Discovery of potent and selective inhibitors of human platelet-type 12-lipoxygenase

Article abstract of DOI:10.1021/jm2005089

We report the discovery of novel small molecule inhibitors of platelet-type 12-human lipoxygenase, which display nanomolar activity against the purified enzyme, using a quantitative high-throughput screen (qHTS) on a library of 153607 compounds. These compounds also exhibit excellent specificity, >50-fold selectivity vs the paralogues, 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity vs ovine cyclooxygenase-1 and human cyclooxygenase-2. Kinetic experiments indicate this chemotype is a noncompetitive inhibitor that does not reduce the active site iron. Moreover, chiral HPLC separation of two of the racemic lead molecules revealed a strong preference for the (-)-enantiomers (IC₅₀ of 0.43 ± 0.04 and 0.38 ± 0.05 μM) compared to the (+)-enantiomers (IC₅₀ of >25 μM for both), indicating a fine degree of selectivity in the active site due to chiral geometry. In addition, these compounds demonstrate efficacy in cellular models, which underscores their relevance to disease modification.

Full text of DOI:10.1021/jm2005089

ARTICLE
pubs.acs.org/jmc
Discovery of Potent and Selective Inhibitors of Human Platelet-Type
12- Lipoxygenase
Victor Kenyon,||,^,# Ganesha Rai,†,^ Ajit Jadhav,† Lena Schultz,† Michelle Armstrong,|| J. Brian Jameson, II,||
Steven Perry,|| Netra Joshi,|| James M. Bougie,† William Leister,† David A. Taylor-Fishwick,‡ Jerry L. Nadler,‡
Michael Holinstat,§ Anton Simeonov,† David J. Maloney,*,† and Theodore R. Holman*,||
†NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center
Drive, MSC 3370 Bethesda, Maryland 20892-3370, United States
‡Department of Internal Medicine, Strelitz Diabetes Center, Eastern Virginia Medical School, Norfolk, Virginia 23501-1980,
United States
§Department of Medicine, Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Pennsylvania
19107, United States
Department of Chemistry and Biochemistry, University of California, Santa Cruz, 250 Physical Sciences Building, Santa Cruz, California
95064, United States
S Supporting Information
b
ABSTRACT: We report the discovery of novel small molecule
inhibitors of platelet-type 12-human lipoxygenase, which display
nanomolar activity against the purified enzyme, using a quanti-
tative high-throughput screen (qHTS) on a library of 153607
compounds. These compounds also exhibit excellent speci-
ficity, >50-fold selectivity vs the paralogues, 5-human lipoxygen-
ase, reticulocyte 15-human lipoxygenase type-1, and epithelial
15-human lipoxygenase type-2, and >100-fold selectivity vs
ovine cyclooxygenase-1 and human cyclooxygenase-2. Kinetic
experiments indicate this chemotype is a noncompetitive inhibitor that does not reduce the active site iron. Moreover, chiral
HPLC separation of two of the racemic lead molecules revealed a strong preference for the (ꢀ)-enantiomers (IC50 of 0.43 ( 0.04
and 0.38 ( 0.05 μM) compared to the (+)-enantiomers (IC50 of >25 μM for both), indicating a fine degree of selectivity in the
active site due to chiral geometry. In addition, these compounds demonstrate efficacy in cellular models, which underscores their
relevance to disease modification.
’ INTRODUCTION
laboratories, remains the only FDA approved drug which targets
a human lipoxygenase.12 Both Pfizer and Merck have developed
potent and selective inhibitors of 5-LOX (PF-419183413 and MK-
0633,14 respectively), however, both of these appear to have been
discontinued fromfurther clinical development.15 Reticulocyte 15-
LOX-1 has been implicated in colorectal16,17 and prostate18ꢀ21
cancers, while epithelial 15-LOX-2 is expressed in hair, prostate,
lung, and cornea22,23 and has been demonstrated to have an in-
verse correlation of expression and prostate cancer.24,25 Mutations
in epidermis-type lipoxygenase-3 and 12-(R)-LOX, which are
expressed in the skin, have been shown to cause nonbullous
congenital ichthyosiform erythroderma.26 Human platelet-type
12-(S)-LOX (12-LOX), for which the current study focuses on,
has been implicated in skin disease,27 pancreatic,28 breast,29,30
prostate cancers,21 diabetes,31 andbloodcoagulation32 and platelet
activation.33,34
Lipoxygenases (LOXs) are nonheme, iron-containing enzymes
that are ubiquitous in the plant and animal kingdoms. LOXs regio-
and stereospecifically peroxidate polyunsaturated fatty acids (e.g.,
arachidonic acid (AA) and linoleic acid (LA)) containing cis,cis-
1,4-pentadiene moieties to form the corresponding hydroperoxy
fatty acids.1 LOXs are the first committed step in a cascade of
metabolic pathways that are implicated in the onset of inflam-
matory diseases such as cancers, heart disease, and asthmas,2ꢀ5
making LOXs ideal candidates for pharmaceutical inhibitory
treatment. However, the discovery of selective, potent inhibi-
tors is critical to providing relevant chemical tools and probes to
investigate LOXs involvement in inflammation and disease states.
Human LOXs are distributed among a variety of tissues and
cellular locations and have been implicated in numerous disease
states. 5-LOX shuttles between the cytosol and nuclear mem-
brane6,7 and has been implicated in cancer8ꢀ10 and asthma.5,7
Despite 5-LOX having been targeted by pharmaceutical com-
panies for many years,11 Zileutin, developed by the Abbott
Received: April 26, 2011
Published: July 08, 2011
r
2011 American Chemical Society
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ARTICLE
Figure 1. Previously reported 12-LOX inhibitors.
Previous studies have demonstrated elevated 12-LOX mRNA
expression levels in cancerous prostate tissue cell lines35 and that
addition of 12-S-hydroxyeicosatetraenoic acid (12-HETE) to hu-
man prostate adenocarcinoma cells increased their motility and
ability to invade neighboring tissues. This study has been supple-
mented with recent data that demonstrates 12-LOX enhances the
excretion and expression of vascular endothelial growth factor
(VEGF)35 under hypoxic conditions by up-regulating the protein
level, mRNA, and functionality of hypoxia inducible factor-1 alpha
(HIF-1R), a transcription factor that up-regulates VEGF activity.36
Taken together, these studies provide a case for the involvement
of 12-LOX in prostate cancer. Additional reports have implicated
12-LOX in breast cancer. 12-LOX mRNA was found to have
an increased expression in breast cancer tissue cell lines versus
noncancerous breast epithelial cell lines, where the addition of
the LOX inhibitor cinnamyl-3,4-dihydroxy-R-cyanocinnamate
dramatically inhibited the growth of the breast cancer cell lines.37
Increased expression of 12-LOX mRNA has also been witnessed
in tissues extracted from patients with breast cancer38ꢀ40 versus
healthy adjacent tissue. 12-LOX is also expressed in human pan-
creatic islets, which increases with inflammatory cytokines and
the 12-HETE generated leads to reduced insulin secretion and
pancreatic β cell damage.31
There have been many attempts to discover selective and
potent12-LOX inhibitorswithlimitedsuccess. Past drug discovery
efforts for human lipoxygenases have utilized traditional medicinal
chemistry41ꢀ49 and natural product isolation,50ꢀ58 yielding com-
pounds that are reductive or promiscuous phenolic/terpene-based
inhibitors. Other reports have focused on pharmacophore virtual
screening59 to yield novel, selective inhibitors for rabbit 15-LOX.
However, there are few 12-LOX inhibitors that are selective or
potent and even fewer which are both.58,60ꢀ62
Several natural products have been shown to exhibit micro-
molar inhibition of 12-LOX, particularly Hinokitiol58 and the
catechins from green tea leaf extract,62 albeit with moderate
selectivity over other human LOXs. Our laboratory has dis-
covered and investigated many nonselective 12-LOX inhibitors.
Investigations of marine derived natural products yielded
brominated aryl phenols,57 and an SAR study, with the known
reductive LOX inhibitor, nordihydroguaiaretic acid (NDGA, a
pan-lipoxygenase inhibitor), yielded a variety of potent derivatives
as shown in Figure 1.63 It should be noted that the mistakenly
identified 12-LOX selective inhibitor, baicalein, was shown,
through steady-state kinetics, to not be selective in vitro and in
fact is equipotent against both 15-LOX-1 and 12-LOX.64
In an attempt to move from screening to more structure-based
design, our laboratory recently identified several nonreductive
inhibitors for 12-LOX and 15-LOX-1 by performing docking to
homology models, however these compounds displayed weak
potency and poor selectivity.65 Our laboratory has also recently
discovered four potent and selective 12-LOX inhibitors while
testing a high-throughput screen (HTS) against the National
Cancer Institute repository and the UC Santa Cruz Marine Extract
Library.60 Unfortunately, these compounds were not drug-like and
not amenable to modification.
Because of the dearth of selective and potent 12-LOX inhi-
bitors and the potential therapeutic benefit for such compounds,
herein we have performed a quantitative HTS (qHTS) against a
large library of 153607 small molecules66 in conjunction with the
NIH Molecular Libraries Probe Production Center Network
(MLPCN) in search of novel, potent, and selective 12-LOX
inhibitors. We report the discovery and SAR of an 8-hydro-
xyquinoline-based scaffold with nanomolar potency that is
selective over the related human isoforms, 5-LOX, reticulocyte
15-LOX-1, epithelial 15-LOX-2, as well as ovine cyclooxygen-
ase-1 and human cyclooxygenase-2. In addition, the mode of
inhibition and the mechanism of action are discussed.
’ CHEMISTRY
The synthesis of analogues (1ꢀ30: see Table 1) could be
rapidly accomplished utilizing the Betti reaction (a type of
Mannich reaction, Scheme 1).67ꢀ69 After optimization of the
reaction conditions, it was found that the reaction worked best in
the absence of solvent (neat) and at temperatures between 120
and 160 ꢀC (160 ꢀC when R2 = F, 150 ꢀC when R2 = Cl or NO2,
and 120 ꢀC when R2 = Br (with overnight heating). One of the
initial challenges that had to be overcome was purification of
these substrates. While ultimately these could be purified via
standard reversed-phase prep-HPLC, we found them to be not
well-behaved and often resulted in having to reprep the samples
to achieve >95% purity. However, we found that recrystallization
using ethanol or a mixture of ethanolꢀDMF provided the pure
product without any chromatographic purification. This method
allowed for the rapid preparation of a wide variety of analogues
for SAR investigations. The synthesis of 31 and 33 (see Table 2)
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Journal of Medicinal Chemistry
ARTICLE
Table 1. 12-LOX Inhibition of Analogues (1ꢀ30)a
Table 2. 12-LOX Inhibition of Analogues (31ꢀ33)a
IC50 (μM)
compd
IC50 (μM) [( SD (μM)]
compd
R1
R2
R3
R4
[( SD (μM)]
31
32
33
>75
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
NO2 thiophene
CH2CH3
CH2CH3
CH3
0.8 [0.2]
1.0 [0.3]
1.0 [0.1]
14 [3.0]
1.0 [0.2]
3.4 [0.6]
2.0 [0.2]
1.2 [0.4]
1.0 [0.2]
3.0 [0.5]
2.0 [0.5]
2.0 [0.3]
5.0 [1]
1.6 [0.3]
3.0 [0.6]
1.2 [0.4]
2.6 [0.4]
>50
>75
2
Cl
Cl
Br
Br
H
F
thiophene
thiophene
thiophene
thiophene
thiophene
thiophene
3.0 [0.7]
a The UVꢀvis-based manual inhibition data were fit as described in the
3
4
CH2CH3
CH3
Methods section.
5
KOH gave the methoxy derivative 31 in good yield. Piperidine
analogue 33 was synthesized via a Rh-catalyzed reduction of 9
under an atmosphere of hydrogen using methanol as a solvent.
Naphthol derivatives, such as compound 32, were synthesized
in a similar manner as analogues 1ꢀ30, except longer reaction
time and lower temperatures were required for optimal yields
(55 ꢀC, 15 h, see Supporting Information for a detailed synthetic
procedure for 31ꢀ33).
6
CH2CH3
CH3
7
8
NO2 furan
CH2CH3
CH2CH3
CH3
9
Cl
Cl
Br
Br
F
furan
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
furan
furan
CH2CH3
CH3
furan
furan
CH3
Cl
Cl
Cl
Cl
Cl
Cl
F
cyclopropane
cyclopropane
isopropyl
isopropyl
methyl
methyl
methyl
H
CH2CH3
CH3
’ RESULTS AND DISCUSSION
To discover novel inhibitors of 12-LOX, we utilized a chro-
mogenic assay to detect the hydroperoxide lipoxygenase reaction
products, as described elsewhere.70 Briefly, after allowing the
lipoxygenase reaction to proceed to the desired completion point,
ferrous Xylenol Orange was added, and the purple product, gen-
erated by the oxidation of the ferrous ions to ferric ions by the
hydroperoxide, was quantitated using visible light absorbance
detection. Prior to the screen, the reaction parameters (concentra-
tions of substrate and enzyme, percent substrate conversion, re-
agent stability under working conditions) were tested and op-
timized to ensure adequate performance (data not shown). The
screen of the entire library involved testing each of the 153607
compounds as a dilution series of seven or more concentrations
spanning the 57 μM to 0.7 nM range. This was accomplished by
testing 936 assay plates in a fully automated robotic system
(Methods). The screen was associated with a high signal-to-
background average of 4.29 and an average Z0 factor of 0.69
(Supporting Information, Figure S1A). NDGA, present on every
screening plate as a 16-point doseꢀresponse, exhibited consis-
tent IC50 throughout the screen.
CH2CH3
CH3
CH2CH3
CH3
>150
CH3
>75
Cl
Cl
Cl
Cl
Cl
CH3
>150
5-Me-thiophene CH2CH3
3.5 [1]
>75
5-bromofuran
furan
CH2CH3
CH3
>75
N(Me)2
furan
CH3
>75
piperidine Cl
furan
CH3
>75
H
H
H
H
Cl
Cl
Cl
Cl
4-Me-Ph
4-F-Ph
furan
CH2CH3
CH2CH3
Ph
>150
>50
>25
furan
4-Me-Ph
>25
a The UVꢀvis-based manual inhibition data (3 replicates) were fit as
described in the Methods section.
Screening data analysis included classification of the concen-
tration responses based on curve shape, presence of asymptotes,
efficacy, and IC50, as described elsewhere.71 In addition to 149407
inactive samples and 2295 inconclusive responses, samples be-
longing to 67 clusters of structurally similar primary screening hits
and 124 singletons were associated with complete concentrationꢀ
response curves (Supporting Information, Figure S1B). Further
prioritization based on potency and synthetic tractability led to
selection of 59 compounds for additional testing. Complete
screening and follow-up data have been made available in Pub-
Chem (PubChem BioAssay identifier 2164). Orthogonal confir-
mation of the hits from the qHTS, using a UVꢀvis-based manual
assay, yielded an 8-hydroxyquinoline (8-HQ) based chemotype,
compound 1 (Figure 2), that displayed micromolar to submicro-
molar potency, with an IC50 of 0.8 ( 0.2 μM. To investigate
Scheme 1. Synthesis of 8-Hydroxyquinoline Analogues
(1ꢀ30)
were conveniently achieved utilizing a common intermediate 9.
Accordingly, treatment of analogue 9 with iodomethane and
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Journal of Medicinal Chemistry
ARTICLE
requirements for inhibition and potency, we prepared derivatives
of the 8-HQ scaffold that were chemically modified at the R1, R2,
R3, and R4 positions (see Table 1). In general, the core scaffold of
1 was tolerant to a variety of changes in size and electrostatics in
the R2, R3, and R4 positions without dramatic changes in potency,
however, there were several combinations of modifications that
were not well tolerated.
Investigations of various substituents at R3 revealed that
compared to the thiophene or furan moiety, both smaller groups
(R3 = Me, entries 18ꢀ20 and R3 = H, entry 21) and larger groups
(R3 = 4-Me-Ph, entry 27 and R3 = 4-F-Ph, entry 28) resulted in
essentially complete loss in activity. However, thiophene, furan,
cyclopropyl, cyclopentyl, and isopropyl groups at this position all
showed comparable activity.
While only limited investigation at R1 was conducted, all
modifications at this position led to loss in activity (entries
24ꢀ26), suggesting that the binding pocket does not tolerate
increased steric bulk in this position. Similarly, increasing the size
of the substituents off the amide (R4) was not beneficial for
improved activity, as shown with analogues 29 and 30 (R = Ph
and 4-Me-Ph, respectively). Additionally, R4 = CH(CH3)2 and
(CH2)2CH3 were synthesized and these analogues also displayed
weak potency (data not shown). In contrast, modifications to R2
was well tolerated, with H, Cl, Br, F, NO2 all showing comparable
activity.
bidentate fashion, given that piperidines are comparable in ligand
strength to pyridine75 and that neither of the monodentate modi-
fications of the 8-HQ core inhibit 12-LOX. This hypothesis is
supported by the fact that modifications at R1 (24, 25, 26) also
lower potency, presumably due to sterics as the 8-HQ moiety
binds the iron. Other small molecule 8-HQs, such as 8-HQ-5-
sulfonic acid and 8-HQ-5-nitro, were found to be weak inhibitors
(IC50 values >80 μM against 12-LOX), however, 5,7-dichloro-8-
HQ (chloroxine) was an exception. It effectively inhibited all
LOX isozymes tested (IC50 values, 5-LOX = 3.6 ( 0.8 μM, 12-
LOX = 1.9 ( 0.4 μM, 15-LOX-1 = 2.4 ( 0.4 μM, 15-LOX-2 = 28
( 7 μM). Interestingly, chloroxine is a topical antimicrobial
agent used in the treatment of seborrheic dermatitis, and while
no studies have linked lipoxygenase to this ailment, inflammation
has been implicated in its treatment, suggesting a possible
connection.76
We have attempted to characterize these inhibitors binding
directly to the active site ferrous and ferric ion, using UV and
fluorescence spectroscopy, however neither experiment was
successful. 12-LOX loses activity dramatically as it concentrates,
thus making it difficult to achieve enzyme concentrations that
would yield a signal of sufficient intensity to observe.
Our final investigation into the SAR around the 8-HQ scaffold
was to determine whether the two enantiomers had differential
potency. As such, we utilized normal-phase chiral HPLC to effec-
tively separate the enantiomers of both analogues 2 and 5 as
shown in Table 3 (HPLC traces, Supporting Information, Figure
S2 and S3). Both (ꢀ)-2 (aka 36) and (ꢀ)-5 (aka 34) displayed
12-LOX inhibition at approximately half the IC50 value of the
racemic mixture (34 (ꢀ)-enantiomer, IC50 = 0.43 ( 0.04 μM;
8-HQ scaffolds are known metal chelators,72 and chelation of
the LOX active site iron has been documented in the litera-
ture previously.73,74 Consequently, we sought to investigate the
chemical requirements for metal chelation by modifying the
oxygen and nitrogen ligands that could bind the active site iron
(Table 2, see compounds 31ꢀ33). Both methylation of the
phenol (entry 31) and removal of the ring nitrogen (analogue
32) resulted in a loss of potency. However, when the pyridine
was reduced to the piperidine (analogue 33), a nonrigid hetero-
cycle, the inhibition was unaffected (IC50 = 3.0 ( 0.7 μM). This
data is consistent with the inhibitors ligating the metal in a
(racemic)-5, IC50 = 1.0 ( 0.2 μM; 36 (ꢀ)-enantiomer, IC50
=
0.38 ( 0.05 μM; (racemic)-2, IC50 = 1.0 ( 0.3 μM). In con-
trast, (+)-2 (aka 37) and (+)-5 (aka 35) displayed no inhibition
(IC50 > 25 μM), indicating a fine degree of selectivity in the active
site due to chiral geometry. We are currently attempting to crystallize
the active enantiomers to determine their absolute stereochemistry.
The above data is suggestive that these inhibitors bind directly
to the active site iron, which raises the possibility that these
inhibitors could have the necessary redox potential to reduce the
active ferric iron to the resting ferrous state, as has been reported
previously.63,64,77,78 To investigate the redox potential of this
chemotype, DPPH, a free radical scavenger, was incubated with
compound 1 and no reduction of DPPH was observed. Stoichio-
metric reduction of DPPH was achieved by the known reductive
LOX inhibitor, NDGA, suggesting that these 8-HQ inhibitors are
Figure 2. Lead compound (1) and positions explored for SAR.
Table 3. 12-LOX Inhibition of Enantiomerically Pure Analogues (34ꢀ37)a
a The UVꢀvis-based manual inhibition data were fit as described in the Methods section.
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not reductive in nature. Unfortunately, measuring the oxida-
tion of the inhibitor (reduction of the active site iron) with the
pseudoperoxidase assay is not possible with 12-LOX due to a lack
of absorbance change at 234 nm. Another mechanism of inhibi-
tion seen for LOX is promiscuous inhibition due to small mole-
cule aggregates.79 Compound 3 was therefore screened with in-
creasing amounts of Triton X-100, from 0.005% to 0.02%, with
negligible changes in the IC50, indicating that small molecule
aggregates are not responsible for inhibition.
In addition to characterizing the inhibition constants, the
mode of binding was investigated with steady state kinetics using
compound 3 by monitoring the formation of 12-HPETE as a
function of substrate and inhibitor concentration in the presence
of 0.01% Triton-X-100. Replots of KM/kcat and 1/kcat versus inhi-
bitor concentration (Supporting Information, Figure S4A,B)
yielded linear plots, with Kic equaling 0.7 ( 0.09 μM and Kiu
equaling 0.8 ( 0.1 μM, which are defined as the equilibrium
constants of dissociation from the enzyme and enzyme substrate
complex, respectively. The similar affinity of inhibitor binding to
both the enzyme and the enzyme substrate complex (Kic is ap-
proximately Kiu) is a rare example of true noncompetitive inhibi-
tion,80 whose inhibitor affinity is not affected by substrate bind-
ing. Regarding the chemical mechanism of inhibition, compound
2 did not display a time-dependent inhibition when it was in-
cubated with 12-LOX, unlike the time dependent inhibition seen
for the bidentate catechol inhibitors against sLO-174 and the 8-HQ
derivatives against macrophage migration inhibitory factor (MIF).81
This data may suggest that compound 2 is not an irreversible
inhibitor, however, this could not be proven due to the inactiva-
tion of 12-LOX upon dialysis.
this chemotype between 12-LOX and 15-LOX-1 is less than that
between 12-LOX and 5-LOX, which may be accounted for by the
similar active sites of 12-LOX and 15-LOX-1.82
Compound 5 was also screened against the mouse 12/15-
lipoxygenase (12/15-LOX), which generates predominately 12-
HPETE, with a smaller percentage of 15-HPETE and it had an
IC50 value of 14 ( 3.0 μM. While this value is over 10-fold larger
than that for 12-LOX, it is still significantly lower than the IC50 of
5 against 15-LOX-1 (IC50 >150 μM), suggesting the active site of
12/15-LOX is more similar to 12-LOX than 15-LOX-1. This was
further supported when a highly specific inhibitor against 15-
LOX-1 (IC50 < 10 nM against 15-LOX-1) showed an IC50 of
greater than 75 μM against 12/15-LOX.70 In addition, compound
5 did not inhibit either cyclooxygenase-1 (COX-1) or COX-2
(IC50 > 100 μM for both), demonstrating a selectivity of greater
than 100-fold for 12-LOX over both COX-1 and COX-2.
To demonstrate the potential for these compounds to be utilized
in more advanced biological systems (e.g., cell-based assays), we
investigated various in vitro ADME properties of a representative
compound (analogue 34) as shown in Table 5. This chemotype
was found to have acceptable kinetic solubility. It should be noted
that these conditions are different from the conditions used for the
IC50 determinations, which had detergent, lower salt concentra-
tions, and higher pH, all leading to greater inhibitor solubility. The
inhibitor also showed good cell permeability and excellent stability
in PBS buffer and mouse plasma. However, the compound was
susceptible to metabolism by mouse liver microsomes with a T1/2
of under 10 min. Despite this result, we were eager to determine
the in vivo PK of this molecule to provide a basis for future
investigations in disease relevant mouse models. As shown in
Table 6, compound 34 had a reasonable plasma T1/2 of 3.5 h and
a Cmax of 288 μM. Importantly, the exposure level exceeded the
purified enzyme assay IC50 for the full 24 h period and IC50 in the
platelet assay (vide infra) for 8 h. Moreover, this compound does
not efficiently cross the bloodꢀbrain barrier (BBB), which for
the treatment of diseases such as diabetes and thrombosis is con-
sidered a desirable result as CNS-active compounds could result
in undesired side effects. These in vitro ADME and in vivo PK
results suggest that the molecules described above should provide
utility in probing the effects of 12-LOX inhibition in both cell-
based assays and possibly in vivo models.
To determine the selectivity of this inhibitor chemotype, a
subset of compounds was screened against a variety of LOX isozymes
(Table 4). In general, the selectivity was excellent, with the potency
difference being 25- to 150-fold against 15-LOX-1, 45- to 1300-fold
against 5-LOX, and 45- to 150-fold against 15-LOX-2. Chiral
analogue, 36, was not only the most potent of the inhibitors but it
also had some of the best selectivity relative to 5-LOX (1300-
fold) and 15-LOX-1 (130-fold). Interestingly, the selectivity of
Table 4. Selectivity Profile of Representative Analoguesa
Other groups have postulated that 7-substituted-8-HQ deri-
vatives, which contain substituted anilines in place of the amide
moiety (at C-9), undergo a retro-Mannich reaction to afford a
reactive species that can form a variety of covalent adducts (see
Figure 3b).83 In agreement with these findings, we too found
such analogues (aniline as opposed to amide group at C-9) were
relatively unstable in both assay buffer and mouse plasma. How-
ever, as shown above, our lead compounds are completely stable
in both PBS buffer (pH 7.4) and mouse plasma over a 48 h
period. Moreover, this particular chemotype displayed favorable
exposure levels in vivo (mouse) as described above. These find-
ings suggest that the retro-Mannich pathway is much less facile
for the amide-containing series, potentially as a result of amide
IC50 (μM)
compd
12-LOX
5-LOX
15-LOX-1
15-LOX-2
1
0.8
ND
>100
>100
>100
>100
>100
>100
>25
>25
>100
>50
>50
>30
>50
ND
3
1.0
ND
5
1.0
>100
>100
>100
ND
6
3.4
9
1.0
34
36
0.43
0.38
ND
a The UVꢀvis-based manual inhibition data (3 replicates) were fit as
described in the Methods section.
Table 5. In Vitro ADME Properties for Representative Analogue (Compound 34)a
aq kinetic soln
Caco-2
efflux ratio
mouse liver microsome stability PBS-pH7.4 stability: mouse plasma stability:
compd (PBS @ pH 7.4) (μM) (Papp10ꢀ6 m/s @ pH 7.4) (BfA)/(AfB)
(T1/2) (min)
% remaining after 48 h % remaining after 48 h
34
14.5
8.8
2.3
<10
100 98.3
a Kinetic solubility measurements were conducted at Analiza Inc. using nitrogen detection methodologies. Caco-2 permeability, microsomal stability,
and mouse plasma stability experiments were conducted at Pharmaron Inc.
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Table 6. In Vivo PK Data for Representative Analogue (Compound 34)a
t1/2
t1/2
Cmax
Cmax
tmax
tmax
compd
(h) [plasma]
(h) [brain]
[brain/plasma] b
0.01
(μM) [plasma]
(μM) [brain]
(h) [plasma]
(h) [brain]
cLogP
2.8
34
3.5
1.7
288
5
0.25
0.5
a Intraperitoneal (IP) administration (30 mg/kg body weight (mpk)), CD1 mice, n = 3, monitored at 8 time points (0.25, 0.5, 1, 2, 4, 8, 12, 24 h).
Compound 34 formulated as a suspension in 50% PEG 200 and 10% Cremophor EL in saline solution. b Calculated based on the average [b/p] ratio over
8 time points (24 h period). Experiments were performed by Pharmaron Inc.
Figure 3. (a) Representative 8-HQ-based ADAMTS-5 inhibitor reported by Wyeth researchers with amide nitrogen at C-9. (b) Proposed mechanism
of covalent modification for 8-HQs with aniline nitrogen at C-9.
nitrogen being less basic than the corresponding aniline nitrogen.
A comparable 8-HQ chemical series was reported by Wyeth
researchers as ADAMTS inhibitors, which like our chemotype
contains the amide moiety at C-9 (Figure 3a). They found that
the compound displayed good ADME properties (CYP inhibi-
tion and microsomal stability), supporting the notion that this
subtle structural difference may have a drastic effect on the overall
stability of this class of compounds.84
A select group of inhibitors, 1, 34, and 35, were then tested for
efficacy in a platelet cellular assay. Human platelets are known to
express large amounts of 12-LOX upon stimulation of the protease-
activated receptor (PAR) with its activating peptide, PAR1-AP.33
We therefore incubated our inhibitors with human platelets,
followed by stimulation with PAR1-AP and measured the change
in 12-HETE production. In three separate experiments, 100 μM
Figure 4. Titration curves of 1 (circles, solid line) and 34 (triangles,
of 1 and 34 showed significant inhibition of PAR1-AP-mediated
12-HETE production, while 35 showed no reduction in 12-HETE
production. This data confirms the in vitro data, where both 1 and
34 are potent (IC50 values of 0.8 and 0.43 μM, respectively), while
35 is inactive (IC50 value >25 μM) and supports the notion that
the potent in vitro inhibitors inhibit 12-LOX intracellularly. A
more thorough titration of inhibition in the platelet cells was then
performed with compounds 1 and 34 (inhibitor concentration
ranging from 1 to 100 μM). The IC50 values were determined to
be 15 ( 10 for 1 and 13 ( 7 for 34 (Figure 4). These cell-based
IC50 values are over 25-fold greater than the isolated enzyme IC50
values, which could be a result of limited solubility and/or intra-
cellular metabolism of the inhibitors. We are currently testing various
modifications of the inhibitor scaffold to improve the cellular
potency with the hopes of applying these inhibitors as anti-
coagulation therapeutics.
The ability of compound 1 to inhibit the activity of 12-LOX in
a diabetic disease relevant model was also assessed using primary
human islets obtained from donated tissues. Upon stimulation
with arachidonic acid and calcium ionophore, production of 12-
HETE in donor islets is significantly increased (Figure 5). This
increase was a consistent response across all donor islets tested,
and the degree of response was compatible with donor variation.
Inclusion of compound 1 at an assay concentration of 10 μM
resulted in an uniform inhibition of 68.9 ( 14.5% across the three
dashed line), against platelets, measuring inhibition of 12-HETE con-
centration by LC-MS-MS. Fitting the data to a simple hyperbolic curve
yielded IC50 values of 15 ( 10 μM for 1 and 13 ( 7 μM for 34.
nondiabetic donors tested (Figure 5A). The basal and stimulated
level of 12-HETE was elevated in islets from confirmed type 2
diabetic donors (Figure 5B). This is consistent with the relevance
of 12-LOX to diabetes disease progression. Again, compound 1
resulted in an equivalent inhibition of stimulated 12-HETE produc-
tion, 70 and 74%, respectively, in the two diabetic donors assayed.
’ CONCLUSION
Because of the absence of selective, drug-like inhibitors of 12-
LOX in the literature, a HTS campaign of 153607 compounds
was performed that identified an 8-HQ chemotype as a novel,
potent, and selective inhibitor of 12-LOX. Modifications to
various positions around the core scaffold led to the development
of an SAR profile. In general, the dynamic interplay between the
various substitutions is not well understood, and while the SAR
was relatively flat, several changes are not at all tolerated. The
most dramatic loss of inhibition was observed at the C-2 position
of the 8-HQ core (R1), where all substitutions resulted in a com-
plete loss of activity. Moreover, loss of activity was observed by
removal of the nitrogen on the pyridine and/or methylation of
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Figure 5. Inhibition of 12-HETE production in human donor islets. (A) 12-HETE levels form three nondiabetic donors (ND-1 to ND-3) either
unstimulated (white bars) or stimulated for 60 min with 100 μM arachidonic acid (AA) and 30 min with 5 μM A23187 (I) in the absence (gray bars) or
presence of 10 μM compound 1 (black bars). (B) shows 12-HETE levels in human islets from two confirmed type 2 diabetic donors (T2D-1 and T2D-2).
Conditions are as described in (A).
the 8-hydroxy group, which strongly suggests the inhibitor is
binding to the catalytic iron in a bidentate fashion. Further in-
vestigations demonstrated the (ꢀ)-enantiomers of this 8-HQ
chemotype are seemingly responsible for the observed activity,
with IC50 values approximately half of the racemic mixture (IC50
of 0.43 ( 0.04 for 34 and 0.38 ( 0.05 μM for 36). 5-LOX, 15-
LOX-1, 15-LOX-2, COX-1, and COX-2 were not inhibited by
this chemotype. Interestingly, this chemotype did show weak
inhibition against the 12/15-LOX (IC50 = 14 μM), suggesting
the active site of 12/15-LOX is more similar to 12-LOX than 15-
LOX-1 (15-LOX-1 IC50 > 150 μM). This is consistent with the
fact that 15-LOX-1 selective inhibitors were inactive against
12/15-LOX and indicates caution when performing lipoxygenase
studies on particular diseases with mouse models. Investigating the
mode of inhibition demonstrated that this class of compounds is
thought to be nonreductive and exhibits noncompetitive inhibition.
We are currently investigating these compounds in cell-based diabetes
and blood coagulation models, and it is our hope that this class of
inhibitors will provide a molecular tool to determine the biological
role of 12-LOX and further validate 12-LOX as a therapeutic target.
HPLC equipped with a Phenomenex Luna C18 reverse phase (5 μm,
30 mm ꢁ 75 mm) column having a flow rate of 45 mL/min. The mobile
phase was a mixture of acetonitrile (0.1% TFA) and H2O (0.1% TFA) at
room temperature.
Samples were analyzed for purity on an Agilent 1200 series LC/MS
equipped with a Luna C18 reverse phase (3 μm, 3 mm ꢁ 75 mm) column
having a flow rate of 0.8ꢀ1.0 mL/min over a 7 min gradient and a 8.5 min
run time. The mobile phase was a mixture of acetonitrile (0.025% TFA)
and H2O (0.05% TFA), and the temperature was maintained at 50 ꢀC.
Purity of final compounds was determined to be >95%, using a 3 μL
injection with quantitation by AUC at 220 and 254 nm (Agilent diode
array detector).
Chiral HPLC Separation of the Enantiomers. Methanol was
added to the sample until the sample was completely dissolved. Sample
dissolution and analysis was performed at room temperature. The analy-
tical analysis was performed on a Chiralcel OD column (4.6 mm ꢁ 150
mm, 5 μm). The mobile phase was 100% methanol at 1.0 mL/min. The
sample was detected with a diode array detector (DAD) at 254 and 330 nm.
Optical rotation (() was determined with an in-line polarimeter (PDR-
Chiral). Preparative purification was performed on a Chiralcel OD
column (2 cm ꢁ 25 cm, 5 μm). The mobile phase was 100% methanol
at 4.5 mL/min. Fraction collection was triggered by UV absorbance
(330 nm). The LC system was limited to 100 μL injections. The frac-
tions containing the requisite enantiomers were combined and concen-
trated under reduced pressure. The enantiomeric purity was determined
by reinjection on the analytical column described above. The enantio-
meric ratios (er) for all separated chiral compounds were found to be
greater than 99:1. The optical rotations of the final compounds were
obtained using a PerkinElmer model 341 polarimeter.
General Procedure for the Synthesis of 8-HQ Analogues
(Scheme 1). Differentially substituted-quinolin-8-ol (1 equiv), amide
(1.05 equiv), and the requisite aldehyde (1.1 equiv) were stirred neat at
120ꢀ160 ꢀC for 15 min to 12 h (depending on the substituent at C-5).
Upon heating, the reaction mixture melted and solid was formed after
completion of the reaction. The solid product was washed with ethyl
acetate, and the crude product was purified by recrystallization from
ethanol or an ethanolꢀDMF mixture.
’ EXPERIMENTAL SECTION
General Chemistry. Unless otherwise stated, all reactions were
carried out under an atmosphere of dry argon or nitrogen in dried glass-
ware. Indicated reaction temperatures refer to those of the reaction bath,
while room temperature (rt) is noted as 25 ꢀC. All solvents were of
anhydrous quality purchased from Aldrich Chemical Co. and used as
received. Commercially available starting materials and reagents were
purchased from Aldrich and were used as received.
Analytical thin layer chromatography (TLC) was performed with
Sigma Aldrich TLC plates (5 cm ꢁ 20 cm, 60 Å, 250 μm). Visualization
was accomplished by irradiation under a 254 nm UV lamp. Chroma-
tography on silica gel was performed using forced flow (liquid) of the
indicated solvent system on Biotage KP-Sil prepacked cartridges and
using the Biotage SP-1 automated chromatography system. 1H and 13
C
NMR spectra were recorded on a Varian Inova 400 MHz spectrometer.
Chemical shifts are reported in ppm with the solvent resonance as the
internal standard (CDCl3 7.26 ppm, 77.00 ppm, DMSO-d6 2.49 ppm,
39.51 ppm for 1H, 13C, respectively). Data are reported as follows: chem-
ical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
brs = broad singlet, m = multiplet), coupling constants, and number of
protons. Low resolution mass spectra (electrospray ionization) were
acquired on an Agilent Technologies 6130 quadrupole spectrometer
coupled to the HPLC system. High resolution mass spectral data was
collected in-house using an Agilent 6210 time-of-flight mass spectro-
meter, also coupled to an Agilent Technologies 1200 series HPLC
system. If needed, products were purified via a Waters semipreparative
Representative Characterization Data. N-((8-Hydroxy-5-
nitroquinolin-7-yl)(thiophen-2-yl)methyl)propionamide (1). LC-MS: rt
(min) = 5.16. 1HNMR(DMSO-d6) δ1.03 (t, J= 7.5 Hz, 3 H), 2.23 (qd, J=
7.6 and 3.1 Hz, 2 H), 6.81 (dt, J = 3.5 and 1.2 Hz, 1 H), 6.89 (dd, J = 8.6 and
1.0 Hz, 1 H), 6.95 (dd, J = 5.1 and 3.5 Hz, 1 H), 7.43 (dd, J = 5.1 and 1.2 Hz,
1 H), 7.90 (dd, J = 8.8 and 4.3 Hz, 1 H), 8.76 (s, 1 H), 9.02 (dd, J = 4.1 and
1.6 Hz, 1 H), 9.09 (d, J = 8.8 Hz, 1 H), and 9.19 (dd, J = 8.8 and 1.6 Hz, 1
H). 13C NMR (DMSO-d6) δ 9.86, 28.38, 45.33, 121.73, 123.84, 125.21,
125.29, 125.35, 126.87, 127.23, 133.05, 134.37, 136.80, 145.17, 149.03,
157.34 and 172.29. HRMS (m/z): [M + H]+ calcd for C17H16N3O4S,
358.0856; found, 358.0861.
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N-((5-Chloro-8-hydroxyquinolin-7-yl)(thiophen-2-yl)methyl)prop-
1
1 H), 8.97 (dd, J = 4.1 and 1.6 Hz, 1 H) and 10.39 (brs, 1 H). 13C NMR
(DMSO-d6) δ 22.51, 44.05, 106.99, 110.39, 118.48, 123.00, 123.05,
125.05, 126.14, 132.52, 138.62, 142.52, 149.19, 149.40, 153.86and168.47.
HRMS (m/z): [M + H]+ calcd for C16H14ClN2O3, 317.0687; found,
317.0689.
N-((5-Bromo-8-hydroxyquinolin-7-yl)(furan-2-yl)methyl)propionamide
(11). LC-MS: rt (min) = 5.39. 1H NMR (DMSO-d6) δ 1.01 (t, J = 7.6 Hz, 3
H), 2.20 (qd, J = 7.5 and 2.2 Hz, 2 H), 6.07 (d, J = 3.1 Hz, 1 H), 6.37 (dd, J =
3.2 and 1.9 Hz, 1 H), 6.71 (d, J = 8.4 Hz, 1 H), 7.59 (dd, J = 1.8 and 0.8 Hz,
1 H), 7.73 (dd, J = 8.6 and 4.1 Hz, 1 H), 7.88 (s, 1 H), 8.42 (dd, J = 8.6 and
1.6 Hz, 1 H), 8.81 (d, J = 8.8 Hz, 1 H), 8.94 (dd, J = 4.1 and 1.6 Hz, 1 H)
and 10.41 (brs, 1 H). 13C NMR (DMSO-d6) δ 9.81, 28.29, 43.96, 106.94,
108.33, 110.39, 123.38, 123.73, 126.35, 129.63, 134.95, 138.86, 142.51,
149.17, 150.03, 153.96, and 172.22. HRMS (m/z): [M + H]+ calcd for
C17H16BrN2O3, 375.0339; found, 375.0344.
N-((5-Bromo-8-hydroxyquinolin-7-yl)(furan-2-yl)methyl)acetamide
(12). LC-MS: rt (min) = 5.03. 1H NMR(DMSO-d6) δ 1.92 (s, 3 H), 6.04
- 6.11 (m, 1 H), 6.37 (dd, J = 3.2 and 1.9 Hz, 1 H), 6.70 (d, J = 8.6 Hz,
1 H), 7.56ꢀ7.62(m, 1 H), 7.73 (dd, J = 8.5and4.2 Hz, 1 H), 7.87 (s, 1 H),
8.43 (dd, J = 8.6 and 1.6 Hz, 1 H), 8.89 (d, J = 8.6 Hz, 1 H), 8.94 (dd, J =
4.1 and 1.6 Hz, 1 H) and 10.42 (brs, 1 H). 13C NMR (DMSO-d6) δ 22.51,
43.99, 106.96, 108.33, 110.39, 123.38, 123.65, 126.35, 129.61, 134.96, 138.86,
142.52, 149.18, 150.00, 153.87, and 168.47. HRMS (m/z): [M + H]+ calcd
for C16H14BrN2O3, 361.0182; found, 361.019.
Methods. Biological Reagents. All commercial fatty acids (Sigma-
Aldrich Chemical Co.) were repurified using a Higgins HAIsil Semi-
Preparative (5 μm, 250 mm ꢁ 10 mm) C-18 column. Solution A was
99.9% MeOH and 0.1% acetic acid; solution B was 99.9% H2O and 0.1%
acetic acid. An isocratic elution of 85% A:15% B was used to purify all fatty
acids, which were stored at ꢀ80 ꢀC for a maximum of 6 months.
Overexpression and Purification of Human 5-Lipoxygenase, Human
12-Lipoxygenase, and the Human 15-Lipoxygenases. Human platelet
12-lipoxygenase (12-LOX), human reticulocyte 15-lipoxygenase-1 (15-
LOX-1), human epithelial15-lipoxygenase-2 (15-LOX-2), wereexpressed
as N-terminally, His6-tagged proteins and purified to greater than 90%
purity, as evaluated by SDS-PAGE analysis.50,85,86 Human 5-lipoxy-
genase was expressed as a nontagged protein and used as a crude
ammonium sulfate protein fraction, as published previously.87 Iron
content of 12-LOX was determined with a Finnegan inductively coupled
plasma mass spectrometer (ICP-MS), using cobalt-EDTA as an internal
standard. Iron concentrations were compared to standardized iron solutions
and used to normalize enzyme concentrations.
ionamide (2). LC-MS: rt (min) = 5.6. H NMR (DMSO-d6) δ 1.03
(t, J = 7.6 Hz, 3 H), 2.16ꢀ2.28 (m, 2 H), 6.75ꢀ6.78 (m, 1 H), 6.89ꢀ6.96
(m, 2 H), 7.40 (dd, J = 5.1 and 1.0 Hz, 1 H), 7.74 (dd, J = 8.6 and 4.1 Hz,
1 H), 7.79 (s, 1 H), 8.50 (dd, J = 8.5 and 1.5 Hz, 1 H), 8.91 (d, J = 8.8
Hz, 1 H), 8.98 (dd, J = 4.1 and 1.4 Hz, 1 H) and 10.42 (brs, 1 H).;
HRMS (m/z): [M + H]+ calcd for C17H16ClN2O2S, 347.0618; found,
347.0621. (ꢀ)-2 (e.g., 36) [R]D23 = ꢀ24 (c = 0.6, CHCl3); (+)-2 (e.g.,
37) [R]D23 = +24 (c = 0.6, CHCl3).
N-((5-Chloro-8-hydroxyquinolin-7-yl)(thiophen-2-yl)methyl)acetamide
(3). LC-MS: rt (min) = 5.28. 1H NMR (DMSO-d6) δ1.94 (s, 3 H), 6.78 (d,
J = 3.5 Hz, 1 H), 6.89 (d, J = 8.8 Hz, 1 H), 6.94 (dd, J = 5.1 and 3.5 Hz, 1 H),
7.41 (dd, J = 5.1 and 1.0 Hz, 1 H), 7.75 (dd, J = 8.5 and 4.2 Hz, 1 H), 7.78 (s,
1 H), 8.51 (dd, J = 8.6 and 1.4 Hz, 1 H), 8.96ꢀ9.02 (m, 2 H) and 10.43 (brs,
1 H)). HRMS (m/z): [M + H]+ calcd for C16H14ClN2O2S, 333.0459;
found, 333.0460.
N-((5-Bromo-8-hydroxyquinolin-7-yl)(thiophen-2-yl)methyl)prop-
ionamide (4). LC-MS: rt (min) = 5.70. 1H NMR (DMSO-d6) δ 1.03 (t,
J = 7.5 Hz, 3 H), 2.16ꢀ2.29 (m, 2 H), 6.76 (d, J = 3.3 Hz, 1 H), 6.87ꢀ6.96
(m, 2 H), 7.40 (dd, J = 5.1 and 1.0 Hz, 1 H), 7.74 (dd, J = 8.5 and 4.2 Hz, 1
H), 7.96 (s, 1 H), 8.43 (dd, J = 8.6 and 1.4 Hz, 1 H), 8.86ꢀ8.99 (m, 2 H)
and 10.45 (brs, 1 H). HRMS (m/z): [M + H]+ calcd for C17H16BrN2O2S,
391.0105; found, 391.0108.
N-((5-Bromo-8-hydroxyquinolin-7-yl)(thiophen-2-yl)methyl)acetamide
(5) ML127. LC-MS: rt (min) = 5.36. 1H NMR (DMSO-d6) δ 1.94 (s, 3 H),
6.78 (dt, J= 3.5 and 1.2 Hz, 1 H), 6.89 (dd, J= 8.9 and 1.1 Hz, 1 H), 6.93 (dd,
J = 5.1 and 3.3 Hz, 1 H), 7.40 (dd, J = 5.1 and 1.4 Hz, 1 H), 7.74 (dd, J = 8.6
and 4.1 Hz, 1 H), 7.95 (s, 1 H), 8.43 (dd, J = 8.5 Hz and 1.5 Hz, 1 H), 8.95
(dd, J = 4.1 and 1.6 Hz, 1 H), 9.00 (d, J = 8.8 Hz, 1 H) and 10.46 (brs, 1 H).
13C NMR (DMSO-d6) δ 22.55, 45.35, 108.50, 123.41, 124.84, 125.08,
125.68, 126.34, 126.78, 129.38, 134.97, 138.89, 145.91, 149.24, 149.71, and
168.39. HRMS (m/z): [M + H]+ calcd for C16H14BrN2O2S, 376.9954;
found, 376.9956. (ꢀ)-5 (e.g., 34) [R]D23 = ꢀ18 (c = 0.3, CHCl3); (+)-
5 (e.g., 35) [R]D23 = +18 (c = 0.3, CHCl3)
N-((5-Fluoro-8-hydroxyquinolin-7-yl)(thiophen-2-yl)methyl)acetamide
(7).LC-MS: rt (min) = 4.76. 1H NMR (DMSO-d6) δ1.93(s,3H),6.77(dt,
J = 3.5 and 1.2 Hz, 1 H), 6.88ꢀ6.95 (m, 2 H), 7.40 (dd, J = 5.0 and 1.3 Hz,
1 H), 7.47 (d, J = 11.2 Hz, 1 H), 7.68 (dd, J = 8.5 and 4.2 Hz, 1 H), 8.44 (dd,
J = 8.5 and 1.7 Hz, 1 H), 8.91ꢀ8.99 (m, 2 H) and 10.07 (brs, 1 H). 13C
NMR (DMSO-d6) δ 22.55, 45.49, 104.54, 109.27, 109.48, 117.53, 117.72,
122.27, 123.67, 123.73, 124.86, 125.04, 126.73, 129.13, 129.16, 137.80,
137.83, 145.91, 146.04, 146.08, 148.17, 149.52, 150.60 and 168.37. HRMS
(m/z): [M + H]+ calcd for C16H14FN2O2S, 317.076; found, 317.0761.
N-(Furan-2-yl(8-hydroxy-5-nitroquinolin-7-yl)methyl)propionamide
(8). LC-MS: rt (min) = 4.96. 1H NMR (DMSO-d6) δ 1.01 (t, J = 7.6 Hz, 3
H), 2.16ꢀ2.27 (m, 2 H), 6.13 (d, J = 3.3 Hz, 1 H), 6.38 (dd, J = 3.1 and 1.8
Hz, 1 H), 6.69 (d, J = 8.4 Hz, 1 H), 7.61 (d, J = 1.0 Hz, 1 H), 7.90 (dd, J =
8.9 and 4.2 Hz, 1 H), 8.68 (s, 1 H), 8.97 (d, J = 8.4 Hz, 1 H), 9.01 (dd, J =
4.1 and 1.4 Hz, 1 H) and 9.17ꢀ9.21 (m, 1 H). HRMS (m/z): [M + H]+
calcd for C17H16N3O5, 342.1084; found, 342.1082.
N-((5-Chloro-8-hydroxyquinolin-7-yl)(furan-2-yl)methyl)propionamide
(9). LC-MS: rt (min) = 5.26. 1H NMR (DMSO-d6) δ 1.01 (t, J = 7.5 Hz, 3
H) 2.20 (qd, J = 7.5 and 2.6 Hz, 2 H) 6.07 (d, J = 3.1 Hz, 1 H) 6.37 (dd, J =
3.0 and 1.9 Hz, 1 H) 6.72 (d, J = 8.6 Hz, 1 H) 7.59 (s, 1 H) 7.66 - 7.89 (m, 2
H) 8.49 (dd, J = 8.5 and 1.5 Hz, 1 H) 8.80 (d, J = 8.8 Hz, 1 H) 8.97 (dd, J =
4.2 and 1.5 Hz, 1 H) and 10.38 (brs, 1 H). 13C NMR (DMSO-d6) δ 9.81,
28.28, 43.99, 106.95, 110.39, 118.47, 123.04, 123.07, 125.04, 126.15,
132.50, 138.62, 142.51, 149.16, 149.42, 153.95, and 172.21. HRMS
(m/z): [M + H]+ calcd for C17H16ClN2O3, 331.0844; found, 331.0849.
N-((5-Chloro-8-hydroxyquinolin-7-yl)(furan-2-yl)methyl)acetamide
(10). LC-MS: rt (min) = 4.83. 1H NMR (DMSO-d6) δ 1.92 (s, 3 H),
6.06ꢀ6.09 (m, 1 H), 6.37 (dd, J = 3.2 and 1.9 Hz, 1 H), 6.70 (d, J = 8.4
Hz, 1 H), 7.59(dd, J = 1.9 and 1.0 Hz, 1 H), 7.71 (s, 1 H), 7.74 (dd, J = 8.5
and 4.2 Hz, 1 H), 8.50 (dd, J = 8.6 and 1.6 Hz, 1 H), 8.88 (d, J = 8.6 Hz,
High-Throughput Screen: Materials. Dimethyl sulfoxide (DMSO)
ACS grade was from Fisher, while ferrous ammonium sulfate, Xylenol
Orange (XO), sulfuric acid, and Triton X-100 were obtained from
Sigma-Aldrich.
Compound Library. A 153607 compound library was screened in
7ꢀ15 concentrations ranging from 0.7 nM to 57 μM. The library in-
cluded 139798 diverse small drug-like molecules that are part of the NIH
Small Molecule Repository. A collection of 2893 compounds from the
Centers of Methodology and Library Development at Boston University
(BUCMLD) and University of Pittsburgh (UPCMLD) were added to
the library. Several combinatorial libraries from Pharmacopeia, Inc. totaled
1649 compounds. Anadditional1981compounds fromthe NCI Diversity
Set were included. Last, 7286 compounds with known pharmacological
activity were added to provide a large and diverse screening collection.
High-Throughput Screening Protocol and HTS Analysis. All screen-
ing operations were performed on a fully integrated robotic system
(Kalypsys Inc., San Diego, CA) as described elsewhere.88 First, 3 μL of
enzyme (approximately 80 nM 12-LOX, final concentration) was
dispensed into 1536-well Greiner black clear-bottom assay plate. Then
compounds and controls (23 nL) were transferred via Kalypsys PinTool
equipped with 1536-pin array. The plate was incubated for 15 min at
room temperature, and then a 1 μL aliquot of substrate solution (50 μM
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arachidonic acid final concentration) was added to start the reaction.
The reaction was stopped after 6.5 min by the addition of 4 μL of FeXO
solution (final concentrations of 200 μM Xylenol Orange (XO) and
300 μM ferrous ammonium sulfate in 50 mM sulfuric acid). After a short
spin (1000 rpm, 15 s), the assay plate was incubated at room tempera-
ture for 30 min. The absorbances at 405 and 573 nm were recorded using
ViewLux high-throughput CCD imager (Perkin-Elmer, Waltham, MA)
using standard absorbance protocol settings. During dispensing, enzyme
and substrate bottles were kept submerged into +4 ꢀC recirculating
chiller bath to minimize degradation. Plates containing DMSO only
(instead of compound solutions) were included approximately every
50 plates throughout the screen to monitor any systematic trend in the
assay signal associated with reagent dispenser variation or decrease in
enzyme specific activity.
radical scavanger, nordihydroguaiaretic acid (NDGA) was used as a
positive control. Then 10 μL of the testing reagent (1 mM), to achieve a
final concentration of 5 μM, was added to 2 mL of 500 μM DPPH,
stirring in a cuvette. Optical absorbance was monitored and recorded at
25 s intervals as described elsewhere.89,90 The decrease in optical absor-
bance at 517 nm was monitored using a Perkin-Elmer Lambda 40 spec-
trometer. The rate of reaction is proportional to the antioxidant potency
of the test compounds.
Steady-State Inhibition Kinetics. Lipoxygenase rates were deter-
mined by monitoring the formation of the conjugated product, 12-
HPETE, at 234 nm (ε = 25 000 Mꢀ1 cmꢀ1) with a Perkin-Elmer
Lambda 40 UV/vis spectrophotometer. Reactions were initiated by
adding approximately 80 nM 12-LOX to a constantly stirring 2 mL
cuvette containing 3ꢀ40 μM AA in 25 mM HEPES buffer (pH 7.5), in
the presence of 0.01% Triton X-100. The substrate concentration was
quantitated by allowing the enzymatic reaction to proceed to comple-
tion. Kinetic data were obtained by recording initial enzymatic rates, at
varied inhibitor concentrations, and subsequently fitted to the Henriꢀ
MichaelisꢀMenten equation, using KaleidaGraph (Synergy) to deter-
Data were analyzed in a similar method as described elsewhere.71
Briefly, assay plate-based raw data were normalized to controls and plate-
based data corrections were applied to filter out background noise. All
concentration response curves (CRCs) were fitted using in-house
developed software (http://ncgc.nih.gov/pub/openhts/). Curves were
categorized into four classes: complete response curves (class 1), partial
curves (class 2), single point actives (class 3), and inactives (class 4).
Compounds with the highest quality, class 1 and class 2 curves, were
prioritized for follow-up.
Lipoxygenase UVꢀVis-Based Manual Assay. The initial one-point
inhibition percentages were determined by following the formation of
the conjugated diene product at 234 nm (ε = 25000 Mꢀ1cmꢀ1) with a
Perkin-Elmer Lambda 40 UV/vis spectrophotometer at one inhibitor
concentration. All reactions were 2 mL in volume and constantly stirred
using a magnetic stir bar at room temperature (23 ꢀC) with approxi-
mately 40 nM for 12-LOX (by iron content), 20 nM of 15-LOX-1 (by
iron content), and 1 μM for 15-LOX-2 (by Bradford). Reactions with
12-LOX were carried out in 25 mM HEPES (pH 8.0) 0.01% Triton
X-100 and 10 μM AA. Reactions with the crude, ammonium sulfate pre-
cipitated 5-LOX were carried out in 25 mM HEPES (pH 7.3), 0.3 mM
CaCl2, 0.1 mM EDTA, 0.2 mM ATP, 0.01% Triton X-100, and 10 μM
AA. Reactions with 15-LOX-1 and 15-LOX-2 were carried out in 25 mM
HEPES buffer (pH 7.5), 0.01% Triton X-100, and 10 μM AA. The
concentration of AA (for 5-LOX, 12-LOX, and 15-LOX-2) was quanti-
tatively determined by allowing the enzymatic reaction to go to com-
pletion. IC50 values were obtained by determining the enzymatic rate at
various inhibitor concentrations and plotted against inhibitor concen-
tration, followed by a hyperbolic saturation curve fit. The data used for
the saturation curves were performed in duplicate or triplicate, depend-
ing on the quality of the data.
mine the microscopic rate constants, Vmax (μmol/min/mg) and Vmax
/
KM (μmol/min/mg/μM). These rate constants were subsequently
replotted and normalized to the iron content (120 nM) as 1/kcat and
KM/kcat, versus inhibitor concentration, to yield Kiu and Kic, respectively.
Incubation Inhibition Kinetics. 12-LOX rates were determined as
above, with the following modifications. First, 1 μL of 1 mM compound
2, in DMSO, was added to 30 μL of 12-LOX (approximately 5 μM), and
allowed to sit on ice for intervals of 2 min, upward to 10 min. Then the
mixture was added at designated time periods to a constantly stirring
2 mL cuvette, containing 10 μM AA in 25 mM HEPES buffer (pH 8.0),
in the presence of 0.01% Triton X-100. The control to this reaction was
the same as above, but only DMSO was added. Incubated samples with
inhibitor were also dialyzed to demonstrate reversible inhibition, but the
enzyme died in the 2 h time frame of the experiment.
Cyclooxygenase Assay. Ovine COX-1 (catalogue no. 60100) and
human COX-2 (catalogue no. 60122) were purchased from Cayman
chemical. Approximately 2 μg of either COX-1 or COX-2 were added to
buffer containing 100 μM AA, 0.1 M Tris-HCl buffer (pH 8.0), 5 mM
EDTA, 2 mM phenol, and 1 μM hematin at 37 ꢀC. Data was collected
using a Hansatech DW1 oxygen electrode chamber. Inhibitors were in-
cubated with the respective COX for 20 min and added to the reaction
mixture, and the consumption of oxygen was recorded. Ibuprofen and
the carrier solvent, DMSO, were used as positive and negative controls,
respectively.
Mouse 12/15-Lipoxygenase Expression, Purification and IC50 Assay.
12/15-mouse lipoxygenase (12/15-LOX), with an N-terminus His-tag
was expressed in SF9 insect cells. 12/15-LOX was PCR amplified from
the pGFP-ALOX15 vector (Gift of Dr. Jerry Nadler) using the following
primers, 50-GGCGCGCGTCGACATGCACCACCATCACCATCA-
TCACGGTGTCTACCGCATCCGCGTCTC-30, 50-GGCTCGAGT-
CTAGATCATTATATGGCCACGCTGTTTTCTACCAG-30, yield-
ing a fragment of approximately 2 kb. The PCR fragment was cut with
SalI and XhoI and ligated into a pFastBac1 vector, cut with the same
enzymes. 12/15LOX-pFastBac was then transposed into a recombinant
pFastBac bacmid using DH10Bac cells and transfected into SF9 cells, as
described in the product literature for pFastBac1. For purification,
infected SF9 cells were harvested after 72 h by centrifugation and stored
at ꢀ20 ꢀC. Thawed cells were dounced in 25 mM HEPES pH 7.5, and
extract was loaded onto a Bio Rad UNO Q1 ion exchange column.
12/15-mLO was eluded with a 0ꢀ1 M sodium chloride linear gradient
using 25 mM HEPES running buffer. Glycerol was added (15%) to
active fractions, which were stored at ꢀ80 ꢀC. It should be noted that the
His-tag purification method could not be used due to rapid inactivation
of the protein. It was unclear as to the cause of this inactivation, but it
appears that certain salts lead to the inactivation. Inhibitor assays were
Fluorescence Iron Binding Assay. Fluorescence readings were
taken with a Perkin-Elmer luminescence spectrometer LS 50 B in the
presence and absence of iron with the excitation maximum λex = 360 nm
and the emission maximum λem = 410 nm. Excitation slit was set at 5 nm,
and the emission slit was set at 10 nm. Because of the low intensity of the
fluorophore, the filter was kept open. As a control, 1 mM 8-HQ-5-sul-
fonic acid was dissolved in 25 mM HEPES buffer (pH 8.5) and aliquoted
to a 2 mL cuvette. In a separate cuvette, 1 mM 8-HQ-5-sulfonic acid and
either 10 μM Fe2+ (as [NH4]2[Fe][SO4]2 6H2O) or Fe3+ (as Fe2-
3
(SO4)3) in 25 mM HEPES buffer (pH 8.5) was added and allowed to
equilibrate for 5 min. For the experimental, 2 mL of 1 mM 1 was dis-
solved in 25 mM HEPES buffer (pH 8.5) and the fluorescence intensity
taken. In a separate cuvette, 1 mM 1 was dissolved in 25 mM HEPES
buffer (pH 8.5) and was incubated with less than 1 μM (by iron content)
of 12-LOX and allowed to equilibrate for 5 min.
DPPH Antioxidant Test. Compound 1 was dissolved in dimethyl sul-
foxide (DMSO) at 1ꢀ20 mM concentration (1000-fold concentrated).
The antioxidant activity of this compound was assayed by monitoring
the quenching of the standard free radical 1,1-diphenyl-2-picrylhydrazyl
(DPPH) upon reaction with the testing compounds.89,90 A known free
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Journal of Medicinal Chemistry
ARTICLE
performed on a Perkin-Elmer Lambda 45 UV/vis spectrophotometer.
Assay buffer consisted of 10 μM arachidonic acid added to 25 mM
HEPES (pH 7.5). These assays were performed in the absence of Triton
X-100 because it was found to inactivate 12/15-LOX. 12/15-LOX
was defrosted on ice and added to the cuvette containing substrate
and inhibitor. Conjugated product formation was monitored at 234 nm.
The one-point inhibition percentage for weak inhibitors was calculated
as 1 ꢀ (maximal experimental rate/maximal control rate) ꢁ 100%. The
multipoint IC50 value was determined by plotting the percent inhibition
vs [inhibitor] and fitting with a hyperbolic equation, as defined before.70
Cellular Inhibition Assay. To determine if these compounds were
effective in a cellular environment, a select few were tested against 12-
LOX in platelets. Human platelets were obtained from healthy volun-
teers at Thomas Jefferson University, and this study was approved by the
Thomas Jefferson University Institutional Review Board. Informed con-
sent was obtained from all donors prior to blood draw. First, 20 mL of
blood was drawn and centrifuged at 200g for 15 min at room tempera-
ture. Then to the plasma, 10% ACD (1:10) and Apyrase (1 μL/2.5 mL)
were added and the solution centrifuged at 2000g for 15 min at room
temperature. The platelets were resuspended with Tyrode’s buffer and
adjusted to a concentration of 3.0 ꢁ 108 platelets/mL. The platelets
were subsequently treated with inhibitor for 10 min at 37 ꢀC prior to
stimulation with 20 μM PAR1-AP (SFLLRN) under stirring conditions
in an aggregometer. Following aggregation, samples were centrifuged at
2000g for 10 min, the supernatant transferred to scintillation vials,
extracted with methylene chloride, reduced with triphenylphosphine,
and evaporated to dryness. The dry samples were then resuspended in
methanol and stored at ꢀ20 ꢀC until analyzed by Finnigan LTQ liquid
chromatographyꢀtandem mass spectrometry (LCꢀMS/MS) system.
An internal control, 12-deuterated(d8)- HETE (12-d8-HETE) was added
to each sample prior to injection on LC-MS to account for the variation in
the detector response, it was assumed that the detector response for 12-
HETE and 12-d8-HETE would be similar. A Thermo Electron Corp.
Aquasil (3 μm, 100 mm ꢁ 2.1 mm) C-18 column was used to detect the
HETEs with an elution protocol consisting of 0.2 mL/min flow rate and a
linear gradient from 54.9% MeCN, 45% H2O, and 0.1% THF to 69.9%
MeCN, 30% H2O, and 0.1% THF. The corresponding 12-HETE and 12-
d8-HETE compounds were detected using selective ion monitoring
analysis (m/z =318.7 to 319.7 and 326.8 to 327.7) in negative ion mode
and then identified by fragmentation pattern (12-HETE, parent ion at m/
z 319 and fragments at m/z 179 and 163; 12-d8-HETE, parent ion at m/z
327 and fragments at m/z 184 and 214) from MSꢀMS.91 The electro-
spray voltage was set to 5.0 kV, and a global acquisition MS mode was
used. The MSꢀMS scan was performed for the five most abundant
precursor ions. The collision induced dissociation (CID) was used for
MS-MS with a collision energy of 35 eV. The peak intensities of 12-
HETEs were normalized to the 12-d8-HETE intensities. The amount of
12-HETE insamples was estimatedusing a standard curve generatedfrom
pure 12-HETE with concentrations ranging from 0 to 2.5 μM along with a
constant amount of 12-d8-HETE added in each sample. The assay is linear
in the concentration range used for the 12-HETE standard curve with a R2
value of 0.997.
for an additional 30 min. Samples were harvested, centrifuged at 1000 rpm
for 5 min, and supernatant drawn off and isletpelletstoredatꢀ80 ꢀC. Cell
pellets were extracted using CHCl3/MeOH and samples dried under
nitrogen gas before reconstitution in 250 μL of ELISA sample buffer. 12-
HETE levels in samples were determined using a 12-HETE ELISA kit
(catalogue no. 901ꢀ050, Assay Design, Plymouth Meeting, PA).
’ ASSOCIATED CONTENT
S
Supporting Information. Additional experimental pro-
b
cedures and spectroscopic data (1H NMR, LC/MS, and HRMS)
for representative compounds. Analytical methods for the chiral
HPLC separation of enantiomers and kinetic inhibition data are
also included. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For D.J.M.: phone, 301-217-4381; fax, 301-217-5736; E-mail,
maloneyd@mail.nih.gov. For T.R.H: phone, 831-459-5884; fax,
831-459-2935; E-mail: holman@ucsc.edu.
Present Addresses
#Department of Molecular Medicine, Beckman Research Insti-
tute at City of Hope, 1500 East Duarte Road, Duarte, California
91010-3000, United States.
Author Contributions
^Both of these authors contributed equally to this work.
’ ACKNOWLEDGMENT
We thank Eric Hoobler for the 5-LOX assay, Norine Kuhn for
islet incubations and the 12-HETE ELISA, Sam Michael for
assistance with the primary HTP screen, and Paul Shinn and
Danielle vanLeer for assistance with compound management and
purification. We also thank Christina Greco for critical reading of
the manuscript. Financial support was from the National Institutes
of Health (R01 GM56062 (T.R.H.), R00 HL089457 (M.H.), R01
DK 55240 (D.T.F., J.L.N.)), the Juvenile Diabetes Research
Foundation (D.T.F., T.R.H., J.L.N.), and the Molecular Libraries
Initiative of the NationalInstitutes of Health Roadmap for Medical
Research (R03 MH081283 (T.R.H.)). Additional financial sup-
port was from NIH (S10-RR20939 (T.R.H.)) and the California
Institute for Quantitative Biosciences (T.R.H.) for the UCSC MS
Facility.
’ ABBREVIATIONS USED
LOX, lipoxygenase; soybean LOX-1, soybean lipoxygenase-1; 15-
LOX-1, human reticulocyte 15-lipoxygenase-1; 15-LOX-2, human
epithelial 15-lipoxygenase-2; 12-LOX, human platelet-type 12-
lipoxygenase; rabbit 15-LOX, rabbit reticulocyte 15-lipoxygenase;
COX, cyclooxygenase;NDGA, nordihydroguaiaretic acid; AA,
arachidonic acid; 15-HPETE, 15-(S)-hydroperoxyeicosatetraenoic
acid; 15-HETE, 15-(S)-hydroxyeicosatetraenoic acid; 12-HPETE,
12-(S)-hydroperoxyeicosatetraenoic acid; 12-HETE, 12-(S)-hy-
droxyeicosatetraenoic acid; LA, linoleic acid; 13-HPODE, 13-(S)-
hydroperoxyoctadecadienoic acid; 13-HODE, 13-(S)-hydroxy-
octadecadienoic acid; ALA, R-linolenic acid;13-HPOTrE, 13-(S)-
hydroperoxyoctadecatrienoic acid; 8-HQ, 8-hydroxyquinoline;
mRNA, messenger ribonucleic acid; Vmax, maximal velocity
(mmol/min);kcat, first-order catalytic rate constant (Vmax/[E]
Inhibition of 12-HETE Production in Human Donor Islets. Studies
with human donor islets had institutional approval. Islets were obtained
from the Integrated Islet Distribution Program. Human donor islets were
incubated overnight in CMRL media (catalogue no. 15ꢀ110-CV Media-
Tech, Inc. Manassas, VA) containing 10% fetal bovine serum + pen/strep.
The islets were serum-starved by incubating in serum free media, CMRL
containing pen/strep and 1% fatty acid free human serum albumin
(catalogue no. A1887 Sigma, St. Louis, MO) for 1 h. Islets were incubated
in serum-free media containing 100 μM arachadonic acid (catalogue no.
BML-FA003ꢀ0100, Enzo Life Sciences Plymouth Meeting, PA), with and
without 10 μM of compound 1 for 60 min at 37 ꢀC. Calcium ionophore, 5
μM of A23187 (catalogue no. C7522, Sigma, St. Louis, MO), was added
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Journal of Medicinal Chemistry
ARTICLE
(sꢀ1)); KM, HenriꢀMichaelisꢀMenten Constant (mM); [E],
total active enzyme concentration; IC50, inhibitor constant at
50% inhibition; DPPH, 1.1-diphenyl-2-picrylhydrazyl; XO, xylenol
orange; HTS, high-throughput screening; MLSMR, Molecular Li-
braries Small Molecule Repository; qHTS, quantitative high-
throughput screening; CRC, concentrationꢀresponse curve; SD,
standard deviation; MLPCN, Molecular Libraries Probe Produc-
tion Center Network
(19) Hsi, L. C.; Wilson, L. C.; Eling, T. E. Opposing Effects of 15-
Lipoxygenase-1 and -2 Metabolites on Mapk Signaling in Prostate
Alteration in Peroxisome Proliferator-Activated Receptor Gamma. J. Biol.
Chem. 2002, 277, 40549–40556.
(20) Shappell, S. B.; Manning, S.; Boeglin, W. E.; Guan, Y. F.;
Roberts, R. L.; Davis, L.; Olson, S. J.; Jack, G. S.; Coffey, C. S.; Wheeler,
T. M.; Breyer, M. D.; Brash, A. R. Alterations in Lipoxygenase and
Cyclooxygenase-2 Catalytic Activity and MRNA Expression in Prostate
Carcinoma. Neoplasia 2001, 3, 287–303.
(21) Shappell, S. B.; Olson, S. J.; Hannah, S. E.; Manning, S.;
Roberts, R. L.; Masumori, N.; Jisaka, M.; Boeglin, W. E.; Vader, V.;
Dave, D. S.; Shook, M. F.; Thomas, T. Z.; Funk, C. D.; Brash, A. R.;
Matusik, R. J. Elevated Expression of 12/15-Lipoxygenase and Cycloox-
ygenase-2 in a Transgenic Mouse Model of Prostate Carcinoma. Cancer
Res. 2003, 63, 2256–2267.
’ REFERENCES
(1) Solomon, E. I.; Zhou, J.; Neese, F.; Pavel, E. G. New Insights
from Spectroscopy into the Structure/Function Relationships of Lipox-
ygenases. Chem. Biol. 1997, 4, 795–808.
(22) Brash, A. R.; Boeglin, W. E.; Chang, M. S. Discovery of a Second
15s-Lipoxygenase in Humans. Proc. Natl. Acad. Sci. U.S.A. 1997,
94, 6148–6152.
(2) Ford-Hutchinson, A. W.; Gresser, M.; Young, R. N. 5-Lipox-
ygenase. Annu. Rev. Biochem. 1994, 63, 383–417.
(3) Kuhn, H.; Chaitidis, P.; Roffeis, J.; Walther, M. Arachidonic Acid
Metabolites in the Cardiovascular System: The Role of Lipoxygenase
Isoforms in Atherogenesis with Particular Emphasis on Vascular Re-
modeling. J. Cardiovasc. Pharmacol. 2007, 50, 609–620.
(4) Pace-Asciak, C. R.; Asotra, S. Biosynthesis, Catabolism, and
Biological Properties of HPETES, Hydroperoxide Derivatives of Ara-
chidonic Acid. Free Radical Biol. Med. 1989, 7, 409–433.
(5) Radmark, O.; Samuelsson, B. 5-Lipoxygenase: Regulation and
Possible Involvement in Atherosclerosis. Prostaglandins Other Lipid
Mediators 2007, 83, 162–174.
(23) Gonzalez, A. L.; Roberts, R. L.; Massion, P. P.; Olson, S. J.; Shyr,
Y.; Shappell, S. B. 15-Lipoxygenase-2 Expression in Benign and Neo-
plastic Lung: An Immunohistochemical Study and Correlation with
Tumor Grade and Proliferation. Hum. Pathol. 2004, 35, 840–849.
(24) Suraneni, M. V.; Schneider-Broussard, R.; Moore, J. R.; Davis,
T. C.; Maldonado, C. J.; Li, H.; Newman, R. A.; Kusewitt, D.; Hu, J.;
Yang, P.; Tang, D. G. Transgenic Expression of 15-Lipoxygenase 2 (15-
Lox2) in Mouse Prostate Leads to Hyperplasia and Cell Senescence.
Oncogene 2010, 29, 4261–4275.
(25) Tang, D. G.; Bhatia, B.; Tang, S.; Schneider-Broussard, R. 15-
Lipoxygenase 2 (15-Lox2) Is a Functional Tumor Suppressor That
Regulates Human Prostate Epithelial Cell Differentiation, Senescence,
andGrowth(Size). Prostaglandins Other Lipid Mediators 2007, 82, 135–146.
(26) Jobard, F.; Lefevre, C.; Karaduman, A.; Blanchet-Bardon, C.;
Emre, S.; Weissenbach, J.; Ozguc, M.; Lathrop, M.; Prud’homme, J. F.;
Fischer, J. Lipoxygenase-3 (Aloxe3) and 12(R)-Lipoxygenase (Alox12b)
Are Mutated in Non-Bullous Congenital Ichthyosiform Erythroderma
(Ncie) Linked to Chromosome 17p13.1. Hum. Mol. Genet. 2002,
11, 107–113.
(27) Hussain, H.; Shornick, L. P.; Shannon, V. R.; Wilson, J. D.;
Funk, C. D.; Pentland, A. P.; Holtzman, M. J. Epidermis Contains
Platelet-Type 12-Lipoxygenase That Is Overexpressed in Germinal
Layer Keratinocytes in Psoriasis. Am. J. Physiol. 1994, 266, C243–C253.
(28) Ding, X. Z.; Iversen, P.; Cluck, M. W.; Knezetic, J. A.; Adrian,
T. E. Lipoxygenase Inhibitors Abolish Proliferation of Human Pancrea-
tic Cancer Cells. Biochem. Biophys. Res. Commun. 1999, 261, 218–223.
(29) Connolly, J. M.; Rose, D. P. Enhanced Angiogenesis and
Growth of 12-Lipoxygenase Gene-Transfected Mcf-7 Human Breast
Cancer Cells in Athymic Nude Mice. Cancer Lett. 1998, 132, 107–112.
(30) Natarajan, R.; Nadler, J. Role of Lipoxygenases in Breast
Cancer. Front. Biosci. 1998, 3, E81–E88.
(6) Brock, T. G. Regulating Leukotriene Synthesis: The Role of
Nuclear 5-Lipoxygenase. J. Cell. Biochem. 2005, 96, 1203–1211.
(7) Newcomer, M. E.; Gilbert, N. C. Location, Location, Location:
Compartmentalization of Early Events in Leukotriene Biosynthesis.
J. Biol. Chem. 2010, 285, 25109–25114.
(8) Ghosh, J. Inhibition of Arachidonate 5-Lipoxygenase Triggers
Prostate Cancer Cell Death through Rapid Activation of C-Jun N-Term-
inal Kinase. Biochem. Biophys. Res. Commun. 2003, 307, 342–349.
(9) Ghosh, J.; Myers, C. E. Inhibition of Arachidonate 5-Lipoxygen-
ase Triggers Massive Apoptosis in Human Prostate Cancer Cells. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 13182–13187.
(10) Nakano, H.; Inoue, T.; Kawasaki, N.; Miyataka, H.; Matsumoto,
H.; Taguchi, T.; Inagaki, N.; Nagai, H.; Satoh, T. Synthesis and
Biological Activities of Novel Antiallergic Agents with 5-Lipoxygenase
Inhibiting Action. Bioorg. Med. Chem. 2000, 8, 373–380.
(11) Radmark, O.; Werz, O.; Steinhilber, D.; Samuelsson, B. 5-
Lipoxygenase: Regulation of Expression and Enzyme Activity. Trends
Biochem. Sci. 2007, 32, 332–341.
(12) Berger, W.; De Chandt, M. T.; Cairns, C. B. Zileuton: Clinical
Implications of 5-Lipoxygenase Inhibition in Severe Airway Disease. In.t
J. Clin. Pract. 2007, 61, 663–676.
(13) Masferrer, J. L.; Zweifel, B. S.; Hardy, M.; Anderson, G. D.;
Dufield, D.; Cortes-Burgos, L.; Pufahl, R. A.; Graneto, M. Pharmacology
of Pf-4191834, a Novel, Selective Non-Redox 5-Lipoxygenase Inhibitor
Effective in Inflammation and Pain. J. Pharmacol. Exp. Ther. 2010,
334, 294–301.
(31) Ma, K.; Nunemaker, C. S.; Wu, R.; Chakrabarti, S. K.; Taylor-
Fishwick, D. A.; Nadler, J. L. 12-Lipoxygenase Products Reduce Insulin
Secretion and {Beta}-Cell Viability in Human Islets. J. Clin. Endocrinol.
Metab. 2010, 95, 887–893.
(32) Thomas, C. P.; Morgan, L. T.; Maskrey, B. H.; Murphy, R. C.;
Kuhn, H.; Hazen, S. L.; Goodall, A. H.; Hamali, H. A.; Collins, P. W.;
O’Donnell, V. B. Phospholipid-Esterified Eicosanoids Are Generated in
Agonist-Activated Human Platelets and Enhance Tissue Factor-Dependent
Thrombin Generation. J. Biol. Chem. 2010, 285, 6891–6903.
(14) Ducharme, Y.; Blouin, M.; Brideau, C.; Chateauneuf, A.;
Gareau, Y.; Grimm, E. L.; Juteau, H.; Laliberte, S.; MacKay, B.; Masse,
F.; Ouellet, M.; Salem, M.; Styhler, A.; Friesen, R. W. The Discovery of
Setileuton, a Potent and Selective 5-Lipoxygenase Inhibitor. ACS Med.
Chem. Lett. 2010, 1, 170–174.
(33) Holinstat, M.; Boutaud, O.; Apopa, P. L.; Vesci, J.; Bala, M.;
Oates, J. A.; Hamm, H. E. Protease-Activated Receptor Signaling in
Platelets Activates Cytosolic Phospholipase A2alpha Differently for
Cyclooxygenase-1 and 12-Lipoxygenase Catalysis. Arterioscler. Thromb.
Vasc. Biol. 2011, 31, 435–442.
(34) Kaur, G.; Jalagadugula, G.; Mao, G.; Rao, A. K. Runx1/Core
Binding Factor A2 Regulates Platelet 12-Lipoxygenase Gene (Alox12):
Studies in Human Runx1 Haplodeficiency. Blood 2010, 115, 3128–3135.
(35) Catalano, A.; Procopio, A. New Aspects on the Role of Lipo-
xygenases in Cancer Progression. Histol. Histopathol. 2005, 20, 969–975.
(15) Company information: www.pfizer.com, www.merck.com.
(16) Jones, R.; Adel-Alvarez, L. A.; Alvarez, O. R.; Broaddus, R.; Das,
S. Arachidonic Acid and Colorectal Carcinogenesis. Mol. Cell. Biochem.
2003, 253, 141–149.
(17) Shureiqi, I.; Lippman, S. M. Lipoxygenase Modulation to
Reverse Carcinogenesis. Cancer Res. 2001, 61, 6307–6312.
(18) Kelavkar, U. P.; Cohen, C.; Kamitani, H.; Eling, T. E.; Badr,
K. F. Concordant Induction of 15-Lipoxygenase-1 and Mutant P53
Expression in Human Prostate Adenocarcinoma: Correlation with
Gleason Staging. Carcinogenesis 2000, 21, 1777–1787.
5495
dx.doi.org/10.1021/jm2005089 |J. Med. Chem. 2011, 54, 5485–5497

Journal of Medicinal Chemistry
ARTICLE
(36) Krishnamoorthy, S.; Jin, R.; Cai, Y.; Maddipati, K. R.; Nie, D.;
Pages, G.; Tucker, S. C.; Honn, K. V. 12-Lipoxygenase and the
Regulation of Hypoxia-Inducible Factor in Prostate Cancer Cells. Exp.
Cell Res. 2010, 316, 1706–1715.
(37) Steele, V. E.; Holmes, C. A.; Hawk, E. T.; Kopelovich, L.; Lubet,
R. A.; Crowell, J. A.; Sigman, C. C.; Kelloff, G. J. Lipoxygenase Inhibitors
as Potential Cancer Chemopreventives. Cancer Epidemiol., Biomarkers
Prev. 1999, 8, 467–483.
(53) Carroll, J.; Jonsson, E. N.; Ebel, R.; Hartman, M. S.; Holman,
T. R.; Crews, P. Probing Sponge-Derived Terpenoids for Human 15-
Lipoxygenase Inhibitors. J. Org. Chem. 2001, 66, 6847–6851.
(54) Fu, X.; Schmitz, F. J.; Govindan, M.; Abbas, S. A.; Hanson,
K. M.; Horton, P. A.; Crews, P.; Laney, M.; Schatzman, R. C. Enzyme
Inhibitors: New and Known Polybrominated Phenols and Diphenyl
Ethers from Four Indo-Pacific Dysidea Sponges. J. Nat. Prod. 1995,
58, 1384–91.
(38) Jiang, W. G.; Douglas-Jones, A.; Mansel, R. E. Levels of
Expression of Lipoxygenases and Cyclooxygenase-2 in Human Breast
Cancer. Prostaglandins, Leukotrienes Essent. Fatty Acids 2003, 69, 275–281.
(39) Mohammad, A. M.; Abdel, H. A.; Abdel, W.; Ahmed, A. M.;
Wael, T.; Eiman, G. Expression of Cyclooxygenase-2 and 12-Lipoxy-
genase in Human Breast Cancer and Their Relationship with Her-2/
Neu and Hormonal Receptors: Impact on Prognosis and Therapy.
Indian .J Cancer 2006, 43, 163–168.
(40) Zeeneldin, A. A.; Mohamed, A. M.; Abdel, H. A.; Taha, F. M.;
Goda, I. A.; Abodeef, W. T. Survival Effects of Cyclooxygenase-2 and 12-
Lipooxygenase in Egyptian Women with Operable Breast Cancer.
Indian J. Cancer 2009, 46, 54–60.
(41) Agrawal, V. K.; Bano, S.; Khadikar, P. V. Qsar Study on
5-Lipoxygenase Inhibitors Using Distance-Based Topological Indices.
Bioorg. Med. Chem. 2003, 11, 5519–5527.
(42) Arockia Babu, M.; Shakya, N.; Prathipati, P.; Kaskhedikar, S. G.;
Saxena, A. K. Development of 3d-QSAR Models for 5-Lipoxygenase
Antagonists: Chalcones. Bioorg. Med. Chem. 2002, 10, 4035–4041.
(43) Basha, A.; Ratajczyk, J. D.; Dyer, R. D.; Young, P.; Carter, G. W.;
Brooks, C. D. W. StructureꢀActivity Relationships of Pyrimido-
Pyrimidine Series of 5-Lipoxygenase Inhibitors. Med. Chem. Res. 1996,
6, 61–67.
(55) Jiang, Z. D.; Ketchum, S. O.; Gerwick, W. H. 5-Lipoxygenase-
Derived Oxylipins from the Red Alga Rhodymenia Pertusa. Phytochem-
istry 2000, 53, 129–133.
(56) Rao, K. C. S.; Divakar, S.; Rao, A. G. A.; Karanth, N. G.;
Suneetha, W. J.; Krishnakantha, T. P.; Sattur, A. P. Asperenone: An
Inhibitor of 15-Lipoxygenase and of Human Platelet Aggregation from
Aspergillus Niger. Biotechnol. Lett. 2002, 24, 1967–1970.
(57) Segraves, E. N.; Shah, R. R.; Segraves, N. L.; Johnson, T. A.;
Whitman, S.; Sui, J. K.; Kenyon, V. A.; Cichewicz, R. H.; Crews, P.;
Holman, T. R. Probing the Activity Differences of Simple and Complex
Brominated Aryl Compounds against 15-Soybean, 15-Human, and 12-
Human Lipoxygenase. J. Med. Chem. 2004, 47, 4060–4065.
(58) Suzuki, H.; Ueda, T.; Juranek, I.; Yamamoto, S.; Katoh, T.;
Node, M.; Suzuki, T. Hinokitiol, a Selective Inhibitor of the Platelet-
Type Isozyme of Arachidonate 12-Lipoxygenase. Biochem. Biophys. Res.
Commun. 2000, 275, 885–889.
(59) Weinstein, D. S.; Liu, W.; Gu, Z.; 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. Tryptamine and Homotryptamine-Based
Sulfonamides as Potent and Selective Inhibitors of 15-Lipoxygenase.
Bioorg. Med. Chem. Lett. 2005, 15, 1435–1440.
(44) Fleiseher, R.; Frohberg, P.; Buge, A.; Nuhn, P.; Wiese, M.
QSAR Analysis of Substituted 2-Phenylhydrazonoacetamides Acting as
Inhibitors of 15-Lipoxygenase. Quant. Struct.ꢀAct. Relationships 2000,
19, 162–172.
(45) Kim, K. H.; Martin, Y. C.; Brooks, C. D. W. Quantitative
StructureꢀActivity Relationships of 5-Lipoxygenase Inhibitors Inhibitory
Potency of Triazinone Analogues in a Broken Cell. Quant. Struct.ꢀActivity
Relationships 1996, 15, 491–497.
(46) Kim, K. H.; Martin, Y. C.; Brooks, D. W.; Dyer, R. D.; Carter,
G. W. Quantitative StructureꢀActivity Relationships of 5-Lipoxygenase
Inhibitors Inhibitory Potency of Pyridazinone Analogues. J. Pharm. Sci.
1994, 83, 433–438.
(47) Mano, T.; Stevens, R. W.; Ando, K.; Nakao, K.; Okumura, Y.;
Sakakibara, M.; Okumura, T.; Tamura, T.; Miyamoto, K. Novel Imida-
zole Compounds as a New Series of Potent, Orally Active Inhibitors of
5-Lipoxygenase. Bioorg. Med. Chem. 2003, 11, 3879–3887.
(48) Redrejo-Rodriguez, M.; Tejeda-Cano, A.; Pinto, M. D.; Macias,
P. Lipoxygenase Inhibition by Flavonoids: Semiempirical Study of the
StructureꢀActivity Relation. J. Mol. Struct.—THEOCHEM 2004, 674,
121–124.
(49) Stewart, A. O.; Bhatia, P. A.; Martin, J. G.; Summers, J. B.;
Rodriques, K. E.; Martin, M. B.; Holms, J. H.; Moore, J. L.; Craig, R. A.;
Kolasa, T.; Ratajczyk, J. D.; Mazdiyasni, H.; Kerdesky, F. A.; DeNinno,
S. L.; Maki, R. G.; Bouska, J. B.; Young, P. R.; Lanni, C.; Bell, R. L.; Carter,
G. W.; Brooks, C. D. StructureꢀActivity Relationships of N-Hydroxyurea
5-Lipoxygenase Inhibitors. J. Med. Chem. 1997, 40, 1955–1968.
(50) Amagata, T.; Whitman, S.; Johnson, T. A.; Stessman, C. C.;
Loo, C. P.; Lobkovsky, E.; Clardy, J.; Crews, P.; Holman, T. R. Exploring
Sponge-Derived Terpenoids for Their Potency and Selectivity against
12-Human, 15-Human, and 15-Soybean Lipoxygenases. J. Nat. Prod.
2003, 66, 230–235.
(51) Benrezzouk, R.; Terencio, M. C.; Ferrandiz, M. L.; Hernandez-
Perez, M.; Rabanal, R.; Alcaraz, M. J. Inhibition of 5-Lipoxygenase
Activity by the Natural Anti-Inflammatory Compound Aethiopinone.
Inflammation Res. 2001, 50, 96–101.
(52) Bracher, F.; Krauss, J.; Laufer, S. Effects of Natural Products
Containing Acylresorcinol Partial Structures on Cyclooxygenases and
5-Lipoxygenase. Pharmazie 2001, 56, 430.
(60) Deschamps, J. D.; Gautschi, J. T.; Whitman, S.; Johnson, T. A.;
Gassner, N. C.; Crews, P.; Holman, T. R. Discovery of Platelet-Type 12-
Human LipoxygenaseSelective InhibitorsbyHigh-Throughput Screening
of Structurally Diverse Libraries. Bioorg. Med. Chem. 2007, 15, 6900–6908.
(61) Vasquez-Martinez, Y.; Ohri, R. V.; Kenyon, V.; Holman, T. R.;
Sepulveda-Boza, S. StructureꢀActivity Relationship Studies of Flavo-
noids as Potent Inhibitors of Human Platelet 12-LO, Reticulocyte
15-LO-1, and Prostate Epithelial 15-LO-2. Bioorg. Med. Chem. 2007, 15,
7408–7425.
(62) Yamamoto, S.; Katsukawa, M.; Nakano, A.; Hiraki, E.; Nishimura,
K.; Jisaka, M.; Yokota, K.; Ueda, N. Arachidonate 12-Lipoxygenases with
Reference to Their Selective Inhibitors. Biochem. Biophys. Res. Commun.
2005, 338, 122–127.
(63) Whitman, S.; Gezginci, M.; Timmermann, B. N.; Holman, T. R.
StructureꢀActivity Relationship Studies of Nordihydroguaiaretic Acid
Inhibitors toward Soybean, 12-Human, and 15-Human Lipoxygenase.
J. Med. Chem. 2002, 45, 2659–2661.
(64) Deschamps, J. D.; Kenyon, V. A.; Holman, T. R. Baicalein Is a
Potent in Vitro Inhibitor against Both Reticulocyte 15-Human and
Platelet 12-Human Lipoxygenases. Bioorg. Med. Chem. 2006, 14, 4295–
42301.
(65) Kenyon, V.; Chorny, I.; Carvajal, W. J.; Holman, T. R.; Jacobson,
M. P. Novel Human Lipoxygenase Inhibitors Discovered Using Virtual
Screening with Homology Models. J. Med. Chem. 2006, 49, 1356–1363.
(66) These compounds are a part of the NIH Small Molecule
Repository: see http://mlsmr.glpg.com/MLSMR_HomePage/.
(67) Betti, M. General Condensation Reaction between B-Naphthol,
Aldehydes and Amines. Gazz. Chim. Ital. 1900, 30, 310–316.
(68) Betti, M. General Condensation Reaction between B-Naphthol,
Aldehydes and Amines. Gazz. Chim. Ital. 1901, 31, 377–393.
(69) Betti, M. General Condensation Reaction between B-Naphthol,
Aldehydes and Amines. Gazz. Chim. Ital. 1901, 31, 170–184.
(70) Rai, G.; Kenyon, V.; Jadhav, A.; Schultz, L.; Armstrong, M.;
Jameson, J. B.; Hoobler, E.; Leister, W.; Simeonov, A.; Holman, T. R.;
Maloney, D. J. Discovery of Potent and Selective Inhibitors of Human
Reticulocyte 15-Lipoxygenase-1. J. Med. Chem. 2010, 53, 7392–7404.
(71) Inglese, J.; Auld, D. S.; Jadhav, A.; Johnson, R. L.; Simeonov, A.;
Yasgar, A.; Zheng, W.; Austin, C. P. Quantitative High-Throughput
5496
dx.doi.org/10.1021/jm2005089 |J. Med. Chem. 2011, 54, 5485–5497

Journal of Medicinal Chemistry
ARTICLE
Screening: A Titration-Based Approach That Efficiently Identifies
Biological Activities in Large Chemical Libraries. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 11473–11478.
(72) Pierre, J. L.; Baret, P.; Serratrice, G. Hydroxyquinolines as Iron
Chelators. Curr. Med. Chem. 2003, 10, 1077–1084.
(73) Liu, Z. D.; Kayyali, R.; Hider, R. C.; Porter, J. B.; Theobald, A. E.
Design, Synthesis, and Evaluation of Novel 2-Substituted 3-Hydroxy-
pyridin-4-Ones: StructureꢀActivity Investigation of Metalloenzyme
Inhibition by Iron Chelators. J. Med. Chem. 2002, 45, 631–639.
(74) Nelson, M. J.; Brennan, B. A.; Chase, D. B.; Cowling, R. A.;
Grove, G. N.; Scarrow, R. C. Structure and Kinetics of Formation of
Catechol Complexes of Ferric Soybean Lipoxygenase-1. Biochemistry
1995, 34, 15219–15229.
Inhibitors as Neuroprotective Reagents. J. Neurosci. Res. 2008, 86,
904–909.
(90) Wang, H.; Li, J.; Follett, P. L.; Zhang, Y.; Cotanche, D. A.;
Jensen, F. E.; Volpe, J. J.; Rosenberg, P. A. 12-Lipoxygenase Plays a Key
Role in Cell Death Caused by Glutathione Depletion and Arachidonic
Acid in Rat Oligodendrocytes. Eur. J. Neurosci. 2004, 20, 2049–2058.
(91) Kim, H. Y.; Sawazaki, S. Structural Analysis of Hydroxy Fatty
Acids by Thermospray Liquid Chromatography/Tandem Mass Spectro-
metry. Biol. Mass Spectrom. 1993, 22, 302–310.
(75) Hahn, C.; Sieler, J.; Taube, R. Synthesis of 2,6-Bis-
(diphenylphosphinomethyl)pyridine-monoligandꢀRhodium(I) Com-
plexes [Rh(Pnp)L]X with L = Pyridine, Ch3cn, Dmso and X =
Cf3so3, Bf4 from the Corresponding Ethylene Complex and Comparison
of the Structures to the Piperidine Complex (L = Piperidine, X = Bf4).
Polyhedron 1998, 17, 1183–1193.
(76) Gupta, A. K.; Bluhm, R. Seborrheic Dermatitis. J. Eur. Acad.
Dermatol. Venereol. 2004, 18, 13–26quiz 19ꢀ20.
(77) Kemal, C.; Louis-Flamberg, P.; Krupinski-Olsen, R.; Shorter,
A. L. Reductive Inactivation of Soybean Lipoxygenase 1 by Catechols: A
Possible Mechanism for Regulation of Lipoxygenase Activity. Biochem-
istry 1987, 26, 7064–7072.
(78) McMillan, R. M.; Walker, E. R. Designing Therapeutically
Effective 5-Lipoxygenase Inhibitors. Trends Pharmacol. Sci. 1992, 13,
323–330.
(79) McGovern, S. L.; Helfand, B. T.; Feng, B.; Shoichet, B. K. A
Specific Mechanism of Nonspecific Inhibition. J. Med. Chem. 2003,
46, 4265–4272.
(80) Cornish-Bowden, A. Fundamentals of Enzyme Kinetics; Portland
Press, Ltd.: Colchester, UK, 1995; p 343.
(81) Crichlow, G. V.; Lubetsky, J. B.; Leng, L.; Bucala, R.; Lolis, E. J.
Structural and Kinetic Analyses of Macrophage Migration Inhibitory
Factor Active Site Interactions. Biochemistry 2009, 48, 132–139.
(82) Sloane, D.; Leung, R.; Barnett, J.; Craik, C.; Sigal, E. Conversion
of Human 15-Lipoxygenase to an Efficient 12-Lipoxygenase—the Side-
Chain Geometry of Amino Acids 417 and 418 Determine Positional
Specificity. Protein Eng. 1995, 8, 275–282.
(83) McLean, L. R.; Zhang, Y.; Li, H.; Li, Z.; Lukasczyk, U.; Choi,
Y. M.; Han, Z.; Prisco, J.; Fordham, J.; Tsay, J. T.; Reiling, S.; Vaz, R. J.;
Li, Y. Discovery of Covalent Inhibitors for Mif Tautomerase via
Cocrystal Structures with Phantom Hits from Virtual Screening. Bioorg.
Med. Chem. Lett. 2009, 19, 6717–6720.
(84) Gilbert, A. M.; Bursavich, M. G.; Lombardi, S.; Georgiadis,
K. E.; Reifenberg, E.; Flannery, C. R.; Morris, E. A. N-((8-Hydroxy-5-
substituted-quinolin-7-Yl)(phenyl)methyl)-2-phenyloxy/Amin O-Acet-
amide Inhibitors of Adamts-5 (Aggrecanase-2). Bioorg. Med. Chem. Lett.
2008, 18, 6454–6457.
(85) Ohri, R. V.; Radosevich, A. T.; Hrovat, K. J.; Musich, C.; Huang,
D.; Holman, T. R.; Toste, F. D. A Re(V)-Catalyzed CꢀN Bond-
Forming Route to Human Lipoxygenase Inhibitors. Org. Lett. 2005,
7, 2501–2504.
(86) Chen, X.-S.; Brash, A.; Funk, C. Purification and Characteriza-
tion of Recombinant Histidine-Tagged Human Platelet 12-Lipoxygen-
ase Expressed in a Baculovirus/Insect Cell System. Eur. J. Biochem. 1993,
214, 845–852.
(87) Robinson, S. J.; Hoobler, E. K.; Riener, M.; Loveridge, S. T.;
Tenney, K.; Valeriote, F. A.; Holman, T. R.; Crews, P. Using Enzyme
Assays to Evaluate the Structure and Bioactivity of Sponge-Derived
Meroterpenes. J. Nat. Prod. 2009, 72, 1857–1863.
(88) Michael, S.; Auld, D.; Klumpp, C.; Jadhav, A.; Zheng, W.;
Thorne, N.; Austin, C. P.; Inglese, J.; Simeonov, A. A Robotic Platform
for Quantitative High-Throughput Screening. Assay Drug Dev. Technol.
2008, 6, 637–657.
(89) van Leyen, K.; Arai, K.; Jin, G.; Kenyon, V.; Gerstner, B.;
Rosenberg, P. A.; Holman, T. R.; Lo, E. H. Novel Lipoxygenase
5497
dx.doi.org/10.1021/jm2005089 |J. Med. Chem. 2011, 54, 5485–5497