Synthesis and evaluation of a novel indoledione class of long chain fatty acid elongase 6 (ELOVL6) inhibitors

Article abstract of DOI:10.1021/jm900391x

Novel indoledione derivatives were synthesized and evaluated as long chain fatty acid elongase 6 (ELOVL6) inhibitors. Systematic optimization of an indole class of lead 1 led to the identification of potent ELOVL6 selective inhibitors. Representative inhibitor 37 showed sustained plasma exposure and good liver penetrability in mice. After oral administration, 37 potently inhibited ELOVL6 activity in the liver in mice.

Full text of DOI:10.1021/jm900391x

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J. Med. Chem. 2009, 52, 3142–3145
Letters
Synthesis and Evaluation of a Novel
Indoledione Class of Long Chain Fatty Acid
Elongase 6 (ELOVL6) Inhibitors
Toshiyuki Takahashi, Tsuyoshi Nagase, Takahide Sasaki,
Akira Nagumo, Ken Shimamura, Yasuhisa Miyamoto,
Hidefumi Kitazawa, Maki Kanesaka, Ryo Yoshimoto,
Katsumi Aragane, Shigeru Tokita, and Nagaaki Sato*
Figure 1. Potency and structure of lead 1.
Scheme 1a
Tsukuba Research Institute, Merck Research Laboratories, Banyu
Pharmaceutical Co., Ltd, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan
ReceiVed March 27, 2009
Abstract: Novel indoledione derivatives were synthesized and evalu-
ated as long chain fatty acid elongase 6 (ELOVL6) inhibitors.
Systematic optimization of an indole class of lead 1 led to the
identification of potent ELOVL6 selective inhibitors. Representative
inhibitor 37 showed sustained plasma exposure and good liver
penetrability in mice. After oral administration, 37 potently inhibited
ELOVL6 activity in the liver in mice.
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 the dysfunction
in those tissues, i.e., insulin resistance.1 Although it is unclear
how increased intracellular lipid content exacerbates tissue and
whole body insulin sensitivity, it has been suggested that
increased levels of long chain fatty acyl-CoA antagonize the
metabolic actions of insulin. Interestingly, recent reports sug-
gested that alternation of the specific fatty acid component (i.e.,
palmitoleate) has significant impact on the insulin sensitivity
of liver and whole body.2,3
a Reagents and conditions: (a) CHCl3, reflux, 2 h; (b) SOCl2, toluene,
reflux, 3 h; (c) CHCl3, room temp, 14 h.
Scheme 2a
With regard to de novo lipid synthesis, the microsomal
enzymes are responsible for the elongation of long chain fatty
acids with chain length >C16 while FAS,a a cytosolic enzyme,
is responsible for the de novo synthesis of the fatty acid with
chain lengths up to C16.4 Fatty acid elongation at microsomal
fractions requires four sequential steps: (1) condensation be-
tween fatty acyl-CoA and malonyl-CoA to generate ꢀ-ketoacyl-
CoA, (2) reduction by ꢀ-ketoacyl-CoA reductase, (3) dehydro-
genation by ꢀ-hydroxyacyl-CoA dehydrogenase, and (4) reduction
by trans-2,3-enoyl-CoA reductase.4,5 ELOVL enzymes are
responsible for the rate-limiting initial condensation reaction.6,7
So far, seven ELOVL enzymes have been identified in
mammals and are designated ELOVL1-7.6-11 Each ELOVL
enzyme exhibits different fatty acid substrate preferences and
tissue distributions, suggesting that they play different physi-
ological roles in vivo.12 Among ELOVL enzymes, ELOVL3
and ELOVL6 show the highest homology to each other (43.7%)
and are expressed in the highly lipogenic tissues such as liver
a Reagents and conditions: (a) neat, room temp, 14 h; (b) toluene, reflux,
2 h; (c) (i) SOCl2, toluene, reflux, 3 h, (ii) 3-anilino-5,5-dimethylcyclohex-
2-en-1-one, CHCl3, room temp, 14 h.
and adipose that play important roles in the regulation of lipid
metabolism.6,9 ELOVL6 regulates the synthesis of stearoyl-CoA
(C18:0) and cis-vaccenoyl-CoA (C18:1).6,7 Regarding the
regulatory mechanism for expression, liver ELOVL6 expression
is up-regulated by refeeding after fasting, by a high carbohydrate
diet, and in obese rodents.6,7,13 At the molecular level, ELOVL6
expression is directly and primarily regulated by sterol regulatory
element-binding protein (SREBP)-1c.14
Recent investigations using gene-deleted mice for ELOVL6
suggest that ELOVL6 regulates the hepatic insulin sensitivity
by modulating the fatty acid components.3 Although increasing
information on the physiological and pathological roles of
ELOVL6 has drawn much attention, a lack of useful chemical
tools makes it difficult to address the pharmacological roles of
ELOVLs and their therapeutic potentials.
* To whom correspondence should be addressed. Phone: 81-29-877-2004.
Fax: 81-29-877-2029. E-mail: nagaaki_sato@merck.com.
a Abbreviations: ELOVL, long chain fatty acid elongase; FAS, fatty acid
synthase; UHTS, ultrahigh-throughput screen; SAR, structure-activity
relationship; SCD-1, stearoyl-CoA desaturase 1.
We recently reported the establishment of a homogeneous
enzyme assay for ELOVL6, which is applicable to UHTS using
10.1021/jm900391x CCC: $40.75
2009 American Chemical Society
Published on Web 04/23/2009

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3143
Table 3. Human ELOVL3 and 6 Inhibitory Activity of 24-38a
Table 1. Human ELOVL6 Inhibitory Activity of 1-17a
a The values represent the mean ( SE for n g 3. b Inhibitory activity of
compounds on human ELOVL6 for palmitoyl-CoA elongation.
Table 2. Human ELOVL6 Inhibitory Activity of 18-23a
a The values represent the mean ( SE for n g 3. b Inhibitory activity of
compounds on human ELOVL6 for palmitoyl-CoA elongation. c Inhibitory
activity of compounds on human ELOVL3 for stearoyl-CoA elongation.
compd
R2
Et
i-Pr
Me
Me
Me
Me
R3, R4
human ELOVL6b (IC50, nM)
corporate chemical collection was screened against human
ELOVL6, resulting in the identification of a novel indoledione
class of lead 1, which has an IC50 of 290 nM for human
ELOVL6 (Figure 1). Systematic SAR studies of the indoledione
lead 1, aiming at optimization of ELOVL6 activity, resulted in
the discovery of potent ELOVL6 inhibitors. In this report,
preliminary results of the SAR studies of the indoledione class
of ELOVL6 inhibitors and the discovery of the potent ELVOL6
selective inhibitor 37 are reported.
18
19
20
21
22
23
R3 ) R4 ) Me
R3 ) R4 ) Me
R3 ) R4 ) H
99 ( 28
>10000
4500 ( 190
704 ( 97
6260 ( 428
123 ( 18
R3 ) H, R4 ) Me
R3 ) H, R4 ) Ph
-(CH2)3-
a The values represent the mean ( SE for n g 3. b Inhibitory activity of
compounds on human ELOVL6 for palmitoyl-CoA elongation.
the recombinant histidine-tagged ACBP as a molecular probe
for the detection of radioactive products of ELOVL6.15 Our
The synthesis of the indoledione ELOVL6 inhibitors 1-38
reported herein is outlined in Schemes 1 and 2. The substituted

3144 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Letters
Table 4. Human ELOVL Subtype Selectivity and Mouse ELOVL3 and 6 Activity of 37a
enzyme
hELOVL1
hELOVL2
hELOVL3
hELOVL5
hELOVL6
mELOVL6
mELOVL3
194 ( 26 nM
activity (IC50)
>10 µM
>10 µM
337 ( 74 nM
>10 µM
8.9 ( 2.7 nM
31 ( 4 nM
a Inhibitory activity of 37 on ELOVLs for their respective substrates. The values are the mean ( SE for n g 3.
Table 5. Plasma Exposure of 37 in Mice
time
2 h
3.4
4 h
2.8
8 h
1.4
24 h
0.4
plasma level (µM)a
a The plasma levels were determined after 1 mg/kg oral administration.
The values represent the mean for n ) 3 animals.
pyrazol-3-one A and methyl 3,3,3-trifluoro-2-oxopropanoate
were coupled to afford B, which was dehydrated using thionyl
chloride to give enone C. Cyclocondensation of the enone C
with the substituted 3-aminocyclohex-2-en-1-one D provided
the desired indoledione derivatives 1-36. The derivatives 37
and 38 were prepared by a slightly modified method (Scheme
2). Ethyl 3-oxobutanoate and methyl 3,3,3-trifluoro-2-oxopro-
panoate were coupled to give methyl 3,5-dideoxy-3-(ethoxy-
carbonyl)-2-C-(trifluoromethyl)pent-4-ulosonate, which was con-
densed with the appropriate substituted hydrazine to give B.
Compound B was dehydrated with thionyl chloride followed
by cyclocondensation with 3-anilino-5,5-dimethylcyclohex-2-
en-1-one to afford 37 or 38.
The compounds were optimized based on human ELOVL6
inhibitory activity, and the potent human ELOVL6 inhibitors
were tested for human ELOVL3 inhibitory activity due to the
high sequence homology among the ELOVL family members.
Substituent effects of R1 of the lead compound 1 were
investigated initially (Table 1). The R1 substituent was essential
for potency, as shown by a 6-fold potency decrease of 2. The
methyl and ethyl derivatives (3 and 4) also showed decreased
activities, while the n-propyl derivative 5 was equipotent to lead
1. The branched isopropyl derivative 6 displayed a decrease in
activity. A series of cycloalkyl derivatives (7-10) were prepared
and evaluated. Of them, the cyclopropyl derivative 7 was slightly
more potent than the parent 1. Benzyl substitution as in 11 was
not tolerated. In light of the substituted phenyl derivatives
(12-17), positional scanning was carried out with electron
withdrawing chloro and electron donating methoxy groups.
However, no noticeable substituent effect was identified, and
the substitution at the meta-position was found to retain potency.
Next, R2-R4substituent effects were studied briefly (Table
2). The ethyl derivative 18 was slightly more potent than the
parent 1; however, the isopropyl derivative 19 showed a
complete loss of potency. Because this portion of the molecule
Figure 2. Effects of 37 on elongase activity in the liver of C57BL/6J
mice. Male C57BL/6J mice were orally administered 0.1, 0.3, and 1
mg/kg 37 (dissolved in 0.5% methylcellulose), and 1 h later [1-14C]palm-
itic acid was interperitoneally administered at 10 µCi/body. At 2 h
postdosing of 37, fatty acids were extracted and measured by radio-
HPLC to calculate the elongation index (C16/C18 ratio).
seems to be sensitive to steric effects, further investigation for
the R2 substituent was not attempted. Regarding the R3 and R4
substituents, desmethylation as in 20 was found to be detrimental
to potency. The monomethyl derivative 21 was less potent than
1, and the phenyl derivative 22 displayed a further decrease in
potency. It was delightful to find that the cyclobutyl derivative
23 displayed an IC50 of 123 nM, which is twice as potent as
lead 1.
Finally, the substituent effect of the R5 group was studied
(Table 3). Methyl substitution as in 24 showed decreased
activity, and the isopropyl and cyclohexyl derivatives (25 and
26) showed complete loss of potency. Hence, we decided to
focus on substituted phenyl derivatives. Positional scanning with
chloro and methoxy groups was carried out. As a result,
substitution of the para-position appeared to be the most
promising. A series of the para-substituted derivatives 33-38
were prepared and evaluated. With the exception of the phenoxy
derivative 38, all the para-substituted phenyl derivatives dis-
played potent ELOVL6 activities. The potent ELVOL6 inhibi-
tors were tested for ELVOL3 inhibitory activity. The test
compounds 29, 32, 34-37 showed moderate to good ELOVL6
selectivity over ELOVL3 as shown in Table 3. The isopropyl
derivative 35 is the least selective where the selectivity (a ratio
Scheme 3. Enzymatic Reactions of Palmitoyl-CoA with ELOVL6 and SCD-1

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3145
and purity for the target compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
of ELVOL3 to ELOVL6) is ∼6-fold. Of them, compound 37
is a significantly more potent and selective inhibitor (Table 3).
Compound 37 was shown to be selective over human ELOVL1,
-2, and -5 enzymes and has a potent inhibitory activity for the
mouse ELOVL6 enzyme as well (Table 4). The selectivity
against mouse ELOVL3 is moderate where the ratio of mouse
ELOVL3 to ELOVL6 is ∼6-fold.
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The plasma and liver levels 2 h following 10 mg/kg oral
administration of 37 in mice were determined to be 30 and 50
µM, respectively; therefore, 37 is demonstrated to be highly
liver penetrable (a liver-to-plasma ratio of 1.7). After oral dosing
at 1 mg/kg in mice, 37 exhibited sustained plasma exposure as
shown in Table 5.
Having demonstrated potent ELOVL6 activity and good
exposure, 37 was evaluated for its effects on the fatty acid profile
in the liver in mice. ELOVL6 is mainly responsible for the
elongation of palmitoyl-CoA. Enzymatic reactions of palmitoyl-
CoA involving ELVOL6 and SCD-1 are depicted in Scheme
3. When ELOVL6 elongation takes place first, palmitoyl-CoA
is elongated to give strearoyl-CoA followed by desaturation by
SCD-1 to give oleoyl-CoA. When the desaturation process by
SCD-1 takes place first, palmitoyl-CoA is converted to palmi-
toleoyl-CoA, which is elongated by ELVOL6 to yield cis-
vaccenoyl-CoA. We defined the elongation index as follows:
elongation index ) C18 fatty acids/C16 fatty acids ) [stearoyl
acid + oleic acid + cis-vaccenic acid]/[palmitic acid +
palmitoleic acid]. 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,
37 potently and dose-proportionally suppressed the elongation
index in the liver in mice (Figure 2).
In conclusion, new indoledione derivatives were synthesized
and evaluated as ELOVL6 inhibitors. After systematic SAR
studies of the R1-R5 substituents of the screen hit 1, several
para-substituted phenyl derivatives were identified as potent
ELOVL6 inhibitors. Representative potent inhibitor 37 was
selective for human ELOVL6 over human ELOVL1, -2, -3, and
-5 enzymes. Compound 37 displayed potent inhibitory activity
for mouse ELOVL6 as well; however, selectivity against mouse
ELOVL3 was moderate. Sustained plasma exposure and ap-
preciable liver penetration were observed after oral administra-
tion of 37 in mice. After oral dosing, 37 potently and dose-
proportionally suppressed the elongation index of fatty acids
in the liver in mice. The potent and selective inhibitor 37 would
be a powerful tool to probe the pharmacology of the ELOVL6
enzyme inhibition in vivo. The updated results will be reported
in due course.
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Acknowledgment. We thank Hirokazu Ohsawa for collect-
ing the high-resolution mass spectral data. We also thank Dr.
Peter T. Meinke (Merck Research Laboratories, Rahway, NJ)
for the editing of this manuscript.
Supporting Information Available: Synthetic procedures for
the preparation of 1-38, biological methods, HPLC retention times,
JM900391X