Inhibition of group IVA cytosolic phospholipase A2by thiazolyl ketones in vitro, ex vivo, and in vivo

Article abstract of DOI:10.1021/jm500192s

Group IVA cytosolic phospholipase A₂(GIVA cPLA₂) is the rate-limiting provider of pro-inflammatory mediators in many tissues and is thus an attractive target for the development of novel anti-inflammatory agents. In this work, we present the synthesis of new thiazolyl ketones and the study of their activities in vitro, in cells, and in vivo. Within this series of compounds, methyl 2-(2-(4-octylphenoxy)acetyl)thiazole-4-carboxylate (GK470) was found to be the most potent inhibitor of GIVA cPLA₂, exhibiting an X_I(50) value of 0.011 mole fraction in a mixed micelle assay and an IC₅₀of 300 nM in a vesicle assay. In a cellular assay using SW982 fibroblast-like synoviocytes, it suppressed the release of arachidonic acid with an IC₅₀value of 0.6 μM. In a prophylactic collagen-induced arthritis model, it exhibited an anti-inflammatory effect comparable to the reference drug methotrexate, whereas in a therapeutic model, it showed results comparable to those of the reference drug Enbrel. In both models, it significantly reduced plasma PGE₂levels.

Full text of DOI:10.1021/jm500192s

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Article
Inhibition of Group IVA Cytosolic Phospholipase A2
by Thiazolyl Ketones In Vitro, Ex Vivo, and In Vivo
George Kokotos, Astrid J. Feuerherm, Efrosini Barbayianni, Ishita Shah, Mari Sæther, Victoria
Magrioti, Thuy Nguyen, Violetta Constantinou-Kokotou, Edward A. Dennis, and Berit Johansen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500192s • Publication Date (Web): 25 Aug 2014
Downloaded from http://pubs.acs.org on August 28, 2014
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Inhibition of Group IVA Cytosolic Phospholipase
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A by Thiazolyl Ketones In Vitro, Ex Vivo, and In
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Vivo
,
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‡,§
‡,†
#
§
George Kokotos,* Astrid J. Feuerherm, Efrosini Barbayianni, Ishita Shah, Mari Sæther,
†
§
║
#
Victoria Magrioti, Thuy Nguyen, Violetta ConstantinouꢀKokotou, Edward A. Dennis, and
,
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Berit Johansen*
†
Laboratory of Organic Chemistry, Department of Chemistry, University of Athens,
Panepistimiopolis, Athens 15771, Greece
§
Department of Biology, Norwegian University of Science and Technology, Nꢀ7491 Trondheim,
Norway
#
Department of Chemistry and Biochemistry and Department of Pharmacology, School of
Medicine, University of California, San Diego, La Jolla, California 92093ꢀ0601
║
Chemical Laboratories, Agricultural University of Athens, Athens 11855, Greece
KEYWORDS. arthritis, inhibitors, phospholipase A , prostaglandin E , thiazolyl ketones
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ABSTRACT. The Group IVA cytosolic phospholipase A (GIVA cPLA ) is the rateꢀlimiting
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provider of proꢀinflammatory mediators in many tissues and is thus an attractive target for the
development of novel antiꢀinflammatory agents. In this work, we present the synthesis of new
thiazolyl ketones and the study of their activities in vitro, in cells and in vivo. Within this series of
compounds, methyl 2ꢀ(2ꢀ(4ꢀoctylphenoxy)acetyl)thiazoleꢀ4ꢀcarboxylate (GK470) was found to
be the most potent inhibitor of GIVA cPLA , exhibiting an X (50) value of 0.011 mol fraction in a
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mixed micelle assay and an IC of 300 nM in a vesicle assay. In a cellular assay using SW982
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fibroblastꢀlike synoviocytes, it suppressed the release of arachidonic acid with an IC value of
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0.6 ꢁM. In a prophylactic collagenꢀinduced arthritis model, it exhibited an antiꢀinflammatory
effect comparable to the reference drug methotrexate, while in a therapeutic model, it showed
results comparable to the reference drug Enbrel. In both models, it significantly reduced plasma
PGE levels.
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INTRODUCTION
The purification, sequence, and cloning of the first human cytosolic phospholipase A (cPLA )
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from the U937 macrophage cell line was reported in 1991. Two years later, arachidonoyl
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trifluoromethyl ketone (1, Figure 1) was reported as the first synthetic inhibitor of this enzyme.
The enzyme has subsequently attracted special attention as a medicinal target, in particular after
the reports that gene targeted mice that lack cPLA are much less prone to inflammatory
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pathological responses to disease, stresses, and physical injuries, which in essence protects them
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from cellular and systemic damage. Wyeth has expended major efforts to develop novel cPLA
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inhibitors as targeted therapeutics for inflammatory diseases. Three indoleꢀbased inhibitors,
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Ecopladib (2a, Figure 1), Efipladib (2b, Figure 1) and Giripladib (2c, Figure 1), entered clinical
trials. Giripladib was the most promising of this indole series as it was advanced into a Phase II
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clinical trial for osteoarthritis, however in 2007 the trial was terminated.
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Phospholipases A constitutes a superfamily of enzymes that currently includes 16 groups and
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many subgroups, most of which differ in their primary sequence and structure, but they also
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exhibit a variety of different mechanisms of action.
Human cPLA has been designated as
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GIVA cPLA and is the most well studied enzyme of this group. Several inhibitors of GIVA
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cPLA have been reported in the literature and are summarized in various review articles.
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Most recently, the inhibitors described in the patent literature have been highlighted. Apart from
the inhibitors developed by Wyeth, Shionogi has developed thiazolidinedione inhibitors based on
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a pyrrolidine template, including pyrrophenone (3, Figure 1).
Pyrrophenone strongly inhibited
arachidonic acid release and prostaglandin E , thromboxane B , and leukotriene B formation in
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human whole blood. Pyrroxyphene (4, Figure 1), which also belongs to this class of inhibitors,
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displayed antiꢀarthritic and antiꢀbone destructive action in a murine arthritis model. Another
important class of GIVA cPLA inhibitors, are indolꢀ1ꢀylꢀpropaneꢀ2ꢀones, for example,
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compound 5 (Figure 1).
One of them was reported to inhibit benzalkonium chlorideꢀinduced
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skin inflammation. Moreover, heterocyclic derivatives have been claimed as GIVA cPLA
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inhibitors. Recently, inhibitors containing an αꢀmethylꢀ2ꢀketothiazole as a metabolically stable
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serine trap have been developed, for example, compound 6 (Figure 1).
During the last decade, we have designed, synthesized and studied novel inhibitors targeting
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various PLA groups. We have developed various 2ꢀoxoamides,
for example 7a and 7b
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(Figure 1) as inhibitors of GIVA cPLA and fluoroketones,
for example FKGK18, (8,
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Figure 1), as inhibitors of the other major intracellular cytosolic phospholipase A (GVIA iPLA )
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enzyme. Some fluoroketones exhibit remarkable inhibition of GIVA cPLA2.
Using a
combination of molecular dynamics simulation and deuterium exchange mass spectrometry, the
interactions of our synthetic inhibitors with the active site of GIVA cPLA and GVIA iPLA were
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studied.
In a continuation of our studies, we have now developed new inhibitors targeting
GIVA cPLA and tested their antiꢀinflammatory effect in vivo. Herein we report the synthesis of
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several thiazolyl ketone derivatives, the study of their in vitro potency and specificity, as well as
their effect on the release of arachidonic acid (AA) and oleic acid (OA) ex vivo, and the first in
vivo studies of the most potent inhibitor of this series in an animal model of collagenꢀinduced
arthritis (CIA).
RESULTS AND DISCUSION
Design and synthesis of inhibitors. GIVA cPLA consists of an Nꢀterminal C2 domain and a Cꢀ
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terminal catalytic domain and utilizes an unusual catalytic dyad (Serꢀ228/Aspꢀ549) located in the
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α/β hydrolase domain to catalyze the hydrolysis of the substrates. We have previously studied
derivatives containing the 2ꢀoxoamide and the polyfluoroketone functionalities targeting the
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active site serine of PLA2.
In both cases, it is proposed that the activated ketone interacts with
the catalytic serine. Although these two functionalities markedly differ in the potency of their
activated carbonyl group, derivatives containing either the oxoamide or the fluoroketone
functionality have been proven to be efficient inhibitors of GIVA cPLA . Apparently, not only
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the potency of the activated carbonyl group, but also the presence of other groups able to present
appropriate hydrophobic and/or hydrophilic interactions contribute to the overall binding of the
inhibitor to the enzyme, determining the inhibitory potency. In the present work, we study
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derivatives containing the ketothiazolyl functionality (Figure 2). The presence of the two
heteroatoms on the heterocyclic ring ensures the activation of the carbonyl group. In addition, the
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presence of an oxygen atom at the βꢀposition enforces the activation. The R group may be either
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an aliphatic or an aromatic group, while a substituent R may be present on the heterocyclic ring.
In fact, a variety of αꢀketoheterocycles have been reported as inhibitors of diverse serine and
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cysteine proteases, as well as fatty acid amide hydrolase. As outlined in the introduction, some
such heterocyclic derivatives have been claimed as agents for the treatment of inflammatory
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diseases, however without providing any in vivo data. Inhibitors containing an αꢀmethylꢀ2ꢀ
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ketothiazole as a metabolically stable serine trap have been presented.
The synthesis of thiazole derivatives 13a,b and 18aꢀc is presented in Schemes 1 and 2. Phenols
9a,b and 14a,b were treated with ethyl bromoacetate. Esters 10a,b were hydrolyzed and
converted to their corresponding Weinreb amides. Treatment of Weinreb amides 12a,b with
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lithium thiazole led to the target derivatives 13a,b. Thiazolyl ketones 18aꢀc were prepared by
another procedure. Alcohols 16a,b were oxidized to aldehydes and treated with lithium thiazole
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or benzothiazole. Compounds 17aꢀc were then oxidized by DessꢀMartin periodinane to the
final compounds.
Substituted thiazoles 24aꢀc and 28 were synthesized as illustrated in Schemes 3 and 4. The keyꢀ
step for their synthesis was the formation of the substituted heterocyclic ring. Alcohols 16b and
19 were oxidized to aldehydes and directly treated with tertꢀbutyldimethylsilyl cyanide
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TBDMSCN). Silylꢀprotected cyanohydrins 20a,b were converted into amides and subsequently
into thioamides by reaction with Lawesson’s reagent. Treatment of 22a,b with ethyl 4ꢀ
chloroacetoacetate, and 22b with ethyl bromopyruvate, according to previously published
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protocols, in the presence of conc. H SO led to heterocyclic derivatives 23aꢀc which were then
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oxidized to the final compounds 24aꢀc. Following another method for the formation of the
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heterocyclic ring, condensation of cysteine methyl ester with nitrile 20a afforded a
diastereomeric mixture of thiazoline 25, which was transformed into thiazole 26 using BrCCl3
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and 1,8ꢀdiazabicycloundecꢀ7ꢀene (DBU). Subsequent removal of the silyl group and Dessꢀ
Martin oxidation led to the thiazolyl ketone 28.
In vitro inhibition of GIVA cPLA , GVIA iPLA and GV sPLA . All synthesized thiazolyl
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ketones were tested for their in vitro activity on recombinant human GIVA cPLA using both
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mixed micelle and vesicle assays. In addition, their selectivity over human GVIA iPLA and GV
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sPLA was studied using mixed micelle assays.
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The in vitro inhibition of human GIVA cPLA , GVIA iPLA and GV sPLA was carried out
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using previously described mixed micelleꢀbased assays.
The inhibition results are presented
in Table 1, either as percent inhibition or as X (50) values. At first, the percent of inhibition for
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each PLA enzyme at 0.091 mole fraction of each inhibitor was determined. Then, the X (50)
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values were measured for compounds that displayed greater than 90% inhibition of GIVA cPLA2.
The X (50) is the mole fraction of the inhibitor in the total substrate interface required to inhibit
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the enzyme activity by 50%.
Thiazolyl ketone 13a as well as its derivatives 13b and 18a containing a fluorine atom or a
phenyl group at the para position, did not present any significant inhibition of GIVA cPLA2
(entries 1ꢀ3, Table 1). However, when a paraꢀnꢀoctyl chain was introduced, a significant
inhibitory activity was observed for 18b (entry 4, Table 1). The replacement of the thiazole ring
by the benzothiazole had as a result the reduction of the inhibitory potency (entry 5 vs entry 4,
Table 1). Derivatives 24b and 24c containing an alkoxy group and a substituted thiazole group
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proved inactive (entries 7 and 8, Table 1). However, the introduction of a paraꢀnꢀoctylꢀphenoxy
group, together with an ester group on the thiazole ring, led to the potent inhibitor of GIVA
cPLA 28 (GK470) showing a X (50) value of 0.011 (entry 9, Table 1). Interestingly, moving the
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ester group one carbon atom away from the heterocyclic ring resulted in a dramatic loss of the
activity for 24a (entry 6, Table 1).
The effect of the thiazolyl ketones synthesized on GIVA cPLA was measured in vesicles as
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previously described
with modifications. The results are presented in Table 1 and are in full
agreement with those using the micellar assay. Compound 28 was found to be the most potent
inhibitor of GIVA cPLA within this series of thiazolyl ketones with an IC value of 0.3 µM
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(entry 9, Table 1). Compound 18b also presented an interesting inhibition in this vesicle assay
with an IC value of 1.2 µM (entry 4, Table 1). From both assays, it is clear that the introduction
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of the ethyl ester group directly on the heterocyclic ring substantially increases the potency of
inhibition. This group presumably develops additional interactions within the enzyme’s active
site. Comparing the results obtained in mixed micelles and in vesicles, it is obvious that inhibitor
28 stands out in this series of thiazolyl ketones.
Ex vivo inhibition of AA and OA release in synoviocytes. The effect of the thiazolyl ketones
synthesized on the release of AA and OA in synoviocytes was evaluated as previously
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described. The percent inhibition of AA and OA was determined at a 10 ꢁM inhibitor
concentration, while for the determination of the IC value the inhibitors were tested in a 0ꢀ20
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M range after interleukinꢀ1β (ILꢀ1β) stimulation for 4 h. No toxic effects were observed for any
inhibitor in the range 0ꢀ20 ꢀM (results not shown). A number of thiazolyl ketones (18a, 18b, 18c,
4a) exhibited significant inhibition of the AA release (Table 1). However, in accordance with
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IC value of 0.6 µM, without displaying equivalent potency on OA release (entry 9, Table 1).
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In vivo prophylactic anti-inflammatory effect of inhibitor 28 in CIA. Inhibitor 28 clearly
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presented a potent inhibitory effect of the GIVA cPLA activity in vitro and a potent suppression
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of the AA release in cells. Thus, studies of its antiꢀinflammatory properties in vivo were designed.
First, a pilot study was performed to evaluate possible toxic effects of inhibitor 28. No toxic or
adverse effects were found at the doses 1, 5, 15, 30 or 60 mg/Kg, following daily intraperitoneal
(ip) injections of the compound for one week (results not shown). Next, the CIA mouse model, a
common autoimmune model of rheumatoid arthritis in which GIVA cPLA activity is
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important,
was employed for the evaluation of the in vivo activity of inhibitor 28.
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The prophylactic effect of inhibitor 28 on the CIA model in male DBA/1 mice following ip
administration, with treatment starting 1 hour prior to the last immunization was studied. A study
comparing naïve mice (healthy, nonꢀCIA, nonꢀtreated), vehicleꢀtreated mice (CIA with DMSO
ip) and CIA mice treated daily with inhibitor 28 (7.5 mg/Kg, ip) or methotrexate (MTX) (0.3
mg/Kg, ip) was performed. In order to capture early disease progression and the effect of
inhibitor 28 and MTX, additional groups of animals (histology groups) enabling comparison of
naïve mice, vehicleꢀtreated mice and CIA mice treated with inhibitor 28 or MTX were designed.
CIA developed rapidly in mice immunized with collagen type II (CII). In the prophylactic study,
a 100% incidence of CIA was observed by day 29 in CIIꢀimmunized mice, with a maximum
arthritis index (AI) of 8.55 observed at 41 days post immunization. The AI and incidence of all
groups increased in a timeꢀdependent mode from day 25 to day 41.
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The AI of the group dosed with inhibitor 28 at 7.5 mg/Kg on Days 32 to 41 was significantly
reduced in comparison to the CIA control group (p<0.005), similar to the effect of MTX (Figure
3). Within the smaller histology groups that were sacrificed at day 32, following 13 days of
treatment, there was no significant difference in the AI value between the histology groups and
main groups (p>0.05). Following 13 days of treatment, one hind paw of mice in each histology
group was collected for histopathology. Compared with the vehicle group, inhibitor 28 at 7.5
mg/Kg significantly reduced articular cavity and peripheral tissue inflammatory cell infiltration
(p<0.03), as well as capillary and synovial hyperplasia (p<0.05), but had no significant effect on
reducing cartilage damage (Figure 4 A). This finding was supported by histopathological
analyses of joints at end of study that also documented reduced inflammatory cell infiltration and
hyperplasia in inhibitor 28ꢀtreated animals compared to vehicle treated group (Figure 4B). In
contrast, MTX did not reduce any of these parameters of joint inflammation and joint damage
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(p>0.05). These data suggest a disease modifying property of inhibitor 28.
In vivo therapeutic anti-inflammatory effect of inhibitor 28 in CIA. The therapeutic effect of
inhibitor 28 on the CIA model in male DBA/1 mice following ip administration with treatment
starting 7 days after the last immunization was explored. CIA developed rapidly in mice
immunized with CII, with maximum AI of 10.2 observed at 39 days post immunization. The AI
and incidence of vehicleꢀ and inhibitor 28 treated groups increased in a timeꢀdependent mode
from days 29 to 41. The AI observed was significantly decreased in comparison to that of the
CIA control group both in the inhibitor 28 treated with 30 mg/Kg group and in the Enbrel group
on days 36 to 41 (p<0.05) (Figure 5). Inhibitor 28 and Enbrel performed equally well and there
were no significant difference between these treatment groups.
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Inhibitor 28 efficiently reduces plasma prostaglandin E (PGE ) levels. PGE is recognized as
2
2
2
5
3
an important contributor to joint inflammation in rheumatoid arthritis, and we investigated if
plasma PGE levels were changed in response to treatment. In the prophylactic study (n=11),
2
0
1
2
3
4
5
6
7
8
9
0
1
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PGE levels in the DMSO treated vehicle group (223 ± 107 ng/mL) were significantly elevated
2
by 3ꢀfold (p<0.001) compared to the nonꢀarthritic healthy mice (70 ± 37 ng/mL) (Figure 6A). The
elevated PGE level was significantly reduced by inhibitor 28 (7.5 mg/mL, 140 ± 92 ng/mL,
2
p<0.03), comparable to the MTX level (0.3 mg/mL, 107 ± 62 ng/mL, p<0.004). There were no
significant differences between the treatment groups (p>0.05). In the therapeutic study (n=10),
similar results were obtained; PGE levels in the DMSO treated vehicle group (231 ± 110 ng/mL)
2
were significantly elevated by 3ꢀfold (p<0.001) compared to the nonꢀarthritic healthy mice (71 ±
36 ng/mL) (Figure 6B). The elevated PGE levels were significantly reduced with inhibitor 28
2
(30 mg/mL, 139 ± 55 ng/mL, p<0.03) treatment, but not with Enbrel (25 mg/Kg, 188 ± 74
ng/mL, nonꢀsignificant p>0.05). In summary, plasma PGE levels in inhibitor 28ꢀtreated animals
2
were significantly reduced by about 40% in both prophylactic and therapeutic modes of the
animal disease models. In the prophylactic model, inhibitor 28 produced PGE plasma level
2
reduction comparable to that of the reference drug MTX. Furthermore, in the therapeutic model,
it appears that inhibitor 28 caused more potent PGE reduction in plasma than that of the
2
reference drug Enbrel.
Pharmacokinetic properties of inhibitor 28. Initial in vitro metabolic studies of inhibitor 28 in
human whole blood and mouse plasma established a t of approximately 190 min for the
1
/2
compound (data not shown). To assess the pharmacokinetic profile of inhibitor 28 in vivo, we
measured maximum compound concentration achieved after a single intraperitoneal
administration of 30 mg/Kg in Swiss male mice. To compare with levels achieved after
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intravenous administration, inhibitor 28 was also injected intravenously at 5 mg/Kg in a different
group of mice (see Materials and Methods). Overall, inhibitor 28 reached a Cmax of 234.5 ng/mL
2 h after ip administration with detectable levels persisting for 24 hours after administration. In
comparison, iv levels of inhibitor 28 peaked at 30 min after administration (2.7 ꢁg/mL) rapidly
declining to background levels at 24h post administration. Calculated AUC(0ꢀt) values over the
same period were 1266.1 and 1861.9 ng/mL*h for ip and iv administration, respectively. These
results collectively indicate that inhibitor 28 administered ip is bioꢀavailable and justified use of a
daily dosing scheme for the compound in further animal studies.
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28
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60
CONCLUSION
Various routes for the synthesis of thiazolyl ketones have been studied. The keyꢀstep for the
synthesis of substituted thiazoles was either the condensation of a thioamide with a halo ketoester
or the condensation of a nitrile with cysteine methyl ester, followed by aromatization. The
compounds synthesized were tested for their in vitro inhibitory activity toward GIVA cPLA2
using both mixed micelle and vesicle assay. Among them, inhibitor 28 was found to be the most
potent inhibitor, with an IC value of 0.6 ꢀM in AA release assay, exhibiting a X (50) value of
5
0
I
0
.011 mol fraction in a mixed micelle assay, and an IC of 300 nM in a vesicle assay. This
50
inhibitor was able to suppress the release of AA in fibroblastꢀlike synoviocytes (IC 600 nM),
50
without significantly effecting potency in OA release. The in vivo activity of this inhibitor was
evaluated in the CIA mouse model. In the prophylactic model at a dose of 7.5 mg/Kg, it exhibited
an antiꢀinflammatory effect comparable to the reference drug methotrexate, while in the
therapeutic model at a dose of 30 mg/Kg, it showed results comparable to the reference drug
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Enbrel. In both the prophylactic and the therapeutic models, the elevated by 3ꢀfold PGE plasma
2
levels were significantly reduced by about 40% using inhibitor 28. Thus, we have identified a
new GIVA cPLA inhibitor, which presents interesting antiꢀinflammatory and diseaseꢀmodifying
2
0
1
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9
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0
in vivo effects in the CIA model and inhibitor 28 may hence represent a new agent for the
treatment of inflammatory diseases.
EXPERIMENTAL SECTION
General. Chromatographic purification of products was accomplished using Merck Silica Gel 60
(70ꢀ230 or 230ꢀ400 mesh). Thinꢀlayer chromatography (TLC) was performed on Silica Gel 60
F254 aluminum plates. Spots were visualised with UV light and/or phosphomolybdic acid in
1
EtOH. Melting points were determined using a Büchi 530 apparatus and were uncorrected. H
13
and C NMR spectra were recorded on a Varian Mercury (200 MHz and 50 MHz respectively)
1
13
in CDCl or as specified. H and C NMR spectra of inhibitor 28 were recorded on a Bruker
3
AVANCE III (600 MHz and 150 MHz respectively) in CDCl . Chemical shifts are given in ppm,
3
and coupling constants (J) in Hz. Peak multiplicities are described as follows: s, singlet, d,
doublet, t, triplet and m, multiplet. Electron spray ionization (ESI) mass spectra were recorded on
a Finnigan, Surveyor MSQ Plus spectrometer. Dichloromethane, diethylether and toluene were
dried by standard procedures and stored over molecular sieves. All other solvents and chemicals
were reagent grade and used without further purification. The purity of all compounds subjected
to biological tests was determined by analytical HPLC, and was found to be ≥95%. HPLC
analyses were carried out on an Agilent 1100 Series apparatus and a Thermo Scientific Hypersil
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Silica column, using 10% iꢀPrOH in hexane, at a flow rate of 1.0 mL/min. HRMS spectra were
recorded on a Bruker Maxis Impact QTOF Spectrometer.
5
4
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56
57
Compounds 10b, 12a, 12b, 15a, have been described elsewhere and their analytical data
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
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58
59
60
are in accordance with literature.
Ethyl 2ꢀ(4ꢀoctylphenoxy)acetate (15b). To a stirred solution of 4ꢀnꢀoctylphenol (1.0 mmol, 206
mg) in acetone (10 mL), K CO (3 mmol, 415 mg) and ethyl bromoacetate (1.1 mmol, 215 mg)
2
3
were added, and the reaction mixture was refluxed for 5 h. Subsequently, the mixture was
filtrated over Celite, and the organic solvent was evaporated under reduced pressure. The residue
was purified by flash column chromatography [EtOAcꢀpetroleum ether (bp 40ꢀ60 °C), 1:9]. Yield
1
9
8%; White oil; H NMR (200 MHz, CDCl ): δ 7.10 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz,
3
2H), 4.60 (s, 2H), 4.28 (q, J = 7.2 Hz, 2H), 2.55 (t, J = 7.8 Hz, 2H), 1.69–1.46 (m, 2H), 1.45–1.12
13
(m, 13H), 0.89 (t, J = 7.0 Hz, 3H); C NMR (50 MHz, CDCl ): δ 169.1, 155.8, 136.1, 129.3,
3
+
114.4, 65.5, 61.3, 35.0, 31.8, 31.6, 29.4, 29.2, 22.6, 14.1; MS (ESI) m/z (%): 293 (90) [M + H] .
Synthesis of alcohols 16a,b. To a stirred solution of esters 15a,b (1 mmol) in dry Et O (10 mL)
2
o
was added DIBALH (2.5 mL, 2.5 mmol, 1.0 M in hexane) at 0 C under Ar atmosphere and the
reaction mixture was stirred for 2 h at room temperature. Water was then added (5 mL), the
mixture was stirred for 30 more minutes and filtrated over Celite. The organic solvent was
evaporated under reduced pressure and the residue was purified by flash column chromatography
[EtOAcꢀpetroleum ether (bp 40ꢀ60 °C), 3:7].
o
1
2ꢀ(Biphenylꢀ4ꢀyloxy)ethanol (16a). Yield 94%; White solid; mp 120ꢀ122 C; H NMR (200
MHz, CDCl ): δ 7.63–7.22 (m, 7H), 7.07–6.94 (m, 2H), 4.19–4.07 (m, 2H), 4.05–3.94 (m, 2H),
3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
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5
5
5
5
5
5
5
5
6
13
1
.91 (br, 1H); C NMR (50 MHz, CDCl ): δ 158.1, 140.6, 134.2, 128.7, 128.2, 126.7, 114.8,
3
69.2, 61.4.
o
1
2ꢀ(4ꢀOctylphenoxy)ethanol (16b). Yield 82%; White solid; mp 40ꢀ42 C; H NMR (200 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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0
CDCl ): δ 7.11 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 4.13−4.02 (m, 2H), 4.01−3.91 (m,
3
2H), 2.56 (t, J = 7.8 Hz, 2H), 2.19 (br, 1H), 1.70–1.48 (m, 2H), 1.45–1.14 (m, 13H), 0.90 (t, J =
13
7
.0 Hz, 3H); C NMR (50 MHz, CDCl ): δ 156.5, 135.5, 129.3, 114.2, 69.1, 61.5, 35.0, 31.9,
3
3
1.7, 29.5, 29.2, 22.7, 14.1.
Synthesis of ketones 13a,b. To a stirred solution of thiazole (3 eq.) in dry Et O (20 mL) at ꢀ78
2
o
C under a dry argon atmosphere was added a solution of nꢀBuLi (1.6 M in hexanes, 3 eq.)
dropwise over a period of 10 min. The resulting orange solution was stirred for 45 min. Then, a
solution of amides 12a,b (1 mmol) in dry Et O (2 mL) was slowly added giving the mixture a
2
o
dark brown colour. After stirring for 30 min at ꢀ78 C, the mixture was allowed to warm up to
room temperature over a period of 2 h. Then, saturated aqueous ammonium chloride solution was
added and the mixture was extracted with ether (2 × 10 mL). The combined extracts were washed
with brine and then dried over Na SO and concentrated under reduced pressure. Purification by
2
4
flash chromatography eluting with the appropriate mixture of [EtOAcꢀpetroleum ether (bp 40ꢀ60
C)] afforded the product.
°
o
1
2
ꢀPhenoxyꢀ1ꢀ(thiazolꢀ2ꢀyl)ethanone (13a). Yield 54%; White solid; mp 77ꢀ79 C; H NMR
(
200 MHz, CDCl ): δ 8.08−8.02 (m, 1H), 7.79−7.74 (m, 1H), 7.39–7.22 (m, 2H), 7.07–6.93 (m,
3
1
3
2
H), 5.55 (s, 2H); C NMR (50 MHz, CDCl ): δ 187.4, 164.0, 157.8, 144.9, 129.5, 126.7, 121.6,
3
+
114.8, 70.0; HRMS (ESI) calcd for C H NNaO S [M + Na] : 242.0246. Found: 242.0256.
1
1
9
2
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ꢀ(4ꢀFluorophenoxy)ꢀ1ꢀ(thiazolꢀ2ꢀyl)ethanone (13b). Yield 61%; White solid; mp 74ꢀ77 C; H
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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4
4
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6
NMR (200 MHz, CDCl ): δ 8.08−8.03 (m, 1H), 7.81−7.76 (m, 1H), 7.09–6.86 (m, 4H), 5.51 (s,
3
1
3
2
H); C NMR (50 MHz, CDCl ): δ 187.4, 164.0, 157.8 (d, J = 239.4 Hz), 154.1, 145.0, 126.8,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
16.2, 115.9 (d, J = 15.8 Hz), 70.8; HRMS (ESI) calcd for C H FNNaO S [M + Na] : 260.0152.
1
1
8
2
Found: 260.0159.
Synthesis of compounds 17aꢀc. To a solution of alcohols 16a,b (1.0 mmol) in a mixture of
toluene (3 mL) and EtOAc (3 mL), a solution of NaBr (0.11 g, 1.1 mmol) in water (0.5 mL) was
added followed by 2,2,6,6ꢀtetramethylpiperidineꢀ1ꢀyloxy free radical (TEMPO) (2.2 mg, 0.01
o
mmol). To the resulting biphasic system, which was cooled at 0 C, an aqueous solution of 0.35
M NaOCl (3.1 mL, 1.1 mmol) containing NaHCO (0.25 g, 3 mmol) was added dropwise under
3
o
vigorous stirring, at 0 C over a period of 1 h. After the mixture had been stirred for a further 15
o
min at 0 C, EtOAc (10 mL) and H O (10 mL) were added. The aqueous layer was separated and
2
washed with EtOAc (2 × 10 mL). The combined organic layers were washed consecutively with
5
% aqueous citric acid (10 mL) containing KI (0.04 g), 10% aqueous Na S O (10 mL), and brine
2 2 3
and dried over Na SO . The solvents were evaporated under reduced pressure and the residue was
2
4
used without any further purification.
o
To a stirred solution of thiazole or benzothiazole (3 eq.) in dry Et O (20 mL) at ꢀ78 C under a
2
dry argon atmosphere was added a solution of nꢀBuLi (1.6 M in hexanes, 3 eq.) dropꢀwise over a
period of 10 min. The resulting orange solution was stirred for 45 min. Then, a solution of the
above prepared aldehyde (1 mmol) in dry Et O (2 mL) was slowly added giving the mixture a
2
o
dark brown colour. After stirring for 30 min at ꢀ78 C, the mixture was allowed to warm up to
room temperature over a period of 2 h. Then, saturated aqueous ammonium chloride solution was
added and the mixture was extracted with ether (2 x 10 mL). The combined extracts were washed
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with brine and then dried over Na SO and concentrated under reduced pressure. Purification by
2
4
flash chromatography eluting with the appropriate mixture of EtOAcꢀpetroleum ether (bp 40ꢀ60
°C) afforded the product.
0
1
2
3
4
5
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8
9
0
1
2
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2
ꢀ(Biphenylꢀ4ꢀyloxy)ꢀ1ꢀ(thiazolꢀ2ꢀyl)ethanol (17a). Yield 48%; Pale yellow solid; mp 93ꢀ95 C;
1
H NMR (200 MHz, CDCl ): δ 7.79 (d, J = 3.2 Hz, 1H), 7.67–7.22 (m, 8H), 7.09–6.93 (m, 2H),
3
4
.18−3.90 (br, 1H), 5.44 (dd, J = 3.9 Hz, J = 7.0 Hz, 1H), 4.49 (dd, J = 3.9 Hz, J = 9.7 Hz,
1
2
1
2
1
3
1
H), 4.30 (dd, J = 7.0 Hz, J = 9.7 Hz, 1H); C NMR (50 MHz, CDCl ): δ 170.9, 157.7, 142.4,
1 2 3
140.5, 134.5, 128.7, 128.2, 126.8, 126.7, 119.6, 115.0, 71.6, 70.6; MS (ESI) m/z (%): 298 (100)
+
[
M + H] .
1
2
ꢀ(4ꢀOctylphenoxy)ꢀ1ꢀ(thiazolꢀ2ꢀyl)ethanol (17b). Yield 34%; Yellow oil; H NMR (200 MHz,
CDCl ): δ 7.78 (d, J = 3.2 Hz, 1H), 7.34 (d, J = 3.2 Hz, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.86 (d, J =
3
8
.6 Hz, 2H), 5.40 (dd, J = 3.9 Hz, J = 7.0 Hz, 1H), 4.42 (dd, J = 3.9 Hz, J = 9.7 Hz, 1H), 4.22
1 2 1 2
(dd, J = 7.0 Hz, J = 9.7 Hz, 1H), 2.54 (t, J = 7.8 Hz, 2H), 1.69–1.45 (m, 2H), 1.43–1.10 (m,
1
2
1
3
1
0H), 0.89 (t, J = 7.0 Hz, 3H); C NMR (50 MHz, CDCl ): δ 171.0, 156.1, 142.4, 135.9, 129.3,
3
119.5, 114.5, 71.6, 70.7, 35.0, 31.8, 31.7, 29.4, 29.2, 22.6, 14.1; MS (ESI) m/z (%): 334.2 (100)
+
[M + H] .
1ꢀ(Benzo[d]thiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀoctylphenoxy)ethanol (17c). Yield 42%; Yellow solid; mp 93ꢀ95
o
1
C; H NMR (200 MHz, CDCl ): δ 8.10–7.97 (m, 1H), 7.96–7.82 (m, 1H), 7.56–7.32 (m, 2H),
3
7
.09 (d, J = 8.5 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 5.50 (dd, J = 4.0 Hz, J = 6.8 Hz, 1H), 4.52
1 2
(
dd, J = 4.0 Hz, J = 9.7 Hz, 1H), 4.34 (dd, J = 6.8 Hz, J = 9.7 Hz, 1H), 2.55 (t, J = 7.8 Hz,
1 2 1 2
1
3
2H), 1.70–1.47 (m, 2H), 1.45–1.12 (m, 10H), 0.89 (t, J = 7.0 Hz, 3H); C NMR (50 MHz,
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CDCl ): δ 172.2, 156.0, 152.8, 136.0, 134.9, 129.3, 126.2, 126.1, 125.0, 122.9, 114.5, 71.4, 71.0,
3
+
3
5.0, 31.8, 31.7, 29.4, 29.2, 22.7, 22.6, 14.1; MS (ESI) m/z (%): 384 (100) [M + H] .
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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4
4
4
4
4
4
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5
5
5
5
5
5
5
5
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6
Synthesis of nitriles 20a,b. To a mixture of TBDMSCN (1.0 mmol, 141 mg), potassium cyanide
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(0.2 mmol, 13 mg), and 18ꢀcrownꢀ6 (0.4 mmol, 106 mg) was added dropwise a solution of the
aldehyde derived from alcohol 16b or 19, according to the NaBr/TEMPO protocol mentioned
above, (1.0 mmol) in CH Cl at room temperature under nitrogen over 30 min. After addition was
2
2
complete, the mixture was stirred overnight at room temperature. The organic solvent was
evaporated under reduced pressure and the residue was purified by flash column chromatography
[
EtOAcꢀpetroleum ether (bp 40ꢀ60 °C), 1:9].
2ꢀ(tertꢀButyldimethylsilyloxy)ꢀ3ꢀ(4ꢀoctylphenoxy)propanenitrile (20a). Yield 93%; White oil;
1
H NMR (200 MHz, CDCl ): δ 7.12 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 4.80 (t, J = 5.4
3
Hz, 1H), 4.21−3.98 (m, 2H), 2.56 (t, J = 7.8 Hz, 2H), 1.65−1.42 (m, 2H), 1.40−1.13 (br, 10H),
1
3
1.04−0.75 (m, 12H), 0.24 (s, 3H), 0.19 (s, 3H); C NMR (50 MHz, CDCl ): δ 155.7, 136.2,
3
129.4, 118.1, 114.4, 69.6, 61.5, 35.0, 31.9, 31.7, 29.4, 29.2, 25.4, 22.6, 18.1, 14.1, ꢀ5.3; MS (ESI)
+
m/z (%): 407 (100) [M + NH ] .
4
1
2
ꢀ(tertꢀButyldimethylsilyloxy)ꢀ3ꢀ(dodecyloxy)propanenitrile (20b). Yield 72%; White oil; H
NMR (200 MHz, CDCl ): δ 4.55 (t, J = 6.4 Hz, 1H), 3.62 (d, J = 6.6 Hz, 2H), 3.52 (t, J = 6.6 Hz,
3
1
3
2H), 1.67–1.48 (m, 2H), 1.26 (br, 18H), 0.98–1.82 (m, 12H), 0.19 (s, 3H), 0.17 (s, 3H); C NMR
(
50 MHz, CDCl ): δ 118.7, 72.6, 72.2, 62.0, 31.9, 29.6, 29.5, 29.4, 29.3, 25.9, 25.5, 22.7, 18.1,
3
+
1
4.1, ꢀ5.3; MS (ESI) m/z (%): 387 (100) [M + NH ] .
4
Synthesis of amides 21a,b. To a solution of nitriles 20a,b (1 mmol) and Bu NHSO (0.2 mmol,
4
4
6
8 mg) in CH Cl (10 mL), was added dropwise a solution of 0.5 N aq. NaOH (2.5 mL) and 30%
2 2
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o
H O (4 mmol, 3.5 mL) at 0 C. The biphasic reaction mixture was stirred overnight at room
2
2
temperature. The organic layer was separated, washed with water (2 × 10 mL) and dried over
Na SO . Organic solvent was evaporated under reduced pressure and the residue was purified by
2
4
0
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flash column chromatography [EtOAcꢀpetroleum ether (bp 40ꢀ60 °C)].
2
ꢀ(tertꢀButyldimethylsilyloxy)ꢀ3ꢀ(4ꢀoctylphenoxy)propanamide (21a). Yield 68%; White
o
1
solid; mp 56ꢀ58 C; H NMR (200 MHz, CDCl ): δ 7.08 (d, J = 8.4 Hz, 2H), 6.82 (d, J = 8.8 Hz,
3
2
H), 6.77 (br s, 1H), 6.08 (br s, 1H), 4.51 (dd, J = 7.2 Hz, J = 2.2 Hz, 1H), 4.32 (dd, J = 10.0
1 2 1
Hz, J = 2.2 Hz, 1H), 4.03 (dd, J = 10.0 Hz, J = 7.2 Hz, 1H), 2.53 (t, J = 7.8 Hz, 2H), 1.63−1.42
2
1
2
13
(
m, 2H), 1.39−1.07 (br, 10H), 0.99−0.73 (m, 12H), 0.17 (s, 3H), 0.16 (s, 3H); C NMR (50
MHz, CDCl ): δ 174.2, 156.4, 135.4, 129.2, 114.2, 73.3, 70.7, 35.0, 31.9, 31.7, 29.5, 29.4, 25.8,
3
+
2
2.7, 18.1, 14.1, ꢀ4.50, ꢀ5.33; MS (ESI) m/z (%): 408 (100) [M + H] .
1
2
ꢀ(tertꢀButyldimethylsilyloxy)ꢀ3ꢀ(dodecyloxy)propanamide (21b). Yield 79%; White oil; H
NMR (200 MHz, CDCl ): δ 6.66 (br s, 1H), 6.60 (br s, 1H), 4.24 (dd, J = 6.2 Hz, J = 2.2, 1H),
3
1
2
3
.67 (dd, J = 10.0 Hz, J = 2.2, 1H), 3.52 (dd, J = 10.0 Hz, J = 6.2 Hz, 1H), 3.41 (t, J = 6.2 Hz,
1 2 1 2
1
3
2
H), 1.63–1.45 (m, 2H), 1.24 (br, 18H), 1.02–1.79 (m, 12H), 0.11 (s, 6H); C NMR (50 MHz,
CDCl ): δ 175.0, 74.0, 73.5, 71.6, 31.9, 29.6, 29.5, 29.4, 29.3, 26.0, 25.7, 22.6, 18.1, 14.1, ꢀ4.7, ꢀ
3
+
5
.4; MS (ESI) m/z (%): 388 (100) [M + H] .
Synthesis of thioamides 22a,b. Lawesson’s reagent (0.6 mmol, 243 mg) was added to a solution
of amides 21a,b (1 mmol) in dry toluene (10 mL) under argon atmosphere. The reaction mixture
was stirred at room temperature overnight. The solvent was evaporated under reduced pressure
and the residue was purified by flash chromatography eluting with the appropriate mixture of
EtOAc/petroleum ether (bp 40ꢀ60 °C).
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2ꢀ(tertꢀButyldimethylsilyloxy)ꢀ3ꢀ(4ꢀoctylphenoxy)propanethioamide (22a). Yield 40%; Pale
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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2
2
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1
yellow oil; H NMR (200 MHz, CDCl ): δ 8.22 (br s, 1H), 7.83 (br s, 1H), 7.09 (d, J = 8.8 Hz,
3
2H), 6.84 (d, J = 8.8 Hz, 2H), 4.89 (dd, J = 7.2 Hz, J = 2.2 Hz, 1H), 4.55 (dd, J = 9.9 Hz, J =
1
2
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4
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6
7
8
9
0
2
.2 Hz, 1H), 4.03 (dd, J = 9.9 Hz, J = 7.2 Hz, 1H), 2.54 (t, J = 7.4 Hz, 2H), 1.63−1.41 (m, 2H),
1
2
1
3
1.39−1.05 (m, 10H), 1.02−0.69 (m, 12H), 0.16 (s, 6H) ; C NMR (50 MHz, CDCl ): δ 205.4,
3
1
56.3, 135.5, 129.2, 114.3, 79.2, 72.6, 35.0, 31.9, 31.7, 29.5, 29.3, 25.8, 25.3, 22.6, 18.2, 14.1, ꢀ
+
4
.6, ꢀ5.2; MS (ESI) m/z (%): 424 (100) [M + H] .
2
ꢀ(tertꢀButyldimethylsilyloxy)ꢀ3ꢀ(dodecyloxy)propanethioamide (22b). Yield 39%; Pale
1
yellow oil; H NMR (200 MHz, CDCl ): δ 8.12 (br s, 1H), 7.97 (br s, 1H), 4.65 (dd, J = 6.2 Hz,
3
1
J = 2.6, 1H), 3.88 (dd, J = 10.0 Hz, J = 2.6, 1H), 3.58 (dd, J = 10.0 Hz, J = 6.2 Hz, 1H),
2
1
2
1
2
1
3
3
.51–3.38 (m, 2H), 1.63–1.45 (m, 2H), 1.25 (br, 18H), 1.05–1.82 (m, 12H,), 0.14 (s, 6H); C
NMR (50 MHz, CDCl ): δ 206.3, 80.1, 75.4, 71.7, 31.9, 29.6, 29.5, 29.4, 29.3, 26.0, 25.7, 22.6,
3
+
18.2, 14.1, ꢀ4.7, ꢀ5.3; MS (ESI) m/z (%): 404 (100) [M + H] .
Synthesis of thiazoles 23aꢀc. To a stirred solution of thioamides 22a,b (1.0 mmol) in EtOH (5
mL) was added ethyl bromopyruvate (1.2 mmol, 0.15 mL) or ethyl 4ꢀchloroacetoacetate (1.0
mmol, 0.14 mL) and c. H SO (0.04 mL), and the reaction mixture was refluxed overnight.
2
4
Organic solvent was evaporated under reduced pressure and the residue was purified by flash
column chromatography [EtOAcꢀpetroleum ether (bp 40ꢀ60 °C)].
Ethyl 2ꢀ(2ꢀ(1ꢀhydroxyꢀ2ꢀ(4ꢀoctylphenoxy)ethyl)thiazolꢀ4ꢀyl)acetate (23a). Yield 17%;
1
Yellowish oil; H NMR (200 MHz, CDCl ): δ 7.20 (s, 1H), 7.09 (d, J = 8.4 Hz, 2H), 6.85 (d, J =
3
8.4 Hz, 2H), 5.35 (dd, J = 7.0 Hz, J = 4.0, 1H), 4.38 (dd, J = 9.6 Hz, J = 4.0, 1H), 4.31–4.09
1
2
1
2
(
m, 3H), 3.82 (s, 2H), 2.54 (t, J = 7.4 Hz, 2H), 1.69–1.45 (m, 2H), 1.43–1.18 (m, 13H), 0.88 (t, J
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5
5
5
5
5
5
5
6
13
=
7.0 Hz, 3H); C NMR (50 MHz, CDCl ): δ 170.5, 170.3, 156.1, 148.5, 136.0, 129.3, 116.7,
3
1
14.5, 71.5, 70.6, 61.1, 36.9, 35.0, 31.8, 31.7, 29.4, 29.2, 22.6, 14.1; MS (ESI) m/z (%): 420
+
(100) [M + H] .
0
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Ethyl 2ꢀ(2ꢀ(2ꢀ(dodecyloxy)ꢀ1ꢀhydroxyethyl)thiazolꢀ4ꢀyl)acetate (23b). Yield 26%; Low mp
1
yellow solid; H NMR (200 MHz, CDCl ): δ 7.16 (s, 1H), 5.12 (dd, J = 6.8 Hz, J = 3.8 Hz, 1H),
3
1
2
4
.18 (q, J = 7.4 Hz, 2H), 3.83 (dd, J = 9.8 Hz, J = 3.8 Hz, 1H), 3.80 (s, 2H), 3.70–3.42 (m, 4H),
1 2
13
1
.65–1.46 (m, 2H), 1.40–1.12 (m, 21H), 0.87 (t, J = 6.8 Hz, 3H); C NMR (50 MHz, CDCl ): δ
3
171.3, 170.3, 148.4, 116.3, 74.1, 71.7, 70.9, 61.0, 37.0, 31.9, 29.6, 29.5, 29.4, 29.3, 26.0, 22.6,
+
1
4.1; MS (ESI) m/z (%): 400 (100) [M + H] .
Ethyl 2ꢀ(2ꢀ(dodecyloxy)ꢀ1ꢀhydroxyethyl)thiazoleꢀ4ꢀcarboxylate (23c). Yield 68%; Low mp
1
offꢀwhite solid; H NMR (200 MHz, CDCl ): δ 8.14 (s, 1H), 5.20 (dd, J = 7.0 Hz, J = 3.8 Hz,
3
1
2
1
H), 4.41 (q, J = 7.4 Hz, 2H), 3.92 (dd, J = 9.8 Hz, J = 3.8 Hz, 1H), 3.66 (dd, J = 9.8 Hz, J =
1 2 1 2
7.0 Hz, 1H), 3.60–3.42 (m, 3H), 1.68–1.49 (m, 2H), 1.39 (t, J = 7.2 Hz, 3H), 1.33−1.19 (m, 18H),
1
3
0
.87 (t, J = 6.6 Hz, 3H); C NMR (50 MHz, CDCl ): δ 173.0, 161.4, 147.0, 127.7, 73.7, 71.7,
3
+
7
0.9, 61.4, 31.9, 29.6, 29.5, 29.4, 29.3, 26.0, 22.6, 14.1; MS (ESI) m/z (%): 386 (100) [M + H] .
Methyl 2ꢀ(1ꢀ(tertꢀbutyldimethylsilyloxy)ꢀ2ꢀ(4ꢀoctylphenoxy)ethyl)ꢀ4,5ꢀdihydrothiazoleꢀ4ꢀ
carboxylate (mixture of diastereomers) (25). To a stirred solution of 20a (1.0 mmol, 390 mg)
ꢀ
+
and CH COO NH (3.6 mmol, 277 mg) in MeOH (4 mL), HCl.HꢀLꢀCysꢀOMe (3.0 mmol, 515
3
4
mg) was added, and the mixture was stirred overnight at room temperature. The organic solvent
was evaporated under reduced pressure and the residue was purified by flash column
1
chromatography [EtOAcꢀpetroleum ether (bp 40ꢀ60 °C), 1:9]. Yield 67%; White oil; H NMR
(
200 MHz, CDCl ): δ 7.07 (d, J = 8.4 Hz , 2H), 6.82 (d, J = 8.4 Hz , 2H), 5.25–5.07 (m, 1H),
3
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5.06–4.90 (m, 1H), 4.38–4.15 (m, 1H), 4.14–3.94(m, 1H), 3.82 (s, 3H), 3.63–3.33 (m, 2H), 2.54
4
5
6
7
8
9
1
1
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1
1
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1
1
2
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(
t, J = 7.8 Hz, 2H), 1.70–1.45 (m, 2H), 1.43–1.16 (br, 10H), 1.05–0.80 (m, 12H), 0.22–0.10 (m,
1
3
6H); C NMR (50 MHz, CDCl ): δ 178.6 (178.0), 171.1, 156.4, 135.3, 129.1, 114.3, 78.3 (78.2),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
2.5 (72.4), 71.5, 52.7 (52.6), 35.0, 33.7, 31.8, 31.7, 29.4, 29.2, 25.7, 22.6, 18.2, 14.1, ꢀ4.7, ꢀ5.2;
+
MS (ESI) m/z (%): 508 (100) [M + H] .
Methyl 2ꢀ(1ꢀ(tertꢀbutyldimethylsilyloxy)ꢀ2ꢀ(4ꢀoctylphenoxy)ethyl)thiazoleꢀ4ꢀcarboxylate
(26). To a solution of 25 (1 mmol, 508 mg) in CH Cl (20 mL), BrCCl (6.0 mmol, 0.59 mL) and
2 2 3
o
o
DBU (6.0 mmol, 0.90 mL) were added at 0 C. The reaction was stirred for 2 h at 0 C and
overnight at room temperature. The organic solvent was evaporated under reduced pressure and
the residue was purified by flash column chromatography [EtOAcꢀpetroleum ether (bp 40ꢀ60 °C),
1
1:9]. Yield 82%; White oil; H NMR (200 MHz, CDCl ): δ 8.18 (s, 1H), 7.07 (d, J = 8.6 Hz, 2H),
3
6
7
3
7
.81 (d, J = 8.6 Hz, 2H), 5.53–5.40 (m, 1H), 4.51–4.37 (m, 1H), 4.12–3.90 (m, 4H), 2.54 (t, J =
.8 Hz, 2H), 1.70–1.45 (m, 2H), 1.44–1.14 (br, 10H), 1.07–0.79 (m, 12H), 0.17 (s, 3H), 0.15 (s,
1
3
H); C NMR (50 MHz, CDCl ): δ 174.5, 161.9, 156.5, 146.8, 135.4, 129.2, 128.0, 114.3, 72.7,
3
2.6, 52.4, 35.0, 31.9, 31.7, 29.5, 29.3, 25.7, 22.7, 18.3, 14.1, ꢀ4.5, ꢀ5.2; MS (ESI) m/z (%): 506
+
(100) [M + H] .
Methyl 2ꢀ(1ꢀhydroxyꢀ2ꢀ(4ꢀoctylphenoxy)ethyl)thiazoleꢀ4ꢀcarboxylate (27). Compound 26 (1.0
mmol, 505 mg) was treated with a solution of 4N HCl in MeOH. The organic solvent was
evaporated under reduced pressure and the residue was recrystallized from ether/petroleum ether
o
1
(bp 40ꢀ60 °C). Yield 95%; White solid; mp 84ꢀ86 C; H NMR (200 MHz, CDCl ): δ 8.18 (s,
3
1
H), 7.06 (d, J = 8.0 Hz, 2H), 6.82 (d, J = 8.0 Hz, 2H), 5.60–5.28 (m, 1H), 4.57–4.36 (m, 1H),
4.31–4.12 (m, 1H), 4.08–3.82 (m, 4H), 2.52 (t, J = 7.8 Hz, 2H), 1.69–1.43 (m, 2H), 1.42–1.11
13
(br, 10H), 0.88 (t, J = 6.8 Hz, 3H); C NMR (50 MHz, CDCl ): δ 172.4, 161.7, 155.8, 146.5,
3
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36.0, 129.2, 128.2, 114.4, 71.2, 70.6, 52.5, 34.9, 31.8, 31.6, 29.4, 29.2, 22.6, 14.0; MS (ESI) m/z
+
(%): 392 (100) [M + H] .
Synthesis of thiazoles 18aꢀc, 24aꢀc and 28. To a solution of compounds 17aꢀc, 23aꢀc and 27 (1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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6
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2
3
4
5
6
7
8
9
0
mmol) in dry CH Cl (10 mL) DessꢀMartin periodinane was added (1.5 mmol, 637 mg) and the
2
2
mixture was stirred for 1 h at room temperature. The organic solvent was evaporated under
reduce pressure and Et O (30 mL) was added. The organic phase was washed with saturated
2
aqueous NaHCO (20 mL) containing Na S O (1.5 g, 9.5 mmol), H O (20 mL), dried over
3
2
2
3
2
Na SO , and the organic solvent was evaporated under reduced pressure. The residue was
2
4
purified by column chromatography using petroleum ether (bp 40ꢀ60 °C)/EtOAc as eluent.
o
2ꢀ(Biphenylꢀ4ꢀyloxy)ꢀ1ꢀ(thiazolꢀ2ꢀyl)ethanone (18a). Yield 82%; White solid; mp 130ꢀ133 C;
1
H NMR (200 MHz, CDCl ): δ 8.09–8.03 (m, 1H), 7.80–7.74 (m, 1H), 7.63–7.23 (m, 7H), 7.12–
3
13
7
.00 (m, 2H), 5.57 (s, 2H); C NMR (50 MHz, CDCl ): δ 187.4, 164.4, 157.5, 145.0, 140.6,
3
+
134.8, 128.7, 128.2, 126.8, 115.1, 70.2; HRMS (ESI) calcd for C H NNaO S [M + Na] :
17 13
2
318.0559. Found: 318.0568.
o
1
2ꢀ(4ꢀOctylphenoxy)ꢀ1ꢀ(thiazolꢀ2ꢀyl)ethanone (18b). Yield 79%; White solid; mp 65ꢀ67 C; H
NMR (200 MHz, CDCl ): δ 8.06 (d, J = 3.0 Hz, 1H), 7.76 (d, J = 3.0 Hz, 1H), 7.11 (d, J = 8.4
3
Hz, 2H), 6.92 (d, J = 8.4 Hz, 2H), 5.52 (s, 2H), 2.55 (t, J = 7.8 Hz, 2H), 1.71–1.46 (m, 2H), 1.42–
1
3
1.10 (m, 10H), 0.89 (t, J = 7.0 Hz, 3H); C NMR (50 MHz, CDCl ): δ 187.6, 164.2, 156.0,
3
145.0, 136.2, 129.3, 126.6, 114.7, 70.3, 35.0, 31.9, 31.6, 29.4, 29.2, 22.6, 14.1; HRMS (ESI)
+
calcd for C H NNaO S [M + Na] : 354.1498. Found: 354.1507.
1
9
25
2
1
ꢀ(Benzo[d]thiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀoctylphenoxy)ethanone (18c). Yield 75%; White solid; mp 80ꢀ82
o
1
C; H NMR (200 MHz, CDCl ): δ 8.26–8.17 (m, 1H), 8.08–7.96 (m, 1H), 7.69–7.51 (m, 2H),
3
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7.13 (d, J = 8.7 Hz, 2H), 6.96 (d, J = 8.7 Hz, 2H), 5.64 (s, 2H), 2.56 (t, J = 7.8 Hz, 2H), 1.72–
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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2
2
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4
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4
4
4
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4
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5
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5
5
5
5
5
5
5
6
1
3
1
.46 (m, 2H), 1.43–1.14 (m, 10H), 0.89 (t, J = 7.0 Hz, 3H); C NMR (50 MHz, CDCl ): δ 189.2,
3
163.5, 155.9, 153.4, 136.9, 136.3, 129.4, 128.0, 127.3, 125.5, 122.5, 114.7, 70.5, 35.1, 31.9, 31.6,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
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7
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9
0
1
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
2
9.5, 29.3, 22.7, 14.1; HRMS (ESI) calcd for C H NNaO S [M + Na] : 404.1655. Found:
2
3
27
2
404.1664.
Ethyl 2ꢀ(2ꢀ(2ꢀ(4ꢀoctylphenoxy)acetyl)thiazolꢀ4ꢀyl)acetate (24a). Yield 78%; Low mp white
1
solid; H NMR (200 MHz, CDCl ): δ 7.66 (s, 1H), 7.10 (d, J = 8.2 Hz, 2H), 6.90 (d, J = 8.0 Hz,
3
2H), 5.48 (s, 2H), 4.23 (q, J = 7.2 Hz, 2H), 3.93 (s, 2H), 2.54 (t, J = 7.6 Hz, 2H), 1.66ꢀ1.44 (m,
13
2
1
2
H), 1.40−1.14 (m, 13H), 0.88 (t, J = 7.0 Hz, 3H); C NMR (50 MHz, CDCl ): δ 187.5, 169.8,
3
63.2, 155.9, 151.5, 136.2, 129.3, 124.3, 114.7, 70.3, 61.3, 36.8, 35.0, 31.8, 31.6, 29.4, 29.2,
+
2.6, 14.1; HRMS (ESI) calcd for C H NNaO S [M + Na] : 440.1866. Found: 440.1883.
2
3
31
4
Ethyl 2ꢀ(2ꢀ(2ꢀ(dodecyloxy)acetyl)thiazolꢀ4ꢀyl)acetate (24b). Yield 49%; Low mp white solid;
1
H NMR (200 MHz, CDCl ): δ 7.59 (s, 1H), 4.94 (s, 2H), 4.21 (q, J = 7.0 Hz, 2H), 3.89 (s, 2H),
3
1
3
3
.60 (t, J = 6.6 Hz, 2H), 1.75–1.59 (m, 2H), 1.45–1.18 (m, 21H), 0.88 (t, J = 7.0 Hz, 3H); C
NMR (50 MHz, CDCl ): δ 189.6, 169.8, 163.8, 151.3, 123.7, 73.1, 72.2, 61.3, 36.9, 31.9, 29.6,
3
+
29.5, 29.4, 29.3, 25.9, 22.7, 14.1; HRMS (ESI) calcd for C H NNaO S [M + Na] : 420.2179.
2
1
35
4
Found: 420.2211.
Ethyl 2ꢀ(2ꢀ(dodecyloxy)acetyl)thiazoleꢀ4ꢀcarboxylate (24c). Yield 84%; Pale yellow solid; mp
o
1
5
8ꢀ61 C; H NMR (200 MHz, CDCl ): δ 8.44 (s, 1H), 5.04 (s, 2H), 4.44 (q, J = 7.2 Hz, 2H),
3
13
3
.60 (t, J = 6.6 Hz, 2H), 1.76–1.58 (m, 2H), 1.46–1.17 (m, 21H), 0.86 (t, J = 6.6 Hz, 3H); C
NMR (50 MHz, CDCl ): δ 189.7, 164.9, 160.6, 148.8, 132.9, 73.1, 72.2, 61.9, 31.9, 29.6, 29.5,
3
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+
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9.4, 29.3, 25.9, 22.6, 14.1; HRMS (ESI) calcd for C H NNaO S [M + Na] : 406.2023. Found:
20 33 4
4
06.2040.
Methyl 2ꢀ(2ꢀ(4ꢀoctylphenoxy)acetyl)thiazoleꢀ4ꢀcarboxylate (28). Yield 93%; Pale yellow
0
1
2
3
4
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7
8
9
0
1
2
3
4
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7
8
9
0
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0
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0
o
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solid; mp 69ꢀ71 C; H NMR (600 MHz, CDCl ): δ 8.51 (s, 1H), 7.10 (d, J = 8.6 Hz, 2H), 6.91
3
(
d, J = 8.6 Hz, 2H), 5.57 (s, 2H), 4.00 (s, 3H), 2.54 (t, J = 7.8 Hz, 2H), 1.61–1.53 (m, 2H), 1.35–
1
3
1
1
.21 (m, 10H), 0.88 (t, J = 7.0 Hz, 3H); C NMR (150 MHz, CDCl ): δ 187.6, 164.5, 161.0,
3
55.8, 148.6, 136.3, 133.5, 129.3, 114.8, 70.4, 52.7, 35.0, 31.8, 31.6, 29.4, 29.2, 22.6, 14.1;
+
HRMS (ESI) calcd for C H NNaO S [M + Na] : 412.1553. Found: 412.1556.
2
1
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4
Biology. Recombinant human ILꢀ1β was from Roche (UK). Phosphateꢀbuffered saline solution
3
3
(
PBS) was from Oxoid (UK). Labeled HꢀAA ([5,6,8,9,11,12,14,15ꢀ H]ꢀarachidonic acid
14
14
(
specific activity 180ꢀ240 Ci/mmol)), CꢀOA ([1ꢀ C]ꢀoleic acid (specific activity 40ꢀ60
14
Ci/mmol)), Lꢀαꢀ1ꢀpalmitoylꢀ2ꢀarachidonylꢀ[arachidonylꢀ1ꢀ C]ꢀphosphatidylcholine (specific
activity 40ꢀ60 Ci/mmol), and liquid scintillation cocktail Ultima Gold were from NEN Perkin
Elmer (USA). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), fatty
acidꢀfree bovine serum albumin (fBSA), dimethylꢀsulfoxide (DMSO), gentamicin and Lꢀ
glutamine were from SigmaꢀAldrich (USA). Enzyme immunoassay (EIA) kit for PGE analysis
2
was from Cayman Chemicals (USA).
In vitro mixed micelle assay. The activity of GIVA cPLA , GVIA iPLA and GV sPLA were
2
2
2
2
9ꢀ31
determined using a modified Dole Assay.
The buffer and substrate conditions were optimized
for each enzyme assay as follows: (i) GIVA cPLA substrate mixedꢀmicelles were composed of
2
14
4
00 µM Triton Xꢀ100, 97 µM PAPC, 1.8 µM Cꢀlabeled PAPC, and 3 µM PIP in 100 mM
2
HEPES buffer, pH 7.5, with 90 µM CaCl , 2 mM DTT, and 0.1 mg/mL BSA; (ii) GVIA iPLA2
2
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substrate mixedꢀmicelles were composed of 400 µM Triton Xꢀ100, 98.3 µM PAPC, and 1.7 µM
1
4
Cꢀlabeled PAPC in buffer containing 100 mM HEPES, pH 7.5, 2 mM ATP, and 4 mM DTT;
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(iii) GV sPLA substrate mixedꢀmicelles were composed of 400 µM Triton Xꢀ100, 98.3 µM
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PAPC, and 1.7 µM Cꢀlabeled PAPC in buffer containing 50 mMTris, pH 8.0, and 5 mM 550
CaCl2.
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48
In vitro vesicle assay. GIVA cPLA was measured as described
with modifications. In
2
short, recombinant human GIVA cPLA enzyme was preꢀincubated with DMSO (1%) with or
2
without inhibitor in assay buffer (80 sec at 37 °C, 10 min at 25°C). Lipid vesicles of Lꢀαꢀ1ꢀ
1
4
palmitoylꢀ2ꢀarachidonylꢀ[arachidonylꢀ1ꢀ C]ꢀphosphatidylcholine (4.3 nmol) were dried under a
stream of N (g). The dried lipid was reꢀsuspended in 2 mL assay buffer and sonicated twice (7
2
min, output 3.5 and 50% duty cycles, on ice) in a Branson Sonifier 250 (Branson Ultrasonic
Corporation, Danbury, CT). Sonicated lipid (0.2 ꢁM) was added to the reaction and incubated for
1
h at 37 °C followed by the addition of a chloroform/methanol buffer to terminate the enzymatic
reaction. The reaction mixture was separated by centrifugation (5 min, 1640 x g). The lower
phase was transferred to a glass tube and dried under a steam of N (g), reꢀsuspended in
2
1
4
chloroform/methanol (9:1, by volume) and applied to a silica gel. Free [1ꢀ C]arachidonic acid
1
4
and, Lꢀαꢀ1ꢀpalmitoylꢀ2ꢀarachidonylꢀ[arachidonylꢀ1ꢀ C]ꢀ phosphatidylcholine were separated by
49
thin layer chromatography and analysed as previously described.
Cell culture. The human synovial sarcoma cell line SW982 was from ATCC (UK) and was used
as a model system to monitor AA/OA release and activation of GIVA cPLA . SW982 cells were
2
passaged biꢀweekly by routine trypsin detachment and maintained in a subꢀconfluent state as
5
8
previously described. Experiments were performed at 3 days postꢀconfluency following
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overnight serum deprivation in serumꢀfree DMEM to ensure differentiation and synchronization
of the cells.
Ex vivo cellular AA and OA release assay. AA and OA release was analyzed as previously
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described. At 2 days postꢀconfluency, SW982 cells were serumꢀstarved and labeled overnight
3
14
with HꢀAA (0.4 ꢀCi/mL) and CꢀOA (0.067 µCi/mL) in serumꢀfree DMEM. Prior to the
addition of the thiazolyl ketone inhibitors, the cells were washed twice with PBS containing
fBSA (2 mg/mL) and PBS in order to remove unincorporated radioactivity. Cells were pretreated
(1 h) with thiazolyl ketones prior to ILꢀ1β stimulation (10 ng/mL, 4 h). Following ILꢀ1β
stimulation, the supernatants were cleared of detached cells by centrifugation (13000 rpm, 5
3
14
min). The release of HꢀAA and CꢀOA from the cells was assessed by liquid scintillation
counting (LS 6500 MultiꢀPurpose Scintillation Counter, Beckman Coulter, Inc (USA). Adherent
3
14
cells were dissolved in 1N NaOH in order to determine incorporated HꢀAA and CꢀOA in the
cells by liquid scintillation counting. In all experiments, DMSO was included for vehicle control
(>0.05%). Following treatments, cells were routinely observed by microscopy to ensure unaltered
3
cell morphology, integrity and viability. The results are given as inhibition of released HꢀAA
1
4
3
14
and CꢀOA in the supernatants relative to total HꢀAA and CꢀOA incorporated into the cells,
from at least three independent experiments performed in triplicate.
PGE analysis. PGE EIA analysis of blood plasma from the prophylactic and therapeutic CIA
2
2
studies was performed according to the kit protocol. Plasma samples were diluted 1:1000ꢀ1:6000
in EIA buffer and allowed to hybridize overꢀnight (18 h, 4 °C). The plate was read using a
Multiscan plate reader (Ascent Labsystems) (OD550 nm). The corresponding Ascent software for
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Multiscan, Version 2.4.1 was used to obtain the data. PGE levels for all treatments are shown
2
relative to the DMSOꢀtreated vehicle arthritic mice (n=10ꢀ11 mice in each category ± SD).
In vivo studies of inhibitor 28. All in vivo studies were conducted in accordance with Standard
Operating Procedures (SOP) and generally accepted procedures for the testing of pharmaceutical
compounds.
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Separate prophylactic and therapeutic efficacy studies of inhibitor 28 were performed. MTX
(Jiangsu Hengrui Medicine Co, #11041411), Enbrel (Boehringer Ingelheim Pharma KG,
#F39487), and vehicle (DMSO 100%, Sigma Aldrich #D2650) were administered to all groups
via intraperitoneal injection once daily at a dose volume of 2 mL/Kg. Clinical observations were
conducted daily during the study. Body weights were measured and recorded prior to
randomization and then once daily during the study. Food consumption was measured and
recorded daily. Biopsies for midꢀterm histology analysis were obtained on day 13 in the
prophylactic study. Necropsy examinations were performed at study termination in both
prophylactic and therapeutic study modes; plasma samples were collected, organ weights were
recorded and hindpaws were collected for histopathology analyses.
Induction of CIA. For the prophylactic and the therapeutic studies, CIA was induced in male
DBA/1 mice (except naïve mice) by immunization with 0.1 mL emulsion containing an equal
volume of bovine type II collagen solution (2 mg/mL) and Freuds Complete Adjuvant at the tail
base. The first injection was given on day 0 and the second injection as booster was given on day
50ꢀ52
21.
Inhibitor 28, vehicle (DMSO) and MTX (0.3 mg/Kg) were administered daily, Enbrel (25
mg/Kg) was administrated twice a week. For the prophylactic study, treatment started one hour
before the second collagen injection and continued for 21 days except for the histology groups
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that were sacrificed at day 13 (33 days after immunization). For the therapeutic study, treatment
started at day 28 and continued for 14 days.
CIA assessment and treatment. CIA was assessed in mice by two blinded observers to measure
paw swelling with a capacity measurement method. The occurrence of arthritis was observed by
scoring all paws for severity of erythema and swelling, using a clinical score ranging from 0 (no
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swelling) to 4 (severe swelling and erythema), i.e. yielding maximum AI score 16.
Measurement of the histopathology and clinical observations. At the end of the studies, one
hind foot of mice in each group was collected for histopathology. The foot including the ankle
was fixed in 10% neutral formalin. The ankle joints were decalcified, dehydrated, embedded in
paraffin, sectioned and stained with routine hematoxylinꢀeosin. Arthritis damage (histological
damage score) was evaluated by light microscopy and scored by an investigator blinded for the
treatment regimen. The following histopathology parameters were evaluated: 1) Articular cavity
and peripheral tissue inflammatory cell infiltration; 2) Capillary and synovial hyperplasia; and 3)
Articular cartilage surface damage, each using a 0ꢀ5 grading system: "0" none; "1" minimal; "2"
mild; "3" moderate; "4" marked; "5" severe damage.
Terminal studies. All animals completed the scheduled test periods and were disposed with
carbon dioxide and subjected to necropsy, supervised by a pathologist. The sacrifices were
performed at approximately 5 hours after the last ip injection. A macroscopic examination of the
animal was performed on all sacrificed animals and any abnormality was recorded.
Statistical analysis. Data of groups was examined by oneꢀway analysis of variance, and
individual groups were then compared with Student's unpaired tꢀtest. Data was given as
Mean±SD, if no particular indication was made. p<0.05 was considered significant.
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Pharmacokinetic studies. Compound 28 was tested in pilot pharmacokinetic studies in 5 weeks
old Swiss male mice (around 30 g). Two groups of 12 mice were injected with 5 or 30 mg/Kg of
compound 28 via intravenous (bolus injection via the lateral tail vein) or intraperitoneal routes
respectively (6 mice per route, 2 sampling times per mouse and 2 mice per time point). For iv
administration, an aqueousꢀbased solution of compound 28 (1 mg/mL) was administered to mice
at a final volume of 5 mL/Kg. All ip administrations were done in formulations containing
compound 28 at 15 mg/mL in 100% DMSO at a final volume of 2 mL/Kg. Four additional mice
injected with vehicle controls (2 mice for each administration route) were also included in the
study.
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Blood samples (~200ꢀ300 µL/sample) were collected via sinus retroꢀorbital collection using
capillary tubes containing lithium heparin and were stored on ice until centrifuged for 5 min at
2500 rpm at around 2ꢀ8 °C. The resulting plasma was separated and placed into labeled
polypropylene tubes stored frozen at ꢀ80 °C prior to analysis. Overall, 6 time points were chosen:
at postꢀdose at 30 min, 1 h, 2 h, 4 h, 8 h and 24 h. For vehicle controls, all samples were collected
2
hours after vehicle administration.
Isolated plasma samples were analyzed by standard LCꢀMS/MS; the method had the lowest level
of detection (LLOQ) at 2ng/mL. Each time point analyzed comprised individual measurements
from samples collected from 2 different mice per time point. Two main parameters were
calculated: maximum plasma concentration (Cmax) and Area Under the Curve from the time of
dosing to the last measurable concentration (AUC(0ꢀt)) using nonꢀcompartmental analysis.
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