Discovery of novel benzoxazinones as potent and orally active long chain fatty acid elongase 6 inhibitors

Article abstract of DOI:10.1021/jm900915x

A series of benzoxazinones was synthesized and evaluated as novel long chain fatty acid elongase 6 (ELOVL6) inhibitors. Exploration of the SAR of the UHTS lead 1a led to the identification of (S)-1y that possesses a unique chiral quarternary center and a

Full text of DOI:10.1021/jm900915x

J. Med. Chem. 2009, 52, 7289–7300 7289
DOI: 10.1021/jm900915x
Discovery of Novel Benzoxazinones as Potent and Orally Active Long Chain Fatty Acid Elongase 6
Inhibitors
Takashi Mizutani,* Shiho Ishikawa, Tsuyoshi Nagase, Hidekazu Takahashi, Takashi Fujimura, Takahide Sasaki,
Akira Nagumo, Ken Shimamura, Yasuhisa Miyamoto, Hidefumi Kitazawa, Maki Kanesaka, Ryo Yoshimoto,
Katsumi Aragane, Shigeru Tokita, and Nagaaki Sato
Tsukuba Research Institute, Merck Research Laboratories, Banyu Pharmaceutical Co., Ltd, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan
Received June 22, 2009
A series of benzoxazinones was synthesized and evaluated as novel long chain fatty acid elongase 6
(ELOVL6) inhibitors. Exploration of the SAR of the UHTS lead 1a led to the identification of (S)-1y that
possesses a unique chiral quarternary center and a pyrazole ring as critical pharmacophore elements.
Compound (S)-1y showed potent and selective inhibitory activity toward human ELOVL6 while
displaying potent inhibitory activity toward both mouse ELOVL3 and 6 enzymes. Compound (S)-1y
showed acceptable pharmacokinetic profiles after oral dosing in mice. Furthermore, (S)-1y significantly
suppressed the elongation of target fatty acids in mouse liver at 30 mg/kg oral dosing.
Introduction
So far, seven ELOVL enzymes have been identified in
mammals and are designated ELOVL1-7.7-12 Each ELOVL
enzymeexhibitsdifferentfatty acyl-CoA substrate preferences
and different tissue distributions, suggesting that they play
distinct physiological roles in vivo.13 Among ELOVL family
members, ELOVL6 (known as LCE, FACE) shows the high-
est amino acid sequence homology to ELOVL3 in humans
and mice (46% and 44%, respectively).14a Not surprisingly,
ELOVL3 and ELOVL6 show overlapped substrate specifi-
city: ELOVL6 regulates the elongation of saturated and
monosaturated fatty acyl-CoAs with C12-16,5-8 whereas
ELOVL3 regulates the synthesis of saturated and monounsa-
turated fatty acyl-CoAs with more broad range of chain
length including very long chain fatty acyl-CoAs with
C16-C24.15
The incidence of type 2 diabetes has dramatically increased
over the past decade. Accumulated evidence suggests a strong
correlation between insulin resistance and the development of
type 2 diabetes mellitus. An increase in de novo lipid synthesis
and fat storage in tissues such as liver leads to dysfunction in
those tissues (i.e., insulin resistance).1 Although it is unclear
how an increased intracellular lipid content deteriorates tissue
and whole body insulin sensitivity, it has been suggested that
increased levels of long chain fatty acyl-CoAs antagonize the
metabolic actions of insulin.2 Interestingly, recent reports
suggested that alternation of the specific fatty acid component
(i.e., palmitoleate) has a significant impact on the insulin
sensitivity of the liver and the whole body.3,4
With regard to de novo lipid synthesis, microsomal en-
zymes are responsible for the elongation of long chain fatty
acyl-CoA with chain lengths over C16, while fatty acid
syntheses in the cytosol are responsible for the de novo
synthesis of the fatty acyl-CoAs with chain lengths up to
C16.5,6 Fatty acyl-CoA elongation at microsomal fractions
requires four sequential steps: (1) condensation between fatty
acyl-CoA and malonyl-CoA to generate β-ketoacyl-CoA by
ELOVLa (long chain fatty acid elongase); (2) reduction by
β-ketoacyl-CoA reductase; (3) dehydrogenation by β-hydro-
xyacyl-CoA dehydrogenase; (4) reduction by trans-2,3-enoyl-
CoA reductase.5,6 ELOVL family enzymes are responsible for
the rate-limiting and initial condensation reaction.7,8
While ELOVL3 and ELOVL6 display unique respective
expression patterns, they both are expressed in highly lipo-
genic tissues such as liver and adipose. Regarding the regula-
tory mechanism for expression, liver ELOVL6 expression is
up-regulated by feeding after fasting, high carbohydrate diet,
and obesity in rodents, suggesting that ELOVL6 expression is
controlled by energy balance and nutrient inputs.7,8,16 In
contrast, liver ELOVL3 expression is under circadian control:
ELOVL3 showshighexpressions inthe inactivephaseandlow
expressions in the active phase.17 Given their primary roles in
the elongation of long chain fatty acyl-CoAs (e.g., palmitoyl-
CoA) and expressions in lipogenic tissues, ELOVL3 and
ELOVL6 are considered to play important physiological roles
in the lipid metabolism. In fact, ELOVL6 regulates hepatic
insulin sensitivity by modulating the fatty acid components,4
while ELOVL3 plays important roles in the regulation of
triglyceride formation in skin and brown adipose tissue and
regulates thermogenesis.15,21 Although the increasing infor-
mation regarding the physiological and pathological roles of
ELOVLs garnered much attention from research scientists,
lackofchemicaltools precludedtheirabilitytofurther address
*Phone: 81-29-877-2000. FAX: 81-29-877-2029. Email: miztnitk@
gmail.com.
a Abbreviations: ELOVL, long chain fatty acid elongase; SAR,
structure-activityrelationship; hELOVL, long chain fatty acid elongase
in human; mELOVL, long chain fatty acid elongase in mice; UHTS,
ultra-high-throughput screening; PMB, p-methoxybenzyl; CAN, cerium
ammonium nitrate; SD rat, Sprague-Dawley rat; LDA, lithium diiso-
propylamide; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
HOBt, 1-hydroxybenzotriazole.
r
2009 American Chemical Society
Published on Web 11/02/2009
pubs.acs.org/jmc

7290 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Mizutani et al.
Figure 1
Scheme 1a
a Reagents and conditions: (a) LDA, THF, -40 ꢀC, 1 h; ethyl trifluoroacetate, -78 ꢀC, 1 h, 62%; (b) 4-methoxybenzylamine, K2CO3, toluene, reflux,
12 h, 83%; (c) (i) vinylmagnesium bromide, THF, 0 ꢀC, 1 h; (ii) triphosgene, triethylamine, toluene, 0 ꢀC, 30 min, 83% in 2 steps; (d) O3, CH2Cl2, MeOH,
-78 ꢀC, 2 h; then NaBH4, 0 ꢀC, 10 min, 87%; (e) (i) Tf2O, 2,6-lutidine, CHCl3, THF, 0 ꢀC, 1 h; (ii) NaN3, DMF, 80 ꢀC, 3 h, 71% in 2 steps; (f) (i)
trimethylphosphite, THF, H2O, 60 ꢀC, 12 h; (ii) 4 N HCl in dioxane, rt, 24 h, 71% in 2 steps; (g) 4-fluorobenzoic acid, EDCI HCl, HOBt H2O, TEA,
3
3
DMF, 0 ꢀC, 12 h, 90%; (h) CAN, CH3CN, H2O, rt, 4 h, 90%; (i) cross coupling with aryl boronic acids or aryl zinc reagents.
Scheme 2a
the pharmacological roles of ELOVLs and their therapeutic
potentials.
In order to identify chemical tools for ELOVL enzyme
studies, we recently established a homogeneous enzyme assay
for ELOVL6, which is applicable to UHTS using the recom-
binant histidine-tagged acyl-CoA binding protein as a mole-
cular probe for the detection of radioactive products of
ELOVL6.22 The UHTS of our corporate sample collections
resulted in the discovery of a benzoxazinone class of lead 1a
(Figure 1). Importantly, 1a shares the core benzoxazinone
structure with Efavirenz, a commercial non-nucleoside HIV-1
reverse transcriptase inhibitor, suggesting druggability of the
structure of 1a.23
a Reagents and conditions: (a) CuI, NaI, N,N’-dimethylethylenedi-
amine, 1,4-dioxane, 110 ꢀC, 4.5 h, 78%; (b) (i) nitrogen nucleophiles,
CuI, K3PO4, N,N’-dimethylethylenediamine, DMF, 110 ꢀC, 1 h; (ii)
AlCl3, anisole, 100 ꢀC, 2 h.
group of 5 and reductive workup of the resultant ozonide gave
alcohol 6. Alcohol 6 was converted to the correspond-
ing amine 8 via azide 7. Condensation of the amine 8 with
4-fluorobenzoic acid followed by deprotection of the PMB
group afforded 10. A series of the amide derivatives were
synthesized in a similar manner. Cross coupling of compound
10 with appropriate boronic acids or aryl zinc reagents
furnished a variety of 6-substituted derivatives (Scheme 1).
Alternatively, the bromide 9 was converted to the correspond-
ing iodide 11, which was treated with various nitrogen nucleo-
philes to furnish the target compounds after cleavage of the
PMB group (Scheme 2). Optical resolution of the representa-
tive potent compound 1 and synthetic intermediate 10 was
conducted to identify and evaluate active isomers by using
chiral stationary HPLC.
We have recently reported the discovery of two structurally
diverse indoledione and 3-sulfonyl-8-azabicyclo[3.2.1]octane
derivatives as ELOVL6 selective inhibitors.14b,c In this report,
we describe the discovery of a structurally divergent benzoxa-
zinone class of ELOVL6 inhibitors. The advanced derivative
(S)-1y (Figure 1) was found to possess potent and selective
inhibitory activity toward human ELOVL6 and potent dual
inhibitory activity toward mouse ELOVL3 and ELOVL6. The
in vitro selectivity and in vivo profiling of (S)-1y is also reported.
Chemistry
The general synthetic route for compounds 1 is shown in
Schemes 1 and 2. 4-Bromofluorobenzene (2) was selectively
deprotonated with LDA, followed by trifluoroacetylation to
give trifluoroacetophenone3. The activated fluorineatom of3
was substituted with p-methoxybenzylamine to give 4. Vinyla-
tion of 4, followed by cyclization in the presence of triphos-
gene, gave the benzoxazinone core 5. Ozonolysis of the vinyl
Results and Discussion
Structure-Activity Relationships. SAR exploration of
the benzoxazinone lead 1a began with replacement of the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7291
Table 1. SAR on the Substituents on the Quarternary Carbon 1a-f a
Table 3. SAR on the Right-Hand Phenyl Moiety (1i-s) and Left-Hand
6-Substitution (1t-ae)a
compound
R
hELOVL6 IC50 (nM)
compound
R1
4-F
R2
hELOVL6 IC50 (nM)
1a
1b
1c
1d
1e
1f
CF3
Me
Et
142 ( 16b
1i
Cl
Cl
Cl
Cl
Cl
Cl
26 ( 2
261 ( 20
539 ( 41
38 ( 2
1364c
1j
3-F
2-F
2255c
1k
1l
i-Pr
cyclopropyl
Ph
5031c
4-Me
3-Me
2-Me
1414c
1m
1n
1o
1p
1q
1r
437 ( 89
971 ( 405
558 ( 120
813 ( 362
1869 ( 57
>10 000
>10 000
95 ( 39
1479 ( 107
106 ( 14
121 ( 47
22 ( 3
>10 000c
a Inhibitory activity of compounds on human ELOVL6 for palmi-
toyl-CoA elongation. b The values represent the mean ( SE for n g 3.
c The values are means of two independent experiments.
4-MeO Cl
4-CF3 Cl
4-i-Pr
4-Ph
4-Ms
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
Cl
Cl
Cl
H
Table 2. SAR on the Side Chain Unit 1a and 1g-ia
1s
1t
1u
1v
Ph
1-(2-pyridone)
4-isoxazol
4-pyrazol
1w
1x
1y
1z
3-pyrazol
1-pyrazol
4.0 ( 1
26 ( 1
97 ( 9
572 ( 252
537 ( 239
39 ( 4
1aa
1ab
1ac
1ad
1ae
5-(1,2,4-triazol)
1-(1,2,4-triazol)
2-imidazol
1-(2-pyrrolidone)
3-(1,3-oxazolidin-2-one)
67 ( 7
a Inhibitory activity of compounds on human ELOVL6 for palmi-
toyl-CoA elongation. The values represent the mean ( SE for n g 3.
potent inhibitory activity. Importantly, the amide bond of 1i is
stable after microsomal incubation, while the urea linkage of
1g is relatively unstable (data not shown); therefore, 1i was
selected as a benchmark compound for further optimization.
Next, substituent effects on the benzamide group of 1i were
systemically investigated (1i-s, Table 3). Of the fluoro deri-
vatives (1i-k), the 2- and 3-fluoro derivatives 1j and 1k showed
markedly decreased potency compared to the 4-fluoro deriva-
tive 1i. This trend was also observed in the methyl derivatives
1l-n. These results clearly suggest that substitutions at the
2- and 3-positions are unfavorable. Thus, modification efforts
were focused on the 4-position. A variety of para-substituted
derivatives such as the methoxy (1o), trifluoromethyl (1p),
isopropyl (1q), phenyl (1r), and methanesulfonyl (1s) deriva-
tives displayed substantially decreased potency, indicating that
bulky substituents are not tolerated at the 4-position. Next, the
6-substituent (R2 group) on the core benzoxazinone ring was
intensively modified using 1i as a template (1t-ae, Table 3).
The deschloride derivative 1t showed a 4-fold decrease in
potency compared to 1i, and replacement of the chloride by
a phenyl group as in 1u resulted in a further decrease in
potency. Interestingly, the pyridone derivative 1v regained
hELOVL6 inhibitory activity, which is as potent as the
deschloride derivative 1t. From these observations, we hypo-
thesized that small and relatively polar substituents might be
optimal for this position; hence, introduction of a line of five-
membered heterocycles was examined. The 4-isooxazole deri-
vative 1w was found to have comparable activity to the
pyridone derivative 1v. We found that pyrazole substitution
potentiates hELOVL6 inhibitory activity. The 4-pyrazole
a Inhibitory activity of compounds on human ELOVL6 for palmi-
toyl-CoA elongation. The values represent the mean ( SE for n g 3.
trifluoromethyl group at the unique quaternary center.
Replacement of the trifluoromethyl group with methyl
(1b), ethyl (1c), isopropyl (1d), cyclopropyl (1e), and phenyl
(1f) resulted in a significant loss of potency, demonstrating
that the unique trifluoromethyl-substituted quaternary cen-
ter is essential for ELOVL6 inhibitory activity (Table 1).
Hence, we focused our efforts on optimization of the side
chain unit and substituents on the benzoxazinone core.
The SAR for the right-hand linkage unit was examined
initially (Table 2), because the carbamate linkage of 1a was
found to be metabolically labile (extensive hydrolysis after
incubation with microsomes, data not shown). Replacement
of the oxygen atom on the side chain of 1a, conserving the
bond length, led to the identification of the urea derivative 1g
with a 4-fold enhancement in activity. On the other hand, the
corresponding phenyl acetamide derivative 1h exhibited a
decrease in activity. Contraction by one carbon resulted in
the simple benzamide derivative 1i, which showed the most

7292 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Mizutani et al.
Table 4. Human and Mouse ELOVL6 Enzyme Assay and Mouse
Cellular Assay on Chiral Compoundsa
Table 5. Unbound Fraction Ratio of the Selected Active Isomers in
Mouse Plasmaa
mouse
hepatocyte
compound
unbound fraction (%)
hELOVL6b
IC50 (nM)
mELOVL6b
IC50 (nM)
(-)-1g
(-)-1i
(S)-1y
0.5 ( 0.1
5.3 ( 0.2
10.7 ( 1.1
compound
H2.35d IC50 (nM)
(-)-1g
(-)-1i
(þ)-1i
(þ)-1x
(S)-1y
(þ)-1z
19 ( 1
15 ( 2
31 ( 4.3
26 ( 5.4
NTc
377 ( 152
131 ( 25
NTc
a The values represent the mean ( SE for n = 3. Plasma protein
binding of drug was determined by equilibrium dialysis. See Experi-
mental Section for details.
>10 000
10 ( 1
2.6 ( 0.15
13 ( 2
20 ( 4.9
14 ( 1.2
29 ( 4.8
50 ( 4.4
3.6 ( 0.84
58 ( 3.0
(Table 5). Compound (-)-1i with a 5-fold discrepancy has
5.3% plasma unbound fraction, which is in between those of
(-)-1g and (S)-1y.
a The values represent the mean ( SE for n g 3. b Inhibitory activity of
compounds on human or mouse ELOVL6 for palmitoyl-CoA elonga-
tion. c Not tested.
[
14C]Palmitic acid was used as a radioactive sub-
d
In Vitro and In Vivo Profiles of Compound (S)-1y. The most
potent compound, (S)-1y, was further characterized
(Table 6). Compound (S)-1y was highly selective for hE-
LOVL6 over other human ELOVL family enzymes
(hELOVL1, 2, 3, and 5: over 50-fold). However, (S)-1y was
found to equipotently inhibit mELOVL3 and mELOVL6.
When the amino acid sequence homology between human
and mouse orthologs (e.g., hELOVLs vs mELOVLs) is
examined, hELOVL3 displays relatively low homology to
mELOVL3 (69%) while other hELOVL members show
88-96% homology to the corresponding mouse ortho-
logs.14a Having considered that, we speculate that relatively
low homology between human and mouse ELOVL3 proteins
could explain species-related difference in affinity of (S)-1y
to ELOVL3 enzymes (i.e., low affinity for hELOVL3 while
high affinity for mELOVL3), which consequently resulted in
the discovery of mouse ELOVL3/6 dual inhibitory activity.
To the best of our knowledge, this is the first example of the
ELOVL3/6 dual inhibitor, and (S)-1y could be a unique
pharmacological tool as a potent mouse ELOVL3/6 dual
inhibitor to potently suppress the biosynthesis of a variety of
long chain fatty acids. Compound (S)-1y was screened by
MDS Pharma Services (PanLabs) in a panel of 168 receptor,
enzyme, channel, and transporter assays. No significant
activity was observed (IC50 > 10 μM) against any of the
PanLabs assays.
strate. Elongation activity was monitored as the elongation index (total
amount of C18 fatty acids/total amount of C16 fatty acids). See
Experimental Section for details.
derivative 1x, which has the same substitution pattern as
4-isooxazole, is equipotent to the parent 1i. Furthermore, the
regio-isomeric 3-pyrazole derivative 1y was found to have a low
nanomolar activity. The 1-pyrazole derivative 1z was equipo-
tent to the parent 1i. Whereas introduction of a pyrazole ring
enhanced hELOVL6 inhibitory activities, other five-membered
heteroaromatics such as triazoles 1aa and 1ab and imidazole
1ac showed only moderate to weak inhibitory activities. Non-
aromatic five-membered heterocycles were also briefly exami-
ned. The pyrrolidone (1ad) and oxazolidone (1ae) derivatives
showed relatively potent hELOVL6 inhibitory activities. Next,
optical resolution of the representative potent derivatives 1g,
1i, and 1x-z was conducted to identify and evaluate active
isomers. Racemic compounds and synthetic intermediate 10
were resolved by using chiral stationary HPLC. In all cases,
one isomer was found to be the active isomer, while its opposite
isomer showed a complete loss in potency ((-)-1i and (þ)-1i,
respectively; Table 4). The absolute configuration of synthetic
intermediate (S)-10 was unequivocally assigned by X-ray
crystallography analysis. The chiral intermediate (S)-10 was
successfully converted to (S)-1y, which has an opposite stereo-
chemistry to Efavirenz (Figure 1).23
To evaluate the potential utility of the selected chiral
compounds (-)-1g, (-)-1i, (þ)-1x, (S)-1y, and (þ)-1z as
pharmacological tools in rodents, their inhibitory activities
on mouse ELOVL6 and on the fatty acid elongation (i.e.,
elongation index) in mouse hepatocyte H2.35 cells were
examined (Table 4). Human and mouse ELOVL6s display
96% identity in amino acid sequence.8 Consistent with the
high homology between species, those active isomers pote-
ntly suppressed elongation activities of both hELOVL6 and
mELOVL6. Encouraged by these results, we evaluated
the inhibitory activities of these compounds on the cellular
fatty acids elongation by using mouse hepatocyte H2.35
cells.14a Of the selected five compounds shown in Table 4,
the 6-chloro derivatives ((-)-1g and (-)-1i) showed rela-
tively large discrepancies in inhibitory actions between the
enzyme and cellular assays: the amide derivative (-)-1i
showed a 5-fold discrepancy and the urea (-)-1g displayed
much larger 12-fold shift. On the contrary, a series of
pyrazols ((þ)-1x-z) showed greater consistency between
the assays, and the most potent (S)-1y showed no discre-
pancy. Plasma protein binding seems to be responsible for
the observed discrepancies. Compound (S)-1y with no dis-
crepancy showed relatively high plasma unbound frac-
tion (10.7%), whereas compound (-)-1g with a 12-fold
shift showed substantially lower unbound fraction (0.5%)
In order to assess the utility of (S)-1y as a tool for in vivo
studies, we further examined the in vivo profiles of (S)-1y.
Compound (S)-1y showed acceptable pharmacokinetic pro-
files and appreciable plasma and liver exposure after
oral dosing (Table 7). The plasma and liver levels at 2 h post
10 mg/kg oral administration of (S)-1y in mice were deter-
mined to be 0.66 μM and 12.5 μM, respectively, with a liver to
plasma ratio of 19. These data suggest that (S)-1y efficiently
reaches the liver where the target enzymes such as ELOVL3
and ELOVL6 abundantly exist. Although (S)-1y showed a
relatively short half-life (1.9 h, Table 7), appreciable plasma
exposure up to 8 h postdosing (0.7 μM, Table 8) allowed us to
evaluate in vivo efficacy of (S)-1y in mice.
Initially, the effect of (S)-1y on the elongation index of the
liver lipids in mice was assessed by using [14C]palmitic acid as
a radiotracer where the elongation index was defined as a
ratio of [total amount of C18 fatty acids (C18:0, C18:1) ] to
[total amount of C16 fatty acids (C16:0, C16:1)] (Figure 2).
We found that orally administered (S)-1y significantly
reduces the elongation index of the mouse liver lipids even
at 1 mg/kg (minimum effective dose). The plasma level 2 h
after 1 mg/kg oral dosing was 0.54 μM, demonstrating
potent in vivo activity of (S)-1y (Figure 2). Next, we exami-
ned the effects of (S)-1y on the elongation index of the
net fatty acids in liver and plasma following subchronic

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7293
Table 6. Human ELOVL Subtype Selectivity and Mouse ELOVL3 and 6 Inhibitory Activities of (S)-1ya
enzyme
hELOVL1
hELOVL2
hELOVL3
hELOVL5
hELOVL6
mELOVL3
mELOVL6
activity (IC50
)
7.0 ( 1.7 μM
>10 μM
130 ( 11 nM
>10 μM
2.6 ( 0.15 nM
19 ( 8.8 nM
14 ( 1.2 nM
a Inhibitory activity of (S)-1y on ELOVLs for their respective substrates. The values represent the mean ( SE for n g 3.
Table 7. Pharmacokinetic Parameters of (S)-1y in Sprague-Dawley
plasma fatty acids tended to decrease but did not reach a
statistically significant decrease. Although further studies
need to be conducted, considering the fact that both
ELOVL3 and ELOVL6 are responsible for the elongation
of palmitoyl-CoA, we speculate that (S)-1y might more
potently block the elongation of palmitoyl-CoA than a
selective ELOVL6 inhibitor in mice.
Ratsa
iv (1 mg/kg)
po (3 mg/kg)
CLp (mL/
min/kg)
Vdss
(L/kg)
Cmax
(μM)
AUC0-¥
t1/2 (h)
1.9
(μM h)
F (%)b
3
4.69
11
0.7
2.4
30
a The values represent the mean, n = 3 animals. b Based on AUC0-¥
values after iv and po administration.
Conclusion
Table 8. Plasma Exposure of (S)-1y in Micea
In summary, a series of novel and highly potent benzox-
azinone derivatives was identified as ELOVL6 inhibitors.
Representative compound (S)-1y showed potent and selective
inhibitory activity toward human ELOVL6 while showing
dual inhibitory activities toward mouse ELOVL3 and ELO-
VL6. In the preliminary pharmacodynamic studies, (S)-1y
significantly reduced the elongation index of the liver lipids
even at 1 mg/kg p.o. in mice. Furthermore subchronic expo-
sure of (S)-1y also resulted in a significant decrease of the
elongation index of liver fatty acids, demonstrating the utility
of (S)-1y as a pharmacological tool for in vivo studies. At this
time, the lack of a selective ELOVL3 inhibitor has limited
understanding of physiological roles of ELOVL3 vs ELOVL6.
However, further studies using potent mouse ELOVL3/6 dual
inhibitor (S)-1y and available selective ELOVL6 inhibitors14
would help in understanding the therapeutic applications of
ELOVL3 and 6 inhibition, which might lead to the develop-
ment of novel therapeutics for metabolic disorders.
time
plasma level (μM)
2 h
11.7
4 h
3.7
8 h
0.7
24 h
NDb
a The plasma levels were determined after 30 mg/kg oral administra-
tion. The values represent the mean for n = 3 animals. b Not detected.
Figure 2. The effect of (S)-1y on the elongation index of the liver
lipids in C57BL/6J mice. The elongation index was determined by
the radio-HPLC using [14C]palmitic acid as a radiolabeled substrate
and defined as % of control of the ratio of peak area [total amount
of C18 fatty acids]/total amount of C16 fatty acids]. Four mice were
used for each treatment. See Experimental Section for details.
Experimental Section
Chemistry. General Procedures. Unless otherwise noted, all
solvents, chemicals, and reagents were obtained commercially
and used without purification. The 1H NMR spectra were
obtained at 400 MHz on a MERCURY-400 (Varian) or JMN-
AL400 (JEOL) spectrometer, with chemical shifts (δ, ppm)
reported relative to TMS as an internal standard. Mass spectra
were recorded with electron-spray ionization (ESI) or atmo-
spheric pressure chemical ionization (APCI) on a Waters micro-
mass ZQ, micromass Quattro II, or micromass Q-Tof-2
instrument. Flash chromatography was carried out with pre-
packed silica gel columns (KP-Sil silica) from Biotage. Prepara-
tive thin-layer chromatography was performed on a TLC Silica
gel 60 F (Merck KGaA). Preparative HPLC purification was
carried out on a YMC-Pack Pro C18 (YMC, 30 mm i.d. S-5 μm),
eluting with a gradient of CH3CN/0.1% aqueous CF3CO2H at a
flow rate of 40 mL/min. Purity of the target compounds was
determined by HPLC with the two different eluting methods
as follows. Analytical HPLC was performed on a SPELCO
Ascentis Express (4.6 mm ꢀ 150 mm, s-2.7 μm), eluting with a
gradient of (Method A) 0.1% H3PO4/CH3CN=95/5 to 10/90
over 7 min followed by 10/90 isocratic over 1 min and (Method
B) 10 mM potassium phosphate buffer (pH 6.6)/CH3CN=95/5
to 20/80 over 7 min followed by 20/80 isocratic over 1 min
(detection at 210 nm). Tested compounds had a purity of
>95%, with the exception of 1aa (94.7%). High-resolution
mass spectra were recorded with electron-spray ionization on
a micromass Q-Tof-2 instrument. Optical rotation was mea-
sured using a 1 mL cell with a 50 mm path length on a JASCO
polarimeter P-1020.
Figure 3. Effects of (S)-1y on the elongation index of total fatty
acids in the liver and plasma of C57BL/6J mice after twice-daily
administration for 3 days with vehicle (open column) and 30 mg/kg
(S)-1y (gray column). The elongation index was determined by UV-
HPLC after converting fatty acids to corresponding 2-nitrophenyl-
hydrazides and defined as % of control of the ratio of peak area
[total amount of C18 fatty acids]/total amount of C16 fatty acids].
Six mice per group were used for each treatment. See Experimental
Section for details.
administration. Considering the plasma exposure at 2, 4, 8,
and 24 h post 30 mg/kg acute oral dosing of (S)-1y in mice
(Table 8), we administered 30 mg/kg of (S)-1y twice daily for
3 days to sustain desired plasma levels (i.e., >0.54 μM)
throughout the dosing period. Consistent with the inhibitory
effect on the [14C]palmitic acid tracer elongation, (S)-1y
significantly decreased the elongation index of total fatty
acids in the liver (Figure 3). Similarly, the elongation index of
1-(5-Bromo-2-fluorophenyl)-2,2,2-trifluoroethanone (3). To a
stirred solution of diisopropylamine (56.4 g, 0.55 mol) in THF

7294 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Mizutani et al.
(500 mL) was added n-BuLi (2.66 M, 200 mL) dropwise at
-40 ꢀC, and the resulting mixture was stirred at -40 ꢀC for 1 h
and cooled to -78 ꢀC. To the mixture was added 1-bromo-4-
fluorobenzene (87.5 g, 0.50 mol) in THF (100 mL) dropwise
while keeping the internal temperature below -70 ꢀC, and the
mixture was stirred at -78 ꢀC for 1 h. After addition of ethyl
trifluoroacetate (65.4 mL, 0.55 mol) in THF (100 mL) at -78 ꢀC,
the resultant mixture was allowed to warm to 0 ꢀC, quenched by
addition of saturated aqueous NH4Cl, and partitioned between
EtOAc and water. The layers were separated, and the organic
layer was washed with brine, dried over MgSO4, and concen-
trated. The residue was purified by flash silica gel column
chromatography (EtOAc/Hexanes = 1/4) to give 3 as a dark
yellow oil, which was further purified by distillation under
reduced pressure (1 mmHg, 53 ꢀC) to give 3 (83.8 g, 62%) as a
yellow oil. 1H NMR (400 MHz, CDCl3): δ 7.15 (1H, dd, J=10.2,
8.8 Hz), 7.78 (1H, ddd, J=8.8, 4.4, 2.4 Hz), 7.99 (1H, dd, J=6.3,
2.4 Hz). MS (ESI) m/z 270 (M þ H)þ.
were separated and the organic layer was washed with brine,
dried over MgSO4, and concentrated. The residue was crystal-
lized from EtOH and water to give 6 (62.4 g, 87%) as a colorless
solid. 1H NMR (400 MHz, DMSO-d6): δ 3.71 (3H, s), 3.95 (1H,
d, J=12.2 Hz), 4.33 (1H, d, J=12.2 Hz), 5.07 (1H, d, J=16.6
Hz), 5.14 (1H, d, J=16.6 Hz), 5.81 (1H, brs), 6.90 (2H, d, J=8.8
Hz), 7.06 (1H, d, J=8.8 Hz), 7.20 (2H, d, J=8.8 Hz), 7.60 (1H,
dd, J=8.8, 2.0 Hz), 7.74 (1H, d, J=2.0 Hz). MS (ESI) m/z
445 (M þ H)þ.
4-(Azidomethyl)-6-bromo-1-(4-methoxybenzyl)-4-(trifluoro-
methyl)-1,4-dihydro-2H-3,1-benzoxazin-2-one (7). To a stirred
solution of 6 (61.3 g, 0.137 mol) and 2,6-dimethylpyridine (48.0
mL, 0.275 mol) in CHCl3 (400 mL) was added trifluorometha-
nesulfonic anhydride (46.4 mL, 0.275 mol) at 0 ꢀC, and the
mixture was stirred 0 ꢀC for 1 h. The reaction was quenched by
addition of saturated aqueous NaHCO3. The resulting mixture
was partitioned between EtOAc and water. The layers were
separated, and the organic layer was successively washed with
saturated aqueous NH4Cl and brine, dried over MgSO4, and
concentrated. The residue was combined with NaN3 (26.7 g,
0.411 mol) in DMF (270 mL) and heated to 80 ꢀC for 3 h. After
being cooled to room temperature, the reaction was quenched
by addition of water. The resultant mixture was partitioned
between EtOAc and water. The layers were separated, and the
organic layer was successively washed with 10% aqueous citric
acid, water, and brine, then dried over MgSO4, and concen-
trated. The residue was purified by flash silica gel column
chromatography (EtOAc/hexanes = 2/3) to give 7 (46.0 g,
71%) as a colorless solid. 1H NMR (400 MHz, CDCl3):
δ 3.77 (3H, s), 3.90 (1H, d, J=13.7 Hz), 4.09 (1H, d, J=13.7
Hz), 5.07 (1H, d, J=16.1 Hz), 5.17 (1H, d, J=16.1 Hz), 6.84 (1H,
d, J=8.8 Hz), 6.86 (2H, d, J=8.8 Hz), 7.19 (2H, d, J=8.8 Hz),
7.33 (1H, s), 7.44 (1H, dd, J=8.8, 2.4 Hz). MS (ESI) m/z 470
(M þ H)þ.
1-{5-Bromo-2-[(4-methoxybenzyl)amino]phenyl}-2,2,2-trifluo-
roethanone (4). A stirred mixture of 3 (76.2 g, 0.281 mol), K2CO3
(46.6 g, 0.337 mol), and 4-methoxy benzylamine (73.4 mL, 0.56
mol) in toluene (300 mL) was heated to reflux overnight and
cooled to 0 ꢀC. After successive addition of water (300 mL) and
citric acid monohydrate (118 g, 0.56 mol), the mixture was
stirred at 0 ꢀC for 30 min. The resulting precipitate was collected,
dried in vacuo, and suspended in a mixture of 500 mL of hexanes
and 10 mL of EtOAc. The suspension was stirred at room
temperature for 2 h. The precipitate was collected by filtration
and vacuum-dried to give 4 as an orange solid (75.6 g, 69%). The
mother liquor was concentrated, and the residue was triturated
with a mixture of hexanes and EtOAc to give a second crop (15.9
1
g, 16%) as a dark brown solid. H NMR (400 MHz, CDCl3):
δ 3.81 (3H, s), 4.43 (2H, d, J=5.4 Hz), 6.69 (1H, d, J=9.3 Hz),
6.89 (2H, d, J=8.8 Hz), 7.24 (2H, d, J=8.8 Hz), 7.46 (1H, dd, J=
9.3, 2.0 Hz), 7.87 (1H, dd, J=4.4, 2.0 Hz), 9.06 (1H, s). MS (ESI)
m/z 387 (M þ H)þ.
4-(Aminomethyl)-6-bromo-1-(4-methoxybenzyl)-4-(trifluoro-
methyl)-1,4-dihydro-2H-3,1-benzoxazin-2-one (8). To a stirred
solution of 7 (46.0 g, 98 mmol) in THF (130 mL) and H2O
(65 mL) was added trimethylphosphite (23.1 mL, 195 mmol) at
60 ꢀC, and the mixture was stirred at 60 ꢀC overnight. The
resulting mixture was partitioned between EtOAc and water,
and the layers were separated. The organic layer was washed
with water and brine, dried over MgSO4, and concentrated. The
residue was dissolved in 200 mL of a 4 N solution of hydro-
chloric acid in dioxane and stirred at room temperature for 24 h.
Volatiles were removed under reduced pressure, and the residue
was partitioned between EtOAc and 2 N aqueous NaOH. The
layers were separated, and the organic layer was washed with
water and brine, dried over MgSO4, and concentrated to give 8
(30.9 g, 71%) as a colorless solid. 1H NMR (400 MHz, DMSO-
d6): δ 3.21 (1H, d, J=14.1 Hz), 3.61 (1H, d, J=14.1 Hz), 3.71
(3H, s), 5.05 (1H, d, J=16.6 Hz), 5.14 (1H, d, J=16.6 Hz), 6.89
(2H, d, J=8.8 Hz), 7.04 (1H, d, J=8.8 Hz), 7.22 (2H, d, J=8.8
Hz), 7.59 (1H, dd, J=8.8, 2.4 Hz), 7.69 (1H, d, J=2.4 Hz). MS
(ESI) m/z 444 (M þ H)þ.
6-Bromo-4-ethenyl-1-(4-methoxybenzyl)-4-(trifluoromethyl)-
1,4-dihydro-2H-3,1-benzoxazin-2-one (5). To a stirred solution
of 4 (75.5 g, 0.195 mol) in THF (500 mL) was added vinyl
magnesium bromide (1.0 M in THF, 389 mL) at 0 ꢀC dropwise
over 1 h, and the mixture was stirred at 0 ꢀC for 3 h. After
addition of saturated aqueous NH4Cl (100 mL), the resultant
mixture was stirred at room temperature for 10 min and parti-
tioned between EtOAc and water. The layers were separated,
and the organic layer was washed with brine, dried over MgSO4,
and concentrated. The residue and triethylamine (81.2 mL, 0.59
mol) were combined in toluene (500 mL) and cooled to 0 ꢀC. To
the mixture was added triphosgene (57.9 g, 0.195 mol) portion-
wise, and the resulting mixture was stirred at 0 ꢀC for 30 min and
quenched by addition of saturated aqueous NaHCO3. The
mixture was partitioned between EtOAc and water. The layers
were separated, and the organic layer was washed with brine,
dried over MgSO4, and concentrated. The residual solid was
suspended in hexanes (50 mL) and stirred at room temperature
overnight. The precipitate was collected by filtration, washed
with hexanes, and dried under reduced pressure to give 5 (71.6 g,
83%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3): δ 3.78
(3H, s), 5.10 (2H, s), 5.69 (1H, d, J=11.2 Hz), 5.72 (1H, d, J=
17.1 Hz), 6.21 (1H, dd, J=17.1, 11.2 Hz), 6.80 (1H, d, J=8.8
Hz), 6.86 (2H, d, J=8.8 Hz), 7.17 (2H, d, J=8.8 Hz), 7.40 (1H,
dd, J=8.8, 2.0 Hz), 7.45 (1H, s). MS (ESI) m/z 441 (M þ H)þ.
6-Bromo-4-(hydroxymethyl)-1-(4-methoxybenzyl)-4-(trifluoro-
methyl)-1,4-dihydro-2H-3,1-benzoxazin-2-one (6). Ozone was
bubbled through a stirred solution of 5 (71.5 g, 0.161 mol) in
CH2Cl2 (200 mL) and MeOH (200 mL) at 0 ꢀC for 2 h. After
being purged with nitrogen, the mixture was treated with
NaBH4 (12.2 g, 0.323 mol) at 0 ꢀC and stirred for 10 min.
Volatiles were evaporated under reduced pressure, and the
residue was partitioned between EtOAc and 1 N hydrochloric
acid (the pH of water layer was adjusted to pH=6). The layers
N-{[6-Bromo-1-(4-methoxybenzyl)-2-oxo-4-(trifluoromethyl)-
1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}-4-fluorobenzamide
(9). To a stirred solution of 8 (13.7 g, 30.7 mmol), 4-fluoroben-
zoic acid (4.73 g, 33.8 mmol), HOBt H2O (4.7 g, 30.7 mmol),
and triethylamine (4.69 mL, 33.8 mmol) in DMF (50 mL) was
3
added EDCI HCl (6.47 g, 33.8 mmol) portionwise at 0 ꢀC, and
3
the mixture was allowed to warm up to ambient temperature
and stirred overnight. The resultant mixture was diluted with
H2O and stirred for 2 h. The resulting precipitate was collected
by filtration, washed with water, and dried under reduced
1
pressure to give 9 (15.6 g, 90%) as a colorless solid. H NMR
(400 MHz, DMSO-d6): δ 3.69 (3H, s), 3.91 (1H, dd, J=14.6,
4.4 Hz), 4.63 (1H, dd, J=14.6, 7.8 Hz), 5.05 (1H, d, J=16.6 Hz),
5.13 (1H, d, J=16.6 Hz), 6.83 (2H, d, J=8.8 Hz), 7.02 (1H, d,
J=8.8 Hz), 7.17 (2H, d, J=8.8 Hz), 7.26 (2H, t, J=8.8 Hz),
7.56 (1H, dd, J=8.8, 2.4 Hz), 7.63 (1H, s), 7.75 (2H, dd, J=8.8,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7295
25
to give (-)-1g ([R]D -51.2 (c 0.13, MeOH)) as the second
eluting enantiomer.
5.4 Hz), 8.87 (1H, dd, J = 7.8, 4.4 Hz). MS (ESI) m/z 566
(M þ H)þ.
N-{[6-Bromo-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-4-fluorobenzamide (10). To a stirred
solution of 9 (13.0 g, 22.9 mmol) in CH3CN (50 mL) was added
cerium(IV) ammonium nitrate (37.7 g, 68.7 mmol) in water
(25 mL) dropwise at room temperature, and the mixture was
stirred at room temperature for 4 h. The reaction was quenched
by addition of an excess amount of sodium pyrosulfite. After
being stirred at room temperature for 30 min, the mixture was
partitioned between EtOAc and water, and the layers were
separated. The organic layer was washed with water and brine,
dried over MgSO4, and concentrated. The residue was crystal-
lized from EtOAc and heptanes to give 10 (9.2 g, 90%) as a
colorless solid. 1H NMR (400 MHz, DMSO-d6): δ 3.85 (1H, d,
J=14.6 Hz), 4.58 (1H, d, J=14.6 Hz), 6.86 (1H, d, J=8.8 Hz),
7.26 (2H, t, J=8.8 Hz), 7.54-7.58 (2H, m), 7.75 (2H, dd, J=8.8,
5.4 Hz). MS (ESI) m/z 446 (M þ H)þ.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-2-(4-fluorophenyl)acetamide (1h). Fol-
lowing the procedure described for 9, 1h was prepared from C
and (4-fluorophenyl)acetic acid. 1H NMR (400 MHz, CD3OD):
δ 3.22-3.42 (2H, m), 3.63 (1H, d, J=14.6 Hz), 4.57 (1H, d, J=
14.6 Hz), 6.78-6.91 (3H, m), 6.98 (2H, dd, J = 8.8, 5.4 Hz),
7.27-7.35 (2H, m). MS (ESI) m/z 417 (M þ H)þ. HRMS (M þ
H)þ calcd for C18H14N2O3F4Cl, 417.0629; found, 417.0632.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-4-fluorobenzamide (1i). Following the
procedure described for 9, 1i was prepared from C and 4-
fluorobenzoic acid. 1H NMR (400 MHz, DMSO-d6): δ 3.87
(1H, dd, J=14.4, 4.4 Hz), 4.58 (1H, dd, J=14.4, 7.6 Hz), 6.92
(1H, d, J=8.3 Hz), 7.25 (2H, t, J=9.3 Hz), 7.43-7.50 (2H, m),
7.71-7.78 (2H, m), 8.81 (1H, dd, J=7.6, 4.4 Hz), 10.19 (1H, s).
MS (ESI) m/z 403 (M þ H)þ. HRMS (M þ H)þ calcd for
C17H12N2O3F4Cl, 403.0473; found, 403.0479.
N-{[(4S)-6-Bromo-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-
3,1-benzoxazin-4-yl]methyl}-4-fluorobenzamide ((S)-10). 10 was
resolved by preparative chiral HPLC (column, AD-H column;
eluant, 20% i-PrOH in hexanes; flow rate, 1 mL/min) to obtain
(S)-10 as the second eluting enantiomer.
N-{[(þ)-6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-
3,1-benzoxazin-4-yl]methyl}-4-fluorobenzamide ((þ)-1i) and
N-{[(-)-6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,
1-benzoxazin-4-yl]methyl}-4-fluorobenzamide ((-)-1i). 1i was
resolved by preparative chiral HPLC (column, AD-H column;
eluant, 20% i-PrOH in hexanes; flow rate, 1 mL/min) to obtain
4-fluoro-N-{[6-iodo-1-(4-methoxybenzyl)-2-oxo-4-(trifluoro-
methyl)-1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide
(11). 9 (1.18 g, 2.08 mmol), copper(I) iodide (79 mg, 0.42 mmol),
sodium iodide (0.624 g, 4.16 mmol), and N,N0-dimethylethyl-
enediamine (89 μL, 0.83 mmol) were combined in 1,4-dioxane
(4.2 mL). The mixture was stirred at 110 ꢀC for 4.5 h under a
nitrogen atmosphere before being cooled to room temperature.
The reaction was quenched by addition of saturated aqueous
NH4Cl. The resultant mixture was partitioned between EtOAc
and water. The layers were separated, and the organic layer was
washed with brine, dried over MgSO4, and concentrated under
reduced pressure. The residue was triturated with EtOAc and
hexanes to give 11 (995 mg, 78%) as a colorless solid. 1H NMR
(400 MHz, DMSO-d6): δ 3.68 (3H, s), 3.87 (1H, dd, J=14.6, 3.9
Hz), 4.61 (1H, dd, J=14.6, 7.8 Hz), 5.02 (1H, d, J=16.6 Hz),
5.10 (1H, d, J=16.6 Hz), 6.83 (2H, d, J=8.8 Hz), 6.87 (1H, d, J=
8.8 Hz), 7.17 (2H, d, J=8.8 Hz), 7.25 (2H, t, J=8.8 Hz), 7.68
(1H, dd, J=8.8, 2.0 Hz), 7.72-7.77 (3H, m), 8.85 (1H, dd, J=
7.8, 3.9 Hz). MS (ESI) m/z 614 (M þ H)þ.
25
(þ)-1i ([R]D þ4.9 (c 0.79, MeOH)) as the first eluting enantio-
25
mer and (-)-1i ([R]D -5.7 (c 0.97, MeOH)) as the second
eluting enantiomer.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-3-fluorobenzamide (1j). Following the
procedure described for 9, 1j was prepared from C and 3-
fluorobenzoic acid. 1H NMR (400 MHz, DMSO-d6): δ 3.87
(1H, dd, J=14.6, 4.4 Hz), 4.57 (1H, dd, J=14.6, 7.8 Hz), 6.91
(1H, d, J=8.8 Hz), 7.32-7.39 (1H, m), 7.42-7.53 (6H, m), 8.88
(1H, dd, J=7.8, 4.4 Hz), 10.87 (1H, s). MS (ESI) m/z 403 (M þ
H)þ. HRMS (M þ H)þ calcd for C17H12N2O3F4Cl, 403.0473;
found, 403.0468.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-2-fluorobenzamide (1k). Following the
procedure described for 9, 1k was prepared from C and 2-
fluorobenzoic acid. 1H NMR (400 MHz, DMSO-d6): δ 3.86
(1H, dd, J=14.4, 4.4 Hz), 4.55 (1H, dd, J=14.4, 7.6 Hz), 6.93
(1H, d, J=8.8 Hz), 7.16-7.23 (2H, m), 7.29 (1H, td, J=7.4, 1.8
Hz), 7.42-7.50 (3H, m), 8.78 (1H, dd, J=7.6, 4.4 Hz), 10.85 (1H,
s). MS (ESI) m/z 403 (M þ H)þ. HRMS (M þ H)þ calcd for
C17H12N2O3F4Cl, 403.0473; found, 403.0467.
[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-ben-
zoxazin-4-yl]methyl-(4-fluorophenyl)carbamate (1a). To a stirred
solution of B (30 mg, 0.11 mmol) in CH2Cl2 (1 mL) was added
1-fluoro-4-isocyanatobenzene (15 mg, 0.11 mmol) and TEA
(15 μL, 0.11 mmol) at room temperature, and the mixture was
stirred at room temperature for 17 h. Volatiles were removed
under reduced pressure, and the residue was purified by pre-
parative HPLC to give 1a (23 mg, 51%) as a colorless solid. 1H
NMR (400 MHz, DMSO-d6): δ 7.75-7.73 (1H, brm), 7.52 (1H,
dd, J=8.8, 2.2 Hz), 7.44-7.30 (7H, m), 7.00 (1H, d, J=8.8 Hz),
6.95-6.92 (2H, br m), 5.04 (1H, d, J=12.2 Hz), 5.04 (2H, s), 4.79
(1H, d, J = 12.2 Hz). MS (ESI) m/z 419 (M þ H)þ. HRMS
(M þ H)þ calcd for C17H12N2O4F4Cl, 419.0422; found,
419.0426.
1-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-3-(4-fluorophenyl)urea (1g). Following
the procedure described for 1a, 1g was prepared from C and
1-fluoro-4-isocyanatobenzene. 1H NMR (400 MHz, DMSO-
d6): δ 3.88 (1H, dd, J=14.6, 5.9 Hz), 4.26 (1H, dd, J=14.6, 6.8
Hz), 6.44 (1H, t, J=6.3 Hz), 6.96 (1H, d, J=8.8 Hz), 7.04 (2H, t,
J=8.8 Hz), 7.27-7.35 (2H, m), 7.49 (1H, dd, J=8.3, 2.4 Hz),
7.59 (1H, d, J=2.0 Hz), 8.47 (1H, s), 10.92 (1H, s). MS (ESI) m/z
418 (M þ H)þ. HRMS (M þ H)þ calcd for C17H13N3O3F4Cl,
418.0582; found, 418.0594.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-4-methylbenzamide (1l). Following the
procedure described for 9, 1l was prepared from C and 4-
1
methylbenzoic acid. H NMR (400 MHz, DMSO-d6): δ 2.30
(3H, s), 3.82 (1H, dd, J=14.6, 4.4 Hz), 4.58 (1H, dd, J=14.6, 7.8
Hz), 6.90 (1H, d, J=8.3 Hz), 7.20 (2H, d, J=8.3 Hz), 7.40-7.48
(2H, m), 7.57 (2H, d, J=8.3 Hz), 8.69 (1H, dd, J=7.8, 4.4 Hz),
10.84 (1H, s). MS (ESI) m/z 399 (M þ H)þ. HRMS (M þ H)þ
calcd for C18H15N2O3F3Cl, 399.0723; found, 399.0726.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-3-methylbenzamide (1m). Following
the procedure described for 9, 1m was prepared from C and 3-
1
methylbenzoic acid. H NMR (400 MHz, DMSO-d6): δ 2.29
(3H, s), 3.83 (1H, dd, J=14.6, 4.4 Hz), 4.58 (1H, dd, J=14.6, 7.8
Hz), 6.91 (1H, d, J=8.3 Hz), 7.25-7.31 (2H, m), 7.41-7.49 (5H,
m), 8.73 (1H, dd, J=7.8, 4.4 Hz), 10.86 (1H, s). MS (ESI) m/z
399 (M þ H)þ. HRMS (M þ H)þ calcd for C18H15N2O3F3Cl,
399.0723; found, 399.0723.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-2-methylbenzamide (1n). Following the
procedure described for 9, 1n was prepared from C and 2-
1-{[(-)-6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-
3,1-benzoxazin-4-yl]methyl}-3-(4-fluorophenyl)urea ((-)-1g).
1g was resolved by preparative chiral HPLC (column, AD-H
column; eluant, 20% i-PrOH in hexanes; flow rate, 1 mL/min)
1
methylbenzoic acid. H NMR (400 MHz, DMSO-d6): δ 2.01
(3H, s), 3.76 (1H, dd, J=14.6, 4.4 Hz), 4.62 (1H, dd, J=14.6, 7.8
Hz), 6.93 (1H, d, J=8.8 Hz), 7.04-7.06 (1H, m), 7.13-7.16 (2H,

7296 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Mizutani et al.
m), 7.23-7.29 (1H, m), 7.47-7.50 (2H, m), 8.75 (1H, dd, J=7.8,
4.4 Hz), 10.85 (1H, s). MS (ESI) m/z 399 (M þ H)þ. HRMS (M þ
H)þ calcd for C18H15N2O3F3Cl, 399.0723; found, 399.0726.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-4-methoxybenzamide (1o). Following
the procedure described for 9, 1o was prepared from C and 4-
methoxybenzoic acid. 1H NMR (400 MHz, DMSO-d6): δ 3.76
(3H, s), 3.81 (1H, dd, J=14.4, 4.4 Hz), 4.57 (1H, dd, J=14.4, 7.8
Hz), 6.88-6.95 (3H, m), 7.41-7.45 (2H, m), 7.64-7.68 (2H, m),
8.61 (1H, dd, J=7.8, 4.4 Hz), 10.84 (1H, s). MS (ESI) m/z 415 (M
þ H)þ. HRMS (M þ H)þ calcd for C18H15N2O4F3Cl, 415.0672;
found, 415.0672.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-4-(trifluoromethyl)benzamide (1p). Fol-
lowing the procedure described for 9, 1p was prepared from C
and 4-trifluoromethylbenzoic acid. 1H NMR (400 MHz,
DMSO-d6): δ 3.89 (1H, dd, J=14.6, 4.4 Hz), 4.59 (1H, dd, J=
14.6, 7.8 Hz), 6.91 (1H, d, J=8.5 Hz), 7.45 (1H, dd, J=8.5, 2.2
Hz), 7.48 (1H, s), 7.78-7.84 (4H, m), 9.03 (1H, dd, J=7.8, 4.4
Hz), 10.86 (1H, s). MS (ESI) m/z 453 (M þ H)þ. HRMS (M þ
H)þ calcd for C18H12N2O3F6Cl, 453.0441; found, 453.0448.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-4-(propan-2-yl)benzamide (1q). Fol-
lowing the procedure described for 9, 1q was prepared from C
and 4-isopropylbenzoic acid. 1H NMR (400 MHz, DMSO-d6):
δ 1.16 (6H, d, J=6.8 Hz), 2.84-2.93 (1H, m), 3.82 (1H, dd, J=
14.6, 4.4 Hz), 4.58 (1H, dd, J=14.6, 7.8 Hz), 6.90 (1H, d, J=8.3
Hz), 7.26 (2H, d, J=8.3 Hz), 7.41-7.48 (2H, m), 7.58 (2H, d, J=
8.3 Hz), 8.69 (1H, dd, J=7.8, 4.4 Hz), 10.84 (1H, s). MS (ESI) m/
z 427 (M þ H)þ. HRMS (M þ H)þ calcd for C20H19N2O3F3Cl,
427.1036; found, 427.1037.
HRMS (M þ H)þ calcd for C23H17N2O3F4, 445.1175; found,
445.1184.
4-Fluoro-N-{[2-oxo-6-(2-oxopyridin-1(2H)-yl)-4-(trifluoro-
methyl)-1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide
(1v). Following the procedure described for 1ab, 1v was pre-
pared from 11 and pyridin-2(1H)-one. 1H NMR (400 MHz,
DMSO-d6): δ 3.86 (1H, dd, J=14.6, 4.4 Hz), 4.59 (1H, dd, J=
14.6, 7.8 Hz), 6.34 (1H, td, J=6.8, 1.5 Hz), 6.47 (1H, dd, J=9.8,
1.5 Hz), 6.97 (1H, d, J = 9.3 Hz), 7.24 (2H, t, J = 8.8 Hz),
7.45-7.52 (4H, m), 7.74 (2H, dd, J=8.8, 5.4 Hz), 8.82 (1H, dd,
J=7.8, 4.4 Hz), 10.90 (1H, s). MS (ESI) m/z 462 (M þ H)þ.
HRMS (M þ H)þ calcd for C22H16N3O4F4, 462.1077; found,
462.1077.
4-Fluoro-N-{[6-(isoxazol-4-yl)-2-oxo-4-(trifluoromethyl)-1,4-
dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide (1w). Fol-
lowing the procedure described for 1y, 1w was prepared from
10 and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxa-
zole. 1H NMR (400 MHz, DMSO-d6): δ 3.95 (1H, d,
J =14.5 Hz), 4.59 (1H, d, J=14.5 Hz), 6.92 (1H, d, J =8.2
Hz), 7.19 (2H, t, J=8.9 Hz), 7.64-7.71 (3H, m), 7.74 (1H, brs),
9.04 (1H, brs), 9.32 (1H, brs). MS (ESI) m/z 436 (M þ H)þ.
HRMS (M þ H)þ calcd for C20H14N3O4F4, 436.0920; found,
436.0915.
4-Fluoro-N-{[2-oxo-6-(1H-pyrazol-4-yl)-4-(trifluoromethyl)-
1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide (1x). Fo-
llowing the procedure described for 1y, 1x was prepared from 10
and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyra-
zole. 1H NMR (400 MHz, DMSO-d6): δ 3.87 (1H, dd, J =
14.1, 3.9 Hz), 4.72 (1H, dd, J=14.1, 8.3 Hz), 6.89 (1H, dd, J=
8.3, 1.5 Hz), 7.22 (2H, t, J = 8.8 Hz), 7.57 (1H, dd, J = 8.3,
1.5 Hz), 7.61 (1H, brs), 7.72 (2H, dd, J=8.8, 5.4 Hz), 7.96 (2H,
brs), 8.83 (1H, dd, J=8.3, 3.9 Hz), 10.68 (1H, s). MS (ESI) m/z
435 (M þ H)þ. HRMS (M þ H)þ calcd for C20H15N4O3F4,
435.1080; found,435.1086.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}biphenyl-4-carboxamide (1r). Following
the procedure described for 9, 1r was prepared from C and 4-
1
phenylbenzoic acid. H NMR (400 MHz, DMSO-d6): δ 3.87
4-Fluoro-N-{[(þ)-2-oxo-6-(1H-pyrazol-4-yl)-4-(trifluoro-
methyl)-1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide
((þ)-1x). 1x was resolved by preparative chiral HPLC (column,
AD-H column; eluant, 20% i-PrOH in hexanes; flow rate, 1 mL/
(1H, dd, J=14.6, 4.4 Hz), 4.62 (1H, dd, J=14.6, 7.8 Hz), 6.92
(1H, d, J=8.3 Hz), 7.37-7.49 (5H, m), 7.64-7.79 (6H, m), 8.84
(1H, dd, J=7.8, 4.4 Hz), 10.86 (1H, s). MS (ESI) m/z 461 (M þ
H)þ. HRMS (M þ H)þ calcd for C23H17N2O3F3Cl, 461.0880;
found, 461.0875.
25
min) to obtain (þ)-1x ([R]D þ63.0 (c 0.42, MeOH)) as the
second eluting enantiomer.
N-{[6-Chloro-2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}-4-(methylsulfonyl)benzamide (1s). Fol-
lowing the procedure described for 9, 1s was prepared from C
and 4-(methanesulfonyl)-benzoic acid. 1H NMR (400 MHz,
DMSO-d6): δ 3.22 (3H, s), 3.89 (1H, dd, J=14.6, 4.0 Hz), 4.60
(1H, dd, J=14.6, 7.8 Hz), 6.91 (1H, d, J=8.8 Hz), 7.45 (1H, dd,
J=8.8, 2.4 Hz), 7.48 (1H, s), 7.86 (2H, dd, J=6.8, 2.0 Hz), 7.96
(2H, dd, J=6.8, 2.0 Hz), 9.06 (1H, dd, J=7.6, 4.0 Hz), 10.87 (1H,
s). MS (ESI) m/z 463 (M þ H)þ. HRMS (M þ H)þ calcd for
C18H15N2O5F3SCl, 463.0342; found,463.0343.
4-Fluoro-N-{[2-oxo-6-(1H-pyrazol-5-yl)-4-(trifluoromethyl)-
1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide (1y). 10
(4.0 g, 8.94 mmol), [1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-
5-yl]boronic acid (2.63 g, 13.42 mmol), PdCl2(dppf) (654 mg,
0.894 mmol), and potassium phosphate trihydrate (4.76 g, 17.88
mmol) were combined in a mixture of DMF (27 mL) and water
(2.7 mL) at room temperature, and the mixture was stirred at
120 ꢀC for 15 min under microwave irradiation. After being
cooled to room temperature, the mixture was treated with water
(10 mL) and conc hydrochloric acid (5 mL) and stirred at room
temperature for 2 h. The resulting mixture was partitioned
between EtOAc and water, and the layers were separated. The
organic layer was washed with water and brine, dried over
MgSO4, and concentrated under reduced pressure. The residue
was purified by flash silica gel column chromatography (EtOAc/
4-Fluoro-N-{[2-oxo-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-
benzoxazin-4-yl]methyl}benzamide (1t). 10 (21 mg, 0.047 mmol)
was hydrogenated over 21 mg of 10% Pd/C in a mixture of TEA
(10 μL) and THF (1 mL) under an atmospheric pressure of H2
for 1 h. The mixture was filtered through a pad of Celite, and the
filtrate was concentrated. The residual solid was crystallized
from EtOH and water to give 1t (3.0 mg, 17%) as a colorless
solid. 1H NMR (400 MHz, DMSO-d6): δ 3.78 (1H, d, J=14.7
Hz), 4.57 (1H, d, J=14.7 Hz), 6.87 (1H, d, J=8.2 Hz), 7.01 (1H,
t, J=8.0 Hz), 7.20 (2H, t, J=9.0 Hz), 7.32 (2H, t, J=8.2 Hz),
7.67-7.71 (2H, m). MS (ESI) m/z 369 (M þ H)þ. HRMS (M þ
H)þ calcd for C17H13N2O3F4, 369.0862; found, 369.0864.
4-Fluoro-N-{[2-oxo-6-phenyl-4-(trifluoromethyl)-1,4-dihydro-
2H-3,1-benzoxazin-4-yl]methyl}benzamide (1u). Following the
procedure described for 1y, 1u was prepared from 10 and
1
hexanes=1/1) to give 1y (2.7 g, 70%) as a colorless solid. H
NMR (400 MHz, DMSO-d6): δ 3.90 (1H, dd, J=14.6, 3.9 Hz),
4.66 (1H, dd, J=14.6, 7.3 Hz), 6.65 (1H, d, J=2.0 Hz), 6.95 (1H,
d, J=8.8 Hz), 7.21 (2H, t, J=8.8 Hz), 7.70-7.80 (5H, m), 8.82
(1H, dd, J=7.3, 3.9 Hz), 12.92 (1H, brs). MS (ESI) m/z 435 (M þ
H)þ. HRMS (M þ H)þ calcd for C20H15N4O3F4, 435.1080;
found,435.1087.
4-Fluoro-N-{[(þ)-(4S)-2-oxo-6-(1H-pyrazol-5-yl)-4-(trifluoro-
methyl)-1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide
((S)-1y). 1y was resolved by preparative chiral HPLC (column,
AD-H column; eluant, 20% i-PrOH in hexanes; flow rate, 1 mL/
1
phenylboronic acid. H NMR (400 MHz, DMSO-d6): δ 3.81
(1H, d, J=14.6 Hz), 4.85 (1H, dd, J=14.6, 6.8 Hz), 6.99 (1H, d,
J=8.8 Hz), 7.22 (2H, t, J=8.8 Hz), 7.35 (1H, t, J=7.6 Hz),
7.46 (2H, t, J=7.6 Hz), 7.64-7.68 (4H, m), 7.75 (2H, dd, J=8.8,
5.4 Hz), 8.89-8.90 (1H, m). MS (ESI) m/z 445 (M þ H)þ.
25
min) to obtain (S)-1y ([R]D þ84.2 (c 1.2, MeOH)) as the second
eluting enantiomer. And also following the same procedure to
prepare 1y, (S)-1y was prepared from (S)-10.

Article
4-Fluoro-N-{[2-oxo-6-(1H-pyrazol-1-yl)-4-(trifluoromethyl)-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7297
concentrated under reduced pressure. The residue was combined
with aluminum chloride (271 mg, 2.0 mmol) in anisole (1.0 mL) at
room temperature, and the mixture was stirred at 100 ꢀC for 2 h.
The reaction was quenched by addition of saturated aqueous
NaHCO3 at room temperature. The resultant mixture was parti-
tioned between EtOAc and water, and the layers were separated.
The organic layer was washed with brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by
preparative HPLC to give 1ac (13.3 mg, 14% in 2 steps) as a
colorless solid. 1H NMR (400 MHz, DMSO-d6): δ 4.08 (1H, dd,
J=14.1, 4.9 Hz), 4.42 (1H, dd, J=14.1, 6.3 Hz), 6.96 (2H, d, J=8.8
Hz), 7.21 (3H, t, J=8.8 Hz), 7.23 (1H, brs), 7.71 (2H, dd, J=8.8,
5.4 Hz), 7.92 (1H, dd, J=8.8, 1.5 Hz), 8.02 (1H, brs), 8.74-8.77
(1H, brm), 10.81 (1H, brs), 12.50 (1H, s). MS (ESI) m/z 435 (M þ
H)þ. HRMS (M þ H)þ calcd for C20H15N4O3F4, 435.1080;
found, 435.1078.
1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide (1z). Fo-
llowing the procedure described for 1ab, 1z was prepared from
11 and pyrazole. 1H NMR (400 MHz, DMSO-d6): δ 3.94 (1H,
dd, J=14.4, 4.9 Hz), 4.63 (1H, dd, J=14.4, 7.3 Hz), 6.55 (1H, t,
J=2.4 Hz), 7.00 (1H, d, J=8.8 Hz), 7.21 (2H, t, J=8.8 Hz),
7.69-7.73 (3H, m), 7.82-7.86 (2H, m), 8.39 (1H, d, J=2.4 Hz),
8.83 (1H, dd, J=7.3, 4.9 Hz), 10.84 (1H, s). MS (ESI) m/z 435 (M
þ H)þ. HRMS (M þ H)þ calcd for C20H15N4O3F4, 435.1080;
found, 435.1089.
4-Fluoro-N-{[(þ)-2-oxo-6-(1H-pyrazol-1-yl)-4-(trifluoromet-
hyl)-1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide
((þ)-1z). 1z was separated by preparative chiral HPLC (column,
AD-H column; eluant, 20% i-PrOH in hexanes; flow rate, 1 mL/
25
min) to give (þ)-1z ([R]D þ71.7 (c 0.38, MeOH)) as the second
eluting enantiomer.
4-Fluoro-N-{[2-oxo-6-(1H-1,2,4-triazol-5-yl)-4-(trifluoro-
methyl)-1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide
(1aa). Following the procedure described for 1ac, 1aa was
prepared from 10 and 1-[(benzyloxy)methyl]-1H-1,2,4-triazole.
HPLC purity (94.7%); 1H NMR (400 MHz, DMSO-d6): δ 3.95
(1H, dd, J=14.4, 4.4 Hz), 4.52 (1H, dd, J=14.4, 7.3 Hz), 7.00
(1H, d, J=8.3 Hz), 7.20 (2H, t, J=8.8 Hz), 7.69 (2H, dd, J=8.8,
5.4 Hz), 7.99 (1H, dd, J=8.3, 2.0 Hz), 8.05 (1H, brs), 8.42 (1H,
brs), 8.79 (1H, dd, J=7.3, 4.4 Hz). MS (ESI) m/z 436 (M þ H)þ.
HRMS (M þ H)þ calcd for C19H14N5O3F4, 436.1033; found,
436.1034.
4-Fluoro-N-{[2-oxo-6-(2-oxopyrrolidin-1-yl)-4-(trifluoro-
methyl)-1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide
(1ad). Following the procedure described for 1ab, 1ad was
1
prepared from 11 and pyrrolidin-2-one. H NMR (400 MHz,
DMSO-d6): δ 1.99-2.07 (2H, m), 2.43-2.48 (2H, m), 3.69-3.81
(2H, m), 3.85 (1H, dd, J=14.6, 4.4 Hz), 4.56 (1H, dd, J=14.6, 7.8
Hz), 6.89 (1H, d, J=8.8 Hz), 7.23 (2H, t, J=8.8 Hz), 7.50 (1H, d,
J=2.0 Hz), 7.74 (2H, dd, J=8.8, 5.9 Hz), 7.79 (1H, dd, J=8.8,
2.4 Hz), 8.78 (1H, dd, J=7.8, 4.4 Hz), 10.67 (1H, s). MS (ESI) m/
z 452 (M þ H)þ. HRMS (M þ H)þ calcd for C21H18N3O4F4,
452.1233; found, 452.1240.
4-Fluoro-N-{[2-oxo-6-(1H-1,2,4-triazol-1-yl)-4-(trifluoro-
methyl)-1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide
(1ab). 11 (50 mg, 0.081 mmol), 1,2,4-triazole (16.9 mg, 0.244
mmol), potassium phosphate (25.9 mg, 0.122 mmol), copper
iodide (3.1 mg, 0.016 mmol) and N,N0-dimethylethylenediamine
(6.9 μL, 0.065 mmol) were combined in DMF (0.4 mL), and the
resultant mixture was stirred at 110 ꢀC for 1 h under a nitrogen
atmosphere before being cooled to room temperature, The
reaction was quenched by addition of saturated aqueous
NH4Cl. The resulting mixture was partitioned between EtOAc
and water, and the layers were separated. The organic layer was
washed with brine, dried over MgSO4, and concentrated under
reduced pressure. The residual oil and aluminum chloride (107
mg, 0.80 mmol) were combined in anisole (0.43 mL) at room
temperature, and the mixture was stirred at 100 ꢀC for 2 h. After
being cooled to room temperature, the reaction was quenched
by addition of saturated aqueous NaHCO3. The mixture was
partitioned between EtOAc and water, and the layers were
separated. The organic layer was washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The residue
was purified by preparative TLC (hexanes/EtOAc = 1/1) fol-
lowed by preparative HPLC to provide 1ab (5.2 mg, 15% in 2
4-Fluoro-N-{[2-oxo-6-(2-oxo-1,3-oxazolidin-3-yl)-4-(trifluo-
romethyl)-1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benz-
amide (1ae). Following the procedure described for 1ab, 1ae was
prepared from 11 and 1,3-oxazolidin-2-one. 1H NMR (400
MHz, DMSO-d6): δ 3.85 (1H, dd, J=14.6, 4.4 Hz), 3.95 (1H,
q, J=8.3 Hz), 4.05 (1H, q, J=8.3 Hz), 4.42 (2H, t, J=8.3 Hz),
4.57 (1H, dd, J=14.6, 7.8 Hz), 6.92 (1H, d, J=8.8 Hz), 7.22 (2H,
t, J=8.8 Hz), 7.45 (1H, d, J=2.4 Hz), 7.68 (1H, dd, J=8.8, 2.4
Hz), 7.74 (2H, dd, J=8.8, 5.9 Hz), 8.79 (1H, dd, J=7.8, 4.4 Hz),
10.68 (1H, s). MS (ESI) m/z 454 (M þ H)þ. HRMS (M þ H)þ
calcd for C20H16N3O5F4, 454.1026; found, 454.1026.
Biology. Reagents. Glucose-6-phosphate, β-nicotinamide-
adenine dinucleotide phosphate and glucose-6-phosphate dehy-
drogenase were purchased from Oriental Yeast Co., Ltd.
(Tokyo, Japan). [14C]-Malonyl-CoA was purchased from GE
Healthcare Science (Little Chalfont, UK). [14C]-Palmitoyl-CoA
was purchased from PerkinElmer Japan (Kanagawa, Japan).
[14C]-Stearoyl-CoA was purchased from Muromachi-Yakuhin
(Tokyo, Japan). The oligonucleotide primers were purchased
from Hokkaido System Science (Hokkaido, Japan). Human
and mouse liver microsomes were obtained from BD Bio-
sciences (San Jose, CA) and XENOTECH (Lenexa, KS), re-
spectively. Other reagents were obtained from Sigma (St. Louis,
MO). Male C57BL/6J mice (5-7 weeks of age) and male SD rats
(5-7 weeks of age) were purchased from CLEA Japan (Tokyo,
Japan) and Charles River Japan, respectively (Kanagawa,
Japan).
Cloning and Expression of hELOVL6. The hELOVL6 coding
sequence (accession number: NP_076995) was amplified by RT-
PCR from human liver cDNA library (Clontech) using the
following primers: 50 primer, 50-GGATCCAACATGT-
CAGTGTTGACTT-30 and 30 primer, 50-CTCGAGCTATT-
CAGCTTTCGTTGTT-30. Cloned DNA sequence was ligated
with the double-digested pPICZRB vector (Invitrogen). The
integrity of all PCR products and ligations was confirmed by
DNA sequencing.
Preparation of the Microsome Fraction Expressing hE-
LOVL6. The pPICZR-FLAG-rhELOVL6 expression vector
was transformed into the Pichia pastoris SMD1168 yeast strain
using the Pichia EasyComp Transformation Kit (Invitrogen).
The transformants were selected on YPDS plates (1% yeast
extract, 2% peptone, 2% dextrose, and 1 M sorbitol) containing
100 μg/mL of Zeocin. The cells were grown in BMGY medium
1
steps) as a colorless solid. H NMR (400 MHz, DMSO-d6): δ
3.99 (1H, dd, J=14.6, 4.4 Hz), 4.59 (1H, dd, J=14.6, 6.8 Hz),
7.05 (1H, d, J=8.8 Hz), 7.21 (2H, t, J=8.8 Hz), 7.70 (2H, dd, J=
8.8, 5.4 Hz), 7.85 (1H, dd, J=8.8, 2.4 Hz), 7.95 (1H, s), 8.23 (1H,
s), 8.81 (1H, dd, J=6.8, 4.4 Hz), 9.18 (1H, s), 10.94 (1H, s). MS
(ESI) m/z 436 (M þ H)þ. HRMS (M þ H)þ calcd for
C19H14N5O3F4, 436.1033; found, 436.1030.
4-Fluoro-N-{[6-(1H-imidazol-2-yl)-2-oxo-4-(trifluoromethyl)-
1,4-dihydro-2H-3,1-benzoxazin-4-yl]methyl}benzamide (1ac). To a
stirred solution of 1-[(benzyloxy)methyl]-1H-imidazole (105 mg,
0.56 mmol) in THF (0.3 mL) was added n-BuLi (2.4 M inhexanes,
233 μL) at -78 ꢀC. After 30 min, zinc bromide (151 mg, 0.67
mmol) was added, and the mixture was stirred at -78 ꢀC for an
additional 30 min and allowed to warm to room temperature. To
the mixture was added 10 (100 mg, 0.224 mmol), Pd(PPh3)4 (25.8
mg, 0.022 mmol), and DMF (0.5 mL) at room temperature, and
the mixture was stirred at 80 ꢀC for 2 h before being cooled to
room temperature. The reaction was quenched by addition of
saturated aqueous NaHCO3. The mixture was partitioned bet-
ween EtOAc and water. The layers were separated, and the
organic layer was washed with brine, dried over MgSO4, and

7298 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Mizutani et al.
(1% yeast extract, 2% peptone, 100 mM potassium phosphate
pH 6.0, 1.34% yeast nitrogen base, (4 ꢀ 10-5)% biotin, and 1%
glycerol). Expression of the R-factor signal sequence/FLAG/
hELOVL6 fusion protein was induced in BMMY medium (1%
glycerol is replaced with 0.5% methanol), and the cells were
cultured for 48 h at 30 ꢀC in a rotary shaker (180 rpm).
Preparation of the microsome fraction was performed at 4 ꢀC.
In brief, the yeast cells were harvested by centrifugation at 3000
g for 10 min and washed with cold breaking buffer (50 mM
potassium phosphate (pH 7.4), 1 mM EDTA, 5% glycerol, and
1 tablet/50 mL of protease inhibitor cocktail (Roche)). The cells
were then vigorously broken with glass beads in cold breaking
buffer. The resultant homogenate was centrifuged at 10 000 g for
10 min, and the supernatant was further centrifuged at 100 000 g
for 1 h at 4 ꢀC. The pellet was suspended in resuspension buffer
(50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 20% glycerol, and
1 tablet/50 mL of protease inhibitor cocktail), and again cen-
trifuged at 100 000 g for 1 h at 4 ꢀC. The pellet was suspended in
the resuspension buffer and used as the microsomal fraction
(FLAG-rhELOVL6 microsome) for the elongase assay.
acid (16:0) was added to each well to a final concentration of
0.8 μCi/mL to detect elongase activity. After 4 h incubation at
33 ꢀC, the culture medium was removed, and the labeled cells
were washed with chilled PBS (3 ꢀ 0.5 mL) and dissolved in
250 μL of 2 N sodium hydroxide. The cell lysate was incubated
at 70 ꢀC for 1 h to hydrolyze radio labeled cellular lipids. After
acidification with 100 μL of 5 N HCl, fatty acids were extracted
with 300 μL of acetonitrile. Radiolabeled palmitic acid (16:0),
palmitoleic acid (16:1), stearic acid (18:0), and cis-vaccenic acid
(18:1,n-7) and oleic acid (18:1,n-9) were quantified by reversed-
phase HPLC with a Packard Flow Scintillation Analyzer 500TR
(radio-HPLC) (Amersham Biosciences, Piscataway, NJ). The
identity of the labeled fatty acids was determined by comparing
the retention times with known fatty acid standards. Elongation
activity was monitored as the elongation index. The elongation
index was defined as follows: Elongation index=total amount
of C18 fatty acids/total amount of C16 fatty acids=[stearoyl
acid þ oleic acid þ cis-vaccenic acid]/[palmitic acid þ palmito-
leic acid].
In Vivo [14C]-Palmitoyl-CoA Elongation. Male C57BL/6J
mice (CLEA Japan) and SD rats (Charles River Japan) were
individually housed in plastic cages with ad libitum access to
normal rodent chow (CE2, CLEA Japan) and water. (S)-1y
(dissolved in 0.5% methylcellulose) was orally administered to
mice, and 1 h later, [1-14C]-palmitic acid was interperitoneally
administered at 10 μCi/body. At 2 h postdosing of (S)-1y,
animals were anesthetized with isoflurane (4%) and killed by
blood collection from the vena cava. 50 mg of the liver was
harvested and incubated in potassium hydroxide/ethanol (2 mL/
1.4 mL) at 70 ꢀC for 1 h. The nonacid-lipid was extracted by
4 mL of petroleum ether and discarded. Fatty acids were
extracted by 2 mL of petroleum ether following saponification
by 2 mL of 6 N HCl. The ether phase containing fatty acids
fraction was evaporated under nitrogen gas and reconstituted in
methanol to measure the radioactivity by radio-HPLC. The
radioactivity corresponding to each fatty acid was quantified to
calculate the elongation index as described above. All animal
procedures were conducted according to protocols and guide-
lines approved by the Banyu Institutional Animal Care and Use
Committee.
Fatty Acid Composition in Liver and Plasma. (S)-1y was
administered orally twice daily at 30 mg/kg for 3 days to male
C57BL/6J mice, and then the mice were anesthetized and tissues
immediately isolated, weighed, frozen in liquid nitrogen and
stored at -80 ꢀC until use. The liver samples were incubated in
100-fold volume (w/v) of 5 N NaOH/ethanol (1:1) at 60 ꢀC.
After 2 h incubation, 500 μL of 5 N HCl and C17:0 (internal
standard) fatty acid was added to all hydrolysates. For plasma
samples, 50 μL of the plasma samples were incubated in 200 μL
of 5 N NaOH/ethanol (1:1), 10 μL of C17:0 fatty acid, and
100 μL of 5 N HCl. The fatty acid composition was analyzed by
a previously described method24 with slight modifications.
Briefly, the fatty acids in the tissue hydrolysate were derivatized
with 2-nitrophenylhydrazine, and these derivatives were puri-
fied using an Oasis HLB column. An aliquot (10 μL) of the
eluate was injected into the HPLC apparatus for analysis.
HPLC analysis was performed with a Shimadzu 10Avp system
(Shimadzu, Kyoto, Japan), equipped with a UV detector (SPD-
10Avp), two pumps (LC-10ADvp), an autosampler (SIL-
10ADvp), and a column oven (CTO-10ACvp). The mobile
phase consisted of CH3CN-water (80:20, flow rate, 0.6 mL/
min). The separation was performed with a CAPCELL PAK
C18 MGII (2.0 mm i.d. ꢀ 150 mm, 5 μm) at 35 ꢀC, and the UV
absorbance was subsequently measured at 400 nm. The elonga-
tion index represented the ratio of total amount of C18 fatty
acids to total amount of C16 fatty acids, which was quantified
from each fatty acid amount.
Cloning and Expression of ELOVL Family. The human or
mouse ELOVL family coding sequence was amplified by RT-
PCR from human or mouse liver cDNA library (Clontech)
described in previous reports.14a Cloned DNA sequence was
ligated with the double-digested pPICZRB vector (Invitrogen).
The integrity of all PCR products and ligations was confirmed
by DNA sequencing.
Preparation of the Microsome Fraction Expressing ELOVL.
The pPICZR-FLAG-rELOVL expression vector was trans-
formed into the Pichia pastoris SMD1168 yeast strain using
the Pichia EasyComp Transformation Kit (Invitrogen). Micro-
some fraction was prepared from each transformants as the
enzyme source for the elongase assay of each ELOVL family.
ELOVL6 in Vitro Enzyme Assay. The long chain fatty acyl-
CoA elongation assay was performed as described elsewhere
(Kitazawa, Miyamoto et al., submitted for publication). Briefly,
30 μL of the reaction mixture (100 mM potassium phosphate
buffer (pH 6.5), 200 μM BSA (fatty acid free), 500 μM NADPH,
1 μM rotenone, 20 μM malonyl-CoA, 833 kBq/mL [14C]-mal-
onyl-CoA, and 40 μM palmitoyl-CoA) was used as the substrate
mixture. To start reaction, 20 μL of the ELOVL6 microsomal
protein (3 μg) was added to the substrate mixture, and then
incubated for 1 h at 37 ꢀC with gentle shaking. This reaction step
was performed in a 96-well plate. After 1 h incubation, 100 μL of
5 N HCl was added for the hydrolysis of acyl-CoAs, and then the
reaction mixture was filtered through a Unifilter-96, GF/C plate
(PerkinElmer, Waltham, MA) using a FilterMate cell harvester
(PerkinElmer, Waltham, MA). The 96-well GF/C filter plate
was subsequently washed with distilled water to remove excess
[14C]-malonyl-CoA and dried, after which 25 μL of MICRO-
SCINT 0 was added to each well and radioactivity was measured
using TopCount (PerkinElmer, Waltham, MA). For other
ELOVL subtypes, corresponded acyl-CoA as indicated below
instead of palmitoyl-CoA was used for respective enzyme assay:
ELOVL1, 10 μM stearoyl-CoA; ELOVL2, 10 μM arachido-
noyl-CoA; ELOVL3, 10 μM stearoyl-CoA; ELOVL5, 40 μM
arachidonoyl-CoA. For an enzyme inhibition assay, indicated
concentrations of test compounds or control vehicle (DMSO:
final concentration 1.0%) was included in the substrate mixture.
Residual enzyme activity was determined as the percentage of
the respective control. All data including IC50 values were
analyzed by nonlinear regression using GraphPad Prism version
4.00 (GraphPad Software, Inc.).
In Vitro Hepatocyte Assay. Mouse hepatoma H2.35 cells were
grown on 24-well plates in Dulbecco’s modified Eagle’s medium
(DMEM) (Invitrogen, Carlsbad, CA) supplemented with
200 nM dexamethasone and 4% heat-inactivated fetal bovine
serum (FBS) at 33 ꢀC under 5% CO2 in a humidified incubator.
The test compound dissolved in medium was incubated with
subconfluent H2.35 cells for 60 min at 33 ꢀC. [1-14C]-palmitic
Plasma Protein Binding in Mice Plasma. Mouse (C57BL/6N)
plasma fraction was prepared by standard procedure and its pH
was adjusted to 7.4 just prior to use. Plasma protein binding was

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7299
References
determined by an equilibrium dialysis method using reusable
96-well Micro-Equilibrium Dialysis Device HTD 96b (HT-
Dialysis LLC). In brief, each well was separated by a dialysis
membrane (HTD 96a/b Dialysis Membrane Strips, MWCO
12 000-14 000, HTDialysis LLC) soaked in PBS (phosphate
buffered saline, purchased from Invitrogen Japan, Inc.) to
produce two side-by-side chambers, plasma side and buffer
side. Plasma (150 μL) containing 1 μM of test compounds and
PBS (150 μL) were placed in the plasma side and the buffer side,
respectively. All assay wells were sealed and incubated at 37 ꢀC
with gentle agitation. After 6 h incubation, aliquots (50 μL) of
plasma and dialysate (PBS) sample were transferred to poly-
propylene tubes and mixed with 150 μL of ethanol containing
internal standard. The all samples were centrifuged at 10 000g
for 10 min at 4 ꢀC, and the resultant supernatants were analyzed
by LC-MS/MS. Percentage of plasma protein unbound frac-
tion was calculated as follows: Unbound fraction (%) = Cf/
Cp ꢀ 100, where Cf and Cp represent the drug concentration
on the buffer side and the plasma side post equilibrium,
respectively.
Plasma and Liver Exposure in Mice. Pharmacokinetic char-
acterization was conducted in male C57BL/6J mice following
single oral administration of (S)-1y. Single doses of (S)-1y at 10
or 30 mg/kg body weight were administered orally by gavage in a
vehicle of 0.5% methylcellulose aqueous suspension. Blood
samples from the abdominal vein and liver samples were ob-
tained 2 h after 10 mg/kg oral administration. Blood samples for
the determination of drug plasma concentrations were obtained
at multiple time points up to 24 h after 30 mg/kg oral admin-
istration. Blood samples were centrifuged to separate the plas-
ma. Liver samples were homogenized with phosphate-buffered
saline (pH 7.4). Each sample was deproteinized with ethanol
containing an internal standard. (S)-1y and the internal stan-
dard were detected by liquid chromatography mass spectro-
metry/mass spectrometry (LC-MS/MS) in positive ionization
mode using an electrospray ionization probe, and their precur-
sor to production combinations were monitored using the
Multiple Reaction Monitoring mode.
Pharmacokinetics in SD Rats. Pharmacokinetics characteri-
zation in SD rats was conducted following single oral and single
intravenous administration. Single doses of (S)-1y were admi-
nistered either intravenously in a vehicle of PEG400/EtOH/
H2O = 50/10/40 or orally by gavage in a vehicle of 0.5%
methylcellurose aqueous suspension. Doses of 1 and 3 mg/kg
were used for iv and po, respectively. Blood samples for the
determination of drug plasma concentrations were obtained at
multiple time points up to 24 h after administration. Blood
samples were centrifuged to separate the plasma, and the
plasma samples were deproteinized with ethanol containing
an internal standard. Compound (S)-1y and the internal stan-
dard were detected by LC-MS/MS in a positive ionization mode
using the electrospray ionization probe, and their precursor to
product ion combinations were monitored in Multiple Reac-
tion Monitoring mode.
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