L. Su et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2512–2517
2515
on APN activity, B16BL6 murine melanoma cell,11 Lewis lung carci-
noma cell and SN12P human renal cancer cell invasion.12 Besides,
bestatin can block human umbilical vascular endothelial cell (HU-
VEC) tubular structure formation and B16BL6 induced angiogene-
sis in C57BL/6 mice.13 Furthermore, there are also similar reports
on other natural occurring APN inhibitors such as AHPA-Val,
amastatin, phebestin and lapstatin, etc.14 But those natural prod-
ucts are difficult to be synthesized, because most of them have
more than one chiral centers, such as (2S,3R)-3-amino-2-hydro-
xy-4-phenylbutanic acid (AHPA) subunit in bestatin, probestin,
and AHPA-Val, etc. Several total synthesis methods of bestatin
have been reported, but the yields are far from satisfaction.15 There
are also various synthetic inhibitors reported, such as AHPA deriv-
atives or analogs, organophosphonates, flavones, and hydroxamic
acids, but only their APN inhibitory activity were evaluated.16 In
addition, bestatin is moderately potent. Therefore, more potent
and easy-obtained synthetic APN inhibitors should be developed,
and more profound studies on their anti-invasion and anti-angio-
genesis effects are needed.
two assays were performed on Matrigel, because the cytokines re-
leased from Matrigel could promote endothelial cell proliferation,
and the vascular tubule formation process involved the invasion
of endothelial cells into Matrigel. Therefore, by inhibiting APN
activity, LB-4b should exhibit anti-angiogenesis effects. As shown
in Figure 4, we can see notable new microvessel growth in 6 days,
and it could be significantly suppressed by LB-4b, much more obvi-
ously than bestatin (P <0.01). As shown in Figure 5, HUVECs formed
tubular structure within 8 h, and both of bestatin and LB-4b could
block this phenomenon obviously. During the assay time, no acute
cytotoxicity of the samples treated with bestatin or LB-4b was ob-
served under microscope.
LB-4b as an APN inhibitor with significantly more potent anti-
invasion and anti-angiogenesis effects than bestatin, can be easily
obtained by the synthetic method shown in Figure 6. With benzyl-
amine as the start material, it was transformed into benzyl isocya-
nate with triphosgene, and then coupled with L-leucine methyl
ester to form the ureido group. Finally, the methyl ester was trans-
formed into hydroxamic acid as the zinc binding group (ZBG).
Compared with bestatin, the synthetic method of LB-4b is much
easier, and the intermediates could be used in next steps without
further purification, so the total yield is high (78%).
In our previous work, a series of L-lysine ureido derivatives were
designed, synthesized and preliminarily evaluated for their APN
inhibitory activity and anticancer potency.17 Among them, several
compounds, such as 1, showed moderate potency toward APN
(Fig. 1). In order to improve the potency, we made some modifica-
In order to compare the binding mode of LB-4b with bestatin to
Escherichia coli APN (PDB ID: 2DQM), molecular docking was
adopted. As shown in Figure 7, LB-4b chelated the zinc ion with
its hydroxamate and ureido carbonyl groups. Similar to bestatin,
LB-4b formed hydrogen bonds with Glu 264 and His 301 with
the distance of 2.66 and 2.98 Å (for bestatin 2.63 and 2.73 Å), and
formed hydrophobic interaction with Met 260, Gly 261, Met 263
Tyr 376 and Tyr 381. Figure 7B and C also showed that LB-4b could
also form hydrogen bonds between its hydroxyl group of and His
297 of APN, as well as its phenolic hydroxyl hydrogen of Tyr 381,
with the distance of 3.27 and 3.03 Å, respectively. We can also
see LB-4b had hydrophobic interaction with Met 263, Tyr 376,
Met 260 and Glu 261 residues of the target.
APN inhibitors have been considered as potential anticancer
agents for decades. Natural occurring APN inhibitors are limited
in development and application because of their rare sources and
synthetic difficulty. Hydroxamic acids as important synthetic
zinc-dependent metalloproteinase inhibitors have been developed
as anticancer candidates for years, some of which have been on the
market, such as Zolinza. LB-4b as a potent APN inhibitor, could be
easily synthesized, and showed more significant anti-invasion and
anti-angiogenesis effects than bestatin. Such outcomes prompt our
continued efforts toward the studies of developing synthetic APN
inhibitors as anticancer agents and revealing their potential mech-
anism of action.
tion, and discovered
2 (LB-4b) with approximately ten-fold
improvement compared to 1 (Table 1). And 2 displayed quite high
selectivity to APN over MMP-2 at the assay concentration. In our
efforts to investigate the anti-invasion effect of this synthetic
APN inhibitor, we selected ES-2 human ovarian clear cell carci-
noma cell line, an advanced matastatic cancer, with high APN
expression. Then the inhibitory activity of LB-4b towards APN on
ES-2 cell surface was tested, and found it 10-fold more potent than
bestatin as the positive control. Besides, it showed very weak influ-
ence on ES-2 cell survival (Table 1).
Cancer cell invasion includes ECM degradation and cell migra-
tion, etc. So both of ES-2 cell migration and invasion were assayed.
Transwell chambers with 8 lm micropores were used in cell
migration assay, on which cancer cells cross through the mem-
brane with amoeboid movement. From Figure 2 we can see that
both of bestatin and LB-4b did not exhibit obvious inhibitory ef-
fects on ES-2 cell migration. Transwell chambers coating with
Matrigel were used in cell invasion assay. As an advanced meta-
static cancer cell line, ES-2 could freely invade Matrigel and mi-
grate to the other side of the chamber within 8 h, but it can be
significantly inhibited by both of bestatin and LB-4b with a dose-
dependent tendency, and LB-4b was more potent than bestatin
(P <0.01) (Fig. 3). As the results shown above, LB-4b could inhibit
APN activity on ES-2 cells and block ES-2 cell invasion without
obvious influence on MMP-2 inhibition, ES-2 cell survival and
migration.
Acknowledgments
Furthermore, we also investegated the anti-angiogenesis effects
of LB-4b with bestatin as the positive control. Rat aortic ring micro-
vessel growth and HUVEC tubular structure formation were the
most commonly used angiogenesis models in vitro. Both of the
This work was supported by National Natural Science Founda-
tion Research of China (Grant No. 9071304, No. 30772654), Na-
tional Science and Technology Major Project (Grant No. 2009-
O
O
H
N
iii
i
ii
NH
2
O
N
C O
HO
N
H
N
H
N
H
N
H
O
O
3
4
5
2 (LB-4b)
i) triphosgene, toluene, reflux; ii) L-leucine methyl ester, TEA, DCM, r.t.; iii) NH OK, MeOH, r.t.
2
Figure 6. Synthetic method of 2 (LB-4b).
Su, Li
