Synthesis and biological evaluation of a novel 3-sulfonyl-8-azabicyclo[3.2. 1]octane class of long chain fatty acid elongase 6 (ELOVL6) inhibitors

Article abstract of DOI:10.1021/jm900488k

Long chain fatty acid elongase 6 (ELOVL6) catalyzes the elongation of long chain fatty acyl-CoAs and is a potential target for the treatment of metabolic disorders. The ultrahigh throughput screen of our corporate chemical collections resulted in the iden

Full text of DOI:10.1021/jm900488k

J. Med. Chem. 2009, 52, 4111–4114 4111
DOI: 10.1021/jm900488k
So far, seven subtypes of ELOVL enzyme have been identi-
fied in mammals and are designated ELOVL1-7.6,7,12-15 The
ELOVL enzymes can be divided into two major groups
in terms of substrate preference: (a) enzymes that elongate
saturated and monounsaturated long-chain fatty acids
(ELOVL1, -3, and -6) and (b) enzymes that elongate poly-
unsaturated fatty acids (ELOVL2, -4, and -5). In addition to
these different fatty acid substrate preferences, each ELOVL
enzyme is expressed in different tissues and their expression is
differently regulated, indicating that they play different phy-
siological roles in vivo.7,16,17
Synthesis and Biological Evaluation of a Novel
3-Sulfonyl-8-azabicyclo[3.2.1]octane Class of
Long Chain Fatty Acid Elongase 6 (ELOVL6)
Inhibitors
Tsuyoshi Nagase,* Toshiyuki Takahashi, Takahide Sasaki,
Akira Nagumo, Ken Shimamura, Yasuhisa Miyamoto,
Hidefumi Kitazawa, Maki Kanesaka, Ryo Yoshimoto,
Katsumi Aragane, Shigeru Tokita, and Nagaaki Sato
Recent investigations revealed potential physiological and
pathological roles of ELOVL6. Matsuzaka and co-workers
recently reported that ELOVL6 deficient mice display im-
proved insulin sensitivity and glucose homeostasis compared
with wild-type mice when fed on a high-fat diet.4 Moreover,
mRNA levels of ELOVL6 are up-regulated in obese rodents
by refeeding after fasting and by exposure to a high carbohy-
drate diet.6,7,18 These observations suggest that ELOVL6
might be a new therapeutic target for diabetes. Despite the
increasing information on the physiological and pathological
roles of ELOVL6, a lack of useful chemical tools has made it
difficult to address pharmacological roles of ELOVL6 and its
therapeutic potential.
Tsukuba Research Institute, Merck Research Laboratories,
Banyu Pharmaceutical Co., Ltd., Okubo 3, Tsukuba, Ibaraki
300-2611, Japan
Received April 17, 2009
Abstract: Long chain fatty acid elongase 6 (ELOVL6) catalyzes the
elongation of long chain fatty acyl-CoAs and is a potential target
for the treatment of metabolic disorders. The ultrahigh throughput
screen of our corporate chemical collections resulted in the identifica-
tion of a novel 3-sulfonyl-8-azabicyclo[3.2.1]octane class of ELOVL6
inhibitor 1a. Optimization of lead 1a led to the identification of the
potent, selective, and orally available ELOVL6 inhibitor 1w.
We previously reported the establishment of a homoge-
neous enzyme assay for ELOVL6, which is applicable to the
ultrahigh throughput screening (UHTS) using a recombinant
histidine-tagged acyl-CoA binding protein as a molecular trap
for the detection of radioactive products of ELOVL6.19 The
UHTS of our company chemical library resulted in the
identification of a novel 3-sulfonyl-8-azabicyclo[3.2.1]octane
lead 1a, which has an IC50 of 1750 nM. In this report, we
describe the synthesis and preliminary SAR of the 3-sulfonyl-
8-azabicyclo[3.2.1]octane class of ELOVL6 inhibitors and the
discovery of the potent, selective, and orally available
ELOVL6 inhibitor 1w. Biological profiles of 1w including
cellular activity, off-target profiles, pharmacokinetic para-
meters, and in vivo efficacy in mice are also reported.
The synthesis of 1 and 2 described herein is outlined in
Scheme 1. Commercially available Boc-tropinone 4 was
reduced with sodium borohydride in the presence of cerium
chloride followed by mesylation of the resultant alcohols
5 and 6 to give exo-mesylate 7 and endo-mesylate 8, respec-
tively. The exo-mesylate 7 was coupled with various arylthiols
to give endo-sulfides 9, which were subsequently oxidized
with m-CPBA or KMnO4 to afford sulfoxide 10 or sulfones
11, respectively. Deprotection of the Boc group of 10 and
11 under acidic conditions gave amines 12. Coupling of
12 with various phenyl arylcarbamates or acids furnished
target 1. The exo-isomer 2 was synthesized from the
endo-mesylate 8 using the same procedures for the endo-
derivatives 1.
The incidence of type 2 diabetes has dramatically increased
overthe past decade. Accumulating evidence suggests a strong
correlation between insulin resistance and the development of
type 2 diabetes mellitus. An increase in fat storage in non-
adipose tissues, such as liver, leads to dysfunction of those
tissues (i.e., insulin resistance).1 Although the mechanism by
which increased intracellular lipid content exacerbates tissue
and whole body insulin sensitivity is unclear, it has been
suggested that increased levels of long chain fatty acyl-CoAa
antagonize the metabolic actions of insulin.2 Interestingly,
recent reports have suggested that alteration of the specific
fatty acid component (i.e., palmitoleate) has significant im-
pact on the insulin sensitivity of the liver and whole body.3,4
These observations indicate that modulation of fatty acid
composition may lead to amelioration of insulin resistance.
Biosynthesis of fatty acids is sequentially catalyzed by
acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS),
long chain fatty acid elongase 6 (ELOVL6), and stearoyl-CoA
desaturase (SCD)-1.5 Of these, ELOVL6 (known as LCE,
FACE) is responsible for the elongation reaction of long chain
fatty acyl-CoAs (C12-16 saturated and monounsaturated
fatty acyl-CoAs) using a malonyl-CoA as a two-carbon
donor.6-10 ELOVL6 is a membrane protein and resides in
the endoplasmic reticulum in cells.6 ELOVL6 is abundantly
expressed in lipogenic tissues such as liver and white adipose
tissue (WAT).6 ELOVL6 expression is directly and primarily
regulated by sterol regulatory element-binding protein
(SREBP)-1c.11 Human and mouse ELOVL6 isoforms display
96% identity in amino acid sequence.6
The newly synthesized compounds were first subjected to
the human ELOVL6 enzyme assay. Selected potent com-
pounds were further evaluated for inhibitory activities for
mouse ELOVL6, human and mouse ELOVL3 enzymes, and
their metabolic stability in mouse liver microsomes. ELOVL3
inhibitory activity was assessed because ELOVL6 and
ELOVL3 show the highest homology to each other (44%)
in the amino acid sequence.6
*To whom correspondence should be addressed. Phone: +81-29-
877-2000. Fax: +81-29-877-2029. E-mail: tsnagase@gmail.com.
aAbbreviations: ACC, acetyl-CoA carboxylase; AUC, area under the
curve; CoA, coenzyme A; ELOVL, long chain fatty acid elongase; FAS,
fatty acid synthase, SCD-1, stearoyl-CoA desaturase-1; SD, Sprague-
Dawley; SREBP, sterol regulatory element binding protein; UHTS, ultrahigh
throughput screening; WAT, white adipose tissue.
r
2009 American Chemical Society
Published on Web 06/12/2009
pubs.acs.org/jmc

4112 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Nagase et al.
Scheme 1a
Table 1. SAR around Left-Hand Phenyl Moietya
human ELOVL6
enzyme IC50 (nM)
compd
R1
1a
1b
1c
1d
1e
1f
4-Me-PhNH-
PhNH-
1750 ( 370
>10000
>10000
>10000
>10000
>10000
5070 ( 1090
970 ( 360
32 ( 3
>10000
78 ( 28
1400 ( 240
>10000
2-pyridinyl-NH-
3-pyridinyl-NH-
4-pyridinyl-NH-
4-F-PhNH-
1g
1h
1i
4-MeO-PhNH-
4-Cl-PhNH-
4-(i-Pr)-PhNH-
4-biphenyl-NH-
4-CF3-PhNH-
3-CF3-PhNH-
2-CF3-PhNH-
1j
1k
1l
1m
a The values represent the mean ( SE for n g 3.
a Reagents and conditions: (a) NaBH4, CeCl3 6H2O, H2O, EtOH;
Table 2. SAR around Central 3-Sulfonyl-8-azabicyclo[3.2.1]octane
Corea
3
(b) MsCl, TEA, THF; (c) R2SH, K2CO3, DMF; (d) m-CPBA, CH2Cl2;
(e) KMnO4, acetic acid, acetone; (f) HCl, MeOH; (g) phenyl arylcarba-
mate, TEA, CHCl3; (h) R1CO2H, WSC HCl, pyridine.
3
As lead 1a exhibits only modest intrinsic ELOVL6 activity,
we initiated SAR exploration of 1a to obtain potent ELOVL6
inhibitors. First, the SAR around the left-hand phenyl moiety
of 1a was investigated (Table 1). Removal of the para-methyl
group on the phenyl portion resulted in analogues devoid of
potency as in 1b, while the pyridine derivatives 1c-e showed
no improvement in potency. Given the important role of the
substituent, the various para-substituted derivatives (1f-j)
were synthesized and evaluated. While the para-fluoro and
methoxy derivatives 1f and 1g were less active than 1a, the
para-chloro derivative 1h was twice as potent as 1a. Intrigu-
ingly, the para-trifluoromethyl derivative 1k displayed a 22-
fold increase in inhibitory activity (IC50=78 nM) relative to
1a. With regard to larger substituents, the isopropyl derivative
1i was the most potent (IC50=32 nM), whereas the phenyl
derivative 1j resulted in a marked loss of potency. The ortho-
and meta-trifluoromethyl derivatives 1m and 1l were
much less potent than the para-derivative 1k. The potent
derivatives 1i and 1k were evaluated for metabolic stability
inmouse liver microsomes. The percent remainingof1iand 1k
after 30 min of incubation with mouse liver microsomes at
37 ꢀC was 0% and 38%, respectively;20 therefore, the trifluor-
omethyl derivative 1k was selected for further optimization.
Next, the central 3-sulfonyl-8-azabicyclo[3.2.1]octane core
was investigated (Table 2). With regard to the urea linkage
moiety, replacement of the urea linkage with the amide (1n)
or methyleneamide (1o) resulted in a marked loss of potency,
indicating that the urea linkage is essential for ELOVL6
inhibitory activity. Regarding the 8-azabicyclo[3.2.1]-
octane core, replacement of 8-azabicyclo[3.2.1]octane with
piperidine resulted in a complete loss of potency, as in 3.
As for the sulfone linkage portion, replacement of the sulfone
with a sulfoxide is detrimental to potency as in 1p. The endo
configuration of 1k is essential for potency; the exo-
isomer 2 is devoid of potency. These results indicate that the
human ELOVL6
enzyme IC50 (nM)
compd
R1
X
1k
1n
1o
1p
2
4-CF3-PhNH-
4-CF3-Ph-
-SO2-
-SO2-
-SO2-
-SO-
78 ( 28
1750 ( 370
>10000
>10000
>10000
>10000
4-CF3-Ph-CH2-
4-CF3-PhNH-
3
a The values represent the mean ( SE for n g 3.
3-endo-sulfonyl-8-azabicyclo[3.2.1]octane core is an essential
structure for ELOVL6 inhibitory activity.
Finally, the right-hand phenyl portion was optimized
(Table 3). Meta-substitution with chloro (1u) and methoxy
(1r) groups resulted in 8- to 11-fold decreased potency. These
deleterious effects were more noticeable in the ortho- and
para-substituted derivatives 1q, 1s, 1t, and 1v. These results
suggest that introduction of substituents on the right-hand
phenyl group is unfavorable. Subsequently, the phenyl group
was replaced by a pyridine ring. As a result, the 2-pyridinyl
derivative 1w was tolerated, whereas the 3- and 4-pyridinyl
derivatives 1x and 1y showed a 7-fold decrease in potency.
Intriguingly, the 2-pyridinyl derivative 1w exhibited improved
mouse microsomal stability (% remaining=67%) compared
to 1k (% remaining = 38%).20 Given the potent ELOVL6
inhibitory activity and good microsomal stability, 1w was
selected as a candidate for further in vitro characterization.
Mouse ELOVL6 inhibitory activity, selectivity over the
other ELOVL subtypes, and cellular activity of 1w are sum-
marized in Table 4. Compound 1w showed comparable
inhibitory activities for human and mouse ELOVL6s (IC50
of 79 and 94 nM, respectively). Regarding ELOVL subtype

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4113
Table 3. SAR around Right-Hand Phenyl Moietya
human ELOVL6
enzyme IC50 (nM)
compd
R2
1k
1q
1r
Ph-
78 ( 28
7970 ( 1560
840 ( 90
5630 ( 1500
3390 ( 150
600 ( 70
2850 ( 490
79 ( 10
560 ( 190
510 ( 120
2-MeO-Ph-
3-MeO-Ph-
4-MeO-Ph-
2-Cl-Ph-
1s
Figure 1. Inhibition of ELOVL6 activity by 1w in the mouse
hepatocyte cell line H2.35. The elongation index was determined
by the radio-HPLC as described in Supporting Information and
defined as % of control of the ratio of peak area [C18:0+C18:1,
n-7 + C18:1,n-9]/peak area [C16:0 + C16:1]. The results are
taken from at least three independent tests and express the mean
( SEM.
1t
1u
1v
1w
1x
1y
3-Cl-Ph-
4-Cl-Ph-
2-pyridinyl
3-pyridinyl
4-pyridinyl
a The values represent the mean ( SE for n g 3.
Table 5. Plasma, Liver, and WAT Exposure of 1w in C57BL/6J
Micea
Table 4. In Vitro Profiles of 1wa
time after
WAT
assay
IC50 (nM)
dose (mg/kg) dosing (h) plasma (μM) liver (nmol/g) (nmol/g)
human ELOVL6 enzyme
human ELOVL3 enzyme
79 ( 10
6940 ( 680
>10000
94 ( 19
>10000
30 ( 9
10
30
2
2
4
8
0.46 ( 0.22
1.41 ( 0.46
0.59 ( 0.10
0.52 ( 0.23
4.2 ( 1.4
10.0 ( 2.5
6.0 ( 1.5
5.9 ( 1.8
2.7 ( 1.4
7.3 ( 3.7
2.5 ( 0.2
2.5 ( 1.1
human ELOVL1,2,5 enzyme
mouse ELOVL6 enzyme
mouse ELOVL3 enzyme
a The reported data are an average generated after 10 or 30 mg/kg po
doses in n = 3 animals.
cellular assay in mouse hepatocyte H2.35 cell
a The values represent the mean ( SE for n g 3.
Table 6. Pharmacokinetic Parameters of 1w in SD Ratsa
Scheme 2. Enzymatic Conversion of Palmitoyl-CoA with
ELOVL6 and SCD-1
iv
po
CLp
Vd
compd ((mL/min)/kg) (L/kg) (h)
T1/2 AUC0-¥ Cmax Tmax
F
(μM h) (μM) (h) (%)
3
3.7
1w 19 15.0 10.7
0.4
2.0 54
a The reported data are an average generated after 1 mg/kg iv and 3
mg/kg po doses in n = 3 animals/dose.
used as a surrogate readout for ELOVL6 inhibitory activity:
elongation index = (C18 fatty acids)/(C16 fatty acids) =
[stearoyl acid+oleic acid + cis-vaccenic acid]/[palmitic acid+
palmitoleic acid]. As shown in Figure 1, 1w effectively reduced
the elongation index with an IC50 of 30 nM, indicating that 1w
is a potent inhibitor even at cellular levels. The suitable
lipophilicity of 1w (log D7.4 =3.2)22 for good membrane
penetration partly explains the potent cellular activity.
Next, the pharmacokinetic profile of 1w was investigated.
Compound 1w exhibited sustained exposure in plasma, liver,
and WAT in mice after oral dosing at 30 mg/kg, as shown in
Table 5. The liver-to-plasma and WAT-to-plasma exposure
ratios were 7-12 and 4-6, respectively, demonstrating that
1w is penetrable to the liver and WAT. Compound 1w also
displayed good pharmacokinetic profiles in SD rats (Table 6).
Given the potent ELOVL6 inhibitory activity, selectivity,
and appreciable pharmacokinetic profiles, 1w was evaluated
for its effects on the fatty acid profile in the liver in mice. The
elongation index was used as a surrogate readout for
ELOVL6 inhibitory activity in the liver using [14C]palmitic
acid as a radiotracer. After oral administration, 1w potently
and dose-dependently suppressed the elongation index in the
liver in mice (Figure 2). On the basis of the potent in vivo
activity, excellent selectivity, and good pharmacokinetic pro-
files in rodents, 1w should be an attractive in vivo tool
selectivity, 1w showed excellent selectivity over the other hu-
man ELOVL subtypes (ELOVL1, -2, -3, and -5; IC50>5 μM)
and mouse ELOVL3 (IC50 > 10 μM). In mouse, ELOVL6
selectivity over ELOVL3 of 1w was more than 100-fold.
+
Compound 1w was also selective against the hERG K
channel (IC50 >10 μM). Furthermore, screening of 1w by
MDS Pharma Services (Panlabs) in a panel of 168 other
receptor, enzyme, ion channel, and transporter assays revealed
that 1w has no significant activity at 10 μM. Encouraged by
these results, we examined the cellular activity of 1w in a mouse
hepatocyte cell H2.35 assay. The ELOVL6 inhibitory activity
in the H2.35 cell assay was assessed using [14C]palmitic acid as
a radiotracer. The enzymatic conversion of palmitic acid by
ELOVL6 and SCD-121 is illustrated in Scheme 2. When
ELOVL6 elongation takes place first, palmitoyl-CoA is elon-
gated to give strearoyl-CoA followed by desaturation by SCD-
1 to give oleoyl-CoA. However, when the desaturation process
by SCD-1 takes place first, palmitoyl-CoA is converted to
palmitoleoyl-CoA, which is elongated by ELOVL6 to yield cis-
vaccenoyl-CoA. The elongation index is defined as follows and

4114 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Nagase et al.
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Figure 2. Effects of 1w on ELOVL6 activity in the liver of C57BL/
6J mice. Male C57BL/6J mice were orally administered vehicle
(0.5% methylcellulose) and 1, 3, and 10 mg/kg 1w (suspended
in 0.5% methylcellulose), and 1 h later [1-14C]palmitic acid was
interperitoneally administered at 10 μCi/body. At 2 h postdosing of
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calculate the elongation index. The basal C18/16 ratio 2 h after oral
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In conclusion, optimization of the lead 1a led to the
identification of the potent and selective ELOVL6 inhibitor
1w. Compound 1w displayed excellent selectivity against other
ELOVL subtypes, hERG K+ channel, and a panel of 168
unrelated biological targets. Compound 1w potently reduced
the elongation index in mouse hepatocyte cells H2.35. Further-
more, 1w displayed sustained exposure in plasma, liver, and
white adipose tissues in mice after oral dosing. Oral adminis-
tration of 1w potently and dose-proportionally suppressed the
elongation index of fatty acids in the liver in mice. Taken
together, these findings suggest that 1w is a promising in vivo
tool for the evaluation of pharmacological effects of ELOVL6
inhibition in rodents. Updated results of pharmacological
studies using 1w in rodents will be reported in due course.
Acknowledgment. We thank Naomi Morita and
Dr. Tomoyuki Ohe for collecting pharmacokinetic data,
Hirokazu Ohsawa for collecting the high-resolution mass
spectral data, and Hiroaki Suwa for HPLC purity analyses.
We also thank Dr. Peter T. Meinke (Merck Research Labora-
tories, Rahway, NJ) for the editing of this manuscript.
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Supporting Information Available: Synthetic procedures and
characterization data for all compounds; biological protocols.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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