 5.3, 10.4 Hz), 4.03 (m, 1 H)₁ 3.97 (ddd, 1 H, J = 5.6, 5.9, 15.7 Hz), 3.81 (m, 2H)₁ 3.73 (m, 1 H)₁ 3.67 (d, 1 H, J = 12.2 Hz), 3.62 (m, 1 H), 3.20 (s, 2H), 3.07 (s, 1 H), 2.29 (d, 1 H, J = 11.0 Hz), 2.07 (br s, 3H), 1.93 (br s, 2H), 1.83 (br s, 3H), 1.28-1.68 (m, 10H), 1.17 (d, 3H, J = 5.9 Hz)₁ 1.11 (m, 1 H), 1.01 (s, 3H), 0.99 (s, 9H), 0.83 (s, 3H); ¹³C NMR (CDCI₃, 125 MHz) 6 198.5, 177.1 , 172.8, 171.5, 159.4, 158.0, 133.4, 117.7, 75.5, 68.1, 60.8, 57.4, 55.9, 48.7, 45.4, 42.3, 40.4, 34.8, 32.8, 31.7, 28.6, 27.8, 27.6, 27.4, 26.9, 26.8, 24.7, 24.4, 19.4, 16.2, 13.5 ppm. HRMS calcd for C₃₉H_6IN₆O₃ [M+1]⁺: 741.4551 , found 741.4543. Preparative Example 82

Step A:

The N-Boc amine 75c (60 mg) was dissolved in 10 mL of 4M HCl solution in dioxanes. The resulting solution was stirred at room temperature for 30 min. Ail the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 ⁰C. Then, 20 drops of aqueous saturated sodium bicarbonate solution were added followed by a solution of the isocyanate 80a in toluene (1.2 eq, 0.8 mL of a 0.155M soln in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 3 h. The reaction mixture was diluted with dichloromethane (60 mL) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 2:8 to 6:4) to afford the product 82 (50 mg, 65%) as a white solid. ¹H NMR (CDCI_3, 500 MHz) δ 8.12 (br s, 1 H), 7.38-7.68 (br s, 1H), 6.28 (br s, 1 H), 5.68 (br s, 1 H)₁ 5.39 (br s, 1 H), 4.66 (dd, 1 H, J = 7.5, 7.5 Hz), 4.49 (br s, 1 H)₁ 4.23 (d, 1 H₁ J = 10.4 Hz)₁ 4.06 (dd, 1 H₁ J = 5.0, 10.4 Hz); 3.81 (m, 2H), 3.71 (m, 1H), 3.67 (d, 1H₁ J = 12.2 Hz)₁ 3.61 (m, 1H)₁ 3.19 (br s, 2H)₁ 3.07 (S₁ 1 H)₁ 2.89 (ddd, 1 H₁ J = 3.7, 7.5, 14.8 Hz)₁ 2.29 (d, 1 H, J = 11.0 Hz), 1.98- 2.13 (m, 3H)₁ 1.75-1.96 (m, 6H)₁ 1.26-1.67 (m, 9H), 1.17 (d, 3H, J = 5.9 Hz)₁ 1.10 (m, 1 H)₁ 1.00 (s, 3H)₁ 0.98 (s, 9H), 0.90 (m, 2H)₁ 0.83 (s, 3H), 0.72 (m, 2H); ¹³C NMR (CDCI₃, 125 MHz) δ 198.9, 178.8, 172.8, 171.5, 160.9, 158.0, 75.5, 68.1 , 60.8, 57.4, 55.9, 48.7, 45.4, 40.2, 34.8, 32.8, 31.7, 28.6, 27.8, 27.7, 27.4, 26.9, 26.8, 24.8, 24.4, 23.1, 19.3, 16.2, 13.5, 6.9, 6.8 ppm. Preparative Example 83

Step A:

The N-Boc amine 33 (60 mg) was dissolved in 10 mL of 4M HCI solution in dioxanes.

The resulting solution was stirred at room temperature for 30 min. All the volatiles were removed under reduced pressure and the residue was placed under high vacuum for 3 h. The resulting amine salt was dissolved in 5 mL of dry dichloromethane and cooled to 0 ⁰C. Then, 20 drops of aqueous saturated sodium bicarbonate solution were added followed by a solution of the isocyanate 80a in toluene (1.2 eq, 0.8 mL of a 0.155M so!n in toluene) and stirring was continued for 10 min. The cooling bath was removed and the mixture was stirred at room temp for 3 h.

The reaction mixture was diluted with dichloromethane (60 mL) and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (gradient: acetone/hexanes; 1 :9 to 1 :1) to afford the product 83 (63 mg, 81%) as a white solid.

Example compounds are shown in Tablei. The Ki values for the compounds are rated as follows: Category "A" for Ki values less than 10OnM, category "B" for Ki values greater than or equal to 10OnM but less than 1 μM and category "C" for Ki values greater than or equal to i μM. Table 1

The compounds of Table 1 have the following binding activity. Compounds 1Z-10Z, 12Z-51Z, 53Z-57Z, 59Z-100Z, 103Z-110Z, 112Z, 115Z-117Z, 119Z-122Z, 124Z₁ 125Z₁ 127Z and 128Z have category A binding activity. Compounds 52Z, 58Z, 102Z, 111 Z, 114Z, 118Z₁ and 123Z have category B binding activity. Compound 113Z has category C binding activity.

The present invention relates to novel HCV protease inhibitors. This utility is manifested in their ability to inhibit the HCV NS2/NS4a serine protease as demonstrated by the following in vitro assays. Assay for HCV Protease .inhibitory Activity;

Spectrophotometry Assay: Spectrophotometry assays for the HCV serine protease was performed on the inventive compounds by foliowing the procedure described by R. Zhang et al, Analytical Biochemistry, 270 (1999) 268-275, the disclosure of which is incorporated herein by reference. The assay based on the proteolysis of chromogenic ester substrates is suitable for the continuous monitoring of HCV NS3 protease activity. The substrates were derived from the P side of the NS5A-NS5B junction sequence (Ac-DTEDWX(Nva), where X = A or P) whose C-terminat carboxyl groups were esterified with one of four different chromophoric alcohols (3- or 4-nitrophenol, 7- hydroxy-4-methyl-coumarin, or 4-phenylazophenol). Presented below are the synthesis, characterization and application of these novel spectrophotometric ester substrates to high throughput screening and detailed kinetic evaluation of HCV NS3 protease inhibitors. Materials and Methods:

Materials: Chemical reagents for assay related buffers were obtained from Sigma Chemical Company (St. Louis, Missouri). Reagents for peptide synthesis were from Aldrich Chemicals, Novabiochem (San Diego, California), Applied Biosystems (Foster City, California) and Perseptive Biosystems (Framingham, Massachusetts). Peptides were synthesized manually or on an automated ABl model 431 A synthesizer (from Applied Biosystems). UV/V1S Spectrometer model LAMBDA 12 was from Perkin Elmer (Norwaik, Connecticut) and 96-well UV plates were obtained from Coming (Corning, New York). The prewarmtng block was from USA Scientific {Ocala, Florida) and the 96-well plate vortexer was from Labline Instruments (Meirose Park, Illinois). A Spectramax Plus microtiter plate reader with monochrometer was obtained from Molecular Devices (Sunnyvale, California).

Enzyme Preparation: Recombinant heterodimeric HCV NS3/NS4A protease (strain 1a) was prepared by using the procedures published previously (D. L. SaIi et al, Biochemistry, 37 (1998) 3392-3401 ). Protein concentrations were determined by the Biorad dye method using recombinant HCV protease standards previously quantified by amino acid analysis. Prior to assay initiation, the enzyme storage buffer (50 mM sodium phosphate pH 8.0, 300 mM NaCI, 10% glycerol, 0.05% lauryl maltoside and 10 mM DTT) was exchanged for the assay buffer (25 mM MOPS pH 6.5, 300 mM NaCI, 10% glycerol, 0.05% lauryl maltoside, 5 μM EDTA and 5 μM DTT) utilizing a Biorad Bio-Spin P-6 prepacked column.

Substrate Synthesis and Purification: The synthesis of the substrates was done as reported by R. Zhang et al, (ibid.) and was initiated by anchoring Fmoc-Nva-OH to 2- chlorotrityl chloride resin using a standard protocol (K. Barlos et al, Int. J. Pept Protein Res., 37 (1991), 513-520). The peptides were subsequently assembled, using Fmoc chemistry, either manually or on an automatic ABI model 431 peptide synthesizer. The N-acetylated and fully protected peptide fragments were cleaved from the resin either by 10% acetic acid (HOAc) and 10% trifluoroethanol (TFE) in dichloromethane (DCM) for 30 min, or by 2% trifluoroacetic acid (TFA) in DCM for 10 min. The combined filtrate and DCM wash was evaporated azeotropically (or repeatedly extracted by aqueous Na2CO3 solution) to remove the acid used in cleavage. The DCM phase was dried over Na2SO4 and evaporated.

The ester substrates were assembled using standard acid-alcohol coupling procedures (K. Hoimber et al, Acta Chem, Scand., B33 (1979) 410-412). Peptide fragments were dissolved in anhydrous pyridine (30-60 mg/ml) to which 10 molar equivalents of chromophore and a catalytic amount (0.1 eq.) of para-toluenesulfonic acid (pTSA) were added. Dicyclohexylcarbodiimide (DCC, 3 eq.) was added to initiate the coupling reactions. Product formation was monitored by HPLC and found to be complete following 12-72 hour reaction at room temperature. Pyridine solvent was evaporated under vacuum and further removed by azeotropic evaporation with toluene. The peptide ester was deprotected with 95% TFA in DCM for two hours and extracted three times with anhydrous ethyl ether to remove excess chromophore. The deprotected substrate was purified by reversed phase HPLC on a C3 or C8 column with a 30% to 60% acetonitrile gradient (using six column volumes). The overall yield following HPLC purification was approximately 20-30%. The molecular mass was confirmed by electrospray ionization mass spectroscopy. The substrates were stored in dry powder form under desiccation. Spectra of Substrates and Products: Spectra of substrates and the corresponding chromophore products were obtained in the pH 6.5 assay buffer. Extinction coefficients were determined at the optimal off-peak wavelength in 1-cm cuvettes (340 nm for 3-Np and HMC, 370 nm for PAP and 400 nm for 4-Np) using multiple dilutions. The optimal off-peak wavelength was defined as that wavelength yielding the maximum fractional difference in absorbance between substrate and product (product OD - substrate OD)/substrate OD).

Protease Assay: HCV protease assays were performed at 30⁰C using a 200 μl reaction mix in a 96-well microtiter plate. Assay buffer conditions (25 mM MOPS pH 6.5, 300 mM NaC!, 10% glycerol, 0.05% lauryl maltoside, 5 μM EDTA and 5 μM DTT) were optimized for the NS3/NS4A heterodimer (D. L. SaIi et al, ibid.)). Typically, 150 μl mixtures of buffer, substrate and inhibitor were placed in wells (final concentration of DMSO <4 % v/v) and allowed to preincubate at 30 ⁰C for approximately 3 minutes. Fifty μls of prewarmed protease (12 nM, 30⁰C) in assay buffer, was then used to initiate the reaction (final volume 200 μl).The plates were monitored over the length of the assay (60 minutes) for change in absorbance at the appropriate wavelength (340 nm for 3-Np and HMC, 370 nm for PAP, and 400 nm for 4-Np) using a Spectromax Plus microtiter plate reader equipped with a monochrometer (acceptable results can be obtained with plate readers that utilize cutoff filters). Proteolytic cleavage of the ester linkage between the Nva and the chromophore was monitored at the appropriate wavelength against a no enzyme blank as a control for non-enzymatic hydrolysis. The evaluation of substrate kinetic parameters was performed over a 30-fold substrate concentration range (~6-200 μM). Initial velocities were determined using linear regression and kinetic constants were obtained by fitting the data to the Michaelis- Menten equation using non-linear regression analysis (Mac Curve Fit 1.1, K. Raner). Turnover numbers (/feat) were calculated assuming the enzyme was fully active. Evaluation of Inhibitors and Inactivators: The inhibition constants (Ki*) for the competitive inhibitors Ac-D-<D-GIa)-L-l-(Cha)-C-OH (27), Ac-DTEDWA(Nva)-OH and Ac-DTEDWP(Nva)-OH were determined experimentally at fixed concentrations of enzyme and substrate by plotting Vo/vj vs. inhibitor concentration ([I] ₀) according to the rearranged Michaelis-Menten equation for competitive inhibition kinetics: v_o/vj = 1 ⁺ EU o /(Kj (1 + [S] o /Km)), where VQ is the uninhibited initial velocity, Vj is the initial velocity in the presence of inhibitor at any given inhibitor concentration ([l]o) and [S]o is the substrate concentration used. The resulting data were fitted using linear regression and the resulting slope, 1/(K[(1+[S] c/Km). was used to calculate the Ki* value.

The obtained Kj values for the various macrocycles of the present invention are given in Table 1. From these test results, it would be apparent to the skilled artisan that the compounds of the invention have excellent utility as NS3-serine protease inhibitors.

Claims

CLAIMSWhat is claimed is:

1. A compound exhibiting HCV protease inhibitory activity, or an enantiomer, stereoisomer, rotamer, tautomer, or racemate of said compound, or a pharmaceutically acceptable salt or solvate or ester of said compound or of said enantiomer, stereoisomer, rotamer, tautomer, or racemate, said compound being selected from the group consisting of the compounds of structures listed below:

2. A pharmaceutical composition comprising as an active ingredient at least one compound of claim 1.

3. The pharmaceutical composition of claim 2 for use in treating disorders associated with HCV.

4. The pharmaceutical composition of claim 2 additionally comprising at least one pharmaceutically acceptable carrier.

5. The pharmaceutical composition of claim 4, additionally containing at least one antiviral agent.

6. The pharmaceutical composition of claim 5, still additionally containing at least one interferon.

7. The pharmaceutical composition of claim 6, wherein said at least one antiviral agent is ribavirin and said at least one interferon is α-interferon or pegylated interferon.

8. A method of treating disorders associated with the HCV, said method comprising administering to a patient in need of such treatment a pharmaceutical composition which comprises therapeutically effective amounts of at least one compound of claim 1.

9. The method of claim 8, wherein said administration is oral or subcutaneous.

10. A compound of claim 1 in purified form.