Synthesis of clovamide analogues that inhibit no production in activated BV-2 microglial cells

Article abstract of DOI:10.1248/bpb.b17-00303

A series of methyl ester of clovamide analogues, where the hydroxyl group of catechol moiety in caffeic acid and L-3,4-dihydroxyphenylalanine (L-dopa) was replaced with various functional groups, were synthesized and their inhibitory effects on nitric oxi

Full text of DOI:10.1248/bpb.b17-00303

Biological and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/bpb.b17-00303
Advance Publication
June 8, 2017
Biol. Pharm. Bull.
Regular Article
Synthesis of Clovamide Analogues that Inhibit NO Production in Activated BV-2
Microglial Cells
Ju-Young Parka, Byung-Wook Kimb , Hae Un Leea, Dong-Kug Choib and Sung-Hwa Yoona, *
aDepartment of Molecular Science and Technology, Ajou University; 206 Worldcup-ro,
Yeongtong-gu, Suwon 16499, Republic of Korea.
2Department of Biotechnology, Konkuk University; 268 Chungwon-daero, Chungju 27478,
Republic of Korea
* To whom correspondence should be addressed. E-mail: shyoon@ajou.ac.kr
Ⓒ 2017 The Pharmaceutical Society of Japan

Summary
A series of methyl ester of clovamide analogues, where the hydroxyl group of catechol
moiety in caffeic acid and L-dopa was replaced with various functional groups, were
synthesized and their inhibitory effects on nitric oxide (NO) production and inducible NO
synthase expression in lipopolysaccharide-induced BV2 cells were tested. Among the
synthesized compounds, 3,5-ditrifluoromethyl analogue 9l (IC50 = 2.8 μM) exhibited a
potency about 26.3 times greater than that of the parent compound 9a (IC50 = 73.6 μM) and
suppressed NO production dose-dependently without cytotoxicity. Compound 9l also
inhibited iNOS expression in LPS-induced BV2 cells at 2.5, 5 and 10 μM concentrations.
These results suggested that the dihydroxyl group of catechol moiety in caffeic acid unit is
not essential for the suppression of NO production and that 9l has potential as a potent
inhibitor of NO production.
Keywords: Clovamide methyl ester; trifluoromethyl analogue; microglia; BV2 cell; nitric
oxide
Biological and Pharmaceutical Bulletin Advance Publication

Introduction
Nitric oxide (NO), a biological mediator in living organisms, plays an important role in
cardiovascular, immune and neuronal systems. The highly reactive peroxynitrite anion
(ONOO-), produced by the reaction of NO with superoxide anions, leads to oxidative damage
in host tissue, modification of DNA bases, and disruption of enzyme function and structural
proteins.1-3) Furthermore, the high level of NO production triggered by the transcriptional
activation and production of inducible NO synthase (iNOS) in pathological situations has
been considered an indicator of cytotoxicity in inflammation.4) Therefore, NO production is a
critical step in numerous neurodegenerative diseases, and reduced NO production is a
promising therapeutic strategy for the reduction of neuronal cell injury or death in various
neuroinflammatory and neurodegenerative diseases.5-7)
Clovamide, also known as N-caffeoyl-L-dihydroxyphenyl-alanine, is an amide isostere
of rosmarinic acid and has two distinctive functional moieties (caffeic acid and L-dopa)
linked by an amide bond (Fig. 1).8) Clovamide, which was originally identified in red clover
(Trifolium pretense) and later discovered in cocoa liquor (Theobroma cacao), has been found
in only a few plants in nature, but plays an important role in protecting plants against stress or
9-12)
pathogen attacks via oxidative enzyme as a part of defense system.
In addition to the
possession of anti-oxidant properties,13-14) clovamide also has a binding affinity for the
p56lck SH2 domain, which is relevant to the treatment of immune disorders.15) Moreover,
clovamide has anti-inflammatory effects via three different mechanisms (inhibition of
superoxide anion production, pro-inflammatory cytokine release, and NF-kB activation) in
human monocyte cells,16) protective effects against oxidative stress in the rat cardiomyoblast
cell line,17) as well as a neuroprotective effect in three different in vitro models of cell death
(oxidative stress, excitotoxicity and OGD/reoxygenation).18) Although evidence suggested
that clovamide can be an interesting plant-derived phenolic compound with expected
Biological and Pharmaceutical Bulletin Advance Publication

beneficial effects, until now its physiological activity has not been adequately recognized
because clovamide is not commercially available.
Most previous research related to clovamide was performed on clovamide or clovamide-
type derivatives obtained directly by separation of extracts from plants. Only a few studies
conducted with the synthesized analogues or derivatives, such as lipophilic clovamide–type
derivatives such as N-caffeoyl tyrosine methyl ester, N-ferulyl L-dopa alkyl ester and N-
sinapinyl L-dopa alkyl ester (alkyl = methyl, dodecyl), have revealed potent antioxidant
activities in bulk lipids and moderated protectants in emulsions.8, 19)
As part of our program to find a potent inhibitor of NO production, we recently found
that clovamide derivative, 3-(3,4-dihydroxy-phenyl)-2-[4-(3-trifluoromethylphenyl)-but-2-
enoylamino]-propionic acid methyl ester, revealed anti-inflammatory effect in
lipopolysaccharide (LPS) stimulated rat derived primary cells, as well as murine BV-2
microglial cells.20) With the aim of improving the potency and elucidating the pharmacophore
of clovamide, we modified clovamide by replacing the catechol group in caffeic acid and L-
dopa with various functional groups. This paper provides details on the synthesis of various
clovamide analogues and their inhibitory activities on NO production and iNOS expression
on BV2 cells stimulated with LPS.
Materials and methods
Instruments
Melting points were determined on a Fisher-Johns melting point apparatus and were left
1
uncorrected. H NMR and 13C NMR spectra were recorded on a Varian Gemini 400
spectrometer at 400 MHz and 100 MHz, respectively. The chemical shifts given are relative
to tetramethylsilane. Infrared spectra were recorded on a Nicolet 6700 FT-IR spectrometer.
ESI-MS spectra were obtained by Shimadzu LCMS-2010EV. Column chromatography was
Biological and Pharmaceutical Bulletin Advance Publication

carried out using Merck silica gel 60 (230-400 mesh). All reactions were performed under a
nitrogen atmosphere.
Methyl (2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate hydrochloride (5a) To a
solution of L-dopa (10.0 g, 50.7 mmol) in MeOH (300 mL) was added thionyl chloride (11.1
mL, 152 mmol) dropwise. The mixture was heated to reflux temperature for 8 h. After the
solution was cooled to room temperature, the solvent was evaporated in vacuo to obtain the
title compound as a white foam (12.0 g, 95.2%). 1H-NMR (DMSO-d6) δ: 2.88-2.95 (1H, m),
2.98-3.04 (1H, m), 3.64 (3H, s), 4.04-4.10 (1H, m), 6.40-6.45 (1H, d), 6.40-6.50 (1H, d, J =
13
8.0 Hz), 6.59 (1H, s), 6.66-6.70 (1H, d, J = 7.6 Hz); C-NMR (DMSO-d6) δ: 35.34, 52.55,
53.45, 115.64, 116.55,119.98, 124.67, 144.42, 145.06, 169.18.
Methyl (2S)-2-amino-3-(3,4-difluorophenyl)propanoate hydrochloride (5b) To a
solution of (S)-2-((tert-butoxycarbonyl)amino)-3-(3,4-difluorophenyl)propanoic acid (550 mg,
183 mmol) in 1,4-dioxane (10 mL) was added 4 N HCl in 1,4-dioxane (5 mL). After the
mixture was stirred at room temperature for 4 h, hexane (10 mL) was added and stirred an
additional 0.5 h. The resulting solid was filtered in vacuo and dissolved in MeOH (30 mL)
was added thionyl chloride (397 μL, 152 mmol) dropwise. The mixture was heated to reflux
temperature for 8 h. After the solution was cooled to room temperature, the solvent was
evaporated in vacuo to obtain the title compound as a white foam (330 mg, 71.9%). 1H-NMR
(CDCl3) δ: 2.70-2.80 (1H, m), 2.90-3.00 (1H, m), 3.55-3.63 (1H, m), 3.64 (3H, s), 6.80-6.88
(1H, m), 6.92-7.05 (2H, m); 13C-NMR (CDCl3) δ: 40.13, 52.09, 55.62, 116.96 (d, JC-F = 16.7
Hz), 117.84 (d, JC-F = 16.6 Hz), 125.04, 134.12, 147.88 (d, JC-F = 244.9 Hz), 148.63 (d, JC-F
245.8 Hz); MS m/z: 216.00 ([M+H]+).
=
Biological and Pharmaceutical Bulletin Advance Publication

General procedure for the synthesis of compounds 7a-7b and 9a-9l To a solution of the
corresponding substituted acid (1 equiv, 2.02 mmol) in dichloromethane/DMF (3/1, 20 mL)
was added EDC (1 equiv, 2.02 mmol), DIEA (1 equiv, 2.02 mmol) and HOBt (1 equiv, 2.02
mmol). After the mixture was stirred at room temperature for 30 min, corresponding amine
(1equiv, 2.02 mmol) and TEA (3 equiv, 6.06 mmol) was added. The solution was stirred at
room temperature for 4 h and then extracted with dichloromethane. The organic layer was
washed with 1 N HCl solution, saturated NaHCO3 solution, water and brine, dried over
Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel column
chromatography (ethyl acetate/ hexane, 1/2) to obtain the title compound.
Methyl
(2S)-2-((2E)-3-(3,4-difluorophenyl)acrylamido)-3-(3,4-dihydroxyphenyl)
propanoate (7a): White foam (400 mg, 52.5%); IR (KBr) cm-1: 3358, 1734, 1620; 1H-NMR
(DMSO-d6) δ: 2.76-2.94 (2H, m), 3.62 (3H, s), 4.46-4.56 (1H, m), 6.42-6.48 (1H, d, J = 8.4
Hz), 6.56-6.62 (2H, m), 6.62-6.70 (1H, d, J = 14.8 Hz), 7.32-7.50 (3H, m), 7.60-7.68 (1H, t, J
= 10.4 Hz); 13C-NMR (DMSO-d6) δ: 36.43, 51.95, 54.22, 115.34, 116.14, 116.25, 117.850 (d,
JC-F = 17.4 Hz), 119.72, 122.67, 124.69, 127.61, 132.54, 137.27, 143.83, 144.85, 148.21 (d,
JC-F = 243.4 Hz), 148.55 (d, JC-F = 247.2 Hz), 164.44, 171.94; MS m/z: 378.10 ([M+H]+).
Methyl
3-(3,4-difluorophenyl)-(2S)-2-((2E)-3-(3,4-dihydroxyphenyl)acrylamido)
1
propanoate (7b): White foam (50 mg, 55.6%); IR (KBr) cm-1: 3352, 1744, 1669; H-NMR
(DMSO-d6) δ: 2.90-2.98 (1H, m), 3.04-3.12 (1H, m), 3.62 (3H, s), 4.56-4.68 (1H, m), 6.40-
6.45 (1H, m), 6.70-6.78 (1H, m), 6.80-6.88 (1H, m), 6.92-7.00 (1H, m), 7.05-7.12 (1H, m),
7.20-7.28 (1H, m), 7.30-7.40 (2H, m), 8.40-8.58 (1H, m), 9.20 (1H, s), 9.39 (1H, s); 13C-
NMR (DMSO-d6) δ: 35.83, 51.98, 53.40, 113.74, 115.63, 116.91 (d, J = 16.7 Hz), 117.21,
117.88 (d, J = 16.7 Hz), 120.52, 125.79, 125.94, 134.91, 140.03, 145.36, 146.86 (dd, J =
69.7, 12.1 Hz), 147.37, 149.28 (dd, J = 70.6, 12.1 Hz), 165.24, 171.69; MS m/z: 378.12
([M+H]+).
Biological and Pharmaceutical Bulletin Advance Publication

Methyl
3-(3,4-dihydroxyphenyl)-(2S)-2-((2E)-3-(3,4-dihydroxyphenyl)acrylamido)
propanoate (9a): White foam (250 mg, 33.2%); IR (KBr) cm-1: 3397, 1728, 1603; 1H-NMR
(DMSO-d6) δ: 2.70-2.80 (1H, m), 2.82-2.90 (1H, m), 3.59 (3H, s), 4.40-4.50 (1H, m), 6.34-
6.41 (1H, d, J = 15.6 Hz), 6.42-6.48 (1H, dd, J = 8.0, 2.0 Hz), 6.53-6.62 (2H, m), 6.70-6.74
(1H, d, J = 8.0 Hz), 6.75-6.83 (1H, dd, J = 8.0, 1.6 Hz), 6.92 (1H, s), 7.10-7.25 (1H, d, J =
15.2 Hz), 8.30-8.40 (1H, d, J = 7.2 Hz); 13C-NMR (DMSO-d6) δ: 36.44, 51.79, 54.12, 113.71,
115.23, 115.59, 116.17, 117.50, 119.60, 120.41, 126.00, 127.66, 139.74, 143.73, 144.75,
145.31, 147.27, 165.12, 172.11; MS (ESI) m/z 374.10 (M+H+).
Methyl
3-(3,4-dihydroxyphenyl)-(2S)-2-((2E)-3-(3-hydroxyphenyl)acrylamido)
propanoate (9b): White foam (300 mg, 41.6%); IR (KBr) cm-1: 3358, 1723, 1598; 1H-NMR
(DMSO-d6) δ: 2.72-2.80 (1H, m), 2.84-2.92 (1H, m), 3.60 (3H, s), 4.44-4.52 (1H, q, J = 4.0
Hz), 6.40-6.48 (1H, d, J = 8.0 Hz), 6.54-6.60 (2H, m), 6.60-6.64 (1H, m), 6.72-6.80 (1H, d, J
= 8.0 Hz), 6.89 (1H, s), 6.94-6.98 (1H, d, J = 7.2 Hz), 7.16-7.22 (1H, t, J = 7.2 Hz), 7.24-7.32
13
(1H, d, J = 15.2 Hz); C-NMR (DMSO-d6) δ: 36.44, 51.89, 54.18, 113.65, 115.27, 116.19,
116.72, 118.65, 119.65, 121.02, 127.60, 129.82, 135.80, 139.49, 143.77, 144.79, 157.45,
164.72, 171.99; MS m/z: 358.15 ([M+H]+), 396.10 ([M+K]+).
Methyl
3-(3,4-dihydroxyphenyl)-(2S)-2-((2E)-3-(4-hydroxyphenyl)acrylamido)
propanoate (9c): White foam (250 mg, 34.7%); IR (KBr) cm-1: 3347, 1723, 1598; 1H-NMR
(DMSO-d6) δ: 2.70-2.80 (1H, m), 2.84-2.90 (1H, m), 3.60 (3H, s), 4.44-4.50 (1H, m), 6.42-
6.50 (2H, m), 6.56-6.62 (2H, m), 6.74-6.82 (2H, d, J = 8.0 Hz), 7.24-7.32 (1H, d, J = 16.0
Hz), 7.36-7.40 (2H, d, J = 8.0 Hz); 13C-NMR (DMSO-d6) δ: 36.42, 51.79, 54.09, 115.22,
115.60, 116.15, 117.65, 119.60, 125.53, 127.63, 129.14, 139.34, 143.71, 144.74, 158.71,
165.71, 165.10, 172.07; MS m/z: 358.20 ([M+H]+), 396.15 ([M+K]+).
Methyl
3-(3,4-dihydroxyphenyl)-(2S)-2-((2E)-3-(3-fluorophenyl)acrylamido)
propanoate (9d): White foam (280 mg, 38.6%); IR (KBr) cm-1: 3342, 1734, 1608; 1H-NMR
Biological and Pharmaceutical Bulletin Advance Publication

(DMSO-d6) δ: 2.74-2.84 (1H, m), 2.86-2.94 (1H, m), 3.61 (3H, s), 4.46-4.56 (1H, m), 6.42-
6.70 (1H, m), 6.58-6.64 (2H, m), 6.70-6.78 (1H, d, J = 15.6 Hz), 7.16-7.24 (1H, t, J = 8.8 Hz),
13
7.36-7.48 (4H, m); C-NMR (DMSO-d6) δ: 36.41, 51.94, 54.19, 113.80 (d, JC-F = 22.0 Hz),
115.29, 116.21, 119.66, 122.83, 123.64, 127.55, 130.73, 130.81, 137.12, 137.20, 143.81,
144.82, 161.00 (JC-F = 241.9 Hz), 163.41, 164.42, 171.93; MS m/z: 360.20 ([M+H]+).
Methyl
3-(3,4-dihydroxyphenyl)-(2S)-2-((2E)-3-(4-fluorophenyl)acrylamido)
propanoate (9e): White foam (300 mg, 41.1%); IR (KBr) cm-1: 3352, 1745, 1669; 1H-NMR
(CDCl3) δ: 2.93-3.05 (2H, m), 3.63 (3H, s), 4.80-4.90 (1H, m), 6.15-6.28 (1H, d, J = 16.0 Hz),
6.35-6.50 (2H, m), 6.65-6.75 (1H, d, J = 8.0 Hz), 6.90-6.98 (2H, t, J = 8.8 Hz), 7.25-7.35 (2H,
m), 7.40-7.50 (1H, d, J = 15.2 Hz); 13C-NMR (CDCl3) δ: 36.83, 52.53, 53.73, 115.25, 115.55
(d, JC-F = 22.0 Hz), 116.00, 119.07, 120.95, 127.45, 129.57, 130.40, 140.76, 143.39, 144.07,
162.07 (d, JC-F = 249.5 Hz), 165.96, 172.15; MS m/z: 360.30 ([M+H]+).
Methyl
3-(3,4-dihydroxyphenyl)-(2S)-2-((2E)-3-(3-methoxyphenyl)acrylamido)
1
propanoate (9f): White foam (400 mg, 53.4%); IR (KBr) cm-1: 3348, 1734, 1641. H-NMR
(DMSO-d6) δ: 2.75-2.81 (1H, m), 2.86-2.92 (1H, m), 3.62 (3H, s), 3.78 (3H, s), 4.45-4.55
(1H, m), 6.42-6.50 (1H, m), 6.58-6.64 (2H, m), 6.66-6.74 (1H, d, J = 15.6 Hz), 6.92-6.98 (1H,
m), 7.10-7.16 (2H, m), 7.30-7.40 (2H, m), 8.42-8.50 (1H, d, J = 8.0 Hz); 13C-NMR (DMSO-
d6) δ: 36.41, 51.86, 55.09, 112.38, 115.34, 116.16, 119.60, 119.85, 121.59, 127.55, 129.81,
135.95, 139.17, 143.75, 144.77, 159.30, 164.62, 171.93; MS m/z: 371.14 ([M+H]+).
Methyl 3-(3,4-dihydroxyphenyl)-(2S)-2-((2E)-3-(m-tolyl)acrylamido)propanoate (9g):
1
White foam (300 mg, 41.8%); IR (KBr) cm-1: 3352, 1721, 1638; H-NMR (DMSO-d6) δ:
2.31 (3H, s), 2.75-2.83 (1H, m), 2.86-2.92 (1H, m), 3.62 (3H, s), 4.45-4.55 (1H, m), 6.44-
6.50 (1H, d, J = 7.6 Hz, m), 6.60-6.72 (3H, m), 7.14-7.20 (1H, d, J = 6.8 Hz), 7.25-7.40 (3H,
m), 8.40-8.50 (1H, d, J = 7.2 Hz), 8.76 (1H, s); 13C NMR (DMSO-d6) δ: 20.98, 36.43, 51.85,
Biological and Pharmaceutical Bulletin Advance Publication

54.15, 115.26, 116.18, 119.63, 121.11, 124.63, 127.57, 127.94, 128.66, 130.12, 134.47,
137.91, 139.32, 143.76, 144.78, 164.70, 171.96; MS m/z: 355.14 ([M+H]+).
Methyl
(2S)-2-((2E)-3-(3-chlorophenyl)acrylamido)-3-(3,4-dihydroxyphenyl)
propanoate (9h): White foam (400 mg, 52.7%); IR (KBr) cm-1: 3328, 1714, 1659; 1H-NMR
(DMSO-d6) δ: 2.75-2.85 (1H, m), 2.86-2.95 (1H, m), 3.62 (3H, s), 4.48-4.60 (1H, m), 6.44-
6.50 (1H, d, J = 7.6 Hz), 6.56-6.66 (2H, m), 6.72-6.82 (1H, d, J = 16.0 Hz), 7.32-7.47 (3H,
13
m), 7.50-7.57 (1H, m), 7.63 (1H, s), 8.36-8.58 (1H, d, J = 7.6 Hz), 8.76 (1H, s); C-NMR
(DMSO-d6) δ: 36.39, 51.86, 54.10, 115.52, 116.16, 119.60, 122.95, 125.86, 127.08, 127.46,
129.01, 130.55, 133.47, 136.83, 137.66, 143.76, 144.77, 164.29, 171.82; MS m/z: 375.09
([M+H]+).
Methyl
3-(3,4-dihydroxyphenyl)-(2S)-2-((2E)-3-(3-(trifluoromethyl)phenyl)
acrylamido)propanoate (9i): White foam (300 mg, 36.3%); IR (KBr) cm-1: 3353, 1734,
1614; 1H-NMR (DMSO-d6) δ: 2.74-2.82 (1H, m), 2.86-2.92 (1H, m), 3.62 (3H, s), 4.48-4.54
(1H, m), 6.52-6.55 (1H, d, J = 8.4 Hz), 6.56-6.64 (2H, m), 6.80-6.88 (1H, d, J = 16.8 Hz),
7.44-7.52 (1H, d, J = 16.8 Hz), 7.60-7.66 (1H, t, J = 8.4 Hz), 7.82-7.88 (1H, d, J = 8.4 Hz),
7.88-7.94 (1H, s); 13C-NMR (DMSO-d6) δ: 36.41, 51.93, 54.15, 115.26, 116.17, 119.63,
123.39, 123.82 (d, JC-F = 192.6 Hz), 127.45, 129.39 (d, JC-F = 31.1 Hz), 129.93, 131.22,
135.73, 137.57, 143.80, 144.80, 164.26, 171.85; MS m/z: 410.20 ([M+H]+). 20)
Methyl 3-(3,4-dihydroxyphenyl)-(2S)-2-((2E)-3-(4-(trifluoromethyl)phenyl)acrylamido)
1
propanoate (9j): White foam (400 mg, 48.4%); IR (KBr) cm-1: 3342, 1734, 1682; H-NMR
(CDCl3+ DMSO-d6) δ: 3.02-2.92 (m, 2H), 3.63 (s, 3H), 4.90-4.80 (m, 1H), 6.48-6.40 (d, J =
8.0 Hz, 1H), 6.61 (s, 1H), 6.70-6.65 (d, J = 8.0 Hz, 1H), 7.15-7.05 (m, 1H), 7.55-7.45 (m,
5H); 13C-NMR (CDCl3+ DMSO-d6) δ: 36.69, 51.77, 53.40, 114.62, 115.55, 120.40, 125.04 (d,
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JC-F = 218.5 Hz), 127.23(d, JC-F = 22.7 Hz), 127.45, 137.83, 138.80, 143.06, 143.92, 164.61,
171.36. MS m/z: 410.10 ([M+H]+).
Methyl (2S)-2-((2E)-3-(3,5-bis(trifluoromethyl)phenyl)acrylamido)-3-(3,4-dihydroxy-
phenyl)propanoate (9k): White foam (200 mg, 20.8%); IR (KBr) cm-1: 3348, 1734, 1614;
1H-NMR (DMSO-d6) δ: 2.76-2.82 (1H, m), 2.87-2.96 (1H, m), 3.62 (3H, s), 4.50-4.58 (1H,
m), 6.42-6.48 (1H, d, J = 7.2 Hz), 6.56-6.64 (2H, m), 6.97-7.04 (1H, d, J = 16.4 Hz), 7.54-
13
7.62 (1H, d, J = 16.4 Hz), 8.04 (1H, s), 8.24 (2H, s); C-NMR (DMSO-d6) δ: 36.43, 51.96,
54.15, 115.32, 116.20, 119.67, 121.70 (d, JC-F = 270.7 Hz), 122.29, 125.54, 127.41, 127.82,
130.55 (q, JC-F = 32.7 Hz), 136.04, 137.53, 143.84, 144.84, 163.97, 171.73; MS m/z: 478.20
([M+H]+).
Methyl
(2S)-2-((2E)-3-(3,5-bis(trifluoromethyl)phenyl)propanamido)-3-(3,4-
dihydroxyphenyl)propanoate (9l): White foam (500 mg, 51.7%); IR (KBr) cm-1: 3402,
1740, 1652; 1H-NMR (CDCl3+DMSO-d6) δ: 2.62-2.68 (2H, t, J = 7.6 Hz), 2.86-2.96 (2H, m),
3.04-3.14 (2H, t, J = 7.2 Hz), 3.66 (3H, s), 4.70-4.80 (1H, m), 6.36-6.42 (1H, d, J = 8.0 Hz),
6.62 (1H, s), 6.65-6.73 (1H, d, J = 8.0 Hz), 7.18 (1H, s), 7.56 (2H, s), 7.72-7.78 (2H, m); 13C-
NMR (CDCl3+DMSO-d6) δ: 30.42, 36.13, 36.40, 51.12, 52.90, 119.08, 119.93, 121.29 (d, JC-
= 269.2 Hz), 127.04, 128.11, 130.23 (d, JC-F = 33.4 Hz), 142.81, 143.21, 143.72, 170.41,
F
170.92; MS m/z: 480.15 ([M+H]+).
Immortalized microglia culture The BV2 cell (a mouse microglial cell line) developed
by Dr. V. Bocchini (University of Perugia, Perugia, Italy) was generously provided by Dr. K.
Suk (Kyungpook National University, Daegu, Korea). The immortalized murine BV2 cell
line that exhibits both the phenotypic and functional properties of the reactive microglial cells
was grown and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented
with 5% heat-inactivated fetal bovine serum (FBS), at 37 °C in a humidified incubator under
5% CO2.
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Nitrite quantification The BV2 cells (5×104 cells/ml) in 24-well plates in 500 μL culture
medium were pretreated with different concentrations of the synthesized compounds for 30
min after being stimulated with LPS (100 ng/mL) for 24 h. In brief, 50 μL of culture
supernatant reacted with an equal volume of Griess reagent (part 0.1%
naphthylethylenediamine and part 1% sulfanilamide in 5% H3PO4) in 96-well plates for 10
min at room temperature in the dark. Nitrite concentrations were determined by using
standard solutions of sodium nitrite prepared in cell-culture medium. The absorbance at 540
nm was determined using a microplate reader (Molecular device, USA). Each experiment
was performed in triplicate.20)
MTT cytotoxicity assay The cell viability of the cultured cells was determined by
measuring the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) to formazan. Briefly, cells were seeded and treated with different concentrations of
the synthesized compounds. After incubation with LPS (100 ng/mL) for 24 h, 0.5 mg/mL of
MTT solution was added to each well. After incubation for 2 h at 37 °C and 5% CO2, the
supernatants were removed and the formed formazan crystals in the viable cells were
dissolved in dimethyl sulfoxide (DMSO). The absorbance at 550 nm was determined using a
microplate reader (Molecular device, USA).20)
Immunoblot analysis To obtain the total cell lysate, 100 μL (or 50 μL) of RIPA buffer
[1×PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, with freshly added protease
inhibitor cocktail (Calbiocam, CA, USA)] was added to the BV2 cells cultured in 6-well
plates. The cells were scraped, incubated for 10 min on ice and centrifuged at 14,000×rpm for
10 min at 4 °C. The protein concentration was determined by the DC protein assay from Bio-
Rad (Hercules, CA), and 20 μg of whole cell lysate was separated in 10% sodium dodecyl
sulfate-polyacrylamide gels (SDS-PAGE) and transferred to PVDF membranes (Millipore,
MA, USA) using an electroblotting apparatus (Biorad, CA, USA). The membranes were
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blocked for 1 h in TBS containing 0.1% Tween-20 and 5% dry milk, and were then incubated
overnight with primary antibodies anti-iNOS, 1:5000 (Upstate, NY, USA) followed by
incubation for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:10,000)
(Cell signaling, MA, USA). The optical densities of the antibody-specific bands were
analyzed by a Luminescent Image Analyzer, LAS-3000 (Fuji, Japan).20)
Isolation of total RNA and RT-PCR analysis BV2 cells (5×104 cells/mL) were cultured
in 6-well plates, and the total RNA was isolated by extraction with TRIzol kit (Invitrogen,
CA, USA). For the reverse transcription-polymerase chain reaction (RT-PCR), 2.5 μg of total
RNA from BV2 cell was used for RT-PCR by using a First Strand cDNA Synthesis kit
(Invitrogen, CA, USA). PCR was performed using the above-prepared cDNA as a template.
The PCR conditions were as follows: iNOS, 27 cycles of denaturation at 94 °C for 30 s,
annealing at 55 °C for 30 s, and extension at 72 °C for 30 s; GAPDH, 19 cycles of
denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s.
GAPDH was used as an internal control to evaluate the relative expressions of iNOS. 20)
Results and discussion
Chemistry of clovamide methyl ester’s analogues Since the inhibitory effect on NO
production of clovamide methyl ester 9a was higher than that of clovamide 2 during the
preliminary study, all clovamide analogues possessing the methyl ester group in L-dopa
moiety were synthesized. Two analogues (7a and 7b), where the hydroxyl group in either
caffeic acid moiety or L-dopa moiety was replaced by a fluorine group, were synthesized as
depicted in Chart 1. After carboxylic acids in L-dopa 3 and compound 4 were protected with
methanol using thionyl chloride, the resulting two methyl esters (5a and 5b) were reacted
with
corresponding
cinnamic
acid
in
presence
of
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC), 1-hydroxy-benzotriazole (HOBt) and N,N-
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diisopropylethylamine (DIEA) to produce crude amide products, which were purified by
chromatography on silica gel with ethyl acetate/hexane (v/v, 1/1) to give pure products.
Other clovamide methyl ester analogues (9a-9l) were synthesized from L-dopa methyl
ester hydrochloride 5a by following Chart 2. L-Dopa methyl ester hydrochloride 5a was
reacted with 3-hydroxy cinnamic acid or coumaric acid (8b and 8c) to afford the crude
products of the corresponding amides (9b and 9c). Similarly, clovamide methyl ester
analogues with various substituents such as fluorinated or trifluoromethylated (9d-9l) were
synthesized from L-dopa methyl ester hydrochloride 5a by reacting with various cinnamic
acids or hydrocinnamic acid (8d-8l) in the presence of EDC, HOBt, and DIEA to produce the
crude products of the corresponding amides (9d-9l). The crude products were purified by
chromatography on silica gel with ethyl acetate/hexane (v/v, 1/1) to give pure products.
Anti-inflammatory activity of clovamide methyl ester’s analogues Neuroinflammation
resulting from the activation of microglia plays an important role in the pathology of
neurodegenerative diseases.21) Over-activation of microglia initiates an inflammatory cascade
in the central nervous system, propagates the levels of inflammatory and oxidative stress
molecules and can lead to neuronal dysfunction and neuronal cell death.22) Among the many
inflammatory molecules produced by activated microglia, NO production has been widely
used as a representative measure of inflammatory activation of microglia.23) Therefore,
in this paper, the inhibitory effect on the production of NO in LPS-induced BV2 cells
was evaluated for rosmarinic acid
1, clovamide 2, clovamide methyl ester 9a and its
analogues (7a 7b and 9b 9l). After BV2 cells were co-treated with compounds at a
,
-
concentration of 10 μM and LPS (100 ng/mL) for 24 h, the nitrite concentration was
determined in the supernatant by the Griess assay, and their results are presented in
Table 1.
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The fluorine substitution, as a bioisostere, for hydrogen or hydroxy group in
compounds is a well-known method to change reactivity, metabolic stability, acidity,
and membrane permeability of the molecules. Introduction of fluorine, due to
resistance of the C-F bond and electronegativity difference between the two atoms,
increases the lipophilicity and suppresses the metabolic detoxification by increasing
the in vivo lifetime.24-25) For these reasons, fluorine has played important role in drug
design and fluorinated drugs have been a popular topic in the field of pharmaceutical
science.26-27) Thus, we first investigated the importance of the catechol moiety in both
the L-dopa moiety and caffeic acid moiety on activity by replacing the hydroxyl group
with a fluorine group. The biological activities of 3,4-difluorophenyl derivative 7a
,
where the catechol moiety in caffeic acid moiety was modified, and 3,4,-
difluorophenyl derivative 7b, where the catechol moiety in L-dopa moiety was
modified, were tested. The inhibitory effect of 7a was about 3-fold more potent than
that of 7b with 32.7 ± 0.9% and 10.3 ± 3.4% at a concentration of 10 μM, respectively.
This result indicated that the catechol moiety in caffeic acid can be replaced with
another group while substitution of the catechol moiety in L-dopa could lead to loss of
activity. Therefore, further modification was focused on the catechol moiety in caffeic
acid.
In order to confirm whether both hydroxyl groups in the catechol moiety are
essential for the activity, we tested 3-hydroxyphenyl analogue 9b and 4-
hydroxyphenyl analogue 9c where one hydroxyl group in catechol moiety in 9a was
removed. Both compounds revealed lower activities (23.6 ± 1.0% and 21.4 ± 0.5%
respectively) than the parent compound 9a (27.1 ± 2.8%), which indicated that the
catechol moiety in 9a is important for retaining activity. However, when we replaced
the hydroxyl group in 9b and 9c with a fluorine group, surprisingly, 3-fluorophenyl
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analogue 9d revealed higher activity with 49.8 ± 0.5% inhibition than 3-
hydroxyphenyl analogue 9b (23.6 ± 1.0%) while 4-fluorophenyl analogue 9e (15.6 ±
2.3%) was less active than the parent compound. This result indicated that the proper
group at the meta position of the catechol moiety could enhance the inhibitory effect
on NO production. Therefore, we further evaluated the substitution effect of fluorine
group in 9d on the NO inhibitory activity by replacing it with bioisosteric groups. The
inhibitory effect of 3-methoxyphenyl analogue 9f (19.4 ± 2.3%) and 3-methylphenyl
analogue 9g (34.1 ± 5.3%) was less active than that of 9d (49.8 ± 0.5%). In addition,
the inhibition of 3-chlorophenyl analogue 9h, where the chlorine group was introduced
in meta position, on NO production was reduced to 24.1 ± 2.2%. These results showed
that electron withdrawing effect and size of halogen group are important for retaining
the activity in this series.
On the basis of our result that fluorine substitution at meta position of catechol
moiety enhanced the activity, and previous reports that CF3–bearing molecules could
lead to different reactivity and biological properties due to different metabolic
consequences,28-29) we then investigated the effect of trifluoromethyl substitution in
catechol moiety on the NO inhibitory activity. Inhibition of 3-trifluroromethylphenyl
analogue 9i and 4-trifluroromethylphenyl analogue 9j on NO production in LPS-
treated BV2 cells were 64.3 ± 1.0% and 20.3 ± 3.4%, respectively, which clearly
showed that a trifluoromethyl group is more favorable than the comparable fluorine
group for enhancing activity. As seen with results of fluorophenyl analogues 9d and 9e
a similar pattern was also reproduced with trifluoromethyl substitution since the NO
inhibition of 3-trifluoromethylphenyl analogue 9i is more active than 4-
trifluoromethylphenyl analogue 9j. The enhanced activity of 9i where substitution of a
trifluoromethylphenyl ring at meta-position prompted us to evaluate 3,5-
,
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ditriflurormethylphenyl analogue 9k where an additional trifluoromethyl group is
introduced at meta position of phenyl ring. As expected, 9k further improved activity
(78.0 ± 1.6%), with IC50 value of 4.8 μM.
Although caffeic acid and its reduced form, dihydrocaffeic acid, generally did not
show significant difference in liver lipid peroxidation,30) we tested the importance of
the double bond on the activity by preparing analogue 9l, which possessed two
trifluoromethyl groups at 3,5-position in catechol moiety but is devoid of a double
bond. Surprisingly, the saturated analogue 9l exhibited significantly improved
inhibition (97.4 ± 1.1%) with IC50 value of 2.8 μM, which implied that the saturated
form in caffeic acid moiety is more favorable for activity in this series. From the result
of structure-activity relationship, it was found that additional introduction of
trifluoromethyl group at the meta position of the catechol moiety ring and reduction of the
double bond in the analogue 9i significantly enhance the inhibition of NO production.
Analogue 9l also exhibited a NO inhibitory activity in a dose-dependent manner (Fig.
2).
To determine whether the inhibitory effects of clovamide methyl ester (9a) and its
analogues (7a, 7b and 9b-9l) on NO production were related to their toxicity, the
cytotoxicities of clovamide methyl ester’s analogues were evaluated by MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.31) As shown in Fig. 3,
there was no suppression in the cell viabilities of the BV2 cells after being treated with
clovamide methyl ester and its analogues at the concentrations of 10 μM. This result
confirmed that the inhibitory effects on NO production were due solely to its intrinsic
activities rather than its cytotoxicity.
NO produced from ι-arginine by NOS is present in three different types of isozymes,
such as endothelial NO (eNOS), neuronal NO (nNOS) and inducible NO (iNOS).32)
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eNOS and nNOS produce a small amount of NO whereas iNOS produces a large
amount of NO, which causes various diseases such as stroke, Alzheimer's disease,
Parkinson’s disease and septic shock.33) Since the reduced NO production is mainly
due to the inhibited iNOS expression, we examined whether the NO production was
caused by a decrease in the mRNA level of iNOS. LPS-stimulated BV2 cells were
treated with 9l and then the amount of iNOS mRNA was measured by RT-PCR. The
expression of iNOS mRNA in LPS-treated BV2 cells was significantly increased in
comparison with the non-treated control cells. When the LPS-treated BV2 cells were
treated with 9l at different concentrations (2.5, 5 and 10 μM), the densities of iNOS
mRNA in the cells were decreased (Fig. 4 (A)). To confirm the effects of 9l on iNOS
protein expression, the amount of iNOS protein expression was measured by the
immunoblotting method. As shown in Fig. 4 (B), the unstimulated cells released basal
levels of iNOS protein, while a significant increase of iNOS protein was detected in LPS-
stimulated BV-2 cells. When the LPS-treated BV2 cells were treated with 9l at different
concentrations (2.5, 5 and 10 μM), the densities of iNOS protein in the cells were
decreased in a dose-dependent manner. These results demonstrated that the inhibitory
action of 9l on NO production was related to a modulation of iNOS induction.
Conclusion
We synthesized various clovamide methyl ester analogues where the dihydroxyl
group in the catechol moiety were replaced with various groups, and tested their anti-
inflammatory activities. Among these synthesized clovamide analogues, the
fluorinated analogues (9d, 9i, 9k and 9l) bearing the F or CF3 group at the meta position
of the catechol moiety significantly improved the inhibitory effect on NO production in LPS-
treated BV2 compared to the parent analogue 9a. In particular, 9l, which has two
Biological and Pharmaceutical Bulletin Advance Publication

trifluoromethyl groups at the meta position of the catechol moiety ring and is devoid of
double bond, inhibited not only NO production 26.3 times better than that of the clovamide
methyl ester 9a, but also iNOS expression in LPS-induced BV2 cells without cytotoxicity.
These preliminary structure-activity relationship results of clovamide broaden our
understanding of the pharmacophore of clovamide, and 9l can be used as a possible new anti-
inflammatory agent.
Acknowledgments This research was supported by Basic Science Research Program through
the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education
[NRF-2014R1A1A2057316 (to S. -H Yoon) and (NRF-2012R1A1A2042207 (to J. -Y Park)].
The authors declare no conflict of interest.
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Figure legends
Figure 1. Structure of rosmarinic acid and clovamide
Figure 2. Effects of 9l on LPS-induced NO production in BV2 cells: BV2 cells were
stimulated with LPS (100 ng/mL) or co-treated with LPS (100 ng/mL) and 9l at
concentrations of 2.5, 5.0 and 10 μM for 24 h. The amounts of NO were determined
using the Griess reagent. All the data of each group are expressed as the mean ± S.E.M.
and are representative of results obtained from three independent experiments.
Figure 3. Effects of clovamide methyl ester analogues on the cell viability of BV2 cells:
Viability was evaluated using the MTT assay. Cells were incubated with LPS (100
ng/mL) and each sample (clovamide methyl ester and its analogues) for 24 hours. The
results are displayed as a percentage of the control samples. Each value indicates the
mean ± S.E.M. and is representative of the results obtained from three independent
experiments.
Figure 4. Effects of clovamide methyl ester (9a) and 9l on iNOS production in LPS-
induced BV2 cells: BV2 microglial cells were pretreated with the indicated
concentrations of 9l for 30 min before incubating with LPS (100 ng/mL) for 18 h. The
cells were lysed, and the lysates were prepared for mRNA levels (A) and analyzed by
immunoblotting with an anti-iNOS antibody (B).
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Figure 1
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Figure 2.
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Figure 3.
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Figure 4.
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Chart 1.
Reagents and conditions: (a) SOCl2, MeOH, reflux, 8 h; (b) i) 4 N HCl in dioxane, r.t., 4 h; ii)
SOCl2, MeOH, reflux, 4 h; (c) EDC, HOBt, DIEA, DCM/DMF (4/1), r.t., 4 h.
Biological and Pharmaceutical Bulletin Advance Publication

Chart 2.
Reagents and conditions: (a) EDC, HOBt, DIEA, DCM/DMF (4/1), r.t., 4 h.
Biological and Pharmaceutical Bulletin Advance Publication