Design, synthesis and evaluation of biphenyl imidazole analogues as potent antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2019.07.037

To further explore the structure activity relationships (SARs) of our previously discovered antifungal lead compound (1), a series of biphenyl imidazole analogues were designed, synthesized and evaluated for their in vitro antifungal activity. Many of the

Full text of DOI:10.1016/j.bmcl.2019.07.037

Accepted Manuscript
Design, synthesis and evaluation of biphenyl imidazole analogues as potent an-
tifungal agents
Shizhen Zhao, Liyu Zhao, Xiangqian Zhang, Peng Wei, Mengya Wu, Xin Su,
Bin Sun, Dongmei Zhao, Maosheng Cheng
PII:
S0960-894X(19)30493-7
https://doi.org/10.1016/j.bmcl.2019.07.037
BMCL 26578
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
13 February 2019
19 June 2019
22 July 2019
Please cite this article as: Zhao, S., Zhao, L., Zhang, X., Wei, P., Wu, M., Su, X., Sun, B., Zhao, D., Cheng, M.,
Design, synthesis and evaluation of biphenyl imidazole analogues as potent antifungal agents, Bioorganic &
Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.07.037
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Design, synthesis and evaluation of biphenyl imidazole analogues as
potent antifungal agents
Shizhen Zhaoa,c, Liyu Zhaoa, Xiangqian Zhanga, Peng Weia, Mengya Wua, Xin Sub, Bin Sund,
Dongmei Zhaoa,*, Maosheng Chenga
aKey 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, PR China
bThe School of life Science and Biopharmaceutical, Shenyang Pharmaceutical University, 103
Wenhua Road, Shenhe District, Shenyang, 110016, PR Chinac
cKey Laboratory of Receptors-Mediated Gene Regulation and Drug Discovery, School of
Medicine, Henan University, Kaifeng, China
dInstitute of BioPharmaceutical Research, Liaocheng University, 1 Hunan Road, Liaocheng
252000, PR China
*Corresponding author: Dongmei Zhao, E-mail: medchemzhao@163.com
ABSTRACT:
To further explore the structure activity relationships (SARs) of our previously discovered
antifungal lead compound (1), a series of biphenyl imidazole analogues were designed,
synthesized and evaluated for their in vitro antifungal activity. Many of the synthesized
compounds showed excellent activity against Candida albicans and Candida tropicalis. Among
these compounds, 2-F substituted analogue 12m displayed the most remarkable in vitro activity
against C. albicans, C. neoformans, A. fumigatus and fluconazole-resistant C.alb. strains, which is
superior or comparable to the activity of the reference drugs fluconazole and itraconazole. Notably,
the compound 12m exhibited low inhibition profiles for various human cytochrome P450 isoforms
and showed low toxicity to mammalian A549 cells and U87 cells. The SARs and binding mode
established in this study will be useful for further lead optimization.
KEYWORDS:
Antifungal activity, CYP51, Structure-activity relationship, Azole antifungals
Over the past decades, the incidence of fungal infections has been steadily increasing and
1

presents a serious threat to human health1. Especially, the invasive fungal infections (IFIs) has
greatly increased, leading to high morbidity and mortality in immunocompromised individuals
including patients with AIDS and those receiving immunomodulatory therapies and organ
transplants2-5. There are three major species of human fungal pathogens responsible for the IFIs
including Candida albicans(mortality rate 20-40%), Cryptococcus neoformans(mortality rate
20-70%), and Aspergillus fumigatus(mortality rate 50-90%)6, 7.
The therapeutic antifungal drugs available for systemic clinical use can be divided into four
different classes based on their mode of action: including polyenes (e.g. amphotericin B and
Nystatin), nucleoside analogues (e.g. 5-fluorocytosine), echinocandins (e.g. caspofungi and
micafungin) and azoles(e.g. fluconazole and itraconazole)8, 9. Amphotericin B is a broad-spectrum
antifungal agent with good therapeutic effects, but nephrotoxicity and other serious side effects
restict its use10, 11. Echinocandins have potent antifungal activities but low oral absorption12.
Among these agents, azoles antifungal drugs, such as fluconazole, itraconazole and voriconazole,
are widely used as first-line antifungal therapy (Figure 1). However, several factors have limited
their clinical applications, such as drug resistance, narrow antifungal spectrum, and low
bioavailability13, 14. Therefore, discovering novel, safer and more effective azole antifungal agents
has great value in study and application.
Figure 1. Chemical structures azole antifungal agents and lead compounds.
In our previous studies, a series of biphenyl imidazole derivatives showed excellent
antifungal activity against Candida albicans and Cryptococcus neoformans, with MIC values in
the range of 0.25 µg/mL to 2 µg/mL.15 However, almost all of the target compounds were inactive
2

against Aspergillus fumigatus, which prompted us to continue studying the structural modification
of our potent original compounds to improved anti-Aspergillus fumigatus efficacy. Our previous
study also identified that compounds aromatic isoxazole (2) and 4,5-dihydronaphtho[2,1-d]
isoxazole (3) displayed the most remarkable in vitro activity against A. fumigatus16, 17. Molecular
docking suggests that the reason may be the formation of the hydrogen bonds with between
isoxazole ring of compounds and A. fumigatus CYP51 Tyr122 (Figure 2).
Figure 2. The binding mode of compound 2 and compound 3 in the active site of A. fumigatus
CYP51B(PDB:4YUM).
To further explore the potent and broad antifungal spectrum of biphenyl derivatives,
compound 1 was docked into the active site of A. fumigatus CYP51B (PDB ID:4UYM)16. Based
on the interactions between compound 1 and A. fumigatus CYP51, the middle benzene ring of
compound 1 was investigated with various substituents to get insights into the structure−activity
relationships (SARs) and improved anti-Aspergillus efficacy.
The target compounds 12a-v were synthesized according to our previously reported
procedure which were outlined in Scheme 1 and Scheme 215, 18. Firstly the key intermediates 8a-l
were synthesized by Suzuki coupling. Commercially available the substituted 4-bromobenzoic
acid 6a-h and the substituted phenylboronic acids 7a-c were subjected to a Suzuki coupling in the
presence of Pd(PPh3)4 to afford the key intermediates 8a-l.
3

Scheme 1. Synthesis of intermediates 8a-l. Reagents and conditions: (a) Pd(PPh3)4, K2CO3, reflux, 5h.
Secondly, the L-serine esters 10a-b were synthesised by L-serine and alcohols (isopropanol,
isobutanol) in the presence of SOCl2. The L-serine esters 10a-b were then treated with
intermediates 8a-l in the presence of a condensing agent to give intermediates 11a-v. Finally, the
target compounds 12a-v were successfully obtained by introducing the imidazole group of
intermediates 11a-z using CDI/imidazole.
Scheme 2. Synthesis of target compounds 12a-v. Reagents and conditions: (a) alcohol reagent, SOCl2, reflux, 1–2
h; (b) EDCI, HOBt, DIEA, r.t., 7 h; (c) CDI, imidazole, CH3CN, reflux, 7 h.
The in vitro antifungal activities of target compounds were determined against five important
fungal pathogens according to the protocols from the NCCLS18, 19. The broth microdilution
method was used to determine the minimum inhibitory concentration (MIC) of the target
compounds in 96-well microtest plates. Fluconazole (FLC) and Itraconazole (ITR) were used as
reference drugs.
The in vitro antifungal activities of the target compounds are summarized in Table 1. The
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results showed that many of these compounds exhibited moderate to good antifungal activity with
broad spectrums of activity. Compounds 12i-j, 12m-p and 12s-v showed the most potent activity
against C. albicans and C. tropicalis with MIC values in the range of 0.03125 to 2 µg/mL. Among
these compounds, 2-F substituted analogue 12m displayed the most remarkable in vitro activity
against all of the tested strains, with the exception of Aspergillus fumigatus, and was superior or
comparable to those of the reference drugs FLC and ITR.
Table 1
In vitro antifungal activities of the target compounds (MIC, μg/mL)a.
Compd.
12a
R1
R2
R3
C. alb.(Ⅰ)
C. alb(Ⅱ).
C. neo.
C.tro.
A. fum.
3-CH3
H
-CH(CH3)2
>16
2
>16
0.5
>16
3-CH3
3-OCH3
3-OCH3
2-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
-CH2CH(CH3)2
-CH(CH3)2
>16
8
2
>16
>16
>16
8
0.5
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
2
12b
12c
12d
12e
12f
1
1
-CH2CH(CH3)2
CH(CH3)2
>16
8
4
2
1
8
2-Cl
-CH2CH2CH3
CH(CH3)2
4
8
>16
8
0.25
8
2-CH3
2-CH3
3,5-F2
3,5-F2
3-Cl
12g
12h
12i
>16
>16
0.5
0.5
4
>16
>16
>16
>16
8
-CH2CH(CH3)2
-CH(CH3)2
>16
4
2
0.5
-CH2CH(CH3)2
-CH(CH3)2
2
2
12j
12k
12l
8
0.5
3-Cl
-CH2CH(CH3)2
-CH(CH3)2
4
8
8
0.25
0.03125
0.03125
0.03125
0.03125
2-F
0.5
0.25
0.25
0.25
0.03125
0.03125
0.03125
0.03125
8
12m
12n
12o
12p
2-F
-CH2CH(CH3)2
-CH(CH3)2
>16
>16
>16
4
3-F
4
3-F
-CH2CH(CH3)2
8
5

3-F
3-F
3-F
3-F
2-F
2-F
-
4-CN
4-CN
2-F
-CH(CH3)2
-CH2CH(CH3)2
-CH(CH3)2
-CH2CH(CH3)2
CH(CH3)2
-CH2CH(CH3)2
-
12q
12r
8
4
>16
>16
>16
>16
>16
8
2
0.5
>16
>16
>16
>16
>16
>16
>16
4
>16
0.5
0.125
0.125
0.03125
0.0625
1
0.25
0.25
0.03125
0.0625
1
12s
2-F
12t
0.5
2-F
0.0625
0.25
0.5
12u
12v
FCZ
ITZ
2-F
>16
4
-
-
0.0625
0.25
1
0.5
aAbbreviations: C.alb.(I), Candida albicans (ATCC SC5314); C.alb.(II), Candida albicans (CPCC400523); C.
neo., Cryptococcus neoformans (cgmcc 2.3161); A.fum., Aspergillus fumigatus (cgmcc 3.7795); C.tro., Candida
tropicalis (cgmcc 2.3739); FCZ: Fluconazole; ITZ: Itraconazole.
The fluconazole is the most widely used today and can seriously increase the problem of
azole resistance for treating fungal infections20
. Based on the results of in vitro antifungal
activity assays, the most potent compounds 12m, 12o and 12u-v were further evaluated against
fluconazole-resistant strains of C. alb. (strain 100 and strain 103). As shown in Table 2,
compounds 12m and 12v showed moderate antifungal activities against fluconazole-resistant
strains of C. alb. with MIC values in the range of 2 to 8 µg/mL.
Table 2
In vitro antifungal activities of the target compounds (MIC, μg/mL) a.
C. alb.
Compd.
R1
R2
Strain100
Strain103
2-F
3-F
3-F
3-F
-
H
H
8
16
16
8
2
4
12m
12o
2-F
2-F
-
2
12u
12v
4
>64
>64
FCZ
aAbbreviations: C.alb., Candida albicans; strain 100, fluconazole-resistant strains of Candida albicans; strain 103,
fluconazole-resistant strains of Candida albicans; FCZ: Fluconazole.Strain 100 and strain 103 were provided by
The Second Military Medical University.
Cytochrome P450 (CYP) enzymes play an important role in phase I of xenobiotic
biotransformation. Drug-drug interactions (DDIs) caused by inhibition of cytochrome P450
enzymes can result in dangerous side effects, therefore it is important to evaluate the CYP
6

inhibition of our compounds. The most potent compounds 12m and 12o were tested against the
five major human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4-M). As
shown in Table 3, compound 12m showed no inhibitory activity against CYP1A2, CYP2C9,
CYP2C19, and CYP2D6, whereas showed weak activity against CYP3A4 with IC50 values 4.05
μM. However, compound 12o showed strong inhibition against CYP3A4 with IC50 values 0.0429
μM.
Table 3.
In vitro CYP inhibition assessment of compounds.
IC50 (μM)
Compd.
CYP1A2
CYP2C9
CYP2C19
CYP2D6
CYP3A4-M
>50
>50
>50
43.4
7.44
>50
4.05
12m
12o
4.49
19.1
0.0429
The compounds designed to target fungal cells may cause unwanted toxicity to mammalian
cells. Therefore, it is important to evaluate the toxicity of the compounds to mammalian cells by in
WTT assays. We tested the most potent compounds 12m and 12o against A549 cells and U87 cells.
As shown in Table 4, compounds 12m and 12o exhibited low toxicity in A549 cells and U87 cells
(IC50>50μM).
Table 4
In vitro cytotoxicity of compounds on A549 cells and U87 cells.
IC50 (μM)
Compd.
U87
A549
>50
>50
>50
12m
12o
>50
The structure-activity relationship between various substituents on the middle aromatic ring
was investigated (compounds 12a-p). Compounds with electron-donating groups at the 2-position
or 3-position, such as -CH3 (12a-b and 12g-h) and -OCH3 (12c-d) moieties, resulted in a
significant decrease in antifungal activity. Compounds with halogens such as -F and –Cl (12e-f
and 12k-p) at the 2-position or 3-position, exhibited higher antifungal activity than those with
electron-donating groups. Among these, compounds 12m-p with 2-F or 3-F substituents showed
the most potent activity against all of the tested strains, with the exception of Aspergillus
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fumigatus. Moreover, Compounds 12i-j with multi-halogens substituents showed no advantage.
Based on the results above, we selected 2-F or 3-F substituents compounds as our starting
point for further modification to expand the SAR studies. In vitro antifungal activity tests have
shown that compounds with substituent (2-F and 4-CN) on the terminal benzene ring led to only a
slight decrease in antifungal activity.
To better understand the binding mode of compound 12m, a highly potent compound,
docking simulations were carried out using the CDOCKER program in the Discovery Studio 3.0
software. The published X-ray structure of A. fumigatus CYP51(PDB ID:4UYM, Figure 3) served
as a useful template for generating binding modes. Images depicting the proposed binding modes
were generated using PyMOL. As shown in Figure 6, the imidazole ring of compound 12m
coordinated the iron in the heme group, and the alkyl ester formed a hydrophobic interaction with
Val135, Ala303and Ala307. The biphenyl side chain extended into CYP51 channel could form
favourable van der Waals and hydrophobic interactions with the surrounding residues Ile373,
Leu503 and Phe504. Interestingly, the 2-F substituent of compound 12m could form a hydrogen
bond with the –OH of A. fumigatus Tyr122. Binding studies may provide a good explanation for
the excellent in vitro antifungal activity against A. fumigatus of compound 12m.
Figure 3. The binding mode of compounds 12m in the active site of A. fumigatus CYP51 (PDB ID: 4UYM).
Based on our laboratory´s work we designed and synthesed a series of biphenyl imidazole
derivatives and their in vitro antifungal activity were evaluated. Many of these compounds
exhibited moderate to good antifungal activity against C. albicans and C. tropicalis with MIC
values in the range of 0.03125 to 2 µg/mL. Among these, 2-F or 3-F substituted analogues 12m-p
displayed the most remarkable in vitro activity against all of the tested strains, with the exception
8

of Aspergillus fumigatus, and was superior or comparable to those of the reference drugs FLC and
ITR. Notably, the compound 12m had weak inhibition for various human cytochrome P450
isoforms, which indicated they had a low potential to cause DDIs. Compounds 12m and 12o were
almost nontoxic to mammalian A549 cells and U87 cells. The SARs and binding mode established
in this study will be useful for further lead optimization.
Acknowledgements
The authors thank Prof. Yongbing Cao from School of Pharmacy, the Second Military
Medical University, for providing the fluconazole-resistant strains of Candida albicans(strain 100
and strain 103). This work was supported by Program for Innovative Research Team of the
Ministry of Education and Program for Liaoning Innovative Research Team in University.
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Highlights




22 new compounds with biphenyl imidazole scaffolds were designed and synthesized.
Compound 12m showed better antifungal activity than fluconazole.
The structure-activity relationships of compounds were discussed.
Compound 12m exhibited low inhibition profiles for human cytochrome P450 isoforms.
12