Design, synthesis and evaluation of novel sulfonyl pyrrolidine derivatives as matrix metalloproteinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.04.027

A series of novel sulfonyl pyrrolidine derivatives were 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. Compounds 6a-d were more potent MMP-2 inhibitors than the positive control LY52. The structure-activity relationships were also briefly discussed.

Full text of DOI:10.1016/j.bmc.2008.04.027

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5398–5404
Design, synthesis and evaluation of novel sulfonyl
pyrrolidine derivatives as matrix metalloproteinase inhibitors
Xian-Chao Cheng,a Qiang Wang,a Hao Fang,a Wei Tangb and Wen-Fang Xua,*
aInstitute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, No. 44 Wenhuaxi Road,
Jinan 250012, China
bHepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine,
The University of Tokyo, Tokyo 113-8655, Japan
Received 1 March 2008; revised 9 April 2008; accepted 10 April 2008
Available online 15 April 2008
Abstract—A series of novel sulfonyl pyrrolidine derivatives were 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. Compounds 6a–d were more potent MMP-2 inhibitors than the
positive control LY52. The structure–activity relationships were also briefly discussed.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
interactions with the enzyme subsites, such as S10 pocket
and S20 pocket.6,7
The matrix metalloproteinases (MMPs) are a family of
structurally related zinc-dependent endoproteinases that
degrade and remodel the structural proteins in the extra-
cellular matrix.1 These include more than 20 subtypes,
among which MMP-2 is highly involved in the process
of tumor invasion and metastasis and has been consid-
ered as a promising target for cancer therapy.2,3
Sulfonamide hydroxamates were designed and synthe-
sized as efficient MMP inhibitors since the discovery of
CGS 27023A in 1994. CGS 27023A 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 sulfonyl group was incorporated in
the inhibitor to improve the enzyme-inhibitor binding,
not only by forming hydrogen bonds to the enzyme
but also by properly directing the hydrophobic substitu-
ent to the S10 pocket and enabling it to plunge in deeply.
It has been reported that besides the catalytic activity
center zinc (II) ion of MMP-2, there are two hydropho-
bic 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 sub-
types, and S20 pocket is solvent exposed.4,5 The cur-
rently identified MMP-2 inhibitors shared the
following structural character and binding mode: (1) a
zinc binding group (ZBG, such as hydroxamate and car-
boxylate) capable of chelating the active site zinc ion; (2)
at least one functional group, which provides a hydro-
gen bond interaction with the enzyme backbone; and
(3) one or more side chains, which undergo effective
Our group has been developing pyrrolidine scaffold-
based MMP-2 inhibitors for a number of years, and
numerous compounds have been reported in the litera-
ture.9,10 One of these compounds, LY52 (see Fig. 1)
shows low nanomolar activity for MMP-2. This com-
pound could significantly block the proteolytic activity
of MMP-2. LY52 also suppressed human ovarian carci-
noma cell line SKOV3 invasion in vitro. Furthermore, a
significant inhibition of pulmonary metastasis of Lewis
lung carcinoma cells was observed in LY52-adminis-
trated mice.11
In order to identify more potent MMP-2 inhibitors,
based on our previous work and the role of the sulfonyl
group in MMP inhibitors, we incorporated the sulfonyl
group into pyrrolidine scaffold to form the new inte-
Keywords: Sulfonyl pyrrolidine derivatives; Synthesis; MMP-2 inhib-
itors; IC50.
*
Corresponding author. Tel./fax: +86 531 88382264; e-mail addresses:
chengxianchao2000@yahoo.com.cn; xuwenf@sdu.edu.cn
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.027

X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 5398–5404
5399
O
O
O
O
O
O
N
O
O
O
O
O
S
O
R1
S
N
O
NHOH
R2
S
O
N
O
N
NHOH
N
N
NHOH
R3 R4
NH
N
S
Novel designed sulfonyl
pyrrolidine derivatives
O
CGS-27023A
LY52
Prinomastat
Figure 1. The chemical structures of CGS-27023A, Prinomastat, LY52, and our further designed sulfonyl pyrrolidine derivatives.
grated structural pattern (see Fig. 1). R1SO2 group can
be any of the various sulfonyl groups, such as toluene-
4-sulfonyl, benzenesulfonyl, or methanesulfonyl. COR2
group can be hydroxamate or carboxylate group. R3
(R4) group can be hydroxyl, carbonyl, or ketal group.
R1SO2 group and R3 (R4) group might occupy the S10
and S20 pockets, respectively, while the COR2 group
might chelate the active site zinc ion. The pyrrolidine
scaffold might bond to the enzyme backbone.
using Sybyl 7.0 of Tripos Incorporation and the result
is shown in Figures 2 and 3. The R1 group at the N1 po-
sition of the pyrrolidine ring (toluene-4-sulfonyl and
benzenesulfonyl, respectively) occupied the deep S10
pocket of MMP-2, and the hydroxamate group chelated
the active site zinc ion 166 with a distance of 1.53 and
˚
2.06 A, respectively. Compounds 6a and 6d interacted
well with the MMP-2 active site, especially the deep
S10 pocket and zinc ion 166, consistent with the
MMP-2 assay results.
2. Results and discussion
The selective inhibition might be explained by the FlexX
docking results of the representative compounds 6a and
6d with APN (Figs. 4 and 5). Although compounds 6a
and 6d chelated well with the active site zinc ion 900,
both the ketal group and sulfonyl group at the pyrroli-
dine ring were solvent exposed and could not occupy
the S10 and S20 pockets of APN. MMP-2 was a zinc-
dependent endopeptidase that could cut the peptide to
parts from the specific amino acid residue of the peptide.
However, AP-N was a membrane-bound zinc exopepti-
dase that catalyzed the removal of NH-terminal amino
acid from the peptide. Due to the structural differences
between MMP-2 and AP-N, there were different struc-
tural requirements for their respective inhibitors. As
these pyrrolidine derivatives exhibited highly selective
inhibition against MMP-2, the following structure–
activity relationships (SARs) were mainly discussed
about MMP-2 inhibition.
The target compounds were synthesized via the route as
shown in Scheme 1. Starting from trans-4-hydroxy-L-pro-
line (1) as a chiral template, the important intermediate 1-
sulfonyl-4-oxo-pyrrolidine-2-carboxylic acid methyl es-
ters (4a–c) were prepared via the route of esterification,12
sulfonation, 13 and Jones oxidation.14 Addition reaction
of 4a–c with various diols produced the ketal compounds
5a–f. In this step, the p-toluenesulfonic acid acted as a cat-
alyst and the Dean–Stark trap was used to remove the
water product.15 Ammonolysis of 5a–f with NH2OK pro-
vided the final spiro hydroxamate compounds (6a–f).16
The chemical structures of the target compounds were
confirmed by IR, 1H NMR and ESI-MS.
The newly synthesized pyrrolidine derivatives were
assayed for the inhibitory activities on MMP-2 and
17,18
aminopeptidase N (AP-N).
Similar to MMP-2,
AP-N is also a zinc-dependent metalloproteinase in-
volved in the process of tumor invasion and metastasis.
Thus the assay was performed on both MMP-2 and
AP-N so as to identify the compound selectivity. LY52
was used as the positive control.
Compounds 6a–f were more potent than their predeces-
sors 5a–f. This activity difference was caused by the
ZBG (COR3), which was the only structural difference be-
tween 6a–f and their predecessor. The ZBG is hydroxa-
mate (CONHOH) for 6a–f and carboxylate (COOCH3)
for their predecessor, respectively. Both of these groups
could chelate zinc ion at the catalytic activity center of
the enzyme. However, the hydroxamate group was a more
potent ZBG than the carboxylate group as shown in the
activity order of 6a–f and their predecessor.
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 (Table 1). Compounds
6a–d were more potent MMP-2 inhibitors than the posi-
tive control LY52. The FlexX docking of the most po-
tent compounds 6a and 6d with MMP-2 was done
Among compounds 6a–c, R1SO2 and COR2 groups
were fixed as toluene-4-sulfonyl and hydroxamate,
O
O
O
O
O
O
O
O
O
O
O
O
O
O
R1
S
R1 S
R1
S
R1 S
OH
O
O
O
O
NHOH
Cl
HN
HO
H2N
HO
N
N
N
N
a
b
c
d
e
1
2
3a-c
4a-c
O
O
5a-f
O
O
6a-f
HO
O
n
n
Scheme 1. Reagents: (a) CH3OH, HCl; (b) R1SO2Cl; (c) Jones reagent; (d) HO–(CH2)n–OH; and (e) NH2OK.

5400
X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 5398–5404
Table 1. The structures and IC50 values of pyrrolidine derivatives
O
O
O
R1
S
R2
N
R3 R4
a
No.
R1
R2
R3
R4
IC50 (lM)
MMP-2
0.1 0.02
AP–N
3a
3b
3c
p-CH3C6H4
C6H5
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
NHOH
NHOH
NHOH
NHOH
NHOH
NHOH
OH
OH
H
H
H
38.4 4.6
53.8 3.2
41.6 6.9
0.8 0.1
7.1 0.6
0.4 0.04
OH
4a
4b
4c
p-CH3C6H4
C6H5
CH3
C@O
C@O
C@O
54.9 4.1
2.0 0.1
9.5 1.2
75.8 10.2
92.6 12.4
155.8 10.3
173.7 20.7
124.3 13.5
150.6 18.4
168.1 10.3
134.8 9.5
48.5 6.7
5a
5b
5c
p-CH3C6H4
p-CH3C6H4
p-CH3C6H4
C6H5
O–(CH2)2–O
O–(CH2)3–O
O–(CH2)4–O
O–(CH2)2–O
O–(CH2)3–O
O–(CH2)4–O
O–(CH2)2–O
O–(CH2)3–O
O–(CH2)4–O
O–(CH2)2–O
O–(CH2)3–O
O–(CH2)4–O
0.1 0.01
0.2 0.03
0.3 0.04
0.2 0.04
5d
5e
C6H5
C6H5
1.1 0.1
1.5 0.1
5f
6a
6b
6c
p-CH3C6H4
p-CH3C6H4
p-CH3C6H4
C6H5
0.003 0.0004
0.006 0.0005
0.008 0.001
0.008 0.0008
0.011 0.001
0.012 0.001
0.009 0.0004
61.8 4.9
73.2 11.6
57.8 5.4
6d
6e
C6H5
C6H5
82.7 12.8
106.7 10.4
141.9 11.7
6f
LY52
a IC50 values are mean of three experiments, standard deviation is given.
Figure 3. FlexX docking result of compound 6d with MMP-2.
Figure 2. FlexX docking result of compound 6a with MMP-2.
toluene-4-sulfonyl and benzenesulfonyl, the differences
in the inhibitory activities of these compounds were
caused by various R1SO2 groups. The compound bear-
ing the toluene-4-sulfonyl group was found to be more
potent than the compound bearing benzenesulfonyl
group. This result might be owing to the toluene-4-sulfo-
nyl group, which could interact with the S10 pocket and
plunge in deeply.
respectively, and the ketal group at R3 and R4 positions
was altered at various lengths. So the differences in the
inhibitory activities of these compounds were caused
by various ketal groups. The introduction of a five-
membered ring ketal group (6a) displayed the highest
activity. As compared with 6a, the compound bearing
a six-membered ring ketal group (6b) was in the next
place, and the compound containing a seven-membered
ring ketal group (6c) presented the least activity. The
same rule can also be seen among compounds 5a–c,
5d–f, and 6d–f.
Finally, the binding mode of compound 6d with MMP-
2 was proposed as follows: (1) the hydroxamate chelated
the active site zinc ion; (2) the sulfonyl and the hydroxyl
groups provided hydrogen bond interactions with the
enzyme backbone Ala86 and Glu121, respectively; and
(3) two side chains (benzenesulfonyl and ketal) under-
Among compounds 5a–f and 6a–f, when the ketal group
and ZBG were fixed, and R1SO2 group was altered as

X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 5398–5404
5401
duction of toluene-4-sulfonyl group at the N1 position
and a five-membered ring ketal group at the C4 position
at the pyrrolidine ring favored the inhibitory activity
against MMP-2. The FlexX docking was consistent with
the above SAR results. Further assays of these com-
pounds on cell culture and animal models are underway.
4. Experimental
4.1. Synthetic methods and spectroscopic details
Melting points were determined using X-6 digital dis-
play binocular microscope (uncorrected). Infrared spec-
tra 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 Hz. Mass spec-
tra were recorded on an electro-spray ionization mass
spectrometer as the value m/z. Flash column chromatog-
raphy was performed using 300 mesh silica gel. The
yields were calculated by the last step reaction.
Figure 4. FlexX docking result of compound 6a with APN.
4.1.1. Trans-4-Hydroxy-L-proline methyl ester hydrochlo-
ride (2). A slurry of trans-4-hydroxy-L-proline (1) (100 g,
763.5 mmol) in methanol (650 ml) was treated with dry
hydrogen chloride until homogeneous. The solution
was heated to the reflux temperature for 3 h and concen-
trated in vacuo. Upon cooling, the product was crystal-
lized from the solvent, collected by filtration, washed
with acetone and ether, and dried under reduced pres-
sure to yield trans-4-hydroxy-L-proline methyl ester
hydrochloride (2) as white crystal (120 g, 87%), mp
Figure 5. FlexX docking result of compound 6d with APN.
1
157–160 ꢁC (lit.10 mp 156–160 ꢁC). H NMR (CD3OD,
d ppm): 4.86 (s, 2H, NH2þ, 4.63 (m, 1H, CH), 3.88 (s,
3H, CH3), 3.50 (m, 1H, CH), 3.35 (m, 2H, CH2),
2.47–2.20 (m, 2H, CH2), 1.31 (s, 1H, OH).
went effective interactions with the enzyme subsites S10
pocket and S20 pocket (Fig. 6). The above binding mode
information encouraged us to further design pyrroli-
dine-scaffold-based MMP-2 inhibitors, which would be
reported later.
4.1.2. 1-Sulfonyl-4-hydroxy-pyrrolidine-2-carboxylic acid
methyl ester (3a–c). Trans-4-Hydroxy-L-proline methyl
ester hydrochloride (2) (69 g, 380 mmol) was dissolved
in water/dioxane (1:1, 300 ml) with triethylamine
(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
for 24 h at room temperature. The mixture was then
concentrated and extracted with EtOAc (6· 50 ml).
Layers were separated and the organic layer was washed
with 1 M HCl (2· 100 ml) and brine (1· 100 ml),
dried over Na2SO4, filtered, and evaporated to give
3a–c as solid material.
3. Conclusions
In conclusion, a series of novel sulfonyl pyrrolidine
derivatives were designed and synthesized. These pyrrol-
idine derivatives exhibited highly selective inhibition
against MMP-2 as compared with AP-N. Compounds
6a–d were more potent MMP-2 inhibitors than the posi-
tive control LY52. SAR studies indicated that the intro-
Ala86
N
4.1.2.1. 4-Hydroxy-1-(toluene-4-sulfonyl)-pyrrolidine-
2-carboxylic acid methyl ester (3a). About 75%; white
crystal; mp 78–79 ꢁC; IR (KBr, cmꢀ1): 3512.59 (OH),
2953.46 (CH), 1742.98 (C@O), 1597.79 (C@C),
1343.73 (SO2), 1156.85 (SO2); ESI-MS: 300.5 (M+1).
Glu121
O
H
Zn2+
O H
O
S1' pocket
O
O
O
S
N
N
H
4.1.2.2. 1-Benzenesulfonyl-4-hydroxy-pyrrolidine-2-
carboxylic acid methyl ester (3b). About 69%; white crys-
tal; mp 102–103 ꢁC; IR (KBr, cmꢀ1): 3467.77 (OH),
2956.75 (CH), 1725.82 (C@O), 1347.60 (SO2), 1156.78
(SO2); ESI-MS: 286.2 (M+1).
O
S2' pocket
Figure 6. Proposed binding mode of compound 6d with MMP-2.

5402
X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 5398–5404
4.1.2.3. 4-Hydroxy-1-methanesulfonyl-pyrrolidine-2-
4.1.4.2. 2-(Toluene-4-sulfonyl)-6,10-dioxa-2-aza-spiro-
[4.5]decane-3-carboxylic acid methyl ester (5b). Flash
column chromatography: dichloromethane/acetone =
100:1–10:1; 40%; colorless oil; IR (KBr, cmꢀ1):
2954.88 (CH), 1756.46 (C@O), 1597.96 (C@C),
1347.88 (SO2), 1158.86 (SO2); 1H NMR (CDCl3, d
ppm): 7.77 (d, 2H, Ar-H, J = 8.08 Hz), 7.31 (m, 2H,
Ar-H), 4.35 (t, 1H, CH, J = 7.56 Hz), 3.88–3.46 (m,
6H, 2OCH2 and NCH2), 3.75 (s, 3H, OCH3), 2.43 (s,
3H, ArCH3), 2.32 (m, 2H, CH2), 1.80–1.57 (m, 2H,
CH2); ESI-MS: 356.3 (M+1).
carboxylic acid methyl ester (3c). About 35%; white crys-
tal; mp 85–87 ꢁC; IR (KBr, cmꢀ1): 3468.57 (OH),
2959.32 (CH), 1723.68 (C@O), 1344.44 (SO2), 1147.72
(SO2); ESI-MS: 224.3 (M+1).
4.1.3. 1-Sulfonyl-4-oxo-pyrrolidine-2-carboxylic acid methyl
ester (4a–c). CrO3 (26.7 g, 267 mmol) was dissolved in
H2SO4 (conc.) (23 ml). H2O (77 ml) was added very
slowly to the above solution and Jones reagent was pre-
pared. 1-Sulfonyl-4-hydroxy-pyrrolidine-2-carboxylic
acid methyl ester (3a–c) (100 mmol) was dissolved in
acetone (268 ml) and cooled to 0 ꢁC. Jones reagent
was added (100 ml) (color changed from orange-red to
green), and the mixture was stirred at room temperature
for 24 h. The reaction mixture was diluted with water
(370 ml) and extracted with EtOAc (1· 250 ml). The
organic layers were washed with water (3· 100 ml)
and brine (1· 100 ml), dried over Na2SO4, and evapo-
rated to give the solid, which was then crystallized from
EtOAc to give the desired product as a crystal.
4.1.4.3. 2-(Toluene-4-sulfonyl)-6,11-dioxa-2-aza-spiro-
[4.6]undecane-3-carboxylic acid methyl ester (5c). Flash
column chromatography: dichloromethane/acetone =
100:1–10:1; 34%; colorless oil; IR (KBr, cmꢀ1):
2950.83 (CH), 1755.91 (C@O), 1597.99 (C@C),
1348.01 (SO2), 1158.08 (SO2); 1H NMR (CDCl3, d
ppm): 7.75 (d, 2H, Ar-H, J = 8.24 Hz), 7.31 (d, 2H,
Ar-H, J = 8.04 Hz), 4.33 (t, 1H, CH, J = 7.72 Hz),
3.65–3.23 (m, 6H, 2OCH2 and NCH2), 3.76 (s, 3H,
OCH3), 2.47 (s, 3H, ArCH3), 2.34–2.11 (m, 2H, CH2),
1.61–1.51 (m, 4H, 2CH2); ESI-MS: 370.3 (M+1).
4.1.3.1. 4-Oxo-1-(toluene-4-sulfonyl)-pyrrolidine-2-car-
boxylic acid methyl ester (4a). About 95%; white crystal;
mp 72–73 ꢁC; IR (KBr, cmꢀ1): 2956.12 (CH), 1768.19
(C@O), 1755.58 (C@O), 1598.80 (C@C), 1348.35
(SO2), 1157.32 (SO2); ESI-MS: 298.4 (M+1).
4.1.4.4. 7-Benzenesulfonyl-1,4-dioxa-7-aza-spiro[4.4]-
nonane-8-carboxylic acid methyl ester (5d). Flash column
chromatography: dichloromethane/acetone = 100:1–
10:1; 36%; white crystal; mp 65–67 ꢁC; IR (KBr,
cmꢀ1): 2954.29 (CH), 1749.05 (C@O), 1337.60 (SO2),
4.1.3.2. 1-Benzenesulfonyl-4-oxo-pyrrolidine-2-carbox-
ylic acid methyl ester (4b). About 91%; white crystal; mp
62–64 ꢁC; IR (KBr, cmꢀ1): 2956.07 (CH), 1768.14
(C@O), 1352.74 (SO2), 1159.45 (SO2); ESI-MS: 284.2
(M+1).
1
1157.55 (SO2); H NMR (CDCl3, d ppm): 7.89 (d, 2H,
Ar-H, J = 8.13 Hz), 7.61 (t, 1H, Ar-H, J = 7.43 Hz),
7.54 (t, 2H, Ar-H, J = 8.24 Hz), 4.43 (t, 1H, CH,
J = 8.00 Hz), 3.95–3.42 (m, 6H, 2OCH2 and NCH2),
3.74 (s, 3H, OCH3), 2.25 (m, 2H, CH2); ESI-MS:
328.3 (M+1).
4.1.3.3. 1-Methanesulfonyl-4-oxo-pyrrolidine-2-carbox-
ylic acid methyl ester (4c). About 82%; white crystal; mp
98–99 ꢁC; IR (KBr, cmꢀ1): 2957.46 (CH), 1754.78
(C@O), 1328.08 (SO2), 1146.09 (SO2); ESI-MS: 222.2
(M+1).
4.1.4.5. 2-Benzenesulfonyl-6,10-dioxa-2-aza-spiro-
[4.5]decane-3-carboxylic acid methyl ester (5e). Flash
column chromatography: dichloromethane/acetone =
100:1–10:1; 51%; colorless oil; IR (KBr, cmꢀ1):
2925.72 (CH), 1746.11 (C@O), 1350.36 (SO2), 1160.47
4.1.4. General procedure for the preparation of the ketal
compounds (5a–f). 1-Sulfonyl-4-oxo-pyrrolidine-2-car-
boxylic acid methyl ester (4a or 4b) (10 mmol) was dis-
solved in toluene (100 ml) and various diols (ethylene
glycol, 1,3-propanediol, or 1,4-butanediol) (180 mmol).
p-Toluenesulfonic acid (0.2236 g, 1.3 mmol) was added,
and the resulting mixture was heated under reflux for
24 h in the presence of a Dean–Stark trap. The solvent
was evaporated in vacuo. The final product was purified
by flash column chromatography and 5a–f were
obtained.
1
(SO2); H NMR (CDCl3, d ppm): 7.87 (m, 2H, Ar-H),
7.55 (m, 3H, Ar-H), 4.38 (t, 1H, CH, J = 7.60 Hz),
4.30–3.21 (m, 6H, 2OCH2 and NCH2), 3.74 (s, 3H,
OCH3), 2.33 (d, 2H, CH2, J = 7.56 Hz), 1.76–1.54 (m,
2H, CH2); ESI-MS: 342.2 (M+1).
4.1.4.6. 2-Benzenesulfonyl-6,11-dioxa-2-aza-spiro[4.6]-
undecane-3-carboxylic acid methyl ester (5f). Flash
column chromatography: dichloromethane/acetone =
100:1–10:1; 44%; colorless oil; IR (KBr, cmꢀ1):
2951.84 (CH), 1743.41 (C@O), 1348.69 (SO2), 1159.59
1
4.1.4.1. 7-(Toluene-4-sulfonyl)-1,4-dioxa-7-aza-spiro-
[4.4]nonane-8-carboxylic acid methyl ester (5a). Flash
column chromatography: dichloromethane/acetone =
100:1–10:1; 31%; colorless oil; IR (KBr, cmꢀ1):
2922.81 (CH), 1743.33 (C@O), 1598.02 (C@C),
1347.99 (SO2), 1157.66 (SO2); 1H NMR (CDCl3, d
ppm): 7.75 (d, 2H, Ar-H, J = 8.20 Hz), 7.32 (d, 2H,
Ar-H, J = 8.08 Hz), 4.40 (t, 1H, CH, J = 7.40 Hz),
3.90–3.76 (m, 4H, OCH2CH2O), 3.75 (s, 3H, OCH3),
3.46–3.37 (m, 2H, NCH2), 2.44 (s, 3H, ArCH3), 2.23
(d, 2H, CH2, J = 7.40 Hz); ESI-MS: 342.2 (M+1).
(SO2); H NMR (CDCl3, d ppm): 7.87 (d, 2H, Ar-H,
J = 7.24 Hz), 7.55 (m, 3H, Ar-H), 4.35 (m, 1H, CH),
3.89–3.19 (m, 6H, 2OCH2 and NCH2), 3.75 (s, 3H,
OCH3), 2.55–2.10 (m, 2H, CH2), 1.70–1.25 (m, 4H,
2CH2); ESI-MS: 356.4 (M+1).
4.1.5. General procedure for the preparation of the final
spiro hydroxamate compounds (6a– f). To a solution of
compound 5 (2 mmol) in methanol (7 ml) at room tem-
perature was added dropwise a solution of NH2OK
(6 mmol) in methanol (3.4 ml). The mixture was stirred

X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 5398–5404
5403
at room temperature for 24 h and the solvent was evap-
orated in vacuo. The residue was purified by flash col-
umn chromatography to give 6a–f.
umn chromatography: dichloromethane/methanol
=
10:1; 63%; colorless oil; IR (KBr, cmꢀ1): 3306.18
(OH), 2946.47 (CH), 1673.77 (C@O), 1348.15 (SO2),
1160.01 (SO2); 1H NMR (CD3OD, d ppm): 7.91 (d,
2H, Ar-H, J = 7.32 Hz), 7.71 (m, 1H, Ar-H), 7.63 (t,
2H, Ar-H, J = 7.76 Hz), 4.10 (t, 1H, CH, J = 8.08 Hz),
3.70–2.96 (m, 6H, 2OCH2 and NCH2), 2.18–2.03 (m,
2H, CH2), 1.56–1.30 (m, 4H, 2CH2); ESI-MS: 357.3
(M+1).
4.1.5.1. 7-(Toluene-4-sulfonyl)-1,4-dioxa-7-aza-spiro[4.4]-
nonane-8-carboxylic acid hydroxyamide (6a). Flash
column chromatography: dichloromethane/methanol =
10:1; 51%; colorless oil; IR (KBr, cmꢀ1): 3179.95
(OH), 2973.82 (CH), 1673.68 (C@O), 1345.78 (SO2),
1157.77 (SO2); 1H NMR (CD3OD, d ppm): 7.77 (d,
2H, Ar-H, J = 8.16 Hz), 7.43 (d, 2H, Ar-H,
J = 8.04 Hz), 4.13 (t, 1H, CH, J = 7.64 Hz), 3.91–3.32
(m, 6H, 2OCH2 and NCH2), 2.46 (s, 3H, ArCH3),
2.23–1.97 (m, 2H, CH2); ESI-MS: 343.3 (M+1).
4.2. Biological evaluation
4.2.1. MMP-2 inhibition assay. The pyrrolidine deriva-
tives were assayed for the inhibitory activities against
MMP-2 in 96-well microtiter plates using succinylated
gelatin as the substrate. The compound and the enzyme
were dissolved in sodium borate buffer (pH8.5, 50 mM),
and incubated at 37 ꢁC for 30 min. The substrate was
added and incubated at 37 ꢁC for another 60 min. Then
0.03% picrylsulfonic acid solution was added and incu-
bated at room temperature for an 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%) ꢀ OD 450(compound)]/
[OD450(100%) ꢀ OD450(blank)] · 100%. The IC50 values
were obtained from the above inhibitory rates using the
OriginPro 7.5 software.
4.1.5.2. 2-(Toluene-4-sulfonyl)-6,10-dioxa-2-aza-spiro-
[4.5]decane-3-carboxylic acid hydroxyamide (6b). Flash
column chromatography: dichloromethane/methanol =
10:1; 47%; white crystal; mp 80–81 ꢁC; IR (KBr,
cmꢀ1): 3368.44 (OH), 2925.68 (CH), 1674.74 (C@O),
1346.32 (SO2), 1158.11 (SO2); 1H NMR (CD3OD, d
ppm): 7.79 (d, 2H, Ar-H, J = 8.20 Hz), 7.44 (d, 2H,
Ar-H, J = 8.08 Hz), 4.08 (t, 1H, CH, J = 7.84 Hz),
3.84–3.48 (m, 6H, 2OCH2 and NCH2), 2.46 (s, 3H,
ArCH3), 2.29–2.10 (m, 2H, CH2), 1.70–1.55 (m, 2H,
CH2); ESI-MS: 357.3 (M+1).
4.1.5.3. 2-(Toluene-4-sulfonyl)-6,11-dioxa-2-aza-spiro-
[4.6]undecane-3-carboxylic acid hydroxyamide (6c). Flash
column chromatography: dichloromethane/methanol =
10:1; 47%; colorless oil; IR (KBr, cmꢀ1): 3221.09 (OH),
2922.41 (CH), 1709.27 (C@O), 1346.63 (SO2), 1159.03
4.2.2. AP-N inhibition assay. The pyrrolidine derivatives
were further assayed for the inhibitory activities against
AP-N using L-leucine p-nitroanilide as the substrate.
The compound and the enzyme were dissolved in phos-
phate 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 resulting
solutions were measured under 405 nm to gain OD405
values, which were then used to calculate the inhibitory
1
(SO2); H NMR (CD3OD, d ppm): 7.78 (m, 2H, Ar-
H), 7.40 (m, 2H, Ar-H), 4.08 (t, 1H, CH, J = 7.96 Hz),
3.68–3.04 (m, 6H, 2OCH2 and NCH2), 2.47 (s, 3H,
ArCH3), 2.36–2.04 (m, 2H, CH2), 1.70–1.50 (m, 4H,
2CH2); ESI-MS: 371.3 (M+1).
rates
by
[OD405(100%) ꢀ OD
405(compound)]/
4.1.5.4. 7-Benzenesulfonyl-1,4-dioxa-7-aza-spiro[4.4]-
nonane-8-carboxylic acid hydroxyamide (6d). Flash
column chromatography: dichloromethane/methanol =
20:1–10:1; 33%; colorless oil; IR (KBr, cmꢀ1): 3165.29
(OH), 2897.67 (CH), 1674.55 (C@O), 1342.93 (SO2),
1158.80 (SO2); 1H NMR (CD3OD, d ppm): 7.90 (d,
2H, Ar-H, J = 7.24 Hz), 7.72 (m, 1H, Ar-H), 7.63 (t,
2H, Ar-H, J = 7.88 Hz), 4.14 (t, 1H, CH, J = 7.72 Hz),
3.91–3.47 (m, 6H, 2OCH2 and NCH2), 2.23–2.00 (m,
2H, CH2); ESI-MS: 329.5 (M+1).
[OD405(100%) ꢀ OD405(blank)] · 100%. The IC50 values
were obtained from the above inhibitory rates using the
OriginPro 7.5 software.
Acknowledgements
This work was supported by the National High Tech-
nology Research and Development Program of China
(863 project; Grant No. 2007AA02Z314), the National
Natural Foundation Great Research Program (No.
90713041), the National Natural Foundation Research
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 a
Grant-in-Aid from the Ministry of Education, Science,
Sports, and Culture of Japan.
4.1.5.5. 2-Benzenesulfonyl-6,10-dioxa-2-aza-spiro[4.5]-
decane-3-carboxylic acid hydroxyamide (6e). Flash
column chromatography: dichloromethane/methanol =
10:1; 59%; white crystal; mp 70–72 ꢁC; IR (KBr,
cmꢀ1): 3200.36 (OH), 2972.34 (CH), 1672.04 (C@O),
1344.03 (SO2), 1159.16 (SO2); 1H NMR (CD3OD, d
ppm): 7.92 (d, 2H, Ar-H, J = 7.36 Hz), 7.71 (m, 1H,
Ar-H), 7.63 (t, 2H, Ar-H, J = 7.84 Hz), 4.10 (t, 1H,
CH, J = 7.96 Hz), 3.85–3.32 (m, 6H, 2OCH2 and
NCH2), 2.27–2.14 (m, 2H, CH2), 1.67–1.52 (m, 2H,
CH2); ESI-MS: 343.4 (M+1).
References and notes
1. Murphy, G. J. P.; Murphy, G.; Reynolds, J. J. FEBS Lett.
1991, 289(1), 4.
4.1.5.6. 2-Benzenesulfonyl-6,11-dioxa-2-aza-spiro[4.6]-
undecane-3-carboxylic acid hydroxyamide (6f). Flash col-

5404
X.-C. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 5398–5404
2. Bjorklund, M.; Koivunen, E. Biochim. Biophys. Acta
2005, 1755, 37.
3. Polette, M.; Nawrocki-Raby, B.; Gilles, C.; Clavel, C.;
Birembaut, P. Crit. Rev. Oncol. Hematol. 2004, 49, 179.
4. Verma, R. P.; Hansch, C. Bioorg. Med. Chem. 2007, 15,
2223.
5. Nagase, H.; Visse, R.; Murphy, G. Cardiovasc. Res. 2006,
69, 562.
6. Kontogiorgis, C. A.; Papaioannou, P.; Hadjipavlou-Liti-
na, D. J. Curr. Med. Chem. 2005, 12, 339.
7. Fisher, J. F.; Mobashery, S. Cancer Metastasis Rev. 2006,
25, 115.
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.
14. Cheng, M.; De, B.; Almstead, N. G.; Pikul, S.; Dowty,
M. E.; Dietsch, C. R.; Dunaway, C. M.; Gu, F.; Hsieh,
L. C.; Janusz, M. J.; Taiwo, Y. O.; Natchus, M. G.;
Hudlicky, T.; Mandel, M. J. Med. Chem. 1999, 42,
5426.
15. Smith, E. M.; Swiss, G. F.; Neustadt, B. R.; Gold, E.
H.; Sommer, J. A.; Brown, A. D.; Chiu, P. J. S.;
Moran, R.; Sybertz, E. J.; Baum, T. J. Med. Chem.
1988, 31, 875.
8. Hanessian, S.; Moitessier, N. Curr. Top. Med. Chem. 2004,
4, 1269.
9. Li, Y. L.; Xu, W. F. Bioorg. Med. Chem. 2004, 12, 5171.
10. Li, X.; Li, Y.; Xu, W. Bioorg. Med. Chem. 2006, 14, 1287.
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(5A), 3573.
12. Jordis, U.; Sauter, F.; Siddiqi, S. M. Indian J. Chem. 1989,
28B, 294.
13. Natchus, M. G.; Bookland, R. G.; De, B.; Almstead, N.
G.; Pikul, S.; Janusz, M. J.; Heitmeyer, S. A.; Hookfin, E.
16. 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.
17. 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.
18. Lejczak, B.; Kafarski, P.; Zygmunt, J. Biochemistry 1989,
28(8), 3549.