Lead optimization generates selenium-containing miconazole CYP51 inhibitors with improved pharmacological profile for the treatment of fungal infections

Article abstract of DOI:10.1016/j.ejmech.2021.113337

A series of selenium-containing miconazole derivatives were identified as potent antifungal drugs in our previous study. Representative compound A03 (MIC = 0.01 μg/mL against C.alb. 5314) proved efficacious in inhibiting the growth of fungal pathogens. However, further study showed lead compound A03 exhibited potential hemolysis, significant cytotoxic effect and unfavorable metabolic stability and was therefore modified to overcome these drawbacks. In this article, the further optimization of selenium-containing miconazole derivatives resulted in the discovery of similarly potent compound B17 (MIC = 0.02 μg/mL against C.alb. 5314), exhibiting a superior pharmacological profile with decreased rate of metabolism, cytotoxic effect and hemolysis. Furthermore, compound B17 showed fungicidal activity against Candida albicans and significant effects on the treatment of resistant Candida albicans infections. Meanwhile, compound B17 not only could reduce the ergosterol biosynthesis pathway by inhibiting CYP51, but also inhibited biofilm formation. More importantly, compound B17 also shows promising in vivo efficacy after intraperitoneal injection and the PK study of compound B17 was evaluated. In addition, molecular docking studies provide a model for the interaction between the compound B17 and the CYP51 protein. Overall, we believe that these selenium-containing miconazole compounds can be further developed for the potential treatment of fungal infections.

Full text of DOI:10.1016/j.ejmech.2021.113337

European Journal of Medicinal Chemistry 216 (2021) 113337
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Lead optimization generates selenium-containing miconazole CYP51
inhibitors with improved pharmacological profile for the treatment of
fungal infections
Hang Xu a, Zhong-zuo Yan a, Meng-bi Guo a, Ran An a, Xin Wang a, Rui Zhang a,
,
, *
Yan-hua Mou b, Zhuang Hou a **, Chun Guo a
a Key Laboratory of Structure-Based Drugs Design and Discovery (Ministry of Education), School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang 110016 China
b School of Life Sciences and Biopharmaceutics, Shenyang Pharmaceutical University, 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 selenium-containing miconazole derivatives were identified as potent antifungal drugs in our
Received 23 December 2020
Received in revised form
18 February 2021
Accepted 19 February 2021
Available online 2 March 2021
previous study. Representative compound A03 (MIC ¼ 0.01
mg/mL against C.alb. 5314) proved efficacious
in inhibiting the growth of fungal pathogens. However, further study showed lead compound A03
exhibited potential hemolysis, significant cytotoxic effect and unfavorable metabolic stability and was
therefore modified to overcome these drawbacks. In this article, the further optimization of selenium-
containing miconazole derivatives resulted in the discovery of similarly potent compound B17
(MIC ¼ 0.02
mg/mL against C.alb. 5314), exhibiting a superior pharmacological profile with decreased rate
Keywords:
of metabolism, cytotoxic effect and hemolysis. Furthermore, compound B17 showed fungicidal activity
against Candida albicans and significant effects on the treatment of resistant Candida albicans infections.
Meanwhile, compound B17 not only could reduce the ergosterol biosynthesis pathway by inhibiting
CYP51, but also inhibited biofilm formation. More importantly, compound B17 also shows promising
in vivo efficacy after intraperitoneal injection and the PK study of compound B17 was evaluated. In
addition, molecular docking studies provide a model for the interaction between the compound B17 and
the CYP51 protein. Overall, we believe that these selenium-containing miconazole compounds can be
further developed for the potential treatment of fungal infections.
Miconazole
Selenium
Antifungal
Superior pharmacological profile
CYP51
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
novel antifungal targets (such as AHAS) and modifying existing
drugs (azole drugs) are effective means to develop effective anti-
Due to high incidence and high mortality, invasive fungal in-
fections (IFIs) remain a major global public health problem [1e3].
The current situation of drug therapy for invasive fungal infections
has the huge challenges: low efficacy, drug interaction, significant
toxicity and drug resistance [4e6]. For example, fluconazole is used
widely for treating fungal infections, but the use of fluconazole can
be limited by hepatotoxicity and severe drug resistance, despite its
low cost and fungistatic efficacy [7,8]. In a word, it is imperative to
develop novel, effective and safe antifungal drugs [9]. Developing
fungal drugs [10,11].
CYP51 (sterol 14
a-demethylase) is a cytochrome P450 enzyme
which is a major target for the treatment of fungal infection and
protozoan infections [12e14]. CYP51 has been identified as a po-
tential drug target for the reduction of fungal ergosterol (ergosterol
plays a crucial role in the biosynthesis of fungal cell membranes)
which is encoded by the ERG11 gene, and its inhibitors (Fig. 1) have
demonstrated excellent antifungal activity both in vivo and in vitro
[15e17]. Although some novel CYP51 inhibitors have been devel-
oped, it is still a huge challenge to convert in vitro activity to
therapeutic efficacy due to unfavorable pharmacokinetic profiles
and pharmacological properties [18,19].
* Corresponding author.
** Corresponding author.
E-mail addresses: houzhuang8@sina.com (Z. Hou), chunguo@syphu.edu.cn
(C. Guo).
In a previous paper, the lead finding process of a new class of
selenium-containing miconazole CYP51 inhibitors was described
and these derivatives exhibited excellent inhibitory activity against
https://doi.org/10.1016/j.ejmech.2021.113337
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
most clinically pathogenic fungus [20]. Among all synthesized
compounds, compound A03 (Fig. 2) showed more excellent anti-
fungal activity than miconazole (Table 1). Meanwhile, our previous
results suggested that the molecular target of compound A03 is
CYP51. In particular, compound A03 could inhibit the growth of
biofilms and kill the fungus. In view of its excellent antifungal ac-
tivity and possible progression into a compound suitable for in vivo
evaluation, compound A03 was chosen for further pharmacological
studies.
Firstly, cytotoxicity is a prerequisite for drug safety, so the
cytotoxicity of compound A03 was priority assessed. Our pre-
liminary results showed compound A03 displayed significant
cytotoxicity (Table 2). The IC50 values of compound A03 for inhi-
bition of HL-60, MDA-MB-231 and PC-3 proliferation were 1.02,
9.06 and 13.10 mM, respectively. Secondly, the hemolytic activity is
an important issue that should to be considered in drug design.
Fig. 2. Chemical structures of lead compound A03.
Compound A03 also showed hemolytic activity at moderate doses
(4 mg/mL). Finally, the metabolic stability of compound A03 was
2. Results and discussion
evaluated with mouse liver microsomes prior to the in vivo studies.
However, compound A03 was found to be labile in these liver
fractions with 4.2 min half-life. Therefore, these suboptimal phar-
macological properties limited the further study of compound A03.
In this article, we expected to avoid potential problems further
down the road to a clinical candidate. Therefore, we thus proceeded
to a lead optimization procedure with the aim of discovering novel
derivatives of compound A03 with comparable inhibitory potency
but with an improved pharmacological profile. Therefore, 42 new
analogues were synthesized and their structure-activity relation-
ships were discussed. Much to our relief, among all the target
compounds, compound B17 displayed superior pharmacological
profile than the previous best compound A03.
2.1. Structural modification strategy of lead compound A03
In order to maintain the antifungal activity of lead compound
A03 and make some improvements in terms of cytotoxicity, he-
molysis, and metabolic stability in liver microsomes, less drastic
structural optimization will be performed in this study. Obviously,
the chemical construction of compound A03 fits structural char-
acteristics of classical CYP51 inhibitors. The key pharmacophores of
typical CYP51 inhibitors consist of three obvious sites for structural
modification: (i) the dichlorophenyl (or difluorophenyl) ring, (ii) a
side chain, (iii) the imidazole or 1,2,4-triazole ring. In our previous
study, we had synthesized compounds A17-A30 with difluor-
ophenyl in place of dichlorophenyl, with a premise that the fluorine
Fig. 1. Chemical structures of clinically used azole antifungal agents.
2

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
Table 1
The antifungal activity of the lead compound A03.
Compds
MIC/(
C.alb
m
g/mL)
Compds
MIC/(
m
g/mL)
Compds
MIC/(
C.alb
mg/mL)
Compds
MIC/(
C.alb
mg/mL)
Compds
C.alb
A03
Miconazole
0.02
0.25
0.01
0.03
1
1
0.25
0.5
0.13
0.5
0.5
0.5
0.13
0.25
2
2
Table 2
The physiological properties and pharmacokinetic profiles of the lead compound A03.
Compd.
Minimum hemolytic concentration (
m
g/mL)
IC50 m
M
T1/2 (min)
CL (mL/min/kg)
1311.5
HL-60
1.02
MDA-MB-231
9.06
PC-3
A03
4
13.10
4.2
atom(s) often enhances ADME properties. Indeed, metabolic sta-
bility has not been enhanced by this transformation. Therefore, we
shifted our attention to the modification of imidazole group and
side chain group. Based on the above analysis, derivatives B01eB28
and C01eC14 were designed and synthesized to observe whether
these structural modifications could produce potent antifungal
activity, combined with more safety and favorable metabolic sta-
bility (Fig. 3). Compounds B01eB28 was designed by shorting
benzyl to phenyl. In addition, the replacement of imidazole moiety
with 1,2,4-triazole was carried out to obtain compounds C01eC14.
Then further assay of in vitro pharmacological properties, cytotox-
icity, hemolysis, and metabolic stability of the target compounds
will be investigated.
magnesium bromide (2a-2n) were coupled under the action of
selenium powder to give 3a-3n. The disodium diselenide (6) were
obtained by the alkylation of selenium powder with sodium
borohydride under nitrogen condition. Next, we constructed the
dibenzyl selenide (8a-8p) via chlorobenzyl or substituted chlor-
obenzyl (7a-7p) with disodium diselenide in dichloromethane by
coupling reaction.
The general synthetic route of key intermediates 14a-14d is
shown in Scheme 3. Firstly, the intermediates 11a-11b were syn-
thesized by the acylation of 1,3-dichlorobenzene or 1,3-
difluorobenzene
with
2-chloroacetyl
chloride
under
FriedeleCrafts acylation conditions. Subsequently, intermediates
with an imidazole or 1,2,4-triazole group (12a-12d) were synthe-
sized via a nucleophilic substitution reaction of 11a-11b. Next,
reduction reactions of intermediates 12a-12d with sodium boro-
hydride gave 13a-13d under mild conditions. Finally, intermediates
13a-13d were treated with thionyl chloride to yield the desired
products 14a-14d.
As shown in Scheme 4, intermediates 3a-3n were dealt with
sodium borohydride and the nucleophilic substitution occurred
with 14a-14b to afford target compounds B01eB28. By a similar
method depicted in Scheme 4, intermediates 8a-8p were treated
with 14c-14d in the presence of sodium borohydride to afford
target compounds C01eC14.
2.2. Chemistry
The synthesis of reaction intermediates 3a-3n, 8a-8p and 14a-
14d is showed in Scheme 1, Scheme 2, and Scheme 3, respectively.
The synthetic route of the contain-selenium miconazole analogues
(B01eB28 and C01eC14) is outlined in Scheme 4.
A series of analogues containing diselenide group (3a-3n and
8a-8p) with various substitutions were first synthesized. The syn-
thesis of intermediates diaryl selenide (3a-3n) commenced with
the commercially available magnesium powder and a series of
different bromobenzene by grignard reaction [21,22]. Phenyl
Fig. 3. General strategy for lead optimization.
3

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
Scheme 1. Synthesis of intermediates 3a-3n
Reagents and conditions: (a) Mg, Et2O, 30 ꢀC, 30 min; (b) Se, NH4Cl, 30 ꢀC, 30 min.
Scheme 2. Synthetic routes of intermediates 8a-8p
Reagents and conditions: (a) EtOH, 30 min; (b) CH2Cl2, 40 ꢀC, 10 h.
Scheme 3. Synthetic routes of intermediates 14a-14d
Reagents and conditions: (a) AlCl3, 30 ꢀC, 3 h; (b) imidazole or 1,2,4-triazole, MeOH, 65 ꢀC, 6 h; (c) NaBH4, EtOH, 20 ꢀC, 12 h; (d) SOCl2, 20 ꢀC, 12 h.
2.3. In vitro antifungal activity of synthesized compounds
the following points should be taken into account:
The minimal inhibitory concentrations (MICs) of the target
compounds were determined against Candida albicans, Candida
zeylanoides, Cryptococcus neoformans, Candida kruseii, Candida
glabrata, Candida parapsilosis and Aspergillus fumigatususing micro-
broth dilution method in 96-well plates according to the CLSI M27-
A3 guidelines by using miconazole and fluconazole as the control
drug. The antifungal activities are indicated as the MICs and ob-
tained in vitro antifungal activity of the target compounds are
summarized in Table 3.
1) Compared with fluconazole (MIC: 4
mg/mL), series B compounds
(MIC: 0.03e0.5 g/mL) had an 8e32 folds improvement in
m
antifungal activity in Candida albicans (CPCC 400616). Most of
the compounds B01eB28 showed inhibitory efficiency against
Candida albicans (CPCC 400616) in comparison with the refer-
ence miconazole (MIC: 0.25 mg/mL). Among them, compound
B25 exhibited the best activity against Candida albicans, which
was 8 folds more potent than the reference miconazole (MIC:
0.03
2) Series
Candida albicans (ATCC SC5314) with MIC ranging from 0.02 to
0.13 g/mL which was much more active than fluconazole (MIC:
g/mL). Among them, compounds B15, B17, B18, B21 and B25
(MIC: 0.02 g/mL) demonstrated slightly potent in vitro anti-
fungal activity than miconazole (MIC: 0.03 g/mL).
m
g/mL).
The results showed that almost all series
B
compounds
B
compounds showed promising activities against
possessed broad-spectrum antifungal activity. However, de-
rivatives C01eC14 exhibited moderate to best antifungal activities
against most strains. SAR revealed that the replacement of imid-
azole with 1,2,4-triazole resulted in substantial decrease of the
antifungal activity. As for the antifungal inhibitory data in Table 3,
m
1
m
m
m
4

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
Scheme 4. Synthetic routes of target compoundsReagents
and conditions: 65 ꢀC, 1 h.
3) The in vitro antifungal activities of series B compounds (MIC:
2.4. Determination of MIC against fluconazole-resistant strains of
0.03e1
g/mL) against Cryptococcus neoformans (CGMCC 2.3161),
which was comparable to the reference miconazole (MIC:
0.5 g/mL). Among them, compounds B03 and B28 possessed
excellent potency against C.neo. (MIC: 0.03 g/mL).
mg/mL) were far more potent than fluconazole (MIC:
Candida albicans
4
m
Due to the widespread use of antifungal drugs, the resistance is
becoming widespread in clinic [23]. The clinical occurrence of drug-
resistant strains lead to the significant impediment to the treat-
ment of invasive fungal infection. As a consequence, the evaluation
of antifungal activities of potent compounds against fluconazole-
resistant strains is a validated strategy to combat with drug resis-
tance. In this study, all target compounds were assessed for their
in vitro antifungal activities against fluconazole-resistant clinical
isolates (C. albicans, strains numbers 17#, CaR, 632, 901 and 904).
The results are summarized in Table 4.
m
m
4) The results of MICs against other Candida spp. including Candida
zeylanoides, Candida kruseii, Candida glabrata and Candida par-
apsilosis indicated that most series B compounds exhibited
considerable inhibitory activity with MIC in the range of
0.13e4
respectively.
5) Against Aspergillus fumigatus (CGMCC 3.7795), consistent with
series B (MIC ¼ 2e16 g/mL), series C (MIC ¼ 4e16 g/mL) with
mg/mL, 0.02e1 mg/mL, 0.06e1 mg/mL, 0.13e1 mg/mL,
m
m
All series B compounds displayed potent inhibitory activity on
fluconazole-resistant fungi, whereas fluconazole was completely
inactive. Among all the target compounds, compounds B02, B10,
B17 and B27 revealed most potent effects against fluconazole
imidazole substituents by 1, 2, 4-triazole were the same potent.
6) The imidazole group was essential for the antifungal spectrum,
the change of X and R did not have amajor impact on the
inhibitory activity. For series B compounds, the activity of C. gla.
was slightly increased when the X was Cl. And the activity of
C. zey. was slightly increased when the R was multiple methyl
substitution. R was the electron-donating substituent, the ac-
tivity of series B compounds against A. f. was slightly decreased,
whereas the activity against C. gla. was slightly increased.
resistant C. albicans (MIC ¼ 0.5e2, 0.13e0.5, 0.06e1 and 0.5e2
mg/
mL, respectively). However, most of the analogues C01eC14
showed significantly reduced inhibitory activity against
fluconazole-resistant strains. These data indicated that the mod-
ification of side chain was helpful for promoting the inhibitory ef-
fect on fluconazole-resistant fungi while imidazole replacement
with 1,2,4-triazole lead to the loss of the antifungal activity. Based
on the results of in vitro antifungal activity against different strains
of fungi, compounds B02, B10, B17 and B27 were chosen to carry
out the further study.
7) For series C compounds, the activities of C.alb., C. gla. and A.f.
were slightly increased when the X was F, meanwhile the ac-
tivity of C. zey. was significantly increased. The antifungal ac-
tivity and antifungal spectrum were better when R was 2-Cl. The
antifungal activity decreased obviously when
R was the
electron-withdrawing group, however, the increase of electron-
donating group did not enhance the antifungal activity.
2.5. In vitro cytotoxicity assay
8) As compared with A03, the shorten of the benzyl group caused a
slight increase of the inhibitory activity against C.zey., C. neo.,
C. kru. and C. par.. Series B compounds had a smaller concen-
According to our previous study, lead compound A03 exhibited
significant cytotoxic effect and therefore further pharmaceutical
research was limited. This prompted us to investigate whether
compounds B02, B10, B17 and B27 are cytotoxic on these
mammalian cell lines. The effects compounds B02, B10, B17 and
B27 on the in vitro cytotoxicity of human leukemia cell line (HL-60),
breast cancer cell line (MDA-MB-231), prostate cancer cell line
tration range against C.zey., C. neo., C. kru. and C. par.. (0.13e4
mL, 0.03e1 g/mL, 0.02e1 g/mL, 0.06e1 g/mL, 0.13e1 g/mL,
respectively).
mg/
m
m
m
m
5

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
Table 3
The antifungal activity of the target compounds B01eB28 and C01eC14
C.alb
C.alb(sc5314)
C.zey
C.neo
C.kru
C.gla
C.par
A.f
B01
B02
B03
B04
B05
B06
B07
B08
B09
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
B27
B28
C01
C02
C03
C04
C05
C06
C07
C08
C09
C10
C11
C12
C13
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
F
F
F
F
H
3-F
4-F
0.25
0.25
0.25
0.13
0.25
0.25
0.06
0.25
0.13
0.25
0.13
0.5
0.13
0.06
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.06
0.06
0.03
0.03
0.02
0.03
0.02
0.02
0.03
0.03
0.02
0.13
0.03
0.06
0.02
0.03
0.03
0.13
0.25
0.13
0.13
2
0.25
0.25
0.25
0.5
0.25
0.25
0.25
0.5
0.5
2
0.13
0.13
0.03
0.5
0.25
0.13
0.5
0.25
0.25
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.25
0.13
1
0.5
0.5
0.25
0.25
0.5
1
0.03
64
2
2
16
8
16
32
4
4
2
4
0.5
32
4
0.25
4
0.13
0.13
0.02
0.13
0.06
0.5
0.13
0.06
0.02
0.5
1
1
0.5
0.25
1
0.25
0.5
0.5
0.25
0.5
0.13
0.25
0.13
0.13
1
0.25
0.25
0.25
0.5
0.25
0.5
0.13
0.5
0.25
0.5
8
4
8
16
4
8
8
8
16
16
8
8
8
4
2
8
4
8
8
8
8
4
8
8
8
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
2,5-di-CH3
2,6-di-CH3
3,5-di-CH3
2-CH2CH3
4-CH2CH3
H
4
2
0.5
0.5
0.5
0.5
0.13
1
0.5
0.5
0.5
0.25
0.5
0.25
0.5
0.5
0.5
0.13
1
0.25
0.5
0.13
0.5
0.25
0.5
128
64
16
128
64
32
128
64
8
1
4
4
0.25
0.5
0.5
0.06
0.25
0.13
0.5
0.02
0.06
0.13
0.5
0.06
0.25
0.03
0.13
0.25
0.25
0.13
0.25
0.25
0.13
0.06
0.13
0.03
0.13
64
32
8
64
64
32
64
>128
16
0.5
3-F
4-F
0.5
1
1
1
1
0.25
0.13
0.25
0.5
1
0.5
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
2,5-di-CH3
2,6-di-CH3
3,5-di-CH3
2-CH2CH3
4-CH2CH3
2,4-di-Cl
H
2-Cl
4-Cl
4-F
4-OCH3
4-CH3
2,4-di-Cl
H
2-Cl
4-Cl
0.5
0.25
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
4
4
2
4
4
2
2
2
2
0.13
0.5
1
F
F
F
F
0.25
0.03
0.13
0.06
0.13
0.25
0.13
0.03
16
2
16
16
0.13
0.5
0.03
0.13
1
1
2
0.02
4
0.25
0.13
0.02
0.13
0.06
0.13
128
128
0.06
128
128
64
64
128
4
2
128
8
8
16
8
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
16
16
16
16
16
4
16
16
8
8
8
8
4
0.13
1
2
0.06
0.06
0.13
0.13
0.25
2
0.5
2
2
64
32
>128
128
0.13
4
4-F
4-OCH3
4-CH3
F
F
32
32
1
4
32
C14
2
0.01
1
128
0.13
32
2
8
2
A03
Fluconazole
Miconazole
0.5
32
0.5
>128
2
0.03
1
0.5
0.5
0.25
6

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
Table 4
Table 5
IC50 of tested samples in cancer cell lines.
The antifungal activity of the target compounds B01eB28 and C01eC14.
Compd.
MIC/(
mg/mL)
Compd.
IC50 mM
strain CaR
strain 17#
strain 632
strain 901
strain 904
HL-60
MDA-MB-231
PC-3
MCF-10A
B01
B02
B03
B04
B05
B06
B07
B08
B09
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
B27
B28
C01
C02
C03
C04
C05
C06
C07
C08
C09
C10
C11
C12
C13
C14
A03
FCZ
1
2
8
4
1
32
2
8
8
0.25
4
8
2
8
1
4
1
8
4
2
8
2
2
8
8
2
1
1
2
2
1
4
2
2
2
0.13
4
16
4
8
4
4
0.06
4
8
16
8
4
16
4
0.25
4
0.5
4
>128
64
8
>128
>128
32
>128
64
32
4
16
32
64
64
0.13
128
1
0.5
1
0.5
1
4
2
16
2
0.5
8
16
16
4
2
1
0.5
4
0.5
1
16
1
16
2
1
8
1
1
>128
>128
>128
>128
>128
>128
>128
>128
>128
8
>128
>128
>128
>128
1
2
1
1
0.5
1
2
1
1
0.5
1
16
8
4
8
0.25
8
16
1
15
1
2
0.06
1
1
4
8
8
8
8
2
8
0.5
8
>128
>128
32
>128
>128
128
>128
>128
16
2
B02
B10
B17
B27
8.26
6.95
24.81
16.95
1.02
0.80
e
13.43
6.61
39.53
26.24
9.06
16.96
<0.001
0.53
20.41
7.25
28.55
27,20
59.37
28.11
30.16
e
54.39
30.25
13.10
13.41
<0.025
0.64
A03
8
1
Miconazole
Taxol
Dox
e
16
8
0.25
16
4
4
4
2
2
0.5
2
4
8
4
4
2
4
2
<0.125
2.64
Values are the average of three independent experiments. Relative errors are
generally within 5e10%.
whereas compounds B17 was inactive (MHC/MIC ¼ 64). After
exposure, compounds B27 displayed 93.59% hemolysis in the
sample treated with 4
pound B02 lysed 87.66% of red blood cells at concentrations of 4
mL and compound B10 exhibited 0.89% hemolysis at concentra-
tions of 2 g/mL. The MIC values of target compounds were much
lower than their hemolytic concentrations and compound B17
exhibited the lowest hemolytic activity. In addition, compound B10
showed no significant improvement in cytotoxicity and hemolysis,
further study of compound B10 will not be considered.
m
g/mL (MHC/MIC ¼ 64). Moreover, com-
mg/
m
4
2
2
0.5
1
2.7. Metabolic stabilities assay
>128
>128
>128
>128
>128
128
>128
>128
128
32
128
128
128
>128
0.06
>128
>128
>128
>128
>128
>128
128
>128
>128
16
4
The microsomal metabolic stabilities of three most potential
compounds were tested before investigating in vivo antifungal ac-
tivities (Table 7). Miconazole and lead compound A03 were fast
metabolic (T1/2 ¼ 8.3 min and 4.2 min, respectively) in the mouse
liver microsomal stability test (Table S2). In contrast, compound
B17 (T1/2 ¼ 19.4 min) had a lower clearance rate than miconazole
and compound A03, suggested that it might have good metabolic
stability.
4
8
>128
>128
>128
0.5
>128
64
>128
>128
0.25
>128
2.8. Minimum fungicidal concentration of compounds B02, B17
and B27 and time-kill curves of B17
>128
Considering the excellent antifungal inhibitory activities, com-
pounds B02, B17 and B27 were selected for further evaluations and
compounds B17 was also our focus. As is well known, fluconazole is
a fungistatic clinical agent rather than fungicidal agent and it is
therefore often used as maintenance therapy. The development of
new fungicidal compounds to treat IFIs remains a major chal-
lenging. In our study, the discovery of effective fungicidal agents
with high therapeutic index is of great importance. The minimum
fungicidal activity (MFC), i.e., the lowest concentration that
resulting in at least 99% killing of the inoculum, is used to charac-
terize in vitro fungicidal activity. Kill of C. albicans (CPCC400616)
(PC3) and human normal mammary epithelial cells (MCF-10A)
were investigated. Doxorubicin (DOX) and taxol were used as
controls. All data were summarized in Table 5.
Remarkably, compounds B02, B17 and B27 significantly
decreased the cytotoxicity of HL-60, MDA-MB-231 and PC-3
compared to lead compound A03 and the inhibitory effect of
compound B17 on MCF-10A was one half of that of compound A03.
Among them, compound B17 exhibited relatively weak cytotoxicity
against test cell lines with the IC50 values ranging from 24.8 to
59.4
mM (9.48e22.69 mg/mL), which were about 38e90 times
higher than the MIC value against C.albicans (Table S1).
Table 6
The hemolysis ratio of the target compounds.
2.6. Hemolysis assays
Compd.
Hemolysis ratio
1
m
g/mL
2
m
g/mL
4
m
g/mL
8
m
g/mL
16 mg/mL
Having several potent compounds in hand, we asked whether
any of these compounds show ameliorated hemolysis effects in
comparison to previous lead compound A03. In this study, rabbit
red blood cells were used to for hemolysis assays of tested com-
pounds B02, B10, B17 and B27 (Table 6).
B02
B10
B17
B27
A03
Miconazole
0
0
0
0
0.00
0.16
0
0.89
0
0
3.15
8.55
87.66
84.36
0
93,59
58.87
81.78
94,60
100
0.33
100
93.23
87.34
100
100
100
100
98.23
94.03
All test compounds had different hemolysis effects at high
concentrations (2e16
mg/mL). Miconazole displayed 87.34% he-
Values are the average of three independent experiments. Relative errors are
molysis at 8 g/mL (minimum hemolytic concentration, MHC),
m
generally within 5e10%.
7

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
Table 7
Metabolic stabilities of compounds B02, B17 and B27 in mouse liver microsomes.
Compd.
T1/2 (min)
CL (mL/min/kg)
B02
B17
B27
A03
Miconazole
Propafenone
10.3
19.4
14.9
4.2
8.3
2.3
530.7
282.4
368.2
1311.5
660.1
2433.0
Considering in vitro antifungal activity, cytotoxicity, hemolytic and metabolic sta-
bility, B17 was selected as the focus for further antifungal activity studies.
Fig. 4. Time-kill curve of compound B17 against Candida albicans (CPCC400616). The
detection limit of this assay was 100 CFU/mL.
treated with compounds B02, B17 and B27 was investigated by
determinating the minimum fungicidal concentration (MFC). Spe-
cifically, we evaluated MFC of potent compounds B02, B17 and B27
in 48 h (Table 8).
2.10. Inhibition of the biofilm formation of FCZ-Resistant C. albicans
Biofilm is an important virulence factor contributing to recur-
rent fungal infection and high-level drug resistance in the clinic.
Fungal cells are surrounded by biofilm, a self-produced extracel-
lular polymeric substance, therefore antifungal drugs are difficult to
enter the fungal cells [25,26]. So that biofilm could protect fungal
cells against attack by antifungal agents. Therefore, it’s a significant
therapeutic meaning to develop novel derivatives with anti-biofilm
activity. In this assay, the effect of compounds B02, B17 and B27 on
biofilm formation of C. albicans (CPCC400616) was evaluated by
using miconazole as control. The SMIC50 and SMIC80 values of
compounds B02, B17 and B27 were shown in Table 10.
Meanwhile, time-Kill curves were assayed to further evaluate
the fungicidal activity of compound B17 during a period of 24 h and
miconazole (2
concentrations of compound B17 (0.25
and 2 g/mL) were used in the time-Kill curve assay (Fig. 4).
m
g/mL) was used as control drug. The gradient
m
g/mL, 0.5 g/mL, 1 g/mL
m
m
m
As shown in Table 10, compounds B17 and B27 showed satis-
factory fungicidal activity. As shown in Fig. 4, compound B17 dis-
played rapid fungicidal activity against C. albicans (CPCC 400616),
which could completely kill fungi cells at 2 mg/mL within 8 h in the
viable colony counts (the CFU was reduced from 5 to 2 Log10 CFU/
mL). Here it can be observed that compound B17 could dose-
dependently kill the C. albicans cells. And the fungicidal activity
was much more effective than miconazole. Compound B17
demonstrated potent fungicidal activity in this assay, therefore, it
may have advantages in treating IFIs and drug resistance.
. 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 (SMIC of FCZ
>1024
mg/mL). The prepared biofilm was preincubated with
different concentrations of the tested compounds for 24 h at 37 ꢀC.
Next, the effect of tested compounds on biofilm formation were
investigated by the XTT reduction assay [27] (Fig. 5).
2.9. GC-MS analysis of sterol composition in Candida albicans
As showed in Fig. 5 and Table 12, treatment of 3 h-biofilms with
32
m
g/mL miconazole inhibited 50% of 3 h-biofilms formation. At
Clarifying the mechanism of action of compounds is an impor-
tant step in drug discovery and development. In our previous study,
the mechanism of lead compound A03 had been clarified. Com-
pound A03 mediated ergosterol biosynthetic pathway by inhibiting
the lower concentration (16
mg/mL), B17 inhibited 80% of 3 h-bio-
films formation, indicating that B17 is a more potent antifungal
inhibitor. Notably, target compounds B02, B17 and B27 were shown
to inhibit the formation of C. alb. biofilm in a dosage depended
manner.
sterol 14a-demethylase enzyme. In this study, we thus stared to
determine whether compound B17 possessed the same mechanism
with lead compound A03. Sterol composition of fungal cells was
detected using gas chromatography-mass spectrometry (GC-MS).
Fluconazole and miconazole were employed as the controls [24].
C. albicans (ATCC SC5314) was used to be a model strain and pre-
incubated together with test compounds for 16 h to investigate the
biosynthesis of ergosterol. As shown in Table 9, the content of the
2.11. In vivo antifungal effects
Considering the good antifungal activity, better metabolic sta-
bility, lower hemolytic and cytotoxicity of compound B17, the
in vivo efficacy was evaluated in infectious murine models by
C. albicans ATCC SC5314. Compound A03 (0.6 mg/kg/day) and flu-
conazole (0.6 mg/kg/day) were used as controls. Two different drug
regimens of compound B17 were investigated at the dosage of 0.4
and 0.6 mg/kg/day. Compound B17 showed potent activity in a
murine model of C. albicans. The results of tissue burdens obtained
after 5 days of experiments in kidneys with B17 was showed in
Fig. 6. Experiments use CFU counts of the kidneys of mice infected
intravenously with C. albicans. The results showed that compound
B17 significantly reduced the kidney fungal burden in a murine
lanosterol 14
eburicol) was increasing. It’s worth noting that eburicol displayed
the largest amount of accumulation in all lanosterol 14 -deme-
a-demethylase substrates (lanosterol, obtusifoliol and
a
thylase substrates. Similar to miconazole, B17 produced the accu-
mulation of methylated sterols, and decreased the content of
ergosterol whether at high or low doses. Together, these results
suggested that compound B17 inhibited lanosterol 14a-demethy-
lase (CYP51) in a dose-dependent manner with the same targets
and mechanism as lead compound A03.
Table 8
The MFC of the most potent compounds.
Compd.
MIC against C.alb. (
m
g/mL)
MFC against C.alb. (
m
g/mL)
MFC/MIC
B02
B17
B27
Miconazole
0.25
0.25
0.06
0.25
e
e
4
4
8
1
0.25
2
8

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
Table 9
Analysis of sterol composition in Candida albicans (ATCC SC5314) by GC-MS.
Compd.
concentration (
m
g/mL)
% of total sterols Candida albicans (ATCC SC5314)
Ergosterol
Obtusifoliol
Lanosterol
Eburicol
Fluconazole
0.03125
0.125
0.5
2
86.34
80.8
78.9
19.8
4.2
12.2
8.3
7.1
5.2
4.1
8.7
5.8
1.0
1.6
2.9
9.5
11.4
7.2
17.4
9.0
6.1
7.8
17.0
11.1
9.9
10.8
10.2
0
9.8
2.9
5.3
6.7
12.4
11.6
7.2
14.1
10.0
9.5
8.9
13.6
7.4
4.1
9.9
10.1
9.0
5.7
63.5
70.3
70.6
64.8
75.1
75.2
80.7
70.2
73.2
75.1
78.0
82.0
0
8
Miconazole
B17
0.03125
0.125
0.5
2
8
0.03125
0.125
0.5
2
8
e
4.9
2.2
2.1
98.3
Control
Table 10
1.7
Fig. 7 and the pharmacokinetic parameters of compound B17 are
given in Table 11.
SMIC values of tcompounds against FCZ-Resistant C. albicans CPCC400616 biofilms.
After intravenous administration of 2 mg/kg area under blood
concentration curve (AUC0-t) and the final half-life (T1/2) of com-
Compd.
SMIC50
1.5 h
(
m
g/mL)
SMIC80
1.5 h
(mg/mL)
3 h
6 h
24 h
3 h
6 h
24 h
pound B17 in rats were observed 2249
112 h ng/mL and
B02
B17
B27
Miconazole
4
4
8
16
8
8
16
32
128
64
>128
>128
>128
>128
>128
>128
16
8
128
128
128
16
>128
128
>128
128
>128
>128
>128
>128
>128
>128
1.89 0.01 h, respectively. After intragastric administration of
5 mg/kg area under blood concentration curve (AUC0-t), the final
half-life (T1/2) and bioavailability (%) of compound B17 in rats were
observed 1256 135 h ng/mL, 1.93 0.04 h and 22.3%, respectively.
model of C. albicans.
The kidney fungal burden could be visually reduced by intra-
peritoneal injection of compound B17 (0.4 mg/kg and 0.6 mg/kg
once daily). The results highlighted compound B17 has better
antifungal activity in vivo than fluconazole.
2.13. Molecular docking analysis
To validate the design rationale and determine the binding
modes, compound B17 and miconazole were selected to be docked
into the active site of C.albicans CYP51 (PDB ID: 5tz1) from Protein
Data Bank (PDB). The results revealed that compound B17 shared
the similar binding modes with miconazole. The docking results are
depicted in Fig. 8. The imidazole group of target compounds B17
was coupled to the Fe2þ atom of the heme group of C.albicans
CYP51. CYS470, GLY307, LEU376, SER378, MET508, HEM601 and
THR311 were the main function residues. The side chain extended
into the CYP51 channel to form favorable hydrophobic interactions
and van der Waals with the surrounding residues such as THR311,
LEU376, SER378 and MET508.
2.12. Pharmacokinetic study
Since compound B17 has good antifungal activity in vivo, we
selected compound B17 to evaluate its pharmacokinetic properties
for further study. In order to study the pharmacokinetic charac-
teristics of compound B17 in SD rats, plasma concentration of
compound B17 was measured after single intravenous adminis-
tration (2 mg/kg) and intragastric administration (5 mg/kg). The
plasma concentrations of compound B17 over time are presented in
Fig. 5. Antibiofilm effect of compounds B02, B17, B27, and miconazole against FCZ-Resistant C. albicans CPCC400616 biofilms for 1.5 h (A), 3 h (B), 6 h (C) and 24 h (D).
9

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
obvious improved metabolic stability. The time-kill curve assay
revealed compound B17 possess fungicidal activity and coluld
completely killed C. albicans cells at 2 mg/mL in a concentration-
dependent manner. In vivo efficacy was also demonstrated in a
murine model of systemic C. albicans infection, which compound
B17 possess a significant fungal load reduction in kidneys. Gener-
ally, compound B17 exhibited potent in vitro and in vivo antifungal
activity in treating C. albicans. Herein, the potential applications
and mechanism of action of compound B17 for the treatment of IFIs
were investigated. Like fluconazole and miconazole, compound B17
produced the accumulation of methylated sterols and decrease
ergosterol biosynthetic, which are symbol of the mechanism of
inhibition of CYP51 by antifungal azoles. Moreover, compound B17
showed excellent activity in inhibiting C. albicans biofilm formation
(>80% inhibition at 16 mg/mL), highlighting its potential to treat
biofilm-associated infections. In addition, docking analysis revealed
the potential binding modes between the target compound B17
and C.alb. CYP51. Considering the excellent antifungal activity,
better metabolic stability, lower hemolytic and cytotoxicity, our
results strongly suggest that compound B17 are promising lead for
the development of the antifungal azole analogues. The study of
pharmacokinetics of compound B17 also provides reference and
foundation for our further work and subsequent structural modi-
fication. Our findings will facilitate further drug discovery efforts
toward the identification of a preclinical candidate to treat fungal
infections.
Fig. 6. Therapeutic efficacies of compound A03 (0.6 mg/kg) B17 (0.4 mg/kg and
0.6 mg/kg) and fluconazole in the C.alb. ATCC SC5314 model. (**P < 0.01, ***P < 0.001).
4. Experimental
4.1. Chemistry
All starting materials and analytically pure solvents, unless
otherwise specified, were received from the vendors. Melting
points were recorded on a X-5 melting point apparatus and are
uncorrected. In all chemical reactions, TLC (thin-layer chromatog-
raphy) monitor was performed by using silica gel precoated GF254
plates and then visualized with a UV lamp. All column chroma-
tography separations were performed on silica gel. The 1H NMR and
13C NMR spectra date were collected on a Bruker AV-400 spec-
trometer machine and the solvent was chosen DMSO‑d6 with TMS.
1H NMR spectra were run at 400 MHz and 13C spectra were run at
100 MHz. The Low-resolution electrospray ionization mass spec-
trometry was measured by Agilent 6120 (quadrupole liquid chro-
matography) equipped Agilent 1200 LC/MS. High-resolution mass
spectra experiments were measured on the Agilent Accurate-Mass
Q-TOF 6530 instrument. GC-MS experiments were performed with
the Agilent 6890N-5975 (Agilent, Santa Clara, CA, USA).
Fig. 7. Plasma concentration-time curves of compound B17 intravenous administra-
tion and oral administration.
3. Conclusion
The selenium-containing miconazole A03 we previously re-
ported were identified and validated as the promising antifungal
lead compound. The potent hemolysis, cytotoxicity and rapid
metabolism, however, limit the widespread use as antifungal
agents. To improve this disadvantage, we have synthesized a series
of selenium-containing miconazole derivatives (B01eB28 and
C01eC14) in attempts to maintain their antifungal activity while
reducing their hemolysis rate, cytotoxicity and enhancing meta-
bolic stability. Due to the poor inhibitory activity against some
clinically pathogenic fungi and fluconazole-resistant strains, ana-
logues C01eC14 don’t meet the requirement for further stage
studies on the activity and safety of these compounds. Series B
compounds not only showed excellent inhibitory activities against
various fungal pathogens, but also improved safety of hemolysis
and cytotoxicity. Among them, compound B17 and B27 showed
4.2. The preparation of compounds 3a-3n
The synthesis of intermediate 1, 2-diphenyldiselane (3a) was
chosen as the example: the solution of phenylmagnesium bromide
was prepared from bromobenzene (5.8 mL), magnesium (1.2 g,
0.05 mol), and anhydrous diethyl ether (30 mL). The selenium
power (3.5 g, 0.044 mol) was added in portions at a rate sufficient to
maintain a vigorous reflux. After the mixture was stirred and
heated at reflux for 30 min, saturated solution of ammonium
Table 11
Pharmacokinetic parameters after administration of rats.
route
dose (mg/kg)
AUC0-t (h$ng/mL)
T1/2 (h)
bioavailability (%)
intravenous
per os
2
5
2249 112
1256 135
1.89 0.01
1.93 0.04
e
22.3
10

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
Fig. 8. Miconazole (A, B) and compound B17 (C, D) were docked into the active site of Candida albicans CYP51.
chloride was then added (20 mL). The mixture was filtered and
extracted with ether (3 ꢁ 20 mL). After evaporating the combined
extracts to dryness, the residue was dissolved in hot hexane (10 mL)
and a small amount of insoluble substance was separated by
thermal filtration. Solids precipitate out of solution at 4 ꢀC. The
yellow solid was collected and dried in the air (2.8 g, 43%). m.p.
4.5. The preparation of compounds 12a-12d
The synthesis of intermediate 1-(2,4-difluorophenyl)-2-(1H-
imidazole-1-yl) ethan-1-one (12b) was chosen as the example: The
2-chloroacetyl chloride (10) (6.7 g, 0.06 mol) was added dropwise
to the mixture of 1,3-difluorobenzene (5.7 g, 0.05 mol) and anhy-
drous aluminum trichloride (10.7 g, 0.08 mol) at 25 ꢀC. The reaction
was stirred at room temperature for 4 h. Subsequently, the mixture
was diluted with ice water mixture (100 g) containing concentrated
hydrochloric acid (10 mL). The solution was extracted with
dichloromethane (3 ꢁ 20 mL). The organic layers were combined
and washed with saturated salt water (30 mL). The organic layer
was dried with Na2SO4, then filtered and concentrated under
reduced pressure. The residue was purified by recrystallization
with ethanol to give 11b (93.1%) as a yellow solid. To a stirred so-
lution of intermediate 11b (14.5 g, 0.08 mol) in 70 mL anhydrous
methanol with triethylamine (8.1 g, 0.08 mol), imidazole (10 g,
0.15 mol) was then added at 25 ꢀC. The mixture was magnetically
stirred heated to reflux for 4 h and then concentrated under
reduced pressure to remove methanol. The residue was purified by
recrystallization from ethanol to obtain 1-(2,4-difluorophenyl)-2-
(1H-imidazole-1-yl) ethan-1-one (12b, 90.6%) as a white solid.
HRMS m/z: found 223.0675 [M þ H]þ(calcd. for C11H8N2F2O,
223.0683).
58e61 ꢀC. LC- MS(m/z): 313.9 [M þ H] þ
.
4.3. The preparation of compound 6
Selenium power (7.9 g, 0.1 mol) and NaBH4 (8 g, 0.2 mol) were
added to a solution of ethanol (180 mL) under N2 (g). The mixture
was allowed to stir at 25 ꢀC for 15 min. Then, another part of se-
lenium power (7.9 g, 0.1 mol) was added into the solution and the
mixture was heated to 80 ꢀC. The reaction mixture was then stirred
for another 30 min and filtered. The filtrate was concentrated under
reduced pressure to afford yellow solid Na2Se2 (20.0 g, 98%). m.p.
101e104 ꢀC.
4.4. The preparation of compounds 8a-8p
The Na2Se2 (6, 5.9 g, 0.024 mol) and chlorobenzyl (7a-7p) (2.6 g,
0.02 mol) were added into the solution of methanol (20 mL) with
stirring. The mixture was stirred at 25 ꢀC for 15 h under N2. Then
the solvent was evaporated to dryness, and the residue was purified
4.6. The preparation of compounds 14a-14d
by flash chromatography to afford the white solid (2.8 g, 83.13%).
þ
LC- MS(m/z): 343.0 [M þ H]
.
The
synthesis
of
intermediate
1-(2-chloro-2-(2,4-
11

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
difluorophenyl) ethyl)-1H-imidazole (14b) was chosen as the
example: NaBH4 (6 g, 0.1 mol) was in portions to a stirred solution
of intermediate 12b (11.1 g, 0.05 mol) in 70 mL ethanol. The solution
was stirred at room temperature for 8 h. The reaction mixture was
then poured into water and white solid was collected by filtration.
The residue was purified by silica gel chromatography to obtain 1-
NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.52 (d, J ¼ 1.3 Hz, 1H),
7.50e7.43 (m, 3H), 7.31 (dd, J ¼ 8.5, 2.2 Hz, 1H), 7.27e7.23 (m, 2H),
7.12 (dt, J ¼ 7.6, 2.5 Hz, 1H), 7.09e7.07 (m, 1H), 6.74 (d, J ¼ 1.1 Hz,
1H), 5.08 (dd, J ¼ 10.2, 6.1 Hz, 1H), 4.75 (dd, J ¼ 14.1, 10.1 Hz, 1H),
4.57 (dd, J ¼ 14.2, 6.2 Hz, 1H), 2.24 (s, 3H). 13C NMR (101 MHz,
DMSO)
d (ppm): 141.7, 137.9, 136.4, 135.9, 134.2, 133.0, 130.9, 130.6,
(2,4-difluorophenyl)-2-(1H-imidazole-1-yl)
ethan-1-ol
(13b,
129.4, 129.3, 128.8, 127.8, 127.2, 119.6, 49.6, 22.9.
79.1%). Intermediate 13b (2.2 g, 0.01 mol) was added to thionyl
chloride (6 g, 0.05 mol) and the mixture was stirred at room tem-
perature for 8 h. The reaction mixture was poured into the ice water
mixture and the pH value of water was adjusted to 7e8 by using
sodium hydroxide. After filtration, the residue was purified by silica
gel chromatography to collect 1-(2-chloro-2-(2,4-difluorophenyl)
ethyl)-1H-imidazole (14b, 62.1.0%) as a yellow solid HRMS m/z:
found 243.0504 [M þ H] þ(calcd. for C11H9N2F2Cl, 245.0501).
4.7.5. 1-(2-(2,4-dichlorophenyl)-2-(m-tolylselanyl) ethyl)-1H-
imidazole (B05)
White solid; yield 81.20%; m.p.: 99.1e101.2 ꢀC. ESI-HRMS(m/z):
found 410.9922 [M þ H]þ(calcd. for C18H17N2Cl2Se, 410.9934); 1H
NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.55 (t, J ¼ 1.1 Hz, 1H), 7.50 (d,
J ¼ 2.2 Hz, 1H), 7.43 (d, J ¼ 8.5 Hz, 1H), 7.30 (dd, J ¼ 8.5, 2.2 Hz, 1H),
7.23e7.15 (m, 3H), 7.12 (dq, J ¼ 1.7, 0.9 Hz,1H), 7.10 (t, J ¼ 1.2 Hz,1H),
6.76 (t, J ¼ 1.1 Hz, 1H), 5.08 (dd, J ¼ 9.6, 6.5 Hz, 1H), 4.72 (dd, J ¼ 14.1,
9.7 Hz, 1H), 4.59 (dd, J ¼ 14.1, 6.6 Hz, 1H), 2.23 (s, 3H). 13C NMR
4.7. The preparation of target compounds
(101 MHz, DMSO)
d (ppm): 139.1, 137.9, 136.0, 136.0, 134.3, 132.9,
The synthesis of B15 was chosen as the example: Add 3a (3.14 g,
0.01 mol) into 30 mL ethanol at room temperature and add sodium
borohydride successively (1 g, 0.04 mol). After stirring for 20 min,
14b (4.8 g, 0.02 mol) was added. Then, the mixture was stirred for
1 h at 65 ꢀC. After that, the solution was evaporated to dryness and
the mixture was purified by column chromatography. Compounds
C01eC14 were prepared from intermediate 8a-8p and 14c-14d
using a method which was similar to that of compounds B01eB28.
132.6, 130.8, 129.7, 129.5, 129.3, 128.8, 127.7, 119.7, 49.6, 21.2.
4.7.6. 1-(2-(2,4-dichlorophenyl)-2-(p-tolylselanyl) ethyl)-1H-
imidazole (B06)
White solid; yield 75.75%; m.p.: 101.3e102.8 ꢀC. ESI-HRMS(m/z):
found 410.9922 [M þ H]þ(calcd. for C18H17N2Cl2Se, 410.9934); 1H
NMR (400 MHz, DMSO‑d6)
d (ppm): 7.54e7.49 (m, 2H), 7.40 (d,
J ¼ 8.4 Hz, 1H), 7.29 (dd, J ¼ 9.5, 7.6 Hz, 3H), 7.13e7.07 (m, 3H), 6.74
(s, 1H), 5.02 (dd, J ¼ 9.9, 6.3 Hz, 1H), 4.70 (dd, J ¼ 14.1, 9.9 Hz, 1H),
4.57 (dd, J ¼ 14.1, 6.4 Hz, 1H), 2.29 (s, 3H). 13C NMR (101 MHz,
4.7.1. 1-(2-(2,4-dichlorophenyl)-2-(phenylselanyl) ethyl)-1H-
imidazole (B01)
DMSO) d (ppm): 138.8,137.9,136.0,136.0,134.2,132.90,130.7,130.4,
White solid; yield 75.40%; m.p.: 102.2.1e103.4 ꢀC. ESI-HRMS(m/
z): found 396.9776 [M þ H]þ(calcd. for C17H15N2Cl2Se, 396.9778);
129.4, 128.8, 127.7, 119.7, 49.6, 21.2.
1H NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.55 (d, J ¼ 1.1 Hz, 1H), 7.51
4.7.7. 1-(2-(2,4-dichlorophenyl)-2-((2-methoxyphenyl) selanyl)
ethyl)-1H-imidazole (B07)
(d, J ¼ 2.2 Hz, 1H), 7.44 (d, J ¼ 8.5 Hz, 1H), 7.41e7.38 (m, 2H),
7.37e7.34 (m, 1H), 7.31e7.27 (m, 3H), 7.10 (t, J ¼ 1.3 Hz, 1H), 6.75 (t,
J ¼ 1.1 Hz, 1H), 5.12e5.07 (m, 1H), 4.74 (dd, J ¼ 14.2, 9.9 Hz, 1H), 4.60
White solid; yield 81.69%; m.p.: 105.1e106.8 ꢀC. ESI-HRMS(m/z):
found 426.9881 [M þ H]þ(calcd. for C18H17N2Cl2OSe, 426.9883); 1H
(dd, J ¼ 14.2, 6.4 Hz, 1H). 13C NMR (101 MHz, DMSO)
d
(ppm): 137.9,
NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.53 (d, J ¼ 8.5 Hz, 1H), 7.50 (t,
135.9, 135.6, 134.2, 132.9, 129.7, 129.4, 129.0, 128.8, 127.8, 119.7, 49.6.
J ¼ 1.7 Hz, 2H), 7.37 (dd, J ¼ 7.6, 1.7 Hz, 1H), 7.34e7.28 (m, 2H), 7.05
(t, J ¼ 1.3 Hz,1H), 7.00 (dd, J ¼ 8.3,1.2 Hz,1H), 6.87 (td, J ¼ 7.5,1.2 Hz,
1H), 6.75 (t, J ¼ 1.1 Hz, 1H), 5.19 (dd, J ¼ 9.9, 6.0 Hz, 1H), 4.72 (dd,
4.7.2. 1-(2-(2,4-dichlorophenyl)-2-((3-fluorophenyl) selanyl)
ethyl)-1H-imidazole (B02)
J ¼ 14.2, 10.0 Hz, 1H), 4.53 (dd, J ¼ 14.2, 6.1 Hz, 1H), 3.78 (s, 3H). 13
C
White solid; yield 87.44%; m.p.: 107.2e108.8 ꢀC. ESI-HRMS(m/z):
found 414.9688 [M þ H]þ(calcd. for C17H14N2Cl2FSe, 414.9683); 1H
NMR (101 MHz, DMSO) d (ppm): 158.7, 137.8, 136.1, 134.7, 134.3,
132.9, 131.0, 130.3, 129.3, 128.8, 127.8, 121.7, 119.7, 117.1, 111.7, 56.2,
49.9.
NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.65 (d, J ¼ 1.2 Hz, 1H),
7.62e7.56 (m, 2H), 7.44e7.38 (m, 2H), 7.32e7.23 (m, 3H), 7.21 (t,
J ¼ 1.3 Hz, 1H), 6.83 (t, J ¼ 1.1 Hz, 1H), 5.24 (dd, J ¼ 9.5, 6.7 Hz, 1H),
4.84 (dd, J ¼ 14.2, 9.5 Hz,1H), 4.72 (dd, J ¼ 14.2, 6.8 Hz,1H). 13C NMR
4.7.8. 1-(2-(2,4-dichlorophenyl)-2-((3-methoxyphenyl) selanyl)
ethyl)-1H-imidazole (B08)
(101 MHz, DMSO)
d
(ppm): 163.4, 160.9, 137.9, 135.8, 134.3, 133.1,
White solid; yield 81.75%; m.p.: 107.8e109.3 ꢀC. ESI-HRMS(m/z):
found 426.9884 [M þ H]þ(calcd. for C18H17N2Cl2OSe, 426.9883); 1H
131.4, 131.4, 131.2, 131.2, 130.9, 129.4, 128.8, 127.8, 121.8, 121.6, 119.7,
116.0, 115.8, 49.6.
NMR (400 MHz, DMSO‑d6)
d (ppm): 7.56e7.51 (m, 2H), 7.46 (d,
J ¼ 8.4 Hz, 1H), 7.32 (dd, J ¼ 8.4, 2.2 Hz, 1H), 7.21 (t, J ¼ 7.9 Hz, 1H),
7.10 (d, J ¼ 1.3 Hz, 1H), 6.99 (dt, J ¼ 7.5, 1.3 Hz, 1H), 6.92e6.88 (m,
1H), 6.87e6.85 (m, 1H), 6.76 (d, J ¼ 1.1 Hz, 1H), 5.11 (dd, J ¼ 9.6,
6.5 Hz, 1H), 4.72 (dd, J ¼ 14.2, 9.7 Hz, 1H), 4.61 (dd, J ¼ 14.2, 6.6 Hz,
4.7.3. 1-(2-(2,4-dichlorophenyl)-2-((4-fluorophenyl) selanyl)
ethyl)-1H-imidazole (B03)
White solid; yield 85.60%; m.p.: 107.1e109.1 ꢀC. ESI-HRMS(m/z):
found 414.9678 [M þ H]þ(calcd. for C17H14N2Cl2FSe, 414.9683); 1H
1H), 3.70 (s, 3H). 13C NMR (101 MHz, DMSO)
d (ppm): 159.7, 137.9,
NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.56 (d, J ¼ 1.0 Hz, 1H), 7.52 (d,
136.0, 134.3, 133.0, 130.9, 130.5, 129.4, 128.8, 128.5, 127.8, 127.5,
120.3, 119.7, 115.1, 55.6, 49.6.
J ¼ 2.2 Hz, 1H), 7.42e7.37 (m, 3H), 7.30 (dd, J ¼ 8.4, 2.2 Hz, 1H),
7.16e7.10 (m, 3H), 6.75 (d, J ¼ 1.1 Hz, 1H), 5.06 (dd, J ¼ 9.8, 6.5 Hz,
1H), 4.74e4.59 (m, 2H). 13C NMR (101 MHz, DMSO)
d
(ppm): 163.9,
4.7.9. 1-(2-(2,4-dichlorophenyl)-2-((4-methoxyphenyl) selanyl)
ethyl)-1H-imidazole (B09)
162.2,138.7,138.7,137.9, 135.8, 134.3,132.9,129.4,128.8,127.7, 119.7,
116.9, 116.8, 49.4.
White solid; yield 79.77%; m.p.: 101.6.1e103.9 ꢀC. ESI-HRMS(m/
z): found 426.9903 [M þ H]þ(calcd. for C18H17N2Cl2OSe, 426.9883);
4.7.4. 1-(2-(2,4-dichlorophenyl)-2-(o-tolylselanyl) ethyl)-1H-
imidazole (B04)
1H NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.54 (t, J ¼ 1.2 Hz, 1H), 7.50
(d, J ¼ 2.2 Hz, 1H), 7.34 (d, J ¼ 8.5 Hz, 1H), 7.30e7.26 (m, 3H),
7.10e7.07 (m, 1H), 6.87e6.83 (m, 2H), 6.75 (t, J ¼ 1.0 Hz, 1H), 4.97
(dd, J ¼ 9.9, 6.4 Hz, 1H), 4.68 (dd, J ¼ 14.1, 9.9 Hz, 1H), 4.57 (dd,
White solid; yield 88.90%; m.p.: 98.4e100.1 ꢀC. ESI-HRMS(m/z):
found 410.9931 [M þ H]þ(calcd. for C18H17N2Cl2Se, 410.9934); 1H
12

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
J ¼ 14.1, 6.4 Hz, 1H), 3.75 (s, 3H). 13C NMR (101 MHz, DMSO)
4.7.15. 1-(2-(2,4-difluorophenyl)-2-(phenylselanyl) ethyl)-1H-
imidazole (B15)
d
(ppm): 160.4, 138.4, 137.9, 136.0, 134.2, 132.7, 130.4, 129.3, 128.8,
127.6, 119.6, 115.4, 55.7, 49.5.
White solid; yield 73.15%; m.p.: 83.9e86.0 ꢀC. ESI-HRMS(m/z):
found 365.0362 [M þ H]þ(calcd. for C17H15N2F2Se, 365.0369); 1H
NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.56 (d, J ¼ 1.1 Hz, 1H),
4.7.10. 1-(2-(2,4-dichlorophenyl)-2-((2,5-dimethylphenyl) selanyl)
ethyl)-1H-imidazole (B10)
7.42e7.25 (m, 6H), 7.14e7.07 (m, 2H), 6.99e6.93 (m, 1H), 6.77 (d,
J ¼ 1.1 Hz,1H), 5.00 (dd, J ¼ 9.5, 6.9 Hz, 1H), 4.67 (dd, J ¼ 14.1, 9.5 Hz,
1H), 4.57 (dd, J ¼ 14.1, 6.9 Hz, 1H). 13C NMR (101 MHz, DMSO)
White solid; yield 74.54%; m.p.: 107.6e109.8 ꢀC. ESI-HRMS(m/z):
found 425.0043 [M þ H]þ(calcd. for C19H19N2Cl2Se, 425.0091); 1H
d
(ppm): 163.1, 163.0, 161.6, 161.5, 160.6, 160.5, 159.1, 159.0, 137.9,
NMR (400 MHz, DMSO‑d6) d (ppm): 7.53 (s, 1H), 7.50e7.45 (m, 2H),
135.2, 131.0, 131.0, 130.9, 130.9, 129.7, 128.8, 128.0, 123.2, 123.2,
123.1, 123.1, 119.7, 112.0, 112.0, 111.8, 111.8, 104.6, 104.3, 104.1, 49.6.
7.31 (dd, J ¼ 8.5, 2.2 Hz, 1H), 7.14e7.08 (m, 3H), 7.07e7.04 (m, 1H),
6.75 (s, 1H), 5.07 (dd, J ¼ 10.0, 6.2 Hz, 1H), 4.73 (dd, J ¼ 14.1, 10.0 Hz,
1H), 4.58 (dd, J ¼ 14.1, 6.3 Hz, 1H), 2.21 (d, J ¼ 4.6 Hz, 6H). 13C NMR
(101 MHz, DMSO)
d (ppm): 138.5, 137.9, 137.0, 136.2, 136.0, 134.3,
4.7.16. 1-(2-(2,4-difluorophenyl)-2-((3-fluorophenyl) selanyl)
ethyl)-1H-imidazole (B16)
132.9, 130.9, 130.3, 130.0, 129.2, 128.8, 128.2, 127.7, 119.7, 49.7, 22.4,
20.7.
White solid; yield 75.09%; m.p.: 85.2.1e86.9 ꢀC. ESI-HRMS(m/z):
found 383.0264 [M þ H]þ(calcd. for C17H14N2F3Se, 383.0274); 1H
NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.57 (t, J ¼ 1.1 Hz, 1H), 7.45 (td,
4.7.11. 1-(2-(2,4-dichlorophenyl)-2-((2,6-dimethylphenyl) selanyl)
ethyl)-1H-imidazole (B11)
J ¼ 8.7, 6.5 Hz, 1H), 7.32 (td, J ¼ 8.0, 6.1 Hz, 1H), 7.26e7.20 (m, 2H),
7.20e7.11 (m, 3H), 7.03e6.96 (m, 1H), 6.77 (d, J ¼ 1.1 Hz, 1H), 5.09
(dd, J ¼ 9.3, 7.1 Hz, 1H), 4.72e4.57 (m, 2H). 13C NMR (101 MHz,
White solid; yield 75.19%; m.p.: 107.4e109.4 ꢀC. ESI-HRMS(m/z):
found 425.0044 [M þ H]þ(calcd. for C19H19N2Cl2Se, 425.0091); 1H
DMSO)
d (ppm): 163.4, 163.2, 163.1, 161.7, 161.5, 161.0, 160.8, 160.6,
NMR (400 MHz, DMSO‑d6)
d (ppm): 7.62 (s, 1H), 7.48 (s, 1H),
159.2, 159.1, 137.9, 131.4, 131.3, 131.1, 131.1, 131.0, 131.0, 130.8, 130.8,
130.0, 129.9, 128.8, 123.0, 123.0, 122.9, 122.8, 121.4, 121.2, 119.8,
115.8, 115.6, 112.1, 112.1, 111.9, 111.9, 104.7, 104.4, 104.2, 49.5.
7.45e7.40 (m, 1H), 7.35 (d, J ¼ 8.5 Hz, 1H), 7.18 (dd, J ¼ 8.6, 6.0 Hz,
1H), 7.12 (d, J ¼ 6.5 Hz, 2H), 7.04 (d, J ¼ 1.3 Hz,1H), 6.73 (d, J ¼ 1.1 Hz,
1H), 4.95 (s, 1H), 4.77 (dd, J ¼ 14.0, 10.4 Hz, 1H), 4.51e4.40 (m, 1H),
2.37 (s, 6H). 13C NMR (101 MHz, DMSO)
d (ppm): 144.1, 137.8, 136.4,
134.1, 132.9, 129.8, 128.8, 128.1, 127.9, 119.6, 49.8, 24.6.
4.7.17. 1-(2-(2,4-difluorophenyl)-2-((4-fluorophenyl) selanyl)
ethyl)-1H-imidazole (B17)
White solid; yield 88.56%; m.p.: 88.2e100.2 ꢀC. ESI-HRMS(m/z):
found 383.0271 [M þ H]þ(calcd. for C17H14N2F3Se, 383.0274); 1H
4.7.12. 1-(2-(2,4-dichlorophenyl)-2-((3,5-dimethylphenyl) selanyl)
ethyl)-1H-imidazole (B12)
NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.57 (t, J ¼ 1.2 Hz, 1H),
White solid; yield 80.56%; m.p.: 107.2e109.1 ꢀC. ESI-HRMS(m/z):
found 425.0085 [M þ H]þ(calcd. for C19H19N2Cl2Se, 425.0091); 1H
7.42e7.32 (m, 3H), 7.16e7.08 (m, 4H), 7.00e6.93 (m, 1H), 6.77 (d,
J ¼ 1.1 Hz, 1H), 4.97 (dd, J ¼ 9.3, 7.1 Hz, 1H), 4.70e4.54 (m, 2H). 13
C
NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.55 (d, J ¼ 1.1 Hz, 1H), 7.51 (dd,
NMR (101 MHz, DMSO)
d (ppm): 164.2, 163.1, 163.0, 161.7, 161.6,
J ¼ 2.2, 1.3 Hz, 1H), 7.42 (d, J ¼ 8.5 Hz, 1H), 7.30 (dd, J ¼ 8.5, 2.2 Hz,
1H), 7.10 (d, J ¼ 1.2 Hz,1H), 6.95 (d, J ¼ 5.7 Hz, 3H), 6.76 (d, J ¼ 1.1 Hz,
1H), 5.06 (dd, J ¼ 9.7, 6.5 Hz, 1H), 4.70 (dd, J ¼ 14.2, 9.7 Hz, 1H), 4.58
(dd, J ¼ 14.2, 6.6 Hz, 1H), 2.20 (s, 6H). 13C NMR (101 MHz, DMSO)
161.5, 160.7, 160.5, 159.1, 159.0, 138.4, 138.3, 137.9, 130.9, 130.8,
130.8, 130.8, 128.8, 123.1, 123.0, 122.9, 122.9, 122.7, 122.7, 119.7,
116.9, 116.7, 112.0, 112.0, 111.8, 111.8, 104.6, 104.4, 104.1, 49.4.
d
(ppm): 138.8, 137.9, 136.1, 134.3, 133.1, 132.9, 130.8, 130.4, 129.3,
128.8, 127.6, 119.7, 49.6, 21.1.
4.7.18. 1-(2-(2,4-difluorophenyl)-2-(o-tolylselanyl) ethyl)-1H-
imidazole (B18)
White solid; yield 81.98%; m.p.: 87.6e89.4 ꢀC. ESI-HRMS(m/z):
found 379.0529 [M þ H]þ(calcd. for C18H17N2F2Se, 379.0525); 1H
4.7.13. 1-(2-(2,4-dichlorophenyl)-2-((2-ethylphenyl) selanyl)
ethyl)-1H-imidazole (B13)
White solid; yield 73.38%; m.p.: 99.5e101.7 ꢀC. ESI-HRMS(m/z):
found 425.0095 [M þ H]þ(calcd. for C19H19N2Cl2Se, 425.0091); 1H
NMR (400 MHz, DMSO‑d6)
d
7.55 (t, J ¼ 1.1 Hz, 1H), 7.46e7.39 (m,
2H), 7.25e7.21 (m, 2H), 7.13e7.06 (m, 3H), 6.97 (tdd, J ¼ 8.6, 2.7,
1.0 Hz, 1H), 6.76 (d, J ¼ 1.1 Hz, 1H), 4.96 (dd, J ¼ 9.8, 6.7 Hz, 1H), 4.69
(dd, J ¼ 14.1, 9.8 Hz, 1H), 4.55 (dd, J ¼ 14.1, 6.7 Hz, 1H), 2.24 (s, 3H).
NMR (400 MHz, DMSO‑d6)
d (ppm): 7.53e7.45 (m, 4H), 7.33e7.23
(m, 3H), 7.13 (td, J ¼ 7.3, 1.9 Hz, 1H), 7.07 (d, J ¼ 1.2 Hz, 1H), 6.74 (d,
J ¼ 1.1 Hz, 1H), 5.08 (dd, J ¼ 10.1, 6.1 Hz, 1H), 4.76 (dd, J ¼ 14.1,
10.2 Hz, 1H), 4.57 (dd, J ¼ 14.2, 6.2 Hz, 1H), 2.61 (p, J ¼ 7.3 Hz, 2H),
13C NMR (101 MHz, DMSO)
d (ppm): 163.1, 163.0, 161.6, 161.5, 160.7,
160.5, 159.2, 159.1, 141.3, 138.0, 136.0, 131.1, 131.0, 131.0, 131.0, 130.5,
129.1, 129.0, 128.8, 127.1, 123.2, 123.1, 123.1, 123.0, 119.7, 112.1, 112.0,
111.9, 111.8, 104.6, 104.3, 104.0, 49.6, 22.8.
1.04 (t, J ¼ 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO)
d (ppm): 147.4,
137.8, 136.5, 135.9, 134.2, 133.0, 130.9, 129.5, 129.3, 129.2, 128.8,
127.8, 127.2, 119.6, 49.6, 29.1, 16.1.
4.7.19. 1-(2-(2,4-difluorophenyl)-2-(m-tolylselanyl) ethyl)-1H-
imidazole (B19)
4.7.14. 1-(2-(2,4-dichlorophenyl)-2-((4-ethylphenyl) selanyl)
ethyl)-1H-imidazole (B14)
White solid; yield 82.66%; m.p.: 86.8e89.0 ꢀC. ESI-HRMS(m/z):
found 379.0518 [M þ H]þ(calcd. for C18H17N2F2Se, 379.0525); 1H
White solid; yield 69.28%; m.p.: 99.5e101.7 ꢀC. ESI-HRMS(m/z):
found 425.0092 [M þ H]þ(calcd. for C19H19N2Cl2Se, 425.0091); 1H
NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.56 (d, J ¼ 1.2 Hz, 1H), 7.39 (td,
NMR (400 MHz, DMSO‑d6)
d
(ppm): 7.54 (t, J ¼ 1.1 Hz, 1H), 7.50 (d,
J ¼ 8.7, 6.5 Hz, 1H), 7.21e7.09 (m, 6H), 6.97 (tdd, J ¼ 8.5, 2.6, 1.0 Hz,
1H), 6.77 (t, J ¼ 1.1 Hz, 1H), 4.97 (dd, J ¼ 9.4, 6.9 Hz, 1H), 4.66 (dd,
J ¼ 2.2 Hz, 1H), 7.40 (d, J ¼ 8.5 Hz, 1H), 7.31e7.26 (m, 3H), 7.14e7.11
(m, 2H), 7.09 (t, J ¼ 1.3 Hz, 1H), 6.75 (t, J ¼ 1.1 Hz, 1H), 5.04 (dd,
J ¼ 9.9, 6.3 Hz, 1H), 4.72 (dd, J ¼ 14.2, 9.9 Hz, 1H), 4.57 (dd, J ¼ 14.2,
6.4 Hz, 1H), 2.58 (q, J ¼ 7.6 Hz, 2H), 1.15 (t, J ¼ 7.6 Hz, 3H). 13C NMR
J ¼ 14.1, 9.5 Hz, 1H), 4.55 (dd, J ¼ 14.1, 6.9 Hz, 1H), 2.24 (s, 3H). 13
C
NMR (101 MHz, DMSO)
d (ppm): 163.1, 163.0, 161.6, 161.5, 160.6,
160.5, 159.2, 159.0, 139.0, 137.9, 135.7, 132.2, 131.0, 131.0, 130.9,
130.9, 129.5, 128.8, 127.7, 123.3, 123.2, 123.1, 123.1, 119.7, 112.0, 111.9,
111.8, 111.7, 104.6, 104.3, 104.1, 49.6, 21.2.
(101 MHz, DMSO)
d (ppm): 145.0, 137.9, 136.0, 136.0, 134.2, 132.8,
130.7, 129.3, 129.2, 128.8, 127.7, 119.7, 49.6, 28.3, 15.9.
13

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
4.7.20. 1-(2-(2,4-difluorophenyl)-2-(p-tolylselanyl) ethyl)-1H-
imidazole (B20)
4.7.25. 1-(2-(2,4-difluorophenyl)-2-((2,6-dimethylphenyl) selanyl)
ethyl)-1H-imidazole (B25)
Colourless oil; yield 76.14%. ESI-HRMS(m/z): found 379.0520
Colourless oil; yield 65.38%. ESI-HRMS(m/z): found 393.0674
[M þ H]þ(calcd. for C18H17N2F2Se, 379.0525); 1H NMR (400 MHz,
[M þ H]þ(calcd. for C19H19N2F2Se, 393.0682); 1H NMR (400 MHz,
DMSO‑d6)
d
(ppm): 7.55 (s, 1H), 7.36 (td, J ¼ 8.7, 6.5 Hz, 1H), 7.27 (d,
DMSO‑d6)
d
(ppm): 7.89e7.77 (m, 1H), 7.55 (d, J ¼ 1.1 Hz, 1H), 7.44
J ¼ 8.1 Hz, 2H), 7.14e7.08 (m, 4H), 6.99e6.93 (m, 1H), 6.76 (d,
J ¼ 1.1 Hz, 1H), 4.92 (dd, J ¼ 9.6, 6.8 Hz, 1H), 4.67e4.60 (m, 1H), 4.54
(dd, J ¼ 14.1, 6.8 Hz, 1H), 2.28 (s, 3H). 13C NMR (101 MHz, DMSO)
(td, J ¼ 8.7, 6.5 Hz, 1H), 7.17e7.10 (m, 4H), 7.00e6.94 (m, 1H), 6.75 (t,
J ¼ 1.1 Hz, 1H), 4.76e4.68 (m, 2H), 4.54e4.45 (m, 1H), 2.34 (s, 6H).
13C NMR (101 MHz, DMSO)
d (ppm): 163.1, 163.0, 162.9, 161.7, 161.5,
d
(ppm): 163.1, 162.9, 161.6, 161.5, 160.6, 160.5, 159.1, 159.0, 138.6,
161.2, 161.0, 160.6, 160.5, 159.2, 159.1, 143.9, 137.9, 137.7, 131.2, 131.1,
131.1, 130.4, 129.7, 129.6, 128.8, 128.0, 126.4, 126.3, 126.3, 126.3,
123.4, 123.3, 123.2, 123.2, 119.7, 117.0, 112.0, 112.0, 111.8, 111.8, 109.1,
104.8, 104.4, 104.2, 103.9, 49.6, 24.5.
137.9, 135.7, 130.4, 128.8, 124.1, 120.0, 119.71, 112.0, 111.9, 111.8, 111.7,
104.9, 104.6, 104.3, 104.1, 49.5, 21.2.
4.7.21. 1-(2-(2,4-difluorophenyl)-2-((2-methoxyphenyl) selanyl)
ethyl)-1H-imidazole (B21)
4.7.26. 1-(2-(2,4-difluorophenyl)-2-((3,5-dimethylphenyl) selanyl)
ethyl)-1H-imidazole (B26)
Colourless oil; yield 74.36%. ESI-HRMS(m/z): found 395.0463
[M þ H]þ(calcd. for C18H17N2F2OSe, 395.0474); 1H NMR (400 MHz,
Colourless oil; yield 68.20%. ESI-HRMS(m/z): found 393.0682
[M þ H]þ(calcd. for C19H19N2F2Se, 393.0682); 1H NMR (400 MHz,
DMSO‑d6)
d
(ppm): 7.52 (s,1H), 7.47 (td, J ¼ 8.7, 6.5 Hz,1H), 7.39 (dd,
DMSO‑d6)
d
(ppm): 8.08 (s, 1H), 7.70 (td, J ¼ 8.8, 6.5 Hz, 1H), 7.31
J ¼ 7.6, 1.6 Hz, 1H), 7.29 (ddd, J ¼ 8.2, 7.4, 1.7 Hz, 1H), 7.14e7.08 (m,
2H), 7.00e6.94 (m, 2H), 6.87 (td, J ¼ 7.5, 1.2 Hz, 1H), 6.77 (d,
J ¼ 1.3 Hz, 1H), 5.06 (dd, J ¼ 9.8, 6.4 Hz, 1H), 4.67 (dd, J ¼ 14.1, 9.9 Hz,
1H), 4.53 (dd, J ¼ 14.1, 6.4 Hz, 1H), 3.78 (s, 3H). 13C NMR (101 MHz,
(ddd, J ¼ 11.5, 9.2, 2.6 Hz, 1H), 7.16e7.12 (m, 2H), 6.96 (d, J ¼ 7.9 Hz,
3H), 6.78 (t, J ¼ 1.1 Hz, 1H), 4.96 (dd, J ¼ 9.4, 6.9 Hz, 1H), 4.65 (dd,
J ¼ 14.1, 9.4 Hz, 1H), 4.55 (dd, J ¼ 14.1, 6.9 Hz, 1H), 2.20 (s, 6H). 13
C
NMR (101 MHz, DMSO)
d (ppm): 163.1, 163.08, 161.7, 161.2, 161.0,
DMSO) d (ppm): 163.1, 162.9, 161.7, 161.5, 160.6, 160.5, 159.2, 159.06,
160.7, 160.5, 159.2, 159.1, 158.7, 158.6, 138.8, 137.9, 137.7, 132.7, 132.7,
131.0, 131.0, 130.9, 130.9, 130.4, 130.3, 129.3, 129.2, 129.2, 129.1,
128.8, 127.6, 126.4, 126.3, 126.3, 126.3, 119.8, 119.8, 117.0, 112.7, 112.7,
112.5, 112.5, 111.9, 111.9, 109.1, 105.1, 104.8, 104.6, 104.3, 104.0, 49.6,
21.1.
158.5, 137.8, 134.2, 131.2, 131.2, 131.1, 131.1, 123.0, 128.7, 123.2, 123.1,
123.0, 123.0, 121.7, 119.7, 117.4, 112.0, 112.0, 111.8, 111.8, 111.6, 104.5,
104.3, 104.0, 49.9, 37.2.
4.7.22. 1-(2-(2,4-difluorophenyl)-2-((3-methoxyphenyl) selanyl)
ethyl)-1H-imidazole (B22)
4.7.27. 1-(2-(2,4-difluorophenyl)-2-((2-ethylphenyl) selanyl)
ethyl)-1H-imidazole (B27)
Colourless oil; yield 81.20%. ESI-HRMS(m/z): found 395.0371
[M þ H]þ(calcd. for C18H17N2F2OSe, 395.0474); 1H NMR (400 MHz,
Colourless oil; yield 86.41%. ESI-HRMS(m/z): found 393.0673
DMSO‑d6)
d
(ppm): 7.56 (t, J ¼ 10 Hz, 1H), 7.42 (td, J ¼ 8.7, 6.5 Hz,
[M þ H]þ(calcd. for C19H19N2F2Se, 393.0682); 1H NMR (400 MHz,
1H), 7.22e7.17 (m, 1H), 7.16e7.10 (m, 2H), 7.00e6.95 (m, 2H), 6.89
(dtt, J ¼ 5.1, 2.6, 1.3 Hz, 2H), 6.77 (t, J ¼ 1.1 Hz, 1H), 5.02 (dd, J ¼ 9.4,
7.0 Hz, 1H), 4.66 (dd, J ¼ 14.1, 9.4 Hz, 1H), 4.57 (dd, J ¼ 14.1, 7.0 Hz,
DMSO‑d6)
d
(ppm): 7.54 (d, J ¼ 1.1 Hz, 1H), 7.48e7.39 (m, 2H),
7.29e7.22 (m, 2H), 7.14e7.05 (m, 3H), 6.97 (tdd, J ¼ 8.6, 2.7, 1.0 Hz,
1H), 6.76 (t, J ¼ 1.1 Hz, 1H), 4.95 (dd, J ¼ 9.8, 6.7 Hz, 1H), 4.69 (dd,
J ¼ 14.0, 9.8 Hz, 1H), 4.56 (dd, J ¼ 14.1, 6.7 Hz, 1H), 2.60 (qd, J ¼ 7.4,
3.4 Hz, 2H), 1.04 (t, J ¼ 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO)
1H), 3.71 (s, 3H). 13C NMR (101 MHz, DMSO)
d (ppm): 163.0, 161.6,
161.5, 160.7, 160.6, 159.7, 159.2, 159.1, 137.9, 131.1, 131.0, 131.0, 131.0,
130.1, 128.9, 128.8, 127.1, 123.2, 123.1, 119.9, 119.8, 114.8, 112.0, 112.0,
111.8, 111.8, 104.6, 104.4, 104.1, 55.6, 49.6, 49.1.
d
(ppm): 163.1, 163.0, 161.6, 161.5, 160.6, 160.5, 159.2, 159.0, 147.1,
137.9, 136.2, 131.1, 131.1, 131.0, 131.0, 129.3, 129.1, 128.8, 128.5, 127.2,
123.2, 123.1, 123.0, 123.0, 119.7, 112.1, 112.0, 111.9, 111.8, 104.5, 104.3,
104.0, 49.6, 29.1, 16.0.
4.7.23. 1-(2-(2,4-difluorophenyl)-2-((4-methoxyphenyl) selanyl)
ethyl)-1H-imidazole (B23)
Colourless oil; yield 73.39%. ESI-HRMS(m/z): found 395.0466
4.7.28. 1-(2-(2,4-difluorophenyl)-2-((4-ethylphenyl) selanyl)
ethyl)-1H-imidazole (B28)
[M þ H]þ(calcd. for C18H17N2F2OSe, 395.0474); 1H NMR (400 MHz,
DMSO‑d6)
d
(ppm): 7.56 (t, J ¼ 1.1 Hz, 1H), 7.32e7.25 (m, 3H),
Colourless oil; yield 85.29%. ESI-HRMS(m/z): found 393.0671
[M þ H]þ(calcd. for C19H19N2F2Se, 393.0682); 1H NMR (400 MHz,
7.13e7.07 (m, 2H), 6.95 (tdd, J ¼ 8.5, 2.7, 1.0 Hz, 1H), 6.86e6.83 (m,
2H), 6.76 (t, J ¼ 1.1 Hz, 1H), 4.86 (dd, J ¼ 9.5, 6.9 Hz, 1H), 4.63 (dd,
DMSO‑d6)
d
(ppm): 7.55 (d, J ¼ 1.1 Hz, 1H), 7.36 (td, J ¼ 8.7, 6.5 Hz,
J ¼ 14.1, 9.5 Hz, 1H), 4.53 (dd, J ¼ 14.1, 6.9 Hz, 1H), 3.74 (s, 3H). 13
C
1H), 7.31e7.28 (m, 2H), 7.15e7.09 (m, 4H), 6.98e6.92 (m, 1H), 6.76
(t, J ¼ 1.1 Hz, 1H), 4.94 (dd, J ¼ 9.6, 6.8 Hz, 1H), 4.65 (dd, J ¼ 14.1,
9.6 Hz,1H), 4.55 (dd, J ¼ 14.1, 6.8 Hz,1H), 2.58 (q, J ¼ 7.6 Hz, 2H),1.15
NMR (101 MHz, DMSO)
d (ppm): 163.0, 162.9, 161.6, 161.4, 160.6,
160.4,160.3,159.1,159.0,138.2,138.1,138.0,137.9,130.8,130.7, 130.7,
130.6, 128.9,128.8, 123.3, 123.3, 123.2, 123.1, 120.1, 119.7, 117.5, 115.3,
111.9, 111.8, 111.7, 111.6, 104.6, 104.3, 104.0, 55.7, 49.4.
(t, J ¼ 7.6 Hz, 3H). 13C NMR (101 MHz, DMSO)
d (ppm): 162.7, 162.6,
161.2, 161.1, 161.0, 160.9, 159.5, 159.4, 144.8, 137.9, 135.7, 130.9, 130.9,
130.9, 129.2, 128.8, 124.5, 119.7, 111.9, 111.8, 104.5, 104.3, 49.6, 28.3,
15.9.
4.7.24. 1-(2-(2,4-difluorophenyl)-2-((2,5-dimethylphenyl) selanyl)
ethyl)-1H-imidazole (B24)
Colourless oil; yield 81.37%. ESI-HRMS(m/z): found 393.0677
4.7.29. 1-(2-((2,4-dichlorobenzyl) selanyl)-2-(2,4-dichlorophenyl)
ethyl)-1H-1,2,4-triazole (C01)
[M þ H]þ(calcd. for C19H19N2F2Se, 393.0682); 1H NMR (400 MHz,
DMSO‑d6)
d
(ppm): 7.56 (t, J ¼ 1.1 Hz, 1H), 7.42 (td, J ¼ 8.7, 6.5 Hz,
White solid; yield 79.30%; m.p.: 91.3e92.8 ꢀC. ESI-HRMS(m/z):
found 481.9058 [M þ H]þ(calcd. for C17H14N3Cl4Se, 481.9078); 1H
1H), 7.17 (d, J ¼ 1.9 Hz, 1H), 7.14e7.07 (m, 3H), 7.05e6.93 (m, 2H),
6.77 (t, J ¼ 1.1 Hz, 1H), 4.95 (dd, J ¼ 9.7, 6.8 Hz, 1H), 4.68 (dd, J ¼ 14.1,
9.7 Hz, 1H), 4.56 (dd, J ¼ 14.1, 6.8 Hz, 1H), 2.20 (s, 6H). 13C NMR
NMR (400 MHz, DMSO‑d6) d (ppm): 8.43 (s, 1H), 7.92 (s, 1H), 7.60 (d,
J ¼ 8.5 Hz,1H), 7.56 (d, J ¼ 2.2 Hz,1H), 7.51 (d, J ¼ 2.2 Hz,1H), 7.44 (d,
J ¼ 8.3 Hz,1H), 7.38 (td, J ¼ 6.2, 3.0 Hz, 2H), 4.97 (dd, J ¼ 12.4, 4.8 Hz,
1H), 4.90e4.81 (m, 2H), 4.00 (d, J ¼ 11.9 Hz, 1H), 3.88 (d, J ¼ 11.9 Hz,
(101 MHz, DMSO)
d (ppm): 163.1, 163.0, 161.7, 161.6, 160.7, 160.5,
159.2, 159.1, 138.1, 137.8, 136.5, 136.1, 131.1, 131.0, 131.0, 130.9, 130.2,
129.8, 128.8, 128.7, 123.2, 123.2, 123.1, 123.0, 119.7, 112.0, 112.0, 111.8,
111.7, 104.5, 104.2, 104,0, 49.6, 22.3, 20.7.
1H). 13C NMR (101 MHz, DMSO)
d (ppm): 152.0, 144.9, 136.0, 136.0,
134.3, 133.9, 133.1, 132.8, 132.6, 130.52, 129.6, 129.5, 128.1, 127.9,
14

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
52.4, 25.4.
2H), 3.89 (d, J ¼ 11.5 Hz,1H), 3.76 (d, J ¼ 11.5 Hz,1H), 2.26 (s, 3H). 13
C
NMR (101 MHz, DMSO)
d (ppm): 152.0, 144.8, 136.4, 136.4, 135.7,
4.7.30. 1-(2-(benzylselanyl)-2-(2,4-dichlorophenyl) ethyl)-1H-
1,2,4-triazole (C02)
133.9, 132.9, 130.5, 129.5, 129.5, 129.3, 128.0, 52.4, 28.3, 21.2.
White solid; yield 73.31%; m.p.: 88.3e90.3 ꢀC. ESI-HRMS(m/z):
found 411.9870 [M þ H]þ(calcd. for C17H16N3Cl2Se, 411.9886); 1H
4.7.36. 1-(2-((2,4-dichlorobenzyl) selanyl)-2-(2,4-difluorophenyl)
ethyl)-1H-1,2,4-triazole (C08)
NMR (400 MHz, DMSO‑d6)
d
(ppm): 8.40 (s, 1H), 7.91 (s, 1H), 7.58 (d,
White solid; yield 75.83%; m.p.: 77.1e79.3 ꢀC. ESI-HRMS(m/z):
found 447.9673 [M þ H]þ(calcd. for C17H14N3Cl2F2Se, 447.9698); 1H
J ¼ 8.5 Hz, 1H), 7.50 (d, J ¼ 2.2 Hz, 1H), 7.38 (dd, J ¼ 8.4, 2.3 Hz, 1H),
7.29 (d, J ¼ 4.4 Hz, 4H), 7.24e7.18 (m, 1H), 4.95 (dd, J ¼ 13.7, 9.0 Hz,
1H), 4.84e4.73 (m, 2H), 3.92 (d, J ¼ 11.6 Hz, 1H), 3.81 (d, J ¼ 11.5 Hz,
NMR (400 MHz, DMSO‑d6) d (ppm): 8.41 (s, 1H), 7.91 (s, 1H), 7.57 (d,
J ¼ 2.1 Hz, 1H), 7.50 (td, J ¼ 8.7, 6.4 Hz, 1H), 7.42 (d, J ¼ 8.3 Hz, 1H),
7.36 (dd, J ¼ 8.3, 2.1 Hz, 1H), 7.15 (ddd, J ¼ 10.8, 9.3, 2.6 Hz, 1H), 7.03
(tdd, J ¼ 8.5, 2.6, 1.1 Hz, 1H), 4.91e4.71 (m, 3H), 3.96 (d, J ¼ 12.0 Hz,
1H). 13C NMR (101 MHz, DMSO)
d (ppm): 152.0, 144.8, 138.9, 136.4,
133.9, 132.9, 130.5, 129.5, 129.4, 128.9, 128.0, 127.3, 52.4, 28.5.
1H), 3.87 (d, J ¼ 11.9 Hz, 1H). 13C NMR (101 MHz, DMSO)
d (ppm):
4.7.31. 1-(2-((2-chlorobenzyl) selanyl)-2-(2,4-dichlorophenyl)
ethyl)-1H-1,2,4-triazole (C03)
163.2, 163.1, 161.4, 161.2, 160.8, 160.6, 158.9, 158.8, 152.0, 144.9,
136.2, 134.2, 132.8, 132.5, 130.8, 130.8, 130.7, 130.7, 129.5, 127.9,
123.4, 123.3, 123.2, 123.2, 112.3, 112.2, 112.0, 112.0, 104.8, 104.6,
104.3, 52.5, 35.2, 25.2.
White solid; yield 85.62%; m.p.: 82.8e84.9 ꢀC. ESI-HRMS(m/z):
found 445.9478 [M þ H]þ(calcd. for C17H15N3Cl3Se, 445.9497); 1H
NMR (400 MHz, DMSO‑d6) d (ppm): 8.42 (s, 1H), 7.92 (s, 1H), 7.61 (d,
J ¼ 8.4 Hz, 1H), 7.51 (d, J ¼ 2.2 Hz, 1H), 7.43e7.37 (m, 3H), 7.31e7.25
(m, 2H), 5.00 (dd, J ¼ 15.5, 11.1 Hz, 1H), 4.89e4.82 (m, 2H), 4.00 (d,
J ¼ 11.7 Hz, 1H), 3.89 (d, J ¼ 11.7 Hz, 1H). 13C NMR (101 MHz, DMSO)
4.7.37. 1-(2-(benzylselanyl)-2-(2,4-difluorophenyl) ethyl)-1H-
1,2,4-triazole (C09)
Colourless oil; yield 71.34%. ESI-HRMS(m/z): found 380.0469
d
(ppm): 152.0, 144.8, 136.7, 136.1, 133.9, 133.4, 133.0, 131.5, 130.5,
[M þ H]þ(calcd. for C17H16N3F2Se, 380.0478); 1H NMR (400 MHz,
130.1, 129.6, 129.1, 128.1, 127.8, 52.4, 26.1.
DMSO‑d6)
d
(ppm): 8.39 (s, 1H), 7.91 (s, 1H), 7.47 (td, J ¼ 8.7, 6.5 Hz,
1H), 7.31e7.25 (m, 4H), 7.24e7.18 (m, 1H), 7.14 (ddd, J ¼ 10.8, 9.3,
2.6 Hz, 1H), 7.03 (tdd, J ¼ 8.5, 2.6, 1.0 Hz, 1H), 4.90e4.72 (m, 2H),
4.63 (dd, J ¼ 9.4, 6.7 Hz, 1H), 3.90 (d, J ¼ 11.6 Hz, 1H), 3.80 (d,
4.7.32. 1-(2-((4-chlorobenzyl) selanyl)-2-(2,4-dichlorophenyl)
ethyl)-1H-1,2,4-triazole (C04)
White solid; yield 82.32%; m.p.: 80.1e82.1 ꢀC. ESI-HRMS(m/z):
found 445.9473 [M þ H]þ(calcd. for C17H15N3Cl3Se, 445.9497); 1H
J ¼ 11.6 Hz, 1H). 13C NMR (101 MHz, DMSO)
d (ppm): 163.1, 163.0,
161.3, 161.2, 160.7, 160.5, 158.9, 158.7, 152.0, 144.8, 139.0, 130.8,
130.8, 130.7, 129.3, 128.9, 127.3, 112.3, 112.2, 112.0, 112.0, 104.8,
104.5, 52.5, 34.6, 28.3.
NMR (400 MHz, DMSO‑d6)
d (ppm): 8.41 (s, 1H), 7.92 (s, 1H),
7.59e7.55 (m, 1H), 7.50 (d, J ¼ 2.2 Hz, 1H), 7.40e7.29 (m, 5H),
5.00e4.79 (m, 2H), 4.72 (dd, J ¼ 9.4, 6.5 Hz, 1H), 3.92 (d, J ¼ 11.9 Hz,
1H), 3.82 (d, J ¼ 11.8 Hz, 1H). 13C NMR (101 MHz, DMSO)
d
(ppm):
4.7.38. 1-(2-((2-chlorobenzyl) selanyl)-2-(2,4-difluorophenyl)
ethyl)-1H-1,2,4-triazole (C10)
152.1, 144.8, 138.2, 136.2, 133.8, 133.0, 131.8, 131.2, 130.5, 129.5,
128.8, 128.1, 52.3, 27.5.
Colourless oil; yield 71.04%. ESI-HRMS(m/z): found 414.0068
[M þ H]þ(calcd. for C17H15N3ClF2Se, 14.0088); 1H NMR (400 MHz,
4.7.33. 1-(2-(2,4-dichlorophenyl)-2-((4-fluorobenzyl) selanyl)
ethyl)-1H-1,2,4-triazole (C05)
DMSO‑d6)
d
(ppm): 8.41 (s, 1H), 7.91 (s, 1H), 7.51 (td, J ¼ 8.7, 6.4 Hz,
1H), 7.43e7.35 (m, 2H), 7.30e7.24 (m, 2H), 7.15 (ddd, J ¼ 10.8, 9.2,
2.6 Hz, 1H), 7.03 (tdd, J ¼ 8.5, 2.6, 1.0 Hz, 1H), 4.92e4.84 (m, 1H),
4.82e4.73 (m, 2H), 3.97 (d, J ¼ 11.8 Hz, 1H), 3.89 (d, J ¼ 11.7 Hz, 1H).
White solid; yield 72.70%; m.p.: 72.2e74.0 ꢀC. ESI-HRMS(m/z):
found 429.9759 [M þ H]þ(calcd. for C17H15N3Cl2FSe, 429.9792); 1H
NMR (400 MHz, DMSO‑d6)
d
(ppm): 8.41 (s, 1H), 7.92 (s, 1H), 7.58 (d,
13C NMR (101 MHz, DMSO)
d (ppm): 163.2, 161.4, 161.3, 160.7, 160.6,
J ¼ 8.4 Hz, 1H), 7.50 (d, J ¼ 2.2 Hz, 1H), 7.38 (dd, J ¼ 8.4, 2.3 Hz, 1H),
7.35e7.30 (m, 2H), 7.15e7.08 (m, 2H), 5.00e4.79 (m, 2H), 4.72 (dd,
J ¼ 9.4, 6.5 Hz, 1H), 3.92 (d, J ¼ 11.9 Hz, 1H), 3.82 (d, J ¼ 11.8 Hz, 1H).
158.9, 158.8, 152.0, 144.8, 136.8, 133.3, 131.3, 130.8, 130.8, 130.7,
130.7, 130.0, 129.3, 127.8, 123.4, 123.4, 123.3, 123.3, 112.3, 112.2,
112.0, 112.0, 104.8, 104.6, 104.3, 52.6, 35.1, 25.9.
13C NMR (101 MHz, DMSO)
d (ppm): 162.8,160.3,152.0,144.8,136.3,
135.2,135.2,133.8,132.9,131.3, 131.3, 130.5,129.5,128.1,115.8, 115.5,
52.4, 27.5.
4.7.39. 1-(2-((4-chlorobenzyl) selanyl)-2-(2,4-difluorophenyl)
ethyl)-1H-1,2,4-triazole (C11)
Colourless oil; yield 73.03%. ESI-HRMS(m/z): found 414.0088
4.7.34. 1-(2-(2,4-dichlorophenyl)-2-((4-methoxybenzyl) selanyl)
ethyl)-1H-1,2,4-triazole (C06)
[M þ H]þ(calcd. for C17H15N3ClF2Se, 14.0088); 1H NMR (400 MHz,
DMSO‑d6)
d
(ppm): 8.40 (s, 1H), 7.91 (s, 1H), 7.47 (td, J ¼ 8.7, 6.5 Hz,
White solid; yield 83.43%; m.p.: 75.7e77.9 ꢀC. ESI-HRMS(m/z):
found 441.9867 [M þ H]þ(calcd. for C18H18N3Cl2OSe, 441.9892); 1H
1H), 7.36e7.32 (m, 2H), 7.31e7.27 (m, 2H), 7.14 (ddd, J ¼ 10.8, 9.3,
2.7 Hz, 1H), 7.02 (tdd, J ¼ 8.4, 2.6, 1.0 Hz, 1H), 4.90e4.74 (m, 2H),
4.62 (dd, J ¼ 9.3, 6.7 Hz, 1H), 3.90 (d, J ¼ 11.9 Hz, 1H), 3.80 (d,
NMR (400 MHz, DMSO‑d6) d (ppm): 8.41 (s, 1H), 7.92 (s, 1H), 7.57 (d,
J ¼ 8.4 Hz, 1H), 7.51 (d, J ¼ 2.2 Hz, 1H), 7.38 (dd, J ¼ 8.4, 2.2 Hz, 1H),
7.22e7.18 (m, 2H), 6.87e6.83 (m, 2H), 4.95 (dd, J ¼ 13.8, 9.2 Hz, 1H),
4.84e4.71 (m, 2H), 3.88 (d, J ¼ 11.6 Hz, 1H), 3.76 (d, J ¼ 11.6 Hz, 1H),
J ¼ 11.9 Hz, 1H). 13C NMR (101 MHz, DMSO)
d (ppm): 163.1, 163.0,
161.3, 161.2, 160.7, 160.6, 158.8, 158.7, 152.0, 144.8, 138.3, 131.8, 131.1,
130.8, 130.8, 130.7, 130.7, 128.8, 123.5, 123.4, 123.3, 123.3, 112.3,
112.2, 112.1, 112.0, 104.8, 104.5, 104.3, 52.5, 34.6, 27.4.
3.72 (s, 3H). 13C NMR (101 MHz, DMSO)
d (ppm): 158.7, 152.0, 144.8,
136.5, 133.9, 132.8, 130.5, 130.5, 129.5, 128.03, 114.4, 55.6, 52.4, 28.0.
4.7.40. 1-(2-(2,4-difluorophenyl)-2-((4-fluorobenzyl) selanyl)
ethyl)-1H-1,2,4-triazole (C12)
4.7.35. 1-(2-(2,4-dichlorophenyl)-2-((4-methylbenzyl) selanyl)
ethyl)-1H-1,2,4-triazole (C07)
Colourless oil; yield 61.48%. ESI-HRMS(m/z): found 398.0374
Colourless oil; yield 75.40%. ESI-HRMS(m/z): found 426.0016
[M þ H]þ(calcd. for C17H15N3F3Se, 398.0383); 1H NMR (400 MHz,
[M þ H]þ(calcd. for C18H18N3Cl2Se, 426.0043); 1H NMR (400 MHz,
DMSO‑d6)
d
(ppm): 8.40 (s, 1H), 7.92 (s, 1H), 7.47 (td, J ¼ 8.7, 6.4 Hz,
DMSO‑d6)
d
(ppm): 8.39 (s, 1H), 7.91 (s, 1H), 7.57 (d, J ¼ 8.5 Hz, 1H),
1H), 7.3e7.28 (m, 2H), 7.17e7.08 (m, 3H), 7.03 (tdd, J ¼ 8.5, 2.6,
1.0 Hz, 1H), 4.90e4.73 (m, 2H), 4.61 (dd, J ¼ 9.4, 6.8 Hz, 1H), 3.90 (d,
J ¼ 11.8 Hz, 1H), 3.80 (d, J ¼ 11.8 Hz, 1H). 13C NMR (101 MHz, DMSO)
7.50 (d, J ¼ 2.2 Hz, 1H), 7.38 (dd, J ¼ 8.4, 2.2 Hz, 1H), 7.16 (d,
J ¼ 8.1 Hz, 2H), 7.11e7.07 (m, 2H), 4.97e4.89 (m, 1H), 4.83e4.74 (m,
15

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
d
(ppm): 162.7, 160.3, 158.8, 158.7, 152.0, 144.8, 135.3, 135.3, 131.2,
concentrations of B17 and miconazole together to incubate at 35 ꢀC
131.1,130.8,130.7,130.7,130.6,123.5,123.5,123.4,123.4,115.8,115.5,
112.3, 112.2, 112.1, 112.0, 104.8, 104.5, 104.3, 52.5, 34.5, 27.4.
for 24 h. The control group was not treated with compound. At
designated time points, fungal suspension (10
mL) was obtained
from the RPMI 1640 medium and then was10-fold serial diluted by
using 0.9% saline. Then, the diluted fungal suspension was inocu-
lated into PDA plate culture and incubated at 35 ꢀC for 24 h. At last,
the colony forming units (CFU) were counted. The Log10 CFU/mL
was drawn on a chart as a time function. The detection limit of this
method was 100 CFU/mL. The experiment was carried out in
triplicate.
4.7.41. 1-(2-(2,4-difluorophenyl)-2-((4-methoxybenzyl) selanyl)
ethyl)-1H-1,2,4-triazole (C13)
Colourless oil; yield 73.24%. ESI-HRMS(m/z): found 432.0392
[M þ Na]þ(calcd. for
C
18H17N3F2OSeNa, 432.0403); 1H NMR
(ppm): 8.39 (s, 1H), 7.90 (s, 1H), 7.46 (td,
(400 MHz, DMSO‑d6)
d
J ¼ 8.7, 6.5 Hz, 1H), 7.19 (d, J ¼ 8.7 Hz, 3H), 7.03 (tdd, J ¼ 8.5, 2.7,
1.1 Hz, 1H), 6.87e6.82 (m, 2H), 4.88e4.72 (m, 2H), 4.61 (dd, J ¼ 9.4,
6.7 Hz, 1H), 3.86 (d, J ¼ 11.6 Hz, 1H), 3.75 (d, J ¼ 11.8 Hz, 1H), 3.73 (s,
4.11. GC-MS analysis of sterol composition
3H). 13C NMR (101 MHz, DMSO)
d (ppm): 163.1, 162.9, 161.3, 161.2,
160.6, 160.5, 158.9, 158.7, 158.6, 152.0, 144.8, 130.8, 130.7, 130.7,
130.6, 130.6, 130.4, 114.4, 112.2, 112.2, 112.0, 112.0, 104.8, 104.5,
104.3, 55.5, 52.6, 34.5, 27.8.
C. albicans ATCC SC5314 cells were gathered and inoculated in
fresh YEPD (60 mL) with various concentrations of compounds for
16 h at 35 ꢀC. Cells were harvested by centrifuging at 3000 g for
10 min. Subsequently, the cells were washed by using PBS
(3 ꢁ 10 mL) and saponified at 80 ꢀC for 60 min by using saponifi-
cation medium (3 mL, 15% NaOH solution in 90% ethanol). And the
residue was extracted by petroleum ether (3 ꢁ 6 mL). Petroleum
ether was concentrated under reduced pressure, and the residue
was dissolved in hexamethylene for the next determination. The
sterol composition analysis experiment was carried out by GC-MS.
Analyses were carried out in a non-splitting mode by selecting
helium as carrier gas (constant rate of 1.0 mL/min). The GC oven
was programmed as follows: the injector temperature was 250 ꢀC.
Initial temperature was 100 ꢀC, hold for 1 min, ramp to 300 ꢀC at 10
minꢂ1, hold for 10 min.
4.7.42. 1-(2-(2,4-difluorophenyl)-2-((4-methylbenzyl) selanyl)
ethyl)-1H-1,2,4-triazole (C14)
Colourless oil; yield 51.55%. ESI-HRMS(m/z): found 394.0608
[M þ H]þ(calcd. for C18H18N3F2Se, 394.0634); 1H NMR (400 MHz,
DMSO‑d6)
d
(ppm): 8.38 (s, 1H), 7.90 (s, 1H), 7.47 (td, J ¼ 8.7, 6.5 Hz,
1H), 7.17e7.07 (m, 5H), 7.03 (tdd, J ¼ 8.5, 2.6, 1.1 Hz, 1H), 4.90e4.71
(m, 2H), 4.62 (dd, J ¼ 9.5, 6.7 Hz, 1H), 3.87 (d, J ¼ 11.6 Hz, 1H), 3.75
(d, J ¼ 11.6 Hz, 1H), 2.26 (s, 3H). 13C NMR (101 MHz, DMSO)
d (ppm):
163.1, 163.0, 161.3, 161.2, 160.6, 160.5, 158.9, 158.7, 152.0, 144.8,
136.4, 135.8, 130.8, 130.8, 130.7, 130.7, 129.5, 129.2, 123.6, 123.6,
123.5, 123.4, 112.2, 112.2, 112.0, 112.0, 104.8, 104.5, 104.3, 52.5, 34.6,
28.1, 21.1.
4.12. In vitro biofilm formation assay
4.8. In vitro antifungal testing
Fungal suspension of C. albicans (1 ꢁ 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 after1.5 h, 3 h, 6 h and 24 h. Each
well of 96-well plates was washed with PBS (3 ꢁ 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. XTT reduction method was used to
calculate the semi-quantitative determination of the formed
biofilms.
The target compounds were tested for their inhibitory potential
against eight kinds of fungus strains by the micro-broth dilution
assay according to the CLSI M27-A3 guidelines. The RPMI 1640 test
medium (Gibco, USA) was buffered with morpholinepropane-
sulfonic acid (MOPS, Genyiew, USA). The Fungi cells which was in
growing stage were gathered and inoculated in RPMI 1640 medium
to an initial concentration of 1e5 ꢁ 103 CFU/mL. FCZ, MCZ and test
compounds were dissolved in DMSO respectively as the stock so-
lution. After inoculation, the plates inoculated fungal cells were at
35 ꢀC for 24 h, while those C. neo. and A, f. were inoculated at 35 ꢀC
for 72 h. The MICs were defined as the lowest concentration of a
compound that prevents visible growth. All measurements were
made using biological repetition.
4.13. Hemolytic assays
Fresh white rabbit the red blood cells were used in this assay.
Test compounds were dissolved in DMSO as the stock solution. The
red blood cells suspension was prepared by washing the rabbit red
blood cells with PBS buffer solution (three times). After diluting the
RBCs to 2% concentration with PBS buffer solution (2%), the stock
solution was diluted to double volume by employing PBS. Subse-
quently, the diluted solution of test compounds was mixed with the
2% the red blood cells suspension in the 8-well plates. The plates
should be incubated at 37 ꢀC for 3 h, and the mixtures were
centrifuged at 600 g for 5 min. Subsequently, the supernatant was
moved into 96-well plates. The absorbance was tested on a
microplate reader at 575 nm. Meanwhile, the red blood cells with
PBS served as the negative control (0% lysis), and the red blood cells
which was treated with 2% Triton X-100 were used as controls
(100% lysis).
4.9. Evaluation of MFC
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 (CPCC400616) cells which was in
growing stage were gathered and inoculated in fresh RPMI 1640
medium to an initial concentration of 1e5 ꢁ 103 CFU/mL. Micon-
azole and test compounds were dissolved in DMSO respectively as
the stock solution. The samples and fungal suspension were
cultured at 35 ꢀC for 48 h. Next, PDA plates were plated with fungal
suspension (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.
4.14. Cytotoxicity assay
4.10. Time-kill study
After recovery, the cells were cultured at 37 ꢀC in a humidified
atmosphere with 95% air and 5% CO2. The MDA-MB-231 cells were
grown in DMEM replenished with 10% fetal bovine serum, 100 IU/
Fungi suspension of C. albicans (1 ꢁ 105 CFU/mL) were prepared
in RPMI 1640 medium. Taken fungal suspension and different
16

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
mL of penicillin G sodium and 100
mg/mL of streptomycin sulphate.
Acknowledgments
The HL-60 and PC3 cell lines were grown in RPMI 1640 medium
replenished with 10% fetal bovine serum, 100 IU/mL of penicillin G
sodium and 100 mg/mL of streptomycin sulphate. In this experi-
Funding for this work came from the following sources: the
National Natural Science Foundation of China (Grant No.
81903463), the Career Development Support Plan for Young and
Middle-aged Teachers in Shenyang Pharmaceutical University
(Grant No. ZQN2018018) and China Postdoctoral Science Founda-
tion (Grant No. 2020M673042).
ment, different cell lines were seeded at
a density of
1.5e3 ꢁ 103 cells/well in 96-well microtiter plate, 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
Appendix A. Supplementary data
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.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113337.
4.15. Microsomal stability assays
Abbreviations
The microsomal stability was performed by WuXi AppTec
(https://www.wuxiapptec.com/zh-cn).
C.alb.
Candida albicans (CPCC400616)
C.alb (SC5314) Candida albicans (ATCC SC5314)
C.zey.
C.neo.
C.kru.
C.gla.
C.par.
A.f.
Candida zeylanoides (CGMCC2.3739)
Cryptococcus neoformans (CGMCC2.3161)
Candida kruseii (AS 2.1045)
Candida glabrata (Clinical isolation)
Candida parapsilosis (ATCC 22019)
Aspergillus fumigatus (CGMCC 3.7795)
4.16. In vivo antifungal assay
Mice were assigned into four groups: the control group, the
fluconazole treatment group (0.6 mg/kg/day), the A03 treatment
group (0.6 mg/kg/day) and two B17 treatment group (0.4 and
0.6 mg/kg/day, respectively). Mice in each group were given
strain 17# fluconazole-resistant strain of C. albicans
strain CaR fluconazole-resistant strain of C. albicans
strain 632 fluconazole-resistant strain of C. albicans
strain 901 fluconazole-resistant strain of C. albicans
strain 904 fluconazole-resistant strain of C. albicans
FCZ
MCZ
T1/2
200
m
L C. albicans ATCC SC5314 (5 ꢁ 106 CFU/mL) of by the tail vein.
Fluconazole, A03 and B17 treatment were initiated by intraperito-
neal injection at 2 h after infection. The treatments were admin-
istered daily for 4 days. The kidneys of the mice were removed,
weighed and homogenized. To determine the efficacy of therapy in
murine models, a determination of renal fungal burden as CFU is
commonly used.
Fluconazole
Miconazole
Half-life
Clearance
CL
SMIC50
sessile minimum inhibitory concentration that reduced
the metabolic activity of biofilms by 50%
sessile minimum inhibitory concentration that reduced
the metabolic activity of biofilms by 80%
3-bis (2-hydroxyethylthio) naphthalene-1,4-dione.
4.17. Pharmacokinetic assay
SMIC80
XTT
Six rats fasted for 12 h before the experiment, and had free ac-
cess to water. Orbital blood samples were collected in heparinized
tubes before dosing and at 5 min (only iv), 15 min, 30 min, 1 h, 2 h,
3 h, 4 h, 8 h, 12 h and 24 h after administration. Plasma samples
were harvested immediately by centrifugation of the rat blood at
6000 rpm for 10 min and stored at ꢂ20 ꢀC until analysis. LC-MS/MS
method was used for quantitative determination, and the data were
analyzed by DAS 2.0 (Chinese Pharmacological Society).
References
[1] T. Roemer, D.J. Krysan, Antifungal drug development: challenges, unmet
clinical needs, and new approaches, Csh. Perspect. Med. 4 (2014) a019703-
a019703.
[2] S. Dominique, Emerging Threats in antifungal-resistant fungal pathogens,
Front. Med. 3 (2016) 11.
[3] S. Antinori, L. Milazzo, S. Sollima, M. Galli, M. Corbellino, Candidemia and
invasive candidiasis in adults: a narrative review, Eur. J. Intern. Med. 34 (2016)
21e28.
[4] N.P. Wiederhold, Antifungal resistance: current trends and future strategies to
combat, Infect. Drug Resist. 10 (2017) 249e259.
[5] M.W. Mccarthy, T.J. Walsh, Drug development challenges and strategies to
address emerging and resistant fungal pathogens, Expert Rev. Anti Infect.
Ther. 15 (2017) 577e584.
[6] D.S. Perlin, E. Shor, Y. Zhao, Update on antifungal drug resistance, Curr. Clin.
Micro. Rpt. 2 (2015) 84e95.
4.18. Docking analysis
The crystal structure of CYP51 (PDB code 5tz1) was obtained
from the Protein Data Bank. The 3D structures of the test com-
pounds were generated in ChemBio 3D Ultra 14.0. Test compounds
were docked into the target proteins after removing hetero atoms,
water molecules and co-factors by using Discovery Studio 3.0.
[7] E.L. Berkow, S. R Lockhart, Fluconazole resistance in Candida species: a current
perspective, Infect. Drug Resist. 10 (2017) 237e245.
Declaration of competing interest
[8] M.K. Kathiravan, A.B. Salake, A.S. Chothe, P.B. Dudhe, R.P. Watode, M.S. Mukta,
S. Gadhwe, The biology and chemistry of antifungal agents: a review, Bioorg.
Med. Chem. 20 (2012) 5678e5698.
We declare that we have no financial and personal relationships
with other people or organizations that can inappropriately influ-
ence our work, there is no professional or other personal interest of
any nature or kind in any product, service and/or company that
could be construed as influencing the position presented in, or the
review of, the manuscript entitled, “Lead optimization generates
selenium-containing miconazole CYP51 inhibitors with
improved pharmacological profile for the treatment of fungal
infections” for possible publication in “European Journal of Me-
dicinal Chemistry”.
[9] N. Liu, J. Tu, G. Dong, Y. Wang, C. Sheng, Emerging new targets for the
treatment of resistant fungal infections, J. Med. Chem. 61 (2018) 5484e5511.
[10] R. Wu, T. Ren, J. Gao, L. Wang, Q. Yu, Z. Yao, G. Song, W. Ruan, C. Niu, F. Song,
L. Zhang, M. Li, J. Wang, Chemical preparation, biological evaluation and 3D-
QSAR of ethoxysulfuron derivatives as novel antifungal agents targeting
acetohydroxyacid synthase, Eur. J. Med. Chem. 162 (2019) 348e363.
[11] K.A. Agnew-Francis, Y. Tang, X. Lin, Y. Low, S. Wun, A. Kuo, S.M.A.S.I. Elias,
T. Lonhienne, N.D. Condon, B.N.A.S. Pimentel, C.E. Vergani, M.e T. Smith,
J.A. Fraser, C.M. Williams, L.W. Guddat, Herbicides that target acetohydrox-
yacid synthase are potent inhibitors of the growth of drug-resistant Candida
auris, ACS Infect. Dis. 6 (2020) 2901e2912.
17

H. Xu, Z.-z. Yan, M.-b. Guo et al.
European Journal of Medicinal Chemistry 216 (2021) 113337
[12] G.I. Lepesheva, L. Friggeri, M.R. Waterman, CYP51 as drug targets for fungi and
protozoan parasites: past, present and future, Parasitology 145 (2018) 1e17.
[13] B.C. Monk, A.A. Sagatova, P. Hosseini, Y.N. Ruma, R.K. Wilson, M.V. Keniya,
biological evaluation of novel miconazole analogues containing selenium as
potent antifungal agents, Eur. J. Med. Chem. 198 (2020), 112360.
[21] H. Xu, Y. Hu, M. Guo, A. Huang, X. Su, C. Guo, Design, synthesis and antifungal
activity of novel selenochroman-4-one derivatives, Chem. Pap. 71 (2017)
2455e2463.
[22] H. Xu, X. Su, X. Liu, K. Zhang, Z. Hou, C. Guo, Design, synthesis and biological
evaluation of novel semicarbazone selenochroman-4-ones hybrids as potent
antifungal agents, Bioorg. Med. Chem. Lett 29 (2019), 126726.
[23] C. Ji, N. Liu, J. Tu, Z. Li, G. Han, J. Li, C. Sheng, Drug repurposing of haloperidol:
discovery of new benzocyclane derivatives as potent antifungal agents against
cryptococcosis and candidiasis, ACS Infect. Dis. 6 (2020) 768e786.
[24] L. Zhao, L. Tian, N. Sun, Y. Sun, Y. Chen, X. Wang, S. Zhao, X. Su, D. Zhao,
M. Cheng, Design, synthesis, and structure-activity relationship studies of L-
amino alcohol derivatives as broad-spectrum antifungal agents, Eur. J. Med.
Chem. 177 (2019) 374e385.
Fungal Lanosterol 14a-demethylase: a target for next-generation antifungal
design, Biochim. Biophys. Acta 1868 (2020), 140206.
[14] R.C. Heel, R.N. Brogden, G.E. Pakes, T.M. Speight, G.S. Avery, Miconazole: a
preliminary review of its therapeutic efficacy in systemic fungal infections,
Drugs 19 (1980) 7e30.
[15] J. Zhang, L. Li, Q. Lv, L. Yan, Y. Wang, Y. Jiang, The fungal CYP51s: their
functions, structures, related drug resistance, and inhibitors, Front. Microbiol.
10 (2019).
[16] J. Song, S. Zhang, L. Lu, Fungal cytochrome P450 protein Cyp51: what we can
learn from its evolution, regulons and Cyp51-based azole resistance, Fungal
Biol. Rev. 32 (2018) 131e142.
[17] Y. Zhang, G.L.V. Damu, S. Cui, J. Mi, V.K.R. Tangadanchu, C. Zhou, Discovery of
potential antifungal triazoles: design, synthesis, biological evaluation, and
[25] J. Tessier, M. Golmohamadi, K.J. Wilkinson, A.R. Schmitzer, Anti-staphylo-
coccal biofilm activity of miconazoctylium bromide, Org. Biomol. Chem. 16
(2018) 4288e4294.
preliminary antifungal mechanism exploration, Medchemcomm
8 (2017)
1631e1639.
[18] P. Nenoff, D. Koch, C. Krüger, C. Drechsel, P. Mayser, New insights on the
antibacterial efficacy of miconazole in vitro, Mycoses (2017) 1e6.
[26] S. Wu, Y. Wang, N. Liu, G. Dong, C. Sheng, Tackling fungal resistance by biofilm
inhibitors, J. Med. Chem. 60 (2017) 2193e2221.
[19] D. Allen, D. Wilson, R. Drew, J. Perfect, Azole antifungals: 35 years of invasive
fungal infection management, Expert Rev. Anti-Infe 13 (2015) 787e798.
[20] H. Xu, X. Su, M. Guo, R. An, Y.M.Z. Hou, C. Guo, Design, synthesis, and
[27] I.B. Rosseti, J.B. Rocha, M.S. Costa, Diphenyl diselenide (PhSe)2 inhibits biofilm
formation by Candida albicans, increasing both ROS production and mem-
brane permeability, J. Trace Elem. Med. Biol. 29 (2015) 289e295.
18