Novel indoline-2,3-dione derivatives as inhibitors of aminopeptidase N (APN)

Article abstract of DOI:10.1016/j.bmc.2012.06.024

Aminopeptidase N (APN/CD13), as a zinc-containing ectoenzyme, plays a critical role in the process of tumor angiogenesis, invasion and metastasis. Through the docking-based virtual screening of chemical databases and the further activity assay, we discovered that compound 10c exhibits potent and selective inhibitory ability towards APN. In addition, a series of indoline-2,3-dione derivates have been designed and synthesized as APN inhibitors. The results of preliminary activity evaluation showed that compound 12a (IC₅₀ = 0.074 ± 0.0026 μM) exhibited the best inhibitory activity against APN, which could be used for further anticancer agent research.

Full text of DOI:10.1016/j.bmc.2012.06.024

Bioorganic & Medicinal Chemistry 21 (2013) 2663–2670
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel indoline-2,3-dione derivatives as inhibitors of
aminopeptidase N (APN)
⇑
Kang Jin, Xiaopan Zhang, Chunhua Ma, Yingying Xu, Yumei Yuan, Wenfang Xu
Department of Medicinal Chemistry, School of Pharmacy, Shandong University, 44, West Culture Road, Jinan, Shandong 250012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Aminopeptidase N (APN/CD13), as a zinc-containing ectoenzyme, plays a critical role in the process of
tumor angiogenesis, invasion and metastasis. Through the docking-based virtual screening of chemical
databases and the further activity assay, we discovered that compound 10c exhibits potent and selective
inhibitory ability towards APN. In addition, a series of indoline-2,3-dione derivates have been designed
and synthesized as APN inhibitors. The results of preliminary activity evaluation showed that compound
Received 8 May 2012
Revised 12 June 2012
Accepted 13 June 2012
Available online 19 June 2012
12a (IC50 = 0.074 0.0026
further anticancer agent research.
lM) exhibited the best inhibitory activity against APN, which could be used for
Keywords:
Aminopeptidase N
Inhibitors
Ó 2012 Published by Elsevier Ltd.
Indoline-2,3-dione derivatives
Synthesis
QSAR
1. Introduction
the process of submission now. In this letter, we described the syn-
thesis and biological activity evaluation of these APNIs which
derived from compound 10c. The docking studies of the interaction
were also discussed.
Aminopeptidase N (APN/CD13) is a zinc-dependent metallopro-
tease which is located on cell surface as an ectoenzyme.1 It is a ubiq-
uitous enzyme, which is widely expressed on hematopoietic cells of
myeloid origin and non-hematopoietic cells and tissues, such as
brain cells, fibroblasts, and epithelial cells of the kidney, liver, and
intestine.2–5 It has been certified that the over-expression of APN is
associated with many diseases, such as cancer, viral infection and
inflammation. Especially in the process of tumorigenesis, it played
a crucial role in tumor invasion, metastasis and angiogenesis.1,6,7
Since the first marketed anti-APN drug Bestatin has been
launched in 1976, a lot of APN inhibitors (APNIs) have been re-
ported, for example, probestin,8 lapstatin,9 AHPA-Val,10 etc. have
displayed efficient inhibitory ability. Through the previous re-
search of the structure–activity relationship (SAR) of many APNIs,
it is obvious that a zinc binding group (ZBG), two hydrophobic
2. Chemistry
The target compounds were synthesized efficiently following
the procedures shown in Schemes 1 and 2. Compounds 3a–3e were
easily prepared from substituted anilines 1a–1e through Sandmey-
er reaction. First, substituted anilines reacted with hydroxylamine
hydrochloride and chloral hydrate to give nitroso acetanilides 2a–
2e. Then the compounds 2a–2e were cyclized under the effect of
concentrated sulfuric acid and hydrolyzed in the ice water to afford
substituted isatins 3a–3e. The compounds 4a–4e were synthesized
by condensation of the ketone group of compounds 3a–3e and eth-
ane-1,2-diol under acidic environment. Using different reactant,
such as propane-1,3-diol and butane-1,4-diol, compounds 5a–5e
and 6a–6c were obtained through the same method. Coupled com-
pounds 4a–4e with methyl 2-bromoacetate to get compounds 7a–
7e, which reacted with NH2OK in methanol to generate the target
compounds 10a–10e. Compounds 11a–11e and 12a–12c were ob-
tained via the same procedure.
0
groups and an appropriate group which can occupy the S2 pocket
are usually necessary.11–13 Based on the brief analysis of the char-
acteristic of the structures of the active site and inhibitors, we car-
ried out the virtual screening and obtained 24 qualified
compounds. Though the further enzyme inhibition assay, com-
pound 10c exhibited the best inhibitory activity against APN,
which is better than Bestatin as well. Based on this, a series of AP-
NIs had been designed (Fig. 1). And these previous work were in
Compound 7c was hydrolyzed by KOH/H2O in ethanol to obtain
compound 13, which was coupled with different natural a-amino
acids to get compounds 14a–14f. Finally, compounds 15a–15f
were obtained by treating compounds 14a–14f with NH2OK in
methanol.
⇑
Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: wfxu@yahoo.cn (W. Xu).
0968-0896/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.06.024

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K. Jin et al. / Bioorg. Med. Chem. 21 (2013) 2663–2670
O
O
n
O
O
Br
O
Br
O
X
O
O
O
N
N
N
O
O
O
R
NH
HN
NH
HO
HO
OH
R= amino acids
10c
X= F, Cl or Br;
n= 0, 1 or 2.
Or X= CH3 or H;
n= 0 or 1.
Figure 1. The new APNIs derived from compound 10c.
O
R
R
R
O
a
b
O
N
N
NH2
N
H
OH
H
1a-1e
2a-2e
3a-3e
d
e
c
O
O
O
O
R
O
R
R
O
O
O
O
N
H
N
H
N
H
5a-5e
4a-4e
6a-6c
f
f
f
O
O
O
O
R
R
R
O
O
O
O
O
O
O
O
N
N
N
O
O
O
7a-7e
8a-8e
9a-9c
g
g
g
O
N
O
O
O
R
O
R
R
O
O
O
O
O
O
O
N
N
HN OH
HN
O
H
HN OH
12a-12c
10a-10e
11a-11e
Scheme 1. Reagents and conditions: (a) Cl3CH(OH)2, NH2OH, Na2SO4, 85 °C; (b) H2SO4, 90 °C; (c) ethane-1,2-diol, TsOH, toluene, 130 °C; (d) propane-1,3-diol, TsOH, toluene,
130 °C; (e) butane-1,4-diol, TsOH, toluene, 130 °C; (f) BrCH2COOCH3, TBAB, K2CO3, KI, acetone, 60 °C; (g) NH2OK, CH3OH.
showed a better potency, which also convincingly demonstrated
that the extension of side chain could trigger a reducing of the
activity.
From the data of Table 1, we can see that compounds 10d and
11d displayed worse activities than the other ones listed in the
same form, which means a substituent in R position such as chlo-
rine, fluorine, bromine, or methane could enhance the potency of
inhibitors. And different halogens in R position also have different
3. Results and discussion
All the target compounds were assayed for their potential APN
inhibitory activities and the results are listed in Tables 1 and 2. As
the data shown, the compounds of Table 1 exhibited better
inhibitory activity than the other compounds in Table 2, which
suggested that the side chain which contains a hydroxamate group
should have a certain length. Compared with compound 15a, 10c

K. Jin et al. / Bioorg. Med. Chem. 21 (2013) 2663–2670
2665
O
O
Br
O
O
O
O
Br
Br
O
O
a
b
N
O
O
O
O
N
N
R1
HN
OH
O
O
13
7c
14a-14f
O
O
O
Br
O
O
c
N
R1
HN
OH
NH
15a-15f
O
Scheme 2. Reagents and conditions: (a) EtOH, KOH, HCl; (b) methyl ester of amino acids, isobutyl chloroformate, NMM, THF, ꢀ20 °C; (c) NH2OK, CH3OH.
levels of impacts on their activities. The compounds with a chloride
group on the benzene ring (10a, 11a and 12a) were more potent
than others containing a bromide substituent (10c, 11c and 12c),
followed by the fluorinate inhibitors (10b, 11b and 12b) at last.
This may be a result of different influent effects of electrical
properties and spatial structures of these subgroups. For example,
the negative inductive effect of fluorine is stronger than chlorine
than 10d and 11d but worse than Bestatin and the derivatives with
chlorine and bromide. So the further transformation of the struc-
tures of 10 and 11 was not continued.
On the other hand, compounds possessing ketal spiro five- to
seven-membered rings sat the 3-postion of indolin-2-one skeleton
also exhibited varied activities. For example, seven-membered het-
erocyclic derivatives 12a, 12b and 12c displayed the highest po-
tency for the inhibition of APN followed by the inhibitors 10a,
10b and 10c containing five-membered heterocyclic ring. Yet the
six-membered heterocyclic counterpart (11a, 11b and 11c) could
not perform as strongly as the above correspondent compounds.
This may due to that compared with six-member ring, the confor-
mations of seven-member and five-member rings may be more
and bromide, which makes the
ring reduce. As a result, compared with the chlorinated and bromi-
nated ones, the interaction between fluorinated compounds
p-electron density on the benzene
p–p
and Tyr376 of APN weakened. On the other hand, the ability of elec-
tronic-supplying conjugation of chlorine is a little better than bro-
mine, which made the interaction stronger as result. Besides
halogens, alkyl group in R position could also affect the inhibitory
ability positively. For example, compounds 10e and 11e with a
methyl group in respective structure showed better IC50 values
conductive to the formation of hydrophobic effect with Met260
.
According to the data of Table 2, all the amino acid derivatives
(15a–15f) showed worse activity than compound 10c, which may
indicated that the addition of the amino acid residues in the side
chain could affect the affinity negatively. In this series, compound
15a containing a Gly residue in the side chain also showed an
acceptable activity against the enzyme, yet compounds with other
Table 1
The structures and inhibitory activities of the target compounds 10a–e, 11a–e and
12a–c against APN
Table 2
n
O
O
The structures and inhibitory activities of the target compounds 15a–f and positive
control bestatin against APN
R
O
O
N
O
O
Br
HN OH
O
N
O
a
Compd
R
n
IC50 (lM) APN
R'
HN
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
12a
12b
12c
Cl
F
Br
H
CH3
Cl
F
Br
H
0
0
0
0
0
1
1
1
1
1
2
2
2
0.31 0.076
5.30 0.231
0.36 0.084
209.13 5.32
2.63 0.241
1.30 0.212
6.71 0.360
3.40 0.190
265.84 7.13
3.33 0.150
0.074 0.0026
4.06 0.357
0.086 0.00201
NH
OH
O
a
Compd
R0
IC50 (lM) APN
15a
15b
15c
15d
15e
15f
–H
–CH2Ph
–CH2CH(CH3)2
–CH(CH3)CH2CH3
–Ph
6.60 0.512
26.38 1.86
22.46 1.54
631.22 9.25
>1000
CH3
Cl
F
–CH(CH3)2
226.07 2.11
Br
O
OH
O
OH
NH2
O
NH2
O
Bestatin
1.30 0.085
Bestatin
1.30 0.085
N
H
N
H
OH
OH
a
a
Mean values and standard deviations of triplicate experiments are given.
Mean values and standard deviations of triplicate experiments are given.

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K. Jin et al. / Bioorg. Med. Chem. 21 (2013) 2663–2670
Table 3
the positive control. HDACs are zinc-dependent metalloproteinase
as well and associated closely with the invasion and metastasis of
tumor. Unlike APN, which is an exopeptidase, HDACs mainly exist
near nucleus. As the results shown in Table 3, all the tested com-
pounds but 15a exhibited much lower inhibitory activities against
HDACs than that of APN, which to some verified our APNIs design-
ing strategy. Compared with SAHA, 15a showed a similar inhibi-
tory ability against HDACs, the reason of which may be that 15a
could bind with the active side of HDACs by its side chain going
through the slender channel, just like SAHA. Thus 15a could be
used as a leader to design more potent HDACs inhibitors in the
future.
the structures and inhibitory activities of compounds 10a–c, 11a–c, 12a–c and 15a
against HDAC
n
O
O
R
O
O
N
HN OH
a
Compd
R
n
IC50 (lM) HDAC
10a
10b
10c
11a
11b
11c
12a
12b
12c
Cl
F
Br
Cl
F
Br
Cl
F
0
0
0
1
1
1
2
2
2
>100
>100
>100
>100
>100
>100
>100
>100
>100
Furthermore, the inhibition of compounds 10a–c, 11a–c and
12a–c against human APN were also determined with ES-2 human
ovarian clear cell carcinoma cells high-expressing APN. Results
were presented in Figure 2, from which we can see that all these
compounds, expect for 11b, could inhibit APN on ES-2 cells more
efficiently than Bestatin, and 12a showed the best capability
(IC50 = 3.12 0.56 lM). Similar to the above enzyme inhibitory
Br
activity, the human APN inhibition of these compounds also pre-
sented consistent trends. The chloride substituent in R position en-
hanced the potency more obviously than bromide substituent
followed by fluoride ones at last (10a > 10c > 10b, 11a >
11c > 11b, 12a > 12c > 12b). The same rule as the results of
previous enzymatic inhibition experiment also appeared from the
perspective of these compounds with different heterocyclic rings
(12a > 10a > 11a, 12b > 10b > 11b, 12c > 10c > 11c).
In addition, we also selected compounds 10a–c, 11a–c and 12a–c
to assay their anti-tumor inhibitory activity against ES-2 cell
proliferation compared with Bestatin by using MTT method.
The results confirmed that the anti-tumor inhibitory activities
O
O
Br
O
N
O
15a
0.33 0.076
HN
NH
OH
O
O
H
N
OH
SAHA
0.11 0.03
N
H
O
of compounds (10a IC50 = 167.19 21.47 lM, 10b IC50 = 141.50
11.19
18.52
17.84
l
l
l
M, 10c IC50 = 204.69 32.33
M, 11c IC50 = 137.14 15.21
M) are similar to each other and all of these compounds
l
l
M, 11a IC50 = 233.34
M, 12a IC50 = 212.25
a
Mean values and standard deviations of triplicate experiments are given.
exhibited better anti-proliferative effect against ES-2 cells than Bes-
tation with the IC50 of 631.74 29.70 M (Fig. 3). The assay of 10a–c,
l
11a–c and 12a–c on MDA-MB-231 cell proliferation was also
performed, the results of which were shown in Figure 4. Compounds
10c, 11c, 12a and 12c exhibited the inhibitory effect with IC50
values of 57.45 10.21
and 32.85 5.32 M, which showed similar potency to Bestatin
with IC50 value of 50.6 4.68 M.
lM, 55.12 6.83 lM, 91.52 15.82 lM
l
l
Aiming to investigate the interaction between the target
compounds and APN, the most active compound 12a, was chosen
to be constructed using a Sybyl/Sketch module and optimized via
Figure 2. Inhibitory activity of compounds 10a–c, 11a–c, 12a–c and Bestatin
against APN on ES-2 cells. Data expressed are mean values of three independent
experiments.
amino acids in the same position did not exhibit expected potency
towards APN. This may be explained by that the larger hydropho-
bic isatin group could bind tightly with APN’s S20 active pocket, but
the hydrophobic side chains of amino acids could not occupy the
S10 pocket as originally designed.
Comparing all the target compounds (compounds 10, 11, 12 and
15), compound 12a had
0.0026 M) than the others and control Bestatin (IC50 = 1.3
0.085 M).
a better inhibition (IC50 = 0.074
l
l
In order to test the selectivity, compounds 10a–c, 11a–c, 12a–c
and 15a which showed satisfactory activities against APN were
chosen to be assayed for the inhibition of HDACs with SAHA as
Figure 3. Effects of compounds 10a–c, 11a–c, 12a–c and Bestatin on ES-2 cells
proliferation. The columns represent the mean values of three independent
experiments.

K. Jin et al. / Bioorg. Med. Chem. 21 (2013) 2663–2670
2667
Figure 6. The docking result of compound 15e with APN.
Figure 4. Effects of compounds 10a–c, 11a–c, 12a–c and Bestatin on MDA-MB-231
cells proliferation. The columns represent the mean values of three independent
experiments.
Figure 5. (a) The FlexX docking result of 12a with APN (PDB: 2DQM). (b) The docking result of 12h shown by LIGPLOT.

2668
K. Jin et al. / Bioorg. Med. Chem. 21 (2013) 2663–2670
Powell’s method by the Tripos force field with convergence criterion
set at 0.05 kcal/(Å mol), and assigned with the Gasteiger–Hückel
method. The docking study of 12a and the active site of APN was per-
formed using Sybyl/FlexX module. The active site was defined as
7.0 Å radius circles around Bestatin in the co-crystal structure
(PDB code: 2DQM). Other docking parameters utilized in the pro-
gram were remained default. The result showed in Figure 5a
suggested that the large hydrophobic backbone of 50-chlorospi-
ro[[1,3]dioxepane-2,30-indolin]-20-one can insert into S1 pocket
and the hydroxamic acid group can chelate with the zinc ion which
was the crucial catalytic factor in active site. In addition, a 2D pattern
of detailed binding mode was created as well (Fig. 5b). The large isat-
in moiety of compound 12a can form hydrophobic interaction with
Met260, Tyr376 and Glu320 of S1 pocket and the carbonyl group can
from hydrogen bonds with Gly261, Ala 262, His301, Lys319 and the zinc
ion at the distance of 5.33, 4.32, 5.68, 7.59 and 3.96 Å, respectively.
The compounds with less activities showed in Table 2, may have a
different interaction modes. In order to investigate the causing of
the decline in activity, compound 15e was selected for docking study
by the same method with compound 12a. As illustrated in Figure 6,
product was recrystallized by distilled water to give compound
2a as white solid (10.18 g, yield 85.4%), mp = 173–175 °C.
5.1.2. 5-Chloroindoline-2,3-dione (3a)
Compound 2a (9.93 g, 0.05 mol) was added to concentrated sul-
furic acid (40 mL) in batches at 60 °C. Keep the reaction at 90 °C for
30 min. Cool the mixture to room temperature and then pour the
solution into ice-water (200 mL) with vigorous stirring for 1 h. Or-
ange solid was precipitated as crude product, which was depurated
by column chromatography to get compound 3a (8.18 g, yield
90.1%), mp = 237–239 °C.
5.1.3. 50-Chlorospiro[[1,3]dioxolane-2,30-indolin]-20-one (4a)
A solution of compound 3a (1.00 g, 6.21 mmol), ethylene glycol
(2.3 g, 31.05 mmol) and TsOH (1.07 g, 6.21 mmol) in 150 mL tolu-
ene was stirred for 5 h at 130 °C. The water which was produced
in the reaction system was removed by water separator frequently.
The toluene was removed with a rotary evaporator. The remaining
mixture was dissolved in 50 mL EtOAc and washed with saturated
NaHCO3 (10 mL ꢁ 3) and brine (10 mL ꢁ 2) in turn. The organic
phase was dried over anhydrous MgSO4, filtered and concentrated
with a rotary evaporator to give the crude product. The crude prod-
uct was depurated by column chromatography to afford compound
4a as white solid (0.97 g, yield: 78.2%), mp = 178–179 °C. 1H NMR
(600 MHz, DMSO) d 10.60 (s, 1H), 7.43 (d, J = 2.2 Hz, 1H), 7.39
(dd, J = 8.3, 2.2 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 4.34–4.25 (m, 4H).
ESI-MS m/z: 226.6 [M+1]+.
the phenyl group of L-phenylglycine moiety can slightly interact
with S1 pocket with an unsatisfactory angle. Although the hydroxa-
mic acid group can also chelate the zinc ion, the lager hydrophobic
50-bromospiro[[1,3]dioxolane-2,30-indolin]-20-one moiety cannot
insert into S20 pocket, so compound 15e had a less inhibitory activity
than 12a.
4. Conclusion
5.1.4. Methyl-2-(50-chloro-20-oxospiro[[1,3]dioxolane-2,30-
indoline]-10-yl)acetate (7a)
In summary, the synthesis and properties of one series of indo-
line-2,3-dione derivates as APNIs have been described. The preli-
minary results demonstrated that most of the target compounds
exhibited better inhibition than the control Bestatin. Comparing
with the lead compound 10c, the inhibitory activities toward APN
were also improved mildly. Among them, compound 12a showed
the most effective inhibition, which could be used as a lead for fur-
ther structure transformation in the future APNIs exploration.
To a solution of compound 4a (0.5 g, 2.2 mmol) and methyl-2-
bromoacetate (0.4 g, 2.64 mmol) in 50 mL acetone was added TBAB
(0.07 g, 0.22 mmol) and KI (0.036 g, 0.22 mmol) as catalyst. Then
K2CO3 (0.36 g, 2.64 mmol) was added to the reaction mixture. Keep
the reaction for 6 h at 56 °C. Filtrated and concentrated in vacuum.
The residue was dissolved in 50 mL EtOAc and washed with satu-
rated NaHCO3 (10 mL ꢁ 3) and brine (10 mL ꢁ 2) in turn. The
EtOAc solution was dried over anhydrous MgSO4 and concentrated
with a rotary evaporator to get the crude product. The crude prod-
uct was purified by fast column chromatograph (PE/EtOAc = 10:1)
to afford compound 7a as white solid (0.62 g, yield: 95.4%),
mp = 123–124 °C. 1H NMR (600 MHz, DMSO) d 7.53 (d, J = 2.0 Hz,
1H), 7.49 (dd, J = 8.4, 2.1 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 4.55 (s,
2H), 4.37–4.29 (m, 4H), 3.68 (s, 3H). ESI-MS m/z: 298.4 [M+1]+.
5. Experiment
5.1. Chemistry: general procedures
All the materials involved were purchased from commercial
suppliers. Solvents were distilled prior to use. All the reactions were
monitored by thin-layer chromatography on 0.25 mm silica gel
plates (60GF-254) and visualized with UV light, or chloride ferric.
The products were purified by column chromatography which
was performed using 200–300 mesh silica gel. NMR spectra were
determined on a Brucker Avance 600 spectrometer, d in parts per
million and J in Hertz. TMS was used as an internal standard. ESI-
MS were determined on an API 4000 spectrometer. Measurements
were made in DMSO-d6 solutions. Melting points were tested using
an electrothermal melting point apparatus and were uncorrected.
5.1.5. 2-(50-Chloro-20-oxospiro[[1,3]dioxolane-2,30-indoline]-10-
yl)-N-hydroxyacetamide (10a)
Compound 7a (0.5 g, 1.68 mmol) was dissolved to a solution of
1.5 N NH2–OK in methanol (10 mL). The reaction mixture was stir-
red for 1.5 h, and then concentrated in vacuum to give the yellow
oily liquid. The residue was acidified by 1 N HCl and extracted with
ethyl acetate. The organic phase was dried with Na2SO4, and then
remove the solvent under low pressure to get the crude product.
Finally, compound 10a was separated by column chromatography
(PE/EtOAc = 1:1) as white solid (0.34 g, yield: 68.0%), mp = 165–
166 °C. 1H NMR (600 MHz, DMSO) d 10.87 (s, 1H), 9.04 (s, 1H),
7.52–7.48 (m, 2H), 7.01–6.97 (m, 1H), 4.34–4.31 (m, 4H), 4.20 (s,
5.1.1. N-(4-Fluorophenyl)-2-(hydroxyimino) acetamide (2a)
Anhydrous sodium fulfate (120 g, 0.84 mol) was dissolved in
lukewarm water (350 mL) and an aqueous solution of chloral hy-
drate (14.89 g, 0.09 mol, 30 mL) was added to the solution. A mix-
ture of compound 1a (7.65 g, 0.06 mol), concentrated hydrochloric
acid (15 mL) and distilled water (105 mL) was added dropwise to
the solution. Then an aqueous solution of hydroxylamine hydro-
chloride (13.9 g, 0.20 mol, 60 mL) was added and the reaction mix-
ture was stirred for 2 h at 85 °C. Then cool the mixture to room
temperature and brown solid was precipitated to give crude prod-
uct N-(4-fluorophenyl)-2-(hydroxyimino) acetamide. The crude
2H). HRMS (AP-ESI) m/z Calcd for
C
12H11ClN2O5 [M+H]+
299.0356. Found: 299.0426.
The other compounds (10b–10e, 11a–11e, 12a–12c) were syn-
thesized by the same method.
5.1.6. 2-(50-Fluoro-20-oxospiro[[1,3]dioxolane-2,30-indoline]-10-
yl)-N-hydroxyacetamide (10b)
White solid, yield 73.1%, mp = 175–176 °C. 1H NMR (600 MHz,
DMSO) d 10.87 (s, 1H), 9.03 (s, 1H), 7.34 (dd, J = 7.6, 2.5 Hz, 1H),

K. Jin et al. / Bioorg. Med. Chem. 21 (2013) 2663–2670
2669
7.29 (td, J = 9.1, 2.5 Hz, 1H), 6.97 (dd, J = 8.6, 4.0 Hz, 1H), 4.36–4.33
(m, 2H), 4.30 (dd, J = 8.4, 4.0 Hz, 2H), 4.19 (s, 2H). HRMS (AP-ESI)
m/z Calcd for C12H11FN2O5 [M+H]+ 283.0652. Found: 283.0719.
6.1 Hz, 1H), 1.68 (d, J = 13.5 Hz, 1H). HRMS (AP-ESI) m/z Calcd for
13H14N2O5 [M+H]+ 279.0903. Found: 279.0975.
C
5.1.14. N-Hydroxy-2-(50-methyl-20-oxospiro[[1,3]dioxane-2,30-
indoline]-10-yl)acetamide (11e)
5.1.7. 2-(50-Bromo-20-oxospiro[[1,3]dioxolane-2,30-indoline]-10-
yl)-N-hydroxyacetamide (10c)
White solid, yield 64.1%, mp = 145–146 °C. 1H NMR (600 MHz,
DMSO) d 10.85 (s, 1H), 9.02 (s, 1H), 7.20 (s, 1H), 7.16 (d,
J = 8.0 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 4.74 (td, J = 11.7, 2.5 Hz,
2H), 4.15 (s, 2H), 3.91 (dd, J = 9.8, 5.0 Hz, 2H), 2.27 (s, 3H), 2.20–
2.12 (m, 1H), 1.71–1.64 (m, 1H). HRMS (AP-ESI) m/z Calcd for
White solid, yield 72.3%, mp = 163–164 °C. 1H NMR (600 MHz,
DMSO) d 10.87 (s, 1H), 9.03 (s, 1H), 7.63 (dd, J = 8.4, 2.0 Hz, 1H),
7.60 (d, J = 1.9 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 4.34–4.32 (m, 2H),
4.32–4.30 (m, 2H), 4.19 (s, 2H). HRMS (AP-ESI) m/z Calcd for
C
14H16N2O5 [M+H]+ 293.1059. Found: 293.1127.
C
12H11BrN2O5 [M+H]+ 342.9851. Found: 342.9928.
5.1.15. 2-(50-Chloro-20-oxospiro[[1,3]dioxepane-2,30-indoline]-
10-yl)-N-hydroxyacetamide (12a)
5.1.8. N-Hydroxy-2-(20-oxospiro[[1,3]dioxolane-2,30-indoline]-
10-yl)acetamide (10d)
White solid, yield 53.6%, mp = 153–154 °C. 1H NMR (600 MHz,
DMSO) d 10.86 (s, 1H), 9.03 (s, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.45
(dd, J = 8.4, 2.1 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 4.25 (dd, J = 11.6,
4.6 Hz, 2H), 4.20 (s, 2H), 4.02 (dd, J = 10.9, 5.4 Hz, 2H), 1.71
(t, J = 8.1 Hz, 4H). HRMS (AP-ESI) m/z Calcd for C14H15ClN2O5
[M+H]+ 327.0669. Found: 327.0745.
White solid, yield 69.7%, mp = 143–144 °C. 1H NMR (600 MHz,
DMSO) d 10.88 (s, 1H), 9.03 (s, 1H), 7.44–7.35 (m, 2H), 7.12–7.07
(m, 1H), 6.94 (d, J = 7.9 Hz, 1H), 4.36 (d, J = 3.4 Hz, 2H), 4.29 (d,
J = 3.4 Hz, 2H), 4.18 (s, 2H). HRMS (AP-ESI) m/z Calcd for
C
12H12N2O5 [M+H]+ 265.0746. Found: 265.0817.
5.1.16. 2-(50-Fluoro-20-oxospiro[[1,3]dioxepane-2,30-indoline]-
10-yl)-N-hydroxyacetamide (12b)
5.1.9. N-Hydroxy-2-(50-methyl-20-oxospiro[[1,3]dioxolane-2,30-
indoline]-10-yl)acetamide (10e)
White solid, yield 48.6%, mp = 159–160 °C. 1H NMR (600 MHz,
DMSO) d 10.86 (s, 1H), 9.02 (d, J = 1.2 Hz, 1H), 7.56 (dd, J = 8.1,
2.6 Hz, 1H), 7.24 (td, J = 9.2, 2.6 Hz, 1H), 6.95 (dd, J = 8.7, 4.1 Hz,
1H), 4.25 (t, J = 11.4 Hz, 2H), 4.19 (s, 2H), 4.01 (d, J = 12.1 Hz, 2H),
1.68 (d, J = 37.5 Hz, 4H). HRMS (AP-ESI) m/z Calcd for C14H15FN2O5
[M+H]+ 311.0965. Found: 311.1038.
White solid, yield 75.8%, mp = 157–158 °C. 1H NMR (600 MHz,
DMSO) d 10.85 (s, 1H), 9.01 (s, 1H), 7.21 (d, J = 6.2 Hz, 2H), 6.83
(d, J = 8.5 Hz, 1H), 4.36–4.34 (m, 2H), 4.29–4.26 (m, 2H), 4.14 (s,
2H), 2.28 (s, 3H). HRMS (AP-ESI) m/z Calcd for C13H14N2O5
[M+H]+ 279.0903. Found: 279.0981.
5.1.17. 2-(50-Bromo-20-oxospiro[[1,3]dioxepane-2,30-indoline]-
10-yl)-N-hydroxyacetamide (12c)
5.1.10. 2-(50-Chloro-20-oxospiro[[1,3]dioxane-2,30-indoline]-10-
yl)-N-hydroxyacetamide (11a)
White solid, yield 51.7%, mp = 152–154 °C. 1H NMR (600 MHz,
DMSO) d 10.86 (s, 1H), 9.03 (s, 1H), 7.79 (d, J = 1.8 Hz, 1H), 7.58
(dd, J = 8.4, 1.9 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 4.24 (dd, J = 17.7,
13.2 Hz, 2H), 4.19 (s, 2H), 4.01 (dd, J = 11.1, 5.5 Hz, 2H), 1.72 (q,
White solid, yield 83.5%, mp = 152–153 °C. 1H NMR (600 MHz,
DMSO) d 10.88 (s, 1H), 9.04 (s, 1H), 7.47 (dd, J = 8.4, 2.1 Hz, 1H),
7.41 (d, J = 2.0 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 4.71 (dd, J = 11.7,
9.4 Hz, 2H), 4.21 (s, 2H), 3.94 (dd, J = 11.5, 3.0 Hz, 2H), 2.23–2.12
(m, 1H), 1.69 (d, J = 13.5 Hz, 1H). HRMS (AP-ESI) m/z Calcd for
J = 14.0 Hz, 4H). HRMS (AP-ESI) m/z Calcd for
C14H15BrN2O5
[M+H]+ 371.0164. Found: 371.0237.
C
13H13ClN2O5 [M+H]+ 313.0513. Found: 313.0586.
5.1.18. Methyl-2-(2-(50-bromo-20-oxospiro[[1,3]dioxolane-2,30-
indoline]-10-yl)acetamido)acetate (14a)
5.1.11. 2-(50-Fluoro-20-oxospiro[[1,3]dioxane-2,30-indoline]-10-
yl)-N-hydroxyacetamide (11b)
To a solution of compound 13 (0.50 g, 1.5 mmol) and N-methyl-
morpholine (0.36 mL, 3.3 mmol) in 30 ml anhydrous THF was
added isobutyl chloroformate (0.23 mL, 1.8 mmol) at ꢀ15 °C. The
mixture was stirred for 30 min at the same temperature. A solution
of compound glycine methyl ester hydrochloride (0.21 g,
1.65 mmol) in 20 mL THF was added dropwise to the mixture. Keep
the reaction for 1 h at ꢀ15 °C, and then removed the cooling bath.
The reaction mixture was stirred for another 12 h at room temper-
ature. Filtrated and concentrated with a rotary evaporator. The res-
idue was dissolved in 50 mL EtOAc and washed with saturated
citric acid, saturated NaHCO3 and brine in turn. The EtOAc phase
was dried over Na2SO4 and concentrated with a rotary evaporator
to get crude product. The crude product was purified by fast col-
umn chromatograph (PE/EtOAc = 5:1) to afford compound 14a as
white solid (0.46 g, yield: 76.7%), mp = 174–174.5 °C. 1H NMR
(600 MHz, DMSO) d 7.63–7.59 (m, 2H), 6.88 (d, J = 8.1 Hz, 1H),
4.35 (s, 2H), 4.34 (d, J = 6.7 Hz, 2H), 4.31 (d, J = 3.8 Hz, 2H), 3.89
(d, J = 5.8 Hz, 2H), 3.63 (s, 3H). ESI-MS m/z: 399.2 [M+1]+.
White solid, yield 78.4%, mp = 161–161.5 °C. 1H NMR (600 MHz,
DMSO) d 10.88 (s, 1H), 9.04 (s, 1H), 7.28–7.22 (m, 2H), 6.95 (dd,
J = 8.4, 4.0 Hz, 1H), 4.72 (t, J = 10.6 Hz, 2H), 4.20 (s, 2H), 3.99–3.87
(m, 2H), 2.18 (qd, J = 12.1, 5.8 Hz, 1H), 1.69 (d, J = 13.5 Hz, 1H).
HRMS (AP-ESI) m/z Calcd for C13H13FN2O5 [M+H]+ 297.0808.
Found: 297.0884.
5.1.12. 2-(50-Bromo-20-oxospiro[[1,3]dioxane-2,30-indoline]-10-
yl)-N-hydroxyacetamide (11c)
White solid, yield 83.2%, mp = 149–150 °C. 1H NMR (600 MHz,
DMSO) d 10.87 (s, 1H), 9.04 (s, 1H), 7.59 (dd, J = 8.4, 2.0 Hz, 1H),
7.51 (d, J = 2.0 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.71 (t, J = 10.6 Hz,
2H), 4.20 (s, 2H), 3.94 (d, J = 11.6 Hz, 2H), 2.23–2.11 (m, 1H), 1.69
(d, J = 13.2 Hz, 1H). HRMS (AP-ESI) m/z Calcd for C13H13BrN2O5
[M+H]+ 357.0008. Found: 357.0081.
The final compounds (15a–15f) were obtained via the same
method as compound 10a which had been introduced previously.
5.1.13. N-Hydroxy-2-(20-oxospiro[[1,3]dioxane-2,30-indoline]-
10-yl)acetamide (11d)
White solid, yield 65.6%, mp = 149–150 °C. 1H NMR (600 MHz,
DMSO) d 10.88 (s, 1H), 9.03 (s, 1H), 7.37 (d, J = 7.3 Hz, 2H), 7.06
(t, J = 7.3 Hz, 1H), 6.92 (d, J = 7.8 Hz, 1H), 4.75 (td, J = 11.8, 2.6 Hz,
2H), 4.19 (s, 2H), 3.92 (dd, J = 10.8, 4.1 Hz, 2H), 2.17 (qd, J = 12.2,
5.1.19. 2-(50-Bromo-20-oxospiro[[1,3]dioxolane-2,30-indoline]-
10-yl)-N-(2-(hydroxyamino)-2-oxoethyl)acetamide (15a)
White solid, yield 43.5%, mp = 173–174 °C. 1H NMR (600 MHz,
DMSO) d 10.57 (s, 1H), 8.86 (d, J = 1.3 Hz, 1H), 8.57 (t, J = 5.8 Hz,

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K. Jin et al. / Bioorg. Med. Chem. 21 (2013) 2663–2670
1H), 7.61–7.56 (m, 2H), 6.92–6.90 (m, 1H), 4.35–4.32 (m, 4H),
4.32–4.30 (m, 2H), 3.65 (d, J = 5.8 Hz, 2H). HRMS (AP-ESI) m/z Calcd
for C14H14BrN3O6 [M+H]+ 400.0066. Found: 400.0141.
suspension of ES-2 cells in PBS (1 ꢁ 105/well). After adding the de-
tected compounds, the solution with various concentrations was
incubated with APN at 37 °C for 5 min. Then the solution of
substrate was added into the above mixture, which was incu-
bated for another 30 min at 37 °C. The hydrolysis of the substrate
was measured by following the change in the absorbance
monitored at 405 nm with a plate reader (Varioskan, Thermo,
USA).
5.1.20. 2-(2-(50-Bromo-20-oxospiro[[1,3]dioxolane-2,30-
indoline]-10-yl)acetamido)-N-hydroxy-3-phenylpropanamide
(15b)
White solid, yield 39.7%, mp = 173–174 °C. 1H NMR (600 MHz,
DMSO) d 10.76 (d, J = 12.9 Hz, 1H), 8.93 (s, 1H), 8.71 (d, J = 8.7 Hz,
1H), 7.56 (d, J = 1.8 Hz, 1H), 7.45 (dd, J = 8.3, 1.7 Hz, 1H), 7.30 (t,
J = 7.2 Hz, 2H), 7.25 (t, J = 7.8 Hz, 4H), 6.38 (d, J = 8.4 Hz, 1H),
4.42–4.35 (m, 2H), 4.34–4.26 (m, 4H), 3.00 (dd, J = 13.6, 5.0 Hz,
1H), 2.77 (dd, J = 13.5, 9.8 Hz, 1H). HRMS (AP-ESI) m/z Calcd for
5.2.2. In vitro HDACs inhibition assay
In vitro HDACs inhibitory activity assay was determined by
using Boc-Lys (acetyl)-AMC as substrate and Hela nuclear extract
(containing HDAC1, HDAC3, HDAC5 and HDAC8) as enzymes in
C
21H20BrN3O6 [M+H]+ 490.0535. Found: 490.0607.
15 mM Tris–HCl (PH8.0), at 37 °C. Fist, 10
was added to tested compounds solutions at various concentra-
tions (50 L) and incubated for 5 min at 37 °C. Then 40 L of sub-
strate was added and the mixture continued to incubate for
another 30 min in the same environment. Finally, 100 L of devel-
lL of enzymes solution
5.1.21. 2-(2-(50-Bromo-20-oxospiro[[1,3]dioxolane-2,30-
indoline]-10-yl)acetamido)-N-hydroxy-4-methylpentanamide
(15c)
l
l
l
White solid, yield 38.9%, mp = 170–171 °C. 1H NMR (600 MHz,
DMSO) d 10.73 (d, J = 1.4 Hz, 1H), 8.87 (d, J = 1.6 Hz, 1H), 8.50 (d,
J = 8.3 Hz, 1H), 7.60 (dd, J = 6.8, 2.0 Hz, 2H), 6.86–6.82 (m, 1H),
4.37–4.26 (m, 6H), 4.22–4.14 (m, 1H), 1.55 (td, J = 13.5, 6.7 Hz,
1H), 1.45 (t, J = 7.3 Hz, 2H), 0.89 (d, J = 6.6 Hz, 3H), 0.82 (d,
oper which containing trypsin and TSA was putted into the mix-
ture. Twenty minutes later, fluorescence intensity was measured
at 390 nm excitation and 460 nm emission wavelengths with a
microplate reader.
J = 6.6 Hz, 3H). HRMS (AP-ESI) m/z Calcd for
C18H22BrN3O6
5.2.3. MTT assay
[M+H]+ 456.0692. Found: 456.0769.
ES-2 cell and MDA-MB-231 cell were grown in RPMI1640 med-
ium with 10% FBS at 37 °C in 5% CO2 humidified incubator. Cell pro-
liferation was determined by MTT [(3-[4,5-dimethyl-2-thiazolyl]-
2,5-diphenyl-2H-tetrazolium bromide)] method. In brief, cells
were pated in 96-well plates (5000/well) and cultivated for 4 h,
and then different concentrations of inhibitors were added. Fol-
lowed by another 48 h treatment, 1% MTT was added each well.
Four hours later, DMSO was added and mixed for 15 min. Finally,
the optical density values were monitored at 570 nm.
5.1.22. 2-(2-(50-Bromo-20-oxospiro[[1,3]dioxolane-2,30-
indoline]-10-yl)acetamido)-N-hydroxy-3-methylpentanamide
(15d)
White solid, yield 41.2%, mp = 168–169 °C. 1H NMR (600 MHz,
DMSO) d 10.70 (s, 1H), 8.90 (s, 1H), 8.47 (d, J = 9.0 Hz, 1H), 7.60
(d, J = 6.1 Hz, 2H), 6.85 (d, J = 8.8 Hz, 1H), 4.43–4.25 (m, 6H), 4.00
(dd, J = 20.9, 12.5 Hz, 1H), 1.70 (s, 1H), 1.45 (s, 1H), 1.13–1.01 (m,
1H), 0.82 (t, J = 6.9 Hz, 6H). HRMS (AP-ESI) m/z Calcd for
C
18H22BrN3O6 [M+H]+ 456.0692. Found: 456.0765
Acknowledgments
5.1.23. 2-(2-(50-Bromo-20-oxospiro[[1,3]dioxolane-2,30-
indoline]-10-yl)acetamido)-N-hydroxy-2-phenylacetamide (15e)
White solid, yield 67.3%, mp = 200–202 °C. 1H NMR (600 MHz,
DMSO) d 11.03 (s, 1H), 9.18–8.93 (m, 2H), 7.60–7.53 (m, 1H),
7.46–7.24 (m, 7H), 5.31 (d, J = 8.1 Hz, 1H), 4.42 (d, J = 17.2 Hz,
1H), 4.34–4.26 (m, 2H), 4.06–4.00 (m, 2H), 3.89–3.77 (m, 1H).
HRMS (AP-ESI) m/z Calcd for C20H18BrN3O6 [M+H]+ 476.0379.
Found: 476.0461.
This work was supported by National Science and Technology
Specific Projects of China (No. 2009ZX09103-118) and National
Nature Science Foundation of China (Nos. 90713041, 30772654
and 21172134).
References and notes
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5.1.24. 2-(2-(50-Bromo-20-oxospiro[[1,3]dioxolane-2,30-
indoline]-10-yl)acetamido)-N-hydroxy-3-methylbutanamide
(15f)
White solid, yield 48.4%, mp = 173–174 °C. 1H NMR (600 MHz,
DMSO) d 10.69 (s, 1H), 8.90 (s, 1H), 8.45 (d, J = 8.9 Hz, 1H), 7.60
(d, J = 6.8 Hz, 2H), 6.86 (d, J = 9.0 Hz, 1H), 4.43–4.23 (m, 6H), 3.96
(t, J = 8.3 Hz, 1H), 1.92 (dq, J = 13.8, 6.9 Hz, 1H), 0.85 (dd, J = 11.8,
6.8 Hz, 6H). HRMS (AP-ESI) m/z Calcd for C17H20BrN3O6 [M+H]+
442.0535. Found: 442.0602.
8. Aoyagi, T.; Yoshida, S.; Nakamura, Y.; Shigihara, Y.; Hamada, M.; Takeuchi, T. J.
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13339.
5.2.1. In vitro APN inhibition assay
IC50 values against APN were determined by using L-leu-p-nitro-
anilide as substrate and Microsomal aminopeptidase from Porcine
Kidney Microsomes (Sigma) as enzyme in 50 mM PBS (pH 7.2) or