(4-Piperidinyl)-piperazine: A new platform for acetyl-CoA carboxylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.10.012

Acetyl-CoA carboxylases (ACCs), the rate limiting enzymes in de novo lipid synthesis, play important roles in modulating energy metabolism. The inhibition of ACC has demonstrated promising therapeutic potential for treating obesity and type 2 diabetes mel

Full text of DOI:10.1016/j.bmcl.2009.10.012

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6645–6648
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
(4-Piperidinyl)-piperazine: A new platform for acetyl-CoA carboxylase inhibitors
a
b
c
a
Tomomichi Chonan a, , Takahiro Oi , Daisuke Yamamoto , Miyoko Yashiro , Daisuke Wakasugi ,
*
Hiroaki Tanaka a, Ayumi Ohoka-Sugita b, Fusayo Io b, Hiroko Koretsune b, Akira Hiratate a
a Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan
b Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan
c Research Computer System, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Acetyl-CoA carboxylases (ACCs), the rate limiting enzymes in de novo lipid synthesis, play important
roles in modulating energy metabolism. The inhibition of ACC has demonstrated promising therapeutic
potential for treating obesity and type 2 diabetes mellitus in transgenic mice and preclinical animal mod-
els. We describe herein the synthesis and structure–activity relationships of a series of disubstituted (4-
piperidinyl)-piperazine derivatives as a new platform for ACC1/2 non-selective inhibitors.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 21 August 2009
Revised 1 October 2009
Accepted 3 October 2009
Available online 8 October 2009
Keywords:
Acetyl-CoA carboxylase
Inhibitor
(4-Piperidinyl)-piperazine
Acetyl-CoA carboxylase (ACC) is a biotin-dependent homo olig-
omeric protein composed of a carboxyltransferase (CT), a biotin
carboxyl carrier protein and biotin carboxylase (BC) domains.
ACC is involved in the synthesis of malonyl-CoA from acetyl-CoA
in an ATP-dependent manner. Malonyl-CoA works not only as a
substrate for de novo fatty acid synthesis, but also as an allosteric
inhibitor of carnitine palmitoyl transferase (CPT-1), a key enzyme
that positively regulates mitochondrial b-oxidation. Therefore,
inhibition of ACC is expected to reduce de novo fatty acid synthesis
(FAS) and to enhance fatty acid b-oxidation (FAO) through disinhi-
bition of CPT-1, which might benefit treatment of metabolic disor-
ders such as obesity and diabetes.1
Two ACC isoforms, ACC1 and ACC2, have been cloned in rodents
and humans. ACC1 is predominantly expressed in lipogenic tissues
such as liver and adipose tissue, while ACC2 is predominantly ex-
pressed in oxidative tissues such as liver, skeletal muscle and
heart.2 Consequently, reduction of malonyl-CoA levels in these tis-
sues by ACC1/2 non-selective inhibitors is expected to reduce de
novo fatty acid synthesis and triglyceride (TG)-rich lipoprotein
secretion in liver, while increasing fatty acid b-oxidation in liver
and skeletal muscle. Therefore, an ACC1/2 non-selective inhibitor
might provide a novel therapeutic approach for treating various
metabolic disorders.
metabolic stability based on CP-640186 (Fig. 1), a non-selective
inhibitor of ACC 1 and 2 (rat ACC1 IC50 = 53 nM, rat ACC2
IC50 = 61 nM).1
In order to enhance the intrinsic potency of CP-640186, the
introduction of a new central core structure was deemed essential.
For this purpose, we screened our scaffold collections and identi-
fied a (4-piperidinyl)-piperazine class of compound 1 with an
IC50 of 239 nM, which appeared to be a potential lead structure
for further optimization. The metabolic stability of compound 1
was assessed using a human microsomal incubation assay; only
19% of the intact compound was recovered,3 indicating that com-
pound 1 is metabolically unstable. A metabolite identification
study of 1 revealed that the left-hand anthranyl and the right-hand
morpholyl groups are the main sites metabolized. Having identi-
fied potential lead 1, we initiated detailed SAR studies.
We have initiated a drug discovery program directed towards
the identification of potent ACC inhibitors with appreciable
* Corresponding author. Tel.: +81 48 669 3108; fax: +81 48 652 7254.
E-mail address: tomomichi.chonan@po.rd.taisho.co.jp (T. Chonan).
Figure 1. CP-640186 and compound 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.012

6646
T. Chonan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6645–6648
The co-crystal structure of CP-640186 and yeast ACC has been
reported by Zhang et al.4 CP-640186 tightly associates with the ac-
tive site of the carboxyltransferase (CT) domain of yeast ACC and
blocks the binding of the biotin substrate to the CT domain at
the domain’s dimer interface. The crystal structure shows that
the oxygen of the anthranyl amide of CP-640186 is hydrogen-
bonded to the main chain amide of Glu20260 near the beginning
of helix a6, whereas the carbonyl oxygen adjacent to the morpho-
line is hydrogen-bonded to the main chain amide of Gly19580. Gi-
ven that the amino acid sequence of the active site of the CT
domain is highly conserved across eukaryotic species having mul-
ti-domain ACCs,5 modeling studies were carried out using MOE.6
The homology model of the CT domain of human ACC2 from the
crystal structure of yeast ACC in complex with CP-640186 (PDB
ID: 1W2X) was used. The predicted binding mode of compound 1
(in yellow) is shown in Figure 2, along with an overlay of CP-
640186 (in purple). The predicted binding mode suggests that
the carbonyl oxygen of the morpholinyl amide of 1 is not hydro-
gen-bonded to Gly21620 (yeast Gly19580); nevertheless, the mor-
pholine rings of both CP-640186 and compound 1 occupy the
same pocket.
The general synthesis of the substituted (4-piperidinyl)-pipera-
zine analogues is outlined in Scheme 1. tert-Butoxy piperazine-1-
carboxylate (2) was coupled with the desired acid chloride under
basic conditions, followed by removal of the tert-butoxycarbonyl
(Boc) group to give the substituted piperazine 3. The amino group
of 3 was reductively alkylated with tert-butyl 4-oxopiperidine-1-
carboxylate followed by cleavage of the Boc group to afford 4,
which was coupled with the desired acid chloride or carboxylic
acid to give the target compounds 5–36.
Scheme 1. Reagents and conditions: (a) R1COCl, Et3N, CHCl3, rt; (b) 4 M HCl–AcOEt,
MeOH, rt; (c) 1-Boc-piperidone, CHCl3, rt, then NaBH(OAc)3, CHCl3, rt; (d) 4 M HCl–
AcOEt, MeOH, rt; (e) (i) R2COOH, EDCl, HOBt, Et3N, DMF, rt, or R2COCl, Et3N, CHCl3,
(ii) 4 M HCl–AcOEt, or 1 M maleic acid in AcOEt.
crease in [14C] acetate incorporation into cellular lipids,1a and the
effects on FAO were evaluated by measuring the generation of
T2O in the culture media.7
The right-hand piperidine substituent was modified initially
(compound 5–11, Table 1). The piperidyl, cyclohexyl, and cyclopen-
tyl derivatives (5, 8, and 9) showed slightly increased activity. The
benzyl derivative 7 displayed a substantial decrease in potency.
The phenyl and 3-(2,5-dimethylfuryl) derivatives (6 and 11) showed
improved potency compared to the parent compound 1. Next, the
metabolically labile anthranyl group was replaced by a (2,6-
diphenyl)pyridyl group8 (12–18, Table 1). The (2,6-diphenyl)pyridyl
The derivatives were screened against partially purified human
liver ACC enzymes, and the subtype selectivity of the optimized
compounds was confirmed using recombinant human ACC1 and
ACC2 enzymes. Among the tested compounds, the selected potent
compounds were further evaluated for their ability to decrease
fatty acid synthesis (FAS) and increase fatty acid oxidation (FAO)
in HepG2 cells. FAS inhibition was assessed by measuring the de-
Table 1
In vitro ACC activity and metabolic stability in human liver microsomes of the
disubstituted (4-piperidinyl)-piperazines 1, 5–18
Compd R1
R2
ACC IC50 hMSb
a
(nM)
(%)
1
5
6
7
8
9-Anthranyl
4-Morpholinyl
1-Piperidyl
Phenyl
239
121
66
2112
91
95
161
60
19
9-Anthranyl
9-Anthranyl
9-Anthranyl
9-Anthranyl
9-Anthranyl
9-Anthranyl
9-Anthranyl
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
Benzyl
Cyclohexyl
Cyclopentyl
2-Furyl
9
10
11
12
3-(2,5-Dimethylfuryl)
4-(2,6-Diphenyl)- 4-Morpholinyl
869
pyridyl
13
14
15
16
17
18
4-(2,6-Diphenyl)- 1-Piperidyl
pyridyl
4-(2,6-Diphenyl)- Phenyl
pyridyl
4-(2,6-Diphenyl)- Cyclohexyl
pyridyl
4-(2,6-Diphenyl)- 3-(2,5-Dimethylfuryl)
pyridyl
434
693
244
157
126
155
NDc
96
61
37
79
52
4-(2,6-Diphenyl)- 1-Acetylpiperidin-4-yl
pyridyl
4-(2,6-Diphenyl)- trans-4-Methoxy-cyclohexyl
pyridyl
a
Inhibitory activity of compounds on the malonyl-CoA synthesis of human ACC1/
% Remaining after 15-min incubation with human liver microsomes (1 mg
2.
b
protein/mL).
c
No data.
Figure 2. Optimized binding mode of 1.

T. Chonan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6645–6648
6647
Table 2
derivatives were found to be substantially less potent than the cor-
In vitro activities of the disubstituted 4-(4-piperidinyl)-piperazines 17, 19–28
responding anthranyl derivatives. Of them, compounds 16–18 dis-
played appreciable activity. We speculate that the enhanced
activity of 16–18 is partly due to their interaction with Tyr1974
(Fig. 2). A molecular model of compound 17 (in yellow) is shown in
Figure 3 along with an overlay of CP-640186 (in purple). The pre-
dicted binding mode suggests that the diphenyl pyridine moiety of
17 is trapped in a narrow space where the anthranyl group of CP-
640186 resides, and the adjacent carbonyl group hydrogen-bonds
to the main chain amide of Glu22300 (yeast Glu20260). The left-hand
1-acetylpiperidine moiety of 17 fits into the mostly hydrophobic
area on the surface of the active site, and the acetyl group of the
piperidineis capableof bindingtoTyr1974. In addition togoodactiv-
ity, the potent (2,6-diphenyl) pyridyl derivative 17 was found to
have good microsomal stability (Table 1).
Optimization of the acetyl substituent of 17 was attempted
(Table 2). The benzamide 20, isopropyl carbamate 24 and methane
sulfonamide 28 were substantially less potent than compound 17,
showing that bulky substituents tend to decrease ACC inhibitory
potency. No compounds with noticeably improved characteristics
were identified by this limited modification study.
Finally, substituent effects on the left-hand side phenyl groups
were investigated (Table 3). The fluoro derivatives 29 and 30 were
less potent than the parent 17. The methyl 31 and methoxy 32
derivatives exhibited improved ACC inhibitory activity both in en-
zyme and cell-based assays. The 4-hydroxymethyl derivative 34
displayed the most potent enzyme activity among this series, with
an IC50 value of 32 nM; nevertheless, its cell activity was substan-
tially lower than those of 31 and 32, probably due to their limited
cell permeability. Also the 4-hydroxyl derivative 33 and the 3- and
4-aminocarbonyl derivatives 35 and 36 exhibited significantly re-
duced enzymatic activity in cells, probably for the same reason.
The metabolic stability of derivatives 29–32 were improved com-
pared to compound 17. Derivative 31, which has optimal activity
in the FAS and FAO cell assays, was selected for further pharmaco-
logical profiling.
a
Compd
R
ACC IC50 (nM)
17
126
115
19
20
290
21
22
169
102
The pharmacological profiles of 31 are summarized in Table 4.
Compound 31 displayed more potent activity against human re-
23
24
102
411
25
26
151
95
27
28
169
248
a
Inhibitory activity of compounds on malonyl-CoA synthesis of human ACC1/2.
combinant ACC2 (rhACC2) than human recombinant ACC1
(rhACC1).9 In HepG2 cell assays, compound 31 showed dose-
dependent inhibition of fatty acid synthesis, with an IC50 of
Figure 3. Optimized binding mode of 17.

6648
T. Chonan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6645–6648
Table 3
0.34
EC50 of 0.58
l
M, and caused an increase in fatty acid oxidation, with an
In vitro activities of the disubstituted 4-(4-piperidinyl)-piperazine 17, 29–36
lM. Additionally, compound 31 is metabolically stable
in human and rat liver microsomes and showed no significant
inhibitory effects on the major cytochrome P450 isozymes
(CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4).
In conclusion, we have identified the (4-piperidinyl)-piperazine
scaffold as a new platform for ACC1/2 non-selective inhibitors and
discovered compound 31, which has promising activity. Compound
31 exhibited significantly improved metabolic stability in liver
microsomes as compared with the lead compound 1. Further mod-
ification of this series is ongoing in order to improve ACC inhibitory
potency.
FAOb (%)
FASc (%)
hMSd (%)
a
Compd
R
ACC IC50 (nM)
17
29
30
31
32
33
34
35
36
H
3-F
4-F
4-Me
4-OMe
4-OH
4-CH2OH
3-CONH2
4-CONH2
126
131
184
76
68
52
32
73
67
148
NDe
NDe
176
167
123f
120f
94f
72
79
90
94
87
NDe
NDe
77
Acknowledgments
The authors thank Yoshiki Fukasawa for collecting DMPK data.
The authors also thank Drs. Nagaaki Sato and Shigeru Tokita for
editing this Letter.
77
87
29f
NDe
NDe
NDe
NDe
NDe
NDe
NDe
135f
a
b
c
Supplementary data
Inhibitory activity of compounds on malonyl-CoA synthesis of human ACC1/2.
Activation of fatty acid oxidation in HepG2 cells at 1 M.
Inhibitory activity of fatty acid synthesis in HepG2 cells at 1
l
lM.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.012.
d
% Remaining after 15-min incubation with human liver microsomes (1 mg
protein/mL).
e
No data.
f
Screening concentration at 10 lM.
References and notes
1. (a) Harwood, H. J., Jr.; Petras, S. F.; Shelly, L. D.; Zaccaro, L. M.; Perry, D. A.;
Makowski, M. R.; Hargrove, D. M.; Martin, K. A.; Tracey, W. R.; Chapman, J. G.;
Magee, W. P.; Dalvie, D. K.; Soliman, V. F.; Martin, W. H.; Mularski, C. J.;
Eisenbeis, S. A. J. Biol. Chem. 2003, 278, 37099; (b) Harwood, H. J., Jr. Exp. Opin.
Cardiovasc. Renal. 2005, 9, 267; (c) Corbett, J. W.; Harwood, H. J., Jr. Rec. Pat.
Cardiovasc. Drug Discovery 2007, 2, 162.
2. Abu-Elheiga, L.; Matzuk, M. M.; Abo-Hashema, K. A.; Wakil, S. J. Science 2001,
291, 2613.
3. Metabolic stability in liver microsomes: 5 lM compound was incubated at 37 °C in
Table 4
Pharmacological profiles and plasma exposure of 31 and CP-640186
Pharmacological profiles
31
CP-640186
Enzyme assaya
hACC 1/2
rhACC1
76 nM
101 nM
23 nM
116 nM
456 nM
194 nM
rhACC2
1 mg/mL human or rat microsomes supplemented with 1.5 mM glucose-6-
phosphate, 0.16 mM b-nicotinamide-adenine dinucleotide phosphate, 1 U/mL
glucose-6-phosphate dehydrogenase, 250 mM phosphate buffer, 2.4 mM
magnesium chloride and 69 mM potassium chloride. Concentrations of the
test compound were determined by LC–MS/MS. Metabolic stability was
calculated from the ratio of the test compound concentration at 0 min to its
concentration after15-min incubation.
Cell-based assay
HepG2 Cell FAS (IC50
HepG2 Cell FAO (EC50
Microsomal metabolismc
Human liver microsomes
Rat liver microsomes
Plasma exposure after pod
Cmax (ng/mL)
)
0.34
0.58
l
l
M
M
0.84
NDb
lM
)
87
96
52
NDb
4. Zhang, H.; Tweel, B.; Li, J.; Tong, L. Structure 2004, 12, 1683.
5. Tong, L. Cell. Mol. Life Sci. 2005, 62, 1784.
6. MOE (Molecular Operating Environment); Chemical Computing Group:
Montreal, Quebec, Canada, 2006.
107
4.00
1000
NDb
NDb
NDb
Tmax (h)
AUC (ng/mL h)
7. Anne, M.; William, J. R. J. Clin. Invest. 1987, 79, 59.
8. Shinde, P.; Srivastava, S. K.; Odedara, R.; Tuli, D.; Munshi, S.; Patel, J.; Zambad, S.
P.; Sonawane, R.; Gupta, R. C.; Chauthaiwale, V.; Dutt, C. Bioorg. Med. Chem. Lett.
2009, 19, 949.
9. Dong, C.; Ching-Hsuen, C.; Luping, C.; John, N. F.; Gabe, A. M.; Yuli, W.; Joseph,
W. C.; Mark, R. H.; Gregory, A. L.; Yongmi, A.; James, K. T. Protein Exp. Purif. 2007,
51, 11.
a
Inhibitory activity of compounds on the malonyl-CoA synthesis of human ACC1/
2, recombinant hACC1 and hACC2.
b
No data.
c
% Remaining after 15-min incubation with human liver microsomes (1 mg
protein/mL).
d
Plasma exposure after single oral administration of 31 at a dose of 10 mg/kg to
male Sprague-Dawley rats.