Synthesis and biological evaluation of 4-phenoxy-phenyl isoxazoles as novel acetyl-CoA carboxylase inhibitors

Article abstract of DOI:10.1080/14756366.2021.1936514

Acetyl-CoA carboxylase (ACC) is a crucial enzyme in fatty acid metabolism, which plays a major role in the occurrence and development of certain tumours. Herein, one potential ACC inhibitor (6a) was identified through high-throughput virtual screening (HTVS), and a series of 4-phenoxy-phenyl isoxazoles were synthesised for structure-activity relationship (SAR) studies. Among these compounds, 6g exhibited the most potent ACC inhibitory activity (IC₅₀=99.8 nM), which was comparable to that of CP-640186. Moreover, the antiproliferation assay revealed that compound 6l exhibited the strongest cytotoxicity, with IC₅₀ values of 0.22 μM (A549), 0.26 μM (HepG2), and 0.21 μM (MDA-MB-231), respectively. The preliminary mechanistic studies on 6g and 6l suggested that the compounds decreased the malonyl-CoA levels, arrested the cell cycle at the G0/G1 phase, and induced apoptosis in MDA-MB-231 cells. Overall, these results indicated that the 4-phenoxy-phenyl isoxazoles are potential for further study in cancer therapeutics as ACC inhibitors.

Full text of DOI:10.1080/14756366.2021.1936514

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of 4-phenoxy-
phenyl isoxazoles as novel acetyl-CoA carboxylase
inhibitors
Xin Wu, Yongbo Yu & Tonghui Huang
To cite this article: Xin Wu, Yongbo Yu & Tonghui Huang (2021) Synthesis and
biological evaluation of 4-phenoxy-phenyl isoxazoles as novel acetyl-CoA carboxylase
inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 1236-1247, DOI:
10.1080/14756366.2021.1936514
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 1236–1247
https://doi.org/10.1080/14756366.2021.1936514
RESEARCH PAPER
Synthesis and biological evaluation of 4-phenoxy-phenyl isoxazoles as novel
acetyl-CoA carboxylase inhibitors
Xin Wu, Yongbo Yu and Tonghui Huang
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, China
ABSTRACT
ARTICLE HISTORY
Received 13 April 2021
Revised 12 May 2021
Accepted 22 May 2021
Acetyl-CoA carboxylase (ACC) is a crucial enzyme in fatty acid metabolism, which plays a major role in the
occurrence and development of certain tumours. Herein, one potential ACC inhibitor (6a) was identified
through high-throughput virtual screening (HTVS), and a series of 4-phenoxy-phenyl isoxazoles were syn-
thesised for structure-activity relationship (SAR) studies. Among these compounds, 6g exhibited the most
potent ACC inhibitory activity (IC50¼99.8 nM), which was comparable to that of CP-640186. Moreover, the
antiproliferation assay revealed that compound 6l exhibited the strongest cytotoxicity, with IC50 values of
0.22 mM (A549), 0.26 mM (HepG2), and 0.21 mM (MDA-MB-231), respectively. The preliminary mechanistic
studies on 6g and 6l suggested that the compounds decreased the malonyl-CoA levels, arrested the cell
cycle at the G0/G1 phase, and induced apoptosis in MDA-MB-231 cells. Overall, these results indicated
that the 4-phenoxy-phenyl isoxazoles are potential for further study in cancer therapeutics as
ACC inhibitors.
KEYWORDS
Acetyl-CoA carboxylase;
antitumor; docking; cell
cycle; apoptosis
1. Introduction
Since the first discovery of the mammalian ACC inhibitor CP-
640186 in 2003 (Figure 1), a large amount of ACC inhibitors have
been identified, some of which have even been approved in clin-
ical trials for the treatment of glycolipid metabolic diseases, such
as type 2 diabetes mellitus (T2DM) and non-alcoholic steatohepa-
titis (NASH)19–21. However, ACC inhibitors with effective anticancer
activities were rarely reported. And most of these inhibitors con-
tain thienopyrimidinone or spirocyclic scaffolds, among which the
spiroketopyrazoles (IV) were previously identified by the author to
Fatty acids are one of the essential biological macromolecules for
cell growth and survival, which are widely involved in energy sup-
ply, membrane biosynthesis, and signal transduction1,2. In contrast
to normal cells that preferentially use circulating lipids to satisfy
their requirement for fatty acids, malignant cells primarily undergo
high rates of de novo fatty acid synthesis (FASyn) to support their
continuous proliferation and division3,4. Therefore, elevated FASyn
is accepted as a major characteristic of numerous cancers, includ-
ing those in the prostate, colon, breast, lung, and liver. The drugs
that intervene with the enzymes in FASyn may provide an innova-
display potent ACC1 inhibitory activity and cytotoxicity22–26
.
Therefore, an investigation into the generation of novel ACC
inhibitors was conducted to further expand our research on anti-
tumor agents and increase the diversity of ACC inhibitors. During
the initial stage of this work, compound 6a (Figure 2), an initial
high-throughput virtual screening (HTVS) hit with moderate ACC1
inhibitory potency (57% inhibition at 5 mM), was selected as a lead
compound. Later, the structure–activity relationship (SAR) study of
4-phenoxy-phenyl isoxazoles was performed with the belief that
these new chemical entities would effectively target the ACC
enzyme and provide a new approach for cancer treatment.
Herein, starting from the HTVS hit 6a, a series of 4-phenoxy-
phenyl isoxazole derivatives were identified. The p-benzyloxy
group of 6a was substituted with various alkoxy groups to reduce
the structural rigidity and increase the flexibility of target com-
pounds. Furthermore, diverse amide and urea substituents were
introduced to improve lipophilicity and increase the number of
hydrogen bond donors. All of the compounds were evaluated for
biological activity towards ACC1 enzyme and three different can-
cer cell lines (A549, HepG2, and MDA-MB-231). The preliminary
docking and pharmacological studies of the representative
tive approach for the fight against cancer5–8
.
Acetyl-CoA carboxylase (ACC), a crucial enzyme in FASyn, has
two subcellular specific isoforms, namely ACC1 and ACC2, which
catalyse the carboxylation of acetyl-CoA to malonyl-CoA and dis-
play distinct physiological roles9. ACC1 is a cytosolic enzyme that
mainly controls the FASyn process, with its product malonyl-CoA
extends the fatty acids chain by two carbon increments under the
catalysis of fatty acid synthase10. In contrast, the mitochondrial
isoform ACC2 is primarily responsible for fatty acid oxidation
(FAOxn) through the inhibition of carnitine palmitoyltransferase I
(CPT-1) by localised malonyl-CoA production11. Thus, the func-
tional abnormalities on ACC could provide a viable modality for
disturbing the energy metabolism and causing cell damage, which
facilitates the utilisation of ACC as an attractive therapeutic tar-
get12,13. Currently, cancer therapeutics mainly focuses on the
ACC1 isoform due to the over-expression of ACC1 mRNA in most
human cancers14–18
.
CONTACT Tonghui Huang
tonghhuang@xzhmu.edu.cn
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou
Medical University, Xuzhou 221004, China
Supplemental data for this article is available online at here.
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1237
Figure 1. CP-640186 and representative ACC inhibitors with anticancer activity.
Figure 2. The design strategy of 4-phenoxy-phenyl isoxazoles as novel ACC inhibitors.
compounds were carried out to elucidate the possible action
mechanism of 4-phenoxy-phenyl isoxazoles.
Table 1. The screening results and biological activity of selected candidates.
Compounds
Total scorea
Crasha
Polara
hACC1 IRs (%)b
C450-0875
C522-2022
D074-0032
D098-1432 (6a)
D400-1402
D400-3199
E667-1377
F292-0452
F697-0546
CP-640186
7.37
9.71
7.99
8.07
6.41
6.77
8.84
7.77
9.04
7.18
ꢁ1.58
ꢁ1.21
ꢁ4.43
ꢁ1.25
ꢁ1.13
ꢁ1.05
ꢁ1.91
ꢁ0.95
ꢁ0.81
ꢁ1.05
1.82
4.04
2.70
2.08
0.35
0.99
4.50
2.43
2.62
2.08
13.53
2.84
14.98
57.39
3.44
6.93
12.56
6.98
20.53
86.83
2. Results and discussion
2.1. High-throughput virtual screening
To identify potent ACC inhibitors with new structural skeletons,
1,500,000 commercially available compounds from the ChemDiv
database were docked into the active site of ACC in silico27,28
.
However, the co-crystal structure of ACC inhibitor and hACC1 has
not been resolved. Therefore, we selected the co-crystal structure
of CP-640186 and hACC2 (PDB ID: 3FF6) for HTVS due to the high
degree of identity between ACC1 and ACC2 at the nucleotide
level (60%) and the amino acid level (75%). Based on the docking
results, 500 top-ranked compounds with high total scores were
selected for the second round of screening, which mainly focussed
aTotal score, crash, and polar values were calculated using Sybyl-X 2.1 software.
bInhibition rates (IRs) of compounds for hACC1 enzyme at 5 lM.
To brief, the commercially available 4-fluorobenzaldehyde was
reacted with 1 (phenol or 4-(benzyloxy)phenol) in the presence of
on the binding modes and interactions between the candidates K2CO3 as the base at 120 ꢀC to afford intermediate 2. Then, the
and the active site28. Finally, nine potential hits with new skele-
addition–elimination reactions of 2 and hydroxylamine readily
tons were obtained. Their corresponding properties are summar-
ised in Table 1 (structures are provided in Figure S1). Thereafter,
the selected compounds were purchased from the commercial
vendors and evaluated for their ACC inhibitory activity using a
luminescent ADP detection assay24. As shown in Table 1, com-
pound 6a emerged as a top candidate, exhibiting the strongest
inhibition rate at 5 mM. Thus, compound 6a was selected as a lead
compound, and its benzyloxy and acetyl groups were modified for
SAR studies (Figure 2).
generated the aldoxime 3, which was subsequently chlorinated
with N-chlorosuccinimide (NCS) to give the corresponding chlor-
oaldoxime 4. The [3 þ 2] dipolar cycloaddition of 4 and 2-(but-3-
yn-2-yl)isoindoline-1,3-dione provided the key intermediate 5 in
good yields (5a, 86%; 5b, 80%).
The phthalimide protecting group of 5a was removed by the
Ing-Manske reaction, and the resulting primary amine was con-
densed with acetyl chloride to afford the final compound 6a.
Later, compound 6b was generated via Pd/C-catalysed hydroge-
nolysis. Moreover, the nucleophilic substitution of 6b and appro-
priate iodoalkane using caesium carbonate as catalyst gave the
target compounds 6c–6g. The synthetic routes of the final com-
pounds 6h–6q were similar to that of 6a, namely by the reaction
2.2. Chemistry
The 4-phenoxy-phenyl isoxazole derivatives (6a–6q) were synthes-
ised according to the pathways described in Schemes 1 and 2. of deprotected 5b with various acyl chlorides or isocyanates.

1238
X. WU ET AL.
Scheme 1. Synthetic route of key intermediate 5a and 5b. Reagents and conditions: (a) 4-fluorobenzaldehyde, K2CO3, DMF, 120ꢀC, 12 h; (b) hydroxylamine hydrochlor-
ide, NaOH, H2O/EtOH (3:1), 0 ꢀC to r.t., 2 h; (c) NCS, DMF, r.t., 2 h; (d) 2-(but-3-yn-2-yl)isoindoline-1,3-dione, K2CO3, toluene, reflux, 12 h.
Scheme 2. Synthetic route of target compounds 6a–6q. Reagents and conditions: (a) hydrazine hydrate, CH2Cl2/EtOH (5:1), reflux, 2 h; (b) appropriate acyl chloride or
isocyanate, TEA, CH2Cl2, 0 ꢀC to r.t., 6 h; (c) H2, Pd-C, MeOH, r.t., 3 h; (d) appropriate iodoalkane, Cs2CO3, DMF, reflux, 12 h.
preliminary screening was carried out at two concentrations (5 mM
2.3. Biological evaluation
and 0.1 mM), where CP-640186 was selected as the positive con-
2.3.1. In vitro ACC inhibitory activity
trol29. As shown in Table 2, the derivatives 6b–6d with short-chain
alkoxy groups at R1 exhibited weaker hACC1 inhibitory activity
To explore the SAR of 4-phenoxy-phenyl isoxazoles, the in vitro
enzymatic inhibitory effects of 6a–6q were evaluated. The than 6a. In contrast, the propoxy-substituted 6e displayed 78.95

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1239
Table 2. The hACC1 inhibitory activity and lipophilicity of compounds 6a–6q.
IRs (%)a
Compounds
5 lM
0.1 lM
IC50 (nM)b
NT
NT
NT
cLogPc
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
54.93
26.83
37.32
45.46
78.95
52.76
95.26
36.37
51.83
58.36
72.47
81.42
16.77
44.72
49.82
65.73
73.68
87.84
18.13
8.76
9.89
5.27
3.31
3.66
4.14
4.62
4.40
4.74
3.57
4.07
4.13
5.00
5.08
4.27
4.02
4.36
5.21
5.18
3.59
12.73
31.12
16.69
51.37
8.78
18.51
20.79
28.11
37.86
3.57
13.36
17.90
21.52
28.27
48.87
NT
438.2 16.3
NT
99.8 3.7
NT
NT
NT
626.3 41.6
279.1 13.1
NT
NT
NT
1055.0 179.7
711.1 52.6
108.9 6.2
Figure 3. The dose–response curves of selected compounds on hACC1 enzyme.
weakest on HepG2 cells. Besides, most of the target compounds
displayed significant inhibitory effects on cell proliferation. Among
them, compounds 6a, 6g, and 6l exhibited inhibitory rates greater
than 80% against the three cancer cell lines, which were compar-
able to that of DOX.
6q
CP-640186
aThe data represent the mean values of two independent experiments.
bIC50 values for ACC are presented as mean SD. NT: not test.
bcLogP values were calculated by Molsoft online software.
Based on the good initial screening results, the IC50 values and
calculated logarithm of the octanol-water partition coefficient
(cLogp) of 4-phenoxy-phenyl isoxazoles were determined for fur-
ther SAR studies30. As shown in Table 3, when R2 was fixed as a
methyl group, the derivatives 6a–6h exhibited varying degrees of
antiproliferative activity. Among them, the compounds 6e and 6g
with good ACC inhibitory activities exhibited satisfactory antitu-
mor effects, in which 6g displayed the best inhibition on A549,
HepG2, and MDA-MB-231 cells, with IC50 values of 1.10, 1.73, and
1.50 mM, respectively. Besides, compound 6a with moderate ACC
activity and high lipophilicity showed considerable antiprolifera-
tive activity on the three cancer cell lines. However, the cytotox-
icity of the derivatives (6b–6d, 6h) with relatively weak enzymatic
activity and low lipophilicity decreased remarkably (IC50 >10 mM).
On the other hand, when R1 was fixed as a hydrogen atom
(6h–6q), most of the synthesised compounds exhibited potent in
vitro antiproliferative activity. Of the five amide derivatives (6h–6l),
compound 6l showed the best potency in cancer treatment, with
IC50 values of 0.22, 0.26, and 0.21 mM for A549, HepG2, and MDA-
MB-231 cells, respectively, which were apparently superior to
DOX. The outstanding cell activity of 6l was supposed to be
attributed to its optimal balance between the enzymatic activity
and lipophilicity. Besides, the ureido-substituted compounds
(6n–6q) exhibited remarkable performances towards the inhibition
of MDA-MB-231 cells. The IC50 values of 6p (0.84 mM) and 6q
(0.89 mM) against MDA-MB-231 cells were both less than 1 mM. In
contrast, the carbamate-substituted 6m only provided a faint anti-
tumor effect, suggesting that the carbamate group was detrimen-
tal to bioactivity.
and 31.12% inhibition towards hACC1 at 5 and 0.1 mM, respect-
ively, which indicated that the increase of the chain length is
beneficial to the inhibition of ACC. Among the analogues with dif-
ferent substituents at R1 (6a–6h), compound 6g appeared to be
the most potential ACC inhibitor, with inhibition rates (IRs) of
95.26 and 51.37% at 5 mM and 0.1 mM, respectively.
For 4-phenoxy-phenyl isoxazoles with various amide substitu-
ents at R2 (6h–6l), the analogues bearing aromatic ring showed
better enzymatic activity, wherein compound 6l exhibited the
strongest ACC inhibitory effect (IRs ¼ 81.42 and 37.86%), which
could be partly attributed to its bulky phenylacetamide group.
Besides, the IRs of compound 6q with the benzylurea group (IRs
¼ 73.68 and 28.27%) were the highest among the ureido-substi-
tuted derivatives (6n–6q), although slightly lower than that of the
corresponding amide derivative 6l. The inhibitory activity of the
carbamate-substituted compound 6m was hardly observed. These
results suggested that the introduction of flexible alkoxy groups
(such as propoxy and cyclopropylmethoxy, 6e and 6g) at the left-
hand hydrophobic terminus, or the incorporation of bulky func-
tional groups (such as phenylacetamide and benzylurea, 6l and
6q) at the right-hand polar end of the molecule was conducive to
the inhibition of ACC.
Based on the above results, the IC50 values of the preferred
compounds (6e, 6g, 6k, 6l, 6p and 6q) with IRs greater than 60%
at 5 mM were calculated and summarised in Table 2. Similar to the
preliminary screening results, the cyclopropylmethoxy-substituted
derivative 6g exhibited the optimal ACC inhibitory activity among
the synthesised compounds. The IC50 value of 6g on the hACC1
enzyme was 99.8 nM, which was lower than that of CP-640186
(IC50¼108.9 nM). The dose-response curves of the selected com-
pounds were plotted, as presented in Figure 3.
To investigate the selectivity of the synthesised compounds
towards cancer cells, the non-cancerous HUVEC cell line was
selected for testing24. As shown in Table 2, most of the com-
pounds exhibited weak toxic to HUVEC. Moreover, the compounds
with strong ACC inhibitory and antitumor activities, namely 6e,
6g, 6k, 6l, 6p and 6q, showed less than 20% IRs towards HUVEC,
which indicated the cancer cell selectivity of ACC inhibitors.
Overall, the SAR studies revealed that the ACC inhibitory activity
and lipophilicity of the target compounds play an important role
in their antiproliferative activity.
2.3.2. In vitro cytotoxic activity
The cytotoxicity of 6a–6q on three different cancer cell lines with
a high expression of ACC1 (A549, HepG2, and MDA-MB-231) was
determined using the MTT assay24. The initial screening was per-
formed at a single concentration of 10 mM, where doxorubicin
(DOX) was served as a reference drug. As illustrated in Figure S2, 2.3.3. Effect on intracellular malonyl-CoA levels
the 4-phenoxyphenylisoxazoles showed the best antiproliferative Due to the most potent ACC1 inhibitory activity of 6g and
activity on MDA-MB-231 cells, followed by A549 cells, and the strongest antitumor activity of 6l, these compounds were

1240
X. WU ET AL.
selected for further biological studies. Moreover, MDA-MB-231 to the control group at the maximum test concentration,
was chosen as the model cell line due to the best antiprolifera-
tive effect of 4-phenoxy-phenyl isoxazoles on these cells. As
illustrated in Figure 4(A), compounds 6g and 6l inhibited the
proliferation of MDA-MB-231 cells in a concentration- and time-
dependent manner. Later, the effects of 6g and 6l on the levels
of malonyl-CoA in MDA-MB-231 cells were investigated. The
results suggested that the treatment of the cells with 6g and
6l significantly decreased the intracellular malonyl-CoA levels,
respectively (Figure 4(B)).
2.3.4. Cell-cycle assay
The cell-cycle arrest creates a stopping point at which the cells no
longer participate in the process of duplication and division that
represents an important approach for cancer treatment31,32. To
investigate the cell-cycle progression, a flow cytometric analysis
with an approximate reduction of 23.59 and 25.85% compared was performed on the MDA-MB-231 cells treated with various
Table 3. In vitro anti-proliferative activity of compounds 6a–6q.
IC50 (lM)a
Compounds
A549
HepG2
MDA-MB-231
Cell viability (%) of HUVEC at 10 lMb
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
DOX
1.40 0.22
>10
>10
>10
2.66 0.47
>10
1.10 0.12
>10
6.02 0.63
2.66 0.47
2.61 0.34
0.22 0.07
>10
5.78 0.86
3.12 0.19
5.30 0.46
5.73 0.11
0.52 0.07
1.91 0.66
>10
>10
>10
3.48 0.74
>10
1.73 0.55
>10
4.21 0.53
3.48 0.74
3.23 0.89
0.26 0.02
>10
9.04 1.43
2.31 0.44
1.80 0.62
2.47 0.50
0.67 0.13
1.57 0.09
>10
>10
45.79 1.16
88.14 3.83
82.39 4.85
89.06 6.09
83.68 4.40
34.96 1.66
87.33 6.19
91.53 2.58
70.33 0.25
59.46 0.95
80.00 2.47
89.54 3.21
79.12 4.76
83.23 2.33
77.65 5.67
85.60 7.35
84.92 3.98
18.95 4.02
>10
1.53 0.38
6.77 0.74
1.50 0.58
>10
2.97 1.49
1.13 0.38
0.57 0.18
0.21 0.01
>10
8.63 1.14
1.90 0.52
0.84 0.08
0.89 0.26
0.42 0.09
aIC50 values for A549, HepG2, and MDA-MB-231 cells are presented as mean SD (n ¼ 3).
bCell viability was evaluated after incubation of compounds at a concentration of 10 lM.
Figure 4. (A) Concentration- and time-dependent cytotoxicity of compounds 6g and 6l. The MDA-MB-231 cells were treated with drugs at increasing concentrations
for 12, 24, 36, 48, 60, and 72 h, respectively. (B) Effects of 6g and 6l on the levels of malonyl-CoA in MDA-MB-231 cells. MDA-MB-231 cells were treated with diverse
ꢂ
ꢂꢂ
concentrations of drugs for 48 h, with the intracellular malonyl-CoA quantified. Data were expressed as mean SD (n ¼ 3). p < 0.05, p < 0.01 versus control group.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1241
Figure 5. The effect of compounds 6g (A) and 6l (B) on cell-cycle distribution. MDA-MB-231 cells were treated with 6g and 6l at the indicated concentrations for 48 h,
ꢂꢂ ꢂꢂꢂ
ꢂ
and stained with propidium iodide for flow cytometry. Data are expressed as mean SD (n ¼ 3). p < 0.05, p < 0.01, and p < 0.001 versus control group.
concentrations (1/2 IC50, IC50, and 2 ꢃ IC50) of 6g and 6l for 48 h. and 20.25%, respectively, as depicted in Figure 6. In addition,
As shown in Figure 5, compound 6g led to an obvious increase in
the cells at the G0/G1 phase from 55.32 to 79.97%, accompanied
by a decrease in the cells at the S and G2/M phases from 30.93
and 13.76% to 10.08 and 9.96%, respectively. Moreover, the treat-
ment of the MDA-MB-231 cells with increasing concentrations of
6l (0.1, 0.2, and 0.4 mM) increased the proportion of cells at the
G0/G1 phase from 53.32% to 81.93%, while the proportion of cells
at the S and G2/M phases decreased. These results confirmed that
6g and 6l induced cell-cycle arrest at the G0/G1 phase in a dose-
dependent manner, which may be a possible mechanism for their
antitumor activity.
compound 6l induced early (from 1.55 to 12.20%) and late apop-
tosis (from 1.11 to 17.50%) in a dose-dependent manner. These
data suggested that the antitumor activity of compounds 6g and
6l might be involved in the induction of apoptosis.
2.4. Molecular docking
To gain insight into the specific interactions between the target
compounds and ACC enzyme, molecular docking studies of the
representative compounds (6a and 6g) were conducted using
the co-crystal structure of ACC and CP-640186 (PDB ID: 3FF6) as
the template27. As illustrated in Figure 7, compounds 6a and 6g
were located at the dimer interface of the carboxyltransferase (CT)
domain of ACC and exhibited different binding modes. Compared
with CP-640186, 6a formed one conserved hydrogen bond with
Gly-2162 (2.1 Å), and one additional hydrogen bond between the
oxygen of oxazole and Ala-2125 (2.3 Å) was observed (Figure
7(A,B)). However, there was no hydrogen bond between 6a and
Glu-2230, which partly explained its moderate ACC inhibitory
activity. The substitution of a cyclopropylmethoxy group at R1
(6g) led to the movement of the binding site and formed three
2.3.5. Cell apoptosis assay
Apoptosis is a form of programmed cell death, which plays an
important role in many cancer treatment strategies33,34
. To
explore the relationship between the antiproliferative effects of
4-phenoxy-phenyl isoxazoles and cell apoptosis, Annexin V and
propidium iodide (PI) double staining was performed on the
vehicle- and drug-treated MDA-MB-231 cells. Upon incubation
with compound 6g (0.75, 1.5, and 3 mM) for 48 h, the total per-
centage of the early (bottom right quadrant) and late (upper right
quadrant) apoptotic cells was increased from 2.34 to 8.34, 13.83, hydrogen bonds between 6g and the CT domain (Figure 7(C)).

1242
X. WU ET AL.
Figure 6. The effect of compounds 6g (A) and 6l (B) on cell apoptosis. MDA-MB-231 cells were incubated with 6g and 6l at the indicated concentrations for 48 h, and
ꢂꢂ
ꢂꢂꢂ
p < 0.001 versus control group.
stained with Annexin V-FITC/PI for flow cytometry. Data are expressed as mean SD (n ¼ 3). p < 0.01 and
To our surprise, 6g and CP-640186 did not have the same hydro- MDA-MB-231 cells, respectively, which were superior to that of
gen bond interaction with ACC (Figure 7(D)). In detail, the Thr-
1960 residue formed two hydrogen bonds with the nitrogen
(2.8 Å) and oxygen atoms (2.0 Å) of oxazole, and the Phe-2160 resi-
due formed one hydrogen bond with the nitrogen of amide
(1.9 Å). The stronger interaction of 6g with ACC than that of 6a
might account for its better activity in enzyme inhibition.
DOX. The best antitumor activity of compound 6l might be attrib-
uted to its optimal balance between the enzymatic activity and lip-
ophilicity. Furthermore, treatment of the MDA-MB-231 cells with 6g
and 6l significantly decreased the intracellular malonyl-CoA levels.
The flow cytometry analysis confirmed that 6g and 6l arrested cell
cycle at the G0/G1 phase and induced apoptosis in a dose-depend-
ent manner. Overall, these results indicated that the 4-phenoxy-
phenyl isoxazoles exhibited potent anticancer activity as novel ACC
inhibitors, which demonstrated the causal relationship between
ACC inhibition and loss of cell viability.
3. Conclusions
In this study, a series of structurally novel 4-phenoxy-phenyl isoxa-
zoles were synthesised and evaluated for their antiproliferative
activities towards a panel of human cancer cell lines as ACC inhibi-
tors. The lead compound 6a was discovered from HTVS, and further
SAR studies resulted in the identification of several derivatives with
potent biological activity. Among them, compound 6g, which
formed three hydrogen bonds with the CT domain of ACC, exhib-
ited the most potent hACC1 inhibitory activity (IC50¼99.8 nM).
Compound 6l displayed the strongest antiproliferative activity, with
4. Experimental
4.1. Chemistry
All reagents and solvents were purchased from commercial sour-
ces without further purification. The reaction progress was moni-
tored by TLC on Silica Gel 60 F254 plates, and the target
IC50 values of 0.22, 0.26, and 0.21 mM against A549, HepG2, and compounds were purified by column chromatography with silica

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1243
Figure 7. Molecular docking studies for compounds 6a and 6g (PDB ID: 3FF6). (A) Docking model of compound 6a (green) with the active site of ACC. (B)
Overlapping conformation of 6a (green) with CP-640186 (cyan). (C) Docking model of compound 6g (yellow) with the active site of ACC. (D) Overlapping conformation
of 6g (yellow) with CP-640186 (cyan). All hydrogen bonds are shown with black dashed lines, and related amino acids are highlighted with magenta sticks.
gel (200–300 mesh size). Melting points were measured in open provide 4. Finally, 2-(But-3-yn-2-yl)isoindoline-1,3-dione (10 mmol),
4 (11 mmol), and K2CO3 (30 mmol) was dissolved in toluene
(50 ml) and heated at reflux for 12 h. After cooling to room tem-
perature, the mixture was filtered, concentrated, and purified by a
silica gel flash column to give the key intermediate 5.
capillary tubes using YRT-3 melting point apparatus and were
uncorrected. 1H NMR and 13 C NMR spectra were recorded on
JNM-ECZR 400 MHz spectrometer with tetramethyl silane (TMS) as
an internal standard. High-resolution mass spectra (HRMS) were
obtained from a (UHR-TOF) maXis 4 G instrument. Analytical HPLC
was run on the Agilent 1260 HPLC instrument, equipped with
Agilent SB-C18 column (Agilent Technologies, Palo Alto, CA) and
UV detection at 250 nm. Eluent system was: 70% MeOH in H2O;
flow rate ¼ 0.2 ml/min.
4.1.1.1. 2-(1-(3-(4-(4-(benzyloxy)phenoxy)phenyl)isoxazol-5-yl)ethyl)
isoindoline-1,3-dione (5a). White solid, yield 70%, m.p.
160.5–162.5 ꢀC. 1H NMR (400 MHz, CDCl3): d 7.87–7.85 (m, 2H),
7.77–7.67 (m, 4H), 7.49–7.31 (m, 5H), 7.06–6.92 (m, 6H), 6.55 (d,
J ¼ 0.6 Hz, 1H), 5.70 (q, J ¼ 7.3 Hz, 1H), 5.06 (s, 2H), 1.93 (d,
J ¼ 7.3 Hz, 3H). 13 C NMR (100 MHz, CDCl3): d 170.6, 167.3, 161.9,
160.1, 155.4, 149.4, 136.8, 134.3, 131.7, 128.6, 128.3, 128.0, 127.5,
123.5, 122.9, 121.2, 117.5, 116.0, 100.3, 70.5, 42.3, 16.8. HRMS m/z
calcd for C32H24N2O5 [M þ Na]þ 539.1577, found 539.1577.
4.1.1. General procedure for the preparation of key intermedi-
ate 5a–b
To a solution of phenol or 4-benzyloxy phenol (1, 27.5 mmol) in
DMF (25 ml), K2CO3 (50 mmol) and 4-fluorobenzaldehyde
(27.5 mmol) was added. After stirring at 120 ꢀC for 12 h, the mix-
ture was poured into water (100 ml) and extracted with ethyl acet-
ate (3 ꢃ 25 ml). The combined organics were dried over anhydrous
Na2SO4 and concentrated to give 2. Then, hydroxylamine hydro-
chloride (20 mmol) and 50% NaOH aqueous solution (40 mmol)
was slowly added to a solution of 2 (20 mmol) in H2O/ethanol
(3:1, 20 ml) at 0 ꢀC. After stirring at room temperature for 2 h, the
mixture was acidified to pH 6 by concentrated HCl and extracted
with ethyl acetate (3 ꢃ 5 ml). The organic phases were dried over
anhydrous Na2SO4 and concentrated in vacuo to afford 3. To a
solution of oxime 3 (20 mmol) in DMF (25 ml), N-chlorosuccinimide
(22 mmol) was added slowly at ambient temperature, the solution
was stirred for 2 h before being poured into ice water (100 ml).
4.1.1.2. 2-(1-(3-(4-Phenoxyphenyl)isoxazol-5-yl)ethyl)isoindoline-1,3-
dione (5b). White solid, yield 72%, m.p. 133.5–134.5 ꢀC. 1H NMR
(400 MHz, CDCl3): d 7.87–7.84 (m, 2H), 7.78–7.72 (m, 4H), 7.40–7.33
(m, 2H), 7.15 (t, J ¼ 7.4 Hz, 1H), 7.08–7.01 (m, 4H), 6.57 (s, 1H), 5.70
(q, J ¼ 7.0 Hz, 1H), 1.94 (d, J ¼ 7.3 Hz, 3H). 13 C NMR (100 MHz,
CDCl3): d 170.7, 167.3, 161.9, 159.0, 156.3, 134.3, 131.7, 129.9,
128.4, 123.9, 123.6, 119.5, 118.6, 100.3, 42.3, 16.8. HRMS m/z calcd
for C26H24N2O4 [M þ Na]þ 433.1159, found 433.1167.
4.1.2. General procedure for the preparation of target com-
pounds 6a–6q
(A) Hydrazine monohydrate (8 mmol) was added dropwise to a
The resulting precipitate was collected by filtration and dried to solution of 5 (1.2 mmol) in methylene chloride/ethanol (5:1, 12 ml)

1244
X. WU ET AL.
and refluxed for 2 h. The mixture was extracted with methylene 4.1.2.5. N-(1-(3-(4-(4-Propoxyphenoxy)phenyl)isoxazol-5-yl)ethyl)a-
1
cetamide (6e). White solid, yield 68%, m.p. 118.5–119.5 ꢀC. H NMR
chloride (3 ꢃ 10 ml), dried over anhydrous Na2SO4, and concen-
trated in vacuo to give corresponding amines. The amine was dis-
(400 MHz, CDCl3): d 7.71 (t, J ¼ 8.2 Hz, 2H), 6.99 (t, J ¼ 7.7 Hz, 4H),
solved in methylene chloride (8 ml) under an ice-bath. Then,
triethylamine (3 mmol) and appropriate acyl chloride or isocyanate
(1 mmol) were added sequentially at 0 ꢀC, with the mixture stirred
6.91 (d, J ¼ 8.8 Hz, 2H), 6.40 (s, 1H), 5.95 (d, J ¼ 9.3 Hz, 1H),
5.47–5.28 (m, 1H), 3.92 (t, J ¼ 4.5 Hz, 2H), 2.04 (s, 3H), 1.88–1.75 (m,
2H), 1.58 (d, J ¼ 5.9 Hz, 3H), 1.06 (t, J ¼ 6.2 Hz, 3H). 13 C NMR
for 6 h. After removal of the solvent, the residue was purified by
column chromatography to yield target compounds 6a
and 6h–6q.
(B) To a solution of 6a (10 mmol) in anhydrous methanol
(60 ml), Pd-C catalyst (10%, 1.5 mmol) was added, and the mixture
was hydrogenated for 3 h at atmospheric pressure. Upon comple-
tion of the reaction, the catalyst was removed by filtration and
the solvent was evaporated to give 6b.
(C) A mixture of 6b (1 mmol), Cs2CO3 (2 mmol), and corre-
sponding iodoalkane (1.2 mmol) in DMF (10 ml) was stirred at
reflux for 12 h. After cooling to room temperature, the mixture
was poured into ice water (50 ml) and extracted with methylene
dichloride (3 ꢃ 10 ml). The combined organic phases were dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by column chromatography to
afford target compounds 6c–6g.
(100 MHz, CDCl3): d 172.9, 169.5, 162.1, 160.5, 156.0, 149.1, 128.4,
121.4, 117.9, 117.5, 115.7, 99.2, 70.1, 42.3, 23.3, 22.7, 19.6, 10.6.
HRMS m/z calcd for C22H24N2O4 [M þ Na]þ 403.1628, found
403.1633. Purity: 99.1%.
4.1.2.6. N-(1-(3-(4-(4-Isopropoxyphenoxy)phenyl)isoxazol-5-yl)ethyl)
acetamide (6f). White solid, yield 65%, m.p. 124–126 ꢀC. 1H NMR
(400 MHz, CDCl3): d 7.71 (t, J ¼ 8.9 Hz, 2H), 7.07–6.81 (m, 6H), 6.40
(s, 1H), 5.95 (d, J ¼ 6.8 Hz, 1H), 5.49–5.25 (m, 1H), 4.51 (s, 1H), 2.04
(s, 3H), 1.58 (d, J ¼ 5.4 Hz, 3H), 1.37 (dd, J ¼ 15.2, 5.6 Hz, 6H). 13 C
NMR (100 MHz, CDCl3): d 172.9, 169.5, 162.1, 160.4, 154.7, 149.1,
128.4, 121.4, 118.1, 117.6, 117.3, 99.1, 70.6, 42.2, 23.3, 22.2, 19.6.
HRMS m/z calcd for C22H24N2O4 [M þ Na]þ 403.1628, found
403.1633. Purity: 98.8%.
4.1.2.7. N-(1-(3-(4-(4-(Cyclopropylmethoxy)phenoxy)phenyl) isoxa-
zol-5-yl)ethyl)acetamide (6g). White solid, yield 50%, m.p.
4.1.2.1. N-(1-(3-(4-(4-(Benzyloxy)phenoxy)phenyl)isoxazol-5-yl) ethyl)
acetamide (6a). White solid, yield 70%, m.p. 152.5–153.5 ꢀC. 1H
NMR (400 MHz, DMSO-d6): d 7.74–7.65 (m, 2H), 7.49–7.31 (m, 5H),
7.05–6.94 (m, 6H), 6.39 (s, 1H), 5.94 (d, J ¼ 8.2 Hz, 1H), 5.44–5.33
(m, 1H), 5.06 (s, 2H), 2.03 (s, 3H), 1.57 (d, J ¼ 7.1 Hz, 3H). 13 C NMR
(100 MHz, DMSO-d6): d 173.0, 169.5, 162.0, 160.3, 155.6, 149.6,
137.0, 128.7, 128.4, 128.2, 127.6, 123.0, 121.4, 117.6, 116.1, 99.1,
70.6, 42.3, 23.3, 19.6. HRMS m/z calcd for C26H24N2O4 [M þ Na]þ
451.1628, found 451.1630. Purity: 99.3%.
1
131.5–132.5 ꢀC. H NMR (400 MHz, CDCl3): d 7.71 (t, J ¼ 9.0 Hz, 2H),
7.14–6.81 (m, 6H), 6.40 (s, 1H), 5.89 (d, J ¼ 8.4 Hz, 1H), 5.55–5.24
(m, 1H), 3.84 (dd, J ¼ 33.2, 5.9 Hz, 2H), 2.04 (s, 3H), 1.58 (d,
J ¼ 6.7 Hz, 3H), 1.40–1.19 (m, 1H), 0.73–0.57 (m, 2H), 0.45–0.30 (m,
2H). 13 C NMR (100 MHz, CDCl3): d 172.9, 169.4, 162.0, 160.4, 155.9,
149.2, 128.4, 121.4, 118.0, 117.5, 115.84, 99.1, 73.4, 42.3, 23.3, 19.6,
10.4, 3.3. HRMS m/z calcd for C23H24N2O4 [M þ Na]þ 415.1628,
found 415.1634. Purity: 99.3%.
4.1.2.8. N-(1-(3-(4-Phenoxyphenyl)isoxazol-5-yl)ethyl)acetamide (6h).
White solid, yield 85%, m.p. 118.5–119.5 ꢀC. 1H NMR (400 MHz,
CDCl3): d 7.76–7.68 (m, 2H), 7.40–7.32 (m, 2H), 7.19–7.11 (m, 1H),
7.07–7.00 (m, 4H), 6.40 (s, 1H), 6.01 (d, J ¼ 71.1 Hz, 1H), 5.45–5.31
(m, 1H), 2.03 (s, 3H), 1.57 (d, J ¼ 7.1 Hz, 3H). 13 C NMR (100 MHz,
CDCl3): d 173.0, 169.4, 161.9, 159.2, 156.3, 129.9, 128.4, 124.0,
123.5, 119.6, 118.6, 99.1, 42.2, 23.2, 19.5. HRMS m/z calcd for
C19H18N2O3 [M þ Na]þ 345.1210, found 345.1225. Purity: 96.9%.
4.1.2.2. N-(1-(3-(4-(4-Hydroxyphenoxy)phenyl)isoxazol-5-yl)ethyl)a-
cetamide (6b). White solid, yield 65%, m.p. 103–105 ꢀC. 1H NMR
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 13.5, 8.3 Hz, 2H), 7.12–6.79 (m,
6H), 6.40 (s, 1H), 5.90 (d, J ¼ 16.1 Hz, 1H), 5.46–5.31 (m, 1H), 2.05
(s, 3H), 1.59 (d, J ¼ 5.9 Hz, 3H). 13 C NMR (100 MHz, CDCl3): 13 C
NMR (101 MHz,) d 172.7, 170.1, 162.1, 160.6, 153.2, 148.7, 128.4,
121.6, 117.9, 117.4, 116.6, 99.3, 42.4, 23.3, 19.5. HRMS m/z calcd
for C19H18N2O4 [M
þ
Na]þ 361.1159, found 361.1162.
Purity: 99.2%.
4.1.2.9. N-(1-(3-(4-Phenoxyphenyl)isoxazol-5-yl)ethyl)propionamide
1
(6i). White solid, yield 83%, m.p. 129.5–130.5 ꢀC. H NMR (400 MHz,
4.1.2.3. N-(1-(3-(4-(4-Methoxyphenoxy)phenyl)isoxazol-5-yl)ethyl)a-
1
cetamide (6c). White solid, yield 70%, m.p. 127.5–128.5 ꢀC. H NMR
CDCl3): d 7.78–7.68 (m, 2H), 7.40–7.36 (m, 2H), 7.21–7.12 (m, 1H),
7.09–7.00 (m, 4H), 6.41 (s, 1H), 5.87 (d, J ¼ 7.8 Hz, 1H), 5.46–5.35
(m, 1H), 2.26 (q, J ¼ 7.6 Hz, 2H), 1.59 (d, J ¼ 7.1 Hz, 3H), 1.18 (t,
J ¼ 7.6 Hz, 3H). 13 C NMR (100 MHz, CDCl3): d 173.1, 173.1, 162.0,
159.3, 156.4, 130.0, 128.5, 124.1, 123.7, 119.6, 118.7, 99.1, 42.1,
29.6, 19.6, 9.7. HRMS m/z calcd for C20H20N2O3 [M þ Na]þ
359.1366, found 359.1369. Purity: 98.2%.
(400 MHz, CDCl3): d 7.81–7.60 (m, 2H), 7.03–6.91 (m, 6H), 6.40 (s,
1H), 5.97 (d, J ¼ 6.2 Hz, 1H), 5.50–5.28 (m, 1H), 3.82 (s, 3H), 2.03 (s,
3H), 1.58 (d, J ¼ 6.4 Hz, 3H). 13 C NMR (100 MHz, CDCl3): d 172.9,
169.4, 162.0, 160.4, 156.4, 149.3, 128.4, 121.4, 118.0, 117.5, 115.1,
99.1, 55.8, 42.2, 23.3, 19.6. HRMS m/z calcd for C20H20N2O4 [M þ
Na]þ 375.1315, found 375.1321. Purity: 98.4%.
4.1.2.10. N-(1-(3-(4-Phenoxyphenyl)isoxazol-5-yl)ethyl)cyclopropa-
necarboxamide (6j). White solid, yield 75%, m.p. 168.5–169.5 ꢀC.
1H NMR (400 MHz, CDCl3): d 7.79–7.70 (m, 2H), 7.44–7.33 (m, 2H),
7.18–7.14 (m, 1H), 7.10–7.01 (m, 4H), 6.41 (s, 1H), 6.09 (d,
J ¼ 8.3 Hz, 1H), 5.45–5.38 (m, 1H), 1.60 (d, J ¼ 7.1 Hz, 3H), 1.43–1.32
(m, 1H), 1.06–0.94 (m, 2H), 0.81–0.77 (m, 2H). 13 C NMR (100 MHz,
CDCl3): d 173.3, 162.0, 159.2, 156.4, 130.0, 128.5, 124.1, 123.7,
4.1.2.4. N-(1-(3-(4-(4-Ethoxyphenoxy)phenyl)isoxazol-5-yl)ethyl)ace-
tamide (6d). White solid, yield 72%, m.p. 106.5–107.5 ꢀC. 1H NMR
(400 MHz, CDCl3): d 7.83–7.58 (m, 2H), 7.01–6.89 (m, 6H), 6.40 (s,
1H), 5.98 (d, J ¼ 5.8 Hz, 1H), 5.50–5.25 (m, 1H), 4.07 (q, J ¼ 12.0 Hz,
2H), 2.03 (s, 3H), 1.58 (d, J ¼ 5.9 Hz, 3H), 1.43 (t, J ¼ 6.3 Hz, 3H). 13 C
NMR (100 MHz, CDCl3): d 172.9, 169.5, 162.1, 160.4, 155.8, 149.1,
128.4, 121.4, 118.0, 117.5, 115.7, 99.1, 64.0, 42.2, 23.3, 19.6, 15.0.
HRMS m/z calcd for C21H22N2O4 [M þ Na]þ 389.1472, found 119.6, 118.7, 99.1, 42.3, 19.7, 14.8, 7.7. HRMS m/z calcd for
389.1477. Purity: 97.4%.
C21H20N2O3 [M þ Na]þ 371.1366, found 371.1367. Purity: 100%.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1245
4.1.2.11. N-(1-(3-(4-Phenoxyphenyl)isoxazol-5-yl)ethyl)benzamide 159.0, 156.9, 156.3, 130.8, 129.0, 124.7, 124.1, 120.0, 119.0, 99.0,
(6k). White solid, yield 72%, m.p. 155.5–156.5 ꢀC. 1H NMR
42.7, 41.5, 23.7, 23.7, 20.6. HRMS m/z calcd for C24H21N3O3 [M þ
Na]þ 422.1475, found 422.1480. Purity: 96.7%.
(400 MHz, CDCl3): d 7.80 (dd, J ¼ 5.3, 3.3 Hz, 2H), 7.76–7.71 (m, 2H),
7.56–7.50 (m, 1H), 7.48–7.42 (m, 2H), 7.40–7.34 (m, 2H), 7.19–7.12
(m, 1H), 7.07–7.03 (m, 4H), 6.52 (d, J ¼ 7.7 Hz, 1H), 6.47 (s, 1H),
4.1.2.17. 1-Benzyl-3-(1-(3-(4-phenoxyphenyl)isoxazol-5-yl)ethyl)urea
5.64–5.57 (m, 1H), 1.70 (d, J ¼ 7.1 Hz, 3H). 13 C NMR (100 MHz,
(6q). White solid, yield 76%, m.p. 161.5–162.5 ꢀC. 1H NMR
CDCl3): d 173.0, 166.8, 162.1, 159.3, 156.4, 133.8, 132.0, 130.0,
128.8, 128.5, 127.2, 124.1, 123.6, 119.6, 118.7, 99.3, 42.8, 19.7.
HRMS m/z calcd for C24H20N2O3 [M þ Na]þ 407.1366, found
407.1368. Purity: 99.0%.
(400 MHz, DMSO-d6): d 7.93–7.78 (m, 2H), 7.54–7.39 (m, 2H),
7.35–7.19 (m, 6H), 7.14–7.05 (m, 4H), 6.78 (s, 1H), 6.64 (d,
J ¼ 8.3 Hz, 1H), 6.42 (t, J ¼ 6.0 Hz, 1H), 5.07–4.99 (m, 1H), 4.32–4.14
(m, 2H), 1.45 (d, J ¼ 7.1 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6): d
176.4, 161.6, 159.0, 157.7, 156.3, 141.2, 130.8, 129.0, 128.8, 127.6,
127.2, 124.7, 124.1, 120.0, 119.0, 99.1, 43.5, 43.0, 20.5. HRMS m/z
calcd for C25H23N3O3 [M þ Na]þ 436.1632, found 436.1636.
Purity: 98.9%.
4.1.2.12. N-(1-(3-(4-Phenoxyphenyl)isoxazol-5-yl)ethyl)-2-phenyla-
1
cetamide (6l). White solid, yield 70%, m.p. 164.5–165.5 ꢀC. H NMR
(400 MHz, CDCl3): d 7.70 (d, J ¼ 8.6 Hz, 2H), 7.41–7.26 (m, 7H), 7.16
(t, J ¼ 7.3 Hz, 1H), 7.05 (dd, J ¼ 11.0, 4.4 Hz, 4H), 6.27 (s, 1H), 5.78
(d, J ¼ 8.2 Hz, 1H), 5.41–5.33 (m, 1H), 3.62 (s, 2H), 1.50 (d,
J ¼ 7.0 Hz, 3H). 13 C NMR (100 MHz, CDCl3): d 172.9, 170.4, 161.9,
159.3, 156.4, 134.5, 130.0, 129.5, 129.2, 128.5, 127.7, 124.1, 123.6,
119.7, 118.7, 99.0, 43.8, 42.4, 19.4. HRMS m/z calcd for C25H22N2O3
[M þ Na]þ 421.1523, found 421.1527. Purity: 96.7%.
4.2. HTVS
The ChemDiv database was provided by Topscience Co., and the
HTVS was performed using the Surflex docking module in Sybyl-X
2.128. The crystal complex of ACC and CP-640186 (PDB ID: 3FF6)
was downloaded from the Protein Data Bank. To improve the effi-
ciency of screening, the maximum quantity of conformations was
reduced from 20 to 10, and the maximum quantity of rotatable
bonds was decreased from 100 to 50, with other parameters kept
as usual. Compounds with high total-scores (top 500) were
extracted to observe their binding modes and interactions with
the CT domain. Finally, nine candidates with new scaffolds were
obtained. The cLogp values and drug-likeness scores were calcu-
lated by Molsoft software (http://www.molsoft.com/mprop/).
4.1.2.13. Ethyl (1-(3-(4-phenoxyphenyl)isoxazol-5-yl)ethyl)carba-
mate (6m). White solid, yield 70%, m.p. 106.5–107.5 ꢀC. 1H NMR
(400 MHz, CDCl3): d 7.74 (d, J ¼ 7.5 Hz, 2H), 7.38 (dd, J ¼ 9.2, 4.2 Hz,
2H), 7.29–7.23 (m, 1H), 7.16 (dd, J ¼ 7.7, 6.8 Hz, 1H), 7.10–6.99 (m,
4H), 6.56–6.34 (m, 1H), 5.27–4.83 (m, 1H), 4.15 (q, J ¼ 8.6 Hz, 2H),
1.59 (d, J ¼ 3.0 Hz, 3H), 1.26 (t, J ¼ 6.5 Hz, 3H). 13 C NMR (100 MHz,
CDCl3): d 173.5, 161.9, 159.2, 156.4, 155.7, 130.0, 128.5, 124.1,
123.7, 119.6, 118.7, 98.9, 61.4, 44.2, 19.9, 14.6. HRMS m/z calcd for
C25H22N2O3 [M þ Na]þ 421.1523, found 421.1527. HRMS m/z calcd
for C20H20N2O4 [M
Purity: 99.4%.
þ
Na]þ 375.1315, found 375.1320.
4.3. ACC inhibitory assay
The hACC1 inhibitory activity of target compounds was evaluated
using the ADP-GloTM kinase assay according to our previous
report24. In brief, 4.5 mL of working solution containing recombin-
ant ACC1 (BPS Biosciences, 50200) and 0.5 mL of diluted com-
pound solution were added to a 384-well Optiplate plate (Perkin
Elmer, 6007290, PerkinElmer, Waltham, MA, USA), and incubated
at room temperature for 15 min. Then, 5 mL of substrate mixture
was added to the 384-well plate to start the reaction. After incu-
bating for 60 min at room temperature, 10 mL of ADP-Glo reagents
were added to the 384-well plate, and incubated for another
40 min to deplete the remaining ATP. Finally, 20 mL of kinase
detection reagents were added to the plate and incubated for
40 min to convert ADP to ATP. The luminescent signal of ATP was
determined using an Envision multifunction reader (Perkin Elmer,
2104–0010, PerkinElmer, Waltham, MA, USA). The IC50 values were
calculated using GraphPad Prism 5 software (GraphPad Software,
La Jolla, CA, USA).
4.1.2.14. 1-Ethyl-3-(1-(3-(4-phenoxyphenyl)isoxazol-5-yl)ethyl)urea
(6n). White solid, yield 80%, m.p. 158.5–159.5 ꢀC. 1H NMR
(400 MHz, DMSO-d6): d 7.91–7.80 (m, 2H), 7.50–7.37 (m, 2H),
7.27–7.17 (m, 1H), 7.15–7.04 (m, 4H), 6.78 (s, 1H), 6.48 (d,
J ¼ 8.3 Hz, 1H), 5.87 (t, J ¼ 5.6 Hz, 1H), 5.03–4.96 (m, 1H), 3.03 (q,
J ¼ 3.6 Hz, 2H), 1.42 (d, J ¼ 7.1 Hz, 3H), 1.00 (t, J ¼ 7.2 Hz, 3H). 13 C
NMR (100 MHz, DMSO-d6): d 176.5, 161.6, 159.0, 157.5, 156.3,
130.8, 129.0, 124.7, 124.1, 120.0, 119.0, 99.0, 42.8, 34.7, 20.5, 16.2.
HRMS m/z calcd for C20H21N3O3 [M þ Na]þ 374.1475, found
374.1480. Purity: 98.6%.
4.1.2.15. 1-Isopropyl-3-(1-(3-(4-phenoxyphenyl)isoxazol-5-yl)ethyl)
urea (6o). White solid, yield 75%, m.p. 197.5–198.5 ꢀC. 1H NMR
(400 MHz, DMSO-d6): d 7.91–7.79 (m, 2H), 7.48–7.39 (m, 2H),
7.25–7.18 (m, 1H), 7.16–7.03 (m, 4H), 6.77 (s, 1H), 6.34 (d,
J ¼ 8.4 Hz, 1H), 5.75 (d, J ¼ 7.7 Hz, 1H), 5.08–4.91 (m, 1H), 3.72–3.64
(m, 1H), 1.42 (d, J ¼ 7.1 Hz, 3H), 1.04 (dd, J ¼ 6.5, 1.8 Hz, 6H). 13 C
NMR (100 MHz, DMSO-d6): d 176.4, 161.6, 159.0, 156.9, 156.3,
130.8, 129.0, 124.7, 124.1, 120.0, 119.0, 99.0, 42.7, 41.5, 23.7, 23.7,
20.6. HRMS m/z calcd for C21H23N3O3 [M þ Na]þ 388.1632, found
388.1638. Purity: 100%.
4.4. Molecular docking
The docking study was carried out with the Sybyl-X 2.1 software,
and the crystal complex of ACC and CP-640186 (PDB ID: 3FF6)
was downloaded from the Protein Data Bank. The whole waters
and superfluous ligands were removed, and hydrogen atoms were
added to the original protein structure. For the docked ligands,
they were added with non-polar hydrogens and further energy
was minimised with the Tripos force field. Then, the optimised
ligands were docked to the prepared protein using a Surflex-Dock
4.1.2.16.
1-(1-(3-(4-Phenoxyphenyl)isoxazol-5-yl)ethyl)-3-phenyl-
urea (6p). White solid, yield 73%, m.p. 195.5–196.5 ꢀC. 1H NMR
(400 MHz, DMSO-d6): d 8.48 (s, 1H), 7.94–7.81 (m, 2H), 7.50–7.35
(m, 4H), 7.22 (dd, J ¼ 16.1, 7.8 Hz, 3H), 7.14–7.05 (m, 4H), 6.96–6.86
(m, 2H), 6.80 (d, J ¼ 8.1 Hz, 1H), 5.12–5.07 (m, 1H), 1.51 (d, protocol. The optimal binding pose of the ligand was determined
J ¼ 7.0 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6): d 176.4, 161.6, by the total score and its interaction with ACC. The best docking

1246
X. WU ET AL.
conformations of 6a and 6g, as well as their corresponding over-
lapping conformations with CP-640186 was visualised by PyMOL.
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Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This study was supported by National Natural Science Foundation
of China (81671744), Natural Science Foundation of Jiangsu
Province (BK20171184), and Science and Technology Planning
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