Design, synthesis and biological evaluation of novel 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid derivatives as aminopeptidase N/CD13 inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.08.041

A series of novel 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were designed, synthesized and assayed for their activities against aminopeptidase N (APN/CD13) and MMP-2. The results showed that most compounds exhibited higher inhibitory activities against APN than that of MMP-2. Within this series, compound 12h (IC₅₀ = 6.28 ± 0.11 μM) showed similar inhibitory activities compared with Bestatin (IC₅₀ = 5.55 ± 0.01 μM), and it could be used as novel lead compound for the future APN inhibitors development as anticancer agents.

Full text of DOI:10.1016/j.bmc.2011.08.041

Bioorganic & Medicinal Chemistry 19 (2011) 6015–6025
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel 1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid derivatives as
aminopeptidase N/CD13 inhibitors
⇑
Xiaopan Zhang, Jian Zhang, Lei Zhang, Jinghong Feng, Yingying Xu, Yumei Yuan, Hao Fang, Wenfang Xu
Department of Medicinal Chemistry, School of Pharmacy, Shan Dong University, 44, West Culture Road, Ji’nan 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2011
Revised 18 August 2011
Accepted 19 August 2011
Available online 24 August 2011
A series of novel 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were designed, synthesized
and assayed for their activities against aminopeptidase N (APN/CD13) and MMP-2. The results showed
that most compounds exhibited higher inhibitory activities against APN than that of MMP-2. Within this
series, compound 12h (IC50 = 6.28 0.11
tin (IC50 = 5.55 0.01 M), and it could be used as novel lead compound for the future APN inhibitors
development as anticancer agents.
lM) showed similar inhibitory activities compared with Besta-
l
Keywords:
Aminopeptidase N
Ó 2011 Elsevier Ltd. All rights reserved.
Inhibitors
1,2,3,4-Tetrahydroisoquinoline-3-carboxylic
acid derivatives
Structure-based drug design
1. Introduction
Our group has reported some novel APN inhibitors such as 3-
amino-2-hydroxy-4-phenyl butanoic acid (AHPA) derivatives,18–20
chloramphenicol amine derivatives,21 3-phenylpropane-1,2-
Aminopeptidase N (APN; EC 3.4.11.2) is a 150 kDa membrane-
bond zinc-dependent type II ectopeptidase, and belongs to the
M1 family of MA clan of peptidase.1,2 APN can preferentially re-
lease neutral or basic amino acids from the N-terminal of unsubsti-
tuted oligopeptides, amides and arylamide, with the exception of
peptides with Pro in the penultimate position.3 APN is widely dis-
tributed in most of mammalian tissues such as kidney,4 intestine,5
liver,6 lung,7 brain,8 fibroblasts,9 vessels of smooth muscle,10 hae-
matopoietic system,11 and so on. The widely distribution of APN
associated with its broad substrate specificity suggests that APN
has probably several physiological functions, such as protein mod-
ification, activation, and degradation as well as in the metabolism
of biologically active peptides in tumor metastasis and leuke-
mia.12,13 So the design and synthesis of APN inhibitors may result
in potential therapeutic agents.
To date, many natural or synthetic small molecule APN inhibi-
tors have been reported, such as amastatin, bestatin, probestin,
phebestin, AHPA-Val and so on.14–16 Among these inhibitors, best-
atin is the only one being used in clinical as anti-cancer agent.
Bestatin is a dipeptide immunomodulator which was first isolated
from the culture filtrate of Streptomyces olivoreticuli in 1976,17 and
now is widely used as the positive control in the search of small
molecule APN inhibitors.
diamine derivatives,22,23 -lysine derivatives,24
L L-arginine deriva-
tives,25,26 1,3,4-thiadiazole derivatives27 and so on. In our previous
work, we have reported a series of novel APN inhibitors with
cyclic-imide scaffold, and found that compound 13f showed the best
APN inhibitory activity with the IC50 value to 1.8 l
M.28 In order to
find better APN inhibitors, we modified 13f by replacing the
hydroxamate group with different amino acid benzyl ester, amino
acid or organic amine, but the result was not satisfied.29 The
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) is
a
constrained analogue of phenylalanine with distinct geometrical
conformation and biological activity, has been used for many
compounds design. And our group has successfully used it in the
design of Histone deacetylases (HDACs) inhibitors.30,31 There is a
phenylalanine residue in the structure of 13f, considering the con-
formation constrained of Tic may be beneficial for APN inhibitors,
we introduced Tic for the modification of 13f. Furthermore, we also
used Tic as the key scaffold, by coupling it with natural or unnatural
amino acids or bi-peptides, we got a series of novel APN inhibitors
with 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid scaffold
(Fig. 1).
2. Chemistry
The target compound 9 was synthesized following the proce-
dures as shown in Scheme 1. The key intermediate compound 3
⇑
Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: wfxu@yahoo.cn (W. Xu).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.041

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X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 6015–6025
O
O
O
O
O
O
O
H
N
X
N
OH
N
OH
N
H
OH
N
H
N
H
N
H
N
H
NH
O
NH
O
NH2
O
X= natural or unnatural amino acids
or di-peptides
13f
9
Figure 1. The strategy for the design of APN inhibitors with 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid scaffold.
O
O
O
a
b
OH
Boc
OH
NH.HCl
OH
O
N
O
O
NH2
3
N
1
2
f
N
H
O
Ph
N
O
H2N
O
O
O
Boc
O
d, e
TFA.
Ph
Boc
O
c
N
NH
7
H2N
N
O
H
Boc
OH
O
O
5
N
H
4
6
O
O
O
O
O
O
N
OH
.HCl
i
g, h
N
H
N
H
N
OH
N
N
NH
O
H
H
N
O
Boc
9
8
Scheme 1. Reagents and conditions: (a) HCHO, HCl (concd), 90 °C; (b) NaOH, (Boc)2O; (c) DCC, HOSu, THF, 65 °C; (d) K2CO3, KI, acetone, BrCH2COOCH2Ph, 56 °C; (e) TFA, DCM;
(f) isobutyl chloroformate, NMM, THF, ꢁ20 °C; (g) 10%Pb/C, H2, EtOH; (h) isobutyl chloroformate, NMM, H2NOHꢀHCl, THF, ꢁ20 °C; (i) 3 N HCl–EtOAc.
O
O
O
O
H
N
H
N
X
X
X
O
c
a
b
N
H
OH
OH
Boc
N
H
OH
N
H
NH
O
N
N
O
.HCl
N
O
Boc
11a-u
Boc
10a-u
12a-u
3
Scheme 2. Reagents and conditions: (a) EDCI, HOBT, DCM, NH2-X-COOCH3ꢀHCl; (b) NH2OK, CH3OH; (c) 3 N HCl–EtOAc.
was easily prepared from
L
-Phe 1 via Pictet–Spengler cyclization
extent validating our strategy for designing APN special inhibitors.
This may due to the structure difference between APN and MMP-2
may lead to different requirements for their respective inhibitors.
APN is a membrane-bound zinc exopeptidase, which catalyzed
the removal of NH2-terminal amino acid from the peptide, while
MMP-2 is an endopeptidase which could cut the peptide to parts
from the specific amino acid residue of the peptide. As these
and amidation according to the literature.32 Compound 6 was pre-
pared as our early report.28 Then coupled compound 3 with com-
pound 6 by mixed anhydride method to give compound 7, which
was then catalytically hydrogenated using 10% Pd/C to afford com-
pound 8. Coupled compound 8 with hydroxylamine hydrochloride
and then deprotected the Boc group to give the target compound 9.
Compounds 12a–12u were synthesized according to the meth-
ods described in Scheme 2. Compound 3 coupled with different
methyl ester of amino acids or bi-peptides by classical EDCI/HOBt
method to give compounds 10a–10u. The ester groups of 10a–10u
can be easily converted to hydroximic acid groups by treated 10a–
10u with NH2OK in methanol to get the compounds 11a–11u. Fi-
nally the compounds 12a–12u were obtained by deprotecting
the Boc groups of 11a–11u in 3 N HCl–EtOAc.
1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid
derivatives
exhibited highly selective inhibition against APN, the following
structure–activity relationships (SARs) were mainly focused on
the APN/CD13 inhibition.
Compound 9 did not show expected activity towards APN,
which may indicated that the replacement of the Phe residue of
13f with Tic could not improve the affinity. This may due to the
cyclization of the amino group with the phenyl group could con-
straint the freedom of the amino group (which may be important
for APN interaction). In order to study whether the other substitu-
tion could improve the activity, we replaced the cyclic-imide scaf-
fold of compound 9 with other natural or unnatural amino acids or
dipeptides to get compounds 12a–12u. Of these compounds, com-
pound 12h with phenylglyine residue, gave the best inhibitory
3. Results and discussion
The newly synthesized 1,2,3,4-tetrahydroisoquinoline-3-car-
boxylic acid derivatives were assayed for the enzymatic inhibitions
both on APN from porcine kidney (Microsomal, Sigma) and MMP-2.
The results are listed in Table 1. MMP-2 is also a zinc-dependent
metalloproteinase and associated with the process of tumor inva-
sion and metastasis. The difference between them is that APN is
an exopeptidase while MMP-2 is an endopeptidase. In order to
observe the selectivity, all the target compounds were assayed
for the inhibitory activities both on APN and MMP-2 with bestatin
as the positive control.
activity with the IC50 value to 6.28 0.11 lM. Comparing com-
pounds 12a, 12b and 12c, we can see that the length of X group
was negatively relative with the APN inhibition. And from com-
pounds 12d, 12e, 12f and 12h, we can see that the APN inhibitory
activity is associated with the volume of hydrophobic side chain of
the amino acid, compounds with bigger volume hydrophobic side
chain of amino acid had better APN inhibitory activities. This
may imply that there is a space requirement in the binding pocket
to accommodate the suitable substituent. Comparing compounds
As shown in Table 1, most compounds exhibited higher inhibi-
tory activities against APN than that of MMP-2, which to some

X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 6015–6025
6017
Table 1
The structures and in vitro APN inhibitory activities of the target compounds and positive control bestatin
a
Compd
Structure
IC50
APN
(lM)
MMP-2
NAb
O
.HCl
OH
H
O
O
N
O
9
20.38 0.79
N
H
N
NH
O
O
OH
12a
12b
37.08 0.52
44.50 0.27
516.83 3.49
251.91 0.01
N
H
N
H
NH
O
.HCl
H
N
N
H
OH
NH
O
.
HCl
O
O
H
N
12c
12d
21.68 3.84
83.85 3.12
249.24 0.12
463.38 0.92
N
H
OH
.HCl
NH
O
H
N
N
H
OH
.HCl
NH
O
O
H
N
12e
12f
36.58 0.28
27.52 0.04
307.27 0.96
836.49 3.84
N
H
OH
NH
O
.HCl
O
H
N
N
H
OH
.HCl
NH
O
O
O
H
N
12g
12h
NAb
671.56 2.69
393.14 0.32
N
H
OH
.HCl
NH
O
H
N
6.28 0.11
N
H
OH
NH
O
O
.HCl
O
H
N
12i
12j
12k
12.82 0.16
9.39 0.19
8.77 0.17
939.57 3.27
540.03 3.51
166.11 0.04
N
H
OH
.HCl
NH
OH
O
H
N
N
H
OH
NH
O
S
.HCl
O
H
N
N
H
OH
.HCl
NH
O
HO
O
H
N
12l
40.19 0.08
46.55 1.09
104.41 2.72
267.73 0.31
200.63 0.73
N
H
OH
NH
O
.HCl
OH
O
H
N
12m
12n
N
H
OH
.HCl
NH
O
H
O
N
O
OH
.HCl
157.42 0.79
N
NH
(continued on next page)

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X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 6015–6025
Table 1 (continued)
a
Compd
Structure
IC50 (lM)
APN
MMP-2
HO
12o
12p
NAb
276.44 1.93
H
N
O
N
.HCl
OH
O
NH
OH
NH
H
O
O
15.87 0.11
43.44 0.04
NAb
N
O
N
H
NH
O
.HCl
HN
NAb
O
12q
12r
12s
12t
H
N
.HCl
OH
N
H
NH
O
O
H
N
N
H
480.72 7.86
465.28 1.05
555.15 3.01
NAb
NH
O
O
.HCl
OH
O
NH
OH
O
O
H
N
N
H
N
656.77 3.37
.HCl
H
NH
O
O
O
H
N
OH
NAb
N
H
N
H
.HCl
NH
O
O
.HCl
OH
O
H
N
12u
435.28 0.25
NAb
N
H
N
H
NH
NH2
O
Bestatin
5.55 0.01
163.48 1.13
OH
N
H
OH
O
a
Values are means of three experiments, standard derivation is given.
NA, No activity.
b
12h and 12i, there is only one carbon difference, compound 12h
showed better APN activity, this may indicate that the phenylgly-
ine residue would be more suitable for APN interaction. Compound
12j with a hydroxyl group in the para-position of the phenyl group
showed better activity than that of compound 12i, this may due to
the hydrogen bond formation between the hydroxyl group and
APN could enhance the interaction. Compound 12k with the
methionine residue also showed better APN inhibitory activity
among these compounds. However, the pseudotripeptides 12r–
12u with di-peptide substituents showed very poor APN activity,
this may due to that the large side chain interfered the interaction
of the hydroxamic acid group with the zinc ion in the active site of
APN.
Sybyl/Sketch module and optimized using Powell’s method with
the Tripos force field with convergence criterion set at 0.05 kcal/
(Å mol), and assigned with Gasteiger–Hückel method. The docking
study performed using Sybyl7.3/FlexX module, the residues in a ra-
dius of 7.0 Å around Bestatin in the co-crystal structure (PDB code:
2DQM) were selected as the active site. Other docking parameters
implied in the program were kept default. As diagrammed inFigure
2, the backbone of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid can insert into the S02 pocket, the hydroxamic acid group of
12h can interact with the zinc ion in the active site of APN, and
the phenyl group of L-phenylglycine moiety can insert into the S1
pocket. For a further and detail understanding of the binding mode
of 12h with APN, a 2D picture was also created with the program
In order to investigate the interaction of the target compounds
with APN, the most active compound 12h was constructed with a
LIGPLOT (Fig. 3), we can see that the phenyl group of
L-phenylgly-
cine moiety could form hydrophobic interaction with Tyr376
,

X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 6015–6025
6019
interaction modes with APN. In order to verify the assumption,
the docking study of the representative compound 12j was also
investigated by using the same method as compound 12h. As is
shown in Figure 4, we can see that in contrast to compound 12h,
the backbone 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid of
12j is insert into the S1 pocket, though the hydroxamic acid group
can interact well with the zinc ion in the active site of APN, the Tyr
residue cannot occupy the S1’ pocket, the inhibitory activity of 12j
is less than that of 12h.
Although the computed information partially supported our
assumption, the exact binding mode of the 1,2,3,4-tetrahydroiso-
quinoline-3-carboxylic acid derivatives with APN should be ob-
tained from further X-ray crystal studies.
In order to study the inhibitory activity of the target compounds
towards human APN, we also tested the enzymatic inhibitory
activity of compound 12h towards APN expressed on the surface
of human ovary clear cell carcinoma cell ES-2. The results showed
that, just like bestatin, compound 12h is also a dose-dependent
Figure 2. The FlexX docking result of 12h with the active site of APN (PDB: 2DQM).
Zinc ion is shown as pale sphere.
APN inhibitor in the concentration range of 0.39–400
the IC50 value to 32.69 1.17 M which is similar with that besta-
tin (IC50 = 29.67 0.11 M) (Fig. 5).
lM with
l
l
Met260 and Tyr381 of the S1 pocket the two oxygen atoms of
hydroxamic acid could chelate the zinc ion of APN. The carbonyl
group could form hydrogen bond with His297 at a distance of
3.06 Å, and the hydroxyl group could form hydrogen bond with
Glu264, Glu298 and His301 at the distance of 2.20, 2.25 and 2.94 Å,
respectively. The phenyl group of the Tic moiety could form hydro-
In addition, the effects of compound 12h on the proliferation of
two human tumor cell lines (ES-2 cells and MDA-MB-231cells)
compared with bestatin were further assessed by using MTT assay
(Fig. 6). The results showed that just like bestatin, the anti-prolifer-
ative effect of 12h against ES-2 cells is better than that of MDA-
MB-231cells, which may due to the higher APN expression on
ES-2 cells than that of MDA-MB-231 cells. Additionally, consistent
with the results of enzyme inhibition, compound 12h also showed
slightly lower anti-proliferative effect on ES-2 cells than that of
phobic interaction with the S02 pocket that formed by Leu378, Thr377
,
and Glu382. The other compounds had less activities than com-
pound 12h, which may due to the different binding energy or
Figure 3. The docking result of 12h with APN showed by LIGPLOT. Compound 12h is shown in violet.

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X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 6015–6025
5. Experiment
5.1. Chemistry: general procedures
All the material we used were purchased from commercial sup-
pliers and used without further purification. Solvents were distilled
prior to use and flash chromatography was performed using silica
gel (60 Å, 200 300 mesh). All reactions were monitored by thin-
layer chromatography on 0.25 mm silica gel plates (60GF-254)
and visualized with UV light, or chloride ferric. Melting points were
determined on an electrothermal melting point apparatus and
were uncorrected. Proton nuclear magnetic resonance (1H NMR)
spectra were determined on a Brucker Avance 600 spectrometer
using TMS as an internal standard in DMSO-d6 solutions. Chemical
shifts are reported in delta (d) units, parts per million (ppm) down-
field from trimethylsilane. ESI-MS were determined on an API 4000
spectrometer. High-resolution mass spectral (HRMS) data were
conducted by Shandong Analysis and Test Center, and are reported
as m/z (relative intensity).
Figure 4. The FlexX docking result of 12j with the active site of APN (PDB: 2DQM).
5.1.1. (S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (2)
The title compound was prepared as described by Cheng et al.32
ESI-MS m/z: 178.3 [M+H]+.
5.1.2. (S)-2-(tert-Butoxycarbonyl)-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid (3)
The title compound was prepared as described by Cheng et al.32
ESI-MS m/z: 278.4 [M+H]+.
5.1.3. (S)-Benzyl 2-(3-amino-2,6-dioxopiperidin-1-yl)acetate
trifluoroacetate (6)
The title compound was prepared as described by Li et al.28
Figure 5. Inhibition of APN/CD13 activity on ES-2 cells induced by 12h or bestatin.
Data are expressed as mean values of three independent experiments ( SE).
5.1.4. (S)-tert-Butyl-3-(((S)-1-(2-(benzyloxy)-2-oxoethyl)-2,6-
dioxopiperidin-3-yl)carbamoyl)-3,4-dihydroisoquinoline-
2(1H)-carboxylate (7)
To a solution of compound 3 (0.54 g, 2 mmol) and N-methyl-
morpholine (0.26 ml, 2.2 mmol) in 30 ml anhydrous THF was
added isobutyl chloroformate (0.30 ml, 2.2 mmol) at ꢁ20 °C. The
mixture was stirred for 30 min at the same temperature. A solution
of compound 6 (2 mmol) in DCM (5 ml) (which was previous neu-
tralized with N-methylmorpholine) was added dropwise to the
reaction mixture. Keep the reaction for 1 h at ꢁ20 °C and then re-
move the cooling bath. The reaction was continued for another 4 h
at room temperature. Filtrated and concentrated with a rotary
evaporator. The residue was dissolved in 50 ml EtOAc and washed
with saturated NaHCO3 (10 ml ꢂ 3), saturated citric acid
(10 ml ꢂ 3) and brine (10 ml ꢂ 2) in turn. The organic phase was
dried over anhydrous Na2SO4 and concentrated with a rotary evap-
orator to afford crude product. The crude product was recrystal-
lized by EtOAc/diethyl ether to give compound 7 as white solid
Figure 6. Anti-proliferative activities of compound 12h and bestatin against two
tumor cell lines (ES-2 cells and MDA-MB-231 cells). The columns represent the
mean values of three independent experiments.
(0.81 g, yield 75.6%), mp = 118–120 °C. ½a D20
¼ ꢁ41:2 (c 0.5, DMSO).
ꢃ
1H NMR (600 MHz, DMSO-d6) d 1.38–1.45 (m, 9H), 1.77–1.81 (m,
1H), 1.86–1.93 (m, 1H), 2.67–2.70 (m, 1H), 2.89–2.93 (m, 1H),
2.96–3.00 (m, 1H), 3.12–3.16 (m, 1H), 4.36–4.50 (m, 4H), 4.58–
4.63 (m, 1H), 4.68–4.73 (m, 1H), 5.14 (s, 2H), 7.16–7.20 (m, 5H),
7.34–7.40 (m, 4H), 8.33–8.38 (m, 1H). ESI-MS m/z: 536.6 [M+H]+.
bestatin with the IC50 value of 174.34 0.09
141.89 8.12 M.
lM
versus
l
4. Conclusion
In summary, we have described the synthesis and properties of
a series of novel 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
derivatives as APN inhibitors. Most of the target compounds
showed potent inhibitory activities towards APN, and compound
12h showed the best inhibition both in the enzymatic assay and
cell-based assay, which makes it a good novel lead for future
APN inhibitors development as anticancer agents.
5.1.5. 2-((S)-3-((S)-2-(tert-Butoxycarbonyl)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamido)-2,6-dioxopiperidin-1-
yl)acetic acid (8)
Compound 7 (1.07 g, 2 mmol) and catalytic amount of 10% Pd/C
in methanol (30 ml) was hydrogenated in the presence of H2 at
room temperature. After 16 h, the catalyst was filtered and the sol-
vent was removed under vacuum. The residue was then dissolved

X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 6015–6025
6021
in 30 ml anhydrous THF at ꢁ20 °C, N-methylmorpholine (0.26 ml,
2.2 mmol) was added, 2 min later, isobutyl chloroformate
(0.30 ml, 2.2 mmol) was added to the mixture. The reaction was
then stirred for 30 min at ꢁ20 °C. A solution of hydroxylamine
hydrochloride (0.14 g, 2 mmol) in MeOH (2 ml) (which was previ-
ous neutralized with N-methylmorpholine) was added dropwise to
the reaction mixture. Keep the reaction for 1 h at ꢁ20 °C and then
remove the cooling bath. The reaction was continued for another
4 h at room temperature. Filtrated and concentrated with a rotary
evaporator. The residue was dissolved in 50 ml EtOAc and washed
with saturated NaHCO3 (10 ml ꢂ 3), saturated citric acid
(10 ml ꢂ 3) and brine (10 ml ꢂ 2) in turn. The organic phase was
dried over anhydrous Na2SO4 and concentrated with a rotary evap-
orator. The residue was purified by column chromatography to
give compound 8 in white solid (0.46 g, yield 50.0%), mp = 164–
5.1.7.2. (S)-tert-Butyl-3-((2-methoxy-2-oxoethyl)carbamoyl)-3,
4-dihydroisoquinoline-2(1H)-carboxylate (10c).
Yield 78.5%, mp = 125–126 °C. ESI-MS m/z: 349.5 [M+H]+.
5.1.7.3. (S)-tert-Butyl-3-(((S)-1-methoxy-1-oxopropan-2-yl)car-
bamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (10d).
Yield 68.5%, mp = 84–85 °C. ESI-MS m/z: 363.5 [M+H]+.
5.1.7.4. (S)-tert-Butyl-3-(((S)-1-methoxy-3-methyl-1-oxobutan-2-
yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (10e).
Yield 64.9%, oil. ESI-MS m/z: 391.5 [M+H]+.
5.1.7.5. (S)-tert-Butyl-3-(((S)-1-methoxy-4-methyl-1-oxopentan-2-
yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (10f).
Yield 70.3%, mp = 81–83 °C. ESI-MS m/z: 405.5 [M+H]+.
166 °C. ½a 2D0
ꢃ
¼ ꢁ84:3 (c 0.5, DMSO). 1H NMR (600 MHz, DMSO-d6)
d 1.38–1.48 (m, 9H), 1.75–1.76 (m, 1H), 1.86–1.91 (m, 1H), 2.62–
2.68 (m, 1H), 2.81–2.86 (m, 1H), 2.96–3.00 (m, 1H), 3.12–3.17
(m, 1H), 4.09–4.17 (m, 2H), 4.37–4.51 (m, 2H), 4.57–4.63 (m,
1H), 4.65–4.74 (m, 1H), 7.16–7.20 (m, 4H), 8.27–8.32 (m, 1H),
8.83 (s, 1H), 10.54 (s, 1H). ESI-MS m/z: 461.6 [M+H]+.
5.1.7.6. (S)-tert-Butyl-3-(((2S)-1-methoxy-3-methyl-1-oxopentan-2-
yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (10g).
Yield 73.5%, mp = 73–74 °C. ESI-MS m/z: 405.5 [M+H]+.
5.1.7.7. (S)-tert-Butyl-3-(((S)-2-methoxy-2-oxo-1-phenylethyl) car-
bamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (10h).
Yield 75.3%, mp = 134–135 °C. ESI-MS m/z: 425.5 [M+H]+.
5.1.6. (S)-N-((S)-1-(2-(Hydroxyamino)-2-oxoethyl)-2,6-
dioxopiperidin-3-yl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxamide hydrochloride (9)
Compound 8 (0.2 g, 0.43 mmol) was dissolved in 10 ml HCl–
EtOAc (3 mol/L) at 0 °C. The reaction solution was stirred at 0 °C
for 2 h, and then the temperature was raised to room temperature.
After 5 h, the solvent was filtrated and the precipitate was washed
with EtOAc to get compound 9 as white solid (0.15 g, yield 88.2%),
5.1.7.8. (S)-tert-Butyl-3-(((S)-1-methoxy-1-oxo-3-phenylpropan-2-
yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (10i).
Yield 68.3%, mp = 91–93 °C. ESI-MS m/z: 439.5 [M+H]+.
5.1.7.9. (S)-tert-Butyl-3-(((S)-3-(4-hydroxyphenyl)-1-methoxy-
1-oxopropan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-
carboxylate (10j).
mp = 198–200 °C. ½a D20
ꢃ
¼ ꢁ37:3 (c 0.5, DMSO). 1H NMR (600 MHz,
DMSO-d6) d 2.02–2.09 (m, 2H), 2.75–2.78 (m, 1H), 2.94–3.00 (m,
1H), 3.06–3.11 (m, 1H), 3.36–3.40 (m, 1H), 4.13–4.22 (m, 2H),
4.24–4.25 (m, 1H), 4.30–4.32 (m, 1H), 4.36–4.40 (m, 1H), 4.88–
4.93 (m, 1H), 7.27–7.32 (m, 4H), 9.13 (d, J = 9.0 Hz, 1H), 9.56 (d,
J = 6.6 Hz, 1H), 9.78 (s, 1H), 10.64 (s, 1H). HRMS (AP-ESI) m/z: calcd
for C17H21N4O5 [M+H]+ 361.1507, found 361.1504.
Yield 62.4%, mp = 62–65 °C. ESI-MS m/z: 455.5 [M+H]+.
5.1.7.10.
(S)-tert-Butyl-3-(((S)-1-methoxy-4-(methylthio)-1-
oxobutan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-car-
boxylate (10k).
Yield 60.9%, mp = 148–149 °C. ESI-MS m/z: 423.5 [M+H]+.
5.1.7. (S)-tert-Butyl-3-(3-methoxy-3-oxopropylcarbamoyl)-3,4-
dihydroisoquinoline-2(1H)-carboxylate (10a)
5.1.7.11. (S)-tert-Butyl-3-(((S)-3-hydroxy-1-methoxy-1-oxopro-
pan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxyl-
ate (10l).
To a solution of Compound 3 (1.62 g, 6 mmol), b-Alanine methyl
ester hydrochloride (0.74 g, 7.2 mmol), HOBt (0.97 g, 7.2 mmol)
and DMAP (0.07 g, 0.6 mmol) in dry DCM, was added TEA (1.82 g,
18 mmol). The reaction mixture was gently cooled to 0 °C in ice
bath. To the reaction mixture was added dropwise a solution of
EDCI (2.3 g, 12 mmol) in DCM for 30 min. After removal of the
ice bath, the reaction mixture was stirred at room temperature
for 12 h and filtered to remove the precipitate. The filtrate was
washed with 1 N citric acid solution, saturated NaHCO3 and brine,
dried over Na2SO4, and evaporated in vacuo to give the crude prod-
uct compound 10a (1.4 g, yield 64.5%), mp = 80–82 °C.
Yield 67.6%, mp = 69–71 °C. ESI-MS m/z: 379.5 [M+H]+.
5.1.7.12. (S)-tert-Butyl-3-(((2S)-3-hydroxy-1-methoxy-1-oxob-
utan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxyl-
ate (10m).
Yield 72.5%, mp = 80–81 °C. ESI-MS m/z: 393.5 [M+H]+.
5.1.7.13. (S)-tert-Butyl-3-((S)-2-(carbonyl)pyrrolidine-1-carbonyl)-
3,4-dihydroisoquinoline-2(1H)-carboxylate (10n).
Yield 70.5%, mp = 103–104 °C. ESI-MS m/z: 389.5 [M+H]+.
½
a 2D0
ꢃ
¼ ꢁ70:6 (c 0.5, DMSO). 1H NMR (600 MHz, DMSO-d6) d
1.36–1.45 (m, 9H), 2.25 (s, 1H), 2.36–2.38 (m, 1H), 2.90–2.94 (m,
1H), 3.03–3.07 (m, 1H), 3.17–3.33 (m, 2H), 3.54–3.56 (m, 3H),
4.33–4.36 (m, 1H), 4.46–4.64 (m, 2H), 7.12–7.19 (m, 4H), 7.85–
7.96 (m, 1H). ESI-MS: m/z: 363.5 [M+H]+.
5.1.7.14. (S)-tert-Butyl-3-((2S,4R)-2-(carbonyl)-4-hydroxypyrrol-
idine-1-carbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate
(10o).
Yield 72.4%, mp = 169–170 °C. ESI-MS m/z: 405.5 [M+H]+.
The other compounds (10b–10u) were prepared in the same
procedure as described above.
5.1.7.15. (S)-tert-Butyl-3-(((S)-6-(benzyloxycarbonyl)-1-meth-
oxy-1-oxohexan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-
2(1H)-carboxylate (10p).
5.1.7.1. (S)-tert-Butyl-3-((4-methoxy-4-oxobutyl)carbamoyl)-3,
4-dihydroisoquinoline-2(1H)-carboxylate (10b).
Yield 74.2%, mp = 79–80 °C. ESI-MS m/z: 377.5 [M+H]+.
Yield 73.2%, oil. ESI-MS m/z: 554.7 [M+H]+.

6022
X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 6015–6025
5.1.7.16. (S)-tert-Butyl-3-(((S)-3-(1H-indol-2-yl)-1-methoxy-1-
oxopropan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-
carboxylate (10q).
5.1.8.6.
(S)-tert-Butyl-3-(((2S)-1-(hydroxyamino)-3-methyl-1-
oxopentan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-
carboxylate (11g).
Yield 72.6%, mp = 71–72 °C. ESI-MS m/z: 478.6 [M+H]+.
Yield 28.4%, mp = 97–98 °C. ESI-MS m/z: 406.5 [M+H]+.
5.1.8.7. (S)-tert-Butyl-3-(((S)-2-(hydroxyamino)-2-oxo-1-phen-
ylethyl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate
(11h).
5.1.7.17. (S)-tert-Butyl-3-(((S)-1-((S)-1-methoxy-4-methyl-1-oxop-
entan-2-ylamino)-4-methyl-1-oxopentan-2-yl)carbamoyl)-3,4-
dihydroisoquinoline-2(1H)-carboxylate (10r).
Yield 28.6%, mp = 94–95 °C. ESI-MS m/z: 426.5 [M+H]+.
Yield 67.4%, mp = 79–80 °C. ESI-MS m/z: 518.7 [M+H]+.
5.1.8.8.
(S)-tert-Butyl-3-(((S)-1-(hydroxyamino)-1-oxo-3-phe-
5.1.7.18.
(S)-tert-Butyl-3-(((S)-1-((S)-1-methoxy-1-oxo-3-phe-
nylpropan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-car-
boxylate (11i).
nylpropan-2-ylamino)-4-methyl-1-oxopentan-2-yl)carbamoyl)-
3,4-dihydroisoquinoline-2(1H)-carboxylate (10s).
Yield 26.5%, mp = 86–87 °C. ESI-MS m/z: 440.5 [M+H]+.
Yield 73.6%, mp = 69–71 °C. ESI-MS m/z: 552.7 [M+H]+.
5.1.8.9. (S)-tert-Butyl-3-(((S)-1-(hydroxyamino)-3-(4-hydroxy-
phenyl)-1-oxopropan-2-yl)carbamoyl)-3,4-dihydroisoquino-
line-2(1H)-carboxylate (11j).
5.1.7.19. (S)-tert-Butyl-3-(((S)-1-((S)-1-methoxy-4-methyl-1-oxop-
entan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-3,4-
dihydroisoquinoline-2(1H)-carboxylate (10t).
Yield 25.6%, mp = 203–204 °C. ESI-MS m/z: 456.5 [M+H]+.
Yield 75.3%, mp = 164–165 °C. ESI-MS m/z: 552.7 [M+H]+.
5.1.8.10.
(S)-tert-Butyl-3-(((S)-1-(hydroxyamino)-4-(methyl-
thio)-1-oxobutan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-
2(1H)-carboxylate (11k).
5.1.7.20.
(S)-tert-Butyl-3-(((S)-1-((S)-1-methoxy-1-oxo-3-phe-
nylpropan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-
3,4-dihydroisoquinoline-2(1H)-carboxylate (10u).
Yield 28.3%, mp = 73–74 °C. ESI-MS m/z: 424.5 [M+H]+.
Yield 72.8%, mp = 73–74 °C. ESI-MS m/z: 587.7 [M+H]+.
5.1.8.11. (S)-tert-Butyl-3-(((S)-3-hydroxy-1-(hydroxyamino)-1-
oxopropan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-
5.1.8. (S)-tert-Butyl-3-((3-(hydroxyamino)-3-oxopropyl)
carboxylate (11l)
.
carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (11a)
To a solution of compound 10a (1.08 g, 3 mmol) in methanol
(7 ml) at room temperature was added dropwise a solution of
NH2OK (6 mmol) in methanol (3.4 ml). The mixture was stirred
for 12 h and the solvent was evaporated in vacuum. The residue
was purified by column chromatography to get compound 11a
Yield 26.4%, mp = 82–83 °C. ESI-MS m/z: 380.5 [M+H]+.
5.1.8.12. (S)-tert-Butyl-3-(((2S)-3-hydroxy-1-(hydroxyamino)-1-
oxobutan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-car-
boxylate (11m).
Yield 27.3%, mp = 196–197 °C. ESI-MS m/z: 394.5 [M+H]+.
(0.3 g, yield 27.5%), mp = 120–122 °C. ½a D20
¼ ꢁ74:5 (c 0.5, DMSO).
ꢃ
1H NMR (600 MHz, DMSO-d6) d 1.33–1.47 (m, 9H), 1.97–2.05 (m,
2H), 2.91–2.94 (m, 1H), 3.04 (s, 1H), 3.13–3.28 (m, 2H), 4.31–
4.34 (m, 1H), 4.48–4.64 (m, 2H), 7.19–7.31 (m, 4H), 7.88–7.95
(m, 1H), 10.37–10.42 (m, 1H). ESI-MS m/z: 364.5 [M+H]+.
The other compounds (11b–11u) were prepared in the same
procedure as described above.
5.1.8.13. (S)-tert-Butyl-3-((S)-2-(hydroxycarbamoyl)pyrrolidine-
1-carbonyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (11n).
Yield 26.8%, mp = 95–96 °C. ESI-MS m/z: 390.5 [M+H]+.
5.1.8.14. (S)-tert-Butyl-3-((2S,4R)-4-hydroxy-2-(hydroxycarba-
moyl)pyrrolidine-1-carbonyl)-3,4-dihydroisoquinoline-2(1H)-
carboxylate (11o).
5.1.8.1. (S)-tert-Butyl-3-((3-(hydroxyamino)-4-oxopropyl)car-
bamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (11b).
Yield 25.6%, mp = 79–80 °C. ESI-MS m/z: 378.5 [M+H]+.
Yield 27.3%, mp = 122–124 °C. ESI-MS m/z: 406.5 [M+H]+.
5.1.8.15. (S)-tert-Butyl-3-(((S)-6-(benzyloxycarbonyl)-1-(hydrox-
yamino)-1-oxohexan-2-yl)carbamoyl)-3,4-dihydroisoquino-
line-2(1H)-carboxylate (11p).
5.1.8.2. (S)-tert-Butyl-3-((3-(hydroxyamino)-2-oxopropyl)car-
bamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (11c).
Yield 27.3%, mp = 121–122 °C. ESI-MS m/z: 350.4 [M+H]+.
Yield 29.7%, mp = 77–78 °C. ESI-MS m/z:555.6 [M+H]+.
5.1.8.16. (S)-tert-Butyl-3-(((S)-1-(hydroxyamino)-3-(1H-indol-2-
yl)-1-oxopropan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-
2(1H)-carboxylate (11q).
5.1.8.3. (S)-tert-Butyl-3-(((S)-1-(hydroxyamino)-1-oxopropan-2-yl)
carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (11d).
Yield 26.5%, mp = 116–117 °C. ESI-MS m/z: 364.5 [M+H]+.
Yield 30.6%, mp = 114–115 °C. ESI-MS m/z: 479.6 [M+H]+.
5.1.8.4. (S)-tert-Butyl-3-(((S)-1-(hydroxyamino)-3-methyl-1-oxob-
utan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate
(11e).
5.1.8.17. (S)-tert-Butyl-3-(((S)-1-((S)-1-(hydroxyamino)-4-methyl-
1-oxopentan-2-ylamino)-4-methyl-1-oxopentan-2-yl)carbam-
oyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (11r).
Yield 28.3%, mp = 88–89 °C. ESI-MS m/z: 392.5 [M+H]+.
Yield 30.4%, mp = 172–173 °C. ESI-MS m/z: 519.7 [M+H]+.
5.1.8.5.
(S)-tert-Butyl-3-(((S)-1-(hydroxyamino)-4-methyl-1-
5.1.8.18. (S)-tert-Butyl-3-(((S)-1-((S)-1-(hydroxyamino)-1-oxo-
3-phenylpropan-2-ylamino)-4-methyl-1-oxopentan-2-yl)car-
bamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (11s).
Yield 32.1%, mp = 207–208 °C. ESI-MS m/z: 553.7 [M+H]+.
oxopentan-2-yl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-
carboxylate (11f).
Yield 30.1%, mp = 92–93 °C. ESI-MS m/z: 406.5 [M+H]+.

X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 6015–6025
6023
5.1.8.19. (S)-tert-Butyl-3-(((S)-1-((S)-1-(hydroxyamino)-4-methyl-
5.1.14. (S)-N-((S)-1-(Hydroxyamino)-4-methyl-1-oxopentan-2-
1-oxopentan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)carbam-
oyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (11t).
Yield 35.8%, mp = 198–201 °C. ESI-MS m/z: 553.7 [M+H]+.
yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
hydrochloride (12f)
White solid, yield 92.1%, mp = 207–209 °C. ½a D20
¼ ꢁ94:1 (c 0.5,
ꢃ
DMSO). 1H NMR (600 MHz, DMSO-d6) d 0.88 (d, J = 6.6 Hz, 3H), 0.91
(d, J = 6.6 Hz, 3H), 1.42–1.47 (m, 1H), 1.51–1.56 (m, 1H), 1.59–1.64
(m, 1H), 2.53–3.00 (m, 1H), 3.29–3.37 (m, 1H), 4.13–4.16 (m, 1H),
4.27–4.36 (m, 3H), 7.24–7.29 (m, 4H), 8.84–8.85 (m, 1H), 9.48–9.50
(m, 1H), 9.84 (s, 1H), 10.86 (s, 1H). HRMS (AP-ESI) m/z: calcd for
5.1.8.20. (S)-tert-Butyl-3-(((S)-1-((S)-1-(hydroxyamino)-1-oxo-
3-phenylpropan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)car-
bamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (11u).
Yield 40.2%, mp = 209–211 °C. ESI-MS m/z: 586.7 [M+H]+.
C
16H24N3O3 [M+H]+ 306.1812, found 306.1844.
5.1.9. (S)-N-(3-(Hydroxyamino)-3-oxopropyl)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide hydrochloride (12a)
Compound 11a (0.20 g, 0.55 mmol) was dissolved in 10 ml HCl–
EtOAc (3 mol/L) at 0 °C. The reaction solution was stirred at 0 °C for
2 h, and then the temperature was raised to room temperature.
After 5 h, the solvent was filtrated and the prepicitate was washed
with EtOAc to get compound 12a as white solid (0.15 g, yield
5.1.15. (S)-N-((2S)-1-(Hydroxyamino)-3-methyl-1-oxopentan-2-
yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
hydrochloride (12g)
White solid, yield 95.6%, mp = 219–220 °C. ½a D20
¼ ꢁ98:0 (c 0.5,
ꢃ
DMSO). 1H NMR (600 MHz, DMSO-d6) d 0.84–0.87 (m, 6H), 1.08–
1.13 (m, 1H), 1.51–1.55 (m, 1H), 1.70–1.74 (m, 1H), 2.90–2.95
(m, 1H), 3.27–3.30 (m, 1H), 4.08–4.10 (m, 1H), 4.18–4.20 (m, 1H),
4.28–4.37 (m, 2H), 7.25–7.28 (m, 4H), 8.81–8.83 (m, 1H). HRMS
91.0%), mp = 134–135 °C. ½a D20
¼ ꢁ117:6 (c 0.5, DMSO). 1H NMR
ꢃ
(600 MHz, DMSO-d6) d 2.21 (t, J = 7.2 Hz, 2H), 2.96–3.01 (m, 1H),
3.26–3.30 (m, 1H), 3.36–3.39 (t, J = 7.2 Hz, 2H), 4.11 (dd,
J = 4.8 Hz, 12 Hz, 1H), 4.28–4.36 (m, 2H), 7.23–7.28 (m, 4H). HRMS
(AP-ESI) m/z: calcd for
C
16H24N3O3 [M+H]+ 306.1812, found
306.1855.
(AP-ESI) m/z: calcd for
C
13H18N3O3 [M+H]+ 264.1343, found
264.1375.
5.1.16. (S)-N-((S)-2-(Hydroxyamino)-2-oxo-1-phenylethyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide hydrochloride
(12h)
The other compounds (12b–12u) were synthesized in the same
procedure as described above.
White solid, yield 90.8%, mp = 178–179 °C. ½a D20
¼ ꢁ64:7 (c 0.5,
ꢃ
5.1.10. (S)-N-(4-(Hydroxyamino)-4-oxobutyl)-1,2,3,4-
DMSO). 1H NMR (600 MHz, DMSO-d6) d 2.88–2.93 (m, 1H), 2.99–
3.04 (m, 1H), 4.25–4.36 (m, 3H), 5.45–5.50 (m, 1H), 7.24–7.26
(m, 3H), 7.27–7.29 (m, 1H) 7.32–7.34 (m, 1H), 7.37–7.40 (m, 2H),
7.41–7.47 (m, 2H), 9.12 (s, 1H), 9.32–9.53 (m, 1H), 9.84 (s, 1H),
11.23 (s, 1H). HRMS (AP-ESI) m/z: calcd for C18H20N3O3 [M+H]+
324.1354, found 324.1349.
tetrahydroisoquinoline-3-carboxamide hydrochloride (12b)
Yellow solid, yield 94.8%, mp = 112–115 °C. ½a D20
¼ ꢁ117:6 (c
ꢃ
0.5, DMSO). 1H NMR (600 MHz, DMSO-d6) d 1.67–1.72 (m, 2H),
2.02–2.05 (m, 2H), 2.98–3.03 (m, 1H), 3.14–3.17 (m, 2H), 3.31–
3.35 (m, 1H), 4.11–4.15 (m, 1H), 4.27–4.34 (m, 2H), 7.24–7.32
(m, 4H), 8.77–8.79 (m, 1H), 9.46–9.47 (m, 1H), 9.80–9.82 (m,
1H), 10.48 (s, 1H). HRMS (AP-ESI) m/z: calcd for C14H20N3O3
[M+H]+ 278.1499, found 278.1532.
5.1.17. (S)-N-((S)-1-(Hydroxyamino)-1-oxo-3-phenylpropan-2-
yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
hydrochloride (12i)
5.1.11. (S)-N-(2-(Hydroxyamino)-2-oxoethyl)-1,2,3,4-
White solid, yield 89.5%, mp = 186–188 °C. ½a D20
¼ ꢁ76:5 (c 0.5,
ꢃ
tetrahydroisoquinoline-3-carboxamide hydrochloride (12c)
DMSO). 1H NMR (600 MHz, DMSO-d6) d 2.86–3.00 (m, 3H), 3.34–
3.35 (m, 1H), 4.09–4.12 (m, 1H), 4.24–4.27 (m, 1H), 4.33–4.36
(m, 1H), 4.46–4.50 (m, 1H), 7.21–7.31 (m, 9H), 8.99–9.00 (m,
1H). HRMS (AP-ESI) m/z: calcd for C19H22N3O3 [M+H]+ 340.1656,
found 340.1659.
White solid, yield 90.1%, mp = 175–177 °C. ½a D20
¼ ꢁ117:6 (c 0.5,
ꢃ
DMSO). 1H NMR (600 MHz, DMSO-d6) d 2.68–3.03 (m, 1H), 3.12–
3.35 (m, 1H), 3.71–3.81 (m, 2H), 4.19–4.22 (m, 1H), 4.29–4.32
(m, 1H), 4.36–4.39 (m, 1H), 7.25–7.30 (m, 4H), 8.93–8.95 (m,
1H). HRMS (AP-ESI) m/z: calcd for C12H16N3O3 [M+H]+ 250.1186,
found 250.1214.
5.1.18. (S)-N-((S)-1-(Hydroxyamino)-3-(4-hydroxyphenyl)-1-
oxopropan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxamide hydrochloride (12j)
5.1.12. (S)-N-((S)-1-(Hydroxyamino)-1-oxopropan-2-yl)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide hydrochloride (12d)
White solid, yield 86.8%, mp = 170–172 °C. ½a D20
¼ ꢁ74:5 (c 0.5,
ꢃ
White solid, yield 88.8%, mp = 130–133 °C. ½a D20
ꢃ
¼ ꢁ68:6 (c 0.5,
DMSO). 1H NMR (600 MHz, DMSO-d6) d 2.74–2.77 (m, 1H), 2.85–
2.88 (m, 1H), 2.90–2.95 (m, 1H), 3.34–3.39 (m, 1H), 4.09–4.12
(m, 1H), 4.24–4.27 (m, 1H), 4.33–4.36 (m, 1H), 4.37–4.41 (m,
1H), 6.68 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 7.23–7.28 (m,
4H), 8.94–8.95 (m, 1H). HRMS (AP-ESI) m/z: calcd for C19H22N3O4
[M+H]+ 356.1605, found 356.1637.
DMSO). 1H NMR (600 MHz, DMSO-d6) d 1.27 (d, J = 7.2 Hz, 3H),
2.94–2.99 (m, 1H), 3.30–3.33 (m, 1H), 4.13–4.16 (m, 1H), 4.18–
4.36 (m, 3H), 7.25–7.29 (m, 4H). HRMS (AP-ESI) m/z: calcd for
C
13H18N3O3 [M+H]+ 264.1343, found 264.1375.
5.1.13. (S)-N-((S)-1-(Hydroxyamino)-3-methyl-1-oxobutan-2-
yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
hydrochloride (12e)
5.1.19. (S)-N-((S)-1-(hydroxyamino)-4-(methylthio)-1-
oxobutan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
hydrochloride (12k)
White solid, yield 93.2%, mp = 229–230 °C. ½a D20
¼ ꢁ86:3 (c 0.5,
ꢃ
DMSO). 1H NMR (600 MHz, DMSO-d6) d 0.87–0.94 (m, 6H), 1.90–
1.99 (m, 1H), 2.90–2.95 (m, 1H), 3.27–3.30 (m, 1H), 4.04–4.07 (t,
J = 8.4 Hz, 1H), 4.17–4.21 (m, 1H), 4.28–4.30 (m, 1H), 4.34–4.38
(m, 1H), 7.24–7.28 (m, 4H), 8.77–8.79 (m, 1H), 9.00 (s, 1H), 9.42–
9.44 (m, 1H), 9.73 (s, 1H), 10.80 (m, 1H). HRMS (AP-ESI) m/z: calcd
for C15H22N3O3 [M+H]+ 292.1656, found 296.1687.
White solid, yield 93.5%, mp = 140–143 °C. ½a D20
¼ ꢁ58:8 (c 0.5,
ꢃ
DMSO). 1H NMR (600 MHz, DMSO-d6) d 1.85–1.91 (m, 2H), 2.06 (s,
3H), 2.42–2.54 (m, 2H), 2.94–2.99 (m, 1H), 3.30–3.34 (m, 1H),
4.17–4.20 (m, 1H), 4.29–4.38 (m, 3H), 7.26–7.29 (m, 4H), 8.93–
8.95 (m, 1H). HRMS (AP-ESI) m/z: calcd for C15H22N3O3S [M+H]+
324.1376, found 324.1403.

6024
X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 6015–6025
5.1.20. (S)-N-((S)-3-hydroxy-1-(hydroxyamino)-1-oxopropan-2-
yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
hydrochloride (12l)
5.1.26. (S)-N-((S)-1-((S)-1-(hydroxyamino)-4-methyl-1-
oxopentan-2-ylamino)-4-methyl-1-oxopentan-2-yl)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide hydrochloride (12r)
White solid, yield 92.8%, mp = 153–156 °C. ½a D20
ꢃ
¼ ꢁ68:6 (c 0.5,
White solid, yield 90.8%, mp = 154–156 °C. ½a D20
¼ ꢁ74:5 (c 0.5,
ꢃ
DMSO). 1H NMR (600 MHz, DMSO-d6) d 2.95–2.30 (m, 1H), 3.32–
3.40 (m, 1H), 3.52–3.60 (m, 3H), 4.15–4.19 (m, 1H), 4.28–4.36
(m, 3H), 7.24–7.28 (m, 4H), 8.87–8.89 (m, 1H), 9.48–9.55 (m,
1H), 9.82–9.89 (m, 1H), 10.78 (s, 1H). HRMS (AP-ESI) m/z: calcd
for C13H18N3O4 [M+H]+ 280.1292, found 280.1328.
DMSO). 1H NMR (600 MHz, DMSO-d6) d 0.78–0.93 (m, 12H),
1.38–1.45 (m, 1H), 1.46–1.58 (m, 4H), 1.64–1.66 (m, 1H), 2.91–
2.96 (m, 1H), 3.33–3.40 (m, 1H), 4.16–4.23 (m, 2H), 4.26–4.29
(m, 1H), 4.33–4.35 (m, 1H), 4.37–4.44 (m, 1H), 7.19–7.28 (m,
4H), 8.15 (d, J = 8.4 Hz, 1H), 8.77 (d, J = 8.4 Hz, 1H), 9.47–9.54 (m,
1H), 9.81–9.83 (m, 1H), 10.72 (s, 1H). HRMS (AP-ESI) m/z: calcd
for C22H35N4O4 [M+H]+ 419.2653, found 419.2689.
5.1.21. (S)-N-((2S)-3-hydroxy-1-(hydroxyamino)-1-oxobutan-2-
yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
hydrochloride (12m)
5.1.27. (S)-N-((S)-1-((S)-1-(hydroxyamino)-1-oxo-3-
White solid, yield 94.8%, mp = 148–150 °C. ½a D20
¼ ꢁ82:4 (c 0.5,
ꢃ
phenylpropan-2-ylamino)-4-methyl-1-oxopentan-2-yl)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide hydrochloride (12s)
DMSO). 1H NMR (600 MHz, DMSO-d6) d 1.10 (d, J = 6.6 Hz, 3H),
2.95–3.00 (m, 1H), 3.37–3.40 (m, 1H), 3.91–3.93 (m, 1H), 4.13–
4.15 (m, 1H), 4.23–4.26 (m, 1H), 4.28–4.36 (m, 2H), 7.24–7.29
(m, 4H), 8.80–8.82 (m, 1H). HRMS (AP-ESI) m/z: calcd for
White solid, yield 90.1%, mp = 158–160 °C. ½a D20
¼ ꢁ74:5 (c 0.5,
ꢃ
DMSO). 1H NMR (600 MHz, DMSO-d6) d 0.83–0.93 (m, 6H), 1.38–
1.48 (m, 2H), 1.58–1.62 (m, 1H), 2.82–2.88 (m, 1H), 2.90–2.94
(m, 2H), 3.27–3.30 (m, 1H), 4.13–4.17 (m, 1H), 4.26–4.42 (m,
4H), 7.18–7.31 (m, 9H), 8.33 (d, J = 8.4 Hz, 1H), 8.74 (d, J = 8.4 Hz,
1H), 9.45–9.53 (m, 1H), 9.78–9.79 (m, 1H), 10.74 (s, 1H). HRMS
C
14H20N3O4 [M+H]+ 294.1448, found 294.1479.
5.1.22. (S)-N-hydroxy-1-((S)-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)pyrrolidine-2-carboxamide hydrochloride (12n)
(AP-ESI) m/z: calcd for
C
25H33N4O4 [M+H]+ 453.2496, found
White solid, yield 94.5%, mp = 155–157 °C. ½a D20
¼ ꢁ49:0 (c 0.5,
ꢃ
453.2533.
DMSO). 1H NMR (600 MHz, DMSO-d6) d 1.80–1.84 (m, 1H), 1.86–
1.91 (m, 1H), 2.01–2.10 (m, 1H), 2.11–2.15 (m, 1H), 2.88–2.93
(m, 1H), 3.34–3.40 (m, 1H), 3.60–3.64 (m, 1H), 3.69–3.73 (m,
1H), 4.25–4.05 (m, 1H), 4.32 (s, 2H), 4.60–4.62 (m, 1H), 7.25–7.30
5.1.28. (S)-N-((S)-1-(1-(hydroxyamino)-4-methyl-1-oxopentan-
2-ylamino)-1-oxo-3-phenylpropan-2-yl)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide hydrochloride (12t)
(m, 4H). HRMS (AP-ESI) m/z: calcd for
C
15H20N3O3 [M+H]+
White solid, yield 90.6%, mp = 150–152 °C. ½a D20
¼ ꢁ70:6 (c 0.5,
ꢃ
290.1499, found 290.1537.
DMSO). 1H NMR (600 MHz, DMSO-d6) d 0.85 (d, J = 6.0 Hz, 3H), 0.90
(d, J = 6.0 Hz, 3H), 1.41–1.46 (m, 1H), 1.48–1.52 (m, 1H), 1.54–1.61
(m, 1H), 2.74–2.85 (m, 1H), 2.88–2.97 (m, 1H), 3.05–3.13 (m, 1H),
3.36–3.42 (m, 1H), 4.06–4.10 (m, 1H), 4.20–4.27 (m, 1H), 4.31–4.35
(m, 1H), 4.63–4.66 (m, 1H), 7.20–7.22 (m, 2H), 7.24–7.25 (m, 2H),
7.27–7.30 (m, 1H), 7.32–7.36 (m, 2H), 8.33–8.35 (m, 1H), 8.83–8.84
(m, 1H), 9.41–9.42 (m, 1H), 9.59–9.61 (m, 1H), 10.75 (s, 1H). HRMS
5.1.23. (2S,4R)-N,4-dihydroxy-1-((S)-1,2,3,4-
tetrahydroisoquinoline-3-carbonyl)pyrrolidine-2-carboxamide
hydrochloride (12o)
White solid, yield 87.8%, mp = 169–171 °C. ½a D20
¼ ꢁ94:1 (c 0.5,
ꢃ
DMSO). 1H NMR (600 MHz, DMSO-d6) d 1.87–1.91 (m, 1H), 2.06–
2.10 (m, 1H), 2.85–2.90 (m, 1H), 3.33–3.39 (m, 1H), 3.61–3.63
(m, 1H), 3.68–3.70 (m, 1H), 4.32–4.34 (m, 3H), 4.41 (s, 1H), 4.63–
4.66 (m, 1H), 7.27–7.29 (m, 1H). HRMS (AP-ESI) m/z: calcd for
(AP-ESI) m/z: calcd for
C
25H33N4O4 [M+H]+ 453.2496, found
453.2530.
C
15H20N3O4 [M+H]+ 306.1448, found 306.1487.
5.1.29. (S)-N-((S)-1-((S)-1-(hydroxyamino)-1-oxo-3-
phenylpropan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide hydrochloride (12u)
5.1.24. Benzyl-(S)-6-(hydroxyamino)-6-oxo-5-((S)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamido)hexylcarbamate
hydrochloride (12p)
White solid, yield 90.8%, mp = 171–173 °C. ½a D20
¼ ꢁ56:9 (c 0.5,
ꢃ
DMSO). 1H NMR (600 MHz, DMSO-d6) d 2.74–2.83 (m, 1H), 2.84–
2.90 (m, 2H), 2.94–3.00 (m, 1H), 3.01–3.04 (m, 1H), 3.33–3.36
(m, 1H), 4.06–4.19 (m, 1H), 4.20–4.23 (m, 1H), 4.31–4.54 (m,
1H), 4.61–4.64 (m, 1H), 7.14–7.32 (m, 14H), 8.51 (d, J = 8.4 Hz,
1H), 8.82–8.83 (m, 1H), 9.37–9.38 (m, 1H), 9.59 (s, 1H), 10.74 (s,
1H). HRMS (AP-ESI) m/z: calcd for C28H31N4O4 [M+H]+ 487.2340,
found 487.2370.
White solid, yield 86.9%, mp = 174–175 °C. ½a D20
¼ ꢁ94:1 (c 0.5,
ꢃ
DMSO). 1H NMR (600 MHz, DMSO-d6) d 1.25–1.28 (m, 1H), 1.31–
1.36 (m, 1H), 1.40–1.42 (m, 1H), 1.57–1.62 (m, 1H), 2.92–2.99
(m, 3H), 3.29–3.32 (m, 1H), 4.16–4.36 (m, 4H), 7.25–7.38 (m,
9H), 8.06–8.10 (m, 1H), 8.85–8.93 (m, 1H), 9.47–9.54 (m, 1H),
9.77–9.84 (m, 1H), 10.81–10.85 (m, 1H). HRMS (AP-ESI) m/z: calcd
for C24H31N4O5 [M+H]+ 455.2289, found 455.2296.
5.1.25. (S)-N-((S)-1-(hydroxyamino)-3-(1H-indol-2-yl)-1-
oxopropan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxamide hydrochloride (12q)
5.2. Biological evaluation
5.2.1. In vitro APN inhibition assay
Pink solid, yield 90.3%, mp = 180–182 °C. ½a D20
¼ ꢁ82:4 (c 0.5,
ꢃ
IC50 values against APN were determined as previously de-
DMSO). 1H NMR (600 MHz, DMSO-d6) d 2.92–2.97 (m, 1H), 2.99–
3.03 (m, 1H), 3.10–3.14 (m, 1H), 3.34–3.40 (m, 1H), 4.10–4.12
(m, 1H), 4.25–4.27 (m, 1H), 4.32–4.35 (m, 1H), 4.53–4.56 (m,
1H), 7.00 (t, J = 7.8 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H), 7.19 (s, 1H),
7.22–7.29 (m, 4H), 7.34 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H),
8.98–8.99 (m, 1H). HRMS (AP-ESI) m/z: calcd for C21H23N4O3
[M+H]+ 379.1765, found 379.1801.
scribed29 by using
L-Leu-p-nitroanilide as substrate and Micro-
somal aminopeptidase from Porcine Kidney Microsomes (Sigma)
as enzyme in 50 mM PBS (pH 7.4) or suspension of ES-2 cells in
PBS (1 ꢂ 105/well). After incubation with various concentrations
of detected compounds at 37 °C, hydrolysis of the substrate was
measured using a plate reader (Varioskan, Thermo, USA) by
observing the change of OD values at 405 nm.

X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 6015–6025
6025
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Acknowledgments
This work was supported by National High Technology Re-
search and Development Program of China (863 project; Grant
No. 2007AA02Z314) and National Nature Science Foundation of
China (Nos. 90713041 and 30772654) and National Science and
Technology Major Project (Grant No. 2009ZX09103-118).
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