Design, synthesis, and preliminary evaluation of substituted cinnamic acid esters as selective matrix metalloproteinase inhibitors

Article abstract of DOI:10.1002/ddr.21015

Strategy, Management and Health Policy Preclinical Research Substituted cinnamic acid esters with extended P1' groups were synthesized using microwave irradiation and tested for their inhibitory activities on matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9. Preliminary structure-activity relationship analysis and docking studies showed that hydroxyl groups in the benzene ring and the presence of extended spatial structures in the carboxylic acid played key roles in the MMP-2 and MMP-9 inhibitory activity and selectivity over MMP-1.

Full text of DOI:10.1002/ddr.21015

DRUG DEVELOPMENT RESEARCH •• : ••–•• (2012)
Research Article
Design, Synthesis, and Preliminary Evaluation of
Substituted Cinnamic Acid Esters as Selective Matrix
Metalloproteinase Inhibitors
Zhi-Hao Shi,1,2* Nian-Guang Li,2,3* Qian-Ping Shi,2 Hao-Tang,2 and Yu-Ping Tang2*
1Department of Organic Chemistry, China Pharmaceutical University, Nanjing, Jiangsu 211198,
China
2Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of
Chinese Medicine, Nanjing, Jiangsu 210046, China
3Department of Medicinal Chemistry, Nanjing University of Chinese Medicine, Nanjing, Jiangsu
210046, China
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Technology,
Genomics,
Proteomics
Preclinical
Research
Toxicology, Formulation
Drug Delivery,
Pharmacokinetics
Phases I-III
Regulatory, Quality,
Manufacturing
Postmarketing
Phase IV
ABSTRACT
Substituted cinnamic acid esters with extended P1′ groups were synthesized using micro-
wave irradiation and tested for their inhibitory activities on matrix metalloproteinase (MMP)-1, MMP-2,
and MMP-9. Preliminary structure–activity relationship analysis and docking studies showed that hydroxyl
groups in the benzene ring and the presence of extended spatial structures in the carboxylic acid played
key roles in the MMP-2 and MMP-9 inhibitory activity and selectivity over MMP-1. Drug Dev Res •• : ••–
••, 2012.
© 2012 Wiley Periodicals, Inc.
Key words: matrix metalloproteinases; tumor; caffeic acid; structure–activity relationship
INTRODUCTION
cellular matrix from both tumor cells and surrounding
stromal cells that are stimulated by the tumor. MMP-2
(gelatinase A) and MMP-9 (gelatinase B) appear to play
a key role in these processes [Crabbe et al., 1994; Aimes
and Quigley, 1995].
In the past few years, some potent “broad spec-
trum” MMP inhibitors (MMPIs) have been tested
against tumors [Ray and Stetler-Stevenson, 1996] with
some entering clinical trials. None is yet on the market.
Matrix metalloproteinases (MMPs) are a family of
zinc-dependent endopeptidases involved in the break-
down of components of the extracellular matrix that
facilitates connective tissue remodeling [Nagase and
Woessner, 1999]. This process is important in embry-
onic development, pregnancy, growth, and wound
healing. Normally, MMP activity is tightly controlled
by the balance between synthesis of active MMPs and
the presence of endogenous inhibitors, e.g., the tissue
inhibitor of metalloproteinases [Nagase and Woessner,
1999].
*Correspondence to: Nian-Guang Li and Yu-Ping Tang,
Jiangsu Key Laboratory for High Technology Research of TCM
This balance is lost with tumor progression, where
an increased expression of certain MMPs accompanies Formulae, Nanjing University of Chinese Medicine, 138 Xianlin
Road, Nanjing, Jiangsu 210046, China.
E-mail: linianguang@njutcm.edu.cn; yupingtang@njutcm.edu.cn
the passage from a benign to a malignant phenotype
that is believed to be involved in metastatic tumor dis-
Received 4 June 2012; Accepted 16 June 2012
persion and angiogenesis [MacDougall and Matrisian,
1995; Stetler-Stevenson, 1999]. The MMPs are pro-
Published online in Wiley Online Library (wileyonlinelibrary.
duced and secreted as inactive zymogens in the extra- com). DOI: 10.1002/ddr.21015
© 2012 Wiley Periodicals, Inc.

2
SHI ET AL.
smaller P1′ groups generally leads to broad-spectrum
inhibition.
This prompted us to find new caffeic acid deriva-
O
S
N
O
O
O
O
O
H
HS
N
HO
O
N
N
N
tives as selective MMP-2 and MMP-9 inhibitors. First,
our docking studies of caffeic acid with MMP-2 and
MMP-9 showed that carboxylic acid could reacted with
the S1′ pocket in MMP-2 and MMP-9 to achieve
enhanced inhibitory activities and selectivity over
MMP-1 (Fig. 2). Esters of caffeic acid with extended
P1′ group (such as branched alkanes and cycloalkanes)
improved the inhibitory activity and selectivity. Fur-
H
H
H
O
O
N
N
BMS-275291 (2)
CGS-27023A (1)
Fig. 1. Structures of CGS-27023A (1) and BMS-275291 (2).
One problem was the design of the trials themselves. thermore, in order to investigate the MMPs inhibitory
MMPIs had been successful in animal models of early- effects of the substitution on the benzene ring of caffeic
stage disease but were only tested in late-stage disease acid, its two natural analogs ferulic acid and cinnamic
in humans [Overall and López-Otín, 2002]. Since they acid were also esterified with an extended P1′ group,
were developed as cytostatic agents, they may be used based on their inhibitory activities on MMP-1, MMP-2,
in a prolonged therapy, and therefore, their bioavail- and MMP-9; structure–activity relationship (SAR)
ability and slow long-term toxicity are key [Hodgson, analysis was conducted.
1995]. Another problem was the lack of selectivity with
compounds such as CGS-27023A (1) (Ciba Geigy) and
MATERIALS AND METHODS
BMS-275291 (2) (Bristol-Myers Squibb) (Fig. 1) show-
Synthesis
ing a severe musculoskeletal syndrome, with fibropro-
liferative effects in the joint capsule of the knees
Reagents and solvents were purchased from com-
[Hutchinson et al., 1998; Holmbeck et al., 1999; mercial sources and used without further purification
Steward, 1999]. These effects are thought to occur via unless otherwise specified. Air- and moisture-sensitive
impairment of normal tissue remodeling controlled by liquids and solutions were transferred via syringe or
MMP-1 [Dahlberg et al., 2000]. A lack of activity with stainless steel cannula. Organic solutions were concen-
respect to MMP-1 is considered to be an important trated by rotary evaporation below 45°C at approxi-
factor in reducing some of the side effects found for mately 20 mmHg. All nonaqueous reactions were
“nonselective” MMPIs [Scatena, 2000].
carried out under anhydrous conditions using flame-
In light of these findings, considerable efforts dried glassware within an argon atmosphere in dry,
have been directed to develop a more selective second freshly distilled solvents, unless otherwise noted. Yields
generation of inhibitors against the specific MMPs referred to chromatographically, unless otherwise
believed to be involved in the different pathologies [Li stated. Reactions were monitored by thin-layer chroma-
et al., 2009a]. MMPIs may also be derived from natural tography carried out on 0.15~0.20 mm Yantai silica
resources, e.g., herbs, plants, fruits, and other agricul- gel plates (RSGF 254) (Yantai Chemical Industry
ture products.
Research Institute, Yantai, Shandong Province, China)
Caffeic acid (3) (Fig. 2), which is found in fruits, using ultraviolet (UV) light as the visualizing agent.
vegetables, wine, olive oil, and coffee [Park et al., 2005], Chromatography was performed on Qingdao silica gel
inhibits activity of MMP-9 (half maximal (50%) inhibi- (160~200 mesh) (Qingdao Banke separation materials
tory concentration IC50 = 10–20 nM), but its selectivity Company Limited, Qingdao, Shandong Province,
for MMP-2 and MMP-9 over MMP-1 was not high China) using petroleum ether (60~90) and ethyl acetate
[Chung et al., 2004].
as the eluting solvent (Sinopharm Chemical Reagent
Selectivity could potentially be achieved by taking Company Limited, Shanghai, China).
advantage of differences in size of the S1′ pocket among
1H NMR spectra were obtained using a Bruker
the various MMPs. From X-ray crystallographic, AV-300 (300 MHz) (Bruker Corporation, Germany).
nuclear magnetic resonance (NMR) analysis, and Chemical shifts were recorded in parts per million
homology modeling, MMPs can be classified into two (ppm) downfield from tetramethylsilane. J-values are
broad structural classes: those with a relatively deep given in Hertz (Hz). Abbreviations used were for singlet
S1′ pocket (MMP-2, 3, 8, 9, and 13) and those with (s), doublet (d), triplet (t), quartet (q), broad (b), and
a shallow S1′ pocket (MMP-1 and 7) [Gooley et al., multiplet (m). Electrospray ionisation tandem mass
1994; Lovejoy et al., 1994; Verma and Hansch, 2007]. spectrometry (ESI-MS) spectra were recorded on a
Consequently, incorporation of an extended P1′ group Waters Synapt high definition mass spectrometry
leads to selective inhibition, whereas the presence of (HDMS) spectrometer.
Drug Dev. Res.

SELECTIVE MATRIX METALLOPROTEINASE INHIBITORS
3
O
O
R2
HO
HO
OP1'
OH
R1
Caffeic acid (3)
Docking studies
Design program
S1'
S1'
Docking result of caffeic acid into MMP-2 Docking result of caffeic acid into MMP-9
Fig. 2. The structures of caffeic acid, docking studies, and the design program. [Color figure can be viewed in the online issue which is
available at wileyonlinelibrary.com]
O
O
R3OH, H2SO4
R2
R1
R3
R2
R1
O
OH
Microwave irradiation
1-16
Fig. 3. Synthesis of compounds 4–18 under microwave irradiation.
Cinnamic acid esters 4–8, ferulic acid esters 9–13 compounds 4–18 were obtained in higher yields (>90%)
and caffeic acid esters 14–18 were prepared as shown in (Table 1).
Figure 3. Although these compounds could be synthe-
sized with the acid directly refluxed with alcohols in
EXPERIMENTS
the presence of various catalysts including sulfuric
General Procedures for the Esterification of
Substituted Cinnamic Acid Esters under
acid, hydrogen chloride, boron trifluoride, aluminum
chloride, trifluoroacetic anhydride, polyphosphate ester,
Microwave Irradiation
neodymium oxide, dicyclohexylcarbodiimide, graphite
bisulfate, etc [Olah et al., 1978; Li, 1999; Zhang, 1999],
To a stirring mixture of substituted cinnamic
the disadvantages of using these catalysts include acid (5 mmol) in alcohol (10 ml) was added the con-
long-reaction time, low yield, and expensive reagents. centrated sulfuric acid (0.067 ml, 1.25 mmol), and
In recent years, reactions using microwave irradia- the reaction mixture was refluxed for 3–6 min in a
tion were, in general, not only faster compared with sealed reaction vessel (Discover CEM, Matthews,
the conventional heating reactions but also poten- North Carolina, USA) under microwave irradiation,
tially more efficient, cleaner, and safer [Kappe, 2004]. where the power was set at 200 W, the temperature was
Further improvements offer enhanced reaction rates, set at 10°C above the boiling point of the respective
higher yields, and greater selectivity for the targeted alcohol, and the pounds per square inch (PSI) was set at
product under milder reaction conditions [Li et al., 180. After cooling to 25°C, ethyl acetate was added and
2009b]. A highly efficient synthesis of compounds the solution washed with water and brine. The ethyl
4–18 under microwave irradiation was adopted and acetate layer was dried over MgSO4, filtered, and con-
implemented with reactions times ranging from centrated under reduced pressure. The residue was
3–6 min (Table 1) methods [Nagaoka et al., 2002] and purified by silica gel column chromatography using
Drug Dev. Res.

4
SHI ET AL.
TABLE 1. The Structures, Reaction Temperature, Reaction Time, and Yields of Compounds 4–18 under Microwave Irradiation
Product
R1
R2
R3
Temp. (°C)
Time (min)
Yield (%)
4
5
6
7
8
H
H
H
H
H
H
H
H
CH(CH3)2
92
118
142
150
171
92
118
142
150
171
92
3
3
4
5
5
4
4
5
6
6
4
4
5
6
6
94
94
93
92
91
93
93
91
91
90
93
92
91
90
90
CH2CH(CH3)2
CH2CH2CH(CH3)2
CH(CH2)4
CH(CH2)5
CH(CH3)2
CH2CH(CH3)2
CH2CH2CH(CH3)2
CH(CH2)4
CH(CH2)5
CH(CH3)2
H
H
9
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
OH
10
11
12
13
14
15
16
17
18
CH2CH(CH3)2
CH2CH2CH(CH3)2
CH(CH2)4
118
142
150
171
CH(CH2)5
20% ethyl acetate in petroleum ether to afford the sub-
stituted cinnamic esters.
6.91 (d, J = 8.0 Hz, 1H), 7.03 (m, 2H), 7.60 (d,
J = 15.9 Hz, 1H); ESI-MS m/z: 223 [M + H]+, 245
[M + Na]+.
Isopropyl cinnamate (4) [Maki et al., 2009]: yield
94%; 1H NMR (CDCl3, AV-300), d: 1.31(d, J =
6.2 Hz, 6H), 4.12(m, 1H), 6.42(d, J = 16.1 Hz, 1H),
7.37(m, 3H), 7.52(m, 2H), 7.66(d, J = 16.1 Hz, 1H);
ESI-MS m/z: 191 [M + H]+, 213 [M + Na]+.
Isobutyl cinnamate (5) [Tani et al., 2004]: yield
94%; 1H NMR (CDCl3, AV-300), d: 0.91(m, 6H),
1.71(m, 2H), 3.91(m, 1H), 6.46(d, J = 16.0 Hz, 1H),
7.37(m, 3H), 7.52(m, 2H), 7.68(d, J = 16.0 Hz, 1H);
ESI-MS m/z: 205 [M + H]+, 227 [M + Na]+.
Isopentyl cinnamate (6) [Narasimhana et al., 2004]:
yield 93%; 1H NMR (CDCl3, AV-300), d: 0.92(m,
6H), 1.57(m, 2H), 1.70(m, 1H), 4.19 (m, 2H), 6.47(d,
J = 16.0 Hz, 1H), 7.36(m, 3H), 7.53(m, 2H), 7.67(d,
J = 16.0 Hz, 1H); ESI-MS m/z: 205 [M + H]+, 227
[M + Na]+.
Cyclopentyl cinnamate (7): yield 92%; 1H NMR
(CDCl3, AV-300), d: 1.61(m, 4H), 1.92(m, 4H),
3.93(m, 1H), 6.41(d, J = 15.9 Hz, 1H), 7.37(m, 3H),
7.51(m, 2H), 7.65(d, J = 15.9 Hz, 1H); ESI-MS m/z:
217 [M + H]+, 239 [M + Na]+.
Cyclohexyl cinnamate (8) [Sova et al., 2006]: yield
91%; 1H NMR (CDCl3, AV-300), d: 1.52(m, 2H),
1.73(m, 4H), 1.90(m, 4H), 4.13(m, 1H), 6.53(d, J =
15.9 Hz, 1H), 7.37(m, 3H), 7.52(m, 2H), 7.66(d, J =
15.9 Hz, 1H); ESI-MS m/z: 231 [M + H]+, 253
[M + Na]+.
•
•
•
(E)-isobutyl-3-(3,4-dihydroxyphenyl)acrylate
(10)
•
•
•
•
[Murakami et al., 2000]: yield 93%; 1H NMR
(CDCl3, AV-300), d: 0.98 (d, J = 6.8 Hz, 6H), 2.03
(m, 1H), 3.92 (s, 3H), 3.98 (d, J = 6.6 Hz, 2H), 6.30
(d, J = 15.9 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H), 7.05
(m, 2H), 7.62 (d, J = 15.9 Hz, 1H); ESI-MS m/z: 237
[M + H]+, 259 [M + Na]+.
(E)-isopentyl-3-(3,4-dihydroxyphenyl)acrylate (11)
[Murakami et al., 2000]: yield 91%; 1H NMR
(CDCl3, AV-300), d: 0.95 (d, J = 6.6 Hz, 6H), 1.56
(m, 2H), 1.81 (m, 1H), 3.91 (s, 3H), 4.22 (t, 2H), 6.29
(d, J = 15.9 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H), 7.07
(m, 2H), 7.61 (d, J = 15.9 Hz, 1H); ESI-MS m/z: 251
[M + H]+, 273 [M + Na]+.
(E)-cyclopentyl-3-(4-hydroxy-3-methoxyphenyl)
acrylate (12): yield 91%; 1H NMR (CDCl3, AV-300),
d: 1.62(m, 4H), 1.78(m, 4H), 3.92(s, 3H), 4.13(m,
1H), 6.26(d, J = 15.9 Hz, 1H), 6.91(d, J = 8.2 Hz,
1H), 7.02(d, J = 2.0 Hz, 1H), 7.06(dd, J1 = 2.0 Hz,
J2 = 8.2 Hz, 1H), 7.57(d, J = 15.9 Hz, 1H); ESI-MS:
m/z 263 [M + H]+, 285 [M + Na]+.
•
•
(E)-cyclohexyl-3-(4-hydroxy-3-
methoxyphenyl)acrylate (13): yield 90%; 1H NMR
(CDCl3, AV-300), d: 1.52(m, 2H), 1.73(m, 4H),
1.88(m, 4H), 3.87(m, 1H), 3.92(s, 3H), 6.27(d, J =
15.7 Hz, 1H), 6.90(d, J = 8.2 Hz, 1H), 7.03(d, J =
2.0 Hz, 1H), 7.06(dd, 1H, J1 = 2.0 Hz, J2 = 8.2 Hz,
1H), 7.59(d, J = 15.7 Hz, 1H); ESI-MS: m/z 277
[M + H]+, 299 [M + Na]+.
(E)-isopropyl-3-(3,4-dihydroxyphenyl)acrylate
(9)
•
[Murakami et al., 2000]: YIELD 93%; 1H NMR
(CDCl3, AV-300), d: 1.32 (d, J = 6.2 Hz, 6H),
3.92 (s, 3H), 5.14 (m, 1H), 6.27 (d, J = 15.9 Hz, 1H),
(E)-isopropyl 3-(3,4-dihydroxyphenyl)acrylate (14)
[de Campos Buzzi et al., 2009]: yield 93%; 1H NMR
•
Drug Dev. Res.

SELECTIVE MATRIX METALLOPROTEINASE INHIBITORS
5
(CDCl3, AV-300), d: 1.23 (m, 6H, 2CH3), 4.99 (m,
TABLE 2. The Inhibitory Activity of Compounds 4–18 toward Some
1H, COOCH), 6.23 (d, J = 15.9 Hz, 1H, C = CH),
6.76 (d, J = 8.0 Hz, 1H, Ar-H), 6.98-7.04 (m, 2H,
Ar-H), 7.45 (d, J = 15.9 Hz, 1H, CH = C), 9.11 (s,
1H, OH), 9.56 (s, 1H, OH); ESI-MS: m/z 223
[M + H]+, 245 [M + Na]+.
of the Principal matrix Metalloproteinases (MMPs), the MMP-2
Selectivity,a and the MMP-9 Selectivitya (in Parentheses)
b
IC50 (nM)
Product
MMP-1
MMP-2
MMP-9
(E)-isobutyl 3-(3,4-dihydroxyphenyl)acrylate (15)
[de Campos Buzzi et al., 2009]: yield 92%; 1H NMR
(CDCl3, AV-300), d: 0.92 (d, J = 6.8 Hz, 6H, 2CH3),
1.93 (m, 1H, CH), 3.90 (d, J = 6.6 Hz, 2H,
COOCH2), 6.27 (d, J = 15.9 Hz, 1H, C = CH), 6.76
(d, J = 8.0 Hz, 1H, Ar-H), 7.00–7.05 (m, 2H, Ar-H),
7.48 (d, J = 15.9 Hz, 1H, CH = C), 9.12 (s, 1H, OH),
9.58 (s, 1H, OH); ESI-MS: m/z 237 [M + H]+, 259
[M + Na]+.
(E)-isopentyl 3-(3,4-dihydroxyphenyl)acrylate (16)
[de Campos Buzzi et al., 2009]; yield 91%; 1H NMR
(CDCl3, AV-300), d: 0.90 (m, 6H, 2CH3), 1.52 (m,
2H, CH2), 1.68 (m, 1H, CH), 4.14 (t, J = 6.8 Hz, 2H,
COOCH2), 6.25 (d, J = 15.9 Hz, 1H, C = CH), 6.75
(d, J = 8.0 Hz, 1H, Ar-H), 6.99-7.04 (m, 2H, Ar-H),
7.46 (d, J = 15.9 Hz, 1H, CH = C), 9.12 (s, 1H, OH),
9.58 (s, 1H, OH); ESI-MS: m/z 251 [M + H]+, 273
[M + Na]+.
•
•
4
5
6
7
8
1503.2 Ϯ 18.4
1488.8 Ϯ 12.5
1631.7 Ϯ 16.4
1510.9 Ϯ 11.7
1797.6 Ϯ 12.8
5761.6 Ϯ 21.6
5812.8 Ϯ 22.4
5410.3 Ϯ 24.3
5971.4 Ϯ 25.6
6111.7 Ϯ 31.7
5892.6 Ϯ 26.9
6132.3 Ϯ 32.7
6270.8 Ϯ 41.6
6766.9 Ϯ 37.6
6972.9 Ϯ 48.7
238.9 Ϯ 2.2
84.2 Ϯ 3.0 (18)
71.9 Ϯ 2.9 (21)
89.3 Ϯ 2.4 (18)
68.8 Ϯ 2.0 (22)
62.7 Ϯ 2.4 (29)
61.7 Ϯ 2.9 (93)
44.8 Ϯ 2.1 (129)
49.2 Ϯ 2.1 (110)
38.3 Ϯ 1.9 (157)
30.8 Ϯ 1.8 (197)
20.3 Ϯ 1.3 (295)
16.2 Ϯ 1.0 (383)
17.1 Ϯ 0.5 (369)
12.4 Ϯ 0.5 (564)
10.8 Ϯ 0.4 (633)
24.3 Ϯ 0.4 (10)
98.4 Ϯ 2.1 (15)
78.7 Ϯ 2.4 (19)
86.4 Ϯ 2.8 (19)
71.5 Ϯ 2.4 (21)
58.6 Ϯ 2.1 (30)
88.9 Ϯ 2.7 (65)
41.2 Ϯ 2.1 (142)
65.2 Ϯ 2.3 (83)
48.7 Ϯ 2.4 (122)
28.2 Ϯ 1.6 (218)
53.5 Ϯ 2.4 (111)
25.2 Ϯ 1.7 (245)
66.2 Ϯ 2.6 (95)
13.4 Ϯ 1.4 (521)
6.8 Ϯ 0.7 (996)
21.2 Ϯ 1.3 (11)
9
10
11
12
13
14
15
16
17
18
Caffeic
acid
aSelectivity for MMP-1 over each of the other MMPs, is expressed as
the ratio of the IC50 value for MMPs over the value for MMP-1.
bIC50 values are the mean of four experiments, standard deviation is
given.
(E)-cyclopentyl
3-(3,4-dihydroxyphenyl)acrylate
•
•
enzyme. Then 0.03% picrylsulfonic acid solution was
added and incubated at room temperature for additional
20 min. The resulting solutions were measured at optical
density 450 (OD450) and the values used to calculate the
inhibitory rates using the equation [OD450(100%) -
OD450 (compound)] / [OD450(100%) - OD450(blank)] ¥
100%. IC50 values were obtained from the aforemen-
tioned inhibitory rates using OriginPro 7.5 software
(OriginLab corporation, Hampton, Massachusetts,
USA).
(17): yield 90%; 1H NMR (CDCl3, AV-300), d:
1.52(m, 4H), 1.64(m, 4H), 4.12(m, 1H), 6.26(d,
J = 15.9 Hz, 1H), 6.91(d, J = 8.1 Hz, 1H),7.02(d,
J = 2.0 Hz, 1H), 7.06(dd, 1H, J1 = 2.0 Hz, J2 = 8.1 Hz,
1H), 7.57(d, J = 15.9 Hz, 1H); ESI-MS m/z: 249
[M + H]+, 271 [M + Na]+.
(E)-cyclohexyl 3-(3,4-dihydroxyphenyl)acrylate (18)
[Uwai et al., 2008]: yield 90%; 1H NMR (CDCl3,
AV-300), d: 1.33(m, 4H), 1.56(m, 2H), 1.75(m, 2H),
1.92(m, 2H), 4.14(m, 1H), 6.26(d, J = 15.9 Hz, 1H),
6.88(d, J = 8.3 Hz, 1H), 7.00(dd, 1H, J1 = 2.0 Hz,
J2 = 8.3 Hz, 1H), 7.08(d, J = 2.0 Hz, 1H), 7.57(d,
J = 15.9 Hz, 1H); ESI-MS m/z: 263 [M + H]+, 285
[M + Na]+.
RESULTS AND DISCUSSION
MMP-1, MMP-2, and MMP-9 Inhibitory Activity
Table 2 summarizes the results obtained in the
assay for the inhibitory activities on MMP-1, MMP-2,
and MMP-9; caffeic acid was used as the reference
control, selectivity indices for MMP-2 and MMP-9
BIOLOGICAL SCREENING
MMP-1, MMP-2, and MMP-9 Inhibition Assay
The substituted cinnamic acid esters were assayed over MMP-1 are also reported, expressed as ratios of
for the inhibitory activities against MMP-1 MMP-2, and their inhibitory indices (Table 2). The results showed
MMP-9 in 96-well microtiter plates using succinylated that these substituted cinnamic acid esters exhibited
gelatin as the substrate [Baragi et al., 2000]. The com- highly selective inhibition against MMP-2 and MMP-9
pounds and enzyme were dissolved in sodium borate as compared with MMP-1, thus confirming our
buffer (pH 8.5, 50 mM) and incubated at 37°C for strategy for designing MMP-2, MMP-9 inhibitors
30 min. The substrate was added and incubated at 37°C (Table 2).
for another 60 min. The 100% and blank groups were
For MMP-2, caffeic acid esters 14–18 showed
also carried out in which the 100% group contained good potency compared with caffeic acid, with IC50
no compound and the blank group contained only the values less than 20 nM, while the IC50 value of caffeic
Drug Dev. Res.

6
SHI ET AL.
Fig. 4. Docking result of compound 18 with matrix metalloproteinase 2. [Color figure can be viewed in the online issue which is available at
wileyonlinelibrary.com]
acid was 24.3 nM. Among the caffeic acid esters, the
most active compound on MMP-2 was 18 (IC50 =
10.8 nM), approximately twice as potent as caffeic acid
(IC50 = 24.3 nM). At MMP-2, the ferulic acid esters
9–11 were less active by greater than two orders of
magnitude than caffeic acid, 12 and 13 showed inhibi-
tory activity practically equal to that of caffeic acid.
Among the cinnamic acid esters, no compound was
equal or more active than caffeic acid. At MMP-9, the
caffeic acid cyclohexane ester 18 was the most active of
the synthesized esters (IC50 = 6.8 nM), some threefold
more active than caffeic acid (IC50 = 21.2 nM). The
caffeic acid cyclopentane ester 17 had good inhibitory
activity (IC50 = 13.4 nM), while the isobutane analog 15
had inhibitory activity equal to that of caffeic acid (IC50
= 25.2 nM). Among the ferulic acid esters, only the
cyclohexane ester 13 showed inhibitory activity equally
to that of caffeic acid (IC50 = 28.2 nM). Among the cin-
namic acid esters 4–8, no compound was equal or more
active than caffeic acid and were twofold to fourfold two
less active than caffeic acid. All compounds showed
minimal inhibition of MMP-1 (IC50 = 1488.8 nM [5] to
6972.9 nM [18]). Caffeic acid had an IC50 value of
238.9 nM. An analysis of the selectivity indices reported
in parentheses in Table 2 for the synthesized esters and
Docking Studies
In the MMP inhibition tests, 18 showed the stron-
gest inhibitory activity on MMP-2 and MMP-9 and the
highest selectivity against MMP-1 so 18 was selected
for the subsequent molecular docking experiment with
MMP-2 (Fig. 4) and MMP-9 (Fig. 5). The MMP-2
docking showed that the cyclohexane group in 18 occu-
pied the deep S1′ pocket (composed of Alanine (Ala)
84, Histidine (His)85, Leucine (Leu)116, Valine (Val)
117, His120, Glutamate (Glu)121) of MMP-2, and the
carbonyl group chelated the active site zinc ion with
a distance of 2.19 Å. The MMP-9 docking showed
that the cyclohexane group in 18 occupied the deep
S1′ pocket (composed of His411, Leu418, Tyrosine
(Tyr)420, Proline (Pro)421, Methionine (Met)422) of
MMP-9, and the carbonyl group chelated the active site
zinc ion with a distance of 2.218 Å. These results indi-
cated that compound 18 interacted well with MMP-2
and the MMP-9 active site, especially the deep S1′
pocket and zinc ion, consistent with MMP-2 and
MMP-9 assay results.
Discussion and SAR
In this study, based on docking studies of caffeic
caffeic acid indicated that all the synthesized com- acid with MMP-2 and MMP-9, 15 compounds were
pounds had higher selectivity profiles than caffeic acid, synthesized and investigated for inhibitory activity on
with 18 having the highest selectivity profile (MMP- MMP-1, MMP-2, and MMP-9. Selected for docking
1/MMP-2 = 633, MMP-1/MMP-9 = 996), whereas 17 experiments for the molecular modeling investigation
(similar in its inhibitory potency toward MMP-2 to 18) was 18.
had lower MMP-1/MMP-2 and MMP-1/MMP-9 selec-
tivity ratios that were 564 and 521, respectively.
Compounds 4–8 were cinnamic acid esters with
no OH group or OCH3 group on the benzene ring,
Drug Dev. Res.

SELECTIVE MATRIX METALLOPROTEINASE INHIBITORS
7
Fig. 5. Docking result of compound 18 with matrix metalloproteinase 9. [Color figure can be viewed in the online issue which is available at
wileyonlinelibrary.com]
compounds 9–13 were ferulic acid esters with one OH the presence of extended spatial structures in the car-
group and one OCH3 group on the benzene ring, and boxylic acid played key roles in the MMP-2 and MMP-9
compounds 14–18 were caffeic acid esters with two inhibitory activity and selectivity. These findings would
OH groups on the benzene ring, when OH group in the facilitate the design of compounds with higher potency
benzene ring was replaced by the OCH3 group or by the to serve as selective MMP-2 and MMP-9 inhibitors that
H group, MMP inhibition activities and selectivity could provide information for the exploitation and uti-
reduced, such as 13, with one OCH3 group and one OH lization of caffeic acid as MMPI for metastatic tumor
group in the benzene ring and 8 with H groups in the treatment.
benzene ring, showed weaker MMP inhibition activities
and selectivity as compared with 18, which had two OH
groups in the benzene ring. These results showed that
the two OH groups were important for MMP inhibition
and selectivity.
ACKNOWLEDGMENTS
This work was supported by the National Natural
Science Foundation of China (81001382), Program
for New Century Excellent Talents by the Ministry
of Education (NCET-09-0163), Research Fund for
the Doctoral Program of Higher Education of China
(20093237120012), the Main Training Fund of Nanjing
University of Chinese Medicine (10XPY02), 333 High-
Level Talents Training Project Funded by Jiangsu Prov-
ince, Six Talents Project Funded by Jiangsu Province
(2011-D-078), Program for Outstanding Scientific and
Technological Innovation Team of Jiangsu Higher Edu-
cation (2009), Key Research Project in Basic Science
of Jiangsu College and University (no. 06KJA36022,
07KJA36024, 10KJA360039), Project Funded by the
In the three series, substituted cinnamic acid
esters, when the carboxylic acid group was converted
to cyclic ester, the inhibitory activity for MMP-2 and
MMP-9 increased, e.g., 8, a cyclohexyl ester, had stron-
ger inhibitory activity on MMP-2 and MMP-9 than 5, a
isobutanol ester. This result indicated that the extended
spatial structures, e.g., cyclohexyl and cyclopentyl, were
important for the improvement of the activity and selec-
tivity in inhibiting MMP-2 and MMP-9.
CONCLUSIONS
In this study, we examined the effects of 15 sub- Priority Academic Program Development of Jiangsu
stituted cinnamic acid esters that were synthesized Higher Education Institutions (ysxk-2010), and Con-
using microwave irradiation on MMP-1, MMP-2, and struction Project for Jiangsu Engineering Center
MMP-9 inhibition activity. Preliminary SAR analysis of Innovative Drug from Blood-Conditioning TCM
showed that hydroxyl groups in the benzene ring and Formulae.
Drug Dev. Res.

8
SHI ET AL.
Maki BE, Chan A, Phillips EM, Scheidt KA. 2009. N-heterocyclic
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