Design, synthesis, and in vitro evaluation of novel antifungal triazoles

Article abstract of DOI:10.1016/j.bmcl.2017.03.062

Twenty-nine novel triazole analogues of ravuconazole and isavuconazole were designed and synthesized. Most of the compounds exhibited potent in vitro antifungal activities against 8 fungal isolates. Especially, compounds a10, a13, and a14 exhibited superior or comparable antifungal activity to ravuconazole against all the tested fungi. Structure-activity relationship study indicated that replacing 4-cyanophenylthioazole moiety of ravuconazole with fluorophenylisoxazole resulted in novel antifungal triazoles with more effectiveness and a broader-spectrum.

Full text of DOI:10.1016/j.bmcl.2017.03.062

Accepted Manuscript
Design, Synthesis, and in vitro Evaluation of Novel Antifungal Triazoles
Fei Xie, Tingjunhong Ni, Jing Zhao, Lei Pang, Ran Li, Zhan Cai, Zichao Ding,
Ting Wang, Shichong Yu, Yongsheng Jin, Dazhi Zhang, Yuanying Jiang
PII:
S0960-894X(17)30309-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.062
BMCL 24814
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
30 November 2016
8 March 2017
22 March 2017
Please cite this article as: Xie, F., Ni, T., Zhao, J., Pang, L., Li, R., Cai, Z., Ding, Z., Wang, T., Yu, S., Jin, Y., Zhang,
D., Jiang, Y., Design, Synthesis, and in vitro Evaluation of Novel Antifungal Triazoles, Bioorganic & Medicinal
Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.03.062
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Design, Synthesis, and in vitro Evaluation of Novel
Antifungal Triazoles
FeiXiea,†, Tingjunhong Nia,†, Jing Zhaob,†, Lei Pangb, Ran Lia, Zhan Caia, ZichaoDinga,
Ting Wanga, ShichongYua, YongshengJina, DazhiZhanga,*, YuanyingJiangc,*
aDepartment of Organic Chemistry, School of Pharmacy, Second Military Medical
University, 325 Guohe Road, Shanghai 200433(China)
bSchool of Pharmacy, Fujian University of Traditional Chinese Medicine, 1 Yueyang
Road, Fuzhou 350112(China)
cCenter of New Drug research, School of Pharmacy, Second Military Medical
University, 325 Guohe Road, Shanghai 200433(China)
*Corresponding author
Zhang D.Tel.: 0084-21-81871226, E-mail address: zdzhang_yjhx@smmu.edu.cn
Jiang Y. Tel.:0084-21-81871357, E-mail address: jiangyy@smmu.edu.cn
†These authors contributed equally to this work.

Abstract
Twenty-nine novel triazole analogues of ravuconazole and isavuconazole were designed and
synthesized. Most of the compounds exhibited potent in vitro antifungal activities against 8 fungal
isolates. Especially, compounds a10, a13, and a14 exhibited superior or comparable antifungal
activity to ravuconazole against all the tested fungi. Structure-activity relationship study indicated
that replacing 4-cyanophenylthioazole moiety of ravuconazole with flourophenylisoxazole
resulted in novel antifungal triazoles with more effectiveness and a broader-spectrum.
Keywords: isoxazole; triazole; antifungal activity; structure-activity relationship; synthesis

Global mortality from fungal infection is over 1.35 million people per annum, far more than
are killed by malaria (~0.63 million).1 The high rate of mortality associated with invasive fungal
infections often exceeds 50%.2 Triazole antifungals still keep the leading and progressive position
in antifungal drug research and development. The increased fungal resistance triazole drugs3
makes it urgent to develop novel antifungal azoles with more effectiveness, lower toxicity and
broader-spectrum.
It is widely recognized that the azole antifungal agents bind with the active site of CYP51
(lanosterol 14α-demethylase) and block the synthesis of ergosterol.4 SAR studies5-7 have revealed
a pharmacophore of antifungal triazoles, which contains a triazole ring linked to a diflourophenyl
through a two-carbon chain, as shown in Fig 1. Optimization research on triazoleshas been mainly
focused on their side chains which play important roles to their druglikeness by affecting their
target binding and physicochemical properties.8
Fig 1. Structures oftriazole antifungal drugs and the designed compounds.
In 2015, isavuconazonium sulfate was first launched in the U.S. for the oral and intravenous
treatment of invasive aspergillosis and invasive mucormycosis. Another new drug candidates,
ravuconazole,is similar to isavuconazole with the difference of the flouro substitution on the pheyl
ring.(Fig 1). Both agents have a cyanophenylthizaole moiety as the side chain. Inspired by the

bioisosteric replacement of their thiazole moiety with is oxazole, a series of novel ravuconazole
analoguesa1-21and isavuconazole analogues b1-8 were designed and synthesized (Fig 1).
We constructed the isoxazolemoiety of the target compound (a1-21, b1-8) via 1,3-dipolar
cycloaddition9 of alkynes 7 with various substituted N-hydroxybenzimidoyl chloride 3 in presence
of triethylamine and ZnCl2 in tetrahydrofuran as shown in Scheme 1.
.
Scheme 1.Reagents and conditions:(i) NH2OH HCl, NaHCO3, H2O, methanol, r.t., 2 h; (ii) N-chlorosuccinimide,
dimethylformamide, 35℃, 2 h; (iii) Pd(CH3CN)2Cl2, THF, ZnEt2, PPh3, r.t., 1 h; (iv) DMSO, NaOH, 1H-1,2,4-triazole, 70℃, 4 h;
(v) THF, triethylamine, ZnCl2, 35℃, 16-20 h.
The intermediates 3 were prepared by chlorination of benzaldehyde oximes 2, which was
obtained by hydroxylamination of various substituted benzaldehydes 1. The alkynes 7 were
synthesized starting from 2-chloro-1-(2,4-difluorophenyl)ethanone4 for target compounds a1-21,
and from 2-chloro-1-(2,5-difluorophenyl)ethanone4 for compounds b1-8, respectively. Both the
starting materials 4were treated with (R)-but-3-yn-2-yl-methanesulfonate 5in the presence of
Pd(CH3CN)2Cl2, ZnEt2, and PPh3, to give intermediate 6, which underwent alkylation reaction
with 1,2,4-triazole to give the alkynes 7. Details of all stepand analysis data are described in
supporting information.X-ray crystallographic analysis clearly supports the chiral structure of the
intermediate alkynes 7a containing 2,5-difluorophenyl to construct a1-21. (Fig 2, Table S1 in the
Supplementary material).

Fig 2.The single-crystal structure of alkynes 7a.
The in vitro antifungal activities of compounds a1-21 and b1-8 were evaluated against eight
human pathogenic fungi including Candida albicans Y0109, Candida albicansSC5314, Candida
parapsilosis22019, Cryptococcus neoformans32605, Candida glabrata537, Aspergillus fumigatus
7544, Trichophyton rubrum cmccftla and Microsporumgypseumcmccftla. Antifungal drugs
ketoconazole, fluconazole and ravuconazole were used as controls. The MIC80 values of these
compounds were determined by the standard micro broth dilution method of the NCCLS.10
As shown in Table 1, compounds a11, a17, and b8 against Candida glabrata, and a12
against Candida albicans, exhibited the lowest MIC80 value of 0.0156 µg/mL. Compounds a1,
a2, a9, a10, a11, a12, a14, exhibited MIC80 value of 0.0313 µg/mL against 4 fungal isolates:
Candida albicansY0109, Candida albicansSC5314, Cryptococcus neoformans, and Candida
glabrata, respectively. This concentration is lower than the MIC80 values of the three control
drugs.
Table 1.In vitro antifungal activity of the target compounds.
MIC80 (µg/mL)
Compd
R
Y010
C.alb
SC53
2201
3260
cmcc
A.fum T.rub
cmc
M.gyp
C.alb
C.par
C.neo
537
C.gla
9
14
9
5
7544
ftla
cftla
4-Cl
4-F
0.0313
0.125
0.125
0.25
0.125
0.0625
0.125
0.125
0.125
0.0313
4.0
2.0
0.125
0.0625
1.0
0.5
a1
a2

4-CH3
4-CF3
a3
a4
0.25
0.125
0.125
0.125
0.25
0.0625
0.5
0.125
1.0
0.125
0.125
0.125
0.25
0.25
1.0
8.0
>8.0
>8.0
8.0
0.25
2.0
2.0
2.0
4-NO2
4-SO2CH3
4-CN
a5
0.125
0.125
0.25
0.5
0.5
1.0
2.0
a6
0.125
0.125
0.125
0.5
0.25
1.0
4.0
0.125
0.125
0.125
0.125
0.0313
0.25
0.0625
0.0625
0.0625
0.0313
0.0156
0.25
8.0
0.125
0.125
0.125
0.0625
0.25
0.5
1.0
a7
4-OCHF2
4-OCF3
2-F
a8
0.0625
0.0313
0.0625
0.125
0.0156
0.0625
0.0625
0.0625
0.0625
0.125
0.0625
0.0625
0.125
0.125
0.5
0.0625
0.125
0.0313
0.125
0.0313
0.0625
0.0625
0.0625
0.125
0.125
0.125
0.25
8.0
1.0
a9
8.0
4.0
0.125
0.125
0.5
2.0
0.5
a10
a11
a12
a13
a14
a15
a16
a17
a18
a19
a20
a21
b1
2-F-4-Cl
3-F-4-Cl
2,3-diF
2,4-diF
2,5-diF
2,6-diF
3,4-diF
3,5-diF
3,4-diCl
2,3,6-triF
2,4,6-triF
2-F
8.0
2.0
4.0
>8.0
2.0
0.25
0.125
0.125
0.125
0.125
0.25
0.5
0.125
0.125
0.125
0.125
0.125
0.125
0.25
0.125
0.0313
0.0625
0.0625
0.0156
0.0625
0.25
0.125
0.0625
0.125
0.125
0.5
0.5
0.5
2.0
4.0
1.0
2.0
1.0
4.0
8.0
4.0
0.25
0.5
2.0
>8.0
2.0
4.0
0.25
0.125
0.125
1.0
0.0625
0.0625
0.0625
0.0625
0.125
0.0625
0.125
0.0625
0.125
0.125
0.0625
0.5
0.0625
0.0625
0.0625
0.0625
0.125
0.0625
0.125
0.125
0.0313
0.0156
0.0625
2.0
0.0625
0.5
0.5
0.25
2.0
1.0
>8.0
>8.0
>8.0
4.0
0.5
>8.0
>8.0
>8.0
>8.0
>8.0
>8.0
>8.0
>8.0
4.0
2.0
4-F
0.125
0.5
0.125
0.5
0.25
2.0
1.0
b2
2,3-diF
2,4-diF
2,5-diF
2,6-diF
2-F-4-Cl
3,4-diF
⁄
8.0
b3
0.125
0.5
0.25
1.0
1.0
b4
1.0
1.0
8.0
>8.0
>8.0
8.0
b5
1.0
0.5
1.0
4.0
b6
0.25
0.5
1.0
1.0
b7
0.125
0.25
0.125
0.125
0.5
0.25
0.5
1.0
>8.0
1.0
b8
1.0
KCZ
FCZ
RCZ
⁄
0.25
2.0
>64.0
2.0
8.0
32.0
1.0
⁄
0.0625
0.0625
0.25
0.125
0.125
0.125
Abbreviations: C.alb: Candida albicans; C.par: Candida parasilosis; C.neo: Cryptococcus
neoformans; C.gla: Candida glabrata; A.fumi: Aspergillus fumigatus; T.rub: Trichophyton rubrum;
M.gyp:Microsporumgypseum; KCZ: ketoconazole; FCZ: fluconazole; RCZ: ravuconazole
The MIC80 values of compounds a10, a13, and a14, bearing 2-flourophenyl-,
2,3-diflourophenyl, and 2,4-diflourophenyl isoxazole, respectively, are equal to or lower than that
of ravuconazole against the tested 8 fungal isolates. Moreover, the other fluorophenylisoxazole
compound a15(2,5-diF),a16 (2,6-diF), a20 (2,3,6-triF),and a21 (2,4,6-triF) also showed very good

activity in general. On the contrary, the compounds without flouro substitution on the phenyl ring,
such as a3 (4-CH3), a5 (4-NO2), a6 (4-SO2CH3), and a7 (4-CN), shows lower activity than the
compounds with flourophenyl group. These results give us a clear conclusion that flourophenyl
group is quite favorable to the antifungal activity.
Since compounds a10, a2, a13-16, a11, and a17 in series a1-21 showed very good antifungal
activity, these active compounds were reconstructed by replacing their 2,4-diflourophenyl group
with 2,5-diflourophenyl group to give the series b1-8, respectively, inspired by the structure of
isavuconazole.
Compounds b1-8 exhibited excellent activity against Cryptococcus neoformansand Candida
glabratawith MIC80 values ranging from 0.0156 to 0.125µg/mL, which are much lower than that
of ravuconazole and fluconazole. However, most of the series b1-8 exhibited lower activity
against other 6 tested fungal isolates than ravuconazole.Even worse, most of their MIC80 values
against Aspergillus fumigatus and Microsporumgypseumare more than 8.0 µg/mL. Thus indicated
that 2,4-diflourophenyl group is more favorable to antifungal activity than 2,5-diflourophenyl
group.
In summary, we have synthesized a series of novel isoxazole-containing triazole analogues of
ravuconazole and isavuconazole. Most of the designed compounds exhibited potent in vitro
antifungal activities against 8 fungal isolates, especially, compounds a10, a13, and a14 exhibited
comparable or superior antifungal activity to ravuconazole. SAR study gave us a clear conclusion
that replacing 4-cyanophenylthioazole of ravuconazole with flourophenylisoxazole resulted in
more active and spectrum-widening antifungal agents.
Supplementary material
Supplementary material associated with this article can be foundin the online version.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 81673280;
21272270; 21072226; 815734738).
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Graphical Abstract
Fei Xiea,†, Tingjunhong Nia,†, Jing Zhaob,†, Lei Pangb, Ran Lia, Zhan Caia, Zichao Dinga,
Ting Wanga, Shichong Yua, Yongsheng Jina, Dazhi Zhanga,*, Yuanying Jiangc,*
N
N
N
N
N
N
OH
OH
N
CN
S
O
N
R
R1
R2
R1
R2
target compound:
a: R1, R2 = 2, 4-diF
b: R1, R2 = 2, 5-diF
Ravuconazole: R1, R2 = 2, 4-diF
Isavuconazole: R1, R2 = 2, 5-diF
Twenty-nine novel triazole analogues of ravuconazole and isavuconazole were designed and
synthesized. Structure-activity relationship study indicated that replacing 4-cyanophenylthioazole
of ravuconazole with flourophenylisoxazole resulted in antifungal triazoles with more
effectiveness and a broader-spectrum.