Novel matrix metalloproteinase inhibitors derived from quinoxalinone scaffold (Part I)

Article abstract of DOI:10.1016/j.bmc.2010.01.008

A series of quinoxalinone peptidomimetic derivatives was designed, synthesized, and assayed for their inhibitory activities on metalloproteinase-2 (MMP-2) and aminopeptidase N (APN). The results showed that all of these quinoxalinone derivatives displayed highly selective inhibition against MMP-2 as compared with APN, with IC₅₀ values in the micromole range. Compound A3 showed comparable MMP-2 inhibitory activities than the positive control LY52, which might be used as a potential lead in future research on anticancer agents.

Full text of DOI:10.1016/j.bmc.2010.01.008

Bioorganic & Medicinal Chemistry 18 (2010) 1516–1525
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel matrix metalloproteinase inhibitors derived from quinoxalinone
scaffold (Part I)
*
Yonggang Li, Jian Zhang, Wenfang Xu, Huawei Zhu, Xun Li
School of Pharmaceutical Sciences, Shandong University, No. 44 WenhuaXi Road, Ji’nan, 250012 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:
A series of quinoxalinone peptidomimetic derivatives was designed, synthesized, and assayed for their
inhibitory activities on metalloproteinase-2 (MMP-2) and aminopeptidase N (APN). The results showed
that all of these quinoxalinone derivatives displayed highly selective inhibition against MMP-2 as com-
pared with APN, with IC50 values in the micromole range. Compound A3 showed comparable MMP-2
inhibitory activities than the positive control LY52, which might be used as a potential lead in future
research on anticancer agents.
Received 27 November 2009
Revised 31 December 2009
Accepted 5 January 2010
Available online 11 January 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Quinoxalinone derivatives
MMP-2 inhibitors
Peptidomimetic
Anti-tumor
1. Introduction
are called S01 and S20 pocket, respectively. S01 pocket, the key domain
to characterize the selectivity of various MMPs, is deeper and nar-
Matrix metalloproteinases (MMPs) are a family of structurally
and functionally related zinc-dependent endopeptidases that are
involved in the degradation of the extracellular matrix (ECM) pro-
teins as well as the tumorgenesis process and, consequently, they
play a crucial role in physiological processes such as tissue remod-
eling, apoptosis and wound healing. As the overregulation of MMPs
is involved in many inflammatory, malignant and degenerative
conditions, attempts to design and exploit inhibitors that may
modulate their regulation have become of great interest.1–3
Up to date, many reported broad-spectrum inhibitors exhibit a
poor selectivity as a consequence of the high structural similarity
among the members of the MMP family. Even if the low specificity
does not prevent the use in vivo, it raises a variety of side effects
and it also limits the dose that may be daily administered. As a re-
sult, clinical trials conducted on these broad-spectrum inhibitors
have yielded disappointing results, especially in the cancer pathol-
ogy area. Accordingly, the search of appropriate MMP inhibitor
(MMPI) with high potency and selectivity for certain subtypes still
represents an important pharmaceutical target.4 Among the iden-
tified more than 20 structurally related family members, MMP-2
(gelatinase A) is highly involved in the process of tumor invasion
and metastasis, which has been a promising target for cancer
therapy.5,6
rower than that of most other MMP subtypes, while S02 pocket is
solvent exposed.7,8 Currently identified MMP-2 inhibitors have
several structural characteristics and binding mode: (1) a zinc-
binding group (ZBG) capable of chelating with the zinc ions and
sequentially inhibiting the metastatic spread of tumors;9 (2) at
least one functional group, which provides at least a hydrogen
bond interaction with the enzyme backbone; and (3) one or more
lipophilic residues that can be accommodated in at least one of the
subsites of the enzyme active sites, such as S01 and S20 pockets.10
Enlightened by these findings, our group has previously re-
ported a series of novel MMP-2 inhibitors with remarkable MMP-
2 inhibitory activities in the enzymatic assays.11–16 One of the
inhibitors, LY52 (see Fig. 1), a caffeinoyl pyrrolidine derivative,
manifested satisfactory potency against MMP-2 with IC50 value
in the nanomolar range. This compound could significantly sup-
press the invasion and metastasis of human carcinoma cells via
inhibition of MMP-2 proteolytic activities.17,18 LY52 was thereby
used as a positive control to elucidate the enzymatic activities.
In our continuous program in the search of new potent MMP-2
inhibitors, a new class of molecular heterocyclic scaffold, quinox-
alinone (or quinoxalin-2-one), an important pharmacophore in
numerous bioactive compounds, was chosen to design selective
MMP-2 inhibitors.19 The main features of this scaffold include:
(1) the electron donor and/or acceptor of the quinoxalinone core
might generate effective interactions with the enzyme backbone,
(2) the quinoxalinone moiety can be decorated with a ZBG and a
lipophilic residue to insert into the hydrophobic S1 pocket, (3)
the synthesis comprises only few steps starting from commercially
It has been reported that besides the catalytic activity center
zinc ion of MMP-2, there are two hydrophobic domains, which
* Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: tjulx2004@sdu.edu.cn (X. Li).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.008

Y. Li et al. / Bioorg. Med. Chem. 18 (2010) 1516–1525
1517
O
O
S1'
S1'
Zn2+
Zn2+
Part A
O
O
3'
4'
5'
HN
O
O
N
2'
N
1
H
6'
NHOH
Part B
N
2
N
O
R
3
R= aromatic or
NH
N
aliphatic side chain
4
O
S2'
LY52 (Positive control)
S2'
Novel designed quinoxalinone derivatives
Figure 1. The design concept of new quinoxalinone peptidomimetics.
available enantiopure precursors and further stereochemistry con-
trol can be easily accomplished and, (4) the quinoxalinone core has
a number of positions which can be chemically functionalized
leading to molecular diversity. All these properties, together with
fair molecular weight, were considered in scaffold selection.
Further optimization on the quinoxalinone scaffold was carried
out as follows (Fig. 1): (i) Given many phenylalanine derivatives
have been reported to demonstrate significant antitumor activ-
structures of the target compounds were analytical confirmed by
IR, 1H NMR, and ESI-MS (see Section 5).
In our synthesis, the quinoxalinone scaffold 1 was easily pre-
pared from the commercially available o-phenylenediamine fol-
lowing the reference.22 Subsequent nucleophilic substitution was
accomplished using ethyl 2-oxopropanoate in the presence of
anhydrous ethanol, followed by hydrolization of ester group to
the carboxylic acid 3, which was condensed with L-phenylalanine
ity,20,21
L-phenylalanine moiety was, therefore, introduced to mi-
methyl ester to provide compound 4. This compound was further
hydrolyzed to offer the carboxylic compound 5, and the key inter-
mediate acyl chloride 6 was then obtained via the acylation. This
was followed by coupling with various aromatic or aliphatic pri-
mary amines in the existence of t-BuOK in anhydrous CH2Cl2 to
provide the target compounds 7. In this reaction, apart from
CH2Cl2, other aprotic polar solvents such as benzene can also be
preferable.
mic the caffeinoyl fragment of LY52 (Part A), and construct the
first integrated peptidomimetic pattern (O@C20–N30H) to interact
with the S01 pocket of MMP-2; (ii) Incorporating a range of aromatic
or aliphatic primary amines to connect with the 5’-carboxyl group
of phenylalanine so as to build another peptidomimetic pattern
(Part B), and the carbonyl group (C2’@O) served as the zinc-binding
group.
In the present study, we hereby describe the synthesis and eval-
uation of the enzymatic activities of the corresponding quinoxali-
none peptidomimetic derivatives, as well as the docking studies
of the interaction, with the anticipation of gaining a more effica-
cious tool compound for tumor therapeutic screening.
3. Results and discussion
3.1. Structure–activity relationship (SARs) studies
The target quinoxalinone peptidomimetic derivatives were
evaluated for their enzymatic inhibition on MMP-2 and APN/
CD13, and the results are listed in Table 1. Similar to MMP-2,
APN is also a zinc-dependent metalloproteinase involved in the
process of tumor invasion and metastasis.23,24 Hence the assay
2. Chemistry
The target compounds were synthesized efficiently following
the procedures outlined in Scheme 1. Meanwhile, the chemical
O
O
OH
O
O
O
H
NH2
N
N
N
O
N
d
b
c
a
NH2
N
N
3
1
2
O
O
O
O
H
N
O
Cl
g
OH
N
H
O
N
H
O
N
N
H
O
R
H
O
O
O
O
O
N
N
N
N
e
f
N
7
N
4
N
6
N
5
Scheme 1. Reagents and conditions: (a) ethyl 2-oxopropanoate, anhydrous EtOH; (b) Ethyl chloroacetate, K2CO3, reflux; (c) KOH solution, EtOH, then concentrated HCl; (d) L-
phenylalanine methyl esters, HOBT/EDCI/Et3N, anhydrous CH2Cl2; (e) KOH, EtOH, then concentrated HCl; (f) sulfuryl dichloride, anhydrous CH2Cl2; (g) t-BuOK, anhydrous
CH2Cl2, rt.

1518
Y. Li et al. / Bioorg. Med. Chem. 18 (2010) 1516–1525
Table 1
Table 1 (continued)
In vitro enzymatic assay (MMP-2 and APN) results for target compounds
Compds
R
IC50
MMP-2
(l
M)a
APN/
CD13
NAb
O
3'
4'
5'
HN 6'
O
O
*
N
2'
2
A18
A19
11520 3.3
1
H
O
N
O
R
3
S
N
1920.5 0.9 NAb
O
4
Compds
R
IC50
MMP-2
(l
M)a
APN/
O
O
CD13
O
A1
A2
58.14 3.0
30.05 1.6
*
NAb
O
O
N
LY52
5.6 0.6
NAb
N
NHOH
N
NAb
*
NH
A3
A4
10.49 2.4** NAb
O
a
Values are means standard error of three experiments. * p <0.01, ** p <0.05
compared to the control (LY52).
37.29 5.7
NAb
b
**
Not activity.
was performed on both of MMP-2 and APN so as to identify the
selectivity of target compounds. And else, LY52 was used as the po-
sitive control.
A5
6460.8 1.3 NAb
The enzyme inhibitory results unveiled that all of the tested
quinoxalinone peptidomimetic derivatives (A1–A19) displayed
high selectivity against MMP-2 as compared with APN, thus, to a
certain extent, validating our strategy for designing MMP-2 inhib-
itors. This possibly descript from the differences between the
structures of two enzymes, leading to different requirements for
their respective inhibitors. MMP-2 is a zinc-dependent endopepti-
dase that could cut the peptide to parts from the specific amino
acid residue of the peptide, while APN is a membrane-bound zinc
exopeptidase that catalyzed the removal of NH2-terminal amino
acid from the peptide.1,6 As the above mentioned selectivity
against APN, the following SARs were mainly discussed about
MMP-2 inhibition.
A6
A7
A8
124.32 2.4 NAb
624.48 1.1 NAb
F
Cl
Br
F
2000 7.3
NAb
A9
24.18 6.6** NAb
F
Cl
F
Among these inhibitors, the R groups can be altered from aro-
matic to aliphatic. Generally speaking, compounds with aromatic
side chains (A1–11) were found to be more potent than those ali-
phatic derivatives (A12–19). The possible reason might be due to
*
A10
A11
26.17 1.6
NAb
Cl
Cl
1362.8 2.4 NAb
the
p system of the aromatic ring enhancing the binding interac-
tion with the hydrophobic region of the enzyme.
The activity of A2 was better than A1, which confirmed that
pyridinyl ring is better than phenyl group. This result might be ow-
ing to the contribution of electronegative nitrogen atom, which can
more easily form hydrogen bond with the enzyme backbone, thus
stabilizing the binding between the compound and the enzyme.
Substitution on aromatic ring (A3–11) also has impact on bioac-
tivity. For instance, comparing compounds with mono-substitution
of methyl group at different positions in the aromatic ring (A3–5),
methyl-substitution at the para-position (A3, IC50 = 10.49
NAb
NAb
**
A12
A13
56.6 4.5
106 1.2
A14
A15
105.3 7.8
NAb
9426.5 3.6 NAb
2.4
l
M) displayed the highest affinity, methyl-substitution at the
M) was in the next place,
O
O
ortho-position (A4, IC50 = 37.29 5.7
l
while the meta-one (A5) presented the least activity. Additionally,
comparing compounds with halogen-substitution at the para-posi-
tion in the aromatic ring (A6–8), 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.
A16
A17
1750 2.6
NAb
O
2757.8 1.8 NAb
O

Y. Li et al. / Bioorg. Med. Chem. 18 (2010) 1516–1525
1519
140
120
100
80
*
*
A3
A10
LY52
60
40
*
**
20
*
*
0
Cell
SKOV3
K562
HL-60
Figure 2. Effects of LY52 and compounds A3 and A10 on proliferation of the SKOV3,
K562 and HL-60 cell lines. Data are expressed as mean values of five independent
experiments ( SE). * p <0.01, ** p <0.05 compared to the control (LY52).
Figure 4. FlexX docking result of compound A3 with MMP-2.
As compared with the halogen-substituted compounds, di-sub-
stitution of fluorine or chlorine at the 3-, 4-position of the aromatic
moiety (A9 and A10) manifested improved potencies in compari-
son with the mono-substitution counterparts (A6 and A7). How-
ever, when the same position of the phenyl moiety was replaced
with fluorine and chlorine (A11), severely impaired affinity was
observed.
As to the compounds with aliphatic substituents (A12–19), the
length of the side chain will partly influence the activities of the
compounds. Specifically, to some extent, length of R groups was
negatively relative with the MMP-2 inhibition, suggesting it is
unfavorable to increase the length of substituents along the orien-
tation of the R groups. This might be due to the longer group was
unfavorable to the accommodation with the enzymes’ hydrophobic
domains.
Compound A16 was found to be more potent than A15. This
activity difference was probably caused by the carboxylate
(COOCH3). As a ZBG and potential prodrug, the carboxylate and
its corresponding metabolite carboxylic acid (COOH) can chelate
with zinc at the catalytic center of the enzyme, resulting in signif-
icantly enzymatic inhibition. Furthermore, as compared with the
activities of A17 and A19, the introduction of methylthio (SCH3)
produced enhanced potency, suggesting that methylthio group
was positively related with the inhibitory activity by forming effec-
tive hydrogen bond with the residue of the enzyme.
Finally, the effects of A3 and A10 on the proliferation of three
tumor cell lines (SKOV3, K562 and HL-60), compared with LY52,
were further assessed by using MTT assay, which are shown in Fig-
ure 2. In our assay, tumor cell lines were selected according to their
expression of MMP-2 and APN, and, the concentrations inducing a
50% inhibition of cell growth (IC50) in mM are reported. Among
these three cell lines, HL-60 (myelo-monocytic human acute gran-
ulocytic leukemia cells having high basal APN activity)25 and K562
cells (human chronic myelogenous leukemic cells expressing low
levels of APN)26 were selected to verify the APN inhibitory activi-
ties of target compounds, while SKOV3 cells (human ovarian carci-
noma cells expressing high levels of MMP-2)27 was selected to
investigate the MMP-2 inhibitory activity. The results indicated
that the anti-proliferative effects of these two compounds (A3
and A10) against HL-60 and K562 cells (data were not given) were
significantly lower than the positive control. This result is consis-
tent with the results of enzyme inhibition. As to the SKOV3 cells,
although these two compounds exhibited improved inhibitory ef-
fects on cell proliferation with IC50 values of 1.98 0.08 mM and
3.02 0.12 mM, respectively, they still gave less potency than
LY52 (0.21 0.09 mM).
From the above SAR studies, it can be concluded that although
some compounds, for example, A3, A9, A10, gave comparable
MMP-2 inhibitory activities with LY52, all of the tested compounds
displayed weaker potency in comparison with the positive control.
Thereby, further structural optimization should be attentively
investigated towards the reported compounds.
3.2. Binding mode
Compounds A3 and A10 were selected to study their binding
mode with MMP-2. Firstly, their predicted conformations were
optimized with the Powell Energetic Gradient method built in
the Sketch/Build Edit model (SYBYL6.91, Linux 7.3).28 As compared
with the positive control LY52, the comparable results demon-
strated the affinity to the same spatial regions (S01 and S20 ), implying
the similar MMP-2 inhibitory activities. In precise words, both the
caffeinoyl portion of LY52 and the phenyl group of target com-
pounds can adjust their flexibility to extend into the S01 pocket with
their preponderant conformations, as diagrammed in Figure 3. In
contrast, the similar situation can be seen between the quinoxali-
none fragments of A3 and A10 and the pyridinyl-substituted amide
portion of LY52.
To further understand the inhibitory difference between the
MMP-2 and APN, the preferred pharmacophore docking studies
were carried out via the FlexX flexible-Dock program. The interac-
tions of A3 and A10 with the active sites of MMP-2 (PDB ID:
1HOV)29 and APN (PDB ID: 2DQM) were compared, which are
shown in Figures 4–7. Not surprisingly, both the two compounds
Figure 3. Comparison of preponderant conformations between compounds LY52, A10 and A3.

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Y. Li et al. / Bioorg. Med. Chem. 18 (2010) 1516–1525
could form hydrophobic contacts with His85 and Ala86 of S01 pock-
et and form hydrogen bond with Ala86 (<2.74 Å) by the carbonyl of
quinoxalinone group. The oxygen atom (O15) of carbonyl group
coordinated with the zinc ion of MMP-2 with a distance of
1.90 Å. As far as the His120 residue is concerned, the imidazolyl
of it can interact with the carbonyl group of A10 by hydrogen inter-
action (<3.35 Å) which might be benefit to stabilize of interaction
intermediate with the zinc ion. In addition, the binding interac-
tions were further enhanced by hydrogen bonds with Leu83
(<2.89 Å). Besides, the hydrophobic parts of aromatic rings are in
contact with nonpolar surface areas of MMP-2.
It should be indicated that although the computed information
assay totally supported our assumption, the exact binding model of
the target compounds with MMP-2 should be validated from fur-
ther X-ray crystal studies.
Figure 5. FlexX docking result of compound A10 with MMP-2.
4. Conclusions
In summary, we have developed a new series of quinoxalinone
peptidomimetic derivatives as potential MMP-2 inhibitors. It
should be noted that although all of these compounds display less
activity than that of LY52, they possess significant selectivity
against MMP-2 as compared with APN. This feature may provide
us a critical point for further chemical modification on these qui-
noxalinone derivatives so as to exploit more potent MMP-2
inhibitors.
5. Experimental
5.1. General procedures
Unless specified otherwise, all the starting materials, reagents
and solvents were commercially available. All reactions except
those in aqueous media were carried out by standard techniques
for the exclusion of moisture. All reactions were monitored by
Figure 6. FlexX docking result of compound A3 with APN.
thin-layer chromatography on 0.25-mm silica gel plates (60GF254
)
and visualized with UV light (254 nm), or iodine vapor. Melting
points were determined using X-6 digital display binocular micro-
scope (uncorrected). 1H NMR spectra were determined on a Brucker
Avance DRX-600 spectrometer using TMS as an internal standard, d
in parts per million and J in hertz (Hz). Infrared spectra were mea-
sured on a nicolet nexus 470 FT-IR spectrometer using KBr plate.
Electrospray ionization mass spectrometry (ESI-MS) was per-
formed on an API-4000 triple-stage quadrupole instrument. Mea-
surements were made in D2O or CD3OD solutions. Elemental
analyses were carried out on a Perkin–Elmer C, H, N elemental
analyzer.
5.2. Syntheses
5.2.1. 3-Methylquinoxalin-2(1H)-one (1)
Figure 7. FlexX docking result of compound A10 with APN.
1,2-Phenylenediamine (10.81 g, 0.10 mol) and ethyl pyruvate
(12.77 g, 0.11 mol) were dissolved in 250 ml of anhydrous EtOH
and the mixture was stirred for 24 h at room temperature. The
crude product spontaneously precipitated in the progress which
was collected and recrystallized from anhydrous EtOH to give com-
pound 1 (14.85 g, 92.7%) as yellow needles. Mp 241–243 °C; ESI-
MS: 161.0 [M+H]+.
could not well-orienting occupy the active domain (S01 and S20 pock-
ets) of APN, resulting in sharply impaired APN inhibition (even has
no APN inhibition), consistent with the enzymatic assay results.
Unlike the binding situations with APN, the moderate bulk of the
quinoxalinone fragments as well as the phenyl groups of the two
compounds could undergo torsional motion to allow their suitable
conformations to accommodate the S01 and S20 pockets, respectively.
Besides, the oxygen atom of carbonyl group (O@C2’) coordinate
with the catalytic zinc ion 166.
5.2.2. Ethyl 2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)acetate (2)
A mixture of compound 1 (8.01 g, 0.05 mol), ethyl chloroacetate
(7.353 g, 0.06 mol), anhydrous potassium carbonate (8.29 g,
0.06 mol) and tetrabutyl ammonium bromide (1.61 g, 0.005 mol)
was dissolved in 200 ml of acetone, the mixture was successively
refluxed for 6 h. The obtained product was crystallized from etha-
Finally, to obtain further insight into the interaction of A10 with
MMP-2, a 2-D picture was also created with the program LIGPLOT.
As graphically shown in Figure 8, we can see the backbone of A10

Y. Li et al. / Bioorg. Med. Chem. 18 (2010) 1516–1525
1521
Figure 8. The docking result of A10 with MMP-2 shown by LIGPLOT. Compound A10 is diagrammed in violet.
nol to give compound 2 as yellow powder (9.013 g, 73.2%). Mp
120–122 °C; 1H NMR (CDCl3-d6, ppm): d 1.18 (3H, s), 2.62 (3H, s),
4.01 (2H, q), 5.05 (2H, s), 7.05–7.86 (4H, m), ESI-MS: 247.1 [M+H]+.
(12.63 g, 0.125 mol) were added successively. The resulting mix-
ture was stirred for 24 h at room temperature. After the reaction
complete monitored by TLC, the mixture was then concentrated
and extracted with EtOAc (3 ꢀ 100 ml). Layers were separated
and the organic layer was washed in turn with 1 M HCl
(2 ꢀ 200 ml) and brine (2 ꢀ 200 ml), dried over anhydrous Na2SO4,
filtered, and evaporated in vacuo to give compound 4 as white so-
lid (13.7 g, 72.3%). Mp 209–210 °C, 1H NMR (CDCl3-d6, ppm): d 2.58
(3H, s), 2.97 (1H, dd, J = 6.9, 14.1 Hz), 3.12 (1H, dd, J = 5.7, 14.1 Hz),
3.72(3H, s), 4.66 (1H, d, J = 15.0 Hz), 4.83 (1H, m), 4.99 (1H, d,
J = 15.0 Hz), 6.74(1H, br), 6.83–7.84 (9H, m); ESI-MS m/z: 380.5
[M+H]+.
5.2.3. 2-(3-Methyl-2-oxoquinoxalin-1(2H)-yl)acetic acid (3)
Compound 2 (4.93 g, 0.02 mol) was suspended in a solution of
1 M KOH/EtOH (80 ml), the suspension was then stirred at room
temperature for 6 h. Subsequently, the mixture was allowed to
concentrate in vacuo until the pale solid appeared which was acid-
ified to pH 4.0. The obtained precipitated product was collected
and crystallized from THF to give compound 3 (3.81 g, 87.3%) as
yellow solid. Mp 225–227 °C, ESI-MS: 218.1 [M+H]+.
5.2.5. (R)-2-(2-(3-Methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenylpropanoic (5)
5.2.4. (R)-Methyl2-(2-(3-methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenylpropanoate (4)
Compound 4 (7.59 g, 0.02 mol) was mixed with a solution of
1 M KOH/EtOH (100 ml) and stirred in the room temperature for
6 h. The mixture was then concentrated in vacuo to provide the
pale solid which was acidified to pH 4.0. The obtained precipitated
Compound 3 (10.91 g, 0.05 mol) and HOBT (8.11 g, 0.06 mol)
were suspended in 120 ml of anhydrous DCM. After stirred in the
room temperature for 20 min,
L-phenylalanine methyl ester hydro-
chloride (11.86 g, 0.055 mol), EDC (11.50 g, 0.06 mol), and Et3N

1522
Y. Li et al. / Bioorg. Med. Chem. 18 (2010) 1516–1525
product was then collected and crystallized from EtOH to give
compound 5 (6.23 g, 85.2%) as white solid. Mp 246–248 °C, ESI-
MS: 366.3 [M+H]+.
5.2.11. (S)-2-(2-(3-Methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenyl-N-o-tolylpropan-amide (A4)
Yellow solid, yield: 66.2%, mp 239–242 °C, ½a D25
¼ þ23:431 (c 1,
ꢁ
DMSO), ESI-MS 454.7 [M+H]+, 1H NMR (DMSO-d6, ppm): d 2.07 (s,
3H, CH3), 2.45 (s, 3H, CH3), 2.94 (dd, J = 9.6, 13.8 Hz, 1H, CH), 3.15
(dd, J = 5.4, 13.8 Hz, 1H, CH), 4.79 (m, 2H, NCH2CO), 5.05 (d,
J = 16.8 Hz, 1H, CHCO), 6.96 (d, J = 8.4 Hz, 1H, ArH), 7.08–7.42 (m,
11H, ArH), 7.74 (1H, d, J = 7.8 Hz, ArH), 8.88 (d, J = 8.4 Hz, 1H,
ArH), 9.513 (s, 1H, CONH). Anal. Calcd for C27H26N4O3: C, 71.35;
H, 5.77; N, 12.33. Found: C, 71.27; H, 5.69; N, 12.42.
5.2.6. (R)-2-(2-(3-Methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenylpropanoyl (6)
Compound 5 (0.73 g, 2 mmol) was dissolved in a mixed solution
of SOCl2 (40 ml) and DMF (1 ml), and stirred in the room temper-
ature for 5 h. The resulting solution was rotary evaporated to get
compound 6 as pale yellow crystal (0.68 g, 88.7%), which was used
immediately in the next reaction without further purification.
5.2.12. (S)-2-(2-(3-Methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenyl-N-m-tolylpropan- amide (A5)
5.2.7. General procedures for the synthesis of compounds A1–
A11
Yellow solid, yield: 55.1%, mp 257–258 °C, ½a D25
¼ þ9:481 (c 1,
ꢁ
DMSO), ESI-MS 455.1 [M+H]+, 1H NMR (DMSO-d6, ppm): d 2.282
(3H, s, CH3), 2.443 (s, 3H, CH3), 2.891 (dd, J = 9.6, 13.2 Hz, 1H,
CH), 3.098 (dd, J = 4.8, 13.8 Hz, 1H, CH), 4.682–4.720 (m, 1H, CH),
4.794 (d, J = 16.8 Hz, 1H, NCHCO), 4.011 (d, J = 16.8 Hz, 1H,
NCHCO), 6.889 (d, J = 7.2 Hz, 1H, ArH), 6.968 (d, J = 8.4 Hz, 1H,
ArH), 7.181–7.431 (m, 10H, ArH), 7.731–7.746 (m, 1H, ArH),
8.853 (d, J = 8.4 Hz, 1H, CONH), 10.034 (s, 1H, CONH). Anal. Calcd
for C27H26N4O3: C, 71.35; H, 5.77; N, 12.33. Found: C, 71.24; H,
5.65; N, 12.41.
o-Toluidine (0.11 g, 1 mmol) was dissolved in anhydrous DCM
(20 ml), t-BuOK (0.11 g, 1.02 mmol) was then added dropwise.
After stirred at room temperature for approximately 20 min, com-
pound 6 (0.38 g, 1 mmol) was added. The mixture was stirred for
another 24 h at room temperature. After the reaction was com-
pleted monitoring by TLC, the mixture was then concentrated in
vacuo and extracted with EtOAc (3 ꢀ 50 ml). Layers were separated
and the organic layer was washed successively with 1 M HCl
(2 ꢀ 50 ml) and brine (2 ꢀ 50 ml), dried over anhydrous Na2SO4,
filtered, and the crude compounds A1–A11 were purified by flash
chromatography (PE:EA = 20:1–1:1) to provide the pure target
products.
5.2.13. (S)-N-(4-Fluorophenyl)-2-(3-methyl-2-oxoquinoxalin-
1(2H)-yl)acetamido)-3-phenyl-propanamide (A6)
Yellow solid, yield: 55.2%, mp 248–250 °C, ½a D25
¼ þ32:325 (c 1,
ꢁ
Compounds A1–A11 were prepared following the procedures
described above.
DMSO), ESI-MS 459.3 [M+H]+, 1H NMR (DMSO-d6, ppm): d 2.441 (s,
3H, CH3), 2.878–2.911 (m, 1H, CH2), 3.058–3.009 (m, 1H, CH2),
4.693 (m, 1H, CH), 4.805 (d, J = 16.8 Hz, 1H, NCHCO), 5.002 (d,
J = 16.8 Hz, 1H, NCHCO), 6.970 (d, J = 8.4 Hz, 1H, ArH), 7.241–
7.554 (m, 11H, ArH), 7.726 (d, J = 8.4 Hz, 1H, ArH), 8.798 (d,
J = 7.8 Hz, 1H, CONH), 10.269 (s, 1H, CONH). Anal. Calcd for
C26H23FN4O3: C, 68.11; H, 5.06; N, 12.22. Found: C, 68.04; H,
4.97; N, 12.31.
5.2.8. (S)-2-(2-(3-Methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-N,3-diphenylpropanamide (A1)
Yellow solid, yield: 65.4%. mp 259–261 °C, ½a D25
¼ þ14:234 (c 1,
ꢁ
DMSO), ESI-MS 440.7 [M+H]+, 1H NMR (DMSO-d6, ppm): d 2.441 (s,
3H, CH3), 2.883–2.921 (m, 1H, CH2), 3.089–3.120 (m, 1H, CH2),
4.699–4.737 (m, 1H, CHCO), 4.799 (d, J = 16.4 Hz, 1H, NCHCO),
5.006 (d, J = 16.2 Hz, 1H, NCHCO), 6.967 (d, J = 9 Hz, 1H, ArH),
7.068 (t, 1H, ArH), 7.244–7.331 (m, 8H, ArH), 7.405–7.430 (m,
1H, ArH), 7.582 (d, J = 7.8 Hz, 1H, ArH), 7.738 (dd, J = 1.2, 7.8 Hz,
1H, ArH), 8.876 (d, J = 8.4 Hz, 1H, CONH), 10.132 (s, 1H, CONH).
Anal. Calcd for C26H24N4O3: C, 70.89; H, 5.49; N, 12.72. Found: C,
70.81; H, 5.42; N, 12.85.
5.2.14. (S)-N-(4-Chlorophenyl)-2-(3-methyl-2-oxoquinoxalin-
1(2H)-yl)acetamido)-3-phenyl- propanamide (A7)
Red powder, yield: 39.8%, mp 285–287 °C, ½a D25
¼ ꢂ15:268 (c
ꢁ
1, DMSO), ESI-MS 475.8 [M+H]+, 1H NMR (DMSO-d6, ppm): d
2.440 (s, 3H, CH3), 2.902 (dd, J = 9.6, 13.2 Hz, 1H, CH2), 3.095
(dd, J = 5.4, 13.8 Hz, 1H, CH2), 4.675–4.714 (m, 1H, CH), 4.802
(d, J = 16.8 Hz, 1H, NCH2CO), 5.005 (d, J = 16.8 Hz, 1H, NCH2CO),
6.971 (d, J = 8.4 Hz, 1H, ArH), 7.239–7.436 (m, 9H, ArH), 7.596–
7.616 (m, 2H, ArH), 7.737 (dd, J = 1.2, 7.8 Hz, 1H, ArH), 8.899
(d, J = 8.4 Hz, 1H, CONH), 10.270 (s, 1H, CONH). Anal. Calcd for
C26H23ClN4O3: C, 65.75; H, 4.88; N, 11.80. Found: C, 65.66; H,
4.74; N, 11.93.
5.2.9. (S)-2-(2-(3-Methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenyl-N-(pyridine-3-yl) propanamide (A2)
Pale yellow solid, yield: 34.9%, mp 48–50 °C, ½a D25
¼ þ36:134 (c
ꢁ
1, DMSO), ESI-MS 442.3 [M+H]+, 1H NMR (DMSO-d6, ppm): d 2.442
(s, 3H, CH3), 2.904–2.943 (m, 1H, CH2), 3.106–3.138 (m, 1H, CH2),
4.707–4.745 (m, 1H, CHCO), 4.813 (d, J = 16.8 Hz, 1H, NCHCO),
5.017 (d, J = 16.2 Hz, 1H, NCHCO), 6.982 (d, J = 8.4 Hz, 1H, ArH),
7.233–7.436 (m, 9H, ArH), 7.739 (d, J = 7.8 Hz, 1H, ArH), 8.009 (d,
J = 9 Hz, 1H, ArH), 8.284 (d, J = 4.8 Hz, 1H, ArH), 8.722 (s, 1H,
ArH), 8.928 (d, J = 8.4 Hz, 1H, CONH), 10.357 (s, 1H, CONH). Anal.
Calcd for C25H23N5O3: C, 68.01; H, 5.25; N, 15.86. Found: C,
67.89; H, 5.21; N, 15.97.
5.2.15. (S)-N-(4-Bromophenyl)-2-(3-methyl-2-oxoquinoxalin-
1(2H)-yl)acetamido)-3-phenyl- propanamide (A8)
Yellow solid, yield: 64.2%, mp 269–271 °C, ½a D25
¼ þ20:123 (c 1,
ꢁ
DMSO), ESI-MS 520.4 [M+H]+, 1H NMR (DMSO-d6, ppm): d 2.40 (s,
3H, CH3), 2.882–2.921 (m, 1H, CH2), 3.078–3.109 (m, 1H, CH2),
4.693 (m, 1H, CH), 4.803 (d, J = 16.8 Hz, 1H, NCHCO), 5.004 (d,
J = 16.8 Hz, 1H, NCHCO), 6.973 (d, J = 8.4 Hz, 1H, ArH), 7.249–
7.564 (m, 11H, ArH), 7.737 (d, J = 8.4 Hz, 1H, ArH), 8.898 (d,
J = 7.8 Hz, 1H, CONH), 10.267 (s, 1H, CONH). Anal. Calcd for
C26H23BrN4O3: C, 60.12; H, 4.46; N, 10.79. Found: C, 59.95; H,
4.41; N, 10.87.
5.2.10. (S)-2-(2-(3-Methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenyl-N-p-tolylpropan- amide (A3)
Yellow solid, yield: 46.9%, mp 277–278 °C, ½a D25
¼ ꢂ11:579 (c 1,
ꢁ
DMSO), ESI-MS 455.3 [M+H]+, 1H NMR (DMSO-d6, ppm): d 2.25 (s,
3H, CH3), 2.44 (s, 3H, CH3), 2.89 (dd, J = 9.6, 13.2 Hz, 1H, CH2), 3.09
(dd, J = 5.4, 13.8 Hz, 1H, CH2), 4.75 (m, 2H, NCH2CO), 5.01 (d,
J = 16.2 Hz, 1H, CH), 6.96 (d, J = 8.4 Hz, 1H, ArH), 7.12–7.47 (m,
12H, ArH), 7.74 (d, J = 7.8 Hz, 1H, ArH), 8.86 (d, J = 7.8 Hz, 1H,
ArH), 10.03 (s, 1H, CONH). Anal. Calcd for C27H26N4O3: C, 71.35;
H, 5.77; N, 12.33. Found: C, 71.28; H, 5.68; N, 12.41.
5.2.16. (S)-N-(3,4-Difluorophenyl)-2-(3-methyl-2-
oxoquinoxalin-1(2H)-yl)acetamido)-3- phenylpropanamide
(A9)
Yellow solid, yield: 44.3%, mp 271–273 °C, ½a D25
¼ ꢂ24:413 (c
ꢁ
1, DMSO), ESI-MS 476.7 [M+H]+, 1H NMR (DMSO-d6, ppm): d

Y. Li et al. / Bioorg. Med. Chem. 18 (2010) 1516–1525
1523
2.441 (s, 3H, CH3), 2.886–2.925 (m, 1H, CH2), 3.088–3.104 (m,
1H, CH2), 4.663 (s, 1H, CH), 4.805 (d, J = 17.4 Hz, 1H, NCHCO),
5.006 (d, J = 16.2 Hz, 1H, NCHCO), 6.976 (d, J = 9 Hz, 1H, ArH),
7.253–7.422 (m, 9H, ArH), 7.732–7.743 (m, 2H, ArH), 8.924 (d,
J = 7.8 Hz, 1H, CONH), 10.381 (s, 1H, CONH). Anal. Calcd for
C26H22F2N4O3: C, 65.54; H, 4.65; N, 11.76. Found: C, 65.48; H,
4.56; N, 11.83.
(t, J = 7.2 Hz, 3H, CH3), 1.179–1.241 (m, 2H, CH2), 1.313–1.362
(m, 2H, CH2), 2.440 (s, 3H, CH3), 2.764–3.095 (m, 4H, CH2),
4.465–4.503 (m, 1H, CH), 4.725 (d, J = 16.8 Hz, 1H, NCHCO), 5.02
(d, J = 16.8 Hz, 1H, NCHCO), 6.912 (d, J = 7.8 Hz, 1H, ArH), 7.237–
7.327 (m, 6H, ArH), 7.397 (t, J = 8.4 Hz, 1H, ArH), 7.733 (d,
J = 7.8 Hz, 1H, ArH), 8.000 (t, J = 5.4 Hz, 1H, CONH), 8.695 (d,
J = 9 Hz, 1H, CONH). Anal. Calcd for C24H28N4O3: C, 68.55; H,
6.71; N, 13.32. Found: C, 68.48; H, 6.68; N, 13.47.
5.2.17. (S)-N-(3,4-Dichlorophenyl)-2-(3-methyl-2-oxoquinoxalin-
1(2H)-yl)acetamido)-3- phenylpropanamide (A10)
5.2.22. (S)-N,N-Diethyl-2-(2-(3-methyl-2-oxoquinoxalin-1(2H)-
Yellow solid, yield: 44.5%, mp 251–253 °C, ½a D25
¼ ꢂ16:09 (c 1,
ꢁ
yl)acetamido)-3-phenyl- propanamide (A14)
DMSO), ESI-MS 510.1 [M+H]+, 1H NMR (DMSO-d6, ppm): d 2.443
(s, 3H, CH3), 2.911 (dd, J = 9.6, 13.8 Hz, 1H, CH), 3.1 (dd, J = 5.4,
13.8 Hz, 1H, CH), 4.669 (d, J = 4.2 Hz, 1H, CH), 4.815 (d,
J = 16.2 Hz, 1H, NCH2CO), 5.004 (d, J = 16.8 Hz, 1H, NCHCO), 6.993
(d, J = 8.4 Hz, 1H, ArH), 7.252–7.333 (m, 6H, ArH), 7.427 (t,
J = 8.4 Hz, 1H, ArH), 7.487 (dd, J = 1.8, 8.4 Hz, 1H, ArH), 7.583 (d,
J = 9 Hz, 1H, ArH), 7.74 (dd, J = 1.8, 8.4 Hz, 1H, ArH), 7.956 (d,
J = 2.4 Hz, 1H, ArH), 8.923 (d, J = 8.4 Hz, 1H, CONH), 10.406 (s, 1H,
CONH). Anal. Calcd for C26H22Cl2N4O3: C, 61.31; H, 4.35; N, 11.00.
Found: C, 61.28; H, 4.26; N, 11.17.
White solid, yield: 68.1%, mp 176–178 °C, ½a D25
¼ þ19:481 (c 1,
ꢁ
DMSO), ESI-MS 421.3 [M+H]+, 1H NMR (DMSO-d6, ppm): d 0.933–
0.975 (m, 6H, CH3), 2.438 (s, 3H, CH3), 2.835 (dd, J = 8.4, 13.8 Hz,
1H, CH), 2.966 (dd, J = 6.6, 13.2 Hz, 1H, CH), 3.097–3.379 (m, 4H,
NCH2,), 4.735 (d, J = 16.8 Hz, 1H, NCHCO), 4.811–4.850 (m, 1H,
CH), 4.996 (d, J = 17.4 Hz, 1H, NCHCO), 6.888 (d, J = 7.8 Hz, 1H,
ArH), 7.245–7.328 (m, 6H, ArH), 7.403–7.429 (m, 1H, ArH),
7.727–7.742 (m, 1H, ArH), 8.934 (d, J = 9 Hz, 1H, CONH). Anal. Calcd
for C24H28N4O3: C, 68.55; H, 6.71; N, 13.32. Found: C, 68.64; H,
6.64; N, 13.21.
5.2.18. (S)-N-(3-Chloro-4-fluorophenyl)-2-(3-methyl-2-oxoquino-
xalin-1(2H)-yl)acetamido)-3- phenylpropanamide (A11)
5.2.23. (S)-N-Isopropyl-2-(2-(3-methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenyl- propanamide (A15)
White solid, mp 251–253 °C, ½a D25
¼ þ13:481 (c 1, DMSO), ESI-
ꢁ
White solid, yield: 55.1%, mp 285–287 °C, ½a D25
¼ þ17:270 (c 1,
ꢁ
MS 493.9 [M+H]+, 1H NMR (DMSO-d6, ppm): d 2.45 (s, 3H, CH3),
2.91 (dd, J = 9.6, 13.2 Hz, 1H, CH2), 3.10 (dd, J = 5.4, 13.8 Hz, 1H,
CH2), 4.66 (m, 2H, NCH2CO), 5.00 (d, J = 16.8 Hz, 1H, CH), 6.99 (d,
J = 8.4 Hz, 1H, ArH), 7.24–7.47 (m, 9H, ArH), 7.74 (d, J = 7.8 Hz,
1H, ArH), 7.88 (dd, J = 2.4, 6.6 Hz, 1H, ArH), 8.92 (d, J = 7.8 Hz, 1H,
ArH), 10.33 (s, 1H, CONH). Anal. Calcd for C26H22ClFN4O3: C,
63.35; H, 4.50; N, 11.37. Found: C, 63.28; H, 4.41; N, 12.45.
DMSO), ESI-MS 406.8 [M+H]+, 1H NMR (DMSO-d6, ppm): d 0.973
(d, J = 6.6 Hz, 3H, CH3), 1.045 (d, J = 6.6 Hz, 3H, CH3), 2.441 (s, 3H,
CH3), 2.782 (dd, J = 9.6, 13.8 Hz, 1H, CH), 2.965 (dd, J = 5.4,
13.2 Hz, 1H, CH), 3.788–3.845 (m, 1H, CH), 4.452–4.490 (m, 1H,
CH), 4.74 (d, J = 16.8 Hz, 1H, NCHCO), 4.991 (d, J = 16.8 Hz, 1H,
NCHCO), 6.949 (d, J = 7.8 Hz, 1H, ArH), 7.228–7.431 (m, 7H,
ArH), 7.738 (dd, J = 1.2, 7.8 Hz, 1H, ArH), 7.860 (d, J = 7.8 Hz, 1H,
CONH), 8.646 (d, J = 9 Hz, 1H, CONH). Anal. Calcd for C23H26
N4O3: C, 67.96; H, 6.45; N, 13.78. Found: C, 67.87; H, 6.38; N,
13.84.
-
5.2.19. General procedures for the synthesis of compounds
A12–A19
Compound A16 (0.3 g, 0.82 mmol), HOBT (0.13 g, 0.99 mmol)
were suspended in anhydrous DCM (40 ml) and after stirred in
the room temperature for 20 min, diethylamine (0.07 g,
0.99 mmol), EDC (0.19 g, 0.99 mmol), and Et3N (0.166 g,
1.62 mmol) were added. And the mixture was stirred for 24 h at
room temperature. After the reaction complete, the mixture was
then concentrated and extracted with EtOAc (3 ꢀ 50 ml).Layers
were separated and the organic layer was washed with 1 M HCl
(2 ꢀ 50 ml) and brine (2 ꢀ 50 ml), dried over Na2SO4, filtered, and
the pure target products A16 were obtained by flash chromatogra-
phy (PE:EA = 10:1–1:1).
5.2.24. (R)-Methyl4-methyl-2-((S)-2-(2-(3-methyl-2-
oxoquinoxalin-1(2H)-yl)acetamido)- 3-phenylpropanamide
(A16)
White solid, yield: 35.6%, mp 241–242 °C, ½a D25
¼ þ32:92 (c 1,
ꢁ
DMSO), ESI-MS 492.6 [M+H]+, 1H-NMR (DMSO-d6, ppm):
d
0.8335 (d, J = 6.6 Hz, 3H, CH3), 0.882 (d, J = 6 Hz, 3H, CH3), 1.504–
1.608 (m, 3H, CH, CH2), 2.435 (s, 3H, CH3), 2.762 (dd, J = 10.8,
13.8 Hz, 2H, CH2), 3.064 (dd, J = 4.2, 14.4 Hz, 1H, NCH), 3.628 (s,
3H, CH3), 4.299–4.337 (m, 1H, CHCO), 4.587–4.625 (m, 1H, CHCO),
4.695 (d, J = 16.8 Hz, 1H, NCHCO), 4.984 (d, J = 16.8 Hz, 1H,
NCHCO), 6.829 (d, J = 8.4 Hz, 1H, ArH), 7.247–7.325 (m, 6H, ArH),
7.379 (t, J = 7.2 Hz, 1H, ArH), 7.730 (d, J = 7.8 Hz, 1H, ArH), 8.489
(d, J = 7.8 Hz, 1H, CONH), 8.712 (d, J = 9 Hz, 1H, CONH). Anal. Calcd
for C27H32N4O5: C, 65.84; H, 6.55; N, 13.37. Found: C, 65.78; H,
6.41; N, 13.43.
Compounds of A12–A19 were synthesized following the general
procedure as described above.
5.2.20. (S)-2-(2-(3-Methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenyl-N-propylpropan- amide (A12)
White solid, yield: 48.7%, mp 283.5–285 °C, ½a D25
¼ þ24:019 (c
ꢁ
1, DMSO), ESI-MS 407.4 [M+H]+, 1H NMR (DMSO-d6, ppm): d
0.790 (t, J = 7.2 Hz, 3H, CH3), 1.355–1.391 (m, 2H, CH2), 2.438 (s,
3H, CH3), 2.785 (dd, J = 9.6,13.8 Hz, 1H, CH), 2.969–3.059 (m, 3H,
CH2, CH), 4.469–4.507 (m, 1H, CH), 4.721 (d, J = 16.8 Hz, 1H,
NCHCO), 5.023 (d, J = 16.8 Hz, 1H, NCHCO), 6.904 (d, J = 8.4 Hz,
1H, ArH), 7.230–7.327 (m, 6H, ArH), 7.732–7.410 (m, 1H, ArH),
7.724–7.740 (m, 1H, ArH), 8.022 (t, J = 6 Hz, 1H, CONH), 8.693 (d,
J = 9 Hz, 1H, CONH). Anal. Calcd for C23H26N4O3: C, 67.96; H,
6.45; N, 13.78. Found: C, 67.89; H, 6.38; N, 13.85.
5.2.25. (S)-Methyl 4-(2-(2-(3-methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenylpropan- amido)butanoate (A17)
Yellow solid, yield: 64.5%, mp 213–215 °C, ½a D25
¼ ꢂ21:27 (c 1,
ꢁ
DMSO), ESI-MS 465.1 [M+H]+, 1H NMR (DMSO-d6, ppm): d 1.194
(t, J = 14.4 Hz, 2H, CH2), 1.612–1.624 (m, 2H, CH2), 2.224–2.250
(m, 2H, CH2N), 2.439 (s, 3H, CH3), 2.775–2.813 (m, 1H, CH2),
3.295–3.047 (m, 1H, CH2), 3.585 (s, 1H, CH3O), 4.458–4.466 (m,
1H, CH), 4.745 (d, J = 16.8 Hz, 1H, NCHCO), 5.026 (d, J = 16.8 Hz,
1H, NCHCO), 6.936 (d, J = 8.4 Hz, 1H, ArH), 7.225–7.327 (m, 6H,
ArH), 7.401 (t, J = 8.4 Hz, 1H, ArH), 7.734 (d, J = 7.8 Hz, 1H, ArH),
8.104 (t, J = 5.4 Hz, 1H, CONH), 8.745 (d, J = 8.4 Hz, 1H, CONH).
Anal. Calcd for C25H28N4O5: C, 64.64; H, 6.08; N, 12.06. Found: C,
64.52; H, 5.98; N, 12.14.
5.2.21. (S)-N-Butyl-2-(2-(3-methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenylpropan-amide (A13)
White solid, yield: 58.9%, mp 267–269 °C, ½a D25
¼ þ26:481 (c 1,
ꢁ
DMSO), ESI-MS 420.8 [M+H]+, 1H NMR (DMSO-d6, ppm): d 0.843

1524
Y. Li et al. / Bioorg. Med. Chem. 18 (2010) 1516–1525
5.2.26. (S)-Methyl 6-(2-(2-(3-methyl-2-oxoquinoxalin-1(2H)-
DMSO and mixing for 15 min. The optical density was read with
ELISA reader at 570 nm.
yl)acetamido)-3-phenylpropan- amido)hexanoate (A18)
Yellow solid, yield: 67.1%, mp 236–239 °C, ½a D25
¼ ꢂ22:121 (c 1,
ꢁ
DMSO), ESI-MS 493.4 [M+H]+, 1H NMR (DMSO-d6, ppm): d 1.161–
1.211 (m, 2H, CH2), 1.319–1.367 (m, 2H, CH2), 1.465–1.515 (m,
2H, CH2), 2.262 (t, J = 7.8 Hz, 2H, CH2CO), 2.438 (s, 3H, CH3),
2.762–2.800 (m, 1H, CH2), 2.981–3.022 (m, 2H, CH2N), 3.040–
3.083 (m, 1H, CH2), 3.576 (s, 1H, CH3O), 4.457–4.496 (m, 1H, CH),
4.725 (d, J = 16.8 Hz, 1H, NCHCO), 5.018 (d, J = 16.8 Hz, 1H,
NCHCO), 6.912 (d, J = 8.4 Hz, 1H, ArH), 7.234–7.324 (m, 6H, ArH),
7.385–7.413 (m, 1H, ArH), 7.732 (dd, J = 1.2, 7.8 Hz, 1H, ArH),
8.104 (t, J = 5.4 Hz, 1H, CONH), 8.691 (d, J = 9 Hz, 1H, CONH). Anal.
Calcd for C27H32N4O5: C, 65.84; H, 6.55; N, 11.37. Found: C, 65.72;
H, 6.48; N, 11.41.
5.5. Computational-docking assay
The docking study was performed as follows: The selected com-
pound was constructed with a Sybyl/Sketch module and optimized
using Powell Energetic Gradiet method with a Tripos force field
with the convergence criterion set at 0.05 kcal/mol Å, and assigned
with Gasteiger-Hückel method.32,33 When it comes to the docking
assay of MMP-2, residues in a radius of 4.0 Å around SC-74020 (the
provided ligand of MMP-2 in the crystal structure. PDB code:
1HOV)29 were selected as the active site, including the zinc(II)
ion. As to APN, the residues in a radius of 7.0 Å around bestatin
(the provided ligand of APN in the co-crystal structure, PDB code:
2DQM)34 were selected as the active site, and other docking
parameters implied in the program were kept default.
5.2.27. Methyl 4-((S)-2-(2-(3-methyl-2-oxoquinoxalin-1(2H)-
yl)acetamido)-3-phenylpropan- amido)-4-
(methylthio)butanoate (A19)
Red solid, yield: 51.1%, mp 208–210 °C, ½a D25
¼ þ23:900 (c 1,
ꢁ
Acknowledgements
DMSO), ESI-MS 510.9 [M+H]+, 1H NMR (DMSO-d6, ppm): d 1.827–
2.039 (m, 5H, CH3S, CH2), 2.282–2.436 (m, 5H, CH3, CH2), 2.760–
2.816 (m, 1H, CH), 2.983–3.073 (m, 1H, CH), 3.639 (s, 3H, CH3O),
4.379–4.438 (m, 1H, CH), 4.580–4.652 (m, 1H, CH), 4.703–4.758
(m, 1H, NCHCO), 4.946–5.019 (m, 1H, NCHCO), 6.82 (d, J = 8.4 Hz,
1H, ArH), 7.223–7.332 (m, 7H, ArH), 7.406 (dd, J = 8.4, 16.8 Hz,
1H, ArH), 7.725 (s, 1H, ArH), 8.531 (d, J = 9.6 Hz, 1H, CONH),
8.707 (d, J = 8.4 Hz, 1H, CONH). Anal. Calcd for C26H0N4O5S: C,
61.16; H, 5.92; N, 10.97. Found: C, 61.02; H, 5.85; N, 11.03.
This work was financial supported from the Natural Science
Foundation of China (30701053), the doctoral Foundation of Minis-
try of Education of the People’s Republic of China (20070422061),
and the Natural Science Foundation of Shandong Province
(Y2008C01).
References and notes
1. Kohrmann, A.; Kammerer, U.; Kapp, M.; Dietl, J.; Anacker, J. BMC Cancer 2009, 9,
188.
5.3. Enzymatic inhibition assay (in vitro)
2. Coussens, L. M.; Fingleton, B.; Matrisian, L. M. Science 2002, 295, 2387.
3. Pavlaki, M.; Zucker, S. Cancer Metastasis Rev. 2003, 22, 177.
4. Michaelides, M. R.; Curtin, M. L. Curr. Pharm. Des. 1999, 5, 787.
5. Nishida, Y.; Miyamori, H.; Thompson, E. W.; Takino, T.; Endo, Y.; Sato, H. Cancer
Res. 2008, 68, 9096.
6. Bjorklund, M.; Koivunen, E. Biochim. Biophys. Acta 2005, 1755, 37.
7. Lovejoy, B.; Welch, A. R.; Carr, S.; Luong, C.; Broka, C.; Hendricks, R. T.;
Campbell, J. A.; Walker, K. A.; Martin, R.; Van Wart, H.; Browner, M. F. Nat.
Struct. Biol. 1999, 6, 217.
8. Verma, R. P.; Hansch, C. Bioorg. Med. Chem. 2007, 15, 2223.
9. Becker, J. W.; Marcy, A. I.; Rokosz, L. L.; Axel, M. G.; Burbaum, J. J.; Fitzgerald, P.
M. D.; Cameron, P. M.; Esser, C. K.; Hagmann, W. K.; Hermes, J. D.; Springer, J. P.
Protein Sci. 1995, 4, 1966.
5.3.1. MMP-2 inhibition assay
Gelatinase A (MMP-2) assay was performed as described by
Baragi et al.30 The gelatinase, substrate (succinylated gelatin) and
inhibitor were dissolved in sodium borate buffer (pH 8.5, 50 mM)
and incubated for 30 min at 37 °C, and then 0.03% trinitrobenzene-
sulfonic acid (TNBS, Sigma) was added and incubated for another
20 min, the resulting solution was detected under 450 nm wave-
length to gain absorption.
10. Kontogiorgis, C. A.; Papaioannou, P.; Hadjipavlou-Litina, D. J. Curr. Med. Chem.
2005, 12, 339.
11. Cheng, X. C.; Wang, Q.; Fang, H.; Tang, W.; Xu, W. F. Bioorg. Med. Chem. 2008,
16, 7932.
12. Cheng, X. C.; Wang, Q.; Fang, H.; Tang, W.; Xu, W. F. Bioorg. Med. Chem. 2008,
16, 5398.
13. Cheng, X. C.; Wang, Q.; Fang, H.; Tang, W.; Xu, W. F. Eur. J. Med. Chem. 2008, 43,
2130.
5.3.2. APN inhibition assay
The IC50 values against APN were determined by 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 a UV–vis spectrophotometer, Pharmacia LKB, Bio-
chrom 4060. All the solutions of inhibitors were prepared in the as-
say buffer, and the pH was adjusted to 7.5 by the addition of 0.1 M
HCl or 0.1 M NaOH. All the inhibitors were preincubated with APN
for 30 min at room temperature. The assay mixture, which con-
tained the inhibitor solution (concentration dependent on the
14. Li, X.; Li, Y.; Xu, W. Bioorg. Med. Chem. 2006, 14, 1287.
15. Zhang, L.; Zhang, J.; Fang, H.; Wang, Q.; Xu, W. Bioorg. Med. Chem. 2006, 14,
8286.
16. Li, Y. L.; Xu, W. F. Bioorg. Med. Chem. 2004, 12, 5171.
17. Qu, X. J.; Yuan, Y. X.; Tian, Z. G.; Xu, W. F.; Chen, M. H.; Cui, S. X.; Guo, Q.; Gai,
R.; Makuuchi, M.; Nakata, M.; Tang, W. Int. J. Mol. Med. 2006, 18, 609.
18. Qu, X.; Yuan, Y.; Xu, W.; Chen, M.; Cui, S.; Meng, H.; Li, Y.; Makuuchi, M.;
Nakata, M.; Tang, W. Anticancer Res. 2006, 26, 3573.
19. Li, X.; Yang, K. H.; Li, W. L.; Xu, W. F. Drug Future 2006, 31, 979.
20. Nemoto, H.; Cai, J.; Asao, N.; Iwamoto, S.; Yamamoto, Y. J. Med. Chem. 1995, 38,
1673.
inhibitor), the enzyme solution (4
the assay buffer, was adjusted to 200
l
g/ml final concentration), and
l.
l
21. Otani, T. T.; Briley, M. R. J. Pharm. Sci. 1985, 74, 40.
22. Li, X.; Wang, D. H.; Wu, J. F.; Xu, W. F. Synth. Commun. 2005, 35, 1.
23. Terauchi, M.; Kajiyama, H.; Shibata, K.; Ino, K.; Nawa, A.; Mizutani, S.; Kikkawa,
F. BMC Cancer 2007, 7, 140.
5.4. MTT assay
The cell lines were grown in RPMI1640 medium containing 10%
FBS at 37 °C in a humidified incubator with 5% CO2. Cell prolifera-
tion was determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-
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 lg/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
24. Pasqualini, R.; Koivunen, E.; Kain, R.; Lahdenranta, J.; Sakamoto, M.;
Stryhn, A.; Ashmun, R. A.; Shapiro, L. H.; Arap, W.; Ruoslahti, E. Cancer Res.
2000, 60, 722.
ˇ
´
25. Gabrilovac, J.; Breljak, D.; Cupic, B. Int. Immunopharmacol. 2008, 8, 613.
26. Mishima, Y.; Matsumoto-Mishima, Y.; Terui, Y.; Katsuyama, M.; Yamada, M.;
Mori, M.; Ishizaka, Y.; Ikeda, K.; Watanabe, J.; Mizunuma, N.; Hayasawa, H.;
Hatake, K. J. Natl. Cancer Inst. 2002, 94, 1020.
27. Yuan, Y. X.; Xu, W. F.; Liu, J.; Chen, M. H.; Meng, H.; Qu, X. J. Ai zheng 2006, 25,
663.
28. SYBYL6.91. Tripos Associates. 1699 S. Hanley Road, Suite 303. St. Louis, MO
63144–2913, 2003.

Y. Li et al. / Bioorg. Med. Chem. 18 (2010) 1516–1525
1525
29. Feng, Y.; Likos, J. J.; Zhu, L.; Woodward, H.; Munie, G.; McDonald, J. J.; Stevens,
A. M.; Howard, C. P.; De Crescenzo, G. A.; Welsch, D.; Shieh, H. S.; Stallings, W.
C. Biochim. Biophys. Acta 2002, 1598, 10.
30. Baragi, V. M.; Shaw, B. J.; Renkiewicz, R. R.; Kuipers, P. J.; Welgus, H. G.;
Mathrubutham, M.; Cohen, J. R.; Rao, S. K. Matrix Biol. 2000, 19, 267.
31. Lejczak, B.; Kafarski, P.; Zygmunt, J. Biochemistry 1989, 28, 3549.
32. GALAHADTM is distributed by Tripos Inc., 1699 South Hanley Rd., St. Louis,
Missouri, USA (http://www.tripos.com).
33. Richmond, N. J.; Abrams, C. A.; Wolohan, P. R.; Abrahamian, E.; Willett, P.;
Clark, R. D. J. Comput. Aided Mol. Des. 2006, 20, 567.
34. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.