Inhibitors of serine proteases

FIELD OF THE INVENTION

The present invention relates to compounds that inhibit serine protease activity, particularly the activity of hepatitis C virus NS3-NS4A protease. As such, they act by interfering with the life cycle of the hepatitis C virus and are also useful as antiviral agents. The invention further relates to compositions comprising these compounds either for ex vivo use or for administration to a patient suffering from HCV infection. The invention also relates to methods of treating an HCV infection in a patient by administering a composition comprising a compound of this invention.

BACKGROUND OF THE INVENTION

Infection by hepatitis C virus (“HCV”) is a compelling human medical problem. HCV is recognized as the causative agent for most cases of non-A, non-B hepatitis, with an estimated human sero-prevalence of 3% globally [A. Alberti et al., “Natural History of Hepatitis C,” J. Hepatology, 31 (Suppl. 1), pp. 17-24 (1999)]. Nearly four million individuals may be infected in the United States alone [M. J. Alter et al., “The Epidemiology of Viral Hepatitis in the United States,” Gastroenterol. Clin. North Am., 23, pp. 437-455 (1994); M. J. Alter “Hepatitis C Virus Infection in the United States,” J. Hepatology, 31., (Suppl. 1), pp. 88-91 (1999)].

Upon first exposure to HCV only about 20% of infected individuals develop acute clinical hepatitis while others appear to resolve the infection spontaneously. In almost 70% of instances, however; the virus establishes a chronic infection that persists for decades [S. Iwarson, “The Natural Course of Chronic Hepatitis,” FEMS Microbiology Reviews, 14, pp. 201-204 (1994); D. Lavanchy, “Global Surveillance and Control of Hepatitis C,” J. Viral Hepatitis, 6, pp. 35-47 (1999)]. This usually results in recurrent and progressively worsening liver inflammation, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma [M. C. Kew, “Hepatitis C and Hepatocellular Carcinoma”, FEMS Microbiology Reviews, 14, pp. 211-220 (1994); I. Saito et. al., “Hepatitis C Virus Infection is Associated with the Development of Hepatocellular Carcinoma,” Proc. Natl. Acad. Sci. USA, 87, pp. 6547-6549 (1990)]. Unfortunately, there are no broadly effective treatments for the debilitating progression of chronic HCV.

The HCV genome encodes a polyprotein of 3010-3033 amino acids [Q. L. Choo, et. al., “Genetic Organization and Diversity of the Hepatitis C Virus.” Proc. Natl. Acad. Sci. USA, 88, pp. 2451-2455 (1991); N. Kato et al., “Molecular Cloning of the Human Hepatitis C Virus Genome From Japanese Patients with Non-A, Non-B Hepatitis,” Proc. Natl. Acad. Sci. USA, 87, pp. 9524-9528 (1990); A. Takamizawa et. al., “Structure and Organization of the Hepatitis C Virus Genome Isolated From Human Carriers,” J. Virol., 65, pp. 1105-1113 (1991)]. The HCV nonstructural (NS) proteins are presumed to provide the essential catalytic machinery for viral replication. The NS proteins are derived by proteolytic cleavage of the polyprotein [R. Bartenschlager et. al., “Nonstructural Protein 3 of the Hepatitis C Virus Encodes a Serine-Type Proteinase Required for Cleavage at the NS3/4 and NS4/5 Junctions,” J. Virol., 67, pp. 3835-3844 (1993); A. Grakoui et. al., “Characterization of the Hepatitis C Virus-Encoded Serine Proteinase: Determination of Proteinase-Dependent Polyprotein Cleavage Sites,” J. Virol., 67, pp. 2832-2843 (1993); A. Grakoui et. al., “Expression and Identification of Hepatitis C Virus Polyprotein Cleavage Products,” J. Virol., 67, pp. 1385-1395 (1993); L. Tomei et. al., “NS3 is a serine protease required for processing of hepatitis C virus polyprotein”, J. Virol., 67, pp. 4017-4026 (1993)].

The HCV NS protein 3 (NS3) contains a serine protease activity that helps process the majority of the viral enzymes, and is thus considered essential for viral replication and infectivity. It is known that mutations in the yellow fever virus NS3 protease decrease viral infectivity [Chambers, T. J. et. al., “Evidence that the N-terminal Domain of Nonstructural Protein NS3 From Yellow Fever Virus is a Serine Protease Responsible for Site-Specific Cleavages in the Viral Polyprotein”, Proc. Natl. Acad. Sci. USA, 87, pp. 8898-8902 (1990)]. The first 181 amino acids of NS3 (residues 1027-1207 of the viral polyprotein) have been shown to contain the serine protease domain of NS3 that processes all four downstream sites of the HCV polyprotein [C. Lin et al., “Hepatitis C Virus NS3 Serine Proteinase: Trans-Cleavage Requirements and Processing Kinetics”, J. Virol., 68, pp. 8147-8157 (1994)].

The HCV NS3 serine protease and its associated cofactor, NS4A, helps process all of the viral enzymes, and is thus considered essential for viral replication. This processing appears to be analogous to that carried out by the human immunodeficiency virus aspartyl protease, which is also involved in viral enzyme processing. HIV protease inhibitors, which inhibit viral protein processing, are potent antiviral agents in man indicating that interrupting this stage of the viral life cycle results in therapeutically active agents. Consequently HCV NS3 serine protease is also an attractive target for drug discovery.

There are not currently any satisfactory anti-HCV agents or treatments. Until recently, the only established therapy for HCV disease was interferon treatment. However, interferons have significant side effects [M. A. Walker et al., “Hepatitis C Virus: An Overview of Current Approaches and Progress,” DDT, 4, pp. 518-29 (1999); D. Moradpour et al., “Current and Evolving Therapies for Hepatitis C,” Eur. J. Gastroenterol. Hepatol., 11, pp. 1199-1202 (1999); H. L. A. Janssen et al. “Suicide Associated with Alfa-Interferon Therapy for Chronic Viral Hepatitis,” J. Hepatol., 21, pp. 241-243 (1994); P. F. Renault et al., “Side Effects of Alpha Interferon,” Seminars in Liver Disease, 9, pp. 273-277. (1989)] and induce long term remission in only a fraction (≈25%) of cases [O. Weiland, “Interferon Therapy in Chronic Hepatitis C Virus Infection”, FEMS Microbiol. Rev., 14, pp. 279-288 (1994)]. Recent introductions of the pegylated forms of interferon (PEG-INTRON® and PEGASYS®) and the combination therapy of ribavirin and pegylated interferon (REBETROL®) have resulted in only modest improvements in remission rates and only partial reductions in side effects. Moreover, the prospects for effective anti-HCV vaccines remain uncertain.

Thus, there is a need for more effective anti-HCV therapies. Such inhibitors would have therapeutic potential as protease inhibitors, particularly as serine protease inhibitors, and more particularly as HCV NS3 protease inhibitors. Specifically, such compounds may be useful as antiviral agents, particularly as anti-HCV agents.

SUMMARY OF THE INVENTION

This invention relates to compounds of formula (I)

embedded image

or a pharmaceutically acceptable salt thereof wherein,

each A is —(CX₁X₂)_a—;

each B is —(CX₁X₂)_b—;

each X₁is independently hydrogen, halo, amino, sulfanyl, optionally substituted aliphatic, optionally substituted aryl, or —O—X_1A;

each X₂is independently hydrogen, halo, amino, sulfanyl, optionally substituted aliphatic, optionally substituted aryl, or —O—X_1B;

X_1Aand X_1Bare each independently an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;

or, X₁and X₂together form an oxo group;

each R₁is —Z^AR₄, wherein each Z^Ais independently a bond or an optionally substituted aliphatic wherein up to three carbon units of Z^Aare optionally and independently replaced by —C(O)—, —C(S)—, —C(O)NR^A—, —C(O)NR^ANR^A—, —C(O)O—, —NR^AC(O)O—, —O—, —NR^AC(O)NR^A—, —NR^ANR^A—, —S—, —SO—, —SO₂—, —NR^A—, —SO₂NR^A—, or —NR^ASO₂NR^A— provided that —NR^ANR^A—, —NR^AC(O)NR^A—, or —NR^ASO₂NR^A— is not directly bound to the nitrogen ring atom of formula (I);

each R₄is independently R^A, halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃;

each R^Ais independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;

each R₂is —Z^BR₅, wherein each Z^Bis independently a bond or an optionally substituted aliphatic wherein up to three carbon units of Z^Bare optionally and independently replaced by —C(O)—, —C(S)—, —C(O)NR^B—, —C(O)NR^BNR^B—, —C(O)O—, —NR^BC(O)O—, —NR^BC(O)NR^B—, —NR^BNR^B—, —S—, —SO—, —SO₂—, —NR^B—, —SO₂NR^B—, or —NR^BSO₂NR^B—, provided that —SO—, —SO₂—, or —SO₂NR^B— is not directly bound to the carbonyl of formula (I);

each R₅is independently R^B, halo, —OH, —CN, —NO₂, —NH₂, alkoxy, or haloalkoxy;

Each R^Bis independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;

or, R₁and R₂, together with the atoms to which they are attached, form an optionally substituted heterocycloaliphatic ring;

each R₃is an optionally substituted aliphatic, amino, sulfonyl, sulfanyl, sulfinyl, sulfonamide, sulfamide, sulfo, —O—R_3A, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;

each R_3Ais independently an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;

each Y and Y′ is independently —Z^DR₇, wherein each Z^Dis independently a bond or an optionally substituted straight or branched C_1-6aliphatic chain wherein up to two carbon units of Z^Dare optionally and independently replaced by —C(O)—, —C(S)—, —C(O)NR^D—, —C(O)NR^DNR^D—, —C(O)O—, —NR^DC(O)O—, —O—, —NR^DC(O)NR^D—, —NR^DNR^D—, —S—, —SO—, —SO₂—, —NR^D—, —SO₂NR^D—, —NR^DSO₂—, or —NR^DSO₂NR^D—, or Y and Y′ together form ═O or ═S;

each R₇is independently R^D, halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃;

each R^Dis independently hydrogen, or optionally substituted aryl; and

each of a and b is independently 0, 1, 2, or 3, provided that the sum of a and b is 2 or 3.

The invention also relates to compounds of formula (I) below.

embedded image

In this formula,

each A is —CH₂—;

each B is —CH₂—;

each R₁is —Z^AR₄, wherein each Z^Ais —C(O)—, —C(S)—, —C(O)NR^A—, —C(O)NR^ANR^A—, —C(O)O—, —NR^AC(O)O—, —O—, —S—, —SO—, —SO₂—, —NR^A—, or —SO₂NR^A—;

each R₄is independently R^A, halo, —OH, —CN, —NO₂, —NH₂, alkoxy, or haloalkoxy;

each R₂is —Z^BR₅, wherein each Z^Bis independently a bond or an optionally substituted aliphatic wherein up to four carbon units of Z^Bare optionally and independently replaced by —C(O)—, —C(S)—, —C(O)NR^B—, —C(O)NR^BNR^B—, —C(O)O—, —C(O)C(O)—NR^B—, —NR^BC(O)O—, —NR^BC(O)NR^B—, —NR^BNR^B—, —S—, —SO—, —SO₂—, —NR^B—, —SO₂NR^B—, or —NR^BSO₂NR^B—, provided that —SO—, —SO₂—, or —SO₂NR^B— is not directly bound to the carbonyl in formula (I);

each R₅is independently R^B, halo, —OH, —CN, —NO₂, —NH₂, alkoxy, or haloalkoxy;

each R^Bis independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;

or, R₁and R₂, together with the atoms to which they are attached, form an optionally substituted heterocycloaliphatic ring;

each of Y and Y′ is H.

In some aspects, the invention features a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in an amount effective to inhibit a serine protease; and an acceptable carrier, adjuvant or vehicle. The composition may include an additional agent selected from an immunomodulatory agent; an antiviral agent; a second inhibitor of HCV protease; an inhibitor of another target in the HCV life cycle; and a cytochrome P-450 inhibitor; or combinations thereof. The immunomodulatory agent is α-, β-, or γ-interferon or thymosin; said antiviral agent is ribavirin, amantadine, or telbivudine; or said inhibitor of another target in the HCV life cycle is an inhibitor of HCV helicase, polymerase, or metalloprotease. Cytochrome P-450 inhibitor may be ritonavir.

In other aspects, a method of inhibiting the activity of a serine protease comprising the step of contacting said serine protease with a compound of formula (I). The serine protease may be an HCV NS3 protease. The methods also include treating an HCV infection in a patient by administering a compound of formula (I). The method may also include administering to said patient an additional agent selected from an immunomodulatory agent; an antiviral agent; a second inhibitor of HCV protease; an inhibitor of another target in the HCV life cycle; or combinations thereof; wherein said additional agent is administered to said patient in the same dosage form as the serine protease inhibitor or as a separate dosage form. The immunomodulatory agent is α-, β-, or γ-interferon or thymosin; said antiviral agent is ribavarin or amantadine; or said inhibitor of another target in the HCV life cycle is an inhibitor of HCV helicase, polymerase, or metalloprotease.

In still other aspects, a method of eliminating or reducing HCV contamination of a biological sample or medical or laboratory equipment includes the step of contacting said biological sample or medical or laboratory equipment with a compound of formula (I). The sample or equipment may be selected from blood, other body fluids, biological tissue, a surgical instrument, a surgical garment, a laboratory instrument, a laboratory garment, a blood or other body fluid collection apparatus; a blood or other body fluid storage material.

The compounds of the invention, as described herein, also exhibit advantageous PK properties and/or increased potency.

The invention also relates to compositions that comprise the above compounds and the use thereof; methods of preparing compounds of formula (I), and methods of assaying compounds for serine protease activity. Such compositions may be used to pre-treat devices that are to be inserted into a patient, to treat biological samples, and for direct administration to a patient. In each case, the composition will be used to lessen the risk of or the severity of the HCV infection.

DEFINITIONS

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

As described herein, compounds of the invention may optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention.

As used herein the term “aliphatic” encompasses the terms alkyl, alkenyl, alkynyl, each of which being optionally substituted as set forth below.

As used herein, an “alkyl” group refers to a saturated aliphatic hydrocarbon group containing 1 to 12 (e.g., 1 to 10, 1 to 8, 1 to 6, or 1 to 4) carbon atoms. An alkyl group can be straight or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or 2-ethylhexyl. An alkyl group can be substituted (i.e., optionally substituted) with one or more substituents such as halo, phospho, cycloaliphatic (e.g., cycloalkyl or cycloalkenyl), heterocycloaliphatic (e.g., heterocycloalkyl or heterocycloalkenyl), aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, acyl (e.g., (aliphatic)carbonyl, (cycloaliphatic)carbonyl, or (heterocycloaliphatic)carbonyl), nitro, cyano, amido (e.g., (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl, or heteroarylaminocarbonyl), amino (e.g., aliphaticamino, cycloaliphaticamino, or heterocycloaliphaticamino), sulfonyl (e.g., aliphatic-SO₂—), sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkoxy, alkoxycarbonyl, alkylcarbonyloxy, or hydroxy. Without limitation, some examples of substituted alkyls include carboxyalkyl (such as HOOC-alkyl, alkoxycarbonylalkyl, and alkylcarbonyloxyalkyl), cyanoalkyl, hydroxyalkyl, alkoxyalkyl, acylalkyl, aralkyl, (alkoxyaryl)alkyl, (sulfonylamino)alkyl (such as (alkyl-SO₂-amino)alkyl), aminoalkyl, amidoalkyl, (cycloaliphatic)alkyl, or haloalkyl.

As used herein, an “alkenyl” group refers to an aliphatic carbon group that contains 2 to 12 (e.g., 2 to 8, 2 to 6, or 2 to 4) carbon atoms and at least one double bond. Like an alkyl group, an alkenyl group can be straight or branched. Examples of an alkenyl group include, but are not limited to, allyl, isoprenyl, 2-butenyl, and 2-hexenyl. An alkenyl group can be optionally substituted with one or more substituents such as halo, phospho, cycloaliphatic (e.g., cycloalkyl or cycloalkenyl), heterocycloaliphatic (e.g., heterocycloalkyl or heterocycloalkenyl), aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, acyl (e.g., (aliphatic)carbonyl, (cycloaliphatic)carbonyl, or (heterocycloaliphatic)carbonyl), nitro, cyano, amido (e.g., (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl, or heteroarylaminocarbonyl), amino (e.g., aliphaticamino, cycloaliphaticamino, heterocycloaliphaticamino, or aliphaticsulfonylamino), sulfonyl (e.g., alkyl-SO₂—, cycloaliphatic-SO₂—, or aryl-SO₂—), sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkoxy, alkoxycarbonyl, alkylcarbonyloxy, or hydroxy. Without limitation, some examples of substituted alkenyls include cyanoalkenyl, alkoxyalkenyl, acylalkenyl, hydroxyalkenyl, aralkenyl, (alkoxyaryl)alkenyl, (sulfonylamino)alkenyl (such as (alkyl-SO₂-amino)alkenyl), aminoalkenyl, amidoalkenyl, (cycloaliphatic)alkenyl, or haloalkenyl.

As used herein, an “alkynyl” group refers to an aliphatic carbon group that contains 2 to 12 (e.g., 2 to 8, 2 to 6, or 2 to 4) carbon atoms and has at least one triple bond. An alkynyl group can be straight or branched. Examples of an alkynyl group include, but are not limited to, propargyl and butynyl. An alkynyl group can be optionally substituted with one or more substituents such as aroyl, heteroaroyl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, nitro, carboxy, cyano, halo, hydroxy, sulfo, mercapto, sulfanyl (e.g., aliphaticsulfanyl or cycloaliphaticsulfanyl), sulfinyl (e.g., aliphaticsulfinyl or cycloaliphaticsulfinyl), sulfonyl (e.g., aliphatic-SO₂—, aliphaticamino-SO₂—, or cycloaliphatic-SO₂—), amido (e.g., aminocarbonyl, alkylaminocarbonyl, alkylcarbonylamino, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, cycloalkylcarbonylamino, arylaminocarbonyl, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (cycloalkylalkyl)carbonylamino, heteroaralkylcarbonylamino, heteroarylcarbonylamino or heteroarylaminocarbonyl), urea, thiourea, sulfamoyl, sulfamide, alkoxycarbonyl, alkylcarbonyloxy, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, acyl (e.g., (cycloaliphatic)carbonyl or (heterocycloaliphatic)carbonyl), amino (e.g., aliphaticamino), sulfoxy, oxo, carboxy, carbamoyl, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, or (heteroaryl)alkoxy.

As used herein, an “amido” encompasses both “aminocarbonyl” and “carbonylamino”. These terms when used alone or in connection with another group refers to an amido group such as —N(R^X)—C(O)—R^Yor —C(O)—N(R^X)₂, when used terminally, and —C(O)—N(R^X)— or —N(R^X)—C(O)— when used internally, wherein R^Xand R^Yare defined below. Examples of amido groups include alkylamido (such as alkylcarbonylamino or alkylaminocarbonyl), (heterocycloaliphatic)amido, (heteroaralkyl)amido, (heteroaryl)amido, (heterocycloalkyl)alkylamido, arylamido, aralkylamido, (cycloalkyl)alkylamido, or cycloalkylamido.

As used herein, an “amino” group refers to —NR^XR^Ywherein each of R^Xand R^Yis independently hydrogen, aliphatic, cycloaliphatic, (cycloaliphatic)aliphatic, aryl, araliphatic, heterocycloaliphatic, (heterocycloaliphatic)aliphatic, heteroaryl, carboxy, sulfanyl, sulfinyl, sulfonyl, (aliphatic)carbonyl, (cycloaliphatic)carbonyl, ((cycloaliphatic)aliphatic)carbonyl, arylcarbonyl, (araliphatic)carbonyl, (heterocycloaliphatic)carbonyl, ((heterocycloaliphatic)aliphatic)carbonyl, (heteroaryl)carbonyl, or (heteroaraliphatic)carbonyl, each of which being defined herein and being optionally substituted. Examples of amino groups include alkylamino, dialkylamino, or arylamino. When the term “amino” is not the terminal group (e.g., alkylcarbonylamino), it is represented by —NR^X—. R^Xhas the same meaning as defined above.

As used herein, an “aryl” group used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl” refers to monocyclic (e.g., phenyl); bicyclic (e.g., indenyl, naphthalenyl, tetrahydronaphthyl, tetrahydroindenyl); and tricyclic (e.g., fluorenyl tetrahydrofluorenyl, or tetrahydroanthracenyl, anthracenyl) ring systems in which the monocyclic ring system is aromatic or at least one of the rings in a bicyclic or tricyclic ring system is aromatic. The bicyclic and tricyclic groups include benzofused 2-3 membered carbocyclic rings. For example, a benzofused group includes phenyl fused with two or more C_4-8carbocyclic moieties. An aryl is optionally substituted with one or more substituents including aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic; heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; oxo (on a non-aromatic carbocyclic ring of a benzofused bicyclic or tricyclic aryl); nitro; carboxy; amido; acyl [e.g., (aliphatic)carbonyl, (cycloaliphatic)carbonyl, ((cycloaliphatic)aliphatic)carbonyl, (araliphatic)carbonyl, (heterocycloaliphatic)carbonyl, ((heterocycloaliphatic)aliphatic)carbonyl, or (heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphatic-SO₂— or amino-SO₂—]; sulfinyl [e.g., aliphatic-S(O)— or cycloaliphatic-S(O)—]; sulfanyl [e.g., aliphatic-S—]; cyano; halo; hydroxy; mercapto; sulfoxy; urea; thiourea; sulfamoyl; sulfamide; or carbamoyl. Alternatively, an aryl can be unsubstituted.

Non-limiting examples of substituted aryls include haloaryl [e.g., mono-, di-(such as p,m-dihaloaryl), and tri-(such as halo) aryl]; (carboxy)aryl [e.g., (alkoxycarbonyl)aryl, ((aralkyl)carbonyloxy)aryl, and (alkoxycarbonyl)aryl]; (amido)aryl [e.g., (aminocarbonyl)aryl, (((alkylamino)alkyl)aminocarbonyl)aryl, (alkylcarbonyl)aminoaryl, (arylaminocarbonyl)aryl, and (((heteroaryl)amino)carbonyl)aryl]; aminoaryl [e.g., ((alkylsulfonyl)amino)aryl or ((dialkyl)amino)aryl]; (cyanoalkyl)aryl; (alkoxy)aryl; (sulfamoyl)aryl [e.g., (aminosulfonyl)aryl]; (alkylsulfonyl)aryl; (cyano)aryl; (hydroxyalkyl)aryl; ((alkoxy)alkyl)aryl; (hydroxy)aryl, ((carboxy)alkyl)aryl; (((dialkyl)amino)alkyl)aryl; (nitroalkyl)aryl; (((alkylsulfonyl)amino)alkyl)aryl; ((heterocycloaliphatic)carbonyl)aryl; ((alkylsulfonyl)alkyl)aryl; (cyanoalkyl)aryl; (hydroxyalkyl)aryl; (alkylcarbonyl)aryl; alkylaryl; (trihaloalkyl)aryl; p-amino-m-alkoxycarbonylaryl; p-amino-m-cyanoaryl; p-halo-m-aminoaryl; or (m-(heterocycloaliphatic)-o-(alkyl))aryl.

As used herein, an “araliphatic” such as an “aralkyl” group refers to an aliphatic group (e.g., a C_1-4alkyl group) that is substituted with an aryl group. “Aliphatic,” “alkyl,” and “aryl” are defined herein. An example of an araliphatic such as an aralkyl group is benzyl.

As used herein, an “aralkyl” group refers to an alkyl group (e.g., a C_1-4alkyl group) that is substituted with an aryl group. Both “alkyl” and “aryl” have been defined above. An example of an aralkyl group is benzyl. An aralkyl is optionally substituted with one or more substituents such as aliphatic (e.g., alkyl, alkenyl, or alkynyl, including carboxyalkyl, hydroxyalkyl, or haloalkyl such as trifluoromethyl); cycloaliphatic (e.g., cycloalkyl or cycloalkenyl); (cycloalkyl)alkyl; heterocycloalkyl; (heterocycloalkyl)alkyl; aryl; heteroaryl; alkoxy; cycloalkyloxy; heterocycloalkyloxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; aroyl; heteroaroyl; nitro; carboxy; alkoxycarbonyl; alkylcarbonyloxy; amido (e.g., aminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, or heteroaralkylcarbonylamino); cyano; halo; hydroxy; acyl; mercapto; alkylsulfanyl; sulfoxy; urea; thiourea; sulfamoyl; sulfamide; oxo; or carbamoyl.

As used herein, a “bicyclic ring system” includes 8- to 12- (e.g., 9, 10, or 11) membered structures that form two rings, wherein the two rings have at least one atom in common (e.g., 1 atom or 2 atoms in common). Bicyclic ring systems include bicycloaliphatics (e.g., bicycloalkyl or bicycloalkenyl), bicycloheteroaliphatics, bicyclic aryls, and bicyclic heteroaryls.

As used herein, a “cycloaliphatic” group encompasses a “cycloalkyl” group and a “cycloalkenyl” group, each of which being optionally substituted as set forth below.

As used herein, a “cycloalkyl” group refers to a saturated carbocyclic mono- or bicyclic (fused or bridged) ring of 3-12 (e.g., 5-12) carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, norbornyl, cubyl, octahydro-indenyl, decahydro-naphthyl, spiro[5.5]undecanyl, spiro[2.5]octanyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2.]decyl, bicyclo[2.2.2]octyl, adamantyl, or ((aminocarbonyl)cycloalkyl)cycloalkyl.

A “cycloalkenyl” group, as used herein, refers to a non-aromatic carbocyclic mono- or bicyclic ring of 3 to 12 (e.g., 4 to 8) carbon atoms having one or more double bonds. Examples of cycloalkenyl groups include cyclopentenyl, 1,4-cyclohexa-di-enyl, cycloheptenyl, cyclooctenyl, hexahydro-indenyl, octahydro-naphthyl, cyclohexenyl, cyclopentenyl, spiro[5.5]undec-3-enyl, spiro[2.5]oct-5-enyl, bicyclo[2.2.2]octenyl, or bicyclo[3.3.1]nonenyl.

A cycloalkyl or cycloalkenyl group can be optionally substituted with one or more substituents such as phosphor; aliphatic (e.g., alkyl, alkenyl, or alkynyl); cycloaliphatic; (cycloaliphatic) aliphatic; heterocycloaliphatic; (heterocycloaliphatic) aliphatic; aryl; heteroaryl; alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; amido (e.g., (aliphatic)carbonylamino, (cycloaliphatic)carbonylamino, ((cycloaliphatic)aliphatic)carbonylamino, (aryl)carbonylamino, (araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino, ((heterocycloaliphatic)aliphatic)carbonylamino, (heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylamino); nitro; carboxy (e.g., HOOC—, alkoxycarbonyl, or alkylcarbonyloxy); acyl (e.g., (cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl, (araliphatic)carbonyl, (heterocycloaliphatic)carbonyl, ((heterocycloaliphatic)aliphatic)carbonyl, or (heteroaraliphatic)carbonyl); cyano; halo; hydroxy; mercapto; sulfonyl (e.g., alkyl-SO₂— and aryl-SO₂—); sulfinyl (e.g., alkyl-S(O)—); sulfanyl (e.g., alkyl-S—); sulfoxy; urea; thiourea; sulfamoyl; sulfamide; oxo; or carbamoyl.

As used herein, the term “heterocycloaliphatic” encompasses a heterocycloalkyl group and a heterocycloalkenyl group, each of which being optionally substituted as set forth below.

As used herein, a “heterocycloalkyl” group refers to a 3-12 membered mono- or bicylic (fused or bridged) (e.g., 5- to 12-membered mono- or bicyclic) saturated ring structure, in which one or more of the ring atoms is a heteroatom (e.g., N, O, S, or combinations thereof). Examples of a heterocycloalkyl group include piperidyl, piperazyl, tetrahydropyranyl, tetrahydrofuryl, 1,4-dioxolanyl, 1,4-dithianyl, 1,3-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholyl, octahydrobenzofuryl, octahydrochromenyl, octahydrothiochromenyl, octahydroindolyl, octahydropyrindinyl, decahydroquinolinyl, octahydrobenzo[b]thiopheneyl, 2-oxa-bicyclo[2.2.2]octyl, 1-aza-bicyclo[2.2.2]octyl, 3-aza-bicyclo[3.2.1]octyl, 3-oxaspiro[5.5]undec-3-enyl, 6-oxaspiro[2.5]oct-5-enyl, and 2,6-dioxa-tricyclo[3.3.1.0^3,7]nonyl.

A “heterocycloalkenyl” group, as used herein, refers to a mono- or bicylic (e.g., 5- to 10-membered mono- or bicyclic) non-aromatic ring structure having one or more double bonds, and wherein one or more of the ring atoms is a heteroatom (e.g., N, O, or S).

Monocyclic and bicycloheteroaliphatics are numbered according to standard chemical nomenclature. A heterocycloalkyl or heterocycloalkenyl group can be optionally substituted with one or more substituents such as phosphor; aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic; heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; amido [e.g., (aliphatic)carbonylamino, (cycloaliphatic)carbonylamino, ((cycloaliphatic) aliphatic)carbonylamino, (aryl)carbonylamino, (araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino, ((heterocycloaliphatic) aliphatic)carbonylamino, (heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylamino]; nitro; carboxy [e.g., HOOC—, alkoxycarbonyl, or alkylcarbonyloxy]; acyl [e.g., (cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl, (araliphatic)carbonyl, (heterocycloaliphatic)carbonyl, ((heterocycloaliphatic)aliphatic)carbonyl, or (heteroaraliphatic)carbonyl]; nitro; cyano; halo; hydroxy; mercapto; sulfonyl [e.g., alkylsulfonyl or arylsulfonyl]; sulfinyl [e.g., alkylsulfinyl]; sulfanyl [e.g., alkylsulfanyl]; sulfoxy; urea; thiourea; sulfamoyl; sulfamide; oxo; or carbamoyl.

A “heteroaryl” group, as used herein, refers to a monocyclic, bicyclic, or tricyclic ring system having 4 to 15 ring atoms wherein one or more of the ring atoms is a heteroatom (e.g., N, O, S, or combinations thereof) and in which the monocyclic ring system is aromatic or at least one of the rings in the bicyclic or tricyclic ring systems is aromatic. A heteroaryl group includes a benzofused ring system having 2 to 3 rings. For example, a benzofused group includes benzo fused with one or two 4 to 8 membered heterocycloaliphatic moieties (e.g., indolizyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl, benzo[b]thiophenyl, quinolinyl, or isoquinolinyl). Some examples of heteroaryl are azetidinyl, pyridyl, 1H-indazolyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl, isoquinolinyl, benzthiazolyl, xanthene, thioxanthene, phenothiazine, dihydroindole, 1,2,3,4-tetrahydroisoquinoline, isoindoline, benzo[1,3]dioxole, benzo[b]furyl, benzo[b]thiophenyl, indazolyl, benzimidazolyl, benzthiazolyl, puryl, cinnolyl, quinolyl, quinazolyl, cinnolyl, phthalazyl, quinazolyl, quinoxalyl, isoquinolyl, 4H-quinolizyl, benzo-1,2,5-thiadiazolyl, or 1,8-naphthyridyl.

Without limitation, monocyclic heteroaryls include furyl, thiophenyl, 2H-pyrrolyl, pyrrolyl, oxazolyl, thazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4-H-pranyl, pyridyl, pyridazyl, pyrimidyl, pyrazolyl, pyrazyl, or 1,3,5-triazyl. Monocyclic heteroaryls are numbered according to standard chemical nomenclature.

Without limitation, bicyclic heteroaryls include indolizyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl, benzo[b]thiophenyl, quinolinyl, isoquinolinyl, indolizyl, isoindolyl, indolyl, benzo[b]furyl, bexo[b]thiophenyl, indazolyl, benzimidazyl, benzthiazolyl, purinyl, 4H-quinolizyl, quinolyl, isoquinolyl, cinnolyl, phthalazyl, quinazolyl, quinoxalyl, 1,8-naphthyridyl, or pteridyl. Bicyclic heteroaryls are numbered according to standard chemical nomenclature.

A heteroaryl is optionally substituted with one or more substituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic; heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; oxo (on a non-aromatic carbocyclic or heterocyclic ring of a bicyclic or tricyclic heteroaryl); carboxy; amido; acyl [e.g., aliphaticcarbonyl; (cycloaliphatic)carbonyl; ((cycloaliphatic)aliphatic)carbonyl; (araliphatic)carbonyl; (heterocycloaliphatic)carbonyl; ((heterocycloaliphatic)aliphatic)carbonyl; or (heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl or aminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl]; sulfanyl [e.g., aliphaticsulfanyl]; nitro; cyano; halo; hydroxy; mercapto; sulfoxy; urea; thiourea; sulfamoyl; sulfamide; or carbamoyl. Alternatively, a heteroaryl can be unsubstituted.

Non-limiting examples of substituted heteroaryls include (halo)heteroaryl [e.g., mono- and di-(halo)heteroaryl]; (carboxy)heteroaryl [e.g., (alkoxycarbonyl)heteroaryl]; cyanoheteroaryl; aminoheteroaryl [e.g., ((alkylsulfonyl)amino)heteroaryl and ((dialkyl)amino)heteroaryl]; (amido)heteroaryl [e.g., aminocarbonylheteroaryl, ((alkylcarbonyl)amino)heteroaryl, ((((alkyl)amino)alkyl)aminocarbonyl)heteroaryl, (((heteroaryl)amino)carbonyl)heteroaryl, ((heterocycloaliphatic)carbonyl)heteroaryl, and ((alkylcarbonyl)amino)heteroaryl]; (cyanoalkyl)heteroaryl; (alkoxy)heteroaryl; (sulfamoyl)heteroaryl [e.g., (aminosulfonyl)heteroaryl]; (sulfonyl)heteroaryl [e.g., (alkylsulfonyl)heteroaryl]; (hydroxyalkyl)heteroaryl; (alkoxyalkyl)heteroaryl; (hydroxy)heteroaryl; ((carboxy)alkyl)heteroaryl; (((dialkyl)amino)alkyl)heteroaryl; (heterocycloaliphatic)heteroaryl; (cycloaliphatic)heteroaryl; (nitroalkyl)heteroaryl; (((alkylsulfonyl)amino)alkyl)heteroaryl; ((alkylsulfonyl)alkyl)heteroaryl; (cyanoalkyl)heteroaryl; (acyl)heteroaryl [e.g., (alkylcarbonyl)heteroaryl]; (alkyl)heteroaryl, and (haloalkyl)heteroaryl [e.g., trihaloalkylheteroaryl].

A “heteroaraliphatic” (such as a heteroaralkyl group) as used herein, refers to an aliphatic group (e.g., a C_1-4alkyl group) that is substituted with a heteroaryl group. “Aliphatic,” “alkyl,” and “heteroaryl” have been defined above.

A “heteroaralkyl” group, as used herein, refers to an alkyl group (e.g., a C_1-4alkyl group) that is substituted with a heteroaryl group. Both “alkyl” and “heteroaryl” have been defined above. A heteroaralkyl is optionally substituted with one or more substituents such as alkyl (including carboxyalkyl, hydroxyalkyl, and haloalkyl such as trifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo, hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, an “acyl” group refers to a formyl group or R^X—C(O)— (such as alkyl-C(O)—, also referred to as “alkylcarbonyl”) where R^Xand “alkyl” have been defined previously. Acetyl and pivaloyl are examples of acyl groups.

As used herein, an “aroyl” or “heteroaroyl” refers to an aryl-C(O)— or a heteroaryl-C(O)—. The aryl and heteroaryl portion of the aroyl or heteroaroyl is optionally substituted as previously defined.

As used herein, an “alkoxy” group refers to an alkyl-O— group where “alkyl” has been defined previously.

As used herein, a “carbamoyl” group refers to a group having the structure —O—CO—NR^XR^Yor —NR^X—CO—O—R^zwherein R^Xand R^Yhave been defined above and R^Zcan be aliphatic, aryl, araliphatic, heterocycloaliphatic, heteroaryl, or heteroaraliphatic.

As used herein, a “carboxy” group refers to —COOH, —COOR^X, —OC(O)H, —OC(O)R^Xwhen used as a terminal group; or —OC(O)— or —C(O)O— when used as an internal group.

As used herein, a “haloaliphatic” group refers to an aliphatic group substituted with 1-3 halogen. For instance, the term haloalkyl includes the group —CF₃.

As used herein, a “mercapto” group refers to —SH.

As used herein, a “sulfo” group refers to —SO₃H or —SO₃R^Xwhen used terminally or —S(O)₃— when used internally.

As used herein, a “sulfamide” group refers to the structure —NR^X—S(O)₂—NR^YR^zwhen used terminally and —NR^X—S(O)₂—NR^Y— when used internally, wherein R^X, R^Y, and R^Zhave been defined above.

As used herein, a “sulfonamide” group refers to the structure —S(O)₂—NR^XR^Yor —NR^X—S(O)₂—R^Zwhen used terminally; or —S(O)₂—NR^X— or —NR^X—S(O)₂— when used internally, wherein R^X, R^Y, and R^Zare defined above.

As used herein a “sulfanyl” group refers to —S—R^Xwhen used terminally and —S— when used internally, wherein R^Xhas been defined above. Examples of sulfanyls include aliphatic-S—, cycloaliphatic-S—, aryl-S—, or the like.

As used herein a “sulfinyl” group refers to —S(O)—R^Xwhen used terminally and —S(O)— when used internally, wherein R^Xhas been defined above. Exemplary sulfinyl groups include aliphatic-S(O)—, aryl-S(O)—, (cycloaliphatic(aliphatic))-S(O)—, cycloalkyl-S(O)—, heterocycloaliphatic-S(O)—, heteroaryl-S(O)—, or the like.

As used herein, a “sulfonyl” group refers to —S(O)₂—R^Xwhen used terminally and —S(O)₂— when used internally, wherein R^Xhas been defined above. Exemplary sulfonyl groups include aliphatic-S(O)₂—, aryl-S(O)₂—, (cycloaliphatic(aliphatic))-S(O)₂—, cycloaliphatic-S(O)₂—, heterocycloaliphatic-S(O)₂—, heteroaryl-S(O)₂—, (cycloaliphatic(amido(aliphatic)))-S(O)₂— or the like.

As used herein, a “sulfoxy” group refers to —O—SO—R^Xor —SO—O—R^Xwhen used terminally and —O—S(O)— or —S(O)—O— when used internally, where R^Xhas been defined above.

As used herein, a “halogen” or “halo” group refers to fluorine, chlorine, bromine or iodine.

As used herein, an “alkoxycarbonyl,” which is encompassed by the term carboxy, used alone or in connection with another group refers to a group such as alkyl-O—C(O)—.

As used herein, an “alkoxyalkyl” refers to an alkyl group such as alkyl-O-alkyl-, wherein alkyl has been defined above.

As used herein, a “carbonyl” refer to —C(O)—.

As used herein, an “oxo” refers to ═O.

As used herein, the term “phospho” refers to phosphinates and phosphonates. Examples of phosphinates and phosphonates include —P(O)(R^P)₂, wherein R^Pis aliphatic, alkoxy, aryloxy, heteroaryloxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy aryl, heteroaryl, cycloaliphatic or amino.

As used herein, an “aminoalkyl” refers to the structure (R^X)₂N-alkyl-.

As used herein, a “cyanoalkyl” refers to the structure (NC)-alkyl-.

As used herein, a “urea” group refers to the structure —NR^X—CO—NR^YR^zand a “thiourea” group refers to the structure —NR^X—CS—NR^YR^zwhen used terminally and —NR^X—CO—NR^Y— or —NR^X—CS—NR^Y— when used internally, wherein R^X, R^Y, and R^Zhave been defined above.

As used herein, a “guanidine” group refers to the structure —N═C(N(R^XR^Y))N(R^XR^Y) or —NR^X—C(═NR^X)NR^XR^Ywherein R^Xand R^Yhave been defined above.

As used herein, the term “amidino” group refers to the structure —C═(NR^X)N(R^XR^Y) wherein R^Xand R^Yhave been defined above.

In general, the term “vicinal” refers to the placement of substituents on a group that includes two or more carbon atoms, wherein the substituents are attached to adjacent carbon atoms.

In general, the term “geminal” refers to the placement of substituents on a group that includes two or more carbon atoms, wherein the substituents are attached to the same carbon atom.

The terms “terminally” and “internally” refer to the location of a group within a substituent. A group is terminal when the group is present at the end of the substituent not further bonded to the rest of the chemical structure. Carboxyalkyl, i.e., R^XO(O)C-alkyl is an example of a carboxy group used terminally. A group is internal when the group is present in the middle of a substituent of the chemical structure. Alkylcarboxy (e.g., alkyl-C(O)O— or alkyl-OC(O)—) and alkylcarboxyaryl (e.g., alkyl-C(O)O-aryl- or alkyl-O(CO)-aryl-) are examples of carboxy groups used internally.

As used herein, “cyclic group” or “cyclic moiety” includes mono-, bi-, and tri-cyclic ring systems including cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl, each of which has been previously defined.

As used herein, a “bridged bicyclic ring system” refers to a bicyclic heterocycloalipahtic ring system or bicyclic cycloaliphatic ring system in which the rings are bridged. Examples of bridged bicyclic ring systems include, but are not limited to, adamantanyl, norbornanyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.2.3]nonyl, 2-oxabicyclo[2.2.2]octyl, 1-azabicyclo[2.2.2]octyl, 3-azabicyclo[3.2.1]octyl, and 2,6-dioxa-tricyclo[3.3.1.0^3,7]nonyl. A bridged bicyclic ring system can be optionally substituted with one or more substituents such as alkyl (including carboxyalkyl, hydroxyalkyl, and haloalkyl such as trifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo, hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, an “aliphatic chain” refers to a branched or straight aliphatic group (e.g., alkyl groups, alkenyl groups, or alkynyl groups). A straight aliphatic chain has the structure —[CH₂]_v—, where v is 1-6. A branched aliphatic chain is a straight aliphatic chain that is substituted with one or more aliphatic groups. A branched aliphatic chain has the structure —[CHQ]_v- where Q is hydrogen or an aliphatic group; however, Q shall be an aliphatic group in at least one instance. The term aliphatic chain includes alkyl chains, alkenyl chains, and alkynyl chains, where alkyl, alkenyl, and alkynyl are defined above.

The phrase “optionally substituted” is used interchangeably with the phrase “substituted or unsubstituted.” As described herein, compounds of the invention can optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention. As described herein, the variables A, B, R₁, R₂, R₃, Y and Y′, and other variables contained in formulae described herein encompass specific groups, such as alkyl and aryl. Unless otherwise noted, each of the specific groups for the variables A, B, R₁, R₂, R₃, Y and Y′, and other variables contained therein can be optionally substituted with one or more substituents described herein. Each substituent of a specific group is further optionally substituted with one to three of halo, cyano, oxo, alkoxy, hydroxy, amino, nitro, aryl, cycloaliphatic, heterocycloaliphatic, heteroaryl, haloalkyl, and alkyl. For instance, an alkyl group can be substituted with alkylsulfanyl and the alkylsulfanyl can be optionally substituted with one to three of halo, cyano, oxo, alkoxy, hydroxy, amino, nitro, aryl, haloalkyl, and alkyl. As an additional example, the cycloalkyl portion of a (cycloalkyl)carbonylamino can be optionally substituted with one to three of halo, cyano, alkoxy, hydroxy, nitro, haloalkyl, and alkyl. When two alkoxy groups are bound to the same atom or adjacent atoms, the two alkoxy groups can form a ring together with the atom(s) to which they are bound.

In general, the term “substituted,” whether preceded by the term “optionally” or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. Specific substituents are described above in the definitions and below in the description of compounds and examples thereof. Unless otherwise indicated, an optionally substituted group can have a substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent can be either the same or different at every position. A ring substituent, such as a heterocycloalkyl, can be bound to another ring, such as a cycloalkyl, to form a spiro-bicyclic ring system, e.g., both rings share one common atom. As one of ordinary skill in the art will recognize, combinations of substituents envisioned by this invention are those combinations that result in the formation of stable or chemically feasible compounds.

The phrase “stable or chemically feasible,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 40° C. or less, in the absence of moisture or other chemically reactive conditions, for at least a week.

As used herein, an effective amount is defined as the amount required to confer a therapeutic effect on the treated patient, and is typically determined based on age, surface area, weight, and condition of the patient. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep., 50: 219 (1966). Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970). As used herein, “patient” refers to a mammal, including a human.

Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. Such compounds are useful, for example, as analytical tools or probes in biological assays, or as therapeutic agents.

In other aspects, the invention features certain compounds as described generically and specifically below. Such specific descriptions are illustrative only and are not meant to limit scope of the compounds or uses thereof.

DETAILED DESCRIPTION
I. Compounds

A. Generic Compounds

In some aspects, the invention provides compounds of formula (I) useful for inhibiting serine protease activity and methods of inhibiting serine protease activity. Compounds of formula (I) include:

embedded image

or a pharmaceutically acceptable salt thereof, wherein,

each A is —(CX₁X₂)_a—;

each B is —(CX₁X₂)_b—;

each X₁is independently hydrogen, halo, amino, sulfanyl, optionally substituted C_1-4aliphatic, optionally substituted aryl, or —O—X_1A;

each X₂is independently hydrogen, halo, amino, sulfanyl, optionally substituted C_1-4aliphatic, optionally substituted aryl, or —O—X_1B;

or, X₁and X₂together form an oxo group;

each R₁is —Z^AR₄, wherein each Z^Ais independently a bond or an optionally substituted branched or straight C_1-12aliphatic chain wherein up to three carbon units of Z^Aare optionally and independently replaced by —C(O)—, —C(S)—, —C(O)NR^A—, —C(O)NR^ANR^A—, —C(O)O—, —NR^AC(O)O—, —O—, —NR^AC(O)NR^A—, —NR^ANR^A—, —S—, —SO—, —SO₂—, —NR^A—, —SO₂NR^A—, or —NR^ASO₂NR^A— provided that —NR^ANR^A—, —NR^AC(O)NR^A—, or —NR^ASO₂NR^A— is not directly bound to the nitrogen ring atom of formula (I);

each R₄is independently R^A, halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃;

each R₂is —Z^BR₅, wherein each Z^Bis independently a bond or an optionally substituted branched or straight C_1-12aliphatic chain wherein up to three carbon units of Z^Bare optionally and independently replaced by —C(O)—, —C(S)—, —C(O)NR^B—, —C(O)NR^BNR^B—, —C(O)O—, —NR^BC(O)O—, —NR^BC(O)NR^B—, —NR^BNR^B—, —S—, —SO—, —SO₂—, —NR^B—, —SO₂NR^B—, or —NR^BSO₂NR^B—, provided that SO, SO₂, or —SO₂NR^B— is not directly bound to the carbonyl of formula (I);

each R₅is independently R^B, halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃;

each R^Bis independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;

or, R₁and R₂, together with the atoms to which they are attached, form an optionally substituted heterocycloaliphatic ring;

each R₇is independently R^D, halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃;

each R^Dis independently hydrogen, or optionally substituted aryl; and

each of a and b is independently 0, 1, 2, or 3; provided that the sum of a and b is 2 or 3.

B. Specific Compounds

1. Substituent R₁:

Each R₁is —Z^AR₄, wherein each Z^Ais independently a bond or an optionally substituted branched or straight C_1-12aliphatic chain wherein up to three carbon units of Z^Aare optionally and independently replaced by —C(O)—, —C(S)—, —C(O)NR^A—, —C(O)NR^ANR^A—, —C(O)O—, —NR^AC(O)O—, —O—, —NR^AC(O)NR^A—, —NR^ANR^A—, —S—, —SO—, —SO₂—, —NR^A—, SO₂NR^A—, or —NR^ASO₂NR^A— provided that —NR^ANR^A—, —NR^AC(O)NR^A—, or —NR^ASO₂NR^A— is not directly bound to the nitrogen ring atom of formula (I).

Each R₄is independently R^A, halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃.

Each R^Ais independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.

In several embodiments R₁is optionally substituted with 1 to 4 substituents.

In certain embodiments, R₁is -Q₄-W₄-Q₃-W₃-Q₂-W₂-Q₁; wherein each of W₂, W₃, and W₄is independently a bond, —C(O)—, —C(S)—, —C(O)N(Q₅)-, —C(O)O—, —O—, —N(Q₅)C(O)N(Qs)—, —SO₂—, —N(Q₅)SO₂—, —S—, —N(Q₅)-, —SO—, —OC(O)—, —N(Q₅)C(O)O—, or —SO₂N(Q₅)-; each of Q₁, Q₂, Q₃and Q₄is independently a bond, an optionally substituted C_1-4aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, or a hydrogen when Q₁, Q₂, Q₃, or Q₄is the terminal group of R_I; and each Q₅is independently hydrogen or an optionally substituted aliphatic. In some specific embodiments, Q₄is a bond.

In several embodiments, R₁is an optionally substituted acyl group. In several examples, R₁is an optionally substituted alkylcarbonyl. Additional examples of R₁include (amino)alkylcarbonyl, (halo)alkylcarbonyl, (aryl)alkylcarbonyl, (cycloaliphatic)alkylcarbonyl, or (heterocycloaliphatic)alkylcarbonyl. Included in these examples are embodiments where R₁is (heterocycloalkyl(oxy(carbonyl(amino))))alkylcarbonyl, (heteroaryl(carbonyl(amino(alkyl(carbonyl(amino)))))alkylcarbonyl, (bicycloaryl(sulfonyl(amino)))alkylcarbonyl, (aryl(alkoxy(carbonyl(amino))))alkylcarbonyl, (alkyl(carbonyl(amino)))alkylcarbonyl, (alkenyl(alkoxy(carbonyl(amino))))alkylcarbonyl, (cycloaliphatic(alkyl(amino(carbonyl(amino)))))alkylcarbonyl, (heteroaryl(carbonyl(amino(alkyl(carbonyl(amino))))))alkylcarbonyl, (alkyl(amino(carbonyl(amino))))alkylcarbonyl, or (bicycloaryl(amino(carbonyl(amino))))alkylcarbonyl, each of which is optionally substituted with 1-3 substituents.

In several embodiments, R₁is an optionally substituted carboxy group. In one example, R₁is optionally substituted alkoxycarbonyl. Another example of R₁includes alkoxycarbonyl, or (tricyclic aryl)alkoxycarbonyl, each of which is optionally substituted with 1-3 substituents. Other carboxy groups include (aliphatic(oxy))carbonyl, a (heteroaralkyl(oxy))carbonyl, (heterocycloaliphatic(oxy)carbonyl, (aralkyl(oxy))carbonyl, each of which is optionally substituted with 1-3 of halo, alkoxy, aliphatic, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, or combinations thereof.

In several embodiments, R₁is optionally substituted aminocarbonyl. Examples of R₁include (alkoxy(aryl(alkyl)))aminocarbonyl, (alkyl)aminocarbonyl, or (aryl(alkoxy(carbonyl(alkyl(amino(carbonyl(alkyl)))))))aminocarbonyl, each of which is optionally substituted with 1-3 substituents.

In several embodiments, R₁is optionally substituted heteroaryl. In one example, R₁is an optionally substituted oxazolyl, pyrrolyl, furyl, thiophenyl, triazinyl, pyridinyl, pyrazinyl, pyrimidinyl, or pyridazinyl.

In several embodiments, R₁is an alkylsulfonyl, aminosulfonyl, arylsulfonyl, heteroarylsulfonyl, cycloaliphaticsulfonyl, or heterocycloaliphaticsulfonyl, each of which is optionally substituted with 1-4 substituents.

In several embodiments, R₁is an optionally substituted alkylsulfonyl. Examples of R₁include (aryl)alkylsulfonyl, or (alkyl(amino))alkylsulfonyl, alkylsulfonyl, aminosulfonyl, arylsulfonyl, heteroarylsulfonyl, cycloaliphaticsulfonyl, or heterocycloaliphaticsulfonyl, each of which is optionally substituted 1-3 substituents. In certain embodiments, R₁is an optionally substituted alkyl sulfonyl.

In several embodiments, R₁is an (aryl)alkylsulfonyl, or (alkyl(amino))alkylsulfonyl, each of which is optionally substituted.

In some specific embodiments, R₁is (amino)alkylcarbonyl, (halo)alkylcarbonyl, (aryl)alkylcarbonyl, (cycloaliphatic)alkylcarbonyl, or (heterocycloaliphatic)alkylcarbonyl, (heterocycloalkyl(oxy(carbonyl(amino))))alkylcarbonyl, (heteroaryl(carbonyl(amino(alkyl(carbonyl(amino)))))alkylcarbonyl, (bicycloaryl(sulfonyl(amino)))alkylcarbonyl, (aryl(alkoxy(carbonyl(amino))))alkylcarbonyl, (alkyl(carbonyl(amino)))alkylcarbonyl, (alkenyl(alkoxy(carbonyl(amino))))alkylcarbonyl, (cycloaliphatic(alkyl(amino(carbonyl(amino)))))alkylcarbonyl, (heteroaryl(carbonyl(amino(alkyl(carbonyl(amino))))))alkylcarbonyl, (alkyl(amino(carbonyl(amino))))alkylcarbonyl, or (bicycloaryl(amino(carbonyl(amino))))alkylcarbonyl, each of which is optionally substituted.

In other specific embodiments, R₁is a heteroarylcarbonyl, a (cycloaliphatic(alkyl(amido(alkyl))))carbonyl, a (heterocycloaliphatic(oxy(amido(alkyl))))carbonyl, an (aryl(sulfonyl(amino(alkyl))))carbonyl, an (aralkyl(oxy(amido(alkyl))))carbonyl, an (aliphatic(oxy(amido(alkyl))))carbonyl, a (cycloaliphatic(alkyl(amido(alkyl))))carbonyl, a (heterocycloaliphatic)carbonyl, or a (heteroaryl(amido(alkyl(amido(alkyl))))carbonyl, each of which is optionally substituted with 1-4 of halo, aliphatic, cycloaliphatic, acyl, alkoxy, or combinations thereof.

In still other embodiments, R₁is amido. For example, R₁is (alkoxy(aryl(alkyl)))aminocarbonyl, (alkyl)aminocarbonyl, or (aryl(alkoxy(carbonyl(alkyl(amino(carbonyl(alkyl)))))))aminocarbonyl, each of which is optionally substituted.

In several embodiments, R₁is

embedded image

wherein T is a bond, —C(O)—, —OC(O)—, —NHC(O)—, —S(O)₂N(H)—, —C(O)C(O)— or —SO₂—; each R is independently hydrogen, amino, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; each R₈and R′₈is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; and each R₉is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, or R₈and R₉, bound on adjacent atoms, taken together with the atoms to which they are attached form a 5 to 7 membered, optionally substituted monocyclic heterocycloaliphatic, or a 6 to 12 membered, optionally substituted bicyclic heterocycloaliphatic; or R₈and R′₈, taken together with the atoms to which they are attached form an optionally substituted cycloaliphatic or an optionally substituted heterocycloaliphatic. For clarity, when R₁is QVI, each of R₈, R′₈and R₉in each subunit can be independently selected as described above. The set of R₈, R′₈and R₉variables in one subunit need not necessarily be identical to the same set of R₈, R′₈and R₉variables in the other subunit.

In other embodiments, R₁is QI or QII.

In some embodiments, R in the substituent in QI, QII, QIII, QIV, QV, or QVI is

embedded image

In other embodiments, R₁is QVI and R is

embedded image

In other embodiments, R in the substituent QI, QII, QIII, QIV, QV, or QVI is

embedded image

wherein each R₁₀and R′₁₀is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic, or R₁₀and R′₁₀together with the atom to which they are both bound form an optionally substituted cycloaliphatic or an optionally, substituted heterocycloaliphatic; and each K is independently a bond, C_1-12aliphatic, —O—, —S—, —S(O)₂—, —NR₁₄—, —C(O)—, or —C(O)NR₁₄—, wherein R₁₄is hydrogen or an optionally substituted C_1-12aliphatic; and n is 1-3. For clarity, when more than one R₁₀is present in QI, QII, QIII, QIV, QV, or QVI, each R₁₀can be the same or different. In several embodiments, R₁₀or R′₁₀is [C_3-10cycloalkyl or cycloalkenyl]-C_1-12aliphatic, (3 to 10 membered)-heterocycloaliphatic, (3 to 10 membered)-heterocycloaliphatic-C_1-12aliphatic-, (5 to 10 membered)-heteroaryl, or (5 to 10 membered)-heteroaryl-C_1-12aliphatic-.

In still other embodiments, R in the substituent in QI, QII, QIII, QIV, QV, or QVI is

embedded image

In further embodiments, R in the substituent in QI, QII, QIII, QIV, QV, or QVI is

embedded image

wherein each Z is independently —O—, —S—, —NR₅₀—, or —C(R₅₀)₂—, custom character is independently a single bond or a double bond, and each R₅₀is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic; and n is 1 or 2.

In several embodiments, R₁is

embedded image

wherein T is a bond, —C(O)—, —OC(O)—, —NHC(O)—, —S(O)₂N(H)—, —C(O)C(O)— or —SO₂—; each R is independently hydrogen, amino, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; each R₈and R′₈is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; and each R₉is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, or R₈and R₉, bound on adjacent atoms, taken together with the atoms to which they are attached form a 5 to 7 membered, optionally substituted monocyclic heterocycloaliphatic, or a 6 to 12 membered, optionally substituted bicyclic heterocycloaliphatic, in which each heterocycloaliphatic ring; or R₈and R′₈, taken together with the atoms to which they are attached form an optionally substituted cycloaliphatic or an optionally substituted heterocycloaliphatic; each R₁₁and R′₁₁is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic; or R₁₁and R′₁₁together with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring; and each R₁₂is independently hydrogen or a protecting group.

In some embodiments, R₁₁and R′₁₁together with the atom to which they are attached form a 3 to 7 membered ring. Non-limiting examples include

embedded image

Non-limiting examples of R₈and R₁₁include

embedded image

Alternatively, R₈and R₁₁together with the atoms to which they are attached may form an optionally substituted 5 to 7 membered monocyclic heterocycloaliphatic or an optionally substituted 6 to 12 membered bicyclic heterocycloaliphatic, in which each heterocycloaliphatic or aryl ring optionally contains an additional heteroatom selected from O, S and N.

Also, R₈and R₉together to with the atoms to which they are attached can form a ring, R₇and the ring system formed by R₈and R₉form an optionally substituted 8 to 14 membered bicyclic fused ring system, wherein the bicyclic fused ring system is optionally further fused with an optionally substituted phenyl to form an optionally substituted 10 to 16 membered tricyclic fused ring system.

In several embodiments, R₁is:

embedded image

wherein T is —C(O)—, and R is

embedded image

In several embodiments, R₁is a group selected from:

embedded image

In several embodiments, R₁is

embedded image

wherein U is a bond or —O—; V is an alkyl; T is —C(O)—; and R is an aliphatic, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkoxy or cycloalkoxy. Examples of R₁include

embedded image

in which R, U, and V are as just defined.

In some embodiments, R is an aliphatic, cycloaliphatic or heterocycloaliphatic such that examples of R₁include

embedded image

In other embodiments, R is an alkoxy or cycloalkoxy such that examples of R₁include

embedded image

In further embodiments, R is cyclopropyl optionally substituted with cyano, halo, aliphatic, cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl such that R₁is selected from the group consisting of:

embedded image

In some embodiments, R₁is

embedded image

wherein X₉₉is OR, OC(NH)R, or

embedded image

X₁₀₀is NH, CH₂; and R is defined above.

Additional examples of R₁are illustrated in PCT publications WO 2004/103996 A1, WO 2004/72243 A2, WO 03/064456 A1, WO 03/64455 A2, WO 03/064416 A1, and U.S. Patent Publication US 2005/0090450, as well as those other publications referenced herein, each of which is incorporated in its entirety by reference.

2. Substituent R₂:

In several embodiments, R₂is —Z^BR₅, wherein each Z^Bis independently a bond or an optionally substituted branched or straight C_1-12aliphatic chain wherein up to three carbon units of Z^Bare optionally and independently replaced by —C(O)—, —C(S)—, —C(O)NR^B—, —C(O)NR^BNR^B—, —C(O)O—, —NR^BC(O)O—, —NR^BC(O)NR^B—, —NR^BNR^B—, —S—, —SO—, —SO₂—, —NR^B—, —SO₂NR^B—, or —NR^BSO₂NR^B—, provided that SO, SO₂, or —SO₂NR^B— is not directly bound to the carbonyl of formula (I). Each R₅is independently R^B, halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃. Each R^Bis independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.

In still further embodiments, R₂is —Z₁—V₁—Z₂—V₂—Z₃—V₃each of V₁, V₂, and V₃is independently a bond, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, or a hydrogen when V₁, V₂, V₃is the terminal group of R₂; each of Z₁, Z₂, and Z₃is independently a bond, —C(O)—, —C(O)C(O)—, —C(S)—, —C(O)N(Q₆)-, —N(Q₆)C(O)—, —C(O)C(O)N(Q₆)-, —O—, SO—, —SO₂—, —N(Q₆)SO₂—, —N(Q₆)C(O)N(Q₆)-, —N(Q₆)C(S)N(Q₆)-, —N(Q₆)-, —N(Q₆)SO₂—, —SO₂N(Q₆)-, —C(O)N(Q₆)SO₂—, —SO₂N(Q₆)C(O)—, or hydrogen when Z₁, Z₂, or Z₃is the terminal group of R₂; and each Q₆is independently hydrogen, or an optionally substituted aliphatic.

In other embodiments, R₂is an optionally substituted (aliphatic)amino wherein the aliphatic portion of R₂is —Z₂—V₂—Z₃—V₃or —Z₃—V₃wherein each of Z₂and Z₃is independently a bond, —C(O)—, —N(Q₅)-, —CH(OH)—, —C(O)N(Q₆)-, or —C(O)C(O)N(Q₆)-; V₂is independently a bond, an optionally substituted aliphatic, or an optionally substituted cycloaliphatic; and V₃is hydrogen, an optionally substituted aliphatic, or an optionally substituted cycloaliphatic.

In still further embodiments, Z₂is —CH(OH)—, V₂is a bond, and Z₃is —C(O)N(Q₆)- such that R₂is —N(Q₆)-CH(OH)—C(O)—N(V₃)(Q₆).

In certain embodiments, R₂is an optionally substituted (aliphatic)amino, optionally substituted (cycloaliphatic)amino, an optionally substituted alkoxy, or hydroxy.

In still another embodiment, R₂is an alkoxy optionally substituted with 1-3 of halo, hydroxy, aliphatic, cycloaliphatic, or heterocycloaliphatic.

In several embodiments, R₂is amino. Examples of R₂include a mono-substituted amino. Additional examples of R₂include (cycloaliphatic(carbonyl(carbonyl(alkyl))))amino (amino(carbonyl(carbonyl(aliphatic))))amino, (aliphatic(carbonyl(carbonyl(aliphatic))))amino, or (aryl(amino(carbonyl(carbonyl(aliphatic)))))amino, each of which is optionally substituted with 1 to 3 substituents.

In several embodiments, R₂is —NR_2ZR′_2Z, —SR_2Y, or —NR_2Y—CR_2XR′_2X-L₁-NR_2Z—R_2W, wherein R_2Yis independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; each R_2Wis independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic; each R_exand R′_2Xis independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic; or R_2Xand R′_2Xtogether with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring; each L₁is —CH₂—, —C(O)—, —C(O)C(O)—, —C(O)O—, —S(O)—, or —SO₂—; each R_2Zor R′_2Zis hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; or R_2Zand R′_2Ztogether with the nitrogen to which they are both attached may form an optionally substituted 3 to 7 membered heterocycloaliphatic ring.

In several embodiments, each R_2Xand R′_2Xis independently hydrogen, or optionally substituted aliphatic, optionally substituted cycloaliphatic, or optionally substituted (cycloaliphatic)aliphatic.

In several embodiments, L₁is —C(O)C(O)— or —SO₂—.

In several other embodiments, each R_2Wis hydrogen or optionally substituted cycloaliphatic.

In several embodiments, R₂is —NH—CHR_2X—C(O)—C(O)—N(R_2Z)R_2W.

In several embodiments, R₂is —NH—CHR_2X—CH(OH)—C(O)—N(R_2Z)R_2W.

In several embodiments, R₂is —NH—CHR_2X—C(O)—C(O)—NHR_2Zwherein —NHR_2Zis NH-cyclopropyl, —NH-Me, —NH-Et, —NH-iPr, —NH-nPr.

In several embodiments R₂is —NR_2ZR′_2Z, —SR_2Zwherein each R_2Zand R′_2Zis independently hydrogen, alkyl, cycloalkyl or aralkyl. Non-limiting examples of R_2Zinclude methyl, ethyl, t-butyl, cyclopentyl, cyclohexyl and benzyl.

In other embodiments R₂is (—NH—CR_2XR′_2X-L₁-C(O))_i-M; wherein each M is independently —OH, R_2X, —NR_2ZR′_2Z, or —OR_2X, each i is 1 or 2, and L₁, R_2Z, R_2X, and R′_2Zare defined above.

In several embodiments R₂is (—NH—CR_2ZR′_2Z— L₁-C(O))_i-M wherein L₁is —C(O)—, i is 1, and M is independently R_2X, —N(R_2XR′_2X), —OR_2X, —NHSO₂R_2X, or —SR_2X.

In some embodiments, R′_2Zis H and R_2Zis an aliphatic, (aryl)aliphatic or cycloaliphatic. Non-limiting examples of R_2Xinclude hydrogen,

embedded image

In some embodiments, R′_2Xis H and R_2Xis an optionally substituted aliphatic, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaliphatic or optionally substituted heteroaralkyl. Some non-limiting examples of R_2Xinclude

embedded image

where c is 0-3.

In several embodiments, R₂is

embedded image

wherein R_2Xis

embedded image

and R_2Xis

embedded image

or hydrogen.

In some embodiments, R₂is selected from the group consisting of

embedded image

In some embodiments, R₂is selected from the group consisting of:

embedded image

In some embodiments, R₂is selected from the group consisting of:

embedded image

In some embodiments, R₂is

embedded image

wherein each R₅₆is independently optionally substituted C_1-6aliphatic; optionally substituted aryl, optionally substituted heteraryl, optionally substituted cycloaliphatic, or optionally substituted heterocycloaliphatic; each R₅₇is independently optionally substituted aliphatic, optionally substituted aryl, optionally substituted aliphatic, optionally substituted heteroaryl, optionally substituted aliphatic, optionally substituted cycloaliphatic or optionally substituted amino; and m is 1 or 2; and each R_2Xand R′_2Xis independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; or R_2Xand R′_2Xtogether with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring.

In some other embodiments, R₂is

embedded image

wherein R₅₈and R₅₉are each independently selected from optionally substituted aliphatic, optionally substituted alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted (cycloaliphatic)oxy, optionally substituted (heterocycloaliphatic)oxy optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloaliphatic or optionally substituted amino; and each R_2Xand R′_2Xis independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; or R_2Xand R′_2Xtogether with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring.

In several embodiments, a portion of R₁and a portion of R₂together can form a cyclic structure, e.g., a 5- to 18-membered cycloheteroaliphatic ring. Some non-limiting example include

embedded image

In several embodiments, R₂is one selected from

embedded image

In some specific embodiments, R₂is

embedded image

where X₂₀₀is —OX₂₀₂or —X₂₀₂, and X₂₀₂is aliphatic, cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl.

In other embodiments, R₂is

embedded image

In some other embodiments, R₂is

embedded image

Additional examples of R₂are illustrated in PCT publications WO 2004/103996 A1, WO 2004/72243 A2, WO 03/064456 A1, WO 03/64455 A2, WO 03/064416 A1, and U.S. Patent Publication US 2005/0090450, as well as those other publications referenced herein, each of which is incorporated in its entirety by reference.

3. Substituent R₃:

Each R₃is an aliphatic, a cycloaliphatic, a heterocycloaliphatic, an aryl, or a heteroaryl, each of which is optionally substituted.

In several embodiments, each R₃is independently —Z^CR₆, wherein each Z^Cis independently a bond or an optionally substituted aliphatic wherein up to two carbon units of Z^Care optionally and independently replaced by —C(O)—, —CS—, —C(O)NR^C—, —C(O)NR^CNR^C—, —C(O)O—, —NR^CC(O)O—, —O—, —NR^CC(O)NR^C—, —NR^CNR^C—, —S—, —SO—, —SO₂—, —NR^C—, —SO₂NR^C—, or —NR^CSO₂NR^C—. Each R₆is independently R^C, halo, —OH, —CN, —NO₂, —NH₂, alkoxy, or haloalkoxy. Each R^Cis independently hydrogen, an optionally substituted aliphatic group, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl. However, in many embodiments, when Z^Cis a bond and R₆is R^C, then R^Cis independently an optionally substituted aliphatic group, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.

In still other embodiments, each R₃is an optionally substituted aliphatic, amino, sulfonyl, sulfanyl, sulfinyl, sulfonamide, sulfamide, sulfo, —O—R_3A, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; and each R_3Ais independently an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.

In several embodiments, R₃is an optionally substituted aryl. In some examples, R₃is a monocyclic, bicyclic, or tricyclic aryl, each of which is optionally substituted. For example, R₃is an optionally substituted phenyl, an optionally substituted naphthyl, or an optionally substituted anthracenyl. In other examples, R₃is a monocyclic, bicyclic, or tricyclic aryl, each of which is optionally substituted with 1 to 4 of halo, hydroxy, cyano, nitro, aliphatic, haloaliphatic, (aliphatic)oxy, (halo(aliphatic))oxy, (aliphatic(oxy(aryl)))oxy, aryl, heteroaryl, haloaryl, cycloaliphatic, heterocycloaliphatic, or combinations thereof. In several examples, R₃is an optionally substituted fused bicyclic aryl. In several examples, R₃is an optionally substituted fused tricyclic aryl.

In several embodiments, R₃is an optionally substituted heteroaryl. In several examples, R₃is a monocyclic or bicyclic heteroaryl, each of which is optionally substituted with 1 to 4 of halo, hydroxy, cyano, nitro, aliphatic, haloaliphatic, (aliphatic)oxy, (halo(aliphatic))oxy, (aliphatic(oxy(aryl)))oxy, aryl, heteroaryl, haloaryl, cycloaliphatic, heterocycloaliphatic, or combinations thereof.

In some embodiments, R₃is optionally substituted aliphatic such as methyl, ethyl or propyl, each of which is optionally substituted.

According to other embodiments, R₃is an optionally substituted aliphatic.

According to other embodiments, R₃is an optionally substituted C_1-5aliphatic.

In several examples, R₃is

embedded image

In several embodiments, R₃is one selected from:

embedded image

In some embodiments, R₃is selected from the group consisting of:

embedded image

In some embodiments, R₃is an optionally substituted monocyclic, bicyclic, or tricyclic heteroaryl, each of which is optionally substituted with 1-3 of halo, hydroxy, cyano, nitro, aliphatic, haloaliphatic, (aliphatic)oxy, (halo(aliphatic))oxy, (aliphatic(oxy(aryl)))oxy, aryl, heteroaryl, haloaryl, cycloaliphatic, heterocycloaliphatic, or combinations thereof.

In further embodiments, R₃is selected from the group consisting of:

embedded image

In other embodiments, R₃is an optionally substituted amino or an optionally substituted aryloxy.

In some embodiments, R₃is selected from the group consisting of

embedded image

4. Group A:

Each A is —(CX₁X₂)_a—, wherein each X₁and X₂is independently hydrogen, optionally substituted C_1-4aliphatic, or optionally substituted aryl; or X₁and X₂taken together form an oxo group; and each a is 0 to 3.

In several embodiments, X₁or X₂is hydrogen.

In several embodiments, X₁or X₂is optionally substituted C_1-4aliphatic. Examples of X₁or X₂include trifluoromethyl, or optionally substituted ethyl, propyl, butyl, or isomers thereof.

In several embodiments, X₁or X₂is an optionally substituted aryl. Examples of X₁or X₂include optionally substituted phenyl, naphthyl, or azulenyl.

5. Group B:

Each B is —(CX₁X₂)_b—, wherein each X₁and X₂is independently hydrogen, optionally substituted C_1-4aliphatic, or optionally substituted aryl; or X₁and X₂taken together form an oxo group; and each b is 0 to 3.

In several embodiments, X₁or X₂is hydrogen.

In several embodiments, X₁or X₂is optionally substituted C_1-4aliphatic. Examples of X₁or X₂include trifluoromethyl, or optionally substituted ethyl, propyl, butyl, or isomers thereof. In several additional examples, X₁or X₂is an optionally substituted mono- or di-substituted (amino)-C_1-4aliphatic.

In several embodiments, X₁or X₂is an optionally substituted aryl. Examples of X₁or X₂include optionally substituted phenyl, naphthyl, indenyl, or azulenyl.

6. Substituents Y and Y′

In several embodiments, each Y and Y′ is independently hydrogen, optionally substituted aliphatic, or optionally substituted aryl.

Each Y and Y′ is independently —Z^DR₇, wherein each Z^Dis independently a bond or an optionally substituted straight or branched (C_1-6)-aliphatic chain wherein up to two carbon units of Z^Dare optionally and independently replaced by —C(O)—, —CS—, —C(O)NR^D—, —C(O)NR^DNR^D—, —C(O)O—, —OC(O)—, —NR^DC(O)O—, —O—, —NR^DC(O)NR^D—, —OC(O)NR^D—, —NR^DNR^D—, —NR^DC(O)—, —S—, —SO—, —SO₂—, —NR^D—, —SO₂NR^D—, —NR^DSO₂—, or —NR^DSO₂NR^D—. Each R₇is independently R^D, halo, —OH, —CN, —NO₂, —NH₂, or —OCF₃. Each R^Dis independently hydrogen, or optionally substituted aryl.

In several embodiments, one selected from Y and Y′ is hydrogen.

In several embodiments, one selected from Y and Y′ is optionally substituted aliphatic.

In several embodiments, one selected from Y and Y′ is optionally substituted aryl.

In several embodiments, both Y and Y′ are hydrogen.

In several embodiments, one of Y or Y′ is hydrogen and the other is fluorine.

In several embodiments, both of Y and Y′ are fluorine.

In additional of examples, one of Y or Y′ is hydrogen and the other is methoxycarbonyl; one of Y or Y′ is hydrogen and the other is hydroxy; or together, Y and Y′ form an oxo group or form ═S.

7. Exceptions:

In compounds of formula (I), the sum of a and b is 2 or 3. For example, a is 0 and b is 3; a is 1 and b is 2; a is 2 and b is 1; or a is 3 and b is 0.

C. Sub-Generic Compounds

Another aspect of the present invention provides compounds of formula (I)a useful for inhibiting serine protease activity and methods inhibiting serine protease activity. Compounds of formula (Ia) include:

embedded image

or a pharmaceutically acceptable salt thereof wherein R₃, A, B, Y, and Y′ are defined above in formula (I).

Each R_1ais -Q₄-W₄-Q₃-W₃-Q₂-W₂-Q₁; wherein each of W₂, W₃, and W₄is independently a bond, —C(O)—, —C(S)—, —C(O)N(Q₅)-, —C(O)O—, —O—, —N(Q₅)C(O)N(Q₅)-, —SO₂—, —N(Q₅)SO₂—, —S—, —N(Q₅)-, —SO—, —N(Q₅)C(O)—, —OC(O)—, —N(Q₅)C(O)O—, or —SO₂N(Q₅)-; each of Q₁, Q₂, Q₃and Q₄is independently a bond, an optionally substituted C_1-4aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, or a hydrogen when Q₁, Q₂, Q₃, or Q₄is the terminal group of R₁; and each Q₅is independently hydrogen or an optionally substituted aliphatic.

Each R_2ais —Z₁—V₁—Z₂—V₂—Z₃—V₃each of V₁, V₂, and V₃is independently a bond, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, or a hydrogen when V₁, V₂, V₃is the terminal group of R₂; each of Z₁, Z₂, and Z₃is independently a bond, —C(O)—, —C(O)C(O)—, —C(S)—, —C(O)N(Q₅)-, —N(Q₅)C(O)—, —C(O)C(O)N(Q₅)-, —O—, SO—, —SO₂—, —N(Q₅)SO₂—, —N(Q₅)C(O)N(Q₅)-, —N(Q₅)C(S)N(Q₅)-, —N(Q₅)-, —N(Q₅)SO₂—, —SO₂N(Q₅)-, —C(O)N(Q₅)SO₂—, —SO₂N(Q₅)C(O)—, or hydrogen when Z₁, Z₂, or Z₃is the terminal group of R₂; and each Q₅is independently hydrogen, or an optionally substituted aliphatic.

In several examples, R_2ais an optionally substituted (aliphatic)amino, an optionally substituted alkoxy, or hydroxy.

In several examples, R_2ais an (aliphatic)amino wherein the nitrogen atom is optionally substituted with —Z₂—V₂—Z₃—V₃or —Z₃—V₃wherein each of Z₂and Z₃is independently a bond, —C(O)—, —N(Q₅)-, or —C(O)C(O)N(Q₅)-; and each of V₂and V₃is independently a bond, an optionally substituted aliphatic, or an optionally substituted cycloaliphatic.

Another aspect of the present invention provides compounds of formula (I)_buseful for inhibiting serine protease activity and methods inhibiting serine protease activity. Compounds of formula (I)b include:

embedded image

or a pharmaceutically acceptable salt thereof, wherein R₃, R₈, R, T, A, B, Y and Y′ are defined above in formula (I).

Each G is a 2 to 15 atom optionally substituted aliphatic chain optionally containing 1 to 3 heteroatoms selected from O, S and N.

Examples of compounds of formula (I)_binclude:

embedded image

wherein T, R, and R₃are defined above in formula (I).

Still other examples of formula (I)_bare

embedded image

wherein each R_2Wis independently

embedded image

or hydrogen; each T is independently a bond, —C(O)—, —OC(O)—, —NHC(O)—, —S(O)₂N(H)—, —C(O)C(O)— or —SO₂—; each R is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; and each R₉is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl.

Further specific examples of compounds of formula (I)b are

embedded image

Other examples of compounds of formula (I)b include:

embedded image

Another aspect of the present invention provides compounds of formula (I)I useful for inhibiting serine protease activity and methods inhibiting serine protease activity. Compounds of formula (II) include:

embedded image

or a pharmaceutically acceptable salt thereof, wherein

Each R₃is an optionally substituted aryl or an optionally substituted heteroaryl;

Each R_2Yis independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;

Each R₉is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic;

Each R_2Xand R′_2Xis independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic; or R_2Xand R′_2Xtogether with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring, or R_2Xand R_2Ytogether with the atoms to which they are attached form an optionally substituted 5 to 7 membered heterocycloaliphatic ring;

Each R_1bis —Z^ER₂₁, wherein Z^Eis —CH₂—, —NH—, —CH(R_1Z)—, or —O—, and R₂₁is optionally substituted 6-7 membered cycloaliphatic or optionally substituted tert-butyl;

Each R_1Zis optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocycloaliphatic, optionally substituted aryl, or optionally substituted heteroaryl;

Each R_2Zis hydrogen, optionally substituted cycloaliphatic, optionally substituted heterocycloaliphatic, or optionally substituted aliphatic; and

Each R_2Wis hydrogen, optionally substituted cycloaliphatic, optionally substituted heterocycloaliphatic, or optionally substituted aliphatic, or R_2Zand R_2W, together with the nitrogen atom to which they are attached form an optionally substituted heterocycloaliphatic.

Another aspect of the present invention provides compounds of formula (I) II useful for inhibiting serine protease activity and methods inhibiting serine protease activity. Compounds of formula (III) include:

embedded image

or a pharmaceutically acceptable salt thereof, wherein

R_1eis

embedded image

R_2eis

embedded image

R′_2eis

embedded image

or hydrogen; and

R_3eis optionally substituted aryl or optionally substituted heteroaryl.

Another aspect of the present invention provides compounds of formula (I)V useful for inhibiting serine protease activity and methods inhibiting serine protease activity. Compounds of formula (IV) include:

embedded image

or a pharmaceutically acceptable salt thereof, wherein

R_1eis

embedded image

R_2eis

embedded image

R′_2eis

embedded image

or hydrogen; and

Each of R_3fand R′_3fis independently hydrogen, sulfonamide, sulfonyl, sulfinyl, optionally substituted acyl, optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocycloaliphatic, optionally substituted aryl, or optionally substituted heteroaryl, or R_3fand R′_3ftogether with the nitrogen atom to which they are attached form an optionally substituted, saturated, partially unsaturated, or full unsaturated, 5-8 membered heterocycloaliphatic or heteroaryl.

Another aspect of the present invention provides compounds of formula V useful for inhibiting serine protease activity and methods inhibiting serine protease activity. Compounds of formula (V) include:

embedded image

or a pharmaceutically acceptable salt thereof, wherein R_1e, R_2e, and R′_2eare defined above in formula (III).

Each D is independently —CR₈—, N, S, or O, provided that no more than two D are independently, S, or O, and R₈is defined above in formula (I).

Another aspect of the present invention provides compounds of formula VI useful for inhibiting serine protease activity and methods inhibiting serine protease activity. Compounds of formula (VI) include:

embedded image

or a pharmaceutically acceptable salt thereof, wherein R_1e, R_2e, and R′_2eare defined above in formula (III).

Each R_3gis a substituted aryl or a substituted heteroaryl. In some embodiments, R_3gis

embedded image

Another aspect of the present invention provides compounds of formula VII useful for inhibiting serine protease activity and methods inhibiting serine protease activity. Compounds of formula (VII) include:

embedded image

or a pharmaceutically acceptable salt thereof, wherein R_1e, R_2e, and R′_2eare defined above in formula (III), and R_3gis defined in formula (VI).

Another aspect of the present invention provides compounds of formula VIII useful for inhibiting serine protease activity and methods inhibiting serine protease activity. Compounds of formula (VIII) include:

embedded image

or a pharmaceutically acceptable salt thereof, wherein R_1e, R_2e, and R′_2eare defined above in formula (I)II, and R_3gis defined in formula VI.

Another aspect of the present invention provides compounds of formula (I)X useful for inhibiting serine protease activity and methods inhibiting serine protease activity. Compounds of formula (IX) include:

embedded image

or a pharmaceutically acceptable salt thereof, wherein R_1e, R_2e, and R′_2eare defined above in formula (III), and R_3gis defined in formula (VI).

Another aspect of the present invention provides compounds of formula (X) useful for inhibiting serine protease activity and methods inhibiting serine protease activity. Compounds of formula (X) include:

embedded image

or a pharmaceutically acceptable salt thereof, wherein R_1e, R_2e, and R′_2eare defined above in formula (III), and R_3gis defined in formula (VI).

D. Combinations of Embodiments

Other embodiments include any combination of the aforementioned substituents R₁, R₂, R₃, A, B, Y, and Y′.

E. Exemplary Compounds

The invention is intended to include compounds wherein R₁and R₂contain structural elements of a serine protease inhibitor. Compounds having the structural elements of a serine protease inhibitor include, but are not limited to, the compounds of the following publications: WO 97/43310, US 20020016294, WO 01/81325, WO 01/58929, WO 01/32691, WO 02/08198, WO 01/77113, WO 02/08187, WO 02/08256, WO 02/08244, WO 03/006490, WO 01/74768, WO 99/50230, WO 98/17679, WO 02/48157, WO 02/08251, WO 02/07761, WO 02/48172, WO 02/08256, US 20020177725, WO 02/060926, US 20030008828, WO 02/48116, WO 01/64678, WO 01/07407, WO 98/46630, WO 00/59929, WO 99/07733, WO 00/09588, US 20020016442, WO 00/09543, WO 99/07734, U.S. Pat. No. 6,018,020, U.S. Pat. No. 6,265,380, U.S. Pat. No. 6,608,027, US 20020032175, US 20050080017, WO 98/22496, WO 05/028502, U.S. Pat. No. 5,866,684, WO 02/079234, WO 00/31129, WO 99/38888, WO 99/64442, WO 2004/072243, WO 02/18369, US 2006/046956, US 2005/197301, WO 2005058821, WO 2005051980, WO 2005030796, WO2005021584, WO2005113581, WO2005087731, WO2005087725, WO2005087721, WO2005085275, WO2005085242, US2003216325, WO2003062265, WO2003062228, WO2002008256, WO 2002008198, WO2002008187, WO 2002048172, WO 2001081325, WO 2001077113, U.S. Pat. No. 6,251,583, U.S. Pat. No. 5,990,276, US20040224900, US20040229818, WO2004037855, WO2004039833, WO200489974, WO2004103996, WO2004030670, WO2005028501, WO2006007700, WO2005070955, WO2006007708, WO2006000085, WO2005073195, WO2005073216, WO2004026896, WO2004072243, WO2004113365, WO2005010029, US20050153877, WO2004093798, WO2004094452, WO2005046712, WO2005051410, WO2005054430, WO2004032827, WO2005095403, WO2005077969, WO2005037860, WO2004092161, WO2005028502, WO2003087092, and WO2005037214, each of which is incorporated herein by reference.

Specific exemplary compounds of the invention are shown below in Table 1.

TABLE 1

Exemplary compounds of Formula (I).

Structure
Compound No.

embedded image

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

466

467

468

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585

586

587

588

589

590

591

592

593

594

595

596

597

598

599

600

601

602

603

604

605

606

607

608

609

610

611

612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

649

650

651

652

653

654

655

656

657

658

659

660

661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

678

679

680

681

682

683

684

685

686

687

688

689

690

691

692

693

694

695

696

697

698

699

700

701

702

703

704

705

706

707

708

709

710

711

712

713

714

715

716

717

718

719

720

721

722

723

724

725

726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

763

764

765

766

767

768

769

770

771

772

773

774

775

776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

793

794

795

796

797

798

799

800

801

802

803

804

805

806

807

808

809

810

811

812

813

814

815

816

817

818

819

820

821

822

823

824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

865

866

867

868

869

870

871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

886

887

888

889

890

891

892

893

894

895

896

897

898

899

900

901

902

903

904

905

906

907

908

909

910

911

912

913

914

915

916

917

918

919

920

921

922

923

924

925

926

927

928

929

930

931

932

933

934

935

936

937

938

939

940

941

942

943

944

945

946

947

948

949

950

951

952

953

954

955

956

957

958

959

960

961

962

963

964

965

966

967

968

969

970

971

972

973

974

975

976

977

978

979

980

981

982

983

984

985

986

987

988

989

990

991

992

993

994

995

996

997

998

999

1000

1001

1002

1003

1004

Batch 5 (last 40)

embedded image

1005

1006

1007

1008

1009

1010

1011

1012

1013

1014

1015

1016

1017

1018

1019

1020

1021

1022

1023

1024

1025

1026

1027

1028

1029

1030

1031

1032

1033

1034

1035

1036

1037

1038

1039

1040

1041

1042

1043

1044

II. Synthesis of the Compounds

Compounds of Formula I may be readily synthesized from commercially available starting materials using the exemplary synthetic routes provided below. Exemplary synthetic routes to produce compounds of Formula I are provided below in the Preparations, Methods, Examples, and Schemes. For example, the spiroisoxazoline moiety may be prepared by 1,3-dipolar addition between a nitrile oxide and a methylene proline as reported by Kurth, M. J., et al., in J. Org. Chem., 2002, 67, pp. 5673-5677, and as illustrated in Scheme 1 below. The nitrile oxides can be generated from cholooximes or nitroalkanes using known methods.

Scheme I provides a general representation of processes for preparing compounds of Formula (I). Its overall strategy is to construct a compound of formula 1h followed by selective removal of the protecting group PG₁in the presence of PG₂to provide the intermediate 1j. The substituent R₁may then be coupled to 1j, which provides intermediates of formula 1k containing R₁. In some embodiments, R₁may itself contain a protecting group which may be selectively removed in the presence of PG₂, followed by further elaboration. Subsequent to the addition of the R₁moiety, the PG₂group is removed to provide the intermediate 1m. Coupling of 1m with an R₂moiety then provides the peptidomimetic compounds of Formula (I).

embedded image

Referring again to Scheme 1, in one example, the hydroxy proline 1a is protected as the Boc derivative (i.e., step ia) to provide the protected proline 1b, wherein PG₁is t-butyloxycarbonate, using known methods. See, e.g., T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3^rdedition, John Wiley and Sons, Inc. (1999). Oxidation of 1b (i.e., step ib) provides the keto-pyrrolidine acid 1c. The oxidation is achieved with a suitable reagent such as, for example, sodium hypochlorite in the presence of TEMPO. Next, in step ic, the keto-pyrrolidine acid 1c is reacted with a Wittig reagent such as, for example, a triphenylphosphonium ylid of the formula (Ph)₃P═C(Y)(Y′) and using known conditions, to provide an exomethylene compound of formula 1d. Use of the free acid 1c to provide the corresponding free acid 1d is advantageous as the acid 1d may be expediently purified from neutral or basic by-products by simple extraction of 1d into aqueous basic solution. The acid 1d is subsequently protected (step id) with a suitable protecting group such as, for example, a t-butyl ester under known conditions (ibid) to provide the intermediate 1e.

Reaction of 1e with a nitrile oxide if provides a mixture of the syn and anti isomers of the spiroisoxazolines 1g and 1h. As referred to herein, syn-means that the 2-carboxyl moiety of the proline ring and the oxygen of the isoxazoline ring are on the same side of a plane as described by the proline ring. The term anti-means that the 2-carboxyl moiety of the proline ring and the oxygen of the isoxazoline ring are on the opposite side of a plane as described by the proline ring. Thus, 1g represents a syn-compound of the invention and 1h represents an anti-compound of the invention.

In some embodiments, when PG₁is Boc and PG₂is t-butoxy, selective removal of the protecting group PG₁from 1g and 1h in the presence of the protecting group PG₂may be achieved with a sulfonic acid such as, for example, methane sulfonic acid in a suitable organic solvent at temperatures from about −40° C. to about 40° C., from about −20° C. to about 20° C. and from about −5° C. to about 5° C. Suitable organic solvents include, for example, methylene chloride and tetrahydrofuran.

The isomers 1i and 1j may be separated advantageously by crystallization of a mixture of the corresponding organic acid salts which avoids more complicated methods such as, e.g., chromatography. Suitable organic salts include those of organic carboxylic acids, e.g., acetic acid, optionally substituted benzoic acids, tartaric acid, malonic acid, fumaric acid, oxalic acid, mandelic acid, citric acid, p-toluoyl tartaric acid and maleic acid; organic sulfonic acids, e.g., methane sulfonic acid, optionally substituted benzene sulfonic acids, trifluoromethane sulfonic acid and camphor sulfonic acid.

A single spiroisoxazoline isomer, for example 1j, is coupled with an acid R₁COOH in the presence of a coupling reagent such as, for example, EDCI to provide the intermediate spiroisoxazoline 1k. Selective removal of the protecting group PG₂of 1k to give 1m with minimum racemization or cleavage of the R₁side chain is achieved by a suitable mineral acid in a suitable organic solvent at temperatures from about −40° C. to about 40° C., from about −20° C. to about 20° C. and from about −5° C. to about 5° C. Suitable mineral acids include, for example, concentrated hydrochloric acid or concentrated sulfuric acid. Suitable organic solvents include, for example, methylene chloride and tetrahydrofuran. The spiroisoxazoline 1m is then coupled with an amine moiety R₂to provide the compounds of Formula I.

Referring again to Scheme 1, PG₁(CO)— can be an amine protecting group, wherein PG₁is, for example, methoxycarbonyl, t-butyloxycarbonyl, 9-flourenylmethyloxycarbonyl, or benzyloxycarbonyl. PG₂(CO)— can be an acid or acid protecting group wherein PG₂is, for example, —OH, methoxy, t-butyloxy or benzyloxy.

Each of PG₁and PG₂groups may be incorporated into the core spiroisoxazoline structure either individually or together using known methods and as further described herein. For example, if the desired R₁substituted is a group other than a PG₁group (e.g., a protecting group), the PG₁group may be removed to provide a compound with a free amine group. That amine group and an appropriate moiety may be coupled under known coupling conditions to provide a compound wherein R₁is a moiety of a protease inhibitor. For example, if the PG₂moiety is protected, the protecting group may be removed and an R₂moiety may be incorporated.

Another method for producing compounds of the present invention is illustrated below in Scheme 2.

embedded image

Referring to Scheme 2, the symbol custom character represents a polymeric resin to which reactants are bound by a functionality that allows further modification and subsequent removal of the product from the resin. A suitable resin is a polymer bound dihydropyran (DHP) resin as described by Ellman et. al. in Tetrahedron Letters, 1994, 35, 9333.

In step iia, simultaneous deprotection of both the amine and acid may be achieved by contacting the proline 1e with an acid, for example, trifluoroacetic acid in methylene chloride to give the amino acid 2a. Reaction of 2a, step iib, with an activated Fmoc derivative, for example, N-(9H-Fluoren-9ylmethoxycarbonyloxy)succinimide (Fmoc-OSu), in the presence of a mild inorganic base, such as sodium carbonate, gives the Fmoc derivative 2b.

Preparation of the resin bound peptide 2d may be accomplished by reacting the Fmoc derivative 2b with the DHP resin bound amino-alcohol 2c, step iiic, which reacts with the free acid 2b, in the presence of a coupling reagent such as, for example, O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate (HBTU), a racemization suppressant, such as 1-hydroxybenzotriazole (HOBT) and a tertiary amine, such as di-isopropylethyl amine (DIEA).

As in Scheme 1, an R₃-substituted nitrile oxide if may undergo a dipolar cycloaddition reaction with the resin bound peptide 2d to provide two isomers, syn- and anti-, of the compound 2e. Next in step iid, the Fmoc protecting group is removed by contacting 2e with a secondary amine such as, for example, piperidine in a polar solvent such as dimethylformamide to give 2f. Formation of the peptide 2g, via step iie, can be achieved through reaction of 2f with a carboxylic acid in the presence of a coupling reagent such as HBTU, a racemization suppressant such as HOBt, and a tertiary amine such as DIEA. Cleavage of the peptide-resin 2g, step iif, to give the alpha-hydroxy-amide 2h, can be achieved by contacting 2g with a strong acid such as, for example, trifluoroacetic acid and water.

In the final step, iig, the alpha-hydroxy-amide 2h is oxidized to 2i using a Dess-Martin periodinane oxidation or a Pfitzner-Moffat oxidation.

Alternatively, compounds of Formula I may be prepared using resin bound reagents as illustrated below in Scheme 3.

embedded image

In Scheme 3, the selective removal of the PG₁in the presence of PG₂(step it) provides spiroisoxazoline isomer(s) 1i and/or 1j. Reaction of 1i and/or 1j, in step iiia, with an activated Fmoc derivative, e.g., N-(9H-Fluoren-9-ylmethoxycarbonyloxy)succinimide (Fmoc-OSu), in the presence of a mild inorganic base, such as sodium carbonate, provides the Fmoc derivative 3a.

Preparation of the resin bound peptide 2e may be accomplished by reaction of the Fmoc derivative 3a with the DHP resin bound amino-alcohol 2c, via step iiib, which reacts with a free acid 3b, in the presence of a coupling reagent (e.g., O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate (HBTU)), a racemization suppressant (e.g., 1-hydroxybenzotriazole (HOBT)), and a tertiary amine (e.g., di-isopropylethyl amine (DIEA)).

In step iid, the Fmoc protecting group is removed by contacting 2e with a secondary amine such as, e.g., piperidine in a polar solvent such as dimethylformamide to give 2f. Formation of the peptide 2g can be achieved, e.g., by reacting 2f with a carboxylic acid in the presence of a coupling reagent (e.g., HBTU), a racemization suppressant (e.g., HOBt) and a tertiary amine (e.g., DIEA). Cleavage of the peptide-resin 2g to give the free peptide 2h can be achieved, e.g., by contacting 2g with a strong acid (e.g., trifluoroacetic acid) and water.

In the final step, iig, the alcohol of 2h can be oxidized to 2i, e.g., with Dess-Martin periodinane or sodium hypochlorite and TEMPO.

Scheme 4 below illustrates a synthetic pathway for compounds of Formula I in which R₁and R₂, together with the atoms to which they are attached, form an optionally substituted macrocyclic heterocycloaliphatic.

embedded image

Referring to Scheme 4, the spiroisoxazoline acid E4 reacts with the amino ester H1 in the presence of a coupling reagent to provide the intermediate H2. Macrocyclization of H2 results in compound H3. Hydrolysis of the ester H2 provides acid H4. Reaction of acid H4 with a sulfonamide or sulfamide in the presence of a coupling reagent provides the product H5.

Shown below in Schemes 5, 6, 7, 8, and 9 are examples of total synthesis of compounds of formula (I) according to one of the methods described above.

embedded image

Referring to Scheme 5, the protected t-butyldimethylsilyl-hydroxybenzaldehyde 5b is converted to the hydroxamoyl chloride 5d as previously described. Reaction of 5d with the exomethylene pyrrolidine provides the spiroisoxazoline 5e. Deprotection of 5e to 5f followed by reaction with triflic anhydride provides the triflate 5g. Reaction of 5f with an amine HNU₁U₂provides the intermediate spiroisoxazoline 5h which is converted to compounds of the invention as previously described.

Alternatively, the hydroxy-spiroisoxazoline intermediate 5f may be alkylated to provide the intermediate 5k which may be similarly converted to compounds of the invention.

embedded image

Referring to Scheme 6, reaction of the diprotected pyrrolidinone with difluorodibromomethane in the presence of HMPT and zinc provides the difluoroexomethylene intermediate 6b. Dipolar addition with the nitrile oxide if as previously described provides the difluorospiroisoxazoline 6c. In a similar fashion, the intermediates 6b and 6f are prepared from 6a and 6e respectively and converted to the corresponding substituted isooxazolines 6d and 6g.

In other variations, the intermediate 6h may be brominated to give 6j, alkylated to provide 6k or oxidized to provide 6m using the reagents illustrated.

embedded image

Referring to Scheme 7, dipolar addition of the exomethylene pyrrolidine shown with if wherein R₃is —COOEt, leads to the ester 7a. Hydrolysis of the ethyl ester in 7a, conversion to the acid chloride (not shown) and reaction with ammonia provides the amide 7c. Reaction of 7c with trifluoroacetic anhydride provides the nitrile 7d which is converted to the peptidic intermediate 7e by methods previously described. The intermediate 7e reacts with an azide U₄N₃to provide the tetrazole 7f which is oxidized to a compound of the invention 7g. In a variation of this scheme, the ester 7a may be converted to the triazole 7h and subsequently to compounds of the invention 71.

embedded image

Referring to Scheme 8, dipolar addition as previously described but using hydroxycarbonimidic dibromide provides the bromoisoxazoline 8a. Reaction of 8a with an arylboronic acid in the presence of a palladium catalyst (Suzuki conditions) provides the intermediate 8b which is converted to compounds of the invention by methods previously described. The AR in step 8a and 8b represents aryl or heteroaryl.

embedded image

Referring to Scheme 9, the Wittig product 9a undergoes a dipolar addition to provide the spiroisoxazoline 9b. Reduction of 9b with, for example, DIBAL provides the alcohol 9c which may be alkylated to provide the intermediate 9e which subsequently may be converted to compounds of the invention by methods previously described. Hydrolysis of ester 9b with, e.g., LiOH, will provide carboxylic acid 9d which can be converted to compounds of formula I as described herein.

embedded image

Referring to scheme 10, the diprotected piperidinone 10b undergoes a Wittig type reaction to form the exomethylene compound 10c which undergoes dipolar addition as previously described to provide a 4.5 spiroisoxazoline 10d which may be converted to compounds of the invention as previously described.

III. Formulations, Administrations, and Uses

Another embodiment of this invention provides a pharmaceutical composition comprising a compound of formula (I) or pharmaceutically acceptable salts or mixtures of salts thereof. According to another embodiment, the compound of formula (I) is present in an amount effective to decrease the viral load in a sample or in a patient, wherein said virus encodes a serine protease necessary for the viral life cycle, and a pharmaceutically acceptable carrier.

If pharmaceutically acceptable salts of the compounds of this invention are utilized in these compositions, those salts are preferably derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentane-propionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2 hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2 naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 phenyl propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Base salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine salts, N methyl D glucamine, and salts with amino acids such as arginine, lysine, and so forth.

Also, the basic nitrogen containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides, such as benzyl and phenethyl bromides and others. Water or oil soluble or dispersible products are thereby obtained.

The compounds utilized in the compositions and methods of this invention may also be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol and wool fat.

According to another embodiment, the compositions of this invention are formulated for pharmaceutical administration to a mammal. In one embodiment said mammal is a human being.

Such pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra articular, intra synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally or intravenously.

Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3 butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

In one embodiment, dosage levels of between about 0.01 and about 100 mg/kg body weight per day of the protease inhibitor compounds described herein are useful in a monotherapy for the prevention and treatment of antiviral, particularly anti-HCV mediated disease. In another embodiment, dosage levels of between about 0.5 and about 75 mg/kg body weight per day of the protease inhibitor compounds described herein are useful in a monotherapy for the prevention and treatment of antiviral, particularly anti-HCV mediated disease. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). In one embodiment, such preparations contain from about 20% to about 80% active compound.

When the compositions of this invention comprise a combination of a compound of formula I and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 10 to 100% of the dosage normally administered in a monotherapy regimen. In another embodiment, the additional agent should be present at dosage levels of between about 10 to 80% of the dosage normally administered in a monotherapy regimen.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These may be prepared by mixing the agent with a suitable non irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

Topical application for the lower intestinal tract may be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

In one embodiment, the pharmaceutical compositions are formulated for oral administration.

In another embodiment, the compositions of this invention additionally comprise another anti-viral agent, preferably an anti-HCV agent. Such anti-viral agents include, but are not limited to, immunomodulatory agents, such as α, β, and γ-interferons, pegylated derivatized interferon-α compounds, and thymosin; other anti-viral agents, such as ribavirin, amantadine, and telbivudine; other inhibitors of hepatitis proteases (NS2-NS3 inhibitors and NS3-NS4A inhibitors); inhibitors of other targets in the HCV life cycle, including helicase and polymerase inhibitors; inhibitors of internal ribosome entry; broad-spectrum viral inhibitors, such as IMPDH inhibitors (e.g., compounds of U.S. Pat. Nos. 5,807,876, 6,498,178, 6,344,465, and 6,054,472, WO 97/40028, WO 98/40381, WO 00/56331, and mycophenolic acid and derivatives thereof, and including, but not limited to VX-497, VX-148, and/or VX-944); or combinations of any of the above. See also W. Markland et al., Antimicrobial & Antiviral Chemotherapy, 44, p. 859 (2000) and U.S. Pat. No. 6,541,496.

embedded image

The following definitions are used herein (with trademarks referring to products available as of this application's filing date):

“Peg-Intron” means PEG-INTRON®, peginteferon alfa-2b, available from Schering Corporation, Kenilworth, N.J.;

“Intron” means INTRON-A®, interferon alfa-2b available from Schering Corporation, Kenilworth, N.J.;

“ribavirin” means ribavirin (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif.; described in the Merck Index, entry 8365, Twelfth Edition; also available as REBETROL® from Schering Corporation, Kenilworth, N.J., or as COPEGASUS® from Hoffmann-La Roche, Nutley, N.J.;

“Pagasys” means PEGASYS®, peginterferon alfa-2a available Hoffmann-La Roche, Nutley, N.J.;

“Roferon” mean ROFERON®, recombinant interferon alfa-2a available from Hoffmann-La Roche, Nutley, N.J.;

“Berefor” means BEREFOR®, interferon alfa 2 available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Conn.;

SUMIFERON®, a purified blend of natural alpha interferons such as Sumiferon available from Sumitomo, Japan;

WELLFERON®, interferon alpha n1 available from Glaxo_Wellcome LTd., Great Britain; and

ALFERON®, a mixture of natural alpha interferons made by Interferon Sciences, and available from Purdue Frederick Co., CT.

The term “interferon” as used herein means a member of a family of highly homologous species-specific proteins that inhibit viral replication and cellular proliferation, and modulate immune response, such as interferon alpha, interferon beta, or interferon gamma. The Merck Index, entry 5015, Twelfth Edition.

According to one embodiment of the present invention, the interferon is α-interferon. According to another embodiment, a therapeutic combination of the present invention utilizes natural alpha interferon 2a. Or, the therapeutic combination of the present invention utilizes natural alpha interferon 2b. In another embodiment, the therapeutic combination of the present invention utilizes recombinant alpha interferon 2a or 2b. In yet another embodiment, the interferon is pegylated alpha interferon 2a or 2b. Interferons suitable for the present invention include:

(a) INTRON-A® (interferon-alpha 2B, Schering Plough),

(b) PEG-INTRON®,

(d) ROFERON®,

(e) BEREFOR®,

(f) SUMIFERON®,

(g) WELLFERON®,

(h) consensus alpha interferon available from Amgen, Inc., Newbury Park, Calif.,

(i) ALFERON®;

(j) VIRAFERON®;

(k) INFERGEN®;

(l) ALBUFERON™.

As is recognized by skilled practitioners, a protease inhibitor would be preferably administered orally. Interferon is not typically administered orally. Nevertheless, nothing herein limits the methods or combinations of this invention to any specific dosage forms or regime. Thus, each component of a combination according to this invention may be administered separately, together, or in any combination thereof.

In one embodiment, the protease inhibitor and interferon are administered in separate dosage forms. In one embodiment, any additional agent is administered as part of a single dosage form with the protease inhibitor or as a separate dosage form. As this invention involves a combination of compounds, the specific amounts of each compound may be dependent on the specific amounts of each other compound in the combination. As recognized by skilled practitioners, dosages of interferon are typically measured in IU (e.g., about 4 million IU to about 12 million IU).

Accordingly, agents (whether acting as an immunomodulatory agent or otherwise) that may be used in combination with a compound of this invention include, but are not limited to, Albuferon™ (albumin-Interferon alpha) available from Human Genome Sciences; PEG-INTRON® (peginterferon alfa-2b, available from Schering Corporation, Kenilworth, N.J.); INTRON-A®, (interferon alfa-2b available from Schering Corporation, Kenilworth, N.J.); ribavirin (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif.; described in the Merck Index, entry 8365, Twelfth Edition); REBETROL® (Schering Corporation, Kenilworth, N.J.), COPEGUS® (Hoffmann-La Roche, Nutley, N.J.); PEGASYS® (peginterferon alfa-2a available Hoffmann-La Roche, Nutley, N.J.); ROFERON® (recombinant interferon alfa-2a available from Hoffmann-La Roche, Nutley, N.J.); BEREFOR® (interferon alfa 2 available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Conn.); SUMIFERON® (a purified blend of natural alpha interferons such as Sumiferon available from Sumitomo, Japan); WELLFERON® (interferon alpha n1 available from Glaxo Wellcome Ltd., Great Britain); ALFERON® (a mixture of natural alpha interferons made by Interferon Sciences, and available from Purdue Frederick Co., CT); α-interferon; natural alpha interferon 2a; natural alpha interferon 2b; pegylated alpha interferon 2a or 2b; consensus alpha interferon (Amgen, Inc., Newbury Park, Calif.); VIRAFERON®; INFERGEN®; REBETRON® (Schering Plough, Interferon-alpha 2B+Ribavirin); pegylated interferon alpha (Reddy, K. R. et al. “Efficacy and Safety of Pegylated (40-kd) Interferon alpha-2a Compared with Interferon alpha-2a in Noncirrhotic Patients with Chronic Hepatitis C” (Hepatology, 33, pp. 433-438 (2001); consensus interferon (Kao, J. H., et al., “Efficacy of Consensus Interferon in the Treatment of Chronic Hepatitis” J. Gastroenterol. Hepatol. 15, pp. 1418-1423 (2000); lymphoblastoid or “natural” interferon; interferon tau (Clayette, P. et al., “IFN-tau, A New Interferon Type I with Antiretroviral activity” Pathol. Biol. (Paris) 47, pp. 553-559 (1999); interleukin-2 (Davis, G. L. et al., “Future Options for the Management of Hepatitis C.” Seminars in Liver Disease, 19, pp. 103-112 (1999); Interleukin-6 (Davis et al. “Future Options for the Management of Hepatitis C.” Seminars in Liver Disease, 19, pp. 103-112 (1999); interleukin-12 (Davis, G. L. et al., “Future Options for the Management of Hepatitis C.” Seminars in Liver Disease, 19, pp. 103-112 (1999); and compounds that enhance the development of type 1 helper T cell response (Davis et al., “Future Options for the Management of Hepatitis C.” Seminars in Liver Disease, 19, pp. 103-112 (1999)). Also included are compounds that stimulate the synthesis of interferon in cells (Tazulakhova, E. B. et al., “Russian Experience in Screening, analysis, and Clinical Application of Novel Interferon Inducers” J. Interferon Cytokine Res., 21 pp. 65-73) including, but are not limited to, double stranded RNA, alone or in combination with tobramycin, and Imiquimod (3M Pharmaceuticals; Sauder, D. N. “Immunomodulatory and Pharmacologic Properties of Imiquimod” J. Am. Acad. Dermatol., 43 pp. S6-11 (2000).

Compounds that stimulate the synthesis of interferon in cells (Tazulakhova, E. B. et al., “Russian Experience in Screening, analysis, and Clinical Application of Novel Interferon Inducers” J. Interferon Cytokine Res., 21 pp. 65-73) include, but are not limited to, double stranded RNA, alone or in combination with tobramycin, and Imiquimod (3M Pharmaceuticals; Sauder, D. N. “Immunomodulatory and Pharmacologic Properties of Imiquimod” J. Am. Acad. Dermatol., 43 pp. S6-11 (2000).

Other non-immunomodulatory or immunomodulatory compounds may be used in combination with a compound of this invention including, but not limited to, those specified in WO 02/18369, which is incorporated herein by reference (see, e.g., page 273, lines 9-22 and page 274, line 4 to page 276, line 11).

Still other agents include those described in various published U.S. patent applications. These publications provide additional teachings of compounds and methods that could be used in combination with VX-950 in the methods of this invention, particularly for the treatment of hepatitis. It is contemplated that any such methods and compositions may be used in combination with the methods and compositions of the present invention. For brevity, the disclosure the disclosures from those publications is referred to be reference to the publication number but it should be noted that the disclosure of the compounds in particular is specifically incorporated herein by reference. Exemplary such publications include U.S. Patent Publication No. 20040058982; U.S. Patent Publication No. 20050192212; U.S. Patent Publication No. 20050080005; U.S. Patent Publication No. 20050062522; U.S. Patent Publication No. 20050020503; U.S. Patent Publication No. 20040229818; U.S. Patent Publication No. 20040229817; U.S. Patent Publication No. 20040224900; U.S. Patent Publication No. 20040186125; U.S. Patent Publication No. 20040171626; U.S. Patent Publication No. 20040110747; U.S. Patent Publication No. 20040072788; U.S. Patent Publication No. 20040067901; U.S. Patent Publication No. 20030191067; U.S. Patent Publication No. 20030187018; U.S. Patent Publication No. 20030186895; U.S. Patent Publication No. 20030181363; U.S. Patent Publication No. 20020147160; U.S. Patent Publication No. 20040082574; U.S. Patent Publication No. 20050192212; U.S. Patent Publication No. 20050187192; U.S. Patent Publication No. 20050187165; U.S. Patent Publication No. 20050049220; and U.S. Patent Publication No. US2005/0222236.

This invention may also involve administering a cytochrome P450 monooxygenase inhibitor. CYP inhibitors may be useful in increasing liver concentrations and/or increasing blood levels of compounds that are inhibited by CYP.

If an embodiment of this invention involves a CYP inhibitor, any CYP inhibitor that improves the pharmacokinetics of the relevant NS3/4A protease may be used in a method of this invention. These CYP inhibitors include, but are not limited to, ritonavir (WO 94/14436), ketoconazole, troleandomycin, 4-methylpyrazole, cyclosporin, clomethiazole, cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine, fluoxetine, nefazodone, sertraline, indinavir, nelfinavir, amprenavir, fosamprenavir, saquinavir, lopinavir, delavirdine, erythromycin, VX-944, and VX-497. Preferred CYP inhibitors include ritonavir, ketoconazole, troleandomycin, 4-methylpyrazole, cyclosporin, and clomethiazole. For preferred dosage forms of ritonavir, see U.S. Pat. No. 6,037,157, and the documents cited therein: U.S. Pat. No. 5,484,801, U.S. application Ser. No. 08/402,690, WO 95/07696 and WO 95/09614.

Methods for measuring the ability of a compound to inhibit cytochrome P450 monooxygenase activity are known. See, e.g., U.S. Pat. No. 6,037,157, and Yun, et al. Drug Metabolism & Disposition, vol. 21, pp. 403-407 (1993).

Upon improvement of a patient's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of active ingredients will also depend upon the particular described compound and the presence or absence and the nature of the additional anti-viral agent in the composition.

According to another embodiment, the invention provides a method for treating a patient infected with a virus characterized by a virally encoded serine protease that is necessary for the life cycle of the virus by administering to said patient a pharmaceutically acceptable composition of this invention. In one embodiment, the methods of this invention are used to treat a patient suffering from a HCV infection. Such treatment may completely eradicate the viral infection or reduce the severity thereof. In another embodiment, the patient is a human being.

In an alternate embodiment, the methods of this invention additionally comprise the step of administering to said patient an anti-viral agent preferably an anti-HCV agent. Such anti-viral agents include, but are not limited to, immunomodulatory agents, such as α-, β-, and γ-interferons, pegylated derivatized interferon-α compounds, and thymosin; other anti-viral agents, such as ribavirin, amantadine, and telbivudine; other inhibitors of hepatitis C proteases (NS2-NS3 inhibitors and NS3-NS4A inhibitors); inhibitors of other targets in the HCV life cycle, including but not limited to helicase and polymerase inhibitors; inhibitors of internal ribosome entry; broad-spectrum viral inhibitors, such as IMPDH inhibitors (e.g., VX-497 and other IMPDH inhibitors disclosed in U.S. Pat. Nos. 5,807,876 and 6,498,178, mycophenolic acid and derivatives thereof); inhibitors of cytochrome P-450, such as ritonavir, or combinations of any of the above.

Such additional agent may be administered to said patient as part of a single dosage form comprising both a compound of this invention and an additional anti-viral agent. Alternatively the additional agent may be administered separately from the compound of this invention, as part of a multiple dosage form, wherein said additional agent is administered prior to, together with or following a composition comprising a compound of this invention.

Pharmaceutical compositions may also be prescribed to the patient in “patient packs” containing the whole course of treatment in a single package, usually a blister pack. Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patients supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in traditional prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions.

It will be understood that the administration of the combination of the invention by means of a single patient pack, or patient packs of each formulation, containing within a package insert instructing the patient to the correct use of the invention is a desirable additional feature of this invention.

According to a further aspect of the invention is a pack comprising at least one compound of formula I (in dosages according to this invention) and an information insert containing directions on the use of the combination of the invention. Any composition, dosage form, therapeutic regimen or other embodiment of this invention may be presented in a pharmaceutical pack. In an alternative embodiment of this invention, the pharmaceutical pack further comprises one or more of additional agent as described herein. The additional agent or agents may be provided in the same pack or in separate packs.

Another aspect of this involves a packaged kit for a patient to use in the treatment of HCV infection or in the prevention of HCV infection (or for use in another method of this invention), comprising: a single or a plurality of pharmaceutical formulation of each pharmaceutical component; a container housing the pharmaceutical formulation(s) during storage and prior to administration; and instructions for carrying out drug administration in a manner effective to treat or prevent HCV infection.

Accordingly, this invention provides kits for the simultaneous or sequential administration of a dose of at least one compound of formula I (and optionally an additional agent). Typically, such a kit will comprise, e.g. a composition of each compound and optional additional agent(s) in a pharmaceutically acceptable carrier (and in one or in a plurality of pharmaceutical formulations) and written instructions for the simultaneous or sequential administration.

In another embodiment, a packaged kit is provided that contains one or more dosage forms for self administration; a container means, preferably sealed, for housing the dosage forms during storage and prior to use; and instructions for a patient to carry out drug administration. The instructions will typically be written instructions on a package insert, a label, and/or on other components of the kit, and the dosage form or forms are as described herein. Each dosage form may be individually housed, as in a sheet of a metal foil-plastic laminate with each dosage form isolated from the others in individual cells or bubbles, or the dosage forms may be housed in a single container, as in a plastic bottle. The present kits will also typically include means for packaging the individual kit components, i.e., the dosage forms, the container means, and the written instructions for use. Such packaging means may take the form of a cardboard or paper box, a plastic or foil pouch, etc.

A kit according to this invention could embody any aspect of this invention such as any composition, dosage form, therapeutic regimen, or pharmaceutical pack. The packs and kits according to this invention optionally comprise a plurality of compositions or dosage forms. Accordingly, included within this invention would be packs and kits containing one composition or more than one composition.

In yet another embodiment the present invention provides a method of pre-treating a biological substance intended for administration to a patient comprising the step of contacting said biological substance with a pharmaceutically acceptable composition comprising a compound of this invention. Such biological substances include, but are not limited to, blood and components thereof such as plasma, platelets, subpopulations of blood cells and the like; organs such as kidney, liver, heart, lung, etc; sperm and ova; bone marrow and components thereof, and other fluids to be infused into a patient such as saline, dextrose, etc.

According to another embodiment the invention provides methods of treating materials that may potentially come into contact with a virus characterized by a virally encoded serine protease necessary for its life cycle. This method comprises the step of contacting said material with a compound according to the invention. Such materials include, but are not limited to, surgical instruments and garments (e.g. clothes, gloves, aprons, gowns, masks, eyeglasses, footwear, etc.); laboratory instruments and garments (e.g. clothes, gloves, aprons, gowns, masks, eyeglasses, footwear, etc.); blood collection apparatuses and materials; and invasive devices, such as, for example, shunts and stents.

In another embodiment, the compounds of this invention may be used as laboratory tools to aid in the isolation of a virally encoded serine protease. This method comprises the steps of providing a compound of this invention attached to a solid support; contacting said solid support with a sample containing a viral serine protease under conditions that cause said protease to bind to said solid support; and eluting said serine protease from said solid support. In one embodiment, the viral serine protease isolated by this method is HCV NS3-NS4A protease.

All references cited within this document are incorporated herein by reference.

IV. Methods and Examples

In order that the invention described herein may be more fully understood, the following methods and examples are provided. It should be understood that these methods and examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

A. Preparation of Intermediates for Compounds of Formula I

Set forth below are various methods for preparing intermediates that can be used to synthesize the compound of Formula I.

embedded image

Preparation of 3-(benzyloxycarbonylamino)-4-cyclobutyl-2-hydroxybutanoic acid

A solution of the cyanohydrin prepared according to methods described in WO 04/113294 (1 g, 3.65 mmol) in conc. HCl (12 mL) was heated to reflux for 18 hours. The reaction was concentrated in vacuo to afford the desired amino acid as an HCl salt (1.7 g) which was used in the next step without further purification. A solution of the above HCl salt in THF was treated with DIPEA (2.68 g) and Z—OSu (5.16 g). The reaction mixture was stirred at room temperature for 8 hours. The reaction mixture was diluted with toluene and HCl (12 N, until pH=1). After separation, the organic layer was extracted with sat. NaHCO₃(50 mL, twice). The aqueous layer was made acidic with HCl (6 N) until pH=1 and extracted with EtOAc (200 mL). The combined organic layer was dried and concentrated in vacuo to afford the title compound (0.6 g). (M+1) 308.

embedded image

Preparation of benzyl 1-cyclobutyl-3-hydroxy-4-(methylamino)-4-oxobutan-2-ylcarbamate

To a solution of 3-(benzyloxycarbonylamino)-4-cyclobutyl-2-hydroxybutanoic acid (250 mg, 0.81 mmol) in DCM (20 mL) was added HOSu (140 mg, 1.22 mmol), EDC (234 mg, 1.22 mmol). After stirring for 1 hour, methylamine in THF (2 N, 0.81 mL) was added to the above mixture. The reaction mixture was stirred for 18 hours and then concentrated in vacuo. The residue was purified by Gilson Prep to afford the title compound (135 mg). ¹H-NMR (CDCl₃): δ 7.54-7.28 (m, 5H), 6.67 (NH, 1H), 5.03 (dd, 2H), 3.68 (m, 1H), 2.73 (m, 3H), 2.26 (m, 1H), 1.97-1.31 (m, 9H). (M+1) 321.

embedded image

Preparation of benzyl 1-cyclobutyl-4-(cyclopropylamino)-3-hydroxy-4-oxobutan-2-ylcarbamate

To a solution of 3-(benzyloxycarbonylamino)-4-cyclobutyl-2-hydroxybutanoic acid (600 mg, 1.95 mmol) in DCM (20 mL) was added HOSu (337 mg, 2.93 mmol), EDC (562 mg, 2.93 mmol). After stirring for 1 hour, cyclopropylamine (223 mg, 3.9 mmol) was added to the above mixture. The product was extracted with EtOAc. The combined organic layer was then washed with HCl (1N), water, NaHCO₃, and brine and then concentrated in vacuo to afford benzyl 1-cyclobutyl-4-(cyclopropylamino)-3-hydroxy-4-oxobutan-2-ylcarbamate (530 mg). (M+1) 347.

embedded image

Preparation of 3-amino-4-cyclobutyl-N-cyclopropyl-2-hydroxybutanamide

To a solution of the CBz amide (530 mg, 1.53 mmol) in MeOH (30 mL) was added Pd(OH)₂/C (106 mg). The mixture was stirred under H₂(1 atm) for 18 hours. After filtration, the filtrate was concentrated in vacuo to afford the title compound (300 mg). ¹H-NMR (CDCl₃): δ 3.29 (m, 1H), 2.74 (m, 1H), 2.37-1.66 (m, 9H), 1.40 (m, 1H), 0.78 (m, 2H), 0.51 (m, 2H). (M+1) 213.

The following compounds were prepared in a similar fashion to preparing 3-amino-4-cyclobutyl-N-cyclopropyl-2-hydroxybutanamide by using the appropriate amine:

embedded image

Preparation of 3-amino-N-cyclopropyl-2-hydroxyhept-6-ynamide

embedded image

3-Amino-N-cyclopropyl-2-hydroxyhept-6-ynamide was prepared as described by N. Kobayashi, et al. in US 2003/153788, which is incorporated herein by reference in its entirety. ¹H-NMR (500 MHz, DMSO-d₆): 8.18 (s), 6.34 (s), 4.22 (s), 3.45 (s), 3.17 (s), 2.84 (s), 2.69 (d, J=3.2 Hz), 2.30 (m), 2.24 (m), 1.70 (m), 1.59 (m), 0.62 (d, J=5.0 Hz), 0.53 (s) ppm; FIA m/z 197.01 ES⁺.

Preparation of Cbz-Protected (3S)-3-amino-4-cyclopropyl-2-hydroxy-N-methylbutanamide

embedded image

Step 1: Preparation of benzyl (2S)-1-cyano-3-cyclopropyl-1-hydroxypropan-2-ylcarbamate

embedded image

To a solution of the aldehyde (7.9 g, 32 mmol) in MeOH (50 mL) at 10° C. was added Na₂S₂O₄(6.13 g, 35.2 mmol) and the resulting mixture was warmed to room temperature and stirred for 2 hours then cooled to 10° C. To this reaction mixture, a solution of KCN in water (50 mL) was added. After stirring at room temperature for 18 hours, the mixture was extracted with TBME (100 mL, twice). The combined organic layers were washed with water and brine, dried and concentrated in vacuo to afford the title compound (8 g). (M+1) 275.

Step 2: Preparation of (3S)-methyl 3-(benzyloxycarbonylamino)-4-cyclopropyl-2-hydroxybutanoate

embedded image

To a solution of the cyanohydrin (1 g, 3.65 mmol) in MeOH (15 mL) at −20° C. was bubbled through a stream of dry HCl gas for 30 minutes. The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was purged with nitrogen gas for 30 minutes and then concentrated. The residue at 0° C. was quenched with ice water and then stirred at room temperature for 1 hour. The product was extracted with EtOAc. The combined organic layer was washed with NaHCO₃, water, brine and concentrated in vacuo to afford the title compound (0.5 g). ¹H-NMR (CDCl₃) δ: 7.31-7.30 (m, 5H), 5.09 (d, 2H), 4.44-4.14 (m, 2H), 3.78 (d, 3H), 1.58-1.42 (m, 2H), 0.70 (m, 1H), 0.47 (t, 2H), 0.11-0.01 (m, 2H). (M+1) 308.

Step 3: Preparation of (3S)-3-(benzyloxycarbonylamino)-4-cyclopropyl-2-hydroxybutanoic acid

embedded image

To a solution of the methyl ester of Step 2 (400 mg; 1.3 mmol) in THF (8 mL) and water (6.63 mL) was added LiOH (1 N; 1.37 mL). The reaction mixture was stirred for 30 minutes and then acidified with 1.0 N HCl to pH=3≈4. The mixture was extracted with EtOAc (20 mL, twice). The combined organic layer was washed with water, brine, and then concentrated in vacuo to afford the title compound (370 mg). (M+1) 294.

Step 4: Preparation of benzyl (2S)-1-cyclopropyl-3-hydroxy-4-(methylamino)-4-oxobutan-2-ylcarbamate

embedded image

To a solution of (3S)-3-(benzyloxycarbonylamino)-4-cyclopropyl-2-hydroxybutanoic acid (180 mg, 0.26 mmol) in DCM (20 mL) was added HOSu (105 mg, 0.92 mmol), EDC (175 mg, 0.92 mmol). After stirred for 30 minutes, methylamine in THF (2 N, 0.92 mL) was added to above mixture. The reaction mixture was stirred for 18 hours and then concentrated in vacuo. The residue was purified by Gilson Prep to afford title compound (50 mg). ¹H-NMR (CDCl₃): δ 7.53-7.26 (m, 5H), 6.83 (NH, 1H), 5.25 (NH, 1H), 5.05 (m, 2H), 4.25-3.89 (m, 3H), 2.70 (m, 3H), 1.4 (m, 1H), 0.86 (m, 1H), 0.61 (m, 1H), 0.38 (m, 2H), 0.33 (m, 2H). (M+1) 307.

The following compounds can be prepared in the similar manner by using appropriate amines, followed by hydrogenation.

embedded image

The following compounds can be prepared in the methods described by Perni, R. et al. in WO 01/74768, which is incorporated herein by reference in its entirety.

embedded image

Preparation of (S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoic acid

embedded image

In a 5 L RB flask dissolved t-butyl glycine (74 g, 0.56 mol, 1.02 eq.) in saturated sodium bicarbonate (11 vol). Cyclopentyl 2,5-dioxopyrrolidin-1-yl carbonate (126 g, 0.55 mol, 1 eq.) was dissolved in acetone (5.5 vol) and the solution slowly added via addition funnel at room temperature to the solution of the glycine. The reaction mixture was stirred at room temperature until complete (approximately 4 hours). The acetone was removed under reduced pressure and the remaining aqueous solution was extracted with 30% ethyl acetate in hexanes (thrice, 5.5 vol each). The organic layers were discarded. The pH of the aqueous layer was adjusted to 2 with 2 N HCl and then extracted with ethyl acetate (thrice, 5.5 vol). The combined organic layers were dried (Na₂SO₄), filtered, and the solvent removed under reduced pressure to provide a clear oil the slowly crystallized. The crude product was crystallized from hexanes/ethyl acetate to provide (S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoic acid as a white solid (82 g). The mother liquid was stripped and a second crop of crystals obtained (combined yield 105.54 g).

Preparation of Sulfonyl Compounds

embedded image

Compounds S1, S2, S3, and S4, shown above, were prepared according to procedures described in WO 2005/095403 and PCT/US2005/010494, hereby incorporated by references by their entireties. Specifically, to a solution of chlorosulfonylisocyanate (10 mL, 115 mmol) in CH₂Cl₂(200 mL) at 0° C. was added t-BuOH (11 mL, 1 eq.). The mixture was stirred for 60 minutes, then added via cannula into a solution of cyclopropylamine (6.6 g) in CH₂Cl₂(200 mL) with triethylamine (30 mL) at 0° C. concurrently with a solution of triethylamine (50 mL) in CH₂Cl₂(100 mL) via addition funnel. Internal temperature was maintained below 8° C. Stirred at room temperature after completion of addition for 4 hours. The reaction was then diluted with CH₂Cl₂and transferred to a separatory funnel, washed with 1 N HCl (twice, 400 mL each), brine (300 mL), dried (MgSO₄), filtered and concentrated. The product was recrystallized from ethyl acetate/hexanes to yield 16.8 g (71.3 mmol) of S3. Compound S3 was deprotected with trifluoroacetic acid in CH₂Cl₂to give compound S4.

embedded image

Ammonia gas was bubbled through a gas dispersion tube into THF (40 mL) cooled to 0° C. for 5 minutes. To this solution at 0° C. was added cyclopropylsulfonylchloride (1 gram, 7.1 mmol). The reaction was stirred at room temperature overnight, then filtered through a plug of silica gel, followed by elution with EtOAc to yield 750 mg (6.19 mmol) of cyclopropylsulfonamide. ¹H-NMR (500 MHz, Methanol-d₄): 4.79 (s, 2H), 2.59-2.54 (m, 1H), 1.06-0.96 (m, 4H).

embedded image

To a solution of compound XX5 (1.37 g, 6.41 mmol) in THF (30 mL) at 0° C. was added dropwise borane-dimethylsulfide (3.85 mL, 7.8 mmol, 2.0 M in toluene). The reaction mixture was stirred for 1 h with gradual warming to room temperature, quenched with H₂O (20 mL), and extracted with ethyl acetate (thrice, 30 mL each). The combined organics were dried and concentrated under reduced pressure to provide 1.3 g of a colorless oil which was used without further purification. To oxalyl chloride (2.24 mL, 25.6 mmol) in CH₂Cl₂(15 mL, anhydrous) at −78° C. under inert atmosphere was added dropwise a solution of DMSO (2.73 mL, 38.5 mmol) in CH₂Cl₂(8 mL). After stirring for 10 min, a solution of the alcohol (1.3 g, 6.41 mmol) in CH₂Cl₂(6 mL) was added dropwise. After an additional 10 min, triethylamine (7.15 mL, 51.3 mmol) in CH₂Cl₂was added and the reaction was stirred another 30 min with gradual warming to 0° C. The reaction mixture was washed with 1 M HCl (20 mL) followed by brine (20 mL). The organic layer was dried over MgSO₄and concentrated under reduced pressure. The resulting oil was purified via silica gel chromatography to afford 748 mg (over 2 steps) of aldehyde XX6. ¹H-NMR (500 MHz, CDCl₃): 9.75 (s, 1H), 3.67 (s, 3H), 2.91-2.85 (m, 1H), 2.78-2.74 (m, 1H), 2.56-2.52 (m, 1H), 1.74-1.71 (m, 2H), 1.66-1.58 (m, 4H), 1.27-0.95 (m, 5H).

To a solution of compound XX6 (581 mg, 2.9 mmol) and K₂CO₃(811 mg, 5.9 mmol) in MeOH (15 mL) was added dimethyl 1-diazo-2-oxopropylphosphonate (676 mg, 3.5 mmol, Synlett 1996, p. 521). The reaction was stirred 1 h at room temperature, diluted wth Et₂O (20 mL), and washed with saturated NaHCO₃solution (10 mL, aqueous). The organic layer was dried over MgSO₄and concentrated under reduced pressure to give 600 mg of alkyne XX7 which was used without further purification. ¹H-NMR (500 MHz, CDCl₃): 3.69 (s, 3H), 2.48-2.37 (m), 1.95 (s, H), 1.73-1.60 (m), 1.30-0.94 (m).

To a solution of compound XX7 (600 mg, 2.9 mmol) in a solution of THF/H₂O/MeOH (25 mL, 2:1:2) was added LiOH monohydrate (850 mg, 20.3 mmol). The reaction mixture was stirred 2 h at room temperature, acidified using 1 N HCl (25 mL), and extracted with EtOAc (thrice, 15 mL each). The combined organics were dried over MgSO₄and concentrated to yield 533 mg of carboxylic acid XX8, which was used without further purification.

embedded image

To a solution of compound XX5 (100 mg, 0.5 mmol) in CH₂Cl₂(2.5 mL) was added EDC (107 mg, 0.6 mmol), HOBt (76 mg, 0.6 mmol) and triethylamine (195 μL, 1.4 mmol). To the activated acid solution was added methylamine hydrochloride (38 mg, 0.6 mmol) and the reaction was stirred at room temperature for 12 h. The reaction mixture was washed with H₂O (2 mL), 1 N HCl (2 mL) and saturated NaHCO₃solution (2 mL). The organic layer was dried over MgSO₄and concentrated to give 100 mg of amide XX9, which was used without further purification. ¹H-NMR (500 MHz, CDCl₃) 3.61 (s, 3H), 2.75-2.70 (m, 4H), 2.48-2.42 (m, 1H), 2.28-2.24 (m, 1H), 1.66-1.48 (m, 6H), 1.35-0.90 (m, 5H).

To a solution of compound XX9 (100 mg, 0.5 mmol) in a solution of THF/H₂O/MeOH (3 mL, 2:1:2) was added LiOH monohydrate (124 mg, 3 mmol). The reaction mixture was stirred 2 h at room temperature, acidified using 1 N HCl (4 mL), and extracted with EtOAc (3×5 mL). The combined organics were dried over MgSO₄and concentrated to yield 87 mg of carboxylic acid XX10, which was used without further purification. ¹H-NMR (500 MHz, CDCl₃) 11.32 (s, H), 2.75-2.64 (m, H), 2.52-2.46 (m, H), 2.37-2.33 (m, H), 2.25 (td, J=8.7, 2.9 Hz, H), 1.97 (s, H), 1.79 (s, H), 1.74-1.62 (m, H), 1.59-1.49 (m, H), 1.23-1.12 (m, H), 1.08-0.81 (m, H).

embedded image

Intermediate XX12 was prepared according to the procedure for preparing intermediate XX10 described above, except for using pyrrolidine as a reagent instead of methylamine hydrochloride. ¹H-NMR (500 MHz, CDCl₃) 11.47 (s, 1H), 3.45-3.32 (m, 4H), 2.76-2.72 (m, 1H), 2.64-2.59 (m, 1H), 2.37-2.33 (m, 1H), 1.92-1.76 (m, 4H), 1.71-1.57 (m), 1.22-0.84 (m).

embedded image

To a solution of compound XX5 (1 g, 4.7 mmol) and HgO yellow (1.01 g, 4.7 mmol) in CCl₄(23 mL) at reflux was added dropwise over 30 min a solution of bromine (264 μL, 5.1 mmol) in CCl₄(5 mL). The reaction was stirred at reflux for 1 h, cooled to room temperature, diluted with CH₂Cl₂(20 mL), washed with 1 N HCl (10 mL), H₂O (10 mL), and brine (10 mL). The organic layer was dried over MgSO₄and concentrated under reduced pressure to yield 1.3 g of compound XX13 as a colorless oil that was used without further purification. ¹H-NMR (500 MHz, CDCl₃): 3.67 (s, 3H), 3.52-3.44 (m, 2H), 2.63-2.58 (m, 1H), 1.70-1.64 (m, 3H), 1.60-1.54 (m, 3H), 1.24-0.92 (m, 5H).

To a solution of compound XX13 (578 mg, 2.3 mmol) in DMSO (12 mL) was added sodium borohydride (177 mg, 4.7 mmol). The reaction mixture was stirred at 90° C. for 1 h, diluted with H₂O (10 mL), and extracted with hexanes (3×15 mL). The combined organics were dried over MgSO₄and concentrated under reduced pressure. Purification via silica gel chromatography, eluting with EtOAc/petroleum ether, afforded 204 mg of compound XX14. ¹H-NMR (500 MHz, CDCl₃): 3.59 (s, 3H), 2.18 (m, 1H), 1.69-1.43 (m, 6H), 1.21-0.83 (m, 8H).

Intermediate XX15 was prepared according to the procedure for preparing intermediate XX10, step b, except for using substrate XX14 instead of XX9.

embedded image

To a solution of (S)-2-amino-3,3-dimethylbutanoic acid (787 mg, 6.0 mmol), bromobenzene (632 μL, 6.0 mmol), K₂CO₃(1.24 g, 9.0 mmol) and CuI (114 mg, 0.6 mmol) was added N,N-dimethylacetamide (7.5 mL). The contents were stirred for 16 h at 90° C. in a sealed pressure vessel. The reaction mixture was diluted with H₂O (15 mL), cooled to 0° C., and acidified to pH≈5 using 1 N HCl. The mixture was extracted with EtOAc (3×20 mL), and the combined organics were washed with brine (1×15 mL), dried over MgSO₄, and concentrated under reduced pressure. The resulting residue was purified via silica gel chromatography to provide 150 mg of compound XX16. ¹H-NMR (500 MHz, CDCl₃): 7.11-7.09 (m, 2H), 6.69 (t, J=7.3 Hz, 1H), 6.60-6.59 (m, 2H), 3.69 (s, 1H), 1.02 (s, 9H).

embedded image

Intermediate XX17 was prepared according to the procedure for preparing XX16, except for using 1-bromo-3-methoxybenzene as a reagent instead of bromobenzene. ¹H-NMR (500 MHz, CDCl₃): 6.98 (t, J=8.1 Hz, 1H), 6.24-6.18 (m, 2H), 6.14 (s, 1H), 3.69 (s, 1H), 3.66 (s, 3H), 1.00 (s, 9H).

embedded image

To a solution of (S)-3-(methoxycarbonyl)-4-methylpentanoic acid (200 mg, 1.2 mmol) in CH₂Cl₂(6 mL) was added EDC (264 mg, 1.4 mmol), HOBt (186 mg, 1.4 mmol) and triethylamine (481 μL, 3.5 mmol). To the activated acid solution was added cyclohexylamine (158 μL, 1.4 mmol) and the reaction was stirred 4 hours. The reaction mixture was washed with H₂O (3 mL), 1 NHCl (3 mL), and saturated NaHCO₃solution (3 mL). The organic layer was dried over MgSO₄, and concentrated under reduced pressure to afford 290 mg of compound XX18 which was used without further purification. ¹H-NMR (500 MHz, CDCl₃): 5.78 (d, J=7.5 Hz, 1H), 3.69-3.61 (m, 4H), 2.73-2.69 (m, 1H), 2.45-2.40 (m, 1H), 2.24-2.20 (m, 1H), 1.85 (m, 1H), 1.82-1.76 (m, 2H), 1.63-1.60 (m, 2H), 1.54-1.50 (m, 1H), 1.31-1.22 (m, 2H), 1.12-1.00 (m, 3H), 0.90-0.85 (m, 6H).

Intermediate XX19 was prepared according to the procedure for preparing compound XX10 described above, except for using substrate XX18 as a reagent instead of compound XX9. ES (+) MS: m/e 256 (M+H)⁺.

embedded image

Intermediate XX20 was prepared according to the procedure for preparing compound XX18 or XX19 described above, except for using isopropylamine as a reagent instead of cyclohexylamine. ES (+) MS: m/e 216 (M+H)⁺.

embedded image

Intermediate XX21 was prepared according to the procedure for preparing XX18 or XX19 described above, except for using benzylamine as a reagent instead of cyclohexylamine. ES (+) MS: m/e 264 (M+H)⁺.

embedded image

Glycine methyl ester hydrochloride (50.0 g) was suspended in MTBE (300 mL) at RT. To this was added benzaldehyde (40.5 mL) and anhydrous Na₂SO₄(33.9 g). The suspension was cooled in an ice-water bath for 20 minutes, then triethylamine (80 mL) was added dropwise over 15 minutes. After 5 minutes, the reaction was removed from the ice-water bath, and stirred at RT for 24 hours. The reaction was quenched with 200 mL ice-water mixture and the organic layer was separated. The aqueous layer was extracted with MTBE (200 mL). The organic layers were combined, washed with a 1:1 mixture of brine and saturated NaHCO₃(aq.), dried (MgSO₄), and concentrated to yield 62.83 grams of the N-benzyl imine as a yellow oil. ¹H-NMR (500 MHz, CDCl₃): 8.30 (s, 1H), 7.78-7.77 (m, 2H), 7.45-7.40 (m, 3H), 4.42 (s, 2H), 3.78 (s, 3H).

embedded image

Lithium tert-butoxide (15.13 g) was suspended in dry toluene (200 mL) at room temperature. To this was added dropwise a solution of the N-benzyl imine of glycine methyl ester (16.89 g) and 1,4-dibromo-2-butene (19.28 g) in toluene (100 mL) over 40 minutes. The red solution was stirred for 100 minutes, then quenched with H₂O (200 mL). The contents were transferred to a separatory funnel and diluted with MTBE (200 mL). The layers were separated and the aqueous layer was extracted with MTBE. The combined organic layers were stirred with 1 N HCl (aq.) (500 mL) for 3 hours. The layers were separated and the organic layer was extracted with H₂O (100 mL). The aqueous layers were combined, NaCl (250 g) and MTBE (700 mL) were added and the pH was brought to ≈13 with 10 N NaOH (aq). The organic layer was separated and the aqueous layer was extracted with MTBE (twice, 300 mL each). The organic layers were combined, dried (MgSO₄), and concentrated to a volume of ≈400 mL. To the solution was added di-tert-butyl dicarbonate (25.0 g) and the reaction was stirred for 3 days. Additional di-tert-butyl dicarbonate (5.6 g) was added, followed by heating of the reaction in a 60° C. bath for 1 hour. The reaction was purified by flash silica gel column chromatography with EtOAc/hexane (1:9) as eluent to yield 10.89 g of racemic N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester. See, e.g., WO00/09558 and Beaulieu, P. L. et al., J. Org. Chem., 70 (15), 5869-5879, 2005. ¹H-NMR (500 MHz, CDCl₃): 5.78-5.71 (m, 1H), 5.29-5.26 (m, 1H), 5.11 (dd, J=1.2, 10.3 Hz, 1H), 3.71 (s, 3H), 2.14 (q, J=8.8 Hz, 1H), 1.79 (s, 1H), 1.53-1.45 (m, 10H).

embedded image

Racemic N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester (4.2 g) was dissolved in acetone (80 mL) and then diluted with water (160 mL). The pH was adjusted to 7.8 with 0.2N NaOH (aq). Subtilisin A (product P-5380 from Sigma, St. Louis, Mo., USA) (4.5 g) was added to the solution. Its pH was maintained between 7.4 and 8.7 for 3 days by the dropwise addition of 0.1 N NaOH (aq.). When HPLC analysis (Chiralpak AD from Daicel Chemical Industries, Tokyo, 4.6 mm×250 mm, 0.5 mL/min, 10-85% 2-propanol/hexanes over 10 minutes, monitor 215.4 nm) of the reaction indicated the presence of only the (1R,2S)-enantiomer (retention time of (1R,2S)=6.2 min, (1S,2R)=5.9 min) the pH was brought to 8.5 with 2 N NaOH (aq). The contents of the reaction were transferred to a separatory funnel and extracted with MTBE (3×400 mL). The extracts were washed with saturated NaHCO₃(aq) solution (3×150 mL), water (2×200 mL), and dried (MgSO₄). The solution was filtered, concentrated, diluted with CH₂Cl₂, dried (MgSO₄), filtered, and concentrated to yield 1.95 g of N-Boc-(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester.

embedded image

N-Boc-(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester (125 mg, 0.52 mmol) stirred in CH₂Cl₂/TFA (1:1, 2 mL) at RT for 90 minutes. Solvents removed under vacuum to yield (1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester trifluoroacetic acid salt.

embedded image

Compound XX1 (2.34 g, 9.71 mmol) was stirred with LiOH.H₂O (0.45 g, 10.7 mmol) in THF/H₂O/THF (3:1:0.5, 22 mL) at room temperature overnight. The solvents were evaporated and the remaining solids were taken up in CH₂Cl₂/EtOAc and 1N HCl (aq). The aqueous layer was extracted with CH₂Cl₂and the combined organic extracts were dried (MgSO₄), filtered, and concentrated. This material was dissolved in CH₂Cl₂(10 mL) at room temperature and treated with trifluoroacetic acid (10 mL). HPLC analysis at 70 minutes showed no starting material was present. The solvents were removed in vacuo to yield a viscous light colored oil. This was taken up in additional CH2Cl2 (30 mL) and evaporated on a rotary evaporator to yield a tan solid. This solid was dissolved in saturated NaHCO₃(aq) and acetone (1:1, 50 mL) and treated with Fmoc-Cl (2.65 g, 10.2 mmol). After 4 hours, the contents of the flask were transferred to a separatory funnel with CH₂Cl₂and acidified with 2N HCl (aq). The aqueous layer was extracted with CH₂Cl₂, the combined organic layers were dried (MgSO4) filtered, and concentrated to yield 1.86 g (5.3 mmol) of XX2 as a light yellow solid. (M+1)=350.1

embedded image

PS-Wang resin (2.0 g, 1.0 eq.) swelled in DMF (enough to cover). (1R,2S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-vinylcyclopropanecarboxylic acid (XX3) (922 mg, 1.1 eq.) was stirred in DCM. Diisopropylcarbodiimide (409 uL, 1.1 eq.) was added to the DCM solution and stirred at 4° C. for 2 hours, then added to resin and DMF. Dimethylaminopyridine (29 mg, 0.1 eq.) in DMF was added to resin solution and shaken for 5 hours. Drained and washed with DMF (thrice) and DCM (thrice) to yield Compound XX4.

embedded image

Preparation of 2-(bicyclo[4.1.0]heptan-1-yl)acetic acid X2

Commercially available compound X1 (Aldrich Chemical Co., Milwaukee, Wis., USA) was converted to X2 according to method described by E. J. Kantorowski et al. in J. Org. Chem., 1999, 64, 570-580. ¹H-NMR (CDCl₃, 500 MHz): 9.2 (br s, 1H), 2.23 (m, 2H), 1.92 (m, 1H), 1.76 (m, 2H), 1.58 (m, 1H), 1.34 (m, 1H), 1.18 (m, 4H), 0.85 (m, 1H), 0.52 (dd, 1H), 0.31 (t, 1H) ppm.

embedded image

Preparation of 2-(1-hydroxycyclohexyl)acetic acid X5

Compound X4 was prepared using essentially the procedure described in Bull. Chem. Soc. Jpn., 1971, 44, 1090. Specifically, A solution of ethylbromoacetate (8.3 mL) (Aldrich Chemical Co., Milwaukee, Wis., USA) in toluene was added dropewise at 80° C. over 30 min. to a thoroughly stirred mixture of cyclohexanone X3 (4.9 g) and zinc powder (4.9 g) in toluene. The addition was carefully monitored and the temperature was kept at 80° C. After the addition was completed, the mixture was refluxed for 90 min., cooled, decomposed with 1N aqueous HCl, and extracted with Et₂O. The organics were washed with water, aq. NaHCO₃, dried (MgSO₄) and concentrated in vacuo to yield X4 (5.9 g). ¹H-NMR (CDCl₃, 500 MHz) 4.16 (t, 2H), 3.0 (br s, 1H), 2.46 (s, 2H), 1.40-1.69 (m, 10H), 1.27 (t, 3H) ppm; FIA m/z 187.1 ES⁺.

To a solution of X4 (510 mg) in MeOH was added 1N aqueous NaOH. The reaction mixture was stirred at 60° C. for 1 h, and then concentrated in vacuo. The residue was diluted with water, washed with Et₂O and the aqueous layer acidified with 1N aqueous citric acid and extracted with EtOAc. The organics were dried (MgSO₄) and concentrated in vacuo to yield after recrystallization compound X5 (220 mg): ¹H-NMR (CDCl₃, 500 MHz) 3.63 (s, 1H), 2.45 (s, 2H), 1.22-1.64 (m, 10H) ppm; FIA m/z 157.2 ES⁻.

embedded image

Preparation of 2-(1-methylcyclohexyl)acetic acid (X8)

Commercially available compound X6 (Aldrich Chemical Co., Milwaukee, Wis., USA) was converted to compound X7 according to the method described by N. Asao et al. in Tetrahedron Lett., 2003, 44, 4265. ¹H-NMR (CDCl₃, 500 MHz): 4.12 (q, 2H), 2.22 (s, 2H), 1.30-1.48 (m, 10H), 1.25 (t, 3H), 1.01 (s, 3H) ppm.

To a solution of compound X7 in EtOH was added 1 N aqueous NaOH. The reaction mixture was stirred at 50° C. for 3 hours, and then concentrated in vacuo. The residue was diluted with water, washed with Et₂O and the aqueous layer acidified with 1 N aqueous citric acid and extracted with CH₂Cl₂. The organics were dried (MgSO₄) and concentrated in vacuo to yield compound X8. ¹H-NMR (CDCl₃, 500 MHz): 11.7 (s, 1H), 2.26 (s, 2H), 1.32-1.49 (m, 10H), 1.05 (s, 3H) ppm.

embedded image

Preparation of 2-(4-methyltetrahydro-2H-pyran-4-yl)acetic acid (X12)

To a solution of dihydro-2H-pyran-4(3H)-one (X9) (3.13 g, from Aldrich) in toluene was added (carbethoxymethylene)-triphenylphosphorane (12.0 g, Aldrich). The solution was stirred at 110° C. for 3 days. The resulting dark solution was concentrated in vacuo and the residue directly purified by column over silica gel to yield compound X10 (4.54 g) as a clear liquid. ¹H-NMR (CDCl₃, 500 MHz): 5.66 (s, 1H), 4.16 (q, 2H), 3.98 (s, 4H), 3.00 (t, 2H), 2.38 (m, 2H), 1.77 (m, 4H), 1.27 (t, 3H) ppm.

Compounds X11 and X12 were obtained in a similar manner as described for compounds X7 and X8. ¹H-NMR (CDCl₃, 500 MHz): 3.64-3.73 (m, 4H), 2.35 (s, 2H), 1.65 (ddd, 2H), 1.50 (ddt, 2H), 1.17 (s, 3H) ppm.

embedded image

Preparation of 2-(cis-2,6-dimethyltetrahydro-2H-pyran-4-yl)acetic acid (X16)

Intermediate X13 was prepared from commercially available 2,6-dimethyl-g-pyrone (Aldrich Chemical Co., Milwaukee, Wis., USA). A solution of the g-pyrone was dissolved in EtOH and hydrogenated (2 atm. H₂) with 10% Pd/C over 2 h. The catalyst was subsequently filtered off and the solution was concentrated in vacuo to yield crude X13 which was purified by column chromatography to yield pure compound X13. ¹H-NMR (CDCl₃, 500 MHz): 3.72 (m, 2H), 2.35 (m, 2H), 2.21 (dd, 2H), 1.32 (d, 6H) ppm.

Compound X14 was then obtained from compound X13 in a similar manner as described for compound X10. ¹H-NMR (CDCl₃, 500 MHz): 5.65 (s, 1H), 4.15 (q, 2H), 3.80 (dt, 1H), 3.49 (m, 2H), 2.17 (dt, 1H), 2.07 (dd, 1H), 1.79 (dt, 1H), 1.28 (m, 9H) ppm. LC-MS m/z 199.126 ES⁺.

A solution of compound X14 in EtOAc was then hydrogenated (1 atm. H₂) with 10% wet Pd/C over 1 hour. The catalyst was subsequently filtered off and the solution was concentrated in vacuo to yield crude compound X15 which was used without further purification for the next step. Compound X16 was then prepared from compound X15 in a similar manner as described for compound X8. ¹H-NMR (CDCl₃, 500 MHz) major diastereomer: 3.50 (m, 2H), 2.27 (d, 2H), 2.07 (m, 1H), 1.71 (m, 2H), 1.19 (d, 6H) 0.92 (m, 2H) ppm; major diastereomer: 3.64 (m, 2H), 2.56 (d, 2H), 2.47 (m, 1H), 1.49 (m, 2H), 1.15 (d, 6H), 0.86 (m, 2H) ppm.

embedded image

Preparation of 2-(1,4-dioxaspiro[4.5]decan-8-yl)acetic acid X20

Compound X20 was prepared from compound X17 (from Aldrich) according to the procedures described above for preparing compound X16.

Compound X18: ¹H-NMR (CDCl₃, 500 MHz): 5.66 (s, 1H), 4.15 (q, 2H), 3.98 (s, 4H), 3.00 (m, 2H), 2.38 (m, 2H), 1.77 (m, 4H), 1.27 (t, 3H) ppm.

Compound X19: ¹H-NMR (CDCl₃, 500 MHz): 4.12 (q, 2H), 3.93 (s, 4H), (d, 2H), 1.83 (m, 1H), 1.72 (m, 4H), 1.56 (dt, 2H), 1.33 (m, 2H), 1.30 (m, 3H) ppm.

Compound X20: ¹H-NMR (CDCl₃, 500 MHz): 3.93 (s, 4H), 2.28 (d, 2H), 1.73-1.86 (m, 4H), 1.57 (dt, 2H), 1.35 (m, 2H) ppm.

embedded image

Preparation of 2-(trans-2,6-dimethyltetrahydro-2H-pyran-4-yl)acetic acid 25

Compounds X21 and X22 were prepared according to the method described by S. Danishefsky et al. in J. Org. Chem. 1982, 47, 1597-1598 and D. S. Reddy et al. in J. Org. Chem. 2004, 69, 1716-1719, respectively. Compound X25 was prepared from compound X22 according to the method described above for preparing compound X16.

Compound X23. ¹H-NMR (CDCl₃, 500 MHz): 5.72 (s, 1H), 4.16 (q, 2H), 4.08 (q, 2H), 3.06 (dd, 1H), 2.75 (dd, 1H), 2.39 (dd, 1H), 2.05 (dd, 1H), 1.28 (t, 3H), 1.19 (m, 6H) ppm.

X25: ¹H-NMR (CDCl₃, 500 MHz) 4.24 (m, 1H), 3.78 (m, 1H), 2.25 (m, 3H), 1.71 (m, 1H), 1.53 (m, 1H), 1.46 (m, 1H), 1.29 (d, 3H), 1.13 (d, 3H), 0.90 (m, 1H) ppm.

embedded image

Preparation of 2-(4-hydroxy-4-methylcyclohexyl)acetic acid X27

A solution of compound X20 in dioxane was treated with 4N HCl in dioxane. The reaction solution was stirred at room temperature for 4 hours and concentrated in vacuo to give crude compound X26 which was used without further purification for the next step. To a stirred solution of compound X26 in THF was slowly added MeMgBr (3 N in THF). The resulting mixture was stirred at 40° C. for 3 hours, quenched with 1 N aqueous citric acid and diluted with EtOAc. The phase were separated and the organics were dried (MgSO₄), concentrated in vacuo and purified by chromatography over silica gel to give compound X27 as a mixture of two diastereomers: isomer 1: ¹H-NMR (CDCl₃, 500 MHz): 4.50 (br s), 2.27 (m, 2H), 1.75 (m, 1H), 1.65 (m, 4H), 1.39 (m, 4H), 1.22 (s, 3H) ppm; isomer 2: ¹H-NMR (CDCl₃, 500 MHz): 2.12 (m, 2H), 1.69 (m, 3H), 1.56 (m, 2H), 1.39 (m, 2H), 1.12 (s, 3H), 1.05 (m, 2H) ppm.

embedded image

Preparation of 2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)acetic acid

To a solution of the methyl ester (500 mg; 2.69 mmol) in THF (21.5 mL), MeOH (21.5 mL) and water (10.75 mL) was added LiOH (1 N; 10.75 mL). The reaction mixture was stirred for 3 hours. The reaction was acidified with HCl (1 N, pH=5). The product was extracted with EtOAc (twice, 20 mL each). The combined organic layer was then wash with water, brine and concentrated in vacuo to afford 420 mg of 2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)acetic acid. ¹H-NMR (CDCl₃): δ 3.76-3.67 (m, 2H), 2.56-2.19 (m, 3H), 1.63 (m, 2H), 1.26-1.10 (m, 8H). (M+1) 173.

embedded image

To a solution of compound X30 (64 g, 237 mmol) and EDC (226 g, 1.19 mol) in EtOAc (1.5 L) was added DMSO (400 mL), and the resulting suspension was cooled to 0° C. To this mixture was added a solution of dichloroacetic acid in EtOAc (1:1 v/v, 130 mL) keeping the internal reaction temperature below 25° C. The reaction was warmed to room temperature, stirred for 15 minutes, cooled to 0° C., and quenched with 1 N HCl (1 L). The organic layer was separated, washed with H₂O (2×500 mL), dried over MgSO₄, and concentrated under reduced pressure. The resulting oil was filtered through a plug of silica eluting with EtOAc/hexanes to afford 48 g of compound X31 as a white solid.

To resin X32 (prepared according to the procedure described in WO 00/23421) (100 g, 0.88 mmol/g) was added a solution of X31 (48 g, 179 mmol) in THF (650 mL), followed by AcOH (30 mL). The mixture was shaken for 16 hours, and the resin was filtered, washed with THF (4 times, 400 mL each) and CH₂Cl₂(4 times, 400 mL each) and dried in vacuo. The filtrate and washes were combined and concentrated, and the above procedure was repeated to afford resin X33 with a loading of approximately 0.4 mmol/g.

Preparation of Aldehyde Compounds

5-chloronicotinaldehyde was prepared according to methods described by D. L. Comins et al. in Hetereocycles, 1987, 26 (8), pp. 2159-2164.

Some other aldehydes such as 2-fluoro-5-chlorobenzaldehyde, 2-methoxy-3-methyl benzaldehyde, 2-methoxynicotinaldehyde, 2,3-dihydrobenofuran-7-carbaldehyde can be made from corresponding acid based on following procedure:

embedded image

Preparation of 2,3-dihydrobenzofuran-7-carbaldehyde

2,3-Dihydrobenzofuran-7-carboxylic acid (820 mg, 5 mmol) was dissolved in THF (10 mL). To the solution was added TEA (0.7 mL, 5 mmol) and methylchloroformate (0.43 mL, 5 mmol). The solution was stirred for 0.5 hour. The white precipitates were removed by filtration, the filtrate was added to a solution of NaBH₄(437 mg, 12.5 mmol) in H₂O (5 mL). The resulting solution was stirred overnight. The reaction mixture was neutralized with 2 M aqueous HCl solution and then extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na₂SO₄and concentrated in vacuo. The crude alcohol was dissolved in DCM. To the solution was added PCC (1.83 g, 7.5 mmol). The mixture was stirred for 2 hours at room temperature and diluted with diethyl ether, then ether layers were decanted. Combined organic layer was filtered though a layer of Celite®. The filtrate was concentrated to give crude product. The crude was purified from column with 10% EtOAc/hexane to afford 450 mg of 2,3-dihydrobenzofuran-7-carbaldehyde as a slightly yellow solid. HPLC 4.3 min.

embedded image

Preparation of 4-chloropicolinaldehyde

A suspension of MnO₂(7.3 g, 84 mmol) and (4-chloro-pyrindin-2-yl)methanol (1 g, 7 mmol) in CHCl₃was heated to refulx for 90 minutes. The mixture was filtered though a layer of Celite® and concentrated in vacuo to afford 520 mg of 4-chloropicolinaldehyde as a white solid. HPLC 1.8 minutes and MS 142 as M=1 peak.

embedded image

Preparation of 3-chloro-5-methoxybenzaldehyde

A mixture of 3-chloro-5-methoxybenzyl alcohol (5.0 g, 28.9 mmol) and pyridinium chlorochromate (20% on alumina, 40 g, 37.8 mmol) was allowed to stir for 1.25 hr. Diethyl ether (200 ml) was then added followed by filtration of precipitate. The filtrate was concentrated under reduced pressure and the resulting residue was purified via silica gel chromatography using 40% dichloromethane, 60% petroleum ether as eluant, to give 3.8 g of 3-chloro-5-methoxybenzaldehyde. ¹H-NMR (CDCl₃): 3.84 (s, 3H) 7.13 (s, 1H), 7.28 (s, 1H), 7.41 (s, 1H), 9.89 (s, 1H).

embedded image

Preparation of 1-(bromomethyl)-3-chloro-5-methylbenzene

To a solution of m-chloroxylene (0.96 g, 6.8 mmol) in carbon tetrachloride at reflux was added N-bromosuccinmide (1.4 g, 7.5 mmol) followed by benzoyl peroxide (1.6 g, 6.8 mmol). The reaction was allowed to stir for 20 minutes and cooled to room temperature, filtered off precipitate and the filtrate was concentrated under reduced pressure and the resulting residue was purified via silica gel chromatography using petroleum ether as eluant to give 0.89 g of 1-(bromomethyl)-3-chloro-5-methylbenzene. NMR (CDCl₃): 2.31 (s, 3H) 4.37 (s, 2H) 7.09 (s, 1H) 7.12 (s, 1H) 7.20 (s, 1H).

embedded image

Preparation of 3-chloro-5-methylbenzaldehyde

To a solution of sodium metal (52 mg, 2.3 mmol) in ethanol was added 2-nitropropane (0.23 g, 2.4 mmole) followed by the addition of 3-chloro-5-methybenzylbromide (0.5 g, 2.3 mmol). The reaction was allowed to stir for 3 hours and the precipitate formed was filtered off. The filtrate was concentrated under reduced pressure, redissolved in diethylether and washed with 1N sodium hydroxide (twice), water, and dried over sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The resulting residue was purified via silica gel chromatography using 10% dichloromethane and 90% petroleum ether, to give 0.15 g of 3-chloro-5-methylbenzaldehyde. ¹H-NMR (CDCl₃): 2.46 (s, 3H) 7.43 (s, 1H) 7.56 (s, 1H) 7.68 (s, 1H), 9.92 (s, 1H).

embedded image

3-Chloro-5-fluoro-4-hydroxybenzaldehyde (1.0 gram, 5.7 mmol) in THF (40 mL) was heated at reflux for 17 hours with KOH (534 mg, 9.5 mmol, 1.7 eq) in water (5 mL) and iodoethane (1 mL, 2.2 eq). The reaction was then transferred to a separatory funnel with water and extracted with methylene chloride (thrice, 150 mL each). The combined organic layers were washed with 10% aqueous HCl (40 mL), dried (MgSO₄), and concentrated to a viscous orange liquid to yield 1.13 g of 3-chloro-4-ethoxy-5-fluorobenzaldehyde. ¹H-NMR (500 MHz, CDCl₃): 9.84 (d, J=1.9 Hz, 1H), 7.71 (t, J=1.6 Hz, 1H), 7.53 (dd, J=1.9, 10.7 Hz, 1H), 4.37-4.32 (m, 2H), 1.47-1.40 (m, 3H).

embedded image

4-Ethoxy-3,5-dimethylbenzaldehyde was prepared in a manner similar to that of 3-chloro-4-ethoxy-5-fluorobenzaldehyde. ¹H-NMR (300 MHz, CDCl₃): 9.89 (s, 1H), 7.56 (s, 2H), 3.91 (q, 7 Hz, 1H), 2.34 (s, 6H), 1.44 (t, J=7 Hz, 6H).

embedded image

4-Isopropoxy-3,5-dimethylbenzaldehyde was prepared in a manner similar to that of 4-Ethoxy-3,5-dimethylbenzaldehyde. ¹H-NMR (300 MHz, CDCl₃): 9.88 (s, 1H), 7.55 (s, 2H), 4.31 (q, J=6 Hz, 1H), 2.32 (s, 6H), 1.32 (d, J=6 Hz, 6H).

embedded image

4-(Cyclopropylmethoxy)-3,5-dimethylbenzaldehyde was prepared in a manner similar to that of 4-Ethoxy-3,5-dimethylbenzaldehyde. ¹H-NMR (300 MHz, CDCl₃): 9.87 (s, 1H), 7.55 (s, 2H), 3.69 (d, J=7 Hz, 2H), 2.35 (s, 6H), 1.35-1.23 (m, 1H), 0.67-0.060 (m, 2H), 0.35-0.30 (m, 2H).

Preparation of (S)-1-(tert-butoxycarbonyl)-4-oxopyrrolidine-2-carboxylic acid

embedded image

A solution of (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (1.0 eq.) in isopropyl acetate (5 vol) was cooled to 0° C. and TEMPO (0.05 eq.) was added. A solution of bleach (12.5 wt %, 1.2 eq., 2.6 vol) was then slowly added over 1 hour while maintaining the temperature at 0-5° C. The mixture was stirred and monitored by HPLC for completion, then aqueous 10% KHSO₄(2.5 vol) was added, stirred for 10 minutes, and then the phases were separated. The organic phase was washed with aqueous 5% Na₂SO₃(2 vol) then brine (1 vol) then dried azeotropically and concentrated to afford the title compound as a solid. The solid was triturated with acetonitrile (1.0 vol) to remove residual color and impurities. ¹H-NMR (400 MHz, DMSO): δ 4.54 (m, 1H), 3.82 (m, 1H), 3.67 (m, 1H); 3.15 (m, 1H); ≈2.50 (m, 1H, coincides with DMSO); 1.42 and 1.39 (2 s rotamers, 9H).

Preparation of (S)-1-(tert-butoxycarbonyl)-4-methylenepyrrolidine-2-carboxylic acid

embedded image

To a suspension of methyltriphenylphosphonium bromide (2.2 eq.) in 2-methyl tetrahydrofuran (3 vol) was added rapidly solid potassium tert-butoxide (2.3 eq.) maintaining the temperature around 0° C. The temperature was kept at +20° C. for 2 hours (a suspension remained) and re-cooled to 0° C. Keeping the temperature below 6° C., (S)-1-(tert-butoxycarbonyl)-4-oxopyrrolidine-2-carboxylic acid (1 eq.) was added over 40 minutes. The reaction was warmed to room temperature and stirred for 16 h and then cooled to 0° C. The reaction was quenched with saturated NaHCO₃(5 vol) and water (2 vol) and the aqueous layer was separated. The organic layer was extracted with saturated NaHCO₃/water (1.8 vol/1.8 vol) and the combined aqueous layers were filtered through Celite®. The aqueous layer was acidified with 6 N HCl (2.6 vol) at ambient temperature and extracted twice with isopropyl acetate (16 vol, then 8 vol). The organic phase was dried (MgSO₄) and the solvent removed. The crude product was dissolved in isopropyl acetate (10 vol) and extracted with 0.5 M NaOH (10 vol, then 1 vol). The combined aqueous layers were acidified at ambient temperature with 6 N HCl to pH=3, and extracted twice with ethyl acetate (10 vol, then 8 vol). The combined extracts were dried (Na₂SO₄), the solvent removed and the crude product was recrystallized from cyclohexane (5 vol) to afford the title compound. ¹H-NMR (400 MHz, DMSO): δ 12.9, (broad, 1H); 5.00 (m, 2H); 4.24 (dt, J=1.9H, J=7.3 Hz, 1H), 3.91 (m, 2H); 2.98 (m, 1H); ≈2.50 (m, 1H, coincides with DMSO); 1.41 and 1.36 (2 s rotamers, 9H).

Preparation of (5S,8S)-tert-butyl 3-(3-chlorophenyl)-1-oxa-2,7-diazaspiro[4.4]non-2-ene-8-carboxylate

embedded image

A solution of 3-chloro-N-hydroxybenzimidoyl chloride (175 g, 0.919 moles) in EtOAc (2.1 L) was added to a solution of (S)-di-tert-butyl 4-methylenepyrrolidine-1,2-dicarboxylate (200 g, 0.707 moles) in EtOAc (2.0 L) at room temperature. The mixture was cooled below 10° C. in an ice bath, then triethylamine (128 mL, 0.919 moles) was added slowly. The resultant mixture was stirred overnight then quenched with water (3 L). The phases were separated and the organic phase washed with water (2×1.0 L), dried over MgSO₄, and the solvent removed to afford a mixture of the syn- and anti-spiroisoxazolines as an oil.

The mixture of isomers was dissolved in THF (0.72 L) and cooled to 20° C. Methanesulfonic acid (150 mL) was slowly added maintaining 20 to 30° C. The mixture was stirred at 25° C. and quenched after 7 hours by carefully adding a solution K₂CO₃(300 g) in water (1 L). The phases were separated and the aqueous phase was extracted with isopropyl acetate (1 L). The organic phases were combined and approximately half of the solvent removed under vacuum. The solution was washed with a 1:1 mixture of saturated brine (250 mL) and water (250 mL). The aqueous phase was extracted with isopropyl acetate (200 mL) and the organic phases combined then dried over K₂CO₃and filtered to afford a homogeneous solution. The solution volume was made up to 3 L by adding isopropyl acetate and then a solution of oxalic acid (20 g) in isopropyl acetate (400 mL) was slowly added. The solid was isolated by filtration and dried in a vacuum oven. The solid was suspended in isopropyl acetate (1.5 L) and water (1.0 L) then K₂CO₃was added slowly until the solids fully dissolved. The organic layer was isolated, dried over K₂CO₃, filtered then a solution of oxalic acid (12.5 g) in isopropyl acetate (250 mL) was added slowly. The solid was isolated by filtration and dried in a vacuum oven to give the spiroisoxazolines as a 98:2 anti-:syn-mixture of diastereomers. ¹H-NMR (400 MHz, DMSO-d₆): δ 7.67-7.48 (m, 4H), 4.08 (dd, J=7.9, 8.9 Hz, 1H), 3.55 (s, 2H), 3.27 (d, J=4.0 Hz, 2H), 2.46 (dd, J=7.8, 13.8 Hz, 1H), 2.19 (dd, J=9.1, 13.8 Hz, 1H), 1.46 (d, J=7.5 Hz, 9H).

embedded image

Compound M2B (1.0 g, 1.0 eq) was stirred in 20 mL benzene with benzoylnitromethane (583 mg, 1.0 eq.) and catalytic triethylamine. Phenyl isocyanate (880 uL) was added slowly and stirred for 40 hours. Dark colored precipitate was filtered off and to the filtrate was added 2 mL water and the mixture was stirred for 2 hours. Organics were separated and concentrated, purified by silica gel chromatography (10-90% ethyl acetate/hexanes gradient) to give 350 mg of Compound X37. (M+H=431.2.) ¹H-NMR (500 MHz, CDCl₃): 8.19 (d, 2H), 7.61 (t, 1H), 7.56-7.46 (m, 2H), 4.45-4.36 (m, 1H), 3.99-3.88 (m, 1H), 3.61 (d, 1H), 3.39-3.33 (m, 2H), 2.77 (m, 1H), 2.17-2.12 (m, 1H), 1.49 (s, 9H) 1.46 (s, 9H).

embedded image

Compound X37 (1.35 g, 1.0 eq.) was stirred in 20 mL 1/1 TFA/DCM for 2 hours. The mixture was concentrated and to it was added 20 mL acetone, 20 mL saturated sodium bicarbonate solution, and FMOC-Cl (1.22 g, 1.5 eq.). The mixture was stirred for 3 hours and diluted with ethyl acetate and a 2 N HCl solution until aqueous became acidic. The mixture was stirred, aqueous extracted with ethyl acetate, combined organics, dried over magnesium sulfate, filtered, and concentrated. The concentrate was purified by silica gel chromatography (100% DCM-10% MeOH/DCM gradient) to give compound X38. (M+H=497.1).

embedded image

To a solution of 2,3-dihydrobenzofuran 5-carboxaldehyde (1 g, 6.75 mmol) in ethanol (5 mL) was added a 2.4 M of NH₂OH (3.3 mL, 8.1 mmol) solution and then 1.2 M of Na₂CO₃(3.3 mL, 4.05 mmol). The resulting solution was stirred for 2 hours at room temperature (HPLC showed no starting material left). The reaction mixture was diluted with EtOAc, washed with brine, dried over Na₂SO₄and concentrated under vacuum. This afforded 1.0 g of the product as a white solid. ES-MS 164 as M+1 peak.

embedded image

To a solution of aldoxime (426 mg, 2.6 mmol) in DMF (5 mL) was added NCS (697 mg, 5.2 mmol). The resulting mixture was stirred for overnight at room temperature. To the solution was added (S)-di-tert-butyl 4-methylenepyrrolidine-1,2-dicarboxylate, compound 1 (600 mg, 2.1 mmol) and then a solution of TEA (0.37 mL, 2.6 mmol) in DMF (2 mL) was added over 10 minutes. The reaction mixture was stirred for 4 hr at room temperature and then heated to 50-60° C. for 2 hours. The reaction mixture was diluted with EtOAc (20 mL) and washed with H₂O, brine, dried over Na₂SO₄, concentrated in vacuo. The crude products were purified from flash column chromatography eluted with 30% EtOAc/Hexane, to afford S (500-600 mg) (Rf=0.3) and R isomer (150 mg) (Rf=0.2). ES-MS 479 as M+1 peak.

B. Synthesis of Exemplary Compounds of Formula I

Certain exemplary compounds of Formula I may be prepared by Method 1 as illustrated below.

Method 1

embedded image

Referring to Method 1, the exomethylene compound A1 is deprotected to A2, which is converted to the corresponding Fmoc derivative A3. Reaction of the resin bound aminoalcohol A4 with A3 in the presence of a coupling reagent provides the resin bound product A5. A dipolar addition reaction of A5 with the nitrile oxide 1f, generated in situ, provides the resin bound spiroisoxazoline A6, which is deprotected to provide the resin bound spiroisoxazoline A7. Reaction of A7 with an R₁-carboxylic acid in the presence of a coupling agent provides A8, wherein R₁is R₄C(O)—. Cleavage of the spiroisoxazoline from the resin provides the alcohol A9. Oxidation of A9 with an oxidizing reagent such as Dess-Martin periodinane or sodium hypochlorite in the presence of TEMPO provides the final compound A10.

In some instance, R₄may contain an amine functionality. Where R₄contains a protected amine, deprotection of the protected amine to give a free amine, following by a reaction with an activated acid, provides a further elaborated R₄. Alternatively, a free amine in R₄may be converted to the corresponding p-nitrophenylcarbamate followed by reactions with an amine or alcohol to provide R₄compounds containing carbamate or urea functionarity.

Preparation of Allyl 1-(cyclopropylamino)-2-(6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)-1-oxohexan-3-ylcarbamate (M1B)
Step 1: Allyl 1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-ylcarbamate (M1A)

embedded image

To a solution of (3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide (10 g, 53.7 mmol), DIEA (28 mL, 161 mmol, 3 eq.) in methylene chloride (250 mL) was added dropewise at 0° C. to a solution of allylchloroformate (6.8 mL, 64.4 mmol, 1.2 eq.) in DCM (50 mL). The reaction solution was warmed to room temperature and stirred for 4 hours. Water (300 mL) was then slowly added followed by aqueous HCl (1.0 N, 300 mL). The phases were separated and the organics washed with saturated aqueous NaHCO₃(300 mL), brine (300 mL), dried with MgSO₄, filtered, and concentrated in vacuo. The resulting off-white solid was recrystallized from 30% hexanes in EtOAc (120 mL) to yield the title compound M1A as a white solid. The mother liquor was concentrated, in vacuo, and recrystallized from 50% hexanes in EtOAc to yield another 4.04 g of M1A. The mother liquor from the second recrystallization was concentrated in vacuo on Celite®, and the resulting Celite® plug was purified by flash chromatography (Isco Companion®, SiO₂, DCM to 70% EtOAc in DCM) to give 1.46 g of M1A. The total amount of compound M1A was 13.4 g. (Rf≈0.40 in 1:1 DCM:EtOAc, CAM detection).

Step 2: Allyl 1-(cyclopropylamino)-2-(6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)-1-oxohexan-3-ylcarbamate bound resin (M1B)

embedded image

A 500 mL two neck round bottom flask equipped with an overhead mechanical stirrer and a reflux condenser was charged with M1A (9.08 g, 33.6 mmol, 3 eq.), pyridinium p-toluenesulfonate (5.6 g, 22.4 mmol, 2 eq.), DHP-resin (10.2 g, 11.2 mmol, Novabiochem, Cat#01-64-0192, loading: 1.1 mmol/g), and dichloroethane (84 mL, [0.4]_t). The mixture was gently stirred at 80° C. for 3 days, before being cooled to 50° C. and filtered. The resin was washed with DCM (200 mL) and the combined filtrate were concentrated in vacuo to give the resin M1B, which was additionally washed with DCM (twice), DMF (thrice), DCM-MeOH (thrice in succession), Et₂O, and dried under vacuum overnight to yield a light brown resin. The loading of the resin M1B was determined by cleavage of an aliquot (176 mg) of the resin with 90% aq. TFA. Loading: 0.48 mmol/g.

Preparation of (9H-fluoren-9-yl)methyl 2-(1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-ylcarbamoyl)-4-methylenepyrrolidine-1-carboxylate bound resin (M1E)
Step 1: 3-Amino-N-cyclopropyl-2-hydroxyhexanamide bound resin (M1D)

embedded image

Allyl 1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-ylcarbamate bound resin M1B (30 g, 1.0 eq.) was swollen with DCM. 1,3-Dimethylbarbituric acid (24.17 g, 12 eq.) and tetrakis(triphenylphosphine)palladium (1.49 g, 0.1 eq.) were added and the mixture shaken overnight. The mixture was filtered and washed with DMF and DCM to yield the resin M1D.

Step 2: (9H-Fluoren-9-yl)methyl 2-(1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-ylcarbamoyl)-4-methylenepyrrolidine-1-carboxylate bound resin (M1E)

embedded image

Resin M1D (1.0 g, 1.0 eq.) was stirred in DMF with FMOC-4-exomethyleneproline carboxylic acid (248 mg, 1.1 eq.), HBTU (4.8 mL of 0.5 M DMF solution, 5.0 eq.), HOBt (2.4 mL of 1.0 M DMF solution, 5.0 eq.), DIEA (836 uL, 10.0 eq.) for 3 hours. The resulting mixture was drained and washed with DMF (thrice) and DCM (thrice) to give title compound M1E.

Preparation of Fmoc-Protected Isoxazoline Compound Bound Resin (M1F)

embedded image

The resin M1E (2 g, 0.94 mmol) in THF was shaken with 3-chlorobenzaldoxime (5 eq.) and bleach (5% NaOCl) (15 eq.) for 18 hours. The resin was then filtered and washed with water, DMF, and DCM to yield the resin compound M1F. An aliquot of the resin was cleaved to provide a sample for LC-mass analysis (M+1=671).

Preparation of Fmoc Protected Isoxazoline Bound Resin Compound (M1G)

embedded image

The resin M1F was shaken in 20% piperidine/DMF for 10 minutes, filtered, and washed with DMF and DCM. The THP resin bound spiroisoxazoline proline (0.14 mmol, 0.3 g) was mixed with FMOC-L-3-benzothienyl-ALA (0.56 mmol, 0.25 g), HOBT (0.56 mmol, 0.075 g), N,N-diisopropylethylamine (0.56 mmol, 0.072 g), HBTU (0.56 mmol, 0.21 g) in DMF 2.3 mL and was agitated for 48 hours. The resin was filtered and washed with DMF, dichloromethane, and ether to yield the resin compound M1G.

Preparation of 7-((S)-3-(benzo[b]thiophen-3-yl)-2-(2-cyclohexylacetamido)propanoyl)-3-(3-chlorophenyl)-N-((3R)-1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-yl)-1-oxa-2,7-diazaspiro[4.4]non-2-ene-8-carboxamide (M1H)

embedded image

To the THP-resin bound FMOC protected spiroisoxazoline M1G was added 20% piperidine in DMF (3 mL). The mixture was agitated for 1 hour, filtered, and washed with DMF and dichloromethane. The resin was them mixed with cyclohexylacetic acid (0.56 mmol, 80 mg), HOBT (0.56 mmol, 0.075 g), N,N-diisopropylethylamine (0.56 mmol, 0.072 g), HBTU (0.56 mmol, 0.21 g) in DMF 2.3 mL and was agitated for 48 hr. The resin was filtered and washed with DMF, dichloromethane, and ether. The resin obtained was then mixed with a solution of (50:45:5) trifluoroacetic acid, dichloromethane, and triisopropyl silane (3 mL) and was agitated overnight. The reaction was filtered and washed with dichloromethane. The filtrate was concentrated under vacuum and purified via silica gel chromatography using a gradient of 40% ethyl acetate/60% dichloromethane to 100% ethyl acetate to produce the alcohol M1H.

Example 1
Compound No. 336

embedded image

To a solution of the hydroxyamide M1H (14 mg, 0.018 mmol) in 0.38 mL of ethyl acetate was added EDC (35 mg, 0.18 mmol) followed by DMSO (0.070 mL). The mixture was cooled in an ice bath and dichloroacetic acid (15 mg, 0.12 mmol) in ethyl acetate (0.15 mL) was added. The reaction was warmed to room temperature and allowed to stir for 15 minutes and then cooled in an ice bath and quenched with 1.0 N HCl (0.21 mL). The solution was partitioned between ethyl acetate and water. The organic phase was washed with water and dried over sodium sulfate and evaporated solvent under vacuum. The resulting residue was purified by chromatography over silica gel using ethyl acetate and hexanes (3:1) as eluant to give Compound No. 336 as a white solid.

Preparation of (9H-fluoren-9-yl)methyl-8-((3S)-1-(cyclopropylamino)-2-hydroxy-1-oxohexan-3-ylcarbamoyl)-3-phenyl-1-oxa-2,7-diazaspiro[4.4]non-2-ene-7-carboxylate (M1N)

embedded image

Step 1: Fmoc Protected Phenyl-Substituted Isoxazoline Bound Resin (M1L)

The resin M1E (2 g, 0.94 mmol) in THF was shaken with the oxime (5 eq.) and bleach (5% NaOCl) (15 eq.) for 18 hours. The resin was then filtered and washed with water, DMF, and DCM to give the Fmoc protected phenyl-substituted isoxazoline bound resin M1L. An aliquot of resin was cleaved for LC-mass analysis (M+1=637).

embedded image

The resin M1L (0.47 mmol) was shaken in 20% piperidine/DMF for 10 minutes, and then filtered and washed with DMF and DCM. The resulting resin was shaken overnight with a solution of Fmoc-tBG-OH (480 mg 3.0 eq.), HOBT (2.82 mL of 0.5 M in DMF, 3.0 eq.), HBTU (2.82 mL of 0.5 M in DMF, 3.0 eq.), and DIEA (0.493 mL, 6.0 eq.). The resin was then filtered and washed with DMF and DCM to give the resin compound M1M, which was used in next reaction without further purification.

Step 2: Compound M1N

embedded image

The resin M1M (0.47 mmol) was shaken in 20% piperidine/DMF for 10 minutes. The resin was filtered, washed with DMF and DCM. The resulting resin (140 mg, 0.065 mmol) was shaken overnight with benzylisocyanate (176 mg 20.0 eq.), then filtered and washed with DMF and DCM. The resin was shaken with 90% TFA in water for 30 min. The resulting solution was concentrated in vacuo to give the compound M1N (0.065 mmol), (M+1) 661, which was used in next reaction without further purification.

Example 2
Compound No. 107

embedded image

A solution of amide compound M1N in DCM (3 mL) was stirred with Dess-Martin Periodinane (54 mg, 2 eq.) and t-BuOH (54 uL) for 1 hour, and then sodium thiosulfate was added to the mixture. The product was extracted with EtOAc and the combined organic layer was then washed with water, NaHCO₃, brine and concentrated in vacuo and purified by Gilson Prep HPLC to afford Compound No. 107. (M+1) 659.

Example 3
Compound No. 283

embedded image

The THP resin M1M (0.065 mmol) was shaken in 20% piperidine/DMF for 10 minutes, and then filtered and washed with DMF and DCM. The resulting resin was shaken overnight with a solution of 2-(pyridin-3-yl)acetic acid (0.25 mmol 3.0 eq.), HOBT (0.5 mL of 0.5 M in DMF, 3.85 eq.), HBTU (0.5 mL of 0.5 M in DMF, 3.85 eq.), and DIEA (0.5 mmol, 7.69 eq.). The resin was then filtered and washed with DMF and DCM and was shaken with 90% TFA in water for 30 minutes. The resulting solution was concentrated in vacuo to give the hydroxyl amide compound M1P (0.065 mmol) which was used in the next reaction without further purification. (M+1) 647.

A solution of the hydroxyl amide M1P (0.065 mmol) in DCM (3 mL) was stirred with Dess-Martin Periodinane (41 mg, 1.5 eq.) and t-BuOH (41 uL). After stirred for 1 hour, sodium thiosulfate was added to above mixture. The product was extracted with EtOAc. The combined organic layer was then washed with water, NaHCO₃, brine and concentrated in vacuo and purified by Gilson Prep HPLC to afford Compound No. 283 (4 mg). (M+1) 645.

Example 4
Compound No. 61

embedded image

Compound M1E (750 mg, 1.0 eq.) was stirred in benzene with 1-nitropropane (315 uL, 10.0 eq.), and phenylisocyanate (385 uL, 10.0 eq.). Triethylamine (5 uL) was added, and the resulting mixture was shaken overnight, drained, and washed with DMF (thrice) and DCM (thrice). This process was repeated to yield compound M1Q. (M+H=589.0)

Compound M1Q (750 mg, 1.0 eq.) was then shaken in 20% piperidine/DMF for 10 minutes. The resin was filtered and washed with DMF (thrice) followed by DCM (thrice). This process was repeated. The resulting resin was shaken overnight with a solution of (S)-3,3-dimethyl-2-(((S)-tetrahydrofuran-3-yloxy)carbonylamino)butanoic acid (216 mg, 2.5 eq.), HBTU (1.76 mL of 0.5 M in DMF, 3.0 eq.), HOBt (0.88 mL of 1.0 M in DMF, 2.5 eq.), and DIEA (307 uL, 5.0 eq.) in DMF. The resin was then filtered and washed with DMF (thrice) and DCM (thrice) to give compound M1R. (M+H=593.9)

Compound M1R (750 mg, 1.0 eq.) stirred in 1/1 TFA/DCM for 3 hours. The resin was drained and washed with DCM (thrice). All of the organics were concentrated and DCM was added followed by Dess-Martin Periodinane (50 mg, 3.0 eq.). The resulting mixture was stirred for 1 hour, 1 N Na₂S₂O₃was added, and stirred again. A racemic mixture of Compound No. 61 was purified by silica gel chromatography (10-90% ethyl acetate/hexanes gradient) to yield Compound No. 61 as one diastereomer. (M+H=591.8) ¹H-NMR (500 MHz, CDCl₃): 7.12 (d, 1H), 6.91 (d, 1H), 5.48 (d, 1H), 5.34 (td, 1H), 5.24 (s, 1H), 4.69 (t, 1H), 4.28 (d, 1H), 4.13 (s, 2H), 3.93-3.82 (m, 4H), 3.60 (d, 1H), 3.06 (s, 0.5H), 3.03 (s, 0.5H), 2.95 (d, 1H), 2.90 (d, 1H), 2.78 (td, 1H), 2.51-2.47 (m, 1H), 2.44-2.34 (m, 3H), 2.14-2.10 (m, 1H), 1.94-1.88 (m, 1H), 1.63-1.57 (m, 1H), 1.46-1.36 (m, 2H), 1.17 (t, 3H), 0.98 (s, 9H), 0.95-0.83 (m, 5H), 0.59 (dd, 2H)

Example 5
Compound No. 146

embedded image

Compound M1E (50 mg, 1.0 eq.) was stirred in DCM with (Z)-ethyl 2-chloro-2-(hydroxyimino)acetate (7.1 mg, 2.0 eq.). To this mixture was slowly added TEA (6.6 uL, 2.0 eq.) in DCM and the mixture was shaken for 3 hours, then drained and washed with DMF (thrice) and DCM (thrice). This process was repeated to give compound M1S (M+H=632.4).

embedded image

Compound M1S (1.0 g, 1.0 eq.) was shaken in 20% piperidine/DMF for 10 minutes. The resin was filtered and washed with DMF (thrice) followed by DCM (thrice). This process was repeated. The resulting resin was shaken overnight with a solution of (S)-3,3-dimethyl-2-(((S)tetrahydrofuran-3-yloxy)carbonylamino)butanoic acid (230 mg 2.0 eq.), HBTU (1.88 mL of 0.5 M in DMF, 2.0 eq.), HOBt (0.94 mL of 1.0 M in DMF, 2.0 eq.), and DIEA (327 uL, 4.0 eq.) in 2 mL DMF. The resin was then filtered and washed with DMF (thrice) and DCM (thrice) to give compound M1T (M+H=638.0).

embedded image

Compound M1T (750 mg, 1.0 eq.) was shaken in THF with KOTMS (133 mg, 3.0 eq.) for 3 hours. The mixture was then drained and washed with THF/water (1/1), THF, DMF, and DCM (thrice each) to give compound M1U. (M+H=609.5).

embedded image

Compound M1U (250 mg, 1.0 eq.) was shaken overnight with a solution of ethylamine (22 mg 3.0 eq.), HBTU (0.54 mL of 0.5 M in DMF, 3.0 eq.), HOBt (0.27 mL of 1.0 M in DMF, 3.0 eq.), and DIEA (47 uL, 3.0 eq.) in DMF. The resin was then filtered and washed with DMF (thrice) and DCM (thrice) to give compound M1V. (M+H=637.2).

embedded image

Compound M1V (0.4 g, 1.0 eq.) was stirred in 1/1 TFA/DCM for 2 hours and then drained and washed with DCM (thrice). The organic phases were combined and dried, and to it was added DCM followed by Dess-Martin Periodinane (97 mg, 3.0 eq.). The solution was stirred for 1 hour and to it was added 1 N Na₂S₂O₃and the mixture was further stirred. The solution was purified by silica gel chromatography (10-90% ethyl acetate/hexanes gradient) to yield 6.1 mg of Compound No. 146. (M+H=635.0) ¹H-NMR (CDCl₃): 5.5-5.2 (m, 2H), 5.1-5.0 (m, 1H), 4.9-4.7 (m, 2H), 4.5-4.2 (m, 3H), 4.1 (m, 1H), 3.9-3.7 (m, 3H), 3.6-3.5 (m, 2H), 3.5-3.2 (m, 2H), 2.8-2.4 (m, 2H), 2.1 (m, 1H), 2.0-1.8 (m, 3H), 1.8-1.5 (m, 3H), 1.5-1.3 (m, 3H), 1.3-1.2 (m, 2H), 1.0 (s, 9H), 0.9 (t, 3H), 0.8 (m, 2H), 0.6 (m, 2H).

The following compounds of Formula I were also produced according to Method 1 and the preparations described thereunder.

TABLE 2

Additional compounds of formula I produced by method 1.

Com-

pound
Starting Material
Starting Material
Starting Material

No.
for P¹
for C¹
for R₃

7
N—FMOC-L-tert-
3-(4-fluorophenyl)
3-chlorobenzene-

butylglycine
propanoic acid
carbaldehyde

oxime

12
N—FMOC-L-tert-
Acetic acid
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

14
N—FMOC-L-tert-
cyclopentylmethanol
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

24
N—((S)-
N/A
3-

tetrahydrofuran-3-

Fluorobenzene-

yloxy)carbonyl)-L-

carbaldehyde

tert-butylglycine

oxime

27
N—FMOC-L-tert-
cyclobutylmethanol
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

29
N—FMOC-L-tert-
2-(1-methylcyclohexyl)
3-chlorobenzene-

butylglycine
acetic acid
carbaldehyde

oxime

30
N—FMOC-L-tert-
(S)-5-oxo-1-(thiophen-
3-chlorobenzene-

butylglycine
2-ylmethyl)pyrrol-
carbaldehyde

idine-2-carboxylic acid
oxime

33
N—FMOC-L-tert-
cyclohexyl isocyanate
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

34
N—FMOC-L-tert-
5-hydroxypentan-
3-chlorobenzene-

butylglycine
2-one
carbaldehyde

oxime

37
N—FMOC-L-tert-
2-(tetrahydro-2H-
3-chlorobenzene-

butylglycine
pyran-4-yl)
carbaldehyde

acetic acid
oxime

39
N—FMOC-L-tert-
2-chlorobenzyl
3-chlorobenzene-

butylglycine
chloroformate
carbaldehyde

oxime

44
N—FMOC-L-tert-
4-oxo-pentanoic acid
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

53
N/A
2-(1-(2,6-dichloro-
Benzaldoxime

benzyl)piperidin-4-

yl)acetic acid

61
N—((S)-
N/A
Nitropropane

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

71
N—FMOC-L-tert-
(R)-2,3-dihydro-
3-chlorobenzene-

butylglycine
benzo[b][1,4]dioxine-
carbaldehyde

2-carboxylic acid
oxime

72
N—FMOC-L-tert-
2-cyclopentylacetic
3-chlorobenzene-

butylglycine
acid
carbaldehyde

oxime

75
N—FMOC-L-tert-
2-(2,4-
3-chlorobenzene-

butylglycine
dimethylthiazol-5-yl)
carbaldehyde

acetic acid
oxime

76
N—FMOC-L-tert-
Cyclopropyl
3-chlorobenzene-

butylglycine
isocyanate
carbaldehyde

oxime

85
N—FMOC-L-tert-
2-Fluoroethyl
3-chlorobenzene-

butylglycine
chloroformate
carbaldehyde

oxime

92
N—FMOC-L-tert-
2-(tetrahydro-2H-
3-chlorobenzene-

butylglycine
pyran-4-yl)acetic acid
carbaldehyde

oxime

93
N—FMOC-L-tert-
cyclohexanecarboxylic
3-chlorobenzene-

butylglycine
acid
carbaldehyde

oxime

94
N—FMOC-L-tert-
2-aminoacetamide
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

102
N—FMOC-L-tert-
2-(3-fluoro-4-
3-chlorobenzene-

butylglycine
methylphenyl)acetic
carbaldehyde

acid
oxime

107
N—FMOC-L-tert-
Benzyl isocyanate
Benzaldoxime

butylglycine

108
N—FMOC-L-tert-
cis-4-
3-chlorobenzene-

butylglycine
methoxycyclohexane-
carbaldehyde

carboxylic acid
oxime

110
N—FMOC-L-tert-
Benzyl chloroformate
3-Chloro-4,6-

butylglycine

dimethoxy-

benzald oxime

112
N—FMOC-L-tert-
2-(tetrahydro-2H-
2-nitro-1-phenyl

butylglycine
pyran-4-yl)acetic acid
ethanone

118
N—FMOC-L-tert-
2-(4-fluorophenyl)
3-Chlorobenzene

butylglycine
ethanol
carbaldehyde

oxime

119
N—FMOC-L-tert-
tert-Butyl isocyanate
2-nitro-1-phenyl

butylglycine

ethanone

122
N—FMOC-L-tert-
3-Fluorobenzyl
3-Chlorobenzene

butylglycine
isocyanate
carbaldehyde

oxime

123
N—FMOC-L-tert-
Ethyl isocyanate
3-Chlorobenzene

butylglycine

carbaldehyde

oxime

124
N—FMOC—O-
2-cyclohexylacetic
3-Chlorobenzene

Methyl-L-Threonine
acid
carbaldehyde

oxime

125
(2R,3S)—N—FMOC-
2-cyclohexylacetic
3-chlorobenzene-

2-Amino-3-phenyl-
acid
carbaldehyde

butyric acid

oxime

128
N—FMOC-L-tert-
4-(1H-pyrrole-2,5-
3-chlorobenzene-

butylglycine
dione)phenyl
carbaldehyde

isocyanate
oxime

135
N—FMOC-L-tert-
1-isopropyl-4-oxo-
3-chlorobenzene-

butylglycine
1,4-dihydroquinoline-
carbaldehyde

3-carboxylic acid
oxime

139
N—FMOC-L-tert-
(R)-2-hydroxy-2-
3-chlorobenzene-

butylglycine
phenylpropanoic
carbaldehyde

acid
oxime

146
N—((S)-
N/A
2-Chloro-2-

tetrahydrofuran-3-

hydroximinoacetic

yloxy)carbonyl)-L-

acid ethyl ester

tert-butylglycine

(chlorooxime)

152
N—FMOC-L-tert-
(tetrahydrofuran-3-
3-chlorobenzene-

butylglycine
yl)methanol
carbaldehyde

oxime

154
N-Alloc-L-tert-
N/A
4-Fluorobenzene-

butylglycine

carbaldehyde

oxime

155
N—FMOC-L-tert-
2-(5-fluoro-2-
3-chlorobenzene-

butylglycine
methylphenyl)
carbaldehyde

acetic acid
oxime

156
N—FMOC-L-tert-
Isobutylamine
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

159
N—FMOC-L-tert-
2-(thiophen-3-yl)
3-chlorobenzene-

butylglycine
ethanol
carbaldehyde

oxime

160
N—CBZ-L-tert-
N/A
4-Fluoro

butylglycine

benzene

carbaldehyde

oxime

161
N—FMOC-L-tert-
5-acetamido-2-
3-chlorobenzene-

butylglycine
acetylthiophene-3-
carbaldehyde

acid
carboxylic acid
oxime

164
N—CBZ-L-tert-
N/A
2-Chlorobenzene-

butylglycine

carbaldehyde

oxime

167
N—FMOC-L-tert-
(2-methylpyridin-3-
3-chlorobenzene-

butylglycine
yl)methanol
carbaldehyde

oxime

173
N—FMOC-L-tert-
2,2-
3-chlorobenzene-

butylglycine
difluoroethylamine
carbaldehyde

oxime

174
N—FMOC-L-tert-
m-tolylmethanol
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

180
N—FMOC-L-tert-
Acetic acid
Nitroethane

butylglycine

183
N—CBZ-L-tert-
N/A
3-Fluorobenzene-

butylglycine

carbaldehyde

oxime

185
N—FMOC-L-3-
2-cyclohexylacetic
3-chlorobenzene-

Thienyl-Alanine
acid
carbaldehyde

oxime

193
N—FMOC-L-tert-
Isopropyl
3-chlorobenzene-

butylglycine
chloroformate
carbaldehyde

oxime

199
N—CBZ-L-tert-
N/A
9-Anthraldehyde

butylglycine

oxime

201
N—FMOC-L-tert-
(3-methoxyphenyl)
3-chlorobenzene-

butylglycine
methanol
carbaldehyde

oxime

203
N—FMOC-L-tert-
(3,5-difluorophenyl)
3-chlorobenzene-

butylglycine
methanol
carbaldehyde

oxime

205
N—FMOC-L-tert-
Benzyl isocyanate
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

207
N—FMOC-L-
Ethyl chloroformate
3-chlorobenzene-

Glycine

carbaldehyde

oxime

208
N—CBZ-L-tert-
N/A
2-Naphthaldehyde

butylglycine

oxime

209
N—FMOC-L-tert-
(3-fluorophenyl)
3-chlorobenzene-

butylglycine
methanol
carbaldehyde

oxime

210
N—FMOC-L-tert-
2-chlorobenzyl
3-Chloro-4,6-

butylglycine
chloroformate
dimethoxybenzald

oxime

213
N—FMOC-4-
2-cyclohexylacetic
3-chlorobenzene-

Methoxy-L-
acid
carbaldehyde

Phenylalanine

oxime

216
N—FMOC-L-tert-
5-oxo-1-(thiophen-2-
3-chlorobenzene-

butylglycine
ylmethyl)pyrrolidine-
carbaldehyde

3-carboxylic acid
oxime

235
N—CBZ-L-tert-
N/A
nitrobutane

butylglycine

237
N—FMOC-L-tert-
3-(2-methyl-1H-
3-chlorobenzene-

butylglycine
imidazol-1-yl)
carbaldehyde

propanoic acid
oxime

241
N—FMOC-L-tert-
(S)-1-isopropyl-5-
3-chlorobenzene-

butylglycine
oxopyrrolidine-2-
carbaldehyde

carboxylic acid
oxime

242
N—FMOC-L-tert-
N—FMOC-L-cyclo-
Piperonal oxime

butylglycine
hexylglycine followed

by 2-pyrazine

carboxylic acid

243
N—((S)-
N/A
nitrobutane

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

249
N—FMOC-L-tert-
cyclohexanemethyl
Benzaldoxime

butylglycine
isocyanate

254
N—FMOC-L-tert-
1-(thiophen-2-yl)
3-chlorobenzene-

butylglycine
propan-2-ol
carbaldehyde

oxime

259
N—FMOC-L-tert-
3,4,5-
3-chlorobenzene-

butylglycine
trimethoxybenzyl
carbaldehyde

isocyanate
oxime

260
N—FMOC-L-tert-
2-methoxyethyl
3-chlorobenzene-

butylglycine
chloroformate
carbaldehyde

oxime

261
N—FMOC-L-tert-
benzyl 4-isocyanato-
3-chlorobenzene-

butylglycine
piperidine-1-
carbaldehyde

carboxylate
oxime

262
N/A
4-nitrophenyl
3-chlorobenzene-

choroformate
carbaldehyde

oxime

276
2-((3S,4aS,8aS)-
N/A
3-chlorobenzene-

3-(tert-butyl-

carbaldehyde

carbamoyl)octa-

oxime

hydroisoquinolin-

2(1H)-yl)acetic acid

278
N—FMOC-L-tert-
benzyl chloroformate
2-(4-

butylglycine

Methoxyphenoxy)

benzenecar-

baldehyde oxime

283
N—FMOC-L-tert-
2-(pyridin-3-yl)acetic
Benzaldoxime

butylglycine
acid

287
N—FMOC-L-tert-
2-(3-methoxyphenyl)
3-chlorobenzene-

butylglycine
acetic acid
carbaldehyde

oxime

288
N—FMOC-L-tert-
1-Naphthyl
Benzaldoxime

butylglycine
isocyanate

289
N—FMOC-2-
2-cyclohexylacetic
3-chlorobenzene-

Trifluoromethyl-L-
acid
carbaldehyde

Phenylalanine

oxime

291
N—FMOC-L-tert-
spiro[indene-1,4′-
3-chlorobenzene-

butylglycine
piperidin]-3(2H)-one
carbaldehyde

oxime

294
N—FMOC-L-tert-
2-cyclohexylacetic
3-chlorobenzene-

butylglycine
acid
carbaldehyde

oxime

308
N—FMOC-L-tert-
(tetrahydro-2H-
3-chlorobenzene-

butylglycine
pyran-2-yl)
carbaldehyde

methanol
oxime

311
N—FMOC-L-tert-
2-(pyrrolidine-1-
3-chlorobenzene-

butylglycine
carbonyl)cyclohexane-
carbaldehyde

carboxylic acid
oxime

313
N/A
benzyl isocyanate
3-Chloro-4,6-

dimethoxybenzald

oxime

317
N—FMOC-L-tert-
2-(1-oxoisoindolin-2-
3-chlorobenzene-

butylglycine
yl)propanoic acid
carbaldehyde

oxime

324
N—FMOC-L-tert-
(R)-3-(1-cyanoethyl)
3-chlorobenzene-

butylglycine
benzoic acid
carbaldehyde

oxime

329
N—FMOC-L-tert-
cyclohexylacetic acid
Nitropropane

butylglycine

331
N—FMOC-L-tert-
acetic acid
4-Fluorobenzene

butylglycine

carbaldehyde

oxime

333
N—FMOC-L-tert-
(S)-1-methyl-
3-Chlorobenzene

butylglycine
benzylamine
carbaldehyde

oxime

334
N—FMOC-L-tert-
(S)-2-methyl-3-
3-chlorobenzene-

butylglycine
phenylpropanoic
carbaldehyde

acid
oxime

336
N—FMOC-L-3-
2-cyclohexylacetic
3-chlorobenzene-

Benzothienyl-
acid
carbaldehyde

Alanine

oxime

338
N—FMOC-2-
2-cyclohexylacetic
3-chlorobenzene-

Fluoro-L-
acid
carbaldehyde

Phenylalanine

oxime

340
N—CBZ-L-tert-
N/A
4-Phenylbenzene-

butylglycine

carbaldehyde

oxime

341
N—((S)-
N/A
2-chloro-2-

tetrahydrofuran-3-

hydroximinoacetic

yloxy)carbonyl)-L-

acid ethyl ester

tert-butylglycine

(chlorooxime)

followed by ester

hydrolysis and

coupling of

ethylamine

342
N/A
pyridine 3-methanol
3-chlorobenzene-

carbaldehyde

oxime

345
N-(5-methyl-3-
N/A
3-chlorobenzene-

nitroprydinyl)-L-

carbaldehyde

tert-butylglycine

oxime

349
N—FMOC-L-tert-
2-(4-fluorophenyl)
3-chlorobenzene-

butylglycine
ethanol
carbaldehyde

oxime

352
N—((S)-
N/A
Pyridine-4-

tetrahydrofuran-3-

aldoxime

yloxy)carbonyl)-L-

tert-butylglycine

357
N—FMOC-L-tert-
pyridin-4-ylmethanol
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

358
N—((S)-
N/A
4-Trifluoro-

tetrahydrofuran-3-

methoxybenzene-

yloxy)carbonyl)-L-

carbaldehyde

tert-butylglycine

oxime

365
N—CBZ-L-tert-
N/A
4-Trifluoro-

butylglycine

methoxybenzene-

carbaldehyde

oxime

367
N/A
3,4,5-trimethoxy-
Benzaldoxime

benzyl isocyanate

373
N—FMOC-L-tert-
3-(pyridin-2-yl)
3-chlorobenzene-

butylglycine
propan-1-ol
carbaldehyde

oxime

374
N—FMOC-L-tert-
tetrahydro-2H-
3-chlorobenzene-

butylglycine
pyran-4-ol
carbaldehyde

oxime

377
N—FMOC-L-tert-
(S)-1-(3 chlorobenzyl)-
3-chlorobenzene-

butylglycine
5-oxopyrrolidine-
carbaldehyde

2-carboxylic acid
oxime

378
N—FMOC-L-tert-
pyridin-2-ylmethanol
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

379
N—FMOC-L-tert-
isopropyl isocyanate
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

381
N—FMOC-L-tert-
4-oxo-3,4-
3-chlorobenzene-

butylglycine
dihydrophthalazine-
carbaldehyde

1-carboxylic acid
oxime

383
N—CBZ-L-tert-
N/A
Nitropropane

butylglycine

387
N—FMOC-L-tert-
(3R,3aS,6aR)-
3-chlorobenzene-

butylglycine
hexahydrofuro[2,3-b]
carbaldehyde

furan-3-ol
oxime

389
N—FMOC-L-tert-
3-(pyridin-3-yl)
3-chlorobenzene-

butylglycine
propan-1-ol
carbaldehyde

oxime

390
N—FMOC-L-tert-
cyclohexylacetic acid
2-nitro-1-phenyl

butylglycine

ethanone

398
N—((S)-
N/A
4-Fluorobenzene-

tetrahydrofuran-3-

carbaldehyde

yloxy)carbonyl)-L-

oxime

tert-butylglycine

400
N—FMOC-L-tert-
N—FMOC-L-cyclo-
nitrobutane

butylglycine
hexylglycine followed

by 2-pyrazine

carboxylic acid

402
N—FMOC-L-tert-
2-(5-oxo-2-(thiophen-
3-chlorobenzene-

butylglycine
2-yl)cyclopent-1-enyl)
carbaldehyde

acetic acid
oxime

407
N—FMOC-L-tert-
ethyl chloroformate
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

417
N—FMOC-L-tert-
N—FMOC-L-tert-
4-Fluorobenzene-

butylglycine
butylglycine followed
carbaldehyde

by 2-pyrazine
oxime

carboxylic acid

427
N—FMOC-L-
ethyl chloroformate
3-chlorobenzene-

Phenylalanine

carbaldehyde

oxime

431
N—FMOC-L-tert-
2-o-tolylacetic acid
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

432
N—FMOC-L-tert-
N-methyl ethylamine
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

437
N—FMOC-S-tert-
2-cyclohexylacetic
3-chlorobenzene-

Butyl-L-Cysteine
acid
carbaldehyde

oxime

450
N—FMOC-L-tert-
N—FMOC-L-tert-
Piperonal oxime

butylglycine
butylglycine followed

by 2-pyrazine

carboxylic acid

454
N—FMOC-L-tert-
2-(quinolin-8-ylthio)
3-chlorobenzene-

butylglycine
acetic acid
carbaldehyde

oxime

459
N—FMOC-L-
2-cyclohexylacetic
3-chlorobenzene-

Norleucine
acid
carbaldehyde

oxime

462
N—FMOC-L-tert-
cyclohexylacetic acid
Piperonal oxime

butylglycine

463
N—FMOC-L-tert-
2-phenylethanol
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

465
N-
N/A
4-Fluorobenzene-

(Cyclopentyl-

carbaldehyde

formoyl)-L-tert-

oxime

butylglycine

467
N—FMOC-L-tert-
2-(bicyclo[2.2.1]
3-chlorobenzene-

butylglycine
heptan-2-yl)
carbaldehyde

acetic acid
oxime

471
N—FMOC-L-tert-
p-tolylmethanol
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

474
N—FMOC-L-tert-
2-methyl-3-(3 methyl-
3-chlorobenzene-

butylglycine
1H-pyrazol-1-yl)
carbaldehyde

propanoic acid
oxime

477
N—FMOC-L-tert-
(S)-1-methoxy-3,3-
3-chlorobenzene-

butylglycine
dimethylbutan-
carbaldehyde

2-amine
oxime

484
N—FMOC-L-tert-
Succinic Anhydride
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

487
N—FMOC-L-tert-
2-(6-methoxy-3-oxo-
3-chlorobenzene-

butylglycine
2,3-dihydro-1H-inden-
carbaldehyde

1-yl)acetic acid
oxime

487
N—FMOC-L-tert-
2-(3-oxo-2,3-dihydro-
3-chlorobenzene-

butylglycine
1H-inden-1-yl)acetic
carbaldehyde

acid
oxime

492
N—FMOC-L-tert-
tert-Butyl isocyanate
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

497
N—FMOC-L-tert-
pyridin-3-
3-chlorobenzene-

butylglycine
ylmethylamine
carbaldehyde

oxime

503
N—FMOC-L-tert-
trans-4-
3-chlorobenzene-

butylglycine
methoxycyclohexane-
carbaldehyde

carboxylic acid
oxime

504
N—FMOC-L-tert-
3-(pyridin-3-yl)
3-chlorobenzene-

butylglycine
propanoic acid
carbaldehyde

oxime

505
N—FMOC-L-tert-
3-(2,5-dioxo-
3-chlorobenzene-

butylglycine
imidazolidin-4-yl)
carbaldehyde

propanoic acid
oxime

512
N—FMOC-L-tert-
(2-fluorophenyl)
3-chlorobenzene-

butylglycine
methanol
carbaldehyde

oxime

515
N—FMOC-L-tert-
tetrahydro-2H-
3-chlorobenzene-

butylglycine
pyran-3-ol
carbaldehyde

oxime

517
N—FMOC-L-tert-
N/A
Nitroethane

butylglycine

518
N—FMOC-L-tert-
(S)-1-(3 methylbenzyl)-
3-chlorobenzene-

butylglycine
5-oxopyrrolidine-2-
carbaldehyde

carboxylic acid
oxime

520
N—FMOC-L-tert-
benzyl chloroformate
Benzaldoxime

butylglycine

523
N—FMOC-L-tert-
tetrahydro-2H-pyran-
3-chlorobenzene-

butylglycine
4-carboxylic acid
carbaldehyde

oxime

526
N—FMOC-L-tert-
benzyl chloroformate
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

528
N—FMOC-L-tert-
3-(1H-indazol-1-yl)
3-chlorobenzene-

butylglycine
propanoic acid
carbaldehyde

oxime

532
N—FMOC-L-tert-
3-methylbutanoic acid
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

533
N/A
N/A
4-Fluorobenzene-

carbaldehyde

oxime

538
N—FMOC-L-tert-
2-cyano-2-methyl-3-
3-chlorobenzene-

butylglycine
phenylpropanoic acid
carbaldehyde

oxime

544
N—FMOC-L-tert-
3-(1H-benzo[d]
3-chlorobenzene-

butylglycine
imidazol-1-yl)-2-
carbaldehyde

methylpropanoic acid
oxime

547
N—FMOC-L-tert-
N—FMOC-L-cyclo-
Nitropropane

butylglycine
hexylglycine followed

by 2-pyrazine

carboxylic acid

553
N—FMOC-L-tert-
2-(2,6-dioxo-1,2,3,6-
3-chlorobenzene-

butylglycine
tetrahydropyrimidin-
carbaldehyde

4-yl)acetic acid
oxime

557
N—FMOC-L-tert-
(1R,6S)-6-
3-chlorobenzene-

butylglycine
(methoxycarbonyl)
carbaldehyde

cyclohex-3-
oxime

enecarboxylic acid

558
N—FMOC-L-tert-
phenyl isocyanate
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

559
N—FMOC-L-tert-
tert-Butyl isocyanate
Nitropropane

butylglycine

561
N—FMOC-L-tert-
(2,5-difluorophenyl)
3-chlorobenzene-

butylglycine
methanol
carbaldehyde

oxime

563
N—FMOC-L-tert-
Pyridine 3-methanol
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

566
N—FMOC-L-tert-
2-(tetrahydro-2H-
Nitropropane

butylglycine
pyran-4-yl)acetic acid

576
N—FMOC-L-tert-
3-pyridyl isocyanate
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

580
N—FMOC-L-tert-
ethyl isocyanate
Benzaldoxime

butylglycine

582
N—FMOC-L-tert-
2-(thiophen-2-yl)
3-chlorobenzene-

butylglycine
ethanol
carbaldehyde

oxime

583
N—FMOC-L-tert-
benzyl isocyanate
3-chlorobenzene-

butylglycine

carbaldehyde

oxime

All starting materials for R₃listed in Table 2 and all other tables herein were either commercially available (nitro or oxime) or readily prepared from corresponding aldehyde precursors.

Additionally, Compound Nos. 20, 22, 53, 81, 103, 116, 166, 187, 189, 194, 197, 200, 220, 223, 226, 245, 252, 271, 204, 307, 319, 339, 354, 360, 361, 371, 392, 393, 435, 449, 506, 514, 531, and 585 were also produced by using Method 1.

Certain other compounds of the invention may be prepared as illustrated by Method 2.

Method 2

embedded image

Referring to Method 2, the protected spiroisoxazoline B1 is deprotected to B2 which in turn is converted to the Fmoc derivative B3. Reaction of B3 with the resin bound aminoalcohol A4 provides the resin bound spiroisoxazoline A6 which is converted to A10 as described in Method 1.

Example 6
Compound No. 281

embedded image

Compound M2A (5.0 g, 1.0 eq.) was stirred in 100 mL acetonitrile and to this mixture was added ditertbutyldicarbonate (9.6 g, 2.0 eq.), dimethylaminopyridine (537 mg, 0.2 eq.), and triethylamine (6.13 mL, 2.0 eq.) and stirred overnight. The resulting mixture was concentrated, ethyl acetate was added, and the mixture was washed with 1.0 N HCl, dried over sodium sulfate, concentrated, and purified by silica gel chromatography (10-30% ethyl acetate/hexanes gradient) to yield compound M2B. (M+H=284.0) ¹H-NMR (CDCl₃): 5.0 (m, 2H), 4.3-4.5 (m, 1H), 4.0-4.1 (m, 2H), 2.9-3.0 (m, 1H), 2.5-2.6 (d, 1H), 1.5 (s, 3/9 of 18H), 1.4 (s, 6/9 of 18H).

embedded image

Compound M2B (2.0 g, 1.0 eq.) stirred in 35 mL DCM with benzaldoxime (2.67 g, 2.0 eq.). The solution was cooled on an ice bath and to this bleach (5% NaOCl) (34.9 mL) was slowly added. The mixture was then warmed to room temperature and stirred for 2 hours. The aqueous layer was separated and extracted with DCM twice. The organics were combined and dried over magnesium sulfate, filtered and concentrated. Purified via silica gel chromatography (5-30% ethyl acetate/hexanes gradient) yielded compound M2C. (M+H=403.1)¹H-NMR (500 MHz, CDCl₃): 7.64-7.63 (m, 2H), 7.41-7.40 (m, 3H), 4.43-4.37 (t, 1H), 3.94-3.85 (dd, 1H), 3.62 (t, 1H), 3.44-3.38 (m, 1H), 3.29-3.24 (m, 1H), 2.74 (m, 1H), 2.14-2.10 (m, 1H), 1.49 (s, 9H), 1.46 (s, 9H).

embedded image

Compound M2C was stirred in 1/1 TFA/DCM for 3 hours. The mixture was concentrated. To the concentrated mixture was added 17 mL DMF, 5 mL water, sodium carbonate (713 mg, 2.5 eq.), FMOC-OSu (951 mg, 1.05 eq.) and stirred 3 hours. Then, ethyl acetate was added and the resulting mixture was washed with 1.0 N HCl followed by brine. It was dried over magnesium sulfate, filtered and concentrated to yield compound M2D. (M+H=468.9).

embedded image

Compound M2D (1.26 g, 2.0 eq.) was stirred in DMF with M1D (2.5 g, 1.0 eq.), HBTU (12 mL of 0.5 M in DMF, 5.0 eq.), HOBt (6 mL of 1.0 M in DMF, 5.0 eq.), and Hünig's base (2.09 mL, 10.0 eq.) overnight. The mixture was drained and washed with DMF (thrice) and DCM (thrice) to yield compound M1L. (M+H=637.0).

embedded image

Compound M1L (0.4 g, 1.0 eq.) was shaken in 20% piperidine/DMF for 10 minutes before being filtered and washed with DMF (thrice) followed by DCM (thrice). This process was repeated. The resulting resin was shaken overnight with a solution of FMOC-tert-butylglycine (200 mg 3.0 eq.), HBTU (1.15 mL of 0.5 M in DMF, 3.0 eq.), HOBt (0.58 mL of 1.0 M in DMF, 3.0 eq.), and DIEA (167 uL, 5.0 eq.) in 2 mL DMF. The resin was then filtered and washed with DMF (thrice) and DCM (thrice) to give compound M1M. (M+H=750.1).

embedded image

Compound M1M (0.4 g, 1.0 eq.) was shaken in 20% piperidine/DMF for 10 minutes and the resin was filtered and washed with DMF (thrice) followed by DCM (thrice). This process was repeated to give Compound M2H.

embedded image

Compound M2H (0.4 g, 1.0 eq.) was shaken overnight with a solution of FMOC-cyclohexylglycine (218 mg 3.0 eq.), HBTU (1.15 mL of 0.5 M in DMF, 3.0 eq.), HOBt (0.58 mL of 1.0 M in DMF, 3.0 eq.), and DIEA (167 uL, 5.0 eq.) in 2 mL DMF. The resin was then filtered and washed with DMF (thrice) and DCM (thrice). The resin was then treated with 20% piperidine/DMF for 10 minutes. The resin was filtered and washed with DMF (thrice) followed by DCM (thrice). This process was repeated to give Compound M2I.

embedded image

Compound M2I (0.4 g, 1.0 eq.) was shaken overnight with a solution of pyrazine carboxylic acid (71 mg, 3.0 eq.), HBTU (1.15 mL of 0.5 M in DMF, 3.0 eq.), HOBt (0.58 mL of 1.0 M in DMF, 3.0 eq.), and DIEA (167 uL, 5.0 eq.) in 2 mL DMF. The resin was then filtered and washed with DMF (thrice) and (thrice) to give compound M2J. (M+H=772.9).

embedded image

Compound M2J (0.4 g, 1.0 eq.) was stirred in 1/1 TFA/DCM for 2 hours. The resin was drained and washed with DCM (thrice). The result was concentrated all organics and added DCM followed by Dess Martin Periodinane (97 mg, 3.0 eq.). Stirred for 1 hour and added 1N Na₂S₂O₃and stirred. The solution was purified by silica gel chromatography (10-90% ethyl acetate/hexanes gradient) to yield 42 mg of Compound No. 281. (M+H=771.0). ¹H-NMR (500 MHz, CDCl₃): 9.38 (d, 1H), 8.75 (d, 1H), 8.56 (t, 1H), 8.31 (d, 1H), 7.64-7.62 (m, 2H), 7.42-7.38 (m, 3H), 7.33 (d, 1H), 7.15 (s, 1H), 6.89 (d, 1H), 5.45-5.41 (m, 1H), 4.85 (t, 1H), 4.69 (d, 1H), 4.57-4.54 (m, 1H), 4.26 (d, 1H), 3.76 (d, 1H), 3.46-3.35 (m, 2H), 2.82 (td, 1H), 2.56 (d, 2H), 1.96-1.87 (m, 2H), 1.76 (m, 4H), 1.65-1.59 (m, 2H), 1.48-1.42 (m, 2H), 1.24 (m, 2H), 1.09 (m, 2H), 0.97 (s, 9H), 0.93 (t, 2H), 0.88-0.84 (m, 2H), 0.65 (t, 2H).

Listed below in Table 3 are additional compounds of Formula I prepared by Method 2.

TABLE 3

Additional compounds of formula I produced by method 2.

Com-

pound
Starting Material
Starting Material
Starting Material

No.
for P¹
for C¹
for R₃

40
N—((S)-
N/A
2,6-

tetrahydrofuran-3-

Dichloro-

yloxy)carbonyl)-L-

benzaldoxime

tert-butylglycine

51
N/A
1H-pyrrole-2-
Piperonal oxime

carboxylic acid

80
N/A
1H-pyrrole-2-
Benzaldoxime

carboxylic acid

101
N—FMOC-L-tert-
1-Naphthylsulfonyl
Benzaldoxime

butylglycine
chloride

147
N—FMOC-L-tert-
N—FMOC-L-
2,6-

butylglycine
cyclohexylglycine
Dichloro-

followed by 2-pyrazine
benzaldoxime

carboxylic acid

151
N-Alloc-L-tert-
N/A
Benzaldoxime

butylglycine

202
N—((S)-
N/A
Benzaldoxime

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

228
N—FMOC-L-tert-
Acetic acid
Benzaldoxime

butylglycine

281
N—FMOC-L-tert-
N—FMOC-L-
Benzaldoxime

butylglycine
cyclohexylglycine

followed by 2-pyrazine

carboxylic acid

325
N—FMOC-L-tert-
Acetic acid
Piperonal oxime

butylglycine

327
N-Alloc-L-tert-
N/A
Piperonal oxime

butylglycine

343
N—((S)-
N/A
Piperonal oxime

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

428
N—FMOC-L-tert-
N—FMOC-L-
Benzaldoxime

butylglycine
cyclohexylglycine

followed by 2-pyrazine

carboxylic acid

464
N/A
1H-pyrrole-2-
Piperonal oxime

carboxylic acid

491
N—((S)-
N/A
Benzaldoxime

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

527
N—((S)-
N/A
Piperonal oxime

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

536
N—FMOC-L-tert-
1-Naphthylsulfonyl
Piperonal oxime

butylglycine
chloride

570
N—FMOC-L-tert-
Acetic acid
Piperonal oxime

butylglycine

578
N—FMOC-L-tert-
Acetic acid
Benzaldoxime

butylglycine

584
N/A
1H-pyrrole-2-carboxylic
Benzaldoxime

acid

Certain other compounds of Formula I may be prepared as illustrated by Method 3.

Method 3

embedded image

Referring to Method 3, the resin bound Fmoc exomethylene compound A5, prepared as in Method 1, is deprotected to give Cl. Reaction of Cl with an R₁carboxylic acid in the presence of a coupling reagent provides C2 wherein R₁is R₄C(O)—. Reaction of C2 with the nitrile oxide 1f leads to A8 which is converted to A10 as illustrated in Method 1.

Example 7
Compound No. 239

embedded image

The resin M1E (0.47 mmol) was shaken in 20% piperidine/DMF for 10 minutes and then filtered and washed with DMF and DCM. The resulting resin was shaken again overnight with a solution of Cbz-tBG-OH (374 mg, 3.0 eq.), HOBT (2.82 mL of 0.5 M in DMF, 3.0 eq.), HBTU (2.82 of 0.5 M in DMF, 3.0 eq.), and DIEA (0.493 mL, 6.0 eq.). The resin was then filtered and washed with DMF and DCM to give the resin compound M3A (0.47 g), which was used in next reaction without further purification.

The Cbz resin M3A (0.0611 mmol) in THF was shaken with 3-bromo-phenyl oxime (10 eq.) and bleach (5% NaOH) (20 eq.) for 12 hours. The resin was then filtered and washed with water, DMF, DCM to give the resin M3B.

The resin M3B was shaken with 95% TFA in water for 30 minutes and the resulting solution was concentrated in vacuo to give the compound M3C (0.031 mmol), (M+1) 740, which was used in next reaction without further purification.

A solution of the compound M3C (0.031 mmol) in DCM (3 mL) was stirred with Dess-Martin Periodinane (26 mg, 2 eq.) and t-BuOH (26 uL). After stirring for 1 hour, sodium thiosulfate was added to above mixture. The product was extracted with EtOAc and the combined organic layer was then washed with water, NaHCO₃, brine and concentrated in vacuo and purified by Gilson Prep HPLC to afford Compound No. 239. (M+1) 738.

Example 8
Compound No. 535

embedded image

Compound M1E (10.0 g, 1.0 eq.) was shaken in 20% piperidine/DMF for 10 minutes. The resin was filtered and washed with DMF (thrice) followed by DCM (thrice). This process was repeated. The resulting resin was shaken overnight with a solution of (S)-2,3-dimethyl-2-(((S)-tetrahydrofuran-3-yloxy)carbonylamino)butanoic acid (3.46 g, 3.0 eq.), HBTU (28.2 mL of 0.5 M in DMF, 3.0 eq.), HOBt (14.1 mL of 1.0M in DMF, 3.0 eq.), and DIEA (4.91 mL, 6.0 eq.) in DMF. The resin was then filtered and washed with DMF (thrice) and DCM (thrice) to give compound M3E. (M+H=523.1)

embedded image

Compound M3E (300 mg, 1.0 eq.) was stirred in THF and 2-nitro-1-phenylethanone (272 mg, 10.0 eq.) was added to the mixture followed by phenyl isocyanate (179 uL, 10.0 eq.) and catalytic TEA (10 uL). The resulting mixture was shaken overnight, drained, and washed with DMF, THF, and DCM (thrice each) to give compound M3F (M+H=669.8).

embedded image

Compound M3F (0.4 g, 1.0 eq.) was stirred in 1/1 TFA/DCM for 2 hours. The resin was drained and washed with DCM (thrice), all organics were concentrated, and DCM was added followed by Dess-Martin Periodinane (97 mg, 3.0 eq.). The resulting mixture was stirred for 1 hour, 1 N Na₂S₂O₃was added and again, stirred. The reaction mixture was purified via silica gel chromatography (10-90% ethyl acetate/hexanes gradient) to yield Compound No. 535. M+H=668.1. ¹H-NMR (500 MHz, CDCl₃): 8.19 (d, 2H), 7.61 (t, 1H), 7.47 (t, 2H), 7.19 (d, 1H), 6.93 (d, 1H), 5.52 (d, 1H), 5.37-5.33 (m, 1H, 5.24 (s, 1H), 4.78 (t, 1H), 4.32-4.29 (m, 2H), 3.93-3.79 (m, 4H), 3.70 (d, 1H), 3.48-3.36 (m, 2H), 2.79 (td, 1H), 2.68-2.63 (m, 1H), 2.55-2.50 (m, 1H), 2.12-2.04 (m, 1H), 1.96-1.89 (m, 1H), 1.66-1.59 (m, 1H), 1.47-1.37 (m, 2H), 1.00 (s, 9H), 0.94-0.81 (m, 6H), 0.63-0.57 (m, 2H).

Listed below in Table 4 are additional compounds of Formula I prepared by Method 3.

TABLE 4

Additional compounds of formula I produced by method 3.

Com-

pound
Starting Material
Starting Material

No.
for P¹
for C¹
Starting Material for R₃

4
N—((S)-
N/A
3-Chloro-5-

tetrahydrofuran-3-

fluorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

8
N—((S)-
N/A
3-(Cyclopentyloxy)-4-

tetrahydrofuran-3-

methoxybenzaldehyde

yloxy)carbonyl)-L-

tert-butylglycine

9
N—((S)-
N/A
3,5-

tetrahydrofuran-3-

Di(trifluoromethyl)

yloxy)carbonyl)-L-

benzaldoxime

tert-butylglycine

11
N/A
(S)-2,5-
3-fluoro-4-

dioxopyrrolidin-1-
methylbenzaldoxime

yl tetrahydrofuran-

3-yl carbonate

15
N—((S)-
N/A
3-Chloro-4-

tetrahydrofuran-3-

methoxybenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

16
N—FMOC-L-tert-
2-(tetrahydro-2H-
4-

butylglycine
pyran-4-yl)acetic
Methoxybenzaldoxime

acid

25
N—((S)-
N/A
3,5-

tetrahydrofuran-3-

Difluorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

32
N—((S)-
N/A
3,4-

tetrahydrofuran-3-

Dichlorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

36
N—((S)-
N/A
3,4-

tetrahydrofuran-3-

Dimethylbenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

47
N/A
(S)-2,5-
4-

dioxopyrrolidin-1-
Ethylbenzaldoxime

yl tetrahydrofuran-

3-y1 carbonate

52
N—((S)-
N/A
4-

tetrahydrofuran-3-

Trifluoromethyl-

yloxy)carbonyl)-

benzaldoxime

L-tert-butylglycine

55
N—FMOC-L-
2-cyclohexylacetic
4-

tert-butylglycine
acid
Chlorobenzaldoxime

56
N—FMOC-L-
2-cyclohexylacetic
3,5-Dichloro-

tert-butylglycine
acid
benzaldoxime

64
N—((S)-
N/A
4-Trifluoromethyl-

tetrahydrofuran-3-

benzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

66
N—FMOC-L-tert-
2-cyclohexylacetic
3-Chloro-4-

butylglycine
acid
fluorobenzaldoxime

70
N—((S)-
N/A
Cyclopentane-

tetrahydrofuran-3-

carboxaldehyde

yloxy)carbonyl)-L-

tert-butylglycine

78
N—FMOC-L-tert-
2-(tetrahydro-2H-
3,4-Dichloro-

butylglycine
pyran-4-yl)acetic
benzaldoxime

acid

82
N—FMOC-L-tert-
2-(tetrahydro-2H-
Piperonal oxime

butylglycine
pyran-4-yl)acetic

acid

83
N—((S)-
N/A
3-

tetrahydrofuran-3-

Chlorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

95
N—FMOC-L-tert-
2-cyclohexylacetic
3-Chloro-5-

butylglycine
acid
fluorobenzaldoxime

106
N—((S)-
N/A
3,5-

tetrahydrofuran-3-

Difluorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

109
N—((S)-
N/A
3-Methyl-4-

tetrahydrofuran-3-

chlorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

142
N—((S)-
N/A
Cyclohexane-

tetrahydrofuran-3-

carboxaldehyde

yloxy)carbonyl)-L-

tert-butylglycine

149
N—((S)-
N/A
3-Trifluoromethoxy-

tetrahydrofuran-3-

benzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

150
N—((S)-
N/A
2,2-Dimethylchromane-

tetrahydrofuran-3-

6-carbaldehyde

yloxy)carbonyl)-L-

tert-butylglycine

171
N—FMOC-L-tert-
2-(tetrahydro-2H-
3-Cyanobenzaldoxime

butylglycine
pyran-4-yl)acetic

acid

177
N—FMOC-L-tert-
2-(tetrahydro-2H-
4-Cyanobenzaldoxime

butylglycine
pyran-4-yl)acetic

acid

191
N—((S)-
N/A
3,5-Dimethy1-4-

tetrahydrofuran-3-

methoxybenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

196
N—((S)-
N/A
3,4-Dimethoxy-

tetrahydrofuran-3-

benzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

198
N/A
(S)-2,5-
3,5-Dimethy1-4-

dioxopyrrolidin-1-
methoxybenzaldoxime

yl tetrahydrofuran-

3-yl carbonate

215
N—((S)-
N/A
3,4,5-Trifluoro-

tetrahydrofuran-3-

benzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

222
N/A
(S)-2,5-
2,2-Difluoro-

dioxopyrrolidin-1-
1,3-benzodioxole-5

yl tetrahydrofuran-
carboxaldehyde

3-yl carbonate

224
N—((S)-
N/A
3,5-Dimethy1-4-

tetrahydrofuran-3-

methoxybenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

229
N—((S)-
N/A
Methyl 4-nitrobutyrate

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

234
N—FMOC-L-tert-
2-(tetrahydro-2H-
3-Chloro-5-

butylglycine
pyran-4-yl)acetic
fluorobenzaldoxime

acid

236
N—((S)-
N/A
3-Chloro-4-

tetrahydrofuran-3-

methoxybenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

239
N—CBZ-L-tert-
N/A
3-Bromobenzaldoxime

butylglycine

240
N/A
(S)-
4-Trifluoromethyl-

tetrahydrofuran-3-
benzaldoxime

yl-carbonate

244
N—((S)-
N/A
3-Trifluoromethoxy-

tetrahydrofuran-3-

benzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

251
N—((S)-
N/A
Phenylnitroethane

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

257
N—((S)-
N/A
3-

tetrahydrofuran-3-

Phenylbenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

258
N—((S)-
N/A
3-fluoro-5-

tetrahydrofuran-3-

trifluoromethyl-

yloxy)carbonyl)-L-

benzaldoxime

tert-butylglycine

270
N—FMOC-L-tert-
2-(tetrahydro-2H-
3-Chloro-4-

butylglycine
pyran-4-yl)acetic
fluorobenzaldoxime

acid

274
N/A
(S)-
3,5-

tetrahydrofuran-3-
Dichlorobenzaldoxime

yl-carbonate

279
N—FMOC-L-tert-
2-(tetrahydro-2H-
3-Chloro-4-

butylglycine
pyran-4-yl)acetic
methoxybenzaldoxime

acid

285
N—FMOC-L-tert-
2-(tetrahydro-2H-
3,5-

butylglycine
pyran-4-yl)acetic
Dichlorobenzaldoxime

acid

299
N—FMOC-L-tert-
2-(tetrahydro-2H-
4-Chlorobenzaldoxime

butylglycine
pyran-4-yl)acetic

acid

301
N—((S)-
N/A
3-Chloro-4-

tetrahydrofuran-3-

fluorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

306
N—((S)-
N/A
3,5-

tetrahydrofuran-3-

Dichlorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

314
N—((S)-
N/A
Methyl 4-

tetrahydrofuran-3-

formylbenzoate

yloxy)carbonyl)-L-

tert-butylglycine

316
N—((S)-
N/A
2,2-Difluoro-1,3-

tetrahydrofuran-3-

benzodioxole-

yloxy)carbonyl)-L-

5-carboxaldehyde

tert-butylglycine

318
N—((S)-
N/A
4-Chlorobenzaldoxime

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

322
N—((S)-
N/A
3-Chloro-5-

tetrahydrofuran-3-

fluorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

323
N—FMOC-L-tert-
2-cyclohexylacetic
3,4-Dichloro-

butylglycine
acid
benzaldoxime

330
N-((S)-
N/A
3,5-

tetrahydrofuran-3-

Di(trifluoromethyl)

yloxy)carbonyl)-L-

benzaldoxime

tert-butylglycine

348
N/A
(S)-
3-fluoro-5-

tetrahydrofuran-3-
trifluoromethyl-

yl-carbonate
benzaldoxime

353
N—((S)-
N/A
Methyl 3-

tetrahydrofuran-3-

formylbenzoate

yloxy)carbonyl)-L-

tert-butylglycine

362
N-FMOC-L-tert-
2-cyclohexylacetic
3,5-Dimethy1-4-

butylglycine
acid
methoxybenzaldoxime

363
N—((S)-
N/A
2,2-Difluoro-1,3-

tetrahydrofuran-3-

benzodioxole-5-

yloxy)carbonyl)-L-

carboxaldehyde

tert-butylglycine

364
N—((S)-
N/A
4-

tetrahydrofuran-3-

Chlorophenylglyoxyl-

yloxy)carbonyl)-L-

ohydroxamyl chloride

tert-butylglycine

385
N—((S)-
N/A
4-Methylbenzaldoxime

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

391
N—((S)-
N/A
3,5-Dichloro-

tetrahydrofuran-3-

benzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

403
N—((S)-
N/A
2-Chloro-6-

tetrahydrofuran-3-

fluorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

405
N/A
(S)-
4-Isopropyl-

tetrahydrofuran-3-
benzaldoxime

yl-carbonate

413
N/A
(S)-
3-Methyl-4-

tetrahydrofuran-3-
fluorobenzaldoxime

yl-carbonate

414
N/A
(S)-
3,4,5-

tetrahydrofuran-3-
Trifluorobenzaldoxime

yl-carbonate

423
N—((S)-
N/A
3-fluoro-4-

tetrahydrofuran-3-

methylbenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

425
N—((S)-
N/A
3-(4-Pyridyl)

tetrahydrofuran-3-

benzaldehyde

yloxy)carbonyl)-L-

tert-butylglycine

434
N—CBZ-L-tert-
N/A
2,3-Dimethoxy-

butylglycine

benzaldoxime

436
N—FMOC-L-tert-
2-cyclohexylacetic
3-Methyl-4-

butylglycine
acid
chlorobenzaldoxime

444
N—((S)-
N/A
3-Methylbenzaldoxime

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

448
N—((S)-
N/A
3-(4-chlorophenyl(-2,1-

tetrahydrofuran-3-

benzisoxazole-5-

yloxy)carbonyl)-L-

carbaldehyde oxime

tert-butylglycine

451
N/A
(S)-
3-Trifluoromethyl-4-

tetrahydrofuran-3-
fluorobenzaldoxime

yl-carbonate

455
N—((S)-
N/A
3,4,5-Trifluoro-

tetrahydrofuran-3-

benzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

456
N—((S)-
N/A
1,4-benzodioxan-6-

tetrahydrofuran-3-

carboxaldehyde

yloxy)carbonyl)-L-

tert-butylglycine

472
N—((S)-
N/A
2-Furanaldoxime

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

480
N—((S)-
N/A
Methyl 3-

tetrahydrofuran-3-

formylbenzoate

yloxy)carbonyl)-L-

tert-butylglycine

481
N—((S)-
N/A
3-(Carboxy)

tetrahydrofuran-3-

benzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

482
N—((S)-
N/A
2-Chloro-6-

tetrahydrofuran-3-

fluorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

486
N—FMOC-L-tert-
2-cyclohexylacetic
3-Chloro-4-

butylglycine
acid
methoxybenzaldoxime

490
N—FMOC-L-tert-
2-(tetrahydro-2H-
3-Methyl-4-

butylglycine
pyran-4-yl)acetic
chlorobenzaldoxime

acid

498
N—((S)-
N/A
3-fluoro-5-

tetrahydrofuran-3-

trifluoromethyl-

yloxy)carbonyl)-L-

benzaldoxime

tert-butylglycine

509
N—((S)-
N/A
3-Trifluoromethyl-

tetrahydrofuran-3-

benzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

511
N—((S)-
N/A
4-Nitrobenzaldoxime

tetrahydrofuran-3-

yloxy)carbonyl)-L-

tert-butylglycine

519
N—((S)-
N/A
3,5-

tetrahydrofuran-3-

Dimethylbenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

524
N/A
(S)-
4-Trifluoromethoxy-

tetrahydrofuran-3-
benzaldoxime

yl-carbonate

525
N—FMOC-L-tert-
2-(tetrahydro-2H-
3-

butylglycine
pyran-4-yl)acetic
Methoxybenzaldoxime

acid

530
N—((S)-
N/A
4-

tetrahydrofuran-3-

Hydroxybenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

535
N—((S)-
N/A
2-nitro-1-phenyl

tetrahydrofuran-3-

ethanone

yloxy)carbonyl)-L-

tert-butylglycine

539
N—FMOC-L-tert-
2-(tetrahydro-2H-
3-Methyl-4-

butylglycine
pyran-4-yl)acetic
fluorobenzaldoxime

acid

543
N—((S)-
N/A
4-(Carboxy)

tetrahydrofuran-3-

benzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

545
N—((S)-
N/A
3-Trifluoromethyl-4-

tetrahydrofuran-3-

fluorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

548
N—FMOC-L-tert-
2-cyclohexylacetic
3-Chloro-4-

butylglycine
acid
fluorobenzaldoxime

550
N—((S)-
N/A
3-fluoro-4-

tetrahydrofuran-3-

methylbenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

551
N—((S)-
N/A
Methyl 4-

tetrahydrofuran-3-

formylbenzoate

yloxy)carbonyl)-L-

tert-butylglycine

555
N—((S)-
N/A
3-Methyl-4-

tetrahydrofuran-3-

fluorobenzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

560
N—((S)-
N/A
3-Trifluoromethyl-

tetrahydrofuran-3-

benzaldoxime

yloxy)carbonyl)-L-

tert-butylglycine

572
N—FMOC-L-tert-
2-(tetrahydro-2H-
3,5-Dimethy1-4-

butylglycine
pyran-4-yl)acetic
methoxybenzaldoxime

acid

Certain other compounds of Formula I may be prepared as illustrated by Method 4.

Method 4

embedded image

Referring to Method 4, the Fmoc derivative A3 is prepared as described in Method 1. Reaction of A3 with the resin bound imino amide D1 in the presence of a coupling reagent provides the compound bound resin D2. The resin bound imino amide D1 may be prepared from the diketo compound X31 by reaction with an amino resin such as, for example, a derivatized aminomethylated polystyrene, e.g., X32. Deprotection of D2 provides D3 which reacts with an R₁carboxylic acid in the presence of a coupling reagent to provide D4 wherein R₁is R₄C(O)—. Reaction of D4 with the nitrile oxide if provides D5 which on hydrolysis from the resin provides A10.

Example 9
Compound No. 303

embedded image

To a suspension of resin M4A, which has the same structure as X33, (20 g, 0.4 mmol/g, 8 mmol) in DCM (100 mL) was added PPh₃(21 g, 80 mmol), dimethyl barbituric acid (12.5 g, 80 mmol) and Pd(PPh₃)₄(920 mg, 0.8 mmol). The suspension was shaken overnight, drained, washed with DMF (10 times) and DCM (4 times). N-Fmoc-4-methyleneproline (3.0 g, 8.8 mmol), HBTU (3.3 g, 8.8 mmol) and HOBt (1.1 g, 8.8 mmol) and DIEA (1.6 mL, 8.8 mmol) were dissolved in DMF (100 mL). The solution was added to the resin and shaken overnight. The resin was then drained, washed with DMF (10 times), DCM (4 times) and dried to afford resin M4B.

To the resin M4B (20 g, 8 mmol) was added 20% piperidine in DMF (100 mL), shaken for 1 hour, and then washed with DMF (10 times), DCM (4 times). To the resin was added a mixture of Fmoc-tert-butylglycine (5.6 g, 16 mmol), HBTU (6.1 g, 16 mmol), HOBt (2.2 g, 16 mmol) and (iPr)₂NEt (2.9 mL, 16 mmol) in DMF (100 mL). The suspension was shaken overnight, drained, washes with DMF (10 times), DCM (4 times) and dried to afford the resin M4C.

To the resin M4C (20 g, 8 mmol) was added 20% piperidine in DMF (100 mL), shaken for 1 hour, and then washed with DMF (10 times), DCM (4 times). To the resin was added a mixture of cyclohexylacetic acid (1.42 g, 10 mmol), HBTU (3.8 g, 10 mmol), HOBt (1.35 g, 10 mmol) and (iPr)₂NEt (1.8 mL, 10 mmol) in DMF (100 mL). The suspension was shaken overnight, drained, washes with DMF (10 times), DCM (4 times) and dried to afford the resin M4D.

embedded image

A solution of 3-pyridinealdoxime (M4E) (122 mg, 1 mmol) in DMF (3 mL) was added NCS (134 mg, 1 mmol). The mixture was heated to 50-60° C. for 30 minutes. After cooling down to room temperature, the 3-pyridinechloroxime (M4F) solution was added to a resin M4D (300 mg, 0.12 mmol). To the mixture was added TEA (0.14 mL, 1 mmol) and the reaction mixture was heated to 50-60° C. for 4 hours. The reaction mixture was drained and washed with DMF (6 times) and DCM (6 times). The resin was treated with 95% TFA for 5 hours. The mixture was drained, and washed with DCM. The filtrate was concentrated in vacuo, purified from column 50-100% EtOAc/Hex to afford 7 mg colorless solid as product Compound No. 303. HPLC 5.7-6.4 minutes; MS 651.5 and LC-MS 3.9 minutes.

Listed below in Table 5 are additional compounds produced by Method 4.

TABLE 5

Additional compounds of formula I produced by method 4.

Com-

pound
Starting Material
Starting Material

No.
for P¹
for C¹
Starting Material for R₃

41
N-FMOC-L-tert-
2-Cyclohexylacetic
5-Bromoprydine-3-

butylglycine
acid
carbaldehyde

179
N-FMOC-L-tert-
2-Cyclohexylacetic
5-Bromo-2-Furaldehyde

butylglycine
acid

230
N-FMOC-L-tert-
2-Cyclohexylacetic
2-methylbenzofuran-3-

butylglycine
acid
carbaldehyde

303
N-FMOC-L-tert-
2-Cyclohexylacetic
3-Pyridine-carboxaldehyde

butylglycine
acid

495
N-FMOC-L-tert-
2-Cyclohexylacetic
4-Chloro-1-methyl-1H-

butylglycine
acid
pyrazole-3-carbaldehyde

552
N-FMOC-L-tert-
2-Cyclohexylacetic
2,3-Dihydrobenzo[b]furan-

butylglycine
acid
5-carboxaldehyde

Certain other compounds of the invention may be prepared as illustrated in Methods 5A and 5B.

Method 5A

embedded image

Referring to Method 5a, the exomethylene acid compound A1 is protected to provide the di-t-butyl dicarboxylate E1. Reaction of E1 with nitrile oxide of formula if provides the intermediate B1 which is transformed into amino acid derivative E2. Reaction of E2 with an aminoalcohol E5 provides A9. Compound A9 is converted to A10 as described in Method 1.

Method 5B

embedded image

Referring to Method 5b, the intermediate compound B1 is transformed into amino acid ester E6. Reaction of E6 with an R₁carboxylic acid in the presence of a coupling reagent provides E7 wherein R₁is R₄C(O)—. E7 is deprotected to provide E2 which is converted to A10 as described in Method 1.

Example 10
Compound No. 422

embedded image

Compound 10A (5.0 g, 1.0 eq.) was stirred in 100 mL acetonitrile and to the solution were added ditertbutyldicarbonate (9.6 g, 2 eq.), dimethylaminopyridine (537 mg, 0.2 eq.), and triethylamine (6.13 mL, 2.0 eq.). The mixture was stirred overnight, concentrated, added ethyl acetate, washed with 1.0N HCl, dried over sodium sulfate, concentrated, and purified with silica gel chromatography (10-30% ethyl acetate/hexanes gradient) to give compound M2B. (M+H=284.0). ¹H-NMR (CDCl₃): 5.0 (m, 2H), 4.3-4.5 (m, 1H), 4.0-4.1 (m, 2H), 2.9-3.0 (m, 1H), 2.5-2.6 (d, 1H), 1.5 (s, 3/9 of 18H), 1.4 (s, 6/9 of 18H).

embedded image

Compound 10B (10.0 g, 1.0 eq.) was stirred in 175 mL DCM with piperonaloxime (11.5 g, 2.0 eq.). The solution was cooled on an ice bath and to it added bleach (175 mL) slowly. The mixture was then warmed to room temperature, stirred for 2 hours, separated and its aqueous layer extracted with DCM twice. Organics were combined and dried over magnesium sulfate, filtered and concentrated. The residue was purified and separated diastereomers by silica gel chromatography (5-30% ethyl acetate/hexanes gradient) to yield 4.1 g of Compound 10C. (M+H=446.9.) ¹H-NMR (CDCl₃): 7.25 (m, 1H), 7.0 (d, 1H), 6.8 (d, 1H), 6.0 (s, 2H), 4.6-4.4 (m, 1H), 4.0-3.8 (m, 1H), 3.7-3.6 (m, 1H) 3.4-3.3 (m, 1H), 3.3-3.2 (m, 1H), 2.8-2.7 (m, 1H), 2.3-2.2 (m, 1H), 1.5 (s, 9H), 1.4 (s, 9H).

Alternatively, compound 10B was prepared by the following procedures:

Preparation: (S)-di-tert-butyl 4-methylenepyrrolidine-1,2-dicarboxylate

embedded image

Procedure 1

Triethylamine (2 eq.) was added to a solution of (S)-1-(tert-butoxycarbonyl)-4-methylenepyrrolidine-2-carboxylic acid (1.0 eq.), di-tert-butyldicarbonate (2.0 eq.), and DMAP (0.2 eq.) in acetonitrile (10 vol) at ambient temperature. The reaction mixture was stirred for 16 h, then diluted with isopropyl acetate (25 vol). A wash with water (20 vol., twice) was followed by a filtration over Na₂SO₄and solvent removal. The crude product was purified by filtration through a pad of silica gel (37 vol silica, first flush with heptane (80 vol), second flush with 10% ethyl acetate in heptane (30 vol)). Removal of solvent from the second flush gave compound 10B.

Procedure 2

embedded image

A solution of di-tert-butyl dicarbonate (1.1 eq.) in MTBE (2 vol.) was added to a mixture of (S)-1-(tert-butoxycarbonyl)-4-methylenepyrrolidine-2-carboxylic acid (1.0 eq.) and DMAP (0.2 eq.) in MTBE (8 vol) and t-butanol (1.75 vol.). The mixture was stirred for 1 hour, at which point gas evolution ceased. The mixture was washed with 1 N HCl (3 vol.), then saturated aqueous NaHCO₃(3 vol.) and then brine (3 vol.). The solvent is then removed to afford compound 10B.

embedded image

Compound 10C (4.0 g, 1.0 eq.) was stirred in 1/1 TFA/DCM for 3 hours and the solution was concentrated. To the concentrate was added 100 mL acetone, 100 mL saturated sodium bicarbonate solution, and ditertbutyldicarbonate and the resulting solution was stirred overnight and then acidified with 1.0 N HCl solution and extracted with ethyl acetate (thrice). The organics were washed with brine solution and dried over magnesium sulfate, filtered and concentrated to yield 4.0 g of Compound 10D (M+H=391.1).

embedded image

Compound 10D (50 mg, 1.0 eq.) stirred in 0.5 mL DMF with EDC (37 mg, 1.5 eq.), PS-HOBt (137 mg, 1.5 eq.) and NMM (56 uL, 4.0 eq.), and to the solution was added 0.5 mL DCM to assist in swelling of the resin. To the mixture was added 3-amino-2-hydroxyhexanamide (30 mg, 1.3 eq.) and the mixture was stirred overnight, filtered, diluted with ethyl acetate, washed with 1.0 N HCl, dried over sodium sulfate, filtered, and concentrated. The solution was purified by silica gel chromatography (100% DCM-5% MeOH/DCM gradient) to yield 21 mg of the crude product, which was then stirred in 4.0 N HCl/dioxane for 2 hours and concentrated to yield compound 10E as an HCl salt (M+H=419.0).

embedded image

Compound 10E (21 mg, 1.0 eq.) was stirred in DMF with NMM (13 uL, 1.4 eq.) and to the solution was added a solution of (S)-3,3-dimethyl-2-(((S)-tetrahydrofuran-3-yloxy)carbonylamino)butanoic acid (14 mg, 1.4 eq.), EDC (11 mg, 1.4 eq.), and PS-HOBt (40 mg, 1.4 eq.) in DMF, with enough DCM to swell the resin. The mixture was stirred overnight, filtered, washed with 1.0 N HCl, dried over sodium sulfate, filtered and then concentrated to give compound 10F, which was used without further purification. (M+H=646.4)

embedded image

Compound 10F was stirred in DCM and to the solution was added Dess-Martin Periodinane (≈3.0 eq.). The solution was stirred for 1 hour, added to it 1.0 N Na₂S₂O₃, and stirred. The mixture was purified by silica gel chromatography (10-90% ethyl acetate/hexanes gradient) to yield 9 mg of Compound No. 422 (M+H=644.3). ¹H-NMR (CDCl₃): 7.3 (m, 1H), 7.15 (m, 1H), 6.95 (m, 1H), 6.8 (m, 1H), 6.75 (m, 1H), 6.0 (s, 2H), 5.5-5.4 (m, 2H), 5.4-5.3 (m, 2H), 4.8-4.7 (m, 1H), 4.3 (m, 1H), 4.2 (m, 1H), 4.0-3.8 (m, 3H), 3.7 (m, 1H), 3.4-3.2 (m, 2H), 2.6 (m, 1H), 2.5 (m, 1H), 2.2-2.1 (m, 1H), 2.1-2.0 (m, 1H), 1.9 (m, 1H), 1.6 (m, 1H), 1.5-1.4 (m, 2H), 1.0-0.9 (m, 13H).

Example 11
Compound No. 562

embedded image

To 2,4-dimethoxybenzaldoxime (4.5 g, 24.8 mmol) in DMF (135 mL) was added dropwise over 2 h at room temperature a solution of N-chlorosuccinimide (6.6 g, 49.7 mmol) in DMF (135 mL). The reaction was stirred 14 hours and compound M2B (5.2 g, 18.4 mmol) was added followed by dropwise addition over 1 h of a solution of triethylamine in DMF (2.6 mL, 18.4 mmol, in 15 mL). After stirring for 3 h, the reaction mixture was washed with H₂O and dried over MgSO₄. The resulting residue was purified via silica gel chromatography to afford 5.8 g of compound 11B as a tan solid. ES (+) MS: m/e 497 (M+H)⁺.

To compound 11B (5.5 g, 11.1 mmol) in CH₂Cl₂(30 mL) was added trifluoroacetic acid (30 mL). The reaction mixture was stirred for 90 minutes at room temperature and concentrated under reduced pressure to provide a tan solid, which was dissolved in MeOH (60 mL) and heated to reflux. Concentrated sulfuric acid (≈5 mL) was added dropwise and the reaction was refluxed for 3 hours, after which the solvent was removed under reduced pressure. The resulting residue was dissolved in CH₂Cl₂(75 mL) and carefully treated with a saturated NaHCO₃solution until pH≈9. The organic layer was dried over MgSO₄and concentrated to provide the intermediate amino ester. To N-Boc-tert-butylglycine (3.1 g, 13.6 mmol) in CH₂Cl₂(60 mL) was added EDC (2.6 g, 13.6 mmol), HOBt (1.8 g, 13.6 mmol) and triethylamine (5.5 mL, 39.5 mmol). After stirring 5 minutes, the above amino ester was added and the reaction was stirred at room temperature 14 hours. The reaction mixture was washed with H₂O, 1 N HCl, and saturated NaHCO₃solution. The organic layer was dried over MgSO₄and concentrated in vacuo to provide 5.6 g of compound 11C (over 3 steps) as a brown solid which was used without further purification. ES (+) MS: m/e 568 (M+H)⁺.

To compound 11C (600 mg, 1.1 mmol) in CH₂Cl₂(3 mL) was added trifluoroacetic acid (3 mL). The reaction was stirred for 1 hour and concentrated under reduced pressure to give the desired amine product as the TFA salt. To cyclohexylacetic acid (181 mg, 1.3 mmol) in CH₂Cl₂(6 mL) was added EDC (243 mg, 1.3 mmol), HOBt (171 mg, 1.3 mmol) and triethylamine (516 μL, 3.7 mmol). After stirring for 5 minutes, the above amine was added and the reaction was stirred at room temperature 14 hours. The reaction mixture was washed with H₂O, 1 N HCl, and saturated NaHCO₃solution. The organic layer was dried over MgSO₄and concentrated in vacuo, and the resulting residue was purified via silica gel chromatography to provide 460 mg of compound 11D (over 2 steps) as an off-white solid. ES (+) MS: m/e 592 (M+H)⁺.

To compound 11D (460 mg, 0.8 mmol) in a solution of THF/H₂O (5 mL, 3:1 v/v) was added LiOH monohydrate (82 mg, 1.9 mmol). The reaction mixture was stirred at room temperature 14 hours, acidified using 1 N HCl, and extracted with EtOAc. The organic layer was dried over MgSO₄and concentrated under reduced pressure to provide 405 mg of compound 11E, which was used without further purification. ES (+) MS: m/e 578 (M+H)⁺.

To compound 11E (80 mg, 0.14 mmol) in CH₂Cl₂(1 mL) was added EDC (38 mg, 0.2 mmol), HOBt (27 mg, 0.2 mmol) and triethylamine (68 μL, 0.5 mmol). After stirring for 5 minutes, compound 11F was added and the reaction was stirred at room temperature 14 hours. The reaction mixture was washed with H₂O, 1 N HCl, and saturated NaHCO₃solution. The organic layer was dried over MgSO₄and concentrated in vacuo to provide 95 mg of compound 11G as a brown solid which was used without further purification. ES (+) MS: m/e 718 (M+H)⁺.

To compound 11G (95 mg, 0.14 mmol) in CH₂Cl₂(1 mL) was added Dess-Martin periodinane (71 mg, 0.17 mmol). After stirring for 30 minutes, the reaction was quenched with 1 N Na₂S₂O₃. The organic layer was purified via silica gel chromatography to give Compound No. 562, i.e., compound 11H shown above, as a white solid. ES (+) MS: m/e 716 (M+H)⁺.

Example 12
Compound No. 362

embedded image

4-Methoxy-3,5-dimethylbenzaldehyde (1.86 g, 11.3 mmol) was dissolved in ethanol (30 mL) and stirred with hydroxylamine hydrochloride (2.4 M aq. solution, 5.65 mL, 1.2 eq.) and Na₂CO₃(1.2 M solution, 5.65 mL, 0.6 eq.) at room temperature for 2.5 hours. The mixture was then heated to 60° C. and additional hydroxylamine hydrochloride and Na₂CO₃was added. The mixture was again stirred overnight at 60° C., transferred to a separatory funnel, diluted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO₄, filtered and concentrated. The product was purified by ISCO chromatography with EtOAc/hexanes eluent to yield 1.55 g (8.56 mmol) of 4-methoxy-3,5-dimethylbenzaldehyde oxime as a white solid. M+1=180.0.

To a solution 4-methoxy-3,5-dimethylbenzaldehyde oxime (1.34 g, 7.48 mmol) in DMF (10 mL) was added N-chlorosuccinimide (1.76 g, 13.2 mmol). This solution was stirred until starting material was consumed as indicated by HPLC. To the solution was then added a solution of (S)-di-tert-butyl 4-methylenepyrrolidine-1,2-dicarboxylate (2.1 g, 1.0 eq.) in DMF (5 mL). To the solution was added triethylamine (1.2 eq.) dropwise, and the reaction mixture was stirred for 2 hours. The reaction was then diluted with EtOAc and the organic phase was washed with water, brine, dried (MgSO₄), filtered and concentrated. The product was purified over silica gel on an ISCO Combiflash using EtOAc/hexanes as the eluent to yield 912 mg (1.98 mmol) of compound 12A. M+1=461.4. ¹H-NMR (500 MHz, CDCl₃): 7.30 (s, 2H), 4.40-4.32 (m, 1H), 3.98-3.79 (m, 1H); 3.74 (s, 3H), 3.64-3.58 (m, 1H), 3.40-3.34 (m, 1H), 3.24-3.19 (m, 1H), 2.72 (dd, J=8.7, 12.9 Hz, 1H), 2.29 (s, 6H), 2.11-2.07 (m, 1H), 1.54-1.45 (m, 18H).

embedded image

Compound 12A (910 mg, 1.98 mmol) was stirred in CH₂Cl₂/trifluoroacetic acid (1:1, 20 mL) until HPLC indicated complete deprotection of starting material. The intermediate amino acid was concentrated and then dissolved in methanol (30 mL) and heated to reflux with concentrated H₂SO₄until the starting material was consumed as indicated by HPLC. Concentrated material in vacuo, then dissolved in EtOAc and washed with NaHCO₃, brine, dried over MgSO₄and concentrated to give compound 12B. M+1=319.0

embedded image

Compound 12B (727 mg, 2.28 mmol) was dissolved in DMF (3 mL) with Boc-t-butylglycine (686 mg, 3.0 mmol), EDC.HCl (659 mg, 3.43 mmol), HOBt (460 mg, 3.4 mmol), and DIEA (1.2 mL, 6.89 mmol) and stirred at room temperature overnight. The reaction was then transferred to a separatory funnel and diluted with EtOAc. The organic layer was washed with 1 N HCl (twice, 20 mL each), sat. aq. NaHCO₃(25 mL), water (10 mL), brine (10 mL), dried over MgSO₄and concentrated. The crude product 12C was purified over silica gel on an ISCO Combiflash with EtOAc/Hexanes as eluent to yield 231 mg (0.435 mmol) of compound 12C as a clear colorless oil. LCMS (M+1)=532.45

embedded image

Compound 12C (231 mg, 0.435 mmol) was stirred in 4N HCl in dioxane (15 mL) for 90 minutes at which point TLC analysis indicated no starting material was present in the reaction mixture. The HCl and dioxane were evaporated to yield an off-white foam. A portion of this intermediate (0.35 mmol), EDC.HCl (96 mg, 0.50 mmol.), HOBt (72 mg, 0.53 mmol), and cyclohexaneacetic acid (78 mg, 0.55 mmol) were stirred in DMF (3.5 mL). To this was added DIEA (0.18 mL, 1.0 mmol) and the reaction was stirred overnight. The reaction was then diluted with EtOAc and transferred to a separatory funnel where the layers were separated and the organic phase was washed with 1.0 N HCl, saturated aq. NaHCO₃, brine, dried over MgSO₄and concentrated. The product was purified over silica gel on an ISCO Combiflash with EtOAc/hexane as eluent to yield 219 mg (0.394 mmol) of compound 12D as a clear oil. M+1=556.4

embedded image

Compound 12D (219 mg, 0.394 mmol) in THF/H₂O/MeOH (4:1:1, 6 mL) was stirred with LiOH.H₂O (1.5 eq.) at room temperature overnight. The reaction was then acidified with 1.0 N HCl and extracted with CH₂Cl₂. The organic layer was washed with brine, dried over MgSO₄and concentrated to yield 207 mg (0.382 mmol) of compound 12E. M+1=548.4

embedded image

Compound 12E (207 mg, 0.382 mmol) was stirred with HOBt (107 mg, 0.792 mmol), EDC.HCl (144 mg, 0.764 mmol), and hydroxyamine hydrochloride (168 mg, 0.75 mmol) in DMF (2.0 mL) at room temperature and treated with DIEA (0.400 mL, 2.3 mmol). The reaction was stirred overnight, diluted with EtOAc, washed with 1N HCl, saturated NaHCO₃, and the combined aqueous layers were back extracted with EtOAc. The organic layers were combined, dried over MgSO₄, concentrated and purified over silica gel on an ISCO combiflash with EtOAc/Hexanes as eluent to yield 227 mg (0.320 mmol) of compound 12F as a white solid. (M+TFA) M−1=822.6.

embedded image

Compound 12F (227 mg, 0.320 mmol) was dissolved at room temperature in CH₂Cl₂(4 mL) and treated with Dess-Martin periodinane (142 mg, 1.0 eq.). After 15 minutes, TLC showed the reaction to be complete, and the reaction solution was quenched by the addition of water and stirred vigorously. Additional CH₂Cl₂was added, the organic layer was separated and purified over silical gel on an ISCO combiflash with EtOAc/Hexanes as eluent to yield 159 mg (0.225 mmol) of Compound No. 362. FIA MS (M+1)=708.42. ¹H-NMR (500 MHz, CDCl₃): 7.30 (s, 2H), 7.17 (d, 1H), 6.93 (d, 1H), 6.15 (d, 1H), 5.39-5.33 (m, 1H), 4.72 (t, 1H), 4.66 (d, 1H), 4.25 (d, 1H), 3.74 (s, 3H), 3.74-3.69 (m, 1H), 3.42 (d, 1H), 3.30 (d, 1H), 2.81-2.75 (m, 1H), 2.58-2.46 (m, 2H), 2.29 (s, 6H), 2.16-2.10 (m, 1H), 2.08-2.00 (m, 1H), 1.97-1.88 (m, 1H), 1.85-1.57 (m, 8H), 1.51-1.35 (m, 2H), 1.33-1.22 (m, 2H), 1.20-1.07 (m, 1H), 1.02-0.96 (m, 10H), 0.92 (t, 3H), 0.88-0.80 (m, 2H), 0.66-0.56 (m, 2H).

Example 13
Compound No. 247

embedded image

To a solution of compound 13A (222 mg, 0.5 mmol) was added TEA (0.14 mL) and t-butylisocyanate (0.6 mmol). The resulting solution was stirred overnight and then diluted with EtOAc (20 mL), washed with water (10 mL), dried over Na₂SO₄and concentrated in vacuo. The crude product was purified chromatography on silica gel to afford compound 13B as a white solid (190 mg). HPLC 8.48 min; LC-MS m/z 507.2 ES⁺.

embedded image

Compound 13B was dissolved in THF and the solution was treated with 1.0 N aqueous LiOH and water. The reaction mixture was stirred for 1 hour, and concentrated in vacuo. The residue was then diluted with water, washed with Et₂O and acidified with 1 N aqueous HCl. The resulting mixture was extracted twice with CH₂Cl₂and the combined organics were dried over MgSO₄, filtered and concentrated in vacuo to give crude compound 13C which was used without further purification for the next step. LC-MS m/z 493.22 ES⁺, 491.21 ES⁻.

embedded image

A solution of compound 13C (20.6 mg) in CH₂Cl₂(800 μL) was treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10 mg) and hydroxybenzotriazole (7 mg) for 1 hour. Diisopropylamine (16 μL) and 3-amino-4-cyclobutyl-2-hydroxybutanamide D (10.5 mg) were then added in one portion and the resulting reaction solution was stirred at room temperature for another 16 hours. The mixture was then washed with 1N aqueous HCl, 1:1 solution of 1N aqueous K₂CO₃:1N aqueous NaHCO₃, and brine in succession. The organics were then dried (MgSO₄), concentrated in vacuo and purified by chromatography over silica (0% to 4% MeOH in CH₂Cl₂) to yield compound 130 (11.6 mg). LC-MS m/z 647.25 ES⁺.

embedded image

A solution of compound 13D (11.6 mg) in CH₂Cl₂(1 mL) was charged with Dess-Martin periodinane (8.4 mg) and the reaction mixture was stirred at room temperature for 2 hours. The resulting white mixture was then washed with 1.0 N aqueous Na₂S₂O₃, the phase were separated and the organics were the dried over MgSO₄, concentrated in vacuo and purified by chromatography over silica (30% to 65% EtOAc in hexanes) to yield 6.7 mg of Compound No. 247 as a white solid: ¹H-NMR (500 MHz, CDCl₃): 7.61 (s), 7.52 (d, J=6.1 Hz), 7.39 (d, J=7.8 Hz), 7.34 (t, J=7.8 Hz), 6.87 (s), 6.77 (s), 5.89 (s), 5.67 (s), 5.23-5.19 (m), 4.83-4.79 (m), 4.47 (s), 4.38 (d, J=11.0 Hz), 3.72 (dd, J=3.1, 11.2 Hz), 3.45 (m), 3.30 (d), 2.64 (m), 2.56 (m), 2.44-2.36 (m), 2.08-1.98 (m), 1.86-1.68 (m), 1.64-1.58 (m), 1.33-1.22 (m), 1.05-1.00 (m, H), 0.95-0.92 (m, H) ppm. LC-MS m/z 647.25 ES⁺.

Example 14
Compound No. 57

embedded image

A solution of compound 14A (512 mg) in dioxane was treated with 4 N HCl in dioxane. The reaction solution was stirred at room temperature for 45 minutes and concentrated in vacuo. The resulting residue was dissolved in a small amount of CH₂Cl₂and crystallized from Et₂O/Hexanes to give compound 14B as a white solid (362 mg). LC-MS m/z 468.24 ES⁺.

embedded image

A solution of cycloheptane acetic acid (83 mg, Aldrich Chemical Co., Milwaukee, Wis.) in CH₂Cl₂(4 mL) was treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (103 mg) and hydroxybenzotriazole (72 mg) for 1 hour. Diisopropylamine (160 μL) and intermediate 14B (179 mg) were then added in one portion and the resulting reaction solution was stirred at room temperature for another 2 hours. The mixture was then washed with 1 N aqueous HCl, 1:1 solution of 1 N aqueous K₂CO₃:1 N aqueous NaHCO₃, and brine in succession. The organics were the dried (MgSO₄), concentrated in vacuo and purified by chromatography over silica (15% to 60% EtOAc in hexanes) to yield compound 14C (188 mg). LC-MS m/z 606.25 ES⁺.

embedded image

Compound 14C (186 mg) was dissolved in THF (3 mL) and the solution was treated with 1 N aqueous LiOH (620 μL) and water (1 mL). The reaction mixture was stirred for 45 minutes at room temperature, and concentrated in vacuo. The residue was then diluted with water, washed with Et₂O and acidified with 1 N aqueous HCl. The resulting mixture was extracted twice with EtOAc and the combined organics were dried over MgSO₄, filtered and concentrated in vacuo to give crude compound 14D which was used without further purification for the next step. LC-MS m/z 592.25 ES⁺, 590.35 ES⁻.

embedded image

A solution of compound 14D (89 mg) in CH₂Cl₂(1 mL)/DMF (1 mL) was treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (44 mg) and hydroxybenzotriazole (31 mg) for 1 hour. Diisopropylamine (70 μL) and (3S)-3-amino-4-cyclopropyl-2-hydroxybutanamide (35 mg) were then added in one portion and the resulting reaction solution was stirred at room temperature for another 16 hours. The mixture was then washed with 1 N HCl, 1:1 solution of 1N aqueous K₂CO₃:1N aqueous NaHCO₃, and brine in succession. The organics were the dried over MgSO₄, concentrated in vacuo and purified by chromatography over silica (0% to 5% MeOH in CH₂Cl₂) to yield 96 mg of compound 14F. LC-MS m/z 732.21 ES⁺.

embedded image

A solution of compound 14F (96 mg) in CH₂Cl₂(1.5 mL) was charged with Dess-Martin periodinane (83 mg) and the reaction mixture was stirred at room temperature for 2 hours. The resulting white mixture was then washed with 1 N aqueous Na₂S₂O₃, the phase were separated and the organics were the dried over MgSO₄, concentrated in vacuo and purified by chromatography over silica (10% to 95% EtOAc in hexanes) to yield Compound No. 57 (44 mg) as a white solid. ¹H-NMR (500 MHz, CDCl₃): 7.76 (s), 6.75 (br s), 6.48 (s), 6.07 (d), 5.40 (m), 4.67 (m), 4.22 (d), 3.95 (s), 3.87 (s), 3.75 (d), 3.43 (m), 2.51 (m), 2.10 (m), 1.30-1.87 (m), 1.12-1.28 (m), 0.97 (m), 0.79 (m), 0.15 (m), 0.03 (m) ppm. LC-MS m/z 730.35 ES⁺, 728.35 ES⁻.

Example 15
Compound No. 600

embedded image

Compound 600 has the same structure as compound 266 in Table 1.

To a solution of (R)-2-cyclohexylbut-3-ynoic acid (430 mg, 2.4 mmol) in CH₂Cl₂(10 mL) was added EDC (458 mg, 2.4 mmol), HOBt (324 mg, 2.4 mmol) and triethylamine (836 μL, 6.0 mmol). After stirring for 5 minutes, compound 15A (800 mg, 2.0 mmol) was added and the reaction was stirred 16 hours. The mixture was washed with H₂O, 1 N HCl, and saturated NaHCO₃solution. The organic layer was dried over MgSO₄and concentrated under reduced pressure to provide 1.23 g crude compound 15B, which was purified by silica gel chromatography. ES (+) MS: m/e 570 (M+H)⁺.

To a solution of compound 15B (220 mg, 0.4 mmol) in THF/H₂O (2 mL, 3:1 v/v) was added LiOH monohydrate (115 mg, 3 mmol). The mixture was stirred for 2 hours, acidified using 1 N HCl (6 mL) and extracted with EtOAc (thrice, 10 mL). The combined organics were dried over MgSO₄and concentrated to afford a colorless oil which was used without further purification. The oil was dissolved in CH₂Cl₂(2 mL), then EDC (90 mg, 0.5 mmol), HOBt (63 mg, 0.5 mmol) and triethylamine (163 μL, 1.2 mmol) were added. After stirring for 5 minutes, (3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide (87 mg, 0.5 mmol) was added. The reaction was stirred 12 hours, washed with H₂O, 1 N HCl, and saturated NaHCO₃solution. The organic layer was dried over MgSO₄and concentrated under reduced pressure to provide 215 mg of compound 15C as a colorless oil, which was used without further purification. ES (+) MS: m/e 724 (M+H)⁺.

To a solution of compound 15C (53 mg, 0.07 mmol) in CH₂Cl₂(0.5 mL) was added Dess-Martin periodinane (41 mg, 0.1 mmol). The mixture was stirred for 30 minutes, quenched with 1 Na₂S₂O₃, and separated. The organic layer was purified by silica gel chromatography to provide 20 mg of Compound No. 600. ¹H-NMR (500 MHz, CDCl₃): 7.53 (d, J=1.6 Hz, 1H), 7.43 (d, J=7.6 Hz, 1H), 7.31-7.25 (m, 2H), 6.83 (d, J=3.3 Hz, 1H), 6.24-6.21 (m, 1H), 5.30-5.26 (m, 1H), 4.70-4.58 (m, 2H), 4.23-4.21 (m, 1H), 3.64 (dd, 1H), 3.36-3.20 (m, 2H), 2.70-2.68 (m, 1H), 2.57-2.35 (m), 2.04-1.82 (m), 1.72-1.30 (m, 10H), 1.18-0.75 (m), 0.55-0.40 (m).

Example 16
Compound No. 602

embedded image

Compound 602 has the same structure as compound 212 in Table 1.

To a solution of compound 15C prepared above (20 mg, 0.03 mmol) and azidomethyl pivalate (4 mg, 0.03 mmol, prepared according to Syn. Lett., 2005, 18, pp. 2847-2850) in tert-butanol/H₂O (1204, 1:1 v/v) was added an aqueous solution of sodium ascorbate (10 μL, 0.01 mmol, 1.0 M) followed by an aqueous solution of copper(II) sulfate pentahydrate (5 μL, 0.001 mmol, 0.3 M). The reaction mixture was stirred 12 hours at room temperature, diluted with H₂O, and extracted with EtOAc. The combined organics were washed with 5% ammonium hydroxide followed by brine, and were dried over MgSO₄and concentrated under reduced pressure to provide 25 mg of crude compound 16B, which was used without further purification. ES (+) MS: m/e 881 (M+H)⁺.

To a solution of compound 16B in MeOH (120 μL) was added aqueous NaOH (120 μL, 1 M). The reaction was stirred at room temperature for 2 hours, then treated with 1 M HCl (120 μL) followed by H₂O (120 μL). The mixture was extracted with CH₂Cl₂(thrice, 200 μL each). The combined extracts were washed with brine and concentrated to a volume of approximately 100 μL. To this solution was added Dess-Martin periodinane (17 mg, 0.04 mmol) and the reaction was stirred 30 minutes. The mixture was quenched with 1 M Na₂S₂O₃(150 μL), and the organic layer was separated and purified via silica gel chromatography to afford 3 mg of Compound No. 602. ES (+) MS: m/e 765 (M+H)⁺.

Listed below in Table 6 are additional compounds of Formula I prepared by Methods 5a and 5b.

TABLE 6

Additional compounds of formula I produced by methods 5a and 5b.

Compound
Starting Material for
Starting Material for
Starting Material for

No.
P¹
C¹
R₃

5
N-BOC-L-tert-
2-(4-hydroxy-4-
3-Chlorobenzaldoxime

butylglycine
methylcyclohexyl)acetic

acid

10
(S)-4-(benzylamino)-2-
N/A
3-chlorobenzaldoxime

isopropyl-4-

oxobutanoic acid

13
N-BOC-L-tert-
2-Norbornaneacetic
3-chlorobenzaldoxime

butylglycine
acid

19
N-BOC-L-tert-
2-
3-chlorobenzaldoxime

butylglycine
(bicyclo[4.1.0]heptan-

1-yl)acetic acid

21
(S)-4-
N/A
3-chlorobenzaldoxime

(cyclohexylamino)-2-

isopropyl-4-

oxobutanoic acid

23
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chlorobenzaldoxime

butylglycine
acid

26
N-BOC-L-tert-
N-Benzoyl-L-Proline
3-Chlorobenzaldoxime

butylglycine

27
N-BOC-L-tert-
Cyclobutaneacetic acid
3-Chlorobenzaldoxime

butylglycine

43
N-BOC-L-tert-
2-Cyclohexylacetic
2,4-Dimethoxy-5-

butylglycine
acid
chlorobenzaldoxime

48
N-BOC-L-tert-
2-Norbornaneacetic
2,4-Dimethoxy-5-

butylglycine
acid
chlorobenzaldoxime

50
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chlorobenzaldoxime

butylglycine
acid

59
N-BOC-L-tert-
2-cycloheptylacetic
2,4-Dimethoxy-5-

butylglycine
acid
chlorobenzaldoxime

63
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chlorobenzaldoxime

butylglycine
acid

67
N-BOC-L-tert-
2-(tetrahydro-2H-
3-Chloro-5-fluoro-4-

butylglycine
pyran-4-yl)acetic acid
ethoxybenzaldoxime

86
N-BOC-L-tert-
Isopropyl isocyanate
Piperonal oxime

butylglycine

90
N-BOC-L-tert-
N/A
3-Chlorobenzaldoxime

butylglycine

104
N-BOC-L-tert-
Tert-butylacetic acid
2,4-Dimethoxy-5-

butylglycine

chlorobenzaldoxime

105
N-BOC-L-tert-
N/A
3-Chlorobenzaldoxime

butylglycine

117
N-BOC-L-tert-
4-methyltetrahydro-
3-Chlorobenzaldoxime

butylglycine
2H-pyran-4-carboxylic

acid

121
N-BOC-L-tert-
2-(2,2-
3-Chlorobenzaldoxime

butylglycine
dimethyltetrahydro-2H-

pyran-4-yl)acetic acid

126
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-5-fluoro-4-

butylglycine
acid
ethoxybenzaldoxime

129
N-BOC-L-tert-
2-cycloheptylacetic
2,4-Dimethoxy-5-

butylglycine
acid
chlorobenzaldoxime

131
N-BOC-L-tert-
N/A
2,4-Dimethoxy-5-

butylglycine

chlorobenzaldoxime

136
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-5-fluoro-4-

butylglycine
acid
ethoxybenzaldoxime

145
N-BOC-L-tert-
2-(tetrahydro-2H-
Piperonal oxime

butylglycine
pyran4-yl)acetic acid

153
N-BOC-L-tert-
2-((2R,5R)-2,5-
3-Chlorobenzaldoxime

butylglycine
dimethyltetrahydro-2H-

pyran-4-yl)acetic acid

168
N-BOC-L-tert-
2-Cyclohexylacetic
Thiophene-3-

butylglycine
acid
carboxaldehyde

172
N-Phenyl-L-tert-
N/A
3-chlorobenzaldoxime

butylglycine

178
N-BOC-L-tert-
2-(tetrahydro-2H-
3-Chloro-5-fluoro-4-

butylglycine
pyran-4-yl)acetic acid
ethoxybenzaldoxime

184
N-BOC-L-tert-
4-methyltetrahydro-
3-Chlorobenzaldoxime

butylglycine
2H-pyran-4-carboxylic

acid

188
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chlorobenzaldoxime

butylglycine
acid

192
N-BOC-L-tert-
cyclopentyl 2,5-
2,4-Dimethoxy-5-

butylglycine
dioxopyrrolidin-1-yl
chlorobenzaldoxime

carbonate

195
N-BOC-L-tert-
2-cyclohexylacetic acid
3-chloro-4-methoxy-5-

butylglycine

methylbenzaldoxime

211
2-(tert-
2-cyclohexylacetic acid
3-chlorobenzaldoxime

butoxycarbonylamino)-

2-(1-

methoxycyclopropyl)

acetic acid

212
N-BOC-L-tert-
(S)-2-cyclohexyl-3-
3-chlorobenzaldoxime

butylglycine
(1H-1,2,3-triazol-4-

yl)propanoic acid

214
N-(3-methoxyphenyl)-
N/A
3-chlorobenzaldoxime

L-tert-butylglycine

217
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chlorobenzaldoxime

butylglycine
acid

219
N-BOC-L-tert-
2-cycloheptylacetic
3-Chlorobenzaldoxime

butylglycine
acid

225
N-BOC-L-tert-
cyclopentyl 2,5-
3-Chlorobenzaldoxime

butylglycine
dioxopyrrolidin-1-yl

carbonate

231
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chlorobenzaldoxime

butylglycine
acid

233
N-BOC-L-tert-
2-(1-
3-Chlorobenzaldoxime

butylglycine
hydroxycyclohexyl)

acetic acid

247
N-BOC-L-tert-
tert-Butyl isocyanate
3-Chlorobenzaldoxime

butylglycine

256
N-BOC-L-tert-
2-cyclohexylacetic acid
5-Ethyl-2-furaldoxime

butylglycine

263
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-5-fluoro-4-

butylglycine
acid
ethoxybenzaldoxime

264
N-BOC-L-tert-
N/A
2,4-Dimethoxy-5-

butylglycine

chlorobenzaldoxime

266
N-BOC-L-tert-
(S)-2-cyclohexylpent-
3-chlorobenzaldoxime

butylglycine
4-ynoic acid

268
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chlorobenzaldoxime

butylglycine
acid

273
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chlorobenzaldoxime

butylglycine
acid

280
(S)-2-isopropyl-4-
N/A
3-chlorobenzaldoxime

(isopropylamino)-4-

oxobutanoic acid

282
N-BOC-L-tert-
2-(tetrahydro-2H-
2,4-Dimethoxy-5-

butylglycine
pyran-4-yl)acetic acid
Chlorobenzaldoxime

284
N-BOC-L-tert-
(S)-2-
3-chlorobenzaldoxime

butylglycine
cyclohexylpropanoic

acid

286
N-BOC-L-tert-
2-(4-methyltetrahydro-
2,4-Dimethoxy-5-

butylglycine
2H-pyran-4-yl)acetic
chlorobenzaldoxime

acid

290
N-CBZ-L-tert-
N/A
Piperonal oxime

butylglycine

294
N-BOC-L-tert-
2-cyclohexylacetic acid
3-chlorobenzaldoxime

butylglycine

295
N-((S)-tetrahydrofuran-
N/A
Piperonal oxime

3-yloxy)carbonyl)-L-

tert-butylglycine

297
N-BOC-L-tert-
Tert-butylacetic acid
2,4-Dimethoxy-5-

butylglycine

chlorobenzaldoxime

307
N-BOC-L-tert-
2-Cyclohexylacetic
2,4-Dimethoxy-5-

butylglycine
acid
methoxybenzaldoxime

310
N-BOC-L-tert-
2-(tetrahydro-2H-
3,5-Dimethyl-4-

butylglycine
pyran-4-yl)acetic acid
methoxybenzaldehyde

326
N-BOC-L-tert-
2-(tetrahydro-2H-
2,4-Dimethoxy-5-

butylglycine
pyran-4-yl)acetic acid
Chlorobenzaldoxime

335
N-CBZ-L-tert-
N/A
2,4-

butylglycine

Dimethoxybenzaldoxime

337
N-BOC-L-tert-
cyclopentyl 2,5-
3-Chlorobenzaldoxime

butylglycine
dioxopyrrolidin-1-yl

carbonate

344
N-BOC-L-tert-
N-FMOC-L-
3-Chlorobenzaldoxime

butylglycine
cyclohexylglycine

followed by 2-pyrazine

carboxylic acid

346
N-BOC-L-tert-
2-Norbornaneacetic
3-Chlorobenzaldoxime

butylglycine
acid

351
N/A
Tert-butylacetic acid
3-chlorobenzaldoxime

356
N-BOC-L-tert-
2-(4-methyltetrahydro-
2,4-Dimethoxy-5-

butylglycine
2H-pyran-4-yl)acetic
chlorobenzaldoxime

acid

362
N-BOC-L-tert-
2-Cyclohexylacetic
3,5-Dimethyl-4-

butylglycine
acid
methoxybenzaldehyde

369
N-BOC-L-tert-
cyclopenty 2,5-
3-Chlorobenzaldoxime

butylglycine
dioxopyrrolidin-1-yl

carbonate

375
N-BOC-L-tert-
2-(tetrahydro-2H-
Piperonal oxime

butylglycine
pyran-4-yl)acetic acid

382
N-BOC-L-tert-
isopropylisocyanate
Piperonal oxime

butylglycine

388
N-BOC-L-tert-
Cyclohexylacetic acid
3-Chlorobenzaldoxime

butylglycine

411
N-BOC-L-tert-
2-(tetrahydro-2H-
3-Chloro-5-fluoro-4-

butylglycine
pyran-4-yl)acetic acid
ethoxybenzaldoxime

415
N-CBZ-L-tert-
N/A
Piperonal oxime

butylglycine

418
N-BOC-L-tert-
2-((2S,5R)-2,5-
3-Chlorobenzaldoxime

butylglycine
dimethyltetrahydro-2H-

pyran-4-yl)acetic acid

419
N-BOC-L-tert-
2-(2,2-
3-Chlorobenzaldoxime

butylglycine
dimethyltetrahydro-2H-

pyran-4-yl)acetic acid

440
N-BOC-L-tert-
cyclopentyl 2,5-
2,4-Dimethoxy-5-

butylglycine
dioxopyrrolidin-1-yl
chlorobenzaldoxime

carbonate

442
N-BOC-L-tert-
2-((2S,5R)-2,5-
3-Chlorobenzaldoxime

butylglycine
dimethyltetrahydro-2H-

pyran-4-yl)acetic acid

445
N-BOC-L-tert-
2-(1,4-
3-Chlorobenzaldoxime

butylglycine
dioxaspiro[4.5]decan-

8-yl)acetic acid

446
N-BOC-L-tert-
2-Norbornaneacetic
2,4-Dimethoxy-5-

butylglycine
acid
chlorobenzaldoxime

453
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chlorobenzaldoxime

butylglycine
acid

468
N-BOC-L-tert-
2-((2R,5R)-2,5-
3-Chlorobenzaldoxime

butylglycine
dimethyltetrahydro-2H-

pyran-4-yl)acetic acid

473
N-BOC-L-tert-
Tert-butylacetic acid
2,4-Dimethoxy-5-

butylglycine

chlorobenzaldoxime

485
N-BOC-L-tert-
trans-2-phenyl-1-
3-Chlorobenzaldoxime

butylglycine
cyclopropanecarboxylic

acid

502
N-BOC-L-tert-
N-FMOC-L-
3-Chlorobenzaldoxime

butylglycine
cyclohexylglycine

followed by 2-pyrazine

carboxylic acid

510
N-BOC-L-tert-
2-(tetrahydro-2H-
2,4-Dimethoxy-5-

butylglycine
pyran-4-yl)acetic acid
Chlorobenzaldoxime

516
N-((S)-tetrahydrofuran-
N/A
2,4-

3-yloxy)carbonyl)-L-

Dimethoxybenzaldoxime

tert-butylglycine

522
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chlorobenzaldoxime

butylglycine
acid

529
N-BOC-L-tert-
2-(4-methyltetrahydro-
2,4-Dimethoxy-5-

butylglycine
2H-pyran-4-yl)acetic
chlorobenzaldoxime

acid

541
N-BOC-L-tert-
2-(4-methyltetrahydro-
3-chlorobenzaldoxime

butylglycine
2H-pyran-4-yl)acetic

acid

542
N-BOC-L-tert-
tert-Butyl isocyanate
3-chlorobenzaldoxime

butylglycine

549
N-BOC-L-tert-
(S)-2-cyclohexyl-4-
3-chlorobenzaldoxime

butylglycine
oxo-4-(pyrrolidin-1-

yl)butanoic acid

554
N-BOC-L-tert-
2-(tetrahydro-2H-
5-Ethyl-2-furaldoxime

butylglycine
pyran-4-yl)acetic acid

562
N-BOC-L-tert-
2-Cyclohexylacetic
2,4-Dimethoxy-5-

butylglycine
acid
chlorobenzaldoxime

569
N-BOC-L-tert-
(S)-2-cyclohexyl-4-
3-chlorobenzaldoxime

butylglycine
(methylamino)-4-

oxobutanoic acid

575
N-BOC-L-tert-
2-(4-hydroxy-4-
3-chlorobenzaldoxime

butylglycine
methylcyclohexyl)acetic

acid

577
N-BOC-L-tert-
2-Cyclohexylacetic
2,4-Dimethoxy-5-

butylglycine
acid
chlorobenzaldoxime

581
N-BOC-L-tert-
N/A
2,4-Dimethoxy-5-

butylglycine

chlorobenzaldoxime

589
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-8-

butylglycine
acid
quinolinecarbaldoxime

590
N-BOC-L-tert-
2-Cyclohexylacetic
2-Methoxy-3-

butylglycine
acid
methylbenzaldoxime

Certain other compounds of Formula I may be prepared by Method 6 as illustrated below.

Method 6

embedded image

Referring to Method 6, the intermediate A1 is converted to the Boc-methyl ester F1. Removal of the Boc group from F1 provides the amine-ester F2 which is reacted with an R₁carboxylic acid in the presence of a coupling reagent to provide F3 wherein R₁is R₄C(O)—. F3 reacts with a nitrile oxide if to provide the spiroisoxazoline acid E4 after hydrolysis of the corresponding methyl ester E3. Conversion of E4 to E7 is achieved as described in Method 5a.

Example 17
Compound No. 267

embedded image

4-Hydroxy-3,5-dimethylbenzaldehyde (2.5 g, 16.6 mmol) in THF (100 mL) was treated with KOH (1.5 eq. of 1 N aq. solution, 25 mL) and 2-iodopropane (2.0 eq.) and heated at reflux for 5 days. The reaction was then cooled, transferred to a separatory funnel, diluted with MTBE, washed with H₂O, 1 N NaOH (twice), 0.5 N HCl (aq.), brine, dried over MgSO₄and concentrated. The product was purified over silica gel on an ISCO combiflash to yield 1.99 g (10.34 mmol) 4-isopropoxy-3,5-dimethylbenzaldehyde as a colorless liquid. H¹NMR (300 MHz, CDCl₃) 9.89 (s, 1H), 7.55 (s, 2H), 4.41-4.26 (m, 1H), 2.32 (s, 6H), 1.32 (d, J=6 Hz, 6H).

embedded image

4-(Isopropoxy)-3,5-dimethylbenzaldehyde (1.98 g, 10.3 mmol) in EtOH (60 mL) was heated to 60° C. with hydroxylamine hydrochloride (2.4 M aq. solution, 5.2 mL, 1.2 eq.) and Na₂CO₃(1.2 M solution, 5.2 mL, 0.6 eq.) at room temperature for 2 hours. The reaction was transferred to a separatory funnel, diluted with EtOAc; the organic layer was separated, washed with brine, dried (MgSO₄), filtered and concentrated to yield 710 mg (3.24 mmol) of 4-(isopropoxy)-3,5-dimethylbenzaldehyde oxime as a light yellow oil. ¹H-NMR (500 MHz, CDCl₃): 8.10 (s, 1H), 7.23 (s, 2H), 4.29-4.18 (m, 1H), 2.29 (s, 6H), 1.29 (d, 6H).

embedded image

4-(Isopropoxy)-3,5-dimethylbenzaldehyde oxime (166 mg, 0.801 mmol) in DMF (3 mL) at room temperature was stirred overnight with NCS (130 mg, 0.974 mmol). To this reaction was added the methyl ester (257 mg, 0.679 mmol) in DMF (1.5 mL) and triethylamine (1.2 eq.). This was stirred overnight at room temperature. The reaction was then diluted with EtOAc/Hexanes (4:1) and washed with 1N HCl (aq.). The layers were separated and the aqueous layer was back extracted with EtOAc/Hexanes (4:1). The organic layers were combined, washed with brine, dried (MgSO₄), and concentrated. The compound was purified over silica gel on an ISCO Combiflash with EtOAc/Hexanes as eluent to yield 173 mg (0.296 mmol) of compound 17A as a white solid. LCMS (M+1)=584.3

embedded image

The compound 17A (173 mg, 0.30 mmol) was stirred with LiOH.H₂O (1.1 eq.) in THF/MeOH/H₂O (4:1:1, 3 mL) at RT overnight. The reaction was diluted with EtOAc, acidified with 1N HCl (aq) and the layers were separated. The aqueous layer was back extracted with EtOAc, the organic layers combined, washed with brine, dried (MgSO₄) and concentrated to yield 171 mg (0.30 mmol) of compound 17B as a white solid. FIA MS (M+1)=570.3.

embedded image

Carboxylic acid 17B (83 mg, 0.146 mmol), EDC.HCl (37 mg, 1.3 eq.), HOBt (26 mg, 1.3 eq.), (3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide hydrochloride (64 mg, 2.0 eq.), and DIEA (0.100 mL, 4.0 eq.) were stirred in DMF (0.9 mL) at room temperature overnight. The reaction mixture was then diluted with EtOAc and washed with 1 N HCl (aq) (twice). The aqueous layer was separated and back extracted with EtOAc. The organic layers were combined, washed with brine, dried (MgSO₄), and concentrated. The product was purified over silica gel on an ISCO combiflash to yield 85 mg (0.115 mmol) of compound 17C. LCMS (M+1)=738.3

embedded image

Compound 17C (85 mg, 0.115 mmol) in CH₂Cl₂(1.0 mL) was treated with Dess-Martin periodinane (54 mg, 1.1 eq.) for 30 minutes. The reaction was quenched with equal volumes (≈1 mL) of saturated aqueous NaHCO₃and 1 N Na₂S₂O₃(aq). The organic layer was separated and purified directly over silica gel on an ISCO combiflash to yield 77 mg (0.105 mmol) of Compound No. 267. FIA MS (M+1)=736.2. ¹H-NMR (300 MHz, CDCl₃): 7.33-7.26 (m, 2H), 7.12 (d, 1H), 6.91 (d, 1H), 6.12 (d, 1H), 5.45-5.32 (m, 1H), 4.78-4.63 (m, 2H), 4.29-4.17 (m, 2H), 3.71 (d, 1H), 3.43 (d, 1H), 3.30 (d, 1H), 2.86-2.74 (m, 1H), 2.63-2.42 (m, 2H), 2.29 (s, 6H), 2.19-1.85 (m, 3H), 1.84-0.82 (m, 34H), 0.65-0.58 (m, 2H).

Example 18
Compound No. 556

embedded image

4-Ethoxybenzaldehyde oxime (204 mg, 1.24 mmol), was dissolved in DMF (to 0.2 M) and treated with NCS (1 eq.). The reaction was stirred until starting material was consumed. One half of the reaction volume was removed and treated with additional NCS (1.5 eq.) and stirred overnight. To this solution was then added the methyl ester (200 mg, 0.85 eq.) in DMF (0.3 mL) and triethylamine (0.10 mL, 1.1 eq.). The reaction was stirred overnight at room temperature, then diluted with EtOAc, washed with 1 N HCl (aq.), and washed with brine. The aqueous layer was back extracted with EtOAc and the combined organic layers were washed with brine, dried (MgSO₄), and concentrated to a dark oil. The product was purified over silica gel on an ISCO combiflash to yield 97 mg (0.168 mmol) of compound 18A. LCMS (M+1)=576.3

embedded image

Compound 18A (97 mg, 0.168 mmol) was dissolved in THF/MeOH/H2O (8:1:1, 5 mL) and treated with LiOH.H2O (1.1 eq.) at room temperature overnight. The reaction was concentrated, diluted in EtOAc and methanol and washed with 1N HCl (aq). The aqueous layer was separated and extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO₄and concentrated to yield 76 mg (0.135 mmol) of compound 18B. FIA MS (M−1)=560.4

embedded image

Compound 18B (35 mg, 0.062 mmol), EDC.HCl (15 mg, 1.3 eq.), HOBt (12 mg, 1.3 eq.), an amino alcohol hydrochloride (55 mg, 2.0 eq.), and DIEA (0.044 mL, 4.0 eq.) were stirred in DMF (0.7 mL) at room temperature overnight. The reaction was then diluted with EtOAc and washed with 1 N HCl (aq) (twice). The aqueous layer was separated and back extracted with EtOAc. The organic layers were combined, washed with brine, dried (MgSO₄), anc concentrated. The product was purified over, silica gel on an ISCO combiflash to yield 28 mg (0.038 mmol) of compound 18C. LCMS (M+1)=730.2

embedded image

Compound 18C (28 mg, 0.038 mmol) in CH₂Cl₂(0.7 mL) was treated with Dess-Martin periodinane (18 mg, 1.1 eq.) for 30 minutes. The reaction was quenched with equal volumes (≈1 mL) of saturated aqueous NaHCO₃and 1N Na₂S₂O₃(aq.). The organic layer was separated and purified directly over silica gel on an ISCO Optix 10× to yield 24 mg (0.033 mmol) of Compound No. 556. FIA MS (M+1)=728.2. ¹H-NMR (300 MHz, CDCl₃): 7.65 (d, 1H), 7.48 (dd, 1H), 7.11 (d, 1H), 6.95-6.88 (m, 2H), 6.08 (d, 1H), 5.40-5.31 (m, 2H), 4.78-4.63 (m, 2H), 4.26 (d, 1H), 4.20-4.11 (m, 2H), 3.71 (d, 1H), 3.42 (d, 1H), 3.27 (d, 1H), 2.84-2.73 (m, 1H), 2.63-2.46 (m, 2H), 2.20-1.86 (m, 3H), 1.62-0.85 (m, 30H), 0.66-0.58 (m, 2H)

Listed below in Table 7 are additional compounds of Formula I prepared by Method 6.

TABLE 7

Additional compounds of formula I prepared by method 6.

Com-

pound
Starting Material
Starting Material

No.
for P¹
for C¹
Starting Material for R₃

18
N-BOC-L-tert-
2-Cyclohexylacetic
7-Chloro-2,3-

butylglycine
acid
dihydrobenzo[b]furan-5-

carboxaldoxime

19
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-4-

butylglycine
acid
methylbenzaldoxime

28
N-BOC-L-tert-
2-Cyclohexylacetic
2-Cyanobenzaldoxime

butylglycine
acid

31
N-BOC-L-tert-
2-cyclohexylacetic
8-Quinoline-carbaldoxime

butylglycine
acid

38
N-BOC-L-tert-
2-Cyclohexylacetic
2,5-Dichloro-3-

butylglycine
acid
methoxybenzaldoxime

42
N-BOC-L-tert-
2-Cyclohexylacetic
8-Quinoline-

butylglycine
acid
carboxaldoxime

62
N-BOC-L-tert-
2-Cyclohexylacetic
5-chloro-3-

butylglycine
acid
Thiophenecarboxaldoxime

68
N-BOC-L-tert-
2-cyclohexylacetic
8-Quinoline-carbaldoxime

butylglycine
acid

74
N-BOC-L-tert-
2-Cyclohexylacetic
8-chloro-2,2-

butylglycine
acid
dimethylchromane-6-

carbaldoxime

89
N-BOC-L-tert-
2-Cyclohexylacetic
3-nitrobenzaldoxime

butylglycine
acid

97
N-BOC-L-tert-
2-cyclohexylacetic
3-Chloro-4-

butylglycine
acid
isopropoxybenzaldoxime

111
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-4-methoxy-5-

butylglycine
acid
methylbenzaldoxime

114
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-4-methoxy-5-

butylglycine
acid
methylbenzaldoxime

132
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-nicotinaldoxime

butylglycine
acid

134
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-2,3-

butylglycine
acid
dihydroxybenzo[b]furan-7-

carboxaldoxime

158
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-6-

butylglycine
acid
methoxybenzaldoxime

165
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-2-

butylglycine
acid
methoxynicotinaldoxime

168
N-BOC-L-tert-
2-Cyclohexylacetic
3-

butylglycine
acid
Thiophenecarboxaldoxime

169
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-2-

butylglycine
acid
fluorobenzaldoxime

170
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-2,2-dimethyl-2,3-

butylglycine
acid
dihydrobenzo[b]furan-7-

carboxaldoxime

250
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-5-

butylglycine
acid
methoxybenzaldoxime

267
N-BOC-L-tert-
2-cyclohexylacetic
4-Isopropoxy-3,5-

butylglycine
acid
dimethylbenzaldoxime

292
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-nicotinaldoxime

butylglycine
acid

305
N-BOC-L-tert-
2-Cyclohexylacetic
6-Fluoro-1,3-

butylglycine
acid
benzodioxene-

8-carbaldoxime

312
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-6-

butylglycine
acid
methoxynicotinaldoxime

315
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-4-Methyl-3,4-

butylglycine
acid
dihydro-2H-1,4-

benzoxazine-7-

carbaldoxime

321
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-2,3-dimethoxy-

butylglycine
acid
benzaldoxime

366
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-4-methoxy-2-

butylglycine
acid
methylbenzaldoxime

370
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-piperonal oxime

butylglycine
acid

396
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-5-

butylglycine
acid
methylbenzaldoxime

406
N-BOC-L-tert-
2-cyclohexylacetic
4-Cyclopropylmethoxy-

butylglycine
acid
3,5-dimethylbenzaldoxime

430
N-BOC-L-tert-
2-Cyclohexylacetic
8-Chloro-1-methyl-1,2,3,4-

butylglycine
acid
tetrahydro-quinoline-6-

carboxaldoxime

469
N-BOC-L-tert-
2-Cyclohexylacetic
2-Methoxy-

butylglycine
acid
nicotinaldoxime

478
N-BOC-L-tert-
2-Cyclohexylacetic
5-Chloro-2-

butylglycine
acid
thiophenecarboxaldoxime

494
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-4,5-

butylglycine
acid
dimethoxybenzaldoxime

499
N-BOC-L-tert-
2-Cyclohexylacetic
7-Chloro-2,3-

butylglycine
acid
dihydrobenzo[b]furan-5-

carboxaldoxime

500
N-BOC-L-tert-
2-Cyclohexylacetic
4-Methoxy-3-

butylglycine
acid
methylbenzaldoxime

513
N-BOC-L-tert-
2-cyclohexylacetic
4-Ethoxy-3,5-

butylglycine
acid
dimethylbenzaldoxime

556
N-BOC-L-tert-
2- cyclohexylacetic
3-Chloro-4-

butylglycine
acid
ethoxybenzaldoxime

591
N-BOC-L-tert-
2-Cyclohexylacetic
2-Pyridinecarboxaldoxime

butylglycine
acid

592
N-BOC-L-tert-
2-Cyclohexylacetic
2-Pyridinecarboxaldoxime

butylglycine
acid

593
N-BOC-L-tert-
2-Cyclohexylacetic
4-Chloro-2-

butylglycine
acid
pyridinecarboxaldoxime

594
N-BOC-L-tert-
2-Cyclohexylacetic
3-Chloro-6-

butylglycine
acid
fluorobenzaldoxime

Certain other compounds of the invention may be prepared by Method 7 as illustrated below.

Method 7

embedded image

Referring to Method 7, the Cbz hydroxy acid G1 is converted to the methyl ester G2 and deprotected to provide the amino-ester G3. Reaction of G3 with the spiroisoxazoline acid G4 in the presence of a coupling reagent provides the intermediate G5. Hydrolysis of the methyl ester of G5 provides the hydroxy acid G6 which is oxidized with, for example, Dess-Martin periodinane to provide the ketoacid G7. Reaction of G7 with an amine R₁₃R₁₀NH in the presence of a coupling reagent provides the final product G8.

Example 19
Compound No. 275
Step 1: Preparation of Compound Q

embedded image

1.00 g of acid 19A was dissolved in 14 mL of methanol and heated to reflux. Two drops of concentrated H₂SO₄was added and the reaction refluxed overnight. The mixture was cooled to room temperature, and neutralized with 50 mL of NaHCO₃(sat. aq.). The reaction mixture was extracted three times with 50 mL of ethyl acetate. The combined organic extracts were dried over magnesium sulfate and evaporated to yield 1.01 g of compound 19B as a white powder. Major diastereomer ¹H-NMR (300 MHz, CDCl₃) δ: 7.40-7.31 (m, 5H), 5.12 (s, 2H), 4.99 (d, 1H, J=8.7 Hz), 4.35 (s, 1H), 4.15-4.02 (m, 1H), 3.81 (s, 3H), 3.05 (br s, 1H), 1.67-1.17 (m, 4H), 0.91 (t, 3H, J=6.8 Hz). Minor diastereomer ¹H-NMR (300 MHz, CDCl₃) δ: 7.40-7.31 (m, 5H), 5.07 (s, 2H), 4.90 (d, 1H, J=9.8 Hz), 4.19 (s, 1H), 4.15-4.02 (m, 1H), 3.76 (s, 3H), 3.03 (br s, 1H), 1.67-1.17 (m, 4H), 0.96 (t, 3H, J=7.1 Hz).

1.00 g of CBz-protected methyl ester 19B was dissolved in 11 mL of methanol. 150 mg of Pd(OH)₂(20 wt % on carbon) was added, and the mixture flushed with 1 atm of hydrogen gas and stirred at room temperature for 3 hours. The methanolic solution was filtered through a Celite® plug and the filter pad rinsed with additional methanol. Upon evaporation, a light yellow oil was collected and redissolved in 5 mL of DCM and treated with 1.5 mL of 4 M HCl solution in dioxane. Upon stirring for 1 minute, the reaction was evaporated. 0.65 g of compound 19C was collected as a white powder, and characterized by LCMS (M+1=162.0).

0.80 g of the spiroisoxazoline acid of compound 19D was stirred with 0.33 g of HOBt, 0.81 g of HBTU, and 15 mL of DMF. To the stirring solution was added 807 μL of DIPEA, and stirred for 10 minutes. 0.33 g of the hydrochloride salt 19C was added. The reaction was stirred at room temperature for 3 hours. To the reaction mixture was added 200 mL of EtOAc, and the mixture washed twice with 100 mL of NaHCO₃(sat. aq.), then 100 mL of brine. The organic phase was dried over MgSO₄and evaporated. The crude reaction mixture was purified by elution through silica gel column (40 g column, gradient elution, 40-55% EtOAc:Hexanes) to give 1.02 g of compound 19E as a white powder, which was identified by LCMS (M+1=661.3).

1.04 g of methyl ester 19E was stirred in 6 mL of THF and to this solution was added 3 mL of 1 M LiOH (aq). The reaction was stirred at room temperature for 2 hours where it was determined by HPLC to be complete. The reaction was treated with 6 mL of 1 M HCl, and extracted three times with 15 mL of ethyl acetate. The combined extracts were evaporated to give 1.00 g of compound Q as a beige solid which was carried on to the next step.

Step 2: Preparation of Compound R

embedded image

To a solution of compound Q (0.300 g, 0.46 mmol) in CH₂Cl₂(15 mL) was added 5.58 mL of a 0.16 M solution of Dess Martin periodinane in CH₂Cl₂dropwise. After it was stirred for 4 hours at room temperature, 10 mL of 1M Na₂S₂O₃solution was added and the reaction mixture was stirred for 30 minutes at ambient temperature. The organic layer was separated, washed with water, dried over Na₂SO₄, filtered and concentrated. The crude mixture was redissolved in CH₂Cl₂and precipitated with Hexanes and filtered to give 230 mg of compound R. LC/MS: m/z 645.7 (M+H)⁺ at 1.99 minutes (10-99% CH₃CN (0.035% TFA)/H₂O (0.05% TFA))

Step 3: Preparation of Compound No. 275

embedded image

To a suspension of compound R (20 mg, 0.0.031 mmol) in anhydrous acetonitrile was added pyridine (10 μL, 0.124 mmol), 2-chloro-1-methyl-pyridinium iodide (15.3 mg, 0.06 mmol), HOBt (6.8 mg, 0.05 mmol), followed by the addition of a 504 solution of isopropylamine (3.7 mg, 0.062 mmol) in anhydrous acetonitrile. The reaction was allowed to stir at room temperature and complete after two hours. The reaction mixture was quenched with 1 mL of saturated aqueous sodium bicarbonate solution, the layers were separated and aqueous layer was extracted three times with CH₂Cl₂. The combined organics were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was dissolved in 1.5 mL CH₂Cl₂and purified by normal phase HPLC (10-99% EtOAc/Hexanes) to yield Compound No. 275. LC/MS: m/z 686.7 (M+H)⁺ at 2.01 minutes (10-99% CH₃CN (0.035% TFA)/H₂O (0.05% TFA))

Example 20
Compound No. 181

embedded image

To a suspension of R (20 mg, 0.031 mmol) in anhydrous 1,4-dioxane was added pyridine (7.6 μL, 0.093 mmol), then pentafluorophenyl trifluoroacetate (8.8 μL, 0.05 mmol) and allowed to stir for 1.5 hours at room temperature, upon which 7-amino-4-methyl-1H-quinolin-2-one (14 mg, 0.08 mmol) was added. The reaction was allowed to stir at room temperature and complete after one hour. The reaction mixture was quenched with 1 mL of saturated aqueous sodium bicarbonate solution, the layers were separated and aqueous layer was extracted three times with CH₂Cl₂. The combined organics were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was dissolved in 1.5 mL CH₂Cl₂and purified by normal phase HPLC (10-99% EtOAc/Hexanes) to yield Compound No. 181. LC/MS: m/z 801.7 (M+H)⁺ at 2.06 minutes (10-99% CH₃CN (0.035% TFA)/H₂O (0.05% TFA)).

Example 21
Compound No. 605

embedded image

A mixture of (3S)-3-(5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-1-oxa-2,7-diazaspiro[4.4]non-2-enecarboxamido)-2-hydroxyhexanoic acid (0.02 g, 0.03 mmol), (3,5-dimethoxyphenyl)methanamine (5.68 mg, 0.033 mmol), HOBt (6.8 mg, 0.05 mmol), DIPEA (22 μL, 0.124 mmol) and CH₂Cl₂(70 μL) was stirred at room temperature for 10 minutes. To the mixture was then added a solution of Mukaiyama's reagent (2-chloro-1-[4-(1H,1H,2H,2H-perfluoro-9-methyldecyl)benzyl]pyridinium hexafluorophosphate) in 200 μL of acetonitrile and the reaction was stirred at room temperature. After 5 hours, 1.34 mL of 0.3 M Dess-Martin Periodinane in CH₂Cl₂was added and the mixture stirred. After 2 hours, the oxidant was quenched with 1.0 mL of saturated NaHCO₃, 1 mL of 1N Na₂S₂O₃and stirred vigorously. The organic layer was separated, dried over Na₂SO₄, filtered and concentrated. The residue was dissolved in 1.5 mL CH₂Cl₂and purified by normal phase HPLC (10%-99% Ethyl acetate/Hexanes) to yield Compound No. 605, (5S,8S)-3-(3-chlorophenyl)-7-(S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(3,5-dimethoxybenzylamino)-1,2-dioxohexan-3-yl)-1-oxa-2,7-diazaspiro[4.4]non-2-ene-8-carboxamide. LC/MS: m/z 794.7 (M+H)⁺ at 4.11 minutes (10%-99% CH₃CN (0.035% TFA)/H₂O (0.05% TFA)).

Listed below in Table 8 are reagents used to prepare additional compounds of Formula I by Method 7.

TABLE 8

Reagents used to prepare additional compounds of formula I by method 7.

Compound

No.
R_2zR_2wNH

2
tert-butylamine

6
2-aminoindane

17
benzo[d]thiazol-2-amine

49
3-((tetrahydrofuran-3-

yl)methoxy)azetidine

58
(R)-(+)-1-(3-

methoxyphenyl)ethylamine

69
6-Methoxytryptamine

73
4-1H-pyrazol-1-yl-

benzylamine

77
benzylamine

79
azetidine

84
2,5-dimethoxyaniline

91
(4-(4-

methoxyphenyl)tetrahydro-

2H-pyran-4-yl)methanamine

96
3-cyano-4-methylaniline

99
cyclohexylamine

113
N,N-Diethylamine

120
Phenyl-2-

pyridinemethylamine

127
3′,5′-dimethoxybenzylamine

133
3-Ethoxyazetidine

138
1-(3-(2-aminopropyl)-1H-

indol-5-yl)ethanone

140
Ethylamine

141
2,3-dihydro-1,4-benzodioxin-

2-ylmethylamine

143
Isobutylamine

148
N-(3-

aminophenyl)methanesulfonamide

176
(2-Phenyl-1,3-thiazol-4-

yl)methylamine

181
7-amino-4-methylquinolin-

2(1H)-one

182
N-Methylethylamine

186
(3R)-(+)-3-

acetamidopyrrolidine

206
beta-alanine-4-methoxy-

betanaphthylamide

221
N-ethyl-3,4-

methylenedioxyamphetamine

238
(R)-3-((tetrahydrofuran-2-

yl)methoxy)azetidine

253
Dimethylamine

255
(S)-(−)-1-(3-

methoxyphenyl)ethylamine

265
cyclopropylmethylamine

275
Isopropylamine

277
(S)-(+)-

tetrahydrofurfurylamine

293
3-aminoisoxazole

296
(S)-alpha-methylbenzylamine

298
3-Pyrazol-1-yl-benzamine

300
1-(Ethyl)propylamine

302
5-Methoxytryptamine

347
(R)-(−)-2-

(methoxymethyl)pyrrolidine

350
N-Methyl-N-propylamine

355
3-Aminobenzamide

368
3-(tetrahydrofuran-3-

yloxy)azetidine

372
Cyclopentylamine

399
1-Aminocyclopropane-1-

carboxylic acid methyl ester

401
Cyclobutylamine

404
2-Methoxyethylamine

408
3-(Aminoethyl)pyridine

410
Morpholine

426
3-Hydroxy-3-methylazetidine

429
1-Phenylcyclopropylamine

433
[3-(4-chlorophenyl0-5-

isoxazolyl}methanamine

441
Furfurylamine

447
2-(3-Pyridyl)ethylamine

452
(R)-2-Butylamine

458
3-(2-aminoethyl)indolin-2-

one

461
4-(Aminomethyl)pyridine

479
2-Fluoroethylamine

488
2-

methoxyphenoxyethylamine

493
Methylamine

496
Pyrrolidine

507
(S)-2-Amino-1,1-dihenyl-1-

propanol

508
(S)-(+)-2-

(methoxymethyl)pyrrolidine

521
3,3-difluoro-azetidine

537
Propylamine

540
2-(3-

methoxyphenyl)ethylamine

546
(R)-alpha-methylbenzylamine

565

embedded image

567
2-aminomethyl benzimidazole

568
Pipecoline

573
3,4-Difluoroaniline

588
3-cyanoaniline

Preparation of Non-Commercial Azetidines Listed in Table 8

embedded image

N-Benzyhydryl-3-methanesulfonylazetidine (104 mg) was combined with ethanol (1.0 mL) and heated in a sealed vial at 95° C. overnight. The reaction was monitored by TLC (30% EtOAc:Hexane). Workup was conducted by adding 1 mL of saturated potassium carbonate solution, and extracting twice with 0.5 mL of ethyl acetate. The combined organic extracts were purified on silica (4 g column, gradient elution, 0-30% EtOAc:hexane). Yielded 49 mg of N-benzhydryl-3-ethoxyazetidine as a clear colorless oil. LCMS (M+1=268.2).

N-Benzhydryl-3-ethoxyazetidine (49 mg) was dissolved in 1 mL of methanol. 22 mg of 10% Pd/C (Degussa-type) was added, and the reaction was carried out under a hydrogen atmosphere. The reaction was stirred at room temperature for 64 h. The mixture was filtered through the Celite®, washed thoroughly with methanol, and evaporated to give a yellow oil (30 mg). The oil consists of a mixture of diphenylmethane and the free azetidine. The crude oil mixture was carried onto subsequent transformations and used in excess.

The following azetidines were prepared in a similar fashion as above, by using the corresponding alcohols.

embedded image

The azetidine

embedded image

was prepared in the method described by Frigola, J. et al. in J. Med. Chem., 36 (1993), 801-810.

Certain other compounds of Formula I may be prepared by Method 8 as illustrated below.

Method 8

embedded image

Referring to Method 8, the spiroisoxazoline acid E4 reacts with the amino ester H1 in the presence of a coupling reagent to provide the intermediate H2. Macrocyclization of H2 results in compound H3. Hydrolysis of the ester H2 provides acid H4. Reaction of acid H4 with a sulfonamide or sulfamide in the presence of a coupling reagent provides the product H5.

Example 22
Compound No. 409

embedded image

(S)-2-(tert-butoxycarbonylamino)non-8-enoic acid, purchased from RSp Amino Acid located in Massachusetts, (179 mg, 1.0 eq.) was stirred in DMF with HBTU (376 mg, 1.5 eq.), HOBt (94 mg, 1.05 eq.), and DIEA (345 uL, 3.0 eq.) for 15 minutes. Added compound 22A (194 mg, 1.0 eq.) and stirred overnight. To the solution was added ethyl acetate. The solution was washed with 1 N HCl (thrice) followed by brine, dried over sodium sulfate, filtered, concentrated and purified by silica chromatography (10-30% ethyl acetate/hexanes gradient) to yield compound 22B (253 mg). (M+H=548.2).

embedded image

Compound 22B (253 mg, 1.0 eq.) was stirred in THF (1 mL) and methanol (0.5 mL). To the solution was added lithium hydroxide (97 mg, 5.0 eq.) in water (0.5 mL) and stirred for 2 more hours. The mixture was diluted with ethyl acetate, washed with 1 N HCl, then brine, and the solution was dried over MgSO₄, filtered and concentrated to yield compound 22C (235 mg) as a pure white solid (M+H=534.2).

embedded image

Compound 22C (247 mg, 1.0 eq.) stirred in 1 mL acetonitrile. To the solution was added TBTU (297 mg, 2.0 eq.), DIEA (241 uL, 3.0 eq.), then (1R,2S)-methyl-1-amino-2-vinylcyclopropanecarboxylate (86 mg, 1.2 eq.) and stirred overnight. The solution was diluted with ethyl acetate and washed with 1 N HCl then brine, dried over sodium sulfate, filtered, concentrated and purified by silica chromatography (10-70% ethyl acetate/hexanes gradient) to yield compound 22D (230 mg). (M+H=657.2).

embedded image

Compound 22D (230 mg, 1.0 eq.) was stirred in 70 mL CH₂Cl₂with Hoveyda-Grubbs catalyst (22 mg, 0.1 eq.) at reflux for 1 hour, and the solution cooled to room temperature and purified by silica chromatography (10-70% ethyl acetate/hexanes) to yield compound 22E (172 mg)

embedded image

Compound 22E (172 mg, 1.0 eq.) was stirred in THF (1 mL) and methanol (0.5 mL). To the solution was added LiOH (46 mg, 4.0 eq.) in 0.5 mL water and solution stirred for 2 more hours. To the solution again was added ethyl acetate and washed with 1N HCl and brine, dried over magnesium sulfate, filtered, and concentrated to yield compound 22F (155 mg) as a pure white solid (M+H=617.1).

embedded image

Compound 22F (155 mg, 1.0 eq.) stirred in 1 mL DMF with carbonyldiimidazole (49 mg, 1.2 eq.) at 80° C. for 15 minutes. To the solution was added cyclopropanesulfonamide (49 mg, 1.6 eq.) followed by DBU (36 uL, 1.0 eq.) and stirred for another 10 minutes at 80° C. Then to the solution was added ethyl acetate and solution washed with 1 N HCl and brine, dried over MgSO₄, filtered, and concentrated. The product was purified by silica chromatography (100% DCM to 5% methanol/DCM gradient) to give Compound No. 409 (64 mg, 35%). (M+H=718.1.)

Listed below in Table 9 are additional compounds of Formula I prepared by Method 8.

TABLE 9

Additional compounds of formula I prepared by method 8.

Com-

pound

No.
Starting Material for W
Starting Material for R3

1
OH
7-Chloro-2,3-dihydrobenzo[b]furan-5-

carboxaldoxime

137
OH
3-Chlorobenzaldoxime

163
Cyclopropane sulfonamide
Phenylglyoxylohydroxamyl chloride

232
Cyclopropane sulfonamide
3-Chlorobenzaldoxime

320
OH
Phenylglyoxylohydroxamyl chloride

386
Cyclopropane sulfonamide
7-Chloro-2,3-dihydrobenzo[b]furan-5-

carboxaldoxime

409
Cyclopropane sulfonamide
3-Chlorobenzaldoxime

470
OH
3-Chlorobenzaldoxime

Certain other compounds of Formula I may be prepared in Method 9 as illustrated below.

Method 9

embedded image

Referring to Method 9, the protected spiroisoxazoline B3 (prepared by Method 2) reacts with the resin bound imino-amine D1 to provide the intermediate I1. Deprotection of I1 provides the amine 12 which reacts with an R₁carboxylic acid in the presence of a coupling reagent to provide 13 wherein R₁is R₄C(O)—. Hydrolysis of 13 provides the final compound A10.

A person skilled in the art can use the examples and methods described herein, along with known synthetic methodologies, to synthesize compounds of Formula I according to Method 9 illustrated above.

Listed below in Table 10 are additional compounds of Formula I prepared by Method 9.

TABLE 10

Additional compounds of formula I prepared by method 9.

Com-

pound

No.
P¹

46
5-Bromoindole-2-carboxylic acid

54
Acetyl-D-ethionine

60
2-(R)-[[(4-Methylphenyl)Sulfonyl]amino]-2-phenylacetic acid

65
2-oxo-1-phenylpyrrolidine-3-carboxylic acid

88
Acetyl-D-Methyltyrosine-OH

98
N-Acetyl-L-leucine

100
2-[[(4-Fluorophenyl)Sulfonyl]amino]-3-methylbutanoic acid

157
5,6-dimethoxyindole-2-carboxylic acid

218
Pyr-Val-OH

227
1-carbamoylcyclopropanecarboxylic acid

246
5-(2,4-dimethylphenylamino)-5-oxopentanoic acid

248
4-Chloro-2-(6-methoxypyridin-3-ylamino)benzoic acid

309
3-[[(4-acetamidophenyl)sulfonyl]amino]-3-propanoic acid

328
3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acid

332
(S)-2-acetamido-3-(4-isopropoxyphenyl)propanoic acid

376
3-(2-oxobenzo[d]oxazol-3(2H)-yl)propanoic acid

380
4-trifluoromethoxyphenylacetic acid

395
2-[[(4-Methoxyphenyl)Sulfonyl]amino]-3-methylbutanoic acid

397
2-((S)-2-oxo-4-phenyloxazolidin-3-yl)acetic acid

412
Acetyl-D-tyrosine-OH

416
2-(R)-[[(4-Chlorophenyl)Sulfonyl]amino]-3-methylpentanoic acid

420
3-(2-diethylamino)-2-oxoethyl)1H-indole-2-carboxylic acidi

421
trans-2-Phenyl-1-cyclopropanecarboxylic acid

466
2-[[(4-Fluorophenyl)Sulfonyl]amino]-2-phenylacetic acid

476
2-(S)-[[(4-Methylphenyl)Sulfonyl]amino]-2-phenylacetic acid

483
3-(N-Phenylphenylsulfonamido)propanoic acid

489
2-(R)-[[(4-Methoxyphenyl)Sulfonyl]amino]-3-methylbutanoic acid

501
2-[(PhenylSulfonyl)amino]-2-phenylacetic acid

534
2-(R)-[[(4-Methoxyphenyl)Sulfonyl]amino]-3-methylpentanoic acid

574
2-(1-oxoisoindolin-2-yl)propanoic acid

586
6-(2,5-dimethoxyphenyl)-2-oxo-1,2,3,6-tetrahydropyrimidine-4-

carboxylic acid

587
2-(R)-[[(4-Methoxyphenyl)Sulfonyl]amino]-4-methylpentanoic acid

Certain other compounds of Formula I may be prepared in Method 10 as illustrated below.

Method 10

embedded image

Referring to Method 10, the protected spiroisoxazoline B3 (e.g., R₁is Fmoc) reacts with M10A (R″, e.g., can be methyl or immobilized on PS-Wang resin) to provide intermediate M10B. Hydrolysis of M10B yields the carboxylic acid M10C, which is subsequently coupled with the appropriate sulfonamide to afford the final compound M10D. M10C can also be a final compound of formula I.

Similarly, a person skilled in the art can use the examples and methods described herein, along with known synthetic methodologies, to synthesize compounds of Formula I according to Method 10 illustrated above.

Listed below in Table 11 are additional compounds of Formula I prepared by Method 10.

TABLE 11

Additional compounds of formula I prepared by method 10.

Compound

P1 Starting
C1 Starting

No.
W Starting Material
Material
Material
R3 Starting Material

35
Cyclopropane
N-Boc-L-tert-
NA
3-

sulfonamide
butylglycine

Chlorobenzaldoxime

45
Cyclopropane
N-((S)-
NA
3-

sulfonamide
tetrahydrofuran-

Chlorobenzaldoxime

3-yloxy)

carbonyl)-L-

tert-

butylglycine

57
Cyclopropane
N-Boc-L-tert-
NA
7-Chloro-2,3-

sulfonamide
butylglycine

dihydrobenzo[b]furan-

5-carboxaldoxime

115

embedded image

N-Boc-L-tert- butylglycine
2- cyclohexylacetic acid
3- Chlorobenzaldoxime

130
Cyclopropanesulfonamide
N-Alloc-L-tert-
NA
3-

butylglycine

Chlorobenzaldoxime

144
OH
N-Boc-L-tert-
2-
3-

butylglycine
cyclohexylacetic
Chlorobenzaldoxime

acid

162
Cyclopropane
N-Boc-L-tert-
2-
3-

sulfonamide
butylglycine
cyclohexylacetic
Chlorobenzaldoxime

acid

190
Cyclopropane
N-Boc-L-tert-
cyclopentyl 2,5-
3-

sulfonamide
butylglycine
dioxopyrrolidin-
Chlorobenzaldoxime

1-yl carbonate

269
OH
N-((S)-
NA
3-

tetrahydrofuran-

Chlorobenzaldoxime

3-yloxy)

carbonyl)-L-

tert-

butylglycine

272
OH
N-Boc-L-tert-
cyclopentyl 2,5-
Nitropropane

butylglycine
dioxopyrrolidin-

1-yl carbonate

359
Cyclopropane
N-Boc-L-tert-
2-(tetrahydro-
3-

sulfonamide
butylglycine
2H-pyran-4-
Chlorobenzaldoxime

yl)acetic acid

384
OH
N-Boc-L-tert-
2-(tetrahydro-
3-

butylglycine
2H-pyran-4-
Chlorobenzaldoxime

yl)acetic acid

438
Cyclopropane
N-Boc-L-tert-
cyclopentyl 2,5-
Nitropropane

sulfonamide
butylglycine
dioxopyrrolidin-

1-yl carbonate

439
OH
N-Boc-L-tert-
NA
3-

butylglycine

Chlorobenzaldoxime

443
OH
N-Boc-L-tert-
cyclopentyl 2,5-
3-

butylglycine
dioxopyrrolidin-
Chlorobenzaldoxime

1-yl carbonate

457
OH
N-Boc-L-tert-
tert-
3-

butylglycine
Butylisocyanate
Chlorobenzaldoxime

460
OH
N-Alloc-L-tert-
NA
3-

butylglycine

Chlorobenzaldoxime

Additional compounds of the invention may be prepared as illustrated in the scheme Method 11.

Method 11

embedded image

Referring to Method 11, reaction of the methylene compound M11A with 1,1-dibromoformaldoxime in the presence of a mild base such as, for example, sodium bicarbonate provides the bromospiroisoxazoline M11B. Reaction of M11B with an amine R^XR^YNH or an arylalcohol R^XOH provides the intermediate M11C wherein R₃is an optionally substituted amino or optionally substituted aryloxy. Deprotection of M11C to provide the corresponding acid followed by reaction with an appropriate amino compound according to procedures previously described provides compounds of the invention.

Example 23
Preparation of Intermediates for Compounds Wherein R₃is Amino or Aryloxy
Step 1

embedded image

Compound M11D (6.96 g, 1.0 eq) was stirred in 85 mL ethyl acetate. Sodium bicarbonate (6.30 g 4.4 eq) was added followed by dibromoformaldoxime (4.11 g, 1.2 eq) and the mixture stirred overnight at room temperature. Water (85 mL) was added and the mixture stirred until clear. The phases were separated, the aqueous phase extracted with ethyl acetate and the combined organic phases were dried over sodium sulfate, filtered, and concentrated. Chromatography on silica column with ethyl acetate/hexanes gradient yielded 5.55 grams of Compound M11E and 0.93 grams of its diastereomer (6:1 ratio).

embedded image

Compound M11F (100 mg, 1.0 eq) was stirred in 270 uL of isoindoline at 95° C. overnight. The mixture was diluted with ethyl acetate and washed with 1N HCl. The organic phase was dried over sodium sulfate, filtered and concentrated. Chromatography on silica with ethyl acetate/hexanes yielded 83 mg of Compound M11G.

embedded image

Phenol (20 mg, 1.1 eq) stirred in 350 uL DMF. Sodium hydride (8.3 mg, 1.1 eq, 60%) was added and the mixture stirred for 5 minutes. Compound M11F was added and the mixture stirred at 90° C. overnight. The mixture was partitioned between 1N HCl and ethyl acetate, and the organic phase dried over sodium sulfate, filtered and concentrated to give 70 mg Compound M11H.

ADDITIONAL EXAMPLES
Example 23
Compound No. 610

embedded image

Oxime 23A (6.29 g, 40.4 mmol) was dissolved in DMF (63 mL) and N-chlorosuccinimide (5.39 g, 40.4 mmol) was added portionwise to the stirring solution. Stirring continued for 3 hours at room temperature when conversion was determined to be 56% (by HPLC). The reaction was pushed to completion by gentle heating at 70° C. for 45 minutes. 4-Methyleneproline derivative (8.81 g, 31.1 mmol) was added and rinsed into the solution using DMF (5 mL). Triethylamine (5.7 mL) was carefully added dropwise over 30 minutes. The reaction was then stirred at room temperature for 16 hours overnight. An aliquot was analyzed by HPLC and it was determined to contain a 4:1 ratio of cycloaddition diasteromers. Ethyl acetate (200 mL) was added and the organic phase was washed with water (thrice, 200 mL each) and brine (200 mL). The organic phase was then dried over magnesium sulfate and evaporated. The crude oil was divided into two portions and each was purified using an ISCO combiflash equipped with a 330 g silica column (10-20% EtOAc: pet. ether, 72 minutes). The desired product was the major isomer which eluted from the column ahead of the minor isomer and 9.42 g of 23B was obtained as an orange oil. The minor isomer was also isolated, subjected to a recrystallization from EtOAc:hexane, and obtained as an off-white crystalline powder (1.53 g).

embedded image

Compound 23B (9.42 g) was stirred in trifluoroacetic acid (12 mL) for 2 hours. The solvent was evaporated and replaced with methanol (50 mL). The solution was heated to reflux and H₂SO₄(3.0 mL) was added dropwise. The reaction was refluxed for a total of 6 hours when by HPLC, conversion to the methylester was determined to be greater than 95%. The reaction was cooled and evaporated to remove the excess methanol. The resulting oil was redissolved in CH₂Cl₂(200 mL) and neutralized with saturated sodium bicarbonate (200 mL). The organic phase was collected, and the aqueous phase was extracted with CH₂Cl₂(twice, 100 mL each). The organic extracts were combined, evaporated over magnesium sulfate, and evaporated to give 5.09 g of compound 23C as an oil that was immediately carried onto the next step.

embedded image

The amino ester 23C (1.25 g, 4.24 mmol) was treated with LiOH.H₂O (186 mg, 4.4 mmol) in THF/H₂O (3:1, 10 mL) for 45 minutes. The solvents were removed in vacuo to obtain a solid. This solid was slurried in acetone (20 mL) and saturated NaHCO₃(aq) (20 mL) at room temperature. Fmoc-Cl (1.12 g, 4.33 mmol) was added and the reaction was monitored by HPLC. After 20 minutes, the contents of the reaction flask were transferred to a separatory funnel with CH₂Cl₂and acidified with 2 N HCl (aq.). The aqueous phase was extracted with CH₂Cl₂(twice, 100 mL each). The resulting emulsion was filtered, and the organic layers were combined, dried over MgSO₄, and concentrated to give compound 23D.

embedded image

Compound XX4 was shaken in a solution of 20% piperidine in DMF (20 mL) for 60 minutes. The resin was washed with DMF (thrice), CH₂Cl₂(thrice) and repeated. The resulting resin was then shaken with compound 23D (437 mg, 0.87 mmol), HATU (392 mg, 1.03 mmol), and DIEA (0.300 mL, 1.72 mmol) in DMF (10 mL) overnight. The result compound bound resin 23F was then washed with DMF (thrice), CH₂Cl₂(thrice) and repeated. (M+1)=612.26.

embedded image

The compound bound resin 23F was shaken in 20% piperidine in DMF (8 mL) for 2 hours. The resin was then washed with DMF (thrice), CH₂Cl₂(thrice) and repeated. (M+1)=390.1. This resin was then shaken overnight in DMF with (S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoic acid (3 eq.), HOBT (3 eq.), HBTU (3 eq.), and DIEA (6 eq.). The resin was washed with DMF (thrice) and CH₂Cl₂(thrice) and repeated, then shaken for 100 minutes in TFA (5 mL). The resulting resin was filtered and the filtrate concentrated and purified by reverse phase chromatography to yield 9.4 mg of Compound 443 as a white solid. (M+1)=615.6, ¹H-NMR (500 MHz, DMSO-d₆): 8.63 (s, 1H), 7.67 (s, 1H), 7.63 (d, J=6.7 Hz, 1H), 7.55-7.49 (m, 2H), 6.90 (d, J=8.4 Hz, 1H), 5.77-5.69 (m, 1H), 5.20-5.17 (m, 1H), 5.06 (d, J=10.5 Hz, 1H), 4.93 (brs, 1H), 4.35 (t, J=7.7 Hz, 1H), 4.11 (d, J=8.8 Hz, 1H), 4.06 (d, J=10.9 Hz, 1H), 3.80 (d, J=11.6 Hz, 1H), 3.62-3.50 (m, 2H), 2.63-2.31 (m, 2H), 2.18-2.13 (m, 1H), 2.07-2.01 (m, 1H), 1.87-1.51 (m, 9H), 1.29-1.28 (m, 1H), 0.95-0.91 (brs, 9H).

embedded image

Compound 23G (6.6 mg, 0.011 mmol) was stirred in DMF (0.5 mL) with CDI (2.8 mg, 0.017 mmol) for 1 hour at 80° C. Cyclopropyl sulfonamide (3.8 mg, 0.031 mmol) and DBU (0.01 mL) were added, the heat was removed and the reaction was stirred overnight at room temperature. The reaction was purified by reverse phase chromatography to yield 2.8 mg of Compound No. 190 (0.0039 mmol). (M+1)=718.1. ¹H-NMR (500 MHz, methanol-d₄): 9.26 (s, 0.4H), 9.02 (s, 0.6H), 7.72 (d, J=1.7 Hz, 1H), 7.61 (dd, J=1.3, 7.3 Hz, 1H), 7.47-7.41 (m, 2H), 5.81-5.73 (m, 1H), 5.33-5.30 (m, 1H), 5.14-5.10 (m, 1H), 5.03 (brs, 1H), 4.45-4.41 (m, 1H), 4.31-4.25 (m, 2H), 3.94 (d, J=11.0 Hz, 1H), 3.62-3.53 (m, 2H), 2.99-2.92 (m, 1H), 2.55-2.49 (m, 1H), 2.29-2.23 (m, 2H), 1.89-1.53 (m, 10H), 1.44-1.40 (m, 1H), 1.32-1.24 (m, 1H), 1.19-1.02 (m, 2H), 0.90 (s, 9H).

Example 24
Compound No. 618

embedded image

Carboxylic acid 24A (69 mg, 0.13 mmol), HATU (50 mg, 0.13 mmol), compound 24B (0.13 mmol), and DIEA (0.045 mL, 0.26 mmol) were stirred in acetonitrile (1.5 mL) for 2 hours. The reaction was then diluted in EtOAc, washed with saturated NaHCO₃(aq), brine, dried (MgSO₄), and concentrated. Purification on silica gel yielded 76 mg (0.12 mmol) of compound 24C. LCMS (M+1)=614.4.

embedded image

The methyl ester 24C (76 mg, 0.12 mmol) dissolved in THF/H₂O (5:1, 2 mL) and stirred overnight with LiOH.H₂O (1.5 eq.). Acidified reaction with 1N HCl (aq) and concentrated. Residue was dissolved in CH₂Cl₂/MeOH (93:7) and eluted through a plug of silica gel to yield 75 mg (0.11 mmol) of Compound No. 144. LCMS (M+1=627.4). ¹H-NMR (500 MHz, Methanol-d₄): 7.84 (d, J=9.1 Hz, 0.5H), 7.71 (s, 1H), 7.60 (d, J=7.2 Hz, 1H), 7.45-7.40 (m, 2H), 5.90-5.83 (m, 1H), 5.23 (d, J=1.4 Hz, 1H), 5.07 (d, J=10.3 Hz, 1H), 4.60 (m, 1H), 4.52-4.49 (m, 1H), 4.27 (m, 1H), 3.90 (m, 1H), 3.59-3.48 (m, 2H), 2.58 (dd, J=8.0, 12.6 Hz, 1H), 2.37-2.32 (m, 1H), 2.21-2.12 (m, 4H), 1.76-1.61 (m, 6H), 1.45-1.42 (m, 1H), 1.32-1.14 (m, 4H), 1.05-0.95 (m, 9H), 0.91 (m, 3H).

embedded image

The carboxylic acid 22D (18.5 mg, 0.029 mmol) stirred with CDI (6.0 mg) in DMF (1.5 mL) at 80° C. for 10 minutes. The reaction was cooled to room temperature and compound 24E in DMF (0.15 mL) with DBU (4 eq.) was added and the reaction was heated in an 80° C. bath for 20 minutes. The reaction was purified directly by reverse phase chromatography to yield 7.6 mg of Compound No. 115. LCMS (M+1=745.2), ¹H-NMR (500 MHz, methanol-d₄): 9.30 (s, 0.5H), 8.02 (m, 0.5H), 7.71 (m, 1H), 7.60 (dt, J=7.2, 1.3 Hz, 1H), 7.46-7.41 (m, 2H), 5.86-5.79 (m, 1H), 5.35-5.28 (m, 1H), 5.12-5.10 (m, 1H), 4.65 (m, 1H), 4.42 (dd, J=6.9, 10.6 Hz, 1H), 4.28 (d, J=11.3 Hz, 1H), 3.95 (d, J=11.4 Hz, 1H), 3.62-3.47 (m, 2H), 2.51-2.47 (m, 1H), 2.35-2.31 (m, 1H), 2.25-2.12 (m, 4H), 1.89 (dd, J=5.4, 8.1 Hz, 1H), 1.80-1.64 (m, 6H), 1.45-1.39 (m, 1H), 1.33-1.15 (m, 3H), 1.04-0.97 (m, 11H), 0.73-0.57 (m, 4H).

Listed below in Table 12 are some physical data of exemplary compounds of Formula I.

LC/MS data were acquired using the following:

Mass spectrometers: PESciex API-150-EX or Waters/Micromass ZQ or Waters/Micromass Quattro II, or Waters/Micromass ZMD; Pumps: Shimadzu LC-8A or Agilent 1100; Autosamplers: Gilson 215 or Gilson 819.

The following methods were used: 3.0 mL/min flow rate, 10-99% CH₃CN (0.035% TFA)/H2O (0.05% TFA) gradient, Phenomenex Luna 5m C18 column (50×4.60 mm); 1.5 mL/min flow rate, 10-90% CH₃CN (0.2% Formic acid)/H2O (0.2% Formic Acid) in 3 minutes, YMC-Pack Pro-C18 column (50×4.6 mm); 1.0 mL/min flow rate, 10-90% CH₃CN (0.2% Formic acid)/H₂O (0.2% Formic Acid) in 5 minutes, YMC-Pro-C18 column (50×2.0 mm); 1.5 mL/min flow rate, 10-90% CH₃CN (0.1% TFA))/H2O (0.1TFA) in 3 minutes, YMC-Pack Pro-C18 column (50×4.60 mm).

TABLE 12

Physical data for exemplary compounds of Formula I.

FIA-
FIA-

Compound
LCMS
LCMS
MS
MS

No.
(M + 1)
RT
(M − 1)
(M + 1)
NMR

1

754.4
756.1

2
700.2
3.99

3
698.8
3.84

4

763
765

5
686.3
2.8

6
760.9
2.4

7
710.2
3.35

8

707.9

9

638
640

10

724
726.1

11

662.4
664.3

12
602
2.94

13
668.2
3.6

14

580.9

15

725.1
726.1

16

691.8

17
791.8
2.29

18
752.4
3.74

19
696.3
3.7

H-NMR (500 MHz, CDCl₃)7.61 (d, J = 1.7 Hz,

H), 7.51 (d, J = 7.7 Hz, H),

7.38 (d, J = 8.2 Hz, H), 7.33 (t, J = 7.8 Hz,

H), 7.26 (s, CDCl3), 7.16 (t, J = 6.5 Hz,

H), 6.91 (d, J = 3.3 Hz, H),

6.55-6.50 (m, H), 5.35 (dd, J = 4.1,

8.5 Hz, H), 4.77-4.74 (m, H), 4.66 (d,

J = 9.4 Hz, H), 4.29 (d, J = 11.1 Hz,

H), 3.71 (d, J = 11.2 Hz, H), 3.47 (d,

1H), 3.29 (d, 1H), 2.78 (td, J = 7.3, 3.7 Hz,

H), 2.63-2.59 (m, H),

2.52-2.49 (m, H), 2.07-2.30 (m, 2H),

1.98-1.92 (m, H), 1.77-1.61 (m, H),

1.47-1.40 (m, H), 1.35-1.25 (m, H), 1.21 (dd, J = 6.6,

12.5 Hz, H), 1.01 (s, 9H),

0.98-0.83 (m, H), 0.61-0.51 (m, H), 0.39 (t,

J = 4.8 Hz, H) ppm

20
672
3.24

21

697.9
699.8

22
713
3.9

CDCl₃; 7.77 (d, 2H), 7.58 (m, 4H),

7.41 (t, 2H), 7.32 (3H)

23
710
4.5

24

630.3
632.2

25

684.6
686.5

26
761.4
3.23

27
672.4
3.7

28
675.3
3.44

29
698.361
3.8

30
767.6
3.15

31
701.3
2.74

32

730.3
732.1

33
685
3.5

34

559

35
706.1
3.66

36

675.5
677.3

37
712
3.19

38
748.1
3.86

39
728
3.82

40

655.3
657.2

41
729
3.4

42
701.3
2.75

43

654.1
656.1

44
658
3

45
720.2
3.29

46
670
3.78

47

734.2
736.1

48
728.2
3.47

49
784.2
3.71

50
696.6
3.76

51

548.1
550

52

704

53
696
2.04

54
634
3.24

55

642.3
644.2

56

600.6
602.4

57

755

58
778.9
2.38

59
730.2
3.58

60
734.4
3.67

61
591.8
2.6

62
690.2
3.64

63
698
3.83

64

595

65
634.4
1.8

66

734.4
736.2

67

720.4
722.2

68
675.2
2.6

69
817.7
3.97

70

712.4
714.3

71
722.1
3.59

72
670
3.53

73
800.7
3.97

74

682
684

75
713.1
2.8

76
643
3.09

77
734.2
3.92

78

79
684.2
3.7

80

553.1
555

81
687
2.1

82

611
612.6

83

772.1
774

84
780.9
4.39

85
650
3.26

86

676.5
678.3

87
712
3.2

88
666
3.31

89
695.2
3.53

90

758
759.9

91
848.7
2.3

92
686
3.05

93
670.4
3.56

94
660
2.69

95

583.9
585.7

96
759.3
4.02

97

670.1
672.1

98
602.6
1.63

99
726.7
2.4

100
704
3.64

101

666.1
668.3

102
710.2
3.6

103
657
3.2

104
690
3.24

105

682.1
684.2

106

690.1
691.9

107
659
3.22

108
700
3.32

109

706.4
708.4

110
754
3.6

111

652.1
654

112

684

113
700.7
2.3

114

712.4
714.3

115
745.2
3.73

116

716.3
718

117

653

118
726.4
3.6

119

691.8

120
811.5
3.95

121
714
3.27

122
711
3.42

123
631
3.09

124
686.2
3.4

125
732.2
3.7

126

680.55
682.4

127
794.7
4.11

128
774
3.28

129
758.2
3.78

130
690.2
3.48

131
720
3.53

132
711.4
3.5

133
728.6
3.8

134
726.2
3.7

135
773.1
3.5

136

666
668.1

137

708.2
710.1

138
843.9
2.2

139
708
3.35

140
672.7
2.13

141
792.7
4.13

142

676.2
677.9

143
700.7
2.32

144
627.4
3.43

145

707.8

146
635
2.47

147

690
691.9

148
813.9
2.15

149

606.8

150

713.5
715.2

151

706
707.8

152
688.4
3.2

153
714.3
3.2

154

700.1
701.9

155
709.6
3.59

156
658
3.35

157
650
3.32

158
714.3
3.72

159

688.3
690.3

160

613.5
615.4

161
769.1
3.5

162
730.2
3.7

163

708

164

630.1
632.1

165
715.2
3.73

166
710
3.46

167
709.4
2.2

168
656.3
3.46

169
702.2
3.71

170
754.2
3.96

171

613.1

172

717.1

173
667
3.17

174

642.3
644.3

175
701.2
2.76

176
817.7
4.1

177

787
789

178

576.1
578.1

179
718.2
3.55

180

770.1
772.1

181
801.7
2.06

182
686.7
2.19

183

714
715.9

184
672.2
2.96

185
724.2
3.6

186
755.3
3.41

187
708
3.53

188
698
3.76

189
722
3.5

190
718.1
3.68

191

710.4
712.4

192

683.3
685.3

193
646
3.44
702
703.7

194

672.1
674

195
700.2
3.59

196

672.1
674

197
722
3.39

198

723.8
725.7

199

766.2
768.2

200

746.2
748.1

201

567.1
568.8

202

638
640

203
730.3
3.7

204
734.2
3.85

205
637
3.35

206
871.9
3.96

207
576
2.87

208

718.2
719.95

209

716.3
718.1

210
675
3.5

211
698.2
3.47

212

692.6
694.5

213
748.2
3.6

214

769.3
770.9

215

733.3
735.3

216
767.1
3.1

217
670
3.6

218
657
3

219
670.2
3.65

220

504.1
506

221
792.9
4.1

222

593
594.9

223
678
3.42

224

707.9
710.1

225

795

226
698
3.89

227
558.5
2.91

228

670

229
649.7
2.55

230
704.3
3.7

231
684
3.73

232
718.1
3.82

233
700.34
3.3

1H-NMR (500 MHz, CDCl₃): 7.61 (t, J = 1.6 Hz,

1H), 7.53 (dd, J = 1.2, 7.6 Hz,

1H), 7.39 (dd, J = 1.7, 6.9 Hz,

1H), 7.34 (t, J = 7.8 Hz, 1H), 7.26 (s,

CDCl3), 7.16 (d, J = 7.3 Hz, 1H),

6.91 (d, J = 3.4 Hz, 1H), 6.62 (d, J = 9.2 Hz,

1H), 5.35 (d, J = 4.1 Hz, 1H),

4.76 (t, J = 7.8 Hz, 1H), 4.64 (d, J = 9.3 Hz,

1H), 4.29 (d, J = 10.8 Hz, 1H),

3.71 (d, J = 11.2 Hz, 1H), 3.29-3.49 (dd, J = 2H),

2.78 (m, 1H), 2.63-2.59 (m,

1H), 2.53-2.51 (m, 1H), 2.37 (d, J = 2.2 Hz,

H), 1.90-1.96 (m, 1H),

1.68-1.60 (m, H), 1.47-1.40 (m, H),

1.21-1.32 (m), 0.99 (s, 9H), 0.95-0.83 (m,

H), 0.60 (dd, J = 3.4, 9.5 Hz, H) ppm

234

722
724

235
626.1
3.34

236

716.1
718.2

237
696.2
2.04

238
784.2
3.77

239
738
3.8

240

668.2
670.2

241
713.4
2.94

242

667.4
669.4

243
605.9
2.77

244

653.2

245
682
3.58

246
664.7
3.39

247
645.3
3.3

248
707.4
3.7

249
665
3.49

250
714.2
3.8

251

613.4
615.2

252
682
3.7

253
672.3
3.72

254
728.4
3.6

255
778.9
2.37

256
668.5
3.6

257

626
628

258

566.3
568.1

259
783
3.24

260
662
3.16

261
821
3.21

262
612
3.4

263

608.9
611

264
692
3.31

265
698.3
3.87

266

704.3
706.2

267

575.6
577.4

268
752
3.87

269
617.6
2.71

270

720

271
704
3.61

272

696
698.1

273
644
3.46

274

714.1
716.2

275
686.7
2.21

276

716.1
717.8

277
728.3
3.75

278
782
3.9

279

670.3
672.2

280

638.2
640

281

684.5
686.4

282
772.2
3.19

283
645
2.02

284
698
3.8

285

695.7
697.6

286
760
3.13

287
708
3.4

288
695
3.46

289
786.2
3.7

290

695.7
697.6

291
787
3.02

292
685.4
3.36

293
711.1
3.7

294
684
3.76

295

654.5
656.5

296
748.7
2.37

297
718
3.48

298
800.6
2.3

299

706.6
708.3

300
714.7
2.36

301

692.1
694.1

302
817.9
3.96

303
651.5
2.9

304
727
2.87

305
726.2
3.52

306

757.5
759.5

307
744
3.57

308
702.5
3.4

309
715.3
3.59

310

683
685

311
767.2
3.4

312

789.1
791

313
640
3.1

314

718
720

315
755.3
3.67

316

813
815

317
747.2
3.35

318

644.1
645.9

319
658
3.57

320

775.3
777.1

321
744.3
3.75

322

666

323

746.3

324
716.9
3.5

325

676.5

326

654.1
655.9

327

686.5
688.4

328
746.7
3.65

329

613.4
615.2

330

678

331

726.5
728.3

332
694.5
1.82

333
707
3.43

334
705.8
3.66

335
720
3.5

336
774.2
3.8

337

680.6
682.6

338
736.2
3.6

339
757
3.24

340

682
684

341
697.1
2.86

342
626
2.3

343

559

344

684.2

345
696
3.98

346
710.2
3.52

347
742.7
2.28

348

531.6
533.3

349
730.4
3.5

350
700.3
3.94

351

634.5
636.3

352

675.6
677.3

353

691.9

354

700.6
702.5

355
762.2
3.91

356
760
3.11

357
695
2.24

358

686.1
687.9

359
732.4
3.12

360
698
3.87

361
698
3.83

362

652
654

363

722
724.1

364
701.9
3.21

365

676.1

366
728.1
3.6

367
636
2.8

368
770.2
3.64

369

721.1
723.2

370
728.3
3.62

371
695
3.7

372
712.3
3.98

373
723.4
2.3

374
688.4
3.2

375

656
658

376
636.3
3.36

377
795.4
3.29

378

761.1
763

379
645
3.16

380
649.5
1.97

381
731.4
3.3

382

758.3
760.1

383
612.1
3.2

384
629.4
2.78

385

504.1
506

386

504.1
506

387
716
3.13

388
656
3.42

389
723.4
2.3

390

696.1
698

391

583
584.8

392
660
3.05

393
696
4.05

394
703.351
3.3

395
730
3.55

396
698.25
3.89

397
650.5
1.73

398

696.1
698

399
742.7
2.16

400

583
584.8

401
698.3
3.9

402
764.1
3.4

403

716.9
719

404
702.5
2.07

405

406

670.1
672.2

407
632
3.35

408
735.7
3.14

409

410
714.5
2.12

411

566.10
568

412
652
3

413

717.9
719.8

414

769.1
771

415

639.5
641.5

416
734.5
3.83

417

653.3

418
686.3
3.1

419
714
3.26

420
704
3.56

421
591.6
1.87

422

693.4
695.4

423

714.2
716.1

424
706
3.31

425

691.8
693.8

426
714.2
3.48

427
666
3.38

428

702.1
704

429
760.9
2.38

430
753.2
3.86

431
691.9
3.3

432
645
3.05

433
835.7
4.16

434
720
3.5

435
744
3.56

436

774
776.2

437
730.2
3.7

438

548.2
549.9

439
603.4
3.34

440

602.6
604.4

441
724.9
2.22

442

712.6
714.5

443
615.6
3.25

444

676.2
678.2

445
742.35
3.2

446
756.2
3.68

447
749.7
1.78

448

608.1
610

449
756
351

450

698.3
700.2

451

630

452
700.3
3.94

453
694.3
3.64

454
761.1
3.3

455

724.4

456

710.5
712.2

457
602.4
3.12

458
803.7
3.97

459
684.2
3.6

460
587.5
3.01

461
735.7
1.8

462

610.1
611.9

463
708.4
3.7

464

706.1
708.2

465

740.4
742.2

466
738.6
3.63

467
696.345
3.7

468
686.2
2.98

469
681.3
3.39

470

610.1
612.05

471
708.2
3.5

472

837
839.1

473

706
708.1

474
710.2
3.2

475
714
3.3

476
734.4
3.67

477
717.37
3.3

478
690.2
3.73

479
690.2
3.66

480

672
673.9

481

718
720

482

698.2
700.1

483
734.6
1.87

484
660
1.44

485
676
3.38

486

803.6
805.4

487
762
3.26

488
794.7
4.07

489
716.5
3.59

490

709.4
711.4

491

754

492
659
3.39
734.4
736.2

493
658.3
3.61

494
744.2
3.71

495
688.2
3.3

496
698.3
3.83

497
694
2.16

498

670.3
672.2

499
726.2
3.65

500
694.3
3.64

501
720.5
3.62

502

724.1
725.9

503
700
3.36

504
692.8
2.13

505
713.8
2.73

506
718
1.87

507
854.7
4.15

508
686.7
2.21

509

724.3

510
756.2
2.95

511

680.5
682.54

512

746.4
748.3

513

726.4
728.2

514
635
3.68

515
688.4
3.2

516
700
2.98

517

744.1
746.1

518
775.2
3.3

519

636

520
660
3.5

521
720.1
3.84

522
670
3.59

523
672
3.1

524

525

735.2
737

526
694
3.64

527

746.1
748.1

528
731.9
3.38

529
732
2.89

530

722

531
650
3.46

532
644
3.39

533

694
696

534
730.5
3.67

535
668.2
3

536

705.8
707.9

537
742.7
2.25

538
731.2
3.7

539

743.2
744.2

540
778.9
4.15

541
700.34
3.2

542
685.34
3.5

543

695.7
697.7

544
746.2
2.3

545

696.1
697.9

546
748.7
2.38

547

726.4
728.25

548

682
684

549

696
697.9

550

653.3
654

551

609.3

552
692.3
3.51

553
712.2
2.6

554
670.5
2.9

555

556

557
725.8
3.4

558
679
3.46

559

702
704

560

696.2
698

561
730.4
3.7

562
716
3.41

563
695
2.46

564

707.9

565
762.2
3.55

566

628
630

567
774.7
3.19

568
712.7
2.3

569

671.9

570

656.1
658.2

571
730.2
3.4

572

639.1
641.2

573

694.1

574
634.5
1.7

575
714.4
3.1

576
680
2.2

577
718
3.51

578

680.5
682.4

579
698
3.72

580
597
2.87

581
720
3.51

582
714.4
3.6

583
693
3.35

584

744

585
762
3.68

586
707
3.2

587
730.5
3.68

588
745.7
4.09

589
735.20
3.70

590
694.30
3.65

591
651.30
3.26

592
651.30
3.24

593
685.20
3.53

594
700.20
3.72

Additional compounds, some of their physical data and method of synthesis are provided in Table 13.

TABLE 13

Cmpd
LC/MASS
FIA
Syn.

No.
PLUS
MS+
Method

595
715.23

5B

596
689.24

5B

597
727.20

6

598
775.10

6

599
685.20

6

600

729.1
6

601
729.10

6

602
687.20

5B

603
673.17

5B

604
703.00

5B

605
716.00

5B

606
742.00

5B

607
676.00

5B

608
702.00

5B

609
676.00

5B

610
690.00

5B

611
716.00

5B

612
650.00

5B

613
676.00

5B

614
724.20

6

615
708.20

6

616

744.1
5B

617

684.1
5B

618
704.70

5B

619
677.60

5B

620

688
5B

621

662
5B

622

702
5B

623

676
5B

624

662
5B

625

636
5B

626

664
5B

627

638
5B

628

704
5B

629

678
5B

630

678
5B

631

652
5B

632
688.20

5B

633
686.10

5B

634
702.20

5B

635
700.10

5B

636
674.10

5B

637
688.20

5B

638
735.10

6

639
723.10

6

640
688.20

5B

641
702.20

5B

642
714.20

5B

643
728.10

6

644
651.21

5B

645
704.00

5B

646
678.00

5B

647
663.70

5B

648
689.70

5B

649
728.70

5B

650

690
5B

651

676
5B

652

690
5B

653

664
5B

654

650
5B

655
732.70

5B

656
718.70

5B

657
730.60

5B

658
700.10

5B

659
701.00

6

660
689.00

6

661

714
5B

662

728
5B

663

688
5B

664

702
5B

665

716
5B

666
673.10

6

667
701.19

5B

668
684.12

5B

669

708.2
6

670
687.70

5B

671
665.20

6

672
742.00

6

673
716.00

6

674
728.00

6

675
742.00

6

676
728.00

6

677
700.40

6

678
700.40

6

679
688.10

5B

680
662.10

5B

681
648.20

5B

682
690.10

5B

683
664.10

5B

684
650.10

5B

685
702.10

5B

686
676.10

5B

687
662.10

5B

688
674.10

5B

689
704.00

6

690
690.00

6

691

692.1
5A

692

706.1
5A

693

692.1
5A

694
690.04; Lot 2: 690.10

5B

695
690.04; Lot 2: 690.10

5B

696
704.62

5B

697
704.04

5B

698
676.06

5B

699
676.07

5B

700
675.00

6

701
663.00

6

702
634.00

5B

703
672.00

5B

704
686.10

5B

705
700.10

5B

706

740.1
5B

707
628.10

5B

708

740
5B

709

754
5B

710

728
5B

711

714
5B

712

682.2
5B

713

744
5B

714
696.10

5B

715

682.2
5B

716

670.1
5B

717
684.60

5B

718
658.80

5B

719
644.60

5B

720
670.60

5B

721
656.60

5B

722
682.60

5B

723
668.70

5B

724
694.70

5B

725
680.70

5B

726
696.70

5B

727
682.70

5B

728
708.70

5B

729
694.80

5B

730
682.70

5B

731
656.60

5B

732
642.70

5B

733
668.70

5B

734
654.70

5B

735
674.80

6

736
702.10

6

737
714.10

6

738
696.00

5B

739
696.00

5B

740

684.6
5A

741

708
5B

742
744.60

5B

743
718.70

5B

744
732.70

5B

745
706.60

5B

746
694.50

5B

747
650.10

5B

748
628.10

5B

749
628.10

5B

750
706.00

5B

751
684.10

5B

752
727.00

5B

753
716.00

5B

754
656.10

5B

755
656.10

5B

756
697.00

5B

757
697.00

5B

758
670.90

5B

759
656.90

5B

760
709.00

5B

761
682.90

5B

762
668.90

5B

763
721.00

5B

764
671.00

5B

765
656.90

5B

766
709.00

5B

767
682.90

5B

768
668.90

5B

769
700.00

5B

770
713.00

6

771
674.00

5B

772
660.10

5B

773
648.10

5B

774
634.10

5B

775
696.10

5B

776
682.10

5B

777
670.10

5B

778
656.10

5B

779
720.00

5B

780
734.00

5B

781

718.1
6

782

692.1
6

783

730.2
6

784

704
6

785
685.80

6

786
724.60

5B

787
710.41

5B

788
712.60

5B

789
698.60

5B

790
736.50

5B

791
750.60

5B

792
724.50

5B

793
764.60

5B

794
700.50

5B

795
714.50

5B

796
700.50

5B

797
714.50

5B

798
694.64

6

799
750.50

5B

800
724.50

5B

801
736.30

5B

802
727.80

6

803
772.00

6

804
693.60

5B

805
693.60

5B

806
707.70

5B

807
707.60

5B

808

698
5B

809

684
5B

810
720.00

5B

811
734.00

5B

812
652.90

6

813
658.00

5B

814
742.00

5B

815
728.00

5B

816
716.00

5B

817
758.60

6

818
758.50

6

819
728.60

6

820
640.80

6

821

722.7
5B

822

653.4
5B

823

684.6
5B

824

710.6
5B

825

698.5
5B

826
742.60

5B

827
716.50

5B

828
742.60

5B

829
728.60

5B

830
714.50

5B

831
680.60

5B

832
680.50

5B

833
738.50

5B

834
764.33

5B

835
684.00

5B

836
716.00

5B

837
704.00

5B

838
639.30

5B

839
670.50

5B

840
696.60

5B

841
684.50

5B

842
642.30

5B

843
628.30

5B

844
728.6

5B

845
728.70

5B

846
702.60

5B

847
714.30

6

848
728.30

6

849
682.30

5B

850
644.30

5B

851
682.30

5B

852
646.30

5B

853
682.30

5B

854
724.30

5B

855
748.00

5B

856
742.00

5B

857
672.00

5B

858
698.00

5B

859

710
5B

896
670.30

5B

897
694.20

5B

898
680.50

5B

899
720.10

5B

900
706.10

5B

901
706.20

5B

902
720.10

5B

903

679.3
11

904
654.20

11

905
704.50

5B

906
718.60

5B

907
718.60

5B

908
704.60

5B

909
656.20

5B

910
642.20

5B

911
748.00

5B

912
762.00

5B

913
736.00

5B

914
761.00

5B

915
672.20

5B

916
658.50

5B

917
694.50

5B

918
694.50

5B

919

710.2
5B

920

710.3
5B

921

684.2
5B

922

698.2
5B

923

696.2
5B

924

670.3
5B

925

696.2
5B

926

684.2
5B

927

684.2
5B

928

684.2
5B

929

658.4
5B

930

672.2
5B

931

670.4
5B

932

670.2
5B

933

644.3
5B

934

658.5
5B

935

605.3
11

936

667.3
11

937
734.00

5B

938
722.00

5B

939

708
5B

940

734
5B

941

720
5B

942

732
5B

943

744
5B

944

720
5B

945

746
5B

946

732
5B

947

696
5B

948
744.60

5B

949
730.60

5B

950
708.20

5B

951
700.60

5B

952
696.20

5B

953
696.20

5B

954
694.10

5B

955
738.67

5B

Example
FIA MASS
FIA MASS
LC MASS
LC MASS
Syn.

No.
MINUS
PLUS
MINUS
PLUS
Method

957

686.41
688.06
5B

958
663.50
665.10

665.10
5B

959

706.60
5B

960

720.60
5B

961

704.00
5B

962

691.70
693.30
11

963

645.30
11

964

679.20
11

965

665.20
11

966

690.00
5B

967

709.60
5B

968

734.10
5B

969

708.10
5B

970

734.00
5B

971

706.00

5B

972

730.00

5B

973

718.00

5B

974

670.00

5B

975

694.00

5B

976

682.00

5B

977

672.00
5B

978

658.00
5B

979
770.70
772.10

5B

980
741.70
743.20

5B

981

743.10

5B

982
741.60
743.20

5B

983
706.60
708.20

5B

984
746.50
748.20

5B

985
756.60
758.20

5B

986
727.60
729.10

5B

987
727.50
729.20

5B

988
727.60
729.10

5B

989
692.50
694.20

5B

990
732.60
734.30

5B

991
744.50
746.10

5B

992
680.60
682.30

5B

993
720.60
722.30

5B

994

759.80;
760.20
5B

759.60

995

734.20
5B

996

760.20
5B

997

734.20
5B

998

746.20
5B

999

744.50
746.10
5B

1000
689.50
691.10

691.10
5B

1001
703.60
705.10

705.10
5B

1002
716.40
718.30

718.30
5B

1003
718.60
720.00

720.00
5B

1004
718.60
720.00

720.00
5B

1005

772.10
5B

1006

744.80
746.00
5B

1007
710.70
712.30

5B

1008
724.60
726.20

5B

1009
724.70
726.20

5B

1010
696.50
698.20

5B

1011
710.60
712.20

5B

1012
710.60
712.20

5B

1013
698.70
700.20

5B

1014

876.30

5B

1015
679.70
681.20

11

1016
713.50
715.10

11

1017
693.60
695.10

11

1018

743.60
745.70
5B

1019

676.60
678.00
11

1020
627.70
629.20

11

1021

629.20
11

1022
702.60
704.20

11

1023
626.60
628.30

11

1024

712.30
5B

1025

686.20
5B

1026

698.40
5B

1027

712.20
5B

1028

686.30
5B

1029

698.30
5B

1030

712.20
5B

1031

686.20
5B

1032

698.30
5B

1033

740.00
5B

1034

714.30
5B

1035

726.30
5B

1036

702.00
5B

1037

670.00
5B

1038

698.00
5B

1039

710.00
5B

1040

684.20
5B

1041
696.70
698.20

5B

1042
696.60
698.20

5B

1043
684.70
686.20

5B

1044
698.70
700.20

5B

V. Assays for Detecting and Measuring Inhibition Properties of Compounds
A. HCV Enzyme Assays

1. Construction and Expression of the HCV NS3 Serine Protease Domain

A DNA fragment encoding residues Ala¹-Ser¹⁸¹of the HCV NS3 protease (GenBank CAB46913) was obtained by PCR from the HCV Con1 replicon plasmid, I₃₇₇neo/NS3-3′/wt (re-named as pBR322-HCV-Neo in this study) [V. Lohmann et al., Science, 285, pp. 110-113 (1999)] and inserted into pBEV11 (S. Chamber, et al., personal communication) for expression of the HCV proteins with a C-terminal hexa-histidine tag in E. coli. All constructs were confirmed by sequencing.

The expression constructs for the HCV NS3 serine protease domain was transformed into BL21/DE3 pLysS E. coli cells (Stratagene). Freshly transformed cells were grown at 37° C. in a BHI medium (Difco Laboratories) pplemented with 100 μg/mL carbenicillin and 35 μg/mL chloramphenicol to an optical density of 0.75 at 600 nm. Induction with 1 mM IPTG was performed for four hours at 24° C. The cell paste was harvested by centrifugation and flash frozen at −80° C. prior to protein purification. All purification steps were performed at 4° C. Next, 100 g of cell paste was lysed in 1.5 L of buffer A (50 mM HEPES (pH 8.0), 300 mM NaCl, 0.1% n-octyl-β-D-glucopyranoside, 5 mM β-mercaptoethanol, 10% (v/v) glycerol) and stirred for 30 minutes. The lysate was homogenized using a Microfluidizer (Microfluidics, Newton, Mass.), followed by ultra-centrifugation at 54,000×g for 45 minutes. Imidazole was added to the supernatant to a final concentration of 5 mM along with 2 mL of Ni-NTA resin pre-equilibrated with buffer A containing 5 mM imidazole. The mixture was rocked for three hours and washed with 20 column volumes of buffer A plus 5 mM imidazole. The HCV NS3 protein was eluted in buffer A containing 300 mM imidazole. The eluate was concentrated and loaded onto a Hi-Load 16/60 Superdex 200 column, pre-equilibrated with buffer A. The appropriate fractions of the purified HCV protein were pooled and stored at −80° C.

2. HCV NS3 Protease Domain Peptide Cleavage Assay

This assay is a modification of that described by Landro, et al. (Landro J A, Raybuck S A, Luong Y C, O'Malley E T, Harbeson S L, Morgenstern K A, Rao G and Livingston D L. Biochemistry 1997, 36, 9340-9348), and uses a peptide substrate (NS5AB), based on the NS5A/NS5B cleavage site for genotype 1a HCV. The substrate stock solution (25 mM) was prepared in DMSO containing 0.2 M DTT and stored at −20° C. A synthetic peptide cofactor (KK4A) was used as a substitute for the central core region of NS4A. Peptide sequences are shown in the table below. The reaction was performed in a 96-well microtiter plate format using 25 ηM to 50 ηM HCV NS3 protease domain in buffer containing 50 mM HEPES pH 7.8, 100 mM NaCl, 20% glycerol, 5 mM DTT and 25 μM KK4A. The final DMSO concentration was no greater than 2% v/v. Reactions were quenched by addition of trifluoroacetic acid (TFA) to yield a final concentration of 2.5%.

Peptide Sequences Used with

HCV NS3 Protease Domain

Peptide
Sequence

NS5AB
NH₂-EDVV-(alpha)Abu-CSMSY-COOH

[SEQ ID NO: 2]

KK4A
NH₂-KKGSVVIVGRIVLSGK-COOH

[SEQ ID NO: 3]

The SMSY product was separated from substrate and KK4A using a microbore separation method. The instrument used was a Agilent 1100 with a G1322A degasser, either a G1312A binary pump or a G1311A quaternary pump, a G1313A autosampler, a G1316A column thermostated chamber and a G1315A diode array detector. The column was a Phenomenex Jupiter, 5 μm C18, 300 Å, 150×2 mm, P/O 00F-4053-B0, with a flow-rate of 0.2 mL/min. The column thermostat was at 40° C. Mobile phases were HPLC grade H₂O/0.1% TFA (solvent A) and HPLC grade CH₃CN/0.1% TFA (solvent B). The SMSY product peak was quantitated using the data collected at 210 ηM.

3. Construction and Expression of NS3•4A Protease

Using standard recombinant DNA techniques, a cDNA fragment encoding the sequence for NS3 and NS4A, residues Ala₁₀₂₇to Cys₁₇₁₁from the HCV sub-type strain 1a, containing an N-terminal hexa-histidine sequence, was cloned into the baculoviral transfer vector pVL1392 (Webb N R and Summers M D (1990) Expression of proteins using recombinant baculoviruses, Techniques 2:173-188). Recombinant baculovirus containing NS3•4A was produced by co-transfection of pVL1392-His-NS3•4A with linearized Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA into Spodoptera frugoperda (SD) insect cells. The transfected insect cells containing recombinant baculovirus clones were subsequently isolated by plaque purification. High-titer clonal baculovirus was routinely used to infect SD insect cells for protein production. In production, Sf9 cells were grown at 27° C. until they reached a density of 2.0×10⁶cells/ml. At this point, the insect cells were infected with virus. After 72 hours or when the cell viability was between 70-80% the culture was harvested and the cells were ready for purification.

4. Purification of NS3•4A Protein

The NS3•4A protein (SEQ ID NO:1) was purified as follows. Cell paste was thawed in at least five volumes of Lysis Buffer (50 mM Na₂HPO₄pH 8.0, 10% Glycerol, 300 mM NaCl, 5 mM β-mercaptoethanol, 0.2 mM PMSF, 2.5 μg/ml Leupeptin, 1.0 μg/ml E64, 2.0 μg/ml Pepstatin) per gram of cell paste. The cell paste was then homogenized on ice using a Dounce homogenizer. The cells were next mechanically disrupted by passing once through a microfluidizer (Microfluidics Corporation, Newton, Mass.), and the cell lysate was collected on ice. The cell lysates was centrifuged at 100,000×g for 30 minutes at 4° C. and the supernatants were decanted. Optionally, the pellets were resuspended in wash buffer (Lysis Buffer+0.1% β-octyl glucopyranoside), homogenized using a Dounce homogenizer and centrifuged at 100,000×g for 30 minutes at 4° C. Insoluble NS3° 4A was extracted from the pellets by resuspending in Extraction Buffer (Lysis Buffer+0.5% lauryl maltoside) using 2.5 ml/g cell paste. The mixture was homogenized using a Dounce homogenizer and mixed at 4° C. for three hours or more. The mixture was centrifuged at 100,000×g for 30 minutes at 4° C. The supernatants were decanted and pooled.

The NS3•4A protein was further purified using Nickel-NTA metal affinity chromatography. Imidazole from a 2 M stock, pH 8.0, solution was added to the pooled supernatants so that the final concentration of imidazole was 10 mM. The supernatants were incubated batchwise overnight at 4° C. with Nickel-NTA affinity resin that had been pre-equilibrated with Lysis Buffer+10 mM imidazole. 1 ml of resin per 5 μg of expected NS3-4A was used. The resin was next settled by gravity or by centrifugation at 500×g for five minutes. The resin was next poured into a gravity flow column and washed with 10 or more column volumes of Nickel Wash Buffer (Lysis Buffer+0.1% lauryl maltoside+10 mM imidazole). The column was next eluted with three to four column volumes of Nickel Elution Buffer (Nickel Wash Buffer+300 mM imidazole). The elution fractions were collected on ice and evaluated using SDS-PAGE. To prevent NS3-4A proteolysis, 100 μM DFP protease inhibitor was added to gel samples before adding SDS sample buffer and boiling. The peak fractions were pooled and protein concentration was determined by measuring absorbance at 280 ηm and by dividing by the extinction coefficient (e), which for NS3•4A is 1.01.

The NS3•4A was purified further using gel filtration chromatography. A Superdex 200 26/60 column was equilibrated with Superdex Buffer (20 mM HEPES pH 8.0, 10% glycerol, 300 mM NaCl, 10 mM β-mercaptoethanol, 0.05% lauryl maltoside) at a rate of 3 ml/min. The nickel purified NS3•4A was concentrated in a Centriprep 30 to greater than 2 mg/ml, if necessary, and was filtered through a 0.2 μm syringe filter and up to 10 ml was loaded onto the Superdex 200 column. After 0.3 column volumes passed through, 4-5 ml fractions were collected. Fractions were evaluated by SDS-PAGE. NS3•4A protein elutes in two peaks. Peak 1 contains aggregated NS3•4A and peak 2 contains active protein. The fractions of peak 2 were pooled, aliquoted and frozen at −70° C.

Analysis of NS3·4A protein.

ANALYSIS
ENTIRE PROTEIN

Length
695 amino acids

Molecular Weight
74,347.78

1 microgram
13.450 picot moles

Molar Extinction Coefficient
73430

1 A₂₈₀corresponds to
1.01 mg/ml

Isoelectric Point
6.50

Charge at pH 7
−3.58

5. HCV NS3 Peptide Cleavage Assay

This assay follows the cleavage of a peptide substrate by full-length hepatitis C viral protein NS3•4A. One of three peptide substrates based on the NS5A/NS5B cleavage site for genotype 1a HCV is used to measure enzyme activity. All substrate stock solutions (25 mM) were prepared in DMSO containing 0.2M DTT and stored at −20° C. A synthetic peptide cofactor (NS4A Peptide) was used to supplement NS4A. Peptide sequences are shown below. The hydrolysis reaction was performed in a 96-well microtiter plate format using 100 ηM to 125 ηM HCV NS3•4A in buffer containing 50 mM HEPES pH 7.8, 100 mM NaCl, 20% glycerol, 5 mM DTT and 25 μM NS4A Peptide. The final DMSO concentration was no greater than 2% v/v. Reactions using NS5AB or NS5AB-EDANS as substrate were quenched by the addition of 10% trifluoroacetic acid (TFA) to yield a final TFA concentration of 2.5%. Reactions using FITC-NS5AB-1 as substrate were quenched by the addition of 0.4M formic acid to yield a final concentration of 0.08M acid.

Enzymatic activity was assessed by separation of substrate and products by reverse phase HPLC. The instrument used was a Agilent 1100 with a G1322A degasser, either a G1312A binary pump or a G1311A quaternary pump, a G1313A autosampler, a G1316A column thermostated chamber, a G1321A fluorescence detector and a G1315A diode array detector. The column thermostat was at 40° C. For substrate NS5AB the column was a Phenomenex Jupiter, 5 μm C18, 300 Å, 150×2 mm, P/O 00F-4053-B0, with a flow-rate of 0.2 mL/min using HPLC grade H₂O/0.1% TFA (solvent A) and HPLC grade CH₃CN/0.1% TFA (solvent B) as mobile phases. The C-terminal product peak (NH₂-SMSY-COOH) was quantitated using the absorbance data collected at 210 ηm. For substrate NS5AB-EDANS the column was a Phenomenex Aqua, 5 μm C18, 125 Å, 50×4.6 mm, P/O 00B-4299-E0, with a flow-rate of 1.0 mL/min using HPLC grade H₂O/0.1% TFA (solvent A) and HPLC grade CH₃CN/0.1% TFA (solvent B) as mobile phases. The C-terminal product peak (NH₂-SMSYT-Asp(EDANS)-KKK-COOH) was quantitated using the fluorescence data collected at 350 ηm excitation/490 ηm emission. For substrate FITC-NS5AB-1 the column was a Phenomenex Prodigy, 5 μm ODS(2), 125 Å, 50×4.6 mm, P/O 00B-3300-E0, with a flow-rate of 1.0 mL/min using 10 mM sodium phosphate pH 7.0 in HPLC grade H₂O (solvent A) and 65% HPLC Grade CH₃CN/35% 10 mM sodium phosphate pH 7.0 in HPLC grade H2O (solvent B) as mobile phases. The N-terminal product peak (FITC-Ahx-EDVV-(alpha)Abu-C—COOH) was quantitated using the fluorescence data collected at 440 nm excitation/520 nm emission. Alternatively, the ratio of N-terminal product to unreacted FITC-NS5AB-1 substrate was determined using a Caliper LabChip 3000 with detection at 488 nm excitation/530 nm emission, using a chip buffer of 100 mM Tris pH 7.0, 10 mM EDTA, 0.01% (v/v) Brij-35, and 0.1% (v/v) CR-3.

Peptide sequences used with HCV NS3.

Peptide
Sequence

NS4A
NH₂-KKGSVVIVGRIVLSGKPAIIPKK-COOH

Peptide
[SEQ ID NO: 4]

NS5AB
NH₂-EDVV-(alpha)Abu-CSMSY-COOH

[SEQ ID NO: 2]

NS5AB-
NH₂-EDVV-(alpha)Abu-CSMSYT-

EDANS
Asp(EDANS)-KKK-COOH

[SEQ ID NO: 5]

FITC-
FITC-Ahx-EDVV-(alpha)Abu-CSMSYTKK-NH₂

NS5AB-1
[SEQ ID NO: 6]

6. Determination of Km and Vmax

To determine the kinetic parameters Km and Vmax, the HCV NS3 protease domain or HCV NS3•4A was reacted with peptide substrate under the assay conditions described above. Peptide substrate concentration was varied between 3 μM and 200 μM, with less than 20 percent conversion at all substrate concentrations. The ratio of the product peak area (as determined by reverse phase HPLC) to the reaction time yielded a rate of enzyme catalyzed hydrolysis. These rate vs. substrate concentration data points were fit to the Michaelis-Menten equation using non-linear regression. The value of k_catwas determined from Vmax using the nominal protease concentration and a fully cleaved substrate peptide as an instrument calibration standard.

Kinetic parameters for peptide substrates with HCV NS3

or NS3 protease domain.

Enzyme
Substrate
Km (μM)
k_cat/Km (M⁻¹sec⁻¹)

NS3 Protease
NS5AB
25
3.0 × 10⁴

Domain

NS3·4A
NS5AB
30
7.9 × 10³

NS3·4A
NS5AB-EDANS
56
1.4 × 10³

NS3·4A
FITC-NS5AB-1
15
1.2 × 10³

7. Determination of Compound Potency

To evaluate apparent Ki values, all components except the test compound and substrate were pre-incubated for 5-10 minutes at room temperature. Then, test compound, dissolved in DMSO, was added to the mixture and incubated for either 15 minutes or 60 minutes at 30° C. Neat DMSO was included as a no inhibitor control. The cleavage reaction was initiated by the addition of peptide substrate at a concentration either equal to Km or equal to one-half times Km, and allowed to proceed at 30° C. for 20 minutes. At the end of the reaction the mixture was quenched, and the extent of reaction was determined as described above. Eleven concentrations of compound were used to titrate enzyme activity for inhibition. Activity vs. inhibitor concentration data points were fit to the Morrison equation describing competitive tight-binding enzyme inhibition using non-linear regression (Sculley M J and Morrison J F. Biochim. Biophys. Acta, 1986, 874, 44-53).

The tested compounds of formula I generally exhibited Ki values from about 0.008 to about 20 μM. In some embodiments, the compounds of formula I exhibited Ki values from about 0.008 to about 0.100 μM. In some other embodiments, the compounds of formula I exhibited Ki values from about 0.100 to about 0.500 μM. In still some other embodiments, the compounds of formula I exhibited Ki values from 0.500 to about 5.000 μM.

Examples of activities of the compounds of formulae (I, Ia, and Ib) on inhibiting serine protease receptors are shown below in Table 13. For compound activities for serine protease measured using the HCV Enzyme Assays, serine protease activity is illustrated with “+++” if activity was measured to be less than 0.1 μM, “++” if activity was measured to be from 0.1 μM to 0.5 μM, “+” if activity was measured to be greater than 0.5 μM, and “−” if no data was available. It should be noted that 0% efficacy is the minimum response obtained with the DMSO only control. The Enzyme Assay 1 refers to the HCV NS3 Protease Domain Peptide Cleavage Assay and Enzyme Assay 2 refers to the HCV NS3 Peptide Cleavage Assay.

TABLE 13

HCV Enzymatic Assay Activities and efficacies of exemplary compounds

in accordance to Formulae I.

Compound
Enzyme
Enzyme

No.
Assay 1
Assay 2

1
−
−

2
−
−

3
++
+++

4
++

5
+++
+++

6
+
+

7
+
−

8
+
−

9
+
−

10
+
+

11
+
−

12
++
+++

13
+++
+++

14
+
−

15
++
−

16
+
−

17
+
++

18
+++
+++

19
++
+++

20
+
−

21
+
+

22
−
−

23
+++
++

24
++
−

25
+
−

26
++
+++

27
++
−

28
+
++

29
++
++

30
++
+++

31
−
++

32
++
−

33
+++
−

34
+
−

35
−
−

36
++
−

37
+
−

38
−
++

39
++
−

40
+
−

41
+++
−

42
++
+++

43
++
+++

44
++
−

45
+
++

46
+
+

47
+
−

48
−
−

49
−
+++

50
−
+++

51
+
−

52
++
−

53
+
−

54
+
++

55
++
−

56
++
−

57
−
−

58
+
+

59
−
+++

60
++
++

61
+
−

62
++
++

63
+
+

64
+
−

65
+
+

66
++
−

67
+++
+++

68
−
+++

69
+
++

70
+
−

71
+
−

72
++
−

73
+
++

74
++
+++

75
++
−

76
++
−

77
++
+++

78
++
−

79
++
+++

80
+
−

81
+
−

82
++
−

83
++
−

84
+
+

85
++
−

86
++
−

87
++
++

88
+
++

89
−
++

90
−
+++

91
+
+

92
++
−

93
++
+++

94
+++
−

95
++
−

96
+
++

97
−
+++

98
+
++

99
+
+

100
+
+

101
+
−

102
++
−

103
++
−

104
−
+++

105
−
+++

106
+
−

107
++
−

108
++
−

109
+++
−

110
++
−

111
++
++

112
++
+++

113
+
+

114
+++
+++

115
+
++

116
+
−

117
−
++

118
+
−

119
++
++

120
+
+

121
++
+++

122
++
−

123
+++
−

124
+
++

125
++
++

126
++
++

127
++
++

128
++
−

129
−
+++

130
++
+++

131
−
+++

132
++
++

133
++
++

134
++
+++

135
+
−

136
++
++

137
−
−

138
+
+

139
+
−

140
++
+++

141
+
++

142
+
−

143
++
++

144
+
+

145
++
−

146
+
−

147
+++
−

148
+
+

149
+
−

150
++
−

151
++
−

152
++
−

153
++
++

154
++
−

155
+
−

156
+++
−

157
+
+

158
+++
+++

159
++
−

160
+
−

161
+
−

162
++
+++

163
+++
+++

164
+
−

165
++
+++

166
++
−

167
++
−

168
++
++

169
−
++

170
−
+++

171
+
+

172
+
++

173
+++
−

174
++
−

175
−
−

176
++
++

177
+
++

178
++
++

179
++
−

180
+
−

181
+
+

182
+
+

183
++
−

184
−
+++

185
+
++

186
+
+

187
+
−

188
+
+

189
+
−

190
++
+++

191
+
−

192
−
−

193
++
−

194
+
−

195
−
+++

196
++
−

197
++
−

198
+
−

199
+
−

200
+
−

201
++
−

202
+
−

203
++
−

204
++
++

205
+
−

206
++
++

207
+
−

208
++
−

209
++
−

210
+
−

211
−
+++

212
++
+++

213
+
+

214
+
−

215
++
−

216
+
−

217
+++
−

218
+
+

219
−
+++

220
+
−

221
++
++

222
+
−

223
++
−

224
++
−

225
−
+++

226
++
−

227
+
+

228
+
−

229
+
−

230
++
−

231
++
−

232
−
−

233
++
+++

234
++
−

235
+
−

236
+
−

237
++
−

238
−
+

239
++
−

240
+
−

241
+
++

242
+++
−

243
+
−

244
++
−

245
++
−

246
+
+

247
+++
−

248
+
+

249
++
−

250
−
+++

251
+
−

252
++
+++

253
+
+

254
++
−

255
++
++

256
++
−

257
++
−

258
+
−

259
++
−

260
++
−

261
++
−

262
+
−

263
++
+++

264
−
+++

265
+
++

266
++
++

267
−
−

268
++
+

269
+
+

270
++
−

271
++
+++

272
+
+

273
++
−

274
+
−

275
++
++

276
+
−

277
+
++

278
+
−

279
++
−

280
+
+

281
+++
−

282
+
++

283
+
−

284
++
++

285
++
−

286
−
+++

287
++
−

288
++
−

289
+
++

290
++
−

291
+
−

292
++
+++

293
+
+

294
++
−

295
++
−

296
+
+

297
−
+++

298
++
++

299
+
−

300
++
++

301
++
−

302
++
++

303
+
−

304
++
+++

305
−
++

306
+
−

307
++
+++

308
++
−

309
+
+

310
+
+

311
+
−

312
+++
+++

313
+
−

314
++
−

315
+++
+++

316
++
−

317
+
−

318
++
−

319
++
−

320
+
+

321
++
+++

322
+
−

323
++
−

324
+
−

325
+
−

326
++
++

327
+
−

328
+
+

329
+
+

330
++
−

331
+
−

332
+
++

333
++
−

334
+
−

335
+
−

336
+
+

337
−
+++

338
+
++

339
++
+++

340
++
−

341
+
−

342
+
−

343
+
−

344
−
−

345
+
−

346
−
+++

347
+
+

348
+
−

349
++
−

350
+
+

351
+
+

352
+
−

353
+
−

354
+
−

355
+
+

356
−
+++

357
++
−

358
++
−

359
++
+++

360
+
−

361
++
+++

362
++
+++

363
+
−

364
+
−

365
++
−

366
++
+++

367
+
−

368
−
++

369
−
+++

370
++
+++

371
++
−

372
++
++

373
++
−

374
++
−

375
+
−

376
+
+

377
++
+++

378
++
−

379
+++
−

380
−
+

381
++
−

382
+++
−

383
+
−

384
+
+

385
+
−

386
−
−

387
++
−

388
+++
+++

389
+
−

390
++
+++

391
++
−

392
++
−

393
++
−

394
+++
−

395
+
+

396
−
+++

397
+
+

398
+
−

399
+
+

400
+++
−

401
++
++

402
++
−

403
+
−

404
+
++

405
+
−

406
−
+++

407
++
−

408
+
++

409
−
−

410
+
+

411
++
+++

412
++
++

413
+
−

414
+
−

415
++
−

416
+
++

417
+++
−

418
++
+++

419
++
++

420
+
+

421
−
+

422
++
−

423
+
−

424
++
−

425
+
−

426
+
++

427
+
−

428
++
−

429
+
+

430
++
+++

431
++
−

432
+
−

433
+
+

434
++
−

435
++
−

436
++
−

437
+
+

438
+
++

439
+
+

440
−
−

441
+
++

442
++
+++

443
+
+

444
++
−

445
++
+++

446
−
+++

447
+
++

448
++
−

449
++
−

450
+++
−

451
+
−

452
+
++

453
++
−

454
++
−

455
+
−

456
++
−

457
+
+

458
+
+

459
+
++

460
+
+

461
+
++

462
++
−

463
+
−

464
+
−

465
++
−

466
+
+

467
++
+++

468
−
+++

469
+
++

470
−
−

471
++
−

472
+
−

473
−
−

474
+
−

475
++
−

476
++
++

477
+++
−

478
++
−

479
−
−

480
+
−

481
+
−

482
+
−

483
+
+

484
++
−

485
−
+++

486
++
−

487
++
−

488
+
++

489
+
++

490
++
−

491
+
−

492
+++
−

493
++
+++

494
+++
−

495
+
−

496
+
+

497
++
−

498
++
−

499
+++
+++

500
++
+++

501
+
+

502
−
−

503
++
−

504
++
−

505
++
−

506
+
−

507
+
+

508
++
++

509
+
−

510
++
+++

511
++
−

512
++
−

513
−
+++

514
+
−

515
++
−

516
+
−

517
+
−

518
+++
−

519
++
−

520
+
−

521
++
+++

522
−
+++

523
++
−

524
+
−

525
+
−

526
++
−

527
++
−

528
++
−

529
−
+++

530
+
−

531
++
++

532
+
−

533
+
−

534
+
+

535
++
−

536
+
−

537
+
+

538
+
−

539
++
−

540
++
++

541
++
+++

542
+++
−

543
+
−

544
++
−

545
++
−

546
++
++

547
+++
−

548
++
−

549
+++
−

550
++
−

551
++
−

552
++
−

553
++
−

554
+
−

555
++
−

556
−
+++

557
++
−

558
++
−

559
+
+

560
++
−

561
++
−

562
−
+++

563
++
−

564
+
−

565
+
+

566
+
+

567
+
++

568
+
+

569
+++
−

570
++
−

571
++
−

572
++
−

573
+
+

574
+
+

575
+++
+++

576
++
−

577
−
+++

578
+
−

579
++
−

580
++
−

581
−
+++

582
++
−

583
++
−

584
+
−

585
++
−

586
+
+

587
+
++

588
+
+

589
−
−

590
−
−

591
−
−

592
−
−

593
−
−

594
−
−

595

+++

596

++

597

++

598

++

599

++

600

++

601

+++

602

+++

603

+++

604

++

605

++

606

++

607

++

608

++

609

+++

610

+++

611

+++

612

+++

613

+++

614

+++

615

++

616

+++

617

++

618

+++

619

+++

620

+++

621

+++

622

++

623

+++

624

++

625

+++

626

+++

627

+++

628

++

629

+++

630

++

631

+++

632

+++

633

+++

634

+++

635

+++

636

+++

637

+++

638

+++

639

+++

640

+++

641

+++

642

+++

643

+++

644

++

645

+++

646

+++

647

++

648

++

649

++

650

++

651

++

652

+++

653

++

654

+++

655

++

656

++

657

+++

658

++

659

−

660

+++

661

+++

662

++

663

++

664

+++

665

+++

666

+++

667

+++

668

+++

669

+++

670

+++

671

++

672

++

673

+++

674

+++

675

+++

676

+++

677

+++

678

++

679

+

680

+++

681

++

682

+++

683

+++

684

+++

685

+++

686

+++

687

+++

688

+++

689

+++

690

++

691

++

692

++

693

++

694

++

695

+++

696

+++

697

+++

698

+++

699

+++

700

+++

701

++

702

++

703

+++

704

+++

705

+++

706

+++

707

++

708

+++

709

++

710

++

711

+++

712

+++

713

+++

714

−

715

+++

716

+++

717

+

718

+++

719

+++

720

+++

721

+++

722

+++

723

+++

724

+++

725

+++

726

+++

727

+++

728

+++

729

+++

730

+++

731

+++

732

++

733

+++

734

+++

735

+++

736

++

737

++

738

++

739

+++

740

+++

741

+++

742

++

743

+++

744

+++

745

+++

746

+++

747

+++

748

+++

749

+++

750

+++

751

++

752

+++

753

+++

754

+++

755

+++

756

+++

757

+++

758

+++

759

+++

760

+++

761

+++

762

+++

763

+++

764

++

765

+

766

++

767

++

768

++

769

++

770

+++

771

+++

772

+++

773

++

774

++

775

+++

776

+++

777

+++

778

+++

779

+++

780

+++

781

+++

782

+++

783

+++

784

++

785

+++

786

++

787

+++

788

+++

789

+++

790

+++

791

+++

792

+++

793

+++

794

+++

795

+++

796

+++

797

+++

798

+++

799

+++

800

+++

801

+++

802

+++

803

++

804

++

805

+++

806

+++

807

++

808

+++

809

+++

810

+++

811

+++

812

+++

813

+

814

+++

815

+++

816

+++

817

+++

818

+++

819

+++

820

+++

821

+

822

++

823

+++

824

+++

825

++

826

+++

827

+++

828

+++

829

+++

830

+++

831

+++

832

++

833

+++

834

+++

835

+++

836

+++

837

++

838

+++

839

++

840

++

841

++

842

+++

843

++

844

++

845

+++

846

+++

847

+++

848

+++

849

+++

850

+++

851

+++

852

+++

853

+++

854

+++

855

+++

856

+++

857

+++

858

+++

859

+++

860

+++

861

+++

862

++

863

+++

864

++

865

+++

866

++

867

+++

868

+++

869

+++

870

++

871

+++

872

+++

873

+++

874

+++

875

+++

876

+++

877

+++

878

+++

879

+++

880

+++

881

+++

882

+++

883

+++

884

+++

885

++

886

+++

887

++

888

+++

889

+++

890

+++

891

+++

892

+++

893

+++

894

+++

895

+++

896

+++

897

+++

898

+++

899

+++

900

+++

901

+++

902

+++

903

+++

904

+++

905

++

906

++

907

++

908

+++

909

+++

910

+++

911

+++

912

+++

913

+++

914

+++

915

+++

916

++

917

++

918

++

919

+++

920

++

921

+++

922

+++

923

+++

924

++

925

+++

926

+++

927

+++

928

++

929

+++

930

+++

931

++

932

+++

933

+++

934

+++

935

+++

936

+

937

+++

938

+++

939

+++

940

++

941

+++

942

+++

943

+++

944

+++

945

+++

946

+++

947

+++

948

+++

949

+++

950

+++

951

+++

952

+++

953

+++

954

+++

955

+++

956

+++

957

++

958

+++

959

+++

960

+++

961

+

962

++

963

++

964

++

965

++

966

+

967

+++

968

+++

969

+++

970

+++

971

+++

972

+++

973

+++

974

+++

975

+++

976

+++

977

+++

978

+++

979

−

980

++

981

+++

982

++

983

+++

984

+++

985

+++

986

++

987

−

988

−

989

−

990

−

991

+++

992

+++

993

+++

994

+++

995

+++

996

+++

997

+++

998

−

999

−

1000

−

1001

−

1002

−

1003

−

1004

−

1005

−

1006

−

1007

−

1008

−

1009

−

1010

−

1011

−

1012

−

1013

−

1014

−

1015

−

1016

−

1017

−

1018

−

1019

−

1020

−

1021

−

1022

−

1023

−

1024

−

1025

−

1026

−

1027

−

1028

−

1029

−

1030

−

1031

−

1032

−

1033

−

1034

−

1035

−

1036

−

1037

−

1038

−

1039

−

1040

−

1041

−

1042

−

1043

−

1044

−

B. HCV Cell Assays

uh-7 cells were propagated in Dulbecco's modified Eagle's medium (DMEM, JRH Biosciences, Lenexa, Kans.) supplemented with 10% heat-inactivated FBS (fetal bovine serum), 2 mM L-glutamine, and nonessential amino acids (JRH). The cells were transfected with an in vitro transcribed HCV replicon RNA identical to replicon I377neo/NS3-37 wt as described by Lohmann et al. (1999). Stable cell clones were selected and maintained in the presence of 250 μg/mL G418 (Invitrogen, Carlsbad, Calif.). One of the clones, 24-2, was used in the subsequent HCV replicon assays. The replicon cells were propagated in DMEM supplemented with 10% FBS, 2 mM L-glutamine, nonessential amino acids, and 250 μg/mL G418. The cells were split twice per week in fresh media upon reaching confluence. There are approximately 200-300 copies of HCV RNA per replicon cell.

HCV replicon RNA from cells was measured using the Quantigene Discover XL kit (Panomics Inc., Fremont Calif.) as per the manufacturer's instructions. Briefly, compound-treated replicon cells were lysed and immobilized on to capture plates using HCV specific oligonucleotides over night and the relative amounts of captured RNA was measured using oligonucleotide probe sets as per the manufacturer's instructions.

1. 2-Day HCV Replicon IC₅₀Assay

On the day prior to the assay, 104 replicon cells were plated per well of a 96-well plate and allowed to attach and grow overnight in DMEM (Invitrogen, Carlsbad, Calif.) supplemented with 10% heat-inactivated FBS (JRH Biosciences, Lenexa, Kans.), 2 mM L-glutamine (Invitrogen), nonessential amino acids (Invitrogen) and 250 μg/ml G418 (Invitrogen). Compounds were serially diluted in DMEM plus 2% FBS and 0.5% DMSO (Sigma Chemical Co., St. Louis, Mo.) without G418. HCV replicon RNA from cells was measured using the Quantigene Discover XL kit (Panomics Inc., Fremont Calif.) as per the manufacturer's instructions. Briefly, compound-treated replicon cells were lysed and immobilized on to capture plates using HCV specific oligonucleotides overnight and the relative amounts of captured RNA was measured using oligonucleotide probe sets as per the manufacturer's instructions. Unless indicated otherwise, each data point represents the average of three replicates. The IC₅₀is the concentration of the compound at which the HCV replicon RNA level in cells is reduced by 50% as compared to the untreated replicon cell controls. To monitor the effect of compounds on cell proliferation or cell viability, replicon cells were treated with serially diluted compounds for 48 h, after which cell viability was determined using a CellTiter Glo assay (Promega, Madison, Wis.). Each CC₅₀is derived from three replicates and is the concentration of the compound at which the number of viable cells is reduced by 50% as compared to untreated cell controls. The IC₅₀and CC_sowas determined using 4 parameter curve fitting in the SoftMax Pro program (Molecular Devices, Sunnyvale, Calif.).

2. 5-Day HCV Replicon IC₉₉Assay

On the day prior to the assay, HCV replicon cells were plated at a low density of 2500 cells per well in a 96-well plate so the cells would not reach confluence during 5 days in culture. Compounds were serially diluted in DMEM containing 10% FBS and 0.5% DMSO in the absence of G418. Fresh media and compounds were added to the cells on day 1 and day 3. After the cells were treated with antiviral compounds for 5 days, HCV replicon RNA from cells was measured using the Quantigene Discover XL kit (Panomics Inc., Fremont Calif.) as per the manufacturer's instructions. Briefly, compound-treated replicon cells were lysed and immobilized onto to capture plates using HCV specific oligonucleotides overnight and the relative amounts of captured replicon RNA was measured using oligonucleotide probe sets (Panomics) as per manufacturer's instructions. Each data point represents the average of two replicates. The IC₉₉is the concentration of the compound at which the HCV replicon RNA level in cells is reduced by 2 logs as compared to the untreated cell controls. To monitor the effect of compounds on cell proliferation or cell viability, replicon cells were treated with serially diluted compounds for 5 days, after which cell viability was determined using a CellTiter Glo assay (Promega, Madison, Wis.). Each CC₅₀is derived from two replicates and is the concentration of the compound at which the number of viable cells is reduced by 50% as compared to untreated cell controls. The IC₉₉and CC₅₀were determined by 4 parameter curve fitting method using the Prism software (GraphPad Software Inc., San Diego, Calif.) and Excel program (Microsoft Corporation, Redmond, Wash.).

Using the assays above, compounds of the present invention are determined to be useful serine protease inhibitors.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Number	Date	Country
3211676	Oct 1983	DE
0417721	Mar 1991	EP
0675112	Oct 1995	EP
09124691	May 1997	JP
WO 9212140	Jul 1992	WO
WO 9325574	Dec 1993	WO
WO 9414436	Jul 1994	WO
WO 9507696	Mar 1995	WO
WO 9509614	Apr 1995	WO
WO 9611697	Apr 1996	WO
WO 9717364	May 1997	WO
WO 9740028	Oct 1997	WO
WO 9743310	Nov 1997	WO
WO 9813365	Apr 1998	WO
WO 9817679	Apr 1998	WO
WO 9822496	May 1998	WO
WO 9840381	Sep 1998	WO
WO 9846630	Oct 1998	WO
WO 9907733	Feb 1999	WO
WO 9907734	Feb 1999	WO
WO 9938888	Aug 1999	WO
WO 9950230	Oct 1999	WO
WO 9964442	Dec 1999	WO
WO 0009543	Feb 2000	WO
WO 0009558	Feb 2000	WO
WO 0009588	Feb 2000	WO
WO 0023421	Apr 2000	WO
WO 0031129	Jun 2000	WO
WO 0056331	Sep 2000	WO
WO 0059929	Oct 2000	WO
WO 0102424	Jan 2001	WO
WO 0107407	Feb 2001	WO
WO 0132691	May 2001	WO
WO 0140262	Jun 2001	WO
WO 0140266	Jun 2001	WO
WO 0158929	Aug 2001	WO
WO 0164678	Sep 2001	WO
WO 0174768	Oct 2001	WO
WO 0177113	Oct 2001	WO
WO 0181325	Nov 2001	WO
WO 0207761	Jan 2002	WO
WO 0208187	Jan 2002	WO
WO 0208198	Jan 2002	WO
WO 0208244	Jan 2002	WO
WO 0208251	Jan 2002	WO
WO 0208256	Jan 2002	WO
WO 0218369	Mar 2002	WO
WO 0248116	Jun 2002	WO
WO 0248157	Jun 2002	WO
WO 0248172	Jun 2002	WO
WO 02060926	Aug 2002	WO
WO 02079234	Oct 2002	WO
WO 03003804	Jan 2003	WO
WO 03006490	Jan 2003	WO
WO 03020298	Mar 2003	WO
WO 03062228	Jul 2003	WO
WO 03062265	Jul 2003	WO
WO 03064416	Aug 2003	WO
WO 03064455	Aug 2003	WO
WO 03064456	Aug 2003	WO
WO 03087092	Oct 2003	WO
WO 2004026896	Apr 2004	WO
WO 2004030670	Apr 2004	WO
WO 2004032827	Apr 2004	WO
WO 2004037855	May 2004	WO
WO 2004039833	May 2004	WO
WO 2004072243	Aug 2004	WO
WO 2004089974	Oct 2004	WO
WO 2004092161	Oct 2004	WO
WO 2004093798	Nov 2004	WO
WO 2004094452	Nov 2004	WO
WO 2004103996	Dec 2004	WO
WO 2004113365	Dec 2004	WO
WO 2005007681	Jan 2005	WO
WO 2005010029	Feb 2005	WO
WO 2005021584	Mar 2005	WO
WO 2005028501	Mar 2005	WO
WO 2005028502	Mar 2005	WO
WO 2005030796	Apr 2005	WO
WO 2005035525	Apr 2005	WO
WO 2005037214	Apr 2005	WO
WO 2005037860	Apr 2005	WO
WO 2005042570	May 2005	WO
WO 2005046712	May 2005	WO
WO 2005051410	Jun 2005	WO
WO 2005051980	Jun 2005	WO
WO 2005054430	Jun 2005	WO
WO 2005058821	Jun 2005	WO
WO 2005070955	Aug 2005	WO
WO 2005073195	Aug 2005	WO
WO 2005073216	Aug 2005	WO
WO 2005077969	Aug 2005	WO
WO 2005085242	Sep 2005	WO
WO 2005085275	Sep 2005	WO
WO 2005087721	Sep 2005	WO
WO 2005087725	Sep 2005	WO
WO 2005087731	Sep 2005	WO
WO 2005095403	Oct 2005	WO
WO 2005 107745	Nov 2005	WO
WO 2005113581	Dec 2005	WO
WO 2005123076	Dec 2005	WO
WO 2006000085	Jan 2006	WO
WO 2006007448	Jan 2006	WO
WO 2006007700	Jan 2006	WO
WO 2006007708	Jan 2006	WO
WO 2007016589	Feb 2007	WO
WO 2007025307	Mar 2007	WO
WO 2008106058	Sep 2008	WO

	Number	Date	Country
Parent	11711845	Feb 2007	US
Child	13074772		US

	Number	Date	Country
Parent	11511109	Aug 2006	US
Child	11711845		US

Inhibitors of serine proteases

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE

US Referenced Citations (214)

Foreign Referenced Citations (108)

Non-Patent Literature Citations (129)

Related Publications (1)

Provisional Applications (1)

Continuations (1)

Continuation in Parts (1)

Number	Name	Date	Kind
4499082	Shenvi et al.	Feb 1985	A
4720484	Vincent et al.	Jan 1988	A
4880780	Trainor et al.	Nov 1989	A
5053519	Teetz et al.	Oct 1991	A
5231084	Hock et al.	Jul 1993	A
5371072	Webb et al.	Dec 1994	A
5384410	Kettner	Jan 1995	A
5468858	Berlin et al.	Nov 1995	A
5484801	Al-Razzak et al.	Jan 1996	A
5496927	Kolb et al.	Mar 1996	A
5502061	Hui et al.	Mar 1996	A
5559158	Al-Razzak et al.	Sep 1996	A
5610193	Al-Razzak et al.	Mar 1997	A
5656600	Abelman et al.	Aug 1997	A
5656627	Bemis et al.	Aug 1997	A
5672582	Veber et al.	Sep 1997	A
5716929	Bemis et al.	Feb 1998	A
5725878	Al-Razzak et al.	Mar 1998	A
5736520	Bey et al.	Apr 1998	A
5756466	Bemis et al.	May 1998	A
5760029	Jadhav et al.	Jun 1998	A
5807876	Armistead et al.	Sep 1998	A
5847135	Bemis et al.	Dec 1998	A
5849866	Kolb et al.	Dec 1998	A
5861267	Su	Jan 1999	A
5866684	Attwood et al.	Feb 1999	A
5948436	Al-Razzak et al.	Sep 1999	A
5973111	Bemis et al.	Oct 1999	A
5990276	Zhang et al.	Nov 1999	A
6018020	Attwood et al.	Jan 2000	A
6025147	Bemis et al.	Feb 2000	A
6025516	Ramaswamy et al.	Feb 2000	A
6037157	Norbeck et al.	Mar 2000	A
6046195	Haworth et al.	Apr 2000	A
6054472	Armistead et al.	Apr 2000	A
6060469	Baker et al.	May 2000	A
6103711	Bemis et al.	Aug 2000	A
6117639	Germann et al.	Sep 2000	A
6130315	Kolb et al.	Oct 2000	A
6143715	Llinas-Brunet et al.	Nov 2000	A
6153579	Kim et al.	Nov 2000	A
6172077	Curtis et al.	Jan 2001	B1
6183121	Kim et al.	Feb 2001	B1
6211338	Malcolm et al.	Apr 2001	B1
6225320	Kulagowski et al.	May 2001	B1
6251583	Zhang et al.	Jun 2001	B1
6265380	Tung et al.	Jul 2001	B1
6268207	Bailey et al.	Jul 2001	B1
6274613	Plant et al.	Aug 2001	B1
6303287	Kim et al.	Oct 2001	B1
6323180	Llinas-Brunet et al.	Nov 2001	B1
6329417	Llinas-Brunet et al.	Dec 2001	B1
6344465	Armistead et al.	Feb 2002	B1
6348608	Shi	Feb 2002	B1
6399771	Plant et al.	Jun 2002	B1
6410531	Llinas-Brunet et al.	Jun 2002	B1
6420380	Llinas-Brunet et al.	Jul 2002	B2
6420522	Bemis et al.	Jul 2002	B1
6498178	Stamos et al.	Dec 2002	B2
6528276	Germann et al.	Mar 2003	B1
6534523	Llinas-Brunet et al.	Mar 2003	B1
6541496	Armistead et al.	Apr 2003	B1
6548555	Curatolo et al.	Apr 2003	B1
6608027	Tsantrizos et al.	Aug 2003	B1
6608067	Tung et al.	Aug 2003	B1
6617309	Tung et al.	Sep 2003	B2
6653127	Malcolm et al.	Nov 2003	B1
6653295	Glunz et al.	Nov 2003	B2
6699855	Zhang et al.	Mar 2004	B2
6727366	Han et al.	Apr 2004	B2
6767991	Llinas-Brunet et al.	Jul 2004	B1
6774212	Han	Aug 2004	B2
6800434	Saksena et al.	Oct 2004	B2
6824769	Chaturvedi et al.	Nov 2004	B2
6833442	Shibasaki et al.	Dec 2004	B2
6838475	Arasappan et al.	Jan 2005	B2
6846802	Chen et al.	Jan 2005	B2
6846806	Priestley	Jan 2005	B2
6867284	Matassa et al.	Mar 2005	B1
6872805	Campbell et al.	Mar 2005	B2
6909000	Farmer et al.	Jun 2005	B2
6911428	Zhu et al.	Jun 2005	B2
6914122	Venkatraman et al.	Jul 2005	B2
6919423	Llinas-Brunet et al.	Jul 2005	B2
6939692	Bathe et al.	Sep 2005	B2
6939854	Priestley	Sep 2005	B2
7012066	Saksena et al.	Mar 2006	B2
7034178	Faber et al.	Apr 2006	B2
7091184	Llinas-Brunet et al.	Aug 2006	B2
7105302	Bathe et al.	Sep 2006	B2
7109172	Britt et al.	Sep 2006	B2
7119073	Colarusso et al.	Oct 2006	B2
7122627	Priestley et al.	Oct 2006	B2
7169760	Saksena et al.	Jan 2007	B2
7208600	Cottrell et al.	Apr 2007	B2
7241796	Farmer et al.	Jul 2007	B2
7244721	Saksena et al.	Jul 2007	B2
7250520	Wallace	Jul 2007	B2
7273885	Pitlik et al.	Sep 2007	B2
7288624	Bemis et al.	Oct 2007	B2
7365092	Cottrell et al.	Apr 2008	B2
7371372	Chaturvedi et al.	May 2008	B2
7378422	Perni et al.	May 2008	B2
7381827	Tanoury et al.	Jun 2008	B2
7388017	Tung et al.	Jun 2008	B2
7504378	Llinas-Brunet et al.	Mar 2009	B2
7592316	Njoroge et al.	Sep 2009	B2
20020016294	Venkatraman et al.	Feb 2002	A1
20020016442	Llinas-brunet et al.	Feb 2002	A1
20020032175	Tung et al.	Mar 2002	A1
20020037998	Llinas-Brunet et al.	Mar 2002	A1
20020042046	Kim et al.	Apr 2002	A1
20020045729	Kerres et al.	Apr 2002	A1
20020065248	Zhang et al.	May 2002	A1
20020068702	Lim-Wilby et al.	Jun 2002	A1
20020102235	Arasappan et al.	Aug 2002	A1
20020107181	Chen et al.	Aug 2002	A1
20020111378	Stamos et al.	Aug 2002	A1
20020123468	Han	Sep 2002	A1
20020142449	Kwong et al.	Oct 2002	A1
20020147160	Bhat et al.	Oct 2002	A1
20020160962	Saksena et al.	Oct 2002	A1
20020177725	Priestley et al.	Nov 2002	A1
20020183249	Taylor et al.	Dec 2002	A1
20020187488	Lin et al.	Dec 2002	A1
20030008828	Priestley et al.	Jan 2003	A1
20030036501	Saksena et al.	Feb 2003	A1
20030064962	Glunz et al.	Apr 2003	A1
20030068369	McAllister et al.	Apr 2003	A1
20030083467	Germann et al.	May 2003	A1
20030100768	Han et al.	May 2003	A1
20030119752	Farmer et al.	Jun 2003	A1
20030144257	Biggadike et al.	Jul 2003	A1
20030153788	Kobayashi et al.	Aug 2003	A1
20030181363	Llinas-Brunet et al.	Sep 2003	A1
20030186895	Llinas-Brunet et al.	Oct 2003	A1
20030186952	Crew et al.	Oct 2003	A1
20030187018	Llinas-Brunet et al.	Oct 2003	A1
20030191067	Llinas-Brunet et al.	Oct 2003	A1
20030195362	Kempf et al.	Oct 2003	A1
20030216325	Saksena et al.	Nov 2003	A1
20030236242	Perni et al.	Dec 2003	A1
20040006237	Dolitzky et al.	Jan 2004	A1
20040018986	Pitlik et al.	Jan 2004	A1
20040048774	Saunders et al.	Mar 2004	A1
20040058982	Harris	Mar 2004	A1
20040067901	Bhat et al.	Apr 2004	A1
20040072788	Bhat et al.	Apr 2004	A1
20040077600	Tung et al.	Apr 2004	A1
20040082574	Wang et al.	Apr 2004	A1
20040105820	Weers et al.	Jun 2004	A1
20040110747	Altman	Jun 2004	A1
20040142876	Colarusso et al.	Jul 2004	A1
20040171626	Beaulieu et al.	Sep 2004	A1
20040180815	Nakajima et al.	Sep 2004	A1
20040186125	Poupart et al.	Sep 2004	A1
20040224900	Bailey et al.	Nov 2004	A1
20040229817	Duggal et al.	Nov 2004	A1
20040229818	Llinas-Brunet	Nov 2004	A1
20040266731	Tung et al.	Dec 2004	A1
20050020503	Llinas-Brunet et al.	Jan 2005	A1
20050049220	Stuyver	Mar 2005	A1
20050059606	Saksena et al.	Mar 2005	A1
20050062522	Haider et al.	Mar 2005	A1
20050080005	Llinas-Brunet et al.	Apr 2005	A1
20050080017	Cottrell et al.	Apr 2005	A1
20050090450	Farmer et al.	Apr 2005	A1
20050107304	Britt et al.	May 2005	A1
20050112093	Ette et al.	May 2005	A1
20050119189	Cottrell et al.	Jun 2005	A1
20050119318	Hudyma et al.	Jun 2005	A1
20050120398	Kalkeri et al.	Jun 2005	A1
20050136400	Lin et al.	Jun 2005	A1
20050137139	Perni et al.	Jun 2005	A1
20050153877	Miao et al.	Jul 2005	A1
20050187165	Scola et al.	Aug 2005	A1
20050187192	Fleming et al.	Aug 2005	A1
20050192212	Llinas-Brunet et al.	Sep 2005	A1
20050197299	Babine et al.	Sep 2005	A1
20050197301	Njoroge et al.	Sep 2005	A1
20050215486	Cottrell et al.	Sep 2005	A1
20050222236	Tsantrizos et al.	Oct 2005	A1
20050249702	Njoroge et al.	Nov 2005	A1
20050287514	Byrn	Dec 2005	A1
20060003317	Perni et al.	Jan 2006	A1
20060003942	Tung et al.	Jan 2006	A1
20060046956	Sannigrahi et al.	Mar 2006	A1
20060089385	Cui et al.	Apr 2006	A1
20060105978	Chu et al.	May 2006	A1
20060205672	Saksena et al.	Sep 2006	A1
20060211629	Britt et al.	Sep 2006	A1
20070087973	Tanoury et al.	Apr 2007	A1
20070105781	Lyons et al.	May 2007	A1
20070161789	Cottrell et al.	Jul 2007	A1
20070179167	Cottrell et al.	Aug 2007	A1
20070191381	Tung	Aug 2007	A1
20070212683	Connelly	Sep 2007	A1
20070218012	Bittorf et al.	Sep 2007	A1
20070218138	Bittorf et al.	Sep 2007	A1
20070225297	Perni et al.	Sep 2007	A1
20070231262	Lin et al.	Oct 2007	A1
20070243166	Llinas-Brunet et al.	Oct 2007	A1
20070244334	Tanoury et al.	Oct 2007	A1
20070292933	Pitlik et al.	Dec 2007	A1
20080045480	Farmer et al.	Feb 2008	A1
20080070972	Kadiyala et al.	Mar 2008	A1
20080125376	Cottrell et al.	May 2008	A1
20080167480	Wallace	Jul 2008	A1
20080267915	Lin et al.	Oct 2008	A1
20080311079	Perni et al.	Dec 2008	A1
20090022688	Farmer et al.	Jan 2009	A1
20090143312	Tung et al.	Jun 2009	A1
20090191555	Lin et al.	Jul 2009	A1
20090247468	Bittorf et al.	Oct 2009	A1

Entry
Cheng et al. J. Org. Chem. 2002, 67, 5673-5677.
Akahoshi, F., “Chymase Inhibitors and their Therapeutic Potential”, Drugs of the Future, 27(8) (2009), pp. 765-770.
Anonymous, VPI internet press release Sep. 7, 2004.
Anonymous, newsrx Internet article, May 31, 2004.
Arasappan, A., “Hepatitis C Virus NS3-4A Serine Protease Inhibitors: SAR of P′2 Moiety with Improved Potency”, Bioorg. & Med. Chem. Let., vol. 15, (2005), pp. 4180-4184.
Avolio, S., “Inhibitors of hepatitis C virus NS3/4A: α-Ketoamide based macrocyclic inhibitors,” Bioorganic & Medicinal Chemistry Letters (2009), 19, pp. 2295-2298.
Bastos, M., “Inhibitors of Human Heart Chymase Based on a Peptide Library”, Proc. Natl. Acad. Sci. USA, vol. 92 (1995), pp. 6738-6742.
Beak, P., “Complex Induced Proximity Effects: Enantioselective Syntheses Based on Asymmetric Deprotonations of N-Boc-Pyrrolidines”, J. Amer. Chem. Soc., vol. 116 (1994), pp. 3231-3239.
Behrens, C., “Selective Transformations of 2,3-Epoxy alcohols and Related Derivatives. Strategies for Nucleophilic Attack at Carbon-3 or Carbon-2”, J. Org.Chem., vol. 50 (1985), pp. 5696-5704.
Bergmeier, S.C., “Synthesis of Bicyclic Praline Analogs Using a formal [3+2] Intramolecular Aziridine-Allylsilane Cycloaddition Reaction”, Tetrahedron, vol. 55, No. 26 (1999), pp. 8025-8038.
Blair, W., “5th Antiviral Drug Discovery and Development Summit,” Expert opinion on investigational drugs (2004), 13 (8), pp. 1065-1069.
Blankley, C.J., “Synthesis and Structure-Activity Relationships of Potent New Abgiotensin Converting Enzyme Inhibitors Containing Saturated Bicyclic Amino Acids”, J. of Medicinal Chem., vol. 30 (1987).
Cacciola, J., “The Synthesis of Lysine α-Ketoamide Thrombin Inhibitors via an Epoxy Amide Ring Opening”, Tetrahedron Let, vol. 38, No. 33 (1997), pp. 5741-5744.
Chawla, et al., “Challenges in Polymorphism of Pharmaceuticals”, CRIPS vol. 5, No. 1, Jan.-Mar. 2004, 4 pages.
Chen, S., “Synthesis and Evaluation of Tripeptidyl α-Ketoamides as Human Rhinovirus 3C Protease Inhibitors”, Bioorg. & Med. Chem. Letters, vol. 13, No. 20 (2003), pp. 3531-3536.
Chen, S., “Discovery of Small-Molecule Inhibitors of HCV NS3-4A Protease as Potential Therapeutic Agents against HCV Infection,” Current Medicinal Chemistry (2005), 12(20), pp. 2317-2342.
Chen, S., “P1 and P1′ Optimization of [3,4]-Bicycloproline P2 Incorporated Tetrapeptidyl α-ketoamide Based HCV Protease Inhibitors,” Letters in Drug Design and Discovery (2005), 2(2), pp. 118-123.
Cheng, W., “Stereoselective Synthesis of Unnatural Spiroisoxazolinoproline-Based Acids and Derivatives”, J. Org. Chem., (2002), pp. 5673-5677.
Collado, I., “Stereocontrolled Synthesis of 4-Substituted (±)-Kainic Acids”, Journal of Organic Chem., vol. 63 (1998).
Davis, G. “Future Options for the Management of Hepatitis C”, Seminars in Liver Disease, vol. 19, Supp. 1 (1999), pp. 103-112.
Dixon, S. M., “A Spiroisoazolinoproline-based Amino Acid Scaffold for Solid Phase and One-Bead—One-Compound Library Synthesis” Journal of Combinatorial Chemistry, 9 (2007) pp. 143-157.
Dunsdon, R., “Solid Phase Synthesis of Aminoboronic Acids: Potent Inhibitors of the Hepatitis C Virus NS3 Proteinase”, Bioorganic & Medicinal Chemistry Letters, 10 (2000), pp. 1577-1579.
Elemes, “Synthesis of enantiopure α-deuterated Boc-L amino acids,” J. Chemical Society, Perkin Trans. vol. 1 (1995) pp. 537-540.
Esch, P.M., “Reductive Cyclization of Carbon-Centered Glycine Radicals; A Novel Synthetic route to Cyclic α-Amino Acids”, Tetrahedron, vol. 48, No. 22 (1992), pp. 4659-4676.
Farmer, L., “Inhibitors of Hepatitis C Virus NS3-4A Protease: P2 Proline Variants,” Letters in Drug Design and Discovery (2005), 2, pp. 497-502.
Forestier, Current status of subjects receiving peg-interferon-alfa-2a (PEG-IFN) and ribavirin (RBV) after a 14-day study of the hepatitis C protease inhibitor telaprevir (VX-950), with PEG-IFN, Hepatology, vol. 44, Supp. 2 (2006), p. 614A.
Freireich, E., “Quantitative Comparison of Toxicity of Anticancer Agents in Mouse, Rat, Hamster, Dog, Monkey and Man”, Cancer Chemother. Rep., vol. 50 No. 219 (1966), pp. 219-244.
Gallagher, D., “Complex-Induced Proximity Effects: Evidence for a Prelithiation Complex and a Rate-Determining Deprotonation in the Asymmetric Lithiation of Boc-Pyrrolidine by an i-PrLi/(−) Sparteine Complex”, J. Org. Chem., vol. 60 (1995), pp. 7092-7093.
Gallagher, D., “Chiral Organolithium Complexes: The Effect of Ligand Structure on the Enantioselective Deprotonation of Boc-Pyrrolidine”, J. Org. Chem., vol. 60 (1995), pp. 8148-8154.
Garrison, G., “Novel 3,7-Diheterabicyclo[3.3.1]nonanes that Possess Predominant Class III Antiarrhythmic Activity in 1-4 Day Post Infarction Dog Models: X-ray Diffraction Analysis of 3-[4-(1 H-Imidazol-1-yl)benzoyl]-7-isopropyl-3,7-diazabicyclo[3.3.1]nonane Dihydroperchlorate”, J. Med. Chem, vol. 39, No. 13 (1996), pp. 2559-2570.
Golina, S., “Vulcanisation of Poly(diethyl-n-butylamino) Phosphazenes”, Internat'l. Polymer Science & Tech., vol. 18, No. 3 (1991), pp. T20-T22.
Han, W., “α-Ketoamides, α-Ketoesters and α-Diketones as HCV NS3 Protease Inhibitors”, Bioorganic & Medicinal Chemistry Letters 10 (2000), pp. 711-713.
Janssen, H.L.A., “Suicide Associated with α-Interferon Therapy for Chronic Viral Hepatitis”, J. Hepatol., 21 (1994), pp. 241-243.
Johansson, A., “Acyl Sulfonamides as Potent Protease Inhibitors of the Hepatitis C Virus Full-Length NS3 (Protease-Helicase/NTPase): A comparative Study of Different C-Terminals”, Bioorganic & Medicinal Chemistry, 11 (2003), pp. 2551-2568.
Johansson, P., “Potent inhibitors of the hepatitis C virus NS3 protease: Use of a novel P2 cyclopentane-derived template,” Bioorganic & Medicinal Chemistry (2006), 14, pp. 5136-5151.
KAKEI, H., “Catalytic Asymmetric Epoxidation of α, β-Unsaturated Esters Using an Yttrium-Biphenyldiol Complex”, J. Am. Chem. Soc., vol. 127 (2005), pp. 8962-8963.
Kalkeri, G., “Expression of HCV Protease in the Liver of Mice Results in Liver Injury Which can be Inhibited by VX-950, A Vertex HCV Protease Inhibitor,” AALSD Abstracts, Hepatology (2004), 40(1), pp. 281A.
Kamandi, E., “Die Synthese von β-Phenyl-lsoserinen Durch Ammonolyse von β-Phenyl-Glycidestern, I”, Archiv de Pharmazie, vol. 307 No. 11 (1974), pp. 871-878.
Kao, J.H., “Efficacy of Consensus Interferon in the Treatment of Chronic Hepatitis”, J. Gastroenterol. Hepatol, 15 (2000), pp. 1418-1423.
Kerrick, S., “Asymmetric Deprotonations: Enantioselective Syntheses of 2-Substituted (tert-Butoxycarbonyl) pyrrolldines”, J. Amer. Chem. Soc., vol. 113 (1991), pp. 9703-9710.