RU2845865C2 - Nitrile-containing antiviral compounds - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: invention relates to nitrile-containing antiviral compounds, pharmaceutical compositions containing such compounds, and use thereof in treating coronavirus infection, such as SARS-CoV-2.

EFFECT: nitrile-containing antiviral compounds are disclosed.

6 cl, 15 dwg, 2 tbl, 98 ex

Description

State of the art

The invention relates to compounds and methods for inhibiting viral replication activity comprising contacting a SARS-CoV-2-related 3C-like ("3CL") protease with a therapeutically effective amount of a SARS-CoV-2-related 3C-like protease inhibitor. The invention also relates to methods for treating coronavirus disease 2019 ("COVID-19") in a patient by administering a therapeutically effective amount of a SARS-CoV-2-related 3C-like protease inhibitor to the patient in need thereof. The invention also relates to methods for treating COVID-19 in a patient, wherein the method comprises administering to the patient in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a SARS-CoV-2-related 3C-like protease inhibitor.

The global outbreak of coronavirus disease 2019 ("COVID-19") was attributed to infections that originated in late 2019 in Wuhan, Hubei Province, China. By mid-2020, the COVID-19 outbreak had become a global pandemic, with millions of people confirmed to be infected, resulting in hundreds of thousands of deaths. The causative agent of COVID-19 was identified as a novel coronavirus, named SARS-CoV-2. The genome sequence of SARS-CoV-2 was sequenced from isolates from nine patients in Wuhan, China, and was found to belong to the subgenus Sarbecovirus of the genus Betacoronavirus. Lu, R. et al. The Lancet, 395, 10224, 565-574; online January 29, 2020. The sequence of SARS-CoV-2 was found to share 88% homology with two bat-derived SARS-type coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, which were collected in 2018 in Zhoushan, eastern China. SARS-CoV-2 was also found to share approximately 79% homology with the severe acute respiratory syndrome coronavirus (“SARS-CoV”), the causative agent of the 2002–2003 SARS outbreak, and approximately 50% homology with the Middle East respiratory syndrome coronavirus (“MERS-CoV”), the causative agent of a respiratory virus outbreak that originated in the Middle East in 2012. Based on a recent analysis of 103 sequenced SARS-CoV-2 genomes, it was suggested that SARS-CoV-2 can be divided into two main types (L and S types), with the S type being ancestral and the L type evolving from the S type. Lu, J.; Cui, J. et al. On the origin and continuing evolution of SARS-CoV-2; National Science Review, 7(6), June 2020, 1012–1023, http://doi.org/10.1093/nsr/nwaa036. The S and L types can be clearly defined by only two closely linked SNPs at positions 8782 (orf1ab:T8517C, synonymous) and 28144 (ORF8: C251T, S84L). Of the 103 genomes analyzed, approximately 70% belonged to the L type and approximately 30% to the S type. It is unclear whether the L type evolved from the S type in humans or via a zoonotic intermediate, but the L type appears to be more aggressive than the S type, and human intervention in efforts to contain the outbreak may have shifted the relative abundance of the L and S types shortly after the onset of the SARS-CoV-2 outbreak. The discovery of the proposed S and L subtypes of SARS-CoV-2 raises the possibility that humans could potentially be infected sequentially with separate subtypes or infected with both subtypes simultaneously. In view of this growing threat, there is an urgent need in the art for effective treatments for COVID-19 and methods to inhibit the replication of the SARS-CoV-2 coronavirus.

Recent evidence clearly shows that the newly emerged coronavirus SARS-CoV-2, the causative agent of COVID-19 (Centers for Disease Control, CDC), has acquired the ability to transmit between humans, resulting in community spread of the virus. The sequence of the receptor-binding domain of the SARS-CoV-2 spike protein ("RBD"), including its receptor-binding motif (RBM), which directly contacts the angiotensin-converting enzyme 2, ACE2, receptor, is similar to the RBD and RBM of SARS-CoV, strongly suggesting that SARS-CoV-2 uses ACE2 as its receptor. Wan, Y.; Shang, J.; Graham, R.; Baric, R.S.; Li, F.; Receptor recognition by the novel coronavirus from Wuhan: An analysis based on decade-long structural studies of SARS coronavirus; J. Virol. 2020; doi:10.1128/JVI.00127-20. Several critical residues in the SARS-CoV-2 RBM (notably Gln493) mediate favorable interactions with human ACE2, consistent with the ability of SARS-CoV-2 to infect human cells. Several other critical residues in the SARS-CoV-2 RBM (notably Asn501) are compatible but not ideal for binding human ACE2, suggesting that SARS-CoV-2 exploits ACE2 binding to some extent for human-to-human transmission.

The replication and transcription function of coronavirus is encoded by the so-called “replicase” gene (Ziebuhr, J., Snijder, E.J., and Gorbalenya, A.E.; Virus-encoded proteinases and proteolytic processing in the Nidovirales. J. Gen. Virol. 2000, 81, 853-879; and Fehr, A.R.; Perlman, S.; Coronaviruses: An Overview of Their Replication and Pathogenesis, Methods Mol. Biol. 2015; 1282: 1-23. doi:10.1007/978-1-4939-2438-7_1), which consists of two overlapping polyproteins that are extensively processed by viral proteases. The C-proximal region is processed at eleven conserved interdomain junctions by the core or "3C-like" coronavirus protease (Ziebuhr, Snijder, Gorbalenya, 2000 and Fehr, Perlman et al., 2015). The name "3C-like" protease derives from certain similarities between the coronavirus enzyme and the well-known picornavirus 3C proteases. These include substrate preference, the use of cysteine as an active site nucleophile in catalysis, and the similarity of their putative common polypeptide folds. The sequence of the SARS-CoV-2 3CL protease (accession number YP_009725301.1) was found to have 96.08% homology to that of the SARS-CoV 3CL protease (accession number YP_009725301.1) Xu, J.; Zhao, S.; Teng, T.; Abdalla, A.E.; Zhu, W.; Xie, L.; Wang, Y.; Guo, X.; Systematic Comparison of Two Animal-to-Human Transmitted Human Coronaviruses: SARS-CoV-2 and SARS-CoV; Viruses 2020, 12, 244; doi:10.3390/v12020244. More recently, Hilgenfeld and colleagues published the high-resolution X-ray structure of the main protease of the SARS-CoV-2 coronavirus (3CL) Zhang, L.; Lin, D.; Sun, X.; Rox, K.; Hilgenfeld, R.; X-ray Structure of Main Protease of the Novel Coronavirus SARS-CoV-2 Enables Design of α-Ketoamide Inhibitors; bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952879. The structure reveals differences when comparing SARS-CoV-2 and SARS-CoV 3CL proteases. In SARS-CoV, but not in the SARS-CoV-2 3CL protease dimer, there is a polar interaction between the two domains III involving a 2.60-Å hydrogen bond between the side-chain hydroxyl groups of Thr285 of each protomer, and is supported by a hydrophobic contact between the side-chain of Ile286 and Thr285 of Cγ2. In SARS-CoV-2 3CL, threonine is replaced by alanine and isoleucine by leucine compared to the same residues in SARS-CoV 3CL. The Thr285Ala substitution observed in SARS-CoV-2 3CL protease brings the two domains III somewhat closer to each other (the distance between the Cα atoms of residues 285 in molecules A and B is 6.77 Å in SARS-CoV 3CL protease and 5.21 Å in SARS-CoV-2 3CL protease, and the distance between the centers of mass of the two domains III is reduced from 33.4 Å to 32.1 Å). In the active site of SARS-CoV-2 3CL, Cys145 and His 41 form a catalytic dyad, which, when taken together with the buried water molecule hydrogen bonded to His41, can be considered the catalytic triad of SARS-CoV-2 3CL protease. Given the ongoing spread of SARS-CoV-2, which has caused the current worldwide outbreak of COVID-19, it is desirable to have new ways to suppress SARS-CoV-2 viral replication and treat COVID-19 in patients.

The essence of the invention

The present invention provides novel compounds that act to inhibit or prevent the replication of the SARS-CoV-2 virus and are thus useful for the treatment of COVID-19. The present invention also relates to pharmaceutical compositions containing the compounds and methods for treating COVID-19 and inhibiting the replication of the SARS-CoV-2 virus by administering the compounds of the present invention or pharmaceutical compositions containing the compounds of the present invention. It should be understood that each of the following embodiments of the method of treatment can also be formulated as corresponding embodiments of use. For example, any of the compounds, or pharmaceutically acceptable salts or solvates or hydrates thereof, or pharmaceutically acceptable salts of the compounds, solvates or hydrates, as set forth in any of embodiments E1-E30, E45-E46, E50, E50a, E59-E68 and E80-E83 can be used for use as a medicine or, alternatively, for use in a method of treatment as described in any of embodiments E36-E41, E47-E49, E52-E58a, E69-E74, E77-R79, E85-E93 and E95-E98.

E1 is a compound according to E45 or E59 below, of formula I

I;

or a pharmaceutically acceptable salt thereof; wherein R1 is selected from the group consisting of C1-C6 alkyl which is optionally substituted with cyano or from one to five fluorine atoms; C2-C6 alkynyl; and (C3-C6 cycloalkyl)-C1-C3 alkyl which is optionally substituted with one to two substituents selected from trifluoromethyl and C1-C3 alkyl, or from one to five fluorine atoms; R2 is hydrogen, or R2 and R1, taken together with the nitrogen and carbon atoms to which they are attached, are a pyrrolidine or piperidine ring which is optionally substituted with one to four R2a; R2a is at each occurrence independently selected from the group consisting of fluoro, C1-C6 alkyl optionally substituted with one to three fluoro atoms, and C1-C6 alkoxy optionally substituted with one to three fluoro atoms; or two R2a groups attached to adjacent carbon atoms and taken together with the atoms to which they are attached are a fused C3-C6 cycloalkyl which is optionally substituted with one to four R2b; or two R2a groups attached to the same carbon atom and taken together with the atoms to which they are attached are spiro C3-C6 cycloalkyl which is optionally substituted with one to four R2b; R2b is at each occurrence independently selected from fluoro, C1-C3 alkyl optionally substituted with one to three fluoro atoms, and C1-C3 alkoxy optionally substituted with one to three fluoro atoms; R3 is selected from the group consisting of C1-C8 alkyl, C1-C8 alkoxy, (C1-C6 alkoxy)-C1-C6 alkyl, C2-C6 alkynyl, C2-C6 alkynyloxy, C3-C12 cycloalkyl optionally fused with a 5-6 membered heteroaryl or phenyl, (C3-C12 cycloalkyl)-C1-C6 alkyl, C3-C12 cycloalkoxy, (C3-C12 cycloalkoxy)-C1-C6 alkyl, 4-12 membered heterocycloalkyl which is optionally fused with a 5-6 membered heteroaryl or phenyl, and wherein said heterocycloalkyl contains from one to four heteroatoms independently selected from N, O and S(O)n, (4-12 membered heterocycloalkyl)-C1-C6 alkyl, wherein said heterocycloalkyl group contains from one to four heteroatoms independently selected from N, O and S(O)n, C6-C10 aryl optionally fused with a C4-C6 cycloalkyl or a 4-7-membered heterocycloalkyl, (C6-C10 aryl)-C1-C6 alkyl, 5-10-membered heteroaryl containing from one to five heteroatoms independently selected from N, O and S, which is optionally fused with a C5-C6 cycloalkyl; (5-10-membered heteroaryl)-C1-C6 alkyl, wherein the heteroaryl group contains from one to five heteroatoms independently selected from N, O and S; (C6-C10 aryl)-(5-10 membered heteroaryl)- wherein the heteroaryl group comprises one to five heteroatoms independently selected from N, O and S, (5-10 membered heteroaryloxy)-C1-C6 alkyl, wherein the heteroaryl group comprises one to five heteroatoms independently selected from N, O and S; (5-6 membered heteroaryl)-(5-6 membered heteroaryl)-, wherein each heteroaryl group comprises one to four heteroatoms independently selected from N, O and S; (4-7 membered heterocycloalkyl)-(5-6 membered heteroaryl)-, wherein the heterocycloalkyl group comprises one to three heteroatoms independently selected from N, O and S(O)n and the heteroaryl group comprises one to four heteroatoms independently selected from N, O and S; (5-6-membered heteroaryl)-(4-7-membered heterocycloalkyl)-, wherein the heterocycloalkyl group contains one to three heteroatoms independently selected from N, O and S(O)n and the heteroaryl group contains one to four heteroatoms independently selected from N, O and S; wherein each R3 group is optionally substituted with one to five R4; R4 is at each occurrence independently selected from the group consisting of oxo, halogen, hydroxy, cyano, phenyl, benzyl, amino, (C1-C6 alkyl)amino optionally substituted with one to five fluorine atoms, di(C1-C6 alkyl)amino optionally substituted with one to ten fluorine atoms, C1-C6 alkyl optionally substituted with one to five fluorine atoms, C1-C6 alkoxy optionally substituted with one to five fluorine atoms, C1-C3 alkoxy-C1-C3 alkyl optionally substituted with one to five fluorine atoms, C3-C6 cycloalkyl optionally substituted with one to three fluorine atoms or C1-C3 alkyl, C1-C6 alkyl-C(O)NH- optionally substituted with one to five fluorine, C1-C6 alkyl-S(O)2NH- optionally substituted with one to five fluorine atoms, C1-C6 alkyl-C(O)- optionally substituted with one to five fluorine atoms, C1-C6 alkyl-S(O)n- optionally substituted with one to five fluorine atoms; and

n in each case is independently chosen from 0, 1 and 2.

E2 is a compound according to any one of E1, E45 and E59, wherein R1 is selected from the group consisting of (CH3)2CHCH2-, (CH3)3CCH2-, cyanomethyl, 2-cyanoethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 3,3,3-trifluoropropyl, 3,3,3-trifluoro-2-methylpropyl, cyclopropylmethyl, (2,2-difluorocyclopropyl)methyl, [1-(trifluoromethyl)cyclopropyl]methyl, (2-methylcyclopropyl)methyl, (3,3-difluorocyclobutyl)methyl, cyclopentylmethyl and propynyl; and R2 is hydrogen; or a pharmaceutically acceptable salt thereof.

E3 is a compound according to any one of E1, E45 and E59, wherein R2 and R1, taken together with the nitrogen and carbon atoms to which they are attached, are a pyrrolidine or piperidine ring which is optionally substituted with one to four R2a; or a pharmaceutically acceptable salt thereof.

E4 is a compound according to any one of E1, E45, E59 and E3 wherein R2a at each occurrence is independently selected from the group consisting of fluoro, methyl, isopropyl, trifluoromethyl and tert-butoxy; or two R2a groups attached to adjacent carbon atoms and taken together with the atoms to which they are attached are a fused cyclopentane or cyclopropane which is optionally substituted with one to four R2b; or two R2a groups attached to the same carbon atom and taken together with the atoms to which they are attached are a spirocyclopropane ring which is optionally substituted with one to four R2b; or a pharmaceutically acceptable salt thereof.

E5 is a compound according to E1, E3, E4, E45 and E59, wherein R2b in each occurrence is independently selected from the group consisting of fluoro, methyl and methoxy; or a pharmaceutically acceptable salt thereof.

E6 is a compound according to any one of E1, E2, E45 and E59, selected from the group consisting of formulae Ia - Ig

And ;

or its pharmaceutically acceptable salt.

E7 is a compound according to any of E1, E3, E4, E45 and E59 selected from the group consisting of formulae Ih - Ik

or its pharmaceutically acceptable salt.

E8 is a compound of any one of E1, E3, E4, E7, E45 and E59 selected from the group consisting of

or its pharmaceutically acceptable salt.

E9 is a compound according to any one of E1, E3, E4, E7, E8, E45 and E59 wherein R3 is selected from the group consisting of C1-C6 alkyl and (C3-C6 cycloalkyl)-C1-C3 alkyl; each of which is substituted with one to four R4; or a pharmaceutically acceptable salt thereof.

E10 is a compound according to any one of E1, E3, E4, E7 to E9, E45 and E59 wherein R3 is selected from the group consisting of (CH3)2CHCH(R4)-, (CH3)3CCH(R4)- and (cyclohexyl)CH(R4)-; or a pharmaceutically acceptable salt thereof.

E11 is a compound according to any one of E1, E3, E4, E7-E10, E45 and E59 selected from the group consisting of

or its pharmaceutically acceptable salt.

E12 is a compound according to any one of E1, E3, E4, E7-E11, E45 and E59, wherein R4 is selected from the group consisting of (C1-C6 alkyl)amino optionally substituted with one to five fluorine atoms, C1-C6 alkyl-C(O)NH- optionally substituted with one to five fluorine atoms, and C1-C6 alkyl-S(O)2NH- optionally substituted with one to five fluorine atoms; or a pharmaceutically acceptable salt thereof.

E13 is a compound according to any one of E1, E3, E4, E7-E12, E45 and E59, wherein R4 is selected from the group consisting of CF3C(O)NH-, CF3S(O)2NH-, CH3C(O)NH-, CH3CH2C(O)NH- and CF3CH2NH-; or a pharmaceutically acceptable salt thereof.

E14 is a compound according to any one of E1, E3, E4, E7-E13, E45 and E59, wherein R4 is CF3C(O)NH- or CF3S(O)2NH-; or a pharmaceutically acceptable salt thereof.

E15 is a compound according to any one of E1-E8, E45 and E59, wherein R3 is a 4-12 membered heterocycloalkyl which is optionally fused with a 5-6 membered heteroaryl or phenyl, and wherein said heterocycloalkyl contains from one to four heteroatoms independently selected from N, O and S(O)n, or is a (4-12 membered heterocycloalkyl)-C1-C6 alkyl, wherein said heterocycloalkyl group contains from one to four heteroatoms independently selected from N, O and S(O)n; each of which is optionally substituted with from one to five R4; or a pharmaceutically acceptable salt thereof.

E16 is a compound according to any one of E1-E8, E15, E45 and E59, wherein the 4-12 membered heterocycloalkyl group in R3 is selected from the group consisting of acetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, oxetanyl, tetrahydrofuranyl, pyranyl, 2-oxo-1,3-oxazolidinyl, oxabicyclo[2.2.1]heptyl, 1-oxa-8-azaspiro[4.5]decyl, 1,1-dioxido-1,2-thiazolidinyl and 1,1-dioxido-1,2-thiazinanyl; each of which is optionally substituted with one to three R4; or a pharmaceutically acceptable salt thereof.

E17 is a compound according to any one of E1-E8, E45 and E59, wherein R3 is selected from the group consisting of phenyl, benzyl, phenethyl, 5-10 membered heteroaryl containing one to five heteroatoms independently selected from N, O and S; (5-10 membered heteroaryl)-C1-C6 alkyl, wherein the heteroaryl group contains one to five heteroatoms independently selected from N, O and S; and (5-10 membered heteroaryloxy)-C1-C6 alkyl, wherein the heteroaryl group contains one to five heteroatoms independently selected from N, O and S; each of which is optionally substituted with one to five R4; or a pharmaceutically acceptable salt thereof.

E18 is a compound according to any one of E1 to E8, E17, E45 and E59, wherein the 5-10 membered heteroaryl group in R3 is selected from the group consisting of imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, indolyl, benzimidazolyl, pyridinopyrrolyl, quinolinyl, quinoxalinyl, benzotriazolyl, imidazo[1,2-a]pyridinyl, imidazo[2,1-b][1,3]thiazolyl, 4H-furo[3,2-b]pyrrolyl, 4H-thieno[3,2-b]pyrrolyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,3]triazolo[1,5-a]pyridinyl and naphthyridinyl; each of which is optionally substituted with one to four R4; or a pharmaceutically acceptable salt thereof.

E19 is a compound according to any one of E1-E8, E17-E18, E45 and E59, wherein R3 is indolyl; which is optionally substituted with one to four R4; or a pharmaceutically acceptable salt thereof.

E20 is a compound according to any one of E1-E8, E17-E19, E45 and E59, wherein R3 is indol-2-yl; which is optionally substituted with one to four R4; and R4 at each occurrence is independently selected from the group consisting of fluoro, chloro, bromo, hydroxy, methyl, ethyl, propyl, isopropyl, 1-methylpropyl, butyl, tert-butyl, acetyl, methoxy, ethoxy, propoxy, butoxy, trifluoromethyl, trifluoromethoxy, cyclohexyl and diethylamino; or a pharmaceutically acceptable salt thereof.

E21 is a compound according to any of E1, E2, E6, E9-E10, E12-E20, E45 and E59 of the formula

;

or its pharmaceutically acceptable salt.

E22 is a compound according to any one of E1, E2, E6, E9-E10, E12-E21, E45 and E59, wherein R3 is selected from the group consisting of 1H-indol-2-yl, 7-fluoro-4-methoxy-1H-indol-2-yl, 4-methoxy-7-(trifluoromethyl)-1H-indol-2-yl, 4-methoxy-1H-indol-2-yl, 4-(trifluoromethoxy)-1H-indol-2-yl, 6-(trifluoromethyl)-1H-indol-2-yl, 4-methoxy-3,6,7-tris(trifluoromethyl)-1H-indol-2-yl, 3-fluoro-4-methoxy-1H-indol-2-yl and 3,5-difluoro-4-methoxy-1H-indol-2-yl; or its pharmaceutically acceptable salt.

E23 is a compound according to any one of E1-E8, E21, E45 and E59, wherein R3 is C1-C6 alkoxy; or a pharmaceutically acceptable salt thereof.

E24 is a compound according to any one of E1-E8, E21, E23, E45 and E59, wherein R3 is selected from the group consisting of methoxy, ethoxy and prop-2-oxy; or a pharmaceutically acceptable salt thereof.

E25 is a compound according to any one of E1-E8, E21, E45 and E59, wherein R3 is selected from the group consisting of C3-C12 cycloalkyl optionally fused with a 5-6 membered heteroaryl or phenyl, (C3-C12 cycloalkyl)-C1-C6 alkyl, C3-C12 cycloalkoxy and (C3-C12 cycloalkoxy)-C1-C6 alkyl; each of which is optionally substituted with one to three R4; or a pharmaceutically acceptable salt thereof.

E26 is a compound according to any one of E1-E8, E21, E25, E45 and E59, wherein R3 is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-(cyclohexyloxy)ethyl, cyclohexoxymethyl, cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclobutylethyl, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl and cyclohexylethyl; each of which is optionally substituted with one to three R4; or a pharmaceutically acceptable salt thereof.

E27 is a compound according to any one of E1-E8, E17, E45 and E59, wherein R3 is selected from the group consisting of phenyl, benzyl and phenethyl, each of which is optionally substituted with one to three R4; or a pharmaceutically acceptable salt thereof.

E28 is a compound according to any one of E1-E8, E17, E27, E45 and E59, wherein R4 is selected from the group consisting of fluorine, chlorine, dimethylamino, trifluoromethyl, CF3C(O)NH- and CF3S(O)2NH-; or a pharmaceutically acceptable salt thereof.

E29 is a compound according to any one of E1, E45 and E59, selected from the group consisting of

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-{(2R)-2-(dimethylamino)-2-[4-(trifluoromethyl)phenyl]acetyl}-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-{(2R)-2-(dimethylamino)-2-[3-(trifluoromethyl)phenyl]acetyl}-4-methyl-L-leucinamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-6-(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-6-(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-3,6,7-tris(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-(trifluoromethoxy)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-(trifluoromethoxy)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3-fluoro-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3,5-difluoro-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5,7-difluoro-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-fluoro-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-3,5,7-tris(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-3,7-bis(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-(trifluoromethyl)-1H-indole-2-carboxamide;

7-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-7-methyl-1H-indole-2-carboxamide;

6-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

4-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

5-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-7-(trifluoromethyl)-1H-indole-2-carboxamide;

4,6-dichloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-(trifluoromethyl)-1H-indole-2-carboxamide;

7-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-7-methyl-1H-indole-2-carboxamide;

6-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

4-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

5,7-dichloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

5-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-7-(trifluoromethyl)-1H-indole-2-carboxamide;

4,6-dichloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-methyl-5-(trifluoromethyl)imidazo[2,1-b][1,3]thiazol-2-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[4-methyl-2-(trifluoromethyl)-1,3-thiazol-5-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[5-methyl-2-(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl}-L-leucinamide;

N2-[(4-bromo-1-ethyl-3-methyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-L-leucinamide;

N2-[(4-chloro-1,3-dimethyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-L-leucinamide;

3-acetyl-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3R)-2,5-dioxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-hydroxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-hydroxy-4-methoxy-1H-indole-2-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(3,3-difluorocyclobutyl)acetyl]-4-methyl-L-leucinamide;

N2-[(trans-4-cyanocyclohexyl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide;

N2-[(trans-4-cyanocyclohexyl)carbonyl]-N-{(1R)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[2-(cyclohexyloxy)propanoyl]-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[cyclohexyl(methoxy)acetyl]-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[cyclohexyl(methoxy)acetyl]-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(2S)-2-(dimethylamino)-2-phenylacetyl]-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-(pyrrolidin-1-ylacetyl)-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(2R)-2-(dimethylamino)-2-phenylacetyl]-4-methyl-L-leucinamide;

N2-[(4-chloro-1,3-dimethyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-3-(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-3,7-bis(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-3,5-bis(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-3,6-bis(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-3-(trifluoromethyl)-1H-indole-2-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(cyclohexylcarbonyl)-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(cyclohexylcarbonyl)-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[2-(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-[(propan-2-yloxy)acetyl]-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(cyclohexyloxy)acetyl]-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-(4,4,4-trifluoro-3-methylbutanoyl)-L-leucinamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-methylimidazo[2,1-b][1,3]thiazole-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

N2-[(4-bromo-1-ethyl-3-methyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(2,6-dichlorobenzoyl)-4-methyl-L-leucinamide;

(2S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4,4-dimethyl-1-[3-methyl-N-(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

3-methyl-N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-4-(trifluoromethyl)-L-prolinamide;

(2S,4S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-{3-methyl-N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-{[2-(trifluoromethyl)-1,3-thiazol-5-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(2S)-2-(dimethylamino)-2-phenylacetyl]-4-methyl-L-leucinamide;

N2-[(trans-4-cyanocyclohexyl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide;

N2-[(trans-4-cyanocyclohexyl)carbonyl]-N-{(1R)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide;

N-{(1R)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[2-(cyclohexyloxy)propanoyl]-4-methyl-L-leucinamide;

(2S,4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[N-(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

5-(butan-2-yl)-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(4,5-dichloro-1H-imidazol-2-yl)carbonyl]-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(4,5-dichloro-1H-pyrazol-3-yl)carbonyl]-4-methyl-L-leucinamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-2,3-dimethyl-4H-furo[3,2-b]pyrrole-5-carboxamide;

5-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-pyrrolo[2,3-b]pyridin-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-(trifluoromethyl)-1H-benzimidazol-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-methoxy-1H-pyrrolo[3,2-b]pyridine-2-carboxamide;

5-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-pyrrolo[3,2-b]pyridin-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-fluoro-1H-benzimidazole-2-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[3-(propan-2-yl)-1H-pyrazol-5-yl]carbonyl}-L-leucinamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-fluoro-1H-benzimidazole-2-carboxamide;

5-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-benzimidazole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5,6-difluoro-1H-benzimidazole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[3-(2-methylpropyl)-1H-pyrazol-5-yl]carbonyl}-L-leucinamide;

N2-{[4-(3-chlorophenyl)-1H-imidazol-2-yl]carbonyl}-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide;

N2-[(3-tert-butyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide;

6-bromo-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-benzimidazole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-methyl-1H-benzimidazole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4,5,6,7-tetrahydro-1H-indazol-3-carboxamide;

4,6-dichloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-benzimidazole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-6-(1-methylcyclopropyl)-4-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide;

N2-{[5-(2-chlorophenyl)-4-fluoro-1H-pyrazol-3-yl]carbonyl}-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-2-methyl-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-{[3-(4-methoxyphenyl)-1H-pyrazol-5-yl]carbonyl}-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-{[3-(2-methoxyphenyl)-1H-pyrazol-5-yl]carbonyl}-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-{[4-(4-methoxyphenyl)-1H-imidazol-2-yl]carbonyl}-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[3-(4-methylphenyl)-1H-pyrazol-5-yl]carbonyl}-L-leucinamide;

7-bromo-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-methyl-1H-indole-2-carboxamide;

7-bromo-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

(2S,4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[3-methyl-N-(methylsulfonyl)-L-valyl]piperidine-2-carboxamide;

(2S,4S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[N-(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

(2S,4S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[3-methyl-N-(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

(2S,4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[3-methyl-N-(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

5-[(2S)-butan-2-yl]-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3',3',3'-trifluoro-N-(trifluoroacetyl)-L-isoleucyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-{(2S)-2-cyclohexyl-2-[(trifluoroacetyl)amino]acetyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-{(2S)-2-cyclopentyl-2-[(trifluoroacetyl)amino]acetyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[4-methyl-N-(trifluoroacetyl)-L-leucyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-{(2S)-2-(4,4-difluorocyclogen xyl)-2-[(trifluoroacetyl)amino]acetyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclopentyl-N-(trifluoroacetyl)-L-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclohexyl-N-(trifluoroacetyl)-L-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-leucyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[6,6-difluoro-N-(trifluoroacetyl)-L-norleucyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-{(2S)-4,4,4-trifluoro-2-[(trifluoroacetyl)amino]butanoyl}-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-fluoro-N-(trifluoroacetyl)-L-valyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-{(2S)-2-cyclopropyl-2-[(trifluoroacetyl)amino]acetyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-(3,3-difluorocyclobutyl)-N-(trifluoroacetyl)-L-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-O-(trifluoromethyl)-L-seryl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-{(2S)-2-phenyl-2-[(trifluoroacetyl)amino]acetyl}-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-phenylalanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3,5-difluoro-N-(trifluoroacetyl)-L-phenylalanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-3-(trifluoromethyl)-L-phenylalanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(2,2,2-trifluoroethyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(2S,4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-{(2S)-3-methyl-2-[(trifluoroacetyl)amino]butyl}piperidine-2-carboxamide;

(2S,4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-{(2S)-3-methyl-2-[(2,2,2-trifluoroethyl)amino]butyl}piperidine-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(3,3,3-trifluoropropanoyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-(N-propanoyl-L-valyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(2S,4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[N-(2,2,2-trifluoroethyl)-L-valyl]piperidine-2-carboxamide;

N2-[(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-L-leucinamide;

5-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3-ethyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-cyclohexyl-1H-indole-2-carboxamide;

5-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3-methyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3,5-dimethyl-1H-indole-2-carboxamide;

5-tert-butyl-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-(propan-2-yl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3-ethyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-6-ethyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-ethyl-1H-indole-2-carboxamide;

4-butoxy-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-(trifluoromethoxy)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-6-(diethylamino)-1H-indole-2-carboxamide;

4-bromo-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

5-bromo-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

6-bromo-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3-methyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-6-propoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-7-fluoro-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-7-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-6-fluoro-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-fluoro-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4,5-dimethoxy-1H-indole-2-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-[(4-methyl-1,3-thiazol-5-yl)carbonyl]-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(ethoxycarbonyl)-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(ethoxycarbonyl)-4-methyl-L-leucinamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[2-(trifluoromethyl)-1,3-oxazol-4-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[3-(trifluoromethyl)-1,2-thiazol-4-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[3-(trifluoromethyl)-1,2-oxazol-4-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-{[2-(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-5,5,5-trifluoro-N2-{[2-(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl}-L-norvalinamide;

(4S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-5,5,5-trifluoro-N2-{[2-(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl}-L-leucinamide;

(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-5,5,5-trifluoro-N2-{[2-(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl}-L-leucinamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-3-cyclopentyl-1-oxopropan-2-yl]-2-(trifluoromethyl)-1,3-thiazol-4-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[5-(trifluoromethyl)-1,2-thiazol-4-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[5-(trifluoromethyl)-1,2-oxazol-4-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[2-(trifluoromethyl)-1,3-oxazol-5-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[2-(trifluoromethyl)-1,3-thiazol-5-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-5,5,5-trifluoro-4-methyl-N2-{[2-(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[(2S)-2-methyltetrahydropyrrolidin-2-yl]carbonyl}-L-leucinamide;

N-[(2S,4R)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

N-[(2S,4S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-3-cyclopentyl-1-oxopropan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

5,7-dichloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

5-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-ethyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-cyclohexyl-1H-indole-2-carboxamide;

5-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-methyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3,5-dimethyl-1H-indole-2-carboxamide;

5-tert-butyl-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-(propan-2-yl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-7-(propan-2-yl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-ethyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-6-ethyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-ethyl-1H-indole-2-carboxamide;

4-butoxy-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-(trifluoromethoxy)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-6-(diethylamino)-1H-indole-2-carboxamide;

4-bromo-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

5-bromo-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

6-bromo-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-methyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-6-propoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-methyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-6-methyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-7-fluoro-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-7-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-6-fluoro-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-fluoro-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-6-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4,5-dimethoxy-1H-indole-2-carboxamide;

5-(butan-2-yl)-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-7-(propan-2-yl)-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-methyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-6-methyl-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-6-methoxy-1H-indole-2-carboxamide;

5-(butan-2-yl)-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(2R)-2-cyclohexyl-2-methoxyacetyl]-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(2R)-2-(cyclohexyloxy)propanoyl]-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-(4,4,4-trifluoro-3-methylbutanoyl)-L-leucinamide;

N2-[(trans-4-cyanocyclohexyl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(1-ethyl-4-methyl-1H-pyrazol-5-yl)carbonyl]-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(cyclohexylcarbonyl)-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(cyclohexyloxy)acetyl]-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(3,3-difluorocyclobutyl)acetyl]-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(propan-2-yloxy)acetyl]-L-leucinamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3-methylimidazo[2,1-b][1,3]thiazole-2-carboxamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(2R)-2-cyclohexyl-2-methoxyacetyl]-4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(1-ethyl-4-methyl-1H-pyrazol-5-yl)carbonyl]-4-methyl-L-leucinamide;

N2-[2-chloro-4-(methylsulfonyl)benzoyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(2,6-dichlorobenzoyl)-L-leucinamide;

(1R,2S,5S)-3-[N-(tert-butylsulfonyl)-3-methyl-L-valyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-3-{[(3R)-1-benzyl-5-oxopyrrolidin-3-yl]carbonyl}-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-{[(3R)-5-oxo-1-phenylpyrrolidin-3-yl]carbonyl}-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-3-{[(3R)-1-tert-butyl-5-oxopyrrolidin-3-yl]carbonyl}-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[(3-methylimidazo[2,1-b][1,3]thiazol-2-yl)carbonyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-{[2-(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl}-3-azabicyclo[3.1.0]hexane-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-3-cyclopropyl-1-oxopropan-2-yl]-4-methoxy-1H-indole-2-carboxamide; and

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-3-cyclopropyl-1-oxopropan-2-yl]-1H-indole-2-carboxamide;

or a pharmaceutically acceptable salt thereof.

E30 is a compound according to any one of E1, E45 and E59 selected from the group consisting of

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide;

(2S,4S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[N-(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

(2S,4S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[3-methyl-N-(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-{(2S)-2-cyclohexyl-2-[(trifluoroacetyl)amino]acetyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(2S,4S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-{3-methyl-N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide;

3-methyl-N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-4-(trifluoromethyl)-L-prolinamide; and

(2S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4,4-dimethyl-1-[3-methyl-N-(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

or a pharmaceutically acceptable salt thereof.

E31 is a pharmaceutical composition comprising a pharmaceutically effective amount of a compound according to any one of E1-E30 or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.

E32 is a pharmaceutical composition according to E31, wherein the composition is in an intravenous, subcutaneous, inhalation or oral dosage form

E33 is a pharmaceutical composition according to E31 or E32, wherein the composition is in oral dosage form.

E34 is a pharmaceutical composition according to any of E31-E33, further comprising an additional therapeutic agent.

E35 is a pharmaceutical composition according to any of E31-E34, wherein the pharmaceutical composition further comprises one or more of dexamethasone, azithromycin and remdesivir.

E36 is a method of treating coronavirus infection in a patient, wherein the method comprises administering a therapeutically effective amount of a compound according to any of E1-E30, or a pharmaceutically acceptable salt thereof, to a patient in need thereof.

E37 is a follow-up to E36, where the coronavirus infection is COVID-19.

E38 is a method of treating coronavirus infection in a patient, wherein the method comprises administering a pharmaceutical composition according to any of E31-E35 to a patient in need thereof.

E39 is a follow-up to E38, where the coronavirus infection is COVID-19.

E40 is a method for inhibiting or preventing the replication of the SARS-CoV-2 virus, comprising contacting the 3CL protease of the SARS-CoV-2 coronavirus with a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any of E1-E30.

E41 is a method of inhibiting or preventing replication of the SARS-CoV-2 virus in a patient, comprising administering to a patient in need of inhibition or prevention of replication of the SARS-CoV-2 virus, a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any of E1-E30.

E42 is a use of a compound or a pharmaceutically acceptable salt thereof according to any of E1-E30 for the treatment of coronavirus infection.

E43 is an application of E42 where the coronavirus infection is COVID-19.

E44 is the use of a compound or a pharmaceutically acceptable salt thereof according to any of E1 to E30 for the preparation of a medicinal product used for the treatment of coronavirus infection.

E44a is a use under E44 where the coronavirus infection is COVID-19.

E45 is a compound of formula I’

I';

or a pharmaceutically acceptable salt thereof; wherein R is independently hydroxy or oxo at each occurrence; p is 0, 1 or 2; R1 is selected from the group consisting of C1-C6 alkyl which is optionally substituted with cyano or one to five fluorine atoms; C2-C6 alkynyl; and (C3-C6 cycloalkyl)-C1-C3 alkyl which is optionally substituted with one to two substituents selected from trifluoromethyl and C1-C3 alkyl or one to five fluorine atoms; R2 is hydrogen or R2 and R1 taken together with the nitrogen and carbon atoms to which they are attached are a pyrrolidine or piperidine ring which is optionally substituted with one to four R2a; R2a is at each occurrence independently selected from the group consisting of fluoro, hydroxy, C1-C6 alkyl optionally substituted with one to three fluoro atoms, and C1-C6 alkoxy optionally substituted with one to three fluoro atoms; or two R2a groups attached to adjacent carbon atoms and taken together with the atoms to which they are attached are a fused C3-C6 cycloalkyl which is optionally substituted with one to four R2b; or two R2a groups attached to the same carbon atom and taken together with the atoms to which they are attached are spiro C3-C6 cycloalkyl which is optionally substituted with one to four R2b; R2b in each occurrence is independently selected from fluoro, hydroxy, C1-C3 alkyl optionally independently substituted with one to three fluoro or hydroxy, and C1-C3 alkoxy optionally independently substituted with one to three fluoro or hydroxy; R3 is selected from the group consisting of C1-C8 alkyl, C1-C8 alkoxy, (C1-C6 alkoxy)-C1-C6 alkyl, C2-C6 alkynyl, C2-C6 alkynyloxy, C3-C12 cycloalkyl optionally fused with a 5-6 membered heteroaryl or phenyl, (C3-C12 cycloalkyl)-C1-C6 alkyl, C3-C12 cycloalkoxy, (C3-C12 cycloalkoxy)-C1-C6 alkyl, 4-12 membered heterocycloalkyl which is optionally fused with a 5-6 membered heteroaryl or phenyl and wherein said heterocycloalkyl contains from one to four heteroatoms independently selected from N, O and S(O)n, (4-12 membered heterocycloalkyl)-C1-C6 alkyl, wherein said heterocycloalkyl group contains from one to four heteroatoms independently selected from N, O and S(O)n, C6-C10 aryl optionally fused with a C4-C6 cycloalkyl or a 4-7-membered heterocycloalkyl, (C6-C10 aryl)-C1-C6 alkyl, 5-10-membered heteroaryl containing from one to five heteroatoms independently selected from N, O and S, which is optionally fused with a C5-C6 cycloalkyl; (5-10-membered heteroaryl)-C1-C6 alkyl, wherein the heteroaryl group contains from one to five heteroatoms independently selected from N, O and S; (C6-C10 aryl)-(5-10 membered heteroaryl)-, wherein the heteroaryl group comprises one to five heteroatoms independently selected from N, O and S, (5-10 membered heteroaryloxy)-C1-C6 alkyl, wherein the heteroaryl group comprises one to five heteroatoms independently selected from N, O and S; (5-6 membered heteroaryl)-(5-6 membered heteroaryl)-, wherein each heteroaryl group comprises one to four heteroatoms independently selected from N, O and S; (4-7 membered heterocycloalkyl)-(5-6 membered heteroaryl)-, wherein the heterocycloalkyl group comprises one to three heteroatoms independently selected from N, O and S(O)n and the heteroaryl group comprises one to four heteroatoms independently selected from N, O and S; (5-6-membered heteroaryl)-(4-7-membered heterocycloalkyl)-, wherein the heterocycloalkyl group contains one to three heteroatoms independently selected from N, O and S(O)n and the heteroaryl group contains one to four heteroatoms independently selected from N, O and S; wherein each R3 group is optionally substituted with one to five R4; R4 is at each occurrence independently selected from the group consisting of oxo, halo, hydroxy, cyano, phenyl, benzyl, amino, (C1-C6 alkyl)amino optionally substituted with one to five fluorine atoms, di(C1-C6 alkyl)amino optionally substituted with one to ten fluorine atoms, C1-C6 alkyl optionally substituted with one to five fluorine atoms, C1-C6 alkoxy optionally substituted with one to five fluorine atoms, C1-C3 alkoxy-C1-C3 alkyl optionally substituted with one to five fluorine atoms, C3-C6 cycloalkyl optionally substituted with one to three fluorine atoms or C1-C3 alkyl, C1-C6 alkyl-C(O)NH- optionally substituted with one to five fluorine atoms, C1-C6 alkyl-OC(O)NH-, optionally substituted by one to five fluorine atoms or one R5, C1-C6 alkyl-NHC(O)NH-, optionally substituted by one to five fluorine atoms or one R5, C1-C6 alkyl-S(O)2NH-, optionally substituted by one to five fluorine atoms or one R5, C1-C6 alkyl-C(O)-, optionally substituted by one to five fluorine atoms or one R5, C1-C6 alkyl-S(O)n-, optionally substituted by one to five fluorine atoms or one R5; R5 is selected from phenyl, phenoxy, C3-C6 cycloalkyl, C3-C6 cycloalkoxy, 4-7-membered heterocycloalkyl-, wherein the heterocycloalkyl group contains from one to three heteroatoms independently selected from N, O and S(O)n, and 5-6-membered heteroaryl-, wherein the heteroaryl group contains from one to four heteroatoms independently selected from N, O and S; wherein each R5 is optionally independently substituted with one to three halo, C1-C3 alkyl and C1-C3 alkoxy; and n at each occurrence is independently selected from 0, 1 and 2.

E46 is a compound selected from the group consisting of (2S,4R)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide; (2R,4S)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide; 3-methyl-N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(methylcarbamoyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; methyl {(2S)-1-[(1R,2S,5S)-2-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2-yl}carbamate; and N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide; or a pharmaceutically acceptable salt thereof.

E47 is a method for treating coronavirus infection in a patient, wherein the method comprises administering a therapeutically effective amount of a compound according to any of E45 and E46 or a pharmaceutically acceptable salt thereof to a patient in need thereof.

E48 is a follow-up to E47, where the coronavirus infection is COVID-19.

E49 is a method of treating coronavirus infection in a patient, wherein the method comprises administering a therapeutically effective amount of a compound according to any one of E1-E30 and E45-E46 or a pharmaceutically acceptable salt thereof, wherein an additional therapeutic agent is administered, and the additional therapeutic agent is selected from the group consisting of remdesivir, galidesivir, favilavir/avifavir, molnupiravir, AT-527, AT-301, BLD-2660, favipiravir, camostat, SLV213, emtriktabine/tenofovir, clevudine, dalcetrapib, boceprevir, ABX464, dexamethasone, hydrocortisone, convalescent plasma, gelsolin (Rhu-p65N), regdanvimab (Regkirova), ravulizumab (Ultomiris), VIR-7831/VIR-7832, BRII-196/BRII-198, COVI-AMG/COVI DROPS (STI-2020), bamlanivimab (LY-CoV555), mavrilimab, leronlimab (PRO140), AZD7442, lenzilumab, infliximab, adalimumab, JS 016, STI-1499 (COVIGUARD), lanadelumab (Takhzyro), canakinumab (Ilaris), gimsilumab, otilimab, casirivimab/imdevimab (REGN-Cov2), MK-7110 (CD24Fc/SACCOVID), heparin, apixaban, tocilizumab (Actemra), sarilumab (Kevzara), apilimod dimesylate, DNL758, DC402234, PB1046, dapaglifosine, abivertinib, ATR-002, bemcentinib, acalabrutinib, baricitinib, tofacitinib, losmapimod, famotidine, ritonavir, niclosamide and diminazene.

E50 is the compound (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof.

E50a is the compound (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide.

E51 is a pharmaceutical composition comprising a therapeutically effective amount of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.

E51a is a pharmaceutical composition comprising a therapeutically effective amount of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide together with a pharmaceutically acceptable carrier.

E52 is a method of treating coronavirus infection in a patient, wherein the method comprises administering a therapeutically effective amount of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof to a patient in need of treatment thereof.

E52a is a method of treating coronavirus infection in a patient, wherein the method comprises administering a therapeutically effective amount of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide to a patient in need of treatment thereof.

E53 is a follow-up to E52, where the coronavirus infection is COVID-19.

E53a is a follow-on to E52a, where the coronavirus infection is COVID-19.

E54 is the method according to E52 or E53, wherein (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof, is administered orally.

E54a is the method according to E52a or E53a, wherein (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide is administered orally.

E55 is the method according to E54, wherein 50 mg to 1500 mg of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof, is administered every day.

E55a is the method according to E54a, wherein 50 mg to 1500 mg of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide is administered every day.

E56 is the method according to E55, wherein 380 mg of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof, is administered three times daily.

E56a is the method according to E55a, wherein 380 mg of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof, is administered three times daily.

E57 is the method according to E55, wherein 50 mg to 1500 mg of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof, is administered every day as an oral suspension, capsule or tablet.

E57a is the method according to E55a, wherein 50 to 1500 mg of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof, is administered every day as an oral suspension, capsule or tablet.

E58 is a method similar to E57 where a tablet is administered.

E58a is a method similar to E57a where the tablet is administered.

E59 is a compound of formula I”

I”;

or a solvate or hydrate thereof, or a pharmaceutically acceptable salt of said compound, solvate or hydrate thereof;

Where

R in each case is independently hydroxy or oxo;

q and q’ are each independently chosen from 0, 1 and 2;

p is 0, 1, or 2;

R1 is selected from the group consisting of C1-C6 alkyl which is optionally substituted with cyano or from one to five fluorine atoms; C2-C6 alkynyl; and (C3-C6 cycloalkyl)-C1-C3 alkyl which is optionally substituted with from one to two substituents selected from trifluoromethyl and C1-C3 alkyl, or from one to five fluorine atoms;

R2 is hydrogen or R2 and R1 taken together with the nitrogen and carbon atoms to which they are attached are a pyrrolidine or piperidine ring which is optionally substituted with one to four R2a;

R2a is at each occurrence independently selected from the group consisting of fluoro, hydroxy, C1-C6 alkyl optionally substituted with one to three fluoro atoms, and C1-C6 alkoxy optionally substituted with one to three fluoro atoms; or two R2a groups attached to adjacent carbon atoms and taken together with the atoms to which they are attached are a fused C3-C6 cycloalkyl which is optionally substituted with one to four R2b; or two R2a groups attached to the same carbon atom and taken together with the atoms to which they are attached are spiro C3-C6 cycloalkyl which is optionally substituted with one to four R2b;

R2b in each occurrence is independently selected from fluoro, hydroxy, C1-C3 alkyl optionally independently substituted with one to three fluoro atoms or hydroxy, and C1-C3 alkoxy optionally independently substituted with one to three fluoro atoms or hydroxy;

R3 is selected from the group consisting of C1-C8 alkyl, C1-C8 alkoxy, (C1-C6 alkoxy)-C1-C6 alkyl, C2-C6 alkynyl, C2-C6 alkynyloxy, C3-C12 cycloalkyl optionally fused with a 5-6 membered heteroaryl or phenyl, (C3-C12 cycloalkyl)-C1-C6 alkyl, C3-C12 cycloalkoxy, (C3-C12 cycloalkoxy)-C1-C6 alkyl, 4-12 membered heterocycloalkyl which is optionally fused with a 5-6 membered heteroaryl or phenyl, and wherein said heterocycloalkyl contains from one to four heteroatoms independently selected from N, O and S(O)n, (4-12 membered heterocycloalkyl)-C1-C6 alkyl, wherein said heterocycloalkyl group contains from one to four heteroatoms independently selected from N, O and S(O)n, C6-C10 aryl optionally fused with a C4-C6 cycloalkyl or a 4-7-membered heterocycloalkyl, (C6-C10 aryl)-C1-C6 alkyl, 5-10-membered heteroaryl containing from one to five heteroatoms independently selected from N, O and S, which is optionally fused with a C5-C6 cycloalkyl; (5-10-membered heteroaryl)-C1-C6 alkyl, wherein the heteroaryl group contains from one to five heteroatoms independently selected from N, O and S; (C6-C10 aryl)-(5-10 membered heteroaryl)-, wherein the heteroaryl group comprises one to five heteroatoms independently selected from N, O and S, (5-10 membered heteroaryloxy)-C1-C6 alkyl, wherein the heteroaryl group comprises one to five heteroatoms independently selected from N, O and S; (5-6 membered heteroaryl)-(5-6 membered heteroaryl)-, wherein each heteroaryl group comprises one to four heteroatoms independently selected from N, O and S; (4-7 membered heterocycloalkyl)-(5-6 membered heteroaryl)-, wherein the heterocycloalkyl group comprises one to three heteroatoms independently selected from N, O and S(O)n and the heteroaryl group comprises one to four heteroatoms independently selected from N, O and S; (5-6-membered heteroaryl)-(4-7-membered heterocycloalkyl)-, wherein the heterocycloalkyl group contains one to three heteroatoms independently selected from N, O and S(O)n and the heteroaryl group contains one to four heteroatoms independently selected from N, O and S; wherein each R3 group is optionally substituted with one to five R4;

R4 is at each occurrence independently selected from the group consisting of oxo, halo, hydroxy, cyano, phenyl, benzyl, amino, (C1-C6 alkyl)amino optionally substituted with one to five fluorine atoms, di(C1-C6 alkyl)amino optionally substituted with one to ten fluorine atoms, C1-C6 alkyl optionally substituted with one to five fluorine atoms, (5-6-membered heteroaryl)amino-, wherein the heteroaryl group contains one to four heteroatoms independently selected from N, O and S; (4-7-membered heterocycloalkyl)amino-, wherein the heterocycloalkyl group contains one to three heteroatoms independently selected from N, O and S(O)n, C1-C6 alkoxy optionally substituted with one to five fluorine atoms, C1-C3 alkoxy-C1-C3 alkyl optionally substituted with one to five fluorine atoms, C3-C6 cycloalkyl optionally substituted with one to three fluorine atoms or C1-C3 alkyl, C1-C6 alkyl-C(O)NH-, optionally substituted with one to five fluorine atoms, C1-C6 alkyl-OC(O)NH-, optionally substituted with one to five fluorine atoms or one R5, C1-C6 alkyl-NHC(O)NH-, optionally substituted with one to five fluorine atoms or one R5, C1-C6 alkyl-S(O)2NH-, optionally substituted with one to five fluorine atoms or one R5, C1-C6 alkyl-C(O)-, optionally substituted with one to five fluorine atoms or one R5, C1-C6 alkyl-S(O)n-, optionally substituted with one to five fluorine atoms or one R5;

R5 is selected from phenyl, phenoxy, C3-C6 cycloalkyl, C3-C6 cycloalkoxy, 4-7-membered heterocycloalkyl wherein the heterocycloalkyl group contains one to three heteroatoms independently selected from N, O and S(O)n, and 5-6-membered heteroaryl wherein the heteroaryl group contains one to four heteroatoms independently selected from N, O and S; wherein each R5 is optionally independently substituted with one to three halogen, C1-C3 alkyl and C1-C3 alkoxy; and

n in each case is independently chosen from 0, 1 and 2.

E60 is a compound according to E59, which is (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a solvate or hydrate thereof, or a pharmaceutically acceptable salt of said compound, solvate or hydrate.

E61 is the compound (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, which has the structure

;

or its solvate or hydrate.

E62 is the compound according to E61, which is crystalline (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide.

E63 is the compound according to E62, which is crystalline (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, solid form 1.

E64 is the compound according to E62, which is crystalline (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, solid form 4.

E65 is the compound according to E61, which is amorphous (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide.

E66 is the compound according to E61, which is (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl solvate.

E67 is the compound according to E66, which is crystalline (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl solvate.

E68 is the compound according to E67, which is crystalline (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl solvate, solid form 2.

E69 is a method of treating coronavirus infection in a patient, wherein the method comprises administering a therapeutically effective amount of a compound of any of E61-E68 to a patient in need of treatment therefor.

E70 is a follow-up to E69, where the coronavirus infection is COVID-19.

E71 is a route similar to E70 where the patient is also given ritonavir.

E72 is the method according to E71, wherein the compound according to any of E61-E68 and ritonavir are administered to the patient orally.

E73 is the method according to E72, wherein about 10 mg to about 1500 mg per day of a compound according to any of E61-E68 and about 10 mg to about 1000 mg per day of ritonavir are administered.

E74 is the method according to E73, wherein about 50 mg of a compound according to any one of E61-E68 and about 100 mg of ritonavir each are administered to the patient twice daily.

E75 is a pharmaceutical composition comprising a therapeutically effective amount of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a solvate or hydrate thereof, or a pharmaceutically acceptable salt of said compound, solvate or hydrate together with a pharmaceutically acceptable carrier.

E75a is a pharmaceutical composition comprising a therapeutically effective amount of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a solvate or hydrate thereof together with a pharmaceutically acceptable carrier.

E76 is a pharmaceutical composition according to E75a containing a compound according to any of E62-E68.

E77 is a method according to E69 or E70, wherein about 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg or 750 mg of a compound according to any of E61-E68 is administered to a patient orally twice a day.

E78 is a route similar to E77 where ritonavir is co-administered orally to the patient twice daily.

E79 is the method according to E78, wherein about 300 mg of a compound according to any of E61-E68 and about 100 mg of ritonavir are co-administered to a patient twice daily.

E80 is a compound of E63, which is characterized by a 19F peak with a chemical shift of -73.3±0.1 ppm and 13C peaks with chemical shifts of 31.0±0.1 ppm, 27.9±0.1 ppm and 178.9±0.2 ppm.

E81 is a compound according to E64, which is characterized by one or more peaks selected from the group consisting of a 19F peak with a chemical shift of -73.6±0.1 ppm and 13C peaks at 26.9±0.1 ppm, 21.6±0.1 ppm and 41.5±0.1 ppm.

E82 is the compound N-(Methoxycarbonyl)-3-methyl-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide, which has the structure

;

or its solvate or hydrate.

E83 is the compound according to E82, which is N-(Methoxycarbonyl)-3-methyl-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide.

E84 is a pharmaceutical composition comprising a therapeutically effective amount of N-(Methoxycarbonyl)-3-methyl-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide; or a solvate or hydrate thereof, together with a pharmaceutically acceptable carrier.

E85 is a method for treating coronavirus infection in a patient, wherein the method comprises administering a therapeutically effective amount of compound E82 or E83 to a patient in need of treatment therefor.

E86 is a follow-up to E85, where the coronavirus infection is COVID-19.

E87 is a method according to E85 or E86, where 10 mg to 1500 mg per day of compound E82 or E83 is administered.

E88 is a method according to any of E85-E87 wherein the compound is administered orally.

E89 is a route similar to E88 where 200 mg of the compound is administered twice daily.

E90 is a method of targeting inhibition of SARS-CoV-2 by a compound according to any of E1-E30, E45-E46, E50, E50a, E59-E68 and E80-E83 as a treatment for indications caused by viral infections related to SARS-CoV-2.

E91 is a method for identifying cellular or viral pathways that interfere with the functioning of members that can be used to treat indications caused by SARS-CoV-2 infections by administering a SARS-CoV-2 protease inhibitor compound according to any of E1-E30, E45-E46, E50, E50a, E59-E68 and E80-E83.

E92 is a method of using a SARS-CoV-2 protease inhibitor compound according to any one of E1-E30, E45-E46, E50, E50a, E59-E68 and E80-E83 as a tool for understanding the mechanism of action of other SARS-CoV-2 inhibitors.

E93 is a method of using a SARS-CoV-2 3C-like protease inhibitor compound according to any one of E1-E30, E45-E46, E50, E50a, E59-E68 and E80-E83 to conduct gene profiling experiments to track gene activation or repression for the purpose of identifying inhibitors for the treatment of indications caused by SARS-CoV-2 infections such as COVID-19.

E94 is a pharmaceutical composition for the treatment of COVID-19 in mammals comprising an amount of a SARS-CoV-2 3C-like protease inhibitor compound according to any one of E1-E30, E45-E46, E50, E50a, E59-E68 and E80-E83 that is effective for the treatment of COVID-19, together with a pharmaceutically acceptable carrier.

E95 is a method of treating MERS in a patient, wherein the method comprises administering a therapeutically effective amount of a compound according to any of E1-E30, E45-E46, E50, E50a, E59-E68 and E80-E83 to a patient in need thereof.

E96 is a method of treating MERS in a patient, wherein the method comprises administering a pharmaceutical composition according to any of E31-E35, E51, E51a, E75, E75a, E84 and E94 to a patient in need thereof.

E97 is a method of inhibiting or preventing MERS viral replication comprising contacting the 3CL protease of the SARS-CoV-2 coronavirus with a therapeutically effective amount of a compound according to any one of E1-E30, 45-46, 50, 50a, 59-68 and 80-83.

E98 is a method of inhibiting or preventing MERS viral replication in a patient comprising administering to a patient in need of inhibition or prevention of MERS viral replication a therapeutically effective amount of a compound according to any of E1-E30, 45-46, 50, 50a, 59-68 and 80-83.

E99 use of a compound according to any of E1-E30, 45-46, 50, 50a, 59-68 and 80-83 for the treatment of coronavirus infection.

E100 use as per E99 where the coronavirus infection is COVID-19.

E101 use of a compound according to any of E1-E30, 45-46, 50, 50a, 59-68 and 80-83 in the preparation of a medicinal product.

E102 is a compound according to any one of embodiments E1-E30, or a pharmaceutically acceptable salt thereof, for use as a medicament.

E103 is a compound according to any one of embodiments E1-E30, or a pharmaceutically acceptable salt thereof, for use in a method of treatment, wherein the method is as described in any one of embodiments E36-E41.

Brief description of the drawings

Figure 1: Powder X-ray diffraction pattern of 13, methyl tert-butyl ether solvate, Solid Form 2, from the alternative synthesis of Example 13, methyl tert-butyl ether solvate; preparation of Solid Form 2.

Figure 2: Powder X-ray diffraction pattern of 13, methyl tert-butyl ether solvate, Solid Form 2, from the second alternative synthesis of Example 13, methyl tert-butyl ether solvate; preparation of Solid Form 2

Figure 3: Powder X-ray diffraction pattern of Example 13, Solid Form 1, from recrystallization of Example 13; obtaining Solid Form 1

Figure 4: Single crystal X-ray structure determination of Example 13, Solid Form 1. The ORTEP diagram is shown with substitution parameters at 50% probability.

Figure 5: Superimposition of the powder X-ray diffraction pattern obtained for Example 13, Solid Form 1, from the recrystallization of Example 13; preparation of Solid Form 1 (Figure 3) and the calculated powder X-ray diffraction pattern obtained using the Mercury program from the resolving single crystal X-ray diffraction data of Form 1 (see Single Crystal X-ray Structure Determination of Example 13, Solid Form 1).

Figure 6: Powder X-ray diffraction pattern of Example 13, Solid Form 4, from an alternative recrystallization of Example 13; obtaining Solid Form 4.

Figure 7: Single crystal X-ray structure determination of Example 13, Solid Form 4. ORTEP diagram depicted with substitution parameters at 50% probability.

Figure 8: Superimposition of the powder X-ray diffraction pattern obtained for Example 13, Solid Form 4, from an alternative recrystallization of Example 13; preparation of Solid Form 4 (Figure 6) and a calculated powder X-ray diffraction pattern obtained using the Mercury program from the resolving single crystal X-ray diffraction pattern of Form 4 (see Single Crystal X-ray Structure Determination of Example 13, Solid Form 4).

Figure 9: Powder X-ray diffraction pattern of Example 13, Solid Form 5, from Example 96.

Figure 10: Powder X-ray diffraction pattern of intermediate C16, HCl salt.

Figure 11: Powder X-ray diffraction pattern of intermediate C91.

Figure 12: Single crystal X-ray structure determination of intermediate C91. ORTEP diagram depicted with substitution parameters at 50% probability.

Figure 13: Powder X-ray diffraction pattern of intermediate compound C92.

Figure 14: Powder X-ray diffraction pattern of intermediate compound C42.

Figure 15: Single crystal X-ray structure determination of intermediate C42. ORTEP diagram depicted with substitution parameters at 50% probability.

Detailed description of the invention

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

As used herein, the terms "comprising" and "including" are used in their open-ended, non-limiting sense. As used herein, the term "treating," unless otherwise specified, means reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. For purposes of treating COVID-19, COVID-19 is understood to be a disease caused in patients by infection with the SARS-CoV-2 virus. The SARS-CoV-2 virus includes the initially detected strain of the virus as well as emerging mutant strains, such as, but not limited to, strains such as B.1.1.7 (UK variant), B.1.351 (South Africa variant), P.1 (Brazil variant), and B.1.427/B.1.429 (California variants). As used herein, unless otherwise specified, the term "treatment" refers to the act of treating as "treatment" is defined immediately above.

The term "patient" refers to warm-blooded animals such as, for example, guinea pigs, mice, rats, gerbils, cats, rabbits, dogs, cattle, goats, sheep, horses, monkeys, chimpanzees and humans. With regard to the treatment of COVID-19, the methods of the invention are particularly useful for the treatment of humans.

The term "pharmaceutically acceptable" means that the substance or composition must be compatible, chemically and/or toxicologically, with the other ingredients in the formulation and/or with the mammal being treated with it.

The term "therapeutically effective amount" means an amount of a compound of the present invention that (i) treats or prevents a particular disease, condition, or disorder, (ii) reduces, alleviates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein.

As used herein, the term "alkyl" refers to a straight or branched chain saturated hydrocarbon substituent (i.e., a substituent derived from a hydrocarbon by removal of hydrogen); in one embodiment, containing from one to eight carbon atoms, in another, up to six carbon atoms, and in yet another, up to three carbon atoms. Non-limiting examples of such substituents include methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl, and tert-butyl), pentyl, isoamyl, hexyl, heptyl, octyl, and the like. In another embodiment, containing from one to three carbon atoms and consisting of methyl, ethyl, n-propyl, and isopropyl.

As used herein, the term "alkynyl" refers to a straight or branched chain saturated hydrocarbon substituent that contains a carbon-carbon triple bond (i.e., a substituent derived from a hydrocarbon containing a triple bond by removal of a hydrogen); in one embodiment, containing from two to six carbon atoms. Non-limiting examples of such substituents include prop-2-yn-1-yl, but-3-yn-1-yl, pent-4-yn-1-yl, and hex-5-yn-1-yl.

The term "alkoxy" refers to a linear or branched saturated hydrocarbyl substituent attached to an oxygen radical (i.e., a substituent derived from a hydrocarbon alcohol by removing hydrogen from OH); in one embodiment, containing from one to six carbon atoms. Non-limiting examples of such substituents include methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutoxy, sec-butoxy and tert-butoxy), pentoxy, hexoxy and the like. In another embodiment, containing from one to three carbon atoms and consisting of methoxy, ethoxy, n-propoxy and isopropoxy. An alkoxy group that is attached to an alkyl group is called alkoxyalkyl. An example of an alkoxyalkyl group is methoxymethyl.

The term "alkynyloxy" refers to a linear or branched saturated hydrocarbyl substituent containing a carbon-carbon triple bond attached to an oxygen radical (i.e., a substituent derived from a hydrocarbon alcohol containing a triple bond by removing a hydrogen from an OH group); in one embodiment, containing from three to six carbon atoms. Non-limiting examples of such substituents include propynyloxy, butynyloxy, and pentynyloxy, and the like.

In some cases, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl, cycloalkyl, etc.) is designated by the prefix "Cx-Cy-" or "Cx-y", where x is the minimum and y is the maximum number of carbon atoms in the substituent. Thus, for example, "C1-C8alkyl" or "C1-8alkyl" refers to an alkyl substituent containing from 1 to 8 carbon atoms, "C1-C6alkyl" or "C1-6alkyl" refers to an alkyl substituent containing from 1 to 6 carbon atoms, "C1-C3alkyl" or "C1-3alkyl" refers to an alkyl substituent containing from 1 to 3 carbon atoms. Illustrating further, C3-C6cycloalkyl or C3-6-cycloalkyl refers to a saturated cycloalkyl group containing from 3 to 6 carbon atoms in the ring.

The term "cycloalkyl" refers to a carbocyclic substituent derived by the removal of a hydrogen from a saturated carbocyclic molecule, e.g., one having three to seven carbon atoms. The term "cycloalkyl" includes monocyclic saturated carbocycles. The term "C3-C7cycloalkyl" means a radical of a three- to seven-membered ring system that includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl groups. The term "C3-C6cycloalkyl" means a radical of a three- to six-membered ring system that includes cyclopropyl, cyclobutyl, cyclopentyl, and cycloalkyl groups. Cycloalkyl groups may also be bicyclic or spirocyclic carbocycles. For example, the term "C3-C12cycloalkyl" includes monocyclic carbocycles and bicyclic and spirocyclic groups such as bicyclopentyl, bicyclohexyl, bicycloheptyl, bicyclooctyl, bicyclononyl, spiropentyl, spirohexyl, spiroheptyl, spirooctyl and spirononyl.

The term "C3-C6cycloalkoxy" refers to a three- to six-membered cycloalkyl group attached to an oxygen radical. Examples include cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy.

The term "aryl" refers to a carbocyclic aromatic system. The term "C6-C10aryl" refers to carbocyclic aromatic systems with 3-10 atoms and includes phenyl and naphthyl.

In some cases, the number of atoms in a cyclic substituent containing one or more heteroatoms (e.g., heteroaryl or heterocycloalkyl) is designated by the prefix "x-y-membered," where x is the minimum and y is the maximum number of atoms forming the cyclic group of the substituent. Thus, for example, "4- to 6-membered heterocycloalkyl" refers to a heterocycloalkyl containing from 4 to 6 atoms, including from one to three heteroatoms, in the cyclic portion of the heterocycloalkyl. Similarly, the phrase "5- to 6-membered heteroaryl" refers to a heteroaryl containing from 5 to 6 atoms, and "5- to 10-membered heteroaryl" refers to a heteroaryl containing from 5 to 10 atoms, each of which includes one or more heteroatoms, in the cyclic group of the heteroaryl. In addition, the phrases "5-membered heteroaryl" and "6-membered heteroaryl" refer to a five-membered heteroaromatic ring system and a six-membered heteroaromatic ring system, respectively. The heteroatoms present in these ring systems are selected from N, O, and S.

The term "hydroxy" or "hydroxyl" refers to -OH. When used in conjunction with another term, the prefix "hydroxy" indicates that the substituent to which the prefix is attached is substituted with one or more hydroxy substituents. Compounds bearing a carbon that has one or more hydroxy substituents include, for example, alcohols, enols, and phenol. The terms cyano and nitrile refer to the -CN group. The term "oxo" means oxygen that is attached to a carbon atom by a double bond (i.e., when R4 is oxo, then R4 together with the carbon to which it is attached is the C=O group).

The term "halo" or "halogen" refers to fluorine (which can be depicted as -F), chlorine (which can be depicted as -Cl), bromine (which can be depicted as -Br), or iodine (which can be depicted as -I).

The term "heterocycloalkyl" refers to a substituent derived by the removal of hydrogen from a saturated or partially saturated ring structure containing a total of a stated number of atoms, for example from 4 to 6 ring atoms or from 4 to 12 atoms, wherein at least one of the ring atoms is a heteroatom (i.e., oxygen, nitrogen or sulfur), the remaining ring atoms being independently selected from the group consisting of carbon, oxygen, nitrogen and sulfur. The sulfur may be oxidized [i.e., S(O) or S(O)2] or not. In a group that has a heterocycloalkyl substituent, the ring atom of the heterocycloalkyl substituent that is bonded to the group may be a nitrogen heteroatom or may be a ring carbon atom. Likewise, if the heterocycloalkyl substituent is in turn substituted by a group or substituent, the group or substituent may be bonded to a nitrogen heteroatom or may be bonded to a ring carbon atom. It should be understood that a heterocyclic group can be monocyclic, bicyclic, polycyclic or spirocyclic.

The term "heteroaryl" refers to an aromatic ring structure containing the stated number of ring atoms, wherein at least one of the ring atoms is a heteroatom (i.e., oxygen, nitrogen, or sulfur), and the remaining ring atoms are independently selected from the group consisting of carbon, oxygen, nitrogen, and sulfur. Examples of heteroaryl substituents include 6-membered heteroaryl substituents such as pyridyl, pyrazyl, pyrimidinyl, and pyridazinyl; and 5-membered heteroaryl substituents such as triazolyl, imidazolyl, furanyl, thiophenyl, pyrazolyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, 1,2,3-, 1,2,4-, 1,2,5-, or 1,3,4-oxadiazolyl, and isothiazolyl. The heteroaryl group may also be a bicyclic heteroaromatic group such as indolyl, benzofuranyl, benzothienyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzoisoxazolyl, oxazolopyridinyl, imidazopyridinyl, imidazopyrimidinyl and the like. In a group that has a heteroaryl substituent, the ring atom of the heteroaryl substituent that is bonded to the group may be one of the heteroatoms or may be a carbon atom of the ring. Similarly, if the heteroaryl substituent is in turn substituted with a group or substituent, the group or substituent may be bonded to one of the heteroatoms, or it may be bonded to a carbon atom of the ring. The term "heteroaryl" also includes pyridyl N-oxides and groups containing a pyridine N-oxide ring. In addition, the heteroaryl group may contain an oxo group, such as the group present in the pyridone group. Further examples include furyl, thienyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyridin-2(1H)-onyl, pyridazin-2(1H)-onyl, pyrimidin-2(1H)-onyl, pyrazin-2(1H)-onyl, imidazo[1,2-a]pyridinyl and pyrazolo[1,5-a]pyridinyl. Heteroaryl may be further substituted as defined herein.

Examples of single-ring heteroaryls and heterocycloalkyls include furanyl, dihydrofuranyl, tetrahydrofuranyl, thiophenyl, dihydrothiophenyl, tetrahydrothiophenyl, pyrrolyl, isopyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, isoimidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, triazolyl, tetrazolyl, dithiolyl, oxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, thiaoxadiazolyl, oxathiazolyl, oxadiazolyl (including oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl), pyranyl (including 1,2-pyranyl or 1,4-pyranyl), dihydropyranyl, pyridinyl, piperidinyl, diazinyl (including pyridazinyl, pyrimidinyl, piperazinyl, triazinyl (including sec-triazinyl, as-triazinyl and v-triazinyl), oxazinyl (including 2H-1,2-oxazinyl, 6H-1,3-oxazinyl or 2H-1,4-oxazinyl), isoxazinyl (including o-isoxazinyl or p-isoxazinyl), oxazolidinyl, isoxazolidinyl, oxathiazinyl (including 1,2,5-oxathiazinyl or 1,2,6-oxathiazinyl), oxadiazinyl (including, 2H-1,2,4-oxadiazinyl or 2H-1,2,5-oxadiazinyl), and morpholinyl.

The term "heteroaryl" may also include, when defined as such, ring systems having two rings, where such rings may be fused, and where one ring is aromatic and the other ring is not entirely part of the conjugated aromatic system (i.e., the heteroaromatic ring may be fused to a cycloalkyl or heterocycloalkyl ring). Non-limiting examples of such ring systems include 5,6,7,8-tetrahydroisoquinolinyl, 5,6,7,8-tetrahydroquinolinyl, 6,7-dihydro-5H-cyclopenta[b]pyridinyl, 6,7-dihydro-5H-cyclopenta[c]pyridinyl, 1,4,5,6-tetrahydrocyclopenta[c]pyrazolyl, 2,4,5,6-tetrahydrocyclopenta[c]pyrazolyl, 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazolyl, 6,7-dihydro-5H-pyrrolo[1,2-b][1,2,4]triazolyl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridinyl, 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridinyl, 4,5,6,7-tetrahydro-1H-indazolyl and 4,5,6,7-tetrahydro-2H-indazolyl. It should be understood that if a carbocyclic or heterocyclic group can be linked or otherwise attached to the designated substrate through various ring atoms without specifying a specific point of attachment, then all possible points are intended, whether through a carbon atom or, for example, a trivalent nitrogen atom. For example, the term "pyridyl" means 2-, 3- or 4-pyridyl, the term "thienyl" means 2- or 3-thienyl, and so on.

If substituents are described as "independent", having more than one variable, each substituent is chosen independently of the others from a list of available variables. Therefore, each substituent may be identical or different from another substituent.

When substituents are described as "independently selected" from a group, each substituent is selected independently of the others. Therefore, each substituent may be identical to or different from another substituent.

As used herein, the terms "Formula I", "Formula I'" or "Formula I" may be further referred to as "a compound of the invention", "the present invention" and "a compound of Formula I, I' or I". Such terms are also defined to include all forms of Formula I, I' and I", including hydrates, solvates, isomers, crystalline and non-crystalline forms, isomorphs, polymorphs and metabolites thereof. For example, the compounds of the invention or pharmaceutically acceptable salts thereof may exist in unsolvated and solvated forms. When the solvent or water is tightly associated, the complex will have a well-defined stoichiometry independent of humidity. However, when the solvent or water is loosely associated, as in channel solvates and hygroscopic compounds, the water/solvent content will be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.

The compounds of the invention may exist as clathrates or other complexes. The invention includes complexes such as clathrates, drug-host inclusion complexes, wherein the drug and host are present in stoichiometric or non-stoichiometric amounts. Also included are complexes of the invention comprising two or more organic and/or inorganic components, which may be in stoichiometric or non-stoichiometric amounts. The resulting complexes may be ionized, partially ionized, or non-ionized. A review of such complexes is provided in J. Pharm. Sci., 64 (8), 1269-1288 by Haleblian (August 1975).

The compounds of the invention have asymmetric carbon atoms. The carbon-carbon compounds of the invention may be depicted herein using a solid line ( ), solid wedge ( ) or a dotted wedge ( ). The use of a solid line to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom are included. The use of a solid or dotted wedge to depict bonds to asymmetric carbon atoms means that only the stereoisomer shown is intended to be included. It is possible that compounds of formula I, I', and I" may contain more than one asymmetric carbon atom. In these compounds, the use of a solid line to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers are to be included. For example, unless otherwise indicated, compounds of formula I, I', and I" are intended to exist as enantiomers and diastereomers, or as racemates and mixtures thereof. The use of a solid line to represent bonds to one or more asymmetric carbon atoms in a compound of formula I, I', and I" and the use of a solid or dotted wedge to represent bonds to other asymmetric carbon atoms in the same compound indicates that a mixture of diastereomers is present.

Stereoisomers of formula I, I' and I" include cis and trans isomers, optical isomers such as the R and S enantiomers, diastereomers, geometric isomers, rotational isomers, conformational isomers and tautomers of the compounds of the present invention, including compounds exhibiting more than one type of isomerism; and mixtures thereof (such as racemates and diastereomeric pairs). Also included are acid addition or base addition salts wherein the counterion is optically active, such as D-lactate or L-lysine, or racemic, such as DL-tartrate or DL-arginine.

When any racemate crystallizes, two different types of crystals are possible. The first type is the racemic compound (true racemate) mentioned above, where a single homogeneous crystal form is formed, containing both enantiomers in equimolar amounts. The second type is the racemic mixture or conglomerate, where two crystal forms are formed in equimolar amounts, each containing a single enantiomer.

The compounds of the present invention, such as the compounds of formula I, I' and I", may exhibit the phenomenon of tautomerism; such tautomers are also considered as compounds of the invention. All such tautomeric forms and mixtures thereof are included within the scope of the compounds of formula I, I' and I". The tautomers exist as mixtures of tautomers in solution. In solid form, one tautomer usually predominates. Even though one tautomer may be described, the present invention includes all tautomers of the compounds of formula I, I' and I" and their salts.

As used herein, the phrase "pharmaceutically acceptable salts," unless otherwise indicated, includes salts of acidic or basic groups that may be present in the compounds described herein. The compounds used in the methods of the present invention, which are basic in nature, are capable of forming a wide range of salts with various inorganic and organic acids. Acids which can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those which form non-toxic acid addition salts, i.e. salts containing pharmacologically acceptable anions, such as acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edislate, estolate, esylate, ethyl succinate, fumarate, gluceptate, gluconate, glutamate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandalate, mesylate, methyl sulfate, mucate, napsylate, nitrate, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, theoclate, tosylate, triethiododo and valerate.

With respect to the compounds of the invention used in the methods of the invention, if the compounds also exist in tautomeric forms, the present invention relates to these tautomers and the use of all such tautomers and mixtures thereof.

The present invention also includes compounds and methods for treating coronavirus infections such as COVID-19 and methods for inhibiting SARS-CoV-2 using isotopically labeled compounds that are identical to those listed herein, but wherein one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature. Examples of isotopes that can be included in the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F and 36Cl, respectively. The compounds of the present invention, prodrugs thereof and pharmaceutically acceptable salts of said compounds or said prodrugs that contain the above isotopes and/or isotopes of other atoms are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those incorporating radioactive isotopes such as 3H and 14C, can be used in drug and/or substrate tissue distribution assays. The isotopes tritium, i.e. 3H, and carbon-14, i.e. 14C, are particularly preferred due to their ease of preparation and detection. In addition, substitution with heavier isotopes such as deuterium, i.e. 2H, can provide certain therapeutic advantages due to greater metabolic stability, such as increased half-life in vivo or reduced dosage requirements, and therefore may be preferred in some circumstances. Isotopically labeled compounds used in the methods of the present invention and prodrugs thereof can generally be prepared by following procedures for preparing compounds described in the art, substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

This invention also includes methods using pharmaceutical compositions and methods for treating coronavirus infections, such as COVID-19 infections, by administering prodrugs of the compounds of the invention. Compounds containing free amino, amido, or hydroxy groups can be converted into prodrugs. Prodrugs include compounds in which an amino acid residue or a polypeptide chain of two or more (e.g., two, three, or four) amino acid residues is covalently linked via an ester bond to a hydroxy of the compounds used in the methods of the present invention. Amino acid residues include, but are not limited to, the 20 naturally occurring amino acids commonly designated by three-letter symbols and also include 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine, and methionine sulfone. Additional types of prodrugs are also included. For example, free hydroxy groups can be derivatized with groups including, but not limited to, hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as discussed in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs, sulfonate esters, and sulfate esters of hydroxy groups. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl esters, wherein the acyl group can be an alkyl ester optionally substituted with groups including, but not limited to, ether, amine, and carboxylic acid functional groups, or wherein the acyl group is an amino acid ester as described above, is also encompassed. Prodrugs of this type are described in J. Med. Chem., 1996, 29, 10. Free amines can also be formed as amides, sulfonamides, or phosphonamides. All of these prodrugs can include groups including, but not limited to, ether, amine, and carboxylic acid functional groups.

The compounds of the present invention can be used in the methods of the present invention in combination with other therapeutic agents. For example, administering to a patient infected with the SARS-CoV-2 coronavirus (i.e., a patient with COVID-19) a SARS-CoV-2 coronavirus 3CL protease inhibitor of the invention and an interferon, such as interferon alpha or a pegylated interferon, such as PEG-Intron or Pegasus, can provide greater clinical benefit than dosing with either the interferon or pegylated interferon alone or the SARS-CoV-2 coronavirus inhibitor alone. Other additional agents that can be used in the methods of the present invention include dexamethasone, azithromycin, and remdesivir. Examples of greater clinical benefits can include a greater reduction in COVID-19 symptoms, a faster time to symptom relief, a reduction in lung pathology, a greater reduction in the amount of SARS-CoV-2 coronavirus in the patient (viral load), and a reduction in mortality.

The SARS-CoV-2 coronavirus infects cells expressing P-glycoprotein. Some of the SARS-CoV-2 coronavirus 3CL protease inhibitors of the invention are P-glycoprotein substrates. Compounds that inhibit the SARS-CoV-2 coronavirus that are also P-glycoprotein substrates can be dosed with a P-glycoprotein inhibitor. Examples of P-glycoprotein inhibitors include verapamil, vinblastine, ketoconazole, nelfinavir, ritonavir or cyclosporine. The P-glycoprotein inhibitors act by inhibiting the efflux of the SARS-CoV-2 coronavirus inhibitors of the invention from the cell. Inhibition of P-glycoprotein-based efflux will prevent a decrease in intracellular concentrations of the SARS-CoV-2 coronavirus inhibitor due to P-glycoprotein efflux. Inhibition of P-glycoprotein efflux will result in higher intracellular concentrations of SARS-CoV-2 coronavirus inhibitors. Dosing a patient infected with SARS-CoV-2 coronavirus with the SARS-CoV-2 coronavirus 3CL protease inhibitors of the invention and a P-glycoprotein inhibitor can reduce the amount of SARS-CoV-2 coronavirus 3CL protease inhibitor required to achieve an effective dose by increasing the intracellular concentration of the SARS-CoV-2 coronavirus 3CL protease inhibitor.

Among the agents that can be used to increase the exposure of a compound of the present invention to a mammal are those that can act as inhibitors of at least one isoform of cytochrome P450 (CYP450) enzymes. CYP450 isoforms that can be effectively inhibited include, but are not limited to, CYP1A2, CYP2D6, CYP2C9, CYP2C19, and CYP3A4. Compounds used in the methods of the present invention include compounds that can be substrates of CYP3A4 and are metabolized by CYP3A4. Dosing a SARS-CoV-2 coronavirus-infected patient with a SARS-CoV-2 coronavirus inhibitor that is a CYP3A4 substrate, such as a SARS-CoV-2 coronavirus protease inhibitor 3CL, and a CYP3A4 inhibitor, such as ritonavir, nelfinavir, or delavirdine, will decrease the metabolism of the SARS-CoV-2 coronavirus inhibitor by CYP3A4. This will result in decreased clearance of the SARS-CoV-2 coronavirus inhibitor and increased plasma concentrations of the SARS-CoV-2 coronavirus inhibitor. Decreased clearance and higher plasma concentrations may result in a lower effective dose of the SARS-CoV-2 coronavirus inhibitor.

Additional therapeutic agents that may be used in combination with SARS-CoV-2 inhibitors in the methods of the present invention include the following:

PLpro inhibitors, apilomod, EIDD-2801, ribavirin, valganciclovir, β-thymidine, aspartame, oxprenolol, doxycycline, acetophenazine, iopromide, riboflavin, reproterol, 2,2'-cyclocytidine, chloramphenicol, chlorphenesin carbamate, levodropropizine, cefamandole, floxuridine, tigecycline, pemetrexed, L(+)-ascorbic acid, glutathione, hesperetin, ademetionine, masoprocol, isotretinoin, dantrolene, sulfasalazine antibacterial, silybin, nicardipine, sildenafil, platycodine, chrysin, neohesperidin, baicalin, 9 sugetriol-3,9-diacetate, (-)-epigallocatechin gallate, phairantrin D, 2-(3,4-dihydroxyphenyl)-2-[[2-(3,4-dihydroxyphenyl)-3,4-dihydro-5,7-dihydroxy-2H-1-benzopyran-3-yl]oxy]-3,4-dihydro-2H-1-benzopyran-3,4,5,7-tetrol, 2,2-di(3-indolyl)-3-indolone, (S)-(1S,2R,4aS,5R,8aS)-1-formamido-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)decahydronaphthalen-2-yl 2-amino-3-phenylpropanoate, piceatannol, rosmarinic acid and magnolol.

3CLpro inhibitors, lymecycline, chlorhexidine, alfuzosin, cilastatin, famotidine, almitrine, progabide, nepafenac, carvedilol, amprenavir, tigecycline, montelukast, carminic acid, mimosine, flavin, lutein, cefpiramide, pheneticillin, candoxatril, nicardipine, estradiol, valerate, pioglitazone, conivaptan, telmisartan, doxycycline, oxytetracycline, (1S,2R,4aS,5R,8aS)-1-formamido-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)decahydronaphthalen-2-yl 5-((R)-1,2-dithiolan-3-yl)pentanoate, betulonal, chrysin-7-O-β-glucuronide, andrographizide, (1S,2R,4aS,5R,8aS)-1-formamido-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)decahydronaphthalene-2-yl2-nitrobenzoate, 2β-hydroxy-3,4-secofridelolactone-27-oic acid (S)-(1S,2R,4aS,5R,8aS)-1-formamido-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)decahydronaphthalen-2-yl-2-amino-3-phenylpropanoate, isodecortinol, cerevisterol, hesperidin, neohesperidin, androgranin, 2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylenedecahydronaphthalen-1-yl)ethyl benzoate, cosmosiin, cleistocalton A, 2,2-di(3-indolyl)-3-indolone, biorobin, gnidicin, phyllaemblinol, theaflavin 3,3'-di-O-gallate, rosmarinic acid, kitchenside I, oleanolic acid, stigmast-5-en-3-ol, deacetylcentapicrin and berchemol.

RdRp inhibitors, valganciclovir, chlorhexidine, ceftibuten, fenoterol, fludarabine, itraconazole, cefuroxime, atovaquone, chenodeoxycholic acid, cromolyn, pancuronium bromide, cortisone, tibolone, novobiocin, sibin, idarubicin bromocriptine, diphenoxylate, benzylpennicioryl G, gabigartan etexilate, gnidicin, 2β,30β-dihydroxy-3,4-secofridelolactone-27-lactone, 14-deoxy-11,12-didehydroandrographolide, gniditrin, theaflavin 3,3'-di-O-gallate, (R)-((1R,5aS,6R,9aS)-1,5a-dimethyl-7-methylene-3-oxo-6-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)decahydro-1H-benzo[c]azepin-1-yl)methyl 2-amino-3-phenylpropanoate, 2β-Hydroxy-3,4-secofridelolactone-27-oic acid, 2-(3,4-dihydroxyphenyl)-2-[[2-(3,4-dihydroxyphenyl)-3,4-dihydro-5,7-dihydroxy-2H-1-benzopyran-3-yl]oxy]-3,4-dihydro-2H-1-benzopyran-3,4,5,7-tetrol, phyllaemblicin B, 14-hydroxycyperotundone, andrographiside, 2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-methylenedecahydronaphthalen-1-yl)ethyl benzoate, andrographolide, sugetriol-3,9-diacetate, Baicalin, (1S,2R,4aS,5R,8aS)-1-formamido-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)decahydronaphthalen-2-yl 5-((R)-1,2-dithiolan-3-yl)pentanoate, 1,7-dihydroxy-3-methoxexanthone, 1,2,6-trimethoxy-8-[(6-O-β-D-xylopyranosyl-β-D-glucopyranosyl)oxy]-9H-xanthen-9-one and 1,8-dihydroxy-6-methoxy-2-[(6-O-β-D-xylopyranosyl-β-D-glucopyranosyl)oxy]-9H-xanthen-9-one, 8-(β-D-glucopyranosyloxy)-1,3,5-trihydroxy-9H-xanthen-9-one.

Additional therapeutic agents that can be used in the methods of the present invention include diosmin, hesperidin, MK-3207, venetoclax, dihydroergocristine, bolazine, R428, ditercalinium, etoposide, teniposide, UK-432097, irinotecan, lumacaftor, velpatasvir, eluxadoline, ledipasvir, lopinavir/ritonavir+ribavirin, alferon, and prednisone. Other additional agents used in the methods of the present invention include dexamethasone, azithromycin, and remdesivir, as well as boceprevir, umifenovir, and favipiravir.

Other additional agents that can be used in the methods of the present invention include the α-ketoamide compounds designated 11r, 13a, and 13b shown below, as described in Zhang, L.; Lin, D.; Sun, X.; Rox, K.; Hilgenfeld, R.; X-ray Structure of Main Protease of the Novel Coronavirus SARS-CoV-2 Enables Design of α-Ketoamide Inhibitors; bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952879

Additional agents that may be used in the methods of the present invention include RIG 1 pathway activators such as those described in U.S. Patent No. 9,884,876.

Other additional therapeutic agents include protease inhibitors such as those described in Dai W, Zhang B, Jiang X-M, et al. Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease. Science. 2020;368(6497):1331–1335, including compounds such as those shown below and compounds designated DC402234

.

Another embodiment of the present invention is a method of treating COVID-19 in a patient, wherein in addition to administering a compound of the present invention (i.e., a compound of formula I, I', or I" or a solvate or hydrate thereof, or a pharmaceutically acceptable salt of the compound or solvate or hydrate thereof), an additional agent is administered, and the additional agent is selected from antiviral agents such as remdesivir, galidesivir, favilavir/avifavir, molnupiravir (MK-4482/EIDD 2801), AT-527, AT-301, BLD-2660, favipiravir, camostat, SLV213 emtrictabine/tenofovir, clevudine, dalcetrapib, boceprevir and ABX464, glucocorticoids such as dexamethasone and hydrocortisone, convalescent plasma, recombinant human plasma such as gelsolin (Rhu-p65N), monoclonal antibodies such as regdanvimab (Regkirova), ravulizumab (Ultomiris), VIR-7831/VIR-7832, BRII-196/BRII-198, COVI-AMG/COVI DROPS (STI-2020), bamlanivimab (LY-CoV555), mavrilimab, leronlimab (PRO140), AZD7442, lenzilumab, infliximab, adalimumab, JS 016, STI-1499 (COVIGUARD), lanadelumab (Takhzyro), canakinumab (Ilaris), gimsilumab, and otilimab, antibody cocktails such as casirivimab/imdevimab (REGN-Cov2), recombinant fusion protein such as such as MK-7110 (CD24Fc/SACCOVID), anticoagulants such as heparin and apixaban, IL-6 receptor agonists such as tocilizumab (Actemra) and sarilumab (Kevzara), PIKfyve inhibitors such as apilimod dimesylate, RIPK1 inhibitors such as DNL758, DC10462234, VIP receptor agonists such as PB1046, SGLT2 inhibitors such as dapaglifozine, TYK inhibitors such as abivertinib, kinase inhibitors such as ATR-002, bemcentinib, acalabrutinib, losmapimod, baricitinib, and tofacitinib, H2 blockers such as famotidine, anthelmintics such as niclosamide, furin inhibitors such as diminaschen.

The term “SARS-CoV-2 inhibitory agent” means any SARS-CoV-2 related coronavirus 3C-like protease inhibitor compound described herein, or a pharmaceutically acceptable salt, hydrate, prodrug, active metabolite or solvate thereof, or a compound that inhibits the replication of SARS-CoV-2 by any means.

The term “interfering with or preventing” the replication of a SARS-CoV-2-related coronavirus (“SARS-CoV-2”) in a cell means reducing the replication of SARS-CoV-2 or the production of SARS-CoV-2 components necessary for the progeny of the virus in a cell treated with a compound of the present invention, compared to a cell not treated with a compound of the present invention. Simple and convenient assays for determining a reduction in SARS-CoV-2 viral replication include ELISA for the presence, absence, or reduced presence of anti-SARS-CoV-2 antibodies in the blood of a subject (Nasoff, et al., PNAS 88:5462-5466, 1991), RT-PCR (Yu, et al., in Viral Hepatitis and Liver Disease 574-577, Nishioka, Suzuki and Mishiro (Eds.); Springer-Verlag, Tokyo, 1994). Such methods are well known to those skilled in the art. Alternatively, total RNA from transduced and infected "control" cells can be isolated and analyzed by dot blot or northern blot and probed with SARS-CoV-2-specific DNA to determine whether SARS-CoV-2 replication is reduced. Alternatively, a reduction in SARS-CoV-2 protein expression can also be used as an indicator of inhibition of SARS-CoV-2 replication. A reduction in SARS-CoV-2 replication by more than fifty percent compared to control cells typically quantifies prevention of SARS-CoV-2 replication.

If the SARS-CoV-2 inhibitor compound used in the method of the invention is a base, the desired salt can be prepared by any suitable method known in the art, including treating the free base with an inorganic acid (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like) or with an organic acid (such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidic acid (such as glucuronic acid or galacturonic acid), an alpha hydroxy acid (such as citric acid or tartaric acid), an amino acid (such as aspartic acid or glutamic acid), an aromatic acid (such as benzoic acid or cinnamic acid), a sulfonic acid (such as p-toluenesulfonic acid or ethanesulfonic acid) and the like.

If the SARS-CoV-2 inhibitor compound used in the method of the invention is an acid, the desired salt can be prepared by any suitable method known in the art, including treating the free acid with an inorganic or organic base [such as an amine (primary, secondary or tertiary)], an alkali metal hydroxide or an alkaline earth metal hydroxide. Illustrative examples of suitable salts include organic salts derived from amino acids (such as glycine and arginine), ammonia, primary amines, secondary amines, tertiary amines and cyclic amines (such as piperidine, morpholine and piperazine), as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

In the case of SARS-CoV-2 inhibitor compounds, prodrugs, salts or solvates that are solids, those skilled in the art will appreciate that the compound, prodrugs, salts and solvates used in the method of the present invention may exist in various polymorphic or crystalline forms, all of which are within the scope of the present invention and the formulae provided. In addition, the compound, salts, prodrugs and solvates used in the method of the present invention may exist as tautomers, all of which are intended to be included within the broad scope of the present invention.

Solubilizing agents can also be used with the compounds of the invention to increase the solubility of the compounds in water of physiologically acceptable solutions. These solubilizing agents include cyclodextrins, propylene glycol, diethylacetamide, polyethylene glycol, Tween, ethanol and micelle forming agents. Suggested solubilizing agents are cyclodextrins, especially beta-cyclodextrins and in particular hydroxypropyl beta-cyclodextrin and sulfobutyl ether beta-cyclodextrin.

In some cases, the SARS-CoV-2 inhibitor compounds, salts, prodrugs, and solvates used in the method of the present invention may have chiral centers. When chiral centers are present, the compound, salts, prodrugs, and solvates may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates, and mixtures thereof are intended to be included within the broad scope of the present invention.

As is generally understood by those skilled in the art, an optically pure compound is a compound that is enantiomerically pure. The term "optically pure" as used herein is intended to mean a compound that has at least sufficient activity. Preferably, an optically pure amount of one enantiomer for obtaining a compound having the desired pharmacologically pure compound of the present invention comprises at least 90% of one isomer (80% enantiomeric excess), more preferably at least 95% (90% ee), even more preferably at least 97.5% (95% ee), and most preferably at least 99% (98% ee).

The term "treating" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which the term applies, or one or more symptoms of such condition or disorder. The term "treating" as used herein, unless otherwise indicated, refers to the act of treating, as "treating" is defined immediately above. In a preferred embodiment of the present invention, "treating" or "treating" means at least alleviating a disease state in a human that is alleviated by inhibiting the activity of SARS-COV-2 3C-like protease, which is the primary protease of SARS-COV-2, the causative agent of COVID-19. For patients suffering from COVID-19, fever, fatigue, and dry cough are the main manifestations of the disease, while nasal congestion, runny nose, and other upper respiratory symptoms are rare. Beijing Centers for Disease Control and Prevention indicates that the typical case of COVID-19 causes progressive exacerbation. COVID-19 can be classified into mild, normal, severe, and critical types based on the severity of the disease. National Health Commission of the People’s Republic of China. Diagnosis and Treatment of Pneumonia Caused by 2019-nCoV (Trial Version 4). Available online: http://www.nhc.gov.cn/jkj/s3577/202002/573340613ab243b3a7f61df260551dd4/files/c791e5a7ea5149f680fdcb34dac0f54e.pdf: (1) Mild cases—the clinical symptoms were mild and no pneumonia was detected on chest computed tomography (CT); (2) Normal cases - fever, respiratory symptoms, and were found to have visualized manifestations of pneumonia; (3) Severe cases - one of the following three conditions: respiratory distress, respiratory rate ≥30 times/min (at rest, related to oxygen saturation ≤93%), partial arterial pressure of oxygen (PaO2)/oxygen absorption concentration (FiO2) ≤300 mmHg (1 mmHg = 0.133 kPa); (4) Critical cases - one of the following three conditions: respiratory failure and need for mechanical ventilation, shock, or associated failure of other organs requiring intensive care. Current clinical data show that most deaths occurred in elderly patients. However, severe cases have been reported in young people who have unique factors, particularly chronic diseases such as diabetes or hepatitis B. Those who have been taking hormones or immunosuppressants for a long time and have reduced immune function are likely to be heavily infected.

Methods of treatment for alleviating a disease condition associated with a coronavirus, such as COVID-19, include using one or more compounds of the invention in any conventional manner. According to some preferred embodiments of the invention, the compound or compounds used in the methods of the present invention are administered to a mammal, such as a human, in need thereof. Preferably, the mammal in need thereof is infected with a coronavirus, such as the causative agent of COVID-19, namely SARS-CoV-2.

The present invention also includes prophylactic methods comprising administering an effective amount of a SARS-CoV-2 inhibitor of the invention, or a pharmaceutically acceptable salt, prodrug, pharmaceutically active metabolite or solvate thereof, to a mammal, such as a human, at risk of infection with SARS-CoV-2. According to some preferred embodiments, an effective amount of one or more compounds of the invention, or a pharmaceutically acceptable salt, prodrug, pharmaceutically active metabolite or solvate thereof, is administered to a human at risk of infection with SARS-CoV-2, the causative agent of COVID-19. The prophylactic methods of the invention include using one or more compounds of the invention by any conventional route.

Certain compounds used in the methods of the invention, such as dexamethasone, azithromycin, and remdesivir, are known and can be prepared by methods known in the art.

Recent evidence indicates that the novel coronavirus SARS-CoV-2 is the causative agent of COVID-19. The nucleotide sequence of the SARS-CoV-2 coronavirus, as well as the newly identified L and S subtypes, have recently been determined and made publicly available.

The activity of the inhibitor compounds as inhibitors of SARS-CoV-2 viral activity can be measured by any suitable method available in the art, including in vivo and in vitro assays. The activity of the compounds of the present invention as inhibitors of coronavirus 3C-like protease activity (such as the SARS-CoV-2 coronavirus 3C-like protease) can be measured by any suitable method known to those skilled in the art, including in vivo and in vitro assays. Examples of suitable assays for measuring activity include the antiviral cell culture assays described herein, as well as the antiprotease assays described herein, such as the assays described in the experimental section.

Administration of SARS-CoV-2 inhibitor compounds and pharmaceutically acceptable prodrugs, salts, active metabolites and solvates thereof may be by any of the conventional routes of administration available to those skilled in the art. Illustrative examples of suitable routes of administration include oral, nasal, pulmonary, parenteral, topical, intravenous, injection, transdermal and rectal. Oral, intravenous, subcutaneous and nasal delivery are preferred.

The SARS-CoV-2 inhibitory agent can be administered as a pharmaceutical composition in any suitable pharmaceutical form. Suitable pharmaceutical forms include solid, semi-solid, liquid, or lyophilized formulations such as tablets, powders, capsules, suppositories, suspensions, liposomes, and aerosols. The SARS-CoV-2 inhibitory agent can be prepared as a solution using any of a variety of techniques. For example, the SARS-CoV-2 inhibitory agent can be dissolved in an acid (e.g., 1 M HCl) and diluted with a sufficient volume of 5% dextrose in water (D5W) to obtain the desired final concentration of the SARS-CoV-2 inhibitory agent (e.g., about 15 mM). Alternatively, a D5W solution containing about 15 mM HCl can be used to obtain a solution of the SARS-CoV-2 inhibitory agent at the appropriate concentration. In addition, the SARS-CoV-2 inhibitory agent can be prepared as a suspension using, for example, 1% carboxymethylcellulose (CMC) solution.

Suitable methods for preparing suitable pharmaceutical forms of the pharmaceutical compositions are known or can be routinely determined by those skilled in the art. For example, the pharmaceutical preparations can be prepared according to conventional pharmaceutical chemistry techniques, including steps such as mixing, granulating and pressing, when necessary, for tablet forms, or mixing, filling and dissolving the ingredients, if necessary, to obtain the desired products for intravenous, oral, parenteral, topical, intravaginal, intranasal, intrabronchial, intraocular, intraaural and/or rectal administration.

Typically, a compound of the present invention is administered in an amount effective to treat the condition described herein. The compounds of the invention are administered by any suitable route in the form of a pharmaceutical composition adapted to such route and at a dose effective for the intended treatment. Therapeutically effective doses of the compounds required to treat the progression of a medical condition are readily determined by one of ordinary skill in the art using preclinical and clinical approaches familiar to those skilled in the art.

The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be used, in which the compound enters the bloodstream directly from the mouth.

In another embodiment, the compounds of the invention can also be administered directly into the bloodstream, into a muscle, or into an internal organ. Suitable routes of parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion methods.

In another embodiment, the compounds of the invention can also be administered topically to the skin or mucous membrane, i.e., dermally or transdermally. In another embodiment, the compounds of the invention can also be administered intranasally or by inhalation. In another embodiment, the compounds of the invention can be administered rectally or vaginally. In another embodiment, the compounds of the invention can also be administered directly to the eye or ear.

The dosage regimen of the compounds and/or compositions containing the compounds is based on a variety of factors including the type, age, weight, sex, and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound used. Thus, the dosage regimen can vary widely. Dosage levels of from about 0.01 mg to about 100 mg per kilogram of body weight per day are useful in the treatment of the above conditions. In one embodiment, the total daily dose of a compound of the present invention (administered as a single or divided dose) is typically from about 0.01 to about 100 mg/kg. In another embodiment, the total daily dose of a compound of the present invention is from about 0.1 to about 50 mg/kg, and in another embodiment from about 0.5 to about 30 mg/kg (i.e., mg of compound of the present invention per kg of body weight). In one embodiment, the dosage is from 0.01 to 10 mg/kg/day. In another embodiment, the dosage is from 0.1 to 1.0 mg/kg/day. Unit dosage compositions may contain such amounts or multiples thereof to make up the daily dose. In many cases, administration of the compounds will be repeated multiple times per day (usually not more than 4 times). If desired, multiple doses per day may be used to increase the total daily dose.

For oral administration, the compositions may be presented in the form of tablets containing from about 0.01 mg to about 500 mg of the active ingredient, or, in another embodiment, from about 1 mg to about 100 mg of the active ingredient. For intravenous administration, doses may range from about 0.1 to about 10 mg/kg/min at a constant infusion rate.

Suitable patients according to the present invention include mammal patients. Mammals according to the present invention include, but are not limited to, dogs, cats, cattle, goats, horses, sheep, pigs, rodents, lagomorphs, primates and the like, and include mammals in the womb. In one embodiment, suitable patients are humans. Human patients can be of either sex and at any stage of development.

In another embodiment, the invention includes the use of one or more compounds of the invention for the preparation of a medicament for the treatment of the conditions listed herein.

For the treatment of the conditions mentioned above, the compound of the present invention can be administered as the compound itself. Alternatively, pharmaceutically acceptable salts are suitable for medical use due to their greater solubility in water compared to the parent compound.

In another embodiment, the present invention includes pharmaceutical compositions. Such pharmaceutical compositions comprise a compound of the invention presented with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier includes any suitable dosage form suitable for administration to a patient. The carrier may be solid, liquid, or both, and may be formulated with the compound as a single dose composition, for example, a tablet, which may contain from 0.05% to 95% by weight of the active compounds. The compound of the present invention may be linked to suitable polymers as targeted drug carriers. Other pharmacologically active substances may also be present.

The compounds of the present invention may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such route and in a dose effective for the intended treatment. The active compounds and compositions, for example, may be administered orally, rectally, parenterally or topically.

Oral administration of a solid dosage form can be, for example, presented in discrete units such as hard or soft capsules, pills, cachets, troches or tablets, each containing a predetermined amount of at least one compound of the present invention. In another embodiment, the oral administration can be in the form of a powder or granules. In another embodiment, the oral dosage form is sublingual, such as, for example, a troche. In such a solid dosage form, the compounds of the present invention are typically combined with one or more adjuvants. Such capsules or tablets can contain a controlled release formulation. In the case of capsules, tablets and pills, the dosage form can also contain buffering agents or can be prepared with enteric coatings.

In another embodiment, oral administration can be carried out in a liquid dosage form. Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art (e.g., water). Such compositions can also contain adjuvants such as wetting, emulsifying, suspending, flavoring (e.g., sweetening) and/or perfuming agents.

In another embodiment, the present invention includes a parenteral dosage form. "Parenteral administration" includes, for example, subcutaneous injections, intravenous injections, intraperitoneal injections, intramuscular injections, intrasternal injections and infusions. Injectable preparations (e.g., sterile aqueous or oily injection suspensions) can be formulated according to the known art using suitable dispersing, wetting agents and/or suspending agents.

In another embodiment, the present invention includes a dosage form for topical use. "Topical administration" includes, for example, transdermal administration, such as transdermal patches or iontophoresis devices, intraocular administration, or intranasal or inhalation administration. Compositions for topical use also include, for example, topical gels, sprays, ointments, and creams. The topical formulation may include a compound that enhances the absorption or penetration of the active ingredient through the skin or other affected areas. When the compounds of the present invention are administered by a transdermal device, the administration will be carried out using a patch of either a reservoir type, a porous membrane, or a solid matrix. Exemplary formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages, and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerol, polyethyleneglycol, and propylene glycol. Penetration enhancers may be included; see, for example, J. Pharm. Sci., 88 (10), 955–958, by Finnin and Morgan (October 1999).

Compositions suitable for topical administration to the eye include, for example, eye drops, wherein the compound of the present invention is dissolved or suspended in a suitable carrier. Exemplary formulations suitable for ocular or aural administration may be in the form of drops of a micronized suspension or solution in isotonic pH-adjusted sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g., absorbable gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and particle or vesicle-based systems such as niosomes or liposomes. A polymer such as cross-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, a cellulose polymer such as hydroxypropyl methylcellulose, hydroxyethylcellulose or methylcellulose, or a heteropolysaccharide polymer such as gellan gum may be included along with a preservative such as benzalkonium chloride. Such preparations may also be delivered by iontophoresis.

For intranasal or inhalation administration, the active compounds of the present invention are conveniently delivered in the form of a solution or suspension from a pump-action nebulizer which is squeezed or pumped by the patient, or as an aerosol spray from a pressurized container or nebulizer, using a suitable propellant. Formulations suitable for intranasal administration are typically administered in the form of a dry powder (either alone, as a mixture, such as in a dry mixture with lactose, or as a mixed complex particle, such as mixed with phospholipids such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, nebulizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may contain a bioadhesive agent such as chitosan or cyclodextrin.

In another embodiment, the present invention includes a rectal dosage form. Such a rectal dosage form may be in the form of, for example, a suppository. Cocoa butter is a traditional suppository base, but various alternatives may be used if desired.

Other carrier materials and routes of administration known in the pharmaceutical art may also be used. The pharmaceutical compositions of the present invention may be prepared by any of the well-known pharmaceutical techniques, such as effective formulation techniques and administration procedures. The above considerations regarding effective formulation and administration techniques are well known in the art and are described in standard textbooks. Compounding of drugs is discussed, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

The compounds of the present invention can be used alone or in combination with other therapeutic agents in the treatment of various conditions or disease states. A compound of the present invention and another therapeutic agent can be administered simultaneously (either in the same dosage form or in separate dosage forms) or sequentially. Two or more compounds can be administered simultaneously, concurrently, or sequentially. In addition, concurrent administration can be accomplished by mixing the compounds prior to administration or by administering the compounds at the same time but to different anatomical sites or by different routes of administration. The phrases "concurrent administration," "co-administration," "simultaneous administration," and "administer simultaneously" mean that the compounds are administered in combination.

The present invention includes the use of a combination of a compound of the invention and one or more additional therapeutic agents. If a combination of active agents is administered, they can be administered sequentially or simultaneously, in a separate dosage form or in combination in a single dosage form. Accordingly, the present invention also includes pharmaceutical compositions comprising an amount of: (a) a first agent comprising a compound of the invention or a pharmaceutically acceptable salt of the compound; (b) a second therapeutic agent; and (c) a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention can also include suitable excipients, diluents, carriers and vehicles, as well as other pharmaceutically active agents, depending on the intended use. Solid or liquid pharmaceutically acceptable carriers, diluents, carriers or excipients can be used in the pharmaceutical compositions. Illustrative solid carriers include starch, lactose, calcium sulfate dihydrate, calcium sulfate, sucrose, talc, gelatin, pectin, acacia, magnesium stearate, and stearic acid. Illustrative liquid carriers include syrup, peanut oil, olive oil, saline, and water. The carrier or diluent may include a suitable sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., solution), or non-aqueous or aqueous liquid suspension.

The dose of the pharmaceutical composition may comprise at least a therapeutically effective amount of a SARS-CoV-2 inhibitory agent, and preferably consists of one or more pharmaceutical dosage units. The selected dose may be administered to a mammal, such as a human patient, in need of treatment mediated by inhibition of the activity of a coronavirus related to SARS-CoV-2, by any known or suitable route of dosing, including topical, such as an ointment or cream; oral, rectal, such as a suppository; parenteral, by injection; intravenous; or continuous by intravaginal, intranasal, intrabronchial, intraaural or intraocular infusion.

The phrases "therapeutically effective amount" and "effective amount" are intended to mean an amount of the agent of the invention that, when administered to a mammal in need of treatment, is sufficient to treat the injury or disease state alleviated by inhibition of SARS-CoV-2 virus replication. The amount of a given SARS-CoV-2 inhibitory agent used in the method of the present invention that will be therapeutically effective will vary depending on factors such as the particular SARS-CoV-2 inhibitory agent, the disease state and its severity, the identity and characteristics of the mammal in need thereof, the amount of which can be routinely determined by those skilled in the art.

It should be understood that the actual dosages of the SARS-CoV-2 inhibitory agents used in the pharmaceutical compositions of the present invention will be selected in accordance with the properties of the particular agent used, the particular composition formulated, the route and site of administration, and the host and condition being treated. Optimum dosages for a given set of conditions can be determined by those skilled in the art using routine dosage determination tests. For oral administration, for example, a dosage that can be used is from about 0.01 to about 1000 mg/kg body weight, preferably from about 0.1 to about 500 mg/kg body weight, and even more preferably from about 1 to about 500 mg/kg body weight, with courses of treatment repeated at appropriate intervals. For intravenous administration, a dosage of up to 5 grams per day can be used. Intravenous administration can be intermittent throughout the day or continuous over a 24-hour period.

The terms "cytochrome P450 inhibitory amount" and "cytochrome P450 enzyme activity inhibitory amount" as used herein refer to the amount of a compound required to reduce the activity of cytochrome P450 enzymes or certain cytochrome P450 enzyme isoforms in the presence of such a compound. Whether a particular compound reduces the activity of a cytochrome P450 enzyme and the amount of such compound required to do so can be determined by methods known to those skilled in the art and by methods described herein.

The protein functions required for coronavirus replication and transcription are encoded by the so-called "replicase" gene. Two overlapping polyproteins are translated from this gene and are extensively processed by viral proteases. The C-proximal region is processed at eleven conserved interdomain junctions by the coronavirus core or "3C-like" protease. The name "3C-like" protease derives from certain similarities between the coronavirus enzyme and the well-known picornavirus 3C proteases. These include substrate preferences, the use of cysteine as a nucleophile in the active site in catalysis, and similarities in their putative overall polypeptide folds. Comparison of the amino acid sequence of the SARS-CoV-2-associated coronavirus 3C-like protease with that of other known coronaviruses, such as SARS-CoV, shows that the amino acid sequences share approximately 96% overall homology.

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

The compounds of the present invention can be prepared according to the methods outlined in Reaction Schemes 1-3 below.

The following schemes further illustrate and exemplify the compounds of the present invention and methods for preparing such compounds. It should be understood that the scope of the present invention is in no way limited by the scope of the following examples and preparations. In the following examples, molecules with one chiral center may exist as a single enantiomer or a racemic mixture. These molecules with two or more chiral centers may exist as a single enantiomer, a racemic or other mixture of two enantiomers, or as various mixtures of diastereomers. Such enantiomers, racemates, and diastereomers may be prepared and/or separated by methods known to those skilled in the art. One skilled in the art will appreciate that certain synthetic manipulations may epimerize or racemize a stereocenter, and synthetic conditions may be selected to promote or hinder such epimerization or racemization.

Scheme 1 illustrates a synthetic sequence for the preparation of compounds of formula I as shown, wherein N-BOC methyl ester of formula 1 (WO 2005/113580) is converted to a primary amide of formula 3 (N-BOC is N-tert-butoxycarbonyl). This can be carried out directly, for example by treatment with ammonia (NH3) in a sealed vessel, in a solvent such as methanol or ethanol, for example optionally in the presence of additives such as calcium chloride (CaCl2) or magnesium dimethoxide, Mg(OMe)2.

Scheme 1

The conversion of a compound of formula 1 into a compound of formula 3 can also be carried out by prior conversion into a carboxylic acid of formula 2 (WO 2005/113580). In this case, the compound of formula 2 can be converted into a compound of formula 3 using methods well known to those skilled in the art. For example, a compound of formula 2 can be treated with a reagent such as O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), isobutyl chloroformate, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI) and hydroxybenzotriazole (HOBt) or 1,1'-carbonyldiimidazole (CDI), optionally in the presence of a base such as N,N-diisopropylethylamine (DIEA), 4-methylmorpholine (NMM) or triethylamine (TEA), followed by treatment with NH3, introduced as a gas or as a solution in a reaction-compatible solvent, or an NH3 salt such as ammonium acetate or ammonium chloride in the presence of a base such as N,N-diisopropylethylamine, 4-methylmorpholine or triethylamine. Suitable solvents include, but are not limited to, dichloromethane (CH2Cl2), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), or acetonitrile (CH3CN).

The compound of formula 3 can be deprotected to give an amine of formula 4 using methods well known to those skilled in the art for carrying out such deprotections. Acidic reagents such as hydrochloride, methanesulfonic acid, or trifluoroacetic acid are often used, typically in a reaction-compatible solvent such as CH2Cl2, 1,4-dioxane, 1,2-dichloroethane, or CH3CN. Those skilled in the art will appreciate that the compound of formula 4 is often obtained as an acid addition salt. The compound of formula 4 can then be converted to a compound of formula 6 by treating the compound with an N-protected amino acid of formula 5 under suitable conditions. Such methods are well known to those skilled in the art, and in general standard peptide coupling conditions can be selected.

The compound of formula 6 can be deprotected from N-protection to give an amine of formula 7 using methods well known to those skilled in the art for carrying out such deprotections. Acidic reagents such as hydrochloride, methanesulfonic acid or trifluoroacetic acid are often used, typically in a reaction-compatible solvent such as CH2Cl2, 1,4-dioxane, 1,2-dichloroethane or CH3CN. Those skilled in the art will appreciate that the compound of formula 7 is often obtained as an acid addition salt. The compound of formula 7 can then be converted to a compound of formula 9 by treatment with a carboxylic acid of formula 8 under suitable conditions. Such methods are well known to those skilled in the art. For example, if X = chlorine, the carboxylic acid is known as an acid chloride and the reaction is carried out in the presence of a base to consume the hydrohalide HX produced as a by-product of the reaction. Examples of suitable bases include, but are not limited to, tertiary amines such as 4-methylmorpholine, 2,6-dimethylpyridine, or N,N-diisopropylethylamine, or inorganic bases such as magnesium oxide (MgO), sodium carbonate (Na2CO3), or potassium bicarbonate (KHCO3). Suitable solvents include, but are not limited to, CH2Cl2, DMF, THF, or CH3CN. If X=OH, a reagent or combinations of reagents are typically used to promote the reaction of the carboxylic acid 8. One skilled in the art may choose to use, for example, a carbodiimide reagent such as 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI) or N,N'-dicyclohexylcarbodiimide (DCC), optionally in the presence of an auxiliary nucleophile such as hydroxybenzotriazole (HOBt) or 2-hydroxypyridine-N-oxide (HOPO). Then, if X=OH, one skilled in the art may choose to use reagents that are suitable for forming mixed carboxylic/carboxylic anhydrides such as CDI, isobutyl, or ethyl chloroformate, often in the presence of a base such as described above. Suitable solvents include, but are not limited to, CH2Cl2, THF, or CH3CN. Another approach commonly used by one skilled in the art when X=OH is to treat a carboxylic acid of formula 8 with a carboxylic acid chloride, such as Me3CCOCl, in the presence of a base such as described above to give a mixed carboxylic anhydride of formula R3C(O)O(O)CCMe3. Suitable solvents include, but are not limited to, CH2Cl2, THF, or CH3CN. In many cases, it is possible to use a symmetrical anhydride of the desired carboxylic acid of formula 8 to carry out the reaction, optionally in the presence of a base such as described above, in which case X=O(O)CR3 and therefore the carboxylic acid of formula 8 is R3C(O)O(O)CR3. Suitable solvents include, but are not limited to, CH2Cl2, THF, or CH3CN.

The compound of formula 9 can be converted to the compound of formula I by treatment under dehydration conditions well known to those skilled in the art. Often, this dehydration step can be accomplished using an excess of trifluoroacetic anhydride or phosphorus oxychloride, typically in the presence of a base such as pyridine, N,N-diisopropylethylamine, 4-methylmorpholine, or triethylamine.

One skilled in the art will recognize that N-BOC-protected amino acids of formula 5 are known in the chemical literature, are commercially available, and can be prepared from corresponding known and commercially available amino acids by one skilled in the art using well-known procedures for synthesizing N-protected amino acids. Similarly, one skilled in the art will recognize that carboxylic acid compounds of formula 8 may be known in the chemical literature, and/or commercially available, and/or can be prepared by published methods or in analogy to published methods.

A person skilled in the art will appreciate that the steps of forming the connection in scheme 1 may be performed in a different order with appropriate considerations, for example as shown in scheme 2.

Scheme 2

In Scheme 2, the compound of formula 3 is converted to the compound of formula 10 by treatment with dehydration conditions well known to those skilled in the art. Often, this dehydration step is carried out using an excess of trifluoroacetic anhydride or phosphorus oxychloride, typically in the presence of a base such as pyridine, N,N-diisopropylethylamine, 4-methylmorpholine, or triethylamine. The compound of formula 10 is deprotected from N-protection to give the amine of formula 11 using methods well known to those skilled in the art for carrying out such deprotections. Acidic reagents such as hydrochloride, methanesulfonic acid, or trifluoroacetic acid are often used, typically in a reaction-compatible solvent such as CH2Cl2, 1,4-dioxane, 1,2-dichloroethane, or CH3CN. One skilled in the art will appreciate that a compound of formula 11 is often prepared as an acid addition salt. The compound of formula 11 can then be converted to a compound of formula I by treatment with a compound of formula 12 under suitable conditions. Such methods are well known to one skilled in the art, and in general standard peptide coupling conditions can be selected. Compounds of formula 12 are extremely well known in the chemical literature, and one skilled in the art can choose to prepare any given compound of formula 12 using methods analogous to those described in the chemical literature.

One skilled in the art will appreciate that the bonding steps in Schemes 1 and 2 may be performed in a different order, taking into account relevant factors, such as shown in Scheme 3.

Scheme 3

In Scheme 3, the compound of formula 4 can then be converted to a compound of formula 9 by treatment with a compound of formula 12 under suitable conditions. Such methods are well known to those skilled in the art, and generally standard peptide coupling conditions can be selected. Compounds of formula 12 are extremely well known in the chemical literature, and one skilled in the art can choose to prepare any given compound of formula 12 using methods analogous to those described in the chemical literature. The compound of formula 9 is then converted to a compound of formula I by treatment under dehydration conditions well known to those skilled in the art. Often, this dehydration step can be accomplished using an excess of trifluoroacetic anhydride or phosphorus oxychloride, typically in the presence of a base such as pyridine, N,N-diisopropylethylamine, 4-methylmorpholine, or triethylamine.

One skilled in the art will appreciate that further permutations of the bond formation steps and manipulations of the functional groups in Schemes 1, 2 and 3 may be employed, taking into account the relevant factors. Such permutations in the choice of the order of the steps are well known in the chemical literature, and one skilled in the art may, if desired, consult the chemical literature for further guidance. One skilled in the art will appreciate that other choices of protecting groups and reagents may be made to effect various transformations.

EXAMPLES

Experimental methods

The following illustrates the synthesis of various compounds of the present invention. Additional compounds within the scope of the present invention can be prepared using the methods illustrated in these examples, either alone or in combination with methods generally known in the art. All starting materials in these preparations and examples are either commercially available or can be prepared by methods known in the art or described herein.

All reactions were carried out with continuous stirring under nitrogen or argon unless otherwise noted. When necessary, reaction apparatus were dried under dynamic vacuum using a heat gun and anhydrous solvents (Sure-Seal™ products from Aldrich Chemical Company, Milwaukee, Wisconsin or DriSolv™ products from EMD Chemicals, Gibbstown, NJ). In some cases, commercial solvents were passed through columns packed with 4Å molecular sieves until the following quality control standards for water were achieved: a) <100 ppm for dichloromethane, toluene, N,N-dimethylformamide, and tetrahydrofuran; b) <180 ppm for methanol, ethanol, 1,4-dioxane, and diisopropylamine. For very sensitive reactions, the solvents are further treated with sodium metal, calcium hydride, or molecular sieves and distilled immediately before use. Other commercial solvents and reagents are used without further purification. For reference synthetic procedures in other examples or methods, the reaction conditions (reaction time and temperature) may vary. Typically, the products are dried under vacuum before being used in further reactions or sent for biological testing.

If indicated, reactions are heated by microwave irradiation using a Biotage Initiator or Personal Chemistry Emrys Optimizer microwave. Reaction progress is monitored by thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC), and/or gas chromatography-mass spectrometry (GCMS). TLC is performed on pre-coated silica gel plates with a fluorescence indicator (excitation wavelength 254 nm) and visualized with UV light and/or I2, KMnO4, CoCl2, phosphomolybdic acid, and/or ceric ammonium molybdate spots. LCMS data are acquired on an Agilent 1100 Series with a Leap Technologies autosampler, Gemini C18 columns, acetonitrile/water gradients, and trifluoroacetic acid, formic acid, or ammonium hydroxide modifiers. The column eluate is analyzed using a Waters ZQ mass spectrometer scanning in both positive and negative ion modes from 100 to 1200 Da. Other similar instruments are also used. HPLC data are typically acquired on an Agilent 1100 Series using the specified columns, acetonitrile/water gradients, and trifluoroacetic acid or ammonium hydroxide modifiers. GCMS data are acquired using a Hewlett Packard 6890 oven with an HP 6890 injector, an HP-1 column (12 m × 0.2 mm × 0.33 μm), and helium carrier gas. The sample is analyzed on an HP 5973 mass selective detector scanning from 50 to 550 Da using electron ionization. Cleanup is accomplished by medium-performance liquid chromatography (MPLC) using Isco CombiFlash Companion, AnaLogix IntelliFlash 280, Biotage SP1, or Biotage Isolera One instruments and Isco RediSep or Biotage Snap prepacked silica cartridges. Chiral purification is accomplished by chiral supercritical fluid chromatography (SLC), typically using Berger or Thar instruments; columns such as ChiralPAK-AD, -AS, -IC, Chiralcel-OD, or -OJ columns; and mixtures of CO2 with methanol, ethanol, 2-propanol, or acetonitrile, alone or modified with trifluoroacetic acid or propan-2-amine. UV detection is used to initiate fraction collection. For reference synthesis procedures in other examples or methods, cleanups may vary: in general, the solvents and solvent ratios used for the eluents/gradients are chosen to provide suitable Rf or retention times.

Mass spectrometry data are obtained from LCMS analysis. Mass spectrometry (MS) is performed using atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), electron impact ionization (EI), or electron scattering ionization (ESI) sources. Proton nuclear magnetic spectroscopy (1H NMR) chemical shifts are given in parts per million downfield from tetramethylsilane and are recorded on 300, 400, 500, or 600 MHz Varian, Bruker, or Jeol spectrometers. Chemical shifts are expressed in parts per million (ppm, δ) with reference to the residual peaks of the deuterated solvent (chloroform, 7.26 ppm; CD2HOD, 3.31 ppm; acetonitrile-d2, 1.94 ppm; dimethyl sulfoxide-d5, 2.50 ppm; DHO, 4.79 ppm). Peak shapes are described as follows: s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m, multiplet; brs, broad singlet; viz, visible. Analytical SLC data are typically obtained on a Berger analytical instrument as described above. Optical rotation data are obtained on a PerkinElmer model 343 polarimeter using a 1 dm cell. Microanalyses are performed by Quantitative Technologies Inc. and the data obtained are within 0.4% of the calculated values.

Unless otherwise stated, chemical reactions are carried out at room temperature (approximately 23 degrees Celsius).

Unless otherwise stated, all reagents were obtained commercially and used without further purification or were prepared by methods known in the literature.

The terms concentrated, evaporated, and concentrated in vacuo refer to the removal of solvent under reduced pressure using a rotary evaporator with a bath temperature of less than 60°C. The abbreviations min and h stand for minutes and hours, respectively. The term TLC refers to thin layer chromatography, room temperature or ambient temperature refers to a temperature between 18 and 25°C, GCMS refers to gas chromatography-mass spectrometry, LCMS refers to liquid chromatography-mass spectrometry, UPLC refers to ultra-performance liquid chromatography, HPLC refers to high-performance liquid chromatography, and SFC refers to supercritical fluid chromatography.

Hydrogenation can be performed in a Parr shaker under hydrogen gas pressure or in a Thales-nano H-Cube flow hydrogenation apparatus under full hydrogen and a flow rate of 1-2 ml/min at the set temperature.

Retention times of HPLC, UPLC, LCMS, GCMS and SLC are measured using the methods specified in the procedures.

In some examples, chiral resolution was performed to separate the enantiomers or diastereomers of certain compounds of the invention (in some examples, the separated enantiomers are designated as ENT-1 and ENT-2, according to their elution order; similarly, the separated diastereomers are designated as DIAST-1 and DIAST-2, according to their elution order). In some examples, the optical rotation of an enantiomer is measured using a polarimeter. According to the observed rotation data (or specific rotation data), the enantiomer with clockwise rotation is designated as the (+)-enantiomer, and the enantiomer with counterclockwise rotation is designated as the (-)-enantiomer. Racemic compounds are indicated by either the absence of drawn or described stereochemistry or the presence of (+/-) adjacent to the structure; in this latter case, the stereochemistry indicated represents only one of the two enantiomers that make up the racemic mixture.

The compounds and intermediates described below have been named using the naming convention provided by ACD/ChemSketch 2019.1.1, File Version C05H41, Build 110712 (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada). The naming convention provided by ACD/ChemSketch 2019.1.1 is well known to those skilled in the art, and the naming convention provided by ACD/ChemSketch 2019.1.1 is believed to be generally consistent with the IUPAC (International Union for Pure and Applied Chemistry) recommendations in the Nomenclature of Organic Chemistry and the CAS Index.

Example 1

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (1)

Step 1. Synthesis of methyl (1R,2S,5S)-3-[N-(tert-butoxycarbonyl)-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate (C1).

A 0°C solution of N-(tert-butoxycarbonyl)-L-valine (69.7 g, 321 mmol) in a mixture of acetonitrile and N,N-dimethylformamide (10:1, 1.10 L) was treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 122 g, 321 mmol) then N,N-diisopropylethylamine (127 mL, 729 mmol). The reaction mixture was then stirred for 5 min, methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1,0]hexane-2-carboxylate hydrochloride (60.0 g, 292 mmol) was added and stirring was continued at 0°C for 1 h. The reaction mixture was then diluted with aqueous citric acid (1 N; 50 ml) and water (100 ml), stirred for 2 min, and concentrated in vacuo to approximately half the original volume. The resulting mixture was partitioned between ethyl acetate and water, and the aqueous layer was extracted three times with ethyl acetate. The combined organic layers were then washed three times with water and once with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was stirred in a minimum amount of ethyl acetate and then filtered; the insoluble material was washed with ethyl acetate until it turned white. The combined filtrates were concentrated under reduced pressure and then chromatographed on silica gel (eluent: 1:1 ethyl acetate/heptane) to give C1 as a yellow oil. Yield: 109 g, quantitative. LCMS m/z 369.3 [M+H]+. 1H NMR (400 MHz, chloroform-d ) δ 5.08 (d, J = 9.6 Hz, 1H), 4.45 (s, 1H), 4.11 (dd, J = 9.7, 7.8 Hz, 1H), 3.95 (d, half of the AB quartet, J = 10.1 Hz, 1H), 3.86 (dd, component of the ABX system, J = 10.2, 4.8 Hz, 1H), 3.74 (s, 3H), 2.04-1.93 (m, 1H), 1.50-1.41 (m, 2H), 1.40 (s, 9H), 1.04 (s, 3H), 1.00 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.93 (s, 3H).

Step 2. Synthesis of methyl (1R,2S,5S)-6,6-dimethyl-3-L-valyl-3-azabicyclo[3,1,0]hexane-2-carboxylate, hydrochloride (C2).

A solution of the hydrochloride salt in 1,4-dioxane (4 M; 15 mL, 60 mmol) was added to a 0 °C solution of C1 (1.00 g, 2.71 mmol) in ethyl acetate (50 mL). The reaction mixture was stirred at 0 °C for 2 h, then additional hydrochloride salt in 1,4-dioxane solution (4 M; 10 mL, 40 mmol) was added and stirring was continued at 0 °C for 3 h, then at room temperature for 1 h. The reaction mixture was then treated with a solution of the hydrochloride salt in 1,4-dioxane (4 M; 10 mL, 40 mmol) and methanol (15 mL) and stirred overnight at room temperature. Concentration in vacuo gave C2 as a gum; this product was used for the next chemical work-up without further purification, and the reaction was evaluated as quantitative. LCMS m/z 269.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.24 (br s, 3H), 4.27 (s, 1H), 3.81-3.61 (m, 3H), 3.67 (s, 3H), 2.21-2.06 (m, 1H), 1.63-1.55 (m, 1H), 1.49 (d, component of AB quartet, J=7.6 Hz, 1H), 1.09-0.88 (m, 12H).

Step 3. Synthesis of methyl (1R,2S,5S)-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylate (C3).

Triethylamine (1.55 mL, 11.1 mmol) was added to a 0°C solution of C2 (1.0 g, 3.3 mmol) in dichloromethane (37 mL), followed by dropwise addition of trifluoroacetic anhydride (0.57 mL, 4.0 mmol) over 30 min. The reaction mixture was stirred at 0°C for 30 min, then it was diluted with dichloromethane (100 mL), washed successively with 10% aqueous potassium bisulfate solution (50 mL) and saturated aqueous sodium chloride solution (30 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to afford C3 as a light yellow oil. Yield: 1.2 g, 3.3 mmol, quantitative. LCMS m/z 365.2 [M+H]+. 1H NMR (400 MHz, chloroform-d) δ 7.04 (bd, J=8.8 Hz, 1H), 4.54 (dd, J=8.9, 6.3 Hz, 1H), 4.46 (s, 1H), 3.91 (dd, J=10.1, 5.0 Hz, 1H), 3.80-3.73 (m, 1H), 3.76 (s, 3H), 2.25-2.13 (m, 1H), 1.55-1.47 (m, 2H), 1.09-1.03 (m, 6H), 0.94 (d, J=6.8 Hz, 3H), 0.92 (s, 3H).

Step 4. Synthesis of (1R,2S,5S)-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C4).

Concentrated hydrochloric acid (0.57 mL, 6.6 mmol) was added to a solution of C3 (1.25 g, 3.43 mmol) in a mixture of acetic acid (40.8 mL) and water (8.2 mL). The reaction mixture was heated at 55 °C for 3 days, then it was partitioned between water (50 mL) and ethyl acetate (100 mL). The aqueous layer was extracted with ethyl acetate (2×50 mL), and the combined organic layers were washed with saturated aqueous sodium chloride solution (50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give C4 as a white foam. Yield: 1.00 g, 2.85 mmol, 83%. LCMS m/z 351.2 [M+H]+. 1H NMR (400 MHz, chloroform-d), characteristic peaks: δ 4.56-4.44 (m, 2H), 2.24-2.12 (m, 1H), [1.66 (d, component of AB quartet, J=7.5 Hz) and 1.59-1.47 (m), total 2H], 1.10-1.01 (m, 6H), 0.96-0.91 (m, 6H).

Step 5. Synthesis of tert-butyl {(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}carbamate (C5).

A solution of ammonia in methanol (7.0 M; 150 mL, 1.0 mol) was added to a 0°C solution of methyl N-(tert-butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninate (5.00 g, 17.5 mmol) in methanol (25 mL). The reaction mixture was then stirred at room temperature for 3 days and concentrated in vacuo; the residue was diluted and re-concentrated successively with a mixture of ethyl acetate and heptane (1:1, 4×50 mL) then heptane (50 mL) to give C5 as a solid (5.27 g, quant.) that contained residual solvent. A portion of this product was used in the next step. LCMS m/z 216.2 [(M - 2-methylprop-1-ene)+H]+. 1H NMR (400 MHz, methanol-d4) δ 4.16-3.96 (m, 1H), 3.40-3.27 (m, 2H, predicted; partially obscured by solvent peak), 2.55-2.42 (m, 1H), 2.35 (dddd, J=12.2, 8.6, 6.8, 3.3 Hz, 1H), 2.03 (ddd, J=14.0, 11.0, 4.4 Hz, 1H), 1.93-1.81 (m, 1H), 1.74 (ddd, J=14.2, 10.1, 4.3 Hz, 1H), 1.45 (s, 9H).

Step 6. Synthesis of tert-butyl {(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}carbamate (C6).

2,6-Dimethylpyridine (2 mL, 17 mmol) and trifluoroacetic anhydride (0.94 mL, 6.6 mmol) were added to a 0°C solution of C5 (from the previous step; 1.0 g, ≤3.3 mmol) in dichloromethane (12 mL). The reaction mixture was stirred at room temperature for 1.5 h, where it was treated with hydrochloric acid (1 M; 30 mL) and dichloromethane (60 mL). The organic layer was washed successively with saturated aqueous sodium chloride (30 mL) and saturated aqueous sodium bicarbonate (30 mL), dried over sodium sulfate, and concentrated in vacuo; silica gel chromatography (gradient: 40% to 100% ethyl acetate in heptane) gave C6 as a solid. Yield: 737 mg, 2.91 mmol, 88% over 2 steps. LCMS m/z 254.3 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 4.72 (dd, J=9.3, 6.8 Hz, 1H), 3.39-3.27 (m, 2H, assumed; partially obscured by solvent peak), 2.57-2.46 (m, 1H), 2.36 (dddd, J=12.2, 8.6, 6.3, 3.4 Hz, 1H), 2.21 (ddd, J=13.8, 9.3, 5.6 Hz, 1H), 1.92-1.79 (m, 2H), 1.47 (s, 9H).

Step 7. Synthesis of (2S)-2-amino-3-[(3S)-2-oxopyrrolidin-3-yl]propanenitrile, methanesulfonate (C7).

Methanesulfonic acid (81.2 μL, 1.25 mmol) was added to a solution of C6 (317 mg, 1.25 mmol) in 1,1,1,3,3,3-hexafluoropropan-2-ol (3 mL). The reaction mixture was then stirred at room temperature for 45 min, concentrated in vacuo, then taken up several times in a solvent mixture and reconcentrated: acetonitrile and ethyl acetate (1:1, 2×10 mL) followed by ethyl acetate and heptane (1:1, 2×10 mL). The resulting C7 was obtained as a glass (423 mg), which was free of the nitrile epimer by 1H and 13C NMR analysis. A portion of this product was used in the following reactions without further purification. LCMS m/z 154.2 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 4.78 (t, J=7.3 Hz, 1H), 3.42-3.36 (m, 2H), 2.82-2.68 (m, 1H), 2.70 (s, 3H), 2.50-2.39 (m, 1H), 2.20 (t, J=7.3 Hz, 1H), 2.07-1.80 (m, 2H).

Step 8. Synthesis of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (1).

A mixture of C7 (from the previous step; 98.8 mg, ≤0.292 mmol) and C4 (100 mg, 0.285 mmol) in acetonitrile (1.5 mL) was cooled to 0 °C. O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU, 97%; 112 mg, 0.286 mmol) was added, followed by a solution of 4-methylmorpholine (94.0 μL, 0.855 mmol) in acetonitrile (0.5 mL), and the reaction mixture was stirred at 0 °C for approximately 2 hours. Then saturated aqueous sodium bicarbonate solution (30 ml) was added to the 0°C reaction mixture, followed by dichloromethane (50 ml), and the organic layer was washed with hydrochloric acid (1 M; 30 ml). The combined aqueous layers were extracted with dichloromethane (60 ml), then the combined organic layers were dried over sodium sulfate, concentrated in vacuo, and chromatographed on silica gel (Gradient: 0% to 20% methanol in ethyl acetate). The resulting product was evaluated by NMR and LCMS as contaminated with the epimer product and was then purified by reversed-phase HPLC (Column: Waters Sunfire C18, 19×100 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v); Gradient: 5% - 95% B over 8.54 min, then 95% B for 1.46 min; Flow rate: 25 mL/min) to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (1). Yield: 14.6 mg, 30.1 µmol, 11%. LCMS m/z 486.5 [M+H]+. Retention time: 2.33 minutes (Analytical conditions. Column: Waters Atlantis C18, 4.6×50 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v). Gradient: 5% - 95% B over 4.0 minutes, then 95% B for 1.0 minute. Flow rate: 2 mL/minute).

Alternative C4 synthesis

(1R,2S,5S)-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C4)

Step 1. Synthesis of (1R,2S,5S)-3-[N-(tert-butoxycarbonyl)-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C8).

An aqueous solution of lithium hydroxide (2.0 M; 436 mL, 872 mmol) was added to a solution of C1 (107 g, 290 mmol) in tetrahydrofuran (730 mL). The resulting mixture was then stirred at room temperature for approximately 2 h, diluted with water and ethyl acetate, then treated with 1 M aqueous sodium hydroxide. The aqueous layer was washed with ethyl acetate, and the combined organic layers were extracted three times with 1 M aqueous sodium hydroxide until LCMS analysis showed that C8 had been completely removed from the organic layer. Acidification of the combined aqueous layers to pH 2 was accomplished by the addition of concentrated hydrochloric acid, then the mixture was extracted three times with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated; trituration of the residue with heptane gave C8 as a white solid. Yield: 92.8 g, 262 mmol, 90%. LCMS m/z 355.3 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 4.32 (s, 1H), 4.05 (d, half of AB quartet, J=10.5 Hz, 1H), 4.01 (d, J=9.0 Hz, 1H), 3.88 (dd, component of the ABX system, J=10.4, 5.3 Hz, 1H), 2.03-1.91 (m, 1H), 1.57 (dd, component of the ABX system, J=7.5, 5.2 Hz, 1H), 1.50 (d, half of AB quartet, J=7.5 Hz, 1H), 1.41 (s, 9H), 1.08 (s, 3H), 0.99 (d, J=6.8 Hz, 3H), 0.97-0.94 (m, 6H).

Step 2. Synthesis of (1R,2S,5S)-6,6-dimethyl-3-L-valyl-3-azabicyclo[3,1,0]hexane-2-carboxylic acid, hydrochloride (C9).

To a solution of C8 (82.8 g, 234 mmol) in dichloromethane (230 mL) was added a solution of the hydrochloride in 1,4-dioxane (4.0 M; 409 mL, 1.64 mol). The reaction mixture was stirred overnight at room temperature, then concentrated in vacuo to give C9 as a white foam. This product was used directly in the next step. LCMS m/z 255.3 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 4.42 (s, 1H), 4.05 (d, J=4.8 Hz, 1H), 3.89 (dd, component of the ABX system, J=10.5, 5.2 Hz, 1H), 3.74 (d, half of the AB quartet, J=10.5 Hz, 1H), 2.36-2.25 (m, 1H), 1.62 (dd, component of the ABX system, J=7.5, 5.1 Hz, 1H), 1.57 (d, half of the AB quartet, J=7.6 Hz, 1H), 1.16 (d, J=7.0 Hz, 3H), 1.10 (s, 3H), 1.04 (d, J=6.9 Hz, 3H), 1.01 (c, 3H).

Step 3. Synthesis of (1R,2S,5S)-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C4).

A solution of C9 (from the previous step; ≤234 mmol) in methanol (230 mL) was cooled to 0 °C, treated with triethylamine (66.7 mL, 479 mmol), and stirred for 5 min, then ethyl trifluoroacetate (36.1 mL, 303 mmol) was added slowly. The reaction mixture was then stirred at room temperature for 90 min and concentrated in vacuo. The residue was diluted with water, 1 M aqueous sodium hydroxide, and ethyl acetate, and the resulting organic layer was extracted twice with 1 M aqueous sodium hydroxide. The combined aqueous layers were acidified to pH 2 by adding 1 M hydrochloric acid, then extracted three times with ethyl acetate. The combined organic layers were washed with water and saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated in vacuo to afford C4 as a white foam. Yield: 73.4 g, 210 mmol, 90% over 2 steps. LCMS m/z 351.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.65 (v brs, 1H), 9.82 (d, J=7.7 Hz, 1H), 4.16 (dd, J=9.9, 7.9 Hz, 1H), 4.12 (s, 1H), 3.86 (d, half of AB quartet, J=10.4 Hz, 1H), 3.81 (dd, component of ABX system, J=10.5, 5.0 Hz, 1H), 2.18-2.05 (m, 1H), 1.54 (dd, component of ABX system, J=7.7, 4.6 Hz, 1H), 1.42 (d, half of AB quartet, J=7.5 Hz, 1H), 1.02 (s, 3H), 0.95 (d, J=6.7 Hz, 3H), 0.89 (d, J=6.6 Hz, 3H), 0.84 (s, 3H).

Alternative synthesis of example 1

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (1)

Step 1. Synthesis of methyl 3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninate, methanesulfonate (C10).

Methanesulfonic acid (2.30 mL, 35.4 mmol) was added to a solution of methyl N-(tert-butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninate (10.1 g, 35.3 mmol) in 1,1,1,3,3,3-hexafluoropropan-2-ol (70 mL). The reaction mixture was then stirred at room temperature for 70 min, LCMS analysis showed that the starting material had been converted to C10: LCMS m/z 187.2 [M+H]+. The reaction mixture was concentrated in vacuo and the residue was redissolved twice, then concentrated under reduced pressure in a mixture of acetonitrile and ethyl acetate (1:1, 2×20 mL). The resulting product was placed in a mixture of acetonitrile and ethyl acetate (1:1, 30 mL), concentrated, then redissolved twice in ethyl acetate (2×40 mL) and concentrated. The residue was triturated with ethyl acetate (60 mL) to give C10. Yield: 9.87 g, 35.0 mmol, 99%. 1H NMR (400 MHz, methanol-d4) δ 4.22 (dd, J=9.7, 3.6 Hz, 1H), 3.86 (s, 3H), 3.41-3.36 (m, 2H), 2.84-2.74 (m, 1H), 2.70 (s, 3H), 2.41 (dddd, J=12.3, 8.6, 5.1, 3.6 Hz, 1H), 2.25 (ddd, J=15.1, 4.5, 3.6 Hz, 1H), 1.98 (ddd, J=15.1, 9.6, 9.6 Hz, 1H), 1.87 (dddd, J=12.6, 10.9, 9.2, 9.2 Hz, 1H).

Step 2. Synthesis of methyl N-({(1R,2S,5S)-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexan-2-yl}carbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninate (C11).

To a 0°C solution of C10 (2.76 g, 9.78 mmol) and C4 (3.43 g, 9.79 mmol) in acetonitrile (40 mL) was added 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1.88 g, 9.81 mmol), then pyridine (2.37 mL, 29.3 mmol) was added dropwise. The reaction mixture was stirred at 0°C for 2.25 h, then treated with hydrochloric acid (1 M; 50 mL) and extracted with ethyl acetate (150 mL). The organic layer was washed successively with saturated aqueous sodium chloride (50 mL), saturated aqueous sodium bicarbonate (50 mL), and saturated aqueous sodium chloride (50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was taken up in methyl tert-butyl ether (30 mL) and concentrated under reduced pressure, and the resulting glass was stirred with methyl tert-butyl ether (50 mL) at room temperature overnight. After filtration, the filter cake was washed with methyl tert-butyl ether (3 x 6 mL) to give C11 as a solid which contained significant residual methyl tert-butyl ether by H NMR analysis. A portion of this product was used in the next step. Yield: 3.74 g; corrected for residual methyl tert-butyl ether: 2.94 g, 5.67 mmol, 58%. LCMS m/z 519.5 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 4.55 (dd, J=12.0, 3.8 Hz, 1H), 4.34 (s, 1H), 4.29 (d, J=9.6 Hz, 1H), 3.97 (d, J=3.1 Hz, 2H), 3.74 (s, 3H), 3.37-3.23 (m, 2H, assumed; partially obscured by solvent peak), 2.73-2.62 (m, 1H), 2.32 (dddd, J=12.4, 8.8, 6.7, 2.4 Hz, 1H), 2.21-2.10 (m, 2H), 1.86-1.74 (m, 2H), 1.60 (dt, ABX2 component of the system, J=7.7, 3.1 Hz, 1H), 1.49 (d, half of AB quartet, J=7.6 Hz, 1H), 1.09 (s, 3H), 1.02 (d, J=6.9 Hz, 3H), 0.99-0.95 (m, 6H).

Step 3. Synthesis of (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (C12).

A solution of ammonia in methanol (7.0 M; 5 mL, 40 mmol) was added to a solution of C11 (from the previous step: 205 mg, 0.311 mmol) in methanol (1 mL). The resulting solution was stirred at room temperature for 1.5 h, then a solution of ammonia in methanol (7.0 M; 5 mL, 40 mmol) was added again and stirring was continued overnight. The reaction mixture was then treated a third time with the same amount of ammonia in methanol; after a further 8 h of reaction, it was concentrated in vacuo. The residue was diluted and re-concentrated successively with ethyl acetate (2×20 mL) and a mixture of ethyl acetate and heptane (1:1, 2×20 mL). The resulting product was dissolved in dichloromethane (50 mL), washed with hydrochloric acid (1 M; 30 mL) and saturated aqueous sodium chloride solution (30 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to afford C12 as a solid. Yield: 87 mg, 0.17 mmol, 55%. LCMS m/z 504.5 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 8.68 (d, J=7.9 Hz, <1H, not fully replaced by solvent), 4.44 (ddd, J=11.9, 7.9, 4.0 Hz, 1H), 4.37-4.26 (m, 2H), 4.01 (dd, component of the ABX system, J=10.3, 5.1 Hz, 1H), 3.94 (d, half of the AB quartet, J=10.2 Hz, 1H), 3.39-3.24 (m, 2H, putative; mostly obscured by the solvent peak), 2.72-2.62 (m, 1H), 2.38-2.28 (m, 1H), 2.21-2.08 (m, 2H), 1.90-1.72 (m, 2H), 1.58 (dd, component of the ABX system, J=7.5, 5 Hz, 1H), 1.54 (d, half of the AB quartet, J=7.7 Hz, 1H), 1.08 (s, 3H), 1.02 (d, J=6.7 Hz, 3H), 0.97 (d, J=6.7 Hz, 3H), 0.96 (s, 3H).

Step 4. Synthesis of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (1).

Methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 88.4 mg, 0.371 mmol) was added to a solution of C12 (85.0 mg, 0.17 mmol) in dichloromethane (4.0 mL), and the reaction mixture was stirred at room temperature. After 3 h, methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 20 mg, 84 μmol) was again added; after 30 min, the reaction mixture was diluted with ethyl acetate (60 mL), washed successively with hydrochloric acid (1 M; 30 mL), saturated aqueous sodium bicarbonate (30 mL), and saturated aqueous sodium chloride (30 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was taken up in heptane and re-concentrated followed by purification by silica gel chromatography (Gradient: 0% to 5% methanol in ethyl acetate). (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1,0]hexane-2-carboxamide (1) was isolated as a solid. Yield: 35 mg, 72 μmol, 42%. LCMS m/z 486.5 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 5.04 (dd, J=10.7, 5.4 Hz, 1H), 4.28 (d, J=9.6 Hz, 1H), 4.25 (s, 1H), 4.03-3.94 (m, 2H), 3.35-3.23 (m, 2H, predicted; mostly obscured by solvent peak), 2.72-2.62 (m, 1H), 2.37-2.26 (m, 2H), 2.19-2.08 (m, 1H), 1.93-1.75 (m, 2H), 1.64 (ddd, J=7.6, 4.2, 2.1 Hz, 1H), 1.41 (d, J=7.6 Hz, 1H), 1.09 (s, 3H), 1.02 (d, J=6.8 Hz, 3H), 1.00-0.95 (m, 6H).

Example 2

N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-(pyrrolidin-1-ylacetyl)-L-leucinamide, trifluoroacetate (2)

Step 1. Synthesis of benzyl 4-methyl-L-leucinate, a salt of p-toluenesulfonic acid (C13).

A suspension of 4-methyl-L-leucine (9.5 g, 65 mmol), benzyl alcohol (28.3 g, 262 mmol), and p-toluenesulfonic acid monohydrate (14.9 g, 78.3 mmol) in toluene (200 mL) was heated at reflux overnight; a Dean-Stark trap was used to azeotropically remove the resulting water. The reaction mixture was then concentrated in vacuo, then the residue was diluted with diethyl ether (200 mL) and ethyl acetate (100 mL). The resulting suspension was stirred for 1.5 h and filtered; the filter cake was washed with diethyl ether to give C13 as a white solid. Yield: 24.9 g, 61.1 mmol, 94%. LCMS m/z 236.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.30 (br s, 3H), 7.47 (d, J=8.1 Hz, 2H), 7.44-7.36 (m, 5H), 7.11 (d, J=7.8 Hz, 2H), 5.23 (AB quartet, JAB=12.3 Hz, ΔνAB=13.7 Hz, 2H), 4.02 (dd, J=7.3, 4.5 Hz, 1H), 2.29 (s, 3H), 1.81 (dd, J=14.5, 7.3 Hz, 1H), 1.57 (dd, J=14.5, 4.6 Hz, 1H), 0.90 (s, 9H).

Step 2. Synthesis of benzyl 4-methyl-N-(pyrrolidin-1-ylacetyl)-L-leucinate (C14).

0°C mixture of C13 (800 mg, 1.96 mmol) and pyrrolidin-1-yl acetic acid (254 mg, 1.97 mmol) in N,N-dimethylformamide (4 mL) was treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 746 mg, 1.96 mmol) then with a solution of 4-methylmorpholine (0.496 mL, 4.51 mmol) in dichloromethane (1 mL). The reaction mixture was then stirred at 0°C for 2 h, saturated aqueous sodium bicarbonate solution (30 mL) was added at 0°C; the resulting mixture was extracted with ethyl acetate (2×60 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Purification by silica gel chromatography was carried out twice (gradient: 0% to 20% ethyl acetate in heptane, followed by a second chromatographic purification using 0% to 10% ethyl acetate in heptane) to give C14 as a gum (761 mg). This product was used directly in the next step. LCMS m/z 347.4 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 7.40-7.29 (m, 5H), 5.16 (AB quartet, JAB=12.2 Hz, ΔνAB=11.1 Hz, 2H), 4.56 (dd, J=9.0, 3.1 Hz, 1H), 3.76 (AB quartet, JAB=15.6 Hz, ΔνAB=13.6 Hz, 2H), 3.17-3.06 (m, 4H), 2.03-1.93 (m, 4H), 1.81 (dd, J=14.5, 3.1 Hz, 1H), 1.60 (dd, J=14.5, 9.0 Hz, 1H), 0.95 (s, 9H).

Step 3. Synthesis of 4-methyl-N-(pyrrolidin-1-ylacetyl)-L-leucine (C15).

To a solution of C14 (from the previous step; 760 mg, ≤1.96 mmol) in methanol (5 mL) was added palladium on carbon (76.0 mg). The reaction mixture was stirred at room temperature under hydrogen (50 psi) overnight, then LCMS analysis showed conversion to C15: LCMS m/z 257.4 [M+H]+. The reaction mixture was filtered twice through a 0.15 μm filter, and the filtrate was concentrated in vacuo. The residue was dissolved twice in a mixture of ethyl acetate and heptane (1:1, 2×20 mL), then concentrated under reduced pressure; this gave C15 as a solid (646 mg). Portions of this material were used in subsequent chemical reactions without further purification. 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J=8.3 Hz, 1H), 4.31 (ddd, J=8.9, 8.6, 3.0 Hz, 1H), 3.74-3.60 (m, 2H), 3.00 br (s, 4H), 1.90-1.79 (m, 4H), 1.70 (dd, ABX system component, J=14.3, 3.0 Hz, 1H), 1.56 (dd, ABX system component, J=14.3, 9.2 Hz, 1H), 0.90 (s, 9H).

Step 4. Synthesis of N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-(pyrrolidin-1-ylacetyl)-L-leucinamide, trifluoroacetate (2).

A mixture of C15 (from the previous step; 30 mg, ≤91 μmol) and C7 (from step 7 of Example 1; 35.3 mg, ≤0.104 mmol) in N,N-dimethylformamide (1 mL) was cooled to 0 °C and treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU, 97%; 39.9 mg, 0.102 mmol) followed by a solution of 4-methylmorpholine (28.0 μL, 0.255 mmol) in dichloromethane (0.25 mL). The reaction mixture was then stirred at 0 °C for about 1.5 h, diluted with saturated aqueous sodium bicarbonate (3 mL) at 0 °C and extracted with dichloromethane (4×4 mL). The combined organic layers were concentrated in vacuo and purified by reverse phase HPLC (Column: Waters Sunfire C18, 19×100 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v); Gradient: 5% - 25% B for 8.5 min, then 25% - 95% acetonitrile for 0.5 min, then 95% B for 1.0 min; Flow rate: 25 mL/min) to give N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-(pyrrolidin-1-ylacetyl)-L-leucinamide, trifluoroacetate (2) as gum. Yield: 8.1 mg, 16 µmol, 18% in 3 stages. LCMS m/z 392.6 [M+H]+. Retention time: 1.47 minutes (Analytical conditions. Column: Waters Atlantis C18, 4.6×50 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v). Gradient: 5% - 95% B over 4.0 minutes, then 95% B for 1.0 minute. Flow rate: 2 ml/minute).

Example 3

N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(2,6-dichlorobenzoyl)-4-methyl-L-leucinamide (3)

Step 1. Synthesis of 3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninamide, methanesulfonate (C16).

Methanesulfonic acid (1.83 g, 19 mmol) was added to a solution of C5 (6.13 g, ≤19 mmol) in 1,1,1,3,3,3-hexafluoropropan-2-ol (40 mL). The reaction mixture was stirred at room temperature for 1 h, then it was concentrated in vacuo, resuspended in a mixture of toluene and heptane, and concentrated once more to give a hygroscopic glass (7.47 g). A portion of this product (6.47 g) was diluted and reconcentrated sequentially with the following: a mixture of dichloromethane and ethanol (2:3, 2 × 50 mL); ethyl acetate and ethanol (2:3, 50 mL); ethyl acetate, heptane, and dichloromethane (4:4:1, 2 × 50 mL). The resulting product was dissolved in a mixture of acetonitrile and water (1:1, 22 mL) and lyophilized for 2 days to afford C16 as a glass. Yield: 3.23 g, 12.1 mmol, 73% over 2 steps. LCMS m/z 172.2 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 4.03 (dd, J=9.1, 4.6 Hz, 1H), 3.43-3.35 (m, 2H), 2.82-2.72 (m, 1H), 2.71 (s, 3H), 2.49-2.38 (m, 1H), 2.12-1.96 (m, 2H), 1.94-1.81 (m, 1H).

Step 2. Synthesis of N-(tert-butoxycarbonyl)-4-methyl-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C17).

A 0°C solution of C16 (1.34 g, 5.02 mmol) and N-(tert-butoxycarbonyl)-4-methyl-L-leucine (1.28 g, 5.22 mmol) in N,N-dimethylformamide (7.0 mL) was treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU, 97%; 2.04 g, 5.20 mmol) then with a solution of 4-methylmorpholine (1.43 mL, 13.0 mmol) in dichloromethane (3 mL). The reaction mixture was then stirred at 0°C for 2.25 h, quenched at 0°C by the addition of hydrochloric acid (1 M; 30 mL) and then diluted with dichloromethane (50 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (30 mL) and the combined aqueous layers were extracted with dichloromethane (60 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and suspended/concentrated with heptane (3 x 10 mL). Purification of the residue by silica gel chromatography (Gradient: 0% to 20% methanol in ethyl acetate) gave C17 as a solid. Yield: 1.42 g, 3.56 mmol, 71%. LCMS m/z 399.4 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 6.83 (d, J=7.4 Hz, <1H, not fully replaced by solvent), 4.43 (dd, J=11.2, 4.2 Hz, 1H), 4.11-4.05 (m, 1H), 3.38-3.24 (m, 2H, assumed; partially obscured by solvent peak), 2.52-2.41 (m, 1H), 2.40-2.30 (m, 1H), 2.13 (ddd, J=14.0, 11.2, 4.5 Hz, 1H), 1.91-1.75 (m, 2H), 1.71 (dd, component of the ABX system, J=14.4, 3.2 Hz, 1H), 1.51 (dd, component systems ABX, J=14.4, 9.3 Hz, 1H), 1.45 (s, 9H), 0.97 (s, 9H).

Step 3. Synthesis of 4-methyl-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninamide, methanesulfonate (C18).

Methanesulfonic acid (32.6 µL, 0.502 mmol) was added to a solution of C17 (200 mg, 0.502 mmol) in 1,1,1,3,3,3-hexafluoropropan-2-ol (1.5 mL). The reaction mixture was stirred at room temperature for 40 min, then it was concentrated in vacuo, dissolved in ethyl acetate and concentrated once more to give C18 as a solid (238 mg). Most of this product was used in the next step. LCMS m/z 299.4 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 4.53 (dd, J=10.3, 5.0 Hz, 1H), 3.91 (dd, J=7.6, 5.5 Hz, 1H), 3.41-3.27 (m, 2H, assumed; partially obscured by solvent peak), 2.70 (s, 3H), 2.57-2.47 (m, 1H), 2.41 (dddd, J=12.0, 8.6, 7.0, 3.2 Hz, 1H), 2.15 (ddd, J=14.0, 10.3, 5.0 Hz, 1H), 2.01 (dd, J=14.4, 7.5 Hz, 1H), 1.96-1.85 (m, 1H), 1.78 (dd, J=14.1, 9.1, 5.0 Hz, 1H), 1.59 (dd, J=14.3, 5.5 Hz, 1H), 1.01 (s, 9H).

Step 4. Synthesis of N-(2,6-dichlorobenzoyl)-4-methyl-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C19).

A 0°C suspension of C18 (from the previous step: 234 mg, ≤0.49 mmol) in dichloromethane (2 mL) was treated with triethylamine (170 µL, 1.2 mmol) followed by dropwise addition of a solution of 2,6-dichlorobenzoyl chloride (130 mg, 0.621 mmol) in dichloromethane (0.2 mL). The reaction mixture was stirred at room temperature for 1 h, then diluted with dichloromethane (60 mL), then washed with hydrochloric acid (1 M; 30 mL), then with saturated aqueous sodium bicarbonate (30 mL). The organic layer was dried over sodium sulfate, filtered, concentrated in vacuo, and chromatographed on silica gel (0% to 30% methanol in ethyl acetate) to afford C19. Yield: 120 mg, 0.255 mmol, 52% over 2 steps. LCMS m/z 471.4 (dichlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 8.45 (d, J=7.9 Hz, <1H, not fully replaced by solvent), 7.45-7.35 (m, 3H), 4.59 (dd, J=7.8, 4.5 Hz, 1H), 4.52-4.44 (m, 1H), 3.37-3.24 (m, 2H, assumed; partially obscured by solvent peak), 2.65-2.55 (m, 1H), 2.37 (dddd, J=12.5, 8.8, 6.6, 2.8 Hz, 1H), 2.19 (ddd, J=13.9, 11.3, 4.5 Hz, 1H), 1.91-1.72 (m, 3H), 1.66 (dd, component of the ABX system, J=14.4, 7.8 Hz, 1H), 1.03 (s, 9H).

Step 5. Synthesis of N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(2,6-dichlorobenzoyl)-4-methyl-L-leucinamide (3).

A solution of C19 (90 mg, 0.19 mmol) and 1H-imidazole (33.8 mg, 0.496 mmol) in pyridine (1 mL) was cooled in an acetonitrile/dry ice bath (-35°C). Phosphorus oxychloride (0.100 mL, 1.07 mmol) was added, and the reaction mixture was stirred at -30°C to -20°C. After 30 min, pyridine (2 mL) was added to facilitate stirring; after 1 h, dichloromethane (2 mL) was added for the same reason. After 2 h reaction, phosphorus oxychloride (0.100 ml, 1.07 mmol) was added again and stirring was continued for 30 min at -30 °C, then the reaction mixture was warmed to 0 °C and stirred for a further 40 min. It was then treated with hydrochloric acid (1 M; 30 ml) and extracted with dichloromethane (2 x 60 ml). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo and chromatographed on silica gel (gradient: 0% to 15% methanol in ethyl acetate) to give a solid (67 mg). This product was combined with the product (12 mg) from a similar reaction carried out using C19 (30 mg, 64 μmol) and taken up twice in ethyl acetate (2 x 3 ml), then concentrated under reduced pressure. The residue was stirred with ethyl acetate/heptane (1:3, 4 mL) at room temperature for 40 min and filtered; the filter cake was washed with ethyl acetate/heptane (1:3, 5×2 mL) to give N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(2,6-dichlorobenzoyl)-4-methyl-L-leucinamide (3) as a solid. Combined yield: 70 mg, 0.15 mmol, 59%. LCMS m/z 453.3 (dichlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 7.45-7.34 (m, 3H), 5.05 (dd, J=10.7, 5.4 Hz, 1H), 4.56 (dd, J=7.0, 5.7 Hz, 1H), 3.37-3.23 (m, 2H, assumed; partially obscured by solvent peak), 2.70-2.59 (m, 1H), 2.42-2.29 (m, 2H), 1.95-1.77 (m, 3H), 1.67 (dd, ABX system component, J=14.4, 7.0 Hz, 1H), 1.04 (s, 9H).

Example 4

N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (4)

Step 1. Synthesis of methyl L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninate, hydrochloride (C20).

A solution of methyl N-(tert-butoxycarbonyl)-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninate (see Prior, A. M., et al., Bioorg. Med. Chem. Lett. 2013, 23, 6317–6320; 2.0 g, 5.0 mmol) in a mixture of methanol (2 mL) and a solution of the hydrochloride in ethyl acetate (4 M; 20 mL) was stirred at 25 °C for 1 h. Concentration in vacuo gave C20 as a white solid (1.92 g, quantitative estimate). 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 9.09-8.98 (m, 1H), 8.39 (br s, 3H), 7.69 (s, 1H), 4.44-4.31 (m, 1H), 3.22-3.07 (m, 2H), 2.5-2.38 (m, 1H, assumed; partially obscured by solvent peak), 2.24-2.11 (m, 1H), 2.11-1.99 (m, 1H), 1.78-1.48 (m, 5H), 0.92 (d, J=6.5 Hz, 3H), 0.89 (d, J=6.5 Hz, 3H).

Step 2. Synthesis of methyl N-(4-methoxy-1H-indole-2-carbonyl)-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninate (C21).

O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 494 mg, 1.30 mmol) and N,N-diisopropylethylamine (388 mg, 3.00 mmol) were added to a 0°C solution of C20 (from a smaller scale experiment similar to step 1; 336 mg, ≤0.840 mmol) and 4-methoxy-1H-indole-2-carboxylic acid (159 mg, 0.832 mmol) in N,N-dimethylformamide (6 mL). The solution was stirred at 0°C for 1.5 h, then poured into water/ice (10 mL) and extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (eluent: 10:1 dichloromethane/methanol) gave C21 as a yellow oil. Yield: 380 mg, 0.804 mmol, 97%. LCMS m/z 473.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.59-11.53 (m, 1H), 8.53 (d, J=8.0 Hz, 1H), 8.37 (d, J=8.0 Hz, 1H), 7.65 (s, 1H), 7.37-7.33 (m, 1H), 7.09 (dd, J=8.8 Hz, 1H), 7.00 (d, AB quartet component, J=8.2 Hz, 1H), 6.50 (d, J=7.6 Hz, 1H), 4.56-4.47 (m, 1H), 4.40-4.31 (m, 1H), 3.88 (s, 3H), 3.62 (s, 3H), 3.18-3.05 (m, 2H), 2.41-2.29 (m, 1H), 2.15-2.03 (m, 2H), 1.78-1.49 (m, 5H), 0.93 (d, J=6.3 Hz, 3H), 0.89 (d, J=6.4 Hz, 3H).

Step 3. Synthesis of N-(4-methoxy-1H-indole-2-carbonyl)-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine (C22).

To a stirred mixture of calcium chloride (0.887 g, 7.99 mmol) and sodium hydroxide (0.168 g, 4.20 mmol) in 2-propanol (7 mL) and water (3 mL) was added C21 (1.8 g, 3.8 mmol). The reaction mixture was stirred at 20 °C for 6 h, then it was concentrated in vacuo, diluted with water (4 mL), adjusted to pH 4 with 1 M hydrochloric acid and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered and concentrated in vacuo. Chromatography on silica gel (eluent: 10:1:0.1 dichloromethane/methanol/acetic acid) gave C22 as a yellow solid. Yield: 1.76 g, 3.84 mmol, 100%. LCMS m/z 459.2 [M+H]+. 1H NMR (400 MHz, chloroform-d), characteristic peaks: δ 6.51-6.43 (m, 1H), 4.80-4.66 (m, 1H), 4.60-4.45 (m, 1H), 3.92 (s, 3H), 3.36-3.18 (m, 2H), 2.59-2.44 (m, 1H).

Alternative step 3. Synthesis of N-(4-methoxy-1H-indole-2-carbonyl)-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine (C22).

A solution of C21 (20 mg, 42 μmol) in tetrahydrofuran (0.4 mL) was treated with an aqueous solution containing lithium hydroxide (14.2 mg, 0.593 mmol). The reaction mixture was then stirred at room temperature for 2.5 h, diluted with ethyl acetate, and washed with 10% aqueous potassium bisulfate. The organic layer was then dried over sodium sulfate, filtered, and concentrated in vacuo to afford C22 as a white solid. Yield: 20 mg, quantitative. LCMS m/z 459.2 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 7.27 (s, 1H), 7.14 (dd, component of the ABX system, J=8, 8 Hz, 1H), 7.02 (d, component of the AB quartet, J=8.3 Hz, 1H), 6.50 (d, J=7.7 Hz, 1H), 4.66 (dd, J=9.0, 5.9 Hz, 1H), 4.52 (dd, J=11.7, 3.9 Hz, 1H), 3.92 (s, 3H), 3.30-3.18 (m, 2H), 2.65-2.52 (m, 1H), 2.38-2.26 (m, 1H), 2.21 (ddd, J=14.0, 11.7, 4.1 Hz, 1H), 1.90-1.70 (m, 5H), 1.02 (d, J=6.3 Hz, 3H), 0.99 (d, J=6.3 Hz, 3H).

Step 4. Synthesis of N-(4-methoxy-1H-indole-2-carbonyl)-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C23).

To a 0°C solution of C22 (1.76 g, 3.84 mmol) and ammonium chloride (0.246 g, 4.60 mmol) in N,N-dimethylformamide (15 mL) were added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 1.90 g, 5.00 mmol) and N,N-diisopropylethylamine (1.49 g, 11.5 mmol). The reaction mixture was then stirred at 0 °C for 1.5 h, N,N-diisopropylethylamine (2.3 g, 18 mmol) was used to adjust the pH to 8. The reaction mixture was stirred for a further 30 min, then it was poured into a mixture of hydrochloric acid (1 M; 20 ml, 20 mmol) and ice. The resulting mixture was extracted with ethyl acetate (3 x 10 ml); the combined organic layers were washed successively with hydrochloric acid (1 M; 10 ml) and saturated aqueous sodium chloride solution (10 ml), dried over sodium sulfate, filtered, concentrated in vacuo, and purified by chromatography on silica gel (eluent: dichloromethane/methanol 10:1) to give C23 as a yellow solid. Yield: 1.09 g, 2.38 mmol, 62%. LCMS m/z 458.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.62-11.55 (m, 1H), 8.42 (d, J=7.9 Hz, 1H), 8.04 (d, J=8.4 Hz, 1H), 7.60 (br s, 1H), 7.38-7.26 (m, 2H), 7.10 (dd, component of the ABX system, J=8.8 Hz, 1H), 7.06 (br s, 1H), 7.00 (d, component of the AB quartet, J=8.2 Hz, 1H), 6.51 (d, J=7.7 Hz, 1H), 4.54-4.41 (m, 1H), 4.34-4.22 (m, 1H), 3.88 (s, 3H), 3.17-3.01 (m, 2H), 2.31-1.95 (m, 3H), 1.76-1.45 (m, 5H), 0.92 (d, J=6.1 Hz, 3H), 0.88 (d, J=6.3 Hz, 3H).

Step 5. Synthesis of N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (4).

To a 0°C mixture of C23 (500 mg, 1.09 mmol) and N,N-diisopropylethylamine (565 mg, 4.37 mmol) in tetrahydrofuran (8 mL) was added 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% by weight in ethyl acetate; 2.78 g, 4.37 mmol). The reaction mixture was then stirred at 50°C for 3 h, concentrated in vacuo, diluted with water (5 mL), and extracted with ethyl acetate (3×5 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo; Purification by silica gel chromatography (eluent: 10:1 dichloromethane/methanol) followed by reverse-phase HPLC (Column: YMC-Actus Triart C18, 50×250 mm, 7 μm; Mobile phase A: water containing 0.225% formic acid; Mobile phase B: acetonitrile; Gradient: 18% - 58% B; Flow rate: 25 ml/min) afforded N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (4) as a yellow solid. Yield: 130 mg, 0.296 mmol, 27%. LCMS m/z 440.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.58 (br s, 1H), 8.90 (d, J=8.0 Hz, 1H), 8.47 (d, J=7.7 Hz, 1H), 7.71 (br s, 1H), 7.38-7.35 (m, 1H), 7.09 (dd, component of the ABX system, J=8.8 Hz, 1H), 7.00 (d, component of the AB quartet, J=8.2 Hz, 1H), 6.51 (d, J=7.7 Hz, 1H), 5.02-4.93 (m, 1H), 4.49-4.40 (m, 1H), 3.88 (s, 3H), 3.19-3.05 (m, 2H), 2.41-2.29 (m, 1H), 2.20-2.06 (m, 2H), 1.85-1.62 (m, 4H), 1.58-1.47 (m, 1H), 0.94 (d, J=6.3 Hz, 3H), 0.89 (d, J=6.3 Hz, 3H).

Alternative synthesis of example 4

N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (4)

Step 1. Synthesis of N-[(4-methoxy-1H-indol-2-yl)carbonyl]-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C23).

A solution of ammonia in methanol (7.0 M, 21 mL, 150 mmol) was added to a solution of C21 (500 mg, 1.06 mmol) in methanol (2.0 mL). The reaction mixture was then stirred at room temperature for 6 h, and a solution of ammonia in methanol (7.0 M, 7.0 mL, 49 mmol) was added again and stirring was continued overnight. A solution of ammonia in methanol (7.0 M, 7.0 mL, 49 mmol) was added again and stirring was continued for 24 h, then a final workup was carried out with a solution of ammonia in methanol (7.0 M, 7.0 mL, 49 mmol). The reaction mixture was stirred for another day, then it was concentrated in vacuo. The residue was combined with the product of a similar reaction (350 mg from a recovered 512 mg) using C21 (500 mg, 1.06 mmol), and the mixture was redissolved in ethyl acetate (5×10 mL) and concentrated under reduced pressure to give C23 (835 mg). This product was used directly in the next step. LCMS m/z 458.4 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 7.29 (d, J=0.9 Hz, 1H), 7.15 (dd, component of the ABX system, J=8, 8 Hz, 1H), 7.03 (bd, component of the AB quartet, J=8.3 Hz, 1H), 6.51 (d, J=7.7 Hz, 1H), 4.59 (dd, J=9.7, 5.0 Hz, 1H), 4.45 (dd, J=11.3, 4.2 Hz, 1H), 3.93 (s, 3H), 3.34-3.19 (m, 2H, assumed; partially obscured by the solvent peak), 2.57-2.47 (m, 1H), 2.31 (dddd, J=12.6, 8.5, 6.8, 2.8 Hz, 1H), 2.15 (ddd, J=14.0, 11.4, 4.6 Hz, 1H), 1.88-1.67 (m, 5H), 1.02 (d, J=6.1 Hz, 3H), 0.98 (d, J=6.1 Hz, 3H).

Step 2. Synthesis of N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (4).

A solution of C23 (from the previous step; 835 mg, ≤1.78 mmol) and 1H-imidazole (323 mg, 4.74 mmol) in a mixture of pyridine (4 mL) and dichloromethane (4 mL) was cooled to -35 °C using an acetonitrile/dry ice bath, then phosphorus oxychloride (0.956 mL, 10.2 mmol) was added dropwise over 5 min. The reaction mixture was stirred at -30 °C to -20 °C for about 1.5 h, then treated with hydrochloric acid (1 M; 50 mL) and stirred for 1 h. After extraction with dichloromethane (3×60 mL), the resulting organic layers were combined, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was combined with purified 4 from another crop (75 mg, 0.17 mmol) and chromatographed on silica gel (Gradient: 0% to 5% methanol in ethyl acetate) to give 4 as a solid (800 mg). This product was combined with the product (80 mg) from a similar reaction carried out using C23 (161 mg, 0.352 mmol); The resulting product was stirred in diethyl ether (25 mL) for 3 days and then filtered. The filter cake was washed with a mixture of diethyl ether and heptane (1:1, 4×2 mL) to give N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (4) as a solid. Combined yield: 519 mg, 1.18 mmol, approximately 50% over 2 steps. LCMS m/z 440.5 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.57 (d, J=2.3 Hz, 1H), 8.90 (d, J=8.1 Hz, 1H), 8.46 (d, J=7.7 Hz, 1H), 7.70 (s, 1H), 7.37 (d, J=2.3 Hz, 1H), 7.10 (dd, component of the ABX system, J=8, 8 Hz, 1H), 7.00 (d, component of the AB quartet, J=8.2 Hz, 1H), 6.51 (d, J=7.7 Hz, 1H), 5.03-4.92 (m, 1H), 4.51-4.39 (m, 1H), 3.88 (s, 3H), 3.19-3.05 (m, 2H), 2.42-2.30 (m, 1H), 2.20-2.06 (m, 2H), 1.80 (ddd, J=13.2, 9.3, 6.7 Hz, 1H), 1.75-1.63 (m, 3H), 1.58-1.47 (m, 1H), 0.94 (d, J=6.2 Hz, 3H), 0.89 (d, J=6.2 Hz, 3H).

Examples 5 and 6

N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-3-(trifluoromethyl)-1H-indole-2-carboxamide (5) and N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide (6)

To a pressure-release tube containing zinc(II) trifluoromethanesulfonate (98%, 2.44 mg, 7.21 μmol) is added sequentially a solution of 4 (0.79 mg, 1.8 μmol) in dimethyl sulfoxide (60 μL), trifluoroacetic acid (0.56 μL, 7.3 μmol) and tert-butyl hydrogen peroxide (70% in water; 1.25 μL, 9.03 μmol). The tube is capped and heated to 50°C overnight, then the reaction mixture is cooled and diluted with acetonitrile and 1% formic acid in water to a volume of approximately 2-3 mL. The final solvent composition mixture is such that the resulting mixture is clear, typically approximately 20% - 30% acetonitrile. The entire mixture was subjected to reverse phase HPLC (Column: Phenomenex Luna C18, 10×250 mm, 10 μm; Mobile phase A: 0.5% acetic acid in water; Mobile phase B: 9:1 acetonitrile/methanol; Gradient: 15% B for 5 min, then 15% - 70% B linear gradient over 84 min, then 70% - 95% B for 1 min, then 95% B for 9 min; Flow rate: 2 mL/min). The eluate was passed through a UV/VIS sensor and then split at approximately 15:1 between a fraction collector and an ion trap mass spectrometer. Fractions were collected every 20 s and those potentially containing products of interest were assessed by UPLC-UV-HRMS before pooling. Two products elute at approximately 71 and 75 minutes. The product to elute first is 5{N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-3-(trifluoromethyl)-1H-indole-2-carboxamide}, and the product to elute second is 6{N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide}.

5 - Yield: 0.101 mg, 0.199 µmol, 11%. High-Resolution MS m/z 508.2171 [M+H]+; calculated for C24H29F3N5O4, 508.2172. 1H NMR (600 MHz, DMSO-d6) δ 12.22 (bs, 1H), 9.01 (d, J=7.6 Hz, 1H), 8.96 (d, J=7.9 Hz, 1H), 7.73 (s, 1H), 7.21 (dd, J=8, 8 Hz, 1H), 7.08 (d, J=8.2 Hz, 1H), 6.69 (d, J=7.8 Hz, 1H), 5.03-4.95 (m, 1H), 4.49-4.40 (m, 1H), 3.87 (s, 3H), 3.22-3.08 (m, 2H), 2.43-2.34 (m, 1H), 2.23-2.10 (m, 2H), 1.82 (ddd, J=13.7, 9.3, 6.8 Hz, 1H), 1.78-1.66 (m, 2H), 1.62 (ddd, J=14.6, 9.7, 5.2 Hz, 1H), 1.49 (ddd, J=13.8, 8.8, 5.5 Hz, 1H), 0.97-0.88 (m, 6H). Retention time: 8.43 minutes (Analytical conditions. Column: Phenomenex Kinetex XB-C18, 2.1×100 mm, 2.6 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile; Gradient: 5% B for 0.5 minutes, then 5% - 70% B for 10.5 minutes, then 70% - 95% B for 2 minutes; Flow rate: 0.4 mL/min).

6 - Yield: 14.7 µg, 0.029 µmol, 1.6%. High-Resolution MS m/z 508.2178 [M+H]+; calculated for C24H29F3N5O4, 508.2172. 1H NMR (600 MHz, DMSO-d6) δ 11.47 (brs, 1H), 9.00 (d, J=7.9 Hz, 1H), 8.79 (d, J=7.8 Hz, 1H), 7.70 (s, 1H), 7.55 (d, J=8.2 Hz, 1H), 7.35 (s, 1H), 6.72 (d, J=8.3 Hz, 1H), 5.02-4.94 (m, 1H), 4.56-4.48 (m, 1H), 3.97 (s, 3H), 3.18-3.05 (m, 2H), 2.39-2.30 (m, 1H), 2.18-2.08 (m, 2H), 1.86-1.77 (m, 1H), 1.75-1.64 (m, 3H), 1.61-1.52 (m, 1H), 0.95 (d, J=6.1 Hz, 3H), 0.90 (d, J=6.1 Hz, 3H). Retention time: 8.92 minutes (Analytical conditions are identical to those used for 5).

Alternative synthesis of example 6

N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide (6)

Step 1. Synthesis of trifluoromethylated 4-methoxy-1H-indole-2-carboxylic acid (C24).

A mixture of 4-methoxy-1H-indole-2-carboxylic acid (100 mg, 0.523 mmol) and zinc(II) trifluoromethanesulfinate (120 mg, 0.362 mmol) was treated with dimethyl sulfoxide (1.5 mL) followed by trifluoroacetic acid (56 μL, 0.727 mmol). After cooling the reaction mixture to 0 °C, tert-butyl hydrogen peroxide (70% in water; 143 μL, 1.03 mmol) was added and stirring was continued at 0 °C for 20 min, then at room temperature for 25 min. The reaction mixture was then heated at 52 °C for 2 h, then it was cooled to room temperature and treated dropwise with aqueous sodium bicarbonate solution until bubbling ceased. The resulting mixture was then partitioned between aqueous sodium bicarbonate and ethyl acetate, the aqueous layer was extracted once with ethyl acetate, and the organic layers were discarded. The aqueous layer was then acidified to pH 7 with 1 M hydrochloric acid; ethyl acetate was added, and the mixture was stirred until the pH was adjusted to 1 with 1 M hydrochloric acid. After stirring the biphasic mixture for 10 min, the organic layer was washed with saturated aqueous sodium chloride, dried over magnesium sulfate, filtered, and concentrated in vacuo. By LCMS analysis, the residue (115 mg) contained a mixture of starting material and mono-trifluoromethylated products, as well as a small amount of di-trifluoromethylated product. The volume of this mixture was used in Step 4. Yield: 115 mg, <0.4 mmol. LCMS m/z 189.8, 257.8, 325.8 (minor) [M-H]-. 1H NMR (400 MHz, methanol-d4), characteristic peaks of their three main components: δ 7.07 (bd, J=8.4 Hz), 7.02 (bd, J=8.4 Hz), 6.81 (d, J=7.8 Hz), 6.66 (d, J=7.8 Hz), 6.51 (d, J=7.7 Hz), 4.06 (s, -OMe), 3.93 (s, -OMe), 3.92 (s, -OMe).

Step 2. Synthesis of N-(tert-butoxycarbonyl)-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninamide (C25).

To a 0°C solution of methyl N-(tert-butoxycarbonyl)-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninate (see Prior, A. M., et al., Bioorg. Med. Chem. Lett. 2013, 23, 6317–6320; 1.5 g, 3.8 mmol) in methanol (5 mL) was added a solution of ammonia in methanol (7 M; 43 mL, 300 mmol). After capping the reaction vessel, the reaction mixture was stirred overnight at room temperature. A solution of ammonia in methanol (7 M; 10.7 mL, 74.9 mmol) was added again and the reaction was continued at room temperature for 3 days, then concentrated in vacuo. The residue was taken up twice in diethyl ether (40 mL) and concentrated under reduced pressure to give C25 as a white solid. Yield: 1.46 g, 3.80 mmol, quantitative. LCMS m/z 385.4 [M+H]+. 1H NMR (400 MHz, chloroform-d) δ 8.29-8.17 (m, 1H), 7.23 (brs, 1H), 5.64 (brs, 1H), 5.32 (brs, 1H), 5.02 (d, J=6.1 Hz, 1H), 4.50-4.38 (m, 1H), 4.05 (ddd, J=10.3, 6.3, 4.5 Hz, 1H), 3.44-3.32 (m, 2H), 2.51-2.35 (m, 2H), 2.16-1.98 (m, 2H), 1.97-1.83 (m, 1H), 1.76-1.6 (m, 2H, supposed; partially covered by peak of water), 1.49-1.39 (m, 1H), 1.45 (s, 9H), 0.94 (d, J=6.4 Hz, 3H), 0.94 (d, J=6.3 Hz, 3H).

Step 3. Synthesis of L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninamide, methanesulfonate (C26).

A solution of methanesulfonic acid (0.861 mL, 13.3 mmol) in 1,1,1,3,3,3-hexafluoropropan-2-ol (5 mL) was slowly added to a solution of C25 (5.1 g, 13 mmol) in 1,1,1,3,3,3-hexafluoropropan-2-ol (43 mL). After 30 min, LCMS analysis showed conversion to C26: LCMS m/z 285.3 [M+H]+. The reaction mixture was concentrated in vacuo, then taken up in the following solvent mixture and re-concentrated: a mixture of acetonitrile and ethyl acetate (1:1, 2×20 mL), then a mixture of ethyl acetate and heptane (1:1, 2×20 mL). The resulting solid was azeotroped twice with a mixture of acetonitrile and ethyl acetate, then twice with a mixture of ethyl acetate and heptane to give C26 as a white solid (6.05 g) which retained the solvents by 1H NMR analysis. Yield: quantitative. 1H NMR (600 MHz, methanol-d4) δ 4.50 (dd, J=10.7, 4.9 Hz, 1H), 3.91 (dd, J=8.6, 5.5 Hz, 1H), 3.39-3.28 (m, 2H, assumed; partially obscured by solvent peak), 2.70 (s, 3H), 2.53-2.46 (m, 1H), 2.43-2.36 (m, 1H), 2.14 (ddd, J=14.0, 10.7, 5.0 Hz, 1H), 1.95-1.86 (m, 1H), 1.82-1.71 (m, 3H), 1.70-1.64 (m, 1H), 1.02 (d, J=6.3 Hz, 3H), 1.01 (d, J=6.1 Hz, 3H).

Step 4. Synthesis of N-{[4-methoxy-7-(trifluoromethyl)-1H-indol-2-yl]carbonyl}-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C27).

A solution of C24 (from step 1; 101 mg, <0.35 mmol) and C26 (from the previous step; 204 mg, ≤0.438 mmol) in acetonitrile (1.7 mL) and N,N-dimethylformamide (1 mL) was cooled to 0 °C and treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 163 mg, 0.429 mmol) followed by 4-methylmorpholine (0.129 mL, 1.17 mmol). The reaction mixture was stirred at 0 °C for 40 min, then a 1:1 mixture of aqueous sodium bicarbonate and ice was slowly added until a cloudy precipitate formed. Ethyl acetate was then added and the biphasic mixture was stirred for 5 min. The aqueous layer was extracted once with ethyl acetate and the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification by silica gel chromatography was performed twice (Gradient #1: 0% to 10% methanol in dichloromethane; Gradient #2: 5% to 10% methanol in dichloromethane) to afford C27. The regiochemistry of this product was confirmed by 2D NMR experiments. Yield: 19 mg, 36 µmol, ca. 10%. LCMS m/z 526.5 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 7.53 (bd, J=8.2 Hz, 1H), 7.41 (s, 1H), 6.68 (d, J=8.3 Hz, 1H), 4.60 (dd, J=9.5, 5.1 Hz, 1H), 4.45 (dd, J=11.4, 4.2 Hz, 1H), 4.01 (s, 3H), 3.3-3.21 (m, 2H, assumed; partially obscured by solvent peak), 2.60-2.49 (m, 1H), 2.36-2.26 (m, 1H), 2.15 (ddd, J=14.1, 11.5, 4.6 Hz, 1H), 1.89-1.68 (m, 5H), 1.03 (d, J=6.1 Hz, 3H), 0.99 (d, J=6.2 Hz, 3H).

Step 5. Synthesis of N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide (6).

Methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 17.2 mg, 72.2 μmol) was added to a solution of C27 (19 mg, 36 μmol) in a mixture of dichloromethane (0.5 mL) and acetonitrile (0.2 mL). The reaction mixture was then stirred at room temperature for 1 h, diluted with ethyl acetate and washed with a 1:1 mixture of aqueous sodium bicarbonate and ice. The aqueous layer was extracted with ethyl acetate and the combined organic layers were washed with saturated aqueous sodium chloride and passed through a solid phase extraction cartridge filled with magnesium sulfate. Concentration of the filtrate in vacuo gave a residue which was purified by reversed-phase HPLC (Column: Waters Sunfire C18, 19×100 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v); Gradient: 25% to 65% B over 8.5 min, then 65% to 95% B over 0.5 min, then 95% B for 1.0 min; Flow rate: 25 mL/min) to give N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide (6). Yield: 4.3 mg, 8.5 µmol, 24%. LCMS m/z 508.6 [M+H]+. Retention time: 2.83 minutes (Column: Waters Atlantis C18, 4.6×50 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v); Gradient: 5% - 95% B over 4.0 minutes, then 95% B for 1.0 minute; Flow rate: 2 mL/minute).

Example 7

N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-methylimidazo[2,1-b][1,3]thiazole-2-carboxamide (7)

Step 1. Synthesis of N2-(tert-butoxycarbonyl)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide (C28).

A solution of C17 (560 mg, 1.41 mmol) and 1H-imidazole (249 mg, 3.65 mmol) in a mixture of pyridine (3 ml) and dichloromethane (3 ml) was cooled to -35 °C using an acetonitrile/dry ice bath. Phosphorus oxychloride (0.74 ml, 7.94 mmol) was added dropwise over 4 min, followed by additional dichloromethane (2 ml), and stirring was continued at -30 °C to -20 °C. After 1 h, the reaction mixture was diluted with dichloromethane (2 ml). After approximately 1.5 h, hydrochloric acid (1 M; 30 ml) was added; the resulting mixture was stirred for 30 min and then extracted with dichloromethane (2 x 60 ml). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to afford C28 as a solid. Yield: 492 mg, 1.29 mmol, 91%. LCMS m/z 381.4 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 5.03 (dd, J=10.4, 5.7 Hz, 1H), 4.09 (dd, J=8.7, 4.2 Hz, 1H), 3.39-3.25 (m, 2H, assumed; partially obscured by solvent peak), 2.64-2.52 (m, 1H), 2.40-2.27 (m, 2H), 1.97-1.78 (m, 2H), 1.70 (dd, ABX system component, J=14.3, 4.1 Hz, 1H), 1.54 (dd, ABX system component, J=14.3, 8.7 Hz, 1H), 1.45 (s, 9H), 1.00 (s, 9H).

Step 2. Synthesis of N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-methylimidazo[2,1-b][1,3]thiazol-2-carboxamide (7).

A solution of the hydrochloride in 1,4-dioxane (4.0 M; 0.3 mL, 1.2 mmol) was added to a solution of C28 (100 mg, 0.263 mmol) in a mixture of acetonitrile (1.5 mL) and methanol (1.0 mL). The reaction mixture was stirred at room temperature for 30 min, then it was treated with 4-methylmorpholine (0.144 mL, 1.31 mmol). After removing the solvents in vacuo, the residue was resuspended twice in a mixture of dichloromethane and heptane (1:1, 2×10 mL) and concentrated under reduced pressure. The residue was combined with 3-methylimidazo[2,1-b][1,3]thiazole-2-carboxylic acid (47.9 mg, 0.263 mmol) in N,N-dimethylformamide (3.3 ml), cooled to 0 °C, and treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 99.9 mg, 0.263 mmol) followed by a solution of 4-methylmorpholine (72 μl, 0.655 mmol) in dichloromethane (0.2 ml). The reaction mixture was then stirred at 0 °C for approximately 2 h, treated at 0 °C with hydrochloric acid (1 M; 30 ml), and the resulting mixture was extracted with dichloromethane (2×60 ml). The aqueous layer was then basified to pH 9 by the addition of saturated aqueous sodium bicarbonate solution, then extracted with dichloromethane (3 x 60 mL). The combined organic layers were washed with saturated aqueous ammonium chloride solution (50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. 1H NMR analysis of this product indicated the presence of a minor epimer, presumably arising from partial racemization at the nitrile-bearing center. The major product was isolated using silica gel chromatography (Gradient: 0% to 20% methanol in ethyl acetate) to give N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-methylimidazo[2,1-b][1,3]thiazole-2-carboxamide (7) as a solid. Yield: 56 mg, 0.13 mmol, 49%. LCMS m/z 445.4 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 7.73 (d, J=1.6 Hz, 1H), 7.37 (d, J=1.6 Hz, 1H), 5.04 (dd, J=10.3, 5.9 Hz, 1H), 4.53 (dd, J=7.8, 5.0 Hz, 1H), 3.36-3.24 (m, 2H; predicted; partially obscured by solvent peak), 2.70 (s, 3H), 2.67-2.57 (m, 1H), 2.38-2.27 (m, 2H), 1.93 (ddd, J=14.0, 9.4, 6.0 Hz, 1H), 1.88-1.78 (m, 3H), 1.03 (s, 9H).

Examples 8 and 9

N-{1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[cyclohexyl(methoxy)acetyl]-4-methyl-L-leucinamide, DIAST-1 (8) and N-{1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[cyclohexyl(methoxy)acetyl]-4-methyl-L-leucinamide, DIAST-2 (9)

Step 1. Synthesis of N-{1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide (C29).

To a solution of C28 (114 mg, 0.300 mmol) in a mixture of acetonitrile (1 ml) and methanol (1 ml) was added a solution of the hydrochloride in 1,4-dioxane (4 M; 0.4 ml, 1.6 mmol). The reaction mixture was stirred at room temperature for 30 min, then 4-methylmorpholine (0.165 ml, 1.50 mmol) was added, adjusting the pH to 7–8. The solvents were then removed in vacuo, the residue was taken up twice in a mixture of ethyl acetate and heptane (1:1, 2×10 ml) and concentrated under reduced pressure to give C29 as a solid (269 mg); according to 1H NMR analysis, this consisted of a mixture of epimers, presumably located at the nitrile-bearing centre, in a ratio of 2–3 to 1. Part of this product was used in the next step. LCMS m/z 281.3 [M+H]+. 1H NMR (400 MHz, methanol-d4), characteristic peaks: δ [5.11 (dd, J=8.8, 7.3 Hz, major) and 5.01 (dd, J=6.5, 6.5 Hz, minor), total 1H], [2.75-2.65 (m, minor) and 2.64-2.54 (m, major), total 1H], 2.48-2.38 (m, 1H), 2.30-2.20 (m, 1H), 2.06-1.83 (m, 3H), 1.64 (dd, J=14.1, 4.8 Hz, 1H), [1.04 (s, major), 1.01 (s, minor), total 9H].

Step 2. Synthesis of N-{1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[cyclohexyl(methoxy)acetyl]-4-methyl-L-leucinamide, DIAST-1 (8) and N-{1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[cyclohexyl(methoxy)acetyl]-4-methyl-L-leucinamide, DIAST-2 (9).

To a 0°C solution of C29 (from the previous step; 83.4 mg, ≤93 μmol) and cyclohexyl(methoxy)acetic acid (17.2 mg, 99.9 μmol) in N,N-dimethylformamide (1 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 38.0 mg, 0.100 mmol), followed by a solution of 4-methylmorpholine (30.8 μL, 0.280 mmol) in dichloromethane (0.2 mL). The reaction mixture was then stirred at 0°C for about 2 h, diluted with saturated aqueous sodium bicarbonate (3 mL) at 0°C, and extracted with dichloromethane (4×4 mL). The combined organic layers were concentrated in vacuo; According to LCMS analysis, the residue consists of two components, presumably corresponding to two epimers at the nitrile-bearing center. These diastereomers are separated by reverse-phase HPLC (Column: Waters XBridge C18, 19×100 mm, 5 μm; Mobile phase A: water; Mobile phase B: acetonitrile; Gradient: 5% - 95% B over 8.54 min, then 95% B for 1.46 min; Flow rate: 25 ml/min). The first eluted diastereomer is designated as 8 (N-{1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[cyclohexyl(methoxy)acetyl]-4-methyl-L-leucinamide, DIAST-1), and the second eluted diastereomer as 9 (N-{1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[cyclohexyl(methoxy)acetyl]-4-methyl-L-leucinamide, DIAST-2).

8 - Yield: 12.8 mg, 29.4 μmol, 32% over 2 steps. LCMS m/z 435.6 [M+H]+. Retention time: 2.63 minutes (Analytical conditions. Column: Waters Atlantis C18, 4.6×50 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v). Gradient: 5% to 95% B over 4.0 minutes, then 95% B for 1.0 minute. Flow rate: 2 mL/minute).

9 - Yield: 10 mg, 23.0 µmol, 25% over 2 steps. LCMS m/z 435.6 [M+H]+. Retention time: 2.72 minutes (Analytical conditions identical to those used for 8).

Example 10

N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (10)

Step 1. Synthesis of N-[(4-methoxy-1H-indol-2-yl)carbonyl]-4-methyl-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C30).

To a 0°C solution of C18 (200 mg, ≤0.46 mmol) and 4-methoxy-1H-indole-2-carboxylic acid (88.2 mg, 0.460 mmol) in acetonitrile (2 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 175 mg, 0.460 mmol), followed by a solution of 4-methylmorpholine (0.127 mL, 1.16 mmol) in acetonitrile (0.2 mL). The reaction mixture was stirred at 0°C for 2.5 h, then it was diluted with saturated aqueous sodium bicarbonate solution (30 mL) at 0°C, then extracted with dichloromethane (50 mL). The organic layer was washed with hydrochloric acid (1 M; 30 mL) and the aqueous layers were extracted with dichloromethane (60 mL). The combined organic layers were then dried over sodium sulfate, filtered and concentrated in vacuo, the residue was purified by silica gel chromatography (gradient: 0% to 30% methanol in ethyl acetate) to give C30 as a solid. Yield: 148 mg, 0.314 mmol, 68% over 2 steps. LCMS m/z 472.4 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 7.25 (d, J=0.9 Hz, 1H), 7.15 (dd, J=8.8 Hz, 1H), 7.03 (bd, AB quartet component, J=8.3 Hz, 1H), 6.51 (d, J=7.7 Hz, 1H), 4.65 (dd, J=9.2, 3.4 Hz, 1H), 4.44 (dd, J=11.2, 4.2 Hz, 1H), 3.93 (s, 3H), 3.29-3.15 (m, 2H), 2.54-2.44 (m, 1H), 2.29 (dddd, J=12.6, 8.6, 7.0, 2.7 Hz, 1H), 2.14 (ddd, J=14.0, 11.2, 4.6 Hz, 1H), 1.89 (dd, component of the ABX system, J=14.5, 3.4 Hz, 1H), 1.85-1.74 (m, 3H), 1.02 (s, 9H).

Step 2. Synthesis of N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (10).

A solution of C30 (143 mg, 0.303 mmol) and 1H-imidazole (53.7 mg, 0.789 mmol) in a mixture of pyridine (1 ml) and dichloromethane (1 ml) was cooled in an acetonitrile/dry ice bath (-35 °C). Phosphorus oxychloride (0.159 ml, 1.71 mmol) was added dropwise over 5 min and the reaction mixture was stirred at -30 °C to -20 °C for 2 h, then it was treated with hydrochloric acid (1 M; 30 ml), stirred for 20 min and extracted with dichloromethane (2 x 60 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 10% methanol in ethyl acetate) afforded N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (10) as a solid. Yield: 68 mg, 0.15 mmol, 50%. LCMS m/z 454.5 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 7.24 (d, J=0.9 Hz, 1H), 7.14 (dd, J=8.8 Hz, 1H), 7.02 (bd, AB quartet component, J=8.3 Hz, 1H), 6.51 (d, J=7.7 Hz, 1H), 5.03 (dd, J=10.1, 6.0 Hz, 1H), 4.64 (dd, J=8.6, 4.3 Hz, 1H), 3.93 (s, 3H), 3.30-3.17 (m, 2H), 2.63-2.52 (m, 1H), 2.37-2.21 (m, 2H), 1.95-1.74 (m, 4H), 1.03 (s, 9H).

Example 11

N2-[(4-Bromo-1-ethyl-3-methyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide (11)

To a 0°C suspension of C18 (43.4 mg, ≤0.10 mmol) and 4-bromo-1-ethyl-3-methyl-1H-pyrazole-5-carboxylic acid (23.3 mg, 0.100 mmol) in acetonitrile (1.0 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (GATU; 38.0 mg, 0.100 mmol), followed by a solution of 4-methylmorpholine (30 μL, 0.27 mmol) in acetonitrile (0.2 mL). The reaction mixture was then stirred at 0°C for approximately 80 min, methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 71.5 mg, 0.300 mmol) was added and stirring was continued. After approximately 2.75 h, methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 71.5 mg, 0.300 mmol) was again added and the reaction was allowed to proceed for 1.5 h, then it was treated with saturated aqueous sodium bicarbonate solution (3 mL) at 0°C and extracted with dichloromethane (2×8 mL). The combined organic layers were concentrated in vacuo, then dissolved in acetonitrile (4 mL) and concentrated again using a Genevac evaporator to give the crude product (138 mg). A portion of this product (80 mg) was purified by reverse-phase HPLC (Column: Waters Sunfire C18, 19×100 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v); Gradient: 5% - 95% B over 8.54 min, then 95% B over 1.46 min; Flow rate: 25 mL/min) to give N2-[(4-bromo-1-ethyl-3-methyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide (11). Yield: 24.7 mg, 49.8 μmol, 86% over 2 steps. LCMS m/z 495.5 (bromine isotope pattern observed) [M+H]+. Retention time: 2.48 min (Analytical conditions. Column: Waters Atlantis C18, 4.6×50 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v). Gradient: 5% - 95% B for 4.0 min, then 95% B for 1.0 min. Flow rate: 2 mL/min).

Example 12

N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(3,3-difluorocyclobutyl)acetyl]-4-methyl-L-leucinamide (12)

Step 1. Synthesis of 4-methyl-L-leucyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide, hydrochloride (C18, HCl salt).

A solution of the hydrochloride in 1,4-dioxane (4 M; 1.7 mL, 6.8 mmol) was added to a solution of C17 (260 mg, 0.652 mmol) in acetonitrile (3 mL). The reaction mixture was stirred at room temperature for 1.5 h, then concentrated in vacuo, then redissolved in dichloromethane:heptane (1:1, 3×10 mL) and reconcentrated to give C18, the HCl salt (242 mg) as a glass. A portion of this product was used in the next step. LCMS m/z 299.3 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 4.53 (dd, J=10.3, 5.0 Hz, 1H), 3.91 (dd, J=7.5, 5.4 Hz, 1H), 3.41-3.26 (m, 2H, assumed; partially obscured by solvent peak), 2.57-2.47 (m, 1H), 2.41 (dddd, J=12.0, 8.7, 7.0, 3.1 Hz, 1H), 2.15 (ddd, J=13.9, 10.3, 4.9 Hz, 1H), 2.05-1.97 (m, 1H), 1.97-1.85 (m, 1H), 1.78 (ddd, J=14.1, 9.1, 5.0 Hz, 1H), 1.60 (dd, J=14.3, 5.4 Hz, 1H), 1.01 (s, 9H).

Step 2. Synthesis of N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(3,3-difluorocyclobutyl)acetyl]-4-methyl-L-leucinamide (12).

A suspension of C18, HCl salt (from the previous step; 37.2 mg, ≤0.100 mmol) and (3,3-difluorocyclobutyl)acetic acid (15.8 mg, 0.105 mmol) in tetrahydrofuran (1.0 mL) was treated with 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide trioxide (50% w/v solution in ethyl acetate; 65.5 μL, 0.110 mmol) and 4-methylmorpholine (27.5 μL, 0.250 mmol). The reaction mixture was then stirred at room temperature overnight, heated at 50°C for 4.5 hours, then 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide trioxide (50% by weight in ethyl acetate; 2.2 equivalents) and 4-methylmorpholine (5 equivalents) were added again. The reaction mixture was then stirred at 50°C for 3 additional days, treated with saturated aqueous sodium bicarbonate (3 ml) and extracted with dichloromethane (4×4 ml). The combined organic layers were concentrated in vacuo and purified by reverse phase HPLC (Column: Waters XBridge C18, 19×100 mm, 5 μm; Mobile phase A: water; Mobile phase B: acetonitrile; Gradient: 20% - 40% B over 8.5 min, then 40% - 95% B over 0.5 min, then 95% B for 1.0 min; Flow rate: 25 mL/min) to give N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(3,3-difluorocyclobutyl)acetyl]-4-methyl-L-leucinamide (12). Yield: 10.1 mg, 24.5 μmol, 24% over 2 steps. LCMS m/z 413.5 [M+H]+. Retention time: 1.96 minutes (Analytical conditions. Column: Waters Atlantis C18, 4.6×50 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v). Gradient: 5% - 95% B for 4.0 minutes, then 95% B for 1.0 minute. Flow rate: 2 mL/minute).

Example 13

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (13)

Step 1. Synthesis of methyl (1R,2S,5S)-3-[N-(tert-butoxycarbonyl)-3-methyl-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate (C31).

O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 7.92 g, 20.8 mmol) was added to a 0°C mixture of N-(tert-butoxycarbonyl)-3-methyl-L-valine (4.38 g, 18.9 mmol) and methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1,0]hexane-2-carboxylate hydrochloride (3.9 g, 19 mmol) in N,N-dimethylformamide (95 mL). The reaction mixture was then stirred for 5 min, N,N-diisopropylethylamine (8.25 mL, 47.4 mmol) was added; Stirring was continued at 0°C for 2 h, then aqueous citric acid (1 N, 20 mL) and water (40 mL) were added. The resulting mixture was stirred for 2 min, and then diluted with ethyl acetate (250 mL). The organic layer was washed with water (3 x 150 mL) and saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Purification by silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) gave C31 as an oil. Yield: 3.60 g, 9.41 mmol, 50%. 1H NMR (400 MHz, methanol-d4) δ 6.42 (d, J=9.7 Hz, <1H; not fully replaced by solvent), 4.35 (s, 1H), 4.21 (d, J=9.7 Hz, 1H), 4.02 (d, half of AB quartet, J=10.4 Hz, 1H), 3.91 (dd, component of ABX system, J=10.3, 5.3 Hz, 1H), 3.73 (s, 3H), 1.57 (dd, component of ABX system, J=7.5, 5.1 Hz, 1H), 1.47 (d, half of AB quartet, J=7.5 Hz, 1H), 1.41 (s, 9H), 1.07 (s, 3H), 1.02 (s, 9H), 0.93 (s, 3H).

Step 2. Synthesis of (1R,2S,5S)-3-[N-(tert-butoxycarbonyl)-3-methyl-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C32).

Aqueous lithium hydroxide (1.0 M; 14.7 mmol, 14.7 mL) was added dropwise to a 0 °C solution of C31 (3.60 g, 9.41 mmol) in a mixture of tetrahydrofuran and methanol (1:1, 30 mL). The reaction mixture was then stirred at 0 °C for 1 h, it was warmed to room temperature and stirred for 1 h, then LCMS analysis showed conversion to C32: LCMS m/z 367.3 [M-H]-. Adjustment to pH 3 was done by adding 1 M hydrochloric acid, after which the mixture was diluted with water (30 mL). The aqueous layer was extracted with ethyl acetate (2 x 75 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give C32 as an off-white solid. Yield: 3.10 g, 8.41 mmol, 89%. 1H NMR (400 MHz, methanol-d4) δ 6.39 (d, J=9.7 Hz, ca. 0.5H; not fully replaced by solvent), 4.33 (s, 1H), [4.21 (d, J=9.6 Hz) and 4.21 (s), total 1H], 4.01 (d, half of AB quartet, J=10.5 Hz, 1H), 3.91 (dd, component of ABX system, J=10.4, 5.2 Hz, 1H), 1.56 (dd, component of ABX system, J=7.5, 5.0 Hz, 1H), 1.50 (d, half of AB quartet, J=7.6 Hz, 1H), 1.42 (s, 9H), 1.07 (s, 3H), 1.02 (s, 9H), 0.93 (s, 3H).

Stage 3. Synthesis of tert-butyl {(2S)-1-[(1R,2S,5S)-2-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2-yl}carbamate (C33).

A 0°C mixture of C7 (31.9 mg, ≤94 μmol) and C32 (34 mg, 92 μmol) in acetonitrile (1 mL) was treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU, 97%; 36.2 mg, 92.3 μmol) followed by a solution of 4-methylmorpholine (25 μL, 0.23 mmol) in acetonitrile (0.25 mL). The reaction mixture was then stirred at 0°C for approximately 1 h, diluted with saturated aqueous sodium bicarbonate (3 mL) at 0°C and extracted with dichloromethane (4×4 mL). The combined organic layers were concentrated in vacuo to give C33 as a gum (48 mg). Most of this product was used in the next step. LCMS m/z 504.6 [M+H]+.

Step 4. Synthesis of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (13).

A stock solution of methanesulfonic acid (60 μL) was prepared in 1,1,1,3,3,3-hexafluoropropan-2-ol (940 μL). To a solution of C33 (from the previous step; 47 mg, ≤90 μmol) in 1,1,1,3,3,3-hexafluoropropan-2-ol (1 mL) was added a portion of the stock solution of methanesulfonic acid (0.1 mL; 100 μmol). The reaction mixture was then stirred at room temperature for 1 h, concentrated in vacuo, then taken up in the following solvent mixture and re-concentrated: a mixture of acetonitrile and ethyl acetate (1:1, 2×10 mL), and then a mixture of ethyl acetate and heptane (1:1, 2×10 mL). The residue was dissolved in dichloromethane (1 ml) and treated with 4-methylmorpholine (30.8 μl, 0.280 mmol) then trifluoroacetic anhydride (0.143 ml, 1.01 mmol). The reaction mixture was stirred at room temperature for 40 min, then it was treated with 4-methylmorpholine (30.8 μl, 0.280 mmol) then trifluoroacetic anhydride (0.143 ml, 1.01 mmol); after 30 min, 4-methylmorpholine (30.8 μl, 0.280 mmol) was added again, then trifluoroacetic anhydride (0.143 ml, 1.01 mmol). After another 15 min of stirring, the reaction mixture was treated with hydrochloric acid (1 M; 3 ml) and the resulting mixture was extracted with dichloromethane (3 x 4 ml); the combined organic layers were concentrated in vacuo and purified using reverse phase HPLC (Waters Sunfire C18, 19 x 100 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v). Gradient: 20% - 60% B for 8.5 min, then 60% - 95% B for 0.5 min, then 95% B for 1 min; Flow rate: 25 ml/min) to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (13). Yield: 7.5 mg, 15 µmol, 17% in 2 stages. LCMS m/z 500.5 [M+H]+. Retention time: 2.66 minutes (Analytical conditions. Column: Waters Atlantis dC18, 4.6×50 mm, 5 μm; Mobile phase A: 0.05% trifluoroacetic acid in water (v/v); Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v/v); Gradient: 5.0% - 95% B for 4.0 minutes, then 95% B for 1.0 minute; Flow rate: 2 mL/minute).

Alternative Synthesis of Example 13, Methyl tert-Butyl Ether Solvate; Preparation of 13, Methyl tert-Butyl Ether Solvate, Solid Form 2

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate), Solid Form 2

Step 1. Synthesis of tert-butyl {(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}carbamate (C5).

This experiment was performed in 2 parallel batches. A solution of ammonia in methanol (7 M; 2.4 L, 17 mol) was added to methyl N-(tert-butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alaninate (600 g, 2.10 mol) and the reaction mixture was stirred at 25 °C for 40 h. Concentration in vacuo and combination of the 2 batches gave C5 as a yellow solid. Combined yield: 1.10 kg, 4.05 mol, 96%. 1H NMR (400 MHz, DMSO-d6) δ 7.63 (br s, 1H), 7.29 (br s, 1H), 7.01 (br s, 1H), 6.89 (d, J=8.5 Hz, 1H), 3.96-3.85 (m, 1H), 3.22-3.06 (m, 2H, predicted; partially obscured by water peak), 2.28-2.08 (m, 2H), 1.89 (ddd, J=14.6, 10.8, 4.0 Hz, 1H), 1.74-1.60 (m, 1H), 1.56-1.43 (m, 1H), 1.36 (s, 9H).

Step 2. Synthesis of 3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide, hydrochloride (C16, HCl salt).

This experiment was performed in 3 parallel batches. To a 0°C solution of C5 (840 g, 3.10 mol) in dichloromethane (2.0 L) was added a solution of the hydrochloride salt in 1,4-dioxane (4 M; 2 L, 8 mol). The reaction mixture was stirred at 25°C for 2 h, then concentrated in vacuo; combining the 3 batches gave C16, the HCl salt, as a white solid. Combined yield: 1.20 kg, 5.78 mol, 62%. MS m/z 172.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.52-8.35 (br s, 3H), 8.12 (s, 1H), 7.95 (s, 1H), 7.57 (s, 1H), 3.88-3.76 (m, 1H), 3.24-3.10 (m, 2H), 2.59-2.5 (m, 1H, assumed; partially obscured by solvent peak), 2.35-2.24 (m, 1H), 2.01 (ddd, J=14.9, 9.2, 6.1 Hz, 1H), 1.80-1.68 (m, 2H).

A sample of the C16, HCl salt was triturated in 2-propanol for 1.5 hours, then collected by filtration and washed with 2-propanol. The combined solid was dried overnight under high vacuum to obtain a sample for powder X-ray diffraction analysis. The powder X-ray diffraction pattern for this product is given in Figure 10; the characteristic peaks are listed in Table Q.

Collection of powder X-ray diffraction data

Powder X-ray diffraction analysis was performed using a Bruker AXS D4 Endeavour diffractometer equipped with a Cu radiation source. The divergence slit was set to 0.6 mm while the secondary optics used variable slits. The diffracted radiation was recorded with a PSD-Lynx Eye sensor. The X-ray tube voltage and current were set to 40 kV and 40 mA, respectively. Data were collected in a Theta-2Theta goniometer at a Cu wavelength of 3.0 to 40.0 degrees 2-Theta using a step size of 0.020 degrees and a step time of 0.3 seconds. Samples were prepared by placing them in a silicon low-background sample holder and rotating during collection.

Powder X-ray diffraction analysis was performed using a Bruker AXS D8 Advance diffractometer equipped with a Cu radiation source. Diffracted radiation was recorded with a LYNXEYE_EX motorized slit sensor. Both the primary and secondary were equipped with 2.5-inch slits. The X-ray tube voltage and current were set to 40 kV and 40 mA, respectively. Data were collected in a Theta-Theta goniometer with closed-pair scanning at a Cu K-alpha (average) wavelength from 3.0 to 40.0 degrees 2-Theta in 0.02 degree steps, using a scan rate of 0.5 seconds per step. Samples were prepared by placing them in a silicon low-background sample holder.

Data are acquired by both instruments using Bruker DIFFRAC Plus software and analyses are performed using EVA DIFFRAC plus software. The PXRD data file is not processed prior to peak searching. Using the peak searching algorithm in EVA software, peaks selected with a threshold of 1 are used for preliminary peak assignments. Corrections are made manually to ensure reliability; the output of automatic assignments is visually checked and peak positions are adjusted to the peak maximum. Peaks with a relative intensity ≥3% are typically selected. Peaks that are unresolved or correspond to noise are typically not selected. The typical error associated with PXRD peak position is specified in the USP to +/- 0.2° 2-Theta (USP-941).

Table Q. Selected X-ray powder diffraction peaks for C16, HCl salt Angle (°2 theta) Relative intensity Angle (°2 theta) Relative intensity 9.97 3 29.24 13 11.67 1 30.98 6 14.17 1 31.78 2 16.08 1 32,32 23 16.35 1 32.79 10 17.10 14 33.10 1 17.27 3 33.50 6 18.23 24 33.70 4 19,21 4 33.90 3 20.83 20 35.27 3 22.20 58 36.20 3 22.97 12 36.42 6 23.35 34 36.75 6 23.79 2 36.95 7 24.62 3 37.83 3 25.10 100 38.58 2 26.85 11 39.44 7 28.39 14 39.75 1

Alternative synthesis of 3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide, hydrochloride, C16 salt HCl

An alternative preparation of the C16 compound, the HCl salt, is shown in the reaction scheme below.

To a solution of ammonia in methanol (7.0 M; 100 mL, 725.4 mmol) was added methyl (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate 4-methylbenzenesulfonate (20 g, 55.8 mmol) and magnesium sulfate (6.7 g, 55.8 mmol) at room temperature. After stirring the reaction mixture for 7 h at room temperature, nitrogen was bubbled into the reaction mixture for 1 h to purge excess ammonia. The reaction mixture was then filtered through a pad of Celite® and then concentrated in vacuo, and the resulting (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide 4-methylbenzenesulfonate was used directly in the next step without further purification. A portion of (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanamide 4-methylbenzenesulfonate (10 g, 25.9 mmol) and a solution of the hydrochloride in 1,4-dioxane (4.0 M; 19.4 ml, 77.7 mmol) were added to a solution of dimethylformamide (50 ml, 647 mmol). After stirring for 12 h at room temperature, the suspension was filtered and washed with dimethylformamide (15 ml, 190 mmol). The resulting solid was dried in a vacuum oven at 40°C for 12 h to give 3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide, hydrochloride, C16 HCl salt (2.7 g, 12.4 mmol) as a tan solid (overall yield 48%).

Step 3. Synthesis of methyl (1R,2S,5S)-3-[N-(tert-butoxycarbonyl)-3-methyl-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate (C31).

This experiment was carried out in three parallel batches. To a 0°C solution of methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate hydrochloride (237 g, 1.15 mol) and N-(tert-butoxycarbonyl)-3-methyl-L-valine (293 g, 1.27 mol) in a mixture of N,N-dimethylformamide (400 mL) and acetonitrile (3.6 L) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 481 g, 1.26 mol), then N,N-diisopropylethylamine (601 mL, 3.45 mol) was added dropwise. The reaction mixture was then warmed to 25°C and stirred for 16 h, then it was poured into a mixture of ice water (1 L) and hydrochloric acid (0.5 M; 1 L), pH approximately 5, and stirred for 6 min. The resulting mixture was extracted with ethyl acetate (2 L), and the organic layer was washed with water (2 L), dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified using silica gel chromatography (Gradient: 0% to 50% ethyl acetate in petroleum ether) to give, after combining three crops, C31 as a colorless oil. Combined yield: 1.17 kg, 3.06 mol, 89%. LCMS m/z 383.3 [M+H]+. 1H NMR (400 MHz, chloroform-d) δ 5.10 (d, J=10.2 Hz, 1H), 4.46 (s, 1H), 4.20 (d, J=10.3 Hz, 1H), 3.98 (d, half AB quartet, J=10.2 Hz, 1H), 3.89-3.82 (m, 1H), 3.74 (s, 3H), 1.48-1.41 (m, 2H), 1.38 (s, 9H), 1.03 (s, 3H), 1.01 (s, 9H), 0.89 (s, 3H).

Step 4. Synthesis of (1R,2S,5S)-3-[N-(tert-butoxycarbonyl)-3-methyl-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C32).

This experiment was carried out in three parallel batches. To a solution of C31 (668 g, 1.75 mol) in tetrahydrofuran (2.5 L) was added lithium hydroxide monohydrate (220 g, 5.24 mol) and water (500 mL). The reaction mixture was then stirred at 25 °C for 2 h and concentrated in vacuo to remove most of the tetrahydrofuran; the residue was then adjusted to pH 2 with 1 M hydrochloric acid. The resulting mixture was extracted with ethyl acetate (2 x 500 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (500 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give C32 as a white solid (2.0 kg) after combining the three batches. This product was used directly in the next step. LCMS m/z 313.2 [(M - 2-methylprop-1-ene)+H]+. 1H NMR (400 MHz, chloroform-d) δ 5.14 (d, J=10.2 Hz, 1H), 4.46 (s, 1H), 4.24 (d, J=10.2 Hz, 1H), 4.06 (d, half of the AB quartet, J=10.5 Hz, 1H), 3.82 (dd, component of the ABX system, J=10.5, 5.5 Hz, 1H), 1.75 (d, J=7.7 Hz, 1H), 1.49 (dd, J=7.7, 5.4 Hz, 1H), 1.40 (s, 9H), 1.06 (s, 3H), 1.00 (s, 9H), 0.89 (s, 3H).

Step 5. Synthesis of (1R,2S,5S)-6,6-dimethyl-3-(3-methyl-L-valyl)-3-azabicyclo[3,1,0]hexane-2-carboxylic acid, hydrochloride (C41).

This experiment was performed in 2 parallel batches. A solution of the hydrochloride salt in 1,4-dioxane (4 M; 4.0 L, 16 mol) was added to a solution of C32 (from the previous step; 1.00 kg, ≤2.62 mol) in dichloromethane (1.0 L) and the reaction mixture was stirred at 25 °C for 16 h. Removal of the solvents in vacuo at 50 °C gave C41 as a white solid (1.8 kg) after combining the 2 batches. This product was used directly in the next step. 1H NMR (400 MHz, methanol-d4) δ 4.42 (s, 1H), 4.00 (s, 1H), 3.94 (dd, component of the ABX system, J=10.7, 5.4 Hz, 1H), 3.80 (d, half of the AB quartet, J=10.7 Hz, 1H), 1.62 (dd, component of the ABX system, J=7.7, 5.2 Hz, 1H), 1.56 (d, half of the AB quartet, J=7.6 Hz, 1H), 1.15 (s, 9H), 1.09 (s, 3H), 1.03 (s, 3H).

Step 6. Synthesis of (1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C42).

This experiment was carried out in 3 parallel batches. To a 0°C solution of C41 (from the previous step; 600 g, ≤1.75 mol) in methanol (2 L) was added triethylamine (1.64 L, 11.8 mol) followed by ethyl trifluoroacetate (699 g, 4.92 mol) and the reaction mixture was then warmed to 25°C and stirred for 16 h. It was then concentrated in vacuo at 50°C and the residue was diluted with ethyl acetate (3 L) and adjusted to pH 3–4 with 2 M hydrochloric acid. After extracting the aqueous layer with ethyl acetate (1 L), the combined organic layers were washed with saturated aqueous sodium chloride solution (3 L), dried over sodium sulfate, filtered, and concentrated under reduced pressure. At this point, the 3 batches were combined, the mixture was treated with petroleum ether and ethyl acetate (5:1, 3 L), and stirred at 25°C for 2 h. Filtration gave C42 as a white solid. Combined yield: 1.90 kg, 5.21 mol, 99% over 3 steps. LCMS m/z 365.1 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 8.88 (d, J=8.8 Hz, <1H; not fully substituted), [4.60 (d, J=8.9 Hz) and 4.59 (s), total 1H], 4.35 (s, 1H), 3.96 (dd, component of the ABX system, J=10.5, 5.1 Hz, 1H), 3.90 (d, half of the AB quartet, J=10.4 Hz, 1H), 1.58 (dd, component of the ABX system, J=7.6, 4.9 Hz, 1H), 1.52 (d, half of the AB quartet, J=7.6 Hz, 1H), 1.08 (s, 12H), 0.92 (s, 3H).

Step 7. Synthesis of (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (C43).

This experiment was carried out in four parallel batches. 2-Hydroxypyridine 1-oxide (33.9 g, 305 mmol) was added to a solution of C42 (445 g, 1.22 mol) and C16 HCl salt (256 g, 1.23 mol) in butan-2-one (2.5 L) and the mixture was cooled to 0°C. N,N-Diisopropylethylamine (638 mL, 3.66 mol) was then added, followed by dropwise addition of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (351 g, 1.83 mol). The reaction mixture was stirred at 25 °C for 16 h, then it was diluted with ethyl acetate (1 L) and treated with a mixture of hydrochloric acid (1 M; 1.5 L, 1.5 mol) and saturated aqueous sodium chloride (1 L). The organic layer was washed with a mixture of aqueous sodium hydroxide (1 M; 1.5 L, 1.5 mol) and saturated aqueous sodium chloride (1 L), dried over sodium sulfate, filtered, and concentrated in vacuo. Combination of 4 batches gave C43 as a white solid (2.3 kg). Combined yield: 2.1 kg (corrected for residual ethyl acetate), 4.1 mol, 84%. LCMS m/z 518.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.41 (bd, J=7.7 Hz, 1H), 8.30 (d, J=8.8 Hz, 1H), 7.56 (s, 1H), 7.32 (bd, 1H), 7.04 (bd, 1H), 4.43 (bd, J=7.3 Hz, 1H), 4.35-4.25 (m, 1H), 4.28 (s, 1H), 3.89 (dd, J=10.3, 5.5 Hz, 1H), 3.67 (d, J=10.4 Hz, 1H), 3.17-3.09 (m, 1H), 3.07-2.98 (m, 1H), 2.46-2.35 (m, 1H), 2.19-2.10 (m, 1H), 1.99-1.89 (m, 1H), 1.70-1.58 (m, 1H), 1.55-1.44 (m, 2H), 1.38 (d, half of AB quartet, J=7.6 Hz, 1H), 1.01 (s, 3H), 0.98 (s, 9H), 0.84 (s, 3H).

Step 8. Synthesis of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate), Solid Form 2.

This experiment was carried out in 3 parallel batches. Methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 552 g, 2.32 mol) was added to a solution of C43 (600 g, 1.16 mol) in ethyl acetate (3 L). The reaction mixture was then stirred at 25 °C for 3 h, it was treated with additional methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 27.6 g, 116 mmol), and the reaction mixture was stirred for 1 h. It was then filtered; The filter cake was washed with ethyl acetate (2 x 500 ml) and the combined filtrates were washed successively with aqueous sodium bicarbonate solution (1 M; 2 l), saturated aqueous sodium chloride solution (2 l), hydrochloric acid (1 M; 2 l), and saturated aqueous sodium chloride solution (2 l). The organic layer was then dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was treated with a mixture of ethyl acetate and methyl tert-butyl ether (1:10, 2.5 l) and heated to 50°C; after stirring for 1 h at 50°C, it was cooled to 25°C and stirred for 2 h. The solid was collected by filtration and the 3 batches were combined in ethyl acetate (8 l) and filtered through silica gel (3.0 kg); the silica gel was then washed with ethyl acetate (2 x 2 l). The combined eluates were then concentrated in vacuo and the residue taken up in ethyl acetate (900 ml) and methyl tert-butyl ether (9 l). This mixture was heated to 50°C for 1 h, cooled to 25°C and stirred for 2 h. Filtration gave (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1,0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate) as a white solid. The powder X-ray diffraction pattern for this product, designated Solid Form 2, is shown in Figure 1; characteristic peaks are listed in Table A. Combined yield: 1.41 kg, 2.82 mol, 81%. LCMS m/z 500.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.42 (d, J=8.4 Hz, 1H), 9.03 (d, J=8.6 Hz, 1H), 7.68 (s, 1H), 4.97 (ddd, J=10.9, 8.5, 5.1 Hz, 1H), 4.41 (d, J=8.5 Hz, 1H), 4.16 (s, 1H), 3.91 (dd, J=10.4, 5.5 Hz, 1H), 3.69 (d, J=10.4 Hz, 1H), 3.18-3.10 (m, 1H), 3.08-2.99 (m, 1H), 2.46-2.34 (m, 1H), 2.20-2.03 (m, 2H), 1.78-1.65 (m, 2H), 1.57 (dd, J=7.6, 5.4 Hz, 1H), 1.32 (d, J=7.6 Hz, 1H), 1.03 (s, 3H), 0.98 (s, 9H), 0.85 (s, 3H).

Collection of powder X-ray diffraction data

Powder X-ray diffraction analysis was performed using a Bruker AXS D8 Endeavour diffractometer equipped with a Cu (medium K-α) radiation source. The divergence slit was set to a constant illumination of 15 mm. Diffracted radiation was recorded with a PSD-Lynx Eye sensor with a PSD sensor opening of 2.99 degrees. The X-ray tube voltage and current were set to 40 kV and 40 mA, respectively. Data were collected in a Theta-Theta goniometer at a Cu wavelength of 3.0 to 40.0 degrees 2-Theta using a step size of 0.00998 degrees and a step time of 1.0 second. The anti-scatter screen was set to a fixed distance of 1.5 mm. During collection, samples were rotated at 15/min. Samples were prepared by placing them in a silicon low-background sample holder and rotating during collection. Data are acquired using Bruker DIFFRAC Plus software and analysis is performed using EVA DIFFRAC Plus software. Using the peak finding algorithm in EVA software, peaks selected with a threshold of 1 are used for preliminary peak assignment. Corrections are made manually to ensure reliability; the output of automatic subtractions is checked visually and peak positions are adjusted to the peak maximum. Peaks with a relative intensity ≥3% are typically selected. Peaks that are unresolved or correspond to noise are not selected. The typical error associated with the PXRD peak position specified in USP is up to +/- 0.2° 2-Theta (USP-941).

Table A. Selected Powder X-ray Diffraction Peaks for 13, Methyl tert-Butyl Ether Solvate, Solid Form 2, from the Alternative Synthesis of Example 13, Methyl tert-Butyl Ether Solvate; Preparation of 13, Methyl tert-Butyl Ether Solvate, Solid Form 2 Angle (°2-theta)
+/- 0.2° 2-Theta Relative intensity (%) Angle (°2-theta)
+/- 0.2° 2-Theta Relative intensity (%) 7.1 78 22.7 9 10.5 8 22.9 10 11.3 15 23.1 5 11.8 36 23.4 6 12.5 49 23.7 22 12.9 4 25.3 14 14.2 34 27.3 3 15.7 10 27.9 6 16.0 24 28.3 9 16.8 100 28.5 4 17.0 41 29.1 3 18.5 50 29.4 6 18.8 7 30.2 3 19.1 25 30.8 5 19.9 11 32.0 4 20.2 8 33.3 7 20.8 14 33.8 4 21.1 9 35.4 7 21.4 4 36.4 6 21.7 4 38.1 3 22.2 24

Alternative synthesis of (1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C42).

Second Alternative Synthesis of Example 13, Methyl tert-Butyl Ether Solvate; Creation 13, Methyl tert-Butyl Ether Solvate, Solid Form 2

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate), Solid Form 2

Methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 392 g, 1.64 mol) was added to a solution of C43 (415 g, 802 mmol) in ethyl acetate (2.0 L). The reaction mixture was stirred at 25 °C for 3 h, then methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 86.0 g, 361 mmol) was added again. The reaction mixture was then stirred for 1 hour, filtered, and the filtrate was washed successively with aqueous sodium bicarbonate (1 M; 1.5 L), saturated aqueous sodium chloride (1.5 L), hydrochloric acid (1 M; 1.5 L), and saturated aqueous sodium chloride (1.5 L), dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was treated with a mixture of ethyl acetate and methyl tert-butyl ether (1:10, 2.5 L) and heated to 50°C; after stirring for 1 hour at 50°C, it was cooled to 25°C and stirred for 2 hours. Collection of the solid by filtration afforded (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate) as a crystalline white solid. The X-ray powder diffraction pattern for this product, designated Solid Form 2, is shown in Figure 2; characteristic peaks are listed in Table B. Yield: 338 g, 575 mmol, 72%. LCMS m/z 500.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.43 (d, J=8.4 Hz, 1H), 9.04 (d, J=8.6 Hz, 1H), 7.68 (s, 1H), 4.97 (ddd, J=10.9, 8.5, 5.0 Hz, 1H), 4.41 (d, J=8.5 Hz, 1H), 4.15 (s, 1H), 3.91 (dd, component of the ABX system, J=10.4, 5.5 Hz, 1H), 3.69 (d, half of the AB quartet, J=10.4 Hz, 1H), 3.18-3.10 (m, 1H), 3.08-2.98 (m, 1H), 2.46-2.34 (m, 1H), 2.20-2.02 (m, 2H), 1.77-1.65 (m, 2H), 1.57 (dd, J=7.6, 5.4 Hz, 1H), 1.32 (d, half AB quartet, J=7.6 Hz, 1H), 1.02 (s, 3H), 0.98 (s, 9H), 0.85 (s, 3H); Methyl tert-butyl ether peaks: 3.07 (s, 3H), 1.10 (s, 9H).

The method for collecting powder X-ray diffraction data is described in Alternative Synthesis of Example 13, Methyl tert-butyl ether solvate, Step 8.

Table B. Selected X-ray Powder Diffraction Peaks for 13, Methyl tert-Butyl Ether Solvate, Solid Form 2, from the Alternative Synthesis of Example 13, Methyl tert-Butyl Ether Solvate; Preparation of 13, Methyl tert-Butyl Ether Solvate, Solid Form 2 Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) 7.2 66 20.0 9 27.4 3 10.6 9 20.3 6 28.0 6 11.4 12 20.8 6 28.4 7 11.9 32 20.9 12 29.5 4 12.6 49 21.2 7 30.3 3 13.0 4 21.5 4 30.9 5 14.3 37 21.8 3 32.1 3 15.8 8 22.3 24 33.4 5 16.1 22 22.8 6 33.5 3 16.9 100 23.0 9 35.5 6 17.2 46 23.2 5 36.5 3 18.6 42 23.5 6 38.2 3 18.9 6 23.8 17 19.3 23 25.4 10

Third alternative synthesis of example 13

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (13)

Step 1. Synthesis of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate)

0°C mixture of C42 (90.5% by weight, 5.05 g, 12.5 mmol) and C16, HCl salt (98.9% by weight, 3.12 g, 14.9 mmol) in acetonitrile (50 mL) was treated with 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% by weight solution in acetonitrile; 17 mL, 24.3 mmol) for about 10 min. Then 1-methyl-1H-imidazole (4.0 mL, 50.2 mmol) was slowly added over about 15 min, and the reaction mixture was stirred at 0°C for 3.5 h, then it was warmed to 25°C. 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% by weight in acetonitrile; 17 mL, 24.3 mmol) was added in one portion and the reaction mixture was stirred at 45 °C for 16 h. It was cooled to 25 °C at this point and then treated with aqueous sodium bicarbonate (1.14 M; 35 mL, 40 mmol) for 10 min. After adding ethyl acetate (25 mL) and enough water to dissolve the resulting solids, the organic layer was washed twice with aqueous sodium bicarbonate (1.14 M; 25 mL, 28 mmol). The organic layer was then washed with aqueous sodium chloride (14%, 2×20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was mixed with ethyl acetate (2.1 ml) and treated with methyl tert-butyl ether (19 ml); the resulting suspension was heated with stirring at 50°C for 1 hour, cooled to 25°C over 1 hour, and kept at 25°C for 1.5 hours. The solids were isolated by filtration, washed with methyl tert-butyl ether (2 ml/g), and dried in a vacuum oven overnight at 50°C to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1,0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate) as a crystalline white solid. The volume of this product progressed in the next step. Yield: 3.71 g, 6.31 mmol, 50%. 1H NMR (400 MHz, DMSO-d6) δ 9.40 (d, J=8.4 Hz, 1H), 9.02 (d, J=8.6 Hz, 1H), 7.66 (s, 1H), 4.97 (ddd, J=10.7, 8.6, 5.1 Hz, 1H), 4.41 (d, J=8.4 Hz, 1H), 4.16 (s, 1H), 3.91 (dd, component of the ABX system, J=10.3, 5.5 Hz, 1H), 3.69 (d, half of the AB quartet, J=10.4 Hz, 1H), 3.18-3.10 (m, 1H), 3.09-2.99 (m, 1H), 2.46-2.35 (m, 1H), 2.20-2.04 (m, 2H), 1.78-1.64 (m, 2H), 1.56 (dd, J=7.4, 5.6 Hz, 1H), 1.32 (d, half AB quartet, J=7.6 Hz, 1H), 1.03 (s, 3H), 0.98 (s, 9H), 0.85 (s, 3H); Methyl tert-butyl ether peaks: 3.07 (s, 3H), 1.10 (s, 9H).

Step 2. Synthesis of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (13).

A mixture of propan-2-yl acetate (17 ml) and heptane (17 ml) was added to 13, methyl tert-butyl ether solvate (from the previous step; 3.41 g, 5.80 mmol), and stirring was carried out overnight at 20°C. Heptane (17 ml) was then added over 2 h, and the mixture was stirred overnight at room temperature. The resulting suspension was filtered and the collected solids were washed with a mixture of propan-2-yl acetate (1.36 mL) and heptane (3.73 mL) and dried at 50 °C under vacuum to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1,0]hexane-2-carboxamide (13) as a crystalline solid. A portion of this crop was used as seed material for the recrystallization of Example 13; preparation of solid Form 1 below. Yield: 2.73 g, 5.46 mmol, 94%.

Recrystallization of Example 13; obtaining solid form 1

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (13), Solid form 1

A mixture of 13, methyl tert-butyl ether solvate (from the alternative synthesis of Example 13, methyl tert-butyl ether solvate; Preparation 13, methyl tert-butyl ether solvate, Solid Form 2; 60.1 g, 102 mmol) and propan-2-yl acetate (480 mL) was heated to 60 °C. A sample of 13 (the seed product from the third alternative synthesis of Example 13, Step 2; 1.2 g, 2.4 mmol) was added; after 10 min, the seed product was still present in solid form. Heptane (360 mL) was slowly added to the stirred mixture over 12 h. Additional heptane (360 ml) was added over 4 hours and the resulting mixture was stirred for 30 minutes. It was then cooled to 20°C at a rate of 0.1 degrees/minute and then stirred overnight. The solid was collected by filtration and washed with a mixture of propan-2-yl acetate (72 ml) and heptane (168 ml). It was then dried under vacuum at 50°C to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1,0]hexane-2-carboxamide (13) as a white crystalline solid. The powder X-ray diffraction pattern of this product, designated Solid Form 1, is shown in Figure 3; the characteristic peaks are listed in Table C. Yield: 47.8 g, 95.7 mmol, 94%.

The method for collecting powder X-ray diffraction data is described in Alternative Synthesis of Example 13, Methyl tert-butyl ether solvate, Step 8.

Table C. Selected X-ray powder diffraction peaks for 13, Solid Form 1 Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) 7.6 16 18.9 11 24.7 8 9.8 10 19.7 7 25.3 7 11.4 10 19.9 14 27.0 3 11.9 13 20.5 36 27.2 6 12.7 100 21.0 14 27.9 4 15.7 40 21.7 4 28.1 3 15.8 18 22.2 23 29.5 7 17.3 10 22.5 3 32.6 6 17.8 12 23.1 6 35.7 4 18.3 55 23.6 10 37.0 3

Single crystal X-ray structure determination of example 13, Solid form 1

A sample of Example 13 was crystallized by diffusion using ethyl acetate and hexane. The crystallization vessel was allowed to stand at room temperature while the solvent evaporated; after 2.5 months, X-ray quality crystals appeared. One of these was used for structure determination. The ORTEP diagram for the single crystal data is shown in Figure 4. Mercury software was used to calculate the powder structure from the resolved crystal structure; comparison with the diffraction pattern from Recrystallization of Example 13; yielding Solid Form 1, identifies this product as Solid Form 1 (see Figure 5). The peak characteristics for these calculated data are presented in Table D.

Table D. Powder Diffraction Pattern Data for 13, Solid Form 1, Calculated from Single Crystal X-ray Structure Determination of Example 13, Solid Form 1 Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) 7.6 22 21.0 28 30.1 4 9.8 21 21.6 14 30.3 3 10.4 9 21.7 14 31.5 4 10.8 4 22.2 40 31.7 5 11.4 16 22.5 7 31.9 4 11.9 75 23.1 5 32.7 8 12.7 89 23.6 15 33.4 3 14.6 3 24.3 5 33.6 8 15.7 100 24.8 15 35.7 8 15.9 30 25.4 10 36.6 3 17.4 34 26.4 3 36.6 3 17.9 24 27.0 9 37.0 4 18.3 67 27.3 8 37.3 4 18.9 12 27.9 3 38.3 3 19.7 15 28.1 5 39.4 3 19.9 63 28.7 5 39.6 4 20.5 53 29.5 9 20.8 9 30.0 9

X-ray analysis of single crystals

Data collection is performed on a Bruker D8 Quest diffractometer at room temperature. Data collection consists of omega and phi scans.

The structure was resolved by internal phasing using the SHELX software package in the orthorhombic space group class P212121. The structure was further refined by full-matrix least-squares. All non-hydrogen atoms were found and refined using anisotropic displacement parameters.

Hydrogen atoms located on nitrogen are found from the difference Fourier map and refined with distance constraints. The remaining hydrogen atoms are placed in calculated positions and allowed to move on their carrier atoms. The final refinement includes isotropic displacement parameters for all hydrogen atoms.

The analysis of the absolute structure using probability methods (Hooft, 2008) is carried out using PLATON (Spek). The results show that the absolute structure is assigned correctly. The method calculates that the probability that the structure is assigned correctly is 100%. The Hooft parameter is represented as -0.01 s s t (3), and the Parson parameter is represented as -0.01 s s t (2).

The resulting R-index is 3.3%. The finite difference Fourier map did not reveal any missing or shifted electron density.

The corresponding crystal, data collection and clarifying information are summarized in Table E. The atomic coordinates, bond lengths, bond angles and displacement parameters are listed in Tables F–H.

Software and links

SHELXTL, Version 5.1, Bruker AXS, 1997.

PLATON, A. L. Spek, J. Appl. Christ. 2003, 36, 7-13.

MERCURY, C. F. Macrae, P. R. Edington, P. McCabe, E. Pidcock, G. P. Shields, R. Taylor, M. Towler, and J. van de Streek, J. Appl. Christ. 2006, 39, 453-457.

OLEX2, O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, and H. Puschmann, J. Appl. Christ. 2009, 42, 339-341.

R. W. W. Hooft, L. H. Straver, and A. L. Spek, J. Appl. Christ. 2008, 41, 96-103.

H. D. Flack, Acta Cryst. 1983, A39, 867-881.

Table E. Crystal data and structure refinements for Example 13, Solid Form 1. Empirical formula
Mass formula
Temperature
Wavelength
Crystal system
Space group
Lattice constant
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta interval for data collection
Index ranges
Collected reflections
Independent reflections
Fullness to theta=67.679°
Absorption correction
Method of clarification
Data/constraints/parameters
F2 match
Finite indices R [I>2σ(I)]
R indices (all data)
Absolute structure parameter
Excitation coefficient
Largest differential peaks and troughs C23H32F3N5O4
499.53
296(2) K
1.54178 Å
Orthothrombotic
P212121
a=9.6836(2) Å
b=15.0522(4) Å
c=18.0272(5) Å
2627.64(11) Å 3
4
1.263 mg/m3
0.862 mm-1
1056
0.300×0.280×0.260 mm3
3.826 to 80.042°
-12<=h<=12, -18<=k<=19, -22<=l<=23
79731
5628 [Rint=0.0294]
99.3%
Empirical
Least squares in full matrix approximation for F2
5628/9/358
1,040
R1=0.0326, wR2=0.0906
R1=0.0346, wR2=0.0928
-0.01(3)
n/a
0.112 and -0.121 e.Å-3 α=90°
β=90°
γ=90°

Table F. Atomic coordinates (x 104) and equivalent isotropic substitution parameters (Å2×103) for Example 13, Solid form 1. U(eq) is defined as one third of the trace of the orthogonalized tensor Uij.

Table G. Bond lengths [Å] and angles [°] for Example 13, Solid Form 1.

Symmetrical transformations are used to create equivalent atoms.

Table H. Anisotropic substitution parameters (Å2×103) for Example 13, Solid form 1. The exponent of the anisotropic substitution coefficient takes the form: -2π2[h2 a*2U11+…+2 h k a* b* U12].

Solid state NMR analysis of the compound of Example 13, Forms 1 and 4, was performed on a CPMAS probe mounted in a Bruker-BioSpin Avance III 500 MHz NMR spectrometer (1H frequency). A magic angle spin speed of 15.0 kHz was used. Form 1 spectra were collected at ambient temperature (not temperature controlled) and Form 4 spectra were collected at 15°C.

13C ssNMR spectra were acquired using a proton decoupled cross-polarization magic angle spinning (CPMAS) experiment. A proton decoupling phase-modulated field of 80-100 kHz was used during spectra collection. The cross-polarization contact time was set to 2 ms and the recirculation delay to 3.5 seconds for Form 1 and Form 4. The number of scans was adjusted to obtain an adequate signal-to-noise ratio. The 13C chemical shift scale was referenced using a 13C CPMAS experiment on a crystalline adamantane external standard, setting its upper-field resonance to 29.5 ppm.

19F ssNMR spectra were obtained using a proton decoupled magic angle spinning (MAS) experiment. A phase-modulated proton decoupling field of 80-100 kHz was used during spectra collection. Spectra were collected with a recirculation delay of up to 6 seconds for Form 1 and 5.25 seconds for Form 4. The number of scans was adjusted to obtain an adequate signal-to-noise ratio. For the 19F chemical shift scale, a 19F MAS experiment with trifluoroacetic acid (50%/50% v/v in H2O) as an external standard was used, setting its resonance to -76.54 ppm.

Automated peak picking is performed using Bruker-BioSpin TopSpin software version 3.6. Typically, a threshold of 4% relative intensity is used for peak pre-selection. The output from automated peak picking is visually inspected to ensure reliability and manual adjustments are made if necessary. Although specific solid state NMR peak values are reported here, there is a range in these peak values due to variations in instrumentation, samples and sample preparation. This is common practice in the field of solid state NMR due to variations in peak positions. Typical variability in the 13C chemical shift x-axis value is of the order of plus or minus 0.2 ppm, unless otherwise stated, for a crystalline solid. Variability in the 19F chemical shift x-axis value is of the order of plus or minus 0.1 ppm. Solid state NMR peak heights reported here are relative intensities. The intensity of solid state NMR can vary depending on the actual experimental parameter settings and the thermal history of the sample.

13C solid state NMR of Example 13, Form 1 was prepared as described above and the following list of peaks of Example 13, Form 1 was determined. The variability for 13C chemical shift values is ±0.2 ppm unless otherwise noted.

13C chemical shift (ppm) Relative intensity (%) 13C Chemical Shift (ppm) Relative intensity (%) 13C Chemical Shift (ppm) Relative intensity (%) 178.9 24 62.7 24 33.0±0.1 35 172.3 21 58.6 27 31.0±0.1 31 172.1 25 47.2 26 27.9±0.1 100 169.6 21 40.3 28 26.3 58 156.7 14 39.4 24 26.0 41 123.5 10 39.0 31 20.8±0.1 49 122.6±0.1 6 37.8±0.1 48 13.0±0.1 47 118.5 4 37.4±0.1 41 116.1 4 34.6±0.1 33

19F solid state NMR of the compound of Example 13, Form 1 was obtained and the 19F solid state NMR peak was determined at a chemical shift of -73.3±0.1 ppm.

The characteristic peaks for compound Example 13, Form 1 are the 19F peak with a chemical shift of -73.3±0.1 ppm in combination with the 13C peaks with chemical shifts of 31.0±0.1 ppm, 27.9±0.1 ppm, and 178.9±0.2 ppm.

Alternative recrystallization of Example 13; obtaining solid form 4

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (13), Solid Form 4

Step 1. Recrystallization of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (13) from propan-2-yl acetate and heptane.

A mixture of propan-2-yl acetate (50 mL) and heptane (50 mL) was added to 13, methyl tert-butyl ether solvate, Solid Form 2 (from the second alternative synthesis of Example 13, methyl tert-butyl ether solvate; 10.02 g, 17.0 mmol) and the mixture was stirred at 20 °C and 3500 rpm overnight. Heptane (50 mL) was then slowly added and stirring was continued for 30 min, then the mixture was cooled to 10 °C over 30 min. After stirring for a further 2 h, the suspension was filtered; The filter cake was washed with a mixture of propan-2-yl acetate (4 ml) and heptane (16 ml) and then dried at 55°C under vacuum to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1,0]hexane-2-carboxamide (13) as a crystalline solid. A portion of this product was used in the next recrystallization. Yield: 7.74 g, 15.5 mmol, 91%.

Step 2. Recrystallization of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (13) from water.

A suspension of 13 (from the previous step; 1.0 g, 2.0 mmol) in water (12 mL) was stirred at 5 °C for 21 days, then the solid was collected by filtration. It was then dried under vacuum for 10 min and air dried in a thin layer on paper for 20 min to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1,0]hexane-2-carboxamide (13) as a white crystalline solid. The powder X-ray diffraction pattern of this product, designated Solid Form 4, is given in Figure 6; The characteristic peaks are listed in Table J. Yield: 755 mg, 1.51 mmol, 76%. The method for collecting powder X-ray diffraction data is described in the Alternative Synthesis of Example 13, methyl tert-butyl ether solvate, Step 8.

Table J. Selected X-ray powder diffraction peaks for Example 13, solid form 4 Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) 7.6 15 20.4 43 30.6 3 9.8 14 20.7 44 30.8 5 10.8 7 21.1 42 31.3 3 11.2 4 21.6 9 31.8 6 11.4 4 21.8 16 32.5 6 11.4 8 22.3 53 32.8 4 11.7 5 23.1 14 33.2 4 12.0 6 23.4 11 34.4 9 12.3 82 24.2 9 35.5 12 12.7 61 24.9 12 35.6 7 13.7 4 25.2 8 35.6 7 14.9 3 26.1 4 36.0 3 15.1 5 27.0 5 36.4 3 15.9 100 27.2 15 37.1 6 17.5 47 28.1 17 38.7 3 18.0 5 28.9 4 39.4 3 18.2 57 29.4 9 39.5 3 18.5 21 29.5 4 39.8 4 18.8 37 29.8 5 20.0 12 30.0 21

Single crystal X-ray structural analysis of example 13, solid form 4

A sample of Example 13 was crystallized by diffusion at room temperature using ethyl acetate and pentane; one of the resulting crystals was used for single crystal X-ray structural analysis. The ORTEP diagram for the single crystal data is shown in Figure 7. Mercury software was used to calculate a powder diffraction pattern based on the resolved crystal structure; comparison with the diffraction pattern from an alternative recrystallization of Example 13; obtaining solid Form 4 identifies this product as solid Form 4 (see Figure 8). The characteristic peaks for these calculated data are listed in Table K.

Table K. Powder Diffraction Pattern Data for 13, Solid Form 1, Calculated for Single Crystal X-Ray Structure Analysis of Example 13, Solid Form 4 Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) Angle (°2-theta)
+/-0.2° 2-Theta Relative intensity (%) 7.6 22 20.1 6 29.9 3 9.7 19 20.4 40 30.0 13 10.8 10 20.7 31 30.7 3 11.2 4 21.1 32 30.9 3 11.3 7 21.6 8 31.3 3 11.7 6 21.8 12 31.9 3 12.2 100 22.3 35 32.6 4 12.6 49 22.5 3 33.2 5 13.7 4 23.1 13 34.5 7 14.9 3 23.5 7 35.5 6 15.1 4 24.2 8 35.8 6 15.7 14 24.8 6 37.2 3 15.9 82 24.9 8 39.9 3 17.5 35 25.2 7 18.2 54 26.2 4 18.5 14 27.0 4 18.7 31 27.2 7 19.9 3 28.1 11

X-ray analysis of single crystals

Data collection is performed on a Bruker D8 Venture diffractometer at -100°C. Data collection consists of omega and phi scans.

The structure was resolved by internal phasing using the SHELX software package in the orthorhombic space group class P212121. The structure was further refined by full-matrix least-squares. All non-hydrogen atoms were found and refined using anisotropic displacement parameters.

Hydrogen atoms located on nitrogen are found from the difference Fourier map and refined with distance constraints. The remaining hydrogen atoms are placed in calculated positions and allowed to move on their carrier atoms. The final refinement includes isotropic displacement parameters for all hydrogen atoms.

Absolute structure analysis using probability methods (Hooft, 2008) was performed using PLATON (Spek). Absolute stereochemistry is not determined due to Hooft/Parsons/Flak parameter values outside the specification and standard deviations.

The resulting R-index is 6.3%. The finite difference Fourier map revealed no missing or shifted electron density.

The corresponding crystal, data collection and clarifying information are summarized in Table L. The atomic coordinates, bond lengths, bond angles and displacement parameters are listed in Tables M-P.

Software and links

SHELXTL, Version 5.1, Bruker AXS, 1997.

PLATON, A. L. Spek, J. Appl. Christ. 2003, 36, 7-13.

MERCURY, C. F. Macrae, P. R. Edington, P. McCabe, E. Pidcock, G. P. Shields, R. Taylor, M. Towler, and J. van de Streek, J. Appl. Christ. 2006, 39, 453-457.

OLEX2, O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, and H. Puschmann, J. Appl. Christ. 2009, 42, 339-341.

R. W. W. Hooft, L. H. Straver, and A. L. Spek, J. Appl. Christ. 2008, 41, 96-103.

H. D. Flack, Acta Cryst. 1983, A39, 867-881.

Table L. Crystal data and structure refinements for Example 13, Solid Form 4. Empirical formula
Mass formula
Temperature
Wavelength
Crystal system
Space group
Lattice constant
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta interval for data collection
Index ranges
Collected reflections
Independent reflections
Fullness to theta=54.284°
Absorption correction
Method of clarification
Data/constraints/parameters
F2 match
Finite indices R [I>2σ(I)]
R indices (all data)
Absolute structure parameter
Excitation coefficient
Largest differential peaks and troughs C23H32F3N5O4
499.53
173(2) K
1.54178 Å
Orthothrombotic
P212121
a=9.2114(9) Å
b=15.1607(16) Å
c=18,191(2) Å
2540.5(5) Å3
4
1.306 mg/m3
0.892 mm-1
1056
0.100×0.060×0.040 mm3
3,795-54,284°
-9<=h<=9, -15<=k<=15, -18<=l<=18
15896
3070 [Rint=0.1260]
99.1%
Empirical
Least squares in full matrix approximation for F2
3070/9/349
1,067
R1=0.0625, wR2=0.1515
R1=0.0696, wR2=0.1578
0.14(14)
n/a
0.280 and -0.220 e. Å-3 α=90°
β=90°
γ=90°

Table M. Atomic coordinates (x 104) and equivalent isotropic substitution parameters (Å2×103) for Example 13, Solid Form 4. U(eq) is defined as one third of the trace of the orthogonalized tensor Uij.

Table N. Bond lengths [Å] and angles [°] for example 13, solid form 4.

Symmetrical transformations are used to create equivalent atoms.

Table P. Anisotropic substitution parameters (Å2×103) for example 13, solid form 4. The exponent of the anisotropic substitution factor takes the form: -2π2[h2 a*2U11+...+2 h k a* b* U12].

List of 13C NMR solid state peaks PF-07321332-00 form 4. Variability for 13C chemical shift values is ±0.2 ppm unless otherwise stated.

13C Chemical Shift (ppm) Relative intensity (%) 13C Chemical Shift (ppm) Relative intensity (%) 13C Chemical Shift (ppm) Relative intensity (%) 179.3 16 62.0 17 32.9 16 178.8 11 58.5 26 31.9 23 172.3 20 47.1 26 31.3±0.1 16 169.6 8 41.5 17 29.7 18 168.6 12 40.2 12 27.9±0.1 100 156.7 12 38.8 22 26.9±0.1 36 123.7 4 38.3 27 26.2 28 120.1 6 37.9 43 25.9 18 119.1 4 37.5 39 21.6±0.1 31 118.5 4 37.0 25 20.8±0.1 22 62.4 16 34.4 13 12.9±0.1 46

19F solid state NMR of the compound of Example 13, Form 4 was obtained and the peak at -73.6±0.1 with a relative intensity of 100% was determined.

For compound Example 13, Form 4, six characteristic peaks were identified: 19F chemical shift at -73.6 ± 0.1 ppm. and 13C chemical shifts at 26.9±0.1 ppm, 21.6±0.1 ppm, 41.5±0.2 ppm, 27.9±0.1 ppm, and 12.9±0.1. 19F peak with a chemical shift of -73.6±0.1 ppm. is characteristic of the compound of Example 13, Form 4. 13C peaks at 26.9±0.1 ppm, 21.6±0.1 ppm. and 41.5±0.1 ppm. each is a characteristic peak of the compound of Example 13, Form 4. The 13C peaks at 27.9 ppm and 12.9 ppm are each characteristic of the compound of Example 13, Form 4 when taken in combination with one or more of the peaks selected from the 13C peaks at 21.6 ppm, 26.9 ppm, and 41.5 ppm and the 19F peak at -73.6 ppm.

Fourth alternative synthesis of Example 13, methyl tert-butyl ether solvate

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate)

To a 0°C suspension of C43 (5.0 g, 9.7 mmol) in propan-2-yl acetate (7.6 mL/g, 38 mL) was charged 4-methylmorpholine (4.5 equivalents; 4.8 mL, 44 mmol). To the resulting suspension was charged trifluoroacetic anhydride (2.25 equivalents; 3.1 mL, 22 mmol) over 1 h via a metering pump. After stirring at 0°C for at least 1 h, the reaction mixture was warmed to about 20°C, quenched with water (8 mL/g, 40 mL), and stirred for at least 10 min. After decanting, the lower (aqueous) layer was discarded and water (8 mL/g, 40 mL) was added to the organic layer. After stirring for at least 10 minutes, the layers are separated and the lower (aqueous) layer is discarded. The organic layer is concentrated under reduced pressure to approximately 4 ml/g (about 20 ml), then the vacuum is broken with nitrogen and the solution is heated to approximately 50 °C. Methyl tert-butyl ether (12 ml/g, 60 ml) is slowly added over at least 4 hours via an addition funnel, and the reaction mixture is maintained at 50 °C for at least 1 hour, then cooled to 25 °C for 1 hour. The resulting suspension is maintained at 25 °C overnight, then filtered, washed successively with a mixture of propan-2-yl acetate and methyl tert-butyl ether [1:3 (v/v); 2 ml/g] and methyl tert-butyl ether (2 ml/g, 10 ml), and dried on the filter for at least 30 minutes. The solids were then transferred to a vacuum oven at 50 °C and dried for at least 8 hours to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1,0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate) as an off-white solid. Yield: 3.4 g, 5.8 mmol, 60%.

Fifth alternative synthesis of example 13, methyl tert-butyl ether solvate

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate)

To a suspension of C43 (4.0 g, 7.7 mmol) in acetonitrile (10 ml/g, 40 ml) at room temperature was charged 1-methyl-1H-imidazole (4.6 equivalents; 2.84 ml, 35.6 mmol) and the resulting mixture was heated to approximately 30 °C. A solution of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% by weight in acetonitrile; 2 equivalents; 10.8 ml) was added over at least 6 hours using a pump. The reaction mixture was then stirred for at least 10 hours, cooled to 25 °C and carefully quenched by adding saturated aqueous sodium bicarbonate (7 ml/g, 28 ml) (exothermic and gasless). Acetonitrile is then distilled off under reduced pressure; ethyl acetate (10 ml/g, 40 ml) and additional saturated aqueous sodium bicarbonate solution (5 ml/g, 20 ml) are added to the resulting mixture. After phase separation, the lower (aqueous) layer is discarded and the organic layer is washed with saturated aqueous sodium bicarbonate solution (3.5 ml/g; 14 ml). The aqueous layer is again discarded and the organic layer is concentrated under reduced pressure to approximately 1 ml/g (4 ml). Methyl tert-butyl ether (9 ml/g; 36 ml) is charged and the resulting solution is heated to 50 °C, quickly obtaining a suspension. This suspension is kept at 50 °C for at least 30 minutes, then it is cooled to 25 °C over 1 hour and kept at 25 °C for at least 8 hours. The suspension is then filtered, washed with a mixture of ethyl acetate and methyl tert-butyl ether [1:3 (v/v); 2 ml/g], and then washed with methyl tert-butyl ether (2 ml/g; 8 ml). The combined solid is dried on the filter for at least 30 minutes, transferred to a vacuum oven at about 50°C, and dried for at least 8 hours to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1,0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate) as an off-white solid. Yield: 2.9 g, 4.9 mmol, 64%.

Examples of compositions for the compound of example 13

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide or a hydrate or solvate thereof or a pharmaceutically acceptable salt of the compound, hydrate or solvate are formulated to provide a conventional tablet and an immediate release film-coated tablet for oral administration, wherein dosages range from 100 to 250 mg. As an example, the immediate release formulation is described in the composition table and contains the conventional inactive excipients microcrystalline cellulose and lactose monohydrate (diluents), crospovidone (disintegrant), colloidal silicon dioxide (glidant) and sodium stearyl fumarate (lubricant) agent). The immediate release tablets are film coated using commercially available film coating formulations including Opadry white and Opadry pink. All excipients in the film coated tablet are commonly used and are present at known levels. The formulation provided is an example of an immediate release tablet formulation and as such, one skilled in the art will be able to use readily available conventional techniques using alternative formulation excipients to produce suitable tablets and achieve the desired tablet quality characteristics.

Formulation examples: typical coated tablet formulations for (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide.

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (hereinafter referred to as API) immediate release tablets are manufactured using conventional, standard batch processes for immediate release solid oral tablets. Examples of standard batch processes that can be used to produce immediate release tablets of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide include direct compression, dry granulation, and wet granulation. Alternatively, a continuous manufacturing process can be used. After compression of the tablets, the tablet cores are film coated. Film coating of the tablets can be carried out using a continuous coating process or using a conventional batch film coating process.

Tablet composition: tablet compositions 100 mg, 150 mg and 250 mg (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide

Component Role 100 mg tablet (% w/w) 100 mg tablet (mg/tablet) 150 mg tablet (% w/w) 150 mg tablet (mg/tablet) 250 mg tablet (% w/w) 250 mg tablet (mg/tablet) API Active ingredient 20,00 100,00 20,00 150,00 25,00 250,00 Microcrystalline cellulose Diluent 49.33 246.67 49.33 370,00 46,00 460,00 Lactose monohydrate Diluent 24.67 123.33 24.67 185,00 23.00 230,00 Crospovidone Baking powder 3.00 15.00 3.00 22.50 3.00 30,00 Colloidal silicon dioxide Glidant 1.00 5.00 1.00 7.50 1.00 10.00 Sodium stearyl fumarate (intragranulated) Lubricating agent 1.00 5.00 1.00 7.50 1.00 10.00 Sodium stearyl fumarate (extragranular) Lubricating agent 1.00 5.00 1.00 7.50 1.00 10.00 Total core 100,00 500,00 100,00 750,00 100,00 1000,00 Opadry White
(YS-1-7027-SP) Coating 3.50 17.50 - - - - - - - - Opadry Pink
(058140011) Coating - - - 3.00 22.50 - - - -

Example 14

N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (14)

Step 1. Synthesis of 9H-fluoren-9-ylmethyl [(2S)-1-amino-5,5,5-trifluoro-1-oxopentan-2-yl]carbamate (C34).

Sodium bicarbonate (4.8 g, 57 mmol) was added to a solution of 5,5,5-trifluoro-L-norvalinamide hydrochloride (synthesized using the method described for its enantiomer in J. E. Starrett, PCT Int. Appl., 2010107997, September 23, 2010; 4.0 g, 19 mmol) and 9H-fluoren-9-ylmethyl carbonochloridate (Fmoc chloride; 10.2 g, 39.4 mmol) in water (80 mL). The resulting suspension was stirred at 15°C - 25°C for 24 h, then partitioned between water and dichloromethane. The organic layer was washed successively with water and brine, dried over sodium sulfate, filtered, and concentrated in vacuo to afford C34 as a solid. Yield: 6.2 g, 16 mmol, 83%. LCMS m/z 393.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 7.9 (d, 2H), 7.7 (m, 2H), 7.5 (d, 1H), 7.4 (m, 5H), 7.1 (br s, 1H), 4.3 (m, 3H), 4.0 (m, 1H), 2.2 (m, 2H), 1.9 (m, 1H), 1.7 (m, 1H).

Step 2. Synthesis of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-5,5,5-trifluoro-L-norvaline (C35).

To a solution of C34 (6.2 g, 16 mmol) in 1,4-dioxane (60 mL) was added hydrochloric acid (3 M; 10 mL, 30 mmol), and the reaction mixture was stirred at 80 °C for 16 h. It was then partitioned between water and dichloromethane, and the organic layer was washed with water and saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was triturated with petroleum ether to give C35 as a solid. Yield: 5.5 g, 14 mmol, 88%. LCMS m/z 392.1 [M-H]-. 1H NMR (400 MHz, DMSO-d6) δ 12.83 (brs, 1H), 7.89 (d, J=7.4 Hz, 2H), 7.77-7.67 (m, 3H), 7.46-7.38 (m, 2H), 7.36-7.28 (m, 2H), 4.38-4.28 (m, 2H), 4.26-4.19 (m, 1H), 4.06 (ddd, J=9, 9, 4.9 Hz, 1H), 2.43-2.15 (m, 2H), 2.01-1.89 (m, 1H), 1.89-1.75 (m, 1H).

Step 3. Synthesis of benzyl N-[(9H-fluoren-9-ylmethoxy)carbonyl]-5,5,5-trifluoro-L-norvalinate (C36).

A mixture of C35 (435 mg, 1.11 mmol), benzyl bromide (0.263 mL, 2.21 mmol), and sodium bicarbonate (464 mg, 5.52 mmol) in N,N-dimethylformamide (20 mL) was stirred for 15 h at 25 °C. The reaction mixture was then diluted with water (30 mL) and extracted with ethyl acetate (3 x 30 mL), and the combined organic layers were washed successively with saturated aqueous sodium chloride and 5% aqueous lithium chloride, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (gradient: 0% to 100% ethyl acetate in petroleum ether) gave C36 as a white solid. Yield: 510 mg, 1.05 mmol, 95%. LCMS m/z 506.1 [M+Na+].

Step 4. Synthesis of benzyl 5,5,5-trifluoro-L-norvalinate (C37).

Diethylamine (10 mL) was added to a 0°C mixture of C36 (510 mg, 1.05 mmol) in acetonitrile (25 mL). The reaction mixture was then stirred at 20°C for 2 h, concentrated under reduced pressure, and chromatography on silica gel (gradient: 0% to 10% methanol in dichloromethane) afforded C37 as a colorless oil. Yield: 250 mg, 0.957 mmol, 91%. LCMS m/z 302.9 [M+CH3CN+H]+. 1H NMR (400 MHz, chloroform-d) δ 7.42-7.32 (m, 5H), 5.17 (s, 2H), 3.50 (dd, J=8.4, 5.0 Hz, 1H), 2.32-2.13 (m, 2H), 2.01 (dddd, J=13.7, 10.8, 5.2, 5.2 Hz, 1H), 1.76 (dddd, J=13.6, 10.8, 8.4, 5.3 Hz, 1H).

Step 5. Synthesis of benzyl 5,5,5-trifluoro-N-[(4-methoxy-1H-indol-2-yl)carbonyl]-L-norvalinate (C38).

To a 0°C solution of C37 (250 mg, 0.957 mmol) and 4-methoxy-1H-indole-2-carboxylic acid (220 mg, 1.15 mmol) in N,N-dimethylformamide (10 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 437 mg, 1.15 mmol), then 4-methylmorpholine (194 mg, 1.92 mmol) was added dropwise. Stirring was continued at 0°C - 10°C for 1 h, then the reaction mixture was diluted with water (20 mL) and aqueous citric acid solution (20 mL) and extracted with ethyl acetate (3×20 mL). The combined organic layers were washed successively with saturated aqueous sodium bicarbonate (30 mL), saturated aqueous sodium chloride, and aqueous lithium chloride (5%, 20 mL), then dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified using silica gel chromatography (gradient: 0% to 100% ethyl acetate in petroleum ether) to afford C38 as a white solid. Yield: 350 mg, 0.806 mmol, 84%. LCMS m/z 435.1 [M+H]+. 1H NMR (400 MHz, chloroform-d ) δ 9.09 (br s, 1H), 7.42-7.33 (m, 5H), 7.23 (dd, J=8.8 Hz, 1H), 7.09 (d, J=2.3 Hz, 1H), 7.03 (d, J=8.3 Hz, 1H), 6.76 (bd, J=7.6 Hz, 1H), 6.53 (d, J=7.8 Hz, 1H), 5.25 (AB quartet, JAB=12.1 Hz, ΔνAB=11.4 Hz, 2H), 4.94-4.87 (m, 1H), 3.96 (s, 3H), 2.35-2.14 (m, 2H), 2.14-1.96 (m, 2H).

Step 6. Synthesis of 5,5,5-trifluoro-N-[(4-methoxy-1H-indol-2-yl)carbonyl]-L-norvaline (C39).

A mixture of C38 (350 mg, 0.806 mmol) and palladium on carbon (10%, 85.7 mg, 80.5 μmol) in methanol (10 mL) was hydrogenated for 16 h at 20 °C and 15 psi. The reaction mixture was then filtered and the filter cake was washed with methanol (10 mL); the combined filtrates were concentrated in vacuo and chromatographed on silica gel (eluent: ethyl acetate) to give C39 as a white solid. Yield: 270 mg, 0.784 mmol, 97%. LCMS m/z 345.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.62 (br s, 1H), 8.61 (d, J=7.9 Hz, 1H), 7.32 (d, J=2.2 Hz, 1H), 7.11 (dd, J=8, 8 Hz, 1H), 7.01 (d, half of AB quartet, J=8.2 Hz, 1H), 6.51 (d, J=7.6 Hz, 1H), 4.47 (ddd, J=8.5, 8.5, 4.8 Hz, 1H), 3.89 (s, 3H), 2.5-2.27 (m, 2H, assumed; partially obscured by solvent peak), 2.12-1.92 (m, 2H).

Step 7. Synthesis of 5,5,5-trifluoro-N-[(4-methoxy-1H-indol-2-yl)carbonyl]-L-norvalyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C40).

0°C mixture of C16 (58.2 mg, 0.218 mmol) and C39 (75.0 mg, 0.218 mmol) in N,N-dimethylformamide (4 mL) was treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 99.4 mg, 0.261 mmol) and 4-methylmorpholine (44.1 mg, 0.436 mmol). The reaction mixture was then stirred at 0°C for 1 h, diluted with water (20 mL) and aqueous citric acid (1 M; 20 mL), and extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate (20 mL) and saturated aqueous sodium chloride (3×20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (eluent: 10:1 ethyl acetate/methanol) gave C40 as a white solid. Yield: 72 mg, 0.145 mmol, 66%. LCMS m/z 498.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.60 (br s, 1H), 8.52 (d, J=7.7 Hz, 1H), 8.20 (d, J=8.3 Hz, 1H), 7.61 (s, 1H), 7.42-7.33 (m, 2H), 7.14-7.05 (m, 2H), 7.00 (d, half of AB quartet, J=8.2 Hz, 1H), 6.51 (d, J=7.7 Hz, 1H), 4.58-4.46 (m, 1H), 4.32-4.22 (m, 1H), 3.89 (s, 3H), 3.18-3.02 (m, 2H), 2.45-2.21 (m, 3H), 2.18-2.07 (m, 1H), 2.06-1.88 (m, 3H), 1.73-1.59 (m, 1H), 1.59-1.48 (m, 1H).

Step 8. Synthesis of N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (14).

To a mixture of C40 (52 mg, 0.10 mmol) in dichloromethane (13 mL) was added methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 37 mg, 0.16 mmol). The reaction mixture was stirred at room temperature for 1.5 h, then methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 37 mg, 0.16 mmol) was added again and stirring was continued for 16 h. The final addition of methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 24.9 mg, 0.105 mmol) was then stirred for 2 h, then the reaction mixture was diluted with water (20 mL) and extracted with dichloromethane (3×10 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution (2×20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo; preparative thin-layer chromatography (eluent: 20:1 ethyl acetate/methanol) gave N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (14) as a white solid. Yield: 17.4 mg, 36.3 μmol, 36%. This product was combined with the purified products of two other syntheses 14 (3 mg and 4 mg) and subjected to supercritical fluid chromatography [Column: Chiral Technologies ChiralCel OD-H, 30×250 mm, 5 μm; Mobile phase: 7:3 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide); Flow rate: 60 mL/min] to give N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (14) as a solid. Yield: 11.3 mg, 23.6 μmol, 46% for supercritical fluid chromatography. LCMS m/z 480.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.61 (br s, 1H), 8.96 (d, J=8.0 Hz, 1H), 8.61 (d, J=7.7 Hz, 1H), 7.71 (s, 1H), 7.37 (d, J=2.2 Hz, 1H), 7.11 (dd, J=8.8 Hz, 1H), 7.01 (d, half of AB quartet, J=8.2 Hz, 1H), 6.51 (d, J=7.7 Hz, 1H), 5.03-4.94 (m, 1H), 4.51-4.43 (m, 1H), 3.89 (s, 3H), 3.19-3.07 (m, 2H), 2.43-2.28 (m, 3H), 2.20-2.08 (m, 2H), 2.06-1.92 (m, 2H), 1.86-1.76 (m, 1H), 1.76-1.64 (m, 1H).

Examples 15, 16, 17, 18, and 19

N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide (15), N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-fluoro-4-methoxy-1H-indole-2-carboxamide (16), N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3-fluoro-4-methoxy-1H-indole-2-carboxamide (17), N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5,7-difluoro-4-methoxy-1H-indole-2-carboxamide (18) and N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3,5-difluoro-4-methoxy-1H-indole-2-carboxamide (19)

A mixture of 4 (10.0 mg, 22.8 μmol), tetra-n-butylammonium decatungstate (TBADT; 3.78 mg, 1.14 μmol), and N-fluoro-N-(phenylsulfonyl)benzenesulfonamide (8.61 mg, 27.3 μmol) was treated with acetonitrile (0.75 mL), water (0.5 mL), and trifluoroacetic acid (1.74 μL, 22.6 μmol) under argon. The reaction tube was then sealed, placed in an EvoluChem™ PhotoRedOx Box equipped with a fan, and irradiated with black light (PAR20-18W LG 365 nm, 100-240 VAC) at 25°C for 16 h. Aqueous potassium phosphate solution (1 M, pH 7.45; 1 mL) was added to the reaction mixture, followed by alternate aliquots of water and acetonitrile to maintain the purified solution at a final volume of 18 mL. Aliquots (3 mL) of this mixture were applied to Biotage Isolute C18 solid phase extraction cartridges that had been pretreated with methanol (3 mL) then water (3 mL). The cartridges were washed with water (3 mL) and 20% acetonitrile in 20 mM aqueous ammonium acetate (3 mL), then eluted with acetonitrile (3 mL). After evaporation of the eluates in a vacuum centrifuge, the residues were reconstituted in a mixture of 1% aqueous formic acid and acetonitrile and combined to a total of 6 mL. This solution was divided in half and each half was subjected to reverse-phase HPLC (Column: Phenomenex Luna C18, 10×250 mm, 10 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile; gradient: 15% B for 5 min, then 15% - 70% B for 70 min, then 70% - 95% B for 15 min; flow rate: 2 mL/min). Fractions were collected every 20 seconds, and similar fractions of interest from the two splits were pooled and concentrated. These fractions were then purified by reversed-phase HPLC (Column: Agilent Polaris C18, 4.6×250 mm, 5 μm; Mobile phase A: water containing 10 mM ammonium acetate; Mobile phase B: acetonitrile; Gradient: 10% B for 5 min, then 10% - 35% B for 35 min, then 35% - 60% B for 15 min, then 60% - 95% B for 9 min; Flow rate: 0.8 mL/min). Fractions were collected every 20 seconds to yield N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide (15), N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-fluoro-4-methoxy-1H-indole-2-carboxamide (16), N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3-fluoro-4-methoxy-1H-indole-2-carboxamide (17), N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5,7-difluoro-4-methoxy-1H-indole-2-carboxamide (18), and N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3,5-difluoro-4-methoxy-1H-indole-2-carboxamide (19).

Example No. Retention time, first HPLC purification (minutes) Retention time, second HPLC purification (minutes) 15 57.1 45.1 16 57.7 45.9 17 58.6 46.7 18 59.9 47.9 19 61.7 49.5

15 - First split, fraction numbers 172-174; Second split, fraction numbers 136-137. Yield: 58 µg, 0.13 µmol, 0.6%. High-resolution MS m/z 458.2201 [M+H]+; calculated for C23H29FN5O4, 458.2204. 1H NMR (600 MHz, DMSO-d6) δ 12.05 (brs, 1H), 8.93 (d, J=7.9 Hz, 1H), 8.51 (d, J=7.6 Hz, 1H), 7.70 (s, 1H), 7.37 (d, J=2.3 Hz, 1H), 6.92 (dd, J=10.8, 8.6 Hz, 1H), 6.42 (dd, J=8.4, 2.5 Hz, 1H), 5.01-4.94 (m, 1H), 4.50-4.43 (m, 1H), 3.87 (s, 3H), 3.18-3.07 (m, 2H), 2.40-2.31 (m, 1H), 2.19-2.08 (m, 2H), 1.85-1.76 (m, 1H), 1.76-1.64 (m, 3H), 1.58-1.49 (m, 1H), 0.94 (d, J=6.3 Hz, 3H), 0.89 (d, J=6.3 Hz, 3H). Retention time: 7.90 minutes (Analytical conditions. Column: Phenomenex Kinetex XB-C18, 2.1×100 mm, 2.6 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile; Gradient: 5% B for 0.5 minutes, then 5% - 70% B for 10.5 minutes, then 70% - 95% B for 2 minutes; Flow rate: 0.4 mL/min).

16 - First split, fraction numbers 172-174; Second split, fraction numbers 138-139. Yield: 153 µg, 0.33 µmol, 1.4%. High-Resolution MS m/z 458.2201 [M+H]+; calculated for C23H29FN5O4, 458.2204. 1H NMR (600 MHz, DMSO-d6) δ 11.73 (brs, 1H), 8.95 (d, J=8.0 Hz, 1H), 8.61 (d, J=7.7 Hz, 1H), 7.70 (s, 1H), 7.49 (s, 1H), 7.09-7.03 (m, 2H), 5.01-4.94 (m, 1H), 4.51-4.43 (m, 1H), 4.06 (s, 3H), 3.18-3.07 (m, 2H), 2.40-2.31 (m, 1H), 2.19-2.08 (m, 2H), 1.80 (ddd, J=13.6, 9.2, 7.2 Hz, 1H), 1.76-1.65 (m, 3H), 1.58-1.50 (m, 1H), 0.94 (d, J=6.3 Hz, 3H), 0.89 (d, J=6.3 Hz, 3H). Retention time: 7.94 minutes (Analytical conditions are identical to those used for 15).

17 - First split, fraction numbers 176-177; Second split, fraction numbers 141-142. Yield: 22 µg, 0.048 µmol, 0.21%. High-resolution MS m/z 458.2199 [M+H]+; calculated for C23H29FN5O4, 458.2204. 1H NMR (600 MHz, DMSO-d6) δ 11.45 (s, 1H), 8.94 (d, J=7.8 Hz, 1H), 7.71 (s, 1H), 7.62 (bd, J=7.5 Hz, 1H), 7.16 (dd, J=8, 8 Hz, 1H), 6.95 (bd, J=8.3 Hz, 1H), 6.54 (d, J=7.8 Hz, 1H), 5.02-4.94 (m, 1H), 4.54-4.46 (m, 1H), 3.88 (s, 3H), 3.19-3.07 (m, 2H), 2.41-2.31 (m, 1H), 2.20-2.08 (m, 2H), 1.85-1.77 (m, 1H), 1.76-1.63 (m, 3H), 1.61-1.53 (m, 1H), 0.94 (d, J=6.3 Hz, 3H), 0.91 (d, J=6.3 Hz, 3H). Retention time: 8.06 minutes (Analytical conditions are identical to those used for 15).

18 - First split, fraction numbers 180-181; Second split, fraction number 145. Yield: 17 µg, 0.036 µmol, 0.16%. High-resolution MS m/z 476.2100 [M+H]+; calculated for C23H28F2N5O4, 476.2109. 1H NMR (600 MHz, DMSO-d6) δ 12.23 (s, 1H), 8.96 (d, J=8.0 Hz, 1H), 8.62 (d, J=7.6 Hz, 1H), 7.70 (s, 1H), 7.52-7.48 (m, 1H), 7.13 (dd, J=11, 11 Hz, 1H), 5.02-4.94 (m, 1H), 4.53-4.44 (m, 1H), 4.01 (s, 3H), 3.18-3.07 (m, 2H), 2.38-2.30 (m, 1H), 2.19-2.08 (m, 2H), 1.81 (ddd, J=13.6, 9.1, 7.0 Hz, 1H), 1.76-1.65 (m, 3H), 1.59-1.51 (m, 1H), 0.95 (d, J=6.2 Hz, 3H), 0.90 (d, J=6.3 Hz, 3H). Retention time: 8.20 minutes (Analytical conditions are identical to those used for 15).

19 - First split, fraction numbers 185-187; Second split, fraction numbers 150-151. Yield: 35 µg, 0.074 µmol, 0.32%. High-resolution MS m/z 476.2107 [M+H]+; calculated for C23H28F2N5O4, 476.2109. 1H NMR (600 MHz, DMSO-d6) δ 11.64 (s, 1H), 8.94 (d, J=7.9 Hz, 1H), 7.80 (bd, J=7 Hz, 1H), 7.71 (s, 1H), 7.16 (dd, component of the ABX system, J=11.9, 9.1 Hz, 1H), 7.08 (bd, half of the AB quartet, J=8.5 Hz, 1H), 5.02-4.94 (m, 1H), 4.55-4.47 (m, 1H), 3.99 (s, 3H), 3.19-3.08 (m, 2H), 2.41-2.32 (m, 1H), 2.19-2.10 (m, 2H), 1.81 (ddd, J=13.7, 9.0, 7.2 Hz, 1H), 1.77-1.63 (m, 3H), 1.57 (ddd, J=12.9, 8.4, 4.8 Hz, 1H), 0.94 (d, J=6.4 Hz, 3H), 0.91 (d, J=6.4 Hz, 3H). Retention time: 8.44 minutes (Analytical conditions are identical to those used for 15).

Examples 20, 21, 22, and 23

N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide (20), N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-fluoro-4-methoxy-1H-indole-2-carboxamide (21), N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-fluoro-4-methoxy-1H-indole-2-carboxamide (22) and N-[(2S)-1-({(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5,7-difluoro-4-methoxy-1H-indole-2-carboxamide (23)

A mixture of 10 (10.0 mg, 22.0 μmol), tetra-n-butylammonium decatungstate (TBADT; 3.66 mg, 1.10 μmol), and N-fluoro-N-(phenylsulfonyl)benzenesulfonamide (8.34 mg, 26.4 μmol) was treated with acetonitrile (0.75 mL), water (0.5 mL), and trifluoroacetic acid (1.69 μL, 21.9 μmol) under argon. The reaction tube was then sealed, placed in an EvoluChem™ PhotoRedOx Box equipped with a fan, and irradiated with black light (PAR20-18W LG 365 nm, 100-240 VAC) at 25°C for 16 h. Aqueous potassium phosphate solution (1 M, pH 7.45; 1 mL) was added to the reaction mixture, followed by alternate aliquots of water and acetonitrile to maintain the purified solution at a final volume of 18 mL. Aliquots (3 mL) of this mixture were applied to Biotage Isolute C18 solid-phase extraction cartridges, which had been pretreated with methanol (3 mL) followed by aqueous ammonium acetate (10 mM; 3 mL). The cartridges were washed with aqueous ammonium acetate (10 mM; 3 mL) and 20% acetonitrile in 20 mM ammonium acetate (3 mL), then eluted with acetonitrile (3 mL). After evaporation of the eluates in a vacuum centrifuge, the residues were reconstituted in a mixture of 1% aqueous formic acid and acetonitrile and combined to a total of 6 mL. This solution was divided in half and each half was subjected to reverse-phase HPLC (Column: Phenomenex Luna C18, 10×250 mm, 10 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile; Gradient: 15% B for 5 min, then 15% - 70% B for 70 min, then 70% - 95% B for 15 min; Flow rate: 2 mL/min). Fractions were collected every 20 seconds and similar fractions of interest from two splits were pooled and concentrated. These fractions were then purified by reversed-phase HPLC (Column: Agilent Polaris C18, 4.6×250 mm, 5 μm; Mobile phase A: water containing 10 mM ammonium acetate; Mobile phase B: acetonitrile; Gradient: 10% B for 5 min, then immediately increase to 20% B, then 20% - 40% B for 35 min, then 40% - 60% B for 15 min, then 60% - 95% B for 9 min; Flow rate: 0.8 mL/min). Fractions were collected every 20 seconds to yield N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide (20), N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-fluoro-4-methoxy-1H-indole-2-carboxamide (21), N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-fluoro-4-methoxy-1H-indole-2-carboxamide (22) and N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5,7-difluoro-4-methoxy-1H-indole-2-carboxamide (23).

Example No. Retention time, first HPLC purification (minutes) Retention time, second HPLC purification (minutes) 20 61.2 49.8 21 61.2 50.2 22 62.3 50.8 23 63.5 51.9

20 - First split, fraction numbers 183-185; Second split, fraction numbers 150-151. Yield: 24 µg, 0.051 µmol, 0.23%. High-resolution MS m/z 472.2342 [M+H]+; calculated for C24H31FN5O4, 472.2360. 1H NMR (600 MHz, DMSO-d6) δ 12.05 (brs, 1H), 8.91 (d, J=8.0 Hz, 1H), 8.52 (d, J=8.0 Hz, 1H), 7.69 (s, 1H), 7.37-7.32 (m, 1H), 6.92 (dd, J=10.9, 8.4 Hz, 1H), 6.41 (dd, J=8.5, 2.7 Hz, 1H), 5.00-4.93 (m, 1H), 4.52 (ddd, J=8.5, 8.2, 3.7 Hz, 1H), 3.87 (s, 3H), 3.17-3.05 (m, 2H), 2.38-2.30 (m, 1H), 2.18-2.06 (m, 2H), 1.85-1.64 (m, 4H), 0.94 (s, 9H). Retention time: 8.32 minutes (Analytical conditions. Column: Phenomenex Kinetex XB-C18, 2.1×100 mm, 2.6 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile; Gradient: 5% B for 0.5 minutes, then 5% - 70% B for 10.5 minutes, then 70% - 95% B for 2 minutes; Flow rate: 0.4 mL/minute).

21 - First split, fraction numbers 183-185; Second split, fraction numbers 152-153. Yield: 68 µg, 0.14 µmol, 0.64%. High-resolution MS m/z 472.2344 [M+H]+; calculated for C24H31FN5O4, 472.2360. 1H NMR (600 MHz, DMSO-d6) δ 11.72 (brs, 1H), 8.91 (d, J=8.1 Hz, 1H), 8.59 (d, J=8.0 Hz, 1H), 7.69 (s, 1H), 7.47 (d, J=2.2 Hz, 1H), 7.09-7.04 (m, 2H), 5.00-4.93 (m, 1H), 4.52 (ddd, J=8.5, 8.5, 3.8 Hz, 1H), 4.06 (shs, 3H), 3.17-3.05 (m, 2H), 2.39-2.31 (m, 1H), 2.18-2.06 (m, 2H), 1.84-1.77 (m, 1H), 1.78 (dd, J=13.9, 9.0 Hz, 1H), 1.74-1.64 (m, 2H), 0.94 (s, 9H). Retention time: 8.34 minutes (Analytical conditions are identical to those used for 20).

22 - First split, fraction numbers 187-188; Second split, fraction number 154. Yield: 5 µg, 0.011 µmol, 0.05%. High-resolution MS m/z 472.2354 [M+H]+; calculated for C24H31FN5O4, 472.2360. 1H NMR (600 MHz, DMSO-d6) δ 11.45 (s, 1H), 8.91 (d, J=7.9 Hz, 1H), 7.70 (s, 1H), 7.57 (dd, J=8.2, 3.6 Hz, 1H), 7.15 (dd, J=8, 8 Hz, 1H), 6.95 (bd, J=8.3 Hz, 1H), 6.54 (d, J=7.8 Hz, 1H), 5.00-4.94 (m, 1H), 4.56-4.49 (m, 1H), 3.88 (s, 3H), 3.18-3.07 (m, 2H), 2.40-2.32 (m, 1H), 2.17-2.09 (m, 2H), 1.84-1.77 (m, 1H), 1.76-1.65 (m, 3H), 0.95 (s, 9H). Retention time: 8.51 minutes (Analytical conditions are identical to those used for 20).

23 - First split, fraction numbers 190-192; Second split, fraction numbers 156-157. Yield: 21 µg, 0.043 µmol, 0.19%. High-resolution MS m/z 490.2258 [M+H]+; calculated for C24H30F2N5O4, 490.2266. 1H NMR (600 MHz, DMSO-d6) δ 12.24 (s, 1H), 8.95 (d, J=8.0 Hz, 1H), 8.64 (d, J=8.0 Hz, 1H), 7.69 (s, 1H), 7.49-7.47 (m, 1H), 7.13 (dd, J=11.1, 11.1 Hz, 1H), 5.00-4.93 (m, 1H), 4.54 (ddd, J=8, 8, 4.1 Hz, 1H), 4.00 (s, 3H), 3.17-3.06 (m, 2H), 2.38-2.30 (m, 1H), 2.18-2.07 (m, 2H), 1.85-1.65 (m, 4H), 0.95 (s, 9H). Retention time: 8.65 minutes (Analytical conditions are identical to those used for 20).

Table 1. Synthesis method, structure and physicochemical data for examples 24–74. No.pr. Synthesis method; Non-commercial starting materials Structure 1H NMR (600 MHz, DMSO-d6) δ; Mass spectrum, observed ion m/z [M+H]+ or HPLC retention time; Mass spectrum m/z [M+H]+ (unless otherwise stated) 24 Example 10; C18 1H NMR (400 MHz, methanol-d4) δ 8.58 (s, 1H), 5.04 (dd, J=9.9, 6.1 Hz, 1H), 4.64 (dd, J=8.7, 4.1 Hz, 1H), 3.34-3.23 (m, 2H; predicted; partially obscured by solvent peak), 2.61-2.50 (m, 1H), 2.35-2.24 (m, 2H), 1.96-1.76 (m, 4H), 1.01 (s, 9H); 460.4 25 Examples 8 and 91.2; C28
or

DIAST-1 2.29 minutes3; 391.4 26 Examples 8 and 91.2; C28
or

DIAST-2 2.69 minutes3; 391.4 27 Example 7; C28 2.18 minutes4; 381.4 28 Example 7; C28 2.61 minutes4; 421.5 29 Example 7; C28 2.31 minutes4; 419.4 30 Example 71; C28 1.59 minutes4; 442.55 31 C296
or
2.06 minutes4; 416.5 32 C296
or
2.67 minutes4; 435.6 33 Example 47 11.40 (s, 1H), 9.60 (shs, 1H), 8.90 (d, J=8.1 Hz, 1H), 8.44 (d, J=7.9 Hz, 1H), 7.70 (s, 1H), 7.31 (s, 1H), 6.95 (dd, J=8, 8 Hz, 1H), 6.85 (d, J=8.2 Hz, 1H), 6.36 (d, J=7.5 Hz, 1H), 5.02-4.93 (m, 1H), 4.50-4.41 (m, 1H), 3.17-3.07 (m, 2H), 2.41-2.31 (m, 1H), 2.19-2.08 (m, 2H), 1.80 (ddd, J=13.6, 9.4, 6.8 Hz, 1H), 1.76-1.65 (m, 3H), 1.57-1.48 (m, 1H), 0.94 (d, J=6.3 Hz, 3H), 0.89 (d, J=6.3 Hz, 3H); High-Resolution MS m/z 426.2139 [M+H]+; calcd for C22H28N5O4, 426.2141 34 Example 47 11.34 (s, 1H), 8.91 (d, J=8.1 Hz, 1H), 8.46 (d, J=7.9 Hz, 1H), 7.69 (s, 1H), 7.29 (s, 1H), 6.95 (d, J=8.6 Hz, 1H), 6.78 (d, J=8.6 Hz, 1H), 5.02-4.93 (m, 1H), 4.50-4.41 (m, 1H), 3.91 (s, 3H), 3.18-3.06 (m, 2H, inferred; partially covered by solvent peak), 2.39-2.31 (m, 1H), 2.20-2.07 (m, 2H), 1.84-1.65 (m, 4H), 1.58-1.49 (m, 1H), 0.94 (d, J=6.2 Hz, 3H), 0.89 (d, J=6.2 Hz, 3H); high-resolution MS m/z 456.2238 [M+H]+; calculated for C23H30N5O5, 456.2247 35 Example 118; C18 2.39 minutes4; 437.4 (chlorine isotope pattern observed) 36 Example 11; C18 1H NMR (600 MHz, methanol-d4) δ 7.45-7.40 (m, 2H), 7.34-7.27 (m, 3H), 4.98 (dd, J=10.5, 5.6 Hz, 1H), 4.27 (dd, J=8.4, 4.3 Hz, 1H), 3.70 (s, 1H), 3.28-3.23 (m, 1H), 3.20 (ddd, J=9.5, 9.3, 7.1 Hz, 1H), 2.57-2.49 (m, 1H), 2.27 (ddd, J=13.8, 10.5, 5.2 Hz, 1H), 2.21-2.13 (m, 1H), 2.17 (s, 6H), 1.85 (ddd, J=13.8, 9.7, 5.7 Hz, 1H), 1.79-1.70 (m, 1H), 1.67 (dd, component of the ABX system, J=14.4, 4.3 Hz, 1H), 1.59 (dd, component of the ABX system, J=14.4, 8.4 Hz, 1H), 0.84 (s, 9H); 442.5 37 Examples 5 and 69; Example 10 12.13 (s, 1H), 8.96 (bd, J=7.6 Hz, 2H), 7.69 (s, 1H), 7.21 (dd, J=8, 8 Hz, 1H), 7.08 (d, J=8.2 Hz, 1H), 6.68 (d, J=7.8 Hz, 1H), 5.00-4.92 (m, 1H), 4.52-4.45 (m, 1H), 3.86 (s, 3H), 3.20-3.15 (m, 1H), 3.14-3.08 (m, 1H), 2.43-2.34 (m, 1H), 2.23-2.13 (m, 2H), 1.85-1.78 (m, 1H), 1.78-1.68 (m, 2H), 1.59 (dd, J=14.1, 6.6 Hz, 1H), 0.95 (s, 9H); high-resolution MS m/z 522.2321 [M+H]+; calculated for C25H31F3N5O4, 522.2328; retention time 7.18 min10 38 Examples 5 and 69; Example 10 11.41 (s, 1H), 8.96 (d, J=7.7 Hz, 1H), 8.79 (d, J=8.0 Hz, 1H), 7.65 (s, 1H), 7.55 (d, J=8.2 Hz, 1H), 7.34 (s, 1H), 6.72 (d, J=8.3 Hz, 1H), 5.01-4.94 (m, 1H), 4.62-4.55 (m, 1H), 3.97 (s, 3H), 3.17-3.11 (m, 1H), 3.11-3.04 (m, 1H), 2.38-2.30 (m, 1H), 2.18-2.07 (m, 2H), 1.86-1.78 (m, 1H), 1.77-1.64 (m, 3H), 0.95 (s, 9H); high-resolution MS m/z 522.2316 [M+H]+; calculated for calculated for C25H31F3N5O4, 522.2328; retention time 7.45 minutes10 39 Examples 5 and 69; Example 10 9.08 (shs, 1H), 9.03 (d, J=8.0 Hz, 1H), 7.65 (s, 1H), 7.60 (d, J=8.3 Hz, 1H), 6.84 (d, J=8.2 Hz, 1H), 4.98-4.91 (m, 1H), 4.53-4.47 (m, 1H), 3.95 (s, 3H), 3.20-3.14 (m, 1H), 3.13-3.06 (m, 1H), 2.44-2.36 (m, 1H), 2.23-2.12 (m, 2H), 1.85 (dd, J=13.9, 7.8 Hz, 1H), 1.80 (ddd, J=13.6, 9.6, 6.5 Hz, 1H), 1.76-1.68 (m, 1H), 1.54 (dd, J=13.9, 5.1 Hz, 1H), 0.95 (s, 9H); high-resolution MS m/z 590.2177 [M+H]+; calculated for C26H30F6N5O4, 590.2202; retention time 7.70 min10 40 Examples 5 and 69; Example 10 12.76 (shs, 1H), 9.19-9.10 (m, 1H), 9.06-9.00 (m, 1H), 7.69 (s, 1H), 7.49 (AB quartet, JAB=8.6 Hz, ΔνAB=48.9 Hz, 2H), 5.01-4.93 (m, 1H), 4.54-4.47 (m, 1H), 3.85 (s, 3H), 3.21-3.15 (m, 1H), 3.15-3.07 (m, 1H), 2.43-2.35 (m, 1H), 2.22-2.13 (m, 2H), 1.86-1.78 (m, 1H), 1.78-1.70 (m, 2H), 1.59 (dd, component of the ABX system, J=14.1, 6.5 Hz, 1H), 0.96 (s, 9H); high-resolution MS m/z 590.2181 [M+H]+; calculated for C26H30F6N5O4, 590.2202; retention time 7.79 minutes10 41 Examples 5 and 69, 11; Example 10 12.62 (s, 1H), 9.14 (d, J=7.5 Hz, 1H), 9.03 (d, J=7.8 Hz, 1H), 7.70 (s, 1H), 7.42 (s, 1H), 6.92 (s, 1H), 5.00-4.93 (m, 1H), 4.54-4.47 (m, 1H), 3.96 (s, 3H), 3.21-3.15 (m, 1H), 3.15-3.08 (m, 1H), 2.43-2.35 (m, 1H), 2.22-2.13 (m, 2H), 1.86-1.79 (m, 1H), 1.78-1.69 (m, 2H), 1.59 (dd, component of the ABX system, J=14.0, 6.4 Hz, 1H), 0.96 (s, 9H); high-resolution MS m/z 590.2175 [M+H]+; calculated for C26H30F6N5O4, 590.2202; retention time 7.84 minutes10 42 Examples 5 and 612; Example 36 8.92 (d, J=8.0 Hz, 1H), 8.45 (d, J=7.9 Hz, 1H), 7.71 (s, 2H), 7.67 (d, J=7.6 Hz, 1H), 7.64-7.60 (m, 1H), 7.58-7.52 (m, 1H), 4.99-4.87 (m, 1H), 4.24-4.17 (m, 1H), 3.92 (s, 1H), 3.20-3.10 (m, 1H), 3.09-3.00 (m, 1H), 2.36-2.27 (m, 1H), 2.19-2.00 (m, 2H), 2.08 (s, 6H), 1.81-1.64 (m, 2H), 1.49 (d, J=6.5 Hz, 2H), 0.74 (s, 9H); high-resolution MS m/z 510.2679 [M+H]+; calculated for C25H35F3N5O3, 510.2692; retention time 5.83 min10 43 Examples 5 and 612; Example 36 8.93 (d, J=8.1 Hz, 1H), 8.42 (d, J=8.1 Hz, 1H), 7.71 (s, 1H), 7.63 (AB quartet, JAB=7.6 Hz, ΔνAB=48.8 Hz, 4H), 4.97-4.88 (m, 1H), 4.26-4.19 (m, 1H), 3.91 (s, 1H), 3.19-3.11 (m, 1H), 3.07-3.00 (m, 1H), 2.35-2.26 (m, 1H), 2.18-1.99 (m, 2H), 2.08 (s, 6H), 1.80-1.64 (m, 2H), 1.56-1.44 (m, 2H), 0.77 (s, 9H); high-resolution MS m/z 510.2676 [M+H]+; calculated for C25H35F3N5O3, 510.2692; retention time 5.92 min10 44 Alternative synthesis of example 6; C26 2.85 minutes4; 478.6 45 Alternative synthesis of example 6; C18 2.95 minutes4; 492.6 46 Examples 5 and 613; 4 12.48 (s, 1H), 9.13 (d, J=7.4 Hz, 1H), 8.97 (d, J=7.7 Hz, 1H), 7.70 (s, 1H), 7.62 (d, J=8.3 Hz, 1H), 6.86 (d, J=8.3 Hz, 1H), 5.04-4.95 (m, 1H), 4.53-4.44 (m, 1H), 3.96 (s, 3H), 3.22-3.14 (m, 1H), 3.14-3.06 (m, 1H), 2.44-2.34 (m, 1H), 2.24-2.10 (m, 2H), 1.87-1.77 (m, 1H), 1.77-1.61 (m, 2H), 1.61-1.50 (m, 2H), 0.98-0.89 (m, 6H); high-resolution MS m/z 576.2055 [M+H]+; calculated for C25H28F6N5O4, 576.2045; retention time 6.70 min14 47 Examples 5 and 613; 4 12.61 (s, 1H), 9.23 (d, J=7.6 Hz, 1H), 9.01 (d, J=7.9 Hz, 1H), 7.71 (s, 1H), 7.20 (s, 1H), 5.04-4.95 (m, 1H), 4.54-4.45 (m, 1H), 4.07 (s, 3H), 3.22-3.14 (m, 1H), 3.14-3.06 (m, 1H), 2.43-2.34 (m, 1H), 2.23-2.11 (m, 2H), 1.82 (ddd, J=13.5, 9.0, 7.0 Hz, 1H), 1.78-1.61 (m, 2H), 1.61-1.49 (m, 2H), 0.99-0.89 (m, 6H); high-resolution MS m/z 644.1914 [M+H]+; calculated for C26H27F9N5O4, 644.1919; retention time 7.43 min14 48 Examples 5 and 613; 4 13.21 (s, 1H), 9.28 (d, J=7.5 Hz, 1H), 9.05 (d, J=7.8 Hz, 1H), 7.86 (s, 1H), 7.72 (s, 1H), 5.04-4.96 (m, 1H), 4.54-4.47 (m, 1H), 3.93 (s, 3H), 3.22-3.15 (m, 1H), 3.15-3.07 (m, 1H), 2.43-2.35 (m, 1H), 2.23-2.11 (m, 2H), 1.82 (ddd, J=13.7, 9.0, 6.9 Hz, 1H), 1.78-1.63 (m, 2H), 1.61-1.51 (m, 2H), 0.98-0.91 (m, 6H); high-resolution MS m/z 644.1901 [M+H]+; calculated for C26H27F9N5O4, 644.1919; retention time 7.51 min14 49 Alternative synthesis of example 6; C26 2.95 minutes4; 494.4 50 Alternative synthesis of example 6; C18 3.04 minutes4; 508.4 51 Alternative synthesis of example 6; C18 3.00 minutes4; 492.4 52 Alternative synthesis of example 6; C18 2.84 minutes4; 458.4 (chlorine isotope pattern observed) 53 Alternative synthesis of example 6; C18 2.80 minutes4; 468.5 54 Alternative synthesis of example 6; C18 2.89 minutes4; 458.4 (chlorine isotope pattern observed) 55 Alternative synthesis of example 6; C18 2.90 minutes4; 458.4 (chlorine isotope pattern observed) 56 Alternative synthesis of example 6; C18 2.89 minutes4; 458.4 (chlorine isotope pattern observed) 57 Alternative synthesis of example 6; C18 2.95 minutes4; 492.4 58 Alternative synthesis of example 6; C18 3.16 minutes4; 492.3 (dichlorine isotope pattern observed) 59 Alternative synthesis of example 6; C18 2.99 minutes4; 492.4 60 Alternative synthesis of example 6; C26 2.91 minutes4; 478.4 61 Alternative synthesis of example 6; C26 2.74 minutes4; 444.4 (chlorine isotope pattern observed) 62 Alternative synthesis of example 6; C26 2.70 minutes4; 454.4 63 Alternative synthesis of example 6; C26 2.80 minutes4; 444.4 (chlorine isotope pattern observed) 64 Alternative synthesis of example 6; C26 2.81 minutes4; 444.4 (chlorine isotope pattern observed) 65 Alternative synthesis of example 6; C26 2.80 minutes4; 444.4 (chlorine isotope pattern observed) 66 Alternative synthesis of example 6; C26 2.86 minutes4; 478.4 67 Alternative synthesis of example 6; C26 3.08 minutes4; 478.3 (dichlorine isotope pattern observed) 68 Alternative synthesis of example 6; C26 2.91 minutes4; 478.4 69 Examples 5 and 615; 7 9.00 (d, J=7.9 Hz, 1H), 8.83 (d, J=7.9 Hz, 1H), 8.01 (s, 1H), 7.72 (s, 1H), 5.01-4.93 (m, 1H), 4.44 (ddd, J=8, 8, 4.4 Hz, 1H), 3.20-3.14 (m, 1H), 3.14-3.08 (m, 1H), 2.66 (s, 3H), 2.42-2.34 (m, 1H), 2.20-2.08 (m, 2H), 1.81 (ddd, J=13.5, 9.4, 6.7 Hz, 1H), 1.77-1.64 (m, 3H), 0.95 (s, 9H); high-resolution MS m/z 513.1873 [M+H]+; calculated for C22H28F3N6O3S, 513.1896; retention time 6.88 min10 70 Alternative synthesis of example 6; C18 2.87 minutes4; 474.5 71 Alternative synthesis of example 6; C18 2.70 minutes4; 474.5 72 Alternative synthesis of example 6; C26 2.41 minutes4; 481.5 (bromine isotope pattern observed) 73 Alternative synthesis of example 6; C26 2.30 minutes4; 423.5 (chlorine isotope pattern observed) 74 416 12.20 (s, 1H), 8.99 (d, J=7.9 Hz, 1H), 8.97 (d, J=7.4 Hz, 1H), 7.70 (s, 1H), 7.24 (dd, J=8, 8 Hz, 1H), 7.12 (d, half AB quartet, J=8.2 Hz, 1H), 6.69 (d, J=7.8 Hz, 1H), 5.01-4.93 (m, 1H), 4.47-4.41 (m, 1H), 3.89 (s, 3H), 3.18-3.08 (m, 2H), 2.57 (s, 3H), 2.40-2.32 (m, 1H), 2.19-2.09 (m, 2H), 1.81 (ddd, J=13.5, 9.3, 6.8 Hz, 1H), 1.75-1.63 (m, 2H), 1.59 (dd, J=7.5, 6.8 Hz, 2H), 0.95 (d, J=6.5 Hz, 3H), 0.91 (d, J=6.5 Hz, 3H); high-resolution MS m/z 482.2391 [M+H]+; calculated for C25H32N5O5, 482.2403; retention time 8.38 min10

1. In this case, C28 is deprotected using methanesulfonic acid rather than the hydrochloride.

2. The epimers of Example 25 and Example 26 were separated by supercritical fluid chromatography (Column: Chiral Technologies Chiralpak IB, 21×250 mm, 5 μm; Mobile phase: 9:1 carbon dioxide/methanol; Back pressure: 120 bar, Flow rate: 75 ml/min). The first eluted diastereomer was designated Example 25, and the second eluted diastereomer was designated Example 26.

3. Conditions for analytical HPLC. Column: Chiral Technologies Chiralpak IB, 4.6×100 mm, 5 μm; Mobile phase: 85:15 carbon dioxide/methanol; Back pressure: 120 bar; Flow rate: 1.5 ml/minute.

4. Analytical HPLC conditions. Column: Waters Atlantis dC18, 4.6×50 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v); Gradient: 5.0% - 95% B, linear, for 4.0 minutes, then 95% B for 1.0 minute; Flow rate: 2 mL/minute.

5. 1H NMR of Example 30 before final purification: 1H NMR (400 MHz, methanol-d4) δ 7.48-7.42 (m, 2H), 7.36-7.26 (m, 3H), 4.90 (dd, J=10.5, 5.7 Hz, 1H), 4.37 (dd, J=7.7, 4.9 Hz, 1H), 3.69 (s, 1H), 3.25 (ddd, J=9.9, 8.9, 2.5 Hz, 1H), 3.18 (ddd, J=9.6, 9.0, 7.1 Hz, 1H), 2.40-2.29 (m, 1H), 2.20 (s, 6H), 2.2-2.10 (m, 1H), 2.09-1.99 (m, 1H), 1.80-1.61 (m, 2H), 1.73 (dd, J=14.5, 5.0 Hz, 1H), 1.61 (dd, J=14.4, 7.8 Hz, 1H), 0.95 (s, 9H).

6. The amide coupling with a suitable carboxylic acid is carried out using 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane trioxide.

7. Example 4 (25 μM) is incubated with human cytochrome P450 3A5 (4 nmol) in potassium phosphate buffer (100 mM, pH 7.4; 40 ml) containing magnesium chloride (3.3 mM) and NADPH (1.3 mM). Incubation is carried out for 0.75 hours in a shaking water bath maintained at 37°C. Incubation is terminated by adding an equal volume of acetonitrile, the mixture is then spun at 1700 x g for 5 minutes, and the supernatant is vacuum centrifuged for approximately 1.5 hours. To this mixture were added formic acid (0.5 ml), acetonitrile (0.5 ml) and water to a final volume of 50 ml, and the resulting mixture was centrifuged at 40,000 x g for 30 min. The supernatant was subjected to reverse-phase HPLC (Column: Polaris C18, 4.6×250 mm; 5 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: methanol; Gradient: 15% B for 5 min, then 15% - 35% B for 75 min, then 35% - 95% B for 10 min; Flow rate: 0.8 ml/min). Fractions were collected every 20 s. The first eluted product, impure Example 33, elutes at 54.7 minutes, and Example 34 elutes at 55.3 minutes. Impure Example 33 is re-purified using reverse-phase HPLC (Column: Phenomenex Kinetex XB-C18, 2.1×100 mm, 2.6 μm; Mobile Phase A: water containing 0.5% acetic acid; Mobile Phase B: 9:1 acetonitrile/methanol; Gradient: 10% B for 0.5 minutes, then 10% - 35% for 26.5 minutes, then 35% - 60% B for 3 minutes; Flow rate 0.5 mL/minute); fractions are collected every 15 seconds. On this system, Example 33 has a retention time of 12.7 minutes; Additional example 34 elutes at 13.5 minutes.

8. The required 4-chloro-1,3-dimethyl-1H-pyrazole-5-carboxylic acid can be obtained by hydrolysis of the commercially available ethyl ester.

9. The reaction mixture is diluted with acetonitrile and 1% aqueous formic acid to a volume of approximately 2 ml; the composition of the final solvent is such that the mixture is clear, with an acetonitrile content of approximately 20% - 30%. The components of this mixture are separated by reverse-phase HPLC (Column: Phenomenex Luna C18, 10 x 250 mm, 10 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile; Gradient: 15% B for 5 min, then 15% - 70% B for 70 min, then 70% - 95% B for 15 min; Flow rate: 2 ml/minute); fractions are collected every 20 seconds. Examples 37, 38, 39, 40, and 41 elute with the retention times given below.

Example Hold time (minutes) 37 64.9 38 68.4 39 72.1 40 73.5 41 74.2

10. Analytical HPLC conditions. Column: Phenomenex Kinetex XB-C18, 2.1×100 mm, 2.6 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile; Gradient: 5% B for 0.5 min, then 5% - 70% B for 10.5 min, then 70% - 95% B for 2 min; Flow rate: 0.4 mL/min.

11. The regiochemistry of Example 41 has not been strictly determined; other possible structures for this example are N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-5,6-bis(trifluoromethyl)-1H-indole-2-carboxamide and N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-6,7-bis(trifluoromethyl)-1H-indole-2-carboxamide.

12. The reaction mixture was purified using the conditions described in Note 9. Example 42 eluted at 58.1 minutes and Example 43 eluted at 59.2 minutes.

13. The reaction mixture was diluted with a mixture of acetonitrile (0.3 mL) and 1% aqueous formic acid (0.7 mL). The resulting mixture was centrifuged and the supernatant was subjected to reverse-phase HPLC (Column: Phenomenex Luna C18, 10×250 mm, 10 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile; Gradient: 2% - 10% B for 5.0 min, then 10% - 95% B for 95 min; Flow rate: 2 mL/min); fractions were collected every 20 seconds. Examples 46, 47, and 48 eluted with the retention times given below. Example 5 was also isolated from this reaction, in fractions 189-190.

Example Fraction number 46 207 47 225-226 48 231-232

14. Conditions for analytical HPLC. Column: Phenomenex Kinetex C18, 2.1×50 mm, 1.7 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile containing 0.1% formic acid; Gradient: 5% B for 0.5 min, then 5% - 50% B for 6.0 min, then 50% - 80% B for 1.5 min, then 80% - 95% B for 1.0 min; Flow rate: 0.4 mL/min.

15. Only the specified product is observed in this reaction.

16. A stock solution of Example 4 (5.56 mg, 12.7 μmol) and trifluoroacetic acid (4 μl, 50 μl) in dimethyl sulfoxide (420 μl) was prepared. One sixth of this solution was treated with sodium 1,1-difluoroethanesulfinate (1.3 mg, 8.5 μmol), then tert-butyl hydrogen peroxide (70% in water; 1.4 μl, 10 μmol) and heated at 50°C overnight. The reaction mixture was diluted with acetonitrile and 1% aqueous formic acid to a volume of approximately 2-3 ml; the final solvent composition was such that the mixture was clear, with an acetonitrile content of approximately 20% to 30%. The components of this mixture were separated by reversed-phase HPLC (Column: Phenomenex Luna C18, 10×250 mm, 10 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile; Gradient: 15% B for 5 min, then 15% - 40% B for 70 min, then 40% - 95% B for 15 min; Flow rate: 2 ml/minute); fractions were collected every 20 seconds. Example 74 eluted at 68.6 minutes.

Examples 75 and 76

(2S,4R)-4-tert-Butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide and (2R,4S)-4-tert-Butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide [75 (DIAST-1) and 76 (DIAST-2)

Step 1. Synthesis of tert-butyl N-[(trifluoromethyl)sulfonyl]-L-valinate (C44).

A solution of trifluoromethanesulfonic anhydride (8.88 mL, 52.8 mmol) in dichloromethane (10 mL) was added to a -78°C solution of tert-butyl L-valinate hydrochloride (10.0 g, 47.7 mmol) and triethylamine (18.7 mL, 134 mmol) in dichloromethane (90 mL). The reaction mixture was stirred at -78°C for 2 h, then it was poured into water and acidified to a pH of approximately 4 by the addition of 1 M hydrochloric acid. The resulting mixture was extracted with dichloromethane, and the organic layer was washed with aqueous sodium bicarbonate and saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was combined with the products of two analogous reactions using tert-butyl L-valinate, hydrochloride (1.00 g, 4.77 mmol; 1.00 g, 4.77 mmol) and purified by silica gel chromatography (gradient: 0% to 20% ethyl acetate in petroleum ether) to give C44 as a white solid. Combined yield: 14.0 g, 45.9 mmol, 80%. 1H NMR (400 MHz, DMSO-d6) δ 9.92 (d, J=8.8 Hz, 1H), 3.68 (dd, J=8.8, 6.2 Hz, 1H), 2.16-2.02 (m, 1H), 1.43 (s, 9H), 0.92 (d, J=6.9 Hz, 3H), 0.90 (d, J=6.9 Hz, 3H).

Step 2. Synthesis of N-[(trifluoromethyl)sulfonyl]-L-valine (C45).

Trifluoroacetic acid (85 mL) was added to a solution of C44 (14.0 g, 45.9 mmol) in dichloromethane (85 mL). The reaction mixture was stirred at room temperature for 3 h, then concentrated in vacuo; the residue was washed with petroleum ether to give C45 as a white solid. Yield: 10.9 g, 43.7 mmol, 95%. MS m/z 248.0 [M-H]-. 1H NMR (400 MHz, DMSO-d6) δ 9.86 (bd, J=8.3 Hz, 1H), 3.79-3.71 (m, 1H), 2.19-2.05 (m, 1H), 0.93 (d, J=6.8 Hz, 3H), 0.90 (d, J=6.8 Hz, 3H).

Step 3. Synthesis of methyl cis-4-tert-butylpiperidine-2-carboxylate, hydrochloride (C46).

To a 0°C solution of cis-4-tert-butylpiperidine-2-carboxylic acid, hydrochloride (see R. T. Shuman et al., J. Org. Chem. 1990, 55, 738-741; 4.00 g, 18.0 mmol) in methanol (40 mL) was added thionyl chloride (6.44 g, 54.1 mmol). The reaction mixture was then stirred at 25°C for 16 h and concentrated in vacuo to give C46 as an off-white solid (4.50 g). A portion of this product was used in the next step. LCMS m/z 200.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.46 (br s, 1H), 9.09 (br s, 1H), 4.11-3.96 (m, 1H), 3.76 (s, 3H), 3.4-3.21 (m, 1H, predicted; significantly obscured by the water peak), 2.93-2.77 (m, 1H), 2.07 (br d, J=10.8 Hz, 1H), 1.75 (br d, J=10.6 Hz, 1H), 1.51-1.32 (m, 3H), 0.84 (s, 9H).

Step 4. Synthesis of methyl (2S,4R)-4-tert-butyl 1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxylate and methyl (2R,4S)-4-tert-butyl 1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxylate (C47).

To a 25°C mixture of C45 (300 mg, 1.20 mmol) and C46 (from the previous step; 341 mg, ≤1.36 mmol) in N,N-dimethylformamide (3 mL) was added 4-methylmorpholine (365 mg, 3.61 mmol). The resulting mixture was cooled to 0°C and treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HFRU; 549 mg, 1.44 mmol). The reaction mixture was then purged with nitrogen for 1 min and stirred at 25°C for 12 h. LCMS analysis at this point showed the presence of C47: LCMS m/z 431.1 [M+H]+. The reaction mixture was partitioned between ethyl acetate (20 mL) and water (20 mL), and the aqueous layer was extracted with ethyl acetate (20 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution (4×20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (gradient: 0% to 20% ethyl acetate in petroleum ether) gave C47 as a yellow gum. 1H NMR analysis confirmed that the product contained a mixture of diastereomers. Yield: 320 mg, 0.743 mmol, 62%. 1H NMR (400 MHz, chloroform-d) δ 6.12-5.94 (m, 1H), [4.51 (dd, J=11.7, 6.3 Hz) and 4.32-4.18 (m), total 2H], [3.73 (s) and 3.71 (s), total 3H], [3.63-3.49 (m) and 3.48-3.39 (m), total 2H], 2.18-1.93 (m, 2H), 1.91-1.77 (m, 1H), 1.63-1.37 (m, 2H), 1.37-1.22 (m, 1H), 1.13-1.04 (m, 3H), [0.94 (d, J=6.8 Hz) and 0.91 (d, J=6.8 Hz), total 3H], 0.87 (s, 9H).

Step 5. Synthesis of (2S,4R)-4-tert-butyl-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxylic acid and (2R,4S)-4-tert-butyl-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxylic acid (C48).

A solution of C47 (314 mg, 0.729 mmol) in a mixture of methanol (2 mL) and tetrahydrofuran (2 mL) was treated with a solution of lithium hydroxide monohydrate (91.8 mg, 2.19 mmol) in water (1.4 mL) and the reaction mixture was stirred at 25 °C for 3 h. After removal of the solvent in vacuo, the residue was diluted with water (10 mL) and acidified to pH ∼1 by addition of 1 M hydrochloric acid. The resulting mixture was extracted with ethyl acetate (2 x 20 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (15 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give C48 as a yellow glass. H NMR analysis confirmed that the product contained a mixture of diastereomers. Yield: 304 mg, quantitative. 1H NMR (400 MHz, DMSO-d6) δ [9.82 (d, J=8.7 Hz) and 9.69 (bd, J=8.8 Hz), total 1H], [4.28 (dd, J=11.5, 6.4 Hz), 4.24-4.14 (m), and 4.05-3.96 (m), total 2H], [3.80-3.69 (m) and 3.6-3.2 (m, inferred; essentially covered by water peak), total 2H], 2.06-1.90 (m, 2H), 1.80-1.65 (m, 1H), 1.41-1.17 (m, 3H), [0.96 (d, J=6.8 Hz) and 0.93 (d, J=6.5 Hz), total 3H], [0.89 (d, J=6.9 Hz) and 0.86-0.80 (m), total 12H].

Step 6. Synthesis of (2S,4R)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-4-tert-butyl-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide and (2R,4S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-4-tert-butyl-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide (C49).

To a 25°C mixture of C16 (120 mg, 0.449 mmol) and C48 (144 mg, 0.346 mmol) in N,N-dimethylformamide (3 mL) was added 4-methylmorpholine (100 mg, 0.989 mmol), then the mixture was cooled to 0°C and treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HFRU; 151 mg, 0.397 mmol). The reaction mixture was purged with nitrogen for 1 min and then stirred at 25°C for 12 h. LCMS analysis showed the presence of C49: LCMS m/z 570.3 [M+H]+. The reaction mixture was then partitioned between ethyl acetate (20 mL) and water (20 mL), and the aqueous layer was saturated with solid sodium chloride and extracted with ethyl acetate (5 x 20 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and chromatographed on silica gel (gradient: 0% to 15% methanol in dichloromethane) to give C49 as a white solid. This product contains a mixture of diastereomers, as analyzed by 1H NMR. Yield: 190 mg, 0.334 mmol, 96%. 1H NMR (400 MHz, DMSO-d6), characteristic peaks, integrations are approximate: δ [9.88 (d, J=8.6 Hz) and 9.82-9.68 (m), total 1H], [8.12 (d, J=8.8 Hz) and 8.09-7.98 (m), total 1H], [7.63 (s) and 7.57 (s), total 1H], [7.30 (brs) and 7.18 (brs), total 1H], [7.06 (brs) and 7.03 (brs), total 1H], [4.36 (dd, J=12.0, 6.1 Hz) and 4.32-4.08 (m), total 2H], 2.26-2.05 (m, 2H), 1.81-1.54 (m, 2H), 1.53-1.30 (m, 2H), 0.98-0.87 (m, 6H), 0.86-0.76 (m, 9H).

Step 7. Synthesis of (2S,4R)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide and (2R,4S)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide [75 (DIAST-1) and 76 (DIAST-2)].

A mixture of C49 (190.0 mg, 0.334 mmol) and methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 238 mg, 1.00 mmol) in dichloromethane (10 mL) was stirred at 25 °C for 2 days, then the reaction mixture was diluted with water (20 mL) and extracted with dichloromethane (2×20 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution (10 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (gradient: 0% to 8% methanol in dichloromethane) gave a white solid which, according to LCMS analysis, contained an approximately 3:1 mixture of products: LCMS m/z 552.2 [M+H]+ and LCMS m/z 552.2 [M+H]+. These diastereomers were separated by supercritical fluid chromatography [Column: Chiral Technologies Chiralpak IG, 30×250 mm, 10 μm; Mobile phase: 3:1 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide); flow rate: 70 ml/min]. The first eluted diastereomer, isolated as a white solid, is designated 75 and the second eluted diastereomer, also a white solid, is designated 76 [(2S,4R)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide and (2R,4S)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide].

75 - Yield: 26.2 mg, 47.5 µmol, 14%. 1H NMR (400 MHz, DMSO-d6) δ 9.87 (d, J=8.8 Hz, 1H), 8.87 (d, J=8.4 Hz, 1H), 7.70 (s, 1H), 4.99-4.91 (m, 1H), 4.24 (dd, J=12.3, 6.0 Hz, 1H), 4.18 (dd, J=8.3, 8.3 Hz, 1H), 3.88-3.78 (m, 1H), 3.19-3.00 (m, 2H), 2.46-2.35 (m, 1H), 2.17-2.02 (m, 2H), 1.99-1.85 (m, 2H), 1.79-1.62 (m, 3H), 1.50-1.36 (m, 2H), 1.26-1.12 (m, 2H), 0.97-0.87 (m, 6H), 0.84 (s, 9H). Retention time: 1.30 minutes (Analytical conditions. Column: Chiral Technologies Chiralpak IG-3, 4.6×50 mm, 3 μm; Mobile phase A: carbon dioxide; Mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5% - 40% B for 2 minutes, then 40% B for 1.2 minutes; Flow rate: 4 mL/minute; Back pressure: 1500 psi).

76 - Yield: 8.8 mg, 16 µmol, 5%. LCMS m/z 552.3 [M+H]+. By 1H NMR analysis, this sample of 76 contains impurities. 1H NMR (400 MHz, DMSO-d6), characteristic peaks, integrations are approximate: δ 9.76 (d, J=8.8 Hz, 1H), 8.59 (d, J=8.1 Hz, 1H), 7.72 (s, 1H), 5.02-4.90 (m, 1H), 0.94-0.86 (m, 6H), 0.82 (s, 9H). Retention time: 1.61 minutes (Analytical conditions are identical to those used for 75).

Example 77

3-Methyl-N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide (77)

Step 1. Synthesis of (4R)-1-(tert-butoxycarbonyl)-4-(trifluoromethyl)-L-prolyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C50).

To a -30°C mixture of (4R)-1-(tert-butoxycarbonyl)-4-(trifluoromethyl)-L-proline (429 mg, 1.51 mmol) and C16 HCl salt (346 mg, 1.67 mmol) in N,N-dimethylformamide (7.8 mL) was added N,N-diisopropylethylamine (0.791 mL, 4.54 mmol), followed by O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 633 mg, 1.66 mmol). The reaction mixture was warmed to 0°C for 1 h, then it was diluted with aqueous sodium bicarbonate (30 mL) and extracted with a mixture of 2-butanol and dichloromethane (9:1, 3×7 mL). The combined organic layers were concentrated in vacuo and purified by silica gel chromatography (0% to 100% methanol in dichloromethane) to afford C50 as an off-white foam. According to 1H NMR analysis, this product existed as a race of rotamers and contained impurities derived from the reagents used; a portion of this sample was carried forward to the next step. Yield: 613 mg, 1.40 mmol, 93%. LCMS m/z 459.3 [M+Na+]. 1H NMR (400 MHz, DMSO-d6), characteristic peaks of the product only: δ 8.33-8.18 (m, 1H), [7.65 (br s) and 7.59 (br s), total 1H], [7.39 (br s) and 7.27 br (s), total 1H], 7.05 (br s, 1H), 4.38-4.28 (m, 1H), 4.28-4.17 (m, 1H), 3.46-3.36 (m, 1H), 2.02-1.89 (m, 1H), 1.80-1.45 (m, 2H), [1.39 (s) and 1.32 (s), total 9H].

Step 2. Synthesis of N-(tert-butoxycarbonyl)-3-methyl-L-valyl-(4R)-4-(trifluoromethyl)-L-prolyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C51).

A mixture of C50 (242 mg, 0.554 mmol) and a solution of the hydrochloride in 1,4-dioxane (4 M; 2 ml, 8 mmol) was stirred at room temperature for 5 min, then the reaction mixture was concentrated in vacuo to remove the solvent and residual hydrochloride. The resulting deprotected product was combined with N-(tert-butoxycarbonyl)-3-methyl-L-valine (128 mg, 0.553 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU: 232 mg, 0.610 mmol) in N,N-dimethylformamide (2 ml), and then cooled to -30 °C. N,N-Diisopropylethylamine (0.290 mL, 1.66 mmol) was added and the reaction mixture was warmed to 0 °C for 1 h. After addition of aqueous sodium bicarbonate solution, the resulting mixture was extracted three times with ethyl acetate; the combined organic layers were concentrated in vacuo and purified by silica gel chromatography (gradient: 0% to 30% methanol in dichloromethane) to give C51 as a solid. Yield: 230 mg, 0.418 mmol, 75%. LCMS m/z 550.3 [M+H]+.

Step 3. Synthesis of 3-methyl-N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide (77).

A mixture of C51 (230 mg, 0.418 mmol) and a solution of the hydrochloride salt in 1,4-dioxane (4 M; 2 mL, 8 mmol) was stirred at room temperature for 5 min, then the reaction mixture was concentrated in vacuo to remove the solvent and residual hydrochloride salt. The resulting deprotected product was combined with ethyl trifluoroacetate (595 mg, 4.19 mmol) and N,N-diisopropylethylamine (0.219 mL, 1.26 mmol) in methanol (1.0 mL). The reaction mixture was then stirred at room temperature for 30 min, ethyl trifluoroacetate (60 mg, 0.422 mmol) was added again and stirring was continued for 30 min. Aqueous sodium bicarbonate solution was then added and the resulting mixture was extracted three times with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered, concentrated in vacuo, and dissolved in dichloromethane (3 mL). To this was added ethyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 299 mg, 1.25 mmol), and the reaction mixture was stirred at room temperature for 2 h, then it was treated with additional methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 100 mg, 0.420 mmol), and stirred for an additional 30 min. Dilute aqueous sodium carbonate solution was then added, and the mixture was extracted twice with ethyl acetate; the combined organic layers were dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification by supercritical fluid chromatography (Column: Princeton Dinitrophenyl, 10×250 mm, 5 μm; Mobile phase: 9:1 carbon dioxide/methanol; Back pressure: 120 bar; Flow rate: 80 ml/min) gave the product, which was then suspended in heptane (2.0 ml) at 50 °C for 2 h, cooled to room temperature and collected by filtration to give 3-methyl-N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide (77) as a solid. Yield: 64 mg, 0.121 mmol, 29%. LCMS m/z 528.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.46 (d, J=8.4 Hz, 1H), 9.05 (d, J=8.6 Hz, 1H), 7.67 (s, 1H), 4.96 (ddd, J=11.0, 8.5, 5.0 Hz, 1H), 4.56 (d, J=8.5 Hz, 1H), 4.37 (dd, J=7.5, 7.5 Hz, 1H), 3.98 (dd, ABX system component, J=11.2, 7.5 Hz, 1H), 3.92 (dd, ABX system component, J=11.3, 4.8 Hz, 1H), 3.46-3.35 (m, 1H), 3.19-3.10 (m, 1H), 3.09-3.00 (m, 1H), 2.5-2.38 (m, 1H, predicted; partially obscured by solvent peak), 2.38-2.28 (m, 1H), 2.21-2.04 (m, 3H), 1.78-1.65 (m, 2H), 0.99 (s, 9H).

Example 78

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(methylcarbamoyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (78)

Step 1. Synthesis of methyl (1R,2S,5S)-6,6-dimethyl-3-(3-methyl-L-valyl)-3-azabicyclo[3,1,0]hexane-2-carboxylate, hydrochloride (C52).

To a 0°C solution of C31 (1.00 g, 2.61 mmol) in dichloromethane (20 mL) was added dropwise a solution of the hydrochloride in ethyl acetate (4 M; 20 mL, 80 mmol). The reaction mixture was then stirred at 25°C overnight and concentrated in vacuo to give C52 as a white gum. Yield: 700 mg, 2.20 mmol, 84%. LCMS m/z 283.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.22 (br s, 3H), 4.25 (s, 1H), 3.87-3.77 (m, 2H), 3.72 (d, half of AB quartet, J=10.8 Hz, 1H), 3.67 (s, 3H), 1.59 (dd, component of the ABX system, J=7.7, 5.3 Hz, 1H), 1.49 (d, half of AB quartet, J=7.7 Hz, 1H), 1.03 (s, 9H), 1.02 (s, 3H), 0.96 (s, 3H).

Step 2. Synthesis of methyl (1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(methylcarbamoyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylate (C53).

To a 0°C solution of C52 (320 mg, 1.00 mmol) in dichloromethane (6 mL) were slowly added triethylamine (0.769 mL, 5.52 mmol) and methylcarbamyl chloride (188 mg, 2.01 mmol). The reaction mixture was warmed to 20°C and stirred for 18 h, then it was treated dropwise with saturated aqueous sodium carbonate solution (5 mL) and extracted with dichloromethane (2 x 5 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution (2 x 20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (0% to 10% methanol in dichloromethane) afforded C53 as a light yellow gum. Yield: 190 mg, 0.560 mmol, 56%. LCMS m/z 339.9 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 6.03 (d, J=9.4 Hz, 1H), 5.89 (shq, J=5 Hz, 1H), 4.20-4.14 (m, 2H), 3.91 (d, half of AB quartet, J=10.3 Hz, 1H), 3.79 (dd, component of the ABX system, J=10.3, 5.3 Hz, 1H), 3.65 (s, 3H), 3.17 (d, J=5.3 Hz, 3H), 1.55-1.49 (m, 1H), 1.40 (d, half of AB quartet, J=7.4 Hz, 1H), 1.00 (s, 3H), 0.92 (s, 9H), 0.83 (s, 3H).

Step 3. Synthesis of (1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(methylcarbamoyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C54).

To a 0°C solution of C53 (190 mg, 0.560 mmol) in a mixture of tetrahydrofuran (2 mL), water (4 mL), and methanol (1 mL) was added lithium hydroxide monohydrate (82.0 mg, 1.95 mmol). The reaction mixture was then stirred at 20°C for 2 h, diluted with ethyl acetate (10 mL); the aqueous layer was then cooled to 0°C - 5°C and acidified to pH 2-3 by adding 1 M hydrochloric acid. The aqueous mixture was extracted with ethyl acetate (3 x 15 mL), and the combined ethyl acetate layers were dried over sodium sulfate, filtered, and concentrated in vacuo to afford C54 as a white solid. Yield: 120 mg, 0.369 mmol, 66%. LCMS m/z 348.3 [M+Na+]. 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 6.04 (d, J=9.6 Hz, 1H), 5.89 (d, J=4.7 Hz, 1H), 4.17 (d, J=9.6 Hz, 1H), 4.09 (s, 1H), 3.87 (d, half of AB quartet, J=10.4 Hz, 1H), 3.77 (dd, component of ABX system, J=10.3, 5.4 Hz, 1H), 1.49 (dd, component of ABX system, J=7.6, 5.1 Hz, 1H), 1.38 (d, half of AB quartet, J=7.5 Hz, 1H), 1.00 (s, 3H), 0.92 (s, 9H), 0.82 (c, 3H).

Step 4. Synthesis of (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-[3-methyl-N-(methylcarbamoyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (C55).

To a 0°C - 5°C solution of C54 (120 mg, 0.369 mmol) and C16, HCl salt (75%, 107 mg, 0.387 mmol) in N,N-dimethylformamide (3.0 mL) were added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 154 mg, 0.405 mmol) and 4-methylmorpholine (0.144 mL, 1.31 mmol). The reaction mixture was then heated from 0°C to 20°C over 1.5 h, it was stirred at 20°C for 18 h, then it was diluted with water and treated with solid sodium sulfate until saturation. The resulting mixture was extracted with a mixture of 2-propanol and chloroform (1:4, 3×20 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (gradient: 0% to 20% methanol in dichloromethane) gave C55 (240 mg) as a colorless glass. A portion of this product was used in the next step. LCMS m/z 479.2 [M+H]+. According to 1H NMR analysis, this product was contaminated with a by-product derived from the GATU reagent. 1H NMR (400 MHz, DMSO-d6), characteristic peaks of the product only: δ 8.21 (d, J=8.7 Hz, 1H), 7.53 (br s, 1H), 7.29 (br s, 1H), 7.03 (br s, 1H), 6.02 (d, J=9.6 Hz, 1H), 5.86 (q, J=4.6 Hz, 1H), 4.31-4.23 (m, 1H), 4.21 (s, 1H), 4.15 (d, J=9.6 Hz, 1H), 2.18-2.08 (m, 1H), 1.98-1.88 (m, 1H), 1.68-1.55 (m, 1H), 1.54-1.42 (m, 2H), 1.34 (d, half of AB quartet, J=7.6 Hz, 1H), 1.01 (s, 3H), 0.90 (s, 9H), 0.84 (s, 3H).

Step 5. Synthesis of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(methylcarbamoyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (78).

To a solution of C55 (from the previous step; 190 mg, ≤0.292 mmol) in acetonitrile (12 mL) was added methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 303 mg, 1.26 mmol). The reaction mixture was stirred at 20 °C for 22 h, then combined with a similar reaction carried out using C55 (from the previous step; 50 mg, ≤77 μmol). The resulting solution was concentrated in vacuo, diluted with water (10 mL), and extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo; Purification by reversed-phase HPLC (Column: Boston Prime C18, 30×150 mm, 5 μm; Mobile phase A: 0.225% formic acid in water; Mobile phase B: acetonitrile; Gradient: 23% to 46% B; Flow rate: 25 mL/min) afforded (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(methylcarbamoyl)-L-valyl]-3-azabicyclo[3.1,0]hexane-2-carboxamide (78) as a white solid. Pooled yield: 25 mg, 54 μmol, 15% over 2 steps. LCMS m/z 461.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 8.96 (d, J=8.6 Hz, 1H), 7.65 (s, 1H), 6.02 (d, J=9.5 Hz, 1H), 5.85 (q, J=4.5 Hz, 1H), 4.95 (ddd, J=10.8, 8.4, 5.1 Hz, 1H), 4.13 (d, J=9.6 Hz, 1H), 4.11 (s, 1H), 3.88-3.79 (m, 2H), 3.18-3.09 (m, 1H), 3.07-2.98 (m, 1H), 2.48-2.37 (m, 1H), 2.20-2.02 (m, 2H), 1.77-1.62 (m, 2H), 1.56-1.50 (m, 1H), 1.27 (d, half of AB quartet, J=7.6 Hz, 1H), 1.02 (s, 3H), 0.89 (s, 9H), 0.85 (s, 3H). 1H NMR (400 MHz, chloroform-d), characteristic peaks: δ 8.12 (d, J=7.6 Hz, 1H), 5.78 (br s, 1H), 5.04 (br d, J=9.4 Hz, 1H), 4.99-4.90 (m, 1H), 4.57-4.49 (m, 1H), 4.39 (d, J=9.7 Hz, 1H), 4.25 (s, 1H), 4.01 (d, half of the AB quartet, J=10.2 Hz, 1H), 3.93 (br dd, component of the ABX system, J=10.6, 4.9 Hz, 1H), 3.43-3.25 (m, 2H), 2.71 (d, J=4.8 Hz, 3H), 2.61-2.50 (m, 1H), 2.45-2.30 (m, 2H), 2.03-1.93 (m, 1H), 1.91-1.78 (m, 1H), 1.05 (s, 3H), 0.98 (s, 9H), 0.91 (s, 3H).

Example 79

Methyl {(2S)-1-[(1R,2S,5S)-2-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2-yl}carbamate (79)

Step 1. Synthesis of methyl (1R,2S,5S)-3-[N-(methoxycarbonyl)-3-methyl-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate (C56).

To a 0°C solution of C52 (370 mg, 1.16 mmol) in dichloromethane (6 mL) were added triethylamine (0.647 mL, 4.64 mmol) and methyl chloroformate (335 mg, 3.55 mmol) slowly. The reaction mixture was then stirred at 20°C for 16 h, diluted dropwise with saturated aqueous sodium carbonate (5 mL), and extracted with dichloromethane (2 x 20 mL). The combined organic layers were washed with saturated aqueous sodium chloride (2 x 20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo; silica gel chromatography (gradient: 0% to 100% ethyl acetate in petroleum ether) gave C56 as a white gum. Yield: 115 mg, 0.338 mmol, 29%. LCMS m/z 341.1 [M+H]+. 1H NMR (400 MHz, chloroform-d) δ 5.29 (bd, J=9.6 Hz, 1H), 4.46 (s, 1H), 4.23 (d, J=9.9 Hz, 1H), 3.94-3.86 (m, 2H), 3.74 (s, 3H), 3.63 (bs, 3H), 1.49-1.41 (m, 2H), 1.04 (s, 3H), 1.03 (s, 9H), 0.91 (s, 3H).

Step 2. Synthesis of (1R,2S,5S)-3-[N-(methoxycarbonyl)-3-methyl-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C57).

To a solution of C56 (115 mg, 0.338 mmol) in a mixture of methanol (2.0 mL), tetrahydrofuran (2.0 mL), and water (2 mL) was added lithium hydroxide monohydrate (28.4 mg, 0.677 mmol). The reaction mixture was stirred at room temperature (22°C - 25°C) for 16 h, then concentrated in vacuo. The aqueous residue was partitioned between water (5 mL) and ethyl acetate (20 mL), then the organic layer was discarded, and the aqueous layer was adjusted to pH 1-2 with concentrated hydrochloric acid. The resulting mixture was extracted three times with ethyl acetate; the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to give C57 as a colorless gum. Yield: 100 mg, 0.306 mmol, 91%. LCMS m/z 327.2 [M+H]+. 1H NMR (400 MHz, chloroform-d) δ 5.42 (d, J=9.9 Hz, 1H), 4.46 (s, 1H), 4.26 (d, J=10.0 Hz, 1H), 3.96 (d, half of AB quartet, J=10.5 Hz, 1H), 3.87 (dd, component of ABX system, J=10.3, 5.4 Hz, 1H), 3.64 (s, 3H), 1.68 (d, half of AB quartet, J=7.7 Hz, 1H), 1.50 (dd, component of ABX system, J=7.6, 5.3 Hz, 1H), 1.06 (s, 3H), 1.01 (s, 9H), 0.91 (s, 3H).

Stage 3. Methyl synthesis {(2S)-1-[(1R,2S,5S)-2-({(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}car bamoyl)-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2-yl}carbamate (C58).

To a 0°C solution of C57 (100 mg, 0.306 mmol) and C16, HCl salt (75%, 84.8 mg, 0.306 mmol) in N,N-dimethylformamide (3 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 140 mg, 0.368 mmol), followed by dropwise addition of a solution of 4-methylmorpholine (93 mg, 0.919 mmol) in N,N-dimethylformamide (1 mL). The reaction mixture was then warmed to room temperature (25°C) and stirred for 16 h, followed by addition of water (10 mL). After adding sodium sulfate to saturation, the resulting mixture was extracted with a mixture of chloroform and 2-propanol (4:1, 3×10 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and purified using silica gel chromatography (Gradient: 0% to 30% methanol in dichloromethane) to give C58 as a white solid. Yield: 93 mg, 0.19 mmol, 62%. LCMS m/z 480.0 [M+H]+. 1H NMR (400 MHz, chloroform-d ) δ 8.30 (br s, 1H), 7.18 (br s, 1H), 5.98 (br s, 1H), 5.64 (br s, 1H), 5.58-5.42 (m, 1H), 4.49-4.37 (m, 1H), 4.29 (d, J = 10.0 Hz, 1H), 4.23 (s, 1H), 4.11 (dd, component of the ABX system, J = 10.3, 5.5 Hz, 1H), 3.93 (d, half of the AB quartet, J = 10.3 Hz, 1H), 3.64 (s, 3H), 3.43-3.29 (m, 2H), 2.55-2.33 (m, 2H), 2.15-1.81 (m, 3H), 1.54-1.47 (m, 1H), 1.45 (d, half of AB quartet, J=7.7 Hz, 1H), 1.03 (s, 3H), 1.01 (s, 9H), 0.88 (s, 3H).

Step 4. Methyl synthesis {(2S)-1-[(1R,2S,5S)-2-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2-yl}carbamate (79).

To a suspension of C58 (93 mg, 0.19 mmol) in dichloromethane (5 mL) was added methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 139 mg, 0.583 mmol), and the reaction mixture was stirred at 25 °C for 2 h. It was then diluted with water (10 mL) and extracted with dichloromethane (3 x 10 mL); the combined organic layers were washed with saturated aqueous sodium chloride solution (2 x 10 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (gradient: 0% to 100% ethyl acetate in petroleum ether, then a gradient of 0% to 20% methanol in dichloromethane) gave methyl {(2S)-1-[(1R,2S,5S)-2-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1,0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2-yl}carbamate (79) as a white solid. Yield: 7.0 mg, 15 μmol, 8%. LCMS m/z 462.2 [M+H]+. 1H NMR (400 MHz, chloroform-d) δ 8.13 (bd, J=7.0 Hz, 1H), 5.68 (bd s, 1H), 5.34 (bd, J=9.9 Hz, 1H), 4.95-4.85 (m, 1H), 4.26 (s, 1H), 4.23 (d, J=10.0 Hz, 1H), 3.94 (dd, component of the ABX system, J=10.1, 4.5 Hz, 1H), 3.88 (d, half of the AB quartet, J=10.3 Hz, 1H), 3.63 (s, 3H), 3.45-3.29 (m, 2H), 2.62-2.50 (m, 1H), 2.46-2.28 (m, 2H), 2.02-1.93 (m, 1H), 1.92-1.79 (m, 1H), 1.6-1.49 (m, 2H, expected; partially obscured by water peak), 1.06 (s, 3H), 0.98 (s, 9H), 0.90 (s, 3H).

Example 80

N-(Trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide (80)

Step 1. Synthesis of 2-benzyl 1-tert-butyl (2S,4R)-4-(trifluoromethyl)pyrrolidine-1,2-dicarloxylate (C59).

A mixture of (4R)-1-(tert-butoxycarbonyl)-4-(trifluoromethyl)-L-proline (400 mg, 1.41 mmol), benzyl bromide (0.335 mL, 2.82 mmol), and sodium bicarbonate (593 mg, 7.06 mmol) in N,N-dimethylformamide (8 mL) was stirred for 15 h at 25 °C. The reaction mixture was then diluted with water (30 mL) and extracted with ethyl acetate (3 x 30 mL), and the combined organic layers were washed with saturated aqueous sodium chloride and 5% aqueous lithium chloride, dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (0% to 30% ethyl acetate in petroleum ether) afforded C59 as a colorless oil. According to 1H NMR analysis, this product exists as a mixture of rotamers. Yield: 355 mg, 0.951 mmol, 67%. 1H NMR (400 MHz, chloroform-d) δ 7.44-7.28 (m, 5H), 5.29-5.07 (m, 2H), [4.54 (bd, J=8.6 Hz) and 4.40 (bdd, J=8.5, 2 Hz), total 1H], 3.87-3.70 (m, 1H), [3.58 (dd, J=11.2, 7.4 Hz) and 3.49 (dd, J=11.0, 7.9 Hz), total 1H], 3.13-2.95 (m, 1H), 2.47-2.27 (m, 1H), 2.25-2.11 (m, 1H), [1.46 (s) and 1.33 (s), total 9H].

Step 2. Synthesis of benzyl (4R)-4-(trifluoromethyl)-L-prolinate hydrochloride (C60).

To a 0°C solution of C59 (200 mg, 0.536 mmol) in ethyl acetate (3 mL) was added a solution of the hydrochloride salt in ethyl acetate (4 M; 6 mL, 24 mmol). The reaction mixture was then stirred at room temperature (28°C) for 3 h and concentrated in vacuo to give C60 as a white solid; this product was taken directly to the next step. LCMS m/z 274.0 [M+H]+.

Step 3. Synthesis of benzyl N-(tert-butoxycarbonyl)-L-valyl-(4R)-4-(trifluoromethyl)-L-prolinate (C61).

O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 277 mg, 0.728 mmol) and 4-methylmorpholine (184 mg, 1.82 mmol) were added to a 0°C mixture of C60 (from the previous step; ≤0.536 mmol) and N-(tert-butoxycarbonyl)-L-valine (158 mg, 0.727 mmol) in N,N-dimethylformamide (3 mL). The reaction mixture was stirred at 0°C for 1 h, then it was poured into ice water (15 mL) and extracted with ethyl acetate (2×15 mL). The combined organic layers were washed successively with 1 M hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated in vacuo. Purification by silica gel chromatography (0% to 40% ethyl acetate in petroleum ether) afforded C61 as a colorless gum. Yield: 230 mg, 0.487 mmol, 91% over 2 steps. LCMS m/z 495.0 [M+Na+]. 1H NMR (400 MHz, chloroform-d), characteristic peaks: δ 7.40-7.30 (m, 5H), 5.17 (AB quartet, JAB=12.3 Hz, ΔνAB=12.6 Hz, 2H), 4.21 (dd, J=9.3, 6.8 Hz, 1H), 4.00-3.86 (m, 2H), 3.18-3.04 (m, 1H), 2.36 (ddd, component of ABXY system, J=13.5, 9, 9 Hz, 1H), 2.20 (ddd, component of ABXY system, J=13.4, 7.4, 3.5 Hz, 1H), 2.05-1.94 (m, 1H), 1.42 (s, 9H), 0.98 (d, J=6.7 Hz, 3H), 0.91 (d, J=6.8 Hz, 3H).

Step 4. Synthesis of benzyl L-valyl-(4R)-4-(trifluoromethyl)-L-prolinate, hydrochloride (C62).

To a 0°C solution of C61 (230 mg, 0.487 mmol) in ethyl acetate (2 mL) was added a solution of the hydrochloride salt in ethyl acetate (4 M; 4 mL, 16 mmol). The reaction mixture was stirred at room temperature (28°C) for 1 h, then LCMS analysis showed conversion to C62: LCMS m/z 373.1 [M+H]+. Concentration of the reaction mixture in vacuo gave C62 as a white solid, which was taken directly to the next step.

Step 5. Synthesis of benzyl N-(trifluoroacetyl)-L-valyl-(4R)-4-(trifluoromethyl)-L-prolinate (C63).

A solution of trifluoroacetic anhydride (154 mg, 0.733 mmol) in dichloromethane (0.5 mL) was added to a 0 °C suspension of C62 (from the previous step; ≤0.487 mmol) in dichloromethane (3 mL). After 3 min, a solution of triethylamine (148 mg, 1.46 mmol) in dichloromethane (0.5 mL) was added dropwise and stirring was continued at 25 °C for 3 h. After dilution with dichloromethane (5 mL), the reaction mixture was washed with saturated aqueous sodium carbonate (10 mL) and saturated aqueous sodium chloride (15 mL), dried, filtered, and concentrated in vacuo; silica gel chromatography (Gradient: 0% to 30% ethyl acetate in petroleum ether) gave C63 as a colorless oil. Yield: 129 mg, 0.275 mmol, 56% over 2 steps. LCMS m/z 491.2 [M+Na+].

Step 6. Synthesis of N-(trifluoroacetyl)-L-valyl-(4R)-4-(trifluoromethyl)-L-proline (C64).

To a 28°C solution of C63 (129 mg, 0.275 mmol) in methanol (3 mL) was added palladium on carbon (10%, 29.3 mg, 27.5 μmol), then the mixture was hydrogenated at 15 psi for 16 h. Filtration gave a filter cake that was washed with methanol (10 mL); the combined filtrates were concentrated in vacuo to give C64 as a light yellow solid. Yield: 80 mg, 0.21 mmol, 76%. LCMS m/z 401.0 [M+Na+].

Step 7. Synthesis of N-(trifluoroacetyl)-L-valyl-(4R)-4-(trifluoromethyl)-L-prolyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C65).

O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 88.5 mg, 0.233 mmol) and 4-methylmorpholine (64.2 mg, 0.635 mmol) were added to a 0°C solution of C64 (80 mg, 0.21 mmol) and C16 (76.8 mg, 0.287 mmol) in N,N-dimethylformamide (3 ml). The reaction mixture was then stirred at 0°C for 2 h, it was treated with water (10 ml) and aqueous citric acid solution (1 M; 10 ml, 10 mmol), then extracted with ethyl acetate (3×10 ml). The combined organic layers were washed successively with saturated aqueous sodium bicarbonate (15 mL) and saturated aqueous sodium chloride (15 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Purification by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) gave C65 as a white solid. Yield: 72 mg, 0.14 mmol, 67%. LCMS m/z 532.2 [M+H]+.

Step 8. Synthesis of N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide (80).

Methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 96.9 mg, 0.407 mmol) was added to a mixture of C65 (72 mg, 0.14 mmol) in dichloromethane (5 mL), and the reaction mixture was stirred at room temperature overnight. After dilution with water (15 mL), the mixture was extracted with dichloromethane (3×15 mL), and the combined organic layers were washed with saturated aqueous sodium chloride solution (2×20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) afforded N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide (80) as a white solid. Yield: 30.9 mg, 60.2 μmol, 43%. LCMS m/z 536.1 [M+Na+]. 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 9.89 (d, J=7.8 Hz, 1H), 9.06 (d, J=8.4 Hz, 1H), 7.69 (s, 1H), 4.96 (ddd, J=10.6, 8.4, 5.5 Hz, 1H), 4.38 (dd, J=7.9, 6.3 Hz, 1H), 4.28 (dd, J=9.8, 7.8 Hz, 1H), 4.07-3.94 (m, 2H), 3.20-3.00 (m, 2H), 2.5-2.41 (m, 1H, assumed; partially obscured by solvent peak), 2.38-2.28 (m, 1H), 2.19-2.02 (m, 4H), 1.78-1.61 (m, 2H), 0.92 (d, J=7 Hz, 3H), 0.90 (d, J=6.8 Hz, 3H).

Examples 81-84

Example 81: (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3R)-5-hydroxy-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide

Example 82: (1R,2S,5S,6R)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide

Example 83: (1R,2S,5S,6S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide

Example 84: (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-(hydroxymethyl)-N-(trifluoroacetyl)-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide

The compounds of Examples 81-84 are prepared by biotransformation routes, both in vitro and in vivo, from (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (the compound of Example 13) as follows. In vitro studies, (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide was incubated with mouse, rat, hamster, rabbit, monkey, or human liver microsomes (see Table M1 below) or rat, monkey, or human hepatocytes (see Table M2 below). Alternatively, in in vivo studies, (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide was administered to rats and monkeys. Rat plasma, urine, and bile and monkey plasma samples were obtained. The resulting metabolites are then analyzed using HPLC/MS and the resulting oxidative metabolites of the compounds of Examples 81-84 are determined and obtained. In addition to the compounds of Examples 81-84, an additional metabolite, (1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylic acid, obtained by hydrolytic cleavage, is observed in in vivo studies.

Table M1: Compounds derived from liver microsomes Example Mouse Rat Hamster Rabbit Monkey Human 81 +++ +++ +++ +++ +++ +++ 82 + + + ++ t + 83 + + + + t + 84 + t - t t +

Table M2: Compounds of Examples 81-84 Derived from Hepatocytes Example Mouse Rat Hamster 81 +++ +++ +++ 82 + t t 83 t t t 84 + t -

Table M3: Compounds of Examples 81-84 produced in vivo in mice or monkeys Example Rat plasma Rat urine Rat bile Monkey Plasma 81 + t t ++ 82 t t t t 83 t - - t 84 + - - +

In tables M1, M2 and M3 the following notations are used: -=not determined;+=determined by mass spectrometry and minor UV peak;++=determined by mass spectrometry and medium UV peak;+++=determined by mass spectrometry and major UV peak; t=trace determined only by mass spectrometry

Examples 82, 83, 84, and 81

(1R,2S,5S,6R)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (82), (1R,2S,5S,6S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (83), (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-(hydroxymethyl)-N-(trifluoroacetyl)-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (84) and (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3R)-5-hydroxy-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (81)

Example 13 (25 μm) is combined with human liver microsomes (2 mg/ml) in potassium phosphate buffer (100 mM, pH 7.4; 40 ml) containing magnesium chloride (3.3 mM) and NADPH (1.3 mM). Incubation is carried out for 55 minutes in a shaking water bath maintained at 37°C. The reaction is terminated by adding an equal volume of acetonitrile, the mixture is then spun at 1800 x g for 5 minutes, and the supernatant is vacuum centrifuged for approximately 1.5 hours. Formic acid (0.5 ml), acetonitrile (0.5 ml) and water were added to the residue to a final volume of 50 ml, and the resulting mixture was centrifuged at 40,000 x g for 30 min. The supernatant was applied to an HPLC column (Polaris C18, 4.6×250 mm; 5 μm) at 1 ml/min using a Jasco HPLC pump. After loading, the column was transferred to a Waters Acquity HPLC-UV system coupled to a Thermo LTQ mass spectrometer and a CTC Analytics fraction collector and subjected to reversed-phase HPLC separation (Mobile phase A: water containing 0.1% formic acid (v/v); Mobile phase B: acetonitrile; Gradient: 2% for 5 min, then increased to 15% B, then 15% - 60% B for 80 min, then 60% - 95% B for 5 min; Flow rate: 0.8 mL/min). Fractions were collected every 20 seconds to yield (1R,2S,5S,6R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (82), (1R,2S,5S,6S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (83), (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-(hydroxymethyl)-N-(trifluoroacetyl)-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (84) and (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3R)-5-hydroxy-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (81).

Example number Retention time, HPLC cleanup (minutes) 82 37.2 83 39.3 84 46.6 81 50.5

82 - Yield: 60 µg, 0.12 µmol, 12%. High-Resolution MS m/z 516.2424 [M+H]+; calculated for C23H33F3N5O5, 516.2434. 1H NMR (600 MHz, DMSO-d6), characteristic peaks: δ 9.42 (bd, J=7.9 Hz, 1H), 9.03 (d, J=8.5 Hz, 1H), 7.66 (s, 1H), 5.01-4.93 (m, 1H), 4.69-4.63 (m, 1H), 4.43 (d, J=8.3 Hz, 1H), 4.15 (s, 1H), 3.94 (dd, component of the ABX system, J=10.2, 5.4 Hz, 1H), 3.68 (d, half of the AB quartet, J=10.4 Hz, 1H), 3.21-3.17 (m, 2H), 3.17-3.11 (m, 1H), 3.07-3.00 (m, 1H), 1.69-1.65 (m, 1H), 1.44 (d, J=7.8 Hz, 1H), 0.98 (s, 9H), 0.84 (s, 3H).

83 - Yield: 30 µg, 0.058 µmol, 6%. High-Resolution MS m/z 516.2425 [M+H]+; calculated for C23H33F3N5O5, 516.2434. 1H NMR (600 MHz, DMSO-d6), characteristic peaks: δ 9.37 (d, J=7.0 Hz, 1H), 9.04 (d, J=8.5 Hz, 1H), 7.65 (s, 1H), 5.00-4.94 (m, 1H), 4.54-4.49 (m, 1H), 4.40 (d, J=8.1 Hz, 1H), 4.28 (s, 1H), 3.93 (dd, component of the ABX system, J=10.2, 5.8 Hz, 1H), 3.74 (d, half of the AB quartet, J=10.6 Hz, 1H), 3.3-3.20 (m, 1H, assumed; partially obscured by the water peak), 3.17-3.11 (m, 1H), 3.07-3.00 (m, 1H), 1.75-1.63 (m, 2H), 1.38 (d, half of AB quartet, J=7.3 Hz, 1H), 1.06 (s, 3H), 0.98 (s, 9H).

84 - Yield: 40 µg, 0.078 µmol, 8%. High-Resolution MS m/z 516.2423 [M+H]+; calculated for C23H33F3N5O5, 516.2434. 1H NMR (600 MHz, DMSO-d6), characteristic peaks: δ 9.61-9.51 (m, 1H), 9.00 (d, J=8.3 Hz, 1H), 7.68 (s, 1H), 5.02-4.92 (m, 1H), 4.51-4.43 (m, 1H), 4.16 (s, 1H), 3.92 (bdd, J=10.0, 5.6 Hz, 1H), 3.78 (d, J=10.6 Hz, 1H), 3.51 (d, J=10.1 Hz, 1H), 3.18-3.10 (m, 1H), 3.10-3.03 (m, 1H), 1.73-1.67 (m, 2H), 1.60-1.54 (m, 1H), 1.31 (d, J=7.6 Hz, 1H), 1.03 (s, 3H), 1.01 (s, 3H), 0.88 (shs, 6H).

81 - Yield: 130 µg, 0.252 µmol, 25%. This product was determined to exist as an interconvertible mixture of stereoisomers around the carbinolaine group of pyrrolidone (see Examples 81 and 82). High-Resolution MS m/z 516.2428 [M+H]+; calculated for C23H33F3N5O5, 516.2434. 1H NMR (600 MHz, DMSO-d6) δ [9.40 (d, J=8.4 Hz) and 9.38 (d, J=8.2 Hz), total 1H], [8.99 (d, J=8.5 Hz) and 8.92 (d, J=7.6 Hz), total 1H], [8.37 (s) and 8.25 (s), total 1H], [5.83 (shs) and 5.70 (shs), total 1H], 5.04-4.92 (m, 2H), 4.44-4.38 (m, 1H), [4.19 (s) and 4.15 (s), total 1H], 3.91 (dd, J=10.2, 5.5 Hz, 1H), [3.69 (d, J=10 Hz) and 3.68 (d, J=10.2 Hz), total 1H], [2.65-2.57 (m), 2.43-2.30 (m), and 2.21-2.13 (m), total 2H], [2.08 (ddd, J=13.7, 8.4, 6.2 Hz), 2.00-1.90 (m), and 1.87-1.79 (m), total 2H], [1.78-1.70 (m) and 1.51-1.44 (m), total 1H], 1.60-1.53 (m, 1H), [1.32 (d, J=7.6 Hz) and 1.29 (d, J=7.6 Hz), total 1H], 1.03 (s, 3H), [0.99 (s) and 0.98 (s), total 9H], [0.85 (c) and 0.84 (c), total 3H].

Examples 81 and 85

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3R)-5-hydroxy-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (81) and (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3R)-2,5-dioxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (85)

A mixture of Example 13 (1.0 mg, 2.0 μmol) and tetra-n-butylammonium deca-tungstate (TBADT; 0.33 mg, 0.10 μmol) was treated with acetonitrile (0.15 mL) and hydrochloric acid (1.0 M; 0.05 mL, 50 μmol). A syringe needle (18 gauge) was inserted through the Teflon cap of the tube and the air-accessible mixture was placed in an EvoluChem™ PhotoRedOx Box equipped with a fan and irradiated with black light (PAR20-18W LG 365 nm, 100-240 VAC) at 25°C for 16 h. Aqueous potassium phosphate (1 M, pH 7.5; 0.5 mL) was added to the reaction mixture, followed by water (to a volume of approximately 6 mL), followed by aqueous formic acid (1%, 2 mL) and sufficient acetonitrile to maintain the solution. The resulting solution was divided in half and applied to two 5 g Biotage Isolute C18 solid phase extraction cartridges. The cartridges were washed with aqueous ammonium acetate (10 mM; 3 mL) and 20% acetonitrile in 10 mM aqueous ammonium acetate (3 mL), then eluted with acetonitrile (3 mL). The solvents were removed using a Genevac evaporator and the two residues were reconstituted in a mixture of acetonitrile and 1% aqueous formic acid and combined to a total of 2 mL of solution. This product was subjected to reverse-phase HPLC (Column: Phenomenex Luna C18, 10×250 mm, 10 μm; Mobile phase A: water containing 0.1% formic acid (v/v); Mobile phase B: acetonitrile; Gradient: 2% - 15% B for 5 min, then 15% - 60% B for 80 min, then 60% - 95% B for 5 min; Flow rate: 2 mL/min). Fractions were collected every 20 seconds; the first compound eluted was (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3R)-5-hydroxy-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (81), and the second compound eluted was (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3R)-2,5-dioxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (85).

Yield 81: 0.122 mg, 0.237 µmol, 12%. This product was determined to exist as an interconvertible mixture of stereoisomers around the carbinolamine group of the pyrrolidone, and eluted as a double peak on HPLC. High Resolution MS m/z 516.2413 [M+H]+; calcd for C23H33F3N5O5, 516.2434. 1H NMR (600 MHz, DMSO-d6) δ 9.44-9.34 (m, 1H), [8.99 (d, J=8.5 Hz) and 8.92 (d, J=7.6 Hz), total 1H], [8.37 (s) and 8.25 (s), total 1H], [5.83 (shs) and 5.70 (shs), total 1H], 5.05-4.91 (m, 2H), 4.44-4.37 (m, 1H), [4.19 (s) and 4.15 (s), total 1H], 3.91 (dd, J=10.3, 5.5 Hz, 1H), [3.69 (d, J=10.1 Hz) and 3.68 (d, J=10.3 Hz), total 1H], [2.65-2.57 (m), 2.43-2.30 (m), and 2.17 (ddd, J=14.9, 10.7, 4.7 Hz), total 2H], [2.08 (ddd, J=14.1, 8.5, 6.2 Hz), 2.01-1.90 (m), and 1.83 (ddd, J=13.7, 10.1, 5.7 Hz), 2H total], [1.78-1.70 (m) and 1.51-1.44 (m), 1H total], 1.60-1.53 (m, 1H), [1.32 (d, J=7.6 Hz) and 1.29 (d, J=7.6 Hz), 1H total], 1.03 (s, 3H), [0.99 (s) and 0.98 (s), total 9H], [0.85 (s) and 0.84 (s), total 3H]. Retention time: 7.7 minutes (Analytical conditions. Column: Phenomenex Kinetex XB-C18, 2.1×100 mm, 2.6 μm; Mobile phase A: water containing 0.1% formic acid (v/v); Mobile phase B: acetonitrile; Gradient: 5% B for 0.5 minutes, then 5% - 70% B for 10.5 minutes, then 70% - 95% B for 2 minutes; Flow rate: 0.4 mL/minute).

Yield 85: 0.104 mg, 0.203 µmol, 10%. High-Resolution MS m/z 514.2259 [M+H]+; calculated for C23H31F3N5O5, 514.2277. 1H NMR (600 MHz, DMSO-d6) δ 11.17 (br s, 1H), 9.40 (br s, 1H), 9.08 (d, J=8.9 Hz, 1H), 5.06-4.98 (m, 1H), 4.42-4.36 (m, 1H), 4.13 (s, 1H), 3.91 (dd, J=10.3, 5.6 Hz, 1H), 3.70 (d, half of AB quartet, J=10.4 Hz, 1H), 3.00-2.93 (m, 1H), 2.60 (dd, component of ABX system, J=18.0, 5.9 Hz, 1H), 2.46 (dd, component of ABX system, J=18.1, 9.1 Hz, 1H), 2.25-2.18 (m, 1H), 2.04-1.97 (m, 1H), 1.60-1.55 (m, 1H), 1.35 (d, half of AB quartet, J=7.6 Hz, 1H), 1.03 (s, 3H), 0.95 (s, 9H), 0.86 (s, 3H). Retention time: 8.3 minutes (Analytical conditions are identical to those used for 81).

Example 86

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[5,5,5-trifluoro-2-(2,2,2-trifluoroacetamido)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (86)

Step 1. Synthesis of tert-butyl (1R,2S,5S)-2-({(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-3-carloxylate (C66).

To a 0°C suspension of (1R,2S,5S)-3-(tert-butoxycarbonyl)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (5.25 g, 20.6 mmol), C16 HCl salt (4.70 g, 22.6 mmol) and 2-hydroxypyridine 1-oxide (571 mg, 5.14 mmol) in butan-2-one (108 mL) was added N,N-diisopropylethylamine (7.97 g, 61.7 mmol), followed by 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (4.73 g, 24.7 mmol). The reaction mixture was then stirred at 0°C for 20 min, gradually warmed to room temperature, and then stirred at room temperature overnight, whereupon LCMS analysis showed the presence of C66: LCMS m/z 407.1 [M-H]-. The reaction mixture was diluted with ethyl acetate (100 mL) and washed successively with a mixture of water (50 mL) and saturated aqueous sodium chloride (20 mL), saturated aqueous sodium chloride (70 mL), twice with a mixture of hydrochloric acid (1 M; 50 mL) and saturated aqueous sodium chloride (20 mL), and finally with saturated aqueous sodium chloride (70 mL). Each aqueous layer was extracted with ethyl acetate (100 mL) and the combined organic layers were dried over sodium sulfate and filtered. The combined sodium sulfate was washed with ethyl acetate (2 x 50 mL) and the combined filtrates were concentrated in vacuo, diluted with heptane (50 mL) and concentrated under reduced pressure to give C66 as a colorless glass (6.69 g). Some solvent was present by 1H NMR analysis; the purity was estimated to be approximately 85% by weight. 1H NMR analysis also showed that the product existed as a mixture of rotamers. Yield corrected for solvents: 5.7 g, 14 mmol, 68%. 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 8.23-8.13 (m, 1H), [7.63 (br s) and 7.59 (br s), total 1H], [7.36 (br s) and 7.23 (br s), total 1H], 7.08-7.00 (m, 1H), 4.31-4.19 (m, 1H), [4.03 (s) and 3.99 (s), total 1H], [3.58 (dd, J=10.8, 4.6 Hz) and 3.49 (dd, J=10.8, 3.9 Hz), total 1H], [3.27 (d, J=10.9 Hz) and 3.26 (d, J=10.7 Hz), total 1H]. 3.22-3.00 (m, 2H), 2.38-2.09 (m, 2H), 1.79-1.43 (m, 2H), [1.36 (s) and 1.29 (s), total 9H], 1.00 (s, 3H), 0.89 (s, 3H).

Step 2. Synthesis of (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide, methanesulfonate (C67).

Methanesulfonic acid (0.920 mL, 14.2 mmol) was added to a solution of C66 (approximately 85% w/w, from the previous step; 6.68 g, 14 mmol) in 1,1,1,3,3,3-hexafluoropropan-2-ol (30 mL). The reaction mixture was stirred at room temperature for 3 h, then concentrated in vacuo. The residue was successively taken up in the following solvent systems, then re-concentrated: acetonitrile/ethyl acetate (1:1, 2×10 mL) and ethyl acetate/heptane (1:1, 2×10 mL) to give C67 as a glass (7.18 g). A portion of this product was taken into the next step. LCMS m/z 309.3 [M+H]+. 1H NMR (400 MHz, methanol-d4) δ 4.51 (dd, J=10.8, 4.7 Hz, 1H), 4.21 (brs, 1H), 3.73 (dd, J=12.4, 6.3 Hz, 1H), 3.41-3.22 (m, 3H), 2.70 (s, 3H), 2.58-2.47 (m, 1H), 2.42-2.32 (m, 1H), 2.16 (ddd, J=14.0, 10.8, 4.8 Hz, 1H), 1.97 (bd, J=7.9 Hz, 1H), 1.95-1.84 (m, 1H), 1.84-1.75 (m, 2H), 1.15 (c, 6H).

Stage 3. Synthesis (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[5,5,5-trifluoro-2-(2,2,2-trifluoroacetamido)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (86).

To a solution of 2-[(tert-butoxycarbonyl)amino]-5,5,5-trifluoropentanoic acid (59.0 mg, 0.218 mmol) in a mixture of acetonitrile (0.60 mL) and N,N-dimethylformamide (0.40 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 82.7 mg, 0.218 mmol), followed by 4-methylmorpholine (54.4 μL, 0.495 mmol). The reaction mixture was then stirred for 20 min, C67 (from the previous step; 100 mg, ≤0.19 mmol) was added as a solid. The reaction mixture was stirred for 1.5 h, then partitioned between ethyl acetate and saturated aqueous sodium bicarbonate. The aqueous layer was extracted with ethyl acetate, and the combined organic layers were dried over magnesium sulfate, filtered, and concentrated with a stream of nitrogen. The residue was dissolved in dichloromethane (0.70 ml), treated with trifluoroacetic acid (0.175 ml), and stirred at room temperature. After 2 h, more trifluoroacetic acid (0.10 ml) was added; stirring was continued for a further 3 h, then the reaction mixture was concentrated under a stream of nitrogen and then in vacuo. This material was dissolved in dichloromethane (0.75 ml), cooled in an ice bath, and treated with triethylamine (54.8 μl, 0.393 mmol); Trifluoroacetic anhydride (41.2 μL, 0.292 mmol) was added dropwise and the reaction mixture was stirred at 0°C for 3 h. The volatiles were removed using a Genevac evaporator and the residue was dissolved in dichloromethane (0.90 mL), treated with methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 132 mg, 0.554 mmol), and stirred at room temperature for 2.5 h. The reaction mixture was then concentrated under a stream of nitrogen, diluted with ethyl acetate, and washed with saturated aqueous sodium bicarbonate. The aqueous layer was extracted with ethyl acetate, and the combined organic layers were dried over magnesium sulfate, filtered, and concentrated under a stream of nitrogen. Reverse-phase HPLC purification (Column: Waters Sunfire C18, 19×100 mm, 5 μm; Mobile phase A: 0.05% trifluoroacetic acid in water; Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 5% - 95% B over 8.54 min, then 95% B over 1.46 min; Flow rate: 25 mL/min) gave (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[5,5,5-trifluoro-2-(2,2,2-trifluoroacetamido)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (86). Yield: 14 mg, 26 µmol, 14% in 2 stages. LCMS m/z 540.6 [M+H]+. Retention time: 2.60 minutes (Analytical conditions. Column: Waters Atlantis dC18, 4.6×50 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v); Gradient: 5.0% - 95% B, linear, for 4.0 minutes, then 95% B for 1.0 minute; Flow rate: 2 mL/minute).

Example 87

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[(2S)-2-cyclohexyl-2-{[(trifluoromethyl)sulfonyl]amino}acetyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (87)

Step 1. Synthesis of methyl (1R,2S,5S)-3-{(2S)-2-[(tert-butoxycarbonyl)amino]-2-cyclohexylacetyl}-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate (C68).

To a 0°C solution of methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate hydrochloride (300 mg, 1.46 mmol) and (2S)-[(tert-butoxycarbonyl)amino](cyclohexyl)acetic acid (394 mg, 1.53 mmol) in N,N-dimethylformamide (5 ml) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 610 mg, 1.60 mmol), then 4-methylmorpholine (443 mg, 4.38 mmol) was added dropwise. The reaction mixture was stirred at 0°C for 10 min, then at room temperature (20°C) for 2 h, then it was poured into ice water (30 mL) and extracted with ethyl acetate (3 x 40 mL). The combined organic layers were washed successively with water (40 mL), hydrochloric acid (1 M; 40 mL), saturated aqueous sodium bicarbonate (40 mL), and saturated aqueous sodium chloride, then dried over sodium sulfate, filtered, and concentrated in vacuo to afford C68 as a white foam. Yield: 580 mg, 1.42 mmol, 97%. LCMS m/z 409.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 7.03 (d, J=8.5 Hz, 1H), 4.17 (s, 1H), 4.00 (d, J=10.4 Hz, 1H), 3.88 (dd, J=9, 9 Hz, 1H), 3.75 (dd, J=10.3, 5.3 Hz, 1H), 3.64 (s, 3H), 1.83-1.47 (m, 8H), 1.46-1.28 (m, 2H), 1.33 (s, 9H), 1.18-1.06 (m, 3H), 1.00 (s, 3H), 0.86 (s, 3H).

Step 2. Synthesis of methyl (1R,2S,5S)-3-[(2S)-2-amino-2-cyclohexylacetyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carloxylate, hydrochloride (C69).

A solution of the hydrochloride in 1,4-dioxane (4 M; 15 ml) was added dropwise to a 5°C solution of C68 (580 mg, 1.42 mmol) in 1,4-dioxane (3 ml). The reaction mixture was then stirred at room temperature (20°C) for 1.5 h and concentrated in vacuo. The residue was co-evaporated with dichloromethane to give C69 as a light yellow foam (490 mg), all of which was used in the next experiment. 1H NMR (400 MHz, DMSO-d6) δ 8.22 (br s, 3H), 4.26 (s, 1H), 3.99-3.90 (m, 1H), 3.76 (dd, component of the ABX system, J=10.7, 5.1 Hz, 1H), 3.71 (d, half of the AB quartet, J=10.6 Hz, 1H), 3.66 (s, 3H), 1.83-1.60 (m, 6H), 1.59 (dd, J=7.6, 5.1 Hz, 1H), 1.49 (d, half of the AB quartet, J=7.6 Hz, 1H), 1.27-1.02 (m, 5H), 1.03 (s, 3H), 0.94 (s, 3H).

Step 3. Synthesis of methyl (1R,2S,5S)-3-[(2S)-2-cyclohexyl-2-{[(trifluoromethyl)sulfonyl]amino}acetyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carloxylate (C70).

To a -10°C solution of C69 (from the previous step; 480 mg, ≤1.39 mmol) in dichloromethane (10 mL) was added N,N-diisopropylethylamine (630 mg, 4.87 mmol), followed by dropwise addition of trifluoromethanesulfonic anhydride (0.328 mL, 1.95 mmol). The reaction mixture was stirred at -10°C for 1 h, then at room temperature (20°C) for 1 h, then it was diluted with saturated aqueous sodium chloride solution (50 mL) and extracted with dichloromethane (3×30 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and purified by chromatography on silica gel (eluent: 1:4 ethyl acetate/petroleum ether) to give C70 as a light yellow oil. Yield: 124 mg, 0.282 mmol, 20% over 2 steps. LCMS m/z 441.1 [M+H]+.

Step 4. Synthesis of (1R,2S,5S)-3-[(2S)-2-cyclohexyl-2-{[(trifluoromethyl)sulfonyl]amino}acetyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C71).

To a solution of C70 (120 mg, 0.272 mmol) in a mixture of water (2 mL), methanol (2 mL), and tetrahydrofuran (2 mL) was added lithium hydroxide monohydrate (28.6 mg, 0.682 mmol). The reaction mixture was then stirred at room temperature (20 °C) for 18 h, LCMS showed the reaction to be complete: LCMS m/z 427.2 [M+H]+. The reaction mixture was concentrated in vacuo to remove organic solvents. The residue was diluted with water (5 mL) and then acidified to pH 2–3 by adding 1 M hydrochloric acid; the resulting mixture was extracted with ethyl acetate (2×30 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to afford C71 as a light yellow solid. Yield: 92.0 mg, 0.216 mmol, 79%.

Stage 5. Synthesis (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-3-[(2S)-2-cyclohex yl-2-{[(trifluoromethyl)sulfonyl]amino}acetyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (C72).

To a 0°C solution of C71 (92.0 mg, 0.216 mmol) and C16, HCl salt (72%, 68.8 mg, 0.238 mmol) in N,N-dimethylformamide (3 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 98.4 mg, 0.259 mmol), followed by 4-methylmorpholine (76.4 mg, 0.755 mmol). The reaction mixture was then stirred at 20°C for 2 h before it was poured into ice water (10 mL) and extracted with a mixture of chloroform and 2-propanol (4:1, 4×20 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and purified by silica gel chromatography (0% to 10% methanol in dichloromethane) to afford C72 as a colorless glass. Yield: 100 mg, 0.173 mmol, 80%. LCMS m/z 580.2 [M+H]+.

Stage 6. Synthesis (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[(2S)-2-cyclohexyl-2-{[(trifluoromethyl)sulfonyl]amino}acetyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (87).

A solution of trifluoroacetic anhydride (47.1 mg, 0.224 mmol) in dichloromethane (1 ml) was added dropwise to a 0°C solution of C72 (100 mg, 0.173 mmol) and pyridine (41.7 μl, 0.516 mmol) in dichloromethane (3 ml). The mixture was then stirred for 20 h at room temperature (10°C - 20°C), concentrated in vacuo and redissolved in dichloromethane (3 ml). Methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 103 mg, 0.432 mmol) was added and the reaction mixture was stirred at room temperature (20°C) for 20 h. It was then diluted with saturated aqueous sodium chloride solution (10 ml) and extracted with dichloromethane (3 x 20 ml); the combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and subjected to supercritical fluid chromatography [Column: Chiral Technologies Chiralcel OD, 30 x 250 mm, 10 μm; Mobile phase: 4:1 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide); Flow rate: 60 ml/min]. The fractions containing 87 were concentrated in vacuo below 40 °C to remove the alcohol cosolvent. The residue was diluted with ethyl acetate (50 ml) and dichloromethane (5 ml) and washed successively with hydrochloric acid (1 M; 20 ml), saturated aqueous sodium bicarbonate solution (20 ml), and saturated aqueous sodium chloride solution. The resulting organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo, then mixed with water (20 mL) and acetonitrile (5 mL); this mixture was lyophilized to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[(2S)-2-cyclohexyl-2-{[(trifluoromethyl)sulfonyl]amino}acetyl]-6,6-dimethyl-3-azabicyclo[3.1,0]hexane-2-carboxamide (87) as a white solid. Yield: 27.6 mg, 49.1 μmol, 28%. LCMS m/z 562.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 9.93 (d, J=8.3 Hz, 1H), 9.10 (d, J=8.7 Hz, 1H), 7.70 (s, 1H), 5.03-4.94 (m, 1H), 4.15 (s, 1H), 3.88-3.78 (m, 2H), 3.55 (d, J=10.2 Hz, 1H), 3.20-3.11 (m, 1H), 3.10-3.00 (m, 1H), 2.19-2.08 (m, 1H), 2.06-1.95 (m, 1H), 1.85-1.53 (m, 9H), 1.33 (d, half AB quartet, J=7.7 Hz, 1H), 1.03 (s, 3H), 0.89 (s, 3H).

Examples 88 and 89

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-L-alanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide and (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-D-alanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (88 and 89)

Stage 1. Synthesis of tert-butyl {1-[(1R,2S,5S)-2-({(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}carbamo yl)-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-3-cyclobutyl-1-oxopropan-2-yl}carbamate (C73).

A 0°C solution of C67 (150 mg, 0.371 mmol) and N-(tert-butoxycarbonyl)-3-cyclobutylalanine (99.2 mg, 0.408 mmol) in N,N-dimethylformamide (3 mL) was treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HFTU; 169 mg, 0.444 mmol). 4-Methylmorpholine (131 mg, 1.30 mmol) was then added dropwise and the reaction mixture was warmed to 25°C and stirred overnight. Ice water (10 mL) was added and the resulting mixture was extracted with a mixture of chloroform and 2-propanol (4:1, 4×20 mL); The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (0% to 10% methanol in dichloromethane) was performed twice to afford C73 as a white solid containing a mixture of two diastereomers. Yield: 106 mg, 0.199 mmol, 54%. LCMS m/z 534.2 [M+H]+. 1H NMR (400 MHz, chloroform-d ) δ [8.59 (d, J = 5.5 Hz) and 7.85 (d, J = 7.7 Hz), total 1H], 7.20-7.06 (m, 1H), [5.78 (br s) and 5.67 (br s), total 1H], [5.51 (br s) and 5.40 br (s), total 1H], 5.22-5.12 (m, 1H), [4.49-4.39 (m) and 4.38-4.23 (m), total 3H], 4.17-4.06 (m, 1H), [3.83 (d, J = 10.4 Hz) and 3.50 (d, J = 10.5 Hz), total 1H], 3.42-3.28 (m, 2H), 2.50-2.30 (m, 3H), 2.23-2.00 (m, 4H), 2.00-1.77 (m, 4H), 1.73-1.44 (m, 5H), [1.40 (s) and 1.39 (s), total 9H], [1.07 (s) and 1.03 (s), total 3H], [0.98 (s) and 0.92 (s), total 3H].

Step 2. Synthesis of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-L-alanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide and (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-D-alanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide [88 (DIAST-1) and 89 (DIAST-2)].

A solution of the hydrochloride salt in 1,4-dioxane (4 M; 5 mL, 20 mmol) was added dropwise to a 0 °C solution of C73 (106 mg, 0.199 mmol) in dichloromethane (5 mL). The reaction mixture was stirred at 0 °C for 15 min and then at 25 °C for 2 h, then it was concentrated in vacuo to give the deprotected product as a white solid: LCMS m/z 434.2 [M+H]+. This was dissolved in dichloromethane (3 mL), cooled in an ice bath, and treated with pyridine (79.9 mg, 1.01 mmol) and a solution of trifluoroacetic anhydride (170 mg, 0.809 mmol) in dichloromethane (1.5 mL). The reaction mixture was stirred at 20 °C for 20 h; then pyridine (40.0 mg, 0.506 mmol) was added and stirring was continued for another 12 h at 25 °C. After dilution with dichloromethane (15 mL), the reaction mixture was washed successively with hydrochloric acid (1 M; 10 mL), saturated aqueous sodium bicarbonate solution (3×10 mL), and saturated aqueous sodium chloride solution (10 mL), dried, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 20% methanol in dichloromethane) was followed by supercritical fluid chromatography [Column: Chiral Technologies Chiralpak IC, 30×250 mm, 10 μm; Mobile phase: 3:1 carbon dioxide/(methanol containing 0.1% ammonium hydroxide); Flow rate: 70 ml/minute], obtaining separated diastereomers (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-L-alanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide and (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-D-alanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide. Which of the products contains the D-alanyl configuration and which contains the L-alanyl configuration was not determined; the first eluted diastereomer was designated 88, and the second eluted diastereomer was designated 89. Both were obtained as a white solid.

88 - Yield: 9.3 mg, 18.2 µmol, 9% over 2 steps. LCMS m/z 512.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.75 (bd, J=7.0 Hz, 1H), 8.96 (d, J=8.1 Hz, 1H), 7.71 (s, 1H), 5.00-4.90 (m, 1H), 4.32-4.23 (m, 1H), 4.13 (s, 1H), 3.82 (dd, component of the ABX system, J=10.2, 5.4 Hz, 1H), 3.69 (d, half of the AB quartet, J=10.2 Hz, 1H), 3.20-3.02 (m, 2H), 2.44-2.28 (m, 2H), 2.16-2.04 (m, 2H), 2.04-1.91 (m, 2H), 1.87-1.54 (m, 9H), 1.32 (d, half of AB quartet, J=7.6 Hz, 1H), 1.03 (s, 3H), 0.88 (s, 3H). Retention time: 2.78 minutes (Analytical conditions. Column: Chiral Technologies Chiralpak IC-3, 4.6×150 mm, 3 μm; Mobile phase A: carbon dioxide; Mobile phase B: methanol containing 0.05% diethylamine (v/v); Gradient: 5% - 40% B for 5 minutes, then 40% B for 2.5 minutes; Back pressure: 1500 psi; Flow rate: 2.5 mL/minute).

89 - Yield: 23 mg, 45.0 mmol, 23% over 2 steps. 1H NMR analysis shows that this product exists as a mixture of rotamers. LCMS m/z 512.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ [9.92 w (s) and 9.65 (shs), total 1H], [9.22 (d, J=7.7 Hz) and 8.85 (d, J=8.4 Hz), total 1H], [7.76 (s) and 7.67 (s), total 1H], [5.11-5.00 (m) and 4.98-4.87 (m), total 1H], [4.51 (s) and 4.07 (s), total 1H], [4.47-4.36 (m) and 4.09-4.00 (m), total 1H], [3.90 (dd, J=10.2, 5.3 Hz) and 3.60-3.45 (m), total 2H], 3.21-3.00 (m, 2H), 2.44-2.33 (m, 1H), 2.28-1.98 (m, 3H), 1.98-1.52 (m, 10H), [1.49-1.38 (m) and 1.32 (d, half of AB quartet, J=7.6 Hz), total 2H], [1.04 (s) and 1.02 (s), total 3H], [0.93 (s) and 0.82 (s), total 3H]. Retention time: 4.14 minutes (Analytical conditions are identical to those used for 88).

Example 90

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(pyridin-2-yl)-N-(trifluoroacetyl)-L-alanyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (90)

Stage 1. Synthesis of tert-butyl [(2S)-1-[(1R,2S,5S)-2-({(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}carbam oil)-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-1-oxo-3-(pyridin-2-yl)propan-2-yl]carbamate (C74).

To a 0°C solution of C67 (250 mg, 0.618 mmol) and N-(tert-butoxycarbonyl)-3-pyridin-2-yl-L-alanine (198 mg, 0.744 mmol) in N,N-dimethylformamide (2 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 282 mg, 0.742 mmol), followed by dropwise addition of a solution of 4-methylmorpholine (188 mg, 1.86 mmol) in N,N-dimethylformamide (1 mL). The reaction mixture was then warmed to 20°C and stirred for 2 h, then it was diluted with water (10 mL) and extracted with ethyl acetate (10 mL). Solid sodium sulfate was added to the aqueous layer until saturation was reached, then the aqueous layer was extracted with a mixture of dichloromethane and methanol (10:1, 3×20 mL). The combined organic layers were concentrated in vacuo and purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give C74 as a yellow gum. Yield: 250 mg, 0.449 mmol, 73%. LCMS m/z 557.0 [M+H]+. 1H NMR (400 MHz, methanol-d4), characteristic peaks: δ 8.49 (d, J=5.2 Hz, 1H), 7.78-7.71 (m, 1H), 7.35 (d, J=7.8 Hz, 1H), 7.30-7.25 (m, 1H), 4.73 (dd, J=8.5, 5.2 Hz, 1H), 4.40 (dd, J=11.8, 4.2 Hz, 1H), 4.30 (s, 1H), 4.01-3.90 (m, 1H), 3.26 (dd, J=14.2, 5.6 Hz, 1H), 2.94 (dd, J=14.1, 8.9 Hz, 1H), 2.65-2.53 (m, 1H), 2.37-2.25 (m, 1H), 2.14-2.04 (m, 1H), 1.91-1.78 (m, 2H), 1.61-1.55 (m, 1H), 1.53 (d, half of AB quartet, J=7.7 Hz, 1H), 1.34 (s, 9H), 1.08 (s, 3H), 1.00 (s, 3H).

Step 2. Synthesis of (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-[3-(pyridin-2-yl)-L-alanyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, hydrochloride (C75).

A solution of the hydrochloride salt in 1,4-dioxane (4 M; 6 mL) was added to a 0°C solution of C74 (250 mg, 0.449 mmol) in dichloromethane (2 mL) and the reaction mixture was stirred for 5 h at 20°C. LCMS analysis showed conversion to C75: LCMS m/z 457.1 [M+H]+. Removal of the solvents in vacuo gave C75 as a yellow solid (250 mg); some of this product was used directly in the next step. 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 8.81 (bd, J=5.4 Hz, 1H), 8.43 (d, J=8.3 Hz, 1H), 8.39-8.31 (m, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.87-7.81 (m, 1H), 7.65 (s, 1H), 7.43 (brs, 1H), 7.06 (brs, 1H), 4.77-4.67 (m, 1H), 4.35 (s, 1H), 4.32-4.23 (m, 1H), 3.94-3.86 (m, 1H), 3.80 (d, half of the AB quartet, J=10.6 Hz, 1H), 3.36-3.25 (m, 1H), 3.20-3.08 (m, 2H), 2.35-2.23 (m, 1H), 2.17-2.06 (m, 1H), 2.01-1.90 (m, 1H), 1.55-1.48 (m, 1H), 1.43 (d, half of AB quartet, J=7.6 Hz, 1H), 1.04 (s, 3H), 0.96 (s, 3H).

Stage 3. Synthesis (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(pyridin-2-yl)-N-(trifluoroacetyl)-L-alanyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (90).

To a 0°C solution of C75 (from the previous step; 175 mg, ≤0.314 mmol) in dichloromethane (6 mL) was added pyridine (197 mg, 2.49 mmol), followed by a solution of trifluoroacetic anhydride (186 mg, 0.886 mmol) in dichloromethane (2 mL). The reaction mixture was then stirred at 20°C for 2 h, diluted with water and extracted with a mixture of dichloromethane and methanol (10:1, 3×15 mL). The combined organic layers were concentrated in vacuo and subjected to silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane), then supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AS, 30×250 mm, 10 μm; Mobile phase: 3:1 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide); flow rate: 70 mL/min] to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(pyridin-2-yl)-N-(trifluoroacetyl)-L-alanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (90) as a white solid. Yield: 25 mg, 46.8 μmol, 15% over 2 steps. LCMS m/z 535.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.92 (d, J=7.5 Hz, 1H), 8.91 (d, J=8.0 Hz, 1H), 8.51 (bd, J=5 Hz, 1H), 7.75-7.68 (m, 2H), 7.28 (d, J=7.8 Hz, 1H), 7.25 (dd, J=7.3, 5.1 Hz, 1H), 5.00-4.88 (m, 2H), 4.15 (s, 1H), 3.86 (dd, component of the ABX system, J=10.3, 5.4 Hz, 1H), 3.70 (d, half of the AB quartet, J=10.3 Hz, 1H), 3.21-3.04 (m, 4H), 2.42-2.31 (m, 1H), 2.18-2.04 (m, 2H), 1.76 (ddd, J=13.5, 9.6, 6.6 Hz, 1H), 1.74-1.62 (m, 1H), 1.62-1.53 (m, 1H), 1.33 (d, half of AB quartet, J=7.6 Hz, 1H), 1.03 (s, 3H), 0.89 (s, 3H).

Example 91

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-{N-[(4-fluorophenoxy)acetyl]-3-methyl-L-valyl}-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (91)

Stage 1. Synthesis of tert-butyl {(2S)-1-[(1R,2S,5S)-2-({(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}car bamoyl)-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2-yl}carbamate (C76).

A solution of C32 (15.4 g, 41.8 mmol) and C16 HCl salts (75%, 11.6 g, 41.9 mmol) in N,N-dimethylformamide (380 mL) was cooled to -5°C to 0°C. O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 18.3 g, 48.1 mmol) and 4-methylmorpholine (12.7 g, 126 mmol) were added at -5°C to 0°C. The reaction mixture was then stirred at 0°C for 1.5 h, poured into ice water (400 mL), and the resulting mixture was extracted with ethyl acetate (3×200 mL). The combined organic layers were washed successively with aqueous citric acid (1 M; 120 mL), saturated aqueous sodium bicarbonate (120 mL), and saturated aqueous sodium chloride (3 x 60 mL), dried over sodium sulfate, filtered, concentrated in vacuo, and combined with the crude product from a similar reaction carried out using C32 (1.08 g, 2.93 mmol). Silica gel chromatography (0% to 10% methanol in dichloromethane) gave C76 as a white solid (9.80 g). The combined aqueous layers were extracted with a mixture of chloroform and 2-propanol (4:1, 3 x 100 mL); Concentration of these combined extracts followed by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) gave additional C76 as a white solid (2.3 g). Combined yield: 12.1 gm, 23.2 mmol, 52%. LCMS m/z 522.5 [M+H]+. 1H NMR (400 MHz, chloroform-d) δ 8.28 (bd, J=5.9 Hz, 1H), 7.20 (bd, 1H), 5.71 (bd, 1H), 5.38 (bd, 1H), 5.10 (bd, J=10.3 Hz, 1H), 4.47-4.38 (m, 1H), 4.28-4.20 (m, 2H), 4.12 (dd, component of the ABX system, J=10.2, 4.0 Hz, 1H), 3.99 (d, half of the AB quartet, J=10.2 Hz, 1H), 3.40-3.33 (m, 2H), 2.53-2.35 (m, 2H), 2.17-2.07 (m, 1H), 2.00-1.81 (m, 2H), 1.52-1.4 (m, 2H, expected; significantly obscured by the water and tert-butyl peak signal), 1.39 (s, 9H), 1.02 (s, 3H), 1.01 (s, 9H), 0.88 (s, 3H).

Step 2. Synthesis of (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-(3-methyl-L-valyl)-3-azabicyclo[3,1,0]hexane-2-carboxamide, hydrochloride (C77).

To a 0°C solution of C76 (12.1 g, 23.2 mmol) in dichloromethane (50 mL) was added a solution of the hydrochloride salt in 1,4-dioxane (4 M; 250 mL). The reaction mixture was then stirred at 20°C for 2 h and filtered. The filter cake was stirred with methyl tert-butyl ether (250 mL) for 18 h; filtration gave C77 as a light yellow/white solid (10.89 g). LCMS m/z 422.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J=9.0 Hz, 1H), 8.19 (br s, 3H), 7.57 (br s, 1H), 7.41 (br s, 1H), 7.04 (br s, 1H), 4.35-4.27 (m, 1H), 4.34 (s, 1H), 3.85-3.72 (m, 2H), 3.65 (d, half of AB quartet, J=10.8 Hz, 1H), 3.16-3.09 (m, 1H), 3.05-2.95 (m, 1H), 2.43-2.31 (m, 1H), 2.17-2.04 (m, 1H), 2.00-1.89 (m, 1H), 1.71-1.42 (m, 3H), 1.38 (d, half of AB quartet, J=7.7 Hz, 1H), 1.02 (s, 3H), 1.02 (s, 9H), 0.97 (s, 3H).

Stage 3. Synthesis (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-3-[N-(chloroacetyl)-3-methyl-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (C78).

Triethylamine (2.21 g, 21.8 mmol) was added to a 0°C solution of C77 (from the previous step; 2.50 g, ≤5.33 mmol) in dichloromethane (100 mL). A solution of chloroacetyl chloride (1.23 g, 10.9 mmol) in dichloromethane (9 mL) was added dropwise to the reaction mixture and stirring was continued at 0°C for 1 h. Water (50 mL) was then added and the resulting mixture was extracted with a mixture of chloroform and 2-propanol (4:1, 3×50 mL); the combined organic layers were washed with saturated aqueous sodium chloride solution (50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Purification by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) gave C78 as a white solid. Yield: 1.21 g, 2.43 mmol, 46% over 2 steps. LCMS m/z 498.1 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.30-8.22 (m, 2H), 7.54 (br s, 1H), 7.30 (br s, 1H), 7.03 (br s, 1H), 4.35 (d, J=8.9 Hz, 1H), 4.29 (ddd, J=12.1, 8.7, 3.4 Hz, 1H), 4.24 (s, 1H), 4.11 (AB quartet, JAB=12.4 Hz, ΔνAB=14.3 Hz, 2H), 3.86 (dd, component of the ABX system, J=10.2, 5.4 Hz, 1H), 3.72 (d, half of the AB quartet, J=10.3 Hz, 1H), 3.18-3.08 (m, 1H), 3.07-2.97 (m, 1H), 2.48-2.36 (m, 1H), 2.19-2.08 (m, 1H), 1.99-1.88 (m, 1H), 1.69-1.43 (m, 3H), 1.37 (d, half of AB quartet, J=7.7 Hz, 1H), 1.01 (s, 3H), 0.94 (s, 9H), 0.84 (s, 3H).

Stage 4. Synthesis (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-3-{N-[(4-fluorophenoxy)acetyl]-3-methyl-L-valyl}-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (C79).

4-Fluorophenol (49.5 mg, 0.442 mmol) was added to a mixture of cesium fluoride (67.1 mg, 0.442 mmol) in N,N-dimethylformamide (3 mL) and the mixture was stirred at 65 °C for 1 h, then C78 (110.0 mg, 0.221 mmol) was added and the reaction mixture was stirred at 65 °C for 8 h. It was then combined with a similar reaction carried out using C78 (30 mg, 60 μmol), poured into water (10 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed successively with water (20 mL), hydrochloric acid (1 M; 10 mL), saturated aqueous sodium carbonate (10 mL), and saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (0% to 10% methanol in dichloromethane) afforded C79 as a white glass. Combined yield: 100 mg, 0.174 mmol, 62%. LCMS m/z 574.2 [M+H]+. 1H NMR (400 MHz, chloroform-d ) δ 8.23 (bd, J = 6 Hz, 1H), 7.19 (bd s, 1H), 7.04 (bd, J = 10.2 Hz, 1H), 6.99 (dd, J = 9.2, 8.0 Hz, 2H), 6.85 (dd, ABX system component, J = 9.1, 4.2 Hz, 2H), 5.78 (bd s, 1H), 5.46 (bd s, 1H), 4.65 (d, J = 9.8 Hz, 1H), 4.52-4.38 (m, 3H), 4.22 (s, 1H), 4.14 (dd, ABX system component, J = 10.3, 5.3 Hz, 1H), 3.93 (d, half of AB quartet, J=10.3 Hz, 1H), 3.41-3.32 (m, 2H), 2.55-2.34 (m, 2H), 2.14-2.05 (m, 1H), 2.03-1.80 (m, 3H), 1.03 (s, 3H), 1.00 (s, 9H), 0.86 (s, 3H).

Step 5. Synthesis of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-{N-[(4-fluorophenoxy)acetyl]-3-methyl-L-valyl}-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (91).

A solution of methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 125 mg, 0.524 mmol) in dichloromethane (2 ml) was added dropwise to a 10°C (room temperature) solution of C79 (100 mg, 0.174 mmol) in dichloromethane (4 ml). The reaction mixture was then stirred at 10°C for 16 h, diluted with water (10 ml) and extracted with dichloromethane (3×10 ml). The combined organic layers were washed with saturated aqueous sodium chloride solution (2×10 ml) and concentrated in vacuo; chromatography on silica gel (Gradient: 0% - 10% methanol in dichloromethane), followed by supercritical fluid chromatography [Column: Chiral Technologies Chiralcel OD, 30×250 mm, 10 μm; Mobile phase: 4:1 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide); Flow rate: 60 ml/min] afforded (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-{N-[(4-fluorophenoxy)acetyl]-3-methyl-L-valyl}-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (91) as a white solid. Yield: 55 mg, 99.0 µmol, 57%. LCMS m/z 556.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.01 (d, J=8.5 Hz, 1H), 7.96 (d, J=9.1 Hz, 1H), 7.68 (s, 1H), 7.13-7.04 (m, 2H), 6.90-6.84 (m, 2H), 4.97 (ddd, J=10.9, 8.5, 5.1 Hz, 1H), 4.61-4.50 (m, 2H), 4.39 (d, J=8.9 Hz, 1H), 4.12 (s, 1H), 3.87 (dd, component of the ABX system, J=10.4, 5.5 Hz, 1H), 3.73 (d, half of the AB quartet, J=10.4 Hz, 1H), 3.19-3.09 (m, 1H), 3.09-2.99 (m, 1H), 2.47-2.36 (m, 1H, predicted; partially obscured by solvent peak), 2.21-2.02 (m, 2H), 1.79-1.65 (m, 2H), 1.53 (dd, J=7.6, 5.4 Hz, 1H), 1.29 (d, half of AB quartet, J=7.6 Hz, 1H), 1.00 (s, 3H), 0.93 (s, 9H), 0.75 (s, 3H).

Example 92

3-Methyl-N-[(4-methylphenyl)acetyl]-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide (92)

A mixture of C51 (68.0 mg, 0.12 mmol) and a solution of the hydrochloride in 1,4-dioxane (4 M; 1 mL, 4 mmol) was stirred at room temperature for 5 min, then the reaction mixture was concentrated in vacuo to remove the solvent, then further sucked under high vacuum to remove residual hydrochloride. (4-Methylphenyl)acetic acid (18.6 mg, 0.124 mmol) was added to the residue; the resulting mixture was dissolved in N,N-dimethylformamide (1.0 mL) and cooled to -30 °C. After addition of N,N-diisopropylethylamine (64.7 μL, 0.371 mmol) followed by O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 61.2 mg, 0.161 mmol), the reaction mixture was warmed to room temperature for 1 h and then treated with aqueous sodium bicarbonate. The mixture was extracted 5 times with ethyl acetate and the combined organic layers were concentrated under reduced pressure. The residue was then dissolved in dichloromethane (1 mL), treated with methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 88.5 mg, 0.371 mmol), and stirred at room temperature for 1 h, then the reaction mixture was treated with dilute aqueous sodium carbonate and extracted twice with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered, concentrated in vacuo, and purified by reverse-phase HPLC (Column: Waters XBridge C18, 19×100 mm, 5 μm; Mobile phase A: water; Mobile phase B: acetonitrile; Gradient: 20% - 60% B for 8.5 min, then 60% - 95% B for 0.5 min, then 95% B for 1.0 min; Flow rate: 25 mL/min) to give 3-methyl-N-[(4-methylphenyl)acetyl]-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide (92). Yield: 20.7 mg, 36.7 μmol, 31%. LCMS m/z 564.8 [M+H]+. Retention time: 2.71 minutes (Analytical conditions. Column: Waters Atlantis dC18, 4.6×50 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v); Gradient: 5.0% - 95% B, linear, for 4.0 minutes, then 95% B for 1.0 minute; Flow rate: 2 mL/minute).

Example 93

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(1H-pyrazol-1-yl)-N-(trifluoroacetyl)-L-alanyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (93)

Step 1. Synthesis of (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide, hydrochloride (C67, HCl salt).

To a solution of C66 (9.97 g, 24.4 mmol) in dichloromethane (50 mL) was added a solution of the hydrochloride salt in 1,4-dioxane (4 M; 90 mL). The reaction mixture was stirred at room temperature (25 °C) for 2 h, then LCMS analysis showed conversion to C67: LCMS m/z 309.0 [M+H]+. Concentration in vacuo gave C67, the HCl salt as a white solid. Yield: 8.10 g, 23.5 mmol, 96%. 1H NMR (400 MHz, DMSO-d6) δ 10.20-10.08 (m, 1H), 8.93 (d, J=8.0 Hz, 1H), 8.86-8.71 (m, 1H), 7.68 (br s, 1H), 7.63 (br s, 1H), 7.12 (br s, 1H), 4.30 (ddd, J=10.9, 8.1, 4.1 Hz, 1H), 4.09-4.02 (m, 1H), 3.63-3.53 (m, 1H, assumed; partially obscured by water peak), 3.22-2.99 (m, 3H), 2.34-2.22 (m, 1H), 2.21-2.11 (m, 1H), 2.01 (ddd, J=13.6, 11.1, 3.6 Hz, 1H), 1.80-1.66 (m, 3H), 1.55 (ddd, J=13.6, 11.4, 4.1 Hz, 1H), 1.08 (s, 3H), 1.05 (s, 3H).

Stage 2. Synthesis of tert-butyl [(2S)-1-[(1R,2S,5S)-2-({(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}carbamoy l)-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-1-oxo-3-(1H-pyrazol-1-yl)propan-2-yl]carbamate (C80).

A 0°C solution of C67, HCl salt (300 mg, 0.870 mmol) and N-(tert-butoxycarbonyl)-3-(1H-pyrazol-1-yl)-L-alanine (222 mg, 0.870 mmol) in N,N-dimethylformamide (10 mL) was treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 430 mg, 1.13 mmol) and 4-methylmorpholine (264 mg, 2.61 mmol). The reaction mixture was then stirred at 0°C for 2 h, diluted with water (50 mL) and extracted with ethyl acetate (20 mL). The aqueous layer was then saturated with solid sodium sulfate and extracted with a mixture of dichloromethane and methanol (10:1, 4×30 mL). The combined organic layers were concentrated in vacuo and chromatographed on silica gel twice (Gradient #1: 0% to 10% methanol in dichloromethane; Gradient #2: 0% to 25% methanol in dichloromethane) to afford C80 as a bright yellow solid. Yield: 340 mg, 0.623 mmol, 72%. LCMS m/z 546.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.29 (bd, J=8.6 Hz, 1H), 7.70-7.66 (m, 1H), 7.64 (s, 1H), 7.51 (bd, 1H), 7.38 (bd, J=8.2 Hz, 1H), 7.15-7.06 (m, 2H), 6.23-6.19 (m, 1H), 4.53-4.43 (m, 1H), 4.34-4.18 (m, 3H), 4.17 (s, 1H), 3.76 (d, half of AB quartet, J=10.5 Hz, 1H), 3.65 (dd, component of ABX system, J=10.4, 5.4 Hz, 1H), 3.20-3.06 (m, 2H), 2.36-2.24 (m, 1H), 2.19-2.08 (m, 1H), 2.02-1.90 (m, 1H), 1.71-1.55 (m, 2H), 1.53-1.46 (m, 1H), 1.39 (d, half of AB quartet, J=7.6 Hz, 1H), 1.30 (s, 9H), 1.01 (s, 3H), 0.89 (s, 3H).

Stage 3. Synthesis (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-[3-(1H-pyrazol-1-yl)-L-alanyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, hydrochloride (C81).

A solution of the hydrochloride salt in 1,4-dioxane (4 M; 15 mL) was added to a 0°C solution of C80 (340 mg, 0.623 mmol) in dichloromethane (4 mL). The reaction mixture was then stirred at 20°C for 1 h, filtered, and the filter cake washed with dichloromethane (3×10 mL). The combined filtrates were concentrated in vacuo to give C81 as a white solid. Yield: 244 mg, 0.506 mmol, 81%. LCMS m/z 446.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 8.57-8.48 (m, 3H), 8.42 (bd, J=8.6 Hz, 1H), 7.81 (d, J=2.3 Hz, 1H), 7.65 (bd s, 1H), 7.58 (d, J=1.9 Hz, 1H), 7.35 (bd s, 1H), 7.09 (bd s, 1H), 6.27 (dd, J=2.2 Hz, 1H), 4.59 (dd, component of the ABX system, J=14.4, 4.7 Hz, 1H), 4.54-4.41 (m, 2H), 4.36-4.25 (m, 1H), 4.30 (s, 1H), 3.66-3.60 (m, 1H), 3.42-3.33 (m, 1H), 3.21-3.03 (m, 2H), 2.31-2.20 (m, 1H), 2.18-2.06 (m, 1H), 1.97 (ddd, J=13.5, 11.5, 3.7 Hz, 1H), 1.46 (dd, component of the ABX system, J=7.7, 5.3 Hz, 1H), 1.41 (d, half of the AB quartet, J=7.7 Hz, 1H), 1.01 (s, 3H), 0.92 (s, 3H).

Stage 4. Synthesis (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(1H-pyrazol-1-yl)-N-(trifluoroacetyl)-L-alanyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (93).

A 0°C solution of C81 (120 mg, 0.249 mmol) in dichloromethane (6.0 mL) was treated with pyridine (170 mg, 2.15 mmol) followed by the addition of a solution of trifluoroacetic anhydride (158 mg, 0.752 mmol) in dichloromethane (2.0 mL). The reaction mixture was then warmed to 20°C and stirred for 3 h, then diluted with water (20 mL) and extracted with dichloromethane (3×20 mL). The combined organic layers were washed with hydrochloric acid (1 M; 20 mL) and saturated aqueous sodium chloride (2×20 mL), dried, filtered, and concentrated in vacuo. Chromatography on silica gel (Gradient: 0% to 10% methanol in dichloromethane) followed by supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD, 30×250 mm, 10 μm; Mobile phase: 4:1 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide); Flow rate: 60 ml/min] afforded (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(1H-pyrazol-1-yl)-N-(trifluoroacetyl)-L-alanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (93) as a white solid. Yield: 15.0 mg, 28.6 µmol, 11%. LCMS m/z 524.0 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ 10.03 (br s, 1H), 8.92 (d, J=8.0 Hz, 1H), 7.74 (br s, 1H), 7.69 (d, J=2.3 Hz, 1H), 7.51 (d, J=1.9 Hz, 1H), 6.23 (dd, J=2.2 Hz, 1H), 5.01-4.93 (m, 1H), 4.91-4.83 (m, 1H), 4.49-4.39 (m, 2H), 4.13 (s, 1H), 3.73 (dd, component of the ABX system, J=10.4, 5.4 Hz, 1H), 3.60 (d, half of the AB quartet, J=10.4 Hz, 1H), 3.21-3.09 (m, 2H), 2.40-2.31 (m, 1H), 2.20-2.10 (m, 2H), 1.79 (ddd, J=13.7, 9.5, 6.8 Hz, 1H), 1.76-1.66 (m, 1H), 1.56 (dd, J=7.5, 5.4 Hz, 1H), 1.36 (d, half AB quartet, J=7.6 Hz, 1H), 1.02 (s, 3H), 0.85 (s, 3H).

Example 94

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[(2S)-4,4-difluoro-2- (2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (94)

Stage 1. Synthesis of 9H-fluoren-9-ylmethyl {(2S)-1-[(1R,2S,5S)-2-({(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}car bamoyl)-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-4,4-difluoro-1-oxobutan-2-yl}carbamate (C82).

To a 0°C solution of C67 (230 mg, 0.569 mmol) and (2S)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4,4-difluorobutanoic acid (247 mg, 0.684 mmol) in N,N-dimethylformamide (5 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 281 mg, 0.739 mmol) in one portion; then 4-methylmorpholine (173 mg, 1.71 mmol) was added dropwise. The reaction mixture was then stirred at 0°C for 10 min, warmed to room temperature (20°C) and stirred for 2 h, then the reaction mixture was poured into ice water (15 mL) and extracted with ethyl acetate (2×15 mL). The combined organic layers were washed successively with 1 M hydrochloric acid, saturated aqueous sodium bicarbonate and saturated aqueous sodium chloride, dried over sodium sulfate, filtered, concentrated in vacuo and purified by silica gel chromatography (0% to 10% methanol in dichloromethane) to afford C82 as a white solid. Yield: 245 mg, 0.376 mmol, 66%. LCMS m/z 652.5 [M+H]+. 1H NMR (400 MHz, chloroform-d ) δ 8.86 (br s, 1H), 7.74 (d, J = 7.6 Hz, 2H), 7.58-7.51 (m, 2H), 7.38 (dd, J = 7.4, 7.4 Hz, 2H), 7.33-7.25 (m, 2H, assumed; partially obscured by solvent peak), 7.08 (br s, 1H), 6.49-6.32 (m, 1H), 6.17-5.79 (m, 3H), 4.78-4.67 (m, 1H), 4.41-4.24 (m, 4H), 4.22-4.07 (m, 2H), 3.83 (d, J = 10.5 Hz, 1H), 3.45-3.32 (m, 2H), 2.62-2.36 (m, 3H), 2.26-2.10 (m, 2H), 1.99-1.83 (m, 2H), 1.53 (d, half of AB quartet, J=7.6 Hz, 1H), 1.51-1.44 (m, 1H), 1.03 (s, 3H), 0.89 (s, 3H).

Stage 2. Synthesis (1R,2S,5S)-3-[(2S)-2-amino-4,4-difluorobutanoyl]-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (C83).

To a 0°C suspension of C82 (195 mg, 0.299 mmol) in dichloromethane (3 mL) was added a solution of diethylamine (32.8 mg, 0.448 mmol) in dichloromethane (0.5 mL). The reaction mixture was stirred at 30°C for 16 h, then combined with a similar reaction carried out using C82 (50 mg, 77 μmol) and concentrated in vacuo. Purification by silica gel chromatography [Gradient: 0% to 10% (10:1 mixture of methanol and ammonium hydroxide) in dichloromethane] gave C83 as a colorless gum. Combined yield: 149 mg, 0.347 mmol, 92%. LCMS m/z 452.3 [M+Na+]. 1H NMR (400 MHz, chloroform-d ) δ 8.73 (bd, J = 5.3 Hz, 1H), 7.11 (bd s, 1H), 6.03 (tdd, J = 56.8, 6.2, 3.0 Hz, 1H), 5.64 (bd s, 1H), 5.28 (bd s, 1H), 4.35-4.27 (m, 1H), 4.27 (s, 1H), 4.11 (dd, J = 10.3, 5.5 Hz, 1H), 3.72 (dd, J = 9.3, 4.1 Hz, 1H), 3.61 (d, J = 10.3 Hz, 1H), 3.41-3.35 (m, 2H), 2.53-2.36 (m, 3H), 2.20-2.11 (m, 1H), 2.00-1.84 (m, 3H), 1.6-1.45 (m, 2H, expected; partially obscured by the water peak), 1.06 (s, 3H), 0.94 (s, 3H).

Stage 3. Synthesis (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[(2S)-4,4-difluoro-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide (94).

To a 0°C solution of C83 (99 mg, 0.23 mmol) in dichloromethane (4 ml) were added pyridine (146 mg, 1.85 mmol) and a solution of trifluoroacetic anhydride (194 mg, 0.924 mmol) in dichloromethane (2 ml). The reaction mixture was stirred at room temperature (15°C) for 20 h, treated with additional pyridine (30 mg, 0.38 mmol) and stirred at room temperature (15°C) for a further 16 h. It was then partitioned between dichloromethane (15 ml) and hydrochloric acid (1 M; 15 ml), and the organic layer was washed with saturated aqueous sodium bicarbonate (15 ml) and saturated aqueous sodium chloride (10 ml), dried, filtered, and concentrated in vacuo. The residue was combined with the product from a similar reaction carried out using C83 (50 mg, 0.12 mmol) and purified using reverse-phase HPLC (Column: Waters XBridge BEH C18, 25×150 mm, 5 μm; Mobile phase A: water containing 0.05% ammonium hydroxide (v/v); Mobile phase B: acetonitrile; Gradient: 23% - 63% B). (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[(2S)-4,4-difluoro-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1,0]hexane-2-carboxamide (94) was isolated as a white solid. Combined yield: 28.8 mg, 56.8 μmol, 16%. LCMS m/z 508.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.01 (br s, 1H), 8.96 (d, J=8.0 Hz, 1H), 7.72 (s, 1H), 6.16 (tt, J=55.9, 4.6 Hz, 1H), 4.99-4.90 (m, 1H), 4.65-4.57 (m, 1H), 4.14 (s, 1H), 3.85 (dd, component of the ABX system, J=10.2, 5.4 Hz, 1H), 3.67 (d, half of the AB quartet, J=10.3 Hz, 1H), 3.21-3.04 (m, 2H), 2.42-2.19 (m, 3H), 2.17-2.04 (m, 2H), 1.83-1.64 (m, 2H), 1.60 (dd, J=7.6, 5.3 Hz, 1H), 1.35 (d, half of AB quartet, J=7.6 Hz, 1H), 1.03 (s, 3H), 0.89 (s, 3H).

Example 95

N-(Methoxycarbonyl)-3-methyl-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide (95)

Step 1. Synthesis of (4R)-1-(tert-butoxycarbonyl)-4-(trifluoromethyl)-L-prolyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C50).

N,N-Diisopropylethylamine (14.8 mL, 85.0 mmol) was added to a -30 °C mixture of (4R)-1-(tert-butoxycarbonyl)-4-(trifluoromethyl)-L-proline (8.00 g, 28.2 mmol), C16 HCl salt (6.45 g, 31.1 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 11.8 g, 31.0 mmol) in N,N-dimethylformamide (100 mL). The reaction mixture was warmed to 0 °C for 1 h, then LCMS analysis showed the presence of C50: LCMS m/z 437.3 [M+H]+. Aqueous sodium bicarbonate solution (300 ml) was added and the resulting mixture was extracted with a mixture of 2-propanol and dichloromethane (1:4, 5×100 ml); the combined organic layers were concentrated in vacuo and purified by silica gel chromatography (Gradient: 0% to 100% methanol in dichloromethane) to give C50 as an oil. 1H NMR analysis showed that this product existed as a mixture of rotamers. Yield: 10.9 g, 25.0 mmol, 89%. 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ [8.28 (d, J=8.5 Hz) and 8.22 (d, J=8.2 Hz), total 1H], [7.64 (s) and 7.59 (s), total 1H], [7.38 (shs) and 7.27 (shs), total 1H], 7.05 (shs, 1H), 4.38-4.28 (m, 1H), 4.28-4.17 (m, 1H), 3.45-3.36 (m, 1H), 3.12-3.00 (m, 1H), 2.42-2.03 (m, 4H), 2.02-1.89 (m, 1H), 1.80-1.45 (m, 2H), [1.39 (s) and 1.32 (s), total 9H].

Step 2. Synthesis of N-(tert-butoxycarbonyl)-3-methyl-L-valyl-(4R)-4-(trifluoromethyl)-L-prolyl-3-[(3S)-2-oxopyrrolidin-3-yl]-L-alanine amide (C51).

A solution of the hydrochloride in 1,4-dioxane (4 M; 80 mL) was added to a solution of C50 (7.00 g, 16.0 mmol) in dichloromethane (15 mL). The reaction mixture was then stirred at room temperature for 5 min, concentrated in vacuo to remove the solvent, and then sucked under high vacuum to remove residual hydrochloride. The residue was combined with N-(tert-butoxycarbonyl)-3-methyl-L-valine (4.08 g, 17.6 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (GATU; 6.71 g, 17.6 mmol) in N,N-dimethylformamide (25 mL), cooled to -30 °C and treated with N,N-diisopropylethylamine (8.38 mL, 48.1 mmol). The reaction mixture was warmed to 0 °C for 1 h, then LCMS analysis showed the presence of C51: LCMS m/z 550.4 [M+H]+. The reaction mixture was then diluted with aqueous sodium bicarbonate and extracted three times with a 4:1 mixture of dichloromethane and 2-propanol. The combined organic layers were then concentrated in vacuo and the residue was purified by silica gel chromatography (Gradient: 0% to 30% methanol in dichloromethane) to afford C51 as a solid. Yield: 3.95 g, 7.19 mmol, 45%, 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 8.28 (d, J=8.8 Hz, 1H), 7.55 (s, 1H), 7.29 (br s, 1H), 7.03 (br s, 1H), 6.77 (bd, J=9.1 Hz, 1H), 4.52-4.43 (m, 1H), 4.24 (ddd, J=12.2, 8.7, 3.5 Hz, 1H), 4.12 (d, J=9.2 Hz, 1H), 4.02-3.84 (m, 2H), 3.16-3.08 (m, 1H), 3.08-2.98 (m, 1H), 2.50-2.37 (m, 1H), 2.31-2.20 (m, 1H), 2.19-2.05 (m, 2H), 2.00-1.87 (m, 1H), 1.69-1.55 (m, 1H), 1.55-1.44 (m, 1H), 1.36 (s, 9H), 0.93 (s, 9H).

Step 3. Synthesis of N-(methoxycarbonyl)-3-methyl-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide (95).

A mixture of C51 (230 mg, 0.418 mmol) and a solution of the hydrochloride in 1,4-dioxane (4 M; 2 ml, 8 mmol) was stirred at room temperature for 5 min, then the reaction mixture was concentrated in vacuo to remove the solvent, then sucked under high vacuum to remove residual hydrochloride. The residue was dissolved in dichloromethane (2 ml), cooled to 0 °C and treated with N,N-diisopropylethylamine (0.219 ml, 1.26 mmol) then methyl chloroformate (59.3 mg, 0.628 mmol). The reaction mixture was then stirred at 0 °C for 10 min, it was diluted with aqueous sodium bicarbonate and extracted three times with a 4:1 mixture of dichloromethane and 2-propanol; the combined organic layers were dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue was dissolved in dichloromethane (3 ml); after addition of methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 299 mg, 1.25 mmol), the reaction mixture was stirred at room temperature for 1 h. It was then treated with dilute aqueous sodium carbonate solution and extracted 3 times with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered, and purified by chromatography on silica gel (Gradient: 50% to 100% ethyl acetate in heptane). The resulting product was suspended in heptane (4 ml) at 50°C for 2 h, cooled to room temperature, and stirred at room temperature overnight; collection of the solid afforded N-(methoxycarbonyl)-3-methyl-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide (95) as a solid. Yield: 123 mg, 0.251 mmol, 60%. LCMS m/z 490.4 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.02 (d, J=8.6 Hz, 1H), 7.65 (s, 1H), 7.27 (bd, J=8.7 Hz, 1H), 4.94 (ddd, J=11.1, 8.5, 5.0 Hz, 1H), 4.36 (dd, J=7.3, 7.2 Hz, 1H), 4.14 (d, J=8.7 Hz, 1H), 4.00-3.91 (m, 2H), 3.52 (s, 3H), 3.46-3.34 (m, 1H, assumed; partially obscured by water peak), 3.18-2.98 (m, 2H), 2.5-2.39 (m, 1H, assumed; partially obscured by solvent), 2.35-2.23 (m, 1H), 2.22-2.01 (m, 3H), 1.77-1.63 (m, 2H), 0.94 (s, 9H).

Example 96

(1R,2S,5S)-N-{(1R)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (96) and (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (13), Solid Form 5

To a solution of 13, methyl tert-butyl ether solvate (from the alternative synthesis of Example 13, methyl tert-butyl ether solvate; 15.0 g, 25.5 mmol) in acetonitrile (80 mL) was added methanesulfonic acid (6.4 mL, 99 mmol). The reaction mixture was stirred at room temperature for 4 h, then it was made basic by adding a mixture of saturated aqueous sodium bicarbonate (80 mL) and saturated aqueous sodium chloride (10 mL). The resulting mixture was extracted with dichloromethane (2×100 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Separation of the two epimers was accomplished by supercritical fluid chromatography (Column: Chiral Technologies Chiralcel OX-H, 30×250 mm, 5 μm; Mobile phase: 9:1 carbon dioxide/2-propanol; Back pressure: 100 bar; Flow rate: 80 ml/min). The first product to elute was reduced (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide (13). According to X-ray powder diffraction, this product was amorphous; it is designated as Solid Form 5. The second eluted product is obtained as a glass which is dissolved in dichloromethane, treated with heptane and concentrated in vacuo to give (1R,2S,5S)-N-{(1R)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (96) as a solid.

Recovered 13 - Yield: 6.00 g, 12.0 mmol, 47%. LCMS m/z 500.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.46-9.33 (m, 1H), 9.01 (d, J=8.5 Hz, 1H), 7.66 (s, 1H), 5.03-4.91 (m, 1H), 4.46-4.37 (m, 1H), 4.16 (s, 1H), 3.97-3.86 (m, 1H), 3.69 (d, half of AB quartet, J=10.4 Hz, 1H), 3.19-3.09 (m, 1H), 3.09-2.98 (m, 1H), 2.46-2.33 (m, 1H), 2.21-2.03 (m, 2H), 1.79-1.65 (m, 2H), 1.61-1.53 (m, 1H), 1.32 (d, half of AB quartet, J=7.7 Hz, 1H), 1.03 (s, 3H), 0.98 (s, 9H), 0.85 (s, 3H). Retention time: 3.93 minutes (Analytical conditions. Column: Chiral Technologies Chiralcel OX-H, 4.6×250 mm, 5 μm; Mobile phase A: carbon dioxide; Mobile phase B: 2-propanol; Gradient: 5% B for 1.00 minute, then 5% - 60% B for 8.00 minutes; Back pressure: 120 bar; Flow rate: 3.0 mL/minute). The powder X-ray diffraction pattern for this amorphous product is given in Figure 9. The method for collecting the powder X-ray diffraction data is described in the Alternative Synthesis of Example 13, Methyl tert-butyl ether solvate, Step 8.

96 - Yield: 2.58 g, 5.16 mmol, 20%. LCMS m/z 500.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.42 (d, J=8.4 Hz, 1H), 9.06 (d, J=7.6 Hz, 1H), 7.79 (s, 1H), 4.96-4.86 (m, 1H), 4.41 (d, J=8.7 Hz, 1H), 4.20 (s, 1H), 3.92 (bdd, J=10.7, 5.4 Hz, 1H), 3.66 (d, half of AB quartet, J=10.5 Hz, 1H), 3.22-3.12 (m, 2H), 2.43-2.31 (m, 1H), 2.31-2.20 (m, 1H), 2.16-2.04 (m, 1H), 1.84-1.63 (m, 2H), 1.57-1.49 (m, 1H), 1.32 (d, half of AB quartet, J=7.7 Hz, 1H), 1.00 (brs, 12H), 0.84 (s, 3H). Retention time: 4.20 minutes (Analytical conditions identical to those used for the reduced 13 above).

Example 97

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[2-(2,2,2-trifluoroacetamido)-3-(trifluoromethyl)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, from C86 (DIAST-2) (97)

Step 1. Synthesis of 2-[(tert-butoxycarbonyl)amino]-3-(trifluoromethyl)pentanoic acid (C84).

Aqueous sodium hydroxide solution (1 M; 1.48 mL, 1.48 mmol) was added to a suspension of 2-amino-3-(trifluoromethyl)pentanoic acid (Wang et al., J. Amer. Chem. Soc. 2003, 125, 6900–6906; 137 mg, 0.740 mmol) in 1,4-dioxane (3 mL) and the resulting mixture was cooled to 0 °C. Di-tert-butyl dicarbonate (0.204 mL, 0.888 mmol) was slowly added and the reaction mixture was stirred at room temperature overnight. After dilution with ethyl acetate, the reaction mixture was cooled in an ice bath and then acidified to pH 2 by adding 1 M aqueous potassium hydrogen sulfate solution. The aqueous layer was extracted twice with ethyl acetate, and the combined organic layers were dried over magnesium sulfate, filtered, and concentrated in vacuo to afford C84 as a solid. This product was suspected to be a mixture of 4 diastereomers, potentially also exhibiting rotamers. Yield: 197 mg, 0.690 mmol, 93%. LCMS m/z 284.3 [M-H]-. 1H NMR (400 MHz, DMSO-d6) δ 13.16 (br s, 1H), [7.29 (d, major, J=9.8 Hz) and 6.95-6.85 (m, minor), total 1H], [4.55 (dd, major, J=9.8, 3.3 Hz), 4.46 (bd, minor, J=9.1 Hz), and 4.40 (dd, minor, J=9.4, 4.5 Hz), total 1H], 2.86-2.67 (m, 1H), 1.71-1.47 (m, 2H), 1.39 (br s, 9H), [0.98 (t, minor, J=7.4 Hz) and 0.91 (t, major, J=7.5 Hz), total 3H].

Stage 2. Synthesis of tert-butyl {1-[(1R,2S,5S)-2-({(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}carbamoyl )-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-1-oxo-3-(trifluoromethyl)pentan-2-yl}carbamate, DIAST-1 (C85) and tert-butyl {1-[(1R,2S,5S)-2-({(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}carbamoyl )-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-1-oxo-3-(trifluoromethyl)pentan-2-yl}carbamate, DIAST-2 (C86).

A 0°C solution of C84 (128 mg, 0.449 mmol) in a mixture of acetonitrile (2.7 mL) and N,N-dimethylformamide (1.5 mL) was treated with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 176 mg, 0.463 mmol) and 4-methylmorpholine (0.116 mL, 1.06 mmol). The reaction mixture was then stirred at 0°C for 30 min, C67 (170 mg, 0.420 mmol) was added as a solid, and stirring was continued for 2 h. The reaction mixture was then diluted with ethyl acetate and water, and washed with saturated aqueous sodium bicarbonate. The aqueous layer was extracted twice with ethyl acetate, and the combined organic layers were washed with brine, dried over magnesium sulfate, filtered, and concentrated in vacuo. The resulting oil was azeotroped twice with heptane and twice with methyl tert-butyl ether, then chromatographed on silica gel (Gradient: 0% to 20% methanol in dichloromethane). The first eluted diastereomer was designated C85, and the second eluted diastereomer was designated C86.

C85 (DIAST-1) - Yield: 77.3 mg, 0.134 mmol, 32%. This product contains a mixture of isomers or rotamers by 1H NMR analysis. LCMS m/z 576.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6), characteristic peaks, integrations are approximate: δ [8.35 (d, J=7.9 Hz) and 8.16 (d, J=8.5 Hz), total 1H], 7.62-7.54 (m, 1H), 7.41-7.18 (m, 2H), [7.02 (shs) and 6.98 (shs), total 1H], 4.59-4.50 (m, 1H), 4.29-4.13 (m, 2H), 3.89 (dd, J=10.4, 5.4 Hz, 1H), 3.44 (d, J=10.4 Hz, 1H), 3.20-3.04 (m, 2H), 2.70-2.59 (m, 1H), 2.43-2.31 (m, 1H), 2.21-2.08 (m, 1H), 1.99-1.88 (m, 1H), [1.38 (s) and 1.36 (s), total 9H], 1.01 (br s, 3H), 0.94-0.82 (m, 6H).

C86 (DIAST-2) - Yield: 87.8 mg, 0.153 mmol, 36%. This product is mostly a single isomer by 1H NMR analysis. LCMS m/z 576.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 8.27 (d, J=8.6 Hz, 1H), 7.59 (s, 1H), 7.31 (d, J=9.5 Hz, 1H), 7.22 (br s, 1H), 7.04 (br s, 1H), 4.55 (dd, J=9.5, 5.9 Hz, 1H), 4.30-4.17 (m, 1H), 4.23 (s, 1H), 3.79 (dd, component of the ABX system, J=10.1, 5.2 Hz, 1H), 3.71 (d, half of the AB quartet, J=10.1 Hz, 1H), 3.09-2.99 (m, 1H), 2.68-2.55 (m, 1H), 2.42-2.30 (m, 1H), 2.17-2.04 (m, 1H), 1.97-1.86 (m, 1H), 1.36 (s, 9H), 1.02 (s, 3H), 0.94-0.83 (m, 6H).

Stage 3. Synthesis (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-[2 -(2,2,2-trifluoroacetamido)-3-(trifluoromethyl)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, from C86 (DIAST-2) (C87).

A solution of C86 (DIAST-2) (87.8 mg, 0.153 mmol) in dichloromethane (1 mL) was treated with a solution of the hydrochloride in 1,4-dioxane (4 M; 0.381 mL, 1.52 mmol). The reaction mixture was then stirred at room temperature for 40 min, and methanol (0.5 mL) was added to improve solubility. After a further 40 min, a solution of the hydrochloride in 1,4-dioxane (4 M; 0.10 mL, 0.4 mmol) was added; after 30 min, LCMS analysis showed complete removal of the protecting group: LCMS m/z 476.2 [M+H]+. The reaction mixture was concentrated in vacuo and azeotroped twice with heptane; The residue was triturated twice with diethyl ether, suspended in dichloromethane (1.2 ml) and cooled to 0°C. After the addition of triethylamine (42.4 µl, 0.304 mmol) followed by trifluoroacetic anhydride (47.9 µl, 0.339 mmol), the reaction mixture was stirred at 0°C for 30 min, then removed from the ice bath and partitioned between water and ethyl acetate. The aqueous layer was extracted with ethyl acetate, and the combined organic layers were washed with saturated aqueous sodium bicarbonate and saturated aqueous sodium chloride, dried over magnesium sulfate, filtered, concentrated in vacuo, and azeotroped twice with methyl tert-butyl ether. By 1H NMR and LCMS analysis, this product contains a mixture of C87 and the corresponding methyl ester (LCMS m/z 587.4 [M+H]+). 1H NMR (400 MHz, DMSO-d6), characteristic peaks for C87: δ 8.31 (d, J=8.7 Hz, 1H), 7.59 (s, 1H), 7.26 (s, 1H), 7.05 (s, 1H), 3.59 (d, half of AB quartet, J=10.0 Hz, 1H), 1.97-1.87 (m, 1H), 1.40 (d, half of AB quartet, J=7.6 Hz, 1H), 1.03 (s, 3H), 0.84 (s, 3H). Purification by silica gel chromatography (0% to 20% methanol in dichloromethane), followed by azeotroping of the resulting oil with heptane, followed by azeotroping with diethyl ether/heptane gave C87 as a white solid. Yield: 17.9 mg, 31.3 µmol, 20%. LCMS m/z 572.0 [M+H]+.

Step 4. Synthesis of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[2-(2,2,2-trifluoroacetamido)-3-(trifluoromethyl)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, from C86 (DIAST-2) (97).

Methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 18.8 mg, 78.9 μmol) was added to a solution of C87 (18 mg, 31 μmol) in ethyl acetate (0.8 mL). The reaction mixture was then stirred at room temperature for 1 h, and more methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent) was added on the end of a spatula. Stirring was continued for 2 h, then the reaction mixture was filtered and the filter cake was washed with ethyl acetate. The combined filtrates were washed with saturated aqueous sodium bicarbonate, and the aqueous layer was extracted once with ethyl acetate; the combined organic layers were washed with saturated aqueous sodium chloride, dried over magnesium sulfate, filtered, and concentrated in vacuo to give the crude product. 1H NMR (600 MHz, DMSO-d6), major component: δ 9.90 (d, J=8.8 Hz, 1H), 8.85 (d, J=8.1 Hz, 1H), 7.56 (br s, 1H), 4.97-4.89 (m, 2H), 4.17 (s, 1H), 3.89 (dd, J=10.1, 5.4 Hz, 1H), 3.62 (d, J=10.1 Hz, 1H), 3.21-3.14 (m, 1H), 3.12-3.06 (m, 1H), 2.92-2.82 (m, 1H), 2.43-2.35 (m, 1H), 2.18-2.10 (m, 2H), 1.78 (ddd, J=13.6, 9.6, 6.0 Hz, 1H), 1.75-1.66 (m, 2H), 1.62-1.55 (m, 2H), 1.35 (d, half of AB quartet, J=7.7 Hz, 1H), 1.04 (s, 3H), 0.97-0.92 (t, J=7.6 Hz, 3H), 0.85 (s, 3H). This product was purified by reversed-phase HPLC (Column: Waters Sunfire C18, 19×100 mm, 5 μm; Mobile phase A: 0.05% trifluoroacetic acid in water (v/v); Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v/v); Gradient: 5% - 95% B over 8.54 min, then 95% B over 1.46 min; Flow rate: 25 mL/min) to give (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[2-(2,2,2-trifluoroacetamido)-3-(trifluoromethyl)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, from C86 (DIAST-2) (97). Yield: 8.3 mg, 15 µmol, 48%. LCMS m/z 554.6 [M+H]+. Retention time: 2.72 minutes (Analytical conditions. Column: Waters Atlantis dC18, 4.6×50 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v); Gradient: 5.0% - 95% B, linear for 4.0 minutes, then 95% B for 1.0 minute; Flow rate: 2 mL/minute).

Example 98

(1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[2-(2,2,2-trifluoroacetamido)-3-(trifluoromethyl)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, from C85 (DIAST-1) (98)

Stage 1. Synthesis (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-3-[2-amino-3-(trifluoromethyl)pentanoyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide, hydrochloride, from C85 (DIAST-1) (C88).

A solution of the hydrochloride salt in 1,4-dioxane (4 M; 0.336 mL, 1.34 mmol) was added to a solution of C85 (DIAST-1) (77.3 mg, 0.134 mmol) in dichloromethane (1 mL). The reaction mixture was then stirred at room temperature for 40 min, methanol (0.5 mL) was added to improve solubility. Stirring was continued for 2 h, then LCMS analysis showed complete deprotection: LCMS m/z 476.2 [M+H]+. The reaction mixture was concentrated in vacuo; the residue was azeotroped twice with heptane, then triturated twice with diethyl ether to give C88 as a white solid. Yield: 54.5 mg, 0.106 mmol, 79%. 1H NMR (400 MHz, DMSO-d6), main characteristic peaks: δ 8.53 (br s, 3H), 8.36 (d, J=8.5 Hz, 1H), 7.61 (s, 1H), 7.28 (br s, 1H), 7.06 (br s, 1H), 4.25 (ddd, J=10.9, 8.4, 4.5 Hz, 1H), 4.17 (s, 1H), 4.08 (dd, J=10.5, 5.6 Hz, 1H), 2.80-2.68 (m, 1H), 2.37-2.26 (m, 1H), 2.23-2.13 (m, 1H), 2.05-1.96 (m, 1H), 1.73-1.51 (m, 5H), 1.44 (d, half of AB quartet, J=7.7 Hz, 1H), 1.04 (s, 3H), 0.90 (s, 3H).

Stage 2. Synthesis (1R,2S,5S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-[2 -(2,2,2-trifluoroacetamido)-3-(trifluoromethyl)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, from C85 (DIAST-1) (C89).

A 0°C suspension of C88 (54.5 mg, 0.106 mmol) in dichloromethane (1 mL) was treated with triethylamine (26 μL, 0.19 mmol) followed by trifluoroacetic anhydride (19.5 μL, 29.1 mg, 0.138 mmol). The reaction mixture was then stirred at 0°C for 1 h and 10 min, trifluoroacetic anhydride (1 equivalent) was added; after 30 min, more trifluoroacetic anhydride (9.4 μL, 67 μmol) was added. Stirring was continued for 45 min, then LCMS analysis showed complete conversion to C89: LCMS m/z 572.4 [M+H]+. The reaction mixture was partitioned between water and ethyl acetate, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed successively with saturated aqueous sodium bicarbonate and saturated aqueous sodium chloride, dried over magnesium sulfate, filtered, and concentrated in vacuo to afford C89. Yield: 41.2 mg, 72.1 μmol, 68%. 1H NMR (400 MHz, DMSO-d6), major component, characteristic peaks: δ 10.04 (d, J=9.3 Hz, 1H), 8.23 (d, J=8.5 Hz, 1H), 7.58 (s, 1H), 7.31 (br s, 1H), 7.01 (br s, 1H), 4.92-4.83 (m, 1H), 4.23 (s, 1H), 3.95 (dd, J=10.2, 5.5 Hz, 1H), 2.98-2.86 (m, 1H), 2.38-2.27 (m, 1H), 1.90 (ddd, J=13.5, 11.2, 4.0 Hz, 1H), 1.39 (d, half AB quartet, J=7.7 Hz, 1H), 1.02 (s, 3H), 0.90 (s, 3H).

Step 3. Synthesis of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[2-(2,2,2-trifluoroacetamido)-3-(trifluoromethyl)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, from C85 (DIAST-1) (98).

Methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 42.7 mg, 0.179 mmol) was added to a solution of C89 (41.0 mg, 71.7 μmol) in ethyl acetate (0.8 mL). The reaction mixture was then stirred at room temperature for 1 h, and methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent) was added on the tip of a spatula. Stirring was continued for 2 h, then the reaction mixture was filtered and the filter cake was washed with ethyl acetate. The combined filtrates were washed with saturated aqueous sodium bicarbonate, and the aqueous layer was extracted with ethyl acetate; the combined organic layers were washed with saturated aqueous sodium chloride, dried over magnesium sulfate, filtered, and concentrated in vacuo to give the crude product. 1H NMR (600 MHz, DMSO-d6), major component, characteristic peaks: δ 10.12 (d, J=9.1 Hz, 1H), 8.99 (d, J=8.1 Hz, 1H), 7.70 (s, 1H), 4.94 (ddd, J=9.4, 8.1, 6.5 Hz, 1H), 4.87 (dd, J=9.0, 9.0 Hz, 1H), 4.11 (s, 1H), 3.96 (dd, J=10.2, 5.6 Hz, 1H), 3.52 (d, J=10.0 Hz, 1H), 3.18-3.07 (m, 2H), 2.98-2.88 (m, 1H), 2.40-2.33 (m, 1H), 1.79-1.52 (m, 4H), 1.61 (dd, J=7.6, 5.5 Hz, 1H), 1.33 (d, half of AB quartet, J=7.7 Hz, 1H), 1.04 (s, 3H), 0.91-0.86 (m, 6H).

Purification of this product by reversed-phase HPLC (Column: Waters Sunfire C18, 19×100 mm, 5 μm; Mobile phase A: 0.05% trifluoroacetic acid in water; Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 5% - 95% B over 8.54 min, then 95% B over 1.46 min; Flow rate: 25 mL/min) gave (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[2-(2,2,2-trifluoroacetamido)-3-(trifluoromethyl)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, from C85 (DIAST-1) (98). Yield: 4.3 mg, 7.8 µmol, 11%. LCMS m/z 554.6 [M+H]+. Retention time: 2.80 minutes (Analytical conditions. Column: Waters Atlantis dC18, 4.6×50 mm, 5 μm; Mobile phase A: water containing 0.05% trifluoroacetic acid (v/v); Mobile phase B: acetonitrile containing 0.05% trifluoroacetic acid (v/v); Gradient: 5.0% - 95% B, linear for 4.0 minutes, then 95% B for 1.0 minute; Flow rate: 2 mL/minute).

Preparation of 3-tert-butyl 2-methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2,3-dicaroxylate (C90)

This preparation was carried out using the general procedure described by C. Uyeda and J. Werth, Angew. Chem. Int. Ed. 2018, 57, 13902–13906. A 3-necked flask equipped with a magnetic stirrer, reflux condenser, thermometer, and nitrogen inlet was charged with cobalt(II) bromide (0.15 equivalent; 0.146 g, 0.667 mmol), (1E,1'E)-1,1'-pyridine-2,6-diylbis[N-(2-tert-butylphenyl)ethanimine] (2-t-BuPDI; 0.15 equivalent; 0.284 g, 0.667 mmol), and tetrahydrofuran (11 mL). The thick green suspension was stirred overnight at room temperature and zinc (2.4 equivalents; 0.70 g, 11 mmol) and zinc bromide (1.1 equivalents; 1.1 g, 4.9 mmol) were added. After stirring for 15 min, the reaction mixture turned purple and a solution of 1-tert-butyl 2-methyl (2S)-2,5-dihydro-1H-pyrrole-1,2-dicaroxylate (1.0 equivalent; 1.0 g, 4.4 mmol) in tetrahydrofuran (7.5 mL) and 2,2-dichloropropane (2.0 equivalents; 1.0 g, 8.8 mmol) was added. The reaction mixture was stirred at room temperature for 5 days, then it was filtered through a pad of diatomaceous earth and washed with tetrahydrofuran (10.8 mL). The filtrate was combined with saturated aqueous ammonium chloride (3.5 mL) and ethyl acetate (9.5 mL); the layers were separated and the aqueous phase was extracted with ethyl acetate (8.4 mL). The combined organic extracts were washed with saturated aqueous sodium bicarbonate (10.5 mL), dried over magnesium sulfate, filtered, and concentrated to dryness to give 3-tert-butyl 2-methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1,0]hexane-2,3-dicaroxylate (C90) as a yellow oil. Yield: 0.90 g, 3.3 mmol, 75%. By 1H NMR analysis, this product exists as two carbamate rotamers (~3:2 ratio). 1H NMR (400 MHz, chloroform-d) δ 4.20 & 4.09 (2 s, 1H), 3.74 & 3.75 (2 s, 3H), 3.68-3.60 (m, 1H), 3.44 & 3.38 (2 d, J=10.9 Hz, 1H), 1.43 & 1.38 (2 s, 9H), 1.38-1.34 (m, 2H), 1.03 & 0.98 & 0.96 (3 s, 6H). ESI-MS (pol.) m/z (%)=255.1 (12.5) [M - Me+H]+, 214.1 (100) [M - t-Bu+H]+, 170.2 (50) [M - Boc+H]+.

Alternative production of C42

(1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C42)

Step 1. Synthesis of 3-methyl-N-(trifluoroacetyl)-L-valine (C91).

A solution of sodium methoxide in methanol (25% by weight; 28.5 mL, 124 mmol) was added to a solution of 3-methyl-L-valine (99%, 15 g, 113 mmol) in methanol (30 mL). Ethyl trifluoroacetate (130 mmol) was then added and the reaction mixture was stirred at 40 °C until the reaction was complete (approximately 2.5 h), then it was cooled to 20 °C. After adding hydrochloric acid (1 M; 136 mL, 136 mmol), the mixture was diluted with ethyl acetate (150 mL) and the layers were separated. The organic layer was washed twice with saturated aqueous sodium chloride solution, dried over magnesium sulfate, and filtered. Heptane was added to the filtrate, then the solution was concentrated at 50 °C to a volume of 5 mL/g. This procedure was repeated twice; After a second distillation, seed crystals of C91 (50 mg; see below) were added. The resulting solid was collected by filtration, washed with heptane, and dried at 40°C to give C91 as an off-white solid. Yield: 22.2 g, 97.7 mmol, 86%.

The seed crystals used above are prepared from a similar reaction using 3-methyl-L-valine; the organic layer containing C91 is then dried over magnesium sulfate and filtered, and concentration in vacuo yields a solid. A portion of this solid is used as seed product.

Physicochemical data were obtained on C91 samples prepared from reactions carried out by the same procedure. HRMS-ESI+ (m/z): [M+H]+ Calculated for C8H13F3NO3, 228.0842; Found, 228.0842. The primary ion was observed as C8H11F3NNa2O3 [M+Na+]: Calculated, 272.0481; Found, 272.0482. 1H NMR (600 MHz, DMSO-d6) δ 13.05 (s, 1H), 9.48 (d, J=8.9 Hz, 1H), 4.21 (d, J=8.9 Hz, 1H), 1.00 (s, 9H). 13C NMR (150.8 MHz, DMSO-d6) δ 170.9, 156.6 (q, 2JCF=36.9 Hz), 115.8 (q, 1JCF=287.7 Hz), 61.0, 33.6, 26.5. The powder X-ray diffraction pattern for C91 is shown in Figure 11; the characteristic peaks are listed in Table R.

Table R. Selected X-ray powder diffraction peaks for C91 Corner
(°2-theta) Relative intensity Corner
(°2-theta) Relative intensity Corner
(°2-theta) Relative intensity 9.7 27 28.1 4 37.7 1 11.8 6 28.7 2 38.2 2 12.6 3 28.9 5 38.7 2 13.2 20 29.7 4 39.3 3 14.7 28 29.9 4 39.6 1 15.6 69 30.3 10 40.0 1 17.2 4 30.8 3 40.5 5 17.9 2 31.0 1 40.7 3 18.3 2 31.3 1 40.9 3 19.5 100 32.6 3 41.5 1 20.0 14 33.5 2 41.8 1 20.4 2 34.0 1 42.4 3 21.5 22 34.2 2 43.1 1 23.2 8 34.7 3 44.2 1 23.3 11 36.2 2 44.8 1 23.8 6 36.4 3 45.7 1 25.2 1 36.7 2 45.9 3 25.4 17 37.1 1 46.4 1 25.7 2 37.2 2 47.0 1 26.6 7 37.4 1 47.3 2 26.7 6 37.6 3 49.5 2

The crystal for X-ray crystallography was prepared by recrystallization from ethyl acetate and hexane using seed crystals from the same lot as above. The ORTEP diagram for the C91 single crystal data is shown in Figure 12.

Single crystal X-ray structural analysis of C91

Single crystal X-ray analysis

Data collection is performed on a Bruker D8 Quest diffractometer at -100°C. Data collection consists of omega and phi scans.

The structure was resolved by internal phasing using the SHELX software package in the orthorhombic space group class P212121. The structure was further refined by full-matrix least-squares. All non-hydrogen atoms were found and refined using anisotropic displacement parameters.

Hydrogen atoms located on nitrogen were found from the difference Fourier map and refined with distance constraints. Hydrogen on O2 (H2Z) and O3 (H3Z) was separated as charge and refined as occupancy by 10.5 each. The remaining hydrogen atoms were placed in calculated positions and allowed to move on their carrier atoms. The final refinement includes isotropic displacement parameters for all hydrogen atoms.

The population occupancy disturbance is defined and modeled as a ratio of ~67/33 in the -CF3 segment.

The analysis of the absolute structure using probability methods (Hooft, 2008) is carried out using PLATON (Spek). The results show that the absolute structure is assigned correctly. The method calculates that the probability that the structure is assigned correctly is 100%. The Hooft parameter is represented as -0.02 s s t (4), and the Parson parameter is represented as -0.02 s s t (4).

The resulting R-index is 4.1%. The finite difference Fourier map did not reveal any missing or shifted electron density.

The corresponding crystal, data collection and clarifying information are summarized in Table S. The atomic coordinates, bond lengths, bond angles and displacement parameters are listed in Tables T-V.

A list of the software and references used can be found in Single Crystal X-ray Structure Determination of Example 13, Solid Form 1.

Table S. Crystal data and structure refinement for C91. Empirical formula
Mass formula
Temperature
Wavelength
Crystal system
Space group
Lattice constant
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta interval for data collection
Index ranges
Collected reflections
Independent reflections
Fullness to theta=67.679°
Absorption correction
Method of clarification
Data/constraints/parameters
F2 match
Finite R indices [I>2σ(I)]
R indices (all data)
Absolute structure parameter
Excitation coefficient
Largest differential peaks and troughs C8H12F3NO3
227.19
173(2) K
1.54178 Å
Tetragonal
P41212
a=9.9168(6) Å
b=9.9168(6) Å
c=22.721(2) Å
2234.5(4) Å3
8
1.351 mg/m3
1.184 mm-1
944
0.200×0.170×0.080 mm3
4.866-70.114°
-11<=h<=10, -12<=k<=12, -27<=l<=27
48160
2122 [Rint=0.0392]
99.8%
Empirical
Least squares in full matrix approximation for F2
2122/9/158
1,010
R1=0.0408, wR2=0.1012
R1=0.0429, wR2=0.1039
0.03(4)
n/a
0.280 and -0.215 e. Å-3 α=90°
β=90°
γ=90°

Table T. Atomic coordinates (x 104) and equivalent isotropic substitution parameters (Å2×103) for C91. U(eq) is defined as one third of the trace of the orthogonalized tensor Uij.

Table U. Bond lengths [Å] and angles [°] for C91.

Symmetrical transformations are used to create equivalent atoms.

Table V. Anisotropic substitution parameters (Å2×103) for C91. The exponent of the anisotropic substitution factor takes the form: -2π2[h2 a*2U11+…+2 h k a* b* U12].

Step 2. Synthesis of lithium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate (C92).

Lithium hydroxide monohydrate (29.0 g, 678 mmol) was added to a mixture of methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1,0]hexane-2-carboxylate hydrochloride (68.5 g, 333 mmol) in tetrahydrofuran (950 mL) and water (48 mL). The reaction mixture was stirred at 25 °C until hydrolysis was complete, then the solid was collected by filtration, washed with 5% water in tetrahydrofuran (400 mL) and dried under vacuum at 70 °C to afford C92 as a white/off-white solid. Yield: 47.6 g, 295 mmol, 89%. The physicochemical data obtained for the C92 samples were obtained from reactions carried out according to the same procedure.

HR-ESI+(m/z): [M+H]+Calculated for C8H14NO2, 156.1019; Found, 156.1019. 1H NMR (600 MHz, D2O) δ 3.23 (d, J=1.1 Hz, 1H), 3.09 (dd, J=11.1, 5.2 Hz, 1H), 2.63 (d, J=11.1 Hz, 1H), 1.33-1.24 (m, 2H), 0.86 (s, 2H), 0.83 (s, 3H). 13C NMR (150.8 MHz, D2O) δ 182.7, 62.3, 45.6, 35.5, 30.0, 25.8, 19.3, 12.7. The powder X-ray diffraction pattern for C92 is given in Figure 13; the characteristic peaks are listed in Table W.

Table W. Selected X-ray powder diffraction peaks for C92 Angle (°2-theta) Relative intensity Corner
(°2-theta) Relative intensity Corner
(°2-theta) Relative intensity 5.1 4 20.2 6 30.2 4 6.0 100 20.8 19 30.5 2 7.3 17 21.4 2 31.4 18 8.7 3 21.9 4 32.2 5 10.2 6 22.3 16 32.6 22 12.1 20 22.8 7 33.8 3 12.7 2 23.8 3 34.3 3 13.7 2 24.5 9 35.2 3 15.3 15 24.8 3 35.9 10 15.7 6 25.5 21 36.7 4 16.7 78 26.2 5 37.4 4 17.2 8 26.5 4 37.8 4 18.0 3 28.4 10 39.0 2 18.8 95 29.0 10 39.2 1 19.5 14 29.5 4 39.8 1

Step 3. Synthesis of (1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C42).

A mixture of C91 (1.29 g, 5.68 mmol), 4-(dimethylamino)pyridine (0.60 g, 4.8 mmol) and N,N-diisopropylethylamine (1.70 mL, 9.75 mmol) in tetrahydrofuran (10 mL) was treated with p-toluenesulfonyl chloride (0.99 g, 5.2 mmol). The reaction mixture was then stirred for 2 h at 20 °C, C92 (75.7 wt %, 1.00 g, 4.70 mmol) was charged and stirring was continued overnight at 20 °C. The resulting suspension was mixed with propan-2-yl acetate (10 mL) and washed successively with aqueous citric acid (10%, 10 mL) and water (10 mL). The organic layer was then concentrated, followed by the addition of propan-2-yl acetate (5 mL), followed by the dropwise addition of heptane (15 mL) via an addition funnel. The solids were isolated by filtration and dried under vacuum to afford (1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (C42) as a white solid. Yield: 1.2 g. This compound exhibited two sets of NMR signals. The major and minor sets corresponded to the Z and E isomers of the tertiary amide, respectively, in a molar ratio of 20:1. The sample also contains isopropyl acetate at 37% molar ratio relative to C42, showing 1H resonances at 4.86, 1.96, and 1.17 ppm, and 13C resonances at 169.7, 66.9, 21.5, and 21.0 ppm. The 1H and 13C signals are indicated using the TMS signal set to 0 ppm in both.

HRMS-IER+(m/z): [M+H]+Calculated for C16H24F3N2O4, 365.1683; Found: 365.1684. 1H NMR (600 MHz, DMSO-d6) δ basic: 9.44 (d, J=8.5 Hz, 1H), 4.44 (d, J=8.5 Hz, 1H), 4.15 (s, 1H), 3.85 (dd, J=10.5, 5.4 Hz, 1H), 3.73 (d, J=10.5 Hz, 1H), 1.53 (dd, J=7.6, 5.3 Hz, 1H), 1.43 (d, J=7.6 Hz, 1H), 1.01 (s, 3H), 1.01 (s, 9H), 0.83 (s, 3H); minor: 9.11 (d, J=9.4 Hz, 1H), 4.53 (s, 1H), 4.33 (d, J=9.4 Hz, 1H), 3.53 (dd, J=12.5, 5.3 Hz, 1H), 3.41 (d, J=12.5 Hz, 1H), 1.55 (d, J=7.5 Hz, 1H), 1.41 (dd, J=7.5, 5.3 Hz, 1H), 1.02 (s, 3H), 0.97 (s, 3H), 0.91 (s, 9H). 13C NMR (150.8 MHz, DMSO-d6) δ main: 172.3, 167.5, 156.8 (2JCF=37.0 Hz), 115.7 (1JCF=287.7 Hz), 59.1, 58.0, 47.1, 34.6, 29.6, 26.7, 26.1, 25.6, 18.7, 12.0; minor: 172.3, 168.1, 155.9 (2JCF=36.8 Hz), 115.8 (1JCF=288.1 Hz), 59.9, 57.3, 46.4, 36.2, 32.1, 26.2, 26.0, 24.4, 19.0, 12.7. The powder X-ray diffraction pattern for C42 is shown in Figure 14; the characteristic peaks are listed in Table X.

Crystallization for powder X-ray diffraction and single crystal X-ray structural analysis was carried out as follows. A solution of C42 (2.96 g) in ethanol (9 mL) was heated to 40 °C with stirring (3500 rpm), then water (10.5 mL) was added over 10 min. Further water (16.5 mL) was then added over 4 h, and the mixture was cooled to 10 °C and stirred overnight. After filtration, the filter cake was washed with water (6 mL) and dried at 50 °C to give crystalline C42 (2.6 g).

Table X. Selected X-ray powder diffraction peaks for C42 Corner
(°2-theta) Relative intensity Corner
(°2-theta) Relative intensity Corner
(°2-theta) Relative intensity 7.4 100 24.2 3 37.4 1 9.4 73 24.4 2 38.3 1 12.0 15 25.3 4 39.0 1 12.9 16 25.5 4 39.7 1 14.1 9 27.6 6 40.1 1 14.9 26 28.6 1 41.2 1 17.4 7 29.5 4 41.9 1 17.7 35 31.4 2 42.6 1 19.0 18 31.5 3 46.3 1 19.2 12 32.4 1 19.7 17 33.2 1 20.4 9 34.1 1 20.6 4 34.5 2 22.4 3 35.7 1 23.0 3 36.1 0 23.2 2 36.6 1

The ORTEP diagram for the C42 single crystal data is shown in Figure 15.

Single-crystal X-ray structural analysis of C42

Single crystal X-ray analysis

Data collection is performed on a Bruker D8 Venture diffractometer at room temperature. A special data strategy of 0.3 degrees width per frame is used to separate the domains, thus eliminating any TWIN and pseudosymmetry issues.

The structure is resolved by internal phasing using the SHELX software package in the R3 rhombohedral class group. The structure is further refined by full-matrix least squares. All non-hydrogen atoms are found and refined using anisotropic displacement parameters.

Hydrogen atoms located on nitrogen and oxygen are found from the difference Fourier map and refined with distance constraints. The remaining hydrogen atoms are placed in calculated positions and allowed to move on their carrier atoms. The final refinement includes isotropic displacement parameters for all hydrogen atoms.

The analysis of the absolute structure using probability methods (Hooft, 2008) is carried out using PLATON (Spek). The results show that the absolute structure is assigned correctly. The method calculates that the probability that the structure is assigned correctly is 100%. The Hooft parameter is represented as -0.08 s s e (7), and the Parson parameter is represented as -0.09 s e (6).

The population occupancy disturbance is defined and modeled as a 78:22 ratio in the C1_F1_F2 segment.

The resulting R-index is 5.8%. The finite difference Fourier map did not reveal any missing or shifted electron density.

The corresponding crystal, data collection and clarifying information are summarized in Table Y. The atomic coordinates, bond lengths, bond angles and displacement parameters are listed in Tables Z-BB.

A list of the software and references used can be found in Single Crystal X-ray Structure Determination of Example 13, Solid Form 1.

Table Y. Crystal data and structure refinement for C42. Empirical formula
Mass formula
Temperature
Wavelength
Crystal system
Space group
Lattice constant
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta interval for data collection
Index ranges
Collected reflections
Independent reflections
Fullness to theta=67.679°
Absorption correction
Method of clarification
Data/constraints/parameters
F2 match
Finite R indices [I>2σ(I)]
R indices (all data)
Absolute structure parameter
Excitation coefficient
Largest differential peaks and troughs C16H23F3N2O4
364.36
296(2) K
1.54178 Å
Trigonal
R3
a=14.1740(6) Å
b=14.1740(6) Å
c=14.1740(2) Å
1715.9(4) Å3
3
1.058 mg/m3
0.788 mm-1
576
0.200×0.100×0.100 mm3
6,445-80,034°
-17<=h<=16, -14<=k<=16,
-14<=l<=17
13310
4011 [Rint=0.0369]
98.9%
Empirical
Least squares in full matrix approximation for F2
4011/6/244
1,056
R1=0.0582, wR2=0.1675
R1=0.0611, wR2=0.01710
-0.09(6)
n/a
0.292 and -0.174 e. Å-3 α=114.11°
β=114.11°
γ=114.11°

Table Z. Atomic coordinates (x 104) and equivalent isotropic substitution parameters (Å2×103) for C42. U(eq) is defined as one third of the trace of the orthogonalized tensor Uij.

Table AA. Bond lengths [Å] and angles [°] for C42.

Symmetrical transformations are used to create equivalent atoms.

Table BB. Anisotropic substitution parameters (Å2×103) for C42. The exponent of the anisotropic substitution factor takes the form: -2π2[h2 a*2U11+…+2 h k a* b* U12].

In addition to preparing (1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxylic acid (C42) according to the methods described above, the compound can also be prepared as depicted in the reaction schemes shown immediately below. In step 1, methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate hydrochloride is first treated with triethylamine in a mixture of tetrahydrofuran and water to neutralize the hydrochloride, then the methyl ester is hydrolyzed using sodium hydroxide in a mixture of tetrahydrofuran and water to obtain sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate.

Preparation of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate

In a suitable vessel, tetrahydrofuran (30 mL), water (7.5 mL), triethylamine (7.62 mL, 54.7 mmol), and methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1,0]hexane-2-carboxylate hydrochloride (7.59 g, 36.9 mmol) are added. The mixture is stirred at 25 °C for at least 30 minutes. Stirring is stopped and the layers are separated. In a separate vessel, 28% w/w aqueous sodium hydroxide (4.19 mL, 38.3 mmol) and tetrahydrofuran (71 mL) are added with stirring at 40 °C. 25% of the organic layer containing a solution of methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate in tetrahydrofuran is added after separation and the solution is seeded with sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate (0.1182 g, 0.7336 mmol - previously prepared by a similar procedure). The mixture is kept at 40 °C for at least 15 minutes and the remaining 75% of the organic layer is slowly added. The mixture is maintained with stirring at 40°C for 16 hours, then slowly cooled to 20°C and maintained for at least 4 hours. The resulting solid sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate is isolated by filtration, washed with a solution consisting of tetrahydrofuran (43 ml) and water (2.25 ml). The solid product is dried at 70°C under vacuum to give 6.13 g (93.8%) of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate as a crystalline solid. The PDP is determined by the methods described above.

Selected XRD peaks of crystalline sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate.

Corner
degrees
2-Ɵ
±0.2°2-Ɵ Relative intensity Corner
degrees
2-Ɵ
±0.2°2-Ɵ Relative intensity Corner
degrees
2-Ɵ
±0.2°2-Ɵ Relative intensity 5.5 19 19.0 15 29.8 4 5.9 9 19.3 100 30.3 8 6.6 59 21.3 45 30.8 8 10.7 2 23.2 12 32.0 3 16.0 9 23.5 17 32.5 5 16.3 6 24.2 20 33.8 7 16.7 10 25.8 11 34.6 4 17.0 52 26.0 8 35.8 6 17.3 42 26.7 4 36.6 8 17.7 15 28.1 3 36.9 7 18.5 7 28.9 6 37.6 3

Selected PRD peaks of (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (C91)

Angle degrees
2-θ±0.2°2-θ Relative intensity Angle degrees
2-θ
±0.2°2-θ Relative intensity Angle degrees
2-θ
±0.2°2-θ Relative intensity 9.7 27 28.1 4 37.7 1 11.8 6 28.7 2 38.2 2 12.6 3 28.9 5 38.7 2 13.2 20 29.7 4 39.3 3 14.7 28 29.9 4 39.6 1 15.6 69 30.3 10 40.0 1 17.2 4 30.8 3 40.5 5 17.9 2 31.0 1 40.7 3 18.3 2 31.3 1 40.9 3 19.5 100 32.6 3 41.5 1 20.0 14 33.5 2 41.8 1 20.4 2 34.0 1 42.4 3 21.5 22 34.2 2 43.1 1 23.2 8 34.7 3 44.2 1 23.3 11 36.2 2 44.8 1 23.8 6 36.4 3 45.7 1 25.2 1 36.7 2 45.9 3 25.4 17 37.1 1 46.4 1 25.7 2 37.2 2 47.0 1 26.6 7 37.4 1 47.3 2 26.7 6 37.6 3 49.5 2

Crystal data refinement and structure elucidation for (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (C91).

Identification code E178 Empirical formula
Mass formula
Temperature
Wavelength
Crystal system
Space group
Lattice constant
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta interval for data collection
Index ranges
Collected reflections
Independent reflections
Fullness to theta=67.679°
Absorption correction
Method of clarification
Data/constraints/parameters
F2 match
Finite R indices [I>2σ(I)]
R indices (all data)
Absolute structure parameter
Excitation coefficient
Largest differential peaks and troughs C8H12F3NO3
227.19
173(2) K
1.54178 Å
Tetragonal
P41212
a=9.9168(6) Å
b=9.9168(6) Å
c=22.721(2) Å
2234.5(4) Å 3
8
1.351 mg/m3
1.184 mm-1
944
0.200×0.170×0.080 mm3
4.866-70.114°
-11<=h<=10, -12<=k<=12, -27<=l<=27
48160
2122 [Rint=0.0392]
99.8%
Empirical
Least squares in full matrix approximation for F2
2122/9/158
1,010
R1=0.0408, wR2=0.1012
R1=0.0429, wR2=0.1039
0.03(4)
n/a
0.280 and -0.215 e.Å-3 α=90°
β=90°
γ=90°

In step 2, the resulting sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxylate is then coupled with (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid in the presence of tosyl chloride and dimethylaminopyridine in tetrahydrofuran. The tetrahydrofuran is removed and replaced with isopropyl acetate, then treated with HCl in brine, then treated with water and heptane to give C42.

Crystalline C42 is characterized by PRD and identifies an additional form obtained after prolonged drying or higher temperature.

Selected PRD peaks for C42 - crystalline form obtained after prolonged drying or higher temperature

Corner
degrees
2-Ɵ
±0.2°2-Ɵ Relative intensity Corner
degrees
2-Ɵ
±0.2°2-Ɵ Relative intensity Corner
degrees
2-Ɵ
±0.2°2-Ɵ Relative intensity 10.2 3 25.7 11 33.7 1 10.8 100 26.1 3 34.1 1 12.6 22 26.8 2 34.6 4 13.3 21 27.1 2 35.1 3 13.8 88 27.4 7 35.5 1 16.3 59 27.8 7 36.3 4 17.1 39 28.0 3 36.9 6 18.7 5 28.2 3 38.2 6 18.9 2 29.0 2 38.8 2 19.5 20 29.5 4 39.3 1 19.9 18 29.8 2 39.6 2 20.3 2 30.1 2 39.8 2 20.6 26 30.6 7 40.2 2 20.8 63 30.9 3 42.2 3 21.7 6 31.2 3 44.2 2 22.2 20 32.0 8 44.7 3 23.4 8 32.7 4 47.0 2 23.7 18 32.9 4 47.3 2 24.5 7 33.1 5 25.4 9 33.5 1

Antiviral activity against SARS-CoV-2 infection

The ability of compounds to prevent SARS-CoV-2-induced cell death, or cytopathic effect, can be assessed by cell viability using an assay format that uses luciferase to measure intracellular ATP as an endpoint. Briefly, VeroE6 cells enriched for hACE2 expression are inoculated with SARS-CoV-2 (USA_WA1/2020) at an MOI of 0.002 in a BSL-3 lab. Virus-inoculated cells are then added to assay-ready compound plates at a density of 4,000 cells/well. Following a 3-day incubation, when virus-induced cytopathic effect is 95% under untreated infected control conditions, cell viability is assessed using Cell Titer-Glo (Promega) according to the manufacturer's protocol, which quantifies ATP levels. Compound cytotoxicity is assessed in parallel with uninfected cells. Test compounds are tested either alone or in the presence of the P-glycoprotein (P-gp) inhibitor CP-100356 at 2 μM. The inclusion of CP 100356 is intended to assess whether test compounds are cleared from VeroE6 cells, which have high levels of P-gp expression. The proportion of effect for each test compound is calculated from the values for virus-free wells and virus-containing control wells in each assay plate. The concentration required to produce 50% response (EC50) is determined from these data using a 4-parameter logistic model. EC50 curves are fitted with a Hill slope of 3 at >3 and the highest dose producing ≥50% response. If cytotoxicity is detected at an effect greater than 30%, the corresponding data are excluded from the EC50 determination.

For cytotoxicity plates, the effect proportion for each test compound species is calculated based on the values for the cell-only control wells and the hyamine control wells in each assay plate. The CC50 value is calculated using a 4-parameter logistic model. The TI is then calculated by dividing the CC50 value by the EC50 value.

FRET study and analysis of 3C protease of SARS-CoV-2 coronavirus

The proteolytic activity of the major protease, 3CLpro, of SARS-CoV-2 is monitored using a continuous fluorescence resonance energy transfer (FRET) assay. The SARS-CoV-2 3CLpro assay measures the activity of the full-length SARS-CoV-2 3CL protease by cleaving a synthetic fluorogenic substrate peptide with the following sequence: Dabcyl-KTSAVLQ-SGFRKME-Edans, modeled on the consensus peptide (V. Grum-Tokars et al. Evaluating the 3C-like protease activity of SARS-coronavirus: recommendations for standardized assays for drug discovery. Virus Research 133 (2008) 63–73). Fluorescence of the cleaved Edans peptide (excitation 340 nm/emission 490 nm) was measured using the fluorescence intensity protocol on a Flexstation reader (Molecular Devices). The fluorescent signal was reduced in the presence of PF-835231, a potent inhibitor of SARS-CoV-2 3CLpro. The assay reaction buffer contained 20 mM Tris-HCl (pH 7.3), 100 nM NaCl, 1 mM EDTA, and 25 μM peptide substrate. Enzyme reactions were initiated by the addition of 15 nM SARS-CoV-2 3CL protease and carried out for 60 min at 23°C. The fraction of inhibition or activity was calculated based on no compound (0% inhibition/100% activity) and compound control (100% inhibition/0% activity) wells. The IC50 values were obtained using a four-parameter fitting model using ABASE (IDBS) software. The Ki values were fitted to the Morrison equation with the enzyme concentration parameter set at 15 nM, the Km parameter set at 14 μM, and the substrate concentration parameter set at 25 μM using ABASE (IDBS) software.

The proteolytic activity of the SARS-CoV-2 coronavirus 3CL protease was measured using a continuous fluorescence resonance energy transfer assay. The SARS-CoV-2 3CLpro FRET assay measures the protease-catalyzed cleavage of TAMRA-SITSAVLQSGFRKMK-(DABCYL)-OH to TAMRA-SITSAVLQ and SGFRKMK(DABCYL)-OH. The fluorescence of the cleaved TAMRA peptide (exc. 558 nm/evaluation 581 nm) was measured using a TECAN SAFIRE fluorescence plate reader for 10 min. Typical reaction solutions contained 20 mM HEPES (pH 7.0), 1 mM EDTK, 4.0 μM FRET substrate, 4% DMSO, and 0.005% Tween-20. Assays were initiated by adding 25 nM SARS 3CLpro (nucleotide sequence 9985-10902 of the complete genome sequence of the Urbani strain of SARS coronavirus (NCBI accession number AY278741)). The fraction of inhibition was determined in duplicate at an inhibitor level of 0.001 mM. Data were analyzed using the Kalidagraph nonlinear regression analysis program using the equation:

FU=divergence+(limit)(1-e-(kobs)t)

where the difference is equal to the fluorescence signal of the uncleaved peptide substrate, and the limit is equal to the fluorescence of the fully cleaved peptide substrate. Kobs is the first-order rate constant for this reaction, and in the absence of any inhibitor represents substrate utilization. A reaction is started in the enzyme that contains an irreversible inhibitor, and if the calculated limit is less than 20% of the theoretical maximum limit, the calculated kobs is the rate of inactivation of the coronavirus 3C protease. The slope (kobs/I) of the plot of kobs vs. [1] is a measure of the avidity of the inhibitor for the enzyme. For very fast irreversible inhibitors, kobs/I is calculated from observations for only one or two [1]s, rather than as the slope.

Table 2. Biological activity and IUPAC name for examples 1–84. Example number Geometric mean Ki (µm) Number of Ki used (µm) Geometric mean EC50 (µm) Number of EC50 used (µm) IUPAC name 1 0,013 4 0.246 7 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide 2 1.08 3 7.52 2 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-(pyrrolidin-1-ylacetyl)-L-leucinamide, trifluoroacetate 3 0.439 2 6.74 6 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(2,6-dichlorobenzoyl)-4-methyl-L-leucinamide 4 0.026 3 1.36 15 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide 5 >0.351 2 >3.33 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-3-(trifluoromethyl)-1H-indole-2-carboxamide 6 0.023 2 0.279 4 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide 7 0.798 1 43.1 2 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-methylimidazo[2,1-b][1,3]thiazole-2-carboxamide 8 0.917 3 5.75 2 N-{1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[cyclohexyl(methoxy)acetyl]-4-methyl-L-leucinamide, DIAST-1 9 0.254 4 0.970 4 N-{1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[cyclohexyl(methoxy)acetyl]-4-methyl-L-leucinamide, DIAST-2 10 0.056 4 2.09 4 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide 11 0.297 3 4.78 4 N2-[(4-bromo-1-ethyl-3-methyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide 12 0.539 3 6.33 2 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(3,3-difluorocyclobutyl)acetyl]-4-methyl-L-leucinamide 13 0.003 6 0.075 18 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide 14 0.302 2 N.D,1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide 15 0.002 1 0.360 2 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide 16 0,018 2 >2.53 2 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-fluoro-4-methoxy-1H-indole-2-carboxamide 17 0.053 1 >0.333 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3-fluoro-4-methoxy-1H-indole-2-carboxamide 18 0,019 2 >0.333 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5,7-difluoro-4-methoxy-1H-indole-2-carboxamide 19 0.208 2 >0.333 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-3,5-difluoro-4-methoxy-1H-indole-2-carboxamide 20 0,005 1 >0.333 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide 21 BUT. BUT. >3.17 2 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-fluoro-4-methoxy-1H-indole-2-carboxamide 22 0.066 1 >0.333 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-fluoro-4-methoxy-1H-indole-2-carboxamide 23 0,021 1 >0.333 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5,7-difluoro-4-methoxy-1H-indole-2-carboxamide 24 1.93 2 6.30 6 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[2-(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl}-L-leucinamide 25 BUT. 37 2 N-{1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(cyclohexylcarbonyl)-4-methyl-L-leucinamide, DIAST-1 26 BUT. >100 1 N-{1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-(cyclohexylcarbonyl)-4-methyl-L-leucinamide, DIAST-2 27 4.50 2 44.9 2 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-[(propan-2-yloxy)acetyl]-L-leucinamide 28 1.79 2 7.26 4 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(cyclohexyloxy)acetyl]-4-methyl-L-leucinamide 29 2.23 2 28.1 2 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-(4,4,4-trifluoro-3-methylbutanoyl)-L-leucinamide 30 >10.8 1 9.37 2 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(2S)-2-(dimethylamino)-2-phenylacetyl]-4-methyl-L-leuinamide 31 0.606 3 40.7 2 N2-[(trans-4-cyanocyclohexyl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide
or
N2-[(trans-4-cyanocyclohexyl)carbonyl]-N-{(1R)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide 32 0,690 3 4.90 2 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[2-(cyclohexyloxy)propanoyl]-4-methyl-L-leucinamide
or
N-{(1R)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[2-(cyclohexyloxy)propanoyl]-4-methyl-L-leucinamide 33 0.068 2 >0.333 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-hydroxy-1H-indole-2-carboxamide 34 0.074 2 >0.333 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-hydroxy-4-methoxy-1H-indole-2-carboxamide 35 0.176 3 3.56 4 N2-[(4-chloro-1,3-dimethyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide 36 0.241 4 1.03 2 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-[(2R)-2-(dimethylamino)-2-phenylacetyl]-4-methyl-L-leucinamide 37 0.168 1 >3.33 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-3-(trifluoromethyl)-1H-indole-2-carboxamide 38 0.023 1 >0.333 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide 39 0,111 1 >3.33 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-3,7-bis(trifluoromethyl)-1H-indole-2-carboxamide 40 0.131 1 >0.333 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-3,5-bis(trifluoromethyl)-1H-indole-2-carboxamide 41 0.104 1 >0.333 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-3,6-bis(trifluoromethyl)-1H-indole-2-carboxamide2 42 >0.356 1 >0.333 1 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-{(2R)-2-(dimethylamino)-2-[3-(trifluoromethyl)phenyl]acetyl}-4-methyl-L-leucinamide 43 >0.356 1 >0.333 1 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N2-{(2R)-2-(dimethylamino)-2-[4-(trifluoromethyl)phenyl]acetyl}-4-methyl-L-leucinamide 44 0.302 2 5.88 2 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-6-(trifluoromethyl)-1H-indole-2-carboxamide 45 0.227 2 6.42 2 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-6-(trifluoromethyl)-1H-indole-2-carboxamide 46 0.283 1 >3.33 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-3,7-bis(trifluoromethyl)-1H-indole-2-carboxamide 47 >0.359 1 >3.33 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-3,6,7-tris(trifluoromethyl)-1H-indole-2-carboxamide 48 >0.359 1 >3.33 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-3,5,7-tris(trifluoromethyl)-1H-indole-2-carboxamide 49 0.083 3 2.68 2 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-(trifluoromethoxy)-1H-indole-2-carboxamide 50 0.147 3 3.95 1 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-(trifluoromethoxy)-1H-indole-2-carboxamide 51 0.192 3 BUT. N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-(trifluoromethyl)-1H-indole-2-carboxamide 52 0.032 3 BUT. 7-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide 53 0.025 3 BUT. N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-7-methyl-1H-indole-2-carboxamide 54 0.049 3 BUT. 6-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide 55 0.104 3 BUT. 4-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide 56 0.151 3 BUT. 5-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide 57 0.052 3 BUT. N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-7-(trifluoromethyl)-1H-indole-2-carboxamide 58 0.091 3 BUT. 4,6-dichloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide 59 0.152 3 BUT. N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-(trifluoromethyl)-1H-indole-2-carboxamide 60 0.261 3 BUT. N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-5-(trifluoromethyl)-1H-indole-2-carboxamide 61 0.034 3 BUT. 7-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide 62 0.029 3 BUT. N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-7-methyl-1H-indole-2-carboxamide 63 0.122 3 BUT. 6-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide 64 0.038 3 BUT. 4-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide 65 0.117 3 BUT. 5-chloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide 66 0.073 3 BUT. N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-7-(trifluoromethyl)-1H-indole-2-carboxamide 67 0,041 3 BUT. 4,6-dichloro-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide 68 0.092 3 BUT. N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-(trifluoromethyl)-1H-indole-2-carboxamide 69 0.083 1 >2.67 2 N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-methyl-5-(trifluoromethyl)imidazo[2,1-b][1,3]thiazol-2-carboxamide 70 6.95 2 4.09 2 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[5-methyl-2-(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl}-L-leucinamide 71 0.254 3 4.99 2 N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-N2-{[4-methyl-2-(trifluoromethyl)-1,3-thiazol-5-yl]carbonyl}-L-leucinamide 72 0.173 3 1.35 2 N2-[(4-bromo-1-ethyl-3-methyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-L-leucinamide 73 0.226 3 2.03 2 N2-[(4-chloro-1,3-dimethyl-1H-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-L-leucinamide 74 >0.356 1 >3.33 1 3-acetyl-N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide 75 0,017 2 0.551 4 Diastereomer 1: (2S,4R)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide
or
(2R,4S)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide 76 >8.16 2 >100 1 Diastereomer 2: (2S,4R)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide
or
(2R,4S)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide 77 0.004 2 0.085 4 3-methyl-N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide 78 0,005 3 2.74 2 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(methylcarbamoyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide 79 0,001 4 0,080 4 methyl {(2S)-1-[(1R,2S,5S)-2-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3,1,0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2-yl}carbamate 80 0.037 2 0.158 3 N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide 81 0.003 2 0,690 3 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3R)-5-hydroxy-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide 82 0.139 1 BUT. BUT. (1R,2S,5S,6R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide 83 0.092 1 BUT. BUT. (1R,2S,5S,6S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide 84 0.003 1 BUT. BUT. (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-(hydroxymethyl)-N-(trifluoroacetyl)-L-valyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide 85 0.004 1 0.334 2 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3R)-2,5-dioxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide 86 0,018 2 0.237 2 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[5,5,5-trifluoro-2-(2,2,2-trifluoroacetamido)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide 87 0,021 2 0,230 2 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[(2S)-2-cyclohexyl-2-{[(trifluoromethyl)sulfonyl]amino}acetyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide 88 0.006 2 0,050 4 First-eluting diastereomer: (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-L-alanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide
or
(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-D-alanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide 89 0,007 3 0,040 4 Second-eluting diastereomer: (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-L-alanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide
or
(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-D-alanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide 90 0,015 2 0.571 2 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(pyridin-2-yl)-N-(trifluoroacetyl)-L-alanyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide 91 0,007 2 0,011 2 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-{N-[(4-fluorophenoxy)acetyl]-3-methyl-L-valyl}-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide 92 0.004 2 0,019 4 3-methyl-N-[(4-methylphenyl)acetyl]-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide 93 0,014 2 0.882 2 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(1H-pyrazol-1-yl)-N-(trifluoroacetyl)-L-alanyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide 94 0,011 2 0.379 2 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[(2S)-4,4-difluoro-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide 95 0.003 2 0.098 4 N-(methoxycarbonyl)-3-methyl-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide 96 0.844 1 9,190 2 (1R,2S,5S)-N-{(1R)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide 97 0,011 5 0.082 16 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[2-(2,2,2-trifluoroacetamido)-3-(trifluoromethyl)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, from C86 (DIAST-2) 98 0.188 1 1,738 2 (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[2-(2,2,2-trifluoroacetamido)-3-(trifluoromethyl)pentanoyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide, from C85 (DIAST-1)

1. Not defined.

2. The regiochemistry of Example 41 is not well determined; other possible structures for this example are N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-5,6-bis(trifluoromethyl)-1H-indole-2-carboxamide and N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-6,7-bis(trifluoromethyl)-1H-indole-2-carboxamide.

Predicted pharmacokinetic parameters of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Example 13 compound) in humans

Based on in vitro physiologically based pharmacokinetic (PBPK) modeling of data including CLint from human liver microsomes and CLbile from human hepatocytes under sandwich culture conditions, the predicted human plasma CL and Vss for (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide are 5.9 mL/min/kg and 0.97 L/kg, respectively, and yield an effective half-life, t1/2, of 1.9 hours. The target Ceff of 0.16 mM (unbound plasma concentration) was determined based on antiviral inhibition data obtained from in vitro studies of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide in either VeroE6 cells in the presence of a P-gp inhibitor (EC90 value of 0.156 mM) or in a differentiated normal human bronchial epithelial (dNHBE) assay (EC90 value of 0.149 mM). A dose of 380 mg of (1R,2S,5S)-N-{(1S)-1-Cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide administered orally three times daily (TID) is expected to cover an effective unbound concentration of 0.16 mM at Cmin.

All patents and publications described above are incorporated herein by reference in their entirety. Although the invention has been described in terms of various preferred embodiments and specific examples, the invention is not to be understood as limited by the foregoing detailed description, but as defined by the appended claims and their equivalents.

Claims (28)

1. A compound selected from the group consisting of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; (2S,4R)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide; (2R,4S)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide; 3-methyl-N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(methylcarbamoyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; methyl {(2S)-1-[(1R,2S,5S)-2-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2-yl}carbamate; N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3R)-5-hydroxy-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; (1R,2S,5S,6R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; (1R,2S,5S,6S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-(hydroxymethyl)-N-(trifluoroacetyl)-L-valyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3R)-2,5-dioxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[5,5,5-trifluoro-2-(2,2,2-trifluoroacetamido)pentanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[(2S)-2-cyclohexyl-2-{[(trifluoromethyl)sulfonyl]amino}acetyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-L-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)-D-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(pyridin-2-yl)-N-(trifluoroacetyl)-L-alanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-{N-[(4-fluorophenoxy)acetyl]-3-methyl-L-valyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide; 3-methyl-N-[(4-methylphenyl)acetyl]-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-L-prolinamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(1H-pyrazol-1-yl)-N-(trifluoroacetyl)-L-alanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-3-[(2S)-4,4-difluoro-2-( 2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide; And (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[2-(2,2,2-trifluoroacetamido)-3-(trifluoromethyl)pentanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide. 2. A pharmaceutical composition useful for treating coronavirus infection, comprising a therapeutically effective amount of a compound according to claim 1, comprising a compound according to claim 1 and a pharmaceutically acceptable carrier. 3. Use of a compound according to claim 1 or a pharmaceutical composition according to claim 2 for the treatment of coronavirus infection in a patient in need thereof. 4. Use according to paragraph 3, characterized in that the coronavirus infection is COVID-19. 5. Use of a compound according to claim 1 or a pharmaceutical composition according to claim 2 in the manufacture of a medicinal product for the treatment of coronavirus infection in a patient. 6. The use according to paragraph 5, characterized in that the coronavirus infection is COVID-19.