Synthesis and structure-activity relationship analysis of caffeic acid amides as selective matrix metalloproteinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.01.027

Four series of acid amides were synthesized, and through measurement using a fluorogenic substrate assay with human recombinant MMP-1, MMP-2 and MMP-9, compound 3f showed considerable inhibitory activities against MMP-2, MMP-9 and the best selectivity over MMP-1. Preliminary structure-activity relationship analysis indicated that caffeic acid amides with electron-donating groups at para-position of amino phenyl group showed better inhibitory activities and selectivity than those with electron-withdrawing groups, and the presence of adjacent dihydroxy in the caffeoyl group was very important for the MMP-2 and MMP-9 inhibitory activities.

Full text of DOI:10.1016/j.bmcl.2013.01.027

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1206–1211
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationship analysis of caffeic acid
amides as selective matrix metalloproteinase inhibitors
Zhi-Hao Shi a,c, , Nian-Guang Li a,b, , , Qian-Ping Shi a, Hao Tang a, Yu-Ping Tang a, , Wei Li a,b, Lian Yin a,
⇑
⇑
Jian-Ping Yang a, Jin-Ao Duan a,
⇑
a Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210046, China
b Department of Medicinal Chemistry, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210046, China
c Department of Organic Chemistry, China Pharmaceutical University, Nanjing, Jiangsu 211198, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Four series of acid amides were synthesized, and through measurement using a fluorogenic substrate
assay with human recombinant MMP-1, MMP-2 and MMP-9, compound 3f showed considerable inhib-
itory activities against MMP-2, MMP-9 and the best selectivity over MMP-1. Preliminary structure–activ-
ity relationship analysis indicated that caffeic acid amides with electron-donating groups at para-position
of amino phenyl group showed better inhibitory activities and selectivity than those with electron-with-
drawing groups, and the presence of adjacent dihydroxy in the caffeoyl group was very important for the
MMP-2 and MMP-9 inhibitory activities.
Received 22 August 2012
Revised 22 December 2012
Accepted 7 January 2013
Available online 16 January 2013
Keywords:
Caffeic acid
Matrix metalloproteinase
Tumor
Ó 2013 Elsevier Ltd. All rights reserved.
Structure–activity relationship
Matrix metalloproteinases (MMPs) are a family of zinc depen-
dent endopeptidases involved in the breakdown of components
of the extracellular matrix which facilitates connective tissue
remodeling.1 This process is important in embryonic development,
pregnancy, growth and wound healing. Normally the activity of
MMPs is controlled by a tight balance between synthesis of active
MMPs and the presence of endogenous inhibitors, such as the TIM-
Ps (tissue inhibitor of metalloproteinases).1 During tumor progres-
sion, this balance is lost and the increased expression of certain
MMPs is believed to be involved in metastatic tumor dispersion
and angiogenesis, accompanies the passage from a benign to a
malignant phenotype.2,3 These metallo (zinc) endopeptidases are
produced and secreted as inactive zymogens in the extracellular
matrix by the tumor cells themselves or by surrounding stromal
cells stimulated by the nearby tumor. Two of them, MMP-2 (gela-
tinase A) and MMP-9 (gelatinase B), seem to play important roles
in these processes, considering that they are involved in metastatic
tumor dispersion and angiogenesis.4,5
in animal models of early stage disease but were only tested in
late-stage disease in clinic trials.7 As they were developed as
cytostatic agents, they may be used in a prolonged therapy, and
therefore their bioavailability and slow long-term toxicity are
very important.8 The other problem was the lack of selectivity,
compounds such as CGS-27023A (1) and BMS-275291 (2)
(Fig. 1) have shown a severe musculoskeletal syndrome, with
fibroproliferative effects in the joint capsule of the knees.9–11
These effects are thought to be linked to an impairment of normal
tissue remodeling governed by the MMP-1.12 For these reasons, a
lack of activity with respect to MMP-1 is considered to be an
important factor in reducing some of the side effects found for
‘nonselective’ MMPIs.13
Selectivity could potentially be achieved by taking advantage of
differences in the size of S10 pocket among the various MMPs. From
X-ray crystallography, NMR analysis and homology modeling, the
In the past few years, some potent ‘broad spectrum’ MMP
inhibitors (MMPIs) have been proposed and tested against tu-
mors.6 Some of them have entered clinical trials, but at present
none of them is on the market. One problem was the design of
the clinic trials themselves. MMP inhibitors had been successful
O
S
N
O
O
O
O
O
H
N
HS
HO
O
N
H
N
H
N
H
O
O
N
⇑
Corresponding authors. Tel./fax: +86 25 85811916.
N
BMS-275291 (2)
CGS-27023A (1)
E-mail addresses: linianguang@njutcm.edu.cn (N.-G. Li), yupingtang@njutcm.
edu.cn (Y.-P. Tang), dja@njutcm.edu.cn (J.-A. Duan).
Co-first authors.
Figure 1. Chemical structures of CGS-27023A (1) and BMS-275291 (2).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.027

Z.-H. Shi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1206–1211
1207
O
O
R1
R2
O
n
N
H
HO
HO
R3
HO
HO
OH
O
New designed caffeic
acid amide derivatives
Caffeic acid (CA) (3)
Caffeic acid phenethyl ester (CAPE) (4)
P1'
Figure 2. Chemical structures of caffeic acid, caffeic acid phenethyl ester and our further designed caffeic acid amide derivatives.
Table 1
Reactions of caffeic acid (3), (E)-3-(3-hydroxyphenyl)acrylic acid (5), (E)-3-(4-hydroxyphenyl)acrylic acid (6) and cinnamic acid (7) with a variety of amine
O
O
R1
R2
R1
R2
3
BOP/Et3N
3
4
n
H2N
N
H
n
OH
R3
R3
+
DMF/CH2Cl2
4
3: R1=R2=OH
3a-3r
1
2
5a-5r
6a-6r
7a-7r
5:
6:
R =OH, R =H
1
2
R =H, R =OH
7: R1=R2=H
Entry
R1
R2
R3
n
Time (h)
Prod. (yield)
Entry
R1
R2
R3
n
Time (h)
Prod. (yield)
1
2
3
4
5
6
7
8
9
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
2-OH
3-OH
4-OH
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
H
H
H
2-OH
3-OH
4-OH
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-F
0
1
2
0
0
0
8
8
8
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
8
8
8
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
3a (78)25
3b (83)24
3c (82)26
3d (85)27
3e (84)27
3f (85)28
3g (84)25
3h (83)25
3i (84)25
3j (84)25
3k (84)25
3l (86)25
3m (82)25
3n (75)25
3o (81)25
3p (80)25
3q (76)25
3r (82)25
5a (80)29
5b (85)
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
0
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8
8
8
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
8
8
8
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
6a (77)25
6b (85)31
6c (84)32
6d (86)
6e (84)
6f (86)
6g (85)
6h (83)
6i (85)
6j (84)
2-OH
3-OH
4-OH
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
H
H
H
2-OH
3-OH
4-OH
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-F
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
6k (83)
6l (84)29
6m (81)
6n (74)
6o (82)
6p (81)29
6q (73)30
6r (80)
7a (82)30
7b (88)33
7c (86)34
7d (87)
5c (84)
5d (86)
5e (85)
5f (86)
5g (86)
5h (85)
5i (86)
5j (85)
7e (86)
7f (88)35
7g (87)36
7h (85)37
7i (86)38
7j (86)39
7k (85)30
7l (87)39
7m (83)40
7n (79)41
7o (81)42
7p (83)38
7q (78)30
7r (82)38
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5k (84)
5l (85)29
5m (81)
5n (74)
3-F
4-F
2-Cl
3-Cl
3-F
4-F
2-Cl
3-Cl
5o (80)
5p (82)29
5q (75)30
5r (83)
H
4-Cl
H
4-Cl
MMPs may be classified into two broad structural classes, those
with a relatively deep S10 pocket (MMP-2, 3, 8, 9, and 13) and those
with a shallow S10 pocket (MMP-1 and 7).14,15 Consequently, incor-
poration of an extended P1’ group leads to selective inhibition,
whereas the presence of smaller P1’ groups generally leads to
broad spectrum inhibition.
Considering these data, many efforts were directed to develop a
more selective second generation of inhibitors against the specific
MMPs believed to be involved in the different pathologies since the
late 90s and MMPIs that may be derived from natural resources
such as herbs, plants, fruits, and other agriculture products have
been highlighted in recent years.16
of MMP-9 with the IC50 of 10–20 nM. Furthermore, caffeic acid
phenylester (CAPE) (4) (Fig. 2), which was extracted from honey-
bee propolis18 and has been synthesized by esterification of CA,19
could selectively inhibited MMP-2 and -9 but not -1, -3, -7.20
However, the ester groups in caffeic acid are metabolically very
labile and limited for their use.21–23 Several modified caffeic acid
amides recently exhibited more stable characteristics.24
These findings prompted us to synthesize a series of corre-
sponding caffeic acid amides with extended P1’ group for the pur-
pose to investigate their selective inhibitory activities against
MMP-2 and MMP-9. Furthermore, in order to investigate the MMPs
inhibitory effects of the substitution on the benzene ring of caffeic
acid, three acids such as (E)-3-(3-hydroxyphenyl)acrylic acid (5),
(E)-3-(4-hydroxyphenyl)acrylic acid (6) and cinnamic acid (7)
Caffeic acid (3) (Fig. 2), which was found in fruits, vegetables,
wine, olive oil and coffee,17 has been shown to inhibit the activity

1208
Z.-H. Shi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1206–1211
Table 2
The inhibitory activity of compounds 3–7 towards some of the principal MMPs and the selectivitya (in parentheses)
b
b
Prod.
IC50 (nM)
Prod.
IC50 (nM)
MMP-1
MMP-2
MMP-9
7.14 0.58(453)
MMP-1
MMP-2
MMP-9
7.80 0.61(436)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3235.42 28.19
3246.57 27.32
3301.33 30.14
5616.56 42.89
5769.78 40.47
5833.61 42.16
5247.92 39.28
5285.42 41.85
5319.54 43.79
4712.73 35.84
4799.42 37.92
4829.57 41.47
4246.83 40.91
4302.21 38.67
4366.42 39.52
3706.63 30.19
3808.43 31.24
3963.66 31.97
3401.82 30.78
3602.31 31.59
3595.63 32.63
5207.75 40.52
5275.93 41.68
5206.82 43.57
5127.23 42.49
5162.55 40.91
5219.32 39.76
4741.38 37.63
4767.79 36.18
4889.64 38.92
4117.53 37.69
4213.83 38.57
4387.42 36.64
3256.49 30.12
3352.83 32.18
3496.65 29.57
8.05 0.53(402)
8.22 0.49(395)
8.20 0.51(403)
5.09 0.34(1103)
1587.17 21.31(4)
3.28 0.29(1779)
5.27 0.58(996)
1679.56 22.53(3)
3.42 0.49(1555)
5.97 0.58(789)
1721.82 23.57(3)
3.55 0.51(1360)
8.92 0.62(476)
1820.53 24.60(2)
8.31 0.59(525)
9.11 0.72(407)
1873.02 23.71(2)
8.95 0.73(443)
8.41 0.63(404)
8.44 0.59(427)
8.46 0.58(425)
5.57 0.39(935)
1524.69 22.36(3)
4.18 0.29(1246)
5.62 0.36 (912)
1674.81 24.33(3)
4.32 0.29(1208)
5.73 0.41(827)
1724.94 24.39(3)
4.43 0.43(1104)
9.09 0.52(453)
1867.71 26.48(2)
8.59 0.53(511)
9.41 0.61(346)
1937.23 26.58(2)
9.11 0.63(384)
6a
6b
6c
6d
6e
6f
6g
6h
6i
3404.64 28.37
3136.72 29.31
3219.85 27.63
5058.56 43.19
5347.73 44.57
5516.37 46.71
5114.38 42.77
5217.29 41.69
5293.55 43.88
4788.43 48.69
4879.72 36.81
4831.49 37.62
4233.53 36.75
4426.63 36.81
4538.24 34.69
3448.67 29.38
3570.27 28.64
3574.82 27.51
3057.91 26.77
3303.83 25.89
3252.67 26.72
5201.56 33.43
5393.29 35.62
5403.64 43.57
5182.87 41.29
5255.29 46.73
5301.68 42.69
4515.39 38.55
4585.74 36.71
4670.92 40.91
4173.64 35.55
4194.94 37.13
4261.69 40.21
3159.54 29.53
3451.32 30.18
3501.61 31.47
238.91 2.22
8.30 0.52(410)
8.32 0.60(377)
8.37 0.55(385)
5.47 0.41(925)
1674.23 23.38(3)
3.97 0.29(1390)
5.53 0.66(925)
1764.37 24.58(3)
4.09 0.44(1294)
5.62 0.61(852)
1754.54 25.49(3)
4.19 0.51(1153)
9.01 0.59(470)
1578.64 22.58(3)
8.39 0.44(541)
9.26 0.67(372)
1729.19 24.69(2)
9.04 0.58(395)
8.95 0.49(342)
9.02 0.77(366)
9.13 0.81(356)
6.24 0.42(834)
1785.67 27.58(3)
5.28 0.46(1023)
6.32 0.50(820)
1853.73 28.48(3)
5.37 0.52(987)
6.41 0.67(704)
1945.82 29.39(2)
5.46 0.44(855)
9.66 0.57(432)
1859.32 25.61(2)
9.17 0.77(465)
9.99 0.59(316)
1649.92 24.63(2)
9.72 0.55(360)
7.25 0.51(448)
7.28 0.60(453)
5.28 0.49(1064)
1642.27 22.43(4)
2.35 0.12(2482)
5.67 0.43(926)
1632.42 21.38(3)
3.33 0.42(1597)
5.91 0.53(797)
1753.85 22.61(3)
3.64 0.49(1327)
7.81 0.66(544)
1869.59 22.70(2)
7.29 0.68(599)
8.19 0.77(453)
1921.13 25.71(2)
7.89 0.69(502)
7.52 0.65(452)
7.57 0.58(476)
7.63 0.52(471)
5.71 0.49(912)
1561.56 23.47(3)
3.37 0.26(1545)
5.82 0.21(881)
1658.63 24.26(3)
3.42 0.31(1526)
5.91 0.57(802)
1758.72 25.59(3)
3.51 0.43(1393)
8.02 0.71(513)
1859.82 25.68(2)
7.79 0.65(563)
8.32 0.70(391)
1948.29 27.64(2)
8.22 0.66(425)
7.82 0.58(401)
7.83 0.63(411)
5.82 0.39(869)
1585.79 22.33(3)
4.25 0.28(1298)
5.91 0.66(865)
1659.85 23.54(3)
4.32 0.37(1225)
5.97 0.61(802)
1685.93 23.53(3)
4.41 0.61(1096)
8.93 0.52(474)
1628.58 23.49(3)
8.41 0.62(540)
9.34 0.71(369)
1819.27 26.59(2)
9.19 0.62(389)
7.98 0.73(383)
8.13 0.57(406)
8.19 0.63(397)
7.18 0.54(724)
1796.72 26.39(3)
6.19 0.41(873)
7.29 0.58(711)
1976.83 29.43(3)
6.21 0.57(854)
7.38 0.49(612)
1896.92 28.58(2)
6.38 0.62(732)
10.58 0.73(394)
1910.47 27.77(2)
10.26 0.69(415)
13.87 0.74(228)
1812.91 26.78(2)
12.59 0.68(278)
21.22 1.31(11)
3j
3k
3l
6j
6k
6l
3m
3n
3o
3p
3q
3r
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
6m
6n
6o
6p
6q
6r
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
5m
5n
5o
5p
5q
5r
7m
7n
7o
7p
7q
7r
CA
24.26
0.42 (10)
a
Selectivity 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.
IC50 values are mean of four experiments, standard deviation is given.
b
S1'
S1'
Figure 3. Docking results of caffeic acid with MMP-2 (left) and MMP-9 (right).
(Table 1) were also amidated with extended P1’ group. Based on
their inhibitory activities on MMP-1, MMP-2 and MMP-9, struc-
ture–activity relationship (SAR) analysis was conducted. The re-
sults of this study may be useful to researchers attempting to
find new potential caffeic acid derivatives as antitumor progres-
sion agents.
acid reacted with a variety of aniline in DMF and CH2Cl2 to give the
72 corresponding amide products in good yield (Table 1).25 Among
these compounds, 28 compounds including 5b–5k, 5m–5o, 5r, 6d–
6k, 6m–6o, 6r and 7d–7e are reported for the first time. 1H NMR
and ESI MS spectra were consistent with the assigned structures.
Compounds 3a–3c, 5a–5c, 6a–6c and 7a–7c were acid anilides
with the numbers of methylene were 0, 1 and 2 respectively. Com-
pounds 3d–3r, 5d–5r, 6d–6r and 7d–7r were acid anilides with a
Using benzotriazole-1-yl-oxy-tris-(dimethylamino)-phospho-
nium hexafluorophosphate (BOP) as a condensing reagent, the free

Z.-H. Shi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1206–1211
1209
S1'
S1'
Figure 4. Docking results of compound 3f with MMP-2 (left) and MMP-9 (right).
Figure 5. Total energy (left) and potential energy (right) of compound 3f with MMP-2 and MMP-9.
single different substituent (OH, OCH3, CH3, F or Cl) in ortho-, meta-
, or para-position of benzene ring.
such as fluorine or chlorine resulted in decreased inhibitory activ-
ity against MMP-2, MMP-9 and selectivity over MMP-1. For
example, the para-F substituted compound 3o (MMP-2
IC50 = 8.31 nM, MMP-9 IC50 = 7.29 nM) and the para-Cl substituted
compound 3r (MMP-2 IC50 = 8.95 nM, MMP-9 IC50 = 7.89 nM)
exhibited an inhibitory activity about three times lower than 3f
(MMP-2 IC50 = 3.28 nM, MMP-9 IC50 = 2.35 nM). In addition,
replacement of the hydroxyanil (3f) with aniline (3a, MMP-2
IC50 = 8.05 nM, MMP-9 IC50 = 7.14 nM), benzylamine (3b, MMP-2
IC50 = 8.22 nM, MMP-9 IC50 = 7.25 nM) and phenylethylamine
(3c, MMP-2 IC50 = 8.20 nM, MMP-9 IC50 = 7.28 nM) led to slightly
less inhibitory activity on MMP-2 and MMP-9 than 3f.
Replacing the adjacent dihydroxyl groups at C-3 and C-4 in 3a–
3r with one hydroxyl group at C-3 (5a–5r) or C-4 (6a–6r), as shown
in Table 2, resulted in some loss in inhibitory activity against MMP-
2 and MMP-9. For example, compound 5i (MMP-2 IC50 = 4.32 nM,
MMP-9 IC50 = 3.42 nM), which had one hydroxyl group at C-3 posi-
tion, showed a slightly less inhibitory activity than 3i (MMP-2
IC50 = 3.42 nM, MMP-9 IC50 = 3.33 nM). Compound 6m (MMP-2
IC50 = 9.01 nM, MMP-9 IC50 = 8.93 nM), which had one hydroxyl
group at C-4 position, also showed a slightly less inhibitory activity
than 3m (MMP-2 IC50 = 8.92 nM, MMP-9 IC50 = 7.81 nM). The
requirement of the adjacent dihydroxyl groups was further ex-
plored by replacement of the hydroxyl group in 3a–3r with hydro-
gen atoms shown in 7a–7r. This modification resulted in some loss
in inhibitory activity against MMP-2 and MMP-9. For example,
compound 7d (MMP-2 IC50 = 6.24 nM, MMP-9 IC50 = 7.18 nM),
which had no hydroxyl groups at C-3 position and C-4 position,
The inhibitory activities of series 3a–3r, 5a–5r, 6a–6r and 7a–7r
on human recombinant MMP-1, MMP-2 and MMP-9 were mea-
sured using a fluorogenic substrate assay.43 The compounds were
tested at 0.1–10000 nM following incubation for 30 min, caffeic
acid was used as the positive control,44 and the results were shown
in Table 2. Selectivity indices for MMP-2 and MMP-9 over MMP-1
are also reported, expressed as ratios of their inhibitory indices
(Table 2). These compounds had not significant inhibition activities
against MMP-1 supported by the fact that their IC50s were all over
1000 nM, thus confirming our strategy for designing MMP-2,
MMP-9 inhibitors (Table 2).
Among the synthesized amides, the most active compound
was 3f, its IC50s on MMP-2 and MMP-9 were 3.28 nM and
2.35 nM respectively. Furthermore, compound 3f also showed
the highest selectivity profile (MMP-1/MMP-2 ratio was 1779,
MMP-1/MMP-9 ratio was 2482). The ortho isomer of 3f, com-
pound 3d, showed
a good inhibitory activity on MMP-2
(IC50 = 5.09 nM) and MMP-9 (IC50 = 5.28 nM). While its meta iso-
mer, compound 3e, was scarcely active, with its IC50s on MMP-
2 and MMP-9 were 1587.17 nM and 1642.27 nM respectively.
Replacing the hydroxyl group with electron-donating groups such
as methoxy group or methyl group resulted in little effect on the
inhibitory activity against MMP-2, MMP-9 and selectivity over
MMP-1. For example, the para-MeO substituted compound 3i
(MMP-2 IC50 = 3.42 nM, MMP-9 IC50 = 3.33 nM) and the para-Me
substituted compound 3l (MMP-2 IC50 = 3.55 nM, MMP-9
IC50 = 3.64 nM) proved to be slightly less active than 3f. However,
replacing the hydroxyl group with electron-withdrawing groups
showed
a slightly less inhibitory activity than 3d (MMP-2
IC50 = 5.09 nM, MMP-9 IC50 = 5.28 nM).

1210
Z.-H. Shi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1206–1211
In the MMP inhibition tests, 3f showed the strongest inhibitory
substituent at ortho-positions showed less potent activities,
whereas anilides with a substituent at meta-positions showed
diminished activity. From this fact, it was reasoned that the para-
position was an essential point for the inhibition activities against
MMP-2 and MMP-9, and substituents at the ortho-position de-
creased their inhibition, substituents at the meta-position showed
no inhibitory activities. For example, when a hydroxyl group was
introduced at para-position to form 3f, the inhibitory activities
against MMP-2 and MMP-9 was the most significant, and when a
hydroxyl group was introduced at ortho-position to form 3d, the
activity decreased slightly. However, when a hydroxyl group was
introduced at meta-position to form 3e, the activity decreased
significantly.
In addition, the data in Table 2 indicated that many caffeic acid
amides (3a–3r) showed more active inhibition against MMP-2 and
MMP-9, better selectivity over MMP-1 than cinnamic acid amides
(7a–7r), indicating that the presence of adjacent dihydroxy in the
caffeoyl group was very important for the MMP-2 and MMP-9
inhibitory activities. This could be further supported that (E)-3-
(3-hydroxyphenyl)acrylic acid amides (5a–5r), which had only
one hydroxyl group in position 3 in the benzene ring, and (E)-3-
(4-hydroxyphenyl)acrylic acid (6a–6r), which had only one hydro-
xyl group in position 4 in the benzene ring, showed diminished
inhibitory activity compared with caffeic acid amides (3a–3r).
In summary a series of acid amides 3a–3r, 5a–5r, 6a–6r and 7a–
7r were synthesized, and through measurement using a fluoro-
genic substrate assay with human recombinant MMP-1, MMP-2
and MMP-9, compound 3f showed considerable inhibitory activi-
ties against MMP-2, MMP-9 and best selectivity over MMP-1. Pre-
liminary SAR analysis indicated that caffeic acid anilides with
electron-donating groups at para-position of amino phenyl group
showed better inhibitory activities and selectivity than those with
electron-withdrawing groups. It was also important to note that
the presence of adjacent dihydroxy in the caffeoyl group was very
important for the MMP-2 and MMP-9 inhibitory activities. There-
fore, the findings of this study would facilitate the design of chem-
ical compounds with higher potency to serve as selective MMP-2
and MMP-9 inhibitors, and provide information for the exploita-
tion and utilization of caffeic acid as MMP inhibitor for metastatic
tumor treatment.
activity on MMP-2 and MMP-9, and the highest selectivity over
MMP-1, so the caffeic acid and 3f were selected for the subsequent
molecular docking experiments with MMP-2 and MMP-9. The
docking studies of caffeic acid (Fig. 3) showed that the carboxylic
acid group in caffeic acid could occupy the deep S1’ pocket in
MMP-2 and MMP-9, and the carbonyl group chelated the active
site zinc ion with a distance of 2.210 Å in MMP-2 and 2.347 Å in
MMP-9 respectively. The docking studies of 3f with MMP-2 and
MMP-9 (Fig. 4) indicated that 3f interacted well with these two
MMPs. The MMP-2 docking of 3f showed that the para-hydroxy
aniline group in 3f occupied well the deep S1’ pocket of MMP-2,
and the carbonyl group chelated the active site zinc ion with a dis-
tance of 3.774 Å. The docking studies of 3f with MMP-9 showed
that the para-hydroxy aniline group in 3f occupied well the deep
S1’ pocket of MMP-9, and the carbonyl group chelated the active
site zinc ion with a distance of 2.296 Å. These results indicated that
compound 3f interacted well with MMP-2 and MMP-9 active site,
especially the deep S1’ pocket and zinc ion, consistent with MMP-2
and MMP-9 assay results.
A molecular dynamic (MD) simulation45 was then performed on
the ligand/protein complex to evaluate whether the binding of 3f
with MMP-2 and MMP-9 was stable. As shown in Figure 5 (left), after
150 ps of MD, the system of 3f with MMP-2 reached an equilibrium,
and the total energy for the last 1.5 ns remained constant. The total
energy of the system of 3f with MMP-9 remained constant for the
last 1 ns. The potential energies of the two systems (Fig. 5: right) re-
mained stable in all the simulations which revealed a potentially rel-
atively stable interaction between the ligand and proteins.
Preliminary structure–activity relationship analysis indicated
that the synthesized acid amines exhibited better inhibitory activ-
ities against MMP-2 and MMP-9 than caffeic acid, however, their
IC50s differed significantly. For example, among the acid anilides
3a–3c, 5a–5c, 6a–6c and 7a–7c with no substitutions on the amino
phenyl group, anilides with no carbon atom between the amide
and the phenyl group showed higher inhibitory activities on
MMP-2 and MMP-9 than those with one carbon atom or two car-
bon atoms between the amide and the phenyl group. Such as in
the inhibitory activity on MMP-2, compound 3a with phenyl group
showed a good potency with its IC50 was 8.05 nM, and replacing
the phenyl group with the benzyl group and the phenylethyl
group, as shown in compounds 3b and 3c, resulted in slightly low-
er inhibitory activities with their IC50s were 8.22 nM and 8.20 nM
respectively.
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (81001382, 81274058), Program for New Century
Excellent Talents by the Ministry of Education (NCET-09-0163), Re-
search 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 Province, Six Talents Project Funded by
Jiangsu Province (2011-D-078), Program for Outstanding Scientific
and Technological Innovation Team of Jiangsu Higher Education
(2009), Key Research Project in Basic Science of Jiangsu College
and University (NO. 06KJA36022, 07KJA36024, 10KJA360039), Pro-
ject Funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions (ysxk-2010), and Construc-
tion Project for Jiangsu Engineering Center of Innovative Drug from
Blood-conditioning TCM Formulae.
Furthermore, among all the synthesized amines, the most active
compound was 3f with one hydroxyl group at para-position on the
amino phenyl group, it showed the best inhibitory potency on
MMP-2 and MMP-9 with its IC50s were 3.28 nM and 2.35 nM
respectively. In addition, 3f was the one with the highest selectiv-
ity profile (MMP-1/MMP-2 ratio was 1779, MMP-1/MMP-9 ratio
was 2482). In the series of caffeic acid anilides, replacement of
the hydroxyl group of (3f) at para-position by electron-donating
groups such as methoxy group (3i) or methyl group (3l) resulted
in little effect on the inhibitory activity against MMP-2, MMP-9
and selectivity over MMP-1. However, replacement of the hydroxyl
group of (3f) at para-position by electron-withdrawing groups
such as fluorine (3o) or chlorine (3r) resulted in the decrease of
inhibitory activity against MMP-2, MMP-9 and selectivity over
MMP-1. These effects can also be reflexed in the other three series
of 5d–5r, 6d–6r and 7d–7r. These results suggested that com-
pounds with electron-donating groups at para-position showed
better inhibitory activities and selectivity than those with elec-
tron-withdrawing groups.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.01.027.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
It should be mentioned that anilides with a single substitute at
the para-positions of the benzene ring exhibited the most powerful
inhibitory activities against MMP-2 and MMP-9, anilides with a

Z.-H. Shi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1206–1211
1211
30. Legoabe, L.; Kruger, J.; Petzer, A.; Bergh, J. J.; Petzer, J. P. Eur. J. Med. Chem. 2011,
46, 5162.
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