Design, synthesis, and preliminary activity evaluation of novel peptidomimetics as aminopeptidase N/CD13 inhibitors

Article abstract of DOI:10.1002/ardp.201100109

The synthesis of a series of novel N-α-galloylated isoglutamic acid I-amide peptidomimetics is described. Their enzymatic inhibition against aminopeptidase N (APN/CD13) and matrix metalloproteinase-2 (MMP-2) was tested. The preliminary activity assay revealed that most of the compounds displayed selective inhibition against APN as compared with MMP-2, with IC₅₀ values in a micromolar range. Within this series, compound 4 (IC ₅₀a=10.2a±a.9μM) demonstrated comparable APN inhibition as compared with the positive control bestatin (IC ₅₀a=a13.1aa0.7aμM), which might be a promising lead for further molecular optimizations. A new series of bi-peptide analogues containing amino acid or organic acid residues as elongated hydrophobic substituents inserting into S₁ or S₁ pocket was designed. Most of the target compounds showed potent and selective activities against APN/CD13 as compared with MMP-2. Compound 4 was comparable to bestatin and could be used as a potential lead for further development of APNIs.

Full text of DOI:10.1002/ardp.201100109

494
Arch. Pharm. Chem. Life Sci. 2011, 344, 494–504
Full Paper
Design, Synthesis, and Preliminary Activity Evaluation of
Novel Peptidomimetics as Aminopeptidase N/CD13 Inhibitors
Xun Li, Junli Wang, Lei Zhang, and Wenfang Xu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, Ji’nan,
Shandong Province, P.R. China
The synthesis of a series of novel N-a-galloylated isoglutamic acid g-amide peptidomimetics is
described. Their enzymatic inhibition against aminopeptidase
N (APN/CD13) and matrix
metalloproteinase-2 (MMP-2) was tested. The preliminary activity assay revealed that most of the
compounds displayed selective inhibition against APN as compared with MMP-2, with IC50 values in a
micromolar range. Within this series, compound 4 (IC50 ¼ 10.2 ꢀ 0.9 mM) demonstrated comparable
APN inhibition as compared with the positive control bestatin (IC50 ¼ 13.1 ꢀ 0.7 mM), which might
be a promising lead for further molecular optimizations.
Keywords: Aminopeptidase N inhibitor / Antiproliferative activity / APN/CD13 / Peptidomimetics
Received: March 26, 2011; Revised: April 21, 2011; Accepted: April 29, 2011
DOI 10.1002/ardp.201100109
Introduction
leukemia, which has been a useful tool as positive control in
elucidating many physiological conditions [8].
Aminopeptidase N (APN, EC 3.4.11.2), also known as human
lymphocyte surface cluster differentiation antigen, CD13, is a
150-kDa monomeric or homodimeric type II membrane-
bound glycoprotein which can release neutral or basic amino
acids from the N-terminal end of peptides [1]. It is a zinc-
dependent exopeptidase that is expressed by myeloid, mono-
cytes, epithelial cells of the intestine and kidney, fibroblasts,
and endothelial cells [2]. It is particularly noticeable that
APN/CD13 is overexpressed on the epithelium of the tumor
and plays critical roles in tumor progression, such as inva-
sion, metastasis, and angiogenesis [3]. Accordingly, APN/
CD13 inhibition represents a promising approach to cancer
treatment [4, 5].
Given most of the reported APNIs are pseudodipeptides or
peptide mimics bearing a chelating moiety (such as hydrox-
amate or carboxylate), inhibitors containing zinc-binding
groups (ZBGs), should be optimal selection to inhibit the
enzymatic activity of APN/CD13. Moreover, according to
the reported binding pattern of bestatin with APN from
Escherichia coli (ePepN), there are two hydrophobic subsites
0
beside the catalytic zinc(II) ion, called S1 and S1 pocket,
respectively, which are long and narrow binding domains.
Hence, it is appropriate to introduce one or more hydro-
phobic portion protruding into these pockets so as to gen-
erate effective interactions [9, 10].
In our previous work, we have reported a spectrum of
peptidomimetic L-Isoglutamine derivatives derived from
antineoplaston AS2–5, one of the active degradation prod-
ucts of antineoplaston A-10, which has efficacious antiproli-
ferative activity. The enzymatic evaluation revealed that most
compounds displayed good inhibitory activities against APN/
CD13 [11, 12]. Considering the side chains of these com-
pounds were all aromatic substituents and not long enough,
we herein designed a new series of bi-peptide analogues,
which contains amino acid or organic acid residues as
To date, several natural or synthetic APN inhibitors (APNIs)
have been exploited and some of them are currently inves-
tigated for clinical usage [6]. Among these inhibitors, besta-
tin, an antibiotic of microbial origin and best known APNI [7],
is a well-established anticancer agent employed clinically
in the ancillary treatment of adult acute nonlymphocytic
0
elongated hydrophobic substituents inserting into S1 or S1
Correspondence: Wenfang Xu, School of Pharmaceutical Sciences,
Shandong University, No 44 Wenhua Xi Road, Ji’nan, 250012, Shandong
Province, P.R. China.
E-mail: tjulx2004@sdu.edu.cn
Fax: þ86-531-88382264
pocket. We hereby hope the elongated side chains could
produce improved inhibitory activity towards APN/CD13.
Meanwhile, the newly introduced amino acid methyl esters
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APN/CD13 Inhibitors as Potential Antiproliferative Agents
495
Zn2+
OH
Zn2+
O
O
O
O
OH
amino acid or organic acid
R
aniline or benzyl amine
H
N
H
N
H3CO
H3CO
H3CO
H3CO
N
H
N
H
R
S1'
S1'
O
O
COOCH3
S1
S1
OCH3
OCH3
Previously reported APNIs by our lab
N
ewly synthesized peptidomimetic APNIs
Figure 1. Newly designed peptidomimetic derivatives.
can also serve as possible ZBGs to bind with the catalytic zinc
ion (see Fig. 1).
to provide the final compounds. In this reaction, other
aprotic polar solvent, e.g. benzene, is also preferable. In
particular, as verified by our previous work [13], when
the starting asymmetrically substituted cyclic anhydride
was treated with different nucleophilic agents, only
L-Isoglutamine derivatives 2a–r and 3 were finally obtained.
Furthermore, in order to compare the binding affinities of
free phenolic hydroxyl group with the galloylated counter-
parts, compound 6 was chemically converted to its depro-
tected homologue 7 by using BBr3 in the mixed solvents of
CH2Cl2 and DMF. Finally, ammonolysis of 2a and 2b with
NH2OK gave the corresponding hydroxamate compounds
Results and discussion
Chemistry
The target compounds 2a–r and 3–7, were synthesized effi-
ciently, following the procedures outlined in Scheme 1. In
our synthesis, the starting cyclic anhydride 1 was synthesized
in our previous condition [11]. This was followed by coupling
with various L-amino acid methyl ester hydrochlorides or
primary amines in the presence of Et3N in anhydrous CH2Cl2
COOH
H
3
O
O
O
H3CO
H3CO
O
N
H
H3CO
H3CO
N
R1
1
O
a)
N
H
O
COOCH3
various nucleophilic agents
OCH3
OCH3
1
2a-r, 3
b)
COOH
COOH
O
O
O
H
N
H
N
HO
HO
N
H3CO
H3CO
N
N
H
N
c)
H
O
S
O
S
OH
O
OCH3
7
6
H
N
OH
COOH
O
O
H
N
H
N
H3CO
H3CO
OH
H3CO
H3CO
R1
COOCH3
N
N
H
d)
N
H
H
O
R1
O
OCH3
OCH3
4
5
R1=H
R1=CH3
2a R1=H
2b R1=CH3
Scheme 1. Synthesis of target compounds
from asymmetrically substituted cyclic anhy-
dride 1.
Reagents and conditions: a) Various L-amino acid methyl ester hydrochloride,
anhydrous CH2Cl2, Et3N, rt. b) thiazol-2-amine, acetic acid, rt. c) BBr3, CH2Cl2,
DMF, 0ºC. d) NH2OK in anhydrous CH3OH, then acetic acid (two steps).
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X. Li et al.
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Table 1. The structures and inhibitory activities of target peptidomimetic compounds 2a–2r and 3–7.
COOH
O
H
8
12
R
3
4
H3CO
H3CO
1
N
11
9
N
7
H
O
COOCH3
5
OCH3
Compd.
R
IC50 [mM] a)
APN/CD13
MMP-2 (gelatinase A)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
H
CH3
CH3CH2
CH3CH2CH2
CH3CH2CH2CH2
C6H5CH2
p-F-C6H4CH2
p-Cl-C6H4CH2
p-Br-C6H4CH2
p-HO-C6H4CH2
HOCH2
33.2 ꢀ 2.2
59.3 ꢀ 0.7
108.2 ꢀ 1.5
470.2 ꢀ 5.4
NA b)
110.2 ꢀ 12.1
65.8 ꢀ 2.3
79.2 ꢀ 1.5
88.7 ꢀ 0.9
45.5 ꢀ 3.6
36.8 ꢀ 5.1
50.7 ꢀ 1.9
37.8 ꢀ 2.2
74.7 ꢀ 8.2
312.9 ꢀ 11.8
NA b)
171.4 ꢀ 8.4
209.5 ꢀ 10.7
380.1 ꢀ 3.5
656.7 ꢀ 2.8
NA b)
880.5 ꢀ 33.6
309.7 ꢀ 7.9
206.5 ꢀ 12.4
163.3 ꢀ 4.9
125.0 ꢀ 7.8
203.9 ꢀ 3.7
118.4 ꢀ 6.5
176.8 ꢀ 15.8
152.4 ꢀ 20.1
NA b)
CH3SCH2CH2
C6H5SCH2CH2
Cbz-NHCH2CH2CH2
Cbz-NHCH2CH2CH2CH2
NO2NH(NH)CNHCH2CH2CH2
22.6 ꢀ 10.1
CH2
2q
2r
56.6 ꢀ 1.7
29.7 ꢀ 0.8
110 ꢀ 16.1
179.3 ꢀ 5.7
N
H
Bzl
CH2
N
N
COOH
O Bzl
O
H3CO
H3CO
N
N
H
3
30.2 ꢀ 1.4
95.8 ꢀ 8.3
COOCH3
O
OCH3
H
N
O
HO
O
O
H
OH
4
H3CO
H3CO
N
10.2 ꢀ 0.9
193.9 ꢀ 5.2
N
H
N
H
O
OCH3
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Table 1. (continued )
APN/CD13 Inhibitors as Potential Antiproliferative Agents
497
COOH
O
H
11
8
12
R
3
4
H3CO
H3CO
1
N
9
N
7
H
O
COOCH3
5
OCH3
Compd.
R
IC50 [mM] a)
APN/CD13
MMP-2 (gelatinase A)
H
N
O
HO
O Bzl
O
H3CO
H3CO
N
N
H
5
18.5 ꢀ 2.4
125.7 ꢀ 9.7
O
NH
OH
O
OCH3
COOH
O
H
H3CO
H3CO
N
N
N
H
6 c)
107.6 ꢀ 12.8
369.4 ꢀ 21.4
O
S
OCH3
O
COOH
H
HO
HO
N
N
N
H
7
22.8 ꢀ 5.5
206.6 ꢀ 7.9
O
S
OH
HO
H
N
Bestatin
13.1 ꢀ 0.7
40.5 ꢀ 1.5
O
NH2
HO
O
a)
b)
c)
Values are means of three experiments, standard deviation is given. NA: no activity. The compound has been reported in
our previous work (see [11]).
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(4 and 5), with the purpose of comparing the chelating
effectiveness of different ZBGs. The chemical structures
of the target compounds were analytical confirmed by IR,
1H-NMR, ESI-MS, and elemental analysis.
Comparing 2a–e, R1 groups were fixed as H atom, while R2
was altered as various aliphatic side chains. We can see that
the length of R2 groups was negatively relative with the APN
inhibition, suggesting it is unfavorable to increase the length
of substituents along the orientation of the R2 groups. The
possible reason is that R2 groups did not accommodate the
Preliminary in-vitro evaluation and SAR discussion
The target compounds were evaluated for their enzymatic
inhibition towards APN/CD13 and MMP-2. Similar to APN,
MMP-2 (gelatinase A) is also a zinc-dependent proteinase
involved in the process of tumor invasion and metastasis
[14, 15]. Hence the assay was performed on both of APN/
CD13 and MMP-2 so as to identify the potential leads with
selectivity. All the inhibition results are summarized in
Table 1 and, bestatin was used as the positive control.
The results showed that most compounds, except 2p, dis-
play a better enzymatic inhibition against APN than that of
MMP-2, with IC50 values lying in micromolar level. This
selectivity against APN as compared with MMP-2, to a certain
extent, validated our strategy for designing potential APNIs.
This might be attributed to their different structures, leading
to different requirements for their respective inhibitors.
Specifically, APN is a membrane-bound zinc exopeptidase
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 [14]. As the described selectivity,
the following SARs were mainly focused on the APN/CD13
inhibition.
0
active binding domains (S1 or S1 pocket) of APN/CD13. Thus,
the longer the side chain of R2 was, to some extent, the less
strongly the compounds inhibited the enzyme. The same rule
can also be applied to compounds 2n–p.
Substitution on aromatic ring also has impact on bioac-
tivity. For instance, compounds 2g–j with halogen substitu-
ents at the para-position in the aromatic ring, to some extent,
exhibited obviously enhanced potencies compared with their
predecessor 2f. This result might be owing to the p-p con-
jugative effect between halogen atoms and aromatic ring
which could enhance the interaction with the enzyme.
Moreover, as compared with the fluoro, chloro and bromo
substitutions, it seems that the increased bulk might lead to
impair activity, implying there is a space requirement in the
binding pockets to accommodate the suitable substituents.
Compounds 4 and 5 were found to be more potent than
their precursors (2a and 3). This difference was possibly
caused by the ZBG, which was the only structural difference
between them. It revealed that introduction of hydroxamate
(CONHOH) produced increased potency in comparison with
its precursor, carboxylate (COOCH3) or carboxyl (COOH),
respectively, which is in accordance with our previously
SAR results [11, 12]. In addition, the antiproliferative effect
of these two compounds (4 and 5) on tumor cell HL-60 (myelo-
monocytic human acute granulocytic leukemia cells express-
ing high APN level) [16] were assessed by using MTT method.
The IC50 values of these two compounds (4, 2.17 ꢀ 0.39 mM
and 5, 2.61 ꢀ 0.31 mM) confirmed again that their inhibitory
activities are similar with bestatin (1.50 ꢀ 0.22 mM, see
Fig. 2).
Of these galloylated inhibitors (2aꢁr and 3ꢁ6), compound
4 with dihydroxamate substituents, gave the best inhibitory
activity (IC50 ¼ 10.2 ꢀ 0.9 mM) and displayed comparable
potency with bestatin (IC50 ¼ 13.1 ꢀ 0.7 mM). Compounds
5, 3, 2a, 2k, 2m, and 2r exhibited the subsequent activities
against APN/CD13, with IC50 values ranging from 18.5 ꢀ 2.4
to 37.8 ꢀ 2.2 mM. However, the above six compounds dem-
onstrated less potent in comparison with bestatin.
As to 2aꢁr, generally speaking, compounds containing
the zinc-binding functional groups, say, 2j and 2k (OH), 2l
and 2m (S), 2n, 2o, 2q, and 2r (N), offered relatively better
potencies. This is because, apart from the contributions of
ZBGs, another possible reason might attribute to the electro-
static interaction with the carbonyl group of a specific amino
acid residue in the active site of APN/CD13. An interesting
case can be used to support this conclusion, that is, sulphur-
containing 2l presented higher affinities than its isosteric
aliphatic-containing counterpart 2e, which even has no APN/
CD13 inhibition. Nevertheless, although both containing
sulphur atom, 2m with aromatic side chain gave better
activity than that of aliphatic counterpart (2l). The most
likely reason may due to the p system of the aromatic ring
enhancing the interaction with the hydrophobic region of
the enzyme.
3.5
*
3
2.5
2
**
1.5
1
0.5
0
Compound 4
Compound 5
Control
Figure 2. Effects of bestatin (control) and compounds 4 and 5 on
proliferation of the HL-60 cell line. Data are expressed as
ꢂ
mean values of five independent experiments (ꢀSE). p < 0.01,
ꢂꢂ p < 0.05 compared to the control.
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APN/CD13 Inhibitors as Potential Antiproliferative Agents
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Figure 3. Comparison of preponderant con-
formations between bestatin and compound 4.
Consistent with this result, free phenol hydroxyl-substituted
compound 7 was prepared derived from its galloylated
counterpart (6), with the purpose of comparing the chelating
effect of hydroxyl group as another ZBG. Compound 6 has
been reported in our previous work, (see [15], compound 2C),
for clarity, different denomination was deployed in present
report. It revealed that the de-galloylated compound (7) pro-
vided higher potency than that of the galloylated one (6),
with nearly 5-fold enhancement towards APN/CD13. Besides
the contribution of the phenol hydroxyl group, the hydrogen
bond between hydroxyl group and the enzyme, to a certain
extent, might also play an important role in the enzymatic
inhibition.
Moreover, the amino group of 8 formed a hydrogen bond
with the phenolic hydroxyl group of Tyr381 residue, thus
stabilizing the reaction intermediate with the zinc ion. In
Binding studies
The representative compound 4, which showed the best
affinity in our series, was selected to study its binding mode
with APN. Firstly, the predicted conformation of 4 was opti-
mized with the Powell Energetic Gradiet method built in the
Sketch/Build Edit model (Sybyl 6.91, Linux 7.3) [17]. As com-
pared with bestatin, the comparable results showed the
affinity to the same spatial region, suggesting the similar
APN/CD13 inhibitory potencies. In precise, both the isopropyl
portion of bestatin and the hydroxamate fragment of 4 can
0
adjust their flexibility to occupy the S1 pocket with their
preponderant conformation, as diagrammed in Fig. 3. In
contrast, the similar situation can also be observed between
the phenyl group of bestatin and the galloylated phenyl
portion of 4.
To further understand the interaction of 4 with APN, the
preferred docking study was ongoing performed using a
Sybyl/FlexX module. From Figure 4A, we can see both the
galloylated phenyl portion and the introduced hydroxamate
fragment of 4 can well-orienting interact with the active
0
domain (S1 and S1 pocket) of APN/CD13. Additionally, both
the nitrogen and oxygen atoms in hydroxymate can coordi-
˚
nate with the zinc ion, with a distance of 2.26 and 1.86 A,
respectively. According to Fig. 4B, the hydroxamate group
can form hydrogen bond with His301, one of the essential
amino acids of the conserved sequence (HEXXHX18E) in the
catalytic domain that is well conserved in peptidase M1
Figure 4. FlexX docking results of compound 4 with the active site
of E. coli APN (A & B) and MMP-2 (C).
˚
family [18], at the distance of 2.65 and 2.73 A, respectively.
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Sigma) was added and incubated for another 20 min, the result-
ing solution was detected under 450 nm wavelength to gain
absorption.
addition, the binding interaction was further enhanced by
˚
hydrogen bond with Gly261 (ꢁ3.39 A).
Finally, the selectivity of 4 might be partially explained by
the molecular docking with the active site of MMP-2 (PDB
code: 1HOV) [19]. As shown in Fig. 4C, although two oxygen
atoms of carbonyl groups could coordinate with the zinc ion
MTT assay
The cell lines were grown in RPMI1640 medium containing
10% FBS at 378C in a humidified incubator with 5% CO2. Cell
proliferation was determined by the MTT (3-[4,5-dimethyl-2-thia-
zolyl]-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 1600,
800, 400, 200, or 100 mg/mL of the compounds for 48 h.
Following this, 0.5% MTT solution was added to each well.
After further incubation for 4 h, the formazan formed from
MTT was extracted by adding DMSO and mixing for 15 min.
The optical density was read with ELISA reader at 570 nm.
˚
of MMP-2, with a distance of 2.36 and 2.16 A, respectively,
0
only the hydroxymate group can partially insert into the S1
pocket, causing seriously impaired MMP-2 inhibitory activity,
in accordance with the enzymatic assay results.
Conclusion
In summary, novel peptidomimetic derivatives were devel-
oped and evaluated as potential inhibitors of APN/CD13.
Most of the target compounds showed potent and selective
activities against APN/CD13 as compared with MMP-2. And
else, compound 4 was comparable to bestatin and could be
used as a potential lead for further development of APNIs. It
should be noted that although our preliminary enzymatic
results are not very encouraging (IC50 values in the micro-
mole range), their selective inhibition towards APN/CD13 and
SAR information provides useful clues for further APNIs
exploitation.
Computational-docking
The docking study was performed as follows: The selected com-
pound was constructed with a Sybyl/Sketch module and opti-
mized using Powell Energetic Gradient method with a Tripos
force field with the convergence criterion set at 0.05 kcal/(mol A),
and assigned with Gasteiger-Hu¨ckel method [22, 23]. When it
comes to the docking assay of APN, the residues in a radius of
˚
7.0 A around bestatin in the co-crystal structure (PDB code:
2DQM) [24] were selected as the active site, and other docking
parameters implied in the program were kept as the defaults. As
˚
˚
to MMP-2, residues in a radius of 4.0 A around SC-74020 (the
provided ligand of MMP-2 in the crystal structure, PDB code:
1HOV) [19] were considered as the active site, including the
catalytic zinc(II) ion.
Experimental
Biological evaluation
All synthesized compounds were evaluated for their inhibitory
properties against APN/CD13 versus MMP-2 (gelatinase A) so as to
compare the selectivity. Compounds 4 and 5 were further tested
the antiproliferative activity on the HL-60 cell.
General procedures for chemistry
Silica gel for column chromatography (CC) and TLC plates pre-
coated with silica gel GF254 were commercial available from
Qingdao Haiyang Chemical Company, Qingdao, China.
Reaction courses and product mixtures were routinely moni-
tored by thin-layer chromatography (TLC) on 0.25 mm silica-
gel 60 F254 plates and visualized under ultraviolet (UV) light
(254 nm), or iodine (I2) vapor. Flash-chromatography (FC) was
performed using 200–300 mesh silica gel and the solvent
system was indicated in the procedure. 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 with no correction.
Infrared spectra (IR) were recorded in the range of 4000–
600 cmꢃ1 using a Nicolet Nexus 470FT-IR spectrometer, and
KBr disks were used as indicated. 1H-NMR spectra were deter-
mined on a Bruker 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.
Measurements were made in D2O or CD3OD solutions.
Elemental analyses were carried out on a Perkin-Elmer C, H, N
elemental analyzer. Anhydrous reactions were carried out in
oven-dried glassware under a nitrogen atmosphere, and all anhy-
drous solvents were distilled over CaH2 or Na/benzophenone
prior to use. Yields refer to purified products and are not
optimized.
Enzyme inhibition assay
APN/CD13 inhibition
The IC50 values against APN/CD13 were determined using
L-Leu-p-nitroanilide as substrate and microsomal aminopepti-
dase from Porcine Kidney Microsomes (Sigma) as the enzyme
in 50 mM PBS, pH 7.2 at 378C [20]. 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 adjusted to
7.5 by the addition of 0.1 M HCl or 0.1 M NaOH. All inhibitors
were preincubated with APN for 30 min at room temperature.
The assay mixture, which contained the inhibitor solution (con-
centration dependent on the inhibitor), the enzyme solution
(4 mg/mL final concentration), and the assay buffer, was adjusted
to 200 mL.
MMP-2 inhibition
MMP-2 (gelatinase A) assay was performed as described by Baragi
et al. [21]. The gelatinase, substrate, and inhibitor were dissolved
in sodium borate (pH 8.5, 50 mmol/L) and incubated for 30 min
at 378C, and then 0.03% trinitrobenzenesulfonic acid (TNBS,
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Arch. Pharm. Chem. Life Sci. 2011, 344, 494–504
APN/CD13 Inhibitors as Potential Antiproliferative Agents
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General procedure for the preparation of L-amino acid
methyl ester hydrochloride
(R)-5-((S)-1-Methoxy-1-oxopropan-2-ylamino)-5-oxo-4-
(3,4,5-trimethoxybenzamido) pentanoic acid 2b
The preparation of methyl 2-aminoacetate hydrochloride is
reported here as a representative example: To a suspension of
glycine (15.0 g, 200 mmol) in anhydrous methanol (150 mL) at
08C was pumped slowly in dried HCl gas until solution was
completely clear. Cool in a refrigerator, preferably overnight.
Remove the solvent and small amount methanol was added
again to remove the excess HCl. The collected white precipitate
was recrystallized from methanol/diethyl ether (1:4) to give
methyl 2-aminoacetate hydrochloride (20 g, 80%). M.p. 175–
1768C; IR (KBr, cmꢃ1): 3215.0–3523.0 (NH), 2935.2 (CH), 1676.3
Yield: 35.0%, m.p. 153–1558C, IR (KBr, cmꢃ1): 3304.8 (NH), 2933.5
–
–
–
(CH), 1751.1 & 1719.9 (O C–NH), 1647.7 (O C–O), 1584.2 (C C),
–
–
–
1129.9 (C–O). [a]D ¼ þ28.9 (c 1, MeOH); 1H-NMR (DMSO-d6,
ppm): d 8.74 (d, 1H, J ¼ 7.8 Hz, NH), 8.33 (d, 1H, J ¼ 6.0 Hz,
NH), 7.24 (s, 2H, Ar-H), 4.36 (m, 1H, CH), 4.23 (m, 1H, CH), 3.85
(s, 6H, 2-OCH3), 3.73 (s, 3H, OCH3), 3.60 (s, 3H, COOCH3), 2.23 (t,
2H, J ¼ 7.8 Hz, CH2), 2.06, 1.83 (m, 2H, CH2), 1.23 (d, 3H,
J ¼ 7.2 Hz, CH3). ESI-MS m/z: 425.1 [M þ H]þ. Anal. calcd.
for C19H26N2O9: C, 53.52; H, 6.10; N, 6.57. Found: C, 54.07; H,
6.18; N, 6.46.
25
–
(C O), 1129.3 (C–O).
–
Similarly, reaction of other L-amino acids and anhydrous
methanol under dry HCl atmosphere gave methyl ester hydro-
chloride as follows.
Methyl 2-amino-4-methylpentanoate hydrochloride: Yield:
69%, m.p. 115–1178C
Methyl 2-aminopropanoate hydrochloride: Yield: 84%, m.p.
(R)-5-((S)-1-Methoxy-1-oxobutan-2-ylamino)-5-oxo-4-
(3,4,5-trimethoxybenzamido) pentanoic acid 2c
Yield: 28.5%, m.p. 105–1078C, IR (KBr, cmꢃ1): 3305.2 (NH), 2942.2
–
–
–
(CH), 1746.1 & 1717.9 (O C-NH), 1644.7 (O C–O), 1586.7 (C C),
–
–
–
1127.6 (C–O). [a]D ¼ þ22.6 (c 1, MeOH); 1H-NMR (DMSO-d6,
ppm): d 8.73 (d, 1H, J ¼ 7.8 Hz, NH), 8.37 (d, 1H, J ¼ 6.0 Hz,
NH), 7.00 (s, 2H, Ar-H), 4.36 (m, 1H, CH), 4.27 (m, 1H, CH), 3.73
(s, 6H, 2-OCH3), 3.67 (s, 3H, OCH3), 3.82 (s, 3H, COOCH3), 2.23 (t,
2H, J ¼ 7.0 Hz, CH2), 2.06, 1.83 (m, 2H, CH2), 1.92 (m, 2H, CH2),
0.98 (d, 3H, J ¼ 7.2 Hz, CH3). ESI-MS m/z: 439.4 [M þ H]þ. Anal.
calcd. for C20H28N2O9: C, 54.55; H, 6.36; N, 6.36. Found: C, 54.64;
H, 6.45; N, 6.22.
25
97–1008C
Methyl 2-amino-3-phenylpropanoate hydrochloride: Yield:
70%, m.p. 101–1058C
Methyl 2-amino-3-(4-chlorophenyl)propanoate hydrochloride:
Yield: 66%, m.p. 180–1828C
Methyl 2-amino-3-(4-fluorophenyl)propanoate hydrochloride:
Yield: 79%, m.p. 174–1768C
Methyl 2-amino-4-methylpentanoate hydrochloride: Yield:
(R)-5-((S)-1-Methoxy-1-oxopentan-2-ylamino)-5-oxo-4-
(3,4,5-trimethoxybenzamido) pentanoic acid 2d
80%, m.p. 129–1328C
Methyl 2-amino-3-(4-hydroxyphenyl)propanoate hydrochlo-
ride: Yield: 76%, m.p. 118–1218C
Yield: 23.4%, m.p. 110–1128C, IR (KBr, cmꢃ1): 3307.8 (NH), 2940.6
Methyl
2-(methylamino)acetate
hydrochloride:
Yield:
–
–
–
(CH), 1755.4 & 1718.1 (O C–NH), 1640.9 (O C–O), 1589.2 (C C),
–
–
–
1125.5 (C–O). [a]D ¼ þ25.2 (c 1, MeOH); 1H-NMR (DMSO-d6,
ppm): d 8.88 (d, 1H, J ¼ 7.8 Hz, NH), 8.24 (d, 1H, J ¼ 8.1 Hz,
NH), 6.98 (s, 2H, Ar-H), 4.33 (m, 1H, CH), 4.53 (m, 1H, CH), 3.73
(s, 6H, 2-OCH3), 3.62 (s, 3H, OCH3), 3.72 (s, 3H, COOCH3), 2.10 (t,
2H, J ¼ 15.3 Hz, CH2), 2.11, 1.79 (m, 2H, CH2), 1.32 (m, 2H, CH2),
1.90 (m, 2H, CH2), 0.97 (d, 3H, J ¼ 8.4 Hz, CH3). ESI-MS m/z: 453.3
[M þ H]þ. Anal. calcd. for C21H30N2O9: C, 55.51; H, 6.61; N, 6.17.
Found: C, 55.63; H, 6.70; N, 6.02.
25
52%, m.p. 111–1158C
Methyl 2-amino-3-hydroxypropanoate hydrochloride: Yield:
62%, m.p. 89–928C
Methyl 2-amino-4-(methylthio)butanoate hydrochloride: Yield:
79%, m.p. 187–1938C
Methyl 3-(2-amino-1H-indol-3-yl)propanoate hydrochloride:
Yield: 63%, m.p. 220–2238C
Methyl 2-amino-3-methylbutanoate hydrochloride: Yield:
83%, m.p. 90–958C
(R)-5-((S)-1-Methoxy-1-oxohexan-2-ylamino)-5-oxo-4-
(3,4,5-trimethoxybenzamido) pentanoic acid 2e
(R)-5-(2-Methoxy-2-oxoethylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid 2a
Yield: 61.2%, m.p. 99–1018C, IR (KBr, cmꢃ1): 3321.4 & 3256.9 (NH),
To a suspension of compound 1 (3.23 g, 10 mmol) and methyl 2-
aminoacetate hydrochloride (2.5 g, 20 mmol) in CH2Cl2 (30 mL)
at room temperature was added 5 mL of Et3N. The mixture was
stirred at this temperature until the disappearance of the start-
ing material, checking via TLC. The solvent was evaporated
in vacuo and poured into ice-cold water (50 mL). The mixture
was acidified with 2 N HCl to pH 2. Cool in a refrigerator, collect
the white flaky crystals on a Buchner funnel, wash with ice cold
water, and dry to give 2a (0.94 g, 74%). M.p. 169–1728C; IR (KBr,
–
–
–
2956.2 (CH), 1724.1 (O C–NH), 1647.6 (O C–O), 1584.3 (C C),
–
–
–
1130.5 (C–O). [a]D ¼ þ21.7 (c 1, MeOH); 1H-NMR (DMSO-d6,
ppm): d 7.82 (d, 1H, J ¼ 7.6 Hz, NH), 7.15 (s, 1H, Ar-H), 7.12 (s,
1H, Ar-H), 6.44 (d, 1H, J ¼ 7.2 Hz, NH), 4.93 (m, 1H, CH), 4.60 (m,
1H, CH), 3.92 (s, 6H, 2-OCH3), 3.80 (s, 3H, OCH3), 3.68 (s, 3H,
COOCH3), 2.28 (t, 2H, J ¼ 7.2 Hz, CH2), 2.06 (m, 2H, CH2), 1.92
(m, 2H, CH2), 1.29 (br, 4H, 2-CH2), 0.87 (t, 3H, J ¼ 7.8 Hz, CH3). ESI-
MS m/z: 467.1 [M þ H]þ. Anal. calcd. for C22H32N2O9: C, 56.41; H,
6.84; N, 5.98. Found: C, 56.51; H, 6.90; N, 5.89.
25
cmꢃ1): 3325.1 (NH), 2939.6 (CH), 1745.7 & 1714.5 (O C–NH),
–
–
25
D
–
–
1637.8 (O C–O), 1584.3 (C C), 1131.3 (C–O). [a]
¼ þ39.5 (c
–
–
(R)-5-((S)-1-Methoxy-1-oxo-3-phenylpropan-2-ylamino)-
5-oxo-4-(3,4,5-trimethoxy-benzamido) pentanoic acid 2f
1, MeOH); 1H–NMR (DMSO-d6, ppm): d 8.45 (d, 1H, J ¼ 7.5 Hz,
NH), 8.38 (t, 1H, J ¼ 5.4 Hz, NH), 7.23 (s, 2H, Ar–H), 4.47 (m, 1H,
CH), 3.88 (d, 2H, J ¼ 5.4 Hz, CH2), 3.83 (s, 6H, 2-OCH3), 3.69 (s, 3H,
OCH3), 3.62 (s, 3H, COOCH3), 2.34 (t, 2H, J ¼ 8.0 Hz, CH2), 2.06,
1.92 (m, 2H, CH2). ESI–MS m/z: 412.8 [M þ H]þ. Anal. calcd.
for C18H24N2O9: C, 52.43; H, 5.83; N, 6.80. Found: C, 52.88; H,
5.91; N, 6.72.
Yield: 56.3%, m.p. 141–1448C, IR (KBr, cmꢃ1): 3416.0 NH), 2939.9
–
–
(CH), 1743.7 & 1718.8 (O C–NH), 1646.4 (O C–O), 1584.1 &
25
–
1536.7 (C C), 1128.9 (C–O). [a]
–
¼ þ33.8 (c 1, MeOH); 1H-
–
–
D
NMR (DMSO-d6, ppm): d 8.69 (d, 1H, J ¼ 4.3 Hz, NH), 8.38 (d,
1H, J ¼ 6.8 Hz, NH), 7.24 (m, 2H, Ar-H), 7.18 (m, 5H, Ar-H), 4.44
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

502
X. Li et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 494–504
(m, 1H, CH), 4.34 (m, 1H, CH), 3.85 (s, 6H, 2-OCH3), 3.70 (s, 3H,
OCH3), 3.57 (s, 3H, COOCH3), 2.18 (t, 2H, J ¼ 7.6 Hz, CH2), 2.11 (d,
2H, J ¼ 7.4 Hz, CH2), 1.98, 1.76 (2m, 2H, CH2). ESI-MS m/z: 501.1
[M þ H]þ. Anal. calcd. for C25H30N2O9: C, 59.76; H, 5.98; N, 5.58.
Found: C, 59.85; H, 6.09; N, 5.47.
J ¼ 7.5 Hz, CH2), 2.11 (d, 2H, J ¼ 6.8 Hz, CH2), 2.00, 1.78 (2m, 2H,
CH2). ESI-MS m/z: 517.1 [M þ H]þ. Anal. calcd. for C25H30N2O10: C,
57.92; H, 5.79; N, 5.41. Found: C, 58.03; H, 5.86; N, 5.32.
(R)-5-((S)-3-Hydroxy-1-methoxy-1-oxopropan-2-ylamino)-
5-oxo-4-(3,4,5-trimethoxybenzamido) pentanoic acid 2k
Yield: 60.0%, m.p. 85–878C, IR (KBr, cmꢃ1): 3266.1 (NH), 2941.7
(R)-5-((S)-3-(4-Fluorophenyl)-1-methoxy-1-oxopropan-2-
ylamino)-5-oxo-4-(3,4,5-trimethoxybenzamido)pentanoic
acid 2g
–
–
–
–
(CH), 1744.2 (O C–NH), 1632.2 (O C–O), 1584.3 (C C), 1129.3
–
–
(C–O). [a]D ¼ þ40.8 (c 1, MeOH); 1H-NMR (DMSO-d6, ppm): d
8.76 (d, 1H, J ¼ 6.5 Hz, NH), 8.26 (d, 1H, J ¼ 6.8 Hz, NH), 7.24
(m, 2H, Ar-H), 5.02 (m, 1H, CH), 4.33 (m, 1H, CH), 3.82 (s, 6H, 2-
OCH3), 3.71 (s, 3H, OCH3), 3.61 (s, 3H, COOCH3), 3.05 (d, 2H,
J ¼ 5.5 Hz, CH2), 2.28 (t, 2H, J ¼ 6.0 Hz, CH2), 2.05, 1.83 (2m,
2H, CH2). ESI-MS m/z: 441.1 [M þ H]þ. Anal. calcd.
for C19H26N2O10: C, 51.58; H, 5.88; N, 6.33. Found: C, 51.66; H,
5.95; N, 6.25.
25
Yield: 61.4%, m.p. 107–1118C, IR (KBr, cmꢃ1): 3296.3 (NH), 2942.1
–
–
–
(CH), 1743.0 O C–NH), 1648.6 (O C–O), 1584.2 & 1537.3 (C C),
–
–
–
25
1127.6 (C–O). [a]D ¼ þ45.1 (c 1, MeOH); 1H-NMR (DMSO-d6,
ppm): d 7.78 (d, 1H, J ¼ 6.6 Hz, NH), 7.26 (s, 2H, Ar-H), 7.12 (m,
2H, Ar-H), 6.95 (m, 2H, Ar-H), 6.47 (d, 1H, J ¼ 8.0 Hz, NH), 4.78 (m,
1H, CH), 4.57 (m, 1H, CH), 3.93 (s, 6H, 2-OCH3), 3.86 (s, 3H, OCH3),
3.68 (s, 3H, COOCH3), 3.10 (d, 2H, J ¼ 8.2 Hz, CH2), 2.27 (t, 2H,
J ¼ 7.2 Hz, CH2), 1.89 (m, 2H, CH2). ESI-MS m/z: 519.2 [M þ H]þ.
Anal. calcd. for C25H29N2O9F: C, 57.69; H, 5.58; N, 5.38. Found: C,
57.77; H, 5.65; N, 5.30.
(R)-5-((S)-1-Methoxy-4-(methylthio)-1-oxobutan-2-
ylamino)-5-oxo-4-(3,4,5-trimethoxybenzamido)pentanoic
acid 2l
Yield: 59.1%, m.p. 124–1278C, IR (KBr, cmꢃ1): 3289.3 (NH), 2941.8
(R)-5-((S)-3-(4-Chlorophenyl)-1-methoxy-1-oxopropan-2-
ylamino)-5-oxo-4-(3,4,5-trimethoxybenzamido)pentanoic
acid 2h
–
–
–
(CH), 1750.9 & 1717.8 (O C-NH), 1647.2 (O C–O), 1584.5 (C C),
–
–
–
1129.7 (C–O). [a]D ¼ þ37.3 (c 1, MeOH); 1H-NMR (DMSO-d6,
ppm): d 8.72 (d, 1H, J ¼ 5.7 Hz, NH), 8.32 (d, 1H, J ¼ 7.3 Hz,
NH), 7.24 (s, 2H, Ar-H), 4.47 (m, 1H, CH), 4.37 (m, 1H, CH), 3.78
(s, 6H, 2-OCH3), 3.70 (s, 3H, OCH3), 3.61 (s, 3H, COOCH3), 2.26 (t,
2H, J ¼ 7.8 Hz, CH2), 2.13 (t, 2H, J ¼ 7.4 Hz, CH2), 2.06 (m, 4H, 2-
CH2), 1.83 (s, 3H, CH3). ESI-MS m/z: 486.1 [M þ H]þ. Anal. calcd.
for C21H30N2O9S: C, 51.85; H, 6.17; N, 5.76. Found: C, 51.97; H,
6.24; N, 5.68.
25
Yield: 67.2%, m.p. 143–1458C, IR (KBr, cmꢃ1): 3300.1 (NH), 2942.3
–
–
–
(CH), 1742.9 & 1718.6 (O C–NH), 1647.0 (O C–O), 1584.0 C C),
–
–
–
1129.0 (C–O). [a]D ¼ þ19.8 (c 1, MeOH); 1H-NMR (DMSO-d6,
ppm): d 7.74 (d, 1H, J ¼ 6.4 Hz, NH), 7.29 (s, 2H, Ar-H), 7.22 (m,
2H, Ar-H), 7.10 (m, 2H, Ar-H), 6.54 (d, 1H, J ¼ 7.8 Hz, NH), 4.79 (m,
1H, CH), 4.55 (m, 1H, CH), 3.93 (s, 6H, 2-OCH3), 3.89 (s, 3H, OCH3),
3.72 (s, 3H, COOCH3), 3.12 (d, 2H, J ¼ 7.6 Hz, CH2), 2.31 (t, 2H,
J ¼ 7.6 Hz, CH2), 1.95 (m, 2H, CH2). ESI-MS m/z: 535.1 [M þ H]þ.
Anal. calcd. for C25H29N2O9Cl: C, 55.92; H, 5.41; N, 5.22. Found: C,
56.04; H, 5.52; N, 5.18.
25
(R)-5-((R)-1-Methoxy-1-oxo-3-(phenylthio)propan-2-
ylamino)-5-oxo-4-(3,4,5-trimethoxybenzamido)pentanoic
acid 2m
Yield: 54.0%, m.p. 77–798C, IR (KBr, cmꢃ1): 3272.9 (NH), 2940.6
(R)-5-((S)-3-(4-Bromophenyl)-1-methoxy-1-oxopropan-2-
ylamino)-5-oxo-4-(3,4,5-trimethoxy benzamido)pentanoic
acid 2i
–
–
–
(CH), 1746.5 & 1722.0 (O C–NH), 1648.5 (O C–O), 1584.0 (C C),
–
–
–
1128.4 (C–O). [a]D ¼ þ20.5 (c 1, MeOH); 1H-NMR (DMSO-d6,
ppm): d 8.77 (d, 1H, J ¼ 6.8 Hz, NH), 8.46 (d, 1H, J ¼ 7.2 Hz,
NH), 7.34 (d, 2H, J ¼ 4.1 Hz, Ar-H), 7.30 (m, 5H, Ar-H), 4.47 (m,
1H, CH), 4.20 (m, 1H, CH), 3.83 (s, 6H, 2-OCH3), 3.75 (s, 3H, OCH3),
3.60 (s, 3H, COOCH3), 3.32 (s, 2H, CH2), 3.06 (d, 2H, J ¼ 6.8 Hz,
CH2), 2.26 (t, 2H, J ¼ 7.8 Hz, CH2), 2.07 & 1.84 (2m, 2H, CH2). ESI-
MS m/z: 547.1 [M þ H]þ. Anal. calcd. for C26H32N2O9S: C, 56.93; H,
5.84; N, 5.11. Found: C, 57.04; H, 5.91; N, 5.01.
25
Yield: 50.7%, m.p. 166–1688C, IR (KBr, cmꢃ1): 3303.4 (NH), 2946.1
–
–
(CH), 1744.2 & 1719.0 (O C–NH), 1647.4 (O C–O), 1583.8 &
25
–
1534.6 (C C), 1128.7 (C–O). [a]
–
¼ þ22.5 (c 1, MeOH); 1H-
–
–
D
NMR (DMSO-d6, ppm): d 7.77 (d, 1H, J ¼ 6.8 Hz, NH), 7.26 (s,
2H, Ar-H), 7.18 (m, 2H, Ar-H), 7.05 (m, 2H, Ar-H), 6.62 (d, 1H,
J ¼ 4.5 Hz, NH), 4.83 (m, 1H, CH), 4.57 (m, 1H, CH), 3.91 (s, 6H, 2-
OCH3), 3.80 (s, 3H, OCH3), 3.77 (s, 3H, COOCH3), 3.01 (d, 2H,
J ¼ 8.4 Hz, CH2), 2.39 (t, 2H, J ¼ 5.8 Hz, CH2), 1.98 (m, 2H,
CH2).
ESI-MS
m/z:
580.3
[M þ H]þ.
Anal.
calcd.
(R)-5-((S)-5-(Benzyloxycarbonyl)-1-methoxy-1-
oxopentan-2-ylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid 2n
for C25H29N2O9Br: C, 51.64; H, 4.99; N, 4.82. Found: C, 51.73;
H, 5.06; N, 4.73.
Yield: 43.0%, m.p. 108–1128C, IR (KBr, cmꢃ1): 3333.2 & 3276.9
(R)-5-((S)-3-(4-Hydroxyphenyl)-1-methoxy-1-oxopropan-
2-ylamino)-5-oxo-4-(3,4,5-trimethoxybenzamido)-
pentanoic acid 2j
–
–
(NH), 2945.9 (CH), 1745.0 O C–NH), 1688.4, 1584.1 (C C), 1128.2
–
–
(C–O). [a]D ¼ þ15.8 (c 1, MeOH); 1H-NMR (DMSO-d6, ppm): d 8.74
(d, 1H, J ¼ 5.6 Hz, NH), 8.28 (d, 1H, J ¼ 6.9 Hz, NH), 7.34 (m, 5H,
Ar-H), 7.24 (s, 2H, Ar-H), 4.99 (s, 2H, CH2), 4.36 (m, 1H, CH), 4.19 (m,
1H, CH), 3.83 (s, 6H, 2-OCH3), 3.71 (s, 3H, OCH3), 3.59 (s, 3H,
COOCH3), 2.96 (t, 2H, J ¼ 5.2 Hz, CH2), 2.25 (t, 2H, J ¼ 7.8 Hz,
CH2), 2.06 & 1.83 (2m, 2H, CH2), 1.66 & 1.54 (2m, 2H, CH2), 1.45
(m, 2H, CH2). ESI-MS m/z: 602.1 [M þ H]þ. Anal. calcd.
for C29H37N3O11: C, 57.71; H, 6.14; N, 6.97. Found: C, 57.84; H,
6.21; N, 6.89.
25
Yield: 49.0%, m.p. 87–898C, IR (KBr, cmꢃ1): 3355.2 & 3300.7 (NH),
–
–
2952.3 (CH), 1744.1 (O C–NH), 1647.7 (O C–O), 1585.9 & 1498.7
25
–
(C C), 1257.7 & 1126.5 (C–O). [a]
–
¼ þ33.4 (c 1, MeOH); 1H-NMR
–
–
D
(DMSO-d6, ppm): d 8.72 (d, 1H, J ¼ 7.0 Hz, NH), 8.33 (d, 1H,
J ¼ 6.4 Hz, NH), 7.23 (d, 2H, J ¼ 3.4 Hz, Ar-H), 6.95 (m, 2H, Ar-
H), 6.64 (m, 2H, Ar-H), 4.34 (m, 1H, CH), 3.83 (s, 6H, 2-OCH3), 3.70
(s, 3H, OCH3), 3.56 (s, 3H, COOCH3), 3.49 (m, 1H, CH), 2.20 (t, 2H,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 344, 494–504
APN/CD13 Inhibitors as Potential Antiproliferative Agents
503
(R)-5-((S)-6-(Benzyloxycarbonyl)-1-methoxy-1-oxohexan-
2-ylamino)-5-oxo-4-(3,4,5-trimethoxybenzamido)-
pentanoic acid 2o
(4R)-5-(4-(Benzyloxy)-2-(methoxycarbonyl)pyrrolidin-1-
yl)-5-oxo-4-(3,4,5-trimethoxybenzamido)pentanoic acid 3
Yield: 65.4%, m.p. 98–1018C; IR (KBr, cmꢃ1): 3237.4 (NH), 3099.1 &
Yield: 48.0%, m.p. 147–1508C, IR (KBr, cmꢃ1): 3325.6 (NH), 2940.1
–
–
–
2944.7 (CH), 1745.6 and 1717.7 (O C–NH), 1657.8 (O C–O),
–
–
1484.6 (C C), 1231.7 and 1121.96 (C–O).
–
1577.8
&
–
–
–
(CH), 1720.9 (O C–NH), 1648.8 (O C–O), 1584.8 & 1498.2 (C C),
–
–
–
25
[a]D ¼ þ40.2 (c 1, MeOH); 1H-NMR (DMSO-d6, ppm): d 8.66 (d,
1H, J ¼ 4.0 Hz, NH), 7.26–7.32 (m, 5H, Ar-H), 7.23 (s, 1H, Ar-H),
4.50 (s, 2H, CH2), 4.39 (m, 1H, CH), 4.32 (t, 1H, J ¼ 7.8 Hz, CH), 3.83
(s, 6H, 2-OCH3), 3.72 (s, 3H, OCH3), 3.61 (s, 3H, COOCH3), 3.23 (s,
1H, CH), 2.39 (m, 2H, CH2), 2.32 (m, 1H, CH), 2.12 (d, 1H,
J ¼ 7.8 Hz, CH2), 2.06 (m, 1H, CH), 1.98 (m, 1H, CH), 1.87 (m,
1234.6 & 1126.5 (C–O). [a]D ¼ þ19.0 (c 1, MeOH); 1H-NMR (DMSO-
d6, ppm): d 8.72 (t, 1H, J ¼ 6.0 Hz, NH), 8.24 (t, 1H, J ¼ 7.2 Hz, NH),
7.33 (m, 5H, Ar-H), 7.24 (s, 2H, Ar-H), 7.20 (s, 1H, Ar-H), 4.99 (s, 2H,
CH2), 4.37 (m, 1H, CH), 4.17 (m, 1H, CH), 3.83 (s, 6H, 2-OCH3), 3.74 (s,
3H, OCH3), 3.61 (s, 3H, COOCH3), 2.94 (t, 2H, J ¼ 6.0 Hz, CH2), 2.27
(t, 2H, J ¼ 7.8 Hz, CH2), 2.13 (s, 3H, CH3), 2.05 (m, 1H, CH), 1.85 (m,
1H, CH), 1.56 (m, 2H, CH2), 1.36 (m, 2H, CH2), 1.26 (m, 2H, CH2).
ESI-MS m/z: 616.1 [M þ H]þ. Anal. calcd. for C30H39N3O11: C, 58.35;
H, 6.32; N, 6.81. Found: C, 58.44; H, 6.41; N, 6.75.
25
1H, CH). ESI-MS m/z: 557.1 [M þ H]þ. Anal. calcd. for C28H34N2O10
:
C, 60.22; H, 6.09; N, 5.02. Found: C, 60.31; H, 6.17; N, 4.98.
(R)-N-(5-(Hydroxyamino)-1-(2-(hydroxyamino)-2-
oxoethylamino)-1,5-dioxopentan-2-yl)-3,4,5-
trimethoxybenzamide 4
(R)-5-((S)-1-Methoxy-5-(3-nitroguanidino)-1-oxopentan-2-
ylamino)-5-oxo-4-(3,4,5-trimethoxybenzamido)pentanoic
acid 2p
A warm solution of KOH (5.6 g, 100 mmol) in anhydrous meth-
anol (15 mL) was added to a hot solution of NH2OH ꢄ HCl (4.67 g,
67.2 mmol) in anhydrous methanol (25 mL), maintaining the
temperature at less than 358C. Filtered and the filtrate (solution
of NH2OK in methanol) was allowed to cool to room temperature.
Successively the obtained filtrate was added dropwise to a
solution of compound 6a (0.5 g, 1.2 mmol) in anhydrous meth-
anol (10 mL). After being stirred for 4 h, the mixture was acidi-
fied with a small amount of AcOH to pH 5–6, and extracted with
CH2Cl2. The aqueous layer was evaporated and dried to give
compound 4 (0.4 g, 80%) as white solid, which appeared pruno-
sus in FeCl3 developer. M.p. 176–1798C; IR (KBr, cmꢃ1): 3299.7
Yield: 52.2%, m.p. 123–1268C, IR (KBr, cmꢃ1): 3307.4 (NH), 2943.8
–
–
–
(CH), 1739.8 (O C–NH), 1649.1 (O C–O), 1584.7 & 1499.7 (C C),
–
–
–
1236.4 & 1127.5 (C–O). [a]D ¼ þ34.2 (c 1, MeOH); 1H-NMR
(DMSO-d6, ppm): d 8.66 (d, 1H, J ¼ 6.6 Hz, NH), 8.22 (t, 1H,
J ¼ 6.0 Hz, NH), 7.24 (s, 2H, Ar-H), 4.37 (m, 1H, CH), 4.24 (m,
1H, CH), 3.84 (s, 6H, 2-OCH3), 3.72 (s, 3H, OCH3), 3.61 (s, 3H,
COOCH3), 3.13 (d, 2H, J ¼ 5.4 Hz, CH2), 2.27 (t, 2H, J ¼ 7.2 Hz,
CH2), 2.13 (s, 2H, CH2), 2.07 (m, 1H, CH), 1.85 (m, 1H, CH), 1.71 (m,
1H, CH), 1.58 (m, 2H, CH2), 1.51 (m, 1H, CH). ESI-MS m/z: 555.1
[M þ H]þ. Anal. calcd. for C22H32N6O11: C, 47.48; H, 5.76; N, 15.11.
Found: C, 47.54; H, 5.85; N, 15.03.
25
–
–
(NH), 2959.1 (CH), 1749.4 and 1718.9 (O C–NH), 1645.6 (O C–O),
25
–
–
–
–
1584.1 (C C), 1236.3 and 1128.9 (C–O). [a]
¼ þ20.7 (c 1,
D
(R)-5-((S)-3-(1H-indol-3-yl)-1-methoxy-1-oxopropan-2-
ylamino)-5-oxo-4-(3,4,5-trimethoxybenzamido)pentanoic
acid 2q
MeOH); 1H-NMR (600 MHz, DMSO-d6, ppm): d 10.59 (s, 1H, OH),
10.46 (s, 1H, OH), 8.75 (s, 1H, NH), 8.43 (d, 1H, J ¼ 8.4 Hz, NH),
8.05 (t, 2H, J ¼ 6.0 Hz, 2NH), 6.98 (s, 2H, 2Ar-H), 4.57 (m, 1H, CH),
3.84 (s, 6H, 2-OCH3), 3.70 (s, 3H, OCH3), 4.09 (m, 2H, CH2), 3.58 (m,
2H, CH2), 2.19 (m, 2H, CH2). ESI-MS m/z: 427.1 [M þ H]þ. Anal.
calcd. for C17H24N4O9: C, 47.66; H, 5.61; N, 13.08. Found: C, 47.75;
H, 5.70; N, 12.99.
Yield: 44.1%, m.p. 167–1698C, IR (KBr, cmꢃ1): 3362.5 (NH), 2948.5
–
–
–
(CH), 1739.8 (O C–NH), 1652.4 (O C–O), 1584.7 & 1497.1 (C C),
–
–
–
1233.3 & 1126.5 (C–O). [a]D ¼ þ22.4 (c 1, MeOH); 1H-NMR
(DMSO-d6, ppm): d 8.14 (s, 1H, NH), 8.09 (s, 1H, OH), 7.91 (d,
1H, J ¼ 6.1 Hz, NH), 7.84 (d, 1H, J ¼ 6.3 Hz, NH), 7.51 (t, 2H,
J ¼ 8.4 Hz, Ar-H), 7.16 (s, 2H, Ar-H), 7.14 (s, 2H, Ar-H), 7.08 (m,
25
4-(Benzyloxy)-N-hydroxy-1-(5-(hydroxyamino)-5-oxo-2-
(3,4,5-trimethoxybenzamido)pentanoyl)pyrrolidine-2-
carboxamide 5
–
1H, CH), 4.76 (m, 1H, CH), 4.55 (m, 1H, CH), 3.92 (s, 6H, 2-OCH ),
–
3
3.90 (s, 3H, OCH3), 3.71 (s, 3H, COOCH3), 2.41 (m, 1H, CH), 2.27 (m,
2H, CH2), 2.20 (m, 2H, CH2), 1.96 (m, 1H, CH). ESI-MS m/z: 540.1
[M þ H]þ. Anal. calcd. for C27H31N3O9: C, 59.89; H, 5.73; N, 7.76.
Found: C, 59.95; H, 5.81; N, 7.62.
Yield: 52.2%, m.p. 173–1758C, IR (KBr, cmꢃ1): 3287.3 (NH), 3004.7
–
–
–
& 2954.4 (CH), 1752.7 and 1716.6 (O C–NH), 1640.1 (O C–O),
25
–
–
–
1646.2 & 1580.2 (C C), 1237.1 & 1130.0 (C–O). [a]
¼ þ29.8 (c 1,
D
MeOH), 1H–NMR (DMSO-d6, ppm): d 10.52 (s, 1H, OH), 10.48 (s, 1H,
OH), 8.70 (t, 2H, 2NH), 8.03 (t, 2H, J ¼ 6.7 Hz, 2NH), 7.19 (m, 5H,
5Ar-H), 6.92 (s, 2H, 2Ar-H), 4.68 (s, 2H, CH2), 4.51 (m, 1H, CH), 4.44
(t, 1H, CH), 3.79 (s, 6H, 2-OCH3), 3.73 (s, 3H, OCH3), 3.71 (m, 2H,
CH2N), 3.32 (m, 1H, CH-OCH2), 2.46 (dd, 2H, J ¼ 8.6 Hz,
J ¼ 6.2 Hz, CH2), 2.18 (m, 2H, CH2), 2.01 (m, 2H, CH2). ESI-MS
m/z: 573.2 [M þ H]þ. Anal. calcd. for C27H34N4O10: C, 56.45; H,
5.92; N, 9.76. Found: C, 56.54; H, 6.01; N, 9.62.
(R)-5-((S)-3-(1-(Benzyloxycarbonyl)-1H-imidazol-4-yl)-1-
methoxy-1-oxopropan-2-ylamino)-5-oxo-4-(3,4,5-
trimethoxybenzamido)pentanoic acid 2r
Yield: 56.3%, m.p. 112–1168C, IR (KBr, cmꢃ1): 3241.5 (NH), 2947.2
–
–
–
(CH), 1744.7 (O C–NH), 1661.9 (O C–O), 1584.4 & 1498.5 (C C),
–
–
–
1233.9 & 1125.8 (C–O). [a]D ¼ þ26.1 (c 1, MeOH); 1H-NMR
25
(DMSO-d6, ppm): d 8.40 (d, 1H, J ¼ 5.7 Hz, NH), 8.27 (d, 1H,
–
–
J ¼ 6.0 Hz, NH), 7.35 (s, 2H, Ar-H), 7.34 (s, 1H, CH), 7.52 (d,
1H, J ¼ 5.3 Hz, Ar-H), 7.22 (d, 1H, J ¼ 2.6 Hz, Ar-H), 7.12 (d, 1H,
(R)-5-Oxo-5-(thiazol-2-ylamino)-4-(3,4,5-trimethoxy-
benzamido)pentanoic acid 6
A suspension of compound 1 (3.23 g, 10 mmol) and 4,5-dihydro-
thiazol-2-amine (2.8 g, 11 mmol) in acetic acid (30 mL) was
allowed to reach ambient temperature. After completion of
–
–
J ¼ 6.6 Hz, Ar-H), 6.67 (s, 1H, CH), 5.03 (m, 2H, Ar-CH ), 4.74 (m,
2
1H, CH), 4.59 (m, 1H, CH), 3.92 (s, 6H, 2-OCH3), 3.91 (s, 3H, OCH3),
3.89 (s, 3H, COOCH3), 3.07 (m, 2H, CH2), 2.53 (m, 2H, CH2), 2.46 (m,
2H, CH2). ESI-MS m/z: 581.2 [M þ H]þ. Anal. calcd. for C29H34N4O9:
C, 59.79; H, 5.84; N, 9.62. Found: C, 59.88; H, 5.91; N, 9.53.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

504
X. Li et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 494–504
the reaction, the white precipitate was collected and dried
in vacuo to give 6 (3.45 g, 81.4%). M.p. 204–2058C; IR (KBr,
References
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–
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–
–
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–
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This work was financial supported in part from the Natural Science
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The authors have declared no conflicts of interest.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com