Introduction

Peptides and proteins perform essential task in both unicellular and multicellular organisms and living without them would be impossible (Avan et al. 2014). Among them, short peptides are sorely significant bioactive compounds and exhibiting an extensive variety of biological activities. For example, glutathione operates as an antioxidant, detoxificant, and anti-aging agent. Other examples of short peptides are endomorphin-2 and tetrapeptide GE81112A, which act as natural inhibitor of stress and antibiotic, respectively (Fig. 1) (Jürjens et al. 2018; Vorobyeva et al. 2012).

Fig. 1
figure 1

Examples of some biologically active of short peptides

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Accordingly, drug discovery based on peptides is an extensively investigated area in biomedical research and peptides synthesis has been very much considered in organic chemistry (Comegna et al. 2015). The therapeutic utilization of peptides and proteins is limited by their short-time life, nonselective receptor binding and low absorption (Rabong et al. 2010). Amide bonds are typically provided from the reaction between carboxylic acids and amines; however, these reactions do not proceed spontaneously at ambient temperature because of formation the stable salts. The direct condensation of the salt can occur at high temperature (160–180 °C). For this reason, it is usually necessary to activate the carboxylic acid via a coupling reagent prior to treatment with the amine (Montalbetti and Falque 2005; Valeur and Bradley 2009). Therefore, the synthesis of peptides composes of difficult sequences, with a numerous residues, such as active pharmaceutical ingredients (Carbajo et al. 2019). To overcome the disadvantages mentioned, chemists explored efficient methods to design pseudo-peptides (peptidomimetics), which have better pharmacological properties and maintain the activities of original peptide (Khalesi et al. 2019; Nielsen 2004).

Isocyanide-based multicomponent reactions (I-MCRs) have already been proven as an extremely efficient strategy for the synthesis of pseudo-peptides. This highly convergent approach supplies conspicuous diversity and complexity (Chandgude and Dömling 2016). Therefore, invention of novel approach based on I-MCRs for the synthesis of peptidomimetics has been very much attended (Chandgude and Dömling 2017; Zakharova et al. 2019). The Ugi reaction is known as a potent method for the synthesis of the tripeptide scaffold and has found widespread applications in combinatorial synthesis (Mroczkiewicz and Ostaszewski 2009; Toure and Hall 2009). The Bargellini reaction is an efficient classic MCR in which phenol is reacted with acetone and chloroform in the presence of a strong base to produce α-phenoxy-isobutyric acid (Butcher and Hurst 2009). Since the initial report, variants of nucleophiles have been investigated to increase complexity, functionality and diversity (Alanine et al. 2016; Mahdavi et al. 2012). Recently, Giustiniano et al. reported a successful replacement of phenols with isocyanides in the Bargellini reaction that afford 3-carboxamido-isobutyric acid (Giustiniano et al. 2016). In view of our interests in design of combinatorial I-MCRs, (Shaabani and Hooshmand 2018; Shaabani et al. 2014) herein, we report a novel method for the synthesis of new pseudo-peptides via isocyanide-based consecutive Bargellini/Ugi reactions.

Results and discussion

New pseudo-peptides containing three amide bonds as structurally interesting compounds were simply obtained in two steps. In the first step, isocyanide-based Bargellini reaction was used for the synthesis of 3-carboxamido-isobutyric acids. In the second step, the prepared acids were applied in Ugi reaction. In a pilot experiment, acetone (1), chloroform (2), sodium hydroxide (3) and cyclohexyl isocyanide (4) were mixed and stirred at 0 °C for 30 min and then at room temperature for 20 h to afford 3-(cyclohexylamino)-2,2-dimethyl-3-oxopropanoic acid (5). Then, the prepared acid (5), 4-methylbenzaldehyde (6), (2-chlorophenyl)methanamine (7), and cyclohexyl isocyanide (4) were mixed in EtOH and stirred at room temperature. After completion of the reaction (24 h), the desired product (8a) was isolated in 86% yield (Scheme 1).

Scheme 1
scheme 1

Consecutive Bargellini/Ugi reactions for the synthesis of pseudo-peptide 8a

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The reactions did not need any optimization. To investigate the scope and limitations of this protocol, various primary amines, isocyanides, aliphatic and aromatic aldehydes containing electron-donating as well as electron-withdrawing groups were used (Scheme 2). As shown in Fig. 2, the reactions proceeded very efficiently at room temperature and led to formation of new class of the pseudo-peptides in high yields without using column chromatography. In this process, eight new bonds are formed, such as carbon–carbon, carbon–nitrogen, and carbon–oxygen in two steps, which is significant privilege of this synthetic protocol. It is noteworthy that all prepared compounds are racemates.

Scheme 2
scheme 2

Diversity of substrates for the synthesis of pseudo-peptides

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Fig. 2
figure 2

Structure and isolated yields of the products 8

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The structures of all products were deduced from their IR, 1H NMR, 13C NMR, mass spectra, and CHN analysis data. For example, compound 8b is observed as a mixture of rotamers in its 1H NMR spectrum due to amide rotation. This spectrum consisted of a multiple for NH groups (δ = 7.78–7.60 ppm, 2H), a multiple for aromatic protons (δ = 7.26–6.82 ppm, 10H), two singlets for two benzylic CH groups (δ = 5.51 ppm, 0.4H and δ = 5.35 ppm, 0.6H), a multiple for CH2 of benzyl amine (δ = 4.55–4.46 ppm, 1.4H), a doublet for CH2 of benzyl amine (δ = 4.16 ppm, J = 17.3 Hz, 0.6H), a multiple for the NH–CH of cyclohexyl rings (δ = 3.71–3.41 ppm, 2H), and a multiple for the methylene protons of the cyclohexyl rings and methyl groups (δ = 1.85–0.97 ppm, 26H). In addition, the 1H decoupled 13C NMR spectrum of compound 8b indicated the presence of six amide carbonyls, which confirmed the existence of mixture of rotamers. The mass spectra of these compounds demonstrated molecular ion peaks at the appropriate m/z values. Finally, the structure of compound 8b was verified unambiguously by single-crystal X-ray analysis (Fig. 3).

Fig. 3
figure 3

ORTEP diagram for 8b

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1H NMR spectrum of some derivatives was indicated products as a mixture of rotamers. We decided to investigate the influence of temperature on compound 8a using variable temperature 1H NMR spectroscopy. The spectrum at 25 °C (Fig. 4, spectrum 1) shows two singlets for the benzylic CH group (δ = 5.57 ppm and δ = 5.50 ppm) and one doublet (δ = 4.64 ppm, J = 16.8 Hz) and one multiple for CH2 of benzyl (δ = 4.45–4.34 ppm). Increasing the temperature causes broaden and coalescence of the CH and CH2 resonances (Fig. 4, spectrums 2–7), and they appear as a singlet (CH group) and an AB quartet system (CH2 group) at 80 °C (Fig. 4, spectrum 8). For more simplification, 1H NMR spectrum of 8a was provided in the presence of D2O, due to assign the exchangeable NH groups (Fig. 4, spectrum 9).

Fig. 4
figure 4

Partial of DNMR spectra (25 °C-80 °C) of compound 8a (DMSO-d6, 300 MHz); spectrum 1: 25 °C, spectrum 2: 35 °C, spectrum 3: 40 °C, spectrum 4: 45 °C, spectrum 5: 50 °C, spectrum 6: 60 °C, spectrum 7: 70 °C, spectrum 8: 80 °C, spectrum 9: 25 °C and in the presence of D2O

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A proposed mechanism for the formation of products is demonstrated in Scheme 3. In the Bargellini four-component reaction, sodium hydroxide abstracts a proton from chloroform (2) and generates a carbanion (9). The carbanion (9) attacks acetone (1) to form the unstable carbinol (10), which cyclizes to produce the 2,2-dichloro-3,3-dimethyloxirane intermediate (11). Then, an isocyanide (12) attacks the intermediate (11) to form nitrilium ion (13), which is intercepted by the hydroxide ion to generate the iminol (14) that tautomerizes to the more stable amide (15). Concomitantly, the acyl chloride (15) is transformed to the product (16) (Giustiniano et al. 2016). When the prepared acid is applied in Ugi four-component reaction, according to the commonly accepted Ugi mechanism, (Okandeji et al. 2008) the acid, amine and aldehyde participate in equilibrium with iminium carboxylates (21). The addition of the terminal C atom of the isocyanides (21) onto the iminium group followed by the addition of the carboxylate ion onto the C atom of the nitrillium ion leads to the formation of the adduct (22), which rearranges by Mumm rearrangement to afford the desired product (8) (Scheme 3).

Scheme 3
scheme 3

Possible mechanism for isocyanide-based consecutive Bargellini/Ugi reactions

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Conclusion

In summary, we have introduced an efficient method for the synthesis of novel pseudo-peptides containing three amide bonds by combination of two isocyanide-based Bargellini and Ugi reactions. This approach is catalyst- and additive-free and has expeditious procedure and easy workup without using column chromatography, which meets many green chemistry criteria. In this process, eight new bonds are formed in two steps, which is remarkable from synthetic point of view. The potential utilization of this method in synthetic and medicinal chemistry may be considerable.

Experimental section

General information

All commercially available chemicals and reagents were purchased from Merck Chemical Company and used without further purification. Melting points were measured with an Electrothermal 9200 apparatus. IR spectra were recorded on a Thermo Nicolet NEXUS 470 FT-IR spectrometer in cm−1. 1H NMR spectra were recorded on a BRUKER AVANCE DRX-500 and DRX-300 spectrometer at 500 and 300 MHz. 13C NMR spectra were recorded on BRUKER AVANCE DRX-500 and DRX-300 spectrometers at 125 and 75 MHz. NMR spectra were obtained in CDCl3 and DMSO-d6. Mass spectra of the products were obtained with an HP (Agilent technologies) 5973 Mass Selective Detector. Elemental analyses were performed on an elementar analysensysteme GmbH VarioEL CHNS mode.

General procedure for the synthesis of acid derivatives 5

In a round-bottom flask, acetone (5 mmol), chloroform (7.5 mmol), sodium hydroxide (7.5 mmol), and an isocyanide (1 mmol) were mixed and stirred at 0 °C for 30 min and then at ambient temperature overnight. The reaction mixture was diluted with water, which was acidified to pH 2 with 2 M HCl and extracted with EtOAc (three times). The organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure to afford the pure product (Giustiniano et al. 2016).

General procedure for the synthesis of pseudo-peptide derivatives 8

A solution of an acid derivative (1 mmol), an amine (1 mmol), an aldehyde (1 mmol) and an isocyanide (1 mmol) in EtOH (5 mL) was stirred at room temperature. The reaction progress was monitored by TLC. After 24 h, the solvent was evaporated and the residue was recrystallized in EtOAc to afford the pure product.

Characterization data of 8a–8r

N1-(2-Chlorobenzyl)-N3-cyclohexyl-N1-(2-(cyclohexylamino)-2-oxo-1-(p-tolyl)ethyl)-2,2-dimethylmalonamide (8a) White powders: 487 mg, 86% yield; mp 215–216 °C. IR (ATR) cm−1: 3236, 3064, 2932, 2855, 1659, 1631, 1552. 1H NMR (300 MHz, DMSO-d6): (mixture of rotamers) δ 7.94–7.68 (m, 2H, NH), 7.21–6.96 (m, 7H, HAr), 6.89–6.87 (m, 1H, HAr), 5.57 (s, 0.6H, CH–N), 5.50 (s, 0.4H, CH–N), 4.64 (d, J = 16.8 Hz, 0.5H, CH2–N), 4.45–4.34 (m, 1.5H, CH2–N), 3.74–3.51 (m, 2H, HCN of cyclohexyl), 2.15 (s, 3H, HMe–Ar) 1.82–1.54 (m, 10H, HAliphatic), 1.37–0.93 (m, 16H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): (mixture of rotamers) δ 175.13, 173.91, 173.25, 172.91, 169.94, 168.31, 137.79, 137.32, 135.78, 135.58, 133.53, 131.15, 130.93, 130.83, 130.56, 129.15, 128.98, 128.68, 128.43, 127.53, 126.98, 126.42, 64.43, 63.26, 50.22, 49.10, 48.80, 48.31, 47.77, 32.47, 27.47, 26.06, 25.67, 25.53, 25.26, 24.89, 23.99, 23.51, 20.99. MS m/z: 567 (M+  + 2, 0.63), 565 (M+, 1.55), 439 (12.89), 369 (13.71), 335 (38.05), 315 (34.45), 244 (100), 196 (32.03), 125 (41.59), 83 (59.43), 55 (44.73). Anal. Calcd for C33H44ClN3O3: C, 70.01; H, 7.83; N, 7.42; found C, 69.91; H, 7.99; N, 7.49.

N1-Benzyl-N3-cyclohexyl-N1-(2-(cyclohexylamino)-2-oxo-1-phenylethyl)-2,2-dimethylmalonamide (8b) White powders: 466 mg, 90% yield; mp 210–212 °C. IR (ATR) cm−1: 3282, 3062, 2926, 2850, 1659, 1626, 1557. 1H NMR (500 MHz, DMSO-d6): (mixture of rotamers) δ 7.78–7.60 (m, 2H, NH), 7.26–6.98 (m, 9H, HAr), 6.84–6.82 (m, 1H, HAr), 5.51 (s, 0.4H, CH–N), 5.35 (s, 0.6H, CH–N), 4.55–4.46 (5, 1.4H, CH2–N), 4.16 (d, J = 17.3 Hz, 0.6H, CH2–N), 3.41–3.71 (m, 2H, HCN of cyclohexyl), 1.85–1.74 (m, 6H, HAliphatic), 1.65–1.50 (m, 6H, HAliphatic), 1.38–0.97 (m, 14H, HAliphatic). 13C NMR (125 MHz, DMSO-d6): (mixture of rotamers) δ 173.92, 173.02, 172.08, 168.69, 167.04, 138.13, 137.14, 135.93, 133.83, 129.59, 128.08, 127.44, 127.33, 127.18, 126.95, 126.63, 126.07, 125.92, 125.66, 124.91, 64.08, 62.54, 49.48, 49.24, 48.52, 48.04, 47.73, 47.22, 31.52, 31.42, 31.22, 30.92, 26.17, 24.65, 24.48, 24.26, 23.87, 23.74, 23.03. MS m/z: 517 (M+, 1.91), 391 (17.77), 321 (32.78), 301 (90.06), 196 (100), 91 (45.34), 55 (15.84). Anal. Calcd for C32H43N3O3: C, 74.24; H, 8.37; N, 8.12; found C, 74.16; H, 8.44; N, 8.19.

N1-Benzyl-N1-(1-(3-bromophenyl)-2-(cyclohexylamino)-2-oxoethyl)-N3-(tert-butyl)-2,2-dimethylmalonamide (8c) White powders: 484 mg, 85% yield; mp 188–190 °C. IR (ATR) cm−1: 3305, 3067, 2929, 2845, 1657, 1628, 1552. 1H NMR (300 MHz, DMSO-d6): (mixture of rotamers) δ 8.01–7.88 (m, 2H, NH), 7.31–7.22 (m, 1H, HAr), 7.21–7.03 (m, 7H, HAr), 6.87–6.84 (m, 2H, HAr), 5.42 (s, 0.2H, CH–N), 5.28 (s, 0.8H, CH–N), 4.76 (d, J = 15.0 Hz, 0.2H, CH2–N), 4.50 (d, J = 15.0 Hz, 0.8H, CH2–N), 4.27 (d, J = 15.0 Hz, 0.2H, CH2–N), 4.18 (d, J = 15.0 Hz, 0.8H, CH2–N), 3.53–3.47 (m, 1H, HCN of cyclohexyl), 1.74–1.49 (m, 5H, HAliphatic), 1.39–0.97 (m, 20H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): (major of rotamers) δ 175.02, 173.48, 168.87, 137.70, 137.62, 133.89, 130.96, 130.02, 129.22, 128.42, 127.38, 126.93, 121.13, 64.27, 51.29, 50.88, 48.32, 32.41, 28.88, 28.67, 26.92, 25.66, 24.88, 24.77, 24.20. MS m/z: 572 (M+  + 2, 3.10), 570 (M+, 3.40), 497 (6.63), 471 (9.71), 379 (29.72), 301 (68.77), 274 (52.84), 196 (100), 91 (85.45), 57 (65.47). Anal. Calcd for C30H40BrN3O3: C, 63.15; H, 7.07; N, 7.36; found C, 63.03; H, 7.01; N, 7.44.

N1-(1-(4-Bromophenyl)-2-(tert-butylamino)-2-oxoethyl)-N1-(2-chlorobenzyl)-N3-cyclohexyl-2,2-dimethylmalonamide (8d) White powders: 525 mg, 87% yield; mp 198–199 °C. IR (ATR) cm−1: 3335, 3241, 2929, 2847, 1669, 1628, 1549. 1H NMR (300 MHz, DMSO-d6): (mixture of rotamers) δ 7.83–7.62 (m, 2H, NH), 7.48–7.39 (m, 2H, HAr), 7.28–7.05 (m, 6H, HAr), 5.65 (s, 0.5H, CH–N), 5.41 (s, 0.5H, CH–N), 4.62–4.43 (m, 2H, CH2–N), 3.75 (bs, 0.5H, HCN of cyclohexyl), 3.60–3.56 (m, 0.5H, HCN of cyclohexyl), 1.85–1.57 (m, 5H, HAliphatic), 1.40–1.12 (m, 20H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): (mixture of rotamers) δ 175.06, 174.48, 173.57, 172.67, 169.84, 167.56, 136.26, 135.36, 135.20, 134.37, 132.57, 131.42, 131.12, 130.95, 129.32, 129.21, 129.06, 128.66, 127.88, 126.95, 126.62, 121.75, 121.32, 63.94, 63.55, 51.11, 50.41, 50.33, 49.14, 48.74, 48.09, 47.65, 32.64, 32.43, 32.34, 32.21, 28.60, 28.30, 27.26, 25.78, 25.53, 25.22, 24.73, 23.76. MS m/z: 607 (M+  + 4, 0.34), 605 (M+  + 2, 1.04), 603 (M+, 0.80), 505 (6.97), 409 (10.12), 379 (28.74), 335 (51.47), 310 (63.44), 196 (100), 125 (66.17), 83 (62.33), 57 (81.90). Anal. Calcd for C30H39BrClN3O3: C, 59.56; H, 6.50; N, 6.95; found C, 59.70; H, 6.59; N, 6.82.

N1-(1-(4-Bromophenyl)-2-oxo-2-((2,4,4-trimethylpentan-2-yl)amino)ethyl)-N3-(tert-butyl)-N1-(2-chlorobenzyl)-2,2-dimethylmalonamide (8e) White powders: 501 mg, 79% yield; mp 78–81 oC. IR (ATR) cm−1: 3299, 2965, 2863, 1664, 1636, 1534. 1H NMR (300 MHz, CDCl3): δ 7.44 (bs, 1H, NH), 7.29–7.24 (m, 1H, HAr), 7.16 (d, J = 7.8 Hz, 1H), 7.10–7.04 (m, 3H, HAr), 6.99–6.98 (m, 3H, HAr), 5.33 (s, 1H, CH–N), 4.85 (bs, 1H, NH), 4.69–4.63 (m, 2H, CH2–N), 1.73–1.26 (m, 23H, HAliphatic), 0.86 (s, 9H, HAliphatic). 13C NMR (75 MHz, CDCl3): δ 175.99, 172.87, 167.95, 133.79, 132.50, 132.09, 131.54, 129.29, 128.98, 128.42, 126.32, 122.78, 55.98, 52.69, 51.77, 50.85, 31.45, 31.35, 28.58, 28.53, 28.17, 25.48, 24.96. MS m/z: 637 (M+  + 4, 0.53), 635 (M+  + 2, 0.87), 633 (M+, 0.44), 507 (5.86), 353 (22.85), 309 (43.99), 170 (48.42), 125 (31.33), 57 (100). Anal. Calcd for C32H45BrClN3O3: C, 60.52; H, 7.14; N, 6.62; found C, 60.72; H, 7.23; N, 6.50.

N1-Allyl-N3-(tert-butyl)-N1-(2-(tert-butylamino)-2-oxo-1-(o-tolyl)ethyl)-2,2-dimethylmalonamide (8f) White powders: 352 mg, 82% yield; mp 200–202 °C. IR (ATR) cm−1: 3312, 3271, 2972, 2934, 1669, 1628, 1549. 1H NMR (300 MHz, DMSO-d6): δ 7.66 (s, 1H, NH), 7.25–7.11 (m, 4H, HAr), 6.68 (s, 1H, NH), 5.90 (s, 1H, CH–N), 5.22–5.07 (m, 1H, HAlkene), 4.46–4.33 (m, 2H, HAlkene), 3.79–3.63 (m, 2H, CH2–N), 2.23 (s, 3H, HMe–Ar), 1.36–1.27 (m, 24H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): δ 174.41, 173.23, 170.49, 139.35, 135.23, 134.95, 130.20, 130.00, 128.56, 126.13, 115.04, 60.32, 51.27, 51.21, 50.97, 50.81, 49.30, 28.95, 28.83, 28.54, 27.54, 23.54, 19.37. MS m/z: 429 (M+, 2.46), 357 (7.40), 329 (13.25), 289 (19.62), 259 (22.03), 160 (100), 57 (75.61). Anal. Calcd for C25H39N3O3: C, 69.90; H, 9.15; N, 9.78; found C, 69.75; H, 9.25; N, 9.62.

N1-(tert-Butyl)-N3-(2-(cyclohexylamino)-2-oxo-1-(o-tolyl)ethyl)-N3-(3,4-dichlorophenyl)-2,2-dimethylmalonamide (8g) White powders: 459 mg, 82% yield; mp 155–157 °C. IR (ATR) cm−1: 3338, 3092, 3934, 2850, 1634, 1526. 1H NMR (300 MHz, DMSO-d6): δ 8.17–7.93 (m, 2H, NH), 7.48–6.99 (m, 3H, HAr), 6.82 (bs, 1H, HAr), 6.64–6.55 (m, 2H, HAr), 6.20–6.28 (m, 1H, HAr), 6.19 (s, 1H, CH–N), 3.62 (bs, 1H, HCN of cyclohexyl), 2.39 (s, 3H, HMe–Ar), 1.80–0.96 (m, 25H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): δ 173.25, 172.63, 169.81, 138.96, 138.32, 134.85, 134.01, 133.71, 132.02, 130.83, 130.22, 129.79, 129.52, 128.45, 125.76, 62.49, 52.13, 51.14, 48.53, 32.68, 28.69, 26.10, 25.66, 25.15, 24.98, 24.61, 19.46. MS m/z: 563 (M+  + 4, 2.90), 561 (M+  + 2, 7.90), 559 (M+, 6.97), 390 (14.68), 264 (90.45), 170 (100), 143 (40.18), 114 (18.16), 83 (23.05), 57 (98.60). Anal. Calcd for C30H39Cl2N3O3: C, 64.28; H, 7.01; N, 7.50; found C, 64.19; H, 7.14; N, 7.39.

N1-Cyclohexyl-N3-(2-(cyclohexylamino)-2-oxo-1-phenylethyl)-N3-(3,4-dichlorophenyl)-2,2-dimethylmalonamide (8h) White powders: 497 mg, 87% yield; mp 135–137 °C. IR (ATR) cm−1: 3305, 3069, 2926, 2852, 1659, 1626, 1536. 1H NMR (300 MHz, DMSO-d6): δ 8.04 (bs, 2H, NH), 7.47 (bs, 1H, HAr), 7.17–7.13 (m, 3H, HAr), 6.97–6.89 (m, 3H, HAr), 6.68–6.55 (m, 1H, HAr), 6.00 (s, 1H, CH–N), 3.61–3.58 (m, 1H, HCN of cyclohexyl), 3.18–3.16 (m, 1H, HCN of cyclohexyl), 1.78–1.52 (m, 10H, HAliphatic), 1.30–0.95 (m, 16H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): δ 172.83, 172.42, 169.07, 138.28, 135.54, 134.18, 130.84, 130.67, 129.26, 128.35, 65.54, 51.01, 49.06, 48.76, 48.41, 32.70, 32.59, 32.22, 32.07, 25.68, 25.25, 24.94. MS m/z: 524 (M+—47, 0.41), 507 (10.97), 434 (48.18), 406 (30.75), 301 (38.08), 210 (100), 196 (37.00), 91 (44.55), 57 (34.57). Anal. Calcd for C31H39Cl2N3O3: C, 65.03; H, 6.87; N, 7.34; found C, 65.17; H, 6.97; N, 7.19.

N1-(1-(4-Bromophenyl)-2-(cyclohexylamino)-2-oxoethyl)-N1-(2-chlorobenzyl)-N3-cyclohexyl-2,2-dimethylmalonamide (8i) White powders: 529 mg, 84% yield; mp 217–218 °C. IR (ATR) cm−1: 3243, 3067, 2926, 2845, 1662, 1626, 1544. 1H NMR (300 MHz, DMSO-d6): (mixture of rotamers) δ 8.00 (m, 1H, NH), 7.72 (m, 1H, NH), 7.38–7.05 (m, 8H, HAr), 5.61 (s, 0.4H, CH–N), 5.48 (s, 0.6H, CH–N), 4.71–4.65 (m, 0.3H, CH2–N), 4.42–4.39 (m, 1.7H, CH2–N), 3.71–3.52 (bs, 2H, HCN of cyclohexyl), 1.96–1.72 (m, 6H, HAliphatic), 1.53–1.13 (m, 20H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): (mixture of rotamers) δ 174.96, 173.88, 173.30, 172.83, 169.13, 167.79, 135.84, 135.51, 135.12, 132.57, 131.55, 131.31, 131.02, 130.92, 129.12, 128.77, 128.50, 127.67, 126.46, 121.84, 121.49, 63.82, 62.94, 50.32, 50.22, 49.12, 47.96, 32.41, 26.95, 26.29, 25.64, 25.50, 25.26, 24.89, 23.87, 23.67. MS m/z: 633 (M+  + 4, 0.84), 631 (M+  + 2, 2.04), 629 (M+, 1.41), 505 (8.10), 435 (13.78), 379 (24.87), 335 (64.59), 310 (54.18), 196 (100), 125 (63.27), 83 (94.34), 55 (80.46). Anal. Calcd for C32H41BrClN3O3: C, 60.91; H, 6.55; N, 6.66; found C, 60.69; H, 6.72; N, 6.76.

N1-Benzyl-N1-(2-(tert-butylamino)-2-oxo-1-(o-tolyl)ethyl)-N3-cyclohexyl-2,2-dimethylmalonamide (8j) White powders: 415 mg, 82% yield; mp 196–198 °C. IR (ATR) cm−1: 3335, 3302, 2932, 2857, 1687, 1623, 1526. 1H NMR (300 MHz, DMSO-d6) δ: 1H NMR (300 MHz, DMSO-d6): (mixture of rotamers) δ 8.14–8.11 (m, 1H, NH), 7.84–7.82 (m, 1H, NH), 7.40 (d, J = 7.5 Hz, 1H, HAr), 7.30–7.14 (m, 4H, HAr), 7.07–6.90 (m, 3H, HAr), 6.76–6.64 (m, 1H, HAr), 5.76 (s, 0.4H, CH–N), 5.34 (d, J = 17.0 Hz, 0.6H, CH2–N), 5.18 (s, 0.6H, CH–N), 4.42 (d, J = 17.0 Hz, 0.4H, CH2–N), 4.24–4.16 (m, 1H, CH2–N), 3.80–3.77 (m, 0.6H, HCN of cyclohexyl), 3.60 (bs, 0.4H, HCN of cyclohexyl), 2.09 (s, 1.8H, HMe–Ar), 2.00 (s, 1.2H, HMe–Ar), 1.86–1.62 (m, 6H, HAliphatic), 1.45–1.05 (m, 13H, HAliphatic), 0.91–0.83 (m, 6H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): (mixture of rotamers) δ 176.08, 175.27, 173.69, 172.79, 171.15, 165.94, 138.92, 138.05, 137.51, 137.01, 136.10, 133.57, 130.92, 129.99, 129.49, 128.40, 128.00, 127.78, 127.27, 126.70, 126.49, 126.37, 125.82, 63.94, 60.80, 51.10, 50.70, 50.60, 50.37, 49.90, 49.33, 48.76, 33.28, 32.41, 32.15, 28.71, 28.05, 27.94, 27.56, 25.83, 25.58, 25.46, 25.26, 24.15, 21.71, 19.81, 19.57. MS m/z: 505 (M+, 0.13), 401 (11.13), 355 (12.24), 275 (100), 196 (15.13), 170 (59.51), 91 (53.42), 57 (28.22). Anal. Calcd for C31H43N3O3: C, 73.63; H, 8.57; N, 8.31; found C, 73.75; H, 8.69; N, 8.19.

N1-Benzyl-N1-(1-(4-bromophenyl)-2-(tert-butylamino)-2-oxoethyl)-N3-cyclohexyl-2,2-dimethylmalonamide (8k) White powders: 496 mg, 87% yield; mp 154 °C. IR (ATR) cm−1: 3279, 3090, 2942, 2850, 1667, 1631, 1554. 1H NMR (300 MHz, DMSO-d6): (mixture of rotamers) δ 7.83–7.76 (m, 2H, NH), 7.66 (bs, 1H, HAr), 7.54–7.47 (m, 1H, HAr), 7.26 (d, J = 7.9 Hz, 1H, HAr), 7.16–7.05 (m, 4H, HAr), 6.98 (d, J = 8.1 Hz, 1H, HAr), 6.91–6.88 (m, 1H, HAr), 5.37 (s, 0.5H, CH–N), 5.32 (s, 0.5H, CH–N), 4.71 (d, J = 16.5 Hz, 0.5H, CH2–N), 4.52–4.36 (m, 1H, CH2–N), 4.21 (d, J = 16.5 Hz, 0.5H, CH2–N) 3.62 (bs, 1H, HCN of cyclohexyl), 1.84–1.59 (m, 5H, HAliphatic), 1.39–1.06 (m, 20H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): (mixture of rotamers) δ 174.68, 174.57, 173.58, 172.93, 169.65, 167.27, 138.65, 137.85, 136.53, 134.89, 132.55, 131.35, 130.99, 130.78, 128.37, 127.81, 127.22, 126.99, 126.13, 121.42, 121.12, 64.50, 63.64, 51.01, 50.95, 50.30, 49.42, 49.03, 48.64, 32.66, 32.46, 32.15, 28.66, 28.15, 26.80, 25.74, 25.53, 25.21, 24.53, 24.32. MS m/z: 573 (M+  + 4, 1.26), 571 (M+  + 2, 1.55), 569 (M+, 0.07), 418 (6.97), 376 (14.32), 250 (84.92), 196 (98.42), 169 (100), 83 (87.10), 55 (69.02). Anal. Calcd for C30H40BrN3O3: C, 63.15; H, 7.07; N, 7.36; found C, 63.29; H, 7.14; N, 7.23.

N1-(1-(4-Bromophenyl)-2-(cyclohexylamino)-2-oxoethyl)-N3-(tert-butyl)-N1-(2-chlorobenzyl)-2,2-dimethylmalonamide (8l) White powders: 537 mg, 89% yield; mp 197–198 °C. IR (ATR) cm−1: 3282, 3080, 2850, 1667, 1639, 1547. 1H NMR (300 MHz, DMSO-d6): (mixture of rotamers) δ 8.06–8.00 (m, 2H, NH), 7.37 (d, J = 8.1 Hz, 1H, HAr), 7.27 (d, J = 8.0 Hz, 2H, HAr), 7.20–7.13 (m, 3H, HAr), 7.07–6.96 (m, 2H, HAr), 5.59 (s, 0.7H, CH–N), 5.41 (s, 0.3H, CH–N), 4.72–4.67 (m, 0.3H, CH2–N), 4.49–4.32 (m, 1.7H, CH2–N), 3.52 (bs, 1H, HCN of cyclohexyl), 1.69–1.54 (m, 5H, HAliphatic), 1.41–1.28 (m, 15H, HAliphatic), 1.23–0.94 (m, 5H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): (mixture of rotamers) δ 175.17, 173.50, 173.24, 169.19, 135.65, 135.11, 133.89, 132.74, 131.68, 130.98, 130.91, 129.16, 129.08, 128.77, 128.45, 127.01, 126.48, 121.91, 121.43, 121.43, 63.77, 63.24, 51.60, 51.37, 50.88, 48.74, 48.38, 47.91, 32.48, 32.40, 28.91, 28.90, 28.56, 27.30, 25.63, 25.12, 24.93, 24.81, 23.47. MS m/z: 607 (M+  + 4, 0.35), 605 (M+  + 2, 0.88), 603 (M+, 0.68), 435 (12.32), 353 (22.23), 309 (73.85), 170 (79.92), 125 (57.57), 83 (30.74), 57 (100). Calcd for C30H39BrClN3O3: C, 59.56; H, 6.50; N, 6.95; found C, 59.32; H, 6.38; N, 6.99.

N1-Benzyl-N3-cyclohexyl-N1-(2-(cyclohexylamino)-2-oxo-1-(o-tolyl)ethyl)-2,2-dimethylmalonamide (8m) White powders: 473 mg, 89% yield; mp 196–198 °C. IR (ATR) cm−1: 3246, 3072, 2929, 2844, 1650, 1631, 1550. 1H NMR (300 MHz, DMSO-d6): (mixture of rotamers) δ 8.05–7.98 (m, 1H, NH), 7.80–7.73 (m, 1H, NH), 7.38–6.90 (m, 7H, HAr), 6.77–6.67 (m, 2H, HAr), 5.71 (s, 0.6H, CH–N), 5.31 (d, J = 16.0 Hz, 0.4H, CH2–N), 5.21 (s, 0.4H, CH–N), 4.41 (d, J = 16.0 Hz, 0.6H, CH2–N), 4.24–4.09 (m, 1H, CH2–N), 3.77–3.70 (m, 0.5H, HCN of cyclohexyl), 3.60–3.36 (m, 1H, HCN of cyclohexyl), 2.90 (bs, 0.5H, HCN of cyclohexyl), 2.07 (s, 1.2H, HMe–Ar), 1.99 (s, 1.8H, HMe–Ar), 1.83–1.50 (m, 9H, HAliphatic), 1.37–0.90 (m, 17H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): (mixture of rotamers) δ 175.90, 175.17, 173.72, 172.97, 170.30, 166.58, 138.76, 138.30, 137.62, 136.68, 136.00, 133.45, 131.03, 130.08, 129.68, 128.44, 127.95, 127.86, 127.55, 126.78, 126.59, 126.51, 125.82, 63.08, 60.73, 50.53, 50.43, 49.65, 49.28, 48.86, 48.50, 47.44, 47.42, 32.99, 32.50, 31.79, 30.71, 27.84, 27.36, 25.73, 25.53, 25.31, 25.11, 24.98, 24.47, 23.52, 22.06, 19.74, 19.44. MS m/z: 531 (M+, 3.04), 405 (15.95), 335 (27.76), 301 (55.63), 210 (100), 196 (34.08), 91 (64.94), 83 (56.43), 55 (41.38). Calcd for C33H45N3O3: C, 74.54; H, 8.53; N, 7.90; found C, 74.42; H, 8.66; N, 7.78.

N1-Benzyl-N3-(tert-butyl)-N1-(1-(cyclohexylamino)-1-oxopentan-2-yl)-2,2-dimethylmalonamide (8n) White powders: 353 mg, 77% yield; mp 69–69 °C. IR (ATR) cm−1: 3289, 2926, 2855, 1631, 1529. 1H NMR (300 MHz, CDCl3): (mixture of rotamers) δ 7.38–7.13 (m, 5H, HAr), 6.50–6.47 (m, 0.5H), 5.91 (bs, 0.6H), 5.74 (bs, 0.4H), 4.69–4.22 (m, 2H), 3.88–3.85 (m, 0.5H), 3.71–3.66 (m, 1H), 3.51–3.43 (m, 0.5H), 2.34–2.21 (m, 0.5H), 1.94–0.71 (m, 32H, HAliphatic). 13C NMR (75 MHz, CDCl3): (mixture of rotamers) δ 175.56, 174.76, 173.32, 172.28, 170.03, 167.44, 138.64, 136.22, 128.64, 128.06, 127.35, 126.49, 62.39, 60.25, 53.50, 51.84, 51.68, 50.99, 48.66, 48.30, 47.72, 34.65, 33.13, 32.80, 32.56, 32.27, 29.05, 28.82, 28.52, 27.91, 26.89, 26.07, 25.59, 25.26, 24.81, 24.45, 22.86, 20.89, 20.63, 14.07, 13.87. MS m/z: 457 (M+, 6.01), 359 (43.43), 331 (39.79), 287 (45.86), 162 (100), 91 (59.28), 57 (61.75). Anal. Calcd for C27H43N3O3: C, 70.86; H, 9.47; N, 9.18; found C, 70.99; H, 9.43; N, 9.01.

N1-(tert-Butyl)-N3-(2-(cyclohexylamino)-2-oxo-1-phenylethyl)-N3-(3,4-dichlorophenyl)-2,2-dimethylmalonamide (8o) White powders: 475 mg, 87% yield; mp 183–185 °C. IR (ATR) cm−1: 3323, 3057, 2924, 2842, 1636, 1552, 1526. 1H NMR (300 MHz, DMSO-d6): δ 8.14–8.12 (m, 1H, NH), 7.91 (bs, 1H, NH), 7.13–7.08 (m, 4H, HAr), 7.00–6.96 (m, 3H, HAr), 6.39–6.33 (m, 1H, HAr), 5.98 (s, 1H, CH–N), 3.60 (bs, 1H, HCN of cyclohexyl), 1.99–1.52 (m, 6H, HAliphatic), 1.26–0.99 (m, 19H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): δ 173.10, 172.51, 169.24, 138.94, 135.32, 133.69, 130.89, 130.25, 129.54, 128.30, 66.14, 52.05, 51.16, 48.51, 32.64, 32.59, 28.69, 25.69, 25.07, 24.94. MS m/z: 549 (M+  + 4, 0.48), 547 (M+  + 2, 1.49), 545 (M+, 1.54), 419 (3.93), 392 (6.48), 376 (13.96), 250 (81.74), 170 (80.50), 143 (60.34), 83 (23.78), 57 (100). Anal. Calcd for C29H37Cl2N3O3: C, 63.73; H, 6.82; N, 7.69; found C, 63.51; H, 6.89; N, 7.79.

N1-(2-Chlorobenzyl)-N3-cyclohexyl-N1-(2-(cyclohexylamino)-1-(3-methoxyphenyl)-2-oxoethyl)-2,2-dimethylmalonamide (8p) White powders: 512 mg, 88% yield; mp 188–190 °C. IR (ATR) cm−1: 3317, 3253, 2929, 2852, 1672, 1621, 1539. 1H NMR (300 MHz, DMSO-d6): (mixture of rotamers) δ 8.00–7.71 (m, 2H, NH), 7.24–7.03 (m, 5H, HAr), 6.83–6.65 (m, 3H, HAr), 5.61 (s, 0.6H, CH–N), 5.51 (s, 0.4H, CH–N), 4.73 (d, J = 17.1 Hz, 0.5H, CH2–N), 4.44–4.38 (m, 1.5H, CH2–N), 3.75–3.55 (m, 5H, HCN of cyclohexyl and O-CH3), 1.85–1.55 (m, 10H, HAliphatic), 1.39–0.99 (m, 16H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): (mixture of rotamers): δ 175.18, 173.86, 173.29, 172.89, 169.71, 168.19, 159.42, 159.15, 158.85, 137.85, 135.76, 135.57, 130.86, 129.44, 129.03, 128.65, 128.37, 127.54, 126.95, 122.74, 121.35, 115.40, 114.58, 113.90, 64.60, 63.47, 55.34, 50.28, 49.16, 48.77, 48.45, 48.33, 47.84, 32.43, 27.40, 26.65, 25.65, 25.25, 24.96, 23.28. MS m/z: 583 (M+  + 2, 1.06), 581 (M+, 3.04), 455 (8.81), 385 (15.95), 331 (39.96), 260 (100), 196 (35.63), 125 (41.34), 83 (53.46), 55 (39.55). Anal. Calcd for C33H44ClN3O4: C, 68.08; H, 7.62; N, 7.22; found C, 68.01; H, 7.71; N, 7.05.

N1-Benzyl-N3-(tert-butyl)-N1-(2-(cyclohexylamino)-2-oxo-1-phenylethyl)-2,2-dimethylmalonamide (8q) White powders: 428 mg, 87% yield; mp 224 °C. IR (ATR) cm−1: 3284, 3082, 2929, 2852, 1651, 1626, 1552. 1H NMR (300 MHz, DMSO-d6): (major of rotamers) δ 7.83–7.81 (s, 2H, NH), 7.32 (bs, 1H, HAr), 7.21–7.00 (m, 7 H, HAr), 6.82–6.77 (m, 2H, HAr), 5.34 (s, 1H, CH–N), 4.49 (d, J = 17.5 Hz, CH2–N), 4.14 (d, J = 17.5 Hz, 1H, CH2–N), 3.52 (bs, 1H, HCN of cyclohexyl), 1.76–0.92 (m, 25 H, HAliphatic). 13C NMR (75 MHz, DMSO-d6): (major rotamer) δ 175.19, 173.48, 169.71, 138.15, 134.69, 130.66, 128.36, 127.97, 127.11, 126.56, 64.97, 51.24, 50.82, 50.35, 48.23, 32.45, 28.72, 27.47, 25.65, 24.89, 24.75, 23.85. MS m/z: 491 (M+, 3.91), 393 (13.91), 365 (17.39), 321 (27.08), 275 (95.40), 196 (100), 170 (50.64), 91 (78.43), 83 (22.72), 55 (29.59). Anal. Calcd for C30H41N3O3: C, 73.29; H, 8.41; N, 8.55; found C, 73.48; H, 8.56; N, 8.37.

N1-Benzyl-N3-(tert-butyl)-N1-(2-(tert-butylamino)-1-(4-nitrophenyl)-2-oxoethyl)-2,2-dimethylmalonamide (8r) Yellow powders: 383 mg, 75% yield; mp 88 °C. IR (ATR) cm−1: 3310, 2975, 2934, 1659, 1644, 1513. 1H NMR (300 MHz, CDCl3): δ 7.95 (s, 1H, NH), 7.92 (s, 1H, NH), 7.28–7.21 (m, 3H, HAr), 7.18–7.08 (m, 3H, HAr), 6.97–6.94 (m, 2H, HAr), 6.61 (bs, 1H, HAr), 5.60 (s, 1H, CH–N), 4.62 (d, J = 16.0 Hz, 1H, CH2–N), 4.44 (d, J = 16.0 Hz, 1H, CH2–N), 1.58–1.00 (m, 24H, HAliphatic). 13C NMR (75 MHz, CDCl3): δ 175.32, 172.91, 167.64, 147.25, 142.87, 135.68, 131.03, 128.50, 127.95, 127.76, 123.12, 77.32, 67.01, 54.29, 52.08, 51.76, 50.78, 28.55, 28.45, 25.84, 24.67. MS m/z: 510 (M+, 0.78), 340 (12.23), 320 (23.55), 275 (36.96), 241 (20.47), 170 (62.04), 91 (72.58), 57 (100). Anal. Calcd for C28H38N4O5: C, 65.86; H, 7.50; N, 10.97; found C, 65.90; H, 7.65; N, 10.78.