Mapping the S1 and S1’ subsites of cysteine proteases with new dipeptidyl nitrile inhibitors as trypanocidal agents

Lorenzo Cianni; Carina Lemke; Erik Gilberg; Christian Feldmann; Fabiana Rosini; Fernanda dos Reis Rocho; Jean F. R. Ribeiro; Daiane Y. Tezuka; Carla D. Lopes; Sérgio de Albuquerque; Jürgen Bajorath; Stefan Laufer; Andrei Leitão; Michael Gütschow; Carlos A. Montanari

doi:10.1371/journal.pntd.0007755

Abstract

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. A series of 26 new compounds were designed, synthesized, and tested against the recombinant cruzain (Cz) to map its S1/S1´ subsites. The same series was evaluated on a panel of four human cysteine proteases (CatB, CatK, CatL, CatS) and Leishmania mexicana CPB, which is a potential target for the treatment of cutaneous leishmaniasis. The synthesized compounds are dipeptidyl nitriles designed based on the most promising combinations of different moieties in P1 (ten), P2 (six), and P3 (four different building blocks). Eight compounds exhibited a K_i smaller than 20.0 nM for Cz, whereas three compounds met these criteria for LmCPB. Three inhibitors had an EC₅₀ value of ca. 4.0 μM, thus being equipotent to benznidazole according to the antitrypanosomal effects. Our mapping approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cysteine proteases.

Author summary

Despite many achievements in identifying novel agents for the treatment of tropical and neglected diseases, further research continues to be of fundamental importance. Our research groups have been using the cruzipain cysteine protease in its recombinant form, cruzain (Cz), to identify new trypanocidal agents. Considering the possible interchangeability with other cysteine proteases, the same series of dipeptidyl nitriles was tested in Leishmania mexicana LmCPB. Other potential targets for such inhibitors are human cysteine cathepsins, which are involved in different disease states. Thus, the inhibitors were also tested against cathepsins B, L, K, and S. Our results demonstrate that appropriate structural modifications of dipeptidyl nitriles can lead to inhibition of these cysteine proteases. It was also possible to identify trypanocidal agents, equipotent to benznidazole, the current drug of choice used for the treatment of Chagas disease.

Citation: Cianni L, Lemke C, Gilberg E, Feldmann C, Rosini F, Rocho FdR, et al. (2020) Mapping the S1 and S1’ subsites of cysteine proteases with new dipeptidyl nitrile inhibitors as trypanocidal agents. PLoS Negl Trop Dis 14(3): e0007755. https://doi.org/10.1371/journal.pntd.0007755

Editor: Igor C. Almeida, University of Texas at El Paso, UNITED STATES

Received: August 29, 2019; Accepted: January 30, 2020; Published: March 12, 2020

Copyright: © 2020 Cianni et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: We are indebted to Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (grant #2013/18009-4, grant #2016/07946-5 and grant #2017/17386-0) for financing this project. We also want to acknowledge the National Council for Scientific and Technological Development (CNPq, grant # 304030/2018-0) in Brazil for scholarships. We would also like to thank the CAPES Drug Discovery program (Process #139/2015) that allowed us to evaluate LmCPB inhibitors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Chagas disease, aka American trypanosomiasis, is a severe health and social problem in Latin America and in new non-endemic areas such as Japan, East Europe, and the USA. Chagas disease has an annual incidence of 30,000 new cases and 10,000 deaths per year. An estimated 8 million people are infected with Chagas disease worldwide and more than 25 million people risk acquiring the disease [1].

The etiological agent, the protozoa parasite Trypanosoma cruzi (T. cruzi), is transmitted by blood-sucking reduviid bugs of the subfamily Triatominae [2]. The only two existing drugs in the market, benznidazole and nifurtimox, show strong side effects and inefficiency in the chronic stage of the disease [3,4]. New safe and efficacious drugs are therefore required to address with these still unmet medical needs. Initiatives such as the one launched by the Drugs for Neglected Diseases (DNDi) have led to worldwide collaborative efforts to discover new therapeutic targets [5]. Cruzain (Cz), a recombinant form of the enzyme cruzipain (EC 3.4.22.51) [6], is the most abundant cysteine protease (CP) present in the parasite and essential for its development and survival inside and outside the host cell in all forms of its life cycle. This makes Cz a druggable target for the development of new chemotherapeutic agents against Chagas disease [7,8].

Cz represents a target for irreversible (or suicide) and reversible inhibitors. K777 was at the forefront of the first generation of irreversible Cz inhibitors and initially characterized by the Sandler Center for Research in Tropical Parasitic Disease (University of California, San Francisco) [9]. Despite its ability to rescue mice of a lethal experimental T. cruzi infection and reduce parasite growth in dogs, preclinical safety and toxicology studies revealed substantial side effects of K777 in primates and dogs, even when administered in low doses [10,11]. Current research is being focused on reversible Cz inhibitors, as these are assumed to overcome possible off-target effects [12]. Drug repurposing programs of different pharmaceutical companies have recently put forward reversible inhibitors of Cz as potential drug candidates [13]. Two key compounds, Cz007 and Cz008, exhibited antiparasitic behavior in the low micro-molar range for the amastigote form of the parasite and they were capable of curing T. cruzi acute form infection in an in vivo model. Both compounds were orally active, well-tolerated and worked most effectively at doses of 3 mg/kg [13].

The structure of Cz is closely related to those of mammalian CPs (CatL, CatK and CatS). Three-dimensional (3D) Cz structures with a variety of ligands have already been resolved [14]. The target-based molecular design was applied to generate the first series of dipeptidyl nitrile ligands as reversible Cz inhibitors with trypanocidal activity [14]. In previous work, some of us reported on low micro-molar inhibitors of Cz as modest trypanocidal agents for the amastigotes form of the T. cruzi Tulahuen strain (pEC₅₀ around 4). For the most potent anti-parasite inhibitor with an EC₅₀ value of 28 μM, the activity was not attributed to the general cytotoxicity of the compound [14]. More recently, the feasibility of achieving a higher Cz affinity by benefiting from the S3 structural singularity was underlined [15].

In this study, we designed a new, structurally expanded series of 26 Cz-inhibiting dipeptidyl nitriles, in particular by leveraging the P1-S1/S1`interactions. We explored the structure-activity relationships (SARs), mapped the active site of the target enzyme and evaluated the antichagasic properties of the compounds. Besides that, we have tested them against four human cysteine cathepsins (CatB, CatL, CatK, CatS) all of which constituting important targets for human diseases [16], and against the cysteine protease LmCPB, a novel macromolecular target to fight Leishmania mexicana. As a result of this study, several new low-nanomolar inhibitors of different CPs were discovered and three new anti-T. cruzi agents, equipotent to benznidazole, were characterized.

Methods

Modeling

Putative binding modes of novel dipeptidyl nitrile inhibitors compounds were derived from the crystal structure of N-(2-aminoethyl)-alpha-benzoyl-l-phenylalaninamide (33L) bound to Cz (PDB ID: 4QH6). This ligand-target-complex served as a template for knowledge-based modeling and was preprocessed using the “Structure Preparation” and “Protonate3D”-tools of the modeling software “Molecular Operating Environment” (MOE) [17], version 2018.0101, with default settings. By modification of moieties, the cocrystallized ligand was structurally transformed into the compound of interest. Obtained conformations were optimized using the force field “Amber10:EHT”.

Synthetic chemistry

General considerations.

The synthesis was performed as summarized in Figs 1 and 2. Melting points were determined on a Büchi 510 oil bath apparatus and are uncorrected. Infrared spectra were obtained from FT-IR Thermo Scientific Nicolet 380. Reagents, starting materials and solvents were of commercial quality and were used without further purification unless otherwise stated. All syntheses were started with enantiopure amino acids. TLC analysis was carried out on Merck 60 F₂₅₄ silica gel plates and visualized under UV light at 254 nm and 365 nm or by using a ninhydrin staining solution. Preparative column chromatography was carried out on Grace Davison Davisil LC60A 20–45 micron or Merck Geduran Si60 63–200 micron silica using the Interchim PuriFlash 430 automated flash chromatography system. The purity of all tested compounds was determined with one of the three protocols (A-C) noted below.

A). Purity was determined via RP-HPLC on a Hewlett Packard 1090 Series II LC with a Phenomenex Luna C18 column (150 x 4.6 mm, 5 μm) and detection was performed by a UV DAD (200–440 nm). Elution was carried out with the following gradient: 0.01 M KH₂PO₄, pH 2.30 (solvent A), MeOH (solvent B), 40% B to 85% B in 8 min, 85% B for 5 min, 85% to 40% B in 1 min, 40% B for 2 min, stop time 16 min, flow 1.5 mL/min.
B). Purity was determined using an LC-MS instrument (ABSCIEX API 2000 LC-MS/MS, HPLC Agilent 1100) with a Phenomenex Luna C18 HPLC column (50 x 2.00 mm, 3 μm) and detection was performed by a UV DAD (200–440 nm). Elution was carried out with the following gradient: 0.02 M NH₄CH₃CO₂, pH 7.0 (solvent A), MeOH (solvent B) start with 100%, 10% B in 20 min to 100% B, 10 min 100% B, stop time 20 min, flow 0.25 mL/min.
C). Purity was determined with an LC-MS instrument (AmaZon SL ESI-MS, Shimadzu LC) with a cellulose-2 Phenomenex column (250 x 4.6 mm, 5 μm) or a Diacel column (IC-chiralpak, 250 x 4.6 mm, 5 μm). Isocratic elution with MeCN and water was applied as specified, stop time 60 min, flow 0.5 mL/min.

Download:

Fig 1. General synthesis of compounds 6–19.

Reagents and conditions: a) HATU, DIPEA, 1-amino-1-cyclopropanecarbonitrile, DMF, rt, 18 h; b) formic acid, rt, 18 h; c) HATU or TBTU, DIPEA, carboxylic acid, DMF, rt, 18 h; d) DDQ, CH₂Cl₂, rt, 3–5 days.

https://doi.org/10.1371/journal.pntd.0007755.g001

Download:

Fig 2. General synthesis of compounds 50–60, 65–69.

Reagents and conditions: a) Isobutyl chloroformate, NH₄Cl 2 M, DIPEA, DMF, 0 °C to rt, 20 h; b) TFA, CH₂Cl₂, 0 °C to rt, 2 h; c) HATU, DIPEA, Boc-AA-OH, DMF, rt, 18 h; d) TFA, CH₂Cl₂, 0 °C to rt, 2 h; e)TBTU, DIPEA, 3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxylic acid, DMF/CH₂Cl₂, rt, 18 h; f) Cyanuric chloride, DMF, 0 °C to rt, 0.5 h; g) H₂ (1 atm), Pd/C, rt, 18 h; h) DDQ, CH₂Cl₂, rt, 3–5 days; i) TFAA, DIPEA, THF, 0 °C to rt, 2 h.

https://doi.org/10.1371/journal.pntd.0007755.g002

NMR spectra were recorded on Bruker Avance 200 MHz, Bruker Avance 400 MHz, and Bruker Avance DRX 500 MHz NMR spectrometers. Chemical shifts are reported in ppm relative to TMS or the residual proton peak of the re-protonated deuterated solvent, and the spectra were calibrated against the residual proton peak of the used deuterated solvent. The following symbols indicate spin multiplicities: s (singlet), s br (broad singlet), d (doublet), dd (doublet of doublet), t (triplet), tt (triplet of triplet), q (quartet), sept (septet), and m (multiplet). Standard mass spectra were obtained either as ESI-MS (pos. and/or neg. mode) from a Advion DCMS interface, (settings as follows: ESI voltage 3.50 kV, capillary voltage 187 V, source voltage 44 V, capillary temperature 250 °C, desolvation gas temperature 250 °C, gas flow 5 L/min) or by an API 2000 mass spectrometer (electron spray ion source, ABSCIEX, Darmstadt, Germany) coupled to an Agilent 1100 HPLC system.

HRMS spectra were recorded on a Bruker micrOTOF-Q mass spectrometer connected to a Thermo Scientific Dionex UltiMate 3000 LC via an ESI interface using a Nucleodur C18 Gravity column (50 × 2.0 mm, 3 μm) or were recorded on Thermo Scientific LTQ Velos Orbitrap, in electrospray ionization (ESI) mode by direct injection.

The synthetic route was developed to optimize the set of substituents to be placed in P1, P2, and P3 that have been defined after the planning and design studies. Due to the diversity of building blocks, it was necessary to evaluate different coupling and dehydrating reagents, aiming at the best yield and preventing racemization.

General procedure for amide synthesis.

Method A: Isobutyl chloroformate (790 mg, 0.75 mL, 5.5 mmol, 1.1 equiv) was added dropwise to a solution of Boc-(R or S) amino acid (5.0 mmol, 1.0 equiv.), DIPEA (1.6 g, 2.28 mL, 13.0 mmol, 2.6 equiv.) in dry DMF (20 mL), under argon atmosphere, at -30 °C and it was stirred for 0.5 h. Then, an aqueous 2 M NH₄Cl solution (294 mg, 2.75 mL, 5.50 mmol, 1.1 equiv.) was added. The resulting solution was stirred at room temperature for 20 h. The reaction mixture was dried under reduced pressure. Ethyl acetate (100 mL) was added, and it was washed with a saturated NaHCO₃ solution (3 × 50 mL) and brine (1 × 50 mL). The organic phase was dried over Na₂SO₄ and evaporated under reduced pressure to give a crude residue that was purified by flash column chromatography.

Method B: The free primary amine (1.0 mmol, 1.0 equiv.) was added to a solution of the carboxylic acid (1.3 mmol, 1.3 equiv.), HATU (490 mg, 1.3 mmol, 1.3 equiv.) and DIPEA (364 mg, 0.45 mL, 2.60 mmol, 2.6 equiv.) in dry DMF (5 mL) under argon atmosphere. The resulting solution was stirred at room temperature for 20 h. The reaction mixture was diluted with ethyl acetate (10 mL) and washed with a saturated NaHCO₃ solution (3 × 20 mL) and brine (3 × 20 mL). The organic phase was dried over Na₂SO₄ and evaporated to give a crude residue that was purified by flash column chromatography.

Method C: The free primary amine (1.0 mmol, 1.0 equiv.) was added to a solution of the carboxylic acid (1.3 mmol, 1.3 equiv.), TBTU (410 mg, 1.30 mmol, 1.3 equiv.) and DIPEA (364 mg, 0.45 mL, 2.60 mmol) in dry DMF/CH₂Cl₂ (1:1, 10 mL) under argon atmosphere. The resulting solution was stirred at room temperature for 20 h. The reaction mixture was diluted with ethyl acetate (10 mL) and washed with a saturated NaHCO₃ solution (3 × 20 mL) and brine (3 × 20 mL). The organic phase was dried over Na₂SO₄ and evaporated to give a crude residue that was purified by flash column chromatography.

General procedure for removal of the Boc protecting group.

Method A: The Boc-protected amino compound (0.25 mmol, 1.0 equiv.) was treated with formic acid (2.44 g, 2.0 mL, 47.9 mmol, 47.9 equiv.) at room temperature. The resulting solution was stirred for 18 h. The reaction mixture was evaporated under reduced pressure to get a yellowish oil. It was treated with an aqueous solution of 1.0 M NaOH until pH 9 was reached. The product was extracted with ethyl acetate (4 × 20 mL) and then washed with brine (1 × 20 mL). The organic phase was evaporated to obtain a colorless oil. The formation of the product was confirmed by TLC (ethyl acetate). The product was used for the next step without further purification.

Method B: To a solution of Boc-protected amino compound (1.0 mmol, 1.0 equiv.) in dry CH₂Cl₂ (3 mL) was added TFA (912 mg, 0.91 mL, 8.00 mmol, 8.0 equiv.) at 0 °C. The mixture was stirred and allowed to reach room temperature within exposed mainly h. The progress of the reaction was monitored by TLC (ethyl acetate). The reaction mixture was evaporated under reduced pressure to eliminate the excess of TFA to get a yellowish solid. The product was used for the next step without further purification.

General procedure for dehydration of primary amides to nitriles.

Method A: The primary amide (1.0 mmol, 1.0 equiv.) was dissolved in dry DMF (5 mL) at 0 °C. Then, cyanuric chloride (73 mg, 0.4 mmol, 1.1 equiv.) was slowly added to the solution under argon atmosphere. The resulted solution was stirred for 0.5 h. Saturated NaHCO₃ solution (30 mL) was added and it was stirred at room temperature for 2 h. The product was extracted with ethyl acetate (2 x 50 mL), and then the reunited organic phases were washed with an aqueous solution of 1.0 M KHSO₄ (3 × 20 mL), brine (4 × 30 mL) and dried over Na₂SO₄. The solvent was removed, and the crude product was purified by flash silica gel chromatography.

Method B: The primary amide (1.0 mmol, 1.0 equiv.) was dissolved in dry THF (5 mL) and DIPEA (364 mg, 0.45 mL, 2.6 mmol, 2.6 equiv.) was added. Trifluoroacetic anhydride (273 mg, 0.18 mL, 1.30 mmol, 1.3 equiv.) was added over 5 min, at 0 °C. The mixture was stirred and allowed to reach room temperature within 2 h. Then the reaction was quenched with H₂O (20 mL), THF removed in vacuo, and the product was extracted into ethyl acetate (2 × 50 mL). The organic phase was washed with a solution 1.0 M of KHSO₄ (3 × 20 mL) and with a saturated NaHCO₃ solution (3 × 20 mL) and brine (3 × 20 mL) and dried over Na₂SO₄. The solvent was removed, and the crude product was purified by flash silica gel chromatography.

Method C: The primary amide (1.0 mmol, 1.0 equiv.) was dissolved in dry pyridine (5 mL) at room temperature. Then, p-toluenesulfonyl chloride (572 mg, 3.0 mmol, 3.0 equiv.) was added to the solution under argon atmosphere. The resulting solution was stirred for 3 days. Upon the addition of a saturated NaHCO₃ solution (30 mL), the reaction mixture was stirred at room temperature for 2 h. The solution was dried under reduced pressure. The product was extracted with ethyl acetate (2 × 50 mL), and then the reunited organic phases were washed with a 1.0 M solution of KHSO₄ (2 × 20 mL), brine (4 × 30 mL) and dried over Na₂SO₄. The solvent was removed, and the crude product was purified by flash silica gel chromatography.

General procedure for removal of the benzyl protecting group.

Method A: The corresponding protected threonine (1.0 mmol, 1.0 equiv.) was dissolved in ethanol absolute (20 mL) in an argon atmosphere. Upon addition of 10% Pd/C, H₂ was bubbled in the solution for 0.5 h. The resulting solution was stirred under H₂ atmosphere for 12 h. The progress of the reaction was monitored by TLC (ethyl acetate). The solution was filtered on celite two times and dried under reduced pressure to afford the desired product as a colorless wax. The product was used for the next step without further purification.

Method B: The corresponding protected threonine (1.0 mmol, 1.0 equiv.) was dissolved in dry CH₂Cl₂ (20 mL) under argon atmosphere. Then, DDQ (908 mg, 4.0 mmol, 4.0 equiv.) was added, and the resulting solution was stirred for 4 days at room temperature. The progress of the reaction was monitored by TLC (ethyl acetate). The reaction was quenched with an aqueous 1.0 M solution of NaHSO₃ (20 mL). Then, CH₂Cl₂ was removed under reduced pressure. The product was extracted with ethyl acetate (2 × 50 mL), and the reunited organic phases were washed with an aqueous solution of 1.0 M KHSO₄ (2 × 20 mL), brine (4 × 30 mL) and dried over Na₂SO₄. The solvent was removed, and the crude product was purified by flash silica gel chromatography.

Synthesis and characterization of compound 1–5.

Compounds 1–5 have been synthesized from the corresponding amino acid and 1-amino-1-cyclopropanecarbonitrile following the general procedure for amide synthesis (method B) [15].

(S)-tert-Butyl (1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (1).

Yield 92%. White solid. R_f = 0.9 (ethyl acetate: n-hexane; 7:3). Mp. 146–147 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.27–7.04 (m, 5H), 4.58 (m, 1H), 3.19 (dd, J = 13.8, 5.0 Hz, 1H), 2.88 (dd, J = 9.5, 5.0 Hz, 1H), 1.31 (s, 9H), 1.25 (m, 2H), 1.04 (m, 2H). ¹³C NMR. (125 MHz, CDCl₃) δ 173.06, 155.30, 137.73, 129.35, 128.19, 126.45, 120.80, 78.30, 55.54, 37.36, 28.26, 19.77, 15.80, 15.75. ESI-MS (+) Calc. for [C₁₈H₂₃N₃O₃] 329.39, found: 352.3 [M+Na]⁺.

(S)-tert-Butyl (3-(3-chlorophenyl)-1-((1-cyanocyclopropyl)amino)-1-oxopropan-2-yl)carbamate (2).

Yield 83%. White solid. R_f = 0.7 (ethyl acetate: n-hexane; 6:4). Mp. 146–147 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.26–7.25 (m, 1H), 7.23–7.21 (m, 1H), 7.08–7.03 (m, 2H), 4.27–4.24 (m, 1H), 3.10 (dd, J = 13.8, 5.0 Hz, 1H), 2.83 (dd, J = 9.5, 5.0 Hz, 1H), 1.52–1.44 (m, 2H), 1.41 (s, 9H), 1.13–1.05 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) 172.02, 155.61, 138.23, 134.32, 129.29, 127.49, 127.25, 119.48, 119.48, 80.55, 55.28, 37.87, 28.18, 20.14, 16.68, 16.56. ESI-MS (+) Calc. for [C₁₈H₂₂ClN₃O₃] 363.83, found: 364.3 [M+H]⁺.

(S)-tert-Butyl (1-((1-cyanocyclopropyl)amino)-1-oxo-3-(pyridin-4-yl)propan-2-yl)carbamate (3).

Yield 75%. White solid. R_f = 0.5 (ethyl acetate: n-hexane; 4:6). Mp. 134–135 °C. ¹H NMR (200 MHz, CD₃OD) δ 8.45 (d, J = 4.9 Hz, 2H), 7.34 (d, J = 5.8 Hz, 2H), 4.36–4.23 (m, 1H), 2.96 (dd, J = 14.0, 5.0 Hz, 2H), 1.53–1.46 (m, 1H), 1.38 (s, 9H), 1.20–1.14 (m, 2H). ¹³C NMR (50 MHz, CDCl₃) δ 174.74, 149.95, 149.15, 126.52, 121.09, 80.79, 56.03, 38.88, 38.31, 28.55, 21.21, 17.05. ESI-MS (+) Calc. for [C₁₇H₂₂N₄O₃] 330.38, found: 331.2 [M+H]⁺.

(S)-tert-Butyl (1-((1-cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (4).

Yield 61%. White solid. R_f = 0.7 (ethyl acetate: n-hexane; 4:6). Mp. 162–164 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.76 (s, 1H), 6.86 (d, J = 7.9 Hz, 1H), 3.86 (dt, J = 8.7, 5.5 Hz, 1H), 1.55–1.54 (m, J = 6.6 Hz, 1H), 1.44 (dd, J = 7.9, 5.5 Hz, 2H), 1.37–1.42 (m, 2H), 1.36 (s, 9H), 1.07 (dd, J = 7.7, 5.3 Hz, 2H), 0.84 (2d, J = 6.6 Hz, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ 174.17, 155.50, 120.92, 78.22, 52.58, 40.49, 28.31, 24.38, 23.01, 21.66, 19.91, 15.87, 15.75. ESI-MS (+) Calc. for [C₁₅H₂₅N₃O₃] 295.37, found: 318.3 [M+Na]⁺.

tert-Butyl ((2S,3R)-3-(benzyloxy)-1-((1-cyanocyclopropyl)amino)-1-oxobutan-2-yl)carbamate (5).

Yield 89%. White solid. R_f = 0.7 (ethyl acetate: n-hexane; 4:6). Mp. 88–90 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.36–7.32 (m, 5H), 4.78–4.60 (m, 3H), 4.22–4.12 (m, 1H), 1.57–1.44 (m, 2H), 1.30–1.27 (m, 9H), 1.17–1.13 (m, 5H). ESI-MS (+) Calc. for [C₂₀H₂₇N₃O₄] 373.44, found: 396.4 [M+Na]⁺.

Synthesis of compounds 6–18.

Compounds 6–18 have been synthesized in two steps from compounds 1–5. First, the Boc group was removed (procedure A), and then the free amine was coupled to the carboxylic acid following the general procedure for amide synthesis (method B or method C, as indicated).

Synthesis and characterization of compounds 6, 9 and 11 have been already published elsewhere [14].

(S)-N-(3-(3-Chlorophenyl)-1-((1-cyanocyclopropyl)amino)-1-oxopropan-2-yl)benzamide (7).

Method B. Yield 86%. White solid. R_f = 0.7 (ethyl acetate: n-hexane; 6:4). Mp. 213–215 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.04 (s, 1H), 8.67 (d, J = 8.1 Hz, 1H), 7.84–7.83 (m, 2H), 7.56–7.54 (m, 1H), 7.53 (t, J = 7.7 Hz, 2H), 7.41 (s, 1H), 7.32–7.25 (m, 3H), 4.65–4.60 (m, 1H), 3.09 (dd, J = 13.6, 5.0 Hz, 1H), 3.02 (dd, J = 15.2, 5.0 Hz, 1H), 1.51–1.49 (m, 2H), 1.12–1.06 (m, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 172.57, 166.48, 140.62, 132.82, 131.52, 130.04, 129.18, 128.31, 128.05, 127.58, 126.50, 120.81, 54.47, 36.60, 19.90, 15.82. HRMS (+) Calc. for [C₂₀H₁₉ClN₃O₂]⁺ 368.11658, found: 368.11615 [M+H]⁺. HPLC (protocol B): t_R (min) = 10.29. Purity: 99.6%.

(S)-3-(tert-Butyl)-N-(3-(3-chlorophenyl)-1-((1-cyanocyclopropyl)amino)-1-oxopropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (8).

Method C. Method B. Yield 72%. Yellowish solid. R_f = 0.7 (ethyl acetate: n-hexane; 5:5). Mp. 152–154 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.03 (s, 1H), 8.59 (d, J = 8.4 Hz, 1H), 7.39 (s, 1H), 7.31–7.24 (m, 3H), 6.79 (s, 1H), 4.57–4.53 (m, 1H), 3.88 (s, 3H), 3.08 (dd, J = 13.6, 5.1 Hz, 1H), 2.94 (dd, J = 13.6, 10.2 Hz, 1H), 1.50–1.47 (m, 2H), 1.28 (s, 9H), 1.10–1.05 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.10, 159.46, 158.70, 140.26, 134.95, 132.64, 129.94, 129.15, 127.85, 126.39, 120.63, 103.82, 55.21, 36.38, 31.58, 30.36, 19.75, 15.67, 15.61. HRMS (+) Calc. for [C₂₂H₂₇ClN₅O₂]⁺ 428.18533, found: 428.1864 [M+H]⁺. HPLC (protocol B): t_R (min) = 11.17. Purity: 98.3%.

(S)-3-(tert-Butyl)-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-(pyridin-4-yl)propan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (10).

Method C. Yield 56%. Yellowish oil. R_f = 0.4 (ethyl acetate: n-hexane; 5:5). ¹H NMR (200 MHz, CD₃OD) δ 8.48–8.46 (m, 2H), 7.43 (d, J = 4.9 Hz, 2H), 6.69 (s, 1H), 4.86–4.78 (m, 1H), 3.95 (s, 3H), 3.04–3.02 (m, 1H), 2.89–2.84 (m, 1H), 1.56–1.50 (m, 2H), 1.31 (d, J = 4.3 Hz, 9H), 1.22–1.18 (m, 2H). ¹³C NMR (50 MHz, CD₃OD) δ 172.80, 160.35, 160.05, 148.62, 147.59, 134.80, 125.00, 119.66, 103.60, 53.18, 37.37, 36.45, 31.42, 29.44, 19.92, 15.60, 15.28. FT-IR (KBr, cm^-1) 3297.16, 2966.17, 2242.31, 1670.63, 1601.36, 1531.90, 1425.65, 1352.10, 1278.55, 1241.77, 1049.72, 988.42, 808.63, 755.51, 722.82, 511.19, 506.24, 489.90. HRMS (+) Calc. for [C₂₁H₂₇N₆O₂]⁺ 395.21955, found: 395.21973 [M+H]⁺. HPLC (protocol B): t_R (min) = 3.68. Purity: 94.7%.

N-((2S,3R)-3-(Benzyloxy)-1-((1-cyanocyclopropyl)amino)-1-oxobutan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (12).

Method C. Yield 53%. Yellowish solid. R_f = 0.4 (ethyl acetate: n-hexane; 6:4). Mp. 100–101 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.36–7.32 (m, 5H), 6.45 (s, 1H), 4.76–4.62 (m, 3H), 4.22–4.12 (m, 1H), 4.09 (s, 3H), 1.57–1.44 (m, 2H), 1.30–1.27 (m, 9H), 1.20–1.13 (m, 5H). ¹³C NMR (50 MHz, CDCl₃) δ 170.36, 160.18, 159.74, 137.15, 134.06, 128.48, 127.98, 127.68, 119.25, 103.18, 74.03, 71.48, 54.81, 38.38, 31.66, 30.16, 28.68, 20.24, 16.91, 15.85. FT-IR (KBr, cm^-1) 3288.99, 2953.92, 2917.14, 2214.31, 1646.31, 1589.10, 1495.12, 1049.31, 1294.89, 1200.91, 1033.37, 922.51, 755.51, 681.95. HRMS (+) Calc. for [C₂₄H₃₂N₅O₃]⁺ 437.25051, found: 438.25102 [M+H]⁺. HPLC (protocol B): t_R (min) = 8.77. Purity: 97.0%.

(S)-7-Chloro-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)quinoline-4-carboxamide (13).

Method B. Yield 74%. White solid. R_f = 0.7 (ethyl acetate: n-hexane; 8:2). Mp. 260–262 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.15 (d, J = 10.2 Hz, 1H), 9.13 (s, 1H), 8,97 (d, J = 10.2 Hz, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.72 (d, J = 15 Hz, 1H), 7.56 (dd, J = 15.1, 10.2 Hz, 1H), 7.44 (d, J = 10.2 Hz, 1H), 7.28–7.24 (m, 5H), 4.75–4.73 (m, 1H), 3.11 (dd, J = 13.8, 5.0 Hz, 1H), 2.87 (dd, J = 9.5, 5.0 Hz, 1H), 1.51–1.49 (m, 2H), 1.09–1.07 (m, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.34, 166.33, 151.74, 148.36, 141.88, 137.63, 134.53, 129.45, 128.42, 128.00, 127.86, 126.74, 122.89, 120.91, 119.69, 54.56, 37.26, 20.02, 15.89. FT-IR (KBr, cm^-1) 3254.05, 2926.14, 2247.17, 1672.36, 1668.36, 1523.83, 846.79, 831.36. HRMS (+) Calc. for [C₂₃H₂₀ClN₄O₂]⁺ 418.12748, found: 419.12813 [M+H]⁺. HPLC (protocol C, 50:50 ACN: water): t_R (min) = 17.32. Purity: 99.9%.

(S)-7-Chloro-N-(1-((1-cyanocyclopropyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)quinoline-4-carboxamide (14).

Method B. Yield 49%. Yellowish solid. R_f = 0.3 (ethyl acetate). Mp. 196–197 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.88 (s, 1H), 9.15 (s, 1H), 9.08 (d, J = 10.2 Hz, 1H), 8.97 (d, J = 4.2 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H), 7.54–7.52 (m, 1H), 7.46 (d, J = 4.5 Hz, 1H), 7.38 (d, J = 10.2 Hz 1H), 7.16 (d, J = 2.4 Hz, 1H), 7.09 (t, J = 5.5 Hz, 1H), 7.00 (t, J = 5.8 Hz, 1H), 4.81–4.79 (m, 1H), 3.20 (dd, J = 13.8, 5.0 Hz, 1H), 3.05 (dd, J = 9.5, 5.0 Hz, 1H), 1.51–1.48 (m, 2H), 1.11–1.08 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 173.02, 166.59, 151.94, 148.61, 142.38, 136.57, 134.72, 128.23, 128.05, 128.03, 127.52, 124.56, 123.19, 121.46, 121.26, 119.97, 119.03, 118.76, 111.81, 110.02, 54.26, 27.79, 20.33, 16.20. FT-IR (KBr, cm^-1) 3254.05, 2926.14, 2247.17, 1672.36, 1665.34, 1522.83, 831.30, 732.98. HRMS (+) Calc. for [C₂₅H₂₁ClN₅O₂]⁺ 458.13838, found: 458.13703 [M+H]⁺. HPLC (protocol C, 50:50 ACN: water): t_R (min) = 18.64. Purity: 99.6%.

(S)-7-Chloro-N-(1-((1-cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)quinoline-4-carboxamide (15).

Method B. Yield 68%. White solid. R_f = 0.7 (ethyl acetate). Mp. 169–170 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.10 (s, 1H), 9.05 (s, 1H), 9.02 (d, J = 5.5 Hz, 1H) 8.20 (d, J = 11.5 Hz, 1H), 8.15 (d, J = 2.5 Hz, 1H), 7.72 (dd, J = 11.5, 2.5 Hz, 1H), 7.60 (d, J = 5.5 Hz, 1H), 4.48–4.46 (m, 1H), 1.70–1.63 (m, 3H), 1.52–1.48 (m, 2H), 1.19–1.14 (m, 2H), 0.92 (2d, J = 10.5 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 173.63, 166.83, 152.05, 148.70, 142.06, 134.83, 131.26, 128.38, 123.35, 121.21, 120.20, 51.97, 24.87, 23.38, 21.79, 20.34, 16.22, 16.06. FT-IR (KBr, cm^-1) 3402.4, 3257.7, 3030.1, 2960.7, 2247.0, 1674.2, 1633.7, 1529.5, 1296.1, 831.31. HRMS (+) Calc. for [C₂₀H₂₂ClN₄O₂]⁺ 384.14313, found: 385.14503 [M+H]⁺. HPLC (protocol C, 65:35 ACN: water): t_R (min) = 11.81. Purity: 96.2%.

(S)-3-((1-((1-Cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)phenyl benzoate (16).

Method B. Yield 89%. White solid. R_f = 0.7 (ethyl acetate: n-hexane; 6:4). Mp. 185–187 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.05 (s, 1H), 8.79 (d, J = 8.0 Hz, 1H), 8.18–8.16 (m, 2H), 7.81–7.78 (m, 3H), 7.64 (t, J = 8.5 Hz, 2H), 7.57 (t, J = 8.5 Hz, 2H), 7.50–7.48 (m, 1H), 7.31–7.23 (m, 4H), 7.18 (tt, J = 7.0, 1.5 Hz, 1H), 4.64–4.59 (m, 1H), 3.06 (dd, J = 13.6, 5.0 Hz, 1H), 3.00 (dd, J = 13.5, 8.5 Hz, 1H), 1.49–1.45 (m, 2H), 1.06–1.01 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 173.04, 165.60, 165.02, 150.88, 138.21, 135.72, 134.66, 130.27, 129.98, 129.57, 129.51, 129.16, 128.57, 126.83, 125.69, 125.53, 121.47, 121.15, 55.10, 37.36, 20.18, 16.15, 16.10, 14.55. FT-IR (KBr, cm^-1) 3254.05, 2926.14, 2247.17, 1672.36, 1668.36, 1523.83, 846.79, 831.36. HRMS (+) Calc. for [C₂₇H₂₄N₃O₄]⁺ 453.17668, found: 454.17800 [M+H]⁺. HPLC (protocol C, 65:35 ACN: water): t_R (min) = 19.62. Purity: 99.9%.

(S)-6-Amino-N-(1-((1-cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)nicotinamide (17).

Method C. Yield 48%. Yellowish solid. R_f = 0.3 (ethyl acetate: methanol; 8:2). Mp. 100–101 °C. ¹H NMR (200 MHz, CD₃OD) δ 8.43 (s, 1H), 7.88 (d, J = 8.8, 2.1 Hz, 1H), 6.54 (d, J = 8.9 Hz,1H), 4.53–4.46 (m, 1H), 1.82–1.51 (m, 3H), 1.47–1.43 (m, 2H), 1.24–1.17 (m, 2H), 0.96–0.93 (m, 6H). ¹³C NMR (50 MHz, CD₃OD) δ 176.59, 168.58, 162.86, 149.21, 138.44, 121.42, 119.42, 109.12, 41.60, 26.13 23.54, 21.98, 21.41, 17.15, 16.78. FT-IR (KBr, cm^-1) 3288.99, 2953.92, 2917.14, 2214.31,1647.63, 1496.06, 1409.33, 1294.89, 1202.33, 1075.33, 1030.81, 922.54, 763.49, 667.21. ESI-MS (+) Calc. for [C₁₆H₂₂N₅O₂]⁺ 316.17735, found: 316.17713 [M+H]⁺. HPLC (protocol B): t_R (min) = 8.77. Purity: 97.0%.

(S)-N-(1-((1-Cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (18).

Method C. Yield 45%. White solid. R_f = 0.3 (ethyl acetate: methanol; 8:2). Mp. 79–80 °C. ¹H NMR (200 MHz, CD₃OD) δ 8.71 (d, J = 1.8 Hz, 1H), 8.47 (d, J = 2.0 Hz, 1H), 7.46 (d, J = 3.5 Hz, 1H), 6.57 (d, J = 3.5 Hz, 1H), 4.59–4.57 (m, 1H), 1.91–1.56 (m, 3H), 1.54–1.49 (m, 2H), 1.29–1.25 (m, 2H), 1.01–0. 89 (m, 6H). ¹³C NMR (50 MHz, CD₃OD) δ 176.50, 169.61, 163.45, 150.38, 143.32, 129.52, 128.65, 123.21, 120.94, 102.66, 53.50, 38.92, 26.18, 23.41, 22.02, 21.25, 17.17, 16.49. FT-IR (KBr, cm^-1) 3286.44, 2933.89, 2924.14, 2216.51, 1647.63, 1588.10, 1496.06, 1409.33, 1294.89, 1202.33, 1075.33, 1030.31, 922.54, 763.54, 667.21. ESI-MS (+) Calc. for [C₁₈H₂₂N₅O₂]⁺ 340.17753, found: 340.17689 [M+H]⁺. HPLC (protocol B): t_R (min) = 5.38. Purity: 99.0%.

Synthesis and characterization of compound 19.

Compound 19 has been synthesized from compound 12 by removal of the benzyl protecting group under mild conditions (protocol B).

3-(tert-Butyl)-N-((2S,3R)-1-((1-cyanocyclopropyl)amino)-3-hydroxy-1-oxobutan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (19).

Yield 40%. Yellowish solid. R_f = 0.3 (ethyl acetate). Mp. 201–202 °C. ¹H NMR (200 MHz, CD₃OD) δ 6.81 (s, 1H), 4.40 (d, J = 4.4 Hz, 1H), 4.23–4.18 (m, 1H), 4.04 (s, 3H), 1.52–1.47(m, 2H), 1.32 (s, 9H), 1.29–1.18 (m, 5H). ¹³C NMR (50 MHz, CD₃OD) δ 174.94, 163.34, 162.74, 137.60, 122.22, 106.16, 69.42, 61.17, 39.91, 34.00, 31.94, 22.41, 21.45, 18.11, 17.83. FT-IR (KBr, cm^-1) 3288.89, 2953.92, 2917.14, 2214.31, 1646.31, 1589.10, 1495.12, 1049.31, 1294.89, 1200.91, 1033.97, 922.51, 755.51, 681.95. ESI-MS (-) Calc. for [C₁₇H₂₅N₅O₃]⁺ 348.20356, found: 348.20314 [M+H]⁺. HPLC (protocol B): t_R (min) = 7.74. Purity: 98.0%.

Synthesis of compounds 20–27.

Compounds 20–27 have been synthesized from the equivalent amino Boc-protected amino acid following the general procedure for amide synthesis (method A).

(S)-tert-Butyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate (20).

Yield 77%. White solid. R_f = 0.6 (ethyl acetate). Mp. 146–149 °C. ¹H NMR (200 MHz, CD₃OD) δ 7.26–7.16 (m, 5H), 4.44–4.41 (m, 1H), 3.14 (dd, J = 13.7, 6.0 Hz, 1H), 2.88 (dd, J = 13.7, 8.1 Hz, 1H), 1.23 (s, 9H). ¹³C NMR (50 MHz, CD₃OD) δ 173.98, 155.60, 130.73, 124.93, 122.46, 118.22, 80.20, 54.02, 37.73, 27.94. ESI-MS (+) Calc. for [C₁₄H₂₀N₂O₃] 264.31, found: 287.3 [M+Na]⁺.

(R)-tert-butyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate (21).

Yield 79%. White solid. R_f = 0.6 (ethyl acetate). Mp. 141–142 °C. ¹H NMR (200 MHz, CD₃OD) δ 7.24–7.17 (m, 5H), 4.42–4.37 (m, 1H), 3.14 (dd, J = 13.7, 6.0 Hz, 1H), 2.92 (dd, J = 13.7, 8.1 Hz, 1H), 1.43 (s, 9H). ¹³C NMR (50 MHz, CD₃OD) δ 173.12, 154.48, 133.36, 122.46, 121.75, 119.38, 83.51, 55.61, 38.38, 28.66. ESI-MS (+) Calc. for [C₁₄H₂₀N₂O₃] 264.31, found: 287.3 [M+Na]⁺.

(S)-tert-Butyl (1-amino-4-methyl-1-oxopentan-2-yl)carbamate (22).

Yield 74%. White solid. R_f = 0.4 (ethyl acetate). Mp. 138–141 °C. ¹H NMR (200 MHz, CDCl₃) δ 6.58 (s br, 1H), 6.13 (s br, 1H), 5.25–4.97 (m, 2H), 4.15 (s br, 1H), 1.73–1.43 (m, 3H), 1.41 (s, 9H), 0.92 (d, J = 3.2 Hz, 6H). ¹³C NMR (50 MHz, CDCl₃) δ 172.34, 155.95, 71.56, 28.57, 28.18, 25.08, 23.21, 19.25. ESI-MS (+) Calc. for [C₁₁H₂₂N₂O₃] 230.30, found: 253.3 [M+Na]⁺.

(R)-tert-Butyl (1-amino-4-methyl-1-oxopentan-2-yl)carbamate (23).

Yield 71%. White solid. R_f = 0.4 (ethyl acetate). Mp. 138–141 °C. ¹H NMR (200 MHz, CDCl₃) δ 6.64 (s br, 1H), 6.21 (s br, 1H), 5.21–4.98 (m, 2H), 4.16 (s br, 1H), 1.71–1.45 (m, 3H), 1.42 (s, 9H), 0.93 (d, J = 3.2 Hz, 6H). ¹³C NMR (50 MHz, CDCl₃) δ 172.11, 155.37, 71.64, 28.61, 28.29, 25.15, 23.34, 19.30. ESI-MS (+) Calc. for [C₁₁H₂₂N₂O₃] 230.30, found: 253.3 [M+Na]⁺.

(S)-tert-Butyl (1-amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)carbamate (24).

Yield 74%. White solid. R_f = 0.4 (ethyl acetate). Mp. 111–112 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.26–7.13 (m, 4H), 6.16 (s, 1H), 5.75 (s, 1H), 5.21 (d, J = 7.8 Hz, 1H), 4.42–4.36 (m, 1H), 3.19–2.94 (m, 2H), 1.44 (s, 9H). ¹³C NMR (50 MHz, CDCl₃) δ 173.53, 155.16, 138.90, 134.09, 130.04, 129.44, 127.55, 127.11, 80.31, 37.88, 28.16. ESI-MS (+) Calc. for [C₁₄H₁₉ClN₂O₃] 298.77, found: 321.8 [M+Na]⁺.

(S)-tert-Butyl (1-amino-1-oxo-3-(pyridin-4-yl)propan-2-yl)carbamate (25).

Yield 92%. White solid. R_f = 0.2 (ethyl acetate). Mp. 131–133 °C. ¹H NMR (200 MHz, CDCl₃) δ 8.42 (d, J = 5.7 Hz, 2H), 7.22 (d, J = 5.9 Hz, 2H), 4.35–4.31 (m, 1H), 3.18 (dd, J = 13.7, 6.0 Hz, 1H), 2.81 (dd, J = 13.7, 8.1 Hz, 1H), 1.49–1.33 (s br, 9H). ¹³C NMR (50 MHz, CDCl₃) δ 173.69, 155.86, 148.48, 147.61, 118.42, 80.22, 54.51, 37.77, 27.89. ESI-MS (+) Calc. for [C₁₃H₁₉N₃O₃] 265.31, found: 266.2 [M+H]⁺.

tert-Butyl ((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)carbamate (26).

Yield 65%. White solid. R_f = 0.5 (ethyl acetate). Mp. 145–147 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.37–7.28 (m, 5H), 6.50 (s, 1H), 5.70–5.51 (m, 2H), 4.62 (q, J = 4.6 Hz, 2H), 4.34–4.31 (m, 1H), 4.18–4.11 (m, 1H), 1.45 (s, 9H), 1.19 (d, J = 6.3 Hz, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 172.09, 155.66, 137.78, 128.41, 127.82, 127.73, 80.03, 74.53, 71.61, 57.13, 28.23, 27.84, 18.92. ESI-MS (+) Calc. for [C₁₆H₂₄N₂O₄] 308.37, found: 331.4 [M+Na]⁺.

tert-Butyl ((2R,3S)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)carbamate (27).

Yield 68%. White solid. R_f = 0.5 (ethyl acetate). Mp. 143–145 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.37–7.28 (m, 5H), 6.50 (s, 1H), 5.70–5.51 (m, 2H), 4.62 (q, J = 4.6 Hz, 2H), 4.33 (s br, 1H), 4.27–4.19 (m, 1H), 1.45 (s, 9H), 1.19 (d, J = 6.3 Hz, 3H) .¹³C NMR (125 MHz, CDCl₃) δ 172.74, 156.31, 138.44, 129.06, 128.47, 128.38, 80.68, 77.16, 75.18, 72.26, 57.78, 28.89, 28.49, 19.57. ESI-MS (+) Calc. for [C₁₆H₂₄N₂O₄] 308.37, found: 331.4 [M+Na]⁺.

Synthesis of compounds 28–38.

Compounds 28–38 have been synthesized in two steps from their precursors 20–27. After removal of the Boc-protecting group (method B), the resulting free amine was coupled to the carboxylic acid following the general procedure for amide synthesis (method B).

tert-Butyl ((S)-1-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (28).

Yield 92%. White solid. R_f = 0.8 (ethyl acetate). Mp. 186–188 °C. ¹H NMR (200 MHz, CD₃OD) δ 7.37–7.22 (m, 6H), 7.16–7.09 (m, 4H), 4.76–4.63 (m, 1H), 4.34–4.20 (m, 1H), 3.15–2.95 (m, 2H), 2.91–2.62 (m, 2H), 1.39 (s, 9H). ¹³C NMR (50 MHz, CD₃OD) δ 174.36, 172.35, 156.09, 136.44, 129.04, 128.35, 128.27, 126.78, 126.64, 79.89, 56.00, 53.78, 37.73, 37.24, 27.84. ESI-MS (+) Calc. for [C₂₃H₂₉N₃O₄] 411.49, found: 434.3 [M+Na]⁺.

tert-Butyl ((S)-1-(((R)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (29).

Yield 92%. White solid. R_f = 0.8 (ethyl acetate). Mp. 185–187 °C. ¹H NMR (200 MHz, CD₃OD) δ 7.75–7.60 (m, 6H), 7.56–7.49 (m, 4H), 5.14–5.01 (m, 1H), 4.72–4.57 (m, 1H), 3.53–3.33 (m, 2H), 3.29–3.00 (m, 2H), 1.77 (s, 9H). ¹³C NMR (50 MHz, CD₃OD) δ 174.85, 172.83, 156.58, 136.93, 129.53, 128.83, 128.76, 127.26, 127.13, 80.38, 56.49, 54.26, 38.22, 37.73, 28.33. ESI-MS (+) Calc. for [C₂₃H₂₉N₃O₄] 411.49, found: 434.3 [M+Na]⁺.

tert-Butyl ((S)-1-(((S)-1-amino-4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (30).

Yield 83%. White solid. R_f = 0.8 (ethyl acetate). Mp. 164–165 °C. ¹H NMR (200 MHz, CD₃OD) δ 7.24–7.20 (m, 5H), 4.33–4.28 (m, 1H), 4.11–4.05 (m, 1H), 3.03–2.93 (m, 2H), 1.56–1.38 (m, 2H), 1.36 (s, 9H), 1.26–1.24 (m, 1H), 0.89 (d, J = 2.6 Hz, 6H). ¹³C NMR (50 MHz, CD₃OD) δ 175.39, 154.59, 143.42, 136.55, 128.99, 128.29, 126.65, 84.91, 72.86, 52.36, 51.47, 27.61, 24.41, 22.20, 21.01. ESI-MS (+) Calc. for [C₂₀H₃₁N₃O₄] 377.48, found: 400.5 [M+Na]⁺.

tert-Butyl ((S)-1-(((R)-1-amino-4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (31).

Yield 88%. White solid. R_f = 0.8 (ethyl acetate). Mp. 168–170 °C. ¹H NMR (200 MHz, CD₃OD) δ 7.24–7.21 (m, 5H), 4.31–4.21 (m, 1H), 4.11–4.09 (m, 1H), 3.04–2.94 (m, 2H), 1.57–1.40 (m, 2H), 1.39 (s, 9H), 1.27–1.24 (m, 1H), 0.90 (d, J = 2.6 Hz, 6H). ¹³C NMR (50 MHz, CD₃OD) δ 175.80, 155.00, 143.83, 136.96, 129.40, 128.70, 127.06, 85.32, 77.16, 73.27, 52.78, 51.89, 28.02, 24.82, 22.61, 21.42. ESI-MS (+) Calc. for [C₂₀H₃₁N₃O₄] 377.48, found: 400.5 [M+Na]⁺.

tert-Butyl ((S)-1-(((S)-1-amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (32).

Yield 78%. White solid. R_f = 0.6 (ethyl acetate). Mp. 148–150 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.38–7.26 (m, 7H), 7.10–7.06 (m, 2H), 6.63 (s br, 1H), 6.39 (s br, 1H), 4.81–4.77 (m, 1H), 4.38–4.35 (m, 1H), 3.05–2.97 (m, 4H), 1.41 (s, 9H). ¹³C NMR (50 MHz, CDCl₃) δ 172.83, 161.82, 138.54, 136.14, 134.46, 133.26, 130.05, 129.51, 129.36, 129.02, 127.69, 127.36, 126.78, 80.95, 62.51, 53.21, 52.76, 38.74, 28.23. ESI-MS (+) Calc. for [C₂₃H₂₈ClN₃O₄] 445.94, found: 468.8 [M+Na]⁺.

tert-Butyl ((S)-1-(((S)-1-amino-1-oxo-3-(pyridin-4-yl)propan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (33).

Yield 71%. Yellowish solid. R_f = 0.4 (ethyl acetate). Mp. 155–157 °C. ¹H NMR (200 MHz, CD₃OD) δ 8.63 (d, J = 5.7 Hz, 2H), 7.58–7.30 (m, 7H), 4.94–4.89 (1H, m), 4.55–4.40 (m, 1H), 3.44 (dd, J = 13.8, 5.5 Hz, 2H), 3.28–2.93 (m, 2H), 1.60 (s, 9H). ¹³C NMR (50 MHz, CDCl₃) δ 173.27, 172.38, 148.58, 140.95, 136.44, 135.10, 128.97, 128.32, 126.68, 124.97, 99.74, 55.40, 27.73, 23.92, 13.06, 10.47.ESI-MS (+) Calc. for [C₂₃H₂₈ClN₃O₄] 445.94, found: 468.8 [M+Na]⁺.

tert-Butyl ((S)-1-(((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (34).

Yield 88%. Yellowish solid. R_f = 0.8 (ethyl acetate). Mp. 161–163 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.38–7.26 (m, 7H), 7.10–7.06 (m, 2H), 6.63 (s br, 1H), 6.39 (s br, 1H), 5.65 (s br, 1H), 5.08 (s br, 1H), 4.81–4.77 (m, 1H), 4.38–4.35 (m, 1H), 3.15–2.97 (m, 5H), 1.74–1.47 (m, 3H), 1.41 (s, 9H). ¹³C NMR (50 MHz, CDCl₃) δ 172.98, 171.90, 156.38, 137.70, 136.53, 128.99, 128.42, 128.13, 127.64, 127.58, 126.75, 80.30, 74.03, 71.36, 60.44, 38.05, 27.67, 20.33, 15.75. ESI-MS (+) Calc. for [C₂₅H₃₃N₃O₅] 455.55, found: 478.5 [M+Na]⁺.

tert-Butyl ((S)-1-(((2R,3S)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (35).

Yield 78%. Yellowish solid. R_f = 0.8 (ethyl acetate). Mp. 138–139 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.40–7.37 (m, 7H), 7.11–7.08 (m, 2H), 6.58 (s br, 1H), 6.43 (s br, 1H), 5.61 (s br, 1H), 5.23 (s br, 1H), 4.83–4.78 (m, 1H), 4.34–4.31 (m, 1H), 3.32–2.85 (m, 5H), 1.39 (s, 9H), 1.19–1.09 m, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 173.34, 172.27, 156.74, 138.07, 136.90, 129.35, 128.79, 128.49, 128.01, 127.95, 127.12, 80.66, 74.40, 71.73, 60.81, 38.42, 28.04, 20.70, 16.12.ESI-MS (+) Calc. for [C₂₅H₃₃N₃O₅] 455.55, found: 478.5 [M+Na]⁺.

tert-Butyl ((S)-1-(((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (36).

Yield 76%. Yellowish solid. R_f = 0.8 (ethyl acetate). Mp. 88–91 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.30–7.22 (m, 5H), 7.19–7.15 (m, 1H), 6.24 (s br, 1H), 6.04 (s br, 1H), 5.36 (s br, 2H), 4.57 (s, 2H), 4.56–4.52 (m, 1H), 4.36–4.21 (m, 1H), 4.17–4.11 (m, 1H), 1.77–1.48 (m, 3H), 1.38 (s, 9H), 1.13 (d, J = 6.4 Hz, 3H), 0.94–0.90 (m, 6H). ¹³C NMR (50 MHz, CDCl₃) δ 172.84, 171.86, 155.98, 137.88, 128.29, 127.66, 127.63, 80.38, 73.89, 71.58, 56.40, 54.01, 40.85, 28.12, 24.74, 22.92, 21.62. ESI-MS (+) Calc. for [C₂₂H₃₅N₃O₅] 421.53, found: 444.7 [M+Na]⁺.

tert-Butyl ((S)-1-(((2R,3S)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (37).

Yield 73%. Yellowish solid. R_f = 0.8 (ethyl acetate). Mp. 86–88 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.31–7.22 (m, 5H), 7.20–7.11 (m, 1H), 6.26 (s br, 1H), 6.02 (s br, 1H), 5.36 (s br, 2H), 4.54 (s, 2H), 4.56–4.53 (m, 1H), 4.33–4.28 (m, 1H), 4.16–4.11 (m, 1H), 1.76–1.48 (m, 3H), 1.48 (s, 9H), 1.11 (d, J = 6.4 Hz, 3H), 0.96–0.90 (m, 6H). ¹³C NMR (50 MHz, CDCl₃) δ 172.84, 171.86, 155.98, 137.88, 128.29, 127.66, 127.63, 80.38, 73.89, 71.58, 56.40, 54.01, 40.85, 28.12, 24.74, 22.92, 21.62. ESI-MS (+) Calc. for [C₂₂H₃₅N₃O₅] 421.53, found: 444.5 [M+Na]⁺.

tert-Butyl ((S)-1-(((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-3-(3-chlorophenyl)-1-oxopropan-2-yl)carbamate (38).

Yield 77%. Yellowish solid. R_f = 0.6 (ethyl acetate). Mp. 164–165 °C. ¹H NMR (200 MHz, CD₃OD) δ 7.47–7.22 (m, 9H), 4.55–4.37 (m, 1H), 4.29–4.17 (m, 2H), 3.26 (dd, J = 13.9, 5.1 Hz, 1H), 3.07–2.96 (m, 1H), 2.92 (s, 2H), 1.44 (s, 9H), 1.27 (d, J = 6.3 Hz, 3H). ¹³C NMR (50 MHz, CD₃OD) δ 172.84, 171.76, 156.24, 138.90, 137.56, 136.39,130.04, 128.85, 128.28, 127.99, 127.50, 127.44, 126.61, 80.16, 73.89, 71.22, 60.30, 49.00, 37.92, 27.53, 20.19. ESI-MS (+) Calc. for [C₂₅H₃₂ClN₃O₅] 489.99, found: 513.1 [M+Na]⁺.

Synthesis of compounds 39–49.

Compounds 39–49 have been synthesized in two steps from their precursors 28–38. After removal of the Boc-protecting group (method B), the free amine was coupled to the carboxylic acid following the general procedure for amide synthesis (method C).

N-((S)-1-(((S)-1-Amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (39).

Yield 79%. Yellowish solid. R_f = 0.3 (ethyl acetate). Mp. 109–110 °C. ¹H NMR (200 MHz, CDCl₃) δ7.32–6.96 (m, 10H), .6.65 (s br, 1H), 6.26 (s, 1H), 6.09 (s br, 1H), 4.68–4.58 (m, 2H), 3.81 (s, 3H), 2.87–2.74 (m, 4H), 1.11 (s, 9H). ¹³C NMR (50 MHz, CDCl₃) δ 173.37, 165.56, 162.46, 160.20, 159.99, 136.34, 136.16, 134.40, 129.13, 128.39, 126.87, 126.81, 124.46, 103.30, 54.93, 53.91, 38.44, 36.34, 31.69, 31.28, 30.27. ESI-MS (+) Calc. for [C₂₇H₃₃N₅O₃] 475.58, found: 498.7 [M+Na]⁺.

N-((S)-1-(((R)-1-Amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (40).

Yield 72%. Yellowish solid. R_f = 0.4 (ethyl acetate). Mp. 121–122 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.59–7.13 (m, 10H), 6.88 (s, 1H), 6.50 (s, 1H), 6.32 (s br, 1H), 4.92–4.82 (m, 2H), 4.04 (s, 3H), 3.10–2.91 (m, 4H), 1.34 (s, 9H). ¹³C NMR (50 MHz, CDCl₃) δ 172.94, 165.13, 162.04, 159.77, 159.56, 135.92, 135.74, 133.97, 128.70, 127.97, 126.45, 126.38, 124.04, 102.88, 54.50, 53.48, 38.01, 35.91, 31.26, 30.86, 29.84. ESI-MS (+) Calc. for [C₂₇H₃₃N₅O₃] 475.58, found: 498.7 [M+Na]⁺.

N-((S)-1-(((S)-1-Amino-4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (41).

Yield 85%. Yellowish solid. R_f = 0.3 (ethyl acetate). Mp. 152–153 °C. ¹H-NMR (200 MHz, CDCl₃) δ. 7.42–7.25 (m, 5H), 6.81 (s, 1H), 4.77–4.65 (m, 1H), 4.36–4.08 (m, 1H), 4.08 (s, 3H), 3.22–3.18 (m, 2H), 1.89–1.46 (m, 3H), 1.40 (s, 9H), 0.88–0.80 (m, 6H). ¹³C NMR (50 MHz, CDCl₃) δ 173.57, 168.11, 162.00, 161.25, 137.23, 135.94, 130.02, 129.31, 127.64, 104.97, 57.07, 49.00, 38.87, 32.59, 31.75, 30.78, 24.80, 23.57, 21.42. ESI-MS (+) Calc. for [C₂₄H₃₅N₅O₃] 441.57, found: 464.5 [M+Na]⁺.

N-((S)-1-(((S)-1-Amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (42).

Yield 72%. Yellowish solid. R_f = 0.3 (ethyl acetate). Mp. 131–133 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.45–7.28 (s, 5H), 6.83 (s, 1H), 4.87–4.68 (m, 1H), 4.39–4.11 (m, 1H), 4.11 (s, 3H), 3.25–3.21 (m, 2H), 1.76–1.49 (m, 3H), 1.42 (s, 9H), 0.88–0.83 (m, 6H). ¹³C NMR (50 MHz, CDCl₃) δ 176.49, 171.02, 164.92, 164.17, 140.15, 138.86, 132.94, 132.23, 130.56, 107.89, 76.35, 59.99, 41.79, 35.51, 34.67, 33.70, 27.72, 26.49, 24.34. ESI-MS (+) Calc. for [C₂₄H₃₅N₅O₃] 441.57, found: 464.5 [M+Na]⁺.

tert-Butyl ((S)-1-(((S)-1-amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (43).

Yield 80%. Yellowish solid. R_f = 0.2 (ethyl acetate). Mp. 101–103 °C. ¹H NMR (200 MHz, CDCl₃:CD₃OD 10:1) δ. 7.58–6.99 (m, 9H), 6.47 (s, 1H), 4.77–4.62 (m, 2H), 4.04 (s, 3H), 3.39–2.91 (m, 4H), 1.34 (s, 9H). ¹³C NMR (50 MHz, CDCl₃:CD₃OD 10:1) δ 172.23, 172.21, 161.25, 161.03, 137.40, 136.96, 135.83, 135.15, 130.68, 129.87, 129.23, 128.42, 128.30, 127.69, 117.62, 104.55, 55.12, 49.00, 42.41, 38.76, 38.62, 32.51, 30.78. ESI-MS (+) Calc. for [C₂₇H₃₂ClN₅O₃] 510.03, found: 532.9 [M+Na]⁺.

N-((S)-1-(((S)-1-Amino-1-oxo-3-(pyridin-4-yl)propan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (44).

Yield 66%. Yellowish oil. R_f = 0.2 (ethyl acetate). ¹H NMR (200 MHz, CDCl₃) δ 8.63 (d, J = 5.7 Hz, 2H), 7.58–7.30 (m, 7H), 7.36–7.13 (m, 7H), 6.59 (s, 1H), 4.84–4.68 (m, 2H), 3.89 (s, 3H), 3.35–3.07 (m, 2H), 1.27 (s, 9H) .¹³C NMR (50 MHz, CDCl₃) δ 174.42, 173.04, 161.51, 161.12, 149.40, 148.81, 138.04, 136.11, 129.86, 129.09, 127.45, 126.03, 104.55, 55.73, 54.08, 38.35, 37.93, 37.75, 32.45, 30.45. ESI-MS (+) Calc. for [C₂₆H₃₂N₆O₃] 476.57, found:477.3 [M+H]⁺.

N-((S)-1-(((2S,3R)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (45).

Yield 65%. Yellowish solid. R_f = 0.3 (ethyl acetate). Mp 109–110 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.30–7.08 (m, 9H), 6.40 (d, J = 14.4 Hz, 1H), 6.26 (s, 1H), 5.00–4.76 (m, 1H), 4.52 (s, 2H), 4.17–4.06 (m, 2H), 3.98 (s, 3H), 3.20–3.09 (m, 2H), 1.27 (s, 9H), 1.11 (d, J = 6.2 Hz, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 171.79, 171.19, 160.58, 160.20, 137.80, 136.25, 134.62, 129.26, 128.78, 128.44, 127.84, 127.78, 127.21, 103.32, 74.00, 71.58, 60.39, 56.64, 38.86, 38.61, 31.91, 30.49, 21.02. ESI-MS (+) Calc. for [C₂₉H₃₇N₅O₄] 519.64, found:549.4 [M+Na]⁺.

N-((S)-1-(((2R,3S)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (46).

Yield 61%. Yellowish solid. R_f = 0.3 (ethyl acetate). Mp 119–120 °C. ¹H NMR (200 MHz, CDCl₃) δ.7.27–7.04 (m, 9H), 6.37 (d, J = 14.4 Hz, 1H), 6.22 (s, 1H), 4.97–4.73 (m, 1H), 4.48 (s, 2H), 4.14–4.03 (m, 2H), 3.95 (s, 3H), 3.17–3.06 (dd, J = 11.3, 5.1 Hz, 2H), 1.23 (s, 9H), 1.08 (d, J = 6.2 Hz, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 171.79, 171.19, 160.58, 160.20, 137.80, 136.25, 134.62, 129.26, 128.78, 128.44, 127.84, 127.78, 127.21, 103.32, 74.00, 71.58, 60.39, 56.64, 38.86, 38.61, 31.91, 30.49, 21.02. ESI-MS (+) Calc. for [C₂₉H₃₇N₅O₄] 519.64, found: 549.4 [M+Na]⁺.

N-((S)-1-(((2S,3R)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-3-(tert-butyl)-1-methyl-1H3-pyrazole-5-carboxamide (47).

Yield 73%. Yellowish solid. R_f = 0.3 (ethyl acetate). Mp 177–178 °C. ¹H NMR (200 MHz, CDCl₃) δ. 7.27–7.20 (m, 4H), 7.14 (d, J = 8.5 Hz, 1H), 6.42 (s, 1H), 4.85–4.82 (m, 1H), 4.62–4.52 (s, 2H), 3.96 (s, 3H), 3.87–3.84 (m, 1H), 1.69–1.59 (m, 3H), 1.22 (s, 9H). 1.18 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.0 Hz, 3H), 0.97 (d, J = 6.0 Hz, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 172.35, 171.66, 160.37, 160.31, 137.82, 134.69, 128.57, 128.00, 127.74, 103.21, 74.07, 71.71, 56.24, 52.12, 39.03, 38.69, 32.02, 30.59, 25.00, 23.01, 22.0. ESI-MS (+) Calc. for [C₂₆H₃₉N₅O₄] 485.62, found: 508.5 [M+Na]⁺.

N-((S)-1-(((2R,3S)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-3-(tert-butyl)-1-methyl-1H3-pyrazole-5-carboxamide (48).

Yield 70%. Yellowish solid. R_f = 0.3 (ethyl acetate). Mp 170–171 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.33–7.21 (m, 4H), 7.15 (d, J = 8.5 Hz, 1H), 6.34 (s, 1H), 4.85–4.82 (m, 1H), 4.64–4.53 (s, 2H), 3.98 (s, 3H), 3.88–3.86 (m, 1H), 1.71–1.60 (m, 3H), 1.24 (s, 9H), 1.21 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.89 (d, J = 6.0 Hz, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 171.72, 171.02, 159.73, 159.68, 137.18, 134.05, 127.94, 127.36, 127.10, 102.58, 73.43, 71.08, 55.60, 51.49, 38.39, 38.06, 31.39, 29.95, 24.37, 22.38, 21.44. ESI-MS (+) Calc. for [C₂₆H₃₉N₅O₄] 485.62, found: 508.5 [M+Na]⁺.

N-((S)-1-(((2S,3R)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-3-(3-chlorophenyl)-1-oxopropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (49).

Yield 65%. Yellowish solid. R_f = 0.2 (ethyl acetate). Mp 102–104 °C. ¹H NMR (200 MHz, CD₃OD) δ 8.59 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.26 (dd, J = 5.6, 2.2 Hz, 9H), 6.59 (s, 1H), 4.64–4.45 (m, 3H), 4.16–4.11 (m, 1H), 3.86 (s, 3H), 3.27–3.25 (m, 1H), 3.03 (dd, J = 14.7, 10.8 Hz, 1H), 1.28 (s, 9H), 1.22 (d, J = 6.3 Hz, 3H). ¹³C NMR (50 MHz, CD₃OD) δ 174.44, 173.31, 162.05, 161.35, 140.90, 139.31, 136.28, 134.96, 130.78, 130.33, 129.08, 128.62, 128.52, 128.44, 127.71, 104.84, 75.78, 72.24, 58.45, 55.77, 38.46, 37.34, 32.66, 30.66, 16.58. ESI-MS (+) Calc. for [C₂₉H₃₆ClN₅O₄] 554.08, found: 577.1 [M+Na]⁺.

Synthesis of compounds 50–60.

Compounds 50–60 have been synthesized by dehydration of the corresponding primary amide precursor 39–49 with cyanuric chloride (method A).

3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (50).

Yield 89%. Yellowish solid. R_f = 0.6 (ethyl acetate: n-hexane; 6:4). Mp. 89–90 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.32–7.21 (m, 8H), 7.16 (dd, J = 6.4, 2.8 Hz, 2H), 6.90 (d, J = 8.2 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 6.35 (s, 1H), 5.04–5.02 (m, 1H), 4.82–4.80 (m, 1H), 4.02 (s, 3H), 3.21–3.07 (m, 2H), 3.00–2.98 (m, 2H), 1.33–1.30 (m, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 170.30, 160.25, 160.30, 135.55, 134.04, 133.37, 129.08, 129.04, 128.75, 128.72, 127.71, 127.26, 117.25, 103.06, 54.17, 41.48, 38.77, 38.35, 37.95, 31.75, 30.26. FT-IR (KBr, cm^-1) 3300.81, 2951.21, 2909.60, 2243.70, 1677.69, 1648.55, 1544.51, 1278.15, 1232.37, 986.82, 751.92, 670.52, 524.68, 424.97. HRMS (+) Calc. for [C₂₇H₃₂N₅O₂]⁺ 458.25560, found: 458.2586 [M+H]⁺. HPLC (protocol A): t_R (min) = 9.07. Purity 99.0%.

3-(tert-Butyl)-N-((S)-1-(((R)-1-cyano-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (51).

Yield 92%. Yellowish solid. R_f = 0.6 (ethyl acetate: n-hexane; 6:4). Mp. 98–99 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.34–7.12 (m, 9H), 6.91 (d, J = 7.9 Hz, 1H), 6.34 (s, 1H), 5.09–5.05 (m, 1H), 4.94–4.91 (m, 1H), 4.00 (s, 3H), 3.12 (dd, J = 13.8, 6.4 Hz, 2H), 2.98 (dd, J = 13.8, 6.4 Hz, 2H), 1.30 (s, 9H). ¹³C NMR (50 MHz, CDCl₃) δ 170.75, 160.43, 160.33, 135.98, 134.32, 133.78, 129.42, 129.33, 129.01, 128.86, 127.99, 127.42, 118.87, 103.48, 54.38, 41.82, 38.96, 38.48, 38.26, 31.98, 30.51. FT-IR (KBr, cm^-1) 3350.28, 3256.30, 2953.92, 2165.27, 1638.14, 1511.6, 1307.15, 1160.05, 1086.49, 906.70, 820.89, 694.21, 669.69, 543.02. HRMS (+) Calc. for [C₂₇H₃₂N₅O₂]⁺ 458.25560, found: 458.2586 [M+H]⁺. HPLC (protocol A): t_R (min) = 17.32. Purity 99.3%.

3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-3-methylbutyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (52).

Yield 84%. Yellowish solid. R_f = 0.7 (ethyl acetate: n-hexane; 6:4). Mp 81–82 °C. ¹H NMR (200 MHz, CD₃OD) δ 7.29–7.20 (m, 5H), 6.64 (s, 1H), 4.80–4.69 (m, 2H), 3.90 (s, 3H), 3.23–2.94 (m, 2H), 1.78–1.62 (m, 3H), 1.27 (s, 9H), 0.95–0.91 (m, 6H). ¹³C NMR (50 MHz, CD₃OD) δ 173.33, 161.87, 161.51, 138.07, 136.59, 130.36, 129.56, 127.93, 119.68, 105.02, 56.04, 49.00, 42.00, 40.04, 38.69, 32.86, 30.88, 25.80, 22.35, 22.19. FT-IR (KBr, cm^-1) 3403.41, 3309.42, 3203.18, 2953.92, 2937.57, 1695.35, 1634.05, 1523.72, 1368.44, 1274.46, 1245.86, 1168.22, 835.58, 702.38, 669.69, 567.54. HRMS (+) Calc. for [C₂₄H₃₄N₅O₂]⁺ 423.27125, found: 424.27572 [M+H]⁺. HPLC (protocol A): t_R (min) = 9.16. Purity 97.4%.

3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-3-methylbutyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (53).

Yield 79%. White solid. R_f = 0.7 (ethyl Acetate: n-hexane; 6:4). Mp. 91–94 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.32–7.21 (m, 3H), 6.98 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.34 (s, 1H), 4.92–4.86 (m, 1H), 4.77–4.71 (m, 1H), 3.99 (s, 3H), 3.29–3.19 (m, 2H), 1.63–1.46 (m, 3H), 1.27 (s, 9H), 0.88 (2d, J = 5.9 Hz, 6H). ¹³C NMR (50 MHz, CDCl₃) δ 171.28, 161.15, 161.04, 136.68, 135.04, 129.97, 129.56, 128.05, 119.17, 104.16, 55.16, 41.95, 39.62, 39.12, 32.67, 31.19, 25.31, 22.79, 22.53. FT-IR (KBr, cm^-1) 3350.28, 3256.30, 2953.92, 2165.27, 1638.14, 1511.46, 1307.15, 1160.05, 1086.49, 906.70, 820.89, 694.21, 669.69, 543.02. HRMS (+) Calc. for [C₂₄H₃₄N₅O₂]⁺ 423.27125, found: 424.27572 [M+H]⁺. HPLC (protocol A): t_R (min) = 9.42. Purity 99.1%.

3-(tert-Butyl)-N-((S)-1-(((S)-2-(3-chlorophenyl)-1-cyanoethyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (54).

Yield 50%. Yellowish solid. R_f = 0.6 (ethyl acetate: n-hexane; 6:4). Mp 132–133 °C. ¹H NMR (200 MHz, CD₃OD/ CDCl₃) δ 7.54–7.35 (m, 9H), 6.75 (s, 1H), 5.19–5.11 (m, 1H), 4.51–4.48 (m, 1H), 4.17 (s, 3H), 3.33–3.21 (m, 4H), 1.49 (s, 9H). ¹³C NMR (50 MHz, CD₃OD) δ 172.23, 161.25, 161.03, 137.40, 136.96, 135.83, 135.15, 130.68, 130.07, 129.87, 129.23, 128.42, 128.30, 127.69, 118.18, 104.55, 55.12, 42.41, 38.76, 38.62, 32.51, 30.78. FT-IR (cm^-1) 3288.99, 2925.31, 2855.85, 2161.18, 1666.74, 1634.05, 1544.15, 1507.38, 1450.17, 1266.29, 1221.34, 1074.23, 861.75, 747.33, 706.47, 694.21. HRMS (+) Calc. for [C₂₇H₃₁ClN₅O₂]⁺ 491.21663, found: 492.21034 [M+H]⁺. HPLC (protocol A): t_R (min) = 9.61. Purity > 99.9%.

3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-2-(pyridin-4-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (55).

Yield 35%. Yellowish wax. R = 0.4 (ethyl acetate: n-hexane; 6:4). ¹H NMR (400 MHz, CD₃OD) δ 8.44 (s br, 2H), 7.38 (d, J = 5.3 Hz, 2H), 7.30–7.22 (m, 5H), 6.63 (s, 1H), 5.14 (t, J = 7.5 Hz, 1H), 4.66 (dd, J = 8.6, 6.8 Hz, 1H), 3.94 (s, 3H), 3.26–3.13 (m, 4H), 1.30 (s, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.17, 170.90, 159.29, 158.46, 137.89, 136.81, 135.23, 134.81, 129.27, 128.86, 128.13, 127.92, 126.93, 126.34, 118.94, 60.46, 53.87, 53.24, 41.15, 31.39, 30.18, 13.74. FT-IR (cm^-1) 3264.47, 2953.92, 2913.05, 2851.76, 2161.43, 1662.66, 1605.45, 1548.24, 1466.62, 1204.99, 1115.10, 996.59, 800.45, 735.07, 681.95. HRMS (+) Calc. for [C₂₆H₃₁N₆O₂]⁺ 459.25085, found: 459.25627 [M+H]⁺. HPLC (protocol A): t_R (min) = 6.84. Purity 98.6%.

N-((S)-1-(((1R,2R)-2-(Benzyloxy)-1-cyanopropyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (56).

Yield 80%. White solid. R_f = 0.5 (ethyl acetate: n-hexane; 6:4). Mp. 115–116 °C. ¹H-NMR (200 MHz, CDCl₃) δ 7.35–7.26 (m, 10H), 6.95 (d, J = 8.7 Hz, 1H), 6.61 (d, J = 7.0 Hz, 1H), 6.30 (s, 1H), 4.88–4.85 (m, 2H), 4.62–4.58 (m, 1H), 4.03 (s, 3H), 3.82–3. 78 (m, 1H), 3.29–3.24 (m, 2H), 1.31 (s, 9H), 1.03 (d, J = 6.0 Hz, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 170.30, 159.83, 159.62, 136.42, 135.27, 133.75, 128.67, 128.41, 128.00, 127.60, 127.30, 126.87, 116.63, 102.65, 72.60, 70.91, 53.85, 44.51, 38.33, 37.60, 31.40, 29.94, 15.29. FT-IR (cm^-1) 3354.37, 3252.21, 2181.61, 1654.61, 1654.48, 1540.07, 1486.95, 1290.81, 1151.87, 1086.48, 894.44, 808.63, 710.56, 661.32, 543.02. HRMS (+) Calc. for [C₂₉H₃₆N₅O₃]⁺ 502.28182, found: 502.28095 [M+H]⁺. HPLC (protocol A): t_R (min) = 9.54. Purity 99.4%.

N-((S)-1-(((1S,2S)-2-(Benzyloxy)-1-cyanopropyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (57).

Yield 82%. White solid. R_f = 0.5 (ethyl acetate: n-hexane; 6:4). Mp. 123–124 °C. ¹H NMR (200 MHz, CDCl₃) δ 7.36–7.25 (m, 10H), 6.98 (d, J = 8.7 Hz, 1H), 6.65 (d, J = 7.0 Hz, 1H), 6.32 (s, 1H), 4.91–4.83 (m, 2H), 4.63–4.60 (m, 1H), 4.05 (s, 3H), 3.80 (s br, 1H), 3.24–3.20 (m, 2H), 1.33 (s, 9H), 1.05 (d, J = 6.0 Hz, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 171.01, 160.54, 160.34, 137.14, 135.98, 134.46, 129.39, 129.12, 128.71, 128.32, 128.02, 127.59, 117.34, 103.37, 72.85, 71.15, 54.56, 45.22, 39.04, 38.32, 32.12, 30.65, 16.00. FT-IR (cm^-1) 2376.73, 2958.00, 2169.35, 1642.22, 15400.07, 1499.21, 1442.00, 1290.81, 1225.43, 1111.01, 1033.37, 988.42, 743.25, 690.12. HRMS (+) Calc. for [C₂₉H₃₆N₅O₃]⁺ 502.28182, found: 502.28095 [M+H]⁺. HPLC (protocol A): t_R (min) = 9.43. Purity 98.3%.

N-((S)-1-(((1R,2R)-2-(Benzyloxy)-1-cyanopropyl)amino)-4-methyl-1-oxopentan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (58).

Yield 78%. White solid. R_f = 0.5 (ethyl acetate: n-hexane; 6:4). Mp. 96–97 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (d, J = 8.1 Hz, 1H), 8.43 (d, J = 7.6 Hz, 1H), 7.38–7.29 (m, 5H), 6.88 (s, 1H), 5.07–5.04 (m, 1H), 4.64–4.61 (m, 2H), 4.59–4.56 (m, 1H), 3.97–3.94 (m, 3H), 3.86–3.84 (m, 1H), 1.72–1.64 (m, 2H), 1.50–1.46 (m, 2H), 1.26–1.20 (m, 12H), 0.93 (d, J = 6.3 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.36, 159.56, 158.66, 137.92, 134.92, 128.10, 127.53, 127.45, 117.82, 103.73, 73.39, 70.40, 51.05, 44.75, 38.44, 31.53, 30.31, 24.25, 22.91, 21.19, 15.69. FT-IR (cm^-1) 3378.89, 3350.28, 3186.83, 2958.00, 2116.43, 1674.91, 1650.40, 1531.90, 1368.44, 1262.20, 1160.05, 1057.89, 1033.37, 780.02, 735.07, 649.26, 604.31. HRMS (+) Calc. for [C₂₆H₃₈N₅O₃] 468.29746, found: 468.29785 [M+H]⁺. HPLC (protocol A): t_R (min) = 9.61. Purity 98.3%.

N-((S)-1-(((1S,2S)-2-(Benzyloxy)-1-cyanopropyl)amino)-4-methyl-1-oxopentan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (59).

Yield 70%. White solid. R_f = 0.5 (ethyl acetate: n-hexane; 6:4). Mp. 110–111 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.27–7.20 (m, 5H), 7.14 (d, J = 8.5 Hz, 1H), 6.42 (d, J = 7.8 Hz, 1H), 6.33 (s, 1H), 4.85–4.82 (m, 1H), 4.62–4.52 (m, 3H), 3.96 (s, 3H), 3.87–3.84 (m, 1H), 1.69–1.59 (m, 3H), 1.22 (s, 9H). 1.18 (d, J = 6.3 Hz, 3H) 1.24–1.19 (m, 12H), 0.92–0.88 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 172.10, 160.65, 160.51, 137.21, 134.43, 128.66, 128.23, 127.92, 117.45, 103.16, 73.69, 71.80, 51.75, 45.28, 40.81, 38.06, 32.10, 30.62, 25.04, 22.97, 22.13, 16.36. FT-IR (cm^-1) 3333.94, 3284.90, 2962.09, 2868.10, 2255.16, 1647.44, 1540.07, 1507.38, 1442.00, 1274.46, 1245.86, 988.42, 739.16, 661.52. HRMS (+) Calc. for [C₂₆H₃₈N₅O₃] 468.29746, found: 468.29785 [M+H]⁺. HPLC (protocol A): t_R (min) = 9.56. Purity 96.9%.

N-((S)-1-(((1R,2R)-2-(Benzyloxy)-1-cyanopropyl)amino)-3-(3-chlorophenyl)-1-oxopropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (60).

Yield 50%. Yellowish wax. R_f = 0.4 (ethyl acetate: n-hexane; 6:4). ¹H NMR (400 MHz, CDCl₃) δ 7.30–7.13 (m, 6H), 7.04 (dt, J = 7.0, 1.7 Hz, 1H), 6.62 (dd, J = 17.8, 8.2 Hz, 2H), 6.25 (s, 1H), 4.77–4.71 (m, 2H), 4.48 (dd, J = 36.1, 11.7 Hz, 2H), 3.95 (s, 3H), 3.82–3.74 (m, 1H), 3.11–3.00 (m, 2H), 1.20 (s, 9H), 1.08 (d, J = 6.3 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.73, 160.54, 160.12, 137.80, 136.91, 134.84, 134.30, 130.44, 129.51, 128.71, 128.34, 128.03, 127.84, 127.62, 117.06, 103.35, 73.35, 71.72, 54.28, 45.07, 39.06, 38.13, 32.07, 30.58, 16.21. FT-IR (cm^-1) 3357.24, 3264.21, 2193.15, 1654.33, 1538.29, 1487.88, 1209.18, 1141.78, 1036.43, 863.33, 807.36, 701.44, 658.23, 534.57. ESI-MS (+) Calc. for [C₂₉H₃₄₅ClN₅O₃] 536.24284, found: 536.24235 [M+H]⁺.HPLC (protocol B): t_R (min) = 12.06. Purity 96.5%.

Synthesis of compounds 61–64.

Compounds 61–64 have been synthesized from compounds 34–37 by removal of the benzyl group through hydrogenation on Pd/C (method A).

N-((S)-1-(((2S,3R)-1-Amino-3-hydroxy-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide.(61).

Yield 92%. Colorless wax. R_f = 0.2 (ethyl acetate). ¹H NMR (200 MHz, CDCl₃) δ 8.03 (s, 1H), 7.36–6.94 (m, 5H), 6.54 (s, 1H), 4.44–4.39 (m, 2H), 4.03–4.01 (m, 1H), 3.96 (s, 3H), 3.18–3.14 (m, 1H), 2.87 (s, 1H), 1.30 (s, 9H), 1.03–1.01 (m, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 173.54, 172.15, 161.25, 160.35, 136.25, 135.98, 134.58, 129.36, 128.76, 127.25, 66.60, 58.51, 55.55, 54.30, 38.70, 31.98, 30.51, 18.92. ESI-MS (+) Calc. for [C₂₂H₃₁N₅O₄] 429.51, found:452.4 [M+Na]⁺.

N-((S)-1-(((2R,3S)-1-Amino-3-hydroxy-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide.(62).

Yield 95%. Colorless wax. R_f = 0.2 (ethyl acetate). ¹H NMR (200 MHz, CDCl₃) δ 8.01 (s, 1H), 7.36–6.98 (m, 5H), 6.59 (s, 1H), 4.42–4.36 (m, 2H), 4.04–4.01 (m, 1H), 3.96 (s, 3H), 3.12–3.09 (m, 1H), 2.89 (s, 1H), 1.33 (s, 9H), 1.03–0.99 (m, 3H). ¹³C NMR (50 MHz, CDCl₃) δ 173.54, 172.15, 161.25, 160.35, 136.25, 135.98, 134.58, 129.36, 128.76, 127.25, 66.60, 58.51, 55.55, 54.30, 38.70, 31.98, 30.51, 18.92. ESI-MS (+) Calc. for [C₂₂H₃₁N₅O₄] 429.51, found: 452.4 [M+Na]⁺.

N-((S)-1-(((2S,3R)-1-Amino-3-hydroxy-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide(63).

Yield 90%. Colorless wax. R_f = 0.2 (ethyl acetate). ¹H NMR (200 MHz, CDCl₃) δ 7.37 (d, J = 3.9 Hz, 1H), 6.74–6.71 (m, 2H), 6.42 (s, 1H), 4.71–4.68 (m, 1H), 4.42–4.38 (m, 2H), 4.06 (s, 3H), 1.71–1.68 (m, 3H), 1.27 (s, 9H), 1.14–1.11 (m, 3H), 0.98–0.92 (m, 6H). ESI-MS (+) Calc. for [C₁₉H₃₃N₅O₄] 395.51, found: 418.5 [M+Na]⁺.

N-((S)-1-(((2R,3S)-1-Amino-3-hydroxy-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (64).

Yield 93%. Colorless wax. R_f = 0.2 (ethyl acetate). ¹H NMR (200 MHz, CDCl₃) δ 7.34 (d, J = 3.9 Hz, 1H), 6.79–6.75 (m, 2H), 6.46 (s, 1H), 4.73–4.70(m, 1H), 4.45–4.41 (m, 2H), 4.11 (s, 3H), 1.77–1.74 (m, 3H), 1.28 (s, 9H), 1.19–1.17 (m, 3H), 0.99–0.91 (m, 6H). ESI-MS (+) Calc. for [C₁₉H₃₃N₅O₄] 395.51, found: 418.5 [M+Na]⁺.

Synthesis of compounds 65–68.

Compounds 65–68 have been synthesized by dehydration of the corresponding primary amide precursor 61–64 with trifluoroacetic anhydride (method B).

3-(tert-Butyl)-N-((S)-1-(((1R,2R)-1-cyano-2-hydroxypropyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (65).

Yield 45%. White solid. R_f = 0.7(ethyl acetate). Mp. 159–160 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.32–7.26 (m, 4H), 7.26–7.21 (m, 1H), 6.65 (s, 1H), 4.84–4.79 (m, 2H), 3.96 (s, 3H), 3.88–3.84 (m, 1H), 3.20 (dd, J = 13.6, 7.1 Hz, 1H), 3.10 (dd, J = 13.6, 7.1 Hz, 1H), 1.31 (s, 9H), 1.07 (d, J = 6.3 Hz, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 173.35, 162.06, 161.60, 138.15, 136.69, 130.42, 129.57, 127.98, 118.24, 105.04, 67.67, 56.05, 38.86, 38.68, 32.90, 30.87, 18.89, 18.55. FT-IR (KBr, cm^-1) 3293.08, 2953.92, 2913.05, 2868.10, 1646.10, 1540.07, 1446.08, 1286.72, 1237.68, 1098.75, 922.51, 739.16, 689.30, 469.47. HRMS (+) Calc. for [C₂₂H₃₀N₅O₃]⁺ 412.23486, found: 412.23811 [M+H]⁺. HPLC (protocol A): t_R (min) = 7.65. Purity 97.8%.

3-(tert-Butyl)-N-((S)-1-(((1S,2S)-1-cyano-2-hydroxypropyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (66).

Yield 42%. White solid. R_f = 0.7 (ethyl acetate). Mp. 145–147 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.31–7.23 (m, 5H), 7.07 (d, J = 8.0 Hz, 1H), 6.67 (d, J = 7.5 Hz, 1H), 6.31 (s, 1H), 4.85–4.84 (m, 1H), 4.72–4.68 (m, 1H), 4.15–4.10 (m, 1H), 4.02 (s, 3H), 3.21–3.15 (m, 2H), 1.29–1.27 (m, 9H), 1.08 (d, J = 6.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 172.41, 171.26, 160.58, 135.89, 134.27, 129.38, 129.14, 127.65, 117.25, 103.52, 67.39, 54.76, 47.02, 39.11, 38.38, 32.09, 30.59, 19.02. FT-IR (KBr, cm^-1) 3289.11, 2955.43, 2918.55, 2860.11, 1649.54, 1543.16, 1444.02, 1277.62, 1233.66, 1091.87, 944.77, 745.55, 690.34, 477.11. HRMS (+) Calc. for [C₂₂H₃₀N₅O₃]⁺ 412.23486, found: 412.23811 [M+H]⁺.HPLC (protocol A): t_R (min) = 7.71. Purity 95.7%.

3-(tert-Butyl)-N-((S)-1-(((1R,2R)-1-cyano-2-hydroxypropyl)amino)-4-methyl-1-oxopentan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (67).

Yield 33%. White solid. R_f = 0.5 (ethyl acetate). Mp. 118–119 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.56–7.51 (m, 1H), 6.80–6.75 (m, 1H), 6.43 (s, 1H), 4.87–4.83 (m, 1H), 4.65–4.63 (m, 1H), 4.15–4.11 (m, 1H), 4.06 (s, 3H), 1.31–1.20 (m, 15H), 0.97 (2d, J = 6.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 174.32, 161.62, 162.14, 135.77, 117.22, 104.56, 67.34, 52.68, 41.12, 38.28, 31.46, 32.15, 26.11, 23.56, 21.65, 19.03. FT-IR (KBr, cm^-1) 3297.16, 2953.92, 2913.05, 2868.10, 1646.31, 1535.89, 1503.29, 1462.52, 1364.36, 1282.63, 1172.38, 1131.44, 996.59, 730.99, 592.05. HRMS (+) Calc. for [C₁₉H₃₂N₅O₃]⁺ 378.25071, found: 378.25231 [M+H]⁺.HPLC (protocol A): t_R (min) = 8.08. Purity: 96.7%.

3-(tert-Butyl)-N-((S)-1-(((1S,2S)-1-cyano-2-hydroxypropyl)amino)-4-methyl-1-oxopentan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (68).

Yield 35%. White solid. R_f = 0.5 (ethyl acetate). Mp. 111–113 °C. ¹H NMR (400 MHz, CD₃OD) δ 6.78 (s, 1H), 4.88–4.84 (m, 1H), 4.65–4.60 (m, 1H), 4.07–4.05 (m, 1H), 4.05 (s, 3H), 1.81–1.61 (m, 4H), 1.32 (s, 9H), 1.26 (d, J = 6.3 Hz, 3H), 1.00 (2d, J = 6.3 Hz, 6H). ¹³C NMR (100 MHz,CD₃OD) δ 175.01, 162.46, 161.76, 136.76, 118.59, 105.26, 68.00, 53.28, 41.76, 39.00, 33.07, 31.04, 26.27, 23.54, 21.94, 19.44. FT-IR (KBr, cm^-1) 3293.08, 2962.09, 2917.14, 2847.67, 1646.31, 1540.07, 1458.34, 1278.55, 1131.44, 1078.32, 1000.68, 853.58, 812.71, 780.02, 730.99, 559.36. HRMS (+) Calc. for [C₁₉H₃₂N₅O₃]⁺ 378.25071, found: 378.25231 [M+H]⁺. HPLC (protocol A): t_R (min) = 7.94. Purity: 98.6%.

Synthesis of compound 69.

Compound 69 has been synthesized by removal of the benzyl group from compound 60 with DDQ (method B).

3-(tert-butyl)-N-((S)-3-(3-chlorophenyl)-1-(((1R,2R)-1-cyano-2-hydroxypropyl)amino)-1-oxopropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (69).

Yield 32%. White solid. R_f = 0.3 (ethyl acetate). Mp. 102–103 °C. ¹H NMR (400 MHz, CD₃OD) δ 7.35–7.28 (m, 4H), 6.69 (s, 1H), 4.87–4.83 (m, 2H), 3.99 (s, 3H), 3.90–3.84 (m, 1H), 3.24 (dd, J = 13.6, 7.1 Hz, 1H), 3.08 (dd, J = 13.6, 6.5 Hz, 1H), 1.34 (s, 9H), 1.11 (d, J = 6.3 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 175.23, 163.23, 151.94, 151.48, 128.04, 126.57, 120.30, 120.10, 119.52, 119.45, 117.86, 108.12, 94.92, 57.55, 45.93, 28.74, 28.56, 22.78, 20.75, 18.78. FT-IR (KBr, cm^-1) 3284.90, 3056.07, 2958.00, 2925.31, 2868.10, 1638.14, 1556.41, 1499.21, 1462.43, 1364.36, 1270.37, 1229.51, 1098.75, 878.09, 730.99, 702.38, 453.12. ESI-MS (+) Calc. for [C₂₂H₂₉ClN₅O₃]⁺ 446.19589, found: 446.19745 [M+H]⁺.HPLC (protocol A): t_R (min) = 8.55. Purity 99.7%.

Enzyme inhibition studies

Enzyme inhibition studies for Cz and LmCPB were performed as previously described for Cz [15]. The experimental procedures are described in the Supporting Information (see chapter S3). Cathepsins B, L, S were assayed as reported [18,19].

All experiments were performed in triplicate and for the most potent compounds (pK_i > 7.5) two other independent experiments were performed.

Cathepsin K assay.

Human recombinant cathepsin K was assayed on a FLUOSTAR Optima plate reader at 25 °C with an excitation wavelength of 360 nm and an emission wavelength of 440 nm on a 96 well plate. The enzyme solution (23 μg/mL in 50 mM sodium acetate pH 5.5, 50 mM NaCl, 0.5 mM EDTA, 5 mM DTT) was diluted 1:100 with assay buffer (100 mM sodium citrate buffer pH 5.0, 100 mM NaCl, 1 mM EDTA, 0.01% CHAPS) containing 5 mM DTT and was then incubated at 37 °C for 30 min for activation. A 1.5 mM stock solution of the substrate Z-Leu-Arg-AMC was prepared in DMSO. The final substrate concentration was 6 μM (= 3.05 × K_m). The assay was performed with a final concentration of cathepsin K of 1.73 ng/mL. Stock solutions of inhibitors were prepared in DMSO. The final DMSO concentration was 2% (4 μL). Into a well containing 194.5 μL assay buffer, 0.8 μL of the fluorogenic substrate, DMSO and inhibitor solution (3.2 μL) were added. Upon addition of cathepsin K (1.5 μL), the measurement was started and followed for 20 min.

Mammalian cytotoxicity assay

LLCMK₂ cells were cultured in 96-well plates at a concentration of 5 x 104 cells/ml. After 48 h, the plates were washed twice with PBS and 200 μL RPMI medium was added with serial dilutions of the compounds and benznidazole (1.95 μM to 250 μM) in triplicate. After 72 h at 37 °C, the cytotoxic activity of the compounds was determined by the classical MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] method. Briefly, 50 μL MTT dissolved in PBS (2.0 mg mL^-1) was added to each well and the plates were incubated for 4 h at 37 °C. The formed formazan crystals were dissolved with DMSO (50 μL/well) and the absorbance of the samples was measured by spectrophotometer at 570 nm after 30 min. The cytotoxicity results (CC₅₀) were calculated as a percentage by the formula [(ABScontrol-ABSample / ABS control) * 100], where ABScontrol represents the mean absorbance of the untreated control (viable cells) and ABSample, the absorbance of each cellular treatment.

All experiments were performed in triplicate and for the most potent compounds (EC₅₀ < 100) two other independent experiments were performed.

In vitro trypanocidal activity evaluation on intracellular amastigote forms (Tulahuen strain)

Cells were evaluated in 96-well plates. Cells from the LLCMK₂ lineage were plated at a concentration of 5x10⁴ cells mL^-1. Trypomastigote forms of the Tulahuen LacZ strain were added at a concentration of 5×10⁵ parasites mL^-1 and placed in the incubator at 37°C with 5% CO₂ for 24 h. After the incubation period, the trypomastigote forms present were removed by successive washes with PBS, remaining only as intracellular amastigote forms. Compounds were added at different concentrations (1.95 μM to 250 μM serial dilutions) and incubated for 72 h. At the end of this period, the substrate CPRG (chlorophenol red β-D-galactopyranoside, 400 μM in 0.3% Triton X-100, pH 7.4) was added. After 4 h of incubation at 37 °C, the plates were analyzed in a spectrophotometer at 570 nm to obtain the effective concentration (EC₅₀) to reduce the parasitemia inside the host cell. Benznidazole was used as a positive control in the same concentrations as the substances, and DMSO as a negative control. Compounds were solubilized in DMSO. The selectivity index (SI) was calculated using the formula: SI = EC₅₀/CC₅₀. All statistical analyses were done with the program GraphPad Prism v.5.

All experiments were performed in triplicate and for the most potent compounds (CC₅₀ < 100) two other independent experiments were performed.

Results

Structure-based design, modeling studies, and compound synthesis

Cz, the recombinant form of cruzipain, is a monomeric enzyme, composed of two folded and equally sized domains. These domains are divided by the enzyme’s active site, which is V-shaped and primarily exposed to solvent. A catalytic triad cysteine-histidine-asparagine forms the active site [14]. The main polar interactions between the protein and inhibitor are well conserved involving the residues Gln19, Gly66, Asp161, His162, and Trp184 (residue numbers correspond to the cruzipain sequence) of the enzyme. Cz is a cathepsin L-like cysteine protease and is closely related to the mammalian CPs such as CatB, CatK, CatL, and CatS.

A variety of studies have been conducted on optimization strategies for the interactions of different classes of inhibitors with the S1, S2 and S3 binding sites of cruzain and related cysteine proteases [12,14,15,20]. Nonetheless, far less is known about the attainable interactions at S1′ for dipeptidyl nitrile inhibitors [21]. The high-resolution crystal structure of cruzain shows that there is a large open surface characterized by Trp177 in the primed binding site region (Fig 3) [22]. The design of compounds to exploit this cavity would provide enhanced enzyme-inhibitor interactions. This concept has already been applied for a class of different dipeptidic vinyl sulfone inhibitors [23]. As Fig 3 (left) exemplarily illustrates, the substituents of vinyl sulfone inhibitors predominantly sit on top of the shelf formed by residues Ser139, Met142 and Asp158 rather than adopting an orientation for an intense aromatic–aromatic interactions with Trp177. The nitrile inhibitor 33L does not bear an appropriate substituent that would allow for interaction with the primed binding region of cruzain (Fig 3, right).

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Fig 3. Crystal structures of vinyl sulfone derivative K777 and dipeptidyl nitrile 33L covalently bound to cruzain.

Left. K777 in the active site of cruzain (PDB-ID: 1F2B). Right 33L in the active site of cruzain (PDB-ID 4QH6).

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The nitrile warhead has been applied successfully for a variety of series of cathepsin inhibitors. Peptidic nitriles are known to interact with the active site cysteine by forming a covalent, but reversible thioimidate adduct [24]. The nitrile warhead was also repurposed for Cz inhibition as trypanocidal agents, displaying low toxicity, probably due to the reversible character of interaction [25]. Therefore, starting from our recent study on dipeptidyl nitriles as trypanocidal agents, we expanded our previous inhibitor series to map the S1/S1´ subsites of Cz [14]. By applying a knowledge-based design approach, we have explored different amino acids as possible building blocks for the P1 moiety. Based on a template crystal structure of the dipeptidyl nitrile inhibitor 33L bound to Cz (PDB ID: 4QH6), structural modifications have been executed that might increase the affinity towards the S1’ specificity pocket. Fig 4 shows dipeptidyl nitriles 50, 52, 56, and 58 with different lipophilic substitution patterns at the P1 position, which were assumed to accommodate the S1´ pocket through hydrophobic interactions without interfering in the general mode of binding.

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Fig 4. The putative orientation of P1 moieties in compounds 50, 52, 56, and 58.

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Possible interactions of inhibitors with residues forming the S1 and S1’ pockets of cruzain (PDB ID: 4QH6). Residue numbers are referring to the cruzipain sequence.

Compound 9 (Fig 5) was adopted as an archetype, with the cyclopropyl group at P1 position and phenylalanine in P2 as well as a pyrazole moiety in P3 for advantageous interactions with the non-primed binding region of the target protease. We mainly used different natural and unnatural amino acids for the P1 moiety and maintained the nitrile warhead. Leucine (Leu) and phenylalanine (Phe) were incorporated (compounds 50–54, Fig 6) as molecular sensors in P1 for aliphatic and aromatic interactions. 4-Pyridylalanine was implemented to leverage the affinity by polar interaction with Asp161 (S1´). Thr-O-Bzl, an unusual building block for peptide inhibitors, was used as a chimera in P1 for aliphatic and aromatic interactions in the S1/S1´ area. After removal of the benzyl protecting group from Thr-O-Bzl, the so produced alcoholic moiety should allow the evaluation of whether a hydrogen bond donor is tolerated in the S1/S1´ area. Moreover, it was intended to investigate how the stereochemistry in this region will influence the affinity with Cz.

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Fig 5. Structure representation of compounds 6–19.

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Fig 6. Structure of compounds 50–60 and 65–69.

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3-(tert-Butyl)-1-methyl-1H-pyrazole-5-carboxylic acid is a privileged building block applied for the inhibition of Cz and CatL [26]. Thus, we have explored some possible bioisosteres in order to increase the affinity and the selectivity towards Cz, such as 7-chloroquinoline carboxylic acid, 1H-indole-5-carboxylic acid, or 6-aminonicotinic acid.

Accordingly, we synthesized a new series of dipeptidyl nitriles (Figs 1 and 2). For compounds 6–12 and 14–19 bearing a cyclopropyl moiety in P1, the synthesis was carried out as known from the literature [15]. The peptide coupling reaction was performed twice, first to connect the enantiomerically pure, Boc-protected P2 amino acid with the aminonitrile moiety, and secondly, after removing the Boc group, to introduce the corresponding aroyl acids (Fig 1). Compound 19 was synthesized from compound 12 by removing the benzyl group under mild oxidative conditions (Fig 1) [27].

For the synthesis of compounds 50–60 (Fig 6), we have adopted a different synthetic strategy. In general, the desired dipeptidyl primary amide was synthesized, followed by the dehydration reaction to form the dipeptidyl nitrile. Due to the diversity of building blocks, it was necessary to evaluate different dehydrating reagents, aiming at the best yield and prevention of racemization. For compounds 65–68 the cleavage of the benzyl group was performed by hydrogenolysis before the conversion of the primary amide to the nitrile, while for compound 69, considering the lability of the chlorine atom under hydrogenolysis, we first transformed the primary amide to the nitrile and then removed the benzyl group under mild oxidative conditions [27]. The absolute geometry of the P1 group did not change, but, owing to CIP priority rules, the configuration at the α−carbon for the Thr-O-Bzl building block changed two times: (i) in the dehydration step to form the nitrile group and, (ii) when the catalytic cysteine attacks the carbon atom of the nitrile warhead to form a covalent bond (Fig 7).

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Fig 7. Change in stereochemistry for compounds bearing Thr or Thr-O-Bzl group in P1.

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Structure-activity relationships for inhibition of cysteine proteases by dipeptidyl nitriles

The pK_i values were determined for parasite cysteine proteases (Cz, LmCPB) and also for human cysteine cathepsins (CatB, CatK, CatL, CatS) and are reported in Table 1. Compounds 6, 8, 9 and 11 have already been described as competitive inhibitors that bind reversibly to Cz [14]. Several new compounds are Cz nanomolar inhibitors and exhibit good affinity for LmCPB, CatL, CatS and CatK (pK_i over 7.5). The application of such inhibitors extends to candidates for antiprotozoal action and as inhibitors of cysteine cathepsins.

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Table 1. Number identification, pK_i values for CatB, CatK, CatL, CatS, Cz and LmCPB.

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One crucial question is the cross-reactivity of CP inhibitors, for which an extrathermodynamic relationship can be formulated [28,29]. Ligand-target affinity involves enthalpy and entropy, which, in most cases, results in enthalpy-entropy compensation (EEC) [30]. The compensation effect should lead to an observable linear relationship between enthalpy and entropy when ligands differ by a single molecular modification. EEC is a fundamental extrathermodynamic phenomenon in medicinal chemistry and one of its grand challenges [31]. A thermodynamic analysis is commonly applied to a series of compounds that act on the same target. Here, the ligand is kept invariant while the targets are changed. Therefore, the nature of the ligand-target interaction governed by the thermodynamic parameter of the free energy change (via the estimation of the dissociation constant) results in extrathermodynamic relationships for the set of derivatives.

Hence, we investigated the degree of linear correlation between Cz and the other CPs by plotting the pK_i data against each other. The results (see S1 Fig) indicated an extrathermodynamic relationship between Cz and LmCPB, while this was not observed for all the other CPs. This finding highlights that the mode of inhibition for this series of compounds is similar for Cz and LmCPB, corroborating the fact that all the structural transformations of prototype compounds 9 and 11 affected the affinity towards the two protozoa CPs with the same magnitude (Figs 8 and 9). Therefore, known Cz inhibitors can be repurposed as LmCPB inhibitors, and the knowledge acquired in the design of Cz inhibitors can be translated into future LmCPB inhibition studies.

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Fig 8. SAR summary for P1-S1/S1´ interactions.

Values are reported as differences in pK_i and are color-coded as red (negative), green (positive), grey (no significant difference, ΔpK_i < 0.2).

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Fig 9. SAR summary starting from compound 11.

Values are reported as differences in pK_i and are color-coded as red (negative), green (positive), grey (no significant difference, ΔpK_i < 0.2).

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One common approach to SAR analysis is to examine ΔpK_i values associated with particular structural transformations, and these can be specified concisely using the square bracket notation previously described [32]. For example, the structural transformation of the phenylalanine in P2 of compound 6 to the corresponding 3-chloro-phenylalanine (8) can be noted as [6→8]. As already described for Cz [14], the exchange of benzoyl by 1-methyl-3-tert-butyl-pyrazolyl-carbonyl [6→9] led to a potency increase of 0.5 log units. Following this path, we inserted a meta-benzoic ester in the P3 position in the attempt to design a prodrug analog [15]. The transformation [6→16] unfortunately displayed a slight increment as compared to the transformation [6→13]. Hence, compound 9 (pK_i of 7.4 for Cz) has been used as a prototype for mapping S1-P1 interaction on different targets (Fig 8).

The effects on affinity resulting from stereochemical modifications in P1 of the structural prototype 9 are shown in Fig 8. In general, the stereochemistry of P1 moiety strongly influences the affinity towards all the CPs. The (S) → (R) conversion in [50→51] and [52→53] decrease the pK_i values for Cz and LmCP both by about one log unit. Likewise, the double stereochemical modification from (R,R) benzyl-protected threonine to the (S,S) diastereomer [56→57] led to a complete all-target affinity loss. Instead, the structural transformation from the cyclopropyl unit to CH attached with a benzyl group [9→50] resulted in a significant affinity increment for Cz (pK_i of 7.7) and LmCPB (pK_i of 7.8) of 0.4 and 0.7 log units, respectively. Replacement of the P1 cyclopropane linker with CH attached to isopropyl [9→52], 3-chlorobenzyl [9→54], or even 4-pyridyl [9→55] led to a small increase or no significant difference in affinity against those two protozoa enzymes. Moreover, the insertion of the benzyl protected threonine in P1 [9→56] and [9→57] decreased the affinity for Cz and LmCPB. Remarkably, replacement by the hydroxybutyl residues led to an almost one hundredfold affinity loss for both enzymes. In general, those results are corroborated by the docking studies which display a reasonable accommodation of the aromatic and hydrophobic moieties in the S1´ site without forming any specific interaction with the residues present in this area. While, for smaller moieties as cyclopropane or Thr-OH the interaction is manly in the interface between the S1 and the S1´. Primarily, the same trend in affinity was observed for the four mammalian CPs, when the structural modifications in P1 were realized starting from the prototype compound 9, as illustrated in Fig 8. Singularly, the introduction of a benzyl-protected threonine [9→50] resulted in a pK_i decrease of 0.3 log units towards CatB.

As recently described [14], the effects on affinity when replacing the P2 phenylalanine (9) with leucine (11) appears to depend on the substructural context, and this relates to non-additivity in the SAR. Accordingly, we used compound 11 as a starting prototype for another SAR considering P1, P2, and P3 for structural modifications, as summarized in Fig 9.

Substitution of the in P3 positioned 1-methyl-3-tert-butyl-pyrazole ring (11) with 7-chloro-quinoline (15), or 1H-indole (19) preserved the high affinity towards Cz and LmCPB, and, strikingly, this substitution led to a decrease of 1.0 and 0.5 in the pK_i value for CatL (Fig 9). Insertion of a basic moiety, i.e., 2-amino pyridine [11→18], produced a significant reduction of potency for Cz (-0.7) and LmCPB (-0.5). Compound 19 showed a high affinity for CatK (pK_i of 8.7) with a significant selectivity over the other mammalian CPs (Table 1). At position P2, the transformation of the leucine moiety into tryptophan, phenylalanine, or derivatives thereof resulted in an affinity loss of up to one log unit for Cz. A similar replacement led to a gain in affinity towards CatL and CatB, and it is consistent with previously reported data [33]. For compound 11, as for compound 9, the stereochemistry of the substituent in P1 was vitally for the bimolecular recognition process. Moreover, the transformation [11→58] kept the pK_i in the same range for Cz while increasing it by 0.4 log units for LmCPB.

Non-additivity in SAR is of considerable interest [34], and this is illustrated for the six cysteine proteases in Fig 10. Non-additivity can be quantified by comparing the ΔpK_i value resulting from a pair of substructural transformations with the sum of ΔpK_i values that result from the individual transformations. The Cz ΔpK_i values for [9→11] (0.5) and [9→56] (-1.0) shown in Fig 10A sum up to -0.5. Nevertheless, [9→58], which corresponds to the simultaneous application of the pair transformations, is associated with a ΔpK_i value increase of +0.6, thus indicating that the effects of this pair of transformations on Cz affinity are superadditive. The same was true for the effects of these two transformations on the other five CPs. Analogous analysis of the results in Fig 10B displays the effects of two transformations to be superadditive for the entire CP targets investigated herein. These results entail the P1-S1 and P1’-S1’ interactions to be driven by the molecular recognition in P2.

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Fig 10. Non-additivity of SAR.

Values are reported as differences in pK_i and are color-coded as red (negative), green (positive), grey (no significant difference, ΔpK_i< 0.2).

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Pairwise plots for the selectivity towards Cz in relation to other human cathepsins are provided in Fig 11. It is not trivial to achieve a significant selectivity for Cz inhibitors (ΔpK_i > 1.0) over mammalian CPs due to their high structural similarity of the active site. Undeniably, CatB has a different mode of binding due to the larger S2 and S3 pockets [33]. Compounds 15 (pK_i of 7.8 for Cz) and 67 (pK_i of 7.1) displayed a significant selectivity toward CatL (pK_i of 6.6) and CatS (pK_i of 5.0), respectively. The Cz selectivity in the case of compound 15 is driven by the S3-P3, while that of 67 is driven by S1-P1 interaction. Additionally, the hydrophobic interaction in S1 and S1´ with P1 of compounds 50 (pK_i of 7.7 for Cz), 54 (pK_i of 7.5 for Cz) and 60 (pK_i of 7.2 for Cz) resulted in a good selectivity over CatK (pK_i of 6.3, 6.3 and 6.0, respectively, for 50, 54 and 60). On the other hand, our study highlights two low nanomolar inhibitors of mammalian CPs: compounds 8 (pK_i of 8.6 for CatL) and 19 (pK_i of 8.7 for CatK) which showed an affinity difference of more than one log unit compared to the other mammalian cathepsins.

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Fig 11. Selectivity pairwise plots.

Values are given in pK_i. The X-axis represents the difference in pK_i for the same inhibitor for a pair of CPs. Y-axis represents the mean value of pK_i for the same inhibitor for a pair of CPs. The black dashed line highlights no selectivity. The magenta dashed line highlights a significant selectivity. Positive differences correspond to Cz pK_i values that are greater than those for CatB, CatK, CatL or CatS.

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Biological evaluation

All compounds synthesized were evaluated for their trypanocidal activity against the amastigote form of the Tulahuen T. cruzi strain, and the best results are presented in Table 2. A number of 15 compounds had pEC₅₀ values above 4 against T. cruzi amastigotes and three (52, 57 and 60) were equipotent to the gold standard benznidazole as trypanocidal agents. In particular, compounds 52 and 60 are both low nanomolar Cz inhibitors and one-digit nanomolar inhibitors for CatL. Compound 57 had no affinity for any of the six CPs reported herein, which excluded the possibility that its mechanism of action is similar to compound 52 and 60. Physiochemical properties (logP, MM, TPSA, LogS) play an important role in drug design. As well, for potential trypanocidal agents, which had been designed as protozoan cysteine proteases inhibitors, physicochemical properties can influence their outcome. Therefore, we have included TPSA, calculated logP (ilogP), and LogS (Ali_LogS) in this discussion (Figs 12, 13 and 14) [35].

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Table 2. Biological data for trypanocidal activity (EC₅₀), cytotoxicity (CC₅₀), and selective index (SI) for the series of dipeptidyl nitriles.

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Fig 12. Schematic representation of physicochemical properties and SARs for trypanocidal activity.

EC₅₀ calculated for amastigote forms of T. cruzi (Tuhaluen strain). CC₅₀ calculated for the LLCMK2 strain (host cell). Green areas highlight biochemical results. TPSA, ilogP, and Ali_Logs have been calculated with the swissADME online service [35]. pK_i values are referring to Cz inhibition.

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Fig 13. Schematic representation for non-additivity of SARs for trypanocidal activity.

EC₅₀ calculated for amastigote forms of T.cruzi (Tuhaluen strain). CC₅₀ calculated for the LLCMK2 strain (host cell). Green areas highlight biochemical results. TPSA, ilogP, and Ali_Logs have been calculated with the swissADME online service [29]. pK_i values are referring to Cz inhibition.

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Fig 14. Schematic representation for non-additivity of SARs for compounds 58, 59, 67 and 68.

EC₅₀ calculated for amastigote forms of T.cruzi (Tuhaluen strain). CC₅₀ calculated for the LLCMK2 strain (host cell). Green areas highlight biochemical results. TPSA, ilogP, and Ali_Logs have been calculated with the swissADME online service [35]. pK_i values are referring to Cz inhibition.

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In general, the substitution of the P3 or P2 moieties from the prototype compound (9) did not result in any increment of potency, except in the case of compound 12 (Fig 12). Modification in P1 strongly modulated the trypanocidal effect. Substitution of Phe for (S,R)-Thr-O-Bn led to a modest trypanocidal activity. If we consider the physicochemical properties of the whole set of compounds, there is no consistent correlation with their trypanocidal activities. However, when considering discontinuity in small sets of compounds related to their matched molecular pairs (MMP) [36], we observed, in some specific cases, that modulation in compound lipophilicity yields to high trypanocidal potency. For instance, the existence of the molecular pair between compounds 52 (EC₅₀ = 4.1 μM; ilogP = 2.3) and 9 (EC₅₀ = 71.8 μM; ilogP = 3.2) suggests that the 17.5-fold increase in potency in favor of 52 may be due to its reduced lipophilicity. In addition, single modification in P2 or P1 of compound 9 did not always produce a beneficial effect for the trypanocidal activity. However, considering both modifications [960], compound 60 (EC₅₀ = 4.9 μM) exhibited a 14.5 times higher trypanocidal potency than compound 9 (EC₅₀ = 71.8 μM). Compound 60 displayed activity against the parasite similar to that observed for benznidazole–EC₅₀ of 4.4 (Fig 13). Interestingly, the ilogP of 4.1 for 60 was the highest among the entire series and more than one log unit larger than that of compound 52. Too, compound 57, which also has ilogP of 4.1, is equipotent to compound 52 whose ilogP is only 2.3! It is noteworthy that compound 57, exhibiting the same values for TPSA and ilogP as 60 (a high affinity cruzain inhibitor), had no affinity for any of the CPs studied herein. Assumably, compound 57 has a different mode of action than 60. Furthermore, inhibitor 57 (CC₅₀ ≃ 124.7 μM) showed the highest selectivity and lowest cytotoxicity in our assays. This guarantees an SI (selective index) ratio of over 20, as well as for compounds 60 and 52, making them appealing targets for further in vivo testing against the acute form of Chagas disease [36].

Compounds bearing a leucine moiety in P2 displayed a peculiar behavior (Fig 14). Indeed, the debenzylation of the threonine moiety in P1 led to an increase in potency [58→66] and [59→67]. The trypanocidal potency for this set of compounds seemed not to correlate directly with Cz affinity; but, once again, active compounds had an ilogP value of less than 3.0.

Potential cytotoxicity of inhibitors was assessed with the LLCMK₂ cell-based assay, and compounds were evaluated over three days using benznidazole as a control. Cytotoxicity at the highest concentration tested that did not lead to precipitation (250 μM) was low for the majority of test compounds. The most potent inhibitors of the amastigote T. cruzi Tulahuen strains (52, 57 and 60) showed the same range of cytotoxicity when compared to benznidazole [37]. Based on the data obtained here and supported by previous reports [14,38], the nitrile warhead introduced to target protozoan cysteine protease yields a low cytotoxicity profile. At the same time, our data reflect the difficulty in the translation of biochemical assay results (highlighted here by Cz inhibitors) to trypanocidal action. Since observable in a multitude of systems, this phenomenon is not limited to dipeptidyl nitrile inhibitors, but also known for other compound classes [12,39] targeting the amastigote form of the parasite. The failure of some Cz inhibitors to affect the intracellular amastigote form might be attributed to (i) the interference with the autoproteolytic cruzain activity, (ii) the necessity of crossing two membranes and (iii) the differences of Cz isoforms, e.g., cruzipain 2, with respect to pH stability, substrate specificity, and sensitivity to inhibition by natural and synthetic inhibitors [40]. Hence, even though Cz inhibition is efficient, the parasite would still survive thanks to the action of cruzipain 2. Further insights would be necessary to specify the importance of different cysteine protease forms in the process of target validation for Chagas disease.

Discussion

In this study, we expanded our previous series of dipeptidyl nitrile inhibitors of Cz by leveraging the P1-S1/S1’ interaction. Our synthetic route was feasible for a variety of nitrile-based peptidic inhibitors. In particular, we spotted the Thr-O-Bn moiety in P1 as a malleable building block for the synthesis of such cysteine protease inhibitors. We studied how the P1-S1/S1’ interaction can influence affinity and selectivity for two protozoa (Cz and LmCPB) and four mammalians (CatB, CatK, CatL and CatS) CPs. Furthermore, 15 compounds had pEC₅₀ values in the range of 4.1–5.4 against the T. cruzi amastigote form (pEC₅₀ = 5.4 for benznidazole control drug). Three of them (52, 57 and 60) are equipotent (pEC₅₀ 5.3–5.4) with benznidazole as trypanocidal agents with an SI of over 20, making them attractive targets for further in vivo testing against the acute form of Chagas disease. Our work also contributes to the perception of the extrathermodynamic relationship in ligand affinity between Cz and LmCPB, which can be used in LmCPB inhibitor development targeting Leishmania mexicana. In this study, we disclosed one strong inhibitor (14) of Cz with high selectivity over CatL and two low nanomolar inhibitors of CatL and CatK (compounds 8 and 18) with more than one log unit selectivity over the other mammalian cysteine proteases. In conclusion, our work contributes to the understanding of subtle drug-target interactions and to the discovery of tailored trypanocidal agents equipotent to benznidazole.

Supporting information

S1 Fig. Extrathermodynamic relationship.

Plot of pK_i (Cz) vs. pK_i (LmCPB). A linear trendline fitted points.

https://doi.org/10.1371/journal.pntd.0007755.s001

(PDF)

S2 Fig. Curves dose-response.

Dose curve response for determination of CC₅₀ (LLCMK₂) and EC₅₀ (T. cruzi Tulahuen) for all compounds.

https://doi.org/10.1371/journal.pntd.0007755.s002

(PDF)

S3 Fig. Characterization of final compounds.

¹H, ¹³C NMR spectra and HPLC reports for final compounds.

https://doi.org/10.1371/journal.pntd.0007755.s003

(PDF)

S1 Table. Biological data for trypanocidal activity.

Number identification, Nequimed number, biological data for trypanocidal activity (EC₅₀) and citotoxicity (CC₅₀).

https://doi.org/10.1371/journal.pntd.0007755.s004

(PDF)

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Figures

Abstract

Author summary

Introduction

Methods

Modeling

Synthetic chemistry

General considerations.

General procedure for amide synthesis.

General procedure for removal of the Boc protecting group.

General procedure for dehydration of primary amides to nitriles.

General procedure for removal of the benzyl protecting group.

Synthesis and characterization of compound 1–5.

(S)-tert-Butyl (1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (1).

(S)-tert-Butyl (3-(3-chlorophenyl)-1-((1-cyanocyclopropyl)amino)-1-oxopropan-2-yl)carbamate (2).

(S)-tert-Butyl (1-((1-cyanocyclopropyl)amino)-1-oxo-3-(pyridin-4-yl)propan-2-yl)carbamate (3).

(S)-tert-Butyl (1-((1-cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (4).

tert-Butyl ((2S,3R)-3-(benzyloxy)-1-((1-cyanocyclopropyl)amino)-1-oxobutan-2-yl)carbamate (5).

Synthesis of compounds 6–18.

(S)-N-(3-(3-Chlorophenyl)-1-((1-cyanocyclopropyl)amino)-1-oxopropan-2-yl)benzamide (7).

(S)-3-(tert-Butyl)-N-(3-(3-chlorophenyl)-1-((1-cyanocyclopropyl)amino)-1-oxopropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (8).

(S)-3-(tert-Butyl)-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-(pyridin-4-yl)propan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (10).

N-((2S,3R)-3-(Benzyloxy)-1-((1-cyanocyclopropyl)amino)-1-oxobutan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (12).

(S)-7-Chloro-N-(1-((1-cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)quinoline-4-carboxamide (13).

(S)-7-Chloro-N-(1-((1-cyanocyclopropyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)quinoline-4-carboxamide (14).

(S)-7-Chloro-N-(1-((1-cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)quinoline-4-carboxamide (15).

(S)-3-((1-((1-Cyanocyclopropyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)phenyl benzoate (16).

(S)-6-Amino-N-(1-((1-cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)nicotinamide (17).

(S)-N-(1-((1-Cyanocyclopropyl)amino)-4-methyl-1-oxopentan-2-yl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (18).

Synthesis and characterization of compound 19.

3-(tert-Butyl)-N-((2S,3R)-1-((1-cyanocyclopropyl)amino)-3-hydroxy-1-oxobutan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (19).

Synthesis of compounds 20–27.

(S)-tert-Butyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate (20).

(R)-tert-butyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate (21).

(S)-tert-Butyl (1-amino-4-methyl-1-oxopentan-2-yl)carbamate (22).

(R)-tert-Butyl (1-amino-4-methyl-1-oxopentan-2-yl)carbamate (23).

(S)-tert-Butyl (1-amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)carbamate (24).

(S)-tert-Butyl (1-amino-1-oxo-3-(pyridin-4-yl)propan-2-yl)carbamate (25).

tert-Butyl ((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)carbamate (26).

tert-Butyl ((2R,3S)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)carbamate (27).

Synthesis of compounds 28–38.

tert-Butyl ((S)-1-(((S)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (28).

tert-Butyl ((S)-1-(((R)-1-amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (29).

tert-Butyl ((S)-1-(((S)-1-amino-4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (30).

tert-Butyl ((S)-1-(((R)-1-amino-4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (31).

tert-Butyl ((S)-1-(((S)-1-amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (32).

tert-Butyl ((S)-1-(((S)-1-amino-1-oxo-3-(pyridin-4-yl)propan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (33).

tert-Butyl ((S)-1-(((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (34).

tert-Butyl ((S)-1-(((2R,3S)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (35).

tert-Butyl ((S)-1-(((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (36).

tert-Butyl ((S)-1-(((2R,3S)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (37).

tert-Butyl ((S)-1-(((2S,3R)-1-amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-3-(3-chlorophenyl)-1-oxopropan-2-yl)carbamate (38).

Synthesis of compounds 39–49.

N-((S)-1-(((S)-1-Amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (39).

N-((S)-1-(((R)-1-Amino-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (40).

N-((S)-1-(((S)-1-Amino-4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (41).

N-((S)-1-(((S)-1-Amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (42).

tert-Butyl ((S)-1-(((S)-1-amino-3-(3-chlorophenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (43).

N-((S)-1-(((S)-1-Amino-1-oxo-3-(pyridin-4-yl)propan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (44).

N-((S)-1-(((2S,3R)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (45).

N-((S)-1-(((2R,3S)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (46).

N-((S)-1-(((2S,3R)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-3-(tert-butyl)-1-methyl-1H3-pyrazole-5-carboxamide (47).

N-((S)-1-(((2R,3S)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-3-(tert-butyl)-1-methyl-1H3-pyrazole-5-carboxamide (48).

N-((S)-1-(((2S,3R)-1-Amino-3-(benzyloxy)-1-oxobutan-2-yl)amino)-3-(3-chlorophenyl)-1-oxopropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (49).

Synthesis of compounds 50–60.

3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (50).

3-(tert-Butyl)-N-((S)-1-(((R)-1-cyano-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (51).

3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-3-methylbutyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (52).

3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-3-methylbutyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (53).

3-(tert-Butyl)-N-((S)-1-(((S)-2-(3-chlorophenyl)-1-cyanoethyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (54).

3-(tert-Butyl)-N-((S)-1-(((S)-1-cyano-2-(pyridin-4-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-methyl-1H-pyrazole-5-carboxamide (55).

N-((S)-1-(((1R,2R)-2-(Benzyloxy)-1-cyanopropyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (56).

N-((S)-1-(((1S,2S)-2-(Benzyloxy)-1-cyanopropyl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (57).

N-((S)-1-(((1R,2R)-2-(Benzyloxy)-1-cyanopropyl)amino)-4-methyl-1-oxopentan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (58).

N-((S)-1-(((1S,2S)-2-(Benzyloxy)-1-cyanopropyl)amino)-4-methyl-1-oxopentan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (59).

N-((S)-1-(((1R,2R)-2-(Benzyloxy)-1-cyanopropyl)amino)-3-(3-chlorophenyl)-1-oxopropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide (60).

Synthesis of compounds 61–64.

N-((S)-1-(((2S,3R)-1-Amino-3-hydroxy-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide.(61).

N-((S)-1-(((2R,3S)-1-Amino-3-hydroxy-1-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide.(62).

N-((S)-1-(((2S,3R)-1-Amino-3-hydroxy-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)-3-(tert-butyl)-1-methyl-1H-pyrazole-5-carboxamide(63).