Novel aminopeptidase N (APN/CD13) inhibitors derived from chloramphenicol amine

Article abstract of DOI:10.1016/j.bmc.2011.07.008

Aminopeptidase N (APN) is involved in different physiological and pathological processes of tumor cells, including proliferation, invasion, apoptosis and metastasis. Herein one series of compounds derived from commercially available (1S,2S)-2-amino-1-(4-n

Full text of DOI:10.1016/j.bmc.2011.07.008

Bioorganic & Medicinal Chemistry 19 (2011) 5190–5198
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel aminopeptidase N (APN/CD13) inhibitors derived from
chloramphenicol amine
Meirong Jia a, Kanghui Yang a, Hao Fang a, Yingying Xu a, Siwei Sun b, Li Su a, Wenfang Xu a,
⇑
a Department of Medicinal Chemistry, School of Pharmacy, Shandong University, 44 West Culture Road, Ji’nan, Shandong 250012, PR China
b School of Public Health, Shandong University, 44 West Culture Road, Ji’nan, Shandong 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Aminopeptidase N (APN) is involved in different physiological and pathological processes of tumor cells,
including proliferation, invasion, apoptosis and metastasis. Herein one series of compounds derived from
commercially available (1S,2S)-2-amino-1-(4-nitrophenyl) propane-1,3-diol have been designed and syn-
thesized. Furthermore, preliminary activity evaluation showed that some compounds elicited moderate
Received 16 May 2011
Revised 5 July 2011
Accepted 6 July 2011
Available online 14 July 2011
inhibitory activity against APN with compounds 10e (IC50 = 6.1 0.5
which could be used as the lead compound in the future for anticancer agents research.
Ó 2011 Elsevier Ltd. All rights reserved.
lM) possessing the best efficacy,
Keywords:
Aminopeptidase N inhibitors
Chloramphenicol amine derivatives
Synthesis
QSAR
1. Introduction
O
OH
O2N
O2N
NH2
O
NH2
Aminopeptidase N (APN/CD13: EC 3.4.11.2) is a Zn2+-containing
exopeptidase consisting of 967 amino acids in primary structure
with highly conserved sequence HEXXHX18E in its active sites.1
APN as a ubiquitous enzyme, is widely expressed on different kinds
of cells such as epithelial cells of the intestine and kidney, neuronal
synaptic membranes, macrophages, granulocytes and so on. It was
identified to partially cleave neutral or basic amino acids from the
N-terminus of oligopeptidases. In accordance to its overexpression
in tumor cells, APN has intimate relationships with tumor invasion,
metastasis and angiogenisis.2–4 Therefore, inhibition of APN may
lead to the development of anti-cancer agents.
OH
OH
N
H
OH
(a)
OH
(b)
O2N
NH2
NH2
NH2
OH
(c)
OH
(d)
O
Figure 1. Chemical structures of (a) Bestatin, (b) chloramphenicol amine, (c)
AHNPA, and (d) DANP.
Various of natural and synthetic inhibitors of APN have been
reviewed.5–7 Of these effective inhibitors, Bestatin (Fig. 1(a)) is
the only marketed drug, which was isolated by Umezawa et al.8
from Streptomyces oliuoreticuli. The Escherichia coli’s crystal struc-
ture of APN was first illustrated in ligand free form and enzyme–
Bestatin complex form, respectively, by Kiyoshi et al.9 Our group
have studied mainly functional sites of APN based on this co-crystal
complex published for years and several potent APN inhibitors pos-
sessing various scaffolds have been synthesized and reported.10–15
Generally, the binding sites of APN with Bestatin contain three
counterparts as shown in Figure 2. Viz. a hydrophobic pocket (S1)
interacting with the phenyl group of Bestatin; a zinc binding group
(ZBG) coordinated by the 2-hydroxy group and 1-carbonyl oxygen
of Bestatin, and final another hydrophobic pocket in opposite which
can be divided into two subsites (S10 and S20).
Some previous work focusing on the derivation of (1S,2S)-2-ami-
no-1-(4-nitrophenyl) propane-1,3-diol (chloramphenicol amine)
(Fig. 1(b)) has been conducted in our group.16 Some compounds
sharing (2R,3S)-2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic
acid (AHNPA) (Fig. 1(c)) as the key intermediate have exhibited
moderate inhibitory activity against APN in the micromolar range,
and the docking study further manifested high affinities of AHNPA
with APN.16 Encouraged by this result, another new modification
direction towards chloramphenicol amine is conducted. In this Let-
ter, we described the synthesis and biological activity evaluation of
newly designed compounds sharing (1S,2S)-2,3-diamino-1-(4-
nitrophenyl) propan-1-ol (DANP) (Fig. 1(d)) as the common scaffold
and docking studies of interactions are also discussed.
⇑
Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: wfxu@yahoo.cn (W. Xu).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.008

M. Jia et al. / Bioorg. Med. Chem. 19 (2011) 5190–5198
5191
2. Chemistry
All the compounds are synthesized via the route shown in
Scheme 1. The starting material, commercially available (1S,2S)-
2-amino-1-(4-nitrophenyl) propane-1,3-diol, was firstly protected
by (Boc)2O, then the product was selectively bromized at the pri-
mary hydroxyl group to give the mono-bromide 3. The following
trinitride 4 was prepared with sodium azide in situ, by the next
hydrogenation step leading to the key intermediate compound 5.
Several Boc-protected amino acids or Boc-protected dipeptides
were condensed with compound 5 adopting classic EDCI/HOBt
protocol. The target compounds 9a–9o were readily obtained after
the deprotection step in the presence of saturated HCl/anhydrous
Figure 2. The interaction mode of Bestatin and APN (Bestatin in the X-ray crystal is
showed in capped sticks mode).
O2N
Boc
O2N
Boc
O2N
a
Boc
O2N
d
Boc
O2N
NH
NH
NH
NH
NH2
b
c
OH N3
4
OH Br
3
OH OH
2
OH NH2
5
OH OH
1
O2N
Boc
I
Boc
H2N
Boc
NH
NH
NH
H
N
H
N
H
N
g
e
f
R
R''
R'
OH
O
OH
O
OH
O
6a-6o
8a-8e
7a-7g
h
h
h
O2N
HCl
H2N
I
HCl
HCl
HCl
NH2
NH2
NH2
H
H
H
N
R
N
R'
N
R''
OH
O
OH
10
10a-10g
O
OH
11
O
9
9a-9o
11a-11e
Scheme 1. Reagents and conditions: (a) (Boc)2O, THF; (b) NBS, Ph3P, pyridine, THF; (c) NaN3, DMF, 60 °C; (d) Ph3P, THF, H2O; (e) N-Boc amino acids or N-Boc dipeptides, Et3N,
EDCI, HOBt, DCM; (f) N2H4ꢀH2O (80%), FeCl3ꢀ6H2O, active C; (g) p-TsOH, KI, NaNO2, H2O; (h) HCl/anhydrous EtOAc.
Table 1
The structures and inhibitory activity of target compounds 9a–9o against APN and MMP-2
O2N
NH2
H
N
R
O
OH
a
Compd
R
IC50
(lM)
IC50 (MMP-2)/IC50 (APN)
APN
MMP-2
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
L
-Ser-NH2
-isoSer-NH2
-Cys-NH2
-Leu-NH2
-Lys-NH2
-Phe-NH2
-Tyr-NH2
-Hpro-NH2
422.5 9.9
163.5 4.9
12.4 1.5
80.6 6.0
50.1 3.5
42.2 1.0
39.2 2.2
441.3 1.8
19.6 2.3
9.4 1.6
21.6 3.2
17.6 2.1
133.6 10.1
74.7 5.4
6.5 0.7
331.1 5.6
100.1 3.4
151.6 3.1
142.9 2.9
84.3 1.2
67.4 1.0
61.3 1.5
>1000
83.9 2.2
173.1 6.9
110.1 3.7
108.9 2.8
44.1 0.9
105.6 3.2
40.6 1.7
0.8
0.06
12.7
1.8
1.7
1.6
1.6
>2.3
4.2
19.2
5.0
6.1
L
L
L
L
D
L
L
L
-Leu-
-Phe-
-Ser-
L
-isoSer-NH2
-Leu-NH2
D
L
L
L
D-Phe-NH2
-isoSer-
-Phe- -Phe-NH2
-Leu- -Phe-NH2
-Phe- -isoSer-NH2
D
-Phe-NH2
D
D
0.3
1.4
6.3
L
D
D
L
a
Mean values of triplicate experiments and standard deviations are given.

5192
M. Jia et al. / Bioorg. Med. Chem. 19 (2011) 5190–5198
Table 2
The structures and inhibitory activity of target compounds 10a–10g, 11a–11e and control Bestatin against APN and MMP-2
R1
NH2
H
N
R2
O
OH
a
Compd
R1
R2
IC50
(
lM)
IC50 (MMP-2)/IC50 (APN)
APN
MMP-2
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
11e
NH2
NH2
NH2
NH2
NH2
NH2
NH2
I
I
I
I
I
-
-
-
-
-
-
-
-
-
-
-
-
L
-Leu-NH2
-Lys-NH2
-Phe-NH2
63.2 5.4
135.6 6.7
141.3 18.3
113.7 5.6
6.1 0.5
30.6 3.9
31.6 1.9
30.6 5.6
21.6 1.8
11.3 0.5
>1000
119.3 4.5
157.4 3.9
149.2 2.1
111.3 1.8
133.8 1.6
40.4 0.9
94.0 1.3
119.5 4.6
42.2 2.9
86.6 2.1
107.1 2.8
52.7 1.1
1.9
1.2
1.1
1.0
22.3
1.3
3.0
3.9
2.0
7.7
<0.1
0.5
L
D
L-Tyr-NH2
D
-Phe-
-Phe-
L
-Leu-NH2
-Phe-NH2
-Phe-NH2
D
D
L
L
L
-Leu-
-Leu-NH2
-Tyr-NH2
D
D
-Phe-
-Phe-
L
-Leu-NH2
-Phe-NH2
-Phe-NH2
D
D
L-Leu-
D
105.4 3.9
O2N
NH2
NH2
DANP
893.9 10.8
3.7 0.4
>1000
>1.1
32.5
OH
O
OH
NH2
O
Bestatin
120.4 2.6
N
H
OH
a
Mean values of triplicate experiments and standard deviations are given.
EtOAc. Meanwhile, the anilides 7 produced by reduction were
converted into target compounds 10a–10g after deprotection.
Similarly, expected compounds 11a–11e were successfully
synthesized.
APN, was helpful to increase affinities of target compounds in con-
trary to amino group by comparing the IC50 values of compounds
9d–9e to compounds 10a–10d correspondingly. Also, the corre-
sponding iodides 11a, 11b have better activity than 10a, 10d,
3. Results and discussion
All the target compounds synthesized have been tested for the
potential enzymatic activity and results are exhibited in Tables 1
and 2. Similarly to APN, MMP-2 is also a Zn2+-dependent metallo-
protease related to tumor invasion and metastasis.17,18 Thus the
assay was performed both on APN and MMP-2 as to study the com-
pounds’ selectivities using Bestatin as the positive control.
From data shown in Tables 1 and 2, we can see that most of tar-
get compounds had higher affinities with APN than MMP-2, which
to some extent promoted our strategy for further rational APNIs
design. Moreover, all the compounds assayed possessed better
APN inhibitory activity compared with parent compound DANP
Figure 3. APN inhibitory activity of compounds 9c, 9j, 9l, 9o, 10e and Bestatin
against HL-60 cells. Data given are mean values of three independent experiments.
(IC50 = 893.9 10.8 lM) demonstrating that the structural addi-
tions of mono-amino acids or dipeptides lead to better efficacy in
inhibiting APN.
Comparing compounds 9a–9o, DANP-amino acid derivatives
(compounds 9a–9h) generally displayed worse activity than those
DANP-dipeptide derivatives (compounds 9i–9o), and this may be
explained by longer hydrophobic side chains binding more tightly
with APN’s S10 and S20 active pockets. Furthermore, compound 9c
with IC50 value to 12.4 1.5 lM has the best activity among com-
pounds 9a–9h, which may be contributed to sulfhydryl group’s po-
sitive hydrophobic effect with APN pockets. In contrary, too large
side chain led to comparatively poorer activity of compound 9m
(IC50 = 133.6 10.1 lM) since APN’s active cavities are relatively
deeper requiring ligands having suitable volumes.9
The electronic and steric effects of nitro group of DNAP were
also studied by converting nitro group into amino group and io-
dine, respectively. The enzymatic assay results unveiled that nitro
group, owing to its strong hydrogen-bonding interactions with
Figure 4. Anti-proliferation activity of compounds 9c, 9j, 9l, 9o, 10e and Bestatin
against two tumor cell lines (HL-60 cells and MDA-MB-231 cells). Data given are
mean values of three independent experiments.

M. Jia et al. / Bioorg. Med. Chem. 19 (2011) 5190–5198
5193
respectively. However, this conclusion is not in accordance with
DNAP-dipeptide derivates, anilides 10e–10g showing better activ-
ity. It is speculated that here amino group decreased tripeptides’
obvious steric hindrances, and that compounds 11c–11e sharing
a larger group iodine than nitro group are less efficacious sup-
ported previous speculation in return.
in Table 1, which is assumed owing to distinct characteristics of
APNs among species.9
Additionally, compounds 9c, 9j, 9l, 9o and 10e are detected for
their potential effects on proliferation of two tumor cell lines
(HL-60 cells and MDA-MB-231 cells) with Bestatin as control via
MTT assay. The result depicted in Figure 4 indicated that all com-
pounds’ anti-proliferative effects against HL-60 cells are better
than against MDA-MB-231 cells mainly owing to APN’s higher
expression level on HL-60 cells than the other. Compound 10e
shows the best effect against HL-60 cells with the IC50 of
1.34 0.08 mM complying with the enzymatic assay. However, ex-
cept compound 9l, all of the others exhibited better anti-prolifera-
tion activity against MDA-MB-231 cells than Bestatin as the
positive control. Compared with others,16 compounds with DANP
scaffold may be the main contributions to the more obvious inhib-
itory activity towards tumor cell proliferation, possibly implying a
Considering all the factors concerned, it is expected that com-
pounds 10e has the best inhibition (IC50 = 6.1 0.5
with Bestatin (IC50 = 3.7 0.4 M).
lM) similar
l
Moreover, the enzymatic inhibitory activity of compounds 9c, 9j,
9l, 9o and 10e against APN was also determined with HL-60 cells
high-expressing APN. Results are shown in Figure 3, from which it
can be learned that none of these five inhibitors exhibited better
activity than the control Bestatin which is consistent with the assay
in enzymatic level described above. However, compounds 9c
performed stronger effect than 9j contrary to the previous result
Figure 5. (a) The docking result of 10e with APN (10e is showed in capped sticks mode). (b) The docking result of compound 10e is shown by LIGPLOT.

5194
M. Jia et al. / Bioorg. Med. Chem. 19 (2011) 5190–5198
thin-layer chromatography on 0.25 mm silica gel plates (60GF-
254) and visualized with UV light or ninhydrin. 200–300 Mesh sil-
ica gel was used to purify the products. Proton nuclear magnetic
resonance (1H NMR) spectra were determined on a Brucker DRX
spectrometer (600 MHz), and chemical shifts are reported in delta
(d) units, parts per million (ppm) downfield from trimethylsilane
and hertz. Preparations were made in DMSO-d6 solvent. ESI-MS
were determined on an API 4000 spectrometer. High-resolution
mass spectra (HRMS) spectra were conducted by Shandong Analy-
sis and Test Center. Melting points were tested on an electrother-
mal melting point apparatus (uncorrected).
5.1.1. N-Protected (1S,2S)(+)-2-amino-1-(4-nitrophenyl)-1,3-pro
panediol (2)
A solution of di-tert-butyl dicarbonate (2.93 g, 11 mmol) in THF
(5 mL) was added dropwise to a solution of compound 1 (2.12 g,
10 mmol) in THF (10 mL) and the mixture was stirred for 24 h at
ambient temperature. Then the solvent was evaporated in vacuum
and the residue was recrystallized with EtOAc to afford white solid
Figure 6. The docking result of 9j with APN (9j is showed in capped sticks mode).
different mechanism from interactions with APN requiring further
identification.
(2.75 g), yield: 88%, mp: 115–116 °C, ½a D20
¼ þ23:1 (c = 1, MeOH),
ꢁ
Aiming to investigate the interaction modes of DANP-deriva-
tives with APN, the most active compound 10e was sketched and
docked into active sites of APN (PDB code: 2DQM) using Sybyl
7.0 via Sybyl/Sketch module and optimized using Powell’s method
in presence of the Tripos force field with convergence criterion set
at 0.05 kcal/Å mol, and assigned with Gasteiger–Hückel charges.
The docking study was performed using Sybyl/FlexX module. Ow-
ing to crucial functions of the zinc ion in ligand–receptor interac-
tions, active sites of APN was defined as 13.1 Å radius circles
around the zinc ion. Other docking parameters applied in the pro-
gram were kept default. As illustrated in Figure 5, the crucial cata-
lytic zinc ion was chelated by 10e’s hydroxyl and carbonyl groups.
Besides, aniline group of 10e deeply inserted into S10 pocket of
1H NMR (600 MHz, DMSO-d6) d 1.29 (s, 9H), 3.37–3.49 (m, 2H),
4.55–4.59 (m, 1H), 5.20 (d, J = 5.9 Hz, 1H), 6.09 (d, J = 9.0 Hz, 1H),
7.59 (d, J = 9.0 Hz, 2H), 8.18 (d, J = 9.0 Hz, 2H). ESI-MS m/z: 313.3
[M+H]+.
5.1.2. tert-Butyl(1S,2R)-3-bromo-1-hydroxy-1-(4-nitrophenyl)
propan-2-ylcarbamate (3)
The mixture of compound 2 (3.12 g, 10 mmol) and Ph3P (3.93 g,
15 mmol) was dissolved in newly distilled THF (100 mL) in ice-
bath condition in a flask. Then pyridine of 0.8 mL (0.79 g, 10 mmol)
was added dropwise, subsequently, solid N-bromosuccinimide
(NBS) (2.67 g, 15 mmol) was also added into the flask in three
equal parts by 10 min interval. The solution was stirred at 0 °C
for 1 h, and then continuously stirred at room temperature for
8 h. The solid was filtrated and solvent was evaporated in vacuum
to leave residue, which was washed by ether (100 mL) and solid
produced here was filtrated and rejected. The filtrate was washed
with 1 N H3PO4, saturated NaHCO3, and brine in turn, then the or-
ganic phase was dried with anhydrous MgSO4 and concentrated to
give a maroon oil. Finally, the crude product was depurated by fast
column chromatograph (EtOAc/PE = 3:1) to afford compound 3 as a
APN. Also, D-phenyl moiety of phenylalanine residue and leucine
residue could plunge into S1 and S20 pockets, respectively, via
hydrophobic interaction. It is also noted that compound 10e could
form hydrogen bonds with His297 at a distance of 2.91 Å. Further-
more, the stability of this protein–ligand complex could be rein-
forced by further interactions of amino group of compound 10e
with Tyr381 and Arg825. Notably, the observed hydrophobic interac-
tions of 10e with Glu298, Ala262, Glu382, Met260 contributed much to
improve binding affinities as well. Additionally, the docking study
of 9j was synchronously investigated following the same protocol
as 10e (shown in Fig. 6), through which the hydrophobic and steric
contributions of nitro group to affinities of 9j and APN being fur-
ther verdicted to approve our previous assumption.
white solid (1.87 g), yield: 50%, mp: 116–119 °C, ½a D20
ꢁ
¼ ꢂ34:35; 1H
NMR (600 MHz, DMSO-d6) d 1.21 (s, 9H), 3.34 (s, 1H), 3.51–3.54 (m,
1H), 3.63–3.68 (m, 1H), 4.72–4.79 (m, 1H), 4.93–4.97 (m, 1H), 5.60
(d, J = 5.4 Hz, 1H), 6.19 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H),
8.18 (d, J = 8.4 Hz, 2H); ESI-MS m/z: 375.3, 377.4 [M+1]+.
Finally, although the computed information assay basically sup-
ported our speculation, the exact binding modes of the DANP-
derivatives with APN should be finally confirmed through further
X-ray crystal study.
5.1.3. tert-Butyl(1S,2S)-3-azido-1-hydroxy-1-(4-nitrophenyl)pr
opan-2-ylcarbamate (4)
Solid sodium azide (0.97 g, 15 mmol) was added slowly into the
solution of compound 3 (3.75 g, 10 mmol) in anhydrous DMF
(10 mL). Keep the reaction at 65 °C for 12 h with nitrogen protec-
tion in situ. Then cool the solution to room temperature, and pour
the mixture into ice-water (60 mL), stir quickly with a glass bar to
get white solid precipitation. And separate the solid by filtration in
the reduced pressure and dry it completely in vacuum to get com-
4. Conclusion
In all, one series of compounds containing (1S,2S)-2,3-diamino-
1-(4-nitrophenyl) propan-1-ol block have been synthesized and
evaluated. Most of target compounds elicited moderate and selec-
tive APN inhibitory activity. Among them, 10e possessed the best
inhibition both in the enzymatic assay and cell-based assay, mak-
ing it a good lead for searching better chloramphenicol amine
derivatives as APN inhibitors.
pound 4 (2.93 g), yield: 87%, mp: 143–145 °C, ½a D20
ꢁ
¼ þ46:05; 1H
NMR (600 MHz, DMSO-d6) d 1.22 (s, 9H), 3.34 (s, 1H), 3.50–3.55
(m, 1H), 3.64–3.67 (m, 1H), 4.75–4.80 (m, 1H), 4.92–4.96 (m,
1H), 5.60 (d, J = 5.4 Hz, 1H), 6.19 (d, J = 9.0 Hz, 1H), 7.56 (d,
J = 8.4 Hz, 2H), 8.18 (d, J = 8.4 Hz, 2H); ESI-MS m/z: 338.5 [M+1]+.
5. Experimental section
5.1. Chemistry: general procedures
5.1.4. tert-Butyl(1S,2S)-3-amino-1-hydroxy-1-(4-nitrophenyl)
propan-2-ylcarbamate (5)
All the materials involved were commercially available. All sol-
vents were distilled before used. All reactions were monitored by
To a solution of compound 4 (3.37 g, 10 mmol) in THF (80 mL,
0.5% water), PPh3 (3.14 g, 12 mmol) was added at ambient

M. Jia et al. / Bioorg. Med. Chem. 19 (2011) 5190–5198
5195
temperature and reacted for 24 h, then most of the solvent was
evaporated off, and the residue was redissolved in EtOAc (40 mL).
Then extract the product with 1 N H3PO4 (10 mL ꢃ 3), incorporate
the inorganic phase and adjust the pH to 10, then reextract the
product with EtOAc (20 mL ꢃ 3). Following, the organic phase
was incorporated and concentrated in vacuum to give light yellow
solid, which was recrystallized by EtOAc/petrol ether to give com-
pound 5 in white solid form (2.27 g), yield: 73%, mp: 197–201 °C,
(EtOAc/petrol ether = 3:1) to obtain compound 8a as a stramineous
solid (5.44 g), yield: 90%, mp: 120–122 °C; 1H NMR (600 MHz,
DMSO-d6) d 0.81 (d, J = 6.6 Hz, 6H), 1.07–1.20 (m, 2H), 1.30 (s,
9H), 1.35 (s, 9H), 1.56–1.62 (m, 1H), 2.84–2.89 (m, 1H), 3.51–
3.56 (m, 1H), 3.64–3.67 (m, 1H), 4.74–4.79 (m, 1H), 4.92–4.96
(m, 1H), 5.60 (d, J = 5.4 Hz, 1H), 6.19 (d, J = 9.0 Hz, 1H), 7.77 (d,
J = 8.4 Hz, 2H), 8.31 (d, J = 8.4 Hz, 2H); ESI-MS m/z: 606.3 [M+1]+.
½
a 2D0
ꢁ
¼ þ35:21; 1H NMR (600 MHz, DMSO-d6) d 1.23 (s, 9H), 3.34
5.1.8. (S)-2-Amino-N-((2S,3S)-2-amino-3-hydroxy-3-(4-nitroph
enyl)propyl)-4-methylpentanamide (9d)
(s, 1H), 3.52–3.55 (m, 1H), 3.62–3.67 (m, 1H), 4.75–4.81 (m, 1H),
4.94–4.96 (m, 1H), 5.60 (d, J = 5.4 Hz, 1H), 6.19 (d, J = 9.0 Hz, 1H),
7.56 (d, J = 8.4 Hz, 2H), 8.18 (d, J = 8.4 Hz, 2H); ESI-MS m/z: 312.2
[M+1]+.
A solution of compound 6d (5.25 g, 10 mmol) in distilled EtOAc
was added dropwise to HCl gas saturated EtOAc solution. Keep the
reaction at room temperature for 4 h, and evaporate the liquid to
get the crude product, which was recrystallized to produce a white
solid 9d (3.08 g), yield: 95%, mp: 194–195 °C; 1H NMR (600 MHz,
DMSO-d6) d 0.81 (d, J = 6.6 Hz, 6H), 1.09–1.21 (m, 2H), 1.54–1.60
(m, 1H), 2.83–2.87 (m, 1H), 3.52–3.56 (m, 1H), 3.68–3.71 (m,
1H), 4.76–4.80 (m, 1H), 5.04–5.08 (m, 1H), 6.29 (d, J = 9.0 Hz,
1H), 7.66 (d, J = 8.8 Hz, 2H), 8.18 (d, J = 8.8 Hz, 2H); HRMS (AP-
ESI) m/z Calcd for C15H24N4O4 [M+H]+ 325.1798. Found: 325.1788.
The other compounds of 9 series were synthesized via the same
route as described above.
5.1.5. N-Protected(S)-2-amino-N-((2S,3S)-2-amino-3-hydroxy-3-
(4-nitrophenyl)propyl)-4-methylpentanamide (6d)
Compound 5 (3.11 g, 10 mmol), Boc-Leu (2.54 g, 11 mmol), and
HOBt (1.62 g, 12 mmol) were mixed and dissolved in distilled DCM
(80 mL) in ice-bath condition and then TEA (1.11 g, 11 mmol) was
added followed the addition of EDCI (3.82 g, 20 mmol) slowly.
Then keep the reaction at 0 °C for 1 h, remove the ice bath and stir
the solution for 12 h at room temperature. The solvent was evapo-
rated off in vacuo and the residue was redissolved with EtOAc
(80 mL), which was washed with 1 N citric acid, saturated NaHCO3
and brine, dried over MgSO4, and concentrated to give the crude
product, which was purified by fast column chromatograph
(EtOAc/petrol ether = 2:1) to afford compound 6d as a white solid
(3.67 g), yield: 70%, mp: 200–201 °C; 1H NMR (600 MHz, DMSO-
d6) d 0.81 (d, J = 6.6 Hz, 6H), 1.08–1.23 (m, 2H), 1.30 (s, 9H), 1.35
(s, 9H), 1.56–1.62 (m, 1H), 2.84–2.89 (m, 1H), 3.52–3.57 (m, 1H),
3.63–3.68 (m, 1H), 4.73–4.80 (m, 1H), 4.91–4.97 (m, 1H), 5.60 (d,
J = 5.4 Hz, 1H), 6.19 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H),
8.18 (d, J = 8.4 Hz, 2H); ESI-MS m/z: 525.4 [M+1]+.
5.1.9. (S)-2-Amino-N-((2S,3S)-2-amino-3-hydroxy-3-(4-nitrophe
nyl)propyl)-3-hydroxypropanamide (9a)
White solid, yield: 87%, mp: 175–176 °C; 1H NMR (600 MHz,
DMSO-d6) d 3.30–3.32 (m, 1H), 3.33–3.35 (m, 1H), 3.41–3.44 (m,
1H), 3.75–3.78 (m, 1H), 3.83–3.84 (m, 1H), 3.85–3.87 (m, 1H),
4.95 (d, J = 6.0 Hz, 1H), 6.70 (d, J = 9.0 Hz, 1H), 7.66 (d, J = 8.8 Hz,
2H), 8.18 (d, J = 8.8 Hz, 2H); HRMS (AP-ESI) m/z Calcd for
C
12H18N4O5 [M+H]+ 299.1277. Found: 299.1285.
5.1.10. (S)-3-Amino-N-((2S,3S)-2-amino-3-hydroxy-3-(4-nitrop
henyl)propyl)-2-hydroxypropanamide (9b)
5.1.6. N-Protected(S)-2-amino-N-((2S,3S)-2-amino-3-(4-amino
phenyl)-3-hydroxypropyl)-4-methylpentanamide (7a)
White solid, yield: 88%, mp: 158–160 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.92–2.94 (m, 1H), 3.06–3.09 (m, 1H), 3.22–3.24 (m,
1H), 3.34–3.37 (m, 1H), 3.46–3.49 (m, 1H), 4.17–4.20 (m, 1H),
4.88 (d, J = 6.0 Hz, 1H), 6.45 (d, J = 9.0 Hz, 1H), 7.73 (d, J = 9.0 Hz,
2H), 8.26 (d, J = 9.0 Hz, 2H); HRMS (AP-ESI) m/z Calcd for
Compound 6d (5.25 g, 10 mmol) was mixed with activated car-
bon (0.12 g, 10 mmol) with quantum sufficit FeCl3ꢀ6H2O as catalyst
in situ in MeOH (100 mL). When the solution was heated to boil,
hydrazine hydrate (80%) (9.6 g, 300 mmol) was added dropwise
slowly to it and keep the reaction refluxing for 3 h until the yellow
color disappeared. Then cool down the solution to room tempera-
ture, and solid was filtrated. The filtrate was concentrated by a ro-
tary evaporator and residue was washed with water and saturated
NaHCO3 in turn and extracted by EtOAc (30 mL ꢃ 3). Subsequently,
the organic phase was concentrated to give the crude product,
which was recrystallized with EtOAc/petrol ether to give pure
compound 7a (4.45 g), yield: 90%, mp: 199–200 °C; 1H NMR
(600 MHz, DMSO-d6) d 0.81 (d, J = 6.6 Hz, 6H), 1.09–1.23 (m, 2H),
1.30 (s, 9H), 1.35 (s, 9H), 1.56–1.62 (m, 1H), 2.87–2.89 (m, 1H),
3.51–3.57 (m, 1H), 3.62–3.68 (m, 1H), 4.75–4.79 (m, 1H), 4.93–
4.97 (m, 1H), 5.60 (d, J = 5.4 Hz, 1H), 6.19 (d, J = 9.0 Hz, 1H), 6.47
(d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H); ESI-MS m/z: 495.5
[M+1]+.
C
12H18N4O5 [M+H]+ 299.1277. Found: 299.1265.
5.1.11. (R)-2-Amino-N-((2S,3S)-2-amino-3-hydroxy-3-(4-nitrop
henyl)propyl)-3-mercaptopropanamide (9c)
White solid, yield: 54%, mp: 156–159 °C; 1H NMR (600 MHz,
DMSO-d6) d 3.30–3.34 (m, 1H), 3.33–3.36 (m, 1H), 3.42–3.45 (m,
1H), 3.76–3.79 (m, 1H), 3.83–3.84 (m, 1H), 3.85–3.87 (m, 1H),
4.96 (d, J = 6.0 Hz, 1H), 6.90 (d, J = 9.0 Hz, 1H), 7.73 (d, J = 9.0 Hz,
2H), 8.26 (d, J = 9.0 Hz, 2H); HRMS (AP-ESI) m/z Calcd for
C
12H18N4O4S [M+H]+ 315.1049. Found: 315.1056.
5.1.12. (S)-2,6-Diamino-N-((2S,3S)-2-amino-3-hydroxy-3-(4-nit
rophenyl)propyl)hexanamide (9e)
White solid, yield: 64%, mp: 155–158 °C; 1H NMR (600 MHz,
DMSO-d6) d 1.34–1.38 (m, 2H), 1.56–1.59 (m, 2H), 1.74–1.78 (m,
2H), 2.74–2.76 (m, 2H), 3.17–3.20 (m, 1H), 3.41–3.46 (m, 1H),
3.47–3.50 (m, 1H), 3.79–3.81 (m, 1H), 4.99 (d, J = 7.2 Hz, 1H),
6.93 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 8.27 (d, J = 8.4 Hz,
2H); HRMS (AP-ESI) m/z Calcd for C15H25N5O4 [M+H]+ 340.1907.
Found: 340.1926.
5.1.7. N-Protected(S)-2-amino-N-((2S,3S)-2-amino-3-hydroxy-3-
(4-iodophenyl)propyl)-4-methylpentanamide (8a)
To a solution of p-TsOH (5.16 g, 30 mmol) in chromatographic
pure MeCN (30 mL) was added compound 7a (4.94 g, 10 mmol)
and the mixture was cooled to 10–15 °C. To this suspension, a solu-
tion of NaNO2 (1.38 g, 20 mmol) and KI (4.15 g, 25 mmol) in H2O
(6 mL) was gradually added. The reaction mixture was stirred for
10 min, then allowed to come to room temperature for continu-
ously reacting for 30 min. H2O (100 mL) was then added and pH
was adjusted to about 10 over NaHCO3. The product was then ex-
tracted with EtOAc and purified by fast column chromatograph
5.1.13. (R)-2-Amino-N-((2S,3S)-2-amino-3-hydroxy-3-(4-nitrop
henyl)propyl)-3-phenylpropanamide (9f)
White solid, yield: 87%, mp: 185–187 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.99–3.03 (m, 1H), 3.06–3.07 (m,1H), 3.08–3.13 (m,
1H), 3.31–3.35 (m, 1H), 3.42–3.46 (m, 1H), 4.03–4.05 (m, 1H),
4.92 (d, J = 6.0 Hz, 1H), 7.23–7.32 (m, 5H), 7.72 (d, J = 8.4 Hz, 2H),

5196
M. Jia et al. / Bioorg. Med. Chem. 19 (2011) 5190–5198
8.22 (d, J = 8.4 Hz, 2H), 9.16 (t, J = 5.4 Hz, 1H); HRMS (AP-ESI) m/z
8.71 (t, J = 6 Hz, 1H); HRMS (AP-ESI) m/z Calcd for C21H27N5O6
Calcd for C18H22N4O4 [M+H]+ 359.1641. Found: 359.1655.
[M+H]+ 446.1961. Found: 446.1975.
5.1.14. (S)-2-Amino-N-((2S,3S)-2-amino-3-hydroxy-3-(4-nitro
phenyl)propyl)-3-(4-hydroxyphenyl)propanamide (9g)
White solid, yield: 87%, mp: 189–191 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.99–3.04 (m, 1H), 3.08–3.10 (m, 1H), 3.12–3.13 (m,
1H), 3.32–3.36 (m, 1H), 3.42–3.47 (m, 1H), 4.03–4.05 (m, 1H),
4.92 (d, J = 6.0 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz,
2H), 7.72 (d, J = 8.4 Hz, 2H), 8.25 (d, J = 8.4 Hz, 2H), 9.01 (t,
J = 6.0 Hz, 1H); HRMS (AP-ESI) m/z Calcd for C18H22N4O5 [M+H]+
375.1590. Found: 375.1575.
5.1.20. (R)-2-Amino-N-((R)-1-((2S,3S)-2-amino-3-hydroxy-3-(4-
nitrophenyl)propylamino)-1-oxo-3-phenylpropan-2-yl)-3-
phenylpropanamide (9m)
White solid, yield: 86%, mp: 121–122 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.62–2.65 (m, 1H), 2.72–2.76 (m, 1H), 2.82–2.85 (m,
1H), 3.12–3.14 (m, 1H), 3.29–3.39 (m, 5H), 4.49–4.52 (m, 1H),
6.59 (d, J = 3.6 Hz, 1H), 7.18–7.27 (m, 10H), 7.68 (d, J = 8.4 Hz,
2H), 8.26 (d, J = 8.4 Hz, 1H), 9.12 (d, J = 7.8 Hz, 1H); HRMS (AP-
ESI) m/z Calcd for C27H31N5O5 [M+H]+ 506.2325. Found: 506.2332.
5.1.15. (S)-N-((2S,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)
propyl)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-
carboxamide (9h)
5.1.21. (S)-N-((2S,3S)-2-Amino-3-hydroxy-3-(4-nitrophenyl)
propyl)-2-((R)-2-amino-3-phenylpropanamido)-4-
methylpentanamide (9n)
White solid, yield: 73%, mp: 159–161 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.87–2.89 (m, 1H), 2.90–3.09 (m, 1H), 3.11–3.13 (m,
1H), 3.32–3.35 (m, 1H), 3.36–3.38 (m, 2H), 3.41–3.42 (m, 1H),
4.29–4.40 (m, 1H), 4.88 (d, J = 6.0 Hz, 1H), 6.56 (d, J = 8.4 Hz, 1H),
6.90 (d, J = 7.8 Hz, 1H), 6.92 (s, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.71
(d, J = 8.4 Hz, 2H), 8.25 (d, J = 8.4 Hz, 2H); HRMS (AP-ESI) m/z Calcd
for C19H22N4O5 [M+H]+ 387.1590. Found: 387.1580.
White solid, yield: 80%, mp: 118–119 °C; 1H NMR (600 MHz,
DMSO-d6) d 0.85 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H), 1.05–
1.08 (m, 1H), 1.44–1.49 (m, 2H), 2.94–2.98 (m, 2H), 3.01–3.15
(m, 1H), 3.15–3.18 (m, 1H), 4.02–4.07 (m, 1H), 4.21–4.24 (m,
1H), 4.92 (d, J = 4.2 Hz, 1H), 6.64 (d, J = 4.2 Hz, 1H), 7.29–7.32
(m, 5H), 7.66 (d, J = 7.8 Hz, 2H), 8.25 (d, J = 8.4 Hz, 2H), 8.84 (d,
J = 7.8 Hz, 1H); HRMS (AP-ESI) m/z Calcd for C24H33N5O5 [M+H]+
472.2482. Found: 472.2488.
5.1.16. (S)-2-((S)-3-Amino-2-hydroxypropanamido)-N-((2S,3S)-
2-amino-3-hydroxy-3-(4-nitrophenyl)propyl)-4-
methylpentanamide (9i)
5.1.22. (S)-3-Amino-N-((R)-1-((2S,3S)-2-amino-3-hydroxy-3-(4-
nitrophenyl)propylamino)-1-oxo-3-phenylpropan-2-yl)-2-
hydroxypropanamide (9o)
White solid, yield: 80%, mp: 107–109 °C; 1H NMR (600 MHz,
DMSO-d6) d 0.85 (d, J = 6.0 Hz, 3H), 0.88 (d, J = 6.0 Hz, 3H), 1.49–
1.57 (m, 3H), 2.78–2.81 (m, 1H), 3.05–3.10 (m, 2H), 3.13–3.16
(m, 2H), 4.19–4.21 (m, 1H), 4.26–4.29 (m, 1H), 4.76 (d, J = 4.2 Hz,
1H), 7.62 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.4 Hz, 1H), 8.22 (d,
J = 8.4 Hz, 2H), 8.24 (d, J = 8.4 Hz, 2H); HRMS (AP-ESI) m/z Calcd
for C18H29N5O6 [M+H]+ 412.2118. Found: 412.2132.
White solid, yield: 78%, mp: 210–212 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.49–2.51 (m, 1H), 2.83–2.85 (m, 1H), 2.90–2.94 (m,
1H), 3.16–3.19 (m, 2H), 3.41–3.43 (m, 1H), 3.8 (br s, 1H), 4.16 (t,
J = 4.8 Hz, 1H), 4.50 (d, J = 4.2 Hz, 1H), 4.94 (t, J = 4.8 Hz, 1H),
7.18–7.26 (m, 5H), 7.69 (d, J = 9 Hz, 2H), 8.14 (d, J = 8.4 Hz, 1H),
8.27 (d, J = 9 Hz, 2H), 8.52 (d, J = 6 Hz, 1H); HRMS (AP-ESI) m/z
Calcd for C21H27N5O6 [M+H]+ 446.1961. Found: 446.1971.
5.1.17. (S)-2-Amino-N-((R)-1-((2S,3S)-2-amino-3-hydroxy-3-(4-
nitrophenyl)propylamino)-1-oxo-3-phenylpropan-2-yl)-4-
methylpentanamide (9j)
5.1.23. (S)-2-Amino-N-((2S,3S)-2-amino-3-(4-aminophenyl)-3-
hydroxypropyl)-4-methylpentanamide (10a)
White solid, yield: 82%, mp: 165–166 °C; 1H NMR (600 MHz,
DMSO-d6) d 0.79 (d, J = 6.0 Hz, 3H), 0.82 (d, J = 6.0 Hz, 3H), 1.22–
1.24 (m, 1H), 1.29–1.30 (m, 1H), 1.46–1.48 (m, 1H), 2.76–2.80
(m, 1H), 2.93–2.98 (m, 2H), 3.81–3.82 (m, 1H), 3.89–3.93 (m,
A solution of compound 7a (4.94 g, 10 mmol) in distilled EtOAc
was added dropwise to HCl gas saturated EtOAc solution. Keep the
reaction at room temperature for 8 h, and evaporate the liquid to
get the crude product, which was recrystallized to produce a white
solid 10a (2.65 g), yield: 90%, mp: 184–185 °C; 1H NMR (600 MHz,
DMSO-d6) d 0.83 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H), 1.17–
1.22 (m, 1H), 1.36–1.40 (m, 1H), 1.66–1.71 (m, 1H), 2.70–2.76
(m, 1H), 2.98–3.02 (m, 1H), 3.02–3.13 (m, 1H), 4.15 (br s, 1H),
6.50 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 7.84 (s, 1H); HRMS
(AP-ESI) m/z Calcd for C15H26N4O2 [M+H]+ 295.2056. Found:
295.2064.
1H), 4.49–4.53 (m, 1H), 4.75 (d,
J = 3.4 Hz, 1H), 6.90 (d,
J = 8.4 Hz, 1H), 7.15–7.23 (m, 5H), 7.53 (d, J = 8.4 Hz, 2H), 7.83
(d, J = 7.8 Hz, 1H), 8.17 (d, J = 8.4 Hz, 2H); HRMS (AP-ESI) m/z
Calcd for C24H33N5O5 [M+H]+ 472.2482. Found: 472.2486.
5.1.18. (R)-2-Amino-N-((S)-1-((2S,3S)-2-amino-3-hydroxy-3-(4-
nitrophenyl)propylamino)-3-hydroxy-1-oxopropan-2-yl)-3-
phenylpropanamide (9k)
The other compounds of 10 series were synthesized via the
same route as described above.
White solid, yield: 76%, mp: 103–105 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.59–2.61 (m, 1H), 2.81–2.84 (m, 1H), 2.98–3.04 (m,
2H), 3.07–3.12 (m, 1H), 3.44–3.47 (m, 2H), 3.60–3.63 (m, 1H),
4.21–4.24 (m, 1H), 4.64 (d, J = 4.0 Hz, 1H), 7.18–7.28 (m, 5H),
7.59 (d, J = 9 Hz, 2H), 7.85 (t, J = 5.4 Hz, 1H), 8.07 (d, J = 7.8 Hz,
5.1.24. (S)-2,6-Diamino-N-((2S,3S)-2-amino-3-(4-aminophenyl)-
3-hydroxypropyl)hexanamide (10b)
Light yellow solid, yield: 70%, mp: 167–168 °C; 1H NMR
(600 MHz, DMSO-d6) d 1.33–1.36 (m, 2H), 1.52–1.53 (m, 2H),
1.73–1.78 (m, 2H), 2.64–2.66 (m, 2H), 3.07–3.12 (m, 1H), 3.40–
3.44 (m, 1H), 3.47–3.50 (m, 1H), 3.76–3.81 (m, 1H), 4.78 (d,
J = 6.8 Hz, 1H), 6.53 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H),
8.07 (d, J = 7.8 Hz, 1H); HRMS (AP-ESI) m/z Calcd for C15H27N5O2
[M+H]+ 310.2165. Found: 310.2172.
1H), 8.18 (d,
C
J = 9 Hz, 2H); HRMS (AP-ESI) m/z Calcd for
21H27N5O6 [M+H]+ 446.1961. Found: 446.1962.
5.1.19. (R)-2-Amino-N-((S)-3-((2S,3S)-2-amino-3-hydroxy-3-(4-
nitrophenyl)propylamino)-2-hydroxy-3-oxopropyl)-3-
phenylpropanamide (9l)
White solid, yield: 80%, mp: 174–175 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.93–2.97 (m, 1H), 3.09–3.13 (m, 2H), 3.24–3.27 (m,
1H), 3.30–3.34 (m, 1H), 3.45–3.46 (m, 1H), 3.48–3.52 (m, 1H),
4.03–4.05 (m, 2H), 4.88 (t, J = 4.8 Hz, 1H), 7.24–7.32 (m, 5H),
7.70 (d, J = 9 Hz, 2H), 8.17 (t, J = 6 Hz, 1H), 8.24 (d, J = 9 Hz, 2H),
5.1.25. (R)-2-Amino-N-((2S,3S)-2-amino-3-(4-aminophenyl)-3-
hydroxypropyl)-3-phenylpropanamide (10c)
White solid, yield: 80%, mp: 190–191 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.91–2.93 (m, 1H), 2.96–2.98 (m, 1H), 3.00–3.12 (m,

M. Jia et al. / Bioorg. Med. Chem. 19 (2011) 5190–5198
5197
1H), 3.19 (s, 1H), 3.33–3.38 (m, 1H), 3.99 (d, J = 5.4 Hz, 1H), 4.64 (d,
5.1.31. (S)-2-Amino-N-((2S,3S)-2-amino-3-hydroxy-3-(4-
J = 7.2 Hz, 1H), 7.01 (d, J = 8.4 Hz, 2H), 7.23–7.32 (m, 5H), 7.45 (d,
J = 8.4 Hz, 2H), 9.03 (t, J = 6.0 Hz, 1H); HRMS (AP-ESI) m/z Calcd
for C18H24N4O2 [M+H]+ 329.1899. Found: 329.1889.
iodophenyl)propyl)-3-(4-hydroxyphenyl)propanamide (11b)
White solid, yield: 92%, mp: 175–177 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.83–2.88 (m, 1H), 2.93–3.00 (m, 2H), 3.05–3.10 (m,
1H), 3.89–3.93 (m, 2H), 4.68 (d, J = 5.4 Hz, 1H), 6.70 (d, J = 8.4 Hz,
2H), 7.04 (d, J = 8.4 Hz, 2H), 8.16 (d, J = 9.0 Hz, 2H), 8.33 (d,
J = 9.0 Hz, 2H), 9.01 (t, J = 6.0 Hz, 1H); HRMS (AP-ESI) m/z Calcd
for C18H22IN3O3 [M+H]+ 456.0706. Found: 456.0722.
5.1.26. (S)-2-Amino-N-((2S,3S)-2-amino-3-(4-aminophenyl)-3-
hydroxypropyl)-3-(4-hydroxyphenyl)propanamide (10d)
Yellow solid, yield: 74%, mp: 123–124 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.45–2.47 (m, 1H), 2.63–2.66 (m, 1H), 2.69–2.73 (m,
1H), 2.77–2.80 (m, 1H), 2.98–3.02 (m, 1H), 3.25–2.27 (m, 2H),
4.10 (d, J = 6.0 Hz, 1H), 6.50 (d, J = 9.0 Hz, 2H), 6.65 (d, J = 9.0 Hz,
2H), 6.92 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 7.74 (t,
J = 5.4 Hz, 1H); HRMS (AP-ESI) m/z Calcd for C18H24N4O3 [M+H]+
345.1848. Found: 345.1859.
5.1.32. (S)-2-Amino-N-((R)-1-((2S,3S)-2-amino-3-hydroxy-3-(4-
iodophenyl)propylamino)-1-oxo-3-phenylpropan-2-yl)-4-
methylpentanamide (11c)
White solid, yield: 85%, mp: 140–141 °C; 1H NMR (600 MHz,
DMSO-d6) d 0.80 (d, J = 6.0 Hz, 3H), 0.83 (d, J = 6.0 Hz, 3H), 1.21–
1.22 (m, 1H), 1.25–1.28 (m, 1H), 1.42–1.44 (m, 1H), 2.74–2.78
(m, 1H), 2.93–2.97 (m, 2H), 3.82–3.85 (m, 1H), 3.89–3.95 (m,
1H), 4.59–4.62 (m, 1H), 4.69 (br s, 1H), 7.15 (d, J = 8.4 Hz, 1H),
7.21–7.23 (m, 5H), 7.43 (d, J = 9.0 Hz, 2H), 7.97 (d, J = 9.0 Hz,
2H), 8.83 (d, J = 7.8 Hz, 1H); HRMS (AP-ESI) m/z Calcd for
5.1.27. (S)-2-Amino-N-((R)-1-((2S,3S)-2-amino-3-(4-
aminophenyl)-3-hydroxypropylamino)-1-oxo-3-phenylpropan-
2-yl)-4-methylpentanamide (10e)
White solid, yield: 82%, mp: 156–158 °C; 1H NMR (600 MHz,
DMSO-d6) d 0.77 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H), 1.04–
1.09 (m, 1H), 1.22–1.26 (m, 1H), 1.58–1.60 (m, 1H), 2.64–2.67
(m, 1H), 2.73–2.82 (m, 2H), 2.94–2.99 (m, 2H), 3.11–3.26 (m,
C
24H33IN4O3 [M+H]+ 553.1597. Found: 553.1605.
5.1.33. (R)-2-Amino-N-((R)-1-((2S,3S)-2-amino-3-hydroxy-3-(4-
iodophenyl)propylamino)-1-oxo-3-phenylpropan-2-yl)-3-
phenylpropanamide (11d)
1H), 4.47–4.48 (m, 1H), 6.50 (d,
J = 8.4 Hz, 2H), 6.93 (d,
J = 8.4 Hz, 2H), 7.15–7.25 (m, 5H), 7.77–7.91 (t, J = 5.4 Hz, 1H),
8.05 (d, J = 7.8 Hz, 1H); HRMS (AP-ESI) m/z Calcd for C24H35N5O3
[M+H]+ 442.2740. Found: 442.2754.
White solid, yield: 88%, mp: 148–149 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.56–2.61 (m, 1H), 2.70–2.74 (m, 1H), 2.80–2.83 (m,
1H), 3.08–3.11 (m, 1H), 3.17–3.20 (m, 1H), 3.27–3.32 (m, 2H),
4.03–4.05 (m, 1H), 4.51–4.55 (m, 1H), 4.65–4.88 (m, 1H), 7.18 (d,
J = 8.4 Hz, 2H), 7.22–7.28 (m, 6H), 7.76 (d, J = 8.4 Hz, 2H), 8.06
(br s, 5H), 8.97 (d, J = 8.4 Hz, 1H); HRMS (AP-ESI) m/z Calcd for
5.1.28. (R)-2-Amino-N-((R)-1-((2S,3S)-2-amino-3-(4-
aminophenyl)-3-hydroxypropylamino)-1-oxo-3-phenylpropan-
2-yl)-3-phenylpropanamide (10f)
White solid, yield: 80%, mp: 183–184 °C; 1H NMR (600 MHz,
DMSO-d6) d 2.61–2.65 (m, 1H), 2.73–2.77 (m, 1H), 2.82–2.85 (m,
1H), 3.14–3.17 (m, 1H), 3.20–3.25 (m, 2H), 3.37–3.39 (m, 1H),
4.08–4.12 (m, 1H), 4.42–4.45 (m, 1H), 4.74 (d, J = 6.6 Hz, 1H),
7.18 (m, 1H), 7.21–7.29 (m, 5H), 7.38 (d, J = 8.4 Hz, 2H), 7.49 (d,
J = 8.4 Hz, 2H), 8.20 (br s, 5H), 9.11 (d, J = 9 Hz, 1H); HRMS (AP-
ESI) m/z Calcd for C27H33N5O3 [M+H]+ 476.2583. Found: 476.2589.
C
27H31IN4O3 [M+H]+ 587.1441. Found: 587.1456.
5.1.34. (S)-N-((2S,3S)-2-Amino-3-hydroxy-3-(4-
iodophenyl)propyl)-2-((R)-2-amino-3-phenylpropanamido)-4-
methylpentanamide (11e)
White solid, yield: 84%, mp: 148–150 °C; 1H NMR (600 MHz,
DMSO-d6) d 0.83 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H), 1.06–
1.07 (m, 1H), 1.40–1.42 (m, 2H), 2.84–2.87 (m, 2H), 3.11–3.15
(m, 1H), 3.25–3.29 (m, 1H), 4.06–4.09 (m, 1H), 4.31–4.35 (m,
1H), 4.85 (d, J = 4.2 Hz, 1H), 6.95 (d, J = 5.2 Hz, 1H), 7.27–7.30
(m, 5H), 7.46 (d, J = 9.0 Hz, 2H), 7.92 (d, J = 9.0 Hz, 2H), 8.75 (d,
J = 8.4 Hz, 1H); HRMS (AP-ESI) m/z Calcd for C24H33IN4O3 [M+H]+
553.1597. Found: 553.1610.
5.1.29. (S)-N-((2S,3S)-2-Amino-3-(4-aminophenyl)-3-
hydroxypropyl)-2-((R)-2-amino-3-phenylpropanamido)-4-
methylpentanamide (10g)
White solid, yield: 79%, mp: 176–178 °C; 1H NMR (600 MHz,
DMSO-d6) d 0.83 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H), 1.04–
1.09 (m, 1H), 1.40–1.45 (m, 2H), 2.84–2.87 (m, 2H), 3.01–3.10
(m, 1H), 3.15–3.18 (m, 1H), 4.01–4.06 (m, 1H), 4.20–4.22 (m,
1H), 4.82 (d, J = 4.2 Hz, 1H), 6.50 (d, J = 8.4 Hz, 2H), 6.93 (d,
J = 8.4 Hz, 2H), 7.29–7.32 (m, 5H), 7.91 (d, J = 5.8 Hz, 1H), 8.84
(d, J = 7.8 Hz, 1H); HRMS (AP-ESI) m/z Calcd for C24H35N5O3
[M+H]+ 442.2740. Found: 442.2766.
5.2. Biological screening
5.2.1. In vitro APN inhibition assay
IC50 values against APN were determined as previously de-
scribed19,20 by using
L-Leu-p-nitroanilide as substrate and Micro-
somal aminopeptidase from Porcine Kidney Microsomes (Sigma)
as enzyme in 50 mM PBS (pH 7.2) or suspension of HL-60 cells in
PBS (1 ꢃ 105/well). After incubation with various concentrations
of detected compounds at 37 °C, hydrolysis of the substrate was
measured using a plate reader (Varioskan, Thermo, USA) by
observing the changes of OD values at 405 nm.
5.1.30. (S)-2-Amino-N-((2S,3S)-2-amino-3-hydroxy-3-(4-
iodophenyl)propyl)-4-methylpentanamide (11a)
A solution of compound 8a (6.05 g, 10 mmol) in distilled EtOAc
was added dropwise to HCl gas saturated EtOAc solution. Keep the
reaction at room temperature for 8 h, and evaporate the liquid to
get the crude product, which was recrystallized to produce a white
solid 11a (3.65 g), yield: 89%, mp: 154–155 °C; 1H NMR (600 MHz,
DMSO-d6) d 0.87 (d, J = 6.0 Hz, 3H), 0.89 (d, J = 6.0 Hz, 3H), 1.49–
1.51 (m, 1H), 1.51–1.53 (m, 2H), 2.99–3.03 (m, 1H), 3.44–3.48 (m,
1H), 3.71–3.72 (m, 1H), 4.70 (d, J = 6.0 Hz, 1H), 8.16 (d, J = 10.4 Hz,
2H), 8.36 (d, J = 10.4 Hz, 2H), 9.01 (t, J = 6.0 Hz, 1H); HRMS (AP-ESI)
m/z Calcd for C15H24IN3O2 [M+H]+ 406.0913. Found: 406.0923.
The other compounds of 11 series were synthesized via the
same route as described above.
5.2.2. In vitro MMP inhibition assay
Enzyme MMP-2 and developer TNBS were both purchased from
Sigma, and the substance was synthesized as described by Baragi
et al.21 The enzyme, substance and inhibitor were dissolved in
50 mM sodium borate (pH 8.5) in a 96-well plate and incubated
for 30 min at 37 °C, and then TNBS (0.03%, m/v) was added and incu-
bated for another 20 min at room temperature, the resulting solu-
tion was detected under 450 nm wavelength to gain absorption.

5198
M. Jia et al. / Bioorg. Med. Chem. 19 (2011) 5190–5198
4. Inagaki, Y.; Tang, W.; Zhang, L.; Du, G.; Xu, W.; Kokudo, N. BioSci. Trends 2010, 4,
5.2.3. MTT assay
56.
HL-60 cells (high APN expression) and MDA-MB-231 cells (low
APN expression) were cultured in RPMI1640 medium containing
10% FBS in situ at 37 °C in 5% CO2 humidified incubator. Cell prolif-
eration was determined by MTT [(3-[4,5-dimethyl-2-thiazolyl]-
2,5-diphenyl-2H-tetrazolium bromide)] assay. Briefly, cells were
seeded in a 96-well plate (1 ꢃ 104/well) and cultured for 4 h, fol-
lowed by addition of different concentrations of inhibitors. After
another 48 h treatment, 1% MTT (0.5 mg/mL) solution was added
to form the formazan product. Finally, DMSO was used to solve
the formazan and concentrations were monitored by detecting
OD values at 570 nm through a plate reader (Varioskan, Thermo,
USA).
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Acknowledgment
This work was supported by National Natural Foundation Re-
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