Novel aminopeptidase N inhibitors derived from antineoplaston AS2-5 (Part I)

Article abstract of DOI:10.1016/j.bmc.2009.02.063

Overexpression of zinc-dependent metalloproteinase, aminopeptidase N (APN/CD13), is considered to be involved in the process of tumor invasion and metastasis. Herein we describe the synthesis and in vitro enzymatic inhibition assay of antineoplaston AS2-5 scaffold peptidomimetic compounds. The results demonstrated that most of these l-iso-glutamine derivatives displayed selective inhibitory activity against APN as compared with MMP-2, with IC₅₀ values in the micromole range. The structure-activity relationships were also briefly discussed.

Full text of DOI:10.1016/j.bmc.2009.02.063

Bioorganic & Medicinal Chemistry 17 (2009) 3053–3060
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel aminopeptidase N inhibitors derived from antineoplaston AS2–5 (Part I)
Xun Li a, Junli Wang a, Jinpei Li b, Jifeng Wu c, Yonggang Li a, Huawei Zhu a, Ruifang Fan b, Wenfang Xu a,
*
a School of Pharmaceutical Sciences, Shandong University, No. 44 WenhuaXi Road, Ji’nan, 250012, Shandong Province, PR China
b Liaocheng People’s Hospital, 252000, Liaocheng, Shandong Province, PR China
c Ji’nan Public Security Bureau, 250002, Ji’nan, Shandong Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Overexpression of zinc-dependent metalloproteinase, aminopeptidase N (APN/CD13), is considered to be
involved in the process of tumor invasion and metastasis. Herein we describe the synthesis and in vitro
enzymatic inhibition assay of antineoplaston AS2–5 scaffold peptidomimetic compounds. The results
demonstrated that most of these L-iso-glutamine derivatives displayed selective inhibitory activity
against APN as compared with MMP-2, with IC50 values in the micromole range. The structure–activity
relationships were also briefly discussed.
Received 20 January 2009
Revised 23 February 2009
Accepted 24 February 2009
Available online 6 March 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
L
-iso-Glutamine derivatives
Antineoplaston AS2–5
APN inhibitors
Peptidomimetics
Anti-tumor
1. Introduction
mains, which are called S1 pocket and S01 pocket, respectively
(see Fig. 1). Therefore, one or more hydrophobic side chains should
During the process of tumor invasion and metastasis, proteo-
lytic degradation of the extracellular matrix (ECM) is believed to
be a key step, which involves a class of zinc-dependent metallopro-
teinase, aminopeptidase N (APN, EC 3.4.11.2; gp150), also known
as human lymphocyte surface cluster differentiation antigen
CD13.1,2 It is a 150-kDa monomeric or homodimeric type II mem-
brane-bound glycoprotein which can release neutral or basic ami-
no acids from the N-terminal end of peptides.3 It is particularly
noticeable that overexpression of APN/CD13 is highly correlated
with various pathological disorders, such as inflammatory bowel
diseases, encephalomyelitis, multiple sclerosis, ulcerative colitis,
Crohn’s disease, rheumatoid arthritis, and cancer.4–8 As a conse-
quence, it is useful to develop chemical inhibitors that could block
its enzymatic activity for medical and therapeutic utilization.9
To date, several excellent reviews on natural or small molecule
inhibitors of APN/CD13 have been published.10–12 Amongst these
inhibitors, bestatin, an antibiotic of microbial origin and known
inhibitor of APN,13 is a well-established anticancer agent which
has been a useful tool as positive control in elucidating many phys-
iological conditions.14
be introduced to insert into these pockets to generate effective
interactions. Moreover, given most of the reported APN inhibitors
(APNIs) are pseudodipeptides or peptidemimics bearing zinc-che-
lating functionality (such as hydroxamate or carboxylate), inhibi-
tors containing zinc-binding groups (ZBGs) should be optimal
selection to inhibit the enzymatic activity of APN.17
According to these findings, our group has previously reported a
series of novel APNIs with remarkable APN inhibitory activities in
the enzymatic assays.18–21 In our ongoing work on the exploitation
of potent APNIs, we herein describe the synthesis and enzymatic
evaluation of analogues derived from the antineoplaston AS2-5
(N2-phenylacetyl L
-iso-glutamine),22 one of active metabolites of
the antineoplaston A-10 (3-phenylacetylamino-2,6-piperidinedi-
one), which is known to be the first chemically identified antine-
oplaston with anti-tumor activity (see Fig. 2).23,24
Enzyme
S
S '
S '
1
1
2
++
Recently, the 3D-structures of APN have been investigated by
the X-ray crystallographic studies on the co-crystal of the enzyme
and various inhibitors.15,16 It has showed that beside the catalytic
center zinc(II) ion of APN, there are two hydrophobic binding do-
Zn
O
H N
N
H
COOH
3
OH
* Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: tjulx2004@sdu.edu.cn (W. Xu).
Figure 1. Schematic representation of the interaction of bestatin with APN.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.063

3054
X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3053–3060
H
N
O
O
N
H
O
Antineoplaston 10 (A10)
H
N
COOH
COOH
O
O
NH2
-glutamine (AS2-5)
O
H
N
N -phenylacetyl L-
O
O
iso
COOH
2
N
R
H
HO
O
O
HO
OH
Novel designed L-
-glutamine derivatives
iso
Gallic acid
Figure 2. Chemical structures of gallic acid, antineoplaston 10 and its active metabolite N2-phenylacetyl L-iso-glutamine (AS2-5).
Meanwhile, 3,4,5-trihydroxybenzoic acid (gallic acid; Fig. 2) and
its derivatives are reported to possess significant anti-tumor and
anti-oxidative activities, especially without any toxicities to nor-
mal cells.25,26 According to the ‘combination principle’, we there-
fore envision that the conjunction of antineoplaston AS2–5
scaffold with gallic acid fragment might generate a more effica-
cious scaffold for potential enzymatic activity of APN.
3. Results and discussion
3.1. SAR studies
The target L-iso-glutamine derivatives were evaluated for their
inhibitory activities toward APN/CD13 and MMP-2. Similar to
APN, MMP-2 is also a zinc-dependent metalloproteinase involved
in the process of tumor invasion and metastasis.28,29 Hence the as-
say was performed on both of APN and MMP-2 so as to identify the
2. Chemistry
selectivity of these L-iso-glutamine compounds, and all the inhibi-
tion results are summarized in Table 1. Besides, bestatin was used
as the positive control.
In order to study the SAR of these novel peptidomimetic com-
pounds, different L-iso-glutamine derivatives were designed and
The results showed that most of the L-iso-glutamine derivatives,
synthesized via the route outlined in Scheme 1. Starting from com-
except compound 6u, display a better enzymatic inhibition on APN
than that of MMP-2, with IC50 values lying in micromolar level. The
resulting relatively selective inhibition against APN as compared
with MMP-2, to a certain extent, confirmed our strategy for design-
ing APNIs. This possibly descript from the differences between the
structures of two enzymes, leading to different requirements for
their respective inhibitors. APN is a membrane-bound zinc exopep-
tidase that catalyzed the removal of NH2-terminal amino acid from
the peptide, while MMP-2 is a zinc-dependent endopeptidase that
could cut the peptide to parts from the specific amino acid residue
of the peptide.1,29 As most compounds exhibited selective inhibi-
tion against APN, the following structure–activity relationships
(SARs) were mainly focused on APN inhibition.
mercially available gallic acid 1, the key intermediate (R)-N-(2,6-
dioxo-tetrahydro-2H-pyran-3-yl)-3,4,5-trimethoxybenzamide
5
was obtained via the sequence of methylation, acylation, nucleo-
philic substitution, and dehydration. This was followed by coupling
with various primary amines in the presence of acetic acid to give
target glutamine peptidomimetics 6a–t. As shown in Scheme 1,
when the asymmetric annular acetic anhydride (5) was treated
with different nucleophilic agents, the six-membered anhydride
ring might be opened from two sides. It revealed that only L-iso-
glutamine derivatives were obtained, which have been confirmed
by our previous work.27 Besides, the IR, ESI-MS analytical and 1H
NMR data of all the synthesized compounds were in full agreement
with the proposed structures (see Section 5).
O
O
O
O
COOH
COOH
HO
HO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
OH
a)
OH
O
Cl
N
H
b)
c)
OH
OCH3
OCH3
OCH3
1
2
3
4
COOH
3
O
O
H
N
H3CO
H3CO
O
H3CO
H3CO
N
H
1
O
e)
d)
N
H
R
O
OCH3
OCH3
various nucleophilic agents
5
6a-u
Scheme 1. Reagents and conditions: (a) Me2SO4, 40% NaOH, then 1 N HCl; (b) SOCl2 in benzene; (c) L-Glu-Na, Na2CO3, then 2 N HCl; (d) Ac2O, 55–60 °C; (e) RNH2, AcOH.

X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3053–3060
3055
Table 1 (continued)
Table 1
In vitro enzymatic assay (APN and MMP-2) results for L-iso-glutamine derivatives
6a–u
Compds
R
IC50
(l
M)a
MMP-2
APN
COOH
Cl
Cl
O
H
6n
80.4 5.5
78.5 8.1
122.4 7.2
107.2 8.0
N
O
O
N
H
R
O
O
Cl
F
Compds
R
IC50 (l
M)a
MMP-2
6o
APN
Br
6p
6q
176.9 1.5
65.9 2.0
287.3 4.6
76.8 1.8
H
N
6a
40.1 2.1
55.3 3.8
N
S
S
O
O
O
S
I
6b
6c
6d
102.6 0.7
107.6 2.8
87.7 3.3
628.0 10.1
369.4 11.4
169.4 5.1
NH2
O
F
6r
6s
45.6 8.4
51.2 5.8
52.8 6.1
76.1 0.9
N
Cl
Br
S
F
O
O
N
N
H
S
O
NH2
O
N
O
O
6e
6f
72.4 4.4
24.3 2.4
40.0 1.5
41.9 10.4
nab
114.9 9.0
26.1 1.8
52.3 4.4
54.4 9.0
nab
N
H
N
6t
104.0 10.3
659.6 4.7
H
O
N
F
O
6u
805.1 3.7
501.5 7.1
Cl
6g
6h
6i
Cl
Br
O
Bestatin
13.1 0.7
40.5 1.2
H2N
N
COOH
H
OH
a
Values are means of three experiments, standard deviation is given.
Not activity.
b
NO2
6j
23.4 2.3
65.8 7.5
33.6 1.9
71.2 2.5
NH2
Of these inhibitors, compound 6j, with phenylamine group as
hydrophobic side chain, showed the best inhibitory activity
(IC50 = 23.4 2.3 M). Compounds, 6a, 6f, 6g, 6h and 6r exhibited
the subsequent activities toward APN, with IC50 value from
24.3 2.4 to 45.6 8.4 M. However, all of these compounds dem-
onstrated a little less activity than that of bestatin. Additionally,
compounds with aromatic side chains (6a–t) gave better activities
compared to aliphatic one (6u). The possible reason might be due
6k
l
COOH
COOH
l
6l
57.8 3.9
89.6 1.6
86.3 3.1
OH
F
to the
p system of the aromatic ring enhancing the interaction with
the hydrophobic region of the enzyme.
Substitution on aromatic ring (6a–t) also has impact on bioac-
tivity. For the activities of sulfonylamino-containing compounds,
6a and 6b, the improved activity was observed via introduction
of a five-membered thiazole ring (6a). Comparing thiazole-con-
6m
108.7 7.8
F

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X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3053–3060
the catalytic zinc ion with the distance of 1.96 Å and 1.72 Å,
respectively. In addition, one of the methoxy group and partial car-
bonyl portion insert into S01 and S1 pocket.
According to the structure shown as Figure 4, the carbonyl
group of 6j can form hydrogen bonds with His297 (<2.85 Å) and
His301 (<3.03 Å), which are the essential amino acids of the con-
served sequence (HEXXHX18E) in the catalytic domain that is well
conserved in peptidase M1 family.30 As far as the Tyr381 residue is
concerned, the phenolic hydroxyl group of it can interact with the
carbonyl group of 6j by hydrogen interaction (<2.66 Å) which
might be benefit to stabilize of interaction intermediate with the
zinc ion.15 In addition, the binding interactions were further en-
hanced by hydrogen bonds with Tyr275, Ala262, Gly261, and
Arg293 as well. Finally, the hydrophobic parts of phenyl rings are
in contact with nonpolar surface areas of APN.
Although the computed information partially supported our
assumption, the exact binding model of the target compounds with
APN should be verified from further X-ray crystal studies.
4. Conclusions
In summary, we developed a new series of L-iso-glutamine
Figure 3. FlexX docking result of compound 6j with the active site of Escherichia coli
APN (PDB: 2DQM).
derivatives originated from the scaffold antineoplaston AS2-5.
Most of the compounds possess selective inhibitory activity
against APN as compared with MMP-2. This feature may offer a
critical point in designing selective metalloproteinase inhibitors
so as to optimize the recognition of APN. Besides, further in vitro
and in vivo evaluation of these compounds on anti-tumor activity
is underway.
taining compounds 6a, 6c and 6d, we can conclude that phenyl sul-
fonamide and amide groups were positively relative with the APN
inhibition. This might be due to the formation of hydrogen bond
with the enzyme backbone, thus stabilizing the binding between
the compound and the enzyme. However, replacing five-mem-
bered thiazole ring (6d) with six-membered pyridinyl ring (6e)
produced slightly improved potency.
5. Experimental
When it comes to compounds with mono-substitution at the
para-position in the aromatic ring (6f–k), to some extent, the elec-
tron-withdrawing property of para-substituent was negatively rel-
ative with the inhibitory activities. For example, the introduction
of amino group (6j) displayed the highest activity, the halogen
group (6f–h) was in the next place, following the carboxyl group
(6k), and the nitro group (6i) presented the least activity, even
has no APN and MMP-2 inhibition. Moreover, as compared with
fluoro, chloro and bromo substitutions, it seems that the increased
volume might lead to impair activity, suggesting there is a space
requirement in the binding pockets to accommodate the suitable
substituents. The same rule can also be applied to compound 6t.
As compared with the halogen-substituted compounds, gener-
ally speaking, di-substitution at the phenyl moiety (6m–o) moder-
ately impaired the activity in comparison with the mono-
substitution counterparts (6f–h). However, when we changed the
phenyl ring into benzyl moiety, the reverse rule can be observed.
That is to say, di-substitution at the benzyl moiety (6r and 6s)
showed better inhibitory activity compared with the mono-substi-
tution compounds, 6p and 6q. Interestingly, compound 6l, with a
hydroxyl group ortho- to the phenyl carboxyl core (at the 3- and
4-positions, respectively), was slightly more potent than 6k, sug-
gesting that hydroxyl group was positively related with the inhib-
itory activity by forming effective hydrogen bond with the residue
of the enzyme.
5.1. General procedures
Silica gel for column chromatography (CC) and thin-layer chro-
matography (TLC) plates precoated with Silica Gel GF254 were com-
mercial available from Qingdao Haiyang Chemical Company,
Qingdao, China. Reaction courses and product mixtures were rou-
tinely monitored by TLC on 0.25 mm Silica Gel 60 F254 plates and
visualized under ultraviolet (UV) light (254 nm), or iodine (I2) va-
por. Flash-chromatography (FC) was performed using 200–300
mesh silica gel and the solvent system was indicated in the proce-
dure. All solvents were of reagent grade and, when necessary, were
purified and dried by standard methods. Melting points (Mp) were
determined on an X-6 micro-melting-point apparatus (Beijing Tech
Co., Ltd) with no correction. Infrared spectra (IR) were recorded in
the range of 4000–600 cmꢀ1 using a Nicolet Nexus 470FT-IR spec-
trometer, and KBr disks were used as indicated. 1H NMR spectra
were determined on a Brucker Avance DRX-600 spectrometer, with
chemical shift (d) given in ppm upfield from Me4Si (TMS) as an
internal standard, and coupling constants J were recorded in hertz
(Hz). Electrospray ionization mass spectrometry (ESI-MS) was per-
formed on an API-4000 triple-stage quadrupole instrument. Anhy-
drous reactions were carried out in oven-dried glassware under a
nitrogen atmosphere, and all anhydrous solvents were distilled
over CaH2 or Na/benzophenone prior to use. Yields refer to purified
products and are not optimized.
3.2. Binding mode
5.2. Syntheses
In order to investigate the interaction of our target compound
with APN, the most active compound 6j was selected for molecular
docking study by utilizing SYBYL 7.0. The binding mode of 6j in the
active site of Escherichia coli APN (PDB code: 2DQM) is proposed
based on computational-docking results. The binding studies
(Fig. 3) demonstrated that carbonyl group of 6j can coordinate with
5.2.1. 3,4,5-Trimethoxybenzoic acid (2)
To a stirred solution of gallic acid 1 (5 g, 29.4 mmol) in 50 ml of
4 N NaOH was added (CH3)2SO4 (8.9 g, 71 mmol) dropwise, keep-
ing the inside temperature under 20 °C. The resulting solution
was allowed to stir at 30–40 °C for 20 min, another portion of gallic

X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3053–3060
3057
Ligand bond
Non-ligand bond
Hydrogen bond and its length
Non-ligand residues involved in hydrophobic contact(s)
Corresponding atoms involvedin hydrophobic contact(s)
Figure 4. Diagram (LIGPLOT) of the hydrogen bonds and hydrophobic interactions of 6j with APN.
acid (5 g) and 50 ml of 4 N NaOH were successively added. The
mixture was slowly heated to 90 °C and maintained at this temper-
ature for 1 h. After refluxing for another 2 h, the reaction mixture
was allowed to cool to rt. The solution was then acidified with
2 N HCl to pH 2, filtered, and the solid was washed twice with dis-
tilled water. The crude product was recrystallized with 50% EtOH
to give compound 2 (40.8 g, 65%) as a white needle crystals: mp
168–171 °C. 1H NMR d 3.97 (s, 9H), 7.23 (s, 2H), 12.11 (s, 1H).
ESI-MS: m/z [M+H]+ 165.8.
5.2.3. (R)-2-(3,4,5-Trimethoxybenzamido)pentanedioic acid (4)
To a suspension of anhydrous Na2CO3 (19.6 g, 185 mmol) and
L
-
glutamic acid (16.9 g, 115 mmol) in distilled water (150 ml) at
ꢀ5 °C was added a solution of compound 3 in 200 ml benzene
dropwise over 1 h. Introduce a mechanical stirrer and stir vigor-
ously until the solid has almost completely dissolved (about 5 h).
The mixture was acidified with 2 N HCl to pH 5–6, the layers were
separated, and the aqueous phase was acidified again with 2 N HCl
to pH 3–4, and extracted twice with chloroform. Remove the com-
bined organics, and the obtained aqueous phase was acidified with
2 N HCl to pH 2. Cool and leave in a refrigerator overnight, the
white precipitate was filtered with glass filter, washed with water,
and dried in vacuo to give compound 4 (28.6 g, 89.3%). Mp 120–
121 °C, IR (KBr, cmꢀ1): 3299.7 and 3255.4 (NH), 2942.5 (CH),
1744.7 and 1699.5 (C@O), 1500.7 (d NH), 1235.5 and 1127.7 (C–
O). 1H NMR (DMSO-d6, ppm): d 12.41 (s, 2H, 2-COOH), 8.53 (d,
1H, J = 7.2 Hz, NH), 7.22 (s, 2H, Ar-H), 4.41 (m, 1H, CH), 3.83 (s,
6H, 2-OCH3), 3.71 (s, 3H, OCH3), 2.35 (m, 2H, CH2), 2.21 (m, 1H,
CH), 1.96 (m, 1H, CH). ESI-MS: m/z [M+H]+ 341.8.
5.2.2. 3,4,5-Trimethoxybenzoic chloride (3)
To a stirred solution of 2 (21.2 g, 100 mmol) in benzene
(320 ml) was added dropwise SOCl2 (40 ml, 548 mmol). The
reaction mixture was refluxed for 3 h, and the solvent was re-
moved to obtain compound 3 as pale yellow oil. Successively an-
other 50 ml of benzene was added and evaporated until the
excess SOCl2 was completely removed. The crude product was
used immediately in the next reaction without further
purification.

3058
X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3053–3060
5.2.4. (R)-N-(2,6-Dioxo-tetrahydro-2H-pyran-3-yl)-3,4,5-
trimethoxybenzamide (5)
J = 7.5 Hz), 7.25 (s, 2H, Ar-H), 7.17 (d, 1H, @CH, J = 3.6 Hz), 4.58
(m, 1H, CH), 3.81 (s, 6H, 2-OCH3), 3.73 (s, 3H, –OCH3), 2.44 (m,
1H, CH), 2.29 (m, 1H, CH), 2.11 (m, 2H, CH2). ESI-MS: m/z [M+H]+
466.4.
Place 10 g (2.9 mmol) of compound 4 and 80 ml of redistilled
acetic anhydride in a 250 ml round-bottomed flask and boil the
mixture steadily under 55–60° for 5 h. Remove a small quantity
of insoluble substance by rapidly filtering the hot reaction mixture
through a small plug of cotton wool loosely inserted into the stem
of a preheated glass filter funnel. After 10 ml of anhydrous diethyl
ether was added, the filtrate was allowed to cool to room temper-
ature. The collected white precipitate was dried in vacuo to give 5
as white solid (5.2 g, 55%). Mp 150–152 °C, IR (KBr, cmꢀ1): 3310.0
(NH), 2945.1 (CH), 1777.0 and 1640.7 (O@C–O–C@O), 1504.2 (d
NH), 1239.6 and 1129.6 (C–O). 1H NMR (DMSO-d6, ppm): d 6.99
(s, 2H, Ar-H), 8.13 (d, 1H, J = 7.2 Hz, NH), 4.45 (m, 1H, CH), 3.73
(s, 9H, 3-OCH3), 1.96–2.25 (m, 2H, CH2), 2.18–2.28 (m, 2H, CH2).
ESI-MS: m/z [M+H]+ 323.8.
5.2.9. (R)-5-(2-Nicotinoylhydrazinyl)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6e)
Yield: 67%, mp 237–238 °C, IR (KBr, cmꢀ1): 3297.3 (NH), 2942.3
(CH), 1715.2 (O@C–NH), 1673.0 and 1654.9 (O@C–O), 1498.3
(C@C), 1124.5 (C–O). 1H NMR (DMSO-d6, ppm): d 10.65 (s, 1H,
COOH), 10.07 (s, 1H, NH), 8.76 (d, 2H, Ar-H, J = 4.5 Hz), 8.68 (d,
1H, NH, J = 7.5 Hz), 7.75 (d, 2H, Ar-H, J = 4.5 Hz), 7.23 (s, 2H, Ar-
H), 4.42 (m, 1H, CH), 3.83 (s, 6H, 2-OCH3), 3.71 (s, 3H, –OCH3),
2.37 (m, 2H, CH2), 2.17 (m, 1H, CH), 2.03 (m, 1H, CH). ESI-MS: m/
z [M+H]+ 460.9.
5.2.10. (R)-5-(4-Fluorophenylamino)-5-oxo-4-(3,4,5-
5.2.5. (R)-5-Oxo-5-4-[(1,3-thiazole-2-amino)sulfonyl]
phenylamino-4-[(3,4,5-trimethoxybenzamido)amino]
pentanoic acid (6a)
trimethoxybenzamido)pentanoic acid (6f)
Yield: 60%, mp 205–207 °C, IR (KBr, cmꢀ1): 3424.5 and 3276.9
(NH), 2939.2 (CH), 1715.7 (C@O), 1640.0 and 1508.2 (C@C),
1125.1 (C-O). 1H NMR (DMSO-d6, ppm): d 12.06 (s, 1H, COOH),
10.07 (s, 1H, NH), 8.49 (d, 1H, NH, J = 7.5 Hz), 7.63 (m, 2H, Ar-H),
7.25 (s, 2H, Ar-H), 7.13 (m, 2H, Ar-H), 4.57 (m, 1H, CH), 3.84 (s,
6H, 2-OCH3), 3.72 (s, 3H, –OCH3), 2.39 (m, 2H, CH2), 2.12 (m, 1H,
CH), 2.05 (m, 1H, CH). ESI-MS: m/z [M+H]+ 434.8.
A suspension of compound 5 (3.23 g, 10 mmol) and 4-amino-N-
(thiazol-2-yl)benzenesulfonamide (2.8 g, 11 mmol) in acetic acid
(30 ml) was stirred at room temperature until the disappearance
of the starting material, checking via TLC. Collect the white flaky
crystals on a Buchner funnel, wash with ice-cold water and dry
to give 6a (5.75 g, 78%). Mp 143–145 °C, IR (KBr, cmꢀ1): 3324.3,
3117.5 (NH), 2939.5 (CH), 1706.3 (O@C–NH), 1590.3 (C@C),
1127.0 (C–O). 1H NMR (DMSO-d6, ppm): d 12.6 (s, 1H, COOH),
12.21 (s, 1H, NH), 10.44 (s, 1H, NH), 8.58 (d, 1H, NH, J = 7.2 Hz),
7.75 (d, 2H, Ar-H, J = 9.3 Hz), 7.71 (d, 1H, @CH, J = 4.3 Hz), 7.25 (s,
2H, Ar-H), 7.23 (d, 2H, Ar-H, J = 9.1 Hz), 6.80 (d, 1H, @CH,
J = 4.4 Hz), 4.56 (d, 1H, CH, J = 5.5 Hz), 3.83 (s, 6H, 2-OCH3), 3.71
(s, 3H, -OCH3), 2.41 (m, 1H, CH), 2.34 (m, 1H, CH), 2.09 (m, 1H,
CH), 2.03 (m, 1H, CH). ESI-MS: m/z [M+H]+ 581.4.
5.2.11. (R)-5-(4-Chlorophenylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6g)
Yield: 68%, mp 217–218 °C, IR (KBr, cmꢀ1): 3420.1 and 3265.5
(NH), 2937.8 (CH), 1712.0 (C@O), 1639.5 and 1491.8 (C@C),
1128.4 (C–O). 1H NMR (DMSO-d6, ppm): d 12.14 (s, 1H, COOH),
10.24 (s, 1H, NH), 8.57 (d, 1H, NH, J = 7.4 Hz), 7.65 (m, 2H, Ar-H),
7.36 (m, 2H, Ar-H), 7.25 (s, 2H, Ar-H), 4.56 (m, 1H, CH), 3.84 (s,
6H, 2-OCH3), 3.71 (s, 3H, –OCH3), 2.39 (m, 1H, CH), 2.34 (m, 1H,
CH), 2.09 (m, 1H, CH), 2.03 (m, 1H, CH). ESI-MS: m/z [M+H]+ 450.8.
The other L-iso-glutamine derivatives were synthesized follow-
ing the general procedure as the above mentioned.
5.2.12. (R)-5-(4-Bromophenylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6h)
5.2.6. (R)-5-[4-(Aminosulfonyl)phenylamino]-5-oxo-4-[(3,4,5-
trimethoxybenzamido)amino]pentanoic acid (6b)
Yield: 79%, mp 221–222 °C, IR (KBr, cmꢀ1): 3420.6 (OH), 3291.8
and 3264.5 (NH), 2938.2 (CH), 1712.3 (O@C–NH), 1639.8 (O@C–O),
1489.9 (C@C), 1128.3 (C–O). 1H NMR (DMSO-d6, ppm): d 12.06 (s,
1H, COOH), 10.16 (s, 1H, NH), 8.51 (d, 1H, NH, J = 6.4 Hz), 7.60 (d,
2H, Ar-H, J = 7.8 Hz), 7.60 (d, 2H, Ar-H, J = 7.8 Hz), 7.25 (s, 2H, Ar-
H), 4.57 (m, 1H, CH), 3.84 (s, 6H, 2-OCH3), 3.71 (s, 3H, –OCH3),
2.39 (m, 1H, CH), 2.35 (m, 1H, CH), 2.12 (m, 1H, CH), 2.04 (m, 1H,
CH). ESI-MS: m/z [M+H]+ 497.1.
Yield: 67%, mp 206–208 °C, IR (KBr, cmꢀ1): 3334.7, 3299.9,
3245.2 (NH), 1733.9 (O@C–NH), 1668.3 (O@C–O), 1529.1 (C@C),
1340.9 and 1134.0 (S–O, C–O). 1H NMR (DMSO-d6, ppm): d 12.04
(s, 1H, COOH), 10.37 (s, 1H, NH), 8.54 (d, 1H, NH, J = 7.08 Hz),
7.77 (s, 4H, Ar-H), 7.26 (s, 2H, Ar-H), 7.18 (s, 2H, NH2), 4.59 (d,
1H, CH, J = 6.5 Hz), 3.84 (s, 6H, 2-OCH3), 3.73 (s, 3H, –OCH3), 2.42
(m, 1H, CH), 2.35 (m, 1H, CH), 2.12 (m, 1H, CH), 2.06 (m, 1H, CH).
ESI-MS: m/z [M+H]+ 497.9.
5.2.13. (R)-5-(4-Nitrophenylamino)-5-oxo-4-(3,4,5-
5.2.7. (R)-5-Oxo-5-(thiazol-2-ylamino)-4-(3,4,5-trimethoxy-
benzamido)pentanoic acid (6c)
trimethoxybenzamido)pentanoic acid (6i)
Yield: 71%, mp 228–230 °C, IR (KBr, cmꢀ1): 3392.3 and 3289.7
(NH), 2940.5 (CH), 1708.9 (O@C–NH), 1583.9 and 1499.9 (C@C),
1326.1 and 1130.3 (N–O, C–O). 1H NMR (DMSO-d6, ppm): d 12.18
(s, 1H, COOH), 10.74 (s, 1H, NH), 8.65 (d, 1H, NH, J = 7.2 Hz), 8.24
(d, 1H, Ar-H, J = 9.2 Hz), 7.89 (d, 1H, Ar-H, J = 9.3 Hz), 7.25 (s, 2H,
Ar-H), 4.59 (m, 1H, CH), 3.84 (s, 6H, 2-OCH3), 3.71 (s, 3H, –OCH3),
3.31 (s, 2H, CH2), 2.43 (m, 1H, CH), 2.37 (m, 1H, CH), 2.12 (m, 1H,
CH), 2.06 (m, 1H, CH). ESI-MS: m/z [M+H]+ 461.4.
Yield: 80%, mp 204–205 °C, IR (KBr, cmꢀ1): 3411.7 and 3328.3
(NH), 2940.7 (CH), 1696.1 (O@C-NH), 1583.0 and 1497.8 (C@C),
1128.3 (C–O). 1H NMR (DMSO-d6, ppm): d 12.27 (s, 1H, COOH),
12.20 (s, 1H, NH), 8.62 (d, 1H, NH, J = 7.0 Hz), 7.48 (d, 1H, @CH,
J = 3.5 Hz), 7.25 (s, 2H, Ar-H), 7.22 (d, 1H, @CH, J = 3.6 Hz), 4.63
(m, 1H, CH), 3.83 (s, 6H, 2-OCH3), 3.71 (s, 3H, -OCH3), 2.41 (m,
1H, CH), 2.34 (m, 1H, CH), 2.09 (m, 2H, CH2). ESI-MS: m/z [M+H]+
423.8.
5.2.14. (R)-5-(4-Aminophenylamino)-5-oxo-4-(3,4,5-
5.2.8. (R)-5-Oxo-5-(2-(thiazole-2-carbonyl)hydrazinyl)-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6d)
trimethoxybenzamido)pentanoic acid (6j)
Yield: 75%, mp >255 °C, IR (KBr, cmꢀ1): 3399.8 and 3279.9
(NH), 2939.6 (CH), 1714.0 (O@C–NH), 1637.8 (O@C–O), 1584.1
and 1500.4 (C@C), 1129.0 (C–O). 1H NMR (DMSO-d6, ppm): d
12.13 (s, 1H, COOH), 10.04 (s, 1H, NH), 8.54 (d, 1H, NH,
J = 7.5 Hz), 7.55 (s, 2H, Ar-H), 7.25 (s, 2H, Ar-H), 4.56 (m, 1H,
CH), 3.84 (s, 6H, 2-OCH3), 3.71 (s, 3H, –OCH3), 2.41 (m, 1H,
Yield: 52%, mp 213–214 °C, IR (KBr, cmꢀ1): 3414.2 and 3326.9
(NH), 2944.7 (CH), 1706.4 (O@C–NH), 1671.4 and 1656.0 (O@C–
O), 1497.7 (C@C), 1126.9 (C–O). 1H NMR (DMSO-d6, ppm): d
12.20 (s, 1H, COOH), 12.08 (s, 1H, NH), 8.61 (d, 1H, NH,
J = 3.8 Hz), 8.06 (d, 1H, NH, J = 5.5 Hz), 7.39 (d, 1H, @CH,

X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3053–3060
3059
CH), 2.34 (m, 1H, CH), 2.10 (m, 1H, CH), 2.04 (m, 1H, CH). ESI-
2-OCH3), 3.70 (s, 3H, –OCH3), 2.29 (m, 2H, CH2), 2.06 (m, 1H, CH),
MS: m/z [M+H]+ 431.8.
1.97 (m, 1H, CH). ESI-MS: m/z [M+H]+ 508.8.
5.2.15. (R)-4-(4-Carboxy-2-(3,4,5-trimethoxybenzamido)
butanamido)benzoic acid (6k)
5.2.21. (R)-5-(3-Iodobenzylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6q)
Yield: 64%, mp 248–249 °C, IR (KBr, cmꢀ1): 3512.0 (OH), 3361.4
(NH), 2944.6 (CH), 1710.9 and 1640.1 (O@C-NH), 1589.8 and
1498.4 (C@C), 1120.8 (C–O). 1H NMR (DMSO-d6, ppm): d 12.35
(br, 2H, 2-COOH), 10.34 (s, 1H, NH), 8.53 (d, 1H, NH, J = 7.2 Hz),
7.90 (d, 2H, Ar-H, J = 8.4 Hz), 7.74 (d, 2H, Ar-H, J = 8.4 Hz), 7.25 (s,
2H, Ar-H), 4.61 (m, 1H, CH), 3.84 (s, 6H, 2-OCH3), 3.72 (s, 3H, –
OCH3), 2.41 (m, 1H, CH), 2.35 (m, 1H, CH), 2.12 (m, 1H, CH), 2.06
(m, 1H, CH). ESI-MS: m/z [M+H]+ 462.9.
Yield: 59%, mp 165–167 °C, IR (KBr, cmꢀ1): 3275.5 (NH), 2937.8
(CH), 1708.7 and 1628.8 (O@C–NH, O@C–O), 1582.9 (C@C), 1128.5
(C–O). 1H NMR (DMSO-d6, ppm): d 12.11 (s, 1H, COOH), 8.49 (m,
1H, NH), 8.47 (d, 1H, NH, J = 7.8 Hz), 7.65 (s, 1H, Ar-H), 7.59 (d, 1H,
Ar-H, J = 8.4 Hz), 7.26 (m, 3H, Ar-H), 4.45 (m, 1H, CH), 4.26 (m, 2H,
CH2), 3.83 (s, 6H, 2-OCH3), 3.71 (s, 3H, –OCH3), 2.31 (m, 2H, CH2),
2.07 (m, 1H, CH), 1.95 (m, 1H, CH). ESI-MS: m/z [M+H]+ 556.7.
5.2.22. (R)-5-(4-Chloro-2-fluorobenzylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6r)
5.2.16. (R)-4-(4-Carboxy-2-(3,4,5-trimethoxybenzamido)
butanamido)-2-hydroxybenzoic acid (6l)
Yield: 62%, mp 145–147 °C, IR (KBr, cmꢀ1): 3292.3 (NH), 2940.8
(CH), 1713.2 (O@C), 1583.6 and 1498.9 (C@C), 1128.2 (C–O). 1H
NMR (DMSO-d6, ppm): d 8.63 (d, 1H, NH, J = 6.2 Hz), 8.46 (t, 1H,
NH, J = 7.2 Hz), 7.36 (m, 3H, Ar-H), 7.23 (s, 2H, Ar-H), 4.47 (m,
1H, CH), 4.30 (t, 2H, CH2, J = 6.3 Hz), 3.83 (s, 6H, 2-OCH3), 3.71 (s,
3H, –OCH3), 2.32 (m, 2H, CH2), 2.29 (m, 1H, CH), 2.84 (m, 1H,
CH). ESI-MS: m/z [M+H]+ 482.8.
Yield: 88%, mp 222–224 °C, IR (KBr, cmꢀ1): 3335.2 (NH), 2992.2
(CH), 1718.2 (O@C-NH), 1668.9 and 1604.9 (O@C–O), 1500.4
(C@C), 1130.0 (C–O). 1H NMR (DMSO-d6, ppm): d 13.69 (s, 1H,
COOH), 12.20 (s, 1H, COOH), 11.35 (s, 1H, OH), 10.42 (s, 1H, NH),
8.63 (d, 1H, NH, J = 7.2 Hz), 7.72 (d, 1H, Ar-H, J = 8.7 Hz), 7.39 (s,
1H, Ar-H), 7.21 (s, 2H, Ar-H), 7.10 (d, 1H, Ar-H, J = 7.7 Hz), 4.56
(m, 1H, CH), 3.84 (s, 6H, 2-OCH3), 3.71 (s, 3H, –OCH3), 2.43 (m,
1H, CH), 2.34 (m, 1H, CH), 2.09 (m, 1H, CH), 2.04 (m, 1H, CH).
ESI-MS: m/z [M+H]+ 476.8.
5.2.23. (R)-5-(4-Bromo-2-fluorobenzylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6s)
Yield: 54%, mp 146–148 °C, IR (KBr, cmꢀ1): 3292.3 (NH), 2940.7
(CH), 1712.4 and 1642.3 (O@CO@C), 1583.6 and 1499.5 (C@C),
1128.9 (C–O). 1H NMR (DMSO-d6, ppm): d 8.63 (d, 1H, NH,
J = 7.2 Hz), 8.46 (t, 1H, NH, J = 7.2 Hz), 7.49 (t, 1H, Ar-H, J = 7.8 Hz),
7.38 (m, 2H, Ar-H), 7.23 (s, 2H, Ar-H), 4.28 (m, 1H, CH), 4.24 (m,
2H, CH2), 3.83 (s, 6H, 2-OCH3), 3.70 (s, 3H, –OCH3), 2.32 (m, 2H,
CH2), 2.31 (m, 1H, CH), 2.28 (m, 1H, CH). ESI-MS: m/z [M+H]+ 528.8.
5.2.17. (R)-5-(2,4-Difluorophenylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6m)
Yield: 56%, mp 174–176 °C, IR (KBr, cmꢀ1): 3328.3 (NH), 2940.0
(CH), 1719.1 and 1639.5 (O@C–NH), 1501.2 (C@C), 1131.0 (C–O).
1H NMR (DMSO-d6, ppm): d 12.16 (s, 1H, COOH), 9.84 (s, 1H,
NH), 8.57 (d, 1H, NH, J = 7.5 Hz), 7.76 (m, 1H, Ar-H), 7.32 (m, 1H,
Ar-H), 7.29 (s, 2H, Ar-H), 7.06 (m, 1H, Ar-H), 4.67 (m, 1H, CH),
3.84 (s, 6H, 2-OCH3), 3.71 (s, 3H, –OCH3), 2.37 (m, 2H, CH2), 2.12
(m, 1H, CH), 2.03 (m, 1H, CH). ESI-MS: m/z [M+H]+ 452.8.
5.2.24. N-(1-(2-Aminophenyl)-2,6-dioxopiperidin-3-yl)-3,4,5-
trimethoxybenzamide (6t)
Yield: 70%, mp >230 °C, IR (KBr, cmꢀ1): 3427.3 and 3245.8 (NH),
2939.8 (CH), 1633.8 (O@C–NH), 1584.1 and 1499.7 (C@C), 1127.9
(C–O). 1H NMR (DMSO-d6, ppm): d 12.23 (br, 2H, NH), 8.86 (d,
1H, NH, J = 4.0 Hz), 7.47 (m, 2H, Ar-H), 7.30 (s, 2H, Ar-H), 7.14
(m, 2H, Ar-H), 5.35 (m, 1H, CH), 3.83 (s, 6H, 2-OCH3), 3.71 (s, 3H,
–OCH3), 2.46 (m, 2H, CH2), 2.43 (m, 1H, CH), 2.40 (m, 1H, CH).
ESI-MS: m/z [M+H]+ 411.1.
5.2.18. (R)-5-(2,5-Dichlorophenylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6n)
Yield: 89%, mp 219–220 °C, IR (KBr, cmꢀ1): 3367.8 and 3281.4
(NH), 2942.1 (CH), 1704.9 (O@C), 1587.2 (C@C), 1132.8 (C–O). 1H
NMR (DMSO-d6, ppm): d 12.11 (s, 1H, COOH), 9.60 (s, 1H, NH),
8.62 (d, 1H, NH, J = 7.2 Hz), 7.97 (d, 1H, Ar-H, J = 2.16 Hz), 7.54 (d,
1H, Ar-H, J = 8.6 Hz), 7.26 (s, 3H, Ar-H), 4.71 (d, 1H, CH,
J = 5.5 Hz), 3.85 (s, 6H, 2-OCH3), 3.73 (s, 3H, –OCH3), 2.42 (m, 2H,
CH2), 2.20 (d, 1H, CH, J = 6.1 Hz), 2.07 (d, 1H, CH, J = 6.1 Hz). ESI-
MS: m/z [M+H]+ 407.1.
5.2.25. (R)-5-(2-Chloroethylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6u)
Yield: 62%, mp 158–159 °C, IR (KBr, cmꢀ1): 3292.9 and 3260.9
(NH), 2942.7 (CH), 1727.6 and 1659.4 (O@C-NH), 1578.4 and
1499.8 (C@C), 1128.3 (C–O). 1H NMR (DMSO-d6, ppm): d 12.10
(s, 1H, COOH), 8.42 (d, 1H, NH, J = 7.8 Hz), 8.19 (d, 1H, NH,
J = 5.7 Hz), 7.23 (s, 2H, Ar-H), 4.45 (m, 1H, CH), 3.83 (s, 6H, 2-
OCH3), 3.70 (s, 3H, –OCH3), 3.61 (m, 2H, CH2), 3.45 (m, 1H, CH),
3.35 (m, 1H, CH), 2.31 (m, 2H, CH2), 2.04 (m, 1H, CH), 1.91 (m,
1H, CH). ESI-MS: m/z [M+H]+ 402.8.
5.2.19. (R)-5-(3-Chloro-4-fluorophenylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6o)
Yield: 58%, mp 204–205 °C, IR (KBr, cmꢀ1): 3333.4 (NH), 2940.5
(CH), 1715.7 (O@C), 1641.4 and 1500.4 (C@C), 1130.1 (C–O). 1H
NMR (DMSO-d6, ppm): d 12.10 (s, 1H, COOH), 10.32 (s, 1H, NH),
8.59 (d, 1H, NH, J = 7.3 Hz), 7.94 (m, 1H, Ar-H), 7.52 (m, 1H, Ar-
H), 7.37 (m, 1H, Ar-H), 7.25 (s, 2H, Ar-H), 4.53 (m, 1H, CH), 3.84
(s, 6H, 2-OCH3), 3.71 (s, 3H, –OCH3), 2.39 (m, 1H, CH), 2.35 (m,
1H, CH), 2.10 (m, 1H, CH), 2.03 (m, 1H, CH). ESI-MS: m/z [M+H]+
468.8.
6. Enzymatic inhibiton assay (in vitro)
6.1. In vitro APN assay
5.2.20. (R)-5-(3-Bromobenzylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid (6p)
The IC50 values against APN were determined using L-Leu-p-
nitroanilide as substrate and microsomal aminopeptidase from
Porcine Kidney Mocrosomes (Sigma) as the enzyme in 50 mM
PBS, pH 7.2 at 37 °C.31 The hydrolysis of the substrate was moni-
tored by following the change in the absorbance measured at
405 nm with the UV–vis spectrophotometer Pharmacia LKB, Bio-
chrom 4060. All solutions of inhibitors were prepared in the assay
Yield: 89%, mp 177–179 °C, IR (KBr, cmꢀ1): 3334.8 (NH), 2992.0
and 2940.4 (CH), 1714.4 (O@C–NH), 1635.9 (O@C–O), 1584.2 and
1500.4 (C@C), 1130.6 (C–O). 1H NMR (DMSO-d6, ppm): d 8.59 (s,
1H, NH), 8.51 (s, 1H, NH), 7.47 (s, 2H, Ar-H), 7.41 (s, 2H, Ar-H),
7.27 (s, 4H, Ar-H), 4.45 (m, 1H, CH), 4.29 (s, 2H, CH2), 3.83 (s, 6H,

3060
X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3053–3060
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lg/ml final concentration), and the assay buf-
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This work was financial supported from the Natural Science
Foundation of China (30701053 and 90713041), the Youth Doc-
toral Foundation of Ministry of Education of the PR China
(20070422061), and also the Natural Science Foundation of Shan-
dong Province (Y2008C01).
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