Potent and selective inhibitors of human reticulocyte 12/15-lipoxygenase as anti-stroke therapies

Article abstract of DOI:10.1021/jm401915r

A key challenge facing drug discovery today is variability of the drug target between species, such as with 12/15-lipoxygenase (12/15-LOX), which contributes to ischemic brain injury, but its human and rodent isozymes have different inhibitor specificities. In the current work, we have utilized a quantitative high-throughput (qHTS) screen to identify compound 1 (ML351), a novel chemotype for 12/15-LOX inhibition that has nanomolar potency (IC ₅₀ = 200 nM) against human 12/15-LOX and is protective against oxidative glutamate toxicity in mouse neuronal HT22 cells. In addition, it exhibited greater than 250-fold selectivity versus related LOX isozymes, was a mixed inhibitor, and did not reduce the active-site ferric ion. Lastly, 1 significantly reduced infarct size following permanent focal ischemia in a mouse model of ischemic stroke. As such, this represents the first report of a selective inhibitor of human 12/15-LOX with demonstrated in vivo activity in proof-of-concept mouse models of stroke.

Full text of DOI:10.1021/jm401915r

Article
pubs.acs.org/jmc
Potent and Selective Inhibitors of Human Reticulocyte 12/15-
Lipoxygenase as Anti-Stroke Therapies
Ganesha Rai,‡,∥ Netra Joshi,§,∥ Joo Eun Jung,†,∥ Yu Liu,†,∥ Lena Schultz,‡ Adam Yasgar,‡ Steve Perry,§
Giovanni Diaz,§ Qiangli Zhang,§ Victor Kenyon,§ Ajit Jadhav,‡ Anton Simeonov,‡ Eng H. Lo,†
Klaus van Leyen,*,† David J. Maloney,*,‡ and Theodore R. Holman*,§
†Neuroprotection Research Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School,
Charlestown, Massachusetts 02129, United States
‡National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC 3370,
Bethesda, Maryland 20892, United States
§Department of Chemistry and Biochemistry, University of California−Santa Cruz, Santa Cruz, California 95064, United States
S
* Supporting Information
ABSTRACT: A key challenge facing drug discovery today is variability of the drug target between species, such as with 12/15-
lipoxygenase (12/15-LOX), which contributes to ischemic brain injury, but its human and rodent isozymes have different
inhibitor specificities. In the current work, we have utilized a quantitative high-throughput (qHTS) screen to identify compound
1 (ML351), a novel chemotype for 12/15-LOX inhibition that has nanomolar potency (IC50 = 200 nM) against human 12/15-
LOX and is protective against oxidative glutamate toxicity in mouse neuronal HT22 cells. In addition, it exhibited greater than
250-fold selectivity versus related LOX isozymes, was a mixed inhibitor, and did not reduce the active-site ferric ion. Lastly, 1
significantly reduced infarct size following permanent focal ischemia in a mouse model of ischemic stroke. As such, this
represents the first report of a selective inhibitor of human 12/15-LOX with demonstrated in vivo activity in proof-of-concept
mouse models of stroke.
born periventricular leukomalacia,32 breast cancer,33 and
INTRODUCTION
■
stroke,34,35 with the latter being the focus of this work.
Human lipoxygenases (LOX) are nonheme iron-containing
enzymes that catalyze the dioxygenation of 1,4-cis,cis-
pentadiene-containing polyunsaturated fatty acids (e.g., linoleic
Stroke is the fourth-leading cause of death in the United
States and the leading cause of disability.36,37 However, only
tissue plasminogen activator (tPA) is an FDA-approved drug
acid (LA) and arachidonic acid (AA)) to form hydroperoxy
fatty acids.1,2 The nomenclature of the LOX isozymes is loosely
treatment, and it is used in less than 5% of stroke patients.
Therefore, a novel target for stroke therapy is highly desirable.
based on the carbon position (e.g., 5, 12, or 15) at which they
The first reports of the contributions of 12/15-LOX to
oxidize arachidonic acid to form the corresponding hydro-
peroxyeicosatetraenoic acid (HpETE),3 which is reduced to the
neuronal cell death in oxidative stress models were published
starting in the late 1990s.38−41 Because oxidative stress is a
hydroxyeicosatetraenoic acid (HETE) by intracellular gluta-
thione peroxidases.4 Human lipoxygenases and their metabo-
major neurodegenerative process in ischemic diseases such as
stroke, a role for 12/15-LOX in stroke-induced brain injury
lites have been implicated in numerous diseases. 5-LOX has
seemed reasonable and, indeed, was found.42−44 Later work
showed that 12/15-LOX was detrimental not only to neurons
but also to the brain vasculature after stroke45 via a mechanism
that involves an intracellular attack on mitochondria and
been implicated in cancer,5 asthma,6,7 COPD,8 allergic rhinitis,9
osteoarthritis,10,11 and atherosclerosis,12−14 whereas platelet-
type 12-LOX has been implicated in diabetes,15,16 blood
coagulation,17 psoriasis,18 and cancer.19,20 Human reticulocyte
15-lipoxygenase-1 (12/15-LOX, aka 15-LOX-1) is also an
attractive therapeutic target, particularly for its role in
atherogenesis,21,22 diabetes,23−28 Alzheimer’s disease,29−31 new-
Received: December 13, 2013
Published: March 31, 2014
© 2014 American Chemical Society
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Figure 1. Examples of previously reported human 12/15-LOX (12/15-LOX) and rabbit 12/15-LOX (r12/15-LOX) inhibitors.
translocation of the apoptosis-inducing factor (AIF) to the
nucleus.46−48 Recent studies have shown that 12/15-LOX is
also increased in human stroke.34 In line with these findings,
several LOX inhibitors have been found to reduce infarct
size,34,42,43 leakage of the blood brain barrier and edema
formation,45 and even hemorrhagic transformation following
infusion of tPA.34
LOX inhibitors, which has been met with limited success.
Unfortunately, many of these inhibitors are reductive and/or
promiscuous, such as boswellic acid (IC50 = 1 μM),61 nor-
dihydroguaiaretic acid (NDGA) (IC50 = 0.11 μM),51 and
baicalein (IC50 = 1.6 μM),55 as shown in Figure 1.
Computational docking methods have been used to identify
novel, nonreductive inhibitors, but the potency and selectivity
of these compounds is limited (LOXBlock-1, Figure 1).49 The
most drug-like 12/15-LOX inhibitors that had been published
prior to our investigations were those reported by researchers at
Bristol-Myers Squibb (BMS) as well as Parke-Davis (now
Pfizer) compound PD146176.62 The BMS team identified
tryptamine-based compounds (37l),58 imidazole-based com-
pounds (21n),59 and pyrazole-based compounds (15i),57 as
shown in Figure 1. These inhibitors exhibited low nanomolar
potency versus rabbit 12/15-LOX (r12/15-LOX) and modest
selectivity against both 5-LOX and 12-LOX, but they had
generally unfavorable physical properties (e.g., solubility and
LogP)57−59 and unacceptable PK properties for use in vivo.
Most importantly, none of the above inhibitors, with the
exception of LOXBlock-1, were screened in parallel against
mouse 12/15-LOX, so it is unlikely that any would be amenable
to stroke drug development.
Our previously discovered inhibitor, LOXBlock-1 (Figure 1),
demonstrated good activity in mouse models for stroke;34
however, it is nonselective,49 suggesting that the identification
of novel selective inhibitors to interrogate 12/15-LOX biology
in vivo is desirable. From our original quantitative high-
throughput screen (qHTS) of 74 290 small molecules, the
1,3,4-oxadiazole-2-thiol chemotype (ML094, Figure 1)56 was
optimized for nanomolar potency and selectivity; however, we
were unable to observe activity of ML094 in a cellular
context.56 Therefore, in this work, we present our efforts to
identify a specific 12/15-LOX inhibitor that is potent against
both the human and the mouse homologues of 12/15-LOX. As
such, we rescreened our top hits from the original HTS with
Consequently, these broad implications of 12/15-LOX in
stroke regulation emphasize the need for small molecule
inhibitors that effectively cross the blood brain barrier and
target affected tissue. However, an additional aspect that must
be considered in discovering human 12/15-LOX inhibitors is
interspecies variability. The gene for 12/15-LOX for both
humans and mice is termed ALOX15, reflecting the close
homology (around 78%) and functional equivalence in both
species. 12/15-LOX isozymes can oxidize both C12 and C15,
forming 12-HpETE or 15-HpETE, respectively; however, the
dominant product varies between species, with several isozymes
oxidizing AA at C12, leading to a somewhat confusing
nomenclature. The 12/15-LOX isozymes targeted here have
historically been called 15-LOX, 15-LOX-1, or 15-LO-1 in
humans and L-12-LOX, leukocyte-type 12-LO, or L-12-LO in
mice. The number prefix reflects the preference for 15-HETE
generation by human 12/15-LOX, whereas the preference is
mostly for 12-HETE production for mouse 12/15-LOX.
Unfortunately, this difference in substrate specificity could
mean that an isoform-specific inhibitor that is selective toward
human 12/15-LOX may not target the orthologous mouse 12/
15-LOX, which would preclude the use of small animal stroke
models. Considering that drug discovery relies to a large extent
on rodent models of disease because of availability and cost,
which is compounded by ethical considerations, it is therefore
of great importance to identify bioactive compounds that target
12/15-LOX in both rodents and humans.
Over the last 12 years, our laboratory49−56 and others57−60
have attempted to identify potent and selective human 12/15-
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our cellular mouse model of oxidative stress-related cell death38
and discovered a novel oxazole-4-carbonitrile core scaffold. We
discuss the SAR and biological activity of this novel scaffold,
which has nanomolar potency against human 12/15-LOX,
selectivity over related eicosanoid producing enzymes, and
potency against mouse 12/15-LOX in both a cellular and an in
vivo model of stroke.
The synthesis of compounds 32−40 (Table 3) is outlined in
Scheme 3. Analogues 32−34 were prepared via chlorination of
intermediates 32a−34a with hexachloroethane in the presence
of LDA followed by condensation with methylamine.
Amination of 5-chloro-2-(naphthalen-1-yl)thiazole-4-carbon-
itrile (32b) required microwave heating in a polar solvent,
such as 2-propanol, for complete conversion of the starting
material. The details of the synthesis of intermediate 32a63,64
are described in the Supporting Information. Intermediates 33a
and 34a were synthesized following the literature protocols
described for similar compounds. The reaction of 4-methyl-3-
thiosemicarbazide with 1-naphthoic acid in the presence of
EDC furnished oxadiazole analogue 35, but, surprisingly, when
T3P coupling conditions were used, only thiadiazole analogue
36 was formed. Synthesis of compound 37 was accomplished
utilizing a Cornforth rearrangement of intermediate 37b, which
was prepared from intermediate 37a by coupling with
methylamine, as shown in Scheme 3. Intermediate 37a was
prepared according to a known protocol reported to similar
compounds in the literature.65 The synthesis of analogue 38
from 1-naphthyl boronic acid and intermediate 38a using
classical Suzuki conditions was met with limited success.66
Gratifyingly, the Suzuki coupling reaction was moderately
successful using a silica-bound DPP-Pd catalyst (SiliaCat-DPP-
Pd) under reflux conditions over 24 h. Synthesis of
intermediates 39a−40a was accomplished using a Pd(PPh3)4-
catalyzed microwave-assisted regioselective Suzuki coupling
between 1-naphthyl boronic acid and 2,6-dichloro pyridine or
pyrimidine derivative, respectively. Subsequent condensation of
these intermediates with methylamine furnished desired
analogues 39−40 following HPLC purification.
CHEMISTRY
■
Compounds 1−18 in Table 1 were synthesized as shown in
Scheme 1. Intermediates 2i−18i and 19 were obtained by
a
Scheme 1. Synthetic Route to Compounds 1−18
a
Reagents and conditions: (a) T3P, NEt3, EtOAc, rt, 12 h; (b)
paraformaldehyde, NaOMe, MeOH, 65 °C, 1 h, then NaBH4, rt, 1 h.
propylphosphonic anhydride (T3P)-assisted coupling of the
corresponding commercially available carboxylic acids with 2-
aminomalanonitrile followed by cyclization in one step. This
modified synthesis of 5-amino-2-substituted-1,3-oxazole-4-car-
bonitriles allows facile access to a variety of compounds via a
simple workup and purification from commercially available
carboxylic acids. Because of the low nucleophilicity of the amine
group, our initial attempts to monomethylate the 5-amino-2-
substituted-1,3-oxazole-4-carbonitrile intermediate with several
known methylation methods including methyl iodide, formic
acid, or methyl boronic acid were met with limited success.
However, condensation with paraformaldehyde in the presence
of sodium methoxide in methanol followed by in situ reduction
with sodium borohydride afforded required products 1−18 in
modest yields.
Scheme 2 represents the general methodology utilized for the
synthesis of compounds listed in Table 2. Toward this end, 5-
amino-2-(naphthalen-1-yl)oxazole-4-carbonitrile 19 was con-
verted to an advanced intermediate (19a) via Sandmeyer
reaction by treating with t-butyl nitrite in the presence of
copper(II) chloride. Heating the various amines to reflux with
intermediate 19a in THF afforded required analogues 20−26
and 28−31. Compound 27 was obtained via acetylation of
intermediate 19 with acetic anhydride and cat. DMAP.
RESULTS AND DISCUSSION
■
To investigate structural requirements for optimal 12/15-LOX
inhibition, we conducted a SAR study of the lead molecule, as
shown in Tables 1−3 (1−40). Initially, the 1-naphthyl group
on the left side of the molecule was replaced with various aryl
and heterocyclic groups, as shown in Table 1. The bioisosteric
replacement of 1-naphthyl with 2,3-dichlorophenyl (7) or 3,4-
dichlorophenyl (8) groups showed comparable, albeit slightly
lower, potencies than 1 (IC50 = 0.46, 0.81, and 0.20 μM,
respectively). Replacement with a 2-naphthyl group showed
reduced potency (2, IC50 > 30 μM) compared to the 1-
naphthyl substitution, which, on the basis of space-filling
analysis, agreed with the findings we observed for dichloro
analogues 7 and 8. Several other modifications, including
saturated rings or heterocyclic rings at this region (3−6 and 9−
18), also resulted in reduced potency. Thus, in general, the 1-
naphthyl group appeared to be optimal for 12/15-LOX
inhibition, as variations in size and electrostatics in this region
were not well tolerated. Accordingly, the 1-naphthyl group was
a
Scheme 2. Synthetic Route to Compounds 20−26 and 28−31
a
Reagents and conditions: (a) CuCl2, t-BuNO2, MeCN, 25 °C, 3 h, 35%; (b) THF, reflux, 1−12 h.
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a
Scheme 3. Synthetic Route to Compounds 32−40
a
Reagents and conditions: (a) C2Cl6, LDA, THF, −78 °C, 2−4 h; (b) CH3NH2, i-PrOH, microwave, 120 °C, 0.5 h; (c) CH3NH2, THF, 65 °C, 1−2
h; (d) EDC, DMF, 25 °C, 12 h; (e) T3P, NEt3, DMF, 25 °C, 6 h; (f) (1) (COCl)2, CH2Cl2, (2) CH3NH2, NEt3, CH2Cl2, 25 °C, 1 h; (g) toluene,
110 °C, 12 h; (h) SiliaCat-DPP-Pd, Na2CO3, DME, 105 °C, 24 h; (i) Pd(PPh3)4, Na2CO3, DME, microwave, 150 °C, 45 min.
held constant while other regions of the molecule were
explored for further SAR (Tables 2 and 3).
continued further for the pyridine (39, IC50 > 40 μM) and
pyrimidine analogues (40, IC50 > 40 μM). Attempts to mimic
the interaction of the oxazole core were largely unsuccessful
with the N-methyl/oxadiazole derivative (35, IC50 = 5.4 μM) or
the thiadiazole analogue (36, IC50 > 40 μM). Interchanging the
positions of the 1-naphthyl group and the -NHMe group
(analogues 33−34) also led to complete loss of activity. In
summary, this chemotype showed very tight SAR, and our
efforts to make several changes around the lead molecule
resulted in similar or lower potency, with the exception of
analogues 21−23, which showed marginally improved potency.
Biological Evaluation of ML351. Concurrently with the
SAR investigations against human 12/15-LOX in vitro, we
investigated the potency of 1 against mouse 12/15-LOX,
utilizing a neuronal cell-based assay. Glutamate-induced
oxidative stress67−69 leads to time-dependent cell death
mediated by 12/15-LOX in both immature primary neurons
and mouse hippocampal HT22 cells.38,46,70 Primary neurons
require a lengthy isolation procedure and also show relatively
modest increases in cell death in this assay, indicating that they
are not suited for a high-throughput testing approach. In
contrast, HT22 cells grow quickly and consistently feature low
cell death under control conditions, but a majority of cells die
after glutamate treatment (50−100%). We have now adapted
this assay to 96-well plates, allowing for moderate-to-high
Having explored modifications to the 1-naphthyl moiety, our
next focus was to explore modifications of the N-methyl side
chain at the 5-position, as shown in Table 2 (19−31). Removal
of the methyl group drastically reduced potency (19, IC50 = 25
μM), and dimethylation reduced potency as well (20, IC50 > 30
μM). Modifications of the methyl group with other alkyl
substitutions, such as ethyl, n-propyl, n-butyl, and n-pentyl
groups, were tolerated (21−24), with comparable or even
improved potency being observed. However, replacing the
methyl group with a branched alkyl group (26) or heterocyclic
groups (30−31) significantly reduced the potency. Larger
groups, such as benzyl- (28, IC50 > 30 μM) or phenyl-
substituted analogues (29, IC50 > 30 μM), also showed
diminished 12/15-LOX activity. Overall, these data suggest that
the monoalkylation with straight-chain alkyl groups is critical
for optimal 12/15-LOX inhibition.
Lastly, we turned our attention to optimization of the 1,3-
oxazole core and its various substituents (Table 3). Replacing
the 1,3-oxazole core with a 1,3-thiazole ring (32, IC50 = 0.55
μM) resulted in a 2-fold decrease in potency. This result could
be partly attributed to the difference in size and hardness/
softness of the sulfur and oxygen atoms in the thiazole and
oxazole rings, respectively. The diminished potency trend
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a
Table 1. Variations to Compound 1 (Analogues 2−18)
a
b
IC50 values represent the half-maximal (50%) inhibitory concentration, as determined in the UV−vis cuvette-based assay in triplicate. Compound
possessed low efficacy, with less than 50% maximal inhibition at 25 μM inhibitor.
throughput against the screened compounds. The characteristic
Z′ score used to evaluate the technical quality and suitability of
this approach as a screening tool averaged 0.77 over 10
experiments (SD = 0.13; range = 0.49−0.94), where values
above 0.5 indicate an excellent assay.71
blocked by 12/15-LOX inhibitors that protected HT22 cells
and reduced 12-HETE levels, indicating that both arachidonic
acid metabolites were made by the same enzyme. It is unclear
why this large amount of 15-HETE was generated, and we are
currently working on a different line of research to address this
important result.
To verify that inhibition of oxidative stress-related cell death
in HT22 cells is indeed a good measure of the efficacy of a
given inhibitor against the mouse 12/15-LOX, we decided to
reinvestigate the characteristics of cell injury in this model.
Levels of the mouse 12/15-LOX product, 12-HETE, are known
to be increased in the mouse brain following ischemia,34 and
they are also elevated by glutamate treatment of HT22
cells.34,38 We used HPLC/MS to measure 12-HETE secreted
to the medium from cells incubated under control conditions
or treated with glutamate as well as cotreated with several
compounds with or without inhibitory activity for mouse 12/
15-LOX. Following exclusion of one outlier with extremely
high 12-HETE (but also high levels of cell death), there was a
good correlation between cell death and levels of 12-HETE
secreted: when 12-HETE levels were high, there was increased
cell death, and when 12-HETE levels were low, there was little
cell death (Figure 2A). Interestingly, levels of 15-HETE were
also increased in the medium of glutamate-treated HT22 cells
to a similar extent as 12-HETE (Figure 2B). This was surprising
because mouse 12/15-LOX is generally thought to make only
minor amounts of 15-HETE, typically less than 20% of the 12-
HETE generated. Nonetheless, 15-HETE production was also
Upon testing of compound 1 in HT22 cells, we found a
dose-dependent protection against oxidative glutamate toxicity
(Figure 3A). To verify the on-target efficacy of 1, we also
measured 12-HETE secreted from the cells. The increase in 12-
HETE following glutamate treatment was completely reversed
by incubation in the presence of 10 μM of 1 (Figure 3B). These
results suggest that 1 is capable of reaching its target in the cell
and effectively inhibiting mouse 12/15-LOX, albeit with lower
affinity than seen against human 12/15-LOX in vitro (IC50
=
200 nM).
In addition to analogues that were active in vitro against
human 12/15-LOX, we also tested several inactive analogues
(11, 31, and 34) in HT22 cells, with the goal of identifying a
good negative control (Figure 4). Surprisingly, 11 and 31
featured similar protective qualities at 5 μM, suggesting that
they are able to inhibit the mouse homologue of 12/15-LOX
even though they are inactive against human 12/15-LOX. In
contrast, 5 μM of 34 did not protect HT22 cells, suggesting
that it is inactive against both human and mouse 12/15-LOX
and is a suitable negative control for 1. These data re-emphasize
the importance of screening 12/15-LOX inhibitors against both
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a
a
Table 2. Variations to Compound 1 (Analogues 19−31)
Table 3. Variations to Compound 1 (Analogues 32−40)
a
IC50 values represent the half-maximal (50%) inhibitory concen-
tration, as determined in the UV−vis cuvette-based assay in triplicate.
b
Compound possessed low efficacy, with less than 50% maximal
inhibition at 25 μM inhibitor.
human and mouse 12/15-LOX because activity against one
species does not guarantee activity or inactivity against the
other.
a
IC50 values represent the half-maximal (50%) inhibitory concen-
tration, as determined in the UV−vis cuvette-based assay in triplicate.
Upon the determination that 1 was potent against both in
vitro human 12/15-LOX and ex vivo mouse 12/15-LOX
(HT22 cell assay), we then investigated the selectivity of a few
of our top analogues against related human LOX isozymes (5-
LOX, 12-LOX, and 15-LOX-2). Of the four 12/15-LOX
inhibitors tested, 1, 7, 8, and 32, all displayed excellent
selectivity against all three isozymes (IC50 > 50 μM, Supporting
Information, Table S1). We were encouraged by these findings
because few compounds reported in the literature have
achieved nanomolar potency toward 12/15-LOX while
maintaining excellent selectivity toward other isozymes. More-
over, we investigated whether these analogues inhibited
cyclooxygenase-1 (COX-1) and/or COX-2 and determined
that none of them displayed inhibition (<10% at 15 μM).
Mechanistic Investigations of Compound 1. LOX
inhibitors are known to exhibit a variety of inhibitory
mechanisms, such as chelative, reductive, or competitive. The
UV−vis pseudoperoxidase activity assay was therefore
performed on four selected analogues (1, 7, 8, and 32;
Supporting Information, Table S1) to determine if the
mechanism was reductive in nature. It was observed that the
hydroperoxide product was not degraded, 12/15-LOX was not
irreversibly inhibited, and there was no elongation of the
enzymatic lag phase (data not shown). These data are
b
Compound possessed low efficacy, with less than 50% maximal
inhibition at 25 μM inhibitor.
consistent with a nonreductive inhibitory mechanism. To
investigate the nature of inhibition further, steady-state kinetics
were performed using compound 1 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 yielded
linear plots (Supporting Information, Figure S2), with Ki
equaling 0.1
0.002 μM and Ki′ equaling 1.2
0.02 μM.
These parameters are defined as the equilibrium constants of
dissociation from the catalytic (Ki) and secondary sites (Ki′),
respectively. The Ki value is in good agreement with the IC50
value, and because of the greater than 10-fold difference
between Ki and Ki′, we assume the secondary site to be the
allosteric site,72,73 which is consistent with our previous studies
of 12/15-LOX inhibition.56
In Vitro ADME and in Vivo PK Profile. Our previously
reported small molecule inhibitor for 12/15-LOX inhibition,
ML094, demonstrated excellent potency and selectivity but
lacked sufficient solubility, cell permeability, and microsomal
stability (t1/2 < 2 min).56 Moreover, this compound possessed
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Figure 2. Correlation between arachidonic acid metabolites and cell death. (A) 12-HETE, which is graphed as the percent of 12-HETE secreted
from glutamate-treated cells vs the percent of cell death in glutamate-treated samples. (B) Positive correlation between 15-HETE and cell death.
Figure 3. (A) Protection against glutamate-induced HT22 death by increasing amounts of 1 (** p < 0.01 and *** p < 0.001 vs glutamate only). (B)
Inhibition of 12-HETE in HT22 cells by 10 μM of 1 following treatment with 5 mM glutamate (* p < 0.05 vs glutamate only).
possibly limiting its utility in advanced biological models. Thus,
we were encouraged by the in vitro ADME (Table 4) and in
vivo PK properties (Table 5) of 1, as they represent a vast
improvement over the majority of compounds reported
previously (vide infra). The majority of the compounds possess
a low molecular weight (e.g., 249 Da) and a favorable log D
(pH 7.4) between 2 and 3, which was obtained by Analiza Inc.
using their scaled-down shake flask lipophilicity method, yet
most analogues have poor solubility. The aqueous kinetic
solubility in PBS buffer (pH 7.4) was determined to be 1.2 μM,
which is about 7 times the in vitro IC50 value. Empirically, we
did observe a vast improvement in the solubility in the 15-LOX
assay buffer (data not shown), which was encouraging and
suggests that solubility was not a detrimental factor in the
biochemical studies. Importantly, the compound demonstrated
favorable PAMPA permeability (passive) and acceptable Caco-
2 permeability of >1 (1.5 cm/s−6), with no evidence of efflux
(efflux ratio: 0.7), indicating that the compound is not
susceptible to the action of P-glycoprotein 1 (Pgp), a well-
characterized ABC transporter. Moreover, 1 was stable in
various aqueous solutions (pH 2, 7.4, and 9) (see Supporting
Figure 4. Cellular protection at 5 μM of 1 and some analogues in
HT22 cells. Despite not inhibiting human 12/15-LOX, 11 and 31
show similar protection to 1, indicating that they inhibit the mouse
enzyme, whereas 34 does not protect HT22 cells.
an essential ester moiety that could be susceptible to
intracellular and plasma esterases, rendering it inactive and
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a
Table 4. ADME Profile for 1
kinetic
CYP 2D6
CYP 3A4
solubility
microsomal stability
inhibition at
inhibition at
mouse plasma stability
remaining at 2 h
b
c
d
e
compound
(μM)
t1/2 (min)
3 μM
3 μM
permeability (10−6 cm/s)
723 (PAMPA) 1.5 (Caco-2)
1
1.2
18 (rat) 5.5 (mouse)
10.30%
3.50%
100%
a
b
c
All experiments were conducted at Pharmaron Inc. In PBS buffer (pH 7.4). Represents the stability in the presence of NADPH. The probe
d
e
compound showed no degradation without NADPH present over a 1 h period. Dextromethorphan was used as the substrate. Midazolam was used
as the substrate.
a
Table 5. In Vivo PK (Mouse) of 1 at 30 mg/kg IP
b
c
compound
tissue
t1/2 (h)
tmax (h)
Cmax (μM)
AUCinf (μM h)
brain/plasma
2.8
1
plasma
brain
1.1
1
0.25
0.5
13. 8
28.8
13
35.5
a
All experiments were conducted at Pharmaron Inc. using male CD1 mice (6−8 weeks of age). Data was collected in triplicate at eight time points
b
c
over a 24 h period. Formulated as a solution (10% Solutol, 10% Cremophor EL, and 20% PEG 400 in saline). Brain/plasma ratio [AUClast(brain)/
AUClast(plasma)].
Information) and mouse plasma (Table 4). In addition, 1
exhibited minimal CYP inhibition of the 2D6 and 3A4 isoforms
at 10.3 and 3.5% inhibition, respectively. Microsomal stability
appears to be species-dependent, with 1 possessing modest
stability to rat liver microsomes (18 min) while being less stable
to mouse liver microsomes (5.5 min). The compounds were
completely stable in the absence of NADPH, suggesting CYP-
mediated degradation. Given our plan to test compound 1 in
proof-of-concept (POC) mouse models of stroke, we also
obtained in vivo PK data on 1 (Table 5) and found a suitable
formulation for 1 (10% Solutol, 10% Cremophor EL, and 20%
PEG400 in saline). As anticipated from the microsomal stability
studies, 1 has a fast half-life in both plasma and brain (t1/2 ∼ 1
h), with a Cmax of 13.8 μM in plasma and 28.8 μM in brain.
Figure 5. (A) Activity of compound 1 (50 mg/kg) administered IP in
a mouse distal middle cerebral artery occlusion (MCAO) model of
permanent focal ischemia (** p < 0.01). (B) Typical examples of
sequential (top to bottom represents front to back sections of a single
brain, respectively) TTC-stained brain sections from vehicle- and
compound 1-treated mice. The white areas in the cortex (top right)
Encouragingly, 1 has a brain/plasma ratio of 2.8, which
demonstrates favorable blood−brain barrier permeability and
suggests that this compound is suitable for in vivo POC models
of ischemic stroke (vide infra).
Cell Activity and in Vivo Efficacy. After establishing the
SAR profile against recombinant human 12/15-LOX, determin-
ing the efficacy of 1 in our cellular mouse 12/15-LOX assay,
and measuring its in vitro ADME/in vivo PK properties, we
then sought to determine its efficacy in mouse models of
ischemic stroke. For these initial studies, we chose the
permanent focal ischemia model in mice, which has been
shown to mimic the pathophysiological mechanisms following
ischemic injury.74 The advantage of this model is that it does
not inflict surgical trauma and has a low mortality compared to
other methods. The thrombosis event in the middle cerebral
artery (MCA) is induced by topical application of FeCl3 to the
intact dura mater, leading to a cortical infarct. Laser doppler
flowmetry was used to monitor blood flow reduction, and
infarct size was measured following sacrifice at 24 h by staining
of 1 mm thick brain slices with 2,3,5-triphenylterazolium
hydrochloride (TTC). IP administration of 1 given 2 h after the
induced ischemia resulted in a ∼30% reduction in infarct size (p
< 0.01), demonstrating efficient neuroprotection in this mouse
model of permanent focal ischemia (Figure 5).
indicate nonviable infarcted tissue.
gradually increases over time after experimental stroke,47
providing an opportunity for therapeutic intervention. Our
initial efforts to identify a stroke-relevant 12/15-LOX inhibitor
proved to be unsuccessful because of its inactivity in our mouse
ex vivo model, resulting from either cellular inactivation or
inactivity against mouse 12/15-LOX.56 Fortunately, reinterro-
gation of the HTS data along with simultaneous cellular mouse
12/15-LOX screening revealed another 12/15-LOX inhibitor
(1) with better drug-like properties and, more importantly,
mouse ex vivo activity. Our initial medicinal chemistry efforts,
described herein, provided important SAR to guide future
investigations, but we were unable to improve potency
significantly, and the tolerability for the structural changes is
limited. The most significant loss of inhibition was observed at
the 1-naphthyl position, where almost all substitutions resulted
in significant loss in activity, with the exception of its
bioisosteric dichloro analogues (7 and 8), which only showed
a slight decrease in activity. Modifications of the methyl group
on the N-methyl side chain were not well tolerated either,
indicating that the straight-chain monoalkylated amine was
critical for potency. Moreover, the only substitution tolerated
for 1,3 oxazole core ring was the replacement with a 1,3-
thiazole (32), which resulted in a 2-fold decrease in the
potency. The cyanide group on the oxazole core was found to
CONCLUSIONS
■
12/15-LOX contributes to neuronal cell death in oxidative
stress models38−44 and is also detrimental not only to neurons
but also to the brain vasculature after stroke45 via intracellular
attack on mitochondria and translocation of AIF to the
nucleus.46−48 Moreover, the protein level of 12/15-LOX
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an Agilent diode array detector for methods 1−3. Mass determination
be essential, with modifications to this group completely
abolishing activity. Subsequent selectivity studies of 1 and
related analogues revealed that these compounds have minimal
activity toward 5-LOX, 12-LOX, 15-LOX-2, COX-1, and COX-
2.
was performed using an Agilent 6130 mass spectrometer with
1
electrospray ionization in the positive mode. H NMR spectra were
recorded on Varian 400 MHz spectrometers. Chemical shifts are
reported in ppm, with undeuterated solvent (DMSO-d6 at 2.49 ppm)
as internal standard for DMSO-d6 solutions. All of the analogues tested
in the biological assays have a purity greater than 95% on the basis of
both analytical methods. High-resolution mass spectrometry was
recorded on Agilent 6210 time-of-flight LC/MS system. Confirmation
of molecular formula was accomplished using electrospray ionization
in the positive mode with the Agilent Masshunter software (version
B.02).
Experimental Procedures. General Procedure for the Prepara-
tion of 5-Amino-2-substituted Oxazole-4-carbonitrile Intermediates
(1i−18i and 19). A mixture of carboxylic acid (5.77 mmol, 1 equiv)
and 2-aminomalononitrile·TsOH (5.77 mmol, 1 equiv) in ethyl acetate
(15 mL) was added NEt3 (17.32 mmol, 3 equiv) followed by a 50%
solution of T3P in ethyl acetate (14.44 mmol, 2.5 equiv). The reaction
was allowed to stir at room temperature for 12 h and was then diluted
with ethyl acetate. The organic layer was successively washed with
water, saturated bicarbonate solution, and brine. The organic layer was
then dried with MgSO4 and concentrated under reduced pressure. The
crude product was purified on a biotage flash system, eluting with 50−
100% ethyl acetate in hexanes containing 0.1% triethylamine to
provide pure products.
General Procedure for the Preparation of 2-Substituted-5-
(methylamino)oxazole-4-carbonitrile (1−18). A mixture of 5-
amino-2-substituted oxazole-4-carbonitrile (1.79 mmol, 1 equiv),
paraformaldehyde (0.11 g, 3.57 mmol, 2 equiv), and sodium
methoxide (0.096 g, 1.79 mmol, 1 equiv) in methanol (10 mL) was
stirred at 65 °C for 1 h until a clear mixture was obtained. The reaction
mixture was cooled to room temperature, sodium borohydride (3.57
mmol, 2 equiv) was added slowly, and the mixture was stirred further
at room temperature for 1 h. The crude product was extracted with
ethyl acetate and successively washed with water and brine. The ethyl
acetate layer was dried with MgSO4 and concentrated under reduced
pressure. The crude product was taken up in DMSO and purified on a
reversed-phase HPLC system. Characterization data for key
compounds is given below (see Supporting Information for details
and characterization data for all other compounds)
5-(Methylamino)-2-(naphthalen-1-yl)oxazole-4-carbonitrile (1,
ML351). This compound was prepared starting from 1-naphthoic
acid and aminomalononitrile via intermediate 19 following the above
general procedure. LC-MS retention time: (method 1) 6.011 and
(method 2) 2.42 min. 1H NMR (400 MHz, DMSO-d6) δ 9.15 (dq, J =
8.7 and 0.9 Hz, 1H), 8.44 (brs, 1H), 8.10−7.99 (m, 3H), 7.74−7.57
(m, 3H), 3.07−3.01 (m, 3H). 13C NMR (400 MHz, DMSO-d6) δ
161.9, 161.9, 149.5, 134.1, 134.0, 131.2, 129.2, 128.3, 127.2, 127.2,
127.0, 126.9, 125.8, 125.8, 125.8, 125.7, 122.1, 116.5, 116.5, 84.1, 84.1,
84.1, 29.7, 29.7, 29.6. HRMS (ESI): m/z (M + H)+ calcd for
C15H12N3O, 250.0975; found, 250.0975.
Most importantly, 1 was active in both protecting mouse
neuronal cells (HT22) and in reducing the infarct size of
stroke-induced mice. This is a significant discovery because
identifying therapeutically useful specific inhibitors of 12/15-
LOX is not trivial due to the differences between the human
and mouse 12/15-LOX isozymes. Interestingly, we have
identified analogues of compound 1 (11 and 31) that did not
inhibit human 12/15-LOX but protected HT22 cells against
oxidative stress. These might work in mice to protect against
experimental stroke, yet in human stroke, they would most
likely fail because of their inability to target human 12/15-LOX.
The lesson to be drawn from this is that simply because a given
inhibitor is selective for a LOX isoform in one species its
efficacy cannot be readily extrapolated to other species.
Moreover, the LOX isotype involved in a given process cannot
be reliably inferred from inhibitor experiments without taking
into account the species from which the cells were derived.
In conclusion, compound 1 is the first highly selective 12/15-
LOX inhibitor active against human 12/15-LOX and mouse
12/15-LOX, as seen by its effectiveness in our mouse stroke
model. This attribute of 1 is significant because it maintains the
human 12/15-LOX selectivity, which is beneficial to a human
therapeutic, but is also functional in a mouse stroke model,
which is critical to developing its biological efficacy. Therefore,
using our pipeline approach, we have identified a potent and
selective inhibitor of human 12/15-LOX that is neuroprotective
in a mouse model of stroke and therefore can be utilized in
preclinical studies to develop it as a potential first-line stroke
therapy.
EXPERIMENTAL SECTION
■
General Methods for Chemistry. All air- or moisture-sensitive
reactions were performed under positive pressure of nitrogen with
oven-dried glassware. Anhydrous solvents such as dichloromethane,
N,N-dimethylforamide (DMF), acetonitrile, dioxan, dimethoxyethane,
methanol, and triethylamine were purchased from Sigma-Aldrich.
Tetrakis and a 50% of solution of T3P in DMF or ethyl acetate were
purchased from Strem Chemicals and used as such. SiliaCat
heterogeneous catalyst DPP-Pd (catalog no. R390-100) was purchased
from SiliCycle Inc. Preparative purification was performed on a Waters
semipreparative HPLC system using a Phenomenex Luna C18 column
(5 μm, 30 × 75 mm) at a flow rate of 45 mL/min. The mobile phase
consisted of acetonitrile and water (each containing 0.1% trifluoro-
acetic acid). A gradient of 10−50% acetonitrile over 8 min was used
during the purification. Fraction collection was triggered by UV
detection (220 nm). Analytical analysis was performed on an Agilent
LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7 min
gradient of 4−100% acetonitrile (containing 0.025% trifluoroacetic
acid) in water (containing 0.05% trifluoroacetic acid) was used with an
8 min run time at a flow rate of 1 mL/min. A Phenomenex Luna C18
column (3 μm, 3 × 75 mm) was used at a temperature of 50 °C.
Method 2: A 3 min gradient of 4−100% acetonitrile (containing
0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic
acid) was used with a 4.5 min run time at a flow rate of 1 mL/min. A
Phenomenex Gemini Phenyl column (3 μm, 3 × 100 mm) was used at
a temperature of 50 °C. Method 3: Analysis was performed on an
Agilent 1290 Infinity Series HPLC. UHPLC long gradient equivalent
4−100% acetonitrile (0.05% trifluoroacetic acid) in water was used
over 3.5 min with a run time of 4 min and a flow rate of 0.8 mL/min.
A Phenomenex Kinetex 1.7 μm C18 column (2.1 × 100 mm) was used
at a temperature of 50 °C. Purity determination was performed using
2-(2,3-Dichlorophenyl)-5-(methylamino)oxazole-4-carbonitrile
TFA (7). This compound was prepared starting from 2,3-benzoic acid
and aminomalononitrile via intermediate 7i following the above
general procedure. LC-MS retention time: (method 3) 2.639 and
(method 2) = 3.559 min. 1H NMR (400 MHz, DMSO-d6) δ 8.50 (q, J
= 4.9 Hz, 1H), 7.83 (ddd, J = 7.9, 1.5, 0.4 Hz, 1H), 7.78 (ddd, J = 8.1,
1.6, 0.4 Hz, 1H), 7.54−7.46 (m, 1H), 2.99 (d, J = 4.9 Hz, 3H). HRMS
(ESI): m/z (M + H)+ calcd for C11H8Cl2N3O, 269.0039; found,
269.0031.
2-(3,4-Dichlorophenyl)-5-(methylamino)oxazole-4-carbonitrile
TFA (8). This compound was prepared starting from 3,4-benzoic acid
and aminomalononitrile via intermediate 8i following the above
1
general procedure. LC-MS retention time: (method 2) 3.737 min. H
NMR (400 MHz, DMSO-d6) δ 8.48 (q, J = 4.9 Hz, 1H), 7.95 (td, J =
1.8, 0.7 Hz, 1H), 7.80−7.72 (m, 2H), 3.09−2.91 (m, 3H). HRMS
(ESI): m/z (M + H)+ calcd for C11H8Cl2N3O, 269.0039; found,
269.0043.
2-([1,1′-Biphenyl]-4-yl)-5-(methylamino)oxazole-4-carbonitrile
TFA (11). This compound was prepared starting from 4-phenylbenzoic
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acid and aminomalononitrile via intermediate 11i following the above
general procedure. LC-MS retention time: (method 1) 6.215 and
(method 2) 3.708 min, 1H NMR (400 MHz, DMSO-d6) δ 8.38 (q, J =
4.9 Hz, 1H), 7.92−7.79 (m, 4H), 7.76−7.68 (m, 2H), 7.53−7.46 (m,
2H), 7.43−7.37 (m, 1H), 3.01 (dd, J = 4.8, 0.6 Hz, 3H). HRMS (ESI):
m/z (M + H)+ calcd for C17H14N3O, 276.1131; found, 276.1143.
Synthesis of 5-(azetidin-3-ylamino)-2-(naphthalen-1-yl)oxazole-
4-carbonitrile TFA (31). A mixture of 5-chloro-2-(naphthalen-1-
yl)oxazole-4-carbonitrile (19a) (details of the synthesis are provided in
the Supporting Information) (0.25 mmol, 1 equiv) and 1-boc-3-
(amino)azetidine (1 mmol, 4 equiv) in THF was heated to reflux for
0.5 h. The reaction mixture was then cooled, and the solvent was
removed by forced air. The crude product was taken up in DMSO and
purified via reversed-phase preparative HPLC. This pure product was
suspended in dichloromethane (2 mL) and treated with TFA (0.5
mL). After stirring for 0.5 h at room temperature, the volatiles were
removed by forced air. The crude product was taken up in DMSO and
purified on a preparative HPLC to give pure product (31) as its TFA
salt. LC-MS retention time: (method 1) 4.078 and (method 2) 3.812
min, 1H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H), 9.12 (d, J = 8.6
Hz, 1H), 8.90 (s, 1H), 8.14−8.01 (m, 2H), 7.75−7.59 (m, 3H), 4.77
(p, J = 7.0, 6.0 Hz, 1H), 4.32−4.18 (m, 2H), 4.18−4.08 (m, 2H),
HRMS (ESI): m/z (M + H)+ calcd for C17H15N4O, 291.1252; found,
291.125.
Synthesis of (methylamino)-2-(naphthalen-1-yl)thiazole-4-car-
bonitrile TFA (32). A mixture of 2-bromothiazole-4-carbonitrile
(1.85 g, 9.79 mmol, 1 equiv), naphthalen-1-ylboronic acid (2.52 g,
14.68 mmol, 1.5 equiv), 2 M sodium carbonate (12.23 mL, 24.47
mmol, 2.5 equiv), and Pd(PPh3)4 (1.11 g, 0.979 mmol, 10 mol %) in
dimethoxy ethane (20 mL) was degassed with argon and then heated
under microwave for 45 min at 150 °C. The reaction mixture was
concentrated, taken up in dichloromethane, stirred with palladium
scavenger resin, and filtered through Celite. The crude product
obtained after evaporating the solvent was purified on a biotage flash
system, eluting with 10% ethyl acetate in hexanes to obtain 1.52 g
(yield: 66%) of 2-(naphthalen-1-yl)thiazole-4-carbonitrile (32a) as a
white solid.
(1.064 g, 4.5 mmol, 1.1 equiv) was added in one portion, and the
mixture was allowed to reach room temperature over 4 h. The reaction
mixture was quenched with saturated ammonium chloride and
extracted with ethyl acetate. The organic layer was washed with
water and brine, dried over magnesium sulfate, filtered, and
concentrated under diminished pressure. The crude product was
purified on a Biotage flash system, eluting with 10% ethyl acetate in
hexanes to furnish 0.75 g (yield: 72%) of 2-chloro-5-(naphthalen-1-
yl)oxazole-4-carbonitrile 34b as a white solid.
A solution of 2-chloro-5-(naphthalen-1-yl)oxazole-4-carbonitrile
34b (0.1 g, 0.393 mmol, 1 equiv) in THF (1 mL) was added a 2 M
solution of methanamine in THF (1.0 mL, 1.98 mmol, 5 equiv) and
stirred at 65 °C for 1 h in a sealed tube. The solvent was removed by
forced air to obtain a crude solid. This crude product was taken up in
DMSO and purified on a preparative HPLC to obtain pure product
(34) as its TFA salt. LC-MS retention time: (method 1) 5.509 and
1
(method 2) 3.498 min, H NMR (400 MHz, DMSO-d6) δ 8.17−7.96
(m, 4H), 7.81 (dd, J = 7.3, 1.3 Hz, 1H), 7.71−7.59 (m, 3H), 2.88 (d, J
= 4.8 Hz, 3H), HRMS (ESI): m/z (M + H)+ calcd for C15H12N3O,
250.0975; found, 250.0979.
Materials and Methods. Materials. Different commercial fatty
acids as lipoxygenase substrates, were purchased from Nu Chek Prep,
Inc. (MN, USA). The fatty acids were further repurified using a
Higgins HAISIL (5 μm, 250 × 10 mm) C-18 column. An isocratic
elution of 85% solvent A (99.9% methanol and 0.1% acetic acid)/15%
solvent B (99.9% water and 0.1% acetic acid) was used to purify all the
fatty acids. Post purification, the fatty acids were stored at −80 °C for a
maximum of 6 months. Lipoxygenase product 13-(S)-HPODE was
generated by reacting LA with soybean LOX-1. The product
generation protocol involved reacting 50 μM substrate in 500 mL of
100 mM borate buffer, pH 9.2, with soybean LOX-1. A small sample
from the larger reaction was monitored on the UV−vis spectrometer
until complete turnover was observed. The products were then
extracted using dichloromethane, reduced with trimethylphosphite,
evaporated to dryness, and reconstituted in methanol. The products
were HPLC-purified using an isocratic elution of 75% A (99.9%
methanol and 0.1% acetic acid)/25% B (99.9% water and 0.1% acetic
acid). The products were tested for their purity using LC−MS/MS
and were found to be >98% pure. Ovine COX-1 (catalog no. 60100)
and human COX-2 (catalog no. 60122) were purchased from Cayman
chemicals. All other chemicals were of high quality and used without
further purification.
A solution of the above 2-(naphthalen-1-yl)thiazole-4-carbonitrile
32a (0.5 g, 2.12 mmol, 1 equiv) in THF (10 mL) was added a 2 M
THF solution of LDA (1.16 mL, 2.33 mmol, 1.1 equiv) dropwise at
−78 °C. After stirring for 30 min at −78 °C, perchloroethane (0.551 g,
2.33 mmol, 1.1 equiv) was added in one portion and allowed to warm
to room temperature over 4 h. The reaction was then quenched with
saturated ammonium chloride and extracted with ethyl acetate. The
organic layer was washed with water and brine. The ethyl acetate layer
was subsequently dried over MgSO4 and filtered. The crude product
obtained after concentrating under diminished pressure was purified
on a Biotage flash system, eluting with 50% CH2Cl2 in hexanes to
furnish 4.5 g (yield: 79%) of 5-chloro-2-(naphthalen-1-yl)thiazole-4-
High-Throughput Screen. See Supporting Information.
Overexpression and Purification of Lipoxygenases. Different
lipoxygenases, such as human reticulocyte 15-lipoxygenase-1 (12/15-
LOX), human epithelial 15-lipoxygenase-2 (15-LOX-2), and human
platelet 12-lipoxygenase (12-LOX), were expressed as N-terminal
His6-tagged proteins and were purified via immobilized metal-affinity
chromatography using Ni-NTA resins for 12/15-LOX and 15-LOX-2,
whereas Ni-IDA resin was used for 12-LOX.75,76 The protein purity
was evaluated by SDS-PAGE analysis and was found to be greater than
90%. Human 5-lipoxygenase (5-LOX) was expressed as a nontagged
protein and used as a crude ammonium sulfate protein fraction, as
published previously.77
Lipoxygenase UV−Vis Assay. The inhibitor compounds were
screened initially using one concentration point on a PerkinElmer
Lambda 40 UV−vis spectrometer. The percent inhibition was
determined by comparing the enzyme rates of the control (DMSO
solvent) and the inhibitor sample by following the formation of the
conjugated diene product at 234 nm (ε = 25 000 M−1 cm−1). The
reactions were initiated by adding either ∼40 nM 12-LOX, 40 nM 12/
15-LOX, 0.5 μM 15-LOX-2, or ∼200 nM 5-LOX (ammonium sulfate
suspension) to a cuvette with a 2 mL of reaction buffer that was
constantly stirred using a magnetic stirrer bar at room temperature (22
°C). It should be noted that LOX isozymes are often expressed in the
inactive demetalated form, so it is best to utilize activity to determine
the optimal LOX concentration for the assay (optimal rate of
approximately 0.001 abs/s at 10 μM AA). Reaction buffers used for
various lipoxygenase were as follows: 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
1
carbonitrile 32b as a white solid. H NMR (400 MHz, DMSO-d6) δ
8.67 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.3 Hz, 1H), 8.13−8.05 (m, 1H),
7.94 (dd, J = 7.3, 1.3 Hz, 1H), 7.69 (dddd, J = 14.7, 9.3, 6.9, 1.7 Hz,
3H).
A mixture of 5-chloro-2-(naphthalen-1-yl)thiazole-4-carbonitrile
32b (0.2 g, 0.739 mmol, 1 equiv) and a 2 M THF solution of
methylamine (1.9 mL, 3.69 mmol, 5 equiv) in 2-propanol (1 mL) was
heated via microwave for 30 min at 120 °C. The crude product
obtained after evaporation of the solvent was taken up in DMSO and
purified by reversed-phase preparative HPLC to obtain 5-(methyl-
amino)-2-(naphthalen-1-yl)thiazole-4-carbonitrile (32) as its TFA salt.
LC-MS retention time: (method 3) 2.814 and (method 2) 3.756 min,
1H NMR (400 MHz, DMSO-d6) δ 8.88 (ddt, J = 8.5, 1.4, 0.8 Hz, 1H),
8.17 (q, J = 4.7 Hz, 1H), 8.07−7.98 (m, 2H), 7.77 (dd, J = 7.2, 1.2 Hz,
1H), 7.71−7.53 (m, 3H), 3.01 (d, J = 4.7 Hz, 3H), HRMS (ESI): m/z
(M + H)+ calcd for C15H12N3S, 266.0754; found, 266.0746.
Synthesis of N-methyl-5-(naphthalen-1-yl)oxazol-2-amine (34).
A solution of 5-(naphthalen-1-yl)oxazole-4-carbonitrile 34a (0.9 g,
4.09 mmol, 1 equiv) in THF (15 mL) was added a 1 M THF solution
of LHMDS (4.5 mL, 4.5 mmol, 1.1 equiv) dropwise at −78 °C. The
reaction mixture was stirred for 30 min at −78 °C, perchloroethane
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μM AA for the crude, ammonium sulfate-precipitated 5-LOX; 25 mM
Hepes (pH 8), 0.01% Triton X-100, and 10 μM AA for 12-LOX, and
25 mM Hepes buffer (pH 7.5), 0.01% Triton X-100, and 10 μM AA
for 12/15-LOX and 15-LOX-2. The substrate concentration was
quantitatively determined by allowing the enzymatic reaction to go to
completion in the presence of 15-LOX-2. For the inhibitors that
showed more than 50% inhibition at the one-point screens (25 μM
inhibitor), IC50 values were obtained by determining the enzymatic
rate at various inhibitor concentrations and were plotted against
inhibitor concentration (approximate range: 0.1−25 μM inhibitor)
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 it is otherwise stated in the tables. Inhibitors were stored at −20
°C in DMSO.
Pseudoperoxidase Assay. The pseudoperoxidase activity rates were
determined with BWb70c as the positive control, 13-(S)-HPODE as
the oxidizing product. and 12/15-LOX on a PerkinElmer Lambda 40
UV−vis spectrometer, as described previously.78 Briefly, activity was
determined by monitoring the decrease at 234 nm (product
degradation) in buffer (50 mM sodium phosphate (pH 7.4), 0.3
mM CaCl2, 0.1 mM EDTA, 0.01% Triton X100, and 20 μM 13-(S)-
HPODE). 12/15-LOX was added to buffer (22 °C), and the reaction
initiated by addition of 20 μM inhibitor (1:1 ratio to product). The
percent consumption of 13-(S)-HPODE was recorded, with individual
controls being conducted with inhibitor alone with product and
enzyme alone with product.
Steady-State Inhibition Kinetics. The steady-state kinetics experi-
ments were performed with the parent analogue, compound 1
(ML351), to determine the mode of inhibition, as described
previously.56,79 The inhibitor concentrations of 0, 0.05, 2, and 5 μM
were used. Reactions were initiated by adding approximately 40−60
nM 12/15-LOX to a constantly stirring 2 mL cuvette containing 1−20
μM AA in 25 mM HEPES buffer (pH 7.5) in the presence of 0.01%
Triton X-100. Lipoxygenase rates were determined by monitoring the
formation of the conjugated product, 15-HpETE, at 234 nm (ε = 25
000 M−1 cm−1) with a PerkinElmer Lambda 40 UV−vis spectrometer.
The substrate concentration was quantitatively determined by allowing
the enzymatic reaction to proceed to completion using 15-LOX-2.
Kinetic data were obtained by recording initial enzymatic rates at
varied substrate and inhibitor concentrations, and the data were
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 with 1/Vmax and Km/Vmax versus
inhibitor concentration, yielding Ki′ and Ki, respectively.
Detection Kit (Roche), and the percentage of LDH released to the
medium was calculated after subtracting the corresponding back-
ground value. To determine levels of the 12/15-LOX metabolite, 12-
HETE, we cultured HT22 cells in 75 cm2 flasks in DMEM medium
without phenol red that was supplemented with 5% FBS, and the cells
were treated the next day when they were 50−70% confluent. Twenty-
four hours later, the eicosanoid-containing fraction was isolated via
Sep-Pak C-18 column, and 12-HETE was detected with a 12-HETE
ELISA kit (Enzo Life Sciences), which was used according to the
manufacturer’s instructions. Three independent experiments were
evaluated. For the MS samples, the above procedure was modified
slightly. The eicosanoids containing fractions were transferred to
scintillation vials followed by addition of perdeuterated 13-HODE (13-
d31-HODE) as an internal control for extraction and 1.5% glacial acetic
acid for protein precipitation. The samples were extracted with
methylene chloride, reduced with trimethylphosphite, and evaporated
to dryness. The dry samples were then reconstituted in methanol, and
an internal control 12-deuterated (d8)-HETE (12-d8-HETE) was
added to each sample for detector response variation; it was assumed
that the change in detector response for 12-HETE and 12-d8-HETE
would be similar. The samples were run on and analyzed by a Finnigan
LTQ liquid chromatography−tandem mass spectrometry (LC−MS/
MS) system. 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 a 0.2 mL/min flow rate and a linear gradient
from 54.9% ACN, 45% H2O, and 0.1% THF to 69.9% ACN, 30%
H2O, and 0.1% THF. The electrospray voltage was set to 5.0 kV and a
global acquisition MS mode was used. The MS−MS scan was
performed for the five most abundant precursor ions. The collision-
induced dissociation was used for MS−MS with a collision energy of
35 eV. The corresponding 12-HETE, 12-d8-HETE, and 13-d31-HODE
compounds were detected using selective ion monitoring analysis (m/
z = 318.7−319.7, 326.8−327.7, and 325.8−326.8, respectively) 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 13-
d31-HODE, parent ion m/z 326 and fragments at m/z 213) from MS−
MS. The peak area of 12-d8-HETE for each sample was normalized to
the area of 13-d31-HODE. The peak intensities of 12-HETEs were
normalized to the corrected 12-d8-HETE intensities. The amount of
12-HETE in samples was estimated from the pure 12-(S)-HETE
standard curve.
Distal MCAO Model of Permanent Focal Ischemia in Mice. To
study 1 in a model of distal MCAO,74 C57Bl6J mice were treated with
ferric chloride (FeCl3) to cause occlusion of the distal middle cerebral
artery. Mice were kept under anesthesia with 1.5% isoflurane in a
nitrous oxide/oxygen mixture via facemask. The body temperature was
Cyclooxygenase Assay. About 2−5 μg of either COX-1 or COX-2
were added to buffer containing 0.1 M Tris-HCl buffer (pH 8.0), 5
mM EDTA, 2 mM phenol, and 1 μM hematin at 37 °C. The inhibitors
were added to the reaction cell followed by an incubation of 5 min
with either of the COX enzymes. The reaction was then initiated by
adding 100 μM AA in the reaction cell. Data was collected using a
Hansatech DW1 oxygen electrode, and the consumption of oxygen
was recorded. Indomethacin and the solvent, DMSO, were used as
positive and negative controls, respectively, and the percent inhibition
of the enzyme was calculated by comparing the rates from samples and
the controls.
HT22 Cell Culture Assay. Glutathione depletion was induced in
HT22 cells by glutamate treatment, and LDH release into the medium
was measured to detect cell death, as described previously.38 Briefly,
HT22 cells were cultured in DMEM containing 10% fetal bovine
serum and penicillin/streptomycin (all media from Invitrogen). For
viability experiments, cells were seeded at 1 × 104 cells/well in 96-well
plates (Corning) and were treated 18 h later when the cells were
approximately 50−70% confluent. Treatment consisted of exchanging
the medium to 100 μL of fresh culturing medium and adding 5 mM
glutamate (stock solution 0.5 M in PBS) in the presence or absence of
DMSO (maximum 0.1% final concentration) as control or the
indicated concentrations of 1. LDH content was determined separately
for the cell extracts and corresponding media using a Cytotoxicity
monitored by a rectal probe and maintained at 37
0.3 °C by a
homeothermic blanket control unit. Briefly, mice were placed in a
stereotaxic frame, the scalp was opened, and the right temporal muscle
was dissected. The area between zygomatic arch and squamous bone
was thinned by a high-speed drill and cooled with saline. The trace of
MCA was visualized, and the thin bony film was lifted up by forceps.
After that, a laser doppler flowmetry probe was placed 2 mm posterior
and 6 mm lateral to the bregma to monitor the regional cerebral blood
flow (rCBF). After obtaining a stable epoch of the preischemic rCBF, a
piece of 10% FeCl3-saturated filter paper was placed over the intact
dura mater along the trace of MCA, and the rCBF was continuously
monitored during the next 3 h. After 2 h of ischemia, either 50 mg/kg
of 1 or DMSO vehicle was injected intraperitoneally. Following
sacrifice at 24 h, brains were sectioned into 1 mm slices, and infarct
sizes were determined by staining with TTC using the indirect method
(infarct volume = contralateral volume minus uninfarcted ipsilateral
volume). Both the surgeon carrying out the procedure and the
researcher determining infarct volumes were blinded as to which
treatment the mice received.
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Article
N-benzyl-triethylammonium chloride; BWB70C, N-[3-[3-
(fluorophenoxy)phenyl]-1-methyl-2-propenyl]-N-hydroxyurea;
LDA, lithium diisopropylamide
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional experimental procedures; spectroscopic (1H), LC/
MS, and HRMS data for representative compounds; and qHTS
assay performance. This material is available free of charge via
the Internet at http://pubs.acs.org.
REFERENCES
■
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from Spectroscopy into the Structure/Function Relationships of
Lipoxygenases. Chem. Biol. 1997, 4, 795−808.
AUTHOR INFORMATION
Corresponding Authors
*(K.v.L.) Tel: 617-724-6556; Fax: 617-726-7830; E-mail:
klaus_vanleyen@hms.harvard.edu.
*(D.J.M.) Tel: 301-217-4381; Fax: 301-217-5736; E-mail:
maloneyd@mail.nih.gov.
*(T.R.H.) Tel: 831-459-5884; Fax: 831-459-2935; E-mail:
tholman@chemistry.ucsc.edu.
■
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Author Contributions
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Sam Michael and Richard Jones for automation
support; Paul Shinn and Danielle van Leer for assistance with
compound management; William Leister, Heather Baker, James
Bougie, Elizabeth Fernandez, and Christopher Leclair for
analytical chemistry and purification support; Kimloan Nguyen
for in vitro ADME data; and Eric Hoobler for helping with the
COX assays. Financial support was from the National Institute
of Health (grants R01 GM56062 (T.R.H.), R01 NS049430
(K.v.L.), and R01 NS069939) and the Molecular Libraries
Initiative of the National Institutes of Health Roadmap for
Medical Research (R03 MH081283 (T.R.H.)). G.R., L.S., A.Y.,
A.J., A.S., and D.J.M. were supported by the intramural research
program of the National Center for Advancing Translational
Sciences and the Molecular Libraries Initiative of the National
Institutes of Health Roadmap for Medical Research
(U54MH084681). 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.).
ABBREVIATIONS USED
■
LOX, lipoxygenase; sLOX-1, soybean lipoxygenase-1; 12/15-
LOX, human reticulocyte 15-lipoxygenase-1; 15-LOX-1, human
reticulocyte 15-lipoxygenase-1; 15-LOX-2, human epithelial 15-
lipoxygenase-2; 12-LOX, human platelet 12-lipoxygenase;
rabbit 12/15-LOX, rabbit reticulocyte 12/15-lipoxygenase;
mouse 12/15-LOX, mouse reticulocyte 12/15-lipoxygenase;
COX, cyclooxygenase; NDGA, nordihydroguaiaretic acid; AA,
arachidonic acid; 15-HpETE, 15-(S)-hydroperoxyeicosatetrae-
noic acid; 12-HpETE, 12-(S)-hydroperoxyeicosatetraenoic
acid; LA, linoleic acid; 13-(S)-HpODE, 13-(S)-hydroperox-
yoctadecadienoic acid; 12-HETE, 12-(S)-hydroxyeicosatetrae-
noic acid; 15-HETE, 15-(S)-hydroxyeicosatetraenoic 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; XO, xylenol orange; HTS, high-
throughput screening; MLSMR, Molecular Libraries Small
Molecule Repository; MLPCN, Molecular Libraries Probe
Centers Network; qHTS, quantitative high-throughput screen-
ing; CRC, concentration response curve; EDC, 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide hydrochloride; BTAC,
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