Design, synthesis and evaluation of novel 5-phenylthiophene derivatives as potent fungicidal of Candida albicans and antifungal reagents of fluconazole-resistant fungi

Article abstract of DOI:10.1016/j.ejmech.2021.113740

A series of 5-phenylthiophene derivatives with novel structures were designed and synthesized to combat the increasing incidence of susceptible and drug-resistant fungal infections. The antifungal activity of the synthesized compounds was assessed against seven susceptible strains and six fluconazole-resistant strains. It is especially encouraging that compounds 17b and 17f displayed significant antifungal activities against all tested strains. Furthermore, the potent compounds 17b and 17f could prevent the formation of fungi biofilms and 17f displayed satisfactory fungicidal activity. Preliminary mechanistic studies showed that the potent antifungal activity of compound 17f stemmed from inhibition of C. albicans CYP51. In addition, Compounds 17b and 17f were almost nontoxic to mammalian A549, MCF-7, and THLE-2 cells. These results strongly suggested that compounds 17b and 17f are promising as novel antifungal drugs.

Full text of DOI:10.1016/j.ejmech.2021.113740

European Journal of Medicinal Chemistry 225 (2021) 113740
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and evaluation of novel 5-phenylthiophene
derivatives as potent fungicidal of Candida albicans and antifungal
reagents of fluconazole-resistant fungi
Wenbo Yin a, Yuxin Zhang a, Hengxian Cui a, Hong Jiang a, Lei Liu a, Yang Zheng a,
,
, *
Tianxiao Wu a, Liyu Zhao a, Yin Sun a, Xin Su b, Song Li a **, Dongmei Zhao a
,
Maosheng Cheng a
a Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, China
b The School of Life Science and Biopharmaceutical, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 5-phenylthiophene derivatives with novel structures were designed and synthesized to
combat the increasing incidence of susceptible and drug-resistant fungal infections. The antifungal ac-
tivity of the synthesized compounds was assessed against seven susceptible strains and six fluconazole-
resistant strains. It is especially encouraging that compounds 17b and 17f displayed significant antifungal
activities against all tested strains. Furthermore, the potent compounds 17b and 17f could prevent the
formation of fungi biofilms and 17f displayed satisfactory fungicidal activity. Preliminary mechanistic
studies showed that the potent antifungal activity of compound 17f stemmed from inhibition of
C. albicans CYP51. In addition, Compounds 17b and 17f were almost nontoxic to mammalian A549, MCF-
7, and THLE-2 cells. These results strongly suggested that compounds 17b and 17f are promising as novel
antifungal drugs.
Received 27 May 2021
Received in revised form
30 July 2021
Accepted 1 August 2021
Available online xxx
Keywords:
Azole antifungals
CYP51
Fluconazole-resistance
Fungicidal agents
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
treatment options are limited. Only four classes of antifungals are
clinically available to treat IFIs according to their mode of action:
Invasive fungal infections (IFIs) represent a growing threat with
high morbidity and mortality, mainly because of the increasing
number of immunocompromised individuals [1,2]. Individuals with
compromised immune function, such as those receiving organ
transplantations, cancer chemotherapies, or clinical treatments in
intensive care units, as well as those infected with human immu-
nodeficiency virus, are highly vulnerable to IFIs. It is estimated that
IFIs are responsible for approximately 1.5e2 million deaths every
year. More than 90% of life-threatening IFIs are caused by oppor-
tunistic fungal pathogens such as Candida albicans (mortality rate of
20e40%), Aspergillus fumigatus (mortality rate of 20e70%), and
Cryptococcus neoformans (mortality rate of 20e70%) [3,4]. Despite
the high morbidity and mortality associated with IFIs, the
polyenes (such as amphotericin B and nystatin), azoles [such as
fluconazole (FLC) and itraconazole (ITC)], echinocandins (such as
caspofungin and micafungin), and antimetabolites (such as 5-
fluorocytosine) [5e8].
Lanosterol 14a-demethylase (CYP51) is the primary target of
triazole antifungal agents. Azole agents block the conversion of
lanosterol to ergosterol in fungal cell membranes by inhibiting the
fungistatic activity of CYP51 [9e12]. As a typical azole antifungal
agent, FLC exhibits high oral bioavailability and is used as a front-
line drug for the treatment of fungal infections, but its fungistatic
nature renders FLC inefficient against IFIs [13]. Consequently,
second-generation analogs such as voriconazole (VOR), pos-
aconazole, and albaconazole have been developed (Fig. 1). Azole
antifungal agents, especially FLC, have been used clinically for many
years, and because of the widespread use of antifungal drugs,
resistance is becoming a common observation in the clinic [14e17].
Therefore, the clinical occurrence of drug-resistant strains leads to
significant impediment in the treatment of invasive fungal
* Corresponding author.
** Corresponding author.
E-mail addresses: lis.lisong@gmail.com (S. Li), medchemzhao@163.com
(D. Zhao).
https://doi.org/10.1016/j.ejmech.2021.113740
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
Fig. 1. Chemical structures of azole antifungal agents and lead compound 6.
infections, which remains a major clinical problem in treating
systemic fungal infections. The extensive use of triazole antifungal
agents for the treatment of IFIs has caused severe drug resistance,
which in combination with toxicity concerns, warrants further
research for developing azole antifungal agents [18,19].
In our previous studies, a series of 4,5-dihydronaphtho[2,1-d]
isoxazole derivatives displayed excellent antifungal activity against
C. albicans and C. neoformans, with minimum inhibitory concen-
bromoacetophenones 7a-h were reacted with hexamethylenetet-
ramine and then refluxed to afford the amino derivatives 8a-h in
the acidic condition. The intermediates 8a-h were protected with
(Boc)2O and obtained the compounds 9a-h. The compounds 10a-h
were obtained via an hydroxymethylation reaction of in-
termediates 9a-h and formaldehyde, which were converted to in-
termediates 11a-h through a substitution reaction with imidazole.
And then cleavage of the Boc protecting group of compounds 11a-h
gave amino derivatives 12a-h. The target compounds 14a-j were
synthesized via amide reaction of amino 12a and acid 13a-j in DMF
at room temperature [16].
As depicted in Scheme 2, The 5-phenylthiophene-2-
carboxamide compounds 16a-m and 17a-h were prepared by us-
ing procedures similar to those described above, except that the 5-
phenylthiophene-2-carboxylic acid derivatives were selected as the
core skeleton.
tration values in the range of 0.25e8 mg/mL [20]. Among all the
compounds, compound 6 (Fig. 1) showed better antifungal activity
than FLC. Meanwhile, our previous results suggested that the mo-
lecular target of compound 6 is CYP51.
To further enhance the antifungal activity, we analyzed the
binding mode of compound 6 and the CYP51 protein through
molecular docking. Based on the docking results, we found that the
isopropyl ester fragment of compound 6 did not fully occupy the
adjacent hydrophobic cavity; therefore, we replaced the original
ester fragments with benzoyl fragments to obtain compound 14a.
However, compound 14a displayed moderate antifungal activity
according to the MIC results. As shown in Fig. 2B, the introduced
benzoyl fragments may increase the steric hindrance of the entire
molecule, resulting in the tricyclic fragments colliding with the
narrow hydrophobic channel of the CYP51 protein. To avoid the
clash and increase antifungal activity, we adopted the strategy of
scaffold hopping to investigate the skeleton part of the compound
and determined that the 5-phenylthiophene moiety exhibited
excellent antifungal activity. Finally, we designed and synthesized a
series of 5-phenylthiophene derivatives. To further evaluate the
antifungal efficacy of the compounds, we carried out a series of
experiments, including in vitro antifungal activity, time-kill, biofilm
inhibition, minimum fungicidal concentration, and cytochrome
P450 (CYP) inhibition experiments (see Fig. 3).
2.2. In vitro antifungal activity
The target compounds were evaluated for their in vitro anti-
fungal activity against seven pathogenic fungi according to the CLSI
M27-A3 guidelines by using FLC, ITC, and VOR as the reference
drugs. The results of in vitro antifungal activities are summarized in
Table 1.
We first introduced the benzoyl group to replace the original
ester fragment to obtain compound 14a. The docking results
showed that the benzoyl moiety of compound 14a adequately
occupied the hydrophobic cavity (Fig. 2B); however, compound 14a
showed only moderate antifungal activity. As mentioned above, the
introduction of a benzoyl fragment may increase the steric hin-
drance of the entire molecule, resulting in the tricyclic nucleus of
the compound colliding with the narrow hydrophobic channel of
the CYP51 protein, which decreases the antifungal activity. To
improve the flexibility of the structure, we then investigated the
skeleton part to replace the massive tricyclic structure to improve
the antifungal activity, and compounds 14be14j were designed and
synthesized.
2. Results and discussion
2.1. Chemistry
The synthetic route of the key intermediates 12a-h and the
target compounds 14a-j were illustrated in Scheme 1. The
The results showed that only compounds 14d and 14e exhibited
moderate to good antifungal activities, while the antifungal
2

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
Fig. 2. (A) Predicted binding mode of 6 in Candida albicans CYP51 (PDB: 5TZ1). (B) Predicted binding mode of 14a in Candida albicans CYP51 (PDB: 5TZ1). The distances are shown as
red dashed lines.
Fig. 3. Design strategies of novel antifungal agents.
activities of the other compounds decreased significantly or dis-
appeared. This also suggested that the narrow hydrophobic channel
of the CYP51 protein had higher requirements in terms of the
structure, and a change in one heteroatom may also lead to a sig-
nificant decrease in antifungal activity, such as that observed in
compounds 14c and 14e. Finally, we determined that compound
14e, which had relatively higher antifungal activity to a variety of
pathogenic fungi, was the preferred compound, and further struc-
tural modification was carried out.
3

Scheme 1. General synthesis of the intermediates 12a-h and the target compounds 14a-j. Reagents and conditions: (a) i) hexamethylenetetramine, CHCl3, 50 ꢀC; ii) 37% HCl, EtOH, reflux; (b) (Boc)2O, NaHCO3, MeOH, H2O, r.t.; (c) 37%
CH2O (aq), NaHCO3, EtOH; (d) imidazole, CDI, CH3CN, 70 ꢀC; (e) HCleEtOH, r.t.; (f) HOBt, EDCI, DIEA, DMF, r.t.

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
Scheme 2. Synthesis of the target compounds 16a-m and 17a-h. (f) HOBt, EDCI, DIEA, DMF, r.t.
The results of in vitro antifungal activities are summarized in
Table 2. In the structural modification of A-ring, only compound
16g exhibited moderate inhibitory effect on multiple pathogens.
We speculated that this might be related to the hydrophobic
interaction formed by chlorine atoms. Therefore, we fixed ring A as
para-chlorine substitution and explored the structure-activity
relationship of ring B. It was observed that compounds 17a-h
exhibited moderate to good antifungal activities against C. albicans
and C. tropicalis. Of these, compounds 17b and 17f showed excellent
antifungal activities against C.albicans, C. tropicalis, C. glabrata,
C. parapsilosis, C. neoformans, and C. krusei with the MIC values in
in vitro antifungal activity against the six FLC-resistant strains. The
results are summarized in Table 3. It could be observed that most
compounds showed potent inhibitory effect on FLC-resistant fungi,
whereas FLC was completely inactive. Among all the target com-
pounds, compounds 17b and 17f revealed most potent effects
against FLC-resistant C. albicans (MIC ¼ 0.5e2 and 0.25e1
mg/mL,
respectively).
2.4. GC-MS analysis of sterol composition in C. albicans (SC5314)
5-phenylthiophene derivatives are a series of novel antifungal
agents. To investigate the antifungal mechanism of compound 17f,
gas chromatography-mass spectrometry (GC-MS) was used to
analyse the sterol composition in the cell membrane using flu-
conazole as the reference drug [21,22]. C. albicans (ATCC SC5314)
was used to be a model strain and preincubated together with test
compounds for 16 h to investigate the biosynthesis of ergosterol.
The sterol profile results are summarized in Table 4. Ergosterol was
the main sterol detected in the control group. Compared with the
control group, the ergosterol content in the FLC group (Candida
the range of 0.03e2 mg/mL, which were superior to the reference
drug FLC. Compounds 17d and 17h with 2,4-diF or 4-OCF3
substituted on the ring B were inactive against the fungi, which also
suggested that the hydrophobic cavity where the B ring was located
was relatively narrow, and the introduction of large-volume groups
would not be conducive to the improvement of activity.
2.3. In vitro antifungal activity against FLC-resistant strains of most
potent compounds
albicans cultured on 1640 medium containing 0.25
18 h) decreased significantly and the related sterols, for example
lanosterol, eburicol and 14 -methyl ergosta-8,24(28)-dien-3 ,6
diol, in the ergosterol synthesis pathway accumulated at 34.42%,
29.02% and 22.35%, respectively. These changes in sterol levels were
mg/mL FLC for
Azole antifungal agents have been used clinically for many
years, especially FLC, and because of the widespread use of anti-
fungal drugs, the resistance is becoming widespread in clinic
[14e17]. Therefore, the evaluation of antifungal activities of com-
pounds against FLC-resistant strains is critically important in the
discovery of innovative drugs. Strains 901, 904, 632, 100, 103 and 311
are FLC-resistant strains of C. albicans, which were isolated from
clinic. The potent compounds were selected for evaluation of
a
b a-
caused by inhibiting the sterol 14
the ergosterol biosynthetic pathway. When C. albicans was treated
with compound 17f at sub-MIC levels of 0.015 g/mL, the changes
in sterol composition were consistent with those of the FLC group.
a-demethylase enzyme (CYP51) of
m
5

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
Table 1
In vitro antifungal activities of target compounds (MIC, m
g/mL)a.
Compd.
R2
C. alb.(I)
C. alb.(II)
C. tro.
C. neo.
C. par.
C. gla.
C. kru
14a
8
8
4
16
32
16
>64
14b
14c
32
16
64
16
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
14d
14e
1
2
2
1
8
8
>64
>64
16
32
0.5
0.5
0.5
2
14f
14g
14h
14i
32
>64
2
>64
0.5
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.5
>64
>64
>64
>64
>64
>64
>64
>64
14j
>64
>64
>64
>64
>64
>64
>64
FLC
VOR
ITC
e
e
e
0.5
0.06
0.03
1
0.5
0.06
0.03
4
2
4
8
0.125
0.03
0.5
0.125
0.06
0.125
0.125
0.125
0.125
0.125
a
Abbreviations: C. alb.(I), Candida albicans (ATCC SC5314); C. alb.(II), Candida albicans (CPCC400616); C. neo., Cryptococcus neoformans (CGMCC 2.3161); C. tro., Candida
tropicalis (CGMCC 2.3739); C. kru., Candida krusei (GIM 2.1); C. par., Candida parapsilosis (GIM 2.190); C. gla., Candida glabrata (Clinical isolation); FLC: fluconazole; VOR:
voriconazole; ITC: itraconazole.
The ergosterol content decreased sharply to 5.06%, and the lano-
sterol, eburicol and 14 -methyl ergosta-8,24(28)-dien-3 ,6 -diol
contents increased to 42.91%, 31.02% and 3.20%, respectively. The
results indicated that compound 17f acts by inhibiting the sterol
14a-demethylase enzyme (CYP51), thereby affecting the biosyn-
pathogens embedded in these difficult-to-treat networks. There-
fore, it's a significant therapeutic meaning to develop novel de-
rivatives with anti-biofilm activity. In this assay, the antibiofilm
activity of compounds 17b and 17f were evaluated against biofilms
of C. albicans (CPCC400616) by XTT reduction assay.
a
b a
thesis of ergosterol.
In this assay, C. albicans (CPCC400616) were seeded on the 96-
well plates for 1.5 h, 3 h, 6 h and 24 h to form biofilm. SMIC50 is
defined as the drug concentration required to inhibit the metabolic
activity of the biofilm by 50%. As showed in Table 5, The results of
the test showed that compounds 17b and 17f had better inhibitory
activity against biofilm than FLC and VOR. Notably, target com-
pounds 17b and 17f inhibited the formation of C. albicans biofilm in
a dose-dependent manner, in addition, antibiofilm effect was
decreased when the ripening time of biofilm was increased.
2.5. Inhibition of the biofilm formation of FLC-Resistant C. albicans
Biofilms are complex functional communities of one or more
species of microorganisms that are encased in extracellular poly-
meric substances and attached to both a solid surface and to each
other [2]. Biofilm is an important virulence factor contributing to
recurrent fungal infection and drug resistance in the clinic. Because
of fungi biofilms, antifungal agents have difficulty reaching the
6

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
Table 2
In vitro antifungal activities of target compounds (MIC,
m
g/mL)a.
Compd.
R1
R2
C. alb. (I)
C. alb. (II)
C. tro.
C. neo.
C. par
C. gla.
C. kru.
16a
16b
16c
16d
16e
16f
16g
16h
16i
3-F
4-F
H
H
H
H
H
H
H
H
H
H
H
H
8
1
8
64
32
>64
32
>64
>64
>64
32
0.125
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
2
0.25
>64
2
>64
0.5
>64
>64
4
>64
>64
>64
>64
>64
>64
0.25
>64
>64
8
>64
>64
>64
>64
>64
>64
1
>64
>64
>64
8
>64
>64
>64
1
1
>64
0.5
>64
>64
>64
>64
>64
>64
4
>64
>64
>64
16
>64
>64
1
0.25
>64
>64
>64
1
2,4-diF
4-CN
2-CI
3-CI
4-CI
2,4-diCI
4-OCF3
4-Me
4-CF3
4-OEt
4-OMe
4-CI
4-CI
4-CI
4-CI
4-CI
4-CI
4-CI
4-CI
e
>64
>64
>64
32
0.125
>64
16
1
2
>64
1
0.06
0.125
0.25
>64
0.25
<0.03
0.125
32
>64
>64
16
0.125
>64
0.5
0.125
0.125
>64
0.125
0.06
0.06
0.06
0.5
<0.03
<0.03
<0.03
32
16j
16k
16l
2
0.125
>64
2
>64
0.125
2
>64
1
<0.03
0.06
>64
0.5
4
>64
>64
>64
0.5
>64
>64
0.5
16m
17a
17b
17c
17d
17e
17f
17g
17h
FLC
VOR
ITC
H
2-F
3-F
4-F
2,4-diF
3-CI
4-Me
OMe
4-OCF3
e
0.5
0.25
4
>64
4
0.125
0.125
>64
>64
2
0.06
0.125
8
>64
8
0.125
0.125
0.5
0.06
<0.03
1
e
e
0.125
<0.03
0.06
<0.03
0.5
0.125
e
e
a
Abbreviations: C. alb.(I), Candida albicans (ATCC SC5314); C. alb.(II), Candida albicans (CPCC400616); C. neo., Cryptococcus neoformans (CGMCC 2.3161); C. tro., Candida
tropicalis (CGMCC 2.3739); C. kru., Candida krusei (GIM 2.1); C. par., Candida parapsilosis (GIM 2.190); C. gla., Candida glabrata (Clinical isolation); FLC: fluconazole; VOR:
voriconazole; ITC: itraconazole.
Table 3
especially for the eradication of immune-mediated fungal infection.
In vitro antifungal activities of the potent compounds (MIC, m
g/mL)a.
The development of new fungicidal compounds to treat IFIs re-
mains a major challenging [23]. So, in the follow-up experiment,
the minimum fungicidal concentration (MFC) and the time-kill
curve were used for evaluation of the in vitro fungicidal ability of
the target compounds. Specifically, we evaluated MFC of potent
compounds 17b and 17f in 48 h (Table 6). Meanwhile, we per-
formed concentration-dependent time-kill assays against
C. albicans (CPCC400616) during a period of 24 h by potent com-
pound 17f. Related experimental result is shown in Fig. 4.
Compd. Candida albicans
Strain 901 Strain 904 Strain 632 Strain100 Strain103 Strain311
14e
16g
16k
16m
17a
17b
17c
17e
17f
4
4
8
4
4
8
2
4
4
8
1
4
4
4
4
4
2
4
4
2
2
4
>64
8
>64
2
2
4
2
2
8
8
1
8
>64
>64
2
>64
0.5
4
>64
1
>64
2
4
2
0.5
4
4
2
8
4
1
8
>64
16
>64
0.25
16
>64
Meanwhile, time-Kill curves were assayed to further evaluate
the fungicidal activity of compound 17f during a period of 24 h and
1
4
>64
17g
FLC
8
>64
ITC (4XMIC, 0.125
concentrations of compound 17f (0.06
0.25 g/mL) were used in the time-Kill curve assay.
m
g/mL) was used as control drug. The gradient
>64
m
g/mL, 0.125 g/mL and
m
a
m
Abbreviations: strain 901, FLC-resistant strain of C.albicans; strain 904, FLC-
resistant strain of C. albicans; strain 632, FLC-resistant strain of C. albicans; strain
100, FLC-resistant strain of C. albicans; strain 103, FLC-resistant strain of C. albicans;
strain 311, FLC-resistant strain of C. albicans; FLC: Fluconazole. Strains 901, 904, 632,
100, 103 and 311 were provided by the Second Military Medical University.
As shown in Table 6, compound 17f showed satisfactory fungi-
cidal activity. As shown in Fig. 4, compound 17f displayed rapid
fungicidal activity against C. albicans (CPCC 400616), which could
effectively kill fungi cells at 8XMIC within 16 h in the viable colony
counts (the CFU was reduced from 4 to 2 Log10 CFU/mL). Here it can
be observed that compound 17f could dose-dependently kill the
C. albicans cells. And the fungicidal activity was much more effec-
tive than ITC. This experiment once more proved that our com-
pound had fungicidal effect. Compound 17f demonstrated potent
fungicidal activity in this assay, Therefore, we believe compound
17f will be able to show its advantages in reducing the treatment
time of invasive infections and the probability of drug resistance.
2.6. Minimum fungicidal concentration of compounds 17b and 17f
and time-kill assays of and 17f
Considering the excellent antifungal inhibitory activities, com-
pounds 17b and 17f were selected for further evaluations. As is well
known, fluconazole is a fungistatic clinical agent and it is therefore
often used as maintenance therapy, as matter of fact, the killing of
fungi is very important to the treatment of fungal infection,
7

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
Table 4
Analysis of sterol composition in C. albicans by GC-MSa.
Sterol
% of total sterols (C.alb.)
No drug
FLC
17f
Lanosterol (1)
Ergosterol (2)
Eburicol (3)
ND
96.93
ND
ND
ND
34.42
3.15
29.02
22.35
4.21
42.91
5.06
31.02
3.20
8.64
6.97
2.20
14
a
-methyl ergosta-8,24(28)-dien-3
b
,6
a
-diol (4)
Unknown sterol (5)
Unknown sterol (6)
Unknown sterol (7)
ND
3.07
5.26
1.59
a
Abbreviations: The fungal strain was treated with DMSO (no drug), FLC at 0.25
mg/mL, compounds 17f at 0.015
mg/mL, ND ¼ not detected.
Table 5
SMIC values of compounds against FLC-Resistant C. albicans CPCC400616 biofilms.
Table 6
The MFC of the most potent compounds.
Compd.
SMIC50
3 h
(m
g/mL)a
Compd. MIC against C. alb. (
m
g/mL) MFC against C. alb. (
mg/mL) MFC/MIC
6 h
18 h
24 h
17b
17f
VOR
ITC
0.125
0.03
0.06
0.03
0.5
025
0.125
8
16
>64
2
4
17b
17f
FLC
ITC
2
1
32
16
16
16
8
>64
32
32
>64
16
>64
>64
>64
>64
>64
>64
>64
>64
>128
>128
>128
FLC
VOR
2.7. In vitro cytotoxicity assay
Potent compounds 17b and 17f were selected to evaluate the
toxicity to human normal liver cells (THLE-2), human lung carci-
noma cell line (A549), and breast cancer cell line (MCF-7), VOR was
selected as a control. The results are shown in Table 7, compounds
17b and 17f displayed weak inhibitory activity against the above
three cell lines, therefore, compounds 17b and 17f showed mod-
erate cytotoxicity to A549, MCF-7, and THLE2 cells.
Fungus is a type of eukaryote that is similar to mammalian cells,
so the side effects of antifungal agents on mammalian cells should
also be concerned. Therefore, it is necessary to consider the toxicity
of the compounds to mammalian cells. it is necessary to consider
the toxicity of the compounds to mammalian cells.
8

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
subtypes. Therefore, compounds 17b and 17f are less likely to
produce drug-drug interactions.
2.9. In vitro human plasma stability of compounds 17b and 17f
Human plasma contains a variety of hydrolases, such as
cholinesterase, aldolase and lipase, therefore, the stability of the
compound in plasma plays an important role in drug discovery and
development [25]. Compounds 17b and 17f were selected to eval-
uate their stability in human plasma. The results are shown in
Table 9. After co-incubated with plasma for 120 min, compound
17b exhibited moderate stability in human plasma with a half-life
of 1.8 h. Compound 17f exhibited excellent stability in human
plasma and the half-life value was greater than 4.8 h, indicating
that compound 17f possessed good plasma stability.
Fig. 4. Time-kill curve of compound 17f against C. albicans (CPCC400616). The
detection limit of this assay was 100 CFU/mL.
2.10. Molecular docking model analysis of compounds 17f in the
active site of CYP51
2.8. Cytochrome P450 enzyme inhibition assay
Cytochrome P450s (CYPs) constitute a large class of metal-
containing enzymes involved in the metabolism of a wide variety
of endogenic and xenobiotic compounds. Among them, CYP1A2,
CYP2D6, CYP2C9, CYP2C19 and CYP3A4 are the main drug-
metabolizing enzymes in humans. It is worth mentioning that
CYP3A is involved in 50% of drug metabolism and CYP2D6 in 30% of
drug metabolism [24]. Azole antifungal drugs generally have a
strong inhibitory effect on CYP enzymes because imidazole and
triazole groups can coordinate with the Fe2þ of the porphyrin ring
of CYP enzymes. Compounds 17b and 17f were selected to evaluate
their inhibitory activities against five major drug-metabolizing
enzymes. The results are summarized in Table 8. Compounds 17b
and 17f showed relatively weak inhibitory effects all five CYPs
To validate the design rationale and better understand the
binding mode of compounds with Candida albicans CYP51, com-
pound 17f was docked into the active site of C. albicans CYP51 (PDB
ID: 5tz1) from Protein Data Bank (PDB). The published crystal
structure of C. albicans CYP51 (PDB code 5TZ1) served as a useful
template for generating the binding mode [26]. As shown in Fig. 5B,
the imidazole group of the compound 17f bound to the heme group
through the formation of a coordination bond with the iron atom.
The benzoyl group occupied the hydrophobic cavity and interacted
with the surrounding amino acid residues Phe126, Ile131 and
Tyr132. The phenylthiophene group extended into the narrow hy-
drophobic pocket and mainly formed hydrophobic and van der
Waals interactions with the surrounding residues, such as Tyr64,
Leu376, His377 and Met508. The binding mode of compound 17f to
C.albicans CYP51 was similar to that of compound VT-1161.
Table 7
IC50 of tested samples in human normal and cancer cell lines.
Compd.
IC50 mM
3. Conclusions
THLE-2
A549
MCF-7
17b
17f
FLC
ITC
4.81
16.10
>30
>30
>30
26.46
19.05
>30
>30
>30
>30
>30
>30
>30
>30
Compound 6 displayed moderate antifungal activity against
C. albicans and C. neoformans and no activity against most FLC-
resistant strains. To overcome these disadvantages, we adopted
the strategy of scaffold hopping to investigate the core part of
compound 6 based on the results of molecular docking and syn-
thesized a series of 5-phenylthiophene derivatives.
VOR
Values are the average of three independent experiments. Relative errors are
generally within 5e10%.
The biological assay results revealed that most of the target
compounds exhibited high in vitro antifungal activity against all the
tested strains. Meanwhile, some compounds showed excellent
inhibitory effects against FLC-resistant strains. The potential ap-
plications and mechanism of action of compound 17f in the treat-
ment of IFIs were investigated. Similar to FLC, compound 17f caused
accumulation of methylated sterols and decreased ergosterol
biosynthesis, which indicate the mechanism of CYP51 inhibition by
antifungal azoles. Moreover, compounds 17b and 17f showed
excellent activity in inhibiting C. albicans biofilm formation high-
lighting their potential to treat biofilm-associated infections. In
addition, the time-kill curve assay revealed that compound 17f
produces fungicidal effects in a concentration-dependent manner.
Compounds 17b and 17f were almost nontoxic to A549, MCF-7, and
THLE-2 cells and exhibited excellent stability in human plasma.
Compounds 17b and 17f showed weak inhibitory effects on CYPs
and were less likely to produce drug-drug interactions. Considering
the excellent antifungal activity, better plasma stability, and lower
CYPs inhibition and cytotoxicity, our results strongly suggest that
compounds 17b and 17f are promising leads for the development of
antifungal azole analogs and warrant further drug discovery efforts
to treat fungal infections.
Table 8
In vitro CYPs inhibition of the potent compoundsa.
Compd.
IC50(mM)
CYP1A2
CYP2C9
CYP2C19
CYP2D6
CYP3A4
17b
17f
2.75
12.3
1.45
1.83
1.79
1.83
5.02
3.30
1.17
1.24
a
a
-Naphthoflavone (CYP1A2, 90.2% inhibition), sulfaphenazole (CYP2C9, 84.5%
inhibition), ticlopidine (CYP2C19, 81.5% inhibiton), quinidine (CYP2D6, 96.2% inhi-
bition), and ketoconazole (CYP3A4, 98.8% inhibition) were used as the positive
controls.
Table 9
Assessment of plasma stability of compounds.
Compd.
Time Point(min)
%Remaining
Human
T1/2 (h)
Human
1.85
17b
17f
60
120
60
73.4
47.1
93.1
89.5
>4.8
120
9

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
Fig. 5. (A) Crystal structure of Candida albicans CYP51 in complex with VT-1161 (PDB code 5TZ1). (B) Predicted binding mode of 17f in the active site of Candida albicans CYP51 (PDB
code 5TZ1).
4. Experimental section
4.5. General procedure for the synthesis of intermediates (11a-h)
4.1. Chemistry
Intermediates 11a-h were synthesized according to previously
reported methods [16].
All starting materials and analytically pure solvents, unless
otherwise specified, were received from the vendors. The reactions
were monitored by TLC, which was performed on silica gel plates
with fluorescence F-254 and visualized with UV light. Silica gel of
200e300 mesh was used for column chromatography. The melting
points of the compounds were determined on a BüCHI Melting
Point B-540 melting point apparatus and were uncorrected. The
Low-resolution electrospray ionization mass spectrometry was
measured by Agilent 6120 (quadrupole liquid chromatography)
equipped Agilent 1200 LC/MS. High-resolution mass spectra ex-
periments were measured on the Agilent Accurate-Mass Q-TOF
6530 instrument GCꢁMS analysis was performed on an Agilent
1200 LCꢁMS using ESI mode. Nuclear magnetic resonance (NMR)
spectra were recorded on a Bruker Avance III-600 or Bruker Avance
III-400 instruments (600 or 400 MHz for 1H and 150 or 100 MHz for
13C) with TMS as an internal standard. The coupling constants (J)
are reported in hertz and the peak multiplicities were described as
s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br,
broad peak.
4.6. General procedure for the synthesis of intermediates (12a-h)
The Boc protected compounds 11a-h (1.0 equiv.) was added to
HCleEtOH (4 N, 7.0 equiv.); then the mixture was stirred at room
temperature for 3 h. The mixture was filtered, and the residue was
dried to afford 12a-h as a white solid.
4.7. General procedure for the synthesis of compounds (14a-j)
To a solution of acid 13a-j (1.0 equiv.) in DMF, HOBt (1.1 equiv.)
and EDCI (1.1 equiv.) were added and the mixture was stirred for 1 h
at room temperature. Then the amino salt 12a (1.1 equiv.) and DIEA
(2.2 equiv.) were added. The reaction was stirred and monitored
with TLC. The mixture was poured into water and extracted with
ethyl acetate three times. The combined organic layers were dried
over Na2SO4 and evaporated in a vacuum [16]. The crude product
was
purified
by
silica
gel
column
chromatography
(CH3OH:CH2Cl2 ¼ 40:1) to afford target compounds 14a-j.
4.7.1. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-4,5-
dihydronaphtho[2,1-d]isoxazole-3-carboxamide (14a)
4.2. General procedure for the synthesis of intermediates 8a-h
Light white solid; yield: 62.0%; mp:145.2e147.0 ꢀC. 1H NMR
Intermediates 8a-h were synthesized according to previously
reported methods [16].
(600 MHz, DMSO)
d
9.45 (d, J ¼ 8.5 Hz, 1H), 8.02 (d, J ¼ 7.2 Hz, 2H),
7.69e7.60 (m, 3H), 7.55e7.52 (m, 2H), 7.39 (dd, J ¼ 6.4, 1.3 Hz, 2H),
7.37e7.30 (m, 1H), 7.21 (s, 1H), 6.86 (s, 1H), 5.79e5.75 (m, 1H), 4.59
(dd, J ¼ 14.2, 4.5 Hz, 1H), 4.44 (dd, J ¼ 14.2, 9.5 Hz, 1H), 2.98 (t,
J ¼ 7.9 Hz, 2H), 2.83e2.66 (m, 2H). 13C NMR (151 MHz, DMSO‑d6)
4.3. General procedure for the synthesis of intermediates 9a-h
d
196.63, 166.85, 159.42, 155.31, 137.36, 137.29, 135.21, 134.18,
Intermediates 9a-h were synthesized according to previously
133.43, 129.62 (2C), 129.24 (2C), 128.98, 128.83, 127.73, 127.69,
reported methods [16].
124.20, 121.99, 121.91, 55.10, 46.19, 28.23, 18.09. ESI-HRMS (m/z):
[MþH]þ for C24H20N4O3 calcd. 413.1619; found 413.1625[M þ H] þ
.
4.4. General procedure for the synthesis of intermediates 10a-h
4.7.2. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-2-
phenyloxazole-5-carboxamide (14b)
Intermediates 10a-h were synthesized according to previously
Light white solid; yield: 55.2%; mp:110.4e111.8 ꢀC. 1H NMR
reported methods [16].
(600 MHz, DMSO‑d6)
d
9.01 (d, J ¼ 8.7 Hz, 1H), 8.68 (s, 1H), 8.03 (dd,
10

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
J ¼ 8.4, 1.4 Hz, 2H), 8.01e7.97 (m, 2H), 7.63 (d, J ¼ 14.8 Hz, 1H), 7.59
(s, 1H), 7.56 (dd, J ¼ 5.2, 2.0 Hz, 3H), 7.54e7.50 (m, 2H), 7.17 (s, 1H),
6.83 (s, 1H), 5.81e5.77 (m, 1H), 4.60 (dd, J ¼ 14.2, 4.5 Hz, 1H), 4.47
4.7.7. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-2-
naphthamide (14g)
Light white solid; yield: 62.2%; mp:103.7e105.7 ꢀC. 1H NMR
(dd, J ¼ 14.2, 9.1 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
d
196.96,
(600 MHz, DMSO)
d
9.28 (d, J ¼ 8.4 Hz, 1H), 8.34 (s, 1H), 8.05 (dd,
161.16, 160.13, 143.21, 136.73, 135.27, 134.11, 131.78, 129.82, 129.72,
129.55 (2C), 129.22 (2C), 128.93 (2C), 128.86 (2C), 126.88, 126.79,
54.67, 46.44. ESI-HRMS (m/z): [MþH]þ for C22H18N4O3 calcd.
J ¼ 8.4, 1.2 Hz, 2H), 7.99 (d, J ¼ 7.9 Hz, 1H), 7.96 (d, J ¼ 8.7 Hz, 2H),
7.79 (dd, J ¼ 8.6, 1.8 Hz, 1H), 7.67 (s, 1H), 7.64e7.56 (m, 3H), 7.52 (t,
J ¼ 7.8 Hz, 2H), 7.25 (s, 1H), 6.84 (s, 1H), 5.84e5.80 (m, 1H), 4.62 (dd,
J ¼ 14.1, 4.6 Hz, 1H), 4.45 (dd, J ¼ 14.1, 9.5 Hz, 1H). 13C NMR
387.1476; found 387.1483 [M þ H] þ
.
(151 MHz, DMSO‑d6) d 196.97, 166.06, 134.96, 134.25, 133.56, 131.96,
130.64, 128.88 (2C), 128.84 (2C), 128.69, 128.41, 128.35, 128.23,
127.98, 127.84, 127.73, 127.61, 126.86, 123.95, 54.90, 45.92. ESI-
4.7.3. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
phenylfuran-2-carboxamide (14c)
HRMS (m/z): [MþH]þ for C23H19N3O2 calcd. 370.1551; found
Light white solid; yield: 51.2%; mp:120.4e121.6 ꢀC.1H NMR
þ
370.1555 [M þ H]
.
(600 MHz, DMSO‑d6)
d
9.12 (d, J ¼ 8.9 Hz, 1H), 8.03 (dd, J ¼ 8.4,
1.3 Hz, 2H), 7.87 (dd, J ¼ 8.4, 1.3 Hz, 2H), 7.64 (t, J ¼ 7.4 Hz, 2H),
7.55e7.51 (m, 2H), 7.46 (t, J ¼ 7.8 Hz, 2H), 7.37 (t, J ¼ 7.4 Hz, 1H), 7.22
(s, 1H), 7.16 (d, J ¼ 3.6 Hz, 1H), 7.07 (d, J ¼ 3.6 Hz, 1H), 6.84 (s, 1H),
5.84e5.79 (m, 1H), 4.61 (dd, J ¼ 14.1, 4.6 Hz, 1H), 4.42 (dd, J ¼ 14.1,
4.7.8. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)benzo
[b]thiophene-2-carboxamide (14h)
Light white solid; yield: 56.7%; mp:103.9e105.5 ꢀC. 1H NMR
(600 MHz, DMSO)
d
9.41 (d, J ¼ 8.5 Hz, 1H), 8.06 (s, 1H), 8.04 (dd,
9.5 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
d 197.11, 157.58, 155.44,
J ¼ 8.4, 1.2 Hz, 2H), 7.99 (d, J ¼ 8.0 Hz, 1H), 7.94 (d, J ¼ 7.1 Hz, 1H),
7.66 (s, 1H), 7.65e7.61 (m, 1H), 7.53 (t, J ¼ 7.0 Hz, 2H), 7.48e7.41 (m,
2H), 7.24 (s, 1H), 6.84 (s, 1H), 5.79e5.77 (m, 1H), 4.59 (dd, J ¼ 14.2,
4.6 Hz, 1H), 4.42 (dd, J ¼ 14.2, 9.5 Hz, 1H). 13C NMR (151 MHz,
146.30, 135.24, 134.19, 129.64, 129.46 (2C), 129.36 (2C), 129.24,
128.94 (2C), 128.86, 124.99 (2C), 124.87, 117.21, 108.16, 54.23, 40.53.
ESI-HRMS (m/z): [MþH]þ for C23H19N3O3 calcd. 386.1604; found
386.1621 [M þ H] þ
.
DMSO‑d6)
d 196.61, 161.28, 140.25, 138.90, 138.34, 137.90, 134.80,
133.72, 129.04, 128.77 (2C), 128.48, 128.38 (2C), 126.49, 125.68,
125.35, 125.01, 122.79, 54.75, 45.89. ESI-HRMS (m/z): [MþH]þ for
4.7.4. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-4-
(thiophen-3-yl)benzamide (14d)
þ
C
21H17N3O2S calcd. 376.1119; found 376.1121 [M þ H]
.
Light white solid; yield: 60.2%; mp:161.1e163.1 ꢀC. 1H NMR
4.7.9. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)
benzofuran-2-carboxamide (14i)
(600 MHz, DMSO)
d
9.13 (d, J ¼ 8.4 Hz, 1H), 8.06e8.00 (m, 2H), 8.00
(dd, J ¼ 2.8, 1.3 Hz, 1H), 7.77 (dd, J ¼ 20.0, 8.5 Hz, 4H), 7.69e7.57 (m,
Light white solid; yield: 66.3%; mp:101.7e103.5 ꢀC. 1H NMR
4H), 7.52 (t, J ¼ 7.8 Hz, 2H), 7.22 (s, 1H), 6.84 (s, 1H), 5.76e5.73 (m,
(600 MHz, DMSO)
d
9.38 (d, J ¼ 8.6 Hz, 1H), 8.02 (dd, J ¼ 8.3, 1.1 Hz,
1H), 4.59 (dd, J ¼ 14.1, 4.5 Hz, 1H), 4.42 (dd, J ¼ 14.1, 9.6 Hz, 1H). 13
C
2H), 7.75 (d, J ¼ 7.8 Hz, 1H), 7.65e7.59 (m, 3H), 7.55e7.50 (m, 3H),
7.48e7.42 (m, 1H), 7.32 (t, J ¼ 7.5 Hz, 1H), 7.21 (s, 1H), 6.83 (s, 1H),
5.82e5.78 (m, 1H), 4.60 (dd, J ¼ 14.2, 4.5 Hz, 1H), 4.44 (dd, J ¼ 14.2,
NMR (151 MHz, DMSO‑d6)
d 197.02, 165.67, 140.38, 138.08, 137.90,
134.97, 133.53, 131.60, 128.67 (2C), 128.40, 128.32 (2C), 127.97 (2C),
127.40 (2C), 126.19, 125.85, 122.47, 120.06, 54.92, 45.83. ESI-HRMS
(m/z): [MþH]þ for C23H19N3O2S calcd. 402.1276; found 402.1283
9.6 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
d 196.46, 157.75, 154.24,
147.89, 137.87, 134.79, 133.66, 128.74 (2C), 128.35 (2C), 128.25,
127.17, 126.89, 123.83, 122.90, 120.01, 111.84, 110.44, 54.37, 45.82.
[M þ H] þ
.
ESI-HRMS (m/z): [MþH]þ for C21H17N3O3 calcd. 360.1344; found
þ
360.1348 [M þ H]
.
4.7.5. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
phenylthiophene-2-carboxamide (14e)
Light white solid; yield: 57.2%; mp:132.7e134.3 ꢀC. 1H NMR
4.7.10. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-4-
(4-phenylpiperazin-1-yl)benzamide (14j)
(600 MHz, DMSO‑d6)
d
9.17 (d, J ¼ 8.6 Hz, 1H), 8.05e8.00 (m, 2H),
Light white solid; yield: 70.3%; mp:103.9e105.5 ꢀC. 1H NMR
7.75e7.70 (m, 2H), 7.69e7.66 (m, 2H), 7.66e7.62 (m, 1H), 7.53 (t,
J ¼ 7.8 Hz, 2H), 7.50 (d, J ¼ 3.9 Hz, 1H), 7.43 (t, J ¼ 7.7 Hz, 2H),
7.39e7.33 (m, 1H), 7.25 (s, 1H), 6.87 (s, 1H), 5.79e5.73 (m, 1H), 4.59
(dd, J ¼ 14.1, 4.5 Hz, 1H), 4.40 (dd, J ¼ 14.2, 9.5 Hz, 1H). 13C NMR
(600 MHz, DMSO)
d
8.84 (d, J ¼ 8.4 Hz, 1H), 8.01 (d, J ¼ 7.8 Hz, 2H),
7.65 (d, J ¼ 8.8 Hz, 2H), 7.63e7.57 (m, 2H), 7.50 (t, J ¼ 7.7 Hz, 2H),
7.24 (t, J ¼ 7.8 Hz, 2H), 7.19 (s, 1H), 6.98 (dd, J ¼ 8.3, 5.1 Hz, 4H),
6.85e6.74 (m, 2H), 5.70e5.66 (m, 1H), 4.56 (dd, J ¼ 14.1, 4.5 Hz, 1H),
4.39 (dd, J ¼ 14.0, 9.5 Hz,1H), 3.47e3.36 (m, 4H), 3.29e3.23 (m, 4H).
(151 MHz, DMSO‑d6)
d 196.67, 160.64, 148.19, 137.17, 134.81, 133.71,
132.89, 130.06, 129.31, 129.26 (2C), 128.98, 128.77 (2C), 128.47,
13C NMR (151 MHz, DMSO‑d6)
d 197.25, 165.59, 152.91, 150.82,
128.40 (2C), 125.80 (2C), 125.74, 124.41, 54.50, 46.10. ESI-HRMS (m/
z): [MþH]þ for C23H19N3O2S calcd. 402.1296; found 402.1303 [M þ
137.84, 135.05, 133.41, 128.98 (2C), 128.71 (2C), 128.60 (2C), 128.31
(2C), 128.15, 122.52, 120.02, 119.19, 115.68 (2C), 113.68 (2C), 54.75,
þ
H]
.
48.06 (2C), 47.04 (2C), 45.87. ESI-HRMS (m/z): [MþH]þ for
þ
C
29H29N3O3 calcd. 480.2399; found 480.2406 [M þ H]
.
4.7.6. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-6-
bromobenzo[d]thiazole-2-carboxamide (14f)
4.8. General procedure for the synthesis of compounds 16a-m
Light white solid; yield: 55.9%; mp:152.7e153.9 ꢀC. 1H NMR
(600 MHz, DMSO)
d
9.87 (d, J ¼ 8.6 Hz, 1H), 8.52 (d, J ¼ 1.8 Hz, 1H),
Compounds 16a-m were prepared using a method which was
similar to that of compounds 14a-j.
8.05 (d, J ¼ 8.8 Hz, 1H), 8.01 (d, J ¼ 7.5 Hz, 2H), 7.78 (dd, J ¼ 8.8,
1.9 Hz,1H), 7.64 (t, J ¼ 7.4 Hz,1H), 7.60 (s,1H), 7.53 (t, J ¼ 7.7 Hz, 2H),
7.20 (s,1H), 6.82 (s,1H), 5.82e5.77 (m,1H), 4.61 (dd, J ¼ 14.2, 4.3 Hz,
1H), 4.51 (dd, J ¼ 14.2, 9.4 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
4.8.1. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-(3-
fluorophenyl)thiophene-2-carboxamide(16a)
d
196.38, 164.48, 159.60, 152.00, 138.42, 138.35, 135.19, 134.19,
Light white solid; yield: 50.23%; mp:102.9e104.5 ꢀC. 1H NMR
131.00, 129.26 (2C), 128.82 (2C), 128.77, 126.17, 126.10, 120.58,
(600 MHz, DMSO‑d6)
d
9.20 (d, J ¼ 8.6 Hz, 1H), 8.04e8.00 (m, 2H),
120.46, 55.70, 46.15. ESI-HRMS (m/z): [MþH]þ for C20H15BrN4O2S
7.72 (d, J ¼ 4.0 Hz, 1H), 7.68e7.62 (m, 1H), 7.58 (d, J ¼ 4.0 Hz, 1H),
calcd. 455.0161; found 455.0168 [M þ H] þ
.
7.57e7.45 (m, 6H), 7.24e7.18 (m, 2H), 6.85 (s, 1H), 5.78e5.73 (m,
11

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
1H), 4.58 (dd, J ¼ 14.2, 4.5 Hz, 1H), 4.39 (dd, J ¼ 14.2, 9.5 Hz, 1H). 13
C
4.8.6. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
(3-chlorophenyl)thiophene-2-carboxamide(16f)
NMR (151 MHz, DMSO‑d6)
d
196.66, 161.41 (d, J ¼ 297.8 Hz), 160.61,
147.02, 137.26, 134.80, 133.72, 132.90, 131.12, 130.08, 129.56 (d,
J ¼ 3.3 Hz), 128.99, 128.77 (2C), 128.47, 128.40 (2C), 128.03 (d,
J ¼ 8.2 Hz), 127.94 (d, J ¼ 8.4 Hz), 116.30 (d, J ¼ 7.4 Hz), 116.16 (d,
Light white solid; yield: 53.20%; mp:126.8e128.8 ꢀC. 1H NMR
(600 MHz, DMSO‑d6)
d
9.22 (d, J ¼ 8.6 Hz, 1H), 8.02 (d, J ¼ 7.1 Hz,
2H), 7.75 (t, J ¼ 1.9 Hz, 1H), 7.71 (d, J ¼ 4.0 Hz, 1H), 7.65e7.60 (m,
2H), 7.59 (d, J ¼ 4.0 Hz, 1H), 7.52 (d, J ¼ 7.7 Hz, 2H), 7.50e7.39 (m,
2H), 7.28 (s, 1H), 6.87 (s, 1H), 5.80e5.75 (m, 1H), 4.62 (dd, J ¼ 14.2,
4.5 Hz, 1H), 4.41 (dd, J ¼ 14.2, 9.5 Hz, 1H). 13C NMR (151 MHz,
J ¼ 7.5 Hz), 54.50, 46.11. ESI-HRMS (m/z): [MþH]þ for C23H18F N3O2S
þ
calcd. 420.1204; found 420.1282 [M þ H]
.
DMSO‑d6)
d 196.54, 160.55, 146.22, 138.02, 134.92, 134.76, 134.01,
4.8.2. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
(4-fluorophenyl)thiophene-2-carboxamide (16b)
133.74, 131.11, 131.04, 130.05, 129.00, 128.77 (2C), 128.48, 128.40
(2C), 128.36, 125.58, 125.23, 124.48, 54.50, 46.28. ESI-HRMS (m/z):
Light white solid; yield: 51.20%; mp:102.9e104.5 ꢀC. 1H NMR
[MþH]þ for C23H18ClN3O2S calcd. 436.0913; found 436.0986 [M þ
þ
(600 MHz, DMSO‑d6)
d
9.18 (d, J ¼ 8.6 Hz, 1H), 8.02 (d, J ¼ 7.0 Hz,
H]
.
2H), 7.76e7.69 (m, 4H), 7.64 (t, J ¼ 7.4 Hz, 1H), 7.56e7.50 (m, 2H),
7.47 (d, J ¼ 3.9 Hz, 1H), 7.27 (t, J ¼ 8.8 Hz, 2H), 7.25 (s, 1H), 6.87 (s,
1H), 5.78e5.73 (m, 1H), 4.59 (dd, J ¼ 14.1, 4.6 Hz, 1H), 4.40 (dd,
4.8.7. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
(4-chlorophenyl)thiophene-2-carboxamide (16g)
J ¼ 14.1, 9.5 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
d 196.66, 161.41
Light white solid; yield: 60.20%; mp:109.6e110.0 ꢀC. 1H NMR
(d, J ¼ 297.4 Hz), 160.61, 147.02, 137.26, 134.80, 133.72, 130.08,
129.56 (d, J ¼ 3.3 Hz), 128.99, 128.77, 128.47, 128.40, 128.03 (d,
J ¼ 8.2 Hz), 127.94 (d, J ¼ 8.4 Hz), 116.30 (d, J ¼ 7.4 Hz), 116.16 (d,
J ¼ 7.5 Hz), 54.50, 46.11. ESI-HRMS (m/z): [MþH]þ for C23H18FN3O2S
(600 MHz, DMSO‑d6)
d
9.19 (d, J ¼ 8.6 Hz, 1H), 8.02 (dd, J ¼ 8.4,
1.3 Hz, 2H), 7.72e7.68 (m, 4H), 7.65e7.62 (m, 1H), 7.53e7.52 (m,
2H), 7.51e7.48 (m, 3H), 7.24 (s, 1H), 6.87 (s, 1H), 5.78e5.72 (m, 1H),
4.59 (dd, J ¼ 14.1, 4.6 Hz, 1H), 4.40 (dd, J ¼ 14.2, 9.5 Hz, 1H). 13C NMR
calcd. 420.1199; found 420.1203 [M þ H] þ
.
(151 MHz, DMSO‑d6)
d 196.05, 159.92, 146.02, 137.27, 137.11, 134.19,
133.10, 132.55, 131.20, 129.47, 128.62 (2C), 128.15 (2C), 127.77 (2C),
127.41, 126.83 (2C), 124.45, 119.54, 53.93, 45.38. ESI-HRMS (m/z):
4.8.3. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
(2,4-difluorophenyl)thiophene-2-carboxamide(16c)
[MþH]þ for C23H18ClN3O2S calcd. 435.0917; found 436.0986 [M þ
þ
H]
.
Light white solid; yield: 58.20%; mp:102.7e104.7 ꢀC. 1H NMR
(600 MHz, DMSO‑d6)
d
9.23 (d, J ¼ 8.6 Hz, 1H), 8.04e8.00 (m, 2H),
4.8.8. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
(2,4-dichlorophenyl)thiophene-2-carboxamide (16h)
7.85 (m,1H), 7.76e7.70 (m, 2H), 7.64 (t, J ¼ 7.4 Hz,1H), 7.55e7.50 (m,
3H), 7.44e7.40 (m, 1H), 7.26e7.17 (m, 2H), 6.88 (s, 1H), 5.76 (m, 1H),
4.59 (dd, J ¼ 14.2, 4.5 Hz, 1H), 4.40 (dd, J ¼ 14.1, 9.5 Hz, 1H). 13C NMR
Light white solid; yield: 58.72%; mp:172.6e174.0 ꢀC. 1H NMR
(600 MHz, DMSO‑d6)
d
9.25 (d, J ¼ 8.6 Hz, 1H), 8.03 (d, J ¼ 7.2 Hz,
(151 MHz, DMSO‑d6)
d
196.67, 161.96 (dd, J ¼ 236.1 Hz), 160.57,
2H), 7.76 (dd, J ¼ 16.2, 3.1 Hz, 2H), 7.66 (d, J ¼ 8.6 Hz, 1H), 7.63 (d,
J ¼ 10.3 Hz, 2H), 7.57e7.49 (m, 3H), 7.43 (d, J ¼ 4.0 Hz, 1H), 7.22 (s,
1H), 6.84 (s, 1H), 5.78e5.73 (m, 1H), 4.57 (dd, J ¼ 14.2, 4.5 Hz, 1H),
4.39 (dd, J ¼ 14.2, 9.7 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
158.49 (dd, J ¼ 252.0, 12.7 Hz), 141.11, 136.11, 134.81, 133.74, 132.92,
130.35, 130.26 (dd, J ¼ 10.0, 4.9 Hz), 129.00, 128.79 (2C), 128.49,
128.40 (2C),127.07 (d, J ¼ 3.9 Hz),117.46 (dd, J ¼ 12.8, 4.1 Hz),112.67
(dd, J ¼ 15.6, 4.4 Hz),105.03 (td, J ¼ 26.4, 7.7 Hz), 54.53, 46.01. HRMS
(EI): m/z (Mþ) for C23H17F2N3O2S calcd. 438.1112; found 438.1119
d
196.65, 160.49, 142.55, 139.30, 137.91, 134.81, 133.87, 133.73,
132.52, 131.92, 130.62, 129.97, 129.21, 128.85, 128.79 (2C), 128.40
[M þ H] þ
.
(2C), 128.32, 128.00, 120.05, 54.53, 45.89. ESI-HRMS (m/z): [MþH]þ
for C22H18Cl2N3O2S calcd. 470.0535; found 470.0579 [M þ H] þ
.
4.8.4. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
(4-cyanophenyl)thiophene-2-carboxamide (16d)
4.8.9. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
Light white solid; yield: 58.20%; mp:168.7e169.9 ꢀC. 1H NMR
(4-(trifluoromethoxy)phenyl)thiophene-2-carboxamide (16i)
Light white solid; yield: 58.20%; mp:149.6e151.0 ꢀC. 1H NMR
(600 MHz, DMSO‑d6)
d
9.25 (d, J ¼ 8.6 Hz, 1H), 8.04e8.01 (m, 2H),
(600 MHz, DMSO‑d6)
d
9.23 (d, J ¼ 8.6 Hz, 1H), 8.04 (d, J ¼ 7.3 Hz,
7.88 (d, J ¼ 3.3 Hz, 3H), 7.76 (d, J ¼ 4.0 Hz, 1H), 7.70 (d, J ¼ 4.0 Hz,
1H), 7.66e7.63 (m, 2H), 7.55e7.51 (m, 3H), 7.22 (s, 1H), 6.85 (s, 1H),
5.79e5.74 (m, 1H), 4.58 (dd, J ¼ 14.2, 4.6 Hz, 1H), 4.39 (dd, J ¼ 14.2,
2H), 7.79 (d, J ¼ 8.7 Hz, 2H), 7.74 (d, J ¼ 4.0 Hz, 1H), 7.68 (s, 1H), 7.63
(d, J ¼ 7.6 Hz, 1H), 7.55e7.50 (m, 3H), 7.42 (d, J ¼ 8.6 Hz, 2H), 7.24 (s,
1H), 6.86 (s, 1H), 5.79e5.75 (m, 1H), 4.59 (dd, J ¼ 14.2, 4.6 Hz, 1H),
4.41 (dd, J ¼ 14.2, 9.5 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
9.5 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
d 197.09, 160.85, 146.16,
139.78, 138.41, 137.64, 135.25, 134.21, 133.64 (2C), 130.58, 129.25
(2C), 128.85 (2C), 127.23, 126.81 (2C), 120.56, 119.07, 111.14, 55.05,
46.40. ESI-HRMS (m/z): [MþH]þ for C24H18N4O2S calcd. 427.1253;
d
196.67, 160.55, 148.31 (d, J ¼ 16.1 Hz), 146.33, 138.03, 134.81,
133.71, 132.25, 130.08, 129.00, 128.76 (2C), 128.46, 128.40 (2C),
þ
127.77, 127.68 (2C), 125.32, 121.80 (d, J ¼ 4.7 Hz, 2C), 118.34 (d,
found 427.1282 [M þ H]
J
C
¼
256.6 Hz), 54.55, 45.99. ESI-HRMS (m/z): [MþH]þ for
þ
24H18F3N3O3S calcd. 486.1130; found 486.1136 [M þ H]
.
4.8.5. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
(2-chlorophenyl)thiophene-2-carboxamide (16e)
4.8.10. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
(p-tolyl)thiophene-2-carboxamide (16j)
Light white solid; yield: 51.20%; mp:151.6e153.5 ꢀC. 1H NMR
(600 MHz, DMSO‑d6)
d
9.23 (d, J ¼ 8.6 Hz, 1H), 8.07e8.00 (m, 2H),
Light white solid; yield: 51.70%; mp:153.1e154.5 ꢀC. 1H NMR
7.74 (d, J ¼ 4.0 Hz, 1H), 7.66e7.62 (m, 3H), 7.60e7.57 (m, 1H), 7.54 (t,
J ¼ 7.8 Hz, 2H), 7.43e7.39 (m, 3H), 7.22 (s, 1H), 6.85 (s, 1H),
5.78e5.74 (m, 1H), 4.58 (dd, J ¼ 14.2, 4.5 Hz, 1H), 4.40 (dd, J ¼ 14.1,
(600 MHz, DMSO‑d6)
d
9.14 (d, J ¼ 8.6 Hz, 1H), 8.04e8.00 (m, 2H),
7.69 (t, J ¼ 6.0 Hz, 2H), 7.64 (t, J ¼ 7.4 Hz,1H), 7.57e7.50 (m, 4H), 7.43
(d, J ¼ 3.9 Hz,1H), 7.23 (d, J ¼ 8.0 Hz, 3H), 6.85 (s, 1H), 5.77e5.70 (m,
1H), 4.59 (dd, J ¼ 14.1, 4.6 Hz, 1H), 4.39 (dd, J ¼ 14.1, 9.5 Hz, 1H), 2.31
9.6 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
d 196.68, 160.57, 143.84,
138.90, 137.92, 134.82, 133.72, 131.56, 131.39, 130.98, 130.55, 130.18,
128.85 (2C), 128.81 (2C), 128.78, 128.40, 128.32, 127.83, 120.06,
54.53, 45.90. ESI-HRMS (m/z): [MþH]þ for C23H18ClN3O2S calcd.
(s, 3H). 13C NMR (151 MHz, DMSO‑d6)
d 196.04, 160.02, 147.76,
137.66, 135.91, 134.16, 133.05, 129.40, 129.18, 129.14 (2C), 128.32,
128.11 (2C), 127.81, 127.74 (2C), 125.05, 124.97 (2C), 123.15, 53.84,
45.43, 20.13. ESI-HRMS (m/z): [MþH]þ for C24H21N3O2S calcd.
436.0913; found 436.0921 [M þ H] þ
.
12

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
þ
416.1456; found 416.1463 [M þ H]
.
(600 MHz, DMSO‑d6)
d
9.22 (d, J ¼ 8.4 Hz, 1H), 7.85 (d, J ¼ 7.8 Hz,
1H), 7.79e7.75 (m, 1H), 7.71 (d, J ¼ 4.0 Hz, 1H), 7.70e7.68 (m, 2H),
7.62 (s, 1H), 7.60e7.56 (m, 1H), 7.53 (d, J ¼ 3.9 Hz, 1H), 7.52e7.49 (m,
2H), 7.48 (d, J ¼ 2.0 Hz, 1H), 7.20 (s, 1H), 6.84 (s, 1H), 5.73e5.68 (m,
4.8.11. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
(4-(trifluoromethyl)phenyl)thiophene-2-carboxamide (16k)
Light white solid; yield: 51.70%; mp:137.1e138.5 ꢀC. 1H NMR
1H), 4.57 (dd, J ¼ 14.2, 4.6 Hz, 1H), 4.40 (dd, J ¼ 14.2, 9.5 Hz, 1H). 13
C
(600 MHz, DMSO‑d6)
d
9.24 (d, J ¼ 8.6 Hz, 1H), 8.04e8.00 (m, 2H),
NMR (151 MHz, DMSO‑d6)
d
195.77 (d, J ¼ 2.1 Hz), 162.82 (d,
7.89 (d, J ¼ 8.2 Hz, 2H), 7.78 (d, J ¼ 8.3 Hz, 2H), 7.75 (d, J ¼ 4.0 Hz,
1H), 7.71e7.66 (m, 2H), 7.65e7.62 (m, 1H), 7.53 (t, J ¼ 7.8 Hz, 2H),
7.24 (s,1H), 6.85 (s,1H), 5.79e5.75 (m,1H), 4.59 (dd, J ¼ 14.2, 4.5 Hz,
1H), 4.40 (dd, J ¼ 14.2, 9.6 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
J ¼ 244.6 Hz), 160.63, 146.76, 137.90, 137.51, 137.07 (d, J ¼ 6.3 Hz),
133.20, 131.77, 130.97 (d, J ¼ 7.8 Hz), 130.16, 129.24 (2C), 128.30,
127.45 (2C), 125.08, 124.54 (d, J ¼ 2.6 Hz), 120.57 (d, J ¼ 21.1 Hz),
120.04, 114.83 (d, J ¼ 22.5 Hz), 54.78, 45.75. ESI-HRMS (m/z):
d
196.62, 160.46, 145.97, 141.05, 138.79, 134.78, 133.75, 132.93,
[MþH]þ for C23H17ClFN3O2S calcd. 454.0839; found 454.0832 [M þ
þ
130.11, 129.01, 128.79 (2C), 128.61 (q, J ¼ 21.5 Hz), 128.49, 128.39
H]
.
(2C), 126.47, 126.38 (2C), 126.29e126.11 (m, 2C), 123.19 (q,
J
C
¼
453.2 Hz), 54.56, 46.02. ESI-HRMS (m/z): [MþH]þ for
4.9.3. 5-(4-chlorophenyl)-N-(1-(4-fluorophenyl)-3-(1H-imidazole-
1-yl)-1-oxopropan-2-yl)thiophene-2-carboxamide (17c)
þ
24H18F3N3O2S calcd. 470.1184; found 470.1179 [M þ H]
.
Light white solid; yield: 53.60%; mp:143.6e144.5 ꢀC. 1H NMR
4.8.12. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
(4-ethoxyphenyl)thiophene-2-carboxamide(16l)
(600 MHz, DMSO‑d6)
d
9.19 (d, J ¼ 8.6 Hz, 1H), 8.10 (dd, J ¼ 8.8,
5.5 Hz, 2H), 7.72 (d, J ¼ 4.0 Hz, 1H), 7.69 (d, J ¼ 8.6 Hz, 2H), 7.61 (s,
1H), 7.54 (d, J ¼ 3.9 Hz,1H), 7.49 (d, J ¼ 7.8 Hz, 2H), 7.37 (t, J ¼ 8.8 Hz,
2H), 7.20 (s, 1H), 6.83 (s, 1H), 5.76e5.71 (m, 1H), 4.56 (dd, J ¼ 14.2,
4.6 Hz, 1H), 4.38 (dd, J ¼ 14.2, 9.5 Hz, 1H). 13C NMR (151 MHz,
Light white solid; yield: 55.70%; mp:159.1e160.8 ꢀC. 1H NMR
(600 MHz, DMSO‑d6)
d
9.11 (d, J ¼ 8.6 Hz, 1H), 8.04e8.00 (m, 2H),
7.67 (d, J ¼ 4.0 Hz, 2H), 7.64 (t, J ¼ 7.4 Hz, 1H), 7.60e7.55 (m, 2H),
7.55e7.49 (m, 2H), 7.36 (d, J ¼ 3.9 Hz,1H), 7.23 (s, 1H), 7.01e6.94 (m,
2H), 6.85 (s, 1H), 5.76e5.72 (m, 1H), 4.58 (dd, J ¼ 14.1, 4.6 Hz, 1H),
4.39 (dd, J ¼ 14.1, 9.5 Hz, 1H), 4.05 (q, J ¼ 7.0 Hz, 2H), 1.32 (t,
DMSO‑d6)
d
195.35, 165.24 (d, J ¼ 278.0 Hz), 160.58, 146.71, 137.90,
137.63, 133.19, 131.42 (d, J ¼ 9.6 Hz, 2C), 130.12, 129.27, 129.23 (2C),
128.31, 127.51, 127.44 (2C), 125.06, 120.04, 115.87 (d, J ¼ 21.9 Hz, 2C),
J ¼ 7.0 Hz, 3H). 13C NMR (151 MHz, DMSO‑d6)
d
197.21, 161.18,
54.51, 45.86. ESI-HRMS (m/z): [MþH]þ for C23H17ClFN3O2S calcd.
þ
159.42, 148.89, 136.40, 135.30, 134.15, 130.56, 129.22 (2C), 128.86
(2C), 127.70, 127.61 (2C), 125.89, 123.54, 115.55, 115.52 (2C), 63.73,
454.0823; found 454.0892 [M þ H]
.
63.70, 54.95, 46.48, 15.04. ESI-HRMS (m/z): [MþH]þ for
4.9.4. 5-(4-chlorophenyl)-N-(1-(2,4-difluorophenyl)-3-(1H-
imidazole-1-yl)-1-oxopropan-2-yl)thiophene-2-carboxamide(17d)
Light white solid; yield: 53.60%; mp:123.6e124.9 ꢀC. 1H NMR
þ
C
25H23N3O3S calcd. 446.1568; found 446.1575 [M þ H]
.
4.8.13. N-(3-(1H-imidazole-1-yl)-1-oxo-1-phenylpropan-2-yl)-5-
(4-methoxyphenyl)thiophene-2-carboxamide (16m)
(600 MHz, DMSO‑d6)
d
9.26 (d, J ¼ 7.8 Hz, 1H), 7.90 (q, J ¼ 8.5 Hz,
1H), 7.70 (d, J ¼ 3.6 Hz, 3H), 7.59 (s, 1H), 7.53 (d, J ¼ 4.0 Hz, 1H), 7.49
(d, J ¼ 8.4 Hz, 2H), 7.40 (t, J ¼ 9.0 Hz, 1H), 7.23 (t, J ¼ 7.2 Hz, 1H), 7.15
(s,1H), 6.85 (s,1H), 5.37e5.32 (m,1H), 4.63 (dd, J ¼ 14.2, 4.4 Hz,1H),
4.36 (dd, J ¼ 14.3, 9.2 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
Light white solid; yield: 55.70%; mp:142.1e143.8 ꢀC. 1H NMR
(600 MHz, DMSO‑d6)
d
9.11 (d, J ¼ 8.6 Hz, 1H), 8.02 (dd, J ¼ 8.4,
1.3 Hz, 2H), 7.67 (d, J ¼ 3.9 Hz, 2H), 7.66e7.62 (m, 1H), 7.61e7.58 (m,
2H), 7.56e7.50 (m, 2H), 7.37 (d, J ¼ 4.0 Hz, 1H), 7.23 (s, 1H),
7.03e6.95 (m, 2H), 6.86 (s, 1H), 5.76e5.71 (m, 1H), 4.58 (dd, J ¼ 14.1,
4.6 Hz, 1H), 4.39 (dd, J ¼ 14.1, 9.5 Hz, 1H), 3.78 (s, 3H). 13C NMR
d
194.77, 165.31 (dd, J ¼ 240.9, 11.1 Hz), 164.07 (dd, J ¼ 211.3,
13.1 Hz), 161.33, 148.02, 142.35, 133.43e133.21 (m), 131.61, 130.67,
129.74 (2C),129.70,128.89,127.99 (2C),127.92,122.07 (dd, J ¼ 158.5,
11.1 Hz), 112.53 (dd, J ¼ 45.1, 21.7 Hz), 105.79e105.27 (m), 58.86 (d,
J ¼ 3.5 Hz), 45.75. ESI-HRMS (m/z): [MþH]þ for C23H16ClF2N3O2S
(151 MHz, DMSO‑d6)
d 197.20, 161.17, 160.13, 136.44, 135.29, 134.15,
130.56, 129.42, 129.22, 128.92, 128.85, 127.72, 127.62, 123.61, 115.14,
115.10, 55.78, 55.75, 54.94, 46.50. ESI-HRMS (m/z): [MþH]þ for
calcd. 472.0730; found 472.0722 [M þ H] þ
.
C
24H21N3O3S calcd. 432.1404; found 432.1479 [M þ H] þ
.
4.9.5. 5-(4-chlorophenyl)-N-(1-(3-chlorophenyl)-3-(1H-imidazole-
1-yl)-1-oxopropan-2-yl)thiophene-2-carboxamide (17e)
4.9. General procedure for the synthesis of compounds 17a-h
Light white solid; yield: 50.60%; mp:140.9e142.7 ꢀC. 1H NMR
Compounds 17a-h were prepared using a method which was
similar to that of compounds 14a-j.
(600 MHz, DMSO‑d6)
d
9.23 (d, J ¼ 8.4 Hz,1H), 7.99 (t, J ¼ 1.9 Hz,1H),
7.94 (dd, J ¼ 7.9, 1.4 Hz, 1H), 7.71e7.67 (m, 4H), 7.62 (s, 1H),
7.58e7.52 (m, 2H), 7.48 (dd, J ¼ 8.8, 2.3 Hz, 2H), 7.20 (s, 1H), 6.84 (s,
1H), 5.73e5.66 (m, 1H), 4.57 (dd, J ¼ 14.2, 4.6 Hz, 1H), 4.40 (dd,
4.9.1. 5-(4-chlorophenyl)-N-(1-(2-fluorophenyl)-3-(1H-imidazole-
1-yl)-1-oxopropan-2-yl)thiophene-2-carboxamide (17a)
J ¼ 14.1, 9.5 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
d 195.83, 160.62,
Light white solid; yield: 51.90%; mp:112.1e114.1 ꢀC. 1H NMR
146.76, 137.91, 137.49, 136.78, 133.58, 133.26, 133.20, 131.75, 130.68,
130.15, 129.25, 129.21, 128.29, 127.99, 127.50, 127.42, 126.91, 125.05,
120.05, 54.80, 45.72. ESI-HRMS (m/z): [MþH]þ for C23H17Cl2N3O2S
(600 MHz, DMSO‑d6)
d
9.26 (d, J ¼ 7.6 Hz, 1H), 7.68 (d, J ¼ 9.3 Hz,
3H), 7.61 (d, J ¼ 16.8 Hz, 2H), 7.55e7.45 (m, 4H), 7.32 (q, J ¼ 6.2,
5.5 Hz, 2H), 7.16 (s, 1H), 6.86 (s, 1H), 5.38e5.33 (m, 1H), 4.65 (dd,
J ¼ 14.2, 4.3 Hz, 1H), 4.37 (dd, J ¼ 14.3, 9.3 Hz, 1H). 13C NMR
calcd. 470.0536; found 470.0532 [M þ H] þ
.
(151 MHz, DMSO‑d6)
d
195.51 (d, J ¼ 3.1 Hz), 161.15 (d, J ¼ 256.4 Hz),
4.9.6. N-(3-(1H-imidazole-1-yl)-1-oxo-1-(p-tolyl)propan-2-yl)-5-
(4-chlorophenyl)thiophene-2-carboxamide (17f)
160.86,146.63, 137.81, 137.54,135.13,135.01 (d, J ¼ 9.1 Hz),134.11 (d,
J ¼ 8.6 Hz), 133.16, 129.26, 129.21 (2C), 128.39, 127.50, 127.43 (2C),
125.03, 124.66 (d, J ¼ 3.1 Hz), 119.91, 116.61 (d, J ¼ 22.4 Hz), 58.62,
45.29. ESI-HRMS (m/z): [MþH]þ for C23H17ClFN3O2S calcd.
Light white solid; yield: 57.10%; mp:156.6e158.2 ꢀC. 1H NMR
(600 MHz, DMSO‑d6)
d
9.16 (d, J ¼ 8.6 Hz, 1H), 7.93 (d, J ¼ 8.3 Hz,
2H), 7.72 (d, J ¼ 4.0 Hz, 1H), 7.69 (d, J ¼ 8.6 Hz, 2H), 7.62 (s, 1H), 7.53
(d, J ¼ 3.9 Hz, 1H), 7.49 (d, J ¼ 8.6 Hz, 2H), 7.33 (d, J ¼ 8.0 Hz, 2H),
7.20 (s,1H), 6.83 (s,1H), 5.78e5.70 (m,1H), 4.55 (dd, J ¼ 14.2, 4.6 Hz,
1H), 4.36 (dd, J ¼ 14.2, 9.5 Hz, 1H), 2.35 (s, 3H). 13C NMR (151 MHz,
454.0826; found 454.0833 [M þ H] þ
.
4.9.2. 5-(4-chlorophenyl)-N-(1-(3-fluorophenyl)-3-(1H-imidazole-
1-yl)-1-oxopropan-2-yl)thiophene-2-carboxamide (17b)
DMSO‑d6)
d 196.15, 160.48, 146.61, 144.29, 137.86, 133.16, 132.27,
Light white solid; yield: 61.90%; mp:140.6e142.5 ꢀC. 1H NMR
131.81, 130.01, 129.34, 129.23 (2C), 128.53, 128.28, 127.50, 127.43
13

W. Yin, Y. Zhang, H. Cui et al.
European Journal of Medicinal Chemistry 225 (2021) 113740
(2C), 125.04, 120.03, 54.39, 45.96. ESI-HRMS (m/z): [MþH]þ for
4.12. In vitro biofilm formation assay
þ
C
24H20ClN3O2S calcd. 450.1034; found 450.1071 [M þ H]
.
Fungal suspension of C. albicans (1 ꢂ 106 CFU/mL) was prepared
in RPMI 1640 medium. Then the fungal suspension was moved to
96-well plates and incubated at 37 ꢀC. The non-adherent cells and
culture medium were removed after 3 h, 6 h 18 h and 24 h. Each
well of 96-well plates was washed with PBS (3 ꢂ 0.1 mL) and
different concentrations of compounds in fresh RPMI 1640 medium
were added to the 96-well plates. The plates were continuously
incubated at 37 ꢀC for 24 h. A semiquantitative determination of the
formed biofilms was calculated by XTT reduction assay [27].
4.9.7. N-(3-(1H-imidazole-1-yl)-1-(4-methoxyphenyl)-1-
oxopropan-2-yl)-5-(4-chlorophenyl)thiophene-2-carboxamide
(17g)
Light white solid; yield: 53.10%; mp:147.2e149.2 ꢀC. 1H NMR
(600 MHz, DMSO‑d6)
d
9.17 (d, J ¼ 8.6 Hz, 1H), 8.08e8.01 (m, 2H),
7.76 (d, J ¼ 4.0 Hz, 1H), 7.71e7.69 (m, 2H), 7.62 (s, 1H), 7.54 (d,
J ¼ 4.0 Hz, 1H), 7.50e7.48 (m, 2H), 7.21 (s, 1H), 7.07e7.04 (m, 2H),
6.82 (s, 1H), 5.76e5.71 (m, 1H), 4.53 (dd, J ¼ 14.2, 4.7 Hz, 1H), 4.37
(dd, J ¼ 14.1, 9.5 Hz, 1H), 3.83 (s, 3H). 13C NMR (151 MHz, DMSO‑d6)
d
195.32, 164.08, 160.94, 147.04, 138.39, 133.61, 132.30, 131.34,
4.13. Evaluation of MFC
130.54, 129.69 (2C), 128.70, 128.01, 127.88 (2C), 125.51, 120.51,
114.54, 56.06, 54.56, 46.52. ESI-HRMS (m/z): [MþH]þ for
The pH of RPMI 1640 medium (Gibco, USA) was adjusted to 7.0
by employing NaOH, NaHCO3 and morpholinepropanesulfonic acid
(MOPS, Genyiew, USA). C. albicans cells which was in growing stage
were gathered and inoculated in fresh RPMI 1640 medium to an
initial concentration of 1e5 ꢂ 103 CFU/mL. Fluconazole and test
compounds were dissolved in DMSO respectively as the stock so-
lution. The samples and fungal suspension were cultured at 35 ꢀC
for 48 h. Next, PDA plates were plated with fungal suspension
þ
C
24H20ClN3O3S calcd. 466.1027; found 466.1022 [M þ H]
.
4.9.8. N-(3-(1H-imidazole-1-yl)-1-oxo-1-(4-(trifluoromethoxy)
phenyl)propan-2-yl)-5-(4-chlorophenyl)thiophene-2-carboxamide
(17h)
Light white solid; yield: 62.30%; mp:128.6e130.2 ꢀC. 1H NMR
(600 MHz, DMSO‑d6)
d
9.22 (d, J ¼ 8.4 Hz, 1H), 8.14 (d, J ¼ 8.9 Hz,
2H), 7.72 (d, J ¼ 4.0 Hz, 1H), 7.69 (d, J ¼ 8.6 Hz, 2H), 7.62 (s, 1H),
7.55e7.51 (m, 3H), 7.49 (d, J ¼ 8.6 Hz, 2H), 7.20 (s, 1H), 6.84 (s, 1H),
5.76e5.71 (m, 1H), 4.57 (dd, J ¼ 14.2, 4.6 Hz, 1H), 4.39 (dd, J ¼ 14.2,
(100 mL) from the 96-well plates which were invisible growth. Then
the plates were incubated at 35 ꢀC for 24 h. MFCs corresponds to the
concentration of colonies less than 5 on the SDA plate.
9.5 Hz, 1H). 13C NMR (151 MHz, DMSO‑d6)
d 195.57, 160.63, 151.80,
146.75, 137.51, 133.72, 133.20, 131.78, 131.35, 130.89 (2C), 130.18,
129.28, 129.24 (2C), 127.52, 127.45 (2C), 125.07, 120.76 (2C), 120.62
(q, J ¼ 86.4 Hz), 54.68, 45.77. ESI-HRMS (m/z): [MþH]þ for
4.14. Time-kill assays
A fungal suspension of C. albicans (1 ꢂ 105 CFU/mL) were pre-
pared in RPMI 1640 medium. Taken fungal suspension and different
concentrations of 17f and itraconazole together to incubate at 35 ꢀC
for 24 h. The diluted fungal suspension was inoculated on PDA
plates and incubated at 35 C for 24 h, then the colony forming units
(CFU) were counted. The Log10 CFU/mL was plotted on a graph as a
time function. The detection limit of this assay was 100 CFU/mL.
The experiment was performed in triplicate [28].
C
24H17ClF3N3O3S: calcd. 520.0746; found 520.0754 [M þ H] þ
.
4.10. In vitro antifungal testing
Sevene sensitive pathogenic fungi and six fluconazole-resistant
strains of Candida albicans were selected to determine the in vitro
minimum inhibitory concentrations (MIC) according to the pro-
tocols of the National Committee for Clinical Laboratory Standards
(NCCLS). The RPMI 1640 test medium (Gibco, USA) was buffered
with morpholinepropanesulfonic acid (MOPS, Genyiew, USA). The
Fungi cells which was in growing stage were gathered and inocu-
lated in RPMI 1640 medium to an initial concentration of
1e5 ꢂ 103 CFU/mL. The MIC values were defined as the lowest
concentrations of an antimicrobial that would inhibit the visible
growth of the fungi. Fluconazole and itraconazole were used as
positive control drugs. All of the compounds were dissolved in
DMSO and serially diluted into the growth medium.
4.15. In vitro cytotoxicity assays
A549, MCF-7 and THLE-2 cells were plated in 96-well microtiter
plates at a concentration of 3000 cells per well and incubated for
24 h. Cells grew for 96 h, after treating with 100 mL different con-
centrations of tested compounds. Subsequently, MTT was added at
5 mg/mL in each well and the plates were incubated for 4 h. The
residual medium was removed and DMSO (100 mL) was added into
each well to solubilise the formazan crystals. Absorbance was taken
at 540 nm with a microplate reader. The IC50 value is defined as the
concentration of the inhibitor that caused 50% inhibition [25].
4.11. GC-MS analysis of sterol composition
Candida albicans (SC5314) was treated with compounds 17f
4.16. Cytochrome P450 enzyme inhibition assay
(0.015 mg/mL) and fluconazole (0.25 mg/mL) at sub-MIC values and
incubated in RPMI 1640 medium (50 mL) for ~18 h at 30 ꢀC with
continuous agitation (200 rpm). Cells were harvested by centrifu-
gation at 3000g for 10 min. Then the cells were washed with PBS
three times, and saponified at 80 ꢀC for 60 min with 3 mL of
ethanol, 2 mL of pyrogallol dissolved in ethanol, and 2 mL of KOH
(60%, w/v). The nonsaponifiable sterols were extracted three times
with 5 mL of heptane. The combined extracts were evaporated in a
Cytochrome P450 inhibition was tested in human liver micro-
somes using five probe substrates (CYP3A4, midazolam; CYP2C19,
S-mephenytoin; CYP2C9, diclofenac; CYP1A2, phenacetin; and
CYP2D6, dextromethorphan) in the presence of compound 17b and
17f. 20
wells of the 96-well plate, and 2
inhibitor were added. Then, 158
solution was added. The 96-well plate was prewarmed for
approximately 10 min at 37 ꢀC water bath. 20
L of NADPH cofactor
m
L of CYP substrate solution was added to the corresponding
L of the test compound and CYP
L of human liver microsomal
m
m
vacuum, and the residue was dissolved in 500
mL of heptane and
derivatized with 250 of N-methyl-N-(trimethylsilyl) tri-
mL
m
fluorooacetaminde (MSTFA) at 70 ꢀC for 20 min. The sterols were
analyzed by GC-MS. The GC-MS data were analyzed using Agilent
software (Agilent MSD productivity ChemStation for GC and GC/MS
systems data analysis application) and matched to known MS data
using the NIST Spectrum Database.
was added to all incubated wells. The samples were mixed and
further incubated for 10 min at 37 ꢀC. At this time point, the reac-
tion was terminated by adding 400
mL tolbutamide and labetalol in ACN). The sample solution was
centrifuged at 4000 rpm for 20 min, and 200 L of supernatant was
mL of cold stop solution (200 ng/
m
14

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Frozen human plasma was thawed in a water bath at 37 ꢀC prior
to experiments. Blank plasma (98
solution (100 M) to achieve 2
duplicate, and samples were incubated at 37 ꢀC in a water bath. At
each time point (0, 10, 30, 60 and 120 min), 400 L of stop solution
mL) was mixed with 2
mL of dosing
m
mM of the final concentration in
m
(200 ng/mL tolbutamide and 200 ng/mL labetalol in 50% ACN/
MeOH) was added to terminate the reactions. The sample plates
were centrifuged at 4000 rpm for approximately 10 min. The su-
pernatant (50
mL) was mixed with 100 mL ultrapure water. The
samples were shaken at 800 rpm for 10 min. Samples were
analyzed by LC/MS/MS. Disappearance of test compounds are
assessed base on the peak area ratio of analyte/IS (no standard
curve).
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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Acknowledgements
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The authors thank Prof. Yongbing Cao from School of Pharmacy,
the Second Military Medical University, for providing the
fluconazole-resistant strains of Candida albicans (strains
901,904,632,100,103 and 311). This work was supported by the
Program for Innovative Research Team of the Ministry of Education
and Program for Liaoning Innovative Research Team in University.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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