Discovery of a novel chimeric ubenimex–gemcitabine with potent oral antitumor activity

Article abstract of DOI:10.1016/j.bmc.2016.09.033

Herein, a novel mutual prodrug BC-A1 was discovered by integrating ubenimex and gemcitabine into one molecule. Biological characterization revealed that compound BC-A1 could maintain both the anti-CD13 activity of ubenimex and the cytotoxic activity of ge

Full text of DOI:10.1016/j.bmc.2016.09.033

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of a novel chimeric ubenimex–gemcitabine with potent
oral antitumor activity
Yuqi Jiang a, Jinning Hou a, Xiaoyang Li a, Yongxue Huang b, Xuejian Wang c, Jingde Wu a, Jian Zhang c,
Wenfang Xu a, Yingjie Zhang a,
⇑
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road, 250012 Ji’nan, Shandong, PR China
b Weifang Bochuang International Biological Medicinal Institute, Weifang, Shandong 261061, PR China
c College of Pharmacy, Weifang Medical University, 261053 Weifang, Shandong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, a novel mutual prodrug BC-A1 was discovered by integrating ubenimex and gemcitabine into one
molecule. Biological characterization revealed that compound BC-A1 could maintain both the anti-CD13
activity of ubenimex and the cytotoxic activity of gemcitabine in vitro. Further characterization also
demonstrated that compound BC-A1 exhibited significant anti-invasion and anti-angiogenesis effects
in vitro. The preliminary stability test of BC-A1 revealed that it could release gemcitabine in vitro. The
in vivo anti-tumor results in liver cancer showed that at the same dosage, oral administration of
BC-A1 was as potent as intraperitoneal administration of gemcitabine. This warranted the further
research and development of the orally active prodrug BC-A1 because gemcitabine can not be orally
administrated in clinic.
Received 25 July 2016
Revised 12 September 2016
Accepted 13 September 2016
Available online xxxx
Keywords:
CD13 inhibitor
Ubenimex
Gemcitabine (GEM)
Anti-angiogenesis
Anticancer
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
catalyzed by cytidine deaminase (CDA) and deoxycytidylate deam-
inase (DCTD) and converted to its inactive metabolite, 2,20-diflu-
Aminopeptidase N (APN/CD13), a membrane-bound metallo-pro-
tease widely expressed on diverse cell surfaces,1–3 is universally
regarded as a multifunctional protein which involved in cancer inva-
sion, angiogenesis, metastasis peptides degradation and so on.2,4–6
Moreover, CD13 has been described as a functional biomarker of
angiogenesis and surface marker of semi-dormant liver cancer stem
cells (CSCs) in human liver cancer cell lines.7–10 Ubenimex (Bestatin,
Ube, 1), a natural product, is the only marketed inhibitor of CD13,
which is used as immune enhancement for the treatment of leuke-
mia or act as an adjuvant agent with other anti-cancer drugs.11–13
Gemcitabine (GEM), a nucleoside analogue, is widely used for
treating a variety of solid tumors, such as non-small lung, pancre-
atic, breast, ovarian cancers and so on.14–22 Gemcitabine was first
launched in 1995 by Lilly.14 After that, combination of gemcitabine
and other cytotoxic antitumor agents or targeted drugs in cancer
treatment are already in advanced clinical trials.23–26 Although
gemcitabine is widely used, the clinical use of gemcitabine is also
subjected to some limitations, such as rapid metabolic inactivation,
poor oral bioavailability, drug resistance, and so on.27–30 After
administration of gemcitabine, more than 90% of gemcitabine is
oro-deoxyuridine (dFdU) and its monophosphate derivative
(dFdUMP).31,32 In order to overcome most of the aforementioned
problems, many gemcitabine derivatives were designed and syn-
thesized via modifications of 4-(N)- and 50-sites of gemcitabine
(Fig. 1).33,34 Among these compounds, LY2334737 is developed to
improve the poor oral bioavailability of gemcitabine. LY2334737
is an oral gemcitabine prodrug, and in Phase I clinical trials.35
In our previous report, we presented the design and synthesis of
a novel mutual prodrug BC-01 containing ubenimex and fluo-
rouracil. Biological characterization of BC-01 proved that this novel
mutual prodrug exhibited improved in vitro and in vivo antitumor
efficiency and displayed multifunctional antitumor properties.36
According to the similar mutual prodrug strategy, ubenimex and
gemcitabine were directly integrated into one molecule to get com-
pound BC-A1 (Fig. 2), which was expected to be an orally active pro-
drug of ubenimex and gemcitabine. Besides, based on our previous
results of BC-0136, BC-A1 itself should also possess CD13 inhibitory
activity, possibly endowing BC-A1 with tumor targeting property.
2. Chemistry
The compound 9 was synthesized starting from (3S,4R)-4-
amino-3-hydroxy-2-oxo-5-phenylpentanoic acid (10, AHPA), as
⇑
Corresponding author. Tel.: +86 531 88382009.
E-mail address: zhangyingjie@sdu.edu.cn (Y. Zhang).
http://dx.doi.org/10.1016/j.bmc.2016.09.033
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
Y. Jiang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
O
HN
O
NH2
N
O
HN
PEG
N
O
N
N
O
F
O
N
O
O
N
F
F
F
O
O
O
F
HO
OH
F
4-N-site
HO
NH2
N
OH
OH
CP-4126, 6
LY2334737, 2
PEG-gemcitabine, 3
N
O
NH2
O
O
O
HN
F
N
HN
18
F
5'-site
HO
O
N
F
OH
Gemcitabine, 1
N
N
O
P
NH
O
O
N
F
O
N
O
O
O
F
F
OH
O
O
F
BnO
F
OH
HO
HO
OH
NUC-1031, 7
4
5
Figure 1. Structure of gemcitabine derivatives.
of BC-A1 was slight weaker than the positive control ubenimex.
Furthermore, we also assessed the inhibitory ability of BC-A1 and
ubenimex toward the human CD13, which is deriving from
cultured A549 and PLC/PRF/5 cells. Similar to the above enzyme
inhibitory activity, both BC-A1 and ubenimex showed micromolar
IC50 values (Table 1). These results confirmed that attachment of
gemcitabine to the carboxyl group of ubenimex did not influence
its binding ability to the active site of CD13, just like our previous
reported mutual prodrug BC-01.36
NH2
N
O
N
F
O
HCl
NH2
F
O
H
N
HO
N
H
OH
F
F
1
O
N
N
OH
OH
O
O
9 (BC-A1)
NH2
O
OH
HO
3.2. Antiproliferative activity assay and colony-forming assay
N
H
O
OH
Ubenimex, 8
hydrolysis
11 kinds of solid or hematological tumor cell lines were selected
to test the antiproliferative activity of BC-A1. Results (Table 2)
showed that compared with its parent compound gemcitabine,
BC-A1 exhibited similar antiproliferative activity toward a broad
range of cancer cell types including liver, lung, ovary, colon, breast
and prostate cancer cells. However, the other parent compound of
BC-A1, the CD13 inhibitor ubenimex exhibited no cytotoxicity.
Moreover, the result in KG1 cell line shows that the combination
of ubenimex and gemcitabine showed no synergistic effects.
Collectively, these results indicated that BC-A1 could release gem-
citabine to exhibit antiproliferative activity. Additionally, cytotox-
icity of BC-A1 and gemcitabine against human liver cell HL-7702
was tested. The IC50 value of BC-A1 and gemcitabine were
(in plasma)
NH2
N
O
N
F
NH2
O
F
O
OH
N
H
HO
O
OH
Ubenimex
OH
Gemcitabine
Figure 2. Design strategy, chemical structure and proposed degradation pathway
of targeted compound.
illustrated in Scheme 1. Compound 10 was protected by the
ditert-butyl dicarbonate (Boc group), followed by condensation
with L-leucine give the compound 12. Then deprotection of the
22.06 lM and 5.43 lM, respectively, which revealed the selectivity
of BC-A1 between nontransformed cells and tumor cells.
In order to further confirm the anti-proliferative activities of
BC-A1, PLC/PRF/5 cell was chosen for the colony-forming assay.
Similarly, the result demonstrated that BC-A1 decreased the num-
ber of colonies formed (Fig. 3).
benzyl group gave the free acid 13. Gemcitabine hydrochloride
(14) was protected by the silyl chloride (TBS group) to afford inter-
mediate 15, which was reacted with 13 in the presence 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
and 1-hydroxybenzotriazole (HoBt) to afford the compound 16.
Deprotection of the TBS group gave the intermediate 17. Treatment
of 17 with hydrogen chloride (HCl) in acetidin led to the target
compound 9 (BC-A1).
3.3. Induction of apoptosis in vitro
Flow cytometry analysis was used to determine the effect of
BC-A1 in inducing apoptosis (Fig. 4). Results suggested that at
the dose of 2 lM, BC-A1 could induce more tumor cells apoptosis
than the positive control ubenimex, while displayed less potency
3. Results and discussion
than gemcitabine.
3.1. In vitro CD13 inhibition
3.4. Anti-invasion assay in vitro
The IC50
porcine kidney were determined to be 3.70 and 8.75
respectively (Table 1), which showed that the inhibitory activity
(
l
M) values of ubenimex and BC-A1 toward CD13 from
lM,
Ubenimex could inhibit cancer-cell invasion in a dose-depen-
dent manner, which has been shown to be attributed to inhibition
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Y. Jiang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Boc
Boc
Boc
NH
O
NH
O
NH2
O
NH O
a
c
b
OH
O
N
H
COOH
Boc
N
H
COOH
O
OH
OH
O
OH
OH
11
10
12
13
NH2
N
HCl
O
NH2
N
NH
O
Boc
H
N
NH
O
N
O
H
N
d
O
N
H
f
b
13
e
F
F
F
N
H
N
F
N
O
N
OH
F
F
F
N
O
N
O
O
OH
F
OH
TBS
O
O
O
O
TBS
O
O
HO
16
OH
17
14
15
TBS
O
HO
TBS
HCl
NH2
O
H
N
N
H
F
F
N
O
N
OH
OH
O
O
9(BC-A1)
HO
Scheme 1. Synthesis of compound 9. (Reagents and conditions: (a) (Boc)2O, 1 N NaOH, THF; (b) EDCI, HoBt, DCM; (c) Pd/C, H2, methanol; (d) N-methylimidazole, iodine, silyl
chloride; (e) TBAF, THF and (f) dry HCl in EtOAc.)
HUVECs.38 To investigate the anti-angiogenic effect of BC-A1, the
in vitro HUVECs tube formation assays were performed. Treatment
of HUVECs with compound BC-A1 at 30 lM within 8 h almost
inhibited the tuber formation completely. Meanwhile BC-A1
exhibited more tuber formation inhibitory activity than ubenimex
at the same concentration (Fig. 6a).
Compared with HUVECs tuber formation model, rat thoracic
aorta rings (TARs) assay is more close to in vivo condition in view
of multi-steps involved in angiogenesis: sprouting, proliferation,
migration and differentiation. Therefore, the anti-angiogenic activ-
ity of compound BC-A1 was further evaluated in TARs assay. The
Table 1
CD13 inhibitory activity of BC-A1 and ubenimex
Compd
IC50
(
l
M)a
IC50
(
l
M)a toward
IC50 (l
M)a toward
CD13 on the surface
of PLC/PRF/5 cells
toward CD13
from porcine
kidney
CD13 on the
surface of
A549cells
BC-A1
Ubenimex 3.70 1.06
8.75 2.17
18.30 1.11
17.34 0.10
15.78 0.85
40.65 9.41
a
Assays were performed in replicate (n P 2); data are shown as mean SD.
of CD13.37 In order to examine the anti-invasion effect of the com-
pound BC-A1, an anti-invasion assay was performed on transwell
chambers coated with Matrigel. Treatment of ES-2 cells with BC-
A1 at 5 lg/mL markedly decreased the invasion of cells through
the matrigel basement membrane in comparison with the uben-
results illustrated that at the concentration of 20 lg/mL, BC-A1
decreased the number of microvessels sprouting from aortic rings
isolated from rat. Again, BC-A1 showed better angiogenesis inhibi-
tory activity than ubenimex at the same dosage (Fig. 6b).
imex and the negative control group (Fig. 5).
3.6. The stability of BC-A1 in human plasma
3.5. HUVEC tuber formation and rat thoracic aorta rings (TARs)
assay
BC-A1 is designed to be a targeted anticancer prodrug, so we
need to evaluate the hydrolysis kinetics of BC-A1 in plasma. To this
end, HPLC method was developed to briefly study the cleavage of
compound BC-A1 in human plasma in vitro. We observed that
CD13 is a key target in anti-angiogenesis, and it was reported
that ubenimex could inhibit the tubular structure formation of
Table 2
Antiproliferative activities of BC-A1 against eleven tumor cell lines with gemcitabine and ubenimex as positive control (IC50 in l
Ma)
Cell lines
BC-A1
Gemcitabine
(GEM)
Ubenimex
(Ube)
GEM + Ube
(1:1)
K562
MDA-MB-231
Hela
ES-2
3AO
HEL
A549
PC-3
U266
0.13 0.03
1.56 0.33
5.48 0.39
0.28 0.12
9.73 2.39
0.61 0.05
0.66 0.05
0.26 0.01
0.26 0.09
0.08 0.01
1.41 0.45
0.82 0.09
0.11 0.01
1.67 0.33
2.74 0.09
0.53 0.15
5.73 0.59
0.48 0.05
0.26 0.02
0.22 0.04
0.22 0.14
0.07 0.01
1.53 0.42
0.86 0.06
>500
>500
>500
>500
>500
>500
>500
>500
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
>500
142.63 17.46
>500
NDb
U937
PLC/PRF/5
KG1
NDb
NDb
>500
1.01 0.12
a
Assays were performed in replicate (n P 2); data are shown as mean SD.
Not determined.
b
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Y. Jiang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 3. Colony formation by PLC/PRF/5 cells observed under an inverted microscope. Gemcitabine was used as a positive control. PLC /PRF/5 cells were stained with Giemsa
after treatment with drugs for two consecutive weeks, and images were captured using an inverted microscope.
Figure 4. Induction of apoptosis at 48 h by compounds BC-A1, gemcitabine (positive control) and ubenimex (positive control), in PLC/PRF/5 cells.
Figure 5. BC-A1 inhibited ES-2 cells invasion in a transwell assay. Ubenimex was used as a positive control representative images indicate the inhibition of migration.
BC-A1 was completely cleaved to their parent compounds uben-
imex and gemcitabine within 120 min of incubations at 37 °C
(Fig. 7). This is consistent with our initial design rationale.
into three groups. The mice were treated with an oral administra-
tion of PBS, single intraperitoneal administration of 25 mg/kg
(0.084 mmol/kg) of gemcitabine, or oral administration of 25 mg/
kg (0.042 mmol/kg) of BC-A1. The mice were treated with the test
compounds at the specified dose every 4 days for a total of 4 doses
(Q4D ꢀ 4). The results showed that compound BC-A1 displayed
potent oral antitumor activity, which is comparable with
gemcitabine at the dosage of 25 mg/kg (Fig. 8a and b). It is worth
noting that at the same dosage of 25 mg/kg, the mol concentration
of gemcitabine in BC-A1 (0.042 mmol/kg) is half of the mol
3.7. In vivo antitumor activity assay
Due to the potent in vitro antiproliferative activity of BC-A1,
in vivo antitumor study of BC-A1 was investigated using H22 (mice
hepotoma cell line) tumor transplant model in Kunming mice. In
this experiment, the transplanted mice were randomly divided
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Y. Jiang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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Figure 6. (a). Representative images of the tube formation assay depicting the formation of a HUVEC capillary-like tubular network by treatment with synthesized compound
BC-A1. (b). Representative images of rat aortic rings treated with compound BC-A1, gemcitabine and ubenimex (positive control). The scale bar indicates 200 m of length.
l
confirmed by another human hepotoma (HepG-2) xenograft model
(Figs. 11 and 12).
4. Conclusions
In summary, a mutual prodrug of gemcitabine and ubenimex
was designed and synthesized. The in vitro pharmacology study
on BC-A1 showed that BC-A1 displayed modest inhibition toward
CD13, and inhibited the proliferation of representative cancer cell
lines. The preliminary stability test of BC-A1 revealed that it could
release gemcitabine and ubenimex in human plasma. Meanwhile,
the anti-tumor results indicated that BC-A1 displayed potent oral
antitumor activity, which is comparable with the positive control
gemcitabine at the same dosage. Our comprehensive in vitro and
in vivo antitumor activity evaluation supported further research
and development of BC-A1 as a promising mutual prodrug of gem-
citabine and ubenimex. These results may also help us to develop
novel orally active prodrug of gemcitabine. Evaluation of its phar-
macokinetics in vivo is currently underway in our laboratory.
Figure 7. Stability of compound BC-A1 in human plasma. Points of BC-A1 were
achieved after 5, 15, 30, 60, 90, 120 min.
concentration of gemcitabine (0.084 mmol/kg), which means the
toxicity of BC-A1 may decrease. Considering gemcitabine can not
be orally administrated due to its toxicity35, our result suggests
that BC-A1 can be developed as an orally active prodrug of gemc-
itabine with less toxicity.
Encouraged by the potent oral antitumor activity of BC-A1 in
mouse hepatoma (H22) tumor transplant model, we further tested
the effect of BC-A1 in a human hepatoma (PLC/PRF/5) xenograft
model. As shown in Figures 9 and 10, the antitumor potency of
BC-A1 (T/C = 51%) was similar to that of gemcitabine (T/C = 54%).
Moreover, the in vivo antihepatoma activity of BC-A1 was further
5. Experimental section
5.1. Chemistry
Starting materials, reagents, and solvents were obtained from
commercial suppliers and used without further purification unless
otherwise indicated. All reactions were monitored via thin layer
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Y. Jiang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 8. Antitumor effect of BC-A1 as tested using tumor-bearing Kunming mice with gemcitabine GEM (25 mg/kg/d) as a positive control. (a) Weights of tumors in
different groups. (b) Photographs of tumors obtained from the tumor-bearing mice.
chromatography (TLC) (0.25 mm silica gel plates (60GF-254)). UV
light and ninhydrin were used to visualize the spots. Silica gel
was used for column chromatography purification. 1H NMR spectra
were routinely recorded on
a Bruker DRX spectrometer at
400 MHz, d in parts per million and J in hertz, using Tetramethyl-
silane (TMS) as an internal standard. High-resolution mass spec-
trometry was performed by Shandong Analysis and Test Center
in Ji’nan, China. ESI-MS spectra were recorded on an API 4000
spectrometer. Melting points were determined uncorrected on an
electrothermal melting point apparatus. The purity of target com-
pound was determined by HPLC using an ODS HYPERSIL column
Figure 9. Growth curve of implanted PLC/PRF/5 xenograft in nude mice.
(5
l
m, 4.6 mm ꢀ 250 mm) with UV detection (249 nm) (22%
acetonitrile/78% water (containing 0.1% triethylamine and 0.15%
trifluoroacetic acid) over 30 min) to be 95%.
5.1.1. (3S,4R)-4-((tert-Butoxycarbonyl)amino)-3-hydroxy-2-
oxo-5-phenylpentanoic acid (11)
10 (19.5 g, 100 mmol) was dissolved in 1 N NaOH solution
(200 mL). The mixture was stirred under ice bath. (Boc)2O
(26.2 g, 120 mmol) dissolved in 120 mL of THF was added into
the solution. After 2 h, the ice bath was removed, and the mixture
was stirred for another 24 h. After removing THF under vacuum,
the solution was acidified to pH3 with 1 N HCl solution. The solu-
tion was washed with 30 mL of acetic ether for 3 times. The organic
layer was collected and dried with anhydrous sodium sulfate over
night. After evaporating the solvent, the residue was dried under
vacuum to get white solid 2 (25.3 g, yield: 86%). mp: 105–106 °C.
1H NMR (400 MHz DMSO-d6): d 1.21–1.52 (m, 9H), 2.94–2.96 (m,
2H), 3.92–4.22 (m, 2H), 6.63 (d, J = 6.0 Hz, 1H), 7.23–7.30 (m,
5H). ESI-MS m/z: 296.6 (M+H)+.
Figure 10. Picture of dissected PLC/PRF/5 tumor tissures.
5.1.2. (S)-Benzyl-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-2-
hydroxy-4-phenylbu-tanamido)-4-methyl-pentanoate (12)
11 (6 g, 20.3 mmol) and HOBt (3 g, 22.3 mmol) were dissolved
in dry DCM (200 mL) under ice bath followed by the addition of
EDCI (4.5 g, 22.3 mmol) and stirring for 30 min at room tempera-
ture. L-Leucine benzyl ester toluene-4-sulfonate (8.5 g, 22.3 mmol)
and TEA (3.1 mL) were added into the solution directly. The mix-
ture was stirred for 5 h at room temperature. The DCM solution
was washed with 1 N HCl, saturated NaHCO3 and brine for 3 times,
dried over anhydrous sodium sulfate and evaporated under vac-
uum. The crude product was purified by recrystallization with
EtOAc and petroleum ether to achieve a white pure solid 12
(4.8 g, yield: 47.5%). mp: 148–150 °C. 1H NMR (400 MHz DMSO-
d6): d 0.78–0.87 (m, 6H), 1.16–1.28 (m, 9H), 1.45–1.52 (m, 1H),
Figure 11. Growth curve of implanted HepG2 xenograft in nude mice.
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Y. Jiang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
purified by column chromatography, using PE–EtOAc (4:1) as the
mobile phase, to obtain 7 as white solid (3.15 g, yield: 35%). mp:
108–110 °C, ESI-MS m/z: 882.8 (M+H)+.
5.1.6. tert-Butyl-((2R,3S)-4-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-
4-hydroxy-5-(hydr-oxymethyl)tetrahydrofuran-2-yl)-2-oxo-
1,2-dihydropyrimidin-4-yl)amino)-4-methyl-1-oxopentan-2-yl)
amino)-3-hydroxy-4-oxo-1-phenylbutan-2-yl)carbamate (17)
7 (0.9 g, 1 mmol) was reacted with tetrabutylammonium flu-
oride (TBAF; 1 M in THF, 1.7 mL, 1.7 mmol) in 20 mL of THF for
2 h at room temperature. THF was evaporated under vacuum;
the crude product was dissolved with EtOAc, washed by 1 N
aqueous citric acid, saturated NaHCO3 and brine for 3 times,
dried over anhydrous sodium sulfate over night and the solvent
was evaporated under vacuum. The crude product was purified
via flash chromatography to afford the compound 17 (0.42 g,
yield: 65%). mp: 196–197 °C. 1H NMR (400 MHz DMSO-d6):
0.84–0.90 (m, 6H), 1.15–1.28 (m, 9H), 1.44–1.50 (m, 1H), 1.62–
1.68 (m, 2H), 2.63–2.68 (m, 1H), 2.77–2.82 (m, 1H), 3.33–3.70
(m, 1H), 3.79–3.96 (m, 4H), 4.13–4.23 (m, 1H), 4.54–4.59 (m,
1H), 5.32 (t, J = 5.4 Hz, 1H), 6.05 (d, J = 6.4 Hz, 1H), 6.15–6.19
(m, 2H), 6.33 (d, J = 6.5 Hz, 1H), 7.16–7.29 (m, 6H), 8.07–8.09
(m, 1H), 8.28 (d, J = 7.6 Hz, 1H), 11.17 (s, 1H). HRMS (AP-ESI)
m/z Calcd for C30H41F2N5O9, [M+H]+ 654.2965. Found: 654.2968.
Figure 12. Picture of dissected HepG2 tumor tissures.
1.57–1.75 (m, 2H), 2.62–2.67 (m, 1H), 2.76–2.82 (m, 1H), 3.84–3.95
(m, 2H), 4.39–4.45 (m, 1H), 5.11 (s, 2H), 6.00 (d, J = 6.6 Hz, 1H), 6.16
(d, J = 9.3 Hz, 1H), 7.16–7.27 (m, 3H), 7.29–7.38 (m, 7H), 8.02 (d,
J = 8.5 Hz, 1H). ESI-MS m/z: 499.6 (M+H)+.
5.1.3. (S)-2-((2S,3R)-3-((tert-Butoxycarbonyl)amino)-2-hydroxy-
4-phenylbutan- amido)-4-methylpentanoic acid (13)
To a solution of 12 (4.9 g, 10.0 mmol) in methanol (200 mL), dry
palladium-carbon (0.5 g) was added and the mixture was stirred
under a hydrogen atmosphere overnight. The palladium-carbon
was filtered off over celite, washed with methanol. After evaporat-
ing the solvent, the residue was dried under vacuum to get white
solid 13 (3.8 g, yield: 92.0%). mp: 124–125 °C. 1H NMR (400 MHz
DMSO-d6): d 0.81–0.88 (m, 6H), 1.15–1.29 (m, 9H), 1.44–1.50 (m,
1H), 1.58–1.67 (m, 2H), 2.65–2.67 (m, 1H), 2.77–2.82 (m, 1H),
3.83–3.92 (m, 2H), 4.27–4.32 (m, 1H), 6.04 (d, J = 6.2 Hz, 1H),
6.16 (d, J = 9.2 Hz, 1H), 7.18–7.29 (m, 5H), 7.78 (d, J = 8.6 Hz, 1H),
12.68 (s, 1H). ESI-MS m/z: 409.6 (M+H)+.
5.1.7. (S)-2-((2S,3R)-3-Amino-2-hydroxy-4-phenylbutanamido)-
N-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)
tetrahydrofuran-2-yl)-2-oxo-1,2-dihydro-pyrimidin-4-yl)-4-
methylpentanamide hydrochloride (9, BC-A1)
17 (0.65 g, 1.0 mmol) was dissolved in saturated chloride
hydrogen in dry ethyl acetate (40 mL). The mixture was stirred at
room temperature for 2 h. The filtered precipitate was washed by
diethyl ether to give the compound 9 (BC-A1), a white solid pow-
der (0.43 g, yield: 91%). mp: 156–158 °C, 1H NMR (400 MHz DMSO-
d6): 0.83–0.92 (m, 6H), 1.50–1.70 (m, 3H), 2.89–2.98 (m, 2H), 3.53–
3.67 (m, 2H), 3.79–3.82 (m, 1H), 3.89–3.91 (m, 1H), 4.01–4.06 (m,
1H), 4.10–4.35 (m, 1H), 4.41–4.51 (m, 1H), 6.10–6.23 (m, 1H),
7.20–7.37 (m, 7H), 8.04–8.08 (m, 3H), 8.28–8.33 (m, 2H), 11.28
(s, 1H). HRMS (AP-ESI) m/z Calcd for C25H33F2N5O7, [M+H]+
554.2421. Found: 554.2429. Retention time: 13.6 min, eluted with
22% acetonitrile/78% water (containing 0.1% triethylamine and
0.15% trifluoroacetic acid).
5.1.4. 4-Amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-(((tertbutyldimethy-lsilyl)-oxy)methyl)-3,3-difluorotetra-
hydrofuran-2-yl)pyrimidin-2(1H)-one (15)
A solution of 14 (3.0 g, 10.0 mmol), N-methylimidazole (5.0 g,
30.0 mmol), and iodine (7.6 g, 30.0 mmol) were dissolved in dry
tetrahydrofuran and pyridine (ratio 10:1, 100 mL). Subsequently,
t-butyldimethylchlorosilane (TBSCl) (3.7 g, 25 mmol) was added.
The reaction mixture was stirred at room temperature until com-
plete disappearance of the starting material (TLC analysis). THF
was evaporated with the residue being taken up in EtOAc
(300 mL). The EtOAc solution was washed with saturated Na2S2O3,
saturated NaHCO3 and brine for 3 times, dried over anhydrous
sodium sulfate over night, evaporated. The residue was purified
by column chromatography, using DCM–MeOH (20:1) as the
mobile phase, to obtain 15 as Pale yellow white solid (3.15 g, yield:
64%). mp: 118–120 °C. 1H NMR (400 MHz DMSO-d6): d 0.09–0.11
(m, 12H), 0.87–0.90 (m, 18H), 3.76–3.79 (m, 1H), 3.88–3.96 (m,
2H), 4.27–4.36 (m, 1H), 5.79 (d, J = 7.5 Hz, 1H), 6.15–6.19 (m,
1H), 7.44 (d, J = 7.1 Hz, 2H), 7.55 (d, J = 7.5 Hz, 1H). ESI-MS m/z:
490.7 (MꢁH)ꢁ.
5.2. CD13 inhibition assay
The inhibitory activity of target compound toward CD13 was
determined by using microsomal aminopeptidase from porcine
kidney microsomes (Sigma) as the enzyme in 50 mM PBS, pH 7.2
at 37 °C. L-Leucine-p-nitroanilide was used as substrate. Briefly,
the CD13 solution was added to tested compound solutions at var-
ious concentrations and incubated at 37 °C for 5 min. Then, the
substrate was added and the mixture was incubated for another
30 min at 37 °C. Finally, the hydrolysis of the substrate was mea-
sured by following the change in the absorbance measured at
405 nm with a Micro-plate Reader (Thermo Fisher, Shanghai,
China).
5.1.5. tert-Butyl-((2R,3S)-4-(((S)-1-((1-((2R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)
methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydrop-
yrimidin-4-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-
hydroxy-4-oxo-1-phenyl-butan-2-yl)carbamate (7)
13 (4.1 g, 10.1 mmol) and HOBt (1.6 g, 12.2 mmol) were dis-
solved dry DCM (200 mL) under ice bath followed by the addition
of EDCI (2.3 g, 12.2 mmol) and stirring for 30 min at room temper-
ature. 15 (5.5 g, 11.1 mmol) and TEA (2.1 mL) were added into the
solution directly. The mixture was stirred for 5 h at room temper-
ature. The DCM solution was washed with 1 N aqueous citric acid,
saturated NaHCO3 and brine for 3 times, dried over anhydrous
sodium sulfate and evaporated under vacuum. The residue was
CD13 activity on the surface of ES-2 and PLC/PRF/5 cells was
estimated by measuring the hydrolysis of substrate (L-leucine-p-
nitro-anilide). A cell suspension (2.5 ꢀ 106/mL) in 1ꢀ PBS buffer
was added to a 96-well plate (70 lL) containing various concentra-
tions of test compounds. Then, the solution of substrate was added
to each well and the mixture was incubated for 1 h at 37 °C. CD13
activity was estimated by measuring the absorbance at 405 nm as
described above.
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8
Y. Jiang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5.3. Cell proliferation analysis
gel for 30 min at 37 °C, 5% CO2. Harvested cells in serum-free
RPMI-1640 (400 L) were added into the upper compartment of
the chamber. Subsequently, 100 L of test compounds were added
into the upper compartment of the chamber. 750 L of conditioned
medium, which derived from ES-2 cells, was placed in the bottom
compartment of the chamber. After 24 h incubation at 37 °C and 5%
CO2, the medium was removed from the upper chamber. Cotton
swab was used to scrape off the non-invaded cells on the upper
side of the chamber. The cells, which had migrated from the matri-
gel into the pores of the inserted filter, were stained with crystal
violet solution.
l
All cell lines were maintained in RPMI1640 medium containing
10% FBS at 37 °C in a 5% CO2 humidified incubator. Cell proliferation
assay was determined by the MTT method. Briefly, cells were pas-
saged the day before dosing into a 96-well cell plate, allowed to
grow for 12 h and then treated with different concentrations of
compound sample for 48 h. A 0.5% MTT solution was added to each
well. After incubation for another 4 h, formazan formed from MTT
l
l
was extracted by adding 150 lL of DMSO rocking for 15 min. Absor-
bance was then determined using an ELISA reader at 570 nm. The
IC50 values were calculated according to the inhibition ratios.
5.9. Stability of representative compound in human plasma
5.4. Colony formation assay
Preparation of human plasma was conducted as previously
described.40 Human plasma added to the stock solution of BC-A1
(4 mg/mL in methanol) and incubated at 37 °C for 24 h. At sched-
uled times sample aliquots were collected and the enzymatic reac-
tion was quenched by adding acetonitrile. Human Plasma samples
For the colony formation assay, PLC/PRF/5 cells (0.5 ꢀ 103/well)
were seeded in 6-well plates. After 12 h, cells were exposed to
BC-A1, GEM, and a combination of the GEM and ubenimex (1:1)
at 37 °C. After two weeks, cells were fixed with fresh 4%
paraformaldehyde at room temperature for 15 min and stained
with giemsa. Cell images were captured using an inverted micro-
scope (magnification ꢀ 4, Olympus, Beijing, China). A minimum
of 50 viable cells were scored as a colony.
underwent extraction using 300
The samples were filtered (0.22
l
L acetonitrile and 300
lL water.
l
m) after shocking 30 s and cen-
trifugation at 12,000 rpm for 10 min. Analytical HPLC was per-
formed on Agilent 1200 HPLC instrument using a Alltima C18
column (5
l
m, 4.6 mm ꢀ 250 mm), compounds were eluted with
5.5. Flow cytometry
22% acetonitrile/78% water (containing 0.1% triethylamine and
0.15% trifluoroacetic acid) over 15 min. The absorbance was mea-
sured at 249 nm, the flow rate was 1 mL/min and the quantity of
Flow cytometry was used to evaluate the cell apoptosis. PLC/
PRF/5 cells seeded on 6-well plates (2 ꢀ 105 per well) were treated
with test compounds for 48 h. Subsequently, cells were harvested,
washed twice in cold PBS, centrifuged, and resuspended in 1ꢀ
annexin-binding buffer according to the instruction of the apopto-
sis kit. Annexin V-FITC and propidium iodide were added to each
tube and incubated for 15 min in the dark. The stained cells were
analyzed by flow cytometry. The results were compared with con-
trol groups.
injection was 20 lM.
5.10. In vivo tumor transplant models
All experiments involving in laboratory animals were per-
formed the approval of the local ethics committee. In this experi-
ment, to establish a solid tumor model, mice were inoculated
subcutaneously with injections of 1 ꢀ 107/mL murine H22 cells
(0.2 mL). When the tumor was approximately 0.2 cm3 in size, the
transplanted mice were randomly divided into treatment and con-
trol groups. The mice were treated with the test compounds at the
specified dose for two weeks (every 4 days for a total of 4 doses
(Q4D ꢀ 4)). After treatment, mice were sacrificed and dissected
to weigh the tumor tissues and to examine the internal organ
injury. The body weight was monitored regularly. The tumor inhi-
bitory rate (%) was calculated as (1 ꢁ average tumor weight/aver-
age tumor weight of PBS group) ꢀ 100%.
5.6. In vitro HUVEC tuber formation assay39
The 96-well microplates were coated with 100 lL of Matrigel
(BD Biosciences, NJ) and allowed to gel for 30 min at 37 °C and
5% CO2. Then, HUVECs were seeded at a density of 40,000 cells
per well in M199 (5% FBS) containing the test compounds and
incubated at 37 °C, 5% CO2 for 6 h. The morphological changes of
the cells and the tubular structure formation of HUVECs were
observed under a phase-contrast microscope and photographed
at 200ꢀ magnification.
5.11. In vivo human tumor xenograft models
5.7. Rat thoracic aorta rings (TARs) assay
In vivo tumor xenograft models were established as previously
reported.41 In Brief, two human liver tumor (PLC/PRF/5 and
HepG2) cell lines were cultured in DMEM growth medium con-
taining 10% FBS and maintained in a 5% CO2 humidified incubator
at 37 °C. For in vivo anti-tumor efficacy research, tumor cells were
inoculated subcutaneously in the right flanks of female athymic
nude mice (BALB/c-nu mice, 5–6 weeks old, Slac Laboratory
Aniamal, Shanghai, China). When the tumor was approximately
100 mm3 in size, the mice were randomized into treatment and
control groups. The treatment groups received specified concen-
trations of compounds by oral administration or intravenous
injection, and the blank control group received an equal volume
of PBS solution. During treatment, the body weight was moni-
tored regularly. After treatment, mice were sacrificed and dis-
sected to weigh the tumor tissues and to examine the internal
organ injury. The tumor inhibitory rate (%) was calculated as
(1 ꢁ average tumor weight of treatment group/average tumor
weight of PBS group) ꢀ 100%.
The 96-well tissue culture plates were coated with 100 lL of
Matrigel (BD Biosciences, NJ) and allowed to gel for 30 min at
37 °C, 5% CO2. Then, the thoracic aortas were excised from 8- to
10-week-old male Wistar rats. After careful removal of fibroadi-
pose tissues, the aortas were cut into 1-mm-long cross-sections,
placed on Matrigel-coated wells, and covered with an additional
100 lL of Matrigel. After the second layer of Matrigel set, the rings
were incubated at 37 °C and 5% CO2 for 30 min. Aorta rings were
treated every other day with test compounds for 9 days and pho-
tographed on the 10th day at 200ꢀ magnification. Experiments
were repeated three times using aortas from four different rats.
5.8. Invasion assay
Transwell filters were coated with matrigel (BD Biosciences, NJ)
on the upper surface of a polycarbonic membrane and allowed to
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Y. Jiang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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This work was supported by National Natural Science
Foundation of China (Grant Nos. 81373282, 21302111 and
81503108), National High-tech R&D Program of China, 863
Program (Grant No. 2014AA020523), and National Major Scientific
and Technological Special Project for ‘‘Significant New Drugs
Development” of China (Grant No. 2012ZX09103101-015), Major
Project of Science and Technology of Shandong Province
(2015ZDJS04001), Young Scholars Program of Shandong University
(YSPSDU, Grant no. 2016WLJH33).
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