Synthesis and ABTS radical, MMP-1 inhibitory activity of CAPE analogues

Article abstract of DOI:10.1002/jccs.200800105

In the photoaging process of skin, the ultraviolet (UV)-induced reactive oxygen species (ROS) is the key regulator of matrix metalloproteinase (MMPs) expression. In this study, a series of Caffeic acid phenethyl ester (CAPE) analogues were synthesized by

Full text of DOI:10.1002/jccs.200800105

698
Journal of the Chinese Chemical Society, 2008, 55, 698-703
Note
Synthesis and ABTS Radical, MMP-1 Inhibitory Activity of CAPE
Analogues
Chih-Chia Chianga (
Tsu-Chung Changc* (
), Ling-Yih Hsub* (
) and Hou-Jen Tsaia (
),
)
aDepartment of Applied Chemistry and Materials Science, Institute of Technology,
National Defense University, Ta-shi, Tao-yuan, Taiwan, R.O.C.
bDepartment of Biological Science and Technology, China Institute of Technology,
Nangang, Taipei, Taiwan, R.O.C.
cDepartment of Biochemistry, National Defense Medical Center, Neihu, Taipei, Taiwan, R.O.C.
In the photoaging process of skin, the ultraviolet (UV)-induced reactive oxygen species (ROS) is the
key regulator of matrix metalloproteinase (MMPs) expression. In this study, a series of Caffeic acid
phenethyl ester (CAPE) analogues were synthesized by conjugating the group VI elements (selenium, sul-
fur, oxygen)-containing aliphatic alcohols to polyphenolic acids. Their biological activities were evalu-
ated by in vitro testing of their radical scavenging activity the of ABTS [2,2¢-azinobis-(3-ethyl-benzo-
thiazoline-6-sulfonic acid)] radical and inhibitory effect against the matrix metalloproteinase-1 (MMP-1)
activity of collagen degradation and cytotoxicity of a human dermal fibroblast skin cell. Our results sug-
gest these compounds displayed moderate anti-free radical, potent MMP-1 inhibitory, and low cytotoxic
activities.
Keywords: CAPE; ABTS radical; MMP-1 inhibitor.
INTRODUCTION
The matrix metalloproteinases (MMPs) comprise a
specific inhibitor) or indirectly (by reducing MMP expres-
sion) may provide an effective therapeutic method for
counteracting photoaging.10,11
relatively large and ever growing family. There are now
more than 28 enzymes that are classified as MMPs.1 These
enzymes have both a descriptive name (e.g. interstitial col-
lagenase, an enzyme found in the interstitial space, which
degrades fibrillar collagens) and an MMP number. The
MMPs’ structure related zinc-dependent endopeptidases
can degrade a wide variety of extracellullar matrix compo-
nents and play important roles in tissue remodeling during
developmental morphogenesis, angiogenesis, tissue repair,
arthritic inflammation, tumor invasion and metastasis.2,3
Recent studies showed the development of epidermal hy-
perplasia, the formation of skin wrinkles and significant
enhancement of MMPs’ (i.e. MMP-1, MMP-2 and MMP-
9) activities when human skin is exposed to UV.4-6 Inhibi-
tion of MMPs’ activity by a specific MMP inhibitor sup-
pressed UV-induced epidermal thickness enhancement and
wrinkle formation.7-9 These results suggest that MMPs may
be directly involved in the skin photoaging process. Thus,
inhibition of MMP activity either directly (by means of a
Oxidative stress has been reported to play an impor-
tant role in the development of the various detrimental ef-
fects of UV-radiation. UV was found to increase the level
of hydrogen peroxides and other reactive oxygen species
(ROS) in skin tissues.12-14 The ROS may cause oxidative
damage to lipids, proteins, and DNA directly, but may also
modulate their expressions through signal transduction
pathways and ultimately, lead to skin damage.
CAPE (Fig. 1), a natural flavonoid-like and polyphe-
nolic ester compound, is one of the major components of
honeybee propolis.15 It has been reported that CAPE has
various biological activities, such as antiviral, antiflam-
Fig. 1. Structure of CAPE.
* Corresponding author. E-mail: g970203@mail.ndmctsgh.edu.tw (C-C Chiang) or E-mail: tcchang@ndmctsgh.edu.tw (T-C Chang)

CAPE Analogues as MMP-1 Inhibitor
J. Chin. Chem. Soc., Vol. 55, No. 3, 2008 699
matory, anticarcinogenic and immunomodulatory proper-
ties.16 Likewise, CAPE was shown17 to inhibit lipooxygen-
ase activities, suppress ROS-induced lipid peroxide (LPO)
in tissues, and display antioxidant activity.18 To identify po-
tential antioxidants and anti-UV-induced photo damaging
agents, it is important to study various CAPE analogues on
their ROS scavenging and anti-MMP-1 activities.
carried out according to the Scheme outlined in Fig. 2. Se-
lenium-containing aliphatic alcohol (2-phenylselenoetha-
nol; 1) was prepared from 2-chloroethanol with phenyl-
selenol produced in situ by a reduction of diphenyl dise-
lenide with sodium borohydride. Thus, polyphenolic acids
(2, 3) were reacted directly with 2-phenylselenoethanol (1)
or commercially obtained 2-phenythiol-ethanol (4) and 2-
phenoxy-ethanol (5), respectively, in the presence of tri-
phenylphosphine (TPP), diisopropyl azodicarboxylate
(DIAD), and tetrahydrofuran to give the target compounds
(6a-6c, 7a-7c).
Recently, studies in our laboratory have been directed
at synthetic antioxidants with diverse anti-oxidative func-
tionalities. Based on the similar chemical properties of the
group VI elements (oxygen, sulfur, selenium), we specu-
lated that ester analogues of an aliphatic alcohol containing
group VI elements and polyphenolic acid might provide
synergistic antioxidative activity. In the present study, a se-
ries of oxygen, sulfur, and selenium-containing polyphe-
nolic acid esters was prepared. Their in vitro efficacy as
radical scavengers, inhibition against MMP-1, and cyto-
toxicity in human dermal fibroblasts was investigated.
Free radical ABTS assay
The model of scavenging ABTS free radicals is a con-
venient method20 to evaluate the in vitro antioxidative ac-
tivity of antioxidants. As shown in Table 1, these com-
pounds displayed various degrees of free radical scaveng-
ing activity, with decreasing activity in the following order:
CAPE > Trolox > 7b > 6c > 6b > 7c > 7a > 6a. Among
them, compounds 7b and 6c, were the most potent, having
antiradical effects comparable to that of Trolox. Unfortu-
nately, all modified CAPE analogues showed less free radi-
cal scavenging activity than CAPE. These results indicated
RESULTS AND DISCUSSION
Synthesis of compounds
The synthesis of compounds used in this study19 was
Fig. 2. Synthesis pathway of target compounds.

700 J. Chin. Chem. Soc., Vol. 55, No. 3, 2008
Chiang et al.
Table 1. ABTS Radical cation quenching activity of compounds
Table 2. Relative MMP-1 inhibitory efficiency of compounds
6a-7c versus CAPE and GM-1489
6a-7c Versus Trolox and CAPE
ABTS radical
Compound (20 mM)
Scavenging activity (%)
MMP-1 inhibit
Compound (66 mM)
Activity (%)
Trolox
CAPE
6a
35.36 ± 0.98
67.17 ± 0.48
21.52 ± 2.87
29.70 ± 1.21
31.10 ± 3.67
23.69 ± 2.26
31.26 ± 1.98
28.34 ± 1.77
DMSO
CAPE
6a
1.2 ± 3.52
19.0 ± 8.250
58.3 ± 39.18
70.2 ± 27.04
31.0 ± 45.22
59.8 ± 48.92
33.3 ± 23.78
21.0 ± 15.90
101.2 ± 8.9900
6b
6b
6c
6c
7a
7a
7b
7b
7c
7c
GM-1489
Results represent mean ± SD (n = 3).
Data are calculated from the relative band intensity of Fig. 3.
Lane-Collagen represents 0% MMP-1 activity and Lane DMSO
represents 100% of MMP-1 activity. The relative MMP-1
inhibitory efficiency (%) of each compound = [1-(collagen-
compound)/(collagen-DMSO)] ´ 100.
that the design of an introduction of group VI elements into
CAPE failed to enhance free radical scavenging activity.
Collagen degradation assay
In the collagen degradation assay,14,21 MMP-1 en-
zyme and rat tail collagen were incubated with synthetic
compounds and the potency of these compounds on inhibi-
tion of MMP-1 activity was assessed by SDS-PAGE gel
electrophoresis. As shown in Fig. 3, in the absence of
MMP-1, the original form of the collagen sample has two
major bands. In the presence of MMP-1 but with no inhibi-
tory compound (lane-DMSO), the major collagen bands
were digested into smaller fragments after 18 h of incuba-
tion. Lane-DMSO, with no apparent MMP-1 inhibitory ac-
tivity, was used as a negative control for this assay. The
known MMP-1 inhibitor, GM-1489, was used as a positive
control for protection of collagen from further degradation
(lane-GM-1489). To evaluate the efficacy of these com-
pounds as MMP-1 inhbitiors, the density of the second
band from the top of the gel was quantitated (marked with
an arrow, Fig. 3). The relative MMP-1 inhibitory efficiency
of these compounds is in the following order: GM-1489 >
6b > 7a > 6a > 7b > 6c > 7c > CAPE > DMSO (Table 2).
Luckily, all modified CAPE analogues showed more effec-
tive collagen degradation activity than CAPE. These re-
sults indicated that the introduction of group VI elements
into CAPE may be beneficial on collagen degradation
against MMP-1. It is of interest to note that the modified
selenium and sulfur analogues of CAPE showed very po-
tent activity.
Fig. 3. Inhibition of MMP-1-mediated collagen degra-
dation by compounds 6a-7c. Collagen degrada-
tion. Type I collagen was cleavaged by purified
MMP-1 in the presence of various compounds.
G: GM-1489 (broad-spectrum inhibitor of
MMPs). The relative inhibitory potency of test
compounds on MMP-1 activity are expressed
as relative intensity of the second bands from
top of the gel (indicated by arrow). Each bar is
the mean ± SD of three independent experi-
ments. *P < 0.05; **P < 0.01 compared with
lane-DMSO.
Cytotoxicity of synthetic test compounds 6a-7c
Concentration-dependent cytotoxicity22 of 6a-7c was
determined by a CCK-8 assay on human derma fibroblast
(HDF) cells. Results presented in Fig. 4 show cell viability
upon treatment with increasing concentrations of test com-
pounds after 48 h. With less than 25 mM, these compounds
displayed no apparent cytotoxicity as compared with the

CAPE Analogues as MMP-1 Inhibitor
J. Chin. Chem. Soc., Vol. 55, No. 3, 2008 701
control cells that were treated with 0.01% DMSO. Signifi-
cant cytotoxicity was observed in cells treated with over 50
mM of these compounds.
75 MHz, respectively. Where necessary, deuterium ex-
change experiments were used to obtain proton shift as-
signments. Mass spectra were recorded on a JEOL J.M.S-
300 spectrophotometer. Analytical samples were dried un-
der reduced pressure at 78 °C in the presence of P2O5 for at
least 12 h unless otherwise specified. Elemental analyses
were obtained using a Perkin-Elmer 2400 Elemental Ana-
lyzer.
In summary, we designed and synthesized a series of
CAPE analogues and evaluated these new compounds for
inhibition of free radical and collagenase (MMP-1) activ-
ity. In addition, the cytotoxicity of these compounds in
HDF cells was also determined. Our results suggest that
these compounds displayed moderate anti-free radical, po-
tent MMP-1 inhibitory, and low cytotoxic activities and
may have the potential of being used as functional cosmetic
agents. It should be noted that these compounds can be eas-
ily prepared by a single step; therefore, industrial mass pro-
duction may be possible. In our further studies, the effect of
these newly synthesized CAPE analogues of polyphenol
derivatives on UV-induced MMP-1 activity and the regula-
tory mechanism will be examined in human dermal fibro-
blasts.
2-Phenylselenoethanol (1)
Sodium borohydride (1.2 g, 15 mmol) was added in
portions to a stirred solution of diphenyl diselenide (3.5 g,
11 mmol) in absolute ethanol at 0 °C. To the resulting color-
less solution was added a solution of the appropriate 2-
chloroethanol (1.8 g, 22 mmol) dissolved in the minimum
quantity of ethanol. The mixture was stirred under reflux
for 3 h. Solvent was filtered and removed in a vacuum. The
residue was purified by flash chromatography on silica gel
with n-hexane/ethyl acetate (3/1) to give 2-phenylseleno-
ethanol (4.1 g). Yield 92%, as pale yellow oil, IR (KBr) n
(cm-1): 3452 (OH). 1H-NMR (DMSO-d6) d: 7.487.21 (m,
5H, ArH), 4.95 (s, 1H, OH), 3.60 (t, 2H, J = 6.4 Hz, CH2),
3.00 (t, 2H, J = 14.2 Hz, SeCH2); 13C-NMR (DMSO-d6) d:
131.2, 130.2, 129.2, 126.4, 60.7, 29.4. FAB-MS, m/z: 202
(Ca1cd for C8H10OSe: 200.13). Anal. Ca1cd for C8H10OSe:
C, 47.77; H, 5.01. Found: C, 47.67; H, 5.03.
EXPERIMENTAL SECTION
Instrumentation
Melting points (mp) were taken on a BUCHI 530 ap-
paratus and are uncorrected. Merck Art No. 105554 plates
precoated with Silica gel 60 containing a fluorescent indi-
cator were used for thin-layer chromatography, and Silica
gel 60 (Merck Art No 109385, 230-400 mesh) was em-
ployed for column chromatography. Evaporations were
carried out at < 50 °C using a rotary evaporator at reduced
pressure (water aspirator). 1H- and 13C-NMR spectra were
obtained with a Varian 300 NMR spectrometer at 300 and
Mitsunobu Esterification of Polyphenolic Acids Gen-
eral Procedure
To a solution of polyphenolic acids (2.3 g, 15 mmol)
and 2-phenylselenoethanol (2 g, 10 mmol) in dry tetrahy-
drofuran (35 mL) were added TPP (15 mmol) and DIAP
(15 mmol) at 0 °C. After stirring at room temperature for 48
h, the reaction was worked up by removal of the solvent,
and the residue was partitioned between ethyl acetate and
saturated NaHCO3. The organic phase was washed with
brine and then dried over Na2SO4, and the solvent was
evaporated. The residue was purified by flash chromatog-
raphy on a silica gel column with 1:1 n-hexane/ethyl ace-
tate as eluent to give 6a (1.9 g, 55%). The compounds 6b-
7c were obtained under the same conditions.
4-Dihydroxy-benzoic acid-(2-phenylseleno-ethyl ester)
(6a)
Yield 55%, mp. 90-92 °C (n-hexane/ethyl acetate); IR
(KBr) n (cm-1): 3253 (ArC-OH), 3023 (ArC-H), 1738
(C=O), 1119 (C-O). 1H-NMR (CDCl3) d: 7.62-6.90 (m, 8H,
ArH), 6.41 (s, 2H, OH), 4.53 (t, J = 14.2 Hz, 2H, COOCH2),
3.24 (t, J = 13.2 Hz, 2H, SeCH2). 13C-NMR (CDCl3) d:
165.95, 157.08, 156.41, 133.03, 132.00, 126.18, 108.98,
Fig. 4. Viability of HDF cells treated with different
concentrations of 6a-7c. Results represent mean
± SD (n = 3).

702 J. Chin. Chem. Soc., Vol. 55, No. 3, 2008
Chiang et al.
107.526, 70.31, 64.53, 25.45, 21.86; FAB-MS m/z: 338
(Ca1cd for C15H14O4Se: 337.24). Anal. Ca1cd for
C15H14O4Se: C, 53.42; H, 4.18. Found: C, 53.46; H, 4.22.
3,4-Dihydroxy-benzoic acid-(2-thiolphenoxy-ethyl es-
ter) (6b)
SCH2). 13C-NMR (DMSO-d6) d: 166.31, 151.26, 149.19,
145.10, 131.75, 129.51, 129.41, 126.96, 123.07, 115.32,
111.81, 110.71, 63.35, 55.81, 55.77, 25.07. FAB-MS m/z:
317 (Ca1cd for C17H16O5: 316.38). Anal. Ca1cd for
C17H16O5: C, 64.54; H, 5.1. Found: C, 65.86; H, 5.26.
3,4-Dihydroxy-cinnamic acid-(2-phenoxy-ethyl ester)
(7c)
Yield 42%, mp. 116-118 °C (n-hexane/ethyl acetate);
IR (KBr) n (cm-1): 3296 (ArC-OH), 2927 (ArC-H), 1683
(C=O), 1126 (C-O). 1H-NMR (CDCl3) d: 7.55-7.30 (m, 7H,
ArH), 6.93 (d, J = 8.7 Hz, 2H, ArH), 6.68 (s, 2H, OH), 4.47
(t, J = 7.2 Hz, 2H, OCH2), 3.29 (t, J = 7.2 Hz, 2H, SCH2).
13C-NMR (DMSO-d6) d: 169.30, 155.89, 147.68, 135.09,
130.27, 129.38, 129.12, 129.07, 126.84, 126.65, 125.13,
114.69, 111.89, 64.07, 32.57. FAB MS m/z: 291 (Ca1cd for
C15H14O5S: 290.34 ). Anal. Ca1cd for C15H14O5S: C, 62.05;
H, 4.86. Found: C, 63.23; H, 4.98.
Yield 33%, mp. 127-128 °C (n-hexane/ethyl acetate);
IR (KBr) n (cm-1): 3210 (ArC-OH), 2990 (ArC-H), 1720
(C=O), 1120 (C-O). 1H-NMR (DMSO-d6) d: 7.61-6.73 (m,
8H, ArH), 6.31 (d, J = 15.9 Hz, 1H, CH), 4.09 (t, J = 4.5 Hz,
2H, COOCH2), 4.21 (t, J = 3.9 Hz, 2H, OCH2). 13C-NMR
(DMSO-d6) d: 167.24, 149.24, 146.33, 164.23, 132.67,
132.12, 130.21, 129.49, 129.33, 126.23, 122.08, 121.54,
116.50, 115.64, 115.35, 66.47, 63.07. FAB-MS m/z: 301
(Ca1cd for C17H16O5: 300.31). Anal. Ca1cd for C17H16O5:
C, 67.99; H, 5.37. Found: C, 68.03; H, 5.41.
3,4-Dihydroxy-benzoic acid-(2-phenoxy-ethyl ester)
(6c)
Yield 58%, mp: 118-120 °C (n-hexane/ethyl acetate);
IR (KBr) n (cm-1): 3296 (ArC-OH), 2927 (ArC-H), 1683
(C=O), 1128 (C-O). 1H-NMR (CDCl3) d: 7.66-6.92 (m, 8H,
ArH), 4.67 (t, J = 3.9 Hz, 2H, COOCH2), 4.33 (t, J = 4.8 Hz,
2H, OCH2); 13C-NMR (DMSO-d6) d: 169.03, 134.46, 162.90,
158.37, 131.82, 129.34, 121.03, 114.81, 108.52, 104.05,
65.81, 63.40. FAB-MS m/z: 275 (Ca1cd for C15H14O5:
274.28). Anal. Ca1cd for C15H14O5: C, 65.69; H, 5.15.
Found: C, 64.73; H, 5.19.
Evaluation of the antioxidant activity by ABTS radi-
cal scavenging test
In vitro antioxidant activity of test compounds was
evaluated using an improved ABTS.+ scavenging assay de-
scribed by Re et al.23 Briefly, the purple ABTS.+ solution
(7.5 mM) was prepared by mixing ABTS solution with po-
tassium persulfate (2.5 mM) overnight in the dark at room
temperature. The ABTS.+ solution was diluted with ethanol
or PBS (pH 7.4) to an OD734 of 0.70 ± 0.02 for assay. Six
minutes after the 5 mL of studied compounds was added to
30 mL of ABTS.+ dilution, the amount of ABTS.+ remaining
was determined at 734 nm, and the radical scavenging ac-
tivity was obtained from the following equation:
3,4-Dihydroxy-cinnamic acid-(2-phenylseleno-ethyl es-
ter) (7a)
Yield 32%, mp. 70-72 °C (n-hexane/ethyl acetate); IR
(KBr) n (cm-1): 3251 (ArC-OH), 2979 (ArC-H), 1737
(C=O), 1109 (C-O). 1H-NMR (CDCl3) d: 7.62-7.01 (m, 8H,
ArH), 6.92 (d, J = 10.2 Hz, 1H, COCH), 6.39 (s, 2H, OH),
6.25 (d, J = 8.1 Hz, 1H, CH), 4.45 (t, J = 14.7 Hz, 2H,
COOCH2), 3.19 (t, J = 14.7 Hz, 2H, SeCH2). 13C NMR
(CDCl3) d: 166.95, 156.31, 146.42, 145.08, 144.01, 132.86,
129.13, 127.52, 127.23, 122.33, 115.40, 115.23, 114.26,
70.18, 63.76, 25.58, 21.87. FAB-MS m/z: 364 (Ca1cd for
C17H16O4Se: 363.27). Anal. Ca1cd for C17H16O4Se: C,
56.21; H, 4.44. Found: C, 56.27; H, 4.51.
Radical scavenging activity (%)
= [(ODcontrol - ODsample)/ODcontrol] ´ 100.
Collagen degradation assay
Rat tail collagen 5.9 g/mL (BD Biosciences, San Jose,
CA USA) was diluted to 238 mg/mL in Tris-glucose (0.5 M
Glucose and 0.33 M Tris). 48 mg/mL of purified MMP-1
(Calbiochem, San Diego, CA, USA) was diluted to 6 ´ 10-4
mg/mL with Tris-glucose. Degradation of the native colla-
gen was carried out by mixing 19 mL of MMP-1 solution, 9
mL of collagen solution and 2 mL of 1 mM synthetic com-
pounds and incubated for 18 h at 37 °C. Intact collagen ex-
posed to buffer alone served as control. Collagen fragments
released were resolved by SDS-PAGE (8.5% gel) and
staining with Coomassic brilliant blue R-250. The relative
intensity of the protein bands were measured by an image
3,4-Dihydroxy-cinnamic acid-(2-thiolphenoxy-ethyl es-
ter) (7b)
Yield 32%, mp. 123-126 °C (n-hexane/ethyl acetate);
IR (KBr) n (cm-1): 3212 (ArC-OH), 2988 (ArC-H), 1720
(C=O), 1120 (C-O). 1H-NMR (CDCl3) d: 7.60 (d, J = 15.6
Hz, 1H, COCH), 7.55-6.89 (m, 8H, ArH), 6.27 (d, J = 15.9
Hz, CH), 7.47-6.92 (m, 8H, ArH), 6.74 (d, J = 15.6 Hz, 1H,
CH), 4.07 (t, J = 6.9 Hz, 2H, OCH2), 3.26 (t, J = 7.2 Hz, 2H,

CAPE Analogues as MMP-1 Inhibitor
J. Chin. Chem. Soc., Vol. 55, No. 3, 2008 703
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ACKNOWLEDGMENTS
The authors would like to thank the School of Phar-
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Hsu) and National Defense Medical Center (DOD-96-17 to
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