Design, synthesis, and in vitro evaluation of novel triazole analogues featuring isoxazole moieties as antifungal agents

Article abstract of DOI:10.1016/j.bioorg.2020.103982

In order to develop novel antifungal agents, based on our previous work, a series of (2R,3R)-3-((3-substitutied-isoxazol-5-yl)methoxy)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl) butan-2-ol (a1-a26) were designed and synthesized. All of the compounds exhibited good in vitro antifungal activities against eight human pathogenic fungi. Among them, compound a6 showed excellent inhibitory activity against Candida albicans and Candida parasilosis with MIC₈₀ values of 0.0313 μg/mL. In addition, compounds a6, a9, a12, a13 and a14 exhibited moderate inhibitory activities against fluconazole-resistant isolates with MIC₈₀ values ranging from 8 μg/mL to 16 μg/mL. Furthermore, compounds a6, a12 and a23 exhibited low inhibition profiles for CYP3A4. Clear SARs were analyzed, and the molecular docking experiment was carried out to further investigate the relationship between a6 and the target enzyme CYP51.

Full text of DOI:10.1016/j.bioorg.2020.103982

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Design, synthesis, and in vitro evaluation of novel triazole analogues featur-
ing isoxazole moieties as antifungal agents
Fei Xie, Tingjunhong Ni, Zichao Ding, Yumeng Hao, Ruina Wang, Ruilian
Wang, Ting Wang, Xiaoyun Chai, Shichong Yu, Yongsheng Jin, Yuanying
Jiang, Dazhi Zhang
PII:
S0045-2068(20)31279-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.103982
YBIOO 103982
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Bioorganic Chemistry
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23 May 2020
29 May 2020
Please cite this article as: F. Xie, T. Ni, Z. Ding, Y. Hao, R. Wang, R. Wang, T. Wang, X. Chai, S. Yu, Y. Jin, Y.
Jiang, D. Zhang, Design, synthesis, and in vitro evaluation of novel triazole analogues featuring isoxazole
moieties as antifungal agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103982
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Design, synthesis, and in vitro evaluation of novel
triazole analogues featuring isoxazole moieties as
antifungal agents
Fei Xiea,†, Tingjunhong Nib,†, Zichao Dinga,†, Yumeng Haoa, Ruina Wangc, Ruilian
Wangc, Ting Wanga, Xiaoyun Chaia, Shichong Yua, Yongsheng Jina, Yuanying
Jiangb,*, Dazhi Zhanga,*
a
Department of Organic Chemistry, School of Pharmacy, Second Military Medical University,
325 Guohe Road, Shanghai 200433, China
b Shanghai Tenth People's Hospital, Tongji University School of Medicine, 301 Middle Yanchang
Road, Shanghai 200072, China
c
Center of New Drug research, School of Pharmacy, Second Military Medical University, 325
Guohe Road, Shanghai 200433, China
† These authors contributed equally to this work.
* Corresponding Author
Jiang Y. Tel.: +86-021-81871357, E-mail address: 13761571578@163.com
Zhang D. Tel.: +86-021-81871226, E-mail address: zdzhang_yjhx@smmu.edu.cn
Abstract: In order to develop novel antifungal agents, based on our previous work, a series of
(2R,3R)-3-((3-substitutied-isoxazol-5-yl)methoxy)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)
butan-2-ol (a1-a26) were designed and synthesized. All of the compounds exhibited good in vitro
antifungal activities against eight human pathogenic fungi. Among them, compound a6 showed
excellent inhibitory activity against Candida albicans and Candida parasilosis with MIC80 values
of 0.0313 μg/mL. In addition, compounds a6, a9, a12, a13 and a14 exhibited moderate inhibitory
activities against fluconazole-resistant isolates with MIC80 values ranging from 8 μg/mL to 16
μg/mL. Furthermore, compounds a6, a12 and a23 exhibited low inhibition profiles for CYP3A4.
Clear SARs were analyzed, and the molecular docking experiment was carried out to further
investigate the relationship between a6 and the target enzyme CYP51.
Keywords: Triazole; Antifungal activity; Structure-activity relationship; Synthesis

1. Introduction
During the last three decades, invasive fungal infections (IFIs) continue to pose a serious
threat to human health, especially among people with HIV infection, organ transplant, cancer and
autoimmune diseases1. More than 90% of all reported fungal-related deaths mainly result from
three genera: candidiasis (Candida albicans, mortality: 46%-75%), cryptococcosis (Cryptococcus
neoformans, mortality: 20%-70%), and aspergillosis (Aspergillus fumigates, mortality:
30%-90%)2. Clinically, treatment for IFIs contains three major classes of drugs which include
azoles, polyenes and echinocandins3. However, with the continuous emergence of drug resistance
and the undesired side effects on existing available antifungal drugs, new effective agents with
improved profiles are urgently needed4.
Triazoles are the most used antifungal agents due to their high therapeutic index. Since
fluconazole was launched in 1988, followed by itraconazole (1989), voriconazole (2002),
posaconazole (2005) and isavuconazole (2015) (Fig.1), more than 20 antifungal “conazoles” have
been approved by the FDA in the past 50 years. They target and act by inhibiting the CYP51
(cytochrome P450 14α-demethylase), a necessary enzyme in the biosynthesis of ergosterol5. SAR
studies6-8 of these antifungal triazoles revealed a common pharmacophore which contained a
triazole ring linked to a dihalophenyl ring through a two-carbon chain, and the carbon alpha to the
phenyl ring bore a hydroxyl group. The difference between fluconazole and other azoles mainly
lied in the optimization of the side chains attached to the pharmacophore.
In our previous work9 (Fig.1), isoxazole moiety was introduced into the side chain by
replacing the thiazole ring of ravuconazole and isavuconazole, respectively. Results indicated that
2,4-difluorophenyl is better than 2,5-difluorophenyl, and most of the designed compounds
exhibited potent in vitro antifungal activities against nearly all tested fungi. The introduction of
isoxazole may contribute to the increased efficacy and decreased toxicity, improving some
pharmacokinetics profiles10-12. In this paper, we retained the isoxazole and 2,4-difluorophenyl
moieties and meanwhile extended the side chain properly by adding an ether bond for better
interaction with CYP51 channel residues. Herein, a series of isoxazole methoxyl motif containing
triazoles were rationally designed and synthesized, and their in vitro antifungal activities were
evaluated.

O
N
N
N
N
O
O
N
N
N
N
OH
N
N
N
N
O
N
Cl
F
N
Cl
F
Fluconazole
Itraconazole
O
N
N
N
N
O
O
N
N
N
N
F
OH
OH
N
N
N
F
F
N
N
F
F
Voriconazole
Posaconazole
N
N
N
N
N
N
N
N
N
OH
OH
OH
N
CN
Previous
work
This
work
O
S
O
N
F
O
N
R
R
R1
R1
R2
R2
F
Isoxazole methoxyl moiety
containing triazoles analogues
a1-a26
Isoxazole containing
triazole analogues
Ravuconazole: R1, R2 = 2, 4-diF
Isavuconazole: R1, R2 = 2, 5-diF
Fig.1. Structures of triazole antifungal agents and the designed compounds.
2. Chemistry
The general synthetic methodology for the preparation of the target compounds (a1-a26) is
depicted in Scheme 1. As a key intermediate of our designed compounds, the diol 1 was
synthesized according to the literature13. X-ray crystallographic analysis clearly supports the chiral
structure of alkyne 2 to construct a1–a26 (Fig.2, Table S1 in the supplementary material). The
intermediates 5 were prepared by the chlorination of benzaldehyde oximes 4 which were obtained
by the hydroxylamination of various substituted benzaldehydes 3. Then, intermediates 5 were
reacted with alkyne 2 by the 1,3-dipolar cycloaddition to form a1-a26. Details of all procedure and
data are described in supporting information.

N
N
N
N
N
N
OH
OH
a1 R = 2-Cl
a3 R = 4-Cl
a5 R = 3-F
a2 R = 3-Cl
a4 R = 2-F
a6 R = 4-F
O
OH
O
i
F
F
O
a7 R = 2-CH3
a9 R = 4-CH3
a11 R = 4-OCF3
a13 R = 2-F-4Cl
a15 R = 3-F-4Cl
a17 R = 2, 3-diF
a19 R = 2, 5-diF
a21 R = 3, 4-diF
a23 R = 2, 4, 6-triF
a8 R = 3-CH3
a10 R = 4-CF3
a12 R = 4-CN
a14 R = 2-Cl-4F
a16 R = 3-Cl-4F
a18 R = 2, 4-diF
a20 R = 2, 6-diF
a22 R = 3, 5-diF
a24 R = 2, 3, 4, 5-4F
N
OH
N
N
F
1
iv
F
2
OH
O
F
O
N
R
OH
iii
OH
Cl
O
N
N
F
ii
R
R
R
a25 R = 2, 3, 4, 5, 6-5F a26 R = H
Target compounds
3
4
5
Scheme 1. Reagents and conditions: (i) 3-bromoprop-1-yne, Cs2CO3, CH3CN, 80℃, 12 h; (ii)
.
NH2OH HCl, NaHCO3, H2O, methanol, r.t., 2 h; (iii) N-chlorosuccinimide, DMF, 35℃, 2 h; (iv)
Et3N, ZnCl2, THF, 35℃, 16-20 h.
Fig.2. Single-crystal structure of alkyne 2.
3. Pharmacology
The in vitro antifungal activities of a1-a26 were evaluated against eight human pathogenic
fungi inculding Candida albicans, Candida parapsilosis, Cryptococcus neoformans, Candida
glabrata, Aspergillus fumigatus, Trichophyton rubrum and Microsporum gypseum. The minimum
inhibitory concentration (MIC) determination was measured by serial dilution method in 96-well
microtest plates with RPMI 1640 as the test medium according to the protocols recommended by
National Committee for Clinical Laboratory Standards (NCCLS, USA)14. The MIC80 was defined
as the first well with an approximate 80% reduction in growth compared with the drug-free well.
For assays, the target compounds to be tested were dissolved in dimethyl sulfoxide (DMSO),
serially diluted in growth medium, inoculated and incubated at 35 °C. Growth MIC80 was
determined at 24 h for C. albicans, at 72 h for C. neoformans, and at 5-7 days for three

filamentous fungi. Fluconazole (FCZ) and ravuconazole (RCZ) served as positive controls. The
results of assays are summarized in Table 1 and Table 2. All of our susceptibility tests were
evaluated twice by each antifungal agent.
Compounds a6, a12, a23 were tested to assess in vitro potency in CYP3A4 enzyme assays.
The 50% inhibitory concentration (IC50) values for CYP3A4 enzyme were determined in human
hepatocyte microsomes using 80 μM testosterone as substrate (a concentration equal to the Km
determined in the same lab). Reactions were analyzed for products using LC-MS/MS methods,
and IC50 values (in μM) were determined by fitting a 4-parameter logistical fit to the dose
response data. The results of assays are summarized in Table 3. All of our tests were evaluated
twice by each antifungal agent.

Table 1 In vitro antifungal activities of the target compounds against fluconazole-susceptible
pathogenic fungi.
N
N
N
OH
O
F
O
N
R
F
MIC80 (μg/mL)
Compd
R
C.alb
C.alb
C.par
C.gla
C.neo
A.fum
T.rub
M.gyp
Y0109 SC5314 22019
537
32609
7544 cmccftla cmccftla
a1
a2
2-Cl
3-Cl
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.25
0.25
0.25
0.125
0.125
0.125
0.25
0.5
1
2
8
4
2
1
2
2
1
1
4
4
2
1
1
1
2
2
1
1
2
1
2
2
1
1
2
2
2
1
a3
4-Cl
1
4
2
a4
2-F
0.0625 0.0625 0.0625
0.0313 0.0625 0.0625
0.0313 0.0625 0.0313
1
4
1
a5
3-F
0.5
0.5
1
4
1
a6
4-F
4
0.5
2
a7
2-CH3
3-CH3
4-CH3
4-CF3
4-OCF3
4-CN
2-F-4-Cl
0.0313 0.0625
0.125 0.25
0.0313 0.0625
0.125
0.25
0.125
0.25
0.5
4
a8
2
8
2
a9
0.125
0.25
0.5
0.5
1
4
1
a10
a11
a12
a13
a14
a15
a16
a17
a18
a19
a20
a21
a22
a23
0.125
0.125
0.25
0.5
4
1
1
4
1
0.0313 0.0625
0.125
0.5
0.125
0.5
0.5
2
4
0.5
1
0.25
0.5
0.25
0.5
4
2-Cl-4-F 0.125
3-F-4-Cl 0.125
0.25
0.25
0.5
0.5
1
16
16
16
8
1
0.25
0.5
2
1
3-Cl-4-F
2,3-diF
2,4-diF
2,5-diF
2,6-diF
3,4-diF
3,5-diF
0.5
0.5
1
2
0.25
0.25
0.25
0.125
0.125
0.125
0.25
0.125
0.125
0.5
0.5
1
1
0.125
0.125
0.125
0.25
0.25
0.25
0.25
0.25
0.5
0.5
1
8
2
0.5
2
8
1
0.25
0.5
2
16
16
8
1
2
2
0.25
0.25
0.125
0.25
2
2
2,4,6-triF 0.0625
0.125
0.125
1
4
1
a24 2,3,4,5-4F 0.0625
1
8
1
2,3,4,5,6-
a25
a26
0.0625
0.125
0.125
0.25
0.25
0.25
1
1
8
8
2
2
1
2
5F
H
0.0313 0.0625

FCZ
RCZ
∕
∕
0.25
0.5
1
2
1
>64
2
8
32
1
0.0625 0.0625
0.25
0.125
0.125
0.125
Abbreviations: C.alb, Candida albicans; C.par, Candida parasilosis; C.gla, Candida glabrata;
C.neo, Cryptococcus neoformans; A.fumi, Aspergillus fumigatus; T.rub, Trichophyton rubrum;
M.gyp, Microsporumgypseum; FCZ, Fluconazole; RCZ, Ravuconazole.
Table 2 In vitro antifungal activities of the target compounds against fluconazole-resistant
Candida albicans.
MIC80 (μg/mL)
Compd
a6
R
4-F
C.alb 100 C.alb 103
16
16
>64
8
8
16
>64
8
a9
4-CH3
4-OCF3
4-CN
2-F-4-Cl
2-Cl-4-F
∕
a11
a12
a13
a14
FCZ
8
8
8
16
>64
>64
Abbreviations: C.alb, Candida albicans; FCZ, Fluconazole.
Table 3 In vitro inhibition of CYP3A4 by representative compounds.
C.alb SC5314
CYP3A4
IC50 (µM)a
Compd
Selectivity indexb
MIC80 (μg/mL)
a6
0.0625
0.0625
0.125
1.18
2.49
18.88
39.84
8.80
a12
a23
1.10
RCZ
KCZ
0.0625
0.125
1.01
16.16
0.10
0.0125
Abbreviations: C.alb, Candida albicans; RCZ, Ravuconazole; KCZ, Ketoconazole.
a. Inhibition of CYP3A4 measured in microsomes obtained from pooled human hepatocytes.
b. In vitro selectivity calculated as CYP3A4 IC50 / C.alb SC5314 MIC80.
4. Results and discussion
As shown in Table 1, all the target compounds showed good or moderate activities against
the eight tested fungi compared with positive controls. The MIC80 values against C.alb Y0109 and
C.alb SC5314 are ranging from 0.0313 μg/mL to 0.5 μg/mL, thus indicating the antifungal
activities of these compounds were comparable or superior to FCZ and RCZ. For example,
compounds a5, a6, a7, a9, a12, and a26 exhibited MIC80 values of 0.0313 μg/mL against C.alb
Y0109. This concentration is lower than the MIC80 values of FCZ (MIC80 = 0.25 μg/mL) and RCZ

(MIC80 = 0.0625 μg/mL). On the C.par 22019 strains, except for compounds a11, a13, a16, a17
and a22 (MIC80 = 0.5 μg/mL), the MIC80 values of our compounds are equal to or lower than RCZ
(MIC80 = 0.25 μg/mL). Most of the compounds including fluconazole only showed moderate
activities against C.gla 537, C.neo 32609, T.rub and M.gyp. Among them, on the M.gyp strain,
compounds a6 and a12 with the MIC80 values of 0.5 μg/mL are 64-fold more potent than FCZ
(MIC80 = 32 μg/mL) and comparable to RCZ (MIC80 = 1 μg/mL). FCZ is inactive against A.fumi
7544, while our compounds showed moderate activities (MIC80 range: 4 μg/mL to 16 μg/mL).
Based on the antifungal activities data, preliminary SARs of these novel azoles were obtained.
In compounds a1-a9, we systematically synthesized derivatives with the ortho, meta and para
mono-substituted R groups (R = Cl, F, CH3, respectively). Compounds with F-substituted (a4-a6)
exhibited better antifungal activities against C.alb Y0109, C.alb SC5314 and C.par 22019 (MIC80
range: 0.0313 μg/mL to 0.0625 μg/mL) than compounds with Cl-substituted (a1-a3) (MIC80
=
0.125 μg/mL). CH3-substituted compounds (a7-a9) apart from a8 (m-CH3) showed better
antifungal activities against all the tested fungi compared with RCZ. These results indicate that the
introduction of mono-F and ortho-CH3, para-CH3 to the terminal phenyl group contributes to the
improvement of antifungal effect. Compounds a10-a12, a12 (p-CN) displayed better antifungal
activities against six pathogens (C.alb Y0109, C.alb SC5314 C.par 22019, C.gla 537, C.neo
32609 and M.gyp) than a10 (p-CF3), a11 (p-OCF3). On the other hand, different from our previous
results, increasing the number of fluorine atoms substituted on the phenyl ring (a13-a25) had less
effect on the improvement of antifungal activity. This point may attribute to the extension of the
side chain, so that the fluorine atom on the phenyl ring cannot form a hydrogen bond with
surrounding amino acid residues.
As more and more serious azole-resistance problems are emerging in clinic, it is also worth
further evaluating these novel synthesized compounds against fluconazole-resistant species of
Candida albicans. The inhibitory activities of six representative compounds, a6, a9, a11, a12, a13
and a14, were tested. Results are presented as MIC80 values in Table 2. The MIC80 values of FCZ
against C. albicans 100 and C. albicans 103 were >64 μg/mL. Except compound a11, the
remaining compounds exhibited moderate activities (MIC80 range: 8 μg/mL to 16 μg/mL).
Considering that triazole antifungal agents target fungal CYP51 protein and inhibit human
CYP enzymes, resulting in hepatotoxicity as a side effect, three representative compounds, a6,

a12, and a23, were tested for their inhibition of CYP3A4 in human hepatocytes microsomes. As
shown in Table 3, the IC50 of a6, a12, and a23 was 1.18 µM, 2.49 µM and 1.10 µM, respectively.
Selectivity index (SI) can indicate the safety of a compound to some extent15. The in vitro safety
profiles of them were superior to marketed azoles such as ketoconazole (CYP3A4 IC50 = 0.0125
µM, SI = 0.10) and comparable to ravuconazole (CYP3A4 IC50 = 1.01 µM, SI = 16.16).
In order to further investigate the interactions between the novel compounds and CYP51, a
molecular docking study was carried out. 2,4-difluorophenylisoxazole containing triazole
(compound a’14 in our previous work，Figure S1 in the supplementary material) and a6 were
docked into the active site of CYP51 (PDB ID: 5TZ1) using the SYBYL-X 2.0. As shown in Fig.3
(A), the triazoly group, hydroxyl group and difluorophenyl group of a’14 were considered as
forming nonbonding interactions with Hem601, Phe126, Thr311, Tyr132, and Thr122. The
isoxazole moiety as the side chain extended into CYP51 channel to form hydrophobic and van der
Waals interactions with surrounding residues Leu121 and Leu376, while the terminal
2,4-difluorophenyl group interacted with Met508, Phe380 and Phe233. Especially, due to the
length of side chain, its 2-fluorine atom forms a hydrogen bond with Tyr-118. In the case of a6
showed in Fig.3 (B), it shared a similar binding mode with a’14 in the active site. The introduced
methoxyl moiety appropriately stretched the length of side chain into CYP51 channel. Moreover,
unlike the ways that a’14 forms hydrogen bond, the oxygen atom of isoxazole moiety was found
to form a hydrogen bond with Tyr118 in the distance of 2.1 Å.
Fig.3. 3D presentation of docked triazoles in the active site of CYP51.
(A) The binding mode of compound a’14. (B) The binding mode of compound a6

5. Conclusion
In summary, based on our previous work, we further designed and synthesized a series of
triazole analogues by replacing 4-cyanophenylthioazole in ravuconazole with substituted
phenylisoxazole methoxyls. Most of the designed compounds exhibited moderate in vitro
antifungal activities against eight fungal isolates. Among them, compound a6 showed excellent
inhibitory activity against Candida albicans and Candida parasilosis with MIC80 values of 0.0313
μg/mL. Moreover, compounds a6, a9, a12, a13 and a14 were also tested against
fluconazole-resistant isolates, suggesting that the appropriate derivation on azole drugs might be a
useful approach to combat fungal resistant problem. SARs study demonstrates that mono-F to the
terminal phenyl ring is favorable to the antifungal activity. Docking study indicates that the
methoxyl motif forms a flexible side chain extending into CYP51 channel and supports the
oxygen atom of isoxazole to generate a key hydrogen-bonding interaction with Tyr118. The above
results show the introduction of isoxazole methoxyl moieties bearing medium side chains plays a
significant role in retaining the antifungal activities of target compounds. In addition, some
representative compounds exhibited low inhibition profiles for CYP3A4, indicating these new
triazole analogues have good safety properties. Further optimization work in our group is in
progress.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No.
81673280; 81830106) and National Mega-project for Innovative Drugs (No. 2019ZX09721001).
Supplementary information
Supplementary information associated with this article can be found in the online version.
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J.; Nguyen, N.; Qiu, J.; Olzog, M.; Reiff, E.; Thoraval, D.; Totleben, M.; Vanyo, D.; Vemishetti, P.;
Wasylak, J.; Wei, C., The Process Development of Ravuconazole: An Efficient Multikilogram Scale
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

Graphical Abstract
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Isoxazole methoxyl moiety
containing triazoles analogues
a1-a26
Isoxazole containing
triazole analogues
Ravuconazole: R1, R2 = 2, 4-diF
Isavuconazole: R1, R2 = 2, 5-diF
Twenty-six novel triazole analogues featuring isoxazole moieties were designed
and synthesized by replacing 4-cyanophenylthioazole moiety in ravuconazole with
substituted phenylisoxazole methoxyl. The in vitro antifungal activities demonstrate
the introduction of isoxazole methoxyl moiety bearing medium side chains is
beneficial for retaining the novel compounds antifungal activities.

Research Highlights
 Novel triazole analogues featuring isoxazole moieties were designed and
synthesized.
 All compounds showed potent activity against Candida spp. (MICs ≤0.5 μg/mL).
 The single-crystal data of compound 2 was obtained.
 Compound a6 showed excellent inhibitory activity against Candida albicans and
Candida parasilosis with MIC80 values of 0.0313μg/mL.