EA047572B1 - NITRILE-CONTAINING ANTIVIRAL COMPOUNDS - Google Patents

Description

Prerequisites for the creation of an invention

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

The worldwide outbreak of coronavirus disease 2019 (COVID-19) was attributed to an infection 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 and hundreds of thousands of deaths. The causative agent of COVID19 was identified as a novel coronavirus, which was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The genomic 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-like 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 severe acute respiratory syndrome coronavirus (SARS-CoV), the causative agent of the SARS disease that broke out in 2002–2003, and approximately 50% homology with Middle East respiratory syndrome coronavirus (MERS-CoV), the causative agent of a respiratory viral disease that broke out 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 (orflab:T8517C, synonym) and 28144 (ORF8: C251T, S84L). Of the 103 genomes analyzed, approximately 70% were L-type and approximately 30% were S-type. It is unclear whether the L-type evolved from the S-type in humans or via a zoonotic intermediate, but it appears that the L-type is more aggressive than the S-type, and human interventions in efforts to contain the outbreak may alter the relative prevalence 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 an individual could potentially be infected with separate subtypes sequentially or infected with two subtypes simultaneously. Given this evolving threat, there is an urgent need in the art for effective treatments for COVID-19 and methods for inhibiting 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 from person to person, resulting in community spread of the virus. The sequence of the SARS-CoV-2 spike protein receptor binding domain (RBD), including its receptor binding motif (RBM) that 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 Gln ⁴⁹³ ) 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 Asn ⁵⁰¹ ) are compatible with, but not ideal for, human ACE2 binding, suggesting that SARS-CoV-2 exploits ACE2 binding in some capacity 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-l-4939-2438-7_1), which consists of two overlapping polyproteins that are intensively processed by viral proteases. The C-proximal region is processed at eleven conserved interdomain junctions by the core or 3C-like protease of the coronavirus (Ziebuhr, Snijder, Gorbalenya, 2000 and Fehr, Perlman et al, 2015). The name 3C-like protease derives from the specific

- 1 047572 significant similarities between the coronavirus enzyme and well-known picornavirus 3C-proteases. These include substrate preferences, the use of cysteine as an active site nucleophile in catalysis, and similarity in their putative common polypeptide folds. The sequence of SARS-CoV-2 3C-protease (accession number YP_009725301.1) was found to have 96.08% homology when compared with SARS-CoV 3C-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 a 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 shows that there is a difference when comparing the 3CL proteases of SARS-CoV-2 and SARS-CoV. In SARS-CoV, but not in the dimeric SARS-CoV-2 3CL proteases, there is a polar interaction between the two domains III involving a 2,60-A hydrogen bond between the side chain hydroxyl groups of Thr ²⁸⁵ of each protomer, and is supported by a hydrophobic contact between the side chain of ile ² and Thr ^{2 5C} ₂ . 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 allows the two domains III to come somewhat closer to each other (the distance between the Ca 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 decreases from 33.4 Å to 32.1 Å). In the active site of SARS-CoV-2 3CL, Cys ¹⁴⁵ and His ⁴¹ form a catalytic dyad, which, together with the buried water molecule that is hydrogen-linked to His ⁴¹ , can be considered to form the catalytic triad of SARS-CoV-2 3CL protease. Given the ongoing spread of SARS-CoV-2, which has led to the current global outbreak of COVID-19, it is desirable to have new ways to inhibit 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 in the treatment of COVID-19. The present invention also provides pharmaceutical compositions containing the compounds and methods for treating COVID-19 and inhibiting the replication of the SARS-CoV2 virus by administering the compounds of the invention or pharmaceutical compositions containing the compounds of the invention. It should be understood that each of the embodiments of the method of treatment hereinafter may also be formulated as corresponding embodiments of the use type. For example, any of the compounds, or pharmaceutically acceptable salts, solvates, or hydrates thereof, or pharmaceutically acceptable salts of the compounds, solvates, or hydrates, as depicted in any of embodiments E1 to E30, E45 to E46, E50, E50a, E59 to E68, and E80 to 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 to E41, E47 to E49, E52 to E58a, E69 to E74, E77 to E79, E85 to E93, and E95 to E98.

E1 is a compound E45 or E59, hereinafter referred to as formula I

or a pharmaceutically acceptable salt thereof; wherein R ¹ is selected from the group consisting of C1C6 alkyl optionally substituted with cyano or one to five fluorine atoms; C2-C6 alkynyl; and (C3-C6 cycloalkyl)-C1-C3 alkyl optionally substituted with one to two substituents selected from trifluoromethyl and C1-C3 alkyl, or one to five fluorine atoms; R ² is hydrogen, or R ² and R ¹ , taken together with the nitrogen and carbon atoms to which they are attached, represent a pyrrolidine or piperidine ring optionally substituted with one to four R ^2a ; R ^2a is independently selected at each occurrence from the group consisting of fluorine, C1-C6 alkyl optionally substituted with one to three fluorine atoms, and C1-C6 _alkoxy optionally substituted with one to three fluorine atoms; or two R ^2a groups when attached to adjacent carbon atoms

- 2 047 572 of the kind and taken together with the carbon atoms to which they are attached represent annelated C3- _C6 cycloalkyl optionally substituted with one to four R ^2b ; or two R ^2a groups when attached to the same carbon atom and taken together with the carbon atom to which they are attached represent spiro C3-C6 cycloalkyl optionally substituted with one to four R ^2b ; R ^2b in each occurrence is 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 anelated with a 5- to 6-membered heteroaryl or phenyl, (C3- _C12 cycloalkyl)-C1- _C6 alkyl, C3- _C12 cycloalkoxy, (C3- _C12 cycloalkoxy)-C1- _C6 alkyl, 4- to 12-membered heterocycloalkyl optionally anelated with a 5- to 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 moiety contains one to four heteroatoms independently selected from N, O and S(O)n, _C6 - _C10 -aryl optionally annelated with _C4 - _C6 -cycloalkyl or 4- to 7-membered heterocycloalkyl, ( _C6 - _C10 -aryl)-C1- _C6 -alkyl, 5- to 10-membered heteroaryl containing one to five heteroatoms independently selected from N, O and S, which are optionally annelated with _C5 - _C6 -cycloalkyl; (5- to 10-membered heteroaryl) _-C1C6- alkyl, wherein the heteroaryl moiety contains one to five heteroatoms independently selected from N, O and S; (C ₆ -C ₁₀ aryl)-(5- to 10-membered heteroaryl)-, wherein the heteroaryl moiety comprises one to five heteroatoms independently selected from N, O and S, (5- to 10-membered heteroaryloxy)-C 1 -C ₆ alkyl, wherein the heteroaryl moiety comprises one to five heteroatoms independently selected from N, O and S; (5- to 6-membered heteroaryl)-(5- to 6-membered heteroaryl)-, wherein each heteroaryl moiety comprises one to four heteroatoms independently selected from N, O and S; (4- to 7-membered heterocycloalkyl)-(5- to 6-membered heteroaryl)-, wherein the heterocycloalkyl moiety comprises one to three heteroatoms independently selected from N, O and S(O)n, and the heteroaryl moiety comprises one to four heteroatoms independently selected from N, O and S; (56-membered heteroaryl)-(4-7-membered heterocycloalkyl)-, wherein the heterocycloalkyl moiety contains one to three heteroatoms independently selected from N, O, and S(O) _n , and the heteroaryl moiety contains one to four heteroatoms independently selected from N, O, and S; wherein each ^R3 group is optionally substituted with one to five ^R4 ; wherein R ⁴ is independently selected in each occurrence from the group consisting of oxo, halogen, hydroxy, cyano, phenyl, benzyl, amino, (C ₁ -C ₆ alkyl)amino optionally substituted with one to five fluorine atoms, di(C 1 -C ₆ alkyl)amino optionally substituted with one to ten fluorine atoms, C 1 -C ₆ alkyl optionally substituted with one to five fluorine atoms, C 1 -C ₆ alkoxy optionally substituted with one to five fluorine atoms, C ₁ -C 3 alkoxy-C ₁ -C 3 alkyl optionally substituted with one to five fluorine atoms, C 3 -C 6 cycloalkyl optionally substituted with one to three fluorine atoms or C 1 -C 3 alkyl, C ₁ -C ₆ alkyl-C(O)NH-, optionally substituted with one to five fluorine atoms, C1- _C6 alkyl-N(O) _2MH- , optionally substituted with one to five fluorine atoms, C1- _C6 -alkyl-C(O)-, optionally substituted with one to five fluorine atoms, C1- _C6 -alkyl-N(O) _n- , optionally substituted with one to five fluorine atoms; and n in each case is independently selected from 0, 1 and 2.

E2 is a compound of any one of E1, E45 and E59, wherein R ¹ is selected from the group consisting of (CH3) ₂ CHCH ₂ -, (CH3)3 CCH ₂ -, 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, (2methylcyclopropyl)methyl, (3,3-difluorocyclobutyl)methyl, cyclopentylmethyl and propynyl; and R ² is hydrogen; or a pharmaceutically acceptable salt thereof.

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

E4 is a compound of any one of E1, E45, E59 and E3, wherein R ^2a is at each occurrence independently selected from the group consisting of fluoro, methyl, isopropyl, trifluoromethyl and tert-butoxy; or two R ^2a groups, when attached to adjacent carbon atoms and taken together with the carbon atoms to which they are attached, represent an annelated cyclopentane or cyclopropane optionally substituted with one to four R ^2b ; or two R ^2a groups, when attached to the same carbon atom and taken together with the carbon atom to which they are attached, represent a spirocyclopropane ring optionally substituted with one to four R ^2b ; or a pharmaceutically acceptable salt thereof.

E5 is a compound of E1, E3, E4, E45 and E59, wherein R ^2b is independently selected in each case from the group consisting of fluoro, methyl and methoxy; or a pharmaceutically acceptable salt thereof.

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E6 is any one of E1, E2, E45 and E59, which is selected from the group consisting of formulas Ia-Ig

or a pharmaceutically acceptable salt thereof.

E7 is any one of E1, E3, E4, E45 and E59, which is selected from the group consisting of formulas Ih-Ik

or a pharmaceutically acceptable salt thereof.

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

- 4 047572

or a pharmaceutically acceptable salt thereof.

E9 is a compound of 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 of any one of E1, E3, E4, E7-E9, E45 and E59, wherein R ³ is selected from the group consisting of (CH3)2CHCH(R ⁴ )-, (CH3) ₃ CCH(R ⁴ )- and (cyclohexyl)CH^ ⁴ )-; or a pharmaceutically acceptable salt thereof.

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

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or a pharmaceutically acceptable salt thereof.

E12 is a compound of any one of E1, E3, E4, E7-E11, E45 and E59, wherein R ⁴ is selected from the group consisting of (C1-C6-alkyl)amino optionally substituted with one to five fluorine atoms, C ₁ -C ₆ -alkyl-C(O)NH- optionally substituted with one to five fluorine atoms, and C ₁ -C ₆ -alkyl-S(O) ₂ NH- optionally substituted with one to five fluorine atoms; or a pharmaceutically acceptable salt thereof.

E13 is a compound of 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-, Cl2 _; CI hC(())NI I- and CF3CH2NH-; or a pharmaceutically acceptable salt thereof.

E14 is a compound of any one of E1, E3, E4, E7-E13, E45 and E59, in which R ⁴ is CF ₃ C(O)NH- or CF ₃ S(O) ₂ NH-; or a pharmaceutically acceptable salt thereof.

E15 is any one of E1-E8, E45 and E59, wherein ^R3 is a 4- to 12-membered heterocycloalkyl optionally annelated with a 5- to 6-membered heteroaryl or phenyl, and wherein said heterocycloalkyl contains one to four heteroatoms independently selected from N, O and S(O)n, or is a (4- to 12-membered heterocycloalkyl)C1- _C6 -alkyl, wherein said heterocycloalkyl moiety contains one to four heteroatoms independently selected from N, O and S(O)n; each of which is optionally substituted with one to five ^R4 ; or a pharmaceutically acceptable salt thereof.

E16 is any one of E1-E8, E15, E45 and E59, wherein the 4- to 12-membered heterocycloalkyl moiety in R ³ is selected from the group consisting of azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, oxetanyl, tetrahydrofuranyl, pyranyl, 2oxo-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 R ⁴ ; or a pharmaceutically acceptable salt thereof.

E17 is a compound any one of E1-E8, E45 and E59, wherein ^R3 is selected from the group consisting of phenyl, benzyl, phenethyl, 5- to 10-membered heteroaryl containing one to five heteroatoms independently selected from N, O and S; (-membered heteroaryl)-C1- _C6 -alkyl, wherein the heteroaryl moiety contains one to five heteroatoms independently selected from N, O and S; and (5- to 10-membered heteroaryloxy)-C1- _C6 -alkyl, wherein the heteroaryl moiety 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 any one of E1 to E8, E17, E45 and E59, wherein the 5- to 10-membered heteroaryl moiety in R ³ 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[3,2b]pyrrolyl, 4H-thieno[3,2-b]pyrrolyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,3]triazolo[1,5

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a]pyridinyl and naphthyridinyl; each of which is optionally substituted with one to four R ⁴ ; or a pharmaceutically acceptable salt thereof.

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

E20 is a compound of any one of E1-E8, E17-E19, E45 and E59, wherein ^R3 is indol-2-yl; optionally substituted with one to four ^R4 ; and ^R4 is independently selected at each occurrence 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 of any one of E1, E2, E6, E9-E10, E12-E20, E45 and E59 of the formula

or a pharmaceutically acceptable salt thereof.

E22 is a compound of any one of E1, E2, E6, E9-E10, E12-E21, E45 and E59, wherein ^R3 is selected from the group consisting of N1-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)-N1-indol-2-yl, 6-(trifluoromethyl)-N1-indol-2-yl, 4-methoxy-3,6,7-tri(trifluoromethyl)-1H-indol-2-yl, 3-fluoro-4-methoxy-N1-indol-2-yl and 3,5-difluoro-4-methoxy-1H-indol-2-yl; or a pharmaceutically acceptable salt thereof.

E23 is a compound of any one of E1-E8, E21, E45 and E59, in which ^R3 is _C1 - _C6 -alkoxy; or a pharmaceutically acceptable salt thereof.

E24 is a compound of 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 of any one of E1-E8, E21, E45 and E59, wherein ^R3 is selected from the group consisting of _C3 - _C12 -cycloalkyl optionally annelated with 5- to 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 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 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 of any one of E1-E8, E17, E27, E45 and E59, wherein R ⁴ is selected from the group consisting of fluorine, chlorine, dimethylamino, trifluoromethyl, CF ₃ C(O)NH- and CF ₃ S(O) ₂ NH-; or a pharmaceutically acceptable salt thereof.

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

N-{(1S)-1 -cyano-2- [(3 8)-2-oxopyrrolidin-3 -yl]ethyl } -N ² - { (2K)-2-(dimethylamino)-2- [4(trifluoromethyl )phenyl] acetyl } -4-methyl-b-leucinamide;

N-{(1S)-1 -cyano-2- [(3 8)-2-oxopyrrolidin-3 -yl]ethyl } -N ² - { (2K)-2-(dimethylamino)-2- [3 (trifluoromethyl )phenyl] acetyl } -4-methyl-b-leucinamide;

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N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-6-(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-6-(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-3,6,7-tri(trifluoromethyl)-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-(trifluoromethoxy)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4-(trifluoromethoxy)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-3-fluoro-4-methoxy-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-3,5-difluoro-4-methoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-5,7-difluoro-4-methoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-5-fluoro-4-methoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-3,5,7-tri(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-3,7-bis(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-(trifluoromethyl)-1H-indole-2-carboxamide;

7-chloro-N-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4,4-dimethyl1-oxopentan-2-yl]-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-7-methyl-1H-indole-2-carboxamide;

- 8 047572 b-chloro-N-[(2 S)-1 -( { (1 S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4,4-dimethyl-1 -oxopentan-2-yl]-1 H-indole-2-carboxamide;

4-chloro-M-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4,4-dimethyl1-oxopentan-2-yl]-1 H-indole-2-carboxamide;

-chloro-N-[(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4,4-dimethyl1 -oxopentan-2-yl]-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-7-(trifluoromethyl)-1H-indole-2-carboxamide;

4,6-dichloro-N-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4,4-dimethyl-1-oxopentan-2-yl]-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-5-(trifluoromethyl)-1H-indole-2-carboxamide;

7-chloro-N-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-7-methyl-1H-indole-2-carboxamide;

b-chloro-N-[(2 S)-1 -( { (1 S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-1H-indole-2-carboxamide;

4-chloro-N-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-1H-indole-2-carboxamide;

5,7-dichloro-N-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4-methyl1-oxopentan-2-yl]-1 H-indole-2-carboxamide;

-chloro-N-[(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-7-(trifluoromethyl)-1H-indole-2-carboxamide;

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4,6-d Hxnop-N-[(2 S)-1 -( { (1S)-1 -cyano-2- [(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4-methyl1 -oxopentan-2-yl] -1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4-(trifluoromethyl)-1H-indole-2-carboxamide;

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

N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl- ^M2 -{[4-methyl-2(trifluoromethyl)-1,3-thiazol-5-yl]carbonyl}-L-leucinamide;

N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl- ^M2 -{[5-methyl-2(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl}-L-leucinamide;

N ² -[(4-6poM-1 -ethyl-3 -methyl-1 H-pyrazol-5-yl)carbonyl]-M- { (1S)-1 -cyano-2-[(3 S)-2oxopyrrolidin- 3 -yl]ethyl } -L-leucinamide;

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

-acetyl-M-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

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

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4-hydroxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-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- [(3 8)-2-oxopyrrolidin-3-yl]ethyl } -N ² - [(3,3 difluorocyclobutyl) acetyl]-4-methyl-E-leucinamide;

M ² -[(trans-4-cyanocyclohexyl)carbonyl]-M-{(18)-1-cyano-2-[(3 S)-2-oxo-pyrrolidin-3yl]ethyl}-4-methyl-E-leucinamide;

N ² - [(trans-4-cyano cyclohexyl) carbonyl]-N- { (1R)-1 -cyano-2- [(3 8)-2-oxopyrrolidin-3 yl] ethyl } -4-methyl-E-leucinamide;

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N- { (1S) -1 -cyano-2- [(3 8) -2-oxopyrrolidin-3 -yl] ethyl } -N ² - [2 (cyclohexyloxy) propanoyl] -4-methyl-E-leucinamide;

N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-N ² -[cyclohexyl(methoxy)acetyl]4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-N ² -[cyclohexyl(methoxy)acetyl]4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl} -N ² -[(2 8)-2-(dimethylamino)-2phenylacetyl]-4- methyl-E-leucinamide;

N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1CH ² -(pyrrolide and 1-ylacetyl)-E-leucinamide;

N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl} ^-M2 -[(2I)-2-(dimethylamino)-2phenylacetyl]-4-methyl-E-leucinamide;

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

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-3-(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-3,7-bis(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-3,5-bis(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-3,6-bis(trifluoromethyl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-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-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-^-(cyclohexylcarbonyl)-4methyl-L-leucinamide;

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N-{(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-N ² -(cyclohexylcarbonyl)-4methyl-L-leucinamide;

N- { (1S) -1 -cyano-2- [(3 8) -2-oxopyrrolidin-3 -yl]ethyl } -4-methyl-M ² - {[2- (trifluoromethyl) 1,3 -thiazol-4-yl] carbonyl } -L-leucinamide;

N- { (1S)-1-cyano-2- [(3 8)-2-oxopyrrolidin-3-yl]ethyl }-4-methyl-N ² -[(propan-2-yloxy)acetyl] -L-leucinamide;

N- { (1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl } -Ь>Г ² -[(cyclohexyloxy)acetyl]-4-methyl-N-leucinamide;

N- { (1S)-1-cyano-2- [(35)-2-oxopyrrolidin-3-yl]ethyl }-4-methyl-Λ' ² - (4,4,4-trifluoro-3 methylbutanoyl)-E-leucinamide;

N- [(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4,4-dimethyl-1 oxopentane- 2-yl]-3-methylimidazo[2,1-b][1,3]thiazol-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methylT4-(trifluoroacetyl)-E-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

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

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-UHaHO-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N(trifluoroacetyl)-E-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

N ² -[(4-6poM-1-ethyl-3-methyl-1H-pyrazol-5-yl)carbonyl]-M-{(1S)-1-cyano-2-[(3 S)-2oxopyrrolidin-3-yl]ethyl}-4-methyl-E-leucinamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide;

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N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-N ² -(2,6-dichlorobenzoyl)-4-methyl-leucinamide;

(2 S)-N- { (1 S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } -4,4-dimethyl-1 - [3 -methyl-N(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

- methyl-N-Crp iforoacetyl)-E-val nn-(4R)-N- { (1S)-1-cyano-2- [(3 S)-2-okcopyrrolidin-3 yl]ethyl } -4-methyl -4-(trifluoromethyl)-b-prolinamide;

(2S,4S)-N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-{3-methyl-N[(trifluoromethyl)sulfonyl]-L-valyl]piperidine-2-carboxamide;

N- { (1S)-1 -cyano-2- [(3 8)-2-oxopyrrolidin-3 -yl]ethyl } -N ² - {[2-(trifluoromethyl)-1,3-thiazol5 -yl] carbonyl } -L-leucinamide;

N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-N ² -[(2,8)-2-(dimethylamino)-2-phenylacetyl]-4-methyl-L-leucinamide;

T4 ² -[(trans-4-cyanocyclohexyl)carbonyl]-N-{(18)-1-cyano-2-[(3 S)-2-oxo-pyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide;

N ² - [(trans-4-cyano cyclohexyl) carbonyl]-N- { (1R)-1 -cyano-2- [(3 8)-2-oxopyrrolidin-3 yl] ethyl } -4-methyl-L-leucinamide;

N-{(1R)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-N ² -[2(cyclohexyloxy)propanoyl]-4-methyl-L-leucinamide;

(2 S,4R)-N- { (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl }-4-methyl-1-[N(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

5-(butan-2-yl)-N-[(28)-1-({(1S)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N- { (1S)-1-cyano-2- [(3 8)-2-oxopyrrolidin-3-yl]ethyl } -N ² - [(4,5-dichloro-1 H-imidazole-2yl)carbonyl]-4-methyl-L-leucinamide;

N- { (1S)-1-cyano-2- [(3 8)-2-oxopyrrolidin-3-yl]ethyl } -N ² - [(4,5-dichloro-1 H-pyrazol-3 yl)carbonyl]-4-methyl-L-leucinamide;

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

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-chloro-N-[(2 S)-1 -( { (1 S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4,4-dimethyl-1 -oxopentan-2-yl]-1 H-pyrrolo[2,3 -b]pyridine-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-(trifluoromethyl)-1H-benzimidazole-2-carboxamide;

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

-chloro-N-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4,4-dimethyl1-oxopentan-2-yl]-1 H-pyrrolo[3,2-b]pyridine-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-fluoro-1H-benzimidazole-2-carboxamide;

N- { (1S) -1 -cyano-2- [(3 8) -2-oxopyrrolidin-3 -yl]ethyl } -4-methyl-X ² - { [3 - (propan-2-yl) -1Hpyrazol-5 -yl] carbonyl } -L-leucinamide;

N-[(2 S)-1 -({(1 S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-fluoro-1H-benzimidazole-2-carboxamide;

-chloro-N- [(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4,4-dimethyl1 - oxopentan-2-yl]-1 H-benzimidazole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5,6-difluoro-1H-benzimidazole-2-carboxamide;

N- [(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4,4-dimethyl-1 oxopentane- 2-yl]-4H-thieno[3,2-b]pyrrole-5-carboxamide;

N- { (1S)-1 -cyano-2- [(3 8)-2-oxopyrrolidin-3 -yl]ethyl } -4-methyl-X ² - {[3 - (2-methylpropyl)-

N-pyrazol-5-yl] carbonyl }-L-leucinamide;

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

N ² - [(3-tert-butyl-1 H-pyrazol-5-yl)carbonyl] -N- { (1S)-1-cyano-2- [(3 8)-2-oxopyrrolidin3-yl]ethyl } -4-methyl-L-leucinamide;

6-bromo-X-[(28)-1-({(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-benzimidazole-2-carboxamide;

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N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-methyl-1H-benzimidazole-2-carboxamide;

N- [(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4,4-dimethyl-1 oxopentane- 2-yl]-4,5,6,7-tetrahydro-1H-indazol-3-carboxamide;

4,6-dichloro-M-[(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4,4dimethyl -1-oxopentan-2-yl]-1 H-benzimidazole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 S)-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;

X ² - {[5-(2-chlorophenyl)-4-fluoro-1H-pyrazol-3-yl]carbonyl}-X-{(18)-1-cyano-2-[(38)-2oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide;

N-[(2 S)-1-({(1 S)-1-cyano-2-[(3 S)-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- [(3 8)-2-oxopyrrolidin-3-yl]ethyl } -N ² - {[3 - (4-methoxyphenyl)-1Hpyrazol-5-yl] carbonyl } -4-methyl-L-leucinamide;

N- { (1S)-1-cyano-2- [(3 8)-2-oxopyrrolidin-3-yl]ethyl } -N ² - {[3 - (2-methoxyphenyl) -1Hpyrazol-5-yl] carbonyl } -4-methyl-L-leucinamide;

N- { (1S) -1 -cyano-2- [(3 8) -2-oxopyrrolidin-3 -yl]ethyl } -N ² - { [4- (4-methoxyphenyl) -1-imidazol-2-yl] carbonyl } -4-methyl-L-leucinamide;

N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl- ^M2 -{[3-(4-methylphenyl)1H-pyrazol-5-yl]carbonyl}-L-leucinamide;

7-bromo-N-[(28)-1-({(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-5-methyl-1 H-indole-2-carboxamide;

7-bromo-N-[(28)-1-({(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

(28,4K)-X-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[3-methyl-N(methylsulfonyl)-L-valyl]piperidine-2-carboxamide;

(28,48)-T4-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[N(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

- 15 047572 (2S,4S)-N-{(1 S)-l-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[3-methyl-N(trifluoroacetyl)-E-valyl]piperidine-2-carboxamide;

(2S,4R)-N-{(lS)-l-4HaHO-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[3-methyl-N(trifluoroacetyl)-E-valyl]piperidine-2-carboxamide;

5-[(28)-butan-2-yl]-N-[(28)-1-({(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}amino)4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

(I R,2S,5S)-N-{(18)-1-cyapo-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3',3',3'trifluoro-N-(trifluoroacetyl)-E-isodeucyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-4HaHO-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-3-{(2,8)-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-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-3-{(2 8)-2-cyclopentyl2-[(trifluoroacetyl)amino]acetyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[4-methylT4-(trifluoroacetyl)-E-leucyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-4HaHO-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-3-{(2,8)-2-(4,4-difluorocyclohexyl)-2-[(trifluoroacetyl)amino]acetyl }-6,6-dimethyl-Zazabicyclo[3,1,0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3,8)-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-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclohexyl-N(trifluoroacetyl)-L-alanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-4HaHO-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[b«G (trifluoroacetyl)-L-leucyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-4HaHO-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-3-[6,6-difluoro-L1(trifluoroacetyl)-L-norleucyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

- 16 047572 (lR,2S,5S)-N-{(lS)-l-ijHaHO-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-{(28)4,4,4-trifluoro-2-[(trifluoroacetyl)amino]butanoyl}-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-φτορ-N(trifluoroacetyl)-L-valyl]-6,6-dimethyl-3-azabicyclo[3.1,0]hexane-2-carboxamide;

(1B,28,58)-X-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-3-{(28)-2-cyclopropyl2-[(trifluoroacetyl)amino]acetyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-HHaHO-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-(3,3difluorocyclobutyl)-X -(trifluoroacetyl)-L-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-HHaHO-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl]-6,6-dimethyl-3-[N(trifluoroacetyl)-O-(trifluoromethyl)-L-seryl]-3-azabicyclo[3,1,0]hexane-2-carboxamide;

(1K,28,58)-X-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-{(28)-2phenyl-2-[(trifluoroacetyl)amino]acetyl}-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-HHaHO-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl]-6,6-dimethyl-3-[N(trifluoroacetyl )-b-phenylalanyl]-3-azabicyclo[3.1,0]hexane-2-carboxamide;

(1R,2S,5S)-N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-3-[3,5-difluoro-N(trifluoroacetyl)-L-phenylalanyl]-6,6-dimethyl-3-azabicyclo[3,1,0]hexane-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-HHaHO-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl]-6,6-dimethyl-3-[N(trifluoroacetyl)-3-(trifluoromethyl)-L-phenylalanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-HHaHO-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[T4-( 2,2,2tr iforeth yl)-b-val yl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(2 S,4R)-N- { (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl }-4-methyl-1- { (2 S)-3-methyl-2[(trifluoroacetyl)amino]butyl}piperidine-2-carboxamide;

(2 S,4R)-N- { (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl }-4-methyl-1- { (2 S)-3-methyl-2[(2,2,2-trifluoroethyl)amino]butyl}piperidine-2-carboxamide;

- 17 047572 (lR,2S,5S)-N-{(lS)-l-ijHaHO-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-|ЬР-(3,3,3· trifluoropropanoyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-4HaHO-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-(Npropanoyl-L-valyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(2S,4R)-N-{(lS)-l-4naHO-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[N-(2,2,2-trifluoroethyl)-l-valyl]piperidine-2-carboxamide;

T4 ² -[(4-chloro-1-ethyl-3 -methyl-1H-pyrazol-5-yl)carbonyl]-M-{ (1S)-1-cyano-2-[(3S)-2oxopyrrolidine-3 -yl]ethyl } -L-leucinamide;

-chloro-N-[(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-3 -ethyl-1 H-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;

-chloro-N-[(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-3 -methyl-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-3,5-dimethyl-1H-indole-2-carboxamide;

-tert-butyl-N-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4methyl-1-oxopentan-2-yl]-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-5-(propan-2-yl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-3-ethyl-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-6-ethyl-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-5-ethyl-1H-indole-2-carboxamide;

4-butoxy-M-[(2 S)-1-({ (1 S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4-methyl1 -oxopentan-2-yl]-1 H-indole-2-carboxamide;

- 18 047572

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-5-(trifluoromethoxy)-1H-indole-2-carboxamide;

N- [(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4-methyl-1 oxopentane-2- yl]-6-(diethylamino)-1H-indole-2-carboxamide;

4-6poM-N-[(2 S)-1 -( { (1S)-1 -cyano-2- [(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-1H-indole-2-carboxamide;

-bromo-N-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-1H-indole-2-carboxamide;

b-bromo-N-[(2 S)-1 -( { (1 S)-1 -cyano-2- [(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-3-methyl-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-6-propoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-7-fluoro-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-7-methoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-6-fluoro-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-5-fluoro-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-5-methoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4,5-dimethoxy-1H-indole-2-carboxamide;

- 19 047572

N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl(-4-methyl-N ² -[(4-methyl-1,3-thiazol-5·yl)carbonyl]-L-leucinamide;

N- {(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl (-M ² -(this is xycarbonyl)-L-leucinamide;

N-{(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl (-X ² -(ethoxycarbonyl)-4-methyl-L-leucinamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-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-[(3 8)-2-oxopyrrolidin-3-yl]ethyl(-4-methyl-M ² - {[2-(trifluoromethyl)1,3-oxazol-4-yl]carbonyl(-L-leucinamide;

N- {(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl(-4-methyl-M ² - {[3 -(trifluoromethyl)1,2-thiazol-4-yl]carbonyl(-L-leucinamide;

N- {(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl (-4-methyl-M ² - {[3 -(trifluoromethyl)1,2-oxazol-4-yl]carbonyl (-L-leucinamide;

N-{(1S)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl ( ^-N2 -{[2-(trifluoromethyl)-1,3-thiazol4-yl]carbonyl ( -L-leucinamide;

N- {(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl (-5,5,5-trifluoro-N ² - {[2(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl (-L-norvalinamide;

(4 S)-N- { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl (-5,5,5 -trifluoro-N ² - {[2(trifluoromethyl)-1,3 -thiazol-4-yl]carbonyl (-L-leucinamide;

(4R)-N- { (1S)-1 -cyano-2- [(3 8)-2-oxopyrrolidin-3 -yl]ethyl (-5,5,5 -trifluoro-N ² - {[2(trifluoromethyl)-1,3 -thiazol-4-yl]carbonyl (-L-leucinamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl(amino)-3-cyclopentyl-1-oxopropan-2-yl]-2-(trifluoromethyl)-1,3-thiazole-4-carboxamide;

N- {(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl (-4-methyl-M ² - {[5 -(trifluoromethyl)1,2-thiazol-4-yl]carbonyl (-L-leucinamide;

N- {(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl (-4-methyl-M ² - {[5 -(trifluoromethyl)1,2-oxazol-4-yl]carbonyl (-L-leucinamide;

- 20 047572

N- { (1S) -1 -cyano-2- [(3 8) -2-oxopyrrolidin-3 -yl]ethyl } -4-methyl-M ² - {[2- (trifluoromethyl) 1,3 -oxazol-5 -yl] carbonyl } -L-leucinamide;

N- { (1S) -1 -cyano-2- [(3 8) -2-oxopyrrolidin-3 -yl]ethyl } -4-methyl-M ² - {[2- (trifluoromethyl) 1,3 -thiazol-5 -yl] carbonyl } -L-leucinamide;

N- { (1S)-1-cyano-2- [(3 8)-2-oxopyrrolidin-3-yl]ethyl )-5,5,5-trifluoro-4-methyl-N ² - {[2(trifluoromethyl)-1,3-thiazol-4-yl]carbonyl } -L-leucinamide;

N- { (1S) -1 -cyano-2- [(3 8) -2-oxopyrrolidin-3 -yl]ethyl } -4-methyl-M ² - {[(2S)-2methyltetrahydrofuran-2-yl]carbonyl } -L-leucinamide;

N-[(2 S,4R)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-4methyl-1-oxopentan-2-yl]-4-methoxy-1 H-indole-2-carboxamide;

N-[(2S,4S)-1-({(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl)amino)-5,5,5-trifluoro-4methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

14-[(28)-1-({(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl)amino)-5,5,5-trifluoro-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-3-cyclopentyl-1-oxopropan-2-yl]-4-methoxy-1H-indole-2-carboxamide;

5,7-dichloro-N-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4,4-dimethyl-1-oxopentan-2-yl]-1 H-indole-2-carboxamide;

-chloro-N-[(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4,4-dimethyl1 -oxopentan-2-yl] -3 -ethyl-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-cyclohexyl-1H-indole-2-carboxamide;

-chloro-N-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4,4-dimethyl1-oxopentan-2-yl]-3-methyl-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-3,5-dimethyl-1H-indole-2-carboxamide;

-tert-butyl-N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1 H-indole-2-carboxamide;

- 21 047572

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-(propan-2-yl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-7-(propan-2-yl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-3-ethyl-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-6-ethyl-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-ethyl-1H-indole-2-carboxamide;

4-butoxy-N-[(2 S)-1-({ (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } amino)-4,4-dimethyl-1-oxopentan-2-yl]-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-(trifluoromethoxy)-1H-indole-2-carboxamide;

N- [(2 S)-1 -( { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } amino)-4,4-dimethyl-1 oxopentane- 2-yl]-6-(diethylamino)-1H-indole-2-carboxamide;

4-6poM-N-[(2S)-l-(i(l 8)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1 H-indole-2-carboxamide;

5-6poM-N-[(2S)-l-(i(l 8)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1 H-indole-2-carboxamide;

6-6poM-N-[(2S)-l-(i(l 8)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl[amino)-4,4-dimethyl-1-oxopentan-2-yl]-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-3-methyl-1 H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-6-propoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methyl-1H-indole-2-carboxamide;

- 22 047572

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-methyl-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-6-methyl-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-7-fluoro-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-7-methoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-6-fluoro-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-fluoro-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-6-methoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-methoxy-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4,5-dimethoxy-1H-indole-2-carboxamide;

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

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-7-(propan-2-yl)-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4-methyl-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-5-methyl-1H-indole-2-carboxamide;

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N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-6-methyl-1H-indole-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-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-[(38)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-1H-indole-2-carboxamide;

N-{(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl} -X ² -[(2K)-2-cyclohexyl-2methoxyacetyl]-L-leucinamide;

N- { (1S)-1-cyano-2- [(3 8)-2-oxopyrrolidin-3-yl]ethyl } -N ² - [(2R)-2 (cyclohexyloxy) propanoyl] -L-leucinamide;

N- { (1S)-1-cyano-2- [(35)-2-oxopyrrolidin-3-yl]ethyl }-4-methyl-V ² -(4,4,4-trifluoro-3 methylbutanoyl)-L-leucinamide;

T4 ² -[(trans-4-cyanocyclohexyl)carbonyl]-N-{(18)-1-cyano-2-[(3 S)-2-oxo-pyrrolidin-3yl]ethyl}-L-leucinamide;

N- { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl } -N ² -[( 1 -ethyl-4-methyl-1H-pyrazol-5 yl )carbonyl]-L-leucinamide;

N-{(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-X ² -(cyclohexylcarbonyl)-Eleucinamide;

N- { (1S)-1 -cyano-2-[(3 8)-2-oxopyrrolidin-3 -yl]ethyl }-bT ² -[(cyclohexyloxy)acetyl]-Eleucinamide;

N- { (1S)-1-cyano-2- [(3 8)-2-oxopyrrolidin-3-yl]ethyl } -N ² - [(3,3 difluorocyclobutyl) acetyl]-E-leucinamide;

N-{(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-M ² -[(propan-2-yloxy)acetyl]-Eleucinamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-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-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-14 ² -[(2R)-2-cyclohexyl-2-methoxyacetyl]-4-methyl-E-leucinamide;

- 24 047572

N- { (1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl } -N ² -[(1-ethyl-4-methyl-1H-pyrazol-5yl)carbonyl]-4-methyl-L-leucinamide;

M ² -[2-chloro-4-(methylsulfonyl)benzoyl]-M-[(18)-1-cyano-2-[(3 S)-2-oxo-pyrrolidin-3yl]ethyl}-L-leucinamide;

N- {(1S)-1-cyano-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-N ² -(2,6-dichlorobenzoyl)-leucinamide;

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

(lR,2S,5S)-3-{[(ЗR)-l-benzyl-5-oκcoπippolidin-3-yl]capbonyl}-N-{(lS)-l-cyano-2-[(ЗS)2- oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-HHaHO-2-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-i[(3R)-5oxo-1-phenylpyrrolidin-3-yl]carbonyl}-3-azabicyclo[3.1.0]hexane-2-carboxamide;

(lR,2S,5S)-3-{[(ЗR)-l-τpeτ-bytyl-5-oκcoπppolidin-3-yl]capbonyl}-N-{(lS)-l-cyano-2[(3 8) -2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2carboxamide;

(lR,2S,5S)-N-{(lS)-l-HHaHO-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[(3methylimidazo [ 2,1-b][1,3]thiazol-2-yl)carbonyl]-3-azabicyclo[3.1.0]hexane-2carboxamide;

(lR,2S,5S)-N-{(lS)-l-HHaHO-2-[(3 8)-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-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-3-cyclopropyl-1 oxopropan-2-yl]-4-methoxy-1 H-indole-2-carboxamide; and

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-3-cyclopropyl-1-oxopropan-2-yl]-1H-indole-2-carboxamide;

or a pharmaceutically acceptable salt thereof.

E3O is a compound of any one of El, E45 and E59, which is selected from the group consisting of

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide;

- 25 047572

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-1H-indole-2-carboxamide;

(lR,2S,5S)-N-{(lS)-l-LiHaHO-2-[(3,8)-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-[(3,8)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methylN-(trifluoroacetyl)-E-valyl]-3-azabicyclo[3,1,0]hexane-2-carboxamide;

N-[(2 S)-1-({(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide;

(2S,4S)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-[1N(trifluoroacetyl)-L-valyl]piperidine-2-carboxamide;

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

(lR,2S,5S)-N-{(lS)-l-4HaHO-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-3-{(28)-2-cyclohexyl-

2-[(trifluoroacetyl)amino]acetyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2carboxamide;

(2S ,4S)-N-{(1S)-1-cyano-2-[(3 8)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-1-{3-methyl-N-

[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide;

-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)-E-valyl]piperidine-2-carboxamide;

or a pharmaceutically acceptable salt thereof.

E31 is a pharmaceutical composition comprising a therapeutically 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 the form of an intravenous, subcutaneous, inhalation or oral dosage form.

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

E34 is a pharmaceutical composition according to any one of E31-E33, additionally containing an additional therapeutic agent.

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

E36 is a method of treating coronavirus infection in a patient, the method comprising administering a therapeutically effective amount of a compound of any one of E1E30 or a pharmaceutically acceptable salt thereof to a patient in need thereof.

E37 is a variant of E36, and the coronavirus infection is COVID-19.

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

E39 is the method according to E38, wherein the coronavirus infection is COVID-19.

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

- 26 047572

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

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

E43 represents the use of E42, and the coronavirus infection is COVID-19.

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

E44a represents the use of E44, and the coronavirus infection is COVID-19.

E45 is a compound of formula I'

or a pharmaceutically acceptable salt thereof; wherein R is independently hydroxy or oxo at each occurrence; p is 0, 1 or 2; R ¹ is selected from the group consisting of C1-C6 alkyl optionally substituted with cyano or one to five fluorine atoms; C2-C6 alkynyl; and (C3-C6 cycloalkyl)-C1-C3 alkyl optionally substituted with one to two substituents selected from trifluoromethyl and C1-C3 alkyl or one to five fluorine atoms; R ² is hydrogen or R ² and R ¹ taken together with the nitrogen and carbon atoms to which they are attached represent a pyrrolidine or piperidine ring optionally substituted with one to four R ^2a ; R ^2a are 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 R ^2a groups, when attached to adjacent carbon atoms and taken together with the carbon atoms to which they are attached, represent annelated C3-C6 cycloalkyl optionally substituted with one to four R ^2b ; or two R ^2a groups, when attached to the same carbon atom and taken together with the carbon atom to which they are attached, represent spiro C3-C6 cycloalkyl optionally substituted with one to four R ^2b ; R ^2b 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; R ³ is selected from the group consisting of Cp^-alkyl, Cp^-alkoxy, (C1-C6-alkoxy)-C1- _C6 -alkyl, _C2 -C6-alkynyl, C2-C6-alkynyloxy, _C3 - _C12- cycloalkyl optionally annelated with a 5- to 6-membered heteroaryl or phenyl, ( _C3 - _C12 -cycloalkyl) _-C1 - _C6 -alkyl, _C3 - _C12 -cycloalkoxy, ( _C3 - _C12 -cycloalkoxy) _-C1 - _C6 -alkyl, 4- to 12-membered heterocycloalkyl optionally annelated with a 5- to 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 moiety contains one to four heteroatoms independently selected from N, O and S(O)n, _C6 - _C10 -aryl optionally annelated with _C4 - _C6 -cycloalkyl, or 4- to 7-membered heterocycloalkyl, ( _C6 - _C10 -aryl) _-C1 - _C6 -alkyl, 5- to 10-membered heteroaryl containing one to five heteroatoms independently selected from N, O and S, optionally annelated with _C5 - _C6 -cycloalkyl; (5- to 10-membered heteroaryl)-C1- _C6 -alkyl, wherein the heteroaryl moiety contains one to five heteroatoms independently selected from N, O and S; (C ₆ -C ₁₀ -aryl)-(5- to 10-membered heteroaryl)-, wherein the heteroaryl moiety contains one to five heteroatoms independently selected from N, O and S, (5- to 10-membered heteroaryloxy)-C 1 -C ₆ -alkyl, wherein the heteroaryl moiety contains one to five heteroatoms independently selected from N, O and S; (5- to 6-membered heteroaryl)-(5- to 6-membered heteroaryl)-, wherein each heteroaryl moiety contains one to four heteroatoms independently selected from N, O and S; (4- to 7-membered heterocycloalkyl)-(5- to 6-membered heteroaryl)-, wherein the heterocycloalkyl moiety contains one to three heteroatoms independently selected from N, O and S(O)n, and the heteroaryl moiety contains one to four heteroatoms independently selected from N, O and S; (5-6-membered heteroaryl)-(4-7-membered

- 27 047572 heterocycloalkyl)-, wherein the heterocycloalkyl moiety contains from one to three heteroatoms independently selected from N, O and S(O)n, and the heteroaryl moiety contains from one to four heteroatoms independently selected from N, O and S; wherein each R ³ group is optionally substituted with from one to five R ⁴ ; R ⁴ 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, C6-C6^k^-C(O)NH-, optionally substituted with one to five fluorine, C1-C6-alkyl-OC(O)2H-, optionally substituted by one to five fluorine atoms or one R ⁵ , C1-C6-alkyl-NHC(O)2NH-, optionally substituted by one to five fluorine atoms or one R ⁵ , C1- ^{C6-alkyl-C(O)-, optionally substituted by one to five fluorine atoms or one R 5 ,} C1-C6-alkyl-N(O) _n- , optionally substituted by one to five fluorine atoms or one R ⁵ ; R ⁵ is selected from phenyl, phenoxy, C3-C6-cycloalkyl, C3-C6-cycloalkoxy, 4- to 7-membered heterocycloalkyl-, wherein the heterocycloalkyl moiety contains from one to three heteroatoms independently selected from N, O and S(O)n, and 5- to 6-membered heteroaryl-, wherein the heteroaryl moiety contains from one to four heteroatoms independently selected from N, O and S; wherein each R ⁵ is optionally independently substituted with one to three halogens, C| _-C3 -alkyl and C _{| C3} -alkoxy; and n in 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; (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3methyl-N-(methylcarbamoyl)-N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; methyl {(2S)-1[(^^^)-2-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}carbamoyl)-6,6-dimethyl-3azabicyclo[3.1.0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2-yl}carbamate; and N-(trifluoroacetyl)-N-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 of treating coronavirus infection in a patient, the method comprising administering a therapeutically effective amount of a compound according to any one of E45 and E46 or a pharmaceutically acceptable salt thereof to a patient in need thereof.

E48 is a variant of E47 in which 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 E1E30 and E45-E46 or a pharmaceutically acceptable salt thereof, wherein an additional therapeutic agent is administered, and wherein 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, 9 (COVIGUARD), lanadelumab (Takhzyro), canakinumab (Ilaris), gimsilumab, otilimab, casirivimab/imdevimab (REGN-Cov2), MK-7110 (CD24Fc/SACCOVID), heparin, apixaban, tosilizumab (Actemra), sarilumab (Kevzara), apilimod dimesylate, DNL758, DC402234, PB1046, dapagliifosine, abivertinib, ATR-002, bemcentinib, acalabrutinib, baricitinib, tofacitinib, losmapimod, famotidine, ritonavir, niclosamide and diminazene.

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

E50a is a compound (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-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-[3methyl-N-(trifluoroacetyl)-N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.

- 28 047572

E51a is a pharmaceutical composition comprising a therapeutically effective amount of (N,28,58)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-(C-trifluoroacetyl)-C-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, the method comprising administering a therapeutically effective amount of (1R,28,58)-N-{(18)-1-cyano-2-[(38)-2oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-^-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof to a patient in need of such treatment.

E52a is a method of treating coronavirus infection in a patient, the method comprising administering a therapeutically effective amount of (1R,28,58)-N-{(18)-1-Cyano-2-[(38)-2oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide to a patient in need of such treatment.

E53 is a variant of E52 in which the coronavirus infection is COVID-19.

E53a is a variant of E52a in which the coronavirus infection is COVID-19.

E54 is a method according to E52 or E53, wherein (N,28,58)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-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 (1^28,58)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide is administered orally.

E55 is the method according to E54, wherein 50 mg to 1500 mg of (1^28,58)-N-{(18)-1-cyano-2[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-(C-trifluoroacetyl)-C-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof, is administered every day.

E55a is a method according to E54a, wherein 50 mg to 1500 mg of (1R,28,58)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide is administered every day.

E56 is a method according to E55, wherein 380 mg of (N,28,58)-N-{(18)-1-cyano-2-[(38)-2oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof, is administered three times daily.

E56a is a method according to E55a, wherein 380 mg of (N,28,58)-N-{(18)-1-cyano-2-[(38)-2oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide; or a pharmaceutically acceptable salt thereof, is administered three times daily.

E57 is a method according to E55, wherein 50 mg to 1500 mg of (1^28,58)-N-{(18)-1-cyano-2[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-(C-trifluoroacetyl)-C-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 mg to 1500 mg of (1R,28,58)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-valyl]-3azabicyclo[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 the method according to E57, wherein a tablet is administered. E58a is the method according to E57a, wherein a tablet is administered. E59 is a compound of formula I o

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

- 29 047572 cotton wool or hydrate;

wherein R in each occurrence independently represents hydroxy or oxo;

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

p is 0, 1, or 2;

R ¹ is selected from the group consisting of C ₁ -C ₆ -alkyl optionally substituted with cyano or from one to five fluorine atoms; C ₂ -C ₆ -alkynyl; and (C 3 -C ₆ -cycloalkyl)-C ₁ -C 3 -alkyl optionally substituted with one to two substituents selected from trifluoromethyl and C ₁ -C 3 -alkyl or from one to five fluorine atoms;

R ² is hydrogen, or R ² and R ¹ taken together with the nitrogen and carbon atoms to which they are attached represent a pyrrolidine or piperidine ring optionally substituted with one to four R ^2a ;

R ^2a are 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 R ^2a groups, when attached to adjacent carbon atoms and taken together with the carbon atoms to which they are attached, represent annelated C3-C6 cycloalkyl optionally substituted with one to four R ^2b ; or two R ^2a groups, when attached to the same carbon atom and taken together with the carbon atom to which they are attached, represent spiro _C3 - _C6 cycloalkyl optionally substituted with one to four R ^2b ;

R ² in each occurrence is independently selected from fluoro, hydroxy, C ₁ -C 3 alkyl optionally independently substituted with one to three fluoro atoms or hydroxy, and C 1 -C 3 alkoxy optionally independently substituted with one to three fluoro atoms or hydroxy;

R3 is selected from the group consisting of CG-alkyl, C1-C8-alkoxy, ( _C1 - _C6 -alkoxy) _-C1 - _C6 -alkyl, _C2 - _C6- alkynyl, _C2 - _C6 -alkynyloxy, C3- _C12 -cycloalkyl optionally anelated with a 5- to 6-membered heteroaryl or phenyl, ( _C3 - _C12 -cycloalkyl)-C1- _C6- alkyl, _C3 - _C12 -cycloalkoxy, ( _C3 - _C12- cycloalkoxy)-C1- _C6 -alkyl, 4- to 12-membered heterocycloalkyl optionally anelated with a 5- to 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 moiety contains one to four heteroatoms independently selected from N, O and S(O)n, _C6 - _C10 -aryl optionally annelated with _C4C6 - _cycloalkyl or 4- to 7-membered heterocycloalkyl, ( _C6 - _C10 -aryl)-C1- _C6 -alkyl, 5- to 10-membered heteroaryl containing one to five heteroatoms independently selected from N, O and S, optionally annelated with _C5 - _C6 -cycloalkyl; (5- to 10-membered heteroaryl)-C1- _C6 -alkyl, wherein the heteroaryl moiety contains one to five heteroatoms independently selected from N, O and S; (C ₆ -C ₁₀ aryl)-(5- to 10-membered heteroaryl)-, wherein the heteroaryl moiety contains one to five heteroatoms independently selected from N, O and S, (5- to 10-membered heteroaryloxy)-C 1 -C ₆ alkyl, wherein the heteroaryl moiety contains one to five heteroatoms independently selected from N, O and S; (5- to 6-membered heteroaryl)-(5- to 6-membered heteroaryl)-, wherein each heteroaryl moiety contains one to four heteroatoms independently selected from N, O and S; (4- to 7-membered heterocycloalkyl)-(5- to 6-membered heteroaryl)-, wherein the heterocycloalkyl moiety contains one to three heteroatoms independently selected from N, O and S(O) _n , and the heteroaryl moiety contains one to four heteroatoms independently selected from N, O and S; (5-6-membered heteroaryl)-(4-7-membered heterocycloalkyl)-, wherein the heterocycloalkyl moiety contains one to three heteroatoms independently selected from N, O and S(O)n, and the heteroaryl moiety contains one to four heteroatoms independently selected from N, O and S; wherein each R ³ group is optionally substituted with one to five R ⁴ ;

R ⁴ in each occurrence is 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, (5-6-membered heteroaryl)amino-, wherein the heteroaryl moiety contains one to four heteroatoms independently selected from N, O and S; (4-7-membered heterocycloalkyl)amino-, wherein the heterocycloalkyl moiety 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, G-C6^km-C(O)NH-, optionally substituted with one to five fluorine atoms, G-C6^km-OC(O)NH-, optionally substituted with one to five fluorine atoms or one R ⁵ , C1- _C6- alkyl-NHC(O)NH-, optionally substituted with one to five fluorine atoms or one R ⁵ , G-C6^km-S(O)2NH-, optionally substituted with one to five fluorine atoms or one R ⁵ , C1-C6-alkyl-C(O)-, optionally substituted with one to five fluorine atoms or one R ⁵ , C1- _C6 -alkyl^(O) _n- , optionally substituted with one to five fluorine atoms or one R ⁵ ;

- 30 047572

R ⁵ is selected from phenyl, phenoxy, C3-C6 cycloalkyl, C3-C6 cycloalkoxy, 4- to 7-membered heterocycloalkyl-, wherein the heterocycloalkyl moiety contains from one to three heteroatoms independently selected from N, O and S(O)n, and 5- to 6-membered heteroaryl-, wherein the heteroaryl moiety contains from one to four heteroatoms independently selected from N, O and S; wherein each R ⁵ is optionally independently substituted with one to three halogens, C1-C3 alkyls and C1-C ₃ alkoxy; and n in each occurrence is independently selected from 0, 1 and 2.

E60 is a compound E59, which is (1R^^)-N-{(^)-1-cyano-2[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-C-valyl]-3azabicyclo[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 a compound (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, having the structure

or its solvate or hydrate.

E62 is a derivative of 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)-Lvalyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide.

E63 is a compound E62, which is crystalline (1R,2S,5S)Na{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-Na(trifluoroacetyl)-Cvalyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, solid form 1.

E64 is a compound E62, which is crystalline (1R,2S,5S)Na{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-Na(trifluoroacetyl)-Cvalyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, solid form 4.

E65 is a derivative of E61, which is amorphous (1R,2S,5S)-N-{(1S)1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl^-(trifluoroacetyl)-C-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide.

E66 is a compound E61, which is (1R^^)-N-{(^)-1-cyano-2[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-C-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl solvate.

E67 is a derivative of 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)-Lvalyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl solvate.

E68 is a compound E67, which is crystalline (1R,2S,5S)Na{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-Na(trifluoroacetyl)-Cvalyl]-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, the method comprising administering a therapeutically effective amount of a compound according to any one of E61-E68 to a patient in need of such treatment.

E70 is a method according to E69, wherein the coronavirus infection is COVID-19.

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

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

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

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

E75 is a pharmaceutical composition comprising a therapeutically effective amount of (^^^)-N-{(^)-1-cyano-2-[^)-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methylN-(trifluoroacetyl)-C-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 (^^^)^-{(^)-1-cyano-2-[^)-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)^-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 one of E62-E68.

E77 is a method according to E69 or E70, approximately 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 one of E61-E68 is administered orally to a patient twice a day.

E78 is a variant of E77 in which the patient is co-administered orally with ritonavir twice daily.

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

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

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

E82 is a compound N-(methoxycarbonyl)-3-methyl-L-α-lyl-(4R)-N-{(1S)-1-cyano-2[^)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)^-prolinamide, having the structure

CF ₃ or its solvate or hydrate.

E83 is a derivative of 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 containing a therapeutically effective amount of N-(methoxycarbonyl)-3-methyl-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin3-yl]ethyl}-4-(trifluoromethyl)-N-prolinamide; or a solvate or hydrate thereof, together with a pharmaceutically acceptable carrier.

E85 is a method of treating coronavirus infection in a patient, the method comprising administering a therapeutically effective amount of a compound according to E82 or E83 to a patient in need of such treatment.

E86 is a method according to E85, wherein the coronavirus infection is COVID-19.

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

E88 represents a route according to any one of E85-E87, according to which the compound is administered orally.

E89 represents the route according to E88, according to which 200 mg of the compound is administered twice daily.

E90 is a method of targeting a compound at any one of E1-E30, E45E46, E50, E50a, E59-E68 and E80-E83 to inhibit SARS-CoV-2, as a treatment for indications caused by viral infections associated with 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 to any one 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 of any one of E1-E30, E45-E46, E50, E50a, E59-E68 and E80-E83 as tools to understand 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 monitor up- or down-regulation of genes in order to discover 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,

- 32 047572 containing such 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, which is effective in the treatment of COVID-19 together with a pharmaceutically acceptable carrier.

E95 is a method of treating MERS in a patient, the method comprising administering a therapeutically effective amount of a compound of any one 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, the method comprising administering a pharmaceutical composition of any one of E31-E35, E51, E51a, E75, E75a, E84 and E94 to a patient in need thereof.

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

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

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

E100 represents the use of E99, and the coronavirus infection is COVID-19.

E101 is the use of a compound according to any one 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

Fig. 1: X-ray powder 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.

Fig. 2: X-ray powder 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.

Fig. 3: X-ray powder diffraction pattern of Example 13, Solid Form 1, from recrystallization of Example 13. Preparation of Solid Form 1.

Fig. 4: Structure determination from X-ray diffraction analysis of a single crystal from Example 13, solid form 1. The ORTEP diagram is plotted with the displacement parameters at 50% probability.

Fig. 5: Overlay of the powder diffraction pattern obtained for Example 13, Solid Form 1, from the recrystallization of Example 13; preparation of Solid Form 1 (Fig. 3), and a calculated powder diffraction pattern generated using Mercury software from determined single crystal X-ray diffraction data of Form 1 (see Single Crystal X-ray Diffraction Structure Determination of Example 13, Solid Form 1).

Fig. 6: X-ray powder diffraction pattern of Example 13, solid Form 4, from an alternative recrystallization of Example 13; obtaining solid Form 4.

Fig. 7: Structure determination from X-ray diffraction analysis of a single crystal from Example 13, solid form 4. The ORTEP diagram is plotted with 50% probability shift parameters.

Fig. 8: Overlay of the powder diffraction pattern obtained for Example 13, Solid Form 4, from an alternative recrystallization of Example 13; preparation of Solid Form 4 (Fig. 6) and a calculated powder diffraction pattern generated using Mercury software from determined single crystal X-ray diffraction data for Form 4 (see Single Crystal X-ray Diffraction Structure Determination for Example 13, Solid Form 4).

Fig. 9: X-ray powder diffraction pattern of Example 13, solid form 5, from Example 96.

Fig. 10: X-ray powder diffraction pattern of intermediate compound C16, HCl salt.

Fig. 11: X-ray powder diffraction pattern of intermediate compound C91.

Fig. 12: Structure determination from single crystal X-ray diffraction analysis of intermediate compound C91. The ORTEP diagram is plotted with 50% probability shift parameters.

Fig. 13: X-ray powder diffraction pattern of intermediate compound C92.

Fig. 14: X-ray powder diffraction pattern of intermediate compound C42.

Fig. 15: Structure determination from single crystal X-ray diffraction analysis of intermediate compound C42. The ORTEP diagram is plotted with 50% probability shift parameters.

- 33 047572

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, non-limiting sense. The term treatment, as used herein, unless otherwise specified, means promoting regression, alleviation, inhibition of progression, or prevention of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. In methods of treating COVID-19, it should be understood that COVID-19 is a disease caused in a patient by infection with SARS-CoV-2. The SARS-CoV-2 virus should be understood to encompass the originally discovered strain of the virus as well as mutant strains that are emerging, such as, but not limited to, strains such as B. 1.1.7 (UK variant), B. 1.351 (South African variant), P.1 (Brazilian variant), and B.1.427/B.1.429 (California variants). The term treatment as used herein, unless otherwise specified, refers to the act of treating, in the sense of cure as 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 respect to the treatment of COVID-19, the methods of the invention are particularly suitable for the treatment of a human patient.

The term pharmaceutically acceptable means that the substance or composition must be compatible, chemically and/or toxicologically, with the other ingredients contained in the preparation 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, improves, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the occurrence of one or more symptoms of a particular disease, condition, or disorder described herein.

The term alkyl, as used herein, refers to a straight or branched chain hydrocarbon saturated substituent (i.e., a substituent derived from a hydrocarbon by removal of hydrogen); which, in one embodiment, contains from one to eight carbon atoms, in another from one to six carbon atoms, and in yet another embodiment, from one 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, contain from one to three carbon atoms and consist of methyl, ethyl, n-propyl, and isopropyl.

The term alkynyl, as used herein, refers to a straight or branched chain hydrocarbon saturated substituent containing a carbon-carbon triple bond (i.e., a substituent derived from a hydrocarbon with a triple bond by removal of a hydrogen); which in one embodiment contains 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 straight or branched chain hydrocarbon saturated substituent attached to an oxygen radical (i.e., a substituent derived from a hydrocarbon alcohol by removing hydrogen from OH); in one embodiment, it contains 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, it has from one to three carbon atoms and consists of methoxy, ethoxy, n-propoxy, and isopropoxy. An alkoxy group attached to an alkyl group is called alkoxyalkyl. An example of an alkoxyalkyl group is methoxymethyl.

The term "alkynyloxy" refers to a straight or branched chain hydrocarbon saturated 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 OH); which in one embodiment contains from three to six carbon atoms. Non-limiting examples of such substituents include propynyloxy, butynyloxy, and pentynyloxy, and the like.

In some examples, the number of carbon atoms in a hydrocarbon substituent (i.e., alkyl, cycloalkyl, and the like) is designated by the prefix C _x -Cy- or C x _-y , wherein x represents the minimum and y represents the maximum number of carbon atoms in the substituent. Thus, for example, C^^-alkyl or C^^-alkyl refers to an alkyl substituent containing from 1 to 8 carbon atoms, C1- _C6 -alkyl or _C1 - _C6 -alkyl refers to an alkyl substituent containing from 1 to 6 carbon atoms, C1- _C3 -alkyl or _C1-3- alkyl refers to an alkyl substituent,

- 34 047572 containing from 1 to 3 carbon atoms. Further illustrating, C ₃ -C ₆ -cycloalkyl or C _3-6 cycloalkyl refers to a saturated cycloalkyl group containing from 3 to 6 ring carbon atoms.

The term cycloalkyl refers to a carbocyclic substituent derived from the removal of a hydrogen from a saturated carbocyclic molecule, e.g., a molecule having three to seven carbon atoms. The term cycloalkyl includes monocyclic saturated carbocycles. The term _C3C7 - _cycloalkyl means a radical of a three- to seven-membered ring system including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl groups. The term _C3 - _C6 -cycloalkyl means a radical of a three- to six-membered ring system including cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups. Cycloalkyl groups may also be bicyclic or spirocyclic carbocycles. For example, the term _C3 - _C12- cycloalkyl includes monocyclic carbocycles, and bicyclic and spirocyclic cycloalkyl moieties such as bicyclopentyl, bicyclohexyl, bicycloheptyl, bicyclooctyl, bicyclononyl, spiropentyl, spirohexyl, spiroheptyl, spirooctyl and spirononyl.

The term _C3 - _C6 -cycloalkoxy 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 C ₆ -C ₁₀ -aryl refers to carbocyclic aromatic systems with 3 to 10 atoms, and includes phenyl and naphthyl.

In some examples, the number of atoms in a cyclic substituent containing one or more heteroatoms (i.e., a heteroaryl or heterocycloalkyl) is designated by a prefix from x- to membered, wherein x represents the minimum and y represents the maximum number of atoms that form the cyclic moiety of the substituent. Thus, for example, a 4- to 6-membered heterocycloalkyl refers to a heterocycloalkyl containing 4- to 6 atoms, including one to three heteroatoms, in the cyclic moiety of the heterocycloalkyl. Thus, the phrase 5- to 6-membered heteroaryl refers to a heteroaryl containing 5 to 6 atoms, and 5- to 10-membered heteroaryl refers to a heteroaryl containing 5 to 10 atoms, each including one or more heteroatoms, in the cyclic moiety of the heteroaryl. Moreover, 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, or S.

The term hydroxy or hydroxyl refers to -OH. When used in combination with another term(s), 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 to which one or more hydroxy substituents are attached include, for example, alcohols, enols, and phenol. The terms cyano and nitrile refer to the -CN group. The term oxo refers to oxygen attached to a carbon through a double bond (i.e., when R ⁴ is oxo, then R ⁴ together with the carbon atom to which they are attached represent the moiety C=O).

The term halogen or halogen atom 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 from the removal of a hydrogen from a saturated or partially saturated ring structure containing a total specified number of atoms, such as 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 having a heterocycloalkyl substituent, a ring atom of the heterocycloalkyl substituent attached to the group may be a nitrogen heteroatom, or it may be a ring carbon atom. Similarly, if the heterocycloalkyl substituent is in turn substituted with a group or substituent, the group or substituent may be attached to a nitrogen heteroatom, or it may be attached 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 a specified number of ring atoms in which 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. 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, benzoxa

- 35 047572 zolyl, benzoisoxazolyl, oxazolopyridinyl, imidazopyridinyl, imidazopyrimidinyl and the like. In a group having a heteroaryl substituent, the ring atom of the heteroaryl substituent attached to the group may be one of the heteroatoms, or it may be a ring carbon atom. Similarly, if the heteroaryl substituent is in turn substituted with a group or substituent, the group or substituent may be attached to one of the heteroatoms, or it/it may be attached to a ring carbon atom. 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.

Additionally, 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, pyrazin2(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, or 1,3,4-oxadiazolyl), pyranyl (including 1,2-pyranyl or 1,4-pyranyl), dihydropyranyl, pyridinyl, piperidinyl, diazinyl (including pyridazinyl, pyrimidinyl, piperazinyl, triazinyl (including s-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 n-isoxazinyl), oxazolidinyl, isoxazolidinyl, oxathiazinyl (including 1,2,5-oxathiazinyl or 1,2,6-oxathiazinyl), oxadiazinyl (including 2H-1,2,4oxadiazinyl or 2H-1,2,5-oxadiazinyl), and morpholinyl.

The term heteroaryl may also include, when indicated as such, ring systems having two rings, wherein such rings may be annelated and wherein one ring is aromatic and the other ring is not a complete part of the conjugated aromatic system (i.e., a heteroaromatic ring may be annelated 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[c]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 moiety can be linked or otherwise attached to the designated substrate through various ring atoms without designating a specific point of attachment, when all possible points are designated, either 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 the like.

If substituents are described as independently having more than one variable, each instance of a substituent is selected independently of the other(s) from the list of available variables. Therefore, each substituent may be identical or different from the other(s).

When substituents are described as being independently selected from a group, each instance of a substituent is selected independently of the other(s). Therefore, each substituent may be identical to or different from the other(s).

As used herein, the term Formula I, Formula I' or Formula I may be referred to hereinafter as the compound(s) of the invention, the present invention, and the compound(s) of formula I, I' or I. Such terms are further defined to include all forms of the compound 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 bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is tightly bound, as in channel solvates and hygroscopic compounds, the water/solvent content will be dependent on humidity and dryness conditions. In such cases, a non-stoichiometric ratio will be the norm.

The compounds of the invention may exist as clathrates or other complexes. Included within the scope of the invention are 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 compounds of the present invention comprising two or more organic and/or inorganic components which may be in

- 36 047572 stoichiometric or nonstoichiometric quantities. The resulting complexes may be ionized, partially ionized, or unionized. For a general review of such complexes, see J. Pharm. Sci., 64 (8), 1269–1288, Haleblian (August 1975).

The compounds of the invention have asymmetric carbon atoms. The carbon-carbon bonds of the compounds of the invention may be depicted herein using a solid line (----), a solid wedge line (^^ ), or a dotted wedge line (.........W). 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 either a solid or a dotted wedge line to depict bonds to an asymmetric carbon atom is intended to indicate that only the stereoisomer shown is included. It is possible that compounds of formula I, I', and I may contain more than one asymmetric carbon atom. In such compounds, the use of a solid line to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers are included. For example, unless otherwise indicated, it is envisaged that compounds of formula I, I' and I may 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 line to represent bonds to other asymmetric carbon atoms in the same compound are intended to indicate 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 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 where 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 one homogeneous crystal form is obtained, containing both enantiomers in equimolar amounts. The second type is a racemic mixture or conglomerate, where two crystal forms are obtained in equimolar amounts, each containing one enantiomer.

The compounds of the invention, such as the compounds of formula I, I' and I, may exhibit the phenomenon of tautomerism; such tautomers are also considered to be 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 aggregates in solution. In solid form, one tautomer generally predominates. Even if only one tautomer can be described, the present invention includes all tautomers of the compounds of formula I, I' and I and their salts.

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

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

The subject invention also includes compounds and methods for treating coronavirus infections such as COVID-19 and methods for inhibiting SARS-CoV-2 with isotopically labeled compounds identical to those described herein, but in fact in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into the compounds of the 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 ^36CI , respectively. Compounds

- 37 047 572 compounds of the present invention, their prodrugs, and pharmaceutically acceptable salts of said compounds or said prodrugs containing the above-mentioned isotopes and/or isotopes of other atoms are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, for example those into which radioactive isotopes such as ³ H and ¹⁴ C have been incorporated, are useful in pharmaceuticals and/or tissue substrate distribution studies. Tritium, i.e. ³ H, and carbon-14, i.e. ¹⁴ C, labeled isotopes are particularly preferred due to their ease of preparation and detectability. In addition, substitution with heavier isotopes such as deuterium, i.e. ² H, may provide specific therapeutic advantages resulting in potentially greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in some circumstances. Isotopically labeled compounds used in the methods of the present invention and prodrugs thereof can be prepared by following the procedures for preparing compounds described in the art by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

The present invention also encompasses methods of using pharmaceutical compositions and methods of treating coronavirus infections, such as COVID-19 infections, by administering prodrugs of the compounds of the invention. Compounds having 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 hydroxy compounds that are used in the methods of the present invention. Amino acid residues include, but are not limited to, up to 20 naturally occurring amino acids, which are 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, ormitine, and methionine sulfone. Additional types of prodrugs are also encompassed. For example, free hydroxy groups can be prepared using 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. Also encompassed is the derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers, wherein the acyl group can be an alkyl ether optionally substituted with groups including, but not limited to, ether, amine, and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above. Prodrugs of this type are described in J. Med. Chem., 1996, 29, 10. Free amines can also be derivatized as amides, sulfonamides, or phosphonamides. All of these prodrug moieties can include groups including, but not limited to, ether, amine, and carboxylic acid functionalities.

The compounds of the present invention may be used in the methods of the present invention in combination with other therapeutic agents. For example, dosing a patient infected with the SARS-CoV-2 coronavirus (i.e., a patient with COVID-19) with a SARS-CoV-2 coronavirus 3^-protease inhibitor of the invention and an interferon, such as interferon alpha, or a pegylated interferon, such as PEG-Intron or Pegasus, may provide greater clinical benefits than dosing either the interferon, pegylated interferon, or the SARS-CoV2 coronavirus inhibitor alone. Other additional agents that may be used in the methods of the present invention include dexamethasone, azithromycin, and remdesivir. Examples of greater clinical benefits may include greater reduction in COVID-19 symptoms, faster time to symptom relief, reduced lung pathology, greater reduction in the amount of SARS-CoV-2 coronavirus in the patient (viral load), and reduced mortality.

The SARS-CoV-2 coronavirus infects cells expressing P-glycoprotein. Some of the SARS-CoV-2 coronavirus 3Gb 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 are verapamil, vinblastine, ketoconazole, nelfinavir, ritonavir or cyclosporine. The P-glycoprotein inhibitors act by inhibiting the release of the SARS-CoV-2 coronavirus inhibitors of the invention from the cell. Inhibition of P-glycoprotein-based release will prevent a decrease in intracellular concentrations of the SARS-CoV-2 coronavirus inhibitor due to P-glycoprotein release. Inhibition of P-glycoprotein release will result in increased intracellular concentrations of SARS-CoV-2 coronavirus inhibitors. Dosing a patient infected with SARS-CoV-2 coronavirus with the SARS-CoV-2 coronavirus 3Gb protease inhibitors of the invention and a P-glycoprotein inhibitor can reduce the amount of the SARS-CoV-2 coronavirus 3Gb protease inhibitor required to achieve

- 38 047572 effective doses due to an increase in the intracellular concentration of the SARS-CoV-2 coronavirus CTD protease inhibitor.

Among the agents that can be used to increase the effect on a mammal of a compound of the present invention are those that can act as inhibitors of at least one isoform of cytochrome P450 (CYP450) enzymes. CYP450 isoforms that can be preferentially inhibited include, but are not limited to, CYP1A2, CYP2D6, CYP2C9, CYP2C19, and CYP3A4. Compounds used in the methods of the invention include compounds that can be substrates of CYP3A4 and are metabolized by CYP3A4. Dosing a SARS-CoV-2 inhibitor that is a CYP3A4 substrate, such as a SARS-CoV-2 3DL protease inhibitor, and a CYP3A4 inhibitor, such as ritonavir, nelfinavir, or delavirdine, to a patient infected with SARS-CoV-2 coronavirus will decrease the metabolism of the SARS-CoV-2 inhibitor by CYP3A4. This will result in decreased clearance of the SARS-CoV-2 inhibitor and increased plasma concentrations of the SARS-CoV-2 inhibitor. Decreased clearance and higher plasma concentrations may result in a lower effective dose of the SARS-CoV-2 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, sugetriol-3,9-diacetate, (-)-Epigallocatechin gallate, phenantrin D, 2-(3,4-dihydroxyphenyl)-2-[[2-(3,4-dihydroxyphenyl)-3,4-dihydro-5,7-dihydroxy-2H-1-benzopyran-3yl]oxy]-3,4-dihydro-2H-1-benzopyran-3,4,5,7-tetrol, 2,2-di(3-indolyl)-3-indolone, (c)(1S,2R,4S,5R,8aS)-1-formamido-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofuran-3yl)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-b-glucuronide, andrographiside, (1S,2R,4aS,5R,8aS)-1-formamido-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofurαn-3-yl)ethenyl)decahydronaphthalen-2-yl 2-nitrobenzoate, 2b-hydroxy-3,4-seco-friedelolactone-27-ovuh acid, (c)-(1S,2R,4aS,5R,8aS)-1formamido-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofurαn-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, cosmosin, cleistocaltone A, 2,2-di(3-indolyl)-3-indolone, biorobin, gnidicin, fileamblinol, theaflavin 3,3'-di-O-gallate, rosmarinic acid, quitchenside I, oleanolic acid, stigmast-5-en-3-ol, deacetyl centapicrin and berchemol.

RdRp inhibitors, valganciclovir, chlorhexidine, ceftibuten, fenoterol, fludarabine, itraconazole, cefuroxime, atovaquone, chenodeoxycholic acid, cromolyn, pancuronium bromide, cortisone, tibolone, novobiocin, silybin, idprubicin bromocriptine, diphenoxylate, benzylpenicloyl G, yes bigatran etexilate, betulonal, gnidicin, 2b,30b-dihydroxy-3,4-seco-fridelolactone-27-lactone, 14-deoxy11,12-didehydroandrographolide, gniditrin, theaflavin 3,3'-di-O-gallate, (R)-((1R,5aS,6R,9aS)-1,5-dimethyl-7-methylene-3-oxo-6-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl )ethenyl)decahydro-1H-benzo[s]azepin1-yl)methyl 2-amino-3-phenylpropanoate, 2b-hydroxy-3,4-seco-friedelolactone-27-oic acid, 2-(3,4-dihydroxyphenyl)-2- [[2-(3,4-dihydroxyphenyl)-3,4-dihydro-5,7-dihydroxy-2H-1-benzopyran-3yl]oxy]-3,4-dihydro-2H-1-benzopyran-3,4 ,5,7-tetrol, filamemblicin 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-3yl)ethenyl)decahydronaphthalene -2-yl, 5-(^)-1,2-dithiolan-3-yl)pentanoate, 1,7-dihydroxy-3methoxyxanthone, 1,2,6-trimethoxy-8-(6-O-β-D-xylopyranosyl-β-D-glucopyranosyl)oxy]-9H-xanthen-9one, and 1,8-dihydroxy-6-methoxy-2-[ (6-O-β-D-xylopyranosyl-β-D-glucopyranosyl)oxy]-9H-xanthen-9one, 8-(β-D-glucopyrαnosyloxy)-1,3,5-trihydroxy-9H-xanthen-9- He.

Additional therapeutic agents that may be used in the methods of the invention include diosmin, hesperidin, MK-3207, venetoclax, dihydroergocristine, bolazine, R428, di

- 39 047572 tercalinium, etoposide, teniposide, UK-432097, irinotecan, lumacaftor, velpatasvir, eluxadoline, ledipasvir, lopinavir/ritonavir + ribavirin, alferon and prednisolone. Other additional agents suitable 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 the compound shown below and the compound designated DC402234

Another embodiment of the present invention is a method of treating COVID-19 in a patient, which comprises administering, 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, and wherein 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 MK-7110 (CD24Fc/SACCOVID), anticoagulants such as heparin and apixaban, IL-6 receptor agonists such as tosilizumab (Actemra) and sarilumab (Kevzara), PIKfyve inhibitors such as apilimod dimesylate, RIPK1 inhibitors such as DNL758, DC402234, VIP receptor agonists such as PB1046, SGLT2 inhibitors such as dapagliifosin, 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, such as diminazene.

The term SARS-CoV-2 inhibitory agent means any inhibitor of the SARS-CoV-2-mediated coronavirus 3C-like protease, a 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 “inhibiting or preventing the replication of a SARS-CoV-2-mediated coronavirus (SARS-CoV-2) virus in a cell” means reducing the replication of SARS-CoV-2 or the production of SARS-CoV-2 components necessary for the progeny virus in a cell treated with a compound of the invention compared to a cell not treated with a compound of the invention.

- 40 047572 teniyu. Simple and convenient assays to determine whether SARSCoV-2 viral replication has been reduced include ELISA assays for the presence, absence, or reduction in the presence of anti-SARS-CoV-2 antibodies in a subject's blood (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 greater than fifty percent compared to control cells generally 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 treatment of the free base with an inorganic acid (such as hydrochloric acid, hydrobromide acid, sulfuric acid, nitric acid, phosphoric acid, and the like) or 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 (e.g., glucuronic acid or galacturonic acid), alpha-hydroxy acid (e.g., citric acid or tartaric acid), amino acid (e.g., aspartic acid or glutamic acid), aromatic acid (e.g., benzoic acid or cinnamic acid), sulfonic acid (e.g., p-toluenesulfonic acid or ethanesulfonic acid), and the like.

If the SARS-CoV-2 inhibitor, a 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, one skilled in the art will recognize that the compounds, prodrugs, salts, and solvates used in the method of the invention may exist in various polymorphic or crystalline forms, all of which are intended to be within the scope of the present invention and the specific formulas. In addition, the compounds, salts, prodrugs, and solvates used in the method of the invention may exist as tautomers, all of which are intended to be within the broad scope of the present invention.

Solubilizing agents 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, polyethyleneglycol, Tween, ethanol and micelle forming agents. The proposed 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 invention may have chiral centers. When chiral centers are present, the compounds, salts, prodrugs, and solvates may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates, and mixtures thereof are intended to be within the broad scope of the present invention.

As generally understood by those skilled in the art, an optically pure compound is a compound that is enantiomerically pure. As used herein, the term optically pure is intended to mean a compound that contains at least sufficient activity. Advantageously, the amount of optically pure single enantiomer to obtain a compound that has a pharmacologically pure compound of the invention is at least 90% of the single isomer (80% enantiomeric excess), more advantageously at least 95% (90% ee), even more advantageously at least 97.5% (95% ee), and most advantageously at least 99% (98% ee).

The term treatment as used herein, unless otherwise specified, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

- 41 047572 conditions. The term treatment as used herein, unless otherwise specified, refers to the action of treating as defined immediately above. In an advantageous embodiment of the present invention, treatment or treating means at least alleviating a disease state in a human being that is alleviated by inhibiting the activity of SARS-CoV-2 3C-like protease, which is the main protease of SARS-CoV-2, the causative agent of COVID-19. For patients suffering from COVID-19, fever, fatigue, dry cough are the main manifestations of the disease, while nasal congestion, runny nose and other upper respiratory tract symptoms are rare. Beijing Centers for Disease Control and Prevention indicated that a typical case of COVID-19 has a progressive exacerbation process. 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/c791e5a7ea5149f680fdcb 34dac0f54e.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 patients were found to have visual manifestations of pneumonia; (3) Severe cases - one of the following three conditions: respiratory distress, respiratory rate > 30 breaths/min (at rest, relative to oxygen saturation < 93%), partial arterial pressure of oxygen (PaO2)/oxygen uptake concentration (FiO2) < 300 mmHg (1 mmHg = 0.133 kPa); (4) Critical cases - one of the following three conditions: respiratory failure and the need for mechanical ventilation, shock, or associated failure of other organs requiring intensive care. Current clinical data indicate that most deaths occur in older patients. However, severe cases have been documented in younger adults with individual factors, particularly those with chronic diseases such as diabetes mellitus or hepatitis B. Those who have been taking hormones or immunosuppressants for a long time and who have reduced immune function are likely to have a severe course of the disease.

Methods of treatment for alleviating a coronavirus disease, such as COVID-19, include administering one or more compounds of the invention in any of the conventional ways. According to certain 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. Advantageously, the mammal in need thereof is infected with a coronavirus, such as the causative agent of the COVID-19 disease, in particular 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 certain preferred embodiments, an effective amount of one or more compounds of the invention, or a pharmaceutically acceptable salt, prodrug, pharmaceutically active metabolite, or solvate thereof, is administered to a human at risk 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 in any of the generally acceptable ways.

Specific 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 recently identified L and S subtypes, has recently been identified and released.

The activity of the inhibitory compounds as inhibitors of SARS-CoV-2 viral activity can be measured by any of the suitable methods available in the art, including in vivo and in vitro assays. The activity of the compounds of the present invention as inhibitors of coronavirus 3C-like protease activity (such as SARS-CoV-2 coronavirus 3C-like) can be measured by any of the suitable methods known to those skilled in the art, including in vivo and in vitro assays. Examples of suitable assays for 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.

The administration of SARS-CoV-2 inhibitors, compounds and pharmaceutically acceptable prodrugs, salts, active metabolites and solvates thereof may be carried out in accordance with any of the acceptable routes of administration available to a person skilled in the art. Illustrative examples of acceptable routes of administration include oral, nasal, pulmonary, intravenous ...

- 42 047572 enteral, topical, intravenous, injection, transdermal, and rectal. Oral, intravenous, subcutaneous, and nasal delivery are preferred.

The SARS-CoV-2 inhibitory agent may be administered as a pharmaceutical composition in any suitable pharmaceutical form. Suitable pharmaceutical forms include solid, semi-solid, liquid, or lyophilized preparations such as tablets, powders, capsules, suppositories, suspensions, liposomes, and aerosols. The SARS-CoV-2 inhibitory agent may be prepared as a solution using any of a variety of methodologies. For example, the SARS-CoV-2 inhibitory agent may be dissolved in an acid (e.g., 1 M HCI) 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., approximately 15 mM). Alternatively, a D5W solution containing approximately 15 mM HCI may be used to prepare a solution of the SARS-CoV-2 inhibitory agent at the appropriate concentration. In addition, the SARS-CoV-2 inhibiting agent can be prepared as a suspension using, for example, a 1% carboxymethylcellulose (CMC) solution.

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

Typically, a compound of the invention is administered in an amount effective to treat a condition as 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 in a dosage effective for the intended treatment. Therapeutically effective doses of the compounds required to treat the progression of a medical condition are readily ascertainable by one skilled in the art using preclinical and clinical approaches close to the medical state of 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 may involve buccal or sublingual administration, by which the compound enters the bloodstream directly from the oral cavity.

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

In another embodiment, the compounds of the invention can also be administered locally 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 for the compounds and/or compositions containing the compounds is based on many 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 compounds being used. Thus, the dosage regimen can vary widely. Dosage levels on the order of about 0.01 mg to about 100 mg per kilogram of body weight per day are acceptable in the treatment of the above conditions. In another embodiment, the total daily dose of a compound of the invention (administered in single or divided doses) is typically from about 0.01 to about 100 mg/kg. In another embodiment, the total daily dose of a compound of the 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 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. A dosage unit of the compositions may contain such amounts or subunits thereof, making up a daily dose. In many cases, the administration of the compound will be repeated multiple times per day (usually not more than 4 times). Typically, multiple doses per day may be used to increase the total daily dose, if necessary.

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. Intravenously,

- 43 047572 Doses may range from approximately 0.1 to approximately 10 mg/kg/minute during constant rate infusion.

Suitable patients according to the present invention include mammalian patients. Mammals according to the present invention include, but are not limited to, a dog, a cat, a cow, a goat, a horse, a sheep, a pig, rodents, lagomorphs, primates, and the like, and include mammals in utero. In one embodiment, humans are suitable patients. 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 described herein.

For the treatment of the conditions mentioned above, the compound of the invention can be administered as the compound per se. Alternatively, pharmaceutically acceptable salts are acceptable for medical applications 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 encompasses any acceptable 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, such as a tablet, which may contain from 0.05% to 95% by weight of the active compounds. The compound of the invention may be combined with suitable polymers as carriers in a drug capable of targeting. In addition, other pharmacologically active substances may be present.

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

Oral administration of a solid dosage form can be, for example, presented as discrete units such as hard or soft capsules, dragees, cachets, troches or tablets, each containing a predetermined amount of at least one compound of the present invention. In another embodiment, 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, troches. In such solid dosage forms, the compounds of the invention are typically combined with one or more excipients. Such capsules or tablets can contain a controlled release composition. In the case of capsules, tablets and dragees, the dosage forms can also contain buffering agents or can be made with an enteric coating.

In another embodiment, oral administration may be in liquid dosage form. Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents that are commonly used in the art (e.g., water). Such compositions may also contain auxiliary substances 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, intracranial injections, intramuscular injections, intrasternal injections and infusion. 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 topical dosage form. Topical administration includes, for example, transdermal administration, such as by transdermal patches or iontophoresis devices, intraocular administration, or intranasal or inhalation administration. Compositions for topical administration also include, for example, topical gels, sprays, ointments and creams. Preparations for topical use 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 this invention are administered using a transdermal device, administration will be via a patch or reservoir or porous membrane type or various solid matrices. Typical preparations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, powders, dressings, foams, films, skin patches, capsule implants, implants, sponges, fibers, bandages, and microemulsions. In addition, liposomes may be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerol, polyethylene glycol, and propylene glycol. Penetration enhancers may be included; see,

- 44 047572 e.g. J. Pharm. Sci., 88(10), 955-958, by Finnin and Morgan (October 1999).

Formulations suitable for topical administration to the eye include, for example, eye drops wherein the compound of the invention is dissolved or suspended in an appropriate carrier. A typical formulation suitable for ocular or aural administration may be in the form of drops of a micronized suspension or solution in isotonic, pH-adjustable, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g., gel sponges, collagen, absorbable) and non-biodegradable (e.g., silicone) implants, capsule implants, lenses, and particulate or vesicular 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 together with a preservative such as benzalkonium chloride. Such preparations may also be administered using iontophoresis.

For intranasal or inhalation administration, the active compounds of the invention are typically administered in the form of a solution or suspension from a container with a spray pump compressed or pumped by the patient, or as an aerosol spray from a pressurized container or nebulizer using an appropriate propellant. Compositions suitable for intranasal administration are typically administered as a dry powder (either alone, as a mixture, such as in a dry mixture with lactose, or as mixed particles of the components, 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 necessary.

In addition, other carrier materials and modes of administration known in the pharmaceutical art may be used. The pharmaceutical compositions of the invention can be prepared using any of the methods well known in the art of pharmacy, such as effective formulation and administration procedures. The above considerations regarding effective formulations and administration procedures are well known in the art and are described in standard textbooks. Drug formulation 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, NY, 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 diseases or disease states. The compound(s) of the present invention and the other therapeutic agent(s) 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. Furthermore, 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 using different routes of administration. The phrases concurrent administration, co-administration, simultaneous administration, and administered concurrently mean that the compounds are administered in combination.

The present invention includes the use of a combination of a compound of the present invention and one or more additional therapeutic agent(s). If a combination of active agents is administered, they may be administered sequentially or simultaneously in separate dosage forms or combined in a single dosage form. Accordingly, the present invention also includes pharmaceutical compositions comprising a certain amount of: (a) a first agent comprising a compound of the present 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 may also include acceptable excipients, diluents, carriers, and fillers, as well as other pharmaceutically active agents, depending on the intended use. Solid or liquid pharmaceutically acceptable carriers, diluents, fillers, or excipients may be used in the pharmaceutical compositions. Illustrative solid fillers include starch, lactose, calcium sulfate dihydrate, kaolin, sucrose, talc, gelatin, pectin, acacia, magnesium stearate, and stearic acid. Illustrative liquid fillers include syrup, peanut oil, olive oil, saline, and

- 45 047572 water. The vehicle or diluent may include a suitable sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. When a liquid vehicle 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 the 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 inhibiting the activity of a coronavirus associated with SARS-CoV-2, using any known or acceptable route of dosing, including topical, such as an ointment or cream; oral; rectal, such as a suppository; parenteral, such as an injection; intravenous; or continuous by intravaginal, intranasal, intrabronchial, intraural, 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 that is alleviated by inhibiting the replication of the SARS-CoV-2 virus. The amount of a given SARS-CoV-2 inhibitory agent used in the method of the 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 generally be 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 according to the properties of the particular agent used, the particular formulation, the route of administration, and the particular site, as well as the host and condition being treated. Optimum dosages for a given set of conditions can be determined by those skilled in the art using conventional dose determination studies. 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 treatment courses repeated at appropriate intervals. For intravenous dosing, a dosage of up to 5 grams per day can be used. Intravenous administration can occur periodically throughout the day or directly 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 a particular cytochrome P450 enzyme isoform 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 using methods known to those skilled in the art and the 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 major or 3C-like protease.

The name 3C-like protease derives from certain similarities between the coronavirus enzyme and well-known picornavirus 3C proteases. These include substrate preferences, the use of cysteine as an active site nucleophile in catalysis, and the similarity of their predicted 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 the N to the 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 a glutamine at P1 and a small amino acid at P1' [Journal of General Virology, 83, p. 595-599 (2002)].

The compounds of the present invention can be prepared according to the methods shown in reaction schemes 1-3 below.

The diagrams below further illustrate and provide examples of connections according to

- 46 047572 to 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 otherwise mixture of two enantiomers, or as different mixtures of diastereomers. Such enantiomers, racemates and diastereomers may be prepared and/or separated by methods known to those skilled in the art. Those 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 (wherein N-BOC is N-tert-butoxycarbonyl). This can be achieved directly, for example by treatment with ammonia (NH ₃ ) in a sealed vessel in a solvent such as methanol or ethanol, for example optionally in the presence of additives such as calcium chloride (CaCl ₂ ) 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 accomplished 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)NH4N,N-tetramethylaronium 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 , which is introduced as a gas or a solution in a reaction-compatible solvent, or a salt of _NH3 , such as ammonium acetate or ammonium chloride in the presence of a base such as N,N-diisopropylethylamine, 4-methylmorpholine, or triethylamine. Acceptable solvents include, but are not limited to, dichloromethane ( _CH2Cl2 ), CL'-dimethylformamide (DMF), tetrahydrofuran ( _THF ), or acetonitrile (CH3CN).

The compound of formula 3 can be N-deprotected to provide an amine of formula 4 using methods well known to one skilled in the art for effecting such deprotection. Often, acidic reagents such as hydrogen chloride, methanesulfonic acid, or trifluoroacetic acid are used, typically in a reactive solvent such as CH2Cl2, 1,4-dioxane, 1,2-dichloroethane, or CH3CN. One skilled in the art will appreciate that the compound of formula 4 will often be obtained as an acid addition salt. The compound of formula 4 can then be converted to a compound of formula 6 by treating the N-protected amino acid compound of formula 5 under appropriate conditions. Such methods are well known to one skilled in the art.

- 47 047572 technical field, and general standard conditions for the peptide compound can be selected.

The compound of formula 6 can be N-deprotected to provide an amine of formula 7 using methods well known to one skilled in the art for effecting such deprotection. Often, acidic reagents such as hydrogen chloride, methanesulfonic acid, or trifluoroacetic acid are used, typically in a reactive solvent such as _{CH2Cl2 ,} 1,4-dioxane, 1,2-dichloroethane, or CH3CN. One skilled in the art will appreciate that the compound of formula 7 will often be 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 compound of formula 8 under appropriate conditions. Such methods are well known to one skilled in the art. For example, when X = chlorine, the carboxylic acid compound is known as an acid chloride and the reaction is carried out in the presence of a base to bond the hydrogen of the halide HX, which is formed 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 (Na ₂ CO3), or potassium bicarbonate (KHCO3). Suitable solvents include, but are not limited to, CH ₂ Cl ₂ , DMF, THF, or CH3CN. When X = OH, it is conventional to use a reagent or combination of reagents to facilitate the reaction of the carboxylic acid compound of formula 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 additional nucleophile such as hydroxybenzotriazole (HOBt) or 2-hydroxypyridine-N-oxide (HOPO). Additionally, when X = OH, one skilled in the art may choose to use reagents suitable for forming mixed carboxyl/carbon 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 that is commonly used by those skilled in the art when X=OH is to treat the carboxylic acid compound of formula 8 with a carboxylic acid chloride, such as _Me3CCOCl , in the presence of a base such as described above, to generate 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 carry out the reaction using a symmetrical anhydride of the desired carboxylic acid compound of formula 8, optionally in the presence of a base such as described above, in this case X = O(O)CR ₃ , and thus the carboxylic acid compound 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 excess trifluoroacetic anhydride or phosphorus oxychloride, typically in the presence of a base such as pyridine, N-diisopropylethylamine, 4-methylmorpholine, or triethylamine.

A person skilled in the art will know 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 a person skilled in the art using well-established procedures for the synthesis of N-protected amino acids. Similarly, a person skilled in the art will understand that carboxylic acid compounds of formula 8 may be known in the chemical literature, and/or are commercially available, and/or can be prepared by published methods or by analogy to published methods.

A person skilled in the art will appreciate that the bond formation steps in Scheme 1 may be performed in different orders with appropriate criteria, such as shown in Scheme 2.

- 48 047572

Scheme 2

¹¹ 12 y.

I

In Scheme 2, a compound of formula 3 is converted to a compound of formula 10 by treatment with dehydration conditions well known to one skilled in the art. Often, this dehydration step can be accomplished using excess trifluoroacetic anhydride or phosphorus oxychloride, typically in the presence of a base such as pyridine, N-diisopropylethylamine, 4-methylmorpholine, or triethylamine. The compound of formula 10 is N-deprotected to provide an amine of formula 11 using methods well known to one skilled in the art for accomplishing such deprotection. Often, acidic reagents such as hydrogen chloride, methanesulfonic acid, or trifluoroacetic acid are used, typically in a reactive solvent such as _{CH2Cl2 ,} 1,4-dioxane, 1,2-dichloroethane, or _CH3CN . The skilled artisan will appreciate that the compound of formula 11 will often be obtained as an acid addition salt. The compound of formula 11 can then be converted to the compound of formula I by treatment with a compound of formula 12 under appropriate conditions. Such methods are well known to the skilled artisan, and general standard peptide coupling conditions can be selected. Compounds of formula 12 are exceptionally well known in the chemical literature, and the skilled artisan can choose to prepare any provided compound of formula 12 using methods analogous to those described in the chemical literature.

A person skilled in the art will appreciate that the bonding steps in diagrams 1 and 2 may be performed in additional different orders with appropriate criteria, for example as shown in diagram 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 appropriate conditions. Such methods are well known to one skilled in the art, and general standard peptide coupling conditions can be selected. Compounds of formula 12 are exceptionally well known in the chemical literature, and one skilled in the art can choose to prepare any provided 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 one skilled in the art. Often, this dehydration step can be accomplished using excess trifluoroacetic anhydride or phosphorus oxychloride, typically

- 49 047572 in the presence of a base such as pyridine, N,N-diisopropylethylamine, 4-methylmorpholine, or triethylamine.

One skilled in the art will appreciate that still further permutations of the bond formation step and manipulation of the functional groups in Schemes 1, 2 and 3 may be employed with appropriate criteria. Such permutations in the choice of the sequence of steps are well known in the chemical literature, and one skilled in the art may consult the chemical literature for further guidance if necessary. One skilled in the art will appreciate that other choices of protecting groups and reagents may be made to effect various transformations.

Examples

Experimental procedures.

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 procedures 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 as described herein.

All reactions were carried out with constant stirring under nitrogen or argon gas unless otherwise noted. When appropriate, reaction apparatuses were dried under dynamic vacuum using a heat gun, and anhydrous solvents were used (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 4A molecular sieves in order to achieve the following QC standards for water content: a) <100 ppm for dichloromethane, toluene, Ν,Ν-dimethylformamide, and tetrahydrofuran; b) <180 ppm for methanol, ethanol, 1,4-dioxane, and diisopropylamine. For very sensitive reactions, solvents were further treated with sodium metal, calcium hydride, or molecular sieves, and distilled immediately before use. Other commercial solvents and reagents were used without further purification. For syntheses according to the procedures referenced in other Examples or Methods, reaction conditions (reaction time and temperature) may vary. Products were generally dried in vacuo before further reactions or biological studies.

Reactions were heated by microwave irradiation as indicated using a Biotage Initiator or Personal Chemistry Emrys Optimizer microwave device. Reaction progress was monitored using thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC), and/or gas chromatography-mass spectrometry (GC-MS) analysis. TLC was performed on pre-coated silica gel plates using a fluorescence indicator (excitation wavelength 254 nm) and visualized under UV light and/or using I ₂ , KMnO ₄ , CoCl ₂ , phosphomolybdic acid, and/or cerium ammonium molybdate staining. LC-MS data were acquired on an Agilent 1100 Series instrument with a Leap Technologies autosampler, Gemini C18 columns, acetonitrile/water gradients, and either trifluoroacetic acid, formic acid, or ammonium hydroxide modifiers. Column eluate was analyzed using a Waters ZQ mass spectrometer, scanning in both positive and negative ion modes from 100 to 1200 Da. Other similar instruments were also used. HPLC data were typically acquired on an Agilent 1100 Series instrument using the indicated columns, acetonitrile/water gradients, and either trifluoroacetic acid or ammonium hydroxide modifiers. GC-MS data were acquired using a Hewlett Packard 6890 oven with an HP 6890 injector, an HP-1 column (12 m x 0.2 mm x 0.33 μm), and helium as the carrier gas. The sample was analyzed on an HP 5973 mass selective detector, scanning from 50 to 550 Da using electron ionization. Cleanups were performed using 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 gel cartridges. Chiral cleanups were performed using chiral supercritical fluid chromatography (SFC), 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 was used to initiate fraction collection. For syntheses referenced in other Examples or Methods, purification may vary: in general, the solvents and solvent ratios used for the eluents/gradients were chosen to provide appropriate Rf or retention times.

Mass spectrometry data are presented using LCMS (liquid chromatography mass spectrometry) analysis. Mass spectrometry was performed using chemical sources.

- 50 047572 atmospheric pressure ionization (APCI), electrospray ionization (ESI), electron impact ionization (EI), or electron scattering ionization (ES). Proton nuclear magnetic spectroscopy (1H NMR) chemical shifts are reported in parts per million downfield from tetramethylsilane and are recorded on Varian, Bruker, or Jeol 300, 400, 500, or 600 MHz spectrometers. Chemical shifts are expressed in parts per million (ppm, δ) relative to the residual peaks of the deuterated solvent (chloroform, 7.26 ppm; _CD2HOD , 3.31 ppm; acetonitrile^, 1.94 ppm; dimethyl _sulfoxided5 , 2.50 ppm; DHO, 4.79 ppm). Peak shapes are described as follows: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br s, broad singlet; v., obvious. SFC analytical data were typically obtained on a Berger analytical instrument as described above. Polarization area rotation data were obtained on a PerkinElmer model 343 polarimeter using 1 dm cells. Microanalyses were performed using Quantitative Technologies Inc. and were within 0.4% of calculated values.

Unless otherwise stated, chemical reactions were 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 using methods known in the literature.

The terms concentrated, evaporated, and concentrated in vacuo refer to the removal of solvent under reduced pressure on a rotary evaporator with a bath temperature of less than 60°C. The abbreviations min and h refer to 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, GC-MS refers to gas chromatography-mass spectrometry, LC-MS 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 carried out in a Parr shaker under pressure of hydrogen gas or in a Thales-nano H-Cube flow hydrogenation apparatus with full hydrogen content and a flow rate of 1-2 ml/min, at a given temperature.

Retention times for HPLC, UPLC, LC-MS, GC-MS and SFC were measured using the methods described in the procedures.

In some examples, chiral resolution was performed to separate enantiomers or diastereomers of particular 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 for an enantiomer was measured using a polarimeter. According to its observed rotation data (or its specific rotation data), the enantiomer with clockwise rotation was designated as the (+)-enantiomer, and the enantiomer with counterclockwise rotation was designated as the (-)-enantiomer. Racemic compounds are designated 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 presented in ACD/ChemSketch 2019.1.1, File Version C05H41, Build 110712 (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada). The naming convention presented in ACD/ChemSketch 2019.1.1 is well known to one of skill in the art, and the naming convention presented in ACD/ChemSketch 2019.1.1 is believed to be generally consistent with the International Union for Pure and Applied Chemistry (IUPAC) recommendations for organic chemistry nomenclature and the CAS index rules.

Example 1.

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

- 51 047572 about sn ₃

Step 1. Synthesis of methyl (1H^^)-3-(tert-butoxycarbonyl)-1.-1-syl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (C1).

At 0°C, a solution of N(tert-butoxycarbonyl)-N-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-tetramethylaronium hexafluorophosphate (HATU; 122 g, 321 mmol), followed by treatment with N,N-diisopropylethylamine (127 mL, 729 mmol). After the reaction mixture was stirred for 5 min, methyl (^^^)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, hydrochloride salt (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, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was stirred in a minimum amount of ethyl acetate and then filtered; The undissolved material was washed with ethyl acetate until it turned white. The combined filtrates were concentrated under reduced pressure and then subjected to silica gel chromatography (eluent: 1:1 ethyl acetate/heptane) to give Cl as a yellow oil. Yield: 109 g, quantitative. LC-MS m/z 369.3 [M+H]+.

'H 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,

- 52 047572

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 salt (C2).

A solution of hydrogen chloride in 1,4-dioxane (4 M; 15 mL, 60 mmol) at 0 °C was added to a solution of Cl (1.00 g, 2.71 mmol) in ethyl acetate (50 mL). The reaction mixture was stirred at 0 °C for 2 h, after which additional hydrogen chloride in 1,4-dioxane (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 hydrogen chloride in 1,4-dioxane (4 M; 10 mL, 40 mmol) and methanol (15 mL) and allowed to stir overnight at room temperature. Concentration in vacuo gave C2 as a gum; This material was used in the following chemical reaction without further purification and the reaction was expected to be quantitative. LC-MS m/z 269.3 [M+H]+.

^1H NMR (400 MHz, _DMSOD6 ) δ 8.24 (w 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, AB quartet component, J = 7.6 Hz, 1H), 1 .09-0.88 (m, 12H).

Step 3. Synthesis of methyl (^^^)-6,6-dimethyl-3-[N-(trifluoroacetyl)-B-valyl]-3azabicyclo[3.1.0]hexane-2-carboxylate (C3).

Triethylamine (1.55 mL, 11.1 mmol) was added to a solution of C2 at 0 °C (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, after which 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 (wd, 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), 1.091.03 (m, 6H), 0.94 (d, J = 6.8 Hz, 3H), 0.92 (s, 3H).

Step 4. Synthesis of (^^^)-6,6-dimethyl-3-[N-(trifluoroacetyl)-B-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, after which it was partitioned between water (50 mL) and ethyl acetate (100 mL). The aqueous layer was extracted with ethyl acetate (2 x 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%. LC-MS 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, AB-quartet component, 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-2yl}carbamate (C5).

A solution of ammonia in methanol (7.0 M, 150 mL, 1.0 mol) was added at 0 °C to a solution of methyl H-(tert-butoxycarbonyl)-3-[(N)-2-oxopyrrolidin-3-yl]-D-alaninate (5.00 g, 17.5 mmol) in methanol (25 mL). After the reaction mixture was stirred at room temperature for 3 days, it was concentrated in vacuo; the residue was diluted and reconcentrated successively with a mixture of ethyl acetate and heptane (1:1, 4 x 50 mL) followed by heptane (50 mL) to give C5 as a solid (5.27 g, assumed quantitative) containing residual solvent. Some of this material was used in the next step. LCMS m/z 216.2 [(N-2-methylprop-1-ene)+H]+.

1H NMR (400 MHz, _methanolD4 ) δ 4.16-3.96 (m, 1H), 3.40-3.27 (m, 2H, suspected; 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 (dddd, 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 {(^)-1-cyano-2-[^)-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 at 0 °C to a 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, after which 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. LC-MS m/z 254.3 [M+H]+.

1H NMR (400 MHz, methanol D ₄ ) δ 4.72 (dd, J = 9.3, 6.8 Hz, 1H), 3.39-3.27 (m, 2H, assumed; h

- 53 047572 is closely obscured by the 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 (^)-2-amino-3-[(^)-2-oxopyrrolidin-3-yl]propanenitrile, methanesulfonate salt (C7).

To a solution of C6 (317 mg, 1.25 mmol) in 1,1,1,3,3,3-hexafluoropropan-2-ol (3 mL) was added methanesulfonic acid (81.2 µL, 1.25 mmol). After the reaction mixture was stirred at room temperature for 45 min, it was concentrated in vacuo, then repeatedly charged with a solvent mixture and concentrated again: acetonitrile and ethyl acetate (1:1, 2 x 10 mL) followed by ethyl acetate and heptane (1:1, 2 x 10 mL). The resulting C7 was obtained as a glass (423 mg), which was free of the nitrile epimer by ¹ H and ¹³ C NMR analysis. Some of this material was used in subsequent reactions without further purification. LCMS m/z 154.2 [M+H]+.

1H NMR (400 MHz, methanol) 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 (^^^)-X-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3[X-(trifluoroacetyl)-C-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)-X,X,X',X'-tetramethylaronium hexafluorophosphate (HATU, 97%; 112 mg, 0.286 mmol) was added, followed by the addition of 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 h. Saturated aqueous sodium bicarbonate solution (30 ml) was then added to the reaction mixture at 0°C, 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), after which the combined organic layers were dried over sodium sulfate, concentrated in vacuo, and subjected to silica gel chromatography (Gradient: 0% to 20% methanol in ethyl acetate). Since the resulting material was judged to be an impure epimer of the product by NMR and LC-MS, it was further purified using reversed-phase HPLC (column: Waters Sunfire C18, 19x100 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 8.54 min, then 95% B over 1.46 min; flow rate: 25 mL/min) to give (^^^)-X-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3yl]ethyl}-6,6-dimethyl-3-[X-(trifluoroacetyl)^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (1). Yield: 14.6 mg, 30.1 μmol, 11%. LC-MS m/z 486.5 [M+H]+. Retention time: 2.33 minutes (Analytical conditions. Column: Waters Atlantis C18, 4.6x50 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 over 1.0 minute. Flow rate: 2 mL/min).

Alternative synthesis of C4.

(^^^)-6,6-dimethyl-3-[X-(trifluoroacetyl)^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (C4)

Synthesis

Stage

1.

(1R,2S,5 S)-3-[N-(tert-butoxycarbonyl)^-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 Cl (107 g, 290 mmol) in tetrahydrofuran (730 mL). After the resulting mixture was stirred at room temperature for about 2 h, it was diluted with water and ethyl acetate, then treated with 1 M aqueous sodium hydroxide. The aqueous layer was washed with ethyl acetate, and

- 54 047572 The combined organic layers were extracted three times with 1 M aqueous sodium hydroxide solution until LC-MS analysis showed that C8 was completely removed from the organic layer. Acidification of the combined aqueous layers to pH 2 was accomplished by adding concentrated hydrochloric acid, after which the mixture was extracted three times with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride solution, 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%. LC-MS m/z 355.3 [M+H]+.

1H NMR (400 MHz, methanol-b ₄ ) 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 ABX system, J = 10.4, 5.3 Hz, 1H), 2.03-1.91 (m, 1H), 1.57 (dd, component of 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 (1K^^)-6,6-dimethyl-3-C-valyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, hydrochloride salt (C9).

To a solution of C8 (82.8 g, 234 mmol) in dichloromethane (230 mL) was added a solution of hydrogen chloride in 1,4-dioxane (4.0 M; 409 mL, 1.64 mol). The reaction mixture was stirred overnight at room temperature and then concentrated in vacuo to give C9 as a white foam. This material was used directly in the next step. LC-MS m/z 255.3 [M+H]+.

¹ H NMR (400 MHz, methanol-04) 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 (s, 3H).

Step 3. Synthesis of (1K^^)-6,6-dimethyl-3-(^-(trifluoroacetyl)-C-valyl]-3-azabicyclo[3.1.0]hexane2-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, after which ethyl trifluoroacetate (36.1 mL, 303 mmol) was slowly added. After the reaction mixture was allowed to stir at room temperature for 90 min, it was concentrated in vacuo. The residue was diluted with water, 1 M aqueous sodium hydroxide solution, and ethyl acetate, and the resulting organic layer was extracted twice with 1 M aqueous sodium hydroxide solution. The combined aqueous layers were acidified to pH 2 by the addition of 1 M hydrochloric acid, then extracted three times with ethyl acetate. The combined organic layers were washed with water and brine, 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. LC-MS m/z 351.3 [M+H]+.

1H NMR (400 MHz, DMSO-a ₆ ) δ 12.65 (very br s, 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.

(1K^^)-^{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[N(trifluoroacetyl)^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (1)

- 55 047572

Step 1. Synthesis of methyl 3-[(3-)-2-oxo11-pyrrolidin-3-yl]-1-alaninate methanesulfonate salt (C10).

Methanesulfonic acid (2.30 mL, 35.4 mmol) was added to a solution of methyl N-(tert-butoxycarbonyl)-3-[N-)-2-oxopyrrolidin-3-yl]N-alaninate (10.1 g, 35.3 mmol) in 1,1,1,3,3,3-hexafluoropropan-2-ol (70 mL). After the reaction mixture was stirred at room temperature for 70 min, LC-MS analysis showed that the starting material was converted to C10: LC-MS m/z 187.2 [M+N]+. The reaction mixture was concentrated in vacuo and the residue was redissolved twice, followed by concentration under reduced pressure, in a mixture of acetonitrile and ethyl acetate (1:1, 2 x 20 mL). The resulting material was loaded into a mixture of acetonitrile and ethyl acetate (1:1, 30 mL), concentrated, then redissolved twice in ethyl acetate (2 x 40 mL) and concentrated. The residue was triturated with ethyl acetate (60 mL) to give C10. Yield: 9.87 g, 35.0 mmol, 99%.

1I NMR (400 MHz, methanol-a ₄ ) δ 4.22 (dd, J = 9.7, 3.6 Hz, 1H), 3.86 (s, 3I), 3.41-3.36 (m, 2I), 2.84-2.74 (m, 1H), 2.70 (s, 3I), 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 ^({(^^^)-6,6-dimethyl-3-^-(trifluoroacetyl)-C-valyl]-3azabicyclo[3.1.0]hexan-2-yl}carbonyl)-3-[^)-2-oxopyrrolidin-3-yl]^-alaninate (C11)

To a solution of C10 (2.76 g, 9.78 mmol) and C4 (3.43 g, 9.79 mmol) in acetonitrile (40 mL) at 0 °C was added 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1.88 g, 9.81 mmol), followed by dropwise addition of pyridine (2.37 mL, 29.3 mmol). The reaction mixture was stirred at 0 °C for 2.25 h, after which it was 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 solution (50 ml), saturated aqueous sodium bicarbonate solution (50 ml), and saturated aqueous sodium chloride solution (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, by 1H NMR analysis, contained significant residual methyl tert-butyl ether. Some of this material was used in the next step. Yield: 3.74 g; corrected for residual methyl tert-butyl ether: 2.94 g, 5.67 mmol, 58%. LC-MS m/z 519.5 [M+H] ⁺ .

'i NMR (400 MHz, methanol^) 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 the solvent gel 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, component ABX ₂ systems, J = 7.7, 3.1 Hz, 1H), 1.49 (d, half of the 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 (^^^)-N{^)-1-amino-1-oxo-3-[^)-2-oxopyrrolidin-3-yl]propan-2-yl}6,6-dimethyl-3-^-(trifluoroacetyl)^-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 Cl1 (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, after which a solution of ammonia in methanol (7.0 M; 5 mL, 40 mmol) was again added 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 reaction, it was concentrated in vacuo. The residue was diluted and again concentrated successively with ethyl acetate (2 x 20 mL) and with a mixture of ethyl acetate and heptane (1:1, 2 x 20 mL). The resulting material 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%. LC-MS m/z 504.5 [M+H] ⁺ .

1I NMR (400 MHz, methanol-O ₄ ) δ 8.68 (d, J = 7.9 Hz, <1I, incomplete exchange with solvent), 4.44 (ddd, J = 11.9, 7.9, 4.0 Hz, 1H), 4.37-4.26 (m, 2I), 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, 2I, assumed; largely obscured by the solvent peak), 2.72-2.62 (m, 1H), 2.38-2.28 (m, 1H), 2.21-2.08 (m, 2I), 1.90-1.72 (m,

- 56 047572

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 (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3[N-(trifluoroacetyl)-P-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 Cl2 (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 added again; 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 concentrated again before being purified using silica gel chromatography (Gradient: 0% to 5% methanol in ethyl acetate). (1R,28,58)-N-{(18)-1-Cyano-2[(2)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-(2-(trifluoroacetyl)-P-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide (1) was isolated as a solid. Yield: 35 mg, 72 μmol, 42%. LC-MS m/z 486.5 [M+H]+.

^1H NMR (400 MHz, methanol^) 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, assumed; largely 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-N ^-2- (pyrrolidin-1-ylacetyl)-Sleucinamide, trifluoroacetate salt (2)

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

A suspension of 4-methyl-D-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, after which 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%. LC-MS m/z 236.3 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-4) 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, Δν _AB = 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).

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

At 0°C, a 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)-NH4N,N-tetramethylaronium hexafluorophosphate (HATU; 746 mg, 1.96 mmol), followed by the addition of a solution of 4-methylmorpholine (0.496 ml, 4.51 mmol) in dichloromethane (1 ml). After the reaction mixture was 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 x 60 ml) and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Purification using silica gel chromatography was performed twice (Gradient: 0% to

- 57 047572

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 material was used directly in the next step. LC-MS m/z 347.4 [M+H]+.

¹ H NMR (400 MHz, methanol-^) δ 7.40-7.29 (m, 5H), 5.16 (AB quartet, Jab = 12.2 Hz, Δαβ = 11.1 Hz, 2H), 4.56 (dd, J = 9.0, 3.1 Hz, 1H), 3.76 (AB quartet, Jab = 15.6 Hz, \vAb = 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)-N-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, after which LC-MS analysis showed conversion to C15: LC-MS 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 x 20 mL), followed by concentration under reduced pressure; this gave C15 as a solid (646 mg). Portions of this material were used in subsequent chemical reactions without further purification.

¹ H NMR (400 MHz, DMSO-d..i δ 8.46 (d, J = 8.3 Hz, 1H), 4.31 (dd, J = 8.9, 8.6, 3.0 Hz , 1H), 3.74-3.60 (m, 2H), 3.00 (w s, 4H), 1.90-1.79 (m, 4H), 1.70 (dd, ABX system component , J = 14.3, 3.0 Hz, 1H), 1.56 (dd, component of the ABX system, J = 14.3, 9.2 Hz, 1H), 0.90 (s, 9H).

Step 4. Synthesis of N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-^-(pyrrolidin-1ylacetyl)^-leucinamide, trifluoroacetate salt (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 H2-dimethylformamide (1 mL) was cooled to 0 °C and treated with O-(7-azabenzotriazol-1-yl)-H2N2,N2-tetramethylaronium hexafluorophosphate (HATU, 97%; 39.9 mg, 0.102 mmol), followed by the addition of a solution of 4-methylmorpholine (28.0 μL, 0.255 mmol) in dichloromethane (0.25 mL). After the reaction mixture was stirred at 0 °C for approximately 1.5 h, it was diluted with saturated aqueous sodium bicarbonate (3 mL) at 0 °C and extracted with dichloromethane (4 x 4 mL). The combined organic layers were concentrated in vacuo and purified using reverse phase HPLC (column: Waters Sunfire C18, 19x100 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 25% B over 8.5 min, then 25% to 95% acetonitrile over 0.5 min, then 95% B over 1.0 min; flow rate: 25 mL/min) to give N-{(1S)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-^-(pyrrolidin-1 ylacetyl)^-leucinamide, trifluoroacetate salt (2) as a resin. Yield: 8.1 mg, 16 μmol, 18% over 3 steps. LC-MS m/z 392.6 [M+H]+. Retention time: 1.47 minutes (Analytical conditions. Column: Waters Atlantis C18, 4.6x50 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 over 1.0 minute. Flow rate: 2 mL/min).

Example 3.

№{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-^-(2,6-dichlorobenzoyl)-4-methyl^leucinamide (3) go %G ^CH 3 'OH

-CH ₃

- 58 047572

Step 1. Synthesis of 3-[^)-2-oxopyrrolidin-3-yl]^-alanine amide, methanesulfonate salt, (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, after which it was concentrated in vacuo, resuspended in a mixture of toluene and heptane, and concentrated more than once to give a hygroscopic glass-like material (7.47 g). A portion of this material (6.47 g) was diluted and reconcentrated successively with a mixture of dichloromethane and ethanol (2:3, 2 x 50 mL); ethyl acetate and ethanol (2:3, 50 mL); ethyl acetate, heptane, and dichloromethane (4:4:1, 2 x 50 mL). The resulting material was dissolved in a mixture of acetonitrile and water (1:1, 22 mL) and lyophilized for 2 days to give C16 as a glass-like substance. Yield: 3.23 g, 12.1 mmol, 73% over 2 steps. LC-MS m/z 172.2 [M+H]+.

1H NMR (400 MHz, methanol-O,) 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).

At 0°C, a solution of C16 (1.34 g, 5.02 mmol) and N-(tert-butoxycarbonyl)-4-methyl-N-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-tetramethylaronium hexafluorophosphate (HATU, 97%; 2.04 g, 5.20 mmol), followed by the addition of a solution of 4-methylmorpholine (1.43 ml, 13.0 mmol) in dichloromethane (3 ml). After the reaction mixture was stirred at 0 °C for 2.25 h, it was quenched at 0 °C by 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 using 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-b ₄ ) δ 6.83 (d, J = 7.4 Hz, <1H, incomplete exchange with 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 the 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, ABX system component, J = 14.4, 3.2 Hz, 1H), 1.51 (dd, component of the ABX system, J = 14.4, 9.3 Hz, 1H), 1.45 (s, 9H), 0.97 (s, 9H).

Step 3. Synthesis of 4-methyl-^-leucyl-3-[^)-2-oxopyrrolidin-3-yl]-^-alanine amide, methanesulfonate salt (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, after which it was concentrated in vacuo, dissolved in ethyl acetate, and concentrated more than once to give C18 as a solid (238 mg). Most of this material was used in the next step. LCMS m/z 299.4 [M+H]+.

1H NMR (400 MHz, methanol-ac 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-N-leucyl-3-[N-)-2-oxopyrrolidin-3-yl]-N-alanine amide (C19).

At 0°C, a 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 the 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

- 59 047572 or at room temperature for 1 h, after which it was diluted with dichloromethane (60 mL), then washed with hydrochloric acid (1 M; 30 mL) followed by the addition of a saturated aqueous solution of sodium bicarbonate (30 mL). The organic layer was dried over sodium sulfate, filtered, concentrated in vacuo, and chromatographed on silica gel (Gradient: 0% to 30% methanol in ethyl acetate) to give C19. Yield: 120 mg, 0.255 mmol, 52% over 2 steps. LCMS m/z 471.4 (dichloro isotope pattern observed) [M+H]+.

¹ H NMR (400 MHz, methanol-b ₄ ) δ 8.45 (d, J = 7.9 Hz, <1H, incomplete exchange with 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-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-^-(2,6-dichlorobenzoyl)-4-methyl-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). To this was added phosphorus oxychloride (0.100 mL, 1.07 mmol) and the reaction mixture was stirred at -30 °C to -20 °C. After 30 min, pyridine (2 mL) was added to aid stirring; after 1 h, dichloromethane (2 mL) was added for the same reason. After 2 h of reaction, phosphorus oxychloride (0.100 mL, 1.07 mmol) was again added and stirring was continued for 30 min at -30 °C, after which the reaction mixture was warmed to 0 °C and stirred for an additional 40 min. The mixture 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 subjected to silica gel chromatography (gradient: 0% to 15% methanol in ethyl acetate) to give a solid (67 mg). This material 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) and concentrated under reduced pressure. The residue was stirred with a mixture of ethyl acetate and heptane (1:3, 4 ml) at room temperature for 40 min and filtered; The filtered cake was washed with a mixture of ethyl acetate and heptane (1:3, 5x2 mL) to give N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-N-2-(2,6-dichlorobenzoyl)-4-methyl-N-leucinamide (3) as a solid. Combined yield: 70 mg, 0.15 mmol, 59%. LC-MS m/z 453.3 (dichloro isotope pattern observed) [M+H]+.

¹ H NMR (400 MHz, methanol-6 ₄ ) 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-[(3 S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl] 4-methoxy-III-indole-2-carboxamide (4)

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

- 60 047572 salts (C20).

A solution of methyl N-(tert-butoxycarbonyl)-N-leucyl-3-[(3 S)-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 hydrogen chloride 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, expected quantitative yield).

¹ H NMR (400 MHz, DMSO-de), 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-3yl]-L-alaninate (C21).

O-(7-Azabenzotriazol-1-yl)-N,N-tetramethylaronium hexafluorophosphate (HATU; 494 mg, 1.30 mmol) and N,N-diisopropylethylamine (388 mg, 3.00 mmol) were added at 0 °C to a solution of C2O (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 onto water/ice (10 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (eluent: 10:1 dichloromethane/methanol) gave C21 as a yellow oil. Yield: 380 mg, 0.804 mmol, 97%. LC-MS m/z 473.2 [M+H]+.

1H NMR (400 MHz, DMSO^) 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, component AB quartet, 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, after which it was concentrated in vacuo, diluted with water (4 mL), adjusted to pH 4 by addition of 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. Silica gel chromatography (eluent: 10:1:0.1 dichloromethane/methanol/acetic acid) gave C22 as a yellow solid. Yield: 1.76 g, 3.84 mmol, 100%. LC-MS 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)-2oxopyrrolidin-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). After the reaction mixture was stirred at room temperature for 2.5 h, it was 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. LC-MS m/z 459.2 [M+H]+.

1H NMR (400 MHz, methanol^ ₄ ) δ 7.27 (s, 1H), 7.14 (dd, ABX system component, J = 8, 8 Hz, 1H), 7.02 (d, AB quartet component, 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).

At 0°C, O-(7-azabenzotriazol-1-yl)-Y^{{\text{N}}}^{{\text{2}}},^{{\text{N}}}-tetramethylaronium hexafluorophosphate (HATU; 1.90 g, 5.00 mmol) and N,N-diisopropylethylamine (1.49 g, 11.5 mmol) were added to a solution of C22 (1.76 g, 3.84 mmol) and ammonium chloride (0.246 g, 4.60 mmol) in N,N-dimethylformamide (15 ml). After the reaction mixture was 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 an additional 30 min and then 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 using silica gel chromatography (eluent: 10:1 dichloromethane).

- 61 047572 tan/methanol) to give C23 as a yellow solid. Yield: 1.09 g, 2.38 mmol, 62%. LC-MS m/z 458.0 [M+H]+.

H NMR (400 MHz, DMSO-de) 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 (w s, 1H), 7.38-7.26 (m, 2H), 7.10 (dd, ABX system component, J = 8, 8 Hz, 1H), 7.06 (w s, 1H), 7.00 (d, AB quartet component, 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-oxopyrroludin-3-yl]ethyl}amino)-4-methyl-1oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (4).

At 0°C, 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) was added to a mixture of C23 (500 mg, 1.09 mmol) and H2SO4-diisopropylethylamine (565 mg, 4.37 mmol) in tetrahydrofuran (8 mL). After the reaction mixture was stirred at 50°C for 3 h, it was concentrated in vacuo, diluted with water (5 mL), and extracted with ethyl acetate (3 x 5 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo; Silica gel chromatography (eluent: 10:1 dichloromethane/methanol) followed by purification using reversed-phase HPLC (column: YMC-Actus Triart C18, 50x250 mm, 7 μm; mobile phase A: water containing 0.225% formic acid; mobile phase B: acetonitrile; gradient: 18% to 58% B; flow rate: 25 ml/min) gave N(4-methyl)-1-({(4-methyl)-1-cyano-2-[4-methyl)-2-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (4) as a yellow solid. Yield: 130 mg, 0.296 mmol, 27%. LC-MS m/z 440.2 [M+H]+.

1H NMR (400 MHz, DMSO-ds) 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, ABX system component, J = 8.8 Hz, 1H), 7.00 (d, ABquartet component, 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.

№[^)-1-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]4-methoxy-1 H-indole-2-carboxamide (4)

C21 C23

Step 1. Synthesis of N-[(4-methoxy-N-indol-2-yl)carbonyl]-N-leucyl-3-[N-)-2-oxopyrrolidin-3-yl]-N-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). After the reaction mixture was stirred at room temperature for 6 h, 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, after which a final workup was performed with a solution of ammonia in methanol (7.0 M, 7.0 mL, 49 mmol). The reaction mixture was stirred for over 1 day, at which time it was concentrated in vacuo. The residue was combined with the product of a similar reaction (350 mg from 512 mg isolated) carried out using C21 (500 mg, 1.06 mmol) and the mixture was redissolved in ethyl acetate (5 x 10 mL) and concentrated under reduced pressure to give C23 (835 mg). This material was used directly in the next step. LCMS m/z 458.4 [M+H]+.

¹ H NMR (400 MHz, methanol- ₄ ) δ 7.29 (d, J = 0.9 Hz, 1H), 7.15 (dd, ABX system component, J = 8, 8 Hz, 1H), 7.03 (br d, AB quartet component, 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 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).

- 62 047572

Step 2. Synthesis of N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1oxopentan-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, after which phosphorus oxychloride (0.956 ml, 10.2 mmol) was added dropwise over 5 min. The reaction was stirred at -30 °C to -20 °C for approximately 1.5 h, then treated with hydrochloric acid (1 M; 50 ml) and stirred for 1 h. After extraction with dichloromethane (3 x 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 subjected to silica gel chromatography (Gradient: 0% to 5% methanol in ethyl acetate) to give 4 as a solid (800 mg). This material was combined with the product (80 mg) from a similar reaction carried out using C23 (161 mg, 0.352 mmol); the resulting material was stirred in diethyl ether (25 mL) for 3 days and then filtered. The filtered cake was washed with a mixture of diethyl ether and heptane (1:1, 4 x 2 mL) to give N([DO)-1-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3yl]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-66) 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, ABX system component, J = 8, 8 Hz, 1H), 7.00 (d, AB quartet component, 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-[(3 S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl] 4-methoxy-3-(trifluoromethyl)-III-indole-2-carboxamide (5) and

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

To a pressure release vessel containing zinc(II) trifluoromethanesulfinate (98%, 2.44 mg, 7.21 μmol) were 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 hydroperoxide (70% in water; 1.25 μL, 9.03 μmol). The vessel was capped and heated to 50 °C overnight, after which the reaction mixture was cooled and diluted with acetonitrile and 1% formic acid in water to a volume of approximately 2-3 mL. The final solvent composition was such that the resulting mixture was clear, typically approximately 20% to 30% acetonitrile. The entire mixture was subjected to reverse phase HPLC (column: Phenomenex Luna C18, 10 x 250 mm, 10 μm; mobile phase A: 0.5% acetic acid in water; mobile phase B: 9:1 acetonitrile/methanol; gradient: 15% B over 5 min, then 15% to 70% B linear gradient over 84 min, then 70% to 95% B over 1 min, then 95% B over 9 min; flow rate: 2 mL/min). The eluate was passed through a UV/VIS detector and then split approximately 15:1 between the fraction collector and the ion trap of the mass spectrometer. Fractions were collected every 20 s and those potentially containing products of interest were assessed using UHPLC-HRMS-UFMS before pooling. Two products eluted at approximately 71 and 75 minutes. The first eluting product was 5{Na[^)-1-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-3-(trifluoromethyl)-Na-indole-2-carboxamide} and the second eluting product was 6{Na[^)-1-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-7-(trifluoromethyl)-Na-indole-2-carboxamide}.

- Yield: 0.101 mg, 0.199 µmol, 11%. High Resolution MS m/z 508.2171 [M+H]+; calculated for C ₂ 4H ₂₉ F ₃ N ₅ O ₄ , 508.2172.

¹ H NMR (600 MHz, DMSO-ds) δ 12.22 (br s, 1H), 9.01 (d, J = 7.6 Hz, 1H), 8.96 (d, J = 7.9 Hz, 1H), 7.73

- 63 047572 (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), 4.494.40 (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, 2.1x100 mm, 2.6 µm; mobile phase A: water containing 0.1% formic acid; mobile phase B: acetonitrile; gradient: 5% B over 0.5 minutes, then from 5% to 70% B over 10.5 minutes, then from 70% to 95% B over 2 minutes; flow rate: 0.4 mL/min).

- Yield: 14.7 µg, 0.029 µmol, 1.6%. High Resolution MS m/z 508.2178 [M+H]+; calculated for C ₂₄ H ₂₉ F ₃ N ₅ O ₄ , 508.2172.

^1H NMR (600 MHz, DMSO- _a6 ) δ 11.47 (w s, 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 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)-III-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 the addition of trifluoroacetic acid (56 μL, 0.727 mmol). After the reaction mixture was cooled to 0 °C, tert-butyl hydroperoxide (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, after which it was cooled to room temperature and treated dropwise with aqueous sodium bicarbonate until bubbling ceased. After the resulting mixture was partitioned between aqueous sodium bicarbonate and ethyl acetate, the aqueous layer was extracted once with ethyl

- 64 047572 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, while the pH was adjusted to 1 with 1 M hydrochloric acid. After the biphasic mixture was stirred for 10 min, the organic layer was washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. LC-MS analysis revealed that the residue (115 mg) contained a mixture of starting material and monotrifluoromethylated products, as well as a small amount of ditrifluoromethylated material. The bulk of this mixture was used in Step 4. Yield: 115 mg, <0.4 mmol. LC-MS m/z 189.8, 257.8, 325.8 (less) [M-H] ^- .

1H NMR (400 MHz, methanol^ ₄ ), characteristic peaks from three main components: δ 7.07 (br d, J = 8.4 Hz), 7.02 (br d, 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-alanine amide (C25).

At 0°C, a solution of ammonia in methanol (7 M; 43 mL, 300 mmol) was added to 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; 1.5 g, 3.8 mmol) in methanol (5 mL). After the reaction vessel was capped, 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 allowed to continue at room temperature for 3 days, after which it was 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 (w s, 1H), 5.64 (w s, 1H), 5.32 (w s, 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, assumed; partially obscured by water peak), 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-alanine amide, methanesulfonate salt (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, LC-MS analysis showed conversion to C26: LC-MS m/z 285.3 [M+H]+. The reaction mixture was concentrated in vacuo, then loaded into the following solvent mixture and concentrated again: a mixture of acetonitrile and ethyl acetate (1:1, 2x20 mL), then a mixture of ethyl acetate and heptane (1:1, 2x20 mL). The resulting solid was azeotropically distilled twice with acetonitrile/ethyl acetate, then twice with ethyl acetate/heptane to give C26 as a white solid (6.05 g), retaining solvents by 1H NMR analysis. Yield: expected quantitative.

1H NMR (600 MHz, methanol^) 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)-2oxopyrrolidin-3-yl]-L-alaninamide (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 NH-dimethylformamide (1 mL) was cooled to 0 °C and treated with O-(7-azabenzotriazol-1-yl)-H,H,H',H'-tetramethylaronium hexafluorophosphate (HATU; 163 mg, 0.429 mmol) followed by the addition of 4-methylmorpholine (0.129 mL, 1.17 mmol). The reaction mixture was stirred at 0 °C for 40 min, after which 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 accomplished twice (Gradient #1: 0% to 10% methanol in dichloromethane; Gradient #2: 5% to 10% methanol in dichloromethane) to afford C27. The regiochemistry of this material was confirmed by 2D NMR experiments. Yield: 19 mg, 36 µmol, ca. 10%. LCMS m/z 526.5 [M+H]+.

1H NMR (400 MHz, meganol-f) δ 7.53 (br d, 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

- 65 047572 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). After the reaction mixture was stirred at room temperature for 1 h, it was 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 packed with magnesium sulfate. Concentration of the filtrate in vacuo gave a residue which was purified using reversed-phase HPLC (column: 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: 25% to 65% B over 8.5 min, then 65% to 95% B over 0.5 min, then 95% B over 1.0 min; flow rate: 25 mL/min) to give N-[(2S)-1-({(1S)-1-cyano-2-[(3 S)-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%. LC-MS m/z 508.6 [M+H]+. Retention time: 2.83 min (column: Waters Atlantis C18, 4.6x50 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 min, then 95% B for 1.0 min; flow rate: 2 mL/min).

Example 7.

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

HCI;

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

A solution of C17 (560 mg, 1.41 mmol) and N-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%. LC-MS m/z 381.4 [M+H]+.

¹ H NMR (400 MHz, methanol^) 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[^)-1-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-3-methylimidazo[2,1-b][1,3]thiazole-2-carboxamide (7).

A solution of hydrogen chloride 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 and then treated with 4-methylmorpholine (0.144 mL, 1.31 mmol). After the solvents were removed in vacuo, the residue was resuspended twice in a mixture of dichloromethane and heptane (1:1, 2 x 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-dimethylformamide (3.3 mL), cooled to 0°C, and treated with O-(7-azabenzotriazol-1-yl)-HNH,N-tetramethylaronium hexafluorophosphate (HATU; 99.9 mg, 0.263 mmol) followed by addition of

- 66 047572 by dilution with a solution of 4-methylmorpholine (72 µl, 0.655 mmol) in dichloromethane (0.2 ml). After the reaction mixture had been stirred at 0 °C for approximately 2 h, it was treated at 0 °C with hydrochloric acid (1 M; 30 ml) and the resulting mixture was extracted with dichloromethane (2 x 60 ml). The aqueous layer was then basified to pH 9 by addition of saturated aqueous sodium bicarbonate solution, after which it was 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 material revealed the presence of a smaller epimer, probably resulting 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 (^[^)-1-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3yl]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%. LC-MS m/z 445.4 [M+H]+.

‘H NMR (400 MHz, methanol^) 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; assumed; 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-[^)-2-oxopyrrolidin-3-yl]ethyl}-M ² -[cyclohexyl(methoxy)acetyl]-4-methyl-Sleucinamide, DIAST-1 (8) and

N-(1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N ² -[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 hydrogen chloride in 1,4-dioxane (4 M; 0.4 ml, 1.6 mmol). The reaction mixture was stirred at room temperature for 30 min, after which 4-methylmorpholine (0.165 ml, 1.50 mmol) was added, which adjusted the pH to 7-8. After the solvents were removed in vacuo, the residue was twice loaded with a mixture of ethyl acetate and heptane (1:1, 2 x 10 ml) and concentrated under reduced pressure to give C29 as a solid (269 mg); by 1H NMR analysis, consisting of a mixture of epimers, expected to be present in the nitrile-bearing center in a ratio of 2-3 to 1. A portion of this material was used in the next step. LC-MS m/z 281.3 [M+H]+.

1H NMR (400 MHz, methanol-b ₄ ), 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}-N ² [cyclohexyl(methoxy)acetyl]-4-methyl-N-leucinamide, DIAST-1 (8) and N-{1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-N ² -[cyclohexyl(methoxy)acetyl]-4-methyl-N-leucinamide, DIAST-2 (9).

At 0 °C, O-(7-azabenzotriazol-1-yl)-N-N-,N-,N-tetramethylaronium hexafluorophosphate (HATU; 38.0 mg, 0.100 mmol) was added to a 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), followed by the addition of a solution of 4-methylmorpholine (30.8 μL, 0.280 mmol) in dichloromethane (0.2 mL). After the reaction mixture was stirred at 0 °C for approximately 2 h, it was diluted with saturated aqueous sodium bicarbonate (3 mL) at 0 °C and extracted with dichloromethane (4 x 4 mL). The combined organic layers were concentrated in vacuo; LCMS analysis showed that the residue consisted of two components, presumably corresponding to the two epimers in the nitrile-bearing center. These diastereomers were separated using reverse-phase HPLC (column: Waters XBridge C18, 19x100 mm, 5 μm; mobile phase A: water; mobile phase B: acetonitrile; grade

- 67 047572 ent: from 5% to 95% B over 8.54 min, then 95% B over 1.46 min; flow rate: 25 mL/min). The first eluting diastereomer was designated 8 (N-(1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-^-[cyclohexyl(methoxy)acetyl]-4-methyl-D-leucinamide, DIAST-1), and the second eluting diastereomer as 9 (N-(1-cyano-2-[D)-2-oxopyrrolidin-3-yl]ethyl}-^-[cyclohexyl(methoxy)acetyl]-4-methyl-D-leucinamide, DIAST-2).

- Yield: 12.8 mg, 29.4 μmol, 32% over 2 steps. LC-MS m/z 435.6 [M+H]+. Retention time: 2.63 minutes (Analytical conditions. Column: Waters Atlantis C18, 4.6x50 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/min).

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

Example 10.

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

Step 1. Synthesis of Na[(4-methoxy-1H-indol-2-yl)carbonyl]-4-methyl-C-leucyl-3-[^)-2oxopyrrolidin-3-yl]-L-alaninamide (C30).

At 0°C, O-(7-azabenzotriazol-1-yl)-H2SO4,Na,Na-tetramethylaronium hexafluorophosphate (HATU; 175 mg, 0.460 mmol) was added to a solution of C18 (200 mg, <0.46 mmol) and 4-methoxy-Ni-indole-2-carboxylic acid (88.2 mg, 0.460 mmol) in acetonitrile (2 ml), followed by the addition of 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, after which it was diluted with saturated aqueous sodium bicarbonate (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). After the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo, the residue was purified using silica gel chromatography (Gradient: 0% to 30% methanol in ethyl acetate) to afford C30 as a solid. Yield: 148 mg, 0.314 mmol, 68% over 2 steps. LCMS m/z 472.4 [M+H] ⁺ .

¹ H NMR (400 MHz, methanol- ₄ ) δ 7.25 (d, J = 0.9 Hz, 1H), 7.15 (dd, J = 8.8 Hz, 1H), 7.03 (br d, 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[^)-1-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-N-indole-2-carboxamide (10).

A solution of C30 (143 mg, 0.303 mmol) and N-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, after which 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. Chromatography on silica gel (Gradient: 0% to 10% methanol in ethyl acetate) gave N[^)-1-({(^)-1-cyano2-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2

- 68 047572 carboxamide (10) as a solid. Yield: 68 mg, 0.15 mmol, 50%. LC-MS m/z 454.5 [M+H] ⁺ .

¹ H NMR (400 MHz, methanol^) δ 7.24 (d, J = 0.9 Hz, 1H), 7.14 (dd, J = 8.8 Hz, 1H), 7.02 (br d, 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-1^pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2oxopyrrolidin-3-yl]ethyl}-4-methyl-leucinamide (11)

C18 (43.4 mg, <0.10 mmol) and 4-bromo-1-ethyl-3-methyl-1H-pyrazole-5-

At 0 °C, O-(7-azabenzotriazol-1-yl)-HN-,N-tetramethylaronium hexafluorophosphate (HATU; 38.0 mg, 0.100 mmol) was added to a suspension of the carboxylic acid (23.3 mg, 0.100 mmol) in acetonitrile (1.0 mL), followed by the addition of a solution of 4-methylmorpholine (30 μL, 0.27 mmol) in acetonitrile (0.2 mL). After the reaction mixture was 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 added again and the reaction was allowed to proceed for 1.5 h, after which it was treated with saturated aqueous sodium bicarbonate (3 ml) at 0 °C and extracted with dichloromethane (2 x 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 material (80 mg) was purified using reversed-phase HPLC (column: 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: 5% to 95% B over 8.54 min, then 95% B over 1.46 min; flow rate: 25 ml/min) to give N ² -[(4-6-pom-1-ethyl-3-methyl-N-pyrazol-5-yl)carbonyl]-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4methyl-N-leucinamide (11). Yield: 24.7 mg, 49.8 μmol, 86% over 2 steps. LC-MS m/z 495.5 (bromine isotope pattern observed) [M+H]+. Retention time: 2.48 minutes (Analytical conditions. Column: Waters Atlantis C18, 4.6 x 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/min).

Example 12.

N{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-M2-[(3,3-difluorocyclobutyl)acetyl]-4methyl^-leucinamide (12)

Step 1. Synthesis of 4-methyl-G-leucyl-3-[^)-2-oxopyrrolidin-3-yl]-G-alanine amide, hydrochloride

- 69 047572 salt (C18, HCl salt).

A solution of hydrogen chloride 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, concentrated in vacuo, redissolved in dichloromethane:heptane (1:1, 3 x 10 mL) and concentrated again to give C18, HCl salt (242 mg) as a glass. Some of this material was used in the next step. LC-MS m/z 299.3 [M+H]+.

1H NMR (400 MHz, methanol^) 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-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-^-[(3,3-difluorocyclobutyl)acetyl]-4-methyl-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). After the reaction mixture had been stirred at room temperature overnight, it was heated at 50°C for 4.5 hours, after which 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. After the reaction mixture had been stirred at 50°C for an additional 3 days, it was treated with saturated aqueous sodium bicarbonate (3 ml) and extracted with dichloromethane (4 x 4 ml). The combined organic layers were concentrated in vacuo and purified using reverse-phase HPLC (column: Waters XBridge C18, 19x100 mm, 5 μm; mobile phase A: water; mobile phase B: acetonitrile; gradient: 20% to 40% B over 8.5 min, then 40% to 95% B over 0.5 min, then 95% B over 1.0 min; flow rate: 25 mL/min) to give N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-^-[(3,3-difluorocyclobutyl)acetyl]-4-methyl-4-leucinamide (12). Yield: 10.1 mg, 24.5 μmol, 24% over 2 steps. LC-MS m/z 413.5 [M+H]+. Retention time: 1.96 minutes (Analytical conditions. Column: Waters Atlantis C18, 4.6x50 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/min).

Example 13.

(^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (13)

C31

- 70 047572

Step 1. Synthesis of methyl (^^^)-3-[(tert-butoxycarbonyl)-3-methyl-(d-valyl)-6,6-dimethyl-3azabicyclo[3.1.0]hexane-2-carboxylate (C31).

O-(7-Azabenzotriazol-1-yl)-NH4Na,Na-tetramethylaronium hexafluorophosphate (HATU; 7.92 g, 20.8 mmol) was added at 0 °C to a mixture of N-(tert-butoxycarbonyl)-3-methyl-N-valine (4.38 g, 18.9 mmol) and methyl (Na-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, hydrochloride salt (3.9 g, 19 mmol) in N-dimethylformamide (95 mL). After the reaction mixture was stirred for 5 min, N-diisopropylethylamine (8.25 mL, 47.4 mmol) was added; Stirring was continued at 0°C for 2 h, after which 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%.

¹ H NMR (400 MHz, methanol^ ₄ ) δ 6.42 (d, J = 9.7 Hz, <1H; incomplete exchange with solvent), 4.35 (s, 1H), 4.21 (d, J = 9.7 Hz, 1H), 4.02 (d, half of the AB quartet, J = 10.4 Hz, 1H), 3.91 (dd, component of the ABX system, J = 10.3, 5.3 Hz, 1H), 3.73 (s, 3H), 1.57 (dd, component of the ABX system, J = 7.5, 5.1 Hz, 1H), 1.47 (d, half of the 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 (^^^)-3-[(tert-butoxycarbonyl)-3-methyl-(d-valyl)-6,6-dimethyl-3azabicyclo[3.1.0]hexane-2-carboxylic acid (C32).

An aqueous solution of lithium hydroxide (1.0 M, 14.7 mmol, 14.7 mL) was added dropwise at 0 °C to a solution of C31 (3.60 g, 9.41 mmol) in a mixture of tetrahydrofuran and methanol (1:1, 30 mL). After stirring at 0 °C for 1 h, the reaction mixture was allowed to warm to room temperature and stirred for 1 h, after which LC-MS analysis showed conversion to C32: LC-MS m/z 367.3 [M-H] ^- . Adjustment to pH 3 was accomplished 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 afford C32 as an off-white solid. Yield: 3.10 g, 8.41 mmol, 89%.

1H NMR (400 MHz, methanol^ ₄ ) δ 6.39 (d, J = 9.7 Hz, approximately 0.5H; incomplete exchange with 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).

Step 3. Synthesis of tert-butyl {^)-1-[(^^^)-2-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3yl]ethyl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2yl}carbamate (C33).

At 0 °C, a 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'-tetramethylaronium hexafluorophosphate (HATU, 97%; 36.2 mg, 92.3 μmol) followed by the addition of a solution of 4-methylmorpholine (25 μL, 0.23 mmol) in acetonitrile (0.25 mL). After the reaction mixture was stirred at 0 °C for approximately 1 h, it was diluted with saturated aqueous sodium bicarbonate (3 mL) at 0 °C and extracted with dichloromethane (4 x 4 mL). The combined organic layers were concentrated in vacuo to give C33 as a gum (48 mg). Much of this material was used in the following

- 71 047572 stages. LC-MS m/z 504.6 [M+H]+.

Step 4. Synthesis of (1R.2S,5S)-N-j(1S)-1-iiuaiio-2-[(3S)-2-OKCOiiuppojuguii-3-uj]9Tu.i }-6,6-di\1ethyl-3 [3-methyl^-(trifluoroacetyl)-C-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-2ol (940 μL). To a solution of C33 (from the previous step; 47 mg, < 90 μmol) in 1,1,1,3,3,3-hexafluoropropan-2ol (1 mL) was added a portion of the stock solution of methanesulfonic acid (0.1 mL; 100 μmol). After the reaction mixture was stirred at room temperature for 1 h, it was concentrated in vacuo, then loaded into the following solvent mixture and concentrated again: a mixture of acetonitrile and ethyl acetate (1:1, 2 x 10 mL), and then a mixture of ethyl acetate and heptane (1:1, 2 x 10 mL). The residue was dissolved in dichloromethane (1 ml) and treated with 4-methylmorpholine (30.8 μl, 0.280 mmol), followed by the addition of trifluoroacetic anhydride (0.143 ml, 1.01 mmol). The reaction mixture was stirred at room temperature for 40 min, after which it was treated with 4-methylmorpholine (30.8 μl, 0.280 mmol), followed by the addition of trifluoroacetic anhydride (0.143 ml, 1.01 mmol); after 30 min, 4-methylmorpholine (30.8 μl, 0.280 mmol) was added again, followed by the addition of trifluoroacetic anhydride (0.143 ml, 1.01 mmol). After further 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% to 60% B over 8.5 min, then 60% to 95% B over 0.5 min, then 95% B over 1 min; flow rate: 25 ml/min) to give (1K,28,58)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (13). Yield: 7.5 mg, 15 μmol, 17% over 2 steps. LC-MS m/z 500.5 [M+H] ⁺ . Retention time: 2.66 minutes (Analytical conditions. Column: Waters Atlantis dC18, 4.6 x 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% to 95% B over 4.0 minutes, then 95% B for 1.0 minute; flow rate: 2 mL/min).

An alternative synthesis of Example 13, methyl tert-butyl ether solvate; Preparation 13, methyl tert-butyl ether solvate, solid form 2.

(^^^)^-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl^(trifluoroacetyl)-1-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate), solid form 2

^Ο χΝΗ _ш ^сн ₃ о Н ³ С^О^ Ν 'τ-Γθ'Ω Н ΓΊβΟ U Ν TT L/Пз ^Н О Н О [ А'%.СНз _ Н ₃ с сн ₃ ^НС1 о 9 ^Нз 11 J-СНз НЫ^О^СНз у ° н ₃ с ^л сн ₃ С32 °y-NH °y-NH _CH30 X> ns! _g K> ^MeOH Η ₂ Ν ^χ γ ^{ΝΗ2 н} O O * HCI C5 C16, HCI salt O _CH3 AJ^ch ₃ n ^CH3 niШ ³ _CH3 O _H3 C _>V ..-L _S X) LiOH ---------* ^H3C CH3 I P ----- HATU Q'VcH ₃ g ^CH3 _{X H3} CyN^CH3 _H3 C _CH3 CH3CH3 C31 NH _{2 o} n ₃ s _>ch ,A_o х HCI _H3 C\L Г o _H3 C^O^CF ₃ _____ ^CH3 /M _h.L ____________ V . ^0Н g ^СНз Н ₃ С СНз ^HCI H ₃ C^N^CH ₃ С41 ^СН з ^СН з

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Step 1. Synthesis of tert-butyl {(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2yl}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-[(D)-2-oxopyrrolidin-3-yl]-D alaninate (600 g, 2.10 mol) and the reaction mixture was stirred at 25 °C for 40 h. Concentration in vacuo and combining the 2 batches gave C5 as a yellow solid. Combined yield: 1.10 kg, 4.05 mol, 96%.

1H NMR (400 MHz, DMSOM ₆ ) 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, assumed; 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 salt (C16, HCl salt).

This experiment was performed in three parallel batches. At 0°C, a solution of hydrogen chloride in 1,4-dioxane (4 M; 2 L, 8 mol) was added to a solution of C5 (840 g, 3.10 mol) in dichloromethane (2.0 L). The reaction mixture was stirred at 25°C for 2 h and then concentrated in vacuo. Combination of the three batches gave the C16, 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, DMSOM ₆ ) 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 C16, the HCl salt, was triturated in 2-propanol for 1.5 h, collected by filtration, and washed with 2-propanol. The collected solid was dried overnight under high vacuum to obtain a sample for X-ray powder diffraction analysis. The X-ray powder diffraction pattern for this material is shown in Fig. 10; the characteristic peaks are given in Table Q.

Collection of powder X-ray diffraction data.

Powder X-ray diffraction analysis was performed using a Bruker AXS D4 Endeavor diffractometer equipped with a Cu radiation source. The divergence slit was set to 0.6 mm, while the secondary optics used interchangeable slits. The diffracted radiation was detected by a PSD-Lynx Eye detector. 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, and the samples were prepared by placing them in a silicone 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 detected by a LYNXEYE_EX detector with motorized slits. Both the primary and secondary slits were equipped with 2.5 Soller 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 in locked paired scans at the Cu K-alpha wavelength (average) from 3.0 to 40.0 degrees 2-theta in 0.02 degree increments using a scan rate of 0.5 seconds per step. Samples were prepared by placing them in a silicone low-background sample holder.

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Data were acquired on both instruments using Bruker DIFFRAC Plus software and analysed using EVA DIFFRAC plus software. The PXRD data file was not processed prior to peak searching. Using the peak searching algorithm in EVA software, peaks selected with a threshold of 1 were used to make a preliminary peak assignment. Manual adjustments were made to ensure reliability; the result of the automatic assignment was checked visually and peak positions were adjusted to the peak maximum. In general, peaks with a relative intensity > 3% were selected. Peaks that were unresolved or consistent with noise were not selected. The typical error associated with peak position from PXRD is stated in USP to be within +/- 0.2° 2-Theta (USP-941).

Table Q

Powder X-ray diffraction peaks for C16, HCl salt are selected.

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 And 39,44 7 28,39 14 39.75 1

Alternative synthesis of 3-[^)-2-oxopyrrolidin-3-yl]G-alanine amide, hydrochloride salt, C16 HCl salt.

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

_NH3 (13 equiv.)

_MgSO4 (1.0 equiv.)

MeOH (5 ml/g) 23 °C, 7 hours.

HCI (3 equiv.)

DMF (5 ml/g) 23 °C, 12 h.

48% C16, HCI SALT

Efficiency: 88.3%, Purity ..

Efficiency: 94.7%, Purity

Methyl (c)-2-amino-3-((c)-2-oxopyrrolidin-3-yl)propanoate 4-methylbenzenesulfonate (20 g, 55.8 mmol) and magnesium sulfate (6.7 g, 55.8 mmol) were added to a solution of ammonia in methanol (7.0 M, 100 mL, 725.4 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 remove excess ammonia. After this, the reaction mixture was filtered through a pad of Celite® and then concentrated in vacuo, and the resulting (c)-2-amino-3-((c)-2-oxopyrrolidin-3-yl)propanamide 4-methylbenzenesulfonate was used directly in the next step without further purification. A portion of (c)-2-amino-3-((c)-2-oxopyrrolidin-3-yl)propanamide 4-methylbenzenesulfonate (10 g, 25.9 mmol) and a solution of hydrogen chloride 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-[(3-C)-2-oxopyrrolidin-3-yl]-C-alanine amide, hydrochloride salt, C16 HCl salt (2.7 g, 12.4 mmol) as a tan solid (overall yield 48%).

Step 3. Synthesis of methyl (M^^)-3-[N-(tert-butoxycarbonyl)-3-methylM-valyl]-6,6-dimethyl-3azabicyclo[3.1.0]hexane-2-carboxylate (C31).

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This experiment was carried out in three parallel batches. At 0°C, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylaronium hexafluorophosphate (HATU; 481 g, 1.26 mol) was added dropwise to a solution of methyl (1R,2S,5S)6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate, hydrochloride salt (237 g, 1.15 mol) and N(tert-butoxycarbonyl)-3-methyl-E-valine (293 g, 1.27 mol) in a mixture of N,N-dimethylformamide (400 mL) and acetonitrile (3.6 L). The reaction mixture was then allowed to warm to 25 °C and stirred for 16 h, after which it was poured into a mixture of ice water (1 L) and hydrochloric acid (0.5 M; 1 L), pH ∼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 batches, C31 as a colorless oil. Combined yield: 1.17 kg, 3.06 mol, 89%. LC-MS m/z 383.3 [M+H]+.

1H NMR (400 MHz, chloroform-d) δ 5.10 (d, J = 10 Hz, 1H), 4.46 (s, 1H), 4.20 (d, J = 10.3 Hz, 1H), 3.98 (d, half of 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 (^^^)-3-[H-(tert-butoxycarbonyl)-3-methyl-E-valyl]-6,6-dimethyl-3azabicyclo[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) were added lithium hydroxide monohydrate (220 g, 5.24 mol) and water (500 mL). After the reaction mixture was stirred at 25 °C for 2 h, it was concentrated in vacuo to remove most of the tetrahydrofuran; the residue was then adjusted to pH 2 by the addition of 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 solution (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 material was used directly in the next step. LC-MS 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 (^^^)-6,6-dimethyl-3-(3-methyl-E-valyl)-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, hydrochloride salt (C41).

This experiment was performed in 2 parallel runs. A solution of hydrogen chloride 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 material was used directly in the next step.

1H NMR (400 MHz, methanol-d.!) 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 (^^^)-6,6-dimethyl-3-[3-methyl-H-(trifluoroacetyl)-E-valyl]-3azabicyclo[3.1.0]hexane-2-carboxylic acid (C42).

This experiment was carried out in three parallel batches. At 0 °C, triethylamine (1.64 L, 11.8 mol) was added to a solution of C41 (from the previous step; 600 g, < 1.75 mol) in methanol (2 L), followed by ethyl trifluoroacetate (699 g, 4.92 mol), and the reaction mixture was allowed to warm 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 to 4 by the addition of 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. The 3 batches were combined at this point, treated with petroleum ether: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^ ₄ ) δ 8.88 (d, J = 8.8 Hz, <1H; incomplete exchange), [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 (^^^)-N-{^)-1-amino-1-oxo-3-[^)-2-oxopyrrolidin-3-yl]propan-2-yl}6,6-dimethyl-3-[3-methyl-H-(trifluoroacetyl)-E-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (C43).

This experiment was carried out in 4 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.

- 75 047572 (2.5 L) and the mixture was cooled to 0°C. N,N-Diisopropylethylamine (638 mL, 3.66 mol) was then added, followed by the 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, after which 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 solution (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 four batches gave C43 as a white solid (2.3 kg). Combined yield: 2.1 kg (corrected for residual ethyl acetate), 4.1 mol, 84%. LC-MS m/z 518.3 [M+H]+.

¹ H NMR (400 MHz, DMSO^6) δ 9.41 (w d, J = 7.7 Hz, 1H), 8.30 (d, J = 8.8 Hz, 1H), 7.56 (s , 1H), 7.32 (w s, 1H), 7.04 (w s, 1H), 4.43 (w d, 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), 1.991.89 (m, 1H), 1.70-1.58 (m, 1H), 1.55-1.44 (m , 2H), 1.38 (d, half of the AB quartet, J = 7.6 Hz, 1H), 1.01 (s, 3H), 0.98 (s, 9H), 0.84 (s, 3H).

Step 8. Synthesis of (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3[3-methyl-N-(trifluoroacetyl)-n-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 performed 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). After the reaction mixture was 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 three 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). After the combined eluates were concentrated in vacuo, the residue was 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 (13,6-dimethyl)-{(13)-1-cyano-2-[13-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 material, designated solid form 2, is shown in Fig. 1; characteristic peaks are listed in Table A. Combined yield: 1.41 kg, 2.82 mol, 81%. LC-MS m/z 500.3 [M+H]+.

1H NMR (400 MHz, DMSO-b6) 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).

Acquisition of powder X-ray diffraction data

Powder X-ray diffraction analysis was performed using a Bruker AXS D8 Endeavor diffractometer equipped with a Cu radiation source (K-a average). The slit divergence was set to 15 mm constant radiation. Diffracted radiation was detected with a PSD-Lynx Eye detector, with the PSD detector opening set to 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 shield was set at a fixed distance of 1.5 mm. During collection, the samples were rotated at 15 fpm. Samples were prepared by placing them in a silicone low-background sample holder and rotating during collection. Data were collected using Bruker DIFFRAC Plus software and analyses were performed using EVA DIFFRAC Plus software. Using the peak finding algorithm in EVA software, peaks selected with a threshold of 1 were used to make a preliminary peak assignment. Manual adjustments were made to ensure reliability; the result of the automatic assignment was checked visually and peak positions were adjusted to the peak maximum. In general, peaks with a relative intensity > 3% were selected. Peaks that were not resolved or did not match the noise were not selected. A typical error associated with the peak position from

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PXRD, as specified in the USP, is within +/- 0.2° 2-Theta (USP-941).

Table A

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 (%) 7.1 78 22.7 9 10,5 8 22.9 10 from 15 23.1 5 I,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 AND 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 (1K^^)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-valyl]-3azabicyclo[3.1.0]hexane-2-carboxylic acid (C42).

A second alternative synthesis of Example 13, methyl tert-butyl ether solvate; Preparation 13, methyl tert-butyl ether solvate, solid form 2.

(1K^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N(trifluoroacetyl)^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate), solid form 2

C43 13, methyl tert-butyl ether solvate

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, after which methyl N-(triethylammoniosulfonyl)carbamate inner salt (Burgess reagent; 86.0 g, 361 mmol) was added again. After the reaction mixture had been stirred for 1 hour, it was 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

- 77,047,572 hours. Collection of the solid by filtration gave (1R,2S,5S)-N-{(1S)-1-pyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-C-valyl]-3azabicyclo[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 material, designated solid form 2, is shown in Fig. 2; characteristic peaks are listed in Table B. Yield: 338 g, 575 mmol, 72%. LC-MS m/z 500.3 [M+H]+.

'H NMR (400 MHz, DMSO-46) 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 AB 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), 2.462.34 (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 of 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 the 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 a second 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(°2theta) +/- 0.2° 2-Theta Relative intensity (%) Angle (°2theta) +/- 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 I,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.

(^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-C-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (13)

Ό'

13, methyl tert-butyl ether solvate

About sn ₃

H ₃ cAAcH ₃ heptane

Step 1. Synthesis of (^^^)^-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3[3-methyl-N(trifluoroacetyl)-C-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate).

At 0°C, a mixture of C42 (90.5 wt.%, 5.05 g, 12.5 mmol) and C16, HCl salt (98.9 wt.%, 3.12 g, 14.9

- 78 047572 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 in acetonitrile; 17 mL, 24.3 mmol) over approximately 10 min. 1-Methyl-III-imidazole (4.0 mL, 50.2 mmol) was then added slowly over approximately 15 min and the reaction mixture was allowed to stir at 0 °C for 3.5 h, after which it was warmed to 25 °C. 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% w/w 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 solid, the organic layer was washed twice with aqueous sodium bicarbonate (1.14 M; 25 mL, 28 mmol). After the organic layer was washed with aqueous sodium chloride solution (14%, 2 x 20 ml), it was 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 h, cooled to 25°C over 1 h, and kept at 25°C for 1.5 h. The solid was isolated by filtration, washed with methyl tert-butyl ether (2 ml/g), and dried in a vacuum oven overnight at 50 °C to give (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3yl]ethyl}-6,6-dimethyl-3-[3-methyl^-(trifluoroacetyl)^-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 bulk of this material was used in the next step. Yield: 3.71 g, 6.31 mmol, 50%.

1H NMR (400 MHz, DMSO-66) 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 of 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 (^^^)^-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3[3-methyl-N-(trifluoroacetyl)^-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 in vacuo to give (^^^)^-{(^)-1-cyano-2-[^)-oxopyrrolidin-3yl]ethyl}-6,6-dimethyl-3-[3-methyl-(trifluoroacetyl)-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (13) as a crystalline solid. A portion of this lot was used as seed crystals in 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.

(^^^)-N{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl^(trifluoroacetyl)^-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 (seed crystals, from the third alternative synthesis of Example 13, Step 2; 1.2 g, 2.4 mmol) was added; after 10 min, the seed crystals were 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 h, and the resulting mixture was stirred for 30 min. It was then cooled to 20°C at a rate of 0.1 degrees/minute, after which it was 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 in vacuo at 50°C to give (1R,2S,5S)-N

- 79 047572 {(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)jvalyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (13) as a white crystalline solid. The powder X-ray diffraction pattern for this material, designated solid form 1, is shown in Fig. 3; 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 the alternative synthesis of Example 13, methyl tert-butyl ether solvate, Step 8.

Table C

Powder X-ray diffraction peaks for 13, solid form 1 are selected

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 AND 24.7 8 9.8 10 19.7 7 25.3 7 And,4 10 19.9 14 27,0 3 I,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

Determination of the structure by X-ray structural analysis of the single crystal from 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 was evaporated; after 2.5 months, X-ray quality crystals were present. One of these was used for structural determination. An ORTEP diagram of the single crystal data is shown in Fig. 4. Mercury software was used to calculate a powder diffraction pattern from the solved crystal structure; comparison with the diffraction pattern from the recrystallization of Example 13; yielding solid Form 1, this material identified as solid Form 1 (see Fig. 5). The characteristic peaks for these calculated data are given in Table D.

Table D

Powder diffraction data for 13, solid form 1, calculated from the X-ray diffraction structure determination of a single crystal from . 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 And,4 16 22.5 7 31.9 4 I,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 β 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 ABOUT 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 structural analysis of a single crystal.

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

The structure was solved using internal phasing using SHELX software, valid in the P212121 space group of the orthorhombic system. The structure was then optimized using the least squares method in the full matrix approximation. All non-hydrogen atoms were found and optimized using anisotropic substitution parameters.

The hydrogen atoms located on nitrogen were determined from the Fourier difference map and refined by limited distances. The remaining hydrogen atoms were placed in the calculated positions and given

- 80 047572 to attack their carrier atoms. The final refinement included isotropic substitution parameters for all hydrogen atoms.

The analysis of the absolute structure using probabilistic methods (Hooft 2008) was performed using PLATON (Spek). The results show that the absolute structure is correctly assigned. The method calculates that the probability of such a structure being correct is 100%. The Hooft parameter is represented as -0.01 with esd (estimated standard deviation) (3), and the Parson parameter is represented as -0.01 with esd(2).

The final R-index was 3.3%. The final Fourier difference revealed neither a missing nor a shifted electron density.

The corresponding crystal, data collection and refinement are presented in Table E. Atomic coordinates, bond lengths, bond angles and substitution parameters are given 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.

Cryst. 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 refinement for example 13, solid form 1

Empirical formula C23H32F3N5O4 Mass by formula 499.53 Temperature 296(2)K Wavelength 1.54178 A Crystal system Orthorhombic Space group P212121 Parameters of the unit cell a = 9.6836(2) A a = 90° Ъ= 15.0522(4) A β = 96° c = 18.0272(5) A γ = 90° Volume 2627.64(11) A ³ Z 4

- 81 047572

Density (calculated) 1.263 mg/ ^m3 Absorption coefficient 0.862 mm ¹ F(000) 1056 Crystal size 0.300 x 0.280 x 0.260 mm ³ Theta range for data collection from 3.826 to 80.042° Index ranges -12<=L<=12, -18<=K=19, -22<=/<=23 Collected reflections 79731 Independent reflections 5628 [R _mt = 0.0294] Total to theta = 67.679° 99.3% Absorption correction Empirical Method of clarification Least squares in full-matrix approximation on r Data/restrictions/parameters 5628/9/358 Consistency criterion on g 1,040 Finite R indices \Ι>2σ(Ι)] Fl = 0.0326, wR2 = 0.0906 R indices (all data) Fl = 0.0346, wR2 = 0.0928 Absolute structure parameter -0.01(3) Extinction coefficient But Maximum differential peak and hole 0.112 and -0.121 e.A ³

- 82 047572

Table F

The atomic coordinates (x 10 ⁴ ) and equivalent isotropic substitution parameters (A ² x10 ³ ) for example 13, solid form 1. and (eq.) are defined as one third of the trace of the orthogonalized tensor U ^ij

X y Z and (equiv.)

F(l) 4585(8) 3891(5) 8183(8) 174(4) F(2) 2984(6) 4623(4) 8601(2) 135(2) F(3) 2988(7) 4449(5) 7471(2) 133(3) F(1A) 2622(7) 4494(8) 8158(16) 237(10) F(2A) 4140(20) 3994(5) 7406(4) 167(6) F(3A) 4404(15) 3963(8) 8488(5) 127(5) N(l) 5507(2) 5598(1) 8478(1) 54(1) N(2) 8733(1) 6526(1) 8104(1) 49(1) N(3) 7304(1) 6456(1) 6213(1) 44(1) N(4) 9229(2) 5659(2) 4815(1) 99(1) N(5) 2159(2) 6087(2) 5207(1) MP 0(1) 4297(2) 5900(1) 7426(1) 84(1) 0(2) 8176(2) 5753(1) 9126(1) 70(1) 0(3) 7711(2) 5377(1) 7059(1) 70(1) 0(4) 3393(2) 7171(1) 4635(1) 86(1) C(l) 3848(3) 4543(2) 8022(2) 93(1) C(2) 4597(2) 5424(2) 7941(1) 63(1) C(3) 6284(2) 6425(1) 8485(1) 51(1) C(4) 5739(3) 7084(2) 9082(1) 69(1) C(5) 6747(4) 7872(2) 9133(2) 98(1) C(6) 5652(4) 6670(2) 9851(1) 94(1) C(7) 4309(3) 7402(3) 8847(2) no(i) C(8) 7812(2) 6195(1) 8592(1) 50(1) C(9) 10204(2) 6319(2) 8211(1) 65(1) C(10) 10931(2) 6770(2) 7575(1) 68(1) C(ll) 10769(2) 7754(2) 7454(1) 70(1) C(12) 11879(3) 8175(2) 6970(2) 101(1) C(13) 10220(3) 8359(2) 8049(1) 85(1) C(14) 9842(2) 7109(1) 7047(1) 58(1) C(15) 8435(2) 6868(1) 7361(1) 45(1) C(16) 7781(2) 6149(1) 6870(1) 44(1) C(17) 6994(2) 5848(1) 5610(1) 47(1) C(18) 8256(2) 5732(2) 5157(1) 67(1) C(19) 5822(2) 6180(1) 5115(1) 47(1) C(20) 4454(2) 6159(1) 5519(1) 44(1) C(21) 3297(2) 6544(1) 5059(1) 56(1) C(22) 2355(2) 5356(2) 5718(2) 82(1) C(23) 3911(2) 5237(1) 5704(1) 63(1)

- 83 047572

Table G

Bond lengths [A] and angles [°] for example 13, solid form 1

F(l)-C(l) 1,248(7) F(2)-C(l) 1,342(5) F(3)-C(l) 1,305(4) F(1A)-C(1) 1,215(9) F(2A)-C(1) 1,414(8) F(3A)-C(1) 1,325(11) N(l)-C(2) 1,335(2) N(l)-C(3) 1,455(2) N(1)-H(1X) 0.906(18) N(2)-C(8) 1,348(2) N(2)-C(15) 1,4631(19) N(2)-C(9) 1,471(2) N(3)-C(16) 1,3527(19) N(3)-C(17) 1,452(2) N(3)-H(3X) 0.944(17) N(4)-C(18) 1,132(3) N(5)-C(21) 1,326(3) N(5)-C(22) 1,447(3) N(5)-H(5X) 0.91(2) O(l)-C(2) 1,209(2) O(2)-C(8) 1,222(2) O(3)-C(16) 1,214(2) O(4)-C(21) 1,218(2) C(l)-C(2) 1,518(4) C(3)-C(8) 1,531(2) C(3)-C(4) 1,554(3) C(3)-H(3) 0,9800 C(4)-C(7) 1,525(4) C(4)-C(6) 1,523(3) C(4)-C(5) 1,540(4) C(5)-H(5A) 0,9600 C(5)-H(5B) 0,9600 C(5)-H(5C) 0,9600 C(6)-H(6A) 0,9600 C(6)-H(6B) 0,9600

- 84 047572

C(6)-H(6C) 0,9600 C(7)-H(7A) 0,9600 C(7)-H(7B) 0,9600 C(7)-H(7C) 0,9600 C(9)-C(10) 1,506(3) C(9)-H(9A) 0,9700 C(9)-H(9B) 0,9700 C(10)-C(11) 1,505(3) C(10)-C(14) 1,510(3) C(10)-H(10) 0,9800 C(11)-C(13) 1,505(4) C(11)-C(14) 1,511(3) C(11)-C(12) 1,522(3) C(12)-H(12A) 0,9600 C(12)-H(12V) 0,9600 C(12)-H(12C) 0,9600 C(13)-H(13A) 0,9600 C(13)-H(13B) 0,9600 C(13)-H(13C) 0,9600 C(14)-C(15) 1,520(2) C(14)-H(14) 0,9800 C(15)-C(16) 1,535(2) C(15)-H(15) 0,9800 C(17)-C(18) 1,480(2) C(17)-C(19) 1,528(2) C(17)-H(17) 0,9800 C(19)-C(20) 1,512(2) C(19)-H(19A) 0,9700 C(19)-H(19B) 0,9700 C(20)-C(21) 1,510(2) C(20)-C(23) 1,521(2) C(20)-H(20) 0,9800 C(22)-C(23) 1,518(3) C(22)-H(22A) 0,9700 C(22)-H(22B) 0,9700

- 85 047572

C(23)-H(23A) 0,9700 C(23)-H(23B) 0,9700 C(2)-N(l)-C(3) 121,10(16) C(2)-N(1)-H(1X) 120.7(15) C(3)-N(1)-H(1X) 117.9(15) C(8)-N(2)-C(15) 126.62(13) C(8)-N(2)-C(9) 118.51(14) C(15)-N(2)-C(9) 112.66(14) C(16)-N(3)-C(17) 120.65(13) C(16)-N(3)-H(3X) 122.1(13) C(17)-N(3)-H(3X) 112.1(12) C(21)-N(5)-C(22) 114.42(16) C(21)-N(5)-H(5X) 126.1(19) C(22)-N(5)-H(5X) 119.3(19) F(l)-C(l)-F(3) 117.2(6) F(1A)-C(1)-F(3A) 103.3(11) F(l)-C(l)-F(2) 104.3(6) F(3)-C(l)-F(2) 101.7(4) F(1A)-C(1)-F(2A) 108.6(10) F(3A)-C(1)-F(2A) 91.8(7) F(1A)-C(1)-C(2) 122.6(7) F(l)-C(l)-C(2) 115.8(4) F(3)-C(l)-C(2) 109.1(3) F(3A)-C(1)-C(2) 116.3(6) F(2)-C(l)-C(2) 107.2(3) F(2A)-C(1)-C(2) 109.8(5) O(l)-C(2)-N(l) 126.8(2) O(l)-C(2)-C(l) 118.5(2) N(l)-C(2)-C(l) 114.65(19) N(l)-C(3)-C(8) 107.86(14) N(l)-C(3)-C(4) 112,12(15) C(8)-C(3)-C(4) 112.72(16) N(l)-C(3)-H(3) 108,0 C(8)-C(3)-H(3) 108,0

- 86 047572

C(4)-C(3)-H(3) 108,0 C(7)-C(4)-C(6) 109.3(2) C(7)-C(4)-C(5) 110.5(2) C(6)-C(4)-C(5) 107.2(2) C(7)-C(4)-C(3) 108.5(2) C(6)-C(4)-C(3) 112.81(19) C(5)-C(4)-C(3) 108.52(19) C(4)-C(5)-H(5A) 109.5 C(4)-C(5)-H(5B) 109.5 H(5A)-C(5)-H(5B) 109.5 C(4)-C(5)-H(5C) 109.5 H(5A)-C(5)-H(5C) 109.5 H(5B)-C(5)-H(5C) 109.5 C(4)-C(6)-H(6A) 109.5 C(4)-C(6)-H(6B) 109.5 H(6A)-C(6)-H(6B) 109.5 C(4)-C(6)-H(6C) 109.5 H(6A)-C(6)-H(6C) 109.5 H(6B)-C(6)-H(6C) 109.5 C(4)-C(7)-H(7A) 109.5 C(4)-C(7)-H(7B) 109.5 H(7A)-C(7)-H(7B) 109.5 C(4)-C(7)-H(7C) 109.5 H(7A)-C(7)-H(7C) 109.5 H(7B)-C(7)-H(7C) 109.5 O(2)-C(8)-N(2) 121.60(16) O(2)-C(8)-C(3) 120.04(16) N(2)-C(8)-C(3) 118.28(13) N(2)-C(9)-C(10) 104.93(15) N(2)-C(9)-H(9A) 110.8 C(10)-C(9)-H(9A) 110.8 N(2)-C(9)-H(9B) 110.8 C(10)-C(9)-H(9B) 110.8 H(9A)-C(9)-H(9B) 108.8 C(11)-C(10)-C(9) 120.3(2)

- 87 047572

C(ll)-C(10)-C(14) 60.18(13) C(9)-C(10)-C(14) 107.82(15) C(ll)-C(10)-H(10) 118,0 C(9)-C(10)-H(10) 118,0 C(14)-C(10)-H(10) 118,0 C(10)-C(ll)-C(13) 121.92(19) C(10)-C(ll)-C(14) 60.07(14) C(13)-C(ll)-C(14) 121.66(19) C(10)-C(ll)-C(12) 114.8(2) C(13)-C(ll)-C(12) 113.9(2) C(14)-C(ll)-C(12) 114.14(18) C(11)-C(12)-H(12A) 109.5 C(11)-C(12)-H(12B) 109.5 H(12A)-C(12)-H(12B) 109.5 C(11)-C(12)-H(12C) 109.5 H(12A)-C(12)-H(12C) 109.5 H(12B)-C(12)-H(12C) 109.5 C(11)-C(13)-H(13A) 109.5 C(11)-C(13)-H(13B) 109.5 H(13A)-C(13)-H(13B) 109.5 C(11)-C(13)-H(13C) 109.5 H(13A)-C(13)-H(13C) 109.5 H(13B)-C(13)-H(13C) 109.5 C(10)-C(14)-C(ll) 59.75(14) C(10)-C(14)-C(15) 108.04(15) C(ll)-C(14)-C(15) 120.32(16) C(10)-C(14)-H(14) 118,0 C(11)-C(14)-H(14) 118,0 C(15)-C(14)-H(14) 118,0 N(2)-C(15)-C(14) 104.39(13) N(2)-C(15)-C(16) 111,18(13) C(14)-C(15)-C(16) 108.87(13) N(2)-C(15)-H(15) 110.7 C(14)-C(15)-H(15) 110.7 C(16)-C(15)-H(15) 110.7

- 88 047572

O(3)-C(16)-N(3) 123.70(15) O(3)-C(16)-C(15) 122.40(14) N(3)-C(16)-C(15) 113.89(13) N(3)-C(17)-C(18) 108.50(13) N(3)-C(17)-C(19) 112.62(13) C(18)-C(17)-C(19) 109.24(14) N(3)-C(17)-H(17) 108.8 C(18)-C(17)-H(17) 108.8 C(19)-C(17)-H(17) 108.8 N(4)-C(18)-C(17) 178.6(2) C(20)-C(19)-C(17) 111.27(13) C(20)-C(19)-H(19A) 109.4 C(17)-C(19)-H(19A) 109.4 C(20)-C(19)-H(19B) 109.4 C(17)-C(19)-H(19B) 109.4 N(19A)-C(19)-N(19B) 108,0 C(21)-C(20)-C(19) 112.17(13) C(21)-C(20)-C(23) 102.32(14) C(19)-C(20)-C(23) 115.26(15) C(21)-C(20)-H(20) 108.9 C(19)-C(20)-H(20) 108.9 C(23)-C(20)-H(20) 108.9 O(4)-C(21)-N(5) 126.30(18) O(4)-C(21)-C(20) 125.79(17) N(5)-C(21)-C(20) 107.91(15) N(5)-C(22)-C(23) 102.06(17) N(5)-C(22)-H(22A) 111.4 C(23)-C(22)-H(22A) 111.4 N(5)-C(22)-H(22B) 111.4 C(23)-C(22)-H(22B) 111.4 N(22A)-C(22)-N(22B) 109.2 C(22)-C(23)-C(20) 103.82(18) C(22)-C(23)-H(23A) 111,0 C(20)-C(23)-H(23A) 111,0 C(22)-C(23)-H(23B) 111,0 C(20)-C(23)-H(23B) 111,0 H(23A)-C(23)-H(23B) 109,0

Symmetry transformations used to generate equivalent atoms.

- 89 047572

TableN

Anisotropic substitution parameters (A ² x10 ³ ) for example 13, solid form 1.

The exponent of the anisotropic shift coefficient takes the form: -2n ² [h ² a* ² U ¹¹ +...+ 2hka*b*U ¹² ]

U ¹¹ U ²² U IT IT u F(l) 105(3) 87(3) 329(14) -12(6) -36(6) -6(2) F(2) 113(4) 185(4) 107(3) 29(2) 25(2) -68(3) F(3) 138(4) 170(5) 91(2) 18(3) -44(2) -92(4) F(1A) 55(3) 183(9) 470(30) 64(16) -24(9) -39(4) F(2A) 317(18) 99(4) 86(4) -28(3) -12(6) -34(7) F(3A) 185(11) 94(6) 103(4) 17(3) -12(4) -63(6) N(l) 48(1) 70(1) 44(1) 10(1) -3(1) -5(1) N(2) 42(1) 64(1) 42(1) 7(1) -7(1) 0(1) N(3) 45(1) 45(1) 42(1) 2(1) -6(1) -4(1) N(4) 46(1) 140(2) 110(2) -65(2) 7(1) -2(1) N(5) 40(1) 105(1) 84(1) 12(1) -14(1) -6(1) 0(1) 75(1) H8(l) 59(1) 14(1) -21(1) -4(1) 0(2) 60(1) 92(1) 58(1) 32(1) -8(1) 1(1) 0(3) 92(1) 51(1) 67(1) 12(1) -19(1) -3(1) 0(4) 76(1) 79(1) 102(1) 36(1) -15(1) 14(1) C(l) 86(2) 113(2) 79(2) 7(2) -14(1) -34(2) C(2) 51(1) 90(1) 48(1) 1(1) -3(1) -4(1) C(3) 47(1) 62(1) 43(1) 11(1) 0(1) 1(1) C(4) 70(1) 70(1) 67(1) -2(1) 7(1) 9(1) C(5) 1H(2) 67(1) 117(2) -14(1) 17(2) 0(1) C(6) 117(2) 108(2) 57(1) -ii(i) 18(1) 2(2) C(7) 81(2) 138(3) 1H(2) -8(2) 10(2) 46(2) C(8) 49(1) 58(1) 42(1) 7(1) -6(1) 0(1) C(9) 44(1) 88(1) 65(1) 16(1) -ii(i) 4(1) C(10) 41(1) 99(2) 63(1) 5(1) -4(1) -3(1) C(ll) 57(1) 95(1) 56(1) H(l) -ii(i) -27(1) C(12) 74(2) 150(3) 80(2) 23(2) -9(1) -55(2) C(13) 93(2) 91(2) 72(1) -3(1) -13(1) -32(1) C(14) 47(1) 84(1) 44(1) 3(1) -2(1) -14(1) C(15) 43(1) 54(1) 39(1) 5(1) -4(1) -3(1) C(16) 41(1) 48(1) 44(1) 4(1) -3(1) 1(1) C(17) 39(1) 52(1) 51(1) -5(1) -6(1) -2(1) C(18) 42(1) 85(1) 73(1) -33(1) -9(1) 0(1) C(19) 41(1) 58(1) 41(1) 1(1) -4(1) -5(1) C(20) 40(1) 52(1) 41(1) 1(1) -6(1) -4(1) C(21) 46(1) 62(1) 58(1) 4(1) -9(1) 6(1) C(22) 58(1) 103(2) 84(1) 14(1) 0(1) -28(1) C(23) 60(1) 64(1) 66(1) 19(1) -4(1) -12(1)

Solid-state NMR analysis of Example 13, Forms 1 and 4 was performed using a CPMAS probe mounted on a Bruker-BioSpin Avance III 500 MHz (1H) NMR spectrometer. A magic angle spin rate of 15.0 kHz was used. Spectra of Form 1 were collected at ambient temperature (temperature uncontrolled) and spectra of Form 4 were collected at 15°C.

^13C mtNMR spectra were collected using a cross-polarized proton magic angle spin (CPMAS) experiment. A phase-modulated proton decoupling field of 80-100 kHz was applied during spectral collection. The contact time with the cross-polarized proton

- 90 047572 polarization was set to 2 ms and the recycle delay was set 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 based using the ^13C CPMAS experiment on an external standard of crystalline adamantane, setting its upfield resonance to 29.5 ppm.

¹⁹ F mtNMR spectra were collected using a proton-split magic angle spinning (MAS) experiment. A phase-modulated proton decoupling field of 80-100 kHz was applied during spectral collection. Spectra were collected with a repeat cycle delay of 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. The ¹⁹ F chemical shift scale was based using a ¹⁹ F MAS experiment on an external standard of trifluoroacetic acid (50%/50% v/v in _H2O ), setting its resonance at -76.54 ppm.

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

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

¹³ s Chemical shift (ppm) Relative intensity (%) ¹³ C Chemical shift (ppm) Relative intensity (%) ¹³ C 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

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

The characteristic peaks for the compound of Example 13, Form 1 are ^{the 19} P peak with a chemical shift at -73.3±0.1 ppm in combination with ^{the 13} C peaks with chemical shifts at 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.

(^^^)-I-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-I(trifluoroacetyl)^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (13), solid form 4

- 91 047572 o

O

13, methyl tert-butyl ether solvate

H ₃ CO ³

Step 1. Recrystallization of (1R.2S,5S)-N-j(1S)-1-iiuaiio-2-[(3S)-2-oi<coiiuppoju;iuii-3-uj]3TUjj6,6-dimethyl-3-[3-methyl-^(trifluoroacetyl)^-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 added slowly and stirring was continued for 30 min, after which the mixture was cooled to 10 °C over 30 min. After stirring for an additional 2 h, the suspension was filtered; The filtered cake was washed with a mixture of propan-2-yl acetate (4 ml) and heptane (16 ml) and then dried at 55°C in vacuo to give (^^^)-N{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl(trifluoroacetyl)-^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (13) as a crystalline solid. A portion of this material was used in the next recrystallization. Yield: 7.74 g, 15.5 mmol, 91%.

Step 2. Recrystallization of (^^^)-N{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}6,6-dimethyl-3-[3-methyl-^(trifluoroacetyl)^-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, after which 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 (^^^)-N{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl(trifluoroacetyl)-N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (13) as a white crystalline solid. The X-ray powder diffraction pattern for this material, designated solid form 4, is shown in Fig. 6; characteristic peaks are listed in Table 1. 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

Powder X-ray diffraction peaks for 13, solid form 4 are selected

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 21D 42 31.3 3

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And,2 4 21.6 9 31.8 6 And,4 4 21.8 16 32.5 6 And,4 8 22.3 53 32.8 4 And,7 5 23.1 14 33.2 4 12,0 6 23.4 AND 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

Determination of the structure by X-ray structural analysis of the single crystal from 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 to determine the structure from single crystal X-ray diffraction analysis. An ORTEP plot of the single crystal data is shown in Fig. 7. Mercury software was used to calculate a powder diffraction pattern from the solved crystal structure; compared with the diffraction pattern from an alternate recrystallization of Example 13; yielding solid Form 4 of this material, identified as solid Form 4 (see Fig. 8). The characteristic peaks for these calculated data are given in Table K.

Table K

Powder diffraction pattern data for 13, solid form 1, calculated from the X-ray diffraction structure determination of the single crystal of Example 13, solid form 4

Corner Relative Corner Relative Corner Relative (°2-theta) +/- 0.2° 2-Theta intensity (%) (°2-theta) +/- 0.2° 2-Theta intensity (%) (°2-theta) +/- 0.2° 2-Theta 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 And,2 4 21,1 32 30.9 3 from 7 21.6 8 31.3 3 And,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 AND

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X-ray structural analysis of a single crystal.

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

The structure was solved using internal phasing using SHELX software, valid in the P212121 space group of the orthorhombic system. The structure was then optimized using the least squares method in the full matrix approximation. All non-hydrogen atoms were found and optimized using anisotropic substitution parameters.

Hydrogen atoms located on nitrogen were determined from the Fourier difference map and refined by limited distances. The remaining hydrogen atoms were placed in calculated positions and allowed to run into their carrier atoms. The final refinement included isotropic substitution parameters for all hydrogen atoms.

Absolute structure analysis using probabilistic methods (Hooft 2008) was performed using PLATON (Spek). Absolute stereochemistry was not determined, through Hooft/Parsons/Fleck parameters that were out of specification and standard deviations.

The final R-index was 6.3%. The final Fourier difference revealed neither a missing nor a shifted electron density.

The corresponding crystal, data collection and refinements are presented in Table L. Atomic coordinates, bond lengths, bond angles and substitution parameters are given 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.

Cryst. 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.

TableL

Crystal data and structure refinements for example 13, solid form 4

Empirical formula C23H32F3N5O4 Mass by formula 499.53 Temperature 173(2)K Wavelength 1.54178 A Crystal system Orthorhombic Space group P212121 Parameters of the unit cell « = 9.2114(9) A a = 90' Ъ = 15.1607(16) A /5 = 90' c = 18.191(2) A γ = 90 ^e Volume 2540.5(5) A ³

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Z 4 Density (calculated) 1.306 mg/ ^m3 Absorption coefficient 0.892 mm ¹ F(000) 1056 Crystal size 0.100 x 0.060 x 0.040 mm ³ Theta range for data collection from 3.795 to 54.284° Index ranges ~9<=h<=9, -15<=L<=15, —18<=7<=18 Collected reflections 15896 Independent reflections 3070 [R _mt = 0.1260] Total to theta = 54.284° 99.1% Absorption correction Empirical Method of clarification Least squares in full matrix approximation on F ² Data / restrictions / parameters 3070/9/349 Consistency criterion for F ² 1,067 Finite R indices [Ι>2σ(Ι)} Fl = 0.0625, wR2 = 0.1515 R indices (all data) Fl = 0.0696, wR2 = 0.1578 Absolute structure parameter 0.14(14) Extinction coefficient But Maximum differential peak and hole 0.280 and -0.220 e. A ³

Table M

The atomic coordinates (α10 ⁴ ) and equivalent isotropic substitution parameters (A ² x10 ³ ) for example 13, solid form 4.1 Deq.) are defined as one third of the trace of the orthogonalized tensor U ^ij

X U z and(equiv.) F(l) 4306(16) 6041(7) 2387(4) 174(5) F(2) 2770(10) 5589(8) 1525(8) 181(5) F(3) 4932(11) 6015(5) 1280(6) 114(3) 0(1) 4026(8) 4159(6) 2397(4) 80(2) C(l) 4115(14) 5581(8) 1775(8) 105(3) C(2) 4561(14) 4555(7) 1871(9) 65(3) F(1A) 3230(70) 5710(30) 2210(20) 174(5) F(2A) 3400(40) 5940(30) 1470(40) 181(5)

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F(3A) 4940(60) 6060(30) 1660(30) 114(3) O(1A) 4410(40) 4580(20) 2430(20) 80(2) C(1A) 4210(70) 5320(30) 1790(40) 105(3) C(2A) 4840(70) 4850(40) 1940(40) 65(3) N(l) 5533(5) 4303(3) 1395(2) 57(1) N(2) 8573(4) 3139(3) 1925(2) 53(1) N(3) 6772(4) 3489(3) 3714(2) 51(1) N(4) 8394(7) 4673(5) 5053(4) 103(2) N(5) 1220(5) 3638(4) 4615(3) 80(2) 0(2) 8305(4) 3783(3) 826(2) 76(1) 0(3) 7583(4) 4429(3) 2845(2) 70(1) 0(4) 2759(5) 2668(3) 5176(3) 84(1) C(3) 6097(5) 3407(4) 1448(3) 55(1) C(4) 5356(6) 2775(4) 886(4) 66(2) C(5) 3799(7) 2611(6) 1123(5) 100(2) C(6) 5336(8) 3163(5) 106(3) 80(2) C(7) 6176(8) 1903(5) 876(5) 94(2) C(8) 7747(6) 3468(3) 1378(3) 55(1) C(9) 10166(5) 3230(5) 1885(3) 68(2) C(10) 10729(6) 2754(5) 2548(4) 73(2) C(ll) 10341(7) 1816(5) 2669(3) 73(2) C(12) 9762(8) 1239(5) 2060(4) 89(2) C(13) 11307(8) 1311(5) 3196(4) 96(2) C(14) 9439(6) 2528(4) 3023(3) 62(2) C(15) 8090(5) 2884(4) 2660(3) 51(1) C(16) 7471(5) 3677(3) 3070(3) 49(1) C(17) 6277(6) 4180(4) 4192(3) 61(2) C(18) 7465(7) 4485(4) 4670(4) 78(2) C(19) 5031(6) 3887(4) 4683(3) 62(2) C(20) 3625(6) 3736(4) 4274(3) 59(2) C(21) 2506(6) 3278(4) 4745(3) 61(1) C(22) 1247(7) 4355(5) 4095(4) 80(2) C(23) 2822(6) 4572(4) 4041(4) 71(2)

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TableN

Bond lengths [A] and angles [°] for example 13, solid form 4

F(l)-C(l) 1,326(15) F(2)-C(l) 1,320(15) F(3)-C(l) 1,345(14) O(l)-C(2) 1,233(18) C(l)-C(2) 1,618(18) C(2)-N(l) 1,303(15) F(1A)-C(1A) 1,33(3) F(1A)-F(2A) 1,40(7) F(2A)-C(1A) 1,33(3) F(2A)-F(3A) 1,47(7) F(3A)-C(1A) 1,34(3) F(3A)-C(2A) 1,90(8) O(1A)-C(2A) 1,05(8) O(1A)-C(1A) 1,62(7) C(1A)-C(2A) 0.95(8) C(2A)-N(1) 1,45(6) N(l)-C(3) 1,458(7) N(1)-H(1X) 0.98(3) N(2)-C(8) 1,348(7) N(2)-C(15) 1,462(7) N(2)-C(9) 1,476(7) N(3)-C(16) 1,368(7) N(3)-C(17) 1,436(7) N(3)-H(3X) 0.98(3) N(4)-C(18) 1,140(9) N(5)-C(21) 1,326(8) N(5)-C(22) 1,441(9) N(5)-H(5X) 0.99(3) O(2)-C(8) 1,225(7) O(3)-C(16) 1,215(6) O(4)-C(21) 1,236(7) C(3)-C(8) 1,528(7) C(3)-C(4) 1,559(8)

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C(3)-H(3) 1,0000 C(4)-C(5) 1,518(9) C(4)-C(7) 1,522(9) C(4)-C(6) 1,537(9) C(5)-H(5A) 0,9800 C(5)-H(5B) 0,9800 C(5)-H(5C) 0,9800 C(6)-H(6A) 0,9800 C(6)-H(6B) 0,9800 C(6)-H(6C) 0,9800 C(7)-H(7A) 0,9800 C(7)-H(7B) 0,9800 C(7)-H(7C) 0,9800 C(9)-C(10) 1,497(9) C(9)-H(9A) 0,9900 C(9)-H(9B) 0,9900 C(10)-C(11) 1,483(10) C(10)-C(14) 1,509(8) C(10)-H(10) 1,0000 C(11)-C(14) 1,507(8) C(11)-C(12) 1,509(9) C(11)-C(13) 1,516(10) C(12)-H(12A) 0,9800 C(12)-H(12V) 0,9800 C(12)-H(12C) 0,9800 C(13)-H(13A) 0,9800 C(13)-H(13B) 0,9800 C(13)-H(13C) 0,9800 C(14)-C(15) 1,506(7) C(14)-H(14) 1,0000 C(15)-C(16) 1,525(7) C(15)-H(15) 1,0000 C(17)-C(18) 1,472(10) C(17)-C(19) 1,521(8) C(17)-H(17) 1,0000

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C(19)-C(20) 1,511(8) C(19)-H(19A) 0,9900 C(19)-H(19B) 0,9900 C(20)-C(21) 1,509(8) C(20)-C(23) 1,528(8) C(20)-H(20) 1,0000 C(22)-C(23) 1,491(9) C(22)-H(22A) 0,9900 C(22)-H(22B) 0,9900 C(23)-H(23A) 0,9900 C(23)-H(23B) 0,9900 F(2)-C(l)-F(l) 114.1(13) F(2)-C(l)-F(3) 106.9(11) F(l)-C(l)-F(3) 103.3(11) F(2)-C(l)-C(2) 106.5(12) F(l)-C(l)-C(2) 112.4(11) F(3)-C(l)-C(2) 113.6(11) O(l)-C(2)-N(l) 130.4(10) O(l)-C(2)-C(l) 116.8(11) N(l)-C(2)-C(l) 112.6(11) C(1A)-F(1A)-F(2A) 59(2) C(1A)-F(2A)-F(1A) 58(2) C(1A)-F(2A)-F(3A) 57(2) F(1A)-F(2A)-F(3A) 85(4) C(1A)-F(3A)-F(2A) 56(2) C(1A)-F(3A)-C(2A) 28(3) F(2A)-F(3A)-C(2A) 84(4) C(2A)-O(1A)-C(1A) 34(4) C(2A)-C(1A)-F(1A) 125(8) C(2A)-C(1A)-F(2A) 171(9) F(1A)-C(1A)-F(2A) 63(4) C(2A)-C(1A)-F(3A) 112(7) F(1A)-C(1A)-F(3A) 94(5) F(2A)-C(1A)-F(3A) 67(4)

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C(2A)-C(1A)-O(1A) 38(5) F(1A)-C(1A)-O(1A) 89(4) F(2A)-C(1A)-O(1A) 149(5) F(3A)-C(1A)-O(1A) 131(7) C(1A)-C(2A)-O(1A) 108(8) C(1A)-C(2A)-N(1) 120(8) O(1A)-C(2A)-N(1) 121(5) C(1A)-C(2A)-F(3A) 41(4) O(1A)-C(2A)-F(3A) 129(5) N(1)-C(2A)-F(3A) 110(5) C(2)-N(l)-C(3) 118.3(7) C(2A)-N(1)-C(3) 130(3) C(2)-N(1)-H(1X) 120(4) C(2A)-N(1)-H(1X) 102(5) C(3)-N(1)-H(1X) 121(4) C(8)-N(2)-C(15) 127.0(4) C(8)-N(2)-C(9) 119.4(5) C(15)-N(2)-C(9) 111.8(4) C(16)-N(3)-C(17) 121,1(4) C(16)-N(3)-H(3X) 113(4) C(17)-N(3)-H(3X) 123(4) C(21)-N(5)-C(22) 114.3(5) C(21)-N(5)-H(5X) 120(4) C(22)-N(5)-H(5X) 125(4) N(l)-C(3)-C(8) 107.0(4) N(l)-C(3)-C(4) 111.9(4) C(8)-C(3)-C(4) 114.7(5) N(l)-C(3)-H(3) 107.7 C(8)-C(3)-H(3) 107.7 C(4)-C(3)-H(3) 107.7 C(5)-C(4)-C(7) 109.3(6) C(5)-C(4)-C(6) 108.2(5) C(7)-C(4)-C(6) 109.1(6) C(5)-C(4)-C(3) 109.2(5) C(7)-C(4)-C(3) 108.9(5)

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C(6)-C(4)-C(3) 112,1(5) C(4)-C(5)-H(5A) 109.5 C(4)-C(5)-H(5B) 109.5 H(5A)-C(5)-H(5B) 109.5 C(4)-C(5)-H(5C) 109.5 H(5A)-C(5)-H(5C) 109.5 H(5B)-C(5)-H(5C) 109.5 C(4)-C(6)-H(6A) 109.5 C(4)-C(6)-H(6B) 109.5 H(6A)-C(6)-H(6B) 109.5 C(4)-C(6)-H(6C) 109.5 H(6A)-C(6)-H(6C) 109.5 H(6B)-C(6)-H(6C) 109.5 C(4)-C(7)-H(7A) 109.5 C(4)-C(7)-H(7B) 109.5 H(7A)-C(7)-H(7B) 109.5 C(4)-C(7)-H(7C) 109.5 H(7A)-C(7)-H(7C) 109.5 H(7B)-C(7)-H(7C) 109.5 O(2)-C(8)-N(2) 120.8(5) O(2)-C(8)-C(3) 120.6(5) N(2)-C(8)-C(3) 118.5(5) N(2)-C(9)-C(10) 105.1(5) N(2)-C(9)-H(9A) 110.7 C(10)-C(9)-H(9A) 110.7 N(2)-C(9)-H(9B) 110.7 C(10)-C(9)-H(9B) 110.7 H(9A)-C(9)-H(9B) 108.8 C(ll)-C(l 0)-0(9) 119.9(5) C(ll)-C(10)-C(14) 60.5(4) C(9)-C(10)-C(14) 107.3(4) C(ll)-C(10)-H(10) 118.2 C(9)-C(10)-H(10) 118.2 C(14)-C(10)-H(10) 118.2 C(10)-C(l 1)-0(14) 60.6(4)

- 101 047572

C(10)-C(ll)-C(12) 122.2(6) C(14)-C(ll)-C(12) 122.3(5) C(10)-C(ll)-C(13) 115.9(6) C(14)-C(ll)-C(13) 114.6(5) C(12)-C(ll)-C(13) 112.2(6) C(11)-C(12)-H(12A) 109.5 C(11)-C(12)-H(12B) 109.5 H(12A)-C(12)-H(12B) 109.5 C(11)-C(12)-H(12C) 109.5 H(12A)-C(12)-H(12C) 109.5 H(12B)-C(12)-H(12C) 109.5 C(11)-C(13)-H(13A) 109.5 C(11)-C(13)-H(13B) 109.5 H(13A)-C(13)-H(13B) 109.5 C(11)-C(13)-H(13C) 109.5 H(13A)-C(13)-H(13C) 109.5 H(13B)-C(13)-H(13C) 109.5 C(15)-C(14)-C(ll) 121.6(5) C(15)-C(14)-C(10) 108.5(5) C(ll)-C(14)-C(10) 58.9(4) C(15)-C(14)-H(14) 117.6 C(11)-C(14)-H(14) 117.6 C(10)-C(14)-H(14) 117.6 N(2)-C(15)-C(14) 104.2(4) N(2)-C(15)-C(16) 110.6(4) C(14)-C(15)-C(16) 112,1(4) N(2)-C(15)-H(15) 109.9 C(14)-C(15)-H(15) 109.9 C(16)-C(15)-H(15) 109.9 O(3)-C(16)-N(3) 121.6(5) O(3)-C(16)-C(15) 122.9(5) N(3)-C(16)-C(15) 115.5(4) N(3)-C(17)-C(18) 110.5(4) N(3)-C(17)-C(19) 112.4(5) C(18)-C(17)-C(19) 107.8(5)

- 102 047572

N(3)-C(17)-H(17) 108.7 C(18)-C(17)-H(17) 108.7 C(19)-C(17)-H(17) 108.7 N(4)-C(18)-C(17) 176.2(8) C(20)-C(19)-C(17) 113.7(5) C(20)-C(19)-H(19A) 108.8 C(17)-C(19)-H(19A) 108.8 C(20)-C(19)-H(19B) 108.8 C(17)-C(19)-H(19B) 108.8 N(19A)-C(19)-N(19B) 107.7 C(21)-C(20)-C(19) 112,1(5) C(21)-C(20)-C(23) 102.0(5) C(19)-C(20)-C(23) 115.2(5) C(21)-C(20)-H(20) 109.1 C(19)-C(20)-H(20) 109.1 C(23)-C(20)-H(20) 109.1 O(4)-C(21)-N(5) 126.1(5) O(4)-C(21)-C(20) 125.2(5) N(5)-C(21)-C(20) 108.6(5) N(5)-C(22)-C(23) 103.1(5) N(5)-C(22)-H(22A) 111,1 C(23)-C(22)-H(22A) 111,1 N(5)-C(22)-H(22B) 111,1 C(23)-C(22)-H(22B) 111,1 N(22A)-C(22)-N(22B) 109.1 C(22)-C(23)-C(20) 105.6(5) C(22)-C(23)-H(23A) 110.6 C(20)-C(23)-H(23A) 110.6 C(22)-C(23)-H(23B) 110.6 C(20)-C(23)-H(23B) 110.6 H(23A)-C(23)-H(23B) 108.7

Symmetry transformations used to generate equivalent atoms.

- 103 047572

TableR

Anisotropic substitution parameters (A ² x10 ³ ) for example 13, solid form 4. The exponent of the anisotropic shift coefficient takes the form: -2n ² [h ² a* ² U ¹¹ +...+ 2 h ka*b*U ¹² ]

U ¹¹ U ²² U ³³ U ²³ u ¹³ u F(l) 279(15) 138(7) 106(5) -56(5) -28(7) 100(8) F(2) 69(5) 172(9) 302(12) 75(9) -10(7) 44(5) F(3) 143(5) 83(3) 117(7) -10(5) -24(6) 33(3) 0(1) 77(5) 91(6) 74(3) 1(4) 29(3) 9(4) C(l) H6(7) 76(10) 122(8) 6(9) 25(6) 43(10) C(2) 65(7) 68(9) 63(6) -13(7) -5(5) 18(6) F(1A) 279(15) 138(7) 106(5) -56(5) -28(7) 100(8) F(2A) 69(5) 172(9) 302(12) 75(9) -10(7) 44(5) F(3A) 143(5) 83(3) 117(7) -10(5) -24(6) 33(3) O(1A) 77(5) 91(6) 74(3) 1(4) 29(3) 9(4) C(1A) H6(7) 76(10) 122(8) 6(9) 25(6) 43(10) C(2A) 65(7) 68(9) 63(6) -13(7) -5(5) 18(6) N(l) 49(2) 70(3) 53(3) -1(2) 3(2) 19(2) N(2) 38(2) 75(3) 45(3) -2(2) 3(2) 2(2) N(3) 50(2) 51(2) 52(3) 1(2) 3(2) 4(2) N(4) 60(3) 130(5) H9(5) -49(4) 0(4) -10(3) N(5) 53(3) 105(4) 82(4) 17(3) 15(3) H(3) 0(2) 51(2) 114(3) 62(3) 25(2) 12(2) 6(2) 0(3) 78(2) 55(2) 76(3) 4(2) 18(2) 0(2) 0(4) 82(3) 83(3) 86(3) 30(3) -2(2) -11(2) C(3) 45(3) 70(3) 51(3) 9(3) 4(2) 9(3) C(4) 48(3) 70(4) 79(4) -5(3) -7(3) 5(3) C(5) 56(3) 131(6) 114(6) -16(5) 4(4) -22(4) C(6) 76(4) 107(5) 59(4) -15(4) -9(3) 12(4) C(7) 82(4) 83(5) H7(6) -17(4) -18(4) 6(4) C(8) 44(3) 60(3) 60(4) -2(3) 6(3) 7(2) C(9) 37(3) 95(4) 70(4) -2(4) 1(3) 2(3) C(10) 41(3) 105(5) 73(4) -24(4) -8(3) 5(3) C(ll) 69(4) 82(4) 67(4) -11(4) -12(3) 20(3) C(12) 85(4) 96(5) 87(5) -24(4) -17(4) 34(4) C(13) 82(4) 116(6) 90(5) -25(4) -18(4) 48(4) C(14) 55(3) 74(4) 57(3) -6(3) -6(3) 16(3) C(15) 44(2) 59(3) 51(3) 0(3) -4(2) 2(2) C(16) 39(2) 48(3) 59(3) 2(3) 3(2) -4(2) C(17) 49(3) 62(3) 73(4) -3(3) 2(3) 3(3) C(18) 57(4) 85(4) 92(5) -26(4) 26(4) -8(3) C(19) 59(3) 65(3) 63(4) 0(3) 15(3) 8(3) C(20) 49(3) 61(3) 66(4) 6(3) 2(3) 6(2) C(21) 58(3) 72(4) 53(3) 6(3) 6(3) 2(3) C(22) 65(4) 92(5) 82(5) 13(4) 11(3) 16(3) C(23) 62(3) 74(4) 77(4) 21(3) 7(3) 5(3)

List of ^13C Solid State NMR Peak 4 Form PF-07321332-00. Variability for ^13C chemical shift values is ±0.2 ppm unless otherwise stated.

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^I C Chemical shift (ppm) Relative intensity (%) °C Chemical shift (ppm) Relative intensity (%) ^And C Chemical shift (ppm) Relative intensity (%) 179.3 16 62,0 17 32.9 16 178.8 AND 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

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

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

A fourth alternative synthesis of Example 13, methyl tert-butyl ether solvate.

(^^^)-N{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl^(trifluoroacetyl)-C-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl ether solvate (13, methyl tert-butyl ether solvate)

043 13, methyl tert-butyl ether solvate

At 0 °C, 4-methylmorpholine (4.5 equivalents; 4.8 mL, 44 mmol) was charged into a suspension of C43 (5.0 g, 9.7 mmol) in propan-2-yl acetate (7.6 mL/g, 38 mL). The resulting suspension was charged with trifluoroacetic anhydride (2.25 equivalents; 3.1 mL, 22 mmol) over 1 h using a metering pump. After stirring at 0 °C for at least 1 h, the reaction mixture was warmed to approximately 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 were separated and the lower (aqueous) layer was discarded. The organic layer was concentrated under reduced pressure to approximately 4 ml/g (approximately 20 ml), after which the vacuum was released using nitrogen and the solution was heated to approximately 50 °C. Methyl tert-butyl ether (12 ml/g, 60 ml) was slowly added over at least 4 hours using a dropping funnel, and the reaction mixture was maintained at 50 °C for at least 1 hour before being cooled to 25 °C over 1 hour. The resulting suspension was 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 (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-C-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.

(^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-C-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide, methyl tert-butyl solvate

- 105 047572 ether (13, methyl tert-butyl ether solvate)

13, methyl tert-butyl ether solvate

To a room temperature suspension of C43 (4.0 g, 7.7 mmol) in acetonitrile (10 mL/g, 40 mL) was charged 1-methyl-III-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 h using a pump. After the reaction mixture had been stirred for at least 10 h, it was cooled to 25 °C and carefully quenched by adding saturated aqueous sodium bicarbonate (7 mL/g, 28 mL) (exothermic and degassed). Acetonitrile was then distilled under reduced pressure; ethyl acetate (10 ml/g, 40 ml) and additional saturated aqueous sodium bicarbonate solution (5 ml/g, 20 ml) were added to the resulting mixture. After phase separation, the lower (aqueous) layer was discarded and the organic layer was washed with saturated aqueous sodium bicarbonate solution (3.5 ml/g; 14 ml). The aqueous layer was again discarded and the organic layer was concentrated under reduced pressure to approximately 1 ml/g (4 ml). Methyl tert-butyl ether (9 ml/g; 36 ml) was charged and the resulting solution was heated to 50 °C, which quickly resulted in the formation of a slurry. This slurry was maintained at 50 °C for at least 30 minutes, after which it was cooled to 25 °C over 1 hour and maintained at 25 °C for at least 8 hours. The suspension was 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 collected solid was dried on the filter for at least 30 minutes, transferred to a vacuum oven at approximately 50°C, and dried for at least 8 hours to give (1R,28,58)-N-{(18)-1-zzano-2-[(38)-2oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-valyl]-3azabicyclo[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 the preparation for the compound of Example 13.

(^,28,58)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N(trifluoroacetyl)-N-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 is formulated to obtain a conventional immediate release film-coated tablet for oral administration, with dosages ranging from 100 to 250 mg. As an example, an immediate release formulation is described in the Formulation Table and contains the common inactive excipients microcrystalline cellulose and lactose monohydrate (diluents), crospovidone (disintegrant), colloidal silicon dioxide (glidant), and sodium stearyl fumarate (lubricant). The immediate release tablets are film coated using commercially available film coating formulations including Opadry white and Opadry pink. All excipients used in the film coated tablets are broadly acceptable and present at precedent levels. The composition provided is an example of an immediate release tablet formulation and as such, one skilled in the art would be able to use readily available routine techniques using alternative formulation excipients to prepare suitable tablets and achieve the desired tablet quality characteristics.

Examples of the preparation: typical preparations of film-coated tablets, (1R,28,58)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide.

(^,28,58)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (hereinafter referred to as API) immediate release tablets are manufactured using conventional standard processes for immediate release hard oral tablets. Examples of standard batch processes that can be used to manufacture (^,28,58)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3

- 106 047572 methyl [N-(trifluoroacetyl)-1-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 tablet compression, the tablet cores are film coated. Film coating of the tablets can be carried out by continuous coating or by a conventional batch film coating process.

Table of preparations: tablet preparations with 100 mg, 150 mg and 250 mg of (1R,2S,5S)-N-{(1S)-1cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)G-valyl]-3azabicyclo[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) AFY Active substance 20,00 100,00 20,00 150,00 25,00 250,00 Microcrystalline allic cellulose Thinner 49.33 246.67 49.33 370,00 46,00 460,00 Lactose monohydrate Diluter 24.67 123.33 24.67 185,00 23,00 230,00 Crosspovido n Baking powder 3.00 15,00 3.00 22,50 3.00 30,00 Colloidal dioxide Sliding substance 1,00 5,00 1,00 7.50 1,00 10,00 silicon Sodium stearyl fumarate (in the middle of the granulate) Lubricant 1,00 5,00 1,00 7.50 1,00 10,00 Sodium stearyl fumarate (outside granulate) Lubricant 1,00 5,00 1,00 7.50 1,00 10,00 the core as a whole 100,00 500,00 100,00 750,00 100,00 1000,00 Opadry white (YS-1-7027SP) Coverage 3.50 17.50 Opadry pink (058140011) Coverage - - - 3.00 22,50 - - - -

Example 14.

N[^)-1-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-1-oxopentan2-yl]-4-methoxy-1 H-indole-2-carboxamide (14)

C34 C35

C36 C37

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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-N-norvalinamide, hydrochloride salt (which was 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-9ylmethylcarbonyl chloridate (Fmoc chloride; 10.2 g, 39.4 mmol) in water (80 mL). The resulting suspension was stirred at 15°C to 25°C for 24 h and then partitioned between water and dichloromethane. The organic layer was washed successively with water and saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo to afford C34 as a solid. Yield: 6.2 g, 16 mmol, 83%. LC-MS m/z 393.1 [M+H]+.

^1H NMR (400 MHz, DMSO-ds) δ 7.9 (d, 2H), 7.7 (m, 2H), 7.5 (d, 1H), 7.4 (m, 5H), 7. 1 (w 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%. LC-MS m/z 392.1 [M-H] ^- .

1H NMR (400 MHz, DMSO-66) δ 12.83 (br s, 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 MX-dimethylformamide (20 mL) was stirred for 15 h at 25 °C. After the reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (3 x 30 mL), the combined organic layers were washed successively with saturated aqueous sodium chloride solution and 5% aqueous lithium chloride solution, 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%. LC-MS m/z 506.1 [M+Na ⁺ ].

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

Diethylamine (10 mL) was added at 0 °C to a mixture of C36 (510 mg, 1.05 mmol) in acetonitrile (25 mL). After the reaction mixture was stirred at 20 °C for 2 h, it was concentrated under reduced pressure; silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) then gave C37 as a colorless oil. Yield: 250 mg, 0.957 mmol, 91%. LC-MS m/z 302.9 [M + CH3CN + H] ⁺ .

- 108 047572 ¹ H 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).

At 0°C, O-(7-azabenzotriazol-1-yl)-N,N,I,I,tetramethylaronium hexafluorophosphate (HATU; 437 mg, 1.15 mmol) was added to a solution of C37 (250 mg, 0.957 mmol) and 4-methoxy-N-indole-2-carboxylic acid (220 mg, 1.15 mmol) in N,N-dimethylformamide (10 ml), followed by dropwise addition of 4-methylmorpholine (194 mg, 1.92 mmol). Stirring was continued at 0°C to 10°C for 1 h, after which the reaction mixture was diluted with water (20 ml) and aqueous citric acid (20 ml) and extracted with ethyl acetate (3 x 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 (br d, J = 7.6 Hz, 1H), 6.53 (d, J = 7.8 Hz, 1H), 5.25 (AB quartet, J _AB = 12.1 Hz, Αν ,.. = 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 subjected to silica gel chromatography (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-[N)-2-oxopyrrolidin-3-yl]-L-alanine amide (C40).

At 0°C, a 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'-tetramethylaronium hexafluorophosphate (HATU; 99.4 mg, 0.261 mmol) and 4-methylmorpholine (44.1 mg, 0.436 mmol). After the reaction mixture was stirred at 0°C for 1 h, it was diluted with water (20 ml) and aqueous citric acid (1 M; 20 ml) and extracted with ethyl acetate (3 x 30 ml). The combined organic layers were washed with saturated aqueous sodium bicarbonate (20 mL) and saturated aqueous sodium chloride (3 x 20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (eluent: 10:1 ethyl acetate/methanol) gave C40 as a white solid. Yield: 72 mg, 0.145 mmol, 66%. LC-MS m/z 498.2 [M+H]+.

1H NMR (400 MHz, DMSO^ ₆ ) δ 11.60 (w 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 the 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-[^)-1-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide (14).

Methyl N-(triethylammoniosulfonyl)carbamate inner salt (Burgess reagent; 37 mg, 0.16 mmol) was added to a mixture of C40 (52 mg, 0.10 mmol) in dichloromethane (13 ml). The reaction mixture was stirred at room temperature for 1.5 h, after which 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 carried out with further stirring for 2 h, after which the reaction mixture was diluted with water (20 ml) and extracted with dichloromethane (3 x 10 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; preparative thin layer chromatography (eluent: 20:1 ethyl acetate/methanol) gave N-[(N)-1-({(N)-1-cyano-2-[N)-oxopyrrolidin-3-yl]ethyl}amino)-5,5,5-trifluoro-1-oxopentan2-yl]-4-methoxy-1H-indole-2-carboxamide (14) as a white solid. Yield: 17.4 mg, 36.3 μmol, 36%. This material was combined with the purified products from two other syntheses of 14 (3 mg and 4 mg) and subjected to supercritical fluid chromatography [Column: Chiral Technologies ChiralCel OD-H, 30 x 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-iiuaHO-2-[(3S)-2oxopyrrolidin-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. LC-MS m/z 480.2 [M+H]+.

^1H NMR (400 MHz, DMSO-bb) δ 11.61 (w 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 the 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-[^)-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-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl] 5-fluoro-4-methoxy-1 H-indole-2-carboxamide (16),

N-[(2S)-1-({(1S)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl]3-fluoro-4-methoxy-1H-indole-2-carboxamide (17), N-[^)-1-({(^)-1-cyano-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-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-4-methyl-1-oxopentan-2-yl] 3,5-difluoro-4-methoxy-1 H-indole-2-carboxamide (19)

A mixture of 4 (10.0 mg, 22.8 μmol), tetra-n-butylammonium deca-tungstate (TBADT; 3.78 mg, 1.14 μmol), and ^fluoro-^-(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 vessel 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 (1 M, pH 7.45; 1 mL) was added to the reaction mixture, followed by alternating aliquots of water and acetonitrile to maintain a clear 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 preconditioned with methanol (3 mL) followed by the addition of 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 the eluates were evaporated in a vacuum centrifuge, the residues were diluted to stock concentration in a mixture of 1% aqueous formic acid and acetonitrile and combined to a total of 6 mL. This solution was divided into equal parts and each part was subjected to reverse-phase HPLC (column: Phenomenex Luna C18, 10 x 250 mm, 10 μm; mobile phase A: water containing 0.1% formic acid; mobile

- 110 047572 phase B: acetonitrile; gradient: 15% B over 5 min, then 15% to 70% B over 70 min, then 70% to 95% B over 15 min; flow rate: 2 ml/min). Fractions were collected every 20 s, and similar fractions of interest from the two splits were pooled and concentrated. These fractions were further purified using reversed-phase HPLC (column: Agilent Polaris C18, 4.6 x 250 mm, 5 μm; mobile phase A: water containing 10 mM ammonium acetate; mobile phase B: acetonitrile; gradient: 10% B over 5 min, then 10% to 35% B over 35 min, then 35% to 60% B over 15 min, then 60% to 95% B over 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-arboxamide (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]-5-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]-7-fluoro-4-methoxy-1H-indole-2-carboxamide (18), 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 (19), N-[(2S)-1-({(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}amino)- S)-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}amino)-4-methyl-1-oxopentan-2-yl]-3,5-difluoro-4-methoxy-1H-indole-2-carboxamide (19).

Example number 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]+; calcd for C23H29FN5O4, 458.2204.

^1H NMR (600 MHz, DMSO-06) δ 12.05 (w s, 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, 2.1x100 mm, 2.6 μm; mobile phase A: water containing 0.1% formic acid; mobile phase B: acetonitrile; gradient: 5% B over 0.5 minutes, then 5% to 70% B over 10.5 minutes, then 70% to 95% B over 2 minutes; flow rate: 0.4 mL/min).

- First separation, fraction numbers 172-174; Second separation, fraction numbers 138-139. Yield: 153 µg, 0.33 µmol, 1.4%. High Resolution MS m/z 458.2201 [M+H]+; calcd for C23H29FN5O4, 458.2204.

1H NMR (600 MHz, DMSO-06) δ 11.73 (w s, 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 identical to those used for 15).

- First separation, fraction numbers 176-177; Second separation, fraction numbers 141-142. Yield: 22 µg, 0.048 µmol, 0.21%. High Resolution MS m/z 458.2199 [M+H]+; calcd 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 ( wd, J = 7.5 Hz, 1H), 7.16 (dd, J = 8.8 Hz, 1H), 6.95 (wd, J = 8.3 Hz, 1H), 6.54 ( d, J = 7.8 Hz, 1H), 5.02-4.94 (m, 1H), 4.544.46 (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 identical to those used for 15).

- First separation, fraction numbers 180-181; Second separation, fraction number 145. Yield: 17 µg, 0.036 µmol, 0.16%. High Resolution MS m/z 476.2100 [M+H]+; calcd for C23H28F2N5O4, 476.2109.

1H NMR (600 MHz, DMSO-de) 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), 3.183.07 (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 identical to those used for 15).

- First separation, fraction numbers 185-187; Second separation, fraction numbers 150-151. Yield: 35 µg, 0.074 µmol, 0.32%. High-resolution MS m/z 476.2107 [M+H]+; calc.

- 111 047572 tano for C23H28F2N5O4, 476.2109.

^1H NMR (600 MHz, DMSOhC) δ 11.64 (s, 1H), 8.94 (d, J = 7.9 Hz, 1H), 7.80 (wd, J = 7 Hz, 1H), 7.71 (s, 1H), 7.16 (dd, ABX system component, J = 11.9, 9.1 Hz, 1H), 7.08 (wd, 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 identical to those used for 15).

Examples 20, 21, 22 and 23.

N-[(2S)- 1-({(1S)-1-cyano-2-[(3 S)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2yl]-7-fluoro-4-methoxy-1H-indole-2-carboxamide (20),

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

A mixture of 10 (10.0 mg, 22.0 μmol), tetra-n-butylammonium deca-tungstate (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 vessel 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 (1 M, pH 7.45; 1 mL) was added to the reaction mixture, followed by alternating aliquots of water and acetonitrile to maintain a clear 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 preconditioned with methanol (3 mL) followed by the addition of 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 the eluates were evaporated in a vacuum centrifuge, the residues were diluted to the original concentration in a mixture of 1% aqueous formic acid and acetonitrile and combined to a total of 6 ml. This solution was divided into equal parts and each part was subjected to 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 over 5 min, then 15% to 70% B over 70 min, then 70% to 95% B over 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 further purified using reversed-phase HPLC (column: Agilent Polaris C18, 4.6 x 50 mm, 5 μm; mobile phase A: water containing 10 mM ammonium acetate; mobile phase B: acetonitrile; gradient: 10% B over 5 min, then immediately increasing to 20% B, then 20% to 40% B over 35 min, then 40% to 60% B over 15 min, then 60% to 95% B over 9 min; flow rate: 0.8 mL/min). Fractions were collected every 20 s, yielding N[^)-1-({(^)-1-cyano-2-[^)-2

- 112 047572 oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-7-fluoro-4-methoxy-1H-indol-2carboxamide (20), N-[( 2S)-1-(i(1S)-1-iiuaiio-2-[(3S)-2-OKCOiiuppojuguii-3-uj]9TUjja\iuiio)-4,4-gu\ieTiui-1oxopentan-2-yl]- 5-fluoro-4-methoxy-1 H-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 No. [^)-1-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1oxopentan-2-yl]-5, 7-difluoro-4-methoxy-1H-indole-2-carboxamide (23).

Example number 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

- First separation, fraction numbers 183-185; Second separation, fraction numbers 150-151. Yield: 24 µg, 0.051 µmol, 0.23%. High Resolution MS m/z 472.2342 [M+H]+; calculated for C ₂₄ H ₃₁ FN ₅ O ₄ , 472.2360.

^1H NMR (600 MHz, DMSO-06) δ 12.05 (w s, 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, 2.1x100 mm, 2.6 μm; mobile phase A: water containing 0.1% formic acid; mobile phase B: acetonitrile; gradient: 5 % B over 0.5 minutes, then 5% to 70% B over 10.5 minutes, then 70% to 95% B over 2 minutes; flow rate: 0.4 ml/min).

- First separation, fraction numbers 183-185; Second separation, fraction numbers 152-153. Yield: 68 µg, 0.14 µmol, 0.64%. High Resolution MS m/z 472.2344 [M+H]+; calcd for C24H31FN5O4, 472.2360.

^1H NMR (600 MHz, DMSO-06) δ 11.72 (w s, 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 (w s, 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, 9N). Retention time: 8.34 minutes (Analytical conditions identical to those used for 20).

- First separation, fraction numbers 187-188; Second separation, fraction number 154. Yield: 5 µg, 0.011 µmol, 0.05%. High Resolution MS m/z 472.2354 [M+H]+; calcd for C24H31FN5O4, 472.2360.

^1H NMR (600 MHz, DMSO-06) 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 (wd, 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 identical to those used for 20).

- First separation, fraction numbers 190-192; Second separation, fraction numbers 156-157. Yield: 21 µg, 0.043 µmol, 0.19%. High Resolution MS m/z 490.2258 [M+H]+; calculated for C ₂₄ H ₃₀ F ₂ N ₅ O ₄ , 490.2266.

^1H NMR (600 MHz, DMSO-06) 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 identical to those used for 20).

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Table 1

Synthesis method, structure, and physicochemical data for examples 24-74

Ex. No. Method of synthesis; non-commercial starting materials Structure ¹ H NMR (600 MHz, DMSO-b/ ₆ ) δ; Mass spectrum, ion observed m/z [M+H] ⁺ or HPLC retention time; Mass spectrum m/z [M+H] ⁺ (unless otherwise indicated) 24 Example 10; C18 „ °V-NH ³ λ=Ν _n O o Μ ^Ν \< ^СН 3 Гсн ₃ СНз ^{0 1} H NMR (400 MHz, methanol-1H) 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; assumed; 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 9 ¹ '²; C28 ΝΗ CV44 o 1 Η Ν Chg ^с з Гсн ₃ СНз ⁰ or ^МуН ^IT^nS О = Η N \< ^СН з Гсн ₃ СНз ³ DIAST-1 2.29 minutes ³ ; 391.4 26 Examples 8 and 9 ¹ '²; C28 °γ- ΝΗ С4А4 О ^{н Ν} ЧХНз Гсн ₃ СНз ³ or СЧАч о 1 Η Ν Ч<СНз Гсн ₃ СНз ³ DIAST-2 2.69 minutes ³ ; 391.4 27 Example 7; C28 CH ₃ ^Ο γΝΗ Н ₃ С <3 Н ⁰ SM4 О = Η Ν ^СНз Гсн ₃ СНз ³ 2.18 minutes ⁴ ; 381.4 28 Example 7; C28 Оо„Ч ^Н Х^СНз Гсн ₃ СН ₃ ³ 2.61 minutes ⁴ ; 421.5

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29 Example 7; C28 °y-NH Tsn ₃ ^N hdc ₃ CH ₃ ³ 2.31 minutes ⁴ ; 419.4 30 Example 7 ¹ ; C28 9 _α ? Pre-trial detention center 1 Η N \LN ₃ psnz SNZ ³ 1.59 minutes ⁴ ; 442.5 ⁵ 31 C29 ⁶ °V NH 0 = Η ^Χ Ν S< ^CH 3 psn ₃ СНз ^{3 OR} ° _V NH ι\κ Г) but about ^^γ ^Ν Ύ^Ν ^Λ ^Ν 0 Ξ Η Ν \<СНз psn ₃ СН ₃ ³ 2.06 minutes ⁴ ; 416.5 32 C29 ⁶ a.o A η ₃ ο ^Λ τ ^ν ύ ^Λ ν ^Λ > 0 _Ξ n \<СНз ΓΌη ₃ СНз ³ XX ^or ° _уын О η ₃ ο ^Λ τ ^ν υ ^Λ ν ^λ > О Ξ Η Ш<СНз Ιόη ₃ СНз ³ 2.67 minutes ⁴ ; 435.6

- 115 047572

11.40 (s, 1H), 9.60 (w s, 1H), 8.90 (d, J= 8.1 Hz, 1H), 8.44 (d, 7= 7.9 Hz, 1H) , 7.70 (s, 1H), 7.31 (s, 1H), 6.95 (dd, 7=8, 8 Hz, 1H), 6.85 (d, 7= 8.2 Hz, 1H) , 6.36 (d, 7= 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, 7= 13.6, 9.4, 6.8 Hz, 1H), 1.76 - 1.65 (m, 3H), 1.57- 1.48 (m, 1H), 0.94 (d, 7 = 6.3 Hz, 3H), 0.89 (d, 7 = 6.3 Hz, 3H); high-resolution MS m/z 426.2139 [M+H] ⁺ ; calculated for C22H28N5O4, 426.2141

11.34 (s, 1H), 8.91 (d, 7= 8.1 Hz, 1H), 8.46 (d, 7= 7.9 Hz, 1H), 7.69 (s, 1H), 7.29 (s, 1H), 6.95 (d, 7= 8.6 Hz, 1H), 6.78 (d, 7= 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, assumed; partially obscured 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, 7= 6.2 Hz, 3H), 0.89 (d, 7= 6.2 Hz, 3H); high-resolution MS m/z 456.2238 [M+H] ⁺ ; calculated for C23H30N5O5, 456.2247

2.39 minutes ⁴ ; 437.4 (a picture of chlorine isotopes is observed)

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Example

11; C18

Examples 5 and ⁹ ; Example 10

Example

¹ H NMR (600 MHz, methanol-6/ ₄ ) 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, 7= 13.8, 9.7, 5.7 Hz, 1H), 1.79- 1.70 (m, 1H), 1.67 (dd, component of the ABX system, 7= 14.4, 4.3 Hz, 1H), 1.59 (dd, component of the ABX system, 7= 14.4, 8.4 Hz, 1H), 0.84 (s, 9H); 442.5

12.13 (s, 1H), 8.96 (shd, 7 = 7.6 Hz, 2H), 7.69 (s, 1H), 7.21 (dd, 7 = 8.8 Hz, 1H), 7.08 (d, 7=8.2 Hz, 1H), 6.68 (d, 7= 7.8 Hz, 1H), 5.00 - 4.92 (m, 1H), 4.52 4, 45 (m, 1H), 3.86 (s, ZN), 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, 7 = 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 minutes ¹⁰ 11.41 (s, 1H), 8.96 (d, 7=7.7 Hz, 1H), 8.79 (d, 7= 8.0 Hz, 1H), 7, 65 (s, 1H), 7.55 (d, 7 = 8.2 Hz, 1H), 7.34 (s, 1H), 6.72 (d, 7 = 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), 2.382.30 (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 C25H31F3N5O4, 522.2328; retention time 7.45 minutes ¹⁰

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Examples 5 and b ⁹ '¹¹; Example 10

9.08 (w s, 1H), 9.03 (d, 7=8.0 Hz, 1H), 7.65 (s, 1H), 7.60 (d, J = 8.3 Hz, 1H) , 6.84 (d, 7=8.2 Hz, 1H), 4.98-4.91 (m, 1H), 4.53 -4.47 (m, 1H), 3.95 (s, ZN ), 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, 7= 13.9, 7.8 Hz, 1H), 1.80 (ddd, 7= 13.6, 9.6, 6.5 Hz, 1H), 1.76-1.68 (m, 1H), 1.54 (dd, 7 = 13.9, 5.1 Hz, 1H), 0 .95 (s, 9H);

High resolution MS m/z 590.2177 [M+H] ⁺ ; calculated for C26H30F6N5O4, 590.2202; holding time 7.70 minutes ¹⁰ 12.76 (w s, 1H), 9.19-9.10 (m, 1H), 9.06 - 9.00 (m, 1H), 7.69 (s, 1H), 7.49 (AB quartet, 7dv = 8.6 Hz, APav = 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, 7= 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 minutes ¹⁰ 12.62 (s, 1H), 9.14 (d, 7= 7.5 Hz, 1H), 9.03 (d, 7= 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, 7= 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 minutes ¹⁰

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42 Examples Example 36 F ₃ Cy^ °<^ΝΗ _n 0 0Η ₃ δ ί Η Ν gsn ₃ CH ₃ 8.92 (d, J= 8.0 Hz, 1H), 8.45 (d, 7= 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), 2.192.00 (m, 2H), 2.08 (s, 6H), 1.81 -1.64 (m, 2H), 1.49 (d, 7=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 minutes ¹⁰ 43 Examples Example 36 Άζ ΖΞΕ Ο 04? °5......<° \=/ ζ-ο ό CO ΞΕ 8.93 (d, 7=8.1 Hz, 1H), 8.42 (d, 7=8.1 Hz, 1H), 7.71 (s, 1H), 7.63 (AV quartet, 7dv= 7.6 Hz, APav=48.8 Hz, 4H), 4.974.88 (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 C25H3 ₅ F ₃ N ₅ O3, 510.2692; time retention 5.92 minutes ¹⁰ 44 Alternative synthesis of example 6; C26 °^ΝΗ ^F3C4 Qi ^η ϊ υ Η ξ Η ^Ν '-γΟΗ ₃ CH ₃ 2.85 minutes ⁴ ; 478.6 45 Alternative synthesis of example 6; C18 ^Ο ^ΝΗ ''Yaml^ Η Α ξ Η Ν K^СНз ГСНз СН ₃ 2.95 minutes ⁴ ; 492.6

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Examples 5 and 6 ¹³ ; 4

12.48 (s, 1H), 9.13 (d, 7=7.4 Hz, 1H), 8.97 (d, 7= 7.7 Hz, 1H), 7.70 (s, 1H), 7.62 (d.7 = 8.3 Hz, 1H), 6.86 (d, 7 = 8.3 Hz, 1H), 5.04-4.95 (m, 1H), 4.534.44 (m , 1H), 3.96 (s, ZN), 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 C ₂₅ H ₂₈ F ₆ N ₅ O ₄ , 576.2045; retention time 6.70 minutes ¹⁴

12.61 (s, 1H), 9.23 (d, 7=7.6 Hz, 1H), 9.01 (d, 7= 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, ZN), 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, 7 = 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 C ₂₆ H ₂₇ F ₉ N ₅ O ₄ , 644.1919; retention time 7.43 minutes ¹⁴

13.21 (s, 1H), 9.28 (d,7=7.5 Hz, 1H), 9.05 (d, 7= 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, ZN), 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, 7 = 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 C ₂₆ H ₂₇ F ₉ N ₅ O ₄ , 644.1919; retention time 7.51 minutes ¹⁴

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49 Alternative synthesis of example 6; C26 O-CF ₃ °·^ΝΗ Н Д = Η N Х^СНз CH ₃ 2.95 minutes ⁴ ; 494.4 50 Alternative synthesis of example 6; C18 0-CF3 °y _NH Qi n 0 A n D = η ⁼ \< ^CH z Gsnz CH ₃ 3.04 minutes ⁴ ; 508.4 51 Alternative synthesis of example 6; C18 ^р зС °γ·ΝΗ Ch n = Г Н S · Η N \<СНз Гсн ₃ СН ₃ 3.00 minutes ⁴ ; 492.4 52 Alternative synthesis of example 6; C18 °у-NH Ava-C n D Ξ Η N \<СНз Гсн ₃ СН ₃ 2.84 minutes ⁴ ; 458.4 (a picture of chlorine isotopes is observed) 53 Alternative synthesis of example 6; C18 __, ^0-СНз %-NH Oh Н 0 А \<СНз Гс ₃ СН ₃ 2.80 minutes ⁴ ; 468.5 54 Alternative synthesis of example 6; C18 °^NH Н S Ё η S< ^CH 3 Гсн ₃ CH ₃ 2.89 minutes ⁴ ; 458.4 (chlorine isotope pattern observed) 55 Alternative synthesis of example 6; C18 / ^Cl °·^-ΝΗ Η ο H D Ξ H ⁼ \< ^CH 3 GSN ₃ CH ₃ 2.90 minutes ⁴ ; 458.4 (chlorine isotope pattern observed)

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56 Alternative synthesis of example 6; C18 ^Cl \ °^NH Ob H 0 H J = n b K^CHZ UCH ₃ CH ₃ 2.89 minutes ⁴ ; 458.4 (chlorine isotope pattern observed) 57 Alternative synthesis of example 6; C18 °γ ΝΗ ³ n = n Ν \<СНз 1 сн ₃ сн ₃ 2.95 minutes ⁴ ; 492.4 58 Alternative synthesis of example 6; C18 , ^C| °<^ΝΗ ^С1 ^1 n 0 Х^СНз иСН ₃ СНз 3.16 minutes ⁴ ; 492.3 (the dichloroisotope pattern is observed) 59 Alternative synthesis of example 6; C18 ,CF ₃ °·^ΝΗ pьч Ш<СНз Гсн ₃ СНз 2.99 minutes ⁴ ; 492.4 60 Alternative synthesis of example 6; C26 F ₃ C NH 01noA H 4 E Η N -уСНз CH ₃ 2.91 minutes ⁴ ; 478.4 61 Alternative synthesis of example 6; C26 °γ-ΝΗ N D = Η Ν Ch^CHNz CH ₃ 2.74 minutes ⁴ ; 444.4 (a picture of chlorine isotopes is observed) 62 Alternative synthesis of example 6; C26 О-СН ₃ °γ-ΝΗ HaOVbb АуСН ₃ СН ₃ 2.70 minutes ⁴ ; 454.4

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63 Alternate explicit synthesis of example 6; C26 °у-мн Н Д = Η Ν Х^СНз СН ₃ 2.80 minutes ⁴ ; 444.4 (a picture of chlorine isotopes is observed) 64 Alternative synthesis of example 6; C26 CI °yNH n ° ^N N ⁿ ° uНз ^N СН3 2.81 minutes ⁴ ; 444.4 (a picture of chlorine isotopes is observed) 65 Alternative synthesis of example 6; C26 Cl OyNH chulu N « i Η N 'уСНз СН ₃ 2.80 minutes ⁴ ; 444.4 (a picture of chlorine isotopes is observed) 66 Alternative synthesis of example 6; C26 °yNH N ° ^F =' ^C n J i n ChuNz CH ₃ 2.86 minutes ⁴ ; 478.4 67 Alternative synthesis of example 6; C26 ci °y, NH “ChuaAD N D = Η N Ch^CHZ CH ₃ 3.08 minutes ⁴ ; 478.3 (the dichloroisotope pattern is observed) 68 Alternative synthesis of example 6; C26 _CF ₃ 4—NH chuchu N 4 = Η ^χ Ν -уСНз CH ₃ 2.91 minutes ⁴ ; 478.4

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69 Examples 5 and ¹⁵ ; 7 °VNH iVs n o ° Cn, ^Ν UCH ₃ sn ₃ 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, 7= 8, 8, 4.4 Hz, IH), 3.20-3.14 (m, IH), 3.14 3.08 (μ, 1H), 2.66 (s, 3H), 2.42 - 2.34 (m, 1H), 2.20 - 2.08 (μ, 2H), 1.81 (ddd, J= 13.5, 9.4, 6.7 Hz, IH), 1.77- 1.64 (m, ЗН), 0.95 (s, 9Н); high-resolution MS m/z 513.1873 [М+Н] ⁺ ; calculated for C22H ₂₈ F ₃ N ₆ O3S, 513.1896; retention time 6.88 minutes ¹⁰ 70 Alternative synthesis of example 6; C18 F C °U\ ^{N Nz} \ X ) Xn _n O ° Uh ₃ ⁿ GSN ₃ sn ₃ 2.87 minutes ⁴ ; 474.5 71 Alternative synthesis of example 6; C18 F WITH UT |_| О ^Нзс ° Uh, ^N Гсн ₃ CH ₃ 2.70 minutes ⁴ ; 474.5 72 Alternative synthesis of example 6; C26 _Br °X> ^H JS Ξ H ^X N ^Н з ^с 4^CH ₃ сн ₃ 2.41 minutes ⁴ ; 481.5 (bromine isotope pattern observed) 73 Alternative synthesis of example 6; C26 H ₃ q _c , )5 H ₃ C ο Ξ Η N Х^СНз CH ₃ 2.30 minutes ⁴ ; 423.5 (a picture of chlorine isotopes is observed) 74 4 ^{16 Нз} % Ο °y-NH Η X Ξ Η ⁴ Ν ° \^ΟΗ ₃ сн ₃ 12.20 (s, 1H), 8.99 (d, 7=7.9 Hz, 1H), 8.97 (d, 7= 7.4 Hz, 1H), 7.70 (s, 1H), 7.24 (dd, 7= 8.8 Hz, 1H), 7.12 (d, half of AB quartet, 7= 8.2 Hz, 1H), 6.69 (d, 7= 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, 7= 13.5, 9.3, 6.8 Hz, 1H), 1.75 - 1.63 (m, 2H), 1.59 (dd, 7= 7.5, 6.8 Hz, 2H), 0.95 (d, 7= 6.5 Hz, 3H), 0.91 (d, 7= 6.5 Hz, 3H); high-resolution MS m/z 482.2391 [M+H] ⁺ ; calculated for C25H32N5O5, 482.2403; retention time 8.38 minutes ¹⁰

1. In this case, deprotection of C28 was carried out using methanesulfonic acid rather than hydrogen chloride.

2. The epimers from Example 25 and Example 26 were separated using supercritical fluid chromatography (Column: Chiral Technologies Chiralpak IB, 21x250 mm, 5 μm; Mobile phase: 9:1 carbon dioxide/methanol; Back pressure: 120 bar, Flow rate: 75 ml/min). The first eluting diastereomer was designated as Example 25 and the second eluting diastereomer as Example 26.

3. Conditions for analytical HPLC. Column: Chiral Technologies Chiralpak IB, 4.6x100 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 x 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% to 95% B, linear over 4.0

- 124 047572 minutes, then 95% B over 1.0 minute; flow rate: 2 ml/minute.

5. 1H NMR of example 30 before final purification:

1H NMR (400 MHz, methanol-dd) 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. Amide coupling with the corresponding carboxylic acid was carried out using 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide trioxide.

7. Example 4 (25 μm) was 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 was carried out for 0.75 h on a shaker with a water thermostat, maintained at 37°C. Incubation was stopped by adding the same volume of acetonitrile, after which the mixture was centrifuged at 1700 x g for 5 min, and the supernatant was subjected to vacuum centrifugation for approximately 1.5 h. Formic acid (0.5 ml), acetonitrile (0.5 ml), and water were added to the resulting mixture to make 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 x 250 mm; 5 μm; mobile phase A: water containing 0.1% formic acid; mobile phase B: methanol; gradient: 15% B over 5 min, then 15% to 35% B over 75 min, then 35% to 95% B over 10 min; flow rate: 0.8 ml/min). Fractions were collected every 20 s. The first eluting material, crude Example 33, eluted at 54.7 minutes, and Example 34 eluted at 55.3 minutes. Crude Example 33 was purified again using reverse-phase HPLC (column: Phenomenex Kinetex XB-C18, 2.1 x 100 mm, 2.6 μm; mobile phase A: water containing 0.5% acetic acid; mobile phase B: 9:1 acetonitrile/methanol; gradient: 10% B over 0.5 minutes, then 10% to 35% over 26.5 minutes, then 35% to 60% B over 3 minutes; flow rate 0.5 mL/min); fractions were collected every 15 seconds. On this system, Example 33 had a retention time of 12.7 minutes; Additional example 34 eluted at 13.5 minutes.

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

9. The reaction mixture was diluted with acetonitrile and 1% aqueous formic acid to a volume of approximately 2 ml; the final solvent composition was such that the mixture was clear, containing approximately 20% to 30% acetonitrile. The components of this mixture were separated using 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 over 5 min, then 15% to 70% B over 70 min, then 70% to 95% B over 15 min; flow rate: 2 ml/min); fractions were collected every 20 seconds. Examples 37, 38, 39, 40, and 41 eluted 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, 2.1x100 mm, 2.6 μm; mobile phase A: water containing 0.1% formic acid; mobile phase B: acetonitrile; gradient: 5% B over 0.5 min, then 5% to 70% B over 10.5 min, then 70% to 95% B over 2 min; flow rate: 0.4 mL/min.

11. The regiochemistry of Example 41 has not been precisely determined; other possible structures for this example are N-[(2S)-1-({(^)-1-cyano-2-[^)-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({(^)-1-cyano-2-[^)-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 footnote 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 x 250 mm, 10 μm; mobile phase A: water containing 0.1% formic acid; mobile phase B: acetonitrile; gradient: 2% to 10% B over 5.0 min, then 10% to 95% B over 95 min; flow rate: 2 mL/min); fractions were collected every 20 seconds. Examples 46, 47, and 48 were eluted with a retention time given by

- 125 047572 below. Example 5 was also isolated from this reaction, in fractions 189-190.

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

14. Analytical HPLC conditions. Column: Phenomenex Kinetex C18, 2.1x50 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 over 0.5 min, then 5% to 50% B over 6.0 min, then 50% to 80% B over 1.5 min, then 80% to 95% B over 1.0 min; flow rate: 0.4 mL/min.

15. Only the product shown was observed from this reaction.

16. A stock solution of Example 4 (5.56 mg, 12.7 μmol) and trifluoroacetic acid (4 μL, 50 μmol) 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), followed by the addition of tert-butyl hydroperoxide (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, containing approximately 20% to 30% acetonitrile. The components of this mixture were separated using reversed-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 over 5 min, then 15% to 40% B over 70 min, then 40% to 95% B over 15 min; flow rate: 2 ml/min); 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-cyano2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-1-{N-[(trifluoromethyl)sulfonyl]-L-valyl}piperidine-2-carboxamide [75 (DIAST-1) and 76 (DIAST-2)

Q A P NH ₂ CF ₃ ^x aS'CF n ₃ s.-So sn ₃ ^{3 0 0 sn} z O Gn ^C z ^{Nz NEt} 3 • HCI Η θ Λ'%Η SOCI ₂ (+/-) · HCI „ „„ НзСфСНз ^Μβ0Η СН ₃ % ^XF 3 HN 'δ H3C^-k^O 1 T 0 ^сн здм^.А°,сн ^з Н3сфсН ₃ CH _{3 c} %.CF ₃ HN '0 НзС^-С^О H ₃ C 1 T o ^CH 0%H - НзСфсНз ^СНз C48 - 4..CF3 °b.CF ₃ 3 HN-% CF3COOH _HN -S ^{3 HZi} L ¹ %G ⁱ E< ^uHz * ^{H z s} y'^OH C44 C45 ^Q S' ^CF 3 HN'% Η _{3 0 Ύ} ·< on ,NA ,CH ₃ ^CH 3O П ⁰ C45 (+/-) T * ^HUI HATU * ^Hz A ^Hz gu ^CHz C46 % ^XF 3 HN '0 HzC^-k^O T 9 LiOH + ^CH 3 Nyk ₀ XH ₃ ----- H ₃ C CSN ₃ SN ₃ 47 °<^NH %CF ₃ * % ^XH z _; ^NH Λ h ₂ n\ ^nh 2 ^НСг '0 CH 1 ? ^{0 C16 CH3} _r ^N AoH ------------► H HATU H ₃ C yCH ₃ f^N' ^CH3 CH ₃ 0^

- 126 047572

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

A solution of trifluoromethanesulfonic anhydride (8.88 mL, 52.8 mmol) in dichloromethane (10 mL) was added to -78 °C with a solution of tert-butyl L-valinate, hydrochloride salt (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 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 similar reactions using tert-butyl L-valinate, hydrochloride salt (1.00 g, 4.77 mmol; 1.00 g, 4.77 mmol) and purified using 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-d,,) 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]-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 and 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-de) δ 9.86 (br d, 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 salt (C46).

At 0°C, thionyl chloride (6.44 g, 54.1 mmol) was added to a solution of cis-4-tert-butylpiperidine-2-carboxylic acid, hydrochloride salt (see R. T. Shuman et al., J. Org. Chem. 1990, 55, 738-741; 4.00 g, 18.0 mmol) in methanol (40 mL). After the reaction mixture was stirred at 25°C for 16 h, it was concentrated in vacuo to give C46 as an off-white solid (4.50 g). A portion of this material 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, assumed; largely obscured by the hydro 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).

At 25 °C, 4-methylmorpholine (365 mg, 3.61 mmol) was added to a 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). The resulting mixture was cooled to 0 °C and treated with O-(7-azabenzotriazol-1-yl)-NHN,N-tetramethylaronium hexafluorophosphate (HATU; 549 mg, 1.44 mmol). After sparging the reaction mixture with nitrogen for 1 min, it was stirred at 25 °C for 12 h. LCMS analysis now 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 x 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. H NMR analysis confirmed that this gum contained a mixture of diastereomers. Yield: 320 mg, 0.743 mmol,

- 127 047572

62%.

¹ H 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), overall 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 solution (15 ml), dried over sodium sulfate, filtered, and concentrated in vacuo to give C48 as a yellow glass. H NMR analysis confirmed that this material contained a mixture of diastereomers. Yield: 304 mg, quantitative.

1H NMR (400 MHz, DMSO-D δ [9.82 (d, J = 8.7 Hz) and 9.69 (br d, J = 8.8 Hz), overall 1H], [4.28 (dd, J = 11.5, 6.4 Hz), 4.24-4.14 (m), and 4.05-3.96 (m), overall 2H], [3.80-3.69 (m) and 3.6-3.2 (m, assumed; largely obscured by water peak), overall 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), overall 3H], [0.89 (d, J = 6.9 Hz) and 0.86-0.80 (m), in total 12H].

Step 6. Synthesis of (2R,4S)-N-{(2S)-1-amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-4-tert-butyl-1-{N[(trifluoromethyl)sulfonyl]-N-valyl}piperidine-2-carboxamide and (2R,4S)-N-{(2S)-1amino-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-4-tert-butyl- 1-{N[(trifluoromethyl)sulfonyl]-N-valyl}piperidine-2-carboxamide (C49)

At 25 °C, 4-methylmorpholine (100 mg, 0.989 mmol) was added to a mixture of C16 (120 mg, 0.449 mmol) and C48 (144 mg, 0.346 mmol) in N,N-dimethylformamide (3 mL), the mixture was cooled to 0 °C and treated with O-(7-azabenzotriazol-1-yl)-N,N-tetramethylaronium hexafluorophosphate (HATU; 151 mg, 0.397 mmol). The reaction mixture was sparged with nitrogen for 1 min and then stirred at 25 °C for 12 h. LC-MS analysis indicated the presence of C49: LC-MS 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 subjected to silica gel chromatography (Gradient: 0% to 15% methanol in dichloromethane) to afford C49 as a white solid. This material contained a mixture of diastereomers, by ¹ H NMR analysis. Yield: 190 mg, 0.334 mmol, 96%.

1H NMR (400 MHz, DMSOD). characteristic peaks, integrations are approximate: δ [9.88 (d, J = 8.6 Hz) and 9.82-9.68 (m), overall 1H], [8.12 (d, J = 8, 8 Hz) and 8.09-7.98 (m), overall 1H], [7.63 (s) and 7.57 (s), overall 1H], [7.30 (br s) and 7 ,18 (w s), overall 1H], [7.06 (w s) and 7.03 (w s), overall 1H], [4.36 (dd, J = 12.0, 6.1 Hz) and 4.32-4.08 (m), overall 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 (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 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, after which the reaction mixture was diluted with water (20 mL) and extracted with dichloromethane (2 x 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, by LC-MS analysis, contained an approximately 3:1 mixture of the products: LC-MS m/z 552.2 [M+H]+ and LC-MS m/z 552.2 [M+H]+. These diastereomers were separated using supercritical fluid chromatography [Column: Chiral Technologies Chiralpak IG, 30x250 mm, 10 μm; mobile phase: 3:1 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide); flow rate: 70 mL/min]. The first eluting diastereomer, isolated as a white solid, was designated 75 and the second eluting diastereomer, also as a white solid, was designated 76 (2R,4S)-4-tert-butyl-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-1-{Na[(trifluoromethyl)sulfonyl]-1-valyl}piperidine-2-carboxamide and (2R,4S)-4-tert-butyl-Na{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-1-{Na[(trifluoromethyl)sulfonyl]-1-valyl}piperidine-2-carboxamide.

- 128 047572 valyl}piperidine-2-carboxamide].

- Yield: 26.2 mg, 47.5 µmol, 14%.

1H NMR (400 MHz, DMSO-b6) 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 min (Analytical conditions: Column: Chiral Technologies Chiralpak IG3, 4.6 x 50 mm, 3 µm; Mobile phase A: Carbon dioxide; Mobile phase B: Ethanol containing 0.05% diethylamine; Gradient: 5% to 40% B over 2 min, then 40% B over 1.2 min; Flow rate: 4 mL/min; Back pressure: 1500 psi).

- Yield: 8.8 mg, 16 µmol, 5%. LC-MS m/z 552.3 [M+H]+. According to ¹ H NMR analysis, this sample 76 contained impurities.

1H NMR (400 MHz, DMSO-dA, 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 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)G-prolinamide (77)

H ₃ C

H ₃ SF ^SN z

O^· ⁰ o ^Ν γΛ _0Η %-NH

X> CF ₃ ~ η ₂ ν\- ^ΝΗ2 hatu

O · HCI ^ SN ₃

C16, HCI salt H ₃ C^N^CH ₃

CH ₃ CH ₃

HCI ;

Step 1. Synthesis of (4R)-1-(tert-butoxycarbonyl)-4-(trifluoromethyl)-L-propyl-3-[(3S)-2oxopyrrolidin-3-yl]-L-alanine amide (C50).

At -30°C, N,N-diisopropylethylamine (0.791 mL, 4.54 mmol) was added to a 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 H-dimethylformamide (7.8 mL), followed by the addition of C-(7-azabenzotriazol-1-yl)HN,N-tetramethylaronium hexafluorophosphate (HATU; 633 mg, 1.66 mmol). The reaction mixture was allowed to warm to 0°C over 1 h, after which it was diluted with aqueous sodium bicarbonate (30 mL) and extracted with a mixture of 2-butanol and dichloromethane (9:1, 3 x 7 mL). The combined organic layers were concentrated in vacuo and purified using silica gel chromatography (Gradient: 0% to 100% methanol in dichloromethane) to give C50 as an off-white foam. By 1H NMR ana

- 129 047572 lysis, this material existed as a mixture of rotamers and contained impurities derived from the reagents used; a portion of this sample was used in the next step. Yield: 613 mg, 1.40 mmol, 93%. LC-MS m/z 459.3 [M+Na+].

1H NMR (400 MHz, DMSO-d6), only characteristic peaks of the product: 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-propyl-3[^)-2-oxopyrrolidin-3-yl]^-alanine amide (C51).

A mixture of C50 (242 mg, 0.554 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4 M; 2 mL, 8 mmol) was stirred at room temperature for 5 min, after which the reaction mixture was concentrated in vacuo to remove the solvent and residual hydrogen chloride. The deprotected material was combined with N-(tert-butoxycarbonyl)-3-methyl-N-valine (128 mg, 0.553 mmol) and O-(7-azabenzotriazol-1-yl)-HN-N-,N-tetramethylaronium hexafluorophosphate (HATU: 232 mg, 0.610 mmol) in H-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 using silica gel chromatography (Gradient: 0% to 30% methanol in dichloromethane) to give C51 as a solid. Yield: 230 mg, 0.418 mmol, 75%. LC-MS m/z 550.3 [M+H]+.

Step 3. Synthesis of 3-methyl-N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-^-prolinamide (77).

A mixture of C51 (230 mg, 0.418 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4 M; 2 mL, 8 mmol) was stirred at room temperature for 5 min, after which the reaction mixture was concentrated in vacuo to remove the solvent and residual hydrogen chloride. The resulting deprotected material 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). After the reaction mixture was 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), ethyl N(triethylammoniosulfonyl)carbamate inner salt (Burgess reagent; 299 mg, 1.25 mmol) was added, and the reaction mixture was stirred at room temperature for 2 h, after which it was treated with additional methyl N(triethylammoniosulfonyl)carbamate inner salt (Burgess reagent; 100 mg, 0.420 mmol) and allowed to stir 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 using supercritical fluid chromatography (column: Princeton Dinitrophenyl, 10x250 mm, 5 μm; mobile phase: 9:1 carbon dioxide/methanol; back pressure: 120 bar; flow rate: 80 ml/min) gave material that 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-oxopyrroludin-3-yl]ethyl}-4(trifluoromethyl)-N-prolinamide (77) as a solid. Yield: 64 mg, 0.121 mmol, 29%. LC-MS m/z 528.2 [M+H]+.

1H NMR (400 MHz, DMSO-46) δ 9.46 (d, J = 8.4 Hz, 1H), 9.05 (d, J = 8.6 Hz, 1H), 7.67 (s, 1H ), 4.96 (dd, 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, assumed; 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.

(^^^)-N{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl(methylcarbamoyl)^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (78)

About CH ₃

hnAd^?h ^{3 H3C} >^° ³ HCI H ₃ C$ _I T O --- ^cn z n d Q Ъ'CH3 H3CCH3 C31 ΝΗ ₂ o ίοΥ Ύ° o sLg ^sn z ^{3 sn} back ⁿ g \ G Ъ'СНз V/ NEt ₃ y · HCI ³ H ₃ C CH ₃ C52

- 130 047572

Step 1. Synthesis of the hydrochloride salt of methyl (1H^^)-6,6-dimethyl-3-(3-methyl-k-1%1lyl)-3azabicyclo[3.1.0]hexane-2-carboxylate (C52).

At 0°C, a solution of hydrogen chloride in ethyl acetate (4 M; 20 mL, 80 mmol) was added dropwise to a solution of C31 (1.00 g, 2.61 mmol) in dichloromethane (20 mL). After the reaction mixture was stirred at 25°C overnight, it was concentrated in vacuo to give C52 as a white gum. Yield: 700 mg, 2.20 mmol, 84%. LC-MS m/z 283.1 [M+H]+.

¹ H NMR (400 MHz, DMSO-06) 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 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 (^^^)-6,6-dimethyl-3-[3-methyl-N(methylcarbamoyl)-N-valyl]-3azabicyclo[3.1.0]hexane-2-carboxylate (C53).

At 0°C, triethylamine (0.769 ml, 5.52 mmol) and methylcarbamyl chloride (188 mg, 2.01 mmol) were slowly added to a solution of C52 (320 mg, 1.00 mmol) in dichloromethane (6 ml). The reaction mixture was allowed to warm to 20°C and stirred for 18 h, after which 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. Silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) gave C53 as a light yellow gum. Yield: 190 mg, 0.560 mmol, 56%. LC-MS m/z 339.9 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d..i δ 6.03 (d, J = 9.4 Hz, 1H), 5.89 (b q, 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 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 (^^^)-6,6-dimethyl-3-[3-methyl-N(methylcarbamoyl)-N-valyl]-3azabicyclo[3.1.0]hexane-2-carboxylic acid (C54).

At 0°C, lithium hydroxide monohydrate (82.0 mg, 1.95 mmol) was added to a solution of C53 (190 mg, 0.560 mmol) in a mixture of tetrahydrofuran (2 ml), water (4 ml), and methanol (1 ml). After the reaction mixture was stirred at 20°C for 2 h, it was diluted with ethyl acetate (10 ml); the aqueous layer was then cooled to 0°C to 5°C and acidified to pH 2-3 by the addition of 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 give C54 as a white solid. Yield: 120 mg, 0.369 mmol, 66%. LC-MS m/z 348.3 [M+Na ⁺ ].

¹ H 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 (s, 3H).

Step 4. Synthesis of (^^^)-N-{(2^)-1-amino-1-oxo-3-[(2^)-2-oxopyrrolidin-3-yl]propan-2-yl)6,6-dimethyl-3-[3-methyl-N-(methylcarbamoyl)-N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (C55).

At 0°C to 5°C, O-(7-azabenzotriazol-1-yl)-HN-N-tetramethylaronium hexafluorophosphate (HATU; 154 mg, 0.405 mmol) and 4-methylmorpholine (0.144 mL, 1.31 mmol) were added to a solution of C54 (120 mg, 0.369 mmol) and C16, HCl salt (75%, 107 mg, 0.387 mmol) in H2-dimethylformamide (3.0 mL) at 0°C to 5°C. After the reaction mixture was allowed to warm from 0°C to 20°C over 1.5 h, it was allowed to stir.

- 131 047572 at 20°C for 18 h, after which it was diluted with water and treated with solid sodium sulfate to saturation. The resulting mixture was extracted with a mixture of 2-propanol and chloroform (1:4, 3 x 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 material was used in the next step. LC-MS m/z 479.2 [M+H]+. By ¹ H NMR analysis, this material was contaminated with a by-product derived from HATU reagent.

^1H NMR (400 MHz, DMSO^6), characteristic product peaks only: δ 8.21 (d, J = 8.7 Hz, 1H), 7.53 (w s, 1H), 7.29 (w s , 1H), 7.03 (w s, 1H), 6.02 (d, J = 9.6 Hz, 1H), 5.86 (kv, 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 the AB quartet, J = 7.6 Hz, 1H), 1.01 (s, 3H), 0.90 (s, 9H), 0.84 (s, 3H ).

Step 5. Synthesis of (^^^)-i-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3[3-methyl-i-(methylcarbamoyl)-1-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 and 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 x 10 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo; Purification using reversed-phase HPLC (column: Boston Prime C18, 30x150 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) gave (1R,2S,5S)-N-{(1S)-1-cyano-2-[(1R)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-n-(methylcarbamoyl)-N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (78) as a white solid. Pooled yield: 25 mg, 54 μmol, 15% over 2 steps. LC-MS m/z 461.2 [M+H]+.

^1H NMR (400 MHz, DMSO^), 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 (kv, 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 (w s, 1H), 5.04 (w 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 (wdd, 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)A-[(1 R,2S,5S)-2-({(1S)-1-cyano-2-[(3 S)-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)

O . O HN%' ^CH 3

^Нзі >^° X ГН ^зс снзХ $ ^с|Л °' ^сн : V °' ^СНЗ NEt ₃ У · HCI Н ₃ с СНз С52 °-yNH n Ао-снз ^Нз ?> С16, HCI salt Н3С р|_| I O -----------------► V ^N yA HATU u ^he N ₃ C CHz C57 icY'S^ ^{0 l} ' ^{oh Н} 3 ^с А|_1 О _______► ^CH V ^N vA О Y-СНз H3CCH3 С56, сн _{3 p} HN ° _о %NH Α _ν Λη ₂ И ^Н ° Н ₃ сНз С58

- 132 047572

Step 1. Synthesis of methyl (1K^^)-3-[V-(meth(oxycarb(nyl)-3-methyl-1,-1-yl)-6,6-dimethyl-3azabicyclo[3.1.0]hexane-2-carboxylate (C56).

At 0°C, triethylamine (0.647 ml, 4.64 mmol) and methyl chloroformate (335 mg, 3.55 mmol) were slowly added to a solution of C52 (370 mg, 1.16 mmol) in dichloromethane (6 ml). After the reaction mixture was stirred at 20°C for 16 h, it was diluted dropwise with saturated aqueous sodium carbonate solution (5 ml) and extracted with dichloromethane (2 x 20 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. 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%. LC-MS m/z 341.1 [M+H]+.

^1H NMR (400 MHz, chloroform-d) δ 5.29 (wd, 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 (w s, 3H), 1.49-1.41 (m, 2H), 1 .04 (s, 3H), 1.03 (s, 9H), 0.91 (s, 3H).

Step 2. Synthesis of (^^^)-3-[N-(methoxycarbonyl)-3-methyl-^-valyl]-6,6-dimethyl-3azabicyclo[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 to 25°C) for 16 h, then concentrated in vacuo. The aqueous residue was partitioned between water (5 mL) and ethyl acetate (20 mL), after which the organic layer was discarded, and the aqueous layer was adjusted to pH 1-2 by the addition of 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%. LC-MS m/z 327.2 [M+H]+.

¹ H 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).

Step 3. Synthesis of methyl (^)-1-[(^^^)-2-({DO)-1-amino-1-oxo-3-[^)-2-oxopyrrolidin-3yl]propan-2-yl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2yl}carbamate (C58).

At 0°C, O-(7-azabenzotriazol-1-yl)-HNH,N-tetramethylaronium hexafluorophosphate (HATU; 140 mg, 0.368 mmol) was added to a 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), 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, after which water (10 mL) was added. After solid sodium sulfate was added to saturation, the resulting mixture was extracted with a mixture of chloroform and 2-propanol (4:1, 3 x 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%. LC-MS m/z 480.0 [M+H]+.

¹ H 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. Synthesis of methyl {^)-1-[(^^^)-2-({(^)-1-cyano-2-[^)-2-oxopyrrolidin-3yl]ethyl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2yl}carbamate (79).

Methyl N(triethylammoniosulfonyl)carbamate inner salt (Burgess reagent; 139 mg, 0.583 mmol) was added to a suspension of C58 (93 mg, 0.19 mmol) in dichloromethane (5 ml), 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

- 133 047572 sodium chloride (2x10 ml), dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 100% ethyl acetate in petroleum ether, followed by 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]hexan3-yl]-3,3-dimethyl-1-oxobutan-2-yl}carbamate (79) as a white solid. Yield: 7.0 mg, 15 µmol, 8%. LC-MS m/z 462.2 [M+H]+.

1H NMR (400 MHz, chloroform-d) δ 8.13 (br d, J = 7.0 Hz, 1H), 5.68 (br s, 1H), 5.34 (br d, 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, assumed; partially obscured by the 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)G-prolinamide (80)

Step 1. Synthesis of 2-benzyl 1-tert-butyl (2S,4R)-4-(mfluoromethyl)pyrrolidine-1,2-dicarboxylate (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-dimethylformamide (8 mL) was stirred for 15 h at 25°C. After the reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (3 x 30 mL), 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. Silica gel chromatography (Gradi

- 134 047572 ent: 0% to 30% ethyl acetate in petroleum ether) gave C59 as a colorless oil. According to ¹ H NMR analysis, this material existed as a mixture of rotamers. Yield: 355 mg, 0.951 mmol, 67%.

¹ H NMR (400 MHz, chloroform-d ) δ 7.44-7.28 (m, 5H), 5.29-5.07 (m, 2H), [4.54 (br d, J = 8.6 Hz) and 4.40 (br dd, 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 9N].

Stage 2. Synthesis of benzyl (4R)-4-(trifluoromethyl)-L-prolinate, hydrochloride salt (C60).

At 0°C, a solution of hydrogen chloride in ethyl acetate (4 M; 6 mL, 24 mmol) was added to a solution of C59 (200 mg, 0.536 mmol) in ethyl acetate (3 mL). After the reaction mixture was stirred at room temperature (28°C) for 3 h, it was concentrated in vacuo to give C60 as a white solid; this material was used directly in 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-tetramethylaronium hexafluorophosphate (HATU; 277 mg, 0.728 mmol) and 4-methylmorpholine (184 mg, 1.82 mmol) were added at 0 °C to a mixture of C60 (from the previous step; < 0.536 mmol) and N-(tert-butoxycarbonyl)-N-valine (158 mg, 0.727 mmol) in N,N-dimethylformamide (3 mL). The reaction mixture was stirred at 0 °C for 1 h, then poured into ice water (15 mL) and extracted with ethyl acetate (2 x 15 mL). The combined organic layers were washed successively with

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 (Gradient: 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+].

¹ H NMR (400 MHz, chloroform-d), characteristic peaks: δ 7.40-7.30 (m, 5H), 5.17 (AB quartet, Jab = 12.3 Hz, ΔνΑΒ = 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 salt (C62).

At 0°C, a solution of hydrogen chloride in ethyl acetate (4 M; 4 mL, 16 mmol) was added to a solution of C61 (230 mg, 0.487 mmol) in ethyl acetate (2 mL). The reaction mixture was stirred at room temperature (28°C) for 1 h, after which LC-MS analysis showed conversion to C62: LC-MS m/z 373.1 [M+H]+. Concentration of the reaction mixture in vacuo gave C62 as a white solid, which was used directly in 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 at 0 °C to a 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 solution (10 mL) and saturated aqueous sodium chloride solution (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. LC-MS m/z 491.2 [M+Na+].

Step 6. Synthesis of N-(trifluoroacetyl)-L-valyl-(4R)-4-(Ίfluoromethyl)-L-proline (C64).

At 28°C, palladium on carbon (10%, 29.3 mg, 27.5 μmol) was added to a solution of C63 (129 mg, 0.275 mmol) in methanol (3 mL), and the mixture was hydrogenated at 15 psi for 16 h. Filtration yielded a filter cake, which 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-propyl-3-[(3S)-2oxopyrrolidin-3-yl]-L-alanine amide (C65).

O-(7-Azabenzotriazol-1-yl)-H,H2S,N-tetramethylaronium hexafluorophosphate (HATU; 88.5 mg, 0.233 mmol) and 4-methylmorpholine (64.2 mg, 0.635 mmol) were added at 0 °C to a solution of C64 (80 mg, 0.21 mmol) and C16 (76.8 mg, 0.287 mmol) in N,N-dimethylformamide (3 ml). After the reaction mixture was stirred at 0 °C for 2 h, it was treated with water (10 ml) and aqueous citric acid (1 M; 10 ml, 10 mmol), then extracted with ethyl acetate (3 x 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 using silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) afforded C65 as a white solid. Yield: 72 mg, 0.14

- 135 047572 mmol, 67%. LC-MS m/z 532.2 [M+H]+.

Step 8. Synthesis of N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3yl]ethyl}-4-(trifluoromethyl)N-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 x 15 mL), and 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. 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)N-prolinamide (80) as a white solid. Yield: 30.9 mg, 60.2 μmol, 43%. LC-MS m/z 536.1 [M+Na+].

¹ H NMR (400 MHz, DMSO-d^, 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)-N-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-(trifluoroacetyl)-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide.

Example 83: (1 R,2S,5S,6S)-N-{(1S)-1-cyano-2-[(3 S)-2-oxopyrrolidin-3-yl]ethyl}-6(hydroxymethyl)-6-methyl-3-[3-methyl-(trifluoroacetyl)-1-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide.

Example 84: (^^^)-N{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-(hydroxymethyl)^(trifluoroacetyl)-C-valyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide.

Examples 81-84 were prepared using both in vitro and in vivo biotransformation pathways from (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Example 13 compound) as described below. In vitro studies, (^^^)-N{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3yl]ethyl}-6,6-dimethyl-3-[3-methyl^-(trifluoroacetyl)-C-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 with rat, monkey, or human hepatocytes (see Table M2 below). Alternatively, in vivo studies, (^^^)^-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3[3-methyl-^(trifluoroacetyl)-C-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 were then analyzed using HPLC/MS, and the resulting oxidized metabolites of the compounds of Examples 81-84 were detected and recovered. In addition, compounds of Examples 81-84 were observed in in vivo studies to have an additional metabolite, (^^^)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, which was obtained by hydrolytic cleavage.

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Compounds from Examples 81-84, which were obtained from hepatocytes

Compounds from Examples 81-84, which were obtained in vivo in rats or monkeys

In Tables M1, M2 and M3 the following abbreviations are used: - = not detected; + = detected by mass spectrometry and a small UV peak; ++ = detected by mass spectrometry and a moderate UV peak; +++ = detected by mass spectrometry and a large UV peak; t = traces, detected by mass spectrometry only.

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-^-(trifluoroacetyl)^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (82), (^^^^)^-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3[3-methyl-^-(trifluoroacetyl)^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (83), (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-(hydroxymethyl)^(trifluoroacetyl)^-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-[3methyl^-(trifluoroacetyl)^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (81)

81

Example 13 (25 μm) was 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 was carried out for 55 minutes on a shaker with a water thermostat maintained at 37°C. The reaction was stopped by adding the same volume of acetonitrile, after which the mixture was centrifuged at 1800 x g for 5 minutes, and the supernatant was 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

- 137 047572 li at 40000 x g for 30 min. The supernatant was applied to an HPLC column (Polaris C18, 4.6 x 250 mm; 5 μm) at a flow rate of 1 ml/min using a Jasco HPLC pump. After use, the column was transferred to a Waters Acquity HPLC-UV system coupled to a Thermo LTQ mass spectrometer, and the CTC Analytics fractions were collected and separated using reversed-phase HPLC (Mobile phase A: water containing 0.1% formic acid (v/v); mobile phase B: acetonitrile; gradient: 2% over 5 min, then washed to 15% B, then 15% to 60% B over 80 min, then 60% to 95% B over 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)-C-valyl]-3azabicyclo[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)-C-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide (83), (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-(hydroxymethyl)-N-(trifluoroacetyl)-C-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)-C-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (81).

Example number Retention time, HPLC purification (minutes) 82 37.2 83 39.3 84 46.6 81 50.5

- Yield: 60 µg, 0.12 µmol, 12%. High Resolution MS m/z 516.2424 [M+H]+; calculated for C ₂₃ H33F ₃ N ₅ O ₅ , 516.2434.

^1H NMR (600 MHz, DMSO^ ₆ ), characteristic peaks: δ 9.42 (wd, 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 AB system, J = 10.2, 5.4 Hz, 1H), 3.68 (d, half of the AB quartet, J = 10, 4 Hz, 1H), 3.213.17 (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).

- 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 the AB quartet, J = 7.3 Hz, 1H), 1.06 (s, 3H), 0.98 (s, 9H).

- 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^ ₆ ), 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 (w dd, 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 (w s, 6H).

- Yield: 130 µg, 0.252 µmol, 25%. This material was determined to exist as an interconverted mixture of stereoisomers relative to the carbinolamine moiety of pyrrolidone (see Examples 81 and 82). High-resolution MS m/z 516.2428 [M+H] ⁺ ; calculated for C ₂₃ H ₃₃ F ₃ N ₅ O ₅ , 516.2434.

1H NMR (600 MHz, DMSO-D6) δ [9.40 (d, J = 8.4 Hz) and 9.38 (d, J = 8.2 Hz), overall 1H], [8.99 (d, J = 8.5 Hz) and 8.92 (d, J = 7.6 Hz), overall 1H], [8.37 (s) and 8.25 (s), overall 1H], [5.83 (br s) and 5.70 (br s), overall 1H], 5.04-4.92 (m, 2H), 4.44-4.38 (m, 1H), [4.19 (s) and 4.15 (s), overall 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), overall 1H], [2.65-2.57 (m), 2.43-2.30 (m), and 2.21-2.13 (m), overall 2H], [2.08 (ddd, J = 13.7, 8.4, 6.2 Hz), 2.00-1.90 (m), and 1.87-1.79 (m), overall 2H], [1.78-1.70 (m) and 1.51-1.44 (m), overall 1H], 1.60-1.53 (m, 1H), [1.32 (d, J = 7.6 Hz) and 1.29 (d, J = 7.6 Hz), overall 1H], 1.03 (s, 3H), [0.99 (s) and 0.98 (s), overall 9H], [0.85 (s) and 0.84 (s), overall 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)N-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)N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (85)

- 138 047572

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). An 18-gauge syringe needle was inserted through the Teflon cap of the container, and the air-exposed reaction 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 solution (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 enough acetonitrile to maintain the solution. The resulting solution was divided equally and 5 g Biotage Isolute C18 solid phase extraction cartridges were applied to both portions. 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 diluted to the original concentration in a mixture of acetonitrile and 1% aqueous formic acid and combined to a total of 2 mL of solution. This material was subjected to reverse-phase HPLC (column: Phenomenex Luna C18, 10 x 250 mm, 10 μm; mobile phase A: water containing 0.1% formic acid (v/v); mobile phase B: acetonitrile; gradient: 2% to 15% B over 5 min, then 15% to 60% B over 80 min, then 60% to 95% B over 5 min; flow rate: 2 ml/min). Fractions were collected every 20 s; The first eluting compound 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)-D-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (81), and the second eluting compound was (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3R)-2,5-dioxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N(trifluoroacetyl)-D-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (85).

Yield 81: 0.122 mg, 0.237 µmol, 12%. This material was determined to exist as an interconverted mixture of stereoisomers relative to the carbinolamine moiety of pyrrolidone and eluted as a double peak on HPLC. High Resolution MS m/z 516.2413 [M+H]+; calculated for C23H33F3N5O5, 516.2434.

¹ H NMR (600 MHz, DMSO-dj δ 9.44-9.34 (m, 1H), [8.99 (d, J = 8.5 Hz) and 8.92 (d, J = 7.6 Hz), overall 1H], [8.37 (s) and 8.25 (s), overall 1H], [5.83 (br s) and 5.70 (br s), overall 1H], 5.05-4.91 (m, 2H), 4.44-4.37 (m, 1H), [4.19 (s) and 4.15 (s), overall 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), overall 1H], [2.65-2.57 (m), 2.43-2.30 (m), and 2.17 (ddd, J = 14.9, 10.7, 4.7 Hz), overall 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), overall 2H], [1.78-1.70 (m) and 1.51-1.44 (m), overall 1H], 1.60-1.53 (m, 1H), [1.32 (d, J = 7.6 Hz) and 1.29 (d, J = 7.6 Hz), overall 1H], 1.03 (s, 3H), [0.99 (s) and 0.98 (s), overall 9H], [0.85 (s) and 0.84 (s), overall 3H].

Retention time: 7.7 minutes (Analytical conditions. Column: Phenomenex Kinetex XB-d8, 2.1x100 mm, 2.6 μm; mobile phase A: water containing 0.1% formic acid (v/v); mobile phase B: acetonitrile; gradient: 5% B over 0.5 minutes, then 5% to 70% B over 10.5 minutes, then 70% to 95% B over 2 minutes; flow rate: 0.4 mL/min).

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-d ,.) 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 (m, 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 identical to those used for 81).

- 139 047572

Example 86.

(1I<2S,5S)-N-{(1S)-1-iiuaim-2-[(3S)-2-()i<c()iiupp()ju;uui-3-uj]9TUj}-6 ,6-guvieTUj-3-[5.5.5-Tpu(|)T()p-2(2,2,2-trifluoroacetamido)pentanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide(86)

Step 1. Synthesis of tert-butyl (^^^)-2-({DO)-1-amino-1-oxo-3-[^)-2-oxopyrrolidin-3yl]propan-2-yl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (C66).

At 0°C, N,N-diisopropylethylamine (7.97 g, 61.7 mmol) was added to a suspension of (^^^)-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 1oxide (571 mg, 5.14 mmol) in butan-2-one (108 mL), followed by the addition of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (4.73 g, 24.7 mmol). After the reaction mixture was stirred at 0 °C for 20 min, it was gradually allowed to warm to room temperature and then stirred at room temperature overnight, after which LC-MS analysis indicated the presence of C66: LC-MS 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 collected 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). By ¹ H NMR analysis, some solvents were present; the purity was estimated to be approximately 85% by weight. ¹ H NMR analysis also indicated that this material existed as a mixture of rotamers. Yield, corrected for solvents: 5.7 g, 14 mmol, 68%.

¹ H NMR (400 MHz, DMSO-66), characteristic peaks: δ 8.23-8.13 (m, 1H), [7.63 (br s) and 7.59 (br s), overall 1H], [7.36 (br s) and 7.23 (br s), overall 1H], 7.08-7.00 (m, 1H), 4.31-4.19 (m, 1H), [4.03 (s) and 3.99 (s), overall 1H], [3.58 (dd, J = 10.8, 4.6 Hz) and 3.49 (dd, J = 10.8, 3.9 Hz), overall 1H], [3.27 (d, J = 10.9 Hz) and 3.26 (d, J = 10.7 Hz), overall 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 methanesulfonate salt of (1H^^)^-{^)-1-amino-1-oxo-3-[^)-2oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, (C67).

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Methanesulfonic acid (0.920 mL, 14.2 mmol) was added to a solution of C66 (approximately 85% by weight, 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 and then concentrated in vacuo. The residue was sequentially loaded into the following solvent systems, followed by reconcentration: acetonitrile/ethyl acetate (1:1, 2 x 10 mL) and ethyl acetate/heptane (1:1, 2 x 10 mL) to give C67 as a glass (7.18 g). Some of this material was used in the next step. LCMS m/z 309.3 [M+H]+.

^1H NMR (400 MHz, methanol-^) δ 4.51 (dd, J = 10.8, 4.7 Hz, 1H), 4.21 (w s, 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 (w d, J = 7.9 Hz, 1H) , 1.95-1.84 (m, 1H), 1.84-1.75 (m, 2H), 1.15 (s, 6H).

Step 3. Synthesis of (^^^)-N-{(^)-1-cyano-2-[^)-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'-tetramethylaronium hexafluorophosphate (HATU; 82.7 mg, 0.218 mmol) followed by the addition of 4-methylmorpholine (54.4 μL, 0.495 mmol). After the reaction mixture was stirred for 20 min, C67 (from the previous step; 100 mg, < 0.19 mmol) was added as a solid. The reaction mixture was allowed to stir for 1.5 h, after which it was 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 using 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 an additional 3 h, after which 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 allowed to stir 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. Purification using reversed-phase HPLC (column: Waters Sunfire C18, 19x100 mm, 5 μm; mobile phase A: 0.05% trifluoroacetic acid in water; mobile phase B: 0.05% trifluoroacetic acid in acetonitrile; gradient: 5% to 95% B over 8.54 min, followed by 95% B over 1.46 min; flow rate: 25 mL/min) gave (1R,2S,5S)-N{(^)-1-cyano-2-[^)-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% over 2 steps. LC-MS m/z 540.6 [M+H] ⁺ . Retention time: 2.60 minutes (Analytical conditions: Column: Waters Atlantis dC18, 4.6 x 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% to 95% B, linear over 4.0 minutes, then 95% B over 1.0 minute; flow rate: 2 mL/min).

Example 87.

(1 R,2S,5S)-N-{( 1S)-1-cyano-2-[(3 S)-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) go ^m'CH3

H ₃ C CH3

C68>0 HCI

- 141 047572

methyl (1R,2S,5S)-3-{(2,S)-2-[(tert-butoxycarbonyl)amino]-2Step 1. Synthesis of cyclohexylacetyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (C68).

At 0°C, O-(7-azabenzotriazol-1-yl)-HNH,N-tetramethylaronium hexafluorophosphate (HATU; 610 mg, 1.60 mmol) was added to a solution of methyl (3,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate hydrochloride salt (300 mg, 1.46 mmol) and (394 mg, 1.53 mmol) in H2-dimethylformamide (5 ml), followed by dropwise addition of 4-methylmorpholine (443 mg, 4.38 mmol). The reaction mixture was stirred at 0°C for 10 min, then at room temperature (20°C) for 2 h, then 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%. LC-MS m/z 409.3 [M+H]+.

^1H NMR (400 MHz, DMSO-46) 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 the hydrochloride salt of methyl (^^^)-3-[DO)-2-amino-2-cyclohexylacetyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (C69).

A solution of hydrogen chloride 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). After the reaction mixture was stirred at room temperature (20°C) for 1.5 h, it was concentrated in vacuo. The residue was co-evaporated with dichloromethane to give C69 as a light yellow foam (490 mg), the bulk of which was used in the following experiments.

1H NMR (400 MHz, DMSO-4) 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 (c, 3H).

Step 3. Synthesis of methyl (^28,58)-3-[(28)-2-cyclohexyl-2{[(trifluoromethyl)sulfonyl]amino}acetyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (C7 0).

To a solution at -10 °C of C69 (from the previous step; 480 mg, <1.39 mmol) in dichloromethane (10 mL) was added H2-diisopropylethylamine (630 mg, 4.87 mmol), followed by the 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, after which it was diluted with saturated aqueous sodium chloride solution (50 mL) and extracted with dichloromethane (3 x 30 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and purified by silica gel chromatography (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. LC-MS m/z 441.1 [M+H] ⁺ .

Step 4. Synthesis of (^^^)-3-[^)-2-cyclohexyl-2-{[(trifluoromethyl)sulfonyl]amino}acetyl]6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (C71).

Lithium hydroxide monohydrate (28.6 mg, 0.682 mmol) was added to a solution of C70 (120 mg, 0.272 mmol) in a mixture of water (2 ml), methanol (2 ml), and tetrahydrofuran (2 ml). After the reaction mixture

- 142 047572 was stirred at room temperature (20 °C) for 18 h, LC-MS showed the reaction to be complete: LC-MS 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 addition of 1 M hydrochloric acid; the resulting mixture was extracted with ethyl acetate (2 x 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%.

Step 5. Synthesis of (^^^)-N-{(2^)-1-amino-1-oxo-3-[^)-2-oxopyrrolidin-3-yl]propan-2-yl}3-[^)-2-cyclohexyl-2-{[(trifluoromethyl)sulfonyl]amino}acetyl]-6,6-dimethyl-3azabicyclo[3.1.0]hexane-2-carboxamide (C72).

At 0°C, O-(7-azabenzotriazol-1-yl)-N,N-dimethylaronium hexafluorophosphate (HATU; 98.4 mg, 0.259 mmol) was added to a 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), followed by the addition of 4-methylmorpholine (76.4 mg, 0.755 mmol). The reaction mixture was then stirred at 20°C for 2 h, poured into ice water (10 ml) and extracted with a mixture of chloroform and 2-propanol (4:1, 4 x 20 ml). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and purified using silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to afford C72 as a colorless glass. Yield: 100 mg, 0.173 mmol, 80%. LC-MS m/z 580.2 [M+H]+.

Step 6. Synthesis of (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[(2^)-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 solution at 0 °C of C72 (100 mg, 0.173 mmol) and pyridine (41.7 μl, 0.516 mmol) in dichloromethane (3 ml). After the mixture was stirred for 20 h at room temperature (10 °C to 20 °C), it was 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 before being combined with water (20 mL) and acetonitrile (5 mL); this mixture was lyophilized to give (1N)-N-{(N)-1-cyano-2-[N)-2-oxopyrrolidin-3-yl]ethyl}-3[N)-2-cyclohexyl-2-{[(trifluoromethyl)sulfonyl]amino}acetyl]-6,6-dimethyl-3azabicyclo[3.1.0]hexane-2-carboxamide (87) as a white solid. Yield: 27.6 mg, 49.1 μmol, 28%. LC-MS m/z 562.2 [M+H]+.

^1H NMR (400 MHz, DMSO-66), 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 of ABquartet, J = 7.7 Hz, 1H), 1.03 (s, 3H), 0.89 (s, 3H).

Examples 88 and 89.

(^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)^-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide and (1R,2S,5S)-N-{(1S)1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-N-(trifluoroacetyl)^-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (88 and 89)

О ^Нз 9/ ^Сн з Хп^СНз CL

- 143 047572

1) HCl

88(DIAST-1) and 89 (DIAST-2)

Step 1. Synthesis of tert-butyl !1-[(1R,2S,5S)-2-(j(2S)-1-a\iHiio-1-OKCO-3-[(3S)-2-OKCOiiHppo.iHguii-3yl]propan-2-yl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl]-3-cyclobutyl-1-oxopropan-2yl}carbamate (C73).

At 0°C, a 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)-NH4N,N-tetramethylaronium hexafluorophosphate (HATU; 169 mg, 0.444 mmol). 4-Methylmorpholine (131 mg, 1.30 mmol) was then added dropwise and the reaction mixture was allowed to warm 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 x 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]+.

¹ H 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 (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-(trifluoroacetyl)-C-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide and (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-(trifluoroacetyl)-O-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide [88 (DIAST-1) and 89 (DIAST-2)].

A solution of hydrogen chloride in 1,4-dioxane (4 M; 5 mL, 20 mmol) was added dropwise to a solution of C73 at 0 °C (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, after which it was concentrated in vacuo to give the deprotected material as a white solid: LC-MS m/z 434.2 [M+H] ⁺ . This material 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 an additional 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 (3 x 10 mL), and saturated aqueous sodium chloride (10 mL), dried, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 20% methanol in dichloromethane) was performed, followed by supercritical fluid chromatography [Column: Chiral Technologies Chiralpak IC, 30 x 250 mm, 10 μm; mobile phase: 3:1 carbon dioxide/(methanol containing 0.1% ammonium hydroxide); flow rate: 70 ml/min] to obtain the separated diastereomers (1R,2S,5S)-N-{(1S)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-^-(trifluoroacetyl)-C-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide and (1R,2S,5S)-N-{(^)-1-11-iano-2-[^)-2-oxo11-pyrrolidin-3-yl]ethyl}-3-[3-cyclobutyl-^-(trifluoroacetyl)-O-alanyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide. The material containing the D-alanyl configuration and the material containing the L-alanyl configuration were not detected; the first eluting diastereomer was designated 88 and the second eluting diastereomer was designated 89. Both were obtained as a white solid.

- Yield: 9.3 mg, 18.2 µmol, 9% in 2 stages. LC-MS m/z 512.1 [M+H] ⁺ .

^1H NMR (400 MHz, DMSO-66) δ 9.75 (w d, J = 7.0 Hz, 1H), 8.96 (d, J = 8.1 Hz, 1H), 7.71 (s , 1H), 5.004.90 (m, 1H), 4.32-4.23 (m, 1H), 4.13 (s, 1H), 3.82 (dd, ABX system component, 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 IC3, 4.6 x 150 mm, 3 µm; Mobile phase A: Carbon dioxide; Mobile phase B: Methanol containing 0.05% diethylamine (v/v); Gradient: 5% to 40% B over 5 minutes, then 40% B over 2.5 minutes; Back pressure: 1500 psi; Flow rate: 2.5 mL/min).

- 144 047572

- Yield: 23 mg, 45.0 mmol, 23% over 2 steps. 1H NMR analysis showed that this material exists as a mixture of rotamers. LC-MS m/z 512.1 [M+H]+.

Ή NMR (400 MHz, DMSO- ₆ ) δ [9.92 br (s) and 9.65 (br s), overall 1H], [9.22 (d, J = 7.7 Hz) and 8.85 (d, J = 8.4 Hz), overall 1H], [7.76 (s) and 7.67 (s), overall 1H], [5.11-5.00 (m) and 4.98-4.87 (m), overall 1H], [4.51 (s) and 4.07 (s), overall 1H], [4.47-4.36 (m) and 4.09-4.00 (m), overall 1H], [3.90 (dd, J = 10.2, 5.3 Hz) and 3.60-3.45 (m), overall 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 identical to those used for 88).

Example 90.

(1K^^)-^{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(pyridin-2-yl)Na(trifluoroacetyl)^-alanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (90)

Step 1. Synthesis of tert-butyl [^)-1-[(1K^^)-2-({^)-1-amino-1-oxo-3-[^)-2-oxopyrrolidin3-yl]propan-2-yl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl]-1-oxo-3-(pyridin-2yl)propan-2-yl]carbamate (C74).

At 0°C, C-(7-azabenzotriazol-1-yl)-N-N-tetramethylaronium hexafluorophosphate (HATU; 282 mg, 0.742 mmol) was added to a solution of C67 (250 mg, 0.618 mmol) and N-(tert-butoxycarbonyl)-3-pyridin-2-yl-galanin (198 mg, 0.744 mmol) in N-dimethylformamide (2 mL), followed by dropwise addition of a solution of 4-methylmorpholine (188 mg, 1.86 mmol) in N-dimethylformamide (1 mL). The reaction mixture was then warmed to 20°C and stirred for 2 h, after which 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, after which the aqueous layer was extracted with a mixture of dichloromethane and methanol (10:1, 3 x 20 mL). The combined organic layers were concentrated in vacuo and purified using silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give C74 as a yellow gum. Yield: 250 mg, 0.449 mmol, 73%. LC-MS m/z 557.0 [M+H]+.

1H NMR (400 MHz, methanol- _b4 ), 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 the AB quartet, J = 7.7 Hz, 1H), 1.34 (s, 9H), 1.08 (s, 3H), 1.00 (s, 3H).

Step 2. Synthesis of the hydrochloride salt of (1K^^)-^{^)-1-amino-1-oxo-3-[^)-2oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-[3-(pyridin-2-yl)^-alanyl]-3azabicyclo[3.1.0]hexane-2-carboxamide (C75).

A solution of hydrogen chloride in 1,4-dioxane (4 M; 6 mL) was added at 0 °C to a solution of C74 (250 mg, 0.449 mmol) in dichloromethane (2 mL) and the reaction mixture was stirred for 5 h at 20 °C. LC-MS analysis showed conversion to C75: LC-MS m/z 457.1 [M+H]+. Removal of the solvents in vacuo gave C75 as a yellow solid (250 mg); some of this material was used directly in the next step.

1H NMR (400 MHz, DMSO-b ₆ ), characteristic peaks: δ 8.81 (wd, 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 (w s, 1H), 7.06 (w s, 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 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).

Step 3. Synthesis of (1К^^)-^{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3

- 145 047572

[3-(pyridin-2-yl)-(trifluoroacetyl)-alanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (90).

At 0°C, pyridine (197 mg, 2.49 mmol) was added to a solution of C75 (from the previous step; 175 mg, <0.314 mmol) in dichloromethane (6 ml), followed by the addition of a solution of trifluoroacetic anhydride (186 mg, 0.886 mmol) in dichloromethane (2 ml). After the reaction mixture was stirred at 20°C for 2 h, it was diluted with water and extracted with a mixture of dichloromethane and methanol (10:1, 3 x 15 ml). The combined organic layers were concentrated in vacuo and subjected to silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane), followed by supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AS, 30 x 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)-N-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]+.

¹ H NMR (400 MHz, DMSO-ds) δ 9.92 (d, J = 7.5 Hz, 1H), 8.91 (d, J = 8.0 Hz, 1H), 8.51 (br d, 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), 3 (d, half of AB quartet, J = 7.6 Hz, 1H), 1.03 (s, 3H), 0.89 (s, 3H).

Example 91.

(^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-{N-[(4-fluorophenoxy)acetyl]-3methyl^-valyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (91)

Step 1. Synthesis of tert-butyl (^)-1-[(^^^)-2-({^)-1-amino-1-oxo-3-[^)-2-oxopyrrolidin3-yl]propan-2-yl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl]-3,3-dimethyl-1-oxobutan-2yl}carbamate (C76).

A solution of C32 (15.4 g, 41.8 mmol) and C16 HCl salt (75%, 11.6 g, 41.9 mmol) in N,N-dimethylformamide (380 mL) was cooled to -5 °C to 0 °C. To this was added O-(7-azabenzotriazol-1-yl)-HN,N-tetramethylaronium hexafluorophosphate (HATU; 18.3 g, 48.1 mmol) and 4-methylmorpholine (12.7 g, 126 mmol) at -5 °C to 0 °C. After the reaction mixture was stirred at 0 °C for 1.5 h, it was poured into ice water (400 mL) and the resulting mixture was extracted with ethyl acetate (3 x 200 mL). The combined organic layers were washed successively with aqueous citric acid solution (1 M; 120 ml), saturated aqueous sodium

- 146 047572 bicarbonate (120 mL) and saturated aqueous sodium chloride solution (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 (Gradient: 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 was done followed by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give additional C76 as a white solid (2.3 g). Combined yield: 12.1 g, 23.2 mmol, 52%. LC-MS m/z 522.5 [M+H]+.

¹ H NMR (400 MHz, chloroform-d) δ 8.28 (w d, J = 5.9 Hz, 1H), 7.20 (w s, 1H), 5.71 (w s, 1H), 5 .38 (w s, 1H), 5.10 (w d, J = 10.3 Hz, 1H), 4.47-4.38 (m, 1H), 4.28-4.20 (m, 2H ), 4.12 (dd, component of the AB 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, suggested; largely obscured by the water signal peak and tertiary butyl), 1.39 (s, 9H), 1.02 (s, 3H), 1.01 (s, 9H), 0.88 (s, 3H).

Step 2. Synthesis of hydrochloride salt of (^^^)-N-{^)-1-amino-1-oxo-3-[^)-2oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-(3-methyl-B-valyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (C77).

At 0 °C, a solution of hydrogen chloride in 1,4-dioxane (4 M; 250 mL) was added to a solution of C76 (12.1 g, 23.2 mmol) in dichloromethane (50 mL). After the reaction mixture was stirred at 20 °C for 2 h, it was filtered. The filtered 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-de) δ 8.38 (d, J = 9.0 Hz, 1H), 8.19 (w s, 3H), 7.57 (w s, 1H), 7.41 (w s, 1H), 7.04 (w s, 1H), 4.35-4.27 (m, 1H), 4.34 (s, 1H), 3.85-3.72 (m, 2H ), 3.65 (d, half of the 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), 1.711.42 (m, 3H), 1.38 (d, half of the AB quartet, J = 7.7 Hz, 1H), 1.02 (s, 3H), 1.02 (s, 9H), 0.97 (s, 3H).

Step 3. Synthesis of (^^^)-N-{^)-1-amino-1-oxo-3-[^)-oxopyrrolidin-3-yl]propan-2-yl}-3[N-(chloroacetyl)-3-methyl-N-valyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (C78).

Triethylamine (2.21 g, 21.8 mmol) was added at 0 °C to a 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 x 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 using 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. LC-MS m/z 498.1 (chlorine isotope pattern observed) [M+H]+.

1H NMR (400 MHz, DMSO-D 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, Ar ,.. = 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 the AB quartet, J = 7.7 Hz, 1H), 1.01 (s, 3H), 0.94 (s, 9H), 0.84 (s, 3H).

Step 4. Synthesis of (^^^)-N-{^)-1-amino-1-oxo-3-[^)-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, after which 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 solution (10 ml), and saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) gave C79 as a white glass.

Combined yield: 100 mg, 0.174 mmol, 62%. LC-MS m/z 574.2 [M+H]+.

1H NMR (400 MHz, chloroform-d) δ 8.23 (br d, J = 6 Hz, 1H), 7.19 (br s, 1H), 7.04 (br d, 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 (br s, 1H), 5.46 (br 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), 2.142.05 (m, 1H), 2.03-1.80 (m, 3H), 1.03 (s, 3H), 1.00 (s, 9H), 0.86 (s, 3H).

Step 5. Synthesis of (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-{N-[(4-fluorophenoxy)acetyl]-3-methyl-B-valyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (91).

- 147 047572

A solution of methyl N-(triethylammoniosulfonyl)carbamate inner salt (Burgess reagent; 125 mg, 0.524 mmol) in dichloromethane (2 ml) was added dropwise at 10 °C (room temperature) to a solution of C79 (100 mg, 0.174 mmol) in dichloromethane (4 ml). After the reaction mixture had been stirred at 10 °C for 16 h, it was 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) and concentrated in vacuo; Silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane), followed by supercritical fluid chromatography [Column: Chiral Technologies Chiralcel OD, 30x250 mm, 10 μm; mobile phase: 4:1 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide); flow rate: 60 ml/min] gave (1K,28,58)-N-{(18)-1-cyano-2-[(38)-2oxopyrrolidin-3-yl]ethyl}-3-N-[(4-fluorophenoxy)acetyl]-3-methyl-C-valyl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (91) as a white solid. Yield: 55 mg, 99.0 μmol, 57%. LC-MS 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, ABX component -system, J = 10.4, 5.5 Hz, 1H), 3.73 (d, half of 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, assumed; 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 the 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-3yl]ethyl}-4-(trifluoromethyl)G-prolinamide (92)

1) HCI

A mixture of C51 (68.0 mg, 0.12 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4 M; 1 mL, 4 mmol) was stirred at room temperature for 5 min, after which the reaction mixture was concentrated in vacuo to remove the solvent, then further evaporated under high vacuum to remove residual hydrogen chloride. (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 addition of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylaronium hexafluorophosphate (HATU; 61.2 mg, 0.161 mmol), the reaction mixture was warmed to room temperature over 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, after which the reaction mixture was treated with dilute aqueous sodium carbonate solution and extracted twice with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered, concentrated in vacuo and purified using reverse phase HPLC (column: Waters XBridge C18, 19x100 mm, 5 μm; mobile phase A: water; mobile phase B: acetonitrile; gradient: 20% to 60% B over 8.5 min, then 60% to 95% B over 0.5 min, then 95% B over 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)-2oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)N-prolinamide (92). Yield: 20.7 mg, 36.7 μmol, 31%. LC-MS m/z 564.8 [M+H] ⁺ . Retention time: 2.71 minutes (Analytical conditions. Column: Waters Atlantis dC18, 4.6 x 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% to 95% B, linear over 4.0 minutes, then 95% B over 1.0 minute; flow rate: 2 mL/min).

Example 93.

(^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(III-pyrazol-1yl)-N-(trifluoroacetyl)-alanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (93)

- 148 047572

Step 1. Synthesis of the hydrochloride salt of (1R,2S,5S)-N-{(2S)-1-aMUHO-1-OKCO-3-[(3S)-2oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (C67, HCl salt).

To a solution of C66 (9.97 g, 24.4 mmol) in dichloromethane (50 mL) was added a solution of hydrogen chloride in 1,4-dioxane (4 M; 90 mL). The reaction mixture was stirred at room temperature (25 °C) for 2 h, after which LC-MS analysis showed conversion to C67: LC-MS 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%.

¹ H NMR (400 MHz, DMSO-06) 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).

Step 2. Synthesis of tert-butyl [(DO)-1-[(^^^)-2-({^)-1-amino-1-oxo-3-[^)-2-oxopyrrolidin-3-yl]propan-2-yl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl]-1-oxo-3-(III-pyrazol-1yl)propan-2-yl]carbamate (C80).

At 0°C, a solution of C67, HCl salt (300 mg, 0.870 mmol) and N-tert-butoxycarbonyl)-3-(III-pyrazole-1N-O-adanine (222 mg, 0.870 mmol) in N,N-dimethylformamide (10 mL) was treated with O-(7-azabenzotriazol-1-yl)-N,III',N'-tetramethylaronium hexafluorophosphate (HATU; 430 mg, 1.13 mmol) and 4-methylmorpholine (264 mg, 2.61 mmol). After the reaction mixture was stirred at 0°C for 2 h, it was 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 x 30 mL). The combined organic layers were concentrated in vacuo and subjected to silica gel chromatography twice (Gradient #1: 0% to 10% methanol in dichloromethane; Gradient #2: 0% to 25% methanol in dichloromethane) to afford C80 as a canary yellow solid. Yield: 340 mg, 0.623 mmol, 72%. LC-MS m/z 546.1 [M+H]+.

1H NMR (400 MHz, DMSO-ds) δ 8.29 (br d, J = 8.6 Hz, 1H), 7.70-7.66 (m, 1H), 7.64 (s, 1H), 7.51 (br s, 1H), 7.38 (br d, 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 from AB quartet, J = 7.6 Hz, 1H), 1.30 (s, 9H), 1.01 (s, 3H), 0.89 (s, 3H).

Step 3. Synthesis of the hydrochloride salt of (^^^)-N-{^)-1-amino-1-oxo-3-[^)-2oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-[3-(1 H-pyrazol-1-yl)-N-alanyl]-3azabicyclo[3.1.0]hexane-2-carboxamide (C81).

A solution of hydrogen chloride in 1,4-dioxane (4 M; 15 ml) was added at 0 °C to a solution of C80 (340 mg, 0.623 mmol) in dichloromethane (4 ml). After the reaction mixture was stirred at 20 °C for 1 h, it was filtered and the filter cake was washed with dichloromethane (3 x 10 ml). The combined filtrates were concentrated in vacuo to give C81 as a white solid. Yield:

- 149 047572

244 mg, 0.506 mmol, 81%. LC-MS m/z 446.0 [M+H]+.

1H NMR (400 MHz, DMSO^ ₆ ), characteristic peaks: δ 8.57-8.48 (m, 3H), 8.42 (wd, J = 8.6 Hz, 1H), 7.81 (d , J = 2.3 Hz, 1H), 7.65 (w s, 1H), 7.58 (d, J = 1.9 Hz, 1H), 7.35 (w s, 1H), 7.09 (w s, 1H), 6.27 (dd, J = 2.2 Hz, 1H), 4.59 (dd, ABX system component, 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).

Step 4. Synthesis of (^^^)^-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3[3 -(1 H-pyrazol-1 -yl)^-(trifluoroacetyl)N-alanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (93).

At 0°C, a 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 allowed to stir for 3 h, after which it was diluted with water (20 ml) and extracted with dichloromethane (3 x 20 ml). The combined organic layers were washed with hydrochloric acid (1 M; 20 ml) and saturated aqueous sodium chloride solution (2 x 20 ml), dried, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) followed by supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD, 30 x 250 mm, 10 μm; mobile phase: 4:1 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide); flow rate: 60 ml/min] afforded (^^^)^-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-(1H-pyrazol-1-yl)^-(trifluoroacetyl)-N-alanyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (93) as a white solid. Yield: 15.0 mg, 28.6 μmol, 11%. LC-MS m/z 524.0 [M+H] ⁺ .

¹ H NMR (500 MHz, DMSO) 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 (dd, 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 of AB quartet, J = 7.6 Hz, 1H), 1.02 (s, 3H), 0.85 (s, 3H).

Example 94.

(^^^)^-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[DO)-4,4-difluoro-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (94)

Step 1. Synthesis of 9H-fluoren-9-ylmethyl ({^)-1-[(^^^)-2-({^)-1-amino-1-oxo-3-[^)-2oxopyrrolidin-3-yl]propan-2-yl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl]-4,4-difluoro-1-oxobutan-2-yl}carbamate (C82).

At 0°C, O-(7-α-benzotriαzol-1-yl)-N,N,N',N'-tetramethylaronium hexafluorophosphate (HATU; 281 mg, 0.739 mmol) was added in one portion to a solution of C67 (230 mg, 0.569 mmol) and (2S)-2-{[(9H-fluoren-9ylmethoxy)carbonyl]amino}-4,4-difluorobutanoic acid (247 mg, 0.684 mmol) in N,N-dimethylformamide (5 mL); then 4-methylmorpholine (173 mg, 1.71 mmol) was added dropwise. After the reaction mixture had been stirred at 0°C for 10 min, it was warmed to room temperature (20°C) and stirring was continued for 2 h, after which the reaction mixture was poured into ice water (15 ml) and extracted with ethyl acetate (2 x 15 ml). The combined organic layers were washed successively with 1 M hydrochloric acid.

- 150 047572 that, saturated aqueous sodium bicarbonate solution, and saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, concentrated in vacuo, and purified using silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to give C82 as a white solid. Yield: 245 mg, 0.376 mmol, 66%. LC-MS 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 the AB quartet, J = 7.6 Hz, 1H), 1.51-1.44 (m, 1H), 1.0 3 (s, 3H), 0.89 (s, 3H).

Step 2. Synthesis of (N(^)-3-[^)-2-amino-4,4-difluorobutanoyl]-N(^)-1-amino-1-oxo-3-[^)2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (C83).

At 0 °C, a solution of diethylamine (32.8 mg, 0.448 mmol) in dichloromethane (0.5 mL) was added to a suspension of C82 (195 mg, 0.299 mmol) in dichloromethane (3 mL). The reaction mixture was stirred at 30 °C for 16 h, after which it was combined from 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%. LC-MS m/z 452.3 [M+Na+].

^1H NMR (400 MHz, chloroform-d) δ 8.73 (w d, J = 5.3 Hz, 1H), 7.11 (w s, 1H), 6.03 (tdd, J = 56.8 , 6.2, 3.0 Hz, 1H), 5.64 (w s, 1H), 5.28 (w 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, assumed; partially shaded by the water peak), 1.06 (s, 3H), 0.94 (s, 3H).

Step 3. Synthesis of (Na^^)-Na{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[^)-4,4-difluoro-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (94).

At 0°C, pyridine (146 mg, 1.85 mmol) and a solution of trifluoroacetic anhydride (194 mg, 0.924 mmol) in dichloromethane (2 mL) were added to a solution of C83 (99 mg, 0.23 mmol) in dichloromethane (4 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 an additional 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, 25x150 mm, 5 μm; mobile phase A: water containing 0.05% ammonium hydroxide (v/v); mobile phase B: acetonitrile; gradient: 23% to 63% B). (1R,2S,5S)-N-{(1S)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[^)-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%. LC-MS m/z 508.0 [M+H]+.

1H NMR (400 MHz, DMSO-D 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, ABX system component, J = 10.2, 5.4 Hz, 1H), 3.67 (d, half of 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)

- 151 047572

3)

H3C^I^CH3 CH ₃ CH ₃

Step 1. Synthesis of (^)-1-(tert-butoxycarbonyl)-4-(trifluoromethyl)-L-propyl-3-[(^)-2oxopyrrolidin-3-yl]-L-alanine amide (C50).

N,N-Diisopropylethylamine (14.8 mL, 85.0 mmol) was added to a mixture at -30°C of (4R)-1-(tert-butoxycarbonyl)-4-(trifluoromethyl)N-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'-tetramethylaronium hexafluorophosphate (HATU; 11.8 g, 31.0 mmol) in N-dimethylformamide (100 mL).

The reaction mixture was allowed to warm to 0 °C over 1 h, after which LC-MS analysis indicated the presence of C50: LC-MS m/z 437.3 [M+H]+. Aqueous sodium bicarbonate (300 mL) was added and the resulting mixture was extracted with 2-propanol:dichloromethane (1:4, 5 x 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. ¹ H NMR analysis showed that this material existed as a mixture of rotamers. Yield: 10.9 g, 25.0 mmol, 89%.

1H NMR (400 MHz, DMSO-06), 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 (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), 3.45-3.36 (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-propyl-3[^)-2-oxopyrrolidin-3-yl]-G- alaninamide (C51).

A solution of hydrogen chloride 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). After the reaction mixture was stirred at room temperature for 5 min, it was concentrated in vacuo to remove the solvent and further evaporated using high vacuum to remove residual hydrogen chloride. The residue was mixed with N-(tert-butoxycarbonyl)-3-methyl-valine (4.08 g, 17.6 mmol) and O-(7-azabenzotriazol-1-yl)-N-tetramethylaronium hexafluorophosphate (HATU; 6.71 g, 17.6 mmol) in N-dimethylformamide (25 mL), cooled to -30 °C, and treated with N-diisopropylethylamine (8.38 mL, 48.1 mmol). The reaction mixture was allowed to warm to 0 °C over 1 h, after which LC-MS analysis indicated the presence of C51: LC-MS 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. After the combined organic layers were concentrated in vacuo, the residue was purified using silica gel chromatography (Gradient: 0% to 30% methanol in dichloromethane) to afford C51 as a solid.

- 152 047572 of the substance. Yield: 3.95 g, 7.19 mmol, 45%,

1H NMR (400 MHz, DMSO-de), characteristic peaks: δ 8.28 (d, J = 8.8 Hz, 1H), 7.55 (s, 1H), 7.29 (w s, 1H), 7.03 (w s, 1H), 6.77 (w d, 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)-2oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)N-prolinamide (95).

A mixture of C51 (230 mg, 0.418 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4 M; 2 ml, 8 mmol) was stirred at room temperature for 5 min, after which the reaction mixture was concentrated in vacuo to remove the solvent, then further evaporated under high vacuum to remove residual hydrogen chloride. The residue was dissolved in dichloromethane (2 ml), cooled to 0 °C, and treated with N-diisopropylethylamine (0.219 ml, 1.26 mmol) followed by the addition of methyl chloroformate (59.3 mg, 0.628 mmol). After the reaction mixture was 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 three times with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered, and purified using silica gel chromatography (Gradient: 50% to 100% ethyl acetate in heptane). The resulting material 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 gave N-(methoxycarbonyl)-3-methyl-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)N-prolinamide (95) as a solid. Yield: 123 mg, 0.251 mmol, 60%. LC-MS m/z 490.4 [M+H]+.

¹ H NMR (400 MHz, DMSO-a ₆ ) δ 9.02 (d, J = 8.6 Hz, 1H), 7.65 (s, 1H), 7.27 (br d, 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 peak), 2.35-2.23 (m, 1H), 2.22-2.01 (m, 3H), 1.77-1.63 (m, 2H), 0.94 (s, 9H).

Example 96 (^^^)-^{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-^(trifluoroacetyl)N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (96) and (1 R,2S,5S)-N-{(1S)-1-cyano-2[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-^(trifluoroacetyl)N-valyl]-3azabicyclo[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, after which 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 x 100 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Separation of the epimers was accomplished using supercritical fluid chromatography (column: Chiral Technologies Chiralcel OX-H, 30x250 mm, 5 μm; mobile phase: 9:1 carbon dioxide/2-propanol; back pressure: 100 bar; flow rate: 80 ml/min). The first eluting substance was recovered

- 153 047572 leno as (1R.2S.5S)-N-{(1S)-1-iiuaiio-2-|(3S)-2-oi<coiiuppojuguii-3-uj|'yruj)-6.6-dUMeTUj-3-|3-methyl-N-(trifluoroacetyl)^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (13). According to powder X-ray diffraction analysis, this material was amorphous; It was designated as solid form 5. The second eluent was obtained as a glassy solid which was dissolved in dichloromethane, treated with heptane, and concentrated in vacuo to give (1R,2S,5S)-N{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-gualyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (96) as a solid.

Reduced 13- Yield: 6.00 g, 12.0 mmol, 47%. LC-MS m/z 500.3 [M+H]+.

1H NMR (400 MHz, DMSO) 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 the 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, 4.6x250 mm, 5 μm; mobile phase A: carbon dioxide; Mobile phase B: 2-propanol; gradient: 5% B over 1.00 min, followed by 5% to 60% B over 8.00 min; Back pressure: 120 bar; Flow rate: 3.0 ml /min). The powder X-ray diffraction pattern for this amorphous material is shown in Fig. 9. 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.

- Yield: 2.58 g, 5.16 mmol, 20%. LC-MS m/z 500.3 [M+H]+.

^1H NMR (400 MHz, DMSO-66) 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 (w dd, J = 10.7, 5.4 Hz, 1H), 3.66 (d, half of the 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 (sh s, 12N), 0.84 (s, 3N). Retention time: 4.20 minutes (Analytical conditions identical to those used for the recovery of 13 above).

Example 97.

(^^^)-N-{(^)-1-cyano-2-[^)-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)

/ 11 ~ CH ₃ O\ ⁿ z ^s L Jt nh ₂ \ n ₃ s <e /~o 1 ^z 2 H ₃ C^^° ------► CF ₃ OH NaOH CH ₃ No. ^{NZ <} HN^ O ° _V NH ^НзС %/Х? 1 \ '-'Gz ip H O C84 ν _ί Α _ν \ ^ΝΗ2 ------------*- n (5 HATU CH3SO3H ^N' ^CH 3 H ₃ C ^CH 3 ^{3 3} C67 Η ₃ ι O ^Nz %,CH ₃ Ho ^LsN ' Ο^,ΝΗ ΗΝ „ T\ o ^{1) NS| Hzs/} bg3 ^Ν η ^Λ Χ ^ΝΗ2 o 0 \ ^N ° CPzAAcPz N ₃ C ^CH z NEt ₃ C86 (DIAST-2) CH ₃ LSN ₃ O CH ₃ HN^O H ₃ C^^Y° CF ₃ OH C84 O ^N3S OSН3 _ην ΑΛ ^Η3 ^νη γ ² H ₃ C ^CH3 DIAST-1 C85 + °, ^H3< k ^CH3 dC b ^{s CF ,} <^ 5 Н ₃ С ^СН 3 DIAST-2 С86 О _Н Л- ₃ °v> ^{h Η3} ° ^/ 'θΡ^ΝγΚ _Ν Χ _ι ^ΝΗ ₂ ^{h Н} ° Н ₃ С ^снз С87 [from С86 (DIAST- 2)]

- 154 047572

Step 1. Synthesis of 2-[(tert-butoxycarbonyl)amino]-3-(trifluoromethyl)pentanoic acid (C84).

An aqueous solution of sodium hydroxide (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, 725, 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. 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 material was assumed to be a mixture of 4 diastereomers, also potentially containing rotamers. Yield: 197 mg, 0.690 mmol, 93%. LC-MS m/z 284.3 [M-H] ^- .

¹ H NMR (400 MHz, DMSO-9 δ 13.16 (br s, 1H), [7.29 (d, major, J = 9.8 Hz) and 6.95-6.85 (m, minor), overall 1H], [4.55 (dd, major, J = 9.8, 3.3 Hz), 4.46 (br d, minor, J = 9.1 Hz), and 4.40 (dd, minor, J = 9.4, 4.5 Hz), overall 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), overall 3H].

Step 2. Synthesis of tert-butyl {1-[(^^^)-2-({DO)-1-amino-1-oxo-3-[^)-2-oxopyrrolidin-3yl]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-[(1K,28,58)-2-({(28)-1-amino-1oxo-3-[^)-2-oxopyrrolidin-3-yl]propan-2-yl}carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3yl]-1-oxo-3-(trifluoromethyl)pentan-2-yl}carbamate, DIAST-2 (C86).

At 0 °C, a 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 □-(f-azabenzotriazole-V1-tetramethylaronium hexafluorophosphate (HATU; 176 mg, 0.463 mmol) and 4-methylmorpholine (0.116 mL, 1.06 mmol). After the reaction mixture was 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 saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. The resulting oil was azeotropically distilled twice with heptane and twice with methyl tert-butyl ether, then subjected to silica gel chromatography (Gradient: 0% to 20% methanol in dichloromethane). The first eluting diastereomer was designated C85, and the second eluting diastereomer was designated C86.

C85 (DIAST-1) - Yield: 77.3 mg, 0.134 mmol, 32%. This material contained a mixture of isomers or rotamers by 1H NMR analysis. LC-MS m/z 576.2 [M+H]+.

1H NMR (400 MHz, DMSO-66), characteristic peaks, integration is approximate: δ [8.35 (d, J = 7.9 Hz) and 8.16 (d, J = 8.5 Hz), overall 1H], 7.62-7.54 (m, 1H), 7.41-7.18 (m, 2H), [7.02 (w s) and 6.98 (w s), overall 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), overall 9H], 1.01 (w s, 3H), 0.94-0.82 (m, 6H).

C86 (DIAST-2) - Yield: 87.8 mg, 0.153 mmol, 36%. This material was predominantly one isomer by 1H NMR analysis. LCMS m/z 576.2 [M+H]+.

1H NMR (400 MHz, DMSO-66), 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).

Step 3. Synthesis of (^^^)-N-{^)-1-amino-1-oxo-3-[^)-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 hydrogen chloride in 1,4-dioxane (4 M; 0.381 ml, 1.52 mmol). After the reaction mixture was stirred at room temperature for 40 min, methanol (0.5 ml) was added to improve solubility. After another 40 min, a solution of hydrogen chloride in 1,4-dioxane (4 M; 0.10 ml, 0.4

- 155 047572 mmol); after 30 min, LC-MS analysis showed complete removal of the protecting group: LC-MS m/z 476.2 [M+H]+. The reaction mixture was concentrated in vacuo and azeotropically distilled twice with heptane; the residue was triturated twice with diethyl ether, suspended in dichloromethane (1.2 mL) and cooled to 0 °C. After 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, after which it was 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 LC-MS analysis, this material contained a mixture of C87 and the corresponding methyl ester (LC-MS m/z 587.4 [M+H]+).

1H NMR (400 MHz, DMSO-de), 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 using silica gel chromatography (0% to 20% methanol in dichloromethane) followed by azeotropic distillation of the resulting oil with heptane, followed by azeotropic distillation with diethyl ether/heptane gave C87 as a white solid. Yield: 17.9 mg, 31.3 µmol, 20%. LC-MS m/z 572.0 [M+H]+.

Step 4. Synthesis of (CH^^)-N-{(CH)-1-cyano-2-[CH)-2-oxopyrroldin-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). After the reaction mixture was stirred at room temperature for 1 h, another spatula of methyl N-(triethylammoniosulfonyl)carbamate inner salt (Burgess reagent) was added. Stirring was continued for 2 h, after which the reaction mixture was filtered and the filter cake was washed with ethyl acetate. The combined filtrates were washed with saturated aqueous sodium bicarbonate solution, and the aqueous layer was extracted once with ethyl acetate; the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo to give the crude product.

^1H NMR (600 MHz, DMSOO, main component: δ 9.90 (d, J = 8.8 Hz, 1H), 8.85 (d, J = 8.1 Hz, 1H), 7.56 (w 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), 3.213.14 (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 the 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 material was purified using reversed-phase HPLC (column: Waters Sunfire C18, 19 x 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% to 95% B over 8.54 min, followed by 95% B over 1.46 min; flow rate: 25 mL/min) to give (CH^^)-Ы-{(CH)-1-cyano-2-[^)-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%. LC-MS m/z 554.6 [M+H]+. Retention time: 2.72 minutes (Analytical conditions. Column: Waters Atlantis dC18, 4.6x50 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% to 95% B, linear over 4.0 minutes, then 95% B over 1.0 minute; flow rate: 2 mL/min).

Example 98.

(Щ^^)-Ы-{(Щ)-1-cyano-2-[^)-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)

- 156 047572

C85 (DIAST-1)

C88 [from C85 (DIAST-1)]

C89 [from C85(DIAST-1)]

H ₃ C p

MQ with ^CH 3 N'-S-NX

O _{< CH3CH3}

[from C85(DIAST-1)]

Step 1. Synthesis of the hydrochloride salt of (1R,2S,5S)-N-{(2S)-1-aMUHO-1-OKCO-3-[(3S)-2oxopyrrolidin-3-yl]propan-2-yl}-3-[2-amino-3-(trifluoromethyl)pentanoyl]-6,6-dimethyl-3azabicyclo[3.1.0]hexane-2-carboxamide, from C85 (DIAST-1) (C88).

A solution of hydrogen chloride 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). After the reaction mixture was stirred at room temperature for 40 min, methanol (0.5 mL) was added to improve solubility. Stirring was continued for 2 h, after which LC-MS analysis indicated that deprotection was complete: LC-MS m/z 476.2 [M+H]+. The reaction mixture was concentrated in vacuo;

The residue was azeotropically distilled 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-66), characteristic main peaks: δ 8.53 (w s, 3H), 8.36 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H ), 7.28 (w s, 1H), 7.06 (w 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), 1.731.51 (m, 5H), 1.44 (d, half of AB quartet, J = 7.7 Hz, 1H), 1.04 (s, 3H), 0.90 (s, 3H).

Step 2. Synthesis of (1K^^)-N-{^)-1-amino-1-oxo-3-[^)-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).

At 0 °C, a suspension of C88 (54.5 mg, 0.106 mmol) in dichloromethane (1 mL) was treated with triethylamine (26 μL, 0.19 mmol), followed by the addition of trifluoroacetic anhydride (19.5 μL, 29.1 mg, 0.138 mmol). After the reaction mixture was stirred at 0 °C for 1 h and 10 min, trifluoroacetic anhydride (1 equivalent) was added; after 30 min, trifluoroacetic anhydride (9.4 μL, 67 μmol) was added again. Stirring was continued for 45 min, after which LC-MS analysis indicated complete conversion to C89: LC-MS 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-66), main 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 (w s, 1H), 7.01 (w 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 of 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). After the reaction mixture was stirred at room temperature for 1 h, a spatula of methyl N-(triethylammoniosulfonyl)carbamate inner salt (Burgess reagent) was added. Stirring was continued for 2 h, after which the reaction mixture was filtered and the filter cake was washed with ethyl acetate. The combined filtrates were washed with saturated aqueous sodium bicarbonate solution, and the aqueous layer was extracted with ethyl acetate; the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo to give the crude product.

1H NMR (600 MHz, DMSO-66), 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),

- 157 047572

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 the AB quartet, J = 7.7 Hz, 1H), 1.04 (s, 3H), 0, 91-0.86 (m, 6H).

Purification of this material using reversed-phase HPLC (Column: Waters Sunfire C18, 19 x 100 mm, 5 μm; Mobile Phase A: 0.05% trifluoroacetic acid in water; Mobile Phase B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 5% to 95% B over 8.54 min, followed by 95% B over 1.46 min; Flow Rate: 25 mL/min) afforded (1R,2S,5S)N-{(1S)-1-cyano-2-[(3 S)-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%. LC-MS m/z 554.6 [M+H]+. Retention time: 2.80 minutes (Analytical conditions. Column: Waters Atlantis dC18, 4.6x50 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% to 95% B, linear over 4.0 minutes, then 95% B over 1.0 minute; flow rate: 2 mL/min).

Preparation of 3-tert-butyl 2-methyl (^^^)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2,3-dicarboxylate (C90)

This preparation was accomplished using the general procedure described by S. Uyeda and J. Werth, Angew. Chem. Int. Ed. 2018, 57, 13902–13906. A 3-necked flask equipped with a magnetic stir bar, reflux condenser, thermometer, and nitrogen inlet was charged with cobalt(II) bromide (0.15 equivalents; 0.146 g, 0.667 mmol), (1E,1'E)-1,1'-pyridine-2,6-diyl-6-is[Na(2-tert-butylphenyl)ethanimine] ( ^2-t-Bu PDI; 0.15 equivalents; 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 (D0)-2,5-dihydro-1-pyrrole-1,2-dicarboxylate (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) were added. The reaction mixture was stirred at room temperature for 5 days and then 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 then 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 (1H-3-dimethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2,3-dicarboxylate (C90) as a yellow oil. Yield: 0.90 g, 3.3 mmol, 75%. By H NMR analysis, this material existed 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). ECI-MS (pos.) m/z (%) = 255.1 (12.5) [M-Me + H] ⁺ , 214.1 (100) [M-t-Bu + H] ⁺ , 170. 2 (50) [M-Vos + H] ⁺ .

Alternative production of C42.

(^^^)-6,6-dimethyl-3-[3-methyl^-(trifluoroacetyl)^-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (C42)

¥ o

NzS-^-O-^-SRz JL nN Un

NaOMe uVY if ^НзЬ CH ₃ O

C91

- 158 047572 o

CH ₃ CH3

Step 1. Synthesis of 3-methyl-N-(trifluoroacetyl)-k-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-k-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), after which it was cooled to 20 °C. After addition of 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, after which the solution was concentrated at 50 °C to a volume of 5 mL/g. This procedure was repeated twice; After the 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 were obtained from a similar reaction carried out using 3-methyl-k-valine; after the organic layer containing C91 was dried over magnesium sulfate and filtered, concentration in vacuo gave a solid. A portion of this solid was used as seed crystals.

Physicochemical data were obtained on C91 samples prepared from reactions carried out in the same manner. MCBP3-ESI (m/z): [M+H]+ Calculated for C ₈ H ₁₃ F ₃ NO ₃ , 228.0842; Found, 228.0842. Parent ion observed as C ₈ H11F ₃ NNa ₂ O ₃ [M+Na+]: Calculated, 272.0481; Found, 272.0482.

1H NMR (600 MHz, DMSOLb) 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). ¹³ C NMR (150.8 MHz, DMSO-SH) δ 170.9, 156.6 (kv, ² Jcf = 36.9 Hz), 115.8 (kv, Jcf = 287.7 Hz), 61.0 , 33.6, 26.5. The X-ray powder diffraction pattern for C91 is shown in FIG. 11; characteristic peaks are given in table. R.

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Table R

Powder X-ray diffraction peaks for C91 were selected

Angle (°2-theta) Relative intensity Angle (°2-theta) Relative intensity Angle (°2-theta) Relative intensity 9.7 27 28.1 4 37.7 1 I,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 z 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 z 21,5 22 34.2 2 43.1 1 23.2 8 34.7 3 44.2 1 23.3 AND 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 of the single crystal data for C91 is shown in Fig. 12.

Determination of the structure by X-ray structural analysis of a C91 single crystal.

X-ray structural analysis of a single crystal.

Data collection was performed on a Bruker D8 Quest diffractometer at -100°C. Data collection consisted of omega and phi scans.

The structure was solved using internal phasing using SHELX software, suitable for the chiral space group P4 ₁ 2 ₁ 2 in the tetragonal system. The structure was then optimized using the least squares method in the full matrix approximation. All non-hydrogen atoms were found and optimized using anisotropic substitution parameters.

Hydrogen atoms placed on nitrogen and oxygen were determined from the Fourier difference map and refined by limited distances. Hydrogen on O2(H2Z) and O3(H3Z) was distributed as a charge and refined by 10.5 for each occupancy. The remaining hydrogen atoms were placed in calculated positions and allowed to run into carrier atoms. The final refinement included isotropic substitution parameters for all hydrogen atoms.

The occupancy mismatch as a ratio of ~67/33 in the -CF3 segment was identified and modeled accordingly.

The analysis of the absolute structure using probabilistic methods (Hooft 2008) was performed using PLATON (Spek). The results show that the absolute structure is correctly assigned. The method calculates that the probability of such a structure being correct is 100%. The Hooft parameter is given as 0.02 with esd (estimated standard deviation) (4), and the Parson parameter is given as 0.02 with esd (4).

The final R-index was 4.1%. The final Fourier difference revealed neither a missing nor a shifted electron density.

The corresponding crystal, data collection and refinement are presented in Table S. Atomic coordinates, bond lengths, bond angles and substitution parameters are given in Tables T-V.

A list of the software and references used can be found in the X-ray diffraction structure determination of the single crystal from Example 13, solid form 1.

Table S

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Refining the data and crystal structure for C91

Empirical formula

Mass by formula

Temperature

Wavelength

Crystal system Space group Unit cell parameters

Volume

Z

Density (calculated) Absorption coefficient

F(000)

Crystal size

Theta range for data collection

Index ranges Collected reflections Independent reflections Completeness to theta = 67.679° Absorption correction Refinement method

Data / constraints / parameters Consistency criterion on F ² Final R indices \Ι>2σ(Ι)~\ R indices (all data)

Absolute structure parameter Extinction coefficient Largest differential peak and well

C ₈ Hi ₂ F ₃ NO ₃

227.19

173(2)K

1.54178 A

T etragonal

Р41212 a = 9.9168(6) A a = 90° ό = 9.9168(6) A ^ = 90° c = 22.721(2) A γ = 90° 2234.5(4) A ³

1.351 mg/ ^m3

1.184 mm ¹

944

0.200 x 0.170 x 0.080 mm ³ from 4.866 to 70.114°

-11<=/?<=10, -12<=k=12, -27<=/<=27 48160

2122 [R, _nt = 0.0392]

99.8%

Empirical

Least squares in full matrix approximation on F ² 2122/9/158

1,010

Al = 0.0408, wR2 = 0.1012

Al = 0.0429, wR2 = 0.01039

0.03(4) n/a

0.280 and -0.215 e.A ³

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Table T

Atomic coordinates (x 10 ⁴ ) and equivalent isotropic substitution parameters (A ² x10 ³ ) for C91. and (eq.) are defined as one third of the trace of the orthogonalized tensor U ^ij

x y z and(equiv.) F(l) 9781(4) 8042(4) 5734(2) 107(1) F(2) 10080(4) 7982(5) 6660(2) 100(2) F(3) 9278(3) 6313(2) 6193(2) 87(1) F(1A) 9349(11) 6675(12) 6782(5) 107(1) F(2A) 9431(11) 6825(16) 5889(6) 100(2) F(3A) 10149(10) 8346(8) 6346(8) 87(1) N(l) 6809(2) 7369(2) 6335(1) 32(1) 0(1) 7784(2) 9443(2) 6392(1) 48(1) 0(2) 5226(2) 6038(2) 7066(1) 45(1) 0(3) 3695(2) 7680(2) 7101(1) 55(1) 0(1) 9239(3) 7599(3) 6263(2) 51(1) 0(2) 7850(3) 8227(2) 6339(1) 38(1) 0(3) 5426(2) 7871(2) 6390(1) 31(1) 0(4) 4731(2) 7135(2) 6890(1) 32(1) 0(5) 4628(3) 7777(3) 5796(1) 39(1) 0(6) 5489(3) 8387(4) 5311(1) 55(1) C(7) 3336(3) 8612(4) 5846(1) 58(1) C(8) 4303(4) 6312(3) 5650(1) 61(1)

Table U

Bond lengths [A] and angles [°] for C91

F(l)-C(l) 1,389(5) F(2)-C(l) 1,286(4) F(3)-C(l) 1,286(4) F(1A)-C(1) 1,498(10) F(2A)-C(1) 1,162(10) F(3A)-C(1) 1,183(9) N(l)-C(2) 1,338(3) N(l)-C(3) 1,464(3) N(1)-H(1X) 0.97(2) O(l)-C(2) 1,214(3) 0(2)-0(4) 1,259(3)

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O(2)-H(2Z) 0.98(3) O(3)-C(4) 1,256(3) O(3)-H(3Z) 0.97(3) C(l)-C(2) 1,522(4) C(3)-C(4) 1,516(3) C(3)-C(5) 1,566(3) C(3)-H(3) 1,0000 C(5)-C(6) 1,520(4) C(5)-C(8) 1,525(4) C(5)-C(7) 1,530(4) C(6)-H(6A) 0,9800 C(6)-H(6B) 0,9800 C(6)-H(6C) 0,9800 C(7)-H(7A) 0,9800 C(7)-H(7B) 0,9800 C(7)-H(7C) 0,9800 C(8)-H(8A) 0,9800 C(8)-H(8B) 0,9800 C(8)-H(8C) 0,9800 C(2)-N(l)-C(3) 120.4(2) C(2)-N(1)-H(1X) 121.0(17) C(3)-N(1)-H(1X) 118.6(17) C(4)-O(2)-H(2Z) 113(4) C(4)-O(3)-H(3Z) 116(4) F(2A)-C(1)-F(3A) 114.0(9) F(3)-C(l)-F(2) 111,1(4) F(3)-C(l)-F(l) 101.2(3) F(2)-C(l)-F(l) 105.3(3) F(2A)-C(1)-F(1 A) 99.3(10) F(3A)-C(1)-F(1A) 101.7(9) F(2A)-C(1)-C(2) 120,1(5) F(3A)-C(1)-C(2) 114.6(5) F(3)-C(l)-C(2) 116.6(2) F(2)-C(l)-C(2) 112.7(3)

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F(l)-C(l)-C(2) 108.6(3) F(1A)-C(1)-C(2) 103.1(4) O(l)-C(2)-N(l) 126.2(2) O(l)-C(2)-C(l) 117.8(2) N(l)-C(2)-C(l) 116.0(2) N(l)-C(3)-C(4) 109.07(19) N(l)-C(3)-C(5) 112.25(19) C(4)-C(3)-C(5) 112.80(19) N(l)-C(3)-H(3) 107.5 C(4)-C(3)-H(3) 107.5 C(5)-C(3)-H(3) 107.5 O(3)-C(4)-O(2) 124.7(2) O(3)-C(4)-C(3) 116.8(2) O(2)-C(4)-C(3) 118.4(2) C(6)-C(5)-C(8) 109.9(2) C(6)-C(5)-C(7) 108.0(2) C(8)-C(5)-C(7) 110.8(3) C(6)-C(5)-C(3) 108.5(2) C(8)-C(5)-C(3) 110.6(2) C(7)-C(5)-C(3) 109.1(2) C(5)-C(6)-H(6A) 109.5 C(5)-C(6)-H(6B) 109.5 H(6A)-C(6)-H(6B) 109.5 C(5)-C(6)-H(6C) 109.5 H(6A)-C(6)-H(6C) 109.5 H(6B)-C(6)-H(6C) 109.5 C(5)-C(7)-H(7A) 109.5 C(5)-C(7)-H(7B) 109.5 H(7A)-C(7)-H(7B) 109.5 C(5)-C(7)-H(7C) 109.5 H(7A)-C(7)-H(7C) 109.5 H(7B)-C(7)-H(7C) 109.5 C(5)-C(8)-H(8A) 109.5 C(5)-C(8)-H(8B) 109.5 H(8A)-C(8)-H(8B) 109.5 C(5)-C(8)-H(8C) 109.5 H(8A)-C(8)-H(8C) 109.5 H(8B)-C(8)-H(8C) 109.5

The symmetry transformation used to generate equivalent atoms.

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Table V Anisotropic substitution parameters (A ² x10 ³ ) for C91. The exponent of the anisotropic shift coefficient takes the form: -2n ² [h ² a* ² U11 + ... + 2 hka* b* U ¹² ]

U ¹¹ U ²² U ³³ U ²³ U ¹³ U ¹² F(l) 84(2) 107(3) 129(3) 42(2) 67(2) 41(2) F(2) 54(2) 128(4) 120(3) -64(3) -31(2) 22(2) F(3) 36(1) 29(1) 195(4) -15(2) 21(2) 0(1) F(1A) 84(2) 107(3) 129(3) 42(2) 67(2) 41(2) F(2A) 54(2) 128(4) 120(3) -64(3) -31(2) 22(2) F(3A) 36(1) 29(1) 195(4) -15(2) 21(2) 0(1) N(l) 29(1) 27(1) 40(1) 2(1) 4(1) -1(1) 0(1) 43(1) 30(1) 72(1) -5(1) 14(1) -3(1) 0(2) 52(1) 39(1) 44(1) 13(1) H(l) 3(1) 0(3) 50(1) 60(1) 54(1) 17(1) 23(1) 12(1) C(l) 36(1) 33(1) 83(2) -14(1) 4(1) -5(1) C(2) 38(1) 30(1) 44(1) -5(1) 7(1) -3(1) C(3) 32(1) 28(1) 32(1) 2(1) 6(1) 2(1) C(4) 31(1) 33(1) 32(1) 2(1) 2(1) -2(1) C(5) 42(1) 42(1) 33(1) 6(1) -2(1) 2(1) C(6) 63(2) 67(2) 35(1) 10(1) 8(1) 9(2) C(7) 43(2) 77(2) 55(2) 15(2) -2(1) 14(2) C(8) 77(2) 54(2) 50(2) -4(1) -22(2) -11(2)

Step 2. Synthesis of lithium (^^^)-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 (^^^)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate hydrochloride salt (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, after which the solid was collected by filtration, washed with 5% water in tetrahydrofuran (400 mL), and dried in vacuo at 70 °C to give C92 as a white to off-white solid. Yield: 47.6 g, 295 mmol, 89%. Physicochemical data were obtained on samples of C92 that were prepared from reactions carried out in the same manner.

MCBP3-ESI ⁺ (m/z): [M+H] ⁺ Calculated for C ₈ H ₁₄ NO ₂ , 156.1019; Found, 156.1019.

1H NMR (600 MHz, D2O) δ 3.23 (d, J = 7.7 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 shown in Fig. 13; the characteristic peaks are listed in Table W.

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Table W

Powder X-ray diffraction peaks for C92 were selected

Angle (°2-theta) Relative intensity Angle (°2-theta) Relative intensity Angle (°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 (^^^)-6,6-dimethyl-3-[3-methyl-N(trifluoroacetyl)-L-valyl]-3azabicyclo[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). After the reaction mixture was 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 and propan-2-yl acetate (5 mL) was added followed by dropwise addition of heptane (15 mL) from a dropping funnel. The solids were isolated by filtration and dried in vacuo to afford (CHF)-6-dimethyl-3-[3-methyl-U(trifluoroacetyl)hexane]-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 represent 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 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 were assigned using the TMS signal set to 0 ppm in both cases.

MCBP3-ESI+ (m/z): [M+H] ⁺ Calculated for C ₁₆ H ₂₄ F ₃ N ₂ O4, 365.1683; Found: 365.1684.

1H NMR (600 MHz, DMSO-dg) δ major: 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). ¹³ C NMR (150.8 MHz, DMSO-C,) δ main: 172.3, 167.5, 156.8 ( ² Jcf = 37.0 Hz), 115.7 J = 287.7 Hz), 59.1, 58.0, 47.1, 34.6, 29.6, 26.7, 25.6, 18.7, 12.0; smaller: 172.3, 168.1, 155.9 ( ² Jcf = 36.8 Hz), 115.8 (1 JcF = 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 given in Table X.

Crystallization for both the powder X-ray diffraction work and the single crystal X-ray structure determination 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), after which water (10.5 ml) was added over 10 min. Additional water (16.5 ml) was then added over 4 h, and the mixture was cooled to 10 °C and allowed to stir overnight. After filtration, the filter cake was washed with water (6 ml) and dried at 50 °C to give crystalline C42 (2.6 g).

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Table X

Powder X-ray diffraction peaks for C42 were selected

Angle (°2-theta) Relative intensity Angle (°2-theta) Relative intensity Angle (°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 data diagram of a single crystal for C42 is shown in Fig. 15. X-ray diffraction analysis of a single crystal of C42 X-ray diffraction analysis of a single crystal

Data acquisition was performed on a Bruker D8 Venture diffractometer at room temperature. Data acquisition consisted of omega and phi scans. A special data processing strategy with a width of 0.3 degrees per frame was used to separate the domains, thus eliminating any problems with TWIN and pseudosymmetry.

The structure was solved using internal phasing using SHELX software, suitable for the R3 group in the rhombohedral system. The structure was then optimized using the least squares method in the full matrix approximation. All non-hydrogen atoms were found and optimized using anisotropic substitution parameters.

Hydrogen atoms placed on nitrogen and oxygen were determined from the Fourier difference map and refined by limited distances. The remaining hydrogen atoms were placed in calculated positions and allowed to run into their carrier atoms. The final refinement included isotropic substitution parameters for all hydrogen atoms.

The analysis of the absolute structure using probabilistic methods (Hooft 2008) was performed using PLATON (Spek). The results show that the absolute structure is correctly assigned. The method calculates that the probability of such a structure being correct is 100%. The Hooft parameter is given as -0.08 with esd (estimated standard deviation) (7), and the Parson parameter is given as -0.09 with esd (6).

The infill population ratio violation in the C1_F1_F2 segment as 78:22 ratio was identified and modeled accordingly.

The final R-index was 5.8%. The final Fourier difference revealed neither a missing nor a shifted electron density.

The corresponding crystal, data collection and refinement are presented in Table Y. The atomic coordinates, bond lengths, bond angles and substitution parameters are given in Table Z-BB.

A list of the software and references used can be found in the X-ray diffraction structure determination of the single crystal from Example 13, solid form 1.

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Table Y

Refinement of data and crystal structure for C42

Empirical formula

Mass by formula Temperature Wavelength Crystal system Space group Unit cell parameters

Volume

Z

Density (calculated) Absorption coefficient

F(000)

Crystal size

Theta range for data collection

Index ranges Collected reflections Independent reflections Completeness to theta = 67.679° Absorption correction Refinement method

Data / constraints / parameters Consistency criterion on F ² Final R indices [Ι2σΠ)] R indices (all data)

Absolute structure parameter Extinction coefficient Largest differential peak and well

C16H23F3N2O4

364.36

296(2) K

1.54178 A trigonal

R3 a= 14.1740(6) A a= 114.11°

Ь = 14.1740(6) A β= 114.11° с = 14.1740(6) A γ = 114.11° 1715.9(4) A ³

1.058 mg/ ^m3

0.788 mm ¹

576

0.220 x 0.100 x 0.100 mm ³ from 6.445 to 80.034°

-17<=L<=16, -14<=L<=16, -14<=/<=17 13310

4011 [R _mt = 0.0369]

98.9%

Empirical

Least squares in full matrix approximation on F ² 4011 /6/244

1,056

I1 = 0.0582, wR2 = 0.1675

I1 = 0.0611, wR2 = 0.1710

-0.09(6) n/a

0.292 and -0.174 e.A ³

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Table Z

Atomic coordinates (x 10 ⁴ ) and equivalent isotropic substitution parameters (A ² x10 ³ ) for C42. and (eq.) are defined as one third of the trace of the orthogonalized tensor U ^ij

X U z and(equiv.) C(1) 9738(11) 9749(11) 7504(7) 172(4) F(l) 9100(20) 9860(20) 7899(14) 233(6) F(3) 9757(18) 8823(16) 7411(12) 171(4) F(1A) 9950(80) 10650(90) 8270(60) 233(6) F(3A) 10320(70) 9400(60) 7630(50) 171(4) F(2) 11182(13) 11032(10) 8680(7) 296(6) N(l) 9252(4) 8810(4) 5336(3) 75(1) N(2) 6546(2) 6624(2) 1650(2) 49(1) 0(1) 9065(6) 10407(6) 6229(5) 130(2) 0(2) 7738(3) 6229(3) 2739(3) 88(1) 0(3) 7054(3) 5611(3) -16(3) 79(1) 0(4) 5159(3) 3362(2) -1315(3) 80(1) C(2) 9318(6) 9683(6) 6263(5) 97(2) C(3) 8891(4) 8676(4) 4135(3) 66(1) C(4) 10197(4) 9437(4) 4311(4) 80(1) C(5) 9748(5) 9404(6) 3121(6) 98(1) C(6) 10798(6) 8766(7) 4324(7) 109(2) C(7) 11359(5) 10996(5) 5744(6) 110(2) C(8) 7683(3) 7092(3) 2793(3) 60(1) C(9) 5449(3) 5104(3) 340(3) 50(1) C(10) 5997(3) 4749(3) -338(3) 56(1) C(ll) 4191(3) 4863(3) -607(3) 53(1) C(12) 3540(3) 5182(4) -65(3) 60(1) C(13) 1999(4) 4426(5) -1228(5) 78(1) C(14) 3859(5) 5374(5) 1178(5) 80(1) C(15) 4643(3) 6293(3) 127(3) 54(1) C(16) 6198(3) 7435(3) 1506(3) 59(1)

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Table AA

Length of bonds [A] and angles [°] for C42 C(1)-F(1A) 1.09(5) C(1)-F(3A) 1,12(5) C(l)-F(l) 1,271(14) C(l)-F(3) 1,277(13) C(l)-F(2) 1,404(14) C(l)-C(2) 1,556(9) N(l)-C(2) 1,321(6) N(l)-C(3) 1,463(5) N(1)-H(1X) 0.94(2) N(2)-C(8) 1,333(4) N(2)-C(9) 1,464(4) N(2)-C(16) 1,477(3) O(l)-C(2) 1,230(6) O(2)-C(8) 1,231(4) O(3)-C(10) 1,199(4) O(4)-C(10) 1,311(4) O(4)-H(4Y) 0.98(2) C(3)-C(8) 1,517(5) C(3)-C(4) 1,556(6) C(3)-H(3) 0,9800 C(4)-C(5) 1,512(8) C(4)-C(6) 1,515(6) C(4)-C(7) 1,533(6) C(5)-H(5A) 0,9600 C(5)-H(5B) 0,9600 C(5)-H(5C) 0,9600 C(6)-H(6A) 0,9600 C(6)-H(6B) 0,9600 C(6)-H(6C) 0,9600 C(7)-H(7A) 0,9600 C(7)-H(7B) 0,9600 C(7)-H(7C) 0,9600 C(9)-C(ll) 1,508(4) C(9)-C(10) 1,521(4) C(9)-H(9) 0,9800 C(ll)-C(15) 1,507(4) C(ll)-C(12) 1,510(4) C(ll)-H(ll) 0,9800 C(12)-C(14) 1,496(5) C(12)-C(15) 1,512(5)

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C(12)-C(13) 1,530(5) C(13)-H(13A) 0,9600 C(13)-H(13B) 0,9600 C(13)-H(13C) 0,9600 C(14)-H(14A) 0,9600 C(14)-H(14B) 0,9600 C(14)-H(14C) 0,9600 C(15)-C(16) 1,510(4) C(15)-H(15) 0,9800 C(16)-H(16A) 0,9700 C(16)-H(16B) 0,9700 F(1A)-C(1)-F(3A) 133(4) F(l)-C(l)-F(3) 109.3(14) F(1A)-C(1)-C(2) 105(2) F(3A)-C(1)-C(2) 109.0(19) F(l)-C(l)-C(2) 115.0(7) F(3)-C(l)-C(2) 118.2(6) F(2)-C(l)-C(2) 104.7(9) C(2)-N(l)-C(3) 119.5(3) C(2)-N(1)-H(1X) 112(3) C(3)-N(1)-H(1X) 128(3) C(8)-N(2)-C(9) 118.3(2) C(8)-N(2)-C(16) 128.7(2) C(9)-N(2)-C(16) 113.0(2) C(10)-O(4)-H(4Y) 103(3) O(l)-C(2)-N(l) 127.2(5) O(l)-C(2)-C(l) 118.2(4) N(l)-C(2)-C(l) 114.5(4) N(l)-C(3)-C(8) 106.8(3) N(l)-C(3)-C(4) 113.3(3) C(8)-C(3)-C(4) 113.4(3) N(l)-C(3)-H(3) 107.7 C(8)-C(3)-H(3) 107.7 C(4)-C(3)-H(3) 107.7

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C(5)-C(4)-C(6) 111.0(5) C(5)-C(4)-C(7) 108.8(4) C(6)-C(4)-C(7) 108.5(4) C(5)-C(4)-C(3) 108.7(3) C(6)-C(4)-C(3) 112,1(4) C(7)-C(4)-C(3) 107.5(4) C(4)-C(5)-H(5A) 109.5 C(4)-C(5)-H(5B) 109.5 H(5A)-C(5)-H(5B) 109.5 C(4)-C(5)-H(5C) 109.5 H(5A)-C(5)-H(5C) 109.5 H(5B)-C(5)-H(5C) 109.5 C(4)-C(6)-H(6A) 109.5 C(4)-C(6)-H(6B) 109.5 H(6A)-C(6)-H(6B) 109.5 C(4)-C(6)-H(6C) 109.5 H(6A)-C(6)-H(6C) 109.5 H(6B)-C(6)-H(6C) 109.5 C(4)-C(7)-H(7A) 109.5 C(4)-C(7)-H(7B) 109.5 H(7A)-C(7)-H(7B) 109.5 C(4)-C(7)-H(7C) 109.5 H(7A)-C(7)-H(7C) 109.5 H(7B)-C(7)-H(7C) 109.5 O(2)-C(8)-N(2) 119.3(3) O(2)-C(8)-C(3) 121.0(3) N(2)-C(8)-C(3) 119.8(2) N(2)-C(9)-C(ll) 105.0(2) N(2)-C(9)-C(10) 110.4(2) C(ll)-C(9)-C(10) 112.2(2) N(2)-C(9)-H(9) 109.7 C(ll)-C(9)-H(9) 109.7 C(10)-C(9)-H(9) 109.7 O(3)-C(10)-O(4) 124.6(3) O(3)-C(10)-C(9) 125.1(3)

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O(4)-C(U)-C(9) 110.4(3) C(15)-C(11)-C(9) 107.9(2) C(15)-C(11)-C(12) 60,1(2) C(9)-C(11)-C(12) 118.0(2) C(15)-C(11)-H(11) 118.7 C(9)-C(11)-H(11) 118.7 C(12)-C(11)-H(11) 118.7 C(14)-C(12)-C(11) 121.8(3) C(14)-C(12)-C(15) 121.6(3) C(11)-C(12)-C(15) 59.8(2) C(14)-C(12)-C(13) 113.5(3) C(11)-C(12)-C(13) 114.8(3) C(15)-C(12)-C(13) 115.3(3) C(12)-C(13)-H(13A) 109.5 C(12)-C(13)-H(13B) 109.5 H(13A)-C(13)-H(13B) 109.5 C(12)-C(13)-H(13C) 109.5 H(13A)-C(13)-H(13C) 109.5 H(13B)-C(13)-H(13C) 109.5 C(12)-C(14)-H(14A) 109.5 C(12)-C(14)-H(14B) 109.5 N(14A)-C(14)-N(14B) 109.5 C(12)-C(14)-H(14C) 109.5 H(14A)-C(14)-H(14C) 109.5 H(14B)-C(14)-H(14C) 109.5 C(11)-C(15)-C(16) 108.6(2) C(11)-C(15)-C(12) 60.04(19) C(16)-C(15)-C(12) 120.2(3) C(11)-C(15)-H(15) 117.9 C(16)-C(15)-H(15) 117.9 C(12)-C(15)-H(15) 117.9 N(2)-C(16)-C(15) 104.0(2) N(2)-C(16)-H(16A) 111,0 C(15)-C(16)-H(16A) 111,0 N(2)-C(16)-H(16B) 111,0 C(15)-C(16)-H(16B) 111,0 N(16A)-C(16)-N(16B) 109,0

The symmetry transformation used to generate equivalent atoms.

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Table BB Anisotropic substitution parameters (A ² x10 ³ ) for C42. The exponent of the anisotropic shift coefficient takes the form: -2n ² [h ² a* ² U11 + .. + 2 hka* b* U ¹² ]

U ^{B * * 11} U ²² U ³³ U ²³ U ¹³ u ¹² C(1) 249(10) 244(10) 92(4) 107(6) 116(6) 219(9) F(l) 349(14) 444(17) 236(8) 274(10) 264(10) 348(15) F(3) 299(12) 246(10) 152(5) 168(7) 178(7) 234(10) F(1A) 349(14) 444(17) 236(8) 274(10) 264(10) 348(15) F(3A) 299(12) 246(10) 152(5) 168(7) 178(7) 234(10) F(2) 294(11) 235(8) 94(3) 84(4) 57(5) 147(8) N(l) 80(2) 83(2) 52(1) 41(1) 37(1) 65(2) N(2) 52(1) 49(1) 44(1) 31(1) 30(1) 39(1) 0(1) 170(4) 160(4) 107(3) 88(3) 95(3) 145(4) 0(2) 83(2) 74(2) 73(2) 50(1) 32(1) 59(1) 0(3) 82(2) 56(1) 91(2) 43(1) 69(2) 42(1) 0(4) 71(1) 50(1) 93(2) 32(1) 61(1) 39(1) C(2) 109(3) 117(4) 73(2) 59(3) 57(2) 93(3) C(3) 62(2) 62(2) 51(2) 33(1) 28(1) 47(2) C(4) 62(2) 64(2) 71(2) 36(2) 35(2) 42(2) 0(5) 76(2) 85(3) 94(3) 58(2) 54(2) 44(2) C(6) 97(3) 1H(4) 134(4) 83(4) 80(3) 82(3) 0(7) 69(2) 70(2) 84(3) 30(2) 32(2) 34(2) 0(8) 62(2) 59(2) 56(2) 40(1) 36(1) 46(1) 0(9) 55(1) 48(1) 49(1) 33(1) 35(1) 37(1) 0(10) 56(2) 50(1) 57(2) 34(1) 39(1) 38(1) 0(11) 53(1) 54(1) 45(1) 31(1) 32(1) 38(1) 0(12) 61(2) 69(2) 63(2) 46(2) 44(1) 50(2) 0(13) 62(2) 85(2) 86(2) 57(2) 50(2) 54(2) 0(14) 91(3) 109(3) 89(2) 77(2) 72(2) 77(2) 0(15) 57(2) 57(2) 50(1) 38(1) 33(1) 43(1) 0(16) 60(2) 51(1) 57(2) 35(1) 33(1) 42(1)

In addition, regarding the preparation of (N-(trifluoroacetyl)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-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 scheme shown immediately below. In step 1, methyl (N-(trifluoroacetyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate hydrochloride is initially treated with triethylamine in a mixture of tetrahydrofuran and water to neutralize the hydrochloride salt, followed by hydrolysis of the methyl ester using sodium hydroxide in a mixture of tetrahydrofuran and azabicyclo[3.1.0]hexane-2-carboxylate.

azabicyclo[3.1.0]hexane-2-carboxylate

HCI and water to obtain sodium (Zh^^)-6,6-dimethyl-3Obtaining sodium (1K,28,58)-6,6-dimethyl-3-

i) TED, THF water ii) aqueous NaOH, THF water n

Tetrahydrofuran (30 ml), water (7.5 ml), triethylamine (7.62 ml,

54.7 mmol) and methyl (W^^)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate hydrochloride (7.59 g, 36.9 mmol). The mixture was stirred at 25 °C for at least 30 min. Stirring was stopped and the layers were separated. In a separate container, 28% w/w aqueous sodium hydroxide (4.19 mL, 38.3 mmol) and tetrahydrofuran (71 mL) were 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 was added to the separation solution and the solution was 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 from a similar procedure). The mixture was kept at 40°C for at least 15 min and the remaining 75% of the organic layer was slowly added. The mixture was stirred at 40°C for 16 h, then cooled slowly to 20°C and held for at least 4 h. The resulting solid sodium (^^^)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate was isolated by filtration, washed with a solution of tetrahydrofuran (43 ml) and water (2.25 ml). The solid was dried at 70°C under vacuum to give 6.13 g (93.8%) of sodium (^^^)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate as a crystalline solid. PXRD was determined according to the methods described above.

PXRD peaks of crystalline sodium (^^^)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane2-carboxylate were selected_________________________________________________________________

Angle degrees 2-Θ ± 0.2 °2-0 Relative intensity Angle, degrees 2-0 ± 0.2 °2-0 Relative intensity Angle degrees 2-0 ± 0.2 °2-0 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 AND 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

PXRD peaks of (c)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (C91) were selected

Angle, degrees 2-0 ± 0.2 °2-0 Relative intensity Angle, degrees 2± 0.2 °2-0 Relative intensity Angle, degrees 2-0 ± 0.2 °2-0 Relative intensity 9.7 27 28.1 4 37.7 1 I,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

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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 AND 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 More precisely] trifluoroacetate; nie dash amido)butane(Ident 1ЫХ and oic acid ification structures (C91). code crystal E178 and for (c)-3,3-dim( lil-2-(2,2,2- Empirical formula C8H12F3NO3 Mass by formula 227.19 Temperature 173(2)K Wavelength 1.54178 A Crystal system Tetragonal Space group P4Λ2 Parameters of the unit cell a = 9.9168(6) A a= 90°. Ь = 9.9168(6) A Ь= 90°. c = 22.721(2) A g = 90°. Volume 2234.5(4) A ³ Z 8

Density (calculated) 1.351 mg/ ^m3 Absorption coefficient 1,184 mm‘1

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F(000)

Crystal size

Theta Range for Data Collection Index Ranges Collected Reflections Independent Reflections

Completeness to theta = 67.679° Absorption correction Refinement method

Data / Constraints / Parameters Consistency Criteria on

Final R indices [1>2sigma(1)] R indices (all data)

Absolute structure parameter Extinction coefficient Largest differential peak and well

944

0.200x0.170x0.080 mm ³ from 4.866 to 70.114°.

-1 l<=h<=10, -12<=k<=12, -27<=1<=27 48160

2122 [R(int) = 0.0392]

99.8%

Empirical

Least squares in full matrix approximation on F ³ 2122/9/158

1,010

RI = 0.0408, wR2 = 0.1012

RI = 0.0429, wR2 = 0.1039

0.03(4) n/a

0.280 and -0.215 e.A-3

In step 2, the resulting sodium (^^^)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate is then coupled with (c)-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, followed by treatment with HCl in brine, followed by treatment with water and heptane to give C42.

Crystalline C42 was characterized using PXRD and an additional form was obtained from prolonged drying or higher temperature identified.

PXRD peaks for C42 were selected - the crystalline form was obtained by drying for a long time or at higher temperature.

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Angle degrees 2-Θ ± 0.2 °2-0 Relative intensity Angle degrees 2-0 ± 0.2 °2-0 Relative intensity Angle degree 2-0 ± 0.2 °2-0 Relative intensity 10.2 3 25.7 AND 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 a compound to prevent cell death or cytopathic effect induced by the SARS-CoV-2 coronavirus 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 were serially inoculated with SARS-CoV-2 (USA_WA1/2020) at a multiplicity of infection of 0.002 in a BSL-3 laboratory. Virus-inoculated cells were then added to compound-ready assay plates at a density of 4,000 cells/well. Following a 3-day incubation, the time at which virus-induced cytopathic effect is 95% of untreated, infected control conditions, cell viability was assessed using Cell Titer-Glo (Promega) according to the manufacturer's protocol, which quantifies ATP levels. Cytotoxicity of compounds was assessed in parallel uninfected cells. Test compounds were tested either alone or in the presence of the P-glycoprotein (P-gp) inhibitor CP-100356 at a concentration of 2 μM. The inclusion of CP-100356 was intended to assess whether the test compounds were released from VeroE6 cells, which have a high level of P-gp expression. The percent effect at each concentration of test compound was calculated from the values for the virus-free control wells and the virus-containing control wells in each test plate. The concentration required to achieve a 50% response (EC50) was determined from these data using a 4-parameter logistic model. _EC50 curves had a Hill slope of 3 when >3 and the maximum dose achieved >50% effect. If cytotoxicity was detected at an effect greater than 30%, the corresponding concentration data were extracted from the EC50 determination.

For the cytotoxicity plates, the percent effect at each concentration of test compound was calculated based on the values for the cell-only control wells and the hyamine control wells in each test plate. _CC50 values ( _CC50 stands for: 50% cytotoxicity concentration, which is the concentration of test compound required to reduce cell viability by 50%) were calculated using a 4-parameter logistic model. The TI (therapeutic index) was then calculated by dividing the _CC50 value by the EC50 value (which is the concentration of compound at which an effect equal to half its maximal effect is observed).

Study and analysis of FRET 3C protease of coronavirus SARS-CoV-2.

The proteolytic activity of the main protease, 3CLpro, of SARS-CoV-2 was monitored using

- 178 047572 using continuous fluorescence resonance energy transfer (FRET) analysis. The SARS-CoV-2 3CLpro assay measures the activity of the full-length SARS-CoV-2 3CL protease to cleave a synthetic fluorogenic substrate peptide with the following sequence: DabcylKTSAVLQ-SGFRKME-Edans according to the consensus peptide model (V. Grum-Tokars et al. Evaluating the 3Clike protease activity of SARS-coronavirus: recommendations for standardized assays for drug discovery. Virus Research 133 (2008) 63-73). The fluorescence of the cleaved Edans peptide (excitation 340 nm/emission 490 nm) is measured using the fluorescence intensity protocol on a Flexstation reader (Molecular Devices). The fluorescent signal is reduced in the presence of PF-835231, a potent inhibitor of 3CLpro SARS-CoV-2. 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 adding 15 nM SARS-CoV-2 3OE protease and allowed to proceed for 60 min at 23°C. Percent inhibition or activity was calculated from blank wells containing no compound (0% inhibition/100% activity) and control compound (100% inhibition/0% activity). IC ₅₀ values were generated using four parametric smoothing models using ABASE (IDBS) software. The Ki values were fitted to the Morrison equation with the enzyme concentration parameter fixed at 15 nM, the Km parameter fixed at 14 μM, and the substrate concentration parameter fixed at 25 μM using ABASE (IDBS) software.

The proteolytic activity of the SARS-CoV-2 coronavirus 3OE protease was measured using a continuous fluorescence resonance energy transfer assay. The FRET 3CL ^pro SARS-CoV-2 assay measures the protease-catalyzed cleavage of TAMRA-SITSAVLQSGFRKMK-(DABCIL)-OH to TAMRA-SITSAVLQ and SGFRKMK(DABCIL)-OH. The fluorescence of the cleaved TAMRA peptide (excit. 558 nm/irradiation 581 nm) was measured using a TECAN SAFIRE fluorescence plate reader over 10 min. Typical reaction solutions contained 20 mM HEPES (pH 7.0), 1 mM EDTO, 4.0 μM FRET substrate, 4% DMSO, and 0.005% Tween-20. Studies were started by adding 25 nM 3CL ^pro SARS (nucleotide sequence 9985-10902 of the Urbani strain of the complete genome sequence of SARS coronavirus (NCBI accession number AY278741)). Percent inhibition was determined in duplicate at an inhibitor level of 0.001 mM. Percent 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 = bias + (limit) (1 - e ^- ( ^(kobs) t) where FU are fluorescence units, bias is the fluorescence signal of uncleaved peptide substrate, and limit is the fluorescence of fully cleaved peptide substrate. Kobs is the first-order rate constant for a given reaction, and in the absence of any inhibitor represents the substrate utilization. In an enzyme triggered reaction involving an irreversible inhibitor and where the calculated limit is less than 20% of the theoretical maximum limit, the calculated kobs represents the rate of inactivation of coronavirus 3C protease. The slope (kobs/I) of a plot of kobs versus [I] is a measure of the avidity of the inhibitor for the enzyme. For very fast irreversible inhibitors, kobs/I is calculated from observations of only one or two [I]s, rather than as the slope.

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Table 2

Biological activities and IUPAC names for examples 1-84

Example number Geometric mean Ki (µM) Amount of K used (µM) Geometric mean EC50 (µM) Amount of used xEC ₅₀ (μM) IUPAC name 1 0,013 4 0.246 7 (1 R,2S,5S)-N-{(15)-1-Cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl }-6,6-dimethyl-3-[7V(trifluoroacetyl)-E-valyl]-3azabicyclo[3.1.0]hexane-2carboxamide 2 1.08 3 7.52 2 A-{(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-4methyl-TV ² -(pyrrolidin-1 ylacetyl)-E-leucinamide, trifluoroacetate salt 3 0.439 2 6.74 6 N- {(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-A ² (2,6-dichlorobenzoyl)-4-methyl-Eleucinamide 4 0,026 3 1.36 15 A-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-1-nindole-2-carboxamide 5 >0.351 2 >3.33 1 A-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-3(trifluoromethyl)-17/-indole-2-carboxamide 6 0,023 2 0.279 4 A-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-7(trifluoromethyl)-1 R-indole-2-carboxamide 7 0,798 1 43.1 2 A-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]-3 methylimidazo [2,1 -3\ [ 1,3 ]thiazole-2-carboxamide 8 0,917 3 5.75 2 A-{1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-A ² [cyclohexyl(methoxy)acetyl]-4methyl-L-leucinamide, DIAST-1 9 0.254 4 0,970 4 A-{1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-A ² [cyclohexyl(methoxy)acetyl]-4methyl-L-leucinamide, DIAST-2 10 0,056 4 2.09 4 A-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-1-nindole-2-carboxamide

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11 0.297 3 4.78 4 N ² - [(4-bro m-1-ethyl-3-methyl-l//pyrazol-5 -yl)carbonyl] -N- {(15)1-cyano-2-[(35)-2oxopyrrolidin-3 -yl]ethyl } -4methyl-L-leucinamide 12 0.539 3 6.33 2 #-{(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-A ² [(3,3 -difluorocyclobutyl)acetyl] 4-methyl-L-leucinamide 13 0,003 6 0,075 18 (1Я,25,5S)-N-{(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl }-6,6-dimethyl-3-[3-methylA-(trifluoroacetyl)-N-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide 14 0.302 2 N.D,1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-5,5,5 -trifluoro-1 oxopentan-2-yl]-4-methoxy-1-nindole-2-carboxamide 15 0,002 1 0,360 2 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-7-fluoro-4methoxy-1 H-indole-2-carboxamide 16 0,018 2 >2.53 2 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-5-fluoro-4methoxy-1 H-indole-2-carboxamide 17 0,053 1 >0.333 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-3 -fluoro-4methoxy-1 H-indole-2-carboxamide 18 0,019 2 >0.333 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-5,7-difluoro-4methoxy-1 H-indole-2-carboxamide 19 0.208 2 >0.333 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-3,5-difluoro-4methoxy-1 H-indole-2-carboxamide

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20 0,005 1 >0.333 1 A-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]-7-fluoro-4methoxy-1 H-indole-2-carboxamide 21 BUT. BUT. >3.17 2 A-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]-5-fluoro-4methoxy-1 H-indole-2-carboxamide 22 0,066 1 >0.333 1 A-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]-3 -fluoro-4methoxy-1 H-indole-2-carboxamide 23 0,021 1 >0.333 1 A-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]-5,7-difluoro-4methoxy-1 H-indole-2-carboxamide 24 1.93 2 6.30 6 A-{(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-4methyl-A ² -{[2-(trifluoromethyl)1,3-thiazol-4-yl]carbonyl}-L-leucinamide 25 BUT. 37 2 A-{1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-A ² (cyclohexylcarbonyl)-4-methylL-leucinamide, DIAST-1 26 But. >100 1 A-{1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-A ² (cyclohexylcarbonyl)-4-methylL-leucinamide, DIAST-2 27 4.50 2 44.9 2 N- {(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl }-4methyl-A ² -[(propan-2-yloxy)acetyl]-L-leucinamide 28 1.79 2 7.26 4 N- {(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-A ² [(cyclohexyloxy)acetyl] -4methyl-L-leucinamide 29 2.23 2 28.1 2 N- {(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl }-4methyl-A ² -(4,4,4-trifluoro-3methylbutanoyl)-L-leucinamide

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30 >10.8 1 9.37 2 N- {(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-A ² [(25)-2-(dimethylamino)-2-phenylacetyl]-4-methyl-L-leucinamide 31 0,606 3 40.7 2 A ² -[(m/?sh/s-4-cyanocyclohexyl)carbonyl]-#{(15)-1-cyano-2-[(35)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide or A ² -[(m/?sh/s-4-cyanocyclohexyl)carbonyl]-#{(1R)-1-cyano-2-[(35)-2-oxopyrrolidin-3-yl]ethyl}-4-methyl-L-leucinamide 32 0,690 3 4.90 2 N- {(15)-1-cyano-2-[(35)-2-oxopyrrolidin-3-yl]ethyl}-A ² [2-(cyclohexyloxy)propanoyl] 4-methyl-L-leucinamide or N- {(1R)-1-cyano-2-[(35)-2-oxopyrrolidin-3-yl]ethyl}-A ² [2-(cyclohexyloxy)propanoyl] 4-methyl-L-leucinamide 33 0,068 2 >0.333 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-4-hydroxy-1-nindole-2-carboxamide 34 0,074 2 >0.333 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-5-hydroxy-4methoxy-1 H-indole-2-carboxamide 35 0,176 3 3.56 4 N ² -[(4-chloro-1,3-dimethyl-1-pyrazol-5-yl)carbonyl]-N-{(15)1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-4methyl-L-leucinamide 36 0.241 4 1.03 2 #-{(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-A ² [(27?)-2-(dimethylamino)-2-phenylacetyl]-4-methyl-L-leucinamide 37 0,168 1 >3.33 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl} amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-3(trifluoromethyl)-1 //-indole-2-carboxamide

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38 0,023 1 >0.333 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-7(trifluoromethyl)-3-indole-2-carboxamide 39 0,111 1 >3.33 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl ] amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-3,7 bis(trifluoromethyl)- 1//-indole-2-carboxamide 40 0,131 1 >0.333 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl ] amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-3,5 bis(trifluoromethyl)- 1//-indole-2-carboxamide 41 0.104 1 >0.333 1 U-[(2A)-1-({(15)-1-cyano-2-[(35)2-oxopyrrolidin-3 yl]ethyl]amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-3,6-bis(trifluoromethyl)- 1R-indole-2-carboxamide ² 42 >0.356 1 >0.333 1 N- {(1S)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl]-A ² { (27?)-2-(dimethylamino)-2-[3 (trifluoromethyl)phenyl] acetyl]-4methyl-L-leucinamide 43 >0.356 1 >0.333 1 N- {(15)-1-cyano-2-[(35)-2oxopyrrolidine-3-methyl}-?/ ² {(27?)-2-(dimethylamino)-2-[4(trifluoromethyl)phenyl]acetyl]-4methyl-L-leucinamide 44 0.302 2 5.88 2 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl ] amino)-4-methyl-1 oxopentan-2-yl]-6(trifluoromethyl)-3-indole-2-carboxamide 45 0.227 2 6.42 2 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl ]amino)-4,4-dimethyl-1 oxopentan-2-yl]-6(trifluoromethyl)-3-indole-2-carboxamide 46 0.283 1 >3.33 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl ]amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-3,7 bis(trifluoromethyl)- 1H-indole-2-carboxamide

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47 >0.359 1 >3.33 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy3,6,7-tri(trifluoromethyl)- 1Nindole-2-carboxamide 48 >0.359 1 >3.33 1 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy3,5,7-tri(trifluoromethyl)- 1Nindole-2-carboxamide 49 0,083 3 2.68 2 #-[(25)-1-({(15)-1-cyano-2-[(35)- 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 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]-4(trifluoromethoxy)-1H-indole-2-carboxamide 51 0,192 3 BUT. #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]-5(trifluoromethyl)-1 R-indole-2-carboxamide 52 0,032 3 But. 7-chloro-TV-[(25)-1-({ (15)-1 -cyano2-[(35)-2-oxopyrrolidin-3yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]- 1//-indole-2-carboxamide 53 0,025 3 But. 7U-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]-4-methoxy-7methyl- 1//-indole-2-carboxamide 54 0,049 3 But. 6-chloro-A - [(25)-1-({(15)-1 -cyano2-[(35)-2-oxopyrrolidin-3yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]- 1//-indole-2-carboxamide 55 0.104 3 But. 4-chloro-#- [(25)-1-({(15)-1 -cyano2-[(35)-2-oxopyrrolidin-3yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]- Ш-indole-2-carboxamide

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56 0,151 3 But. 5-chloro-TV-[(25)-1-({ (15)-1 -cyano2-[(35)-2-oxopyrrolidin-3yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]- 1//-indole-2-carboxamide 57 0,052 3 But. TU-[(25)-1-({(15)-1-cyano-2-[(35)2-oxopyrrolidine-3 yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-7(trifluoromethyl)-1 R-indole-2-carboxamide 58 0,091 3 But. 4,6-dichloro-TU-[(25)-1-({(15)-1cyano-2-[(35)-2-oxopyrrolidin3-yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]- 1H-indole-2-carboxamide 59 0,152 3 But. U-[(25)-1-({(15)-1-cyano-2-[(35)2-oxopyrrolidin-3 yl]ethyl}amino)-4,4-dimethyl-1 oxopentan-2-yl]-4(trifluoromethyl)-1 R-indole-2-carboxamide 60 0.261 3 But. U-[(25)-1-({(15)-1-cyano-2-[(35)2-oxopyrrolidin-3 yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-5(trifluoromethyl)-1 R-indole-2-carboxamide 61 0,034 3 But. 7-hl op-TV-[(25)-1-({ (15)-1 -cyano2-[(35)-2-oxopyrrolidin-3yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]- 1H-indole-2-carboxamide 62 0,029 3 But. U-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl} amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-7methyl- 1//-indole-2-carboxamide 63 0,122 3 But. 6-chloro-X-[(28)-1-({(1S)-1-cyano2-[(3 8)-2-oxopyrrolidin-3yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-1H-indole-2-carboxamide 64 0,038 3 But. 4-chloro-U-[(25)-1-({(15)-1-cyano2-[(35)-2-oxopyrrolidin-3yl]ethyl}amino)-4-methyl-1 oxopentan-2-yl]-1H-indole-2-carboxamide

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65 0,117 3 But. 5-chloro-TV-[(25)-1-({ (15)-1 -cyano2-[(35)-2-oxopyrrolidin-3yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]- 1H-indole-2-carboxamide 66 0,073 3 But. U-[(25)-1-({(15)-1-cyano-2-[(35)2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-7(trifluoromethyl)-1 //-indole-2-carboxamide 67 0,041 3 But. 4, b-dichloro-TV-[(25)-1 -({(15)-1 ’ cyano-2-[(35)-2-oxopyrrolidin3 -yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]- 1H-indole-2-carboxamide 68 0,092 3 But. #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-4(trifluoromethyl)-1 //-indole-2-carboxamide 69 0,083 1 >2.67 2 #-[(25)-1-({(15)-1-cyano-2-[(35)- 2-oxopyrrolidin-3 yl]ethyl } amino)-4,4-dimethyl-1 oxopentan-2-yl]-3-methyl-5(trifluoromethyl)imidazo[2,1 Ь\[ 1,3 ]thiazole-2-carboxamide 70 6.95 2 4.09 2 N- {(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl }-4methyl-TV ² -{[5-methyl-2(trifluoromethyl)-1,3-thiazol-4yl]carbonyl }-L-leucinamide 71 0.254 3 4.99 2 N- {(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl }-4methyl-TV ² -{[4-methyl-2(trifluoromethyl)-1,3-thiazol-5-yl]carbonyl }-L-leucinamide 72 0,173 3 1.35 2 N ² -[(4-bromo-1-ethyl-3-methyl-1-pyrazol-5-yl)carbonyl] -N- {(15)1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl } -L-leucinamide 73 0.226 3 2.03 2 N ² -[(4-chloro-1,3-dimethyl-Spyrazol-5-yl)carbonyl] -N- {(15)1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl } -L-leucinamide

- 187 047572

74 >0.356 1 >3.33 1 3-acetyl-TV- [(25)-1 -({(15)-1 cyano-2-[(35)-2-oxopyrrolidin3 -yl]ethyl } amino)-4-methyl-1 oxopentan-2-yl]-4-methoxy-1-nindole-2-carboxamide 75 0,017 2 0.551 4 Diastereomer 1: (2S,4R)-4-methylbutyl-V-{(15)-1-cyano-2-[(35)-2-oxopyrrolidin-3-yl]ethyl}-1{V-[(trifluoromethyl)sulfonyl]L-valyl} piperidine-2-carboxamide or (25,45)-4-methylbutyl-V-{(15)-1-cyano-2-[(35)-2-oxopyrrolidin-3-yl]ethyl}-1-{V-[(trifluoromethyl)sulfonyl]L-valyl} piperidine-2-carboxamide 76 >8.16 2 >100 1 Diastereomer 2: (2S,4R)-4-methylbutyl-Jv-{(15)-1-cyano-2-[(35)-2-oxopyrrolidin-3-yl]ethyl}-1{V-[(trifluoromethyl)sulfonyl]L-valyl} piperidine-2-carboxamide or (25,45)-4-/7?/7c/7?-butyl-5-{(15)-1-cyano-2-[(35)-2-oxopyrrolidin-3-yl]ethyl}-1-{V-[(trifluoromethyl)sulfonyl]-L-valyl} piperidine-2-carboxamide 77 0,004 2 0,085 4 3-methyl-Λ-(trifluoroacetyl)-Lvalyl-(45)-U- {(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl }-4-(trifluoromethyl )-Lprolinamide 78 0,005 3 2.74 2 (15,25,5S)-N- {(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl }-6,6-dimethyl-3-[3-methyl-5-(methylcarbamoyl)-L-valyl]-3 azabicyclo[3.1.0]hexane-2-carboxamide 79 0,001 4 0,080 4 methyl {(25)-1-[(15,25,55)-2({(15)-1-cyano-2-[(35)-2oxopyrrolidin-3 yl]ethyl } carbamoyl)-6,6-dimethyl3 -azabicyclo[3.1.0]hexan-3 -yl]- 3,3 -dimethyl-1 -oxobutan-2yl}carbamate 80 0,037 2 0,158 3 U-(trifluoroacetyl)-L-valyl-(45)- U-{(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl}-4(trifluoromethyl)-E-prolinamide

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81 0,003 2 0,690 3 (1R,2S,5S)-N-{(15)-1-cyano-2[(37?)-5-hydroxy-2oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-75- (trifluoroacetyl)-L-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide 82 0.139 1 BUT. BUT. (1R,2S,5S,6R)-N-{(15)-1-cyano-2[(35)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6methyl-3-[3-methyl-75(trifluoroacetyl)-L-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide 83 0,092 1 But. But. (1R,2S,55,6S)-N-{(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl}-6-(hydroxymethyl)-6methyl-3-[3-methyl-75(trifluoroacetyl)-L-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide 84 0,003 1 But. But. ( 17?, 25,55)-75- {(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl}-3-[3-(hydroxymethyl)- A-(trifluoroacetyl)-L-valyl]-6,6-dimethyl-3azabicyclo[3.1.0]hexane-2-carboxamide 85 0,004 1 0.334 2 (17?,25,55)-75-{(15)-1-cyano-2[(37?)-2,5-dioxopyrrolidin-3yl]ethyl }-6,6-dimethyl-3-[3-methyl75-(trifluoroacetyl)-E-valyl]-3azabicyclo[3.1.0]hexane-2carboxamide 86 0,018 2 0.237 2 (17?,25,55)-75-{(15)-1-cyano-2[(35)-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 (17?,25,55)-75- {(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl } -3 -[(25)-2-cyclohexyl2- {[(trifluoromethyl)sulfonyl]amino } acetyl] -6,6-dimethyl-3 azabicyclo [3.1.0] hexane-2-carboxamide

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88 0,006 2 0,050 4 First eluted diastereomer: (1R,2S,5S)-N-{(1S)1-cyano-2-[(35)-2-oxopyrrolidin-3-yl]ethyl }-3-[3-cyclobutyl-A-(trifluoroacetyl)- L-alanyl]-6,6-dimethyl-3azabicyclo [3.1.0] hexane-2-carboxamide or (1R,2S,5S)-N-{(15)-1-cyano-2[(35)-2-oxopyrrolidin-3-yl]ethyl }-3-[3-cyclobutyl-TV(trifluoroacetyl)-O-alanyl]-6,6-dimethyl-3azabicyclo [3.1.0] hexane-2-carboxamide 89 0,007 3 0,040 4 Second eluted diastereomer: (1R,2S,5S)-N-{(1S)1-cyano-2-[(35)-2-oxopyrrolidin-3-yl]ethyl }-3-[3-cyclobutyl-A-(trifluoroacetyl)- L-alanyl]-6,6-dimethyl-3azabicyclo [3.1.0] hexane-2-carboxamide or (1R,2S,5S)-N-{(15)-1-cyano-2[(35)-2-oxopyrrolidin-3-yl]ethyl }-3-[3-cyclobutyl-TV(trifluoroacetyl)-O-alanyl]-6,6-dimethyl-3azabicyclo [3.1.0] hexane-2-carboxamide 90 0,015 2 0,571 2 (1 A, 25', 5S)-N- {(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl } -6,6-dimethyl-3-[3 (pyridin-2-yl)-A(trifluoroacetyl )-y-alanyl]-3 azabicyclo[3.1.0]hexane-2-carboxamide 91 0,007 2 0,011 2 (17?,25',55)-V- {(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl } -3 - {#-[(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-#-[(4methylphenyl)acetyl]-L-valyl(4R)-N- {(15)-1-cyano-2-[(35)-2oxopyrrolidin-3-yl]ethyl }-4(trifluoromethyl)-L-prolinamide

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93 0,014 2 0,882 2 (1R, 2S, 5S)-N- {(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl } -6,6-dimethyl-3 - [3 -(177pyrazol-1 -yl)-7Y(trifluoroacetyl)-b-alanyl]-3 azabicyclo[3.1.0]hexane-2carboxamide 94 0,011 2 0.379 2 (17?,25,5S)-N-{(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl }-3-[(25)-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 7/-(methoxycarbonyl)-3-methyl-Lvalyl-(47?)-7U-{(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl }-4-(trifluoromethyl )-Lprolinamide 96 0,844 1 9,190 2 (17?,25,5S)-N-{(17?)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl}-6,6-dimethyl-3-[3-methyl7/-(trifluoroacetyl)-L-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide 97 0,011 5 0,082 16 (17?,25,55)-A-{(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl }-6,6-dimethyl-3-[2-(2,2,2-trifluoroacetamido)-3(trifluoromethyl)pentanoyl]-3azabicyclo[3.1.0]hexane-2carboxamide, from C86 (DIAST-2) 98 0,188 1 1,738 2 (17?,25,55)-A-{(15)-1-cyano-2[(35)-2-oxopyrrolidin-3 yl]ethyl}-6,6-dimethyl-3-[2-(2,2,2-trifluoroacetamido)-3(trifluoromethyl)pentanoyl]-3azabicyclo[3.1.0]hexane-2carboxamide, from C85 (DIAST-1)

1. Not determined.

2. The regiochemistry of Example 41 has not been precisely determined; other possible structures for this example are N-(28)-1-({(18)-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-[(38)-2-oxopyrrolidin-3-yl]ethyl}amino)-4,4-dimethyl-1-oxopentan-2-yl]-4-methoxy-6,7bis(trifluoromethyl)-1H-indole-2-carboxamide.

Predicted pharmacokinetic parameters of (^,28,58)-N-{(18)-1-cyano-2-[(38)-2oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-N-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide (the compound from Example 13) in humans.

Based on physiologically based pharmacokinetic (PBPK) modeling of in vitro data including CL _int from human liver microsomes and CL _bile from human hepatocytes under sandwich culture conditions, the predicted human plasma CL and V _ss of (^,28,58)-N-{(18)-1-cyano-2[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3azabicyclo[3.1.0]hexane-2-carboxamide are 5.9 mL/min/kg and 0.97 L/kg, respectively, providing an effective elimination half-life, t1/ ₂ , of 1.9 hours. The target Ceff of 0.16 μM (unbound plasma concentration) was determined based on antiviral inhibition data obtained in vitro with (N,28,58)-N-{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide in or VeroE6 cells in the presence of a P-gp inhibitor (EC ₉₀ values of 0.156 μM) or in a differentiated normal human bronchial epithelial (dNHBE) cell assay (EC ₉₀ values of 0.149 μM). A dose of 380 mg of (1R,28,58)-N{(18)-1-cyano-2-[(38)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide administered orally three times daily (TID) is designed to cover an effective unbound concentration of 0.16 μM at C _min .

All patents and publications described herein 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 should be understood as

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Claims (16)

such as is not limited by the above detailed description, but as defined by the formula which is appended and their equivalents. CLAUSE OF THE INVENTION 1. A compound selected from the group consisting of where R ⁴ is selected from the group consisting of (C1-C ₆ -alkyl)amino optionally substituted with one to five fluorines, Q-^^km-C^NH-, optionally substituted with one to five fluorines, and Ο ₁ -Ο ₆ alkyl^(O) ₂ NH-, optionally substituted with one to five fluorines; or a solvate thereof, or a pharmaceutically acceptable salt of said compound or solvate. 2. The compound according to claim 1, wherein R ⁴ is selected from the group consisting of CF3C(O)NH-, CF3S(O)2NH-, - 192 047572 CH ₃ C(O)NH-, CH ₃ CH ₂ C(O)NH- and CF3CH2NH-; or a solvate thereof, or a pharmaceutically acceptable salt of said compound or solvate. 3. The compound according to claim 2, wherein R ⁴ is CF ₃ C(O)NH- or CF ₃ S(O) ₂ NH-; or a solvate thereof. 4. The compound N-(methoxycarbonyl)-3-methyl-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-G-prolinamide, having the structure CF ₃ or its solvate. 5. The compound according to claim 4, which is N-(methoxycarbonyl)-3-methyl-L-valyl-(4R)-N{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-4-(trifluoromethyl)-G-prolinamide. 6. A pharmaceutical composition comprising a compound according to any one of claims 1-5 or a solvate thereof and a pharmaceutically acceptable carrier. 7. Use of a compound according to any one of claims 1-5 or a solvate thereof, or a pharmaceutical composition according to claim 6 for the treatment of coronavirus infection in a patient. 8. The use according to paragraph 7, where the coronavirus infection is COVID-19. 9. Use according to item 8, wherein the patient is simultaneously administered ritonavir. 10. Use according to claim 9, wherein the compound according to any one of claims 1-5 and ritonavir are administered to the patient orally. 11. The use according to claim 10, wherein the patient is administered from about 10 mg to about 1500 mg per day of the compound according to any one of claims 1-5 and from about 10 mg to about 1000 mg per day of ritonavir. 12. The use according to claim 11, wherein the patient is orally administered twice a day approximately 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 the compound according to any one of claims 1 to 5. 13. Use according to item 12, wherein the patient is simultaneously administered ritonavir orally twice a day. 14. The use according to claim 13, wherein the patient is simultaneously administered orally twice a day approximately 300 mg of the compound according to any one of claims 1-5 and approximately 100 mg of ritonavir. 15. Use of a compound according to any one of claims 1-5 or a solvate thereof in the manufacture of a medicinal product for the treatment of coronavirus infection in a patient. 16. A compound selected from the group consisting of (^^^)-N-{(^)-1-cyano-2-[N-(trifluoroacetyl)-N-valyl]-3-azabicylo[3.1.0]hexane-2-carboxamide; (2S,4R)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolgin-3-yl]ethyl}-1-{N[(trifluoromethyl)sulfonyl]N-valyl}piperidine-2-carboxamide; (2R,4S)-4-tert-butyl-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolgin-3-yl]ethyl}-1-{N[(trifluoromethyl)sulfonyl]N-valyl}piperidine-2-carboxamide; 3-methyl-N-(trifluoroacetyl)-L-valyl-(4R)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-4(trifluoromethyl)G-prolinamide; (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(methylcarbamoyl)-N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; methyl{^)-1-[(^^^)-2-({(^)-1-cyano-2-[^)-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)N-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)-N-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-(trifluoroacetyl)-N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; (^^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-6-(hydroxymethyl)-6-methyl-3[3-methyl-N-(trifluoroacetyl)-6-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; (^^^)-N-{(^)-1-cyano-2-[^)-2-oxopyrrolidin-3-yl]ethyl}-3-[3-(hydroxymethyl)-N-(trifluoroacetyl)-N-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)-N-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide; (^^^)-N{(^)-1-cyano-2-[^)-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; - 193 -