Design, synthesis and preliminary activity evaluation of novel 3-amino-2-hydroxyl-3-phenylpropanoic acid derivatives as aminopeptidase N/CD13 inhibitors

Figures & data

Figure 1.  Novel aminopeptidase N inhibitors with 3-amino-2-hydroxy-3-phenylpropanoic acid scaffold.

Scheme 1.  Reagents and conditions: (a) (Boc)₂O, NaOH, THF; (b) EDCI, HOBT, DCM, NH₂-AA-COOCH₃; (c) NaOH, CH₃OH; (d) 3N HCl-EtOAc; (e) NH₂OK, CH₃OH;

Scheme 2.  Reagents and conditions: (a) EDCI, HOBT, DCM, NH²R⁴; (b) 3N HCl-EtOAc

Table 1. The structures and in vitro APN inhibitory activities of the target compounds.

Compound	R1	R2	IC50^a(μM)
			APN	MMP-2
5a	−CH2CH(CH3)2	−OH	351.71 ± 0.70	214.87 ± 0.49
5b	−CH(CH3)2	−OH	NA^b	287.42 ± 0.93
5c	−CH3	−OH	342.42 ± 0.45	273.08 ± 0.25
5d	−CH(CH3)CH2CH3	−OH	NA^b	264.1 ± 0.5
5e		−OH	197.57 ± 11.02	254.11 ± 0.25
5f		−OH	NA^b	497.15 ± 0.91
5g		−OH	NA^b	407.25 ± 1.89
5h		−OH	NA^b	561.79 ± 2.55
5i			590.39 ± 10.61	690.44 ± 1.92
5j			512.91 ± 23.91	424.18 ± 1.18
5k			NA^b	185.90 ± 0.36
5l			NA^b	NA^b
5m			NA^b	NA^b
5n			104.53 ± 0.21	NA^b
5o			NA^b	NA^b
7a	−CH2CH(CH3)2	−NHOH	56.71 ± 0.01	64.60 ± 0.79
7b	−CH(CH3)2	−NHOH	76.95 ± 0.43	128.12 ± 0.14
7c	−CH3	−NHOH	NA^b	292.22 ± 2.08
7d	−CH(CH3)CH2CH3	−NHOH	82.89 ± 2.67	207.11 ± 1.32
7e		−NHOH	1.26 ± 0.01	212.97 ± 1.35
7f		−NHOH	44.00 ± 2.32	249.42 ± 0.94
7g		−NHOH	172.87 ± 1.99	298.79 ± 1.48
7h		−NHOH	157.64 ± 0.58	292.35 ± 1.97
9a			NA^b	NA^b
9b			362.76 ± 0.04	NA^b
9c			NA^b	NA^b
9d			NA^b	NA^b
Bestatin			2.55 ± 0.11	163.48 ± 1.13

^aValues are means of three experiments and standard derivation.

^bbNA, no activity.

Figure 2.  The FlexX docking result of 7e with the active site of aminopeptidase N (PDB: 2DQM).

Figure 3.  The docking result of 7e with aminopeptidase N showed by LIGPLOT.

Figure 4.  Inhibition of aminopeptidase N (APN)/CD13 activity on ES-2 induced by 7e or bestatin. OVCA cells (1 × 10⁵) seeded in 96-well plates were exposed to various concentrations of 7e or bestatin at 37°C, and the substrate L-leucine-p-nitroanilide was added. APN/CD13 activity was estimated by measuring absorbance at 405 nm using a microplate reader after 60 min of incubation. The inhibition rate was calculated by comparing to the control (without drug exposure). The bars indicate means ± standard deviation (n = 3).

Figure 5.  In vitro anti-proliferative activities of compound 7e and bestatin against ES-2 cells. The bars indicate means ± standard deviation (n = 3).