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

Article abstract of DOI:10.1016/j.bmc.2009.03.017

As our ongoing work, a series of peptidomimetic l-iso-glutamine derivatives derived from antineoplaston AS2-5 scaffold were prepared and their APN/CD13 and MMP-2 inhibitory activities were evaluated hereby. The results displayed that these compounds exhibited selective inhibition against APN as compared with MMP-2, with IC₅₀ values in micromole range. Compounds A1 and A2 showed comparable APN inhibitory activities than the positive control bestatin.

Full text of DOI:10.1016/j.bmc.2009.03.017

Bioorganic & Medicinal Chemistry 17 (2009) 3061–3071
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 II)
Xun Li a, Yazhou Wang a, Jifeng Wu b, Yonggang Li a, Qiang Wang a, Wenfang Xu a,
*
a School of Pharmaceutical Sciences, Shandong University, No. 44 WenhuaXi Road, Ji’nan, 250012, Shandong Province, PR China
b 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:
As our ongoing work, a series of peptidomimetic L-iso-glutamine derivatives derived from antineoplaston
Received 6 February 2009
Revised 5 March 2009
Accepted 6 March 2009
Available online 14 March 2009
AS2–5 scaffold were prepared and their APN/CD13 and MMP-2 inhibitory activities were evaluated
hereby. The results displayed that these compounds exhibited selective inhibition against APN as com-
pared with MMP-2, with IC50 values in micromole range. Compounds A1 and A2 showed comparable
APN inhibitory activities than the positive control bestatin.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
L
-iso-Glutamine derivatives
Antineoplaston AS2–5
Ferulic acid
APN inhibitors
Peptidomimetics
Anti-tumor
1. Introduction
In fact, many natural or small molecule inhibitors of APN/CD13
have been reported previously.9–11 Amongst these inhibitors, best-
Satisfactory curative chemotherapeutic agents with high affin-
ity and selectivity are expected to be rationally designed against
specific tumor-associated targets. Given that aminopeptidase N
(APN) is over-expressed on tumor cells and plays a pivotal role in
tumor invasion, growth and angiogenesis,1 it has been regarded
as an attractive target for anticancer drugs design.
atin, an antibiotic of microbial origin with APN inhibitory activ-
ity,12 has become a useful tool as positive control in elucidating
many physiological conditions.13 Almost all these compounds are
pseudodipeptides bearing zinc-chelating functionality. As a conse-
quence, inhibitors containing zinc-binding groups (ZBGs) should
be optimal selection in the designed molecules. This kind of inhib-
itors can interact with the catalytic zinc ions of zinc-dependent
metalloenzymes and sequentially inhibit the metastatic spread of
tumors and block the processes of tumor neovascularization.14
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 Accordingly, the interactions of bestatin
with the active sites of APN can be illustrated by a simplified model
(see Fig. 1). It revealed that beside the catalytic center zinc(II) ion
of APN, there are two hydrophobic binding domains, which are
As a zinc-dependent endopeptidase, APN is also known as CD
13. It is a 150-kDa myeloid cell surface glycoprotein belonging to
the M1 family, which preferentially releases neutral and basic ami-
no acids from the N-terminal end of peptides.2 It is noticeable that
tumor cells which over-express APN, for example, melanoma cells,
acute lymphocytic leukemic cells, as well as urological cancer cells,
are highly motile and capable of migration through extracellular
matrix.3,4 Moreover, angiogenesis emanating from microvascular
endothelial cells plays a central role in tumor growth, invasion
and metastasis.5 In case the mRNA of the APN/CD13 was knocked
out, the level of angiogenesis decreased dramatically.1 On the other
hand, APN inhibitors (such as CD13 monoclonal antibodies or best-
atin) could significantly block induced-retinal neovascularization
in mice and in chorioallantoic membrane angiogenesis in vitro.6,7
Accordingly, it is useful to develop potential APN inhibitors (APNIs)
to block its enzymatic activity for medical and therapeutic
utilization.8
called S1 pocket and S0 pocket, respectively. Therefore, one or more
1
Enzyme
S
S '
S '
1
1
2
++
Zn
O
H N
N
H
COOH
3
(Bestatin)
OH
* Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: tjulx2004@sdu.edu.cn (Wenfang 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.2009.03.017

3062
X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3061–3071
H
N
O
O
H3CO
HO
OCH3
OH
O
O
N
H
O
Antineoplaston 10 (AS 10)
Curcumin
COOH
O
COOH
NH2
N
O
H
H
N
O
H3CO
H3CO
N
H
R
N2-phenylacetyl L-iso-glutamine (AS 2-5)
O
O
H3CO
OH
Novel designed L-iso-glutamine derivatives
HO
Ferulic acid
Figure 2. Chemical structures of ferulic acid, curcumin, AS-10, AS-25 and the newly designed compound.
hydrophobic side chains should be introduced to insert into these
pockets to generate effective interactions.
icant anti-oxidative and chemopreventive activities.25,26 For in-
stance, it was found that curcumin (see Fig. 2), a ferulic acid-
based natural product, has shown irreversible APN inhibitory
activity.27 Hence, according to the ‘combination principle’, we
therefore envision that the conjunction of antineoplaston AS-25
scaffold with ferulic acid fragment might generate a more effica-
cious scaffold for potential enzymatic activity of APN/CD13.
According to these findings, our group has previously reported a
series of novel APNIs with remarkable APN inhibitory activities in
the enzymatic assays.17–20 In our ongoing efforts to identify more
potent APNIs, we herein describe the synthesis and enzymatic
evaluation of analogues derived from the antineoplaston AS2-5
(N2-phenylacetyl L
-iso-glutamine),21,22 one of the active degrada-
tion product of antineoplaston A-10 (3-phenylacetylamino-2,6-
piperidinedione, NSC-648539), which is known to be the first
chemically identified antineoplaston with anti-tumor activity
(see Fig. 2).23,24
2. Chemistry
In order to study the SAR of these novel peptidomimetic com-
pounds, different L-iso-glutamine derivatives were designed and
Meanwhile, we noticed that phenolic (E)-3-(4-hydroxy-3-
methoxyphenyl)acrylic acid (ferulic acid; Fig. 2), one of efficient
biologically component of Chinese traditional medicine herbs
Angelica sinensis, Rhizoma Chuanxiong, and Asafetida, possess signif-
synthesized via the route outlined in Scheme 1. Starting from com-
mercially available ferulic acid 1, the key intermediate (S,E)-2-(3-
(3,4-dimethoxyphenyl)acrylamido)pentanedioic acid 5 was ob-
tained via the sequence of methylation, acylation, nucleophilic
O
O
O
H3CO
H3CO
H3CO
H3CO
HO
OH
Cl
OH
a
b
H3CO
1
3
2
5
COOCH3
COOH
O
4
O
3
H3CO
H3CO
N
COOCH3
e
H3CO
H3CO
c
2
d
N
H
COOH
H
1
4
5
2eq RNH2
O
1eq RNH2
COOH
H
N
1eq RNH2
O
H
O
O
N
R
R
R
H
N
H
N
O
H3CO
H3CO
H3CO
H3CO
N
H3CO
H3CO
N
H
R
N
H
COOH
H
O
O
A1-A18
B1-B7
Scheme 1. Reagents and conditions: (a) Me2SO4, NaOH; (b) SOCl2, C6H6; (c) L-dimethyl 2-aminopentanedioate, CH2Cl2; (d) Na2CO3, H2O; (e) RNH2 (1 equiv, for A1–A18;
2 equiv, for B1–B7), DCC, CHCl3.

X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3061–3071
3063
Figure 3. HMBC spectrum of compound A1.
substitution, and dehydration. This was followed by coupling with
various primary amines (1 equiv and 2 equiv, respectively) in the
presence of DCC to afford the primary amide peptidomimetics
A1–A18 and B1–B7.
over, this conclusion could be elucidated as Figure 4, which has
been shown by arrows. Besides, the IR, ESI-MS analytical and 1H
NMR data of other target compounds were in full agreement with
the proposed structures (see Section 5).
3.2. Structure–activity relationship studies(in vitro) for series A
compounds
3. Results and discussion
3.1. Structure confirmation for series A compounds A1–A18
The target compounds were evaluated for their inhibitory activ-
ities toward APN/CD13 and MMP-2. Similar to APN, MMP-2 is also
a zinc-dependent metalloproteinase involved in the process of tu-
mor invasion and metastasis.28,29 Thereby the assay was per-
formed on both of APN and MMP-2 so as to identify their
selectivity and, all the inhibition results are summarized in Tables
1 and 2. And else, bestatin was used as the positive control.
As shown in Table 1, it is worthy to note that these mono-
substituted amide derivatives (A1–A18) displayed a better enzy-
matic inhibition towards APN as compared with MMP-2, with
IC50 values lying in micromole level. The results, to a certain extent,
validated our strategy for rational designing of potential APNIs. As
the above mentioned selectivity against APN, the following SARs
were mainly discussed about APN inhibition.
As shown in Scheme 1, theoretically speaking, when compound
5 was treated with 1 equiv of nucleophilic agents, both C5 and C1
center might be attacked to produce mixture compounds. In fact, it
revealed that only C1-based amide derivatives (A1–A18) were ob-
tained, this might be due to the more adjacent to NH group of C1 in
comparison with C5, resulting in lower electron cloud density. As a
result, when the electronegative nitrogen atom of NH2 attacks the
electropositive carbon atom of a carbonyl group, it is inclined to at-
tack C1 to get target compounds.
Take compound A1 as an example, the corresponding chemical
structure was further validated by IR, 1H NMR, 13C NMR, mass
spectrometry (MS) and HMBC spectrum. Figure 3 diagrammed
the HMBC spectrum (DMSO-d6, 500 MHZ, ppm) of A1. There are
cross-peaks between N5–H (10.04) and C4 (170.8), C3–H (4.54)
and C4 (170.8), N2–H (8.27) and C1 (165.3), as well as C7–H
(2.29) and C8 (173.1). Whilst N5–H (10.04) and C8 (dissociative
carboxyl 173.1) have no correlative cross-peak. This information
indicated that the NH2 moiety was attacked at the C4 position,
the dissociative carboxyl group was at the C8 position and, struc-
tures of compounds A1–A18 were confirmed to be correct. More-
Among these inhibitors, generally speaking, compounds with
aromatic side chain showed better activity compared to aliphatic
derivatives. The possible reason may be due to the
p system of
the aromatic ring enhancing the interaction with the hydrophobic
region of the enzyme.
Substitution on aromatic ring (A1–8) also has impact on bioac-
tivity. Compound A1, with methyl-substitution at the para-posi-
tion in the aromatic ring displayed the best APN inhibitory
activity among series A compounds. Comparing compounds with
halogen-substitution at the para-position in the aromatic ring
(A2–4), it seems that the increased bulk of halogen substituents
leads to impaired activity, suggesting there is a space requirement
in the binding pockets to accommodate the suitable substituents.
Additionally, mono-substitution of fluorine at the para-position
in the aromatic ring (A2) displayed higher affinities
173.1
8
COOH
H
2.29
7
O
6
H
165.3
10.04
2
N
H
3
H3CO
H3CO
N
4
1
5
170.8
H
4.54
O
8.27
(IC50 = 23.4 3.2
56.8 6.4 M). For the chlorine counterparts, the para-substituted
derivative (A3) gave the highest activity, the ortho-one (A7) was in
l
M)
than
at
the
ortho-position
(A6,
l
Figure 4. Diagram of the main correlative relations of A1.

3064
X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3061–3071
Table 1 (continued)
Table 1
The inhibitory activities of series A compounds
Compds
R
IC50
APN/CD13
(l
M)a
MMP-2
O
OH
OCH3
OCH3
OCH3
O
A14
50.4 2.3
55.1 1.8
69.2 3.9
79.6 3.0
83.2 0.7
76.8 4.5
H
N
O
O
N
R
H
O
O
Compds
A1
R
IC50 (l
M)a
A15
APN/CD13
20.7 0.6
MMP-2
O
O
37.8 4.2
A16
OCH3
O
A2
23.4 3.2
34.5 3.8
58.1 3.3
98.6 4.3
56.8 6.4
38.3 4.2
80.5 2.5
47.6 0.8
59.9 2.3
71.2 0.3
116.3 0.6
71.4 5.1
64.3 1.8
120.8 4.8
A17
A18
60.5 2.6
101.5 8.9
F
OCH3
O
A3
F
Cl
43.7 2.1
13.1 0.7
76.5 0.9
40.5 1.2
OCH3
A4
O
HO
Br
F
H
N
Bestatin
O
NH2
HO
O
A5
a
Values are means standard error of three experiments.
F
F
the next place, and the meta-one (A8) presented the least activity.
On the other hand, di-substitution of fluorine at the 3-, 5-position
of the aromatic ring exhibited impaired potency than at the 2-, or
4-position. However, the introduction of benzyl moiety (A9)
caused obviously decreased potency.
When it comes to compounds with aliphatic side chain (A10–
13), to some extent, length of R groups was negatively relative with
the APN inhibition, suggesting it is unfavorable to increase the
length of substituents along the orientation of the R groups. This
might be due to longer groups were unfavorable to the accommo-
dation with the enzymes’ hydrophobic domains.
A6
Cl
A7
Cl
A8
Comparing A14–18, the introduction of carbomethoxy residue
(COOCH3) as a ZBG produced improved potency. As ZBGs, both car-
bomethoxy and its corresponding metabolite carboxylic acid
(COOH) can chelate with zinc at the catalytic center of the enzyme,
resulting in significantly enzymatic inhibition.
Finally, although compounds A1 and A2 gave comparable APN
inhibitory activities with bestatin, all of the tested compounds
demonstrated weaker potency in comparison with the positive
control. Therefore, further structural optimization should be atten-
tively investigated towards series A.
A9
177.6 7.9
125.8 8.4
184.3 5.7
237.5 0.8
207.2 6.6
235.8 4.3
159.1 6.6
211.4 3.0
283.6 8.4
256.1 1.3
A10
A11
A12
A13
3.3. Structure–activity relationship studies(in vitro) for series B
compounds
Overall, the SAR patterns of B series (B1–B7) were similar to
those of A series (A1–A18), as shown in Table 2. However, these
di-substituted amide derivatives displayed obviously weaker APN
inhibition as compared with mono-substituted ones. Thereby, ex-
cept compounds listed in Table 2, no further chemical modification
was conducted towards series B. The possible reason might be that
too bulky side chains might lead to the difficulties to accommodate
the active domain of APN. For example, disubstitution of fluoro

X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3061–3071
3065
Table 2
group at the 3-, 5-position of the aromatic ring exhibited sharply
impaired potency than mono-substitution of fluorine atom at the
2-position, even has no APN or MMP-2 inhibitory potency. Or in
other words, carboxyl group, as an essential ZBG, should be consid-
ered to be reserved when designing potent APNIs.
The inhibitory activities of series B compounds
H
N
O
R
R
O
H
N
3.4. Binding mode
O
N
H
O
Compound A1, which showed the best affinity in our target
compounds, was selected to study its binding mode with APN.
Firstly, the predicted conformation of A1 was optimized with the
Powell Energetic Gradient method built in the Sketch/Build Edit
model (SYBYL6.91, Linux 7.3).30 As compared with the known APNI
bestatin, the comparable results showed the affinity to the same
spatial regions (S1 and S01), implying the similar APN inhibitory
activities. In precise words, both the isopropyl group of bestatin
and the introduced toluene fragment of A1 can adjust their flexibil-
ity to extend into the S01 pocket with their preponderant conforma-
tions, as shown in Figure 5. In contrast, the similar situation can be
seen between the phenyl group of bestatin and the di-methoxy
phenyl portion of A1.
To further understand the APN inhibitory difference between
series A and series B, the preferred pharmacophore docking
studies were carried out via the FLEXX flexible-Dock program.
The interactions of A1 and B1 with active site of Escherichia coli
APN (PDB: 2DQM) were compared, which are shown in Figure
6. Not surprisingly, both the di-methoxy phenyl fragment and
the introduced di-substituted amide portion of B1 could not
well-orienting interact with the active domain (S1 and S01) of
APN. Unlike B1, the moderate bulk of mono-substituted amide
portion of A1 could undergo torsional motion to allow their
suitable conformations to accommodate the S1 and S01 pockets,
respectively.
O
Compds
B1
R
IC50
(l
M)a
MMP-2
APN/CD13
334.7 7.2
397.6 8.9
368.1 11.3
470.6 13.8
B2
371.3 9.5
405.2 15.7
577.6 10.2
405.2 12.4
F
B3
Cl
Br
b
B4
na
F
B5
470.7 10.1
Finally, the selective inhibition of target compounds might also
be explained by the molecular docking method of the representa-
tive compounds A1 and B1 with the active site of MMP-2 (PDB ID:
1HOV). As diagrammed in Figure 7, both the two compounds could
not occupy the S1 and S01 pockets of MMP-2, resulting weaker
MMP-2 inhibitory activities. In addition, the comparable docking
results also graphically validated the importance of carboxyl group
(as a ZBG) when designing potent APNIs. This is due to the effective
interaction of A1 with the catalytic zinc ion 166 of MMP-2, while
B1 was far from it, leading to sharply decreased MMP-2 inhibition,
consistent with enzymatic assay results. The binding mode infor-
mation encouraged us to further design antineoplaston AS2-5 scaf-
fold based APNIs.
F
B6
B7
nab
nab
F
OCH3
O
213.2 7.6
340.8 9.0
OCH3
O
HO
H
N
Bestatin
13.1 0.7
40.5 1.2
O
It should be pointed out that although the computed informa-
tional assay totally supported our assumption, the exact binding
model of the target compounds with APN should be verified from
further X-ray crystal studies.
NH2
HO
O
a
Values are means standard error of three experiments.
Not activity.
b
Figure 5. Comparison of preponderant conformations between bestatin and compound A1.

3066
X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3061–3071
Figure 6. Comparison of FlexX docking results of compounds A1 and B1 with the active site of E. coli APN (PDB: 2DQM).
Figure 7. Comparison of FlexX docking results of compounds A1 and B1 with the active domain of MMP-2 (PDB ID: 1HOV).

X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3061–3071
3067
was removed to obtain compound 3 as pale yellow oil. Then
another 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.
4. Conclusion
In conclusion, we have developed two series of novel peptidom-
imetic L-iso-glutamine derivatives based on the antineoplaston
AS2-5 scaffold. All 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 inhib-
itors 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.
5.2.3. (R)-2-(3,4,5-Trimethoxybenzamido)pentanedioic acid (4)
Preparation of L-glutamine acid dimethylesters: L-glutamine
acid (20 g) was added to a cooled solution of and anhydrous meth-
anol (140 ml). Successively 16 ml of SOCl2 was added at ꢀ5 °C in
small portions over 1 h. The resulting suspension was allowed to
stir overnight at room temperature. The insoluble material was fil-
tered off and the filtrate was concentrated in vacuo. The following
mixture was basified with NH3ꢁH2O to pH 5–6, extracted twice
with chloroform, then dried over anhydrous Na2SO4, filtered, and
evaporated to give colorless oil (18.8 g), which was further used
without further purification.
5. Experimental
5.1. General procedures
Silica gel for column chromatography (CC) and analytical thin-
layer chromatography (TLC) plates precoated with Silica Gel
GF254 were commercial available from Qingdao Haiyang Chemical
Company, Qingdao, China. Reaction courses and product mixtures
were routinely monitored by TLC on 0.25 mm Silica Gel 60 F254
plates and visualized under ultraviolet (UV) light (254 nm), or io-
dine (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 neces-
sary, 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 spectrometer, and KBr disks were used as indicated. 1H
NMR spectra were determined on a Brucker Avance DRX-600 spec-
trometer, with chemical shift (d) given in ppm upfield from Me4Si
(TMS) as an internal standard, and coupling constants J were re-
corded in hertz (Hz). Peak multiplicities are abbreviated: single,
s; double, d; triple, t; quartet, q; multiplet, m; broad, br. Electro-
spray ionization mass spectrometry (ESI-MS) was performed on
an API-4000 triple-stage quadrupole instrument. Anhydrous reac-
tions 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.
To a solution of
L-glutamine acid dimethylesters (19.25 g,
110 mmol) in CH2Cl2 (100 ml) at ꢀ5 °C was added a solution of
compound 3 in 100 ml CH2Cl2 in small portions. The mixture was
stirred at this temperature until the solid has almost completely
dissolved (about 1 h). The resulting residue was purified by flash
chromatography using hexane/EA (4:1–1:1) as an eluant to yield
compound 4 (23.9 g, 65.5%). Mp 151.0–152.5 °C, ESI-MS: m/z (rel
intensity) 365.5 [M+1].
5.2.4. (E)-2-(3-(3,4-Dimethoxyphenyl)acrylamido)pentanedioic
acid (5)
A suspension of compound 4 (18.25 g, 50 mmol) and 1 N
Na2CO3 (100 ml) was stirred at 75–80 °C until the disappearance
of the starting material and the clarification of the reaction mix-
ture, checking via TLC. The resulting mixture was acidified with
1 N HCl to pH 2, extracted with EA (50 ml ꢂ 5), then dried over
anhydrous Na2SO4, filtered, and evaporated the organic solvent.
The obtained residue was dissolved by a small amount of acetone
and allowed to place at rt. Collect the white columnar crystals on a
Buchner funnel, then dried to give 5 (12.5 g, 82%). Mp 104.0–
105.3 °C, ESI-MS: m/z (rel intensity) 337.3 [M+1].
5.2.5. (S,E)-5-(p-Toluidino)-4-(3-(3,4-dimethoxyphenyl)acry-
lamido)-5-oxopentanoic acid (A1)
To a solution of compound 5 (3.5 g, 10 mmol) in CHCl3 (20 ml)
at ꢀ5 °C was added a solution of 1,3-dicyclohexylcarbodiimide
(DCC, 2.06 g, 1 equiv, 10 mmol) in 50 ml CHCl3 in small portions.
5.2. Syntheses
After completion the DCC,
a solution of p-toluidine (1.07 g,
5.2.1. (E)-3-(3,4-Dimethoxyphenyl)acrylic acid (2)
10 mmol) in 20 ml CHCl3 was added dropwise. Then the mixture
was stirred at this temperature until the solid has almost com-
pletely dissolved (about 5 h). The insoluble material (dicyclohexyl-
urea, DCU) was filtered off and washed with cold 1 N Na2CO3. The
eluant was acidified with 1 N HCl to pH 2, extracted with EA
(30 ml ꢂ 3), and subsequently the solvent was removed under re-
duced pressure. The resulting crude residue was recrystallized
from ethanol and dried to afford the target compound as white so-
lid 1.98 g, yield: 46.5%. Mp 192–193 °C, IR (KBr, cmꢀ1): 3344 (NH),
3297 (OH), 1735 (O@C–OH), 1650 (O@C–NH), 1599 and 1514 (Ar),
1267 (C–N), 1161 and 1145 (C–O). 1H NMR (DMSO-d6, ppm): d
12.16 (s, 1H, COOH), 10.04 (s, 1H, NH), 8.27 (d, 1H, NH,
J = 7.92 Hz), 7.48 (d, 2H, Ar-H, J = 8.4 Hz), 7.37 (d, 1H, C@CH,
J = 9.72 Hz), 7.16 (d, 1H, Ar-H, J = 4.32 Hz), 7.11 (m, 3H, Ar-H),
6.98 (d, 1H, Ar-H, J = 8.4 Hz), 6.68 (d, 1H, C@CH, J = 15.72 Hz),
4.57 (m, 1H, CH), 3.79 (s, 3H, –OCH3), 3.78 (s, 3H, –OCH3), 2.29
(m, 2H, CH2), 2.08 (s, 3H, CH3), 1.99 (m, 1H, CH2), 1.88 (m, 1H,
CH2). ESI-MS: 427.6 [M+1].
To a stirred solution of ferulic acid (1, 19.4 g, 100 mmol) in
60 ml of 4 N NaOH was added (CH3)2SO4 (18.9 g, 150 mmol) drop-
wise, keeping the inside temperature under 20 °C. The resulting
solution was allowed to stir at 20–30 °C for 20 min, another por-
tion of (CH3)2SO4 (150 mmol) and 40 ml of 4 N NaOH were succes-
sively added. The mixture was slowly heated to 90 °C and
maintained at this temperature 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 distilled water. The crude product was
recrystallized with 70% EtOH to give compound
2 (17.5 g,
84.13%) as white needle crystals: Mp 179–181 °C. 1H NMR d
12.11 (s, 1H, COOH), 7.61 (Ar-CH), 6.77 (s, 1H, Ar-H), 6.65 (s, 1H,
Ar-H), 6.45 (CH–COO), 3.83 (s, 6H, CH3O–). ESI-MS: m/z [M+H]+
209.2 [M+1].
5.2.2. 3,4,5-Trimethoxybenzoic chloride (3)
To a stirred solution of 2 (20.8 g, 100 mmol) in benzene
(250 ml) was added dropwise with SOCl2 (40 ml, 548 mmol).
The reaction mixture was refluxed for 4 h, and the solvent
The following L-iso-glutamine derivatives A2–A13 were pre-
pared according to the general procedure as described for the prep-
aration of compound A1.

3068
X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3061–3071
5.2.6. (S,E)-4-(3-(3,4-Dimethoxyphenyl)acrylamido)-5-(4-fluor-
ophenylamino)-5-oxopentanoic acid (A2)
5.2.11. (S,E)-5-(2-Chlorophenylamino)-4-(3-(3,4-dimethoxy-
phenyl)acrylamido)-5-oxopentanoic acid (A7)
Yield: 32.7%, mp 171.0–173.0 °C, IR (KBr, cmꢀ1): 3318 (NH),
3289 (OH), 1703 (O@C–OH), 1672 (O@C–NH), 1599 and 1510
(Ar), 1265 (C–N), 1159 and 1141 (C–O). 1H NMR (DMSO-d6,
ppm): d 12.12 (s, 1H, COOH), 10.05 (s, 1H, NH), 8.26 (d, 1H, NH,
J = 7.86 Hz), 7.62 (q, 2H, Ar-H), 7.38 (d, 1H, C@CH, J = 15.72 Hz),
7.14 (m, 4H, Ar-H), 6.98 (d, 1H, Ar-H, J = 8.34 Hz), 6.68 (d, 1H,
C@CH, J = 15.78 Hz), 4.54 (m, 1H, CH), 3.80 (s, 3H, –OCH3), 3.79
(s, 3H, –OCH3), 2.31 (m, 2H, CH2), 1.90 (m, 1H, CH2), 1.82 (m, 1H,
CH2). ESI-MS: 431.7 [M+1].
Yield: 39.3%, mp 125.0–126.0 °C, IR (KBr, cmꢀ1): 3421 (NH), 3336
(OH), 1748 (O@C–OH), 1651 (O@C–NH), 1596 and 1512 (Ar), 1264
(C–N), 1162 and 1141 (C–O). 1H NMR (DMSO-d6, ppm): d 12.10 (s,
1H, COOH), 9.88 (s, 1H, NH), 8.68 (d, 1H, NH, J = 1.84 Hz), 7.76 (d,
1H, Ar-H, J = 7.92 Hz), 7.49 (d, 1H, Ar-H, J = 8.01 Hz), 7.39 (d, 1H,
C@CH, J = 15.72 Hz), 7.32 (d, 1H, Ar-H, J = 7.35 Hz), 7.18 (m, 1H, Ar-
H), 7.13 (d, 1H, Ar-H, J = 6.79 Hz), 6.99 (d, 1H, Ar-H, J = 8.36 Hz),
6.66 (d, 1H, C@CH, J = 15.74 Hz), 4.63 (m, 1H, CH), 3.79 (s, 3H, –
OCH3), 3.78 (s, 3H, –OCH3), 2.28 (m, 2H, CH2), 2.03 (m, 1H, CH2),
1.92 (m, 1H, CH2). ESI-MS: 447.6 [M+1].
5.2.7. (S,E)-5-(4-Chlorophenylamino)-4-(3-(3,4-dimethoxy-
phenyl)acrylamido)-5-oxopentanoic acid (A3)
5.2.12. (S,E)-5-(3-Chlorophenylamino)-4-(3-(3,4-dime-
Yield: 36.1%, mp 187.0–188.0 °C, IR (KBr, cmꢀ1): 3316 (NH),
3281 (OH), 1701 (O@C–OH), 1652 (O@C–NH), 1597 and 1514
(Ar), 1265 (C–N), 1160 and 1143 (C–O). 1H NMR (DMSO-d6,
ppm): d 12.13 (s, 1H, COOH), 10.23 (s, 1H, NH), 8.27 (d, 1H,
NH, J = 7.8 Hz), 7.64 (d, 2H, Ar-H, J = 8.88 Hz), 7.39 (m, 3H, Ar-
H), 7.38 (d, 1H, C@CH, J = 9.72 Hz), 7.11 (m, 4H, Ar-H), 6.98
(d, 1H, Ar-H, J = 2.4 Hz), 6.68 (d, 1H, C@CH, J = 21.78 Hz), 4.54
(m, 1H, CH), 3.80 (s, 3H, –OCH3), 3.79 (s, 3H, –OCH3), 2.30
(m, 2H, CH2), 2.08 (m, 1H, CH2), 1.99 (m, 1H, CH2). ESI-MS:
447.6 [M+1].
thoxyphenyl)acrylamido)-5-oxopentanoic acid (A8)
Yield: 37.6%, mp 163.0–164.0 °C, IR (KBr, cmꢀ1): 3316 (NH),
3281 (OH), 1701 (O@C–OH), 1652 (O@C–NH), 1597 and 1514
(Ar), 1265 (C–N), 1160 and 1143 (C–O). 1H NMR (DMSO-d6,
ppm): d 12.19 (s, 1H, COOH), 10.36 (s, 1H, NH), 8.35 (d, 1H, NH,
J = 1.38 Hz), 7.82 (d, 1H, Ar-H, J = 1.74 Hz), 7.43 (m, 3H, Ar-H),
7.39 (d, 1H, C@CH, J = 7.68 Hz), 7.12 (m, 3H, Ar-H), 6.99 (d, 1H,
Ar-H, J = 8.34 Hz), 6.66 (d, 1H, C@CH, J = 15.72 Hz), 4.51 (m, 1H,
CH), 3.79 (s, 3H, –OCH3), 3.78 (s, 3H, –OCH3), 2.33 (m, 2H, CH2),
1.97 (m, 1H, CH2), 1.90 (m, 1H, CH2). ESI-MS: 447.5 [M+1].
5.2.8. (S,E)-5-(4-Bromophenylamino)-4-(3-(3,4-dimethoxy-
phenyl)acrylamido)-5-oxopentanoic acid (A4)
5.2.13. (S,E)-5-(Benzylamino)-4-(3-(3,4-dimethoxyphenyl)-
acrylamido)-5-oxopentanoic acid (A9)
Yield: 33.9%, mp 186.0–187.2 °C, IR (KBr, cmꢀ1): 3334 (NH),
3288 (OH), 1741 (O@C–OH), 1653 (O@C–NH), 1596 and 1514
(Ar), 1271 (C–N), 1163 and 1145 (C–O). 1H NMR (DMSO-d6,
ppm): d 12.12 (s, 1H, COOH), 10.23 (s, 1H, NH), 8.27 (d, 1H,
NH, J = 5.87 Hz), 7.58 (d, 2H, Ar-H, J = 8.82 Hz), 7.49 (d, 2H,
Ar-H, J = 8.76 Hz), 7.37 (d, 1H, C@CH, J = 9.72 Hz), 7.16 (d, 1H,
Ar-H, J = 1.14 Hz), 7.12 (d, 1H, Ar-H, J = 8.88 Hz), 6.99 (d, 1H,
Ar-H, J = 8.34 Hz), 6.67 (d, 1H, C@CH, J = 15.72 Hz), 4.53 (m,
1H, CH), 3.80 (s, 3H, –OCH3), 3.79 (s, 3H, –OCH3), 2.30 (m,
2H, CH2), 2.09 (m, 1H, CH2), 2.01 (m, 1H, CH2). ESI-MS: 491.5
[M+1].
Yield: 43.1%, mp 180.0–181.0 °C, IR (KBr, cmꢀ1): 3302 (NH),
1708 (O@C–OH), 1666 (O@C–NH), 1599 and 1511 (Ar), 1264 (C–
N), 1159 and 1142 (C–O). 1H NMR (DMSO-d6, ppm): d 12.08 (s,
1H, COOH), 8.48 (s, 1H, NH), 8.12 (d, 1H, NH, J = 8.04 Hz), 7.36 (d,
1H, C@CH, J = 15.78 Hz), 7.32 (t, 1H, Ar-H), 7.23 (q, 1H, Ar-H),
7.16 (d, 1H, Ar-H, J = 1.14 Hz), 7.11 (d, 1H, Ar-H, J = 8.28 Hz), 6.99
(d, 1H, Ar-H, J = 8.34 Hz), 6.66 (d, 1H, C@CH, J = 15.72 Hz), 4.43
(m, 1H, CH), 4.28 (d, 2H, CH2), 3.79 (s, 3H, –OCH3), 3.78 (s, 3H, –
OCH3), 2.26 (m, 2H, CH2), 1.98 (m, 1H, CH2), 1.82 (m, 1H, CH2).
ESI-MS: 427.6 [M+1].
5.2.14. (S,E)-4-(3-(3,4-Dimethoxyphenyl)acrylamido)-5-
(methylamino)-5-oxopentanoic acid (A10)
5.2.9. (S,E)-5-(3,5-Difluorophenylamino)-4-(3-(3,4-dimethoxy-
phenyl)acrylamido)-5-oxopentanoic acid (A5)
Yield: 53.1%, mp 108.0–109.0 °C, IR (KBr, cmꢀ1): 3300 (NH),
1707 (O@C–OH), 1668 (O@C–NH), 1606 and 1513 (Ar), 1261 (C–
N), 1155 and 1139 (C–O). 1H NMR (DMSO-d6, ppm): d 12.10 (s,
1H, COOH), 8.09 (s, 1H, NH), 7.99 (d, 1H, NH, J = 1.78 Hz), 7.35 (d,
1H, C@CH, J = 8.4 Hz), 7.17 (d, 1H, Ar-H, J = 6.45 Hz), 7.16 (d, 1H,
Ar-H, J = 7.92 Hz), 6.79 (d, 1H, Ar-H, J = 15.72 Hz), 6.84 (d, 1H,
C@CH, J = 8.24 Hz), 4.53 (m, 1H, CH), 3.77 (s, 3H, –OCH3), 3.73 (s,
3H, –OCH3), 2.23 (m, 2H, CH2), 2.06 (m, 1H, CH2), 1.71 (s, 3H,
CH3). ESI-MS: 351.5 [M+1].
Yield: 35.3%, mp 190.0–191.0 °C, IR (KBr, cmꢀ1): 3322 (NH),
3280 (OH), 1710 (O@C–OH), 1657 (O@C–NH), 1617 and 1514
(Ar), 1266 (C–N), 1161 and 1140 (C–O). 1H NMR (DMSO-d6,
ppm): d 12.17 (s, 1H, COOH), 10.54 (s, 1H, NH), 8.36 (d, 1H, NH,
J = 7.56 Hz), 7.38 (d, 1H, C@CH, J = 15.72 Hz), 7.35 (m, 2H, Ar-H),
7.16 (d, 1H, Ar-H, J = 1.8 Hz), 7.12 (q, 1H, Ar-H), 7.06 (d, 1H, Ar-H,
J = 8.4 Hz), 6.99 (d, 1H, Ar-H, J = 1.8 Hz), 6.66 (d, 1H, C@CH,
J = 15.78 Hz), 4.50 (m, 1H, CH), 3.80 (s, 3H, –OCH3), 3.78 (s, 3H, –
OCH3), 2.32 (m, 2H, CH2), 2.01 (m, 1H, CH2), 1.90 (m, 1H, CH2).
ESI-MS: 449.6 [M+1].
5.2.15. (S,E)-4-(3-(3,4-Dimethoxyphenyl)acrylamido)-5-
(ethylamino)-5-oxopentanoic acid (A11)
Yield: 40.7%, mp 112.0–113.0 °C, IR (KBr, cmꢀ1): 3298 (NH),
1706 (O@C–OH), 1661 (O@C–NH), 1597 and 1510 (Ar), 1259 (C–
N), 1156 and 1137 (C–O). 1H NMR (DMSO-d6, ppm): d 12.11 (s,
1H, COOH), 8.09 (s, 1H, NH), 7.98 (d, 1H, NH, J = 7.56 Hz), 7.56 (d,
1H, C@CH, J = 8.01 Hz), 6.97 (d, 1H, Ar-H, J = 7.2 Hz), 6.78 (d, 1H,
Ar-H, J = 1.8 Hz), 6.67 (d, 1H, Ar-H, J = 9.2 Hz), 6.61 (d, 1H, C@CH,
J = 15.78 Hz), 4.53 (m, 1H, CH), 3.75 (s, 3H, –OCH3), 3.73 (s, 3H, –
OCH3), 3.24 (m, 2H, CH2), 2.23 (m, 2H, CH2), 2.05 (m, 1H, CH2),
1.28 (m, 3H, CH3). ESI-MS: 365.6 [M+1].
5.2.10. (S,E)-4-(3-(3,4-Dimethoxyphenyl)acrylamido)-5-(2-
fluorophenylamino)-5-oxopentanoic acid (A6)
Yield: 30.5%, mp 168.0–169.0 °C, IR (KBr, cmꢀ1): 3420 (NH),
3337 (OH), 1748 (O@C–OH), 1651 (O@C–NH), 1594 and 1512
(Ar), 1264 (C–N), 1162 and 1141 (C–O). 1H NMR (DMSO-d6,
ppm): d 12.18 (s, 1H, COOH), 9.89 (s, 1H, NH), 8.32 (d, 1H,
NH, J = 7.92 Hz), 7.81 (m, 1H, Ar-H), 7.38 (d, 1H, C@CH,
J = 15.72 Hz), 7.38 (d, 1H, Ar-H, J = 10.8 Hz), 7.25 (m, 1H, Ar-
H), 7.18 (m, 1H, Ar-H), 7.13 (d, 1H, Ar-H, J = 1.62 Hz), 6.99 (d,
1H, Ar-H, J = 8.34 Hz), 6.67 (d, 1H, C@CH, J = 7.56 Hz), 4.67 (m,
1H, CH), 3.79 (s, 3H, –OCH3), 3.78 (s, 3H, –OCH3), 2.34 (m,
2H, CH2), 2.03 (m, 1H, CH2), 1.90 (m, 1H, CH2). ESI-MS: 431.7
[M+1].
5.2.16. (S,E)-4-(3-(3,4-Dimethoxyphenyl)acrylamido)-5-oxo-5-
(propylamino)pentanoic acid (A12)
Yield: 37.0%, mp 126.0–127.0 °C, IR (KBr, cmꢀ1): 3282 (NH),
1708 (O@C–OH), 1643 (O@C–NH), 1611 and 1512 (Ar), 1262 (C–

X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3061–3071
3069
N), 1160 and 1141 (C–O). 1H NMR (DMSO-d6, ppm): d 12.09 (s, 1H,
COOH), 8.11 (s, 1H, NH), 7.99 (d, 1H, NH, J = 5.7 Hz), 7.35 (d, 1H,
C@CH, J = 15.72 Hz), 7.16 (d, 1H, Ar-H, J = 1.26 Hz), 7.11 (d, 1H,
Ar-H, J = 8.28 Hz), 6.98 (d, 1H, Ar-H, J = 8.34 Hz), 6.66 (d, 1H, C@CH,
J = 15.06 Hz), 4.35 (m, 1H, CH), 3.78 (s, 3H, –OCH3), 3.72 (s, 3H, –
OCH3), 3.00 (m, 2H, CH2), 2.22 (m, 2H, CH2), 1.93 (m, 1H, CH2),
1.87 (m, 1H, CH2), 1.39 (m, 2H, CH2), 0.83 (m, 3H, CH3). ESI-MS:
379.7 [M+1].
5.2.20. (4S,E)-4-(3-(3,4-Dimethoxyphenyl)acrylamido)-5-(1-
methoxy-3-methyl-1-oxobutan-2-ylamino)-5-oxopentanoic acid
(A16)
Yield: 39.5%, mp 143.1–144.5 °C, IR (KBr, cmꢀ1): 3326 (NH),
3284 (OH), 1740 (O@C–OH), 1651 (O@C–NH), 1616 and 1514
(Ar), 1262 (C–N), 1169 and 1140 (C–O). 1H NMR (DMSO-d6,
ppm): d 12.00 (s, 1H, COOH), 8.22 (s, 1H, NH), 8.13 (d, 1H, NH,
J = 8.03 Hz), 7.36 (d, 1H, C@CH, J = 15.71 Hz), 7.16 (d, 1H, Ar-H,
J = 1.41 Hz), 7.11 (q, 1H, Ar-H), 6.98 (d, 1H, Ar-H, J = 8.37 Hz),
6.68 (d, 1H, C@CH, J = 15.73 Hz), 4.18 (m, 1H, CH), 3.79 (s, 3H, –
OCH3), 3.78 (s, 3H, –OCH3), 3.61 (s, 3H, –OCH3), 2.50 (m, 2H,
CH2), 2.08 (m, 1H, CH), 1.93 (m, 1H, CH2), 1.81 (m, 1H, CH2), 0.90
(s, 3H, CH3), 0.88 (s, 3H, CH3). ESI-MS: 451.3 [M+1].
5.2.17. (S,E)-4-(3-(3,4-Dimethoxyphenyl)acrylamido)-5-
(isopropylamino)-5-oxopentanoic acid (A13)
Yield: 38.8%, mp 157.0–158.0 °C, IR (KBr, cmꢀ1): 3373 (NH),
3327 (OH), 1737 (O@C–OH), 1642 (O@C–NH), 1609 and 1516
(Ar), 1262 (C–N), 1160 and 1136 (C–O). 1H NMR (DMSO-d6,
ppm): d 12.13 (s, 1H, COOH), 8.05 (s, 1H, NH), 7.90 (d, 1H, NH,
J = 7.68 Hz), 7.35 (d, 1H, C@CH, J = 15.72 Hz), 7.16 (m, 1H, Ar-H),
7.10 (d, 1H, Ar-H, J = 1.32 Hz), 6.98 (d, 1H, Ar-H, J = 8.34 Hz), 6.68
(d, 1H, C@CH, J = 15.72 Hz), 4.36 (m, 1H, CH), 4.03 (m, 1H, CH),
3.82 (s, 3H, –OCH3), 3.79 (s, 3H, –OCH3), 2.22 (m, 2H, CH2), 1.88
(m, 1H, CH2), 1.77 (m, 1H, CH2). ESI-MS: 379.7 [M+1].
5.2.21. (4S,E)-5-(1,4-Dimethoxy-1,4-dioxobutan-2-ylamino)-4-
(3-(3,4-dimethoxyphenyl)acrylamido)-5-oxopentanoic acid
(A17)
Yield: 41.2%, mp 155.0–156.0 °C, IR (KBr, cmꢀ1): 3290 (NH),
3284 (OH), 1731 (O@C–OH), 1644 (O@C–NH), 1613 and 1512
(Ar), 1263 (C–N), 1165 and 1142 (C–O). 1H NMR (DMSO-d6,
ppm): d 12.07 (s, 1H, COOH), 8.52 (s, 1H, NH), 8.12 (d, 1H, NH,
J = 8.19 Hz), 7.39 (d, 1H, C@CH, J = 2.06 Hz), 7.16 (s, 1H, Ar-H),
7.11 (d, 1H, Ar-H, J = 8.31 Hz), 6.97 (d, 1H, Ar-H, J = 8.39 Hz),
6.69 (d, 1H, C@CH, J = 4.25 Hz), 4.68 (m, 1H, CH), 4.47 (m, 1H,
CH), 3.78 (s, 3H, –OCH3), 3.76 (s, 3H, –OCH3), 3.64 (s, 3H,
OCH3), 3.60 (s, 3H, –OCH3), 2.86 (m, 1H, CH), 2.76 (m, 1H, CH),
2.28 (m, 2H, CH2), 1.90 (m, 1H, CH2), 1.80 (m, 1H, CH2). ESI-MS:
481.4 [M+1].
5.2.18. (S,E)-4-(3-(3,4-Dimethoxyphenyl)acrylamido)-5-(2-
methoxy-2-oxoethylamino)-5-oxopentanoic acid (A14)
Preparation of L-Amino acid methyl ester hydrochloride: To a
suspension of glycine (15.0 g, 200 mmol) in anhydrous methanol
(150 ml) at 0 °C 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%). Mp 175–176 °C, IR
(KBr, cmꢀ1): 3215.0–3523.0 (NH), 2935.2 (CH), 1676.3 (C@O),
1129.3 (C–O).
To a solution of compound 5 (3.5 g, 10 mmol) in CHCl3 (20 ml)
at ꢀ5 °C was added a solution of DCC (2.06 g, 10 mmol) in 50 ml
CHCl3 in small portions. After completion the DCC, a solution of
methyl 2-aminoacetate hydrochloride (10 mmol) in 20 ml CHCl3
was added dropwise. Then the mixture was stirred at this temper-
ature for 5 h. Filtered off the DCU and washed with dilute Na2CO3
solution. The eluant was acidified with 1 N HCl to pH 2, extracted
with EA (30 ml ꢂ 3), and subsequently the solvent was removed
under reduced pressure. The resulting crude residue was recrystal-
lized from ethanol and dried to afford the target compound as
white solid, yield: 46.1%. Mp 172.0–173.0 °C, IR (KBr, cmꢀ1):
3326 (NH), 3286 (OH), 1754 (O@C–OH), 1651 (O@C–NH), 1597
and 1513 (Ar), 1264 (C–N), 1160 and 1140 (C–O). 1H NMR
(DMSO-d6, ppm): d 12.06 (s, 1H, COOH), 8.40 (s, 1H, NH), 8.13 (d,
1H, NH, J = 8.1 Hz), 7.36 (d, 1H, C@CH, J = 15.72 Hz), 7.16 (s, 1H,
Ar-H), 7.11 (q, 1H, Ar-H), 6.99 (d, 1H, Ar-H, J = 8.34 Hz), 6.66 (d,
1H, C@CH, J = 15.72 Hz), 4.46 (m, 1H, CH), 3.80 (s, 3H, –OCH3),
3.78 (s, 3H, –OCH3), 3.63 (s, 3H, –OCH3), 3.60 (s, 3H, –OCH3), 2.28
(m, 2H, CH2), 1.96 (m, 1H, CH2), 1.81 (m, 1H, CH2). ESI-MS: 409.6
[M+1].
5.2.22. (4S,E)-4-(3-(3,4-Dimethoxyphenyl)acrylamido)-5-(3-(4-
fluorophenyl)-1-methoxy-1-oxopropan-2-ylamino)-5-
oxopentanoic acid (A18)
Yield: 35.2%, mp 116.0–117.0 °C, IR (KBr, cmꢀ1): 3149 (NH), 3263
(OH), 1721 (O@C–OH), 1750 (O@C–NH), 1616 and 1512 (Ar), 1264
(C–N), 1160 and 1140 (C–O). 1H NMR (DMSO-d6, ppm): d 12.01 (s,
1H, COOH), 8.27 (s, 1H, NH), 8.01 (d, 1H, NH, J = 8.4 Hz), 7.36 (d,
1H, C@CH, J = 15.66 Hz), 7.25 (m, 2H, Ar-H), 7.16 (d, 1H, Ar-H,
J = 1.86 Hz), 7.10 (m, 1H, Ar-H), 7.02 (m, 2H, Ar-H), 6.99 (d, 1H, Ar-
H, J = 8.4 Hz), 6.65 (d, 1H, C@CH, J = 15.72 Hz), 4.45 (m, 1H, CH),
4.43 (m, 1H, CH), 3.79 (s, 3H, –OCH3), 3.78 (s, 3H, –OCH3), 3.06 (m,
1H, CH2), 2.86 (m, 1H, CH2), 2.03 (m, 2H, CH2), 1.76 (m, 1H, CH2),
1.63 (m, 1H, CH2). ESI-MS: 503.3 [M+1].
5.2.23. (S,E)-2-(3-(3,4-Dimethoxyphenyl)acrylamido)-N1,N5-
dip-tolylpentanediamide (B1)
To a solution of compound 5 (3.5 g, 10 mmol) in CHCl3 (20 ml)
at ꢀ5 °C was added a solution of DCC (4.12 g, 2 equiv, 20 mmol)
in 100 ml CHCl3 in small portions. After completion the DCC, a
solution of p-toluidine (2.16 g, 2.05 eq, 21 mmol) in 30 ml CHCl3
was added dropwise. Then the mixture was stirred at this temper-
ature until the solid has almost completely dissolved (about 5 h).
The insoluble material DCU was filtered off and washed with cold
1 N Na2CO3. The eluant was purified by flash column chromatogra-
phy with cyclohexane–EA (4:1 to 1:1) to provide 1.68 g of white
solid, yield 32.6%, mp 240.0–241.0 °C, IR (KBr, cmꢀ1): 3273 (NH),
1652 (O@C–NH), 1600 and 1514 (Ar), 1260 (C–N), 1160 and
1138 (C–O). 1H NMR (DMSO-d6, ppm): d 10.08 (s, 1H, NH), 9.85
(s, 1H, NH), 8.29 (d, 1H, NH, J = 8.04 Hz), 7.51 (d, 2H, Ar-H,
J = 8.4 Hz), 7.44 (d, 2H, Ar-H, J = 8.4 Hz), 7.38 (d, 1H, C@CH,
J = 15.72 Hz), 7.16 (d, 1H, Ar-H, J = 7.86 Hz), 7.11 (d, 3H, Ar-H,
J = 8.4 Hz), 7.06 (d, 2H, Ar-H, J = 8.4 Hz), 6.98 (d, 1H, Ar-H,
J = 8.4 Hz), 6.71 (d, 1H, C@CH, J = 15.72 Hz), 4.57 (m, 1H, CH),
3.79 (s, 3H, –OCH3), 3.78 (s, 3H, –OCH3), 2.38 (m, 2H, CH2), 2.25
(s, 3H, CH3), 2.24 (s, 3H, CH3), 2.07 (m, 1H, CH2), 1.96 (m, 1H,
CH2). ESI-MS: 516.8 [M+1].
5.2.19. (4S,E)-4-(3-(3,4-Dimethoxyphenyl)acrylamido)-5-(1-
methoxy-1-oxopropan-2-ylamino)-5-oxopentanoic acid (A15)
Yield: 38.8%, mp 163.0–164.0 °C, IR (KBr, cmꢀ1): 3326 (NH), 3300
(OH), 1755 (O@C–OH), 1650 (O@C–NH), 1590 and 1518 (Ar), 1244
(C–N), 1164 and 1141 (C–O). 1H NMR (DMSO-d6, ppm): d 12.21 (s,
1H, COOH), 8.46 (s, 1H, NH), 8.11 (d, 1H, NH, J = 8.20 Hz), 7.37 (d,
1H, C@CH, J = 15.71 Hz), 7.16 (d, 1H, Ar-H, J = 1.43 Hz), 7.11 (q, 1H,
Ar-H), 6.97 (d, 1H, Ar-H, J = 8.39 Hz), 6.68 (d, 1H, C@CH,
J = 15.75 Hz), 4.49 (m, 1H, CH), 3.80 (s, 3H, –OCH3), 3.78 (s, 3H, –
OCH3), 3.62 (s, 3H, –OCH3), 2.30 (m, 2H, CH2), 1.98 (m, 1H, CH2),
1.83 (m, 1H, CH2), 1.30 (s, 3H, CH3). ESI-MS: 423.5 [M+1].

3070
X. Li et al. / Bioorg. Med. Chem. 17 (2009) 3061–3071
The other
L
-iso-glutamine derivatives B2–B7 were prepared
–OCH3), 2.47 (m, 2H, CH2), 2.12 (m, 1H, CH2), 1.98 (m, 1H, CH2).
ESI-MS: 560.5 [M+1].
according to the general procedure as described for the preparation
of compound B1.
5.2.29. (S,E)-2-[3-(3,4-Dimethoxyphenyl)acrylamido]-N1,N5-
[(R)-1,4-dimethoxy-1,4-dioxobutan]pentanediamide (B7)
Yield: 42.3%, mp 179.0–181.0 °C, IR (KBr, cmꢀ1): 3291 (NH),
1645 (O@C–NH), 1613 and 1514 (Ar), 1264 (C–N), 1161 and
5.2.24. (S,E)-2-(3-(3,4-Dimethoxyphenyl)acrylamido)-N1,N5-
bis(4-fluorophenyl)pentanediamide (B2)
Yield: 30.0%, mp 188.0–189.0 °C, IR (KBr, cmꢀ1): 3277 (NH),
1659 (O@C–NH), 1615 & 1509 (Ar), 1261 (C–N), 1161 and 1145
(C–O). 1H NMR (DMSO-d6, ppm): d 10.25 (s, 1H, NH), 10.00 (s,
1H, NH), 8.34 (d, 1H, NH, J = 6.12 Hz), 7.65 (q, 2H, Ar-H), 7.58 (q,
2H, Ar-H), 7.38 (d, 1H, C@CH, J = 15.72 Hz), 7.16 (t, 3H, Ar-H),
7.11 (t, 3H, Ar-H), 6.98 (d, 1H, Ar-H, J = 2.34 Hz), 6.70 (d, 1H, C@CH,
J = 9.72 Hz), 4.57 (m, 1H, CH), 3.79 (s, 3H, –OCH3), 3.78 (s, 3H,
–OCH3), 2.45 (m, 2H, CH2), 2.09 (m, 1H, CH2), 1.96 (m, 1H, CH2).
ESI-MS: 524.7 [M+1].
1140 (C–O). 1H NMR (DMSO-d6, ppm):
d 8.47 (d, 1H, NH,
J = 7.91 Hz), 8.32 (d, 1H, NH, J = 7.88 Hz), 8.07 (d, 1H, NH,
J = 8.21 Hz), 7.36 (d, 1H, C@CH, J = 15.71 Hz), 7.16 (d, 1H, Ar-H,
J = 1.72 Hz), 7.10 (d, 1H, Ar-H, J = 6.58 Hz), 6.98 (d, 1H, Ar-H,
J = 8.35 Hz), 6.67 (d, 1H, C@CH, J = 15.85 Hz), 4.62 (m, 1H, CH),
4.32 (m, 1H, CH), 3.79 (s, 3H, –OCH3), 3.78 (s, 3H, –OCH3), 3.63
(s, 3H, –OCH3), 3.58 (s, 3H, –OCH3), 2.82 (m, 2H, CH2), 2.76 (m,
2H, CH2), 2.18 (m, 2H, CH2), 1.93 (m, 1H, CH2), 1.73 (m, 1H, CH2).
ESI-MS: 624.6 [M+1].
5.2.25. (S,E)-N1,N5-Bis(4-chlorophenyl)-2-(3-(3,4-
dimethoxyphenyl)acrylamido)pentanediamide (B3)
6. Enzymatic inhibiton assay (in vitro)
6.1. In vitro APN assay
Yield: 38.3%, mp 206.0–207.0 °C, IR (KBr, cmꢀ1): 3271 (NH),
1652 (O@C–NH), 1596 and 1514 (Ar), 1261 (C–N), 1160 and
1138 (C–O). 1H NMR (DMSO-d6, ppm): d 10.28 (s, 1H, NH), 10.04
(s, 1H, NH), 8.30 (d, 1H, NH, J = 7.86 Hz), 7.66 (d, 2H, Ar-H,
J = 8.22 Hz), 7.59 (d, 2H, Ar-H, J = 2.82 Hz), 7.38 (m, 3H, Ar-H),
7.32 (d, 1H, C@CH, J = 8.88 Hz), 7.15 (d, 1H, Ar-H, J = 1.62 Hz),
7.11 (d, 1H, Ar-H, J = 1.62 Hz), 6.99 (d, 1H, Ar-H, J = 8.34 Hz), 6.68
(d, 1H, C@CH, J = 15.78 Hz), 4.58 (m, 1H, CH), 3.79 (s, 3H, –OCH3),
3.78 (s, 3H, –OCH3), 2.45 (m, 2H, CH2), 2.09 (m, 1H, CH2), 1.97
(m, 1H, CH2). ESI-MS: 556.5 [M+1].
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
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 (concentration dependent on the inhibitor), the
5.2.26. (S,E)-N1,N5-Bis(4-bromophenyl)-2-(3-(3,4-dimethoxy-
phenyl)acrylamido)pentanediamide (B4)
Yield: 37.7%, mp 210.0–212.0 °C, IR (KBr, cmꢀ1): 3268 (NH),
1648 (O@C–NH), 1591 and 1515 (Ar), 1262 (C–N), 1160 and
1138 (C-O). 1H NMR1H NMR (DMSO-d6, ppm): d 10.28 (s, 1H,
NH), 10.04 (s, 1H, NH), 8.30 (d, 1H, NH, J = 7.86 Hz), 7.60 (q, 2H,
Ar-H), 7.54 (d, 2H, Ar-H, J = 8.88 Hz), 7.50 (d, 2H, Ar-H,
J = 8.88 Hz), 7.44 (d, 2H, Ar-H, J = 7.02 Hz), 7.38 (d, 1H, C@CH,
J = 15.78 Hz), 7.16 (d, 1H, Ar-H, J = 1.8 Hz), 7.11 (q, 1H, Ar-H),
6.99 (d, 1H, Ar-H, J = 8.34 Hz), 6.68 (d, 1H, C@CH, J = 15.72 Hz),
4.58 (m, 1H, CH), 3.80 (s, 3H, –OCH3), 3.78 (s, 3H, –OCH3), 2.43
(m, 2H, CH2), 2.09 (m, 1H, CH2), 1.97 (m, 1H, CH2). ESI-MS: 644.3
[M+1].
enzyme solution (4
lg/ml final concentration), and the assay buf-
fer, was adjusted to 200
lL.
6.2. In vitro MMP-2 assay
MMP-2, also called as Gelatinase A, assay was performed as de-
scribed by Baragi et al.32 The gelatinase, substrate and inhibitor
were dissolved in sodium borate (pH 8.5, 50 mmol/L) and incu-
bated for 30 min at 37 °C, and then 0.03% trinitrobenzenesulfonic
acid (TNBS, Sigma) was added and incubated for another 20 min,
the resulting solution was detected under 450 nm wavelength to
gain absorption.
5.2.27. (S,E)-2-(3-(3,4-Dimethoxyphenyl)acrylamido)-N1,N5-
bis(2-fluorophenyl)pentanediamide (B5)
Yield: 38.6%, mp 187.0–188.0 °C, IR (KBr, cmꢀ1): 3326 (NH),
1626 (O@C–NH), 1575 and 1515 (Ar), 1261 (C–N), 1159 and
1139 (C–O). 1H NMR (DMSO-d6, ppm): d 9.87 (s, 1H, NH), 9.72 (s,
1H, NH), 8.31 (d, 1H, NH, J = 7.92 Hz), 7.76 (m, 2H, Ar-H), 7.41 (d,
1H, C@CH, J = 15.72 Hz), 7.23 (m, 2H, Ar-H), 7.15 (m, 3H, Ar-H),
7.15 (m, 3H, Ar-H), 7.10 (m, 3H, Ar-H), 6.99 (d, 1H, Ar-H,
J = 8.34 Hz), 6.70 (d, 1H, C@CH, J = 15.72 Hz), 4.72 (m, 1H, CH),
3.79 (s, 3H, –OCH3), 3.78 (s, 3H, –OCH3), 2.53 (m, 2H, CH2), 2.13
(m, 1H, CH2), 1.98 (m, 1H, CH2). ESI-MS: 524.5 [M+1].
Acknowledgments
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).
References and notes
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dimethoxyphenyl)acrylamido)pentanediamide (B6)
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