Discovery of potent and selective inhibitors of human reticulocyte 15-lipoxygenase-1

Article abstract of DOI:10.1021/jm1008852

There are a variety of lipoxygenases in the human body (hLO), each having a distinct role in cellular biology. Human reticulocyte 15-lipoxygenase-1 (15-hLO-1), which catalyzes the dioxygenation of 1,4-cis,cis-pentadiene- containing polyunsaturated fatty acids, is implicated in a number of diseases including cancer, atherosclerosis, and neurodegenerative conditions. Despite the potential therapeutic relevance of this target, few inhibitors have been reported that are both potent and selective. To this end, we have employed a quantitative high-throughput (qHTS) screen against ～74000 small molecules in search of reticulocyte 15-hLO-1 selective inhibitors. This screen led to the discovery of a novel chemotype for 15-hLO-1 inhibition, which displays nM potency and is >7500-fold selective against the related isozymes, 5-hLO, platelet 12-hLO, epithelial 15-hLO-2, ovine cyclooxygenase-1, and human cyclooxygenase-2. In addition, kinetic experiments were performed which indicate that this class of inhibitor is tight binding, reversible, and appears not to reduce the active-site ferric ion.

Full text of DOI:10.1021/jm1008852

7392 J. Med. Chem. 2010, 53, 7392–7404
DOI: 10.1021/jm1008852
Discovery of Potent and Selective Inhibitors of Human Reticulocyte 15-Lipoxygenase-1
Ganesha Rai,†,§ Victor Kenyon,‡,§ Ajit Jadhav,† Lena Schultz,† Michelle Armstrong,‡ J. Brian Jameson, II,‡ Eric Hoobler,‡
William Leister,† Anton Simeonov,† Theodore R. Holman,*,‡ and David J. Maloney*,†
†NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, MSC
3370, Bethesda, Maryland 20892, and ‡Department of Chemistry and Biochemistry, University of California, Santa Cruz, 250 Physical Sciences
Building, Santa Cruz, California 95064. § Both of these authors contributed equally to this work.
Received July 9, 2010
There are a variety of lipoxygenases in the human body (hLO), each having a distinct role in cellular
biology. Human reticulocyte 15-lipoxygenase-1 (15-hLO-1), which catalyzes the dioxygenation of
1,4-cis,cis-pentadiene-containing polyunsaturated fatty acids, is implicated in a number of diseases
including cancer, atherosclerosis, and neurodegenerative conditions. Despite the potential therapeutic
relevance of this target, few inhibitors have been reported that are both potent and selective. To this end,
we have employed a quantitative high-throughput (qHTS) screen against ∼74000 small molecules
in search of reticulocyte 15-hLO-1 selective inhibitors. This screen led to the discovery of a novel
chemotype for 15-hLO-1 inhibition, which displays nM potency and is >7500-fold selective against the
related isozymes, 5-hLO, platelet 12-hLO, epithelial 15-hLO-2, ovine cyclooxygenase-1, and human
cyclooxygenase-2. In addition, kinetic experiments were performed which indicate that this class
of inhibitor is tight binding, reversible, and appears not to reduce the active-site ferric ion.
Introduction
and various types of cancers.4-6 5-hLO7 has been implicated
in cancer3e,8 and asthma,9 while platelet-type 12-LO10 has
been implicated in psoriasis11 and pancreatic,12 breast,13,14
and prostate cancers.15,16 Reticulocyte 15-hLO-1 (15-hLO-1)
is less straightforward since it has been implicated both in
resolvingandpromotinghumandisease.17 Inprostatetumors,
cancer cells have a higher expression of 15-hLO-1 compared
with normal adjacent tissue, and this expression positively
correlates with the virulence of the tumor.18-20 In contrast,
15-hLO-2 is expressed in normal prostate tissue, but poorly
expressed in prostate tumors, with an inverse correlation with
the virulence of the tumor.21 This opposing effect between
15-hLO-1 and 15-hLO-2 is thought to be due to the difference
in product generation. While one of the major 15-hLO-1
products, 13-(S)- hydroperoxy-9,11-(Z,E)-octadecadienoic
acid (13-HPODE) from LA, up-regulates the MAP kinase
signaling pathway, the major 15-hLO-2 product, 15-(S)-
HPETE from AA, down-regulates MAP kinase. In colon
cancer, however, 15-hLO-1 has been proposed to have a
beneficial role. Downregulation of 15-hLO-1 is linked to
colorectal tumorigenesis and restoring 15-hLO-1 expression
in colon cancer in vivo xenografts downregulates antiapoptotic
proteins and inhibits cell growth.22
Other potential therapeutic benefits of 15-hLO-1 inhibitors
include asthma,23 cardiovascular disease,24 and minimizing
the brain damage that occurs after a stroke. One of the major
features of neuronal cell death after a stroke event is the
accumulation of reactive oxygen species (ROS).25 Recently, it
has been reported that 15-hLO-1 damages the mitochondria,
which leads to the breakdown of the membrane potential, the
production of ROS, and cytochrome c release, suggesting that
15-hLO-1 is the central executioner in an oxidative stress-
related neuronal death program.26 These broad implications
in disease regulation underscore the need for small molecule
Lipoxygenases (LOsa) are nonheme, iron-containing en-
zymes found in both the plant and animal kingdoms. LOs
catalyze the dioxygenation of 1,4-cis,cis-pentadiene-contain-
ing polyunsaturated fatty acids (e.g., linoleic acid (LA) and
arachidonic acid (AA)) to form hydroperoxy-fatty acids.1 The
mechanism for this reaction is the abstraction of a hydrogen
atom from the 1,4-cis,cis-pentadiene by an active site ferric
ion.2 Inhibitors of lipoxygenases can target this unique reac-
tion via a variety of mechanisms such as reductive, chelation,
competitive, and/or allosteric.3
Human lipoxygenases (hLOs) have been implicated in
several diseases involving inflammation, immune disorders,
*To whom correspondence should be addressed. 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.
a Abbreviations: LO, lipoxygenase; sLO-1, soybean lipoxygenase-1;
15-hLO-1, human reticulocyte 15-lipoxygenase-1; 15-hLO-2, human
epithelial 15-lipoxygenase-2; 12-hLO, human platelet 12-lipoxygenase;
15-rLO, rabbit reticulocyte 15-lipoxygenase; COX, cyclooxygenase;
NDGA, nordihydroguaiaretic acid; AA, arachidonic acid; 15-HPETE,
15-(S)-hydroperoxyeicosatetraenoic acid; 15-HETE, 15-(S)-hydroxyei-
cosatetraenoic acid; 12-HPETE, 12-(S)-hydroperoxyeicosatetraenoic
acid; 12-HETE, 12-(S)-hydroxyeicosatetraenoic acid; LA, linoleic acid;
13-HPODE, 13-(S)-hydroperoxyoctadecadienoic acid; 13-HODE,
13-(S)-hydroxyoctadecadienoic acid; ALA, alpha-linolenic acid;
13-HPOTrE, 13-(S)-hydroperoxyoctadecatrienoic acid; Vmax; maximal
velocity (μmol/min); KM, Henri-Michaelis-Menten constant (μM);
[E], total active enzyme concentration; IC50, inhibitor constant at 50%
inhibition; Kiapp, apparent inhibition constant when [E] . Kiapp; DPPH,
1.1-diphenyl-2-picrylhydrazyl; XO, xylenol orange; HTS, high-through-
put screening; MLSMR, Molecular Libraries Small Molecule Reposi-
tory; MLPCN, Molecular Libraries Probe Production Center Network;
qHTS, quantitative high-throughput screening; CRC, concentration
response curve; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride; BTAC, N-benzyl-triethylammonium chloride.
r
pubs.acs.org/jmc
Published on Web 09/24/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7393
inhibitors against 15-hLO-1. However, to date, relatively few
inhibitors have been reported that are both potent and
selective.
(IC50 = 2 μM),27a and bacterial hopanoids (IC50 = 10 μM).30
The literature is also replete with other 15-LO inhibitors, but
these have only been screened against sLO-1,31-33 which has
been found to be a poor model for 15-hLO-1 inhibition.27a,b,34
Recently, we were able to identify nonreductive and selec-
tive inhibitors utilizing computational docking; however, the
potency of these compounds were only in the low micromolar
range.35 The most promising 15-hLO-1 inhibitors thus far are
the tryptamine36 (3) and imidazole-based37 (4) derivatives,
which have low nanomolar potency and selectivity against
both 5-hLO and 12-hLO (Figure 1).38
Given the limited number of potent and selective 15-hLO-1
inhibitors and the potential therapeutic benefit for such
compounds, we sought to provide the scientific community
with additional small molecule biochemical probes to study
this important target. As such, we conducted a quantitative
high-throughput screen (qHTS) in 1536-well format using a
library containing 74290 small molecules39 as part of the NIH
Molecular Libraries Probe Production Center Network
(MLPCN) in search of novel potent and selective 15-hLO-1
inhibitors. In this report, we discuss the discovery, synthesis,
and structure-activity relationships (SAR) of a novel che-
motype, which displayed nanomolar potency and is highly
selective against 15-hLO-1. Moreover, preliminary investiga-
tions into the mechanism of action are presented.
Over the last 10 years, our laboratory3a,27 and others28-30
have attempted to identify potent and selective 15-hLO-1
inhibitors, but with limited success (submicromolar potency,
with approximately 40-fold selectivity against 12-hLO).
Unfortunately, the majority of these compounds are reductive
inhibitors and/or are promiscuous polyphenolic/terpene based
terrestrial natural products such as boswellic acid (1) (IC50 =
1 μM),28 hexamethoxyflavone (IC50 = 50 μM),29 nor-dihy-
droguaiaretic acid (NDGA) (IC50 = 0.5 μM),3a baicalein (2)
Chemistry
Despite the fact that we screened a relatively large number of
compounds belonging to a diverse chemical library, few hits
exhibited potent and selective inhibition for 15-hLO-1. However,
the 1,3,4-oxadiazole-2-thiol chemotype (5) (Figure 2D) showed
potent (19 nM) inhibition against 15-hLO-1 and was selec-
tive against related isozymes (5-hLO, 12-hLO, and 15-hLO-2).
Figure 1. Representative examples of previously reported 15-hLO-
1 inhibitors.
Figure 2. (A) qHTS concentration response profiles of 74290 compounds screened against 15-hLO-1. (B) Top actives from the primary screen
are shown in blue, and inconclusive or weak inhibitors are shown in green. (C) Distribution of activity response in the primary screen. (D) The
lead compound 5 from qHTS, which was chosen for optimization.

7394 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Scheme 1. Synthetic Route of Compounds 5, 9-43a
Rai et al.
a Reagents and conditions: (a) anhydrous hydrazine (5 equiv), EtOH, reflux, 16 h, 92%; (b) CS2 (2.5 equiv), KOH (2.3 equiv), EtOH, reflux, 2 h, then
acidified, 90%; (c) 4-chlorobut-2-yn-1-ol, K2CO3, acetone, reflux, 1 h, 86%; (d) EDC (2 equiv), cat. DMAP, various carboxylic acid derivatives (1.2
equiv), DMF, room temperature, 1 h.
To further investigate the linker region, we attempted the
synthesis of both the Z-alkene analogue and E-alkene analo-
gue 64, however, under several conditions, we found that the
Z-olefin rapidly isomerized to the E-olefin so we were unable
to isolate this compound. Synthesis of (E-alkene) 64 was
achieved via alkylation of 8 with (E)-1,3-dichloropropene
Figure 3. Synthesis of Analogues 43-55.
using N-benzyl-triethylammonium chloride (BTAC) and
sodium hydroxide followed by hydrolysis using 0.1 M HCl/
DMSO to afford the allylic alcohol 63, which was esterified
using standard conditions. Propargylic alcohol derivative 65
was synthesized by alkylation of 8 with 4-chlorobut-2-yn-1-ol,
which was further modified using 4-fluorobenzyl bromide to
provide compound 66.
Moreover, this novel scaffold is chemically tractable and
amenable to chemical modification at various positions of the
molecule, allowing for rapid exploration of the SAR profile.
Our initial round of analogues was focused around mod-
ification of the 2-thiophene carboxylic acid moiety in our lead
compound 5. As such, the synthesis commenced with treat-
ment of the commercially available ethyl 1-naphthoate (6)
with anhydrous hydrazine in ethanol at reflux to afford the
desired hydrazide 7 in high yield. (Scheme 1). Cyclization was
accomplished using carbon disulfide in the presence of KOH
in ethanol followed by acidification of the resulting thiolate to
provide 8. Alkylation of 8 with 4-chlorobut-2-yn-1-ol with
K2CO3 in acetone provided the key propargylic alcohol
intermediate from which a wide variety of ester derivatives
(9-42) were prepared in a facile manner using EDC and cat.
DMAP in DMF at room temperature.
The synthesis of analogues 43-55 was more laborious, as
the divergent point for each analogue is in the first step of the
sequence as shown in Figure 3. A variety of commercially
available aromatic and heteroaromatic esters were carried
through a similar synthetic route with one exception. To make
the route as convergent as possible, 4-chlorobut-2-yn-1-ol was
first coupled with either 2-thiophene carboxylic acid or
4-fluorobenzoic acid followed by subsequent coupling with
intermediate 8 to provide the desired analogues 43-55.
Last, we explored the effects of changing various heteroa-
toms throughout the core scaffold as well as altering the
nature of the linker and terminal ester moiety. As such, the
synthesis of the amide analogue 56 was accomplished via
alkylation of 8 with N-(4-chlorobut-2-ynyl)thiophene-2-car-
boxamide using potassium carbonate in DMF as shown in
Scheme 2. The requisite carboxamide was prepared using
EDC mediated coupling of thiophene-carboxylic acid to
commercially available 4-chlorobut-2-yn-1-amine. Analogues
58 and 60 were obtained from common intermediate 7, either
by treatment with carbon disulfide in the presence of KOH
followed by cyclization with conc H2SO4 to provide the thi-
adiazole intermediate 57 or cyanogen bromide to give the
oxadiazole 2-amine 59, which upon alkylation with 4-chlorobut-
2-ynyl thiophene-2-carboxylate afforded 58 and 60, respectively.
Synthesis of analogues 61 (alkyne reduced to alkane) and 62
(alkyne replaced with phenyl) involved alkylation of inter-
mediate 8 with chloro-1-butanol and 4-(chloromethyl)benzyl
alcohol, respectively, followed by EDC and cat. DMAP medi-
ated esterification with 4-fluorobenzoic acid.
Results and Discussion
In the search for novel small molecule inhibitors, we
performed a high-throughput screen, testing a diverse collec-
tion of 74290 compounds arrayed as dilution series. The 15-
hLO-1 HTS assay utilized a colorimetric method for detecting
the hydroperoxide reaction products of lipoxygenase.40 The
extent of product formation was monitored by a secondary
chromogenic reaction in which Xylenol Orange (XO) binds to
the ferric ions produced from the reaction between the
hydroperoxideproduct and the ferrous ionadded tothe assay.
The resulting Fe(III)-XO complex is characterized by a red-
shifted absorbance in the 560-580 nm region (orange to
purple color). The assay was initially developed for a 384-well
format and further miniaturized to 4 μL enzymatic reaction
volume in a 1536-well format. Enzyme and substrate concen-
trations, as well as reaction time, were optimized in order to
achieve the highest signal-to-background ratio without reach-
ing reaction plateau. The stability of working concentrations
of the assay components, 15-hLO-1 enzyme, and substrate at
þ4 °C, and the chromogenic reagent FeXO prepared in dilute
acid at room temperature permitted the implementation of an
unattended overnight screening operation.
The full-collection screen utilized 463 assay plates run in an
uninterrupted robotic screening sequence of approximately
52 h. The average signal-to-background ratio was 5.5 and the
average Z0 screening factor was 0.64 (Supporting Information
Figure S1), indicating a robust performance of the screen. The
known lipoxygenase inhibitor, nordihydroguaiaretic acid
(NDGA), was included as an intraplate control on each assay
plate (16-point dilution series in duplicate between 57.5 μM
and 1.8 nM), to further ascertain screening quality. The
resulting average IC50 of NDGA was 0.283 ( 0.09 μM (n =
926) with a minimum significant ratio (defined by Eastwood
et al.41) of 1.9, thus indicating a stable run (Supporting
Information Figure S2). In the present screen, 74290 com-
pounds were tested in at least seven concentrations, ranging
from 57.5 μM to 0.7 nM; the screen yielded a range of active
samples associated with different potencies (IC50) and con-
centration-response curve (CRC) quality (Figure 2A,B).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7395
Scheme 2. Synthetic Route for Compounds 56-66
a Reagents and conditions: (a) N-(4-chlorobut-2-ynyl)thiophene-2-carboxamide (1.1 equiv), K2CO3 (5 equiv), DMF, 40 °C, 2 h; (b) CS2 (2.3 equiv),
KOH (1 equiv), EtOH, rt, 4 h, filter then add to conc H2SO4, 0 °C f rt, 2 h, 61%; (c) 4-chlorobut-2-ynyl thiophene-2-carboxylate (1.1 equiv), NaH (2
equiv), DMF, 0 °C, 1 h then rt, 5 h; (d) cyanogen bromide (1.2 equiv), EtOH, reflux, 1 h, 84%; (e) 4-chlorobutan-1-ol, (1.1 equiv), K2CO3 (4 equiv),
DMF, 40 °C, 1.5 h, 63%; (f) 4-fluorobenzoic acid (1.1 equiv), EDC (1.5 equiv), cat. DMAP, DMF; (g) 4-(chloromethyl)benzyl alcohol, (1.1 equiv),
K2CO3 (4 equiv), DMF; (h) 4-fluorobenzoic acid (1.1 equiv), EDC (1.5 equiv), cat. DMAP, DMF; (i) (E)-1,3-dichloropropene (1.5 equiv), BTAC (0.15
equiv), 1:1 (1% aq NaOH/CHCl3), rt, 4 h; (j) 2:1 (0.1 M aq HCl/DMSO), rt; (k) 4-chlorobut-2-yn-1-ol, (1.1 equiv), K2CO3 (4 equiv), DMF, 40 °C, 1.5 h;
(l) 4-fluorobenzyl bromide, (2.2 equiv), NaH (4 equiv), DMF, 10 h.
Following the qHTS, the CRC data were subjected to a
classification scheme to rank the quality of the CRCs, as
described by Inglese and co-workers.42 Briefly, the CRCs were
placed in four classes. Class 1 contains complete concentration-
response curves showing both upper and lower asymp-
totes and R2 values greater than 0.9. Class 2 contains
incomplete CRCs lacking the lower asymptote and shows
r2 > 0.9. Class 3 curves are of the lowestconfidence as they are
defined by a single concentration point where the minimal
acceptance activity is set 3 SD of the mean activity, calculated
as described above. Finally, class 4 contains compounds
that do not show any CRCs and are therefore classified as
inactives. Of the 74290 screened compounds, 69447 were
regarded as inactives (Figure 2C) and 3789 as inconclusive.
There were 1054 compounds that were classified as actives,
belonging to curve classes -1.1, -1.2, -2.1, or -2.2. The
complete screening results have been made available in Pub-
Chem (PubChem Assay ID 887). Selected compounds that
were active in the primary screen, and associated with high-
quality complete concentration-response curves (presence of
both asymptotes and maximum efficacy of greater than 80%
inhibition) were reacquired and tested in the original HTS
assay to confirm activity. Prioritized hits were then further
characterized in the standard cuvette-based kinetic assay for
lipoxygenase activity, which measures product formation by
monitoring the increase in absorbance at 234 nm.40b On the
basis of the retest and the confirmatory cuvette-based kinetic
measurements, a novel chemotype (compound 5, Figure 2D),
which displayed nM potency against the enzyme, and
was inactive in enzymatic assays against the related isozymes
15-hLO-2, 12-hLO, and 5-hLO, was selected for further
characterization.
To date, an X-ray structure of 15-hLO-1 has yet to be
reported. However, crystallization of rabbit reticulocyte
15-LO (15-rLO), which has approximately 80% sequence
identity with the human variant, has been achieved.43 As
mentioned above, utilization of a homology model based on
the 15-rLO has been used successfully in the identification of
15-hLO-1 inhibitors and thus could be exploited in our
investigations. However, initially we aimed to define the
SAR profile through systematic modification of the various
functional groups on the lead molecule 5 (Figure 2D). Our
efforts toward this goal commenced with derivatization of
the thiophene ester moiety. This position allows for rapid
exploration ofSAR via a late-stage esterification ofa common
intermediate (Scheme 1). As shown in Table 1, we prepared
molecules with varying substitution on the thiophene core
(analogues 9-11), carbocycles (13-15, 18), and various

7396 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Rai et al.
Table 1. SAR of Various Ester Derivatives: Lead Compound 5 and Analogues (9-23)
a The inhibition data were fit as described in the Methods section, where the weak inhibitors (greater than 1 μM) were fit with a standard hyperbolic
equation and the moderate inhibitors (1 μM to 10 nM) were fit using the Morrison equation. The potent inhibitors (less than 10 nM) could not be fit using
the Morrison equation due to the high dependence of the Kiapp on active enzyme concentration. b Maximum inhibition was 60%.
heterocycles (12, 16, 17, 19-23). Our first observation was
that modification of this terminal ester moiety is well tolerated
asthemajority ofthe analoguesmaintained potency. Itshould
be noted that due to limitations in assay detection and enzyme
concentration, we are unable to accurately differentiate
potencies lower than 10 nM, thus limiting our ability to evaluate
SAR of these more potent analogues (see the Experimental
Section for a more detailed explanation). Given that the
potency of our lead compound 5 is near the limit of detection,
we are not able to clearly define improved potency over the
qHTS hit, however, we are able to determine which structural
changes are not well tolerated and which compounds should
be progressed into further studies. As such, some clear SAR
trends emerged from the initial round of analogues, the first
being that sulfur containing heterocycles appear to be pre-
ferred over their oxygen counterpart. This is highlighted by
2-thiophene analogue (5) and 2-furan analogue (12), which
resulted inan 8-foldloss in activity (19-170nM, respectively).
Moreover, a moderate loss in potency was observed when
comparing the 2-benzothiophene analogue 21 (<10 nM) and
the corresponding 2-benzofuran derivative 19 (15 nM). Com-
parison of analogues 13, 14, and 15 reveals a preference
for increased hydrophobicity in this portion of the binding
pocket as potency improved proportionally to ring size
(IC50 = cyclopropane (13)>cyclobutane (14)>cyclopentane
(15)). The most pronounced loss in potency was observed with
the imidazole analogue 16, which is approximately 70 times
less active than the original lead 5 whereas the indole analogue
20 was approximately 4-fold less active, consistent with hydro-
phobicity playing an important role. Taken together, these
results suggest that sulfur is preferred over oxygen in the
heterocycle and that potency is generally improved with
increased hydrophobicity of this moiety (vide infra).
observed, which istobe expected given the results of ourinitial
ester modifications (Table 1). While not explored in detail, it
appears that ortho-substituted electron-donating groups are
not favorable given the drastic loss in potency (470 nM) for
analogue 33 (R = 2-OMe). However, this effect could also be
a result of an unfavorable steric interaction of the 2-methoxy
group disrupting a key hydrogen bond interaction of the
carbonyl oxygen on the ester moiety.
Through these initial SAR investigations (see Tables 1 and 2),
we learned that the terminal ester moiety is fairly amendable to
substitution with hydrophobic groups and could possibly be
exploited later to engineer a molecule with improved properties
such as aqueous solubility.
We next investigated changes to the various heteroatoms
throughout the core scaffold and also the “left-hand” portion
of the molecule (i.e., naphthalene moiety). Replacing the
naphthalene group with other carbocycles and heterocycles
(analogues 43-55) was generally well tolerated. Interestingly,
analogues 43 (R = 2-naphthalene, <10 nM), 47 (R = 4-F-
Ph, <10 nM), and 49 (R = 4-Cl-Ph, <10 nM) are more
potent than the ortho-substituted analogues 45 (R = 2-F-Ph,
43 nM) and 48 (R = 2-Cl-Ph, 57 nM), which may suggest that
the more linear analogues (43, 47, and 49) are able to acquire
favorable interactions by extending into a hydrophobic region
in the active site. Future efforts will aim to exploit this SAR by
preparing analogues such as (R = p-bisphenyl) and (R =
6-quinoline or 2-quinoline) to test this binding hypothesis.
Unlike the relatively flat SAR observed for the ester
modifications, variation of the heteroatoms throughout the
core scaffold resulted in significant loss of potency in most
cases, as shown in Table 3. Given the potential hydrolysis of
modified ester derivatives by intracellular esterases, we first
examined the more hydrolytically stable amide analogue 56.
However, this change resulted in a drastic loss in activity (100-
fold), with the amide 56 having an IC50 of 2 μM compared to
the corresponding ester analogue 5 (IC50 = 19 nM). For
analogue 58, in which the oxadiazole is changed to a thiadia-
zole, we also observe a loss in potency, but this analogue was
noticeably less soluble, which complicated the biochemical
characterization of this compound. We postulated that
switching the sulfur for a nitrogen, at the 2-position of the
Having explored various heterocyclic changes to the ester
moiety, we turned our attention to benzoic acid derivatives, as
we noticed that the simple benzoyl analogue (24) exhibited
comparable if not improved potency to the lead compound 5
(<10 and 19 nM, respectively). As shown in Table 2, we
explored various electron-donating and electron-withdrawing
groups at different positions on the ring. Generally, all sub-
stitutions were well tolerated and relatively flat SAR was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7397
Table 2. SAR of Benzoic Acid Derivatives: Analogues (24-42)
a The inhibition data were fit as described in the Methods section, where the weak inhibitors (greater than 1 μM) were fit with a standard hyperbolic
equation and the moderate inhibitors (1 μM to 10 nM) were fit using the Morrison equation. The potent inhibitors (less than 10 nM) could not be fit using
the Morrison equation due to the high dependence of the Kiapp on active enzyme concentration.
Table 3. SAR of Analogues (43-56, 58 and 60)
a The inhibition data were fit as described in the Methods section, where the weak inhibitors (greater than 1 μM) were fit with a standard hyperbolic
equation and the moderate inhibitors (1 μM to 10 nM) were fit using the Morrison equation. The potent inhibitors (less than 10 nM) could not be fit using
the Morrison equation due to the high dependence of the Kiapp on active enzyme concentration.
oxadiazole (analogue 60), might result in an additional
hydrogen-bond (donating) interaction; however, this too
was unfavorable, with a potency of 1.3 μM being obtained.
Accordingly, this SAR investigation indicates that linear
hydrophobic moieties are favored for the “left-hand” portion
of the molecule and other heterocycles in this position are
relatively well tolerated, but heteroatom changes to the core
structure are generally unfavorable.
Finally, we investigated the tolerance for changes in the
linker region and the role of the carbonyl oxygen on the
terminal ester moiety. Preliminary docking-based binding
hypothesis of this chemotype in the active site of 15-hLO-1
suggested that the alkyne occupies a narrow constriction
linking two hydrophobic pockets, presenting a “barbell-type”
shape. To test this hypothesis, we synthesized alkane deriva-
tive (61), E-alkene (64), and replaced the alkyne moiety with a
phenyl group (62), which should maintain the planar nature
of the appended groups. As mentioned above, attempts to
synthesize the Z-alkene derivative were unsuccessful, as under
numerous conditions rapid isomerization to the seemingly
more stable E-alkene occurred. As shown in Table 4, a
moderate loss of potency was observed for alkane derivative
61 (600 nM), however, the E-alkene analogue (64) had almost
no loss in potency (26 nM) compared to the alkyne (19 nM). In
contrast, complete loss in activity occurred with the phenyl
analogue 62 (>100 μM), suggesting that while the alkyne
moiety is not required for activity, more bulky linkers (i.e.,
switching alkyne for phenyl) are unfavorable, presumably
because they cannot fit within a narrow region in the active site.
We next synthesized the ether analogue 66 to investigate the
importance of the ester carbonyl and observed complete loss
in activity, indicating the necessity of a hydrogen bond
acceptor in this position. Additionally, this key interaction
appears to be easily disrupted, as was seen with the loss of
activity with the ortho-substituted benzoic acid derivative 33
and the amide 56. The ketone analogue 67 was also prepared
but unfortunately had reduced solubility, which hampered
the ability to obtain an accurate IC50. Given the apparent

7398 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Table 4. SAR of Analogues (61, 62, 64-67)
Rai et al.
a The inhibition data were fit as described in the Methods section, where the weak inhibitors (greater than 1 μM) were fit with a standard hyperbolic
equation and the moderate inhibitors (1 μM to 10 nM) were fit using the Morrison equation. The potent inhibitors (less than 10 nM) could not be fit using
the Morrison equation due to the high dependence of the Kiapp on active enzyme concentration. b Maximum inhibition was 70%.
class of compounds. To ensure that the inhibition was not an
effect of small molecule aggregates, the concentration of
Triton X-100 was varied from 0.005% to 0.02%, with no
change in the Kiapp for compound 5 witnessed.44 To investi-
gate if the inhibition resulted from reduction of the active site
iron, DPPH, a free radical scavenger, was incubated with a
representative group of inhibitors, and no reduction of the
DPPH was observed (NDGA was used as a positive control).
Additionally, there was no elongation of enzymatic lag phase
or resumption of activity over time when these inhibitors were
used, suggestive of a nonreductive inhibitory mechanism.
A competitive substrate experiment was also performed to
probe if the inhibition was allosteric in nature, but no change
in product distribution was observed.3d
Table 5. Lipoxygenase Isozyme Selectivity of the Top 15-hLO-1
Inhibitors
analogue
15-hLO-1a
15-hLO-2a
12-hLOa
5-hLOa
5
0.019
<0.010
<0.010
<0.010
<0.010
<0.010
<0.010
<0.010
<0.010
<0.010
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>15
>15
>75
>20
>50
>30
>15
>30
>10
>75
11
21
24
26
29
39
41
43
49
a IC50 values are reported in μM and estimated from three inhibitor
concentrations, due to their lack of potency.
To investigate the reversibility of inhibition, 15-hLO-1 was
incubated with the low nanomolar inhibitor 25, the less potent
33 and an equivalent volume of DMSO as a positive control.
After 10 min of incubation, the control displayed activity,
whereas activity was abolished in the samples with inhibitor
present. All three samples were then dialyzed for 2 h. Upon
checking the activity of the three samples, activity had been
restored in the samples with inhibitor present, indicating that
the inhibition is a reversible process.
Steady-state kinetics were performed using compound 16
by monitoring the formation of 15-HPETE as a function of
substrate and inhibitor concentration in the presence of
0.01% Triton X-100. Replots of KM/Vmax and 1/Vmax versus
inhibitor concentration (Supporting Information, Figures S3
and S4) yielded linear plots, with Ki equaling 0.87 ( 0.07 μM
and Ki0 equaling 8.1 ( 0.9 μM, which are defined as the
equilibrium constants of dissociation from the catalytic and
secondary sites, respectively. The secondary site could be the
allosteric site due to its approximately 100-fold difference in
binding from the catalytic site, which is consistent with our
previous studies of 15-hLO-2,45 however, no change in substrate
preference was observed when inhibitor was added to 15-hLO-1
(vide supra). It should be noted that we were not able to
determine the inhibitor mechanism for the more potent inhibi-
tors due to the small Kiapp to enzyme ratio, however, we assume
their mechanisms are comparable due to their similar structures.
necessityof a hydrogen-bond acceptor atthis position and our
interest in improving the inherent stability of the lead mole-
cule, we also prepared several ester bioisosteres which we
expected would be more hydrolytically stable yet maintain
potency. These analogues included 1,2,4- and 1,3,4-oxadia-
zoles, sulfonamides, and reversed esters, however, the potency
of these derivatives was greatly decreased, with none having
an IC50 of less than 1 μM (data not shown).
With the commencement of our initial rounds of SAR
explorations, which provided compounds with low nM
potency against 15-hLO-1, we were then eager to determine
the selectivity of our top analogues against related hLO
isozymes (5-hLO, 12-hLO, and 15-hLO-2). Gratifyingly, all
of our most potent analogues displayed excellent selectivity
against all three isozymes, with only 5-hLO having IC50 values
below100 μM (Table5). These resultswere quiteencouraging,
as few compounds reported in the literature have achieved
both nM potency toward 15-hLO-1 and selectivity against
other isozymes, with the exception of chemotypes 3 and 4
(Figure 1). In addition, we explored whether this chemotype
could inhibit cyclooxygenase-1 (COX-1) and/or COX-2 and
found that compound 5 did not inhibit either COX at 10 μM
(inhibition less than 5% of total activity), demonstrating a
selectivityof greater than1000-foldfor thischemotype against
15-hLO-1 over either COX-1 or COX-2.
Conclusion
As previously mentioned, the X-ray structure of 15-hLO-1
has yet to be reported, therefore, several biochemical methods
were employed to test the mechanism of inhibition for this
An HTS campaign of ∼74290 compounds, aimed at iden-
tifying small molecule 15-hLO-1 inhibitors, uncovered the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7399
5-substituted-1,3,4-oxadiazole-2-thiol chemotype, which was
further optimized and elaborated through synthetic efforts to
provide potent and selective inhibitors of 15-hLO-1. Modifi-
cations of the core scaffold helped us develop an SAR profile.
Replacement of the thiophene and naphthalene moieties of
the lead compound 5 with other aromatic and heteroaromatic
groups was well tolerated. Consideration of the binding site
and SAR of key analogues indicate the presence of a “barbell-
like” binding pocket, which helps explain the requirement of
a narrow linker region between the oxadiazole and terminal
ester groups. Gratifyingly, our best compounds are potent
(single digit nM) and exhibit >7500-fold selectivity over
similar hLO isozymes (5, 12, and 15-hLO-2) and COX
isozymes. Additionally, this class of compounds was found
to be nonreductive and exhibited reversible inhibition, with
inhibitor binding occurring at both the catalytic site and
possibly the allosteric site. Current efforts are focused on
utilizing these molecules in cell-based systems across several
therapeutic areas including cancer and stroke models. These
results, along with other ongoing investigations, will be
reported in due course. It is our hope that these reagents will
provide a general, molecular tool to validate 15-hLO-1 as
a therapeutic target.
in absolute ethanol (50 mL) was added anhydrous hydrazine
(50 mmol), and the reaction mixture was refluxed overnight. The
solid which precipitated upon cooling was collected by filtra-
tion. The collected precipitate was washed with water, then cold
ethanol, and dried to provide aryl hydrazides (yield: 89-92%).
The products were used without further purification.
General Procedure for the Formation of 2-Aryl-5-mercapto-
1,3,4-oxadiazoles (Scheme 1). Potassium hydroxide (40 mmol)
was dissolved in ethanol (50 mL), and then aryl hydrazide
(17 mmol) and carbon disulfide (42 mmol) were added. The
reaction mixture was heated at reflux for 2 h. Solvent was
evaporated, and the residue was acidified with 10% HCl. The
precipitate was collected by filtration, washed with water, and dried
(88-99%). The product(s) were used without further purification.
5-(Naphthalen-1-yl)-1,3,4-oxadiazole-2-thiol (8). 1H NMR
(DMSO-d6) δ 7.63-7.77 (m, 3 H), 8.06-8.16 (m, 2 H), 8.22
(d,J= 8.2 Hz, 1H), and 8.83 (dd, J= 8.5 and 1.1 Hz, 1H). 13CNMR
(DMSO-d6) δ 118.51, 124.81, 125.32, 126.87, 128.35, 128.51,
128.62, 129.01, 132.96, 133.34, 160.30 and 176.90. HRMS (m/z):
[M þ H]þ calcd for C12H9N2OS, 229.0430; found, 229.0432.
4-(5-(Naphthalen-1-yl)-1,3,4-oxadiazol-2-ylthio)but-2-yn-1-ol
(65). A solution of 5-(naphthalen-1-yl)-1,3,4-oxadiazole-2-thiol
(8) (10 mmol), 4-chlorobut-2-yn-1-ol (10.2 mmol), and potassium
carbonate (50 mmol) in acetone (50 mL) was refluxed for 1 h. The
reaction mixture was filtered, and the filtrate was evaporated via
reduced pressure. The crude residue was purified on a Biotage
silica gel column. Elution with 30% ethyl acetate in hexanes gave
the product. Yield: 86%. 1H NMR (DMSO-d6) δ 4.09 (dd, J =
5.6 and 2.4 Hz, 2 H), 4.29-4.33 (m, 2H), 5.21 (td, J = 5.8 and 2.4
Hz, 1 H), 7.63-7.78 (m, 3 H), 8.09 (d, J = 8.0 Hz, 1 H), 8.18-8.24
(m, 2 H), and 9.03 (d, J = 8.6 Hz, 1 H). 13C NMR (DMSO-d6) δ
21.18, 48.99, 78.61, 84.04, 119.27, 125.29, 125.36, 126.84, 128.28,
128.68, 128.90, 128.98, 132.72, 133.39, 162.48 and 165.37. HRMS
(m/z): [M þ H]þ calcd for C16H13N2O2S, 297.0692; found,
297.0691.
4-(5-(Naphthalen-1-yl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
Thiophene-2-carboxylate (5). A solution of alcohol 65 (0.20
mmol), 2-thiophene carboxylic acid (0.22 mmol), EDC (0.40
mmol), and DMAP (0.10 mmol) in DMF (1 mL) was stirred at
room temperature for 1 h. The product was purified directly by
preparative HPLC (see General Chemistry for details). LC-MS: rt
(min) = 6.91. 1H NMR (CDCl3) δ 4.15-4.22 (m, 2 H), 4.89-4.96
(m, 2 H), 7.03-7.10 (m, 1 H), 7.51-7.64 (m, 3 H), 7.64-7.74 (m,
1 H), 7.77-7.84 (m, 1 H), 7.93 (d, J = 8.2 Hz, 1 H), 8.03 (d, J= 8.2
Hz, 1 H), 8.14 (dd, J = 7.3 and 1.1 Hz, 1 H) and 9.18 (d, J= 8.6Hz,
1 H). 13C NMR (CDCl3) δ 21.45, 52.69, 78.61, 80.55, 119.86,
124.81, 126.06, 126.73, 127.77, 128.18, 128.34, 128.64, 129.78,
132.61, 132.71, 132.99, 133.75, 134.08, 161.33, 162.44, and166.17.
HRMS (m/z): [M þ H]þ calcd for C21H15N2O3S2, 407.0519;
found, 407.0517.
Experimental Section
General Chemistry. Unless otherwise stated, all reactions
were carried out under an atmosphere of dry argon or nitrogen
in dried glassware. 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 A, 250 μm).
Visualization was accomplished by irradiation under a 254 nm
UV lamp. Chromatography 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 auto-
mated chromatography system. 1H and 13C 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
1
ppm, 39.51 ppm for H, 13C, respectively). Data are reported
as follows: chemical 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
spectrometer, also coupled to an Agilent Technologies 1200
series HPLC system. If needed, products were purified via a
Waters semipreparative 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 (0.025% TFA) and H2O (0.05% TFA), and the
temperature was maintained at 50 °C.
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 0.8-1.0 mL/min over a
7 min gradient and a 8.5 min run time. 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).
N-(4-Chlorobut-2-ynyl)thiophene-2-carboxamide. A mixture
of thiophene-2-carboxylic acid (1.0 g, 7.8 mmol) and EDC
(2.2 g, 11.7 mmol) in CH2Cl2 (40 mL) was stirred for 30 min,
and 4-chlorobut-2-yn-1-aminium chloride hydrochloride (1.3 g,
8.6 mmol) was added and stirred at room temperature for 2 h.
The reaction mixture was diluted with dichloromethane and
washed with water. The organic layer was dried over sodium
sulfate. The crude residue was purified on a Biotage silica gel
column. Elution with 20% ethyl acetate in hexanes gave the
product. Yield: 1.32 g (79%). 1H NMR (DMSO-d6) δ 4.13 (dt,
J = 5.6 and 2.1 Hz, 2 H), 4.46 (t, J = 2.0 Hz, 2 H), 7.15 (dd, J =
4.9 and 3.7 Hz, 1 H), 7.74-7.80 (m, 2 H) and 8.97 (t, J = 5.5 Hz,
1 H). 13C NMR (DMSO-d6) δ 28.47, 31.07, 77.21, 83.81, 127.97,
128.45, 131.19, 139.14, and 160.84. HRMS (m/z): [M þ H]þ
calcd for C9H9ClNOS, 214.0088; found, 214.0090.
N-(4-(5-(Naphthalen-1-yl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl)-
thiophene-2-carboxamide (56). To a mixture of 8 (0.040 g,
0.18 mmol) and N-(4-chlorobut-2-ynyl)thiophene-2-carboxamide
(0.041 g, 0.19 mmol) in DMF (1 mL) was added potassium
carbonate (0.12 g, 0.88 mmol) and was stirred at 40 °C for 2 h.
General Procedure for the Syntheses of Aryl Hydrazides
(Scheme 1). To a solution containing the aryl ester (10 mmol)

7400 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Rai et al.
The resulting crude product was purified by preparative HPLC
(see General Chemistry for HPLC purification conditions). LC-
MS: rt (min) = 6.15. H NMR (CDCl3) δ 4.12 (t, J = 2.2 Hz,
4-(5-(Naphthalen-1-yl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
Benzofuran-2-carboxylate (19). LC-MS: rt (min) = 7.05. H
1
1
NMR (DMSO-d6) δ 4.40 (t, J = 2.1 Hz, 2 H), 5.06 (t, J = 2.0 Hz,
2 H), 7.31-7.40(m,1 H), 7.51 (ddd, J =8.4, 7.1, and1.3Hz, 1 H),
7.61-7.69 (m, 4 H), 7.69-7.77 (m, 2 H), 8.06 (d, J = 8.0 Hz, 1 H),
8.13-8.23 (m, 2 H), and 9.03 (dd, J = 8.7 and 0.9 Hz, 1 H). 13C
NMR (DMSO-d6) δ 20.95, 52.73, 52.97, 77.85, 82.44, 112.10,
114.81, 119.22, 123.26, 124.04, 125.26, 125.29, 126.41, 126.80,
128.14, 128.24, 128.59, 128.87, 128.94, 132.68, 144.10, 155.05,
157.79, 162.26, and 165.44. HRMS (m/z): [M þ H]þ calcd for
C25H17N2O4S, 441.0904; found, 441.0907.
4-(5-(Naphthalen-1-yl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
1H-Indole-4-carboxylate (20). LC-MS: rt (min) = 6.70. 1H
NMR (CDCl3) δ 4.20 (t, J = 2.1 Hz, 2 H), 5.03 (t, J = 2.1 Hz,
2 H), 7.17-7.23 (m, 2 H), 7.31-7.36 (m, 1 H), 7.50-7.61 (m,
3 H), 7.67 (ddd, J = 8.5, 6.9, and 1.4 Hz, 1 H), 7.92 (dd, J = 7.6
and 1.0 Hz, 2 H), 8.01 (d, J = 8.2 Hz, 1 H), 8.13 (dd, J = 7.3 and
1.3 Hz, 1 H), 8.43 (br s, 1 H), and 9.19 (dd, J = 8.5 and 0.9 Hz,
1 H). 13C NMR (CDCl3) δ 21.56, 52.30, 79.31, 80.07, 103.94,
116.31, 119.96, 120.63, 121.11, 123.77, 124.84, 126.12, 126.47,
126.72, 127.51, 128.16, 128.33, 128.63, 129.81, 132.64, 133.77,
136.53, 162.51, 166.17, and 166.65. HRMS (m/z): [M þ H]þ
calcd for C25H18N3O3S, 440.1069; found, 440.1064.
4-(5-(Naphthalen-1-yl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Fluorobenzoate (27) (aka ML094). LC-MS: rt (min) = 7.12.
1H NMR (CDCl3) δ 4.18 (t, J = 2.2 Hz, 2 H), 4.94 (t, J = 2.1 Hz,
2 H), 7.01-7.10 (m, 2 H), 7.51-7.63 (m, 2 H), 7.68 (td, J = 7.7
and 1.4 Hz, 1 H), 7.93 (d, J = 8.0 Hz, 1 H), 8.03 (dt, J = 8.9 and
2.7 Hz, 3 H), 8.13 (dd, J = 7.3 and 1.1 Hz, 1 H), and 9.19 (d, J =
8.6 Hz, 1 H). 13C NMR (CDCl3); δ 21.45, 52.75, 78.64, 80.57,
115.42, 115.63, 119.89, 124.78, 126.07, 126.73, 128.16, 128.25,
128.63, 129.75, 132.26, 132.36, 132.67, 133.75, 162.29, 164.60,
164.74, 166.16, and 167.13. HRMS (m/z): [M þ H]þ calcd for
C23H16FN2O3S, 419.0865; found, 419.0864.
4-(5-(Naphthalen-1-yl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Chlorobenzoate (31). LC-MS: rt (min) = 7.38. 1H NMR
(CDCl3) δ 4.18 (t, J = 2.1 Hz, 2 H), 4.94 (t, J = 2.2 Hz, 2 H),
7.35 (m, 2 H), 7.58 (m, 2 H), 7.68 (ddd, J = 8.6, 6.9, and 1.6 Hz,
1 H), 7.93 (m, 3 H), 8.03 (d, J = 8.2 Hz, 1 H), 8.13 (dd, J = 7.3
and 1.3 Hz, 1 H), and 9.19 (dd, J = 8.6 and 1.0 Hz, 1 H). 13C
NMR (CDCl3); δ 21.47, 52.88, 78.58, 80.70, 119.94, 124.82,
126.11, 126.78, 127.76, 128.22, 128.31, 128.68, 128.75, 129.81,
131.13, 132.73, 133.80, 139.79, 162.32, 164.91, and 166.24.
HRMS (m/z): [M þ H]þ calcd for C23H16ClN2O3S, 435.0565;
found, 435.0570.
2 H), 4.27 (dt, J = 5.3 and 2.2 Hz, 2 H), 6.30-6.39 (m, 1 H), 7.03
(dd, J = 4.9 and 3.7 Hz, 1 H), 7.46 (dd, J = 5.1 and 1.2 Hz, 1 H),
7.52-7.57 (m, 2 H), 7.57-7.64 (m, 1 H), 7.68 (ddd, J = 8.5, 6.9,
and 1.4 Hz, 1 H), 7.90-7.95 (m, 1 H), 8.03 (d, J = 8.2 Hz, 1 H),
8.13 (dd, J = 7.3 and 1.3 Hz, 1 H), and 9.19 (dd, J = 8.6 and 0.8
Hz, 1 H). 13C NMR (CDCl3) δ 21.03, 21.56, 29.95, 80.44, 119.87,
124.84, 126.04, 126.75, 127.62, 128.19, 128.34, 128.49, 128.68,
129.78, 130.33, 132.74, 133.78, 138.01, 161.48, 162.50 and 166.23.
HRMS(m/z):[M þ H]þ calcdforC21H16N3O3S, 406.0678; found,
406.0675.
2-(1-Naphthyl)-5-mercapto-1,3,4-thiadiazole (57). 1-Naphtho-
hydrazide (7) (4.0 g, 21.5 mmol) and potassium hydroxide (1.2 g,
21.5 mmol) in ethanol (100 mL) was stirred for 30 min, and then
carbon disulfide (3.0 mL, 49.4 mmol) was added. The reaction
mixture was stirred at room temperature for 4 h. The yellow
precipitate was collected by filtration, and the product was
washed with ether. This product was slowly added to sulfuric
acid (20 mL, 375 mmol) at 0 °C and then stirred at rt for 2 h. The
reaction mixture was poured into ice, and the product was
collected by filtration. Recrystallization from ethanol gave pure
yellow product (3.2 g, 61%). 1H NMR (DMSO-d6) δ 7.60-7.74
(m, 3 H), 7.81 (d, J = 7.2 Hz, 1 H), 8.07 (d, J = 7.8 Hz, 1 H), 8.16
(d, J = 8.2 Hz, 1 H) and 8.60 (d, J = 8.2 Hz, 1 H). 13C NMR
(DMSO-d6) δ 124.69, 124.98, 125.42, 126.87, 128.07, 128.78,
129.16, 129.55, 131.83, 133.46, 161.13, and 174.68. HRMS (m/
z): [M þ H]þ calcd for C12H9N2S2, 245.0202; found, 245.0204.
4-(5-(Naphthalen-1-yl)-1,3,4-thiadiazol-2-ylthio)but-2-ynyl
Thiophene-2-carboxylate (58). To a solution of 57 (0.20 mmol)
in DMF (1 mL) at 0 °C was added NaH (0.40 mmol) followed by
4-chlorobut-2-ynyl thiophene-2-carboxylate (0.22 mmol), the
reaction mixture was stirred at 0 °C for 1 h and then at room
temperature for 5 h. The product was purified in a preparative
HPLC (see General Chemistry for details). LC-MS: rt (min) =
7.10. 1H NMR (CDCl3) δ 4.23 (t, J = 2.2 Hz, 2 H), 4.89-4.96 (m,
2 H), 7.02-7.08 (m, 1 H), 7.50-7.65 (m, 4 H), 7.73-7.78 (m, 1 H),
7.80-7.84 (m, 1 H), 7.91-7.95 (m,1 H), 8.00 (d, J = 8.2 Hz, 1 H)
and 8.68-8.76 (m, 1 H). 13C NMR (CDCl3) δ 22.55, 52.72, 78.49,
81.02, 93.24, 109.67, 124.89, 125.55, 126.66, 127.70, 127.77,
127.78, 128.39, 129.63, 129.68, 130.35, 131.47, 132.87, 133.84,
133.98, 161.27, 163.72, and 164.67. HRMS (m/z): [M þ Na]þ
calcd for C21H15N2O2S3, 445.0108; found, 445.0107.
2-(1-Naphthyl)-5-amino-1,3,4-oxadiazole (59). A mixture of
1-naphthohydrazide (7) (2.0 g, 10.7 mmol) and cyanogen bro-
mide (1.4 g, 13 mmol) in EtOH (107 mL) was heated at reflux for
1 h. The solvent was removed, and the product was purified by
recrystallization from ethanol to yield 1.9 g (84%) of pure
4-(5-(Naphthalen-1-yl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Methoxybenzoate (34). LC-MS: rt (min) = 7.03. H NMR
1
(CDCl3) δ 3.84 (s, 3 H), 4.18 (t, J = 2.1 Hz, 2 H), 4.92 (t, J = 2.1
Hz, 2 H), 6.87 (m, 2 H), 7.58 (m, 2 H), 7.68 (ddd, J = 8.6, 6.9, and
1.6 Hz, 1 H), 7.97 (m, 4 H), 8.14 (dd, J = 7.2 and 1.2 Hz, 1 H) and
9.20 (d, J = 8.6 Hz, 1 H). 13C NMR (CDCl3) δ 21.54, 52.40,
55.42, 79.12, 80.20, 105.02, 113.64, 119.99, 121.72, 124.85, 126.15,
126.75, 128.19, 128.33, 128.65, 129.84, 131.85, 132.69, 133.80,
162.42, 165.50, and 166.19. HRMS (m/z): [M þ H]þ calcd for
C24H19N2O4, 431.1060; found, 431.1064.
4-(5-Phenyl-1,3,4-oxadiazol-2-ylthio)but-2-ynyl 4-Fluorobenzoate
(44). LC-MS: rt (min) = 6.65. 1H NMR (CDCl3) δ 4.12 (t, J = 2.1
Hz, 2 H), 4.92 (t, J = 2.1 Hz, 2 H), 7.03-7.12 (m, 2 H), 7.43-7.55
(m, 3 H), and 7.94-8.07 (m, 4 H). 13C NMR (CDCl3) δ 21.46,
52.74, 78.62, 80.51, 115.46, 115.67, 123.41, 125.56, 125.59, 126.68,
129.02, 131.77, 132.30, 132.40, 162.50, 164.64, 164.74, 166.18, and
167.17. HRMS (m/z): [M þ H]þ calcd for C19H14FN2O3S,
369.0709; found, 369.0710.
4-(5-(2-Fluorophenyl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Fluorobenzoate (45). LC-MS: rt (min) = 6.55. 1H NMR
(CDCl3) δ 4.13 (t, J = 2.1 Hz, 2 H), 4.92 (t, J = 2.2 Hz, 2 H),
7.05-7.14 (m, 2 H), 7.17-7.25 (m, 1 H), 7.27-7.33 (m, 1 H),
7.49-7.57 (m, 1 H), and7.98-8.09(m, 3 H). 13C NMR(CDCl3) δ
21.48, 52.78, 78.69, 80.47, 111.93, 112.05, 115.49, 115.71, 116.15,
116.37, 116.88, 117.08, 124.63, 124.67, 124.99, 125.60, 125.63,
1
product 59. H NMR (DMSO-d6) δ 7.36 (s, 2 H), 7.59-7.71
(m, 3 H), 7.95-7.99 (m, 1 H), 8.03 (d, J = 8.0 Hz, 1 H), 8.08 (d,
J = 8.2 Hz, 1 H), and 9.13 (d, J = 8.6 Hz, 1H). 13C NMR
(DMSO-d6) δ 120.62, 125.35, 125.80, 126.44, 126.57, 127.68,
128.69, 128.91, 130.96, 133.48, 157.28, and 163.52.
4-(5-(Naphthalen-1-yl)-1,3,4-oxadiazol-2-ylamino)but-2-ynyl
Thiophene-2-carboxylate (60). To a solution of 5-(naphthalen-
1-yl)-1,3,4-oxadiazol-2-amine (59) (0.23 g, 1.1 mmol) in DMF
(3 mL) was added NaH (0.07 g, 2.2 mmol) and stirred for 1 h at
0 °C, and then 4-chlorobut-2-ynyl thiophene-2-carboxylate
(0.26 g, 1.2 mmol) was added. The reaction mixture was stirred
for 5 h at room temperature and then product was poured into
satd NH4Cl (aq) and extracted with ethyl acetate, washed with
water and then brine, and dried (MgSO4). The crude product
was purified in HPLC. LC-MS: rt (min) = 4.80. 1H NMR
(DMSO-d6) δ 5.09 (d, J = 2.0 Hz, 2 H), 5.21 (d, J = 1.4 Hz,
2 H), 7.24-7.26 (m, 1 H), 7.68-7.81 (m, 3 H), 7.85-7.87 (m,
1 H), 8.01-8.03 (m, 1 H), 8.07-8.10 (m, 1 H), 8.15 (d, J = 8.0
Hz, 1 H), 8.32 (d, J = 8.4 Hz, 1 H), 8.77-8.83 (m, 1 H) and
10.45 (brs, 1H). HRMS (m/z): [M þ H]þ calcd for C21H16N3-
O3S, 390.0907; found, 390.0912.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7401
129.53, 132.34, 132.43, 133.24, 133.34, 133.55, 133.63, 158.55,
161.13, 162.94, 163.14, 164.68, 164.79, 165.43, 167.21, and167.99.
HRMS (m/z): [M þ H]þ calcd for C19H13F2N2O3S, 387.0615;
found, 387.0610.
4-(5-(3-Hydroxynaphthalen-2-yl)-1,3,4-oxadiazol-2-ylthio)-
but-2-ynyl 4-Fluorobenzoate (53). LC-MS: rt (min) = 6.50. 1H
NMR (DMSO-d6) δ 4.07 (s, 2 H), 4.19 (t, J = 2.1 Hz, 2 H),
7.44-7.53 (m, 2 H), 7.64-7.77 (m, 2 H), 8.04 (d, J = 8.0 Hz, 1
H), 8.10 (s, 1 H), 8.19-8.23 (m, 1 H), 8.25-8.31 (m, 2 H), and
8.79 (s, 1 H). 13C NMR (DMSO-d6) δ 21.07, 48.97, 78.42, 84.04,
115.48, 116.05, 116.27, 121.87, 125.55, 125.58, 127.30, 127.42,
128.84, 129.16, 130.36, 130.61, 132.98, 133.08, 134.42, 144.24,
162.69, 162.83, 164.14, 164.30, and 166.82. HRMS (m/z): [M þ
H]þ calcd for C23H16FN2O4S, 435.0814; found, 435.0812.
4-(5-(1H-indol-2-yl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Fluorobenzoate (54). LC-MS: rt (min) = 6.26. 1H NMR (400
MHz, DMSO-d6) δ 4.29 (t, J = 2.1 Hz, 2 H), 4.98 (t, J = 2.1 Hz,
2 H), 7.19-7.30 (m, 3 H), 7.53 (dd, J = 6.9 and 1.5 Hz, 1 H),
7.87-7.95 (m, 2 H), 8.02-8.09 (m, 1 H), 8.16 (d, J = 2.9 Hz,
1 H), and 12.03 (brs, 1 H). 13C NMR (DMSO-d6) δ 21.13, 52.78,
78.19, 82.10, 99.02, 112.43, 115.74, 115.96, 120.10, 121.24,
122.87, 123.87, 128.40, 132.01, 132.10, 136.39, 159.20, 163.51,
and 163.96. HRMS (m/z): [M þ H]þ calcd for C21H15FN3O3S,
408.0818; found, 408.0813.
4-(5-(Quinolin-5-yl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Fluorobenzoate (55). LC-MS: rt (min) = 5.60. 1H NMR
(DMSO-d6) δ 4.38 (t, J = 2.1 Hz, 2 H), 4.98 (t, J = 2.0 Hz, 2 H),
7.14-7.23 (m, 2 H), 7.77 (dd, J = 8.7 and 4.2 Hz, 1 H),
7.81-7.88 (m, 2 H), 7.92 (dd, J = 8.5 and 7.3 Hz, 1 H),
8.23-8.31 (m, 2 H), 9.06 (dd, J = 4.2 and 1.7 Hz, 1 H), and
9.41-9.48 (m, 1 H). 13C NMR (DMSO-d6) δ 21.02, 52.72, 78.37,
81.96, 115.68, 115.90, 119.70, 123.12, 124.52, 128.74, 129.22,
131.90, 132.00, 133.13, 134.21, 147.21, 151.07, 162.55, 163.83,
164.58, and 166.34.
4-(5-(3-Fluorophenyl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Fluorobenzoate (46). LC-MS: rt (min) = 6.74. 1H NMR
(CDCl3) δ 4.13 (t, J = 2.1 Hz, 2 H), 4.92 (t, J = 2.1 Hz, 2 H),
7.06-7.13 (m, 2 H), 7.23 (td, J = 8.4 and 2.6 Hz, 1 H), 7.47 (td,
J = 8.1 and 5.6 Hz, 1 H), 7.70 (dt, J = 9.2 and 2.1 Hz, 1 H), 7.80
(d, J = 7.8 Hz, 1 H), and 8.01-8.08 (m, 2 H). 13C NMR (CDCl3)
δ 21.50, 52.74, 78.78, 80.39, 113.65, 113.89, 115.49, 115.72,
118.77, 118.98, 122.46, 122.49, 125.24, 125.32, 125.57, 125.60,
130.88, 130.97, 132.33, 132.42, 161.55, 163.05, 164.01, 164.69,
164.76, 165.17, 165.20, and 167.22. HRMS (m/z): [M þ H]þ
calcd for C19H13F2N2O3S, 387.0615; found, 387.0612.
4-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Fluorobenzoate (47). LC-MS: rt (min) = 6.68. 1H NMR
(CDCl3) δ 4.12 (t, J = 2.1 Hz, 2 H), 4.92 (t, J = 2.1 Hz, 2 H),
7.06-7.13 (m, 2 H), 7.14-7.21 (m, 2 H), and 7.97-8.09 (m, 4 H).
13C NMR (CDCl3) δ 21.50, 52.75, 78.70, 80.45, 115.50, 115.73,
116.32, 116.54, 119.78, 119.81, 125.59, 125.62, 128.99, 129.07,
132.33, 132.43, 162.59, 163.53, 164.70, 164.77, 165.40, 166.05,
and 167.23. HRMS (m/z): [M þ H]þ calcd for C19H13F2N2O3S,
387.0615; found, 387.0612.
4-(5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Fluorobenzoate (48). LC-MS: rt (min) = 6.80. 1H NMR
(CDCl3) δ 4.14 (t, J = 2.1 Hz, 2 H), 4.93 (t, J = 2.1 Hz, 2 H),
7.06-7.14 (m, 2 H), 7.36-7.42 (m, 1 H), 7.46 (td, J = 7.6 and 1.8
Hz, 1 H), 7.51-7.56 (m, 1 H), 7.95 (dd, J = 7.8 and 1.8 Hz, 1 H),
and 8.01-8.10 (m, 2 H). 13C NMR (CDCl3) δ 21.48, 52.78,
78.72, 80.46, 115.49, 115.72, 122.76, 127.07, 130.98, 131.26,
132.35, 132.44, 132.47, 133.07, 163.26, 164.54, 164.69, and
164.80. HRMS (m/z): [M þ H]þ calcd for C19H13ClFN2O3S,
403.0319; found, 403.0317.
4-(5-(Naphthalen-1-yl)-1,3,4-oxadiazol-2-ylthio)butyl 4-Fluoro-
1
benzoate (61). LC-MS: rt (min) = 7.39. H NMR (CDCl3) δ
1.92-2.18 (m, 4 H), 3.44 (td, J = 7.1 and 2.8 Hz, 2 H), 4.36-4.46
(m, 2 H), 7.04-7.14 (m, 2 H), 7.52-7.64 (m, 2 H), 7.66-7.74 (m,
1 H), 7.93 (d, J = 8.2 Hz, 1 H), 7.99-8.10 (m, 3 H), 8.10-8.18
(m, 1 H), and 9.21 (d, J = 8.6 Hz, 1 H). 13C NMR NMR
(CDCl3) δ 26.11, 27.74, 32.15, 64.25, 115.41, 115.64, 120.12,
124.83, 126.17, 126.38, 126.73, 128.14, 128.66, 129.83, 132.06,
132.15, 132.55, 133.82, 163.97, 164.49, 165.56, 165.76, and
167.01. HRMS (m/z): [M þ H]þ calcd for C23H20FN2O3S,
423.1178; found, 423.1176.
4-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Fluorobenzoate (49). LC-MS: rt (min) = 7.00. 1H NMR
(CDCl3) δ 4.13 (t, J = 2.1 Hz, 2 H), 4.92 (t, J = 2.1 Hz, 2 H),
7.06-7.14 (m, 2 H), 7.46 (m, 2 H), 7.94 (m, 2 H) and 8.01-8.07
(m, 2 H). 13C NMR (CDCl3) δ 21.50, 52.73, 78.73, 80.41, 115.50,
115.72, 121.90, 125.56, 125.59, 127.97, 129.46, 132.33, 132.41,
138.10, 162.82, 164.69, 164.76, 165.39, and 167.23. HRMS
(m/z): [M þ H]þ calcd for C19H13ClFN2O3S, 403.0319; found,
403.0320.
4-((5-(Naphthalen-1-yl)-1,3,4-oxadiazol-2-ylthio)methyl)-
benzyl 4-Fluorobenzoate (62). LC-MS: rt (min) = 7.59. H
1
4-(5-(Furan-2-yl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl 4-Fluoro-
benzoate (50). LC-MS: rt (min) = 6.22. 1H NMR (DMSO-d6) δ
4.29 (s, 2 H), 4.97 (s, 2 H), 6.77 (dd, J = 3.4 and 1.7 Hz, 1 H),
NMR (CDCl3) δ 4.59 (s, 2 H), 5.35 (s, 2 H), 7.06-7.14 (m, 2 H),
7.44 (d, J = 8.0 Hz, 2 H), 7.51-7.63 (m, 4 H), 7.65-7.72 (m,
1 H), 7.93 (d, J = 8.0 Hz, 1 H), 8.03 (d, J = 8.0 Hz, 1 H),
8.05-8.13 (m, 3 H), and 9.19 (d, J = 8.6 Hz, 1 H). 13C NMR
NMR (CDCl3) δ36.41, 66.35, 115.44, 115.66, 120.08, 124.82,
126.16, 126.22, 126.25, 126.73, 128.14, 128.19, 128.64, 129.48,
129.82, 132.21, 132.30, 132.58, 133.81, 135.87, 135.93, 163.50,
164.57, 165.39, 165.89, and 167.10. HRMS (m/z): [M þ H]þ
calcd for C27H20FN2O3S, 471.1178; found, 471.1175.
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. LO products
were generated by reacting substrate with the appropriate LO
isozyme (13-HPODE from sLO-1 and LA, 13-HPOTrE from
sLO-1 and ALA, 15-HPETE from sLO-1 and AA, and 12-
HPETE from 12-hLO and AA). Product generation was per-
formed as follows. An assay of 100 mL of 50-100 μM substrate
was run to completion, reactions were quenched with 5 mL acetic
acid, extracted twice with 50 mL of dichloromethane, evaporated
to dryness, and reconstituted in MeOH for HPLC purification.
All products were tested with sLO-1 to show that no residual
substrate was present, and demonstrated, by both analytical
HPLC and LC/MS/MS, to have greater than 98% purity.
7.28-7.40 (m, 3 H), 7.92-8.00 (m, 2 H), and 8.03 (s, 1 H). 13
C
NMR (DMSO-d6) δ 21.19, 52.74, 78.36, 81.77, 112.60, 114.76,
115.85, 116.07, 125.48, 132.08, 132.18, 138.08, 147.06, 158.35,
161.67, 163.96, and 166.49. HRMS (m/z): [M þ H]þ calcd for
C17H12FN2O4S, 359.0502; found, 359.0500.
4-(5-(Thiophen-2-yl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Fluorobenzoate (51). LC-MS: rt (min) = 6.48. 1H NMR
(CDCl3) δ 4.10 (t, J = 1.8 Hz, 2 H), 4.91 (t, J = 2.2 Hz, 2 H),
7.07-7.15 (m, 3 H), 7.53 (dd, J = 4.8 and 1.2 Hz, 1 H), 7.70 (dd,
J = 3.6 and 1.2 Hz, 1 H), and 8.03-8.06 (m, 2 H). 13C NMR
(CDCl3); δ 21.5, 52.7, 76.7, 77.0, 77.3, 78.7, 80.4, 115.5, 115.7,
124.6, 125.5, 125.6, 128.1, 129.8, 130.2, 132.3, 132.4, 161.9,
162.4, 164.6, 164.7, and 167.2. HRMS (m/z): [M þ H]þ calcd
for C17H12FN2O3S2, 375.0271; found, 375.0267.
4-(5-(2-Methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)but-2-ynyl
4-Fluorobenzoate (52). LC-MS: rt (min) = 6.40. 1H NMR
(CDCl3) δ 3.95 (s, 3 H), 4.12 (t, J = 2.2 Hz, 2 H), 4.92 (t, J =
2.2 Hz, 2 H), 7.01-7.13 (m, 4 H), 7.49 (ddd, J = 8.6, 7.3, and 1.8
Hz, 1 H), 7.87 (dd, J = 7.9 and 1.5 Hz, 1 H), and 8.02-8.08 (m, 2
H). 13C NMR (CDCl3) δ 21.43, 52.81, 55.95, 78.51, 80.67,
111.91, 112.58, 115.48, 115.70, 120.72, 125.62, 125.65, 130.27,
132.34, 132.43, 133.18, 157.76, 162.19, 164.67, 164.80, 164.94,
and 167.21. HRMS (m/z): [M þ H]þ calcd for C20H16FN2O4S,
399.0815; found, 399.0820.

7402 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Rai et al.
The reduced products were generated by selectively reducing the
98% pure peroxide product to the alcohol, with trimethylpho-
sphite. The purity of the reduced hydroxy products was then
confirmed with LC-MS/MS. All other chemicals were reagent
grade or better and were used without further purification.
Overexpression and Purification of 15-Human Lipoxygenase-1
and 12-Human Lipoxygenase. Human reticulocyte 15-lipoxygen-
ase-1 (15-hLO-1), human epithelial 15-lipoxygenase-2 (15-hLO-
2), and human platelet 12-lipoxygenase (12-hLO) were expressed
as N-terminally, His6-tagged proteins and purified to greater than
90% purity, as evaluated by SDS-PAGE analysis.27c,34,46 Human
5-lipoxygenase was expressed as a nontagged protein and used
as a crude ammonium sulfate protein fraction, as published
previously.47 Iron content of 15-hLO-1 was determined with a
Finnigan inductively coupled plasma mass spectrometer (ICP-
MS), using cobalt-EDTA as an internal standard. Iron concen-
trations were compared to standardized iron solutions and used
to normalize enzyme concentrations.
conjugated diene product at 234 nm (ε = 25000 M-1cm-1) 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 approximately 40 nM for 12-hLO, 20 nM of 15-
hLO-1 (by iron content), and 1 μM for 15-hLO-2. Reactions
with the crude, ammonium sulfate precipitated 5-hLO 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, 10 μM AA and
with 12-hLO in 25 mM Hepes buffer (pH 8), 0.01% Triton
X-100, and 10 μM AA. Reactions with 15-hLO-1 and 15-hLO-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-hLO, 12-
hLO, and 15-hLO-2) and LA (for 15-hLO-1) were quantitatively
determined by allowing the enzymatic reaction to go to comple-
tion. IC50 values were obtained by determining the enzymatic
rate at various inhibitor concentrations and plotted against
inhibitor concentration, followed by a hyperbolic saturation
curve fit (assuming total enzyme concentration [E] , Kiapp, so
IC50 ∼ Kiapp). It should be noted that all of the potent inhibitors
displayed greater than 80% maximal inhibition unless stated in
the tables. For a number of inhibitors, the Kiapp value ap-
proached the total active enzyme concentration ([E]), indicating
that hyperbolic fitting of the data was inappropriate. The Kiapp
values were then determined by plotting the fractional velocity
as a function of the inhibitor concentration, followed by a
quadratic fit using the Morrison equation.49 To determine the
average Kiapp and the associated error, the enzyme concentra-
tion in the Morrison equation was varied from the maximal [E]
(as measured by the metal content) to 0.01 nM. It should be
noted that the total active enzyme concentration ([E]) depends on
the iron content and varies with enzyme preparation. The sub-
sequent Kiapp values were averaged and the standard deviation
determined. Inhibitors that displayed a standard deviation in
Kiapp less than 50% are presented in the tables with their standard
deviation, whereas compounds with greater than 50% standard
deviation in their Kiapp were considered beyond the limit of our
assay and thus the maximal Kiapp value was set to half the [E],
or <10 nM. Inhibitors were stored at -20 °C in DMSO.
Lag-Phase Kinetic Analysis. The lag-phase of 15-hLO-1 was
examined with the addition of compounds 5, 13, 39, and 44 with
our standard kinetic analysis (vide supra). Inhibitor concentra-
tions were varied and the reactions allowed to progress for an
extensive period of time in order to determine if the inhibitory
lag-phase could be overcome. If the mechanism of the inhibitor
is reductive, it is common to see resumption of enzyme activity
after a period of time due to the destruction of the inhibitor by
the pseudoperoxidase activity of the LO.50
DPPH Antioxidant Test. Compounds 22 and 30 were dis-
solved in dimethyl sulfoxide (DMSO) at 1-20 mM concentra-
tion (1000-fold concentrated). The antioxidant activity of these
compounds was assayed by monitoring the quenching of the
standard free radical 1.1-diphenyl-2-picrylhydrazyl (DPPH)
upon reaction with the testing compounds.51,52 A known free
radical scavanger, nordihydroguaiaretic acid (NDGA), was
used as a positive control. Ten μL of 1 mM testing reagents
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.51,52 The decrease in optical absorbance at 517 nm
was monitored using a P-E Lambda 40 spectrometer. The rate of
reaction is proportional to the antioxidant potency of the test
compounds.
Reversibility of Inhibition. Three separate 1 mL samples of 15-
hLO-1 were incubated with 10 μL of 25 (final concentration of
50 μM), 33 (final concentration of 50 μM), and DMSO followed
by dialysis in 25 mM HEPES, 150 mM NaCl, and 10% glycerol
at pH 7.5 for 2 h. Activity was recorded by monitoring the
formation of the conjugated product before and after the
dialysis.
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 74290 compound library was screened
in 7-15 concentrations ranging from 0.7 nM to 57 μM. The
library included 61548 diverse small drug-like molecules that are
part of the NIH Small Molecule Repository. A collection of
1372 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 2419
compounds. An additional 1963 compounds from the NCI
Diversity Set were included. Last, 6925 compounds with known
pharmacological activity were added to provide a large and
diverse screening collection.
High-Throughput Screening Protocol and HTS Analysis. All
screening operations were performed on a fully integrated
robotic system (Kalypsys Inc., San Diego, CA) as described
elsewhere.48 First, 3 μL of enzyme (40 nM 15-hLO-1, final
concentration) was dispensed into a 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 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 temperature 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 þ4 °C recirculating
chiller bath to minimize degradation. Plates containing DMSO
only (instead of compound solutions) were included approxi-
mately 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.
Data were analyzed in a similar method as described else-
where.42 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). Com-
pounds with the highest quality, Class 1 and Class 2 curves, were
prioritized for follow-up.
Lipoxygenase UV-vis Assay. The initial one-point inhibition
percentages were determined by following the formation of the

Article
Steady-State Inhibition Kinetics. Lipoxygenase rates were
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7403
for human reticulocyte and epithelial 15-lipoxygenases reveal
allosteric product regulation. Biochemistry 2008, 47, 7364–7375.
(e) 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.
determined by monitoring the formation of the conjugated
product, 15-HPETE, at 234 nm (ε = 25 000 M-1 cm-1) with a
Perkin-Elmer Lambda 40 UV/vis spectrophotometer. Reac-
tions were initiated by adding approximately 80 nM 15-hLO-1
to a constantly stirring 2 mL cuvette containing 3-40 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 completion. Kinetic data
were obtained by recording initial enzymatic rates, at varied
inhibitor concentrations, and subsequently fitted to the Henri-
Michaelis-Menten equation, using KaleidaGraph (Synergy) to
determine the microscopic rate constants, Vmax (μmol/min/mg)
and Vmax/KM (μmol/min/mg/μM). These rate constants were
subsequently replotted, 1/Vmax and KM/Vmax versus inhibitor
concentration, to yield Ki0 and Ki, respectively.
Allosteric Inhibition. To assess if this chemotype affected the
substrate specificity by binding to the allosteric site, we per-
formed the competitive substrate capture method, as previously
described.3d Briefly, 15-hLO-1 was allowed to react with a
mixture of AA and LA and the product ratio determined by
HPLC. This experiment was repeated with inhibitor present and
the product ratio compared.
(4) Samuelsson, B.; Dahlen, S. E.; Lindgren, J. A.; Rouzer, C. A.;
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Acknowledgment. We thank Paul Shinn, Danielle van
Leer, and James Bougie for assistance with compound
management and purification. We also thank Dr. Matthew
Jacobson and Chakrapani Kalyanaraman for helpful discus-
sions during the preparation of this manuscript. Financial
support was from the National Institute of Health grants R01
GM56062 (T.R.H.) and the Molecular Libraries Initiative of
the National Institutes of Health Roadmap for Medical
Research (R03 MH081283 (T.R.H.)). Additional financial
support was from NIH (S10-RR20939 (T.R.H.)) and the
California Institute for Quantitative Biosciences for the
UCSC MS Facility (T.R.H.).
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Elevated expression of 12/15-lipoxygenase and cyclooxygenase-2
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2003, 63, 2256–2267.
Supporting Information Available: Additional experimental
procedures and spectroscopic data (1H NMR, LC/MS, and
HRMS) for representative compounds; qHTS assay perfor-
mance figures. This material is available free of charge via the
Internet at http://pubs.acs.org.
(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.
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Concordant induction of 15-lipoxygenase-1 and mutant p53
expression in human prostate adenocarcinoma: correlation with
Gleason staging. Carcinogenesis 2000, 21, 1777–1787.
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