Design, synthesis and bioactivity evaluation of novel arylalkene-amide derivatives as dual-target antifungal inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112645

Ergosterol as the core component of fungal cell membrane plays a key role in maintaining cell morphology and permeability. The squalenee epoxidase (SE) and 14-demethylase (CYP51) are the important rate-limiting enzymes for ergosterol synthesis. In the study, these active fragments, which is derived from the structural groups of the common antifungal agents, were docked into the active sites of dual targets (SE, CYP51), respectively. Some of active fragments with the matching MCSS_Score values were selected and connected to construct three different series of novel arylalkene-amide derivatives as dual-target (SE, CYP51) antifungal inhibitors. Subsequently, these compounds were further synthesized, and their bioactivity was evaluated. Most of compounds showed a certain degree of antifungal activity in vitro. It was worth noting that the target compounds 17a and 25a with excellent antifungal activity (0.125–4 μg/mL) can inhibit the fluconazole-resistant Candida Strain 17#, CaR, 632, and 901 in the range of MIC values (4–8 μg/mL). Furthermore, their molecular mechanism, structural stability and low toxicity were further confirmed. The molecular docking and ADMET properties were predicted to guide the subsequent optimization of target compounds.

Full text of DOI:10.1016/j.ejmech.2020.112645

Journal Pre-proof
Design, synthesis and bioactivity evaluation of novel arylalkene-amide derivatives as
dual-target antifungal inhibitors
Bin Sun, Yue Dong, Yunfei An, Min Liu, Jun Han, Liyu Zhao, XinYong Liu
PII:
S0223-5234(20)30617-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112645
EJMECH 112645
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 May 2020
Revised Date: 19 June 2020
Accepted Date: 5 July 2020
Please cite this article as: B. Sun, Y. Dong, Y. An, M. Liu, J. Han, L. Zhao, X. Liu, Design, synthesis and
bioactivity evaluation of novel arylalkene-amide derivatives as dual-target antifungal inhibitors, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112645.
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Graphical Abstract

Design, synthesis and bioactivity evaluation of novel
arylalkene-amide derivatives as dual-target antifungal inhibitors
Bin Sun a, b, 1, Yue Dong b, 1, Yunfei An b, Min Liu b, Jun Han b, Liyu Zhao c, XinYong Liu a,
a
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West Culture
Road, Jinan 250012, PR China
b
Institute of BioPharmaceutical Research, Liaocheng University, 1 Hunan Road, Liaocheng 252000, PR China
c
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang
110016, PR China
1
Both authors contributed equally to this study
*
Corresponding anthor: XinYong Liu, E-mail: xinyongl@sdu.edu.cn
ABSTRACT
Ergosterol as the core component of fungal cell membrane plays a key role in
maintaining cell morphology and permeability. The squalenee epoxidase (SE) and
14-demethylase (CYP51) are the important rate-limiting enzymes for ergosterol
synthesis. In the study, these active fragments, which is derived from the structural
groups of the common antifungal agents, were docked into the active sites of dual
targets (SE, CYP51), respectively. Some of active fragments with the matching
MCSS_Score values were selected and connected to construct three different series of
novel arylalkene-amide derivatives as dual-target (SE, CYP51) antifungal inhibitors.
Subsequently, these compounds were further synthesized, and their bioactivity was
evaluated. Most of compounds showed a certain degree of antifungal activity in vitro.
It was worth noting that the target compounds 17a and 25a with excellent antifungal
activity (0.125-4 μg/mL) can inhibit the fluconazole-resistant Candida Strain 17#,
CaR, 632, and 901 in the range of MIC values (4-8 µg/mL). Furthermore, their
molecular mechanism, structural stability and low toxicity were further confirmed.
The molecular docking and ADMET properties were predicted to guide the
subsequent optimization of target compounds.
Key Words: Antifungal; Molecular docking; Dual-Target Inhibitors; Molecular
mechanism

1. Introduction
At present, fungal infection showed a rapid growth trend with the drug abuse and
the environmental deterioration [1]. In clinically, the major pathogenic fungi are
Candida spp. and Aspergillus spp., which account for 78.9% of fungal diseases [2,3].
At the same time, they were becoming more and more difficult to treat, especially in
the invasive fungal infection of immune-compromised patients and the emergence of
fungal resistance, the annual morbidity and mortality rate have increased dramatically
to 16% and 33% [4,5]. Therefore, it is very important task to discover an effective
therapeutic drug for fungal diseases.
The conventional antifungal agents in clinical practice are mainly the ergosterol
synthetase inhibitors [6], they can be classified into three categories according to their
action modes, which includes the SE inhibitor (e.g., naftifine, terbinafine), CYP51
inhibitor (e.g., fluconazole, itraconazole and voriconazole) and antibiotics (e.g.,
nystatin, amphotericin B) [7,8]. It is worth noting that SE and CYP51 inhibitors can
produce the obvious synergistic effect in the treatment of deep fungal infection and
anti-resistant fungal disease [9] (Figure 1). When their rate-limiting enzymes activity
was inhibited, some toxic intermediates and upstream components were accumulated
rapidly in fungal cells, these materials can cause the changes in permeability and
fluidity of cell membrane, and it will eventually lead to the cracking and death of
fungal cells [11,12]. However, there have some problems in the curative effect of
combination therapy with SE and CYP51 inhibitors, such as unbalanced absorption,
low bioavailability, nephrotoxicity and hepatotoxicity [13]. In order to solve the
problems, the concept of dual-target drug design was introduced to develop the novel
antifungal inhibitors [14]. The kind of inhibitors can co-regulate the key links of
pathogenicity by adjusting the dual-targets activity. Therefore, it can reduce the
occurrence of drug side effects, especially the emergence of fungal resistance [15,16].
Based on this principle, we expect to construct the novel dual-target (SE, CYP51)
antifungal inhibitors though analyzing their active sites and structural features.
Figure. 1. Chemical structures of acrylamines and azole antifungal agents.
In the study, the corresponding fragment library based on the reported antifungal
agents was constructed, and these organic fragments were docked into the active sites
of SE and CYP51 through the fragments docking (MCSS) program, respectively.
These common active fragments with the higher MCSS_Score values were selected to
construct the different series of dual-target (SE, CYP51) antifungal inhibitors. Their
structures were further synthesized, the antifungal activity and action mechanism
were evaluated. This study will provide a new method and direction to design the

novel antifungal target compound.
2. Results and discussion
2.1. Dual-target docking study based on active fragments
At present, the technology of molecular docking has become a familiar
computer-aided drug design method [17,18]. However, when the size and flexibility
of the ligand molecules increase, it is difficult to define the correct location,
orientation and conformation of the ligands in the active site [19]. The fragment-based
drug design method can be used to more accurately locate the corresponding active
fragment in the target binding site.
Figure. 2. Construction of the antifungal compound fragment library and the fragment screening
based on dual-target (SE, CYP51) features
In this study, MCSS algorithm was selected as a classical important fragment
docking method, and the MCSS_Score can accurately evaluate the binding ability of
organic fragments in each active site. First, the two different compound sets were
constructed, which contain a series of SE inhibitors and CYP51 inhibitors (Figure 2).
Their organic fragments were extracted to constitute the common antifungal fragment
library. Subsequently, these fragment groups were docked in the active sites of
dual-target (SE, CYP51), and these optimal position fragments with high
MCSS_Score were determined. They are scored and sorted independently, which was
listed in Table 1.
Table 1. The scoring values for preferred docking fragments
Name
Fragment
structure
Potential
Energy
12.5839
Common
Docking region
A
MCSS_Score
SE
CYP51
1. Naphthyl
14.8733 41.9067
2. 2,3-dihydrobenzo [b]
[1,4]dioxine
14.3584
3.3422
A
A
A
14.2299 38.475
12.2299 32.5382
15.3445 28.2268
3. Tetrahydronaphthyl
4. Biphenyl
10.3049
5. Benzofuran
4.86303
A
11.5248 21.6914

6. Pyridyl
4.32576
4.16902
7.01813
2.84007
24.1744
B, C
B
16.5241 73.7736
18.4921 31.2876
13.1828 14.2328
15.3187 34.7495
15.1674 30.9729
7. Phenyl
8. Cyclohexyl
9. Imidazole
10. Triazole
B
C
C
The structure of the docking fragments can form the proper superposition
phenomenon with ligand molecules (naftifine, VT1602) in the corresponding active
sites (SE and CYP51), and their combined conformation was analyzed and
determined, which was shown in Figure 3. We found that the different kinds of active
fragments were distributed in the different active regions (A, B, C). Naphthyl,
2,3-dihydrobenzo [b] [1,4] dioxine, tetrahydronaphthyl, biphenyl and benzofuran as
the main chain groups were mainly located in region A, the scores of naphthyl and
2,3-dihydrobenzo [b] [1,4] dioxine are higher than other main chain groups, and they
can be well superimposed to the main chain structure of naftifine and VT1602.
Therefore, the naphthyl and 2,3-dihydrobenzo [b] [1,4] dioxine were selected to
design the novel target compounds, which can play the main chain function. Pyridyl
and phenyl with the higher MCSS_Score were mainly distributed in the region B, they
can be matched to the phenyl group of naftifine and VT1602, and produce
hydrophobic interaction with the surrounding key amino acid residues. In addition,
the nitrogen-containing groups, which contain the pyridinyl, imidazole and triazoles
groups, were distributed in the region C. It was noteworthy that these
nitrogen-containing groups can be bound to heme through the formation of
coordination bond interaction, and block the oxidation function in the CYP51 active
site.
Figure. 3. The combination pattern of active fragments in the active sites of SE (A) and CYP51
(B).
2.2. The Design of new Dual-Target Antifungal Inhibitors

On this basis, three different dual-target candidate compounds were designed based
on the binding of active fragments (Figure 4). At first, the naphthyl and 2,3-
2,3-dihydrobenzo[b][1,4]dioxine group with proper MCSS_Score were selected as the
main chain groups in the region A. The pyridine group can replace the phenyl group
of naftifine in the region B of SE and the azole group of VT1602 in the region C of
CYP51. The different connecting methods were performed to construct the series 1
and 2 target compounds (Figure 4 A1, A2). In addition, the phenyl group can also be
bound into the region B of dual-target (SE, CYP51), the imidazole group was located
in the region C, which can form coordination bond interactions with heme in the
region C of CYP51. Finally, the series 3 target compounds can be constructed by
connecting their core groups as a whole (Figure 4 A3). Subsequently, their structures
were further modified and optimized, and the biological activity was evaluated. We
expected that these compounds may have the dual-target antifungal activity.
Figure. 4. Three different series (1, 2, 3) of novel arylalkene-amide derivatives as
dual-target (SE, CYP51) inhibitors.
2.3. Chemistry
The series 1 target compounds (7a-c, 8a-c, 11a-c, 12a-c) were synthesized
according to the route summarized in Scheme 1. The starting material L-glycine (1)
was dissolved in ethanol and treated with SOCl2 to form the glycine ethyl ester
hydrochloride (2). 2-naphthaldehyde, 1-naphthaldehyde, 1,4-benzodioxan-6-carbox
-aldehyde (3a-c) were selected to react with 1,3-propanedioic acid, which can form
these intermediates (4a-c) [20,21]. Next, these intermediates were separately treated
with the glycine ethyl ester hydrochloride (2) in the condition of TBTU and DIEA to
give the key intermediates products (5a-c). They can be directly hydrolyzed to obtain
the corresponding organic acids (6a-c). Moreover, these intermediates products can
also be introduced the methyl group through methylation reaction, and then further
hydrolyzed to generate the corresponding organic acids (10a-c). Finally, these target
compounds (7a-c, 8a-c, 11a-c and 12a-c) were successfully obtained by introducing
the aminomethyl pyridine groups to intermediates 10a-c via amidation reaction.

Scheme 1. Reagents and conditions: (a) SOCl2, ethanol, reflux, 2-3h. (b) 1,3-propanedioic acid,
pyridine, piperidine, 80℃, 10h. (c) HATU, DIEA, DMF, 80℃, 6h. (d) MeOH, 2N NaOH, 60°C, 2
h; (e) PyBOP, HOBt, NEt3, aminomethyl pyridine, DMF, 80°C,7 h. (f) CH3I, K2CO3, THF, 60 °C,
5 h.
O
H
N
R
N
N
H
R
O
O
O
N
O
H
3a-c
H
d
c
N
17a-c
b
N
R
N
OH
R
N
O
O
O
O
H
H
a
O
O
N
N
O
O
O
R
N
N
H
N
NH2
16a-c
15a-c
O
O
O
13
14
18a-c
Scheme 2. Reagents and conditions:(a) Hydrazine hydrate, ethanol, rt., overnight. (b) 60°C, 5h.
(c) MeOH, 2N NaOH, 60°C, 2 h. (d) PyBOP, HOBt, NEt3, aminomethyl pyridine, DMF, 80°C,7 h.
The synthetic route of the series 2 target compounds (17a-c, 18a-c) was illustrated
in Scheme 2. First, the diethyl oxalate and hydrazine hydrate were mixed into
anhydrous ethanol to produce compound (14) [23,24]. 2-naphthaldehyde,
1-naphthaldehyde, 1,4-benzodioxan -6-carboxaldehyde (3a-c) were added and reacted
with compound 14 to obtain the intermediate products (15a-c) via condensation
reaction, respectively. These corresponding organic acids (16a-c) were generated by
the hydrolysis of intermediate products (15a-c) in alkaline conditions. Finally, the
target compounds 17a-c, 18a-c were synthesized from organic acids and
aminomethylpyridine.
The series 3 target compounds (22a-c, 24a-c, 25a-c) were synthesized according to
the route summarized in Scheme 3. The starting material L-serine (19) was dissolved
in ethanol and refluxed with SOCl2 to generate the serine ethyl ester hydrochloride
(20) [25]. Next, it was separately treated with these intermediate compounds (4a-c) to
obtain the required products (21a-c) via amidation reaction. The imidazole group was
introduced to afford the target compounds 22a-c. Subsequently, they were further
converted to the corresponding organic acids (23a-c) through hydrolysis reaction.
Finally, these organic acids were treated separately with the benzylamine or aniline in
the presence of condensing agents to give the target compounds (24a-c, 25a-c).
Scheme 3. Reagents and conditions: (a) SOCl2, ethanol, reflux, 2-3h. (b) HATU, DIEA, DMF,

60℃, 6h. (c) CDI, imidazole, reflux, 3h. (d) MeOH, 2N NaOH, 60°C, 2 h. (e) PyBOP, HOBt,
amine (aniline or benzylamine), DMF, 80°C,7 h.
NEt3
,
2.4. The bioactivity of target compounds against different pathogenic fungi in vitro
In the study, all target compounds were selected and evaluated their antifungal
activity against four common pathogenic Candida strains (C. alb., Candida albicans;
C. kru., Candida krusei; C. tro., Candida tropicalis; C. gla., Candida glabrata) and
one Aspergillus strain (A. fum., Aspergillus fumigatus) [26]. The protocols from
NCCLS (National Committee for Clinical Laboratory Standards) was determined
using two-fold broth dilution method in 96-well micro-test plates, and fluconazole and
naftifine were used as the positive control drugs.
The results of antifungal activities were summarized and deposited in Table 2. It
revealed that the most of target compounds exhibited the moderated to excellent
antifungal activity by inhibiting the proliferation of Candida strains, which suggests
the design rationality for novel target compounds. These target compounds (7a-c,
8a-b, 11a-c, 12a-b, 17a-c, 18a-c, 24a, 24c, 25a-c) exhibited the antifungal properties
against Candida strains in the range of 0.125-2 μg/mL, which were similar potency
with fluconazole and naftifine. Of these, the compounds (7a, 11a, 11b, 12a, 17a, 17b,
18a, 25a, 25b) with the range of MIC value (0.125-0.5 μg/mL) against C.alb, C.gla.
and C.tro. showed the more efficient than these control drugs. The compounds (11b,
12a, 17b and 18a) also exhibited the excellent antifungal activities against C.kru. with
0.25 μg/mL. However, their inhibitory effect on A.fum. was not ideal. It was worth
nothing that compounds 11b, 17a and 25a show the excellent antifungal activity with
the range of MIC value (0.125-8 μg/mL) against the Candida spp. and A.fum.
Subsequently, the QSAR (Quantitative structure–activity relationship) of target
compounds was preliminarily analyzed and summarized. The main chain groups of
different series compounds with 1-naphthyl group is better than other main chain
groups, their structure with 3-pyridine group can display a slightly decrease in
antifungal activities against Candida strains. At the same time, these groups are
connected linked by the acylhydrazone structure, which show the obvious structural
advantages over other linking methods.
Table 2 The MIC value of the target compounds in vitro.
Compd
R
Nitrogen-
MIC, µg/mL
containing groups
C.alb.
C.gla.
C.kru.
C.tro.
A.fum
7a
7b
7c
8a
8b
0.25
0.25
0.5
0.25
0.25
1
4
4-picolinylamine
0.25
0.5
0.5
0.5
1
1
0.5
1
>16
>16
8
4-picolinylamine
4-picolinylamine
3-picolinylamine
3-picolinylamine
0.5
0.5
2
0.5
0.5
2
>16

8c
3-picolinylamine
4-picolinylamine
4-picolinylamine
4-picolinylamine
3-picolinylamine
3-picolinylamine
3-picolinylamine
4-picolinylamine
4-picolinylamine
4-picolinylamine
3-picolinylamine
3-picolinylamine
3-picolinylamine
Imidazole
2
0.125
0.25
0.5
0.5
0.5
2
4
0.25
0. 125
1
4
0.5
0.25
1
2
0.25
0.25
0.5
0.5
1
>16
4
11a
11b
11c
12a
12b
12c
17a
17b
17c
18a
18b
18c
24a
24b
24c
25a
25b
25c
8
>16
>16
>16
>16
2
0.5
1
0.25
0.5
4
0. 5
0.25
0.5
1
0. 5
0.125
0.5
1
0.125
0.25
0.5
0.5
1
0.5
0.25
0.5
0.25
0.5
2
>16
8
0.5
0. 25
0.5
0.5
0.5
1
0.5
0.25
1
>16
8
0.5
0.5
2
>16
>16
8
2
0.5
0.5
0.5
0. 25
0.5
0.25
Imidazole
4
Imidazole
0.5
2
>16
4
Imidazole
0.125 0.125
0.25
0.5
1
Imidazole
0.5
0.25
0.5
8
Imidazole
0.25
>16
Fluconazole
Naftifine
-
-
-
-
0.25
0.5
0.5
1
1
0.5
2
>16
4
0.5
2.5. The bioactivity of target compounds against different fluconazole-resistant
strain of C.alb. in vitro.
At present, fluconazole as a representative antifungal agents have been widely
applied in clinical antifungal practice, which has produced an excellent therapeutic

effect. However, the fluconazole-treated Candida strains are constantly showing the
drug resistance, which has become a serious threat to human health. Therefore, it is
very urgent task to further improve their antifungal activity against fluconazole-
resistant strains. In the study, the optimization compounds 11b, 17a and 25a were
selected to evaluate their bioactivity against the different fluconazole-resistant strains
in vitro, and the result was deposited in Table 3. It can be observed that the target
compound 11b with the MIC values > 16 µg/mL was inactive against the
fluconazole-resistant strains 17#, 901 and 904. Compounds 17a, 25a with the range of
MIC values (4-8 µg/mL) displayed the moderate or remarkable activity against Strain
17#, CaR, 632 and 901. Noticeably, the compounds 17a exhibited the best bioactivity
against the Strain 17#, CaR and 901 with MIC values 4 µg/mL, respectively. At the
same time, it also displayed a certain degree of antifungal effect on the strains 632 and
904.
Table 3. The MIC value of the target compounds against the fluconazole-resistant strains in
vitro.
Compd
MIC, µg/mL
Strain 17#
Strain CaR
Strain 632
Strain 901
Strain 904
˃16
11b
˃16
4
8
8
˃16
4
17a
4
8
8
25a
4
8
8
8
˃16
8
Naftifine
8
˃16
˃16
8
˃16
˃16
Fluconazole ˃16
˃16
˃16
Abbreviations: fluconazole-resistant strains of Candida strains: strain 17#, strain CaR, strain
632, strain 901, strain 904; These strains were provided by the Shenyang Pharmaceutical
University.
2.6. Density analysis of Candida albicans in different treatment groups
Assessing the density change of fungal cell is an important indicator, which can
reflect the effect of different treatment groups on the proliferation capacity of fungal
cells. In the study, the untreated group and treated groups with positive control drugs
(fluconazole, naftifine) and target compounds (17a, 25a) were set, respectively. The
polarizing microscopy was used to observe the changes of cell density in different
treatment groups (Figure 5).

Figure 5. The cell density changes of C.alb. (ATCC SC5314) in different treatment groups
(Untreated: A1-A3; fluconazole: B1-B3; naftifine: C1-C3; Compound 17a: D1-D3; Compound 25a:
E1-E3).
In the untreated group, the cell density of C.alb. (ATCC SC5314) showed a rapid
growth trend, and some fungal cells can be directly observed the phenomenon of cell
germination and spores, which shows these fungal cells have the significant
proliferation ability. In the treated group with fluconazole, we found that the fungal
cells did not show a rapid proliferation phenomenon, their density was significantly
lower than the untreated group at the same time, which indicated that the proliferation
ability of fungal cells was inhibited. In the treatment groups with naftifine, compound
17a and compound 25a, the fungal cell density decreased rapidly, and the cleavage of
some fungal cells can be directly observed. The possible reason is that the antifungal
mechanism of the target compounds is similar with naftifine, they can not only
effectively inhibit the fungal cell proliferation, but also kill the existing fungal cells.
2.7. The changes of C.alb. cell structure and morphology
C.alb. (ATCC SC5314) was selected and treated with the preferred target
compound 17a, and the structure and morphology changes of fungal cell was
observed through the method of TEM (Transmission electron microscopy), which can
directly reflect the effect of the target compound on the fungal cell [27]. In the early
stage, we can observe that the cell structure of C.alb. exhibits the elliptical
characteristics. The fungal cell wall and membrane with the transparent and smooth
properties were distributed on the cell edge, and they can form the clear boundary
between cell communities. The internal substance surrounded by the cytoplasmic
membrane is composed of genetic material and cytoplasm (Figure 6 A1-A2). When
C.alb. was treated to the third day, we found that some smooth surfaces of fungal cell
transform into the irregular structure, and their boundaries disappear. The
permeability of fungal cells was weakened, and the endogenous substances leaked to
the surrounding regions of fungal cell. Moreover, the fungal cytoplasm area also
occurred some special phenomena, such as vesicles formation, edema cavitation and
nuclear membrane cleavage (Figure 6 B1-B2).With the further prolongation of
processing time (6th day), the fungal cells have completely cracked, only residual
fungal cell wall and membrane structure were left (Figure 6 C1-C2). The preliminary
analysis has confirmed that the target compounds can affect the surface function of
fungal cell and eventually lead to the lysis of cell structures.

Figure 6. The morphological changes of C.alb. (ATCC SC5314) treated with target compound
17a in different time periods.
2.8. The action mechanism of target compounds by analyzing sterol composition
The target enzymes SE and CYP51 can synergistically catalyze the synthesis of
ergosterol [28]. When these target enzymes are inhibited, the content of their
intermediate components will accumulate rapidly. Therefore, the change of sterol
composition can reflect the activity of target enzymes. In the study, C.alb. SC5314
cells was selected as the test strain. The untreated group, the fluconazole-treated
group and the naftifine-treated group were set as the positive control. The results were
summarized in Table 4.
In the untreated control group, it could be directly observed that ergosterol is the
main sterol component in fungal cell, which account for 79.43%. The other
components (lanosterol, eburicol, squalene) were also detected, and they accounted
for 6.62%, 4.67% and 2.25%, respectively. In the fluconazole-treated group, the
content of eburicol was accumulated from 4.67% to 65.51%, and the ergosterol
decreased significantly to 7.62%. These changes of sterol contents indicate that
fluconazole can inhibit CYP51 and block the ergosterol biosynthetic pathway. In the
naftifine-treated group, the content of eburicol did not obviously change, and the
content of squalene showed a dramatic increase, which account for 52.48%. While the
content of ergosterol sharply decreased to 15.39%, which indicate that naftifine can
exert the inhibitory activity of SE to block ergosterol synthesis. In the compounds 17a
and 25a-treated group, the content of ergosterol decreased rapidly to 8.27% and
10.73%, respectively. At the same time, the content of eburicol increased to 32.81%
and 40.15%, and the content of squalene increased to 48.26% and 29.14%. The results
indicated that compounds 17a and 25a can synergistically inhibit the activity of
double-target enzymes (SE, CYP51) at the same time, and affect the biosynthetic
pathway.
Table 4. Analysis the composition change of sterol in C.alb.
Contents.
% of total sterols (Candida albicans)
Control Fluconazole Naftifine Compound 17a
Compound 25a

79.43
6.62
4.67
2.25
7.03
7.62
13.93
65.51
3.68
15.39
10.26
14.65
52.48
7.22
8.27
11.35
32.81
42.86
4.71
10.73
14.52
40.15
29.14
5.46
Ergosterol
Lanosterol
Eburicol
Squalene
9.26
Unidentified
2.9. Study on the stability of target compounds in vitro human plasma
The structural stability of target compounds in vitro human plasma is an essential
index for medicinal property of compound [29]. Based on their excellent biological
activity in vitro, the target compounds 17a and 25a were selected and incubated with
human Plasma, which was summarized in Table 5, compounds 17a and 25a in human
plasma demonstrated the high structural stability, which account for 86.5% and 84.1%
content at 120 min, respectively. The result indicate that these compounds can
maintain their medicinal efficacy in vivo for a long time.
Table 5. In vitro human plasma stability of compounds 17a and 25a.
Compd
Stability in Human Blood Plasma
% Incubating for 60 min % Incubating for 120 min
17a
25a
92.3
89.7
86.5
84.1
2.10. In vivo study of toxicity and therapeutic Efficacy
The in vivo latent toxicity and therapeutic efficacy have always been a problem in
the development of novel antifungal agent. Therefore, it is of great significance to
evaluate the pharmacodynamic properties of compounds 17a and 25a through the
mice fungal infection model [30]. Firstly, the body weights of mice in different
treatment groups were monitored during the whole treatment period, and their weights
did not show the significant difference. We further analyzed the in vivo antifungal
efficacy of compound 17a and 25a, H&E (hematoxylin-eosin staining) was performed
to observe the morphology of tissue in different treatment groups, which was shown
in Figure 7.
Figure 7. The morphological change of fungal infections tissues in different treatment groups (A:
Saline; B: Compound 17a; C: Compound 25a).
In the treated group with saline, some endothelial tissue cells exhibit the
phenomenon of damage and irregular shape with shrinking nucleus. A large number
of nuclear gathers in the infected region of endothelial tissue, which indicates that the
pathogenic fungal infection has appeared, and the corresponding immune response

has been produced. In the treated groups with target compounds 17a and 25a, we
found that these infected tissue cells have gradually returned to the complete state and
uniform distribution characteristics, the infection positions completely disappeared at
the 7 days, the aggregation phenomenon of cell nuclear has disappeared,
which suggested that these target compounds can significantly treat the endogenous
fungal infection. These H&E results further demonstrated the synergistic therapeutic
effect of the compounds.
Figure 8. The histological images of H/E staining (lung, liver, spleen, kidney and heart) from the
different treatment groups. (A1-5: Saline; B1-5: Compound 17a; C1-5: Compound 25a).
Subsequently, the safety of the target compounds in vivo was further evaluated. All
main organs (heart, liver, spleen, kidney, and lung) in the different treatment groups
were collected and analyzed. They did not show significant morphological changes,
and the phenomenon of pathology has not been found (Figure 80. Moreover, the
alanine aminotransferase (ALT), aspartate aminotransferase (AST) and blood urea
nitrogen (BUN) were the most common parameters to evaluate the function of liver
and kidney. The result was showed in the Table 6, the AST, ALT and BUN levels in
the different treatment groups didn't display the significant difference (p>0.05),
indicating the compounds 17a and 25a had no obvious hepatic and renal toxicity. In
summary, the target compounds have the antifungal activity in vivo, and they can
significantly inhibit the proliferation of fungi in mice with low toxicity.
Table 6 The analysis of plasma biochemical levels with different treatment groups
Group
AST(IU/L)
109.4±4.8
112.7±9.3
101.5±6.5
ALT(IU/L)
102±9.2
BUN (mmol/L)
6.7±0.3
Saline
Compound 17a
Compound 25a
117±13.4
105±4.9
7.1±0.7
6.9±0.5
2.11. Molecular Docking
In order to better understand the binding mode of target compounds with active
site, molecular docking was performed to investigate the interaction between the
target compound 17a and the dual-target enzymes (CYP51, SE) using the CDOCKER
program in the Discovery Studio 3.5 software, and it can provide the guide
information for further structural optimization of compound [31]. The published

crystal structure of CYP51 and the homology model of SE from C.alb. were selected
as the useful templates to construct their binging mode, which was shown in Figure 9.
The compound 17a can be properly docked in the active site of SE, and its
structure can form the interaction with the key residues (Figure 9 A1-3). The phenyl
group of compounds 17a occupies the upper region of active pocket, the π-alkylation
and π-π interactions were formed with the residues (Arg 134, Val 240 and Leu 434).
The pyridine group of compounds 17a was bound in the bottom region, and it can
form the van der Waals and hydrophobic interactions with Pro 430 and Leu 398,
respectively. At the same time, the compounds 17a can also be docked in the active
site of CYP51 (Figure 9 B1-3 ), the 1-naphthyl group extends into the upper region of
CYP51 channel, and it can produce the π-π and π-alkylation interaction with the
surrounding key residues, such as Tyr 118, Pro230, Phe233, Leu376, His377 and Met
508. The pyridine group can form the coordination bond interaction with the iron
atom of heme group in the bottom region of the active site (The coordinate bond
length is 2.513 Å). In summary, the result can confirm that the target compounds can
form the stable combination mode with dual-target (SE, CYP51).
Figure 9. (A1) The molecular docking of compound 17a with SE; (A2) The binding conformation
of compound 17a in the active site of SE; (A3) The 2D interactions map of compound 17a with SE.
(B1) The molecular docking of compound 17a with CYP51; (B2) The binding conformation of
compound 17a in the active site of CYP51; (B3) The 2D interactions map of compound 17a with
CYP51.
2.12. The prediction of ADMET properties
In the early stages of drug development, the ADMET properties of compounds can
guide the optimization of lead compounds [32]. It is very necessary to improve the
success rate of drug development and reduce the funds waste. Therefore, the ADMET
properties of target compounds was predicted, which was shown in Figure 10.

Figure 10. The Plot of 95% and 99% confidence limit ellipses that correspond to the
blood-brain barrier and intestinal absorption models for the target compounds. Abbreviations:
ADME/T, absorption, distribution, metabolism, excretion and toxicity; PSA, polar surface
area; 2D, two-dimensional; A log P, the logarithm of the partition coefficient between
octanol and water; BBB, blood brain barrier.
The blood brain barrier (BBB) and human intestinal absorption (HIA) of
permeation models can be expressed by two similar 95% and 99% confidence ellipses.
All the target compounds and control drugs (naftifen, fluconazole) were located in the
99% confidence interval of BBB permeation and HIA, and a few number
of compounds and naftifine were excluded from the 95% confidence ellipses of
BBB-95 and Absorption-95, which indicate that most of target compounds display the
appropriate HIA and BBB properties.
Table 7. In silico ADME/T prediction of the potential amidopyridine compounds and
amidoimidazole compounds compared with the naftifine and fluconazole.
ADME/T
parameters
A log P98 a
PSAb
Series 1
compounds
0.99~2.112
Series 2
Series 3
Naftifine
Fluconazole
compounds
0.591~1.714
compounds
1.812~3.209
4.863
3.352
2
0.750
76.556
4
71.483~89.343 82.806~100.666 76.831~94.6916
Aqueous
3
3
2, 3
solubility
BBB c
CYP450 2D6 d
2, 3
0
3
0
2, 3
0
0
0
3
1
Hepatotoxicity
Non-Toxic
Non-Toxic
Non-Toxic,
Toxic
Non-Toxic
Toxic
PPB e
Highly bound
Highly bound
Highly bound
Highly bound
Highly bound
a. A log P98 (atom-based log P) (≦-2.0 or ≧7.0: very low absorption). b. PSA (polar surface area) (>140:
very low absorption). c. BBB (blood brain barrier), level blood brain barrier penetration prediction: 0 (very high
penetrate), 1 (high), 2 (medium), 3 (low), 4 (undefined). d. Prediction cytochrome P4502D6 enzyme inhibition
(0: non-inhibitor; 1: inhibitor). e. PPB, plasma protein binding.
These detailed ADME/T properties of target compounds were summarized in Table
7. A logP and PSA are important indicators related to the drug bioavailability. They

can reflect the hydrophobicity and oral bioavailability of the target compounds. The A
logP < 5 and PSA <140 of these compounds are in the limit range, which indicates
that these target compounds have proper permeability properties and oral
bioavailability. At the same time, they also can remain the low-level BBB
permeability and Non-Hepatotoxicity. In addition, we found that all the target
compounds had no significant inhibitory activity on metabolizing enzymes (CYP450
2D6=0, non-inhibitor), they showed the optimal solubility level, which could be
effectively absorbed in human intestinal tract and highly bound to plasma protein.
3. Conclusions
At present, the frequent emergence of endogenous fungal infection and the
drug-resistant fungi have become a serious threat to human health. SE and CYP51 are
important antifungal targets, and their inhibitors can synergistically block the
synthesis of ergosterol and improve the treatment effect of fungal diseases. Therefore,
the dual-target (SE, CYP51) inhibitors was regarded as an effective coping strategy.
In this study, the corresponding fragment library was composed of the core groups of
SE and CYP51 inhibitors, and they were docked into the dual-target enzymes. Some
of core groups with the higher MCSS_Score values were selected as the basis of
fragment-based drug design. Subsequently, three series of target compounds were
constructed and synthesized, their corresponding antifungal activity was evaluated.
The results showed that most compounds have a certain activity against the different
pathogenic fungi, especially compounds 17a and 25a with excellent antifungal
activity (0.125-4 μg/mL) also has the bioactivity activity against fluconazole-resistant
Candida Strain 17#, CaR, 632, and 901 in the range of MIC values (4-8 µg/mL).
Electron microscopic observation and sterol composition analysis have confirmed that
these target compounds can destroy the integrity of the fungal cell surface by
inhibiting the activity of dual-target (SE, CYP51). Notably, they also exhibit the
excellent antifungal activity in vivo with low toxicity effects and blood plasma
stability. The molecular docking and ADMET properties were further performed, and
these target compounds have the potential to be further optimized as the lead
compound.
4. Experimental section
4.1. General Methods for Chemistry
All reagents used in the experiment were procured from Energy Chemical, Aladdin,
Bide and Sinopharm Company (except special instruction) without additional
purification, and the solvents were purifified according to the standard procedures.
The reactions were monitored by thin layer chromatography (TLC). and the analysis
was performed on Silica Gel 60 F254 plates (Jiangyou, Yantai), which was used to
monitor the reaction process. The silicagel (200-300 mesh) from Qingdao Ocean
Chemicals (Qingdao, Shandong, China) was used for column chromatography. The
melting points of all compounds were determined and corrected with a Büchi Melting
Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland). The liquid
chromatograph mass spectrometer (LC-MS) were determined in ESI mode on a
Shimadzu 8040 LC-MS (Shimadzu, Japan). Nuclear magnetic resonance (1H-NMR
13
and C-NMR) spectra were recorded on a Bruker 500 MHz NMR spectrometer with

TMS as an internal standard. The coupling constants (J) are reported in Hertz, and the
peak multiplicities were expressed as follows: s, singlet; d, doublet; t, triplet; q,
quartet; dd, doublet of doublet; dt, doublet of triplet; td, triplet of doublet; ddd,
doublet of doublet of doublet; m, multiplet; br, broad peak.
4.2. General procedure for the synthesis of amino acid ethyl ester hydrochloride
hydrochloride (2, 20)
L-amino acids (Glycine, Serine; 1.0 equiv) was dissolved in ethanol solution,
respectively. Thionyl chloride (3.0 equiv) was slowly dripped into the mixed solution
at 0 °C. The mixed solution was heated to reflux for 2-3 h. When the reaction liquid
becomes transparent, the reaction mixture was concentrated under reduced pressure to
give the white solid (2, 20).
4.3. General procedure for the synthesis of intermediate compounds (4a-c)
Aryl formaldehyde (1-naphthaldehyde, 2-naphthaldehyde, 2, 3-dihydrobenzo [b]
[1,4] dioxine-6-carbaldehyde; 1equive) was dissolved in pyridine solution, and
piperidine was slowly dripped into the mixed solution. The mixed solution was heated
at 80 ℃ for 10 h. The reaction process was monitored by TLC analysis. After the
completion of reaction, pH was adjusted to 1-2 by the dilute hydrochloric acid
solution, and white solid was filtered and dried.
4.4. General procedure for the synthesis of compounds (5a-c)
The appropriate organic acids (4a-c; 1 equiv) and HATU (1.2 equiv) were added in
anhydrous DMF, respectively. The mixture was stirred at room temperature for 2 h.
Then, glycine ethyl ester hydrochloride (2; 1.0 equiv) and DIEA (3.0 equiv) were
added, the mixture was heated to 85 °C for 6 h. The reaction process was monitored
by TLC analysis. After the completion of reaction, the reaction mixture was poured
into ice water, and extracted with ethyl acetate, the organic phase was dried over
Na2SO4 overnight. Finally, the desired compounds were obtained by vacuum
distillation.
4.5. General procedure for the synthesis of compounds (9a-c)
The compounds (5a-c; 1.0 equiv), K2CO3 (3 equiv) and CH3I (2 equiv) were
dissolved in tetrahydrofuran. Then, the reaction mixture was stirred at 60 °C for 5 h.
The reaction process was monitored by TLC analysis. After the completion of
reaction, the mixture was poured into ice water, and extracted with ethyl acetate, the
organic phase was dried over Na2SO4 overnight. Finally, the desired compounds were
obtained by vacuum distillation.
4.6. General procedure for the synthesis of compounds (6a-c, 10a-c)
The compounds (5a-c, 9a-c; 1.0 equiv) were dissolved into methanol solution (10
mL), the solution of 2N sodium hydroxide (20 mL) was added. Then, the reaction
mixture was stirred at 60 °C for 7 h. The reaction process was monitored by TLC
analysis. After the completion of reaction, the methanol solution was removed by
vacuum distillation, pH was adjusted to 2-3 by dilute hydrochloric acid solution, and
white solid was filtered and dried to give the desired compounds.
4.7. General procedure for the synthesis of target compounds (7a-c, 8a-c, 11a-c,
12a-c)
The key intermediate (6a-c, 10a-c; 1 equiv), PyBOP (1.2 equiv), HOBT (3 equiv)

and triethylamine (4 equiv) were added into the solution of DMF, respectively. The
reaction mixture was stirred at room temperature for 10 min. Then, the aminomethyl
pyridine (1.0 equiv) were added, the reaction mixture was heated at 80 °C, and stirred
for 7 h. The reaction process was monitored by TLC analysis. After the completion of
reaction, the reaction mixture was poured into ice water, the resulting solid was
filtered and dried to give the desired compound. The crude product was purified by
silica gel column chromatography (CH2Cl2: MeOH =100:3).
4.8. General procedure for the synthesis of monoethyl oxalate hydrazide (14)
Diethyl oxalate 13 (3 equiv) and hydrazine hydrate (1 equiv) were dissolved in the
ethanol, respectively. The hydrazine hydrate was slowly added dropwise into the the
mixed solution. Then, the mixture was stirred at room temperature overnight.
Filtration was performed to remove the white insoluble impurities, and concentrate
the residual mixture at reduced pressure. The desired compound 14 was separated
from the excess ester by throwing away the organic phase and collecting the aqueous
one. Compound 14 was temporarily saved in water.
4.9. General procedure for the synthesis of intermediate compounds (15a-c)
The corresponding aryl formaldehyde 3a-c (1-naphthaldehyde, 2-naphthaldehyde, 2,
3-dihydrobenzo [b] [1,4]dioxine-6-carbaldehyde; 1equive) were added into the
aqueous solution of monoethyl oxalate hydrazide 14 (1 equiv), Then, the reaction
mixture was stirred at 60 °C for 5 h. Solids were collected by filtration and filtrate
was washed by water. The product was drained by vacuum pump to afford desired
compounds 15a-c.
4.10. General procedure for the synthesis of compounds (16a-c)
The compounds (15a-c; 1.0 equiv) were dissolved into methanol solution (10 mL),
the solution of 2N sodium hydroxide (20 mL) was added. Then, the reaction mixture
was stirred at 60 °C for 2 h. The reaction process was monitored by TLC analysis.
After the completion of reaction, the methanol solution was removed by vacuum
distillation, pH was adjusted to 2-3 by dilute hydrochloric acid solution, and white
solid was filtered and dried to give the desired compounds.
4.11. General procedure for the synthesis of target compounds (17a-c, 18a-c)
The key intermediate (16a-c; 1 equiv), PyBOP (1.2 equiv), HOBT (3 equiv) and
triethylamine (4 equiv) were added into the solution of DMF, respectively. The
reaction mixture was stirred at room temperature for 10 min. Then, the aminomethyl
pyridine (1.0 equiv) were added, the reaction mixture was heated at 80 °C, and stirred
for 7 h. The reaction process was monitored by TLC analysis. After the completion of
reaction, the reaction mixture was poured into ice water, the resulting solid was
filtered and dried to give the desired compounds (17a-c, 18a-c). The crude product
was purified by silica gel column chromatography (CH2Cl2: MeOH =100:3).
4.12. General procedure for the synthesis of compounds (21a-c)
The appropriate organic acids (4a-c; 1 equiv) and HATU (1.2 equiv) were added in
anhydrous DMF, respectively. The mixture was stirred at room temperature for 2 h.
Then, glycine ethyl ester hydrochloride (20; 1.0 equiv) and DIEA (3.0 equiv) were
added, the mixture was heated at 75 °C for 7 h. The reaction process was monitored
by TLC analysis. After the completion of reaction, the reaction mixture was poured

into ice water, and extracted with ethyl acetate, the organic phase was dried over
Na2SO4 overnight. Finally, the desired compounds were obtained by vacuum
distillation.
4.13. General procedure for the synthesis of target compounds (22a-c)
The intermediates 21a-c (1.0 equiv.), CDI (1.2 equiv.) and imidazole (1.0 equiv.)
were added to acetonitrile. The mixture was heated to reflux for 3 h and then cooled to
room temperature. The solvent was removed under vacuum. The residue was
dissolved in ethyl acetate, washed with water thrice, dried over Na2SO4 and filtered.
The filtrate was concentrated to afford the target compounds (22a-c) as a white solid.
The crude product was purified by silica gel column chromatography (CH2Cl2: MeOH
=100:3).
4.14. General procedure for the synthesis of compounds (23a-c)
The compounds (22a-c; 1.0 equiv) were dissolved into methanol solution (10 mL),
the solution of 2N sodium hydroxide (20 mL) was added. Then, the reaction mixture
was stirred at 60 °C for 2 h. The reaction process was monitored by TLC analysis.
After the completion of reaction, the methanol solution was removed by vacuum
distillation, pH was adjusted to 2-3 by dilute hydrochloric acid solution, and white
solid was filtered and dried to give the desired compounds.
4.15. General procedure for the synthesis of target compounds (24a-c, 25a-c)
The key intermediate (23a-c; 1 equiv), PyBOP (1.2 equiv), HOBT (3 equiv) and
triethylamine (4 equiv) were added into the solution of DMF, respectively. The
reaction mixture was stirred at room temperature for 10 min. Then, the amine (aniline
or benzylamine; 1.0 equiv) was added, the reaction mixture was heated at 80 °C, and
stirred for 7 h. The reaction process was monitored by TLC analysis. After the
completion of reaction, the reaction mixture was poured into ice water, the resulting
solid was filtered and dried to give the desired compounds (24a-c, 25a-c). The crude
product was purified by silica gel column chromatography (CH2Cl2: MeOH =100:3).
4.15.1. (E)-3-(naphthalen-1-yl)-N-(2-oxo-2-((pyridin-4-ylmethyl)amino)ethyl) acryl
-amide (7a)
1
The product was obtained as a white solid; yield: 73.6%; mp: 181.4–184.8℃. H
NMR (500 MHz, DMSO) δ 8.72 – 8.40 (m, 4H), 8.23 (dd, J = 15.3, 12.2 Hz, 2H),
7.99 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 7.2 Hz, 1H), 7.60 (ddd, J = 15.3, 13.8, 7.2 Hz,
3H), 7.29 (d, J = 5.4 Hz, 2H), 6.80 (d, J = 15.6 Hz, 1H), 4.35 (d, J = 6.0 Hz, 2H), 3.96
13
(d, J = 5.8 Hz, 2H). C NMR (126 MHz, DMSO) δ 169.80, 165.89, 149.92, 148.92,
135.92, 133.84, 132.43, 131.30, 130.10, 129.15, 127.38, 126.72, 126.24, 125.44,
124.96, 123.67, 122.55, 42.90, 41.61. ESI-MS m/z: 346 [M-H]+. HPLC purity 99.2%.
Retention time: 4.3 min, eluted with 20% purified water/80% methanol.
4.15.2.(E)-3-(naphthalen-2-yl)-N-(2-oxo-2-((pyridin-4-ylmethyl)amino)ethyl)acryla
mide (7b)
1
The product was obtained as a white solid; yield: 75.8%; mp: 182.4–186.7℃. H
NMR (500 MHz, DMSO) δ 8.59 – 8.42 (m, 4H), 8.09 (s, 1H), 7.97 – 7.92 (m, 3H),
7.75 (d, J = 8.5 Hz, 1H), 7.63 (d, J = 15.8 Hz, 1H), 7.55 (dd, J = 6.1, 3.2 Hz, 2H),
7.28 (d, J = 5.4 Hz, 2H), 6.88 (d, J = 15.8 Hz, 1H), 4.34 (d, J = 6.0 Hz, 2H), 3.94 (d, J
= 5.8 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 169.81, 165.95, 149.91, 148.91,

139.44, 133.86, 133.52, 132.92, 129.26, 129.05, 128.76, 128.14, 127.40, 127.20,
123.98, 122.87, 122.54, 42.89, 41.60. ESI-MS m/z: 346 [M-H]+. HPLC purity 99.3%.
Retention time: 4.0 min, eluted with 20% purified water/80% methanol.
4.15.3.(E)-N-(2-oxo-2-((pyridin-4-ylmethyl)amino)ethyl)-3-(2,3,4a,8a-tetrahydroben
zo[b][1,4]dioxin-6-yl)acrylamide (7c)
1
The product was obtained as a white solid; yield: 76.1%; mp: 186.7–191.2℃. H
NMR (500 MHz, DMSO) δ 8.50 (dd, J = 10.0, 5.7 Hz, 3H), 8.30 (t, J = 5.7 Hz, 1H),
7.34 (d, J = 15.8 Hz, 1H), 7.26 (d, J = 5.5 Hz, 2H), 7.07 (d, J = 9.6 Hz, 2H), 6.89 (d, J
= 8.1 Hz, 1H), 6.56 (d, J = 15.8 Hz, 1H), 4.32 (d, J = 6.0 Hz, 2H), 4.26 (d, J = 1.1 Hz,
4H), 3.89 (d, J = 5.8 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 169.86, 166.08, 149.84,
149.02, 145.24, 144.02, 139.05, 128.82, 122.55, 121.50, 120.57, 117.98, 116.44,
64.75, 64.48, 42.82, 41.58. ESI-MS m/z: 354 [M-H]+. HPLC purity 99.0%. Retention
time: 3.8 min, eluted with 20% purified water/80% methanol.
4.15.4. (E)-3-(naphthalen-1-yl)-N-(2-oxo-2-((pyridin-3-ylmethyl)amino)ethyl)acryl
-amide (8a)
The product was obtained as a white solid; yield: 74.1%; mp: 180.4–185.82℃. 1H
NMR (500 MHz, DMSO) δ 8.51 (d, J = 5.5 Hz, 2H), 8.43 (t, J = 6.0 Hz, 1H), 8.33 (t,
J = 5.6 Hz, 1H), 7.94 (s, 1H), 7.87 (d, J = 5.0 Hz, 4H), 7.74 – 7.69 (m, 2H), 7.51 (dd,
J = 6.2, 3.2 Hz, 2H), 7.27 (d, J = 5.4 Hz, 2H), 6.84 (d, J = 15.7 Hz, 1H), 4.43 (d, J =
6.0 Hz, 2H), 4.05 (d, J = 5.7 Hz, 2H). 13C NMR (126 MHz, HDMSO) δ 170.68,
167.21, 151.50, 148.05, 141.09, 136.28, 134.53, 133.94, 129.10, 128.87, 127.79,
127.15, 126.76, 124.12, 123.92, 43.31, 42.47. ESI-MS m/z: 346 [M-H]+. HPLC purity
98.9%. Retention time: 4.2 min, eluted with 20% purified water/80% methanol.
4.15.5.(E)-3-(naphthalen-2-yl)-N-(2-oxo-2-((pyridin-4-ylmethyl)amino)ethyl)acryl
-amide (8b)
The product was obtained as a white solid; yield: 69.2%; mp: 183.5–189.5 ℃. 1H
NMR (500 MHz, DMSO) δ 8.50 (d, J = 5.5 Hz, 2H), 8.42 (t, J = 6.0 Hz, 1H), 8.33 (t,
J = 5.6 Hz, 1H), 7.93 (s, 1H), 7.86 (s, 3H), 7.74 – 7.68 (m, 2H), 7.53 – 7.49 (m, 2H),
7.26 (d, J = 5.4 Hz, 2H), 6.85 (d, J = 15.7 Hz, 1H), 4.42 (d, J = 6.0 Hz, 2H), 4.11 –
3.99 (m, 2H). 13C NMR (126 MHz, HDMSO) δ 169.83, 166.35, 149.68, 139.85,
133.79, 133.35, 132.66, 129.10, 128.68, 128.44, 127.79, 126.76, 123.61, 122.33,
122.04, 43.12, 41.82. ESI-MS m/z: 346 [M-H]+. HPLC purity 99.1%. Retention time:
4.1 min, eluted with 20% purified water/80% methanol.
4.15.6.(E)-N-(2-oxo-2-((pyridin-4-ylmethyl)amino)ethyl)-3-(2,3,4a,8a-tetrahydroben
zo[b][1,4]dioxin-6-yl)acrylamide(8c)
1
The product was obtained as a white solid; yield: 76.1%; mp: 191.7–194.3℃. H
NMR (500 MHz, DMSO) δ 8.53 – 8.40 (m, 3H), 8.28 (t, J = 5.7 Hz, 1H), 7.66 (d, J =
7.8 Hz, 1H), 7.33 (dd, J = 14.0, 8.6 Hz, 2H), 7.07 (d, J = 9.7 Hz, 2H), 6.89 (d, J = 8.1
Hz, 1H), 6.56 (d, J = 15.8 Hz, 1H), 4.32 (d, J = 5.9 Hz, 2H), 4.26 (s, 4H), 3.86 (d, J =
5.8 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 169.68, 166.04, 149.18, 148.49, 145.23,
144.01, 139.05, 135.47, 135.34, 128.81, 123.85, 121.51, 120.56, 117.97, 116.43,
64.75, 64.48, 42.79. ESI-MS m/z: 354 [M-H]+. HPLC purity 99.0%. Retention time:
4.0 min, eluted with 20% purified water/80% methanol.
4.15.7.(E)-N-methyl-3-(naphthalen-1-yl)-N-(2-oxo-2-((pyridin-4-ylmethyl)amino)et

hyl) acryl -amide (11a)
1
The product was obtained as a white solid; yield: 71.8%; mp: 178.3–184.5℃. H
NMR (500 MHz, CDCl3) δ 8.56 (dd, J = 12.7, 10.7 Hz, 3H), 8.19 (d, J = 8.2 Hz, 1H),
7.90 (t, J = 8.5 Hz, 2H), 7.75 (d, J = 7.1 Hz, 1H), 7.53 (ddd, J = 22.3, 12.5, 7.4 Hz,
4H), 7.20 (d, J = 5.5 Hz, 2H), 6.97 (d, J = 15.1 Hz, 1H), 4.49 (d, J = 6.2 Hz, 2H), 4.21
(s, 2H), 3.34 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 169.51, 167.65, 150.03, 147.33,
141.67, 133.69, 132.38, 131.47, 130.47, 128.75, 126.94, 126.35, 125.42, 124.85,
123.49, 122.12, 118.91, 53.55, 42.17, 37.24. ESI-MS m/z: 360 [M-H]+. HPLC purity
99.4%. Retention time: 4.0 min, eluted with 20% purified water/80% methanol.
4.15.8.(E)-N-methyl-3-(naphthalen-1-yl)-N-(2-oxo-2-((pyridin-4-ylmethyl)amino)et
hyl) acryl amide (11b)
1
The product was obtained as a white solid; yield:67.4%; mp: 182.9–186.3℃. H
NMR (500 MHz, CDCl3) δ 8.59 (s, 1H), 8.55 (s, 1H), 8.54 (s, 1H), 8.19 (d, J = 8.2 Hz,
1H), 7.91 (t, J = 8.6 Hz, 2H), 7.73 (d, J = 7.4 Hz, 1H), 7.55 (dt, J = 20.6, 11.4 Hz, 4H),
7.23 (d, J = 5.5 Hz, 2H), 6.98 (d, J = 15.6 Hz, 1H), 4.47 (d, J = 6.2 Hz, 2H), 4.28 (s,
2H), 3.39 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 171.59, 168.56, 150.69, 148.04,
143.87, 135.21, 133.47, 130.23, 130.02, 126.55, 124.14, 123.22, 121.04, 120.22,
117.79, 53.55, 43.08, 39.00. ESI-MS m/z: 360 [M-H]+. HPLC purity 99.4%.
Retention time: 4.2 min, eluted with 20% purified water/80% methanol.
4.15.9.(E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-methyl-N-(2-oxo-2-((pyridin-4
-ylmethyl)amino)ethyl)acrylamide (11c)
1
The product was obtained as a white solid; yield:68.4%; mp: 187.9–192.3℃. H
NMR (500 MHz, CDCl3) δ 8.54 (d, J = 4.3 Hz, 2H), 7.58 (d, J = 15.3 Hz, 1H), 7.48 (s,
1H), 7.27 (s, 2H), 7.08 – 7.00 (m, 2H), 6.86 (d, J = 8.3 Hz, 1H), 6.73 (d, J = 15.3 Hz,
1H), 4.48 (d, J = 5.8 Hz, 2H), 4.28 (dd, J = 9.0, 5.1 Hz, 4H), 4.17 (s, 2H), 3.29 (s, 3H).
13C NMR (126 MHz, CDCl3) δ 169.73, 167.98, 149.53, 148.34, 145.59, 143.88,
143.71, 128.36, 122.62, 122.06, 117.73, 116.53, 114.12, 64.56, 64.24, 53.39, 42.13,
37.21. ESI-MS m/z: 368 [M-H]+. HPLC purity 99.1%. Retention time: 4.1 min, eluted
with 20% purified water/80% methanol.
4.15.10.(E)-N-methyl-3-(naphthalen-1-yl)-N-(2-oxo-2-((pyridin-3-ylmethyl)amino)e
thyl)acryl -amide (12a)
1
The product was obtained as a white solid; yield: 71.8%; mp: 178.3–184.5℃. H
NMR (500 MHz, CDCl3) δ 8.55 – 8.48 (m, 3H), 8.18 (d, J = 8.0 Hz, 1H), 7.88 (d, J =
7.5 Hz, 2H), 7.73 (d, J = 7.0 Hz, 1H), 7.64 (d, J = 7.4 Hz, 1H), 7.58 – 7.47 (m, 4H),
7.17 (s, 1H), 6.95 (d, J = 15.1 Hz, 1H), 4.49 (d, J = 5.8 Hz, 2H), 4.19 (s, 2H), 3.32 (s,
3H). 13C NMR (126 MHz, CDCl3) δ 169.33, 167.56, 148.82, 141.51, 135.43, 133.67,
130.39, 128.72, 126.89, 126.31, 125.41, 124.83, 123.63, 123.55, 119.00, 53.40, 40.88,
37.17. ESI-MS m/z: 360 [M-H]+. HPLC purity 98.4%. Retention time: 4.3 min, eluted
with 20% purified water/80% methanol.
4.15.11. (E)-N-methyl-3-(naphthalen-2-yl)-N-(2-oxo-2-((pyridin-3-ylmethyl) amino)
ethyl) acrylamide (12b)
1
The product was obtained as a white solid; yield:67.4%; mp: 182.9–186.3℃. H
NMR (500 MHz, CDCl3) δ 8.55 – 8.48 (m, 2H), 7.93 – 7.82 (m, 5H), 7.66 (dd, J =
17.4, 8.1 Hz, 2H), 7.57 – 7.40 (m, 3H), 7.16 (s, 1H), 6.99 (d, J = 15.4 Hz, 1H), 4.47 (d,

J = 6.0 Hz, 2H), 4.17 (s, 2H), 3.33 (s, 3H).13C NMR (126 MHz, CDCl3) δ 169.37,
167.73, 148.90, 148.66, 144.36, 135.55, 134.14, 133.32, 132.19, 129.84, 128.68,
128.55, 127.77, 127.22, 126.76, 123.64, 123.51, 116.10, 53.44, 40.84, 37.16. ESI-MS
m/z: 360 [M-H]+. HPLC purity 98.7%. Retention time: 4.2 min, eluted with 20%
purified water/80% methanol.
4.15.12.(E)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-methyl-N-(2-oxo-2-((pyridin-
3-ylmethyl) amino) ethyl) acrylamide (12c)
1
The product was obtained as a white solid; yield:68.4%; mp: 187.9–192.3℃. H
NMR (500 MHz, CDCl3) δ 8.53 – 8.48 (m, 2H), 7.63 (d, J = 7.7 Hz, 1H), 7.58 (d, J =
15.3 Hz, 1H), 7.22 (d, J = 17.0 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.3 Hz,
1H), 6.71 (d, J = 15.3 Hz, 1H), 4.45 (d, J = 6.0 Hz, 2H), 4.30 – 4.27 (m, 4H), 4.14 (s,
2H), 3.26 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 169.48, 167.86, 148.83, 148.55,
145.54, 143.88, 143.68, 135.57, 134.04, 128.42, 123.64, 122.04, 117.69, 116.54,
114.08, 64.55, 64.23, 53.40, 40.78, 37.06. ESI-MS m/z: 368 [M-H]+. HPLC purity
99.5%. Retention time: 4.3 min, eluted with 20% purified water/80% methanol.
4.15.13.(E)-2-(2-(naphthalen-1-ylmethylene)hydrazinyl)-2-oxo-N-(pyridin-4-yl meth
-yl) acetamide (17a)
1
The product was obtained as a white solid; yield: 70.5%; mp: 221.3–226.8℃. H
NMR (500 MHz, DMSO) δ 12.26 (s, 1H), 9.68 (t, J = 6.2 Hz, 1H), 9.33 (s, 1H), 8.69
(d, J = 8.5 Hz, 1H), 8.55 (dd, J = 28.0, 9.3 Hz, 2H), 8.16 – 7.88 (m, 3H), 7.72 – 7.55
13
(m, 3H), 7.39 – 7.21 (m, 2H), 4.53 – 4.35 (m, 2H). C NMR (126 MHz, DMSO) δ
160.71, 156.68, 150.91, 150.06, 147.92, 133.96, 131.51, 130.93, 129.67, 129.33,
127.90, 127.78, 126.82, 126.08, 124.20, 122.64, 42.01. ESI-MS m/z: 333 [M-H]+.
HPLC purity 99.2%. Retention time: 4.2 min, eluted with 20% purified water/80%
methanol.
4.15.14.(E)-2-(2-(naphthalen-2-ylmethylene)hydrazinyl)-2-oxo-N-(pyridin-4-ylmeth
yl)acetamide (17b)
1
The product was obtained as a white solid; yield:67.4%; mp: 224.7–230.5℃. H
NMR (500 MHz, DMSO) δ 12.32 (s, 1H), 9.69 (t, J = 6.3 Hz, 1H), 8.79 (s, 1H), 8.57
(d, J = 5.5 Hz, 2H), 8.19 (s, 1H), 8.06 – 8.01 (m, 3H), 7.64 (dd, J = 8.9, 5.2 Hz, 3H),
7.35 (d, J = 5.7 Hz, 2H), 4.48 (d, J = 6.3 Hz, 2H). 13C NMR (126 MHz, DMSO) δ
160.72, 151.37, 150.04, 147.92, 134.44, 133.27, 132.11, 129.84, 129.06, 128.95,
128.25, 127.87, 127.30, 123.16, 122.68, 42.02. ESI-MS m/z: 333 [M-H]+. HPLC
purity 99.5%. Retention time: 3.9 min, eluted with 20% purified water/80% methanol.
4.15.15.(E)-2-(2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methylene)hydrazinyl)-2-oxo
-N-(pyridin-4-ylmethyl)acetamide (17c)
1
The product was obtained as a white solid; yield:68.4%; mp: 230.2–236.6℃. H
NMR (500 MHz, DMSO) δ 12.07 (s, 1H), 9.61 (t, J = 6.4 Hz, 1H), 8.51 (d, J = 5.9 Hz,
2H), 8.43 (s, 1H), 7.28 (d, J = 5.8 Hz, 2H), 7.20 – 7.16 (m, 2H), 6.93 (t, J = 7.1 Hz,
1H), 4.40 (d, J = 6.4 Hz, 2H), 4.29 (dd, J = 9.0, 5.0 Hz, 4H).13C NMR (126 MHz,
DMSO) δ 160.74, 156.56, 150.97, 150.03, 147.94, 146.17, 144.12, 127.73, 122.62,
121.64, 118.02, 115.90, 64.81, 64.48, 41.95. ESI-MS m/z: 341 [M-H]+. HPLC purity
99.3%. Retention time: 3.9 min, eluted with 20% purified water/80% methanol.
4.15.16. (E)-2-(2-(naphthalen-1-ylmethylene)hydrazinyl)-2-oxo-N-(pyridin-4-ylmeth

-yl) acetamide (18a)
1
The product was obtained as a white solid; yield: 74.2%; mp: 231.7–236.4℃. H
NMR (500 MHz, DMSO) δ 12.22 (s, 1H), 9.54 (d, J = 4.4 Hz, 1H), 9.35 (d, J = 3.3
Hz, 1H), 8.71 – 8.64 (m, 1H), 8.59 (s, 1H), 8.47 (d, J = 3.2 Hz, 1H), 8.03 (t, J = 4.8
Hz, 1H), 7.98 – 7.91 (m, 2H), 7.74 (d, J = 6.5 Hz, 1H), 7.64 – 7.52 (m, 3H), 7.31 (dt,
J = 8.0, 4.1 Hz, 1H), 4.49 (d, J = 6.1 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 160.31,
156.62, 150.67, 149.50, 148.56, 135.73, 134.31, 133.79, 131.13, 131.07, 129.69,
128.93, 127.41, 126.37, 125.55, 124.06, 123.58, 40.75. ESI-MS m/z: 333 [M-H]+.
HPLC purity 98.7%. Retention time: 4.0 min, eluted with 20% purified water/80%
methanol.
4.15.17.(E)-2-(2-(naphthalen-2-ylmethylene)hydrazinyl)-2-oxo-N-(pyridin-4-ylmeth
yl)acetamide (18b)
1
The product was obtained as a white solid; yield:66.9%; mp: 226.5–232.7℃. H
NMR (500 MHz, DMSO) δ 12.18 (s, 1H), 9.63 (s, 1H), 9.38 (s, 1H), 8.71 (d, J = 8.8
Hz, 1H), 8.57 (d, J = 5.7 Hz, 2H), 8.07 – 7.96 (m, 3H), 7.61 – 7.56 (m, 3H), 7.32 (d, J
= 5.4 Hz, 2H), 4.46 (d, J = 6.7 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 161.79,
158.09, 151.14, 148.52, 146.31, 134.39, 131.98, 130.45, 129.67, 129.33, 128.30,
126.54, 124.81, 123.45, 121.03, 42.70. ESI-MS m/z: 333 [M-H]+. HPLC purity 99.1%.
Retention time: 4.0 min, eluted with 20% purified water/80% methanol.
4.15.18.(E)-2-(2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methylene)hydrazinyl)-2-oxo
-N-(pyridin-4-ylmethyl)acetamide (18c)
1
The product was obtained as a white solid; yield:70.9%; mp: 231.1–237.9℃. H
NMR (500 MHz, DMSO) δ 11.70 (s, 1H), 9.09 (d, J = 4.3 Hz, 1H), 8.59 (d, J = 1.9
Hz, 1H), 8.50 (dd, J = 4.7, 1.5 Hz, 1H), 8.39 (d, J = 4.3 Hz, 1H), 7.75 – 7.67 (m, 1H),
7.36 – 7.19 (m, 3H), 6.84 (dd, J = 8.3, 4.4 Hz, 1H), 4.57 – 4.45 (m, 2H), 4.34 – 4.22
(m, 4H). 13C NMR (126 MHz, DMSO) δ 160.15, 156.21, 151.29, 149.43, 148.67,
146.02, 143.74, 135.68, 133.76, 127.28, 123.49, 121.81, 117.39, 116.37, 64.53, 64.15,
40.83. ESI-MS m/z: 341 [M-H]+. HPLC purity 99.3%. Retention time: 4.0 min, eluted
with 20% purified water/80% methanol.
4.15.19.(E)-N-(1-(benzylamino)-3-(1H-imidazol-1-yl)-1-oxopropan-2-yl)-3-(naphth
alen-1-yl)acrylamide (24a)
1
The product was obtained as a white solid; yield: 64.3%; mp: 162.3–168.6℃. H
NMR (500 MHz, CDCl3) δ 8.85 (d, J = 14.6 Hz, 1H), 8.54 (d, J = 15.4 Hz, 1H), 8.21
(d, J = 8.3 Hz, 1H), 7.91 – 7.85 (m, 2H), 7.73 –7.53 (dt, J = 12.8, 12.3 Hz, 9H), 7.03
(d, J = 18.8 Hz, 3H), 6.85 (s, 1H), 6.62 (d, J = 15.4 Hz, 1H), 5.08 – 4.97 (m, 1H), 4.56
13
(d, J = 3.7 Hz, 2H), 4.28 (pd, J = 6.5, 3.6 Hz, 2H). C NMR (126 MHz, CDCl3) δ
169.25, 165.83, 142.79, 140.85, 137.67, 135.03, 134.17, 133.30, 131.78, 131.52,
130.73, 129.91, 129.25, 128.71, 128.55, 127.78, 127.21, 126.73, 125.09, 123.47,
121.61, 119.65, 119.32, 62.71, 53.72, 47.60. ESI-MS m/z: 425 [M-H]+. HPLC purity
99.0%. Retention time: 4.0 min, eluted with 20% purified water/80% methanol.
4.15.20.(E)-N-(1-(benzylamino)-3-(1H-imidazol-1-yl)-1-oxopropan-2-yl)-3-(naphth
alen-2- yl)acrylamide (24b)
1
The product was obtained as a white solid; yield:63.1%; mp: 159.2–164.2℃. H
NMR (500 MHz, CDCl3) δ 8.74 (d, J = 12.1 Hz, 1H), 8.56 (d, J = 15.4 Hz, 1H), 8.22

(d, J = 8.3 Hz, 1H), 7.85 – 7.81 (m, 2H), 7.67 – 7.44 (m, 9H), 7.08 (s, 3H), 6.82 (s,
1H), 6.65 (d, J = 15.4 Hz, 1H), 5.06 – 5.03 (m, 1H), 4.62 (d, J = 3.7 Hz, 2H), 4.32 (dt,
J = 7.0, 4.2 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 169.85, 164.85, 143.90, 140.83,
137.40, 135.98, 135.19, 132.56, 130.61, 129.76, 128.99, 128.55, 127.12, 126.26,
124.52, 123.47, 119.65, 119.32, 61.16, 52.05, 44.80. ESI-MS m/z: 425 [M-H]+. HPLC
purity 99.5%. Retention time: 4.0 min, eluted with 20% purified water/80% methanol.
4.15.21.(E)-N-(1-(benzylamino)-3-(1H-imidazol-1-yl)-1-oxopropan-2-yl)-3-(2,3-dihy
drobenzo[b][1,4]dioxin-6-yl)acrylamide (24c)
1
The product was obtained as a white solid; yield:68.5%; mp: 167.4–172.1℃. H
NMR (500 MHz, DMSO) δ 8.71 (t, J = 5.8 Hz, 1H), 8.64 (d, J = 8.5 Hz, 1H), 7.62 (s,
1H), 7.42 (t, J = 8.2 Hz, 2H), 7.32 (m, J = 7.5 Hz, 3H), 7.26-7.24 (m, 4H), 7.16 (s,
1H), 7.00 (d, J = 8.1 Hz, 1H), 6.82 (s, 1H), 4.46 (td, J = 10.1, 4.3 Hz, 1H), 4.32 (dd, J
13
= 13.9, 4.2 Hz, 2H), 4.29 (d, J = 6.0 Hz, 2H), 4.06 – 3.96 (m, 4H). C NMR (126
MHz, DMSO) δ 169.33, 164.28, 148.92, 148.70, 146.08, 142.85, 138.42, 136.28,
128.78, 128.23, 127.18, 121.92, 119.92, 117.18, 117.04, 65.33, 64.51, 57.57, 54.78,
51.46. ESI-MS m/z: 425 [M-H]+. HPLC purity 99.2%. Retention time: 3.8 min, eluted
with 20% purified water/80% methanol.
4.15.22.(E)-N-(3-(1H-imidazol-1-yl)-1-oxo-1-(phenylamino)propan-2-yl)-3-(naphth
alen-1-yl)acrylamide (25a)
The product was obtained as a white solid; yield: 63.9%; mp: 156.5–161.7℃. 1H
NMR (500 MHz, DMSO) δ 8.54 (d, J = 15.4 Hz, 1H), 8.30 (d, J = 13.5 Hz, 1H), 7.97
(d, J = 6.5 Hz, 1H), 7.71 – 7.59 (m, 5H), 7.47 (m, 3H), 7.26 (dd, J = 42.6, 14.9 Hz,
6H), 7.05 (s, 1H), 6.69 (d, J = 9.5 Hz, 1H), 5.14 – 4.84 (m, 1H), 4.41 – 4.21 (m, 2H).
13C NMR (126 MHz, DMSO) δ 168.36, 164.88, 140.36, 138.93, 136.63, 135.06,
133.16, 131.53, 131.29, 130.55, 129.93, 129.56, 128.31, 128.29, 128.02, 127.65,
127.24, 127.23, 124.93, 122.06, 120.58, 61.61, 53.73. ESI-MS m/z: 411 [M-H]+.
HPLC purity 98.9%. Retention time: 4.0 min, eluted with 20% purified water/80%
methanol.
4.15.23.(E)-N-(3-(1H-imidazol-1-yl)-1-oxo-1-(phenylamino)propan-2-yl)-3-(naphth
alen-2-yl)acrylamide (25b)
1
The product was obtained as a white solid; yield:63.1%; mp: 158.3–163.8℃. H
NMR (500 MHz, DMSO) δ 8.54 (d, J = 15.4 Hz, 1H), 8.30 (d, J = 13.5 Hz, 1H), 7.97
(d, J = 6.5 Hz, 1H), 7.71 – 7.59 (m, 5H), 7.47 (m, 3H), 7.26 (dd, J = 42.6, 14.9 Hz,
6H), 7.05 (s, 1H), 6.69 (d, J = 9.5 Hz, 1H), 5.14 – 4.84 (m, 1H), 4.41 – 4.21 (m, 2H).
13C NMR (126 MHz, DMSO) δ 168.36, 164.88, 140.36, 138.93, 136.63, 135.06,
133.16, 131.53, 131.29, 130.55, 129.93, 129.56, 128.31, 128.29, 128.02, 127.65,
127.24, 127.23, 124.93, 122.06, 120.58, 61.61, 53.73. ESI-MS m/z: 411 [M-H]+.
HPLC purity 99.1%. Retention time: 4.0 min, eluted with 20% purified water/80%
methanol.
4.15.24.(E)-N-(3-(1H-imidazol-1-yl)-1-oxo-1-(phenylamino)propan-2-yl)-3-(2,3-Dih
ydrobenzo[b][1,4]dioxin-6-yl)acrylamide (25c)
1
The product was obtained as a white solid; yield:68.5%; mp: 160.6–165.3℃. H
NMR (500 MHz, DMSO) δ 8.65 (d, J = 8.5 Hz, 1H), 8.46 (s, 1H), 7.62 (s, 1H), 7.38
(dt, J = 15.0, 12.2 Hz, 5H), 7.25 (d, J = 7.3 Hz, 2H), 7.16 (m, 3H), 6.99 (d, J = 8.4 Hz,

1H), 6.78 (d, J = 16.3 Hz, 1H), 4.84 (td, J = 10.1, 4.3 Hz, 1H), 4.52 – 4.33 (m, 2H),
4.13 (t, J = 23.1 Hz, 4H). 13C NMR (126 MHz, DMSO) δ 171.70, 167.20, 149.14,
148.72, 147.55, 144.19, 139.27, 136.06, 131.13, 128.78, 127.77, 127.45, 125.56,
123.81, 121.52, 117.74, 117.02, 65.70, 65.53, 58.41, 50.40. ESI-MS m/z: 419 [M-H]+.
HPLC purity 99.4%. Retention time: 3.7 min, eluted with 20% purified water/80%
methanol.
4.16. Protein structure preparation and ligand preparation
Protein structure preparation: The homology model of SE and protein crystals
structure of CYP51 (5TZ1) were processed with the protein preparation module in the
DS 3.5. The program was performed as follows: Added all hydrogen atoms, optimized
the side chain of amino acid residues, completed the loop area and protonated the
structure at the specified pH value. Fragment and Ligand preparation: Active fragment
group and target compounds were optimized using LigPrep, which included adding all
hydrogen atoms, adjusting the bond order and atom types, adjusting the appropriate
protonation and generating the conformations with low energy.
4.17. Docking simulations
In order to evaluate the binding modes between active fragments (or target
compound 17a) with target enzymes (SE, CYP51), the molecular docking was
performed using MCSS (or CDOCKER), which is the semi-flexible docking program.
First, the binding region of co-crystallized ligand was selected as the active center of
target enzyme (The Site Sphere of the SE homology model-grid is -19.799, 73.637,
55.144); The position of the 5V5Z-grid is -37.5166, -17.4582,26.2275, 15.3922), and
the parameter of active sphere was set to 9 Å. Then, the active fragments and target
compound 17a were docked into the active sites. The maximum save conformation
was set to 10, and all other options were kept as the default settings during the
docking process. Finally, the docking result was obtained.
4.18. Antifungal activity test in vitro
The antifungal activity of target compounds was tested using the standard guidelines,
which described in the National Committee for Clinical Laboratory Standards
(NCCLS), and the in vitro minimum inhibitory concentrations (MIC) value was defined
as the lowest concentration of antifungal inhibitor with inhibitory effect. Naftifine and
fluconazole were selected as positive control drugs. In the study, the pathogenic fungi
were cultured using the Sabouraud medium, and the fungal solution was added into
24-well culture plate. The different concentration of target compounds solution was
serially dripped into the growth medium with different strains, and the concentration
gradient was set to 0.0625, 0.125, 0.5, 1, 2, 4, 8 and 16 μg/mL, respectively.
Subsequently, they were statically cultured in a 35 °C incubator, and the MIC results
were observed and recorded.
4.19. The Experiment of Transmission Electron Microscopy (TEM)
According to the test protocol of NCCLS, C.alb. SC5314 was selected as the test
strain. The concentration of target compound 17a in the bacterial solution was adjusted
to 4 ug/mL, and they were incubated at 35℃. Subsequently, the bacterial solution was
sampled at different time points (72h), and they were fixed in 4℃ refrigerators with
3% glutaric acid, washed with PBS solution. Finally, these samples were further stained

with uranium osmium for 30 min, they were observed using transmission electron
microscopy.
4.20. The analysis experiment of the components of C. albicans cells
In this process, C.alb. SC5314 was selected as the test strain. Naftifine and
fluconazole were purchased as the positive control drug. In the study, the concentration
of target compounds was set to 4 μg/mL. After 16 hours of cultivation, the different
groups of wet bacteria are concentrated and washed with PBS, and the sterol
component was extracted using petroleum ether. The solvent was removed under
reduced pressure. Finally, the product was dissolved in methanol solution (10mL),
filtered and detected by LC-MS. The chromatographic conditions were selected with
Phenomenex Luna C18 (2.50 mm×4.6 mm, 5μm) column. The mobile phase was
methanol-water (98:2), the flow rate was 1 mL/min-1, and the detection wavelength was
210 nm. The peak area ratio of each component is calculated.
4.21. In Vivo Therapeutic Efficacy
These mice with abdominal Candida infection were randomly divided into 3
groups: (1) Saline solution-injected group; (2) Compound 17a-injected group; (3)
Compound 25a-injected group. Therapy was continued through abdominal injection
for 7 days. Finally, these mice tissues, including the heart, livers, spleen, lung, kidney,
and endothelial tissue in abdominal infection, were harvested and collected
immediately for H/E histology analysis. All of the animal experiments were
conducted under approved protocols of the Institutional Animal Care and Use
Committee of Liaocheng University.
4.22. ADME/T prediction
The pharmacokinetic properties and toxicity (ADME/T) are an important index of
the drug-forming property of target compounds. In the study, the target compounds
were predicted using the DS ADMET program in Discovery Studio 3.5. The specific
operation process was performed as follows: Frist, the small molecule compound
files containing target compounds were imported. Then, the “ADMET descriptors”
module was selected and opened, the prediction items (aqueous solubility, blood
brain barrier penetration, CYP2D6 binding, hepatotoxicity, intestinal absorption and
plasma protein binding) were set as research objects in the parameter browser,
respectively. Finally, the program was run to obtain the corresponding results.
Declaration of interest
The authors report no declarations of interest.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(Grant No. 81703357, Grant No. 21473085, Grant No.31701827), the Shandong
Provincial Natural Science Foundation (Grant No. ZR2017BH102) and the Open
Project of Shandong Cllaborative Innovation Center for Antibody Drugs (Grant No.
CIC-AD1837and the Open Project of Shandong Collaborative Innovation Center for
Antibody Drugs, and Tai-Shan Scholar Research Fund of Shandong Province of
China. This work was also technically supported by Engineering Research Center for
Nanomedicine and Drug Delivery Systems.
References

[1] D.A. Enoch, H. Yang, S.H. Aliyu, C. Micallef, The changing epidemiology of
invasive fungal infections, Methods Mol. Biol. 1508 (2017) 17-65.
[2] Y. Wu, S. Du, J.L. Johnson, H.Y. Tung, C.T. Landers, Y. Liu, B.G. Seman, R.T.
Wheeler, M. Costa-Mattioli, F. Kheradmand, H. Zheng, D.B. Corry, Microglia and
amyloid precursor protein coordinate control of transient Candida cerebritis with
memory deficits, Nat. Commun. 10 (2019) 58-73.
[3] C.M. Calvet, D.F. Vieira, J.Y. Choi, D. Kellar, M.D. Cameron, J.L. Siqueira-Neto,
J. Gut, J.B. Johnston, L. Lin, S. Khan, J.H. McKerrow, W.R. Roush, L.M. Podust,
4-Aminopyridyl-Based Cyp51 Inhibitors as Anti-Trypanosoma Cruzi Drug Leads
with Improved Pharmacokinetic Profile and in Vivo Potency, J. Med. Chem. 57 (2014)
6989-7005.
[4] N.M. Revie, K.R. Iyer, N. Robbins, L.E. Cowen, Antifungal drug resistance:
evolution, mechanisms and impact, Curr. Opin. Microbiol. 45 (2018) 70-76.
[5] B. Sun, W.X. Huang, M. Liu, Evaluation of the combination mode of azoles
antifungal inhibitors with CACYP51 and the influence of site-directed mutation, J.
Mol. Graph. Model. 73 (2017) 157-165.
[6] R.S. Shapiro, N. Robbins, L.E. Cowen, Regulatory circuitry governing fungal
development, drug resistance, and disease, Microbiol. Mol. Biol. Rev. 75 (2011)
213-267.
[7] M.K. Kathiravan, A.B. Salake, A.S. Chothe, P.B. Dudhe, R.P. Watode, M.S.
Mukta, S. Gadhwe, The biology and chemistry of antifungal agents: a review, Bioorg.
Med. Chem. 20 (2012) 5678-5698.
[8] Y.F. An, Y. Dong, M. Liu, L.Y. Zhao, D.M. Zhao, J. Han, B. Sun, Construction
and Evaluation of Molecular Models: Guide and Design of Novel SE Inhibitors, ACS
Med. Chem. Lett. 11(2020) 1152-1159.
[9] T.Y. Hargrove, L. Friggeri, Z. Wawrzak, A. Qi, W.J. Hoekstra, R.J. Schotzinger,
J.D. York, F. Peter Guengerich, G.I. Lepesheva, Structural analyses of Candida
albicans sterol 14a-demethylase complexed with azole drugs address the molecular
basis of azole-mediated inhibition of fungal sterol biosynthesis, J. Biol. Chem. 292
(2017) 6728-6743.
[10] M. Nowosielski, M. Hoffmann, L.S. Wyrwicz, P. Stepniak, D.M. Plewczynski,
M. Lazniewski, K. Ginalski, L. Rychlewski, Detailed Mechanism of Squalene
Epoxidase Inhibition by Terbinafine, J. Chem. Inf. Model. 51(2011) 455-462.
[11] V. Moudgal, J. Sobel, Antifungals to treat Candida albicans, Expert. Opin.
Pharmacother. 11 (2010) 2037-2048.
[12] D.A. Enoch, H.A. Ludlam, N.M. Brown, Invasive fungal infections: a review of
epidemiology and management options, J. Med. Microbiol. 55 (2006) 809-818.
[13] P. Pooja, P. Prasher, K. Singh, K.S. Pawar, N. Vikramdeo, S.S. Mondal, Komath,
Synthesis of amino acid appended indoles: appreciable anti-fungal activity and
inhibition of ergosterol biosynthesis as their probable mode of action, Eur. J. Med.
Chem. 80 (2014) 325-339.
[14] Z. Zhang, Z.W. Bai, Y. Ling, L.Q. He, P. Huang, H.X. Gu, R.F. Hu, Design,
synthesis and biological evaluation of novel furoxan-based coumarin derivatives as
antitumor agents, Med. Chem. Res. 27 (2018) 1198-1205.

[15] R.I. Benhamou, M. Bibi, K.B. Steinbuch, H. Engel, M. Levin, Y. Roichman, J.
Berman, M. Fridman, Real-time imaging of the azole class of antifungal drugs in live
Candida cells, ACS Chem. Biol. 12 (2017) 1769-1777
[16] M.V. Papadopoulou, G.I. Bloomer, H.S. Lepesheva, M. Rosenzweig, M. Kaiser,
B. Aguilera-Venegas, S.R. Wilkinson, E. Chatelain, J.R. Ioset, Novel 3-
nitrotriazole-based amides and carbinols as bifunctional antichagasic agents, J. Med.
Chem. 58 (2015) 1307-1319.
[17] C.W. Murray, M.L. Verdonk, D.C. Rees, Experiences in fragment-based drug
discovery, Trends Pharmacol. Sci. 33 (2012) 224-232.
[18 B. Sun, W. Huang, M. Liu, K. Lei, Comparison and analysis of the structures and
binding modes of antifungal SE and CYP51 inhibitors, J. Mol. Graph. Model. 77
(2017) 1-8.
[19] B. Sun, H. Zhang, Y. Dong, L. Zhao, M. Liu, Evaluation of the combination
mode and features of p38 MAPK inhibitors: construction of different pharmacophore
models and molecular docking. Mol. Simulat. 45 (2019) 975-984.
[20] T.M. Gonzalez Santiago, B. Pritt, L.E. Gibson, N.I. Comfere, Diagnosis of deep
cutaneous fungal infections: Correlation between skin tissue culture and
histopathology, J. Am. Acad. Dermatol. 71 (2014) 293-301.
[21] S. Zhao, L. Zhao, X. Zhang, C. Liu, C. Hao, H. Xie, B. Sun, D. Zhao, M. Cheng,
Design, synthesis, and structure-activity relationship studies of benzothiazole
derivatives as antifungal agents, Eur. J. Med. Chem. 123 (2016) 514-522.
[22] B. Sun, Y. Dong, K. Lei, J. Wang, L. Zhao, M. Liu, Design, synthesis and
biological evaluation of amide-pyridine derivatives as novel dual-target (SE, CYP51)
antifungal inhibitors, Bioorg. Med. Chem. 27 (2019) 2427-2437.
[23] D. Zhao, S. Zhao, L. Zhao, X. Zhang, P. Wei, C. Liu, C. Hao, B. Sun, X. Su, M.
Cheng, Discovery of biphenyl imidazole derivatives as potent antifungal agents:
design,synthesis, and structure-activity relationship studies, Bioorg. Med. Chem. 25
(2017) 750-758.
[24] I.D. Kuntz, Structure-based strategies for drug design and discovery, Science.
257 (1992) 1078-1082.
[25] S.Z. Zhao, X.Q. Zhang, P. Wei, X. Su, L.Y. Zhao, M.Y. Wu, C.Z. Hao, C.C. Liu,
D.M. Zhao, M.S. Cheng, Design, synthesis and evaluation of aromatic heterocyclic
derivatives as potent antifungal agents, Eur. J. Med. Chem. 137(2017) 96-107.
[26] S.C. Chen, M.A. Slavin, T.C. Sorrell, Echinocandin antifungal drugs in fungal
infections: a comparison, Drugs. 71 (2011) 11-41.
[27] S.H. Chen, C.Q. Sheng, X.H. Xu, Y.Y. Jiang, W.N. Zhang, C. He, Expression
and detection the enzyme activity of the wild and mutation type of CYP51 protein of
candida albicans, Microbiology. 36 (2009) 1564-1570.
[28] Y. Dong, M. Liu, J. Wang, Z. Ding, B. Sun, Construction of antifungal dual-
target (SE, CYP51) pharmacophore models and the discovery of novel antifungal
inhibitors, RSC Adv. 9 (2019) 26302-26314.
[29] L.Y.Zhao, L.F. Tian, N.N. Sun, Y. Sun, Y.X. Chen, X.R. Wang, S.Z. Zhao, X.
Su, D.M. Zhao, M.S. Cheng, Design, synthesis, and structure-activity relationship
studies of l-amino alcohol derivatives as broad-spectrum antifungal agents, Eur. J.