Synthesis, biological evaluation and molecular docking studies of piperidinylpiperidines and spirochromanones possessing quinoline moieties as Acetyl-CoA carboxylase inhibitors

Article abstract of DOI:10.3390/molecules200916221

Acetyl-coenzyme A carboxylases (ACCs) play critical roles in the regulation of fatty acid metabolism and have been targeted for the development of drugs against obesity, diabetes and other metabolic diseases. Two series of compounds possessing quinoline moieties were designed, synthesized and evaluated for their potential to inhibit acetyl-CoA carboxylases. Most compounds showed moderate to good ACC inhibitory activities and compound 7a possessed the most potent biological activities against ACC1 and ACC2, with IC50 values of 189 nM and 172 nM, respectively, comparable to the positive control. Docking simulation was performed to position compound 7a into the active site of ACC to determine a probable binding model.

Full text of DOI:10.3390/molecules200916221

Molecules 2015, 20, 16221-16234; doi:10.3390/molecules200916221
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Biological Evaluation and Molecular Docking Studies
of Piperidinylpiperidines and Spirochromanones Possessing
Quinoline Moieties as Acetyl-CoA Carboxylase Inhibitors
Tonghui Huang †,*, Jie Sun †, Qianqian Wang, Jian Gao and Yi Liu
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy,
Xuzhou Medical College, Xuzhou 221004, Jiangsu, China; E-Mails: xincheng127@sina.cn (J.S.);
ruofei16888@163.com (Q.W.); gaojian.gucas@gmail.com (J.G.); cbpeliuyinew@163.com (Y.L.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: hancp@iccas.ac.cn;
Tel.: +86-516-8326-2137; Fax: +86-516-8326-2251.
Academic Editor: Derek J. McPhee
Received: 11 August 2015 / Accepted: 1 September 2015 / Published: 7 September 2015
Abstract: Acetyl-coenzyme A carboxylases (ACCs) play critical roles in the regulation of
fatty acid metabolism and have been targeted for the development of drugs against obesity,
diabetes and other metabolic diseases. Two series of compounds possessing quinoline
moieties were designed, synthesized and evaluated for their potential to inhibit acetyl-CoA
carboxylases. Most compounds showed moderate to good ACC inhibitory activities and
compound 7a possessed the most potent biological activities against ACC1 and ACC2,
with IC50 values of 189 nM and 172 nM, respectively, comparable to the positive control.
Docking simulation was performed to position compound 7a into the active site of ACC to
determine a probable binding model.
Keywords: acetyl-CoA carboxylase; inhibitors; piperidinylpiperidines; spirochromanones;
synthesis; molecular docking

Molecules 2015, 20
16222
1. Introduction
Obesity is a chronic disease related to many other metabolic diseases such as type 2 diabetes mellitus
(T2DM) and dyslipidemia [1,2]. Acetyl-coenzyme A carboxylases (ACCs) are key enzymes that
catalyze the formation of malonyl coenzyme A (CoA), which contributes to the regulation of fatty acid
biosynthesis and oxidation in humans and most other living organisms [3,4]. There are two isoforms of
ACC in mammals identified as ACC1 and ACC2. ACC1 is a cytosolic enzyme and primarily expressed in
lipogenic tissues responsible for the committed step in fatty acid biosynthesis. ACC2 is a mitochondrial
enzyme that is highly expressed in oxidative tissues associated with mitochondrial fatty acid oxidation [2].
Thus, inhibition of the ACCs gives the potential for managing both long chain fatty acid biosynthesis
and fatty acid oxidation, which has been regarded as a novel approach to treat obesity type 2 diabetes
and other diseases associated with metabolic syndrome [5].
Wakil and coworkers [2] have reported that the ACC2-deficient mice exhibited increased fatty acid
oxidation and reduced fat accumulation in skeletal muscle and heart tissue when placed on a high
fat/high calorie diet. In contrast, liver ACC1-deficient mice showed a significant reduction in fatty acid
synthesis as compared to wild type mice [6]. In the past few years, several classes of small molecules
that inhibit both isoforms of mammalian ACC have been reported, such as spirochromanones [7],
piperidinylpiperidines [8] and benzthiazolylamides [9], examples of which are shown in Figure 1.
Among them is compound CP-640186 (I, Figure 1) which is the first well documented example from
Pfizer’s drug discovery program targeting ACCs with IC50 values of about 50 nM against ACC1 and
ACC2 from mammalia [8,10]. In a subsequent publication, Pfizer described a series of spirochromanone
ACC inhibitors (II, Figure 1) and discussed efforts to improve the properties [7]. Further work by Takeda
focused on improving the compound of CP-640186 by replacing the anthracene ring and morpholine
amide resulting in a benzthiazolylamides compound (III, Figure 1) [9]. Moreover, Pfizer is currently
pursuing the clinical development of their clinical candidate PF-05175157 (structure not disclosed)
targeting ACC in phase 2 [11].
O
N
O
N
HN
R
N
N
O
N
O
O
I
II
O
S
N
O
N
NH
O
NH
O
O
III
Figure 1. Chemical structures of representative ACC inhibitors.
It has been demonstrated that a spirochromanone and CP-640186 bound to the same active site at the
interface of a dimer of ACC carboxyl transfer (CT) domain [12–14]. The shared binding pattern reveals
that these two classes of inhibitors both interact with conserved residues Gly-2162 (yeast Gly-1958) and
Glu-2230 (yeast Glu-2026). The crystal structure of the complex of ACC CT with CP-640186 indicates
that the side chains of Glu-2230, Glu-2232, Ala-1964 and Lys-1967 are located near the anthracene ring

Molecules 2015, 20
16223
(Figure 2) [14]. We propose that the conversion of the anthracene ring into a heterocyclic ring may
enhance the interaction of ligand and the binding site, resulting in increased ACC inhibitory activities.
Designs based on the anthracene regions and piperidinylpiperidine core are shown in Figure 3. To increase
the structural diversity and discover more potent inhibitors of ACC, we describe herein the synthesis
of novel piperidinylpiperidines and spirochromanones bearing a quinoline moiety and evaluated their
enzyme inhibitory activities against ACCs. Additionally, molecular docking was carried out to investigate
the possible binding mode of representative compounds using the Sybyl software.
Figure 2. Stereographic drawing showing the binding site for CP-640186.
R2
Hydrophobic
pocket
Anthracene
N
Replacements
N
N
O
O
N
R1
O
N
O
R2
N
N
O
O
N
O
O
Figure 3. Design strategy of the target compounds.
2. Results and Discussion
2.1. Chemistry
The synthetic routes for the novel target piperidinylpiperidine and spirochromanone derivatives are
outlined in Schemes 1 and 2. As shown in Scheme 1, substituted 1-(quinoline-4-carbonyl)piperidin-
4-ones 3 were obtained by the reaction of substituted quinoline-4-carboxylic acids 1 with piperidin-4-
one hydrochloride (2) in the presence of HATU and triethylamine at room temperature. Derivatives
of 3 then underwent reductive amination with (R)-ethyl piperidine-3-carboxylate hydrochloride (4) in
the presence of sodium triacetoxyborohydride to afford substituted (R)-ethyl 1′-(quinoline-4-carbonyl)-
[1,4′-bipiperidine]-3-carboxylates 5. Derivatives of 5 further underwent hydrolysis and amidation to
give the desired quinoline substituted piperidinylpiperidine derivatives 7.

Molecules 2015, 20
16224
Scheme 1. Synthesis of piperidinylpiperidines.
Scheme 2. Synthesis of spirochromanones.
As shown in Scheme 2, the spirochromanones 12a–12g were synthesized via a three-step procedure.
The key intermediate tert-butyl 4-oxospiro[chroman-2,4′-piperidine]-1′-carboxylate (10) was prepared
by the reaction of 1-(2-hydroxyphenyl)ethanone (8) with N-Boc-4-piperidinone (9) in a base catalyzed
spirocyclization according to the reported procedure [7]. The Boc group on the intermediate was removed
with trifluoroacetic acid (TFA) at room temperature, and the resulting spiro[chroman-2,4′-piperidin]-4-one
(11) was further coupled with substituted quinoline-4-carboxylic acids 1 in the presence of HATU and
triethylamine to afford the corresponding spirochromanone derivatives 12.
The structures of all the synthesized piperidinylpiperidines 7 and spirochromanones 12 have been
confirmed by NMR, IR and MS, and all the corresponding data can be found in the Experimental section.
2.2. Biological Activities
The in vitro inhibitory activities of compounds 7a–7i and 12a–12g against both ACC1 and ACC2
are listed in Table 1. Among the tested compounds, the selected potent compounds (ACC IC50 < 1000 nM)
were further evaluated for their cytotoxicity against human embryonic lung fibroblast (HELF) cells

Molecules 2015, 20
16225
using the 3-[4,5-dimethylthylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) method [15]. For the
convenience of structure-activity relationship analysis, compounds 7a–7i and 12a–12g were defined as
piperidinylpiperidine derivatives and spirochromanone derivatives, respectively. As shown in Table 1,
we can conclude that most of the compounds showed moderate to good activities against ACC1 and
ACC2 and low toxic effects against normal human embryonic lung fibroblasts. For example, 7a–7g,
12a, 12b and 12d showed promising ACC2 inhibitory activities with IC50 values ranging from 172 nM
to 940 nM and low cytotoxic activities (>100 μM). 7a–7e, 7g, 12a and 12b showed promising ACC1
inhibitory activity with IC50 values below 1000 nM. Moreover, 7a–7g and 12d displayed more potent
ACC2 inhibitory activity compared to their anti-ACC1 activity and exhibited a relative selectivity. It is
worth mentioning that the most active compound, the piperidinylpiperidine derivative 7a displayed
comparable inhibitory activity against ACC1/2 as the parent compound CP-640186. The octanol/water
partition coefficients (miLogP) and drug-likeness model scores were also computed for all the compounds
using the online molinspiration LogP calculation program and molsoft software, respectively. All the
synthesized compounds showed moderate to good drug-likeness score ranging from 0.41 to 1.61, which
is higher than CP-640186 (0.27). The logP values of most of the test compounds range from 3.18 to
5.0 within the acceptable criteria.
Table 1. LogP measurements, drug-likeness model scores, in vitro cytotoxicity assay and
inhibitory activities of ACC1 and ACC2.
Comp.
7a
ACC1 IC50 (nM) a ACC2 IC50 (nM) a TC50 (μM) a,b LogP d Drug-Likeness Model Score e
189 (±3)
750 (±9)
940 (±5)
620 (±12)
750 (±11)
>1000
172 (±7)
360 (±10)
205 (±9)
294 (±3)
382 (±15)
920 (±14)
810 (±11)
>1000
>100
>100
>100
>100
>100
N.T. c
>100
N.T. c
N.T. c
>100
>100
N.T. c
N.T. c
N.T. c
N.T. c
N.T. c
>100
3.48
5.03
4.38
3.48
4.03
4.54
3.42
4.10
4.05
4.84
4.43
5.18
5.41
5.46
3.91
3.18
3.59
1.14
1.61
1.49
1.38
1.4
7b
7c
7d
7e
7f
1.37
0.85
1.13
0.92
1.07
1.05
0.73
0.81
1.04
0.65
0.41
0.27
7g
860 (±3)
>1000
7h
7i
>1000
>1000
12a
12b
12c
12d
12e
12f
12g
600 (±7)
760 (±11)
>1000
650 (±12)
820 (±14)
>1000
>1000
940
>1000
>1000
>1000
>1000
>1000
>1000
CP-640186
173 (±4)
185 (±5)
a
b
Values are the arithmetic means of three experiments, standard deviation is given in parentheses; TC50:
Concentrations which reduce the cell viability by 50%; c N.T.: not test; d Calculated by [16]; e Calculated by [17].
2.3. Molecular Docking Study
The ACCase is a multienzyme complex composed of three function domains: biotin carboxylase
domain (BC), biotin carboxyl carrier protein domain (BCCP) and carboxyltransferase domain (CT) [18].

Molecules 2015, 20
16226
The CT domain catalyzes the transfer of an activated carboxyl group to acetyl CoA to form malonyl
CoA. The crystal structures of the CT domain in complex with CP-640186 [12], coenzyme A [19], and
pinoxaden [20] have been reported, respectively. The cocrystal structures of CP-640186 reveal that the
active site of the CT domain resides at the dimeric interface of the N and C domains. The binding
pattern suggests that the anthracenyl moiety is sandwiched between helix α6 in the N domain and helix
α6′ in the C domain near the dimer interface; the carbonyl oxygen atom adjacent to the anthracene is
hydrogen bonded to Glu-2026 and the carbonyl oxygen atom adjacent to the morpholine is hydrogen
bonded to Gly-1958.
In order to investigate the interactions of these two series of compounds with ACC, a molecular
docking study was performed by docking of several representative compounds into the active site of
CT domain, using the Sybyl 7.1 program package (Tripos International, St. Louis, MO, USA). The
three-dimensional structure of CT domain of human ACC was taken from the Protein Data Bank
(PDB ID: 3FF6) [14]. The synthesized compounds were energy minimized and docked into the active
site, which formed by residues Ala-1964, Lys-1967, Val-1968, Leu-1965, Gly-2162, Ser-2161, Phe-2160,
Arg-2158, Glu-2230, Glu-2232, Glu-2236 and Ile-2237. The clustering of docked compounds 7a, 7g, 7h,
7i, 12a, 12e and CP-640186 is shown in Figure 4. These compounds exhibited similar conformations and
binding modes as CP-640186. The best inhibitor 7a with a higher docking score (8.01) in comparison
to CP-640186 (7.18) and binds at the same site as the ligand. Two hydrogen bonds were observed between
the carbonyl groups with residues Glu-2230 and Gly-2162 (Figure 4, right), respectively. The quinoline
moiety is surrounded by the side chain of Ala-1964, Lys-1967, Val-1968, Gly-2233, Glu-2236 and
Ile-2237. More importantly, the oxygen atom associated with 2-phenylquinoline moiety interacts with
the residue Lys-1967 by an additional hydrogen bond. We speculate that the enhanced activity of 7a is
partly due to the additional hydrogen bond with Lys-1967.
Figure 4. (Left) Overlay of the CT domain of hACC2 with the compounds of 7a, 7g, 7h, 7i
(green), 12a, 12e (yellow) and CP-640186 (red), respectively. Red dashed lines denote the
hydrogen bond. Molecular surface of the binding site is colored in white for the N domain and
cyan for the C domain; (Right) Molecular docking model for compound 7a (green and stick)
with the active site of hACC2, highlighting the hydrogen bonds (red dashed lines) coordination
between the oxygen atoms in 7a and the amino acid residues Glu-2230, Gly-2162and Lys-1967.

Molecules 2015, 20
16227
3. Experimental Section
3.1. General Information
All solvents were dried according to standard methods before use. Melting points were measured on
a Buchi B-545 melting point apparatus and are uncorrected. IR spectra were determined using KBr disks
with a PE-983 infrared spectrometer and only major absorptions are listed. NMR spectra were recorded
on a Mercury-Plus 400 spectrometer in CDCl3 using TMS as an internal reference. LC-MS data were
obtained on an API 2000 liquid chromatography-tandem mass spectrometer. Unless otherwise noted,
all materials were commercially available and were used directly without further purification.
3.1.1. General Procedure for the Preparation of Substituted 1-(Quinoline-4-carbonyl)piperidin-4-ones 3
To a solution of piperidin-4-one hydrochloride (2, 3.24 g, 24 mmol) and triethylamine (4.86 g,
48 mmol) in DMF (40 mL) was added substituted quinoline-4-carboxylic acid (1, 20 mmol) followed
by HATU (9.12 g, 24 mmol). The reaction mixture was stirred at room temperature for 6 h and then
poured into ice-cold water (50 mL). The precipitate was filtered and dried to give the corresponding
substituted 1-(quinoline-4-carbonyl)piperidin-4-one 3. This product was used directly in the next reaction
step without further purification.
3.1.2. General Procedure for the Preparation of Substituted (R)-Ethyl 1′-(Quinoline-4-carbonyl)-[1,4′-
bipiperidine]-3-carboxylates 5
A solution of (R)-ethyl piperidine-3-carboxylate hydrochloride (4, 2.32 g, 12 mmol) in
1,2-dichloroethane (20 mL) was treated with a substituted 1-(quinoline-4-carbonyl)piperidin-4-one 3
(10 mmol) followed by sodium triacetoxyborohydride (3.2 g, 15 mmol) and then acetic acid (4.5 mL).
The suspension was stirred at room temperature for 2 h and then at 60 °C for 6ꢀh. After cooling to
room temperature, the reaction mixture was quenched with 1 N NaOH (5 mL), and stirred for 20 min.
The suspension was extracted with CH2Cl2 (100 mL) and washed with water (50 mL). The organic
layer was dried over Na2SO4, filtered, and concentrated to give a yellow residue which was purified by
flash chromatography using EtOAc/petroleum ether as eluent.
3.1.3. General Procedure for the Preparation of Substituted (R)-1′-(Quinoline-4-carbonyl)-[1,4′-
bipiperidine]-3-carboxamides 7a–7i
To a solution of substituted (R)-ethyl 1′-(quinoline-4-carbonyl)-[1,4′-bipiperidine]-3-carboxylate
5, (5 mmol) in ethanol (15 mL) was added 1 N NaOH (20 mL). The reaction mixture was stirred
at room temperature and monitored by TLC. Upon completion, the mixture was concentrated and
acidified with 2 N HCl. The resulting solids were filtered, washed with H2O, and dried to provide
(R)-1′-(quinoline-4-carbonyl)-[1,4′-bipiperidine]-3-carboxylic acid 6 which was used directly in the next
step with no further purification. To a solution of the abovementioned acid 6 (5 mmol), and the amine
(6 mmol) in DMF (5 mL) was added HATU (2.28 g, 6 mmol) followed by NEt3 (0.61 g, 6 mmol).
The reaction mixture was stirred at room temperature for 6 h, then diluted with water (30 mL) and

Molecules 2015, 20
16228
extracted with EtOAc (60 mL). The organics were dried with Na2SO4 and concentrated in vacuo to
give crude compound 7, which were purified by flash chromatography.
(R)-(2-(4-Methoxyphenyl)quinolin-4-yl)(3-(morpholine-4-carbonyl)-[1,4′-bipiperidin]-1′-yl)methanone
1
(7a). Pale yellow solid, m.p. 94–96 °C. H-NMR δ (ppm): 8.17–8.11 (m, 3H), 7.85–7.71 (m, 3H), 7.51
(d, J = 8.0 Hz, 1H), 7.04 (t, J = 8.0 Hz, 2H), 4.99 (t, J = 10.0 Hz, 1H), 3.88 (s, 3H), 3.67–3.59 (m, 4H),
3.53–3.42 (m, 6H), 3.01–2.95 (m, 5H), 2.21–2.08 (m, 3H), 1.78–1.56 (m, 7H). IR ν: 3448.5, 1635.5,
1448.4, 1249.8, 842.8 cm−1. LC-MS calcd for C32H39N4O4 (m/z) [M + H]+ 543.30; found 543.28.
(R)-N-Benzyl-1'-(2-(4-methoxyphenyl)quinoline-4-carbonyl)-N-methyl-[1,4'-bipiperidine]-3-carbox-amide
(7b). White solid, m.p. 90–92 °C. 1H-NMR δ (ppm): 8.26–8.13 (m, 3H), 7.89–7.80 (m, 3H), 7.75–7.70
(m, 2H), 7.64–7.47 (m, 4H), 7.06–7.01 (d, J = 8.0 Hz, 2H), 5.10 (t, J = 10.0 Hz, 1H), 4.20 (s, 2H),
3.87 (s, 3H), 3.68–3.60 (m, 4H), 3.53–3.38 (m, 4H), 3.08–2.90 (m, 3H), 2.03–1.93 (m, 5H), 1.78–1.56
(m, 4H). IR ν: 3442.7, 2936.5, 1631.7, 1448.4, 1109.0, 841.0 cm−1. LC-MS calcd for C36H40N4O3 (m/z)
[M + H]+ 577.22; found 577.21.
(R)-N,N-Diethyl-1'-(2-(4-methoxyphenyl)quinoline-4-carbonyl)-[1,4'-bipiperidine]-3-carboxamide (7c).
1
White solid, m.p. 101–102 °C. H-NMR δ (ppm): 8.16–8.11 (m, 3H), 7.81–7.70 (m, 3H), 7.51 (d,
J = 8.0 Hz, 1H), 7.05 (t, J = 6.0 Hz, 2H), 5.00 (t, J = 10.0 Hz, 1H), 3.88 (s, 3H), 3.70–3.62 (m, 4H),
3.46–3.32 (m, 4H), 2.91–2.43 (m, 3H), 2.21–2.01 (m, 3H), 1.78–1.56 (m, 7H), 1.17 (t, J = 8.0 Hz, 3H),
1.09 (t, J = 8.0 Hz, 3H). IR ν: 3415.7, 3361.0, 1631.7, 1450.4, 1251.2, 842.8 cm−1. LC-MS calcd for
C32H40N4O3 (m/z) [M + H]+ 529.32; found 529.30.
(R)-(2-(4-Methoxyphenyl)quinolin-4-yl)(3-(4-methylpiperazine-1-carbonyl)-[1,4'-bipiperidin]-1'-yl)-
methanone (7d). White solid, m.p. 108–110 °C. 1H-NMR δ (ppm): 8.19–8.14 (m, 3H), 7.74–7.65 (m, 3H),
7.30 (d, J = 8.0 Hz, 1H), 7.23–6.99 (m, 2H), 4.97 (t, J = 10.0 Hz, 1H), 3.85 (s, 3H), 3.47–3.14 (m, 4H),
3.11–2.97 (m, 6H), 2.97–2.90 (m, 5H), 2.11–2.08 (m, 6H), 1.90–1.87 (m, 4H), 1.35–1.23 (m, 3H). IR
ν: 3440.8, 1636.5, 1438.8, 1251.8, 769.5 cm−1. LC-MS calcd for C33H41N5O3 (m/z) [M + H]+ 556.31;
found 556.29.
(R)-(2-(4-Methoxyphenyl)quinolin-4-yl)(3-(pyrrolidine-1-carbonyl)-[1,4'-bipiperidin]-1'-yl)methanone
1
(7e). White solid, m.p. 127–129 °C. H-NMR δ (ppm): 8.19–8.11 (m, 3H), 7.79 (d, J = 8.0 Hz, 1H),
7.75–7.65 (m, 2H), 7.57–7.48 (m, 1H), 7.05 (d, J = 8.0 Hz, 2H), 4.98 (t, J = 10.0 Hz, 1H), 3.89 (s, 3H),
3.46–3.39 (m, 6H), 3.10–2.85 (m, 5H), 2.22–2.06 (bs, 2H), 1.96–1.90 (m, 4H), 1.85–1.71 (m, 8H); IR ν:
3415.7, 1623.9, 1452.3, 1251.7, 1026.1, 842.8 cm−1. LC-MS calcd for C32H38N4O3 (m/z) [M + H]+
527.26; found 527.25.
(R)-(2-(4-Methoxyphenyl)quinolin-4-yl)(3-(piperidine-1-carbonyl)-[1,4′-bipiperidin]-1′-yl)methanone
(7f). Pale yellow solid, m.p. 89–91 °C. 1H-NMR δ (ppm): 8.21–8.12 (m, 3H), 7.78 (d, J = 8.0 Hz, 1H),
7.76–7.64 (m, 2H), 7.60–7.51 (m, 1H), 7.08 (d, J = 8.0 Hz, 2H), 4.99 (t, J = 10.0 Hz, 1H), 3.92 (s, 3H),
3.52–3.43 (m, 6H), 3.12-2.84 (m, 5H), 2.31–2.24 (m, 2H), 2.02–1.92 (m, 4H), 1.80–1.64 (m, 10H) ; IR ν:
3426.8, 1632.6, 1460.9, 1258.6, 1034.5, 856.7 cm−1. LC-MS calcd for C32H38N4O3 (m/z) [M + H]+
541.42; found 541.42.

Molecules 2015, 20
16229
(R)-(3-(Morpholino-4-carbonyl)-[1,4'-bipiperidin]-1'-yl)(2-phenylquinolin-4-yl)methanone (7g).
White solid, m.p. 92–93 °C. 1H-NMR δ (ppm): 8.24 (d, J = 8.8 Hz, 2H), 7.91–7.80 (m, 3H), 7.67–7.63
(m, 1H), 7.55–7.52 (m, 2H), 7.39–7.33 (m, 2H), 4.97 (t, J = 10.0 Hz, 1H), 3.70–3.35 (m, 4H),
3.49–3.40 (m, 6H), 3.20–2.92 (m, 5H), 2.20–2.13 (m, 3H), 1.80–1.64 (m, 7H). IR ν: 3418.5, 1641.2,
1476.3, 1260.7, 1042.9, 867.6 cm−1. LC-MS calcd for C31H36N4O3 (m/z) [M + H]+ 513.29; found 513.31.
(R)-(2-(4-Chlorophenyl)quinolin-4-yl)(3-(morpholine-4-carbonyl)-[1,4'-bipiperidin]-1'-yl)methanone
(7h). Pale yellow solid, m.p. 87–89 °C. 1H-NMR δ (ppm): 8.28–8.21 (m, 3H), 7.84 (d, J = 8.0 Hz, 1H),
7.70–7.65 (m, 2H), 7.65–7.58 (m, 1H), 7.36 (d, J = 8.0 Hz, 2H), 4.98 (t, J = 10.0 Hz, 1H), 3.76–3.48 (m,
4H), 3.50–3.42 (m, 6H), 3.25–2.96 (m, 5H), 2.24–2.16 (m, 3H), 1.87–1.66 (m, 7H). IR ν: 3420.6,
1646.8, 1464.5, 1270.6, 1038.4, 853.4 cm−1. LC-MS calcd for C31H36ClN4O3 (m/z) [M + H]+ 548.25;
found 547.26.
(R)-(2-(2-Chlorophenyl)quinolin-4-yl)(3-(morpholine-4-carbonyl)-[1,4'-bipiperidin]-1'-yl)methanone (7i).
1
White solid, m.p. 93–95 °C. H-NMR δ (ppm): 8.24–8.16 (m, 3H), 7.86–7.70 (m, 3H), 7.64–7.56
(m, 1H), 7.42–7.38 (m, 2H), 5.00 (t, J = 10.0 Hz, 1H), 3.78–3.48 (m, 4H), 3.52–3.43 (m, 6H), 3.42–3.12
(m, 5H), 2.30–2.21 (m, 3H), 1.92–1.72 (m, 7H); IR ν: 3452.6, 1642.8, 1426.9, 1247.5, 780.4 cm−1.
LC-MS calcd for C31H35ClN4O3 (m/z) [M + H]+ 547.25; found 547.26.
3.1.4. General Procedure for the Preparation of Tert-butyl 4-oxospiro[chroman-2,4' piperidine]-1'-
carboxylate (10)
The synthesis was performed according to the reported methodwith some modifications [7]. Generally,
a solution of 1-(2-hydroxyphenyl)ethanone (8, 4.08 g, 30 mmol) in ethanol (80 mL) was added pyrrolidine
(2.04 g, 30 mmol). The solution was stirred for 2 h at room temperature and then, the reaction mixture
was added tert-butyl 4-oxopiperidine-1-carboxylate (9, 5.97 g, 30 mmol) and stirred at room temperature
for 10 h. The mixture was then concentrated in vacuo to give crude product which was purified via flash
chromatography to afford tert-butyl 4-oxospiro[chroman-2,4′-piperidine]-1′-carboxylate 10 (6.84 g,
72% yield) as a white solid.
3.1.5. General Procedure for the Preparation of Substituted 1′-(Quinoline-4-carbonyl)spiro[chroman-
2,4′-piperidin]-4-ones 12a–12g
To a solution of tert-butyl 4-oxospiro[chroman-2,4′-piperidine]-1′-carboxylate (1.6 g, 5 mmol) in
dichloromethane (20 mL) was added trifluoroacetic acid (2.5 mL). The reaction mixture was stirred
at room temperature for 2 h and the solvent was removed under reduced pressure to give crude
spiro[chroman-2,4′-piperidin]-4-one 11. The resulting solid was used directly for the next step reaction
without further purification. A mixture of substituted quinoline-4-carboxylic acid (1, 5 mmol), NEt3
(0.61 g, 6 mmol) and HATU (1.9 g, 5 mmol) in DMF (10 mL) was stirred at room temperature for
0.5 h, followed by the abovementioned spiro[chroman-2,4′-piperidin]-4-one 11. The reaction mixture
was stirred overnight at room temperature after which time it was diluted with EtOAc (100 mL) and
washed with water (60 mL × 2). The organic extract was dried over Na2SO4, filtered and concentrated

Molecules 2015, 20
16230
under reduced pressure. The crude material was purified by chromatography to afford substituted
1′-(quinoline-4-carbonyl)spiro[chroman-2,4′-piperidin]-4-ones 12.
1'-(2-(4-Methoxyphenyl)quinoline-4-carbonyl)spiro[chroman-2,4'-piperidin]-4-one (12a). White solid, m.p.
113–114 °C. 1H-NMR δ (ppm): 8.18–8.12 (m, 3H), 7.86 (d, J = 4.0 Hz, 1H), 7.80–7.71 (m, 3H), 7.55 (t,
J = 10.0 Hz, 1H), 7.50–7.48 (m, 1H), 7.06–6.99 (m, 4H), 3.89 (s, 3H), 3.51–3.39 (m, 2H), 3.27 (m,
2H), 2.76 (s, 2H), 1.98–1.80 (m, 2H), 1.57–1.46 (m, 2H). IR ν: 1689.5, 1637.5, 1606.6, 1461.9, 1249.8,
763.76 cm−1. LC-MS calcd for C30H26N2O4 (m/z) [M + H]+ 479.19; found 479.18.
1'-(2-(3,4-Dimethoxyphenyl)quinoline-4-carbonyl)spiro[chroman-2,4'-piperidin]-4-one (12b). White solid,
m.p. 121–123 °C. 1H-NMR δ (ppm): 8.20 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.81–7.77 (m, 3H),
7.68 (d, J = 8.0 Hz, 1H), 7.59–7.50 (m, 2H), 7.02 (q, J = 6.7 Hz, 3H), 4.77 (t, J = 12.0 Hz, 1H), 4.08
(s, 3H), 3.98 (s, 3H), 3.46 (t, J = 10.0 Hz, 2H), 3.28 (m, 2H), 2.73 (s, 2H), 1.98–1.83 (m, 2H), 1.60–1.48
(m, 2H). IR ν: 2929.7, 1637.5, 1461.9, 1261.4, 812.0, 765.7 cm−1. LC-MS calcd for C31H28N2O5 (m/z)
[M + H]+ 509.22; found 509.23.
1'-(2-(o-Tolyl)quinoline-4-carbonyl)spiro[chroman-2,4'-piperidin]-4-one (12c). White solid, m.p.
1
126–127 °C. H-NMR δ (ppm): 8.19 (d, J = 8.0 Hz, 1H), 7.87–7.78 (m, 3H), 7.62 (t, J = 8.0 Hz, 1H),
7.52–7.45 (m, 3H), 7.37–7.32 (m, 3H), 7.01–6.99 (m, 2H), 3.53–3.40 (m, 2H), 2.75 (s, 2H), 2.43 (s, 3H),
2.32–2.26 (m, 2H), 1.97–1.82 (m, 2H), 1.60–1.46 (m, 2H). IR ν: 2923.9, 1697.2, 1635.5, 1483.9, 1228.6,
761.8 cm−1. LC-MS calcd for C30H26N2O3 (m/z) [M + H]+ 463.20; found 463.20.
1'-(2-(2-Chlorophenyl)quinoline-4-carbonyl)spiro[chroman-2,4'-piperidin]-4-one (12d). White solid,
1
m.p. 130–132 °C. H-NMR δ (ppm): 8.21 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.81–7.77
(m, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.68–7.62 (m, 2H), 7.52–7.50 (m, 2H), 7.45–7.39 (m, 2H), 7.02 (t,
J = 8.0 Hz, 2H), 3.56–3.49 (m, 2H), 3.48–3.40 (m, 2H), 2.77 (s, 2H), 1.99–1.83 (m, 2H), 1.69–1.62
(m, 2H). IR ν: 3438.8, 1693.4, 1639.4, 1463.9, 1226.6, 761.8 cm−1. LC-MS calcd for C29H23ClN2O3
(m/z) [M + H]+ 483.15; found 483.14.
1'-(2-(4-Chlorophenyl)quinoline-4-carbonyl)spiro[chroman-2,4'-piperidin]-4-one (12e). White solid,
128–130 °C. 1H-NMR δ (ppm): 8.21 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 8.0 Hz, 2H), 7.88 (d, J = 8.0 Hz,
1H), 7.83–7.77 (m, 3H), 7.62 (t, J = 8.0 Hz, 1H), 7.54–7.51 (m, 3H), 7.03 (q, J = 6.7 Hz, 2H), 3.53–3.41
(m, 2H), 3.31–3.24 (m, 2H), 2.78 (s, 2H), 2.06–1.83 (m, 2H), 1.62–1.46 (m, 2H). IR ν: 2923.8, 1648.5,
1461.9, 1226.6, 835.1, 763.8 cm−1. LC-MS calcd for C29H23ClN2O3 (m/z) [M + H]+ 483.15; found;
483.14 [M + H]+.
1'-(2-Chloroquinoline-4-carbonyl)spiro[chroman-2,4'-piperidin]-4-one (12f). Pale yellow solid, m.p.
121–123 °C. 1H-NMR δ (ppm): 8.09 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.83–7.79 (m, 2H),
7.66–7.62 (m, 1H), 7.55–7.51 (m, 1H), 7.36–7.33 (m, 1H), 7.07–7.02 (m, 2H), 3.56–3.43 (m, 2H),
3.23–3.21 (m, 2H), 2.79 (s, 2H), 2.10–1.96 (m, 2H), 1.65–1.47 (m, 2H). IR ν: 3436.9, 3064.7,
1639.4, 1461.9, 1228.6, 758.0 cm−1. LC-MS calcd for C23H19ClN2O3 (m/z) [M + H]+ 407.12; found;
407.12 [M + H]+.

Molecules 2015, 20
16231
1'-(2-Morpholinoquinoline-4-carbonyl)spiro[chroman-2,4'-piperidin]-4-one (12g). White solid, m.p.
138–140 °C. 1H-NMR δ (ppm): 8.11 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.62–7.50 (m, 3H),
7.32–7.30 (m, 1H), 7.06–6.99 (m, 2H), 6.93–6.88 (m, 1H), 3.88–3.84 (m, 4H), 3.77-3.68 (m, 4H),
3.48–3.44 (m, 2H), 3.29–3.23 (m, 2H), 2.78 (s, 2H), 1.94–1.81 (m, 2H), 1.57–1.47 (m, 2H). IR ν:
3417.6, 3381.0, 1604.7, 1444.6, 1116.71, 761.8 cm−1. LC-MS calcd for C27H27N3O4 (m/z) [M + H]+
458.20; found; 458.20.
3.2. Molecular Docking
The molecular docking was performed and analyzed with the Sybyl 7.1 program package [21].
The X-ray structure of CT domain bounded with CP-640186 was taken from the Protein Data Bank
(PDB ID: 3FF6). All water molecules and ligands were removed from the original enzyme structure.
For docked ligands, non-polar hydrogens were added and Gasteiger-Huckl charges were assigned.
To validate our docking procedure, the co-crystallized ligand was redocked into the active site of the
hACC2 and the reasonable RMSD value of 1.9620 Å was obtained. Dock scores were evaluated by
Consensus Score (CScore), which integrates a number of popular scoring functions for ranking the
affinity of ligands bound to the active site of a receptor.
3.3. ACC1 and ACC2 in Vitro Assay
To investigate the biological activities of the synthesized compounds, the in vitro inhibitory activities
of these compounds against both rat ACC1 and ACC2 were evaluated according to the previous
experimental method with some changes [8]. The ACC1 and ACC2 enzymes were supplied by Huawei
Pharmaceutical Co. Ltd (Shanghai, China). The compound CP-640186 was supplied by Selleck Chemicals
Co. Ltd. (Houston, TX, USA) Briefly, the test compounds were diluted in 10% DMSO at 10 μM. Then,
the solution was diluted with buffer solution until it reached the required concentration (1 nM to 10 μM)
and 5μL of the dilution was added to a 50 μL reaction mixture containing 30 mM HEPES, pH 7.4,
2 mM MgCl2, 2 mM sodium citrate, 1 mM DTT, 25 μM ATP, 16 mM NaHCO3 and 15 μM acetyl-CoA.
All of the enzymatic reactions were conducted at 30 °C for 60 min. The assay was performed using
Kinase-Glo Plus luminescence kinase assay kit (Beijing, China), which measures kinase activity by
quantifying the amount of ATP remaining in solution following a kinase reaction. The luminescent
signal from the assay is correlated with the amount of ATP present and is correlated with the amount
of kinase activity. The same concentrations of DMSO were tested in parallel and used as background
control. The inhibition percentage was expressed as the average value from three independent experiments.
CP-640186, an ACC1/ACC2 dual inhibitor developed by Pfizer, was used as a control.
3.4. In Vitro Cytotoxic Activities
In the cytotoxicity experiment, tetrazolium-based colorimetric assay (MTT test) was used to measure
the cytotoxic effect of these selected potent compounds on normal cells [15,22]. Human embryonic lung
fibroblast cell line (HELF, WI-38) was purchased from the Cell Resource Center, Chinese Academy of
Medical Sciences (Beijing, China). Briefly, cells (5000 per well) were collected and seeded in 96-well
plates. After incubation for 24 h, cells were exposed to fresh medium containing various concentrations

Molecules 2015, 20
16232
of test compound at 37 °C. After incubation for 48 h, 20 μL of MTT tetrazolium salt dissolved in
phosphate buffered saline (PBS) at a concentration of 5 mg/mL was added to each well and incubated
in a CO2 incubator for 4 h. The medium was then aspirated from each well and 150 μL of DMSO was
added to dissolve formazan crystals. The absorbance of each well was measured by ELx808 Microplate
Reader (BioTek, Winooski, VT, USA) at a test wavelength of 490 nm. TC50, the drug concentration
which reduces the cell viability by 50% was calculated by the Logit method.
4. Conclusions
In conclusion, we have designed and synthesized two series of novel ACC inhibitors bearing a quinoline
moiety. The molecular docking study revealed that compounds from these two series possessed similar
conformations and binding modes as CP-640186 in the CT domain of human ACC. The preliminary
in vitro assay indicated that most of the synthesized compounds possessed considerable ACC1/2
inhibitory activities. Moreover, compound 7a displayed comparable inhibitory activity against ACC2 as
the parent compound CP-640186, which may partly due to the additional hydrogen bond with the side
chain amide of Lys-1967. Further studies in animal models are in progress in our laboratory.
Acknowledgments
The authors are grateful to the financial support from National Natural Science Foundation of China
(No. 81302629). We thank Professor Mingjuan Ji in University of Chinese Academy of Sciences (UCAS)
for providing access to the SYBYL 7.1 software package in her laboratory.
Author Contributions
Tonghui Huang designed the research and wrote the paper. Tonghui Huang, Jie Sun and Qianqian Wang
synthesized the compounds and carried out the biological experiments. Jie Sun, Jian Gao and Yi Liu
carried out the computational studies. All authors have read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Tsai, S.; Clemente-Casares, X.; Revelo, X.S.; Winer, S.; Winer, D.A. Are obesity-related insulin
resistance and type 2 diabetes autoimmune Diseases? Diabetes 2015, 64, 1886–1897.
2. Abu-Elheiga, L.; Wu, H.; Gu, Z.; Bressler, R.; Wakil, S.J. Acetyl-CoA carboxylase 2−/− mutant
mice are protected against fatty liver under high-fat, high-carbohydrate dietary and de novo
lipogenic conditions. J. Biol. Chem. 2012, 287, 12578–12588.
3. Zu, X.Y.; Zhong, J.; Luo, D.X.; Tan, J.J.; Zhang, Q.H.; Wu, Y.; Liu, J.H.; Cao, R.X.; Wen, G.B.;
Cao, D.L. Chemical genetics of acetyl-CoA carboxylases. Molecules 2013, 18, 1704–1719.
4. Lee, J.; Walsh, M.C.; Hoehn, K.L.; James, D.E.; Wherry, E.J.; Choi, Y. Regulator of fatty acid
metabolism, acetyl Coenzyme A carboxylase 1, controls T cell. J. Immunol. 2014, 192, 3190–3199.

Molecules 2015, 20
16233
5. Wang, J.X.; Hudson, R.; Sintim, H.O. Inhibitors of fatty acid synthesis in prokaryotes and eukaryotes
as anti-infective, anticancer and anti-obesity drugs. Future Med. Chem. 2012, 4, 1113–1151.
6. Harada, N.; Oda, Z.; Hara, Y.; Fujinama, K.; Okawa, M.; Ohbuchi, K.; Yonemoto, M.; Ikeda, Y.;
Ohwaki, K.; Aragane, K.; et al. Hepatic de novo lipogenesis is present in liver-specific ACC1-deficient
mice. Mol. Cell. Biol. 2007, 27, 1881−1888.
7. Freeman-Cook, K.D.; Amor, P.; Bader, S.; Buzon, L.M.; Coffey, S.B.; Corbett, J.W.; Dirico, K.J.;
Doran, S.D.; Elliott, R.L.; Esler, W.P.; et al. Maximizing lipophilic efficiency: The use of free-wilson
analysis in the design of inhibitors of Acetyl-CoA carboxylase. J. Med. Chem. 2012, 55, 935−942.
8. Harwood, H.J.; 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.; et al. Isozyme-nonselective
N-substituted bipiperidylcarboxamide acetyl-CoA carboxylase inhibitors reduce tissue malonyl-CoA
concentrations, inhibit fatty acid synthesis and increase fatty acid oxidation in cultured cells and in
experimental animals. J. Biol. Chem. 2003, 278, 37099–37111.
9. Kamata, M.; Yamashita, T.; Kina, A.; Funata, M.; Mizukami, A.; Sasaki, M.; Tani, A.; Funami, M.;
Amano, N.; Fukatsu, K. Design, synthesis, and structure-activity relationships of novel spiro-piperidines
as acetyl-CoA carboxylase inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 3643−3647.
10. Bourbeau, M.P.; Bartberger, M.D. Recent advances in the development of acetyl-CoA carboxylase
(ACC) inhibitors for the treatment of metabolic disease. J. Med. Chem. 2015, 58, 525–536.
11. Pfizer Pipeline. As of 8 May 2014. Available online: http://www.pfizer.com/sites/default/files/
product-pipeline/May%208%2C%202014%20Pipeline%20Update_Final_to%20BT.pdf (accessed on
10 August 2015).
12. Zhang, H.; Tweel, B.; Li, J.; Tong, L. Crystal structure of the carboxyltransferase domain of
acetyl-coenzyme A carboxylase in complex with CP-640186. Structure 2004, 12, 1683–1691.
13. Corbett, J.W.; Freeman-Cook, K.D.; Elliott, R.; Vajdos, F.; Rajamohan, F.; Kohls, D.; Marr, E.;
Zhang, H.L.; Tong, L.; Tu, M.H.; et al. Discovery of small molecule isozyme non-specific inhibitors
of mammalian acetyl-CoA carboxylase 1 and 2. Bioorg. Med. Chem. Lett. 2010, 20, 2383–2388.
14. Madauss, K.P.; Burkhart, W.A.; Consler, T.G.; Cowan, D.J.; Gottschalk, W.K.; Miller, A.B.;
Short, S.A.; Tran, T.B. Williams, S.P. The human ACC2 CT-domain C-terminus is required for
full functionality and has a novel twist. Acta Crystallogr. D Biol. Crystallogr. 2009, 65, 449–461.
15. Park, E.H.; Kim, H.S; Eom, S.J.; Kim, K.T. Paik, H.D. Antioxidative and Anticanceric Activities
of Magnolia (Magnolia denudata) Flower Petal Extract Fermented by Pediococcus acidilactici
KCCM 11614. Molecules 2015, 20, 12154–12165.
16. Brylinski, M.; Waldrop, G.L. Computational redesign of bacterial biotin carboxylase inhibitors
using structure-based virtual screening of combinatorial libraries. Molecules 2014, 19, 4021–4045.
17. Molinspiration Cheminformatics. Available online: http://www.molinspiration.com (accessed on
10 August 2015).
18. Drug-Likeness and Molecular Property Prediction. Available online: http://www.molsoft.com/
mprop/ (accessed on 10 August 2015).
19. Zhang, H.L.; Yang, Z.; Shen, Y. Crystal structure of the carboxyltransferase domain of acetyl-coenzyme
A carboxylase. Science 2003, 299, 2064–2067.
20. Yu, L.P.; Kim, Y.S.; Tong, L. Mechanism for the inhibition of the carboxyltransferase domain of
acetyl-coenzyme A carboxylase by pinoxaden. Proc. Natl. Acad. Sci. USA 2010, 107, 22072–22077.

Molecules 2015, 20
16234
21. Lang, P.T.; Brozell, S.R.; Mukherjee, S.; Pettersen, E.T.; Meng, E.C.; Thomas, V.; Rizzo, R.C.;
Case, D.A.; James, T.L.; Kuntz, I.D. Dock 6: Combining technique to model RNA-small molecule
complexes. RNA 2009, 15, 1219–1230.
22. Wang, X.K.; Lin, J.; Chen, Y.; Zhong, W.; Zhao, G.M.; Liu, H.Y.; Li, S.; Wang, L.L.; Li, S. Novel
fatty acid synthase (FAS) inhibitors: Design, synthesis, biological evaluation, and molecular docking
studies. Bioorg. Med. Chem. 2009, 17, 1898–1904.
Sample Availability: Samples of the compounds 7a–7i and 12a–12g are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).