We describe the synthesis of fluorogenic arylureas and amides and their interaction with primary or secondary amines under air and light in organic-aqueous mixtures to give rise to a new class of persistent organic radicals, described on the basis of their electron paramagnetic resonance (EPR), as well as UV–vis, fluorescence, NMR, and quantum mechanics calculations, and their prospective use as multi-signal reporters in a smart label for fish freshness. Persistent organic radicals are of immense importance for the preparation of new hybrid materials with spin-probe and spin-label functions , magnetiThe synthesis of arylureas used in this study was performed using an easy Suzuki reaction between 5-bromoindanone and 4-aminobenzeneboronic acid pinacol ester, followed by a Knoevenagel reaction with malononitrile and a reaction with selected mono- and bis-isocyanates . To comp1–7 were prepared  from ecstasy tablets • was optimized by using the Becke three-parameter functional •, and therefore, the radical species that could form from the amine were reversed in the presence of water and air compared to the neutral amine, which appeared unchanged in all NMR experiments.From previous studies and the new experiments, we assumed that compound transfer  from thetransfer , giving 1 was measured in DMSO in the presence of pyrrolidine, used as a reference, and then histamine, putrescine, or cadaverine, immediately after the addition of the amine. The reference was quinine sulfate in H2SO4 (0.05 M)  . The resScomber scombrus) samples were bought in a local market, the fish were cleaned, the spines were extracted, and the muscle was ground (Philips HR 28308/B). Then, fish samples of 10 ± 0.2 g were placed in 20 mL vials and sealed with a septum. Half of the samples were incubated at 4 °C, and the other half were incubated at 25 ± 1 °C. Then, 3 mL of the gas phase of the vial was taken with a syringe and bubbled into a probe solution at different times as the fish decayed over time. The absorbance and fluorescence of the samples were measured at their maximum peaks, and the differences between the obtained values and a reference solution were plotted  urea1. 5-(4-Aminophenyl)indan-1-one: Pd(PPh3)4  was added under nitrogen to a solution of 5-bromoindanone  in a toluene/butanol/water 4:1:0.4 mixture (50 mL), and the mixture was stirred for 30 min at room temperature. Then, solid 4-aminobenzeneboronic pinacol ester  and Na2CO3  were added, and the mixture was heated under reflux for 16 h. Then, the solvent was evaporated under reduced pressure, and the residue was added to water (20 mL) and extracted with CH2Cl2 (5 × 40 mL). The combined organic extracts were dried (Na2SO4) and evaporated, and the residue was purified by flash chromatography , CH2Cl2, to obtain 5-(4-aminophenyl)indan-1-one  as a pale yellow solid, mp 250–251 °C. IR : 3429, 3335, 1678 (C=O), 1592, 1297, 1278, 811. 1H NMR  δ: 7.71 , 7.60 , 7.48 , 6.68 , 5.44 , 3.10 , 2.62 . 13C NMR and DEPT  δ: 205.44 (C=O), 156.14 (C-NH2), 149.53, 146.91, 134.05, 127.90 (CHar), 125.84, 124.60 (CHar), 123.25 (CHar), 122.79 (CHar), 114.13 (CHar), 36.10 (CH2), 25.44 (CH2). MS (EI) m/z (%): 224 , 223 , 195 (18), 167 (8), 152 (9), 139 (4), 117 (7), 96 (3). HRMS (EI): calcd. for C15H13NO: 223.0997 (M+); found: 223.0998. 1-Dicyanomethylene-5-(4-aminophenyl)indane: Malononitrile  and DABCO  were added under nitrogen to 5-(4-aminophenyl)indan-1-one  dissolved in toluene (50 mL). The mixture was heated under reflux for 8 h and then added to water (30 mL), stirred for 5 min, and extracted with dichloromethane (5 × 30 mL). The combined organic extracts were dried (Na2SO4), and the solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography , from CH2Cl2 to CH2Cl2:AcOEt (15:1), to obtain 1-dicyanomethylene-5-(4-aminophenyl)indene  as an orange solid, mp: >250 °C (decomp.), IR : 3491, 3382, 2221 (CN), 1627, 1600, 1561 (Car-Car). 1H NMR  δ: 8.15 , 7.75 , 7.54 , 6.66 , 5.62 , 3.23 , 3.11 . 13C NMR and DEPT  δ: 179.83 (C=C(CN)2), 156.43, 150.35, 147.42, 132.53, 128.20 (CHar), 125.34 (CHar), 124.90 (CHar), 124.72, 121.69 (CHar), 114.36 (CN), 114.15 (CHar), 113.95 (CN), 69.86 (C(CN)2), 34.65 (CH2), 29.41 (CH2). MS (EI) m/z (%): 272 , 271 , 243 (3), 214 (2), 206 (1), 204 (2), 189 (2), 135 (2). HRMS (EI): calcd. for C18H13N3: 271.1109 (M+); found: 271.1108. 1-(4-methoxyphenyl-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea 1: 1-Dicyanomethylene-5-(4-aminophenyl)indane  was added to 4,4′-diisocyanatodiphenylether  dissolved in CHCl3 (20 mL), and the mixture was stirred at room temperature for 48 h. Cyclohexane (4 mL) was then added, and the solid formed was filtered off, washed with CHCl3 (2 × 4 mL), and dried. Compound 1-(4-methoxyphenyl-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea 1  was obtained as a dark yellow solid, mp: >250 °C. IR : 3316, 2221 (C≡N), 1654 (C=O), 1598, 1557, 1510, 1239, 1184, 827. 1H NMR  δ: 8.90 , 8.73 , 8.25 , 7.91 , 7.87 , 7.77 , 7.61 , 7.46 , 6.96 , 3.30 , 3.19 . 13C NMR and DEPT  δ: 178.08 (C=C(CN)2), 156.27, 152.46, 151.95, 146.39, 140.92, 134.93, 133.73, 131.31, 127.81 (CHar), 125.95 (CHar), 125.40 (CHar), 123.26 (CHar), 120.11 (CHar), 118.82 (CHar), 118.41 (CHar), 114.09 (CN), 113.67 (CN), 71.23 (C(CN)2), 34.71 (CH2), 29.51 (CH2). MS (ESI) m/z (%): 795 . HRMS (ESI): calcd. for C50H34N8O3 + H+: 795.2827 (M+ + 1); found: 795.2832.Synthesis of 1,3-bisurea3. 1-Dicyanomethylene-5-(4-aminophenyl)indane  was added to p-methoxyphenyl isocyanate  dissolved in CHCl3 (10 mL), and the mixture was stirred at room temperature for 48 h. Cyclohexane (3 mL) was then added, and the solid formed was filtered off, washed with CHCl3 (2 × 2 mL), and dried. Compound 1-(4-methoxyphenyl-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea 3  was obtained as a dark yellow solid, mp: 232–233 °C. IR : 3320, 2221 (CN), 1658 (C=O), 1596, 1557, 1510, 1239, 1184, 1029, 823. 1H NMR  δ: 8.85 , 8.56 , 8.23 , 7.90 , 7.86 , 7.76 , 7.58 , 7.37 , 6.87 , 3.82 , 3.28 , 3.18 . 13C NMR and DEPT  δ: 180.04 (C=C(CN)2), 156.22, 154.52, 152.47, 146.33, 141.02, 133.63, 132.44, 131.05, 127.74 (CHar), 125.86 (CHar), 125.34 (CHar), 123.15 (CHar), 120.07 (CHar), 118.23 (CHar), 114.06 (CN), 113.94 (CHar), 113.63 (CN), 71.12 (C(CN)2), 55.13 (OCH3), 34.63(CH2), 29.45 (CH2). MS (EI) m/z (%): 421 , 420 , 297 (100), 271 (87), 149 (17), 123 (31). HRMS (EI): calcd. for C26H20N4O2: 420.1586 (M+); found: 420.1582.Synthesis of 1-[4-phenyl]-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea4. 1-Dicyanomethylene-5-(4-aminophenyl)indane  was added to p-N,N-dimethylaminophenyl isocyanate  dissolved in CHCl3 (10 mL), and the mixture was stirred at room temperature for 48 h. Cyclohexane (3 mL) was then added, and the solid formed was filtered off, washed with CHCl3 (2 × 2 mL), and dried. Compound 1-[4-phenyl]-3-[4-(1-(dicyanomethylene-indan-5-yl)phenyl]urea 4  was obtained as a dark yellow solid, mp: 178–179 °C. IR : 3320, 2221 (C≡N), 1654 (C=O), 1596, 1565, 1518, 1320, 1242, 1185, 823. 1H NMR  δ: 8.81 , 8.41 , 8.25 , 7.92 , 7.88 , 7.76 , 7.60 , 7.29 , 6.72 , 3.30 , 3.20 , 2.84 . 13C NMR and DEPT  δ: 180.06 (C=C(CN)2), 156.49, 152.63, 146.60, 141.28, 133.67, 130.96, 129.26, 127.80 (CHar), 125.89 (CHar), 125.42 (CHar), 123.16 (CHar), 120.32 (CHar), 118.22 (CHar), 117.54, 114.15 (CN), 113.73 (CN), 113.16 (CHar), 71.07 (C(CN)2), 40.64 (N(CH3)2), 34.61 (CH2), 29.43 (CH2). MS (EI) m/z (%): 433 , 297 (100), 271 (92), 162 (35), 136 (37). HRMS (EI): calcd. for C27H23N5O: 433.1903 (M+); found: 433.1907.Synthesis of 1-(4-methylthiophenyl)-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea5. 1-Dicyanomethylene-5-(4-aminophenyl)indane  was added to p-methylthiophenyl isocyanate  dissolved in CHCl3 (10 mL), and the mixture was stirred at room temperature for 48 h. Cyclohexane (3 mL) was then added, and the solid formed was filtered off, washed with CHCl3 (2 × 2 mL), and dried. Compound 1-(4-methylthiophenyl)-3-[4-(1-(dicyanomethyleneindan-5-yl)-phenyl]urea 5  was obtained as a dark yellow solid, mp: 209–210 °C. IR : 3317, 2221 (CN), 1654 (C=O), 1596, 1546, 1495, 1324, 1281, 1192, 827. 1H NMR  δ: 8.91 , 8.77 , 8.25 , 7.91 , 7.87 , 7.77 , 7.60 , 7.44 , 7.23 , 3.30 , 3.19 , 2.44 . 13C NMR and DEPT  δ: 180.05 (C=C(CN)2), 156.23, 152.28, 146.35, 140.80, 137.20, 133.72, 131.36, 130.17, 127.78 (CHar), 127.70 (CHar), 125.94 (CHar), 125.38 (CHar), 123.25 (CHar), 119.06 (CHar), 118.42 (CHar), 114.06 (CN), 113.64 (CN), 71.23 (C(CN)2), 34.69 (CH2), 29.50 (CH2), 15.94 (CH3). MS (EI) m/z (%): 436 , 297 (100), 271 (93), 165 (32). HRMS (EI): calcd. for C26H20N4OS: 436.1358 (M+); found: 436.1359.Synthesis of N,N-bis-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]pyridine-2,6-dicarboxamide6. 2,6-Pyridinedicarbonyl dichloride  dissolved in dichloromethane (10 mL) was added dropwise to a chilled and stirred solution of 1-dicyanomethylene-5-(4-aminophenyl)indane  and N,N-diisopropylethylamine  in dichloromethane (50 mL). The mixture was left to warm to room temperature and stirred for 24 h. The solid formed was filtered off, washed with CHCl3 (2 × 3 mL), and dried. Compound N,N-bis-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]pyridine-2,6-dicarboxamide 6  was obtained as a dark yellow solid, mp: >250 °C. IR : 3344, 2221 (C≡N), 1682 (C=O), 1565, 1526, 1340, 1188, 819. 1H NMR  δ (ppm): 10.79 , 8.51 , 8.42 , 8.16 , 8.10 , 7.72 , 3.30 , 3.24 . 13C NMR and DEPT  δ: 179.95 (C=C(CN)2), 161.74, 156.13, 148.69, 146.00, 139.99 (CHar), 139.00, 134.04, 133.80, 127.65 (CHar), 127.05, 126.20 (CHar), 125.35 (CHar), 123.61 (CHar), 121.09 (CHar), 113.93 (CN), 113.52 (CN), 71.50 (C(CN)2), 34.65 (CH2), 29.48 (CH2). MS (ESI) m/z (%): 674 . HRMS (EI): calcd. for C43H27N7O2 + H+: 674.2299 (M+); found: 674.2295.Synthesis of 5,5′-bis)bis)bis(indan-1-one)7. 6-(Piperidin-1-yl)--2′,6′-dicarboxylic acid: Pd(PPh3)4  was added under nitrogen to a solution of dimethyl 4-bromopyridine-2,6-dicarboxylate  in tetrahydrofuran (120 mL), and the mixture was stirred for 30 min at room temperature. Then, solid 2-(piperidin-1-yl)-5-pyridine  and cesium carbonate  dissolved in water (15 mL) were successively added, and the mixture was heated under reflux for 24 h. Then, the solvent was evaporated under reduced pressure, and the residue was suspended in water (10 mL). Then, NaOH solution (5% w/v in H2O) was added dropwise until pH = 10, and the mixture was dissolved in tetrahydrofuran (60 mL) and heated under reflux for 1 h until complete hydrolysis of the diester and monoester mixture. Then, the mixture was chilled in an ice bath, and HCl solution (35% w/v in water) was added dropwise until pH = 1. Then, the solvent was evaporated until reduced pressure was achieved, and the solid residue was dried under vacuum. The solid residue was suspended in anhydrous dichloromethane (125 mL) under nitrogen, oxalyl chloride  and anhydrous dimethylformamide (0.2 mL) were added, and the mixture was stirred overnight. Then, the solvent and the excess of oxalyl chloride were evaporated under reduced pressure, anhydrous methanol  in dichloromethane (60 mL) was added under nitrogen, and the mixture was stirred overnight. Then, the solvent was evaporated under reduced pressure, and the residue was purified by flash chromatography , from CH2Cl2 to CH2Cl2/MeOH 9:1 v/v, to obtain dimethyl 6-(piperidin-1-yl)-2′,6′-dicarboxylate as a light yellow solid , mp: 146–148 °C, Rf : 0.83; 1H RMN : δ = 8.63 ; 8.47 ; 7.87–7.83 ; 6.75–6.72 ; 4.04 ; 3.66 ; 1.68 ; 13C RMN : δ = 165.4 (C=O), 159.6 (CAr), 148.7 (CHAr), 148.6 (CHAr), 147 (CHAr), 135.5 (CAr), 123.6 (CHAr), 119.1 (CAr), 106.5 (CHAr), 53.4 (CH3), 45.9 (CH2), 25.5 (CH2), 24.7 (CH2); MS (EI) m/z (%): 355 , 326 (55), 312 (30), 299 (20), 272 (32), 84 (28); HRMS (EI): calcd. for C19H21N3O4: 355.1532; found 355.1535. The diester  was dissolved in a mixture of tetrahydrofuran (10 mL) and water (1 mL), a solution of NaOH (5% w/v in H2O) was added dropwise until pH = 10, and the mixture was heated under reflux for 1 h until complete hydrolysis of the diester. Then, the mixture was chilled in an ice bath, and HCl solution (35% w/v in water) was added dropwise until pH = 1. Then, the solvent was evaporated until reduced pressure was achieved, and the solid residue was extracted several times with dichloromethane/methanol 1:1 until complete extraction of the diacid. Evaporation of the solvent under reduced pressure afforded pure 6-(piperidin-1-yl)--2′,6′-dicarboxylic acid as a light yellow solid , mp: 244–246 °C (decomp.); 1H RMN : δ = 8.49 ; 8.43 ; 8.28 ; 8.14–8.11 ; 7.10–7.07 ; 3.66 ; 1.69 ; 13C RMN : δ = 167.4 and 166.4 (C=O), 150.8 (CAr), 149.9 (CHAr), 140.2 (CHAr), 131.2 (CHAr), 128.8 (CAr), 124.7 (CHAr), 121.1 (CAr), 111.4 (CHAr), 26.6 (CH2), 25.2 (CH2); IR (KBr): 3609 (OH), 2786, 2167, 1648, 1340, 1108, 752 cm−1; MS (EI) m/z (%): 327 , 283 (92), 239 (45), 157 (41), 84 (100). HRMS (EI): calcd. for C17H17N3O4 + H+: 328.1292; found: 328.1291. 7: Oxalyl chloride  was added to a suspension of 6-cyclohexyl--2′,6′-dicarboxylic acid  in CH2Cl2 (100 mL) with three drops of DMF as a catalyst. The mixture was stirred at room temperature for 24 h, and then the solvent and the excess of oxalyl chloride were evaporated under reduced pressure. A solution of 1-dicyanomethylene-5-(4-aminophenyl)indane  in DMF (55 mL) was added over the white solid in an ice bath. Then, N,N-diisopropylethylamine  and a spatula tip of 4-dimethylaminopyridine (DMAP) were added, and the mixture was stirred at 0 °C for 15 min and then at room temperature for 24 h. A yellow precipitate formed that was filtered off. The filtered solid was washed with CHCl3 (3 × 10 mL) and CH2Cl2 (3 × 5 mL). Finally, the solid was collected, and traces of the solvent were evaporated under reduced pressure to obtain 5,5′-bis)bis)bis(indan-1-one) 7  as an orange solid, almost insoluble in all common solvents, mp: 212-213 °C (decomp.); IR : 3626 (NH), 3069, 2360, 2218, 1648 (C=O), 1558, 1527, 1337, 1188, 1122, 1005 cm−1; 1H NMR  δ: 11.21 , 8.72 , 8.61 , 8.31 , 8.21 , 8.17 , 8.00 , 7.95 , 7.08 , 3.70 ), 3.35 , 3.24 , 1.68 , 1.61 ; MS (FAB) m/z (%): 833 , 834 (4), 835 (10), 836 (6), 837 (3). HRMS (EI): calcd. for C53H39N9O2 + H+: 834.3299; found: 834.3298.5,5′-bis)bis)bis(indan-1-one) In conclusion, we describe a rare class of persistent radicals formed through the interaction of fluorogenic arylurea derivatives and primary or secondary amines in the presence of light, water, and air. The most persistent species of the series was studied using spectroscopic techniques as well as quantum mechanics calculations. All results pointed to a rare case of photoinduced proton-coupled electron transfer, although the slow kinetics of formation and the evolution of the radical species from a formerly fluorescent transient intermediate rendered the system more complicated than classic examples. The two best examples of the formation of radicals having a well-resolved EPR hyperfine structure and high intensity came from a well-known selective fluorogenic probe for biogenic amines or a fluorescent probe for ω-amino acids, therefore adding a new perspective to these and other to-be-obtained fluorescent probes. These chemical probes are the first examples of multi-signal reporters for the discrimination between primary, secondary, and tertiary amines in which the usual UV–visible or fluorescent signal is complemented by paramagnetic signals of the same samples at different reporting times. The selectivity of the chemical probes for different types of amines and the large types of signals offered by these probes, now including paramagnetic species, make them good starting points for the preparation of multi-signaling chemical probes for the detection of important organic metabolites. As an immediate application, we developed a multi-signal smart label for the quick inspection of freshness in fish.


Due to solubility problems, reactions must be carried out either in THF using tetrabutylammonium fluoride (TBAF) or in DMF using cesium fluoride (CsF), respectively. The reactive sulfurizing reagents are in situ generated, nucleophilic fluoropolysulfide anions FS(8−x)−, which react with the C=S bond according to the carbophilic addition mode. Dithiiranes formed thereby, existing in an equilibrium with the ring-opened form  are key-intermediates, which undergo either a step-wise dimerization to afford 1,2,4,5-tetrathianes or an intramolecular insertion, leading in the case of thioxo derivatives of 2,2,4,4-tetramethylcyclobutane-1,3-dione to ring enlarged products. In reactions catalyzed by TBAF, water bounded to fluoride anion via H-bridges and forming thereby its stable hydrates is involved in secondary reactions leading, e.g., in the case of 2,2,4,4-tetramethyl-3-thioxocyclobutanone to the formation of some unexpected products such as the ring enlarged dithiolactone and ring-opened dithiocarboxylate. In contrast to thioketones, the fluoride anion catalyzed sulfurization of their α,β-unsaturated analogues, i.e., thiochalcones is slow and inefficient. However, an alternative protocol with triphenylphosphine (PPh3) applied as a catalyst, offers an attractive approach to the synthesis of 3H-1,2-dithioles via 1,5-dipolar electrocyclization of the in situ-generated α,β-unsaturated thiocabonyl S-sulfides. All reactions occur under mild conditions and can be considered as attractive methods for the preparation of sulfur rich heterocycles with diverse ring-size.Fluoride anion was demonstrated as a superior activator of elemental sulfur (S Under these conditions, 3-thioxo-1,2-dithiole 9 was obtained as the product of a cascade reaction and isolated in good yield (84%). A series of analogous Se-heterocycles was also obtained using elemental selenium instead of sulfur -cycloaddition reactions.Unlike thioketones, similar sulfurization of αlusively  (Scheme 1a) and cycloaliphatic thioketones 2a–2c. In extension of the study with thioketones, first sulfurization reactions of selected thiochalcones 3a–3d were carried out by using elemental sulfur (S8) in the presence of a nucleophilic catalyst. Elucidation of the mechanisms of the studied reactions, leading to the formation of sulfur-rich heterocycles with diverse ring size, was of primary interest.The main goal of the present study was examination of the utility of the fluoride anion as a new type of a catalyst for sulfurization of thiobenzophenone (1a) and cycloaliphatic thioketones 2a–2c upon treatment with catalytic amounts of TBAF in THF solution were reported  (Entry A) led to a mixture of the isomeric thiolactone 14 (63% yield) and the ring enlarged five-membered thiolactone 16a (11%)  , Table 11H-NMR spectrum eight signals with approximately the same intensities located at 1.32, 1.35, 1.51, 1.51, 1.69, 1.71, 1.74, and 1.75 ppm. More important information was found in the 13C-NMR spectrum, which revealed the presence of two isomeric compounds with very similar patterns of absorptions. The most characteristic ones were those found at 248.18 and 248.39 ppm, which were attributed to two C=S groups of the dithiocarboxylate type. In addition, two signals at 78.54 and 78.71 ppm corresponded to non-equivalent Csp3 atoms connected with two sulfur atoms. In analogy to the 1H-NMR, eight signals between 26.32 and 30.66 ppm suggested the presence of structurally similar 16 Me groups. The elemental analysis confirmed the molecular formula C16H24S6, which can be attributed to a 1:1-mixture of the isomeric bis-spiro-1,3-dithietane derivatives meso- and d,l-17. All attempts to separate this mixture to obtain pure diastereoisomers, either by chromatography or fractional crystallization, were unsuccessful.Enhancement of the sulfur amount in the 1:1 experiment (Entry B) resulted in a drastic change of composition of the products in the reaction mixture. The main component isolated as the most polar fraction showed in the 1H-NMR spectrum showed the presence of only two broadened singlets located at 1.42 and 1.69 ppm. More information delivered the 13C-NMR spectrum with two low field absorptions at 243.0 and 216.6 ppm. Whereas the first signal could be attributed to the C=S group incorporated in the four-membered ring of cyclobutane, the second one was identified as the C=O unit of a cyclobutane moiety. In addition, two closely located signals with low intensity, found at 79.86 and 79.91 ppm, respectively, revealed the presence of two Csp3-atoms attached to two S-atoms like in the molecule of a 1,2,4,5-tetrathiane. As a matter of fact, elemental analysis confirmed the molecular formula C16H24OS5, which corresponds to the structure of the ‘non-symmetric’ dispiro-1,2,4,5-tetrathiane 6e (6d (not isolated) underwent hydrolysis in the reaction solution and one C=S group was converted into the C=O moiety.The less polar fraction formed a yellowish oil, which in the hiane 6e , Table 116a (8%)  .1H-NMR spectrum showed a complicated pattern of signals located in the 1.30–2.00 ppm region. In the spectrum registered at 90 °C in CHCl2–CHCl2 solution, four broadened signals emerged at 1.92, 1.98. 2.08, and 2.20 ppm, respectively, indicating thereby the existence of dynamic phenomena, like in the case of pentathiepane 7a described by Huisgen et al. [16H24S7 expected for the new pentathiepane 7c and allowed to determine the yield of the isolated product to 46%. In another experiment carried out with 2a and [S] in a ratio 1:2 (Entry C), pentathiepane 7c was isolated in a lower yield (23%) and the already described sulfur heterocycles 14 (9%), 16a (8%), and 18 (5%) were formed as side products.Increase of the molar excess of sulfur to 4 mol-equiv. (Entry D) changed dramatically the composition of the product mixture, and in this case the major fraction consisted of an orange oil, which in the 18  was calculated to 1:2.Notably, the same pentathiepane 2a, its monothione analogue 2b was studied extensively by Huisgen and Rapp in sulfurization reactions with elemental sulfur using potassium thiophenolate as a catalyst  in a molar ratio 1:0.5 (Entry A) or 1:1 (Entry B), the only sulfur-rich compound identified in the crude mixture of products was 6b accompanied by the above reported 3-oxopentanedithioate 15 and dithiolactone 16b. In the 1:1 experiment, the yield of tetrathiane 6b was higher (40%)  in DMF solution and starting with a 1:2 ratio of 2b and atomic [S], the latter heterocycle was isolated in 43% yield (Entry C′). Spectroscopic and physico-chemical data of 7a fitted well with those reported earlier [7a was redetermined by X-ray analysis and the crystallographic characterization of this sulfur-rich heterocycle could be refined thereby.In contrast to the hitherto unexplored dithione catalyst . The maier 40%) . Further0% . Furt earlier . In the 2a or 2b and starting with a 1:0.5 molar ratio of a thioketone 2 and elemental sulfur [S], no traces at all of the corresponding 1,2,4-trithiolane of type 5 could be detected in the mixtures of products. These findings allow to exclude participation of thiocarbonyl S-sulfides (thiosulfines) 4A–C derived from the studied thioketones 2 as reactive intermediates in the herein presented sulfurizations. Instead, these observations suggest to consider dithiiranes 21 and their ring opened forms, i.e., diradicals 22 (or zwitterions 23), as elusive species involved in the formation of the isolated sulfur-rich heterocycles , sulfurization reactions of easily available thioketones, such as the cycloaliphatic adamantanethione (2c) and the aromatic thiobenzophenone (1a), were also tested in the fluoride anion catalyzed sulfurization reactions. In both cases, the reactions were carried out with TBAF as a catalyst in THF at rt, and required remarkably longer times for completion  or 1:2 (Entry C), only the symmetric 1,2,3,5,6-pentathiepane 7b was obtained in moderate yields (37–51%). However, in the experiment with a 1:0.5 molar ratio , 1,2,4-trithiolane 5c was found in the mixture of products as the major component (59%). The same product was formed in DMF solution at rt exclusively, when the reaction was performed with CsF (Entry C′). Its presence suggests the appearance of adamantane-2-thione S-sulfide and subsequent trapping by another molecule of 2c according to the [3+2]-cycloaddition mode as suggested in our earlier study [5c follows a hitherto unknown mechanism of the reaction of fluoride anion with 2c mentioned in our earlier publication [7b can results from the mechanism initiated by the attack of fluoropolysulfide anion onto the C=S bond according to the pathways presented in 2c and obtained by Huisgen and Rapp [In extension of the study performed with cycloaliphatic thioketones tion see . Analysier study . Limitedlication . Neverth1a) was performed starting with 1:1 or 1:2 molar ratio of 1a to [S] . In both cases colorless products precipitated from THF solution, and the analysis of the obtained crude mixtures demonstrated that the literature known, symmetric 3,3,6,6-tetraphenyl-1,2,4,5-tetrathiane (6a) -cycloaddition with a ‘non-sulfurized’ molecule of the starting thiochalcone. Therefore, in none of the experiments formation of a respective 1,2,4-trithiolane was observed. Participation of thiocarbonyl S-sulfides in the [3+2]-cycloaddition with thiocarbonyl dipolarophiles is an important method for the synthesis of sulfur-rich heterocycles and diverse methods for the generation of this type of ‘sulfur-centered 1,3-dipoles’ are known  (5c). Yields: Procedure I—(a) Entry A: 27 mg (59% based on S/30% based on 1c); (b) Procedure II—Entry C′: 54 mg (59%) (method B). Colorless solid, m.p. 200–201 °C (methanol/CH2Cl2) , 2.15–2.40  ppm. 13C-NMR: δ 26.6, 26.9 (4CH), 36.7, 37.2, 37.9 , 39.4 (4CH), 90.3  ppm.2) . Yields: Procedure I—(a) Entry B: 32 mg (28%) (method B); (b) Entry C: 60 mg (52%) (method B); (c) Procedure II—Entry C′: 65 mg (56%) (method B). Colorless crystals, m.p. 210–212 °C  (methanol/CH2Cl2) , 7.55–7.59  ppm. 13C-NMR: δ 71.4 , 127.9, 128.5, 128.6 (20CHarom.), 141.7 (4Carom.) ppm.1,1,3,3,8,8,10,10-Octamethyl-5,6,11,12-tetrathiadispiro-[3.2.3.3]dodecane-2,9-dione (6b). Yields: Procedure I—(a) Entry A: 12% (method A); (b) Entry B: 40% (method A), 30 mg (32%) (method B). Colorless solid, m.p. 170–172 °C (methanol/CH2Cl2) , 1.54  ppm. 13C-NMR: δ 20.9, 24.9 (4CH3 each), 67.1 , 71.5 , 217.9 (2C=O) ppm.1,1,3,3,8,8,10,10-Octamethyl-5,6,11,12-tetrathiadispiro-[3.2.3.3]-9-oxododecane-2-thione (6e). Yield: Procedure I—(a) Entry B: 15 mg (15%) (method B). Orange oil. 1H-NMR: δ 1.42 , 1.69  ppm. 13C-NMR: δ 27.8, 28.1 (4CH3 each), 61.3 , 63.4 , 77.86, 77.91 , 216.6 (C=O), 243.0 (C=S) ppm.C16H24OS5 (392.69): calculated, C 48.94, H 6.16, S 40.83; found, C 48.96, H 6.33, S 40.58.1,1,3,3,8,8,10,10-Octamethyl-5,6,11,12,13-pentathiadispiro-[3.2.3.3]tridecane-2,9-dione (7a). Yields: Procedure I—(a) Entry C: 35 mg (34%) (method B); (c) Entry D: 53 mg (52%) (method B); (d) Entry E: 61 mg (60%) (method B); Procedure II—(e) Entry C′: 44 mg (43%) (method B). Colorless crystals, m.p. 146–148 °C (methanol/CH2Cl2)  ppm. 13C-NMR: δ 21.0–26.0 , 67.3 , 71.5 , 216.2 (2C=O) ppm. C16H24O2S5 (408.69): calculated, C 47.02, H 5.92, S 39.23; found, C 47.13, H 6.07, S 39.34.: 35 mg 3% (method2) -pentathiepane-7′,2”-adamantane] (7b). Yields: Procedure I—(a) Entry A: 20 mg (37% based on S/19% based on 2c) (method B); (b) Entry B: 50 mg (47%) (method B); (c) Entry C: 55 mg (51%) (method B). Colorless solid, m.p. > 236 °C (decomposition) (methanol/CH2Cl2)  ppm. 13C-NMR: δ 27.1, 33.7, 34.1, 35.4, 37.6, 38.6 (18C), referred weak signal at 84 ppm  is missing . Yields: Procedure I—(a) Entry C: 25 mg (23%) (method B); (b) Entry D: 51 mg (46%) (method B); Procedure II—(c) Entry C′: 59 mg (54%) (method B); red-orange viscous oil. 1H-NMR: δ 1.30–2.00  ppm; 1H-NMR : δ 1.92, 1.98, 2.08, 2.20  ppm. 13C-NMR: δ 24.0–29.0 , 50.9  53.5 , 276.0 (2C=S) ppm. C16H24S7 (440.82): calculated, C 43.59, H 5.49, S 50.92; found, C 43.37, H 5.38, S 50.97.3,3-Dimethyl-4-(propan-2-ylidene)thietane-2-thione (14). Yields: Procedure I—(a) Entry A: 63% (method A); (b) Entry B: 9% (method A); (c) Entry C: 3% (method A).2′,2′,4′,4′-Tetramethyl-3′-oxocyclobutyl 2,2,4-trimethyl-3-oxopentanedithioate (15). Yields: Procedure I—(a) Entry A: yield: 30% (method A); (b) Entry B: 20% (method A).3,3,5,5-Tetramethylthiolane-2,4-dithione (16a). Yields: Procedure I—(a) Entry A: 11% (method A); (b) Entry B: 8 mg (8%) (method B); (c) Entry C: 8% (method A), 8 mg (8%) (method B); (d) Entry D: 5% (method A); 3 mg (3%) (method B). Red-orange oil , 1.83  ppm. 13C-NMR: δ 33.5 (2CH3), 33.7 (2CH3), 71.0 (C-3), 77.8 (C-5), 245.3 (S=C-2), 267.4 (S=C-4) ppm.3,3,5,5-Tetramethyl-2-thioxothiolane-4-one (16b). Yields: (a) Entry A: 10% (method A); (b) Entry B: 23% (method A), 16 mg (17%) (method B).17), and 1,1,4,4,8,8,11,11-Octamethyl-2,6,10,12-tetrathiadispiro[4.1.4.1]dodecane-3,9-di-thione . Yield: Procedure I—(a) Entry B: 44 mg (43%) (method B). Yellow crystals, m.p. 130–138 °C (methanol/CH2Cl2). 1H-NMR: δ 1.32 , 1.35 , 1.51 , 1.51 , 1.69 , 1.71 , 1.74 , 1.75  ppm. 13C-NMR: δ 26.32 (2CH3), 26.36 (2CH3), 26.57 (2CH3), 26.61 (2CH3), 29.72 (2CH3), 29.80 (2CH3), 30.66 (4CH3), 58.20 (2C), 58.57 (2C), 64.22 (2C), 64.51 (2C), 78.54 (2C-5), 78.71 (2C-7), 248.18 (2C=S), 248.39 (2C=S) ppm. C16H24S6 (408.75): calculated, C 47.01, H 5.92, S 47.07; found, C 46.91, H 6.15, S 46.81.1,1,4,4,8,8,11,11-Octamethyl-2,6,9,12-tetrathiadispiro[4.1.4.1]dodecane-3,10-dithione -1,2-dithiolane-3-thione (18). Yields: Procedure I—(a) Entry C: yield: 5% (method A); (b) Entry D: yield: 2% (method A). Yellow-orange oil , 1.99 , 2.02  ppm. 13C-NMR: δ 21.4 (CH3), 26.4 (CH3), 29.6 (2CH3), 64.2 (C-4), 128.2 (=C), 137.10 (=C), 247.9 (C=S) ppm.il . Yield: Procedure I—(a) Entry D: 7 mg (4%) (method B). Pink solid, m.p. 111‒114 °C (MeOH/CH2Cl2). 1H-NMR: δ 1.54  ppm. 13C-NMR: δ 22.5, 28.6 (2CH3 each), 70.6 , 95.6 (C-4), 274.2 (C=S) ppm. C8H12S7 (332.64): calculated, C 28.89, H 3.64, S 67.48; found, C 29.11, H 3.70, S 67.18.3a–d, 0.125 mmol of elemental sulfur (S8)  and 0.125 mmol of triphenylphosphine (Ph3P) were placed and 2 mL of dry acetone were added. The resulting mixture was heated to the reflux under condenser and under argon and stirred for the appropriate time (discoloring). Then, the solvent was evaporated in vacuo and the residue was purified, firstly by short column chromatography and afterwards by thin-layer chromatography, by using SiO2 as the absorbent and a mixture of petroleum ether and dichloromethane (9:1) as the eluent, yielding the corresponding 3H-1,2-dithiole derivative 26a–d. Reported yields refer either to isolated amounts of products 26 (Method A) or to amounts calculated on the basis of standard analysis performed with weighted amounts of Cl2CH–CHCl2 added to the crude mixture (method B).In a two-necked round-bottomed flask (10 mL) equipped with a magnetic stirrer 0.5 mmol of the corresponding thiochalcone 3a was reacted with elemental sulfur in the presence of cat. amounts (0.06 mol-equiv.) of potassium thiophenolate (PhSK) in boiling acetone .3a was reacted with elemental sulfur in the presence of cat. amounts of PPh3 using butanone as a solvent.In analogy to Procedure I, thiochalcone 3a or 3c, (1 mmol) and 64 mg (2.0 mmol) of sulfur were stirred magnetically in dry DMF (2.5 mL) with cat. amounts of freshly calcined cesium fluoride (CsF). After 72h the reaction solution was diluted with dichloromethane (25 mL) and extracted several times with small portions of water for removal of DMF. Combined organic layers were dried over MgSO4, filtered and purified by column chromatography on silica gel using a 95:5 mixture of petroleum ether and dichloromethane as eluent.Thiochalcones 3,5-Diphenyl-3H-1,2-dithiole (26a). Yields: (a) Procedure I—51 mg (40%) (method A), 63% (method B); (b) Procedure II—53 mg (41%) (method A); (c) Procedure III—19 mg (15%) (method A); Procedure IV: 38 mg (15%) (method A). Orange-red solid, m.p. 78–81 °C. 1H-NMR: δ 5.87 ; 6.19 ; 7.34–7.38 ; 7.39–7.44 ; 7.49–7.53 ; 7.57–7.61  ppm. 13C-NMR: δ 63.7 (C-3); 120.7 (C-4); 127.4, 127.5, 128.4, 128.8 129.0, 129.3 (10Carom.); 132.9 (C-5); 140.8, 145.2 (2Carom.) ppm. C15H12S2 (256.39): calculated, C 70.27, H 4.72, S 25.01; found: C 70.20, H 4.68, S 25.01.3-(4-Chlorophenyl)-5-phenyl-3H-1,2-dithiole (26b). Yield: (a) Procedure I—22 mg (15%) (method A). Red oil. 1H-NMR: δ 5.80 ; 6.15 ; 7.36 , 7.39–7.45 , 7.56–7.59  ppm. 13C-NMR: δ 62.7 (C-3); 120.1 (C-4); 127.4, 128.8, 129.1, 129.4 (9Carom.); 132.7 (C-5); 134.2, 139.4, 145.7 (3Carom.) ppm. C15H11S2Cl (290.83): calculated, C 61.95, H 3.81, S 22.05; found: C 61.68, H 3.85, S 22.01.3-(4-Methylphenyl)-5-phenyl-3H-1,2-dithiole (26c). Yield: (a) Procedure I—27 mg (20%) (method A); Procedure IV—46 mg (17%) (method A). Red solid, m.p. 79–82 °C. 1H-NMR: δ 2.39 ; 5.86 ; 6.18 ; 7.21, 7.59 , 7.38–7.42  ppm. 13C-NMR: δ 21.2 (CH3), 63.5 (C-3), 120.9 (C-4), 127.4, 128.7, 129.2, 129.6, (9Carom.); 133.0 (C-5), 137.8 138.3, 145.0 (3Carom.) ppm. C16H14S2 (270.41): calculated, C 71.07, H 5.22, S 23.72; found: C 70.92, H 5.43, S 23.61.3-(4-Methoxyphenyl)-5-phenyl-3H-1,2-dithiole (26d). Yield: (a) Procedure I—17 mg (12%) (method A). Red oil. 1H-NMR: δ 3.84 ; 5.87 ; 6.17 ; 6.93 , 7.38–7.44 , 7.57–7.60  ppm. 13C-NMR: δ 55.4 (OCH3), 63.3 (C-3), 114.3 (C-4), 121.0, 127.4, 127.7, 127.8, 129.21 (9Carom.); 132.9 (C-5), 133.0, 144.8, 159.7 (3Carom.) ppm.The presented study showed that fluoride anion can be applied as an excellent activator of elemental sulfur in sulfurization reactions of easily available cycloaliphatic thioketones derived from 2,2,4,4-tetramethylcyclobutane-1,3-dione. Depending on the molar ratio of the starting materials, sulfur-rich heterocycles with variable ring size can be obtained in satisfactory to good yields. In situ-generated fluoropolysulfide anions act as powerful sulfurizing reagents. As a key intermediate in the studied reactions, three-membered, reactive dithiiranes, formed via the addition of the latter to the C=S group, are postulated for both cycloaliphatic and aromatic thioketones.H-1,2-dithiole derivatives, can be performed using triphenylphosphine (PPh3) as a catalyst. The latter heterocycles are of interest as unique starting materials for the preparation of sulfur-heterocycles based persistent radicals [H-1,2-dithioles and their conversion into the stable organic radicals are rarely reported and they are described only in rather remote literature [In contrast to thioketones, their α,β-unsaturated analogues, i.e., thiochalcones, do not undergo the sulfurization reaction with sulfur in the presence of fluoride anion. On the other hand, their sulfurization with elemental sulfur, leading to 3radicals ,33. Howeterature .S-heterocycles  in sulfurization reactions of diverse organic compounds, including stable thioketones [The developed protocols of sulfurization in the presence of fluoride anion can be of interest not only for the synthesis of sulfur-rich heterocycles, such as 1,2,4-trithiolanes , 1,2,4,5oketones ,22,24,30oketones ,41.


Several anti-inflammatory agents based on pyrazole and imidazopyrazole scaffolds and a large library of substituted catechol PDE4D inhibitors were reported by us in the recent past. To obtain new molecules potentially able to act on different targets involved in inflammation onset we designed and synthesized a series of hybrid compounds by linking pyrazole and imidazo-pyrazole scaffolds to differently decorated catechol moieties through an acylhydrazone chain. Some compounds showed antioxidant activity, inhibiting reactive oxygen species (ROS) elevation in neutrophils, and a good inhibition of phosphodiesterases type 4D and, particularly, type 4B, the isoform most involved in inflammation. In addition, most compounds inhibited ROS production also in platelets, confirming their ability to exert an antiinflammatory response by two independent mechanism. Structure–activity relationship (SAR) analyses evidenced that both heterocyclic scaffolds (pyrazole and imidazopyrazole) and the substituted catechol moiety were determinant for the pharmacodynamic properties, even if hybrid molecules bearing to the pyrazole series were more active than the imidazopyrazole ones. In addition, the pivotal role of the catechol substituents has been analyzed. In conclusion the hybridization approach gave a new serie of multitarget antiinflammatory compounds, characterized by a strong antioxidant activity in different biological targets. Inflammation is a very complex response of mammals’ body to different external injuries, including traumatic events, contact with irritant substances, pathogens invasion and more. There are different stages in inflammation evolution: the acute phase includes the movement of leukocytes from the blood vessel to the tissues and the immune system activation, aiming to damage control. Then, the prolongation of this initially protective response can lead to a state of chronic inflammation that is characterized by the intervention of other type of cells (such as macrophages and lymphocytes) and injured tissues destruction. Several pathologies are caused by chronic inflammation.Neurodegenerative disorders are typically multi-factorial diseases. Neuroinflammation is one among the different pathological alteration responsible for their onset and, as other multi-target pathologies, is difficult to treat by a mono-therapy. To get around this problem a new medicinal chemistry paradigm suggests the use of multi-targeted compounds, single small molecules able to act with different mechanism of action against two or more target ,2. Polyp1 and imidazopyrazoles 2 -induced chemotaxis while imidazopyrazoles 2 inhibited only formylmethyl-leucyl-phenylalanine (fMLP)-induced chemotaxis. The molecular stiffening of previous compounds 1 generates a new class of imidazo-pyrazole derivatives where the urea group is in part inserted in the cycle and in part on the lateral chain  or by overlapping two pharmacophore fragments (obtaining “fused” or “hybrid” molecule). Our previous research on the antiinflammatory agent topic allowed us to identify different pyrazolylureas azoles 2  able to ar range ,4,5. Pyrar range , by inhiar range .GEBR-54 pyrazole-7-carboxylic acid ethyl ester  and hydrazine monohydrate  was stirred at 120 °C for 8 h. After cooling to room temperature, water (10 mL) was added and the obtained solids were purified by Florisil column cromatography using a diethyl ether/methanol mixture (9:1) as the eluent. The obtained solids were then recrystallized by ethanol/methanol (2:1).A mixture of 5-amino-1-(2-hydroxy-2-phenylethyl)-15-Amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (7). White solid. Yield 48%. Mp: 183–184 °C. IR (KBr) cm−1: 3513, 3389 (NH2), 3316 (OH), 1603 (C=O). 1H-NMR (CDCl3): δ 3.90–4.08 , 4.21 , 4.86–5.03 , 5.72 , 6.04 , 7.21–7.47 , 7.66 , 8.96 . Analysis (%) calcd. for C12H15N5O2.2-Phenyl-2,3-dihydro-1H-imidazopyrazole-7-carbohydrazide (12). White solid. Yield 66%. Mp 250–252 °C. IR (KBr) cm−1: 3296 (NH2), 1627 (C=O). 1H-NMR (DMSO-d6) δ: 3.77 , 4.26 , 4.57 , 5.42 , 7.04 , 7.26–7.52 , 7.67 , 8.80 . Analysis (%) calcd. for C12H13N5O.H-pyrazole-4-carboxylic acid ethyl ester (6) pyrazole-7-carbohydrazide (12) (2 mmol) solved in the same mixture solvent (15 mL). The reaction mixture was stirred at 100 °C for 18 h, cooled to room temperature and poured into ice water (10 mL). The white solids obtained were simply dried, if pure at TLC check ), or recrystallized by absolute ethanol or ethyl acetate.A solution of a suitable benzaldehyde (E)-5-Amino-N′-(4-(difluoromethoxy)-3-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (4a). Yield 63%. White solid. Mp: 119–121 °C. 1H-NMR (DMSO-d6): δ 3.91 , 3.94–4.15 , 4.94–5.11 , 5.72 , 6.77 , 7.10–7.70 , 7.82–8.20 , 11.20 . Anal calcd. for C21H21F2N5O4.(E)-5-Amino-N′-(2-(cyclopentyloxy)-3-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (4b). Yield 46%. White solid. Mp: 180–181 °C. IR (KBr) cm−1: 3427 (OH), 3321, 3323, 3139 (NH2 + NH), 1634 (C=O), 1572 (C=N). 1H-NMR (DMSO-d6): δ 1.40–2.00 , 3.78 , 3.90–4.80 , 4.80–4.90 , 4.95–5.05 , 5.65–5.75 , 6.20–6.50 , 6.68 and 7.12 , 7.20–7.50 , 7.80–8.20 , 11.03 . Anal calcd. for C25H29N5O4.(E)-5-Amino-N′-(3-(sec-butoxy)-4-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (4c). Yield 64%. White solid. Mp: 190–193 °C. IR (KBr) cm−1: 3427 (OH), 3325, 3234, 3196 (NH2 + NH), 1644 (C=O), 1614 (C=N). 1H-NMR (DMSO-d6): δ 0.91–0.94 , 1.18–1.35 , 1.40–1.80 , 3.30–3.40 , 3.79 , 3.90–4.20 , 4.95–5.05 , 5.60–5.80 , 6.15–6.55 , 6.90–7.50 , 7.70–8.30 , 11.01 . Anal calcd. for C24H29N5O4.(E)-5-Amino-N′-(3-(cyclopentyloxy)-4-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (4d). Yield 78%. White solid. Mp: 193–195 °C. IR (KBr) cm−1: 3324, 3219 (NH), 2970 (OH), 1615 (C=N), 1642 (CONH). 1H-NMR (CDCl3): δ 1.48–2.10 , 3.89 , 4.02–4.33 , 4.78–4.99 , 5.11–5.32 , 5.82 , 6.80–7.01 , 7.04–7.22 , 7.22–7.61 , 7.75–8.38 , 9.00 . Anal calcd. for C25H29N5O4.(E)-5-Amino-N′-(3-(cyclopentyloxy)-4-(difluoromethoxy)benzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (4e). Yield 96%. White solid. Mp: 178–180 °C. IR (KBr) cm−1: 3450–3200 (NH + OH), 1635 (CONH). 1H-NMR (DMSO-d6): δ 1.51–2.04 , 3.30–3.40 , 3.90–4.21 , 4.84–5.10 , 5.75 , 6.42 , 7.06–7.60 , 7.83–8.35 , 11.20 . Anal calcd. for C25H27F2N5O4.(E)-5-Amino-N′-(4-(difluoromethoxy)-3-phenoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (4f). Yield 38%. White solid. Mp: 184–186 °C. 1H-NMR (CDCl3): δ 3.92–4.26 , 5.08–5.23 , 6.60–6.78 , 7.17–7.38 , 7.97–8.38 , 9.18–9.40 . Anal calcd. for C26H23F2 N5O4.(E)-5-Amino-N′-(3-(benzyloxy)-4-(difluoromethoxy)benzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (4g). Yield 61%. White solid. Mp: 144–146 °C. 1H-NMR (DMSO-d6): δ 3.95–4.20 , 4.92–5.10 , 5.26 , 5.75 , 6.40 , 7.16–7.70 , 7.83–8.35 , 11.25 . Anal calcd. for C27H25F2N5O4.(E)-5-Amino-1-(2-hydroxy-2-phenylethyl)-N′--1H-pyrazole-4-carbohydrazide (4h). Yield 34%. White solid. Mp: 176–178 °C. IR (KBr) cm−1: 3490 (OH), 3435, 3391, 3341, 3239 (NH2 +NH), 1640 (CO), 1561 (C=N). 1H-NMR (DMSO-d6): δ 3.36 , 3.71 , 3.83 , 4.40–4.10 , 4.95–5.05 , 5.70–5.80 , 6.25–6.55 , 6.98 , 7.25–7.50 , 7.80–8.20 , 11.18 . Anal calcd. for C22H24N4O5.(E)-5-Amino-1-(2-hydroxy-2-phenylethyl)-N′-(3-methoxy-2-phenoxybenzylidene)-1H-pyrazole-4-carbohydrazide (4i). Yield 66%. White solid. Mp: 207–209 °C. IR (KBr) cm−1: 3395 (OH), 3324, 3287, 3206, 3239 (NH2 +NH), 1646 (CO), 1551 (C=N). 1H-NMR (DMSO-d6): δ 3.69 , 3.85–4.15 , 4.90–5.05 , 5.69 , 6.20–6.50 , 6.78 , 7.00 , 7.15–7.45 , 7.55 , 7.60–8.40 , 11.20 . Anal calcd. for C26H25N5O4.(E)-5-Amino-N′-(2-(benzyloxy)-3-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (4j). Yield 71%. White solid. Mp: 198–200 °C. IR (KBr) cm−1: 3415 (OH), 3352, 3151, 3034 (NH2 +NH), 1627 (CO), 1564 (C=N). 1H-NMR (DMSO-d6): δ 3.87 , 3.90–4.20 , 4.85–5.10 , 4.99 , 5.71 , 6.20–6.60 , 7.00–7.55 , 7.80–8.10 , 8.35–8.55 , 11.19 . Anal calcd. for C27H27N5O4.(E)-5-Amino-N′-(3-(cyclopentyloxy)-4-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbohydrazide (4k). Yield 80%. White solid. Mp: 206–207 °C. IR (KBr) cm−1: 3364, 3309 (NH), 2951 (OH), 1598 (C=N), 1656 (CONH). 1H-NMR (DMSO-d6): δ 1.45–2.15 , 3.88 , 3.97–4.19 , 4.75–4.97 , 5.17–5.33 , 6.57 , 6.73 , 6.78–7.18 , 7.22–7.61 , 8.38 , 9.85 . Anal calcd. for C29H31N5O4.(E)-N′--2-phenyl-2,3-dihydro-1H-imidazopyrazole-7-carbohydrazide (5a). Yield 70%. White solid. Mp: 206–207 °C. IR (KBr) cm−1: 3432–3213 (NH), 1700–1600 (CONH + C=N). 1H-NMR (DMSO-d6): δ 3.70–3.92 , 4.63 , 5.48 , 7.02 , 7.09 , 7.24–7.53 , 7.60–8.40 , 11.10 . Anal calcd. for C21H21N5O3.(E)-N′-(2-(Cyclopentyloxy)-3-methoxybenzylidene)-2-phenyl-2,3-dihydro-1H-imidazopyrazole-7-carbohydrazide (5b). Yield 78%. White solid. Mp: 150–151 °C. IR (KBr) cm−1: 3392–3234 (NH), 1644 (CONH), 1594 (C=N). 1H-NMR (DMSO-d6): δ 1.47–2.20 , 3.72–3.95 , 4.62 , 4.75–4.92 , 5.48 , 7.00 , 7.18 , 7.23–7.52 , 7.99 , 11.07 . Anal calcd. for C25H27N5O3.(E)-N′-(3-(sec-Butoxy)-4-methoxybenzylidene)-2-phenyl-2,3-dihydro-1H-imidazopyrazole-7-carbohydrazide (5c). Yield 62%. White solid. Mp: 130–131°C. IR (KBr) cm−1: 3373 (NH), 1631 (CONH), 1594 (C=N). 1H-NMR (DMSO-d6): δ 0.95 , 1.25 , 1.45–1.80 , 3.60–3.95 , 4.20–4.40 , 4.60 , 5.40 , 6.90–7.50 , 7.95 , 11.10 . Anal calcd. for C24H27N5O3.(E)-N′-(3-(cyclopentyloxy)-4-methoxybenzylidene)-2-phenyl-2,3-dihydro-1H-imidazopyrazole-7-carbohydrazide (5d). Yield 93%. White solid. Mp: 120–124 °C. IR (KBr) cm−1: 3415, 3324 (NH), 1581 (C=N), 1633 (CONH). 1H-NMR (CDCl3): δ 1.47–2.20 , 3.90 , 4.07 , 4.62 , 4.79–4.95 , 5.50 , 6.83 , 7.09 , 7.29 , 7.32–7.52 , 7.82 , 8.25 , 9.18 , 9.85 . Anal calcd. for C25H27N5O3.(E)-N′-(3-(cyclopentyloxy)-4-(difluoromethoxy)benzylidene)-2-phenyl-2,3-dihydro-1H-imidazo-pyrazole-7-carbohydrazide (5e). Yield 62%. Red solid. Mp: 104–105 °C. IR (KBr) cm−1: 3228, 2961 (NH), 1642 (CONH), 1599 (C=N). 1H-NMR (DMSO-d6): δ 1.52–2.00 , 3.82 , 4.64 , 4.87–4.99 , 5.48 , 7.07 , 7.17–7.54 , 7.99 , 11.20 . Anal calcd. for C25H25F2N5O3.(E)-N′-(3-(Cyclohexyloxy)-4-(methoxy)benzylidene)-2-phenyl-2,3-dihydro-1H-imidazopyrazole-7-carbohydrazide (5f). Yield 65%. White solid. Mp: 119–120 °C. IR (KBr) cm−1: 3584 (NH), 1633 (CONH), 1581 (C=N). 1H-NMR (DMSO-d6): δ 1.20–2.20 , 3.80–4.10 , 4.25 , 4.52 , 5.39 , 6.60–7.60 , 7.90 , 8.20 , 10.15 . Anal calcd. for C26H29N5O3.(E)-N′--2-phenyl-2,3-dihydro-1H-imidazopyrazole-7-carbohydrazide (5g). Yield 93%. White solid. Mp: 230–233 °C. IR (KBr) cm−1: 3400, 3227 (NH), 1638 (CONH), 1608 (C=N). 1H-NMR (DMSO-d6): δ 3.82 , 4.62 , 5.43 , 4.96 , 6.10 , 7.00 , 7.14 , 7.26 , 7.32–7.52 , 7.92–8.20 , 11.05 . Anal calcd. for C20H17N5O.(E)-2-Phenyl-N′--2,3-dihydro-1H-imidazopyrazole-7-carbohydrazide (5h). Yield 74%. White solid. Mp: 120–121 °C. IR (KBr) cm−1: 3400–3200 (NH), 1643 (CONH), 1611 (C=N). 1H-NMR (DMSO-d6): δ 3.60–3.90 , 4.60 , 5.45 , 6.90–7.00 , 7.18  7.20–7.50 , 7.90 , 11.10 . Anal calcd. for C22H23N5O4.(E)-N′-(2-(Phenoxy)-3-(methoxy)benzylidene)-2-phenyl-2,3-dihydro-1H-imidazopyrazole-7-carbo- hydrazide (5i). Yield 81%. White solid. Mp: 265–266 °C. IR (KBr) cm−1: 3261, 3060 (NH), 1629 (CONH), 1580 (C=N). 1H-NMR (DMSO-d6): δ 3.32 , 3.62–3.82 , 4.60 , 5.40 , 6.75–7.60 , 7.86 , 8.28 , 11.19 . Anal calcd. for C26H23N5O3.(E)-N′-(2-(Benzyloxy)-3-(methoxy)benzylidene)-2-phenyl-2,3-dihydro-1H-imidazopyrazole-7-carbohydrazide (5j). Yield 78%. Light yellow solid. Mp 200–202 °C. IR (KBr) cm−1: 3602, 3173 (NH), 1644 (CONH), 1578 (C=N). 1H-NMR (DMSO-d6): δ 3.78–3.96 , 4.61 , 4.99 , 5.44 , 7.00–7.20 and 7.11–7.60 , 7.93 , 8.20–8.50 , 11.23 . Anal calcd. for C27H25N5O3.5d  and sodium acetate  in acetic anhydride (5 mL) was stirred at 40–50 °C for 4 h. After cooling to room temperature, the reaction was poured into water (20 mL) and extracted with dichloromethane (3 × 10 mL). The organic phase was washed with brine (3 × 10 mL), dried (MgSO4) and concentrated under reduced pressure to give an oil that was crystallized by absolute ethanol to give a light yellow solid. Yield: 49%. Mp: 140–141 °C. IR (KBr) cm−1: 2959 (NH), 1650 (C=O), 1596 (C=O). 1H-NMR (CDCl3): δ 1.51–2.20 , 3.91 , 3.97 , 4.15–4.25 , 4.78–5.03 , 5.78–5.91 , 6.85 , 7.00 , 7.15–7.25 , 7.35–7.60 , 8.15 , 8.30 , 9.87 . Elemental Analysis (%) calculated for C27H29N5O4.A suspension of 4 and 5 were diluted in saline from a stock DMSO solution immediately before each experiment. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) and thrombin were purchased from Sigma-Aldrich/Merck Millipore.Compounds g for 20 min. The obtained platelet-rich plasma was then centrifuged at 1100× g for 15 min. Pellet was washed once with pH 5.2 ACD solution , centrifuged at 1100× g for 15 min and then re-suspended in pH 7.4 Hepes buffer .Freshly drawn venous blood from healthy volunteers of the “Centro Trasfusionale, Ospedale San Martino” in Genoa was collected into 130 mM aqueous trisodium citrate anticoagulant solution (9:1). The donors claimed to have not taken drugs known to interfere with platelet function during two weeks prior to blood collection and gave their informed consent. Washed platelets were prepared centrifuging whole blood at 100× 8/mL), pre-incubated with saline, compounds 4 or 5, for 15 min at 37 °C, were stimulated by 0.1 U/mL thrombin. Incubation was stopped by cooling samples in ice bath and then samples were immediately analyzed in a Merck Millipore Bioscience Guava easyCyte flow cytometer . The reported IC50 value is the molar concentration of the compound able to obtain 50% inhibition of the maximal aggregation induced by the agonist and is calculated by the percentage of inhibition that is the inhibition of the maximal aggregation measured in the presence of the agent compared with that measured in a control sample containing saline, carried out under the same conditions.ROS production was quantified as previously reported ,23 by DC8/mL) were pre-incubated for 3 min at 37 °C with saline, compound 4 or 5 before the addition of 0.1 U/mL thrombin. The IC50 values were calculated as above detailed.Platelet aggregation was performed in a Bio-Data Aggregometer  according to Born’s method  and quanHeparinized  venous blood was obtained from healthy male volunteers after written informed consent. Neutrophils were isolated by dextran sedimentation , and subsequent centrifugation on a Ficoll-Hypaque density gradient , as described ,40. Cont4d and 5d at different concentration. Thereafter, neutrophils were pre-treated with 2′-7′-dichlorofluorescein-diacetate  (5 mM), During the incubation time, DCFH-DA entered into the cells, and it was cleaved by intracellular esterases to give non-fluorescence DCFH trapped within the cells. After washing in PBS, the cells were incubated for 15 min at room temperature in the presence or absence of N-formyl-l-methionin-l-leucyl-l-phenylalanine  (1 mM) for 30 min at 37 °C. During this period, intracellular ROS oxidized DCFH to give a green fluorescence DCFH. The reaction was stopped by keeping the samples on ice before being analyzed by flow cytometry .Flow cytometric analysis of neutrophil oxidative metabolism was carried out according to literature method . Briefly4d (10 µM) was assessed by propidium iodide (PI) method [In the same test, cell vitality in presence of ) method ,41,42 atSpodoptera frugiperda (Sf9) cells using a baculovirus expression system (PDE enzyme at 0.5 U/mL in 20 mM Tris.HCl pH 7.4) as previously reported [PDE4 tests were performed by Scottish Biomedical Drug Discovery  using human recombinant enzyme obtained from reported ,43. IMAP4d and 5d were solved in DMSO at 10−2 M concentration and then diluted with water to the final suitable concentrations. All our subsequent assays were performed in 3% DMSO . All the compounds were tested preliminary at 10 µM concentration, in duplicate.Title compounds In all the experiments, Rolipram as reference compound (at 10 μM for PDE4B2 and 1 μM for PDE4D3) was tested at nine concentrations, in duplicate, to obtain an inhibition curve in order to validate this experiment.4d and 5d have been further tested in the same assay at five different concentrations (5 × 10−8–10−4 M) and IC50 values were determined by nonlinear regression analysis of the inhibition curve, using Hill equation curve fitting .Being inhibition >50%, 4d and 5d. In addition, the same derivatives showed a good PDE4 inhibition, and also against PDE4B, isoform most involved in inflammation. Results strongly suggested that the PDE4 inhibition is related to antioxidant activity in neutrophils [Starting from previous anti-inflammatory pyrazole and imidazopyrazole scaffolds and PDE4Is, we designed and synthesized a series of hybrid molecules in order to obtain new antiinflammatory agents able to act on different biological targets. Preliminary tests evidenced a great antioxidant activity in neutrophils for compounds trophils . In addiIn conclusion, the hybridization between pyrazole and imidazopyrazole scaffolds and substituted catechol ring through an acylhydrazone linker, gave a new series of multitarget antiinflammatory compounds, characterized by a strong antioxidant activity in different biological system.


S. mansoni and I. ricinus legumains. The crystal structure of caspase-3 in complex with our caspase-specific aza-peptide methyl ketone inhibitor with an aza-Asp residue at P1 revealed a covalent linkage between the inhibitor carbonyl carbon and the active site cysteinyl sulphur. Aza-peptide aldehydes and ketones showed no cross-reactivity towards cathepsin B or chymotrypsin. The initial in vitro selectivity of these inhibitors makes them suitable candidates for further development into therapeutic agents to potentially treat multiple myeloma, neurodegenerative diseases, and parasitic infections.Aza-peptide aldehydes and ketones are a new class of reversible protease inhibitors that are specific for the proteasome and clan CD cysteine proteases. We designed and synthesised aza-Leu derivatives that were specific for the chymotrypsin-like active site of the proteasome, aza-Asp derivatives that were effective inhibitors of caspases-3 and -6, and aza-Asn derivatives that inhibited  Aza-peptides are peptidomimetics where the α-carbon centre of one or more of the natural amino acids in a peptide chain is replaced with a nitrogen atom . This mo,Incorporation of the aza-peptide motif in protease inhibition was first introduced by Dolle and co-workers1 residue , and these enzymes play essential roles in inflammation and neurodegeneration,,cat/Km values in generalCaspases are a family of cysteine proteases that belong to a small clan, the clan CD cysteine proteases. They have a strict requirement for peptides containing an Asp residue at the P1 position in order to cleave peptide substrates,Schistosoma mansoniTrichomonas vaginalisIxodes ricinusHaemaphysalis longicornisS. mansoni and I. ricinus, legumains contribute to digestion of the blood meal proteinstrans-activate other protease zymogens involved in protein digestionLegumains are also clan CD cysteine proteases, sharing a common fold with caspases that is unique to this clan. Legumains are acidic lysosomal enzymes that strictly require an asparagine residue at the PS. Mansoni and I. ricinus.Herein, we show that aza-peptide aldehydes and ketones are selective inhibitors with considerable activity against the proteasome, caspases-3 and -6, as well as the legumains derived from Purification of Common Laboratory Chemicals. Product purification by flash chromatography was carried out using normal phase 40–64 µm 60 Å silica gel from Sigma Aldrich or using a Teledyne Isco CombiFlash RF + UV auto column system. Dichloromethane, tetrahydrofuran, N,N-dimethylformamide, diethyl ether, and pentane solvents were obtained from a solvent purification system . Triethylamine and diisopropylethylamine were freshly distilled over CaH2 prior to use and stored under an argon atmosphere. NMR experiments were performed on Bruker 400, 700 or 850 MHz spectrometers. Chemical shifts are expressed in parts per million  while coupling constant values (J) are given in Hertz (Hz). Residual solvent protons were used as internal standards: for 1H NMR spectra CDCl3 = 7.26 ppm, DMSO-d6 = 2.50 ppm and D2O = 4.79 ppm while for 13C NMR spectra CDCl3 = 77.0 ppm and DMSO-d6 = 41.23 ppm; CDCl3 and DMSO-d6 were purchased from Cambridge Isotope Laboratories. High resolution mass spectra were recorded on a Bruker MicroTOF II instrument with internal sodium formate calibrant under electrospray ionisation (ESI) conditions. Hydrogenations were carried out using a Parr hydrogenation apparatus. Kinetic assays were performed on a Molecular Devices SpectraMax i3.All described reactions were carried out in flame/oven-dried glassware under a nitrogen or argon atmosphere, unless otherwise noted. Starting chemical reagents were purchased from reputable suppliers  and used without further purification or purified in accordance with procedures described in 2-OCO-; Pz, pyrazinyl; DCM, dichloromethane; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EtOAc, ethyl acetate; iBCF, isobutyl chloroformate; MeOH, methanol; NMM, N-methylmorpholine; NTA, nitrilotriacetic acid; RT, room temperature; TEV, tobacco etch virus; THF, tetrahydrofuran.Abbreviations. The following abbreviations have been used: AMC, 7-amino-4-methyl coumarin; AAla, aza-alanine residue; AAsp, aza-aspartic acid residue; AAsn, aza-asparagine residue; AGly, aza-glycine residue; ALeu, aza-leucine residue; Cbz, Ph-CHL-leucyl-L-leucinate (Cbz-Leu-Leu-OMe). Cbz-Leu-OH  was dissolved in dry THF (200 ml) and cooled to −20 °C. NMM  and iBCF  were added dropwise and the mixture was allowed to react for 30 min. Separately, H-Leu-OMe  was dissolved in dry THF (200 ml) and cooled to −20 °C and NMM  was added dropwise. This mixture was allowed to stir for 15 min. The two mixtures were then combined and allowed to stir for 1 h at −20 °C. Following, the solution was allowed to stir while gradually warming to RT for 18 h. The solvent was removed in vacuo, and the resulting residue was dissolved with EtOAc, washed with 1 M HCl, H2O, saturated NaHCO3, and saturated brine. The organic layer was dried over Na2SO4, and concentrated in vacuo to afford the product as a white solid . 1H NMR : 8.19 , 7.39–7.28 , 5.00 , 4.33–4.25 , 4.12–4.01 , 3.59 , 1.71–1.36 , 0.93–0.81 .Methyl ((benzyloxy)carbonyl)-L-alanyl-L-alaninate (Cbz-Ala-Ala-OMe). See procedure and workup of Cbz-Leu-Leu-OMe. A white solid was obtained in 83% yield. 1H NMR : 8.26 , 7.40 , 7.36 , 5.01 , 4.27 , 4.08 , 3.62 , 1.29 , 1.20 .Methyl ((benzyloxy)carbonyl)-S,8S,11S)-5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-(tert-butoxy)-3-oxopropyl)-11-isopropyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oate (Cbz-Asp(OtBu)-Glu(OtBu)-Val-OMe). See procedure and workup of Cbz-Leu-Leu-OMe. A white foam was obtained in 86% yield. 1H NMR : 8.12 , 7.92 , 7.60 , 7.48–7.23 , 5.04 , 4.42–4.30 , 4.00–4.19 , 3.62 , 2.65 , 2.43 , 2.22 , 2.04 , 1.94–1.81 , 1.79–1.67 , 1.40 , 1.37 , 0.88 .Methyl (5tert-Butyl (S)-4-(((benzyloxy)carbonyl)amino)-5-(((S)-1-methoxy-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (Cbz-Glu(OtBu)-Val-OMe). See procedure and workup of Cbz-Leu-Leu-OMe. The product was obtained as a colourless oil in quantitative yield. 1H NMR : 0.88 , 1.39 , 1.72 , 1.85 , 2.05 , 2.25 , 3.63 , 4.16 , 5.02 , 7.35 , 7.42 , 8.10 .tert-Butyl (S)-4-(((benzyloxy)carbonyl)amino)-5--3-(tert-butoxy)-1-methoxy-1-oxobutan-2-yl)amino)-5-oxopentanoate (Cbz-Glu(OtBu)-Thr(OtBu)-OMe). At −15 °C a solution of Cbz-Glu(OtBu)-OH  in THF (200 ml) was treated with NMM  and allowed to stir for 10 min. The solution was then treated with iBCF  and allowed to stir for 30 min at −15 °C. A second solution of H-Thr(tBu)-OMe  in THF (200 ml) at −15 °C was treated with NMM  and stirred for 15 min. The solutions were combined and stirred for 1 h at −15 °C, then allowed to stir while gradually warming to RT for 16 h. The solvent was removed in vacuo and the resulting residue was dissolved in EtOAc, washed with 1 M HCl, H2O, saturated Na2HCO3, and saturated brine. The organic layer was dried over Na2SO4 and removed in vacuo to afford the product as a white solid . 1H NMR  δ: 7.67 , 7.53 , 7.41–7.27 , 5.03 , 4.35 , 4.24–4.12 , 3.61 , 2.35–2.21 , 1.97–1.83 , 1.78–1.66 , 1.38 , 1.07 ; HRMS (ESI) calcd for [C26H40N2O8+Na]+, 531.2685, found 531.2699 (M + 23).tert-Butyl (S)-4-amino-5--3-(tert-butoxy)-1-methoxy-1-oxobutan-2-yl)amino)-5-oxopentanoate (H-Glu(OtBu)-Thr(OtBu)-OMe). A solution of tert-butyl (S)-4-(((benzyloxy)carbonyl)amino)-5--3-(tert-butoxy)-1-methoxy-1-oxobutan-2-yl)amino)-5-oxopentanoate  in MeOH (300 ml) was treated with 10% Pd/C and placed under H2 at 40 psi for 2 h. The solution was filtered over Celite and concentrated in vacuo to afford the product as a white solid . 1H NMR  δ: 8.03 , 4.30 , 4.16 , 3.63 , 3.24 , 3.17 , 2.30 , 1.98–1.86 , 1.57 , 1.39 , 1.08 ; HRMS (ESI) calcd for [C18H34N2O6 +H]+, 375.2495, found: 375.2486 (M + 1).S,8S,11S)-8–(3-(tert-butoxy)-3-oxopropyl)-11-((S)-1-(tert-butoxy)ethyl)-5-((R)-s-butyl)-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oate tert-butyl (S)-4-(((benzyloxy)carbonyl)amino)-5--3-(tert-butoxy)-1-methoxy-1-oxobutan-2-yl)amino)-5-oxopentanoate (Cbz-Ile-Glu(OtBu)-Thr(OtBu)-OMe). A solution of Cbz-Ile-OH  in THF (100 ml) was cooled to −15 °C followed by the addition NMM  and allowed to stir for 10 min. The solution was then treated with iBCF  and allowed to stir for 30 min at −15 °C. Separately, a solution of tert-butyl (S)-4-amino-5--3-(tert-butoxy)-1-methoxy-1-oxobutan-2-yl)amino)-5-oxopentanoate  in THF (100 ml) at −15 °C was treated with NMM  and stirred for 15 min. The solutions were combined and allowed to stir for 1 h at −15 °C. Following, the solution was allowed to stir while gradually warming to RT for 16 h. The solvent was removed in vacuo, the resulting residue was dissolved in EtOAc, washed with 1 M HCl, H2O, saturated Na2HCO3, and saturated brine. The organic layer was dried over Na2SO4 and concentrated in vacuo to afford the product as a white solid . 1H NMR  δ: 8.11 , 7.68 , 7.41–7.25 , 5.03 , 4.52–4.42 , 4.36 , 4.18–4.11 , 3.93 , 3.62 , 2.35–2.15 , 1.99–9.80 , 1.79–1.62 , 1.49–1.31 , 1.14–1.02 , 0.93–0.73 ; HRMS (ESI) calcd for [C32H51N3O9+Na]+, 644.3525, found: 644.3485 (M + 23).Methyl (5S)-1-(((S)-1-hydrazineyl-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (Cbz-Leu-Leu-NHNH2). Cbz-Leu-Leu-OMe  was dissolved in MeOH (150 ml) and hydrazine was added dropwise . The solution was allowed to stir for 18 h at RT. The solvent was removed in vacuo to afford Cbz-Leu-Leu-NHNH2 as white solid . 1H NMR : 9.18 , 7.86 , 7.44 , 7.44–7.27 , 5.03 , 4.32–4.23 , 4.10–4.00 , 1.68–1.51 , 1.50–1.28 , 0.82–0.88 .Benzyl ((S)-4-methyl-1-(((S)-4-methyl-1–(2-((E)-2-methylpropylidene)hydrazineyl)-1-oxopentan-2-yl)amino)-1-oxopentan-2-yl)carbamate (Cbz-Leu-Leu-NHN = CHCH(CH3)2). Cbz-Leu-Leu-NHNH2  was dissolved in dry THF (200 ml) followed by the addition of isobutyraldehyde  and catalytic acetic acid (1 drop). The solution was allowed to stir for 18 h at RT. The reaction was quenched with saturated NaHCO3, extracted with DCM. The organic layer was dried over Na2SO4 and removed in vacuo to afford Cbz-Leu-Leu-NHN = CHCH(CH3)2 as a white solid . 1H NMR : 10.81,11.01 , 7.93, 7.77 , 7.74 , 7.41 , 7.40–7.24 , 5.02 , 4.12–4.00 , 1.48–1.31 , 1.08–0.99 , 0.93–0.75 .Benzyl ((S)-1-(((S)-1–(2-isobutylhydrazinyl)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (Cbz-Leu-Leu-NHNHCHCH(CH3)2). A solution of benzyl ((S)-4-methyl-1-(((S)-4-methyl-1–(2-((E)-2-methylpropylidene)hydrazinyl)-1-oxopentan-2-yl)amino)-1-oxopentan-2-yl)carbamate  in MeOH (25 ml) was treated with acetic acid (0.2 ml) followed by sodium cyanoborohydride . The reaction was stirred at RT for 16 h, then the pH was adjusted to 10 by addition of 1 M NaOH. The solvent was removed in vacuo, and the residue was dissolved in DCM. The organic layer was washed with water, saturated brine, dried over Na2SO4 and concentrated in vacuo to afford the product as a white solid . 1H NMR  δ: 9.25 , 7.87 , 7.30 , 7.28–7.39 , 6.47 , 5.10 , 4.33–4.42 , 4.10–4.19 , 2.60 , 1.42–1.82 , 0.8–1.05 . HRMS (ESI) calcd for [C24H40NO4+Na]+ 471.2927, Found: 471.2950 (M + 23).Benzyl ((3: Benzyl ((S)-1-(((S)-1–(2-isobutyl-2–(2-oxoacetyl)hydrazinyl)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate. (Cbz-Leu-Leu-ALeu-CHO). A 0 °C solution of benzyl ((S)-1-(((S)-1–(2-isobutylhydrazinyl)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate  in dry DCM (2 ml) was treated with a solution of 2-oxoacetyl chloride  in DCM (0.5 ml) followed by DIPEA . The reaction was warmed to RT and stirred for 2 h. The reaction was quenched with H2O and extracted with DCM. The organic layer was washed with saturated NaHCO3, dried over Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel chromatography (2% MeOH/DCM) to afford the product as a white solid . 1H NMR  δ 9.08 , 7.96 , 7.49–7.24 , 5.01 , 4.37–4.20 , 4.14–4.01 , 3.78–3.66 , 1.77–1.32 , 0.94–0.71 ; 13C NMR  δ (ppm) = 189.9, 173.9, 173.0, 157.6, 138.8, 130.2, 129.4, 129.3, 67.0, 54.6, 54.3, 42.5, 42.1, 25.3, 24.2, 23.2, 22.0; HRMS (ESI) calcd for [C27H44N4O7+Na]+ 559.3108, found 559.3098 Compound 4: Benzyl ((S)-1-(((S)-1–(2-isobutyl-2–(2-oxopropanoyl)hydrazinyl)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (Cbz-Leu-Leu-ALeu-COMe). A 0 °C solution of pyruvic acid  in DCM (10 ml) was treated with oxalyl chloride  followed by DMF (1 drop). The reaction mixture was warmed to RT and stirred 2 h. The solvent was removed in vacuo, and residue was dissolved in fresh DCM (5 ml) and chilled to 0 °C. A solution of benzyl ((S)-1-(((S)-1–(2-isobutylhydrazinyl)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate  was added, followed by the addition of DIPEA . The reaction was stirred 1 h at 0 °C, then warmed to RT and stirred for an additional 16 h. The reaction mixture was diluted with H2O and extracted with EtOAc. Organic extracts were dried over Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel chromatography (20–40% EtOAc/Hexanes) to afford the product as a white solid . 1H NMR  δ 9.12 , 7.28–7.47 , 6.38 , 5.24 , 5.13 , 4.45 , 4.05–4.22 , 3.21–3.56 , 2.44 , 1.87–1.98 ,1.40–1.75 , 0.86–0.98 .13C NMR : δ (ppm) = 200.3, 174.1, 157.6, 138.8, 130.0, 129.5, 129.3, 67.0, 55.1, 54.5, 51.1, 42.3, 41.7, 28.9, 25.8, 24.7, 24.3, 23.2, 21.5; HRMS (ESI) calcd for [C27H42N4O6+Na]+ 541.2997, found: 541.2981.Compound 5: Benzyl ((S)-1-(((S)-1–(2-isobutyl-2–(2-oxo-3-phenylpropanoyl)hydrazinyl)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (Cbz-Leu-Leu-ALeu-COBn). To a solution of phenylpyruvic acid  in DCM (2 ml) oxalyl chloride  was added, followed by DMF (1 drop). The reaction mixture was stirred at RT for 2 h, then concentrated in vacuo. The resulting residue was dissolved in fresh DCM (3 ml) and chilled to 0 °C. To this chilled solution was added a solution of benzyl ((S)-1-(((S)-1–(2-isobutylhydrazinyl)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate  in DCM (1 ml) followed by DIPEA . The reaction was warmed to RT and stirred 16 h. The reaction mixture was diluted with H2O and extracted with DCM. The organic layer was dried over NasSO4 and concentrated in vacuo. The crude product was purified by silica gel chromatography (5% MeOH/DCM) and recrystallised from EtOAc/Et2O to afford the pure product as a white solid . 1H NMR  δ: 8.92 , 7.27–7.41 , 7.15–7.20 , 6.06 , 5.04–5.14 , 4.31–4.92 , 3.92–4.20 , 3.40–3.65 , 3.20–3.37 , 1.84–1.99 , 1.49–1.71 , 1.22–1.35 , 0.81–0.95 . 13C NMR : δ (ppm) = 198.4, 173.9, 156.3, 137.6, 130.4, 130.1, 129.0, 128.9, 128.8, 128.7, 128.2, 128.1, 65.8, 53.9, 53.3, 42.2, 41.1, 24.6, 23.5, 23.3, 22.0, 21.9, 21.1, 20.3; HRMS (ESI) calcd for [C33H46N4O6+Na]+ 617.3316, found: 617.3308.Compound tert-Butyl (S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanamido)-5-(((S)-1-hydrazinyl-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (Cbz-Asp(OtBu)-Glu(OtBu)-Val-NHNH2). A solution of methyl -5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-(tert-butoxy)-3-oxopropyl)-11-isopropyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oate  in) MeOH (80 ml) was treated with hydrazine . The reaction was stirred at RT for 18 h. The solvent and excess hydrazine were removed in vacuo to afford the product as a white solid. This was used for further transformations without additional purification . 1H NMR  δ: 9.16 , 7.98 , 7.80 , 7.64 , 7.44–7.26 , 5.13–4.95 , 4.46–4.11 , 4.04 , 2.69–2.36 , 2.30–2.14 , 1.96–1.81 , 1.52–1.30 , 0.89–0.76 ; HRMS (ESI) calcd for [C30H48N5O9]+ 622.3447, found: 622.3415.tert-Butyl -5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-(tert-butoxy)-3-oxopropyl)-11-isopropyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate (Cbz-Asp(OtBu)-Glu(OtBu)-Val-NHNHCH2CO2tBu). A −15 °C solution of tert-butyl (S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanamido)-5-(((S)-1-hydrazinyl-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate  in DMF (6 ml) was treated with the dropwise addition of t-butyl bromoacetate . The reaction was stirred for 30 min, then warmed to RT and stirred for 16 h. The solvent was removed in vacuo, and the residue was purified by silica gel chromatography (4% MeOH/DCM) to afford the title product as a white solid ; 1H NMR  δ: 9.48 , 7.95 , 7.85 , 7.62 , 7.47–7.22 , 5.18–5.11 , 5.10–4.98 , 4.51–4.19 , 4.11–3.99 , 3.41–3.38 , 2.27–2.16 , 1.96–1.69 , 1.46–1.34 , 0.86–0.77 .tert-Butyl -5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-(tert-butoxy)-3-oxopropyl)-11-isopropyl-3,6,9,12-tetraoxo-14–(2-oxopropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate (Cbz-Asp(OtBu)-Glu(OtBu)-Val-AAsp(OtBu)-COMe). Prepared following the general procedure for coupling of aza-peptide to pyruvic acid as previously described; ; 1H NMR  δ: 9.02 , 7.90 , 7.46–7.33 , 6.11–5.99 , 5.22–5.10 , 4.55–4.45 , 4.37–4.25 , 4.24–4.16 , 3.01–2.74 , 2.57–2.39 (m 2H), 2.37 , 2.33–2.24 , 2.19–1.99 , 1.49–1.44 , 0.97–0.92 ; HRMS (ESI) calcd for [C39H59N5O13+Na]: 828.4002, found: 828.3984.tert-Butyl -5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-(tert-butoxy)-3-oxopropyl)-11-isopropyl-3,6,9,12-tetraoxo-14–(2-oxo-3-phenylpropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate (Cbz-Asp(OtBu)-Glu(OtBu)-Val-AAsp(OtBu)-COBn). This product was prepared following the general procedure for coupling of an aza-peptide to phenylpyruvic acid as previously described: ; 1H NMR  δ: 11.07 , 8.00 , 7.93 , 7.60 , 7.40–7.15 , 5.12–4.96 , 4.41–4.28 , 4.21–4.12 , 4.10–3.96 , 2.70–2.57 , 2.47–2.39 , 2.26–2.14 , 1.94–1.80 , 1.77–1.65 , 1.44 , 1.37–1.32 , 0.81–0.75 ; HRMS (ESI) calcd for [C45H63N5O13+Na]: 904.4315, found: 904.4282.6: -8–(2-Carboxyethyl)-5-(carboxymethyl)-11-isopropyl-3,6,9,12-tetraoxo-14–(2-oxopropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oic acid . A solution of tert-butyl -5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-(tert-butoxy)-3-oxopropyl)-11-isopropyl-3,6,9,12-tetraoxo-14–(2-oxopropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate  in DCM (0.5 ml) was treated with trifluoroacetic acid (0.5 ml). The reaction was stirred 2 h, then concentrated to dryness in vacuo to afford the product as a white solid . 1H NMR  δ: 11.02 , 8.17–7.78 , 7.73–7.53 , 7.42–7.29 , 5.04 , 4.42–4.29 , 4.13 , 2.70–2.57 , 2.48–2.43 , 2.29–2.18 , 1.96–1.82 , 0.82 . 13C NMR : δ (ppm) = 199.7, 175.7, 173.4, 173.2, 172.9, 172.8, 172.5, 170.4, 170.0, 157.5, 138.6, 130.1, 129.5, 129.4, 67.2, 57.8, 53.3, 53.0, 50.3, 37.9, 31.7, 31.6, 29.1, 20.5, 19.6; HRMS (ESI) calcd for [C27H35N5O13+Na]+ 660.2124, found 660.2116 (M + 23).Compound 7: -8–(2-Carboxyethyl)-5-(carboxymethyl)-11-isopropyl-3,6,9,12-tetraoxo-14–(2-oxo-3-phenylpropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oic acid . tert-Butyl -5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-(tert-butoxy)-3-oxopropyl)-11-isopropyl-3,6,9,12-tetraoxo-14–(2-oxo-3-phenylpropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate  was dissolved in a 1:1 DCM/trifluoroacetic acid mixture (1.5 ml). The mixture was stirred for 2.5 h, then solvent was removed, and product was dried in vacuo to give the title compound as a white solid . 1H NMR  δ: 11.06 , 8.02–7.91 , 7.61 , 7.42–7.15 , 5.03 , 4.44–4.27 , 4.21–4.12 , 4.06–3.94 , 2.70–2.60 , 2.80–2.18 , 1.95–1.84 , 1.80–1.70 , 0.84–0.70 ; 13C NMR : δ (ppm) = 199.3, 173.4, 173.3, 173.0, 172.5, 170.4, 157.5, 138.6, 131.2, 131.7, 131.6, 130.0, 129.9, 129.5, 129.4, 128.6, 67.2, 57.8, 53.4, 53.0, 50.3, 46.8, 37.9, 31.7, 26.4, 20.7, 19.5; HRMS (ESI) calcd for [C33H39N5O13+Na]+ 736.2437, found: 736.2415 (M + 23).Compound tert-Butyl (S)-4--2-(((benzyloxy)carbonyl)amino)-3-methylpentanamido)-5--3-(tert-butoxy)-1-hydrazineyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (Cbz-Ile-Glu(OtBu)-Thr(OtBu)-NHNH2). A solution of methyl -8–(3-(tert-butoxy)-3-oxopropyl)-11-((S)-1-(tert-butoxy)ethyl)-5-((R)-s-butyl)-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oate  in MeOH (10 ml) was treated with hydrazine . The reaction mixture was stirred at RT for 16 h. Excess hydrazine and MeOH were removed in vacuo to afford the product as a white solid . 1H NMR  δ: 8.88 , 8.12 , 7.46 , 7.46–7.35 , 5.02 , 4.38 , 4.25  4.15 , 3.40 , 2.30–2.12 , 1.93–1.79 , 1.76–1.63 , 1.13–1.06 , 1.10 , 0.99–0.93 , 0.77–0.82 ; HRMS (ESI) calcd for [C31H51N5O8+Na]+, 644.3638, found: 644.3624 (M + 23).tert-Butyl -8–(3-(tert-butoxy)-3-oxopropyl)-11-((S)-1-(tert-butoxy)ethyl)-5-((R)- -butyl)-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate (Cbz-Ile-Glu(OtBu)-Thr(OtBu)-NHNHCH2CO2tBu). A -15 °C solution of tert-butyl (S)-4--2-(((benzyloxy)carbonyl)amino)-3-methylpentanamido)-5--3-(tert-butoxy)-1-hydrazineyl-1-oxobutan-2-yl)amino)-5-oxopentanoate  in dry DMF (100 ml) was treated with NMM  and left to stir 15 min. The solution was then treated with t-butyl bromoacetate  and allowed to stir for 30 min at -15 °C. Following, the solution was allowed to stir while gradually warming to RT for 16 h. The reaction mixture was concentrated in vacuo and the crude product was purified by silica gel chromatography (10% MeOH/DCM) to afford the product as a white solid . 1H NMR  δ: 9.14 , 8.11 , 7.55 , 7.36 , 5.12 , 5.02 , 4.42–4.31 , 4.18 , 3.92–3.88 , 3.38 , 2.36–2.09 , 1.96–1.79 , 1.77–1.58 , 1.47–1.34 , 1.10 , 1.00–0.93 , 0.90–0.74 ; HRMS (ESI) calcd for [C37H61N5O10 +Na]+ 758.4318, found 758.4301 (M + 23).tert-Butyl -8–(3-(tert-butoxy)-3-oxopropyl)-11-((S)-1-(tert-butoxy)ethyl)-5-((R)-s-butyl)-3,6,9,12-tetraoxo-14–(2-oxopropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate (Cbz-Ile-Glu(OtBu)-Thr(OtBu)-AAsp(OtBu)-COMe). A solution of pyruvic acid  in dry DCM (50 ml) was treated with oxalyl chloride  followed by 1 drop of DMF (cat). The reaction was stirred at RT for 3 h, then concentrated in vacuo. The resulting residue was dissolved in dry DCM (50 ml), chilled to 0 °C and treated dropwise with a solution of tert-butyl -8–(3-(tert-butoxy)-3-oxopropyl)-11-((S)-1-(tert-butoxy)ethyl)-5-((R)-s-butyl)-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate  in dry DCM (20 ml) and DIPEA . The reaction was stirred for 16 h at RT. The reaction was quenched with H2O, and washed with saturated brine. The organic layer was dried over Na2SO4 and concentrated in vacuo The crude product was purified by silica gel chromatography (10% MeOH/DCM) to afford product as a white solid . 1H NMR  δ: 10.77 , 8.05 , 7.75 , 7.42–7.24 , 5.02 , 4.54–4.44 , 4.27 , 3.92 , 3.95–3.89 , 2.23 , 1.85 , 1.70 , 1.88–1.39 , 1.11 , 0.95 , 0.86–0.74 ; HRMS (ESI) calcd for [C40H63N5O12+Na]+ 828.4373, found 828.4360 (M + 23).8: -5-((R)-s-butyl)-8–(2-carboxyethyl)-11-((S)-1-hydroxyethyl)-3,6,9,12-tetraoxo-14–(2-oxopropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oic acid (Cbz-Ile-Glu-Thr-AAsp-COMe). tert-butyl -8–(3-(tert-butoxy)-3-oxopropyl)-11-((S)-1-(tert-butoxy)ethyl)-5-((R)-s-butyl)-3,6,9,12-tetraoxo-14–(2-oxopropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate  was dissolved in 1:1 DCM/TFA (20 ml) at 0 °C and allowed to stir 30 min the warmed to RT and stirred an additional 1 h. The solution was concentrated in vacuo to afford the product as a white solid . 1H NMR  δ: 10.82 , 8.00 , 7.80 , 7.35 , 5.02 , 4.89 , 4.39 , 4.17 , 3.92 , 2.24 , 1.90 , 1.70 , 1.39 , 1.22 , 0.99 , 0.80 ; 13C NMR  δ (ppm) = 201.4, 178.6, 176.2, 172.2, 170.1, 167, 163, 149.1, 145.3, 135.9, 130.0, 129.9, 129.4, 67.1, 65.3, 62.3, 60.8, 57.4, 36.6, 31.6, 25.9, 21.2, 18.2, 12.7, 11.8. HRMS (ESI) calcd for [C28H39N5O12+Na]+ 660.2487, found 660.2485 (M + 23).Compound S)-1-(((S)-1-hydrazinyl-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (Cbz-Ala-Ala-NHNH2). To a solution of methyl ((benzyloxy)carbonyl)-L-alanyl-L-alaninate (Cbz-Ala-Ala-OMe)  in MeOH (50 ml), hydrazine  was added dropwise. The reaction was allowed to stir at RT for 18 h. MeOH and excess hydrazine were removed in vacuo and the resulting white solid was washed with EtOAc to afford a white solid . 1H NMR  δ 9.05 , 7.90 , 7.42 , 7.41–7.26 , 4.97–5.07 , 4.20–4.26 , 4.04–4.11 , 1.19 , 1.18 .Benzyl ((S)-2-((S)-2-(((benzyloxy)carbonyl)amino)propanamido)propanamido)glycinate (Cbz-Ala-Ala-NHNHCH2CO2Et). To solution of benzyl ((S)-1-(((S)-1-hydrazinyl-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate  and NMM  in DMF (8 ml) at −10 °C ethyl bromoacetate  was added dropwise. The reaction was stirred at −10 °C for 30 min, then warmed to RT and stirred for an additional 16 h. The reaction was then concentrated in vacuo, and the resulting residue was purified by silica gel chromatography (1:9 MeOH/DCM) to afford a white solid . 1H NMR:  δ 9.34 , 7.92 , 7.41–7.47 , 7.28–7.41 , 5.18 , 5.02 , 4.18–4.26 , 4.01–4.14 , 3.48 , 1.35–1.44 , 1.15–1.22 .Ethyl ((S)-1-(((S)-1–(2-(2-amino-2-oxoethyl)hydrazinyl)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (Cbz-Ala-Ala-NHNHCH2CONH2). A 7 M solution of ammonia in MeOH  was added to a 0 °C solution of ethyl ((S)-2-((S)-2-(((benzyloxy)carbonyl)amino)propanamido)propanamido)glycinate  in DMF (0.4 ml). NaCN  was added, and the reaction vessel was sealed. The mixture was stirred at 0 °C for 2 h, then warmed to RT and stirred for 48 h. The reaction was concentrated in vacuo and the product was precipitated with 9:1 DCM/MeOH to afford a white solid . 1H NMR  δ 9.33 , 7.98 , 7.30–7.47 , 7.12 , 5.22 , 4.97–5.08 , 4.15–4.24 , 4.01–4.11 , 3.20 , 1.20 , 1.18 .Benzyl ((10: Benzyl ((S)-1-(((S)-1–(2–(2-amino-2-oxoethyl)-2-(2-oxopropanoyl)hydrazinyl)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (Cbz-Ala-Ala-AAsn-COMe). A 0 °C of pyruvic acid  in DCM (10 ml) was treated with oxalyl chloride , followed by one drop of DMF (cat). The reaction was stirred at 0 °C for 30 min, then warmed to RT and stirred an additional 1.5 h. To this mixture was added DIPEA  followed by a solution of benzyl ((S)-1-(((S)-1–(2-(2-amino-2-oxoethyl)hydrazinyl)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate  in 1:2 DCM/DMF (1 ml). The reaction was stirred for 16 h, then concentrated in vacuo and purified by silica chromatography (1:9 MeOH/DCM) to afford a white solid . 1H NMR  δ: 10.41 , 9.05 , 8.11–8.00 , 7.55–7.26 , 5.03 , 4.13–4.05 , 3.18 , 2.03–1.98 , 1.55–1.42 ; HRMS (ESI) calcd for [C19H25N5O7+Na]+ 458.1652, Found: 458.1634 (M + 23).Compound 11: Benzyl ((S)-1-(((S)-1–(2–(2-amino-2-oxoethyl)-2-(2-oxo-3-phenylpropanoyl)hydrazinyl)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (Cbz-Ala-Ala-AAsn-COBn). A solution of phenylpyruvic acid  in DCM (3 ml) was treated with oxalyl chloride , followed by DMF (1 drop), and the reaction was stirred for 1.5 h at RT, then concentrated in vacuo. The resulting residue was taken up in fresh DCM (5 ml). To this solution was added a solution of benzyl ((S)-1-(((S)-1–(2-(2-amino-2-oxoethyl)hydrazinyl)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate  in DCM (1.5 ml), followed by DIPEA . The reaction was stirred at RT for 16 h, then concentrated in vacuo and purified by silica gel chromatography (2–10% MeOH/DCM) then recrystallised from EtOAc/Et2O to afford the product as a white solid . 1H NMR  δ: 8.13 , 7.17–7.40 , 4.94–5.08 , 4.17–4.31 , 4.05–4.13 , 4.00 , 1.05–1.30 ; 13C NMR  δ (ppm) = 197.0, 168.4, 161.8, 157.4, 130.1, 130.0, 129.97, 129.94, 129.9, 129.5, 129.4, 67.0, 60.0, 59.5, 51.5, 45.9, 45.5, 19.8; HRMS (ESI) calcd for [C25H29N5O7+Na]+ 534.1965, Found: 534.1975 (M + 23).Compound ex = 380 nm, λem = 442 nm. 18 µL of 2 µM proteasome solution was diluted with 425 µL H2O to make a 0.06 µM stock enzyme solution for kinetic assays. In a 96-well plate suitable for fluorometric assays was added 86 µL assay buffer, 2 µL inhibitor, 2 µL enzyme substrate, and 10 µL enzyme stock solution (0.06 nM enzyme concentration in well). Fluorescence at 442 nM was monitored for 10 min. The enzyme activity was measured in triplicate at varying concentrations of inhibitor  and substrate  by converting the slope of the plot of fluorescence intensity at 442 nm vs time for time points between 4 and 8 min using Beer-Lambert’s law. IC50 values were obtained from non-linear fitting of the data to a competitive inhibition model using GraphPad Prism 7.0 software. The proteasome β5 chymotrypsin-like active site activity assay was performed as previously describedHuman 20S proteasome, 700 KDa, was purchased from Boston Biochem ) in 50 mM HEPES buffer . Assay buffer was prepared as follows: 20 mM HEPES buffer pH 7.8, 0.5 mM EDTA, 0.037% SDS. Proteasome specific substrate Suc-LLVY-AMC was purchased from Boston Biochem and used as a fluorogenic substrate; λ2O were added resulting in the caspase assay buffer. Studies were performed at 37 °C.Caspase-3 and caspase-6 were generous gifts from Prof. Guy Salvesen’s laboratory and then used at Ohio State University. Caspase activities were measured in 100 mM HEPES, 10% sucrose, 0.1% CHAPS, 10 mM DTT, pH 7.5 at 37 °C. To 5,000 µL of this buffer, 100 µL of freshly prepared 1 M DTT and 4,900 µL DI Hex = 353 nm, λem = 442 nm. 2 µL of 33 µM caspase-3 solution was diluted with 4998 µL caspase assay buffer to make a 13.2 nM enzyme stock solution. In a 96-well plate suitable for fluorometric assays, 80 µL assay buffer, 5 µL inhibitor, 5 µL enzyme substrate (0.1 mM), and 10 µL enzyme stock solution were added. Fluorescence at 442 nM was monitored for 10 min. The enzyme activity was measured in triplicate at varying concentrations of inhibitor  and substrate (0.1 mM in DMSO) by converting the slope of the plot of fluorescence intensity at 442 nm vs time for the time points between 4 and 8 min using Beer-Lambert’s law. IC50 values were obtained from non-linear fitting of the data to a competitive inhibition model using GraphPad Prism 7.0 software.Caspase-3 specific substrate Ac-DEVD-AMC was purchased from Cayman Chemical Company and used as fluorogenic substrate; λex = 400 nm, λem = 505 nm. To 45 µL of assay buffer, 5 µL of the 19.7 µM enzyme was added. This resulting enzyme solution was further diluted 1:5 with assay buffer resulting in a 0.32 µM solution. In a 96-well plate suitable for fluorometric assays was added 80 µL assay buffer, 5 µL inhibitor, 5 µL enzyme substrate (0.1 mM), and 10 µL enzyme stock solution (32 nM enzyme concentration in well). The enzyme activity was measured in triplicate at varying concentrations of inhibitor  and substrate (0.1 mM in DMSO) by converting the slope of the plot of fluorescence intensity at 442 nm vs time for the time points between 4 and 8 min using Lambert-Beer’s law. IC50 values were obtained from non-linear fitting of the data to a competitive inhibition model using GraphPad Prism 7.0 software.Caspase-6 specific substrate Ac-VEID-AFC was purchased from ChemCruz and used as a fluorogenic substrate; λSchistosoma mansoni and Ixodes ricinus legumain zymogens were expressed in the yeast, Pichia pastoris, as described,Ir) or overnight (Sm) at room temperature in 0.1 M Na-Ac pH 4.0; 5 mM DTT. Activities were measured in 0.1 M Na-Ac pH 5.5; 2.5 mM DTT, 0.1% PEG 6000 buffer. The substrate used was Cbz-AAN-AMC with 50 µM final concentration in the assay. Inhibitors were prepared in DMSO (1% DMSO in activity assay). Measurements were taken with an incubation period of 10 min at room temperature, and the enzyme activity was measured in triplicate. Asparaginyl Endopeptidase (AE) activity assay was performed as previously describedex = 380 nm, λem = 442 nm. The assay buffer was prepared as follows 0.1 M K3PO4, 1.25 mM EDTA, 0.01% Brij 35 with a pH of 6 was made with 1 mM DTT added immediately prior to assay. To 100 µL of assay buffer, 5 µL of the received 0.427 mg/mL Cathepsin B solution was added. The resulting solution was further diluted 1:6 of Cathepsin B to assay buffer for a final concentration of 2.9 µg/mL. In a 96-well plate suitable for fluorometric assays was added 85 µL assay buffer, 5 µL inhibitor, 5 µL enzyme substrate, and 5 µL enzyme stock solution (0.145 µg/mL enzyme concentration in well). The enzyme activity was measured in triplicate at varying concentrations of inhibitor  and substrate (0.1 mM in DMSO) by converting the slope of the plot of fluorescence intensity at 442 nm vs time for the time points between 4 and 8 min using Lambert-Beer’s law. IC50 values were obtained from non-linear fitting of the data to a competitive inhibition model using GraphPad Prism 7.0 software.Human Liver Cathepsin B (purified) was purchased from Enzo Life Sciences . The Cathepsin B activity assay is performed in 0.1 M phosphate, 1.25 mM EDTA, 0.01% Brij, pH 6.0 buffer at 37 °C. Cathepsin B specific substrate Cbz-Arg-Arg-AMC was purchased from Sigma Aldrich and used as fluorogenic substrate; λex = 353 nm, λem = 442 nm. In a 96-well plate suitable for fluorometric assays was added 80 µL assay buffer, 5 µL inhibitor, 5 µL enzyme substrate, and 10 µL enzyme stock solution (0.5 µg/mL enzyme concentration in well). The enzyme activity was measured in triplicate at varying concentrations of inhibitor  and substrate (0.1 mM in DMSO), by converting the slope of the plot of fluorescence intensity at 442 nm vs time for the time points between 4 and 8 min using Lambert-Beer’s law. IC50 values were obtained from non-linear fitting of the data to a competitive inhibition model using GraphPad Prism 7.0 software.α-Chymotrypsin from bovine pancreas, type II lyophilised powder, >40 units/mg protein activity was purchased from Sigma Aldrich and was tested in 50 mM sodium phosphate, pH 7.5 buffer at 37 °C. To prepare the enzyme stock solution, 1.6 mg α-chymotrypsin was dissolved in 1,600 µL assay buffer. The resulting stock enzyme solution was further diluted 1:200 stock to assay buffer for a final stock concentration of 5 µg/mL. α-Chymotrypsin specific substrate Suc-LLVY-AMC was purchased from Boston Biochem and used as a fluorogenic substrate; λ,E. coli (GenScript) and placed in a pET15b vector (Invitrogen). The protein was expressed in E. coli at 30 °C for 24 h after 200 mM IPTG induction. Cells were pelleted by centrifugation and lysed by sonication. The lysate was cleared by centrifugation and purified by Ni-NTA  and Q-Sepharose  chromatography. TEV protease (1.25 mg) was added to the purified protein during overnight dialysis at 4 °C and the TEV protease and free His-tag were removed by reverse Ni-NTA purification. Any remaining impurities were removed and the buffer was exchanged by size-exclusion chromatography on a Superdex 200 Increase column equilibrated with 20 mM Tris, 20 mM NaCl, 10 mM DTT, pH = 7.5 yielding protein with purity >95% by SDS-PAGE. The protein was concentrated to 5 mg/mL by ultrafiltration , flash frozen, and stored at −80 °C until ready to use.The expression and purification of caspase-3 was performed as previously describedThe protein was inhibited by adding 20 mM Cbz-DEVaD-COMe inhibitor in DMSO to a final concentration of 2.1 mM giving a final concentration of 4 mg/ml caspase-3/inhibitor complex. Protein crystallisation was performed by hanging-drop vapour diffusion at 298 K with 1:1 ratio of mother liquor  to the protein/inhibitor complex. Crystals were observed within one day and reached their maximum dimensions within 7 days. Crystals were cryoprotected in a 1:4 solution of glycerol:mother liquor and frozen on the instrument in the cryo-stream at 100 K. X-ray diffraction data were collected in-house on a Compact HomeLab X-ray diffractometer (Rigaku) with MicroMax 003i generator, AFC-11 4-axis goniometer, and Pilatus 200 K detector.121 space group with a maximum resolution of 2.73 Å. Phases were determined by molecular replacement with 2H5ISupplemental information.Diffraction images were indexed, integrated, and scaled with HKL3000  in the P22in situ with thionyl chloride) for aldehydes and to methyl or benzyl pyruvic acid chlorides for ketones, respectively.The general preparation of aza-peptide aldehydes and ketones involves the synthesis of the substituted hydrazide precursor2 amino acid and the carboxybenzyl protected P3 amino acid  The methyl group is the smallest group possible for our proposed aza-peptide ketone design, and (b) the phenyl group, if tolerated, can be derivatised further to make more specific interactions at the prime site. Coupling of the warheads was performed by conversion of readily available pyruvic acid or phenylpyruvic acid to the corresponding acid chloride intermediates via reaction with oxalyl chloride, and then acyl substitution of the acid chloride with the aza-peptide hydrazide intermediates, thereby yielding the final aza-peptide ketones -Glu(OtBu)-Val-NHNH2 and Z-Ile-Glu(OtBu)-Thr(OtBu)-NHNH2 were synthesised using the general peptide coupling and hydrazide formation conditions described for the proteasome inhibitors above. The aza-aspartate residue was synthesised according to tert-butyl bromoacetate gave the tert-butyl aspartate protected intermediates of the general structure 9. Analogous warhead coupling methods to those described in tert-butyl groups, affording the final inhibitors 6, 7, and 8.The tri-peptide hydrazides Z-Asp. The results of the inhibition of the various aza-peptide aldehydes and ketones are summarised in We synthesised a total of eight final compounds. Each compound was characterised by 3, 4 and 5 are designed to target the β5, chymotrypsin-like (CT-L) active site of the human 20S proteasome, and thus are based on the ideal tripeptidyl sequence Leu-Leu-Leu3, 4 and 5 showed inhibition in the mid-µM range, suggesting that the proteasome active site can actually tolerate the aza-modification at the P1 position . MG132 is a very potent, but non-selective, proteasome inhibitor. In our assay, we have determined the Ki value of MG132 of the β5, chymotrypsin-like (CT-L) active site of the 20S proteasome as 14.28 ± 3.06 nM. This value compares well with the previously reported Ki values of MG132 as 2–4 nMCompounds position . Inhibit6 and 7 were designed as tetrapeptides to target human caspase-3 with the ideal sequence Asp-Glu-Val-Asp and compound 8 targets human caspase-6 with its optimal sequence Ile-Glu-Thr-Asp as caspases require a minimum of four amino acids in their recognition sequence6, 7, and 8 show low-to-mid µM range inhibition with their target enzyme, where the best inhibitor is ketone 6 with an IC50 value of 7.74 µM against caspase-3 .Compounds µM range . Compouni values of 5–12 nM54 in addition to the proteasomein vitro cross-reactivity and high selectivity among different families of proteases . While in vitro and in vivo selectivities do not always correlate, initial observation of in vitro selectivity is valuable information, and then, one can interpret subsequent in vivo behaviour in light of in vitro data more precisely. The initial in vitro selectivity is a desired and promising asset to these compounds, rendering their further development into potential drug candidates both compelling and exciting.In addition to screening all of the compounds with their respective target enzymes, we also tested them for possible cross-reactivities with human liver cathepsin B and bovine pancreas α-chymotrypsin . Catheps6, Cbz-Asp-Glu-Val-AAsp-COMe . X-ray crystallographic data with caspase-3 revealed a tetrahedral adduct with compound 6, providing insight into the mechanism of inhibition. The micromolar range of inhibitory potency for these aza-peptide aldehydes and ketones could be attributed to the fact that the point of the nucleophilic attack is one chemical bond away towards the C-terminus from where the scissile bond normally would reside (Eight novel compounds were synthesised and evaluated for their ability to inhibit their respective target proteases d reside . Howeverin vitro selectivity and reversible nature of these inhibitors makes them more desirable drug candidates, as any possible cross reactivity would be reversible, arguably resulting in less severe side effects.We hypothesise that the potency of aza-peptide ketones can be improved by increasing the inductive effect on the prime site’s alkyl group which will render the ketone carbonyl to be more electrophilic. Our future, second generation compounds will be designed and synthesised accordingly. Overall, the Click here for additional data file.