Symmetrical approach of spiro-pyrazolidinediones as acetyl-CoA carboxylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.05.062

Spiro-pyrazolidinedione derivatives without quaternary chiral center were discovered by structure-based drug design and characterized as potent acetyl-CoA carboxylase (ACC) inhibitors. The high metabolic stability of the spiro-pyrazolo[1,2-a]pyridazine sc

Full text of DOI:10.1016/j.bmcl.2012.05.062

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4769–4772
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Symmetrical approach of spiro-pyrazolidinediones as acetyl-CoA
carboxylase inhibitors
⇑
Makoto Kamata , Tohru Yamashita, Asato Kina, Michiko Tawada, Satoshi Endo, Atsushi Mizukami,
Masako Sasaki, Akiyoshi Tani, Yoshihide Nakano, Yuuki Watanabe, Naoki Furuyama, Miyuki Funami,
Nobuyuki Amano, Kohji Fukatsu
Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 2-26-1, Muraoka-higashi, Fujisawa, Kanagawa 251-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Spiro-pyrazolidinedione derivatives without quaternary chiral center were discovered by structure-
based drug design and characterized as potent acetyl-CoA carboxylase (ACC) inhibitors. The high meta-
bolic stability of the spiro-pyrazolo[1,2-a]pyridazine scaffold and enhancement of the activity by incor-
poration of a 7-methoxy group on the benzothiophene core successfully led to the identification of
compound 4c as an orally bioavailable and highly potent ACC inhibitor. Oral administration of 4c signif-
icantly decreased the values of the respiratory quotient in rats, indicating the stimulation of fatty acid
oxidation.
Received 10 April 2012
Revised 8 May 2012
Accepted 15 May 2012
Available online 19 May 2012
Keywords:
Acetyl-CoA carboxylase
ACC
Ó 2012 Elsevier Ltd. All rights reserved.
Spiro-pyrazolidinedione
Spiro-pyrazolo[1,2-a]pyridazine
Obesity
Fatty acid oxidation
Acetyl-CoA carboxylase (ACC) catalyzes the biotin-dependent
carboxylation of acetyl-CoA to form malonyl-CoA,1 which is a
known substrate for fatty acid synthase in de novo lipogenesis as
well as an allosteric inhibitor of carnitine palmitoyltransferase 1
(CPT-1).2 ACC inhibitors, which could reduce fatty acid synthesis
and accelerate fatty acid oxidation by lowering malonyl-CoA levels,
between the logD value and metabolic stability.5 This time we
made an attempt to adopt a symmetrical approach for spiro struc-
tures as one of the solutions. Herein, we describe the synthesis and
structure-activity relationship (SAR) of spiro-pyrazolidinediones
derivatives and the excellent metabolic stability of the spiro-pyraz-
olo[1,2-a]pyridazine scaffold in human hepatic microsomes.
Me
4
O
MeO
7
N
Me
Me
N
N
N
N
O
S
S
O
S
N
N
N
O
N
N
O
O
Me
O
NH
O
O
NH
O
O
NH
O
Me
NH
NH
NH
Et
(S
)-1
(S
)-2
4c
Et
Me
are widely known to be promising therapeutic targets for the treat-
ment of obesity.3 We have previously reported on spiro series as
novel ACC inhibitors.4,5 In particular, spiro-lactone (S)-1 containing
a ureidobenzothiophenyl group was designed by structure-based
drug design, and its potent inhibitory activity has been demon-
strated.4 Moreover, we successfully identified highly potent and
orally bioavailable inhibitor (S)-2 through optimization of the ben-
zothiophene and spiro-piperidine groups by using the relationship
Spiro-pyrazolidinediones 3a, b were synthesized following the
route illustrated in Scheme 1. Carboxylation of ester 5 with benzyl
chloroformate gave diester 6. After the benzyl group of 6 was
deprotected under reductive conditions, amide 8 was obtained
by condensation with 7 and Z-hydrazine. Removal of the Z group
in 8 by catalytic hydrogenation followed by cyclization under basic
and heat conditions afforded spiro-pyrazolidinedione 9. Dialkyla-
tion of 9 afforded N,N-dialkyl derivatives 10a, b with moderate
yield, which were easily separated from byproduct N,O-dialkyl
compounds 11a, b by using silica gel column chromatography.
Deprotection of the Boc groups in 10a, b followed by reductive
⇑
Corresponding author. Tel.: +81 466 32 1130; fax: +81 466 29 4451.
E-mail address: Kamata_Makoto@takeda.co.jp (M. Kamata).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.062

4770
M. Kamata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4769–4772
O
O
O
O
d
a
c
b
O
N
NHNHZ
OEt
N
OH
OEt
N
N
OBzl
OEt
N
OEt
Boc
Boc
Boc
NH
Boc
Boc
N
N
O
O
O
O
O
Boc
H
5
6
7
8
9
13
O
R2
O
O
O
N
N R2
g
f
N
e
N
N R2
N
S
Boc
N
N
Boc
N R2
N
N
O
N
S
O
R2
N
OH
N
Boc
R2
O
O
O
R2
NH
NH
O
Me
Byproduct
H2N
O
R1
10a (R2 = Me)
10b (R2 = Et)
11a (R2 = Me)
11b (R2 = Et)
12a (R2 = Me)
12b (R2 = Et)
3a (R1 = Et, R2 = Me)
3b (R1 = Me, R2 = Et)
14
Scheme 1. Reagents and conditions: (a) 1.1 M LHMDS, ZCl, cyclopentylmethyl ether, À78 °C, quant.; (b) H2/Pd-C, THF, 90%; (c) ZNHNH2, WSC, HOBt, DMF, quant.; (d) (i) H2/
Pd-C, THF, 90%; (ii) pyridine, 100 °C, 91%; (e) MeI or EtI, NaH, DMF, 53% (10a), 50% (10b); (f) (i) 4 M HCl–EtOAc, 94% (R2 = Me), quant. (R2 = Et); (ii) 13, NaBH(OAc)3, THF–AcOH,
30% (12a), 55% (12b); (g) (i) 4 M HCl–EtOAc, 92% (R2 = Me), quant. (R2 = Et); (ii) 14, WSC, HOBt, Et3N, DMF, 72% (R2 = Me), 85% (R2 = Et); (iii) R1NCO, pyridine, 80 °C, 77% (3a),
66% (3b).
O
O
O
2HCl
N
O
N
d
c
b
a
N
N
S
O
N
N
N
N
N
O
9
(
)n
Boc
N
S
N
N
O
HN
N
O
N
N
O
( )n
NH
NH
O
(
)n
O
Boc
(
)n
O
R1
NH
NH
O
3c (n = 1, R1 = Et)
15c (n = 1)
15d (n = 2)
15e (n = 3)
15f (n = 4)
16c (n = 1)
16d (n = 2)
16e (n = 3)
17c (n = 1)
17d (n = 2)
17e (n = 3)
3d (n = 2, R1 = Me)
3e (n = 2, R1 = Et)
3f (n = 3, R1 = Me)
3g (n = 3, R1 = Et)
3h (n = 4, R1 = Et)
Et
18
e
Scheme 2. Reagents and conditions: (a) Br(CH2)nBr, NaH, DMF, 48% (15c), 70% (15d), 70% (15e), 14% (15f); (b) (i) 4 M HCl–EtOAc, 87% (n = 1), 87% (n = 2); (ii) 13, NaBH(OAc)3,
THF or CH2Cl2, 30% (16a), 55% (16b), 71% (2 steps, 16c); (c) 4 M HCl–EtOAc, 92% (n = 1), quant. (n = 2), 90% (n = 3); (d) (i) 14, WSC, HOBt, Et3N, DMF, 65% (n = 1), 32% (n = 2); (ii)
R1NCO, pyridine, 70 °C, 71% (3c), 34% (3d), 83% (3e), 29% (2 steps, 3f), (e) (i) 4 M HCl–EtOAc; (ii) 18, NaBH(OAc)3, Et3N, CH2Cl2, 51% (2 steps, 3g), 37% (2 steps, 3h).
Table 1
alkylation with Boc-4-piperidone (13) provided compounds 12a, b,
which were converted to desired compounds 3a, b by deprotection
of the Boc groups, acylation with 2-amino-3-benzothiophenecarb-
Acetyl-CoA carboxylase (ACC) inhibitory activity, human metabolic stability (hMS),
and logD values of spiro-pyrazolidinediones 3a–h
oxylic acid (14), and formation of an ureido group using alkyl
isocyanate.
Other spiro-pyrazolidinediones (3c–f) were also easily synthe-
sized (Scheme 2), in a manner similar to the one shown in Scheme
1. Only 3g, h were directly prepared by reductive alkylation of
deprotected 15e, f with 1-ethyl-3-(3-[(4-oxopiperidin-1-yl)car-
bonyl]-1-benzothiophen-2-yl)urea (18).6
O
N
N R2
S
N
N
O
R2
O
NH
O
NH
R1
Substituents
IC50 (nM)
hMSa
logDb
Synthesis of 4a–c with 7-methoxybenzothiophenyl derivatives
is shown in Scheme 3. Starting from commercially available thio-
R1
R2
ACC1
ACC2
3a
3b
3c
3d
3e
3f
Et
Me
Et
Me
Et
Me
Et
Me
Et
120
41
170
200
130
48
20
8.1
33
36
23
8.7
7.5
5.1
12
120
25
12
18
71
100
200
2.03
2.33
1.93
2.14
2.45
2.14
2.48
2.73
phene 19,
a-keto bromide 20 was prepared according to the
known protocol.7 Successive aromatization and alkylation of phe-
nol 21 afforded 22, which was hydrolyzed to give carboxylic acid
23. Compound 23 was converted to 4a–c by condensation with
17d and subsequent formation of an ureido group.
–(CH2)3–
–(CH2)4–
–(CH2)4–
–(CH2)5–
–(CH2)5–
–(CH2)6–
3g
3h
43
22
The spiro compounds prepared in this study were evaluated for
their inhibitory activities toward human ACC1/2 by using the mal-
achite green method.8 The results of ACC inhibition, human meta-
bolic stability,9 and lipophilicity (logD) are summarized in Tables
1–3. Because the 50% inhibitory concentration (IC50) ratios of
ACC2 and ACC1 inhibitory activities were nearly the same, we pri-
marily discuss the SAR for ACC2 inhibition in the present report.
As previously reported,4 the co-crystal data of human ACC2 and
(S)-1 revealed that the carbonyl group of the spiro-lactone ring
interacts with the backbone of Gly2162. In fact, the chiral resolu-
Et
a
In vitro metabolic clearance in human hepatic microsomes
protein).
LogD values were obtained using high-performance liquid chromatography
(HPLC) at pH 7. IC50 = 50% inhibitory concentration.
(
l
L/min/mg
b
tion of 1 demonstrated that (S)-1 (ACC2: IC50 = 5.4 nM) was more
potent than (R)-1 (ACC2: IC50 = 1,600 nM). Moreover, a wide space
filled with water at the periphery of the 4-position (methylene) in
spiro-lactone (S)-1 was also observed (Fig. 1). The result suggests
O
Br
MeO
O
MeO
HO
MeO
MeO
N
a
b
c
d
O
f
e
N
N
S
N
N
N
O
S
S
S
S
N
N
O
S
S
O
CO2Et
CO2Et
CO2H
NH
O
CO2Et
19
CO2Et
20
H2N
O
AcNH
21
AcNH
22
H2N
23
R1
H2N
AcNH
NH
4a (R1 = H)
24
4b (R1 = Me)
4c (R1 = Et)
Scheme 3. Reagents and conditions: (a) (i) Ac2O, AcOH; (ii) Ce(SO4)2, AcOH, CHCl3, 45%; (iii) Br2, CHCl3, reflux, 89%; (b) Li2CO3, DMF, 120 °C, 41%; (c) MeI, K2CO3, DMF, 59%; (d)
8 M NaOH, EtOH, reflux, 68%; (e) 17d, WSC, HOBt, Et3N, DMF, 81%; (f) Cl3CCONCO, THF then 7 M NH3 in MeOH, 77% (4a) or R1NCO, pyridine, 70 °C, 85% (4b), 51% (4c).

M. Kamata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4769–4772
4771
Table 2
Acetyl-CoA carboxylase (ACC) inhibitory activity, human metabolic stability (hMS),
and logD values of spiro-pyrazolo[1,2-a]pyridazines 4a–c
O
MeO
N
N
S
N
N
O
O
NH
O
NH
R1
R1
IC50 (nM)
hMSa
logDb
ACC1
ACC2
4a
4b
4c
H
Me
Et
22
25
21
5.9
6.1
4.9
15
6
35
1.76
2.08
2.33
Figure 2. Relationships between logD values and metabolic stability of spiro-
pyrazolidinediones 3a–h. Red diamonds correspond to pyrazolo[1,2-a]pyridazine
derivatives 3d and 3e and black diamonds correspond to the compounds. hMS:
human metabolic stability.
a
In vitro metabolic clearance in human hepatic microsomes
protein).
(l
L/min/mg
b
LogD values were obtained by using high-performance liquid chromatography
(HPLC) at pH 7. IC50 = 50% inhibitory concentration.
Figure 3. Detailed crystal structure of the benzothiophene part in (S)-1 with the
carboxyl terminal (CT) domain of human acetyl-CoA carboxylase 2 (ACC2).
Figure 1. Detailed crystal structure of the spiro-lactone core in (S)-1 with the
carboxyl terminal (CT) domain of human acetyl-CoA carboxylase 2 (ACC2).
membered ring suggested that the logD values inversely correlated
with metabolic stability in human microsomes except for those of
3d, e (Fig. 2). Interestingly, pyrazolo[1,2-a]pyridazine derivatives
(red diamonds: 3d, e) exhibited higher metabolic stability, though
the logD values of 3d, e were the same as those of 3b and 3f–h.
Therefore, we selected the pyrazolo[1,2-a]pyridazine core and
modified the benzothiophene moiety to enhance the activity.
The results of the spiro-compounds possessing the pyrazolo[1,2-
a]pyridazine moiety are summarized in Table 2. As the X-ray co-
crystal data of ACC2 with (S)-1 also suggest the presence of a small
cavity formed by Glu2236, Lys1967, and Ala1964 at the 7-position
of the benzothiophene ring (Fig. 3), introduction of a 7-methoxy
group on the ring was examined as to whether it potentiates the
that incorporation of a polar group, for example, an oxo group at
the 4-position in the spiro-lactone, would be favorable for ACC2
inhibition and prompted us to design a symmetrical spiro-pyrazo-
lidinedione scaffold. The results of the in vitro activities of the
spiro-pyrazolidinedione derivatives are summarized in Table 1.
All synthesized compounds showed activities in the order of 10À8
to 10À9 M. As introduction of a more lipophilic and bulky substitu-
ent on the imidazolone or lactam core potentiated the activity,5
compounds 3b and 3f–h displayed potencies of nanomolar order.
In addition, we could identify a high metabolic property of
pyrazolidinediones. Examination of substituents on the spiro-5-
Table 3
Pharmacokinetic profile of 4ca
i.v. (0.1 mg/kg)
p.o. (1 mg/kg)
b
c
e
f
CLtotal (L/h/kg)
Vss (L/kg)
MRTd (h)
1.3
Cmax (ng/mL)
Tmax (h)
AUCg (ng.h/mL)
Fh (%)
4c
0.78
0.97
71
0.25
180
14
a
b
c
Rat cassette dosing (n = 3); nonfasted Crl: CD(SD)(IGS) rats.
Total clearance.
Volume of distribution at steady state.
Mean residence time.
Maximal plasma concentration.
Time of maximal concentration.
Area under the plasma concentration versus time curve.
Bioavailability.
d
e
f
g
h

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M. Kamata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4769–4772
Figure 4. Decrease in the respiratory quotient (RQ) values by 4c. Wistar fatty rats (n = 6, male) were administered with either vehicle or compound 4c (0.3, 1, 3 mg/kg). (a)
Times course of RQ. (b) Average RQ values at 3 h after administration. Values are presented as means SEM. #p < 0.025 (Williams test).
ACC2 inhibitory activity. In fact, the 7-methoxybenzothiophene
derivatives 4a–c exhibited potencies of nanomolar order. More-
over, all compounds retained excellent metabolic stability indepen-
dent of their logD values. Among these compounds, 4c with the
highest ACC inhibitory activity had a favorable pharmacokinetic
profile in rat cassette dosing 10 as described in Table 3.
The selected compound 4c also exhibited potent rat ACC inhib-
itory activity (rACC1: IC50 = 28 nM, rACC2: IC50 = 13 nM) and was
tested at doses of 0.3, 1, and 3 mg/kg in Wistar fatty rats to confirm
increased fatty acid oxidation by lowering the respiratory quotient
(RQ). The compound 4c-induced decrease in RQ values was dose-
dependent and reached a significant level at a dose of 1 mg/kg,
indicating stimulation of fatty acid oxidation (Fig. 4).11
complex with (S)-1. The authors appreciate the helpful discussion
with Dr. Yu Momose.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.062.
References and notes
1. (a) Munday, M. R.; Hemingway, C. J. Adv. Enzyme Regul. 1999, 39, 205; (b)
Ruderman, N. B.; Saha, A. K.; Vavvas, D.; Witters, L. A. Am. J. Physiol. 1999, 276,
E1; (c) Ruderman, N. B.; Saha, A. K.; Kraegen, E. W. Endocrinology 2003, 144,
5166.
2. Rudermen, N.; Prentki, M. Nat. Rev. Drug Disc. 2004, 3, 340.
3. Harwood, H. J. Expert Opin. Ther. Targets 2005, 9, 267.
In conclusion, we designed and synthesized various spiro-
pyrazolidinedione derivatives without an asymmetric center. The
compounds displayed high ACC inhibitory activities and their logD
values inversely correlated with their metabolic stability in human
microsomes. Only pyrazolo[1,2-a]pyridazines 3d and 3e showed
excellent metabolic stability independent of their logD values.
Moreover, X-ray co-crystal data showed that the introduction of
a methoxy group at the 7-position of the benzothiophene core in-
creased the activity. We identified orally bioavailable and highly
potent ACC inhibitor 4c, which stimulated fatty acid oxidation in
rats, by combining the pyrazolo[1,2-a]pyridazine scaffold and the
7-methoxybenzothiophene moiety.
4. Yamashita, T.; Kamata, M.; Endo, S.; Yamamoto, M.; Kakegawa, K.; Watanabe, H.;
Miwa, K.; Yamano, T.; Funata, M.; Sakamoto, J.; Tani, A.; Mol, C. D.; Zou, H.;
Dougan, D. R.; Sang, B.; Snell, G.; Fukatsu, K. Bioorg. Med. Chem. Lett. 2011, 21, 6314.
5. Kamata, M.; Yamashita, T.; Kina, A.; Funata, M.; Mizukami, A.; Sasaki, M.; Tani,
A.; Funami, M.; Amano, N.; Fukatsu, K. Bioorg. Med. Chem. Lett. 2012, 22, 3642.
6. Synthesis of 1-ethyl-3-{3-[(4-oxopiperidin-1-yl)carbonyl]-1-benzothiophen-2-
yl}urea 18 was performed by acylation of 4-piperidone with 14 followed by
formation of ureido group using ethyl isocyanate.
7. (a) Cauquil-Caubère, I.; Kamenka, J-M. Eur. J. Med. Chem. 1998, 33, 867; (b)
Pinna, G. A.; Curzu, M. M.; Cignarella, G.; Barlocco, D.; D’Amico, M.; Filippelli,
A.; De Novellis, V.; Rossi, F. Eur. J. Med. Chem. 1994, 29, 447.
8. Bernal, C.; Palacin, C.; Boronat, A.; Imperial, S. Anal. Biochem. 2005, 337, 55.
9. Koike, T.; Takai, T.; Hoashi, Y.; Nakayama, M.; Kosugi, Y.; Nakashima, M.;
Yoshikubo, S.; Hirai, K.; Uchikawa, O. J. Med. Chem. 2011, 54, 4207.
10. Negoro, N.; Sasaki, S.; Ito, M.; Kitamura, S.; Tsujihata, Y.; Ito, R.; Suzuki, M.;
Takeuchi, K.; Suzuki, N.; Miyazaki, J.; Santou, T.; Odani, T.; Kanzaki, N.; Funami,
M.; Tanaka, T.; Yasuma, T.; Momose, Y. J. Med. Chem. 2012, 55, 1538.
11. 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.
Acknowledgments
The authors thank Clifford D. Mol, Hua Zou, Douglas R. Dougan,
BiChing Sang, and Gyorgy Snell for acquisition of the X-ray co-crys-
tal data of the carboxyl terminal (CT) domain of human ACC2 in