Design, synthesis, and evaluation of novel galloyl pyrrolidine derivatives as potential anti-tumor agents

Article abstract of DOI:10.1016/j.bmc.2005.09.031

A series of novel galloyl pyrrolidine derivatives were synthesized as potential anti-tumor agents. Their inhibiting activities on gelatinase (MMP-2 and -9) were tested with succinylated gelatin as the substrate. Structure-activity analyses demonstrate that introduction of longer and more flexible side chains at the C₄ position of the pyrrolidine ring brings higher activity against gelatinase. Free phenol hydroxyl group is more favorable than the methylated one, which confirms the important role of the phenol hydroxyl group when inhibitors interact with gelatinase. In particular, (2S,4S)-4-(3-(3,4-dimethoxyphenyl)acrylamido)-N-hydroxy-1-(3,4,5- trimethoxybenzoyl)pyrrolidine-2-carboxamide (18) stood out as the most attractive compound (IC₅₀ = 0.9 nM). The anti-metastasis model of mice bearing H₂₂ tumor cells was used to evaluate their anti-tumor activities in vivo. The assay in vivo revealed that most of these inhibitors displayed favorable inhibitory activities (inhibitory rate >35%) and no significant toxic effects were observed. The inhibition for 62.37% of 19 indicates the strategy used to design MMP inhibitors (MMPIs) of galloyl pyrrolidine derivatives as potential anti-tumor agents is promising.

Full text of DOI:10.1016/j.bmc.2005.09.031

Bioorganic & Medicinal Chemistry 14 (2006) 1287–1293
Design, synthesis, and evaluation of novel galloyl
pyrrolidine derivatives as potential anti-tumor agents
Xun Li, Yalin Li and Wenfang Xu*
College of Pharmacy, Shandong University, JiÕnan 250012, PR China
Received 5 August 2005; revised 13 September 2005; accepted 14 September 2005
Available online 4 October 2005
Abstract—A series of novel galloyl pyrrolidine derivatives were synthesized as potential anti-tumor agents. Their inhibiting activities
on gelatinase (MMP-2 and -9) were tested with succinylated gelatin as the substrate. Structure–activity analyses demonstrate that
introduction of longer and more flexible side chains at the C4 position of the pyrrolidine ring brings higher activity against gelatin-
ase. Free phenol hydroxyl group is more favorable than the methylated one, which confirms the important role of the phenol
hydroxyl group when inhibitors interact with gelatinase. In particular, (2S,4S)-4-(3-(3,4-dimethoxyphenyl)acrylamido)-N-hy-
droxy-1-(3,4,5- trimethoxybenzoyl)pyrrolidine-2-carboxamide (18) stood out as the most attractive compound (IC50 = 0.9 nM).
The anti-metastasis model of mice bearing H22 tumor cells was used to evaluate their anti-tumor activities in vivo. The assay in vivo
revealed that most of these inhibitors displayed favorable inhibitory activities (inhibitory rate >35%) and no significant toxic effects
were observed. The inhibition for 62.37% of 19 indicates the strategy used to design MMP inhibitors (MMPIs) of galloyl pyrrolidine
derivatives as potential anti-tumor agents is promising.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
To date, at least 26 structurally related members have
been found in the mammal MMPs gene family, and they
manipulate so many physiological processes that block-
ing all MMPs is usually not favored as a positive thera-
py. It is desirable to find MMPIs that are highly selective
for certain MMPs associating with cancers.10 MMP-2
and -9 (gelatinase) are among those meeting this require-
ment.11,12 Herein, we designed galloyl pyrrolidine deriv-
atives as novel MMPIs with the activity of inhibiting
both MMP-2 and -9.
Satisfactory curative chemotherapeutic agents with nov-
el action mechanisms against metastatic cancer are still
urgently needed. By sequencing the human genome,
we have identified an enormous number of targets asso-
ciated with cancers. Based on this historically global
work, anti-tumor drugs with high affinity and selectivity
are expected to be rationally designed against the corre-
sponding targets. Matrix metalloproteinases (MMPs)
have been suggested to be one of the important targets
for cancer therapy.1,2
2. Inhibitor design
MMPs, a family of calcium- and zinc-dependent endo-
peptidases, have been proved to play crucial roles in
some physiological conditions of arthritis, periodontitis,
heart failure, endothelial cell configuration, tumor pro-
liferation, invasion, angiogenesis, and metastasis.3,4
Importantly, they are also found to be present at high
levels in malignant tumor cells and associated with an
aggressive malignant phenotype and poor prognosis in
cancer patients.5–8 Therefore, moderately manipulating
the over-expression of MMPs may be useful in the con-
trol of cancer at various stages.9
Inhibitors containing zinc-binding groups (ZBGs)
should be designed to inhibit the activity of MMPs. This
class of inhibitors can interact with the zinc ions of zinc-
dependent metalloenzymes and sequentially inhibit the
metastatic spread of tumors and block the processes of
tumor neovascularization.13,14
There are two hydrophobic domains beside the catalytic
activity center of MMPs, called pockets S01 and S20 ,
respectively.15 S10 is the key domain to characterize the
selectivity of various MMPs and appears to be responsi-
ble for much of the observed substrate specificity of a
given MMP.16 Herein, we designed MMPIs with galloyl
functional group to extend into the S01 pocket.
Keywords: Galloyl pyrrolidine derivatives; Gelatinase inhibitor;
MMPI; Anti-tumor agents.
*
Corresponding author. Tel./fax: +86 531 88382264; e-mail:
tjulx2004@sdu.edu.cn
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.031

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X. Li et al. / Bioorg. Med. Chem. 14 (2006) 1287–1293
4-Hydroxyproline is the major specific amino acid of
collagens, which are substrates of MMPs. So, deriva-
tives of 4-hydroxyproline may specifically recognize
and interact with the active sites of MMPs in a compet-
itive manner. On the other hand, gallic acid-bearing
polyphenols have been known to have broad biological
activities, especially anti-tumor and antioxidative prop-
erties.17,18 According to the Ôcombination principlesÕ, if
we link hydroxyproline with gallic acid, the resulting
galloyl pyrrolidine scaffold should be capable of inhibit-
ing the enzymatic activity of gelatinase.
docked with the cavity of S01 pocket by superposing 16
and I52 molecules. We compared the binding modes of
docking R1 group or gallic acid portion into the S10
pocket (Fig. 2). Binding energies of the two interaction
modes were calculated. The lower binding energy of
R1 group indicated that it is reasonable to insert the
R1 group into the S01 pocket. Moreover, the flexible
mobility of R1 could undergo torsional motion to allow
their individual low-energy conformation and well-ori-
ented interaction with the catalytic domain of MMP,
and the bulk of R1 was also required to be moderate
to accommodate the S01 pocket.
3. Chemistry
Although the computed information partially supported
our assumption, the exact binding model of the galloyl
pyrrolidine derivatives with MMP should be obtained
from further X-ray crystal studies.
The synthesis of target compounds was carried out effi-
ciently following the procedures presented in Scheme 1.
It was accomplished upon acylation of 3,4,5-trimethoxy-
benzoic acid with excess SOCl2 (reflux, 3 h) and subse-
quent nucleophilic substitution with the esterified
4-L-hydroxyproline (Py/CH2Cl2, 1.1 equiv, Et3N,
ꢀ5 °C, 3.5 h) to give the key intermediate 4. The use of
sulfonyl chloride for mesylation to provide the desired
product 5 and subsequent SN2 reaction upon treatment
with the azide group (anhydrous DMF, 55 °C, 10 h)
caused the conformational reversal of the C4* chirality
to produce exclusive compound 6. The preparation of
the amino group by catalytic hydrogenation of the azide
(–N3) (5% Pd-C/CaCO3, rt, 10 h) was carried out to af-
ford 7 with the unaltered conformation of C4*, and the
acylation of 7 with various hydrophobic substituents
(halogenated alkylcarbonyl or sulfonyl) (CH2Cl2, Et3N,
2 equiv) to provide the desired compounds 8–17.
5. Structure–activity relationship studies of the galloyl
pyrrolidine series
5.1. Effect of the length of the side chains linked to the
pyrrolidine ring at C4
As demonstrated in Table 1, to some extent, the longer
the R1 the more strongly the compounds inhibit the en-
zyme. The side chain of 16 is much longer than that of 6,
so 16 (IC50 = 2.1) can insert into the S01 pocket and inter-
act with the enzyme preferably.
5.2. Effect of H-bond interactions of the R1 groups with
the enzyme
Considering that the inhibitors designed should have
good ZBG to interact with zinc ions, we prepared sever-
al compounds with hydroximic acid functional group by
treating with NH2OK (CH3OH, rt, 24 h) upon ester ex-
change reaction. Besides, toward the goal of comparing
the inhibitory activity of free phenol hydroxyl group
against gelatinase, compound 16 with free polyphenols
was also prepared to contrast with 15.
The R1 group of 4 (hydroxyl) is too small to occupy the
S01 pocket entirely, thus it cannot form steady H-bond
interactions with the active domain of enzyme. There-
fore, compound 4 cannot chelate with zinc commend-
ably (IC50 > 1000 nM).
The activity of 8 is better than that of 10, which implies
that the sulfonyl amide was better than that of acryl.
This is also because the sulfonyl group is easier to form
hydrogen bonds with the active sites of the enzyme. So,
it is favorable to introduce an appropriate side chain of
R1 that could establish few H-bond interactions with the
active binding sites of the enzyme.
4. Molecular modeling studies
R/S configuration of C4* was optimized with the Powell
Energetic Gradient method built in the Sketch/Build
Edit model (SYBYL6.91,19 Linux 7.3). The predicted
conformation of 5 was comparable with the known
MMPI (marimastat), suggesting affinity to the same spa-
tial regions (S01 and S20 ). As shown in Figure 1, both
the methyl sulfonyl ester group of 5 and the methyl
amide group of marimastat can adjust their flexibility
to extend into the S01 pocket with their preponderant
conformations.
5.3. Effect of the flexibility of the side chain linked to the
pyrrolidine ring at C4
Compounds 11 and 19 showed higher activities, which
might be owing to the flexibility of the hexanyl group,
which is flexible enough to match the 3-D structure of
the enzyme.
5.4. Effect of the ZBG
To further understand the interactions of hydrophobic
R1 group with the S01 pocket, the preferred pharmaco-
phore docking studies were out via the FlexX flexible-
Dock program. An X-ray crystallographic structure of
MMP-2 complexed with a hydroximic acid inhibitor
SC-74020 (I52)20,21 was used as a template, the ligands
Comparing 15 and 16, it is believed that the free phenol
hydroxyl group is favored. An explanation was that the
phenol hydroxyl group as ZBG was easier to bind zinc
ions. As per our initial designing, compounds bearing
hydroxamate groups (18–20) gave the most satisfactory

X. Li et al. / Bioorg. Med. Chem. 14 (2006) 1287–1293
MeO MeO
MeO
1289
HO
HO
O
O
O
a
b
d
OH
MeO
MeO
Cl
OH
MeO
HO
2
3
O
.
H HCl
H
c
N
N
OH
OMe
O
HO
HO
1
f
O
O
O
MeO
MeO
MeO
MeO
MeO
MeO
O
O
O
OMe
OMe
OMe
e
N
MeO
MeO
N
N
4*
4*
4*
MeO
OH
H3CO3SO
N3
6
5
4
O
O
MeO
MeO
MeO
O
MeO
MeO
MeO
R2
O
OMe
NH2
h
i
g
N
N
4*
4*
HN R1
8 - 20
7
8
R1=SO2CH3
R2=OCH3
R2=OCH3
R2=OCH3
R2=OCH3
R2=OCH3
9
R1=SO2C6H4CH3-p
11 R1=CO(CH2)4CH3
13 R1=CO(CH2)2COOH
R2=OCH3
R2=OCH3
R2=OCH3
R2=OCH3
R2=OCH3
R2=NHOH
10 R1=COCH2CH3
12 R1=COC5H4N-3'
14 R1=COCH2C6H5COOH
16 R1=COCH=CHC6H3(OH)2-3',4'
15 R1=COCH=CHC6H3(OMe)2-3',4'
17 R1=COC6H2(OH)3-3',4',5'
19 R1=NHCO(CH2)4CH3
18 R1=COCH=CHC6H3(OMe)2-3',4' R2=NHOH
20 R1=SO2C6H4CH3-p R2=NHOH
Scheme 1. The synthesis of galloyl pyrrolidine derivatives. Reagents and conditions: (a) (CH3)2SO4, NaOH; (b) SOCl2, benzene; (c) MeOH, HCl (g);
(d) dry Py, Et3N; (e) MsCl, Et3N, CH2Cl2; (f) NaN3, DMF; (g) 5% Pd-C/H2, EtOH, rt; (h) Et3N, CH2Cl2, R-Cl (R = alkylcarbonyl or sulfonyl
group); (i) NH2OK, MeOH.
S2’
S2’
S1’
S1’
Compound 5
Marimastat
Figure 1. Comparison of preponderant conformations between compound 5 and marimastat.
inhibitory activities in vitro with IC50 values of 0.9–
6.7 nmol.
6. Conclusions
It can be concluded that the galloyl pyrrolidine deriv-
atives designed are a new class of gelatinase (MMP-2,
-9) inhibitors endowed with significant anti-tumor
activity. Several compounds are interesting as they
display favorable inhibitory potency (inhibitory rate
>35%) and no obvious toxic effects were observed.
Free phenol hydroxyl substituents (16) gave more potent
activity than compounds bearing methoxy substituents
(15), with a 30-fold improvement in gelatinase inhibition.
But it seemed that more phenol hydroxyl substituents did
not result in further enhancement in potency (15 vs 17).

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X. Li et al. / Bioorg. Med. Chem. 14 (2006) 1287–1293
In particular, compound 18 shows the best inhibitory
a
activity in the anti-metastasis assay in vivo, and thus
can be regarded as a lead in further development of
anticancer galloyl pyrrolidine derivatives.
2.25Å
Zn166
7. Materials and methods
7.1. General
Reaction courses and product mixtures were routinely
monitored by thin-layer chromatography (TLC) on sili-
ca gel (precoated GF254 plates) and visualized with
iodine. Flash chromatography was performed using
200–300 mesh silica gel and the solvent system was indi-
cated in the procedure. All solvents were of reagent
grade and, when necessary, were purified and dried by
standard methods. Melting points were determined with
a capillary apparatus with no correction. Infrared spec-
tra were recorded in the range of 4000–600 cmꢀ1 using a
Nicolet Nexus 470FT spectrometer, and KBr disks were
b
Zn166
49Å
1
used as indicated. H NMR spectra were recorded on a
Bruker AM-300 spectrometer, with chemical shifts (d)
given in ppm upfield from Me4Si as internal standard,
and the spectra were recorded in CDCl3 solvents.
Electrospray ionization mass spectrometry (ESI-MS)
was performed on an API 4000 spectrometer.
Figure 2. Predicted binding mode by superposing 16 (color of red-
cyan) and I52 (color of red-orange) when bound to the cavity of the S10
pocket of MMP-2 in the cavity depth (CD) on surface map. (a) R1
group of 16 inserts into the S01 pocket; (b) it is unreasonable to insert
the gallic acid portion into the S01 pocket.
7.2. Chemistry
7.2.1. (2S,4R)-Methyl 4-hydroxy-2-pyrrolidinecarboxyl-
ate hydrochloride (1). The synthesis of compound 1 was
accomplished according to the published procedure.22
Table 1. Structure–activity relationship of the galloyl pyrrolidine derivatives 4–20
O
O
R2
1
N
H3CO
H3CO
2
3
4*
OCH3
R1
Compound
R1
R2
IC50 (nmol)
logP
4*
MR
8.3548
4
5
OH
OSO2CH3
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
NHOH
NHOH
NHOH
>1000
4.14eꢀ002
S
S
S
S
S
S
S
S
S
R
R
R
R
R
R
R
R
541.4 22.6
162.3 11.5
nda
ꢀ0.4158
9.6911
9.1304
a
6
N3
NH2
7
nda
8. 9571
9.9067
8
NHSO2CH3
NHSO2C6H4CH3-p
NHCOCH2CH3
NHCO(CH2)4CH3
NHCOC5H4N-30
NHCO(CH2)2CO2H
NHCOCH2C6H5CO2H
NHMc
262.5 17.6
113.1 13.4
451.7 23.5
15.7 1.5
71.9 12.3
76.1 9.8
103.4 13.4
62.0 5.5
2.1 0.4
0.5908
2.332
9
12.4179
9.9975
10
11
12
13
14
15
16
17
18
19
20
0.2467
1.4986
0.1534
ꢀ0.7391
1.4348
1.579
11.3889
11.3700
10.6501
12.0449
14.0211
13.0935
12.0404
11.9498
11.2938
12.3267
NHCa
NHGa
1.0528
0.322
49.3 5.3
0.9 0.2
7.7 0.4
NHMc
NHCO(CH2)4CH3
NHSO2C6H4CH3-p
0.6875
0.7513
1.4258
6.7 0.17
Mc, COCH@CHC6H3(OCH3)2-30,40; Ca, COCH@CHC6H3(OH)2-30,40; Ga, COC6H2(OH)3-30,40,50; 4* is the chiral center; MR was determined by
the Chem3D Ultra 7.0 program.
a nd, not determined.

X. Li et al. / Bioorg. Med. Chem. 14 (2006) 1287–1293
1291
7.2.2. 3,4,5-Trimethoxybenzoic acid (2). To a stirred
solution of gallic acid (5 g, 29.4 mmol) in 50 mL of
4 N NaOH was added (CH3)2SO4 (8.9 g, 71 mmol)
dropwise, keeping the inside temperature under 20 °C.
The solution was allowed to stir at 30–40 °C for
20 min, another gallic acid (5 g) and 50 mL of 4 N
NaOH were successively added. The resulting mixture
was slowly heated to 90 °C and maintained at this tem-
perature for 1 h. After refluxing for another 2 h, the
reaction mixture was allowed to cool to rt. The solution
was acidified with 2 N HCl to pH 2, filtered, and the
solid was washed with water. The crude product was
recrystallized with 50% EtOH to give compound 2
(40.8 g, 65%) as a white needle crystal: mp 168–171 °C.
1H NMR d 3.97 (s, 9H), 7.23 (s, 2H), 12.11 (s, 1H).
ed in one portion. Then the reaction mixture was heated
at 55 °C under nitrogen atmosphere for 10 h. After the
reaction mixture was cooled to 23 °C, it was partitioned
between ethyl acetate (50 mL) and water (20 mL). The
organic portion was washed with water (10 mL) and
brine (10 mL), dried over anhydrous MgSO4, filtered,
and concentrated. The residue oil was purified by flash
chromatography (hexane/ethyl acetate 5:1 to 3:1) to af-
ford compound 6 (2.3 g, 63.2%) as a pale yellow oil. IR
(KBr): m 2937 (CH3), 2103.45 (N3), 1727 (C@O), 1644
1
and 1586, 1513 (C@C). H NMR d 1.92–1.95 (m, 1H),
2.25–2.26 (m, 2H), 3.47–3.50 (m, 2H), 3.88 (s, 9H), 4.22
(s, 1H), 4.501 (m, 1H), 4.62 (t, J = 2.8 Hz, 1H), 7.24 (s,
2H). ESI-MS: m/z (rel intensity) 363.1.
7.2.7. (2S,4S)-Methyl-4-amino-1-(3,4,5-trimethoxyben-
zoyl)pyrrolidine-2-carboxylate (7). Compound 6 (11 g,
30 mmol) in EtOH (120 mL) was hydrogenated over 5%
palladium on calcium carbonate (3 g, 30 mmol) at rt un-
der an atmospheric pressure of hydrogen for 10 h. The
reaction mixture was filtered through a Celite pad, and
the filtrate was concentrated in vacuo to give the crude
product as a brown oil, which was purified by flash chro-
matography (chloroform/ethanol 4:1 to 1:4), to afford 7
(2.32 g, 60.8%) as a sheet crystal: mp 179–180 °C. IR
(KBr): m 3345.94, 3257.87 (NH2), 2936 (CH3), 1726,
7.2.3. 3,4,5-Trimethoxybenzoic chloride (3). To a stirred
solution of 2 (21.2 g, 100 mmol) in benzene (320 mL)
was added dropwise SOCl2 (40 mL, 548 mmol). The
reaction mixture was refluxed for 3 h, and the solvent
was evaporated to give 3,4,5-trimethoxybenzoic chloride
as a pale yellow oil.
7.2.4.
(2S,4R)-Methyl-4-hydroxy-1-(3,4,5-trimethoxy-
benzoyl)pyrrolidine-2-carboxylate (4). A mixture of com-
pound 1 (1.85 g, 11 mmol) and Et3N (3 mL, 22 mmol) in
pyridine (20 mL) was stirred at rt for 20 min and filtered.
The filtrate was cooled to ꢀ5 °C, and then was added
dropwise a solution of 3 (2.30 g, 10 mmol) in CH2Cl2
(10 mL). After the mixture was stirred at ꢀ5 °C for
3.5 h, the white precipitate was removed and concentrat-
ed to dryness. The residue was purified by flash chroma-
tography (petroleum ether/ethyl acetate 4:1 to 1:4) to
give 4 (2.7 g, 79.6%) as a pale yellow solid: mp 139.2–
1
1641 (C@O) and 1590, 1514 (C@C). H NMR d 2.04–
2.06 (m, 1H), 2.56 (m, 1H), 3.23 (s, 3H), 3.86 (s, 9H),
4.12 (m, 1H), 4.57 (d, J = 7.0 Hz, 2H), 4.80–4.90 (b,
2H), 6.91 (s, 2H). ESI-MS: m/z (rel intensity) 339.4.
7.2.8.
(2S,4S)-Methyl-4-(methylsulfonamido)-1-(3,4,5-
trimethoxybenzoyl)pyrrolidine-2-carboxylate (8). Com-
pound 7 (338 mg, 1 mmol) was dissolved in anhydrous
CH2Cl2 (2 mL) and Et3N (0.5 mL) under an atmospheric
pressure of nitrogen. A solution of methanesulfonyl chlo-
ride (236 mg, 2 mmol) in 2 mL CH2Cl2 was added drop-
wise at 0 °C. The resulting solution was stirred at rt for
another 3 h. The reaction mixture was partitioned be-
tween CH2Cl2 and water, the organic portion was washed
successively with 1% HCl, 5% Na2CO3, and water, dried
over anhydrous MgSO4, filtered, and concentrated in vac-
uum to give the crude product as a yellow solid, which was
purified by flash chromatography (petroleum ether/
EtOAc 4:1 to 1:4) to give compound 8 (301 mg, 72.4%)
as white crystal: mp 52.5–54.5 °C. 1H NMR d 1.26–1.36
(m, 1H), 2.09–2.12 (m, 1H), 2.96 (s, 3H), 3.86 (s, 3H),
3.89 (s, 9H), 3.92–3.96 (m, 2H), 4.11–4.17 (m, 1H), 4.64
(s, 1H), 6.76 (s, 2H). ESI-MS: m/z (rel intensity) 415.0.
1
140.4 °C. H NMR d 2.03–2.16 (m, 2H), 3.55–3.60 (m,
1H), 3.77 (s, 3H), 3.81–3.91 (m, 2H), 3.85 (m, 9H),
3.97 (s, 1H), 4.50 (m, 1H), 4.81 (t, J = 7.8 Hz, 1H),
6.79 (s, 2H). ESI-MS: m/z (rel intensity) 338.5.
7.2.5. (2S,4R)-Methyl-4-(methoxysulfonyloxy)-1-(3,4,5-
trimethoxybenzoyl)pyrrolidine-2-carboxylate (5). Com-
pound 4 (3.39 g, 10 mmol) was dissolved in CH2Cl2
(5 mL) at 0 °C under nitrogen atmosphere. Et3N
(4.5 mL, 32 mmol) and methanesulfonyl chloride
(1.26 g, 11 mmol) were successively added. The mixture
was stirred at rt for another 4 h. The solution was parti-
tioned between ethyl acetate (50 mL) and water
(20 mL). The organic phase was washed with saturated
NaHCO3 solution (15 mL), water (10 mL), and brine
(10 mL), and dried over anhydrous MgSO4. The solvent
was removed in vacuo to give the crude product as a pale
yellow oil, which was purified by flash chromatography
(petroleum ether/ethyl acetate, 4:1 to 1:2) to afford 8
7.2.9. (2S,4S)-Methyl-4-(4-methylphenylsulfonamido)-1-
(3,4,5-trimethoxybenzoyl)pyrrolidine-2-carboxylate (9).
1
Yield 69.3%, mp 68.8–70.3 °C. H NMR d 1.82–1.85
1
(3.5 g, 83.9%) as a white crystal: mp 156.7–158.9 °C. H
(m, 2H), 2.39 (s, 3H), 3.52 (d, J = 10.5 Hz, 2H), 3.80
(s, 3H), 3.85–3.86 (m, 9H), 3.92–4.00 (m, 1H), 4.49
(1H), 6.74 (s, 2H), 7.23 (d, J = 7.5 Hz, 2H), 7.68 (d,
J = 7.5 Hz, 2H). ESI-MS: m/z (rel intensity) 491.6.
NMR d 2.33 (m, 1H), 2.02–2.04 (m, 1H), 3.03 (s, 3H),
3.81 (s, 3H), 3.86–3.87 (m, 9H), 3.91–4.11 (m, 2H), 4.83
(d, J = 6.6 Hz, 1H), 5.29 (m, 1H), 6.78 (s, 2H). ESI-MS:
m/z (rel intensity) 415.8.
7.2.10.
(2S,4S)-Methyl-4-propionamido-1-(3,4,5-tri-
7.2.6.
benzoyl)pyrrolidine-2-carboxylate (6). Compound
(4.17 g, 10 mmol) was dissolved in anhydrous DMF
(15 mL) and ground NaN3 (695 mg, 10.7 mmol) was add-
(2S,4S)-Methyl-4-azido-1-(3,4,5-trimethoxy-
5
methoxybenzoyl)pyrrolidine-2-carboxylate (10). Yield
75.7%, mp 142.5–143.9 °C. 1H NMR d 1.08 (t,
J = 7.5 Hz, 3H), 2.13 (q, J = 7.5 Hz, 2H), 2.51–2.56
(m, 2H), 3.56 (t, J = 6.9 Hz, 1H), 3.83 (s, 9H), 3.88–

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X. Li et al. / Bioorg. Med. Chem. 14 (2006) 1287–1293
3.91 (m, 1H), 4.62 (t, J = 8.7 Hz, 2H), 6.77 (s, 2H), 6.87
(d, J = 6.9 Hz, 1H). ESI-MS: m/z (rel intensity) 393.5.
2.06 (m, 1H), 2.55–2.60 (m, 1H), 3.29 (s, 3H), 3.44–
3.53 (m, 2H), 3.70 (s, 3H), 3.80 (s, 6H), 3.85–3.87
(m, 2H), 6.76 (s, 1H), 6.80 (s, 1H). ESI-MS: m/z (rel
intensity) 489.6.
7.2.11.
(2S,4S)-Methyl-4-hexanamido-1-(3,4,5-tri-
methoxybenzoyl)pyrrolidine-2-carboxylate (11). Yield
62.3%, mp 72.5–74.6 °C. 1H NMR d 0.85 (t, J = 7.2 Hz,
3H), 1.22–1.32 (m, 4H), 1.52–1.62 (m, 2H), 1.93–2.04
(m, 2H), 2.12 (t, J = 4.8 Hz, 2H), 3.59 (t, J = 8.1 Hz,
2H), 3.85 (s, 9H), 4.60–4.69 (m, 2H), 6.74 (s, 2H), 6.86
(d, J = 6.0 Hz, 1H). ESI-MS: m/z (rel intensity) 435.5.
7.2.17. (2S,4S)-4-(3-(3,4-Dimethoxyphenyl)acrylamido)-
N-hydroxy-1-(3,4,5-trimethoxybenzoyl)pyrrolidine-2-car-
boxamide (18). Compound 15 (858 mg, 2 mmol) was
dissolved in anhydrous MeOH (7 mL) and a solution
of NH2OK in MeOH (1.5 mL) was added. The result-
ing solution was stirred at rt for 24 h, then 1.5 g silica
gel was added and evaporated to give a pale yellow
powder, which was purified by flash chromatography
(CH2Cl2/MeOH 50:1 to 1:50) to afford the target com-
pound 21 (537 mg, 50.8%) as a pale yellow crystal,
which appeared red in FeCl3. 1H NMR d 1.82–1.90
(m, 2H), 3.30 (s, 1H), 3.69–3.80 (15H), 4.26 (s, 1H),
4.38 (s, 1H), 6.43 (d, J = 15.6 Hz, 2H), 6.82 (s, 2H),
6.96 (d, J = 8.4 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H),
7.13 (s, 1H), 7.33 (d, J = 15.6 Hz, 2H), ESI-MS: m/z
(rel intensity) 528.3.
7.2.12.
(2S,4S)-Methyl-4-(nicotinamido)-1-(3,4,5-tri-
methoxybenzoyl)pyrrolidine-2-carboxylate (12). Yield
72.9%, mp 55.0–57.1 °C. 1H NMR d 2.03–2.30 (m,
2H), 3.79 (s, 3H), 3.83–3.84 (m, 9H), 3.93–4.08 (m,
1H), 4.66 (d, J = 7.8 Hz, 1H), 4.97 (s, 1H), 6.74 (s,
2H), 7.40 (dd, J = 4.5, 7.8 Hz, 1H), 8.03 (d, J = 7.8 Hz,
1H), 8.24 (d, J = 6.0 Hz, 1H), 8.72 (d, J = 4.5 Hz, 1H),
9.06 (s, 1H). ESI-MS: m/z (rel intensity) 442.5.
7.2.13.
methoxybenzoyl)pyrrolidin-3-ylamino)propanoic
3-((3S,5S)-5-(Methoxycarbonyl)-1-(3,4,5-tri-
acid
1
(13). Yield 70.0%, mp 72.5–75.5 °C. H NMR d 1.97–
2.04 (m, 2H), 2.45 (t, J = 6.3 Hz, 2H), 2.65 (t,
J = 6.3 Hz, 2H), 3.83 (s, 3H), 3.84–3.86 (m, 9H), 3.91–
3.97 (m, 2H), 4.66 (m, 2H), 6.76 (s, 2H), 7.13 (s, 1H).
ESI-MS: m/z (rel intensity) 437.5.
7.2.18. (2S,4S)-4-Hexanamido-N-hydroxy-1-(3,4,5-tri-
methoxybenzoyl)pyrrolidine-2-carboxamide (19). Yield
53.6%, mp 183.3–185.3 °C. 1H NMR d 0.82 (t,
J = 3.9 Hz, 3H), 1.21–1.27 (m, 4H), 1.41–1.50 (m,
2H), 1.71–1.78 (m, 1H), 2.02 (t, J = 4.5 Hz, 2H),
2.41–2.46 (m, 1H), 2.49–2.50 (m, 2H), 3.69 (s, 3H),
3.79 (s, 6H), 4.12 (s, 1H), 4.31–4.33 (m, 1H), 6.79
(s, 1H), 8.05 (d, J = 5.4 Hz, 1H). ESI-MS: m/z (rel
intensity) 436.4.
7.2.14. 4-(2-((3S,5S)-5-(Methoxycarbonyl)-1-(3,4,5-tri-
methoxybenzoyl)pyrrolidin-3-ylamino)-2-oxoethyl)benzo-
icacid(14).Yield62.3%, mp73.0–74.5 °C. 1HNMRd2.50
(m, 2H), 3.48 (s, 1H), 4.59 (m, 2H), 5.27 (s, 1H), 6.71 (s,
1H), 7.22–7.31 (m, 5H). ESI-MS: m/z (rel intensity) 455.6.
7.2.19. (2S,4S)-N-Hydroxy-4-(4-methylphenylsulfonami-
do)-1-(3,4,5-trimethoxybenzoyl)pyrrolidine-2-carboxam-
ide (20). Yield 55.3%, mp 101.5–104.5 °C. 1H NMR
d 2.05–2.27 (m, 2H), 3.61 (m, 1H), 3.78 (s, 3H),
3.81 (s, 6H), 3.88–3.93 (m, 2H), 4.31 (t, J = 5.8 Hz,
1H), 6.55 (d, J = 15.3 Hz, 1H), 6.97 (d, J = 7.8 Hz,
1H), 7.19 (d, J = 7.8 Hz, 1H), 7.30 (s, 1H), 7.35
(d, J = 15.3 Hz, 1H), 7.50 (d, J = 8.1 Hz, 2H), 7.80
(d, J = 8.1 Hz, 2H). ESI-MS: m/z (rel intensity)
488.4.
7.2.15. (2S,4S)-Methyl-4-(3-(3,4-dimethoxyphenyl)acryl-
amido)-1-(3,4,5-trimethoxybenzoyl)pyrrolidine-2-carbox-
1
ylate (15). Yield 68.9%, mp 176.5–177.0 °C. H NMR d
2.56–2.66 (m, 2H), 3.61–3.74 (m, 2H), 3.84–3.94 (m,
14H), 6.22 (d, J = 15.3 Hz, 1H), 6.76 (s, 1H), 6.85 (d,
J = 8.4 Hz, 1H), 7.02 (d, J = 1.8 Hz, 1H), 7.08 (dd,
J = 1.8, 8.4 Hz, 1H), 7.13 (d, J = 8.4 Hz), 7.54 (d,
J = 15.3 Hz, 1H). ESI-MS: m/z (rel intensity) 527.8.
(2S,4S)-Methyl-4-(3-(3,4-dihydroxyphenyl)acrylamido)-
1-(3,4,5-trimethoxybenzoyl)pyrrolidine-2-carboxylate (16).
The same procedure, as described for the synthesis of 8,
was followed by using (E)-3-[3,4-di(acetyloxy)phenyl]-2-
propanoic chloride instead of methanesulfonyl chloride.
The crude product was purified by flash chromatogra-
phy (petroleum ether/EtOAc/acetic acid 30:10:1 to
10:30:1) to give the target compound 16 (58.5%) as a
white crystal, which appeared blue in FeCl3 and Fe
7.3. In vitro gelatinase inhibitory assay
The galloyl pyrrolidine derivatives were submitted for
the assessment of the inhibition against gelatinase
(MMP-2, -9) in vitro in a 96-well microtiter plate using
L-leucyl-p-nitroaniline (L-Leu-pNA) as a substrate,
which was synthesized according to the method of
Vijaykumar et al.23 Inhibitions were evaluated with
IC50 values (Table 1). The gelatinase (sigma) was a mix-
ture of gelatinase A (MMP-2) and gelatinase B (MMP-
9), which are both closely correlated with the tumor. So
the mixture was directly used in the in vitro test without
separation (see Table 2).
1
(SCN)3. mp 137.7–141.3 °C. H NMR d 1.58 (s, 3H),
2.05–2.55 (m, 2H), 2.89 (s, 1H), 2.96 (s, 1H), 3.45–3.52
(m, 1H), 3.81 (s, 3H), 3.88 (s, 6H), 3.92–3.95 (m, 1H),
4.09–4.16 (m, 1H), 6.23 (d, J = 15.3 Hz, 1H), 6.69 (s,
1H), 6.84 (d, J = 8.1 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H),
7.46 (d, J = 15.3 Hz, 1H), 8.02 (s, 1H). ESI-MS: m/z
(rel intensity) 499.5.
The gelatinase, substrate, and inhibitors were dissolved
in sodium borate buffer (pH 8.5, 50 mM) and incubated
for 30 min at 37 °C. Then 0.03% 2,4,6-trinitrobenzene
sulfonic acid (TNBS, 50 lL) was added and incubated
at 37 °C for another 20 min. Absorbance at 450 nm
was measured.
7.2.16. (2S,4S)-Methyl-4-(3,4,5-trihydroxybenzamido)-1-
(3,4,5-trimethoxybenzoyl)pyrrolidine-2-carboxylate (17).
Yield 47.1%, mp 115.6–117.8 °C. 1H NMR d 2.02–

X. Li et al. / Bioorg. Med. Chem. 14 (2006) 1287–1293
Table 2. Anti-tumor activity assay of galloyl pyrrolidine derivatives
1293
Compound
Mice (n)
Body weight (g)
Lung weight (g)
Metastasized nodes on lung surface (n)
Inhibitory rate (%)
Control
10
23.50 3.42
18.08 3.27
21.36 2.42
21.83 1.79
23.17 1.16
22.58 4.25
17.54 3.12
22.90 3.74
20.00 2.62
0.168 0.021
0.165 0.013
0.155 0.051
0.154 0.012
0.150 0.042
0.164 0.022
0.168 0.080
0.174 0.052
0.157 0.089
46.5 2.12
27.1 9.34
17.9 0.96
25.3 8.32
19.5 0.96
28.0 1.17
28.7 9.63
19.8 5.43
17.5 5.68
8 (100 mg/kg/day)
12 (100 mg/kg/day)
13 (100 mg/kg/day)
14 (100 mg/kg/day)
15 (100 mg/kg/day)
16 (100 mg/kg/day)
18 (50 mg/kg/day)
19 (50 mg/kg/day)
9 (10)a
9 (10)a
10
41.72
61.51
45.66
58.06
39.78
38.28
57.42
62.37
10
10
10
10
9 (10)a
a The animal number in parentheses is the original number, logP was determined by the Chem 3D Ultra 7.0 program.
5. Sounni, N. E.; Janssen, M.; Foidart, J. M. Matrix Biol.
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A further assay in vivo was performed in the anti-metas-
tasis model of mice bearing H22 carcinoma cells to eval-
uate anti-tumor activities of the compounds.
Suspension of inhibitors was prepared by homogenizing
the tested compounds in excipient (0.5% sodium carb-
oxymethyl cellulose solution, 0.9% benzyl alcohol, and
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animals of the control group were treated with the same
volume of excipient, while the other groups were given
the inhibitors by oral administration, at a dose of 50
(hydroxamates) or 100 mg/kg/day (carboxylates), 6
days/week for 2 weeks. The mice were then weighed
and sacrificed for autopsy immediately. The lungs with
tumor nodes were removed, weighed, and then placed
in bouin stationary solution (saturated 2,4,6-trinitrophe-
nol solution/formaldehyde/glacial acetic acid = 15:5:1).
One day later, the metastasized nodes on the surface
of lungs were counted.
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Acknowledgments
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We thank the financial support from Natural Science
Foundation of China (Grant No. 20272033) and the
Doctoral Foundation of Ministry of Education of the
PeopleÕs Republic of China (Grant No. 20020422024).
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