ES2357494T3 - MACROCYCLIC PEPTIDES AS INHIBITORS OF HEPATITIS C. - Google Patents

Abstract

A compound of formula (I) ** Formula ** or a pharmaceutically acceptable enantiomer, diastereomer or salt thereof, wherein R1 is selected from alkoxy, hydroxy and -NHSO2R7; R2a and R2b are independently selected from hydrogen and methyl; R3 is selected from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, heterocyclyl and heterocyclylalkyl; R4 is -OR8; R5 is selected from hydrogen, alkyl and cycloalkyl; R6 is selected from hydrogen, alkyl, alkoxycarbonyl, alkylaminocarbonyl, alkylcarbonyl, aminocarbonyl, aryloxycarbonyl, cycloalkyloxycarbonyl, dialkylaminocarbonyl, haloalkoxycarbonyl, haloalkyl, haloalkylcarbonyl, heterocyclyloxycarbonyl and (NRaRb) sulfonyl; R7 is selected from alkyl, aryl, cycloalkyl, (cycloalkyl) alkyl, dialkylaminocarbonyl, dialkylaminocarbonylalkyl, heterocyclyl, heterocyclylcarbonyl and -NRaRb; the cycloalkyl and the cycloalkyl part of the (cycloalkyl) alkyl being optionally substituted with one, two or three groups independently selected from alkenyl, alkoxy, alkoxyalkyl, alkyl, arylalkyl, arylcarbonyl, cyano, cycloalkenyl, (cycloalkyl) alkyl, halo, haloalkoxy, haloalkyl and (NReRf) carbonyl; and wherein Ra and Rb are independently selected from hydrogen, alkoxy, alkyl, aryl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, haloalkyl, heterocyclyl and heterocyclylalkyl; and wherein Re and Rf are independently selected from hydrogen, alkyl, aryl, arylalkyl and heterocyclyl; wherein the aryl, the aryl part of the arylalkyl and the heterocyclyl are optionally substituted with one or two substituents independently selected from alkoxy, alkyl and halo; and R8 is selected from alkoxyalkyl, alkyl, alkylcarbonyl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, cycloalkylcarbonyl, haloalkoxyalkyl, haloalkyl, (NRcRd) carbonyl and -P (O) (OR '') 2; Rc and Rd being selected independently of hydrogen, alkyl and arylalkyl; or Rc and Rd, together with the nitrogen atom to which they are attached, form a five or six membered monocyclic heterocyclic ring that optionally contains an additional heteroatom selected from O, NRx and S; where Rx is selected from hydrogen and alkyl; and where R '' is selected from hydrogen and alkyl; and Q is a saturated or unsaturated C3-9 chain, which optionally contains one to three heteroatoms independently selected from O, S (O) and NR9, where m is 0, 1 or 2 and R9 is selected from hydrogen, alkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylsulfonyl, aminocarbonyl, arylsulfonyl, cycloalkyl, (cycloalkyl) alkyl, cycloalkyloxy, dialkylaminocarbonyl, dialkylaminocarbonylalkyl, haloalkyl and heterocyclylcarbonyl.

Description

The present disclosure is directed, in general, to antiviral compounds, and more specifically it is directed to compounds that inhibit the function of the NS3 protease (also referred to herein as "serine protease") encoded by the hepatitis C virus ( HCV), compositions comprising said compounds and methods for inhibiting the function of the NS3 protease.

HCV is an important human pathogen that infects about 170 million people worldwide (approximately five times the number of people infected by the human immunodeficiency virus type 1). A substantial fraction of these HCV-infected individuals develop severe progressive liver disease, including cirrhosis and hepatocellular carcinoma.

At present, the most effective HCV therapy employs a combination of interferon alfa and ribavirin, leading to substantial efficacy in 40% of patients. However, clinical results show that pegylated interferon alfa is superior to unmodified alpha interferon as monotherapy. However, even with experimental therapeutic guidelines that involve combinations of pegylated interferon alpha and ribavirin, a substantial fraction of patients do not experience a sustained reduction in viral load. Thus, there is a clear and unsatisfied need to develop effective therapeutic procedures for the treatment of HCV infection.

HCV is a positive polarity RNA virus. Based on the comparison of the deduced amino acid sequence and the extensive similarity in the 5 ’untranslated region, HCV has been classified as a separate genus in the Flaviviridae family. All members of the Flaviviridae family have encapsulated virions that contain a positive polarity RNA genome that encodes all known virus-specific proteins by translating an open, uninterrupted and unique reading frame.

Within the sequence of nucleotides and amino acids encoded throughout the HCV genome is considerable heterogeneity. At least six major genotypes have been characterized and more than 50 subtypes have been described. The main HCV genotypes differ in their distribution worldwide and the clinical significance of the genetic heterogeneity of HCV remains difficult to achieve despite numerous studies of the positive effect of genotypes on pathogenesis and treatment.

The HCV single stranded RNA genome is approximately 9,500 nucleotides in length and has a single open reading frame (ORF) that encodes a single large polyprotein of approximately 3,000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural proteins (NS). In the case of HCV, the generation of mature non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) is carried out by two viral proteases. The first is cleaved at the NS2 – NS3 junction; the second is a serine protease contained within the N-terminal region of NS3 and a half all subsequent cleavage downstream of NS3, both cis, at the NS3-NS4A cleavage site, and trans, for the remaining NS4A sites– NS4B, NS4B – NS5A, NS5A – NS5B. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly aiding in the localization of the NS3 membrane and other components of the viral replicase. The formation of the NS3 protein complex with NS4A is essential to effectively process polyproteins, enhancing proteolytic cleavage at all sites. The NS3 protein also shows nucleoside triphosphatase and RNA helicase activities. NS5B is an RNA-dependent RNA polymerase that is involved in HCV replication. US 2005/070955 describes macrocyclic compounds that are useful as hepatitis C virus protease inhibitors.

The present disclosure provides peptide compounds that can inhibit the functioning of the NS3 protease, for example in combination with the NS4A protease. In addition, the present disclosure describes the administration of combination therapy to a patient for which a compound according to the present disclosure can be administered, which is effective by inhibiting HCV NS3 protease, with one or two additional compounds having activity against the HCV

In a first aspect, the present disclosure provides a compound of formula (I)

image 1

or a pharmaceutically acceptable salt thereof; in which

R1 is selected from alkoxy, hydroxy and -NHSO2R7;

R2a and R2b are independently selected from hydrogen and methyl;

R3 is selected from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, heterocyclyl and heterocyclylalkyl;

R4 is –OR8;

R5 is selected from hydrogen, alkyl and cycloalkyl;

R6 is selected from hydrogen, alkyl, alkoxycarbonyl, alkylaminocarbonyl, alkylcarbonyl, aminocarbonyl, aryloxycarbonyl, cycloalkyloxycarbonyl, dialkylaminocarbonyl, haloalkoxycarbonyl, haloalkyl, haloalkylcarbonyl, heterocyclyloxycarbonyl and (NRaRb) sulfonyl;

R7 is selected from alkyl, aryl, cycloalkyl, (cycloalkyl) alkyl, dialkylaminocarbonyl, dialkylaminocarbonylalkyl, heterocyclyl, heterocyclylcarbonyl and -NRaRb; the cycloalkyl and the cycloalkyl part of the (cycloalkyl) alkyl being optionally substituted with one, two or three groups independently selected from alkenyl, alkoxy, alkoxyalkyl, alkyl, arylalkyl, arylcarbonyl, cyano, cycloalkenyl, (cycloalkyl) alkyl, halo, haloalkoxy, haloalkyl and (NReRf) carbonyl; and wherein Ra and Rb are independently selected from hydrogen, alkoxy, alkyl, aryl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, haloalkyl, heterocyclyl and heterocyclylalkyl; and wherein Re and Rf are independently selected from hydrogen, alkyl, aryl, arylalkyl and heterocyclyl; the aryl, the aryl part of the arylalkyl and the heterocyclyl being optionally substituted with one or two substituents independently selected from alkoxy, alkyl and halo; Y

R8 is selected from alkoxyalkyl, alkyl, alkylcarbonyl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, cycloalkylcarbonyl, haloalkoxy-alkyl, haloalkyl, (NRcRd) carbonyl and -P (O) (OR ’) 2; Rc and Rd being independently selected from hydrogen, alkyl and arylalkyl; or Rc and Rd, together with the nitrogen atom to which they are attached, form a five or six membered monocyclic heterocyclic ring that optionally contains an additional heteroatom selected from O, NRx and S; where Rx is selected from hydrogen and alkyl; and where R 'is selected from hydrogen and alkyl; Y

Q is a C3–9 saturated or unsaturated chain, which optionally contains one to three heteroatoms independently selected from O, S (O) and NR9, where m is 0, 1 or 2 and R9 is selected from hydrogen, alkoxy, alkoxycarbonyl, alkyl , alkylcarbonyl, alkylsulfonyl, aminocarbonyl, arylsulfonyl, cycloalkyl, (cycloalkyl) alkyl, cycloalkyloxy, dialkylaminocarbonyl, dialkylaminocarbonylalkyl, haloalkyl and heterocyclylcarbonyl.

In a first embodiment of the first aspect, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R1 is -NHSO2R7.

In a second embodiment of the first aspect, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 7 is cycloalkyl.

In a third embodiment of the first aspect, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R2a and R2b are hydrogen.

In a fourth embodiment of the first aspect, the present disclosure provides a compound of formula

5

10

fifteen

twenty

25

30

(I) or a pharmaceutically acceptable salt thereof, wherein Q is an unsaturated C5-7 chain that contains no heteroatom. In a fifth embodiment Q is an unsaturated C6 chain that does not contain any heteroatoms.

In a second aspect, the present disclosure provides a compound of formula (II):

image 1

or a pharmaceutically acceptable salt thereof; in which

R1 is –NHSO2R7;

R2a and R2b are hydrogen;

R3 is selected from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, heterocyclyl and heterocyclylalkyl;

R4 is –OR8;

R5 is hydrogen.

R6 is alkoxycarbonyl;

R7 is selected from alkyl, aryl, cycloalkyl, (cycloalkyl) alkyl, dialkylaminocarbonyl, dialkylaminocarbonylalkyl, heterocyclyl, heterocyclylcarbonyl and -NRaRb; Ra and Rb being selected independently of hydrogen, alkoxy, alkyl, aryl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, haloalkyl, heterocyclyl and heterocyclylalkyl;

R8 is selected from alkoxyalkyl, alkyl, alkylcarbonyl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, cycloalkylcarbonyl, haloalkoxy-alkyl, haloalkyl, (NRcRd) carbonyl and -P (O) (OR ’) 2; Rc and Rd being independently selected from hydrogen, alkyl and arylalkyl; or Rc and Rd, together with the nitrogen atom to which they are attached, form a five or six membered monocyclic heterocyclic ring that optionally contains an additional heteroatom selected from O, NRx and S; where Rx is selected from hydrogen and alkyl; and where R 'is selected from hydrogen and alkyl; Y

Q is a C3–9 saturated or unsaturated chain, which optionally contains one to three heteroatoms independently selected from O, S (O) and NR9, where m is 0, 1 or 2 and R9 is selected from hydrogen, alkoxy, alkoxycarbonyl, alkyl , alkylcarbonyl, alkylsulfonyl, aminocarbonyl, arylsulfonyl, cycloalkyl, (cycloalkyl) alkyl, cycloalkyloxy, dialkylaminocarbonyl, dialkylaminocarbonylalkyl, haloalkyl and heterocyclylcarbonyl.

In a first embodiment of the second aspect, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 7 is cycloalkyl.

In a second embodiment of the second aspect the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein Q is an unsaturated C6 chain that does not contain any heteroatom.

In a third aspect the present disclosure provides a compound that is or a pharmaceutically acceptable salt thereof.

image 1

In a fourth aspect, the present disclosure provides a composition comprising the compound of formula (I), or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In a first embodiment of the fourth aspect, the composition further comprises at least one additional compound having activity against HCV. In a second embodiment of the fourth aspect, at least one of the additional compounds is an interferon or a ribavirin. In a third embodiment of the fourth aspect, interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, and tau lymphoblastoid interferon.

In a fourth embodiment of the fourth aspect, the present disclosure provides a composition comprising the compound of formula (I), or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier and at least one additional compound having activity against HCV, in which at least one of the additional compounds is selected from interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of an auxiliary T lymphocyte response type 1, interfering RNA, antisense RNA, imiqimod, ribavirin, a Inosine 5'-monophosphate dehydrogenase, amantadine and rimantadine inhibitor.

In a fifth embodiment of the fourth aspect, the present disclosure provides a composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier and at least one additional compound having activity against HCV, in the that at least one of the additional compounds is effective by inhibiting the function of a selected target of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, exit from HCV, HCV NS5A protein and IMPDH for the treatment of an HCV infection.

In a fifth aspect the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in a procedure to treat an HCV infection in a patient. Said method comprises administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In a first embodiment of the fifth aspect, the process further comprises administering at least one additional compound having activity against HCV before, after or simultaneously with the compound of formula (I) or a pharmaceutically acceptable salt thereof. In a second embodiment, at least one of the additional compounds is an interferon or a ribavirin. In a third embodiment the interferon is selected from interferon alfa 2B, pegylated interferon alpha, consensus interferon, interferon alfa 2A and interferon lymphoblastoid tau.

In a fourth embodiment of the fifth aspect the present disclosure provides a compound of formula

(I)

 or a pharmaceutically acceptable salt thereof for use in a procedure to treat an HCV infection in a patient. Said method comprises administering to the patient a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof and at least one additional compound having activity against HCV before, after, or simultaneously with, the compound of formula

(I)

 or a pharmaceutically acceptable salt thereof, wherein at least one of the additional compounds is selected from interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T lymphocyte response, interfering RNA, Antisense RNA, imiqimod, ribavirin, an inhibitor of inosine 5'-monophosphate dehydrogenase, amantadine and rimantadine.

In a fifth embodiment of the fifth aspect the present disclosure provides a compound of formula

(I)

 or a pharmaceutically acceptable salt thereof for use in a procedure to treat an HCV infection in a patient. Said method comprises administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one additional compound having activity against HCV, before, after, or simultaneously with, the compound of formula

(I)

 or a pharmaceutically acceptable salt thereof, wherein at least one of the additional compounds is effective in inhibiting the function of a selected target of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein , HCV input, HCV assembly, output

HCV, HCV NS5A protein and IMPDH for the treatment of an HCV infection.

In a sixth aspect, the present disclosure provides a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, one, two, three, four or five additional compounds having activity against HCV and a pharmaceutically acceptable carrier. . In a first embodiment of the sixth aspect, the composition comprises three or four additional compounds having activity against HCV. In a second embodiment of the sixth aspect the composition comprises one or two additional compounds having activity against HCV.

In a seventh aspect the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in a method of treating an HCV infection in a patient. Said method comprises administering to the patient a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof and one, two, three, four or five additional compounds having activity against HCV before, after, or simultaneously with, the compound of formula (I) or a pharmaceutically acceptable salt thereof. In a first embodiment of the seventh aspect, the method comprises three or four additional compounds having activity against HCV. In a second embodiment of the seventh aspect the method comprises administering one or more additional compounds having activity against HCV.

Other aspects of the present embodiment may include suitable combinations of embodiments disclosed herein.

Other aspects and embodiments can be found in the description provided in this document.

The description of the present embodiment in this document would be interpreted in a manner consistent with the laws and principles of chemical bond formation. In some cases it may be necessary to remove a hydrogen atom in order to accommodate a substituent at any given location.

It will be understood that the compounds comprised by the present embodiment are those that are suitably stable for use as a pharmaceutical agent.

As used herein, the following terms have the indicated meanings:

The term "alkenyl," as used herein, refers to a linear or branched chain group of two to six carbon atoms containing at least one carbon-carbon double bond.

The term "alkoxy," as used herein, refers to an alkyl group attached to the main molecular moiety through an oxygen atom.

The term "alkoxyalkyl," as used herein, refers to an alkyl group substituted with one, two or three alkoxy groups.

The term "alkoxycarbonyl," as used herein, refers to an alkoxy group attached to the main molecular moiety through a carbonyl group.

The term "alkyl," as used herein, refers to a group derived from a saturated straight or branched chain hydrocarbon containing one to six carbon atoms.

The term "alkylamino," as used herein, refers to -NHR, where R is an alkyl group.

The term "alkylaminocarbonyl," as used herein, refers to an alkylamino group attached to the main molecular moiety through a carbonyl group.

The term "alkylcarbonyl," as used herein, refers to an alkyl group attached to the parent molecular moiety through a carbonyl group.

The term "alkylsulfonyl," as used herein, refers to an alkyl group attached to a parent molecular moiety through a sulfonyl group.

The term "amino," as used herein, refers to -NH2.

The term "aminocarbonyl," as used herein, refers to an amino group attached to the main molecular moiety through a carbonyl group.

The term "aryl," as used herein, refers to a phenyl group or a fused bicyclic ring system in which one or both of the rings is a phenyl group. The condensed bicyclic ring systems are constituted by a condensed phenyl group with a four to six membered aromatic or non-aromatic carbocyclic ring. The aryl groups of the present disclosure may be attached to the main molecular moiety through any substitutable carbon atom in the group. Representative examples of aryl groups include, but are not limited to, indanyl, indenyl, naphthyl, phenyl and tetrahydronaphthyl. The aryl groups of the present disclosure may be optionally substituted with one, two, three, four or five substituents independently selected from alkenyl, alkoxy, alkoxycarbonyl, alkyl, a second aryl group, arylalkyl, aryloxy, cyano, cyanoalkyl, halo, haloalkoxy, haloalkyl, heterocyclyl, heterocyclylalkyl, nitro and oxo; wherein the second aryl group, the aryl part of the arylalkyl and the aryloxy, the heterocyclyl and the heterocyclyl part of the heterocyclylalkyl may be additionally optionally substituted with one, two, three, four or five substituents independently selected from alkenyl, alkoxy, alkyl, cyano, halo, haloalkoxy, haloalkyl, nitro and oxo.

The term "arylalkyl," as used herein, refers to an alkyl group substituted with one, two or three aryl groups. The term "arylcarbonyl," as used herein, refers to an aryl group attached to a major molecular moiety through a carbonyl group.

The term "aryloxy," as used herein, refers to an aryl group attached to the main molecular moiety through an oxygen atom.

The term "aryloxycarbonyl," as used herein, refers to an aryloxy group attached to the main molecular moiety through a carbonyl group.

The term "arylsulfonyl," as used herein, refers to an aryl group attached to a major molecular moiety through a sulfonyl group.

The term "carbonyl," as used herein, refers to -C (O) -. The term "cyano", as used herein, refers to -CN.

The term "cyanoalkyl," as used herein, refers to an alkyl group substituted with one, two or three cyano groups. The term "cycloalkenyl," as used herein, refers to a partially saturated, non-aromatic, monocyclic, bicyclic or tricyclic ring system having three to fourteen carbon atoms and no heteroatom. Representative examples of cycloalkenyl groups include, but are not limited to, cyclohexenyl, octahydronaphthalenyl and norbornylenyl. The term "cycloalkyl," as used herein, refers to a monocyclic, bicyclic or tricyclic saturated hydrocarbon ring system having three to fourteen carbon atoms and no heteroatom. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl, bicyclo [3.1.1] heptyl and adamantyl.

The term "(cycloalkyl) alkyl", as used herein, refers to an alkyl group substituted with one, two, or three cycloalkyl groups.

The term "cycloalkylcarbonyl," as used herein, refers to a cycloalkyl group attached to the parent molecular group through a carbonyl group.

The term "cycloalkyloxy," as used herein, refers to a cycloalkyl group attached to the main molecular moiety through an oxygen atom.

The term "cycloalkyloxycarbonyl," as used herein, refers to a cycloalkyloxy group attached to the main molecular moiety through a carbonyl group.

The term "dialkylamino," as used herein, refers to -NR2, where each R group is an alkyl group. The two R groups can be the same or different.

The term "dialkylaminocarbonyl," as used herein, refers to a dialkylamino group attached to the parent molecular group through a carbonyl group.

The term "dialkylaminocarbonylalkyl," as used herein, refers to an alkyl group substituted with one, two or three dialkylaminocarbonyl groups.

The terms "halo" and "halogen," as used herein, refer to F, Cl, Br, or I.

The term "haloalkoxy", as used herein, refers to a haloalkyl group attached to the parent molecular moiety through an oxygen atom.

The term "haloalkoxyalkyl," as used herein, refers to an alkyl group substituted with one, two, or three haloalkoxy groups.

The term "haloalkoxycarbonyl," as used herein, refers to a haloalkoxy group attached to the main molecular moiety through a carbonyl group.

The term "haloalkyl," as used herein, refers to an alkyl group substituted with one, two, three or four halogen atoms. The term "haloalkylcarbonyl," as used herein, refers to a haloalkyl group attached to the parent molecular group through a carbonyl group.

The term "heterocyclyl," as used herein, refers to a five, six or seven membered ring containing one, two or three heteroatoms independently selected from nitrogen, oxygen and sulfur. The five-member ring has zero to two double bonds and the six and seven-member ring have zero to three double bonds. The term "heterocyclyl" also includes bicyclic groups in which the heterocyclyl ring is fused to an aromatic or non-aromatic carbocyclic ring or another monocyclic heterocyclyl group of four to seven members, preferably four to six members. The heterocyclyl groups of the present disclosure may be linked to a parent molecular moiety through a carbon atom or a nitrogen atom of the group. Examples of heterocyclyl groups include, but not limited to, benzothienyl, furyl, imidazolyl, indolinyl, indolyl, isothiazolyl, isoxazolyl, morpholinyl, oxazolyl, piperazinyl, piperidinyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrrolopyridinyl, pyrrolyl, thiazolyl, thienyl, and thiomorpholinyl. The heterocyclyl groups of the present disclosure may be optionally substituted with one, two, three, four or five substituents independently selected from alkenyl, alkoxy, alkoxycarbonyl, alkyl, aryl, arylalkyl, aryloxy, cyano, cyanoalkyl, halo, haloalkoxy, haloalkyl, a second heterocyclyl, heterocyclylalkyl, nitro and oxo group; wherein the second aryl group, the aryl part of the arylalkyl and the aryloxy, the second heterocyclyl and the heterocyclyl part of the heterocyclylalkyl may be additionally optionally substituted with one, two, three, four or five substituents independently selected from alkenyl, alkoxy, alkyl, cyano , halo, haloalkoxy, haloalkyl, nitro and oxo.

The term "heterocyclylalkyl," as used herein, refers to an alkyl group substituted with one, two or three heterocyclyl groups.

The term "heterocyclylcarbonyl," as used herein, refers to a heterocyclyl group attached to the main molecular moiety through a carbonyl group.

The term "heterocyclyloxy," as used herein, refers to a heterocyclyl group attached to a major molecular moiety through an oxygen atom.

The term "heterocyclyloxycarbonyl," as used herein, refers to a heterocyclyloxy group attached to the main molecular moiety through a carbonyl group. The term "hydroxyl," as used herein, refers to -OH. The term "nitro", as used herein, refers to -NO2.

The term "-NRaRb," as used herein, refers to two groups, Ra and Rb, which are attached to a parent molecular moiety through a nitrogen atom. Ra and Rb are independently selected from hydrogen, alkoxy, alkyl, aryl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, haloalkyl, heterocyclyl and heterocyclylalkyl;

The term "(NRaRb) sulfonyl," as used herein, refers to a group -NRaRb linked to the main molecular moiety through a sulfonyl group.

The term "-NReRf," as used herein, refers to two groups, Re and Rf, which are attached to the main molecular moiety through a nitrogen atom. Re and Rf are independently selected from hydrogen, alkyl, aryl, arylalkyl and heterocyclyl; wherein the aryl, the aryl part of the arylalkyl and the heterocyclyl are optionally substituted with one or two substituents independently selected from alkoxy, alkyl and halo.

The term "(NReRf) carbonyl," as used herein, refers to a group -NReRf attached to the main molecular moiety through a carbonyl group. The term "oxo," as used herein, refers to = O. The term "sulfonyl," as used herein, refers to SO2.

The compounds of the present disclosure may exist as prodrugs. The term "prodrug", as used herein, represents compounds that are rapidly transformed in vivo into the main compounds by hydrolysis in the blood. The prodrugs of the present disclosure include esters of hydroxy groups in the main molecule, esters of carboxy groups in the main molecule and amide amides of the main molecule.

The compounds of the present discussion may exist as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt", as used herein, represents bipolar ionic salts or forms of the compounds of the present disclosure that are soluble or dispersible in water or oil, which are, within the scope of a medical trial. Well, suitable for use in contact with the patient's tissues without excessive toxicity, irritation, allergic response or other problem or complication proportional to a reasonable benefit / risk ratio, and are effective for their desired use. The salts can be prepared during the final isolation and purification of the compounds or in a separate way by reacting a suitable basic functionality with a suitable acid. Representative addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, canforate, canphosulfonate; digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, iodhydrate, 2– hydroxyethanesulfonate, lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2– naphthalectaninate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxylate phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate and undecanoate. Examples of acids that can be used to form pharmaceutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric and phosphoric, and organic acids such as oxalic, maleic, succinic and citric acids.

Basic addition salts may be prepared during the final isolation and purification of the compounds by reacting an acid group with a suitable base such as hydroxide, carbonate or bicarbonate of a metal cation or with ammonia or a primary, secondary or tertiary organic amine. Pharmaceutically acceptable salt cations include lithium, sodium, potassium, calcium, magnesium and aluminum, as well as non-toxic quaternary amine cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine , N, N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N, N-dibenzylphenethylamine and N, N'-dibenzylethylenediamine. Other representative organic amines useful for the formation of basic addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine and piperazine.

As used herein, the term "against HCV activity" means that the compound is effective in treating the HCV virus.

The term "compounds of the disclosure" and the equivalent terms mean that they encompass compounds of formula (I), and pharmaceutically acceptable enantiomers, diastereomers and salts thereof. Similarly, references to intermediates mean that they cover their salts where the context allows.

The term "patient" includes humans and other animals.

The term "pharmaceutical composition" means a composition comprising a compound of the disclosure in combination with at least one additional pharmaceutical carrier, ie adjuvant, excipient or vehicle, such as diluents, preservatives, fillers, flow regulators, agents disintegrants, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents, depending on the nature of the mode of administration and the dosage forms. For example, the ingredients listed in Remington’s Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, USA (1999) can be used.

The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of a thorough medical trial, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response or other problem or complication proportional to a reasonable relationship between benefit and risk.

The term "therapeutically effective amount" means the total amount of each active component that is sufficient to show a significant patient benefit, for example, a sustained reduction in viral load. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the terms refer to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, in series or simultaneously.

The terms "treatment" and "treat" refer to: (i) prevent a disease, disorder or condition from occurring in a patient, who may be predisposed to the disease, disorder and / or condition, but that has not yet been diagnosed to have it; (ii) inhibit the disease, disorder or condition, that is, stop its development; and / or (iii) relieve the disease, disorder or condition, that is, cause the disease, disorder or condition to regress .

When used in compounds mentioned herein, the designations P1 ', P1, P2, P2 *, P3 and P4, as used herein, map the relative positions of the amino acid residues of binding of a protease inhibitor concerning the binding of a cleavage substrate of a natural peptide. The cleavage takes place in the natural substrate between P1 and P1 'where the non-preferential positions of the aforementioned amino acids begin at the C-terminal end of the natural cleavage site of the peptide and extend towards the N-terminus, while the preferential positions emanate from the terminal end N of the designation of the cleavage site and extend towards the end C. For example, P1 'refers to the first position away from the end to the right of the terminal C end of the cleavage site (i.e., first position of the N terminal end); while P1 begins the numbering from the left side of the cleavage site of the C-terminal end, P2: second position from the C-end, etc.). (see Berger A. and Schechter I., Transactions of the Royal Society London series (1970), B257, 249-264).

There are asymmetric centers in the compounds of the present disclosure. For example, the compounds may include a cyclopropyl P1 element of the formula

image 1

wherein C1 and C2 each represent an asymmetric carbon atom in positions 1 and 2 of the cyclopropyl ring.

image2

5 It should be understood that the disclosure encompasses all stereochemical forms, or mixtures thereof, that possess the ability to inhibit HCV protease.

Certain compounds of the present discussion may also exist in different stable conformational forms that may be separable. Torsional asymmetry due to restricted rotation on an asymmetric individual bond, for example due to its steric hindrance or ring tension, may allow separation of

10 different shapers. The present disclosure includes each conformational isomer of these compounds and mixtures thereof.

Certain compounds of the present discussion may exist in hybrid form and the present disclosure includes each hybrid form of these compounds and mixtures thereof.

When it is possible that, for use in therapy, amounts can be administered therapeutically

Effective of a compound of formula (I), as well as pharmaceutically acceptable salts thereof can be administered as the raw material, it is possible to present the active ingredient as a pharmaceutical composition. Accordingly, the discussion further provides pharmaceutical compositions, which include therapeutically effective amounts of compounds of formula (I) or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients. The compounds of formula (I) and pharmaceutically acceptable salts thereof, are as described above. The vehicle (s), diluent (s), or excipient (s) must be acceptable in the sense that they are compatible with the other ingredients of the formulation and not harmful to the recipient thereof. In accordance with another aspect of the discussion there is provided a process for the preparation of a pharmaceutical formulation including mixing a compound of formula (I), or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. In a monotherapy for the prevention and treatment of HCV-mediated disease, dose levels ranging from about 0.01 to about 250 milligrams per kilogram ("mg / kg") of body weight per day are typical, preferably about 0, 05 to about 100 mg / kg body weight per day of the compounds of the disclosure. Typically, the pharmaceutical compositions of the present disclosure will be administered from about 1 to about 5 times a day or alternatively, in the form of continuous infusion. Such administration can be used as chronic or acute therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single pharmaceutical form will vary depending on the condition being treated, the severity of the condition, the time of administration, the route of administration, the rate of excretion of the compound used, duration of treatment and age, sex, weight and condition of the patient. Single dose formulations are those containing a daily dose or sub-dose, as indicated hereinbefore, or an appropriate fraction thereof of an active substance. Generally, treatment is initiated with small doses substantially below the optimal dose of the compound. Then, the dose is increased in small increments until the optimum effect is reached in the given circumstances. In general, the compound is administered, most desirably at a concentration level that will generally achieve antivirally effective results without causing harmful or harmful side effects.

When the compositions of this disclosure comprise a combination of a compound of the disclosure and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent are usually present at dose levels of about 10 to 150%, and more preferably between approximately 10 and 80% of the dose normally administered in a monotherapy regimen.

Pharmaceutical formulations may be adapted for administration by any appropriate route, for example orally (including oral or sublingual), rectal, nasal, topical (including oral, sublingual, or transdermal), vaginal, or parenteral (including injections or subcutaneous, intracutaneous, intramuscular, intraarticular, intrasynovial, intraesternal, intrathecal, intralesional, intravenous, or intradermal infusions). Such formulations can be prepared by any method known in the art of pharmacy, for example by binding in association with the active ingredient with the vehicle (s) or excipient (s).

Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or shakes; or oil in water emulsions or water in oil emulsions.

For example, for oral administration in the form of a tablet or a capsule, the active drug component may be combined with a non-toxic, oral pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. The powders were prepared by grinding the compound to a suitable fine size and mixing with a similarly ground pharmaceutical carrier such as an edible carbohydrate, such as, for example, starch or mannitol. A flavoring, preservative, dispersing and coloring agent may also be present.

The capsules are made by preparing a powder mixture, as described above and filled formed jelly covers. Gliding agents and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol can be added to the powder mixture before the loading operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate, or sodium carbonate may also be added to increase the availability of the medicament when the capsule is ingested.

In addition, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth gum or sodium alginate, carboxymethyl cellulose, polyethylene glycol, waxes, and the like. The lubricants used in these dosage forms include sodium oleate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. The tablets were formulated, for example, by preparing a powder mixture, granulating or welding with incomplete fusion, adding a lubricant and disintegrant, and pressing into tablets. A powder mixture was prepared by mixing the compound, suitably ground, with a diluent or base as described above, and optionally, with a ligand such as carboxymethyl cellulose, an alginate, gelatin, or polyvinylpyrrolidine, a solution retarder such as paraffin, a accelerated resorption such as a quaternary salt and / or absorption agent such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, gum arabic mucilage, or solutions of cellulosic or polymeric materials and forcing it to pass through a sieve. As an alternative to granulate, the powder mixture can be passed through the compressor machine and the result is imperfectly broken posts formed in granules. The granules can be lubricated to prevent them from sticking to the tablet forming molds by the addition of stearic acid, a stearate salt, talc, or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present disclosure can also be combined with an inert carrier that flows free and can be compressed into tablets directly without going through the granulation or post-forming stages. A transparent or opaque protective coating consisting of a sealant of shellac, a coating of sugar or polymeric material, and a polished coating of wax can be provided. Dyes can be added to these coatings to distinguish different unit dosages.

Oral fluids such as a solution, syrups and elixirs can be prepared in unit dosage form such that a given amount contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs were prepared by the use of a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, aromatic additive such as peppermint oil or natural sweeteners, or saccharin or other artificial sweeteners and the like can also be added.

Where appropriate, unit dosage formulations for administration can be encapsulated. The formulation can also be prepared to prolong or maintain release such as by coating or embedding particulate material in polymers, waxes, or the like.

The compounds of formula (I), and pharmaceutically acceptable salts thereof, can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

The compounds of formula (I) and pharmaceutically acceptable salts thereof can also be administered by the use of monoclonal antibodies as individual transporters to which the compound molecules are coupled. The compounds may also be coupled with soluble polymers as transporters of drugs that are labeled as targets. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl methacrylamidephenol, polyhydroxyethylapartamidaphenol, or polyethylene oxide polylysine substituted with palitoyl residues. In addition, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyortoesters, polyacetals, polyhydropyran, polycyanoacrylates, and crosslinked hydrophilic block copolymers .

Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches that are desired to remain in intimate contact with the epidermis of a recipient for a prolonged period of time. For example, the active ingredient can be administered from the iontophoresis patch as generally described in Pharmaceutical Research, 3 (6), 318 (1986).

Pharmaceutical formulations adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

For treatments of the eye or other external tissues, for example mouth and skin, the formulations are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient can be used either with a paraffinic base or with a water-mixable ointment base. Alternatively, the active ingredient can be formulated in a cream with an oil-in-water base or with a water-in-oil base.

Pharmaceutical formulations adapted for topical administrations for the eye include eye drops in which the active ingredient is dissolved or suspended in a suitable vehicle, especially an aqueous solvent.

Pharmaceutical formulations adapted for topical administration in the mouth include tablets, pills and mouthwashes.

Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or as enemas.

Pharmaceutical formulations adapted for nasal administration in which the vehicle is solid include a coarse powder having a particle size for example in the range of 20 to 500 micrometers that is administered in the manner in which the monkfish is inhaled, is say by rapid inhalation through the nasal passages from a dust container that is attached close to the nose. Suitable formulations in which the excipient is a liquid, for administration as a nasal spray or as nasal drops, include aqueous solutions.

or oily solutions of the active ingredient.

Formulations adapted for administration by inhalation include dusts or fine particle mists, which can be generated by various types of aerosols, nebulizers, or pressurized insufflators in measured doses.

Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, buffers, creams, gels, pastes, foams or spray formulations.

Pharmaceutical formulations adapted for parenteral administration include sterile aqueous and non-aqueous injection solutions that may contain antioxidants, buffers, bacteriostats and solutes that return to the isotonic formulation with the blood of the desired recipient; and sterile aqueous and non-aqueous suspensions that may include suspending agents and thickening agents. The formulations can be presented in unit dose or multidose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition that requires only the addition of the sterile liquid vehicle, for example water for injections, immediately before to use Injection solutions and suspensions can be prepared

10 extemporaneous from sterile powders, granules and tablets.

It should be understood that in addition to the ingredients mentioned in particular above, the formulations may include other conventional agents in the art that have been considered for the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

15 Table 1 below lists some illustrative examples of compounds that can be administered with the compounds of this disclosure. The compounds of the disclosure can be administered with other compounds with activity against HCV in combination therapy, either together or separately or by combining the compounds to form a composition.

Table 1

Tradename

Physiological class Type of inhibitor or target Supplier company

NIM811

Cyclophilin inhibitor Novartis

Zadaxin

Immunomodulator Sciclone

Suvus

Methylene blue Bioenvision

Actilon (CPG10101)

TLR9 agonist Coley

Batabulin (T67)

Anticancer Β-tubulin inhibitor Francisco, Tularik Inc., South San CA

ISIS 14803

Antiviral Antisense ISIS Pharmaceuticals Inc, Carlsbad, CA / Elan Phamaceuticals Inc., New York, NY

Summetrel

Antiviral Antiviral Endo Pharmaceutical s Holdings Inc., Chadds Ford, PA

GS – 9132 (ACH – 806)

Antiviral HCV inhibitor Achillion / Gilead

Pyrazolopyrimidine compounds and salts of WO-2005047288 May 26, 2005

Antiviral HCV inhibitors Arrow Therapeutics Ltd.

Levovirin

Antiviral IMPDH inhibitor Ribapharm Inc., Costa Mesa, CA

Merimepodip (VX – 497)

Cambridge antiviral, IMPDH inhibitor Vertex Pharmaceutical s Inc., MA

XTL – 6865 (XTL – 002)

Antiviral Monoclonal antibody XTL Biophamaceuti cals Ltd., Rehovot, Israel

Telaprevir (VX – 950, LY– 570310)

Antiviral NS3 serine protease inhibitor Vertex Pharmaceuticals Inc., Cambridge, MA / Eli Lilly and Co. Inc., Indianapolis, IN

Tradename

Physiological class Type of inhibitor or target Supplier company

HCV – 796

Antiviral NS5B Replicase Inhibitor Wyeth / Viropharma

NM – 283

Antiviral NS5B Replicase Inhibitor Idenix / Novartis

GL – 59728

Antiviral NS5B Replicase Inhibitor Gene Labs / Novartis

GL – 60667

Antiviral NS5B Replicase Inhibitor GeneLabs / Novartis

2’C MeA

Antiviral NS5B Replicase Inhibitor Gilead

PSI6130

Antiviral NS5B Replicase Inhibitor Roche

R1626

Antiviral NS5B Replicase Inhibitor Roche

2’C Methyl adenosine

Antiviral NS5B Replicase Inhibitor Merck

JTK – 003

Antiviral RdRp inhibitor Japan Tobacco Inc., Tokyo, Japan

Levovirin

Antiviral Ribavirin ICN Pharmaceuticals, Costa Mesa, CA

Ribavirin

Antiviral  Ribavirin Schering – Plow Corporation, Kenilworth, NJ

Viramidine

Antiviral Ribavirin Prodrug Ribapharm Inc., Costa Mesa, CA

Heptazyme

Antiviral Ribozyme Ribozyme Pharmaceuticals Inc., Boulder, CO

BILN – 2061

Antiviral Serine protease inhibitor Boehringer Ingelheim Pharma KG, Ingelheim, Germany

SCH 501034

Antiviral Serine protease inhibitor Schering Plow

Zadazim

Immunomodulator Immunomodulator SciClone Pharmaceuticals Inc., San Mateo, CA

Ceplene

Immunomodulator Immunomodulator Maxim Pharmaceuticals Inc., San Diego, CA

Cellcept

Immunosuppressant HCV immunosuppressive IgG F. Hoffmann – La Roche LTD, Basel, Switzerland

Civacir

Immunosuppressant HCV immunosuppressive IgG Nabi Biopharmaceuticals Inc., Boca Raton, FL

Albuferon - α

Interferon IFN-α2b albumin Human Genome Sciences Inc., Rockville, MD

Infergen A

Interferon IFN alfacon – 1 InterMune Pharmaceuticals Inc., Brisbane, CA

Tradename

Physiological class Type of inhibitor or target Supplier company

Omega IFN

Interferon JFN – ω Intarcia Therapeutics

IFN – β and EMZ701

Interferon  IFN – β and EMZ701 Transition Therapeutics Inc., Ontario, Inc., Ontario, Canada

Rebif

Interferon IFN – β1a Serono, Geneva, Switzerland

Roferon A

Interferon IFN – α2a F. Hoffmann – La Roche LTD, Basel, Switzerland

Intron A

Interferon IFN – α2b Schering – Plow Corporation, Kenilworth, NJ

Intron A and Zadaxin

Interferon IFN – α2b / α1 – thymosin RegeneRx Biopharmiceuti cals Inc., Bethesda, MD / SciClone Pharmaceuticals Inc, San Mateo, CA

Rebetron

Interferon IFN – α2b / ribavirin Schering – Plow Corporation, Kenilworth, NJ

Actimmune

Interferon INF –  InterMune Inc., Brisbane, CA

Interferon – β

Interferon Interferon – β – 1a Serono

Multiferon

Interferon Long-acting IFN Viragen / Valentis

Wellferon

Interferon IFN – αn1 lymphoblast GlaxoSmithKline plc, Uxbridge, United Kingdom

Omniferon

Interferon IFN –  natural Viragen Inc., Plantation, FL

Pegasys

Interferon Pegylated IFN – α2a F. Hoffmann – La Roche LTD, Basel, Switzerland

Pegasys and Ceplene

Interferon IFN – α2a PEGylated / immunomodulator Maxim Pharmaceuticals Inc., San Diego, CA

Pegasys and Ribavirin

Interferon IFN – α3a PEGylated / ribavirin F. Hoffmann – La Roche LTD, Basel, Switzerland

PEG – Intron

Interferon IFN – α2b PEGylated Schering – Plow Corporation, Kenilworth, NJ

PEG – Intron / ribavirin

Interferon IFN – α2b PEGylated / ribavirin Schering – Plow Corporation, Kenilworth, NJ

IP – 501

Liver protection Antifibrotic Indevus Pharmaceuticals Inc., Lexington, MA

IDN – 6556

Liver protection Caspase Inhibitor Idun Pharmaceuticals Inc., San Diego, CA

Tradename

Physiological class Type of inhibitor or target Supplier company

ITMN – 191 (R – 7237)

Antiviral Serine protease inhibitor InterMune Pharmaceuticals Inc., Brisbane, CA

GL – 59728

Antiviral NS5B Replicase Inhibitor Genelabs

ANA – 971

Antiviral TLR – 7 agonist Anadys

TMC – 465350

Antiviral Serine protease inhibitor Medivir / Tibotec

5

10

fifteen

twenty

25

30

35

40

Four. Five

The compounds of the disclosure can also be used as laboratory reagents. The compounds can be a decisive contribution to provide research tools to design virus replication assays, validation of animal test systems and structural biology studies to further enhance awareness of the mechanisms of HCV disease. In addition, the compounds of the present disclosure are useful for establishing or determining the binding site of other antiviral compounds, for example, by competitive inhibition.

The compounds of this disclosure may also be useful for treating or preventing viral contamination of materials and therefore reducing the risk of viral infection by laboratory or medical personnel or patients coming into contact with such materials, for example, blood, tissues, instruments and surgical garments, instruments and laboratory garments, and apparatus and materials for blood collection or transfusion.

The present disclosure is intended to include compounds having the formula (I) when they are prepared by synthesis procedures or by metabolic procedures that include those produced in the human body

or animal (in vivo) or procedures that occur in vitro.

The chemical abbreviations commonly used to identify chemical compounds disclosed herein include Bn: benzyl; Boc: tert-butyloxycarbonyl {Me3COC (O)}; BSA: bovine serum albumin; CDI: carbonyldiimidazole; DBU: 1.8 – diazabicyclo [5.4.0] –undec – 7 – eno; CH2Cl2 = DCM: methylene chloride; TBME: methyl tert-butyl ether; DEAD: diethyl azodicarboxylate; DIAD: diisopropyl azodicarboxylate; DIEA: diisopropylethylamine; DIPEA: diisopropylethylamine; 4-DMAP: 4-dimethyl-aminopyridine; DCC: 1,3-dicyclohexylcarbodiimide; DMF: dimethylformamide; DMSO: dimethylsulfoxide; DPPA: diphenylphosphoryl azide; Et: ethyl; EtOH: ethanol; EtOAc: ethyl acetate; Et2O: diethyl ether; Grubb catalyst: bis (tricyclohexylphosphine) benzylidene ruthenium (IV) dichloride; second generation Grubb catalyst: tricyclohexylphosphine dichloride [1,3-bis (2,4,6-trimethylphenyl) -4,5- dihydroimidazol-2-ylidene] [benzylidene] ruthenium (IV); HATU: [O– (7-azabenzotriazol-1-yl) –N, N, N ’, N’– tetramethyluronium hexafluorophosphate]; HBTU: [O– (1H-benzotriazol-1-yl) -N, N, N ’, N’-tetramethyluronium hexafluorophosphate]; HOBT, 1– hydroxybenzotriazole; HOAT, 1-hydroxy-7-azabenzotriazole; HPLC: high performance liquid chromatography; MS: mass spectrometry; Me: methyl; MeOH: methanol; NMM: N-methylmorpholine; NMP: N-methylpyrrolidine; Pr: propyl; PPA: poly (phosphoric acid); TBAF: tetra-n-butylammonium fluoride; 1,2-DCE or DCE: 1,2-dichloroethane; TFA: trifluoroacetic acid; THF: tetrahydrofuran.

The starting materials useful for synthesizing the compounds of the present discussion are known to those skilled in the art and can be easily made or commercially available.

The following procedures set forth below are provided for illustrative purposes and are not intended to limit the scope of the claims. It will be recognized that it may be necessary to prepare a compound in which a functional group is protected using a conventional protective group then to remove the protective group to provide a compound of the present disclosure. Details concerning the use of protecting groups in accordance with the present disclosure are known to those skilled in the art.

As shown in Scheme 1, the intermediates of the present invention, such as dipeptide 1, can be used for the preparation of compounds of formula (I). In the first stage of this process, the Boc protected nitrogen of 1 is deprotected using an acid such as HCl in a solvent such as ether, providing the corresponding free amine 2. The amine 2 can then be coupled to an amino acid 3 using a coupling agent such as HATU in a solvent such as dichloromethane to provide intermediate tripeptide 4. It will be appreciated that in some cases, intermediates such as 3 are commercially available and as an alternative, such compounds can be prepared racematically or chirally by methods known in the art. A key transformation in the construction of compounds of formula (I) is the microcycling process in which intermediates of general structure 4 become intermediates of general structure 5. In the cited general example, the conversion of intermediate 4 to 5 can be effect by an intramolecular olefin metathesis reaction. This kind of reaction is well documented in the art and as such, they have been developed and are

A series of olefin metathesis catalysts are commercially available. For example, the conversion of diene 4 to macrocycle 5 could be carried out by treating 4 with a sufficient amount of Grubb olefin metathesis catalyst first generation, in a solvent such as dichloromethane or dichloroethane. In some examples for the conversion of 4 to 5, it may be necessary to heat the reaction mixture in order to effect this cyclization process. Intermediate 5 is then converted to the compounds of formula (I) such as 7 by a two step process. In the first stage of this process, the ester functionality of intermediate 5 is hydrolyzed to the corresponding carboxylic acid 6. This transformation can be carried out by a saponification reaction in which 5 is treated with a base such as lithium hydroxide in a mixture of THF, methanol and water. The resulting acid 6 can be converted to a compound of formula (I) by a simple coupling reaction

10 with a sulfonamide derivative as shown. For example, it is well documented in the art that the treatment of a carboxylic acid such as 6, with CDI in a solvent such as methylene chloride, generates in situ a reactive intermediate that, when treated with a sulfonamide, provides 7, a compound of formula (I).

 Scheme 1

image3

If, in the previous final coupling process, that is, in the conversion from 6 to 7, the entity R8SO2NH2 is a sulfamide derivative, such as RaRbNSO2NH2, then an alternative coupling procedure can be used. In this, the sulfamine 1 intermediate (Scheme 2) is first deprotonated using a base such as lithium bishexamethyldisilane in a solvent such as THF. The resulting THF solution of sulfamide anion 2 is then added to the aforementioned reaction mixture containing the activated carboxylic acid. This reaction mixture is then stirred for several hours to provide the required acylsulfamide 7.

image4

The compounds of formula (I) can also be converted into other compounds of Formula I as described herein. An example of such a process is shown in Scheme 3, in which a compound of Formula I (I) having a Boc group in the P4 position is converted to a compound of Formula I (3) in which said compound has a urea group in position P4. The conversion of 1 to 3 can be carried out in a two-stage process, the first of which is the conversion of 1 to the amine 2 by treatment of 1 with a

Acid such as TFA in a solvent such as methylene chloride. The resulting TFA amine salt can be treated with an isocyanate in the presence of a base equivalent to provide a compound of Formula I (3) in which the P3 moiety is capped with a urea. As indicated above, one skilled in the art will recognize that intermediate 2 can be used as a starting material for the preparation of compound of Formula I in which the P3 group is capped with an amide or a carbamate. The construction of sayings

Compounds of Formula I can be achieved using conventional conditions for the formation of said P4 functionalities in amines.

image5

Exemplary procedures for preparing P2 intermediates and compounds of formula (I) are shown in the following Schemes. Said intermediates, reaction conditions and procedures given in the specific examples are broadly applicable to compounds with other substitution models. For example, the synthesis

5 of P2 elements found in compounds of formula (I) of Scheme IV can be prepared following the defined synthesis route. In this, N-Boc-4-oxo-L-proline is treated commercially, with an organometallic agent such as a Grignard reagent (or, alternatively, a kind of alkyl or aryl lithium, or, alternatively alternatively, a kind of alkyl or aryl zinc) providing Intermediate (2) in which the C4 position of the proline has a substituent R3 and a free tertiary hydroxy group. The alcohol functionality of intermediate 2 can be

10 functionalize to provide the desired R8 functionality. In this process, the alcohol of intermediate 2 can be introduced in a series of reactions well established in the art. For example, the alcohol of 2 can be acylated to provide esters, carbamates or carbonates; rent to provide ethers and phosphonate to provide phosphates. For the conversion of intermediate 2 to intermediate 3 of Scheme IV it may be necessary to first protect the carboxylic acid group of 2 as shown.

image6

Chemistry for the functionalization of alcohols is described in conventional texts in the field such as: Comprehensive Organic Transformations: A Guide to Functional Group Preparations. Second Edition, by Richard Larock. This text is edited by Wiley and Sons. It underlines references and reviews

5 that a person skilled in the art can easily use for conversion of intermediate 2 of Scheme IV to intermediate 3. For example, conditions and references relevant for the formation of ethers from alcohols can be found on pages 883 to 929 of the text of Larock More specifically, the conditions and references cited on pages 890 to 894 are the most pertinent for carrying out the present invention. Similarly, conditions for the conversion of alcohols to the corresponding ester derivatives can be

10 found on pages 1952 and 1955 of Larock's text. In addition, the chemistry described in Journal of Organic Chemistry 2001, volume 66, page 8926 and the pertinent references cited therein are useful for practicing the present invention.

It will be appreciated that the addition of organometallic agents to the ketone moiety of proline derivative I (Scheme VI) is well established in the art. For example, Hruby et al. (J. Org. Chem. 2001, 66, 3593) have described the addition of phenylmagnesium bromide to intermediates of general structure 1 of Scheme IV. These findings provide evidence that the optimum yields of the desired 1,2 addition products (2, from Scheme VI) are obtained when a tert-butyl ether group is used as the protecting group of the C2 carboxyl moiety. In addition, this work provided clear evidence in the form of X-ray crystallography in regard to the stereochemical results of this addition reaction. Specifically, as a result of the addition of the aforementioned Grignard to ketone 1, a single product was obtained in which the C4 hydroxyl group and the C2 carboxyl group assumed a relative orientation syn around the five-membered ring. From the determination of this structure the selectivity of faces in the addition of R3M to the ketone of I which was e alpha was deduced in the context of structure I of Scheme IV. That is, the organometallic selectivity adds to the face re (bottom face) of the carbonyl in 1 providing the corresponding tertiary alcohol (2) with the stereochemistry

25 shown.

Scheme VI

image 1

The aforementioned work by Hruby describes the addition of a specific Grignard reagent to derivatives of I (Scheme VI). However, the addition of a variety of Grignard reagents to proline 1 is included in the present disclosure. The bibliographic corpus that describes the addition of organometallic agents,

5 including Grignard reagents, ketones is considerable and is summarized in general descriptions in the art such as: Comprehensive Organic Functional Group Transformations. Volume 2: Synthesis: Carbon with one heteroatom attached by a single bond. Editor in Chief Alan. R. Katritzky, et al. 1995. Chapter 2.02, page 37. This kind of reaction is also described in Comprehensive Organic synthesis. Editor in Chief Barry M Trost, Volume 1: Additions to C – X pi – bonds (part 1). 1991

Recent research in the art provides conditions for the subsequent optimization of Grignard reagents in ketone addition reactions and these works may be useful in the present disclosure. For example, Ishihara et al. (Org. Lett. 2005, Vol. 7, No. 4, 573) recently describe the formation and utility of "ate" magnesium complexes (magnesiates). The "ate" magnesium complexes, R3MgLi, are derived from Grignard and alkyl lithium reagents. As described by Ishihara these complexes provide excellent yields of 1.2 addition products in reactions to ketones. In a separate study, Knochel et al. (Angew. Chem Int. Ed. 2006, 45, 497) have described the use of soluble lanthanide salts such as LnCl3 in combination with organomagnesium reagents. The presence of these lanthalide salts results in an improvement in the efficiency of the 1,2 addition reaction to carbonyl compounds. These works, and the references cited therein, establish the state of the art regarding the optimization of the Grignard reaction in simple additions to carbonyl compounds and serve as an important source of information in the present disclosure.

It will be appreciated that a range of organometallic reagents participate in ketone addition reactions. Included in this corpus of work are reagents such as aryl lithium, alkyl lithium and heteroaryl lithium reagents, which are well known to add carbonyl moieties in a 1,2 form. For example, a recent study by Dondoni et al. (J. Org. Chem. 2005, 70, 9257) lithium benzothiazole using BuLi and the resulting C2-lithium species is added in a 1.2 form to a lactone. By way of analogy, one might expect to add a lithiated benzothiazole of a 1,2 form to a ketone I of Scheme IV to provide an intermediate such as 2a.

One skilled in the art will recognize that organometallic reagents derived from heterocycles such as oxazoles and thiazoles and imidazoles can also participate in reactions of addition 1,2 to ketone 1. There is a considerable bibliographic corpus that defines the specific conditions used for each of these systems heterocyclic and this formation is readily available to one skilled in the art. For example, the use of organometallic reagents derived from benzoxazole or oxazole, in ketone addition reactions requires the use of lithium magnesiates. The specificity of this recent study by Bayh et al. Is described in J. Org. Chem., 2005, 70, 5190. The addition of benzoxazole to ketone 1 of Scheme VI would provide access to intermediates such as 2b.

There is significant preceding literature for the addition to ketones using a wide range of organometallic reagents derived from heterocycles. For example, the work of Behinda et al. (Tet. Lett. 42, 2001, 647) describes the formation of a lithiated benzimidazole and its addition to a simple lactone. Similarly, the use of this lithiated benzimidazole in ketone 1 addition reactions of Scheme VI would provide access to intermediates such as 2c. In addition, a recent study by Kawasaki et al. (Bioorganic and Medicinal Chem. Lett. 13, 2003, 87) describes the formation of a series of lithiated heteroaromatic compounds and their addition reactions to activated amides. Similarly, the use of these lithiated heteroaromatic intermediates in ketone 1 addition reactions of Scheme VI would provide access to the 2d-2k intermediates.

The use of organometallic derivatives of biaryl, or heteroaryl-aryl systems in addition reactions 1,2 to ketone 1 is also relevant for the present disclosure. The addition of this class of organometallic reagents to ketone 1 would provide access to intermediates such as 21 and 2m. It will be appreciated that in the exemplification of the present invention, it may be necessary to synthesize baryl or heteroaryl organometallic for subsequent use in ketone 1 addition reactions of Scheme VI. One skilled in the art will recognize the significant bibliographic corpus that describes the preparation of organometallic of this type and its precursors. For example, a recent review by Chinchilla et al. (Chem. Rev. 2004, 104, 2667) describes the preparation of metalated heterocycles and their usefulness. The basic chemistry for the preparation of biaryl or heteroaryl-aryl systems often employs Suzuki-type coupling reactions. A bibliographic corpus described by Gregory Fu describes the state of the art in said coupling reactions and a subset of these references are as follows: JAGS 2004, 126, 1340; JACS, 2002, 124, 13662; Angew Chem. Int. Ed. 2002, 41, No. 11, 1945; Angew Chem, Int. Ed. 2002, 41, No. 20, 3910; JACS 2002, 122, 4020; JACS 2001, 123, 10099; Org. Lett. 2001, Vol. 3, No. 26, 4295; Angew Chem. Int. Ed. 1998, 37, No. 24, 3387. In addition to this study of critical reviews in the area, others such as Rossi in Synthesis 2004 are available,

EXAMPLES

The present disclosure will now be described in relation to certain embodiments that are not intended to limit its scope. Thus, the following examples, which include specific embodiments, will illustrate a practice of the present disclosure, it being understood that the examples are for purposes of illustration of certain embodiments and are presented to provide what is believed to be the most useful and easily understood description of its procedures and conceptual aspects.

The following examples 1 to 37 and 86 are reference examples. Examples 87 and 88 refer to embodiments of the invention. Solution percentages express a weight-to-volume ratio, and solution ratios express a volume-to-volume ratio, unless otherwise indicated. Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker 300, 400 or 500 MHz spectrometer; chemical shifts (δ) are expressed in parts per million. Flash chromatography was carried out on silica gel (SiO2) according to Still's rapid chromatography technique (J. Org. Chem., 1978, 43, 2923).

EXAMPLE 1

Sulfamide Preparation

image 1

The intermediate sulfamoyl chloride was prepared by adding water (1 equiv) in THF to a cold stirred solution (-20 ° C) of chlorosulfonyl isocyanate (1 equiv) in THF and the resulting solution was allowed to warm to 0 ° C. To this solution was added anhydrous Et3N (1 equiv) followed by the required secondary amine (1 equiv). The

The reaction mixture was heated to room temperature, then filtered and the filtrate was rotary evaporated to provide the desired sulfonamides. Said sulfonamide was then coupled to a carboxylic acid to provide the desired acylsulfamide.

EXAMPLE 2

10 Specific procedure for the preparation of an acylsulfamide intermediate

image7

To a solution of (1R, 2S) 1-tert-butoxycarbonylamino-2-vinyl-cyclopropanecarboxylic acid (217 mg, 1,194

mmol) in THF (5 ml), CDI (290 mg, 1,791 mmol) was added and the reaction mixture was heated at reflux for 45

min. In another round bottom flask, LiHMDS (1.0 M solution in hexanes, 2.4 ml, 2.4 mmol) was added to a

Solution of N-ethylmethylsulfamide (330 mg, 2,388 mmol) in THF (5 ml) and the reaction mixture was stirred at rt for 1 hour. Two reaction mixtures were added together and stirred at rt for 2 hours. Water was added to quench the reaction and the reaction solution was extracted with EtOAc. The organic phase was separated and dried over MgSO4. Evaporation of the solvent gave the crude product that was purified by HPLC Prep. providing the desired N-acylsulfamide. N-acylsulfamide was then dissolved in 4N HCl solution in dioxane (2 ml) and stirred at rt for 4

20 hours. Evaporation of the solution gave brownish oil as HCl salt. (112 mg, 33% yield). 1H NMR (400Mz, CD3OD) δ 1.16 (t, J = 7.2 Hz, 3 H), 1.68 (dd, J = 10.03, 7.83 Hz, 1 H), 2.15 (m, 1H), 2.37 (m, 1 H), 2.89 (s, 3 H), 3.30 (m, 2 H), 5.31 (d, J = 10.27 Hz, 1 H), 5.42 (d, J = 17.12 Hz, 3 H), 5.68 (m, 1 H). LC-MS (retention time: 0.883 min.), MS m / z 270 (M + Na +).

EXAMPLE 3:

Preparation of racemic ethyl ester hydrochloride (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid (Procedure A and Procedure B)

image 1

The title compound was racemic prepared by each of the following procedures A and B.

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PROCEDURE A

Preparation of N-benzyl imine of the glycine ethyl ester

0 ° C using an ice-water bath. Triethylamine (455 ml, 3.26 mol) was added dropwise over 30 min and the mixture was stirred for 48 h at rt. The reaction was then quenched by the addition of ice-cold water (1 liter) and the organic phase was separated. The aqueous phase was extracted with tert-butylmethyl ether (0.5 L) and the combined organic phases were washed with a mixture of saturated aqueous NaHCO3 (1 L) and brine (1 L). The solution was dried over MgSO4, concentrated in vacuo to afford 392.4 g of the N-benzyl imine product as a dense yellow oil that was used directly in the next step. 1H NMR (CDCl3, 300 MHz) δ 1.32 (t, J = 7.1 Hz, 3H), 4.24 (q, J = 7.1 Hz, 2H), 4.41 (d, J = 1.1 Hz, 2H), 7.39–7.47 (m, 3H), 7.78–7.81 (m, 2H), 8.31 (s, 1H).

Preparation of ethyl ester of N-Boc acid (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropane carboxylic propane

image8

To a suspension of lithium tert-butoxide (84.06 g, 1.05 mol) in dry toluene (1.2 L), a mixture of the N-benzyl imine of the glycine ethyl ester (100) was added dropwise , 4 g, 0.526 mol) and trans – 1,4 – dibromo – 2 – butene (107.0 g, 0.500 mol) in dry toluene (0.6 l) for 60 min. After completion of the addition, the deep red mixture was quenched by the addition of water (1 L) and tert-butylmethyl ether (TBME, 1 L). The aqueous phase was separated and extracted a second time with TBME (1 L). The organic phases were combined, 1 N HCl (1 L) was added and the mixture was stirred at room temperature for 2 h. The organic phase was separated and extracted with water (0.8 L). The aqueous phases were then combined, saturated with salt (700 g), TBME (1 L) was added and the mixture was cooled to 0 ° C. The stirred mixture was then basified to pH 14 by the dropwise addition of 10 N NaOH, the organic phase was separated and the aqueous phase was extracted with TBME (2 x 500 ml). The combined organic extracts were dried (MgSO4) and concentrated to a volume of 1 L. To this solution of the free amine, BOC2O or di-tert-butyl dicarbonate (131.0 g, 0.6 mol) was added and the mixture was stirred 4 days at rt. More di-tert-butyl dicarbonate (50 g, 0.23 mol) was added to the reaction, the mixture was refluxed for 3 hours and then allowed to cool to room temperature overnight. The reaction mixture was dried over MgSO4 and concentrated in vacuo to afford 80 g of crude material. This residue was purified by flash chromatography (2.5 Kg of SiO2 eluted with 1% to 2% MeOH / CH2Cl2) to provide 57 g (53%) of N-Boc acid ethyl ester (1R, 2S) / (1S , 2R) –1 – amino – 2– racemic vinyl cyclopropanecarboxylic acid as a yellow oil that solidified at rest in the refrigerator: 1H NMR (CDCl3, 300 MHz) δ 1.26 (t, J = 7.1 Hz, 3H), 1.46 (s, 9H), 1.43-1.49 (m, 1H), 1.76-1.82 (wide m, 1H), 2.14 (q, J = 8.6 Hz, 1H ), 4.18 (q, J = 7.2 Hz, 2H), 5.12 (dd J = 10.3, 1.7 Hz, 1H), 5.25 (wide s, 1H), 5.29 (dd, J = 17.6, 1.7 Hz, 1H), 5.77 (ddd, J = 17.6, 10.3, 8.9 Hz, 1H); MS m / z 254.16 (M – 1).

Preparation of racemic ethyl ester hydrochloride (1R, 2S) / (1S, 2R) 1-amino-2-vinylcyclopropanecarboxylic acid

image9

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N-Boc- (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester (9.39g, 36.8mmol) was dissolved in 4N HCl / dioxane (90ml, 360 mmol) and stirred for 2 hours at rt. The reaction mixture was concentrated to provide (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester hydrochloride in quantitative yield (7 g, 100%). 1H NMR (methanol-d4) δ 1.32 (t, J = 7.1, 3H), 1.72 (dd, J = 10.2, 6.6 Hz, 1H), 1.81 (dd, J = 8.3, 6.6 Hz, 1 H), 2.38 (q, J = 8.3 Hz, 1H), 4.26-4.34 (m, 2H), 5.24 (dd, 10.3, 1.3 Hz, 1H) 5.40 (d, J = 17.2, 1H), 5.69-5.81 (m, 1H).

PROCEDURE B

Preparation of racemic N-Boc-1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester hydrochloride

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commercially available dibenzyl imine of the glycine ethyl ester (25.0 g, 93.53 mmol) in THF (112 ml). The reaction mixture was heated to 0 ° C, stirred for 40 minutes and then cooled again to -78 ° C. To this solution was added trans-1,4-dibromo-2-butene (20.0 g, 93.50 mmol), the mixture was stirred for 1 hour at 0 ° C and cooled again to -78 ° C. Potassium tert-butoxide (11.55 g, 102.9 mmol) was added, the mixture was immediately heated to 0 ° C and stirred an additional hour before concentrating in vacuo. The crude product was taken up in Et2O (530 ml), 1N aqueous HCl solution (106 ml, 106 mmol) was added and the resulting biphasic mixture was stirred for 3.5 h at rt. The phases were separated and the aqueous phase was washed with Et2O (2x) and basified with a saturated aqueous solution of NaHCO3. The desired amine was extracted with Et2O (3x) and the combined organic extract was washed with

Brine, dried (MSO4) and concentrated in vacuo to obtain the free amine. This material was treated with a solution of 4N HCl in dioxane (100 ml, 400 mmol) and concentrated to provide the acid ethyl ester hydrochloride (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid as a brown semi-solid (5.3 g, 34% yield) identical to the material obtained by procedure A, except for the presence of a small amount of unidentified aromatic impurity (8%).

25 EXAMPLE 4

Resolution of N-Boc- (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester

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Resolution A

To an aqueous solution of sodium phosphate buffer (0.1 M, 4.25 liters ("l"), pH 8) housed in a 12-liter jacketed reactor, maintained at 39 ° C and stirred at 300 rpm, 511 grams of Alcalase 2.4L 5 (approximately 425 ml) (Novozymes North America Inc.). When the temperature of the mixture reached 39 °, the pH was adjusted to 8.0 by the addition of 50% NaOH in water. A solution of the racemic N-Boc acid ethyl ester (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid (85 g) in 850 ml of was then added over a period of 40 minutes DMSO The reaction temperature was then maintained at 40 ° C for 2.5 hours, during which time the pH of the mixture was adjusted to 8.0 at times 1.5 h and 19.5 h using 50% NaOH in water. After 24.5 h, the enantiomeric excess was determined to be 97.2% and the reaction was cooled to room temperature (26 ° C) and stirred overnight (16 h), after which it was determined that the enantiomeric excess was 100%. The pH of the reaction mixture was then adjusted to 8.5 with 50% NaOH and the resulting mixture was extracted with MTBE (2 x 2 L). The combined MTBE extract was then washed with 5% NaHCO3 (3 x 100 ml), water (3 x 100 ml), and evaporated in vacuo to give the N-Boc acid ethyl ester (1R, 2S) / - 1 – amino – 2–

15 enantiomerically pure vinylcyclopropanecarboxylic acid as a light yellow solid (42.55 g; purity: 97% @ 210 nanomolar ("nM"), acid-free; enantiomeric excess ("ee") 100%.

The aqueous phase of the extraction process was then acidified to pH 2 with 50% H2SO4 and extracted with MTBE (2 x 2 L). The MTBE extract was washed with water (3 x 100 ml) and evaporated to give the acid as a light yellow solid (42.74 g; purity: 99% @ 210 nM, not containing ester).

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1R, 2S – 1S ester, 2R – acid

ester

acid

High Resolution Mass Spectrometry

(+) ESI, C13H22NO4, [M + H] +, calculated 256.1549, found 256.1542 (-) ESI, C11H16NO4, [M – H] -, calculated 226,1079, found 226,1089

NMR chemical shifts observed Solvent: CDCl3 (proton δ 7.24 rpm, C – 13 δ 77.0 ppm) Bruker DRX – 500C: proton 500.032 MHz, carbon 125.746 MHz

Position

Proton (model) ppm C – 13 ppm Proton (model) ppm C – 13 ppm

one

––– 40.9 –––– 40.7

2

2.10 (q, J = 9.0 Hz) 34.1 2.17 (q, J = 9.0 Hz) 35.0

3rd

1.76 (width) 23.2 1.79 (width) 23.4

3b

1.46 (width) 1.51, (width)

4

–––– 170.8 –––– 175.8

5

5.74 (ddd, J = 9.0, 10.0, 17.0 Hz) 133.7 5.75 (m) 133.4

6th

5.25 (d, J = 17.0 Hz) 117.6 5.28 (d, J = 17.0 Hz) 118.1

6b

5.08 (dd, J = 10.0, 1.5 Hz) 5.12 (d, J = 10.5 Hz)

7

–––– 155.8 –––– 156.2

8

–––– 80.0 –––– 80.6

9

1.43 (s) 28.3 1.43 (s) 28.3

10

4.16 (m) 61.3 –––– ––––

eleven

1.23 (t, J = 7.5 Hz) 14.2 –––– ––––

Resolution B

To 0.5 ml of 100 millimolar Heps • Na buffer ("mM") (pH 8.5) in a well of a 24-well plate (capacity: 10 ml / well), 0.1 ml of Savinase 16.0 was added L (Bacillus clausii protease) (Novozymes North 5 America Inc.) and a solution of the N-Boc acid ethyl ester (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid (10 mg) in 0.1 ml of DMSO. The plate was sealed and incubated at 250 rpm at 40 ° C. After 18h, the enantiomeric excess of the ester was determined to be 44.3% as follows: 0.1 ml of the reaction mixture was extracted and mixed well with 1 ml of ethanol; after centrifugation, 10 microliters ("µl") of the supernatant was analyzed with chiral HPLC. To the rest of the reaction mixture, 0.1 ml of DMSO was added and the plate was incubated for another 3 days at

10 250 rpm at 40 ° C, after which four ml of ethanol was added to the well. After centrifuging, 10 µl of the supernatant was analyzed with chiral HPLC and the enantiomeric excess was determined to be 100%.

Resolution C

To 0.5 ml of 100 mM Heps • Na buffer (pH 8.5) in a well of a 24-well plate (capacity: 10 ml / well), 0.1 ml of Esperasa 8.0L, (protease of Bacillus halodurans) (Novozymes North America 15 Inc.) and a solution of N-Boc acid ethyl ester (1R, 2S / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid (10 mg) in 0.1 ml of DMSO The plate was sealed and incubated at 250 rpm at 40 ° C. After 18 hours, the enantiomeric excess of the ester was determined to be 39.6% as follows: 0.1 ml of the reaction mixture was extracted and mixed well with ethanol; after centrifugation, 10 μl of the supernatant was analyzed with chiral HPLC. To the remaining reaction mixture, 0.1 ml of DMSO was added and the plate was incubated for another 3 days at 250 rpm at 40 ° C,

After which four ml of ethanol was added to the well. After centrifuging, 10 µl of the supernatant was analyzed with chiral HPLC and the enantiomeric excess of the ester was determined to be 100%.

The analysis of the samples was carried out as follows:

1) Sample preparation: Approximately 0.5 ml of the reaction mixture was mixed well with 10 volumes of EtOH. After centrifuging, 10 µl of the supernatant was injected into the HPLC column.

25 2) Conversion determination: Column: YMC ODS A, 4.6 x 50 mm ("mm"), S – 5 µm

Solvent: A, 1 mM HCl in water; B, MeCN

Gradient: 30% of B for 1 min; 30% to 45% B for 0.5 min; 45% B for 1.5 min; 45% to 30% B for 0.5 min.

Flow rate: 2 ml / min

UV detection: 210 nM

Retention time: acid, 1.2 min; ester, 2.8 min.

3) Determination of enantiomeric excess for the ester:

Column: CHIRACEL OD – RH, 4.6 x 150 mm, S – 5 µm

Mobile phase: 50 mM MeCN / HClO4 in water (67/33)

Flow rate: 0.75 ml / min.

UV detection: 210 nM.

Holding time:

(1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid 5.2 min;

Racemate of ethyl ester of acid (1R, 2S) / (1S, 2R) -1-amino-2-vinylcyclopropanecarboxylic acid 18.5 min and 20.0 min;

ethyl ester of (1R, 2S) -1-amino-2-vinylcyclopropanecarboxylic acid 18.5 min.

Resolution D

5 L of 0.3 M sodium phosphate buffer (pH 8) was maintained at 38 ° C in a 20 liter jacketed reactor, stirred at 130 rpm. Four liters of Alcalasa 2.4L (Novozymes North America Inc.) and 1 liter of DI water were added to the reactor. When the temperature of the mixture approached 38 ° C, the pH was adjusted to 7.8 with 10 N NaOH. A solution of N-Boc acid ethyl ester solution was added to the reactor over a period of 1 hour. (1R, 2S) / (1S, 2R) –1 – amino – 2 – racemic vinylcyclopropanecarboxylic acid (500 grams) in 5 liters of DMSO. The reaction temperature was then adjusted to 48 ° C. After 21 hours, the enantiomeric excess of the ester reached 99.3%. Heating was stopped at 24 hours and the reaction was slowly cooled to room temperature (approximately 25 ° C) and stirred overnight. The pH of the reaction mixture was adjusted to 8.5 with 10 N NaOH and the mixture was extracted with MTBE (2 x 4 L). The combined MTBE extract was washed with 5% NaHCO3 (3 x 400 ml) and water (3 x 400 ml), and evaporated to give N-Boc acid ethyl ester (1R, 2S) / - 1-amino- 2- enantiomerically pure vinylcyclopropanecarboxylic acid as a light yellow crystal (259 g; purity: 96.9% @ 210 nM, which did not contain acid; 100% ee).

Resolution E

10 L of 0.1 M sodium phosphate buffer (pH 8) was maintained at 40 ° C in a 20 liter jacketed reactor, stirred at 360 rpm. 1.5 liters of Alcalasa 2.4L (Novozymes North America Inc.) was added to the reactor. When the temperature of the mixture approached 38 ° C, the pH was adjusted to 8.0 with 10 N NaOH. A solution of the N-Boc acid ethyl ester was added to the reactor over a period of one hour. (1R, 2S) / (1S, 2R) –1 – amino – 2 – racemic vinylcyclopropanecarboxylic acid (200 grams) in 2 liters of DMSO. The reaction temperature was then adjusted to 40 ° C. After 3 hours, the pH was adjusted to 8.0 with 10 N NaOH. After 21 hours the reaction was cooled to 25 ° C. The pH of the reaction mixture was adjusted to 8.5 with 10 N NaOH and the mixture was extracted with MTBE (2 x 5 L). The combined MTBE extract was washed with 5% NaHCO3 (3 x 500 ml) and water (3 x 200 ml), and evaporated to give 110 g of yellow oil. The oil was left at room temperature under vacuum of the installation and gave the enantiomerically pure N-Boc– (1R, 2S) / - 1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester as a colorless long rod crystal (10 g; purity: 97.9% @ 210 nM, which did not contain acid; 100% ee).

The enantiomerically pure N-Boc– (1R, 2S) / - 1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester of crystalline structure had been characterized by single crystal analysis (X-ray NB #: 52795-093, reference code: 634592N1 ). The absolute configuration is not determined by lack of a known chiral center or heavier atoms. A chain structure is formed along the axis to crystallographic through intermolecular hydrogen bonds between the amide group and the carbonyl oxygen atom (N ... O 3,159 Å).

N-Boc– (1R, 2S) -1-amino-2-vinylcyclopropane-carboxylic acid ethyl ester structure:

image 1

Structure of the N-Boc– (1R, 2S) -1-amino-2-vinylcyclopropane-carboxylic acid ethyl ester:

image 1

Crystal Data: Chemical formula: C13H21N1O4 Crystal system: Orthorhombic Space group: P212121 a = 5,2902 (1) Å  = 90 ° b = 13.8946 (2) Å  = 90 ° c = 19.9768 (3) Å  = 90 ° V = 1468.40 (4) Å3 Z = 4 dx = 1,155 g cm – 3 No. of reflections for network parameters: 6817

0.0776) Range θ for network parameters (°): 2.2–65.2

Resolution F

Experimental:

Crystallization

Crystal source: MTBE Crystal description: Colorless rods Crystal size (mm): 0.12 X 0.26 X 0.30

Data collection

Temperature (K): 293 θmax (°): 65.2 (Cu K) Number of reflections measured: 7518 Number of independent reflections: 2390 (Rint = Number of reflections observed (I ≥ 2σ: 2284 Absorption correction (Tmin –Tmax): 0.688–1,000

5 5 l of 0.2 M sodium borate buffer (pH 9) was maintained at 45 ° C in a 20 liter jacketed reactor, stirred at 400 rpm. Three liters of DI water and four liters of Savinase 16L, type EX (Novozymes North America Inc.) were added to the reactor. When the temperature of the mixture approached 45 ° C, the pH was adjusted to 8.5 with 10 N NaOH. A solution of N-acid ethyl ester solution was added to the reactor over a period of 40 minutes. Boc– (1R, 2S) / (1S, 2R) –1 – amino – 2 – racemic vinyl cyclopropanecarboxylic acid (200 grams) in 2

10 liters of DMSO. The reaction temperature was then adjusted to 48 ° C. After 2 hours, the pH was adjusted

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to 9.0 with 10 N NaOH. At 18 hours, the enantiomeric excess of the ester reached 72%, the pH was adjusted to 9.0 with 10 N NaOH. At 24 hours, the temperature was reduced to 35 ° C. At 42 hours, the temperature rose to 48 ° C and the pH was adjusted to 9.0 with 10 N NaOH. Heating was stopped at 48 hours and the reaction was slowly cooled to room temperature (approximately 25 ° C) and He stirred overnight. At 66 hours, the pH of the reaction mixture was 8.6. The mixture was extracted with MTBE (2 x 4 L). The combined MTBE extracts were washed with 5% NaHCO3 (6 x 300 ml) and water (3 x 300 ml), and evaporated to give N-Boc acid ethyl ester (1R, 2S) / - 1-amino- 2-Vinyl cyclopropanecarboxylic enantiomerically pure as a light yellow crystal (101A g; purity: 95.9% @ 210 nM, which did not contain acid; 98.6% ee).

EXAMPLE 5

Stage 1: Preparation of hydrochloride 1 (R) –amino – 2 (S) –ethyl vinylcyclopropanecarboxylate

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1 (R) -amino-2 (S) -vinylcyclopropanecarboxylate hydrochloride (8.5 g, 33.3 mmol) was stirred under an N2 atmosphere with 200 ml of 4 N HCl / dioxane (Aldrich) at t.a. for 3 h. The solvent was removed under reduced pressure keeping the temperature below 40 ° C. This gave 6.57 g (~ 100%) of 1 (R) -amino-2 (S) -ethyl vinylcyclopropanecarboxylate hydrochloride as a light tan solid. 1H NMR (300 MHz, CD3OD) δ 1.31 (t, J = 7.0 Hz, 3 H), 1.69-1.82 (m, 2 H), 2.38 (c, J = 8 , 8 Hz, 1 H), 4.29 (c, J = 7.0 Hz, 2 H), 5.22 (d, J = 10.3 Hz, 1H), 5.40 (d, J = 17 , 2 Hz, 1 H), 5.69-5.81 (m, 1 H). MS: m / z 156 (M ++ 1).

Stage 2: Preparation of 1 (R) - [1-tert-butoxycarbonyl-4 (R) -hydroxypyrrolidine-2 (S) -carboxyamide] -2 (S) -vinylcycle-ethyl propanecarboxylate

image 1

A stirred suspension of Boc-L-4-hydroxyproline ((2S, 4R) -hydroxyproline of N-Boc) (10 g, 43.3 mmol) in 400 ml of methylene chloride was treated sequentially with N-methyl-morpholine ( 9.3 ml, 84.7 mmol), HATU (19.5 g, 51.3 mmol) and ethyl 1 (R) -amino-2 (S) hydrochloride -vinylcyclopropanecarboxylate (9.1 g, 47.5 mmol ). The homogeneous gold solution was stirred at t.a. in N2 for 18 h and then concentrated in vacuo to give a brown oil. This was partitioned between ethyl acetate and saturated aqueous NaHCO3. The organic phase was washed with brine, dried (MSO4) and concentrated in vacuo to give 15 g (94%) of 1 (R) - [1-tert-butoxycarbonyl-4 (R) -hydroxypyrrolidine-2 (S) - carboxamido] –2 (S) –vinyl– ethyl cyclopropanecarboxylate as an off-white solid: LC-MS (Xterra HPLC column: 3.0 x 50 mm in length. Gradient: 100% solvent A / 0% solvent a 0% solvent A / 100% solvent B. Gradient time: 3 min Retention time: 1 minute Flow rate: 5 ml / min Detector Wavelength: 220 nM Solvent

A: 10% MeOH / 90% H2O / 0.1% TFA. Solvent B: 10% H2O / 90% MeOH / 0.1% TFA). (Retention time: 2.09 min), MS: m / z 369 (M ++ 1).

Stage 3: Preparation of 1 (R) - [4 (R) –hydroxypyrrolidine hydrochloride – 2 (S) –carboxamido] –2 (S) –vinylcycle – propane ethyl carboxylate

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A stirred suspension of 1 (R) - [1-tert-butoxycarbonyl-4 (R) -hydroxypyrrolidine-2 (S) -carboxamido] -2 (S) -ethyl vinylcyclopropanecarboxylate (5.0 g, 13.6 mmol) It was treated with 4N HCl / dioxane (20 ml) for 3 h. The reaction mixture was concentrated in vacuo to give 4.5 g (97%) of 1 (R) - [4 (R) -hydroxypyrrolidine-2 (S) -carboxamido] -2 (S) -vinylcyclopropanecarboxylate hydrochloride as a white solid: 1 H NMR (300 MHz, CD3OD) δ 1.26 (t, J = 7.14 Hz, 3 H), 1.46 (dd, J = 9.70, 5.31 Hz, 1 H ), 1.80 (dd, J = 8.23, 5.31 Hz, 1 H), 2.00 - 2.15 (m, 1 H), 2.18 - 2.30 (m, 1H), 2.45 (dd, J = 13.36, 7.50 Hz, 1 H), 3.36-3.48 (m, 1 H), 4.11-4.24 (m, 2 H), 4 , 44 (dd, J = 10.25, 7.68 Hz, 1H), 4.58-4.65 (m, 1 H), 4.84-4.94 (m, 1 H), 5.17 (d, J = 1.83 Hz, 1 H), 5.27-5.42 (m, 1 H), 5.67-5.89 (m, 1 H).

EXAMPLE 6

Preparation of cyclopropylsulfonamide. Procedures A and B.

Procedure A:

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A solution of 100 ml of THF cooled to 0 ° C was bubbled in gaseous ammonia until saturation was reached. To this solution was added a solution of 5 g (28.45 mmol) of cyclopropylsulfonyl chloride (purchased from Array Biopharma) in 50 ml of THF, the solution was heated to t.a. for a whole night and an additional day was stirred. The mixture was concentrated to 1-2 ml of the remaining solvent, applied on a 30 g short bed of SiO2 (eluted with 30% 60% EtOAc / Hexanes) to provide 3.45 g (100%) of cyclopropylsulfonamide as a white solid 1H NMR (Methanol-d4) δ 0.94-1.07 (m, 4H), 2.52-2.60 (m, 1H); 13 C NMR (methanol-d4) δ 5.92, 33.01.

Procedure B:

Stage 1: Preparation of N-tert-butyl- (3-chloro) propylsulfonamide

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Tert-Butylamine (3.0 mol, 315.3 ml) was dissolved in THF (2.5 L). The solution was cooled to –20 ° C. 3-Chloropropanesulfonyl (1.5 mol, 182.4 ml) was added slowly. The reaction mixture was allowed to warm to t.a. and stirred for 24 hours. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was dissolved in CH2Cl2 (2.0 L). The resulting solution was washed with 1 N HCl (1.0 L), water (1.0 L), brine (1.0 L) and dried over Na 2 SO 4. This was filtered and concentrated in vacuo to give a slightly yellow solid, which crystallized from hexane to give the product as a white solid (316.0 g, 99%). 1H NMR (CDCl3) δ 1.38 (s, 9H), 2.30-2.27 (m, 2H), 3.22 (t, J = 7.35 Hz, 2H), 3.68 (t , J = 6.2 Hz, 2H), 4.35 (b, 1H).

Stage 2: Preparation of cyclopropanesulfonic acid tert-butylamide

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To a solution of N-tert-butyl- (3-chloro) propylsulfonamide (2.14 g, 10.0 mmol) in THF (100 ml) was added n-BuLi (2.5 M in hexane, 8.0 ml , 20.0 mmol) at –78 ° C. The reaction mixture was allowed to warm to room temperature for a period of 1 hour. Volatiles were removed in vacuo. The residue was partitioned between EtOAc and water (200 ml, 200 ml). The separated organic phase was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was recrystallized from hexane to provide the desired product as a white solid (1.0 g, 56%). 1 H NMR (CDCl 3) δ 0.98-1.00 (m, 2H), 1.18-1.19 (m, 2H), 1.39 (s, 9H), 2.48-2.51 (m , 1H), 4.19 (b, 1H).

Stage 3: Preparation of cyclopropylsulfonamide

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5 A solution of cyclopropanesulfonic acid tert-butylamide (110.0 g, 0.62 mol) in TFA (500 ml) was stirred at room temperature for 16 hours. The volatile product was removed in vacuo. The residue was recrystallized from EtOAC / hexane (60 ml / 240 ml) to provide the desired product as a white solid (68.5 g, 91%). 1 H NMR (DMSO – d6) δ 0.84–0.88 (m, 2H), 0.95–0.98 (m, 2H), 2.41–2.58 (m, 1H), 6, 56 (b, 2H).

EXAMPLE 7

10 Preparation of N-tert-butyl- (1-methyl) cyclopropylsulfonamide.

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Stage 1 Preparation of N-tert-butyl- (3-chloro) propylsulfonamide.

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As shown above.

Stage 1b Preparation of N-tert-butyl- (1-methyl) cyclopropylsulfonamide.

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A solution of N-tert-butyl- (3-chloro) propylsulfonamide (4.3 g, 20 mmol) was dissolved in dry THF (100 ml) and

cooled to –78 ° C. To this solution was added n-BuLi (17.6 ml, 44 mmol, 2.5 M in hexane) slowly. Ice bath

Dry was removed and the reaction mixture was allowed to warm to t.a. over a period of 1.5 h. This mixture cooled

20 at -78 ° C and a solution of n-BuLi (20 mmol, 8 ml, 2.5 M in hexane) was added. The reaction mixture was heated to t.a., cooled to -78 ° C for a period of 2 hours and a net solution of methyl iodide (5.68 g, 40 mmol) was added. The reaction mixture was allowed to warm to t.a. overnight, it was deactivated with saturated NH4Cl (100 ml) at t.a. This was extracted with EtOAc (100 ml). The organic phase was washed with brine (100 ml), dried (MgSO4) and concentrated in vacuo to give a yellow solid that crystallized from hexane to provide the

25 product as a slightly yellow solid (3.1 g, 81%): 1 H NMR (CDCl 3) δ 0.79 (m, 2H), 1.36 (s, 9H), 1.52 (m, 2H ), 1.62 (s, 3H), 4.10 (sa, 1H).

Stage 1c: Preparation of 1-methylcyclopropylsulfonamide

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A solution of N-tert-butyl- (1-methyl) cyclopropylsulfonamide (1.91 g, 10 mmol) was dissolved in TFA (30 ml) and the reaction mixture was stirred at t.a. for 16 hours The solvent was removed in vacuo to give a yellow oil that crystallized from EtOAc / hexane (1: 4, 40 ml) to give Example 3, 1-methylcyclopropylsulfonamide, as a white solid (1.25 g, 96%): 1H NMR (CDCl3) δ 0.84 (m, 2H), 1.41 (s, 9H), 1.58 (m, 3H), 4.65 (sa, 2H). Anal. Calcd For C4H9NO2S: C, 35.54; H, 6.71; N, 10.36. Found: C, 35.67; H, 6.80; N, 10.40.

EXAMPLE 9

Preparation of 1-propylcyclopropylsulfonamide

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Stages 1b: Preparation of N-tert-butyl- (1-benzyl) cyclopropyl-sulfonamide.

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This compound was prepared using the procedure described for the preparation of 1-methylcyclopropylsulfonamide except that propyl halide was used instead of methyl iodide in the second stage of this procedure.

EXAMPLE 10

Preparation of N-tert-butyl- (1-allyl) cyclopropylsulfonamide.

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This compound, N-tert-butyl- (1-allyl) cyclopropylsulfonamide, was obtained in 97% yield according to the procedure described in the synthesis of N-tert-butyl- (1-methyl) cyclopropylsulfonamide unless 1.25 equivalents of allyl bromide were used as electrophile. The compound was taken directly in the following reaction without purification: 1H NMR: (CDCl3) δ (CDCl3) δ 0.83 (m, 2H), 1.34 (s, 9H), 1.37 (m, 2H) , 2.64 (d, J = 7.3 Hz, 2H), 4.25 (sa, 1H), 5.07–5.10 (m, 2H), 6.70–6.85 (m, 1H ).

Preparation of 1-allylcyclopropylsulfonamide.

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This compound, 1-allylcyclopropylsulfonamide, was obtained in 40% yield from N-tert-butyl- (1-allyl) cyclopropylsulfonamide according to the procedure described in the synthesis of 1-methylcyclopropylsulfonamide. The compound was purified by column chromatography on SiO2 using 2% MeOH in CH2Cl2 as the eluent: 1 H NMR (CDCl3) δ 0.88 (m, 2 H), 1.37 (m, 2 H), 2, 66 (d, J = 7.0 Hz, 2 H), 4.80 (s, 2 H), 5.16 (m, 2 H), 5.82 (m, 1 H); 13C NMR (CDCl3) δ 11.2, 35.6, 40.7, 119.0, 133.6.

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EXAMPLE 15

Preparation of cyclopropylsulfonamine tert-butyl-carbamate, a key intermediate in the repair of C1-substituted cyclopropylsulfonamides.

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Stage 1: Preparation of 3-chloropropylsulfonamide

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A solution of 3-chloropropanesulfonyl chloride (55 g, 310.7 mmol) was dissolved in THF (200 ml) and added dropwise over 30 minutes to a solution of NH4OH (200 ml) cooled to 0 ° C. The reaction mixture was heated to room temperature, stirred 1 hour and the aqueous phase was fractionated multiple times with dichloromethane (4 x 500 ml). The dichloromethane phase was washed with 1N HCl (150 ml), water (150 ml), dried over MgSO4, filtered and concentrated in vacuo. The crude solid was recrystallized from the minimum amount of dichloromethane in hexanes to provide 3-chloropropylsulfonamide as a white solid (45.3 g, 93%). 1H NMR (CDCl3) δ 2.34 (m, 2H), 3.32 (t, J = 7.3 Hz, 2H), 3.70 (t, J = 6.2 Hz, 2H), 4, 83 (s, 2H); 13C NMR (CDCl3) δ 27.10, 42.63, 52.57.

Stage 2: Preparation of 3-chloropropylsulfonylamine tert-butylcarbamate

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To a solution of 3-chloropropylsulfonamide (30.2 g, 191.5 mmol), triethylamine (30.2 ml, 217.0 mmol) and 4-DMAP (2.40 g, 19.6 mmol) in dichloromethane (350 ml) cooled to 0 ° C, a solution of i-tert-butyldicarbonate (47.2 g, 216.9 mmol) in dichloromethane (250 ml) was added slowly over 30 minutes. The reaction mixture was allowed to warm to room temperature, stirred for an additional 3 hours and fractionated with 1 N HCl (300 ml), water (300 ml), brine (300 ml), dried over MgSO4, filtered and filtered. concentrated in vacuo to provide the crude product. This material was triturated with 70 ml of 5% dichloromethane in hexanes to provide 3-chloropropylsulfonylamine tert-butylcarbamate as an off-white solid (47.2 g, 96%): 1 H NMR (CDCl 3) δ 1.51 (s, 9H), 2.33 (m, 2H), 3.60 (t, J = 7.3 Hz, 2H), 3.68 (t, J = 6.21 Hz, 2H); 13C NMR (CDCl3) δ 26.50, 27.95, 42.37, 50.40, 84.76, 149.53.

Stage 3: Preparation of cyclopropylsulfonylamine tert-butylcarbamate

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A solution of n-butyllithium (74.7 ml, 119.5 mmol, 1.6 M in hexane) was dissolved in dry THF (105 ml) and cooled to -78 ° C under an argon atmosphere. To this solution was added a solution of 3-chloropropylsulfonylamine tert-butylcarbamate (14 g, 54.3 mmol) in dry THF (105 ml) dropwise over 20-30 minutes. The dry ice bath was removed and the reaction mixture was allowed to warm to t.a. over a period of 2 hours. The reaction mixture was quenched with glacial acetic acid (3.4 ml), concentrated in vacuo and partitioned between dichloromethane (100 ml) and water (100 ml). The organic phase was washed with brine (100 ml), dried (MgSO4), filtered and concentrated in vacuo to afford the cyclopropylsulfonylamine tert-butylcarbamate as a whitish waxy solid (12.08 g, 100%): NMR of 1H (CDCl3) δ 1.10 (m, 2H), 1.34 (m, 2H), 1.50 (s, 9H), 2.88 (m, 1H), 7.43 (s, 1H). 13C NMR (CDCl3) δ 6.21, 28.00, 31.13, 84.07, 149.82.

EXAMPLE 16

Preparation of 1-methoxy-methylcyclopropyl-sulfonamide

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Stage 1: Preparation of 1-methoxymethylcyclopropylsulfonylamine tert-butylcarbamate

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To a solution of cyclopropylsulfonylamine tert-butylcarbamate (1.0 g, 4.5 mmol) dissolved in THF (30 ml)

5 cooled to -78 ° C, n-butyllithium (6.4 ml, 10.2 mmol, 1.6 M in hexane) was added and the reaction mixture was stirred for 1 hour. To this solution was added a net solution of chloromethylmethyl ether (0.40 ml, 5.24 mmol) and the mixture was added slowly at room temperature overnight. The solution pH was adjusted to 3 using 1N aqueous HCl and then extracted with ethyl acetate (4 x 50 mL portions). The combined extracts were dried (MgSO4), filtered and concentrated to provide 1-methoxymethylcyclopropylsulfonylamine tert-butylcarbamate, as a

Cereal solid (1.20 g, 100%) that was taken directly in the following reaction without further purification: 1 H NMR (CDCl 3) δ 1.03 (m, 2H), 1.52 (s, 9H), 1.66 (m, 2H), 3.38 (s, 3H), 3.68 (s, 2H), 7.54 (s, 1 H); 13C NMR (CDCl3) δ 11.37, 28.29, 40.38, 58.94, 73.43, 83.61, 149.57.

Stage 2: Preparation of 1-methoxymethylcyclopropylsulfonamide

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A solution of 1-methoxymethylcyclopropylsulfonylamine tert-butylcarbamate (1.14 g, 4.30 mmol) was dissolved in a 50% TFA / dichloromethane solution (30 ml) and stirred at room temperature for 16 hours. The solvent was removed in vacuo and the residue was chromatographed on 80 g of SiO2 (eluting with 0% 60% ethyl acetate / hexanes at 1-methoxymethylcyclopropylsulfonamide as a white solid (0.55 g, 77% in general for two steps)): 1 H NMR (CDCl 3) δ 0.95 (m, 2H), 1.44 (m, 2H), 3.36 (s, 3H), 3.65 (s, 2H), 4.85 (s, 2H); 13C NMR (CDCl3) δ

20 11.17, 40.87, 59.23, 74.80; LREM m / z 183 (M ++ NH4).

EXAMPLE 17

Preparation of 1-cyclopropylmethylcyclopropylsulfonamide

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Step 1. Preparation of 1-cyclopropylmethylcyclopropylsulfonylamine tert-butylcarbamate

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1-Cyclopropylmethylcyclopropylsulfonylamine tert-butylcarbamate was obtained in 92% yield according to the procedure described in the synthesis of tert-butylcarbamate 1-methoxymethylcyclopropylsulfonylamine, except for 1.10 equivalents of cyclopropylmethyl bromide which were used as electrophile. The compound was taken directly in the following reaction without purification: 1 H NMR (CDCl 3) δ 0.10 (m, 2H), 0.51 (m, 2H), 0.67 (m, 1H),

30 1.10 (m, 2H), 1.49 (s, 9H), 1.62 (m, 2H), 1.87 (d, J = 7.0 Hz, 2H).

Stage 2: Preparation of 1-cyclopropylmethylcyclopropylsulfonamide

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This compound was obtained in 65% yield of 1-cyclopropylmethylcyclopropylsulfonylamine tert-butylcarbamate according to the procedure described for the synthesis of 1-methoxymethylcyclopropylsulfonamide. The compound was purified by column chromatography on SiO2 using 0% to 60% ethyl acetate in hexanes as the eluent: 1 H NMR (CDCl 3) δ 0.15 (m, 2H), 0.51 (m, 2H) , 1.01 (s, 2H), 1.34 (s, 3H), 1.86 (d, J = 7.0 Hz, 2H), 4.83 (s, 2H); 13C NMR (CDCl3) δ 4.65, 7.74, 11.26, 35.62; LREM m / z 193 (M ++ NH4).

EXAMPLE 19

Preparation of 1– (3,5-dimethylisoxazol-4il) carbamoylcyclopropanesulfonamide

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Stage 1: Preparation of 1– (3,5-dimethylisoxazol-4-yl) carbamoylcyclopropanesulfonamide tert-butylcarbamate

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This compound was obtained in a 100% crude yield according to the procedure described for the synthesis of 1-methoxymethylcyclopropylsulfonylamine tert-butylcarbamate except that 1.20 equivalents of 3,5-dimethylisoxazol-4-isocyanate were used as the electrophile . The compound was taken directly in the next reaction without purification.

Stage 2: Preparation of 1– (3,5-dimethylisoxazol-4-yl) carbamoylcyclopropanesulfonamide

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This compound was obtained in 50% yield (580 mg) from 1.62 g (4.52 mmol) of 1– (3,5-dimethylisoxazol-4-yl) carbamoylcyclopropanesulfonamide tert-butylcarbamate using 30 ml (120 mmol) of HCl 4

20 N / dioxanes, stirring overnight, concentration and chromatography on a 40 M Biotage column (eluting with 0% to 5% methanol / dichloromethane: 1 H NMR (methanol-d4) δ 1.57 (m, 2H ), 1.1 (m, 2H), 2.15 (s, 3H), 2.30 (s, 3H), 4.84 (s, 3H); 13 C NMR (methanol – d4) δ 9.65 , 10.94, 15.01, 46.11, 114.82, 159.45, 165.55, 168.15; LREM m / z 260 (M ++ H).

EXAMPLE 20

25 Preparation of cyclobutyl sulfonamide from cyclobutyl bromide

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To a solution of 5.0 g (37.0 mmol) of cyclobutyl bromide in 30 ml of anhydrous diethyl ether (Et2O) cooled to -78 ° C was added 44 ml (74.8 mmol) of tert-butyllithium 1, 7 M in pentanes and the solution was slowly heated at -35 ° C for 1.5 hours. This mixture was slowly cannulated in a solution of 5.0 g (37.0 mmol) of freshly distilled sulfuryl chloride in 100 ml of hexanes cooled to –40 ° C, heated at 0 ° C for 1 hour and carefully concentrated in the void. This mixture was redissolved in Et2O, washed once with some ice water,

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It was dried (MgSO4) and concentrated carefully. This mixture was redissolved in 20 ml of THF, added dropwise to 500 ml of NH3 saturated in THF and allowed to stir overnight. The mixture was concentrated in vacuo to a crude yellow solid and recrystallized from the minimum amount of CH2Cl2 in hexanes with 1-2 drops of MeOH providing 1.90 g (38%) of cyclobutyl sulfonamide as a white solid. 1H NMR (CDCl3) δ 1.95-2.06 (m, 2H), 2.30-2.54 (m, 4H), 3.86 (p, J = 8 Hz, 1H), 4.75 (sa, 2H); 13C NMR (CDCl3) δ 16.43, 23.93, 56.29. HREM m / z (M – H) - calc. for C4H8NSO2: 134.0276; found 134.0282.

EXAMPLE 21

Preparation of cyclopentylsulfonamide

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A solution of 18.5 ml (37.0 mmol) of 2M cyclopentyl-magnesium chloride in ether was added dropwise to a solution of 3.0 ml (37.0 mmol) of freshly distilled sulfuryl chloride ( obtained from Aldrich) in 100 ml of hexanes cooled to –78 ° C. The mixture was heated at 0 ° C for 1 hour and then carefully concentrated in vacuo. This mixture was redissolved in Et2O (200 ml), washed once with some ice water (200 ml), dried (MgSO4) and carefully concentrated. This mixture was redissolved in 35 ml of THF, added dropwise to 500 ml of NH3 saturated in THF and allowed to stir overnight. The mixture was concentrated in vacuo to a crude yellow solid, the residue was filtered through 50 g of silica gel using 70% EtOAc-hexanes as the eluent and the solution was then concentrated. The residue was recrystallized from the minimum amount of CH2Cl2 in hexanes with 1-2 drops of MeOH providing 2.49 g (41%) of cyclopentylsulfonamide as a white solid. 1H NMR (CDCl3) δ 1.58–1.72 (m, 2H), 1.74–1.88 (m, 2H), 1.94–2.14 (m, 4H), 3.48– 3.59 (m, 1 H), 4.80 (sa, 2 H); RMA of 13C (CDCl3) δ 25.90, 28.33, 63.54; MS m / e 148 (M – H) -.

EXAMPLE 22

Preparation of cyclohexylsulfonamide

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A solution of 18.5 ml (37.0 mmol) of 2M cyclohexyl magnesium chloride (TCI Americas) in ether was added dropwise to a solution of 3.0 ml (37.0 mmol) of freshly distilled sulfuryl (obtained from Aldrich) in 100 ml of hexanes cooled to –78ºC. The mixture was heated at 0 ° C for 1 hour and then concentrated in vacuo. This mixture was redissolved in Et2O (200 ml), washed once with some ice water (200 ml), dried (MgSO4) and carefully concentrated. This mixture was redissolved in 35 ml of THF, added dropwise to 500 ml of NH3 saturated in THF and allowed to stir overnight. The mixture was concentrated in vacuo to a crude yellow solid, the residue was filtered through 50 g of silica gel using 70% EtOAc-hexanes as the eluent and concentrated. The residue was recrystallized from the minimum amount of CH2Cl2 in hexanes with 1–2 drops of MeOH providing 1.66 g (30%) of cyclohexylsulfonamide as a white solid: 1H NMR (CDCl3) δ 1.11-1, 37 (m, 3H), 1.43-1.56 (m, 2H), 1.67-1.76 (m, 1H), 1.86-1.96 (m, 2H), 2.18– 2.28 (m, 2H), 2.91 (tt, J = 12, 3.5 Hz, 1H), 4.70 (sa, 2H); 13 C NMR (CDCl 3) δ 25.04, 25.04, 26.56, 62.74; MS m / e 162 (M – 1) -.

EXAMPLE 23

Preparation of Neopentilsulfonamide

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Following the procedure for the preparation of cyclohexylsulfonamide, 49 ml (37 mmol) of 0.75 M neopentylmagnesium chloride (Alpha) was converted to 1.52 g (27%) diethyl ether of neopentylsulfonamide as a white solid. 1H NMR (CDCl3) δ 1.17 (s, 9H), 3.12 (s, 2H), 4.74 (s.a., 2H); 13C NMR (CDCl3) δ 29.46, 31.51, 67.38; MS m / c 150 (M – 1) -.

EXAMPLE 24

Preparation of cyclobutylcarbonylsulfonamide

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A solution of 12.3 g (83 mmol) of cyclobutylcarbinyl bromide (Aldrich) and 13.7 g (91 mmol) of sodium iodide in 150 ml of acetone was refluxed overnight and then cooled to temperature ambient. Inorganic solids were emilinated by filtration and acetone and cyclopropylcarbonyl iodide (8.41 g,

5 to 46%) were removed by distillation under ambient conditions and at 19995 pascals (150 torr) at 80 ° C, respectively.

A solution of 4.0 g (21.98 mmol) of cyclobutylcarbinyl iodide in 30 ml of anhydrous diethyl ether (diethyl ether) cooled to -78 ° C was cannulated in a solution of 17 ml (21.98 mmol) of sec - 1.3 Mbutyllithium in cyclohexanes and the solution was stirred for 5 minutes. To this mixture a solution of 3.0 g (21.98 mmol) of chloride was cannulated

10 freshly distilled sulfuryl in 110 ml of hexanes cooled to –78 ° C, the mixture was heated at room temperature for 1 hour and then carefully concentrated in vacuo. This mixture was redissolved in Et2O, washed once with some ice water, dried (MgSO4), filtered and carefully concentrated. This mixture was redissolved in 30 ml of THF, added dropwise to 500 ml of NH3 saturated in THF and allowed to stir overnight. The mixture was concentrated in vacuo to a crude yellow solid and recrystallized from the minimum amount of

Dichloromethane in hexanes with 1–2 drops of methanol providing 1.39 g (42%) of cyclobutyl sulfonamide as a white solid. 1H NMR (CDCl3) δ 1.81-2.03 (m, 4H), 2.14-2.28 (m, 2H), 2.81-2.92 (m, 1H), 3.22 ( d, J = 7 Hz, 2H), 4.74 (sa, 1H); 13C NMR (CDCl3) δ 19.10, 28.21, 30.64, 60.93; MS m / e 148 (M – H) -.

EXAMPLE 25

Preparation of cyclopropylcarbinilsu / fonamide

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Using the procedure used for the preparation of cyclobutylcarbonylsulfonamide, cyclopropylcarbonylsulfonamide was prepared from cyclopropylcarbinyl bromide (Aldrich) (see also JACS 1981, p. 442-445). 1H NMR (CDCl3) δ 0.39–0.44 (m, 2H), 0.67–0.76 (m, 2H), 1.13–1.27 (m, 1H), 3.03 ( d, J = 7.3 Hz, 2H), 4.74 (sa, 1H); 13C NMR (CDCl3) δ 4.33, 5.61, 59.93; MS m / e 134 (M – 1).

25 EXAMPLE 26

Preparation of 2-thiophenesulfonamide

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It was prepared from 2-thiophenesulfonyl chloride (purchased from Aldrich) using the Justus Liebigs Ann procedure. Chem., 501, 1933, p. 174–182.

EXAMPLE 27

Preparation of 4-bromobenzenesulfonamide

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4-Bromophenylsulfonamide was prepared by treatment of commercially available 4-bromosulfonyl chloride with THF saturated ammonia.

EXAMPLE 28

Preparation of HCl salt of (1– (R) –amino – 2– (S) –vinylcyclopropanecarbonyl) amide of cyclopropanesulfonic acid

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Step 1. Preparation of 1 (R) -terc-butoxycarbonylamino-2 (S) -vinylcyclopropanecarboxylic acid

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To a solution of 1 (R) -terc-butoxycarbonylamino-2 (S) -vinylcyclopropanecarboxylic acid ethyl ester (3.28 g, 13.2 mmol) in THF (7 ml) and methanol (7 ml) suspension was added of LiOH (1.27 g, 53.0 mmol) in water (14 ml). The mixture was stirred overnight at room temperature and quenched with 1 N NaOH (15 ml) and water (20 ml). The resulting mixture was washed with ethyl acetate (20 ml) and the organic phase was extracted with 20 ml of 0.5 N NaOH. The combined aqueous phases were acidified with 1 N HCl to pH 4 and extracted with ethyl acetate ( 3 x 40 ml). The combined organic extracts were washed with brine, dried (MgSO4), filtered and concentrated to give the title compound as a white solid (2.62 g, 87%). 1H NMR: (DMSO – d6) δ 1.22–1.26 (m, 1H), 1.37 (s, 9H), 1.50–1.52 (m, 1H), 2.05 (c , J = 9 Hz, 1H), 5.04 (d, J = 10 Hz, 1H), 5.22 (d, J = 17 Hz, 1 H), 5.64–5.71 (m, 1H) , 7.18, 7.53 (s, NH (rotamers), 12.4 (sa, 1H)); MS m / z 228 (M ++ H).

Stage 2: Preparation of (1– (R) –terc-butoxycarbonylamino – 2– (S) –vinylcyclopropanecarbonyl) –amide of cyclopropanesulfonic acid

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A solution of the product of Step 1 (2.62 g, 11.5 mmol) and CDI (2.43 g, 150 mmol) in THF (40 ml) was heated at reflux for 50 minutes under nitrogen. The solution was cooled to room temperature and transferred by cannula to a solution of cyclopropylsulfonamide (1.82 g, 15.0 mmol) in THF (10 ml). To the resulting solution was added DBU (2.40 ml, 16.1 mmol) and stirring was continued for 20 hours. The mixture was quenched with 1 N HCl at pH 1 and THF was concentrated in vacuo. The suspension was extracted with ethyl acetate (2 x 50 ml) and the combined organic extracts were dried (Na2SO4), filtered and concentrated. Purification by recrystallization from hexanes - ethyl acetate (1: 1) provided the title compound (2.4 g) as a white solid. The mother liquors were purified by a Biotage 40S column (eluted 9% acetone in dichloromethane) to give a second batch of the title compound (1.1 g). Both lots were combined (92% total yield). 1H NMR (DMSO-d6) δ 0.96-1.10 (m, 4H), 1.22 (dd, J = 5.5, 9.5 Hz, 1H), 1.39 (s, 9H) , 1.70 (t, J = 5.5 Hz, 1 H), 2.19–2.24 (m, 1H), 2.90 (m, 1H), 5.08 (d, J = 10 Hz , 1 H), 5.23 (d, J = 17 Hz, 1 H), 5.45 (m, 1H), 6.85, 7.22 (s, NH (rotamers)); MS m / z 331 (M ++ H).

Step 3: Preparation of HCl salt of (1– (R) –amino – 2– (S) –vinylcyclopropanecarbonyl) cyclopropanesulfonic acid amide

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A solution of the product of Step 2 (3.5 g, 10.6 mmol) in dichloromethane (35 ml) and TFA (32 ml) was stirred at room temperature for 1.5 hours. The volatiles were removed in vacuo and the residue was suspended in 1 N HCl in diethyl ether (20 ml) and concentrated in vacuo. This procedure was repeated once. The resulting mixture was triturated from pentane and filtered to give the title compound as a whitish, hygroscopic solid (2.60 g, 92%). 1 H NMR: (DMSO – d6) δ 1.01–1.15 (m, 4H), 1.69–1.73 (m, 1H), 1.99–2.02 (m, 1H), 2, 38 (c, J = 9 Hz, 1H), 2.92-2.97 (m, 1H), 5.20

(d, J = 11 Hz, 1H), 5.33 (d, J = 17 Hz, 1H), 5.52-5.59 (m, 1H), 9.17 (s.a., 3H); MS m / z 231 (M ++ H). EXAMPLE 29

Preparation of acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18-hydroxy-2.15-dioxo-3.16- diazatricyclo [14.3.0.04.6] nonadec-7 -Eno-4-carboxylic, Example 29.

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Stage 1: Preparation of methyl ester 1– (2 (S) -terc-butoxycarbonylamino-non-8-enoyl) -4 (R) -hydroxy-pyrrolidine-2 (S) -carboxylic

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A solution of 2 (S) -terc-butoxycarbonylamino-8-nonenoic acid (purchased from RSP Amino Acids) (3.5 g,

10 12.9 mmol) in 200 ml of DCM was treated sequentially with 4 (R) -hydroxypyrrolidine-2 (S) -carboxylic acid methyl ester hydrochloride (2.15 g, 11.8 mmol), N-methyl- morpholine (4.25 ml, 38.6 mmol) and HATU (5.37 g, 14.1 mmol). The reaction mixture was stirred at t.a. in N2 for 3 days and then concentrated in vacuo. The residue was partitioned between ethyl acetate and buffer at pH 4 (biftalate). The organic phase was washed with saturated aqueous NaHCO3, dried (MgSO4) and concentrated in vacuo to give the crude product. Flash chromatography (ethyl acetate at

50% / hexane to 100% ethyl acetate) gave 4.7 g (~ 100%) of 1– (2 (S) -terc-butoxycarbonylamino-non-8-enoyl acid methyl ester) -4 (R ) -Hydroxy-pyrrolidine-2 (S) -carboxylic as a colorless solid: 1H NMR (500 MHz, CD3OD) δ 1.33-1.50 (m, 8 H), 1.46 (s, 9 H) , 1.57 (m, 1 H), 1.72 (m, 1 H), 2.08 (m, 2 H), 2.28 (m, 1 H), 3.72 (s, 3 H) , 3.75-3.87 (m, 2 H), 4.36 (m, 1 H), 4.51 (sa, 1 H), 4.57 (t, J = 8.2 Hz, 1 H ), 4.95 (d, J = 10.4 Hz, 1 H), 5.01 (m, 1 H), 5.83 (m, 1 H); MS m / z 399 (M ++ 1).

Step 2: Preparation of 1 - {[1– (2 (S) –terc-butoxycarbonylamino-non-8-enoyl) -4 (R) -hydroxy-pyrrolidine-2 (S) carbonyl acid] ethyl ester (- 1R) –amino} –2 (S) –vinyl – cyclopropanecarboxylic

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Methyl ester of 1– (2 (S) –terc-butoxycarbonylamino-non-8-enoyl) -4 (R) -hydroxy-pyrrolidine-2 (S) -carboxylic acid (4.7 g, 11.8 mmol) it was dissolved in THF (80 ml), methanol (20 ml) and water (40 ml). Powdered lithium hydroxide (5.6 g, 233 mmol) was added. The light yellow suspension was stirred at t.a. in N2 for 16 hours and then concentrated in vacuo. The residue was partitioned between ether and water. The ether phase was discarded, and the aqueous phase was treated with 1 N HCl until the pH was 4. This acid solution was extracted with EtOAc (3x). The combined EtOAc extracts were dried (MgSO4) and concentrated in vacuo to give 4.36 g (96%) of 1– (2 (S) -terc-butoxycarbonylamino-8-nonenoyl) -

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4 (R) -hydroxy-pyrrolidine-2 (S) -carboxylic as a white solid. This acid was then dissolved in 150 ml of DMF and then (1R, 2S) -1-amino-2-vinylcyclopropanecarboxylic acid hydrochloride (2.61 g, 13.6 mmol), N-methyl-morpholine were added (2.5 ml, 22.6 mmol) and HATU (5.2 g, 13.7 mmol). The reaction mixture was stirred at t.a. in N2 for 16 hours and then concentrated in vacuo. The residue was partitioned between ethyl acetate and buffer at pH 4 (biftalate). The organic phase was washed with saturated aqueous NaHCO3, dried (MgSO4) and concentrated in vacuo to give the crude product. Flash chromatography (60% -80% ethyl acetate / hexane) gave 6.0 g (98%) of 1 - {[1– (2 (S) -terc-butoxycarbonylamino-non-8– ethyl ester enoyl) –4 (R) –hydroxy-pyrrolidine – 2 (S) carbonyl] - (1R) –amino} –2 (S) –vinyl– cyclopropanecarboxylic acid as a white solid: 1 H NMR (500 MHz, CD3OD) δ 1 , 25 (t, J = 7.2 Hz, 3 H), 1.33-1.80 (m, 10 H), 1.46 (s, 9 H), 2.09 (m, 3 H), 2.25 (m, 2 H), 3.76 (m, 2 H), 4.14 (m, 2 H), 4.27 (dd, J = 8.5, 5.2 Hz, 1 H) , 4.50 (m, 2 H), 4.94 (d, J = 10.1 Hz, 1 H), 5.01 (dd, J = 17.1, 1.8 Hz, 1 H), 5 , 11 (dd, J = 10.4, 1.8 Hz, 1 H), 5.30 (d, J = 15.6 Hz, 1 H), 5.80 (m, 2 H), 8.57 (s, 1 H); MS m / z 522 (M ++ 1).

Stage 3: Preparation of ethyl ester of acid (1S, 4R, 6S, 14S, 18R) –7 – cis – 14-tert-butoxycarbonylamino – 18 – hydroxy– 2.15 – dioxo – 3.16 – diazatricyclo [14.3.0.04 .6] nonadec – 7 – eno – 4 – carboxylic

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A solution of 1 - {[1– (2 (S) -terc-butoxycarbonyl-amino-non-8-enoyl) -4 (R) -hydroxy-pyrrolidine-2 (S) carbonyl acid] - (1R ) -Amino} -2 (S) -vinylcyclopropane-carboxylic acid (800 mg, 1.53 mmol) in 2 l of methylene chloride was purged with N2 for 0.5 h. Then trichlorhexylphosphine [1,3-bis (2,4,6-trimethyl-phenyl) -4,5-dihydroimidazol-2-yl-deno] [benzylidene] -ruthenium (IV) (Strem) dichloride (64 mg) was added , 0.075 mmol) and the mixture was purged with N2 for another 10 min. The light orange homogeneous solution was refluxed for 2 hours giving a dark orange solution. The reaction mixture was cooled to t.a. and concentrated in vacuo giving an orange oil. Flash chromatography (ethyl acetate) gave 460 mg (61%) of acid ethyl ester (1S, 4R, 6S, 14S, 18R) –7 – cis – 14– tert-butoxycarbonylamino – 18 – hydroxy – 2.15– dioxo – 3.16 – diazatricyclo [14.3.0.04.6] –nonadec – 7 – eno – 4 – carboxylic as a gray solid. 1 H NMR (500 MHz, CDCl 3) δ 1.19 (t, J = 7.2 Hz, 3 H), 1.42 (s, 9 H), 1.22–1.8 (m, 8 H) , 1.87 (m, 2 H), 2.03– 2.22 (m, 4 H), 2.63 (m, 1 H), 3.65 (m, 1 H), 4.09 (m , 3 H), 4.45 (m, 1 H), 4.56 (s, 1 H), 4.82 (m, 1 H), 5.23 (m, 1 H), 5.51 (s , 1 H), 7.16 (s, 1 H); MS m / z 494 (M ++ 1).

Stage 4: Acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18-hydroxy-2.15-dioxo-3.16-diazatricyclo [14.3.0.04.6] nonadec– 7 – eno – 4 – carboxylic

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To an ethyl ester solution of (1S, 4R, 6S, 14S, 18R) –7-cis-14-tert-butoxycarbonylamino-18-hydroxy-2.15- dioxo-3.16-diazatricyclo [14.3.0.04.6 ] -Nonadec-7-ene-4-carboxylic acid (493 mg, 1.0 mmol) in THF (4 ml), methanol (1 ml) and water (2 ml), lithium hydroxide powder (480 mg, 20 mmol) and the yellow suspension was stirred at rt on N2 for 16 hours. The mixture was concentrated in vacuo and the residue was partitioned between ether and water. The ether phase was discarded, and the aqueous phase was treated with 1 N HCl until the pH was 4. This acid solution was extracted with EtOAc three times. The combined extracts were dried (MgSO4) and concentrated in vacuo to give 460 mg (98%) of Example 18, acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18-hydroxy –2.15 – dioxo – 3.16 – diazatricyclo [14.3.0.04.6] –nonadec – 7– eno – 4 – carboxylic as a gray solid. 1H NMR (500 MHz, CD3OD) δ ppm 1.26 (t, J = 7.2 Hz, 3 H), 1.35–1.52 (m, 15 H), 1.57–1.68 ( m, 3H), 1.79 (m, 1 H), 2.04 (m, 1H), 2.16–2.41 (m, 3 H), 3.80 (dd, J = 10.7, 4.3 Hz, 1 H), 3.88 (m, 1 H), 4.38 (dd, J = 8.9, 3.1 Hz, 1 H), 4.55 (m, 2 H), 5.39 (t, J = 9.8 Hz, 1 H), 5.58 (m, 1H); MS m / z 466 (M ++ 1).

EXAMPLE 30

Preparation of tert-butyl ester of (4-cyclopropanesulfonylaminocarbonyl-18-hydroxy-2.15-dioxo-3.16-diaza– tricycle [14.3.0.04.6] nona-dec-7-en-14-yl) -carbamic

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Stage 1: Peparation of 1 - {[1– (2-tert-butoxycarbonylamino-non-8-enoyl) -4- (tert-butyl-dimethyl-silanyloxy) -pyrrolidine-2-carbonyl] -amino} ethyl ester –2 – vinylcyclopropanecarboxylic

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To a mixture of 1 - {[1– (2 (S) -terc-butoxycarbonylamino-non-8-enoyl) -4 (R) -hydroxy- ethyl ester

pyrrolidine-2 (S) carbon-yl] - (1R) -amino} -2 (S) -vinyl-cyclopropanecarboxylic acid (1.5 g, 2.87 mmol) in 10 ml of DMF is

added imidazole (0.25 g, 3.67 mmol) and tert-butyl dimethylsilyl chloride (516 mg, 3.44 mmol). The mixture was stirred at t.a.

10 for two days. The reaction mixture was then concentrated in vacuo and the residue was dissolved in ethyl acetate. This solution was washed with water, dried over magnesium sulfate and concentrated in vacuo to obtain a crude solid. Purification by purge chromatography (eluting with 20% ethyl acetate in hexane) gave 1.43 g (78%) of 1 - {[1– (2-tert-butoxycarbonylamino-non-8-enoyl) -4 acid - (tert-Butyldimethyl-silanyloxy) -pyrrolidine-2-carbonyl] -amino} -2-vinylcyclopropanecarboxylic acid as a white solid.

1 H NMR (300 MHz, CD3OD) δ 0.10 (s, 6 H), 0.89 (s, 9 H), 1.22 (m, 3 H), 1.31–1.48 (m , 16 H), 1.50–1.75 (m, 3 H), 2.06 (m, 3 H), 2.11–2.33 (m, 2 H), 3.70 (m, 2 H), 4.03-4.19 (m, 2 H), 4.21 (m, 1 H), 4.45 (t, J = 7.87 Hz, 1 H), 4.59 (m, 1 H), 4.91 (d, J = 9.15 Hz, 1 H), 4.98 (d, J = 17.20 Hz, 1 H), 5.08 (dd, J = 10.25, 1.83 Hz, 1 H), 5.27 (dd, J = 17.38, 1.65 Hz, 1 H), 5.65-5.87 (m, 2 H); MS m / z 636 (M ++ 1).

Stage 2: Preparation of ethyl ester of 14-tert-butoxycarbonylamino-18- (tert-butyl-dimethylsilanyloxy) -2.15-dioxo– 20 3.16-diaza-tricycle [14.3.0.04.6] nonadec-7– eno – 4 – carboxylic

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To a solution of 1 - {[1– (2-tert-butoxycarbonylamino-non-8-enoyl) -4– (tert-butyldimethyl) ethyl ester

silanyloxy) -pyrrolidine-2-carbonyl] -amino} -2-vinylcyclopropanecarboxylic acid (1.63 g, 2.56 mmol) in 640 ml of chloride

methylene 215 mg (0.26 mmol) of tricyclohexylphosphine dichloride [1,3-bis (2,4,6-tri [benzylidene] ruthenium (IV)) was added.

The mixture was heated at reflux for 15 minutes. The residue was concentrated in vacuo and then purified by chromatography.

ultrafast eluting with 30% ethyl acetate / hexane. Further decolorizing the sample, the raw product

chromatographed a second time eluting with 50% ether in hexane giving 1.5 g (96%) of acid ethyl ester

14-tert-butoxycarbonylamino-18- (tert-butyldimethyl-silanyloxy) -2.15-dioxo-3.16-diaza-tricycle [14.3.0.04.6] nonadec-7-eno-4–

carboxylic as a white solid. 1 H NMR (500 MHz, CD 3 Cl) δ 0.06 (s, 3 H), 0.07 (s, 3 H), 0.86 (s, 9 H), 1.18–1.24

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(m, 6 H), 1.34–1.64 (m, 14 H), 1.86–1.96 (m, 3 H), 2.02–2.09 (m, 1 H), 2 , 11–2.17 (m, 1 H), 2.19–2.28 (m, 1 H), 2.57– 2.63 (m, 1 H), 3.50–3.54 (m , 1 H), 3.71 (dd, J = 10.22, 6.26 Hz, 1 H), 4.06-4.17 (m, 2 H), 4.52-4.58 (m, 2 H), 4.75 (d, J = 8.55 Hz, 1 H), 5.21 (t, J = 9.92 Hz, 1 H), 5.35 (d, J = 7.63 Hz , 1 H), 5.45-5.50 (m, 1 H), 6.94 (s, 1 H); MS m / z 608 (M ++ 1).

Stage 3: Preparation of ethyl ester of 14-tert-butoxycarbonylamino-18- (tert-butyl-dimethylsilanyloxy) -2.15-dioxo– 3.16-diaza-tricycle [14.3.0.04.6] nonadec-7-eno –4 – carboxylic

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To a solution of ethyl ester of 14-tert-butoxycarbonylamino-18- (tert-butyl-dimethyl-silanyloxy) -2.15-dioxo-3.16-diaza-tricycle [14.3.0.04.6] nonadec-7– eno-4-carboxylic acid (1.5 g, 2.47 mmol) in a mixed solvent system of THF (4 ml), methanol (1 ml) and water (2 ml), lithium hydroxide powder hydroxide monohydrate ( 1.0 g, 50 mmol). The light yellow suspension a was stirred at t.a. in N2 for 4 h. The mixture was then concentrated in vacuo and the residue was partitioned between ether and water. The ether phase was discarded and the aqueous phase was treated with 1 N HCl until the pH was 4. This acid solution was extracted with EtOAc (3x). The combined EtOAc extracts were dried (MgSO4) and concentrated in vacuo to give 1.2 g (84%) of 14-tert-butoxycarbonylamino-18- (tert-butyl-dimethyl-silanyloxy) -2.15-dioxo– 3.16 – diaza – tricycle [14.3.0.04.6] nonadec – 7 – eno – 4 – carboxylic as an off-white solid. 1H NMR (300 MHz, CD3OD) 0.12 (s, 6 H), 0.89 (s, 9 H), 1.23–1.64 (m, 17 H), 1.70–1.87 (m, 1 H), 1.90–2.49 (m, 6 H), 3.70–3.80 (m, 1 H), 3.83–3.90 (m, 1 H), 4 , 28–4.36 (m, 1 H), 4.47–4.55 (m, 1 H), 4.65 (s, 1 H), 5.30–5.39 (m, 1 H) , 5.53-5.62 (m, 1 H); MS m / z 580 (M ++ 1).

Stage 4: Preparation of tert-butyl acid ester [18– (tert-butyl-dimethyl-silanyloxy) -4-cyclopropane sulfonylamino carbonyl-2.15-dioxo-3.16-diaza-tricycle [14.3.0.04.6] nonadec – 7 – en – 14 – il] –carbamic

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14-tert-Butoxycarbonylamino-18- (tert-butyl-dimethyl-silanyloxy) -2.15-dioxo-3.16-diaza-tricycle [14.3.0.04.6] nonadec-7-ene-4-carboxylic acid was dissolved (500 mg, 0.86 mmol) in 25 ml of THF and treated with CDI (180 mg, 1.12 mmol). (Care was taken to avoid moisture using oven-dried glassware and maintaining a dry N2 atmosphere.) After the reaction mixture was refluxed for 2 hours, it was cooled to t.a. and treated sequentially with cyclopropylsulfonamide (135 mg, 1.12 mmol) and DBU (170 mg, 1.12 mmol). The reaction mixture was stirred for 4 hours at t.a. and THF was removed by rotary evaporation. The residue was partitioned between ethyl acetate and pH 4 buffer. The organic phase was dried (MgSO4) and concentrated in vacuo to give the crude product. This was then purified by flash chromatography (eluting with 33% ethyl acetate in hexane) to give 300 mg (51%) of tert-butyl acid ester [18– (tert-butyldimethyl-silanyloxy) -4-cyclopropanesulfonylaminocarbonyl-2, 15 – dioxo – 3.16– diaza – tricycle [14.3.0.04.6] nonadec – 7 – en – 14-il] –carbamic as a white solid. 1H NMR (300 MHz, CD3OD) δ 1H 0.07 (s, 3 H), 0.08 (s, 3 H), 0.85 (s, 9 H), 0.87–1.49 (m , 21 H), 1.73–1.95 (m, 3 H), 2.08–2.16 (m, 1 H), 2.25–2.36 (m, 2 H), 2.42 –2.56 (m, 1 H), 2.85–2.93 (m, 1 H), 3.65–3.74 (dd, J = 10.61, 3.66 Hz, 1 H), 3.89 (d, J = 10.25 Hz, 1 H), 4.34 (m, J = 9.70, 9.70 Hz, 1 H), 4.43 (t, J = 7.87 Hz , 1 H), 4.57 (s, 1 H), 4.94–5.01 (m, 1 H), 5.10 (d, J = 8.78 Hz, 1 H), 5.66– 5.75 (m, 1 H), 6.55 (s, 1 H), 10.13 (s, 1 H); MS m / z 683 (M ++ 1).

Step 5: Preparation of Example 19, tert-butyl ester of (4-cyclopropanesulfonylaminocarbonyl-18-hydroxy-2.15-dioxo-3.16-diaza-tricycle [14.3.0.04.6] nonadec-7-en-14 –Il) –carbamic

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To a mixture of [18- (tert-butyl-dimethylsilanyloxy) -4-cyclopropane sulfonyl aminocarbonyl-2.15-dioxo-3.16-diaza-tricyclo [14.3.0.04.6] nonadec-7 acid tert-butyl ester -En-14-yl] -carbamic (330 mg, 0.48 mmol) in 25 ml of THF was added tetrabutylammonium fluoride (150 mg, 0.54 mmol). The reaction mixture was stirred at t.a. for 18

5 hours and then THF was removed by rotary evaporation. The residue was partitioned between ethyl acetate and water. The organic phase was dried (MgSO4) and concentrated in vacuo to give the crude product. It was then purified by triturating with hexane to provide 200 mg (73%) of tert-butyl ether of the (4-cyclopropanesulfonylaminocarbonyl-18-hydroxy-2.15-dioxo-3.16-diaza-tricycle [14.3.0.04.6] - nonadec – 7 – en – 14 – il) –carbamic, Example 19, as a white solid. 1H NMR (500 MHz, CD3Cl) δ 1.87–1.64 (m, 21 H), 1.70–1.98 (m, 3 H), 2.15–2.56 (m, 5 H ), 2.85-2.94 (m, 1 H), 3.71 (d, J = 13.91

10 Hz, 1 H), 4.10-4.26 (m, 2 H), 4.51 (t, J = 7.87 Hz, 1 H), 4.62 (s, 1 H), 4, 98 (m, 1 H), 5.06 (d, J = 8.78 Hz, 1 H), 5.64-5.71 (m, 1 H), 6.72 (s, 1 H), 10 , 24 (s, 1 H); MS m / z 569 (M ++ 1).

The following macrocyclic alcohol intermediates were prepared using the procedures described in examples 29 and 30:

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The following macrocyclic alcohol intermediates could be prepared using the procedures described in examples 29 and 30:

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EXAMPLE 31

Preparation of Example 31, 2 (S) –terc-butoxycarbonylamino-3-pent-4-enylsulfanylpropionic acid

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Step 1: To a solution of N-Boc-cysteine methyl ester (3.36 g, 0.014 mol) in methanol (166 ml) at t.a. triethylamine (10.8 ml) and 1-bromopent-4-ene (3.19 g, 21 mmol, 1.5 equivalents) were added and the resulting solution was stirred at room temperature overnight. The mixture was then concentrated in vacuo and the resulting residual mixture was purified using flash chromatography (hexane, ethyl acetate gradient) to provide 1.76 g (41%) of the desired thioether. 1H NMR (500 MHz, CDCl3) δ 1.43 (s, 9H), 1.64 (m, 2H), 2.11 (m, 2H), 2.51 (m, 2H), 2.95 ( m, 2H), 3.75 (s, 3H), 4.51 (m, 1H), 4.95-5.03 (m, 2H), 5.34 (m, 1H), 5.80 (1 H, m); MS m / z 304 (M ++ 1).

Step 2: the thioether product from step 1 (9.51 g, 31.4 mmol) was added to a mixture of 1 M LiOH in water (200 ml) and THF (200 ml) and the resulting mixture was stirred at room temperature overnight. The reaction mixture was acidified using 1N hydrochloric acid and the resulting mixture was extracted several times with ethyl acetate. The extracts were combined, dried over magnesium sulfate and concentrated in vacuo to provide the desired acid, Example 20, which is used as in the following reaction.

EXAMPLE 32

Preparation of Example 32, N-tert-butoxycarbonyl-3- (4-pentenylthio) -L-valine

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Stop 1: Preparation of N-tert-butoxycarbonyl-3- (4-pentenylthio) -L-valine methyl ester

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To a solution of 7.12 g (48 mmol, 1.0 eq.) Of L-penicillamine in 100 ml of 1,4-dioxane and 25 ml of water at room temperature were added 9.60 ml (96 mmol, 2.0 eq.) of aqueous sodium hydroxide solution ION, followed by the dropwise addition of 12.00 ml (101 mmol, 2.1 eq.) of 5-bromo-1-pentene for several minutes. The resulting mixture was stirred at room temperature for 68 hours. At this point, 12.50 g (57 mmol, 1.2 eq.) Of di-tert-butyl dicarbonate were added and the mixture was stirred at room temperature for another 6 hours. The mixture was concentrated in vacuo and the residue was dissolved in water. The aqueous mixture was washed with diethyl ether, adjusted to pH 3 using 1 N hydrochloric acid and then extracted with ethyl acetate. The combined extracts were washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo.

The crude product (12.20 g) was dissolved in 120 ml of anhydrous dimethylsulfoxide. To this solution, 10.50 g (76 mmol) of potassium carbonate and 4.70 ml (76 mmol) of iodomethane were added and the resulting mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined extracts were washed with water (2x) and brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Column chromatography silica gel (elution: 2-10% ethyl acetate / hexane) provided 8.54 g of N-tert-butoxycarbonyl-3– (4-pentenylthio) -L-valine methyl ester as a colorless solid. NMR (300 MHz, CDCl3.): Δ 5.76 (d of d of t, 1 H, J = 17.2, 10.3, 6.6 Hz), 5.35 (da, 1 H, J = 9.0 Hz), 5.05– 4.94 (m, 2 H), 4.27 (da, 1 H, J = 9.0 Hz), 3.73 (s, 3 H), 2.52 (m, 2 H), 2.13 (quartet, 2 H, J = 7.3 Hz), 1.61 (quintuple, 2 H, J = 7.3 Hz), 1.43 (s, 9 H) , 1.35 (s, 3 H), 1.33 (s, 3 H).

Step 2: Preparation of Example 32, N-tert-butoxycarbonyl-3- (4-pentenylthio) -L-valine

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To a solution of 8.52 g (25.7 mmol) of N-tert-butoxycarbonyl-3– (4-pentenylthio) -L-valine methyl ester in 200 ml of tetrahydrofuran at room temperature was added a solution of 1, 10 g (26.2 mmol) of lithium hydroxide monohydrate in 50 ml of water. The resulting mixture was stirred at room temperature for 65 hours. To

The reaction mixture was then added 28 ml of 1.00 N hydrochloric acid. The mixture was diluted with diethyl ether, washed with water (3X) and brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. providing 8.10 g of N-tert-butoxycarbonyl-3- (4-pentenylthio) -L-valine as a colorless oil.

NMR (300 MHz, CDCl3): δ 5.75 (d of d of t, 1 H, J = 17.2, 10.3, 6.6 Hz), 5.40 (sa, 1 H), 5, 05–4.94 (m, 2 H), 4.28 (sa, 1 H), 2.56 (m, 2 H), 2.13 (quartet, 2 H, J = 7.3 Hz), 1 , 63 (quintuple, 2 H, J = 7.3 Hz), 1.44 (s, 9 H), 1.39 (s, 3 H),

10 1.37 (s, 3 H).

EXAMPLE 33

Preparation of Example 33, 5-allyloxy-2 (S) - (tert-butoxycarbonylamino) pentanoic acid

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Stage 1: Preparation of pyrrolidin-5-one-2 (S) -isopropyl carboxylate

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A solution of L-pyroglutamic acid (Aldrich, 25.0 g, 195 mmol) and paratoluenesulfonic acid monohydrate (3.71 g, 19.5 mmol) was refluxed in isopropanol (40 ml) in nitrogen for hours using a Dean – Stark trap variation (condensate returns through a Soxhlet extractor loaded with 4 Å molecular sieves). After cooling to room temperature, the reaction was diluted with

20 ether, washed with saturated aqueous sodium bicarbonate and then with saturated aqueous NaCl, dried (MgSO4) and evaporated to give a colorless syrup. This crystallized after adjusting. Grinding the crystalline residue in hexane provided 31.9 g (96%) of pyrrolidin-5-one-2 (S) -isopropyl carboxylate as white prisms: 1 H NMR (300 MHz, Chloroform-D) δ 6, 35 (sa, 1 H), 5.04 (septuple 1 H, J = 6.2 Hz), 4.18 (dd, 1 H, J = 8.4, 5.3 Hz), 2.51–2, 28 (m, 3 H), 2.27– 2.12 (m, 1 H), 1.24 (d, 6 H, J = 6.2 Hz). LCMS m / z 172 (M + H) +.

Step 2: Preparation of 1– (tert-butoxycarbonyl) -pyrrolidin-5-one-2 (S) -isopropyl carboxylate

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A solution of pyrrolidin-5-one-2 (S) -isopropyl carboxylate (product of step 26A, 31.9 g, 188 mmol), di-tert-butyl dicarbonate (48.6 g, 225 mmol) and DMAP (2.30 g, 8.8 mmol) in acetonitrile (300 ml) was stirred at room temperature in N2 for 30 minutes. The reaction was evaporated to approximately 100 ml, diluted with ether, washed with 1 N HCl then with saturated aqueous NaCl, dried (MgSO4) and evaporated to give 1– (tert-butoxycarbonyl) pyrrolidin-5-one-2 ( S) - Isopropyl carboxylate as a light yellow oil, 50.1 g (99%): 1 H NMR (300 MHz, Chloroform – D) δ 5.06 (septet, 1 H, J = 6.2 Hz), 4.53 (dd, 1 H, J = 9.5, 2.9 Hz), 2.66-2.40 (m, 2H), 2.36-22 (m, 1 H), 2, 03–1.93 (m, 1 H), 1.47 (s, 9 H), 1.26 (d, 3 H, J = 6.2 Hz), 1.24 (d, 3 H, J = 6.2 Hz). LCMS m / z 272

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(M + H) +. Step 3: Preparation of 2 (S) - (tert-butoxycarbonylamino) -5-isopropyl hydroxypentanoate

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To a solution of 1– (tert-butoxycarbonyl) pyrrolidin-5-one – 2 (S) -isopropyl carboxylate (product of step 26B, 49.5 g, 183 mmol) in methanol (300 ml) was added sodium borohydride (10.0 g, 263 mmol) in portions of ~ 1 g for 1.5 hours. The reaction was stirred under nitrogen for another 10 minutes. This was diluted with water, extracted with ether, the combined organic fractions were washed with saturated aqueous NaCl, dried (MgSO4) and evaporated to give a light yellow oil. Flash chromatography (silica gel, 20-30% ethyl acetate / hexane) gave 31.8 g (64%) of 2 (S) - (tert-butoxycarbonylamino) -5-isopropyl hydroxypentanoate as a colorless syrup: 1 H NMR (300 MHz, Chloroform – D) δ 5.16 (da, 1 H, J = 7.3 Hz), 5.03 (septet, 1 H, J = 6.2 Hz), 4.28 ( sa, 1 H, J = 6.2 Hz), 3.67 (dda, J = 10.2, 5.5 Hz), 1.94–1.79 (m, 2 H), 1.76–1 , 67 (m, 1 H), 1.66–1.56 (m, 2 H), 1.43 (s, 9 H), 1.25 (d, 3 H, J = 6.2 Hz), 1.23 (d, 3 H, J = 6.2 Hz). LCMS m / z 276 (M + H) +.

Stage 4: Preparation of isopropyl-5-allyloxy-2 (S) - (tert-butoxycarbonylamino) pentanoate

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A degassed mixture of 2 (S) - (tert-butoxycarbonylamino) -5-isopropyl hydroxypentanoate (product of step 26C, 17.6 g, 63.9 mmol), allylmethyl carbonate (24.0 ml, 213 mmol), Pd2 (dba) 3 (1.62 g, 1.78 mmol) and BINAP (4.42 g, 7.10 mmol) in THF (150 ml) was refluxed under nitrogen for 3 hours. After cooling to room temperature, the reaction was diluted with ether, filtered through celite and evaporated to give a dark brown syrup. Flash chromatography of the residue (silica gel, 30% ether / hexane) gave isopropyl 5-allyloxy-2 (S) - (tert-butoxycarbonylamino) pentanoate as a colorless viscous oil, 16.3 g (81%): 1 H NMR (300 MHz, Chloroform – D) δ 5.88 (ddt, 1 H, 17.4, 10.4, 5.5), 5.28 (m, 1 H), 5.22–5, 11 (m, 1 H), 5.02 (septet, 1 H, J = 6.2 Hz), 4.21 (ta, 1 H, J = 6.7 Hz), 3.94 (dt, 2 H , J = 5.9, 1.5 Hz), 3.42 (t, 2 H, J = 5.9 Hz), 1.90–1.82 (m, 1 H), 1.75–1, 57 (m, 3 H), 1.42 (s, 9 H), 1.21 (d, 3 H, J = 6.2 Hz), 1.19 (d, 3 H, J = 6.2 Hz ). LCMS m / z 316 (M + H) +.

Stage 5: Preparation image21 from Example 33, 5-allyloxy-2 (S) - (tert-)

butoxycarbonylamino) pentanoic acid

A mixture of 5-allyloxy-2 (S) - (tert-butoxycarbonylamino) isopropyl pentanoate (product of step 26D, 16.1 g, 51.1 mmol) and lithium hydroxide hydrate (4.19 g, 102 mmol ) in THF / water (100 ml / 20 ml) was stirred at room temperature under nitrogen for 16 hours. The reaction was diluted with water, washed with ether, the pH of the aqueous fraction was adjusted to ~ 4, extracted with ether, the combined fractions washed with NaCl, dried (MgSO4) and evaporated to give 5-allyloxy acid. –2 (S) - (tert-butoxycarbonylamino) pentanoic as a light yellow syrup: 1 H NMR (300 MHz, Chloroform – D) δ 5.89 (ddt, 1 H, J = 17.4, 10.4, 5 , 5), 5.25 (dd, 1 H, J = 17.4, 1.6 Hz), 5.17 (dd, 1 H, J = 10.4, 1.6 Hz), 4.30 ( gives, 1 H, J = 6.2), 3.96 (dt, 2 H, J = 5.9, 1.5 Hz), 3.46 (t, 2 H, J = 5.9 Hz), 1.96-1.86 (m, 1 H), 1.85-1.77 (m, 1 H), 1.75-1.64 (m, 2 H), 1.43 (s, 9 H ). LCMS m / z 274 (M + H) +.

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EXAMPLE 34

General Procedure for the Preparation of Example 34

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Example 23 was prepared by adding a DMF solution of N-trityl protected threonine to a DMF solution of sodium hydride cooled to -15 ° C. The reaction mixture was stirred for 30 minutes at -15 ° C after which 5-bromo-1-pentene was added and the resulting mixture was heated to -5 ° C. The reaction mixture was maintained at -5 ° C for 3 days after the reaction was quenched by the addition of 1N HCl and was stimulated using standard extraction procedures as described above. Example 23 was obtained in pure form by standard chromatographic procedures.

EXAMPLE 35:

Preparation of Example 35, N-tert-butoxycarbonyl-O- (4-pentenyl) -L-serine

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Stage 1: Preparation of N-tert-butoxycarbonyl-O- (4-pentenyl) -L-serine methyl ester

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To a solution of 10.26 g (50 mmol, 1.0 eq) of N-tert-butoxycarbonyl-L-serine in 500 ml of anhydrous dimethyl sulfoxide at room temperature was added 2.00 g (50 mmol, 1, 0 eq.) Of 60% sodium hydride in mineral oil. This mixture was stirred at room temperature for 0.5 hours until the evolution of the gas had ceased. To the resulting solution were added 6.00 ml (50 mmol, 1.0 eq.) Of 5-bromo-1-pentene followed immediately by another 2.00 g (50 mmol, 1.0 eq) of sodium hydride at 60% in mineral oil. The reaction mixture was then stirred at room temperature for 16 hours. The mixture was diluted with 2000 ml of water, adjusted to pH 3-4 by the addition of 50 ml of 1.00 N hydrochloric acid and extracted with ethyl acetate. The organic phase was washed with water (2x) and brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Removing the residual mineral oil, the resulting material was dissolved in a dilute aqueous sodium hydroxide solution. This aqueous solution was washed with hexane and then adjusted to pH 4 using hydrochloric acid and extracted with ethyl acetate. The organic phase was washed with water (2x) and brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.

The crude product (7.70 g) was dissolved in 100 ml of anhydrous dimethylsulfoxide. To this solution, 7.80 g (56 mmol) of potassium carbonate and 4.70 ml (56 mmol) of iodomethane were added and the resulting mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined extracts were washed with water (2x) and brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Column chromatography silica gel (elution: 2-10% ethyl acetate / hexane) provided 6.70 g of N-tert-butoxycarbonyl-O- (4-pentenyl) -L-serine methyl ester as a colorless solid. NMR (300 MHz, CDCl3): δ 5.78 (d of d of t, 1 H, J = 17.2, 10.2, 6.6 Hz), 5.34 (da, 1 H, J = 8 , 0 Hz), 5.03– 4.92 (m, 2 H), 4.40 (m, 1 H), 3.81 (d d, 1 H, J = 9.5, 2.9 Hz ), 3.74 (s, 3 H), 3.61 (d d, 1 H, J = 9.5, 3.5 Hz), 3.42 (m, 2 H), 2.06 (quartet , 2 H, J = 7.3 Hz), 1.61 (quintuple, 2 H, J = 7.3 Hz), 1.44 (s, 9 H).

Step 2: Preparation of Example 35, N-tert-butoxycarbonyl-O- (4-pentenyl) -L-serine

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To a solution of 6.65 g (23 mmol) of N-tert-butoxycarbonyl-O- (4-pentenylthio) -L-serine methyl ester in

5 500 ml of tetrahydrofuran at room temperature was added a solution of 1.95 g (46 mmol) of lithium hydroxide monohydrate in 100 ml of water. The resulting mixture was stirred at room temperature for 40 hours. To the reaction mixture was then added 46 ml of 1.00 N hydrochloric acid. The mixture was diluted with ethyl acetate, washed with water (3X) and brine, dried over anhydrous sodium sulfate, filtered and concentrated. under vacuum providing 6.30 g of N-tert-butoxycarbonyl-O- (4-pentenyl) -L-valine as a colorless oil. NMR (300 MHz,

10 CDCl3): δ 5.77 (d of d of t, 1 H, J = 17.2, 10.2, 6.6 Hz), 5.37 (da, 1 H, J = 8.0 Hz) , 5.03-4.92 (m, 2 H), 4.42 (m, 1 H), 3.87 (d d, 1 H, J = 9.5, 2.6 Hz), 3, 63 (d d, 1 H, 4.0 Hz), 3.45 (m, 2 H, J = 6.6 Hz), 2.07 (quartet, 2 H, J = 7.3 Hz), 1 , 64 (quintuple, 2 H, J = 7.3 Hz), 1.44 (s, 9 H).

EXAMPLE 36

Preparation of Example 36, (S) -4-allyloxy-2- (tert-butoxycarbonylamino) butyric acid

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To a mixture of sodium hydride (913 mg, 22.8 mmol) in DMF at 0 ° C was added N-t-Boc-L-homoserine (2 g, 9.13 mmol). This reaction mixture was stirred at 0 ° C for 15 minutes and then allyl bromide (1.38 g, 11.4 mmol) was added. The mixture was heated to t.a. and stirred for 2 hours. This was concentrated in vacuo. The residue was diluted with water and washed sequentially with hexane and ether. The organic phases were discarded and the aqueous phase was adjusted

20 carefully at pH 3 with 1N HCl. This acidic aqueous solution was extracted with ethyl acetate. The organic phase was dried (MgSO4) and concentrated in vacuo to provide 2.2 g (93%) of (S) -4-allyloxy-2- (tert-butoxycarbonylamino) butyric acid as a colorless oil. 1H NMR (300 MHz, CD3OD) δ 1.42 (s, 9 H), 1.80–1.90 (m, 1 H), 2.04–2.16 (m, 1 H), 3, 50–3.54 (m, 2 H), 3.97 (d, J = 4.39 Hz, 2 H), 4.23 (dd, J = 8.78, 4.39 Hz, 1 H), 5.15 (d, J = 10.25 Hz, 1 H), 5.26 (dd, J = 17.38, 1.65 Hz, 1 H), 5.84–5.97 (m, 1 H ).

25 EXAMPLE 37

Preparation of Example 37, (S) -2- (tert-butoxycarbonyl) -3- (2-nitro-N- (pent-4-enyl) phenylsulfonamido) propanoic acid

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Stage 1: Preparation of methyl 3-amino-2 (S) - (tert-butoxycarbonylamino) propanoate

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To a mixture of i (Boc-DAP-OH) (3.0 g 14.7 mmol) in 50 ml of methylene chloride was added 5 ml of methanol. To this solution was added slowly (trimethylsilyl) diazomethane (2M in ether, 7.9 ml, 15.8 mmol). The mixture was stirred at t.a. for 2 hours until all the solid dissolved and the solution turned light yellow. This was concentrated to provide 3.2 g (99%) of methyl 3-amino-2 (S) - (tert-butoxycarbonylamino) propanoate ii as a colorless solid. 1H NMR (CD3OD, 300 MHz) δ 1.46 (s, 9 H), 2.82-3.00 (m, 2 H), 3.71 (s, 3 H), 4.14 (sa, 1 HOUR).

Preparation of methyl 2 (S) - (tert-butoxycarbonylamino) -3- (2-nitrophenylsulfonamido) propanoate iii:

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To a mixture of methyl 3-amino-2 (S) - (tert-butoxycarbonylamino) propanoate ii (1.6 g, 7.3 mmol) in DCM

10 (50 ml) DIPEA (1.64 ml, 9.4 mmol) and 2-nitrobenzene chlorurosulfonyl (1.62 g, 7.3 mmol) were added. The mixture was stirred at t.a. for 2 h. It was then concentrated, dissolved in ethyl acetate, which was then washed with saturated sodium bicarbonate, brine and dried over magnesium sulfate. This was then filtered, concentrated to provide 2.9 g (98%) of methyl 2 (S) - (tert-butoxycarbonylamino) -3- (2-nitrophenylsulfonamido) propanoate iii as a yellow foam. 1H NMR (CD3OD, 300 MHz) δ 1.41 (s, 9 H), 3.36-3.51 (m, 2 H), 3.71 (s, 3 H), 4.22 (m, 1 H), 7.80–7.90

15 (m, 3 H), 8.07–8.10 (m, 1 H).

Preparation of methyl 2 (S) - (tert-butoxycarbonylamino) -3- (2-nitro-N- (pent-4-enyl) phenylsulfonamido) propanoic acid iv:

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To a mixture of methyl 2 (S) - (tert-butoxycarbonylamino) -3– (2-nitrophenylsulfonamido) propanoate iii (150 mg, 0.37 mmol) in 3 ml of DMF was added potassium carbonate (102 mg, 0 , 74 mmoL). This mixture was stirred at t.a. 20 for 20 minutes followed by the addition of 5-bromo-1-pentene (65 μl, 0.55 mmol). The reaction mixture was stirred at

t.a. for 2 days. This was then filtered, concentrated and purified by silica gel chromatography (eluting with 25% ethyl acetate in hexane) to give 75 mg (43%) of 2 (S) - (tert-butoxycarbonylamino) -3– ( Methyl 2-nitro-N– (pent-4-enyl) phenylsulfonamido) propanoate iv as a yellow solid. 1H NMR (CD3OD, 300 MHz) δ 1.42 (s, 9 H), 1.54–1.64 (m, 2 H), 1.97 (c, J = 7.20 Hz, 2 H) , 3.37 (m, 2 H), 3.57–3.80 (m, 2 H), 3.72 (s, 3 H), 4.42 (dd, J = 8.60, 5.31 Hz, 1

25 H), 4.91–5.01 (m, 2 H), 5.69–5.79 (m, 1 H), 7.75–7.85 (m, 3 H), 8.04 ( m, 1 H); MS m / z 372 (M ++ 1-Boc).

Preparation of Example 37. (S) –2– (tert-butoxycarbonyl) –3– (2-nitro-N– (pent-4-enyl) phenylsulfonamido) propanoic acid v:

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2– (tert-Butoxycarbonyl) -3– (2-nitro-N- (pent-4-enyl) phenylsulfonamido) -propanoate of (S) -methyl iv (500 mg, 1.06 mmol) was dissolved in the solvent system mixed: THF (4 ml), methanol (1 ml) and water (2 ml). Powdered lithium hydroxide (250 mg, 10.4 mmol) was added. The light yellow suspension was stirred at t.a. for 15 hours and then

5 was concentrated in vacuo. The residue was partitioned between ether and water. The ether phase was discarded and the aqueous phase was treated with 1 N HCl until the pH was 4. This acid solution was extracted with ethyl acetate four times. The combined ethyl acetate extracts were dried (MgSO4) and concentrated in vacuo to give 430 mg (89%) of 2– (tert-butoxycarbonylamino) -3– (2-nitro-N– (pent-4-enyl) Phenylsulfonamido) propanoic acid (Example 26) as a yellow oil. 1H NMR (CD3OD, 300 MHz) δ 1.38 (s, 9 H), 1.51–1.60 (m, 2 H), 1.89–1.98 (m, 2 H), 3, 28–3.32 (m, 2 H), 3.59–3.64

10 (dd, J = 14.95, 9.46 Hz, 1 H), 3.71–3.74 (m, 1 H), 4.33 (dd, J = 9.61, 4.43 Hz, 1 H), 4.87–4.94 (m, 2 H), 5.63–5.72 (m, 1 H), 7.71–7.77 (m, 3 H), 8.01 ( dd, J = 7.48, 1.37 Hz, 1 H); MS m / z 358 (M ++ 1-Boc).

EXAMPLE 86

Preparation of tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) - [7-cis-4-cyclopropanesulfonylaminocarbonyl-12- cyclopropyl-18-hydroxy-2.15-dioxo-3.12,16-triaza –Tricycle [14.3.0.04.6] nonadec – 7 – en – 14 – il] carbamic (Example 86)

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Stage 1: Synthesis of N– (pent – 4-enyl) cyclopropanamine.

Using an addition funnel, a solution of 5-bromopentene (15.75 g, 106 mmol) in 50 ml of methanol was added over the course of 5 minutes to a solution of cyclopropylamine (20.6 g, 361 mmol) in 200 ml of methanol This solution was allowed to stir at t.a. for 72 hours the time it was refluxed for 1 hour. Methanol and

The excess cyclopropylamine was removed by distillation. The residue, hydrobromide salt of the product, was partitioned between ether and 4N NaOH. The aqueous phase was washed with ether (2x). The combined ether extracts were dried (MgSO4), filtered and concentrated to give 8 g (60%) of N- (pent-4-enyl) cyclopropanamine as a yellow solid: 1H NMR (500 MHz, CDCl3) δ 0 , 31–0.36 (m, 2 H) 0.40 - 0.46 (m, 2 H) 1.53 - 1.63 (m, 2 H) 1.87 (sa, 1 H) 2.05 - 2.10 (m, 2 H) 2.10 - 2.14 (m, 1 H) 2.69 (t, J = 7.32 Hz, 2 H) 4.91 - 5.07 (m, 2 H) 5.72-5.88 (m, 1 H).

Step 2: Synthesis of 2 (S) - (tert-butoxycarbonylamino) -3– [N-cyclopropyl-N- (pent-4-enyl) amino] propanoic acid

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N- (pent-4-enyl) cyclopropanamine (668 mg, 5.30 mmol) in 20 ml of acetonitrile was added to a suspension of N-t-butoxycarbonyl-L-serine- (3-lactone (1.0 g, 5.30 mmol) in 40 ml of acetonitrile The mixture was stirred in N2 at rt for 5 days and then concentrated in vacuo to give acid 2 (S) - (tert-butoxycarbonylamino) -3- [N-cyclopropyl-N- (pent-4-enyl) amino] crude propanoic acid This material was used in Stage 3 without purification LC-MS (Phenomenex micrometer C18 10 HPLC column (Vm "): 3.0 x 50 mm in length. Gradient : Solvent A 100% / Solvent B 0% a

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Solvent A 0% / Solvent B 100%. Gradient time: 3 min. Retention time: 1 min. Flow rate: 4 ml / min. Detector Wavelength: 220 nM. Solvent A: 10% MeOH / 90% H2O / 0.1% TFA. Solvent B: 10% H2O / 90% MeOH / 0.1% TFA. (Retention time: 2.50 min), MS m / z 313 (M ++ 1).

Stage 3: Synthesis of 1 (R) - [1– [2 (S) - (tert-butoxycarbonylamino) –3– [N-cyclopropyl-N– (pent-4-enyl) amino] propinoyl] –4 (R) - hydroxypyrrolidine – 2 (S) –carboxamido] –2 (S) –ethyl vinylcyclopropanecarboxylate.

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A solution of 2 (S) - (tert-butoxycarbonylamino) -3– [N-cyclopropyl-N- (pent-4-enyl) amino] propanoic acid (1.47 g, 4.71 mmol) in 20 ml of DCM was treated sequentially with 1 (R) - [4 (R) -hydroxypyrrolidine-2 (S) -carboxamido] -2 (S) -vi-nylcyclopropanecarboxylate hydrochloride (prepared in Example 5) (1.44 g, 4.71 mmol), N-methylmorpholine (1.80 ml, 16.34 mmol) and HATU (2.14 g, 5.53 mmol). The reaction mixture was stirred at t.a. in N2 for 3 hours and then concentrated in vacuo. The residue was dissolved in water and 1 N HCl was added until pH = 5. This aqueous solution was extracted with EtOAc (3x). The organic phase was washed with saturated aqueous NaHCO3, dried (MgSO4) and concentrated in vacuo to give the crude product. Flash chromatography (50% ethyl acetate / 100% hexane) gave 1.55 g (58%) of 1 (R) - [1– [2 (S) - (tert-butoxycarbonylamino) –3– [N –Cyclopropyl – N– (pent – 4 – enyl) amino] propanoyl] –4 (R) - hydroxypyrrolidine – 2 (S) –carboxamido] –2 (S) –ethyl vinyl cyclopropanecarboxylate as a white foam:

LC-MS (HP10 Shen Phenomenex – Luna column: 3.0 x 50 mm in length. Gradient: 100% Solvent A / 0% Solvent B at 0% Solvent A / 100% Solvent B. Gradient Time : 2 min Retention time: 1 minute Flow rate: 4 ml / min Detector Wavelength: 220 nM Solvent A: 10% MeOH / 90% H2O / 0.1% TFA Solvent B: 10% H2O / 90% MeOH / 0.1% TFA). (Retention time: 1.38 min), MS m / z 564 (M ++ 1).

Stage 4: Synthesis of 1 (R) - [1– [2 (S) - (tert-butoxycarbonylamino) –3– [N-cyclopropyl-N– (pent – 4-enyl) amino] propinoyl] –4 (R) - ethyl (tert-butyldimethylsilyloxy) pyrrolidine-2 (S) -carboxamido] -2 (S) -carboxamido] -vinylcyclopropanecarboxylate.

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To a mixture of 1 (R) - [1– [2 (S) - (tert-butoxycarbonylamino) –3– [N-cyclopropyl-N– (pent-4-enyl) amino] propanoyl] - 4 (R) - hydroxypyrrolidine – 2 (S) –carboxamido] –2 (S) –ethyl vinylcyclopropanecarboxylate (1.55 g, 2.75 mmoL) in 10 ml of DMF was added imidazole (0.47 g, 6.88 mmol) and chloride of tert-butyldimethylsilyl (826 mg, 5.50 mmol). The mixture was stirred at

t.a. for 18 hours, it was concentrated in vacuo and partitioned between ethyl acetate and water. The organic phase was dried over magnesium sulfate and concentrated in vacuo to obtain an off-white solid. Flash chromatography (eluting with methylene chloride and then with ethyl acetate) gave 1 (R) - [1– [2 (S) - (tert-butoxycarbonylamino) –3– [N- cyclopropyl-N– (pent – 4 -Enyl) amino] propanoyl] -4 (R) - (tert-butyldimethylsilyloxy) pyrrolidine-2 (S) -carboxamido] -2 (S) -ethyl vinylcyclopropanecarboxylate as a white solid (1.75g, 94%) : LC-MS (Phenomenex C18 10 micromolar HPLC column (Vm "): 3.0 x 50 mm in length. Gradient: 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B % Gradient time: 2 min Retention time: 1 minute Flow rate: 5 ml / min Detector Wavelength: 220 nM Solvent A: 10% MeOH / 90% H2O / 0.1 TFA % Solvent B: 10% H2O / 90% MeOH / 0.1% TFA) (Retention time: 2.51 min), MS m / z 677 (M ++ 1).

Stage 5: Synthesis of ethyl ether of acid (1S, 4R, 6S, 14S, 18R) –7 – cis – 14-tert-butoxycarbonylamino – 18– (tert-butyldimethylsilyloxy) –2.15 – dioxo – 3.12 – triazatricyclo [14.3.0.04.6] nonadec – 7 – eno – 4 – carboxylic

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To a solution of 1– (R) - [1– [2 (S) - (tert-butoxycarbonylamino) –3– [N-cyclopropyl-N– (pent-4-enyl) amino] propanoyl] - 4 (R) - (tert-Butyldimethylsilyloxy) pyrrolidine-2 (S) -carboxamido] -2 (S) -vinylcyclopropanecarboxylate ethyl (1.45 g, 2.14 mmol) in 1 L of methylene chloride was added 181 mg (0.21 mmol) of the second generation catalyst of Grubb [(1,3-bis- (2,4,6-trimethylphenyl) -2-imidazolidinylidene) dichloro (phenylmethylene) (tricyclohexylphosphine) ruthenium]. The mixture was heated at reflux for 1 hour. A second fraction of the catalyst (50 mg, 0.058 mmol) was added and the mixture was stirred at

t.a. for a whole night. The residue was concentrated in vacuo and then purified by flash chromatography eluting with 50% ether / hexane to give 0.84 g (62%) of acid ethyl ester (1S, 4R, 6S, 14S, 18R) -7-cis –14 – tert- butoxycarbonylamino – 18– (tert-butyldimethyl-silyloxy) –2.15-dioxo – 3.12,16-triazatricyclo [14.3.0.04.6] nonadec-7-eno-4– carboxylic, as a solid white: LC-MS (Phenomenex C18 10 micromolar HPLC column: 3.0 x 50 mm in length. Gradient: 100% Solvent A / 0% Solvent B at 0% Solvent A / 100% Solvent B. Time gradient: 2 min Retention time: 1 min Flow rate: 5 ml / min Detector Wavelength: 220 nM Solvent A: 10% MeOH / 90% H2O / 0.1% TFA. B: 10% H2O / 90% MeOH / 0.1% TFA). (Retention time: 2.43 min), MS m / z 649 (M ++ 1).

Stage 6: Synthesis of acid (1S, 4R, 6S, 14S, 18R) –7 – cis – 14-tert-butoxycarbonylamino-18– (tert-butyldimethylsilyloxy) –2.15– dioxo – 3.12,16-triazatricyclo [ 14.3.0.04.6] nonadec – 7 – eno – 4 – carboxylic.

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To a solution of ethyl ester of acid (1S, 4R, 6S, 14S, 18R) –7 – cis-14-tert-butoxycarbonylamino-18– (tert-butyldimethylsilyloxy) –2.15-dioxo – 3.12,16– triazatricyclo [14.3.0.04.6] nonadec-7-ene-4-carboxylic (0.84 g, 1.30 mmol) in THF (30 ml), methanol (15 ml) and water (4 ml) was added hydrate pulverized lithium hydroxide (0.31 g, 12.90 mmol). The resulting light yellow suspension was stirred at t.a. on N2 for a whole night. The mixture was concentrated in vacuo, and partitioned between hexane / ether (1: 1) and water. The organic phase was discarded and the aqueous phase was treated with 1 N HCl until the pH was 5. This acid solution was extracted with EtOAc (3x). The combined EtOAc extracts were dried (MgSO4) and concentrated in vacuo to give 0.495 g (61%) of acid (1S, 4R, 6S, 14S, 18R) -7-cis-14-tert-butoxycarbonylamino-18- (tert –Butyldimethylsilyloxy) –2.15 – dioxo – 3.12,16 – triazatricyclo [14.3.0.04.6] nonadec – 7 – eno – 4– carboxylic acid as an off-white solid: LC – MS (Phenomenex C18 10 micromolar HPLC column: 3.0 x 50 mm in length Gradient: 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B. Gradient time: 2 min Retention time: 1 minute. Flow rate: 5 ml / min Detector Wavelength: 220 nM Solvent A: 10% MeOH / 90% H2O / 0.1% TFA Solvent B: 10% H2O / 90% MeOH / 0.1% TFA). (Retention time: 2.36 min), MS m / z 621 (M ++ 1).

Stage 7: Synthesis of tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) - [7-cis-4-cyclopropanesulfonylaminocarbonyl– 12-cyclopropyl-18– (tert-butyldimethylsilyloxy) –2.15-dioxo– 3.12,16 – triaza – tricycle [14.3.0.04.6] nonadec – 7 – en – 14 – il] carbamic

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Acid (1S, 4R, 6S, 145, 18R) -7-cis-14-tert-butoxycarbonylamino-18- (tert-butyldimethylsilyloxy) -2.15-dioxo- 3.12,16-triazatricyclo [14.3.0.04, 6] nonadec-7-ene-4-carboxylic (490 mg, 0.79 mmol) was dissolved in 15 ml of THF and treated with CDI (179 mg, 1.10 mmol). (Care was taken to avoid moisture using oven-dried glassware and maintaining a dry N2 atmosphere.) After the reaction mixture was refluxed for 2 hours, it was cooled to t.a. and treated sequentially with cyclopropylsulfonamide (134 mg, 1.10 mmol) and DBU (168 mg, 1.10 mmol). After stirring overnight at t.a., THF was removed by rotary evaporation. The residue was dissolved in water and 1 N HCl was added until pH = 5. This aqueous solution was extracted with EtOAc (3x). The combined EtOAc extracts were dried (MgSO4) and concentrated in vacuo to give the crude product. Purification by column chromatography, eluting with 3% methanol in methylene chloride, gave 300 mg (53%) of tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) - [7-cis-4– cyclopropanesulfonylaminocarbonyl-12-cyclopropyl-18- (tert-butyldimethylsilyloxy-2.15-dioxo- 3.12,16-triaza-tricycle [14.3.0.04.6] nona-dec-7-en-14-i] carbamic as a white solid: LC-MS (Phenomenex 10 micromolar C18 HPLC column: 3.0 x 50 mm in length. Gradient: 100% solvent A / 0% solvent B to 0% solvent A / 100% solvent B. Gradient time: 2 min Retention time: 1 minute Flow rate: 5 ml / min Detector Wavelength: 220 nM Solvent A: 10% MeOH / 90% H2O / 0.1 TFA % Solvent B: 10% H2O / 90% MeOH / 0.1% TFA) (Retention time: 2.40 min), MS m / z 724 (M ++ 1).

Stage 8: Synthesis of tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) - [7-cis-4-cyclopropanesulfonylaminocarbonyl– 12-cyclopropyl-18-hydroxy-2.15-dioxo-3.12, 16 – triaza – tricycle [14.3.0.04.6] nonadec – 7 – en – 14 – il] carbamic (Example 86).

image26

To a mixture of tert-butyl ester compound of the acid (1S, 4R, 6S, 14S, 18R) - [7-cis-4- cyclopropanesulfonylaminocarbonyl-12-cyclopropyl-18- (tert-butyldimethylsilyloxy) –2.15-dioxo –3,12,16 – triaza– tricycle [14.3.0.04.6] nonadec – 7 – en – 14-yl] carbamic (250 mg, 0.35 mmol) in 15 ml of THF was added tetrabutylammonium fluoride (129 mg , 0.46 mmol). The mixture was stirred at t.a. for 18 h. THF was removed by rotary evaporation and the residue was partitioned between ethyl acetate and water. The organic phase was dried (MgSO4) and concentrated in vacuo to give the crude product. Purification by triturating with hexane provided 200 mg (94%) of tert-butyl acid ester (1S, 4R, 6S, 14S, 18R) - [7-cis-4-cyclopropanesulfonylaminocarbonyl-12-cyclopropyl-18-hydroxy-2, 15 – dioxo – 3.12,16 – triaza– tricycle [14.3.0.04.6] nonadec – 7 – en – 14 – il] carbamic as a white solid: LC – MS (retention time: 2.32 min), MS m / z 610 (M ++ 1).

(HPLC C18 10 micromolar Phenomenex column: 3.0 x 50 mm in length. Gradient: 100% Solvent A / 0% Solvent B at 0% Solvent A / 100% Solvent B. Gradient time: 3 min Retention time: 1 min Flow rate: 4 ml / min Detector Wavelength: 220 nM Solvent A: 10% MeOH / 90% H2O / 0.1% TFA Solvent B: 10 H2O % / 90% MeOH / 0.1% TFA). (Retention time: 2.32 min), MS m / z 610 (M ++ 1).

EXAMPLE 87:

Preparation of (2R, 6S, 13aS, 14aR, 16aS, Z) –2– (biphenyl – 4-yl) –14a– (cyclopropylsulfonylcarbamol) –2 – methoxy – 5.16 – dioxo– 1,2,3,5, 6,7,8,9,10,11,13a, 14,14a, 15,16,16a-hexadecahydrocyclopropa [e] pyrrolo [1,2-a] [1,4] diazacyclopentadecin-6-tert-butyl ilcarbamate , Compound 1

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image 1

Stage 1.

To methylsulfoxide solution (23.90 ml, 337 mmol) in DCM (100 ml) at –78 ° C, oxalyl chloride (2

5 M in DCM, 84 ml, 168 mmol) drop by drop. The solution formed was stirred at this temperature for 30 min. A solution of 4-hydroxypyrrolidine-1,2-dicarboxylate of (2S, 4R) -1-benzyl 2-methyl (21.38 g, 77 mmol) in DCM (100 ml) was added dropwise at -78 ° C . The suspension formed was stirred at -78 ° C for 2 hours before the addition of N, N-diisopropylethylamine (66.7 ml, 383 mmol) dropwise. The final solution was stirred at room temperature 3 h. The mixture was washed with ice cold 1M HCl, 5% citric acid and then brine, dried over MgSO4, filtered and

10 evaporated. The residual light brown oil was purified by silica gel chromatography, eluted with hexane-EtOAc 4: 1, 3: 1, then with 2: 1 to provide 4-oxopyrrolidine-1,2-dicarboxylate of (S) -1 Benzyl-2-methyl (14.8 g, 70% yield) as a light brown viscous oil.

1H NMR (CDCl3) δ 2.58-2.63 (m, 1 H), 2.90-2.99 (m, 1 H), 3.62, 3.77 (s, 3 H, rotamers) , 3.95–4.02 (m, 2 H), 4.82– 4.89 (m, 1 H), 5.11–5.24 (m, 2 H), 7.32–7.39 (m, 5 H).

15 Stage 2.

To a solution of 4-oxopyrrolidine-1,2-dicarboxylate of (S) -1-benzyl-2-methyl (14.0 g, 50.5 mmol) in toluene (500 ml) at 0 ° C was added bromide of Biphenyl-4-ilmagnesium (152 ml, 0.5 M in THF, 75.75 mmol) drop by drop. The light yellow solution formed was stirred at this temperature for 1 hour. It was deactivated with NH4Cl, the organic phase was separated. The aqueous phase was extracted with EtOAc. The combined organic extracts were washed with brine,

20 were dried over MgSO4, filtered and evaporated. The residue was purified by passing through a short bed of silica gel, eluted with hexane-EtOAc 4: 1, 3: 1 after 2: 1 and finally 3: 2 providing 11.70 g of white solid, which was recrystallized from EtOAc-Hexane (50 ml – 150 ml) to provide 7.8 g of 4– (biphenyl-4-yl) –4– hydroxypyrrolidine – 1,2-dicarboxylate of (2S, 4R) –1-benzyl– 2-methyl as small needles. The mother liquors were concentrated and purified by ultrafast column, eluted with hexane-EtOAc 4: 1, 3: 1, then 2: 1 and finally

3: 2 providing an additional 2.41 g of the desired product.

1H NMR (CDCl3) δ 2.39-2.45 (m, 1 H), 2.70-2.75 (m, 1 H), 3.66, 3.86 (s, 3 H, rotamers) , 3.80–3.90 (m, 1 H), 4.00– 4.07 (m, 1 H), 4.62 (dd, J1.2 = 9.5, 28 Hz, 1 H), 5.09–5.15 (m, 1 H), 5.21–5.25 (m, 1 H), 7.31–7.38 (m, 6 H), 7.42–7.45 ( m, 2 H), 7.54-7.59 (m, 6 H);

LC-MS (retention time: 2.77 min, procedure B), MS m / z 414 (M + - H2O), (M + - H2O - CO2) Stage 3.

To a solution of 4– (biphenyl – 4-yl) –4-hydroxypyrrolidine – 1,2-dicarboxylate of (2S, 4R) –1-benzyl-2-methyl (8.08 g, 18.73 mmol) in DMF (150 ml) at 0 ° C sodium hydride (0.520 g, 20.60 mmol) was added. The light brown solution formed was stirred at this temperature for 30 min. Dimethyl sulfate (1,949 ml, 20.60 mmol) was added dropwise at 0 ° C. The

The final solution was stirred at room temperature for 2 hours. It was deactivated with 5% citric acid, extracted with EtOAc. The organic part was washed with brine, dried over MgSO4, filtered and evaporated. The residue was purified by flash column chromatography on silica gel, eluted with hexane-EtOAc 4: 1, 3: 1, then 2: 1 to give 1.45 g of the desired product, which was recrystallized from MeOH (10 ml). providing 1.20 g (yield 14.38%) as a white solid. 4.50 g of starting material was also recovered during purification of

10 flash chromatography. 1H NMR (CDCl3) δ 2.51-2.56 (m, 1H), 2.85-2.89 (m, 1 H), 2.95, 2.97 (s, 3 H, rotamers), 3.67, 3.80 (s, 3 H, rotamers), 3.69–3.86 (m, 1H), 4.02–4.08 (m, 1 H), 4.62 (dd, J1 , 2 = 9.5, 28 Hz, 1 H), 5.09–5.17 (m, 1 H), 5.20–5.29 (m, 1 H), 7.29–7.46 (m , 10 H), 7.57-7.60 (m, 4 H);

LC-MS (retention time: 2.92 min, procedure B), MS m / z 446 (M ++ H), 414 (M + -MeOH), 370 (M + -MeOH-CO2).

15 Stage 4.

To a Parr shaker vessel with ice containing 4– (biphenyl – 4-yl) –4 – methoxypyrrolidine – 1,2-dicarboxylate solution of (2S, 4R) –1-benzyl-2-methyl (1.29 g, 2.90 mmol) in MeOH (30 ml) palladium (0.308 g, 0.290 mmol) in carbon (10%, wet) was added. The vessel was placed on a Parr stirrer in hydrogen with a pressure of

172,369 pascals (25 psi) for 5 hours. Deactivated with celite, filtered, evaporated yielding 0.811 g

20 (91%) of the desired product as an off-white powder. This material was used for the following coupling reaction without further purification; LC-MS (retention time: 1.92 min, procedure B), MS m / z 312 (M ++ H), 280 (M + -MeOH).

image 1

Stage 1: Preparation of 4– (biphenyl – 4-yl) –1 - ((S1–2– (tert-butoxycarbonylamino) non – 8 – enoyl) –4 – methoxypyrrolidine – 2– carboxylate of (2S, 4R) –methyl

image 1

To a solution of 4– (biphenyl – 4-yl) –4 – methoxypyrrolidine – 2-carboxylate of (2S, 4R) –methyl (150 mg, 0.482 mmol), acid (S) –2– (tert-butoxycarbonylamino) non –8 – enoic (144 mg, 0.530 mmol) and HATU (260 mg, 0.723 mmol) in DCM (5 ml) N, N-diisopropylethylamine (0.222 ml, 1,445 mmol) was added at 0 ° C. The reaction mixture was allowed to warm to t.a. Y

5 stirred for 18 hours. It was then diluted with DCM, washed with 5% citric acid and brine, dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by prep-HPLC to provide 4– (biphenyl-4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) non-8-enoyl) –4-methoxypyrrolidine-2-carboxylate of (2S , 4R) -methyl (140 mg, 51.5% yield) as white foam. 1 H NMR (CD3OD) δ 1.31–1.57 (m, 15 H), 1.62–1.65 (m, 1 H), 1.78–1.82 (m, 1 H), 2 , 11–2.13 (m, 2 H), 2.66–2.69 (m, 1 H), 2.84–2.89 (m, 1 H), 3.00 (s, 3 H) , 3.76 (s, 3 H), 4.16 (s, 2 H), 4.30–4.35 (m, 1 H), 4.79–

10 4.81 (m, 1 H), 4.95 (d, J = 12 Hz, 1 H), 5.03 (d, J = 18.5 Hz, 1 H), 5.83–5.87 (m, 1 H), 7.32–7.39 (m, 1 H), 7.45–7.56 (m, 4 H), 7.64–7.71 (m, 4 H); LC-MS (retention time: 3.20 min, procedure B), MS m / z 565 (M + + H).

Preparation (step) of (2S, 4R) –4– (biphenyl – 4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) non – 8 – enoyl) –4 – methoxypyrrolidine – 2– carboxylate methyl

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To a solution of 4– (biphenyl – 4 – yl) –1 - ((S) –2– (tert-butoxycarbonylamino) non – 8 – enoyl) –4 – methoxypyrrolidine – 2– carboxylate of (2S, 4R) –methyl (166 mg, 0.294 mmol) in THF (2 ml) and MeOH (2 ml) a pre-prepared solution of lithium hydroxide monohydrate (37 mg, 0.882 mmol) in water (2 ml) was added. This cloudy solution was stirred at t.a. for 18 hours and then concentrated in vacuo. The residue was dissolved in water and acidified with 1 N HCl at pH 2. This

The aqueous solution was extracted with EtOAc. The organic phase was washed with 5% citric acid and brine, dried over MgSO4, filtered and concentrated in vacuo to give 148 mg (91%) of acid (2S, 4R) -4- (biphenyl-4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) non-8-enoyl) -4-methoxypyrrolidine-2-carboxylic acid as a white solid. No further purification was carried out. LC-MS (retention time: 3.14 min, procedure B), MS m / z 551 (M + + H).

Stage 3: Preparation of 1 - ((2S, 4R) –4– (biphenyl – 4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) non – 8 – enoyl) –4 – methoxypyrrolidine– 25 2-carboxylate of (1R, 2S) -ethyl

image 1

A mixture of acid (2S, 4R) –4– (biphenyl-4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) non – 8 – enoyl) –4 – methoxy

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Pyrrolidine-2-carboxylic acid (68.2 mg, 0.124 mmol), ethyl 1-amino-2-vinylcyclopropanecarboxylate, HCl (26.1 mg, 0.136 mmol), HATU (56.5 mg, 0.149 mmol) and Base stirred Hunig (0.076 ml, 0.433 mmol) in DCM (3 ml) at rt for a whole night. The reaction mixture was concentrated in vacuo. The residue was dissolved in EtOAc and washed with dilute HCl, then with saturated aqueous NaHCO3 and water. The organic phase was dried over MgSO4, filtered and concentrated in vacuo to afford 120 mg of the crude product as a yellow oil. Purification by Biotage eluting with 40% EtOAc / hexane gave 67 mg (79%) of 1 - ((2S, 4R) –4– (biphenyl – 4 – yl) –1 - ((S) –2– (tert– butoxycarbonylamino) non-8-enoyl) -4-methoxypyrrolidine-2-carboxamido) -2-vinylcyclopropanecarboxylate of (1R, 2S) -ethyl as a white solid. LC-MS: MS m / z 688 (M + 1).

Stage 4: Preparation of 2– (biphenyl – 4-yl) –6– (tert-butoxycarbonylamino) –2 – methoxy – 5.16 – dioxo – 1,2,3,5,6,7,8,9, 10 , 11,13a, 14,14a, 15,16,16a – hexadecahydrocyclopropa [e] pyrrolo [1,2 – a] [1,4] diazacyclopentadecin – 14a – carboxylate of (2R, 6S, 13aS, 14aR, 16aS, Z )-ethyl

image 1

A mixture of 1 - ((2S, 4R) –4– (biphenyl – 4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) non – 8 – enoyl) –4 – methoxypyrrolidine– 2 – carboxamido ) -2-Vinylcyclopropanecarboxylate of (1R, 2S) -ethyl (60 mg, 0.087 mmol) and Grubbs II (14.81 mg, 0.017 mmol) in DCM (100 ml) was refluxed for 4 h. At this time another 0.2 equivalents of Grubbs II (14.81 mg, 0.017 mmol) was added and the mixture was refluxed for another 4 hours. The reaction mixture was concentrated in vacuo and the crude product was purified by Biotage eluting with 40% EtOAc / hexane to give 56 mg (97%) of 2– (biphenyl-4-yl) –6– (tert-butoxycarbonylamino) –2 – methoxy – 5.16 – dioxo – 1,2,3,5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a– hexadecahydrocyclopropa [e] pirrolo [1 , 2-a] [1,4] diazacyclopentadecin-14a-carboxylate of (2R, 6S, 13aS, 14aR, 16aS, Z) -ethyl as a dark brown solid. LC-MS: MS m / z 628 (M + 1-MeOH).

1H NMR (300 MHz, d4 – MeOH) δ ppm 1.19-1.57 (m, 19 H), 1.75 (d, J = 8.78 Hz, 3 H), 1.81-1, 96 (m, 1 H), 2.15 - 2.34 (m, 3 H), 2.60 - 2.82 (m, 2 H), 3.09 (s, 3 H), 4.07 - 4.24 (m, 3 H), 4.39 - 4.54 (m, 2 H), 4.78 - 4.86 (m, 1 H), 5.25 - 5.38 (m, 1 H ), 5.54-5.69 (m, 1 H), 7.36 (t, J = 7.32 Hz, 1 H), 7.46 (t, J = 7.50 Hz, 1 H), 7.58-7.72 (m, 6 H).

Stage 5: Preparation of acid (2R, 6S, 13aS, 14aR, 16aS, Z) –2– (biphenyl) –6– (tert-butoxycarbonylamino) –2 – methoxy – 5.16– dioxo – 1,2,3, 5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a-hexadecahydrocyclopropanopyrrolo [1,2-a] [1,4] diazacyclopentadecin– 14a-carboxylic

image 1

A mixture of (2R, 6S, 13aS, 14aR, 16aS, Z) –ethyl – 2– (biphenyl – 4-yl) –6– (tert-butoxycarbonylamino) –2 – methoxy – 5.16– dioxo – 1.2 , 3,5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a – hexadecahydrocyclopropa [e] pirrolo [1,2– a] [1,4] diazacyclopentadecin – 14a– carboxylate (56 mg, 0.085 mmol), LiOH monohydrate (40.7 mg, 1.70 mmol) in tetrahydrofuran (2 ml) / water (0.5 ml) / MeOH (1 ml) was stirred at rt for 18 hours This was then concentrated in vacuo and washed with ether. The aqueous phase was adjusted to pH = 4 using 1 N HCl and extracted with EtOAc. The EtOAc extract was dried over MgSO4, filtered and concentrated in vacuo to give 52 mg (97%) of acid (2R, 6S, 13aS, 14aR, 16aS, Z) -2- (biphenyl-4-yl) -6 - (tert-butoxycarbonylamino) -2-methoxy-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a - hexadecahydrocyclopropa [e] pyrrolo [1,2-a] [1,4] diazacyclopentadecin-14a-carboxylic acid as an off-white solid.

LC-MS: MS: m / z 654 (M ++ 1 + Na).

1 H NMR (500 MHz, d4 – MeOH) δ ppm 1.25 - 1.64 (m, 15 H), 1.66 - 1.84 (m, 3 H), 1.88 - 2.03 (m , 1 H), 2.10 - 2.43 (m, 3H), 2.58 - 2.86 (m, 2 H), 3.09 (s, 3 H), 4.06 - 4.27 ( m, 1 H), 4.33-4.57 (m, 2 H), 4.77-4.86 (m, 1 H), 5.29-5.45 (m, 1H), 5.52 - 5.72 (m, 1 H), 7.37 (t, J = 7.48 Hz, 1 H), 7.47 (t, J = 7.63 Hz, 2 H), 7.53 - 7 , 84 (m, 6 H).

Stage 6: Preparation of (2R, 6S, 13aS, 14aR, 16aS, Z) –2– (biphenyl – 4-yl) –14a– (cyclopropylsulfonylcarbamoyl) –2 – methoxy – 5.16– dioxo – 1,2,3 , 5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a-hexadecahydrocyclopropa [e] pyrrolo [1,2-a] [1,4] diazacyclopentadecin– 6-ylcarbamate tert-butyl

image 1

A mixture of acid (2R, 6S, 13aS, 14aR, 16aS, Z) –2– (biphenyl-4-yl) –6– (tert-butoxycarbonylamino) –2 – methoxy – 5.16– di – oxo – 1, 2,3,5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a – hexadecahydrocyclopropa [e] pirrolo [1,2–

10 a] [1,4] diazacyclopentadecin-14a-carboxylic acid (50 mg, 0.079 mmol) and CDI (755 mg, 4.66 mmol) in tetrahydrofuran (5 ml) was heated at reflux for 1 hour. It was then cooled to t.a. and then cyclopropanesulfonamide (11.51 mg, 0.095 mmol) was added followed by DBU (0.042 ml, 0.277 mmol). This reaction mixture was stirred at t.a. for 18 hours and then concentrated in vacuo to give the crude product as a light colored oil. Purification by preparative HPLC gave 15 mg (26%) of (2R, 6S, 13aS, 14aR, 16aS, Z) –2– (biphenyl-4-yl) –14a– (cyclopropylsulfonylcarbamoyl) –2 – methoxy–

15 5.16 – dioxo – 1,2,3,5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a – hexadecahydrocyclopropa [e] pirrolo [1,2– a ] [1,4] tert-butyl diazacyclopentadecin-6-ylcarbamate as a white solid. LC-MS: MS: m / z 757 (M ++ 1 + Na).

1 H NMR (500 MHz, MeOD) ppm 0.86 - 0.97 (m, 1 H), 0.99 - 1.09 (m, 1 H), 1.08 - 1.74 (m, 19 H ), 1.80 (dd, J = 7.78, 5.95 Hz, 1 H), 1.87-2.00 (m, 1 H), 2.01 - 2.17 (m, 1 H) , 2.34 - 2.44 (m, J = 8.24 Hz, 2 H), 2.63 - 2.77 (m, 2 H), 2.90 - 3.01 (m, 1 H), 3.14 (s, 3 H), 4.05 (d, J = 10.38 Hz, 1 H), 4.35 (t, J = 7.32 Hz, 1 H), 4.40-4, 51 (m, 1 H), 4.76 (d, J = 9.77

20 Hz, 1 H), 5.14 (t, J = 9.46 Hz, 1 H), 5.61 - 5.77 (m, 1 H), 7.37 (t, J = 7.32 Hz , 1 H), 7.46 (t, J = 7.63 Hz, 2 H), 7.55 - 7.66 (m, 6 H).

Example 88: Preparation of Compound 2

Preparation of (2R, 6S, 13aS, 14aR, 1 6aS, Z) –2– (biphenyl – 4-yl) –14a– (cyclopropylsulfonylcarbamoyl) –2 – methoxy – 8– (2– nitrophenylsulfonyl) –5.16 – dioxo –1,2,30,5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a – hexadecahydrocyclopropa [n] pirrolo [2,1– c] [1,4] tert-butyl diazacyclopentadecin-6-ylcarbamate, Compound 2.

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image 1

Compound 2.

Scheme 3.

image 1

Stage 1: Preparation of 4– (biphenyl – 4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) –3– (2-nitro – N– (pent – 4-enyl) phenylsulfonamido) propanoyl) -4-methoxypyrrolidine-2-carboxylate of (2S, 4R) -methyl

image 1

5

A mixture of 4– (biphenyl – 4-yl) –4 – methoxypyrrolidine – 2-carboxylate of (2S, 4R) –methyl (100 mg, 0.287 mmol), acid (S) –2– (tert-butoxycarbonylamino) –3 - (2-nitro-N– (pent-4-enyl) phenylsulfonamido) propanoic acid (145 mg, 0.316 mmol), Hunig's base (0.176 ml, 1.01 mmol) and HATU (131 mg, 0.345 mmol) in DCM ( 3 ml) stirred at rt for 18 hours This was then concentrated in vacuo. The residue was dissolved in EtOAc and washed with dilute HCl, followed by saturated NaHCO3

10 aqueous and water. The organic phase was dried over MgSO4, filtered and concentrated in vacuo to afford 250 mg of

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Raw product as a brown oil. Purification by Biotage eluting with 60% EtOAc / hexane gave 188 mg (87%) of 4– (biphenyl-4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) –3– (2– nitro-N- (pent-4-enyl) phenylsulfonamido) propanoyl) -4-methoxypyrrolidine-2-carboxylate of (2S, 4R) -methyl as an off-white foam. LC-MS: MS m / z 751 (M + 1).

1 H NMR (500 MHz, MeOD) δ ppm 1.28 - 1.52 (m, 9 H), 1.51 - 1.74 (m, 2 H), 1.90 - 2.07 (m, 2 H), 2.58-2.71 (m, 1 H), 2.78-2.91 (m, 1 H), 2.95, 3.01 (s, 3 H, rotamers), 3.39 - 3.54 (m, 2 H), 3.56–3.68 (m, 2 H), 3.77 - 3.80 (m, 4 H), 4.00 –4.17 (m, 1 H), 4.34 (d, J = 11.29 Hz, 1 H), 4.74-4.81 (m, 1 H), 4.91 - 5.06 (m, 2 H), 5, 66-5.85 (m, 1 H), 7.37 (t, J = 7.32 Hz, 1 H), 7.44-7.54 (m, 4 H), 7.61-7.73 (m, 4 H), 7.75-7.90 (m, 3 H), 8.06-8.17 (m, 1 H).

Stage 2: Preparation of acid (2S, 4R) –4– (biphenyl – 4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) –3– (2 – nitro – N– (pent – 4 - enyl) phenylsulfonamido) propanoyl) -4-methoxypyrrolidine-2-carboxylic

image 1

A mixture of 4– (biphenyl – 4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) –3– (2-nitro – N– (pent – 4 – enyl) –phenylsulfonamido) propanoyl) - (2S, 4R) -methyl 4-methoxypyrrolidine-2-carboxylate (185 mg, 0.246 mmol) and LiOH monohydrate (118 mg, 4.93 mmol) in tetrahydrofuran (2 ml) / MeOH (0.5 ml) / Water (1 ml) was stirred at rt for 18 hours The reaction mixture was then concentrated in vacuo and diluted with 5 ml of water, and then washed with ether. The aqueous phase was adjusted to pH = 4 using 1 N HCl, and extracted with EtOAc, dried over MgSO4, filtered and concentrated in vacuo to afford 160 mg (87%) of acid (2S, 4R) -4- ( biphenyl – 4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) –3– (2-nitro-N– (pent – 4– enyl) phenyl) sulfonamido) propanoyl) –4 – methoxypyrrolidine – 2 –Carboxylic as a white solid. LC-MS: MS m / z 737 (M + 1).

1 H NMR (500 MHz, d4 – MeOH) δ ppm 1.29 - 1.73 (m, 11 H), 1.90 - 2.12 (m, 2 H), 2.67 (dd, J = 13 , 12, 9.46 Hz, 1 H), 2.88 (d, J = 13.43 Hz, 1 H), 3.04 (s, 3 H), 3.40 - 3.55 (m, 2H ), 3.56-3.71 (m, 2 H), 3.80 (dd, J = 15.41, 3.81 Hz, 1 H), 4.06 (d, J = 10.99 Hz, 1 H), 4.26 - 4.38 (m, 1 H), 4.70 - 4.81 (m, 1 H), 4.95 - 5.06 (m, 2 H), 5.59 - 5.90 (m, 1 H), 7.37 (t, J = 7.48 Hz, 1 H), 7.42-7.58 (m, 4 H), 7.62-7.75 (m , 4 H), 7.76-7.92 (m, 3 H), 8.01-8.21 (m, 1 H).

Stage 3: Preparation of 1 - ((2S, 4R) –4– (biphenyl – 4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) –3– (2 – nitro – N– (pent –4– enyl) phenylsulfonamido) propanoyl) -4-methoxypyrrolidine-2-carboxamido) -2-vinyl (1R, 2S) -ethyl vinylcyclopropanecarboxylate

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A mixture of acid (2S, 4R) –4– (biphenyl – 4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) –3– (2 – nitro – N– (pent – 4– enil ) phenyl-sulfonamido) propanoyl) -4-methoxypyrrolidine-2-carboxylic acid (160 mg, 0.217 mmol), ethyl 1-amino-2-vinylcyclopropanecarboxylate, HCl salt (49.9 mg, 0.261 mmol), HATU (99 mg , 0.261 mmol) and Hunig Base (0.133 ml, 0.760 mmol) in DCM (3 ml) was stirred at rt for 18 hours This was then concentrated in vacuo. The residue was dissolved in EtOAc and washed with dilute HCl, then with saturated aqueous NaHCO3 and water. The organic phase was then dried over MgSO4, filtered, concentrated in vacuo to provide 180 mg of the crude product as a yellow oil. Purification by Biotage eluting with 40% EtOAc / hexane gave 100 mg (53%) of 1 - ((2S, 4R) –4– (biphenyl – 4 – yl) –1 - ((S) –2– (tert -Butyloxycarbonylamino) -3- (2-nitro-N- (pent-4-enyl) phenylsulfonamido) propanoyl) -4-methoxypyrrolidine-2-carboxamido) -2-vinylcyclopropanecarboxylate of (1R, 2S) -ethyl as a white solid. LC-MS: MS m / z 874 (M + 1).

1 H NMR (300 MHz, DMSO-D6) δ ppm 1.14 (t, J = 7.14 Hz, 3 H), 1.18 - 1.42 (m, 12 H), 1.60 - 1, 69 (m, 2 H), 1.83 - 1.96 (m, 2 H), 2.11 (c, J = 8.17 Hz, 1 H), 2.51 - 2.61 (m, 2 H), 2.96 (s, 3 H), 3.33-3.41 (m, 2 H), 3.43-3.65 (m, J = 9.51 Hz, 2 H), 3, 81-3.91 (m, 1 H), 3.98-4.10 (m, J = 6.83, 6.83, 6.83 Hz, 3 H), 4.58-4.72 (m , J = 9.33, 4.21 Hz, 1 H), 4.86 -

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5.01 (m, 2 H), 5.09 (d, J = 13.17 Hz, 1 H), 5.22 (d, J = 17.20 Hz, 1 H), 5.50-5, 81 (m, 2 H), 7.07 (d, J = 9.15 Hz, 1 H), 7.34-7.41 (m, 1 H), 7.47 (t, J = 7.32 Hz, 4 H), 7.61 - 7.74 (m, 4 H), 7.77 - 7.93 (m, 2 H), 7.94 - 8.08 (m, 2 H), 8, 43 (s, 1 H).

Stage 4: Preparation of 2– (biphenyl – 4-yl) –6– (tert-butoxycarbonylamino) –2 – methoxy – 8– (2-nitrophenylsulfonyl) –5.16 – dioxo– 1,2,3,5,6 , 7,8,9,10,11,13a, 14,14a, 15,16,16a-hexadecahydrocyclopropa [n] pyrrolo [2,1-c] [1,4,8] triazacyclopentadecin - 14a-carboxylate of (2R , 6S, 13aS, 14aR, 16aS, Z) -ethyl

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A mixture of 1 - ((2S, 4R) –4– (biphenyl – 4-yl) –1 - ((S) –2– (tert-butoxycarbonylamino) –3– (2 – nitro – N– (pent – 4 - enyl) phenylsulfonamidopropanoyl) -4-methoxypyrrolidine-2-carboxamido) -2-vinylcyclopropanecarboxylate (1R, 2S) -ethyl (95 mg, 0.109 mmol) and Grubbs II (18.46 mg, 0.022 mmol) in DCM (200 ml ) was refluxed for 4 h The reaction mixture was then concentrated in vacuo The residue was purified by Biotage eluting with 60% EtOAc / hexane isolating 65 mg (71%) of 2– (biphenyl-4-yl) –6– (tert-butoxycarbonylamino) –2-methoxy-8– (2-nitrophenylsulfonyl) –5.16-dioxo– 1,2,3,5,6,7,8,9,10,11,13a, 14 , 14a, 15,16,16a-hexadecahydrocyclopropa [n] pyrrolo [2,1-c] [1,4,8] triazacyclopentadecin-14a- carboxylate of (2R, 6S, 13aS, 14aR, 16aS, Z) -ethyl as a whitish solid.

LC-MS: MS m / z 868 (M ++ 1 + Na).

1 H NMR (500 MHz, d4 – MeOH) ppm 1.37 - 1.53 (m, 13 H), 1.57 - 1.71 (m, 1 H), 1.74 (dd, J = 9, 77, 5.19 Hz, 1 H), 1.76 1.87 (m, 1 H), 1.98 - 2.05 (m, 1 H), 2.09-2.22 (m, J = 10.99 Hz, 1 H), 2.38 (c, J = 9.46 Hz, 1 H), 2.53 - 2.73 (m, 2 H), 3.07 3.14 (m, 3 H), 3.35-3.46 (m, 2 H), 3.50-3.59 (m, 1 H), 3.62-3.74 (m, 1 H), 3.99 (d , J = 10.99 Hz, 1 H), 4.14 - 4.21 (m, 2 H), 4.26 (t, J = 7.63 Hz, 1 H), 4.46 (d, J = 10.38 Hz, 1 H), 4.82 - 4.86 (m, 1 H), 5.59 (t, J = 10.38 Hz, 1 H), 5.63 - 5.72 (m , 1 H), 7.37 (t, J = 7.32 Hz, 1 H), 7.47 (t, J = 7.63 Hz, 2 H), 7.53 - 7.58 (m, 2 H), 7.59-7.72 (m, 4 H), 7.78-7.92 (m, 3 H), 8.08-8.10 (m, 1 H).

Step 5: Preparation of acid (2R, 6S, 13aS, 14aR, 16aS, Z) –2– (biphenyl-4-yl) –6– (tert-butoxycarbonylamino) –2 – methoxy – 8– (2– nitrophenylsulfonyl) - 5.16 – dioxo – 1,2,3,5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a – hexadecahydrocyclopropa [n] pirrolo [2,1– c] [1,4,8] triazacyclopentadecin-14a-carboxylic

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A mixture of (2R, 6S, 13aS, 14aR, 16aS, Z) –ethyl – 2– (biphenyl – 4-yl) –6– (tert-butoxycarbonylamino) –2 – methoxy – 8– (2– nitrophenylsulfonyl) –5 , 16 – dioxo – 1,2,3,5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a – hexadecahydrocyclopropa [n] pirrolo [2,1– a] [ 1,4] diazacyclopentadecin-14a-carboxylate (60 mg, 0.071 mmol) and LiOH monohydrate (34.0 mg, 1,419 mmol) in tetrahydrofuran (2 ml) / water (0.5 ml) / MeOH (1 ml) stirred to ta for 18 hours This was concentrated in vacuo and partitioned between water and ether. The aqueous phase was adjusted to pH = 4 using 1 N HCl and extracted with EtOAc. The organic phase was then dried over MSO4, filtered and concentrated by isolating 52 mg of the crude product as an off-white solid. Preparation of acid (2R, 6S, 13aS, 14aR, 16aS, Z) -2- (biphenyl-4-yl) -6- (tert-butoxycarbonylamino) -2-methoxy-8- (2-nitrophenylsulfonyl) –5.16 –Dioxo – 1,2,3,5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a – hexadecahydrocyclopropa [n] pirrolo [2,1– c] [1, 4,8] triazacyclopentadecin-14a-carboxylic acid as a white solid. LC-MS: MS mlz 818 (M + 1 + Na). 1 H NMR (500 MHz, d4 – MeOH) ppm 1.29 - 1.71 (m, 11 H), 1.72 - 1.96 (m, 2 H), 2.09 - 2.27 (m, 2 H), 2.30 - 2.46 (m, 1 H), 2.80

- 2.90 (m, 1 H), 3.03 (s, 3 H), 3.06 - 3.13 (m, 1 H), 3.45 - 3.62 (m, 2 H), 3 , 72 (dd, J = 14.95, 3.66 Hz, 1 H), 3.78 (d, J = 12.21 Hz, 1 H), 4.04 (d, J = 12.51 Hz, 1 H), 4.08-4.18 (m, 1 H), 4.53 (dd, J = 11.44, 3.51 Hz, 1 H), 4.98 - 5.03 (m, 1 H), 5.60-5.80 (m, 2 H), 7.37 (t, J = 7.32 Hz, 1 H), 7.43-7.58 (m, 4 H), 7, 62-7.73 (m, 4 H), 7.76-7.90 (m, 3 H), 8.07-8.16 (m, 1 H).

Stage 6: Preparation of Compound 2: (2R, 6S, 13aS, 14aR, 16aS, Z) –2– (biphenyl-4-yl) –14a– (cyclopropylsulfonylcarbamoyl) –2– methoxy – 8– (2-nitrophenylsulfonyl) - 5.16 – dioxo – 1,2,3,5,6,7,80,9,10,11,13a, 14,14a, 15,16,16a– hexadecahydrocyclopropa [n] pirrolo [2,1 – c] [1,4,8] tert-butyl diazacyclopentadecin-6-ylcarbamate

A mixture of acid (2R, 6S, 13aS, 14aR, 16aS, Z) –2– (biphenyl-4-yl) –6– (tert-butoxycarbonylamino) –2 – methoxy – 8– (2– nitro-phenylsulfonyl) - 5.16 – dioxo – 1,2,3,5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a – hexadecahydrocyclopropa [n] pirrolo [2,1– c] [1,4,8] triazacyclopentadecin-14a-carboxylic acid (13 mg, 0.016 mmol) and CDI (3.61 mg, 0.022 mmol) in tetrahydrofuran (2 ml) was refluxed for 1 h. It was then cooled to t.a. and cyclopropanesulfonamide (2.70 mg, 0.022 mmol) was added followed by DBU (8.39 µl, 0.056 mmol). The reaction mixture was stirred at t.a. at 18 hours and then concentrated in vacuo to provide a light yellow oil. To this oil 5 ml of water was added, adjusting to pH = 4 using 1 N HCl. The white precipitate was collected by filtration and washed with water to give 15 mg of the crude product as a white solid. Purification by flash chromatography eluting with 2% MeOH / CH2Cl2 gave 8 mg of (2R, 6S, 13aS, 14aR, 16aS, Z) –2– (biphenyl-4-yl) –14a– (cyclopropylsulfonylcarbamoyl) –2 – methoxy –8– (2-nitrophenylsulfonyl) –5.16– dioxo – 1,2,3,5,6,7,8,9,10,11,13a, 14,14a, 15,16,16a – hexadecahydrocyclopropa [n ] pyrrolo [2,1-c] [1,4,8] tert-butyl triazacyclopentadecin-6-ylcarbamate as a white solid. LC-MS: MS m / z 889 (M + 1-MeOH). (This product was contaminated with an equal part of the P1 methyl ester by-product).

Biological studies

In the present disclosure HCV NS3 / 4A protease complex enzyme assays and cell-based HCV replicon assays were used, and prepared, conducted and validated as follows:

Generation of recombinant HCV NS3 / 4A protease complex

HCV NS3 protease complexes, derived from BMS strains, H77 strains or J4L6S strains, were generated as described below. These purified recombinant proteins were generated for use in a homogeneous assay (see below) to provide an indication of the efficacy of the compounds of the present disclosure in inhibiting the proteolytic activity of HCV NS3.

The serum of a patient infected with HCV was obtained from Dr. T. Wright, San Francisco Hospital. A full-length cDNA template (complementary deoxyribonucleic acid) designed from the HCV genome (BMS strain) was constructed from DNA fragments obtained by reverse transcription-PCR (TI-PCR) of serum RNA (ribonucleic acid) and using primers selected based on homology among other strains of genotype 1a. From the determination of the entire genome sequence, a genotype 1a was assigned to the isolated HCV according to the classification of Simmonds et al. (See P Simmonds, KA Rose, S Graham, SW Chan, F McOmish, BC Dow, EA Follett, PL Yap and H Marsden, J. Clin. Microbiol., 31 (6), 1493-1503 (1993)). The amino acid sequence of the non-structural region, NS2-5B, was identical in a value> 97% to HCV genotype 1a (H77) and 87% identical to genotype 1b (J4L6S). Infectious clones, H77 (genotype 1a) and J4L6S (genotype 1b) were obtained from R. Purcell (NIH) and the sequences were published in Genbank (AAB67036, see Yanagi, M., Purcell, RH, Emerson, SU and Bukh, J. Proc. Natl. Acad. Sci. USA 94 (16), 8738-8743 (1997); AF054247, see Yanagi, M., St Claire, M., Shapiro, M., Emerson, SU, Purcell, RH and Bukh, J, Virology 244 (1), 161-172. (1998)).

Strains H77 and J4L6S were used for the production of recombinant NS3 / 4A protease complexes. The DNA encoding the recombinant HCV NS3 / 4A protease complex (amino acids 1027 to 1711) for these strains was manipulated as described by P. Gallinari et al. (see Gallinari P, Paolini C, Brennan D, Nardi C, Steinkuhler C, De Francesco R. Biochemistry. 38 (17): 5620–32, (1999)). Succinctly, a solubilizing tail of three lysines was added to the 3 'end of the NS4A coding region. The cysteine of the P1 position of the NS4A-NS4B cleavage site (amino acid 1711) was changed to a glycine to avoid proteolytic cleavage of the lysine label. In addition, a cysteine to serine mutation was introduced by PCR at amino acid position 1454 to prevent autolytic cleavage in the NS3 helicase domain. The variant DNA fragment was cloned into the bacterial expression vector pET21b (Novagen) and the NS3 / 4A complex was expressed in strain BL21 (DE3) of Escherichia coli (Invitrogen) following the protocol described by P. Gallinari et al. (see Gallinari P, Brennan D, Nardi C, Brunetti M, Tomei L, Steinkuhler C, De Francesco R., J Virol. 72 (8): 6758–69 (1998)) with modifications. Briefly, expression of the NS3 / 4A protease complex was induced with 0.5 millimolar (mT) isopropyl-D-1-thiogalactopyranoside (IPTG) for 22 hours (h) at 20 ° C. A typical fermentation (1 liter (l)) produced approximately 10 grams

(g) of wet cell paste. The cells were resuspended in lysis buffer (10 ml / g) consisting of N- (2-Hydroxyethyl) Piperazine-N '- (2-Sulfonic Ethane) (HEPES) 25 mM, pH 7.5, 20% glycerol , 500 mM Sodium Chloride (NaCl), 0.5% Triton X – 100, 1 microgram / ml (/g / ml) lysozyme, 5 mM Magnesium Chloride (MgCl2), 1 µg / ml DnaseI, p– 5 mM mercaptoethanol ((3ME), Protease Inhibitor - Free Tetraacetic Ethylenediamine Acid (EDTA) (Roche), homogenized and incubated for 20 minutes (min) at 4 ° C. The homogenate was subjected to ultrasound and cleared by ultrafiltration at 235000 g for 1 hour (h) at 4 ° C. Imidazole was added to the supernatant to a final concentration of 15 mm and the pH was adjusted to 8.0 The crude protein extract was loaded on a nickel-nitroloacetic acid column ( Ni-NTA) pre-equilibrated with buffer B (25 mM HEPES, pH 8.0, 20% glycerol, 500 mM NaCl, 0.5% Triton X – 100, 15 mM imidazole, 5 mM ME). loaded with a flow 1 ml / min. The column was washed with 15 column volumes of buffer C (similar to buffer B, but with 0.2% Triton X-100). The protein was eluted with 5 column volumes of buffer D (similar to buffer C, but with 200mM imidazole).

Fractions containing NS3 / 4A protease complex were pooled and loaded onto a pre-equilibrated Superdex-S200 desalination column with buffer D (25mM HEPES, pH 7.5, 20% glycerol, 300mM NaCl, Triton X-100 at 0 , 2%, ME 10 mM). The sample was charged with a flow rate of 1 ml / min. Fractions containing NS3 / 4A protease complex were pooled and concentrated at approximately 0.5 mg / ml. The purity of the NS3 / 4A protease complexes, derived from strains BMS, H77 and J4L6S, was determined to be greater than 90% by SDS-PAGE and mass spectroscopy analysis. The enzyme was stored at -80 ° C, thawed on ice and diluted before using in the assay buffer.

FRET peptide assay to control the NS3 / 4A proteolytic activity of HCV

The purpose of this in vitro assay was to measure the inhibition of HCV NS3 protease complexes, derived from BMS, H77 or J4L6S strains, as described below, by compounds of the present disclosure. This assay provides an indication of the efficacy of the compounds of the present disclosure in the inhibition of HC3 NS3 protein activity.

To control the activity of HCV NS3 / 4A protease, an NS3 / 4A peptide substrate was used. The substrate was RET S1 (Resonant Energy Transfer Depsipeptide Substrate; AnaSpec, Inc. # cat 22,991) (FRET peptide), described by Taliani et al. in Anal. Biochem 240 (2): 60–67 (1996). The sequence of this peptide is based approximately on the NS4A / NS4B natural cleavage site for HCV NS3 protease, with the exception that there is a steric linkage instead of an amide linkage at the cleavage site. The peptide also contains a fluorescence donor, EDANS, near one end of the peptide, and a fluorescence acceptor, DABCILO, near the other end. The fluorescence of the peptide is inactivated by intermolecular resonance energy transfer (RET) between the donor and the acceptor, but since the NS3 protease cleaves the peptide, the products are released from the RET inactivation and the donor fluorescence becomes apparent.

The peptide substrate is incubated with one to three recombinant NS3 / 4A protease complexes, in the absence or presence of a compound of the present disclosure. The inhibitory effects of a compound are determined by monitoring the formation of fluorescent reaction products in real time using a Cytofluor Series 4000.

The reagents were the following: HEPES and glycerol (Ultrapure) were obtained from GIBCO-BRL. Dimethyl sulfoxide (DMSO) was obtained from Sigma. P-mercaptoethanol was obtained from Bio Rad.

Assay buffer: 50 mM HEPES, pH 7.5; 0.15 M NaCl; 0.1% tritol; 15% glycerol; ME 10 mM. Substrate: 2 µM final concentration (from a 2 mM stock solution in DMSO stored at –20 ° C). Type 1a (1b) of HCV NS3 / 4A protease, 2–3 nM final concentration (from a 5 madreM stock solution in 25 mM HEPES, pH 7.5, 20% glycerol, 300 mM NaCl, Triton –X100 at 0.2%, ME 10 mM). For compounds with potencies that approach the limit of the assay, the assay becomes more sensitive by adding bovine serum albumin 50 µg / ml (Sigma) to the assay buffer and reducing the final protease concentration to 300 pM.

The test was performed on a black 96-well polystyrene plate from Falcon. Each well contained 25 µl of NS3 / 4A protease complex in assay buffer, 50 µl of a compound of the present disclosure in 10% DMSO / assay buffer and 25 µl of substrate in assay buffer. A control (without compound) was also prepared on the same test plate. The enzyme complex was mixed with compound or control solutions for 1 min before starting the enzymatic reaction by adding substrate. The test plate was read immediately using the Cytofluor Series 4000 (Perspective Biosystems). The instrument was calibrated to read at an emission of 340 nm and excitation of 490 nm at 25 ° C. The reactions were generally followed for approximately 15 minutes.

The percent inhibition was calculated with the following equation:

100 - [(FInh / FCon) x 100]

where F is the change in fluorescence over the linear range of the curve. A non-linear curve adjustment was applied to the inhibition – concentration data, and the 50% effective concentration (IC50) was calculated using the Excel XLfit program using the equation, y = A + ((B – A) / ( 1 + ((C / x) ^ D))).

Specificity tests

Specificity assays were performed to demonstrate the in vitro selectivity of the compounds of the present disclosure in the inhibition of NS3 / 4A protease complex of HCV compared to other serine proteases or cysteines.

The specificities of compounds of the present disclosure were determined against a variety of serine proteases: human neutrophil elastase (HNE), porcine pancreatic elastase (PPE) and human pancreatic chymotrypsin and a cysteine protease: human hepatic cathepsin B. In all cases, a 96-well plate format protocol was used using a specific amino-methyl-coumarin (AMC) substrate for each enzyme as previously described (PCT Patent Application No. WO 00/09543) with some modifications to serine protease assays. All enzymes were purchased from Sigma, EMDbiosciences, while the substrates were purchased from Bachem, Sigma and EMDbiosciences.

Compound concentrations varied from 100 to 0.4 µM depending on their potency. Each of the enzymatic assays was initiated by adding substrate to the pre-incubated inhibitor for 10 min at room temperature and hydrolysis up to 15% conversion as a measure of cytofluor.

The final conditions for each trial were performed as follows:

50 mM tris (hydroxymethyl) aminomethane hydrochloride (Tris – HCl), pH 8, 0.5 M sodium sulfate (Na2SO4), 50 mM NaCl, 0.1 mM EDTA, 3% DMSO, Tween – 20 at 0, 01% with LLVY – AMC 5 M and 1 nM chymotrypsin.

50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mM EDTA, 3% DMSO, 0.02% Tween-20, 5 µM suAP-AAPV-AMC and 20 nM HNE or 8 nM PPE;

100 mM NaOAC (sodium acetate), pH 5.5, 3% DMSO, 1 mM TCEP (2-carboxyethyl) phosphine hydrochloride, 5 nM cathepsin B (buffer activated enzyme stock solution containing TCEP 20 mM before use), and Z – FR – AMC 2 M diluted in H2O.

The percent inhibition was calculated using the formula:

[1 - ((UVinh - UVwhite) / (UVcontrol - UVwhite))] x 100

A non-linear curve adjustment was applied to the inhibition-concentration data, and the 50% effective concentration (IC50) was calculated using the Excel XLfit program.

HCV Replicon Generation

A complete HCV replicon cell system was established as described by Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R., Science 285 (5424): 110-3 (1999). This system allowed us to evaluate the effects of our HCV protease compounds on the replication of HCV RNA. Briefly, using the sequence of HCV strain 1b described in Lohmann's article (Award number: AJ238799), an HCV cDNA was synthesized by Operon Technologies, Inc. (Alameda, CA. USA), and The full length replicon was then assembled into plasmid pGem9zf (+) (Promega, Madison, WI) using standard molecular biology techniques. Replicon consists of (i) the 5 'UTR of HCV condensed to the first 12 amino acids of the capsid protein, (ii) the neomycin phosphotransferase (neo) gene, (iii) the IRES of the encephalomyocarditis virus (EMCV) , and (iv) NS3 to NS5B genes of HCV and 3 'UTR of HCV. Plasmid DNAs were linearized with ScaI and RNA transcripts were synthesized in vitro using the T7 MegaScript transcription kit (Ambion, Austin, TX, USA) according to the manufacturer's guidelines. In vitro transcription of the cDNA was transfected into a human hepatoma cell line, HUH-7. Selection for cells constitutively expressing HCV replicon was achieved in the presence of a selectable marker (G418). The resulting cell lines were characterized by the production of positive and negative chain RNA and protein production over time.

HCV Replicon FRET Assay

The FRET HCV replicon assay was developed to check the inhibitory effects of compounds described in the present disclosure on HCV viral replication. HUH-7 cells, constitutively expressing HCV replicon, were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco-BRL) containing 10% fetal calf serum (FCS) (Sigma) and 1 mg / ml of G418 (Gibco – BRL). The cells were seeded the night before (1.5 x 104 cells / well) in sterile 96-well tissue culture plates. Controls with compound and without compound were prepared in DMEM containing 4% FCS, 1: 100 penicillin / streptomycin (Gibco-BRL), 1: 100 L-glutamine and 5% DMSO on the dilution plate (DMSO at 0 , 5% final concentration of the test). Compound / DMSO mixtures were added to the cells and incubated for 4 days at 37 ° C. After 4 days, the cells were evaluated by cytotoxicity using Alamar Blue (Trek Diagnotstic Systems) for a CC50 reading. The toxicity of the compound (CC50) was determined by adding 1 / 10th volume of blue Alamar to the incubation medium of the cells. After 4 h, the fluorescence signal from each well was read,

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with an excitation wavelength of 530 nm and an emission wavelength of 580 nm, using the Cytofluor Series 4000 (Perspective Biosystems). The plates were then washed thoroughly with phosphate buffered saline (PBS) (3 times 150 µl). The cells were lysed with 25 µl of a lysis test reagent containing HCV protease substrate (5X cell luciferase cell culture lysis reagent (Promega No. E153A) diluted to 1X with distilled water, added NaCl at a final 150 mM concentration, the FRET peptide substrate (as described for the previous enzymatic assay) diluted to a final concentration of 10 M from a 2 mM stock solution in 100% DMSO. placed on the Cytofluor 4000 instrument, adjusted to 340 nm excitation / 490 nm emission, automatically for 21 cycles and the plate was read in kinetic mode.The EC50 determinations were carried out as described for the determinations of IC50

HCV Replicon Luciferase Indicator Assay

As a secondary test, EC50 determinations from the FRET replicon assay were confirmed in a replicon luciferase indicator assay. The use of a luciferase replicon indicator assay was first described by Krieger et al (Krieger N, Lohmann V and Bartenschlager R, J. Virol. 75 (10): 4614-4624 (2001)). The replicon construct described by our FRET assay was modified by inserting cDNA encoding a humanized form of the Renilla luciferase gene and a condensed binding sequence directly to the 3 ′ end of the luciferase gene. This insert was introduced into the replicon construct using an Asc1 restriction site located in the center, directly upstream of the neomycin marker gene. The adaptive mutation was also introduced at position 1179 (serine to isoleucine) (Blight KJ, Kolykhalov, AA, Rice, CM, Science 290 (5498): 1972-1974). A stable cell line constitutively expressing this HCV replicon construct was generated as described above. The luciferase indicator assay was adjusted as described for the HCV replicon FRET assay with the following modifications. After 4 days in a 37 ° C / 5% CO2 incubator, the cells were analyzed to determine Renilla Luciferase activity using the Promega Dual – Glo Luciferase Assay System. Media (100 µl) were removed from each well containing cells. To the remaining 50 µl of media, 50 µl of Dual-Glo luciferase reagent was added and the plates were shaken for 10 minutes at 2 hours at room temperature. Dual-Glo Stop & Glo reagent (50 µl) was added to each well and the plates were gently shaken for an additional time of 10 min at 2h at room temperature. The plates were read on a Packard TopCount NXT using a luminescence program.

The percent inhibition was calculated using the formula:

Mean luciferase signal in experimental wells (+ compound)% control =

medium luciferase signal in control wells with DMSO (-composite)

A graph with the values was made and analyzed using XLfit to obtain EC50 values.

Note that using the example number of the patent and the compound number of the patent shown in Table 2, the structures of the compounds can be found here.

Table 2

Compound number

IC50 interval EC50 (nM)

one

2 5

It will be apparent to one skilled in the art that the present disclosure is not limited to the preceding illustrative examples, and that it can be done in other specific ways without departing from its essential attributes. Therefore, it is desired that the examples be considered in all respects as illustrative and not restrictive, with reference to the appended claims, rather than the previous examples, and therefore it is intended that all changes within the meaning and scope of equivalence of the claims are included in them.

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Claims (13)

1. A compound of formula (I) image 1 or a pharmaceutically acceptable enantiomer, diastereomer or salt thereof, in which R1 is selected from alkoxy, hydroxy and -NHSO2R7; R2a and R2b are independently selected from hydrogen and methyl; R3 is selected from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, heterocyclyl and heterocyclylalkyl; R4 is –OR8; R5 is selected from hydrogen, alkyl and cycloalkyl; R6 is selected from hydrogen, alkyl, alkoxycarbonyl, alkylaminocarbonyl, alkylcarbonyl, aminocarbonyl, aryloxycarbonyl, cycloalkyloxycarbonyl, dialkylaminocarbonyl, haloalkoxycarbonyl, haloalkyl, haloalkylcarbonyl, heterocyclyloxycarbonyl and (NRaRb) sulfonyl; R7 is selected from alkyl, aryl, cycloalkyl, (cycloalkyl) alkyl, dialkylaminocarbonyl, dialkylaminocarbonylalkyl, heterocyclyl, heterocyclylcarbonyl and -NRaRb; the cycloalkyl and the cycloalkyl part of the (cycloalkyl) alkyl being optionally substituted with one, two or three groups independently selected from alkenyl, alkoxy, alkoxyalkyl, alkyl, arylalkyl, arylcarbonyl, cyano, cycloalkenyl, (cycloalkyl) alkyl, halo, haloalkoxy, haloalkyl and (NReRf) carbonyl; and wherein Ra and Rb are independently selected from hydrogen, alkoxy, alkyl, aryl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, haloalkyl, heterocyclyl and heterocyclylalkyl; and wherein Re and Rf are independently selected from hydrogen, alkyl, aryl, arylalkyl and heterocyclyl; wherein the aryl, the aryl part of the arylalkyl and the heterocyclyl are optionally substituted with one or two substituents independently selected from alkoxy, alkyl and halo; Y R8 is selected from alkoxyalkyl, alkyl, alkylcarbonyl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, cycloalkylcarbonyl, haloalkoxyalkyl, haloalkyl, (NRcRd) carbonyl and -P (O) (OR ’) 2; Rc and Rd being selected independently of hydrogen, alkyl and arylalkyl; or Rc and Rd, together with the nitrogen atom to which they are attached, form a five or six membered monocyclic heterocyclic ring that optionally contains an additional heteroatom selected from O, NRx and S; where Rx is selected from hydrogen and alkyl; and where R 'is selected from hydrogen and alkyl; Y Q is a C3–9 saturated or unsaturated chain, which optionally contains one to three heteroatoms independently selected from O, S (O) and NR9, where m is 0, 1 or 2 and R9 is selected from hydrogen, alkoxy, alkoxycarbonyl , alkyl, alkylcarbonyl, alkylsulfonyl, aminocarbonyl, arylsulfonyl, cycloalkyl, (cycloalkyl) alkyl, cycloalkyloxy, dialkylaminocarbonyl, dialkylaminocarbonylalkyl, haloalkyl and heterocyclylcarbonyl.

2.

A compound of claim 1, or a pharmaceutically acceptable enantiomer, diastereomer or salt thereof, wherein R1 is -NHSO2R7.

3.

A compound of claim 2, or a pharmaceutically acceptable enantiomer, diastereomer or salt thereof, wherein R 7 is cycloalkyl.

Four.

A compound of claim 1, 2 or 3, or a pharmaceutically acceptable enantiomer, diastereomer or salt thereof, wherein R2a and R2b are hydrogen.

5.

A compound of claim 1, 2, 3 or 4, or a pharmaceutically acceptable enantiomer, diastereomer or salt thereof, wherein Q is an unsaturated C6 chain that does not contain any heteroatom.

6. A compound of formula (II) of claim 1, image 1 or a pharmaceutically acceptable enantiomer, diastereomer or salt thereof, in which R1 is –NHSO2R7; R2a and R2b are hydrogen; R3 is selected from alkenyl, alkyl, aryl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, heterocyclyl and heterocyclylalkyl; R4 is –OR8; R5 is hydrogen; R6 is alkoxycarbonyl; R7 is selected from alkyl, aryl, cycloalkyl, (cycloalkyl) alkyl, dialkylaminocarbonyl, dialkylaminocarbonylalkyl, heterocyclyl, heterocyclylcarbonyl and NRaRb; wherein Ra and Rb are independently selected from hydrogen, alkoxy, alkyl, aryl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, haloalkyl, heterocyclyl and heterocyclylalkyl; R8 is selected from alkoxyalkyl, alkyl, alkylcarbonyl, arylalkyl, cycloalkyl, (cycloalkyl) alkyl, cycloalkylcarbonyl, haloalkoxyalkyl, haloalkyl, (NRcRd) carbonyl and -P (O) (OR ’) 2; wherein Rc and Rd are independently selected from hydrogen, alkyl and arylalkyl; or Rc and Rd, together with the nitrogen atom to which they are attached, form a five to six membered monocyclic heterocyclic ring that optionally contains an additional heteroatom selected from O, NRx and S; wherein Rx is selected from hydrogen and alkyl; and where R 'is selected from hydrogen and alkyl; Y Q is a C3–9 saturated or unsaturated chain, which optionally contains one to three heteroatoms independently selected from O, S (O) and NR9, where m is 0, 1 or 2 and R9 is selected from hydrogen, alkoxy, alkoxycarbonyl , alkyl, alkylcarbonyl, alkylsulfonyl, aminocarbonyl, arylsulfonyl, cycloalkyl, (cycloalkyl) alkyl, cycloalkyloxy, dialkylaminocarbonyl, dialkylaminocarbonylalkyl, haloalkyl and heterocyclylcarbonyl.

7.

A compound of claim 6, or a pharmaceutically acceptable enantiomer, diastereomer or salt thereof, wherein

 R7 is cycloalkyl; Y Q is an unsaturated C6 chain that does not contain any heteroatoms.

8.

A compound of claim 1 which is:

image 1 or a pharmaceutically acceptable enantiomer, diastereomer or salt thereof. 9. A composition comprising the compound of any one of claims 1 to 8, or a enantiomer, diastereomer or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

10.

The composition of claim 9, further comprising at least one additional compound having activity against HCV.

eleven.

The composition of claim 10, wherein at least one of the additional compounds is an interferon or a ribavirin.

12. The composition of claim 10, wherein at least one of the additional compounds is selected from interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of the type 1 helper T lymphocyte response, RNA of interference, antisense RNA, imiqimod, ribavirin, an inhibitor of inosine 5'-monophosphate dehydrogenase, amantadine and rimantadine. 13. A compound of any one of claims 1 to 8, or a pharmaceutically acceptable enantiomer, diastereomers or salt thereof for use in the treatment of an HCV infection. 14. The compound for use of claim 13 further comprising at least one additional compound having activity against HCV to be administered before, after, or simultaneously with, the compound of any one of claims 1 to 8 , or a pharmaceutically acceptable enantiomer, diastereomer or salt thereof. 15. The compound for use of claim 14, wherein at least one of the additional compounds is an interferon or a ribavirin.