Design and synthesis of novel chloramphenicol amine derivatives as potent aminopeptidase N (APN/CD13) inhibitors.

Herein we report a series of novel chloramphenicol amine derivatives as aminopeptidase N (APN)/CD13 inhibitors. All compounds were synthesized starting from commercially available (1S,2S)-2-amino-1-(4-nitrophenyl) propane-1,3-diol. The preliminary biological screening showed that some compounds exhibited potent inhibitory activity against APN. It should be noted that one compound, 13b (IC(50)=7.1 microM), possess similar APN inhibitory activity compared with Bestatin (IC(50)=3.0 microM).

Introduction

Aminopeptidase N (APN/CD13) is a Zn2+-dependent metalloectopeptidase existing in a wide variety of human tissues and cell lines (endothelial, epithelial, fibroblast, leukocyte, etc.). APN expression is dysregulated in inflammatory diseases and in cancers (solid and hematologic tumors). APN is also a receptor for coronaviruses, the pathogenesis of upper respiratory infection. In recent years, the identification of activities of anti-APN/CD13 monoclonal antibody and natural/synthetic APN inhibitors suggests that APN plays a crucial role in modulating bioactive peptide responses (pain management, vasopressin release) and in influencing immune functions and major biological events (cell proliferation, secretion, invasion, angiogenesis). Therefore, the development of APN inhibitors may be of clinically significance for the discovery of anti-cancer and anti-inflammatory agents.

For most APN inhibitors reported in literatures, so far there are four possible binding sites, including a zinc binding group (ZBG), two hydrophobic groups interacting with the S1 and subsites in APN, and one appropriate group interacting with the subsite surrounded by several Arg residues.3, 4, 5 It should be emphasized that among these four sites, at least three ones are necessary for a potent inhibitor. For example, AHPA ((2S,3R)-3-amino-2-hydroxyl-4-phenylbutanoic acid) is a common scaffold in many natural APN inhibitors. The co-crystal complex of APN and Bestatin (AHPA-Leu) from Escherichia coli indicated that the hydroxyl and carbonyl belong to zinc binding group (ZBG). The phenyl ring in AHPA fragment can insert into S1 pocket and amino group in AHPA can interact with Glu 350 (Fig. 1 ).

Figure 1

The binding mode of bestatin to the active sites of APN.

Our group has previously reported several kinds of new APN inhibitors, such as l-lysine derivates, 3-galloylamido-N′-substituted 2,6-piperidinedione-N-acetamide peptidomimetics, 1,3,4-thiadiazole derivates, and AHPA (β-amino-α-hydroxyl-phenylbutanoic acid) derivates. In our recent screening, compound (2R,3S)-2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic acid (1, AHNPA) developed from commercially available chloramphenicol amine 2, exhibited a moderate APN inhibition (IC50  = 140.2 μM). Considering the structural similarity between compound 1 and AHPA, this molecule could act as a new lead compound for further chemical modification and optimization. Therefore, different amino acids and other functional groups can be coupled with compound 1 to generate diverse dipeptide and tripetide derivatives. In this article, we would like to describe the synthesis, enzymatic evaluation and possible binding mode of the chloramphenicol amine derivates.

Chemistry

The synthetic route of target compounds is shown in Scheme 1, Scheme 2 . Chloramphenicol amine (1S,2R)-2-amino-1-(4-nitrophenyl)propane-1,3-diol, 2) was firstly protected by (Boc)2O, then was selectively oxidized at the terminal hydroxyl group to give the N-Boc-protected acid 4. The compound 4 was easily deprotected with 3 N HCl in EtOAc to provide product 1. The key intermediate 4 can be coupled with different substituted amine by classical EDCI/HOBt method, and then converted into compound 6a–6m after deprotection. Using the same procedure, different moieties, such as ω-amino acids, natural α-amino acids and dipeptides, were coupled with intermediate 4 to obtain AHNPA derivatives 7a–7b, 9a–9e, 12a–12b. Finally, the esters were hydrolyzed to carboxylic acid by NaOH/H2O in methanol, and then converted into target compounds 8a–8b, 10a–10e, and 13a–13b by deprotecting Boc group. Compounds 11a–11b containing hydroximic acid groups were obtained from 9a–9b by reacting with NH2OK and then cleaving Boc.

Scheme 1

Reagents and conditions: (a) (Boc)2O/THF; (b) 13% w/w NaOCl, 5% NaHCO3,TEMPO/CH3COCH3, H2O, 0 °C; (c) HCl/anhydrous EtOAc, Na2CO3.

Scheme 2

Reagents and conditions: (a) EDCI, HOBt/anhydrous THF, 0 °C to room temperature; (b) HCl/anhydrous EtOAc, Na2CO3; (c) (i) 4% NaOH/CH3OH; (ii) HCl/anhydrous EtOAc, Na2CO3; (d) (i) NH2OKa/anhydrous CH3OH; (ii) HCl/anhydrous EtOAc, Na2CO3.

Results and discussion

All the target compounds were tested for their inhibition against APN as listed in Table 1 . As a result, the structural addition of different alkylamine, simple benzylamine and phenethylamine can not significantly enhance the inhibitory activity compared with parent compound AHNPA (IC50  = 140.2 μM). It seems that the substitution of phenyl ring in phenethyl group of R position can slightly contribute to the affinity. For example, para-hydroxy substitution shows up to 50 μM IC50 value.

Table 1

The structure and inhibitory activities of compounds 6a–6m, 8a–8b against APN

Compound	R	APN/IC50 (μM)
6a	–CH2CH2CH3	183.2
6b	–CH2(CH3)2	250.7
6c	–CH2CH2CH3	232.6
6d	–CH2CH2CH3	521.7
6e		251.3
6f		169.9
6g		302.5
6h		144.7
6i		106.3
6j		136.8
6k		51.4
6l		116.3
6m		90.3
8a		2891.2
8b		1388.6

On the other hand, the incorporation of natural amino acids would generate compounds with similar or better inhibitory activities compared with parent compound AHNPA, while ω amino acid derivative showed very poor inhibition (Table 2 ). These results suggest that the introduction of natural amino acids to AHNPA scaffold would be better than of simple nonpeptide fragment. As a zinc binding group (ZBG), hydroximate group has been used in many enzyme inhibitors especially Zn2+-dependent enzyme to improve affinity. In our case, the AHNPA-amino acid derivatives can also enhance their activity after converting terminal carboxylate to hydroximate group. For instance, compounds 11a and 11b shows more potent inhibition than compounds 10a and 10b.

Table 2

The structure and inhibitory activities of AHNPA-amino acid and AHPNPA-dipeptide derivatives against APN

Compound	R1	R2	APN/IC50 (μM)
10a		–OH	85.6
10b		–OH	60.4
10c		–OH	159.9
10d		–OH	102.8
10e		–OH	162.2
11a		–NHOH	54.0
11b		–NHOH	31.3
13a			34.6
13b			7.10
Bestatin			3.00

The enzyme inhibitory results unveiled that AHNPA-dipeptide derivative 13b shows the best inhibition among all target compounds. This phenomenon suggests that the dipeptide side chain would be helpful to increase the interaction with APN binding sites. Furthermore, compounds 13a and 13b were further assessed on their inhibition against HL-60 cell proliferation by using MTT method. The IC50 values of these two compounds (13a 2.02 ± 0.13 mM and 13b 2.21 ± 0.11 mM) confirmed again that their inhibitory activities are similar with Bestatin (1.65 ± 0.09 mM) (Fig. 2 ).

Figure 2

Effects of bestatin and compound 13a–13b on the HL-60 cell line proliferation. Each column represents the mean values with S.E values for five independent experiments.

In order to determine the interaction between AHNPA derivatives and APN, compound 13b was docked into the active sites of APN (PDB entry: 2DQM) using sybyl 7.0. The docking results showed that the carbonyl group and amine group of compound 13b could chelate with the zinc ion in APN. The nitrophenyl group of the 13b could insert to the pocket S1 by forming hydrophobic interactions with this subsite. The phenyl ring in the terminal amino acid residue could also insert to the pocket , in the meantime another phenyl group closed to the AHNPA scaffold could have interaction with the pocket (Fig. 3 ). His297, Glu298 and His301 are the essential amino acids of the conserved sequence (HEXXHX18E) in the catalytic domain of peptidase M1 family. Compound 13b could form hydrogen bonds with these three residues at the distance of 2.40 Å, 3.06 Å, 2.79 Å, respectively. In addition, the carboxylate group of 13b could interact with Arg825 and Tyr381 residue in the pocket by hydrogen bond for stabilizing the reaction intermediate with the zinc ion (Fig. 4 ).

Figure 3

The docking result of 13b is showed by sybyl7.0 (Bestatin in the X-ray crystal is showed in red).

Figure 4

The docking result of 13b is showed by Ligplot.

Conclusions

In summary, we reported the synthesis and biological evaluation of a series of novel chloramphenicol amine derivatives as potent APN inhibitors. Compound 13b is the most active. Among these compounds, compound 13b not only exhibited similar enzymatic inhibition compared with natural APN inhibitor Bestatin, but also showed good cell-based inhibitory activity. Therefore, 13b could be a lead compound to search new chloramphenicol amine derivatives as APN inhibitors.

Experimental

APN inhibition assay

IC50 values against APN were determined by using l-Leu-p-nitroanilide as substrate and Microsomal aminopeptidase from Porcine Kidney Microsomes (Sigma) as the enzyme in 50 mM PBS, pH 7.2, at 37 °C. The hydrolysis of the substrate was monitored by following the change in the absorbance measured at 405 nm with the UV–vis spectrophotometer Pharmacia LKB, Biochrom 4060. All solutions of inhibitors were prepared in the assay buffer, and pH was neutralized to 7.5 by the addition of 0.1 M HCl or 0.1 M NaOH. All inhibitors were pre-incubated with APN for 30 min at room temperature. The assay mixture, which contained the inhibitor solution (concentration dependent on the inhibitor), the enzyme solution (4 μg/mL final concentration), and the assay buffer, was adjusted to 200 μL.

MTT assay

HL-60 Cell was grown in RPMI1640 medium containing 10% FBS at 37 °C in 5% CO2 humidified incubator. Cell proliferation was determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-2.5-diphenyl-2H-tetrazolium bromide) assay. Briefly, cells were plated in a 96-well plate at 10,000 cells per well, cultured for 4 h in complete growth medium, then treated with 2000, 1000, 500, 250, 125 μg/mL of compounds for 48 h. 0.5% MTT solution was added to each well. After further incubation for 4hr, formazan formed from MTT was extracted by adding DMSO and mixing for 15 min. Optical density was read with an ELISA reader at 570 nm.

Chemistry: general procedures

The starting material 2 is a white powder (mp = 159–162 °C, (c 1, MeOH)), purchased from Wuhan Kaitong Fine Chemical Co., Ltd, China. Unless otherwise specified, other materials were purchased from commercial suppliers and used without further purification. Solvents were distilled prior to use and flash chromatography was performed using silica gel (60 Å, 200 ± 300 mesh).Melting points are uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra were recorded at either 300 MHz. Chemical shifts are reported in delta (δ) units, parts per million (ppm) downfield from trimethylsilane. High-resolution mass spectral (HRMS) data are reported as m/e (relative intensity). Elemental analyses for compounds were performed using an elementar vario EL III CN analyzer (Germany).

N-Protected (1S,2S)(+)-2-amino-1-(4-nitrophenyl)-1,3-propanediol (3)

To a solution of compound 2 (2.12 g, 10 mmol) in THF (10 mL) was added dropwise a solution of di-tert-butyl dicarbonate (2.93 g, 11 mmol) in THF (5 mL). After 24 h stirring at room temperature the solvent was concentrated under vacuum to leave a residue. The crude product was recrystallized with EtOAc to give white solid (2.80 g), yield: 90%, mp = 115–116 °C, (c 1, MeOH). ESI-MS m/z: 313.3 (M+H); 1H NMR (DMSO-d 6) δ 1.214 (s, 9H), 3.478–3.704 (m, 2H), 4.771–4.790 (m, 1H), 5.607 (d, J  = 5.40, 1H), 6.194 (d, J  = 9.00, 1H), 7.564 (d, J  = 8.40, 2H), 8.181 (d, J  = 8.40, 2H). Anal. Calcd for C14H20N2O6: C, 53.84; H, 6.45; N, 8.97. Found: C, 53.79; H, 6.31; N, 8.77.

(2R,3S)-2-(tert-Butoxycarbonylamino)-3-hydroxy-3-(4-nitrophenyl) propanoic acid (4)

To a solution of compound 3 (3.12 g, 10 mmol) in acetone (50 mL) containing TEMPO (0.16 g, 1 mmol) was added a 5% aqueous NaHCO3 solution (50 ml). The mixture was cooled to 0 °C and NaOCl (20.5 mL, ca. 10% w/w) was added dropwise over 15 min while following the reaction by TLC. The solution was concentrated under vacuum to remove acetone. The remaining mixture was washed with EtOAc (2 × 10 mL), acidified to pH 1–2 by a 1 N citric acid solution, extracted with EtOAc (3 × 20 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash column chromatography(DCM/MeOH, 20:1 (V/V)) to give the desired compound 4 as a white solid (0.97 g), yield: 30%, mp = 80–82 °C, (c 1, MeOH). ESI-MS m/z: 327.5 (M+H); 1H NMR (DMSO-d 6) δ 1.224 (s, 9H), 4.344 (dd, J  = 9.60, J  = 3.00, 1H), 5.265 (d, J  = 3.00, 1H), 6.483 (d, J  = 9.60, 1H), 7.658 (d, J  = 8.40, 2H), 8.190 (d, J  = 8.40, 2H). Anal. Calcd for C14H18N2O7: C, 51.53; H, 5.56; N, 8.59. Found: C, 51.49; H, 5.62; N, 8.37.

(2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)propanoic acid (1)

To a solution of compound 4 (3.26 g, 10 mmol) in dry EtOAc at 0 °C was added dropwise a solution of EtOAc (10 mL) saturated with dry HCl gas. The reaction solution was stirred at 0 °C for 2 h, and then the temperature is raised to room temperature and the reaction proceeds for 5 h before being concentrated in vacuo. The residue was dissolved in water (5 mL), and then washed with EtOAc (2 × 5 mL). 1 N NaOH was added to the mixture until pH 6–7, and the solid was crystallized out. The solution was removed by filtration, and the remaining solid was sequentially washed with water and dried overnight, then recrystallized with MeOH to give a yellow crystals, yield: 74.2%, mp = 187–189 °C, (c 1, MeOH). ESI-MS m/z: 227.3 (M+H); 1H NMR (DMSO-d 6) δ 3.425 (d, J  = 3.90, 1H), 5.193 (d, J  = 3.90, 1H), 7.659 (d, J  = 8.70, 2H), 8.194 (d, J  = 8.70, 2H). Anal. Calcd for C9H10N2O5: C, 47.79; H, 4.46; N, 12.39. Found: C, 47.58; H, 4.31; N, 12.27.

(2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)-N-propylpropanamide (6a)

To a 150 mL solution of Compound 4 (3.26 g, 10 mmol), 1-propylamine (0.71 g, 12 mmol), and HOBt (1.62 g, 12 mmol) in dry THF, was added TEA (1.11 g, 11 mmol). The reaction mixture was gently cooled to 0 °C in ice bath. To the reaction mixture was added dropwise a solution of EDCI (3.82 g, 20 mmol) in THF for 1 h. After removal of the ice bath, the reaction mixture was stirred at room temperature for 12 h and filtered to remove the precipitate. The filtrate was washed with 1 N citric acid solution, saturated NaHCO3 and brine, dried over Na2SO4, and evaporated in vacuo to give the crude product compound 5a (3.15 g, yield 86%). This product was used for the following reaction without further purification, yield 84%, mp = 104–106 °C, (c 1, MeOH). ESI-MS m/z: 368.2 (M+H); 1H NMR (DMSO-d 6) δ 0.828–0.835 (m, 3H), 1.251 (s, 9H), 1.301–1.405 (m, 2H), 2.843–2.904 (m, 2H), 3.203 (d, J  = 3.90, 1H), 5.009 (d, J  = 3.90, 1H), 7.895 (m, 2H), 7.621 (d, J  = 8.70, 2H), 8.187 (d, J  = 8.70, 2H). Anal. Calcd for C17H25N3O6: C, 55.58; H, 6.86; N, 11.44. Found: C, 55.67; H, 6.59; N, 11.83.

To a solution of compound 5a (1.83 g, 5 mmol) in dry EtOAc at 0 °C was added dropwise a solution of EtOAc (10 mL) saturated by dry HCl gas. The reaction solution was stirred at 0 °C for 2 h, and then the temperature is raised to room temperature and the reaction proceeds for 5 h before being concentrated in vacuo. The residue was dissolved by saturated Na2CO3 (10 mL), the aqueous solution was extracted with ethyl ether (3 × 30 mL). The extractions were combined, washed with brine, dried over Na2SO4, and evaporated in vacuo. The residue was purified by flash column chromatography(DCM/MeOH, 6/1) to give the desired compound 6a as a white solid (1.12 g), yield 84%, mp = 118–120 °C, (c 1, MeOH). ESI-MS m/z: 268.3 (M+H); 1H NMR (DMSO-d 6) δ 0.738–0.787 (m, 3H), 1.290–1.407 (m, 2H), 2.898–3.104 (m, 2H), 3.234 (d, J  = 3.90, 1H), 5.018 (d, J  = 3.90, 1H), 7.894 (t, J  = 6.00, 1H), 7.613 (d, J  = 8.70, 2H), 8.187 (d, J  = 8.70, 2H). Anal. Calcd for C12H17N3O4: C, 53.92; H, 6.41; N, 15.72. Found: C, 78.67; H, 7.70; N, 4.12.

The other compounds of 6 series were synthesized following the general procedure as described above.

(2R,3S)-2-Amino-3-hydroxy-N-isopropyl-3-(4-nitrophenyl)-propanamide (6b)

White solid, yield 79%, mp = 132–134 °C, (c 1, MeOH). ESI-MS m/z: 268.3 (M+H);1H NMR (DMSO-d 6) δ 0.926 (d, J  = 6.60, 3H), 1.033 (d, J  = 6.60, 3H), 3.193 (d, J  = 4.20, 1H), 3.748–3.862 (m, 1H), 4.963 (d, J  = 4.20, 1H), 7.591 (d, J  = 8.70, 2H), 7.651 (d, J  = 7.80, 1H), 8.182 (d, J  = 8.70, 2H). Anal. Calcd for C12H17N3O4: C, 53.92; H, 6.41; N, 15.72. Found: C, 53.89; H, 6.48; N, 15.91.

(2R,3S)-2-Amino-N-butyl-3-hydroxy-3-(4-nitrophenyl)propanamide (6c)

White solid, yield 83%, mp = 88–89 °C, (c 1, MeOH). ESI-MS m/z: 282.4 (M+H); 1H NMR (DMSO-d 6) δ 0.786–0.835 (m, 3H), 1.082–1.204 (m, 2H), 1.242–1.333 (m, 2H), 2.908–3.233 (m, 2H), 3.234 (d, J  = 3.90, 1H), 4.982 (d, J  = 3.90, 1H), 7.591 (d, J  = 8.70, 2H), 7.854 (t, J  = 6.00, 1H), 8.188 (d, J  = 8.70, 2H). Anal. Calcd for C13H19N3O4: C, 55.50; H, 6.81; N, 14.94. Found: C, 55.31; H, 6.76; N, 14.83.

(2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)-N-pentylpropanamide (6d)

White solid, yield 83%, mp = 112–144 °C, (c 1, MeOH). ESI-MS m/z: 296.4 (M+H); 1H NMR (DMSO-d 6) δ 0.795–0.842 (m, 3H), 1.098–1.319 (m, 6H), 2.908–2.970 (m, 1H), 3.044–3.108 (m, 1H), 3.228 (d, J  = 3.60, 1H), 4.968 (d, J  = 3.60, 1H), 7.597 (d, J  = 8.70, 2H) 7.849 (t, J  = 6.00, 1H.), 8.176 (d, J  = 8.70,2H). Anal. Calcd for C14H21N3O4: C, 56.94; H, 7.17; N, 14.23. Found: C, 56.92; H, 7.07; N, 14.29.

(2R,3S)-2-Amino-N-cyclohexyl-3-hydroxy-3-(4-nitrophenyl)propanamide (6e)

White solid, yield 83%, mp = 127–129 °C, (c 1, MeOH). ESI-MS m/z: 308.5 (M+H); 1H NMR (DMSO-d 6) δ 0.679–1.312 (m, 6H), 1.437–1.990 (m, 4H), 3.417 (d, J  = 4.50, 1H), 3.393–4.064 (m, 1H), 4.988 (d, J  = 4.50, 1H), 7.659 (d, J  = 9.00, 1H), 8.238 (d, J  = 9.00, 2H), 8.283 (d, J  = 7.80, 1H). Anal. Calcd for C15H21N3O4: C, 58.62; H, 6.89; N, 13.67. Found: C, 58.42; H, 6.40; N, 13.99.

(2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)-N-propylpropanamide (6f)

White solid, yield 88%, mp = 125–127 °C, (c 1, MeOH). ESI-MS m/z: 316.3 [M+H]; 1H NMR (DMSO-d 6) δ 3.355 (d, J  = 3.90, 1H). 4.217 (dd, J  = 5.70, J  = 15.70, 1H), 4.326 (dd, J  = 5.70, J  = 15.70, 1H), 5.040 (d, J  = 3.90, 1H), 7.181–7.285 (m, 5H), 7.618 (d, J  = 8.70, 2H), 8.157 (d, J  = 8.70, 2H), 8.442 (t, J  = 5.70, 1H). Anal. Calcd for C16H17N3O4: C, 60.94; H, 5.43; N, 13.33. Found: C, 60.44; H, 5.73; N, 12.97.

(2R,3S)-2-Amino-N-(4-fluorobenzyl)-3-hydroxy-3-(4-nitrophenyl) propanamide (6g)

White solid, yield 81.5%, mp = 115–118 °C, (c 1, MeOH). ESI-MS m/z: 334.3 (M+H); 1H NMR (DMSO-d 6) δ 3.327 (d, J  = 3.60, 1H), 4.193 (dd, J  = 6.00, J  = 15.00, 1H), 4.294 (dd, J  = 6.00, J  = 15.00, 1H), 5.053 (d, J  = 3.60, 1H), 7.052–7.111 (m, 2H), 7.187–7.235 (m, 2H) 7.593 (d, J  = 8.70, 2H), 8.155 (d, J  = 8.70, 2H), 8.458 (t, J  = 6.30,1H). Anal. Calcd for C16H16FN3O4: C, 57.56; H, 4.84; N, 12.61. Found: C, 57.09; H, 4.82; N, 12.30.

(2R,3S)-2-Amino-3-hydroxy-N-(4-methoxybenzyl)-3-(4-nitrophenyl) propanamide (6h)

White solid, yield 66.7%, mp = 141–143 °C, (c 1, MeOH). ESI-MS m/z: 346.3 (M+H); 1H NMR (DMSO-d 6) δ 3.330 (d, J  = 3.30, 1H), 3.717 (s, 3H), 4.114 (dd, J  = 6.30, J  = 15.70, 1H), 4.225 (dd, J  = 6.30, J  = 15.70, 1H), 5.014 (d, J  = 3.30, 1H), 7.061 (d, J  = 8.70, 2H), 7.592 (d, J  = 8.70, 2H), 7.907 (d, J  = 8.70, 2H), 8.147 (d, J  = 8.70,2H), 8.325 (t, J  = 6.30, 1H). Anal. Calcd for C17H19N3O5: C, 59.12; H, 5.55; N, 12.17. Found: C, 59.15; H, 5.61; N, 12.48.

(2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)-N-phenethylpropanamide (6i)

White solid, yield 76%, mp = 126–128 °C, (c 1, MeOH). ESI-MS m/z: 330.3 (M+H); 1H NMR (DMSO-d 6) δ 2.664 (t, J  = 7.50, 2H), 3.246 (d, J  = 3.60, 1H), 3.286–3.349 (m, 2H), 5.059 (d, J  = 3.60, 1H), 7.167–7.213 (m, 3H), 7.225–7.306 (m, 2H), 7.593 (d, J  = 8.70, 2H), 8.037 (t, J  = 5.70, 1H), 8.182 (d, J  = 8.70, 2H). Anal. Calcd for C17H19N3O4: C, 62.00; H, 5.81; N, 12.76. Found: C, 62.13; H, 5.80; N, 12.65.

(2R,3S)-2-Amino-N-(2-chlorophenethyl)-3-hydroxy-3-(4-nitrophenyl) propanamide (6j)

White solid, yield 72.3%, mp = 140–142 °C, (c 1, MeOH). ESI-MS m/z: 364.2 (M+H); 1H NMR (DMSO-d 6) δ 2.730 (t, J  = 6.30, 1H), 3.220–3.391 (m, 2H), 3.398 (d, J  = 3.9, 1H), 5.041 (d, J  = 3.90, 1H), 7.226–7.260 (m, 3H), 7.400–7.432 (m, 1H), 7.601 (d, J  = 8.70, 2H), 8.184 (t, J  = 5.70, 1H), 8.213 (d, J  = 8.70,2H). Anal. Calcd for C17H18ClN3O4: C, 56.13; H, 4.99; N, 11.55. Found: C, 56.52; H, 5.01; N, 11.54.

(2R,3S)-2-Amino-3-hydroxy-N-(4-hydroxyphenethyl)-3-(4-nitrophenyl) propanamide (6k)

White solid, yield 83%, mp = 99–100 °C, (c 1, MeOH). ESI-MS m/z: 346.3 (M+H); 1H NMR (DMSO-d 6) δ 2.538–2.563 (m, 2H), 3.145–3.164 (m, 2H), 3.188 (d, J  = 3.90, 1H), 5.067 (d, J  = 3.90, 1H), 6.657 (d, J  = 8.70, 2H), 6.953 (d, J  = 8.70, 2H), 7.591 (d, J  = 8.70, 2H), 7.988 (t, J  = 6.00, 1H), 8.187 (d, J  = 8.70, 2H). Anal. Calcd for C17H19N3O5: C, 59.12; H, 5.55; N, 12.17. Found: C, 59.08; H, 5.57; N, 12.46.

(2R,3S)-2-Amino-3-hydroxy-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-3-(4-nitrophenyl)propanamide (6l)

White solid, yield 80.3%, mp = 133–134 °C, (c 1, MeOH). ESI-MS m/z: 360.3 (M+H); 1H NMR (DMSO-d 6) δ 3.259–2.809 (m, 2H), 3.176–3.343 (m, 2H), 3.882 (d, J  = 3.30, 1H), 5.047 (d, J  = 3.30, 1H), 7.151–7.176 (m, 3H), 7.223–7.272 (m, 2H), 7.593 (d, J  = 8.70, 2H), 7.901 (d, J  = 8.70, 1H), 8.164 (d, J  = 8.70, 2H). Anal. Calcd for C18H21N3O5: C, 60.16; H, 5.89; N, 11.69. Found: C, 60.19; H, 5.88; N, 11.48.

(2R,3S)-2-Amino-N-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-3-hydroxy-3-(4-nitrophenyl) propanamide (6m)

White solid, yield 85.9%, mp = 136–137 °C, (c 1, MeOH). ESI-MS m/z: 374.4 (M+H); 1H NMR (DMSO-d 6) δ 2.581 (t, J  = 7.20, 2H), 3.171–3.214 (m, 2H), 3.236 (d, J  = 3.30, 1H), 5.060 (d, J  = 3.30, 1H), 5.961 (s, 2H), 6.622 (d, J  = 7.80, 1H), 6.756–6.819 (m, 2H), 7.696 (d, J  = 8.70, 2H), 7.998 (t, J  = 5.40, 1H), 8.183 (d, J  = 8.70 Hz, 2H). Anal. Calcd for C18H19N3O6: C, 57.90; H, 5.13; N, 11.25. Found: C, 57.84; H, 5.11; N, 11.20.

Compounds 8a and 8b were synthesized following the general procedure as described bellow (preparation of 12a).

4-((2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)propanamido)butanoic acid (8a)

White solid, yield 43.3%, mp = 141–143 °C, (c 1, MeOH). ESI-MS m/z: 312.3 (M+H); 1H NMR (DMSO-d 6) δ 1.186–1.210 (m, 2H), 2.137 (t, J  = 7.50, 2H), 2.996–3.020 (m, 2H), 3.581 (d, J  = 5.10, 1H), 5.024 (d, J  = 5.10, 1H), 7.649 (d, J  = 8.70, 2H), 8.204 (d, J  = 8.70, 2H), 8.292 (d, J  = 7.80, 1H). Anal. Calcd for C13H17N3O6: C, 50.16; H, 5.50; N, 13.50. Found: C, 50.12; H, 5.53; N, 13.60.

6-((2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)propanamido)hexanoic acid (8b)

White solid, yield 43.3%, mp = 150–153 °C, (c 1, MeOH). ESI-MS m/z: 340.3 (M+H); 1H NMR (DMSO-d 6) δ 1.066–1.165 (m, 2H), 1.214–1.430 (m, 4H), 2.134 (t, J  = 7.50, 2H), 3.006–3.055 (m, 2H), 3.583 (d, J  = 4.80, 1H), 5.028 (d, J  = 4.80, 1H), 7.633 (d, J  = 8.70, 2H), 8.204 (d, J  = 8.70,2H), 8.259 (d, J  = 7.00,1H). Anal. Calcd for C15H21N3O6: C, 53.09; H, 6.24; N, 12.38. Found: C, 53.04; H, 6.28; N, 12.36.

(S)-2-((2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)propanamido)-4-methylpentanoic acid (10a)

To a 150 mL solution of compound 4 (3.26 g, 10 mmol), l-leucine methyl ester hydrochloride (1.81 g, 10 mmol), and HOBt (1.62 g, 12 mmol) in dry THF, was added TEA (2.22 g, 22 mmol). The reaction mixture was gently cooled to 0 °C in ice bath, to the reaction mixture was added dropwise a solution of EDCI (3.82 g, 20 mmol) in THF for 1 h. After removal of the ice bath, the reaction mixture was stirred at room temperature for 12 h. and filtered to remove the precipitate. The filtrate was washed with 1 M citric acid solution, saturated NaHCO3 and brine, dried over Na2SO4, and evaporated in vacuo. The residue was purified by flash column chromatography (EtOAc/PE, 1:4(V/V)) to give the desired compound 9a as a white solid (4.08 g), yield 88.7%, mp = 141–143 °C, (c 1, MeOH). ESI-MS m/z: 454.3 (M+H); 1H NMR (DMSO-d 6) δ 0.789 (d, J  = 20.40, 6H), 1.235 (s, 9H), 1397–1.423 (m, 1H), 1.442–1.529 (m, 2H), 3.628 (s, 3H), 4.234 (d, J  = 4.50, 1H), 4.313–4.359 (m, 1H), 5.033 (d, J  = 4.50, 1H), 6.423 (s, J  = 9.60, 1H), 7.625 (d, J  = 8.40, 2H), 8.194 (d, J  = 8.40, 2H), 8.472 (d, J  = 7.80 Hz, 1H). Anal. Calcd for C21H31N3O8: C, 55.62; H, 6.89; N, 9.27. Found: C, 55.42; H, 6.83; N, 9.23.

To a solution of compound 9a (4.53 g, 10 mmol) in MeOH at 5 °C was added dropwise a solution of 1 N NaOH (12 mL). The reaction was warmed slowly to room temperature and stirred for 5 h. The solvent was concentrated under vacuum to remove the MeOH. The remaining mixture was added water (10 ml) and acidified to pH 1–2 by a 1 N citric acid solution. The precipitate was collected, washed with water (20 mL), and dried overnight. The crude product was then recrystallized with EtOAc to give the desired compound as white solid (4.39 g), yield 93.1%, mp = 128–130 °C, (c 1, MeOH). ESI-MS m/z: 440.7 (M+H); 1H NMR (DMSO-d 6) δ 0.809 (d, J  = 18.60, 6H), 1.231 (s, 9H), 1.330–1.420 (m, 1H), 1.421–1.439 (m, 2H), 4.165 (d, J  = 4.50, 1H), 4.318–4.363 (m, 1H), 5.054 (d, J  = 4.50, 1H), 6.398 (d, J  = 9.60, 1H), 7.633 (d, J  = 8.40, 2H), 8.197 (d, J  = 8.40, 2H), 8.332 (d, J  = 8.10, 1H). Anal. Calcd for C20H29N3O8: C, 54.66; H, 6.65; N, 9.56. Found: C, 54.51; H, 6.62; N, 9.57.

The compound 10a was synthesized from following the general procedure as described above (preparation of 1), white solid, yield 60%, mp = 203–205 °C, (c 1, MeOH). ESI-MS m/z: 340.4 (M+H); 1H NMR (DMSO-d 6) δ 0.731 (d, J  = 12.10, 6H), 1.121–1.142 (m, 1H), 1.336–1.354 (m, 2H), 3.417 (d, J  = 4.50, 1H), 4.094–4.113 (m, 1H), 4.882 (d, J  = 4.50, 1H), 7.631 (d, J  = 8.70, 2H), 8.092 (d, J  = 7.50, 1H), 8.180 (d, J  = 8.70, 2H). Anal. Calcd for C15H21N3O6: C, 53.09; H, 6.24; N, 12.38. Found: C, 53.50; H, 6.43; N, 12.30.

The other compounds of 10 series were synthesized following the general procedure as described above (preparation of 10a).

(S)-2-((2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)propanamido)-3-phenylpropanoic acid (10b)

White solid, yield 85.9%, mp = 215–218 °C, (c 1, MeOH). ESI-MS m/z: 374.4 (M+H); 1H NMR (DMSO-d 6) δ 2.854–3.013 (m, 2H), 3.462 (d, J  = 3.90, 1H), 4.375–4.442 (m, 1H), 5.061 (d, J  = 3.90, 1H), 7.079–7.105 (m, 2H), 7.132–7.243 (m, 3H), 7.606 (d, J  = 8.70, 2H), 8.165 (d, J  = 8.70, 2H), 8.272 (d, J  = 7.20, 1H). Anal. Calcd for C18H19N3O6: C, 57.90; H, 5.13; N, 11.25. Found: C, 57.85; H, 5.16; N, 11.23.

(S)-2-((2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)propanamido)-3-methylbutanoic acid (10c)

White solid, yield 85.9%, mp = 179–180 °C, (c 1, MeOH). ESI-MS m/z: 326.3 (M+H); 1H NMR (DMSO-d 6) δ 0.688–0.731 (m, 6H), 1.912–1.919 (m, 1H), 3.493 (d, J  = 3.00, 1H), 4.055–4.059 (m, 1H), 5.001–5.003 (d, J  = 3.00, 1H), 7.632 (d, J  = 8.70, 2H), 8.087 (d, J  = 6.00, 1H), 8.177 (d, J  = 8.70, 2H). Anal. Calcd for C18H19N3O6: C, 51.69; H, 5.89; N, 12.92. Found: C, 51.67; H, 5.49; N, 12.61.

(S)-2-((2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)propanamido)-3-methylbutanoic acid (10d)

White solid, yield 85.9%, mp = 178–179 °C, (c 1, MeOH). ESI-MS m/z: 340.3 (M+H); 1H NMR (DMSO-d 6) δ 0.685 (d, J  = 6.60, 3H), 0.752 (t, J  = 7.50, 3H), 0.939–0.940 (m, 1H), 1.210–1.213 (m, 1H), 1.591–1.594 (m, 1H), 3.504 (d, J  = 3.00, 1H), 4.095–4.098 (m, 1H), 4.964 (d, J  = 3.00, 1H), 7.632 (d, J  = 8.70, 2H), 8.081 (d, J  = 6.00, 1H), 8.195 (d, J  = 8.70, 2H). Anal. Calcd for C15H21N3O6: C, 53.09; H, 6.24; N, 12.38. Found: C, 53.51; H, 6.19; N, 12.40.

(S)-2-((2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)propanamido)-3-(1H-indol-2-yl) propanoic acid (10e)

White solid, yield 85.9%, mp = 179–180 °C, (c 1, MeOH). ESI-MS m/z: 413.4 (M+H); 1H NMR (DMSO-d 6) δ 3.064–3.113 (m, 2H), 3.163 (d, J  = 3.00, 1H), 4.487–4.490 (m, 1H), 5.106 (d, J  = 3.00, 1H), 6.965–7.030 (m, 1H), 7.054–7.078 (m, 2H), 7.327 (d, J  = 8.10, 1H), 7.504 (d, J  = 7.50, 1H), 7.599 (d, J  = 8.70, 2H), 8.132 (d, J  = 8.70, 2H), 8.275 (d, J  = 6.50, 1H). Anal. Calcd for C20H20N4O6: C, 58.25; H, 4.89; N, 13.59. Found: C, 58.60; H, 4.73; N, 13.54.

(S)-2-((2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)propanamido)-N-hydroxy-4-methylpentanamide (11a)

To a solution of compound 10a (4.53 g, 10 mmol) in MeOH at room temperature was added dropwise a solution of 1.5 N H2N–OK (12 mL), The reaction mixture was stirred for 1 h, and then acidified to pH 7 by acetic acid. The solvent was evaporated in vacuo to give the yellow solid. The crude product was purified by flash column chromatography (DCM/MeOH, 40:1 (V/V)) to give the desired compound as a white solid (3.15 g), yield 70.7%, mp = 153–155 °C, (c 1, MeOH). ESI-MS m/z: 455.3 (M+H); 1H NMR (DMSO-d 6) δ 0.545–0.610 (m, 6H), 0.703–0.801 (m, 1H), 0.934–1.187 (m, 2H), 3.954 (d, J  = 3.50, 1H), 4.062–4.082 (m, 1H), 4.903 (d, J  = 3.50, 1H), 7.652 (d, J  = 8.70, 2H), 8.246 (d, J  = 8.70, 2H), 8.746 (m, 2H).

The compound 11a was synthesized following the general procedure as described above (preparation of 1), yield 74.2%, mp = 212–213 °C. (c 1, MeOH). ESI-MS m/z: 355.6 (M+H); 1H NMR (DMSO-d 6) δ 0.548–0.609 (m, 6H), 0.701–0.811 (m, 1H), 0.911–1.198 (m, 2H), 3.967 (d, J  = 3.50, 1H), 4.063–4.081 (m, 1H), 4.968 (d, J  = 3.50, 1H), 7.651 (d, J  = 8.70, 2H), 8.244 (d, J  = 8.70, 2H), 8.746 (d, J  = 8.10, 2H). Anal. Calcd for C15H22N4O6: C, 50.84; H, 6.26; N, 15.81. Found: C, 50.90; H, 6.06; N, 16.17.

(2R,3S)-2-Amino-3-hydroxy-N-((S)-1-(hydroxyamino)-1-oxo-3-phenylpropan-2-yl)-3-(4-nitrophenyl)propanamide (11b)

The compound 11b was synthesized following the general procedure as described above, yield 74.2%, mp = 215–217 °C, (c 1, MeOH). ESI-MS m/z: 389.5 (M+H); 1H NMR (DMSO-d 6) δ 2.553–2.763 (m, 2H), 4.071 (d, J  = 3.30, 1H), 4.317–4.366 (m, 1H), 5.063 (d, J  = 3.30, 1H), 7.043–7.065 (m, 2H), 7.118–7.268 (m, 3H), 7.636 (d, J  = 8.70, 2H), 8.199 (d, J  = 8.70, 2H), 8.958 (d, J  = 8.10, 2H). Anal. Calcd for C18H20N4O6: C, 55.67; H, 5.19; N, 14.43. Found: C, 55.34; H, 5.16; N, 14.30.

(S)-2-((S)-2-((2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)propanamido)-4-methylpentanamido)-3-phenylpropanoic acid (13a)

Compounds 13a, 13b were synthesized following the general procedure as described above (preparation of 10a). White solid, yield 85.9%, mp = 115–116 °C, (c 1, MeOH). ESI-MS m/z: 487.4 (M+H); 1H NMR (DMSO-d 6) δ 0.692 (d, J  = 12.10, 6H), 1.023–1.110 (m, 1H), 1.218–1.287 (m, 2H), 2.849–3.053 (m, 2H), 3.742 (d, J  = 4.80, 1H), 4.114–4.225 (m, 1H), 4.252–4.295 (m, 1H), 4.919 (d, J  = 4.80, 1H), 7.179–7.279 (d, J  = 8.70, 5H), 7604 (d, J  = 8.70, 2H), 8.021 (t, J  = 7.50, 1H), 8.150 (t, J  = 7.30, 1H), 8.185 (d, J  = 8.70, 2H). Anal. Calcd for C24H30N4O7: C, 59.25; H, 6.22; N, 11.52. Found: C, 59.19; H, 6.34; N, 11.28.

(S)-2-((S)-2-((2R,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)propanamido)-3-phenylpropanamido)-3-phenylpropanoic acid (13b)

White solid, yield 85.9%, mp = 169–170 °C, (c 1, MeOH). ESI-MS m/z: 521.3 (M+H); 1H NMR (DMSO-d 6) δ 2.722–2.933 (m, 2H), 2.941–3.097 (m, 2H), 3.427 (d, J  = 3.60, 1H), 4.322–4.441 (m, 1H), 4.457–4.511 (m, 1H), 5.004 (d, J  = 3.60, 1H), 7.088–7.563 (m, 10H), 7.498 (d, J  = 8.70, 2H). 8.139 (d, J  = 8.70, 2H), 8.281 (t, J  = 7.30, 2H). Anal. Calcd for C27H28N4O7: C, 62.30; H, 5.42; N, 10.76. Found: C, 62.28; H, 5.37; N, 11.12.