Synthesis of new sulfonyl pyrrolidine derivatives as matrix metalloproteinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.07.073

A series of new sulfonyl pyrrolidine derivatives was designed, synthesized, and assayed for their inhibitory activities on matrix metalloproteinase 2 (MMP-2) and aminopeptidase N (AP-N). The results showed that these pyrrolidine derivatives exhibited highly selective inhibition against MMP-2 as compared with AP-N. The compounds 4c, 4j, 5a, and 5b were equally or more potent MMP-2 inhibitors than the positive control LY52. The FlexX docking was done to explain the reason for the different potency between MMP-2 and AP-N. Structure-activity relationships were also briefly discussed.

Full text of DOI:10.1016/j.bmc.2008.07.073

Bioorganic & Medicinal Chemistry 16 (2008) 7932–7938
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of new sulfonyl pyrrolidine derivatives as matrix
metalloproteinase inhibitors
Xian-Chao Cheng a, Qiang Wang a, Hao Fang a, Wei Tang b, Wen-Fang Xu a,
*
a Institute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, No.44 Wenhuaxi Road, Jinan 250012, China
b Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo 113-8655, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new sulfonyl pyrrolidine derivatives was designed, synthesized, and assayed for their inhibi-
tory activities on matrix metalloproteinase 2 (MMP-2) and aminopeptidase N (AP-N). The results showed
that these pyrrolidine derivatives exhibited highly selective inhibition against MMP-2 as compared with
AP-N. The compounds 4c, 4j, 5a, and 5b were equally or more potent MMP-2 inhibitors than the positive
control LY52. The FlexX docking was done to explain the reason for the different potency between MMP-2
and AP-N. Structure–activity relationships were also briefly discussed.
Received 26 May 2008
Revised 23 July 2008
Accepted 24 July 2008
Available online 30 July 2008
Keywords:
Pyrrolidine derivatives
Synthesis
Ó 2008 Elsevier Ltd. All rights reserved.
MMP-2 inhibitors
IC50
1. Introduction
X-ray crystallography data disclosed the important role of sulfonyl
group of these inhibitors in the MMP inhibition potency. The pres-
Matrix metalloproteinases (MMPs) are a family of structurally
related zinc-containing endopeptidases that are involved in the
degradation of the macromolecular components in the extracellu-
lar matrix (ECM) of connective tissue.1 This family of enzymes is
composed of more than 20 known members, among which
MMP-2 is highly involved in the process of tumor invasion and
metastasis and has attracted tremendous attention as a promising
target for cancer therapy.2,3
It has been reported that besides the catalytic activity center
zinc (II) ion of MMP-2, there are two hydrophobic domains, which
are called S10 pocket and S20 pocket, respectively. S10 pocket, the
key domain of MMP-2, is deeper and narrower than that of most
other MMP subtypes, and S20 pocket is solvent exposed.4,5 The
majority of MMP inhibitors contain an effective zinc binding group
(ZBG, such as hydroxamate and carboxylate), which interact with
the active site zinc ion. To improve selectivity, MMP inhibitors
are substituted with side chains that interact with the enzyme sub-
sites, such as S10 pocket and S20 pocket.6,7
ent of the sulfonyl group is crucial, enabling the establishment of
hydrogen bonds with the enzyme backbone, and also orienting
the hydrophobic substituent in the S10 pocket.
Our group has been developing pyrrolidine scaffold-based
MMP-2 inhibitors for a number of years, and numerous compounds
have been reported in the literature.9,10 One of these compounds,
LY52 (see Fig. 1) showed high inhibitory activity against MMP-2
with IC50 values in the nanomolar range. LY52 also suppressed hu-
man ovarian carcinoma cell line SKOV3 invasion in vitro. Further-
more, a significant inhibition of pulmonary metastasis of Lewis
lung carcinoma cells was observed in LY52-administered mice.11
Further design and synthesis of LY52 analogues as MMP-2
inhibitors were conducted in our lab. Based on the role of the sul-
fonyl group in MMP inhibitors, we incorporated the sulfonyl group
into pyrrolidine scaffold to form the new integrated structural pat-
tern (see Fig. 1). R1SO2 group can be various sulfonyl groups, such
as toluene-4-sulfonyl, benzenesulfonyl or methanesulfonyl. R2
group can be hydrogen or various acyl groups. COR3 group can
be hydroxamate or carboxylate group. R1CO group and R2 group
might occupy the S10 and S20 pockets, respectively, while the
COR3 group might chelate the active site zinc ion. The pyrrolidine
scaffold might bond to the enzyme backbone.
Sulfonamide hydroxamate CGS-27023A was designed and syn-
thesized as efficient MMP inhibitors in 1994. This compound has
been used in clinical trials for the treatment of cancer. Cyclic vari-
ants, including prinomastat have also been reported (see Fig. 1).8
The extensive structure–activity relationship (SAR) studies and
2. Results and discussion
* Corresponding author. Tel./fax: +86 531 88382264.
E-mail addresses: chengxianchao2000@yahoo.com.cn (X.-C. Cheng), xuwenf@
sdu.edu.cn (W.-F. Xu).
The target compounds were synthesized via the route as shown
in Scheme 1. Starting from trans-4-hydroxy-L-proline (1) as a chiral
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.073

X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7932–7938
7933
O
O
O
O
O
O
S
O
S
N
O
N
O
O
O
O
O
R1
N
NHOH
R3
S
N
O
N
O
NHOH
N
NHOH
N
NH
R2
O
S
New designed sulfonyl
pyrrolidine derivatives
O
CGS-27023A
LY52
Prinomastat
Figure 1. Chemical structures of CGS-27023A, Prinomastat, LY52 and our further designed sulfonyl pyrrolidine derivatives.
Table 1
template, the intermediate trans-4-hydroxy-L-proline methyl ester
hydrochloride (2) was prepared according to the literature.12 1-
Sulfonyl-4-hydroxy-pyrrolidine-2-carboxylic acid methyl ester
(3a–c) was prepared by sulfonation of compound 2 with various
sulfonyl chlorides in the presence of 4-(dimethylamino) pyridine
as catalyst.13 Esterification of 3a–c afforded the target compounds
4a–r and ammonolysis of 3a–c with NH2OK provided the target
compounds 5a–c.13,14 The chemical structures of the target com-
pounds were confirmed by IR, 1H NMR, and ESI-MS.
The structures and IC50 values of pyrrolidine derivatives
O
O
O
R1
S
R3
N1
2
5
3
4
R2
O
a
No.
R1
R2
R3
IC50
(l
M)
The newly synthesized pyrrolidine derivatives were assayed for
the inhibitory activities on MMP-2 and AP-N.15,16 Similarly as
MMP-2, AP-N is also a zinc-dependent metalloproteinase involved
in the process of tumor invasion and metastasis. Thus, the assay
was performed on both of MMP-2 and AP-N so as to identify the
compound selectivity. LY52 was used as the positive control.
The results showed that these pyrrolidine derivatives exhibited
highly selective inhibition against MMP-2 as compared with AP-N,
thus confirming our strategy for designing MMP-2 inhibitors (Ta-
ble 1). The compounds 4c, 4j, 5a, and 5b were equally or more po-
tent MMP-2 inhibitors than the positive control LY52. The FlexX
docking of these compounds with MMP-2 was done using Sybyl
7.0 of Tripos Incorporation and the result was shown in Figures
2–4. The R1 group at N1 position of pyrrolidine ring (toluene-4-sul-
fonyl, benzenesulfonyl, respectively) occupied the deep S10 pocket
of MMP-2, and the ZBG chelated the active site zinc ion 166 with a
distance of 2.16, 2.19, and 1.47 Å, respectively. These compounds
interacted well with MMP-2 active site, especially the deep S10
pocket and zinc ion 166, consistent with MMP-2 assay results.
The selective inhibition might be explained by the FlexX dock-
ing results of representative compound 5b with AP-N (Fig. 5).
Although compound 5b chelated well with the active site zinc
ion 900, the R2 and R1SO2 groups at pyrrolidine ring were both sol-
vent exposed and could not occupy the S10 and S20 pockets of AP-N.
MMP-2 was a zinc-dependent endopeptidase that could cut the
peptide to parts from the specific amino acid residue of peptide.
MMP-2
0.1 0.02
0.8 0.1
7.1 0.6
3.2 0.3
2.2 0.2
0.007 0.001
0.05 0.006
0.2 0.01
0.04 0.008
3.5 0.4
AP-N
3a
3b
3c
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
5a
5b
5c
LY52
p-CH3C6H4
C6H5
CH3
p-CH3C6H4
p-CH3C6H4
p-CH3C6H4
p-CH3C6H4
p-CH3C6H4
p-CH3C6H4
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
CH3
CH3
CH3
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
38.4 4.6
53.8 3.2
41.6 6.9
24.8 2.6
51.2 6.1
101.4 10.7
74.6 9.4
41.4 3.5
168.6 14.2
43.8 2.6
36.4 4.7
61.3 8.2
47.3 1.5
39.5 7.3
134.7 11.2
53.6 5.2
47.5 3.9
68.9 7.1
28.4 4.5
31.6 2.9
114.3 12.6
21.7 1.9
33.5 4.1
26.3 3.2
141.9 11.7
C6H5CO
p-ClC6H4CO
p-CH3C6H4SO2
C6H5SO2
CH3SO2
(E)C6H5CH@CHCO
C6H5CO
p-ClC6H4CO
p-CH3C6H4SO2
C6H5SO2
CH3SO2
(E)C6H5CH@CHCO
C6H5CO
p-ClC6H4CO
p-CH3C6H4SO2
C6H5SO2
CH3SO2
(E)C6H5CH@CHCO
H
2.7 0.2
0.04 0.003
0.01 0.001
1.3 0.1
0.09 0.01
5.2 0.4
1.5 0.2
0.2 0.01
0.7 0.06
8.9 1.0
CH3
CH3
CH3
p-CH3C6H4
C6H5
0.5 0.07
NHOH
NHOH
NHOH
0.002 0.0002
0.004 0.0003
0.02 0.001
H
H
CH3
0.009 0.0004
a
IC50 values are mean of three experiments, standard deviation is given.
O
S
N
O
R1 S
O
O
O
O
O
O
R1
OH
O
O
O
Cl
HN
HO
H2N
HO
N
a
b
c
1
2
3a-c
4a-r
HO
d
R2 O
O
S
O
O
R1
NHOH
N
5a-c
HO
Scheme 1. Reagents: (a) CH3OH, HCl; (b) R1SO2Cl; (c) R2COCl or R2SO2Cl; (d) NH2OK.

7934
X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7932–7938
Figure 5. FlexX docking result of compound 5b with AP-N.
Figure 2. FlexX docking result of compound 4c with MMP-2.
The ZBG is hydroxamate (CONHOH) for 5a–c and carboxylate
(COOCH3) for their predecessor, respectively. Both of these two
groups could chelate zinc ion at catalytic activity center of the en-
zyme. However, the hydroxamate group was a more potent ZBG
than carboxylate group as shown in the activity order of 5a–c
and their predecessor.
Among compounds 4a–f, R1SO2 and COR3 groups were fixed as
toluene-4-sulfonyl and carboxylate, respectively, and R2 group was
altered as various length and substitutions. So the differences in
the inhibitory activities of these compounds were caused by vari-
ous R2 groups. For compound 4c, R1SO2 and R2 groups were both
toluene-4-sulfonyl, thus forming a butterfly like structure. The
high potency of 4c might be due to the symmetrical structure,
which could adjust the conformation to extend into the catalytic
activity center of the enzyme, respectively. Substitution on aro-
matic ring also has impact on bioactivity. For example, compound
4b which was substituted with chlorine in para position showed a
slightly better inhibitory activity compared with 4a. Increasing
conjugate system between ester and aromatic ring (4a) produced
4f with improved potency.
Figure 3. FlexX docking result of compound 4j with MMP-2.
Among compounds 4g–l, R1SO2 and COR3 groups were fixed as
benzenesulfonyl and carboxylate, respectively, and R2 group was
altered as various length and substitutions. So the differences in
the inhibitory activities of these compounds were caused by vari-
ous R2 groups. The SARs were similar to compounds 4a–f. For com-
pound 4j, R1SO2 and R2 groups were both benzenesulfonyl, thus
forming a butterfly like structure. The high potency of 4j might
be due to the symmetrical structure, which could adjust the con-
formation to extend into the catalytic activity center of the en-
zyme, respectively. Substitution on aromatic ring also has impact
on bioactivity. For example, compound 4h which was substituted
with chlorine in para position showed a slightly better inhibitory
activity compared with 4g. Increasing conjugate system between
ester and aromatic ring (4g) produced 4l with improved potency.
However, compounds 4g–l was less potent than its counterpart
4a–f, which indicated that benzenesulfonyl substitution at N1 po-
sition of pyrrolidine ring was less favorable than the toluene-4-sul-
fonyl substitution.
Among compounds 4m–r, R1SO2 and COR3 groups were fixed as
methanesulfonyl and carboxylate, respectively, and R2 group was
altered as various length and substitutions. So the differences in
the inhibitory activities of these compounds were caused by vari-
ous R2 groups. However, the SARs were not similar to compounds
4a–f and 4g–l. For compound 4q, R1SO2 and R2 groups were both
methanesulfonyl, thus forming a butterfly like structure. However,
this compound did not present high potency. This might be due to
the too small methanesulfonyl group, which could not match the
Figure 4. FlexX docking result of compound 5b with MMP-2.
However, AP-N was a membrane-bound zinc exopeptidase that
catalyzed the removal of NH-terminal amino acid from peptide.
Due to the structural differences between MMP-2 and AP-N, there
were different structural requirements for their respective inhibi-
tors. As these pyrrolidine derivatives exhibited highly selective
inhibition against MMP-2, the following structure-activity rela-
tionships (SARs) were mainly discussed about MMP-2 inhibition.
Compounds 5a–c was more potent than their predecessor 3a–c.
This activity difference was caused by the ZBG (COR3), which was
the only structural difference between 5a–c and their predecessor.

X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7932–7938
7935
160 °C (lit.10 mp 156–160 °C). 1H NMR (CD3OD, d ppm): 4.86 (s,
2H, NH2+), 4.63 (m, 1H, 2-CH), 3.88 (s, 3H, CH3), 3.50 (m, 1H, 4-
CH), 3.35 (m, 2H, 5-CH2), 2.47–2.20 (m, 2H, 3-CH2), 1.31 (s, 1H,
OH).
Ala86
N
H
Glu121
O
Zn2+
O H
N
H
O
O
O
4.1.2. 1-Sulfonyl-4-hydroxy-pyrrolidine-2-carboxylic acid
methyl ester (3a–c)
S
N
Trans-4-hydroxy-L-proline methyl ester hydrochloride (2) (69 g,
S1' pocket
380 mmol) was dissolved in water/dioxane (1:1, 300 ml) with tri-
ethylamine (135 ml, 960 mmol). Various sulfonyl chlorides
(420 mmol) were added along with 4-(dimethylamino) pyridine
(4.6 g, 38 mmol) and the mixture was stirred 24 h at room temper-
ature. The mixture was then concentrated and extracted with
EtOAc (6ꢀ 50 ml). Layers were separated and the organic layer
was washed with 1 M HCl (2ꢀ 100 ml), and with brine
(1ꢀ 100 ml), dried over Na2SO4, filtered, and evaporated to give
3a–c as solid material.
HO
O
Ala84
Figure 6. Proposed binding mode of compound 5b with MMP-2.
catalytic activity center of the enzyme. Compound 4o displayed
high potency, and this could be explained by the toluene-4-sulfo-
nyl at C4 position of pyrrolidine ring, which was large enough to
plunge in the S10 pocket.
Finally, the binding mode of the compound 5b with MMP-2 was
proposed as follow: (1) the hydroxamate chelated the active site
zinc ion; (2) the sulfonyl, and hydroxyl groups provided hydrogen
bond interactions with the enzyme backbone Ala86, Ala84, and
Glu121; (3) the side chain (benzenesulfonyl) underwent effective
interactions with the enzyme S1’ pocket (Fig. 6). The above binding
mode information encouraged us to further design pyrrolidine-
scaffold-based MMP-2 inhibitors, which would be reported later.
4.1.2.1. 4-Hydroxy-1-(toluene-4-sulfonyl)-pyrrolidine-2-car-
boxylic acid methyl ester (3a).
Yield: 75%; white crystal; mp
78–79 °C; IR (KBr, cmꢁ1): 3513 (OH), 2953 (CH), 1743 (C@O), 1598
(C@C), 1344 (SO2), 1157 (SO2); ESI-MS: 300.5 [M+1]+.
4.1.2.2. 1-Benzenesulfonyl-4-hydroxy-pyrrolidine-2-carboxylic
acid methyl ester (3b).
Yield: 69%; white crystal; mp 102–
103 °C; IR (KBr, cmꢁ1): 3468 (OH), 2957 (CH), 1726 (C@O), 1348
(SO2), 1157 (SO2); ESI-MS: 286.2 [M+1]+.
4.1.2.3. 4-Hydroxy-1-methanesulfonyl-pyrrolidine-2-carbox-
ylic acid methyl ester (3c).
Yield: 35%; white crystal; mp 85–
87 °C; IR (KBr, cmꢁ1): 3469 (OH), 2959 (CH), 1724 (C@O), 1344
3. Conclusions
(SO2), 1148 (SO2); ESI-MS: 224.3 [M+1]+.
In conclusion, a series of new sulfonyl pyrrolidine derivatives
was designed and synthesized. These pyrrolidine derivatives
exhibited highly selective inhibition against MMP-2 as compared
with AP-N. The compounds 4c, 4j, 5a, and 5b were equally or more
potent MMP-2 inhibitors than the positive control LY52. SAR stud-
ies indicated that introduction of butterfly like symmetrical struc-
ture (toluene-4-sulfonyl group) at pyrrolidine ring favored the
inhibitory activity against MMP-2. The FlexX docking was consis-
tent with the above SAR results. Further assays of these com-
pounds on cell culture and animal models are underway.
4.1.3. General procedure for the preparation of 1-(toluene-4-
sulfonyl)-4-(acyloxy or sulfonyloxy)-pyrrolidine-2-carboxylic
acid methyl ester (4a–f)
4-Hydroxy-1-(toluene-4-sulfonyl)-pyrrolidine-2-carboxylic
acid methyl ester (3a) (3 g, 10 mmol) was dissolved in dichloro-
methane (50 ml) and triethylamine (1.01 g, 10 mmol) was added.
A solution of acyl chloride or sulfonyl chloride (10 mmol) in dichlo-
romethane (50 ml) was added dropwise at 0 °C. The resulting mix-
ture was stirred overnight at room temperature. The solvent was
evaporated in vacuo. The residue was purified by flash column
chromatography to give 4a–f.
4. Experimental
4.1.3.1. 4-Benzoyloxy-1-(toluene-4-sulfonyl)-pyrrolidine-2-car-
boxylic acid methyl ester (4a). Flash column chromatography:
dichloromethane/acetone = 100:1; 45%; white crystal; mp 95–
97 °C; IR (KBr, cmꢁ1): 2953 (CH), 1720 (C@O), 1599 (C@C), 1350
(SO2), 1160 (SO2); 1H NMR (CDCl3, d ppm): 7.72 (d, 2H, 1-SO2Ar-H,
J = 8.28 Hz), 7.63 (d, 2H, 1-SO2Ar-H, J = 8.36 Hz), 7.55 (m, 1H, 4-
OCOAr-H), 7.36 (t, 2H, 4-OCOAr-H, J = 7.76 Hz), 7.14 (d, 2H, 4-
OCOAr-H, J = 8.09 Hz), 5.29 (s, 1H, 4-CH), 4.40 (m, 1H, 2-CH), 3.80
(s, 3H, OCH3), 3.89–3.73 (m, 2H, 5-CH2), 2.51–2.30 (m, 2H, 3-CH2),
2.20 (s, 3H, ArCH3); ESI-MS: 404.4 [M+1]+.
4.1. Synthetic methods and spectroscopic details
Melting points were determined using X-6 digital display binoc-
ular microscope (uncorrected). Infrared spectra were measured on
a nicolet nexus 470 FT-IR spectrometer using smear KBr crystal or
KBr plate. 1H NMR spectra were recorded on a Bruker Avance
(400 MHz) spectrometer; J values are in Hertz. Mass spectra were
recorded on an electro-spray ionization mass spectrometer as the
value m/z. Flash column chromatography was performed using
300 mesh silica gel. The yields were calculated by the last step
reaction.
4.1.3.2. 4-(4-Chloro-benzoyloxy)-1-(toluene-4-sulfonyl)-pyrrol-
idine-2-carboxylic acid methyl ester (4b). Flash column
chromatography: dichloromethane/acetone = 100:1; 57%; white
crystal; mp 122–124 °C; IR (KBr, cmꢁ1): 2924 (CH), 1724 (C@O),
1593 (C@C), 1350 (SO2), 1159 (SO2); 1H NMR (CDCl3, d ppm): 7.71
(d, 2H, 4-OCOAr-H, J = 8.25 Hz), 7.57 (d, 2H, 1-SO2Ar-H,
J = 9.03 Hz), 7.33 (d, 2H, 4-OCOAr-H, J = 8.60 Hz), 7.14 (d, 2H, 1-
SO2Ar-H, J = 8.05 Hz), 5.36 (s, 1H, 4-CH), 4.39 (m, 1H, 2-CH), 3.82 (s,
3H, OCH3), 3.88–3.73 (m, 2H, 5-CH2), 2.52–2.30 (m, 2H, 3-CH2),
2.23 (s, 3H, ArCH3); ESI-MS: 438.3 [M+1]+.
4.1.1. Trans-4-hydroxy-
L
-proline methyl ester hydrochloride (2)
-proline (1) (100 g, 763.5 mmol) in
A slurry of trans-4-hydroxy-
L
methanol (650 ml) was treated with dry hydrogen chloride until
homogeneous. The solution was heated to the reflux temperature
for 3 h and concentrated in vacuo. Upon cooling, the product was
collected by filtration, washed with acetone and ether, and dried
under reduced pressure to yield trans-4-hydroxy-
L-proline methyl
ester hydrochloride (2) as white crystal (120 g, 87%), mp 157–

7936
X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7932–7938
4.1.3.3. 1-(Toluene-4-sulfonyl)-4-(toluene-4-sulfonyloxy)-pyrrol-
4.1.4.2. 1-Benzenesulfonyl-4-(4-chloro-benzoyloxy)-pyrroli-
idine-2-carboxylic acid methyl ester (4c). Flash column chro-
dine-2-carboxylic acid methyl ester (4h). Flash column chro-
matography:
dichloromethane/acetone = 100:1; 42%; white
matography:
dichloromethane/acetone = 100:1; 64%; white
crystal; mp 85–86 °C; IR (KBr, cmꢁ1): 2945 (CH), 1748 (C@O),
1599 (C@C), 1354 (SO2), 1171 (SO2); 1H NMR (CDCl3, d ppm): 7.70
(d, 2H, 4-OSO2Ar-H, J = 8.24 Hz), 7.61 (d, 2H, 1-SO2Ar-H,
J = 8.32 Hz), 7.32 (t, 4H, 4-OSO2Ar-H and 1-SO2Ar-H, J = 8.68 Hz),
4.97 (m, 1H, 4-CH), 4.27 (t, 1H, 2-CH, J = 7.92 Hz), 3.75 (s, 3H,
OCH3), 3.77–3.55 (m, 2H, 5-CH2), 2.46 (s, 3H, ArCH3), 2.44 (s, 3H,
ArCH3), 2.38–2.16 (m, 2H, 3-CH2); ESI-MS: 454.2 [M+1]+.
crystal; mp 120-122 °C; IR (KBr, cmꢁ1): 2923 (CH), 1755 (C@O),
1725 (C@O), 1593 (C@C), 1348 (SO2), 1163 (SO2); 1H NMR (CDCl3, d
ppm): 7.87 (m, 2H, 1-SO2Ar-H), 7.52 (d, 2H, 4-OCOAr-H,
J = 6.80 Hz), 7.43 (m, 3H, 1-SO2Ar-H), 7.32 (d, 2H, 4-OCOAr-H,
J = 6.74 Hz), 5.40 (s, 1H, 4-CH), 4.41 (m, 1H, 2-CH), 3.80 (s, 3H,
OCH3), 3.89–3.73 (m, 2H, 5-CH2), 2.53–2.31 (m, 2H, 3-CH2); ESI-
MS: 424.2 [M+1]+.
4.1.3.4. 4-Benzenesulfonyloxy-1-(toluene-4-sulfonyl)-pyrroli-
4.1.4.3. 1-Benzenesulfonyl-4-(toluene-4-sulfonyloxy)-pyrroli-
dine-2-carboxylic acid methyl ester (4d).
Flash column
dine-2-carboxylic acid methyl ester (4i).
Flash column chro-
chromatography: dichloromethane/acetone = 100:1; 61%; white
crystal; mp 95–97 °C; IR (KBr, cmꢁ1): 2941 (CH), 1745 (C@O),
1597 (C@N), 1351 (SO2), 1187 (SO2); 1H NMR (CDCl3, d ppm):
7.75 (d, 2H, 4-OSO2Ar-H, J = 8.08 Hz), 7.70 (d, 2H, 1-SO2Ar-H,
J = 8.27 Hz), 7.67 (m, 1H, 4-OSO2Ar-H), 7.55 (t, 2H, 4-OSO2Ar-H,
J = 7.95 Hz), 7.29 (d, 2H, 1-SO2Ar-H, J = 8.10 Hz), 5.02 (m, 1H, 4-
CH), 4.28 (t, 1H, 2-CH, J = 7.78 Hz), 3.73 (s, 3H, OCH3), 3.72–3.56
(m, 2H, 5-CH2), 2.43 (s, 3H, ArCH3), 2.40–2.17 (m, 2H, 3-CH2);
ESI-MS: 440.3 [M+1]+.
matography: dichloromethane/acetone = 100:1–10:1; 46%; white
crystal; mp 62-63 °C; IR (KBr, cmꢁ1): 2956 (CH), 1747 (C@O),
1599 (C@C), 1353 (SO2), 1173 (SO2); 1H NMR (CDCl3, d ppm):
7.81 (d, 2H, 1-SO2Ar-H, J = 7.24 Hz), 7.59 (m, 3H, 1-SO2Ar-H),
7.51 (t, 2H, 4-OSO2Ar-H, J = 7.84 Hz), 7.32 (d, 2H, 4-OSO2Ar-H,
J = 8.16 Hz), 4.99 (m, 1H, 4-CH), 4.28 (t, 1H, 2-CH, J = 7.92 Hz),
3.73 (s, 3H, OCH3), 3.71–3.58 (m, 2H, 5-CH2), 2.45 (s, 3H, ArCH3),
2.41–2.17 (m, 2H, 3-CH2); ESI-MS: 440.3 [M+1]+.
4.1.4.4. 1-Benzenesulfonyl-4-benzenesulfonyloxy-pyrrolidine-
4.1.3.5. 4-Methanesulfonyloxy-1-(toluene-4-sulfonyl)-pyrrol-
2-carboxylic acid methyl ester (4j).
Flash column chroma-
idine-2-carboxylic acid methyl ester (4e).
Flash column
tography: dichloromethane/acetone = 100:1–10:1; 68%; white
crystal; mp 75–76 °C; IR (KBr, cmꢁ1): 2925 (CH), 1741 (C@O),
1360 (SO2), 1168 (SO2); 1H NMR (CDCl3, d ppm): 7.81 (d, 2H,
4-OSO2Ar-H, J = 7.32 Hz), 7.71 (m, 2H, 4-OSO2Ar-H), 7.66 (d, 1H,
4-OSO2Ar-H, J = 7.48 Hz), 7.54 (m, 5H, 1-SO2Ar-H), 5.03 (s, 1H, 4-
CH), 4.29 (t, 1H, 2-CH, J = 7.96 Hz), 3.73 (s, 3H, OCH3), 3.70–3.60
(m, 2H, 5-CH2), 2.43-2.16 (m, 2H, 3-CH2); ESI-MS: 426.1 [M+1]+.
chromatography: dichloromethane/acetone = 100:1; 35%; color-
less oil; IR (KBr, cmꢁ1): 2955 (CH), 1754 (C@O), 1598 (C@C),
1348 (SO2), 1160 (SO2); 1H NMR (CDCl3, d ppm): 7.77 (d, 2H, Ar-
H, J = 8.28 Hz), 7.35 (d, 2H, Ar-H, J = 8.09 Hz), 5.20 (m, 1H, 4-CH),
4.38 (t, 1H, 2-CH, J = 8.07 Hz), 3.76 (s, 3H, OCH3), 3.74 (m, 2H, 5-
CH2), 2.82 (s, 3H, CH3SO2), 2.56–2.50 and 2.32–2.25 (m, 2H, 3-
CH2), 2.44 (s, 3H, ArCH3); ESI-MS: 378.3 [M+1]+.
4.1.4.5. 1-Benzenesulfonyl-4-methanesulfonyloxy-pyrrolidine-
2-carboxylic acid methyl ester (4k). Flash column chromatog-
raphy: dichloromethane/acetone = 100:1; 74%; colorless oil; IR
(KBr, cmꢁ1): 2955 (CH), 1751 (C@O), 1351 (SO2), 1173 (SO2); 1H
NMR (CDCl3, d ppm): 7.89 (d, 2H, 1-SO2Ar-H, J = 7.25 Hz), 7.63 (m,
1H, 1-SO2Ar-H), 7.57 (t, 2H, 1-SO2Ar-H, J = 7.80 Hz), 5.20 (s, 1H, 4-
CH), 4.40 (t, 1H, 2-CH, J = 8.18 Hz), 3.76 (s, 3H, OCH3), 3.79–3.74
(m, 2H, 5-CH2), 2.80 (s, 3H, CH3SO2), 2.55–2.29 (m, 2H, 3-CH2); ESI-
MS: 364.2 [M+1]+.
4.1.3.6. 4-(3-Phenyl-acryloyloxy)-1-(toluene-4-sulfonyl)-pyr-
rolidine-2-carboxylic acid methyl ester (4f).
Flash column
chromatography: dichloromethane/acetone = 100:1; 74%; white
crystal; mp 110–112 °C; IR (KBr, cmꢁ1): 2953 (CH), 1745 (C@O),
1716 (C@O), 1636 (C@O), 1345 (SO2), 1159 (SO2), 1022 (@CH);
1H NMR (CDCl3, d ppm): 7.75 (d, 2H, 1-SO2Ar-H, J = 8.20 Hz), 7.56
(m, 1H, 4-Ar-H), 7.47–7.39 (m, 4H, 4-Ar-H), 7.25 (d, 2H, 1-SO2Ar-
H, J = 8.16 Hz), 6.46 (d, 1H, 4-OCOC@CH, J = 16.00 Hz), 5.91 (d,
1H, 4-OCOCH@C, J = 16.00 Hz), 5.26 (s, 1H, 4-CH), 4.37 (m, 1H, 2-
CH), 3.80 (s, 3H, OCH3), 3.83–3.65 (m, 2H, 5-CH2), 2.47–2.39 (m,
2H, 3-CH2), 2.16 (s, 3H, ArCH3); ESI-MS: 430.3 [M+1]+.
4.1.4.6. 1-Benzenesulfonyl-4-(3-phenyl-acryloyloxy)-pyrroli-
dine-2-carboxylic acid methyl ester (4l). Flash column chro-
matography: dichloromethane/acetone = 50:1; 43%; white crystal;
mp 85–86 °C; IR (KBr, cmꢁ1): 2953 (CH), 1744 (C@O), 1714 (C@O),
1640 (C@O), 1347 (SO2), 1162 (SO2), 1021 (@CH); 1H NMR (CDCl3, d
ppm): 7.87 (m, 2H, Ar-H), 7.42 (m, 8H, Ar-H), 6.45 (d, 1H, 4-
OCOC@CH, J = 16.00 Hz), 5.81 (d, 1H, 4-OCOCH@C, J = 16.08 Hz),
5.26 (s, 1H, 4-CH), 4.37 (m, 1H, 2-CH), 3.78 (s, 3H, OCH3), 3.84-3.65
(m, 2H, 5-CH2), 2.45–2.24 (m, 2H, 3-CH2); ESI-MS: 416.3 [M+1]+.
4.1.4. General procedure for the preparation of 1-benzenesulfonyl-
4-(acyloxy or sulfonyloxy)-pyrrolidine-2-carboxylic acid methyl
ester (4g–l)
1-Benzenesulfonyl-4-hydroxy-pyrrolidine-2-carboxylic
acid
methyl ester (3b) (2.86 g, 10 mmol) was dissolved in dichloro-
methane (50 ml) and triethylamine (1.01 g, 10 mmol) was added.
A solution of acyl chloride or sulfonyl chloride (10 mmol) in dichlo-
romethane (50 ml) was added dropwise at 0 °C. The resulting mix-
ture was stirred overnight at room temperature. The solvent was
evaporated in vacuo. The residue was purified by flash column
chromatography to give 4g–l.
4.1.5. General procedure for the preparation of 1-methanesul-
fonyl-4-(acyloxy or sulfonyloxy)-pyrrolidine-2-carboxylic acid
methyl ester (4m–r)
4-Hydroxy-1-methanesulfonyl-pyrrolidine-2-carboxylic
acid
methyl ester (3c) (2.23 g, 10 mmol) was dissolved in dichloro-
methane (50 ml) and triethylamine (1.01 g, 10 mmol) was added.
A solution of acyl chloride or sulfonyl chloride (10 mmol) in dichlo-
romethane (50 ml) was added dropwise at 0 °C. The resulting mix-
ture was stirred overnight at room temperature. The solvent was
evaporated in vacuo. The residue was purified by flash column
chromatography to give 4m–r.
4.1.4.1. 1-Benzenesulfonyl-4-benzoyloxy-pyrrolidine-2-carbox-
ylic acid methyl ester (4g).
Flash column chromatography:
dichloromethane/acetone = 100:1; 54%; white crystal; mp 110-
111 °C; IR (KBr, cmꢁ1): 2954 (CH), 1751 (C@O), 1710 (C@O),
1599 (C@C), 1335 (SO2), 1158 (SO2); 1H NMR (CDCl3, d ppm):
7.86 (m, 2H, 4-OCOAr-H), 7.56 (m, 3H, 4-OCOAr-H), 7.39 (m, 5H,
1-SO2Ar-H), 5.40 (s, 1H, 4-CH), 4.40 (m, 1H, 2-CH), 3.80 (s, 3H,
OCH3), 3.91–3.73 (m, 2H, 5-CH2), 2.53–2.31 (m, 2H, 3-CH2); ESI-
MS: 390.2 [M+1]+.
4.1.5.1. 4-Benzoyloxy-1-methanesulfonyl-pyrrolidine-2-car-
boxylic acid methyl ester (4m).
Flash column chromatogra-

X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7932–7938
7937
phy: dichloromethane/acetone = 100:1; 52%; white crystal; mp 65-
66 °C; IR (KBr, cmꢁ1): 2957 (CH), 1745 (C@O), 1714 (C@O), 1326
(SO2), 1147 (SO2); 1H NMR (CDCl3, d ppm): 8.00 (d, 2H, Ar-H,
J = 7.20 Hz), 7.58 (t, 1H, Ar-H, J = 7.44 Hz), 7.45 (m, 2H, Ar-H),
5.54 (s, 1H, 4-CH), 4.71 (t, 1H, 2-CH, J = 8.12 Hz), 3.79 (s, 3H,
OCH3), 3.93–3.75 (m, 2H, 5-CH2), 3.03 (s, 3H, CH3SO2), 2.70–2.39
(m, 2H, 3-CH2); ESI-MS: 328.3 [M+1]+.
The residue was purified by flash column chromatography to give
5a–c.
4.1.6.1. 4-Hydroxy-1-(toluene-4-sulfonyl)-pyrrolidine-2-car-
boxylic acid hydroxyamide (5a).
Flash column chromatogra-
phy: dichloromethane/methanol = 10:1; 36%; white crystal; mp
130–132 °C; IR (KBr, cmꢁ1): 3331 (OH), 2924 (CH), 1682 (C@O),
1599 (C@O), 1332 (SO2), 1157 (SO2); 1H NMR (CD3OD, d ppm):
7.98 (d, 1H, 2-CONH, J = 8.48 Hz), 7.75 (d, 2H, Ar-H, J = 8.27 Hz),
7.39 (m, 2H, Ar-H), 4.32 (m, 1H, 2-CH), 4.09 (t, 1H, 4-CH,
J = 7.68 Hz), 3.73 (s, 1H, 4-OH), 3.30 (s, 1H, 2-CONOH), 3.62–3.21
(m, 2H, 5-CH2), 2.41 (s, 3H, ArCH3), 2.11–1.93 (m, 2H, 3-CH2);
ESI-MS: 301.4 [M+1]+.
4.1.5.2. 4-(4-Chloro-benzoyloxy)-1-methanesulfonyl-pyrrol-
idine-2-carboxylic acid methyl ester (4n). Flash column chro-
matography:
dichloromethane/acetone = 100:1; 51%; white
crystal; mp 105–107 °C; IR (KBr, cmꢁ1): 2959 (CH), 1746 (C@O),
1716 (C@O), 1593 (C@C), 1329 (SO2), 1146 (SO2); 1H NMR (CDCl3, d
ppm): 7.95 (d, 2H, Ar-H, J = 6.72 Hz), 7.43 (d, 2H, Ar-H, J = 6.74 Hz),
5.53 (s, 1H, 4-CH), 4.74 (t, 1H, 2-CH, J = 8.07 Hz), 3.80 (s, 3H, OCH3),
3.97–3.71 (m, 2H, 5-CH2), 3.05 (s, 3H, CH3SO2), 2.70–2.34 (m, 2H, 3-
CH2); ESI-MS: 362.2 [M+1]+.
4.1.6.2. 1-Benzenesulfonyl-4-hydroxy-pyrrolidine-2-carboxylic
acid hydroxyamide (5b).
Flash column chromatography:
dichloromethane/methanol = 10:1; 41%; white crystal; mp 155–
157 °C; IR (KBr, cmꢁ1): 3216 (OH), 2948 (CH), 1668 (C@O), 1332
(SO2), 1158 (SO2); 1H NMR (CD3OD, d ppm): 7.89 (m, 2H, Ar-H),
7.68 (m, 1H, Ar-H), 7.60 (t, 2H, Ar-H, J = 7.84 Hz), 4.33 (m, 1H, 2-
CH), 4.13 (t, 1H, 4-CH, J = 7.76 Hz), 3.66–3.28 (m, 2H, 5-CH2),
2.13–1.94 (m, 2H, 3-CH2); ESI-MS: 287.3 [M+1]+.
4.1.5.3. 1-Methanesulfonyl-4-(toluene-4-sulfonyloxy)-pyrrol-
idine-2-carboxylic acid methyl ester (4o). Flash column chro-
matography: dichloromethane/acetone = 100:1-10:1; 73%; white
crystal; mp 95-96 °C; IR (KBr, cmꢁ1): 2959 (CH), 1747 (C@O), 1596
(C@C), 1338 (SO2), 1155 (SO2); 1H NMR (CDCl3, d ppm): 7.79 (d, 2H,
Ar-H, J = 8.28 Hz), 7.38 (d, 2H, Ar-H, J = 8.12 Hz), 5.08 (s, 1H, 4-CH),
4.48 (t, 1H, 2-CH, J = 8.08 Hz), 3.75 (s, 3H, OCH3), 3.80–3.60 (m, 2H,
5-CH2), 2.95 (s, 3H, CH3SO2), 2.47 (s, 3H, ArCH3), 2.62–2.55 and
2.27–2.16 (m, 2H, 3-CH2); ESI-MS: 378.3 [M+1]+ and 395.2
[M+NH4]+.
4.1.6.3. 4-Hydroxy-1-methanesulfonyl-pyrrolidine-2-carbox-
ylic acid hydroxyamide (5c). Flash column chromatography:
dichloromethane/methanol = 10:1; 46%; white crystal; mp
150–151 °C; IR (KBr, cmꢁ1): 3416 and 3285 (OH), 2926
(CH), 1678 (C@O), 1336 (SO2), 1145 (SO2); 1H NMR (CD3OD,
d
ppm): 4.43 (s, 1H, 2-CH), 4.24 (t, 1H, 4-CH, J = 8.12 Hz),
4.1.5.4. 4-Benzenesulfonyloxy-1-methanesulfonyl-pyrrolidine-
2-carboxylic acid methyl ester (4p). Flash column chromatog-
raphy: dichloromethane/acetone = 100:1-10:1; 58%; white crystal;
mp 78-80 °C; IR (KBr, cmꢁ1): 2956 (CH), 1751 (C@O), 1340 (SO2),
1151 (SO2); 1H NMR (CDCl3, d ppm): 7.91 (d, 2H, Ar-H, J = 8.68 Hz),
7.72 (t, 1H, Ar-H, J = 7.48 Hz), 7.60 (t, 2H, Ar-H, J = 7.96 Hz), 5.12 (s,
1H, 4-CH), 4.48 (t, 1H, 2-CH, J = 8.12 Hz), 3.74 (s, 3H, OCH3), 3.80-
3.61 (m, 2H, 5-CH2), 2.95 (s, 3H, CH3SO2), 2.62–2.05 (m, 2H, 3-CH2);
ESI-MS: 364.2 [M+1]+ and 381.3 [M+NH4]+.
3.62-3.46 (m, 2H, 5-CH2), 3.33 (m, 2H, 4-OH and 2-CONOH),
2.96 (s, 3H, CH3), 2.33–2.15 (m, 2H, 3-CH2); ESI-MS: 225.4
[M+1]+.
4.2. Biological evaluation
4.2.1. MMP-2 inhibition assay
The pyrrolidine derivatives were assayed for the inhibitory
activities against MMP-2 in 96-well microtiter plates using succ-
inylated gelatin as the substrate. The compound and enzyme were
dissolved in sodium borate buffer (pH 8.5, 50 mM), and incubated
at 37 °C for 30 min. The substrate was added and incubated at
37 °C for another 60 min. The 100% and blank groups were also car-
ried out, in which the 100% group contained not compound and the
blank group contained only the enzyme. Then 0.03% picrylsulfonic
acid solution was added and incubated at room temperature for
additional 20 min. The resulting solutions were measured under
450 nm to gain OD450 values, which were then used to calculate
the inhibitory rates by [OD450(100%) ꢁ OD450(compound)]/
[OD450(100%) ꢁ OD450(blank)] ꢀ 100%. The IC50 values were ob-
tained from the above inhibitory rates using OriginPro 7.5
software.
4.1.5.5. 1-Methanesulfonyl-4-methanesulfonyloxy-pyrrolidine-
2-carboxylic acid methyl ester (4q).
Flash column chroma-
tography: dichloromethane/acetone = 100:1; 32%; yellow oil; IR
(KBr, cmꢁ1): 2940 (CH), 1748 (C@O), 1336 (SO2), 1173 (SO2); 1H
NMR (CDCl3, d ppm): 5.28 (s, 1H, 4-CH), 4.64 (t, 1H, 2-CH,
J = 8.16 Hz), 3.79 (s, 3H, OCH3), 4.02–3.66 (m, 2H, 5-CH2), 3.11 (s,
3H, 4-OSO2CH3), 3.04 (s, 3H, 1-SO2CH3), 2.76–2.17 (m, 2H, 3-
CH2); ESI-MS: 302.3 [M+1]+ and 319.2 [M+NH4]+.
4.1.5.6. 1-Methanesulfonyl-4-(3-phenyl-acryloyloxy)-pyrroli-
dine-2-carboxylic acid methyl ester (4r).
Flash column
chromatography: dichloromethane/acetone = 50:1; 43%; white
crystal; mp 70–72 °C; IR (KBr, cmꢁ1): 2954 (CH), 1747 (C@O),
1713 (C@O), 1634 (C@O), 1336 (SO2), 1151 (SO2), 1026 (@CH);
1H NMR (CDCl3, d ppm): 7.71 (d, 1H, 4-OCOC@CH, J = 16.00 Hz),
7.54 (m, 2H, Ar-H), 7.41 (m, 3H, Ar-H), 6.41 (d, 1H, 4-OCOCH@C,
J = 16.00 Hz), 5.43 (s, 1H, 4-CH), 4.67 (t, 1H, 2-CH, J = 8.04 Hz),
3.80 (s, 3H, OCH3), 3.87–3.70 (m, 2H, 5-CH2), 3.04 (s, 3H, CH3SO2),
2.64–2.29 (m, 2H, 3-CH2); ESI-MS: 354.2 [M+1]+ and 371.3
[M+NH4]+.
4.2.2. AP-N inhibition assay
The pyrrolidine derivatives were further assayed for the inhibi-
tory activities against AP-N using L-leucine p-nitroanilide as the
substrate. The compound and enzyme were dissolved in phosphate
sodium buffer (pH 7.2, 50 mM), and incubated at 37 °C for 30 min.
The substrate was added and incubated at 37 °C for another
60 min. The 100% and blank groups were also carried out, in which
the 100% group contained not compound and the blank group con-
tained only the enzyme. The resulting solutions were measured
under 405 nm to gain OD405 values, which were then used to cal-
culate the inhibitory rates by [OD405(100%) ꢁ OD405(compound)]/
[OD405(100%) ꢁ OD405(blank)] ꢀ 100%. The IC50 values were ob-
tained from the above inhibitory rates using OriginPro 7.5
software.
4.1.6. General procedure for the preparation of 4-hydroxy-1-
sulfonyl-pyrrolidine-2-carboxylic acid hydroxyamide (5a–c)
To a solution of compound 3 (2 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 at room
temperature for 24 h and the solvent was evaporated in vacuo.

7938
X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 7932–7938
4. Verma, R. P.; Hansch, C. Bioorg. Med. Chem. 2007, 15, 2223–2268.
Acknowledgments
5. Nagase, H.; Visse, R.; Murphy, G. Cardiovasc. Res. 2006, 69, 562–573.
6. Kontogiorgis, C. A.; Papaioannou, P.; Hadjipavlou-Litina, D. J. Curr. Med. Chem.
2005, 12, 339–355.
7. Fisher, J. F.; Mobashery, S. Cancer Metastasis Rev. 2006, 25, 115–136.
8. Hanessian, S.; Moitessier, N. Curr. Top. Med. Chem. 2004, 4, 1269–1287.
9. Li, Y. L.; Xu, W. F. Bioorg. Med. Chem. 2004, 12, 5171–5180.
10. Li, X.; Li, Y.; Xu, W. Bioorg. Med. Chem. 2006, 14, 1287–1293.
11. Qu, X.; Yuan, Y.; Xu, W.; Chen, M.; Cui, S.; Meng, H.; Li, Y.; Makuuchi, M.;
Nakata, M.; Tang, W. Anticancer Res. 2006, 26, 3573–3578.
12. Jordis, U.; Sauter, F.; Siddiqi, S. M. Indian J. Chem. 1989, 28B, 294–296.
13. Natchus, M. G.; Bookland, R. G.; De, B.; Almstead, N. G.; Pikul, S.; Janusz, M. J.;
Heitmeyer, S. A.; Hookfin, E. B.; Hsieh, L. C.; Dowty, M. E.; Dietsch, C. R.; Patel,
V. S.; Garver, S. M.; Gu, F.; Pokross, M. E.; Mieling, G. E.; Baker, T. R.; Foltz, D. J.;
Peng, S. X.; Bornes, D. M.; Strojnowski, M. J.; Taiwo, Y. O. J. Med. Chem. 2000, 43,
4948–4963.
This work was supported by the National High Technology Re-
search and Development Program of China (863 project; Grant No.
2007AA02Z314), the National Natural Foundation Great Research
Program (No. 90713041), the National Natural Foundation Re-
search Grant (Grant No. 30772654), the Doctoral Foundation of
Ministry of Education of the People’s Republic of China (Grant
No. 20060422029) and the Shandong Province Natural Science
Foundation (Grant No. Y2004C02). This work was also supported
in part by Grant-in-Aid from the Ministry of Education, Science,
Sports and Culture of Japan.
14. Pikul, S.; Dunham, K. L. M.; Almstead, N. G.; De, B.; Natchus, M. G.; Anastasio,
M. V.; McPhail, S. J.; Snider, C. E.; Taiwo, Y. O.; Chen, L.; Dunaway, C. M.; Gu, F.;
Mieling, G. E. J. Med. Chem. 1999, 42, 87–94.
References and notes
15. Baragi, V. M.; Shaw, B. J.; Renkiewicz, R. R.; Kuipers, P. J.; Welgus, H.
G.; Mathrubutham, M.; Cohen, J. R.; Rao, S. K. Matrix Biol. 2000, 19,
267–273.
1. Murphy, G. J. P.; Murphy, G.; Reynolds, J. J. FEBS Lett. 1991, 289, 4–7.
2. Bjorklund, M.; Koivunen, E. Biochim. Biophys. Acta 2005, 1755, 37–69.
3. Polette, M.; Nawrocki-Raby, B.; Gilles, C.; Clavel, C.; Birembaut, P. Crit. Rev.
Oncol. Hematol. 2004, 49, 179–186.
16. Lejczak, B.; Kafarski, P.; Zygmunt, J. Biochemistry 1989, 28, 3549–3555.