Discovery of selective imidazole-based inhibitors of mammalian 15-lipoxygenase: Highly potent against human enzyme within a cellular environment

Article abstract of DOI:10.1016/j.bmcl.2007.07.011

A series of 2,4,5-tri-substituted imidazoles has proven to be highly potent in inhibiting mammalian 15-lipoxygenase (15-LO) with excellent selectivity over human isozymes 5- and P-12-LO. Non-symmetrical sulfamides (e.g., 21a-n) were found to be suitable r

Full text of DOI:10.1016/j.bmcl.2007.07.011

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5115–5120
Discovery of selective imidazole-based inhibitors of mammalian
15-lipoxygenase: Highly potent against human enzyme within
a cellular environment
David S. Weinstein,a,* Wen Liu,a, Khehyong Ngu,a Charles Langevine,a
Donald W. Combs,a Shaobin Zhuang,b Cindy Chen,b Cort S. Madsen,b
Timothy W. Harperc and Jeffrey A. Robla
aDepartment of Discovery Chemistry, Bristol-Myers Squibb, Research and Development, PO Box 4000, Princeton,
NJ 08543-5400, USA
bDepartment of Cardiovascular Biology, Bristol-Myers Squibb, Research and Development, PO Box 4000, Princeton,
NJ 08543-5400, USA
cDepartment of Drug Metabolism, Bristol-Myers Squibb, Research and Development, PO Box 4000, Princeton, NJ 08543-5400, USA
Received 8 May 2007; revised 2 July 2007; accepted 5 July 2007
Available online 13 July 2007
Abstract—A series of 2,4,5-tri-substituted imidazoles has proven to be highly potent in inhibiting mammalian 15-lipoxygenase (15-
LO) with excellent selectivity over human isozymes 5- and P-12-LO. Non-symmetrical sulfamides (e.g., 21a–n) were found to be
suitable replacements for the earlier arylsulfonamide-containing members of this series (e.g., 2, 14a–p). Several members of these
series also demonstrated potent inhibition of human 15-LO in a cell-based assay.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The lipoxygenase family of enzymes has received consid-
erable attention for its role in the etiology of human
disease. For example, as a mediator of leukotriene
biosynthesis, 5-lipoxygenase (5-LO) has been an active
therapeutic target for the control of asthma.1 Human
15-lipoxygenase (h15-LO) has also emerged as an inter-
esting target for drug discovery, with reports implicating
its role in the progression of numerous diseases includ-
ing cancer,2 Alzheimer’s disease,3 and, most notably,
cardiovascular disease.4 However, convincing pre-clini-
cal or clinical evidence for therapeutic effects as a result
of 15-LO inhibition has yet to be reported. Also, recent
reports suggesting anti-inflammatory and anti-neoplas-
tic roles for 15-LO cast some uncertainty on the poten-
tial for this target in treating human disease.5,6 Herein
we describe our continued efforts to discover potent,
selective, and orally bioavailable inhibitors of h15-LO,
the identification of which is expected to assist in eluci-
dating the role of this enzyme in disease progression.
We previously reported the discovery of a series of trypt-
amine and homotryptamine-based sulfonamides as
inhibitors of mammalian (rabbit) 15-LO.7 This series
of compounds potently inhibited the enzyme with high
selectivity over the closely related isozymes 5- and plate-
let-derived 12-LO (P-12-LO). The p-methoxyphenyl-
substituted tryptamine derivative
1 (Fig. 1) was
previously reported to inhibit rabbit reticulocyte-derived
15-LO with IC50’s of 37 nM and 164 nM, employing
linoleic acid (LA) or arachidonic acid (AA) as enzyme
substrates, respectively.8 Despite the favorable inhibi-
tion potency and selectivity profiles of this lead series,
the generally unfavorable physical chemical properties
(solubility, logP) of its members generally precluded
their evaluation in vivo. A campaign was thus initiated
to improve these properties while retaining the activity
profile of the lead series. To this end, it was found that
the diaryl histamine-based sulfonamide 2, conceptually
derived from 1 by fragmentation of its indole core and
introduction of one nitrogen atom, showed improved
inhibitory potency against 15-LO while maintaining
Keywords: 15-Lipoxygenase; Inhibition; Imidazole; Sulfonamide; Sul-
famide; Arachidonic acid; Linoleic acid.
*
Corresponding author. Tel.: +1 609 252 3060; fax: +1 609 252
6804; e-mail: david.weinstein@bms.com
Current address: Amgen Inc., 1120 Veterans Blvd., South San
Francisco, CA 94080, USA.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.011

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D. S. Weinstein et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5115–5120
O
S
An initial survey of substituted phenyl groups at C-5 of
HN
n-Pentyl
the imidazole was carried out. Compounds were
prepared as described in Scheme 1. Treatment of the
commercially available c-chloro butyrophenones 3a–e
with sodium azide, followed by a-bromination of the
resulting ketones, gave bromo azides 4a–e. Imidazole
formation was accomplished by condensation with ben-
zamidine hydrochloride to provide the imidazole azides
5a–e, which were subsequently hydrogenated to the pri-
mary amines and converted to the 4-n-pentyl benzene
sulfonamides 2 and 6a–d. The 15-LO inhibitory activi-
ties of the differentially substituted C-5 phenyl imidaz-
oles are depicted in Table 1. The diphenyl imidazole
6a proved to be two- to threefold less potent than 2.
Electron-withdrawing groups at the para position of
the C-5 phenyl ring decreased potency relative to meth-
oxy, as evidenced by the trifluoromethyl and fluoro-con-
taining compounds 6b and 6c, respectively. Placement of
the methoxy group in the meta position of the C-5 phe-
nyl appendage (6d) also led to a significant loss in
potency.
O
OMe
N
r15-LO IC50 = 37 nM (LA), 164 nM (AA)
5-LO IC50 = 8.9 μM
P-12-LO IC50 > 10 μM
H
1
O
HN
S
n-Pentyl
O
N
OMe
N
r15-LO IC50 = 14 nM (LA), 50 nM (AA)
5-LO IC50 > 3 μM
P-12-LO IC50 > 3 μM
H
2
Figure 1. Tryptamine-derived sulfonamide 1 and 2,5-diarylhistamine
sulfonamide 2.
selectivity over 5- and P-12 LO’s. In addition, 2 demon-
strated potent inhibition of 15-LO in Chinese hamster
ovary (CHO) cells overexpressing human recombinant
enzyme (vide infra).
A series of sulfonamide analogs of the imidazole 2,
maintaining the p-methoxyphenyl group at C-5, was
O
N3
HN
S
n-Pentyl
O
Br
a, b
c
d,e
N
R
R
N
R
N
H
Cl
N3
R
N
H
O
O
3a-e
5a-e
2, 6a-d
4a-e
O
S
HN
n-Pentyl
X
Br
X
O
b
n
N
n
MeO
MeO
OMe
O
O
N
11
g,d,e
3a (n = 1, X = Cl)
7 (n = 1, X = N3)
8 (n = 2, X = Cl)
4a (n = 1, X = N3)
10 (n = 2, X = NPhth)
a
f
9 (n = 2, X = NPhth)
h
O
X
HN
n
S
R2
d,e
or
O
n
j, i
N
N
OMe
OMe
R1
R1
N
N
H
H
12a-n (n = 1, X = N3)
14 a-p (R2
=
n
-Pentyl)
13a,b (n = 2, X = NPhth,
15 (n = 1, R1 = Ph, R2 = Me)
R1 = Ph, 2-thienyl)
O
O
S
HN
S
n
-Pentyl
HN
n-Pentyl
O
O
Me
N
N
5e
+
k, d, e
OMe
OMe
(R = 4-OMe)
N
N
Me
16
17
Scheme 1. Reagents and conditions: (a) NaN3, DMF (95%); (b) Br2, 1,4-dioxane (99%); (c) benzamidine hydrochloride, potassium carbonate,
CH3CN (35–54%); (d) H2, Pd/C (10% w/w), MeOH (100%); (e) 4-n-pentylbenzenesulfonyl chloride, triethylamine, CH2Cl2 (90–95%); (f) potassium
phthalimide, DMF (81%); (g) 2-aminopyridine, EtOH (99%); (h) R1C(@N)NH2 hydrochloride, potassium carbonate, DMF; (i) 4-MePhSO2Cl,
triethylamine, CH2Cl2 (90–95%); (j) hydrazine hydrate, THF (60%); (k) MeI, NaH, DMF (96%).

D. S. Weinstein et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5115–5120
5117
Table 1. 15-LO inhibitory activities of sulfonamides 2, 6a–d Table 2. 15-LO Inhibitory activities of sulfonamides 2, 14a–p, 15
Compound
R
r15-LO IC50 (lM)
LAa AAb
Compound
n
R1
r15-LO IC50
(lM)
LAa
AAb
2
4-OMe
H
0.014
0.033
0.072
0.059
0.082
0.05
6a
6b
6c
6d
0.162
0.372
0.261
0.531
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
1
Ph
0.014
>10
>3
0.05
—
4-CF3
4-F
3-OMe
14a
14b
14c
14d
14e
14f
14g
14h
14i
4-OMe-Ph
4-Me-Ph
4-Cl-Ph
—
1.1
0.21
0.099
>10
3-NO2-Ph
H
Me
1.1
—
a Enzyme inhibition measured in the presence of linoleic acid as
substrate.
b Enzyme inhibition measured in the presence of arachidonic acid as
substrate.
>10
>10
—
Cyclopropyl
tert-Butyl
4-Pyridyl
2-Pyridyl
3-Pyridyl
2-Pyrazinyl
4-(2-Me)-thiazoyl
2-Thienyl
Ph
>10
>10
3211
5322
—
1.92
0.087
0.97
0.56
2.81
0.72
0.006
0.140
0.053
0.364
14j
14k
14l
then prepared according to the chemistry described in
Scheme 1. Treatment of c-chloro p-methoxy butyrophe-
none 3a with sodium azide provided azide 7. The
one-carbon homolog, phthalimide analog of 7 (9), was
prepared by treatment of chloride 8 with potassium
phthalimide. Treatment of the ketones with bromine
provided bromides 4a and 10. The azide 4a was con-
verted to imidazo [1,2-a]pyridine 11 by condensation
with 2-aminopyridine followed by reduction of the azide
and subsequent sulfonylation with 4-pentylbenzene sul-
fonyl chloride. Bromides 4a and 10 were condensed with
the appropriate amidines to provide the imidazole azides
12a–n and phthalimides 13a,b. Hydrogenation of the
azide or hydrazinolysis of the phthalimide afforded the
primary amines which were then converted to the sul-
fonamides 14a–p and 15. The regioisomeric N-methyl
imidazoles 16 and 17 were prepared by methylation of
the intermediate imidazole 5e with methyl iodide,
followed by subsequent separation of the regioisomers,
hydrogenation of the azides, and sulfonylation of the
resultant amines.
—
—
14m
14n
14o
14p
15
0.013
0.914
0.396
2.80
2-Thienyl
Ph (R2 = Me)
a Enzyme inhibition measured in the presence of linoleic acid as
substrate.
b Enzyme inhibition measured in the presence of arachidonic acid as
substrate.
of 2 (approximate 7 fold loss in activity with LA as
substrate, 60 fold loss with AA), the corresponding
2- and 3- pyridyl isomers (14j and 14k), however, were
less tolerated. The 2-pyrazinyl analog 14l and 4-(2-
methyl)thiazolyl analog 14m also showed reduced po-
tency versus 2. Isosteric replacement of the C-2 phenyl
group with 2-thienyl gave 14n, which proved to be
most potent within this series, providing single
digit nanomolar inhibition of 15-LO (LA as sub-
strate). In addition, 14n maintained selectivity over
5-LO (IC50 > 10 lM) and P-12-LO (IC50 = 9.7 lM).
The effect of one-carbon homologation of the imida-
zole-sulfonamide linker was also studied. In contrast
to previous observations in the tryptamine-based ser-
ies,7 homologation of the ethylene chain adjacent to
the sulfonamide nitrogen proved deleterious. Thus,
the one-carbon homologs of 2 and 14n (14o and
14p, respectively) were both an order of magnitude
less potent than their ethylene-linked counterparts.
The requirement for the imidazole N-1 hydrogen
was addressed by comparison of imidazo[1,2-a]pyri-
dine 11 and N-methylated regioisomers 16 and 17
with the related indole 1 and imidazole 2 . Removal
of the N–H pharmacophore resulted in a dramatic
loss in potency (IC50 > 10 lM) versus 15-LO. A sim-
ilar loss of activity was observed upon methylation
of the previously reported tryptamine-based series
of 15-LO inhibitors7 and is consistent with the
requirement for a hydrogen-bond donor at this posi-
tion of the molecule.
Despite its promising potency and isozyme selectivity,
14n did little to enhance the physicochemical limitations
of the tryptamine-based series, having both suboptimal
aqueous solubility and hydrophilicity (<1 lg/mL at
pH 6.5, logD > 6). Not surprisingly, a single, 7 mg/kg
oral dose of 14n to male Sprague Dawley rats resulted
in limited systemic plasma exposure (Cmax = 0.6 lM,
AUC = 1.60 lM h).
A variety of groups were explored at the imidazole
C-2 position (Table 2). Little tolerance was observed
for substitution at the para position of the C-2 phenyl
group. Introduction of methoxy or methyl groups (14a
and 14b, respectively) led to a significant loss of inhib-
itory activity. Some tolerance was noted for para-
chlorophenyl and meta-nitrophenyl groups (14c and
14d, respectively). The C-2 unsubstituted compound
14e proved to be inactive as did the C-2 methyl and
cyclopropyl analogs (14f and 14g). Incorporation of
the tert-butyl group (14h) resulted in only micromolar
potency. Several heterocyclic replacements for the C-2
phenyl group were also explored. While the 4-pyridyl
analog 14i maintained some of the inhibitory potency
It is also noteworthy that in contrast to the previously
reported indole-based series,7 the presence of the
p-methoxyphenyl group at C-5 of the imidazole core
of 2 (analogous to the C-2 indole substituent of 1) did
not allow for a significant reduction in the size of the
aryl sulfonamide moiety. Thus, the p-tolyl sulfonamide
15 proved to be significantly less potent than the corre-

5118
D. S. Weinstein et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5115–5120
O
O
S
O
NR1R2
OMe
O
HN
HN
S
N
NH2
O
O
S
Cl
O
N
a
Cl
NH
O
b
+
N
N
OMe
O
OMe
O
N
Ar
N
N
Ar
H
Ar
H
H
19a, Ar = 2-thienyl
19b, Ar =phenyl
18
20a,b
21a-l
Scheme 2. Reagents and condition: (a) Et3N, CH2Cl2; (b) NHR1R2, CH3CN (75–98% for two steps).
sponding n-pentyl benzene sulfonamide 2. On the basis
of these findings, significant additional modifications
would need to be incorporated into the imidazole-based
series to further improve its physicochemical properties.
14n, an improvement in systemic plasma exposure after
a single 7 mg/kg oral dose of 21a to rats was observed
(Cmax = 1.7 lM, AUC = 4.4 lM h) in part attributed
to an increased aqueous solubility of 21a by the presence
of the sulfamide group. The chlorinated phenyl pipera-
zines 21c,d proved to be of similar potency to alkyl pip-
erazines 21a,b and more potent than the 2-phenyl
substituted imidazoles 21e and 21f. Given the promising
activity of the piperazine sulfamides, a solution-phase li-
brary of sulfamides utilizing amine 19b and diamines
(primary or secondary amines bearing pendant tertiary
amines) was prepared. From this work, it was deter-
mined that sulfamides derived from 1-benzyl-3-amino-
pyrrolidines (21g–i) were found to be suitable
replacements for the piperazine sulfamides, with some
preference for the R isomer observed. The sulfamide
21g derived from R-1-benzyl-3-aminopyrrolidine proved
to be threefold (with both substrates) more potent than
the corresponding S isomer 21h. An additional threefold
boost in potency was observed upon methylation of the
exocyclic amine with maintained preference for the R
enantiomer (21i) over the S isomer (21j). Replacement
of the phenyl substituent at the imidazole C-2 of 21i with
thiophene (21k) maintained activity, consistent with
observations in the sulfonamide series (cf., 14n and 2).
Incorporation of chlorine atoms at the 3- and 4-posi-
tions of the benzylic phenyl group (21l) continued to im-
prove potency by another 3-fold over the simple benzyl
pyrrolidine 21k. Bearing in mind the propensity of ben-
zylic amines to undergo metabolic oxidative de-alkyl-
ation, the gem-dimethyl benzyl pyrrolidine 21m and
the p-fluoro phenethyl pyrrolidine 21n were prepared.
21n proved to be the most potent 15-LO inhibitor in
the series, with an IC50 of 3 nM (LA).
A significant improvement in the physicochemical prop-
erties of the lead series was realized by incorporation of
tertiary amine-containing sulfamides as replacements
for the sulfonamide functionality in 2. Non-symmetrical
sulfamides were prepared by condensation of carbamoyl
chlorosulfamate 18 with either amine 19a or 19b in the
presence of triethylamine to give the sulfamoyl oxazolid-
inones 20a,b (Scheme 2). Displacement of the oxazolid-
inone leaving group with either primary or secondary
amines then provided the sulfamides 21a–l.9 The
3-aminopyrrolidines employed for the preparation
of examples 21j–l were prepared as described in
Scheme 3. Thus, N-acetyl-D-aspartic acid anhydride
22 was condensed with either cumylamine (for 21k) or
1-(4-fluorophenyl)ethanamine (for 21l) to provide the
imides 23 and 24.10 Reduction of the imides provided
the 3-ethylaminopyrrolidines 25 and 26. For the prepa-
ration of 21j, alkylation of (3R)-(+)-3-(Boc-amino)pyr-
rolidine 27 with 3,4-dichlorobenzyl bromide in the
presence of potassium carbonate, followed by removal
of the Boc group and reductive methylation, provided
the homochiral 3-methylaminopyrrolidine 28.
N-Alkyl-substituted piperazine sulfamides were first
explored as replacement for the phenyl sulfonamides
(Table 3). The 4-n-butyl piperazine 21a provided potent
inhibition of the enzyme, with a significant loss in
potency observed upon chain elongation to the 4-n-pen-
tyl piperazine 21b. As compared to phenyl sulfonamide
Representative sulfonamide and sulfamide-bearing com-
pounds were assayed for inhibitory activity over human
5- and P-12-LOs (Table 4). Only modest inhibition of
these isozymes with select compounds at relatively high
concentrations (10 or 3 lM) was observed, consistent
with the selectivity noted for the tryptamine series of
15-LO inhibitors.7
O
O
AcHN
AcHN
EtHN
a, b
c
O
N
R
N R
O
O
22
23 R = C(Me)2Ph
24 R = CH(Me)[(4-F)-Ph]
25 R = C(Me)2Ph
26 R = CH(Me)[(4-F)-Ph
H
O
N
Cl
Given the promising potencies of these histamine-based
sulfonamides and sulfamides in inhibiting isolated rab-
bit-derived 15-LO enzyme, select compounds were also
assayed for their abilities to inhibit the activity of the
human enzyme in a cellular assay (Table 5). Chinese
hamster ovary (CHO) cells overexpressing recombinant
h15-LO were employed in this study, with enzymatic
d, e, f, c
MeHN
N
NH
O
Cl
27
28
Scheme 3. Reagents and conditions: (a) NHR, acetone; (b) Ac2O,
reflux (85% for two steps); (c) LAH, THF (74%); (d) 3,4-dichlorob-
enzyl bromide, K2CO3, DMF (98%); (e) TFA, CH2Cl2 (95%); (f)
methyl chloroformate, K2CO3, CH2Cl2, H2O (65%).
inhibition
measured
by
reverse-phase
HPLC

D. S. Weinstein et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5115–5120
5119
Table 3. 15-LO inhibitory activities of sulfamides 21a–l
Table 4. Selectivity data for selected compounds
Compound
% Inhibitiona
Compound Ara NR1R2
r-15-LO IC50
(lM)
5-LO
P-12-LO
LAb
AAc
2b
6a
15
39
45
41
67
45
32
47
50
42
21a
21b
21c
21d
21e
21f
21g
A
A
A
A
B
B
B
0.014 0.05
0.091 0.646
0.045 0.132
0.036 0.133
0.055 0.378
0.212 3.45
0.083 0.719
N
N
N
N
N
N
N
N
N
N
N
N
n-Bu
6c
14n
21l
(n-Pentyl)
(2-Cl)Ph
a Measured at 10 lM inhibitor concentration, unless specified
otherwise.
b Measured at 3 lM inhibitor concentration.
Table 5. CHO cell 15-hLO inhibitory activities of selected compounds
(3,4-diCl)Ph
(3,4-diCl)Ph
Compound
15-LO inhibition
% Inhibition (10 lM)
IC50 (lM)
2
0.402
0.147
3.13
83
98
70
84
96
14n
21i
21d
21n
0.433
0.051
Ph
Bn
N
HN
HN
H
In conclusion, a series of 2,4,5-tri-substituted imidazoles
evolved from previously described tryptamine-based
compounds7 has provided potent inhibitors of mamma-
lian 15-LO. Replacement of the aryl sulfonamide moiety
with sulfamides was well tolerated, retaining potency of
the former while showing promise in improving physico-
chemical and pharmacokinetic properties. As observed
in our previous work,7 inhibitor potency depends on
the choice of substrate employed in the assay (LA vs
AA). The Km values reported for both substrates against
15-LO isolated from human cultured keratinocytes are
very similar (10.6 lM and 9.5 lM for AA and LA,
respectively),12 making it difficult to reconcile the differ-
ence in inhibitor potency based on these values alone.
Additional studies, beyond the scope of this investiga-
tion, will be required to adequately explain these find-
ings. Selectivity over isozymes 5- and P-12-LO was
also demonstrated. Importantly, representative exam-
ples proved to be potent in inhibiting h15-LO in a cellu-
lar assay. The ability of these compounds to inhibit both
the human and rabbit enzymes is consistent with their
overall sequence homology (80%).13 While an X-ray
crystal structure of h15-LO has yet to be reported, the
structural similarity of the rabbit and human enzymes
is also supported by a recent report which demonstrates
the utility of a homology model based on the crystal
structure of rabbit-derived 15-LO in identifying inhibi-
tors of the human enzyme.14 The identification of potent
and selective inhibitors of 15-LO may enable better val-
idation of this target and ultimately new therapeutic
treatments for human disease.
Bn
Bn
H
N
21h
21i
B
B
B
A
0.272 2.41
0.031 0.186
0.080 0.822
0.038 0.375
N
N
Me
H
Bn
Bn
H
N
21j
N
N
Me
Me
N
N
21k
H
(3,4-DiCl)Ph
21l
A
A
A
0.011 0.083
0.010 0.132
0.003 0.075
N
Me
H
Me
Ph
Me
N
21m
21n
N
Et
Et
H
Me
(4-F)Ph
N
N
H
a A, 2-thienyl; B, phenyl.
b Enzyme inhibition measured in the presence of linoleic acid as
substrate.
c Enzyme inhibition measured in the presence of arachidonic acid as
substrate.
quantitation of the extent of generation of 15-HETE
from exogenous AA.11 The sulfonamides 2 and 14n both
provided potent (submicromolar) inhibition of human
15-LO in this cellular context. The sulfamides 21i, 21d,
and 21n also provided potent inhibition in the CHO as-
say, with 21n proving the most potent, consistent with
its high inhibitory activity against the isolated rabbit
enzyme.
References and notes
1. (a) Larsen, J. S.; Acosta, E. P. Ann. Pharmacother. 1993,
27, 898; (b) Ford-Hutchinson, A. W. New Drugs Asthma
1992, 2, 94c, 1968, 90, 8234.

5120
D. S. Weinstein et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5115–5120
2. (a) Kelavkar, U.; Glasgow, W.; Eling, T. E. Curr. Urol.
10. (a) Witiak, D. T.; Vishnuvajjala, B. R.; Cook, W. L.;
Minatelli, J. A.; Gupta, T. K.; Gerlad, M. C. J. Med.
Chem. 1977, 20, 801; (b) Hudkins, R. L.; Dehvan-
Hudkins, D. L.; Doukas, P. Bioorg. Med. Chem. Lett.
1997, 7, 979.
11. Stably transfected Chinese hamster ovary (CHO) cells
overexpressing human 15-lipoxygenase cDNA were gen-
erated. In a 48-well format (triplicate wells per data
point), CHO/15-LO cells were pretreated with com-
pound, supplemented with arachidonic acid (5 lM final
concentration), whereupon the 15-HETE released by the
cell into the surrounding serum-free medium was ana-
lyzed by reverse-phase HPLC on a Waters 600 chro-
Rep. 2002, 3, 207; (b) Nie, D.; Che, M.; Grignon, D.;
Tang, K.; Honn, K. V. Cancer Metast. Rev. 2001, 20, 195.
3. Pratico, D.; Zhukareva, V.; Yao, Y.; Uryu, K.; Funk, C.
D.; Lawson, J. A.; Trojanowski, J. Q.; Lee, V. M.-Y. Am.
J. Pathol. 2004, 164, 1655.
4. (a) Zhao, L.; Grosser, T.; Fries, S.; Kadakia, L.; Wang,
H.; Zhao, J.; Falotico, R. Expert Rev. Clin. Immunol.
2006, 2, 649; (b) Zhao, L.; Funk, C. D. Trends Cardiovasc.
Med. 2004, 14, 191; (c) Cornicelli, J. A.; Trivedi, B. K.
Curr. Pharmaceut. Design 1999, 5, 11.
5. (a) Gronert, K.; Maheshwari, N.; Khan, N.; Hassan, I. R.;
Dunn, M.; Laniado Schwartzman, M. J. Biol. Chem. 2005,
280, 15267; (b) Serhan, C. N.; Jain, A.; Marleau, S.; Clish,
C.; Kantarci, A.; Behbehani, B.; Colgan, S. P.; Stahl, G.
L.; Merched, A.; Petasis, N. A.; Chan, L.; Van Dyke, T. E.
J. Immunol. 2003, 171, 6856.
6. (a) Fuerstenberger, G.; Krieg, P.; Muller-Decker, K.;
Habenicht, A. J. Int. J. Cancer 2006, 119, 2247; (b)
Middleton, M. K.; Zukas, A. M.; Rubinstein, T.; Jacob,
M.; Zhu, P.; Zhao, L.; Blair, I.; Pure, E. J. Exp. Med.
2006, 203, 2529.
7. Weinstein, D. S.; Liu, W.; Gu, Zh.; Langevine, C.; Ngu,
K.; Fadnis, L.; Combs, D. W.; Sitkoff, D.; Ahmad, S.;
Zhuang, S.; Chen, X.; Wang, F.-L.; Loughney, D. A.;
Atwal, K. S.; Zahler, R.; Macor, J. E.; Madsen, C. S.;
Murugesan, N. Bioorg. Med. Chem. Lett. 2005, 15, 1435.
8. See footnote 17 of Ref. 7 for a description of the 15-LO
enzyme inhibition colorimetric assay.
9. Ducry, L.; Reinelt, S.; Seiler, P.; Diederich, F.; Bolin, D.
R.; Campbell, R. M.; Olson, G. L. Helv. Chim. Acta 1999,
82, 2432.
matography system using
a
Luna C18 column
(2 · 50 mm). The column was developed at a flow rate
of 0.4 ml/min by a gradient program using two solvents
(A and B) set at 70% B for 0–6 min, 100% B for
7–13.5 min, where A was 50% MeOH/H2O/0.1%TFA/
0.05%TEA and
B
was 90%MeOH/H2O/0.1%TFA/
0.05%TEA. The HPLC eluate was monitored using a
Waters 996 photodiode array (PDA) detector set at
235 nm for monoHETE. In order to record the UV
spectra for each individual peak, the PDA was set for
data acquisition from 200 to 300 nm. Products were
quantitated based on absorbance at 235 nm using
Millennium32 (Waters) software.
12. Burrall, B. A.; Cheung, M.; Chiu, A.; Goetz, E. J.
J. Invest. Dermatol. 1988, 91, 294.
13. Sigal, E.; Grunberger, D.; Craik, C. S.; Caughey, G. H.;
Nadel, J. A. Adv. Prostag. Thromb. L. 1989, 19, 156.
14. Kenyon, V.; Chorny, I.; Carvajal, W. J.; Holman, T. R.;
Jacobson, M. P. J. Med. Chem. 2006, 49, 1356.