Synthesis and Biological Evaluation of N-aryl-4-aryl-1,3-Thiazole-2-Amine Derivatives as Direct 5-Lipoxygenase Inhibitors

Article abstract of DOI:10.1111/j.1747-0285.2012.01371.x

Biological evaluation of N-aryl-4-aryl-1,3-thiazole-2-amine derivatives was examined for anti-inflammatory activity in in vitro and in vivo assays. The thiazole compounds showed direct inhibition of 5-lipoxygenase (LOX) that is a key enzyme of leukotrienes synthesis and involved in the inflammation-related diseases, including asthma and rheumatoid arthritis. To optimize biological activity, we synthesized 1,3-thiazole-2-amine derivatives and investigated for structure and activity relationship. Especially, N-(3,5-dimethylphenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine was shown to have a potent anti-inflammatory activity as a 5-LOX inhibitor.

Full text of DOI:10.1111/j.1747-0285.2012.01371.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01371.x
Chem Biol Drug Des 2012; 80: 90–99
Research Article
Synthesis and Biological Evaluation of N-aryl-
4-aryl-1,3-Thiazole-2-Amine Derivatives as Direct
5-Lipoxygenase Inhibitors
Jeehee Suh1,2, Eul Kgun Yum2, Hyae
Gyeong Cheon3,* and Young Sik Cho4,*
search for selective COX-2 inhibitors (COXibs). In the beginning, a
variety of approaches targeting cyclooxygenase-2 (COX-2) have been
successful for interfering with inflammatory progression, but found
soon to be inappropriate because of the adverse effect of heart
1Center for Metabolic Syndrome Therapeutics, Bio-Organic Science
Division, Korea Research Institute of Chemical Technology, PO Box
107, Yuseong-gu, Daejeon 305-600, Korea
attacks (3,4). Afterward, special attentions on alternative pathway
to produce inflammatory messengers have been given to promote
the drug development for the treatment of inflammation-related dis-
eases. In fact, LTs produced by LOX have potent pro-inflammatory
properties including bronchoconstriction and chemotaxis of polymor-
phonuclear leukocytes (5,6). LTB4 is a well-known chemoattractant
for inflammatory cells (7), and other three LTC4, D4, and E4, which
contain glutathione conjugation, are collectively referred as the
slow-reacting substances of anaphylaxis (8). Besides most notable
asthma, rheumatoid arthritis and inflammatory bowel disease (9–
11), these LTs can also play a role in the generation of psoriasis
and atopic dermatitis, in which topical application of LOX products
causes psoriasis-like lesions, suggesting their inflammatory roles in
the pathogenesis of skin disorders (12,13).
2Department of Chemistry, Chungnam National University, Yuseong-
gu, Daejeon 305-764, Korea
3Department of Pharmacology and Pharmaceutical Sciences, Gachon
University of Medicine and Science, Incheon 406-799, Korea
4Department of Pharmacy, Keimyung University, 1000 Sindang-dong,
Dalseo-gu, Daegu 704-701, Korea
*Corresponding authors: Young Sik Cho, yscho123@kmu.ac.kr;
Hyae Gyeong Cheon, hgcheon@gachon.ac.kr
Biological evaluation of N-aryl-4-aryl-1,3-thiazole-2-
amine derivatives was examined for anti-inflammat
ory activity in in vitro and in vivo assays. The
thiazole compounds showed direct inhibition of 5-li
poxygenase (LOX) that is a key enzyme of leukotri-
enes synthesis and involved in the inflammation-
related diseases, including asthma and rheumatoid
arthritis. To optimize biological activity, we synthe-
sized 1,3-thiazole-2-amine derivatives and investi-
gated for structure and activity relationship.
Especially, N-(3,5-dimethylphenyl)-4-(4-chlorophen
yl)-1,3-thiazole-2-amine was shown to have a potent
anti-inflammatory activity as a 5-LOX inhibitor.
5-Lipoxygenase (5-LOX) is a key enzyme involved in the first step of
the LTs synthesis, and its dysregulation causes various inflammatory
diseases mentioned earlier (14,15). Therefore, 5-LOX inhibitors to
intervene LTs production can be developed as the effective thera-
peutic agents to treat inflammatory diseases. Presently, the only
drug approved by FDA as a direct 5-LOX inhibitor is zileuton (16),
while there are mostly drugs based on the abrogation of the
ligand–receptor interaction or indirect interference in the process of
5-LOX activation (17,18).
Key words: 5-lipoxygenase, anti-inflammation, leukotriene B4, neu-
trophil, thiazole scaffold
In an effort to discover new chemical entities with selective inhibitory
activities of 5-LOX, in-house chemical library was screened by high-
throughput screening (HTS) method as described previously (19). As a
result, we identified a new series of aminothiazole derivatives, and
further characterized biochemical and pharmacological profiles of a
representative compound, N-(3,5-dimethylphenyl)-4-(4-chlorophenyl)-
1,3-thiazole-2-amine, 14. The potent compound 14 used at 1 micro-
molar inhibited 5-LOX enzyme activity in vitro and LTB4 production
from intact RBL cells by 96% and 98%, respectively, (Table 1). Struc-
ture and activity relationship (SAR) study revealed that the presence
of either OH or NH2 group at 2- or 4-position of N-aryl moiety of am-
inothiazole scaffold was mandatory for its inhibitory activity, while 4-
aryl moiety of it was relatively tolerable to a variety of substituents.
Intriguingly, the presence of 3,5-diCH3 group adjacent to OH group
attached to N-aryl of aryl aminothiazole was more potent than was
that of 3,5-diCl or 3-Cl-5-CH3, although compound 13 and 16 bearing
Abbreviations: 5-LO, 5-lipoxygenase; LTB4, leukotriene B4; SAR,
structure and activity relationship; AA, arachidonic acid; COX-2, cycloox-
ygenase-2; RBL-1, rat basophilic leukemia-1.
Received 13 February 2011, revised 20 February 2012 and accepted for
publication 25 February 2012
Starting from arachidonic acid (AA) released from cytoplasmic mem-
brane upon external stimulation, inflammatory chemical mediators
are generated by a cascade of enzymes (1). Among them, prosta-
glandins (PGs) and leukotrienes (LTs) are, respectively, generated by
cyclooxygenase (COX) and lipoxygenase (LOX) at the bifurcation of
metabolic pathway using AA (2). Usage of classical NSAIDs was
limited because of their non-selective inhibition of COX enzymes,
which leads to side-effects like ulceration that resulted in the
90

Design of Novel 5-LO Inhibitors
Table 1: Inhibitory effects of N-aryl-4-aryl-1,3-thiazole-2-amines on enzymatic 5-lipoxygenase (LOX) activity
R1
N
NH
X
S
R2
In vitro assaya
Cell-based assayc
Compounds
R1
R2
X
% inhibition (at 1 lM)
IC50 (nM)b
% inhibition (at 1 lM)
Zileuton
4131JH0380
3
75
84
97
92
98
94
91
96
90
19
91
98
90
96
88
92
90
74
39
25
15
2
39
91
91
87
77
73
79
13
73
84
180
110
99
82
35
25
81
65
77
62
99
82
52
78
96
89
81
H
4-F
4-Br
4-Cl
4-NO2
2-F
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
2-OH
3-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
H
H
H
H
H
H
H
H
H
H
H
4
5
6
7
8
3-F
9
4-Cl
4-Cl
2,5-diCl
2,4-diF
4-Cl
4-Cl
4-F
4-F
4-F
4-F
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
H
140
ND
92
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
81
93
99
98
93
81
75
103
85
3,5-diCl
3,5-diCH3
3-Cl-5-CH3
3,5-diCl
2,5-diCH3
2,3,5-triCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
146
127
63
93
104
224
ND
ND
ND
ND
ND
120
105
201
160
92
4-OH
4-OCH3
4-CO2CH3
4-CF3
4-CH2OH
4-NHCOCH3
4-NH2
4-NH2
4-NH2
4-NH2
4-NH2
2-NH2
3-NH2
4-NH2
4-NH2
4-F
4-Br
4-Cl
4-NO2
4-Cl
4-Cl
2,5-diCl
2,4-diF
421
ND
60
70
ND, not determined.
a5-LOX enzymatic activities were examined in the reaction mixtures including chemical derivatives at 1 lM, and compared with that of vehicle control.
b5-LOX activities were assayed for potent chemical derivatives in a wide range of concentrations. IC50 was calculated by fitting the dose–response curves for
% inhibition using GraFit software (Erithacus Software Ltd, Surrey, UK).
cCell-based assay was determined by LTB4 amounts released in RBL-1 cells stimulated with A23187. Cells were preloaded with selected compounds prior to
A23187 stimulation to compare their inhibitory potencies. LTB4 levels, indicative of 5-LOX activity, were quantitatively measured according to the protocol pro-
vided by a manufacturer.
Cl and F group at 4-aryl moiety of N-aryl-4-aryl-1,3-thiazole-2-amine
analogs remained still effective. Apart from thiazole derivatives, a
class of thiazolinone scaffold has been studied as selective 5-LOX
inhibitors from other researchers (20).
rather than oral route proposed a potential use as a therapeutic
agent for inflammation-related skin disorders.
Methods and Materials
Consistent with those in vitro and cell-based assays, topical appli-
cation of the compound 14 onto skin in AA-induced ear edema
model improved inflammatory skin lesions as determined by pheno-
typic and biochemical parameters. Furthermore, it was demon-
strated that the compound 14 had a longer-lasting effect upon
topical application than zileuton. Its successful topical application
Cell culture and animal care
RBL-1 cells were obtained from American Type Culture Collection
(Manassas, VA, USA) and maintained in Dulbecco's Modified Eagle's
Medium (DMEM) supplemented with 10% heat-inactivated fetal calf
serum, glutamine, penicillin, and streptomycin. For in vivo efficacy,
Chem Biol Drug Des 2012; 80: 90–99
91

Suh et al.
Balb ⁄ c mice (male, 8 weeks of age) were bred, cared in the barrier
system under temperature- and humidity-controlled room with a
12:12 h light cycle, and acclimated for 1 week before experiments.
All animal experiments were carried out in accordance with the
Guide for the Care and Use of Laboratory Animals (NIH) and
approved by the Institutional Animal Care and Use Committee of the
Korea Research Institute of Chemical Technology (Daejeon, Korea).
13C NMR (75 MHz, DMSO-d6) d 102.76, 115.48, 119.31, 120.40,
127.64, 131.50, 133.18, 133.86, 148.85, 152.40, 164.40.
HR MS (M+) calcd. for C15H11 BrN2OS: 345.9775; found: 345.9758.
MS (EI) m ⁄ e 346 (100%) [M]+, 348 (98%), 134 (15%), 89 (20%).
Synthesis of N-(4-hydroxyphenyl)-4-(4-
chlorophenyl)-1,3-thiazole-2-amine (5)
1H NMR (300 MHz, DMSO-d6) d 6.75 (d, J = 8.8 Hz, 2H), 7.28 (s,
1H), 7.45 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.89 (d,
J = 8.6 Hz, 2H), 9.12 (s, 1H), 9.92 (s, 1H).
Reagents and enzymes
Chemicals and enzymes used in this experiment were available com-
mercially or were prepared by in-house synthesis or partial isolation.
Xylenol, iron sulfate, ATP, hexadecyltrimethylammonium bromide
(HTAB), a-phophatidyl choline (PC) were obtained from Sigma (St.
Louis, MO, USA). AA and A23187 were obtained from Calbiochem (La
Jolla, CA, USA). The LT B4 (LTB4) enzyme immunoassay (EIA) kit was
obtained from Amersham (Piscataway, NJ, USA). Zileuton and a series
of chemical derivatives were synthesized in Korea Research Institute
of Chemical Technology (KRICT; Daejeon). Soybean 15-LOX enzyme
was obtained from Cayman Chemical (Ann Arbor, MI, USA), and 12-
LOX was prepared from platelets-rich plasma as described before
(21,22). All other reagents were of analytical grade from Sigma.
13C NMR (75 MHz, DMSO-d6) d 102.66, 115.48, 119.30, 127.32,
128.59, 131.81, 133.19, 133.52, 148.80, 152.40, 164.39.
HR MS (M+) calcd. for C15H11 ClN2OS: 302.0281; found: 302.0274.
MS (EI) m ⁄ e 302 (100%) [M]+, 304 (33%), 168 (17%), 134 (23%).
Synthesis of N-(4-hydroxyphenyl)-4-(4-
nitrophenyl)-1,3-thiazole-2-amine (6)
Synthesis of N-(4-hydroxyphenyl)-4-(4-
1H NMR (300 MHz, DMSO-d6) d 6.68 (d, J = 8.8 Hz, 2H), 7.40 (d,
J = 8.8 Hz, 2H), 7.55 (s, 1H), 8.07 (d, J = 8.9 Hz, 2H), 8.21 (d,
J = 8.9 Hz, 2H), 9.09 (s, 1H), 9.97 (s, 1H).
fluorophenyl)-1,3-thiazole-2-amine (3)
About 0.60 g (2.76 mmol) of 2-bromo-4¢-fluoroacetophenone was
dissolved in 10 mL of ethanol, 0.29 g (3.59 mmol) of sodium thio-
cyanate and 0.33 g (3.04 mmol) of p-aminophenol were added at
room temperature, and the mixture was refluxed at 80 ꢀC for 12 h.
After the reaction was completed, the reaction mixture was diluted
with 10 mL of ethyl acetate, washed with water, dried over anhy-
drous magnesium sulfate, and concentrated under a reduced pres-
sure. The residue was purified using silica gel column
chromatography (hexane:ethyl acetate = 2:1) to obtain the title com-
pound (0.490 g, 62%).
13C NMR (75 MHz, DMSO-d6) d 106.89, 115.53, 119.50, 124.10,
126.43, 132.98, 140.62, 146.15, 148.04, 152.59, 164.66.
HR MS (M+) calcd. for C15H11 N3O3S: 313.0521; found: 313.0526.
MS (EI) m ⁄ e 313 (100%) [M]+, 267 (25%), 89 (18%).
1H NMR (300 MHz, DMSO-d6) d 6.76 (d, J = 8.73 Hz, 2H), 7.19–
7.27 (m, 3H), 7.48 (d, J = 8.73 Hz, 2H), 7.90–7.95 (m, 2H), 9.13 (s,
1H), 9.90 (s, 1H).
Synthesis of N-(4-hydroxyphenyl)-4-(2-
fluorophenyl)-1,3-thiazole-2-amine (7)
1H NMR (300 MHz, DMSO-d6) d 6.74 (d, J = 8.8 Hz, 2H), 7.13 (s,
1H), 7.23–7.36 (m, 3H), 7.45 (d, J = 8.8 Hz, 2H), 8.09 (m, 1H), 9.16
(s, 1H), 9.93 (s, 1H).
13C NMR (75 MHz, DMSO-d6) d 101.55, 115.26, 115.48, 115.55,
119.25, 127.54, 127.65, 131.29, 131.33, 133.25, 149.00, 152.35,
159.93, 163.17, 164.35.
13C NMR (75 MHz, DMSO-d6) d 106.42, 106.62, 115.49, 115.81,
116.11, 119.32, 122.12, 122.27, 124.69, 124.74, 128.91, 129.03,
129.54, 129.58, 133.19, 143.70, 143.73, 152.41, 157.89, 161.19,
163.55.
HR MS (M+) calcd. for C15H11 FN2OS: 286.0576; found: 286.0580.
MS (EI) m ⁄ e 286 (100%) [M]+, 152 (25%), 134 (10%).
HR MS (M+) calcd. for C15H11 FN2OS: 286.0576; found: 286.0570.
MS (EI) m ⁄ e 286 (100%) [M]+, 152 (18%).
The following compounds were prepared according to the general
procedures described previously employing the appropriate 2-bromo-
acetophenone.
Synthesis of N-(4-hydroxyphenyl)-4-(3-
fluorophenyl)-1,3-thiazole-2-amine (8)
1H NMR (500 MHz, DMSO-d6) d 6.74 (d, J = 8.9 Hz, 2H), 7.11 (m,
1H), 7.34 (s, 1H), 7.45 (m, 3H), 7.64 (d, J = 10.8 Hz, 1H), 7.72 (d,
J = 8.7 Hz, 1H), 9.14 (s, 1H), 9.93 (s, 1H).
Synthesis of N-(4-hydroxyphenyl)-4-(4-
bromophenyl)-1,3-thiazole-2-amine (4)
1H NMR (300 MHz, DMSO-d6) d 6.54 (d, J = 8.8 Hz, 2H), 7.08 (s,
1H), 7.24 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.5 Hz, 2H), 7.62 (d,
J = 8.5 Hz, 2H), 8.90 (s, 1H), 9.71 (s, 1H).
92
Chem Biol Drug Des 2012; 80: 90–99

Design of Novel 5-LO Inhibitors
13C NMR (75 MHz, DMSO-d6)
104.12, 104.47, 104.82,
105.85, 106.04, 111.69, 112.01, 115.50, 118.99, 119.04, 119.19,
119.36, 130.63, 130.69, 130.76, 130.82, 133.14, 142.90, 152.45,
157.77, 157.93, 159.40, 159.56, 161.11, 161.27, 162.67, 162.83,
163.66.
13C NMR (75 MHz, DMSO-d6) d 103.36, 111.94, 112.24, 113.94,
114.23, 115.51, 119.35, 120.05, 121.66, 130.53, 130.64, 133.16,
137.01, 137.12, 148.74, 148.78, 152.44, 160.91, 164.13, 164.36.
d
HR MS (M+) calcd. for C15H11 FN2OS: 286.0576; found: 286.0576.
MS (EI) m ⁄ e 286 (100%) [M]+, 152 (18%).
HR MS (M+) calcd. for C15H10 F2N2OS: 304.0482; found:
304.0475.
Synthesis of N-(2-hydroxyphenyl)-4-(4-
chlorophenyl)-1,3-thiazole-2-amine (9)
1H NMR (300 MHz, DMSO-d6) d 6.83 (m, 3H), 7.33 (s, 1H), 7.46 (d,
J = 8.52 Hz, 1H), 7.89 (d, J = 8.52 Hz, 1H), 8.26 (m, 1H), 9.50 (s,
1H), 9.89 (d, 1H).
MS (EI) m ⁄ e 304 (100%) [M]+, 170 (20%).
Synthesis of N-(3,5-dichloro-4-hydroxyphenyl)-
4-(4-chlorophenyl)-1,3-thiazole-2-amine (13)
1H NMR (300 MHz, DMSO-d6) d 7.44 (s, 1H), 7.51 (d, J = 8.4 Hz,
2H), 7.68 (s, 2H), 7.90 (d, J = 8.4 Hz, 2H), 9.71 (br, 1H),
13C NMR (75 MHz, DMSO-d6) d 104.06, 115.00, 119.03, 119.26,
122.41, 127.24, 128.64, 129.19, 131.81, 133.49, 146.39, 148.289,
164.31.
10.33 (s, 1H).
HR MS (M+) calcd. for C15H11 ClN2OS: 302.0281; found: 302.0281.
MS (EI) m ⁄ e 302 (100%) [M]+, 304 (33%), 269 (40%), 165 (403%).
13C NMR (75 MHz, DMSO-d6) d 104.15, 116.89, 122.74, 127.20,
128.75, 132.10, 133.24, 134.46, 143.30, 148.77, 162.97.
HR MS (M+) calcd. for C15H9 Cl3N2OS: 369.9501; found: 369.9503.
MS (EI) m ⁄ e 370 (83%) [M]+, 168 (33%), 133 (53%), 89 (100%).
Synthesis of N-(3-hydroxyphenyl)-4-(4-
chlorophenyl)-1,3-thiazole-2-amine (10)
1H NMR (300 MHz, DMSO-d6) d 6.37 (d, J = 8.1 Hz, 1H), 7.01 (d,
J = 8.1 Hz, 1H), 7.11 (t, J = 8.1 Hz, 1H), 7.32 (s, 1H), 7.39 (s, 1H), 7.48
(d, J = 8.7 Hz, 2H), 7.95 (d, J = 8.7 Hz, 2H), 9.43 (s, 1H), 10.17 (s, 1H).
Synthesis of N-(3,5-dimethyl-4-hydroxyphenyl)-
4-(4-chlorophenyl)-1,3-thiazole-2-amine (14)
1H NMR (300 MHz, CDCl3) d 2.27 (s, 6H), 7.29 (s, 1H), 7.36 (s,
1H), 7.56 (d, J = 8.3 Hz, 2H), 7.99 (d, J = 8.3 Hz, 2H), 8.04 (s, 1H),
9.91 (s, 1H).
13C NMR (75 MHz, DMSO-d6) d 103.62, 104.08, 107.87, 108.58,
127.42, 128.62, 129.64, 131.94, 133.40, 142.12, 148.84, 157.95,
163.20.
13C NMR (75 MHz, DMSO-d6) d 17.00, 102.51, 118.21, 124.94,
127.27, 128.62, 131.80, 133.18, 133.57, 148.34, 148.79, 164.62.
HR MS (M+) calcd. for C15H11 ClN2OS: 302.0281; found: 302.0282.
MS (EI) m ⁄ e 302 (100%) [M]+, 304 (33%), 168 (17%), 134 (23%).
HR MS (M+) calcd. for C17H15 ClN2OS: 330.0594; found:
330.0609.
Synthesis of N-(4-hydroxyphenyl)-4-(2,5-
MS (EI) m ⁄ e 330 (100%) [M]+, 332 (33%), 162 (9%).
dichlorophenyl)-1,3-thiazole-2-amine (11)
1H NMR (500 MHz, DMSO-d6) d 6.73 (d, J = 8.8 Hz, 2H), 7.36–7.42
(m, 4H), 7.56 (d, J = 8.6 Hz, 1H), 7.93 (s, 1H), 9.19 (s, 1H), 9.95 (s, 1H).
Synthesis of N-(3-chloro-4-hydroxy-5-dimethyl
phenyl)-4-(4-fluorophenyl)-1,3-thiazole-2-amine
(15)
1H NMR (300 MHz, CDCl3) d 2.25 (s, 3H), 4.68 (s, 1H), 6.67 (s,
1H), 6.95–6.96 (m, 1H), 7.02–7.29 (m, 4H), 7.74–7.79 (m, 2H), 7.97
(m, 1H).
13C NMR (75 MHz, DMSO-d6) d 108.01, 115.50, 119.61, 128.54,
129.31, 130.32, 131.79, 132.17, 133.00, 134.70, 145.16, 152.61, 163.75.
HR MS (M+) calcd. for C15H10Cl2N2OS: 335.9891; found: 335.9897.
MS (EI) m ⁄ e 336 (100%) [M]+, 338 (79%), 340 (19%), 301 (17%),
166 (17%).
13C NMR (75 MHz, DMSO-d6) d 17.21, 41.64, 102.29, 112.79,
115.38, 115.67, 115.73, 115.91, 116.21, 118.55, 120.73, 127.47,
127.57, 127.67, 131.17, 131.21, 131.67, 131.80, 134.03, 145.42,
148.98, 160.01, 163.25, 163.53, 167.29, 190.98.
Synthesis of N-(4-hydroxyphenyl)-4-(2,4-
difluorophenyl)-1,3-thiazole-2-amine (12)
1H NMR (300 MHz, DMSO-d6) d 6.72 (d, J = 8.67 Hz, 2H), 7.11 (s,
1H), 7.20 (t, 1H), 7.34 (t, 1H), 7.47 (d, J = 8.67 Hz, 2H), 8.11 (q,
J = 8.1 Hz, 1H), 9.15 (s, 1H), 9.95 (s, 1H).
HR MS (M+) calcd. for C16H12Cl FN2OS: 334.0343; found:
334.0340.
MS (EI) m ⁄ e 334 (100%) [M]+, 336 (33%), 152 (25%), 123 (22%).
Chem Biol Drug Des 2012; 80: 90–99
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Suh et al.
Synthesis of N-(3,5-dichloro-4-hydroxyphenyl)-
4-(4-fluorophenyl)-1,3-thiazole-2-amine (16)
1H NMR (300 MHz, DMSO-d6) d 7.15 (s, 1H), 7.20 (m, 2H), 7.69
(s, 2H), 7.83 (m, 2H), 10.2 (s, 1H).
Synthesis of N-(4-aminophenyl)-4-(4-
fluorophenyl)-1,3-thiazole-2-amine (24)
Synthesis of N-(4-acetaminophenyl)-4-(4-
fluorophenyl)-1,3-thiazole-2-amine
13C NMR (75 MHz, DMSO-d6) d 103.05, 115.45, 115.73, 116.84,
122.74, 127.44, 127.55, 134.52, 143.25, 148.95, 162.90.
About 0.45 g (2.00 mmol) of 2-bromo-4¢-fluoroacetophenone was
dissolved in 10 mL of ethanol, 0.21 g (2.59 mmol) of sodium thiocy-
anate and 0.33 g (2.20 mmol) of p-aminophenol were added at
room temperature, and the mixture was refluxed at 80 ꢀC for 12 h.
After the reaction was completed, the reaction mixture was diluted
with 10 mL of ethyl acetate, washed with water, dried over anhy-
drous magnesium sulfate, and concentrated under a reduced pres-
sure. The residue was purified using silica gel column
chromatography (hexane:ethyl acetate = 1:1) to obtain the title com-
pound (0.38 g, 58%).
HR MS (M+) calcd. for C15H9Cl2 FN2OS: 353.9797; found:
353.9789.
MS (EI) m ⁄ e 354 (81%) [M]+, 356 (52%), 358 (11%), 152 (100%).
Synthesis of N-(2,5-dimethyl-4-hydroxyphenyl)-
4-(4-fluorophenyl)-1,3-thiazole-2-amine (17)
1H NMR (300 MHz, CDCl3) d 2.18 (s, 3H), 2.21 (s, 3H), 6.62 (s,
1H), 6.72 (s, 1H), 7.04 (m, 2H), 7.15 (s, 1H), 7.67 (s, 1H), 7.75 (m,
2H), 8.55 (s, 1H).
1H NMR (300 MHz, DMSO-d6) d 2.08 (s, 3H), 7.28 (m, 3H), 7.58 (d,
2H), 7.66 (d, 2H), 7.99 (dd, 1H), 9.89 (s, 1H), 10.22 (s,1H).
MS (EI) m ⁄ e 327 (100%) [M]+, 285 (60%), 152 (13%).
13C NMR (75 MHz, DMSO-d6) d 15.67, 17.48, 25.13, 30.42, 67.02,
101.25, 115.18, 115.47, 116.59, 121.96, 127.43, 127.54, 127.66,
130.61, 131.29, 131.48, 131.52, 149.24, 153.24, 159.86, 163.09,
168.94.
Synthesis of N-(4-aminophenyl)-4-(4-
fluorophenyl)-1,3-thiazole-2-amine (24)
About 0.23 g (0.70 mmol) of N-(4-acetaminophenyl)-4-(4-fluorophe-
nyl)-1,3-thiazole-2-amine was dissolved in 2 mL of 6 N HCl solution.
The mixture was refluxed at 100 ꢀC under a nitrogen atmosphere
for 2 h. After cooling to ambient temperature, the mixture was
basified with 2 N NaOH, extracted into ethyl acetate, dried over
MgSO4, and filtered and distilled under a reduced pressure. The
resulting residue was purified using silica gel column chromatogra-
phy (n-hexane:ethyl acetate = 1:1) to obtain the title compound
(0.19 g, 96%).
HR MS (M+) calcd. for C17H15 FN2OS: 314.0889; found: 314.0895.
MS (EI) m ⁄ e 314 (100%) [M]+, 281 (37%), 160 (26%).
Synthesis of N-(2,3,5-trimethyl-4-
hydroxyphenyl)-4-(4-fluorophenyl)-1,3-thiazole-
2-amine (18)
1H NMR (300 MHz, CDCl3) d 2.22 (s, 6H), 2.24 (s, 3H), 6.59 (s,
1H), 7.02 (m, 3H), 7.73 (m, 2H).
1H NMR (300 MHz, DMSO-d6)
d 4.90 (br, 2H), 6.57 (d,
J = 8.37 Hz, 2H), 7.13 (s, 1H), 7.20 (m, 4H), 7.90 (m, 2H), 9.68 (s,
1H).
13C NMR (75 MHz, DMSO-d6) d 12.94, 14.41, 16.63, 101.15,
115.16, 115.44, 122.41, 124.22, 125.36, 127.43, 127.54, 130.80,
131.13, 132.21, 149.31, 151.21, 169.63.
13C NMR (75 MHz, DMSO-d6) d 101.05, 114.36, 115.22, 115.51,
120.11, 127.49, 127.59, 130.78, 131.38, 131.42, 144.20, 149.03,
159.94, 163.20, 165.29.
HR MS (M+) calcd. for C18H17FN2OS: 328.1046; found: 328.1043.
MS (EI) m ⁄ e 328 (100%) [M]+, 295 (20%).
HR MS (M+) calcd. for C15H12 FN3S: 285.0736; found: 285.0738.
MS (EI) m ⁄ e 285 (100%) [M]+, 152 (10%), 133 (22%).
Synthesis of N-(4-aminophenyl)-4-(phenyl)-1,3-
thiazole-2-amine (23)
1H NMR (300 MHz, DMSO-d6) d 4.94 (br, 2H), 6.60 (d, J = 8.4 Hz,
2H), 7.17 (s, 1H), 7.30 (m, 3H), 7.43 (t, J = 7.53 Hz, 2H), 7.89 (d,
J = 8.4 Hz, 2H), 9.69 (s,1H).
Synthesis of N-(4-aminophenyl)-4-(4-
bromophenyl)-1,3-thiazole-2-amine (21)
1H NMR (300 MHz, DMSO-d6) d 4.86 (br, 2H), 6.57 (d, J = 8.6 Hz,
2H), 7.23 (s, 1H), 7.27 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.4 H, 2H),
7.82 (d, J = 8.4Hz, 2H), 9.70 (s, 1H).
13C NMR (75 MHz, DMSO-d6) d 101.33, 114.41, 120.09, 125.61,
127.39, 128.56, 130.88, 134.79, 144.16, 150.11, 165.20.
13C NMR (75 MHz, DMSO-d6) d 102.24, 114.36, 120.18, 120.31,
127.60, 130.70, 131.47, 133.96, 144.26, 148.88, 165.35.
HR MS (M+) calcd. for C15H13 N3S: 267.0830; found: 267.0846.
MS (EI) m ⁄ e 267 (100%) [M]+, 133 (18%).
HRMS (M+) calcd. for C15H12BrN3S: 344.9935; found: 344.9939.
Chem Biol Drug Des 2012; 80: 90–99
94

Design of Novel 5-LO Inhibitors
MS (EI) m ⁄ e 345 (100%) [M]+, 347 (98%), 164 (12%), 133 (30%).
Synthesis of N-(4-aminophenyl)-4-(2,5-
dichlorophenyl)-1,3-thiazole-2-amine (31)
1H NMR (300 MHz, DMSO-d6)
4.87 (s, 2H), 6.56 (d,
d
Synthesis of N-(4-aminophenyl)-4-(4-
chlorophenyl)-1,3-thiazole-2-amine (22)
1H NMR (300 MHz, DMSO-d6) d 4.89 (br, 2H), 6.58 (d, J = 8.6 Hz,
2H), 7.22 (s, 1H), 7.29 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H),
7.87 (d, J = 8.5 Hz, 2H), 9.70 (s, 1H).
J = 8.6 Hz, 2H), 7.22 (d, J = 8.6 Hz, 2H), 7.33 (s, 1H), 7.43 (d,
J = 8.5 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.95 (s, 1H), 9.73 (s,
1H).
13C NMR (75 MHz, DMSO-d6) d 107.53, 114.41, 120.47, 128.40,
129.22, 130.32, 130.54, 131.76, 132.15, 134.68, 144.40, 145.14,
164.68,
13C NMR (75 MHz, DMSO-d6) d 102.13, 114.36, 120.15, 127.27,
128.54, 130.71, 131.71, 133.61, 144.23, 148.82, 165.33.
HRMS (M+) calcd. for C15H11 Cl2N3S: 335.0051; found: 335.0052.
MS (EI) m ⁄ e 335 (100%) [M]+, 337 (79%), 379 (19%).
HR MS (M+) calcd. for C15H12 ClN3S: 301.0440; found: 301.0439.
MS (EI) m ⁄ e 301 (100%) [M]+, 303 (33%), 168 (13%).
Synthesis of N-(4-aminophenyl)-4-(2,4-
Synthesis of N-(4-aminophenyl)-4-(4-
difluorophenyl)-1,3-thiazole-2-amine (32)
nitrophenyl)-1,3-thiazole-2-amine (25)
1H NMR (300 MHz, DMSO-d6)
d 4.86 (s, 2H), 6.57 (d,
1H NMR (300 MHz, DMSO-d6)
d
4.67 (brs, 2H), 6.36 (d,
J = 8.6 Hz, 2H), 7.04 (s, 1H), 7.16–7.36 (m, 4H), 8.04 (m, 1H), 9.72
J = 8.6 Hz, 2H), 7.05 (d, J = 8.6 Hz, 2H), 7.31 (s, 1H), 7.89 (d,
J = 8.7 Hz, 2H), 8.04 (d, J = 8.7 Hz, 2H), 9.59 (s, 1H).
(s, 1H).
13C NMR (75 MHz, DMSO-d6) d 104.08, 104.43, 104.78 105.35,
105.54, 111.69, 111.96, 114.36, 119.05, 119.21, 119.25, 120.21,
130.66, 131.69, 131.76, 131.82, 133.14, 142.90, 144.30,
158.73, 158.93, 160.34, 160.56, 162.11, 162.27, 163.67, 163.83,
164.59.
13C NMR (75 MHz, DMSO-d6) d 106.407, 114.38, 120.37, 124.08,
126.38, 130.47, 140.72, 144.47, 146.09, 148.10, 165.63.
HR MS (M+) calcd. for C15H12 N4O2S: 312.0681; found: 312.0682.
MS (EI) m ⁄ e 312 (100%) [M]+, 266 (33%), 133 (8%).
HR MS (M+) calcd. for C15H11 F2N3S: 303.0642; found: 303.0655.
MS (EI) m ⁄ e 303 (100%) [M]+, 133 (25%).
Synthesis of N-(2-aminophenyl)-4-(4-
chlorophenyl)-1,3-thiazole-2-amine (26)
1H NMR (300 MHz, DMSO-d6) d 5.00 (s, 2H), 6.61 (t, J = 7.1 Hz,
1H), 6.75 (d, J = 7.1 Hz, 1H), 6.91 (t, J = 7.1 Hz, 1H), 7.25 (s, 1H),
7.44 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 7.1 Hz, 1H), 7.86 (d,
J = 8.5 Hz, 1H), 9.11 (s, 1H).
In vitro 5-LOX assay
5-LOX enzyme assay was carried out with some modifications of
ferric oxidation of xylenol orange (FOX) assay, which is based on
the complex formation of Fe3+ ⁄ xylenol orange with absorption at
visible light (19,25). The enzyme sources were prepared from insect
cell (Sf21) lysates highly expressing rat 5-LOX as described previ-
ously (19). Cell lysates (15 lg protein) were preincubated with vari-
ous concentrations of either some selected chemicals or zileuton (in
DMSO) in 50 lL of 50 mM Tris-HCl buffer (pH 7.4) containing
0.4 mM CaCl2, 24 lg ⁄ mL phosphatidylcholine (PC) and 20 lM ATP
at room temperature for 4 min. The reactions were started by the
addition of 40 lM AA, kept for another 4 min, and terminated by
the addition of 100 lL FOX reagent: sulfuric acid (25 mM), xylenol
orange (100 lM), iron (II) sulfate (100 lM), methanol:water (9:1,
v ⁄ v). Blank assay was carried out without substrate during incuba-
tion, after that substrate was added before mixing with FOX
reagent.
HR MS (M+) calcd. for C15H11 FN2OS: 301.0440; found:
301.0428.
MS (EI) m ⁄ e 301 (88%) [M]+, 303 (33%), 268 (100%), 164 (84%).
Synthesis of N-(3-aminophenyl)-4-(4-
chlorophenyl)-1,3-thiazole-2-amine (27)
1H NMR (300 MHz, DMSO-d6)
d 5.13 (s, 2H), 6.22 (d,
J = 8.7 Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 6.93 (m, 2H), 7.35 (s,
1H), 7.45 (d, J = 8.5 Hz, 2H), 7.94 (d, J = 8.5 Hz, 2H), 9.98 (s,
1H).
13C NMR (75 MHz, DMSO-d6) d 103.72, 107.65, 111.69, 112.10,
127.52, 128.58, 129.09, 131.92, 133.39, 141.39, 148.96, 163.15,
168.29.
12- and 15-lipoxygenases assays for the
determination of specificity
Catalytic activities of 12-LOX and 15-LOX were tested as the same
way as 5-LOX assay except that ATP was absent in the assay sys-
tem. As reference compounds of 12- and 15-LOXs, hinokitiol, and
ebselen were used, respectively.
HR MS (M+) calcd. for C15H12ClN3S: 301.0440; found: 301.0454.
MS (EI) m ⁄ e 301 (88%) [M]+, 303 (33%), 268 (100%), 164 (84%).
Chem Biol Drug Des 2012; 80: 90–99
95

Suh et al.
LTB4 secretion in RBL-1 cells activated by
A23187
Cell-based assay was carried out according to the methods as pre-
viously reported (26). In brief, RBL-1 cells were plated at a density
of 4 · 104 cells into 96 well plates. Various concentrations of
either compounds or zileuton dissolved in DMSO (final 0.5%) was
added to plates and kept incubation for 10 min at 37 ꢀC prior to
the stimulation with Ca2+ ionophore A23187 (final 10 lM in DMSO).
After further incubation for 15 min, culture media were collected
and LTB4 content in the supernatant was measured by using an EIA
kit (Amersham).
Scheme 1: Sodium thiocyanate, EtOH, reflux, 40–83%.
aryl-4-aryl-1,3-thiazole-2-amine analogs 3–32 were synthesized
with substituent variation at phenyl groups as described in Experi-
mental Section.
Arachidonic acid-induced ear inflammation in
mice
Biological activity
In this work, we pursued to identify a novel scaffold that is able to
inhibit directly the enzyme activity of 5-LOX from in-house chemical
library of Korea Research Institute of Chemical Technology (KRICT;
Daejeon). Primarily, a highly diverse chemical library was tested
using enzymatic assay as previously described (19). Eventually, we
identified N-aryl-4-aryl-1,3-thiazole-2-amine derivatives as the
potent inhibitor of 5-LOX catalytic activity. For further SAR analyses,
chemical derivatives bearing a variety of diverse substituents were
synthesized and then their inhibitory potencies against enzymatic 5-
LOX were listed in Table 1. From the in vitro enzymatic results
above, we found that either OH or NH2 group at N-aryl moiety of
N-aryl-4-aryl-1,3-thiazole-2-amine analogs was critical for conferring
inhibitory activity against 5-LOX. By comparing IC50, the compounds
3–18 with OH group at N-aryl moiety present in N-aryl-4-aryl-1,3-
thiazole-2-amine analogs exhibited a little more potent in vitro
inhibitory activity for 5-LOX than did the compounds 24–32 bearing
NH2 group (Table 1). On the other hand, a variety of substituents (F,
Cl, Br, OCH3, NO2, CH3) at 4-aryl moiety of N-aryl-4-aryl-1,3-thia-
zole-2-amine analogs did not show any drastic changes with regard
to 5-LOX inhibition. As OH group was differentially introduced at N-
aryl moiety in thiazole compounds (5, 9 and 10), the inhibitory
activity on 5-LOX was significantly influenced by OH position at N-
aryl. Specifically, 4-substituted OH at N-aryl-1,3-thiazole compound
5 (98% inhibition at 1 lM) showed good inhibitory activity. How-
ever, 3-substituted OH at N-aryl compound 10 (19% inhibition at
1 lM) abolished completely the 5-LOX inhibitory potency. On the
other hand, 2-substituted OH at N-aryl compound 9 (90% inhibition
at 1 lM) was comparable with compound 5 regarding 5-LOX inhibi-
tory power. Variations in position of NH2 group into N-aryl exhibited
inhibitory profiles similar to those observed in OH group attached
to N-aryl. Placement of NH2 at 2- or 4-position of N-aryl moiety
remained active, whereas attachment of it at 3-position severely
diminished its inhibitory activity. Based on these results, we found
that OH or NH2 group at the 2- or 4-position of N-aryl moiety was
pharmacologically necessary for 5-LOX inhibition. Aside from NH2 or
OH, the introduction of –OCH3, –CO2CH3, –CF3, –CH2OH or –NHC-
OCH3 group at 4-position of N-aryl compounds 19–23 deteriorated
severely their inhibitory activities against 5-LOX. The synthesized
N-(aminophenyl)-4-aryl-1,3-thiazole-2-amines analogs 24–32 with
various substituents at 4-aryl moiety were also examined. When
NH2 group was located at a 4-position of N-aryl moiety, the SAR in
these analogs revealed a very similar trend to the N-hydroxyphenyl-
4-aryl-1,3-thiazole-2-amine analogs. Interestingly, the positioning of
To induce inflammatory response, 2 mg of AA in 20 lL of acetone
was painted to the inner surface of the right ear of Balb ⁄ c mouse
(8 weeks of age, male, n = 5–6 ⁄ group). A potent derivative 14 and
zileuton were painted directly on the ears or orally given to mice
1 h before AA treatment. For vehicle control of p.o administration,
equal volume of 0.5% CMC to treated groups was given. One hour
after AA stimulation, ear thickness of the treated- and untreated-
ears was measured with a dial thickness gauge (Mitutoyo, Tokyo,
Japan). For long-lasting effect of topical application against ear
edema animal model, mouse ears were preloaded topically with
either zileuton or compound 14 over the indicated times, followed
by AA administration. The effects of chemical derivatives were cal-
culated as the percent difference in ear thickness from the mean
values of the contralateral untreated ear. Myeloperoxidase (MPO)
activity in the inflamed ear tissue was determined according to the
method described by Bradley et al. (27). Briefly, punched ears
(2 mm in diameter) were chopped and homogenized in 300 lL of
50 mM potassium phosphate buffer, pH 6.0 containing 0.5% HTAB.
The homogenate was then centrifuged and the resulting superna-
tant (100 lL) was allowed to react with 0.167 mg ⁄ mL o-dianisidine
and 0.0005% H2O2 in order to determine the absorbance at
450 nm.
Statistical analysis
Results were expressed as means € SEM of two experiments each
carried out as triplicates. Statistical significance was evaluated
using Student's t-test, and p < 0.05 was considered as statistically
significant.
Results and Discussion
Chemistry
To delineate the pharmacological moiety for 5-LOX inhibition, a ser-
ies of 1,3-thiazole derivatives were synthesized and evaluated by
in vitro enzymatic assay and further cell-based format. The N-aryl-
4-aryl-1,3-thiazole-2-amine analogs 3–32 were prepared according
to Hantzsch thiazole synthesis (28) and a literature procedure (29)
as shown in Scheme 1. Briefly, A series of compounds were pre-
pared from the substitution of 2-bromoacetophenone derivatives
1 with excess sodium thiocynate and sequential cyclization
with various aniline derivatives 2 in refluxing ethyl alcohol. The N-
96
Chem Biol Drug Des 2012; 80: 90–99

Design of Novel 5-LO Inhibitors
Table 2: The inhibitory effects of N-aryl-4-aryl-1,3-thiazole-2-
amines on LTB4 secretion from A23187-stimulated RBL-1 cells
Table 3: Comparative in vivo potency of compounds via p.o and
topical applications.
Compounds
IC50 (nM)a
Ear edema (% inhibition)b
Zileuton
13
14
430
77
25
348
324
576
121
Compoundsa
Thickness
MPO
Zileuton
14
75
45
82
62
15
16
Compounds (100 lg)
Ear edema (% inhibition)c
Thickness
17
18
MPO
aCellular 5-lipoxygenase activities were assayed for potent chemical deriva-
tives in a wide range of concentrations. IC50 was calculated by fitting the
dose–response curves using GraFit software, achieving relative potencies of
compounds under investigation.
Zileuton
14
26
60
38
60
aMice (5–6 mice ⁄ group) were orally given with each compound at a dose of
100 mg ⁄ kg (mpk) 30 min before arachidonic acid (AA) painting onto ear.
bAA-treated ear tissues were assessed by two inflammation indications
reflecting tissue swelling and neutrophil infiltration, so that ears applied
with test compounds were relatively compared with those treated with vehi-
cle.
an electron donor NH2 at N-aryl of compound 27 (IC50 = 160 nM,
77% inhibition at 1 lM) was remarkable in improving IC50 value,
unlike compound 23 containing NHCOCH3 at N-aryl (39% inhibition
at 1 lM). Additionally, an in vitro enzymatic assay demonstrated
that the 2,5-, or 3,5-disubstituted compounds 13–17 with electron
donors or electron acceptors around OH group at N-aryl did not
affect significantly 5-LOX inhibitory activity, comparable with parent
compound (4131JH0380) and 2,3,5-trisubstituted compound 18.
cTo test topical effects of chemical derivatives, 100 lg of each compound
were directly applied to ears of mice, and 30 min later, AA was painted to
induce inflammation. Ear edema and neutrophil infiltration were measured
by ear thickness and myeloperoxidase (MPO) activity in the extracted tissues
and then evaluated by the inhibitory power in chemical-treated groups rela-
tive to vehicle-treated group.
Subsequently, the potent N-hydroxyphenyl-4-aryl-1,3-thiazole-2-
amine analogs selected through in vitro enzymatic assay were fur-
ther validated in a cell-based assay (Tables 1 and 2), in which LTs
B4 (LTB4) is released into culture media from Ca2+ionophore
(A23187)-stimulated rat basophilic leukemia-1 (RBL-1) cells. We sim-
ply reasoned that additional methyl group around OH of R2 can
make readily penetrable to cell membrane. Importantly, pharmaco-
phore that we found is the structure bearing thiazole ring with OH
or NH2 at 4 position of R2 and the presence of this functional group
is especially crucial for 5-LO inhibitory activity. It may be expected
that substituents can improve uptake into cells or protect from
intracellular metabolism of OH or NH2 of benzene ring because OH
or NH2 group of R2 is likely to be metabolized in vivo (our unpub-
lished pharmacokinetics data). Based on this rationale, selected
compounds 13–18 were tested as a function of dose in a cell-
based format to achieve the IC50 values of those compounds
(Table 2). Some of the derivatives were comparable or superior to
zileuton with respect to interference with LTB4 generation. Intrigu-
ingly, we found that modification of substituent around OH group at
the 4- position of N-aryl moiety influenced very strongly the ability
of compounds to inhibit LTB4 production. As shown in Table 2, com-
pound 14 (IC50 = 25 nM, 98% inhibition at 1 lM) with 3,5-diCH3
group around OH group was shown to be the most potent in this
series of 1,3-thiazole analogs, while compound 17 with 2,5-diCH3
was relatively weak. However, compound 13 (IC50 = 77 nM, 99%
inhibition at 1 lM), 15 (IC50 = 348 nM, 93% inhibition at 1 lM) and
16 (IC50 = 324 nM, 81% inhibition at 1 lM) with Cl group at N-aryl
moiety were less potent than the compound 14, suggesting that
introduction of halogen group may interfere with being permeable
to cell membrane or may affect its cellular metabolism. As shown
in in vivo efficacies of the compound 14 via different routes
(Table 3), the compound 14 had a protective effect against AA-
induced edema when topically, but not orally administered, as
predicted strongly from pharmacokinetic data demonstrating that
derivatives with pharmacological moiety of thiazole did not exhibit
good bioavailability in rat (30). It is demonstrated that a specific
compound KR-33749, chosen through in vitro and cell-based assays,
improves inflammatory skin lesions such as atopic dermatitis. This
study highlights a perspective series of processes from screening,
SAR through in vivo efficacy over previous report performed in our
group. Through SAR study, specifically, we could propose pharmaco-
phore responsible for inhibitory potency. Typically, administration of
selected compound 14 via p.o route was less effective than that
via topical application or intraperitoneal route (data not shown),
implying that the compound 14 is readily susceptible to hepatic
metabolism. Topical application of the compound 14 to mice
reduced the inflammatory responses such as ear swelling and MPO
activity following AA spreading onto ear. More intriguingly, it not
only had more potent anti-inflammatory activity than had zileuton,
but also exhibited sustainable protective effect in AA-inflamed ear
model. The in vivo efficacy of compound 14 in mice given at a low
dose considerably improved the two indications of acute inflamma-
tion such as MPO and ear edema. Of note, preapplied zileuton did
not retain its anti-inflammatory effect, while compound 14 remained
relatively potent even if applied topically 2 days before AA painting.
It suggests that compound 14 resides longer around skin tissue or
is metabolically stable compared with zileuton (Figure 1). Lastly, we
checked the selectivity of compound 14 for 5-LOX over the other
isozymes (Table 4) because other LOXs shares partly with 5-LOX
with regard to the structural or non-heme iron constraint for their
activities. More favorably, it inhibited preferentially 5-LOX to 12-
LOX or 15-LOX so that IC50 values were 10–100 times higher for
12-(15-)LOX than that for 5-LOX, thereby minimizing side-effects. On
the other hand, 12-LOX and 15-LOX have, respectively, been known
Chem Biol Drug Des 2012; 80: 90–99
97

Suh et al.
on 5-LOX in mice given at low doses considerably improved two
indications of acute inflammation such as MPO and ear edema,
superior to zileuton which is the only drug approved by FDA as a
direct 5-LOX inhibitor. Although its precise mechanism remains
unclear, it might be estimated that skin penetration and metabolic
transformation of 14 would be primary for its therapeutic success.
In fact, we have not checked the mechanism yet. We will further
elucidate the action mechanism of compound 14 by which it
would inhibit in either reversible or irreversible. Regarding redox
properties, we examined indirectly the possibilities of redox inhibi-
tor by carrying out methemoglobin induction (data not shown).
Compound 14 did not induce methemoglobin formation, indicating
that it does not inhibit 5-LO in a redox manner. Besides in vivo
efficacy, it exerted more selective inhibition against 5-LOX over
other isozymes (Table 4). Therefore, it is encouraged from its selec-
tivity and in vivo efficacy data that this compound will be applica-
ble to a wide range of skin models associated with 5-LOX in the
future.
Figure 1: The sustainable anti-inflammatory activity of com-
pound 14 and zileuton when applied topically to ear. Chemical dis-
solved in acetone was applied on the right ear 30 min before
arachidonic acid (AA) painting (2 mg in 20 lL acetone) on the same
ear. One hour after AA application, ear thickness of inflamed right
ear was measured and then inflamed ears were taken for myeloper-
oxidase (MPO) activity. Ear thickness and MPO activity in ear homo-
genates. Data are mean € SEM from three experiments (n = 5 in
each experiment). To determine the duration of action of compound
14, the compound was applied and kept for 48, 24 or 0.5 h. After
the indicated times, AA was applied, and ear thickness and MPO
activity were measured 1 h later.
Acknowledgments
The present research has been conducted by the Settlement
Research Grant of Keimyung University in 2011.
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Table 4: Selective inhibition of compound 14 against 12-lipoxy-
genase (LOX) and 15-LOX.
IC50 (lM)a
Compound
12-LOXb
12.5
15-LOXb
>100
14
aIC50 of compound 14 for LOXs was calculated from % inhibitory activity as
a function of concentration.
bBoth 12-LOX and 15-LOX assays were carried out according to protocol as
previously reported (19).
to be implicated in tumor angiogenesis and colorectal tumorigenesis
(23,24). Therefore, dual inhibition of 5-LOX ⁄ 12-LOX or 5-LOX ⁄ 15-
LOX might be beneficial than single inhibition of 5-LOX under spe-
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