Design, synthesis and preliminary evaluation of α-sulfonyl γ-(glycinyl-amino)proline peptidomimetics as matrix metalloproteinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.12.025

A series of novel α-sulfonyl γ-(glycinyl-amino)proline peptidomimetic derivatives were designed, synthesized and assayed for their activities against matrix metalloproteinase-2 (MMP-2), aminopeptidase N (APN)/CD13 and HDACs. The results indicated that all the compounds exhibited highly selective inhibition against MMP-2 as compared with APN and HDACs. The antiproliferative activities of some compounds against SKOV3, HL60 and A549 cells were also investigated. Comparing with the control LY52, compound 12u, with excellent activity both in the enzymatic inhibition assay and cell-based assay, could be used as lead compound for the further development of MMP inhibitors.

Full text of DOI:10.1016/j.bmc.2013.12.025

Bioorganic & Medicinal Chemistry 22 (2014) 3055–3064
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and preliminary evaluation of
-(glycinyl-amino)proline peptidomimetics as matrix
metalloproteinase inhibitors
a-sulfonyl
c
Jian Zhang a, Xiaoyang Li a, Yuqi Jiang a, Jinhong Feng b, Xiaoguang Li a, Yingjie Zhang a, Wenfang Xu a,
⇑
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44, West Wenhua Road, Ji’nan, Shandong 250012, China
b Shandong Analysis and Test Center, Shandong Academy of Sciences, Ji’nan, Shandong 250012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel
a
-sulfonyl
c
-(glycinyl-amino)proline peptidomimetic derivatives were designed, syn-
Received 26 September 2013
Revised 5 December 2013
Accepted 7 December 2013
Available online 21 December 2013
thesized and assayed for their activities against matrix metalloproteinase-2 (MMP-2), aminopeptidase
N (APN)/CD13 and HDACs. The results indicated that all the compounds exhibited highly selective inhi-
bition against MMP-2 as compared with APN and HDACs. The antiproliferative activities of some com-
pounds against SKOV3, HL60 and A549 cells were also investigated. Comparing with the control LY52,
compound 12u, with excellent activity both in the enzymatic inhibition assay and cell-based assay, could
be used as lead compound for the further development of MMP inhibitors.
Keywords:
Matrix metalloproteinase-2
Aminopeptidase N
HDACs
Ó 2014 Published by Elsevier Ltd.
Inhibitors
Hyp-Gly derivatives
Synthesis
1. Introduction
With the rapid increased studies on the solution and the crystal
structures of MMP-inhibitor complexes, the detailed descriptions
The zinc-dependent and calcium-containing family of endopep-
tidases called matrix metalloproteinases (MMPs) play a crucial role
in the degradation of extracellular matrix (ECM) and tissue remod-
eling. To date, more than 26 subfamily members have been identi-
fied and can be loosely grouped into five classes according to their
primary structure and function, substrate specificity, as well as
their cellular sources: collagenases (MMP-1, MMP-8 and MMP-
13), gelatinases (MMP2 and MMP-9), stromelysins (MMP-3,
MMP-7, MMP-10, MMP-11 and MMP-12), membrane-type MMPs
(MT1-MMP, MT2-MMP, MT3-MMP and MT4-MMP), and other
enzymes.1–3 Under normal physiological conditions, MMPs are
minimally expressed and well-regulated by endogenous tissue
inhibitors of metalloproteinases (TIMPs), while in the presence of
specific stimuli exemplified by growth factors and cytokines, they
are up-regulated destroying the balance between MMPs and TIMPs
that can lead to a variety of pathological disorders ranging from
cardiovascular disease to cancer.4–6 Among all the identified
MMP subtypes, MMP-2 is an attractive target for cancer research
and development of anticancer drugs because of its tight correla-
tion with tumor progression.7–10
of MMP structure have clearly been revealed. Briefly, except for
the conserved catalytic center Zn2+ of MMP-2 enzyme, there are
0
0
two hydrophobic domains, which are called S1 and S2 pocket,
0
respectively. S1 pocket, a hydrophobic cavity positioned to the
right of the catalytic zinc, offered a degree of individuality to MMPs
0
that can be exploited to ascertain specificity, While S2 pocket is a
solvent-exposed cleft.11–14 Accordingly, typical MMP inhibitors
shared two structural characters: (1) a ‘warhead’ for zinc coordina-
tion also known as zinc binding group (ZBG), such as hydroxamate;
(2) at least one side chain effectively interacting with the enzyme
subsites, such as S1 and S2 pocket.13–16 Except for these two main
pharmacophores discussed above, it has also been reported that
one or more functional groups (e.g., carbonyl group or amino
group) could provide hydrogen bond interactions with enzyme
backbone.16
0
0
The vast number of relevant research indicated that the sulfon-
amide group played an important role in MMP inhibitory activities,
exemplified by the discovery of CGS 27023A (see Fig. 1).17 Other N-
arylsulfonyl hydroxamate derivatives including AG 3340 (see
Fig. 1) have also been reported.18,19 The sulfonamide group was
incorporated into the MMP inhibitors to improve the enzyme-
inhibitor binding by forming effective hydrogen bonds as well as
⇑
Corresponding author. Tel./fax: +86 531 88382264.
E-mail addresses: zhangjian_3323@163.com (J. Zhang), wenfxu@gmail.com (W.
properly accommodation to the S1 pocket of the enzyme.20
0
Xu).
http://dx.doi.org/10.1016/j.bmc.2013.12.025
0968-0896/Ó 2014 Published by Elsevier Ltd.

3056
J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 3055–3064
O
O
O
O
O
S
O
O
O
S
N
O
N
NHOH
O
S
N
R1
NHOH
O
O
N
O
N
O
O
N
NHOH
O
NHOH
ClH3N
NH
NH
N
H
S
N
R2
O
O
Novel designed alpha-sulfonyl gamma
-(glycinyl-amino)proline derivatives
LY52
CGS 27023A
AG 3340
Figure 1. The structures of CGS27023A, AG3340, LY52 and novel designed
a
-sulfonyl
c
-(glycinyl-amino)proline derivatives.
O
O
O
O
O
O
O
O
O
O
O
R1
S
R1
S
R1
S
OH
OH
OCH3
OCH3
OCH3
OCH3
HN
N
ClH2N
N
N
a
b
d
c
5
OH
OH
OMs
N3
O
OH
3
1
2
4
e
O
O
O
O
BocHN
g
BocHN
OCH3
R1
S
a
f
H2N
HCl
OCH3
H2N
OH
N
H
OCH3
N
H
N
R2
R2
O
O
O
O
7
8
9
10
NH2
6
f
O
O
O
O
j
O
O
R1
S
R1
S
NHOH
OCH3
O
N
h
N
O
R2
R2
H
H
N
N
N
H
NH3Cl
N
NHBoc
H
O
O
12
11
Scheme 1. Reagents and conditions: (a) MeOH, CH3COCl; (b) RC6H5SO2Cl, DCM; (c) CH3SO2Cl; (d) NaN3, DMF; (e) 10% Pd–C/H2; (f) IBCF, NMM; (g) NaOH, CH3OH; (h) HCl/
EtOAC; (j) NH2OK, MeOH.
In our laboratory, intensive efforts have been devoted in recent
years to identify pyrrolidine derivatives as effective MMP inhibi-
tors. Most compounds, such as LY52 (see Fig. 1) showed high inhi-
bition against MMP-2.21,22 LY52 was also proved to inhibit tumor
invasion and metastasis via suppression of matrix metalloprotein-
tant intermediate (2S,4S)-1-arylsulfonyl-4-amino-pyrrolidine-2-
carboxylate (6) was prepared starting from optically pure trans-
4-hydroxy-L-proline (1), followed by the sequence of esterification,
acylation, sulfonation, SN2 nucleophilic substitution and catalytic
hydrogeonation.25 The condensation of compound 6 with various
biopeptides containing glycine led to compounds 11a–x, which
removed the Boc protecting groups after being treated with NH2OK
in anhydrous methanol to yield the target compounds 12a–x.
ase activity.23 Moreover, trans-4-hydroxy-
L-proline and glycine
residues are the principle amino acid residue of collagens that
are specific substrates of gelatinases (MMP-2 and MMP-9).24 Thus,
cis-c-amino-L-proline was chosen on the basis that derivatives or
analogues of 4-hydroxyproline, hold the promise of recognizing
its substrate and subsequently interacting with the active sites of
MMPs in a competitive manner. Enlightened by these findings,
together with the role of the sulfonyl group in MMP inhibitors,
3. Results and discussion
The newly synthesized
a-sulfonyl c-(glycinyl-amino)proline
peptidomimetic derivatives were assayed for their inhibitory activ-
ities toward MMP-2, APN/CD13 and HDACs, and all the inhibition
results were summarized in Table 1. Similar to MMP-2, APN and
HDACs are both zinc-dependant metalloproteinase and closely
associated in tumor invasion and metastasis. Thus the assay was
performed on MMP-2, APN and HDACs so as to identify the com-
pounds’ selectivity.26–28 LY52 was used as the positive control to
MMP-2, while Bestatin and SAHA were used for APN and HDACs,
respectively.
we thereof design the
domimetics, wherein the arylsulfonyl group and some biopeptides
containing glycine are introduced into the cis- -amino- -proline
a-sulfonyl c-(glycinyl-amino)proline pepti-
c
L
scaffold (see Fig. 1). R1 group can be H–, CH3–, CH3O–, Cl–. R2 group
can be various hydrophobic amino acid residues, which might
0
occupy S2 pocket, resulting effective enzyme interaction.
In the present study, we hereby described the synthesis and
biological activity of all these a-sulfonyl c-(glycinyl-amino)proline
peptidomimetic derivatives, as well as the docking studies of the
interaction. The structure–activity relationships have also been
established.
Notably, all these
a-sulfonyl c-(glycinyl-amino)proline pepti-
domimetic derivatives displayed better inhibitory activities against
MMP-2 compared with APN and HDACs, which confirmed our
strategy for designing effective MMP-2 inhibitors to some degree.
The different inhibitory profile may be explained by the structural
2. Chemistry
differences of activity sites of three enzymes. MMP-2 is a Zn2+
-
The target compounds were designed and synthesized follow-
ing the procedures as illustrated in Scheme 1. Briefly, the impor-
dependent endopeptidase that could cut the peptide to parts from
the specific amino acid residue of the peptide, while, APN is a

J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 3055–3064
3057
Table 1
The structures and inhibitory activities of the target compounds against MMP-2, APN and HDACs
O
O
O
N
NHOH
R1
S
O
NH
ClH3N
N
H
R2
O
a
Compd
Structure
R2
IC50 (lM)
R1
MMP-2
APN
HDACs
12a
12b
12c
12d
12e
12f
12g
12h
12i
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
CH3
CH (CH3)2
7.43 0.56
5.76 0.43
1.52 0.17
5.21 0.39
2.07 0.24
2.32 0.28
4.36 0.46
2.95 0.42
0.87 0.09
2.05 0.24
0.98 0.10
1.03 0.11
4.19 0.49
2.71 0.35
0.52 0.04
1.94 0.22
0.72 0.06
0.76 0.07
3.36 0.34
1.81 0.25
0.27 0.02
1.28 0.13
0.43 0.03
0.46 0.04
>100
>100
82.31 2.43
>100
94.47 3.56
>100
>100
96.24 4.72
75.81 1.89
92.63 4.25
87.95 3.06
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
CH2CH (CH3)2
CH(CH3)CH2CH3
CH2C6H5
CH2CH2SCH3
CH3
CH (CH3)2
CH2CH (CH3)2
CH(CH3)CH2CH3
CH2C6H5
CH2CH2SCH3
CH3
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
12t
12u
12v
12w
12x
>100
CH (CH3)2
90.76 3.71
83.57 2.53
89.95 3.32
93.82 4.15
>100
CH2CH (CH3)2
CH(CH3)CH2CH3
CH2C6H5
CH2CH2SCH3
CH3
>100
Cl
Cl
Cl
Cl
CH (CH3)2
79.58 2.18
72.29 2.16
86.43 2.84
89.95 2.97
95.3 4.65
CH2CH (CH3)2
CH(CH3)CH2CH3
CH2C6H5
Cl
CH2CH2SCH3
O
O
O
O
NHOH
N
N
O
LY52
0.95 0.09
>100
>100
ND
NH
O
NH2
Bestatin
SAHA
>100
ND
1.64 0.12
OH
N
H
OH
O
O
H
N
OH
ND
0.12 0.03
N
H
O
ND: not determined.
a
IC50 values are mean of three experiments and standard deviation is given.
membrane-bound zinc exopeptidase that catalyzed the removal of
NH2-terminal amino acid from the peptide and HDACs are a class
ter inhibition than the others. Moreover, When the R2 group is
same, the methyl-substituted or methoxy-substituted at C-4 posi-
tion of arylsulfonyl group makes no great difference to the inhibi-
tory activity, but the methoxy-substituted ones showed a little
better inhibitory activity (e.g., 12g and 12m, 12h and 12n). Among
of metalloproteinase that removed acetyl groups from an
e-N-
acetyl lysine amino acid on a histone. As the above mentioned
selectivity against MMP-2, the following structure–activity rela-
tionships (SARs) mainly focus on MMP-2 inhibition.
all the compounds, compound 12u (IC50 = 0.27 0.02
more potent than the others and control LY52 (IC50 = 0.95 0.09 -
M). Compound 12o, 12w and 12x also have comparable activities
(IC50 = 0.52, 0.43 and 0.46 M, respectively).
lM) was
Keeping R1 at the C-4 position of arylsulfonyl group constant, a
survey of a variety of amino acid (e.g., compounds 12s–x) revealed
that compounds containing Leu, Phe or Met residue (12u, 12w and
12x) were more potent than those containing Ala, Val or Ile residue
(12s, 12t and 12v). A similar observation was found in compounds
12a–f, 12g–l and 12m–r. While keeping R2 group constant (e.g.,
12u, 12o, 12i and 12c), compounds containing substituted aryl-
sulfonyl group (12u, 12o and 12i) displayed more potent inhibitory
activities compared with those benzenesulfonyl derivatives (12c),
especially, of which, the chloro-substituted compounds had a bet-
l
l
Compounds 12i, 12o, 12q, 12r, 12u, 12w and 12x, of which the
IC50 values are lying in nanomole level, were chosen to test against
human MMP-2 deriving from cultured SKOV3 human ovarian car-
cinoma cells expressing high level of MMP-2. As can be seen in Fig-
ure 2, all the tested compounds exhibited better inhibitory
activities against MMP-2 on SKOV3 cell than LY52
(IC50 = 43.75 1.12 lM), especially compound 12u showed the

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J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 3055–3064
Figure 2. Inhibitory activities of compounds 12i, 12o, 12q, 12r, 12u, 12w, 12x and
LY52 against MMP-2 on SKOV-3 cells. Data are expressed as mean values of three
experiments.
Table 2
Anti-proliferative activities of compounds 12u, 12w, 12x and LY52 against SKOV3,
Figure 4. The binding mode of compound 12u with MMP-2.
HL60, and A549 cells
a
Compd
IC50
(
lM)
SKOV3
HL60
A549
result showed in Figure 3 suggested that the hydroxamic acid
group can chelate with Zn2+ which was crucial for catalytic active
site and the arylsulfonyl group can insert into S10 pocket, while,
the Leu residues can insert into S20 pocket. The picture of H-bond
interactions between the inhibitor and the residues lined in the
active site of the protein was also created (Fig. 4). The sulfonyl
group of compound 12u can form hydrogen bonds with Leu83
and Ala84, while the carbonyl group and amino group of the glycin
12u
12w
12x
196.37
285.46
321.72
697.14
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
LY52
a
Mean values and standard deviation of triplicate experiments are given.
best capability (IC50 = 18.42 0.36 lM). Similar to the previous
enzymatic inhibition assay, the human MMP-2 inhibition of these
compounds also presented consistent trends.
residues of compound 12u can form hydrogen bonds with Ala86
.
However, it should be kept in mind that the exact binding mode
of the compound 12u with MMP-2 could be obtained from further
X-ray crystal studies.
The potent anti-MMP-2 activities of compounds 12u, 12w and
12x promoted us to evaluate their in vitro antiproliferative activi-
ties against human ovarian tumor cell line (SKOV3), leukemia cell
line (HL60), and lung cancer cell line (A549) with MTT method.
Among these three cell lines, HL60 and A549 cell were over-
expressed APN, while SKOV-3 cell was over-expressed MMP-2.
The results presented in Table 2 indicated that all compounds’
anti-proliferative effects against SKOV3 cells were better than that
against HL60 and A549 cells, which may owe to the higher MMP-2
expression on SKOV3 cells than the other two. Compound 12u also
showed slightly lower antiproliferative effect on SKOV3 cells than
4. Conclusion
In summary, we report here the synthesis and biological evalu-
ation of a series of a-sulfonyl c-(glycinyl-amino)proline peptidom-
imetic derivatives as MMP-2 inhibitors. Most of the target
compounds showed more inhibitory activities against MMP-2 than
the positive control LY52. Especially, compound 12u, with excel-
lent activity both in the enzymatic inhibition assay and cell-based
assay, could be used as a lead for further structure optimization in
the future MMPIs exploration.
that of LY52 (with the IC50 values of 196.37 and 697.14 lM, respec-
tively), which was consistent with the previous results of enzyme
inhibition.
5. Experimental section
To fully investigate the interaction between the target com-
pounds and MMP-2, the most potent compound 12u, was chosen
to be carried out docking studies using Sybyl 7.3 of Tripos Incorpo-
ration. The docking study of 12u with the active site of MMP-2 was
performed by using Syble/FlexX module. The active site was
defined as 10.0 Å radius circles around Zn2+ (PDB: 1HOV). The
5.1. Chemistry: general procedures
Unless otherwise noted, all the materials were commercial
available and used without further purification. All reactions were
monitored by TLC with 0.25 mm silica gel plates (60GF-254) and
visualized with UV light, iodine vapor or chloride ferric. Flash col-
umn chromatography was performed using 200–300 mesh silica
gel. Melting points were determined on an electrothermal melting
point apparatus (uncorrected). Proton NMR spectra were deter-
mined on a Brucker DRX spectrometer (600 MHz), d in parts per
million and J in Hertz, using TMS as an internal standard. Measure-
ments were made in DMSO-d6 solutions. ESI-MS were determined
on an API 4000 spectrometer. HR-MS were determined on an Agi-
lent Q-TOF-6250 spectrometer in Shandong Analysis and Test Cen-
ter in Ji’nan, China. Anhydrous reactions were carried out in over-
dried glassware under a nitrogen atmosphere.
5.1.1. trans-4-Hydroxy-
L-proline methylester hydrochloride (2)
26.2 g (200 mmol) trans-4-hydroxy-L-proline (1) in methanol
(300 ml) was treated with dry hydrogen chloride until homoge-
neous. The solution was heated to the reflux temperature for 2 h
Figure 3. The FlexX docking of compound 12u with MMP-2.

J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 3055–3064
3059
and concentrated in vacuo. Upon cooling, the product was crystal-
lized from the solvent, collected by filtration, washed with acetone
(30 ml ꢀ 5). The organic phase was washed with 0.1 M HCl and
brine, dried over Na2SO4. Evaporation of EtOAc gave the com-
pounds 5a–d.
and ether, and dried to yield trans-4-hydroxy- -proline methylester
L
hydrochloride (2) as white crystal (32.7 g, 90%), mp 159–162 °C.
5.1.4.1. (2S,4S)-Methyl 4-azido-1-benzenesulfonylpyrrolidine-2-
5.1.1.1. Compound 8 was synthesized following the procedure
carboxylate (5a).
Colorless oil: ESI-MS m/z: 311.4(M+H)+.
described above.
About 90%, white crystal: mp 172–175 °C.
5.1.4.2. (2S,4S)-Methyl 4-azido-1-tosylpyrrolidine-2-carboxylate
(5b).
White solid: mp 69–71 °C. ESI-MS m/z: 325.5(M+H)+. 1H
5.1.2. (2S,4R)-Methyl 4-hydroxy-1-sulfonylpyrrolidine-2-
carboxylate (3a–d)
NMR (DMSO-d6): 2.03 (m, 1H), 2.22 (m, 1H), 2.41 (s, 3H), 3.25 (m,
1H), 3.34 (m, 1H, CH2), 3.67 (s, 3H, CH3), 4.24 (m, 1H), 4.42 (m, 1H),
7.47, 7.60 (4H, C6H6).
9.1 g (50 mmol) trans-4-hydroxy-L-proline methylester hydro-
chloride (2) was dissolved in DCM with Et3N (15 ml, 106 mmol),
and added with various sulfonyl chloride (55 mmol) at 0 °C. The
mixture was stirred at room temperature for 12 h, washed with
1 M citric acid, saturated NaHCO3 solution and brine, and dried
over Na2SO4. Evaporation of DCM gave birth to white solid material
(3a–d).
5.1.4.3. (2S,4S)-Methyl 4-azido-1-((4-methoxphenyl)sulfonyl)
pyrrolidine-2-carboxylate (5c).
White solid: mp 56–58 °C.
ESI-MS m/z: 341.4(M+H)+.
5.1.4.4. (2S,4S)-Methyl 4-azido-1-((4-chlorophenyl)sulfonyl)
5.1.2.1. (2S,4R)-Methyl 4-hydroxy-1-benzenesulfonylpyrroli-
pyrrolidine-2-carboxylate (5d).
White solid: mp 67–69 °C.
dine-2-carboxylate (3a).
White crystal: mp 102–104 °C.
ESI-MS m/z: 345.7(M+H)+.
ESI-MS m/z: 286.2 (M+H)+.
5.1.5. (2S,4S)-Methyl 4-amino-1-sulfonylpyrrolidine-2-
5.1.2.2. (2S,4R)-Methyl 4-hydroxy-1-tosylpyrrolidine-2-carbox-
carboxylate (6a–d)
ylate (3b).
White crystal: mp 92–94 °C. ESI-MS m/z: 300.5
(2S,4S)-Methyl
4-azido-1-sulfonylpyrrolidine-2-carboxylate
(M+H)+.
(5a–d) (20 mmol) in MeOH (100 ml) was hydrogenated in the
presence of catalytic amount of 10% Pd–C at room temperature
and 1 atm pressure. After 24 h, the catalytic was filtered and the
solvent was removed under vacuum to give compounds 6a–d.
5.1.2.3. (2S,4R)-Methyl 4-hydroxy-1-((4-methoxphenyl)sulfo-
nyl) pyrrolidine-2-carboxylate (3c).
White crystal: mp 97–
99 °C. ESI-MS m/z: 316.3(M+H)+.
5.1.5.1. (2S,4S)-Methyl 4-amino-1-benzenesulfonylpyrrolidine-
5.1.2.4. (2S,4R)-Methyl 4-hydroxy-1-((4-chlorophenyl)sulfonyl)
2-carboxylate (6a).
Colorless oil: ESI-MS m/z: 285.3(M+H)+.
pyrrolidine-2-carboxylate (3d).
White crystal: mp 124–
126 °C. ESI-MS m/z: 320.4(M+H)+.
5.1.5.2. (2S,4S)-Methyl 4-amino-1-tosylpyrrolidine-2-carboxyl-
ate (6b).
Colorless oil: ESI-MS m/z: 299.4(M+H)+.
5.1.3. (2S,4R)-Methyl 4-((methylsulfonyl)oxy)-1-
sulfonylpyrrolidine-2-carboxylate (4a–d)
5.1.5.3. (2S,4S)-Methyl 4-amino-1-((4-methoxphenyl)sulfonyl)
(2S,4R)-Methyl 4-hydroxy-1-sulfonylpyrrolidine-2-carboxylate
(3a–d) (40 mmol) was dissolved in anhydrous DCM with Et3N
(7 ml, 44 mmol) at 0 °C, and treated with MsCl (3.5 ml, 44 mmol)
in 10 ml anhydrous DCM. After 12 h at 0 °C, the mixture was
washed with 1 M citric acid, saturated NaHCO3 solution and brine,
and dried over Na2SO4. Evaporation of DCM gave colorless oil (4a–
d).
pyrrolidine-2-carboxylate (6c).
Colorless oil: ESI-MS m/z:
315.5(M+H)+.
5.1.5.4. (2S,4S)-Methyl 4-amino-1-((4-chlorophenyl)sulfonyl)
pyrrolidine-2-carboxylate (6d).
Colorless oil: ESI-MS m/z:
319.8(M+H)+.
5.1.6. Methl-2-(2((tert-butoxycarbonyl)amino)propanamido)
acetate (9a)
5.1.3.1.
(2S,4R)-Methyl
4-((methylsulfonyl)oxy)-1-benze-
nesulfonylpyrrolidine-2-carboxylate (4a).
Colorless oil: ESI-
2((tert-Butoxycarbonyl)amino)propanoic acid (9.45 g, 50 mmol)
and NMM (2 equiv) were dissolved in 100 ml anhydrous THF. To
this stirring solution was added IBCF (1.05 equiv) followed by com-
pound 8. After 6 h, the mixture was condensed, dissolved in EtOAC
and washed with saturated NaHCO3 solution, 1 M citric acid and
brine. Evaporation of EtOAc gave compound 9a. White solid: yield
82.7%, mp: 79–80 °C. 1H NMR (DMSO-d6): 1.20 (q, 3H, CH3), 1.38 (s,
9H, CH3), 3.62 (s, 3H, CH3), 3.84 (m, 2H, CH2), 4.01 (m, 1H), 7.47(d,
J = 7.8 Hz, 1H, NH), 8.17 (t, 1H, NH).
MS m/z: 364.2(M+H)+.
5.1.3.2. (2S,4R)-Methyl 4-((methylsulfonyl)oxy)-1-tosylpyrroli-
dine-2-carboxylate (4b).
Colorless oil: ESI-MS m/z: 378.3
(M+H)+.
5.1.3.3. (2S,4R)-Methyl 4-((methylsulfonyl)oxy)-1-((4-methox-
phenyl)sulfonyl) pyrrolidine-2-carboxylate (4c).
Colorless
oil: ESI-MS m/z: 394.2(M+H)+.
5.1.6.1. Compounds 9b–f were synthesized following the pro-
cedure described above. 5.1.6.1.1. Methl-2-(2((tert-butoxycar-
bonyl)amino)-3-methylbutanamido) acetate (9b). White solid:
yield 85.2%, mp: 103–105 °C.
5.1.3.4. (2S,4R)-Methyl 4-((methylsulfonyl)oxy)-1-((4-chloro-
phenyl)sulfonyl) pyrrolidine-2-carboxylate (4d).
Colorless oil:
ESI-MS m/z: 398.3(M+H)+.
5.1.6.1.2. Methl-2-(2((tert-butoxycarbonyl)amino)-4-methylpen-
tanamido) acetate (9c). White solid: yield 87.9%, mp: 128–130 °C.
5.1.6.1.3. Methl-2-(2((tert-butoxycarbonyl)amino)-3-methylpen-
tanamido) acetate (9d). White solid: yield 86.4%, mp: 73–75 °C.
5.1.6.1.4. Methl-2-(2((tert-butoxycarbonyl)amino)-3-phenylprop-
anamido) acetate (9e). White solid: yield 84.7%, mp: 99–101 °C.
5.1.4. (2S,4S)-Methyl 4-azido-1-sulfonylpyrrolidine-2-
carboxylate (5a–d)
(2S,4R)-Methyl 4-((methylsulfonyl)oxy)-1-sulfonylpyrrolidine-
2-carboxylate (4a–d) (40 mmol) and 6.5 g NaN3 (100 mmol) were
stirred in DMF (40 ml) at 60–65 °C overnight. The mixture was
mixed with 20 ml ice-water and extracted with ethyl acetate
5.1.6.1.5.
Methl-2-(2((tert-butoxycarbonyl)amino)-4-(methyl-

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J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 3055–3064
thio)butanamido) acetate (9f). White solid: yield 81.5%, mp: 84–
86 °C.
ESI-MS m/z: 555.4(M+H)+. 1H NMR (DMSO-d6): 0.78(d, J = 6.6 Hz,
3H, CH3), 0.80 (d, J = 6.6 Hz, 3H, CH3), 1.07 (m, 1H), 1.36 (s, 9H,
CH3), 1.40 (m, 1H), 1.65 (m, 1H), 1.84 (m, 1H), 2.37 (m, 1H), 3.10
(m, 1H), 3.56 (m, 1H), 3.61 (m, 2H), 3.67 (s, 3H, CH3), 3.76 (m,
1H), 3.83 (m, 1H), 4.28 (t, 1H), 6.82 (d, J = 8.4 Hz, 1H, NH), 7.65,
7.74, 7.85 (5H, C6H6), 7.88 (d, J = 6.6 Hz, 1H, NH), 8.06 (t, 1H, NH).
5.1.8.1.4. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-3-
phenylpropanamido)acetamido)-1-(phenylsulfonyl)pyrrolidine-2-car-
boxylate (11e). White solid, 4.38 g, yield 74.5%, mp 173–175 °C.
ESI-MS m/z: 589.4(M+H)+. 1H NMR (DMSO-d6): 1.28 (s, 9H, CH3),
1.85 (m, 1H), 2.38 (m, 1H), 2.71 (m, 1H), 2.97 (m, 1H), 3.11 (m,
1H), 3.56 (m, 1H), 3.60 (m, 2H), 3.67 (s, 3H, CH3), 3.85 (m, 1H),
4.14 (m, 1H), 4.28 (t, 1H), 6.98 (d, J = 0.6 Hz, 1H, NH), 7.18, 7.25
(5H, C6H6), 7.65, 7.74, 7.85 (5H, C6H6), 7.90 (d, J = 7.2 Hz, 1H,
NH), 8.16 (t, 1H, NH).
5.1.8.1.5. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-4-
(methylthio)butanamido) acetamido)-1-(phenylsulfonyl)pyrrolidine-
2-carboxylate (11f). White solid, 4.05 g, yield 70.8%, mp 120–
122 °C. ESI-MS m/z: 573.4(M+H)+. 1H NMR (DMSO-d6): 1.36 (s,
9H, CH3), 1.75 (m, 1H), 1.85 (m, 2H), 2.02 (s, 3H, CH3), 2.37 (m,
1H), 2.43 (m, 2H, CH2), 3.10 (m, 1H), 3.53 (m, 1H), 3.61 (m, 2H),
3.69 (s, 3H, CH3), 3.83 (m, 1H), 3.96 (m, 1H), 4.28 (t, 1H), 7.07 (d,
J = 0.6 Hz, 1H, NH), 7.65, 7.74, 7.84 (5H, C6H6), 7.87 (d, J = 6.6 Hz,
1H, NH), 8.05 (t, 1H, NH).
5.1.7. 2-(2((tert-Butoxycarbonyl)amino)propanamido) acetic
acid (10a)
Compound 9a (2.6 g, 10 mmol) in 50 ml methanol was treated
with 1 M NaOH (30 ml), and stirred for 3 h at room temperature.
The solvent was evaporated in vacuum and the residue was
adjusted to PH 2–3 with 1 M HCl. The mixture was extracted 3
times by EtOAc. The organic phase was dried over Na2SO4. Evapo-
ration of EtOAc provided compound 10a. White solid: yield 92.4%,
mp: 102–105 °C.
5.1.7.1. Compounds 10b–f were synthesized following the
procedure described above. 5.1.7.1.1. 2-(2((tert-Butoxycar-
bonyl)amino)-3-methylbutanamido) acetic acid (10b). White solid:
yield 90.8%, mp: 130–133 °C.
5.1.7.1.2. 2-(2((tert-Butoxycarbonyl)amino)-4-methylpentanami-
do) acetic acid (10c). White solid: yield 93.1%, mp: 162–165 °C.
5.1.7.1.3. 2-(2((tert-Butoxycarbonyl)amino)-3-methylpentanami-
do) acetic acid (10d). White solid: yield 91.5%, mp: 116–120 °C.
5.1.7.1.4. 2-(2((tert-Butoxycarbonyl)amino)-3-phenylpropanami-
do) acetic acid (10e). White solid: yield 94.6%, mp: 158–161 °C.
5.1.7.1.5. 2-(2((tert-Butoxycarbonyl)amino)-4-(methylthio)buta-
namido) acetic acid (10f). White solid: yield 87.2%, mp: 109–
112 °C.
5.1.8.1.6.
(2S,4S)-Methyl
4-(2-(2-((tert-butoxycar-
bonyl)amino)propanamido)acetamido)-1-tosylpyrrolidine-2-carbox-
ylate (11g). White solid, yield 72.9%. Mp 162–164 °C. 1H NMR
(DMSO-d6): 1.16 (q, 3H, CH3), 1.36 (s, 9H, CH3), 1.85 (m, 1H),
2.36 (m, 1H), 2.41 (s, 3H, CH3), 3.09 (m, 1H), 3.49 (m, 1H), 3.60
(m, 2H, CH2), 3.67 (s, 3H, CH3), 3.81 (m, 1H), 3.91 (m, 1H), 4.23
(t, 1H), 7.06 (d, J = 6.6 Hz, 1H, NH), 7.29, 7.81 (4H, C6H6), 7.81 (d,
J = 6.6 Hz, 1H, NH), 8.05 (t, 1H, NH). HRMS m/z: calcd for C23H34N4-
O8S [M+H]+ 527.2176, found 527.2177.
5.1.8.1.7. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-3-
methylbutanamido)acetamido)-1-tosylpyrrolidine-2-carboxylate
(11h). White solid, yield 74.6%. Mp 168–170 °C. 1H NMR (DMSO-
d6): 0.82 (dd, J = 6.6 Hz, 6H, CH3), 1.37 (s, 9H, CH3), 1.83 (m, 1H),
1.91 (m, 1H), 2.41 (s, 3H, CH3), 3.09 (m, 1H), 3.49 (m, 1H), 3.61
(m, 2H, CH2), 3.67 (s, 3H, CH3), 3.74 (m, 1H), 3.81 (m, 1H), 4.22
(t, 1H), 6.77 (d, J = 8.4 Hz, 1H, NH), 7.45, 7.88 (4H, C6H6), 7.88 (d,
J = 6.6 Hz, 1H, NH), 8.06 (t, 1H, NH). HRMS m/z: calcd for C25H38N4-
O8S [M+H]+ 555.2489, found 555.2482.
5.1.8. (2S,4S)-Methyl 4-(2-(2-((tert-
butoxycarbonyl)amino)propanamido) acetamido)-1-
(phenylsulfonyl)pyrrolidine-2-carboxylate (11a)
Compound 10a (2.46 g, 10 mmol) and NMM (2 equiv) were dis-
solved in 100 ml anhydrous THF. To this stirring solution was
added IBCF (1.05 equiv) followed by compound 6a. After 6 h, the
mixture was condensed, dissolved in EtOAC and washed with sat-
urated NaHCO3 solution, 1 M citric acid and brine. 3.29 g 11a was
obtained by flash columm chromatography, yield 64.2%. Mp 158–
160 °C. ESI-MS m/z: 513.5(M+H)+. 1H NMR (DMSO-d6): 1.16 (d,
J = 7.2 Hz, 3H, CH3), 1.36 (s, 9H, CH3), 1.85 (m, 1H), 2.38 (m, 1H),
3.10 (m, 1H), 3.53 (m, 1H), 3.59 (m, 2H), 3.63 (s, 3H, CH3), 3.84
(m, 1H), 3.89 (m, 1H), 4.28 (t, 1H), 7.05 (d, J = 6.6 Hz 1H, NH),
7.65, 7.74, 7.84 (5H, C6H6), 7.81 (d, J = 7.2 Hz, 1H, NH), 8.03 (d,
J = 7.2 Hz, 1H, NH).
5.1.8.1.8. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-4-
methylpentanamido)acetamido)-1-tosylpyrrolidine-2-carboxylate
(11i). White solid, yield 75.3%. Mp 164–167 °C. 1H NMR (DMSO-
d6): 0.84 (dd, J = 6.6 Hz, 6H, CH3), 1.36 (s, 9H, CH3), 1.40 (m, 2H,
CH2), 1.58 (m, 1H), 1.83 (m, 1H), 2.36 (m, 1H), 2.41 (s, 3H, CH3),
3.09 (m, 1H), 3.49 (m, 1H), 3.60 (m, 2H, CH2), 3.66 (s, 3H, CH3),
3.81 (m, 1H), 3.89 (m, 1H), 4.22 (t, 1H), 6.99 (d, J = 7.8 Hz, 1H,
NH), 7.45, 7.73 (4H, C6H6), 7.85 (d, J = 7.2 Hz, 1H, NH), 8.03 (t,
1H, NH). HRMS m/z: calcd for C26H40N4O8S [M+H]+ 569.2645, found
569.2650.
5.1.8.1.9. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-3-
methylpentanamido)acetamido)-1-tosylpyrrolidine-2-carboxylate
(11j). White solid, yield 76.8%. Mp 157–159 °C. 1H NMR (DMSO-
d6): 0.79 (m, 6H, CH3), 1.08 (m, 1H), 1.39 (s, 9H, CH3), 1.40 (m, 1H),
1.65 (m, 1H), 1.83 (m, 1H), 2.36 (m, 1H), 2.41 (s, 3H, CH3), 3.08 (m,
1H), 3.50 (m, 1H), 3.63 (m, 2H, CH2), 3.67 (s, 3H, CH3), 3.78 (m, 1H),
3.82 (m, 1H), 4.22 (t, 1H), 6.81 (d, J = 7.8 Hz, 1H, NH), 7.45, 7.73
(4H, C6H6), 7.87 (d, J = 6.6 Hz, 1H, NH), 8.06 (t, 1H, NH). HRMS m/
z: calcd for C26H40N4O8S [M+H]+ 569.2645, found 569.2647.
5.1.8.1.10. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
3-phenylpentanamido)acetamido)-1-tosylpyrrolidine-2-carboxylate
(11k). White solid, yield 73.4%. Mp 177–179 °C. 1H NMR (DMSO-
d6): 1.28 (s, 9H, CH3), 1.85 (m, 1H), 2.36 (m, 1H), 2.41 (s, 3H, CH3),
2.72 (m, 1H), 2.98 (m, 1H), 3.10 (m, 1H), 3.51 (m, 1H), 3.63 (m, 2H,
5.1.8.1. Compounds 11b–x were synthesized following the
procedure described above. 5.1.8.1.1. (2S,4S)-Methyl 4-(2-(2-
((tert-butoxycarbonyl)amino)-3-methylbutanamido)acetamido)-1-
(phenylsulfonyl)pyrrolidine-2-carboxylate
(11b). White
solid,
3.69 g, yield 68.3%, mp 122–124 °C. ESI-MS m/z: 541.4(M+H)+. 1H
NMR (DMSO-d6): 0.81(d, J = 6.6 Hz, 3H, CH3), 0.83 (d, J = 6.6 Hz,
3H, CH3),1.37 (s, 9H, CH3), 1.84 (m, 1H), 1.92 (m, 1H), 2.37 (m,
1H), 3.10 (m, 1H), 3.53 (m, 1H), 3.62 (m, 2H), 3.66 (s, 3H, CH3),
3.74 (m, 1H), 3.82 (m, 1H), 4.28 (t, 1H), 6.77 (d, J = 8.4 Hz 1H,
NH), 7.65, 7.74, 7.84 (5H, C6H6), 7.89 (d, J = 6.6 Hz, 1H, NH), 8.06
(t, 1H, NH).
5.1.8.1.2. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-4-
methylpentanamido)acetamido)-1-(phenylsulfonyl)pyrrolidine-2-car-
boxylate (11c). White solid, 3.97 g, yield 71.6%, mp 137–139 °C.
ESI-MS m/z: 555.3(M+H)+. 1H NMR (DMSO-d6): 0.84(d, J = 6.6 Hz,
3H, CH3), 0.85 (d, J = 6.6 Hz, 3H, CH3),1.36 (s, 9H, CH3), 1.40 (m,
1H), 1.58 (m, 1H), 1.84 (m, 1H), 2.37 (m, 1H), 3.10 (m, 1H), 3.54
(m, 2H), 3.62 (m, 1H), 3.67 (s, 3H, CH3), 3.83 (m, 1H), 3.89 (m,
1H), 4.28 (t, 1H), 6.99 (d, J = 7.8 Hz, 1H, NH), 7.65, 7.74, 7.84 (5H,
C6H6), 8.03 (t, 1H, NH).
5.1.8.1.3. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-3-
methylpentanamido)acetamido)-1-(phenylsulfonyl)pyrrolidine-2-car-
boxylate (11d). White solid, 3.73 g, yield 67.4%, mp 114–116 °C.

J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 3055–3064
3061
CH2), 3.68 (s, 3H, CH3), 3.83 (m, 1H), 4.14 (m, 1H), 4.23 (t, 1H), 6.98
(d, J = 8.4 Hz, 1H, NH), 7.18, 7.25 (5H, C6H6), 7.45, 7.73 (4H, C6H6),
7.90 (d, J = 6.6 Hz, 1H, NH), 8.16 (t, 1H, NH). HRMS m/z: calcd for
3.96 (m, 1H), 4.20 (t, 1H), 7.08 (d, J = 7.2 Hz, 1H, NH), 7.15, 7.77
(4H, C6H6), 7.87 (d, J = 6.6 Hz, 1H, NH), 8.05 (t, 1H, NH).
5.1.8.1.18.
(2S,4S)-Methyl
4-(2-(2-((tert-butoxycar-
C
29H38N4O8S [M+H]+ 603.2489, found 603.2488.
bonyl)amino)propanamido)acetamido)-1-((4-chlorophenyl)sulfo-
nyl)pyrrolidine-2-carboxylate (11s). White solid, 3.85 g, yield
70.4%, mp 172–174 °C. ESI-MS m/z: 547.6(M+H)+. 1H NMR
(DMSO-d6): 1.16 (d, J = 7.2 Hz, 3H, CH3), 1.36 (s, 9H, CH3), 1.85
(m, 1H), 2.38 (m, 1H), 3.10 (m, 1H), 3.59 (m, 1H), 3.63 (m, 2H),
3.67 (s, 3H, CH3), 3.84 (m, 1H), 3.90 (m, 1H), 4.28 (m, 1H), 7.05
(d, J = 5.4 Hz, 1H, NH), 7.74, 7.84 (4H, C6H6), 7.81 (d, J = 6.6 Hz,
1H, NH), 8.04 (t, 1H, NH).
5.1.8.1.19. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
3-methylbutanamido)acetamido)-1-((4-chloroyphenyl)pyrrolidine-2-
carboxylate (11t). White solid, 4.14 g, yield 72.1%, mp 153–
156 °C. ESI-MS m/z: 575.5(M+H)+. 1H NMR (DMSO-d6): 0.81 (d,
J = 6.6 Hz, 3H, CH3), 0.83 (d, J = 6.6 Hz, 3H, CH3), 1.37 (s, 9H, CH3),
1.85 (m, 1H), 1.90 (m, 1H), 2.39 (m, 1H), 3.10 (m, 1H), 3.54 (m,
1H), 3.63 (m, 2H), 3.67 (s, 3H, CH3), 3.74 (m, 1H), 3.90 (m, 1H),
4.30 (m, 1H), 6.77 (d, J = 7.8 Hz, 1H, NH), 7.72, 7.85 (4H, C6H6),
7.89 (d, J = 6 Hz, 1H, NH), 8.06 (t, 1H, NH).
5.1.8.1.20. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
4-methylpentanamido)acetamido)-1-((4-chlorophenyl)pyrrolidine-2-
carboxylate (11u). White solid, 4.23 g, yield 71.8%, mp 161–
163 °C. ESI-MS m/z: 589.5(M+H)+. 1H NMR (DMSO-d6): 0.83 (d,
J = 6 Hz, 3H, CH3), 0.85 (d, J = 6 Hz, 3H, CH3), 1.36 (s, 9H, CH3),
1.41 (m, 2H), 1.58 (m, 1H), 1.85 (m, 1H), 2.39 (m, 1H), 3.10 (m,
1H), 3.54 (m, 1H), 3.62 (m, 2H), 3.67 (s, 3H, CH3), 3.83 (m, 1H),
3.90 (m, 1H), 4.30 (m, 1H), 6.99 (d, J = 7.8 Hz, 1H, NH), 7.74, 7.85
(4H, C6H6), 7.87 (m, 1H, NH), 8.03 (t, 1H, NH).
5.1.8.1.21. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
3-methylpentanamido)acetamido)-1-((4-chlorophenyl)pyrrolidine-2-
carboxylate (11v). White solid, 4.09 g, yield 69.4%, mp 145–
147 °C. ESI-MS m/z: 589.5(M+H)+. 1H NMR (DMSO-d6): 0.78 (d,
J = 7.2 Hz, 3H, CH3), 0.80 (d, J = 7.2 Hz, 3H, CH3), 1.07 (m, 1H),
1.36 (s, 9H, CH3), 1.40 (m, 1H), 1.66 (m, 1H), 1.85 (m, 1H), 2.41
(m, 1H), 3.10 (m, 1H), 3.57 (m, 2H), 3.65 (m, 1H), 3.67 (s, 3H,
CH3), 3.77 (m, 1H), 3.90 (m, 1H), 4.31 (m, 1H), 6.82 (d, J = 7.8 Hz,
1H, NH), 7.73, 7.85 (4H, C6H6), 7.88 (m, 1H, NH), 8.06 (t, 1H, NH).
5.1.8.1.22. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
3-phenylpropanamido)acetamido)-1-((4-chlorophenyl)pyrrolidine-2-
carboxylate (11w). White solid, 4.65 g, yield 74.7%, mp 176–
178 °C. ESI-MS m/z: 623.6(M+H)+. 1H NMR (DMSO-d6): 1.28 (s,
9H, CH3), 1.85 (m, 1H), 2.41 (m, 1H), 2.72 (m, 1H), 2.98 (m, 1H),
3.10 (m, 1H), 3.59 (m, 2H), 3.65 (m, 1H), 3.67 (s, 3H, CH3), 3.83
(m, 1H), 4.13 (m, 1H), 4.23 (t, 1H), 6.98 (d, J = 7.8 Hz, 1H, NH),
7.18, 7.25 (5H, C6H6), 7.74, 7.85 (4H, C6H6), 7.91 (d, J = 6.6 Hz, 1H,
NH), 8.16 (t, 1H, NH).
5.1.8.1.11. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
4-(methylthio)butanamido) acetamido)-1-tosylpyrrolidine-2-carbox-
ylate (11l). White solid, yield 71.2%. Mp 146–149 °C. 1H NMR
(DMSO-d6): 1.37 (s, 9H, CH3), 1.75 (m, 1H), 1.83 (m, 2H, CH2),
2.02 (s, 3H, CH3), 2.36 (m, 1H), 2.41 (s, 3H, CH3), 2.45 (m, 2H,
CH2), 3.09 (m, 1H), 3.50 (m, 1H), 3.60 (m, 2H, CH2), 3.68 (s, 3H,
CH3), 3.81 (m, 1H), 3.96 (m, 1H), 4.23 (t, 1H), 7.08 (d, J = 7.2 Hz,
1H, NH), 7.45, 7.73 (4H, C6H6), 7.87 (d, J = 6.6 Hz, 1H, NH), 8.05
(t, 1H, NH). HRMS m/z: calcd for C25H38N4O8S2 [M+H]+
587.2209, found 587.2209.
5.1.8.1.12.
(2S,4S)-Methyl
4-(2-(2-((tert-butoxycar-
bonyl)amino)propanamido)acetamido)-1-((4-methoxyphenyl)sulfo-
nyl)pyrrolidine-2-carboxylate (11m). White solid, 3.52 g, yield
64.8%, mp 157–160 °C. ESI-MS m/z: 543.6(M+H)+. 1H NMR
(DMSO-d6): 1.15 (q, 3H, CH3), 1.36 (s, 9H, CH3), 1.87 (m, 1H),
2.36 (m, 1H), 3.09 (m, 1H), 3.49 (m, 1H), 3.62 (m, 2H, CH2), 3.69
(s, 3H, CH3), 3.82 (m, 1H), 3.86 (s, 3H, CH3), 3.91 (m, 1H), 4.21 (t,
1H), 7.05 (d, J = 6.0 Hz, 1H, NH), 7.15, 7.78 (4H, C6H6), 7.84 (d,
J = 6.6 Hz, 1H, NH), 8.06 (t, 1H, NH).
5.1.8.1.13. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
3-methylbutanamido)acetamido)-1-((4-methoxyphenyl)pyrrolidine-
2-carboxylate (11n). White solid, 3.84 g, yield 67.3%, mp 144–
145 °C. ESI-MS m/z: 571.6(M+H)+. 1H NMR (DMSO-d6): 0.82 (dd,
J = 6.6 Hz, 6H, CH3), 1.37 (s, 9H, CH3), 1.82 (m, 1H), 1.92 (m, 1H),
2.36 (m, 1H), 3.08 (m, 1H), 3.49 (m, 1H), 3.62 (m, 2H, CH2), 3.68
(s, 3H, CH3), 3.74 (m, 1H), 3.82 (m, 1H), 3.86 (s, 3H, CH3), 4.20 (t,
1H), 6.76 (d, J = 8.4 Hz, 1H, NH), 7.16, 7.75 (4H, C6H6), 7.90 (d,
J = 6.6 Hz, 1H, NH), 8.06 (t, 1H, NH).
5.1.8.1.14. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
4-methylpentanamido)acetamido)-1-((4-methoxyphenyl)pyrrolidine-
2-carboxylate (11o). White solid, 4.18 g, yield 71.5%, mp 160–
162 °C. ESI-MS m/z: 585.6(M+H)+. 1H NMR (DMSO-d6): 0.88 (dd,
J = 6.6 Hz, 6H, CH3), 1.36 (s, 9H, CH3), 1.40 (m, 2H, CH2), 1.54 (m,
1H), 1.87 (m, 1H), 2.35 (m, 1H), 3.13 (m, 1H), 3.49 (m, 1H), 3.60
(m, 1H), 3.69 (s, 3H, CH3), 3.70 (m, 1H), 3.80 (m, 1H), 3.86 (s, 3H,
CH3), 3.89 (m, 1H), 4.20 (t, 1H), 6.99 (d, J = 7.8 Hz, 1H, NH), 7.16,
7.78 (4H, C6H6), 7.86 (d, J = 7.2 Hz, 1H, NH), 8.03 (t, 1H, NH).
5.1.8.1.15. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
3-methylpentanamido)acetamido)-1-((4-methoxyphenyl)pyrrolidine-
2-carboxylate (11p). White solid, 4.07 g, yield 69.7%, mp 149–
151 °C. ESI-MS m/z: 585.5(M+H)+. 1H NMR (DMSO-d6): 0.79 (m,
6H, CH3), 1.07 (m, 1H), 1.39 (s, 9H, CH3), 1.40 (m, 1H), 1.66 (m,
1H), 1.83 (m, 1H), 2.36 (m, 1H), 3.08 (m, 1H), 3.50 (m, 1H), 3.63
(m, 2H, CH2), 3.67 (s, 3H, CH3), 3.75 (m, 1H), 3.81 (m, 1H), 3.86
(s, 3H, CH3), 4.20 (t, 1H), 6.81 (d, J = 7.8 Hz, 1H, NH), 7.15, 7.78
(4H, C6H6), 7.87 (d, J = 6.6 Hz, 1H, NH), 8.06 (t, 1H, NH).
5.1.8.1.16. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
3-phenylpropanamido)acetamido)-1-((4-methoxyphenyl)pyrrolidine-
2-carboxylate (11q). White solid, 4.55 g, yield 73.6%, mp 169–
171 °C. ESI-MS m/z: 619.7(M+H)+. 1H NMR (DMSO-d6): 1.28 (s,
9H, CH3), 1.85 (m, 1H), 2.36 (m, 1H), 2.72 (m, 1H), 2.98 (m, 1H),
3.10 (m, 1H), 3.48 (m, 1H), 3.64 (m, 2H, CH2), 3.69 (s, 3H, CH3),
3.83 (m, 1H), 3.86 (s, 3H, CH3), 4.13 (m, 1H), 4.20 (t, 1H), 6.98 (d,
J = 8.4 Hz, 1H, NH), 7.23, 7.31 (5H, C6H6), 7.15, 7.78 (4H, C6H6),
7.88 (d, J = 6.6 Hz, 1H, NH), 8.06 (t, 1H, NH).
5.1.8.1.23. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
4-(methylthio)butanamido) acetamido)-1-((4-chlorophenyl)pyrroli-
dine-2-carboxylate (11x). White solid, 4.29 g, yield 70.6%, mp
149–151 °C. ESI-MS m/z: 607.5(M+H)+. 1H NMR (DMSO-d6): 1.37
(s, 9H, CH3), 1.76 (m, 1H), 1.86 (m, 2H, CH2), 2.02 (s, 3H, CH3),
2.37 (m, 1H), 2.45 (m, 2H, CH2), 3.10 (m, 1H), 3.53 (m, 1H), 3.61
(m, 2H, CH2), 3.67 (s, 3H, CH3), 3.82 (m, 1H), 3.96 (m, 1H), 4.30
(m, 1H), 7.08 (d, J = 7.8 Hz, 1H, NH), 7.73, 7.85 (4H, C6H6), 7.87
(d, J = 6 Hz, 1H, NH), 8.05 (t, 1H, NH).
5.1.9. (2S,4S)-4-(2-(2-Aminopropanamido) acetamido)-N-
hydroxy-1-(phenylsulfonyl)pyrrolidine-2-carboxamide
hydrochloride (12a)
To a solution of compound 11a (0.51 g, 1 mmol) in 10 ml anhy-
drous methanol at room temperature was added a solution of NH2-
OK (3 mmol) in methanol (1.7 ml). The mixture was stirred for 2 h
and the solvent was evaporated in vacuum. The residue was
adjusted to PH 2–3 with 1 M citric acid and the mixture was
extracted 3 times by EtOAc. The organic phase was dried over Na2-
5.1.8.1.17. (2S,4S)-Methyl 4-(2-(2-((tert-butoxycarbonyl)amino)-
4-(methylthio)butanamido) acetamido)-1-((4-methoxyphenyl)pyrrol-
idine-2-carboxylate (11r). White solid, 4.24 g, yield 70.3%, mp
136–138 °C. ESI-MS m/z: 603.5(M+H)+. 1H NMR (DMSO-d6): 1.37
(s, 9H, CH3), 1.76 (m, 1H), 1.84 (m, 2H, CH2), 2.02 (s, 3H, CH3),
2.37 (m, 1H), 2.46 (m, 2H, CH2), 3.09 (m, 1H), 3.48 (m, 1H), 3.60
(m, 2H, CH2), 3.68 (s, 3H, CH3), 3.82 (m, 1H), 3.86 (s, 3H, CH3),

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J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 3055–3064
SO4. Evaporation of EtOAc and de-protection of Boc group provide
the target compound 12a (0.17 g). yield 39.4%. Mp 178–180 °C. 1
HRMS m/z: calcd for C17H25N5O6S [M+H]+ 428.1664, found
428.1667.
H
NMR (DMSO-d6): 1.36 (d, J = 7.2 Hz, 3H, CH3), 1.55 (m, 1H), 2.14 (m,
1H), 3.19 (m, 1H), 3.25 (m, 1H), 3.59 (m, 1H), 3.73 (m, 1H), 3.82 (m,
1H), 4.01 (m, 1H), 4.20 (m, 1H), 7.65, 7.75, 7.89 (5H, C6H6), 8.30 (s,
3H, NH2), 8.62 (d, J = 7.2 Hz, 1H, NH), 8.99 (t, 1H, NH), 9.69 (s, 1H,
NH), 11.54 (s, 1H, OH). HRMS m/z: calcd for C16H23N5O6S [M+H]+
414.1517, found 414.1520.
5.1.9.1.7. (2S,4S)-4-(2-(2-Amino-3-methylbutanamido) acetam-
ido)-N-hydroxy-1-(tosylsulfonyl)pyrrolidine-2-carboxamide hydro-
chloride (12h). White solid, 0.23 g, yield 48.6%, mp 177–180 °C.
1H NMR (DMSO-d6): 0.97 (d, J = 6.6 Hz, 6H, CH3), 1.91 (m, 1H),
2.08 (m, 1H), 2.37 (m, 1H), 2.41 (s, 3H, CH3), 3.15 (m, 1H), 3.50
(m, 1H), 3.62 (m, 2H, CH2), 3.73 (m, 1H), 3.85 (m, 1H), 4.12 (m,
1H), 7.46, 7,73 (4H, C6H6), 8.24 (s, 3H, NH2), 8.67 (d, J = 8.4 Hz,
1H, NH), 8.99 (s, 1H, NH), 11.53 (s, 1H, OH). HRMS m/z: calcd for
5.1.9.1. Compounds 12b–x were synthesized following the
procedure described above. 5.1.9.1.1. (2S,4S)-4-(2-(2-Amino-3-
methylbutanamido) acetamido)-N-hydroxy-1-(phenylsulfonyl)pyrrol-
idine-2-carboxamide hydrochloride (12b). White solid, 0.19 g,
yield 40.6%, mp 183–185 °C. 1H NMR (DMSO-d6): 0.93 (d,
J = 6.6 Hz, 3H, CH3), 0.96 (d, J = 6.6 Hz, 3H, CH3), 1.53 (m, 1H),
2.07 (m, 1H), 2.16 (m, 1H), 3.23 (m, 2H), 3.59 (m, 1H), 3.69 (m,
1H), 3.85 (m, 1H), 4.00 (m, 1H), 4.24 (m, 1H), 7.68, 7.75, 7.88
(5H, C6H6), 8.29 (s, 3H, NH2), 8.63 (d, J = 8.4 Hz, 1H, NH), 9.00 (t,
1H, NH), 9.77 (s, 1H, NH), 11.60 (s, 1H, OH). HRMS m/z: calcd for
C
19H29N5O6S [M+H]+ 456.1917, found 456.1913.
5.1.9.1.8. (2S,4S)-4-(2-(2-Amino-4-methylpentanamido) acetam-
ido)-N-hydroxy-1-(tosylsulfonyl)pyrrolidine-2-carboxamide hydro-
chloride (12i). White solid, 0.26 g, yield 52.3%, mp 200–202 °C.
1H NMR (DMSO-d6): 0.89 (t, 3H, CH3), 0.92 (d, J = 6 Hz, 3H, CH3),
1.50 (m, 1H), 1.58 (m, 2H, CH3), 1.62 (m, 1H), 2.13 (m, 1H), 2.41
(s, 3H, CH3), 3.16 (m, 1H), 3.21 (m, 1H), 3.56 (m, 1H, CH2), 3.86
(m, 1H, CH2), 3.93 (m, 1H), 4.10 (m, 1H), 4.29 (m, 1H), 7.48, 7.78
(4H, C6H6), 8.39 (s, 3H, NH2), 8.63 (d, J = 8.4 Hz, 1H, NH), 9.16 (s,
1H, NH), 9.75 (s, 1H, NH), 11.64 (s, 1H, OH). HRMS m/z: calcd for
C
18H27N5O6S [M+H]+ 442.1840, found 442.1839.
5.1.9.1.2. (2S,4S)-4-(2-(2-Amino-4-methylpentanamido) acetam-
C
20H31N5O6S [M+H]+ 470.2103, found 470.2087.
ido)-N-hydroxy-1-(phenylsulfonyl)pyrrolidine-2-carboxamide hydro-
chloride (12c). White solid, 0.22 g, yield 45.3%, mp 198–200 °C.
1H NMR (DMSO-d6): 0.89 (d, J = 6.0 Hz, 3H, CH3), 0.92 (d,
J = 6.0 Hz, 3H, CH3), 1.55 (m, 1H), 1.63 (m, 2H,CH2), 1.85 (m,1H),
2.15 (m, 1H), 3.20 (m, 2H), 3.55 (m, 1H), 3.68 (m, 1H), 3.85 (m,
1H), 4.04 (m, 1H), 4.27 (m, 1H), 7.66, 7.76, 7.90 (5H, C6H6), 8.39
(s, 3H, NH2), 8.70 (d, J = 9.6 Hz, 1H, NH), 9.16 (s, 1H, NH), 9.83 (s,
1H, NH), 11.72 (s, 1H, OH). HRMS m/z: calcd for C19H29N5O6S
[M+H]+ 456.1917, found 456.2009.
5.1.9.1.3. (2S,4S)-4-(2-(2-Amino-3-methylpentanamido) acetam-
ido)-N-hydroxy-1-(phenylsulfonyl)pyrrolidine-2-carboxamide hydro-
chloride (12d). White solid, 0.20 g, yield 41.7%, mp 178–181 °C.
1H NMR (DMSO-d6): 0.86 (d, J = 6.6 Hz, 3H, CH3), 0.89 (d,
J = 6.6 Hz, 3H, CH3), 1.14 (m, 1H), 1.55 (m, 2H, CH2), 1.84 (m, 1H),
2.15 (m, 1H), 3.22 (m, 2H), 3.56 (m, 1H), 3.74 (m, 1H), 3.85 (m,
1H), 4.03 (m, 1H), 4.24 (m, 1H), 7.67, 7.76, 7.90 (5H, C6H6), 8.34
(s, 3H, NH2), 8.65 (d, J = 8.4 Hz, 1H, NH), 9.01 (s, 1H, NH), 11.68
(s, 1H, OH). HRMS m/z: calcd for C19H29N5O6S [M+H]+ 456.1917,
found 456.1884.
5.1.9.1.4. (2S,4S)-4-(2-(2-Amino-3-methylpentanamido) acetam-
ido)-N-hydroxy-1-(phenylsulfonyl)pyrrolidine-2-carboxamide hydro-
chloride (12e). White solid, 0.26 g, yield 48.5%, mp 192–195 °C.
1H NMR (DMSO-d6): 1.54 (m,1H), 2.16 (m, 1H), 3.01 (m, 1H), 3.08
(m, 1H), 3.22 (m, 2H), 3.46 (m, 1H), 3.77 (m, 1H), 3.99 (m, 1H),
4.25 (m, 1H), 7.66, 7.76, 7.33 (5H, C6H6), 7.66, 7.76, 7.87 (5H,
C6H6), 8.34 (s, 3H, NH2), 8.61 (d, J = 9.0 Hz, 1H, NH), 8.98 (s, 1H,
NH),9.71 (s, 1H, NH), 11.56 (s, 1H, OH). HRMS m/z: calcd for C22H27-
N5O6S [M+H]+ 490.1860, found 490.1846.
5.1.9.1.5. (2S,4S)-4-(2-(2-Amino-(4-methylthio)butanamido) acet-
amido)-N-hydroxy-1-(phenylsulfonyl)pyrrolidine-2-carboxamide
hydrochloride (12f). White solid, 0.19 g, yield 37.6%, mp 168–
171 °C. 1H NMR (DMSO-d6): 1.54 (m,1H), 2.01 (m, 2H, CH2), 2.06
(s, 3H, CH3), 2.15 (m, 1H), 2.53 (m, 2H, CH2), 3.23 (m, 2H), 3.58
(m, 1H), 3.70 (m, 1H), 3.83 (m, 1H), 4.02 (m, 1H), 4.20 (m, 1H),
7.66, 7.76, 7.90 (5H, C6H6), 8.42 (s, 3H, NH2), 8.63 (d, J = 8.4 Hz,
1H, NH), 9.08 (s, 1H, NH), 9.74 (s, 1H, NH), 11.59 (s, 1H, OH). HRMS
m/z: calcd for C18H27N5O6S2 [M+H]+ 474.1481, found 474.1487.
5.1.9.1.6. (2S,4S)-4-(2-(2-Aminopropanamido) acetamido)-N-
hydroxy-1-(tosylsulfonyl) pyrrolidine-2-carboxamide hydrochloride
(12g). White solid, 0.22 g, yield 47.2%, mp 188–191 °C. 1H NMR
(DMSO-d6): 1.35 (d, J = 7.2 Hz, 3H, CH3), 1.53 (m, 1H), 2.14 (m,
1H), 2.41 (s, 3H, CH3), 3.18 (m, 1H), 3.20 (m, 1H), 3.39 (m, 1H),
3.59 (m, 1H, CH2), 3.83 (m, 1H, CH2), 3.99 (m, 1H), 4.22 (m, 1H),
7.46, 7.78 (4H, C6H6), 8.27 (s, 3H, NH2), 8.61 (d, J = 8.4 Hz, 1H,
NH), 8.99 (s, 1H, NH), 9.69 (s, 1H, NH), 11.50 (s, 1H, OH).
5.1.9.1.9. (2S,4S)-4-(2-(2-Amino-4-methylpentanamido) acetam-
ido)-N-hydroxy-1-(tosylsulfonyl)pyrrolidine-2-carboxamide hydro-
chloride (12j). White solid, 0.28 g, yield 54.7%, mp 176–178 °C.
1H NMR (DMSO-d6): 0.85 (t, 3H, CH3), 0.90 (d, J = 6.6 Hz, 3H,
CH3), 1.13 (m, 1H, CH2), 1.54 (m, 1H), 1.82 (m, 1H), 2.15 (m, 1H),
2.41 (s, 3H, CH3), 3.17 (m, 1H), 3.20 (m, 1H), 3.58 (m, 1H, CH2),
3.74 (m, 1H), 3.86 (m, 1H, CH2), 3.97 (m, 1H), 4.26 (m, 1H), 7.45,
7.77 (4H, C6H6), 8.31 (s, 3H, NH2), 8.65 (d, J =9.0 Hz, 1H, NH),
9.01 (1H, NH), 9.82 (s, 1H, NH), 11.65 (s, 1H, OH). HRMS m/z: calcd
for C20H31N5O6S [M+H]+ 470.2103, found 470.2113.
5.1.9.1.10. (2S,4S)-4-(2-(2-Amino-3-methylpentanamido) acetam-
ido)-N-hydroxy-1-(tosylsulfonyl)pyrrolidine-2-carboxamide hydro-
chloride (12k). White solid, 0.30 g, yield 56.4%, mp 196–198 °C.
1H NMR (DMSO-d6): 1.53 (m, 1H), 2.15 (m, 1H), 2.41 (s, 3H, CH3),
3.06 (m, 1H), 3.10 (m, 1H), 3.18 (m, 2H, CH2), 3.44 (m, 1H, CH2),
3.76 (m, 1H, CH2), 3.97(m, 1H), 4.13 (m, 1H), 4.22 (m, 1H), 7.26,
7.32 (5H, C6H6), 7.45, 7.78 (4H, C6H6), 8.43 (s, 3H, NH2), 8.67 (d,
J = 9.0 Hz, 1H, NH), 9.01 (1H, NH), 9.85 (s, 1H, NH), 11.68 (s, 1H,
OH). HRMS m/z: calcd for C23H29N5O6S [M+H]+ 504.1917, found
504.1937.
5.1.9.1.11.
(2S,4S)-4-(2-(2-Amino-(4-methylthio)butanamido)
acetamido)-N-hydroxy-1-(tosylsulfonyl)pyrrolidine-2-carboxamide
hydrochloride (12l). White solid, 0.22 g, yield 43.8%, mp 185–
188 °C. 1H NMR (DMSO-d6): 1.81 (m, 1H), 1.98 (m, 2H, CH2), 2.05
(s, 3H, CH3), 2.38 (m, 1H), 2.41 (s, 3H, CH3), 2.53 (m, 2H, CH2),
3.10 (m, 1H), 3.49 (m, 1H), 3.70 (m, 2H, CH2), 3.77 (m, 1H), 3.88
(m, 1H), 4.10 (m, 1H), 7.45, 7.73 (4H, C6H6), 8.26 (s, 3H, NH2),
8.62 (d, J = 8.4 Hz, 1H, NH), 9.07 (1H, NH), 9.73 (s, 1H, NH), 11.56
(s, 1H, OH). HRMS m/z: calcd for C19H29N5O6S2 [M+H]+ 488.1638,
found 488.1668.
5.1.9.1.12.
(2S,4S)-4-(2-(2-Aminopropanamido)acetamido)-N-
hydroxy-1-((4-methoxyphenyl) sulfonyl)pyrrolidine-2-carboxamide
hydrochloride (12m). White solid, 0.21 g, yield 43.6%, mp 186–
188 °C. 1H NMR (DMSO-d6): 1.37 (d, J = 6.6 Hz, 3H, CH3), 1.54 (m,
1H), 2.14 (m, 1H), 3.18 (m, 2H), 3.58 (m, 1H), 3.80 (m, 1H), 3.86
(s, 3H, CH3), 3.97 (m, 1H), 3.99 (m, 1H), 4.22 (m, 1H), 7.17, 7.83
(4H, C6H6), 8.32 (s, 3H, NH2), 8.63 (d, J = 9.0 Hz, 1H, NH), 9.02 (s,
1H, NH), 11.55 (s, 1H, OH). HRMS (AP-ESI) m/z: calcd for C17H25N5-
O7S [M+H]+ 444.1553, found 444.1543.
5.1.9.1.13. (2S,4S)-4-(2-(2-Amino-3-methylbutanamido) acetam-
ido)-N-hydroxy-1-((4-methoxyphenyl)sulfonyl)pyrrolidine-2-carbox-
amide hydrochloride (12n). White solid, 0.19 g, yield 38.2%, mp
171–174 °C. 1H NMR (DMSO-d6): 0.93 (d, J = 6.6 Hz, 3H, CH3),
0.96 (d, J = 6.6 Hz, 3H, CH3), 1.84 (m, 1H), 2.15 (m, 1H), 2.36 (m,
1H), 3.18 (m, 2H), 3.60 (m, 1H), 3.69 (m, 1H), 3.83 (s, 3H, CH3),

J. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 3055–3064
3063
3.87 (m, 1H), 3.99 (m, 1H), 4.25 (m, 1H), 7.18, 7.83 (4H, C6H6), 8.33
(s, 3H, NH2), 8.65 (d, J = 9.0 Hz, 1H, NH), 9.03 (t, 1H, NH), 9.77 (s,
1H, NH), 11.65 (s, 1H, OH). HRMS m/z: calcd for C19H29N5O7S
[M+H]+ 472.1866, found 472.1865.
(m,1H), 2.23 (m, 1H), 3.22 (m, 2H), 3.57 (m, 1H), 3.85 (m, 1H),
3.87 (m, 1H), 4.02 (m, 1H), 4.30 (m, 1H), 7.77, 7.90 (5H, C6H6),
8.37 (s, 3H, NH2), 8.70 (d, J = 8.4 Hz, 1H, NH), 9.15 (s, 1H, NH),
9.83 (s, 1H, NH), 11.71 (s, 1H, OH). HRMS m/z: calcd for C19H28ClN5-
O6S [M+H]+ 490.1597, found 490.1563.
5.1.9.1.21. (2S,4S)-4-(2-(2-Amino-3-methylpentanamido) acetam-
ido)-N-hydroxy-1-((4-methoxyphenylsulfonyl)pyrrolidine-2-carbox-
amide hydrochloride (12v). White solid, 0.25 g, yield 47.5%, mp
193–196 °C. 1H NMR (DMSO-d6): 0.86 (dd, J = 7.2 Hz, 3H, CH3),
0.89 (d, J = 6.6 Hz, 3H, CH3), 1.14 (m, 1H), 1.56 (m, 2H, CH2), 1.84
(m, 1H), 2.23 (m, 1H), 3.23 (m, 2H), 3.58 (m, 1H, CH2), 3.75 (m,
1H), 3.85 (m, 1H), 4.05 (m, 1H), 4.26 (m, 1H), 7.76, 7.92 (4H,
C6H6), 8.35 (s, 3H, NH2), 8.67 (d, J = 9.0 Hz, 1H, NH), 9.02 (t, 1H,
NH), 9.80 (s, 1H, NH), 11.73 (s, 1H, OH). HRMS m/z: calcd for C19-
H28ClN5O6S [M+H]+ 490.1597, found 490.1653.
5.1.9.1.14. (2S,4S)-4-(2-(2-Amino-4-methylpentanamido) acetam-
ido)-N-hydroxy-1-((4-methoxyphenyl)sulfonyl)pyrrolidine-2-carbox-
amide hydrochloride (12o). White solid, 0.27 g, yield 51.6%, mp
199–202 °C. 1H NMR (DMSO-d6): 0.89 (d, J = 6.0 Hz, 3H, CH3),
0.92 (d, J = 6.0 Hz, 3H, CH3), 1.52 (m, 2H,CH2), 1.58 (m, 1H), 1.63
(m,1H), 2.13 (m, 1H), 3.16 (m, 1H), 3.20 (m, 1H), 3.55 (m, 1H),
3.85 (m, 1H), 3.87 (s, 3H, CH3), 3.98 (m, 1H), 4.29 (m, 1H), 7.18,
7.83 (4H, C6H6), 8.38 (s, 3H, NH2), 8.70 (d, J = 9.0 Hz, 1H, NH),
9.17 (s, 1H, NH), 9.83 (s, 1H, NH), 11.70 (s, 1H, OH). HRMS m/z:
calcd for C20H31N5O7S [M+H]+ 486.2022, found 486.2026.
5.1.9.1.15. (2S,4S)-4-(2-(2-Amino-3-methylpentanamido) acetam-
ido)-N-hydroxy-1-((4-methoxyphenyl)sulfonyl)pyrrolidine-2-carbox-
amide hydrochloride (12p). White solid, 0.23 g, yield 43.5%, mp
181–183 °C. 1H NMR (DMSO-d6): 0.84 (dd, J = 7.2 Hz, 3H, CH3),
0.89 (d, J = 6.6 Hz, 3H, CH3), 1.13 (m, 1H), 1.54 (m, 2H, CH2), 1.83
(m, 1H), 2.16 (m, 1H), 3.17 (m, 2H), 3.58 (m, 1H, CH2), 3.74 (m,
1H), 3.85 (m, 1H), 3.87 (s, 3H, CH3), 3.97 (m, 1H), 4.27 (m, 1H),
7.16, 7.83 (4H, C6H6), 8.31 (s, 3H, NH2), 8.65 (d, J = 9.6 Hz, 1H,
NH), 9.01 (t, 1H, NH), 9.79 (s, 1H, NH), 11.64 (s, 1H, OH). HRMS
m/z: calcd for C20H31N5O7S [M+H]+ 486.2022, found 486.2041.
5.1.9.1.16. (2S,4S)-4-(2-(2-Amino-3-methylpentanamido) acetam-
ido)-N-hydroxy-1-((4-methoxyphenyl)sulfonyl)pyrrolidine-2-carbox-
amide hydrochloride (12q). White solid, 0.31 g, yield 55.8%, mp
210–212 °C. 1H NMR (DMSO-d6): 1.54 (m,1H), 2.15 (m, 1H), 3.08
(m, 2H), 3.17 (m, 2H), 3.46 (m, 1H), 3.85 (m, 1H), 3.87 (s, 3H,
CH3), 4.01 (m, 1H), 4.13 (m, 1H), 4.23 (m, 1H), 7.26, 7.32 (5H,
C6H6), 7.16, 7.84 (4H, C6H6), 8.47 (s, 3H, NH2), 8.65 (d, J = 8.4 Hz,
1H, NH), 9.02 (s, 1H, NH), 9.74 (s, 1H, NH), 11.65 (s, 1H, OH). HRMS
m/z: calcd for C23H29N5O7S [M+H]+520.1886, found 520.1887.
5.1.9.1.22. (2S,4S)-4-(2-(2-Amino-3-methylpentanamido) acetam-
ido)-N-hydroxy-1-((4-methoxyphenylsulfonyl)pyrrolidine-2-carbox-
amide hydrochloride (12w). White solid, 0.32 g, yield 57.1%, mp
208–211 °C. 1H NMR (DMSO-d6): 1.55 (m,1H), 2.21 (m, 1H), 3.07
(m, 2H), 3.22 (m, 2H), 3.45 (m, 1H), 3.85 (m, 1H), 4.02 (m, 1H),
4.13 (m, 1H), 4.24 (m, 1H), 7.26, 7.32 (5H, C6H6), 7.72, 7.89 (4H,
C6H6), 8.43 (s, 3H, NH2), 8.64 (d, J = 9.0 Hz, 1H, NH), 9.01 (s, 1H,
NH), 9.74 (s, 1H, NH), 11.64 (s, 1H, OH). HRMS m/z: calcd for C22-
H26ClN5O6S [M+H]+ 524.1401, found 524.1405.
5.1.9.1.23.
(2S,4S)-4-(2-(2-Amino-(4-methylthio)butanamido)
acetamido)-N-hydroxy-1-(phenylsulfonyl)pyrrolidine-2-carboxamide
hydrochloride (12x). White solid, 0.24 g, yield 44.2%, mp 192–
194 °C. 1H NMR (DMSO-d6): 1.56 (m, 1H), 2.01 (m, 2H, CH2), 2.06
(s, 3H, CH3), 2.23 (m, 1H), 2.53 (m, 2H, CH2), 3.20 (m, 1H), 3.25
(m, 1H), 3.59 (m, 1H), 3.71 (m, 1H), 3.84 (m, 1H), 4.01 (m, 1H),
4.22 (m, 1H), 7.76, 7.90 (5H, C6H6), 8.41 (s, 3H, NH2), 8.63 (d,
J = 9.0 Hz, 1H, NH), 9.08 (s, 1H, NH), 9.74 (s, 1H, NH), 11.60 (s,
1H, OH). HRMS m/z: calcd for C18H26ClN5O6S2 [M+H]+ 508.1097,
found 508.1110.
5.1.9.1.17.
(2S,4S)-4-(2-(2-Amino-(4-methylthio)butanamido)
acetamido)-N-hydroxy-1-((4-methoxyphenyl)pyrrolidine-2-carbox-
amide hydrochloride (12r). White solid, 0.19 g, yield 39.2%, mp
195–198 °C. 1H NMR (DMSO-d6): 1.53 (m, 1H), 2.01 (m, 2H, CH2),
2.06 (s, 3H, CH3), 2.15 (m, 1H), 2.53 (m, 2H, CH2), 3.19 (m, 2H),
3.59 (m, 1H), 3.71 (m, 1H), 3.83 (m, 1H), 3.97 (m, 1H), 4.22 (m,
1H), 7.16, 7.82 (4H, C6H6), 8.24 (s, 3H, NH2), 8.62 (d, J = 8.4 Hz,
1H, NH), 9.08 (t, 1H, NH), 9.73 (s, 1H, NH), 11.56 (s, 1H, OH). HRMS
m/z: calcd for C19H29N5O7S2 [M+H]+ 504.1597, found 504.1607.
5.1.9.1.18. (2S,4S)-4-(2-(2-Aminopropanamido) acetamido)-N-
5.2. Biological evaluation
5.2.1. In vitro MMP-2 inhibition assay
IC50 values against MMP-2 were determined by using succiny-
lated gelatin as substrate and MMP-2 (Gelatinase A, Sigma) as
enzyme or suspension of SKOV-3 cells in PBS (1 ꢀ 105/well). The
enzyme and inhibitors were dissolved in sodium borate (pH 8.5,
50 mmol/L) and incubated in 96-well microtiter plates for 10 min
at 37 °C. The substrate was added and incubated for another
30 min at 37 °C. Then 0.03% TNBS was added and incubated for
an additional 20 min. The resulting solution was detected under
450 nm wavelength to gain OD450 values with a plate reader
(Varioskan, Thermo). Data analysis was performed using OriginPro
7.5 software and the IC50 values were obtained.
hydroxy-1-((4-chlorophenyl)
sulfonyl)pyrrolidine-2-carboxamide
hydrochloride (12s). White solid, 0.21 g, yield 42.7%, mp 196–
199 °C. 1H NMR (DMSO-d6): 1.35 (d, J = 7.2 Hz, 3H, CH3), 1.56 (m,
1H), 2.24 (m, 1H), 3.19 (m, 1H), 3.25 (m, 1H), 3.50 (m, 1H), 3.60
(m, 1H, CH2), 3.81 (m, 1H, CH2), 3.96 (m, 1H), 4.23 (m, 1H), 7.68,
7.87 (4H, C6H6), 8.21 (s, 2H, NH2), 8.59 (d, J = 9.0 Hz, 1H, NH),
8.94 (s, 1H, NH), 9.66 (s, 1H, NH), 11.46 (s, 1H, OH). HRMS m/z:
calcd for C16H22ClN5O6S [M+H]+ 448.1058, found 448.1053.
5.1.9.1.19. (2S,4S)-4-(2-(2-Amino-3-methylbutanamido) acetam-
ido)-N-hydroxy-1-((4-methoxyphenylsulfonyl)pyrrolidine-2-carbox-
amide hydrochloride (12t). White solid, 0.25 g, yield 48.3%, mp
204–206 °C. 1H NMR (DMSO-d6): 0.93 (d, J = 6.6 Hz, 3H, CH3),
0.97 (d, J = 6.6 Hz, 3H, CH3), 1.54 (m, 1H), 2.08 (m, 1H), 2.24 (m,
1H), 3.20 (m, 1H), 3.25 (m, 1H), 3.60 (m, 1H), 3.70 (s, 3H, CH3),
3.86 (m, 1H), 4.01 (m, 1H), 4.26 (m, 1H), 7.71, 7.87 (4H, C6H6),
8.30 (s, 3H, NH2), 8.64 (d, J = 8.4 Hz, 1H, NH), 9.01 (t, 1H, NH),
9.77 (s, 1H, NH), 11.64 (s, 1H, OH). HRMS m/z: calcd for C18H26ClN5-
O6S [M+H]+ 476.1431, found 476.1438.
5.2.2. In vitro APN inhibition assay
Microsomal aminopeptidase from Porcine Kidney Microsomes
as enzyme and L-Leu-p-nitroanilide as substrate were purchased
from Sigma. The enzyme and inhibitors were dissolved in PBS
(50 mM, pH 7.2) and incubated in 96-well microtiter plates for
5 min at 37 °C. Then the solution of substrate was added into the
above mixture, which was incubated for another 30 min at 37 °C.
The hydrolysis of the substrate was monitored by following the
change in the absorbance measured at 405 nm with a plate reader
(Varioskan, Thermo). Data analysis was performed using OriginPro
7.5 software and the IC50 values were obtained.
5.1.9.1.20. (2S,4S)-4-(2-(2-Amino-4-methylpentanamido) acetam-
ido)-N-hydroxy-1-((4-methoxyphenylsulfonyl)pyrrolidine-2-carbox-
amide hydrochloride (12u). White solid, 0.30 g, yield 56.4%, mp
210–212 °C. 1H NMR (DMSO-d6): 0.89 (d, J = 6.6 Hz, 3H, CH3),
0.92 (d, J = 6.6 Hz, 3H, CH3), 1.55 (m, 2H,CH2), 1.60 (m, 1H), 1.64
5.2.3. In vitro HDACs inhibition assay
In vitro HDACs inhibition assays were performed as described
by Zhang, Y. J. et al. Briefly, 10
extract) and various concentrations of tested compound (50
l
l enzyme solution (Hela nuclear
l)
l

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Acknowledgements
This work was supported by National Science and Technology
Major Project of China (Grant Nos. 2009ZX09103-118 and
2011ZX09401-015), National Natural Science Foundation of China
(Grant Nos. 21172134 and 30772654) and Doctoral Foundation
of Shandong University (Grant No. 21310070613253).
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