Novel naphthylamide derivatives as dual-target antifungal inhibitors: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2020.112991

Fungal infections have become a serious medical problem due to the high infection rate and the frequent emergence of drug resistance. Squalene epoxidase (SE) and 14α-demethylase (CYP51) are considered as the important antifungal targets, they can show the synergistic effect on antifungal therapy. In the study, a series of active fragments were screened through the method of De Novo Link, and these active fragments with the higher Ludi_Scores were selected, which can show the obvious binding ability with the dual targets (SE, CYP51). Subsequently, three series of target compounds with naphthyl amide scaffolds were constructed by connecting these core fragments, and their structures were synthesized. Most of compounds showed the antifungal activity in the treatment of pathogenic fungi. It was worth noting that compounds 10b-5 and 17a-2 with the excellent broad-spectrum antifungal properties also exhibited the obvious antifungal effects against drug-resistant fungi. Preliminary mechanism study has proved these target compounds can block the biosynthesis of ergosterol by inhibiting the activity of dual targets (SE, CYP51). Furthermore, target compounds 10–5 and 17a-2 with low toxicity side effects also demonstrated the excellent pharmacological effects in vivo. The molecular docking and ADMET prediction were performed, which can guide the optimization of subsequent lead compounds.

Full text of DOI:10.1016/j.ejmech.2020.112991

Journal Pre-proof
Novel naphthylamide derivatives as dual-target antifungal inhibitors: Design,
synthesis and biological evaluation
Yunfei An, Yue Dong, Min Liu, Jun Han, Liyu Zhao, Bin Sun
PII:
S0223-5234(20)30963-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112991
EJMECH 112991
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 September 2020
Revised Date: 15 October 2020
Accepted Date: 31 October 2020
Please cite this article as: Y. An, Y. Dong, M. Liu, J. Han, L. Zhao, B. Sun, Novel naphthylamide
derivatives as dual-target antifungal inhibitors: Design, synthesis and biological evaluation, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112991.
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Graphical Abstract

Novel naphthylamide derivatives as dual-target antifungal
inhibitors: Design, synthesis and biological evaluation
a, 1
a, 1
a
a
b
a,∗
Yunfei An , Yue Dong , Min Liu , Jun Han , Liyu Zhao , Bin Sun
a
Institute of BioPharmaceutical Research, Liaocheng University, 1 Hunan Road, Liaocheng 252000, PR China
b
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: Bin Sun, E-mail: sunbin19840104@163.com
ABSTRACT:
Fungal infections have become a serious medical problem due to the high infection
rate and the frequent emergence of drug resistance. Squalene epoxidase (SE) and
1
4α-demethylase (CYP51) are considered as the important antifungal targets, they can
show the synergistic effect on antifungal therapy. In the study, a series of active fragments
were screened through the method of De Novo Link, and these active fragments with the
higher Ludi_Scores were selected, which can show the obvious binding ability with the
dual targets (SE, CYP51). Subsequently, three series of target compounds with naphthyl
amide scaffolds were constructed by connecting these core fragments, and their structures
were synthesized. Most of compounds showed the antifungal activity in the treatment of
pathogenic fungi. It was worth noting that compounds 10b-5 and 17a-2 with the excellent
broad-spectrum antifungal properties also exhibited the obvious antifungal effects against
drug-resistant fungi. Preliminary mechanism study has proved these target compounds
can block the biosynthesis of ergosterol by inhibiting the activity of dual targets (SE,
CYP51). Furthermore, target compounds 10-5 and 17a-2 with low toxicity side effects
also demonstrated the excellent pharmacological effects in vivo. The molecular docking
and ADMET prediction were performed, which can guide the optimization of subsequent
lead compounds.
Key Words: Fungal infections; Dual-Target; Inhibitors; Organic synthesis; Antifungal
activity
`

1
. Introduction
Over the past two decades, the incidence of fungal infections has been increasing
dramatically, especially the emergence of systemic fungal infection and fungal resistance,
it has presented a serious threat to human health [1,2,3]. These infections occurrence has
resulted from many factors, such as the widespread abuse of broad-spectrum antibiotics,
immunosuppressants, radiotherapy and chemotherapy drugs in clinic, which seriously
destroyed the normal physiological function in human body [4-7]. However, we have
lacked of the effective treatment for these fungal diseases. This requires us to constantly
develop new antifungal drugs to cope with the growing problems.
At present, the clinically available antifungal agents can be divided into two different
classes based on their structure characteristics, including antibiotics (e.g., polyenes,
echinocandins) and small molecular compounds (e.g., azoles, acrylamines and
5
-fluorocytosine) [8-11]. Of those, acrylamines and azole antifungal agents as small
molecular compounds are currently most widely used in first-line antifungal therapy
Figure 1), especially there has still a large number of azoles compounds reported
(
constantly [12-15]. They can block the ergosterol biosynthesis by inhibiting separately
the activity of squalene epoxidase (SE) and 14α-demethylase (CYP51) [16,17,18]. When
the ergosterol content decreased, fungal cell permeability and osmotic pressure would
change [19,20]. At the same time, some toxic endogenous intermediates (e.g., lanosterol
and 14a-methyl-3-6-diol) also were accumulated rapidly in the fungal cells, which
eventually lead to fungal death [21,22]. In addition, azoles (e.g., miconazole, fluconazole,
albaconazole) and acrylamides (e.g., butenafine, terbinafine, naftifine) drugs can also be
selected as the combination drugs to treat fungal infection, especially for the
drug-resistant emergence of pathogenic fungi [23,24]. They exhibited the obvious
synergistic effects and excellent antifungal activity in vitro [25]. However, several factors
have limited their practical applications, such as uneven absorption, cytotoxicity,
drug resistant and low bioavailability [26,27]. Therefore, there is still an urgent task to
develop novel antifungal agents. Dual-target drugs or multi-target drugs can avoid these
disadvantages through the simultaneous inhibition of signaling pathways [28,29].
Dual-target or multi-target drugs can regulate multiple links in the disease network
system by combining more than two targets. The resulting synergy effect improve the
efficacy of drugs and reduce the development of drug resistance. In addition, these
inhibitors generally have weak affinity with biological targets, which can greatly reduce
the occurrence of adverse drug reactions [30,31]. Based on this principle, we aimed to
construct the novel dual-target antifungal inhibitors by analyzing the active sites of
dual-target (SE, CYP51).

Figure. 1 Structure of target enzymes (SE, CYP51) and the representative acrylamines (SE inhibitors)
and azole antifungal agents (CYP51 inhibitors).
In the study, we studied the structural characteristics of acrylamine and azole drugs,
and constructed the different series of novel dual-target (SE, CYP51) antifungal
inhibitors using the method of fragment-based drug discovery (FBDD). Subsequently,
these target compounds were further synthesized, and their antifungal activity were
evaluated in vivo and vitro. At the same time, the preliminary action mechanism was also
explored and proved using cell-based and biochemical assays. This study will provide a
new direction to design the novel dual-target antifungal inhibitors.
2
. Results and discussion
2
.1. Dual-target docking study based on active fragments
With the development of protein structure diffraction technology, the crystal structure
of CYP51 has been resolved and studied [32]. At the same time, the homology model of
SE has also been constructed in our previous study [33]. However, it is still a great
challenge to discover the corresponding target inhibitors. This is because their binding
conformation is difficult to be determined in the conventional molecular screening
process. De novo drug design is a brand-new method of fragment-based drug discovery
(
FBDD) [34,35]. It can be used to more accurately determine the location, orientation and
conformation of the core fragments in the different active sites.

Figure. 2. The screening of active fragments based on the active sites of dual-target enzymes (SE,
CYP51).
The drug-like fragment library was constructed, which contain a series of different
kinds of organic fragments. De Novo Link, which can attach new fragments based on the
existing active groups, was selected as a classical important fragment docking method,
and the value of Ludi_Score can accurately reflect the binding ability of docking
fragments in active site (Figure 2). In the study, these active fragments in library were
docked into the active sites of dual-target (SE, CYP51). Some of organic fragments
demonstrated the strong binding ability, they can simultaneously produce this strong
interaction force with the key amino acid residues, which distributed in the different
active sites. Ten preferred fragments with high value of Ludi_Score were selected and
determined, which were summarized in Table 1.
Table 1. The scoring values for preferred docking fragments
Name
Fragment
structure
Potential Energy
Ludi_Score
SE
CYP51
1. 1-naphthoic acid
104.9261
331
348
2. 5-Amino-1-benzofuran
3. Aminomethyl pyridine
4. 9H-pyrido[3,4-b]
98.9786
83.0628
49.2029
279
308
287
316
324
301
indole
5
. 1-ethyl-1H-imidazole
97.1579
72.7368
301
284
298
296
6. N-methyl-1H-imidazole-1
-
carboxamide
7
. 2-Aminoacetophenone
103.2519
49.0356
314
252
291
280
8. N-phenylmethanetriamine
9
. Cyclohexaneamine
0. Resorcinol
55.6181
86.6885
279
249
264
237
1

Subsequently, we analyzed the structures of the inhibitors (SE inhibitor: naftifine;
CYP51 inhibitor: compound 5) [30,33] and their binding mode in their corresponding
active sites. These screening fragments (1, 2, 4) were located in their upper region of
active sites (SE, CYP51). Among them, the naphthyl fragment (1) with the highest value
of Ludi_Score (SE: 331; CYP51: 348) can form the most stable interactions with the
surrounding key amino acid residues, which were distributed in the active regions (A)
1 3
(Figure 3 A -A ). Moreover, it also is the main chain group of compound 5 (CYP51
inhibitor) and naftifine (SE inhibitor). Therefore, we discarded the active fragments (2, 4),
and selected to retain the naphthyl fragment as the main chain group to design the new
target compound. In addition, we further analyzed the active site characteristics of the
dual target, it can be seen that their hydrophobic feature groups were distributed in the all
central region of active sites (SE, CYP51), which indicates that organic fragments should
1 2
retain a certain of hydrophobic properties (Figure 3 B -B ). Based on the above result,
we found that these screening fragments (3, 5, 7, 9) with the proper scoring value can
satisfy a certain degree of hydrophobic properties, they were distributed in the active
regions (B, C), and produced the proper superposition phenomenon with ligand
molecules in the active sites of dual-target (SE, CYP51). It is noteworthy that these
nitrogen-containing fragments (3, 5) can also form the key coordination bonds interaction
with heme in active site of CYP51 (Figure 3 C -C ).
1
2
Figure. 3. (A
1 3
-A ) The superposition model based on the structure of terbinafine and compound 5.
(
B -B ) The distribution of feature groups in SE and CYP51 active site. (C -C ) The combination
1
2
1
2
pattern of active fragments in the active regions of SE and CYP51.
2
.2. The design of antifungal agents
On this basis, three series of dual-target target compounds with the common naphthyl
fragment as main chain group were designed based on the different splicing methods of
active fragments (Figure 4). At first, the pyridyl fragment (3) can replace the phenyl

group of naftifine and the azole group of compound 5, and exert their corresponding
biological functions in the different active sites. Therefore, the pyridyl fragment (3) was
selected as the core groups of dual-target target compounds. This specific connection
method was performed to construct the series 1 target compounds (Figure 4 A ). In
1
addition, the phenyl and cyclohexane fragments (7, 9) were bound into the region A of SE
and the region B of CYP51, the imidazole fragment (5) was docked in the region B of SE
and the region A of CYP51. Finally, these fragment combinations were connected as a
whole, and the series 2, 3 target compounds were produced though the different
2 3
connecting methods (Figure 4 A , A ).
Figure 4. Design of naphthylamide derivatives as novel dual-target antifungal inhibitors using the
method of FBDD.
In order to further verify the design rationality, these target compounds were docked
into the active sites of SE and CYP51, respectively. They can be combined in the active
sites of dual-target, and these core fragments were properly distributed in the key regions
of the active sites (Figure 5 A , A ). At the same time, these target compounds can also
1
2
be superimposed with ligand molecules (SE inhibitor: naftifine; CYP51 inhibitor:
compound 5), and they show the higher matching degree. The result indicates that these
target compounds can simultaneously satisfy the distribution characteristics of dual-target
inhibitors (Figure 5 B , B , B ). Based on the detailed analysis of dual-target (SE, CYP51)
1
2
3
docking model and molecular superposition model, their structures were further modified
and optimized as the leading compounds. We expected that these compounds may have
the dual-target antifungal activity, and the subsequent biological activity evaluation has
confirmed the design rationality.

Figure 5. (A -A ) Docking results of these representative target compounds with dual-target (SE,
1
2
CYP51); (B -B ) Molecular superposition model of target compounds with ligand molecular (naftifine,
1
3
compound 5).
2
.3. Chemistry
The synthetic route of key intermediates 4a, b-1, 2, 3, 4 was illustrated in Scheme 1.
The commercially available L-Glycine, Glycine, Alanine, Valine, Serine (2-1, 2, 3, 4)
were selected as the starting material, and they were dissolved in ethanol, and refluxed
with SOCl for 5 h, the amino acid ethyl ester hydrochloride (3-1, 2, 3, 4) were obtained
2
[
1
36]. Next, the amino acid ester hydrochloride was separately treated with the
-naphthoic acid (1a) and 2-naphthoic acid (1b) in the presence of condensing agent to
give the key intermediates (4a, b-1, 2, 3, 4). Target compounds 7a-1, 2, 3, 4 and 7b-1, 2,
, 4 were prepared according the procedures, which was shown in Scheme 2. The methyl
3
group was introduced into the key intermediates (4a, b-1) through methylation reaction,
and formed the intermediates products (5a, b-1). Next, they can be directly hydrolyzed to
obtain the corresponding organic acids (6a, b-1). Finally, the target compounds 7a, b-1, 2,
3
, 4 were synthesized via amination reaction.
Scheme 1. Reagents and conditions: (a) Ethanol, SOCl
h.
2
, reflux, 2–5 h; (b) EDCI, HOBt, DIEA, 80 °C,
7

3 2 3
Scheme 2. Reagents and conditions: (a) CH I, K CO , THF, 60 °C, 5 h; (b) 2 N NaOH solution,
MeOH, 60 °C, 2 h; (c) HATU, DIEA, DMF, amine (aminopyridine or aminomethyl pyridine), 80 °C, 7
h.
The synthetic route of the target compounds 10a, b-1, 2, 3, 4, 5 was illustrated in
Scheme 3. First, the imidazole group was introduced into the key intermediates (4a, b-4)
to afford the intermediate products 8a, b [37]. Subsequently, they were further converted
into the corresponding organic acids (9a, b) through hydrolysis reaction. Finally, the
target compounds 10a, b-1, 2, 3, 4, 5 were synthesized from organic acids and
aminomethylpyridine via amidation reaction.
Scheme 3. Reagents and conditions: (a) CDI, imidazole, 70 °C, 3-5h; (b) 2 N NaOH solution, MeOH,
6
0 °C, 5 h; (c) HATU, DIEA, DMF, amine, 80 °C, 7 h.
The synthetic route of the target compounds 17a, b-1, 2, 3 and 18 a, b was illustrated
in Scheme 4. First, the key intermediates (4a, b-1, 2, 3) were hydrolyzed and converted
into the corresponding organic acids (11a, b-1, 2, 3). Next, these organic acids (6a, b-1;
11a, b-1, 2, 3) were separately treated with the aminoacetophenone to obtain the key
intermediates (13a, b-1, 2, 3; 14a, b). Subsequently, the treatment of intermediates was
treated with formaldehyde and NaHCO in MeOH/H O to produce the hydroxyl
3
2
compounds (15a, b-1, 2, 3; 16a, b) [38]. Finally, these compounds (15a, b-1, 2, 3; 16a, b)
were further reacted with CDI and imidazole to obtain the target compounds (17a, b-1, 2,
3
; 18a, b).

Scheme 4. Reagents and conditions: (a) 2N NaOH solution, MeOH, 60 °C, 2 h; (b) EDCI, HOBt,
DIEA, 80 °C, 7h; (c) 37% CH O (aq), NaHCO , EtOH, 6 h; (d) Imidazole, CDI, CH CN, 70 °C, 5 h.
2
3
3
2
.4. In vitro antifungal activity of target compounds
In the study, all target compounds were selected to evaluate their in vitro antifungal
activity against pathogenic fungi through the protocols of the National Committee for
Clinical Laboratory Standards (NCCLS) [39,40], and the tested strain contains four
pathogenic Candida (C. alb., Candida albicans; C. gla., Candida glabrata; C. kru.,
Candida krusei; C. tro., Candida tropicalis) and one Aspergillus (A. fum., Aspergillus
fumigatus). Naftifine and fluconazole (FCZ) were selected as the reference drugs.
The antifungal activities of target compounds were summarized in Table 2. It was
observed that most of compounds exhibited the antifungal activity against C. alb., C. gla.,
C. kru. and C. tro. in vitro. These target compounds, which contain 7a-2, 7b-2, 7b-4,
1
0a-4, 10a-5, 10b-1, 10b-4, 10b-5, 17a-1, 17a-2, 17b-2, 18a and 18b, showed a certain
of antifungal activity against Candida in the range of 0.125-0.5 μg/mL. Of these, these
target compounds (7b-2, 10b-4, 10b-5, 17a-1, 17a-2 and 18b) with the range of MIC50
value (0.125-0.25 μg/mL) against C.alb, C.gla. and C.tro., which can show the similar
potency with the control drugs (fluconazole: 0.25 μg/mL, naftifine: 0.25-0.5 μg/mL). In
addition, the target compounds (7b-2, 7b-4, 10b-4, 10b-5, 17a-2, 17b-2 and 18b) also
exhibited the obvious antifungal activities against C.kru. with 0.25 μg/mL. However,
most of compounds have an unsatisfactory inhibitory effect on A. fum. It was
noteworthy that compounds 7b-2, 10b-5 and 17a-2 shows the most potent activity against
A.fum. with MIC50 values 4 µg/mL. Subsequently, we preliminarily analyzed the
structure-activity relationship of target compounds. It should be mentioned that the
different position of naphthyl fragment as main chain can retain a certain effect on
antifungal activity. At the same time, the change of side chain fragments can increase the

antifungal activity and the antibacterial spectrum. It was worth noting that the pyridine
group can also replace azole group to maintain the antifungal activity.
Table 2. In vitro antifungal activities of the target compounds (MIC , μg/mL).
5
0
Compd
R
MIC50, µg/mL
C.gla. C.kru. C.tro. A.fum.
C.alb.
0.5
7a-1
7a-2
7a-3
7a-4
7b-1
7b-2
7b-3
7b-4
4-aminopyridine
3-aminopyridine
4-pyridinemethaneamine
3-pyridinylmethylamine
4-aminopyridine
3-aminopyridine
4-pyridinemethaneamine
3-pyridinylmethylamine
cyclohexanamine
cyclopentanamine
phenylmethanamine
4-methylaniline
4-pyridinemethaneamine
cyclohexanamine
cyclopentanamine
phenylmethanamine
4-methylaniline
4-pyridinemethaneamine
H
0.5
0.25
1
0.5
0.5
2
1
8
>16
8
0.25
1
0.25
0.5
0.5
2
0.5
1
0.25
0.25
0.125
1
>16
8
0.25
0.125
0.25
0.5
0.5
0.25
2
0.25
0.5
0.25
0.5
0.5
1
4
8
0.5
0.25
2
0.25
1
>16
>16
>16
8
10a-1
10a-2
10a-3
10a-4
10a-5
10b-1
10b-2
10b-3
10b-4
10b-5
17a-1
17a-2
17a-3
17b-1
17b-2
17b-3
0.5
0.5
0.5
0.5
0.5
0.25
0.5
0.25
0.25
1
0.25
0.5
0.25
0.25
0.5
0.5
0.5
0.5
1
>16
8
0.25
0.5
>16
8
0.25
0.5
0.5
1
0.5
>16
8
0.125
0.25
0.25
0.125
0. 25
0.25
0.25
0.5
0.25
0.125
0.25
0.25
0.5
1
0.25
0.25
0.5
0.25
1
0.25
0.25
0.125
0.125
0.5
4
8
methyl
4
Isopropyl
>16
>16
8
H
0.5
0.25
1
0.25
0.125
0.5
methyl
0.5
1
Isopropyl
>16
8
1
8a
H
0.5
0.25
0.25
0.25
0.25
0.5
0.25
1
0.25
0.25
0.25
0.5
1
8b
H
0.125
0.25
0.5
8
Fluconazole
Naftifine
-
>16
8
-
2
Abbreviations: C. alb., Candida albicans (ATCC 10231); C. gla., Candida glabrata (ATCC
001); C. kru., Candida krusei (ATCC 6258); C. tro., Candida tropicalis (ATCC 1369); A.
0
fum., Aspergillus fumigatus (KM8001).
2
.5. In vitro antifungal activity against fluconazole-resistant strains of Candida
albicans
Currently, azole antifungal agents, especially fluconazole, have been widely applied in
the clinical practice for many years, they have produced the excellent treatment effect.

However, the emergence of drug-resistant fungi has become more and more frequent,
which has been an important medical problem for treating shallow and systemic fungal
infection. Therefore, it's very important to further investigate the biological activity of
target compounds against fluconazole-resistant strains. In the study, these fluconazole-
resistant strain 17#, CaR, 632, 901 and 904 of C.alb. were selected as the test strains. Of
there, strain 17# and strain CaR were isolated from AIDS patients. The result was shown
in Table 3. The potent compounds (7b-2, 10b-5, 17a-2) and the reference drugs
(fluconazole, naftifine) were selected to evaluate their antifungal activity. Fluconazole
was inactive against the fluconazole-resistant strains with the MIC50 values > 16 µg/mL.
Compounds (7b-2, 10b-5, 17a-2) and naftifine exhibited the moderate antifungal
activities against strains 17# and CaR with MIC50 values in the range of 2-8 μg/mL. In
addition, compounds 10b-5 and 17a-2 also displayed a certain of antifungal activity
against strains 632 and 901 with MIC50 values in the range of 4-8 μg/mL. It's worth
noting that compound 17a-2 showed the the best antifungal activity against
fluconazole-resistant strains 17#, CaR and 901 with MIC50 values in the range of 2-4
μg/mL.
Table 3. In vitro antifungal activities of the target compounds (MIC , μg/mL).
50
Compd
MIC , µg/mL
50
Strain 17#
Strain CaR
Strain 632
Strain 901
Strain 904
7
b-2
8
4
4
8
˃16
8
˃16
4
˃16
˃16
4
1
0b-5
7a-2
1
2
4
8
4
Naftifine
8
˃16
˃16
8
˃16
˃16
8
Fluconazole
˃16
˃16
˃16
Abbreviations: strain 17#, CaR, 632, 901, 904, fluconazole-resistant strain of Candida albicans;
Strains 17# and CaR were provided by Institute of Microbiology, Chinese Academy of Sciences.
Strains 632, 901 and 904 were provided by the Second Military Medical University.
2
.6. Density analysis of Candida albicans in different treatment groups
Observing the change of fungal cell density with different target compounds is an
important index to evaluate the antifungal efficacy of target compounds. In the study, the
untreated group and treated groups with positive control drugs (fluconazole, naftifine)
and target compounds (10b-5, 17a-2) were set, respectively. Their density was explored
by polarizing microscopy in the particular concentrations (10 nm/mL).
In the untreated groups, C. alb. (ATCC SC5314) exhibited the significant proliferation
ability, the density of fungal cells increased rapidly with the prolong of treatment time,
and a large number of cell germination and spores were observed in nutrient solution
(
Figure. 6 A ). In the treated groups with positive control drugs and target compounds,
1, 2
the tendency of cell proliferation was obviously inhibited, and cell density has gradually
decreased with the prolong of treatment time. The proportion of Candida cells in the
division phase was significantly lower than the untreated group, and some cells even have
the fracture phenomenon (Figure. 6 B -E1, 2). These results indicated that the target
1
, 2

compounds (10b-5, 17a-2) show the same antifungal effect with the positive control
drugs (fluconazole, naftifine), they can significantly affect the changes of fungal cell
density by inhibiting the fungal cells proliferation.
Figure 6. The density changes of fungal cell in different treatment groups. (A -A : Untreated; B -B :
1
2
1
2
Fluconazole; C -C : Naftifine; D -D : Compound 10b-5; E -E : Compound 17a-2).
1
2
1
2
1
2
In order to further evaluate the antifungal efficacy of target compounds (10b-5, 17a-2).
The survival and mortality rates of C. alb. in different treatment groups were studied. The
results showed that the survival rate of fungal cells was as high as 97% in untreated group,
and cell size exhibits the state of uniform distribution (Figure 7). In the treated groups
with target compounds (10b-5, 17a-2), their survival rates of fungal cells fall to the 16%
and 11%, respectively. Moreover, the sizes of living fungal cells also decreased to 7.53
μm and 7.51 μm from the 8.29 μm. The result further confirmed that compounds 10b-5
and 17a-2 can significantly affect the physiological functions of fungal cell, they not only
can effectively inhibit the fungal cell proliferation, but also can kill the existing fungal
cells.
Figure 7. The survival and mortality rate of C. alb. cells in different treatment groups (A: untreated; B:
compound 10b-5; C: and compound 17a-2).
2
.7. Morphological analysis of Candida albicans in the treatment groups with target
compounds 17a-2
In order to further understand the effect of preferred compound 17a-2 on the cell
morphology, the morphological changes of the Candida cells were observed using
transmission electron microscopy (TEM) [41,42]. At the early stage, the structure of C.
alb., cells mostly exists in the shape of ellipse. Cytoplasm was uniformly distributed in
the inner region of the fungal cells. The fungal cell membrane and wall were located at

the edge of cells. Their boundary can be clearly distinguished, and show the smooth and
transparent characteristics (Figure 8 A -A ). When the fungal cells were treated with
1
2
compound 17a-2 for 72h, the clear boundary between the colony cell population began to
disappear, and the smooth surfaces changed into the irregular structure. At the same time,
some specific phenomena occurred in the cytoplasmic regions, such as edema cavitation,
disordered ridge arrangement, and exfoliation of rough endoplasmic reticulum particles
(
Figure 8 B
have completely cracked, only residual fungal cell wall and membrane structure were left
Figure 8 C -C ). The preliminary result suggests that the target compound 17a-2 can
1 2
-B ). With the further prolongation of treated time (144h), the fungal cells
(
1
2
destroy the structure of cell membrane and affect the osmotic pressure of fungal cell. It
can eventually lead to the rupture and death of fungal cells.
Figure 8. The morphological structure changes of C. alb. cells treated with target compound 17a-2 in
1 2 1 2 1 2
the different time periods. (A -A : 0 h; B -B : 72h; C -C : 144h).
2
.8. Molecular mechanism and enzyme activity of target compounds
In order to further study the antifungal mechanism of target compounds (10b-5, 17a-2),
the sterol composition change of C. alb. SC5314 cells in different treated groups were
analyzed. The untreated group and positive control drugs (fluconazole, naftifine)-treated
group were set as reference. The assay has been successfully used in studying the action
mechanism of antifungal agents on the sterol biosynthesis pathways [35, 42]. The sterol
profifile results were summarized in Table 4.
a
Table 4. Analysis of sterol composition in C.alb. by LC-MS.
Compd.
Concentration
μg/mL)
% of total sterols (C. alb.)
Lanosterol Eburicol Unknown
sterol
(
Ergosterol
Squalene
b
1
1
0b-5
0.125
68.4
35.6
13.2
66.4
9.2
14.5
20.5
7.9
3.8
7.2
13.6
37.9
49.3
17.5
5.0
4.8
6.1
5.4
0.5
4
10.9
2.8
7a-2
0.125

0
.5
29.9
10.8
73.5
40.8
15.9
78.5
43.9
12.7
95.1
21.6
27.9
16.8
39.2
56.8
-
8.7
12.5
3.7
9.4
12.5
5.1
8.9
16.3
-
34.0
42.6
1.9
5.8
6.2
4.1
4.9
5.1
3.6
4.7
5.2
3.5
4
c
Naftifine
0.125
0.5
5.7
4
9.7
Fluconazole 0.125
12.8
42.5
64.5
1.4
0
4
-
.5
-
1.3
-
d
Control
a
Abbreviations: C.alb., Candida albicans (ATCC SC5314).
Treated with target compounds 10b-5, 17a-2.
Treated with naftifine.
b
c
d
Control (no drug).
In the untreated control, the content of ergosterol in the fungal cell membrane
contained 95.1% of the sterol fraction, while squalene and lanosterol was not observed,
and other unknown sterols contained only 3.5%. When C. alb. was treated with
fluconazole at 0.125-4 µg/mL for 48 h, the content of ergosterol in the cell membrane
decreased to 12.7% from 95.1%, the content of eburicol increased sharply to 56.8%,
while squalene and lanosterol contents didn't increase significantly. These changes of this
components are due to the competitive inhibition activity of target enzyme CYP51, which
are consistent with previously published studies. In the treated group with naftifine, the
content of ergosterol reduced to 15.9% from 95.1%, and the contents of squalene,
lanosterol and eburicol were accumulated. In particular, the content of squalene increased
sharply to 56.8%. These changes were caused by the competitive inhibition of CASE in C.
alb. The ergosterol content was significantly reduced to 13.2% and 10.8%, respectively.
Interestingly, the treatment with compounds 10b-5 and 17a-2 also resulted in the
noticeable accumulation of squalene (20.5%, 27.9%) and eburicol (49.3%, 42.6%), These
results suggested that these novel compounds can caused the potent disruption of
ergosterol biosynthesis by inhibiting the double-target (SE, CYP51) activity in C. alb.,
which was similar with the accepted mechanism of fluconazole and naftifine.
Subsequently, the dual-target (SE, CYP51) enzymatic activity of compounds 10b-5 and
1
7a-2 was tested (Table 5). Compared with naftifine (SE, IC50 = 0.284 μM) and
fluconazole (CYP51, IC50 = 0.116 μM), they displayed a certain degree of dual-target
enzymatic activity with IC50 values ranging from 0.537-1.153 μM, the study further
proved their dual-target inhibitory activity.
Table 5. The dual-target (SE, CYP51) enzymatic activity of compounds 10b-5 and 17a-2
Compd
IC 50 (μM)
SE
CYP51
1
0b-5
7a-2
Naftifine
0.685
0.961
0.284
1.153
0.537
-
1

Fluconazole
-
0.116
a
Used as positive control.
2
.9. In vitro human plasma stability assay
The stability of the target compound in human plasma is an important indicator to
maintain the medicinal property [42]. Based on their antifungal activities in vitro.
Compounds 10b-5 and 17a-2 were further selected to evaluate its stability in human
plasma. In the study, these target compounds 10b-5 and 17a-2 were incubated in human
plasma for 60 min and 120 min, respectively. Their result was shown in Table 6, these
target compounds exhibited the excellent metabolic stability at same times (120 min:
remaining 91.6% and 92.1%, respectively), which indicate that these compounds can
maintain their medicinal efficacy in vivo for a long time.
Table 6. In vitro human plasma stability of compounds 10b-5 and 17a-2.
Compd.
Stability in Human Blood Plasma
Remaining at 60 min % Remaining at 120 min
%
1
0b-5
7a-2
94.5
96.3
91.6
92.1
1
2
.10. In vivo study of toxicity and therapeutic efficacy
The potential toxicity and therapeutic effect of new target compounds in vivo is an
important index during the process of new drug development. The pharmacodynamic
properties of the preferred compound 10b-5 and 17a-2 were further evaluated in mice
[42].
In this study, we constructed the fungal infection mouse models by subcutaneous
injection, and the body weights of mouse in these different treatment groups were
monitored, and their body weights did not display the significant differences during the
whole treatment period. H&E (hematoxylin–eosin staining) was performed to observe the
morphology of tissue after in the different treated groups, which was shown in Figure 9.
Figure 9. The morphological change of fungal infections tissues in different treatment groups (A:
Saline; B: Compound 10b-5; C: Compound 17a-2).
In the treated group with saline, the arrangement of endothelial tissue cells begins to
display the irregular distribution characteristics, the space of some endothelial cells
increases significantly, and their configuration also undergoes a certain change. At the
same time, a large number of nuclear gathers in the infected region was observed, which
indicates that the pathogenic fungal infection has appeared, and the region has produced
the corresponding immune response. In the treated groups with target compounds 10b-5

and 17a-2, we found that these tissue cells reappeared the uniform distribution
characteristics and the complete state in the fungal infection region. It is worth noting that
the aggregation of cell nuclear in the infected region has also disappeared,
which suggested that these target compounds can significantly treat the endogenous
fungal infection, and the infected tissues have gradually returned to normal state. These
H&E results further demonstrated the synergistic therapeutic effect of the compounds.
Figure 10. The histological images of H/E staining (lung, liver, spleen, kidney and heart) from the
different treatment groups. (A : Saline; B : Compound 10b-5; C : Compound 17a-2).
1-5
1-5
1-5
Subsequently, the in vivo safety of the compounds was further evaluated. Their main
organs (heart, liver, spleen, kidney, and lung) were separated and collected in the different
treatment groups. The structures of the visceral tissue cells in these different treatment
groups were similar, and they didn’t show the obvious morphological changes or cell
damage Figure 10. Moreover, the blood urea nitrogen (BUN), aspartate aminotransferase
(AST) and the alanine aminotransferase (ALT) are important indexes that can directly
reflect the function of kidney and liver. We can see that the AST, ALT and BUN levels
didn't show the significant difference (p>0.05) in the different treatment groups, which
indicated the compounds 10b-5 and 17a-2 did not have the obvious hepatic and renal
toxicity (Table 7). 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 7. The analysis of plasma biochemical levels with different treatment groups
Group
AST(IU/L)
104.7±5.2
108.6±6.3
112.3±5.9
ALT(IU/L)
105±8.3
BUN (mmol/L)
6.8±0.4
Saline
Compound 10b-5
Compound 17a-2
102±12.6
106±7.4
7.3±0.9
6.4±0.7
2
.11. Molecular docking
In order to better understand the binding mode of target compound 17a-2, it was
docked into the dual-targets enzymes (SE and CYP51) of C. alb., using the CDOCKER
program [43,44], the SE homology model and CYP51 (PDB: 5TZ1) served as the

docking templates to generate the binding mode. Images depicting the proposed binding
modes were generated using PyMOL, and the results were shown in Table 8 and Figure
1
1.
Table 8. Molecular docking results of target compound 17a-2 with dual-target (SE, CYP51)
Compd
-CDOCKER
ENERGY
-CDOCKER
Absolute Energy Target Enzyme
INTERACTION ENERGY
1
1
7a-2
7a-2
24.53
21.15
34.65
27.72
42.85
39.92
47.72
44.76
57.44
49.51
60.67
65.94
SE
CYP51
SE
Naftifine
Fluconazole
CYP51
In the SE docking model, the binding energies of target compound (17a-2)
CDOCKER ENERGY: −24.53 kcal/mol; CDOCKER INTERACTION ENERGY:
42.85 kcal/mol) were lower than naftifine (CDOCKER ENERGY: −21.15 kcal/mol;
(
−
CDOCKER INTERACTION ENERGY: −39.92 kcal/mol), which indicated the target
compounds can form the more stable interactions than positive control naftifine. It can be
seen that the active site of SE exhibited the long column shape, compound 17a-2 was
properly docked into the active site, and its docking conformation were displayed in
Figure 11 A -A . The naphthyl group of compounds 17a-2 can bind in the upper region
1
3
of the active site, and form π-alkylation with the key amino acid residues (Tyr 77, Arg
34, Leu 249 and Tyr 251). In addition, the phenyl and imidazole groups were located in
the bottom region, they can produce the hydrophobic bond interaction with Leu 398, Cys
16, Val 427 and Pro 430, respectively. At the same time, the compound 17a-2 also can
properly docked into the active chamber of the CYP51 (Figure 11 B -B ). The
1
4
1
3
CDOCKER ENERGY and CDOCKER INTERACTION ENERGY values were predicted
as −24.65 and −45.38 kcal/mol, respectively, which is close to the docking value of
fluconazole (CDOCKER ENERGY: −21.15 kcal/mol; CDOCKER INTERACTION
ENERGY: −39.92 kcal/mol). The naphthyl group was surrounded with the key
hydrophobic residues (Pro 230, Phe 235, Leu376, His377, Tyr 505, and Ser 507), the
interaction of π-alkylation with Pro 230 and Leu 376 was produced. It is worth noting
that the imidazole group plays an important role, which occupied the bottom region of the
3
+
active site. It can form the key coordination bonds with Fe of heme (The coordinate
bond length is 2.707 Å). Additionally, the phenyl group can form the π-π interaction with
Phe 126. In summary, the docking result have confirmed that the target compounds can
match well in the binding cavity of dual-target (SE, CYP51), and t form the stable
combination mode.

Figure. 11. Docking models of representative compound 17a-2 with dual-target (SE, CYP51). (A1)
The binding mode of the preferred compounds 17a-2 with CASE. (A ) The interaction mode of
2
3
compound 17a-2 in the active site of SE. (A ) The interaction between the compound 17a-2 and SE on
the 2D diagram. (B ) The docking results of the preferred compounds 17a-2 with CYP51. (B ) The
1
2
interaction mode of compound 17a-2 in the active site of CYP51. (B
3
) The interaction mode of
compound 17a-2 in the active site of CYP51.
2
.12. Theoretical evaluation of ADME/T properties
The properties of ADMET can reflect the process of the body's absorption, distribution,
metabolism, and excretion of foreign chemicals. Therefore, the evaluation of ADMET
properties is an important condition to ensure the drug-ability of the target compounds
[45]. In the study, the ADME/T properties of target compounds were predicted, which
was shown in Figure 12.

Figure 12. The evaluation plot of Alog P and PSA for the all target compounds. The 95% and
9% confidence limit ellipses that correspond to the intestinal absorption and BBB (blood-brain
9
barrier) models. Abbreviations: ADME/T, absorption, distribution, metabolism, excretion and
toxicity; A log P, the logarithm of the partition cofficient between octanol and water; PSA, polar
surface area; 2D, two-dimensional.
The analogous 95% and 99% confidence ellipses for the blood-brain barrier (BBB)
penetration and human intestinal absorption (HIA) was represented in the biplot Figure
1
2. All the target compounds and the control drug (fluconazole) were positioned in the
confidence regions for HIA, which indicate these compounds have appropriate HIA
properties. At the same time, most of compounds also have a certain permeability of
blood-brain barrier, it reflects that these compounds have a certain therapeutic effect on
fungal infection of nervous system.
Table 9. The ADME/T prediction of target compounds compared with naftifine and fluconazole.
ADME/T parameters
Series 1
Series 2
Series 3
Naftifine
Fluconazole
compounds
compounds
compounds
a
A log P98
1.638~1.645
62.025
2.139~2.741
2.139~3.466
4.863
0.750
b
PSA
76.831
84.674, 94.132
3.352
76.556
Aqueous solubility
3
2, 3
2, 3
2
4
d
HIA
0
0
0
1
0
e
PPB
Highly bound
3
Highly bound
Highly bound
Highly bound
Highly bound
f
BBB penetration
3
3
0
3
g
CYP450 2D6 binding
0
0
0
0
1
Hepatotoxicity
Non-Toxic
Toxic,
Non-Toxic
Toxic
Non-Toxic
Toxic
Non-Toxic
Toxic
Toxic
Toxic
h
DTP
Non-Toxic
Non-
i
FDA rodent
Non-
Non-
Non-
Non-
carcinogenicity
carcinogen
Non-mutagen
Non-irritant
carcinogen
Non-mutagen
Non-Irritant,
Irritant
carcinogen
Non-mutagen
Non-Irritant,
Irritant
carcinogen
Non-mutagen
Irritant
carcinogen
Non-mutagen
Irritant
Ames mutagenicity
Skin sensitization
Skin irritating
Non-irritant
Non-irritant
Non-irritant
Mild-irritant
Non-irritant
a. A log P98 (atom-based log P) (≦ -2.0 or ≦ 7.0: very low absorption). b. PSA (polar surface area) (>150: very low
absorption). c. Level of aqueous solubility predicted: 0 (extremely low), 1 (very low, but possible), 2 (low), 3
(good), 4 (optimal), 5 (too soluble), 6 (warning: molecules with one or more unknown A log P calculations). d. HIA
(human intestinal absorption), level of human intestinal absorption prediction: 0 (good), 1 (moderate), 2 (poor), 3
(very poor). e. PPB, plasma protein binding. f. BBB (blood brain barrier), level blood brain barrier penetration
prediction: 0 (very high penetrate), 1 (high), 2 (medium), 3 (low), 4 (undefined). g. Prediction cytochrome P4502D6
enzyme inhibition (0: non-inhibitor; 1: inhibitor). h. DTP, development toxicity potential. i. FDA, food and drug
administration.
These detailed values of ADME/T properties were summarized Table 9. The A logP
and PSA of target compounds are the important properties of drug bioavailability. We can

see that the target compounds with PSA < 140, -2.0 < A logP < 7.0 have high oral
bioavailability, these compounds have proper absorption or permeation, and they can be
effectively absorbed in human intestine and highly bound with plasma protein. At the
same time, those target compounds can also remain the Non-Hepatotoxicity, Low
inhibitory activity on CYP450 2D6, which indicated that the target compound can
maintain structural stability during the phase of metabolism. For toxicity risk, these
compounds are non-carcinogenic, non-mutagenic and non-irritating. Although some of
compounds demonstrate a certain degree of DTP and Skin sensitization, we believe that
the further structural modifications can reduce the compounds' toxicity profiles.
3
. Conclusions
At present, the phenomenon of invasive fungal infection and drug-resistant fungi is
more and more frequent in clinical practice, which seriously threatens people's health.
The design of dual-target (SE, CYP51) antifungal inhibitors was considered as an
effective coping strategy. In the study, the corresponding fragment library was
constructed by extracting the active groups of common antifungal inhibitors, and they
were docked into the active sites of dual-target though the method of De Novo Link.
These core groups with the higher MCSS_Score values were selected to design the
fragment-based novel antifungal inhibitor. Subsequently, three series of target compounds
were constructed and synthesized, their corresponding antifungal activity was evaluated.
The most of target compounds exhibit the moderate or excellent antifungal activity.
Among these, compounds 10b-5 and 17a-2 not only displayed the most remarkable in
vitro activity against Candida spp. and A. fum., but also had the obvious inhibitory effect
on fluconazole-resistant C.alb. strains. In order to further understand the action
mechanism of target compounds, the analysis of sterol composition and the study of
dual-target enzyme inhibition were performed. The target compounds can block the
biosynthesis of ergosterol and destroy the structure of fungal cell membranes by
inhibiting the activity of dual-target (SE, CYP51). Notably, the compounds 10b-5 and
1
7a-2 with low toxicity and the excellent blood plasma stability also exhibited the
obvious antifungal activity in vivo. At the same time, the target compounds can match
well with the dual-target (SE, CYP51) binding cavity. In summary, these results
demonstrate that these target compounds have the potential to be developed as the novel
antifungal inhibitor.
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).
1
13
Nuclear magnetic resonance ( H-NMR and C-NMR) spectra were recorded on a Bruker
00 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;
5
(
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
3
.2. General procedure for the synthesis of amino acid ethyl ester hydrochloride (3-1, 2,
, 4)
Thionyl chloride (3.0 equiv) was slowly dripped into the ethanol solution. Then,
L-amino acids (Glycine, Alanine, Valine, Serine) (1.0 equiv) were dissolved in the mixed
solution, respectively. The mixed solution was heated to reflux for 3-6 h. When the
reaction liquid becomes transparent, the reaction mixtures were concentrated under
reduced pressure to give the white solid (3-1, 2, 3 and 4).
4
4
.3. General procedure for the synthesis of key intermediate compounds (4a-1, 2, 3, 4;
b-1, 2, 3, 4)
The appropriate 1-naphthoic acid and 2-naphthoic acid (1 equiv) and HATU (1.2 equiv)
were added into anhydrous DMF, respectively. The mixture was stirred at room
temperature for 2 h. Then, amino acid ethyl ester hydrochloride (3-1, 2, 3, 4;1.0 equiv)
and DIEA (3.0 equiv) were added, the mixed solution was heated at 80 °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 though Na
2
4
SO overnight. Finally, the desired intermediate compounds
(4a-1, 2, 3, 4; 4b-1, 2, 3, 4) were obtained under vacuum distillation.
4
.4. General procedure for the synthesis of compounds (5a-1; 5b-1)
The intermediate compounds (4a-1 or 4b-1; 1.0 equiv), K CO (3 equiv) and CH I (2
2
3
3
equiv) were dissolved in tetrahydrofuran solution. Then, the mixed solution was stirred in
the conditions of room temperature 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 by Na SO overnight. Finally,
2
4
the desired compounds were obtained under vacuum distillation.
.5. General procedure for the synthesis of compounds (6a-1; 6b-1)
The compounds (5a-1 or 5b-1; 1.0 equiv) were dissolved into methanol solution (10
4
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 under vacuum
distillation, pH was adjusted to 2-3 by dilute hydrochloric acid solution, and the white
solid was filtered and dried to give the desired compounds.
4
3
.6. General procedure for the synthesis of target compounds (7a-1, 2, 3, 4 and 7b-1, 2,
, 4)

The key intermediate (6a-1 or 6b-1; 1.0 equiv) and HATU (1.2 equiv) were added into
DMF, respectively. The mixture was stirred at room temperature for 2 h. The
4
-aminopyridine, 3-aminopyridine, 4-pyridinemethaneamine or 3-pyridinylmethyl amine
(1.2 equiv) and DIEA (2.0 equiv) were added, and the mixture was heated at 80 °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 by Na
vacuum. The resulting solid was dried to give the desired compounds. The crude product
was purified by silica gel column chromatography (CH Cl : MeOH =100:3).
.7. General procedure for the synthesis of target compounds (8a; 8b)
The intermediates (4a-4 or 4b-4; 1.0 equiv), CDI (1.2 equiv.) and imidazole (1.0 equiv.)
2 4
SO overnight, and the solvent was removed under
2
2
4
were added to acetonitrile. The mixture was heated at 70 °C for 2 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 Na SO and filtered. The filtrate was
2
4
concentrated to afford the target compounds (8a, 8b) as a white solid. The crude product
was purified by silica gel column chromatography (CH Cl : MeOH =100:3).
2
2
4
.8. General procedure for the synthesis of key intermediate compounds (9a; 9b)
The compounds (8a or 8b; 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 under 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
5
.9. General procedure for the synthesis of key intermediate compounds (10a-1, 2, 3, 4,
; 10b-1, 2, 3, 4, 5)
The appropriate key intermediate organic acid (9a or 9b;1 equiv) and HATU (1.2 equiv)
were added into anhydrous DMF, respectively. The mixture was stirred at room
temperature for 2 h. Then, amino intermediates (1.1 equiv) and DIEA (3.0 equiv) were
added, the mixed solution was heated at 80 °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
Na SO overnight. Finally, the desired compounds were obtained under vacuum
2
4
distillation.
.10. General procedure for the synthesis of key intermediate compounds (11a-1, 2, 3;
1b-1, 2, 3; 6a-1; 6b-1)
4
1
The intermediates (4a-1, 2, 3, 4 or 4b-1, 2, 3, 4; 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 under 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 (11a-1, 2,

3
4
1
; 11b-1, 2, 3; 6a-1; 6b-1).
.11. General procedure for the synthesis of key intermediates (13a-1, 2, 3; 13b-1, 2, 3;
4a; 14b)
The appropriate key intermediate organic acids (11a-1, 2, 3; 11b-1, 2, 3; 6a-1; 6b-1; 1
equiv) and HATU (1.2 equiv) were added into anhydrous DMF, respectively. The mixture
was stirred at room temperature for 2 h. Then, aminoacetophenone (1.1 equiv) and DIEA
(3.0 equiv) were added, the mixed solution was heated at 80 °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 Na SO overnight. Finally, the desired compounds were obtained under
2
4
vacuum distillation.
4
1
.12. General procedure for the synthesis of key intermediates (15a-1, 2, 3; 15b-1, 2, 3;
6a; 16b)
To a solution of the key intermediate compounds (13a-1, 2, 3; 13b-1, 2, 3; 14a; 14b;
.0 equiv) in ethanol, NaHCO (0.5 equiv) and 37% formaldehyde solution (1.5 equiv)
1
3
were added. The mixture was stirred at 50 °C for 48h, and then the organic solvent was
removed under vacuum. The residue was dissolved in DCM and washed with water, brine
sequentially. The organic phase was dried over Na SO and filtered. The filtrate was
2
4
concentrated and purified by silica column to give the compounds (15a-1, 2, 3; 15b-1, 2,
3
4
1
; 16a; 16b) as a white solid.
.13. General procedure for the synthesis of target compounds (17a-1, 2, 3; 17b-1, 2, 3;
8a; 18b)
The intermediates (15a-1, 2, 3; 15b-1, 2, 3; 16a; 16b; 1.0 equiv), CDI (1.2 equiv.) and
imidazole (1.0 equiv) were added to acetonitrile. The mixture was heated at 70 °C for 2 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 Na
filtered. The filtrate was concentrated to afford the target compounds (17a-1, 2, 3; 17b-1,
, 3; 18a; 18b) as a white solid. The crude product was purified by silica gel column
chromatography (CH Cl : MeOH =100:3).
2 4
SO and
2
2
2
4
.13.1. N-methyl-N-(2-oxo-2-(pyridin-4-ylamino)ethyl)-1-naphthamide (7a-1)
1
The product was obtained as a white solid; yield: 76.1%; mp: 155.2–157.9°C. H NMR
500 MHz, DMSO) δ 10.97 (s, 1H), 8.48 (d, J = 6.0 Hz, 1H), 8.38 (d, J = 6.1 Hz, 1H),
(
8
2
.11 (d, J = 8.2 Hz, 1H), 8.01 (d, J = 8.1 Hz, 2H), 7.68 (d, J = 6.1 Hz, 2H), 7.62 – 7.57 (m,
H), 7.48 (d, J = 6.9 Hz, 1H), 7.45 (d, J = 6.1 Hz, 1H), 3.17 (s, 2H), 2.82 (s, 3H). C
1
3
NMR (126 MHz, DMSO) δ 170.76, 168.73, 150.94, 150.83, 146.01, 134.91, 133.39,
29.30, 128.77, 127.50, 126.97, 125.89, 125.53, 124.18, 113.70, 113.60, 50.92, 38.38.
1
+
+
+
ESI-MS m/z: 318 [M-H] ; 320 [M+H] ; 342 [M+Na] . HPLC purity 98.4%. Retention
time: 5.550 min, eluted with 20% purified water/80% methanol.
4
.13.2. N-methyl-N-(2-oxo-2-(pyridin-3-ylamino)ethyl)-1-naphthamide (7a-2)
1
The product was obtained as a white solid; yield: 75.3%; mp: 161.5–164.1°C. H NMR
500 MHz, DMSO) δ 10.96 (s, 1H), 8.89 (d, J = 2.0 Hz, 1H), 8.66 (d, J = 2.1 Hz, 1H),
(

8
4
1
1
3
.30 (d, J = 4.6 Hz, 1H), 8.12 (d, J = 8.2 Hz, 1H), 8.01 (d, J = 8.1 Hz, 2H), 7.71 – 7.54 (m,
H), 7.42 – 7.39 (m, 1H), 3.17 (s, 2H), 2.82 (s, 3H). C NMR (126 MHz, DMSO) δ
1
3
70.71, 168.07, 144.67, 141.21, 136.21, 135.00, 133.38, 129.26, 128.75, 127.48, 126.95,
+
+
26.53, 125.89, 125.57, 124.15, 50.65, 38.33. ESI-MS m/z: 318 [M-H] ; 320 [M+H] ;
+
42 [M+Na] . HPLC purity 99.1%. Retention time: 5.823 min, eluted with 20% purified
water/80% methanol.
4
.13.3. N-methyl-N-(2-oxo-2-((pyridin-4-ylmethyl)amino)ethyl)-1-naphthamide (7a-3)
The product was obtained as a white solid; yield: 72.4%; mp: 158.2–165.3°C. H NMR
1
(500 MHz, DMSO) δ 8.74 (t, J = 6.0 Hz, 1H), 8.52 (d, J = 5.8 Hz, 1H), 8.45 (d, J = 5.7
Hz, 1H), 8.12 – 8.06 (m, 1H), 8.02 – 7.98 (m, 2H), 7.60 – 7.54 (m, 2H), 7.34 (d, J = 5.5
1
3
Hz, 1H), 7.02 (d, J = 5.4 Hz, 1H), 4.44 (d, J = 5.9 Hz, 2H), 3.16 (s, 2H), 2.78 (s, 3H). C
NMR (126 MHz, DMSO) δ 170.62, 168.98, 149.99, 149.91, 148.89, 135.06, 133.36,
1
29.22, 128.71, 127.39, 126.92, 125.85, 124.17, 122.58, 122.43, 53.51, 50.08, 38.36.
+
+
+
ESI-MS m/z: 332 [M-H] ; 334 [M+H] ; 356 [M+Na] . HPLC purity 98.8%. Retention
time: 4.930 min, eluted with 20% purified water/80% methanol.
4
.13.4. N-methyl-N-(2-oxo-2-((pyridin-3-ylmethyl)amino)ethyl)-1-naphthamide (7a-4)
The product was obtained as a white solid; yield: 72.6%; mp: 162.2–166.7°C. H NMR
1
(
1
500 MHz, DMSO) δ 8.69 (t, J = 5.8 Hz, 1H), 8.56 (d, J = 1.5 Hz, 1H), 8.48 (dd, J = 4.7,
.3 Hz, 1H), 8.11 – 8.06 (m, 1H), 7.99 (d, J = 8.7 Hz, 2H), 7.74 (d, J = 7.9 Hz, 2H), 7.58
s, 1H), 7.46 (d, J = 7.1 Hz, 2H), 7.38 (d, J = 3.1 Hz, 1H), 4.43 (d, J = 5.8 Hz, 2H), 3.14
(
(
1
3
s, 2H), 2.77 (s, 3H). C NMR (126 MHz, DMSO) δ 170.58, 168.75, 149.21, 149.16,
1
1
48.60, 135.55, 135.09, 133.36, 129.48, 129.20, 128.70, 127.40, 126.92, 125.67, 123.96,
23.88, 53.54, 50.02, 38.31. ESI-MS m/z: 332 [M-H] ; 334 [M+H] ; 356 [M+Na] .
+
+
+
HPLC purity 99.0%. Retention time: 5.005 min, eluted with 20% purified water/80%
methanol.
4
.13.5. N-methyl-N-(2-oxo-2-(pyridin-4-ylamino)ethyl)-2-naphthamide(7b-1)
1
The product was obtained as a white solid; yield: 72.3%; mp: 156.8–160.5°C. H NMR
500 MHz, CDCl ) δ 9.50 (s, 1H), 8.68 (s, 1H), 8.32 (dd, J = 4.7, 1.0 Hz, 1H), 8.10 (d, J
8.0 Hz, 1H), 8.01 (s, 1H), 7.92 – 7.85 (m, 3H), 7.56 (s, 3H), 7.22 (dd, J = 8.3, 4.8 Hz,
(
=
3
1
3
1
1
1
H), 4.36 (s, 2H), 3.21 (s, 3H). C NMR (126 MHz, CDCl ) δ 173.19, 167.53, 145.13,
3
41.30, 134.91, 134.05, 132.51, 131.61, 128.54, 127.86, 127.75, 127.62, 126.99, 124.21,
+
+
+
23.62, 53.69, 39.61. ESI-MS m/z: 318 [M-H] ; 320 [M+H] ; 342 [M+Na] . HPLC
purity 98.9%. Retention time: 5.947 min, eluted with 20% purified water/80% methanol.
.13.6. N-methyl-N-(2-oxo-2-(pyridin-3-ylamino)ethyl)-2-naphthamide(7b-2)
4
1
The product was obtained as a white solid; yield: 79.4%; mp: 163.7–168.2°C. H NMR
500 MHz, CDCl ) δ 9.53 (s, 1H), 8.68 (s, 1H), 8.31 (dd, J = 4.9, 1.0 Hz, 1H), 8.11 (d, J
(
3
=
9.3 Hz, 1H), 8.03 (s, 1H), 7.92 – 7.87 (m, 3H), 7.56 (s, 3H), 7.18 (dd, J = 8.6, 4.9 Hz,
1
3
1
1
1
H), 4.38 (s, 2H), 3.23 (s, 3H). C NMR (126 MHz, CDCl ) δ 172.32, 167.28, 145.55,
40.74, 128.80, 127.87, 127.65, 123.50, 123.35, 122.89, 122.32, 122.20, 121.88, 121.55,
19.46, 52.07, 41.22. ESI-MS m/z: 318 [M-H] ; 320 [M+H] ; 342 [M+Na] . HPLC
3
+
+
+
purity 99.3%. Retention time: 5.671 min, eluted with 20% purified water/80% methanol.

4
.13.7. N-methyl-N-(2-oxo-2-((pyridin-4-ylmethyl)amino)ethyl)-2-naphthamide(7b-3)
The product was obtained as a white solid; yield: 71.9%; mp: 161.8–166.2°C. H NMR
1
(500 MHz, DMSO) δ 8.51 (d, J = 20.1 Hz, 3H), 7.96 – 7.84 (m, 3H), 7.71 (s, 1H), 7.62 –
7
3
1
3
.48 (m, 3H), 7.30 (s, 1H), 7.16 (s, 1H), 4.44 (d, J = 38.4 Hz, 2H), 4.01 (s, 1H), 3.15 (s,
H). C NMR (126 MHz, HDMSO) δ 171.80, 168.98, 149.69, 148.45, 133.60, 133.12,
1
3
32.51, 128.39, 127.83, 127.21, 126.81, 126.33, 124.56, 124.33, 122.44, 54.50, 51.05,
+
+
+
8.60. ESI-MS m/z: 332 [M-H] ; 334 [M+H] ; 356 [M+Na] . HPLC purity 98.5%.
Retention time: 5.011 min, eluted with 20% purified water/80% methanol.
4
.13.8. N-methyl-N-(2-oxo-2-((pyridin-3-ylmethyl)amino)ethyl)-2-naphthamide(7b-4)
The product was obtained as a white solid; yield: 77.4%; mp: 169.4–173.0°C. H NMR
1
(500 MHz, DMSO) δ 8.50 (d, J = 16.4 Hz, 3H), 7.90 – 7.86 (m, 3H), 7.73 (s, 1H), 7.57 –
7
3
1
1
.52 (m, 3H), 7.38 (s, 1H), 7.15 (s, 1H), 4.46 (d, J = 27.6 Hz, 2H), 4.06 (s, 1H), 3.13 (s,
H). C NMR (126 MHz, HDMSO) δ 172.26, 168.79, 149.86, 148.12, 136.42, 133.67,
1
3
33.52, 133.12, 132.53, 131.92, 128.49, 128.33, 128.10, 127.83, 127.69, 127.28, 127.06,
+
+
26.33, 124.38, 122.75, 54.23, 50.55, 39.29. ESI-MS m/z: 332 [M-H] ; 334 [M+H] ; 356
+
[
M+Na] . HPLC purity 99.5%. Retention time: 5.150 min, eluted with 20% purified
water/80% methanol.
.13.9.N-(1-(cyclohexylamino)-3-(1H-imidazol-1-yl)-1-oxopropan-2-yl)-1-naphthamide
10a-1)
4
(
1
The product was obtained as a white solid; yield: 70.8%; mp: 132.6–138.1°C. H NMR
(500 MHz, DMSO) δ 8.79 (d, J = 8.5 Hz, 1H), 8.07 (d, J = 7.7 Hz, 1H), 8.01 (d, J = 8.2
Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.62 (s, 1H), 7.57 – 7.45 (m, 4H), 7.20 (s, 1H), 6.92 (s,
1
1
1
1
H), 4.92 (td, J = 9.4, 4.8 Hz, 1H), 4.29 (ddd, J = 23.7, 13.9, 7.3 Hz, 2H), 3.69 – 3.51 (m,
1
3
H), 1.87 – 1.66 (m, 4H), 1.33 – 1.12 (m, 6H). C NMR (126 MHz, DMSO) δ 169.09,
68.27, 138.27, 134.58, 133.52, 130.46, 130.11, 128.74, 128.57, 127.12, 126.72, 125.86,
25.73, 125.37, 120.26, 54.72, 48.28, 47.63, 32.69, 25.65, 24.90. ESI-MS m/z: 389
+
+
+
[
M-H] ; 391 [M+H] ; 413 [M+Na] . HPLC purity 99.5%. Retention time: 5.484 min,
eluted with 20% purified water/80% methanol.
.13.10.N-(1-(cyclopentylamino)-3-(1H-imidazol-1-yl)-1-oxopropan-2-yl)-1-naphthami
de (10a-2)
4
1
The product was obtained as a white solid; yield: 69.5%; mp: 137.2–140.3°C. H NMR
(500 MHz, DMSO) δ 8.78 (d, J = 8.9 Hz, 1H), 8.24 (d, J = 11.3 Hz, 1H), 8.03 – 7.95 (m,
3
1
H), 7.64 (s, 1H), 7.56 – 7.47 (m, 4H), 7.21 (s, 1H), 6.94 (s, 1H), 4.91 (td, J = 7.5, 6.2 Hz,
H), 4.29 (ddd, J = 18.6, 12.7, 9.8 Hz, 2H), 4.10 – 4.01 (m, 1H), 1.85 – 1.46 (m, 8H). C
1
3
NMR (126 MHz, DMSO) δ 166.03, 163.99, 139.83, 135.61, 133.37, 130.21, 129.43,
28.02, 127.71, 127.52, 123.90, 119.84, 53.77, 52.03, 47.46, 32.67, 32.54, 24.74.
1
+
+
+
ESI-MS m/z: 375 [M-H] ; 377 [M+H] ; 399 [M+Na] . HPLC purity 99.3%. Retention
time: 5.004 min, eluted with 20% purified water/80% methanol.
4
.13.11.N-(1-(benzylamino)-3-(1H-imidazol-1-yl)-1-oxopropan-2-yl)-1-naphthamide
(10a-3)

1
The product was obtained as a white solid; yield: 65.3%; mp: 141.6–145.8°C. H NMR
(500 MHz, DMSO) δ 8.97 (d, J = 9.8 Hz, 1H), 8.75 (t, J = 7.3 Hz, 1H), 8.48 (s, 1H), 8.03
–
6
2
1
4
7.99 (m, 3H), 7.93 – 7.86 (m, 1H), 7.78 (s, 1H), 7.65 – 7.63 (m, 2H), 7.31 – 7.24 (m,
H), 6.84 (s, 1H), 4.99 (td, J = 11.4, 6.8 Hz, 1H), 4.42 – 4.40 (m, 2H), 4.36 (t, J = 7.9 Hz,
1
3
H). C NMR (126 MHz, DMSO) δ 170.19, 167.98, 140.01, 138.61, 135.45, 133.00,
30.94, 129.54, 129.15, 128.47, 128.06, 127.48, 127.04, 124.07, 120.75, 55.66, 48.23,
+
+
+
4.51. ESI-MS m/z: 397 [M-H] ; 399 [M+H] ; 421 [M+Na] . HPLC purity 99.1%.
Retention time: 5.108 min, eluted with 20% purified water/80% methanol.
.13.12.N-(3-(1H-imidazol-1-yl)-1-oxo-1-(p-tolylamino)propan-2-yl)-1-naphthamide
10a-4)
4
(
1
The product was obtained as a white solid; yield: 54.9%; mp: 137.6–143.9°C. H NMR
(500 MHz, DMSO) δ 8.96 (d, J = 5.9 Hz, 1H), 8.77 (t, J = 6.3 Hz, 1H), 8.46 (s, 1H), 8.05
–
=
4
1
1
8.03 (m, 3H), 7.97 – 7.83 (m, 1H), 7.73 (s, 1H), 7.62 (dd, J = 9.9, 3.9 Hz, 2H), 7.37 (t, J
7.4 Hz, 2H), 7.23 (dd, J = 15.1, 5.7 Hz, 4H), 6.91 (s, 1H), 4.99 (td, J = 9.7, 4.5 Hz, 1H),
.54 – 4.38 (m, 2H), 1.23 (s, 1H). C NMR (126 MHz, DMSO) δ 170.54, 166.43, 140.06,
1
3
37.16, 136.37, 133.12, 131.18, 130.65, 129.33, 128.76, 128.31, 128.23, 128.11, 127.65,
+
+
27.30, 124.13, 119.80, 56.05, 48.66, 29.27. ESI-MS m/z: 399 [M+H] ; 421 [M+Na] .
HPLC purity 99.3%. Retention time: 5.108 min, eluted with 20% purified water/80%
methanol.
4
.13.13.N-(3-(1H-imidazol-1-yl)-1-oxo-1-((pyridin-4-ylmethyl)amino)propan-2-yl)-1-na
phthamide (10a-5)
1
The product was obtained as a white solid; yield: 72.7%; mp: 145.2–149.6°C. H NMR
(500 MHz, DMSO) δ 8.91 (d, J = 7.3 Hz, 1H), 8.72 (t, J = 6.8 Hz, 1H), 8.42 (s, 1H), 8.00
–
7
7.97 (m, 3H), 7.89 (d, J = 7.4 Hz, 1H), 7.75 (s, 1H), 7.56 (m, 2H), 7.28 – 7.22 (m, 4H),
.02 (s, 1H), 6.84 (s, 1H), 4.94 (td, J = 9.7, 4.5 Hz, 1H), 4.53 – 4.36 (m, 2H), 4.07 (s, 3H).
C NMR (126 MHz, DMSO) δ 169.99, 164.28, 145.49, 142.31, 139.98, 135.36, 133.93,
31.07, 128.74, 127.91, 127.29, 124.69, 120.36, 117.48, 56.59, 51.70, 47.99. ESI-MS
1
3
1
+
+
m/z: 400 [M+H] ; 422 [M+Na] . HPLC purity 99.3%. Retention time: 5.108 min, eluted
with 20% purified water/80% methanol.
4
.13.14.N-(1-(cyclohexylamino)-3-(1H-imidazol-1-yl)-1-oxopropan-2-yl)-2-naphthamid
e (10b-1)
1
The product was obtained as a white solid; yield: 70.2%; mp: 131.6–135.4°C. H NMR
(500 MHz, DMSO) δ 8.84 (d, J = 8.5 Hz, 1H), 8.45 (s, 1H), 8.11 (d, J = 7.8 Hz, 1H), 8.07
–
7
3
1
1
3
7.95 (m, 4H), 7.90 (dd, J = 8.6, 1.5 Hz, 1H), 7.62 (td, J = 7.2, 1.3 Hz, 2H), 7.31 (s, 1H),
.02 (s, 1H), 4.93 (td, J = 9.1, 4.9 Hz, 1H), 4.41 (ddd, J = 23.5, 13.9, 7.3 Hz, 2H), 3.58 –
1
3
.54 (m, 1H), 1.79 – 1.65 (m, 4H), 1.32 – 1.12 (m, 6H). C NMR (126 MHz, DMSO) δ
68.20, 166.89, 137.79, 134.73, 132.51, 131.48, 129.35, 128.34, 128.29, 128.25, 128.11,
27.29, 124.69, 120.94, 54.68, 48.33, 48.23, 32.68, 32.65, 25.63, 24.98. ESI-MS m/z:
+
+
+
89 [M-H] ; 391 [M+H] ; 413 [M+Na] . HPLC purity 99.6%. Retention time: 5.484 min,
eluted with 20% purified water/80% methanol.
.13.15.N-(1-(cyclopentylamino)-3-(1H-imidazol-1-yl)-1-oxopropan-2-yl)-2-naphthami
4

de (10b-2)
1
The product was obtained as a white solid; yield: 67.2%; mp: 137.6–141.8°C. H NMR
(500 MHz, DMSO) δ 8.77 (d, J = 8.5 Hz, 1H), 8.14 (d, J = 7.1 Hz, 1H), 8.01 (d, J = 8.1
Hz, 1H), 7.96 (dd, J = 7.7, 5.2 Hz, 2H), 7.62 (s, 1H), 7.53 (ddd, J = 22.4, 13.2, 2.4 Hz,
4
7
H), 7.19 (s, 1H), 6.92 (s, 1H), 4.92 (td, J = 9.5, 4.8 Hz, 1H), 4.29 (ddd, J = 23.7, 13.9,
.3 Hz, 2H), 4.06 (dd, J = 13.4, 6.5 Hz, 1H), 1.89 – 1.51 (m, 8H). C NMR (126 MHz,
13
DMSO) δ 168.66, 166.87, 137.88, 134.72, 132.51, 131.50, 129.35, 128.34, 128.28,
28.11, 127.29, 124.69, 120.75, 54.66, 51.01, 48.07, 32.67, 32.54, 23.95, 23.92. ESI-MS
1
+
+
+
m/z: 375 [M-H] ; 377 [M+H] ; 399 [M+Na] . HPLC purity 99.4%. Retention time: 5.583
min, eluted with 20% purified water/80% methanol.
4
.13.16.N-(1-(benzylamino)-3-(1H-imidazol-1-yl)-1-oxopropan-2-yl)-2-naphthamide
(
10b-3)
1
The product was obtained as a white solid; yield: 69.4%; mp: 143.6–148.3°C. H NMR
(500 MHz, DMSO) δ 8.95 (d, J = 8.5 Hz, 1H), 8.74 (t, J = 5.9 Hz, 1H), 8.45 (s, 1H), 8.15
–
6
7.95 (m, 3H), 7.94 – 7.81 (m, 1H), 7.75 (s, 1H), 7.69 – 7.51 (m, 2H), 7.43 – 7.17 (m,
H), 6.87 (s, 1H), 4.97 (td, J = 9.7, 4.5 Hz, 1H), 4.60 – 4.25 (m, 4H). C NMR (126
1
3
MHz, DMSO) δ 169.50, 167.05, 139.50, 138.12, 134.72, 132.49, 131.55, 129.33, 128.76,
28.31, 128.23, 128.11, 127.65, 127.30, 124.75, 120.43, 55.08, 47.53, 42.76. ESI-MS
1
+
+
m/z: 399 [M+H] ; 421 [M+Na] . HPLC purity 99.2%. Retention time: 5.350 min, eluted
with 20% purified water/80% methanol.
4
.13.17.N-(3-(1H-imidazol-1-yl)-1-oxo-1-(p-tolylamino)propan-2-yl)-2-naphthamide
(
10b-4)
1
The product was obtained as a white solid; yield: 50.4%; mp: 143.6–148.3°C. H NMR
(500 MHz, DMSO) δ 8.96 (d, J = 9.2 Hz, 1H), 8.79 (d, J = 7.9 Hz, 1H), 8.46 (s, 1H), 8.06
–
7
2
1
3
5
4
7.95 (m, 4H), 7.82 – 7.71 (m, 1H), 7.62 (s, 1H), 7.54 (ddd, J = 30.5, 14.8, 4.8 Hz, 5H),
.22 (s, 1H), 6.94 (s, 1H), 4.94 (td, J = 9.4, 4.8 Hz, 1H), 4.28 (ddd, J = 23.7, 13.9, 7.3 Hz,
H), 1.15 (s, 3H). C NMR (126 MHz, DMSO) δ 170.36, 167.76, 140.40, 138.56,
1
3
35.71, 133.14, 131.23, 128.76, 128.31, 128.23, 127.65, 127.30, 121.30, 56.76, 48.75,
+
+
1.07. ESI-MS m/z: 399 [M+H] ; 421 [M+Na] . HPLC purity 99.2%. Retention time:
.034 min, eluted with 20% purified water/80% methanol.
.13.18.N-(3-(1H-imidazol-1-yl)-1-oxo-1-((pyridin-4-ylmethyl)amino)propan-2-yl)-2-na
phthamide(10b-5)
1
The product was obtained as a white solid; yield: 66.1%; mp: 146.2–150.6°C. H NMR
(500 MHz, DMSO) δ 8.82 (d, J = 5.9 Hz, 1H), 8.45 (d, J = 8.1 Hz, 1H), 8.17 (d, J = 6.2
Hz, 1H), 7.98 – 7.93 (m, 3H), 7.79 (s, 1H), 7.62 (d, J = 8.1Hz, 1H),7.52 (ddd, J = 13.5,
9
1
1
1
4
.8, 7.8 Hz, 5H), 7.19 (s, 1H), 6.87 (s, 1H), 4.94 (td, J = 9.4, 4.8 Hz, 1H), 4.26 (ddd, J =
7.3, 10.5, 5.8 Hz, 2H), 4.05 (s, 3H). C NMR (126 MHz, DMSO) δ 170.31, 165.70,
1
3
41.48, 137.11, 136.06, 135.67, 133.68, 133.12, 131.08, 129.63, 128.23, 128.11, 127.91,
+
27.30, 125.56, 123.53, 122.01, 118.39, 56.59, 48.83, 43.37. ESI-MS m/z: 400 [M+H] ;
+
22 [M+Na] . HPLC purity 99.4%. Retention time: 4.124 min, eluted with 20% purified
water/80% methanol.

4
.13.19.N-(2-((3-(1H-imidazol-1-yl)-1-oxo-1-phenylpropan-2-yl)amino)-2-oxoethyl)-1-
naphthamide(17a-1)
1
The product was obtained as a white solid; yield: 63.7%; mp: 161.2–164.9°C. H NMR
(500 MHz, DMSO) δ 8.81 – 8.63 (m, 2H), 8.28 – 8.16 (m, 1H), 8.01 (d, J = 7.6 Hz, 3H),
7
6
6
1
1
.97 – 7.94 (m, 1H), 7.66 (d, J = 7.3 Hz, 1H), 7.61 (d, J = 2.2 Hz, 1H), 7.59 – 7.48 (m,
H), 7.19 (s, 1H), 6.85 (s, 1H), 5.64 (td, J = 8.3, 4.7 Hz, 1H), 4.40 (ddd, J = 22.6, 14.2,
1
3
.5 Hz, 2H), 3.86 (qd, J = 16.3, 6.0 Hz, 2H). C NMR (126 MHz, DMSO) δ 197.11,
69.42, 169.37, 138.38, 135.15, 134.67, 134.19, 133.58, 130.43, 130.29, 129.24, 128.95,
28.74, 128.54, 127.08, 126.66, 126.20, 125.80, 125.35, 120.58, 54.81, 46.69, 42.82.
+
+
+
ESI-MS m/z: 425 [M-H] ; 427 [M+H] ; 449 [M+Na] . HPLC purity 99.4%. Retention
time: 4.841 min, eluted with 20% purified water/80% methanol.
4
.13.20.N-(1-((3-(1H-imidazol-1-yl)-1-oxo-1-phenylpropan-2-yl)amino)-1-oxopropan-2
-
yl)-1-naphthamide(17a-2)
The product was obtained as a white solid; yield: 72.5%; mp: 165.2–169.1°C. H NMR
1
(500 MHz, DMSO) δ 8.86 (d, J = 8.5 Hz, 1H), 8.71 (d, J = 8.4 Hz, 1H), 8.62 (d, J = 6.9
Hz, 1H), 8.19 – 8.15 (m, 1H), 7.99 (d, J = 7.4 Hz, 4H), 7.78 (d, J = 7.5 Hz, 1H), 7.54 (d,
J = 9.9 Hz, 6H), 7.24 (s, 1H), 6.86 (s, 1H), 5.50 (td, J = 8.9, 3.9 Hz, 1H), 4.45 (t, J = 7.2
1
3
Hz, 1H), 4.36 – 4.28 (m, 2H), 1.00 (d, J = 7.2 Hz, 3H). C NMR (126 MHz, DMSO) δ
1
1
4
97.30, 182.47, 168.98, 138.51, 135.35, 134.74, 133.51, 133.14, 132.54, 130.22, 129.36,
29.12, 128.98, 128.66, 128.53, 127.07, 126.62, 126.06, 125.84, 125.33, 120.68, 55.00,
9.35, 46.40, 17.32. ESI-MS m/z: 441 [M+H] ; 463 [M+Na] . HPLC purity 98.9%.
+
+
Retention time: 4.879 min, eluted with 20% purified water/80% methanol.
.13.21.N-(1-((3-(1H-imidazol-1-yl)-1-oxo-1-phenylpropan-2-yl)amino)-3-methyl-1-oxo
butan-2-yl)-1-naphthamide(17a-3)
4
1
The product was obtained as a white solid; yield: 69.4%; mp: 168.7–171.5°C. H NMR
(500 MHz, DMSO) δ 8.90 (d, J = 8.4 Hz, 1H), 8.44 (d, J = 8.7 Hz, 1H), 7.99 (ddd, J =
1
(
8
7.5, 9.2, 8.2 Hz, 5H), 7.63 (d, J = 16.6 Hz, 2H), 7.58 – 7.45 (m, 6H), 7.23 (s, 1H), 6.85
s, 1H), 5.70 (td, J = 8.5, 4.6 Hz, 1H), 4.51 (dt, J = 14.1, 4.8 Hz, 1H), 4.32 (ddd, J = 12.4,
.5, 5.7 Hz, 2H), 1.95 – 1.82 (m, 1H), 0.82 (d, J = 6.7 Hz, 6H). C NMR (126 MHz,
1
3
DMSO) δ 197.19, 171.38, 168.98, 138.42, 135.28, 134.98, 134.14, 133.63, 133.51,
1
1
30.24, 129.15, 129.02, 128.91, 128.66, 128.55, 127.09, 126.62, 125.96, 125.77, 125.35,
+
+
20.49, 59.19, 54.68, 46.73, 30.58, 19.50. ESI-MS m/z: 467 [M-H] ; 469 [M+H] ; 491
+
[
M+Na] . HPLC purity 99.2%. Retention time: 5.991 min, eluted with 20% purified
water/80% methanol.
.13.22.N-(2-((3-(1H-imidazol-1-yl)-1-oxo-1-phenylpropan-2-yl)amino)-2-oxoethyl)-2-
naphthamide (17b-1)
4
1
The product was obtained as a white solid; yield: 69.1%; mp: 159.3–164.8°C. H NMR
500 MHz, DMSO) δ 8.91 (d, J = 5.7 Hz, 1H), 8.74 (d, J = 8.3 Hz, 1H), 8.47 (s, 1H), 7.99
dt, J = 17.0, 8.0 Hz, 6H), 7.68 – 7.57 (m, 4H), 7.53 (t, J = 7.7 Hz, 2H), 7.22 (s, 1H), 6.88
s, 1H), 5.58 (td, J = 8.3, 4.7 Hz, 1H), 4.53 – 4.23 (m, 2H), 3.86 (ddd, J = 42.1, 16.5, 5.9
(
(
(
1
3
Hz, 2H). C NMR (126 MHz, DMSO) δ 197.09, 169.52, 167.04, 135.19, 134.67, 134.15,