Design, synthesis and biological evaluation of novel spiro-pentacylamides as acetyl-CoA carboxylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.03.014

Acetyl-CoA carboxylase (ACC) catalyzes the rate-determining step in de novo lipogenesis and plays an important role in the regulation of fatty acid oxidation. Therefore, ACC inhibition offers a promising option for intervention in nonalcoholic fatty liver disease (NAFLD), type 2 diabetes (T2DM) and cancer. In this paper, a series of spiropentacylamide derivatives were synthesized and evaluated for their ACC1/2 inhibitory activities and anti-proliferation effects on A549, H1975, HCT116, SW620 and Caco-2 cell lines in vitro. Compound 6o displayed potent ACC1/2 inhibitory activity (ACC1 IC₅₀ = 0.527 μM, ACC2 IC₅₀ = 0.397 μM) and the most potent anti-proliferation activities against A549, H1975, HCT116, SW620 and Caco-2 cell lines, with IC₅₀ values of 1.92 μM, 0.38 μM, 1.22 μM, 2.05 μM and 5.42 μM respectively. Further molecular docking studies revealed that compound 6o maintained hydrogen bonds between the two carbonyls and protein backbone NHs (Glu-B2026 and Gly-B1958). These results indicate that compound 6o is a promising ACC1/2 inhibitor for the potent treatment of cancer.

Full text of DOI:10.1016/j.bmc.2018.03.014

Accepted Manuscript
Design, synthesis and biological evaluation of novel spiro-pentacylamides as
acetyl-CoA carboxylase inhibitors
Qiangqiang Wei, Liankuo Mei, Yifei Yang, Hui Ma, Hongyi Chen, Huibin
Zhang, Jinpei Zhou
PII:
S0968-0896(18)30255-4
https://doi.org/10.1016/j.bmc.2018.03.014
BMC 14252
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 February 2018
7 March 2018
8 March 2018
Please cite this article as: Wei, Q., Mei, L., Yang, Y., Ma, H., Chen, H., Zhang, H., Zhou, J., Design, synthesis and
biological evaluation of novel spiro-pentacylamides as acetyl-CoA carboxylase inhibitors, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.03.014
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Design, synthesis and biological evaluation of novel spiro-pentacylamides as
acetyl-CoA carboxylase inhibitors
Qiangqiang Weia, Liankuo Meib, Yifei Yanga, Hui Mab, Hongyi Chena, Huibin Zhangb*, Jinpei
Zhoua*
a Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, PR China1
Abstract
Acetyl-CoA carboxylase (ACC) catalyzes the rate-determining step in de novo lipogenesis and
plays an important role in the regulation of fatty acid oxidation. Therefore, ACC inhibition offers a
promising option for intervention in nonalcoholic fatty liver disease (NAFLD), type 2 diabetes
(T2DM) and cancer. In this paper, a series of spiro-pentacylamide derivatives were synthesized
and evaluated for their ACC1/2 inhibitory activities and anti-proliferation effects on A549, H1975,
HCT116, SW620 and Caco-2 cell lines in vitro. Compound 6o displayed potent ACC1/2 inhibitory
activity (ACC1 IC50 = 0.527 µM, ACC2 IC50 = 0.397 µM) and the most potent anti-proliferation
activities against A549, H1975, HCT116, SW620 and Caco-2 cell lines, with IC50 values of 1.92
µM, 0.38 µM, 1.22 µM, 2.05 µM and 5.42 µM respectively. Further molecular docking studies
revealed that compound 6o maintained hydrogen bonds between the two carbonyls and protein
backbone NHs (Glu-B2026 and Gly-B1958). These results indicate that compound 6o is a
promising ACC1/2 inhibitor for the potent treatment of cancer.
1. Introduction
In contrast to normal differentiated cells, which satisfy their requirement for fatty acids by
importing them from the circulation, cancer cells undergo high rates of de novo lipogenesis (DNL)
to support cell division and continuous proliferation[1, 2]. A wide number of cancers such as those
of the prostate[3], hepatoma[4], bladder, lung, colon and ovary[5] have been shown to have a high
rate of fatty acid synthesis (FASyn) which is reflected by the increased expression of lipogenic
enzymes[6-9]. Modulation of lipogenic enzymes such as cytoplasmic acetyl-CoA synthetase
(ACSS2)[10, 11], ATP citrate lyase (ACLY)[12], acetyl-CoA carboxylase (ACC)[13-15]and fatty acid
synthase (FASN)[16] have demonstrated inhibition of cell growth and proliferation in cancer
models both in vitro and in vivo.
Acetyl-CoA carboxylase (ACC) is a biotin-dependent protein, composed of a carboxyl
transferase domain (CT), biotin carboxy carrier protein (BCCP) and biotin carboxylase domain
(BC), which catalyzes the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA, the
rate-limiting and first committed reaction in FASyn[17, 18]. There are two characterized isoforms of
1 ∗Corresponding authors. Center of Drug Discovery, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Tel: +86-25-83271302; Fax:
+86-25-83271480. E-mail: zhanghb80@cpu.edu.cn
∗Corresponding authors. Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, PR China. Tel: +86-25-83271302; Fax: +86-25-83271480. E-mail: jpzhou668@163.com

mammalian ACC (known as ACC1 and ACC2), which are encoded by separate genes and display
distinct cellular distributions[19]. ACC1 is located in the cytosol and primarily expressed in lipid
rich tissues (liver, adipose)[20]. In contrast, the second ACC isoform, ACC2 is a mitochondrially
associated isozyme present in oxidative tissues (heart, skeletal muscle)[21]. ACC1 converts
acetyl-CoA into malonyl-CoA for DNL while the ACC2 isoform carries out the same reaction to
generate malonyl-CoA for the inhibition of carnitine palmitoyl transferase 1 (CPT-1)[22], the
protein that catalyzes the conjugation of free fatty acyls to carnitine from entering the
mitochondrial membrane for subsequent β-oxidation[23]. Thus, malonyl-CoA production by ACC2
activity serves to directly inhibit fatty acid oxidation. Considering the roles of the ACC in both the
synthesis and oxidation of fatty acids, inhibition of ACC1/2 has the potential to favorably affect a
variety of metabolic diseases including T2DM, obesity and NAFLD by reducing lipid
accumulation and improving insulin sensitization[24, 25]. In addition, given that the tumor cells rely
on FASyn for energy storage, membrane formation and production of signaling molecules[26] and
that both ACC and FASN mRNAs are upregulated in a number of cancers, FASyn has been
postulated to offer a therapeutic window. In this paper, efforts to target cancer cells that bear
elevated rates of lipogenesis have focused on attempts to chemically inhibit ACC1/2.
To date, several classes of small molecule ACC1/2 inhibitors have been reported and the
representative structures are presented in Fig.1 [5, 15, 27-29]. Pfizer has had a long-standing interest in
the development of ACC inhibitors and reported a series of potent, nonselective and orally
bioavailable spirochormanone ACC1/2 dual inhibitors as exemplified by PF-1 and the co-crystal
structure of the humanized yeast ACC carboxyl transferase (CT) domain in complex with PF-2
was performed (PDB: 4WYO, Fig. 2). Compound PF-2 was oriented in the channel generated at
the dimeric interface of the N and C domains, and the pyrazolopyranone group was filling a
narrow, deep, hydrophobic pocket formed by a cluster of hydrophobic residues (Leu-A1762,
Val-A1765, Leu-A1766, Ala-B1920, Val-B1923, and Phe-B1925) from each domain. The
co-crystal data also indicated that the amide carbonyl (right side) interacts with the backbone of
Glu-B2026 and the ketone carbonyl (left side) is bound to the backbone of Gly-B1958, which
made significant contributions to binding potency. Notably, further co-crystal structure published
by Pfizer of compound PF-3 ( Fig. 2 ) demonstrated that the proper fixation of the ketone
carbonyl (left side) direction may lead to a nearly identical hydrogen bonding interaction as
compared to PF-2. In addition, the structural rigidity imparted by the spirocyclic ring system was
essential to binding by reducing the entropic penalty for properly orienting the hydrogen bond
acceptors[30]. Herein, we report the discovery of a spirolactam derivative 6o bearing a
spiro-pentacylamide ring formed by focusing interactions with Gly-B1958 and Glu-B2026 to
explore inhibitory activity of ACC.

OMe
O
Me
Me
HN
O
O
O
OMe
O
O
OH
O
N
O
O
OH
N
F
OH
Me OMe
Cl
pinoxaden
Soraphen A
BAY ACC002 (Bayer)
O
OH
N
N
O
O
O
O
N
O
S
H
N
O
N
O
O
N
H
NH2
N
O
O
N
N
N
O
O
O
O
ND-630 (GS-0976,Gilead)
PF-1 (Pfizer)
Ta-1 (Takeda)
Fig. 1. Representative inhibitors of ACC
On the basis of spirochromanone scaffold reported by Pfizer, our modification strategy was
focused on the pyrazolopyranone region of compound PF-2 as shown in Fig. 3. Spatial orientation
of the amide carbonyl (right side) and ketone carbonyl (left side) were retained to form the key
hydrogen-bonds with Glu-B2026 and Gly-B1958, respectively. On the other hand, we focused on
searching for substituents that would provide an optimal fit in the binding pocket composed
primarily of hydrophobic side chains. In consideration of the above two points, the
spiro-pentacylamide scaffold was designed. Meanwhile a range of phenyl substituents were
introduced via standard Buchwald coupling conditions to occupy the hydrophobic pocket.
A
B
O
O
O
N
N
H
N
H
N
N
N
N
N
N
N
O
O
PF-2
PF-3
ACC1 IC50 = 111 nM
ACC2 IC50 = 9.8 nM
ACC1 IC50 = 62 nM
ACC2 IC50 = 30 nM
Fig. 2. (A). ACC inhibitors PF-2 and PF-3. (B). Co-crystal structure of PF-2 (orange) bound in the CT-domain of
ACC, overlaid with the bound conformation of PF-3 (magenta). PDB accession codes: PF-2, 4WYO; PF-3,
4WZ8.

O
O
N
H
N
H
N
R
R
N
N
N
N
O
O
Fig. 3. Design strategy of spiro-pentacylamide derivatives.
Herein, we would like to describe our efforts on the biological evaluation and structural
optimization of the novel spiro-pentacylamide-based ACC1/2 inhibitors. The synthesized
compounds were evaluated for their biological activities in vitro toward five different human
tumour cell lines, and ACC enzymes. Among these derivatives, compound 6o with the most potent
ACC inhibition activity (ACC1 IC50 = 0.527 µM, ACC2 IC50 = 0.397 µM) and the most potent
anti-proliferation activity against A549, H1975, HCT116, SW620 and Caco-2 cell lines (1.92 µM,
0.38 µM, 1.22 µM, 2.05 µM and 5.42 µM), respectively, was considered to be a promising lead
compound worthy of further investigation. The binding interactions between ACC and compound
6o are presented within this paper as well.
2. Chemistry
The synthetic route with high to moderate yields for accessing the desired compounds 6a-6t
is described in Scheme 1. The commercially available tert-butyl 4-oxopiperidine-1-carboxylate
and methyl (triphenylphosphoranylidene)acetate were refluxed in toluene to afford 1[31]. The
intermediate 2 was prepared from 1 using nitromethane in the presence of tetrabutylammonium
fluoride (TBAF) in tetrahydrofuran. The key intermediate 3 was obtained by treating 2 with
Raney-nickel under an hydrogen atmosphere at room temperature. Next, 3 was subjected to
Buchwald-Hartwig C-N coupling reaction with substituted bromophenyl reagents in the presence
of potassium phosphate and catalytic amount of 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
and palladium catalyst in dioxane at 100°C to give the desired compound 4a-e[32]. The removal of
the Boc group of 4a-e by treatment with HCl in ethyl acetate yielded 5a-e at room temperature.
Finally, the target compounds 6a-6e and 6g-6t were synthesized by the classical method of amide
formation involving condensation between 5a-e with a variety of carboxylic acid derivatives in
dimethylformamide (DMF) under room temperature. In addition, the target compound 6f was
obtained by hydrolysis of compound 6a with 2 M aqueous NaOH in MeOH/water.

Scheme 1. Synthetic route for target compounds 6a-6t. Reagents and conditions: (a) methyl
(triphenylphosphoranylidene)acetate, toluene, reflux; (b) nitromethane, TBAF, THF, rt-70°C; (c) Raney-Ni, H2,
EtOH,
rt;
(d)
substituted
bromophenyl
reagents,
Pd2(dba)3,
K3PO4,
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, dioxane, 100°C; (e) (i) HCl gas, ethyl acetate, rt; (ii) 2M
aqueous NaOH, H2O; (f) corresponding carboxylic acid derivatives, 1-hydroxybenzotriazole (HOBt),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), Et3N, DMF, 40°C. (g) 2M aqueous NaOH,
MeOH/H2O, 40°C.
3. Results and discussion
3.1. In vitro ACC inhibitory activities
Taking PF-1 as reference compound, the target compounds 6a-6t were tested for in vitro ACC
inhibitory activities and the results are summarized in Tables 1 and 2. We initially varied the
groups on the N-phenyl moiety to afford compounds 6a-6f. The most potent analog from this work
was (trifluoromethyl)phenyl derivative 6b (ACC1 IC50 = 1.202 µM, ACC2 IC50 = 0.944 µM).
Next, we selected the (trifluoromethyl)phenyl as an ‘anchor’ group, and the amide tail region SAR
was explored by coupling 5b to several carboxylic acids and the results are shown in Table 2.
5-Carboxybenzimidazole derivatives showed more activity than the indole, indazole and quinoline
analogs, which were similar to spirochromanone-based ACC inhibitors reported by Pfizer. Of the
three benzimidazole derivatives prepared (6g, 6h and 6o), 6o (ACC1 IC50 = 0.527 µM, ACC2 IC50
= 0.397 µM) exhibited the best potency. Therefore, further optimization efforts around the
benzimidazole moiety led to the synthesis of compounds 6p-6t. Unexpectedly, all compounds
exhibited modestly lower ACC inhibitory potency than 6o. To understand the binding mode of this
series, we performed docking experiments with Glide docking in Schrodinger. Comparison of the
interactions of PF-2 and 6o bound in the CT-domain of ACC showed that both molecules

maintained hydrogen bonds between the two carbonyls and protein backbone NHs (Fig. 4).
Notably, however, the bound position of the carbonyl of 6o and its hydrogen bond distance (3.7 Å)
were clearly distinct from PF-2, which may resulted in lower potency of spiro-pentacylamide
derivatives compounds. Another interesting phenomenon observed was that the
2-phenylbenzimidazole moiety of 6o was directed toward an open pocket formed by backbone
atoms of residues Ile-B2033, Glu-B2032, Gly-B2029, Lys-C1954, Ser-C1976 and Gly-C1758 (for
clarity, not shown in Fig. 4), which may offer a promising option for compound design.
Table 1
Chemical structures of target compounds 6a-6f and their ACC inhibitory activities.
Compound
R
IC50 (µM)a
ACC1
ACC2
2.241 0.013
1.882 0.019
0.944 0.017
> 5
6a
6b
1.202 0.006
3.990 0.024
2.011 0.022
3.559 0.018
2.792 0.011
0.032 0.009
6c
3.772 0.024
> 5
6d
6e
4.646 0.009
0.008 0.013
6f
PF-1b
a IC50 values for ACC1 and ACC2 activities presented are the mean SD values of three independent determinations
b Used as positive control.
Table 2
Chemical structures of target compounds 6g-6t and their ACC inhibitory activities.

Compound
R
IC50 (µM)a
ACC1
ACC2
2.221 0.008
1.687 0.017
> 5
3.021 0.0012
6g
6h
6i
1.944 0.006
> 5
4.955 0.015
4.726 0.009
4.923 0.016
> 5
> 5
6j
2.738 0.019
> 5
6k
6l
> 5
6m
6n
6o
6p
6q
> 5
> 5
0.527 0.022
1.147 0.013
2.649 0.016
0.397 0.014
0.764 0.015
1.237 0.024

1.054 0.021
1.948 0.022
4.265 0.018
0.056 0.011
0.976 0.006
1.112 0.019
3.477 0.023
0.014 0.015
6r
6s
6t
PF-1b
a IC50 values for ACC1 and ACC2 activities presented are the mean SD values of three independent determinations
b Used as positive control.
3.2. Anticancer evaluation against cancer cell lines in vitro
The anti-proliferation activity of the compounds was screened against A549, H1975, HCT116,
SW620 and Caco-2 cell lines using the MTT assay. ACC1 was highly expressed in all five cell
lines[13], and ACC2 had expression levels ranging from low to undetectable. The bioactivity data is
summarized in Table 3. Compound PF-1 was used as positive control.
As illustrated in Table 3, most of the target compounds 6a–b, 6h, 6o-s were shown to have
anti-proliferation activities. Overall, all the compounds showed higher activity against H1975 cell
line and moderate activity against A549 cell line, but lower activity against HCT116, SW620 and
Caco-2 cell lines. Two selected compounds (6h and 6o) showed comparable activity against
H1975, HCT116 and SW620 cells lines with the positive drug PF-1. The most promising
compound 6o exhibited the best activity against A549, H1975, HCT116, SW620 and Caco-2 cell
lines with the IC50 values of 1.92 µM, 0.38 µM, 1.22 µM, 2.05 µM and 5.42 µM respectively.
These results indicated that compound 6o could be a promising lead compound for anti-cancer.
Table 3
The anti-proliferation activities of 6a–o against A549, H1975, HCT116, SW620 and Caco-2 cell
lines in vitro.
Compound
IC50 (µM)a
HCT116
>10
A549
2.11 0.41
>10
H1975
5.64 0.81
>10
SW620
>10
Caco-2
>10
6a
6b
8.67 2.78
4.21 0.17
1.22 0.11
6.21 0.25
7.64 1.49
>10 0.63
8.67 0.18
2.43 0.44
>10
>10
>10
0.27 0.02
0.38 0.03
>10
1.82 0.61
2.05 0.07
>10
>10
6h
6o
1.92 0.05
1.03 0.73
3.49 0.94
7.96 0.13
>10
5.42 0.69
>10
6p
0.39 1.22
0.72 0.98
n.d.
7.24 1.91
3.26 1.11
4.74 0.94
3.19 0.49
>10
n.d.b
6q
6r
n.d.
6s
PF-1c
0.74 0.22
0.21 0.05
6.51 2.12
a
The data were expressed as the means SD, representing the relative levels of anti-proliferation from three independent
experiments.

b n.d. = not determined.
c Used as positive control.
A
B
C
D
Fig. 3 (A) Docking conformation of PF-2 with ACC CT domain (PDB id: 4WYO). (B) Docking conformation of
6o with ACC CT domain. (C) (D) Superimposition docking conformation of PF-3 (Green) and 6o (Pink) with
ACC CT domain.
4. Conclusions
In summary, on the basis of the spiroketone scaffold reported by Pfizer, we designed and
synthesized a series of novel spiro-pentacylamide derivatives as ACC inhibitors. All the
compounds were initially screened for ACC enzyme inhibition and some selected compounds
were further evaluated for the activity against A549, H1975, HCT116, SW620 and Caco-2 cell
lines. Two compounds (6h and 6o) showed comparable activity against H1975, HCT116 and
SW620 cells lines with the positive control PF-1. The most promising compound, 6o (ACC1 IC50
= 0.527 µM, ACC2 IC50 = 0.397 µM), had IC50 values of 1.92 µM, 0.38 µM, 1.22 µM, 2.05 µM
and 5.42 µM against A549, H1975, HCT116, SW620 and Caco-2 cell lines, respectively.
Moreover, SAR study indicated that substituting group at the 2-position on the terminal
benzimidazole ring was beneficial in terms of ACC inhibitory and anti-proliferation activities.
Furthermore, docking studies demonstrated that compound 6o formed the key hydrogen-bonds
with Glu-B2026 and Gly-B1958. Combined results from enzymatic assays and molecular docking
analysis therefore indicated that compound 6o was a potent ACC inhibitor and ACC inhibition, by
reducing DNL and increasing mitochondrial oxidation rates, may have therapeutic utility for the
suppression of tumor growth. The structural modification of 6o as well as the SAR will be carried

out in the follow-up study.
5. Experimental section
5.1. General information
Solvents and reagents were purchased from Bide Pharmatech Ltd., Energy Chemical, Accela
ChemBio Co., Ltd., Shanghai Chemical Reagent Co., Ltd., and TCI, and were used without
further purification. Reaction progress was monitored by TLC using HSGF 254 (150–200µm
thickness; Yantai Jiangyou Co., China) with detection by UV. Silica gel 200-300 was used for
1
column chromatography. H NMR and 13C NMR spectra were recorded on Bruker AV-300
spectrometer at 25°C and referenced to TMS. Chemical shifts were given in ppm (δ) downfield
from tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d), triplet
(t), quartet (q), multiplet (m), and broad (br). Melting points were measured on capillary tube and
were uncorrected. Purity of the target compounds were determined by HPLC analysis (UV
detector, wavelength: 272 nm). Mass spectrometry (MS) was performed using an Hewlett-Packard
1100 LC/MSD spectrometer;
5.2. Chemical synthesis
5.2.1. tert-Butyl 4-(2-methoxy-2-oxoethylidene)piperidine-1-carboxylate (1)
A solution of tert-butyl tert-butyl 4-oxopiperidine-1-carboxylate (5 g, 25.1 mmol) and methyl
(triphenylphosphoranyl)acetate (8.4 g, 25.1 mmol) in toluene (100 mL) was heated to reflux and
stirred for 17 h. The reaction mixture was then cooled to room temperature and evaporated under
reduced pressure and purified by silica gel column chromatography, eluting with DCM/petroleum
1
ether (1/1, v/v) to obtain 1 as a white solid (5.44 g, 85% yield). H NMR (300 MHz, CDCl3) δ
5.72 (s, 1H), 3.70 (s, 3H), 3.50 (dd, J = 12.6, 7.2 Hz, 4H), 2.94 (t, J = 5.6 Hz, 2H), 2.33 – 2.22 (m,
2H), 1.49 (d, J = 6.5 Hz, 9H).
5.2.2. tert-Butyl 4-(2-methoxy-2-oxoethyl)-4-(nitromethyl)piperidine-1-carboxylate (2)
To a stirred solution of compound 1 (4 g, 15.62 mmol) and nitro methane (1.05 g, 17.22 mmol)
in THF (20 mL), 1.0 M solution of TBAF in THF (23.53 mL, 23.53 mmol ) was added drop wise
at 0 °C for 20 min. The reaction mixture was refluxed at 70°C for 16 h. The reaction mixture was
partitioned between H2O and ethyl acetate, and the organic layers were combined and dried
(Na2SO4). The solvent was removed in vacuo and the residue was purified by column
chromatography (0 to 30% EtOAc in petroleum ether) to afford 2 as a white solid (3.79 g, 76%
yield). 1H NMR (300 MHz, DMSO) δ 4.77 (s, 2H), 3.60 (s, 3H), 3.49 – 3.27 (m, 4H), 2.58 (s, 2H),
1.67 – 1.47 (m, 4H), 1.39 (s, 9H).
5.2.3. tert-Butyl 3-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (3)
A mixture of compound 2 (3 g, 9.45 mmol) and Ranet-nickel (4 g) in 50 mL EtOH was stirred
under hydrogen at room temperature for 12 h. The reaction mixture was filtered through Celite,
washed with EtOH and the filtrate was evaporated to afford compound 3 as an off-white solid
(1.95 g, 81% yield). 1H NMR (300 MHz, CDCl3) δ 6.19 (s, 1H), 3.60 – 3.43 (m, 2H), 3.38 – 3.24
(m, 2H), 3.22 (s, 2H), 2.25 (s, 2H), 1.62 (t, J = 5.2 Hz, 4H), 1.47 (s, 9H).

5.2.4. General procedure for preparation of intermediates (4a–e )
To a round bottom flask was added compound 3 (0.5 g, 1.97 mmol), K3PO4 (1.25 g, 5.9 mmol),
Pd2(dba)3 (0.018 g, 0.02 mmol), 9,9-dimethyl-4,5-bis(diphenylphosphino)-xanthene (0.023 g,
0.039 mmol), corresponding bromophenyl substituents (2.36 mmol) and 10 mL dioxane. The
reaction mixture was thoroughly degassed with nitrogen, and heated at 100 °C overnight. After
cooling to room temperature, the reaction mixture was partitioned between H2O and ethyl acetate.
The organic layers were combined and concentrated. The resulting crude material was purified via
silica gel chromatography by eluting with a gradient of 1:3 to 2:1 ethyl acetate/ petroleum ether to
give the title compounds 4a-e.
5.2.4.1. tert-Butyl 2-(4-(methoxycarbonyl)phenyl)-3-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate
(4a). Off white solid, yield 88%; 1H NMR (300 MHz, CDCl3) δ 8.06 (d, J = 7 Hz, 2H), 7.73 (d, J
= 8.9 Hz, 2H), 3.93 (s, 3H), 3.71 (s, 2H), 3.64 (b, 2H), 3.35 (m, 2H), 2.59 (s, 2H), 1.71 (m, 4H),
1.49 (s, 9H).
5.2.4.2. tert-Butyl 3-oxo-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspiro[4.5]decane-8-carboxylate
(4f). White solid, yield 91%; 1H NMR (300 MHz, CDCl3) δ 7.76 (d, J = 7.6 Hz, 2H), 7.64 (d, J =
7.6 Hz, 2H), 3.69 (s, 4H), 3.33 (d, J = 7.0 Hz, 2H), 2.58 (s, 2H), 1.69 (s, 4H), 1.50 (s, 10H).
5.2.4.3. tert-Butyl 2-(4-cyanophenyl)-3-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (4c). White
solid, yield 89%; 1H NMR (300 MHz, CDCl3) δ 7.77 (d, J = 8.9 Hz, 2H), 7.65 (d, J = 8.9 Hz, 2H),
3.70 – 3.55 (m, 4H), 3.37 – 3.23 (m, 2H), 2.58 (s, 2H), 1.68 (t, J = 5.6 Hz, 4H), 1.47 (s, 10H).
5.2.4.4. tert-Butyl 2-(4-cyano-3-methylphenyl)-3-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate
(4d) White solid, yield 80%; 1H NMR (300 MHz, CDCl3) δ 7.66 – 7.53 (m, 3H), 3.68 – 3.56 (m,
4H), 3.36 – 3.24 (m, 2H), 2.57 (d, J = 5.3 Hz, 5H), 1.68 (t, J = 5.6 Hz, 4H), 1.47 (s, 9H).
5.2.4.5. tert-Butyl 2-(4-cyano-2-fluorophenyl)-3-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate
1
(4e). White solid, yield 85%; H NMR (300 MHz, CDCl3) δ 7.85 (dd, J = 7.0, 1.9 Hz, 1H), 7.58
(ddd, J = 8.6, 4.5, 2.1 Hz, 1H), 7.31 (s, 1H), 3.67 (s, 2H), 3.62 – 3.49 (m, 2H), 3.48 – 3.29 (m, 2H),
2.52 (s, 2H), 1.70 (dd, J = 12.1, 6.3 Hz, 4H), 1.48 (s, 9H).
5.2.5. General procedure for preparation of intermediates (5a–e )
To a solution of an appropriate intermediates 4a-e (2 mmol) in ethyl acetate (5 mL) was added 4
N HCl in ethyl acetate (10 mL), and the resulting mixture was stirred at room temperature for 2 h.
The resulting precipitate was collected by filtration and washed with ethyl acetate. Subsequently,
the precipitate was resolved and neutralized to afford the target compounds 4a-e as an off white
solid.
5.2.5.1. Methyl 4-(3-oxo-2,8-diazaspiro[4.5]decan-2-yl)benzoate (5a) Off-white solid, yield 91%;
1H NMR (300 MHz, DMSO-d6) δ 7.96 (d, J = 8.9 Hz, 2H), 7.83 (d, J = 8.9 Hz, 2H), 3.83 (s, 3H),
3.69 (s, 2H), 2.81 – 2.59 (m, J = 14.3 Hz, 4H), 2.48 (s, 2H), 1.49 (m, J = 14.3 Hz, 4H).
5.2.5.2. 2-(4-(Trifluoromethyl)phenyl)-2,8-diazaspiro[4.5]decan-3-one (5b) White solid, yield

88%; 1H NMR (300 MHz, DMSO-d6) δ 7.90 (d, J = 8.3 Hz, 2H), 7.76 (d, J = 8.3 Hz, 2H), 3.81 (s,
2H), 3.07 (m, J = 12.3 Hz, 4H), 2.61 (s, 2H), 1.85 (m, J = 12.3 Hz, 4H).
1
5.2.5.3. 4-(3-Oxo-2,8-diazaspiro[4.5]decan-2-yl)benzonitrile (5c) Off white solid, yield 80%; H
NMR (300 MHz, DMSO-d6) δ 7.86 (dd, J = 16.2, 8.0 Hz, 4H), 3.69 (s, 2H), 2.67 (m, J = 5.2 Hz,
4H), 2.43 (s, 2H), 1.50 (m, J = 5.2 Hz, 4H).
5.2.5.4. 2-Methyl-4-(3-oxo-2,8-diazaspiro[4.5]decan-2-yl)benzonitrile (5d) Off white solid, yield
1
90%; H NMR (300 MHz, DMSO-d6) δ 7.83 – 7.64 (m, 3H), 3.65 (s, 2H), 2.65 (m, J = 12.6, 6.4 Hz,
4H), 2.46 (s, 5H), 1.48 (m, J = 5.0 Hz, 4H).
5.2.5.5. 3-Fluoro-4-(3-oxo-2,8-diazaspiro[4.5]decan-2-yl)benzonitrile (5e) White solid, yield 81%;
1H NMR (300 MHz, DMSO-d6) δ 7.84 – 7.66 (m, 3H), 3.61 (s, 2H), 2.62 (m, J = 13.9 Hz, 4H), 2.42 (s,
2H), 1.46 (m, J = 13.9 Hz, 4H).
5.2.6. General procedure for preparation of target compounds (6a–6e, 6g-6t)
A mixture of intermediates 4a-e (0.60 mmol), EDCI (138 mg, 0.72 mmol), corresponding
carboxylic acid derivatives (0.60 mmol) and HOBt (97 mg, 0.72 mmol) in
N,N-dimethylformamide (6.0 mL) was stirred for 20 h. The reaction mixture was diluted with
ethyl acetate and washed with brine, the organic layer was dried over magnesium sulfate and
concentration in vacuo. The residue was purified by silica gel column chromatography (5%
methanol/chloroform) to afford the title compounds 6a-t as a white solid.
5.2.6.1.
Methyl
4-(8-(7-methyl-1H-indazole-5-carbonyl)-3-oxo-2,8-diazaspiro[4.5]decan-2-yl)benzoate
(6a)
White solid; yield 70%; m.p.: 199-201°C; 1H NMR (300 MHz, DMSO-d6) δ 13.35 (s, 1H), 8.13 (s,
1H), 7.97 (d, J = 8.9 Hz, 2H), 7.82 (d, J = 8.9 Hz, 2H), 7.64 (s, 1H), 7.16 (s, 1H), 3.83 (s, 3H), 3.78 (s,
2H), 3.49 (m, 4H), 2.59 (s, 2H), 2.54 (s, 3H), 1.65 (s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 173.1,
169.6, 165.7, 143.6, 140.1, 134.5, 129.9, 128.5, 124.7, 124.3, 121.8, 120.2, 118.5, 116.9, 57.6, 51.9,
43.6, 34.2, 16.7. ESIMS m/z [M + H]+ 447.1; Anal. calcd. For C25H26N4O4: C, 67.25; H, 5.87; N,
12.55. Found: C, 67.29; H, 5.85; N, 12.51.
5.2.6.2.
3-Fluoro-4-(8-(7-methyl-1H-indazole-5-carbonyl)-3-oxo-2,8-diazaspiro[4.5]decan-2-yl)benzonitri
1
le (6b) White solid; yield 81%; m.p.: 221-223°C; H NMR (300 MHz, DMSO-d6) δ 13.35 (s, 1H),
8.14 (s, 1H), 7.86 (d, J = 5.4 Hz, 4H), 7.64 (s, 1H), 7.16 (s, 1H), 3.78 (s, 2H), 3.69 – 3.39 (m, 4H), 2.60
(s, 2H), 2.51 (s, 3H), 1.65 (s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 173.1, 169.6, 142.8, 140.1, 134.5,
128.5, 125.8, 125.8, 124.7, 121.8, 120.2, 119.1, 116.9, 57.6, 43.6, 34.3, 16.7. ESIMS m/z [M + H]+
457.1; Anal. calcd. For C24H23F3N4O2: C, 63.15; H, 5.08; N, 12.27. Found: C, 63.11; H, 5.12; N,
12.24.
5.2.6.3.
4-(8-(7-Methyl-1H-indazole-5-carbonyl)-3-oxo-2,8-diazaspiro[4.5]decan-2-yl)benzonitrile
(6c)

1
White solid; yield 90%; m.p.: 217-219°C; H NMR (300 MHz, DMSO-d6) δ 13.36 (s, 1H), 8.14 (s,
1H), 7.89 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.7 Hz, 2H), 7.65 (s, 1H), 7.16 (s, 1H), 3.79 (s, 2H), 3.4Z9 (s,
2H), 3.33 (s, 2H), 2.60 (s, 2H), 2.55 (s, 3H), 1.67 (s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 173.4,
169.6, 143.3, 140.1, 134.5, 132.9, 128.4, 124.7, 121.8, 120.2, 119.1, 118.9, 116.9, 105.4, 57.5, 43.6,
34.2, 16.7. ESIMS m/z [M + H]+ 414.1; Anal. calcd. For C24H23N5O2: C, 69.72; H, 5.61; N, 16.94.
Found: C, 69.69; H, 5.65; N, 16.97.
5.2.6.4.
2-Methyl-4-(8-(7-methyl-1H-indazole-5-carbonyl)-3-oxo-2,8-diazaspiro[4.5]decan-2-yl)benzonitri
le (6d) White solid; yield 72%; m.p.: 237–239°C; 1H NMR (300 MHz, DMSO-d6) δ 13.35 (s, 1H),
8.13 (s, 1H), 7.75 (s, 2H), 7.71 (s, 1H), 7.64 (s, 1H), 7.16 (s, 1H), 3.76 (s, 2H), 3.66 – 3.40 (m, 4H),
2.58 (s, 2H), 2.54 (s, 3H), 2.47 (s, 3H), 1.65 (s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 173.3, 169.6,
143.1, 142.3, 140.1, 134.5, 133.1, 128.4, 124.7, 121.8, 120.2, 119.8, 118.1, 116.9, 116.7, 106.0, 57.5,
43.7, 34.2, 20.2, 16.7. ESIMS m/z [M + H]+ 428.1; Anal. calcd. For C25H22N4O6S3: C, 70.24; H,
5.89; N, 16.38. Found: C, 70.25; H, 5.92; N, 16.43.
5.2.6.5.
3-Fluoro-4-(8-(7-methyl-1H-indazole-5-carbonyl)-3-oxo-2,8-diazaspiro[4.5]decan-2-yl)benzonitri
1
le (6e) White solid; yield 65%; m.p.: 241–243°C; H NMR (300 MHz, DMSO-d6) δ 13.37 (s, 1H),
8.15 (s, 1H), 8.10 (d, J = 6.9 Hz, 1H), 7.87 (d, J = 6.8 Hz, 1H), 7.66 (s, 1H), 7.59 (t, J = 9.6 Hz, 1H),
7.18 (s, 1H), 3.72 (s, 2H), 3.52 (s, 4H), 2.56 (s, 3H), 2.52 (s, 2H), 1.71 (s, 4H). 13C NMR (75 MHz,
DMSO-d6) δ 172.4, 169.6, 160.8, 157.4, 140.1, 134.5, 132.7, 131.8, 131.8, 128.5, 127.7, 124.7, 121.8,
120.1, 118.4, 118.1, 117.5, 116.9, 107.8, 107.8, 58.8, 58.7, 36.1, 16.7. ESIMS m/z [M + H]+ 432.1;
Anal. calcd. For C24H22FN5O2: C, 66.81; H, 5.14; N, 16.23. Found: C, 66.79; H, 5.15; N, 16.21.
5.2.6.6.
8-(2-Methyl-1H-benzo[d]imidazole-5-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspiro[4.5]
decan-3-one (6g) White solid; yield 75%; m.p.: 203-205°C; 1H NMR (300 MHz, DMSO-d6) δ 12.47
(s, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H), 7.62 – 7.41 (m, 2H), 7.15 (dd, J = 8.3, 1.5
Hz, 1H), 3.77 (s, 2H), 3.46 (s, 4H), 2.57 (s, 2H), 2.50 (s, 3H), 1.64 (s, 4H). 13C NMR (75 MHz,
DMSO-d6) δ 173.1, 169.8, 153.1, 142.8, 129.1, 125.84, 125.8, 125.7, 123.9, 123.5, 122.5, 120.4, 119.1,
57.6, 43.6, 34.3, 14.6. ESIMS m/z [M + H]+ 457.1; Anal. calcd. For C24H23F3N4O2: C, 63.15; H,
5.08; N, 12.27. Found: C, 63.21; H, 5.04; N, 12.29.
5.2.6.7.
8-(7-Methyl-2-propyl-1H-benzo[d]imidazole-5-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazas
piro[4.5]decan-3-one (6h) White solid; yield 85%; m.p.: 234-236°C; 1H NMR (300 MHz,
DMSO-d6) δ 7.88 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H), 7.32 (s, 1H), 6.97 (s, 1H), 3.78 (s, 2H),
3.55 (d, J = 44.3 Hz, 4H), 2.80 (t, J = 7.5 Hz, 2H), 2.58 (s, 2H), 2.50 (s, 3H), 1.80 (q, J = 7.4 Hz, 2H),
1.65 (s, 4H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 173.1, 167.0, 156.0, 142.8,
129.0, 126.1, 125.8, 125.8, 123.9, 123.5, 120.7, 119.1, 57.6, 43.6, 34.3, 30.5, 21.0, 16.7, 13.7. ESIMS
m/z [M + H]+ 499.2; Anal. calcd. For C27H29F3N4O2: C, 65.05; H, 5.86; N, 11.24. Found: C, 65.01;
H, 5.82; N, 11.25.

5.2.6.8. 8-(1H-Indole-5-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspiro[4.5]decan-3-one
(6i) White solid; yield 78%; m.p.: 2432-234°C;1H NMR (300 MHz, DMSO-d6) δ 11.27 (s, 1H), 7.89
(d, J = 8.6 Hz, 2H), 7.73 (d, J = 8.6 Hz, 2H), 7.66 – 7.58 (m, 1H), 7.53 – 7.40 (m, 2H), 7.13 (dd, J =
8.3, 1.6 Hz, 1H), 6.49 (ddd, J = 3.0, 1.9, 0.9 Hz, 1H), 3.78 (s, 2H), 3.57 (d, J = 43.6 Hz, 4H), 2.59 (s,
2H), 1.65 (d, J = 6.7 Hz, 4H). 13C NMR (75 MHz, DMSO-d6) δ 173.1, 170.5, 142.8, 136.2, 126.9,
126.6, 126.5, 125.8, 125.8, 125.8, 125.7, 123.9, 123.4, 120.2, 119.2, 119.1, 111.1, 101.6, 57.6, 43.6,
34.3, 14.0. ESIMS m/z [M + H]+ 442.2; Anal. calcd. For C24H22F3N3O2: C, 65.30; H, 5.02; N, 9.52.
Found: C, 65.33; H, 5.01; N, 9.55.
5.2.6.9. 8-(1H-Indole-6-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspiro[4.5]decan-3-one
1
(6j) White solid; yield 68%; m.p.: 229-231°C; H NMR (300 MHz, DMSO-d6) δ 11.30 (s, 1H), 7.89
(d, J = 8.6 Hz, 2H), 7.74 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.1 Hz, 1H), 7.50 – 7.42 (m, 2H), 7.03 (dd, J
= 8.1, 1.4 Hz, 1H), 6.54 – 6.44 (m, 1H), 3.79 (s, 2H), 3.58 (d, J = 51.8 Hz, 4H), 2.60 (s, 2H), 1.66 (s,
4H). 13C NMR (75 MHz, DMSO-d6) δ 173.1, 170.3, 142.8, 135.0, 128.5, 128.4, 127.1, 125.9, 125.8,
125.8, 125.7, 119.6, 119.1, 117.8, 110.5, 101.1, 57.6, 43.6, 34.3. ESIMS m/z [M + H]+ 442.1; Anal.
calcd. For C24H22F3N3O2: C, 65.30; H, 5.02; N, 9.52. Found: C, 65.31; H, 5.05; N, 9.51.
5.2.6.10.
8-(1H-Indazole-6-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspiro[4.5]decan-3-one
(6k)
White solid; yield 61%; m.p.: 244–246°C; 1H NMR (300 MHz, DMSO-d6) δ 13.22 (s, 1H), 8.13 (s,
1H), 7.88 (d, J = 8.5 Hz, 2H), 7.82 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 8.6 Hz, 2H), 7.54 (s, 1H), 7.11 (dd,
J = 8.3, 1.3 Hz, 1H), 3.79 (s, 2H), 3.49 (s, 4H), 2.59 (s, 2H), 1.68 (s, 4H). 13C NMR (75 MHz,
DMSO-d6) δ 173.1, 169.2, 142.8, 139.1, 133.8, 133.6, 125.9, 125.8, 125.8, 125.7, 123.0, 120.7, 119.1,
118.9, 108.5, 57.6, 43.6, 34.2. ESIMS m/z [M + H]+ 443.3; Anal. calcd. For C23H21F3N4O2: C, 62.44;
H, 4.78; N, 12.66. Found: C, 62.45; H, 4.75; N, 12.69.
5.2.6.11. 8-(Quinoline-7-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspiro[4.5]decan-3-one
(6l) White solid; yield 84%; m.p.: 237-239°C; 1H NMR (300 MHz, DMSO-d6) δ 8.97 (d, J = 3.9 Hz,
1H), 8.42 (d, J = 8.3 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 8.00 (s, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.73 (d, J
= 8.6 Hz, 2H), 7.60 (dd, J = 8.4, 4.5 Hz, 2H), 3.80 (s, 2H), 3.52 (d, J = 67.8 Hz, 3H), 2.60 (s, 2H), 1.71
(s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 173.1, 168.2, 151.4, 147.0, 142.8, 137.1, 136.0, 128.6, 128.1,
126.6, 125.9, 125.8, 125.8, 125.7, 124.8, 122.3, 119.1, 57.6, 43.5, 34.2. ESIMS m/z [M + H]+ 454.1;
Anal. calcd. For C25H22F3N3O2: C, 66.22; H, 4.89; N, 9.27. Found: C, 66.21; H, 4.87; N, 9.25.
5.2.6.12. 8-(Quinoline-6-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspiro[4.5]decan-3-one
(6m) White solid; yield 77%; m.p.: 216–218°C; 1H NMR (300 MHz, DMSO-d6) δ 8.96 (dd, J = 4.2,
1.7 Hz, 1H), 8.44 (dd, J = 8.5, 1.7 Hz, 1H), 8.17 – 7.98 (m, 2H), 7.89 (d, J = 8.6 Hz, 2H), 7.84 – 7.67
(m, 3H), 7.59 (dd, J = 8.3, 4.2 Hz, 1H), 3.80 (s, 2H), 3.44 (s, 4H), 2.60 (s, 2H), 1.71 (s, 4H). 13C NMR
(75 MHz, DMSO-d6) δ 173.1, 168.4, 151.5, 147.5, 142.8, 136.5, 134.1, 129.2, 127.8, 127.3, 126.4,
126.1, 125.8, 125.8, 123.9, 123.5, 122.1, 119.1, 57.5, 43.6, 34.2. ESIMS m/z [M + H]+ 454.2; Anal.
calcd. For C25H22F3N3O2: C, 66.22; H, 4.89; N, 9.27. Found: C, 66.23; H, 4.85; N, 9.22.

5.2.6.13. 8-(Quinoline-3-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspiro[4.5]decan-3-one
(6n) White solid; yield 74%; m.p.: 239-241°C; 1H NMR (300 MHz, DMSO-d6) δ 8.92 (d, J = 2.2 Hz,
1H), 8.45 (d, J = 2.2 Hz, 1H), 8.09 – 8.01 (m, 2H), 7.95 – 7.83 (m, 3H), 7.70 (dd, J = 18.6, 8.3 Hz, 3H),
3.80 (s, 4H), 3.49 (s, 4H), 2.61 (s, 2H), 1.72 (s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 173.0, 166.7,
148.4, 147.4, 142.8, 134.3, 130.6, 129.1, 128.7, 128.6, 127.3, 126.6, 125.8, 125.8, 119.1, 57.5, 43.5,
34.2. ESIMS m/z [M + H]+ 454.1; Anal. calcd. For C25H22F3N3O2: C, 66.22; H, 4.89; N, 9.27.
Found: C, 66.27; H, 4.84; N, 9.27.
5.2.6.14.
8-(2-Phenyl-1H-benzo[d]imidazole-5-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspiro[4.5]
decan-3-one (6o) White solid; yield 80%; m.p.: 212-214°C; 1H NMR (300 MHz, DMSO-d6) δ 13.11
(s, 1H), 8.29 – 8.10 (m, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.7 Hz, 3H), 7.55 (q, J = 9.1, 7.9 Hz,
4H), 7.27 (d, J = 9.4 Hz, 1H), 3.79 (s, 2H), 3.59 (d, J = 46.5 Hz, 4H), 2.60 (s, 2H), 1.68 (s, 4H). 13C
NMR (75 MHz, DMSO-d6) δ 173.1, 169.6, 142.8, 140.1, 134.5, 128.4, 125.8, 125.8, 125.7, 125.7,
124.7, 121.8, 120.2, 119.0, 116.9, 57.6, 43.5, 34.2, 16.7. ESIMS m/z [M + H]+ 519.1; Anal. calcd.
For C29H25F3N4O2: C, 67.17; H, 4.86; N, 10.80. Found: C, 67.13; H, 4.88; N, 10.81.
5.2.6.15.
8-(2-(4-Isopropylphenyl)-1H-benzo[d]imidazole-5-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-di
1
azaspiro[4.5]decan-3-one (6p) White solid; yield 77%; m.p.: 197-199°C; H NMR (300 MHz,
DMSO-d6) δ 13.15 (s, 1H), 8.13 (d, J = 8.0 Hz, 2H), 7.88 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 8.6 Hz, 2H),
7.69 – 7.57 (m, 2H), 7.42 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 8.3 Hz, 1H), 3.77 (s, 2H), 3.51 (s, 4H), 2.96
(dd, J = 13.9, 7.1 Hz, 1H), 2.59 (s, 2H), 1.67 (s, 4H), 1.24-1.24 (m, 6H). 13C NMR (75 MHz, DMSO-d6)
δ 173.1, 169.7, 152.7, 150.6, 142.7, 127.4, 126.8, 126.6, 125.8, 125.8, 125.7, 125.7, 119.0, 57.6, 43.5,
34.2, 33.3, 31.0, 29.7, 28.9, 23.5. ESIMS m/z [M + H]+ 561.1; Anal. calcd. For C32H31F3N4O2: C,
68.56; H, 5.57; N, 9.99. Found: C, 68.59; H, 5.58; N, 9.98.
5.2.6.16.
8-(2-(Pyridin-4-yl)-1H-benzo[d]imidazole-5-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspi
ro[4.5]decan-3-one (6q) White solid; yield 83%; m.p.: 222-224°C; 1H NMR (300 MHz, DMSO-d6)
δ 13.51 (s, 1H), 8.78 (d, J = 5.1 Hz, 2H), 8.20 – 8.03 (m, 2H), 7.89 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 8.7
Hz, 4H), 7.32 (d, J = 8.4 Hz, 1H), 3.79 (s, 2H), 3.51 (s, 4H), 2.60 (s, 2H), 1.69 (s, 4H). 13C NMR (75
MHz, DMSO-d6) δ 173.1, 169.4, 150.5, 150.2, 142.8, 136.7, 125.8, 125.7, 123.8, 122.7, 121.2, 120.3,
119.0, 117.9, 111.8, 110.6, 57.6, 43.5, 34.2, 31.4, 31.2, 31.0, 28.9. ESIMS m/z [M + H]+ 520.1; Anal.
calcd. For C28H24F3N5O2: C, 64.73; H, 4.66; N, 13.48. Found: C, 64.75; H, 4.67; N, 13.49.
5.2.6.17.
8-(2-(Furan-2-yl)-1H-benzo[d]imidazole-5-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspir
o[4.5]decan-3-one (6r) White solid; yield 65%; m.p.: 221-223°C;1H NMR (300 MHz, DMSO-d6) δ
13.18 (s, 1H), 7.97 (d, J = 1.7 Hz, 1H), 7.89 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 8.7 Hz, 2H), 7.60 (d, J =
8.1 Hz, 2H), 7.25 (p, J = 3.0, 2.5 Hz, 2H), 6.75 (dd, J = 3.5, 1.8 Hz, 1H), 3.79 (s, 2H), 3.51 (s, 4H),
2.60 (s, 2H), 1.68 (s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 173.1, 169.5, 145.1, 144.9, 144.8, 142.8,
130.0, 126.0, 125.8, 125.7, 123.8, 123.4, 122.4, 119.0, 112.3, 111.0, 57.6, 43.6, 34.2, 31.0, 29.7, 28.9.
ESIMS m/z [M + H]+ 509.1; Anal. calcd. For C27H23F3N4O3: C, 63.78; H, 4.56; N, 11.02. Found:

C, 63.79; H, 4.57; N, 11.01.
5.2.6.18.
8-(2,3,4,9-Tetrahydro-1H-carbazole-6-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspiro[4.5
]decan-3-one (6s) White solid; yield 80%; m.p.: 204–205°C; 1H NMR (300 MHz, DMSO-d6) δ
10.89 (s, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.5 Hz, 2H), 7.40 (s, 1H), 7.27 (d, J = 8.2 Hz, 1H),
7.05 (dt, J = 8.2, 1.6 Hz, 1H), 3.78 (s, 2H), 3.57 (d, J = 50.5 Hz, 4H), 2.75 – 2.54 (m, 6H), 1.81 (s, 4H),
1.65 (s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 173.1, 170.8, 142.8, 136.0, 135.8, 126.5, 125.84, 125.7,
125.7, 119.3, 119.0, 116.4, 110.0, 108.7, 57.6, 43.6, 34.3, 22.8, 22.7, 20.4. ESIMS m/z [M + H]+ 496.2;
Anal. calcd. For C28H28F3N3O2: C, 67.87; H, 5.70; N, 8.48. Found: C, 67.89; H, 5.74; N, 8.49.
5.2.6.19.
8-(1,2,3,4-Tetrahydrocyclopenta[b]indole-7-carbonyl)-2-(4-(trifluoromethyl)phenyl)-2,8-diazaspir
o[4.5]decan-3-one (6t) White solid; yield 85%; m.p.: 196-198°C; 1H NMR (300 MHz, DMSO-d6) δ
11.03 (s, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.5 Hz, 2H), 7.35 (s, 1H), 7.29 (d, J = 8.3 Hz, 1H),
7.01 (dd, J = 8.2, 1.7 Hz, 1H), 3.76 (s, 2H), 3.46 (s, 4H), 2.80 (t, J = 6.4 Hz, 2H), 2.71 (d, J = 7.2 Hz,
2H), 2.57 (s, 2H), 2.49 (p, J = 1.9 Hz, 2H), 1.63 (s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 173.1, 170.7,
145.6, 142.8, 141.3, 126.2, 125.8, 125.7, 125.7, 123.3, 119.0, 118.8, 118.1, 117.0, 111.1, 57.6, 43.6,
34.3, 28.2, 25.2, 23.8. ESIMS m/z [M + H]+ 482.2; Anal. calcd. For C27H26F3N3O2: C, 67.35; H,
5.44; N, 8.73. Found: C, 67.39; H, 5.47; N, 8.73.
5.2.6.20. 4-(8-(7-Methyl-1H-indazole-5-carbonyl)-3-oxo-2,8-diazaspiro[4.5]decan-2-yl)benzoic
acid（6f）
A solution of this compound 6a (100 mg, 0.22 mmol) in MeOH/water (1:1) (10 mL) was
mixed with NaOH (2 M in water, 2 mL). The mixture was heated at 50 ° C for 1 h. The mixture
was then cooled and MeOH was evaporated. The resultant was diluted with water, and acidified
with 2 M aqueous HCl. The title compound was obtained by filtered as a white solid (81.4 mg,
92% yield). m.p.: 217-219°C; 1H NMR (300 MHz, DMSO-d6) δ 13.30 (s, 1H), 8.13 (s, 1H), 7.95 (d, J
= 8.5 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.64 (s, 1H), 7.16 (s, 1H), 3.78 (s, 2H), 3.49 (s, 4H), 2.59 (s,
2H), 2.54 (s, 3H), 1.66 (s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 172.9, 169.6, 166.9, 143.1, 140.2,
134.4, 130.0, 128.5, 125.8, 124.7, 121.8, 120.2, 118.4, 116.9, 57.7, 43.6, 34.2, 16.7. ESIMS m/z [M +
H]+ 433.1; Anal. calcd. For C24H24N4O4: C, 66.65; H, 5.59; N, 12.96. Found: C, 66.69; H, 5.64; N,
12.92.
5.3. Biological evaluation
5.3.1. In Vitro ACC1 and ACC2 Inhibition Assay.
We commissioned Pharmaron Beijing Co. Ltd., to carry out the experiments. Assay kit used
is ADP-Glo™ Kinase Assay from Promega. The ADP-Glo™ Kinase Assay is a luminescent ADP
detection assay to measure enzymatic activity by quantifying the amount of ADP produced during
the enzymatic first half-reaction. Specifically, 4.5 µL of assay buffer containing either recombinant
hACC1 (BPS Bioscience, Catalog no. 50200) or recombinant hACC2 (BPS Biosciences catalog
no. 50201) were added to the wells of a 384-well Optiplate (PerkinElmer, 6007290) followed by
0.5 µL of DMSO or DMSO containing inhibitor. After 15 min incubation at room temperature, 5
µL of substrate solution was added to each well to start reaction. Final assay concentrations were 1

nM hACC1 or 0.5 nM hACC, 20 µM ATP, 10 µM (hACC1 assay) or 20 µM (hACC2 assay)
acetyl-CoA, 30 mM (hACC1 assay) or 12 mM (hACC2 assay) NaHCO3, 0.01% Brij35, 2 mM
DTT, 5% DMSO, inhibitor in half-log increments between 100 µM and 0.0017 µM[18]. After 60
min incubation at room temperature, 10 µL ADP-Glo Reagent was added to terminate the reaction,
and plates were incubated at room temperature for 40 min to deplete remaining ATP. Then Kinase
Detection Reagent, 20 µL, was added, and plates were incubated for 40 min at room temperature
to convert ADP to ATP. ATP was measured via a luciferin/luciferase reaction using a PerkinElmer
EnVision 2104 plate reader to assess luminescence.
4.2. Docking studies
For docking purposes, the crystal complex (PDB id: 4WYO) was recovered from RCSB
Protein Data Bank. The docking studies were processed with the Glide docking protocol.
Hydrogen atoms were added to the structure and the water was removed. Then a 60-Å box
centered on the geometrical center of the ligand binding site was generated for grid calculation.
All ligand molecules were drawn in ChemDraw 2014, and saved as sdf style. Then ligands were
processed at a simulated pH of 7.4 1.0 to generate all possible tautomers, stereoisomers, and
protonation states and were finally minimized at the OPLS 2005 force field with Ligand
preparation protocol of Maestro 10.2. After docking finished, only one docking conformation was
saved for every compound.
4.3.2. Cell culture and proliferation inhibition assays.
The anti-proliferation activities of selected compounds were evaluated against A549, H1975,
HCT116, SW620 and Caco-2 cell lines in vitro using a standard MTT assay, with PF-1 as the
positive controls. The tumor cells were seeded in 96-well plates at a concentration of 1 × 104 cells
per well and cultured in RPMI 1640 medium containing 10% (v/v) fetal bovine serum, 100 U
penicillin/mL and 100 mg streptomycin/mL under 5% CO2 at 37°C for 24 h. Then 50 µL of which
containing various concentrations of compounds (triple diluted) was added and the cells were
incubated for a further 48 h in FBS-free media. MTT solution was added to each well at the
terminal concentration (0.5 mg/mL) followed by incubation for 4 h at 37°C. Living cells
containing MTT formazan crystals were solubilized in 200 µL DMSO. The spectrophotometric
absorbance of each well was measured by a multi-detection microplate reader at a wavelength of
450 nm. The inhibition rate was calculated as ((A450 treated - A450 blank) / (A450 control - A450
blank)) × 100. The results, expressed as IC50 values, was calculated by GraphPad Prism 5
statistical software.
Acknowledgements
This study was supported by the Natural Science Foundation of Jiangsu Province (No. BK
20141349) and the China National Key HiTech Innovation Project for the R&D of Novel Drugs
(No.2013ZX09301303-002).
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