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

Article abstract of DOI:10.1016/j.ejmech.2007.12.020

A series of novel 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. The hydroxamates 8a-c were equally or more potent MMP-2 inhibitors than the positive control LY52. The binding mode of the most potent compound 8a with MMP-2 was proposed. Structure-activity relationships were also briefly discussed.

Full text of DOI:10.1016/j.ejmech.2007.12.020

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European Journal of Medicinal Chemistry 43 (2008) 2130e2139
http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and preliminary evaluation of novel 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, University of Tokyo 113-8655, Japan
Received 28 June 2007; received in revised form 28 November 2007; accepted 6 December 2007
Available online 31 December 2007
Abstract
A series of novel 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. The hydroxamates 8aec were equally or more potent MMP-2 inhibitors than the positive control LY52.
The binding mode of the most potent compound 8a with MMP-2 was proposed. Structureeactivity relationships were also briefly discussed.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Pyrrolidine derivatives; Synthesis; MMP-2 inhibitors; IC50
1. Introduction
which undergo effective interactions with the enzyme subsites,
such as S10 and S20 pockets [6,7].
The matrix metalloproteinases (MMPs) are a family of
zinc-dependent endopeptidases that are involved in the degra-
dation of all components of the extracellular matrix [1].
Currently, more than 20 mammalian MMPs have been discov-
ered, among which MMP-2 is highly involved in the process
of tumor invasion and metastasis and has been considered 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]. Currently identified MMP-2 inhibitors
shared the following structural character and binding mode:
(1) a zinc binding group (ZBG, such as hydroxamate and
carboxylate) capable of chelating the active site zinc ion; (2)
at least one functional group provides a hydrogen bond inter-
action with the enzyme backbone; (3) one or more side chains
LY52 (see Fig. 1) is a caffeinoyl pyrrolidine derivative
designed in our lab to fit and extend into the active pocket of
MMP-2 [8]. This compound could significantly block the pro-
teolytic activity of MMP-2. LY52 also suppressed human ovar-
ian carcinoma cell line SKOV3 invasion in vitro. Furthermore,
a significant inhibition of pulmonary metastasis of Lewis lung
carcinoma cells was observed in LY52-administrated mice [9].
We might conclude from these results that LY52 might sup-
press invasion and metastasis of carcinoma cells via inhibition
of MMP-2 proteolytic activities.
In order to identify more potent MMP-2 inhibitors, based
on our previous work, we introduced some functional groups
(R1CO, R2 and COR3) into trans-4-hydroxy-L-proline to
form the new integrated structural pattern (see Fig. 1). R1CO
group can be various acyl groups, such as acetyl, (E )-3-(3,4-
dimethoxyphenyl)acryloyl or 3,4-dimethoxyphenylpropanoyl.
R2 group can be hydroxyl, amino, sulfonyl, or imide group,
with R or S configuration. COR3 group can be hydroxamate,
carboxyl or carboxylate group. R1CO and R2 groups might
occupy the S10 and S20 pockets, respectively, while the
COR3 group might chelate the active site zinc ion. The pyrro-
lidine scaffold might bond to the enzyme backbone.
* Corresponding author. Tel./fax: þ86 531 88382264.
E-mail addresses: chengxianchao2000@yahoo.com.cn (X.-C. Cheng),
xuwenf@sdu.edu.cn (W.-F. Xu).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.12.020

X.-C. Cheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2130e2139
2131
(10). The chemical structures of the target compounds were
1
confirmed by IR, H NMR and ESI-MS.
O
O
N
O
N
O
O
R
1
NHOH
R
3
O
3. Biological evaluation and discussion
N
The newly synthesized pyrrolidine derivatives were assayed
for the inhibitory activities on MMP-2 and AP-N [16,17]. Sim-
ilar to MMP-2, AP-N is also a zinc-dependent metalloprotei-
nase 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.
NH
R
2
O
Fig. 1. Chemical structures of LY52 (left) and our further designed pyrrolidine
derivatives (right).
2. Chemistry
The results showed that these pyrrolidine derivatives
exhibited highly selective inhibition against MMP-2 as com-
pared with AP-N, thus confirming our strategy for designing
MMP-2 inhibitors (Table 1). The hydroxamates 8aee were
equally or more potent MMP-2 inhibitors than the positive
control LY52. The FlexX docking of the most potent com-
pounds 8aec with MMP-2 was done using Sybyl 7.0 of Tripos
Incorporation and the result is shown in Figs. 2e4. The R2
group at C4 position of pyrrolidine ring (3,4,5-trimethoxyben-
zamido, nicotinamido, cinnamamido groups, respectively)
occupied the deep S10 pocket of MMP-2, and the hydroxamate
group chelated the active site zinc ion 166 with a distance of
The target compounds were synthesized via the route shown
in Scheme 1. Starting from trans-4-hydroxy-L-proline (1) as
a chiral template, the important intermediate (2S,4S )-methyl
1-acyl-4-aminopyrrolidine-2-carboxylates (6aec) were pre-
pared via the route of esterification [10], acylation, sulfonation
[11], SN2 nucleophilic substitution [12] and catalytic hydroge-
nation [13]. In the nucleophilic substitution step, (3R,5S )-5-
(methoxycarbonyl)-1-acylpyrrolidin-3-yl sulfonates (4a,b)
reacted with sodium azide to produce configurationally reversed
azides (5a,b), which were then catalytically hydrogenated using
5% PdeCaCO3 to afford the intermediates 6aec. Acylation of
6aec with various acyl chlorides provided compounds,
(2S,4S )-methyl 4-acylamido-1-acylpyrrolidine-2-carboxylates
(7aem) [14], some of which were further ammonolyzed or
hydrolyzed to obtain the target compounds (2S,4S )-1-acyl-4-
(acylamido)-N-hydroxypyrrolidine-2-carboxamides (8aee) and
(2S,4S )-4-(3,4,5-trimethoxybenzamido)-1-acetylpyrrolidine-
2-carboxylic acid (9) [15]. Further coupling of compound 9 with
glycine methyl ester gave methyl 2-((2S,4S )-4-(3,4,5-trime-
thoxybenzamido)-1-acetylpyrrolidine-2-carboxamido)acetate
˚
0.95, 2.13 and 2.05 A, respectively. Compounds 8aec inter-
acted 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
docking results of representative compounds 8aec with AP-
N (Figs. 5e7). Although compounds 8aec chelated well
with the active site zinc ion 900, the R2 and R1CO groups at
pyrroliding ring were both solvent 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
O
N
O
N
O
O
O
O
R1
R1
OH
O
O
O
H2N
HO
Cl
HN
HO
a
c
b
d
R2
HO
R1
S
O
O2
1
2
3a-b
4a-b
O
O
N
O
O
N
O
O
O
O
R1
R1
R1
O
O
O
NHOH
N
N
g
e
f
H2N
R2
NH
R2
NH
N3
O
O
5a-b
6a-c
7a-m
8a-e
h
O
O
N
O
O
R1
R1
R2
NH
O
O
OH
N
i
R2
NH
NH
10
9
O
O
Scheme 1. Reagents: (a) CH3OH, HCl; (b) R1COCl; (c) R2SO2Cl; (d) NaN3; (e) 5% PdeCaCO3/H2; (f) R2COCl; (g) NH2OK; (h) NaOH/HCl; (i)
H2NCH2COOCH3.

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X.-C. Cheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2130e2139
Table 1
The structures and IC50 values of pyrrolidine derivatives
O
N
O
R
1
R
3
R
2
a
IC50 (mM)
Compound
no.
R1
R2
R3
4*
MMP-2
AP-N
3a
3b
4a
4b
5a
5b
6a
6b
6c
7a
7b
7c
7d
7e
7f
CH3
CH]CHC6H3(OCH3)2-30,40(E )
CH3
CH]CHC6H3(OCH3)2-30,40(E )
CH3
CH]CHC6H3(OCH3)2-30,40(E )
CH3
CH]CHC6H3(OCH3)2-30,40(E )
OH
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
NHOH
NHOH
NHOH
NHOH
NHOH
OH
R
R
R
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
2.0 ꢀ 0.2
2.9 ꢀ 0.2
38.6 ꢀ 4.1
81.8 ꢀ 10.4
30.4 ꢀ 4.9
46.6 ꢀ 4.9
73.7 ꢀ 9.4
71.3 ꢀ 5.5
24.1 ꢀ 4.5
70.6 ꢀ 8.2
68.4 ꢀ 6.4
122.5 ꢀ 9.6
121.7 ꢀ 23.4
149.3 ꢀ 15.2
72.3 ꢀ 5.5
98.4 ꢀ 12.3
78.4 ꢀ 8.4
74.8 ꢀ 8.7
82.6 ꢀ 4.9
135.3 ꢀ 26.9
161.8 ꢀ 18.3
137.3 ꢀ 13.0
196.4 ꢀ 23.5
232.7 ꢀ 42.8
33.5 ꢀ 4.8
29.8 ꢀ 1.4
56.1 ꢀ 5.9
62.2 ꢀ 7.3
96.6 ꢀ 8.3
51.4 ꢀ 6.4
121.5 ꢀ 18.4
141.9 ꢀ 11.7
OSO2C6H4CH3-p
OSO2CH3
N3
0.3 ꢀ 0.1
1.1 ꢀ 0.04
2.6 ꢀ 0.3
N3
NH2
3.4 ꢀ 0.4
1.5 ꢀ 0.1
NH2
NH2
ND
CH2CH2C6H3(OCH3)2-30,40
CH3
2.1 ꢀ 0.3
NHCOC6H5
NHCOC6H4Cl-p
NHCOC6H4Br-p
NHCOC6H4NO2-p
NHCOC6H2(OCH3)3-30,40,50
NHCOC5H4N-30
2.7 ꢀ 0.3
CH3
CH3
4.2 ꢀ 0.4
6.1 ꢀ 1.0
CH3
CH3
5.2 ꢀ 0.6
0.1 ꢀ 0.01
1.0 ꢀ 0.1
CH3
CH3
7g
7h
7i
NHCOC5H4N-40
NHCOC4H3N2-20,50
2.2 ꢀ 0.2
CH3
CH3
2.1 ꢀ 0.3
NHCOCH]CHC6H5(E )
0.1 ꢀ 0.01
0.8 ꢀ 0.1
7j
CH3
CH]CHC6H3(OCH3)2-30,40(E )
CH2CH2C6H3(OCH3)2-30,40
CH2CH2C6H3(OCH3)2-30,40
CH3
NHCOCH]CHC6H3(OCH3)2-30,40(E )
NHCOC5H4N-30
7k
7l
3.0 ꢀ 0.04
2.2 ꢀ 0.2
NHCOC5H4N-30
7m
8a
8b
8c
8d
8e
9
NHCOCH]CHC6H5(E )
NHCOC6H2(OCH3)3-30,40,50
NHCOC5H4N-30
1.6 ꢀ 0.3
0.001 ꢀ 0.0001
0.005 ꢀ 0.0004
0.004 ꢀ 0.0005
0.012 ꢀ 0.002
0.011 ꢀ 0.001
0.016 ꢀ 0.002
0.109 ꢀ 0.012
0.009 ꢀ 0.0004
CH3
CH3
NHCOCH]CHC6H5(E )
NHCOC5H4N-30
CH2CH2C6H3(OCH3)2-30,40
CH2CH2C6H3(OCH3)2-30,40
NHCOCH]CHC6H5(E )
NHCOC6H2(OCH3)3-30,40,50
NHCOC6H2(OCH3)3-30,40,50
CH3
CH3
10
LY52
NHCH2COOCH3
ND: not determined.
a
Mean value of three experiments, and standard deviation is given.
parts from the specific amino acid residue of peptide. The
motif HEFGHAMGLEH in the catalytic domain where the
zinc was coordinated by three histidine residues, and the ‘cys-
teine switch’ motif PRCGNPD in the propeptide were con-
served structural signatures [2]. However, AP-N was
a membrane-bound zinc exopeptidase that catalyzed the
removal of NH-terminal amino acid from peptide. AP-N con-
tained the HELAH conserved motif where the zinc was coor-
dinated by two histidine residues and one glutamate residue.
The glutamate of the conserved motif acted as a base in catal-
ysis [18]. Due to the structural differences between MMP-2
and AP-N, there were different structural requirements for
their respective inhibitors. As these pyrrolidine derivatives
exhibited highly selective inhibition against MMP-2, the
following structureeactivity relationships (SARs) were
mainly discussed about MMP-2 inhibition.
Compounds 8aee were more potent than their predecessors
7e, 7f, 7i, 7l and 7m. This activity difference was caused by
the ZBG (COR3), which was the only structural difference be-
tween 8aee and their predecessors. The ZBG is hydroxamate
(CONHOH) for 8aee and carboxylate (COOCH3) for their
predecessors. Both these groups could chelate zinc ion at cat-
alytic activity center of the enzyme. However, the hydroxa-
mate group was a more potent ZBG than carboxylate group
as shown in the activity order of 8aee and their predecessors.
Compound 9 was the only compound containing carboxyl
(COOH) ZBG, which exhibited almost equal potency to the
hydroxamates. The FlexX docking of 9 with MMP-2 was

X.-C. Cheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2130e2139
2133
Fig. 4. FlexX docking result of compound 8c with MMP-2.
with the active domain of enzyme. As compared with 7a,
chloro, bromo and nitro substitutions at the phenyl moiety
(7bed) moderately impaired the inhibitory activity, while
replacement of the phenyl moiety by heterocyclic moiety
(7feh) favored the inhibitory activity. Moreover, the R or S
configuration of these compounds did not seem important to
inhibitory activities.
Fig. 2. FlexX docking result of compound 8a with MMP-2.
also done to elucidate its potency (Fig. 8). The 3,4,5-trime-
thoxybenzamido group at C4 position of pyrrolidine ring inter-
acted well with the deep S10 pocket of MMP-2, and the
carboxyl group chelated the active site zinc ion 166 with a dis-
Among compounds 7f, 7k and 7l, R2 and COR3 groups
were fixed as nicotinamide and carboxylate, respectively,
and R1CO group was altered as acetyl, (E )-3-(3,4-dimethoxy-
phenyl)acryloyl and 3,4-dimethoxyphenylpropanoyl. So the
differences in the inhibitory activities of these compounds
were caused by various R1CO groups. The compound bearing
the shorter R1CO group (acetyl, 7f) was found to be the most
potent one, thus we might conclude that compounds with
small R1CO group would exhibit high activities. The com-
pound bearing 3,4-dimethoxyphenylpropanoyl group (7l)
was in the next place, and the compound containing (E )-3-
(3,4-dimethoxyphenyl)acryloyl group (7k) presented the least
activity. This result might be owing to the flexibility of the 3,4-
dimethoxyphenylpropanoyl group, which could adjust the con-
formation to match the catalytic activity center of the enzyme.
Finally, the binding mode of the most potent compound 8a
with MMP-2 was proposed as follows: (1) the hydroxamate
chelated the active site zinc ion; (2) the carbonyl, amido and
hydroxyl groups provided hydrogen bond interactions with
˚
tance of 1.96 A. In comparison with the docking result of
counterpart 8a, some structureeactivity relationship informa-
tion concluded that the hydroxamate group was a better
ZBG than carboxyl group.
Among compounds 3a, 4a, 5a, 6a and 7aej, R1CO and
COR3 groups were fixed as acetyl and carboxylate, respec-
tively, and R2 group was altered as various length, configura-
tions and substitutions. So the differences in the inhibitory
activities of these compounds were caused by various R2
groups. Introduction of longer R2 group (7i) displayed higher
activity. Compound 3a contained a hydroxyl group at C4 posi-
tion of pyrrolidine ring and it was too small to occupy the S10
pocket entirely. The inhibitory activity of 3a might be caused
by the formation of hydrogen bond with the enzyme backbone,
thus stabilizing the binding between the compound and the en-
zyme. Sulfonation of 3a produced 4a with increased activity,
due to the sulfonyl group easier to form a hydrogen bond
Fig. 3. FlexX docking result of compound 8b with MMP-2.
Fig. 5. FlexX docking result of compound 8a with AP-N.

2134
X.-C. Cheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2130e2139
Fig. 6. FlexX docking result of compound 8b with AP-N.
Fig. 8. FlexX docking result of compound 9 with MMP-2.
the enzyme backbone Ala86, Ala84 and Glu121; (3) two side
chains (3,4,5-trimethoxybenzamido and acetyl) underwent
effective interactions with the enzyme subsites, S10 and S20
pockets (Fig. 9). The above binding mode information encour-
aged us to further design pyrrolidine-scaffold-based MMP-2
inhibitors, which would be reported later.
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 spectra were recorded on an electro-
spray ionization mass spectrometer as m/z value. Flash column
chromatography was performed using 300 mesh silica gel. The
yields were calculated in the last step reaction.
4. Conclusions
A series of novel pyrrolidine derivatives were designed and
synthesized. These pyrrolidine derivatives exhibited highly
selective inhibition against MMP-2 as compared with AP-N.
The hydroxamates 8aec were equally or more potent MMP-
2 inhibitors than the positive control LY52. SAR studies
indicated that moderately long R2 group and short R1 group
at pyrrolidine ring favored the inhibitory activity against
MMP-2. The FlexX docking was consistent with the above
SAR results. Further assays of these compounds on cell cul-
ture and animal models are underway.
5.1.1. trans-4-Hydroxy-L-proline methyl ester
hydrochloride (2)
A
slurry of trans-4-hydroxy-L-proline (1) (100 g,
763.5 mmol) in methanol (650 ml) was treated with dry hydro-
gen 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 157e160 ꢁC (lit. [10]
1
mp 156e160 ꢁC). H NMR (CD3OD, d ppm): 4.86 (s, 2H,
5. Experimental
NHþ2 ), 4.63 (m, 1H, CH), 3.88 (s, 3H, CH3), 3.50 (m, 1H,
CH), 3.35 (m, 2H, CH2), 2.47e2.20 (m, 2H, CH2), 1.31 (s,
1H, OH).
5.1. Synthetic methods and spectroscopic details
Melting points were determined using X-6 digital display
binocular microscope (uncorrected). Infrared spectra were
Glu121
2+
Zn
S2' pocket
O
O
H
O
O
N
N
H
Ala86
N
H
HN
Ala84
O
O
O
O
O
S1' pocket
Fig. 7. FlexX docking result of compound 8c with AP-N.
Fig. 9. Proposed binding mode of compound 8a with MMP-2.

X.-C. Cheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2130e2139
2135
5.1.2. (2S,4R)-Methyl 1-acyl-4-hydroxypyrr
olidine-2-carboxylates (3a,b)
1751.25 (C]O), 1647.97 (C]O), 1596.11 (C]C), 1336.45
(SO2), 1199.34 (CeO), 1172.48 (SO2); 1H NMR (CDCl3,
d ppm): 7.79 (d, 2H, AreH, J ¼ 8.28 Hz), 7.37 (d, 2H, Are
H, J ¼ 8.00 Hz), 5.14 (m, 1H, CH), 4.52 (m, 1H, CH), 3.80
(m, 2H, CH2), 3.72 (s, 3H, OCH3), 2.47 (s, 3H, ArCH3), 2.16
(m, 2H, CH2), 2.07 (s, 3H, CH3CO); ESI-MS: 342.5 (M þ 1).
To a mixture of (2S,4R)-methyl 4-hydroxy-1-((E )-3-(3,4-di-
methoxyphenyl)acryloyl)pyrrolidine-2-carboxylate (3b) (6.72 g,
20 mmol) and Et3N (2.02 g, 20 mmol) in dichloromethane
(100 ml) at 0 ꢁC was added dropwise a solution of methane-
sulfonyl chloride (2.3 g, 20 mmol) in dichloromethane
(100 ml). The mixture was stirred for 4 h at room temperature,
then washed with saturated NaHCO3 solution (2 ꢂ 70 ml) and
H2O (2 ꢂ 70 ml). The organic phase was dried to give
(3R,5S )-5-(methoxycarbonyl)-1-((E )-3-(3,4-dimethoxypheny-
l)acryloyl)pyrrolidin-3-yl methanesulfonate (4b) as pale yel-
To a stirred solution of trans-4-hydroxy-L-proline methyl
ester hydrochloride (2) (64.6 g, 360 mmol) in dioxaneeH2O
(1:1, 200 ml) at 0 ꢁC was added NaHCO3 (62.8 g,
750 mmol) in small portions over 10 min, together with acetic
anhydride (37 ml, 390 mmol), dropwise over 30 min. After 2 h
at 23 ꢁC, the resulting solution was concentrated, diluted with
H2O (100 ml) and saturated with NaCl, and extracted with
chloroform (10 ꢂ 100 ml). The pooled extracts were dried, fil-
tered, and concentrated to a solid that was recrystallized from
ethyl acetateeether to yield (2S,4R)-methyl 1-acetyl-4-hy-
droxypyrrolidine-2-carboxylate (3a) as white crystal (51.9 g,
78%); mp 81e82 ꢁC (lit. [11] mp 80e81 ꢁC). IR (KBr,
cmꢃ1): 3367.34 (OH), 2938.12 (CH), 1746.51 (C]O),
1
1613.95 (C]O), 1196.52 (CeO); H NMR (CDCl3, d ppm):
1
4.56 (t, 1H, CH, J ¼ 7.8 Hz), 3.78 (m, 2H, CH2), 3.73 (s,
3H, OCH3), 3.51 (m, 1H, CH), 2.32 (m, 2H, CH2), 2.08 (s,
3H, CH3CO), 1.97 (s, 1H, OH); ESI-MS: 188.5 (M þ 1).
trans-4-Hydroxy-L-proline methyl ester hydrochloride (2)
(18.5 g, 100 mmol) was suspended in pyridine (200 ml), and
Et3N (30 ml) was added. After stirring for 20 min at room
temperature, the resulting mixture was cooled to ꢃ5 ꢁC, and
a solution of (E )-3-(3,4-dimethoxyphenyl)acryloyl chloride
(22.7 g, 100 mmol) in dichloromethane (1000 ml) was added.
After stirring overnight at room temperature, the resulting
mixture was filtered and the filtrate was evaporated to give
crude oil. The crude oil was dissolved in dichloromethane
(2000 ml), washed with 3 M HCl and H2O, and dried to give
yellow solid, which was then recrystallized from dichlorome-
thane to provide (2S,4R)-methyl 4-hydroxy-1-((E )-3-(3,4-di-
methoxyphenyl)acryloyl)pyrrolidine-2-carboxylate (3b) as
yellow crystal (27.7 g, 81%); mp 60e62 ꢁC. IR (KBr,
cmꢃ1): 3416.25 (OH), 2951.09 (CH), 1744.64 (C]O),
1645.18 (C]O), 1513.81 (C]C), 1267.46 (CeO), 1023.50
(]CH); 1H NMR (CDCl3, d ppm): 7.61 (d, 1H, ]CH,
J ¼ 15.36 Hz), 7.05 (d, 1H, AreH, J ¼ 7.60 Hz), 6.99 (s, 1H,
AreH), 6.80 (d, 1H, AreH, J ¼ 8.28 Hz), 6.51 (d, 1H,
]CH, J ¼ 15.40 Hz), 4.72 (t, 1H, CH, J ¼ 7.92 Hz), 4.62 (s,
1H, CH), 3.94 (m, 2H, CH2), 3.91 (s, 6H, ArOCH3), 3.75 (s,
3H, COOCH3), 2.35 (m, 1H, OH), 2.12 (m, 2H, CH2); ESI-
MS: 336.6 (M þ 1).
low solid (6.6 g, 80%); mp 139e140 ꢁC. H NMR (CDCl3,
d ppm): 7.69 (d, 1H, ]CH, J ¼ 15.36 Hz), 7.11 (d, 1H, Are
H, J ¼ 8.32 Hz), 7.03 (s, 1H, AreH), 6.86 (d, 1H, AreH,
J ¼ 8.32 Hz), 6.49 (d, 1H, ]CH, J ¼ 15.40 Hz), 5.38 (s, 1H,
CH), 4.73 (t, 1H, CH, J ¼ 7.88 Hz), 4.12 (m, 2H, CH2), 3.93
(s, 6H, ArOCH3), 3.79 (s, 3H, COOCH3), 3.07 (s, 3H,
CH3SO2), 2.33 (m, 2H, CH2); ESI-MS: 414.5 (M þ 1).
5.1.4. (2S,4S )-Methyl 1-acyl-4-azidopyrrolidine-2-
carboxylates (5a,b)
(3R,5S )-5-(Methoxycarbonyl)-1-acylpyrrolidin-3-yl sulfo-
nate (4a or 4b) (9.04 mmol) and NaN3 (46.2 mmol) were
stirred in dry DMF (10 ml) at 45e55 ꢁC overnight. The
solvent was evaporated, and the residue was mixed with ethyl
acetate (25 ml) and H2O (15 ml). After separation of the
layers, the organic phase was washed with H2O until neutral
and then with 0.1 M HCl and dried. Evaporation of ethyl
acetate gave (2S,4S )-methyl 1-acetyl-4-azidopyrrolidine-2-car-
boxylate (5a) or (2S,4S )-methyl 4-azido-1-((E )-3-(3,4-dime-
thoxyphenyl)acryloyl)pyrrolidine-2-carboxylate (5b), which
was suitable for the next step. Azide 5a: colorless oil; 95%;
IR (KBr, cmꢃ1): 2954.70 (CH), 2108.36 (N3), 1747.83
(C]O), 1654.99 (C]O), 1205.16 (CeO); 1H NMR
(CDCl3, d ppm): 5.03 (m, 1H, CH), 3.84 (m, 2H, CH2), 3.81
(s, 3H, OCH3), 2.52 (m, 1H, CH), 2.24 (m, 2H, CH2), 2.09
(s, 3H, CH3CO); ESI-MS: 213.4 (M þ 1). Azide 5b: yellow
oil; 93%; IR (KBr, cmꢃ1): 2952.70 (CH), 2105.74 (N3),
1748.66 (C]O), 1649.85 (C]O), 1513.90 (C]C), 1266.96
5.1.3. (3R,5S )-5-(Methoxycarbonyl)-1-acylpyrrolidin-3-yl
sulfonates (4a,b)
1
(CeO), 1023.57 (]CH); H NMR (CDCl3, d ppm): 7.61 (d,
To a solution of p-toluene sulfonyl chloride (12.5 g,
65.6 mmol) in pyridine (25 ml) at 0 ꢁC was added (2S,4R)-
methyl 1-acetyl-4-hydroxypyrrolidine-2-carboxylate (3a)
(11.2 g, 59.9 mmol). After 15 h at 0 ꢁC, the mixture was poured
into 1 M HCl (180 ml), and the solution was extracted with
ethyl acetate (3 ꢂ 60 ml). The pooled extracts were washed
with 1 M HCl (3 ꢂ 30 ml), saturated NaHCO3 (2 ꢂ 30 ml)
and brine (2 ꢂ 30 ml), and then dried and concentrated to
a tan oil that was crystallized from ether to yield (3R,5S )-5-
(methoxycarbonyl)-1-acetylpyrrolidin-3-yl 4-methylbenzene-
sulfonate (4a) as white crystal (11.3 g, 55%); mp 70e71 ꢁC
(lit. [11] mp 70e72 ꢁC). IR (KBr, cmꢃ1): 2923.81 (CH),
1H, ]CH, J ¼ 15.47 Hz), 7.25 (d, 1H, AreH, J ¼ 8.21 Hz),
7.07 (s, 1H, AreH), 6.86 (d, 1H, AreH, J ¼ 8.36 Hz), 6.44
(d, 1H, ]CH, J ¼ 15.38 Hz), 5.04 (s, 1H, CH), 4.23 (m, 2H,
CH2), 3.85 (s, 6H, ArOCH3), 3.71 (s, 3H, COOCH3), 2.71
(m, 1H, CH), 2.21 (m, 2H, CH2); ESI-MS: 361.4 (M þ 1).
5.1.5. (2S,4S )-Methyl 1-acyl-4-aminopyrrolidine-2-
carboxylates (6aec)
(2S,4S )-Methyl 1-acyl-4-azidopyrrolidine-2-carboxylate
(5a or 5b) (7.8 mmol) in methanol (20 ml) was hydrogenated
in the presence of catalytic amount of 5% PdeCaCO3 at room
temperature and 1 atm pressure. After 16 h, the catalyst was

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X.-C. Cheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2130e2139
1
1640.35 (C]O), 1540.70 (C]C), 1204.25 (CeO); H NMR
filtered off and the solvent was removed under vacuum to
give (2S,4S )-methyl 1-acetyl-4-aminopyrrolidine-2-carboxyl-
ate (6a) or (2S,4S )-methyl 4-amino-1-((E )-3-(3,4-dimethoxy-
phenyl)acryloyl)pyrrolidine-2-carboxylate (6b). When 5b was
hydrogenated in the presence of excessive catalyst, both the
acryloyl and azido groups were hydrogenated to give (2S,4S )-
methyl 1-(3-(3,4-dimethoxyphenyl)propanoyl)-4-aminopyrro-
lidine-2-carboxylate (6c). Compound 6a: white solid; 97%;
mp 55e57 ꢁC; IR (KBr, cmꢃ1): 3268.58 (NH2), 2954.78
(CDCl3, d ppm): 8.01 (d, 1H, NH, J ¼ 8.64 Hz), 7.79 (d, 2H,
AreH, J ¼ 8.45 Hz), 7.41 (d, 2H, AreH, J ¼ 8.40 Hz), 4.96
(m, 1H, CH), 4.52 (m, 1H, CH), 3.79 (s, 3H, OCH3), 3.86e
3.67 (m, 2H, CH2), 2.58e2.02 (m, 2H, CH2), 2.10 (s, 3H,
CH3CO); ESI-MS: 325.5 (M þ 1).
5.1.6.3. (2S,4S )-Methyl 4-(4-bromobenzamido)-1-acetylpyrro-
lidine-2-carboxylate (7c). Flash column chromatography:
chloroformeacetone ¼ 10:1e4:1; 57%; colorless oil; IR
(KBr, cmꢃ1): 3308.78 (NH), 2953.23 (CH), 1747.17 (C]O),
1
(CH), 1744.78 (C]O), 1652.40 (C]O), 1205.29 (CeO); H
NMR (CDCl3, d ppm): 4.97 (m, 1H, CH), 4.41 (m, 1H, CH),
3.75 (m, 2H, CH2), 3.65 (s, 3H, OCH3), 2.26 (m, 2H, CH2),
2.09 (s, 3H, CH3CO), 1.83 (m, 2H, NH2); ESI-MS: 187.3
(M þ 1). Compound 6b: yellow solid; 80%; mp 60e62 ꢁC; IR
(KBr, cmꢃ1): 3205.41 (NH2), 2952.71 (CH), 1744.66 (C]O),
1651.18 (C]O), 1513.74 (C]C), 1264.35 (CeO), 1023.18
(]CH); 1H NMR (CDCl3, d ppm): 7.74 (d, 1H, ]CH,
J ¼ 15.31 Hz), 7.23 (d, 1H, AreH, J ¼ 8.27 Hz), 7.08 (s, 1H,
AreH), 6.79 (d, 1H, AreH, J ¼ 8.35 Hz), 6.43 (d, 1H, ]CH,
J ¼ 15.43 Hz), 5.17 (s, 1H, CH), 4.77 (t, 1H, CH,
J ¼ 7.75 Hz), 4.03 (m, 2H, CH2), 3.97 (s, 6H, ArOCH3), 3.74
(s, 3H, COOCH3), 2.38 (m, 2H, CH2), 1.87 (m, 2H, NH2);
ESI-MS: 335.7 (M þ 1). Compound 6c: yellow oil; 82%; IR
(KBr, cmꢃ1): 3346.24 (NH2), 2953.17 (CH), 1744.65 (C]O),
1
1640.45 (C]O), 1540.62 (C]C), 1204.02 (CeO); H NMR
(CDCl3, d ppm): 7.96 (d, 1H, NH, J ¼ 8.41 Hz), 7.71 (d, 2H,
AreH, J ¼ 8.52 Hz), 7.57 (d, 2H, AreH, J ¼ 8.56 Hz), 4.94
(m, 1H, CH), 4.52 (m, 1H, CH), 3.79 (s, 3H, OCH3), 3.86e
3.67 (m, 2H, CH2), 2.58e2.02 (m, 2H, CH2), 2.09 (s, 3H,
CH3CO); ESI-MS: 371.4 (M þ 2).
5.1.6.4. (2S,4S )-Methyl 4-(4-nitrobenzamido)-1-acetylpyrroli-
dine-2-carboxylate (7d). Flash column chromatography:
chloroformeacetone ¼ 10:1e4:1; 48%; yellow oil; IR (KBr,
cmꢃ1): 3307.94 (NH), 2954.37 (CH), 1746.17 (C]O),
1652.65 (C]O), 1526.40 and 1348.65 (NO2), 1204.88 (Ce
O); 1H NMR (CDCl3,
d ppm): 8.28 (d, 1H, NH,
1
1656.07 (C]O), 1515.45 (C]C), 1261.62 (CeO); H NMR
J ¼ 6.70 Hz), 8.04 (d, 2H, AreH, J ¼ 7.46 Hz), 7.98 (d, 2H,
AreH, J ¼ 8.24 Hz), 4.99 (m, 1H, CH), 4.56 (m, 1H, CH),
3.81 (s, 3H, OCH3), 3.91e3.73 (m, 2H, CH2), 2.62e2.03
(m, 2H, CH2), 2.12 (s, 3H, CH3CO); ESI-MS: 336.5 (M þ 1).
(CDCl3, d ppm): 7.19 (d, 1H, AreH, J ¼ 8.25 Hz), 7.00 (s,
1H, AreH), 6.72 (d, 1H, AreH, J ¼ 8.39 Hz), 5.15 (m, 1H,
CH), 4.65 (m, 1H, CH), 4.05 (m, 2H, CH2), 3.89 (s, 6H, Ar-
OCH3), 3.71 (s, 3H, COOCH3), 2.95e2.42 (m, 6H, 3CH2),
1.87 (m, 2H, NH2); ESI-MS: 337.7 (M þ 1).
5.1.6.5. (2S,4S )-Methyl 4-(3,4,5-trimethoxybenzamido)-1-ace-
tylpyrrolidine-2-carboxylate (7e). Flash column chromatogra-
phy: chloroformeacetone ¼ 4:1; 56%; white crystal; mp 150e
152 ꢁC; IR (KBr, cmꢃ1): 3439.94 (NH), 2948.89 (CH),
1743.28 (C]O), 1655.76 (C]O), 1586.03 (C]C), 1127.10
(CeO); 1H NMR (CDCl3, d ppm): 7.92 (d, 1H, NH,
J ¼ 8.67 Hz), 7.13 (s, 2H, AreH), 4.99 (d, 1H, CH,
J ¼ 3.98 Hz), 4.56 (d, 1H, CH, J ¼ 9.79 Hz), 3.94 (s, 3H,
OCH3), 3.92 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.81 (s,
3H, OCH3), 3.71 (m, 2H, CH2), 2.53 (m, 2H, CH2), 2.10 (s,
3H, CH3CO); ESI-MS: 381.4 (M þ 1).
5.1.6. General procedure for the preparation of (2S,4S)-
methyl 4-acylamido-1-acylpyrrolidine-2-carboxylates(7aem)
To a mixture of (2S,4S )-methyl 1-acyl-4-aminopyrrolidine-
2-carboxylate (6a, 6b, or 6c) (5 mmol) and N-methyl morpho-
line (5 mmol) in dichloromethane (50 ml) at room temperature
was added dropwise a solution of acyl chloride (5 mmol) in di-
chloromethane (50 ml). The mixture was stirred at room tem-
perature for 10 h and the solvent was evaporated in vacuo. The
final product was purified by flash column chromatography
and 7aem was obtained.
5.1.6.6. (2S,4S )-Methyl 1-acetyl-4-(nicotinamido)pyrrolidine-
2-carboxylate (7f). Flash column chromatography: chloro-
formeacetone ¼ 1:1; 57%; colorless oil; IR (KBr, cmꢃ1):
3303.87 (NH), 2953.60 (CH), 1746.25 (C]O), 1652.53
(C]O), 1541.27 (C]N), 1202.86 (CeO); 1H NMR
(CDCl3, d ppm): 9.06 (s, 1H, AreH), 8.71 (s, 1H, AreH),
8.22 (d, 1H, AreH, J ¼ 8.43 Hz), 8.15 (d, 1H, NH,
J ¼ 7.68 Hz), 7.40 (t, 1H, AreH, J ¼ 6.18 Hz), 4.95 (s, 1H,
CH), 4.52 (d, 1H, CH, J ¼ 7.92 Hz), 3.78 (s, 3H, OCH3),
3.89e3.70 (m, 2H, CH2), 2.58e2.02 (m, 2H, CH2), 2.09 (s,
3H, CH3CO); ESI-MS: 292.4 (M þ 1).
5.1.6.1. (2S,4S )-Methyl 1-acetyl-4-(benzamido)pyrrolidine-2-
carboxylate (7a). Flash column chromatography: chloro-
formeacetone ¼ 10:1e4:1; 56%; colorless oil; IR (KBr,
cmꢃ1): 3314.68 (NH), 2952.91 (CH), 1747.36 (C]O),
1
1652.10 (C]O), 1537.68 (C]C), 1203.62 (CeO); H NMR
(CDCl3, d ppm): 7.83 (d, 1H, NH, J ¼ 9.49 Hz), 7.81 (t, 2H,
AreH, J ¼ 1.75 Hz), 7.46 (m, 3H, AreH), 4.98 (m, 1H,
CH), 4.53 (m, 1H, CH), 3.77 (s, 3H, OCH3), 3.84e3.65 (m,
2H, CH2), 2.55e2.47 (m, 2H, CH2), 2.07 (s, 3H, CH3CO);
ESI-MS: 291.3 (M þ 1).
5.1.6.2. (2S,4S )-Methyl 4-(4-chlorobenzamido)-1-acetylpyrro-
lidine-2-carboxylate (7b). Flash column chromatography:
chloroformeacetone ¼ 10:1e4:1; 46%; colorless oil; IR
(KBr, cmꢃ1): 3312.24 (NH), 2953.12 (CH), 1747.23 (C]O),
5.1.6.7. (2S,4S )-Methyl 1-acetyl-4-(isonicotinamido)pyrroli-
dine-2-carboxylate (7g). Flash column chromatography:
chloroformeacetone ¼ 1:1; 68%; yellow oil; IR (KBr,
cmꢃ1): 3295.63 (NH), 2954.80 (CH), 1745.57 (C]O),

X.-C. Cheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2130e2139
2137
1
1652.24 (C]O), 1541.28 (C]N), 1203.96 (CeO); H NMR
(CDCl3, d ppm): 8.71 (m, 2H, AreH), 8.33 (d, 1H, NH,
J ¼ 8.30 Hz), 7.70 (m, 2H, AreH), 4.92 (m, 1H, CH), 4.54
(m, 1H, CH), 3.79 (s, 3H, OCH3), 3.91e3.70 (m, 2H, CH2),
2.60e2.02 (m, 2H, CH2), 2.09 (s, 3H, CH3CO); ESI-MS:
292.5 (M þ 1).
(m, 1H, CH), 4.67 (m, 1H, CH), 3.90 (s, 3H, OCH3), 3.89
(s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.99e3.78 (m, 2H,
CH2), 2.63e2.12 (m, 2H, CH2); ESI-MS: 440.7 (M þ 1).
5.1.6.12. (2S,4S )-Methyl 1-(3-(3,4-dimethoxyphenyl)propa-
noyl)-4-(nicotinamido)pyrrolidine-2-carboxylate (7l). Flash
column chromatography: dichloromethaneeacetone ¼ 10:1e
1:1; 39%; white crystal; mp 40e42 ꢁC; IR (KBr, cmꢃ1):
3326.38 (NH), 2952.93 (CH), 1744.85 (C]O), 1651.39
(C]O), 1516.08 (C]N), 1262.30 (CeO); 1H NMR
(CDCl3, d ppm): 8.98 (s, 1H, AreH), 8.65 (m, 1H, AreH),
8.05 (m, 2H, AreH and NH), 7.31 (m, 1H, AreH), 6.68 (m,
3H, AreH), 4.86 (m, 1H, CH), 4.46 (m, 1H, CH), 3.78 (s,
3H, OCH3), 3.74 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.87e
3.60 (m, 2H, CH2), 2.85e2.81 (m, 2H, CH2), 2.54e2.44 (m,
4H, CH2CH2); ESI-MS: 442.6 (M þ 1).
5.1.6.8. (2S,4S )-Methyl 1-acetyl-4-(pyrazine-2-carboxamido)-
pyrrolidine-2-carboxylate (7h). Flash column chromatogra-
phy: chloroformeacetone ¼ 10:1e1:1; 37%; white crystal;
mp 51e52 ꢁC; IR (KBr, cmꢃ1): 3347.47 (NH), 2954.33
(CH), 1741.40 (C]O), 1652.27 (C]O), 1529.54 (C]N),
1204.19 (CeO); 1H NMR (CDCl3, d ppm): 9.39 (d, 1H,
AreH, J ¼ 5.28 Hz), 8.77 (d, 1H, AreH, J ¼ 2.40 Hz), 8.73
(d, 1H, AreH, J ¼ 8.79 Hz), 8.59 (d, 1H, NH, J ¼ 3.92 Hz),
4.98 (m, 1H, CH), 4.59 (m, 1H, CH), 3.80 (s, 3H, OCH3),
3.97e3.76 (m, 2H, CH2), 2.64e2.58 (m, 2H, CH2), 2.11 (s,
3H, CH3CO); ESI-MS: 293.3 (M þ 1).
5.1.6.13. (2S,4S )-Methyl 1-(3-(3,4-dimethoxyphenyl)propa-
noyl)-4-(cinnamamido)pyrrolidine-2-carboxylate (7m). Flash
column chromatography: dichloromethaneeacetone ¼ 10:1e
1:1; 53%; yellow crystal; mp 49e51 ꢁC; IR (KBr, cmꢃ1):
3293.07 (NH), 2951.62 (CH), 1746.11 (C]O), 1656.55
(C]O), 1516.24 (C]C), 1205.72 (CeO), 1028.00 (]CH);
1H NMR (CDCl3, d ppm): 8.00 (s, 1H, NH), 7.60 (d, 1H,
]CH, J ¼ 15.68 Hz), 7.51 (m, 2H, AreH), 7.36 (m, 3H,
AreH), 7.24 (d, 1H, AreH, J ¼ 8.61 Hz), 6.76 (m, 2H, Are
H), 6.41 (d, 1H, ]CH, J ¼ 15.68 Hz), 4.77 (m, 1H, CH),
4.47 (m, 1H, CH), 3.87 (s, 3H, OCH3), 3.79 (s, 3H, OCH3),
3.78 (s, 3H, OCH3), 3.72e3.68 (m, 2H, CH2), 2.94e2.86
(m, 2H, CH2), 2.63e2.43 (m, 4H, CH2CH2); ESI-MS: 467.6
(M þ 1).
5.1.6.9. (2S,4S )-Methyl 1-acetyl-4-(cinnamamido)pyrrolidine-
2-carboxylate (7i). Flash column chromatography: chloro-
formeacetone ¼ 10:1e4:1; 64%; colorless oil; IR (KBr,
cmꢃ1): 3285.26 (NH), 2952.54 (CH), 1747.59 (C]O),
1656.66 (C]O), 1544.07 (C]C), 1206.76 (CeO), 981.86
(]CH); 1H NMR (CDCl3, d ppm): 7.63 (d, 1H, ]CH,
J ¼ 15.70 Hz), 7.49 (m, 2H, AreH), 7.43 (d, 1H, NH,
J ¼ 8.51 Hz), 7.35 (m, 3H, AreH), 6.45 (d, 1H, ]CH,
J ¼ 15.69 Hz), 4.84 (m, 1H, CH), 4.49 (m, 1H, CH), 3.76 (s,
3H, OCH3), 3.88e3.58 (m, 2H, CH2), 2.57e2.01 (m, 2H,
CH2), 2.09 (s, 3H, CH3CO); ESI-MS: 317.5 (M þ 1).
5.1.6.10. (2S,4S )-Methyl 4-((E )-3-(3,4-dimethoxyphenyl)acry-
lamido)-1-acetylpyrrolidine-2-carboxylate (7j). Flash column
chromatography: chloroformeacetone ¼ 10:1e4:1; 43%;
white crystal; mp 61e63 ꢁC; IR (KBr, cmꢃ1): 3288.56 (NH),
2953.27 (CH), 1746.92 (C]O), 1651.71 (C]O), 1514.28
5.1.7. General procedure for the preparation of (2S,4S )-
1-acyl-4-(acylamido)-N-hydroxypyrrolidine-2-
carboxamides (8aee)
To a solution of compound 7 (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. The residue was purified by flash column chromatogra-
phy to give 8aee.
1
(C]C), 1261.91 (CeO), 1023.44 (]CH); H NMR (CDCl3,
d ppm): 7.51 (d, 1H, ]CH, J ¼ 15.61 Hz), 7.10 (d, 1H,
AreH, J ¼ 8.82 Hz), 7.05 (m, 1H, NH), 6.99 (m, 1H, AreH),
6.81 (d, 1H, AreH, J ¼ 8.32 Hz), 6.21 (d, 1H, ]CH,
J ¼ 15.57 Hz), 4.84 (m, 1H, CH), 4.46 (m, 1H, CH), 3.87 (s,
3H, OCH3), 3.86 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.81e
3.56 (m, 2H, CH2), 2.51e1.95 (m, 2H, CH2), 2.06 (s, 3H,
CH3CO); ESI-MS: 377.5 (M þ 1).
5.1.7.1. (2S,4S )-4-(3,4,5-Trimethoxybenzamido)-1-acetyl-N-
hydroxypyrrolidine-2-carboxamide (8a). Flash column chro-
matography: chloroformemethanol ¼ 10:1e1:1; 42%; white
crystal; mp 98e100 ꢁC; IR (KBr, cmꢃ1): 3238.49 (OH and
NH), 2940.34 (CH), 1633.03 (C]O), 1584.66 (C]C),
5.1.6.11. (2S,4S )-Methyl 1-((E )-3-(3,4-dimethoxyphenyl)acry-
loyl)-4-(nicotinamido)pyrrolidine-2-carboxylate (7k). Flash
column chromatography: dichloromethaneeacetone ¼ 10:1e
1:1; 55%; yellow crystal; mp 70e72 ꢁC; IR (KBr, cmꢃ1):
3377.87 (NH), 2952.67 (CH), 1744.19 (C]O), 1648.06
(C]O), 1513.59 (C]N), 1269.94 (CeO), 1024.37 (]CH);
1H NMR (CDCl3, d ppm): 9.10 (s, 1H, AreH), 8.72 (d, 1H,
AreH, J ¼ 5.99 Hz), 8.30 (d, 1H, AreH, J ¼ 8.71 Hz), 8.15
(d, 1H, AreH, J ¼ 7.97 Hz), 7.66 (d, 1H, ]CH,
J ¼ 15.34 Hz), 7.38 (q, 1H, NH, J ¼ 4.22 Hz), 7.08 (d, 1H,
AreH, J ¼ 8.34 Hz), 7.03 (s, 1H, AreH), 6.83 (d, 1H,
AreH, J ¼ 8.31 Hz), 6.54 (d, 1H, ]CH, J ¼ 15.35 Hz), 5.05
1
1126.40 (CeO); H NMR (CD3OD, d ppm): 10.49 (s, 1H,
NH), 8.63 (d, 1H, NH, J ¼ 6.52 Hz), 7.15 (s, 2H, AreH),
4.78e4.72 (m, 2H, CH), 3.93 (s, 3H, OCH3), 3.91 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 3.79e3.69 (m, 2H, CH2),
2.57e2.31 (m, 2H, CH2), 2.11 (s, 1H, OH), 2.08 (s, 3H,
CH3CO); ESI-MS: 382.5 (M þ 1).
5.1.7.2. N-((3S,5S )-5-(Hydroxycarbamoyl)-1-acetylpyrrolidin-
3-yl)nicotinamide (8b). Flash column chromatography: di-
chloromethaneemethanol ¼ 10:1e1:1; 59%; white crystal;

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X.-C. Cheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2130e2139
mp 160e162 ꢁC; IR (KBr, cmꢃ1): 3381.51 (OH and NH),
evaporated in vacuo. The residue was purified by flash column
chromatography (chloroformemethanol ¼ 5:1) to give 9 as
white crystal; 87%; mp 246e248 ꢁC; IR (KBr, cmꢃ1):
3388.34 (OH and NH), 2941.41 (CH), 1732.34 (C]O),
1
2935.07 (CH), 1629.01 (C]O), 1545.37 (C]N); H NMR
(CD3OD, d ppm): 8.98 (t, 1H, AreH, J ¼ 9.52 Hz), 8.68 (s,
1H, AreH), 8.26 (m, 1H, AreH), 7.98 (s, 1H, NH), 7.95 (s,
1H, NH), 7.54 (m, 1H, AreH), 4.72 (m, 1H, CH), 4.39 (m,
1H, CH), 3.95e3.65 (m, 2H, CH2), 3.30 (s, 1H, OH), 2.76e
2.35 (m, 2H, CH2), 2.09 (s, 1H, CH3CO); ESI-MS: 293.4
(M þ 1).
1
1628.52 (C]O), 1585.08 (C]C), 1126.63 (CeO); H NMR
(CDCl3, d ppm): 8.01 (s, 1H, NH), 7.59 (s, 1H, OH), 7.06
(s, 2H, AreH), 4.84 (d, 1H, CH, J ¼ 5.82 Hz), 4.68 (d, 1H,
CH, J ¼ 4.41 Hz), 3.92 (s, 3H, OCH3), 3.91 (s, 3H, OCH3),
3.87 (s, 3H, OCH3), 3.83e3.67 (m, 2H, CH2), 2.95e2.33
(m, 2H, CH2), 2.16 (s, 3H, CH3CO); ESI-MS: 367.5 (M þ 1).
5.1.7.3. (2S,4S )-1-Acetyl-4-(cinnamamido)-N-hydroxypyrroli-
dine-2-carboxamide (8c). Flash column chromatography:
dichloromethaneemethanol ¼ 10:1e1:1; 61%; white crystal;
mp 185e187 ꢁC; IR (KBr, cmꢃ1): 3281.59 (OH), 3223.28
(NH), 2928.65 (CH), 1679.29 (C]O), 1653.98 (C]O),
1624.68 (C]O), 1534.83 (C]C), 1209.10 (CeO), 974.10
(]CH); 1H NMR (CD3OD, d ppm): 8.09 (d, 1H, NH,
J ¼ 7.64 Hz), 7.54e7.51 (m, 3H, AreH, ]CH and NH),
7.41e7.26 (m, 4H, AreH), 6.45 (m, 1H, ]CH), 4.74 (m,
2H, CH), 3.85e3.49 (m, 4H, CH2), 2.17 (s, 1H, OH), 2.09
(s, 3H, CH3CO); ESI-MS: 318.5 (M þ 1).
5.1.9. Procedure for the preparation of methyl 2-((2S,4S)-4-
(3,4,5-trimethoxybenzamido)-1-acetylpyrrolidine-2-
carboxamido)acetate (10)
Compound 9 (5 mmol) was dissolved in tetrahydrofuran
(10 ml), then N-hydroxysuccinimide (5 mmol) and N,N0-dicy-
clohexylcarbodiimide (5 mmol) were added. The mixture was
stirred at 0 ꢁC for 8 h, then glycine methyl ester hydrochloride
(5 mmol) and N-methyl morpholine (5 mmol) were added.
The mixture was stirred at 0 ꢁC for another 8 h. The solvent
was evaporated in vacuo and the residue was purified by flash
column chromatography (chloroformemethanol ¼ 10:1) to
give 10 as white crystal; 59%; mp 50e52 ꢁC; IR (KBr,
cmꢃ1): 3303.73 (NH), 2944.39 (CH), 1751.98 (C]O),
5.1.7.4. N-((3S,5S )-5-(Hydroxycarbamoyl)-1-(3-(3,4-dimetho
xyphenyl)propanoyl)pyrrolidin-3-yl)nicotinamide (8d). Flash
column
chromatography:
dichloromethaneemeth-
1
anol ¼ 40:1e10:1; 64%; white crystal; mp 90e92 ꢁC; IR
(KBr, cmꢃ1): 3253.73 (OH and NH), 2935.30 (CH), 1647.80
(C]O), 1515.91 (C]N), 1263.02 (CeO); 1H NMR
(CD3OD, d ppm): 10.82 (s, 1H, NH), 9.18 (d, 1H, NH,
J ¼ 7.38 Hz), 8.98 (s, 1H, AreH), 8.62 (d, 1H, AreH,
J ¼ 3.48 Hz), 8.11 (d, 1H, AreH, J ¼ 8.02 Hz), 7.32 (m, 1H,
AreH), 6.67 (m, 3H, AreH), 4.75 (m, 1H, CH), 4.64 (m,
1H, CH), 3.80 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.91e
3.61 (m, 2H, CH2), 3.44 (s, 1H, OH), 2.85e2.81 (m, 2H,
CH2), 2.69e2.32 (m, 4H, CH2CH2); ESI-MS: 443.7 (M þ 1).
1651.83 (C]O), 1585.09 (C]C), 1126.51 (CeO); H NMR
(CDCl3, d ppm): 8.77 (d, 1H, NH, J ¼ 6.63 Hz), 8.26 (t, 1H,
NH, J ¼ 5.33 Hz), 7.14 (s, 2H, AreH), 4.86 (d, 1H, CH,
J ¼ 8.78 Hz), 4.72 (m, 1H, CH), 3.90 (s, 3H, OCH3), 3.89
(s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.68 (s, 3H, OCH3),
4.04e3.65 (m, 4H, CH2), 2.51e1.99 (m, 2H, CH2), 2.07 (s,
3H, CH3CO); ESI-MS: 438.5 (M þ 1).
5.2. Biological evaluation
5.2.1. MMP-2 inhibition assay
5.1.7.5. (2S,4S )-1-(3-(3,4-Dimethoxyphenyl)propanoyl)-4-(cin
namamido)-N-hydroxypyrrolidine-2-carboxamide (8e). Flash
column chromatography: dichloromethaneemethanol ¼ 40:1e
10:1; 43%; white crystal; mp 85e87 ꢁC; IR (KBr, cmꢃ1):
3257.07 (OH and NH), 2934.53 (CH), 1655.92 (C]O),
1624.51 (C]O), 1515.95 (C]C), 1262.66 (CeO), 1027.60
The pyrrolidine derivatives were assayed for the inhibitory
activities against MMP-2 in 96-well microtiter plates using suc-
cinylated 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 carried 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 incu-
bated 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%) ꢃ OD450(compound)]/[OD450(100%) ꢃ OD450
(blank)] ꢂ 100%. The IC50 values were obtained from the above
inhibitory rates using OriginPro 7.5 software.
1
(]CH); H NMR (CD3OD, d ppm): 10.72 (s, 1H, NH), 8.22
(d, 1H, NH, J ¼ 7.68 Hz), 7.58 (d, 1H, ]CH, J ¼ 15.68 Hz),
7.48 (t, 2H, AreH, J ¼ 3.74 Hz), 7.30 (m, 3H, AreH), 6.72
(m, 3H, AreH), 6.44 (d, 1H, ]CH, J ¼ 15.70 Hz), 4.72 (m,
1H, CH), 4.53 (m, 1H, CH), 3.82 (s, 3H, OCH3), 3.77 (s, 3H,
OCH3), 3.64e3.52 (m, 2H, CH2), 3.46 (s, 1H, OH), 2.83 (m,
2H, CH2), 2.59e2.16 (m, 4H, CH2CH2); ESI-MS: 468.5
(M þ 1).
5.1.8. Procedure for the preparation of (2S,4S )-4-(3,4,
5-trimethoxybenzamido)-1-acetylpyrrolidine-2-carboxylic
acid (9)
To a stirred solution of ester 7e (2 mmol) in methanol
(5 ml) at 0 ꢁC was added 1 M NaOH (2.1 ml, 2.1 mmol).
The mixture was slowly warmed to 23 ꢁC and treated with
1 M HCl (2.1 ml, 2.1 mmol). After 15 min, the solvent was
5.2.2. AP-N inhibition assay
The pyrrolidine derivatives were further assayed for the in-
hibitory 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

X.-C. Cheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2130e2139
2139
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 contained only the enzyme. The resulting
solutions were measured under 405 nm to gain OD405 values,
which were then used to calculate the inhibitory rates by
[OD405(100%) ꢃ OD405(compound)]/[OD405(100%) ꢃ OD405
(blank)] ꢂ 100%. The IC50 values were obtained from the
above inhibitory rates using OriginPro 7.5 software.
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Acknowledgments
This work was supported by the National High Technology
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 Grant-in-Aid from the Ministry of
Education, Science, Sports and Culture of Japan.
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